Developments in Plant Genetics and Breeding, 4
Biology of Brassica Coenospecies
Developments in Plant Genetics and Breeding 1A ISOZYMES IN PLANT GENETICS AND BREEDING, PART A edited by S.D. Tanksley and T.J. Orton 1983 x +516 pp. 1B ISOZYMES IN PLANT GENETICS AND BREEDING, PART B edited by S.D. Tanksley and T.J. Orton 1983 viii +472 pp. 2A CHROMOSOME ENGINEERING IN PLANTS: GENETICS, BREEDING, EVOLUTION, PART A edited by P.K. Gupta and T. Tsuchiya 1991 xv + 639 pp. 2B CHROMOSOME ENGINEERING IN PLANTS: GENETICS, BREEDING, EVOLUTION, PART B edited by T. Tsuchiya and P.K. Gupta 1991 vi + 630 pp. 3 GENETICS IN SCOTS PINE edited by M. Giertych and Cs. M,~tyas 1991 280 pp. 4 BIOLOGY OF BRASSICA COENOSPECIES edited by C. G6mez-Campo 1999 x + 490 pp.
Developments in Plant Genetics and Breeding, 4
Biology of Brassica Coenospecies edited by
Cesar Gbmez- Campo Departamento de Biologia Vegetal Universidad Po/itecnica de Madrid E. T.S. Ingenieros Agronomos Ciudad Universitaria Madrid, Spain
1999 ELSEVIER Amsterdam
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FOREWORD
Brassica crop species a n d their allies (Raphanus, Sinapis, Eruca, etc.) are i m p o r t a n t s o u r c e s of edible roots, stems, leaves, b u d s a n d inflorescences, as well as of edible or i n d u s t r i a l oils, c o n d i m e n t s a n d forage. Many well k n o w n n a m e s of p l a n t s or p l a n t p r o d u c t s , s u c h as kale, cabbage, broccoli, cauliflower, B r u s s e l s s p r o u t s , kohl-rabi, Chinese cabbage, turnip, rape, r u t a b a g a , swede, colza or rapeseed, canola, m u s t a r d , rocket, etc. are directly a s s o c i a t e d to this botanical group. M a n y are also a s s o c i a t e d to o u r everyday life. Historically, m o s t cultivated forms a n d some of their wild relatives were u s e d in medicine as anti-escorbutic; a m o d e r n version of their medicinal value is the anti-carcinogenic effect of some of their c o n s t i t u e n t s . Some m e m bers of the g r o u p have also proved to be very effective in the control of soil n e m a t o d e s . O t h e r s have been positively tested for the c o n c e n t r a t i o n of heavy metals from c o n t a m i n a t e d soils. O t h e r s are a c t u a l or potential s o u r c e s of natural anti-fungic s u b s t a n c e s . In turn, m a n y wild relatives have traditionally been collected for different p u r p o s e s . In m a n y cases, t h e s e are an a c t u a l or potential s o u r c e of useful genes to t r a n s f e r to the cultivated forms. The development of highly productive lines for seed oil, where u n p l e a s a n t or u n h e a l t h y c o m p o n e n t s are completely removed, h a s rocketed the economic importance a n d the acreage of some Brassica crops. In some c o u n t r i e s of the n o r t h e r n t e m p e r a t e zone, for instance, r a p e s e e d occasionally displaces w h e a t from the first place, d e p e n d i n g u p o n the yearly prices. The scientific i n t e r e s t for this botanical g r o u p h a s r u n parallel to its economical i m p o r t a n c e , a n d r e s e a r c h a c h i e v e m e n t s in o u r d a y s would have certainly a p p e a r e d u n i m a g i n a b l e only two d e c a d e s ago. As the end of the c e n t u r y a p p r o a c h e d , entirely new fields (transformation, somatic fusion, etc.) have been a d d e d to the classical ones. T h u s , n o b o d y can d o u b t the o p p o r t u n e n e s s of this book, in which the m o s t recent a d v a n c e s in the biology of this group are compiled a n d p r e s e n t e d . The a u t h o r s a n d the editor only expect to have m a d e a useful w o r k which, at least partially, is in line with the above brilliant c i r c u m s t a n c e s . The editor w i s h e s to t h a n k Dr. S. P r a k a s h who first s u g g e s t e d the idea of p r e p a r i n g this book a n d collaborated in its initial planning. He is also very grateful to Miss E s p e r a n z a Garcia-Lanza, for her valuable help in the edition work. The editor
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vii
CONTENTS Chapter 1. TAXONOMY (C. G6mez-Campo) Cultivated B r a s s i c a species B. o l e r a c e a wild relatives The genus B r a s s i c a Other related genera The tribe B r a s s i c e a e References
3 4 12 14 16 19 23
Chapter 2. ORIGIN a n d DOMESTICATION (C. G6mez-Campo and S. Prakash) The phylogeny of B r a s s i c a and allied genera Domestication of cultivated brassicas and allies References
33 34 43 52
Chapter 3. CYTOGENETICS (by S. Prakash, Y. Takahata, P. B. Kirti and V. L. Chopra) T h e B r a s s i c a coenospecies Crop brassicas: cytogenetic architecture Genome manipulation Cytogenetics of wild allies: wide hybridizations Introgression of genes Cytoplasm divergence and genome homoeology Chromosome addition lines References
59 60 65 68 74 84 84 87 90
Chapter 4. SOMATIC HYBRIDIZATION (K. Glimelius) Protoplast technology Somatic hybrids produced between different B r a s s i c a species Intergeneric somatic hybrids within the tribe B r a s s i c e a e Limited gene transfer via protoplast fusion Cytological investigations using in s i t u hybridization The utilization of protoplast fusion to modify the cytoplasm Modification of cytoplasmic traits via protoplast fusion Conclusions References
107 108 110 116 123 124 126 132 135 136
Chapter 5. SELF-INCOMPATIBILITY (M. Watanabe and K. Hinata) Morphology and physiology Classical genetics and dominance relationships The S-multigene family Signal perception and signal transduction Molecular analysis of self-compatibility
149 149 150 151 160 164
viii Evolutionary aspects Related studies with future prospects References
165 166 168
Chapter 6. MALE STERILITY (R. Delourme and F. Budar) Genic male sterility Cytoplasmic male sterility Use for the production of commercial hybrids References 203
185 185 186 198
Chapter 7. GENOME STRUCTURE a n d MAPPING (C. F. Quiros) Linkage m a p s Structure of the B r a s s i c a genomes Cyclic amphiploidy and the origin and evolution of the B r a s s i c a species A r a b i d o p s i s as a model for a simpler genome Applications of the m a p s in breeding References
217 218 224
Chapter 8. IIAPLOIDY (C. E. Palmer and W. A. Keller) Historical overview Methodology Factors influencing microspore culture Developmental aspects of microspore embryogenesis Utilization of microspore-derived embryos of B r a s s i c a Conclusions and future prospects References
247 248 249 249 258 262 267 268
Chapter 9. G E ~ I C
Gene transfer methods Types of genes transferred Field tests of transgenic plants Legal issues Transgenic B r a s s i c a crops now being commercialized F u t u r e prospects References
287 287 288 290 293 300 300 302 303
Chapter I0. C,IIIt, M I C A I , COMPOSITION (E. A. S. Rosa) The importance of the B r a s s i c a and allies in h u m a n and animal diet The chemical composition of B r a s s i c a crops General components Secondary plant metabolites: the glucosinolates Other compounds References
315 318 319 319 323 342 346
ENGIgEERING (E. D. Earle and V. C. Knauf)
B r a s s i c a species transformed
232 234 234 236
ix C h a p t e r 11. PHYSIOLOGY {P. Hadley and S. Pearson) Germination Vegetative growth The transition from vegetative to reproductive development Hormonal control of flowering in Brassica Progress to crop m a t u r i t y Yield determining factors References
359 359 360 361 365 365 366 369
C h a p t e r 12. DISEASES (J. P. Tewari and R. F. Mithen) Blackspot or grey leaf c a u s e d by Altemaria brassicae a n d d a r k leaf spot caused by A. brassicicola Stem c a n k e r or blackleg caused by Leptosphaeria maculans Stem rot c a u s e d Sclerotinia sclerotiorum White r u s t a n d s t a g h e a d disease caused by Albugo candida Light leaf spot c a u s e d by Pyrenopeziza brassicae Downy mildew c a u s e d by Peronospora parasitica Verticillium wilt c a u s e d by Verticillium dahliae Clubroot c a u s e d by Plasmodiophora brassicae Other fungal diseases References
375 375 378 384 386 388 389 390 391 393 394
C h a p t e r 13. BREEDING: AN OVERVIEW (H. C. Becker, H. L6ptien a n d G. R6bbelen Breeding objectives Genetical resources Operational steps for breeding Breeding m e t h o d s Breeding results F u t u r e developments References
413 415 425 427 431 442 445 448
C h a p t e r 14. GENETIC RESOURCES (I. W. B o u k e m a a n d T. J. L. v a n Hintum) Strategies for conservation Availability S u m m a r i e s of Brassica genetic resources collections I m p o r t a n t collections Concluding r e m a r k s References
461 462 467 467 473 475 476
SUBJECT INDEX
481
Biology of Brassica Coenospecies
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Biology of Brassica Coenospecies C. G6mez-Campo (Editor) 91999 Elsevier Science B.V. All rights reserved.
TAXONOMY C6sar G 6 m e z - C a m p o
Dept. Biologia Vegetal, Universidad Polit~cnica de Madrid. 28040 - Madrid. Spain. Coenospecies is not a t a x o n o m i c category b u t a cytogenetical concept whose taxonomic c o u n t e r p a r t could c o n s i s t of a g r o u p of "allies" or "relatives" to a given taxon. Here, it is applied to the species a n d s u b s p e c i e s of the g e n u s Brassica, together with those of its closest related genera. Where s h o u l d the limits be placed? Special e m p h a s i s is p u t on g e n e r a Erucastrum,
Diptotaxis, Hirschfeldia, Eruca, Sinapis, Sinapidendron, Coincya, Raphanus and Trachystoma, together with Brassica itself. However, a brief i n c u r s i o n into the rest of the tribe Brassiceae will cover o t h e r possible wild relatives.
Some y e a r s ago, a book edited by T s u n o d a , H i n a t a a n d G 6 m e z - C a m p o (1980) was p u b l i s h e d with a similar scope to the p r e s e n t one. Almost two d e c a d e s later, trying to review new d e v e l o p m e n t s c o n c e r n i n g the t a x o n o m y of this group, p r o d u c e s a mixture of positive, negative a n d c o n f u s i n g feelings. On the one h a n d , a lot h a s been written on the s u b j e c t a n d references at the end of this article include only a part, t h a t t h o u g h t to be the m o s t significant, of all w h a t h a s been p r o d u c e d in this period. On the o t h e r h a n d , it is p e r h a p s too early to profit a d e q u a t e l y from the c o n t r i b u t i o n s t h a t cytogenetic a n d m o l e c u l a r m e t h o d s are m a k i n g to the knowledge of this botanical group as they do for m a n y others. In short, this book m a y be o p p o r t u n e as a whole, b u t a c h a p t e r on the t a x o n o m y of Brassica a n d allies would have benefited from being p o s t p o n e d p e r h a p s a decade or so to receive - a n d digest- f u r t h e r i n p u t s from o t h e r fast developing fields to become more useful a n d complete. A significant p a r t of recent w o r k on the t a x o n o m y of Brassica coenospecies is directly or indirectly related to a series of m o n o g r a p h i c t h e s e s which are worth listing together in advance" Baillargeon (1986) on Sinapis, Gladis (1989), E a s t w o o d (1996), Lann6r (1997) a n d Ls (1997) on Brassica, Leadlay (see Leadley a n d Heywood, 1990) on Coincya, Martinez-Laborde (1988a) on Diplotaxis, Pistrick (1987) on Raphanus, S a l m e e n (1979) on Brassica a n d S~nchez-Y61amo (1990) on the Diplotaxis-Erucastrum-Brassica complex. In addition to c u r r e n t literature a n d several new local floras, the publication of E u c a r p i a Cruciferae Newsletter h a s proved to be a positive factor in
s t i m u l a t i n g r e s e a r c h a n d c o m m u n i c a t i o n in this discipline a n d in m a n y ot h e r s related to Brassica. At the s a m e time, a n u m b e r of related w o r k s h o p s a n d meetings, periodic or not, at least in three c o n t i n e n t s , have also played a significant part. This c h a p t e r s t a r t s with a s h o r t reference to the already well known six cultivated species of the classical U-triangle (see c h a p t e r 3) to proceed to the wild relatives of Brassica oleracea, t h e n to the g e n u s Brassica as a whole, to its m o s t related g e n e r a and, finally, beyond the coenospecies limits, giving a few c o m m e n t s on the tribe Brassiceae as a whole. U p d a t e d s y n o p s e s are provided for the s u b g e n e r a , sections, species a n d s u b s p e c i e s of the ten genera m e n t i o n e d so far (Table 1.1) as well as for all the tribe g e n e r a (Table 1.2).
Cultivated B r a s s i c a species Regarding the infraspecific variability of B. oleracea, there is an unwritten a g r e e m e n t to describe this with t a x o n o m i c categories below the range of s u b s p e c i e s . This is r e a s o n a b l e b e c a u s e a l t h o u g h this variability is very high, it originated u n d e r d o m e s t i c a t i o n in a relatively s h o r t period of time. A detailed a c c o u n t is b e y o n d the scope of this c h a p t e r , b u t the r e a d e r is referred to Dickson a n d Wallace (1986) or to several articles grouped within the section "cole crops" in Kalloo a n d Berg (1993). A p r e l i m i n a r y information can be found in tables 10.1, 10.3 a n d 10.6 within the p r e s e n t book.
B. oleracea wild types are only truly wild along the E u r o p e a n Atlantic c o a s t s a n d t h e y have often been referred to var. sylvestris L. They are close to cultivated forms (Gustafsson a n d L a n n ~ r - H e r r e r a , 1997a) and almost morphologically i n d i s t i n g u i s h a b l e from m a n y kales. O t h e r so called "sylvestris" in other p a r t s of the world, are mere e s c a p e s from cultivation. Two o t h e r closely related t a x a originally c o n s i d e r e d as species, n a m e l y B. bourgeaui a n d B. alboglabra, s h o u l d p r o b a b l y merit an infraspecific s t a t u s within B. oleracea variation. B. bourgeaui, from the C a n a r y Islands, was included in the g e n u s Sinapidendron in early literature a n d t h o u g h t to be extinct for m a n y years. It was re-found by Borgen et al. (1979) who ascribed it to the n = 9 Brassica group. They only found two p l a n t s in La Palma Island. Today, m o s t opinions e m p h a s i z e its close relation to B. oleracea (Lann~r, 1998) t h o u g h , p e r h a p s , some possible differential c h a r a c t e r s in leaves a n d pod b e a k s s h o u l d be s t u d i e d more carefully. A second population h a s been found more recently (Marrero, 1989) in E1 Hierro Island. B. alboglabra, a n old cultivated form from China, h a s puzzled r e s e a r c h e r s for years bec a u s e of the p h y t o g e o g r a p h i c a l problem it poses. B o t h m e r et al. (1995) s u m marize the r e a s o n s w h y it s h o u l d be kept within B. oleracea. Although it h a d always been believed to have r e a c h e d C h i n a from the M e d i t e r r a n e a n region, conclusive evidence only a p p e a r e d w h e n H a m m e r et al. (1992) found it locally cultivated in S o u t h Italy. Flowers are n o r m a l l y white a l t h o u g h they m a y be yellow a n d they distinctively a p p e a r in early wintertime.
T a b l e 1 . 1 A t a x o n o m i c synopsis of B r a s s i c a and its allied genera with indication o f subgenera, sections, species and subspecies. B R A S S I C A L.
Subgen. Brassica Sect. Brassica B. oleracea L. B. montana Pourret B. incana Ten. subsp, incana B. i. subsp, cazzae (Ginz. and Teyb.) Trinajstic B. villosa Biv. subsp, villosa B. v. subsp, bivoniana (Mazzola and Raimondo) R.and M. B. v. subsp, drepanensis (Caruel) Raimondo and Mazzola B. v. subsp, tinei (Lojac.) Raimondo and Mazzola B. rupestris Rafin subsp, rupestris B. r. subsp, brevisiliqua Raimondo and Mazzola B. r. subsp, hispida Raimondo and Mazzola B. macrocarpa Guss. B. insularis Moris B. cretica Lam. subsp, cretica B. c. subsp, aegaea (Heldr. and Hal.) Snogerup et al. B. c. subsp, laconica Gustafsson and Snogerup B. botteri Vis. subsp, botteri B. b. subsp, mollis (Vis.) Trinajstic B. hilarionis Post. B. carinata Braun B. balearica Pets.
Sect. Rapa (Miller) Salmeen B. rapa L. subsp, rapa B. r. subsp, campestris (L.) Clapman B. r. subsp, chinensis (L.) Hanelt B. r. subsp, dichotoma (Roxb.) Hanelt B. r. subsp, narinosa (Bailey) Hanelt B. r. subsp, nipposinica (Bailey) Hanelt B. r. subsp, pekinensis (Lour.) Hanelt B. r. subsp, trilocularis (Roxb.) Hanelt B. napus L. B. juncea (L.) Czern.
Sect. Micropodium DC. B. fruticulosa Cyr. subsp, fruticulosa B. f subsp, djafarensis Blanco and Matarranz B . f subsp, dolichocarpa Emberger and Maire B . f subsp, glaberrima (Pomel) Maire
B. f subsp, mauritanica (Cosson) Maire B. f subsp, numidica Maire B. f subsp, pomeliana Maire B. f subsp, radicata (Desf.) Maire B. nigra (L.) Koch B. cossoniana Boiss. and Reuter B. spinescens Pomel B. maurorum Durieu B. procumbens (Poiret) O.E.Schulz B. cadmea O.E.Schulz B. desertii Danin and Hedge Sect. Brassicoides Boiss. B. deflexa Boiss.
Sect. Sinapistrum Willkomm B. barrelieri (L.) Janka B. oxyrrhina Coss. B. tournefortii Gouan
Sub~en. Brassicaria (Godr.) G6mez-Campo Sect. Brassicaria (Godr.) Cosson B. repanda (Willd.) DC. subsp, repanda B.r. subsp, africana (Maire) Greuter and Burdet B.r. subsp, almeriensis G6mez-Campo B.r. subsp, blancoana (Boiss.) Heywood B.r. subsp, cadevallii (Font Quer) Heywood B.r. subsp, cantabrica (Font Quer) Heywood B.r. subsp, confusa (Emb. and Maire) Heywood B.r. subsp, galissieri (Giraud) Heywood B.r. subsp, glabrescens (Poldini) G6mez-Campo B.r. subsp, gypsicola G6mez-Campo B.r. subsp, latisiliqua (Boiss. and Reut.) Heywood B.r. subsp, nudicaulis (Lag.) Heywood B.r. subsp, saxatilis (DC.) Heywood B.r. subsp, silenifolia (Emberger) Greuter and Burdet B. desnotesii Emb. and Maire B. gravinae Ten. B. jordanoffii O.E.Schulz B. loncholoma Pomel B. nivalis Boiss. and Heldr. B. elongata Ehrh. subsp, elongata B. e. subsp, integrifolia (Boiss.)Breistr. B. e. subsp, imdrahsiana Quezel B. e. subsp, pinnatifida (Smal'g) Greuter and Burdet B. e. subsp, subscaposa Maire and Weiller B. setulosa (Boiss. and Reuter) Cosson
B. somaliensis Hedge and Miller
Sect. Nasturtiops (Pomel) Salmeen B. souliei (Batt.) Batt. subsp, souliei B. s. subsp, amplexicaulis (Desf.) Greuter and Burdet B. dimorpha Coss. and Dur.
ERUCASTRUM C. Presl. E. gallicum O.E.Schulz E. nasturtiifolium (Poiret) O.E.Schulz subsp, nasturtiifolium E. n. subsp, sudrei Vivant E. leucanthum Coss. and Dur. E. palustre (Pir.) Vis. E. virgatum C.Presl. subsp, virgatum E. v. subsp, baeticum (Boiss.) G6mez-Campo E. v. subsp, brachycarpum (Rouy) G6mez-Campo E. v. subsp, pseudosinapis (Lange) G6mez-Campo E. varium Durieu subsp, varium E. v. subsp, mesatlanticum Maire and Wilczek E. v. subsp, subsiifolium Maire E. littoreum (Pau and F.Quer) Maire subsp, littoreum E. l. subsp, brachycarpum (Maire and Weiller)G6mez-Campo E. l. subsp, glabrum (Maire) G6mez-Campo E. rifanum (Emb. and Maire) G6mez-Campo E. elatum (Ball.) O.E.Sehulz E. brevirostre (Maire) G6mez-Campo E. canariense Webb and Berth. E. cardaminoides (Webb ex Christ.) O.E.Schulz E. ifniense G6mez-Campo E. arabicum Fisch. and Mey. E. elgonense Jonsell E. meruense Jonsell E. abyssinicum (A. Rich.) O.E.Schulz E. pachypodum (Chiov.) Jonsell E. rostratum (Balf. f.)G6mez-Campo E. strigosum (Thunb.) O.E.Schulz E. griquense (Brown) O.E.Schulz DIPL 0 TAXIS D C.
Subgen. Diplotaxis D. tenuifolia (L.)DC. subsp, tenuifolia D. t. subsp, cretacica (Kotov)Sobrino-Vesperinas D. muralis (L.) DC. subsp, muralis D. m. subsp, ceratophylla (Batt.) Mart.-Lab. D. viminea (L.) DC. D. simplex (Viv.) Spr.
Subgen. Hesperidium (DC.) Mart.-Lab. D. harra (Forsk.) Boiss. subsp, harra D. h. subsp, confusa Mart.-Lab. D. h. subsp, crassifolia (Rafin) Maire D. h. subsp, lagascana (DC.) O.Bol6s and J.Vigo D. antoniensis Rustan D. glauca (Schmidt) O.E.Schulz D. gorgadensis Rustan subsp, gorgadensis D. g. subsp, brochmanii Rustan D. gracilis (Webb) O.E.Schulz D. hirta (Chev.) Rustan and Borgen D. sundingii Rustan D. varia Rustan D. vogelii (Webb) Cout. D. pitardiana Maire D. acris (Forsk.) Boiss. D. villosa Boul. and Jail. D. griffithii (Hook.f. and Thorns.) Boiss. D. nepalensis Hara Subgen. Rh~nchocarpum (Prantl) Mart.-Lab. Sect. Rhynchocarpum Prantl D. assurgens (Del.) Gren. D. tenuisiliqua Del. subsp, tenuisiliqua D. t. subsp, rupestris (J. Ball.) Mart.-Lab. D. brachycarpa Godr. D. virgata (Cav.) DC. subsp, virgata D. v. subsp, australis Mart.-Lab. D. v. subsp, rivulorum (Br.-B1. and Maire) Mart.-Lab. D. v. subsp, sahariensis (Coss.) Mart.-Lab. D. berthautii Br.-B1. and Maire D. catholica (L.) DC. D. ollivieri Maire D. siifolia G. Kunze. subsp, siifolia D. s. subsp, bipinnatifida (Coss.) Mart.-Lab. D. s. subsp, vicentina (P.Cout.) Mart.-Lab. Sect. Heterocarpum Mart.-Lab. D. D. D. D.
ibicensis (Pau) G6mez-Campo siettiana Maire brevisiliqua (Coss.) Mart.-Lab. ilorcitana (Sennen) Aedo and Mart.-Lab.
Sect. Erucoides Mart.-Lab. D. erucoides (L.) DC. subsp, erucoides D. e. subsp, longisiliqua (Coss.)G6mez-Campo
SINAPIS L.
Sect. Sinapis S. alba L. subsp, alba S. a. subsp, mairei (H.Lindb.) Maire S. a. subsp, dissecta (Lag.) Bonnier S. flexuosa Poir.
Sect. Ceratosinapis DC. S. arvensis L. subsp, arvensis S. a. subsp, allioni (Jacq.) Baillargeon S. a. subsp, nilotica (O.E.Schulz) Baillargeon
Sect. Hebesinapis DC. S. pubescens L. subsp, pubescens S. p. subsp, virgata (Batt.) Baillargeon S. boivinii Baillargeon S. indurata Coss. S. aristidis Coss.
Sect. Chondrosinapis O.E.Schulz S. aucheri (Boiss.) O.E.Schulz ER UCA Mill. E. vesicaria (L.)Cav. subsp, vesicaria E. v. subsp, sativa (Miller) Thell. E. v. subsp, pinnatifida (Desf.) Emb.and Maire COINCYA Rouy C. richeri (Vill.) Greuter and Burdet C. wrightii (O.E.Schulz) Stace C. monensis (L.) Greuter and Burdet subsp, monensis C.m. subsp, cheiranthos (Vill.) Aedo, Leadlay and Mufioz-Garm. C.m. subsp, nevadensis (Willk.) Leadlay C.m. subsp, orophila (Franco) Aedo, Leadlay and Mufioz-Garm. C.m. subsp, puberula (Pau) Leadlay C. transtagana (Cout.) Clem.-Mufioz and Hern.-Bermejo C. longirostra (Boiss.) Greuter and Burdet C. rupestris Porta and Rigo subsp, rupestris C. r. subsp, leptocarpa (Gonz.-Albo) Leadlay RAPHANUS L. R. raphanistrum L. subsp, raphanistrum R. r. subsp, landra (DC.) Bonnier and Layens R. r. subsp, maritimus (Sm.) Thell. R. r. subsp, microcarpus (Lange) Thell.
10 R. r. subsp, rostratus (DC.) Thell. R. sativus L. HIRSCHFELDIA Moench
H. incana (L.) Lagr6ze-Fossat subsp, incana H. i. subsp, incrassata (Thell.) G6mez-Campo SINAPIDENDR ON Lowe
S. angustifolium (DC.) Lowe S. frutescens (Sol.) Lowe subsp.frutescens S. f subsp, succulentum (Lowe) Rustan S. gymnocalyx (Lowe) Rustan S. rupestre Lowe S. sempervivifolium Mnzs. TRACHYSTOMA O.E. Schulz
7". aphanoneurum (Maire and Weiller) M.and W. 7". balli O.E.Schulz T. labasii Maire
The s a m e r e a s o n to avoid u s i n g the s u b s p e c i e s category in B. oleraceaa r e c e n t history in cultivation - s h o u l d be logically applied to B. napus, since this is t h o u g h t to be a n amphidiploid derived from crosses B. oleracea x B. rapa which, m o s t likely, o c c u r r e d in cultivation. However, the s u b s p e c i e s category h a s b e e n traditionally applied to some g r o u p s of B. n a p u s cultivars s u c h as napobrassica, rapifera, pabularia, etc. (not included in Table 1.1). Infraspecific variability of B. rapa (syn. B. campestris) is given a different t r e a t m e n t , p e r h a p s in a c c o r d a n c e with its older history in domestication. For a time, the species category was widely applied (B. chinensis, B. p e k i n e n s i s , B. japonica, etc.) b u t this is now c o n s i d e r e d an excessive license. The s u b s p e c i e s r a n k is only r e c o m m e n d e d for the m o s t significant v a r i a n t s (Oost, 1985) t h o u g h a n intensification of the u s e of n a m e s according to the I n t e r n a t i o n a l Code of N o m e n c l a t u r e for Cultivated Plants (Trehane et al., 1995) would be desirable (Oost, p e r s o n a l c o m m u n i c a t i o n ) . The prioritary n a m e for the species is B. rapa (after the first c o m b i n a t i o n by Metzger in 1833) while the n a m e s u b s p , c a m p e s t r i s s h o u l d be reserved for non specialized semi-wild forms with slender root (Toxopeus et al., 1984). As a m a t t e r of fact, B. c a m p e s t r i s h a s been a very widely u s e d n a m e and, for many, it m i g h t take some effort to a d a p t to the new correct n o m e n c l a t u r e . In the Far East, d o m e s t i c a t i o n led to a wide variety of forms, parallel to those obtained in the M e d i t e r r a n e a n with B. oleracea (except cauliflower!). In the West, the i m p o r t a n c e of B. rapa h a s also been c o n s i d e r a b l e b u t more specialized final
11 forms developed d u e to the "competition" by a n extensive u s e of B. oleracea. The n u m b e r of e p i t h e t s u s e d , either from the E a s t or from the West is very high b u t Oost (1985) s u g g e s t s t h a t only 10-12 s h o u l d merit subspecific rank. Table 1.1 only includes the seven t h a t have been formally c o m b i n e d (Hanelt, 1986), b u t o t h e r epithets as dubiosa, japonica, perviridis, purpurar/a, etc. m i g h t p e r h a p s be equal c a n d i d a t e s for as m a n y s u b s p e c i e s . Upper a c u t e widely e m b r a c i n g leaves a n d long n a r r o w b e a k s in the pods are the best c h a r a c t e r s to d e t e c t B. rapa individuals (B. n a p u s is more g l a b r o u s a n d glaucous, its b e a k is thicker a n d its leaves are only loosely embrasing). An i n t e r n a t i o n a l effort to e s t a b l i s h a c c e p t a b l e criteria in the n o m e n c l a t u r e of Brassica crops s e e m s highly n e c e s s a r y .
B. juncea, a n a m p h i d i p l o i d B. rapa x B. nigra is a n i m p o r t a n t source of edible oil in S. Asia, of vegetables in C h i n a a n d of m u s t a r d c o n d i m e n t elsewhere in the World. Its variation h a s b e e n t h o r o u g h l y s t u d i e d over m a n y y e a r s a n d s o m e s u b s p e c i e s (juncea, crispifolia, foliosa, integrifolia, napiformis; not included in Table 1.1) have often been recognized. Some r e c e n t DNA a n a lyses are of relevance for its taxonomic differentiation (see below). The book by C h o p r a a n d P r a k a s h (1996) is mostly c e n t e r e d on this species a n d is a good source of l i t e r a t u r e references. B. carinata derives by a m p h i d i p l o i d y from one or several B. oleracea x B. nigra c r o s s e s a n d is cultivated locally in E t h i o p i a where it h a s a wide a r r a y of uses" s o u r c e of oils a n d spices, medicinal, vegetable, etc. The valid n a m e for this species is the widely u s e d B. carinata B r a u n a n d not its alternative B. integrifolia (West) Rupr., as it h a s been s o m e t i m e s p r o p o s e d (see Jonsell, 1982, p. 69). IBPGR, now IPGRI (International Plant Genetic R e s o u r c e s Institute) (1987, 1990) h a s p u b l i s h e d d e s c r i p t o r s for B. rapa a n d B. oleracea (+ R a p h a nus) respectively with the aim of helping p r o p e r c h a r a c t e r i z a t i o n of cultivars in this group of species. The six B r a s s i c a species of the U-triangle are u s u a l l y referred to as "crop b r a s s i c a s " , a f o r t u n a t e d e n o m i n a t i o n b e c a u s e B r a s s i c a species outside this g r o u p have a limited a g r i c u l t u r a l i m p o r t a n c e (only B. tournefortii does not belong to the U-triangle a n d is cultivated for oil in India) . However, it s h o u l d be n o t e d t h a t the correct Latin p l u r a l for B r a s s i c a is B r a s s i c a e (not "Brassicas"), so t h a t "brassicas" s h o u l d be only u s e d as a c o m m o n nonL i n n e a n n a m e in o t h e r l a n g u a g e s , w i t h o u t italics or a n initial capital. For f u r t h e r r e a d i n g on this group of species the review by P r a k a s h a n d H i n a t a (1980), is a u s e f u l d a t a source on its biology a n d use. F u r t h e r useful reviews in the s a m e line c a n be found in C h o p r a a n d P r a k a s h (1990, 1996). The infra-specific variability of these cultivated species h a s b e e n the object of diverse molecular, biochemical or morphologically b a s e d a n a l y s e s within the last decade either for B. oleracea (Kresowich et al., 1992; Dias et al., 1992; Dias et al., 1993; Arfls et al., 1987) for B. rapa (Lamboy et al., 1994) or for B. j u n c e a (Jain et al., 1994; B h a t i a et al., 1995).
12
B. o l e r a c e a
wild relatives
B. oleracea, a n Atlantic plant, h a s a n u m b e r of closely related species which grow a r o u n d the M e d i t e r r a n e a n basin. All of t h e m have n = 9 chrom o s o m e s a n d are m o r e or less interfertile a m o n g t h e m s e l v e s a n d with B. oleracea. Knowledge of these h a s been s t i m u l a t e d in the r e c e n t y e a r s as a c o n s e q u e n c e of active IBPGR s u p p o r t e d seed collecting activities (G6mezC a m p o a n d G u s t a f s s o n , 1991) which supplied not only living material b u t also a b u n d a n t geographical a n d ecological data. An i m p o r t a n t review by S n o g e r u p et al. (1990) describes with m u c h detail the t a x o n o m y a n d geograp h y of these taxa. These a u t h o r s propose some n o m e n c l a t u r a l readjustm e n t s , m a i n l y for B. cretica subspecies. The m a x i m u m morphological diversity of the group clearly o c c u r s in Sicily where at least four species - B. macrocarpa, B. villosa, B. rupestris a n d B. incana, from west to e a s t - are present. R a i m o n d o a n d Mazzola (1997), confer the r a n k of s u b s p e c i e s to some other Sicilian t a x a w h i c h are closely related to either B. rupestris or B. villosa (see Table 1.1).
The affinities a m o n g this group of species have been studied from different points of view. S t o r k et al. (1980) describe in detail a n u m b e r of seed c h a r a c t e r s a n d m i c r o - c h a r a c t e r s s u c h as t e s t a layers, surface s t r u c t u r e s a n d mucilage cells t h r o u g h o u t the species complex. G 6 m e z - C a m p o et al. (1999) c o m p a r e a n o t h e r m i c r o - c h a r a c t e r , the morphology of epicuticular wax c o l u m n s , a n d find a distinctive type for B. oleracea, B . a l b o g l a b r a a n d B. bourgaei a n d two o t h e r s for the other wild species. Mithen et al. (1987) analyzed eighteen p o p u l a t i o n s belonging to eleven specific or subspecific taxa for their c o n t e n t s of nine glucosinolates. Some taxonomically meaningful differences were found for wild vs. cultivated B. oleracea, as well as a m o n g wild Sicilian species, a n d also for S a r d i n i a n vs. T u n i s i a n p o p u l a t i o n s of B. insular/s. Aguinagalde et al. (1992) u s e d flavonoids, seed proteins a n d five isozyme s y s t e m s a n d found a comparatively high p h y t o c h e m i c a l diversity in B. cretica, even h i g h e r t h a n for the Sicilian group of species. No differences were found between wild a n d cultivated B. oleracea. In t u r n , B. bourgeaui closely r e s e m b l e s the g r o u p formed by B. oleracea a n d B. m o n t a n a (all lacking isor h a m n e t i n ) , while B. oleracea a n d B. alboglabra, considered co-specific by m o s t a u t h o r s , c a n be d i s t i n g u i s h e d by their flavonoid p a t t e r n s . S t u d i e s with RAPDs a n d isozymes on twenty-two p o p u l a t i o n s belonging to fifteen specific a n d sub-specific t a x a (Ls a n d Aguinagalde, 1998 a,b) clearly d i s t i n g u i s h e s a group with the Sicilian species except B. incana, a second W e s t e r n g r o u p including B. oleracea, B. m o n t a n a a n d also B. i n c a n a a n d B. b o u r g e a u i and, finally, a third group including B. alboglabra (suggesting some gene influx) together with B. cretica a n d B. hilarionis. O t h e r importa n t s t u d i e s c o m p a r i n g either species or p o p u l a t i o n s are those by H o s a k a et al. (1990), G u s t a f s s o n a n d L a n n ~ r - H e r r e r a (1997a) a n d Lann~r (1998). Evaluations of interfertility a m o n g all these taxa (Snogerup, 1996) show t h a t r e d u c e d fertility m e a s u r e d by pollen stainability often occurs, b u t is only loosely correlated to morphological differences.
13 Other works focus on single species. Aguinagalde et al. (1991) study eight populations belonging to all the three existing subspecies of B r a s s i c a cretica. By analyzing seed storage proteins and five isoenzyme systems the a u t h o r s find again a high inter-population diversity. The only exception is provided by subsp, taconica (Gustafsson and Snogerup, 1983) whose populations seem to be more homogeneous. Maselli et al. (1996) st udy five enzyme systems on seven populations of B. insularis from Tunisia, Sardinia and Corsica. Though it does not appear too clearly in their dendrogram, a clinal variation from S o u t h to North is evident if the p a t h followed by the clustering process is closely observed. This agrees with the expectations derived from morphological analyses. A few years before, Widler and Bocquet (1979) had recognized four differentiated varieties of this species in Corsica alone. G u s t a f s s o n a n d Lann~r-Herrera (1997b) compare wild B. oleracea populations u s i n g different criteria while Lann~r-Herrera et al. (1996) perform a similar c o m p a r i s o n on eighteen popul at i ons u s i n g isozyme and RAPDs analyses. Though interesting results were obtained on the intra- and inter-populational variation, no evident correlation with the geographical origin (Spain, France and Great Britain) was found. The truly wild s t a t u s of B. oleracea populations in Noth Spain has been s u p p o r t e d by Fern~ndez-Prieto and Herrera-Gall~stegui (1992) on phytosociological grounds. Regarding B. m o n t a n a , a good detailed a c c o u n t of the French populations with morphological analyses and a study of several enzymatic s y s t e m s was produced by Cauwet-Marc et al. (1993). The investigations by Rac a n d Lovric (1991) and by Eastwood (1996) help considerably to u n d e r s t a n d some imperfectly known variability roughly related to B. incana existing in the Adriatic coasts and islands. Trinajsic and Dubravec (1986) recognized four different taxa with the range of subspecies, one part ascribed to B. i n c a n a itself and a n o t h e r part to B. botteri, a differentiated form which is recognized to merit specific rank. A critical additional species B. balearica, from Majorca Island, was first considered a small-sized m e m b e r of this group a n d later excluded from it. In fact, it is an d it is not. Snogerup and Persson (1983) studied it cytologically and found t h a t the n = 9 genome is present as part of its 2n = 32 chromosome complement. They suggest t h a t an ancient hybridization involving B. insularis gave rise to this species by amphidiploidy. G6mez-Campo (1993a) suggests t h a t the other p a r e n t might have been a m e m b e r of Subgen. Brassicaria (see below). Seed and seedling morphology, the general a p p e a r a n c e of the plant a n d some phytogeographical considerations are in favor of this hypothesis a l t h o u g h it is necessary to a c c o u n t for some c h r o m o s o m e losses. In Table 1.1, B. balearica is included in Sect. B r a s s i c a , together with a n o t h e r hybrid species, B. carinata, for which one of the p a r e n t s belongs to the n = 9 genome group. Though B. n a p u s is a similar case, it is placed in Sect. R a p a due to its capacity to form t u b e r o u s roots.
14
The genus Brassica D i s t r i b u t i o n s into sections a n d lists of Brassica species have been proposed in very s e p a r a t e times (Schulz, 1936; Salmeen, 1979) a n d with very different a m p l i t u d e - the former a u t h o r a d m i t s three sections a n d the second nine. These w o r k s have been u s e d as b a c k g r o u n d for the s h o r t analysis which follows. In t u r n , Table 1.1 provides a n u p d a t e d version where m o s t r e c e n t r e s e a r c h r e s u l t s have been incorporated. U-triangle species a n d their r e l a t i v e s - here all included in Subgen. Brassica - s h a r e a few c o m m o n c h a r a c t e r s s u c h as the p r e s e n c e of seeds in their pod b e a k s (or at least the potentiality for it), spherical or quasi spherical seeds, n o t c h e d cotyledons a n d well defined l y r a t e - p i n n a t i p a r t i t e b a s a l a n d m e d i a n leaves, where s i n u s e s r e a c h the mid-nerve at least toward the leaf base. D o u b t s in the i n t e r p r e t a t i o n s o m e t i m e s arise b e c a u s e some of the above c h a r a c t e r s m a y a p p e a r in a regressive form a n d b e c o m e difficult to detect. For i n s t a n c e , a p a t i e n t observation of m a n y pods of B. oleracea m a y be n e e d e d to find only a few seeded beaks. It m a y be even more difficult with the slender long b e a k s of B. rapa, t h o u g h at least some seed primordia can be discovered w h e n green pods are observed. In B. nigra, the p r o n o u n c e d s h o r t e n i n g of the whole fruit m i g h t r e n d e r the s a m e t a s k impossible. However, all o t h e r c h a r a c t e r s agree with those of the group. In o t h e r taxa, leaves m i g h t show a r e d u c t i o n in the n u m b e r of their s e c o n d a r y s e g m e n t s so t h a t w h e n these are a b s e n t (as in B. carinata) the r e s u l t a n t simple silhouette can h a r d l y be a s s o c i a t e d with a lyrate leaf. Five sections have been recognized in Subgen. Brassica (Table 1.1). Sect Brassica h a s a l r e a d y been d i s c u s s e d above. While S a l m e e n (1979) considers t h a t B. rapa alone deserves a Sect. Rapa, the a m p h i d i p l o i d s B. napus a n d B. juncea, have b e e n a d d e d to this, since all three have the capacity to form t u b e r o u s roots even if this c h a r a c t e r is not always expressed. However, it is recognized t h a t Sect. Rapa a n d Sect. Brassica are akin. The B. f r u t i c u l o s a / s p i n e s c e n s / maurorum complex, all with n = 8, together with B. procumbens from Algeria a n d B. cadmea clearly s h a r e the s a m e c h a r a c t e r s mentioned above for b e a k s , seeds, cotyledons a n d leaves. They m i g h t r e p r e s e n t the closest relatives of B. nigra (Truco a n d Quir6s, 1995) a n d they are all p u t together in Sect. Micropodium. B. dej~exa deserves a special section b a s e d on its deflexed siliques, some cytological c o n s i d e r a t i o n s (unique Brassica species with n = 7) a n d a n i n d u m e n t u m with very long p a t e n t h a i r s which is s o m e h o w atypical. B. assyriaca is c o n s i d e r e d to fall within the variability of B. dej~exa. The trio B. tournefortii / B. barrelieri / B. oxyrrhina also shows spherical seeds, seeded b e a k a n d lyrate leaves, a l t h o u g h b e c a u s e of their t e n d e n c y to form c o m p a c t basal rosettes the leaves of B. barrelieri a n d B. oxyrrhina are b e t t e r described as r u n c i n a t e . In Table 1.1 they have been conservatively g r o u p e d together u n d e r Sect. Sinapistrum a l t h o u g h some recent r e s e a r c h s u g g e s t s t h a t the trio is not h o m o g e n e o u s (Horn a n d V a u g h a n , 1983; P r a d h a n et al. 1992). Their similitudes a n d differences can be well d e s c r i b e d by c o n s i d e r i n g all possible pairs of species v e r s u s the r e m a i n i n g
15 third species. T h u s , B. barrelieri a n d B. oxyrrhina are identical in the vegetative stage by their a d p r e s s e d r o s e t t e s a n d can be well d i s t i n g u i s h e d from B. tournefortii. B. tournefortii a n d B. oxyrrhina s h a r e small sized flowers probably a s s o c i a t e d with a u t o g a m y while B. barrelieri flowers are bigger. In t u r n , B. barrelieri a n d B. tournefortii b e a k s are s h o r t e r t h a n t h o s e of B. oxyrrhina. The last species is also u n i q u e in s h o w i n g white (against yellow) flowers. Let u s finally a d d a n o n - t a x o n o m i c a l fact: B. tournefortii is often cultivated in India as a s o u r c e of oil while the two o t h e r s are only wild. A n o t h e r quite different g r o u p - Subgen. Brassicaria -exemplified by B. repanda (n = 10) always s h o w s a seedless beak, their seeds are flattened to a greater or lesser extent, a n d their leaves c a n rarely be described as properly lyrate b u t r a t h e r as s i n u a t e or pinnatifid (sinuses never or very rarely reach the mid-nerve). Their t e n d e n c y to be s c a p o s e a n d / o r to show s h o r t s u b t e r r a n e a n s t e m s with leaf s c a r s (as if they were small b u r i e d b u s h e s ) is also characteristic. B. repanda h a s a l m o s t fifteen, often poorly defined, s u b s p e c i e s from the M a r o c c a n Atlas to the E u r o p e a n Alps. Galland (1988) found highly polyploid forms in the Great Atlas, a u n i q u e case for the g e n u s Brassica. B. glabrescens from N. Italy s h o u l d be c o n s i d e r e d as an additional s u b s p e c i e s of B. repanda. Significantly, B. repanda is a b s e n t from the Balearic Islands. F u r t h e r to the E a s t in Europe, B. jordanoffii a n d B. nivalis s h a r e the s a m e set of c h a r a c t e r s a n d are very local in Bulgaria a n d Greece, respectively. Akeroyd a n d Leadlay (1991) s u g g e s t e d t h a t b o t h s h o u l d be only s e p a r a t e d at the subspecific level, b u t they s h o w sufficient morphological differences a n d different c h r o m o s o m e n u m b e r s (n = 11 a n d n= 10, respectively). In t u r n , B. elongata is very c o m m o n from the B a l k a n s to I r a n a n d it occasionally o c c u r s to the West (Roux a n d G u e n d e , 1995). In fact, two s u b s p e cies are local in Morocco. It is not always completely s c a p o s e a n d c o n t a i n s an n = 11 genome. In North Africa, there are other m e m b e r s of the group s u c h as B. gravinae, B. setulosa and B. loncholoma from Algeria a n d B. desnottesii local in Morocco, the last three close to B. repanda. Also, the original description of B. somaliensis strongly a d v o c a t e s for it to be included in Subgen. Brassicaria. Most r e c e n t r e s u l t s t e n d to confirm t h a t m e m b e r s of Subgen. Brassicarla are very s e p a r a t e from Subgen. Brassica. For i n s t a n c e , Clemente-Mufioz a n d H e r n ~ n d e z - B e r m e j o (1980) o b t a i n seven d e n d r o g r a m g r o u p i n g s in Brassica, in w h i c h all s e e d l e s s - b e a k e d species fall into the s a m e group, separate from all o t h e r m e m b e r s of the genus. Curiously, they did not s t u d y fruits at all b u t only the m o r p h o l o g y of the sterile p a r t s of the flower, sepals, petals, nectaries, which is considered to be very c o n s t a n t t h r o u g h o u t the whole Brassicaceae family. F u r t h e r s u p p o r t for the s a m e idea can be found in m a n y r e c e n t morphologically or biochemically b a s e d w o r k s ( T a k a h a t a a n d Hinata, 1986; Horn a n d V a u g h a n , 1983, etc.). However, some of these d a t a seem to s u g g e s t a s e p a r a t e section for B. elongata.
16
B. souliei from NW Africa a n d Sicily a n d B. dimorpha from Algeria do not easily fit in a n y of the above sections. Both are morphologically closer to Subgen. Brassicaria b u t they c o n s t a n t l y a p p e a r very s e p a r a t e from other Brassica t a x a in m o s t recently p r o d u c e d m o l e c u l a r - b a s e d d e n d r o g r a m s . Two d e c a d e s ago, S a l m e e n (1979) placed t h e m in Sect. Nasturtiops which is here recognized a n d located within Subgen. Brassicaria.
Other related genera Erucastrum a n d Diplotaxis have traditionally b e e n considered as very close relatives of Brassica, a n d several r e c e n t investigations s t u d y the relative positions of the t h r e e genera. S~nchez-Y~lamo (1990) a n d S~nchez-Y~lamo et al. (1992) s u c c e s s f u l l y differentiate t h e m on the b a s i s of seed proteins. S~nchez-Y~lamo a n d Aguinagalde (1996) i n c o r p o r a t e d s t u d i e s with flavonoids. M a n y o t h e r c o m p a r i s o n s a m o n g these three g e n e r a have been carried out within s t u d i e s on larger g r o u p s of t a x a (below). Some specific a s p e c t s of the t a x o n o m y of Erucastrum have been studied by G 6 m e z - C a m p o (1982, 1983, 1984). F o r m e r t a x a k n o w n u n d e r E. laevigaturn are d i s t r i b u t e d b e t w e e n E. littoreum a n d E. virgatum. A new species, E. ifniense, is newly d e s c r i b e d as a c o n t i n e n t a l v i c a r i a n t of the M a c a r o n e s i a n group (E. canariense a n d E. cardaminoides). A n o t h e r controversial taxon from Morocco formerly k n o w n u n d e r several generic d e n o m i n a t i o n s is t r a n s ferred into this g e n u s as E. rifanum. Hirschfeldia incana a n d Erucastrum littoreum are p r o p o s e d as p a r e n t s of the amphidiploid species E. elatum, a hypothesis later confirmed by S~nchez-Y~lamo (1992) b a s e d on isoenzyme a n a lysis. The recognition of sections within Erucastrum r e m a i n s an open possibility. If we restrict ourselves to the c i r c u m - m e d i t e r r a n e a n taxa, E. gallicum, E. leucanthum a n d E. nasturtiifolium show c h a r a c t e r s closer to Diplotaxis (thinner pods, smaller s e e d s a n d shallowly n o t c h e d cotyledons) while E. virgatum, E. littoreum or E. varium are s o m e h o w closer to Brassica. The species of E a s t a n d S o u t h Africa are a p p a r e n t l y closer molecularly to the Diplotaxislike trio so t h a t f u r t h e r detailed morphological or caryological c o m p a r i s o n s between the species of each biogeographic a r e a would be convenient to clarify the possibility of e s t a b l i s h i n g sections. J o n s e l l (1982) h a s described two new species, E. elgonense in U g a n d a a n d E. m e r u e n s e in Tanzania.
Hirschfeldia is very similar to Erucastrum b u t its a m p l i t u d e as a g e n u s h a s been differently interpreted. The r e a s o n lies in its definition by a combin a t i o n of c h a r a c t e r s w h i c h are often feeble a n d do n o t always occur together even in the type species H. incana. In o u r opinion, H. incana should p e r h a p s r e m a i n as s u c h b u t H. rostrata from S o k o t r a Island s h o u l d be ascribed to Erucastrum. H. incana s u b s p , incrassata is in m a n y r e s p e c t s intermediate between Hirschfeldia a n d Erucastrum. Diplotaxis h a s a t t r a c t e d m u c h interest, as j u d g e d by the n u m b e r of papers p r o d u c e d . G 6 m e z - C a m p o (1981a) gives specific s t a t u s to D. ibicensis
17 a n d ascribes a frequently confused yellow f o w e r e d taxon from Algeria to D. erucoides- as s u b s p , longisiliqua. The work by Martinez-Laborde (1988ab, 1989, 1991a, 1991b, 1991c, 1992 a n d 1993) is i m p o r t a n t b e c a u s e it not only brings to light a n d gives a proper specific s t a t u s to some o b s c u r e taxa s u c h as D. ilorcitana, D. brevisiliqua, D. brachycarpa, etc. a n d p r o p o s e s several infraspecific taxa within some variable species s u c h as D.virgata, D. harra and D.siifotia, b u t also p r e s e n t s an u p d a t e d s y s t e m for the genus. This system is f u r t h e r developed by the s a m e a u t h o r within a recent p a p e r by G6m e z - C a m p o a n d Martinez-Laborde (1998) a n d is fully reflected in Table 1.1. In turn, B r o c h m a n n et al., (1997) exhaustively refer to the frequently neglected taxa of Cape Verde Islands (also included in Table 1.1). S~nchezY~lamo (1994) s t u d i e s flavonoid c o n t e n t of m a n y Diplotaxis species w i t h o u t clearly d i s c r i m i n a t i n g the s u b g e n e r a , while the work by S~nchez-Y~lamo a n d Martinez-Laborde (1991) on the isozymes of Subgen. Diplotaxis s h e d s light on the p a r e n t s of the amphidiploid species D. muralis. S o b r i n o - V e s p e r i n a s (1985, 1993, 1996) refers to some p u n c t u a l p r o b l e m s of this genus. In the work by Warwick et al. (1992) based on chloroplast DNA, a lack of congruity between m o l e c u l a r a n d morphological d a t a is reported. However, it is noteworthy (Table 1.1) that, at least within the s u b g e n e r a a n d sections of Diplotaxis, there is a high degree of congruity between morphological a n d molecular results. In Diplotaxis, leaves are always pinnatifid or p i n n a t i s e c t (sinuses never reach the mid-nerve) a n d seeds are always small a n d more or less ovoid or elliptic. Two of the s u b g e n e r a (Diplotaxis a n d Hesperidium) c o n s t a n t l y include species with seedless beak. Together with Brassica, Diplotaxis is u n i q u e in containing b o t h k i n d s of taxa - with seedless a n d seeded beaks. It is to be noted t h a t the presence of seeded b e a k s (hetero-arthrocarpic fruits) is an exclusive d e v e l o p m e n t of the tribe Brassiceae, p r e s e n t only in p a r t of the genera. It is believed t h a t m u c h of the general variability p r e s e n t in Brassica a n d Diplotaxis including molecular heterogeneity is, in fact, a s s o c i a t e d to this b e a k duality. Exclusively b a s e d on floral m e a s u r e m e n t s , Hern~ndezBermejo a n d Clemente (1986) found a m a x i m u m variability in Brassica, followed by Diplotaxis a n d t h e n by Erucastrum, well above t h a t of sixteen other related genera. The s a m e three genera p r e s e n t the h i g h e s t variability at the molecular level (Warwick a n d Black, 1991, 1993). However, Erucastrum does not show a n y convincing signs of b e a k duality.
Raphanus is treated in an extensive morphological study, by Pistrick (1987). Two species are recognized viz. Raphanus sativus a n d R. raphanistrum, the latter including several subspecies. The origin a n d the infraspecific variability of Raphanus sativus h a s merited special a t t e n t i o n a n d a m o n g s t the m o s t recent p a p e r s on this species, those by Crisp (1995) a n d Rabbani et al. (1998) s h o u l d be m e n t i o n e d . The i n d e h i s c e n t fruit of Raphanus is i n t e r p r e t e d as a fully developed b e a k i.e. w i t h o u t valvar portion. However, at least in R. raphanistrum, a stereomicroscope can often help to
18
detect two small scales at the base of the fruit vestigially representing the missing valves.
Sinapis is the object of a detailed thesis by Baillargeon (1986). This author distinguishes four sections (Table 1.1) and introduces some interesting novelties s u c h as placing all cultivated forms of S. alba u n d e r subsp, alba and all wild ones u n d e r subsp, mairei. He also describes or recombines some other taxa by s u b o r d i n a t i n g them u n d e r S. arvensis or S. pubescens (Baillargeon, 1983). The morphological and molecular heterogeneity present in genus Sinapis can easily be correlated with the existence of two basic chromosome n u m b e r s , n = 9 and n = 12. All the species are hetero-arthrocarpic. Coincya h a s been studied in a n o t h e r thesis (see Leadlay and Heywood, 1990) soon after u p d a t e d for Spanish material by Leadlay (1993). Both works constitute a significant step toward u n d e r s t a n d i n g this difficult heteroarthrocarpic genus. The t r e a t m e n t is synthetic and gets rid of a n u m b e r of superfluous n a m e s t h a t had produced m u c h confusion, mostly in the Iberian P e n in s u la where the genus reaches its m a x i m u m variability. SobrinoVesperinas (1991) carried out hybridizations with material from Sierra Morena (S.C. Spain) to s t u d y the inheritance of fruit shape. An extensive phytochemical s t u d y of the lipids by Vioque-Pefia (1992) h a s no significant systematic implications. Molecular studies (Warwick and Black, 1993) show a distinct and f u n d a m e n t a l l y hom ogeneous genus. Trachystoma, with three species, has been the object of some intergeneric crosses (see c h a p t e r s 3 and 4 of this book) and also of anatomic studies on its strongly hetero-arthrocarpic fruit (Giberti, 1984). It is known (Maire and S a m u e l s s o n , 1937) to hybridize s p o n t a n e o u s l y with Ceratocnemum. In a way, this challenges the presently defined limits for the ~coenospecies" and invites an exploration of several other genera with shortened fruits. Eruca h a s become a monotypic genus after two poorly known former Eruca species (setulosa and loncholoma) from North Africa are ascribed to Brassica Subgen. Brassicaria (Table 1.1). The uni que species E. vesicaria is an a n n u a l n o n -het er o- ar t hr ocar pi c plant, one of whose subspecies (subsp. sativa) h as reached c i r c u m - m e d i t e r r a n e a n distribution and is cultivated in m a n y other parts of the World. The difficulty to distinguish the three (or perh a p s four) subspecies of E. vesicaria using pressed material has been noted by G6mez-Campo (1993b), but it is expected t hat further characterization work would clarify this. Sobrino-Vesperinas (1995) observed partial sterility between subsp, sativa and subsp, vesicaria. Sinapidendron is a n o t h e r apparently seedless-beaked genus with five species plus one subspecies restricted to Madeira Island (Hansen and Sunding, 1993). Former S. palmense should be referred to Sinapis pubescens (Rustan, 1980). Sinapidendron is woody and the only non-heteroarthrocarpic genus which belongs to the so called ~nigra" molecular lineage (see next ch a p ter or Warwick and Black 1991; for B. nigra itself see above). Testing an obvious possible interpretation, the a u t h o r of this chapter stu-
19 died several h u n d r e d b e a k s in s e a r c h for vestigial seed primordia, so far w i t h o u t success.
The tribe Brassiceae Table 1.2 tries to u p d a t e previous lists with all the g e n e r a of the tribe Brassiceae t o g e t h e r with their e s t i m a t e d n u m b e r of species a n d the type of fruit exhibited. S u b g e n e r a for Brassica a n d Diplotaxis are also included. Novelties c o n s i s t of Quidproquo a n d Dolichorhynchus, while Reboudia is det a c h e d after its u n i q u e species was placed u n d e r Erucaria by G r e u t e r et al. (1986). We have only tentatively re-included Calepina, very distinct, b u t of doubtful position elsewhere in the family Brassicaceae. It is obvious t h a t not all these 54 g e n e r a (with r o u g h l y 240 species) have received the s a m e attention in the p a s t two decades. Only those not t r e a t e d above b u t considered in recent s t u d i e s will be briefly c o m m e n t e d , s o m e t i m e s with still briefer references to s o m e of their m o s t relevant relatives.
Moricandia is often considered to belong to the Brassica coenospecies on the basis of morphological a n d cytogenetical similarities. The g e n u s pres e n t s its m a x i m u m variability in N. Africa (where M. suffruticosa, M. spinosa, M. foleyii a n d M. nitens are endemic) a n d in the Iberian P e n i n s u l a (where M. moricandioides a n d M. foetida are endemic). S o b r i n o - V e s p e r i n a s (1983) suggests t h a t so called M. arvensis var. robusta Batt. from the C o n s t a n t i n e a r e a in Algeria is exactly the s a m e form which once e s c a p e d a n d h a s now invaded the M e d i t e r r a n e a n b a s i n as a weed. The s a m e a u t h o r p o s t u l a t e s the identity of M. sinaica (W. Asia) with M. arvensis var. g a r a m a n t u m Maire from the Hoggar m a s s i f (S. Algeria). The m o s t e a s t e r n m e m b e r of the tribe Brassiceae, Douepia is very similar to Moricandia except for its capitate stigma a n d its s a b a n a - o r i e n t e d fenology. A m m o s p e r m a a n d Pseuderucaria are a d a p t e d to s a n d y s u b d e s e r t i c h a b i t a t s a n d are very similar to each other. Quezeliantha was described u n d e r Quezelia by Scholz (1966) b u t the original generic n a m e was later a d j u s t e d (1982) by the s a m e a u t h o r . This grows in Tibesti massif. Vella w a s revised by G 6 m e z - C a m p o (198 lb). Crespo (1992) describes a new distinct species V. lucentina in SE Spain, not far from Alicante. More recently, Crespo et al. (1999) have p r o d u c e d a c o m b i n e d a n a l y s i s of the subtribe Vetlinae involving ITS ribosomal s e q u e n c e s a n d morphological data. In t u r n , Ponce-Diaz (1998) h a s s t u d i e d the s a m e g r o u p with i s o e n z y m e s a n d ISSR m a r k e r s . Warwick a n d A1-Shehbaz (1998) propose to include Boleum a n d Euzomodendron within Vella. However, the three g e n e r a exhibit very distinct sets of adaptive c h a r a c t e r s in a c c o r d a n c e with their respective mec h a n i s m s for seed dispersal. Quidproquo is a new g e n u s p r o p o s e d by G r e u t e r a n d B u r d e t (1983) for material from L e b a n o n a n d Israel t h a t h a d been e r r o n e o u s l y included in Sinapis aucheri. A s t u d y by Baillargeon (1985) h a s d i s s i p a t e d some previous d o u b t s on its t a x o n o m i c s t a t u s a n d geographical distribution. Its fruit s h o w s
20 a well developed seeded beak. Dolichorhynchus was described by Hedge and Kit (1987) from W e s t e r n Arabia. It is a desert a d a p t e d p l a n t resembling Moricandia b u t exhibiting seeded beaks.
Crambe h a s often a t t r a c t e d the a t t e n t i o n of b o t a n i s t s b e c a u s e of its evolved simplified fruits ( n u c a m e n t a c e o u s , derived from one-seeded beaks) a n d the a n t i q u i t y s u g g e s t e d by the E-W geographic d i s j u n c t i o n it presents. Using flavonoids, Aguinagalde a n d G 6 m e z - C a m p o (1984) reflect s u c h disj u n c t i o n at the biochemical level. Khalilov (1991) r e a d j u s t s the sections of this g e n u s after i n c l u d i n g two new species. More recently Warwick a n d Black (1997) s t u d y c h l o r o p l a s t DNA a n d F r a n c i s c o - O r t e g a et al. (1999) ribosomal ITS s e q u e n c e s with similar results. Crambe s e e m s to be a well defined g e n u s with clearcut limits a n d no evident close relatives. Division of the tribe Brassiceae into s u b t r i b e s h a s been an unresolved issue for a long time (Schulz, 1919; J a n c h e n , 1942) mainly b e c a u s e it is difficult to e s t a b l i s h a d e q u a t e criteria. Today, m o l e c u l a r m e t h o d s can be very useful at providing a rich source of h y p o t h e s e s (Yanagino et al. 1987; Song et al., 1990; Warwick a n d Black, 1991, 1994, 1997). Clustering m e t h o d s a r o u n d r e p r e s e n t a t i v e genera might be the way to define indubitable n a t u r a l groups, b u t as morphological, biochemical or m o l e c u l a r phenetic distances become greater, the b o u n d a r i e s of these defined g r o u p s become more and more diffuse. The case of the Vellinae is a good example of this situation (Warwick a n d Black, 1994; Crespo et al., 1999 a n d Ponce-Diaz, 1998). It s e e m s obvious t h a t Boleum, Euzornodendron a n d Carrichtera are the closest allies to Vella, followed by Succowia a n d Psichyne in this order. On the contrary, Schouwia, s h o w i n g a winged fruit similar to t h a t of Psichyne, is normally rejected as a relative of this group. All these genera show seedless b e a k a n d u s u a l l y s h o r t e n e d fruits a n d perennial habit, being a d a p t e d to either semiarid or m e s o p h y t i c s t e p p e s or m o u n t a i n s . Thus, the subtribe Vellinae s e e m s to c o r r e s p o n d to a n a t u r a l a n d c o h e r e n t group where Euzomodendron might r e p r e s e n t the long-fruited a r c h e t y p e a n d Succowia an evolved a n n u a l representative. It is u n f o r t u n a t e t h a t Kremeriella was not t a k e n into a c c o u n t b e c a u s e its a s y m m e t r i c u n i l o c u l a r fruit might well have derived from a bilocular one by lateral abortion. A fusion of s u b t r i b e s VeUinae a n d Savignynae was proposed by the a u t h o r of this article on the basis of their seed wing (sometimes vestigial) b u t molecular d a t a (Ponce-Diaz, 1998) suggest t h a t they s h o u l d be kept separate. Henophyton (perennial) a n d Savignya (annual), are not too d i s t a n t from each o t h e r a n d are b o t h a d a p t e d to more arid e n v i r o n m e n t s in North Africa. Quezeliantha, from Tibesti, might be also ascribed to the subtribe Savignynae. Therefore, this subtribe is small a n d its limits are not well defined. The s a m e o c c u r s with Cakilinae, where the s u p p r e s s i o n of Reboudia h a s left the pair Erucaria a n d Cakile alone. The isolated position of Crambe a n d its evolved strongly hetero-arthrocarpic fruit m i g h t well deserve a monotypic s u b t r i b e (Crambinae). The E-W
21 T a b l e 1.2 Genera and subgenera of the tribe Brassiceae (Cruciferae).
+ H H H H H H H H + H
H H H H H ?
Ammosperma (1) Boleum (1) Brassica Subgen. Brassica (27) Subgen. Brassicaria (11) Cakile (7) Calepina (1) Carrichtera (1) Ceratocnemum (1) Chalcanthus (1) Coincya (6) Conringia (6) Cordylocarpus (1) Crambe (35) Crambella (1) Didesmus (2) Diplotaxis Subgen. Diplotaxis (4) Subgen. Hesperidium (14) Subgen. Rhynchocarpum (13) Dolichorrhynchus (1) Douepia (1) Enarthrocarpus (5) Eremophyton (1) Eruca (1) Erucaria (6) Erucastrum (21) Euzomodendron (1) Fezia (1) Foleyola (1)
In parentheses: estimated number of species. H = heteroarthrocarpic genera or subgenera. + = genera with species of both types. ? = fruit status doubtful.
H H H H ? H H H H
H H H H
H H
?
Fortuynia (2) Guiraoa (1) Hemicrambe (1) Henophyton (2) Hirschfeldia (1) Kremeriella (1) Moricandia (7) Morisia (1) Muricaria (1) Orychophragmus (1) Otocarpus (1) Physorrhynchus (2) Pseuderucaria (3) Pseudofortuynia (1) Psychine (1) Quezeliantha (1) Quidproquo (1) Raffenaldia (2) Raphanus (2) Rapistrum (2) Rytidocarpus (1) Savignya (2) Schouwia (2) Sinapidendron (5) Sinapis (8) Succowia (1) Trachystoma (3) Vella (5) Zilla (2)
22 M e d i t e r r a n e a n geographic d i s j u n c t i o n it p r e s e n t s is i n t e r p r e t e d as an indication of antiquity. At first view, Crambella or Muricaria fruits are similar to those of Crambe b u t m o l e c u l a r r e s u l t s (Warwick a n d Black, 1997; FranciscoO r t e g a et al., 1999) do not s u p p o r t the existence of a close relationship. A similar s i t u a t i o n o c c u r s with Hemicrambe. F u r t h e r s t u d i e s are n e c e s s a r y to find a suitable position for t h e s e genera. Also, m a n y a u t h o r s have found similitude between Crambe a n d Calepina b a s e d on their n u c a m e n t a c e o u s fruits. In o u r opinion, the u n i q u e p e n d e n t seed found in Calepina fruit m e a n s t h a t a d r a m a t i c r e d u c t i o n a n d loss of d e h i s c e n c e of the valvar portion h a s t a k e n place while the u n i q u e erect seed of Crambe fruit m e a n s t h a t we are faced with a r o u n d e d o n e - s e e d e d beak. In o t h e r words, it is a case of mere convergence w h e r e the fruit of Calepina is derived from the valves a n d t h a t of Crambe from the beak. On the other h a n d , it is d o u b t f u l t h a t Calepina a m e m b e r of the tribe Brassiceae. Were it ever fully a c c e p t e d in the tribe, it would merit a n o t h e r monotypic s u b t r i b e for itself. All m e m b e r s of the s u b t r i b e ZiUinae (Zilla, Foleyola, Physorrhynchus a n d Fortuynia) s h a r e c o m m o n d e s e r t - a d a p t i v e morphological traits (Schulz, 1936) a n d are a p p a r e n t l y close molecularly (Warwick a n d Black, 1994, Crespo, pers. comm.}. F r u i t s in the E a s t e r n g e n e r a (Physorrhynchus a n d Fortuynia) are clearly h e t e r o - a r t h r o c a r p i c . However, seeds in the w e s t e r n genera (Zilla, Foleyola) are p e n d e n t , strongly s u g g e s t i n g t h a t their simplified fruits might not c o r r e s p o n d to b e a k s b u t to i n d e h i s c e n t valvar portions. If this is confirmed t h r o u g h a n a t o m i c a l studies, the s t r u c t u r e of this s u b t r i b e s h o u l d p e r h a p s be t h o r o u g h l y reconsidered. For the r e m a i n i n g s u b t r i b e s Brassicinae, Raphaninae a n d Moricandinae, it is p r e m a t u r e to try to modify the p r e s e n t s t a r e s b e c a u s e their limits are u n d e r c o n s t a n t a s s a u l t by the r e s u l t s of new r e s e a r c h a n d interpretations. B e a k d e v e l o p m e n t in size or s h a p e a n d / o r fruit s h o r t e n i n g are agile evolutionary t r e n d s w h o s e variation c a n often be observed within a single g e n u s (for i n s t a n c e Coincya, Trachystoma, etc.) while n a t u r a l h y b r i d s are frequently found between long fruited g e n e r a - T r a c h y s t o m a a n d Cordylocarpus- a n d s h o r t fruited ones s u c h as Ceratocnemum a n d Rapistrum respectively. Thus, s u b - t r i b e Raphaninae h a s p r o b a b l y derived polyphyletically from different m e m b e r s of the s u b t r i b e Brassicinae a n d a c l e a r - c u t limit between both cannot really be e s t a b l i s h e d . The Moricandinae are also close to the Brassicinae in m a n y respects, their differences being mostly a s s o c i a t e d with a d a p t a t i o n of the former to more xerophytic h a b i t a t s . We s h o u l d note t h a t while subtribe Moricandinae only c o n t a i n s n o n - h e t e r o - a r t h r o c a r p i c g e n e r a (with seedless beaks) a n d s u b t r i b e Raphaninae only c o n t a i n s h e t e r o - a r t h r o c a r p i c gen e r a (with seeded beaks), s u b t r i b e Brassicinae c o n t a i n s both types of genera. F u r t h e r m o r e , both t r e n d s are p r e s e n t within Diplotaxis a n d Brassica. If this m o r p h o g e n e t i c c h a r a c t e r is eventually recognized a higher relative taxonomic i m p o r t a n c e with r e g a r d to other merely adaptive c h a r a c t e r s (see next chapter) this would m e a n a split not only for s u b t r i b e Brassicinae, b u t also for the g e n e r a Diplotaxis a n d Brassica. If this idea is t a k e n further, this could
23 lead to the recognition of only two subtribes (genera with seedless beak and genera with seeded beak) but this seems somewhat exaggerated. However, giving an excessive or u n b a l a n c e d emphasis to molecular data might equally lead to similar exaggerated situations. Future developments should be based on the intelligent integration of the new information which is continuously arriving from different directions.
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32 Warwick, S. I. and Black, L. D. 1997. Phylogenetic implications of chloroplast DNA restriction site variation in subtribes R a p h a n i n a e and Cakilinae (Brassicaceae, tribe Brassiceae). Can. J. Bot. 75,960-973. Warwick, S. I., Black, L. D. and Aguinagalde, I. 1992. Molecular systematics of Brassica and allied genera (subtribe Brassicinae, Brassiceae) chloroplast DNA variation in the genus Diplotaxis. Theor. Appl. Genet. 83, 839-850. Widler, B. E. and Bocquet, G. 1979. Brassica insularis Moris: Beispiel eines messinischen Verbreitunsmusters. Candollea 34, 133-151. Yanagino, T., Takahata, Y. and Hinata, K. 1987. Chloroplast DNA variation among diploid species in Brassica and allied genera" J a p a n J. Genet. 62, 119-125.
Biology of Brassica Coenospecies C. Gdmez-Campo (Editor) 91999 Elsevier Science B.V. All rights reserved.
33
2 ORIGIN
and
DOMESTICATION
C~sar G 6 m e z - C a m p o (1) a n d S h y a m P r a k a s h (2)
(1) Dept. Biologia Vegetal, Universidad Potit~cnica de Madrid. 2 8 0 4 0 Madrid. Spain. (2) National R e s e a r c h Centre on Plant Biotechnology. Indian Agricultural Research Institute. N e w Delhi - 110012. India. Inferences from antiquity, excavated p l a n t parts, references in a n c i e n t literature of v a r i o u s civilizations, c o m p a r a t i v e t a x o n o m y , n a t u r a l distribution, cytogenetical a n d biochemical evidence and, in r e c e n t years, m o l e c u l a r m a r k e r s have greatly helped in r e c o n s t r u c t i n g the p a s t events of origin a n d evolution of Brassica a n d its allied genera. However, the origin of a n y cultivated p l a n t involves two s e p a r a t e aspects. On the one h a n d , the origin of the taxon itself before d o m e s t i c a t i o n , as a result of the evolutionary process in the wild. On the other, its origin in cultivation or, in o t h e r words, the h i s t o r y of its d o m e s t i c a t i o n a n d s u b s e q u e n t u s e a n d diversification. For wild allies - w h o s e c o n s i d e r a t i o n is of great i m p o r t a n c e n o w a d a y s - the first case would always a n d u n i q u e l y apply. B u t conversely, some p l a n t species were p r e s u m a b l y b o r n in cultivation - s u c h as Brassica napus, R a p h a n u s sativus, etc. - a n d lack previous evolutionary history. Evolution o c c u r s in b o t h cases, b u t a p a r t from m u t a t i o n it is the r e s u l t of n a t u r a l selection in the first case a n d of h u m a n influence a n d artificial selection in the second. H u m a n influence h a s also been exerted t h r o u g h the practice of p l a n t collection not only before b u t also d u r i n g the practice of agriculture, b u t its evaluation is extremely difficult a p a r t from a highly p r o b a b l e selection a g a i n s t the m o s t edible forms. M a n y p l a n t s are also a d a p t e d to m a n w i t h o u t being c u l t i v a t e d - s u c h as weeds, for instance. The r e l a t i o n s h i p between crops a n d weeds is not only one of competition. Adaptation to d i s t u r b e d h a b i t a t s a n d vigorous h a b i t have m a d e m a n y weeds s o m e h o w p r e - a d a p t e d to d o m e s t i c a t i o n (for instance, weedy a n d cultivated Daucus carota are the s a m e species). On the contrary, m a n y c o n t e m p o r a r y weeds are often crop p l a n t s e s c a p e d from cultivation (Medicago sativa). There are r e a s o n s to believe (see below) that, chronologically, the first d o m e s t i c a t e d Brassica species was the diploid B. rapa (turnip rape). Also the u s e s of B. nigra (black m u s t a r d ) s e e m to be very a n t i q u e a n d the s a m e can
34 p r o b a b l y be said of B. j u n c e a (Indian m u s t a r d ) an a m p h i d i p l o i d originated by c r o s s e s between b o t h species. In t u r n , B. oleracea (kale, cabbage) seems to have entered cultivation later b e c a u s e its n a t u r a l a r e a (Atlantic coasts) was too far from the i m p o r t a n t c e n t e r s of domestication. Therefore, amphidiploid species in which B. oleracea intervenes as a parent, n a m e l y B. n a p u s (rape) a n d B. carinata (Ethiopian m u s t a r d ) , s h o u l d have been the last b r a s s i c a s to be i n c o r p o r a t e d into agriculture. We will first c o m m e n t on the evolutionary origin of the m e m b e r s of B r a s s i c a coenospecies a n d will follow the above chronological order in o u r s u b s e q u e n t a c c o u n t on domestication.
The p h y l o g e n y o f B r a s s i c a and allied g e n e r a . Morphologically b a s e d c o n c e p t s a n d / o r morphologically based numerical t a x o n o m y s t u d i e s of relevance for the phylogenetic relations between B r a s s i c a species a n d its wild allies, are relatively f r e q u e n t in the literature of the p a s t two d e c a d e s (G6mez-Campo, 1980, 1999; T a k a h a t a and Hinata, 1980, 1986.). Many o t h e r biochemically b a s e d p u b l i c a t i o n s relevant to their phylogeny have already b e e n m e n t i o n e d in c h a p t e r 1 of this book and they roughly s u p p o r t ideas t h a t could also be derived from morphology. Incongruities of m o r p h o l o g y with reproductive or cytogenetic d a t a have often been detected in the past, b u t they were not paid m u c h attention. After all, sexual compatibility, for i n s t a n c e , is of less taxonomic or phylogenetic importance in p l a n t s t h a n in a n i m a l s b e c a u s e p l a n t evolution h a s often occurred with the protection of geographic barriers. In an extreme bizarre situation, nobody d o u b t s t h a t self-incompatibility does not imply the existence of two species in a single individual! Equally, different c h r o m o s o m e n u m b e r is not e n o u g h to s e p a r a t e two species w h e n all other visible c h a r a c t e r s are homogeneous: autopolyploidy or disploidy are j u s t a first step toward a possible speciation b u t they do not m e a n speciation by themselves. T a x o n o m y h a s been s u p p o s ed to follow p h y l o g e n y as m u c h as possible b u t it h a s also been expected to be p r u d e n t w h e n p h y l o g e n y is not well e s t a b l i s h e d - as o c c u r s in so m a n y cases. B u t above all, t a x o n o m y s h o u l d be pragmatic, practical and easy to use.
The a d v e n t of m o l e c u l a r biology h a s resulted in the addition of massive a p p a r e n t incongruities with e s t a b l i s h e d taxonomy, so t h a t frequent interm i x t u r e s of taxa in newly p r o d u c e d d e n d r o g r a m s raises a certain discomfort to a n y taxonomist. However, at least with o u r material, we can see t h a t incongruities are not so w o r r i s o m e and, on the other h a n d , there can be m a n y e x p l a n a t i o n s for the i n t e r m i x t u r e of taxa. S a m p l e d genes are evidently different for each case. While m o l e c u l a r m e t h o d s have aimed to s t u d y a few genes localized in organelles or a larger r a n d o m sample of the n u c l e a r genome where a large p r o p o r t i o n of genes are r e d u n d a n t or encode for primary metabolic p r o c e s s e s - classical t a x o n o m y h a s r a t h e r been b a s e d on a handful of specific genes w h o s e effects are externally visible.
35 In Brassica, m o l e c u l a r biology s t a r t e d with exploration of the origin of amphidiploid species via cpDNA (Palmer et al. 1983; E r i k s s o n et al. 1983; I c h i k a w a a n d Hirai, 1983). Shortly after, Yanagino et al. (1987) c o m p a r e d for the first time cpDNA of a set of eleven species a n d found clear incongruities with t a x o n o m y , since a group of four Brassica species a p p e a r e d split a n d intermixed in the d e n d r o g r a m with other five allied genera. The correlation with c h r o m o s o m e pairing (Mizushima, 1980) was very high, while t h a t with some n u m e r i c a l t a x o n o m y r e s u l t s ( T a k a h a t a a n d Hinata, 1986) w a s low or not significant. Soon after, Song. et al. (1990) s t u d i e d n u c l e a r RFLPs of thirty-eight a c c e s s i o n s belonging to fifteen species of Brassica a n d of three additional g e n e r a a n d found similar incongruities. Warwick a n d Black (1991, 1993, 1994, 1997) a n d Warwick et al. (1992) with cpDNA a n d P r a d h a n et al. (1992) with cp a n d mtDNA extend this type of s t u d y to m a n y other m e m b e r s of the tribe Brassiceae with c o m p a r a t i v e l y similar results. According to t h e s e a u t h o r s , at least in the a r e a of the phylogenetic tree where Brassica a n d its closest allies are found, two lineages c a n be distinguished: '~nigra" a n d " r a p a / o l e r a c e a " lineages - a c c o r d i n g to the n o m e n c l a ture u s e d by W a r w i c k a n d Black. M e m b e r s of large g e n e r a viz. Brassica, Diplotaxis, Erucastrum a n d Sinapis are r e p r e s e n t e d in both the lineages while monotypic or smaller g e n e r a are only r e p r e s e n t e d in one of these lineages. Cp a n d m t DNA RFLP information is therefore in favor of recognizing a polyphyletic origin, at least for these genera. C o m p a r a t i v e r e s u l t s by three aut h o r s r e g a r d i n g the d i s t r i b u t i o n of several species in t h e s e two lineages are s h o w n in Table 2.1. Let u s p u t forward t h a t s u b g e n e r a a n d sections recognized in Brassica, Diplotaxis a n d Sinapis on strictly morphological g r o u n d s (Schulz, 1936; Salmeen, 1979; G 6 m e z - C a m p o , in this book) a l r e a d y provide a basis on which s u c h p o l y p h y l e t i s m c a n be envisaged a n d u n d e r s t o o d . In general, m o l e c u l a r d a t a s u p p o r t the existence of s u c h large t a x o n o m i c divisions a n d act as a g e n e r a t o r of h y p o t h e s e s on how the evolution of p a r t i c u l a r t a x a or g r o u p s of t a x a might have occurred.
The "nigra" and "rapa" lineages Within Brassica Subgen. Brassica, the ~nigra" lineage involves a group of species i. e. r o u g h l y those included in Sect. Micropodium, w h o s e affinities with the g e n e r a Sinapis a n d Hirschfeldia a n d with some Erucastrum or Diplotaxis species are h i g h e r t h a n with the o t h e r m e m b e r s of Brassica belonging to sections Brassica, Rapa or Brassicoides). Sinapis species are all i n c l u d e d in ~nigra" lineage except Sect. Chondrosinapis w h o s e u n i q u e m e m b e r is S. aucheri, a t a x o n w h i c h h a s always b e e n c o n s i d e r e d to be very s e p a r a t e from the others. Within Diplotaxis, only the m e m b e r s of Subgen. Rhynchocarpum (except Sect. Erucoides) s e e m to belong to this lineage. In the case of Erucastrum w h e r e no sections have been recognized, m o s t species fall within this lineage (as it does Hirschfeldia, very close to Erucastrum) a n d only a few
36
Table 2.1 A select list of genera and species of the tribe Brassiceae comprising the two lineages - Brassica (Rapa) and Sinapis (Nigra) - as reported by various authors. Compiled by M. Lakshmikumaran. Song & aL 1990 TAG 79:
Warwick & Black 1991 TAG
Pradhan & al. 1992 TAG
497-526 (nuclear RFLP)
82:81-92 (cp RFLP) and 1992
85:331-340 (cp RFLP)
TAG 83:839-850 (cp RFLP) Sinapis lineage
Brassica lineage
Sinapis lineage
Brass&a
lineage
lineage
Sinapis lineage
Brassica rapa
B.futiculosa
B. rapa
B. Higra
B. rapa
B. Fligra
B.oleracea
B.nigra
B.oleracea
Xarvensis
B.juncea
B.carinata
Diplotaxis erucoides
S.arvens&
D.erucoides
S.alba
B.oleracea
S.arvensis
B.tournefortii
S.alba
B.deflexa
S.flexuosa
B.napus
S.flexuosa
Eruca sativa
S.aucheri
D.siettiana
D.erucoides
S.alba
Hirschfeldia incana
Raphanus sativus
B.fruticulosa
R.sativus
D.siettiana
B.oxyrrhina
S.pubescens
S.aucheri
D.siifolia
B.barrelieri
B.tournefortii
B.gravinae
S.pubescens
D.harra
H. incana
D.muralis
H. incana
E.sativa
D.siifolia
B.oxyrrhina
B.maurorum
B.gravinae
E.sativa
B.spinescens
D.muralis
D.harra
B.fruticulosa
Erucastrum abyssinicum
B.tournefortii
Moricandia arvensis
Erucastrum. varium
Brassica
B.souliei
37 (see below) are apart. The g e n u s Trachystoma a n d two s t u d i e d Sinapidendron species also belong to this lineage. Coincya is closer to "nigra" t h a n to "rapa" lineages b u t occupies a s e p a r a t e position, p e r h a p s s u g g e s t i n g a lineage of its own. The "rapa" lineage (short for "rapa/oleracea") will therefore contain all m e m b e r s of Sect. Brassica, Sect. Rapa a n d Sect. Brassicoides. Within Sect. Sinapistrum, B. barrelieri a n d B. oxyrrhina s h o u l d also be included. In t u r n , B. tournefortii h a s offered c o n t r a d i c t o r y r e s u l t s w h e n n u c l e a r or chloroplast DNA were u s e d , as d i s c u s s e d below. Significantly, "rapa" lineage also contains m o s t t a x a with seedless beak: not only m e m b e r s of Brassica s u b g e n u s Brassicaria b u t also all Diplotaxis species of the Diplotaxis a n d Hesperidium s u b g e n e r a as well as the g e n u s Eruca. As for Erucastrum, the "rapa" lineage c o n t a i n s the g r o u p gallicum \ nasturtiifolium \ l e u c a n t h u m - all morphologically close to e a c h other as indicated in c h a p t e r 1 - as well as some e a s t e r n a n d S. African species. G e n u s R a p h a n u s also falls within this lineage. We can see t h a t m o l e c u l a r d a t a are not as i n c o n g r u o u s with t a x o n o m y as initially t h o u g h t , since at least the larger t a x o n o m i c g r o u p s s h o w no difficulties in being m a t c h e d to DNA lineages. In t u r n , lineages offer a new view of evolution where not only m o r p h o g e n e t i c genes with visible p h e n o t y p i c effect are involved b u t s a m p l e s of a m u c h larger a n d / o r different sets of genes are manifested. B e c a u s e of its low c h r o m o s o m e n u m b e r , Hirschfeldia incana (n = 7) h a s been p r o p o s e d as an a n c e s t o r for the "nigra" lineage. However, Hirschfeldia could be seen as a n Erucastrum with specialized fruit, so t h a t o t h e r less specialized species of Erucastrum with n = 7 as E. virgatum or E. varium might better play t h a t role. Nonetheless, as the "nigra" lineage goes b a c k to the Sect. Rhynchocarpum of Diplotaxis a n d f u r t h e r b a c k into the g e n u s Sinapidendron - this case showing primitive seedless b e a k - p e r h a p s the real archaeotype for the "nigra" lineage s h o u l d be traced t h a t far.
S. arvensis h a s been r e g a r d e d as primitive within its genus, a n d its closeness to B. nigra is s u p p o r t e d by s t r o n g evidence. Both species show high homology in their n u c l e a r DNA (Song et al., 1988ab; P o u l s e n et al., 1994; Kapila et al., 1996), chloroplast DNA (Warwick a n d Black, 1991; Pradh a n et al., 1992), fraction I protein (Uchmiya a n d W i l d m a n 1978) a n d seed proteins ( V a u g h a n a n d Denford, 1968). Both species have 3 pairs of satellited c h r o m o s o m e s (Cheng a n d Heneen, 1995). The c h r o m o s o m e s of these two species p o s s e s s similar genic s e q u e n c e s as e x p r e s s e d in the high degree of pairing in the inter-generic hybrid B. nigra x S. arvensis (up to 8 bivalents, Mizushima, 1950). F u r t h e r evolution of Sinapis s e e m s to have o c c u r r e d in two directions, one line toward S. p u b e s c e n s a n d its relatives a n d a n o t h e r tow a r d S. alba a n d S. j~exuosa. The last two are very similar morphologically, cytologically a n d molecularly. On the o t h e r h a n d , S. aucheri m i g h t recognize a different origin.
3g Molecular d a t a also s u p p o r t a high degree of coherence for Sect. Microp o d i u m of Brassica. A close relationship exists between B. nigra and B. fruticulosa (n = 8) in regard to n u c l e a r DNA (Song et al., 1990), cp DNA (Warwick a n d Black, 1991; P r a d h a n et al., 1992), fraction I protein (Uchimiya a n d Wildman, 1978), a n d c h r o m o s o m e pairing (up to 7 bivalents in the hybrid. B. nigra x B. fruticulosa (Takahata a n d Hinata, 1983). Another three species viz. B. fruticulosa, B. maurorum a n d B. s p i n e s c e n s are ascribed to the same B. fruticulosa cytodeme (Harberd, 1976; T a k a h a t a a n d Hinata, 1983). Hybrids between t h e m are o b t a i n e d with m u c h ease (Takahata a n d Hinata, 1983) alt h o u g h their c h r o m o s o m e behavior reveals meiotic irregularities suggesting these species are u n d e r differentiation. Among them, B. maurorum is an unspecialized weed, while B. fruticulosa (with several subspecies) a n d B. spin e s c e n s have developed morphological c h a r a c t e r s of a d a p t a t i o n to coastal habitats. B. cossoniana w a s proposed to be an a u t o t e t r a p l o i d of B. fruticulosa (Harberd, 1972) b u t it s e e m s to show closer similarities in cp a n d m t DNA with B. maurorum ( P r a d h a n et al., 1992). B. p r o c u m b e n s is morphologically similar to other m e m b e r s of this group b u t it h a s recently been shown to have n = 9 c h r o m o s o m e s ( Baldini, 1998). Its lineage r e m a i n s u n k n o w n . For the rapa/oleracea lineage, Diplotaxis erucoides (n = 7) h a s been p r o p o s e d to be the closest ancestor, b a s e d on cp DNA (Warwick a n d Black, 1991; P r a d h a n et al., 1992). High homologies of repeat s e q u e n c e s between D. erucoides a n d B. rapa a n d B. oleracea (96 a n d 94% homology respectively, H a r b i n d e r a n d L a k s h m i k u m a r a n , 1990) s u b s t a n t i a t e this view. According to this, stocks r e p r e s e n t e d by a part of the genera Diplotaxis, Erucastrum and Brassica, plus the complete genera Eruca a n d R a p h a n u s evolved from this species. However, we would like to m a k e again o u r point to this scheme. It is obvious t h a t "rapa" lineage is well rooted in the group of taxa with a s p e r m beak, since those from Brassica s u b g e n u s Brassicaria, Diplotaxis s u b g e n u s Diplotaxis a n d s u b g e n u s Hesperidium a n d also the g e n u s Eruca belong to it. This m e a n s t h a t s u c h primitive a s p e r m - b e a k stock could never have derived from an a n c e s t o r similar to D. e r u c o i d e s - m u c h more evolved - b u t from more primitive a n c e s t o r s . These might tentatively be identified with Cape Verde Diplotaxis of Subgen. Hesperidium. S o m e c a s e s for f u r t h e r s t u d y
B. tournefortii (n = 10) belongs to the "nigra" lineage. Although Song et al. (1990) held the view t h a t this species evolved from a "rapa/oleracea" a n c e s t o r with n u c l e a r introgression from B. nigra, studies by Warwick and Black (1991) a n d P r a d h a n et al. (1992) on cp DNA suggest the opposite: it a p p e a r s t h a t B. tournefortii evolved from a B. fruticulosa-like a n c e s t o r with strong n u c l e a r a n d cytoplasmic introgression from B. r a p a / R a p h a n u s material (both s y m p a t r i c in their distribution) as reflected in the high a m o u n t of c h r o m o s o m e pairing p r e s e n t in the h y b r i d s (Mizushima, 1968).
39 Sinapis aucheri (n = 7) origin a n d evolution have been related to t h a t of R a p h a n u s (n = 9) b a s e d on their strongly h e t e r o a r t h r o c a r p i c fruits a n d their cp DNA similarity (Warwick a n d Black, 1991; P r a d h a n et al., 1992). However, we believe t h a t fruit similarity is a mere morphological convergence while cp DNA similarity is j u s t t h a t showed by two m e m b e r s of the "rapa" lineage. C h r o m o s o m e n u m b e r , leaf i n d u m e n t u m a n d shape, geographic distribution, etc. all advocate for a relation between Sinapis Sect. C h o n d r o s i n a p i s a n d B r a s s i c a Sect. Brassicoides. In other simpler words, Sinapis aucheri m i g h t be to B r a s s i c a dej~exa w h a t R a p h a n u s s a t i v u s is to B r a s s i c a rapa or B. oleracea. They both r e p r e s e n t the evolution of long seminiferous b e a k s from a n c e s t o r s with m o d e r a t e l y developed ones. Cultivated taxa
From i n t e r p r e t a t i o n of cytogenetic evidence, diploid B r a s s i c a species B. nigra, B. oleracea a n d B. r a p a - were t h o u g h t to have evolved in a n ascending order from a c o m m o n a r c h e t y p e (R6bbelen, 1960; P r a k a s h a n d Hinata, 1980). However, recent m o l e c u l a r investigations clearly d i s c a r d a monophyletic origin a n d s u g g e s t t h a t B. rapa a n d B. oleracea evolved from one progenitor while B. nigra evolved from another. In t u r n , several sets of evidence point to a c o m m o n origin for B. oleracea a n d B. rapa. These include: close similarities in their c y t o p l a s m s (Palmer et al., 1983; E r i c k s o n et al., 1983; Yanagino et al., 1987; Warwick a n d Black, 1983; P r a d h a n et al., 1992); high c h r o m o s o m e pairing in the interspecific hybrid B. rapa x B. oleracea (up to 9 bivalents, Olsson, 1960a; Namai, 1976); serological similarities in seed p r o t e i n s (Vaughan et at., 1966); n u c l e a r DNA RFLPs (Song et al., 1988a) a n d the presence of one satellite c h r o m o s o m e in b o t h g e n o m e s (Wang et al., 1989; C h e n g et al., 1995). The r e l a t i o n s h i p s of B. nigra within the "nigra" lineage h a s already been d i s c u s s e d . According to R6bbelen (1960) the B. o l e r a c e a / B , rapa real a r c h e t y p e is now extinct a n d h a d six pairs of c h r o m o s o m e s . Evidence in s u p p o r t of this view comes from s e c o n d a r y association of bivalents, c h r o m o s o m e pairing in haploids (see P r a k a s h a n d Hinata, 1980) a n d the presence of d u p l i c a t e d loci for rDNA genes (Quiros et al., 1985, 1987). Diploid species evolved from this prototype by two m e c h a n i s m s (i) selective c h r o m o s o m e d o u b l i n g i.e. secondary polyploidy, a n d (ii) c h r o m o s o m e re-patterning. RFLP d a t a s u b s t a n t i a t e this view of c h r o m o s o m e duplication a n d also s u g g e s t c h r o m o s o m e re-organization t h r o u g h localized sequence duplication a n d s e q u e n c e t r a n s p o s i t i o n (Song et al., 1991; Chyi et al., 1992; S n o w d o n et al., 1997). Recent r e s e a r c h on genome m a p p i n g (see c h a p t e r 7) h a s a d d e d m u c h information a n d new views to this subject. It s h o u l d be a d d e d t h a t a n y c o n s i d e r a t i o n on the origin of B. oleracea c a n n o t be s e p a r a t e d from the origin of its n = 9 relatives, since B. oleracea itself s h o u l d be considered as having evolved in close relation to t h a t group. As the m a x i m u m diversity of the whole group o c c u r s in Sicily, this Island a p p e a r s to be the m o s t probable c a n d i d a t e for its center of origin.
40
Additionally, Sicilian B. villosa and B. i n c a n a show several primitive characters such as hairy leaves and beaks with 1-3 seeds (primitive only for this material!). From there, five lines of evolution involving hair loss and wax development gave rise to the other taxa, after extending geographically through the Mediterranean and Atlantic coasts and islands (G6mez-Campo and Gustafsson, 1991). Alloploid species - B. carinata, B. j u n c e a and B. n a p u s - originated following multiple natural interspecific hybridizations (Olsson, 1960ab; Prakash, 1973, 1974; Song and Osborn, 1992). Natural hybridizations were always unidirectional as revealed by the studies on Fraction-I protein (Uchimiya and Wildman, 1978) and cp DNA restriction patterns, which conclusively established that B. nigra and B. rapa are the cytoplasmic donors of B. c a r i n a t a and B. j u n c e a respectively (Erickson et al., 1983; Ichikawa and Hirai, 1983; Palmer et al., 1983; Warwick and Black, 1991; Pradhan et al., 1992), while in B. n a p u s there is a slightly altered B. oleracea cytoplasm (Palmer et al., 1983). Similarities in cp genomes of diploids and their derived alloploids indicated that cp genomes have been well conserved in B. j u n c e a and B. c a r i n a t a since their origin (Erickson et al., 1983) while in B. n a p u s it has diverged slightly from the maternal B. oleracea cp genome. These studies also revealed that mt and cp genomes were co-inherited during their evolution. The close similarity in cp DNA of diploids and alloploids suggests that alloploids are of recent origin. A survey of rDNA of the allotetraploid species led Quiros et al. (1985) to propose that B. j u n c e a was the first to evolve and B. n a p u s and B. c a r i n a t a originated later. This agrees with the delayed entrance of B. oleracea in the agricultural world postulated in another part of this chapter. Amount of DNA in tetraploids has not changed significantly since their origin though there has been a reduction in nuclear size, probably due to higher DNA density resulting from greater condensation of chromosome material (Verma and Rees, 1974). Nuclear DNA composition of alloploid species is more closely related to the maternal cytoplasmic donors than to the male parents (Song et al., 1988a). Nuclear genomes of male progenitors in B. j u n c e a and B. carinata have undergone extensive changes while the maternal parents have preserved genomic integrity during evolutionary history (Song et al., 1988a). This suggests co-evolution of nuclear and cytoplasmic genomes. Only in B. n a p u s are these changes in nuclear genomes minimal (Parkin and Lydiate, 1997). It is enigmatic that each monogenomic species has not contributed cytoplasm to more than one alloploid species in spite of the fact that alloploids are of polyphyletic origin involving several interspecific hybridization events. Song et al. (1993) suggested that low seed fertility might be the cause for the elimination in nature of alloploids having opposite cytoplasm. The presence of different types of cytoplasm in various accessions of B. n a p u s strongly supports the concept of multiple origins of B. n a p u s (Song and Osborn, 1992).
41
Raphanus p r o b a b l y s h a r e s a c o m m o n a n c e s t r y with B. rapa/oleracea s t o c k s as indicated by the close c h r o m o s o m e homology between B. oleracea a n d Raphanus g e n o m e s - u p to 7 bivalents in R. sativus x B. oleracea (2n = 18) a n d u p to 7 bivalents in R. sativus x B. rapa (2n = 19) (Richharia, 1937). Many other similarities exist a m o n g Raphanus, B. oleracea a n d B. rapa. They can easily form intergeneric h y b r i d s a n d it is n o t e w o r t h y t h a t a m o n g the diploid species of Brassica coenospecies, only two - Raphanus a n d B. rapa s u b s p , rapa (turnip) - form t u b e r o u s roots. R. sativus is t h o u g h t to have derived from an a n c i e n t d o m e s t i c a t i o n of R. raphanistrum. Similarities in n u clear DNA RFLPs a n d morphological c h a r a c t e r s s u c h as flower size between Eruca a n d Raphanus led Song et at. (1990) to s u g g e s t t h a t b o t h h a d evolved from a c o m m o n ancestor. However, P r a d h a n et al. (1992) d i s c o u n t e d this in view of large differences in their cp DNA. Eruca h a s cp DNA similarities with Diplotaxis tenuifolia a n d D. pitardiana (all with n = 11 a n d a s p e r m beaks) while Raphanus h a s n = 9 a n d strongly h e t e r o a r t h r o c a r p i c fruit. The " i s t h m u s " c o n c e p t . I n t r o d u c e d by G 6 m e z - C a m p o (1999) it a t t e m p t s to help a better u n d e r s t a n d i n g of the evolution of the tribe Brassiceae as a whole. It c o n s i s t s of giving an a n g u l a r i m p o r t a n c e to the p r e s e n c e of seeds within the stylar cavity in m a n y of its m e m b e r s as a s i n g u l a r a c h i e v e m e n t t h a t can only be f o u n d within this tribe, while it is a b s e n t in all other Crucifer tribes. Within the tribe Brassiceae, r o u g h l y one half of the g e n e r a do not s h o w s u c h develo p m e n t , so they resemble o t h e r Crucifers at this respect. The other half s h o w seeded stylar portions (heteroarthrocarpy), a n d they often develop this t r e n d into bizarre types of seeded beaks. Two successive evolutionary r a d i a t i o n s are p o s t u l a t e d (Figure 2.1). The first involves t h o s e g e n e r a where the fruit b e a k keeps its primitive seedless condition ( n o n - h e t e r o a r t h r o c a r p i c fruit) s u c h as Sinapidendron, Eruca, Euzo-
modendron, Vella, Boleum, Carrichtera, Succowia, Moricandia, Rytidocarpus, Douepia, Conringia, Chalcanthus, Pseudofortuynia, Ammosperma, Pseuderucarla, Savignya, Henophyton, Quezeliantha, etc. as well as to the s u b g e n e r a Diplotaxis a n d Hesperidium of Diplotaxis a n d s u b g e n u s Brassicaria of Brassica. An a r c h e t y p e of the tribe m i g h t have r e s e m b l e d p r e s e n t M a d e i r a n Sinapidendron (but with biseriate i n s t e a d of u n i s e r i a t e seeds) or p r e s e n t Cape Verde Diplotaxis of subgen. Hesperidium (but with p e r h a p s woodier stems). The a c h i e v e m e n t of h e t e r o a r t h r o c a r p i c fruits (with seeded beaks) m i g h t have o c c u r r e d with the a d v e n t of s u b g e n u s Rhynchocarpum of Diplotaxis, so t h a t Diplotaxis s t a y s at b o t h sides of the i s t h m u s or bridge between b o t h radiations. F r o m here, a second evolutionary r a d i a t i o n originated Erucastrum, Hirschfetdia, Brassica (Subgen. Brassica), Sinapis, Coincya, Erucaria,
Trachystoma, Raphanus, Enarthrocarpus, Cakile, Ceratocnemum, Cordylocarpus, Crambe, Didesmus, Eremophyton, Fortuynia, Guiraoa, Hemicrambe, Muricaria, Otocarpus, Physorrhynchus, Rapistrum, Fezia, etc. All of t h e m s h o w
Guiraoa / Otocarpus / Ceratocnemum I
-0
Morisia
Brassica - Sinapis / Trachystoma / Coincya /
Sinapidendron / Brassicaria (Archetype)
I
< -I
rn
Fezia
v)
Erucastrum / Hirschfeldia / Cordylocarpus - Rapistrum ===
B)
Moricandia - Douepia Rytidocarpus / Conringia Pseudofortuynia Ammosperma - Pseuderucaria
Rhynchocarpum
Dolichorhynchus
-
-
-
-
-
X
rn ;[I
0 Erucaria - Cakile Didesmus / Eremophyton
Henophyton / Quezeliantha Savignya /Schouwia Foleyola - Zilla?
v)
Raphanus / Enarthrocarpus / Raffenaldia / Quidproquo
Eruca
Hesperidiurn ---------- Diplotaxis
rn 0
Psychine Succowia / Kremeriella? Vella - Boleum Euzomodendron / Carrichtera
A)
E
Hemicrambe / Crambe Crambella, Muricaria
Calepina?
-0
I
< --I
rn
cn Physorrhynchus - Fortuynia
Figure 2.1 The “isthmus” concept is exemplified by tentatively placing fifty genera (and five subgenera, underlined) into two separate evolutionary branches: A) genera with seedless beak (non-hetero-arthrocarpic), and B) genera with seeded beak (heteroarthrocarpic). Within each of these radiations, taxa are roughly grouped by their morphological or ecological affinities.
i3
43 h e t e r o - a r t h r o c a r p i c fruits with very different degrees of b e a k development, a c c o m p a n i e d or not with overall fruit reduction. It is believed t h a t m a n y previous c o n f u s i o n s derived from the joint consideration of b o t h r a d i a t i o n s a n d from the a b u n d a n t c a s e s of adaptive convergence involved. The i s t h m u s c o n c e p t provides a n u m b e r of h y p o t h e s e s to be either confirmed or rejected. Its c o n g r u i t y with p r e s e n t t a x o n o m y is high. While the c o n g r u i t y between morphological a n d m o l e c u l a r d a t a is not always high, it is believed t h a t this c o n c e p t m i g h t help to a b e t t e r i n t e r p r e t a t i o n of the existing divergences. For i n s t a n c e , the duality of lineages found by molec u l a r m e t h o d s in Diplotaxis a n d B r a s s i c a largely c o r r e s p o n d s with the d i s t r i b u t i o n of their s u b g e n e r a a n d sections on both sides of the i s t h m u s . However, it is obvious t h a t the s e c o n d r a d i a t i o n m a y not be completely monophyletic since both lineages seem to go a c r o s s the i s t h m u s .
Domestication Brassica
rapa
of cultivated brassicas and allies L.
B r a s s i c a rapa L. (syn. B. campestris) s e e m s to have grown n a t u r a l l y from the West M e d i t e r r a n e a n region to Central Asia, a n d is still p r e s e n t t h r o u g h o u t this area, in general a s s o c i a t e d to weedy h a b i t a t s . Its wide availability m a d e it p r o b a b l y the first d o m e s t i c a t e d Brassica, p e r h a p s several millenia ago, as a m u l t i p u r p o s e crop (roots in turnip, leaves in Chinese cabbage, y o u n g flowering s h o o t s in Galician "grelos", seeds in original colza or r a p e s e e d , etc.) a n d it h a s b e e n widely u s e d by all civilizations developed in t h a t ample region.
Nonetheless, t r u e a n t i q u i t y of B. rapa is not well d o c u m e n t e d . The earliest reference to a B. rapa ecotype is of yellow s a r s o n in S a n s k r i t literat u r e U p a n i s a d a s a n d B r a h a m a n a s (c. 1500 BC) w h e r e it w a s referred to as 'Siddhartha' (Prakash, 1961; Watt, 1989). Some B. rapa s e e d s have b e e n recovered from the s t o m a c h of Tollund m a n (Renfrow, 1973). Burkill (1930) c o n s i d e r e d E u r o p e as the place w h e r e B. rapa w a s first d o m e s t i c a t e d as a biennial p l a n t from w h i c h a n n u a l forms arose t h r o u g h selection. A c o m p a r ative morphological s t u d y led S u n (1946) to propose the existence of two lines: Western, w h i c h i n c l u d e s oilseed forms a n d t u r n i p , a n d are d i s t r i b u t e d t h r o u g h o u t E u r o p e , Central Asia a n d the I n d i a n s u b - c o n t i n e n t ; a n d E a s t e r n , c o m p r i s i n g E a s t Asian vegetable forms. Isozyme d i s t r i b u t i o n p a t t e r n s (Denford a n d V a u g h a n , 1977) a n d RFLP a n a l y s i s (Song et al., 1988b) s u p p o r t S u n ' s views. Evidence from morphology, geographic d i s t r i b u t i o n , isozymes a n d n u c l e a r RFLPs indicate t h a t t h e s e g r o u p s r e p r e s e n t two i n d e p e n d e n t c e n t e r s of origin. E u r o p e c o n s t i t u t e s the p r i m a r y c e n t e r for oleiferous forms a n d turnip. E a s t e r n forms evolved in the n o r t h - w e s t of I n d i a in the oleiferous direction, while Chinese forms differentiated as leafy vegetables in S o u t h China.
44 T u r n i p (B. rapa s u b s p , rapa) is believed to have evolved in Europe. Carbonized t u r n i p s have been recovered from Neolithic sites (Hyams, 1971). DeCandolle (1886) p r o p o s e d its cultivation in Europe a r o u n d 2 5 0 0 - 2 0 0 0 BC a n d its s p r e a d to Asia after 1000 BC. The Chinese book 'Shih-ching'by Confuceous (551-479 BC) m e n t i o n e d t u r n i p (Keng, 1974). T h e o p h r a s t u s (370285 BC) m e n t i o n e d t u r n i p in 'Enquiry into Plants'. He also m e n t i o n e d a wild p o p u l a t i o n with long n a r r o w roots having small hairy leaves. Similarly, Rom a n Cato (234-149 BC) referred to t u r n i p in 'On Agriculture' as a vegetable. Columella (42 AD) a n d recorded its uses. Plinius (23-79 AD) wrote extensively a b o u t t u r n i p in 'Natural History' a n d referred to it as rapa and napus. He stated t h a t Greeks d i s t i n g u i s h 3 k i n d s of turnip: a fiat one, a r o u n d one a n d a wild form with very long root. Columella (c. 60 AD), a u t h o r of one of the best R o m a n h a n d b o o k s on agriculture gave significant information on t u r n i p s . He m e n t i o n e d long 'Roman', 'Round' from Spain, the 'Syrian', the 'White' a n d the 'Egyptian'. The existence of m a n y Semitic, Greek a n d Slavic n a m e s for t u r n i p is significant a n d indicative of the antiquity of its domestication. In Middle English, napus b e c a m e nep a n d this together with turn (made round) gave the n a m e turnip (Boswell, 1949) which a p p e a r e d only after 1400 AD. Leafy forms are believed to have differentiated in C h i n a from oilseed forms of B. rapa after its i n t r o d u c t i o n t h r o u g h w e s t e r n Asia or Mongolia in the 1st c e n t u r y AD. Pak-choi (subsp. chinensis) with a n a r r o w or wide greenwhite petioles was the first to evolve in central C h i n a (Li, 1982). Its antiquity is s u g g e s t e d by a vast range of morphological diversity (Li, 1982) a n d high levels of DNA p o l y m o r p h i s m (Figdore et al., 1988; Song et al., 1988b). This was also the m o s t primitive form of East-Asian group from which s u b s p parachinensis developed in central China. The history a n d origin of Chinese cabbage - B. r. s u b s p , p e k i n e n s i s - is well d o c u m e n t e d (Li, 1982). The primitive loose-leaved form a p p e a r e d in the 10th c e n t u r y as a hybrid between p a k - c h o i (subsp. chinensis) a n d t u r n i p (subsp. rapa) in the city of YoungChow. This information is based on a reference in the book Ben-Cao-Tou-Jing (The Classics of Illustrated Medical Herbs). Its hybrid origin is also s u p p o r t e d by RFLP analysis (Song et al., 1988b). This primitive form is still grown in s o u t h e r n China. The h e a d i n g form with thick petioles a p p e a r e d for the first time in n o r t h e r n C h i n a a n d is recorded in 12th c e n t u r y literature. Better agronomy, irrigation a n d n u t r i e n t s u p p l y helped the a p p e a r a n c e of semih e a d i n g forms. S u b s e q u e n t l y , solid h e a d forms evolved t h r o u g h selection. A 14th c e n t u r y book Shua-Pu-Tsa-Su (Miscellanea of Gardening) mentioned s u c h forms. These forms were f u r t h e r improved by g a r d e n e r s a n d solid h e a d s with fluffy tops or fully solid h e a d s were developed. These types were described in Shuin-Tian-Fu-Tse (Local Records of Shuin 7~an FU) in the 17th century. Oleiferous B. rapa is d i s t r i b u t e d from Europe to China. It is believed t h a t E u r o p e a n forms developed in the M e d i t e r r a n e a n area (Sinskaia, 1928). On the other h a n d , Asian forms originated in the region comprising Central
45 Asia, A f g h a n i s t a n a n d adjoining n o r t h - w e s t India. In the I n d i a n s u b c o n t i n e n t there are 3 ecotypes of oleiferous B. rapa: b r o w n s a r s o n , toria a n d yellow sarson. Of t h e s e brown s a r s o n a p p e a r s to be the oldest (Singh, 1958). Two views r e g a r d i n g its origin exist: a) it evolved in the n o r t h - w e s t of the I n d i a n s u b - c o n t i n e n t from the original B. rapa stock (Sinskaia, 1928) a n d b) it r e a c h e d n o r t h - w e s t of India t h r o u g h Iran a l r e a d y in the s u b - d i f f e r e n t i a t e d s t a t e (Alam, 1945), from w h e r e it m i g r a t e d e a s t w a r d s a n d differentiated into o t h e r ecotypes. In t u r n , Toria is a n early m a t u r i n g crop very similar to b r o w n s a r s o n in m o r p h o l o g y except for the growing period a n d the size of the plant. It is believed to have been selected from a brown s a r s o n p o p u l a t i o n in the s u b - m o u n t a n e o u s t r a c t of the H i m a l a y a s . Yellow s a r s o n is c h a r a c t e r i z e d by yellow colored seeds a n d self-compatibility. M a n y of the cultivars have 3-4 valved siliquae a n d for this r e a s o n it w a s once n a m e d B. trilocularis (Roxburg, 1832). However, this c h a r a c t e r does not s e e m intrinsic to it as forms with bilocular fruits also o c c u r in n a t u r e . It is believed to have evolved from brown s a r s o n as a m u t a n t a n d have survived b e c a u s e of its self-compatible n a t u r e . It m i g h t have b e e n selected by f a r m e r s for its a t t r a c t i v e yellow seed color a n d a bigger seed size. It w a s first m e n t i o n e d as Siddhartha in S a n s krit l i t e r a t u r e from c. 1 0 0 0 - 8 0 0 BC i n d i c a t i n g t h a t it w a s well e s t a b l i s h e d by t h a t time. H i n a t a a n d P r a k a s h (1984) s u g g e s t e d c. 1200 BC a n d n o r t h - w e s t of India as the tentative date a n d place of origin.
B r a s s i c a nigra (L.) K o c h . This species w a s m e n t i o n e d by H i p p o c r a t e s (480 BC) for its m e d i c i n a l value. The New T e s t a m e n t m e n t i o n s ~ m u s t a r d " as a '~plant growing fast a n d high u p from a small seed, b r a n c h i n g a n d allowing birds to n e s t on its b r a n ches" in a c o m p a r i s o n with the growth of the Kingdom of God. This m i g h t n o t be too e x a g g e r a t e d since in good c o n d i t i o n s this a n n u a l species c a n easily b e c o m e t h r e e to four m e t e r s high in only a few weeks. Its n a t u r a l a r e a is c i r c u m - m e d i t e r r a n e a n e x t e n d i n g into C e n t r a l Asia a n d the Middle East, so it w a s easily available for d o m e s t i c a t i o n by m a n y Old World civilizations. PreC o l u m b i a n M e d i t e r r a n e a n civilizations were p a r t i c u l a r l y k e e n for spices to m a k e food m o r e tasteful, a n d c o n d i m e n t s b a s e d on Cruciferae, Labiatae or Umbelliferae were very i m p o r t a n t . The s t r o n g t a s t e of Brassica nigra (black m u s t a r d ) or Sinapis alba (white m u s t a r d ) seed m e a l s w a s therefore m u c h a p p r e c i a t e d . E i t h e r cultivated or s i m p l y collected from the wild, t h e s e two species have also been the object of m e d i c i n a l u s e s (as in s i n a p i s m s ) since very a n c i e n t times. The word m u s t a r d is t h o u g h t to come from Latin ~mustus ardens", b u r n i n g juice in English. Nowadays, m u s t a r d u s e s of B. nigra (black m u s t a r d ) have d i m i n i s h e d in favor of B. juncea (Indian m u s t a r d ) or in a growing scale B. carinata (Ethiopian m u s t a r d ) . F u r t h e r m o r e , B. nigra h a s also benefited m a n k i n d by p a r t i c i p a t i n g as a p a r e n t in t h e s e two a m p h i diploids.
46
Brassicajuncea (L.) C z e r n . Prain (1898) held t h a t B. juncea originated in China, a view also supported by Sinskaia (1928) who believed t h a t E a s t - E u r o p e a n B. juncea is also of Chinese origin from where it migrated naturally t h r o u g h the Kirgiz steppes. In evidence, she stated t hat it grows wild along this route. She further proposed t h a t forms with lyrate-pinnatisect leaves are the most primitive from which evolution occurred in three directions" bipinnate leaves dissected into th r ead like s e g m e n t s (in the E a s t Asian forms), crisp leaves (in the Chinese forms) and non-divided leaves (comprising the Central Asian and Indian forms). However, Burkill (1930) and Sun (1970) discard its Chinese origin placing it in the Middle East. S u n (1970) argued t h a t since the parental species are not found naturally in China, B. juncea had to be introduced from outside. Vavilov (1949) proposed Afghanistan and adjoining regions as the primary center of its origin and favored Central and Western China, Eastern India and Asia Minor t h r o u g h Iran as the secondary centers. However, the cytogenetical, biochemical and, in recent years, molecular evidence, point to a polyphyletic origin (Olsson, 1960b; Prakash, 1973a; Vaughan, 1977; Song et al., 1988a) at m a n y places where the parental species have a sympatric distribution. Due to the occurrence of wild forms of B. rapa and B. nigra, the region of the Middle East has strongly been favored as its original place of origin (Olsson, 1960b; Mizushima and Tsunoda, 1967). Wild forms of B. juncea still grow in this region particularly in the plateau of Asia Minor a n d s o u t h e r n Iran (Tsunoda and Nishi, 1968). Regions of southwestern China and north-west India constitute two secondary centers showing e n o r m o u s variations. Biochemical studies of V a u g h a n et al. (1963) and V a u g h a n an d Gordon (1973) provided strong evidence for the existence of two geographical races viz. Chinese, where the seeds have a marked mucilagenous epidermis and produce allylisothiocynates, and Indian which produce 3-butenyl isothiocynate. RFLP studies (Song et al., 1988a) also give s u p p o r t to two main centers of origin: a) Middle East-Indian region where p r e d o m i n a n t l y oil forms evolved; a n d b) China, where evolution occurred mainly towards leafy forms. China h a s a long history of B. juncea cultivation (Wen, 1980; Chen, 1982). Its first mention is in literature from the Chou Dynasty (1122-247 BC). Its use as a flavouring agent is recorded during the West Han Dynasty (206 BC-24AD). Dai in his work Liji (The book of Rites) referred to a "sliced j a m of fish with mustard". Uses for its seeds and leaves are also mentioned in C h i a - s s u - h s i c h ' s book Himin-yao-shu of late 5th or early 6th century (Li, 1969). F r e q u en t references are found in Su's (10-61 AD) work Tu-Jin-Bin-Cao (Illustrated Book of Medicinal Herbs) indicating that m u s t a r d was a popular crop of the time. Wang (1576-1588) mentioned root forms in his work 'Gua Guo Shz~ (Explanations of Cucurbits and Vegetable Crops). The famous work by Li (1578) described m a n y forms which were used as leaves or shoots during Ming dynasty.
47 A fascinating a c c o u n t r e g a r d i n g the origin of v a r i a t i o n s in Chinese B.
juncea h a s b e e n p r e s e n t e d by Wen (1980). Description in a n c i e n t literature
from the 5 t h c e n t u r y AD indicate t h a t the primitive type w a s a small a n n u a l p l a n t with poor leaf growth cultivated for its p u n g e n t seeds. S u b s e q u e n t variations of leaf s h a p e , size a n d colour, petiole width, h e a d i n g type a n d s p r e a d of leaves evolved a n d were selected. F o r m s with l u x u r i o u s b r o a d leaves were developed in the Tang D y n a s t y (618-907 AD) a n d u s e d as greens in t e m p e r a t e a n d h u m i d s o u t h China. A form with deeply dissected leaves a d a p t e d to arid e n v i r o n m e n t s was developed in n o r t h e r n China. This, in t u r n , p r o d u c e d a tillering form which was more productive, b r a n c h e d early d u r i n g vegetative growth a n d was good for pickles. F o r m s with broad, thick mid-ribs a n d petioles were developed d u r i n g the Chin d y n a s t y (1644-1911 AD). Later, h e a d e d forms with leaves with fleshy m i d r i b s a n d petioles evolved. Types with swollen s t e m were also bred. F l e s h y root forms evolved i n d e p e n d e n t l y from b r o a d leaved forms p r e s u m a b l y after the 12th century.
B. juncea w a s also a c o m p o n e n t of the a g r i c u l t u r e of the I n d u s Valley civilization w h i c h flourished a r o u n d 2 3 0 0 - 1 7 5 0 BC. The a r t of e x t r a c t i n g oil w a s k n o w n to this civilization. In fact, seeds of B. juncea have been excavaed from C h a n h u d a r o , a site of this civilization (Allchin, 1969: Mackey, 1943). W h e n the A r y a n s c a m e to India c. 1500 BC, they a d o p t e d B. juncea oil as a preservative. Its u s e w a s later e x t e n d e d to cooking a n d m a s s a g e p u r p o s e s . A r o u n d 1000 BC, it s p r e a d e a s t w a r d s with m i g r a t i n g people. Reports of Chinese travelers H u e n T s a n g (c. 640 AD) a n d Itsing (c. 690 AD) reveal t h a t it was e s t a b l i s h e d as a n oil crop in the I n d o - G a n g e t i c Plains by 700 AD. T h o u g h conflicting views have been e x p r e s s e d r e g a r d i n g the route of e n t r y of B. juncea into India, it s e e m s t h a t B. juncea r e a c h e d n o r t h - w e s t India from the Middle East, its place of origin, t h r o u g h A f g h a n i s t a n b e t w e e n 5 5 0 0 - 2 3 0 0 BC (Hinata a n d P r a k a s h , 1984). Intensive differentiation of m a n y agro-ecotypes was later c o m p l e m e n t e d by new h y b r i d i z a t i o n s b e t w e e n the constit u e n t p a r e n t s in n o r t h - w e s t India. Brassica
o l e r a c e a L.
B. oleracea L., grows wild in the Atlantic c o a s t s of Europe, where it m i g h t have b e e n cultivated by Celts in its primitive form (kales). W h e n it was eventually b r o u g h t to the E a s t M e d i t e r r a n e a n region (estimatedly by the limit between the first a n d second millenia BC) it b e c a m e fully d o m e s t i c a t e d a n d s t a r t e d an explosive diversification giving rise to a n e n o r m o u s r a n g e of cultivated forms. The m o s t widely k n o w n forms or g r o u p s of forms are: 1) kales w h i c h develop a s t r o n g m a i n s t e m a n d are u s e d for their edible foliage. These are old cultivated forms a n d include green curly kales, n a r r o w s t e m kale, a n d giant J e r s e y kale. Land r a c e s of these kales are widely scattered.
48
2) cabbages are charaterized by formation of h e a d s of tightly packed leaves and are represented by head cabbage, savoy cabbage and Brussels sprouts. 3) Kohlrabi is grown for its thickened stem. 4) Inflorescence kales are u s e d for their edible inflorescences. Major forms are cauliflower, broccoli and calabrese. 5) Chinese albogtabra kale, u s e d for its leaves. Earlier, it was widely held t h a t wild oleracea kales which are found along the Mediterranean coasts, were progenitors of the cultivated forms (see P r a k a s h an d Hinata, 1980), b u t the pr es ent concept is the opposite: Mediterr a n e a n kales are mere escapes from early cultivations. More recently, a polyphyletic origin by incorporation of genes into the B. oleracea genome from different wild Mediterranean species (Gustafsson, 1979; Snogerup, 1980; Mithen et al., 1987) was suggested. The species preferently considered in this respect were B. cretica, B. rupestris, B. insularis, and B. montana. However, Hosaka et al. (1990) do not find molecular evidence to suggest specific wild ancestors for the different B. oleracea types. Today, the tendency is to minimize the possible introgression by other species, t h u s returning again to the Atlantic B r a s s i c a oleracea the main role in the development of cultivated forms b u t admitting t h a t introgression of genes from wild species has probably been responsible for increasing the variability and adaptability of cultivated B. oleracea. The earliest cultivated B. oleracea was most likely a leafy kale which gave rise to a wide variety of kales along the coasts of the Medit e r r a n e a n an d Atlantic from Greece to Wales (Song et al., 1990). Diverse forms were developed in different areas primarily due to selection in different climates, n a t u r a l hybridization and gene introgression. Macro- and microm u t a t i o n a l events and c h r o m o s o m a l changes also played a substantial role (Chiang an d Grant, 1975; Kianian and Quiros, 1992). Song et al. (1988b, 1990) considered Chinese kale (var. alboglabra) to be very close to the primitive type which spread to the center of the East Mediterranean and eventually reached China. The extent to which Celts domesticated B. oleracea in western and n o r t h w e s t e r n Europe, as believed by DeCandolle (1886), is an open question, since it is also likely t h a t invading Celts a r o u n d the VI-VIII centuries BC found cabbage already domesticated by aborigines and adopted it from them. Another open question is that of how a wild plant from the Atlantic coasts could reach the centers of civilization, at that time Egypt and Mesopotamia. A slow diffusion t h r o u g h France by 1000 BC seems improbable. However, a rapid way could have been provided by the tin route, linking the ~Casiteride" Islands (British Islands) with the East Mediterranean by sea (G6mez-Campo and Gustafsson, 1991). Tartessians, from SW Spain, were exploiting the tin mines of Cornualles a n d selling the ore to the Phoenicians in Gades (now Cadiz). The Phoenician maritime commercial network then at its peak in the Mediterranean coasts did the rest. The early nam e of B. oleracea (~krambe")
49 u s e d by the Greeks, w a s probably of Phoenician origin. Brassica itself h a s been linked to Celt n a m e s , b u t it p r o b a b l y comes from "prasikein", vegetable in Greek. Greek T h e o p h r a s t u s (370-285 BC) a n d R o m a n Cato (234-149 BC) a n d Plinus (23-79 AD) already described s t e m k a l e s a n d h e a d e d cabbages. It is believed t h a t an a r r a y of cultivated coles was available to R o m a n s at the time of Christ. Cauliflower a n d broccoli evolved in the e a s t e r n M e d i t e r r a n e a n (Hyams, 1971; S n o g e r u p , 1980). T h o u g h s p r o u t i n g forms of c a b b a g e were m e n t i o n e d by early G r e e k s a n d R o m a n s a distinction was not a p p a r e n t between cauliflower a n d broccoli, indicating t h a t the differences did not exist or t h a t the two were c o n s i d e r e d as v a r i a n t s of the s a m e form. A S p a n i s h - A r a b i c a u t h o r , Ibn-al-Awam, (c.1140) m a d e the first clear distinction between h e a d i n g a n d s p r o u t i n g forms in his book Kitab-al-Falaha, wherein he devoted a s e p a r a t e c h a p t e r to cauliflower (Hyams, 1971). He u s e d the n a m e 'quarnabit', the present day Arabic word for cauliflower. Herbalist D o d o e n s (1578) referred to cauliflower as B. cypria indicating its origin in Cyprus. H y a m s (1971), b a s e d on the o b s e r v a t i o n s of Ibn-al-Awam who referred to it as Syrian or Mosul cabbage, c o n s i d e r e d Syria as the place of its origin. Cauliflowers are generally r e g a r d e d as derived from broccolies (Crisp, 1982; Gray, 1982). Crisp (1982), b a s e d on his w o r k on hybridization between broccoli a n d cauliflower, c o n c l u d e d t h a t a single m a j o r gene m u t a t i o n in broccoli gave rise to cauliflower. Broccoli is an Italian word derived from the Latin '~brachium" which m e a n s a n a r m or b r a n c h (Boswell, 1949). It includes h e a d i n g forms with a single large t e r m i n a l inflorescence. Another form is s p r o u t i n g broccoli, a b r a n c h e d type in which the y o u n g edible inflorescences are referred to as sprouts. Broccoli p r o b a b l y originated between 4 0 0 - 6 0 0 y e a r s BC w h e n the a n c e s t r a l forms of m o d e r n varieties were selected (Schery, 1972). Dalec h a m p ' s (1586) description in the 16th c e n t u r y h e r b a l Historia Generalis Plantarum c o n s t i t u t e s the first d o c u m e n t e d report. Broccolis were introd u c e d into Italy from the e a s t e r n M e d i t e r r a n e a n where diversification took place a n d m a n y forms, including h e a d i n g a n d s p r o u t i n g ones arose. The regular h a n d s o m e inflorescence of recently commercialized '~Romanesco" broccoli is often cited by the s t u d e n t s of fractal geometry. B r u s s e l s s p r o u t s were developed n e a r B r u s s e l s in Belgium d u r i n g the 14th century. A kale r e s e m b l i n g B r u s s e l s s p r o u t s , b u t with finely dissected leaves a n d n u m e r o u s b u d s , is d e s c r i b e d by Gerarde (1597) as ~persil cabbage" (Henslow, 1908). These s p r o u t s were reportedly served at a wedding feast in 1481 (Hyams, 1971). Brassica
napus
L.
Brassica napus is not k n o w n to occur truly wild in n a t u r e t h o u g h it often o c c u r s as an escape. The first reference to r a p e s e e d (B. napus s u b s p . oleifera) was by D o d o e n s (1578) in 'Cruydt Boeck' wherein he referred to Slo-
50 o r e n being grown for oilseeds (Toxopeus, 1979). This was probably an early form of winter rape. As a crop, it a p p e a r e d a r o u n d the y e a r 1600. Its oil was k n o w n as "raepolie" a n d was u s e d in l a m p s for lighting, as a food a n d in soap making. S u m m e r rape developed a r o u n d the y e a r 1700 and is cultivated in places where winter is not too severe. Some older records have been o b s c u r e d by its similarity to B. rapa.
S i n s k a i a (1928) a n d S c h i e m a n n (1932) p r o p o s e d t h a t it originated in the M e d i t e r r e n e a n region of s o u t h - w e s t E u r o p e where the two contributing p a r e n t s , B. o l e r a c e a a n d B. rapa, overlap in their n a t u r a l distribution (Olsson, 1960b; T s u n o d a , 1980). However, s u c h overlapping did not probably exist. Wild B. r a p a is very poorly r e p r e s e n t e d today in S p a i n (only in Girona, the n o r t h e a s t e r n extreme) a n d it is difficult to envisage it a p p r o a c h i n g the Atlantic m a r i t i m e cliffs where B. o l e r a c e a was growing wild. Thus, we believe t h a t it m a y have originated elsewhere, even outside the Mediterranean region, b u t obviously in a n agricultural environment. In fact, the B. o l e r a c e a • B. r a p a cross h a s o c c u r r e d several times a n d in both directions. Palmer et al. (1983) explored the m a t e r n a l lines in the U s c h e m e t h r o u g h chloroplast analysis. C o n t r a d i c t o r y phylogenies for two lines of B. n a p u s might indicate either f u r t h e r introgressive hybridization or multiple origins. Ar~s, Baladr6n a n d Ord~s (1987) u s e d isozymes to analyze B. n a p u s a n d their parents. Aguinagalde (1988) detected additive profiles of flavonoids not only for B. n a p u s b u t for all the three hybrid species. An alternative view on the origin of B. n a p u s h a s been provided by Song a n d O s b o r n (1992) b a s e d on chloroplast a n a l y s e s of several B. n a p u s accessions. His r e s u l t s suggest t h a t B. nap u s cDNA is closer to t h a t of B. m o n t a n a t h a n t h a t of B. oleracea. R u t a b a g a s or swedes (B. n a p u s s u b s p , rapifera) are also believed to be of recent origin. According to Boswell (1949), the h e r b a l i s t B a u h i n mentioned swede for the first time t h o u g h no a c c u r a t e reference is provided. Zwinger (1696) described a form S t e c k r u e b e n - k o h l which in all probability r e p r e s e n t s a n early form of r u t a b a g a . It b e c a m e p o p u l a r in Scandinavia a n d later s p r e a d to E n g l a n d in the late 18th c e n t u r y (McNaughton and Thow, 1972). Cultivated r a p e s e e d or colza started with oleiferous varieties of B. r a p a (which are still in use), b u t B. n a p u s h a s progressively t a k e n the s u p r e m a c y in this role. This h a s o c c u r r e d to a point t h a t B. n a p u s , only 400 years old as a k n o w n species, h a s now climbed to the second or third places in economic i m p o r t a n c e a m o n g edible crops in several c o u n t r i e s s u c h as C a n a d a a n d a few in Central Europe. D e p e n d i n g u p o n the yearly prices, it sometimes surp a s s e s w h e a t in relative importance. Double-zero varieties (without erucic acid a n d w i t h o u t glucosinolates) have given a new e n o r m o u s impulse to its use. For f u r t h e r information a b o u t recent scientific a n d applied developm e n t s involving B. n a p u s , the reader is referred to o t h e r c h a p t e r s of this book, especially to n u m b e r 13.
51 B. c a r i n a t a Braun
An a m p h i d i p l o i d derived from c r o s s e s b e t w e e n B. oleracea a n d B. nigra, it h a s b e e n g r o w n for c e n t u r i e s locally in Ethiopia. M i z u s h i m a a n d T s u n o d a (1967) failed to locate a n y wild t y p e s in t h e E t h i o p i a n p l a t e a u , b u t t h e y often o b s e r v e d t h e p a r e n t s growing close to e a c h o t h e r in c u l t i v a t i o n or a s escapes. The u s e s of t h i s p l a n t in E t h i o p i a are m u l t i p l e (Astley, 1982; Riley a n d B e l a y n e h , 1982): oil is e x t r a c t e d , the c a k e is u s e d a s a m e d i c i n e , c r u s h e d s e e d s are c o n s u m e d in s o u p s or as spice, leaves are boiled a n d e a t e n , etc. Brassica carinata is n o w b e c o m i n g m o r e a n d m o r e p o p u l a r in o t h e r p a r t s of the World as a p r o m i s i n g oilcrop a n d s o m e i m p o r t a n t b r e e d i n g p r o g r a m s a r e u n d e r w a y (Rakow a n d Getinet, 1998).
O t h e r g e n e r a o f agricultural s i g n i f i c a n c e
R a p h a n u s sativus L. is n o t k n o w n in the wild s t a t u s ( t h o u g h it h a s bec o m e w e e d y in m a n y a r e a s , e s p e c i a l l y in t e m p e r a t e S o u t h America). It is t h o u g h t to h a v e o r i g i n a t e d by d o m e s t i c a t i o n a n d selection of t h e wild s p e c i e s R a p h a n u s raphanistrum. Mainly u s e d for its root, t h i s h a s r e a c h e d a h i g h m u l t i p l i c i t y of s h a p e s , sizes a n d colors. B u t leaf forage v a r i e t i e s are also c u l t i v a t e d a n d t h e long fruits of var. caudatus are often a n object of h u m a n c o n s u m p t i o n in s o m e p a r t s of Asia. Eruca vesicaria (L.) Cav., t h e rocket, h a s four c o n s p i c u o u s s u b s p e c i e s , t h r e e r e s t r i c t e d to the W e s t M e d i t e r r a n e a n region. The fourth, s u b s p , sativa ( c o m m o n l y referred to a s E. sativa) s h o w s a c i r c u m - m e d i t e r r a n e a n a r e a w h i c h e x t e n d s (in its wild form) into I r a n i a n plains. C u l t i v a t i o n h a s f u r t h e r e x t e n d e d t h i s a r e a into the I n d i a n s u b c o n t i n e n t , C h i n a a n d N. a n d S. America. C u l t i v a t e d f o r m s are easily d i s t i n g u i s h e d b e c a u s e t h e i r p o d s are c o m p a r a t i v e l y m o r e r o b u s t a n d t h e i r s e e d s are bigger. S a i n t Isidoro of Seville (VIII c e n t u r y ) a n d Ibn a l - A w w a m (XII c e n t u r y ) m e n t i o n its c u l t i v a t i o n in Spain, b u t t h i s is t o d a y c o m p l e t e l y m a r g i n a l . O t h e r w i s e , r o c k e t is very p o p u lar in Italy a s a s o u r c e of p u n g e n t s a l a d , or in T u r k e y a n d Egypt, w h e r e n o n p u n g e n t v a r i e t i e s are a s a p p r e c i a t e d a s l e t t u c e b u t c h e a p e r . In India, '~taramira" s e e d s are a c o m m o n s o u r c e of oil for b u r n i n g a n d also for h u m a n consumption. O t h e r Eruca vesicaria s u b s p e c i e s are n o t c u l t i v a t e d b u t t h e y s h o w som e p r o m i s i n g traits. For i n s t a n c e , s u b s p , vesicaria from S p a i n is h i g h l y p u n gent a n d its odor c a n even be d e t e c t e d in the field from a few m e t e r s away. S u b s p . pinnatifida grows in b a r r e n a r e a s close to NW African d e s e r t s , showing a h i g h r e s i s t a n c e to d r o u g h t c o n d i t i o n s a n d a very s h o r t life cycle. S o m e Diplotaxis s p e c i e s are also c u l t i v a t e d , m o s t l y in Italy, for u s e s sim i l a r to t h o s e of Eruca. T h e s e are a l m o s t exclusively D. tenuifolia a n d D. muralis, t h o u g h the g e n u s Diplotaxis c o n t a i n s m a n y o t h e r species, m a i n l y w i t h i n t h e s u b g e n u s Diplotaxis t h a t c o u l d be e q u a l l y u s e d to provide p u n gency to s a l a d s a n d o t h e r d i s h e s .
52
Acknowledgement The a u t h o r s are indebted to Dr M. L a k s h m i k u m a r a n for her permit to reproduce the content of Table 2.1.
References Aguinagalde, I. 1988. Flavonoids in Brassica nigra (L.) Koch, B. oleracea L., B. campestris L. and their n a t u r a l amphidiploids. Bot. Mag. Tokyo, 101, 55-60. Alam, Z. 1945. Nomenclature of oleiferous brassicas cultivated in Punjab. Indian J. Agr. Sci. 15, 173-181. Allchin, F. R. 1969. Early cultivated plants in India and Pakistan. In: Ueko, P.J. and Dimbleby G.W. (eds.). The domestication and exploitation ofplants and animals. Duckworth, London, pp. 323-328. Arfls, P., Baladrdn, J . J . and Ord/ts, A. 1987. Species identification of cultivated b r a s s i cas with isozyme electroforesis. Cruciferae Newsl. 12, 26. Astley, D. 1982. Collecting in Ethiopia. Cruciferae Newsl. 7, 3-4. Baldini, R. M. 1998. Rediscovery of Brassica procumbens (Poir.) O.E.Schulz (Cruciferae) in Italy with some systematic and distributional observations. Webbia 53, 57-68. Boswell, V. R. 1949. Our vegetable travellers. Nat. Geogr. Magaz. 96, 134217. Burkill, I. H. 1930. The Chinese m u s t a r d s in the Malay Peninsula. Gad's
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Chen, S. R. 1982. The origin and differentiation of m u s t a r d varieties in China. Cruciferae Newsl. 7, 7-10. Cheng, B. F. and Heneen, W. K. 1995. Satellited c h r o m o s o m e nucleolus organizer regions and nucleoli of Brassica campestris L., B. nigra (L.) Koch. and Sinapis arvensis L. Hereditas 122, 113-118. Cheng, B. F., Heneen, W. K. and Pedersen, C. 1995. Ribosomal RNA gene loci and their nucleolar activity in Brassica alboglabra Bailey. Hereditas 123, 169-173. Chiang, B.Y. and Grant, W. F. 1975. A putative heterozygous interchange in the cabbage (Brassica oleracea var capitata} cultivar ~Badger Shipper'. Euphytica 24, 581-584. Chyi, Y.S., Hoenecke, M. E. and Sernyk, J. L. 1992. A genetic linkage map of restriction fragment length polymorphism loci for Brassica rapa (syn. campestris). Genome 35, 746-757. Crisp, P. 1982. The use of an evolutionary scheme for cauliflowers in the screening of genetic resources. Euphytica 3 1 , 7 2 5 - 7 3 4 .
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57 Song, K. M., Suzuki, J. Y., Slocum, M. K., Williams, P. H. and Osborn, T. C. 1991. A linkage map of Brassica rapa (syn. campestris) based on restriction fragment length polymorphism loci. Theor. Appl. Genet. 82, 296-304. Song, K. M. and Osborn, T.C. 1992. Polyphyletic origins of Brassica napus: new evidence based on organelle and nuclear RFLP analyses. Genome 35, 992-1001. Song, K. M., Tang, K. and Osborn, T. C. 1993. Development of synthetic Brassica amphidiploids by reciprocal hybridization and comparison to natural amphidiploids. Theor. Appl. Genet. 86, 811-821. Sun, V. G. 1946. The evaluation of taxonomic characters of cultivated Brassica with a key to species and varieties. I. The characters. Bull. Torrey Bot. Cl. 73, 244-281. Sun, V. G. 1970. Breeding plants of Brassica. J. Agr. Assoc. China, 71, 4152. Takahata, Y. and Hinata, K. 1980. A variation study of subtribe Brassicinae by principal component analysis. In: Tsunoda, S., Hinata, K. and G6mez-Campo, C. (eds.), Brassica crops and wild allies. Biology and breeding, J a p a n Scientific Societies Press, Tokyo, pp. 33-49. Takahata, Y. and Hinata, K. 1983. Studies on cytodemes in the subtribe Brassicineae. Tohoku, J. Agri. Res. 33, 111-124. Takahata, Y. and Hinata, K. 1986. A consideration of the species relationships in subtribe Brassicinae (Cruciferae) in view of cluster analysis of morphological characters. Pl. Sp. Biol. 1, 79-88. Toxopeus, H. 1979. The domestication of Brassica crops. Proc. Eucarpia Conference on the breeding of Cruciferous crops., 47-56. Tsunoda, S. 1980. Ecophysiology of wild and cultivated forms in Brassica and allied genera. In: Tsunoda, S., Hinata, K. and G6mez-Campo, C. (eds.), Brassica crops and wild allies. Biology and breeding, Japan Scientific Societies Press, Tokyo, pp. 109-120. Tsunoda, S. and Nishi, S. 1968. Origin, differentiation and breeding of cultivated Brassica. Proc. XII Int. Congr. Genet. 2, 77-88. Uchimiya, H. and Wildman, S. G. 1978. Evolution of fraction I protein in relation to origin of amphidiploid Brassica species and other member of Cruciferae. J. Heredity 69, 299-303. Vaughan, J. G. 1977. A multidisciplinary study of the taxonomy and origin of Brassica crops. Bio. Sci. 27, 35-40. Vaughan, J. G. and Denford, K. E. 1968. An acrylamide gel electrophoretic study of the seed proteins of Brassica and Sinapis species, with special reference to their taxonomic value. J. Exp. Bot. 19, 724732.
58 V a u g h a n , J. G. a n d Gordon, E. I. 1973. A taxonomic s t u d y of Brassica juncea u s i n g the t e c h n i q u e s of electrophoresis, gas-liquid chromatog r a p h y a n d serology. Ann. Bot., 37, 167-184. V a u g h a n , J. G., Hemingway, J. S. a n d Schofield, H. J. 1963. Contributions to a s t u d y of variations in Brassica juncea Coss et. Czern. Bot. Journal of the Linnean Society 58, 435-447. V a u g h a n , J. G., Waite, A. Boulter, D. a n d Waiters, S. 1966. Comparative s t u d i e s of the seed proteins of Brassica campestris, B. oleracea a n d B. nigra. J. Exp. Bot., 17, 332-343. Vavilov, N. I. 1949. The origin, variation, i m m u n i t y a n d breeding of cultivated plants. Chron. Bot. 13, 1-364. Verma, S. C. a n d Rees, H. 1974. Nuclear DNA a n d the evolution of allotetraploid Brassicae. Heredity 33, 61-68. Wang, X. H., Luo, P. a n d Shu, J. J. 1989. Giemsa N - b a n d i n g pattern in Cabbage a n d Chinese kale. Euphytica 41, 17-21. Warwick, S. I. a n d Black, L. D. 1991. Molecular s y s t e m a t i c s of Brassica a n d allied genera (Subtribe Brassicinae Brassicae) - chloroplast genome a n d cytodeme congruence. Theor. Appl. Genet. 82, 81-92. Warwick, S. I. a n d Black, L. D. 1993. Molecular r e l a t i o n s h i p s in subtribe Brassicinae (Cruciferae, tribe Brassiceae). Can. J. Bot. 7 1 , 9 0 6 - 9 1 8 . Warwick, S. I. a n d Black, L. D. 1994. Evaluation of the s u b t r i b e s Moricandiinae, Savignynae, Vellinae a n d Zillinae (Brassicaceae, Brassiceae) u s i n g chloroplast DNA restriction site variation. Can. J. Bot. 72, 1692-1701. Warwick, S. I. a n d Black, L. D. 1997. Phylogenetic implications of chloroplast DNA restriction site variation in s u b t r i b e s Raphaninae a n d Cakilinae (Brassicaceae, tribe Brassiceae). Can. J. Bot. 75, 960973. Warwick, S. I., Black, L. D. a n d Aguinagalde, I. 1992. Molecular systematics of Brassica a n d allied genera (subtribe Brassicinae, Brassiceae) c h l o r o p l a s t DNA variation in the g e n u s Diplotaxis. Theor. Appl. Genet. 83, 839-850. Watt, G. 1989. Brassica. In: Dictionary of Economic Products of India. I., Calcutta, pp. 520-534. Wen, L. C. 1980. Classification a n d evolution of m u s t a r d crops (Brassicajuncea) in China. Cruciferae Newsl. 5, 33-35. Yanagino, T., T a k a h a t a , Y. a n d Hinata, K. 1987. Chloroplast DNA variations a m o n g diploid species in Brassica a n d allied genera. Japan J. Genet. 62, 119-125. Zwinger, F. 1696. Theatricum Botanicum. Basel.
Biology of Brassica Coenospecies C. G6mez-Campo(Editor) 91999 Elsevier Science B.V. All rights reserved.
59
3 CYTOGENETICS S h y a m P r a k a s h (1}, Yoshihito T a k a h a t a (2), P u l u g u r t h a B. Kirti (1) a n d Virender L. C h o p r a (1) (1) National R e s e a r c h Centre on Plant Biotechnology. I n d i a n Agricultural R e s e a r c h Institute. N e w Delhi - 11 O012. India. (2) Faculty o f Agriculture. I w a t e University. Morioka 020. J a p a n
D e t e r m i n a t i o n of c h r o m o s o m e n u m b e r for B r a s s i c a rapa (syn. B. campestris) by a J a p a n e s e r e s e a r c h e r T a k a m i n e in 1916 w a s the beginning of cytogenetical r e s e a r c h in Brassica, a l t h o u g h interspecific a n d intergeneric h y b r i d s were reported m u c h earlier ( R a p h a n u s s a t i v u s x B. oleracea, Sageret, 1826; B. n a p u s x B. rapa, Herbert, 1847). Hybridizations between different species a n d s t u d y of their meiotic behavior led Morinaga (1928-1934) to u n r a v e l the genetic a r c h i t e c t u r e of crop brassicas. A r o u n d t h a t time, Manton (1932) carried o u t a n extensive g e n o - s y s t e m a t i c a l survey of Cruciferae a n d d e t e r m i n e d the c h r o m o s o m e n u m b e r s for a n u m b e r of taxa - a s t u p e n d o u s pioneering t a s k indeed. During the 1970s, wild g e r m p l a s m of B r a s s i c a a n d related genera was extensively collected a n d cytogenetical s t u d i e s were initiated. While the t h e m e of r e s e a r c h in the early p h a s e centered a r o u n d polyploid breeding, later the priorities shifted to exploitation of wild allies for introgression of n u c l e a r genes for desirable agronomic traits, cytoplasmic s u b s t i t u t i o n s a n d c o n s t r u c t i o n of c h r o m o s o m e m a p s , t a k i n g a d v a n t a g e of d e v e l o p m e n t in the a r e a s of somatic cell a n d m o l e c u l a r genetics in recent years. Use of m o l e c u l a r t e c h n i q u e s h a s considerably helped in c o n s t r u c t i n g linkage m a p s by applying restriction f r a g m e n t length p o l y m o r p h i s m (RFLP), r a n d o m amplified polymorphic DNA (RAPD) a n d DNA fingerprinting. These investigations have yielded i m p o r t a n t information on g e n o m e organization, extent of gene duplications, c h r o m o s o m e s t r u c t u r a l c h a n g e s a n d intergenomic gene introgression. Recent years have seen a s p e c t a c u l a r d e v e l o p m e n t in in vitro t e c h n i q u e s s u c h as ovary a n d embryo culture, a n d somatic hybridization. Somatic hybridization n o t only overcomes reproductive barriers b u t also generates cytoplasmic variability which is n o t possible t h r o u g h the conventional m e t h o d of sexual hybridization. T h o u g h some of these recent d e v e l o p m e n t s are more t h r o u g h l y d i s c u s s e d in other p a r t s of this book, we
60 will m a k e some occasional reference to t h e m from the cytogenetical point of view.
The Brassica
coenospecies
The Brassica coenospecies e n c o m p a s s e s those genera a n d species which are genetically related to crop b r a s s i c a s a n d are capable of exchanging genetic material with t h e m (Harberd, 1976). T a x o n o m i s t s in the last 200 y e a r s have collected a n d described m a n y species a n d have often classified the s a m e species u n d e r different n a m e s resulting in large-scale synonymy. A c o m p r e h e n s i v e t a x o n o m i c investigation was a t t e m p t e d by Schulz (1919, 1936). H a r b e r d (1976) for the first time classified this g e r m p l a s m biologically into c y t o d e m e s or c r o s s i n g g r o u p s taking into c o n s i d e r a t i o n c h r o m o s o m e n u m b e r a n d pairing, a n d extent of fertility in the hybrids. He included 91 species belonging to 9 g e n e r a of the s u b t r i b e Brassicinae of Schulz (1919) viz. Brassica, Diplotaxis, Erucastrum, Eruca, Sinapis, Coincya (syn. Hutera), Hirschfeldia, Trachystoma, a n d Sinapidendron b u t excluded the g e n u s Reboudia. Two more g e n e r a from the related subtribe Raphaninae viz. Raphanus a n d Enarthrocarpus were also included. H a r b e r d recognized 44 cytodem e s including the crop species. Six c y t o d e m e s have two species each a n d a large n u m b e r c o n t a i n only one single species. Two c y t o d e m e s include more t h a n six species each - these are Coincya (syn. Hutera a n d Rhynchosinapis), a n d B. oleracea. T a k a h a t a a n d H i n a t a (1983) f u r t h e r extended this s t u d y a n d a d d e d a few more c y t o d e m e s in recent years. S y s t e m a t i s t s have investigated the t a x o n o m i c s t a t u s a n d r e l a t i o n s h i p s of a wider g e r m p l a s m beyond the b o u n d a r i e s of the s u b t r i b e Brassicinae employing m o l e c u l a r techniques which include nuclear, m i t o c h o n d r i a l a n d chloroplast DNA restriction fragm e n t length p o l y m o r p h i s m . This r e s e a r c h h a s s u b s t a n t i a t e d the earlier proposed t a x o n o m i c s t a t u s a n d cytogenetical r e l a t i o n s h i p s of m a n y taxa and h a s also s u g g e s t e d new r e l a t i o n s h i p s between g e n e r a a n d species. These investigations (Warwick a n d Black, 1994) proposed, for instance, the close relationship of three more genera viz. Moricandia, Rytidocarpus a n d Pseuderucaria from the related s u b t r i b e Moricandiinae with crop b r a s s i c a s suggesting their inclusion in the Brassica coenospecies. T h u s , Figure 3.1 and Table 3.1 refer to the coenospecies in its b r o a d e s t sense. The lowest gametic c h r o m o s o m e n u m b e r in the group is n = 7 and is c h a r a c t e r i s t i c of seven cytodemes. H a r b e r d (1976) was of the view t h a t all c y t o d e m e s of n = 13 or less are diploids (only one, t h a t of Diplotaxis harra is n = 13), while c y t o d e m e s with n = 14 or higher c h r o m o s o m e s are polyploids. A total of 43 c y t o d e m e s are diploids where every n u m b e r from n = 7 to n = 13 is r e p r e s e n t e d . A r o u n d 50% of these c y t o d e m e s have gametic chromosome n u m b e r n = 9 a n d n = 10. Polyploidy is not u n c o m m o n a n d is characteristic of 20 additional c y t o d e m e s c o m p r i s i n g both a u t o a n d allopolyploids (Table 3.2). Level of ploidy is k n o w n to exceed tetraploidy only in Moricandia (6x, 8x; S o b r i n o - V e s p e r i n a s , 1980) a n d Brassica repanda (Galland, 1988).
61
Family:
Tribe
Subtribe:
Genus
Cruciferae
Brassiceae
9
Brassicinae
I
: Brassica (20)
Diplotaxis (13) Erucastrum (11)
Raphaninae
Moricandiinae
Raphanus (1)
Moricandia (4)
Enarthrocarpus (1)
Rytidocarpus ( 1)
I
I
Pseuderucaria (1)
Eruca (1) Hirschfeldia (1) Sinapis (5) Coincya (2) Trachystoma (1) Sinapidendron (1)
Figure 3.1 Architecture of Brassica coenospecies (number of cytodemes in brackets)
62 T a b l e 3. 1 Cytodemes in Brassica coenospecies sensu lato (originally based
In Harberd with modifications by Takahata and Hinata (1983), W a r w i c k and Black (1994) and G 6 m e z - C a m p o (pers. comm.).
n
Principal species
1
7
2
7
Brassica deflexa Boiss. Diplotaxis erucoides (L.) DC.
3
7
Erucastrum virgatum C. Presl.
4
7
Erucastrum varium Durieu
5
7
Sinapis aucheri (Boiss.) O.E. Schulz
6
7
Hirschfeldia incana (L.) Lagreze-Fossat
7
7
Pseuderucaria spp. O.E. Schulz
8
8
Brassica nigra (L.) Koch
9
8
Brassica fruticulosa Cyr. (+ maurorum + spinescens)
10
8
Diplotaxis siettiana Maire
11
8
Erucastrum abyssinicum (A. Rich.) O.E. Schulz
12
8
E. nasturtiifolium (Poiret) O.E. Schulz (+ leucanthum)
13
8
Erucastrum strigosum (Thunb.) O.E. Schulz
14
8
Trachystoma spp.
15
9
Brassica oleracea L. and 8 wild Mediterranean allied species
16
9
Brassica oxyrrhina Coss.
17
9
Diplotaxis assurgens (Del.) Gren.
18
9
Diplotaxis catholica (L.) DC.
19
9
Diplotaxis tenuisiliqua Del.
20
9
Diplotaxis virgata (Cav.) DC.
21
9
Diplotaxis berthautii Braun-Blanq. & Maire
22
9
Erucastrum cardaminoides Webb & Berth. (+ canariense+ ifniense)
23
9
Raphanus L. all species and subspecies
24
9
Sinapis arvensis L. (+ allioni)
25
9
Sinapis pubescens L.
26
10
Brassica tournefortii Gouan.
27
10
Brassica barrelieri (L.) Janka
28
10
Brassica gravinae Ten.
29
10
Brassica repanda (Willd.)DC. (+ desnottesii)
63 30
10
Brassica rapa L. (+ many cultivated subspecies)
31
10
Diplotaxis siifolia G. Kunze
32
10
33
10
Diplotaxis viminea (L.) DC Enarthrocarpus spp.
34
10
35
11
Sinapidendron spp. Brassica souliei Batt.
36
11
37
11
Diplotaxis acris (Forsk.) Boiss. Brassica elongata Ehrh.
38
11
Diplotaxis tennuifolia (L.) DC. (+ pitardiana)
39
11
40
12
Eruca spp. Mill. Coincya spp. (syn. Hutera and Rhynchosinapis)
41
12
Sinapis alba L.
42
12
43
13
44
14
Sinapis flexuosa Poir. Diplotaxis harra (Forsk.) Boiss. (+ several subsps.) Erucastrum virgatum C. Presl. (subsp. pseudosinapis)
45
14
Moricandia arvensis (L.) DC.
46
14
Moricandia moricandioides (Boiss.) Heywood
47
14
Rytidocarpus moricandioides Coss.
48
15
49
15
Erucastrum gallicum (Willd.) O.E. Schulz Erucastrum elatum (Ball.) O.E. Schulz
50
16
51
16
Brassicafruticulosa (Boiss.& Reut.) N. African subpecies (+ B. cossoniana) Brassica balearica Pers.
52
16
Erucastrum nasturtiifolium (Poiret) O.E. Schulz (4x)
53
16
Erucastrum abyssinicum (A. Rich.) O.E. Schulz (4x)
54
17
Brassica carinata A. Braun
55
18
Brassicajuncea (L.) Czem & Coss.
56
19
Brassica napus L.
57
20
58
21
Brassica gravinae Ten. (4x) Diplotaxis muralis (L.) DC.
59
22
Brassica dimorpha Coss. & Dur.
60
24
Coincya spp. (4x)
61
28
62
42
63
80?
Moricandia suffruticosa (Desf.) Coss. & Dur. Moricandia spinosa Pomel Brassica repanda (Willd.) DC. (High Atlas)
64 Table 3.2
Polyploid cytodemes in B r a s s i c a coenospecies
Alloploids
Diploid progenitors
Reference
Brassica carinata, n = l 7
B. nigra, B. oleracea
U, 1935
Brassica juncea, n = 18
B. rapa, B. nigra
U, 1935
Brassica napus, n = 19
B. oleracea, B. rapa
U, 1935
Diplotaxis muralis, n=21
D. tenuifolia, D. viminea
Harberd and McArthur, 1972
Erucastrum gallicum, n=15
E. leucanthum x sp?
Harberd,1976
Erucastrum elatum, n = 15
Hirschfeldia incana x Erucastrum sp.
G6mez-Campo, 1983
Brassica balearica, n = 16
B. oleracea group x
Snogerup and Persson,1983
another species
Tentative autopoll:ploids
Diploid homologue
Reference
Moricandia arvensis, n = 14
unknown
Harberd, 1976
Moricandia moricandioides, n =14
unknown
Harberd, 1976
Rytidocarpus moricandioides, n=14 unknown
Harberd, 1976
Erucastrum virgatum, n = 14 (subsp. pseudosinapis) Brassica cossoniana, n - 16
E. virgatum, n = 7
Harberd, 1976
B. maurorum, n--8
Pradhan et al., 1992
Erucastrum abyssinicum, n--16
E. abyssinicum, n=8
Harberd,1976
Erucastrum nasturtiifolium, n=16
E. nasturtiifolium, n=8
Harberd,1976
Brassica gravinae, n = 20
B. gravinae, n= l O
Takahata and Hinata, 1983
Brassica dimorpha, n=22
B. souliei, n=l l
G6mez-Campo, 1980
Coincya spp., n=24
Coincya, n=12
Harberd, 1976
Moricandia suffruticosa, n - 2 8
Moricandia sp., n = 14
Sobrino-Vesperinas,1980
Moricandia spinosa, n=42
Moricandia sp., n=14
Sobrino-Vesperinas,1980
Brassica repanda n - 8 0
B. repanda, n=l 0
Galland, 1988
65
Crop brassicas: cytogenetic architecture Morinaga (1928-1934) pioneered genome analysis and unravelled the cytogenetic structure of cultivated B r a s s i c a species. These investigations established that crop brassicas consist of six species. Of these, B. nigra (n = 8), B. oteracea (n = 9) and B. rapa (n = 10) are diploid monogenomic elementary species. The other three viz. B. carinata (n = 17), B. j u n c e a (n = 18) and B. n a p u s (n = 19) are high chromosome digenomic species which evolved in n a t u r e through convergent alloploid evolution following hybridization between any two of the diploid species. This proposal was subsequently verified by experimental synthesis of B. n a p u s by U (1935). The relationship amongst crop species was presented in a diagram which is commonly referred to as U's triangle (Figure 3.2). The diploid species represent an ascending aneuploid series (Manton, 1932) and are regarded as secondary balanced polyploids. The earlier view held that they evolved from a common prototype with x = 6 (Mizushima, 1950; R6bbelen, 1960). However, recent investigation on nuclear, mitochondrial and chloroplast DNA restriction fragment length polymorphism established their evolution from two prototypes: B. nigra from one prototype and B. oleracea and B. rapa from the other. The evolutionary diversion is also reflected in their cytoplasm (Palmer, 1988; Warwick and Black, 1991; P r a d h a n et al., 1992). B. oleracea and B. rapa cytoplasms are closer to each other than either is to B. nigra (Palmer, 1988). Although variations were observed in the cytoplasms of B. rapa and B. oleracea in cp and mt DNA patterns (Song and Osborn, 1992), the mitochondrial genome of B. rapa was more variable t h a n that of B. oleracea. Pachytene chromosome analysis by R6bbelen (1960) and Venkateswarlu and Kamala (1973) revealed that diploids have 6 basic types of chromosomes. The B. rapa genome is represented by AABCDDEFFF (tetrasomic for chromosomes A and D; and hexasomic for chromosome F), B. oleracea by ABBCCDEEF (tetrasomic for chromosome B C and E) and B. nigra by AABCDDEF (tetrasomic for chromosomes A and D). Chromosomes of each type have lost homology due to structural a n d / o r genic alterations during their long evolutionary history (see chapter 7 in this book). However, all the three genomes are partially homologous as revealed by cytogenetic (Mizushima, 1950; Atria and R6bbelen, 1986; P r a k a s h and Hinata, 1980), and molecular evidence (Hosaka et al., 1990; Teutonico and Osborn, 1994). It is also held that the genetic information in all the three genomes is similar, only its organization and distribution on the chromosomes is different (Truco et al., 1996). Chromosomal duplications and translocations have played a pivotal role in differentiating the chromosomes (Quiros et al., 1988; Hosaka et al., 1990; McGrath et al., 1990; Truco and Quiros, 1994). Deletions may also have contributed in repatterning the chromosomes (Hu and Quiros, 1991). Moreover, because of the secondary balanced n a t u r e of B r a s s i c a genomes, these changes were tolerated and adjusted (Kianian and Quiros, 1992a).
66 The high c h r o m o s o m e species B. carinata, B. j u n c e a a n d B. n a p u s are chromosomally balanced with a regular bivalent forming regime devoid of any homoeologous pairing. Their alloploid n a t u r e has been confirmed by m a n y reports on their artificial synthesis (see P r a k a s h and Chopra, 1991), nuclear DNA content (Verma a n d Rees, 1974), r RNA genes (Quiros et al., 1987), nuclear DNA m a r k e r s (Song et al., 1988; H osaka et al., 1990; Demeke et al., 1992) an d genomic in situ hybridization (Snowdown et at., 1997b). Research involving Fraction-1 protein (Uchimiya and Wildman, 1978) and chloroplast a n d mitochondrial DNA analysis (Palmer et al., 1983; Erickson et al., 1983; Warwick and Black, 1991; P r a d h a n et al., 1992) has revealed the directionality of n a t u r a l hybridizations. These studies established B. nigra and B. rapa as the cytoplasmic donors of B. carinata and B. j u n c e a respectively. Chloroplast DNA restriction patterns for B. n a p u s were different from both B. rapa and B. oleracea b u t were more similar to B. oleracea. (Palmer et al., 1983; Erickson et al., 1983). A detailed s t u d y by Song and Osborn (1992) based on the RFLP pattern of cp an d mt DNAs provided strong evidence for multiple origins of B. n a p u s and suggested t h a t a closely related ancestral species of B. rapa and B. oleracea identical to B. m o n t a n a was the cytoplasmic donor of most of the B. n a p u s accessions. The cp genomes have been conserved in B. carinata and B. juncea, however it is slightly altered in B. n a p u s (Palmer et al., 1983). There are also indications of coinheritance of m t a n d cp genomes in all the 3 species (Palmer, 1988). It is also observed t h a t when the parental diploid species of the amphidiploid had highly differentiated cytoplasm as in B. j u n c e a and B. carinata, the nuclear genomes of the alloploids contributed by the male parents were altered considerably compared to the nuclear genomes of the female p a r e n t s (Song et al., 1988; 1995). Extensive s t r u c t u r a l r e a r r a n g e m e n t s of c h r o m o s o m e s have occurred since the evolution of these alloploid species (Slocum et al., 1990; Song et al., 1991; Kianian a n d Quiros, 1992b; Harrison and Heslop-Harrison, 1995). However, the average c h r o m o s o m e size remained u n c h a n g e d (Parkin and Lydiate, 1997). N u c l e a r DNA
Nuclear DNA content in crop species has been estimated by several investigators (Yamaguchi and Tsunoda, 1969; Verma and Rees, 1974; Arumug u n a t h a n an d Earle, 1991, Figure 3.2). An appreciable reduction in DNA content and nuclear volume in autotetraploids of B. oleracea and natural B. n a p u s led Yamaguchi and T s u n o d a (1969) to believe that nuclear DNA had been lost with evolution. Verma and Rees (1974) on the other h a n d were of the view t h a t there is a diminution in DNA content in allotetraploid subseq u e n t to their formation and lower DNA values in tetraploids were associated
67
III
(Solid and broken lines represent female and male parents, respectively, DNA values Mbpc/IC)
Figure 3.2 Cytogenetic relationships of crop brassicas (U, 193 5)
68 to reduced nuclear volume. Higher DNA density of nuclei in natural alloploids might be due to chromosome condensation at interphase because of switching off the r e d u n d a n t gene copies in tetraploids.
S a t e l l i t e c h r o m o s o m e s and rDNA loci Among the diploid species, B. r a p a has been reported to have one pair of chromosomes showing secondary constriction and satellites (Olin-Fatih and Heneen, 1992; Cheng et al., 1995). B. oleracea also has one pair of satellited chromosomes (Wang et al., 1989; Cheng et al., 1995) while B. nigra has three pairs (Cheng and Heneen, 1995). This is not in accordance with previous observations by Lan et al. (1991) who reported two satellited chromosome pairs. In situ hybridization with r DNA probes, however, revealed five pairs of loci in B. r a p a (Maluzynska and Heslop-Harrison, 1993; Fukui et al., 1998). Cheng and Heneen (1995) were of the opinion that four pairs are inactive in nucleolus formation and since it has 2 nucleolar organizing region bearing chromosomes, it represents one pair of active rDNA loci. B. nigra has two pairs of rDNA loci (Maluzynska and Heslop-Harrison, 1993) supporting the earlier observations of R6bbelen (1960) who found 2 pairs of chromosomes associated with nucleoli at pachytene. Variations in n u m b e r of rRNA gene loci have been observed in B. oteracea. T w o major and one minor pairs of loci were observed by Maluzynska and Heslop-Harrison (1993), and McGrath et al. (1990) indentifying two synteny groups linked to strong rDNA signals and one synteny group linked to weak signals. Similar observations were found by Kianian and Quiros (1992 ab) reporting three pairs of rRNA gene loci. However, Cheng et al. (1995) found two major and two minor rRNA gene loci located on satellited chromosome pair 9 and non-satellited pair 8 respectively and concluded that only two pairs of chromosomes are involved in nucleoli formation in agreement with earlier observations of R6bbelen (1960) and Kamala (1976) who found two bivalents associated with nucleoli at meiosis in B. oleracea and also of Delseny et al. (1990) who reported two pairs of rRNA gene loci. The alloploids have fewer rDNA loci than the s u m of their parents. B. j u n c e a has five major pairs and one minor pair, one less than those of the parental sum. B. n a p u s possess six major rDNA loci while B. carinata has four loci. Maluzynska and Heslop-Harrison (1993) and Snowdon et al. (1997
a) believed that the n u m b e r of loci has been reduced during evolution of these alloploid species and might be ascribed to chromosome rearrangments leading to loss of rDNA site.
Genome
manipulation
Genome manipulation is one of the ways to achieve breeding goals. It supplements nature's role in crop evolution by designed synthesis of the alloploid species through selectively choosing the donor progenitors. Such
69 experimental synthesis of a B r a s s i c a alloploid species dates back to 1935 when U d e m o n s t r a t e d the successful artificial synthesis of B. n a p u s . Subsequent literature during the last 60 years a b o u n d s in reports on synthesis not only on the naturally occurring alloploid species B. n a p u s , B. j u n c e a a n d B. c a r i n a t a b u t also on new alloploids combining entirely new genomes using a variety of methods. The early syntheses were entirely academic in n a t u r e aiming at verifying the proposals of Morinaga (1934). However, in the 1960s the priorities shifted to comprehensive programs of designed e n h a n c e m e n t of genetic variability of breeding value. Extensive variability in highly polymorphic B. r a p a and B. o l e r a c e a h a s been exploited in the synthesis. The objectives in these syntheses varied and included: development of productive B. j u n c e a forms (Olsson, 1960a; Prakash, 1973a); early a n d productive B. c a r i n a t a (Prakash et al., 1984); high seed yielding B. n a p u s (Olsson, 1960b); early developing B. n a p u s (Prakash, 1980; P r a k a s h a n d Raut, 1983; Akbar, 1987); synthesizing fodder forms of B. n a p u s (Namai a n d Hosoda, 1967, 1968; Ellerstrom and Sjodin, 1973); synthesis of root forming r u t a b a g a s or swedes (Olsson et al., 1955; Namai a n d Hosoda, 1968; Kato et al., 1968); a new head forming type (Shinohara and Kanno, 1961) a n d developing yellow seeded B. n a p u s (Chen et al., 1988). Synthesis Synthetic alloploids have been obtained t h r o u g h conventional sexual hybridizations from reciprocal cross (Table 3.3). It h a s been generally accepted that B. j u n c e a a n d B. c a r i n a t a are easier to synthesize t h a n B. n a p u s . In general, the cross B. o l e r a c e a • B. r a p a is very difficult to obtain followed by B. nigra x B. r a p a due to the operation of strong post-fertilization barriers. Failure of normal e n d o s p e r m development is the major c a u s e of interspecific hybrid embryo abortion (Inomata, 1975; Wojciechowski, 1985). Various embryo rescue techniques viz. ovary, ovule and embryo culture have been developed in recent years leading to the obtention of a large n u m b e r of hybrids (see Inomata, 1993; Shivanna, 1996). Cytoplasm type plays a crucial role in the success of producing synthetic alloploids. Synthetic B. j u n c e a with B. nigra cytoplasm a n d B. n a p u s with B. o l e r a c e a cytoplasm are very difficult to obtain. It is reported t h a t crosses with the same cytoplasm donor as the natural alloploids yield more hybrids t h a n the others (Song et al., 1993). A few reports indicated e n h a n c e d crossability when hybridization was carried out between tetraploids (Olsson, 1960b). In recent years alloploid species have been synthesized following protoplast fusion which not only helped in nuclear g e r m p l a s m e n h a n c e m e n t b u t also generated novel cytoplasmic organelle combinations. The first a t t e m p t was by S c h e n c k a n d R6bbelen (1982) on B. n a p u s . Somatic hybrid plants were intermediate between the p a r e n t s in general morphology a n d growth pattern and comparable to n a t u r a l accessions. However, some floral abnormalities were observed. This subject is throughly treated in c h a p t e r 4 of this
70
Table 3.3 Major investigations on artificial synthesis of natural alloploid species B. c a r i n a t a , B. j u n c e a and B. n a p u s through sexual hybridization Species B. carinata B. nigra x B. oleracea
and reciprocals
Frandsen, 1974; Mizushima, 1950b; Pearson, 1972; Prakash et al., 1984; Song et al., 1993
B. juncea B.rapa x B. nigra
and reciprocals
Frandsen, 1943; Ramanujam and Srinivasachar, 1943; Olsson, 1960a; Prakash, 1973a,b; Campbell et al., 1991; Song et al., 1993
B. napus B. rapa x B. oleracea
and reciprocals Oil rape
U, 1935; Karpechenko and Bogdanova, 1937; Frandsen, 1947; Rudorf, 1950; Hoffmann and Peters, 1958; Olsson, 1960b; Gland, 1982; Prakash and Raut, 1983; Akbar, 1987; Chen et al., 1988; Mithen and Magrath, 1992; Song et al., 1993
Forage rape
Hosoda, 1950, 1953, 1961; Hosoda et al., 1969; Sarashima 1967, 1973; Nishi, et al., 1970
Rutabaga
Olsson et al., 1955; Olsson, 1960b, Hosoda et al., 1963, 1969; Namai and Hosoda, 1967, 1968; Kato et al., 1968
Heading form
Shinohara and Kanno, 1961 ; Takada, 1986
71 book, so t h a t we will restrict ourselves to c o m m e n t some cytogenetical aspects.
Meiosis and fertility Meiosis is generally disturbed in sexually obtained synthetic taxa in early generations due to the occurrence of multivalents a n d univalents (Table 3.4). With advancing generations these abnormalities decrease and complete meiotic stabilization is achieved by AT-As generations. Multivalents (upto 4 quadrivalents) were frequently observed in B. c a r i n a t a (Mizushima, 1950b) and B. n a p u s (upto 2 IV + 1 III; S a r a s h i m a , 1973; P r a k a s h and Raut, 1983). Interestingly, higher associates were a b s e n t or rare in B. j u n c e a (Olsson, 1960a; P r a kas h, 1973a). Occurrence of univalents was of c o m m o n observance in all the synthetics in early generations. Synthetics invariably show reduced pollen and seed fertility in early generations (Table 3.4). Selections resulted in a considerable improvement accompanied by stabilization of c h r o m o s o m e pairing. Synthetic plants generally achieve complete fertility by As-A6 generations. C h r o m o s o m e analysis of somatic hybrid pl ant s revealed t h a t besides forms with n o r m a l c h r o m o s o m e s , hypo a n d hyperdiploids were also obtained in B. n a p u s (Schenck and R6bbelen, 1982; Terada et al., 1987; S u n d b e r g et al., 1987). High c h r o m o s o m e plants probably resulted from multiple fusions. Meiosis was by and large regular with formation of 17 bivalents in A1 generation plants of B. c a r i n a t a (Narasimhulu et al., 1992) and 19 bivalents in B. n a p u s (Schenck a n d R6bbelen, 1982; Rosen, et al., 1988). However, multivalents were also noticed - up to 3 quadrivalents in B. c a r i n a t a (Narasimhulu et al., 1992) a n d in B. n a p u s (Schenck and R6bbelen, 1982). Somatic hybrids had varied fertility. Pollen and seed fertility in B. carin a t a obtained by N a r a s i m h u l u et al. (1992), ranged from 36-87% and 18% respectively while those obtained by J o u r d a n a n d Salazar (1993) had 4-98% fertile pollen. However, these a u t h o r s reported t h a t pollen was ineffective in producing seed on selfing and on crossing to n a t u r a l B. c a r i n a t a seed set was very poor. In B. n a p u s , Schenck and R6bbelen (1982) reported low fertility, while Rosen et al. (1988) observed very low or no seed set. But pollination with n a t u r a l B. n a p u s gave a high seed set. Although S u n d b e r g et at. (1987) observed 38-70% pollen fertility, seed set was very poor. Similar resuits were also obtained by Heath and Earle (1996, 1997). In spite of regular meiosis, the pl ant s were highly sterile, the r e a s o n s for which are unknown. Analysis of cytoplasmic organelles indicated t h a t besides chloroplasts and mitochondrial combinations similar to n a t u r a l forms, other situations s u c h as recombined mitochondria and novel c o m b i n a t i o n s of chloroplasts and mitochondria were obtained. For example B. c a r i n a t a with B. o l e r a c e a cp and B. nigra m t (Narasimhulu et al., 1992; J o u r d a n and Salazar, 1993) and
Table 3.4 Chromosome pairing and seed fertility in synthetic Brussicu alloploids in different alloploid generations. Alloploid
B. curinata
Chromosome pairing
Seed fertility
Al
A2
17II-I6II+2I
-
17II-4IV+9II
171I-4IV+91I Mostly 1711 Poor
A3
Al
A2
Very poor Very low 2 1.3
A3
A4
A5
A6
27-53
References Frandsen, 1947
47-62
87
92
Mizushima and Katsuo, 1953 Prakash (unpub.)
B. juncea
B. napus
18I1-1611+41
1811 mostly
1811
6.2
26.6
3 1-64
43-92
Fully fertile
Fully fertile
Olsson, 1960a
18II-I4II+8I
1611+41-1811
1811 mostly
26.1
45.8
64
96
Fully fertile
Fully fertile
Prakash, 1973a
1911 occasional univalents
1911
1911
7.9
Highly fertile Highly fertile Highly fertile Highly fertile Normal fertile Olsson, 1960b
21V+2111+711
21V+1511-1911 1911 mostly
8.8
24
36
59
31V+9II+8I
1911 mostly
7-29
19-52
58-92
Highly fertile Highly fertile Highly fertile
1911 mostly
79
Sarashima, 1973 Prakash and
73 B. n a p u s with B. oleracea cp and B. rapa mt were reported (Sundberg, 1987; Rosen et al., 1988).
Agronomic potential of synthetic alloploids A survey of synthetic alloploids reveals t hat research with B. j u n c e a is concentrated in India whereas with B. carinata and B. n a p u s research takes place in several countries, primarily Europe and C a n a d a and also India. Synthetics in general are inferior to n a t u r a l cultivars in productivity. Since they r ep res ent new genetic variability, these have been u s e d to advantage as genetic stocks in breeding p r o g r a m m e s and as bridging material for introgressing disease resistance. As a result, several cultivars have been developed. Synthetic B. j u n c e a forms originating from leafy B. rapa have vigorous vegetative growth with quick growing large sized leaves which m ake t hem suitable for fodder. In contrast, plants involving oily forms of B. rapa have their leaves of small size b u t are productive and rich in oil content (Prakash, 1973a). Oil rape B. n a p u s h a s attracted considerable attention in Europe and of late in India while fodder rape and swedes have been exploited in Europe and J a p a n . Several promising oil rape forms were released in Sweden (Olsson, 1986). These include Svalof Panter, m a r k e t e d for its higher oil yield and comparatively rapid growth at low t em per atures; Svalof Norde which possess high seed a n d oil yield and considerable resistance to Peronospora and Verticillium; Brink and J u p i t e r for their high seed yields and very low erucic acid content. Early and productive selections of oil seed B. n a p u s have been found to be very promising in India (Prakash and Raut, 1983) a n d Bangla Desh (Zaman, 1989). Synthetic B. n a p u s derived from clubroot r e s i s t a n t B. rapa a n d B. oleracea p o s s e s s e d very effective resistance to clubroot (Diederichsen a n d Sacristan, 1996). Synthetic forms developed from B. rapa x wild B. oleracea were resistant to blackleg disease caus ed by L e p t o s p h a e r i a m a c u l a n s . Resistance was ascribed to a high level of alkenyl glucosinolates in the leaves (Mithen and Magrath, 1992). Heath a n d Earle (1996) recovered somatic hybrid B. n a p u s plants which were dwarf with n o n - s h a t t e r i n g pods and bold seeds. They also possessed a large a m o u n t of erucic acid. Fodder rape forms have been bred from synthetics originated from leafy and root forms of B. rapa viz. subsp, chinensis, narinosa, nipposinica, p e k i n e n s i s an d rapa. Hosoda (1953) bred a fodder rape-CO which was cultivated in J a p a n for its vigorous growth and winter hardiness. A synthetic head forming type (which does not exist in nature) developed from the cross B. oleracea var. capitata • B. rapa subsp, p e k i n e n s i s was m a r k e t e d in J a p a n
74 u n d e r the n a m e H a k u r a n in 1968. It h a s soft leaves with less fibres, tastes like h e a d lettuce a n d p o s s e s s e s a high degree of r e s i s t a n c e to soft rot (Takada, 1986).
C y t o g e n e t i c s of wild allies: wide hybridizations. Genetic e n r i c h m e n t of crop species from their wild relatives is currently one of the major a p p r o a c h e s in crop i m p r o v e m e n t p r o g r a m s . Extensive genetic diversity occurs in the wild a n d weedy g e r m p l a s m of B r a s s i c a allies for n u c l e a r a n d cytoplasmic genes. This g e r m p l a s m occurs in n a t u r e in a vast stretch from the w e s t e r n M e d i t e r r a n e a n area to the e a s t e r n end of the Sahara desert in the n o r t h - w e s t of India. It occupies very diverse ecological habits s u c h as coastal d u n e s , slopes of coastal volcanos, stony p a s t u r e s and arid to semi-arid regions (Tsunoda, 1980). Wild allies p o s s e s s a n u m b e r of agronomically useful traits which include n u c l e a r gene encoded resistance to diseases a n d insects; i n t e r m e d i a t e C3-C4 p h o t o s y n t h e t i c activity, tolerance to cold, salt a n d heat; a n d m i t o c h o n d r i a l genes for i n d u c i n g cytoplasmic male sterility. B r a s s i c a crops h o s t an array of p a t h o g e n s a n d p e s t s a n d since wild taxa are potentially capable of exchanging genetic material with them, cytogeneticists a n d b r e e d e r s are t u r n i n g more a n d more t o w a r d s this variability for incorporating desirable genes into existing crops. As we have already mentioned, M a n t o n (1932) d e t e r m i n e d the c h r o m o s o m e n u m b e r for m a n y of the species which were s u p p l e m e n t e d s u b s e q u e n t l y (see G 6 m e z - C a m p o and Hinata, 1980). R e s e a r c h with wild g e r m p l a s m was initiated by Mizushima in the 1950s (Mizushima, 1950a, 1968) a n d consisted of hybridizations, studying c h r o m o s o m e pairing in hybrids a n d interpreting g e n o m e homologies. A major step was the collection expeditions a r o u n d the Mediterranean particularly in Spain, Portugal, Morocco a n d Algeria by S p a n i s h and J a p a n e s e scientists from 1968 o n w a r d s . Harberd (1972, 1976) carried o u t an extensive cytotaxonomic survey a n d grouped this g e r m p l a s m into cytodemes (crossing groups). This s t u d y w a s further extended by T a k a h a t a a n d Hinata (1983), a n d Warwick a n d Black ( 1991, 1993, 1994) as already d i s c u s s e d .
Hybridization between wild a n d crop b r a s s i c a s is a pre-requisite for their exploitation in a n y breeding program. As the wild g e r m p l a s m belong to the s e c o n d a r y a n d tertiary gene pool, several kinds of hybridization barriers operate. The hybridization process is a complex p h e n o m e n o n culminating into seed formation. A close coordination between pollen a n d pistil during pollen-pistil interaction, a n d developing embryo a n d e n d o s p e r m after fertilization is essential. A b r e a k at a n y level results in incompatibility. Barriers operate either at the time of fertilization or after fertilization depending u p o n the extent of reproductive isolation - the wider the distance, earlier is the stage of operation of barriers. Pre-fertilization barriers include pollen germination, pollen t u b e entry in the stigma, a n d growth of the pollen tube t h r o u g h the style. Many crosses show unilateral incompatibility i.e. pollina-
75 tion is effective only in one direction while the reciprocal c r o s s e s show strong pre-fertilization barriers (see S h i v a n n a , 1996). In general, it is observed t h a t in the majority of crosses, s u c c e s s is greater w h e n wild species are female parents. Post-fertilization barriers c a u s e embryo abortion leading to formation of shrivelled or r u d i m e n t a r y seeds, w i t h o u t embryo. Several forms of m a n i p u l a t i o n s have been carried o u t to obtain sexual h y b r i d s s u c h as grafting, mixed pollinations, b u d pollinations a n d s t u m p pollinations. In recent years in vitro fertilization h a s effectively been u s e d to raise several intergeneric h y b r i d s (Zenkteller, 1990). E m b r y o rescue h a s been the m o s t effective technique, pioneered by J a p a n e s e scientists, to overcome post-fertilization barriers a n d is very widely u s e d to raise hybrids. This p h e n o m e n o n covers all the t e c h n i q u e s which are u s e d to p r o m o t e the growth of hybrid e m b r y o a n d includes ovary culture, ovule culture a n d sequential culture (see S h i v a n n a , 1996). As a result, a large n u m b e r of h y b r i d s have been obtained a n d extensively studied for their meiotic c h r o m o s o m e behavior (Table 3.5). One of the characteristic features of meiosis of these wide h y b r i d s is the occurrence of a low to very low level of c h r o m o s o m e pairing. Bivalents wherever they occur, are in general, rod s h a p e d m o n o c h i a s m a t e s . Multivalents in h y b r i d s between diploid species are either a b s e n t or occur rarely. However, a high n u m b e r of bivalents a n d frequent t r i v a l e n t / q u a d r i v a l e n t are formed in triploid (tetraploid x diploid) or tetraploid (tetraploid x tetraploid) hybrids. Simply b a s e d on o c c u r r e n c e of bivalents in diploid hybrids, it is difficult to arrive at the extent of auto a n d allosyndesis. We have very limited information on a u t o s y n d e s i s b a s e d on pairing in the h a p l o i d s which m a y help in inferring homologies between the different genomes. In previous years, M i z u s h i m a (1950a, 1968, 1980) considered c h r o m o some pairing in the h y b r i d s as an index of genome homoeology a n d p r o p o s e d a partially h o m o l o g o u s relationship between g e n o m e s belonging to Brassica, Sinapis, Diplotaxis, Eruca, Hirschfeldia (syn. B. adpressa) a n d Raphanus. This relationship c a n now be e x t e n d e d to other genera of coenospecies as Diplotaxis, Erucastrum, Enarthrocarpus, Sinapidendron, etc. Many of the wild species are genetically very d i s t a n t a n d sexually incompatible with the crop species, t h u s m a k i n g the genes of wild t a x a inaccessible. Somatic hybridization overcomes the incompatibility barriers in s u c h situations. Cell fusion also allows the generation of extensive cytoplasmic heterogeneity t h r o u g h organelle reorganization a n d r e c o m b i n a t i o n . Many somatic h y b r i d s have been o b t a i n e d c o m b i n i n g wild a n d crop species r e p r e s e n t i n g interspecific intergeneric a n d intertribe c o m b i n a t i o n s (Tables 4.3 a n d 4.4 in the next chapter). T h o u g h initial s y n t h e s e s were for d e m o n s tration, s u b s e q u e n t l y the objectives shifted t o w a r d s their practical utilization to introduce n u c l e a r a n d cytoplasmic genes. The desirable traits for introgression include the Ca-C4 p h o t o s y n t h e t i c s y s t e m (Moricandia arvensis), high nervonic acid c o n t e n t (Thtaspi perfoliatum), club root r e s i s t a n c e (Raphanus
Table 3.5 Wide hybrids in Brussica coenospecies and their meiotic behaviour Hybrids Diplotaris erucoides (n=7) x Hirschfeldia incana (n=7) x Brassica nigra (n=8) x Sinapis pubescens (n=9) x Brassica oleracea (n=9) x Brassica rapa (n= 10) x Brassica juncea (n= 18) x
Brassica napus (n=19)
Erucastrum varium (n=7) x Brassica nigra (n=8) Erucastrum virgatum (n=7) x Sinapis pubescens (n=9) Hirschfeldia incana (n=7) x Brassica nigra (n=8) x Brassica napus (n= 19) Brassicafruticulosa (n=8) x Brassica nigra (n=8) x
x x x
Erucastrum littoreurn (n=8) ssp. glabrum Erucastrum cardaminoides (n=8) Brassica barrelieri (n=10) Brassica rapa (n=l 0 )
Brassica maurorum (n=8) x Erucastrum varium (n=7)
2n
Chromosome pairing at M1 of meiosis
Reference
14 15 16 25 17 25 25 45
141-611 + 21 151-711 + 11 1 6 1 4 1 1 + 81 7II+ 111 171-1111 + 311 + 81 111 + 231-1IV + 611 + 91 311 + 191-511 + 151 1111+ 1911 + 41 1IV + 1111 + 5-1311 + 6 2 3 1
Quiros etal., 1988 Quiros etal., 1988 Harberd and McArthur, 1980 Vyas et al., 1995 Vyas etal., 1995 Vyas etal., 1995 Inomata, N., 1998 Vyas etal., 1995 Delourme et al., 1989
15
151-511
16
1 6 1 4 1 1 + 81
+ 51
Takahata and Hinata, 1983 Harberd and McArthur, 1980 Mattsson, 1988 Kerlan et al.. 1993
15 26 161-711 + 21 1 6 1 4 1 1 + 81 711 + 21-811
Mizushima, 1968 Truco and Quiros, 1991 Takahata and Hinata, 1983
17 18 18
171-311 + 111 8 1 4 1 1 + 101 81-711 + 41 81-511 + 81
Takahata and Hinata, 1983 Harberd and McArthur, 1980 Mizushima, 1968 Takahata and Hinata, 1983 Nanda-Kumar et al., 1988
15
51-411 + 71
16 16
Takahata and Hinata, 1983
4
m
+ 21
x
Brassica nigra (n=S)
16
161-711
x
Sinapis arvensis (n=9) Brassica barrelieri (n=10) Brassica rapa (n=lO) Raphanus sativus (n=9)
17 18 18 17
171-311 + 111 181-211 + 141 181-511 + 81 17&6II+SI
15 15 16
111 + 1 3 1 4 1 1 + 71 15I--IIII+5II + 21 161-211+ 121
16 16 17 17
11 + 151-811
16 18
-
16 18
-
16 16
161- 411 + 81 611 + 41-161
18 21 21
181-511 + 81 211 + 171-711 + 71 111 + 191-511 + 111
x x x
Brassica nigra (n=8) x Erucastrurn virguturn (n=7) x Hirschfeldia incana (n=7) x Brassicafruticulosa (n=8)
x
x x
x
Brassica rnaurorum (n=8) Brassica spinescens (n=8) Raphanus sativus (n=9) Sinapis arvensis (n=9)
Brassica spinescens (n=8) x Brassica nigra (n=8) x Brassica rupa (n= 10) Diplotaxis siettiana (n=8) x Brassica nigra (n=8) x Brassica rapa (n= 10) Brassica oleracea (n=9) x Diplotaxis erucoides (n=7) x Hirschfeldia incana (n=7) x Raphnanus sativus (n=9) x Sinapis arvensis (n=9) x Coincya monensis (n=12) x Sinapis alba (n=12) x Moricandia arvensis (n=14)
+ 11
167-411 + 81 1 6 1 4 1 1 + 41
-
Takahata and Hinata, 1983 Truco and Quiros, 1991 Truco and Quiros, 1991 Takahata and Hinata, 1983 Takahata and Hinata, 1983 Bang et al., 1997 Harberd and McArthur, 1980 Quiros etal., 1988 Harberd and McArthur, 1980 Prakash et aL, 1982; Mattson, 1988 Prakash etal., 1982 Prakash etal., 1982 Matsuzawa and Sarashima, 1986 Mizushima, 1950a Harberd and McArthur, 1980 Bing el al., 1991, Mattson, 1988 Truco and Quiros, 1991 Takahata and Hinata, 1983 Nanda-Kumar and Shivanna, 1993 Mizushima, 1980 Quiros et aL, 1988 Sarashima et aL, 1980 Mizushima, 1950a Harberd and McArthur, 1980 Harberd and McArthur, 1980 Ape1 et al., 1984
J.
4
x Diplotaxis muralis (n=2 1) Brassica oxyrrhina (n=9) x Brassica nigra (n=8) x Brassica oleracea (n=9) x Sinapis pubescens (n=9) x Raphanus sativus (n=9) x
x x
Brassica barrelieri (n= 10 ) Brassica rapa (n=10) Brassica tournefortii (n=lO)
Diplotaxis catholica (n=9) x Brassica rapa (n=10) x Brassica juncea (n=l 8) Diplotaxis virgata (n=9) x Brassica rapa (n= 10) x Brassica juncea (n=l 8) Erucastrum canariense (n=9) x Brassica oleracea (n=9) Erucastrum cardaminioides (n=9) x Brassica oleracea (n=9) Raphanus sativus (n=9) x Brassica nigra (n=8) x Brassica oleracea (n=9) x Brassica rapa (n= 10) Sinapis arvensis (n=9) x Brassica nigra (n=8) x Raphanus sativus (n=9) x Brassica napus (n= 19) Brassica barrelieri (n=lO) x Brassica fruticulosa (n=8) x Brassica nigra (n=8) x Brassica oxyrrhina (n=9)
30
111 + 281-611 + 181
Harberd and McArthur, 1980
17 18 18 18
-
Prakash et al., 1982 Harberd and McArthur, 1980 Harberd and McArthur, 1980 Kaneda and Kato, 1997 Bang et al., 1997 Mattsson, 1988 Prakash and Chopra, 1990 Mattsson. 1988
211 + 141-611 + 61 111+ 161-411 + 101 181- 311 + 121 181- 111 + 611 + 61 -
19
191411 + 111
19 27
211 + 151-1 111 + 311 + 101 131+ 711-2111 + 711 + 71
Mohanty, 1996 Mohanty, 1996
19 27
191411 + 71 111+ 251-811 + 111
Takahata and Hinata, 1983 Harberd and McArthur, 1980
18
1I1 + 161-811
+ 21
Harberd and McArthur, 1980
-
18
1111+111-91-11V+1111+111+91
Mohanty, 1996
-
-
18 19
181-1111 + 611 + 31 1I1 + 171-711 + 51
Matsuzava and Sarashima, 1986 McNaughton, 1973 Mizushima, 1950a
-
Bing et al., 1991 Mizushima, 1950a Mathias, 1991
181411 + 101 181-411 + 101
Takahata and Hinata, 1983 Takahata and Hinata, 1983
181-311 18 18
+ 121
Sinapis pubescens (n=9) Brassica rapa (n=10) Brassica rapa (n= 10) X Hirschfeldia incana (n=7) X Brassicafiuticulosa (n=8) x
x
X
X X
X X X
X X X
X
Brassica spinescens (n=8) Erucastrum leucanthum (n=8) Brassica atlantica (n=9) Brassica bourgeaui (n=9) Brassica cretica (n=9) Raphanus sativus (n=9) Sinapis arvensis (n=9) Brassica barrelieri (n= 10) Brassica tournefortii (n=l 0 ) Eruca sativa (n=l 1)
X Moricandia arvensis (n= 14) Brassica tournefortii (n=lO) x Brassica fruticulosa (n=8) x Brassica nigra (n=8) x Brassica oleracea (n=9)
19 20
191-4511+ 71 201-4511 + 81
Harberd and McArthur, 1980 Takahata and Hinata. 1983
17 18
171-511 181-711
+ 71
18 18 19 19 19 19
-
19
191-511
Mizushima, 1968 Mizushima, 1968 Harberd and McArthur, 1980; Nanda-Kumar et al., 1988; Prakash et al., 1982 Prakash et al., 1982 Harberd and McArthur, 1980 Mithen and Herron, 1991 Inomata, 1986 Inomata, 1986 Mizushima, 1950a Harberd and McArthur, 1980 Mizushima, 1950a Mattsson, 1988 Prakash and Narain, 1971 Mizushima, 1950a Harberd and McArthur, 1980 Takahata and Takeda, 1990
+ 41
111+ 161-511
+ 81
191- 0-511 + 9
-
+ 91
20 21
2 0 1 4 1 1 + 121 211-811 + 51
24
241-511
18 18 19
-
181-311 191-311
+ 141 + 121
+ 131
x x
Brassica oxyrrhina (n=9) Raphanus sativus (n=9)
19 19
-
191-711
+ 51
x
Sinapis arvensis (n=9)
19
191-511
+ 91
20
201-511
+ 101
x Brassica rapa (n=l 0 ) Diplotaxis siifolia (n=lO)
Prakash et al., 1982 Narain and Prakash, 972 Mizushima, 1968 Narain and Prakash, 972 Mattsson, 1988 Mizushima, 1968 Harberd and McArthur, 1980 Mizushima, 1968 Harberd and McArthur, 1980 Sikka, 1940, Mizushima, 1968 I . \o
Brassica rapa (n= 10) Brassica juncea (n=l8) x Brassica napus (n= 19) Diplotaxis viminea (n=lO) x Diplotaris tenufolia (n=l 1) x Brassica carinata (n= 17) x Brassica napus (n=19) Enarthrocarpus lyratus (n=10) x Brassica oleracea (n=9) x Brassica rapa (n=lO) x Raphanus sativus (n=9) x Erucastrum abysinicum (n=16) x Brassica carinata (n=17) x Brassica napus (n=19) Sinapidendronfi-utescens (n= 10) x Brassicafruticulosa (n=8) x Sinapis pubescens (n-8) x Brassica juncea (n= 18) Diplotaxis tennuifolia (n=l 1) x Erucastrum virgatum (n=7) x Hirschfeldia incana (n=7) x Brassica nigra (n=8) x Brassica oleracea (n=9) x Brassica rapa (n=lO) x Brassica elongata (n=l 1) x Coincya leptocarpa (n= 12) x Brassica juncea (n=l8) Eruca sativa (n=l 1) x Brassica oleracea (n=9) x x
20 28 29
-
Batra et al., 1990 Batra et al., 1990 Batra et al., 1990
21 27 29
211-411 + 131 3111 + 411 + 101 21V + 1111 + 211 + 141
Harberd and McArthur, 1980 Mohanty, 1996 Mohanty, 1996
19 20 25 26 37 29
191-1111 +4II + 81 201-2111 + 411 + 61 211 + 2 11-911 + 71 611 + 141-1011 + 61 1111 + 1311 + 81 291-1 IV + 1111 + 611 + 101
Gundimeda et al., 1992 Gundimeda et al., 1992 Bang et al., 1997 Harberd and McArthur, 1980 Gundimeda et al., 1992 Gundimeda et al., 1992
18 19 28
311 + 121-911 111 + 171-611 + 71 211 + 2 4 1 4 1 1 + 121
Harberd and McArthur, 1980 Harberd and McArthur, 1980 Harberd and McArthur, 1980
18 18 18 20 21 22 23 29
181-711 + 41 1 8 1 4 1 1 + 61
Harberd and McArthur, Harberd and McArthur, Salisbury, 1989 Harberd and McArthur, Salisbury, 1989 Harberd and McArthur, Harberd and McArthur, Salisbury, 1989
20
x
Raphanus sativus (n=9)
20
x
Brassica rapa (n= 10)
21
281
-
201-211
+ 161
-
221-411 231-511
+ 141 + 131
-
201-311
+ 141
00
0
1980 1980 1980 1980 1980
Matsuzawa and Sarashima, 1986 U etal., 1937 Dayal, 1987 Matsuzawa and Sarashima, 1986 Agnihotri et al., 1988
x Diplotaxis tennuifolia (n=l 1) Sinapis alba (n=l2) x Brassica oleracea (n=9) x Brassica napus (n=l9) Eurcastrum laevigatum (n=14) x Hirschfeldia incana (n=7) Moricandia arvensis (n= 14) x Brassica nigra (n=8) x Brassica oleracea (n=9) x Raphanus sativus (n=9)
Brassica rapa (n=10) x Brassica juncea (n=l 8) x Brassica napus ( ~ 1 9 ) Moricandia moricandioides (n=l4) x Brassica juncea (n=l8) Erucastrum gallicum (n=l5) x Diplotaxis erucoides (n=7) x Erucastrum virgatum (n=7) x Hirschfeldia incana (n=7) x E. nasturtiifolium (n=8) x
Raphanus sativus (n=9) Sinapis arvensis (n=9) x Sinapis pubescens (n=9) x Brassica barrelieri (n=10) x Enarthrocarpus lyratus (n=10) x Sinapidendronfrutescens (n=lO) x Brassica juncea (n=18) x Brassica napus (n=l9) Brassica balearica (n=16) x Brassica oleracea (n=9) var. alboglabra x
x
22 21
221-511
+ 121
211
U etal., 1937 Ripley and Arnison, 1990
-
21
311 + 151-911
22 23 23 23 24 32 33
221--1III+ 5II+91 231-2111 + 611 + 51 1111 + 0-511 + 13-231
+ 31
-
241-511
+ 141
32 22 22 22 23 24 24 24 25 25 25 33 38 25
Takahata and Hinata, 1983
Harberd and McArthur, 1980 Takahata and Takeda, 1990 Takahata, 1990 Bang etal., 1995 Takahata et al., 1993 Takahata and Takeda, 1990 Takahata et al., 1993 Takahata et al.. 1993 Takahata et al., 1993
211 + 181-811 + 61 111 + 201-711 + 81 111 + 201-711 + 81 511 + 131-1011 + 31 711 + 91-811 + 71 311 + 181-811 + 81 211 + 201-911 + 61 411 + 161-911 + 61 311 + 191-811 + 91 311 + 171- 811 + 91 311 + 171-911 + 71 3111 + 1I1 + 221 3111 + 611 + 171
Harberd and McArthur, 1980 Harberd and McArthur, 1980 Harberd and McArthur, 1980 Harberd and McArthur, 1980 Takahata and Hinata, 1983 Harberd and McArthur, 1980 Harberd and McArthur, 1980 Harberd and McArthur, 1980 Harberd and McArthur, 1980 Harberd and McArthur, 1980 Harberd and McArthur, 1980 Batra etal., 1989 Batra etal., 1989 Snogeroup and Persoon, 1983
2
Brassica oleracea (n=9) var. insularis Brassica cossoniana (n=l6) x Brassica napus (n=19) Erucastrum abyssinicum (n=16) x Erucastrum virgatum (n=7) x E. leucanthum (n=8) x E. nasturtiifolium (n=8) x Brassica oleracea (n=9)
25
911 + 71
35
1311 + 91-1611
23 24 24 25
711 + 91-911 + 511 511 + 141-811 + 81 511 + 141-1011 + 41 111 + 231-911 + 71
Brassica rapa (n=l 0 ) Brassica carinata (n= 17) Brassica juncea (n= 1 8) Brassica carinata (n=l7) x Brassicafruticulosa (n=8) x Diplotaxis assurgens (n=9) x Diplotaxis tenuisiliqua (n=9) x Diplotaxis virgata (n=9) x Raphanus sativus (n=9) x Sinapis arvensis (n=9)
26 33 34
-
25 26 26 26 26 26
311 + I ~ I - - ~ I I171 + 311 + 201-1011 + 61 1I1 + 241-1 011 + 61 411 + 181-1 1I1 + 41 211 + 221-911 + 81 261 + 811 + 101
26
3-711
x
x x x
x
Sinapis pubescens (n=9)
Orychophragmus violaceus (n=12) 34 Erucastrum gallicum (n=15) 32 Brassica juncea (n=18) x Diplotaxis virgata (n=9) 27
Snogeroup and Persoon, 1983
711 +I21-1011+
+ 31
61
Harberd and McArthur, Harberd and McArthur, Harberd and McArthur, Harberd and McArthur, Rao et al., 1994 Harberd and McArthur, Rao et al., 1994 Rao et al., 1994
1980 1980 1980 1980 1980
5-1 211 + 8-221
Harberd and McArthur, 1980 Harberd and McArthur, 1980 Harberd and McArthur, 1980 Harberd and McArthur, 1980 Harberd and McArthur, 1980 Mizushima, 1950 Bing etal., 1991 Harberd and McArthur, 1980 Bing eral., 1991 Li et al., 1998 Harberd and McArthur, 1980
0 - 1 IV + %-2III+ 7 -1211 +
Inomata, 1994
+ 12-201
x x
Harberd and McArthur, 1980
12-201 27 27 27
-
x
Raphanus sativus (n=9) Sinapis arvensis (n=9) Sinapis arvensis (n=9)
x
Sinapispubescens (n=9)
27
0 4 1 1 + 15-271
x
x
7-1011
+ 7-131
Fukushima, 1945 Bing et al., 1991 Harberd and McArthur, 1980 Mizushima, 1950 Inomata, 1991
Brassica gravinae (n= 10) Brassica tournefortii (n=10) x Enarthrocarpus lyratus (n=10) x Sinapis alba (n=l2) x Orychophragmus violaceus (n=12) x Crambe abyssinica ( ~ 4 5 ) Brassica napus (n= 19) x Diplotaxis erucoides (n=7) x Hirschfeldia incana (n=7) x Raphanus raphanistrum (n=9) x Raphanus sativus (n=9) x Sinapis arvensis (n=9) x
x
Sinapis pubescens (n=9) Brassica gravinae (n=10) x Eruca sativa (n= 1 1) x Orychophragmus violaceus (n=12) x Brassica cossoniana (n=l6) Diplotaxis muralis (n=2 1) x Brassica rapa (n= 10) x Sinapidendronfrutescens (n=lO) x Diplotaxis harra (n=13) x Erucastrum gallicum (n=l5) x Brassica napus (n=19) x
x
38 38 28 30 24-36
281-1811
+ 21
Nanda-Kumar et al., 1989 Yadav etal., 1991 Gundimenda et al., 1992 Bajaj, 1990 Li et al., 1998 Wang, 1997
-
281-711
+ 141
-
__
-
26 26 28
3-1011 + 6-201 0-1IV + 1-711 + 10-241 0-1IV + 4-1011 + 8-201
28
0-2111
28 39 30 31 35
0-1111 + 2-1 111 + 5-241 391-1011 + 191
--
31 31 34 36 40
+ 0-1011 + 6-281
-
12-1611
+ 3-1
1I
311-511 + 211 1I1 + 291-511 + 2 1I 341-4511+ 221 311 + 301-1011 + 161 -
Harberd and Mc Arthur, 1980 Kerlan et al., 1993 Kerlan et al., 1993 Takeshita et al., 1980 Kerlan et al., 1993 Mizushima, 1950a Inomata, 1994 Nanda-Kumar et al., 1989 Bijral and Sharma, 1996 Li and Luo, 1993 Harberd and McArthur. 1980 Harberd and McArthur, Harberd and McArthur, Harberd and McArthur, Harberd and McArthur, Ringdhal et al., 1987 Fan etal., 1985
1980 1980 1980 1980
00 W
84 sativus), alternaria leaf spot resistance (Sinapis alba, Camelina sativa), nematode resistance (Sinapis alba) and cytoplasmic genes for inducing male sterility.
Introgression of g e n e s Several fungal diseases cause heavy losses to seed yield in Brassica crops. The major ones are white rust caused by Albugo candida, alternaria leaf spot (Alternaria spp.), blackleg or stem canker (Leptosphaeria maculans), and sclerotinia stem rot (Sclerotinia sclerotiorum). Nuclear genes conferring resistance to these diseases (see chapter 12) and for other desirable agronomic traits have been incorporated from related sources exploiting non-homologous recombination following sexual / somatic hybridization and also by generating alien chromosome addition lines. Successful introgression of genes following sexual hybridization are black leg resistance from B. juncea to B. napus (Roy, 1984) and from B. nigra to B. napus (Chevre et al., 1996, 1997); clubroot resistance from B. napus to cabbage (Chiang et al., 1977); self-incompatibility alleles from B. rapa to forage rape, earliness to oil rape leading to release of cultivars like Norin 16 and Asahi-Natane in J a p a n (Shiga, 1970; Namai et al., 1980); resistance to pod shattering from B. juncea to B. napus (Prakash and Chopra, 1990a); chlorosis corrections and fertility restoring genes from Raphanus sativus to CMS B. napus (Paulmann and R6bbelen, 1988) and resistance against Phoma lingam from B. juncea to B. napus (SacristAn and Gerdemann, 1986) and from B. nigra to B. napus (Struss et al., 1996). Examples of introgression through somatic hybridization include beet cyst nematode resistance (Heterodera schachtiz) from Sinapis alba and R a p h a n u s sativus to B. napus (Lelivelt et al., 1993; Lelivelt and Krens, 1992); alternaria leaf spot resistance from S. alba to B. napus (Primard et al., 1988); clubroot resistance (Plasmodiophora brassicae) from Rap h a n u s sativus to B. oleracea var. botrytis (Hagimori et al., 1992), blackleg resistance from B. nigra to B. napus (Gerdemann et al., 1994) and fertility restoring genes from Raphanus to B. napus (Sakai et al., 1996) and from Trachystoma baUii and Moricandia arvensis to B. juncea (Kirti et al., 1997; Prakash et al., 1998b).
Cytoplasm divergence and g e n o m e h o m o e o l o g y The taxa in Brassica coenospecies are classified into 2 distinct lineages based on their chloroplast DNA RFLPs (Warwick and Black, 1991; Pradhan et al., 1992). Each lineage is further separated into groups and four major groups have been recognized within each lineage. Five genera viz. Brassica, Diplotaxis, Erucastrum, Sinapis and Trachystoma are represented in both the lineages, t h u s indicating the incongruence between cp DNA data and the morphology based taxonomy. An interesting observation is that the taxa of these genera show more cp DNA homologies among them than to cogeneric
85 taxa. The lowest level of divergence was observed between taxa belonging to the s a m e cytodeme, e . g . B , oleracea, B. fruticulosa, E r u c a s t r u m virgatum a n d Coincya. On the contrary, divergence was h i g h e s t for B. rapa - B. oleracea v e r s u s B. nigra; B. rapa v e r s u s S i n a p i s arvensis, S. p u b e s c e n s a n d Coincya. Chloroplast g e n o m e similarity was h i g h e s t between B. nigra a n d S. arvensis. B. rapa a n d B. oleracea are also very close. This close affinity is also reflected in c h r o m o s o m e pairing in their h y b r i d s (B. nigra x S. arvensis, 2n = 17, 1-8 II, Mizushima, 1950a; H a r b e r d a n d McArthur, 1980; B. rapa x B. oleracea, 2n = 19; 9II + 1I, Olsson, 1960b). However, c h l o r o p l a s t genome divergence is not always c o n s i s t e n t with n u c l e a r genome divergence as the c h r o m o s o m e pairing between the taxa within a lineage is not always higher t h a n a c r o s s the lineage. At times, chrom o s o m e pairing could be as high or even higher in h y b r i d s a c r o s s the lineage. This high extent of pairing m a y be ascribed to f r e q u e n t hybridization leading to gene introgression, since a large n u m b e r of t h e m are s y m p a t r i c in their distribution. A few s u c h e x a m p l e s are the interlineage hybrids: Diplotaxis erucoides ( r a p a / o l e r a c e a lineage) x B. nigra (nigra lineage), 2n = 15, 7II (Quiros et al., 1988); B. f r u t i c u l o s a x B. rapa 2n = 18, 7II + 4I, (Mizushima, 1968); E r u c a s t r u m c a n a r i e n s e • B. oleracea, 2n = 18, 811 + 2I, (Harberd a n d McArthur, 1980); B. tournefortii • R a p h a n u s s a t i v u s 2n = 19, 7II + 5I, (Harberd a n d McArthur, 1980); Diplotaxis tennuifolia x E r u c a s t r u m virgatum 2n = 18, 7II + 4I, (Harberd a n d McArthur, 1980). On the o t h e r h a n d , some of the h y b r i d s between the t a x a within the lineage show little c h r o m o s o m e pairing indicating a high degree of genetic differentiation b e c a u s e of repatterning. E x a m p l e s include B. f r u t i c u l o s a • E r u c a s t r u m c a r d a m i n o i d e s , 2n = 17, 3II+11 I, (Harberd a n d McArthur, 1980); B. tournefortii • B. nigra, 2n = 18, 31I + 12I, (Narain a n d P r a k a s h , 1972); Dip l o t a x i s tenuifolia • B. oleracea, 2n = 20, 2II + 16I, (Harberd a n d McArthur, 1980). An interesting feature of these h y b r i d s was the o c c u r r e n c e of a high degree of c h r o m o s o m e pairing in the intertribe h y b r i d s involving Moricandia a r v e n s i s a n d B r a s s i c a spp. 1III + 5II in M. a r v e n s i s • B. nigra, 2n = 22 a n d 2III + 6II in M. a r v e n s i s • B. oleracea, 2n = 23 ( T a k a h a t a a n d Takeda, 1990; T a k a h a t a , 1990). Similarly, the somatic hybrid M. a r v e n s i s + B. j u n c e a (2n = 64) exhibited u p t o 1 q u a d r i v a l e n t a n d 3 trivalents (Kirti et al., 1992). These higher a s s o c i a t i o n s m i g h t be an e x p r e s s i o n of h o m o e o l o g y between chromos o m e s of the two species. This c l o s e n e s s is also reflected in their chloroplast g e n o m e s as Moricandia h a s affinities with B. rapa a n d B. oleracea. (Warwick a n d Black, 1994). These o b s e r v a t i o n s led these a u t h o r s to s u g g e s t the inclusion of Moricandia in B r a s s i c a coenospecies a n d also indicate t h a t recognition of Moricandiinae as a s e p a r a t e s u b t r i b e from s u b t r i b e B r a s s i c i n a e m a y be artificial. At the morphological level they b o t h s h o w elongated or siliquose d e h i s c e n t fruits.
86 Cytoplasmic substitutions Maternally inherited pollen sterility is of major importance as a pollination control m e c h a n i s m in developing hybrid cultivars. This trait - the cytoplasmic male sterility (CMS) - is encoded in mitochondrial genome and arises either as a mutation in mt DNA or can be engineered by placing the crop nucleus in alien cytoplasm -the alloplasmics (see chapter 6). Wild germplasm in B r a s s s i c a coenospecies is a rich repository of diverse cytoplasms as revealed by cp and mt DNA RFLPs (Warwick and Black, 1991, 1994; Pradhan et al., 1992). By combining their cytoplasms, alloplasmics of crop species have been synthesized exhibiting stable pollen sterility. These were obtained following sexual a n d / o r somatic hybridization. A major difference is that in the former the cytoplasm is exclusively contributed by the female parent and is unaltered while in the latter, organelle a s s o r t m e n t and mitochondrial DNA r e a r r a n g e m e n t s are of frequent occurrence. The first case of CMS in Brassica was reported in a wild population of R a p h a n u s (Ogura, 1968). This sterility inducing cytoplasm was later introduced to several B r a s s i c a spp. (Bannerot, 1974; Kirti et al., 1995a). Pearson (1972) developed a CMS system in broccoli (B. oleracea var. italica) by placing its nucleus in B. nigra cytoplasm. A little later, Hinata and Konno (1979) combined the cytoplasm of a wild species Diplotaxis muralis with B. rapa subsp, c h i n e n s i s nucleus (a leafy type). Subsequently, a n u m b e r of CMS systems have been developed in Brassica spp. carrying the cytoplasms of related wild species as B r a s s i c a oxyrrhina (Prakash and Chopra, 1990b) or Diplotaxis siifolia (Rao et al., 1994), Trachystoma ballii (Kirti et al., 1995b) and Moricandia a r v e n s i s (Prakash et al., 1998b). Somatic hybridization has added m a n y other possibilities (see Prak a s h et al., 1998a and chapter 4).
C y t o g e n e t i c c o n s t r u c t i o n of fertility restorers Fertility restorer lines for some of the CMS systems have been developed through chromosome manipulations. Restorer genes for ogu system were located in synthetic R a p h a n o b r a s s i c a (R. s a t i v u s x B. napus) and R. s a t i v u s x B. oteracea progeny by Heyn (1976) and Rouselle and Dosba (1985) respectively. Heyn (1976, 1979) introgressed these genes into B. napus, however, the fertility of restored plants decreased considerably. Pellan-Delourme and Renard (1988) observed that although restored B. n a p u s plants had the normal 38 chromosomes, 0-3 quadrivalents, 0-3 trivalents and upto 8 univalents per cell formed frequently. Decrease in fertility was attributed to high degree of ovule abortion. However, intensive selection led to developing restored lines with normal fertility. P a u l m a n n and R6bbelen (1988) also introgressed such genes for Ogu CMS in B. napus. Their method consisted of synthesis of hexaploid (ogu) R a p h a n u s s a t i v u s • B. n a p u s (2n = 56, RRAACC) and backcrossing it to B. rapa thus allowing recombination between the A and C genome chromosomes in BC1 hybrid AACR and s u b s e q u e n t introgression of restorer genes into B. napus. Stiewe et al. (1995) introduced such
87 genes in CMS (tournefortiz) B. n a p u s from B. tournefortii usi ng the synthetic alloploid B. tournefortii • B. rapa as a bridge species. Similarly, restorer genes for CMS (Moricandia) B. j u n c e a were transferred from Moricandia a r v e n s i s (Prakash et al., 1998b). Individual c h r o m o s o m e of M. arvensis was added to CMS B. j u n c e a and introgression was achieved t h r o u g h homoeologous chrom o s o m e recombination. In a n o t h e r study, fertility restorers were constructed for CMS (Trachystoma) B. j u n c e a by allowing intergenomic recombination between T. baUii a n d B. j u n c e a c h r o m o s o m e s in the backcross progeny of somatic hybrid T. ballii + B. j u n c e a (Kirti et al., 1997).
Chromosome
addition lines
Generation of c h r o m o s o m e addition lines have opened up the possibility of studying genome organization and evolution, identifying gene linkage groups and assigning species specific char act ers to a particular c h r o m o s o m e and comparing gene synteny between related species. They also have considerable value in transferring desirable cha ract ers of agronomic value from alien species to crop cultivars. All the three basic diploid genomes viz. B. nigra, B. oleracea a n d B. rap a have been dissected and a series of m on osom i c and disomic addition lines have been generated. As B r a s s i c a c h r o m o s o m e s are very small and lack morphological a n d cytological l a n d m a r k s , they have been characterized t h r o u g h genome specific m a r k e r s s u c h as isozymes, rDNA, RFLP and RAPDs. A general observation on these addition lines was that, unlike other genera, they did not show characteristic morphological features specific for a particular c h r o m o s o m e and they were rarely distinguishable from each other. It may well be t h a t the b a c k g r o u n d genome m a s k s the effect of the alien chromosome. Or the homoeologous chromosome, because of secondary polyploid nature, may nullify the effect of the alien chromosome. Another significant observation was t h a t high t r a n s m i s s i o n of alien c h r o m o s o m e t h r o u g h ovules and pollen was observed in the b a c k g r o u n d of amphidiploid species in c o n t r a s t to alien lines developed in a diploid backg ro u n d where t r a n s m i s s i o n occurs mostly t h r o u g h ovules. B. nigra addition lines B. nigra c h r o m o s o m e c o m p l e m e n t was dissected and m o n o s o m i c / d i s o mic addition lines were generated in the b a c k g r o u n d of the B. n a p u s genome following the r e c u r r e n t backcrossing of the trigenomic AB hybrid with B. nap u s (Jahier et al., 1989; Chevre et al., 1991; S t r u s s et al., 1991). Disomic addition lines were developed either on selfing or following a n t h e r culture and characterized by isozymes, fatty acid and RFLP m a r k e r s (Jahier et al., 1989). Six enzymes viz. 6-PGDH, GOT, TPI, PGM, PGI and ADH were able to discriminate B. n a p u s and B. nigra genomes. Therefore, loci coding for these
88
e n z y m e s were u s e d for identification of B. nigra c h r o m o s o m e s . The results s u g g e s t e d t h a t 6 PGDH-2 a n d GOT-5 formed p a r t of the s a m e s y n t e n y group. A similar s i t u a t i o n exists for TPI-1 a n d PGM-3. S y n t e n y g r o u p 1 displayed high levels of linoleic a n d linolenic acids in the seeds of B. nigra p a r e n t s ; s y n t e n y group 3 a c c u m u l a t e d higher levels of eicosenoic a n d erucic acid t h a n B. nigra. RFLP m a r k e r s also confirmed the six s y n t e n y groups. The p r e s e n c e of multiple f r a g m e n t s for several probes a n d a different d i s t r i b u t i o n in two c h r o m o s o m e s s u g g e s t the existence of duplicated c h r o m o s o m e s e g m e n t s in B. nigra. Meiosis revealed the formation of 19II+ 1I in m o n o s o m i c addition (Struss et al., 1991) a n d 19II+ 1II in disomic additions (Jahier et al., 1989). Transm i s s i o n of alien c h r o m o s o m e w a s high both t h r o u g h ovules a n d pollen ranging from 50 to 100 per cent. Very little c h r o m o s o m e pairing w a s noticed between B. nigra a n d B. n a p u s c h r o m o s o m e s t h u s confirming the earlier views t h a t B. nigra g e n o m e is genetically d i s t a n t from A a n d C genomes. B. oleracea addition lines B. oleracea m o n o s o m i c a n d disomic c h r o m o s o m e addition lines were g e n e r a t e d in the b a c k g r o u n d of B. rapa (Quiros et al., 1987; McGrath et al., 1990; C h e n et al., 1992) by r e c u r r e n t b a c k c r o s s i n g of n a t u r a l or synthetic B. n a p u s with B. rapa. They were c h a r a c t e r i z e d by genome specific m a r k e r s . F o u r e n z y m e s y s t e m s viz. 6-PGD, PGI, LAP a n d PGM, rDNA genes a n d RFLP m a k e r s were u s e d to identify individual c h r o m o s o m e s . T h e s e m a r k e r s revealed the existence of a high level of gene duplication in B. oleracea genome (McGrath et al., 1990) s u p p o r t i n g the earlier h y p o t h e s i s of its s e c o n d a r y polyploid origin. Addition lines were, in general, morphologically undisting u i s h a b l e from e a c h other. Only one morphological c h a r a c t e r , rugose or p u c k e r e d leaf w a s c h a r a c t e r i z e d for a B. oleracea c h r o m o s o m e which was always a s s o c i a t e d with a n isozyme m a r k e r 6-PGD-1. No o t h e r c h a r a c t e r s could be a s s o c i a t e d with other B. oleracea c h r o m o s o m e s . C h e n et al. (1992) developed five B. oleracea var. alboglabra c h r o m o s o m e addition lines. One of these was r e p o r t e d to have 3 loci viz. EC, WC a n d L a p - l C controlling the b i o s y n t h e s i s of erucic acid, white flower color, a n d faster migrating b a n d of leucine a m i n o p e p t i d a s e respectively on s a m e c h r o m o s o m e . The alien chrom o s o m e mostly r e m a i n s u n i v a l e n t b u t also u n d e r g o e s pairing with B. rapa c h r o m o s o m e s forming a trivalent (Chen et al., 1992). K a n e k o et al. (1987) developed B. oleracea var. a c e p h a l a m o n o s o m i c c h r o m o s o m e addition lines in the b a c k g r o u n d of R a p h a n u s s a t i v u s by r e c u r r e n t b a c k c r o s s i n g of synthetic R a p h a n o b r a s s i c a (2n=36 RRCC) with R. sativus. Seven addition lines could be d i s t i n g u i s h e d by morphological a n d physiological c h a r a c t e r s s u c h as seedling, leaf, root a n d pod traits a n d growth habit. C h r o m o s o m e configuration of 9II + 1I w a s p r e d o m i n a n t in these addition lines, a n d formation of a trivalent or a q u a d r i v a l e n t w a s rarely observed. Since one of these addition lines showed r e s i s t a n c e to t u r n i p mosaic virus, the r e s i s t a n c e gene is consi-
89 dered to locate on the added c h r o m o s o m e of B. oleracea (Kaneko et al., 1996).
B. ox.yrrhina addition lines Seven different B. oxyrrhina c h r o m o s o m e a d d i t i o n s were identified with reliable morphological m a r k e r s in the b a c k g r o u n d of B. rapa s u b s p , oleifera (Srinivasan et al., 1998). Novel p h e n o t y p e s s u c h as c u p p e d leaves a n d crinckled petals were observed which p r e s u m a b l y r e s u l t e d from intergenomic interactions between both genomes. However, no deleterious effect on floral morphology or seed fertility was appreciated. Since these lines were developed on alien cytoplasm, they were all pollen sterile except s y n t e n y group 6 which showed 12-16 % pollen fertility. Four m o n o s o m i c additions showed trivalent formation suggesting allosyndetic c h r o m o s o m e associations. The m a t e r n a l t r a n s m i s s i o n frequency a m o n g the a d d i t i o n s indicated r e d u c t i o n in the ovule t r a n s m i s s i o n frequency a g a i n s t theoretical expectations. RAPD analysis of these a d d i t i o n s revealed u n i q u e B. oxyrrhina specific b a n d s r e p r e s e n t i n g each a d d i t i o n s except for s y n t e n y group 6. S a m e sized PCR p r o d u c t s from a single primer, r e p r e s e n t i n g different B. oxyrrhina c h r o m o s o m e s indicated the possibility of i n t r a g e n o m i c r e c o m b i n a t i o n s a m o n g B. oxyrrhina c h r o m o s o m e s .
Concluding remarks Brassica a n d allied genera form a gene pool which can be easily manip u l a t e d genetically a n d chromosomally. As these are very a m e n a b l e to in vitro t e c h n i q u e s as d e m o n s t r a t e d by m a n y studies, new a n d exciting develo p m e n t s can be expected in developing s u p e r i o r a g r o n o m i c types. Some of the s u g g e s t i o n s in this regard are: A major limitation in a breeding p r o g r a m is a lack of a d e q u a t e variability. It is widely recognized t h a t genetically a n d geographically d i s t a n t genotypes p r o d u c e a m u c h superior progeny. P r o d u c t i o n of synthetic s t r a i n s of B. juncea, B. n a p u s a n d B. carinata exploiting large variations of the const i t u e n t p a r e n t s will r e s u l t in useful variability. Since n a t u r a l h y b r i d s were always unidirectional, s y n t h e t i c s with new c y t o p l a s m s , opposite to n a t u r a l ones a n d somatic h y b r i d s with new c o m b i n a t i o n s of cytoplasmic organelles will give more variations. New variability can also be o b t a i n e d exploiting nonh o m o l o g o u s r e c o m b i n a t i o n in F1 interspecific h y b r i d s a n d s u b s e q u e n t functioning of duplication-deficiency g a m e t e s (Prakash, 1973b). Wild g e r m p l a s m will be of major i m p o r t a n c e in a n y crop i m p r o v e m e n t p r o g r a m in the future. They not only p o s s e s s n u c l e a r gene controlled desirable agronomic traits, b u t also their c y t o p l a s m s also carry DNA s e q u e n c e s which control c h a r a c t e r s of significance. These include m a t e r n a l l y inherited male sterility, herbicide r e s i s t a n c e a n d p h o t o s y n t h e t i c activity. We are fortunate in having a large gene pool of wild taxa p o s s e s s i n g e n o r m o u s varia-
90 bility in c h l o r o p l a s t a n d m i t o c h o n d r i a l g e n o m e s (Warwick a n d Black, 1991) a n d where n u c l e a r genes for useful c h a r a c t e r s are liberally distributed. A s p e c t r u m of male steriles can be p r o d u c e d for developing heterotic hybrids b a s e d on these wild taxa. With the a d v a n c e m e n t of in vitro techniques, it is even possible to obtain intertribe hybrids. Wide hybrids of every possible c o m b i n a t i o n s h o u l d be obtained for c h r o m o s o m e m a n i p u l a t i o n . S u s t a i n e d efforts s h o u l d be directed t o w a r d s developing alien chromosome addition lines particularly from the g e n o m e s of wild allied g e r m p l a s m to locate the genes of i m p o r t a n c e on a specific c h r o m o s o m e . These will be of i m m e n s e value in introgressing c h a r a c t e r s of agronomic value t h r o u g h cytogenetic m a n i p u l a t i o n s a n d also for the application of genetic engineering t e c h n i q u e s for crop improvement. Brassica c h r o m o s o m e s are small and lack cytogenetic l a n d m a r k s . However, in situ hybridization techniques, RFLPs a n d RAPDs provide the m e a n s to identify the specific c h r o m o s o m e as these allow a large n u m b e r of molecular m a r k e r s to be associated with them. Efforts in this direction are very e n c o u r a g i n g since molecular m a p s for B. nigra, B. oleracea, B. j u n c e a a n d B. n a p u s are now available (see c h a p t e r 7).
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Biology of Brassica Coenospecies C. G6mez-Campo(Editor) 91999Elsevier Science B.V. All rights reserved.
107
4 SOMATIC HYBRIDIZATION Kristina Glimelius
Department of Plant Breeding Research, The Swedish University of Agricultural Sciences, Box 7003, 750 07 Uppsala, Sweden Many efforts have been made to improve Brassica crops by sexual crosses designed to enrich their g e r m p l a s m t h r o u g h artificial resynthesis and wide hybridization of naturally existing amphidiploids (for reviews see Olsson and Ellerstr6m, 1980; Prakash, 1980; Namai et al., 1980; Downey a n d R6bbelen, 1989; Buzza,1995). However, even though there is a large tolerance for interspecific hybridization between the Brassica crop species a n d several of the wild species related to Brassica species, successful use of sexual hybridization is limited. A n u m b e r of pre- and post-fertilization barriers limit the diversity of different genomic combinations. F u r t h e r m o r e , in sexual hybrids, the haploid gametes a n d t h u s the haploid genomes of each p a r e n t are combined, which usually results in problems of pairing of non-homologous c h r o m o s o m e s during meiosis. Poor pairing results in the presence of large n u m b e r s of univalent chromosomes, a n d even some tri- a n d quadrivalent c h r o m o s o m e s (Chopra et al., 1996). Besides the reduction in fertility c a u s e d by d i s t u r b a n c e s in chromosome pairing, m a t e r n a l inheritance of organelles can result in cytoplasmic incompatibility barriers which further restrict the possibilities for hybridization. Dysfunctional n u c l e a r - c h o n d r i o m e a n d / o r unclear-plastom combinations may be expressed in s u c h defects as cytoplasmic male-sterility (CMS; Edwardson, 1970), leaf chlorosis (Bannerot et al., 1977), floral abnormalities (Beillard et al., 1978; Bonnett et al., 1991), poorly developed nectaries (Bannerot et al., 1977) and reduced female fertility (Rawat and Anand, 1979). Methods to overcome these restrictions have been developed, viz interspecific hybrids t h r o u g h embryo, ovule, a n d ovary culture. At present, sexual hybridization is limited to combinations between certain species a n d genotypes representing closely related genera s u c h as Bras-
sica, Diplotaxis, Eruca, Erucastrum, Enarthrocarpus, Hirschfeldia, Coincya, Raphanus, Sinapis, Sinapidendron a n d Trachystoma (for a review see Chopra et al., 1996). Protoplast fusion can circumvent the limitations on sexual hybridization, so t h a t more taxonomically divergent species may be employed in breeding programs. Fusing protoplasts can b y p a s s problems with non-
108 homologous chromosome pairing, as well as create new nuclear-cytoplasmic combinations. In this review, a survey is presented of the results from using protoplasts as artificial gametes for the production of somatic hybrids and cybrids in the family B r a s s i c a c e a e .
Protoplast technology Isolation of protoplasts from the genus B r a s s i c a was first reported by Wenzel (1973). One year later a presentation on callus formation and plant regeneration from B. n a p u s was published by Kartha et al. (1974). However, it was not until the beginning of the 1980's that reproducible protocols for plant regeneration from isolated B r a s s i c a protoplasts were fully developed (Schenck and R6bbelen, 1982; Pelletier et al., 1983; Glimelius, 1984). These protocols expanded the possibilities of protoplast technology for plant breeding purposes. Reports have continued to be published on successful culture methods of different B r a s s i c a species (see Wamling and Glimelius, 1990). Efficient m a s s fusions of protoplasts isolated from different sources of tissues make it possible to combine genetic material independently of species and sexual compatibility (Wallin et al., 1974; Kao and Michayluck, 1974). The main procedure used for protoplast fusion is the polyethylene glycol (PEG) method, first described by Wallin et al. (1974) and Kao and Michayluck (1974) and adapted for B r a s s i c a species by, for example, Pelletier et al. (1983), Sundberg and Glimelius (1986), Morgan and Maliga (1987) and Robertson et al. (1987). High frequencies of heterodimeric fusion products can be obtained following PEG treatment. Successful utilization of electric stimulation has also been reported to promote fusion (Zimmermann, 1982). However, selection of the desired heterokaryons from the mass population of protoplasts after fusion is still a bottleneck. Several different techniques are used to enrich and select fused protoplasts. The most efficient method is flow cytometry combined with cell sorting (Glimelius et al., 1986). However, the most commonly used method is the donor-recipient fusion method, originally developed by Sidorov et al. (1981), in which one of the parental protoplasts is pretreated with iodoacetate or iodoacetamide (IOA) and the other with irradiation. A method in which one parent exhibiting poor growth and regeneration capacity is fused with a pretreated parent has also been used successfully in several instances (Terada et al., 1987; Gerdemann-Kn6rck et al., 1994; Waiters and Earle, 1993; O'Neill et al., 1996). Other methods include m a n u a l selection of fusion products using the micromanipulator (Sundberg and Glimelius, 1986; Lelivelt et at., 1993) or simply utilising special growth requirements for the protoplasts, which allow for sustained growth and development of the hybrid but not the parental protoplasts (Schenck and R6bbelen, 1982; Ros6n et al., 1988). To verify the hybrid nature of the regenerated plants, isoenzyme analysis has proven to be an easy and efficient method (Sundberg and Glimelius, 1986). RFLP-analysis using nuclear probes is even more efficient. Hybridi-
109
f spe ies B
Ng
speciesA
//
~~
!!
~ ~ ~-~
@
~
~
[-//-] laserlight ~ ........ detector, _ ~ +§ computer o 4- ~ o% droplet ~( o ~) deflector I~ ~ . B " selectionby dfl~ s~ wast~e ! 0
0r
protoplasts
droplet chargesignal
cuitu~plate " (~sel~t!o.n ~ ~ ~ oy,,sning .....CMturepiate
~
Analyses; fertility, seedset
'""'"~
"'
~ ~else;tned ~7~ products
~ ;~I'
division
isoenzyme& molecular markers ABHHP RFLP, chloroplast & mitochondrial _ _ = = DNA
~
~~~~
, ~
callus
~ ~
DNA-cont. measurements chromosome counting
nuclei p~
"
~
shoot regeneration
~
root tip
in situ hybridization squash
Figure 4.1 Schematic drawing of the different steps used for production of somatic hybrids and for analysis of the obtained plants.
110 zation of probes which are either species-specific or giving species-specific patterns is useful for hybrid confirmation. Repetitive species-specific sequences which assay for large parts of the parental genome have proven to be very useful (Fahleson, 1993; Fahleson et al., 1994a) and can even be used for a quantitative estimate of the proportion of genomic material from each parent in asymmetric hybrids (Imamura et al., 1987; Piastuch and Bates, 1990). RFLP analysis is also used to determine the organellar composition of hybrid plants (Beillard et al., 1978; Aviv et al., 1980; Pelletier et al., 1983). Steps used for the production and analysis of somatic hybrids are presented in Figure 4.1.
Somatic hybrids produced between different B r a s s i c a species Resynthesis of B r a s s i c a n a p u s Cytological experiments have shown that B r a s s i c a n a p u s is a hybrid between B. rapa (syn. B. c a m p e s t r i s , AA genome) and B. oleracea (CC genome) (U, 1935). These results prompted researchers and breeders to re-synthesize rapeseed by sexual hybridization from the progenitor species (Namai et al., 1980). For breeding purposes, the main goal of producing B. n a p u s hybrids is to broaden genetic diversity of the cultivated crop. However, hybrid production often requires that B. rapa is used as the female parent, and sexual hybridization between some genotypes never results in hybrid production (Namai et al., 1980). Thus, the restrictions on sexual hybridization have stimulated researchers to resynthesize rapeseed by somatic hybridization. A substantial number of investigations have described production of somatic hybrids between B. oleracea and B. rapa (Table 4.1). Several of the regenerated hybrids had the expected chromosome n u m b e r of 38, which will result from fusing somatic ceils with the diploid chromosome number from B. rapa (2n = 20) and B. oleracea (2n = 18). However, deviations from the expected chromosome n u m b e r were also recorded in several hybrids. This could be due to triple fusions involving one B. rapa protoplast with two B. oleracea protoplasts, or vice versa, resulting in hexaploid plants containing either the genome composition AAAACC or AACCCC, as discussed by Terada et al. (1987) and Heath and Earle (1996a). Aneuploid n u m b e r s were also obtained in several cases, which most likely reflected chromosome elimination during regeneration and development (Sundberg et at., 1987; Terada et al., 1987). Nevertheless, most of the plants could set seed, either after selfing or crossing to an established rapeseed cultivar (Table 4.1). Rapeseed hybrids from protoplast fusion generally exhibited lower self fertility than hybrids obtained by sexual hybridization (Schenck and R6bbelen, 1982; Sundberg et al., 1987; Ros~n et al., 1988; Ozminkowski and Jourdan, 1993). Reduction in self fertility could be attributed to chromosomal alterations induced by the in vitro culture or from genetic factors such as incompatibility (Sundberg et al., 1987; Heath and Earle, 1996a). Another factor which could affect the
Table 4.1 A summary of the results obtained from resynthesis of Brassica napus via somatic hybridization of B. rapa (+) B. oleracea where regeneration of hybrid plants per number of calli, chromosome number, traits and fertility are presented. Hybrid plants 12
Calli
Chromosome number
Trait
Fertility
Reference
139
38, 36,54, 18
ND*
F
Schenck and Rlibbelen 1982
4
ND
38
vegetable properties
F
Taguchi and Kameya 1986.1987
10
ND
23
38, 33,36,49, 56, 57
ND*
F, MS
Terada et al. 1987
2300
38,50-60
ND*
F, MS
Sundberg et al. 1987
1
1089
36-38
CMS
ND
Robertson et al. 1987
5
1980
38
earliness, winter hardiness
F
Rosen et al. 1988
34
1136
38,58, aneuploidy
atrazinR CMS
F, MS
Jourdan et al. 1989
72
ND
38,58
fertility, SI-genes organelle segregation
F
Ozminkowski and Jourdan 1993; 1994 a; b
109
3 80
38
erucic acid, shattering and lodging resistance; seed size.
F
Heath and Earle 1995
39
110
38, 56, 5 8
CMS, cold tolerance
F, MS
Heath and Earle 1996a
F = fertile,
MS = male sterility,
ND = not determined,
CMS = cytoplasmic male sterility,
* basic investigations of hybrid features and fertility,
112 fertility of the hybrids is the expression of self-incompatibility (SI) genes from the parental genomes (Ozminkowski and J o u r d a n , 1993).
Comparison of resynthesized rapeseed produced by somatic and sexual hybridization. In a thorough investigation Ozminkowski a n d J o u r d a n (1993, 1994ab) have compared the results obtained from resynthesis of rapeseed by interspecific somatic a n d sexual hybridization. The same parental material was u s e d for hybrid production in both experiments. They found that more hybrids were obtained by somatic hybridization. In addition, when combining different genotypes of the parental species successful results were more likely via somatic hybridization. Only one of the chosen B. rapa genotypes gave progeny after sexual hybridization. Furthermore, the time required to obtain somatic hybrids was shorter (7 months) t h a n for sexual hybrids obtained by embryo rescue (usually a r o u n d 1 year). Time to flowering was shorter for the somatic hybrids t h a n for the sexual hybrids. Some of the latter required vernalization to flower, while the somatic hybrids did not. Hybrids derived from protoplast fusions are not uniform and they often differ in ploidy level, morphology, fertility a n d cytoplasmic composition (Schenck and R6bbelen, 1982; S u n d b e r g et al., 1987; Terada et al., 1987). Nevertheless, Ozminkowski a n d J o u r d a n could establish t h a t within each fusion experiment, it was possible to obtain some hybrid plants with the expected and balanced c h r o m o s o m e n u m b e r of n = 19, utilizing protoplasts from one genotype of each parent. These plants displayed uniformity in morphology and fertility, were expected to contain all the aUelic combinations present in the p a r e n t s and, thus, were considered clones for the unclear-encoded characteristics. However, additional investigations, such as characterization of hybrids utilizing RFLP m a r k e r s would be needed before drawing further conclusions. In c o n t r a s t to nuclear traits, the organelle composition differed a m o n g the somatic hybrids, whereas the sexual hybridization resulted in m a t e r n a l inheritance of the organelles. Heath a n d Earle (1996a) have also m a d e a comparison of resynthesized rapeseed produced from the same parental material by sexual and somatic hybridization. Their investigation included a comparison between somatic hybrids a n d sexual hybrids produced by s p o n t a n e o u s seed development as well as by embryo rescue. The hybrids compared in their study differed in several characters, s u c h as leaf morphology, flower color, fertility, DNA content and organelle composition. Their investigations revealed more variability in the somatic hybrids than in the sexual hybrids, especially for leaf morphology, b u t also for flower color. When analysing the sexual hybrids they found less segregation in the different traits in the spontaneously obtained hybrids t h a n in the hybrids obtained via embryo rescue. Their conclusion was t h a t the s p o n t a n e o u s l y obtained hybrids may have been derived from u n r e d u c e d gametes rather t h a n via hybridization. Another difference was
113 that the somatic hybrids displayed a higher DNA cont ent t h a n the expected DNA co n te n t in a hybrid, possibly due to a three-way fusion involving one B. c h i n e n s e s a n d two B. o l e r a c e a protoplasts. All the sexually produced hybrids had a DNA c o n t e n t similar to the amphidiploid DNA content in rapeseed. Fertility investigations of the material revealed t h a t the pollen viability was high in the sexually produced hybrids, s o m e w h a t lower in the putative hexaploid somatic hybrids, while it was low for all the other somatic hybrids having aneuploid or multiple genome compositions. Only the s p o n t a n e o u s sexually derived hybrids set seeds after selfing while the hybrids obtained via embryo rescue were self and sib-incompatible. However, they were able to set seeds when crossed with anot her B. n a p u s cultivar. None of the somatic hybrids could be selfed, b u t two of the putative hexaploid hybrids were able to set seeds after elimination of the B. c h i n e n s i s genome. Regarding the chloroplast composition in the somatic hybrids a strong bias towards the B. chin e n s i s chloroplasts was found. This might have been due to the fact t h a t most hybrids were hexaploid and contained two AA genomes. From these investigations it can be concluded t h a t somatic hybridization can be beneficial for hybrid production of some cultivars whereas for others sexual hybridization is better. The reasons for the variations in somatic hybrid plants may depend on the cultivars combined or the m e t h o d s u s e d for hybridization and regeneration. Nevertheless, new breeding materials of potential importance can be p r oduced via somatic hybridization. Several of the somatic rapeseed hybrids are being evaluated in field trials for agronomic traits such as earliness, winter hardiness, erucic acid content, lodging and shattering resistance, CMS, atrazine resistance and cold tolerance (Table 4.1). Furthermore, the vegetable "Hakuran" or r u t a b a ga, has also been produced by somatic hybridization of cabbage with Chinese cabbage (Taguchi and Kameya, 1986). An area where somatic hybridization might be of practical importance is to modify the fatty acid composition in rapeseed. As an example, efforts have been mad e to resynthesize B. n a p u s with a high content of erucic acid. Fatty acids consisting of long carbon chains with 20 or more carbon atoms, like erucic acid, are of great importance in the industrial applications for production of polyethylene films, polymers a n d nylon p r o d u c t s (Sonntag, 1995). Thus, experiments were conducted fusing a B. o l e r a c e a cultivar exhibiting high levels of erucic acid (C22:1) with a B. r a p a cultivar also with high erucic acid content. This resulted in a hybrid line exhibiting as m u c h as 57.4% erucic acid (Heath and Earle, 1995). A c o n t e n t of 56.6% was recorded a m o n g the progeny obtained after selfing and culture in field conditions. However, to be of real practical importance levels higher t h a n 66% should be obtained. This could be the case if erucic acid is esterified to all 3 positions in the glycerol molecule (Uppstr6m, 1995). Thus, a transfer of the genes responsible for esterification of erucic acid to all 3 carbon positions in the glycerol molecule would result in the m o s t optimal combination (Taylor et al., 1994).
114
Combinations of other Brassica
g e n o m e s into s o m a t i c hybrids
Besides the resynthesis of rapeseed a large n u m b e r of somatic hybrids have been produced by combination of the other Brassica genomes (Figure 4.2, Table 4,2). Somatic hybridization of the different Brassica progenitor species within the triangle of U (1935) have resulted in the production of all three amphidiploid species, B. napus, B. carinata (Jourdan and Salazar, 1993; N a r a s h i m h u l u et al., 1994) and B. juncea (Campbell et al., 1990). Furthermore, the combination of all three Brassica genomes, A, B, C, has been performed by the production of B. naponigra (Sj6din and Glimelius, 1989a). Several of the combinations have been produced to improve disease resistance in the cultivated species B. napus, B. juncea or B. oleracea (Table 4. 2). Numerous efforts have been made to breed for resistance to blackleg caused by the pathogen Phoma lingam with the perfect stage Leptoshaeria maculans (Desm). This fungus causes blackleg disease in all cruciferous crops and is a severe problem in Europe, Australia and Canada. Resistance to the pathogen has been found in several different germplasms of B. carina-ta, B. nigra and B. juncea (Sj6din and Glimelius, 1988), but also in B. tournefortii (Salisbury, 1991) and Sinapis alba (syn. B. hirta) (Pl~mper and Sacristan, 1995). Disease-resistant hybrid plants have been obtained from most fusion experiments (see review by Dixelius and Glimelius, 1995). Analysis of some hybrid progenies obtained after backcrossing to rapeseed have confirmed presence of resistant lines after 10 to 12 generations, indicating stable inheritance of the resistance genes (Axelsson and Dixelius, 1994). Other diseases of Brassica crops include beet cyst nematode (Heterodera schachtil), clubroot (Plasmodiophora brassiceae), black spot (Alternaria brassiceae), white rust (Albugo candida) and black rot (Xanthomonas campestris pv. campestris). High levels of resistance have been found in the primary hybrids produced between rapeseed and the resistant Sinapis alba (syn. B. hirta) for beet cyst nematode (Lelivelt et al., 1993). Clubroot resistance was derived from B. nigra (Gerdemann-Kn6rck et al., 1994), black spot resistance from the donor Sinapis alba (Chevre et al., 1994) and black rot resistance was transferred from B. napus to B. oleracea (Hansen and Earle, 1995). The primary hybrids have been utilized as bridges for transferring the genes for resistance to the cultivated crop by backcrossing. In several cases, for example clubroot, black spot and black rot, BC1 progenies have been obtained displaying disease resistance. This confirms that transfer and inheritance of the resistance genes can be established via protoplast fusion.
C o m p a r i s o n of s e x u a l v e r s u s sion of alien genes~
s o m a t i c hybridization for introgres-
Chevre et al. (1994) performed an investigation comparing the efficiency of protoplast fusion to sexual crosses as a means to introduce new traits in a crop. Cytogenetic and molecular characterizations of somatic hybrids produced between B. napus (AACC) and Sinapis alba (Sal) were compared with
115
Genus Brassica
B.oleracea
Figure 4.2 Somatic hybrids produced between different
Brassica species. The somatic hybrids are represented by grey circles. Arrows indicate which species were combined. The number of circles represents the number of genomes in the species and hybrids. The Brassica species are arranged according to the triangle of U (U, 1935) with solid black lines showing the phylogenetic relation between the allotetraploid and diploid species.
116 sexually produced hybrids from the same species. The molecular studies revealed t h a t all the somatic hybrids except one had the complete genomes of both parents. This was confirmed by chr om osom e n u m b e r analysis. However, the c h r o m o s o m e n u m b e r varied from 42 to 54 after backcrossing to rapeseed. Thus, instead of obtaining the expected c h r o m o s o m e n u m b e r 50, which would be the result from the combination AACCSal, a variation was observed. This might be due to d i s t u r b a n c e s during meiosis. Nevertheless, in the b a c k c r o s s e d plants, meiotic abnormalities were found, s u c h as trivalents a n d quadrivalents, indicating homologies which enabled c h r o m o s o m e s from the two p a r e n t s to pair, and possibly to recombine. From the meiotic analysis it was confirmed t h a t c h r o m o s o m e r e a r r a n g e m e n t s occurred more frequently in the somatic t h a n the sexual hybrids. The conclusion from these results is t h a t r e a r r a n g e m e n t s induced by protoplast fusion might facilitate introgression of new traits from taxonomically d i s t a n t species to a crop. This seems to have occurred t hr ough recombination rat her t h a n translocation.
I n t e r g e n e r i c s o m a t i c hybrids w i t h i n t h e tribe B r a s s i c e a e Intergeneric hybrids have been produced between all the different Brassica species in the U triangle and species belonging to the genera Eruca, Sinapis, Raphanus, Moricandia, Diplotaxis and Trachystoma (Figure 4.3, Table 4.3). All intergeneric combinations resulted in hybrid plants. Some of these hybrids h a d c h r o m o s o m e n u m b e r s representing the s u m of the parental species, while large variations, ranging from c h r o m o s o m e n u m b e r s slightly higher t h a n the c h r o m o s o m e n u m b e r of the Brassica species to multiples of the expected s u m of the two species were found. According to isozyme and RFLP analysis hybrid plants lacking c h r o m o s o m e s also lacked some of the genetic markers. However, even t h o u g h some of the genetic material from the "donor" parental line was missing, partial hybridity was confirmed by presence of some m a r k e r s from both species. Thus, in the hybrids with fewer c h r o m o s o m e s t h a n expected, and even in the plants with a chromosome n u m b e r equal to the Brassica species (regarded as the "acceptor"), alien DNA was present. In a s t u d y performed by Sun dberg and Glimelius (1991a) the correlation between the frequency of somatic hybrids with eliminated chrom o s o m e s and the genetic distance between the species in each combination was investigated. They concluded t h a t a larger genetic distance between the two species resulted in a greater degree of c h r o m o s o m e elimination. This was significantly greater after fusing species with different ploidy levels.
Intertribal somatic hybrids The great and u n i q u e potential of protoplast fusion technology is that it produces species combinations t h a t could never be obtained via sexual hybridizations, even when using the embryo rescue and in vitro fertilization techniques. The very first somatic hybrids produced between different spe-
Table 4.2 A summary of the somatic hybrids produced between different Brassica species within the tribe Brassiceae. The traits of interest to modify with the somatic hybridization and fertility have been listed. Somatic hybrid
Trait
Fertility
References
Brassica napus
Brassica nigra
Phoma lingamR
F
Sjodin and Glimelius 1989 a; b
Brassica napus Brassica napus
Brassica nigra Brassica nigra
?
Sacristan et al. 1989 Gerdemann-Knorck et al. 1995
Brassica napus Brassica napus
Brassicajuncea Brassica carinata
Phoma lingamR Phoma lingam' Plasmodiophora brassiceae' Phoma lingamR Phoma lingamR
Brassica napus
Sinapis alba (syn. B. hirta)
Brassica napus
Brassica oleracea
Brassica napus Brassica napus
Brassica oleracea Brassica tournefortii
Brassica napus Brassica oleracea
Brassica tournefortii Brassica nigra
Brassica oleracea
Brassica napus
Brassica juncea
Brassica spinescens
F = fertility,
MS = male sterility,
Alternaria brassiceae' Heterodera shachtii' Drought tolerance CMS triazinR, seedling chlorosis ND* Phoma lingam' CMS CMS Albugo candid2 A lternaria brassiceaeR Xanthomonas campestriis pv campestrisR,CMS, atrazinR Albugo candid2 salt tolerance
CMS = cytoplasmic male sterility.
S, F(few) F F F S F MS, FF
Sjodin and Glimelius 1989 b Sjiidin and Glimelius 1989 b Plumper and Sacristan 1995 Primard et al. 1988 Lelivelt et al. 1993 Chevre et al. 1994 Kao et al. 1992
F F MS F, MS F FF, MS F
Stiewe and Riibbelen 1994 Narasimhulu et al. 1992 Jourdan and Salazar 1993 Hansen and Earle 1995
FF, MS
Kirti et al. 1991
Sundberg et al. 199 1 Liu et al. 1995
*basic investigation of chromosome elimination and chloroplast segregation
118
Tribe Brassiceae
Genus Moricandia
Genus Eruca
Moricandia arvensis Brassica
B. juncea
B. oleracea
muralis Diplotaxis catholica
Smapls
Figure 4.3. Intergeneric somatic hybrids produced within the tribe Brassiceae. Hybrids, the number of genomes and the phylogenetic relationships are marked as in Figure 4.2.
Table 4.3 A summary of the somatic hybrids produced between species from different genera from the tribe Brassiceae where the desired traits and fertility are listed. Somatic hybrid
Trait
Fertility
Reference
Brassica napus
Diplotaxis muralis
CMS
?
McLellan et al. 1987
Brassica napus
Diplotaxis harra
CMS
S
Klimazewska and Keller 1988
Brassica napus
Eruca saliva
Drought tolerance, AphidR, erucic acid
F
Fahleson et al. 1988,1997
Brassica napus
Raphanus sativus
CMS
MS
Sundberg and Glimelius 1991b
Brassica napus
Raphanus sativus
Heterodera schachtiiR
S
Lelivelt and Krens 1992
Brassica napus
Moricandia arvensis
Alternaria brassiceaeR, Phyllotreta crucijereaeR,, Plasmidiophora brassiceaeR
F
O’Neill et al. 1996
Brassica juncea
Eruca saliva
Drought tolerance
F
Sikdar et al. 1990
Brassica juncea
Diplotaxis muralis
ND*
F
Chatterjee et al. 1988
Brassica juncea
Diplotaxis catholica
AphidR,Alternaria brassiceaeR
F
Kirti et al. 1995c
Brassica juncea
Moricandia arvensis
Photorespiration
MS
Kirti et al. 1992a
Brassica juncea
Trachystoma ballii
Alternaria brassiceaeR, hard pods, CMS
S
Kirti et al. 1992b
Brassica oleracea
Sinapis turgida
ND
ND
Toriyama et al. 1987b
Brassica oleracea
Moricandia arvensis
Photorespiration, Albugo candid2
?
Toriyama et al. 1987a,1988
Brassica nigra
Sinapis turgida
ND
ND
Toriyama et al. 1987b
Brassica oleracea
Raphanus sativus
Plasmodiophora brassiceaceR
F
Hagimori et al. 1992
CMS = cytoplasmic male sterility,
MS = male sterility,
ND = not determined,
S = sterile,
F = fertile
*basic cytogenetic investigations
120 cies in the family of B r a s s i c a c e a e were made between B. rapa (syn. B. campestris) and A r a b i d o p s i s thaliana, two species from different tribes of the family (Gleba and Hoffman, 1979, 1980). Their success in obtaining intertribal somatic hybrids with genetic material from both parental species, encouraged other researchers to try other species from different genera and tribes. As presented in Figure 4.4 and Table 4.4, intertribal combinations between species derived from five different tribes have been produced. In all the experiments reported, hybrid or partial hybrid plants have been obtained according to molecular m a r k e r analysis of the regenerated plant material. However, evaluations of chromosome n u m b e r s , isoenzyme content, and RFLPpatterns revealed that a higher degree of asymmetric hybrids were obtained among the intertribal hybrids than among the intrageneric and intergeneric hybrids. Surprisingly, no differences in the frequency of hybrid fusion products and hybrid shoots were recorded, when comparing the intertribal hybrids with inter or intrageneric hybrid. Only after attempts to culture and establish the hybrid material in the greenhouse was a clear difference noted. The intertribal combinations were, in general, more difficult to root and culture to m a t u r e plants outside in vitro conditions (Fahleson et al., 1994a). These difficulties were especially pronounced in fusions between B. n a p u s and B a r b a r e a vulgaris, which, in spite of leading to hybrid plants that could be cultured in vitro, never resulted in plants that could grow under ordinary greenhouse conditions. According to Oikairinen and Ry6ppy (1992), fertile plants were obtained after fusing B. rapa with B a r b a r e a vulgaris. However, clear evidence that these plants were real hybrids has not yet been reported. The problem of establishing somatic hybrids between species from different tribes was also encountered by other researchers. Hybrids between B. n a p u s (+) A. thaliana (Bauer-Weston et al. 1993) and between B. n a p u s (+) Lesquerella f e n d l e r i (Skarzinskaya et al., 1996) were difficult to root and culture in the greenhouse. Similar problems were observed in the hybrids produced between B. rapa and A. thaliana by Gleba and Hoffmann (1979, 1980). Success in obtaining hybrids that could be rooted and established in soil in the greenhouse was increased by partially eliminating DNA from the wild species using irradiation (Bauer-Weston et al., 1993; Skarzhinskaya et al., 1996). Furthermore, the irradiation improved fertility of the hybrid plants. For example, all L. fendleri (+) B. n a p u s hybrids obtained from symmetric fusions were self-sterile, while 38% of the asymmetric hybrids could be selfed (Skarzinskaya et al., 1996). A clear significant positive correlation between degree of a s y m m e t r y and seed set after selfing was obtained in asymmetric hybrids produced between B. n a p u s (+) A. thaliana (Forsberg et at., 1998b). Even though no positive effect of irradiation on fertility was found in hybrids between Thlaspi perfoliatum (+) B. napus, it improved the efficiency of hybrid production (Fahleson et al., 1994a). It is also interesting to note that some of the somatic hybrids produced between B. n a p u s (+) A. thaliana developed to fully differentiated plants that could be cultured in the greenhouse as well as set seeds even after selfing (Forsberg et al., 1994). The reasons for these
121
Tribe)
Lepideae ,
Family
Brassicaceae
Tribe
Sisymbrieae Ara~is thaliana
Brassiceae
Arabideae
Figure 4.4 Intertribal somatic hybrids within the family Brassicaceae. Hybrids are illustrated by the grey circles and the different species combined are marked with arrows.
Table 4.4 Intertribal somatic hybrids produced within the family Brassicaceae Somatic hybrid Parent A (tribe Brassiceae) Parent B
Tribe
Brassica rapa
Arabidopsis thaliana
Brassica napus
Trait of interest
Fertility
Reference
Sisymbrieae
ND
ND
Gleba and H o h a n n 1979; I980
Arabidopsis thaliana
Sisymbrieae
acetolactate-synthase
MS
Bauer-Weston et al. 1993
Brassica napus
Arabidopsis thaliana
Sisymbrieae
Phoma lingamR
F
Forsberg et al. 1994
Brassica carinata
Camelina sativa
Sisymbrieae
Alternaria brassiceaeR
ND
Narasimhulu et al. 1994
Brassica rapa
Barbarea stricta
Arabideae
cold tolerance
MS
Oikarinen and Ryoppy 1992
Brassica rapa
Barbarea vulgaris
Arabideae
cold tolerance
MS
Oikarinen and Ryoppy 1992
Brassica napus
Barbarea vulgaris
Arabideae
cold tolerance
S
Fahleson ei al. 1994b
Brassica napus
Thlaspi perfoliatum
Lepideae
nervonic acid
MS
Fahleson ei al. 1994a
Brassica napus
Lesquerella fendleri
Drabeae
lesquerolic acid
F
Skarzhinskaya et al. 1996
F = fertile,
MS = male sterility,
ND = not determined,
S = sterile
123 differences are unclear, but could be the result of the genotypes combined, as well as differences in the fusion and culture conditions.
Modification of fatty acid c o m p o s i t i o n by intertribal hybridization The practical importance of utilizing somatic hybridization between species from different tribes would be to modify the composition of the fatty acids in rapeseed. Several wild and distantly related species within the Brassicaceae exhibit fatty acids in their seeds which would be valuable to utilize for production of technical oils and replace the use of mineral oil. Thus, efforts to modify the fatty acid composition in rapeseed in order to obtain high enough quantities to be of economic importance is of interest to investigate. With that purpose in mind Fahleson et al. (1994a) performed hybridization experiments between rapeseed and Thlaspi perfoliatum. Seeds of T. perfoliatum have high levels of nervonic acid (C24:1), which is an oil of value as a lubricant. Some of the somatic hybrids were able to set seeds when backcrossed with rapeseed pollen. Seeds from the backcrossed progeny had an increased level of nervonic acid compared to rapeseed, reaching 4.9%. However, this content is still m u c h lower than the 20% level in T. perfoliaturn. Nevertheless, the results from these investigations clearly show that gene(s) coding for synthesis of nervonic acid have been transferred via protoplast fusion and are expressed during seed development. The relatively low level of nervonic acid in hybrid plants indicates that additional genes may be responsible for the elongation of C22 to C24 fatty acids, or that expression of the genes is modified in the hybrid material. The somatic hybrids produced between Lesquerella fendleri (+) B. napus were also made with the aim to tranfer genes coding for valuable fatty acids {Skarzinskaya et al., 1996). In Lesquerella the presence of high levels of hyd r o x y - u n s a t u r a t e d fatty acids has been recorded (Muuse et al., 1992). Since castor oil is the main source for hydroxylated derivates of oleic acid (C 18:1OH), breeders have sought other crops containing hydroxy fatty acids which are possible to cultivate in temperate regions. Actually, several attempts to domesticate L. fendleri have been made. Another approach would be to identify and transfer the genes coding for the elongation and hydroxilation steps into rapeseed, using protoplast fusion. Progeny from somatic hybrids obtained between L. fendleri and rapeseed have been investigated for their fatty acid composition (Schr6der-Pontoppidan et al., 1998) and material producing high levels (64%) of very long chain fatty acids (VLCFA) have been obtained.
Limited gene transfer via protoplast
fusion
Induced a s y m m e t r y in combination with a selection pressure To facilitate the transfer of specific traits after somatic hybridization, the use of a selection pressure for the trait of interest is a suitable way to succeed. In the case of improving resistance to Phoma lingam in B. napus a che-
124 mically characterized toxin, sirodesmin, isolated from culture filtrates of in vitro fungal cultures, was u s e d as a selective agent. Analysis of resistant and susceptible Brassica species revealed the selective properties of the toxin (Sj6din et al., 1988). To promote transfer of the resistance trait, donor materials of r e s i s t a n t B. nigra, B. juncea or B. carinata were irradiated prior to fusion (Sj6din and Glimelius, 1989b). The small cell colonies developed from the fusion p r o d u c t s were exposed to concentrations of the toxin in which only the r es is tan t het er okar yon cells could divide and differentiate into plants. Most of the hybrid pl ant s exhibited resistance to Phoma lingam, indicating t h at transfer of the resistance genes had occurred. According to chromosome analysis, isoenzyme a n d RFLP patterns, parts of the alien donor genome were p r e s en t in the material (Sj6din and Glimelius, 1989b). By backcrossing the asymmetric hybrids to rapeseed and screening for resistance to pycnospores from Phoma lingam, resistant lines have been selected exhibiting both cotyledon a n d adult leaf resistance (Wahlberg and Dixelius, 1997). These lines have been investigated in greenhouse and field conditions and represent valuable material both for studying the molecular regulation of P. lingain resistance and for breeding resistant rapeseed for agricultural use.
Selection of desired traits by using marker genes Another a p p r o a c h to select for desirable traits in asymmetric hybrids is the utilization of selectable m a r k e r genes t hat have been transferred to the donor species via transformation. This approach was first utilized by MUller and Schieder (1987) who showed that m a r k e r genes introduced by Agrobacterium tumefaciens t r a n s f o r m a t i o n were transferred from protoplasts of an inactivated donor species into recipient protoplasts. Besides the T-DNA, other genetic material of the donor genome was also transferred. This approach h a s later been u s e d by Sacristan et al. (1989) and GerdemannKn6rck et al. (1994, 1995). As donor material in the fusion experiments they u s ed a B. nigra line t h a t was resistant to Phoma lingam and Plasmodiophora brassiceae (Sacristan et al., 1989). This line also contained the gene for hygromycin p h o s p h o t r a n s f e r a s e (HPT), which was inserted into the genome by t r a n s f o r matio n with Agrobacterium tumefaciens. By screening for tolerance to hygromycin, asymmetric hybrids could be selected after fusion of irradiated B. nigra lines with B. napus. Besides presence of the m a r k e r gene in the hybrids, the genes coding for resistance to Plasmodiophora brassicae and Phoma lingam were also present, even t hough the genes were not coupled. However, to be certain to transfer the desired traits of agronomic importance, the m a r k e r gene and agronomically valuable genes have to be closely linked.
Cytological investigations using in situ hybridization Somatic hybridization between distantly related species is mainly performed to obtain a somatic hybrid which can serve as a bridge between the
125 alien gene d o n o r and the cultivated crop. Thus, it is of interest to confirm the transfer by cytological investigations, since the desired result is transfer and stable inheritance of specific genes. The combination of two alien chromosome sets in the same cell, obtained after protoplast fusion, might result in the production of a new species, like R a p h a n o b r a s s i c a (Olsson and Ellerstr6m, 1980), or in partial hybrids in which c h r o m o s o m e s and traits of interest are either p r e s e n t or eliminated (Glimelius et al., 1991). To follow the chromosome constitution and inheritance of specific c h r o m o s o m e s after somatic hybridization, genetic studies utilizing in situ hybridization have been performed on two different somatic hybrid c o m b i n a t i o n s in our laboratory. Progeny from the intergeneric hybrids between Eruca sativa (+) B. n a p u s (Fahleson et al., 1997), a n d the hybrids between Lesquerella f e n d l e r i (+) B. n a p u s (Skarzinskaya et al., 1998) have been studied. To confirm the presence of alien c h r o m o s o m e s , species specific repetitive DNA sequences and total DNA have been u s e d as probes for genomic in situ hybridization (GISH). In addition, the c h r o m o s o m e constitution was analysed by morphological studies. GISH analysis of B. n a p u s c h r o m o s o m e s revealed t h a t total DNA from B. n a p u s hybridized preferentially to the centromeric regions (Fahleson et al., 1997). Other species investigated with the GISH technique have shown more uniform labelling of c h r o m o s o m e s (Parokonny et al., 1992; A n a m t h a w a t - J 6 n son et al., 1995; Hoan et al., 1989). The difference might be due to the generally low c o n t e n t of dispersed repeats and high cont ent of t a n d e m l y organized centromeric repeats found in B r a s s i c a species. In contrast, GISH analysis of E. s a t i v a (Fahleson et al., 1997) a n d L. f e n d l e r i c h r o m o s o m e spreads revealed uniform labelling of all c h r o m o s o m e s . Thus, we were able to analyse the c h r o m o s o m e constitution in some of the hybrids a n d their progeny. From the studies of progeny of the E. sativa (+) B. n a p u s hybridization we found the presence of one or two extra E. sativa c h r o m o s o m e s . These plants m ost likely r e p r e s e n t addition lines in which the genetic material from E. sativa will probably not be stably inherited. In contrast, in the L. f e n d l e r i (+) B. n a p u s hybrids, we were able to confirm the presence of c h r o m o s o m e s made up of c h r o m o s o m e s egm ent s from both parents, t hus, constituting recombined a n d t r a n s l o c a t e d chromosomes. In the L. fendleri (+) B. n a p u s results, asymmetric hybrids were produced after irradiation of the L. fendleri genome, while the E. sativa (+) B. n a p u s hybrids were produced w i t hout irradiation. These results indicate t h a t it might be beneficial to induce chromosome fragmentation by irradiation in order to introgress alien genetic material into the acceptor genome. Whether the translocated c h r o m o s o m e s will persist, achieving stable introgression of alien DNA r e m a i n s to be investigated. Another m e t h o d to determine w h e t h e r introgression h a s t aken place in somatic hybrids is to compare their RFLP p a t t e r n s with the p a t t e r n s found in m a p p e d genomes s u c h as B. n a p u s , B. nigra, B. j u n c e a and A. thaliana. We have b e g u n a s t u d y in our laboratory of the asym m et ri c B. n a p u s (+) A. thaliana somatic hybrids (Forsberg a n d Glimelius, 1995), and of the B. nap u s (+) B. nigra, B. n a p u s (+) B. carinata, and B. n a p u s (+) B. j u n c e a hybrids
126 (Sj6din an d Glimelius, 1989b). The pur pose of the analysis is to follow the inheritance of the alien B r a s s i c a and A. t h a l i a n a DNA in the somatic hybrids. An additional p u r p o s e in the study involving the B. n a p u s (+) A. t h a l i a n a hybrids is to investigate w het her different p r e t r e a t m e n t s of the donor material with X-ray, UV a n d restriction enzymes will result in specific breakpoints in the genome, and increase transfer of specific c h r o m o s o m e fragments. The results from these investigations have clearly shown t h a t analyses of RFLPm a r k e r s spread over the c h r o m o s o m e s are very suitable for studying presence of alien DNA and c h r o m o s o m e segments in the somatic hybrids (Forsberg et al., 1998a). The investigations revealed t h a t U V - and X - irradiation proved to be efficient m e a n s of inducing asymmetry, while the treatment with restriction enzymes did not result in any significant elimination of DNA from the donor protoplasts. Furthermore, besides fragmentation and elimination of donor c h r o m o s o m e s intergenomic translocations were most probably obtained according to presence of RFLP-band deviating from those in the A. t h a l i a n a p a r e n t (Forsberg et al., 1998b).
The utilization of protoplast fusion to modify the cytoplasm Sexual hybridization prevents the mixing and exchange of cytoplasmic genetic material, since the chloroplast and mitochondrial genomes are generally maternally inherited. In contrast, somatic hybridization allows mixing and exchange, leading to unique combinations of organelles and even to organelles with recombined genetic material (Nagy et al., 1981; Boeshore et al., 1983; Pelletier, 1986). Since plant organelles encode several traits of agronomic importance including CMS, herbicide resistance, nectar production, resistance to fungal toxins and cold tolerance, m a n i p u l a t i o n of the cytoplasmic genomes is of interest for plant breeding purposes.
Production of cybrids Fusion of protoplasts of different parental species always results in a mixing of the cyt opl as m s into a single cell. However, nucl ear fusion does not always follow cell fusion, so t hat cells may form with the nucl eus from only one p a r e n t in a hybrid cytoplasm. Such a cell is called a cybrid. A more successful way to obtain cybrids is to eliminate the n u c l e u s from one of the fusion partners. Several m e t h o d s have been applied with that purpose. Zelcer et al. (1978) u s e d X-rays to destroy the nuclei of the protoplast from one parent. The irradiated nuclei are unable to divide b u t the cell can still contribute organelles to the fusion product. A combination of X-ray treatm e n t of one p a r t n e r with t r e a t m e n t of the other fusion partner with the metabolic inhibitor, IOA, increases the possibility t h a t the nucleus from the IOA pretreated p a r e n t will be combined with the organelles from the irradiated parent. An even more sophisticated way to obtain cybrids is to enucleate the protoplasts from one fusion par t ner before fusion, for example, by centri-
127 fugation a n d / o r chemical treatment (Wallin et al., 1978; L6rz, 1985), or to enrich for spontaneously formed or plasmolytically induced cytoplasts, i.e., protoplasts without nuclei (Sundberg and Glimelius, 1991 b).
Organelle segregation and recombination after protoplast fusion. From the initial mixture of organelles obtained by protoplast fusion, segregation and sorting out of the organelles will take place during cell division and plant differentiation. Usually the chloroplasts sort out during subsequent mitoses, resulting in an establishment of one or the other parental type (Pelletier 1986). In rare cases a mixture of the two chloroplast types has been found in the hybrid plants and their progeny. It has not been confirmed that this mixture will persist in s u b s e q u e n t generations. Even more rare is recombination between combined chloroplast genomes. Nevertheless, Medgeyesy et al., (1985) demonstrated, by using two Nicotiana m u t a n t lines, that recombination had taken place after fusion and selection for the traits coded by m a r k e r genes present in the chloroplast genomes in the m u t a n t lines. In the investigation of different cybrids and hybrids produced between species in the family of B r a s s i c a c e a e it is difficult to draw conclusions and generalize about factors influencing segregation of chloroplasts. However, in a systematic investigation performed by Sundberg and Glimelius (1991a), the influence of genetic divergence and ploidy level on chloroplast segregation was studied. In this investigation the hybrids were produced with uniform and reproducible methods, with no pretreatment of the cells or directed selection. This study was extended by investigating hybrids from B. n a p u s (+) T. perfoliatum (Fahleson et al., 1994a), B. n a p u s (+) A. thaliana (Forsberg et al., 1994) and B. n a p u s (+) L. f e n d l e r i (Skarzhinskaya et al., 1996). The resuits are reported in Table 4.5. The methods to produce and enrich the fusion products, as well as culture and regenerate the plants were the same in all these studies. Chloroplasts from rapeseed were favored in almost all of the somatic hybrids. The most r a n d o m segregation of chloroplasts appeared in fusions between B. oleracea (+) B. rapa. These species represent closely related species, and the hybrid nuclei contained the complete nuclear genome of both parents. In all the other combinations the nuclear genome of the hybrids was dominated by the B. n a p u s genome, since a varying n u m b e r of chromosomes from the diploid species B. rapa, B. oleracea, B. nigra, R. sativus, E. sativa, T. perfoliatum, A. thaliana and L. f e n d l e r i had been preferentially eliminated. The biased segregation might be due to genetic divergence between the nuclear genomes of the species resulting in chromosome elimination of one species and t h u s incompatability reactions between nuclei and chloroplasts. The bias towards B. n a p u s chloroplasts could also reflect ploidy level differences between the species combined. A higher ploidy level of a species usually leads to larger n u m b e r s of chloroplasts per cell (Butterfass, 1989). Thus, the amphidiploid rapeseed protoplasts might provide more chloroplasts to the fusion products t h a n the protoplasts from the diploid
Table 4.5 Organelle composition in somatic hybrids produced between different species within the family of Brassicaceae Hybrid combination
No of hybrids
Parent B
Parent A
Frequency (YO) of hybrids with chloroplasts mitochondria from Parent A Cp Mt
Parent B CP Mt
Mix recom. Cp Mt
References
Intrageneric hybrids
B. napus"
(+)
B. oleracea
B. rapa B. napus" B. napus" B. napus"
(+) (+) (+) (+)
B. oleracea B. nigra B. juncea (tour) B. juncea (rap)
58
72
ND
38
ND
0
ND
II
45 92 ND 100
10
0 22 ND
55 8 ND 0
0 5 17 ND
0 0 ND 0
90 95 61 ND
91 88
33 56
9 12
8 0
0 0
59 44
94
50 57 26
6 0 21
0 0 9
0 0
50 43 65
24
18 6
Sundberg and Glimelius 1991a
Intergeneric hybrids
B. napus" B. napus"
(+) (+)
R. sativus (Ogura) E. sativa
12
A . thaliana T. perfoliatum L. fendleri
16 7 34
17
Intertribal hybrids B. napus"
B. napus" B. napus"
a
(+) (+) (+)
The B. napus cultivar contained B.rapa cytoplasm,
100
16
ND = not determined
3
Forsberg et a/. 1994 Fahleson et a[. 1994a Skarzhinskaya et al. 1996
129 species. In a n o t h e r s t udy performed by S u n d b e r g et al. (1991) the effects of cell type on chloroplast segregation was investigated. No significant correlation between the type of tissue u s e d for isolating protoplasts and segregation was found. However, even t h o u g h the segregation and sorting out of chloroplasts is u s u a l l y biased the r e a s o n s for this n o n - r a n d o m segregation have not been elucidated. This indicates t hat it is very difficult to control which chloroplast type will be established in the hybrid plants. The fate of the mitochondrial genome after protoplast fusion differs from t h at of the chloroplast. Besides segregation and sorting out t h a t takes place during cell division and development, the heteroplasmic state often results in intergenomic recombination between the mitochondrial genomes. The hybrid mitochondrial genome contains DNA fragments characteristic of both parents, as well as novel and u n i q u e fragments not found in the parental mt DNA (Beillard et al., 1979). Segregation of the m i t ochondri a in the somatic hybrids produced in our laboratory was biased in all hybrid c o m b i n a t i o n s (Table 4.5) an d favored the m i t ochondr i a from rapeseed (Landgren and Glimelius, 1994). This c o n t r a s t s to the segregation of chloroplasts, which was generally more r a n d o m when closely related species were hybridized. This bias to rapeseed (B. rapa) m i t o c h o n d r i a was proposed to result from i nherent mitochondrial d e t e r m i n a n t s since no clear effects of differences in genetic divergence or ploidy level were found on mitochondrial segregation (Landgren and Glimelius, 1994; Landgren et al., 1994). As for the chloroplast segregation the cell type u s e d for protoplast isolation did not influence the segregation of m i t o c h o n d r i a (Landgren et al., 1994).
The effect of p r e t r e a t m e n t of protoplasts on organelle segregation Since the organelles encode traits of agricultural importance, scientists desire to control the tranfer of these traits by somatic hybridization. Therefore, several studies have been u n d e r t a k e n to influence the sorting out and segregation of the organelles after fusion. These investigations have focused on the effects of IOA t r e a t m e n t combined with irradiation. The m o s t substantial studies on B r a s s i c a materials have been performed by Morgan and Maliga (1987) a n d Waiters and Earle (1993). In these studies, protoplast fusions were induced between a male-fertile and a CMS line of B. n a p u s (Morgan and Maliga, 1987) and a male-fertile a n d CMS line of B. oleracea (Waiters a n d Earle, 1993). In the latter investigation protoplasts isolated from different tissues were fused with protoplasts subjected to different pret r e a t m e n t s , in a variety of combinations. The results from both investigations, in which a large n u m b e r of calli was analysed, revealed t h a t segregation of chloroplasts was biased in favor of the fertile parent. According to Morgan an d Maliga (1987) segregation of chloroplasts was complete after 19 to 22 cell divisions, so t h a t no calli were found to have a mixture of chloroplasts. On the contrary, Waiters a n d Earle (1993) found both types of chloroplasts in calli, indicating a mixed cytoplasm. It c a n n o t be excluded, how-
Table 4.6 A presentation of selected cybrids with the desired features produced via protoplast fusion within the Brassicaceae family including their organelle composition and modified features. ~~~
N. of selected
~
Nuclear donor
Cytoplasm donor
Cytoplasmic trait
plants/ regenerated
Organelle composition Cp Mt
B. napus
B. napus CMS
Ogura-CMS AtrazinR
71131
Bn
Ogu-rec
4
B. napus (cam) atrR
B. napus
1/85
Bc
Ogu-rec
1
B. napus (cam)
B. napus CMS
16/36
Pol Pol Bc Bc
Pol Bc Bc Pol
21 5 10
Ogu
ow
2
Bn
ogu
2 2
B. napus
B. napus
1987
MF
CMS
B. napus
B. napus
MF
CMS
B.rapa ATR~
B. oleracea
MF
CMS
Polima-CMS
Ogura-CMS
411
Number of plants
Reference Pelletier et al. 1983 Chetrit et al. 1985
Barsby et al. 1987
0
Ogura-CMS
2/87
Bn
OP
AtrazinR Ogura-CMS
114 10134
Bc
Ogu-rec
1 10
Morgan and Maliga
Jar1 and Bornman 1988, Jar1 et al. 1989
Robertson et al. 1987 Jourdan et al. 1989 Temple et al. 1992
B. n a p
B. napus
ATR
AtrazinR Polima-CMS
11261
Bn
Pol-rec
1
Chuong el al. 1988
CMS
B. napus
R. sativus
Kosena-CMS
10117
ND
Kos-res
1
Sakai and Imamura
ND ND
rec Bn
3 6
Bn
Ogu-rec
3
Kao et al. 1992
Br Br
2 1
Earle and Dickson 1994
rec
1990
CMS B. oleracea
B. napus
Ogura-CMS
419
CMS B. oleracea CMS
B. rapa atrR
:Y-CMS3/62
e W
0
Ogura-CMS
B. oleracea Dicksson 1994 MF
B. oleraca
B. napus MF amR
B. oleracea CMS
Ogura-CMS
I ix
Bo
Ogu
1
B. oleracea
B. napus CMS
Polima-CMS
313
ND
rec
3
Wang et al. 1995a
B. juncea MF
B. napus CMS
Ogura-CMS
41123
Bj
rec
4
Kirti el al. 1995a
B. juncea MF
Trachvsfoma ballii
CMS
1/10
rec(?)
rec
1
Kirti et al. 1995b
313
Bo
Ogu 3 Earle and
CMS
MF B. napus (cam) MF
B. tournefortii MF
Tour-CMS
611674
Bc
Bt
4
Stiewe and Robb. 1994
B. napus MF
B. tournefortii
Tour-CMS
7/25
Bt Bn Bt
Bt Bt rec
4
Liu et al. 1996
1
2
B. oleracea Ppt
B. juncea HygR CMS MF
Tour-CMS
B. rapa MF atrR
B. oleracea CMS
Ogura-CMS
2 1/53
Br
Ogu
21
B. oleracea
B . rapa
Tour-CMS
17141
Bo Bo Bo Bt Bt
Bo Bt rec Bo Bt
1 1 1
CMS
39/78
Bo Bo
Bt rec
25 14
Arumugam et al. 1996
Heath and Earle 1996b Cardi and Earle 1997
0
6
atrR= Atrazin resistant chloroplasts, Ppt'= Phosphinotricin resistance, Bn = B. napus, Br = B. rapa, Bc = B. rapa subsp. campestris, ole-racea, Bj = B. juncea, Bt = B. tournefortii, rec = recombined mitochondria1 DNA, ND = not determined.
BO= B.
132 ever, t h a t the calli were chimeric. They did not find a n y correlation to age or size of the callus. Nevertheless, the r e g e n e r a t e d p l a n t s c o n t a i n e d either the Brassica or the R a p h a n u s c h l o r o p l a s t s revealing t h a t the segregation a n d sorting o u t p r o c e s s e s were complete in the differentiated p l a n t a n d can occur both d u r i n g the first cell divisions a n d formation of the callus as well as during the early s t a g e s of differentiation of a shoot p r i m o r d i u m . Since, neither the p r o t o p l a s t source, cell type, nor the p r e t r e a t m e n t by itself affected chlor o p l a s t segregation the b i a s e d segregation a g a i n s t the R a p h a n u s chloroplasts w a s s u g g e s t e d as a n effect of the n u c l e a r - p l a s t i d incompatibility. Analysis of the m i t o c h o n d r i a l composition in the calli revealed t h a t the mtDNA was r e c o m b i n a n t a n d t h a t the segregation was t o w a r d s the Raphan u s m i t o c h o n d r i a . F r o m the s t u d i e s where the effects of cell type on segregation w a s investigated in correlation with p r e t r e a t m e n t of the protoplasts with IOA it w a s found t h a t the p r e t r e a t m e n t of hypocotyl p r o t o p l a s t s resulted in a r e d u c e d m i t o c h o n d r i a l c o n t r i b u t i o n to the fusion products. This r e s u l t w a s also found by H e a t h a n d Earle (1996b) a n d Liu et al. (1996). However, in the e x p e r i m e n t s performed by O z m i n k o w s k i a n d J o u r d a n (1994b) utilizing m e s o p h y l l p r o t o p l a s t s from both p a r e n t s in the fusions, biased segregation of m i t o c h o n d r i a t o w a r d s the IOA t r e a t e d material w a s not found. The c o n c l u s i o n s from t h e s e investigations are t h a t chloroplasts a n d m i t o c h o n d r i a segregate, after fusion a n d r e g e n e r a t i o n of calli a n d plants, in a n i n d e p e n d e n t , b u t biased way. F u r t h e r m o r e , the segregation of chlorop l a s t s was not affected by IOA p r e t r e a t m e n t . In the case of mitochondria, the r e s u l t s s u g g e s t t h a t IOA h a s the effect of c a u s i n g a r e d u c t i o n of the mitoc h o n d r i a from the p r e t r e a t e d hypocotyl p r o t o p l a s t s in the hybrids. The generality a n d significance of t h e s e r e s u l t s r e m a i n to be proven, however. With r e s p e c t to irradiation of the p r o t o p l a s t s before fusion, no significant effects on segregation a n d e s t a b l i s h m e n t of the organelles have been recorded.
M o d i f i c a t i o n o f c y t o p l a s m i c traits via p r o t o p l a s t fusion Cytoplasmic male sterility (CMS) Protoplast fusion provides a n excellent tool to create novel combinations of organelles, w h i c h modify cytoplasmic traits. In spite of the rapidity with which t r a n s f o r m a t i o n technology h a s been developed, the practical a n d most suitable way to genetically modify the organelle c o m b i n a t i o n s a n d organellar DNA so far is to utilize p r o t o p l a s t fusion. Unique c o m b i n a t i o n s of cytoplasmic traits m a y arise as a r e s u l t of novel c o m b i n a t i o n s of chloroplasts a n d m i t o c h o n d r i a a n d of r e c o m b i n a t i o n s of organellar DNA (Pelletier, 1986). An elegant d e m o n s t r a t i o n of s u c h a modification is the i m p r o v e m e n t of the CMS B. n a p u s lines c o n t a i n i n g the "Ogura" c y t o p l a s m from R a p h a n u s sativus. Even t h o u g h this male sterility h a s been found to be highly stable in B. n a p u s g e n o t y p e s it suffers from chlorophyll deficiency, which is expressed especially at low t e m p e r a t u r e s . Moreover, the male-sterile plants have
133 low nectar production, which causes the flowers to be less attractive to honey bees (Pelletier et al., 1988). By producing hybrids between the B. n a p u s cultivar and a rapeseed line with the "Ogura" cytoplasm, an exchange of chloroplasts was obtained in some lines resulting in normal chlorophyll production, while retaining the male-sterile trait. Furthermore, a b u n d a n t nectar production was recovered in some cybrids (Pelletier et al., 1986). By this procedure a most valuable material was obtained, which has been used for further breeding to improve existing CMS cultivars (Renard et al., 1992). The material has also been very valuable for molecular analysis of malesterility (Bonhomme et al., 1992; Grelon et al., 1994). Similar experiments intended to restore chlorophyll levels and nectar production in cytoplasmic male-sterile B. n a p u s lines were carried out by Jarl and B o r n m a n (1988). These experiments have been followed by several other investigations, successfully transfering the '~Ogura" mitochondria coding for the cytoplasmic male-sterility and replacing the "Ogura" chloroplasts with the chloroplasts from the nuclear donor line in, for example, different cultivars of B. napus, B. j u n c e a and B. oleracea (for references see Table 4.6). Other cytoplasms known to code for cytoplasmic male sterility in Brassica species are the "Polima" (Fang and Mc Vetby, 1989) and '~CMS juncea" ("Anand", "Tour") (Rawat and Anand, 1979). Both these cytoplasms have been transferred via protoplast fusions to a nuclear acceptor line for which the CMS trait was desired (for references see Table 4.6). Also, in these studies material was produced which is utilized for commercial production of CMS cultivars. The '~CMS juncea" cytoplasm is reported to be derived from B. toumefortii (Pradhan et al., 1991; Szasz et al., 1991). To investigate whether the '~CMS juncea" was of alloplasmic origin and whether cytoplasm from fertile lines of B. toumefortii could induce male sterility in B. n a p u s , asymmetric fusion experiments between B. n a p u s and B. toumefortii have been carried out (Stiewe and R6bbelen, 1994: Liu et al., 1996). From these experiments lines of B. n a p u s were recovered, exhibiting the CMS trait and containing the B. toumefortii mitochondria, or mitochondria with recombined mt-DNA from the two parental genomes (Stiewe and R6bbelen, 1994; Liu et al., 1996). Phenotypically, flower morphology closely resembled the malesterile B. n a p u s plants obtained after sexual transfer of the cytoplasm of the original CMS B. j u n c e a line (Sodhi et al., 1994). Besides the male-sterile cybrids exhibiting floral abnormalities like narrow petals, swollen pistils, and abnormal or degenerated anthers, male-sterile cybrids with a more normal flower morphology was also obtained. In the cybrids displaying a more normal flower morphology the mt-DNA was recombined (Liu et al., 1996). A r u m u g a m et al., (1996) synthesized a hexaploid (AABBCC) somatic hybrid between B. j u n c e a (AABB), carrying the cytoplasm of B. toumefortii, and B. oleracea (CC), in order to produce a bridging material. From these fusions both male-sterile and fertile plants have been obtained with different compositions of organelles, including recombined mt-genomes. The hybrids are also represented by male-sterile lines that besides the male-sterility are free
134 from other floral abnormalities. These results are in agreement with the findings of Liu et al. (1996), indicating t h a t mt-DNA recombinations can rectify the a b n o r m a l floral features. Similarly, Cardi and Earle (1997) have produced a new CMS-B. oleracea by transferring the "CMS-tour" cytoplasm from B. rapa via protoplast fusion. A new alloplasmic CMS line of putative importance was produced via protoplast fusion between B. j u n c e a and T r a c h y s t o m a ballii (Kirti et al., 1992b). Backcrossing with a B. j u n c e a cultivar as the male parent, stable inheritance of the CMS trait was achieved (Kirti et al., 1995b). Molecular analysis revealed t h a t the CMS-line had r e c o m b i n a n t mitochondrial mt-DNA and suggested t h a t the cp-DNA was also recombinant, although more detailed investigations are required (Kirti et al., 1995b). Nevertheless, a new sterilityinducing cytoplasm was transferred to B. j u n c e a by protoplast fusion, which will be evaluated as a putative CMS-system for B. napus. A s y m m e t r i c h y b r i d s as a t o o l to t r a n s f e r n u c l e a r f e r t i l i t y r e s t o r e r g e n e s to CMS l i n e s
Even t h o u g h production of cybrids is performed mainly to create novel nuclear-cytoplasmic combinations, the nuclear genome can also be modified. If the cytoplasm donor cells are treated with ionizing radiations (X or gamma) prior to fusion, fragmentation of c h r o m o s o m e s is promoted, resulting in a limited transfer of a nuclear DNA (Dudits, 1987). This can be of practical importance aiming at producing cytoplasmic lines with nuclear restorer genes. Sakai a n d I m a m u r a (1990, 1992) have reported on the production of CMS lines of B. n a p u s obtained by introducing CMS from radish ( R a p h a n u s s a t i v u s cv. Kosena). This line differs from the R a p h a n u s lines containing the "Ogura" cytoplasm with respect to the CMS-inducing genes, and might therefore require different nuclear genes to restore the male sterility (Sakai et al., 1996). A stable CMS-line was used as a source of protoplasts to fuse with a restorer line of radish by the donor-recipient fusion m e t h o d (Sakai et al., 1996). Male-fertile lines were obtained from asymmetric hybrids, in spite of the presence of the CMS-inducing Kosena cytoplasm. Genetic analysis was performed on one of the lines, which gave progeny segregating to male-fertile and male-sterile plants after back-crossing. Several successive backcrosses to rapeseed were performed and the monogenic inheritance of fertility in the BC2S1 plants was interpreted to result from an integration of the restorer gene from radish in the nuclear genome of rapeseed. F u r t h e r studies will be expectedly performed to clarify the genomic organization and m ap the locus of the putatively integrated restorer gene in the acceptor rapeseed line. Similarly, in the studies performed by Liu et al. (1996) in which the alloplasmic CMS-lines of rapeseed were produced by introducing the mitochondria from B. tournefortii, the asymmetric hybrids segregated into fertile and male-sterile lines. The fertile lines are being u s e d as pollinators to establish w h eth er they can restore rapeseed lines containing the CMS-inducing cyto-
135 p l a s m of B. tournefortii. Besides, this m a t e r i a l is of clear i n t e r e s t for genetic a n d m o l e c u l a r s t u d i e s of C M S - i n d u c i n g a n d restoring genes. H e r b i c i d e r e s i s t a n c e or t o l e r a n c e
A n o t h e r trait of i n t e r e s t in b r e e d i n g of B r a s s i c a crops is triazine or a t r a zine tolerance. An atrazine t o l e r a n t B. rapa line w a s discovered in C a n a d a ( S o u z a - M a c h a d o et al., 1978) a n d explained by a m u t a t i o n in the c h l o r o p l a s t 32 KD QB protein (Hirschberg et al., 1984). Since b r o a d leaf w e e d s in c a n o l a fields are a s e r i o u s problem, a n extensive s e a r c h for h e r b i c i d e s to control t h e s e weeds h a s b e e n made. The c h l o r o p l a s t coded trait of a t r a z i n e tolerance c a n only be c o m b i n e d with the m i t o c h o n d r i a l coded CMS trait via p r o t o p l a s t fusion. In Table 4.6, different c o m b i n a t i o n s , bringing together a t r a z i n e toler a n t c h l o r o p l a s t s with CMS i n d u c i n g m i t o c h o n d r i a , are listed. As s h o w n in the table, a large n u m b e r of a t r a z i n e t o l e r a n t CMS lines have b e e n created. It m i g h t be a p p r o p r i a t e to a d d some c o m m e n t s a b o u t the trait of atrazine tolerance in this context. Triazine herbicides are not c o n s i d e r e d environm e n t a l l y a p p r o p r i a t e due to p r o b l e m s with the d e g r a d a t i o n of the herbicide. Their u s e will be limited in the future, a n d t h e y are a l r e a d y strictly regulated in several countries. F u r t h e r m o r e , a t r a z i n e t o l e r a n t c h l o r o p l a s t s in B. nap u s c a u s e a yield p e n a l t y (Reboud a n d Till-Bottraud, 1991) a l t h o u g h no yield p e n a l t y h a s b e e n found in B. oleracea vegetables (Christey a n d Earle, 1991). However, as d i s c u s s e d by Earle a n d Dickson (1994), a t r a z i n e r e s i s t a n t lines m i g h t be benefitial w h e n p l a n t i n g in soil with high levels of triazine residues. Conclusions
F r o m this overview of the l i t e r a t u r e r e g a r d i n g utilization of p r o t o p l a s t technology to improve the crops within the family of B r a s s i c a c e a e , it is clear t h a t s u b s t a n t i a l p r o g r e s s h a s been m a d e d u r i n g the last 15 years. Although the first r e p o r t s of p r o t o p l a s t isolation were from the b e g i n n i n g of the 1970's, it w a s not until the middle of the 80's t h a t real efforts were m a d e to utilize the p r o t o p l a s t technology to improve crops. It is obvious t h a t p r o t o p l a s t fusion c a n be u s e d as a m e t h o d to b y p a s s b a r r i e r s restricting s e x u a l hybridization of different species. Although h y b r i d p l a n t s c a n be o b t a i n e d in m o s t of the somatic hybridization e x p e r i m e n t s performed, low fertility severely restricts the likelihood of seed p r o d u c t i o n u p o n self-fertilization. However, seeds c a n u s u a l l y be o b t a i n e d by pollinating the h y b r i d s with pollen from one of the p a r e n t s . T h u s , the i m p o r t a n c e of the m e t h o d of somatic hybridization is not so m u c h to p r o d u c e a m p h i d i p l o i d s , c o m b i n i n g the complete g e n o m e s from the two species, as to utilize the somatic h y b r i d as a bridge for t r a n s f e r of desirable traits between the alien gene d o n o r a n d the crop. The i n h e r i t a n c e of certain desired traits, t r a n s f e r r e d to different B r a s sica crops, h a s b e e n followed in several investigations. In o r d e r to improve B r a s s i c a crops, traits like disease r e s i s t a n c e to different p a t h o g e n s , modifi-
136 cations of fatty acid composition, cytoplasmic male sterility, herbicide tolerance and repair of seedling chlorosis or sensitivity to cold have been modified and stably inherited over several consecutive generations of back-crosses. Thus, several lines have been derived from somatic hybrids, that are of great potential value for further breeding. However, more detailed investigations of the inheritance of the alien DNA carrying the desired genes and how to enhance the introgression of the alien DNA is also of importance to study. An introgression of DNA might occur via intergenomic translocations, recombination between homologous chromosomes, or even between nonhomologous chromosomes. Tools to detect intergenomic translocations have been developed; GISH and Prince (Koch et al., 1993) analyses will most probably become powerful techniques to elucidate configuration and constitution of chromosomes in the somatic hybrids. Besides these methods RFLP analysis of the linkage groups of the genomes in the somatic hybrids and the parents will most probably reveal whether recombinations a n d / o r translocations between the genomes have occurred. Of even greater importance would be the possibility to promote introgression of certain alien genes in a directed way. However, production of useful breeding material and new cultivars derived from somatic hybrids will be the final proof of the importance of the technique, as is the case for the improved Ogura-CMS-lines produced.
Acknowledgements. Thanks are due to all the research partners and students in my group performing the work with production of somatic hybrids and for discussions and suggestions of improvements of the manuscript. Special acknowledgements are devoted to Dr. E. Sundberg for allowing me to modify and utilize figures from her thesis. Thanks are also due to the Swedish Research Council for Forestry and Agricultural Research for support to this research.
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Biology of Brassica Coenospecies C. G6mez-Campo (Editor) 91999 Elsevier Science B.V. All rights reserved.
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Masao Watanabe (1) and Kokichi Hinata
(2)
(1) Faculty of Agriculture, Iwate University, Ueda 3-18-8, Morioka 020-8550, Japan (2) Faculty of Agriculture, Tohoku University, Sendai 981-8555, Japan Self-incompatibility is an elaborated breeding system for securing outcrossing and m a x i m u m recombination in the angiosperms. It is classified into heteromorphic and homomorphic types with respect to flower morphology. The homomorphic type is comprised of gametophytic and sporophytic types according to the phase (gametophyte or sporophyte) of S gene expression. The self-incompatibility in the Brassicaceae is classified into the homomorphic sporophytic type. According to Bateman's survey (1955), out of 182 species in the Brassicaceae, 80 species express self-incompatibility. In Brassica species and its closest allies, 50 species were self-incompatible out of 57 species examined (Hinata et al., 1994). The self-incompatibility system has probably played an important role in the differentiation and adaptation of species in this group. A n u m b e r of economically important vegetables are included in the Brassiceae (Brassica oleracea, B. rapa (syn. B. campestris), Rap h a n u s sativus etc.) and hybrid variety seeds made by using self-incompatibility are highly evaluated. Recently, self-incompatibility-aided hybrid breeding has been conducted in oleiferous B. napus (Pink, 1993). Investigations on the mechanism of self vs. non-self recognition and of resulting pollen-tube rejection may greatly contribute to the understanding of reproduction in plants, as well as to the further development of breeding techniques. A few short recently published review articles on this subject (de Nettancourt, 1997; Nasrallah, 1997; Suzuki et al., 1997d; Charlesworth and Awadalla, 1998) may be useful for general understanding of this field.
Morphology and physiology The stigma of Brassicaceae belongs to the so-called "dry stigma" group and is covered with one layer of papilla cells on its surface. The papilla is a typical secretory cell, in which endoplasmic reticulum and Golgi a p p a r a t u s are developed (Kishi-Nishizawa et al., 1990). The cell wall of papilla cell is
150 composed of an inner cellulose layer and an outer cuticle layer, on which waxes are deposited. Yellow colored pollen grains are generally transferred by bees. They have three germ slits, and the exine is covered with a lipoidal coating, which is called a pollen kit, tryphine or pollen coat (Dickinson and Lewis, 1973; Roggen, 1974). The coating is believed to include some other substances that may provide the signal of pollen identity. In cross-pollination, pollen grains absorb water from papilla cells, resulting in germination, and the pollen tubes, penetrating the outer cuticle layer, grow in the cellulose layer toward the stylar tissue. In self-pollination, absorption of water and consequently germination are disturbed (reviewed in Dickinson, 1995). When ambient humidity is sufficiently high, the pollen grains can germinate and the pollen tubes penetrate the cuticle layer, but they cannot grow into the cellulose layer. Therefore, self-incompatibility in this family is considered to derive from an interaction between a pollen grain (or pollen tube) and the cell wall of papilla (Kanno and Hinata, 1969). Upon self-pollination, callose deposition is observable at the plasmalemma of papilla. The site is limited to the attached portion with pollen and pollen tubes. Callose is also recognizable at the tips of pollen tubes. It has not yet been determined whether callose deposition is a cause or a result of pollen tube arrest (reviewed in Hinata et al., 1993).
Classical g e n e t i c s and d o m i n a n c e relationships Bateman (1952, 1954, 1955) first proposed that self-incompatibility in
Iberis a m a r a was controlled by a sporophytic multiple allelic system at a single locus, S, and the general applicability of this proposal to B r a s s i c a
species was also pointed out. In this sense, self-incompatibility is equivalent to self vs. non-self recognition between S alleles. Since then, a number of studies have supported this scheme (Thompson, 1957; Sampson, 1964; Thompson and Taylor, 1966; Okazaki and Hinata, 1984; Nou et al., 1991, 1993a, b), although there are different opinions as to two-locus systems with multiple alleles (Verma et al., 1977; Lewis 1977) and the joint expression of both sporophytic and gametophytic systems (Zuberi and Lewis, 1988; Lewis et al., 1988). In the sporophytic system, the behavior of pollen tubes is determined by the phenotype of the sporophyte that produced the pollen. Therefore, the phenotype of the pollen and the stigma of heterozygous plants depends upon the outcome of complex dominant / recessive allelic interactions (Thompson and Taylor, 1966). Dominance relationships between S alleles have been investigated for several species in the family B r a s s i c a c e a e : I. a m a r a (Bateman, 1954, 1955), B. o l e r a c e a and R. s a t i v u s (Haruta, 1962), B. oleracea (Thompson and Taylor, 1966; Ockendon, 1975; Visser et al., 1982; Wallace, 1979), R. r a p h a n i s t r u m (Sampson, 1964, 1967), S i n a p i s a r v e n s i s (Stevens and Kay, 1989) and recently, in B. r a p a (Hatakeyama et al., 1998a). Among the cha-
151 racteristic features of d o m i n a n c e r e l a t i o n s h i p s in these species are that: (a) c o d o m i n a n t r e l a t i o n s h i p s occur more frequently t h a n d o m i n a n t / recessive ones; (b) d o m i n a n t / recessive r e l a t i o n s h i p s occur more frequently in the pollen t h a n in the stigma; (c) the d o m i n a n t / recessive r e l a t i o n s h i p s are not identical for S alleles between stigma a n d pollen; a n d (d) n o n - l i n e a r domin a n c e r e l a t i o n s h i p s are observed more frequently in the s t i g m a t h a n in the pollen. N u m b e r s of S alleles were e s t i m a t e d by several s t u d i e s (Sampson, 1967; O c k e n d o n , 1974, 1980; Ford a n d Kay, 1985, Stevens a n d Kay, 1988, 1989; Karron et al., 1990; Nou et al., 1991, 1993a, b). Generally speaking, a b o u t 30 or more S alleles have been found in a population. Nou et al., (1993a) e s t i m a t e d t h a t there are more t h a n one h u n d r e d S alleles in B. rapa t h r o u g h o u t the world.
The S-multigene family Since the discovery of SLG, several genes whose s e q u e n c e s are similar to S L G have been isolated a n d characterized. They are c o n s i d e r e d to form an S-multigene family. A brief e x p l a n a t i o n for t h e m in Table 5.1 m a y provide an overview. SLG
(S-locus glycoprotein)
Detection a n d protein c h e m i s t r y of S L G The p r e s e n c e of S-specific a n t i g e n s in s t i g m a w a s first detected by immunological m e t h o d s (Nasrallah a n d Wallace, 1967; H i n a t a et al., 1982). Isoelectric focusing (IEF) a n a l y s i s of s t i g m a proteins revealed t h a t the Sspecific glycoproteins h a d different pI values c o r r e s p o n d i n g to respective S alleles (Nishio a n d Hinata, 1977). The c o n t e n t of the c o r r e s p o n d i n g S-glycoproteins in the stigma of S-heterozygotes was a b o u t half t h a t of the stigma of S-homozygotes (Hinata a n d Nishio, 1978). These S-glycoproteins (SLGs) cosegregated with S alleles w i t h o u t exception (Hinata a n d Nishio, 1978; Nou et al., 1991, 1993a). The S-glycoproteins were p r o d u c e d in s t i g m a s a few days before flower a n t h e s i s a n d the expression was coincident with the expression of self-incompatibility (Nishio a n d Hinata, 1977). This line of evidence s u p p o r t e d the idea t h a t the S-glycoproteins were the p r o d u c t s of S genes a n d they were c o n s i d e r e d to be the m o s t likely s u b s t a n c e to be involved in self vs. non-self recognition of self-incompatibility. Three S L G s were isolated from the s t i g m a s of S 8, S 9 a n d S12-homozy gotes of B. r a p a (Takayama et al., 1987; Isogai et al., 1987). Ninety-eight percent of the a m i n o acid s e q u e n c e of SLG s (B.r.) w a s d e t e r m i n e d . The three SLGs (B.r.) (SLG s, S L G 9 a n d S L G lz) h a d two k i n d s (A type a n d B type) of Nlinked oligosaccharide c h a i n s in c o m m o n . The ratios of the A type to the B were very similar, a n d no discernible differences were found in the N-glycosidic c a r b o h y d r a t e c h a i n s a m o n g these three SLGs ( T a k a y a m a et al., 1989).
152
Table 5.1
Brief explanation
of t h e m e m b e r s
of S - m u l t i g e n e family.
SLG (S-locus glycoprotein)
A secreting glycoprotein in the m a t u r e papilla cells, co-segregates with S allele. This w a s called S-protein by Nasrallah a n d Wallace (1967), a n d Sglycoprotein by Nishio a n d Hinata (1977). This protein group is highly variable; each h a s different pI value c o r r e s p o n d i n g to respective S allele a n d possibly involved in the self vs. non-self recognition of self-incompatibility. The n u m b e r of s u p e r s c r i p t s denotes the protein or gene t h a t is isolated from the S homozygote having its n u m b e r . S alleles have been d e n o t e d by s u b s c r i p t s widely, b u t in this report they are s h o w n by s u p e r s c r i p t s to clear t h a t they are controlled by allelic genes.
SRK (S-receptor kinase) A t r a n s m e m b r a n e protein k i n a s e ( s e r i n e / t h r e o n i n e type), whose receptor d o m a i n is highly h o m o l o g o u s with SLG, existing in the m a t u r e papilla cells. The S L G a n d S R K of the same S allele are considered to play a recognition role as a haplotype. SLR1 (S-locus related gene 1)
This was first described as NS glycoprotein in the m a t u r e papilla cells by Isogai et al. (1988), a n d the cDNA cloned as SLR1 by Lalonde et al. (1989) is c o n s i d e r e d to c o r r e s p o n d to the NS glycoprotein. This protein is not so variable as SLG; only 4 types being found in B. rapa. It does not participate in the recognition reaction, so far as the d a t a are concerned. C h r o m o s o m a l location of this gene is i n d e p e n d e n t of S allele. This was cited as SRA by H i n a t a et al. (1993). S L R 2 (S-locus related ~ene 2)
This was cloned to be linked with SLR1 by Boyes et al. (1991). Its DNA s e q u e n c e is highly h o m o l o g o u s with the Class II SLG. This was cited as S R B by H i n a t a et al. (1993). S L R 3 (S-locus related gene 3)
This gene cloned by Cock et al. (1995) was not linked to the S locus. The putative S L R 3 protein lacks a few cysteine r e s i d u e s t h a t are conserved in the other proteins of the S-multigene family. The expression of this was found in petals, sepals, a n d vegetative apices, in addition to stigmas a n d anthers.
153 Therefore, the specificity is considered to be determined by the protein portions of SLGs, a l t hough there is a possibility t hat the variation of the N-glycosylation sites might have significance for S-specificity (Takayama et al., 1987; Isogai et al., 1987). SLC~ protein was produced by a transgenic tobacco cell line with the c o n s t r u c t encoding S L G driven by duplicated CaMV 35S promoter. The characteristics of the SLC~ protein produced by this transgenic tobacco were very similar to those from B r a s s i c a stigmas in molecular m a s s and pI value (Perl-Treves et al., 1993).
Cloning of S L G cDNA clones of S L G were isolated from eight S-homozygotes of B. olerac e a (Nasrallah et al., 1985, 1987; Lalonde et al., 1989; Trick and Flavell, 1989; Chen a n d Nasrallah, 1990; Scutt and Croy 1992; Delorme et al., 1995 a), seven S-homozygotes of B. r a p a (Yamakawa et al., 1994; W a t a n a b e et al., 1994; M a t s u s h i t a et al., 1996; H a t a k e y a m a et al., 1998b; H a t a k e y a m a et al., 1998c) and two S-homozygotes of B. n a p u s (Goring et al., 1992a, 1992b), which contained d o m i n a n t and recessive alleles. These different S L G s so far cloned were classified into two groups. The first group c o n t a i n s pollen-domin a n t SLGs (Class I SLG) and the second contains pollen-recessive S L G s (Class II SLG). The amino acid sequence homology of S L G s within each class is a b o u t 78-90%, but t h a t between classes is a b o u t 65%. Several genomic clones derived from d o m i n a n t S alleles of B. r a p a and B. o l e r a c e a had no intron (Nasrallah et al., 1988; Dwyer et al., 1991; Suzuki et al., 1995; Delorme et al., 1995a). Pollen-recessive S L G 2 (Class II SLG) of B. o l e r a c e a had a small intron. This gene produced two transcripts: a secreted glycoprotein a n d a m e m b r a n e - a n c h o r e d protein. Transcripts of the m e m b r a n e - a n c h o r e d type were not detected in the S L G s of the pol l en-dom i nant group (Tantikanj a n a et al., 1993). On the other hand, three genes of the Class II S L G in B. r a p a so far cloned contained an intron b u t encoded only secreted glycoproreins (Hatakeyama et al., 1998b). The m e m b r a n e - a n c h o r e d form of S L G is not necessary for the pollen-recessive n a t u r e in B r a s s i c a species. Each sequence contains twelve conserved cysteine residues at the carboxyl terminal. These residues may be involved in the e s t a b l i s h m e n t of the tertiary configuration of this protein (Isogai et al., 1987; Nasrallah et al., 1987). The p a r t s having relatively different amino acid sequences a m o n g S L G s are located in 180-200, 250-280 residues (Yamakawa et al., 1994). These parts are relatively highly hydrophilic and m ay be responsible for the specificity of S L G s (Isogai et al., 1987; Nasrallah et al., 1987). The sequence of EP1 (embryonic protein 1), whose cDNA was isolated from non-embryonic carrot s u s p e n s i o n cells, h a d a region of high homology with SLG. Beside sequence homology, EP1 s har ed other characteristics with SLGs: all were 5060 kDa in size, they contained several glycosylation sites in the N-terminal two-thirds of the protein, and the C-terminal domain was cysteine-rich (van
154 Engelen et al., 1993). In the plant kingdom, there seem to be several genes or DNA clones whose sequence is similar to SLG. Expression and localization of S L G The mRNA t r a n s c r i p t s and protein products of SLG were not detected in the early p h as e of stigma development, b u t were expressed in m a t u r e papilla cells of the stigma surface (Nasrallah et al., 1988; Mariac et al., 1992; Cappadocia et al., 1993). A very small a m o u n t of SLG protein has also been identified in the style of B. oleracea (Kleman-Mariac et al., 1995), although the papilla cell is the recognition site for self-incompatibility. Electron microscopic observation using a n t i - S L G a n t i s e r u m has revealed t h a t S L G a c c u m u l a t e s in the m a t u r e papilla cell wall, where inhibition of self-pollen tube development occurs (Kandasamy et al., 1989; Kishi-Nishizawa et al., 1990). SLG was secreted via the route t h a t involved the r u m e n of ER, Golgi bodies and the small vesicles (Kishi-Nishizawa et al., 1990; Kandas a m y et al., 1991). Promoter analysis of SLG A chimeric toxic gene consisting of the diphtheria toxin A chain gene fused to 3.65kb SLG 13 promoter was us ed to detect expression in transgenic A r a b i d o p s i s and B. n a p u s . In the transgenic A r a b i d o p s i s , the papilla cells were s t u n t e d and became biologically inactive. Anther development was also impaired by toxic gene expression. The combined defects of pistil and ant her rendered the t r a n s f o r m a n t s self-sterile. However, the t r a n s f o r m a n t s were cross-fertile with u n t r a n s f o r m e d plants: the viable pollen of ablated plants was rescued by the wild-type stigmas, and the ablated papilla cells allowed the growth of wild-type pollen (Thorsness et al., 1993). In the transgenic B. n a p u s , flower morphology was normal except for a b e r r a n t papilla cell develo p m e n t and partial pollen sterility. Papilla cells lost their ability to elongate, to synthesize cell-specific proteins, and to s u p p o r t pollen germination after self- and cross-pollination (Kandasamy et al., 1993). A r a b i d o p s i s was transformed with a chimeric gene consisting of the 3.65-kb promoter region of an SLG 13 (B.o.) fused to the reporter GUS (~-glucronidase) gene. GUS activity was found in a different pattern between stigm a s an d anthers. In stigmas, the time and distribution of GUS activity was similar to t h a t described for SLG gene expression in B r a s s i c a . In anthers, however, the expression of GUS activity was detected in the sporophytic t a p e t u m tissues at an earlier stage of flower development (Toriyama et al., 199 lb). Another transgenic plant, B. oleracea, with SLG 13 (B.o.)-GUS showed a slightly different expression p a t t e r n than the transgenic Arabidopsis. Histochemical and fluorometric a s s a y s revealed that, in addition to its primary site of expression in the stigmatic papilla, this gene was expressed in the t r a n s m i t t i n g tissue of the stigma, style and ovary in pistils. Furthermore,
155 in anthers, the SLG-promoter was active not only in the t a p e t u m cells, with sporophytic expression, but also in the haploid microspores (Sato et al., 1991). For analysis of the promoter region, tobacco was transformed with truncated versions of the S L G 13 (B.o) promoter fused to the GUS reporter gene. The promoter had a modular organization and consisted of separable DNA elements that independently specify the gene expression in pistil and pollen. A 196-bp region was sufficient to confer stigma and style specificity to the marker gene. Two distinct, but functionally r e d u n d a n t domains allowed specific expression of the gene in pollen. The functional domains identified within the S L G 13 promoter contained several sequence elements (Box I to Box V) that were highly conserved in different alleles of the S L G (Dzelzkalns et al., 1993). In Sg-homozygote of B. rapa, the promoter regions of S L G 9 and S R I ~ were completely identical for 1,407 bp u p s t r e a m from their respective initiaion codons. The five sequence elements were also found in these clones, although one conserved element (Box III) lacked 7 of 11 bp. The transgenic tobacco plants (Nicotiana t a b a c u m ) transformed with S L G 9 and S R I ~ separately expressed their transcripts in the pistil tissues. This indicates that box III is not always necessary for their expression in pistil (Suzuki et al., 1995, 1996, 1997a). Transformation of S L G In order to analyze the function of SLG, gain of function and loss of function would be powerful strategies. In a transgenic B. r a p a with antisense S L G driven by the S L G promoter, the transcripts of S L G and S R K decreased, and the transformant became self-compatible (Shiba et al., 1995). A similar p h e n o m e n o n was also observed in different materials, but the progeny recovered its self-incompatibility (Takasaki et al., 1998). These experiments clearly show that S L G a n d / o r S R K function in self / non-self recognition reaction. Another interesting point is whether the S-specificity of recipient plants can be changed by the introduction of the S L G gene. Transgenic B. o t e r a c e a with the SLC~ (B.r.) altered their pollen-stigma interaction and became fully self-compatible. Reciprocal pollinations between transgenic and u n t r a n s formed plants showed that the stigma reaction changed in one recipient strain, whereas the pollen reaction was altered in the others (Toriyama et al., 199 l a). Self-compatible B. n a p u s , which is an amphidiploid between B. r a p a and B. oleracea, was transformed with four different SLGs. In these transgenic plants, the newly produced SLGs had pI values and molecular weights similar to those of donor plants. However, the production of S L G in the transgenic B. n a p u s was lower in quantity t h a n that of donor self-incompatible plants, and introduction of the S L G to the B. n a p u s cv. Westar did not alter the self-incompatibility phenotype (Nishio et al., 1992).
156 Takasaki et al., (1998) transformed a self-incompatible B. rapa with SLC~ and S L G 9 t h a t were isolated from the same species. The expression of one S allele in the host plant was c o - s u p p r e s s e d by the introduced SLGs. However, this c o - s u p p r e s s i o n was not observed in the progeny. An S L G gene of self-incompatible B. r a p a u n d e r control of a tapetumspecific promoter was introduced into self-compatible B. n a p u s . A pollination test indicated t h a t the pollen of the transgenic B. n a p u s did not gain the self-incompatibility phenotype (Sasaki et al., 1998). Simple identification m e t h o d s of S allele by S L G in breedinR programs Identification of S genotypes was d e m o n s t r a t e d by polymerase chain reaction (PCR) of genomic S L G followed by restriction analysis. Primers homologous to the conserved regions near the 5' and 3' ends of the SLG coding sequence were u s e d to amplify the SLGs. The S genotypes of the plants t h u s determined agreed well with the results based on pollen tube growth tests (Brace et al., 1993). When applying this m e t h o d to 50 different S-homozygotes in B. oleracea, RFLP using six restriction enzymes showed a u niq u e p a tter n for each S-allele with two exceptions (Brace et al., 1994). In a n o t h e r case, SLG-specific primers permitted amplification of SLG without any c o n t a m i n a t i o n of S L R 1 , SLR2, and other m e m b e r s of the S multigene family. This was applied to several cultivars and 27 different S-homozygote tester lines in B. rapa. The use of combinations of specific primers and restriction enzymes is r e c o m m e n d e d for registering S alleles (Nishio et al., 1994, 1996). Application of this system for R a p h a n u s species and some o r n a m e n t a l plants in the B r a s s i c a c e a e suggested t hat the diversification of the S L G alleles predates generic differentiation (Sakamoto et al., 1998). Another strategy for the identification of the S genotypes is the observation of single-strand conformation polymorphism in the PCR products of SLG. This technique distinguishes the PCR products derived from different S-homozygotes by electrophoretic mobility (Delorme et al., 1995b).
SRK (S-receptor kinase) Identification and cloning of S R K Walker an d Z hang (1990) pointed out t hat the amino acid sequence of the extracellular dom ai n of a serine / threonine type putative t ransm em brane protein kinase from maize had high homology to t hat of SLG. This finding suggested t hat a t r a n s m e m b r a n e protein kinase may play an import a nt role in self-incompatibility, since m a n y kinds of protein kinase are involved in signal t r a n s d u c t i o n in animals (Pawson, 1991; Trewavas and Gilroy, 1991). Stein et al. (1991) cloned S R K 6 from the genome of B. oleracea. Its extracellular domain, showing 89% homology to SLG ~, was connected via a single-pass t r a n s m e m b r a n e domain to a protein kinase catalytic center. This S R K gene comprised seven exons and had two stop codons, one in the first
157 intron at the e n d of the extracellular d o m a i n a n d one at the end of the seventh exon. Two S-locus linked genes, SLUr 2A a n d SLUr2B, w e r e isolated from the S2-homozygote, a n d this SLC_r2B w a s c o n s i d e r e d to be a n S R K 2 (Chen a n d Nasrallah, 1990; Stein et al., 1991). B e c a u s e b o t h S L G a n d S R K are considered to p a r t i c i p a t e in the recognition reaction a n d m a p to the S locus, a n S allele is referred to as a n S haplotype (Nasrallah a n d Nasrallah, 1993). Since t h e n , cDNA or genomic DNA clones of S R K have been isolated u n der different cloning strategies from two S-homozygotes of B. o l e r a c e a (Kum a r a n d Trick, 1994; Delorme et al., 1995a), five S-homozygotes of B. r a p a (Watanabe et al., 1994; Y a m a k a w a et al., 1995; S u z u k i et al., 1995; H a t a k e y a m a et al., 1998b; H a t a k e y a m a et al., 1998c), a n d two S-homozygotes of B. n a p u s (Goring a n d Rothsetin, 1992; Glavin et al., 1994). In e a c h case, S L G a n d S R K were found to be tightly linked to e a c h other, a n d they were also linked with S g e n o t y p e s d e t e r m i n e d by pollen t u b e behavior (Stein et al., 1991; W a t a n a b e et al., 1994; Delorme et al., 1995a). The positions of i n t r o n s were well c o n s e r v e d in e a c h S R K gene, t h o u g h the length of the first intron varied (Stein et al., 1991; K u m a r a n d Trick, 1994; Delorme et at., 1995a; Suzuki et al., 1997a; H a t a k e y a m a et al., 1998b). The homology between S L G a n d the S d o m a i n e n c o d e d by S R K varied from 85 to 90% in m a n y cases. A rapid identification s y s t e m for the diversity of the S R K genes w a s d e m o n s t r a t e d recently by PCR-RFLP by u s i n g a set of specific p r i m e r s (Nishio et al., 1997). E x p r e s s i o n a n d localization of S R K The t r a n s c r i p t of S R K w a s m a i n l y detected in s t i g m a tissue. Even in s t i g m a tissue, the e x p r e s s i o n of S R K was extremely low relative to S L G (Stein et al., 1991; W a t a n a b e et al., 1994; Glavin et al., 1994; Delorme et al., 1995a). The time of the e x p r e s s i o n of S R K w a s similar to t h a t of S L G (Glavin et al., 1994; Stein et al., 1996). B e c a u s e the S R K genomic clones h a d a n inframe stop codon, S R K s e e m e d to direct the s y n t h e s i s of several t r a n s c r i p t s (Stein et al., 1991; K u m a r a n d Trick, 1994; S u z u k i et at., 1995, 1996; Delorme et al., 1995a). These t r a n s c r i p t s were a p p a r e n t l y g e n e r a t e d by a c o m b i n a t i o n of alternative splicing a n d the u s e of alternative p o l y a d e n y l a t i o n signals (Stein et al., 1991; S u z u k i et al., 1996; G i r a n t o n et al., 1995). The t r u n c a t e d S R K w h i c h were derived from the S d o m a i n were detected as a protein a n d as a n RT-PCR p r o d u c t (Giranton et al., 1995). Recently, several n a t u r a l a n t i s e n s e t r a n s c r i p t s of S R K were identified in B. oleracea. W h e n different RNase protection p r o b e s were u s e d , regions of the promoter, exon I a n d intron I of SRK, were t r a n s c r i b e d in a n a n t i s e n s e direction. These antisense t r a n s c r i p t s would be correlated with a lower level e x p r e s s i o n of S R K t h a n t h a t of S L G (Cock et al., 1997). An e l e c t r o p h o r e s i s e x p e r i m e n t with s t i g m a p r o t e i n s s h o w e d t h a t S R K was a glycoprotein targeted to the p l a s m a m e m b r a n e (Delorme et al., 1995a). The s a m e r e s u l t w a s d e m o n s t r a t e d u s i n g t r a n s g e n i c tobacco p l a n t s with
158 S R K ~ (Stein et in E. coli, was late on serine and Nasrallah,
al., 1996). The product of SRK, expressed as a fusion protein a functional protein kinase, and was able to autophosphoryand threonine residues (Goring and Rothstein, 1992, Stein 1993).
Transformation of S R K A genomic clone of S R K 6 (B.o.) was introduced into S2-homozygotes. Although S R K was expressed in both stigmas and a n t h e r s of the transgenic plants, the self-incompatibility phenotype was not altered (Stein et al., 1991). Further transgenic approaches were u n d e r t a k e n using several chimeric genes. The transgenes led to a dramatic reduction in the expression of the endogenous S locus and related genes. The homology-dependent silencing of endogenous genes was associated, in at least some cases, with increased cytosine methylation. The silencing of SLG a n d / o r S R K genes in self-incompatible host plants results in the breakdown of self-incompatibility, whereas a reduction in SLG a n d / o r S R K gene transcripts in anthers does not affect pollen phenotype (Conner et al., 1997). A self-incompatible B. n a p u s was transformed with an inactive copy of the SRK gene. The transformants became self-compatible because of co-suppression and dominant-negative effects. The change of the S phenotype was only observed in stigma, but not in pollen (Stahl et al., 1998). Molecular characterization of S locus
The long genomic regions spanning the S locus were analyzed in some S haplotypes. These analyses have revealed that several genes exist on the flanking region of S L G and S R K genes. In self-incompatible B. n a p u s , two genes, SLL1 (S-locus linked gene 1) and SLL2 (S-locus liked gene 2), were located between the S L G and S R K genes, and expressed in the anthers. However, SLL1 did not have allelic specificity and SLL2 was also expressed in stigmas, indicating that these genes might not be the pollen S gene (Yu et al., 1996). In S 8 haplotype of B. rapa, two nonpolymorphic and vegetatively expressed sequences, 2 9 8 and 299, were located at the 3'-flanking region of the SLG 8. It was found that the 2 9 9 encoded SLL2 gene, and the 2 9 8 encoded ClpP (Clp protease) homologue (Boyes et al., 1997; Conner et al., 1998; Letham and Nasrallah, 1998). Using P 1-derived artificial chromosome (PAC) vector, Suzuki et al. (1997 c) directly cloned an 80-kb M/ul genomic fragment containing both SLG and S R K genes of S 9 haplotype in B. rapa. They have found more than ten genes are located in this genomic region, and this is currently being studied more throughly. The physical localization of the S locus (SLG and SRIO in the chromosome of B. rapa was visualized by multi-color fluorescent in situ hybridization. The S L G gene is localized at the interstitial region close to the end of the chromosome (lwano et al., 1998).
159 For the S 8 haplotype of B. rapa, a 100-kb region s p a n n i n g the S locus was m a p p e d with several cDNA and genomic DNA clones of A r a b i d o p s i s . Comparative m a p p i n g between the S locus region of B r a s s i c a and its homologous region in A r a b i d o p s i s revealed t h a t no sequences similar to S locus in B r a s s i c a were detected in the A r a b i d o p s i s genome (Conner et al., 1998).
Other members of S-multigene family S o u t h e r n blot analysis of B r a s s i c a genome with the S L G cDNA probe exhibited S haplotype-associated restriction site p o l y m o r p h i s m (Nasrallah et al., 1985; Nasrallah et al., 1988; Nou et al., 1993b). A part of these hybridized b a n d s have been isolated a n d characterized as S L G a n d SRK. In addition to these, self-incompatible B r a s s i c a species possess S related genes called SLR1 (Isogai et al., 1988, 1991; Lalonde et al., 1989; Trick a n d Flavell, 1989; Trick, 1990; Y am akaw a et al., 1993; W a t a n a b e et al., 1998), S L R 2 (Scutt et al., 1990; Boyes et al., 1991; T a n t i k a n j a n a et al., 1996), a n d S L R 3 (Cock et al., 1995). These genes are not linked to the S locus (Lalonde et al., 1989; Boyes et al., 1991; W a t a n a b e et al., 1992; Cock et al., 1995). Considerable evidence suggests t h a t the SLR1 gene does not participate in the selfvs. non-self recognition events, at least not directly (Franklin et al., 1996). Anyhow, together these genes form a large S multigene family (Dwyer et al., 1989). Observation of the SLR1 a n t i s e n s e transgenic B. n a p u s revealed t h a t it reduced the a d h e s i o n between pollen a n d stigma, and indicated t h a t SLR1 is one of the factors involved in the pollen-stigma adhesi on (Luu et al., 1997). When the region encoding the S R K catalytic d o m a i n was u s e d as a probe, m a n y genomic clones hybridized a n d some of t h e m were cloned (Kum a r and Trick,1993). One clone was identified as a pseudogene, b u t some others encoded functional protein kinases. Three clones each of 5 functional protein k in a s es cross-hybridized with an SLCr 29 cDNA probe, indicating the presence of u p s t r e a m receptor d o m a i n s t h a t m ay be homologous to the S L G gene. The previously reported S sequence complexity m ay be ascribed to a large gene family of receptor kinase (Kumar and Trick, 1993). In some of these clones, the t r a n s c r i p t s were detected in several t i ssues by RT-PCR analysis. RFLP analysis revealed t h a t one or two clones co-segregated with the S locus (Kumar and Trick, 1994; Oldknow and Trick, 1995). In self-incompatible B. rapa, 12 groups of genomic clones were isolated to obtain SLG-homologous regions. Of these groups, two corresponded to S L G and SRK, respectively. Of the remaining ten groups, four were SLG-like clones and six S R K - l i k e clones. Two clones with two S R K - l i k e sequences and one with an SLG-like sequence co-segregated with the S locus (Suzuki et al., 1995; Suzuki et al., 1997b). A pulse-field gel electrophoresis s t u d y in conjunction with DNA blot analysis d e m o n s t r a t e d t h a t S L G and S R K were separat ed by a m a x i m u m distance of 2 2 0 k b to 350kb. C o m p a r i s o n of the d a t a from the two genotypes, S 2 an d S~, revealed t h a t a high level of p o l y m o r p h i s m existed across
160 the entire S locus (Boyes a n d Nasrallah, 1993). Kianian a n d Quiros (1992) showed t h a t the S m u l t i g e n e family w a s organized in a linkage group of three loci by RFLP analysis. The p r e s e n c e of three linked loci indicates t h a t the self-incompatibility reaction m a y be the r e s u l t of the concerted action or interaction of several loci. F u r t h e r m o l e c u l a r a n a l y s i s will be n e c e s s a r y to d e t e r m i n e w h e t h e r all three m e m b e r s of the S m u l t i g e n e family are expressed a n d w h e t h e r t h e y have r e s u l t e d from the t a n d e m duplication of an ancestral locus (Kianian a n d Quiros, 1992). S u z u k i et al., (1997a) showed t h a t the p r o m o t e r s e q u e n c e s of S L G 9 a n d S R K 9 are completely identical to each other. This identity m a y suggest the r e c e n t o c c u r r e n c e of gene conversion in this locus.
Signal perception and signal transduction signal of pollen The recognition reaction of self-incompatibility is ascribed to the identity of S genes in pollen a n d stigma. Therefore, a s u b s t a n c e which correlates with S genes is expected to be p r e s e n t in pollen. The first possibility is t h a t the p r o d u c t of S L G or S R K functions as a n S gene d e t e r m i n a n t in pollen. Another possibility is t h a t the p r o d u c t of a gene t h a t is tightly linked to SLG a n d S R K f u n c t i o n s as a n S gene d e t e r m i n a n t in pollen. In e a c h case, genes m u s t m a p to the S locus b e c a u s e the self vs. non-self recognition reaction is r e g u l a t e d by the S locus. F u r t h e r m o r e , in the case of sporophytic self-incompatibility, this s u b s t a n c e would be p r o d u c e d before meiosis in pollen m o t h e r cells, or, if later, in the sporophytic a n t h e r wall from which it is t r a n s f e r r e d to pollen. The t r a n s c r i p t s of a recessive S L G were detected not only in the stigma b u t also in the a n t h e r , b u t the levels of t r a n s c r i p t i o n were different between s t i g m a a n d a n t h e r ( T a n t i k a n j a n a et al., 1993). Fully spliced a n d unspliced S R K 6 (B.o.) t r a n s c r i p t s were m a x i m a l l y e x p r e s s e d in a n t h e r s at the binucleate pollen stage (Stein et al., 1991). In the S 2 haplotype of B. oleracea, a gene, S L A 2 located in the 3' flanking region of S L G 2 gene, t r a n s c r i b e d two c o m p l e m e n t a r y anther-specific t r a n s c r i p t s by two promoters. One was spliced a n d the o t h e r unspliced. They a c c u m u l a t e d in a n antiparallel m a n n e r in developing m i c r o s p o r e s a n d a n t h e r s . S e q u e n c e s related to S L A (S locus anther gene) were not detected in either DNA or RNA in other p l a n t s carrying S h a p l o t y p e s o t h e r t h a n S 2 (Boyes a n d Nasrallah, 1995). In f u r t h e r studies the p r e s e n c e of a f u n c t i o n - d i s r u p t e d S L A gene by a large insertion was identified in a n S h a p l o t y p e of self-incompatible B. o l e r a c e a (Pastuglia et al., 1997b). Therefore, this gene does not seem to be required for the self-incompatibility response. In a t r a n s g e n i c A r a b i d o p s i s t h a t carried a r e p o r t e r G U S gene with the p r o m o t e r of S L G 13 (B.o.) G U S activity was found in a n t h e r tapetal cells at the u n i n u c l e a t e m i c r o s p o r e stage (Toriyama et al., 199 l b). In the t r a n s g e n i c B.
161
oleracea with the s a m e reporter gene (SLG 13 (B.o.)-GUS), e x p r e s s i o n of GUS was detected in the t a p e t a l cells a n d in the pollen g r a i n s at the bi- a n d trinucleate pollen s t a g e s (Sato et al., 1991). These o b s e r v a t i o n s s u p p o r t the presence of an SLG-derived t r a n s c r i p t in a n t h e r s . The p r e s e n c e in y o u n g a n t h e r s of the t r a n s c r i p t h o m o l o g o u s with SLG was s u g g e s t e d by the aid of PCR m e t h o d with c o m b i n a t i o n s of primers. Several t r a n s c r i p t s differing in size were detected in s e x u a l as well as in vegetative t i s s u e s (Guilluy et al., 1991). The p r e s e n c e of a n a n t h e r - s u b s t a n c e t h a t c r o s s - r e a c t e d with anti-SLGS(B.r.) a n t i s e r u m w a s r e p o r t e d by W a t a n a b e et al., (1991). This s u b s t a n c e , SAP (S-glycoprotein-like a n t h e r protein), generated a single distinct b a n d at pl 5.0 on a n I E F - i m m u n o b l o t . SAP w a s found in a n t h e r walls at the uni- a n d b i n u c l e a t e stages r a t h e r t h a n in pollen. This observation m a y also s u g g e s t t h a t SLG-like p r o d u c t s are p r o d u c e d on the ant h e r side. However, correlation between t h e s e s u b s t a n c e s a n d S genotypes h a s not yet b e e n d e m o n s t r a t e d . Several pollen coat proteins (PCI~ have b e e n looked for a n d isolated. W h e n a m i x t u r e of s t i g m a e x t r a c t s with pollen coat p r o t e i n s w a s applied to isoelectric focusing analysis, one b a n d was newly detected; this w a s considered to be a p r o d u c t formed by the i n t e r a c t i o n b e t w e e n a certain pollen coat protein a n d SLGs. This pollen s u b s t a n c e , d e s i g n a t e d PCPT, w a s a 7 - k D a nonglycosylated peptide (Doughty et al., 1993). In a n o t h e r e x p e r i m e n t , PCP1 was isolated from a n t h e r mRNA u s i n g RT-PCR. PCP1 c o n t a i n e d a single intron a n d e n c o d e d a small, basic peptide c o m p o s e d of 83 a m i n o acids containing a h y d r o p h o b i c signal peptide s e q u e n c e . Eight cysteine residues, which have high homology to a n u m b e r of o t h e r anther-specific genes, were found in the c e n t r a l p a r t a n d C - t e r m i n a l region. T r a n s c r i p t s of PCP1 were detected in the c y t o p l a s m of the t r i n u c l e a t e pollen grain, b u t not in the t a p e t u m . The PCP g e n e s formed a large m u l t i g e n e family, c o m p o s e d of 30 to 40 copies per g e n o m e of B. oleracea, b u t s h o w e d no b a n d linked to the S locus (Stanchev et al., 1996). The cDNA clones h o m o l o g o u s to PCP1 were also isolated from a cDNA library of i m m a t u r e a n t h e r s by u s i n g polyclonal a n t i s e r u m raised a g a i n s t the e x t r a c e l l u a r pollen p r o t e i n s (Toriyama et al., 1998). Recently, by u s i n g a b i o a s s a y s y s t e m , addition of PCP-A {renamed from PCP7) w a s e x a m i n e d on s t i g m a surface. W h e n "self" PCP-A fraction was u s e d , the s u c c e s s of compatible cross-pollination w a s prevented, while a "cross" PCP-A fraction could i n d u c e the g e r m i n a t i o n a n d growth of selfpollen. This s u g g e s t s a possibility t h a t a m e m b e r of the PCP-A protein family could be a d e t e r m i n a n t at the pollen side in the B r a s s i c a self-incompatibility s y s t e m ( S t e p h e n s o n et al., 1997; D o u g h t y et al., 1998).
Signal transduction cascade via protein phosphorylation By analogy with a n i m a l growth factors, one could imagine t h a t S R K accepts a signal from pollen a n d t r a n s d u c e s the signal into papilla cells via a protein p h o s p h o r y l a t i o n c a s c a d e (Figure 5.1).
162 Using a yeast two-hybrid system, proteins interacting with SRK-910 kinase domain were screened. Two different kinds of cDNA clones were isolated and characterized. One of them included two thioredoxin-h-like clones, THL-1 and THL-2. These clones specifically interacted with the kinase domain of SRK-910. THL-1 was expressed in a variety of tissues, but THL-2 preferentially expressed in floral tissues. Thioredoxin may possibly be one of the effector molecules in the signal cascade of self-incompatibility (Bower et al., 1996). Another cDNA clone was ARC1 (Arm Repeat Containing) gene. The ARC 1 specifically interacted with the kinase domain of SRK, but not with the kinase domains taken from different kind of Arabidopsis receptor-like kinases. The interaction was phosphorylation dependent (Gu et al., 1998). Two independent experiments have been reported on phosphorylation using inhibitors of serine / threonine protein phosphatase. One showed that treatment with okadaic acid via transpiration stream against the newly opened flower bud was sufficient to overcome self-incompatibility, though the magnitude of this effect was S genotype dependent. At the higher concentrations used, pollen tube growth was arrested before the pollen tubes reached the ovary, b u t this effect was also observed in cross-pollinated styles treated in the same m a n n e r (Scutt et al., 1993). In contrast, the treatment of mature flowers with p h o s p h a t a s e inhibitors, okadaic acid and microcystin, had no effect on the self-incompatibility response of four different S genotypes, although the treatment of flower b u d s j u s t prior to anthesis allowed self-pollen tube invasion of papilla cells (Rundle et al., 1993). A cDNA clone encoding type 1 serine / threonine protein phosphatase (BoPP1) was isolated from B. oleracea stigmas. It was suggested that the arrest of self-pollen development might result from activation of the SRK and the phosphorylation of specific protein substrates, whereas to allow the growth of compatible pollen tube, protein p h o s p h a t a s e activity might be required to dephosphorylate these substrates (Rundle and Nasrallah, 1992). A new A r a b i d o p s i s m u t a n t (pop1) gives us an insight into the pollen tube arrest in self-pollination. Stigma cells that had been in contact with the m u t a n t pollen produced callose, a It-1,3-glucan. The m u t a n t pollen failed to germinate because it did not absorb water from the stigma; yet it germinated in vitro, indicating it was viable (Preuss et al., 1993). In these points, the similarity between the pop1 mutation and the self-incompatibility studied in Brassica species is striking. Both regulate pollen germination at the stigma surface, often through control of pollen hydration. In addition, there are m a n y examples of callose production in stigma cells that are in direct contact with incompatible self-pollen. Singh et al. (1989) showed that self-pollen had higher levels of chlorotetracycline fluorescence and higher calcium content than cross-pollen in energy-dispersive analysis of X-rays. Callose deposition was found to be a calcium dependent process in pollination, by using a calcium channel antagonist and a calcium ionophore. However, pretreatment of pistils with 2-
163
Figure 5.1 A hypothetical picture drawing the recognition reaction. A pollen specific substance (unknown) is transferred onto the stigma papilla cell as a ligand. The ligand is accepted by SLG and/or the receptor domain of SRK at the cell wall of papilla. The accepted signal is transferred to the cytoplasm of the papilla cell by SRK, and then through protein phosphorylation cascade the incompatibility reaction would be controlled.
164 deoxy-D-glucose abolished the callose formation in self-pollination, whereas self-pollen remained inhibited and cross-pollen grew normally. This suggests that callose formation in the stigma is not an essential factor preventing the growth of pollen tubes in the self-incompatibility (Singh and Paolillo, 1990). Furthermore, transgenic plants with the [t-1, 3-glucanase gene were raised. In the plants, little or no callose was detected in the papilla cells, though the self-incompatibility system appeared to be unaffected. This result significantly indicates that callose deposition is not required for the rejection of incompatible pollen (Sulaman et al., 1997).
Molecular analysis of self-compatibility A self-compatible (Sc) line of B. oleracea possessed SLG sc which had a similar spatial and temporal expression to that of self-incompatible B r a s s i c a plants. Immunological analysis of an F2 population revealed that the SLG sc segregated with the self-compatibility phenotype, suggesting that the changes in SLG were responsible for the self-compatibility character. The deduced amino acid sequence of an SLG sc cDNA clone showed a high level of homology with that of pollen-recessive SLC~ (Gaude et al., 1993). From selfcompatible B. n a p u s cv. Westar, two different stigma-specific cDNA clones homologous to S L G were isolated. One of these sequences, S L G - w s l , showed high homology to Class I SLG, whereas the other, S L G - w s 2 , to Class II SLG. Both genes were expressed at high level in stigmas following a developmental pattern typical to SLG in the self-incompatible diploids, B. rapa and B. oleracea (Robert et al., 1994). A single s u p p r e s s o r gene caused the reduction of SLG content (Nasrallah and Nasrallah, 1989). This self-compatibility trait is not tightly linked to the S locus, and the genetic data suggested action of a single recessive gene. The genetic locus defined by the self-compatible mutation was designated as s c f l (stigma compatibility factor 1). The s c f l mutation affected the RNA expression level of secreted type glycoproteins, SLG, SLR1 and SLR2, but not the receptor protein kinase, S R K and, therefore, it is considered that this disrupted a regulatory gene which possibly encodes a trans-acting factor required for high-level expression of the secreted-type glycoproteins in stigma (Nasrallah et al., 1992). In one self-compatible B. oleracea, transcripts of S R K were not detected, though SLG was normally expressed. The analysis of S R K genomic clones demonstrated that the first and the second exons were deleted (Nasrallah et al., 1994). On the other hand, in spontaneous self-compatible B. rapa var. y e l l o w s a r s o n (C636), self-compatibility was mainly explained by a recessive epistatic gene, m. The transcript of SLG was less a b u n d a n t than in self-incompatible strains, though SLR1 and S L R 2 were normally expressed. Furthermore, the transcript of S R K was not detected. This down-regulation of SLG and S R K may be related to this self-compatibility (Watanabe et al., 1997). Similar trends were observed in different strains of y e l l o w s a r s o n
165 (Lalonde et al., 1989, Nasrallah et al., 1994). Recently, the self-compatible rood (renamed from m) locus was dissected with molecular techniques. The phenotype was associated with the absence of t r a n s c r i p t s encoded by an aquaporin-related gene. This may suggest t h a t a water c h a n n e l is required for the self-incompatibility response of B r a s s i c a species (Ikeda et al., 1997). Besides, the importance of pollen coat lipids for pollen germination was also stressed (Wolter-Arts et al., 1998). An isolated S R K from self-compatible B. n a p u s h a d a 1-bp deletion toward the 3' end of the S domain. This deletion would a p p e a r to lead to the p r e m a t u r e t e r m i n a t i o n of translation and the production of a t r u n c a t e d SRK. An active S R K might be required for the expression of self-incompatibility (Goring et al., 1993). Genes homologous with SLR1 were cloned from several self-compatible species (Oldknow et al., 1995, Lalonde et al., 1989). So far, self-compatibility seems to be connected with the down regulation of SLG and SRK, t h o u g h the main p a t h w a y involved h a s not yet been determined.
Evolutionary aspects The role of self-incompatibility in plants is considered to promote outbreeding, t h r o u g h which plants maximize genetic recombination and maintain the genetic heterogeneity. Uyenoyama (1988) proposed t h a t the evolutionary function of self-incompatibility s yst em s is to serve as a eugenic mec h a n i s m for the improvement of offspring quality. Production of offspring t h a t are heterozygous at specific antigen loci is adaptive if the expected viability or fertility of those offspring is greater. U y e n o y a m a (1989) considered conditions for the origin of partial sporophytic self-incompatibility, a s s u m i n g two quantitative models, which differ with respect to the determination of offspring viability. In both cases, the origin of self-incompatibility requires t h a t the relative change in the n u m b e r s of offspring derived by the two reproductive m o d e s c o m p e n s a t e for the twofold cost of outcrossing. Sporophytic self-incompatibility systems could arise in response to identity disequilib r iu m between modifiers of incompatibility a n d a locus subject to overdom i n a n t viability selection. Recent a d v a n c e s of studies on SLG, S R K and related genes have made it possible to d i s c u s s the variation of S alleles in view of evolutionary trends. Hinata et al. (1995) compared s y n o n y m o u s and n o n s y n o n y m o u s base substitutions in the S dom ai n and kinase d o m a i n separately between SRKs. Acc u m u l a t i o n of s y n o n y m o u s and n o n s y n o n y m o u s base s u b s t i t u t i o n s per site in the S d o m a i n was mostly comparable with those of SLGs, respectively. In the kinase domain, however, the level of n o n s y n o n y m o u s base s u b s t i t u t i o n was as low as half of those of the S domain, while the level of s y n o n y m o u s base s u b s t i t u t i o n was mostly comparable between these two domains. This may indicate t h a t the n o n s y n o n y m o u s base s u b s t i t u t i o n of the kinase domain is c o n s t r a i n e d because this d o m a i n should keep its kinase activity,
166 while the S d o m a i n could diversify b e c a u s e the c h a n g e of s t r u c t u r e would be recognized as different S alleles. Analysis of nucleotide s e q u e n c e s on S L G a n d related genes h a s been c o n d u c t e d by Trick a n d H e i z m a n n (1992), U y e n o y a m a (1995), Hinata et al. (1995) a n d King a n d Lynn (1995). It was s u g g e s t e d t h a t the age of the sporophytic self-incompatibility s y s t e m expressed in B r a s s i c a exceeds species divergence within the g e n u s by four to five folds. The extraordinarily high levels of s e q u e n c e diversity exhibited by S alleles a p p e a r s to reflect their ancient derivation, with the alternative h y p o t h e s i s of h y p e r m u t a b i l i t y rejected by the a n a l y s i s (Uyenoyama, 1995). According to d e n d r o g r a m s c o n s t r u c t e d on the basis of s y n o n y m o u s a n d n o n s y n o n y m o u s s u b s t i t u t i o n s , it was considered t h a t SLR1 differentiated first, followed by S L R 2 (Figure 5.2). The differentiation of S L G a n d the S d o m a i n of SRK, b o t h of which occurred coincidentaUy, is one of the prerequisite factors for the e s t a b l i s h m e n t of self-incompatibility. The allelic differentiation was e s t i m a t e d to have occurred more t h a n tens of million y e a r s ago (Hinata et al., 1995). Phylogenetic analysis of m a n y S L G a n d S R K alleles suggested t h a t intragenic r e c o m b i n a t i o n was involved in the evolution of S L G a n d t h a t gene conversion might have often occurred between S L G a n d SRK. The generation of a large n u m b e r of SLG a n d S R K alleles m a y have been c a u s e d not only by point m u t a t i o n b u t also by i n t r a / i n t e r genic recombination. Since S L G a n d S R K alleles are classified into Class I a n d Class II b a s e d on their s e q u e n c e similarity, it is possible t h a t self-incompatibility was first expressed in a two-haplotype system with a d o m i n a n t Class I a n d a recessive Class II haplotype (Kusaba et al., 1997). The c u r r e n t p a r a d i g m for the origin of self-incompatibility p o s t u l a t e s multiple episodes of r e c r u i t m e n t a n d modification of pre-existing genes in each major m e c h a n i s m of self-incompatibility. U y e n o y a m a (1997) a n d S c h i e r u p et al. (1997, 1998) have d i s c u s s e d evolutionary d y n a m i c s a n d the allelic genealogies t h r o u g h stochastic simulation.
R e l a t e d s t u d i e s with future p r o s p e c t s In higher plants, self vs. non-self recognition reactions are only known in the pollination system, involving self- a n d interspecific incompatibility. Besides, gene-to-gene recognition reaction is k n o w n in p l a n t - p a t h o g e n interactions. Hodgkin et al. (1988) have c o m p a r e d B r a s s i c a self-incompatibility a n d p l a n t - p a t h o g e n interactions. Both reactions have m a n y features in common: both c o n c e r n cell surface c o m p o n e n t s s u c h as glycoproteins a n d both are active p r o c e s s e s requiring enzyme synthesis. The m o s t obvious differences between these two s y s t e m s are t h a t self-incompatibility in B r a s s i c a is m e d i a t e d t h r o u g h the s u p p l y of water a n d does not exhibit cross-protection reactions. A characteristic feature of self-incompatibility is a pre-programmed process in p l a n t s r a t h e r t h a n a n i n d u c e d r e s p o n s e to external events.
167 SLG 13 Bo SLG 8 Bc SLG 29 Bo
47
, SLG 14 Bo
4 98
SLG 6 Bo , SRK 6 Bo(RD) r S L G Bc loo
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b: Nonsynonymous substitution per site
Figure 5.2 Dendrograms of 17 OTUs constructed by neighbour-joining method based on synsynonymous and non-synonymous base substitution per site. The SLR1 differentiated first, followed by SLR2. The differentiation of SLG as well as the receptor domain of SRK is one of the prerequisite factors for the establishment of self-incompatibility in the Brassicaceae. (From Hinata et al. (1995); reproduced with permission from the Genetic Society of America).
168 Recently, several resistance genes against p a t h o g e n s were isolated (reviewed in Bent, 1996). Two of them, PTO and X a 2 1 , were encoded membrane b o u n d serine / threonine protein kinase (Martin et al., 1993, Song et al., 1995). In this respect, both self-incompatibility a n d pathogen-plant interaction are regulated by similar m e c h a n i s m s via phosphorylation cascade in the signal transduction. Another different protein kinase had an important role in plant morphogenesis. Erecta (er) m u t a n t of A r a b i d o p s i s Landsberg showed a compact inflorescence, blunt fruits, and short petioles. The er gene was encoded by a receptor type serine / threonine protein kinase (Torii et al., 1996). Several receptor type protein kinase genes were isolated and characterized, although their biological functions remain u n k n o w n (Chang et al., 1992; Kohorn et al., 1992; Tobias et al., 1992; Dwyer et al., 1994; Walker, 1992; Zhang a n d Walker, 1993; Wang et al., 1996; Pastuglia et al., 1997a). The elucidation of signal t r a n s d u c t i o n in these physiological traits would contribute to the u n d e r s t a n d i n g of the m e c h a n i s m of signal transduction of self / non-self recognition in self-incompatibility.
Acknowledgements We t h a n k Dr. M. K. Uyenoyama, Duke University, U.S.A., Dr. E. Newbigin, University of Melbourne, Australia, and Dr. A. Isogai, Nara Institute of Science a n d Technology, J a p a n , for helpful c o m m e n t s and improving the manuscript. This work was supported in part by Grants-in-Aid for Special Research on Priority Areas (nos. 07281102 a n d 07281103; Genetic Dissection of Sexual Differentiation and Pollination Process in Higher Plants) from the Ministry of Education, Science, Culture and Sports, J a p a n .
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177 quired for the self-incompatibility response in Brassica oleracea. Plant Cell 9, 2065-2076. Pawson, T. 1991. Signal transduction in the control of cell growth and development. Trend. Genet. 7, 343-345. Perl-Treves, R., Howlett, B. and Nasrallah, M. E. 1993. Self-incompatibility related glycoproteins of Brassica are produced and secreted by transgenic tobacco cell cultures. Plant Sci. 92, 99-110. Pink, D. A. C. 1993. Swede and turnip Brassica napus L. var. napobrassica, B. rapa L. var. glabra. In: Kalloo, G. and Bergh, B. O. (eds), Genetic improvement of vegetable crops, pp. 511-519, Pergamon Press. Preuss, D., Lemieux, B., Yen, G. and Davis, R. W. 1993. A conditional sterile mutation eliminates surface components from Arabidopsis pollen and disrupts cell signaling during fertilization. Genes Dev. 7, 974985. Robert, L. S., Allard, S., Franklin, T. M. and Trick, M. 1994. Sequence and expression of endogenous S-locus glycoprotein genes in self-incompatible Brassica napus. Mol. Gen. Genet. 242, 209-216. Roggen, H. 1974. Pollen washing influences (in)compatibility in Brassica oleracea varieties. In: Linskens, H.F. (ed.) Fertilization in higher plants, pp 273-278, North-Holland. Rundle, S. J. and Nasrallah, J. B. 1992. Molecular characterization of type 1 serine/threonine p h o s p h a t a s e s from Brassica oleracea. Plant Mol. Biol. 20, 367-375. Rundle, S. J., Nasrallah, M. E. and Nasrallah, J. B. 1993. Effects of inhibitors of protein serine / threonine p h o s p h a t a s e s on pollination in Brassica. Plant Physiol. 103, 1165-1171. Sakamoto, K., Kusaba, M. and Nishio, T. 1998. Polymorphism of the S-locus glycoprotein gene (SLG) and the S-locus related gene (SLR1) in Rap h a n u s sativus L. and self-incompatible ornamental plants in the Brassicaceae. Mol. Gen. Genet. 258, 397-403. Sampson, D. R. 1964. A one-locus self-incompatibility system in R a p h a n u s raphanistrum. Can. J. Genet. Cytol. 6, 435-445. Sampson, D. R. 1967. Frequency and distribution of self-incompatibility alleles in R a p h a n u s raphanistrum. Genetics 56, 241- 251. Sasaki, Y., Iwano, M., Matsuda, N., Suzuki, G., Watanbe, M., Isogai, A. and Toriyama, K. 1998. Localization of an SLG protein expressed u n d e r the regulation of a tapetum-specific promoter in a n t h e r s of transgenic Brassica napus. Sex. Plant Reprod. 1 1 , 2 4 5 - 2 5 0 . Sato, T., Thorsness, M. K., Kandasamy, M. K., Nishio, T., Hirai, M., Nasrallah, J. B. and Nasrallah, M. E. 1991. Activity of an S locus gene
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179 Stein, J. C. a n d Nasrallah, J. B. 1993. A plant receptor-like gene, the S-locus receptor kinase of Brassica oleracea L., encodes a functional serine / threonine kinase. Plant Physiol. 101, 1103-1106. Stein, J. C., Howlett, B., Boyes, D. C., Nasrallah, M. E. a n d Nasrallah, J. B. 1991. Molecular cloning of a putative receptor protein kinase gene encoded at the self-incompatibility locus of Brassica oleracea. Proc. Natl. Acad. Sci. USA, 88, 8816-8820. Stein, J. C., Dixit, R., Nasrallah, M. E. and Nasrallah, J. B. 1996. SRK, the stigma-specific S locus receptor kinase of Brassica, is targeted to the p l a s m a m e m b r a n e in transgenic tobacco. Plant Cell 8, 429-445. Stephenson, A. G., Doughty, J., Dixon, S., Elleman, C., Hiscock, S. a n d Dickinson, H. G. 1997. The male d e t e r m i n a n t of self-incompatibility in Brassica oleracea is located in the pollen coating. Plant J. 12, 1351-1359. Stevens, J. P. a n d Kay, Q.O.N. 1988. The n u m b e r of loci controlling the sporophytic self-incompatibility system in Sinapis arvensis. Heredity
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180 Suzuki, G., Watanabe, M. and Hinata, K. 1997d. Molecular genetics of selfincompatibility in Brassica. Recent Res. Dev. Agri. Biol. Chem., 1, 235-242. Takasaki, T., H a t a k e y a m a , K., Ojima, K., Watanabe, M., Toriyama, K. and Hinata, K. 1998. Alteration of self-incompatibility phenotype by sense and antisense t r a n s g e n e of S-locus glycoprotein in Brassica rapa L. (in preparation). Takayama, S., Isogai, A., Tsukamoto, C., Ueda, Y., Hinata, K., Okazaki, K. a n d Suzuki, A. 1987. Sequences of S-glycoproteins, products of the Brassica campestris self-incompatibility locus. Nature 326, 102104. Takayama, S., Isogai, A., Tsukamoto, C., Shiozawa, H., Ueda, Y., Hinata, K., Okazaki, K., Koseki, K. a n d Suzuki, A. 1989. Structure of N-glycosidic saccharide chains in S-glycoproteins, products of S-genes associated with self-incompatibility in Brassica campestris. Agric. Biol. Chem. 53, 713-722. Tantikanj ana, T., Nasrallah, M. E., Stein, J. C., Chen, C-H. and Nasrallah, J. B. 1993. An alternative transcript of the S-locus glycoprotein gene in a class II pollen-recessive self-incompatibility haplotype of Brassica oleracea encodes a m e m b r a n e - a n c h o r e d protein. Plant Cell 5, 657-666. Tantikanjana, T., Nasrallah, M. E. a n d Nasrallah, J. B. 1996. The Brassica S gene family: molecular characterization of the SLR2 gene. Sex. Plant Reprod. 9, 1O- 116. Thompson, K. F. 1957. Self-incompatibility in n a r r o w - s t e m kale, Brassica oleracea var. acephala. I. demonstration of a sporophytic system. J. Genet. 55, 45-60. Thompson, K. F. a n d Taylor, J. P. 1966. Non-linear dominance relationships between S alleles. Heredity 2 1 , 3 4 5 - 3 6 2 . Thorsness, M. K., Kandasamy, M. K., Nasrallah, M. E. and Nasrallah, J. B. 1993. Genetic ablation of floral cells in Arabidopsis. Plant Cell. 5, 253-261. Tobias, C. M., Howlett, B. and Nasrallah, J. B. 1992. An Arabidopsis thaliana gene with sequence similarity to the S-locus receptor kinase of Brassica oleracea: Sequence and expression. Plant Physiol. 99, 284 -290. Torii, K. U., Mitsukawa, N., Oosumi, T., Matsuura, Y., Yokoyama, R., Whittier, R. F. and Komeda, Y. 1996. The Arabidopsis erecta gene encodes a putative receptor protein kinase with extracellular leucinerich repeat. Plant Cell 8, 735-746.
181 Toriyama, K., Stein, J. C., Nasrallah, M. E. and Nasrallah, J. B. 1991a. Transformation of B r a s s i c a oleracea with an S-locus gene from B. c a m p e s t r i s changes the self-incompatibility phenotype. Theor. Appl. Genet. 8 1 , 7 6 9 - 7 7 6 . Toriyama, K., Thorsness, M. K., Nasrallah, J. B. and Nasrallah, M. E. 1991 b. A B r a s s i c a S-locus gene promoter directs sporophytic expression in the a n t h e r t a p e t u m of transgenic Arabidopsis. Dev. Biol. 143, 427431. Toriyama, K., Hanaoka, K., Okada, T. and Watanabe, M. 1998. Molecular cloning of a cDNA encoding a pollen extracellular protein as a potential source of a pollen allergen in B r a s s i c a rapa. F E B S Lett. 424, 234-238. Trewavas, A. and Gilroy, S. 1991. Signal transduction in plant cells. Trend. Genet. 7, 356-361. Trick, M. 1990. Genomic sequence of a B r a s s i c a S-locus-related gene. Plant Mol. Biol. 15, 203-205. Trick, M. and Flavell, R. B. 1989. A homologous S genotype of B r a s s i c a oleracea expresses two S-like genes. Mol. Gen. Genet. 218, 112-117. Trick, M. and Heizmann, P. 1992. Sporophytic self-incompatibility systems: B r a s s i c a S gene family. Inter. Rev. Cytol. 140, 485524. Uyenoyama, M. K. 1988. On the evolution of genetic incompatibility systems: Incompatibility as a m e c h a n i s m for the regulation of outcrossing distance. In Michod, R. E. and Levin, B. R. (eds.) The evolution o f sex: A n e x a m i n a t i o n o f current ideas, pp 212-232, Sinauer, Sunderland, MA. Uyenoyama, M. K. 1989. On the evolution of genetic incompatibility systems. V. Origin of sporophytic self-incompatibility in response to overdominance in viability. Theor. Pop. Biol. 36:339-365. Uyenoyama, M. K. 1995. A general least-squares estimate for the origin of sporophytic self-incompatibility. Genetics 139, 975-992. Uyenoyama, M. K. 1997. Genealogical structure among alleles regulating self-incompatibility in natural populations of flowering plants. Genetics 147, 1389-1400. Van Engelen, F. A., Hartog, M. V., Thomas, T. L., Sturm, A., Van Kammen, A. and De Vries, S. C. 1993. The carrot secreted glycoprotein gene EP1 is expressed in the epidermis and has sequence homology to B r a s s i c a S-locus glycoproteins. Plant J. 4, 855- 862. Verma, S. C., Malik, R. and Dhir, I. 1977. Genetics of the incompatibility systems in the crucifer Eruca sativa. Proc. R. Soc. Lond. B., 196, 131159.
182 Visser, D. L., Hal, J. G. Van. and Verhoeven, W. 1982. Classification of Salleles by their activity in S-heterozygotes of Brussels sprouts (Brassica oleracea var. gemmifera (DC.) Schultz). Eupytica 3 1 , 6 0 3 611. Walker, J. C. 1992. Receptor-like protein kinase genes of Arabidopsis thaliana. Plant J. 3, 451-456. Walker, J. C. and Zhang, R. 1990. Relationship of a putative receptor protein kinase from maize to the S-locus glycoproteins of Brassica. Nature 345, 743-746. Wallace, D. H. 1979. Interactions of S-alleles in sporophytically contorolled self-incompatibility of Brassica. Theor. Appl. Genet. 54, 193-201. Wang, X., Zafian, P., Choudhary, M. and Lawton, M. 1996. The PR5K receptor protein kinase from Arabidopsis thaliana is structurally related to a family of plant defense proteins. Proc. Natl. Acad. Sci. USA, 93, 2598-2602. Watanabe, M., Shiozawa, H., Isogai, A., Suzuki, A., Takeuchi, T. and Hinata, K. 1991. Existence of S-glycoprotein-like proteins in anthers of self-incompatible Brassica species. Plant Cell Physiol. 32, 10391047. Watanabe, M., Nou, I. S., Takayama, S., Yamakawa, S., Isogai, A., Suzuki, A., Takeuchi, T. and Hinata, K. 1992. Variations in and inheritance of NS-glycoprotein in self-incompatible Brassica campestris L. Plant Cell Physiol. 33, 343-351. Watanabe, M., Takasaki, T., Toriyama, K., Yamakawa, S., Isogai, A., Suzuki, A. and Hinata, K. 1994. A high degree of homology exists between the protein encoded by SLG and the S receptor domain encoded by SRK in self-incompatible Brassica campestris L. Plant Cell. Physiol. 35, 1221-1229. Watanabe, M., Ono, T., Hatakeyama, K., Takayama, S., Isogai, A. and Hinata, K. 1997. Molecular characterization of SLG and S-related genes in a self-compatible Brassica campestris L. var. yellow sarson. Sex. Plant Reprod. 10, 332-340. Watanabe, M., Watanabe, M., Suzuki, G., Shiba, H., Takayama, S., Isogai, A. and Hinata, K. 1998. Seqeunce comparison of four SLR1 alleles in Brassica campestris (syn. B. rapa). Sex. Plant Reprod. 11,295-296. Wolters-Arts, M., Lush, W. M. and Mariani, C. 1998. Lipids are required for directional pollen-tube growth. Nature 392, 818-821. Yamakawa, S., Watanabe, M., Isogai, A., Takayama, S., Satoh, S., Hinata, K. and Suzuki, A. 1993. The cDNA sequence of NS3-glycoprotein from Brassica campestris and its homology to related proteins. Plant Cell Physiol. 34, 173-175.
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Biology of Brassica Coenospecies C. G6mez-Campo (Editor) 91999 Elsevier Science B.V. All rights reserved.
185
6 MALE STERILITY R6gine D e l o u r m e (1) a n d Fran~oise B u d a r (2)
(1) INRA, Station d'Am~lioration d e s Plantes, BP 29, 3 5 6 5 3 Le Rheu Cedex, France (2) INRA, Station de G~n~tique et d'Am~lioration d e s Plantes, Route de St-Cyr, 7 8 0 2 6 Versailles Cedex, France The value of h y b r i d s in the breeding of Brassica crops h a s s t i m u l a t e d interest in r e s e a r c h on male sterility. This h a s r e s u l t e d in a large a m o u n t of literature c o n c e r n i n g all a s p e c t s of male sterility (cytological a n d physiological analysis, m o l e c u l a r analysis, interspecific transfer). These s t u d i e s have d r a w n benefit from f e a t u r e s of the Brassicaceae family (some of which are covered in o t h e r c h a p t e r s of this publication) including the relative ease with which it c a n be m a n i p u l a t e d in a b r o a d r a n g e of genetic a n d biotechnological e x p e r i m e n t s , s u c h as i n t e r s p e c i f i c / i n t e r g e n e r i c crosses, in vitro culture, somatic hybridization, a n d genetic engineering. The fact t h a t m i t o c h o n d r i a l gen o m e s of Brassica are a m o n g the s m a l l e s t a n d the s i m p l e s t k n o w n a m o n g Angiosperms, h a s u n d o u b t e d l y facilitated m o l e c u l a r a n a l y s e s of cytoplasmic male sterility. Some good reviews on male sterility in Brassica have b e e n p u b l i s h e d in the p a s t y e a r s (Kaul, 1988; Shiga, 1980; Stiewe et al., 1995b). In this c h a p ter we will specifically focus on s t u d i e s on male sterility or its application in r e c e n t years.
Genic male sterility The first genic male sterility d e s c r i b e d in Brassica species w a s found in green s p r o u t i n g broccoli (Anstey a n d Moore, 1954). It h a s b e e n later described in m o s t of the cultivated b r a s s i c a s (Table 6.1) a n d in the majority of c a s e s it is i n h e r i t e d as a monogenic recessive c h a r a c t e r . Only four monogenic d o m i n a n t male sterilities have b e e n reported, two in B. oleracea (Dunem a n n a n d G r u n e w a l d t , 1991; Ruffio-Chable et al., 1993), one in B. rapa (Van der Meer, 1987) a n d one in B. n a p u s (Mathias, 1985a). A digenic male sterility s y s t e m h a s been identified in C h i n a (Li et al., 1988): one d o m i n a n t gene (Ms) i n d u c e s male sterility a n d male fertility c a n be r e s t o r e d by a n o t h e r
186 dominant gene (Rf). Other cases of genic male sterility reported in China are controlled by two or three recessive genes (Fu and Yang, 1995). The male sterile plants have mainly arisen as spontaneous mutants. However, one recessive male sterility in B. n a p u s (Takagi, 1970) and one dominant male sterility in B. o l e r a c e a (Dunemann and Grunewaldt, 1991) were obtained through mutagenic treatment. More recently, Plant Genetic Systems has developed a genic male sterility system by genetic engineering. Mariani et al. (1990) fused the tapetum-specific promoter T A 2 9 from tobacco (Goldberg, 1988) to the coding regions of two genes encoding RNases: barna s e from B a c i l l u s a m y l o l i q u e f a c i e n s and R N a s e T1 from A s p e r g i l l u s oryzae. Male sterile B. n a p u s plants were produced by transferring these chimaeric genes into the plant genome via A g r o b a c t e r i u m t u m e f a c i e n s . The male sterile plants with the b a r n a s e construction can be restored to fertility by crossing to restorer lines carrying the b a r s t a r gene, encoding a specific inhibitor for b a r n a s e (Mariani et al., 1992). The T A 2 9 - b a r n a s e construct was also introduced into B. oleracea var botrytis and male sterile transformants were obtained (Reynaerts et al., 1993). In most histologically described male sterilities, microsporogenesis breaks down at the tetrad stage and sometimes at the uninucleate vacuolate microspore stage (Table 6.1). These two stages of degeneration are observed in the d o m i n a n t male sterility of Mathias (1985a) and Ruffio-Chable et al. (1993) depending on the temperature conditions (Theis and R6bbelen, 1990). The digenic male sterility of Li et al. (1988) shows a specific degeneration process: the two meiotic divisions and the s u b s e q u e n t cytodieresis are disturbed (micro-nuclei formation) or stopped (Zhou, 1990). When male sterility is complete, the meiosis is completely stopped and the disturbed nuclear envelope does not perfectly separate the nuclear chromatin from the surrounding cytoplasm. In the engineered male sterility of Mariani et al. (1990), the disruption of microsporogenesis is correlated with the activity of the chimeric T A 2 9 - R n a s e or b a r n a s e genes (De Block and Debrouwer, 1993; Denis et al., 1993). Degeneration of the tapetal RNA takes place after microspore release from the tetrads and is immediately followed by the disappearance of the RNA from the microspores, after which they collapse. This confirms the intimate relationship of the tapetum and microspores during their development.
Cytoplasmic male sterility Many cytoplasmic male sterility (CMS) systems have been elaborated in the cultivated B r a s s i c a species. Table 6.2 summarizes the extensive research work that has been developed in this field mainly during the last 25 years. As cytoplasmic male sterility is the result of specific nuclear/mitochondrial interactions, the association of cytoplasm and nucleus from different species often results in total or partial male sterility, sometimes associated with other striking effects on floral morphology. In this section we will describe how the most popular CMS systems were obtained, their morphology, and
Table 6.1 Genic male sterilities in the Brassiceae (Cruciferae) Species
References
Inheritance
Origin
Anstey and Moore (1954); Cole (1959)
1 recessive gene ms-1
spontaneous
Dickson (1970)
1 recessive gene ms-6
spontaneous
Dunemann and Grunewaldt (1991)
1 dominant gene
mutagenic treatment
Nieuwhof (1961)
1 recessive gene ms-5
spontaneous
Borchers (1 966)
1 recessive gene ms-4
spontaneous
Chatterjee and Swarup (1972)
1 recessive gene
spontaneous
Ruffo-Chable et al. (1993)
1 dominant gene
spontaneous
Reynaerts er al. (1993)
1 dominant gene
genetic engineering
Nishi and Hiraoka (1958)
1 recessive gene
spontaneous
Rundfeldt (1960)
1 recessive gene
spontaneous
Johnson (1958)
1 recessive gene ms-2
spontaneous
Nieuwhof ( 1961; 1968)
1 recessive gene
spontaneous
Das and Pandey ( 1961)
1 recessive gene
spontaneous
Chowdhury and Das (1967; 1968)
recessive genes
spontaneous
Microsporogenesis breakdown stage
Brassica oleracea
var italica
var botrytis
var capitata
var gemmifera
after microspore release
tetrad - microspore *
tetrad
tetrad
Brassica rapa
brown sarson
tetrad tetrad / anther indehiscence
yellow sarson
Chowdhury and Das (1966; 1968)
1 recessive gene
spontaneous
var pekinensis
Van der Meer (1987)
1 dominant gene
spontaneous
Takahata ei al. (1996)
1 recessive gene
spontaneous
uninucleate microspore
Brassica juncea
Banga and Labana (1 983)
I recessive gene
spontaneous
vacuolate microspore
Brassica napus
Takagi (1 970)
2 recessive genes
mutagenic treatment
Heyn (1973)
2 recessive genes
spontaneous
Mathias (1985a)
1 dominant gene
spontaneous
Li et al. (1988)
digenic
spontaneous
Mariani ei a/. ( 1 990; 1992)
digenic
genetic engineering
Theis and Robbelen (1990)
1 recessive gene
N P Z system
vacuolate microspore
Raphanus salivus
Tokumasu (1951; 1957)
1 recessive gene
spontaneous
late prophase I of meiosis
* Ruffo-Chable, 1994
** Theis and Robbelen (1990)
*** Zhou,
**** Denis eial.. 1993
1990
tetrad
tetrad
**
tetrad or vacuolate microspore meiosis
**
***
vacuolate microspore
** ** **
189 nuclear restoration of fertility. We will go on to describe the recently acquired knowledge on the molecular basis of three of these systems. Establishment
of CMS
Most of these CMS s ys t em s have been obtained after transfer of the n u c l e u s of the studied species into the cytoplasm of an alien species, and t h u s result from alloplasmy. Only in a few cases, CMS h a s arisen spontaneously within the species as in Chinese cabbage (Okhawa and Shiga, 1981) a n d in rapeseed i.e. 'Polima' system (Fu, 1981) and ' S h a a n 2A' system (Li, 1986 in Fu an d Yang, 1995). The male sterility system first described by Ogura (1968) in an open pollinated radish cultivar h a s been recently reported to be widespread in wild J a p a n e s e populations of radish and in some specimens of wild R a p h a n u s raphanistrum (Yamagishi and Terachi, 1997). This probably corresponds to a s p o n t a n e o u s male sterility in this species. Another CMS system occurred s p o n t a n e o u s l y in a field trial of B. j u n c e a in India (Rawat and Anand, 1979) but, recently, molecular analyses of the cytoplasm showed t h a t this system probably results from s p o n t a n e o u s intergeneric hybridization between B. tournefortii and B. j u n c e a (Pradhan et al., 1991). In a few other cases, CMS h a s been obtained after intraspecific crosses. The first reports of CMS in rapeseed originated from intraspecific crosses using 'Bronowski' (Thompson, 1972) or 'Hokuriku 23' (Shiga and Baba, 1973) as male parents. This CMS is know n as the nap system. In the same way, O k h a w a (1984) identified a CMS in B. rapa close to this nap system. All the other reported CMS s y s t e m s result from interspecific or intergeneric crosses. In some cases they have been obtained by transferring a previously identified male sterility-inducing cytoplasm to the desired species. Thus, the male sterility-inducing cytoplasm of radish originally found by Ogura (1968) was successfully transferred to B. oleracea, B. n a p u s (Bannerot et al., 1974) a n d B. j u n c e a (Labana and Banga, 1989) and induced CMS in all these species. CMS p la n ts of B. n a p u s were also produced by crossing a male sterile radish line to B. n a p u s (Paulmann and R6bbelen, 1988). In radish, this male sterility was decribed as genic male sterility (Tokumasu, 1951). Its transfer to B. n a p u s resulted in cytoplasmic male sterility. Two h y p o t h e s e s might be drawn: either it was a non identified CMS in radish or the resulting CMS is due to the created alloplasmic situation. CMS s y s t e m s can also arise after crossing two male fertile species and can then be transferred to a n o t h e r species. The CMS obtained in B. oleracea with the cytoplasm of B. nigra (Pearson, 1972), in B. rapa with the cytoplasm of Diplotaxis muralis (Hinata and Konno, 1979) or in B. j u n c e a with the cytop l a s m s of B. tournefortii (Rawat and Anand, 1979) and Diplotaxis siifolia (Rao et al., 1994) were then transferred to B. n a p u s (Mathias, 1985b; Pellan-Delourme et al., 1987; Rao and Shivanna, 1996). These interspecific crosses were performed t h r o u g h sexual hybridization. More recently, protoplast fu-
190 sion between more or less related species was used by Kameya et al. (1989) in B. oleracea, Sakai and I m a m u r a (1990) in B. napus, Mukhopadhyay et al. (1994) in B. carinata, and by Kirti et al. (1995a) and Prakash et al. (1995, 1998a) in B. j u n c e a to acquire new CMS systems. Protoplast fusion was used by Yarrow et al. (1990) to transfer the 'Polima' cytoplasm from B. n a p u s to B. oleracea and by Cardi and Earle (1997) to transfer the B. tournefortii cytoplasm from B. rapa to B. oleracea. Protoplast fusion was also extensively used to induce new combinations of cytoplasmic traits. Male sterility has often been obtained by introducing the genome of one species into an alien cytoplasm and results from mitoc h o n d r i a / n u c l e u s interactions, but at the same time some defects may appear from chloroplast/nucleus or m i t o c h o n d r i a / n u c l e u s incompatibilities. When the male sterility-inducing cytoplasm of radish (Ogura, 1968) was introduced into B. oleracea, B. n a p u s , B. rapa and B. juncea, male sterile lines were obtained, b u t these showed a severe chlorophyll deficiency and low nectar secretion. This CMS system was first improved in B. n a p u s by protoplast fusion (Pelletier et al., 1983). Male sterile cybrids with normal photosynthesis and improved nectar secretion were obtained through chloroplast exchange and mitochondrial recombination. One of these cybrid cytoplasms was introduced to B. rapa and B. j u n c e a to produce male sterile lines (Delourme et al., 1994b). Then, similar experiments were performed on 'Ogura' CMS in B. n a p u s (Jarl and Bornman, 1988; Jarl et al. 1989; Menczel et al., 1987) as well as in B. oleracea (Kao et al., 1992; Pelletier et al., 1989; Waiters et al., 1992; Waiters and Earle, 1993) and in B. j u n c e a (Kirti et al., 1995a). Improved 'Ogura' cytoplasms were then transferred to vegetable B. rapa (Heath et al., 1994) and cabbage (Sigareva and Earle, 1997). Likewise, male sterile plants of B. rapa and B. j u n c e a carrying B. oxyrrhina cytoplasm have delayed flowering and chlorotic leaves (Prakash and Chopra, 1990). Chlorosis correction in B. j u n c e a was achieved through protoplast fusion (Kirti et al., 1993). Stiewe and R6bbelen (1994) and Liu et at. (1996) fused B. n a p u s and X-ray treated B. tournefortii protoplasts and obtained cybrids with B. n a p u s chloroplasts and B. tournefortii or recombined mitochondria. Development of improved CMS lines through protoplast fusion is also in progress in B. j u n c e a for B. tournefortii and B. oxyrrhina CMS (Pradhan et al., 1995). Protoplast fusion was also an efficient way of combining atrazine resistant chloroplasts of B. rapa with the CMS trait of B. nigra cytoplasm in B. oleracea (Christey et al., 1991) and with nap cytoplasm (Yarrow et al., 1986), 'Polima' cytoplasm (Barsby et al., 1987) or 'Ogura' cytoplasm (Jourdan et al., 1989) in B. napus.
Flower morphology and microsporogenesis Two main stages of microsporogenesis inhibition have been observed. For the nap, 'Polima' and tournefortii CMS, a premeiotic inhibition of microspore division and cell differentiation is already observed in the archespore
191 tissue when male sterility is complete (Theis and R6bbelen, 1990; Mishra and Anand, 1985; Gourret and Delourme, u n p u b l i s h e d results). In these systems, male sterile flowers are also characterized by narrow petals, and the stability of the male sterility is largely dependent on the environment or on maintainer lines. Fertile pollen grains may be produced at temperatures higher than 25-30~ in nap and 'Polima' systems (Burns et al., 1991; Fan and Stefansson, 1986) or at low temperatures depending on the maintainer lines in 'Polima' system (Fu et al., 1990). After protoplast fusion, Stiewe and R6bbelen (1994) obtained a cybrid line whose male sterility was more stable at high temperature than that of plants with the original B. tournefortii cytoplasm, but its petals were also narrower. For the B. oxyrrhina system, flowers have a normal appearance but short and u n d e h i s c e n t anthers; microsporogenesis breaks down after the tetrad stage (Prakash and Chopra, 1990). In the CMS system induced by 'Tokumasu' cytoplasm in B. n a p u s , degeneration also begins after the tetrad stage leading to empty exins and t a p e t u m walls in the a n t h e r s (Theis and R6bbelen, 1990). Gourret et al. (1992) compared the expression of 'Ogura' CMS in cybrids of B. n a p u s and in male sterile lines of B. n a p u s with the original 'Ogura' cytoplasm (from radish, R a p h a n u s sativus). Their results confirmed the observations made by Ogura (1968) in radish i.e. an excessive vacuolization of the tapetal cells leading to their degeneration prior to the sudden collapse of the microspores at the uninucleate stage. This expression of male sterility was attributed to the 'Ogura' male sterility already present in radish. In addition, reduction in n u m b e r and size of the microsporangia and feminization of the androecium (carpelloidy) were observed in the B. n a p u s plants with 'Ogura' cytoplasm and in some cybrids (Gourret et al., 1992). Polowick and Sawhney (1987; 1990; 1991) have shown that low or moderate temperature during bud development favors feminization of the androecium of B. n a p u s with 'Ogura' cytoplasm. This feminization was related to the expression of a second independent male sterility due to alloplasmy (Gourret et al., 1992). These two CMS determinants i.e. 'Ogura' male sterility and alloplasmic male sterility have been hypothetized after the study of the different cybrids produced (Pelletier et al., 1987; Primard et al., 1992). Other alloplasmic male sterilities also show an abortive pattern that includes feminization of the androecium e.g. Diplotaxis muralis CMS in B. n a p u s (PellanDelourme and Renard, 1987), B. tournefortii CMS in B. j u n c e a (Mishra and Anand, 1985) and in B. n a p u s (Gourret and Delourme, u n p u b l i s h e d data) or B. nigra CMS in B. n a p u s (Pellan-Delourme et al., 1987). In this latter CMS, petaloid s t a m e n s were also observed in cabbage B. oleracea (Pearson, 1972) and in rape B. n a p u s (Pellan-Delourme et al., 1987). Some male sterile plants with petaloid s t a m e n s were also produced in B. rapa by Mekinayon et al. (1994, 1995) with the cytoplasm of rocket (Eruca vesicaria subsp, sativa or in B r a s s i c a j u n c e a by Kirti et al. (1995b) with the cytoplasm of wild Trachystom a ballii.
192
Male fertility r e s t o r a t i o n The genetics of male fertility r e s t o r a t i o n is indicated in Table 6.2. When r e s t o r e r genes have b e e n identified, they always act as d o m i n a n t genes. The d e t e r m i n i s m is m o n o g e n i c or oligogenic according to the male sterility-inducing cytoplasm. For the nap CMS system, various n u m b e r s of r e s t o r e r genes have been found according to the cultivars s c r e e n e d (Rousselle a n d Renard, 1982; Shiga, 1976). The genetics of the 'Polima' s y s t e m is m u c h more simple since it is monogenic. Restorer genes have been found in a s u m m e r oilseed rape variety 'Italy', in the line 'UM2353' (Fang a n d McVetty, 1989) a n d in B. j u n c e a 'Zem' variety (Fan a n d S t e f a n s s o n , 1986). In C h i n a a n d India, several restoring lines were selected in B. n a p u s species (Banga a n d Gurjeet, 1994; Yang a n d Fu, 1990). F a n g a n d McVetty (1989) reported t h a t the two r e s t o r e r genes originating from 'Italy' a n d 'UM2353' were non allelic a n d unlinked. Recently, J e a n et al. (1997) identified DNA m a r k e r s linked to these two genes (named R f p l a n d Rfp2, respectively) a n d to a restorer gene of the nap s y s t e m (Rfn) (Jean, 1995). One of t h e s e m a r k e r s co-segregated perfectly with R f p l , Rfp2 a n d Rfn, indicating t h a t the three genes m u s t be at least tightly linked to one a n o t h e r a n d m a y reside at the s a m e locus. The b a s i s of the d i s c r e p a n c y between these r e s u l t s a n d those of McVetty et al. (1989) is still not clear. If the three genes reside at the s a m e locus, the allele of Rfn capable of restoring the nap c y t o p l a s m would be a m a i n t a i n e r allele for the 'Polima' CMS (Jean, 1995). For the B. tournefortii system, genotypes partially restoring male fertility have been found in B. nigra a n d B. rapa for male sterile lines of B. j u n c e a (Anand et al., 1986). These restorer genes have been b r o u g h t together in the B. j u n c e a g e n o m e a n d completely restoring g e n o t y p e s have been obtained (Angadi a n d A n a n d , 1988). In B. napus, genotypes partially restoring the male fertility of this CMS have been identified, s u c h as Asian genotypes 'Mokae' a n d 'Yudar (Delourme et al., u n p u b l i s h e d data) or a fodder winter variety 'Arvor' ( B a r t k o w i a k - B r o d a et al., 1991). The genetic d e t e r m i n i s m seems to be oligogenic. In the r e s t o r e r lines isolated by B a n g a et al. (1994), one or two r e s t o r e r genes are hypothezized. Sodhi et al. (1994) also identified restorer genes in 'Yudar as well as in a n o t h e r Asian variety 'Mangun', a n d concluded t h a t the i n h e r i t a n c e w a s monogenic. With these latter two genotypes, Stiewe et al. (1995a) o b t a i n e d restored p l a n t s with i n t e r m e d i a t e fertility a n d the det e r m i n i s m did not s e e m to be simple. These genotypes also partially restored the male sterile cybrid line developed by Stiewe a n d R6bbelen (1994). Stiewe et al. (1995a) a n d Pental et al. (1995) also a t t e m p t e d to t r a n s f e r restorer genes from B. tournefortii to B. n a p u s or to B. j u n c e a via B. tournefortii / B. rapa h y b r i d s or via somatic hybridization. Plants with nearly n o r m a l fertility were found in the BC 1 generation (Stiewe et al., 1995a).
193 For the 'Ogura' CMS, no restorer has been found in any of the Brassica species (Rousselle, 1982). Restorer genes were identified in E u r o p e a n radish varieties (Bonnet, 1975). They were introduced into rapeseed from a R a p h a nobrassica (Heyn, 1976). Segregation studies with the derived B. n a p u s restorers showed t h a t the original 'Ogura' cytoplasm needed several restorer genes to be restored to fertility b u t subsequently, protoplast fusion t h r o u g h mitochondrial recombination m ade it possible to simplify the genetics of the CMS system. Fully restored plants with only one d o m i n a n t restorer allele (Rfo) could be selected on the best cybrid cytoplasms (Pellan-Delourme, 1986; Pelletier et al., 1987). Those cybrids have been shown to bear the det e r m i n a n t inducing sterility in the original 'Ogura' radish, whereas a more complex genetic system for restoration is e n c o u n t e r e d w hen this 'Ogura' det e r m i n a n t is combined with the alloplasmic sterility due to the association of a B r a s s i c a n u c l e u s with radish type mitochondria (Pelletier et al., 1987; Prim a r d et al., 1992). The introduction of the Rfo gene into CMS lines of B. rapa and B. j u n c e a (with 'Ogura' cybrid cytoplasm) is in progress (Delourme et al., 1994b). An isozyme (Pgi-2) gene (Delourme and Eber, 1992) a n d DNA markers (Delourme et at., 1994a) were found to be perfectly linked to the Rfo gene. Such m a r k e r s can be us ed for m a r k e r assisted selection of restorer lines (Hansen et al., 1997). Recently the radish introgression carrying the Rfo gene was characterized (Delourme et al., 1998). This Rfo gene h a s been found to be widely distributed in the wild radish populations which the Ogura cytop l as m is s u p p o s e d to originate from (Yamagishi, 1998). In the other CMS B. n a p u s systems derived from radish, restorer gene(s) were also introduced from radish. P a u l m a n n and R6bbelen (1988) introduced restorer genes t h r o u g h intergeneric crosses for the 'Tokumasu' cytoplasm. Sakai et al. (1996) transferred a restorer gene by protoplast fusion for the 'Kosena' CMS system. More studies are needed to know w het her this restorer gene is the same. For m o s t of the other CMS systems, the genetics of restoration is u n k n o w n or no restorers are yet available, especially for those derived from wide interspecific or intergeneric crosses. Nevertheless, restorer genes could recently be introgressed into B. j u n c e a or B. n a p u s from the cytoplasm donor species i.e. T r a c h y s t o m a baltii (Kirti et al., 1997), Moricandia arvensis (Prak a s h et al., 1998b) and B. oxyrrhina (Banga and Banga, 1998). Molecular features of some cytoplasmic
male sterilities
The 'O~ura' a n d 'Kosena' systems. Although the mitochondrial genome of the 'Ogura' radish has been extensively analysed and compared to the normal fertile radish one (Makaroff et al., 1989; 1990; 1991; Makaroff and Palmer, 1988) the identification of the mitochondrial d e t e r m i n a n t for this CMS has been possible only after Brassica cybrids were obtained via the protoplast fusion experiments described above (Pelletier et al., 1983).
Table 6.2 Cytoplasmic male sterilities in the Brussicaceue Transfer method
Fertility restoration
B. rapa
lnterspecific cross
2 dominant genes
Pearson (1972) Christey et a/. (1991)
B nigra
lnterspecific cross Protoplast fusion
2 dominant genes
Chiang and Crete ( 1 987)
B. napus
lnterspecific cross
unknown **
Species
References
Brassica aleracea
Nishi and Hiraoka (1958)
Yarrow et a/. (1 990) McCollum (1981) Bannerot et al. (1974) Pelletier ef al. (1989) Kao el 01. (1992) Walters et al. (1992) Kameya ef al. (1989) Brassica rapa
B.napus (Polima) R.sativus ('Early Scarlet Globe') R. sofivus (Ogura)
R. safivus ('Shougoin')
-*
Protoplast fusion Intergeneric cross Intergeneric cross Protoplast fusion
unknown no restorer
Protoplast fusion
Okhawa and Shiga (1981)
B. napus
spontaneous
2 dominant genes
Okhawa (1984)
Brapa '14'
lntraspecific cross
unknown
B. oxyrrhina
Interspecific cross
Prakash and Chopra ( 1990) Delourme et a/. (1994)
Brassica juncea
Cytoplasm origin
modified 'Ogura' in B. napus
lnterspecific cross
1 restorer gene unknown
Hinata and Konno (1979)
Diplofaris muralis
Intergeneric cross
Mekiyanon eta/. (1994; 1995)
Eruca sativa
Intergeneric cross
Rawat and h a n d (1979) h a n d et al. (1986)
B. fournefortii
Alloplasmy
Prakash and Chopra (1990) Kirti ef a/. ( I 993)
B. oxyrrhina
lnterspecific cross Protoplast fusion
Labana and Banga (1989) Kirti et al. (1995a) Delourme et al. (1994)
R. sativus (Ogura)
Intergeneric cross Protoplast fusion Interspecific cross
no restorer
Intergeneric cross
unkonwn
Rao et al. (1994)
modified 'Ogura' in B. napus Diplotaxis siifolia
4 dominant genes
I restorer gene
Brassica juncea
Brassica napus
Kirti et al. (1995b)
Trachystoma ballii
Protoplast fusion
no restorer
Prakash et al. (1995)
Moricandia arvensis
Protoplast fusion
no restorer
Prakash et al. (1995)
Diplotaxis catholica
Protoplast fusion
no restorer
Prakash et al. (1995)
Sinapis alba
Protoplast fusion
no restorer
Thompson (1 972) Rousselle and Renard (1982) Shiga and Baba(1973); Shiga(1976)
B. napus
Intraspecific cross
1 to 2 genes
Fu (1981); Yang and Fu (1990) Barshy et al. (1987)
Polima
spontaneous Protoplast fusion
1 dominant gene
Li(1986)inFuandYang(1995)
Shaan 2A
spontaneous
unknown
Pellan-Delourme et al. (1987)
B.nigra
Interspecific cross
unknown
Mathias (1985b) Battkowiak-Broda et al. (1991) Sodhi et al. (1 994) Stiewe and Robbelen (1994) Liu et al. ( I 996)
B.tournefortii
Interspecific cross
Bannerot et al. (1974) Pelletier el al. (1983; 1987) Jar1 et al. ( 1988) Jourdan et al. (1989) Paulmann and Rdbbelen (1988) Sakai and Imamura (1990); Sakai et al. (1 996)
Brassica carinata
1 to 4 genes
oligogenic I dominant gene Protoplast fusion Protoplast fusion R.sativus (Ogura)
Intergeneric cross Protoplast fusion
oligogenic 1 dominant gene
R.sativus (Tokumasu) R.sativus (Kosena)
Intergeneric cross Protoplast fusion
unknown 1 dominant gene
Pellan-Delourme and Renard (1987)
Diplotaxis muralis
Intergeneric cross
unknown
Rao and Shivanna (1996)
Diplotaxis siijolia
Interspecific cross
no restorer
h a n d (1987)
B. tournefortii
Interspecific cross Protoplast fusion
unknown unknown
Rmtivus (Ogura)
spontaneous
1 dominant gene
Mukhopadhyay et al. (1994) Raphanus sativus
Ogura (1968)
* not studied
** restorer genes have been identified but the genetics was not determined
196 These fusion e x p e r i m e n t s not only gave the B. n a p u s a n d B. oleracea cytotypes now m o s t extensively u s e d for the c o m m e r c i a l p r o d u c t i o n of hybrid seeds, b u t also offered the best m a t e r i a l for the m o l e c u l a r c h a r a c t e r i z a t i o n of the sterility d e t e r m i n a n t . B o n h o m m e et al. (1991; 1992) identified a mitoc h o n d r i a l gene, orf138, originally found in the 'Ogura' r a d i s h mitochondrial genome, a n d w h o s e e x p r e s s i o n at the RNA level is strictly correlated with the sterile p h e n o t y p e of B. n a p u s a n d B. oleracea cybrids (in the a b s e n c e of a n y r e s t o r e r gene). E x p r e s s i o n of this m i t o c h o n d r i a l gene h a s since been associated with the sterile p h e n o t y p e of 'Ogura' r a d i s h ( K r i s h n a s a m y and Makaroff, 1993). The orf138 gene is e x p r e s s e d in r a d i s h a n d m o s t B. napus cybrids on a bicistronic RNA also encoding orfB. The orf138 gene p r o d u c t is a m e m b r a n e b o u n d protein which is p r o b a b l y a s s o c i a t e d in oligomers in the m i t o c h o n d r i a l m e m b r a n e (Grelon et al., 1994; K r i s h n a s a m y a n d Makaroff, 1994). The p l a n t s carrying the orf138 gene in their m i t o c h o n d r i a l genome exhibit the O R F 1 3 8 protein in all the organs. However, in male-sterile radish, the O R F 1 3 8 protein h a s been r e p o r t e d to be highly a c c u m u l a t e d in roots relative to the a l f a - s u b u n i t of m i t o c h o n d r i a l ATPase ( K r i s h n a s a m y a n d Makaroff, 1994) w h e r e a s in B. n a p u s cybrids, its a c c u m u l a t i o n seems to follow the a b u n d a n c e of n o r m a l m i t o c h o n d r i a l proteins in the different o r g a n s of the p l a n t (Bellaoui et al., in preparation). The r e s t o r a t i o n of fertility by n u c l e a r r e s t o r e r s h a s a d r a m a t i c effect on the a c c u m u l a t i o n of the ORF138 protein in b u d s a n d leaves in r a d i s h ( K r i s h n a s a m y a n d Makaroff, 1994). In B. napus, r e s t o r a t i o n is a c c o m p a n i e d with the abolition of ORF138 a c c u m u lation in a n t h e r s (Bellaoui et al., in preparation). In both species, the restoration of fertility does not affect at all the level of a c c u m u l a t i o n of the mitochondrial m e s s e n g e r RNA of the orf138 gene, indicating t h a t the m e c h a n i s m of r e s t o r a t i o n is acting either on the t r a n s l a t i o n efficiency of the m e s s e n g e r or on the stability of the ORF138 protein ( K r i s n a s a m y a n d Makaroff, 1994; Bellaoui et al., in preparation). Most if not all of m i t o c h o n d r i a l male sterility a s s o c i a t e d genes described so far p r e s e n t a c h i m a e r i c s t r u c t u r e where p a r t s of n o r m a l mitochondrial genes c a n still be recognized, strongly suggesting t h a t they occured by genetic r e c o m b i n a t i o n inside the m i t o c h o n d r i a l genome, even if some of their s e q u e n c e is of u n k n o w n origin (Braun et al., 1992; H a n s o n , 1991). The orf 138 gene h a s long been entirely of " u n k n o w n origin", since its sequence does not significantly r e s e m b l e a n y t h i n g in the d a t a b a s e s (Bonhomme et al., 1992). The complete s e q u e n c i n g of the m i t o c h o n d r i a l genome of Arabidopsis thaliana recently allowed the identification of the end of the coding sequence a n d 3' flanking region of the orf138 gene as identical (70 nucleotides including the last 12 codons) to the 3' u n t r a n s l a t e d region of Arabidopsis thaliana orf557 p r o b a b l y encoding an NADH d e h y d r o g e n a s e s u b u n i t (Bellaoui et al., 1998). Recently, the B. n a p u s orf577 gene, homologous to the bacterial ccll gene h a s been described by H a n d a et al. (1996) a n d M e n a s s a et al., (1997). This gene is very similar to the A. thaliana orf557 a n d also p r e s e n t s in its 3' u n t r a n s l a t e d region the s a m e s h o r t a n d perfect homology to orf138. So the orf138 gene m i g h t also be a r e s u l t of m i t o c h o n d r i a l genetic recombination.
197 The O R F 1 3 8 protein p o s s e s s e s a h y d r o p h o b i c N-terminal region, probably c o m p r i s i n g a m e m b r a n e s p a n n i n g domain, a n d a highly hydrophilic Ct e r m i n a l region s t r u c t u r e d in three repetitions of 13 a m i n o - a c i d s . The 'Kosena' cultivar of radish, as well as some wild s a m p l e s exhibiting O g u r a - t y p e m i t o c h o n d r i a , p o s s e s s a deleted orf138 gene where one repetition of the hydrophilic region of the protein h a s been lost (Sakai et al., 1995; Yamagishi a n d Terachi, 1996). However, it is not clearly e s t a b l i s h e d w h e t h e r this t r u n c a t e d ORF138 is responsible for the male sterility p h e n o t y p e in the 'Kosena' cultivar. The 'Polima' system. The pol m i t o c h o n d r i a l genome shows some r e a r r a n g e m e n t s c o m p a r e d to the n o r m a l B. napus (nap) one (Handa a n d Nakajima, 1992; Singh a n d Brown, 1991; Witt et al., 1991). Differences in the t r a n s c r i p t p a t t e r n of the atp6 gene b e t w e e n sterile, fertile a n d r e s t o r e d p l a n t s were detected a n d directed i n t e r e s t t o w a r d s this region of the genome as the possible determ i n i n g locus of 'Polima' sterility. In the pol m i t o c h o n d r i a l genome, the atp6 gene is c o t r a n s c r i b e d with (and d o w n s t r e a m of) a new gene c a p a b l e of encoding a 224 a m i n o a c i d protein, orf224. The first 58 codons of the orf224 gene are highly similar to the a m i n o - t e r m i n a l coding region of the orfB gene, followed by 43 bp of homology with the last exon 1 of rps3, the r e s t of the seq u e n c e r e m a i n i n g of u n k n o w n origin. Therefore, this C M S - a s s o c i a t e d gene h a s probably been g e n e r a t e d by m i t o c h o n d r i a l genetic r e c o m b i n a t i o n s . The putative protein p r o d u c t of the orf224 gene h a s not been detected in sterile p l a n t s so far. In the p r o g e n y of crosses segregating sterile a n d r e s t o r e d p l a n t s with the pol cytoplasm, the bi-cistronic t r a n s c r i p t orf224-atp6 h a s b e e n correlated with the sterile p h e n o t y p e of the plants, w h e r e a s in fertile r e s t o r e d plants, a m o n o c i s t r o n i c atp6 t r a n s c r i p t p r o d u c e d by RNA p r o c e s s i n g is a c c u m u l a t e d . Singh et al. (1996} have elegantly d e m o n s t r a t e d t h a t the r e s t o r e r gene of 'Polima' c y t o p l a s m is a n allele of a locus influencing RNA p r o c e s s i n g of some m i t o c h o n d r i a l t r a n s c r i p t s in B. napus.It Mapping e x p e r i m e n t s on the n o r m a l B. napus c y t o p l a s m s u g g e s t e d t h a t s e q u e n c e s similar to orf224 are also pres e n t in these m i t o c h o n d r i a , b u t not a s s o c i a t e d with the atp6 gene (L'Homme a n d Brown, 1993}. Hence, the e m e r g e n c e of the sterile p h e n o t y p e could be d u e to the a s s o c i a t i o n of the orf224 coding s e q u e n c e with e x p r e s s i o n signals of the atp6 gene Recently, the e x p r e s s i o n of a new gene, orf522, a s s o c i a t e d with the nad 5c region a n d potentially encoding a peptide which s h a r e s 79% s e q u e n c e similarity to the predicted p r o d u c t of orf224 h a s b e e n a s s o c i a t e d with nap CMS in B. napus (L'Homme et al., 1997). Even more interesting, it a p p e a r s t h a t the locus previously a s s o c i a t e d with 'Polima' fertility r e s t o r a t i o n a n d involved in m i t o c h o n d r i a l t r a n s c r i p t m a t u r a t i o n is also allelic to the r e s t o r e r for nap CMS (Li et al., 1998}. Hence, this is the first e x a m p l e of two CMS
198 s y s t e m s being r e s t o r e d by two different alleles of the s a m e locus a n d associated with m i t o c h o n d r i a l genes which are probably evolutionarily related. The B. tournefortii system. The m o l e c u l a r b a s i s of this s y s t e m h a s been explored by a survey of the e x p r e s s i o n of m i t o c h o n d r i a l genes in p l a n t s with the s a m e B. tournefortii c y t o p l a s m b u t in different n u c l e a r b a c k g r o u n d s , where the sterility is either e x p r e s s e d (B. juncea, B. napus) or not (B. tournefortii, B. napus restored). L a n d g r e n et al. (1996) t h u s showed t h a t the e x p r e s s i o n of the mitochondrial B. tournefortii g e n o m e is extensively affected by the n u c l e a r b a c k g r o u n d . They have correlated alterations of the atp6 t r a n s c r i p t p a t t e r n with the e x p r e s s i o n of the male sterile p h e n o t y p e in the plants, the B. napus a n d B. juncea sterile alloplasmic lines exhibiting a larger RNA molecule revealed with atp6 probe in addition to the n o r m a l m o n o c i s t r o n i c atp6 t r a n s c r i p t found in all the plants. This larger RNA is actually a bi-cistronic m e s s e n g e r which carries a n open reading frame, orf263, d o w n s t r e a m of atp6. The orf 263 gene is, therefore, p r e s e n t b u t not e x p r e s s e d in n o r m a l B. tournefortii plants. It potentially e n c o d e s a 2 9 k D a protein. About one third of the orf263 s e q u e n c e s h o w s homology to a p a r t of the nad5 gene. T h u s , orf263 also s e e m s to be a chimaeric m i t o c h o n d r i a l gene. In organello s y n t h e s e s of proteins revealed a 3 2 k D a polypeptide w h o s e expression s e e m s to be correlated with the sterile p h e n o t y p e of the plants. However, no evidence h a s yet been o b t a i n e d t h a t this polypeptide is indeed the p r o d u c t of the orf263 gene. Analysis of r e s t o r e d B. napus progeny segregating male sterile a n d male fertile p l a n t s showed t h a t the p r e s e n c e of the 32 k D a protein in in organello s y n t h e s e s is correlated with the sterile phenotype, b u t the bicistronic RNA atp6-orf263 is p r e s e n t in all the progeny (Landgren et al., 1996). F u r t h e r e x p e r i m e n t s are now n e c e s s a r y to e n s u r e t h a t the 3 2 k D a protein is the protein e n c o d e d by the orf263 gene. In t h a t case, the r e s t o r a t i o n of fertility in the s t u d i e d B. napus progeny would be effective by a control on protein accum u l a t i o n w i t h o u t affecting the RNA level, which is r e m i n i s c e n t of the situation in the 'Ogura' system. However, one can a s s u m e t h a t n o r m a l B. tournefortii p l a n t s do not exhibit sterility b e c a u s e they can efficiently process the bicistronic m e s s e n g e r atp6-orf263 a n d only a c c u m u l a t e the monocistronic atp6 RNA. In this case, we can predict t h a t B. tournefortii p o s s e s s e s restorer gene(s) which act on the p r o c e s s i n g of the atp6 m e s s e n g e r , r e m i n i s c e n t of the s i t u a t i o n in the 'Polima' system.
Use for t h e p r o d u c t i o n o f c o m m e r c i a l hybrids As we have seen above, a large n u m b e r of male sterility s y s t e m s have been p r o p o s e d for the p r o d u c t i o n of hybrid Brassica seeds. However, m a n y of these s y s t e m s have been s h o w n to p o s s e s s i n h e r e n t d i s a d v a n t a g e s when u s e d in breeding p r o g r a m s .
199
Genic male sterility The u s e of genic male sterility is limited b e c a u s e in m a n y c a s e s the male sterility p h e n o t y p e is u n s t a b l e a n d b e c a u s e male fertile p l a n t s m u s t be d i s c a r d e d j u s t before flowering in h y b r i d seed p r o d u c t i o n fields. However, in B. oleracea var botrytis, the selected male sterile p l a n t s c a n be vegetatively p r o p a g a t e d (in vivo or in vitro). T h u s , it h a s b e e n possible to u s e recessive male sterility for the c o m m e r c i a l p r o d u c t i o n of F1 h y b r i d s a n d a d o m i n a n t male sterility is now u s e d in the s a m e way (Ruffio-Chable, 1994). The digenic male sterility identified by Li et al. (1988) h a s been applied in C h i n a to p r o d u c e 100% male fertile F1 h y b r i d r a p e s e e d varieties from a 100% male sterile line (Figure 6.1). This implied, however, the m a n u a l elimin a t i o n of the male fertile p l a n t s in the female strips d u r i n g m a i n t e n a n c e a n d multiplication of the A line. These h y b r i d varieties have b e e n cultivated on 4 0 , 0 0 0 h a over the last five y e a r s (Li S.L., pers. comm.). O t h e r h y b r i d s b a s e d on different genic male sterilities are registered a n d u s e d for p r o d u c t i o n in C h i n a (Fu a n d Yang, 1995). In the e n g i n e e r e d digenic male sterility of Mariani et al. (1990; 1992), a herbicide r e s i s t a n c e gene is linked to the Ms gene. The male sterile p l a n t s c a n t h e n be selected in a l t e r n a t i n g strip seed p r o d u c t i o n fields by s p r a y i n g the herbicide. Elite Ms a n d Rf alleles have been selected in s u m m e r B. n a p u s after trials p e r f o r m e d in different locations a n d y e a r s a n d have been t r a n s ferred to w i n t e r B. n a p u s a n d also to B. rapa a n d B. j u n c e a (De Both, 1995). Two s u m m e r B. n a p u s F1 h y b r i d s were a c c e p t e d for r e g i s t r a t i o n a n d commercial scale p r o d u c t i o n in F e b r u a r y 1996 in C a n a d a (Joos, 1997). Breeding w i n t e r B. n a p u s a n d B. j u n c e a F1 h y b r i d s is in progress. The d e v e l o p m e n t of s u c h t r a n s g e n i c varieties will d e p e n d on their r e g u l a t o r y approval.
Cytoplasmic male sterility The a c h i e v e m e n t of a n effective CMS s y s t e m h a s often been slowed down or i m p e d e d by various difficulties s u c h as the instability of the male sterility, the a b s e n c e of one of the c o m p o n e n t s of the s y s t e m i.e. the a b s e n c e of m a i n t a i n e r or r e s t o r e r lines, or negative effects of the male sterility-inducing cytoplasm. Instability of male sterility e x p r e s s i o n u n d e r v a r i o u s e n v i r o n m e n t a l conditions is the r e a s o n w h y the nap s y s t e m is not u s e d in B. n a p u s a n d w h y m u c h work h a d to be done on the 'Polima' s y s t e m to obtain good m a i n t a i n e r genotypes. The selection of very few m a i n t a i n e r lines could be achieved by s c r e e n i n g a great n u m b e r of lines in different e n v i r o n m e n t a l conditions ( B a r t k o w i a k - B r o d a et al., 1991) or u s i n g haplodiploidization (BartkowiakB r o d a et al., 1995). In some s y s t e m s s u c h as B. nigra CMS or D. muralis CMS in B. napus, the a b s e n c e or the too rare o c c u r r e n c e of m a i n t a i n e r lines have
200 p r e v e n t e d their u s e for F1 h y b r i d breeding (Pellan-Delourme et al., 1987; P e l l a n - D e l o u r m e a n d Renard, 1987). The male sterility-inducing c y t o p l a s m m a y have negative effects on chlorophyll content, n e c t a r secretion, flower morphology a n d yield. For the 'Ogura' s y s t e m , the chlorophyll deficiency was corrected by obtaining cybrid cytop l a s m s in B. n a p u s a n d B. oleracea. Male sterile cybrid r a p e s e e d plants also s h o w e d improved n e c t a r secretion (Mesquida et al., 1991). T h u s , high yielding male sterile lines c a n be u s e d efficiently in F1 hybrid seed production (Renard et al., 1991, 1992). In the B. tournefortii CMS system, the cybrids p r o d u c e d in B. n a p u s m a y also be useful to correct the light-green color of the leaves of the male sterile p l a n t s with the original B. tournefortii cytoplasm. B. tournefortii c y t o p l a s m h a s been s h o w n to i n d u c e negative effects on some yield c o m p o n e n t s in B. j u n c e a b u t heterotic c o m b i n a t i o n s can be found (Sheikh a n d Singh, 1996). The 'Polima' c y t o p l a s m was also reported to i n d u c e yield p e n a l t y in B. n a p u s (McVetty et al., 1990). Nevertheless, it is possible to get h y b r i d s which exhibit a superior p e r f o r m a n c e c o m p a r e d to the c o n v e n t i o n a l varieties ( B a r t k o w i a k - B r o d a et al., 1995; McVetty et al., 1990). Some male sterile p l a n t s have flowers with small a n d n a r r o w petals e . g . B , n a p u s p l a n t s with 'Polima' or B. tournefortii cytoplasm. This h a s to be t a k e n into c o n s i d e r a t i o n for pollination efficiency since these floral morphology c h a n g e s greatly increase the n u m b e r of "sideworking" bees. Studies carried o u t by McVetty et al. (1989) revealed t h a t this did not lead to a decrease of yield on the male sterile lines b u t this r e s u l t could be due to the design u s e d (alternating A- a n d B-lines) a n d to the flooding of the trials with leaf c u t t e r bees, e n s u r i n g n u m e r o u s visits to each flower. However, these floral morphology c h a n g e s m a k e pollination more u n c e r t a i n in the seed production d e s i g n s generally used, which d e c r e a s e s F1 hybrid seed yield. This led McVetty et al. (1995) to propose a 3A:3R row ratio design a n d 50,000 leaf c u t t e r b e e s . h a -1 for efficient F1 h y b r i d seed p r o d u c t i o n in W e s t e r n Canada. The last r e q u i r e m e n t which s h o u l d be met for oilseed B r a s s i c a crops is the availability of efficient restorer lines. As decribed above, restorer genes have been identified in B. n a p u s for 'Polima' c y t o p l a s m a n d can be easily i n t r o d u c e d in elite lines b e c a u s e of a monogenic inheritance. For B. tournefortii CMS, the genetic d e t e r m i n i s m is not so clear b u t it s e e m s t h a t good r e s t o r e r lines of B. j u n c e a a n d B. n a p u s could be selected (Angadi a n d Anand, 1988; B a n g a et al., 1995). The restoration b r o u g h t by the genotypes 'Mangun', 'Mokae' or 'Yudal' is not complete a n d its t r a n s f e r in double low genetic b a c k g r o u n d s is not very easy. The transfer of a r e s t o r e r gene from B. tournefortii to B. n a p u s is in p r o g r e s s a n d the crosses are being followed by RAPD a n a l y s i s to find selectable m a r k e r s for r e s t o r a t i o n a n d to identify p l a n t s c o n t a i n i n g the m i n i m u m a m o u n t of T genome b a c k g r o u n d (Stiewe et al., 1995a). In 'Ogura' CMS, one d o m i n a n t r e s t o r e r gene is available for the cybrids selected by Pelletier et al. (1983). However, the i n t r o d u c t i o n of this restorer gene from r a d i s h to r a p e s e e d was a c c o m p a n i e d by a d e c r e a s e in seed set
201
Step 1"
Maintenance of the A line
A
I ~s~s~ 50% F
A
I
I ~s~s~, I
I MsMsRfrf I 50% F
50% S
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discarded
MsMsrfrf I
Ms MsRfrf I 50% F
Step 2:
50% S
Multiplication of the A line
A
A
B
msmsffff I 50% F
50% S
discarded
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Step 3:
Production of F1 hybrid seeds A
R
[ Msms rfrf I
X
[ ms ms RfRfl
lOO% s
/
lOO% F
F1 hybrid Ms ms Rf rf ms ms Rf rf 100% F
Figure 6.1 Seed multiplication scheme using a digenic male sterility system.
50% S
202 (Pellan-Delourme and Renard, 1988). It was a s s u m e d t hat restored plants had retained too m u c h radish genetic information a r o u n d the restorer gene or elsewhere in the genome. Improvement of the restorer material was achieved t h r o u g h b ackcr os s and pedigree breeding a n d restorer lines with good female fertility have been obtained (Delourme et al., 1991). However, the length of the introgressed radish segment and the fact t h a t a linkage was found between this introgression and glucosinolate content have slowed down the use of these restorer lines in double low F1 hybrid breeding (Delourme et al., 1995). This led the breeders to propose varietal associations which do not use male fertility restoration (Renard et al., 1992). Recently, double low restored F1 hybrids were obtained (Renard et al., 1998). For m o s t of the other CMS systems, no restorers are yet available. Thus, until now, commercial F1 hybrid production based on CMS has been achieved in B. oleracea us i ng the improved 'Ogura' cytoplasm obtained by Pelletier et al. (1989). F1 hybrids of various B. oleracea types (cauliflower, s a u e r k r a u t cabbage, garden cabbage and Savoy cabbage) have recently been registered (Leviel, 1998). Other improved 'Ogura' cytoplasms are available in B. oleracea (broccoli, cauliflower and cabbage) as well as in vegetable B. rapa (Chinese cabbage and pak choi) and are being tested for use in commercial hybrid production in seed companies worldwide (Earle and Dickson, 1995). In B. n a p u s , s u m m e r F1 hybrid varieties have been registered in Canada (Downey, 1994), in France (Pinochet, 1995), in Australia, in China and in India using the 'Polima' system. In China, the most cultivated F1 hybrid 'Qinyou 2' was developed with the 'Shaan 2A' CMS system. A hybrid based on B. tournefortii CMS was released for general cultivation in Punjab during 1994 (Banga et al., 1995). Hybrid and line composite varieties of winter and spring B. n a p u s based on the improved 'Ogura' CMS (named Ogu-INRA CMS) have been registered and cultivated in Europe since 1994 and, more recently, restored F1 hybrids have been also registered (Renard et al., 1998). In B. j u n c e a , development of the first hybrid on a commercial scale was achieved in India with B. tournefortii CMS (Angadi and Anand, 1988). In the years to come, the n u m b e r of commercial hybrid varieties in B r a s s i c a crops will certainly increase significantly due to the intensive work made in this field all a r o u n d the world. New systems are always appearing, like the rapeseed F1 hybrids recently registered in Europe, which are based on a system ('NPZ' system) developed in a seed company. Development of new engineered male sterilities are also in prospect.
Aknowledgements We t h a n k F. Eber, M. Renard, V. Ruffio and I. Small for helpful criticism of the m a n u s c r i p t . We are grateful to B.S. Landry and T. Sakai for providing their m a n u s c r i p t s prior to publication.
203
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212 Pelletier, G., Primard, C., Vedel, F., Chetrit, P., Renard, M.-D., R and Mesquida, J.1987. Molecular, phenotypic and genetic characterization of mitochondrial recombinants in rapeseed. Proc. 7th Int. Rapeseed Conference, Poznan, Poland, pp. 113-118. Pelletier, G., Ferrault, M., Lancelin, D. and Boulidard, L. 1989. CMS Brassica oleracea cybrids and their potential for hybrid seed production. 12th Eucarpia Congress, G~ttingen 11(7), 15. Pental, D., Pradhan, A. K., S o d h i , Y. S., Arumugam, M. and Mukhopadhyay, A. 1995. Heterosis breeding in m u s t a r d (Brassica juncea) and rapeseed (B. napus) by a combination of molecular and conventional methods. Proc. 9th Int. Rapeseed Congress, Cambridge, UK, pp. 122-124. Pinochet, X. 1995. Arriv~e de materiel de types hybrides en France. GCIRC Bulletin. 11, 32-37. Polowick, P. L. and Sawhney, V. K. 1987. A scanning electron microscopic study on the influence of temperature on the expression of cytoplasmic male sterility in Brassica napus. Can. J. Bot. 65, 807-814. Polowick, P. L. and Sawhney, V. K. 1990. Microsporogenesis in a normal line and in the ogu cytoplasmic male-sterile line of Brassica napus. I The influence of high temperature. Sex Plant Reprod. 3, 263-276. Polowick, P. L. and Sawhney, V. K. 1991. Microsporogenesis in a normal line and the ogu cytoplasmic male-sterile line of Brassica napus. II. The influence of intermediate and low temperatures. Sex Plant Reprod. 4, 22-27. Pr~adhan, A. K., Mukhopadhyay, A. and Pental, D. 1991. Identification of putative cytoplasmic donor of CMS system in Brassica juncea. Plant Breeding 106, 204-208. Pradhan, A. K, Arumugam, N., Mukhopadhyay, A., Gupta, B. S, Yadav, J. K, Verma, J. K. and Pental, D. 1995. Development of improved cytoplasmic male sterile lines in Brassica through somatic cell hybridization. In: Proc. 9th Int. Rapeseed Congress, Cambridge, UK, pp. 52-54. Prakash, S. and Chopra, V. L. 1990. Male sterility caused by cytoplasm of Brassica oxyrrhina in B. campestris and B. juncea. Theor. Appl. Genet. 79, 285-287. Prakash, S., Kirti, P. B. and Chopra, V. L. 1995. Cytoplasmic male sterility (CMS) systems other than ogu and Polima in Brassicae: current status. Proc. 9th Int. Rapeseed Congress, Cambridge, UK, pp. 44-48. Prakash, S., Kirti, P. B. and Chopra, V. L. 1998a. Development of cytoplasmic male sterility fertility restoration systems of variable origin in m u s t a r d - Brassica juncea. In: Thomas, G. and Monteiro, A. (eds.)
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Brassica 97, International symposium on Brassicas, Rennes, Acta Hort. 4 5 9 , 299-304. Prakash, S., Kirti, P. B., Bhat, S. R., Gaikwad, K., Kumar, V. D. a n d Chopra, V. L. 1998b. A Moricandia arvensis- based cytoplasmic male sterility a n d fertility restoration system in Brassica juncea. Theor. Appl. Genet. 97, 488-492. Primard, C., Delourme, R., Eber, F., Lancelin, D. a n d Pelletier, G. 1992. Identification of three types of CMS after somatic fusion between a fertile a n d a CMS B. napus. XIIIth EUCARPIA Congress, Angers, France, pp. 113-114. Rao, G. U. a n d Shivanna, K.R. 1996. Development of a new alloplasmic CMS Brassica napus in the cytoplasmic b a c k g r o u n d of Diplotaxis siifolia. Cruciferae Newsl. 18, 68-69. Rao, G. U., Batra Sarup, V., Prakash, S. a n d Shivanna, K. R. 1994. Development of a new cytoplasmic male-sterility system in Brassica juncea t h r o u g h wide hybridization. Plant Breeding 112, 171-174. Rawat, D. S. a n d Anand, I. J. 1979. Male sterility in Indian m u s t a r d . Indian J. Genet. 39, 412-415. Renard, M., Mesquida, J., Delourme, R. a n d Vallee, P. 1991. Les contraintes de la production de semences hybrides de colza. Bulletin Semences 117, 63-65. Renard, M., Delourme, R., Mesquida, J., Pelletier, G., Primard, C., Boulidard, L., Dore, C., Ruffio, V., Herve, Y. and Morice, J. 1992. Male sterility and F1 hybrids in Brassica. XIIIth EUCARPIA Congress:, Reproductive Biology a n d Plant Breeding, Angers, France, pp. 107-119. Renard, M., Delourme, R., Vallee, P. a n d Pierre, J. 1998. Hybrid rapeseed breeding a n d production. In: Thomas, G. a n d Monteiro, A. (eds.) Brassica 97, International s y m p o s i u m on Brassicas, Rennes, Acta Hort. 4 5 9 , 291-298. Reynaerts, A., Vandewiele, H., Desutter, G. a n d J a n s s e n s , J. 1993. Engineered genes for fertility a n d their application in hybrid seed production. Scientia Hort. 55, 125-139. Rousselle, P. 1982. Premiers r6sultats d'un p r o g r a m m e d'introduction de l'androst6rilit6 "Ogura" du radis chez le colza. Agronomie 2, 859864. Rousselle, P. a n d Renard, M. 1982. Int6r6t du cultivar "Bronowski" pour l'obtention de plantes m~le-st6riles cytoplasmiques chez le colza (Brassica napus L.). Agronomie 2, 951-956. Ruffio-Chable, V. 1994. Les s y s t ~ m e s d'hybridations chez le chou-fleur (Brassica oleracea L. van botrytis L.). Application ~t l'am~lioration g~n~tique., Thesis, ENSA de Rennes.
214 Ruffio-Chable, V., Bellis, H. a n d Herve, Y. 1993. A d o m i n a n t gene for male sterility in cauliflower (Brassica oleracea var. botrytis). Phenotype expression, inheritance a n d use in F1 hybrid production. Euphytica 67, 9-17. Rundfeldt, H. 1960. U n t e r s u c h u n g e n zur Z ~ c h t u n g des Kopfkohls (B. oleracea L. var capitata). Z. Pflanzenzficht. 44, 30-62. Sakai, T. a n d I m a m u r a , J. 1990. Intergeneric transfer of cytoplasmic male sterility between Raphanus sativus (CMS line) a n d Brassica napus t h r o u g h cytoplast-protoplast fusion. Theor. Appl. Genet. 80, 421427. Sakai, T., Iwabuchi, M., Kohno-Murase, J., Liu, H. J. and I m a m u r a , J. 1995. Transfer of radish CMS-restorer gene into Brassica napus by intergeneric protoplast fusion. Proc. 9th Int. Rapeseed Congress, Cambridge, UK, pp. 3-5. Sakai, T., Liu, H. J., Iwabuchi, M., Kohno-Murase, J. and I m a m u r a , J. 1996. Introduction of a gene from fertility restored radish (Raphanus sativus) into Brassica napus by fusion of X-irradiated protoplasts from a radish restorer line and iodacetoamide-treated protoplasts from a cytoplasmic male-sterile cybrid of B. napus. Theor. Appl. Genet. 9;t, 373-379. Sheikh, I. A. a n d Singh, J. N. 1996. Exploitation of male sterility in Indian m u s t a r d . Cruciferae Newsl. 18, 70-71. Shiga, T. 1976. Cytoplasmic male sterility a n d its utilization for heterosis breeding in rapeseed, Brassica napus L. Japan Agric. Res. Quarterly 10, 178-182. Shiga, T. 1980. Male sterility a n d cytoplasmic differenciation. In: Tsunoda, S., Hinata, K. and G6mez-Campo, C., (eds.), Brassica crops and wild allies. Biology and breeding, J a p a n Scientific Societies Press, Tokyo, pp. 205-221. Shiga, T. a n d Baba, S. 1973. Cytoplasmic male sterility in oilseed rape (Brassica napus L.), a n d its utilization to breeding. Japan. J. Breed. 23, 187-197. Sigareva, M. A. a n d Earle, E. D. 1997. Direct transfer of a cold-tolerant Ogura male sterile cytoplasm into cabbage (Brassica oleracea ssp. capitata) via protoplast fusion. Theor. Appl. Genet. 94, 213-220. Singh, M. a n d Brown, G.G. 1991. Suppression of cytoplasmic male sterility by n u c l e a r genes alters expression of a novel mitochondrial gene region. The Plant Cell 3, 1349-1362. Singh, M., Hamel, N., Menassa, R., Li, X.-Q., Young, B., J e a n , M., Landry, B. S. a n d Brown, G.G. 1996. Nuclear genes associated with a single
215
Brassica CMS restorer locus influence transcripts of three different mitochondrial gene regions. Genet. 143, 505-516. Sodhi, Y. S., Pradhan, A. K., Verma, J. K., Arumugam, N., Mukhopadhyay, A. and Pental, D. 1994. Identification and inheritance of fertility restorer genes for 'tour' CMS in rapeseed (Brassica napus L.). Plant Breeding 112, 223-227. Stiewe, G. and R6bbelen, G. 1994. Establishing cytoplasmic male sterility in Brassica napus by mitochondrial recombination with B. tournefortii. Plant Breeding 113, 294-304. Stiewe, G., Sodhi, Y. S. and R6bbelen, G. 1995a. E s t a b l i s h m e n t of a new CMS-system in Brassica napus. Proc. 9th Int. Rapeseed Congress, Cambridge, UK, pp. 49-51. Stiewe, G., Witt, U., Hansen, S., Theis, R., Abel, W. O. and R6bbelen, G. 1995b. Natural and experimental evolution of CMS for rapeseed breeding. Genetic m e c h a n i s m s for hybrid breeding. Adv. in Plant Breeding 18, 59-76. Takagi, Y. 1970. Monogenic recessive male sterility in oilseed rape (Brassica napus L.) induced by g a m m a irradiation. Z. Pflanzenzfichtg. 64, 242-247. Takahata, Y., Nagasaka, M., Kondo, H. and Kaizuma, N. 1996. Genic male sterility in Brassica campestris L. In: Dias, J. S., Crute, I. and Monteiro, A.A. (eds.), Proc. Int. Syrup. on Brassicas, Ninth Crucifer Genetics Workshop. Acta Hort. 407, 147-150. Theis, R. a n d R6bbelen, G. 1990. Anther and microspore development in different male sterile lines of oilseed rape (Brassica napus L.). A n g e w a n d t e Botanik 64, 419-434. Thompson, K. F. 1972. Cytoplasmic male sterility in oilseed rape. Heredity 29, 253-257. Tokumasu, S. 1951. Male sterility in J a p a n e s e radish (Raphanus sativus L.). Sci. Bull Fac. Agric. Kyushu Univ. 13, 83-89. Tokumasu, S. 1957. Histological studies on pollen degeneration in male sterile J a p a n e s e radish (Raphanus sativus L.). Japan J. Breed. 6, 4550. Van Der Meer, Q. P. 1987. Chromosomal monogenic d o m i n a n t male sterility in chinese cabbage (Brassica rapa subsp, pekinensis (Lour.) Hanelt). Euphytica 36, 927-931. Waiters, T. W and Earle, E. D. 1993. Organellar segregation, r e a r r a n g e m e n t and recombination in protoplast fusion-derived Brassica oleracea calli. Theor. Appl. Genet. 85, 761-769.
216 Walters, T. W., Mutschler, M. A. and Earle, E. D. 1992. Protoplast fusionderived Ogura male sterile cauliflower with cold tolerance. Plant Cell Rep. 10, 624-628. Witt, U., Hansen, S., Albaum, M. and Abel, W. O. 1991. Molecular analyses of the CMS-inducing 'Polima' cytoplasm in Brassica napus L. Curr. Genet. 19, 323-327. Yamagishi, H. 1998. Distribution and allelism of restorer genes for Ogura cytoplasmic male sterility in wild and cultivated radishes. Genes and Genetic Systems 73, 79-83. Yamagishi, H. a n d Terachi, T. 1996. Molecular a n d biological studies on male-sterile cytoplasm in the Cruciferae. III. Distribution of Oguratype cytoplasm among j a p a n e s e wild r a d i s h e s and Asian radish cultivars. Theor. Appl. Genet. 93, 325-332. Yamagishi, H. a n d Terachi, T. 1997. Molecular a n d biological studies on male sterile cytoplasm in the Cruciferae. IV. Ogura-type cytoplasm found in the wild radish, Raphanus raphanistrum. Plant Breeding 116, 323-339. Yang, G. a n d Fu, T. 1990. The inheritance of polima cytoplasmic male sterility in Brassica napus L. Plant Breeding 104, 121-124. Yarrow, S. A., Wu, S. C., Barsby, T. L., Kemble, R. J. a n d Shepard, J. F. 1986. The introduction of CMS mitochondria to triazine tolerant Brassica napus L., var."Regent", by micromanipulation of individual heterokaryons. Plant Cell Rep. 5, 415-418. Yarrow, S. A., Burnett, L. A., Wildeman, R. D. and Kemble, R. J. 1990. The transfer of Polima cytoplasmic male sterility from oilseed rape (Brassica napus) to broccoli (B. oleracea) by protoplast fusion. Plant Cell Rep. 9, 185-188. Zhou, X. 1990. La st~rilit~ mole dig~nique dominante du colza (Brassica napus). Recherche du g~ne restaurateur R f et ~tude cytologique en microscopie ~lectronique. M6moire D.E.A., Labo. de Biologie Cellulaire. Universit6 Rennes I.
Biology of Brassica Coenospecies C. G6mez-Campo (Editor) 91999 Elsevier Science B.V. All rights reserved.
217
7 GENOME STRUCTURE AND MAPPING Carlos F. Quiros
Department of Vegetable Crops. University of California. Davis, CA 95616, U.S.A. Most of the m a p p i n g work in Brassica h a s t a k e n place d u r i n g the p a s t ten years. This activity h a s been focused mostly on r a p e s e e d B. napus a n d on all three diploid cultivated species, B. nigra, B. oleracea a n d B. rapa. More recently, m a p p i n g h a s been e x p a n d e d to include B. juncea. The m a p s p r o d u c e d in Brassica crops are b a s e d mainly on F2 progenies developed by various laboratories, which will require in the future their integration for a more efficient use. In addition to linkage maps, a few synteny m a p s based on alien addition lines have been created, allowing chromosome assignment of some of the existing linkage groups. The m a r k e r m a p s are being u s e d to locate genes d e t e r m i n i n g traits of economic interest, including quantitative trait loci. Another i m p o r t a n t application of the maps, which is quite active at the p r e s e n t time, is on the s t u d y of the structure, origin a n d evolution of the Brassica genomes. Recent reviews of m a p p i n g activity in Brassica are those of Quiros et al. (1994), Altenbach et al. (1995) a n d Paterson (1997). Relatedness between the three basic Brassica genomes, A (n = 10), B (n = 8) a n d C (n = 9), h a s been cytogenetically investigated by u s i n g digenomic diploids from interspecific hybrids (Attia a n d R6bbelen, 1986), from amphidiploids through haploidy (Morinaga a n d F u k u s h i m a , 1933; Olsson a n d Hagberg, 1955), a n d digenomic triploids from crosses between amphidiploids a n d diploids (Morinaga, 1929, 1934; Attia et al., 1987). In general, the three genomes are considered to be mutually a n d partially homologous and p r e s u m a b l y derived from a common ancestral genome (Mizushima, 1950). The advent of modern molecular techniques is playing an i m p o r t a n t role in u n d e r s t a n d i n g the organization a n d relationships of the Brassica genomes. Results from these studies not only confirmed the origin of the amphidiploid species, b u t also suggested t h a t the A a n d C genomes originated from a single lineage, whereas the B genome is genetically dist a n t to both A a n d C genomes forming a separate lineage (Song et al., 1990; Warwick a n d Black, 1991). A c o m m o n a s s u m p t i o n is t h a t the n = 8, 9 a n d 10 cultivated species have evolved in a n a s c e n d i n g diploid series from a c o m m o n
218 primitive genome, "Urgenome' (Haga, 1938). Although there are no known B r a s sica species in n a t u r e with genomes of less t h a n n = 7 chromosomes, Catcheside (1934), Sikka (1940) a n d R6bbelen (1960) postulated t h a t the ancestral genome for these species consisted of five or six basic c h r o m o s o m e s , which through polysomy originated the p r e s e n t day cultivated genomes ranging from n = 8 to n = 10 chromosomes. T h u s , as a corollary of these hypotheses, the cultivated diploids can be considered s e c o n d a r y polyploids (Prakash a n d Hinata, 1980). Genetic m a p s in B r a s s i c a serve a double purpose" a) u n d e r s t a n d i n g the relationship a m o n g the genomes of the B r a s s i c a cultivated diploid species, and b) utilization in applied genetics and breeding of the n u m e r o u s B r a s s i c a crops.
Linkage m a p s B. o l e r a c e a maps Several m a p s have been developed independently for this species involving crosses between different crops. Slocum et al. (1990) reported an extensive RFLP m a p of 258 m a r k e r s covering 820 recombination u n i t s in nine linkage groups, with average intervals of 3.5 units. This is a proprietary m a p developed from a broccoli • cabbage cross. Landry et al. (1992) c o n s t r u c t e d a m a p consisting of 201 RFLP m a r k e r s distributed on nine major linkage g r o u p s coveting 1112cM. The F2 progeny u s e d to c o n s t r u c t this m a p was developed by crossing a cabbage line r e s i s t a n t to clubroot to a rapid cycling stock (Figure 7.1). The cabbage line was derived from an interspecific cross involving B. o l e r a c e a a n d B. n a p u s , followed by a series of b a c k c r o s s e s to B. oleracea. According to the authors, it is likely t h a t the resulting cabbage line h a d two c h r o m o s o m e segments of the A genome carrying the disease resistant genes. These h a d been introgressed or s u b s t i t u t e d in the C genome of the cabbage line. Kianian a n d Quiros (1992) developed a m a p comprising 108 markers, spread in 11 linkage groups and coveting 747 cM. This m a p was b a s e d on three intra-specific a n d one inter-specific F2 populations, namely: collard • cauliflower, collard • broccoli, kale • cauliflower and kohlrabi • B. insularis. The majority of the markers in the map are RFLP loci, with a few morphological a n d isozyme markers. Later, a few RAPD markers were added a n d the m a p was r e d r a w n based only on the three intra-specific crosses using JOINMAP (Stare, 1993). It consists of 82 markers distributed on eight major linkage g r o u p s covering 431 cM (Quiros et al., 1994). R e c o m b i n a n t inbreds by single seed d e s c e n t are u n d e r development for the collard by cauliflower and collard by broccoli progenies. C a m a r g o et al. (1997) recently reported a m a p developed in an F2 population of c a b b a g e • broccoli. It includes 112 RFLP a n d 47 RAPD loci and a selfincompatibility locus on nine m a i n linkage groups. Kearsey et al. (1996) and R a m s a y et al. (1996) c o n s t r u c t e d a linkage m a p based on backcross progenies obtained from double haploid lines of broccoli a n d B. alboglabra. Recombination of m a r k e r s was higher in the BC2 t h a n in the BCI generation. These differences were a t t r i b u t e d to differential c h i a s m a frequency in female a n d male meiosis.
LG 2
LG 1 WG6M
E m % WG2E12 wMB2b AAlOD W G W . AAlSC
EcIDll A834 WwD7 WGEASb'
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51 21 14
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17
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MlW ABlbE AUB ACIC' TG.?f2 l
Figure 7.1 B. oleruceu linkage map produced by an F2 from a cross cabbage x broccoli. It includes 112 RRFLP and 47 RAPD loci and a self-incompatibility locus on 9 main linkage groups. Letters in parenthesis after some of the markers indicate recessive alleles originating from cabbage (A) or broccoli parent (B). Asterisks indicate loci with significant segregation distortion (Cmargo et al., 1997).
220 B o u h o n et al. (1996) u s e d this m a p for aligning the C genome linkage groups of B. oleracea a n d B. n a p u s . Lagercrantz a n d Lydiate (1995) also observed sex dep e n d e n t recombination rates in B. nigra. In this species, however, recombination rate in each sex varied for some c h r o m o s o m e segments. If differential recombination rates in male a n d female gametes proves to be a widespread phenomenon, it posses a n o t h e r complexity to consider when applying linkage information to breeding problems. We have aligned m o s t of the linkage groups of four of these m a p s (Hu et al., 1998). For this purpose, a linkage m a p was c o n s t r u c t e d from an F2 population of 69 individuals with s e q u e n c e s previously m a p p e d i n d e p e n d e n t l y in three linkage m a p s of this species. These were the m a p s p u b l i s h e d by Kianian and Quiros (1992), Landry et al. (1992) a n d C a m a r g o et al. (1997). The base m a p developed in this s t u d y consisted of 167 RFLP loci in nine linkage groups, plus eight m a r k e r s in four linkage pairs, covering 1738 cM. Linkage group alignment was also possible with a fourth m a p p u b l i s h e d by R a m s a y et al. (1996), containing c o m m o n loci with the m a p of Camargo et al. (1997). In general, cons i s t e n t linear order a m o n g m a r k e r s were m a i n t a i n e d , a l t h o u g h often the dist a n c e s between m a r k e r s varied from m a p to map. A linkage group in Landry's m a p carrying a clubroot resistance QTL was found to be rearranged, consisting of m a r k e r s from two other linkage groups. This was not surprising considering t h a t the resistance gene was introgressed from B r a s s i c a n a p u s . The extensively duplicated n a t u r e of the C genome w a s revealed by 19 s e q u e n c e s detecting duplicated loci within c h r o m o s o m e s a n d 17 sequences detecting duplicated loci between c h r o m o s o m e s . The variation in m a p p i n g distances between linked loci pairs on different c h r o m o s o m e s d e m o n s t r a t e d t h a t sequence r e - a r r a n g e m e n t is a distinct feature of this genome. Although the consolidation of all linkage groups in the four B. oleracea m a p s compared was not possible, a high n u m b e r of m a r k e r s to c o r r e s p o n d i n g linkage g r o u p s were added. Some c h r o m o s o m e s e g m e n t s were enriched with m a n y m a r k e r s which m a y be useful for future r e s e a r c h in gene tagging or cloning. The a s s i g n m e n t B. oleracea linkage groups to their respective chromosomes h a s been partially accomplished. This t a s k h a s been based on the development of alien addition lines allowing the construction of synteny maps. We developed two synteny m a p s for the nine C genome chromosomes including isozyme, RFLP and RAPD markers. One m a p has approximately 194 markers and was constructed using a set of alien addition lines B. rapa-oleracea extracted from artificial B. n a p u s "Hakuran' (McGrath and Quiros 1990; McGrath et al., 1990). The second m a p h a s approximately 103 m a r k e r s a n d was assembled from a set of addition lines B. rapa-oleracea extracted from natural B. n a p u s (Quiros et al., 1987). Chen et al. (1992) a n d Cheng et al. (1994ab, 1995) also developed B. rapa-oleracea {alboglabra) alien addition lines, allowing the identification of the chromosome carrying genes for seed a n d flower color. Later, various c h r o m o s o m e s of these lines were characterized by molecular m a r k e r s (Jorgensen et al., 1996). Although it was possible in most cases to physically assign linkage groups to chromosomes using these sets of lines, two major complications arising from this activity
221 deserve comment. The first one is the frequent lack of p o l y m o r p h i s m of inter specific m a r k e r s of the alien c h r o m o s o m e s in the intraspecific crosses u s e d to develop the linkage maps. Low coincidence of p o l y m o r p h i s m between these two sets of materials m a k e s c h r o m o s o m e a s s i g n m e n t tedious a n d time consuming. The second complication is the instability of the alien c h r o m o s o m e s . We have addressed this problem by following groups of syntenic m a r k e r s in the progeny of monosomic addition lines of B. rapa-oleracea (Hu a n d Quiros 1991). Data have been obtained from two progenies of approximately 100 plants each, derived from two monosomic addition lines of B. rapa-oleracea for chromosomes C4 and C5 (2n = 21). After following several m a r k e r s located on each c h r o m o s o m e , the alien c h r o m o s o m e s were found not to be always stable. All the expected m a r k e r s were recovered in approximately 50% of the plants carrying the alien chromosome. The remaining 2n = 21 plants h a d one or more m a r k e r s missing. These observations were extended to other six c h r o m o s o m e s of the C genome in the "Hakuran' derived lines, with a larger n u m b e r of m a r k e r s (Hu and Quiros, unpublished). The study d e m o n s t r a t e d that this is widespread in the C genome a n d seems to occur to all chromosomes. Most of the deletions were terminal, with a few exceptions. The deletions were found useful to physically individual m a r k e r s of linkage segments to specific c h r o m o s o m e a r m s (Hu and Quiros 1991). Another anomaly of the alien c h r o m o s o m e s is their ability to undergo n o n - h o m o l o g o u s recombination at a low frequency when present in the same cytoplasm in double or triple monosomic condition or forming part of similar hyperploids. This type of situation provides the opportunity for association of homoeologous segments resulting from duplicated segments. Although we were u n a b l e to quantify this type of recombination precisely, it is estimated to occur at a frequency ranging between 5 to 10% (Hu a n d Quiros, u n p u b l i s h e d ; S t r u s s et al., 1996). B. r a p a
maps
The two m o s t extensive m a p s created for this species are proprietary. The first one, developed by Chyi et al. (1992) resulted by crossing s a r s o n by canola. It includes 360 loci on 10 linkage groups covering 1876 recombination units. The average distance between m a r k e r s is 5.2 m a p units. The second one developed by Slocum (1989) a n d Song et al. (1991) was obtained by crossing Chinese cabbage x spring broccoli. This m a p includes 273 loci covering 1455 recombination u n i t s in 10 linkage groups with average intervals of 5 units. An u p d a t e d version was used to locate genes involving 28 phenotypic traits (Song et al., 1995a). Kole et al. (1996) created a m a p involving a white r u s t resistance line for the purpose of mapping the resistance gene. Other m a p s have been developed by Schilling and Bernatzky (Schilling, 1991) including 58 RFLP loci covering approximately 700 cM after crossing the oilseed cultivar "Candle' by a rapid cycling strain. McGrath and Quiros (1991) developed another m a p from an F2 of turnip x Pak Choi containing 49 m a r k e r s in eight linkage groups a n d covering a total of 262 cM. Both m a p s have been consolidated by exchanging probes a n d DNA from the m a p p i n g populations (Hu, Vernatzky a n d Quiros, unpublished).
222 B. n i g r a m a p
Truco and Quiros (1994) developed a map for this species based on a single F2 population of 83 plants, involving two parental individuals from geographically divergent populations. The map constructed with the program Mapmaker (Lander et al., 1988), has 67 markers arranged in eight major linkage groups which may correspond to the eight B. nigra chromosomes, plus two small groups. The markers include RFLP's, RAPD's and a few isozymes. The RFLPs are based on EcoRI digestions. The map covers 561 cM with average intervals of 8.4 cM (Figure 7.2). Lagercrantz and Lydiate (1995) developed a RFLP map in a backcross population of B. nigra, consisting of 288 loci covering a length of 855 cM. A trend of higher recombination rates was observed in male gametes for the distal portions of the linkage groups. Also, recombination rates tended to higher in the proximal regions of some of the linkage groups. This map was recently expanded by Lagercrantz (1998) who incorporated 284 additional loci based on A. thaliana probes. It has been possible to assign only four linkage groups of the first map to their respective chromosomes by synteny mapping based on alien addition lines (Chevre et al., 1991). Different species combinations for developing these lines have been used. These include a B. oleracea-nigra series extracted from B. carinata (Quiros et al., 1986) for five of the eight B genome chromosomes. The second set consists of Diplotaxis erucoides-B, nigra covering seven of the eight chromosomes in the B genome (Quiros et a/., 1988; This eta/., 1990). The third and fourth sets are in tetraploid background, B. napus-nigra lines, and were developed independently in France (jahier et al., 1989; Chevre et al., 1991) and Germany (Struss et al., 1991, 1996). Chevre's set covers at least seven of the eight B genome chromosomes. The alien chromosomes are in disomic condition and contain RFLP, RAPD and isozyme markers. Also in this set the chromosomes carrying genes for fatty acid chain elongation and desaturation and possibly a chromosome carrying resistance to blackleg have been identified. The addition lines developed by Struss et al. (1991) include B genome chromosomes extracted from B. nigra, B. carinata and B. juncea. Struss et al. (I 996) constructed synteny map for most of these B genome alien with isozyme, RAPD and RFLP and phenotypic markers. Although they found extensive intra- and intergenomic recombination in these chromosomes, it was possible to construct a consensus synteny map for the B genomes extracted from all three sources. In this map, chromosomes carrying genes for erucic acid, sinigrin and possible blackleg resistance were identified. B. n a p u s m a p
Most of the mapping activity in Brassica has been focused to this species because of its economic importance. This has resulted in the construction of at least six independent maps. Several maps for this amphidiploid species have been reported by various laboratories. The first map was developed by Landry et al. (1991), and resulted from crossing two rapeseed cultivars, "Westar' and "Topaz', based on single-enzyme digestions by four enzymes, BamHI, EcoRI, EcoRV and HindIII. It included 120 loci arranged in 19 linkage groups covering 1413 recom-
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Figure 7.3 (continued from the previous page) Linkage map of B. napus resulting from double haploid lines of the F1 resulting from crossing the winter rapeseed cv "Mansholt' with cv. Samourai". Mapped genes and QTLs are underlined: Pale (pale yellow flower), ACP (acyl-carrier-protein), KAS (beta-ketoacyl-ACP synthase) I, gls (see glucosinolate content QTLs) (after Uzunova et al., 1995). bination units. Hoenecke a n d Chyi, (1991) reported the s a m e year at the International Congress of Rapeseed a proprietary m a p developed by crossing two breeding lines BN0011 • BN0031 based on EcoRI digestions. This m a p consisted of 125 m a r k e r s a r r a n g e d in 19 linkage g r o u p s covering 1350 m a p units. In the p a s t three years, four other m a p s have been developed b a s e d on F2 progenies of double haploid lines. Ferreira et al. (1994) c o n s t r u c t e d a m a p by crossing the a n n u a l canola cultivar ' S t e l l a r ' b y the biennial cultivar 'Major'. Double haploid lines generated from the resulting F1 served to construct a m a p of 132 loci covering 1016 cM in 22 linkage groups. A partial m a p was also c o n s t r u c t e d from the F2 progeny of the two p a r e n t a l lines. For the c h r o m o s o m e s e g m e n t s compared, no significant differences on linkage associations were observed between the two maps. Uzunova et al. (1995) constructed a m a p based on double haploid F1 lines by crossing the two winter rapeseed varieties, ~lVlansholt's H a m b u r g e r Raps' a n d 'Samourai'. It consisted in 204 RFLP a n d two RAPD m a r k e r s distributed in 19 linkage g r o u p s covering 1441 cM (Figure 7.3). S h a r p e et al. (1995) reported an integrated m a p based on two populations, one of which included a resynthesized B. n a p u s line crossed to a rapeseed cultivar. The c h r o m o s o m e instability of the resynthesized line a d d e d to the complexity of these maps. Parkin et al. (1995)
226 published also a map based only on this synthetic population, consisting of 399 RFLP markers. The integration of the maps by Sharpe et al. (1995), based in the combination of the two populations resulted in a reduction of over 100 loci. Most recently, Foisset et al. (1996) constructed a map based on RFLP, RAPD and isozyme markers developed from a double haploid progeny of the F1 generated by crossing a dwarf rapeseed isogenic line 'Darmor-bzh' of cultivar 'Darmor' and ~Yudal', a spring Korean line. The map based on 153 double haploid lines had 254 markers on 19 linkage groups coveting 1765 cM. Several economically important genes were located on it.
B. j u n c e a map The activity in this species is quite recent. Sharma et al. (1994) developed a small map based on a F2 mapping population resulting of crossing 5/aruna', a brown seeded cultivar and BEC 144, a yellow seeded accession. This map has only 25 markers on nine linkage groups covering 243 cM. A more extensive map developed by Cheung et al. (1996) is based on a double haploid progeny from a canola quality line by a high oil content line. The map was developed with probes from B. n a p u s , consisting of 343 loci on 18 main linkage groups covering 2073 cM.
Structure of the Brassica genomes General attributes A great deal of information has been gathered during the past few years on the genomic structure of all three cultivated genomes. Diverse analysis of synteny maps for the B and C genomes (McGrath et al. 1990; Struss et al., 1996), and the linkage maps for the A, B and C genomes (Slocum et al., 1990; Song et al., 1991; McGrath and Quiros 1991; Kianian and Quiros, 1992, Truco and Quiros, 1994) reveal extensive sequence duplication. For example, Slocum et al. (1990) reported for B. oleracea that 35% of the genomic clones produced more than one locus, and 56% besides disclosing single locus segregations often produced other monomorphic fragments that may represent duplications. Kianian and Quiros (1992) also in B. oleracea found 50% of their cDNA sequences mapping to more than one locus. McGrath et al. (1990) reported over 40% sequence duplications in B. oleracea chromosomes represented in a series of addition lines. Song et al. (1991) reported in B. rapa that 36% of their genomic clones produced segregating RFLPs at more than one locus and 41% detected sequences segregating as single locus but also revealed additional monomorphic fragments. Truco and Quiros (1994) found approximately 40% of RFLP loci duplicated in B. nigra. This finding was confirmed by Lagercrantz and Lydiate (1995) who reported that "essentially every chromosomal region [is present] in three copies" in this species. Thus, these reports demonstrate that close to 50% of the loci in all three cultivated genomes are duplicated, supporting the hypothesis that the B r a s s i c a diploid species are secondary polyploids (Prakash and Hinata, 1980; Quiros et al., 1994). In general, these duplications are distributed on more than one chromosome. Some of the
227 linkage and synteny groups have sequences present in more than one group. When linkage arrangement is conserved, the distances are usually changed. Rerrangements of linkage groups may be explained by chromosomal translocations due in part to homoeologous recombination. Translocations are of common occurrence in B r a s s i c a and have been reported by independent investigators as a widespread event in various species (Songerup, 1980; Quiros et al., 1988, Kianian and Quiros, 1992). In addition to duplications and linkage rearrangements, deletions seem to be another important molding force of the B r a s s i c a genomes. The four independent F2 linkage studies cited above have detected a large n u m b e r of loci containing dominant markers, where one allele is apparently null, which may be due to deletions. This phenomenon, however, may be also due to the masking of common alleles in duplicated loci (Uzunova et al., 1995). Sometimes these loci are assembled in linkage blocks implying large deletions in some chromosomes (McGrath and Quiros, 1991; Song et al., 1991). The presence of deletions has been demonstrated cytologically for the C genome in alien addition lines (Hu and Quiros, 1991). Finally, inversions have also been observed in the F2 linkage maps of B. o l e r a c e a by Song et al. (1991) and Kianian and Quiros (1992). Similar levels of genome duplication have been reported in B. n a p u s . Landry et al. (1991) found that 88% of the probes disclosed more than one locus. B. n a p u s being an amphidiploid, these duplications are expected to correspond to both intra-genomic and inter-genomic sequences. This indeed seems to be the case, because some of the probes disclosed three or four segregating loci located on different chromosomes (Landry and al., 1991; Hoenecke and Chyi, 1991). The same trend was observed by Uzunova et al. (1995), detecting ten different clusters of two to four duplicated loci in 11 linkage groups. Ferreira et al. (1994) also detected a similar level of duplicated loci in B. n a p u s . Another interesting attribute of the B r a s s i c a maps in general is the relatively large number of loci deviating from Mendelian segregation, which often cluster in linkage blocks (McGrath and Quiros 1991; Figdore et al., 1993; Landry et al., 1991; Kianian and Quiros 1992; Chry et al., 1992; Ferreira et al., 1994; Uzunova et al., 1995; Foisset et al., 1996). The deviations in some cases may be due to genomic divergence between the parents involved in the crosses (McGrath and Quiros 1991). This conjecture is supported by the fact that the number of deviating loci increases with the level of divergence of the parents (Kianian and Quiros, 1992). In androgenic plant material such as double haploid lines, often used in B. n a p u s for mapping purposes, loci with biased segregation ratios can reach up to 35% (Ferreira et al., 1994; Foisset et al., 1996). Inclusion of loci with distorted segregation ratios in linkage maps is risky, since caution is necessary to avoid disclosing pseudolinkages due to biased statistical tests (Foisset et al., 1996).
Plasticity of the B r a s s i c a g e n o m e s The highly duplicated nature of the genomes have important implications in structural changes of the chromosomes. The homoeologous regions arising by duplications u n d e r certain situations, such as those imposed by hybridization,
228 will facilitate intra-genomic and inter-genomic recombination events. This is especially true in Brassica, where hybridization often results in aneuploidy and amphiploidy. Because of their plasticity, the B r a s s i c a genomes are prone to frequent structural changes.
Intragenomic homoeologus recombination This type of recombination has been detected mostly in aneuploids, such as alien addition lines and newly synthesized amphidiploids. Evidence from the C genome derived addition lines (McGrath et al., 1990) indicates that non-homologous recombination may take place in the B. oleracea genome when two or more of these chromosomes are present in single copies as alien additions. Furthermore, in addition to the nine expected synteny groups resolved from the B. n a p u s C genome, four recombinant groups including markers from different chromosomes were recovered (Quiros et al., 1994). The chromosomes most often involved in these recombination events were 4C, 5C and 6C, indicating that some chromosomes are more recombinogenic than others. Comparison of chromosomes of addition lines derived from natural and synthetic B. n a p u s "Hakuran' dissecting the C genome (McGrath et al., 1990) disclosed also possible syntenic differences. Although chromosomes 3, 4, 5 and 6 aligned well in both sets, chromosomes 1, 2 and 7 displayed rearrangements of the markers. The synteny changes observed in the alien addition lines occurred in 12% of the plants carrying more t han one alien chromosome. Variation in synteny has also been observed for the B genome chromosomes in addition lines from independent origins (This et al., 1990; Chevre et al., 1991; Struss et al., 1996). Extensive intergenomic recombination was detected for addition lines originating from trigenomic hybrids of constitution ABC. In this type of hybrid the chromosomes of the three genomes have maximum opportunity for auto and allosyndetic pairing resulting in intra and inter-genomic recombination events (Struss et al., 1996). Other instances of probable intergenomic recombination was disclosed by comparative mapping in B. rapa and B. n a p u s (Hoenecke and Chyi, 1991). They found significant linkage arrangement differences between the A genomes from the diploid and amphidiploid species. However, it was still possible to identify major conserved linkage groups. The structural changes observed in the three cultivated genomes indicate that this p h e n o m e n o n is widespread, not exclusive of a single genome. Although amphiploidy and interspecific aneuploidy may serve to induce these genomic changes, some rearrangements might take place in the diploid environment.
Intergenomic homoeologous recombination Occasionally a few of the diploid individuals derived from B. rapa-oleracea monosomic addition plants were found to carry a few C-genome-specific markers present in the alien chromosomes of the parental plant, indicating that inter-
229 genomic recombination had taken place. Earlier during the development of these lines, we detected intergenomic recombination between the A and C genome chromosomes for the isozyme locus Pgi-2 (Quiros et al., 1987). Recently we have observed intergenomic recombinants for rDNA sequences, where B. r a p a individuals display EcoRI fragments typical of B. oleracea (Hu et al., unpublished). Another line of evidence for this type of recombination has been obtained by Sharpe et al. (1995) who followed segregation of genome specific RFLP markers in B. n a p u s progenies. Parkin et al. (1995), detected non-homoeologous recombination, resulting in non reciprocal homoeologous translocations in B. n a p u s cultivars at approximately 0.3%. In resynthezided B. n a p u s recombination of this type was approximately 10%. Early generations of synthetic B r a s s i c a amphiploids after several cycles of selfing displayed genomic changes. Some of these changes could be attributed to inter genomic recombination (Song et al., 1995b). Other instances of intergenomic recombination have been reported (Struss et al., 1996; Plieske et al., (1998) in B. n a p u s - n i g r a addition lines. AACC euploids derived from some of the lines carried genes for resistance to balckleg, erucic acid and sinigrin content from the B genome. It is unknown whether the B genome chromosome segments translocated to either the A or C genome or both. A similar situation was reported by Landry et al. (1991), where resistance genes from B. rapa were transferred to the C genome by crossing and backcrossing B. n a p u s to B. oleracea.
Genomic relationships Comparative mapping of the genomes extracted from diploid and amphidiploid species is j u s t beginning. Hoenecke and Chry (1991) comparing linkage groups for the A genome of B. rapa and B. n a p u s found mostly conserved loci associations. This allowed the identification of the A genome linkage groups in the amphidiploid species. However, it was also possible to detect linkage arrangement differences between the A genomes from the diploid and amphidiploid species. Teutonico and Osborn (1994) found good coincidence for most of the linkage groups of the A and C genomes from B. rapa and B. oleracea, respectively, with those of B. n a p u s . Because of the small n u m b e r of loci used in the comparative study, it was no possible to trace the linkage groups of B. n a p u s to the respective parental diploid species. In contrast, Lydiate et al. (1993) and Parkin et al. (1995) reported that the A and C genomes of B. rapa a n d B. oleracea, respectively, conserve virtually the same loci arrangement as in natural amphidiploid B. n a p u s . However, in a parallel study from the same laboratory. Sharpe et al. (1995) reported altered linkage arrangement at low frequencies due to nonreciprocal homoeologous translocations. Comparative mapping for all three basic genomes from the cultivated diploid species is at its infancy in B r a s s i c a . Previous work on this subject was limited to comparison of the A and C genomes (Slocum 1989; McGrath and Quiros 1991, Camargo 1994). Only recently comparative mapping for the A, B and C genomes has been reported, allowing to draw inferences on the origin of the three genomes based on their relationships (Lagercrantz and Lydiate 1996; and Truco
230 et al., 1996). All three Brassica species share regions of homology in their genomes (Figure 7.4). Often a single linkage group showed regions of homology with more than one group of the other species. This is in agreement with a comparative study between m aps of B. rapa and B. oleraeea by Slocum (1989), who found that in some cases it was possible to align linkage groups from one to the other species. Some of the linkage groups, however, also shared homologous regions that were separated into more t h a n one group in the other species. It is evident that extensive gene reordering has taken place during the evolution of Brassica species, even though there is considerable conservation among certain chromosome regions within and among the three genomes. This results in complex intra and intergenomic chromosomal relationships where co-linearity is maintained for some segments, but broken up for other chromosomal regions. The extensive map of B. rapa produced by Chyi et al. (1992) illustrates this p h e n o m e n o n quite well for intragenomic homology, where six of the ten chromosomes, representing approximately half of the genome, associate in this fashion. Similarly, in B. oleracea we found seven of the nine chromosomes showing homologous segments, covering also approximately 40% of the genome. On the basis of marker arrangement conservation, we have drawn phylogenetic relationships among the chromosomes of the three genomes. This allowed u s to postulate the possible n u m b e r of chromosomes in the hypothetical ancestral genome originating the A, B and C genomes u n d e r the two following assumptions: 1) The A genome is related to the C genome and possibly derived from it. 2) Two main lineages originated the diploid cultivated Brassica species, the B. rapa/B, oleracea and B. nigra lineage. These a s s u m p t i o n s are supported by taxonomic studies based on chloroplast (Warwick and Black 1991; Pradhan eta/., 1992) and nuclear DNA (Song et al., 1990). Based on the above a s s u m p t i o n s , no more t h a n seven ancestral chromosomes can be postulated to explain the existing linkage groups and their homologous relationships (Figure 7.5). However, analysis of the comparative mapping data of Lagercrantz and Lydiate (1996) in a similar fashion suggests that this n u m b e r could be as low as four. Although hypothetical, chromosomal relationships derived in this fashion serve as a framework to characterize the Brassica chromosomes and determine the m e c h a n i s m s leading to their origin. For example, the higher level of intergenomic homology between the A and C genomes supports the conjecture t h at the former derived later on from an already established C genome. More recently Gale and Devos (1998) based mostly on Lagercrantz and Lydiate (1996) presented an hypothetical alignment of all Brassica chromosomes of the A, B and C genomes. However, the a m o u n t of mapping data available at this time for the three diploid cultivated Brassica species is not sufficient to support this scheme.
On the origin o f the g e n o m e s Based on the mapping data disclosing extensive loci duplication, it is clear that the three diploid Brassica cultivated species are indeed ancient polyploids as suggested earlier by Prakash and Hinata (1980). The reiteration of chromosomes within the genomes certainly agrees with the hypotheses that the existing Brassica genomes derive from a smaller ancestral genome. The n u m b e r of chromo-
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beta-ketocyl-CoA synthase
Simmmonhia chinensis (jojoba)
Lassner et al. 1996
Trierucin (22: 1) triacylglycerols
lysophosphatidic acid acyltransferase
Limnanthes alba (meadowfoam)
Lassner et al. 1995
I -acyl-sn-glycerol-3-phosphate acyltransferase
L. douglasii
Brough et al. 1996
ketoacyl-CoA synthase
Lunaria
Lassner, pers. c o r n .
napin
B. napus
Radke el al. 1988
increased cruciferin
anti-sense napin
B. napus
Kohno-Murase el al. 1994
increased napin
anti-sense cruciferin
B. napus
Kohno-Murase et al. 1995
increased methionine
2 s albumin
Bertholletia excelsa (Brazil nut)
increased lysine
dapA (for lysine-insensitive dihydroxy-picolinic acid synthase)
Corynebacterium
Guerche et al. 1990 Altenbach et al. 1992 Denis et al. 1995 Falco el al. 1995
tryptophan decarboxylase
Catharanthus roseus
Chavedej et al. 1994
Induction of male sterility
barnase
Bacillus amyloliquefaciens
Mariani et al. 1990 Denis et al. 1993
Restoration of male fertility
barstar
B. amyloliquefaciens
Mariani et al. 1992
No effect on male fertility
antisense tapetum-specific A9 gene
B. napus
Turgut el al. 1994
24:1 fatty acid
Seed Proteins
Secondary Metabolites Decreased indole glucosinolates
Reproductive Characters
Self-incompatibility
S-locus genes
B. oleracea, B. campestris
Nishio et al. 1992
Receotivitv to pollination
toxin, A chain
Coynebacterium diphtheriae
Kandasamy et al. 1993
Medical
enkephalin (neuropeptide)
Plastics
phaB, phaC
coding sequence added to Arabidop- Vanderkerckhove et al. 1989 sis 2s albumin Afcafigeneseufrophus cited in Poirier ef al. 1995
Taraetina proteins to oil bodies
oleosin
Zea mays;
Lee et 01. 1991;
hirudin + oleosin
Arabidopsis
Van Rooijen and Moloney
xylanase + oleosin
Hirudo medicinalis
Parmenter et al. 1996
Neocallimastix patriciarum
Liu et al. 1997
oleosin gene
Arabidopsis
Plant el al. 1994
low temperature responsive gene
B. napus
White et al. 1994
S-locus glycoprotein gene
Brassica
Sat0 ef al. 1991
Medical or industrial products
Regulation of Promoters
1995
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298 M o v e m e n t of t r a n s g e n i c Brassica m a t e r i a l s from the laboratory a n d g r e e n h o u s e into the field h a s been a c c o m p a n i e d by c o n c e r n s a b o u t i m p a c t s on h e a l t h a n d the e n v i r o n m e n t a n d the a p p r o p r i a t e levels of regulatory oversight. Most of the i s s u e s raised are similar to those raised with other t r a n s genic crops, b u t some are p a r t i c u l a r l y relevant to Brassica: 1) Are the t r a n s g e n i c p l a n t s directly d a n g e r o u s to h u m a n s , animals, or the e n v i r o n m e n t ? This is a p p r o p r i a t e l y studied on a case by case basis, d e p e n d i n g on the specific t r a n s g e n e a n d crop use. For example, e x p r e s s i o n of the Brazil n u t 2S a l b u m i n protein in s o y b e a n m a y c a u s e allergic r e a c t i o n s in people who are allergic to Brazil n u t s (Goldberg, 1994). The s a m e gene h a s been introduced into r a p e s e e d seed proteins to i n c r e a s e m e t h i o n i n e levels in animal feed (Altenbach et al., 1992; Denis et al., 1995). 2) C a n the t r a n s g e n i c p l a n t s c a u s e indirect u n d e s i r a b l e e n v i r o n m e n t a l effects? This also m u s t be a s s e s s e d with a t t e n t i o n to the t r a n s g e n e a n d the cropping s y s t e m used. Availability of herbicide t o l e r a n t t r a n s g e n i c p l a n t s could lead to either i n c r e a s e d or d e c r e a s e d u s e of herbicides or to shifts toward more e n v i r o n m e n t a l l y benign chemicals. W i d e s p r e a d d e p l o y m e n t of t r a n s g e n i c crops c a r r y i n g Bacillus thuringiensis endotoxin genes for insect control h a s the potential to accelerate d e v e l o p m e n t of r e s i s t a n t insects, t h e r e b y m a k i n g these crops ineffective a n d also h a m p e r i n g the u s e of Bt s p r a y s (McGaughey a n d Whalon, 1992). This issue is of i m m e d i a t e concern for crops like cotton a n d corn for which large a c r e a g e s of Bt-transgenic p l a n t s are a l r e a d y being grown in the U.S., yet little relevant experimental information is available. B t - t r a n s g e n i c broccoli p l a n t s are being u s e d in g r e e n h o u s e a n d field e x p e r i m e n t s to test r e s i s t a n t m a n a g e m e n t strategies involving refuges a n d seed m i x t u r e s (Metz et al., 1995b). The insect u s e d is the d i a m o n d b a c k m o t h (PtuteUa xylosteUa), a crucifer p e s t from which resist a n t s t r a i n s have a l r e a d y been recovered following e x p o s u r e to Bt sprays. 3) Are the t r a n s g e n i c p l a n t s t h e m s e l v e s likely to become weeds? This h a s been e x a m i n e d in a variety of s t u d i e s c o m p a r i n g s u c h plants either for c o m p e t i t i v e n e s s t h r o u g h o u t the whole life cycle or at specific stages, s u c h as seedling e m e r g e n c e (e.g., F r e d s h a v n et al., 1995). British trials have s h o w n t h a t t r a n s g e n i c r a p e s e e d carrying tolerance to k a n a m y c i n or the herbicide BASTA is not more invasive or p e r s i s t e n t t h a n controls (Crawley et al., 1993). Generally similar r e s u l t s have been o b t a i n e d in o t h e r studies on d o r m a n c y , survival, emergence, or growth of seedlings from transgenic plants, including ones modified to have high l a u r a t e or high s t e a r a t e levels in the seeds (Linder a n d Schmitt, 1995). Results of s u c h oilseed mo-
299 difications are of p a r t i c u l a r interest since they seem more likely to alter seedling p e r f o r m a n c e t h a n m a n y other types of t r a n s g e n e s . Some differences were seen in tests of the s a m e lines in different geographic areas, California a n d Georgia (Linder a n d Schmitt, 1995), indicating t h a t e v a l u a t i o n s s h o u l d be done at multiple locations e n c o m p a s s i n g the areas in which the crops are to be grown. 4) Will t r a n s g e n i c p l a n t s t r a n s f e r ~enes to wild relatives, leadin~ to new weed p r o b l e m s ? C o n c e r n s are often raised a b o u t possible m o v e m e n t of t r a n s g e n e s from the crops to related plants. This is particularly relevant to B. napus b e c a u s e of its k n o w n potential for genetic e x c h a n g e with wild or weedy relatives. M u c h of the earlier work in this area dealt with m a n u a l pollination, sometimes followed by embryo rescue, or somatic hybridization. In more recent studies, s p o n t a n e o u s hybridization between r a p e s e e d a n d wild relatives s u c h as B. rapa s u b s p , campestris (syn. B. campestris), R a p h a n u s raphanistrum or Hirschfeldia incana h a s been m o n i t o r e d in field tests, u s i n g either t r a n s g e n e m a r k e r s or o t h e r t e c h n i q u e s to recognize h y b r i d s (Baranger et al., 1993; Leckie et al., 1993; Darmency, 1994; Eber et al., 1994; J ~ r g e n s o n a n d Andersen, 1994; Scheffler et al., 1994). It is clear t h a t s p o n t a n e o u s hybridization can in any case occur, b u t the rates vary considerably d e p e n d i n g b o t h on the specific lines of r a p e s e e d or wild species a n d on the test conditions used. Although fertility of the inter-specific h y b r i d s is u s u a l l y low, seeds c a n often be recovered from them. Competitiveness a n d persistence of s u c h seeds t h e n b e c o m e s the e x p e r i m e n t a l key question. While m u c h of this work is motivated by regulatory i s s u e s a b o u t a p p r o p r i a t e u s e of t r a n s g e n i c r a p e s e e d crops, there is clearly potential for productive interaction of ecologists a n d molecular biologists to a d d r e s s more general biological a n d ecological questionso Various strategies to decrease gene flow from t r a n s g e n i c crops to wild relatives have been tested or proposed. One is u s e of b o r d e r a r e a s t h a t lack vegetation, c o n t a i n p l a n t s t h a t are n o t pollinated by insects or t h a t will be destroyed, or c o m b i n a t i o n s of the two p r o c e d u r e s (Morris et al., 1994; Scheffler et al., 1995). Genetic strategies include u s e of male sterile p l a n t s t h a t produce no pollen or i n t r o d u c t i o n of t r a n s g e n e s into c h l o r o p l a s t DNA r a t h e r t h a n into the n u c l e a r genome (McBride et al., 1995). The latter app r o a c h s h o u l d prevent pollen t r a n s m i s s i o n b e c a u s e pollen does not c o n t a i n chloroplasts. To date, chloroplast t r a n s f o r m a t i o n h a s been achieved only in tobacco (Nicotiana tabacum) b e c a u s e special c o n s t r u c t s a n d selection proc e d u r e s are required, b u t work is in p r o g r e s s on d e v e l o p m e n t of the proc e d u r e s required for both Arabidopsis a n d Brassica (P. Maliga, p e r s o n a l communication).
300
Legal i s s u e s M a n y a s p e c t s of gene t r a n s f e r technology have been p a t e n t e d , including Agrobacterium tumefac~ens s y s t e m s , m a n y of the widely u s e d promoters a n d selectable m a r k e r s , genes of interest, c o n c e p t s (e.g., a n t i s e n s e technology, u s e of coat protein or replicase a p p r o a c h e s to virus resistance), etc. Calgene holds a p a t e n t on Agrobacterium tumefaciens-mediated t r a n s f o r m a tion of Brassica (United States P a t e n t No. 5,188,958, Feb. 23, 1993). Permission to u s e p a t e n t e d or p r o p r i e t a r y m a t e r i a l s for r e s e a r c h p u r p o s e s can often be obtained; however, the licensing a n d financial a r r a n g e m e n t s required for c o m m e r c i a l u s e m a y delay or p r e v e n t the a g r i c u l t u r a l u s e of t r a n s f o r m e d p l a n t s p r o d u c e d in a c a d e m i c r e s e a r c h p r o g r a m s or small companies.
Transgenic Brassica crops now being commercialized The first c o m m e r c i a l planting of a t r a n s g e n i c Brassica crop took place in the S o u t h e r n United S t a t e s in the A u t u m n of 1994. Approximately 2.5 million p o u n d s of seed from a n e n g i n e e r e d B. napus t e r m e d "Laurate Canola" were h a r v e s t e d , c r u s h e d by the following s u m m e r , a n d the resulting oil delivered to a n u n - n a m e d c o m p a n y for i n d u s t r i a l p u r p o s e s . L a u r a t e Canola was developed by Calgene (Voelker et al., 1996) by Agrobacterium-mediated transfer of a gene c o n s t r u c t e d with a cDNA cloned from seed of the California bay tree, Umbellularia californica. This chimeric gene c o n s t r u c t conifers the trait of lauric acid a c c u m u l a t i o n in the seed oil. The oil composition trait in L a u r a t e Canola is specific to the seed storage lipid as e x p r e s s i o n of the t r a n s g e n e is driven by a p r o m o t e r element derived from a highly e x p r e s s e d n a p i n storage protein gene from B. rapa (Kridl et al., 1993). To limit t r a n s g e n e effects specifically to the seed of oilseed b r a s s i c a s , n a p i n p r o m o t e r s are often u s e d a l t h o u g h p r o m o t e r elements derived from cruciferin (Sjodahl et al., 1995), oleosin (Lee et al., 1991), a n d o t h e r embryo-specific genes have also been employed. L a u r a t e Canola is physically i n d i s t i n g u i s h a b l e from r e g u l a r canola in the field. Within a few cycles of selection u s i n g haploid b r e e d i n g t e c h n i q u e s , crop yields have r e a c h e d e s s e n t i a l parity with top open pollinated varieties in C a n a d i a n field trials. L a u r a t e C a n o l a is a notable e x a m p l e of the u s e of t r a n s g e n e s to enrich
Brassica g e r m p l a s m , since l a u r a t e molecule is n o r m a l l y p r e s e n t only in trace
a m o u n t s in crucifer seed oils. Years of screening for v a r i a n t s a n d / o r mut a n t s of oilseed b r a s s i c a s t h a t m i g h t p r o d u c e lauric acid have not been fruitful. Moreover, the pre-existing c o m m e r c i a l s o u r c e s of l a u r a t e oils, coconut a n d p a l m kernel, c o n t a i n oil triacylglycerols (TAG) with lauric acid in a n y of the three positions of the lipid molecule (as does U. californica). Laurate C a n o l a t e n d s to exclude lauric a n d other s a t u r a t e d fatty acids from the second position of the TAG molecule (Del Vecchio, 1996). T h u s its oil is chemically different from c o c o n u t or p a l m kernel oils, a n d L a u r a t e Canola oil t u r n s out to be u n i q u e l y a n d significantly better for certain high value food applications~
301 Other transgenic B. n a p u s with a modified oil composition a n d in advanced development include lines in which fatty acid d e s a t u r a s e enzymes are s u p p r e s s e d in seed via antisense (Knutzon et al., 1992) or co-suppression m e t h o d s (Reiter et al., 1994). Down-regulation of the stearoyl-ACP desaturase is an example where m u t a t i o n breeding in oilseed rape (and also A r a b i d o p s i s thaliana) failed to identify lesions in the stearoyl-ACP desaturase gene or the expected corresponding increase in levels of stearic acid. This may be because there are multiple genes for this enzyme, and knocking out any one of t h e m will have no phenotypic effect. Alternatively, lesions in a locus significantly affecting stearoyl-ACP d e s a t u r a s e activity may be lethal. However, if multiple stearoyl-ACP d e s a t u r a s e genes share e n o u g h DNA homology a n d ant i s ens e control is limited to the p h a s e of seed development during storage lipid biosynthesis, it m a y be possible to engineer higher levels of stearic acid in transgenic canola oil. This is indeed the case (Knutzon et al., 1992), a l t h o u g h it r e m a i n s to be seen if the trait will be stable over m a n y generations in multiple environments. Regarding the d e s a t u r a s e activities t h a t convert oleic acid to linoleic acid and linoleic acid to linolenic acid ("D 12" and "D 15", respectively), mutation breeding h a s succeeded in developing lines with lowered polyunsaturated fatty acids. Perhaps the m o s t interesting involves increasing oleic acid by s u p p r e s s i n g activity of the D 12 d e s a t u r a s e . High oleic lines with seed oil containing a b o u t 75% oleic acid have shown good agronomic properties; however, efforts to raise oleic levels above 80% by combining two non-allelic high oleic m u t a t i o n s have resulted in yield penalties and poor agronomic characters. This a p p e a r s to be due to D 12 d e s a t u r a s e activity deficiencies in leaf and other parts of the plant. The DuPont group (Reiter et al., 1994) has addressed this problem by transgenic s u p p r e s s i o n of D 12 activity specifically in the seed. Field trials are currently u n d e r w a y with materials having seed oils considerably above 80% oleic. The second transgenic rapeseed p r o d u c t to be commercialized was the sale of seed from a variety of oilseed rape developed by Agrevo to be resistant to the herbicide glufosinate. The t r a n s g e n e basis underlying the herbicide resistance derives from observations (DeBlock et al., 1989) t h a t a Strept o m y c e s gene can be engineered to express in B. n a p u s and B. oleracea an enzyme t h a t degrades glufosinate. G l y p h o s a t e - r e s i s t a n t oilseed rape developed by Mo n s a nt o d e p e n d s partly on a bacterial gene coding for an enzyme active in the degradation of glyphosate (Barry et al., 1992). These transgenic approaches were able to take advantage of procaryotic genetic elements selected in bacterial environments. Another transgenic development t h a t highlights a synergistic contribution to both B r a s s i c a g e r m p l a s m and more conventional breeding approaches is engineered nuclear male-sterility (Mariani et al., 1990, 1992). Plant Genetic S y s t e m s scientists and breeders are close to commercializing hybrid canola seed in C a n a d a made possible with a male-sterile female p a r e n t t h a t can be directly selected after pollination. Selection against the male p a r e n t
302 during hybrid seed production is accomplished with the herbicide glufosinate; the above m e n t i o n e d herbicide resistance t r a n s g e n e is directly linked to the t r a n s g e n e encoding the nuclear male-sterility trait. The female parent of the hybrid cross is male-sterile due to a transgenic RNAse (again from a bacterial source) engineered to express specifically in the t a p e m m tissue. When expressed, the RNAse kills the hos t cell and t h u s blocks normal pollen development. Fertility can be restored with a transgene encoding an effective inhibitor of the RNAse enzyme; contribution of this gene from the male p a r en t allows efficient seed set by plants grown from F1 hybrid seed. Whereas both nuclear a n d cytoplasmic male-sterile g e r m p l a s m is available in m a n y Brassica species, the transgene system promises to be more efficient with no likely yield penalty as observed in some other systems. In addition, the transgene system m a y be easily transferred to plant species within and outside the crucifers, wherever transformation syst em s are available. Other examples of commercially interesting t r a n s g e n e s in the crucifers (Table 9.3) include an altered acetolactate s y n t h a s e enzyme conferring resistance to sulfonyl u r e a herbicides; Bacillus thurigiensis toxin genes conferring insect feeding resistance; a short chain acyl-ACP thioesterase from Cuphea hookeriana t h a t allows synthesis of C8:0 and C 10:0 fatty acids; a palmitoylACP thioesterase from C. hookeriana t h a t allows synt hesi s of high levels of palmitic acid; a lysophosphatidic acid acyltransferase cDNA from meadowfoam t h a t allows s y n t h e s i s of trierucin triacylglycerols; a ketoacyl-CoA synthase cDNA from Lunaria t hat allows B. napus to m a k e a C24"1 fatty acid; and a castor bean hydroxylase cDNA t h a t allows synt hesi s of ricinoleic acid by Arabidopsis. These examples and still others to come underscore the potentially wide impact of us i ng transgenes in Brassica a n d related genera.
Future p r o s p e c t s Work in progress with the model crucifer Arabidopsis thaliana has major implications for genetic engineering of Brassica crops. Many additional genes for tests in Brassica will become available t h r o u g h complete sequencing of the Arabidopsis genome, analysis of m u t a n t s , and gene isolation efforts. Genes t h a t e n h a n c e resistance to crucifer diseases will be of particular interest. The impact of genetic engineering is likely to be greatest with the oilseed b r a s s ic as because of the large acreages grown a n d their high commercial value. Resistance to herbicides, pathogens, and pests can enhance performance in the field a n d reduce costs to growers. Alteration of oil or protein quality permits production in rapeseed of improved p r o d u c t s or ones previously obtained in other ways. This may, of course, result in loss of revenue for the former sources of such pr oduct s (e.g., producers of tropical oils). Production of n o n - p l a n t products within seeds of B. napus ("molecular farming") could expand the us e of the crop still further.
303 The impact of transgenic Brassica vegetables is harder to predict. Because the vegetables are so diverse, less support is available for work on any given one. Obtaining the data required for regulatory and legal approval is onerous and expensive, especially for minor crops. On the other hand, transgenic resistance to herbicides, diseases or insects could have important environmental benefits for vegetable production by allowing use of modern herbicides and reduction in application of insecticides. Such changes could be especially beneficial in developing countries where applications of chemicals are often high and poorly regulated. Opportunities for use of other genes already identified in other systems include control of pollination, alteration of growth habits, or extension of shelf life (provided cost comparisons to other approaches are favorable). Current attention to Brassica vegetables as anticarcinogens m a k e s manipulation of nutrient and flavor components another attractive target for genetic manipulation. Quite apart from commercial applications, current studies of crucifer genomes combined with the ability to produce genetically altered plants provide m a n y opportunities to gain basic information about developmental biology, physiology, biochemistry, molecular biology, population genetics, and other aspects of Brassica species.
Acknowledgment The a u t h o r s t h a n k Dr. M.C. Christey, New Zealand Institute for Crop and Food Research, Ltd., for helpful comments about Agrobacterium rhizogenes-mediated transformation and for access to papers in press.
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312 Reiter, R. S., Mauvais, C. J., Ripp, K. G., Yadav, N. S., Kinney, A. J., Chen, Z., Debonte, L. R. and Hitz, W. D. 1994. Transgenic modification of seed lipid desaturation in Brassica napus. 4th International Congress of Plant Molecular Biology. Amsterdam, Abstract 1469. Sato, T., Thorsness, M. K., Kandasamy, M. K., Nishio, T., Hirai, M., Nasrallah, J. B. and Nasrallah, M. E. 1991. Activity of an S locus gene promoter in pistils and anthers of transgenic Brassica. The Plant Cell 3, 867-876. Scheffler, J. A., Parkinson, R. and Dale, P. J. 1995. Evaluating the effectiveness of isolation distances for field plots of oilseed rape (Brassica napus) using a herbicide-resistance transgene as a selectable marker. Z. Pflanzenzucht. 114, 317-321. Sjodahl, S., Gustavsson, H. O., Rodin, J. and Rask, L. 1995. Deletion analysis of the Brassica napus cruciferin gene crul promoter in transformed tobacco. Planta 197, 264-271. Srivastava,V., Reddy, A. S. and Guha-Mukherjee, S. 1988. Transformation and regeneration of Brassica oleracea mediated by an oncogenic Agrobacterium tumefaciens. Plant Cell Rep. 7, 504-507 Stewart, C. N. Jr., Adang, M. J., All, J. N., Raymer, P. L., Ramachandran, S. and Parrott, W. A. 1996. Insect control and dosage effects in transgenic canola containing a synthetic Bacillus thuringiensis crylAc gene. Plant Physiol. 112, 115-120. Swanson, E. B. and Erickson, L. R. 1989. Haploid transformation in Brassica napus using an octopine-producing strain of Agrobacterium turnefaciens. Theor. Appl. Genet. 78, 831-85. Tepfer, D. 1989. Ri T-DNA from Agrobacterium rhizogenes: a source of genes having applications in rhizospere biology and plant development, ecology and evolution. In: Kosuge, T. and Nester, E. W. (eds.), Plant-Microbe Interactions. Molecular and Genetic Perspectives, Vol. 3, McGraw-Hill, Inc. pp. 294-342. Thomzik, J. E. 1993. Transformation in oilseed rape (Brassica napus L.). In: Bajaj, Y. P. S. (ed.), Biotechnology in Forestry and Agriculture, Vol. 23, Plant Protoplasts and Genetic Engineering I V , Springer-Verlag, pp. 170-182. Thomzik, J. E. and Hain, R. 1990. Transgenic Brassica napus plants obtained by cocultivation of protoplasts with Agrobacterium tumefaciens. Plant Cell Rep. 9, 233-236 Toriyama, K., Stein, J. C., Nasrallah, M. E. and Nasrallah, J. B. 1991. Transformation of Brassica oleracea with an S-locus gene from B. campestris changes the self-incompatibility phenotype. Theor. Appl. Genet. 8 1 , 7 6 9 - 7 7 6 .
313 Turgut, K., Barsby, T., Craze, M., Freeman, J., Hodge, R., Paul, W. and Scott, R. 1994. The highly expressed tapetum-specific A9 gene is not re-quired for male fertility in Brassica napus. Plant Molec. Biol. 24, 97-104. Vanderkerckhove, J, Damme, J. Van, Lijsebettens, M. Van, Bottermanm J., Block, M. De, Vandewiele, M, Clercq, A. de, Leemans, J., Montagu, M. Van and Krebbers, E. 1989. Enkephalins produced in transgenic plants using modified 2S seed storage proteins. Bio/Technology 7, 929-932. Van Rooijen, G. J. H.,Van and Moloney, M. M. 1995. Plant seed oil-bodies as carriers for foreign proteins. Bio/Technology 13, 72-77. Verwoert, I.I.G.S., Linden, K. H. Van Der., Walsh, M. C., Nijkamp, H.J.J. and Stuitje A. R. 1995. Modification of Brassica napus seed oil by expression of Escherichia coli fabH gene, encoding 3-ketoacyl-acyl carrier protein synthase III. Plant Molec. Biol. 27, 875-886. Voelker, T. A., Worrell, A. C. anderson, L., Bleibaum, J., Fan, C., Hawkins, D. J., Radke, S. E. and Davies, H. M. 1992. Fatty acid biosynthesis redirected to medium chains in transgenic oilseed plants. Science 257, 72-74. Voelker, T. A., Hayes, T. R., Cranmer, A. C., Turner, J. C. and Davies, H. M. 1996. Genetic engineering of a quantitative trait: metabolic and genetic parameters the accumulation of laurate in rapeseed. The Plant Journal 9, 229-241. White, T.C, Simmonds, D., Donaldson, P. and Singh J. 1994. Regulation of B N l l 5 , a low-temperature-responsive gene from winter Brassica napus. Plant Physiol. 106, 917-928. Yadav, R. C., Konishi, H., Kamada, H. and Kikuchi, F. 1996. Transformation of Brassica carinata A. Braun using Agrobacterium rhizogenes. Cruciferae Newsl. 18, 44-45. Zaccomer, B., Cellier, F., Boyer, J. C., Haenni, A. L. and Tepfer, M. 1993. Transgenic plants that express genes including the 3' untranslated region of the turnip yellow mosaic virus (TYMV) genome are partially protected against TYMV infection. Gene 136, 87-94.
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Biology of Brassica Coenospecies C. G6mez-Campo (Editor) 91999 Elsevier Science B.V. All rights reserved.
315
10 CHEMICAL COMPOSITION E d u a r d o A. S. Rosa Horticulture Section. Universidade de Tras os Montes e Alto Douro. 5 0 0 1 - 9 0 9 Vila Real, Portugal.
Several species of Brassica a n d ally g e n e r a w h i c h are given a wide range of uses. A p a r t from direct h u m a n a n d a n i m a l c o n s u m p t i o n i n d u s t r i a l u s e s include the m a n u f a c t u r e of r a p e s e e d oil a n d the p r e p a r a t i o n of m u s t a r d products. R a p e s e e d oil r a n k s third b e h i n d s o y b e a n a n d oil palm, showing the i m p o r t a n c e of this product. After extraction of the oil from r a p e s e e d (Brassica n a p u s a n d B. rapa), the r e s i d u a l m e a l w h i c h is high in protein, is u s e d in a n i m a l feeds. Those w h i c h are u s e d for h u m a n c o n s u m p t i o n are often subjected to p r o c e s s e s s u c h as: blanching, freezing, freeze-drying, boiling or fermentation. D e p e n d i n g on the species c o n c e r n e d , a l m o s t a n y p a r t of the p l a n t p a r t including the roots, s t e m s a n d petioles, leaves, inflorescences a n d b u d flowers c a n be u s e d (Table 10.1). The p e r capita c o n s u m p t i o n of vegetables r a n k s in third place worldwide after cereals a n d o t h e r n o n - p l a n t sources (Table 10.2). Within the vegetables, Brassica crops r a n k third a m o n g the major vegetable p r o d u c i n g botanical g r o u p s in developed c o u n t r i e s after potatoes a n d t o m a t o e s , r a n k i n g also in third place in c o n s u m p t i o n (FAO, 1992). P r o d u c t i o n a n d c o n s u m p t i o n in developing c o u n t r i e s could be h i g h e r b u t take s e c o n d place to high calorie grain crops. In m o s t Brassica species, after the h a r v e s t of the m a i n p l a n t part, the r e m a i n i n g b i o m a s s m a y be u s e d for fodder. For i n s t a n c e , in cauliflower, broccoli a n d B r u s s e l s s p r o u t s , the p a r t s which are u s e d for h u m a n c o n s u m p t i o n r e p r e s e n t only a small portion of the total b i o m a s s production. T h u s , a c h e m i c a l c h a r a c t e r i z a t i o n of t h e s e "residual" p a r t s is i m p o r t a n t in order to evaluate their e v e n t u a l u s e as fodder. C o m p r e h e n s i v e information on the c h e m i c a l c o m p o s i t i o n of Brassica crops is also r e q u i r e d w h e n o t h e r u s e s are envisaged s u c h as medicinal applications a n d in the p r o d u c t i o n of a r o m a t i c s , c o s m e t i c s a n d additives a n d more recently for the control of soil-borne d i s e a s e s a n d n e m a t o d e s . With the d e v e l o p m e n t of analytical c h e m i s t r y , new m e t h o d s have been i n t r o d u c e d t h a t p e r m i t a more c o m p r e h e n s i v e knowledge of the chemical composition of the plants. In the past, food c o m p o s i t i o n t a b l e s were consi-
316 T a b l e 10. 1 Brassica species and their potential uses.
Species
Human consumption
Fodder
White cabbage (B. oleracea var. capitata)
Heads
Leaves and petioles
Savoy cabbage (B. oleracea var. bulata)
Heads
Leaves and petioles
Caulifower
Inflorescences
Leaves and petioles
Broccoli (B. oleracea var. italica)
Inflorescences
Leaves and petioles
Leaves
Leaves and petioles
Vegetative buds
Leaves and petioles
Roots
Roots, leaves and petioles
Loose heads
Leaves and petioles
Roots
Roots, leaves and petioles
(B. oleracea var. botrytis)
Kale, collards (B. oleracea var. acephala) Brussels sprouts (B. oleracea var. gemmifera)
Turnips (B. napus) Portuguese cabbage (B. oleracea var. tronchuda) Swede (B. napus var rapifera)
dered to s u p p l y e n o u g h information a b o u t the quality of a p a r t i c u l a r vegetable. Today, however, information on s e c o n d a r y p l a n t metabolites is often required. I n t e r e s t e x t e n d s beyond basic information a b o u t the levels of protein, fat, c a r b o h y d r a t e s , v i t a m i n s a n d minerals, a n d the s e c o n d a r y plant metabolites are a group of s u b s t a n c e s t h a t can explain some of the effects of vegetables in h u m a n s a n d a n i m a l s which in m a n y c a s e s h a d been already d e s c r i b e d by the a n c i e n t R o m a n a n d Greek writers b a s e d on empirical knowledge t h r o u g h the c e n t u r i e s . For i n s t a n c e , Plinius s t a t e d t h a t "Cruciferae are s u p e r i o r to o t h e r vegetables in their n a t u r a l properties". E u r o p e a n policy h a s focused on the quality of vegetables to the d e t r i m e n t of high yields a n d al-
317 T a b l e 10. 2
World food sources and average annual per capita food consumption, and its daily calorie, protein and fat contribution (Rubatzky and Yamaguchi, 1996).
Food source
Annual volume
Protein
Fat
(kg)
(cal/day)
(cal/day)
(cal/day)
Cereals
188
1371
33.4
5.7
62
141
2.0
0.3
Vegetables
69
46
2.5
0.4
Sugar
25
237
0.1
0.0
Pulses
6
58
3.6
0.4
Nuts and oils
7
50
2.4
3.6
Fruits
53
64
0.8
0.4
188
710
25.6
55.5
Starchy roots and tubers
Meats and non-plant sources
m o s t every c o u n t r y is c o n c e r n e d with diet a n d its effect on h u m a n h e a l t h with the objective of r e d u c i n g costs in h e a l t h care services. For instance, it is expected t h a t the h i g h e s t c a u s e of d e a t h in the world, h e a r t disease, which is responsible for 7.2 millions every year m i g h t be r e d u c e d with the improvem e n t of diet which s h o u l d include greater a m o u n t s of fruit a n d vegetables (50 th HWO Assembly, 1998). Nutrition is b e c o m i n g a n increasingly i m p o r t a n t factor in the choice of food p r o d u c t s and, as a c o n s e q u e n c e , a c h a n g e in eating habits, to e n s u r e a more h e a l t h y diet, h a s been n o t e d in m a n y countries. For i n s t a n c e Americans are m a k i n g food choices b a s e d on fat c o n t e n t ( J o h n s t o n , 1998). More c o m p r e h e n s i v e food labeling, indicating the major c o n s t i t u e n t s of the food together with the m o s t i m p o r t a n t beneficial effects is probably the next step. In fact, other c o n c e p t s have been recently i n t r o d u c e d which in the future migh also apply to B r a s s i c a p r o d u c t s , including "functional foods", "novel foods", "probiotics", "prebiotics", a n d "design foods". In this chapter, the chemical c o m p o s i t i o n of the m a i n B r a s s i c a crops will be a d d r e s s e d focusing on the information available from food composi-
318 tion tables. There will be special e m p h a s i s on s e c o n d a r y p l a n t metabolites, particularly the glucosinolates, a m a j o r group of c o m p o u n d s p r e s e n t in the family Brassicaceae (=Cruciferae) which have been the subject of several c o m p r e h e n s i v e reviews.
T h e i m p o r t a n c e o f t h e B r a s s i c a a n d a l l i e s in h u m a n a n d animal diet This group of p l a n t s are well a d a p t e d to a wide range of climatic conditions which p e r m i t s t h e m to grow in different latitudes t h r o u g h o u t the world. In a p a r t i c u l a r site u s i n g different species a n d primitive cultivars it is a l m o s t possible to grow Brassica p l a n t s t h r o u g h o u t the year. Good examples are kale a n d collards which might be available as fresh green vegetables the whole year a r o u n d even at higher n o r t h e r n latitudes, a situation that can be u s e d to a d v a n t a g e in the p r o d u c t i o n of fodder kales, a crop with a high u n tritive value within the Brassica group, as seen in Table 10.1. Brassica crops in general, could play a n even more i m p o r t a n t role in the nutritional quality of diets with better d i s s e m i n a t i o n of information a b o u t their nutritive value together with c h a n g e s in eating h a b i t s t h a t will benefit people, especially those of m a r g i n a l diets. However, in some countries, due to limited food availability, the t e n d e n c y is to meet caloric d e m a n d s first, regardless of other n u t r i t i o n a l n e e d s leading generally to u n d e r n o u r i s h m e n t or malnutrition. Dietary fibre (either soluble a n d insoluble), a low caloric value, the vitamin a n d m i n e r a l c o n t e n t s a n d the protein quality are some of the a r g u m e n t s to increase Brassica c o n s u m p t i o n . O t h e r nutritional factors p r e s e n t in this group of plants, which will be d i s c u s s e d later, also justify the recommendation for their increased c o n s u m p t i o n .
Brassica crops have also been grown as catch crops for forage (Sheldrick et al., 1981; Rosa a n d Heaney, 1996). By definition, a catch crop occupies the land for only p a r t of the growing season. They are utilized in the aut u m n a n d early winter as grazing either for sheep or, occasionally, for dairy cows. The dry m a t t e r c o n t e n t of b r a s s i c a s d e p e n d s on the crop species and growing season, varying between 2.0 % a n d 12.0 % a n d is considered to be highly n u t r i t i o u s (Kay 1971). Cruciferous crops, a l t h o u g h m u c h bulkier t h a n cereals, are capable of supplying high yields or energy per hectare with digestibility values of a r o u n d 0.7 a n d N c o n c e n t r a t i o n s between 20.0 to 39.0 g.kg -1 dry m a t t e r (Sheldrick et al., 1981). The a m i n o acids, lysine, methionine, cystein a n d t r y p t o p h a n , which are likely to limit growth in y o u n g animals are found in greater a m o u n t s in the green t h a n in the root crops (either Brassica napus, B. rapa or even s o m e t i m e s B. j u n c e a a n d also R a p h a n u s sativus). The crude protein c o n t e n t of root crops (10-13%) is also lower t h a n in the green crops (13-16%), being largely degradable in the r u m e n suggesting s u p p l e m e n t a t i o n with a protein which is only slowly degradable (Kay et al., no date).
319
The c h e m i c a l c o m p o s i t i o n o f B r a s s i c a crops During this c e n t u r y several food composition tables have been developed to supply information on the m os t diverse products, facilitating an improvement in diets and generally for a healthier population with less health risks. The information on vegetables is now comprehensive enough to take advantage of their characteristics to improve the diet, a l t h o u g h in m a n y countries due to social-economic c o n s t r a i n t s the food composition tables are not used e n o u g h or even at all. The B r a s s i c a c e a e is a major family of plants generally u s e d worldwide, including m e m b e r s which in m a n y situations are seen as having a low food value. In the following pages the compositional characteristics of this group of plants will be treated with e m p h a s i s in the genus Brassica.
General c o m p o n e n t s The major food composition aspects of B r a s s i c a oleracea plants m o s t commonly u s e d for h u m a n c o n s u m p t i o n are show n in Table 10.3. The high water content and the low fat a n d carbohydrate values are relevant features, being responsible for the low caloric value, a characteristic of these products. When comparing B r a s s i c a vegetables with other of high water content, the levels of fibre and protein are relatively high. Protein levels in Brussels s p r o u t s and kale are high each having over 4 % of fresh edible portion. B r a s s i c a are also a good source of minerals particularly, p o t a s s i u m a n d calcium, the latter being a b u n d a n t in kales and broccoli. These two brassicas together with Brussels s p r o u t s also have i m p o r t a n t levels of vitamins, particularly the two carotenoid anti-oxidants and vitamin C. In several cultivars of Chinese cabbage, vitamin C c o n t e n t was between 22 and 97 m g / 1 0 0 g of fresh weight whilst the carotenoid content (expressed as gcaroten) varied between 31 and 219 lug/100g fresh weight (Bajaj et al., 1991). Broccoli and B r u s s e l s s p r o u t s are an i m p o r t a n t source of lutein (Table 10.4). Increased bioavailability of carotenoids to h u m a n s was reported after the typical "in home" cooking (Table 10.4) (Scott a n d Hart, 1995). Kale an d Br us s el s s p r o u t s have the highest amino acid content followed by broccoli. Special attention should to be paid to the nine essential amino acids which a n i m a l s are strictly d e p e n d e n t on. Among these, methionine permits the entry of s u l p h u r in animal cells a n d is involved in the synthesis of cysteine a n d other s u l p h u r c o m p o n e n t s with major functions for cell metabolism (protein biosynthesis, catalytic activities, detoxification processes, etc.) (Gaki~re et al., 1999).Of the two s u l p h u r amino acids, methionine and cysteine, the first is c o m m o n to all B. oleracea forms, whilst cysteine was the only one reported in white and red cabbages a n d in kale which has more t h a n twice the a m o u n t pr es ent in the other two varieties (Table 10.3). The other major group of crops within the g e n u s B r a s s i c a are the oilseed species (B. n a p u s , B. rapa and B. juncea) generally k n o w n as rapeseed.
Table 10.3 a - Major constituents of the most commonly used Brussicu oleruceu types for human consumption. Brassica crops
Water
(“w
Protein Fat Carbohydrates Fibre
Na
K
Mg
Ca
P
Fe
Se
(mg) (mg) (mg) (mg) (mg) (mg) (pg)
1
B
(w) (m)
Mn
Cu
Zn
(vg)
(pg)
(
(8)
(g)
(g)
89.70 3.30 89.20 3.60 88.20 4.40
0.20 0.35 0.90
2.51 5.70 1.80
3.00 1.40 2.60
19.0 23.0 8.0
373.0 389.0 370.0
24.0 105.0 23.0 101.0 22.0 56.0
91.60 2.46 91.10 2.60 88.40 3.60
0.28 0.22 0.90
2.34 5.20 3.00
2.92 0.88 1.80
16.0 16.0 9.0
328.0 328.0 380.0
17.0 20.0 17.0
20.0 25.0 21.0
54.0 0.63 0.94 0.64 150.0 170.0 - 57.0 1.00 - 300.0 64.0 0.70 Trace Trace
41.92
257.0
30.00
600.0
85.00 4.45 84.80 4.20 84.30 3.50
0.34 0.40 1.40
3.29 8.60 4.10
4.40 1.60 4.10
9.6 22.0 6.0
387.0 404.0 450.0
22.0 26.0 8.0
31.0 61.0 26.0
83.6 0.60 40.00 0.70 270.0 260.0 - 80.0 1.50 77.0 0.70 - 200.0 ND 1.00
64.86
590.0
20.00
500.0
86.30 4.30 85.00 4.00 2.93 -
0.90 0.80
2.54 6.80
4.20 1.40
42.0 55.0
31.0 212.0 35.0 177.0 21.4 117.1
87.0 1.90 71.0 2.00 78.4 1.07
55.60
330.0
-
490.0 383.0 357.0
(g)
W h)
0
Font
broccoli [ B. oleracea
convar. bottytis var. italica ]
82.0 77.0 87.0
1.30 0.70 15.00 160.0 260.0 126.00 605.0 - 1.20 - 1.70 Trace 2.00 - 200.0 20.00 600.0
1
2 3
cauliflower [ B. oleracea
convar. bottytis var. botrytis ]
-
-
I 2 3
Brussels sprouts [ B. oleracea
convar. oleracea var. gemmi$ra ]
-
-
I 2 3
kale [ B. oleracea
convar. acephala ]
-
-
-
1.37 12.00 240.0 550.0
-
-
- 495.0
-
- 1470
Portuguese kale [ B. oleracea var. tronchuda 0..costata
- _ - 855.0 -
840.0
42.36
239.0
-
-
-
-
310.5
31.1 486.0
69.0
1.70
-
91.80 1.50 0.18 91.70 1.60 0.22
3.54 6.10
2.50 1.00
4.0 17.0
266.0 257.0
18.0 35.0 17.0 43.0
30.0 0.92 36.0 0.70
100.0
90.49 1.37 0.20 92.50 1.40 0.22 90.70 1.40 0.20
4.16 5.10 5.00
2.96 0.09 2.10
11.7 17.0 7.0
208.0 272.0 240.0
23.0 16.0 6.0
46.0 49.0 49.0
27.5 2.51 37.00 1.93 600.0 100.0 - 29.0 0.50 - 200.0 29.0 0.50 Trace 2.00
33.31
90.00 2.95 90.50 2.50
2.41 4.90
2.57 0.60
9.0 20.0
252.0 262.0
12.0 20.0
47.0 54.0
55.6 1.23 22.00 51.0 0.80
34.68
-
4.00
I 2 4
4
red cabbage [ B. oleracea
convar. capitate var. rubra ]
-
5.20 250.0
-
-
100.0
-
-
-
1
2
white cabbage [ B. oleracea convar. capitata var alba ]
224.0
1
10.00 200.0
2 3
-
-
Savoy cabbage [ B. oleracea
convar. capitate var. sabauda I
0.38 0.24
Units- d l 0 0 g edible portion, I-Adaptedfrom Souci et a l ( l 9 8 4 ) 2-Adaptedfrom Rubatzky and Yamaguchi ( I 996) 3-Adaptedfrom 4-Adaptedfrom E.Rosa and R.Heaney (1996)
5Ih
-
300.0 200.0
-
-
-
262.0
-
suppl. t o McCance and Widdowson ‘s (1991)
1
2
Table 10.3 b - Major constituents of the most commonly used Brassica oleracea types for human consumption.
broccoli [ B. oleracea convar. botrytis var. italica 1
cauliflower [ B. oleracea
convar. botrytis var. botrytis ]
Brussel sprouts [ B. oleracea convar. oleracea var. gemmiferal
kale
[ B. oleracea
convar. acephala ]
red cabbage [ B. oleracea convar. capitate var. rubra 1
white cabbage [ B. oleracea
convar. capitate var alba ]
Savoycabbage [ B. oleracea
convar. capitate var. sabauda 1
846.0 -
575.0
10.4
174.0
98.5
- 100.0 -
-
110.0 100.0
50.0
-
-
447.0
275.0
126.0
-
215.0 5170.0
-
167.0
-
1.00
-
- _
100.0 100.0
0.60
-
- _
I .29
280.0 0.50
_
-
-
140.0
ND
1.01
200.0
1.50
-
-
280.0 1.50
-
-
- - - - -
- - - - -
- I
- - 2
- - 3
-
I .oo
370.0 0.40
100.0 110.0
250.0 210.0
2.10
-
250.0 0.50
-
187.0 105.0 300.0 69.0 240.0 140.0 250.0 52.0 140.0 130.0 64.0 230.0 180.0 69.0 I - 119.0 - - - - - - - - - - - - 2
0.32
150.0 2.00
35.0
-
-
71.8
79.5
48.6 60.0
37.3 50.0
0.32
0.26
-
-
-
59.2 60.0
-
-
-
0.43
-
-
336.0 0.40
50.0 50.0
39.0
140.0 110.0 170.0 48.0 77.0 110.0 34.0 150.0 35.0
-
68.0 80.0
40.0
66.0
73.0 110.0 49.0 75.043.0 - -
-
-
_
125.0
_
0.67
24.5
_
-
0.60
_
15.0
_
111.0 115.0 190.0 63.0 150.0 130.0 160.0 50.0 120.0 120.0 37.0 170.0 - - I 109.0- - - - - - - - - 2 90.0 87.0 - - - - - - - - - - - - 3
_
134.0 150.0
- 110.0 817.0
178.0 200.0
-
- _
63.8 60.0
-
_
-
-
-
-
-
174.0 3.08
182.0 112.0 280.0 110.0 250.0 210.0 230.0 40.0 150.0 160.0 50.0 240.0 98.0- - - - 135.0 115.0 - - - - - - - - - - - -
_
-
31.0
- - _
0.21
180.0 0.10
34.0
0.33
0.21
156.0 0.10
90.0
-
-
-
-
-
- - _ _ _ _
50.0 110.0 27.0 58.0 - -
71.0 -
-
45.2 100.0 25.0 50.035.0 -
65.0
39.0
49.4 150.0 47.0 47.0-
92.0
-
-
43.0
-
-
-
-
-
-
56.0 13.0 30.0 38.0 - 42.0
- -
-
- - _
- 27.0
- _ - _ _ -
-
-
_
-
-
100.0 190.0 27.0 120.0 110.0 32.0 140.0 -
2 3
- 30.0 I - - 2
61.0 14.0 32.0 42.0 12.0 46.0
-
- I
_ _ _
-
I 2
3
- - I - - 2
Units- @I00 g edible portion. I - Adaptedfrom Souci et al(l984) 2 - Adaptedfrom Rubatzky and Yamaguchi (1996) 3 - Adaptedfrom 5Ihsuppl to McCance and Widdowson's (1991)
322
Table 10.4 Carotenoid content (pg/100g wet weight, as eaten) of some Brassica oleracea varieties (Scott and Hart, 1995)
Vegetable
Type
Lutein
Brussels sprouts
Raw
610
441
112
Brussels sprouts
Cooked
621
411
144
Broccoli-fresh
Raw
1614
800
119
Broccoli-fresh
Cooked
1949
1125
256
White cabbage
Raw
80
51
8
White cabbage
Cooked
111
65
6
Savoy cabbage
Raw
103
50
nd
Savoy cabbage
Cooked
341
240
33
Savoy cabbage
Outside 14457
10020
1829
Leaves
Zeax 13-cryp Lyco m-car 13-car cis 0-car
Cauliflower
Raw
Tr
nd
nd
Cauliflower
Cooked
Tr
nd
nd
The seed of these three species contain 40 to 50% of oil which was originally u s ed for lighting and a lubricant, and now, after refining mainly for edible purposes. The fatty acid composition of seed oils affects their chemical, physical and nutritive characteristics (Rudloff and Wehling, 1998). One of the major fatty acids in oilseed, erucic acid (C22:1), responsible in part for the antinutritive properties of rapeseed oil, has been reduced by selective breeding from 45% to less t h a n one percent in the so called "double low" cultivars of rapeseed (low in erucic acid and glucosinolates). The fatty acid composition of these cultivars h a s higher (55-65% and could be up to 80%) oleic acid (C18:1) an d lower (20-23% and even down to 3%) linoleic acid (C18:2) contents t h a n m o s t other vegetable oils whilst the linolenic acid (C 18:3) content is higher (8-12%) t h a n in sunflower, olive or poppy oils and similar to soybean (8%) (Krzymafiski, 1998; Rudloff and Wehling, 1998). The quality of the oil can be increased by new techniques involving the fusion of protein / p e p t i d e s rich in essential amino acids, enzymes or t h e r a p e u t i c / p h a r m a c e u tical proteins to oleosins (structural proteins closely associated with the oil body, the n a t u r a l oil storage organelle of the plant seed) (van Rooijen et al., 1998). After oil extraction the residual rapeseed meal const i t ut es 52-58% of the original seed weight. This meal has a protein content of 38 to 46% (levels
323 in B. j u n c e a being slightly higher (45.9%) than B. n a p u s and B. rapa (44.6 and 43.1%, respectively)) (Newkirk et al., 1997), whilst levels of the amino acids lysine, methionine, cysteine, threonine and tryptophan compared favorably to other oil meals and cereals (Krzymafiski, 1998). The seed protein content of either B. n a p u s and B. j u n c e a might be increased by nitrogen and sulphur fertilizations (Trivedi and Sharma, 1997; Wang et al., 1997; Aulakh et al., 1995). The fibre content (11%) of the seed, mainly localized in the hulls and accounting for 27 to 30% of the rapeseed meal, is higher t h a n that of soybean seed (7%). The meals from B. j u n c e a contained less (27%) total dietary fibre t h a n B. n a p u s and B. rapa (29.5 and 29.7%, respectively) (Newkirk et al., 1997). The utilization of these high-quality products is more or less reduced due to oil mill processing but the aqueous enzymatic extraction procedure h a s been further improved to allow more optimal utilization of the various high-quality products in oilseed rape, resulting in a "Green Chemistry" biorefining process (Bagger et al., 1998). Another important aspect of rapeseed chemical composition is the chlorophyll content of the seed. The presence of as little as 2% of green seeds resuits in a downgrading of the crop (Morissette et al., 1998). Thus, seeds of B. rapa and B. n a p u s have been submitted to breeding programmes and genetic transformation in order to reduce chlorophyl content, seed coat and levels of polyphenols resulting in reduced off-flavors and odors and improved shelf life of the oil (Morissette et al., 1998). Other species such as Indian (B. juncea) and Ethiopian (B. carinata) mustards, with reported levels of erucic of between 0 to 50% of the total fatty acid content, might be used as oilseeds for h u m a n and industrial purposes u n d e r Mediterranean or semi-arid conditions, due to their resistance to drought, to pod shattering and to a wide range of diseases and pests (Velasco et al., 1998; Getinet et al., 1996).
S e c o n d a r y plant m e t a b o l i t e s : t h e g l u c o s i n o l a t e s Occurrence and distribution When considering the secondary metabolites of Crucifers, there can be little doubt t h a t the glucosinolates, a class of sulphur-containing glucosides are by far the most important. Although more than 100 such compounds are known, only a r o u n d 15-16 occur in significant a m o u n t s t h r o u g h o u t the brassicas a n d within any particular species the n u m b e r is usually m u c h lower. Glucosinolates are present to some extent in the seeds, roots, stems, leaves and flowers of all B r a s s i c a species in which they co-exist with an enzyme called myrosinase (thioglucoside glucohydrolase, E.C. 3.2.3.1.) which mediates their breakdown to a range of physiologically active compounds. The presence of high levels of such c o m p o u n d s in rapeseed meal, a by-product of the rape-oil industry, rendered the meal unsuitable for use in m a n y
324 potential a n i m a l feeding applications t h u s s t i m u l a t i n g i n t e r n a t i o n a l r e s e a r c h p r o g r a m s a i m e d at r e d u c i n g the levels of these c o m p o u n d s . F u r t h e r r e s e a r c h i m p e t u s was g e n e r a t e d w h e n s u l p h u r c o m p o u n d s derived from g l u c o s i n o l a t e s were shown to be partly r e s p o n s i b l e for the flavor of B r a s s i c a vegetables a n d t h a t selection of varieties for breeding p u r p o s e s , b a s e d on glucosinolate criteria might afford a m e a n s of d e t e r m i n i n g this imp o r t a n t a t t r i b u t e . R e s e a r c h was f u r t h e r s t i m u l a t e d w h e n studies showed t h a t glucosinolates are closely involved in the prevention of insect a n d fungal a t t a c k in p l a n t s a n d m o r e recently, t h a t t h e y m a y have a n i m p o r t a n t role in the control of biological p r o c e s s e s a s s o c i a t e d with the initiation and progression of carcinogenic p r o c e s s e s in m a n .
Chemistry In general, glucosinolates conform to the basic s t r u c t u r e shown in Figure 10.1 (Fenwick et al., 1983a). The s t r u c t u r a l diversity of this large group of c o m p o u n d s is due a l m o s t entirely to the different s u b s t i t u e n t s possible at the s i d e c h a i n position R, a l t h o u g h some exceptions have been reported (Sor e n s e n , 1990), n o t a b l y esterification of the s u g a r moiety with sinapic, caffeic or malic acids.
R - C - S - 13 - D - Glucose II N " OSO3
Figure 10.1 General s t r u c t u r e of glucosinolates. The R s u b s t i t u e n t m a y be a n alkyl or alkenyl side c h a i n which itself m a y c o n t a i n s u b s t i t u e n t hydroxyl g r o u p s or s u l p h u r in various oxidation states. Alternatively, the R s u b s t i t u e n t m a y be an a r o m a t i c or a hetero-aromatic group, the possible hydroxy or m e t h o x y s u b s t i t u e n t s which f u r t h e r complicate the picture. As will be s h o w n below, this variability in the sidec h a i n at R is d u e to the biosynthetic p a t h w a y s involved in the formation of the glucosinolate a n d as a c o n s e q u e n c e of these v a r i a t i o n s a great n u m b e r of b r e a k d o w n p r o d u c t s are possible. The s t r u c t u r e s a n d n a m e s of the glucosinolates m o s t c o m m o n l y found in b r a s s i c a s (particularly in B. oleracea cultivars, B. rapa a n d in the a m p h i diploid B. napus) are given in Table 10.5 (Bjerg a n d S o r e n s e n , 1987). In addition to t h e s e m a j o r c o m p o u n d s , a f u r t h e r 15-16 glucosinolates are commonly detected b u t t h e s e are u s u a l l y p r e s e n t only in m i n o r a m o u n t s (< 10 p m o l / 1 0 0 g fresh weight).
325
Table 10. 5 Structure of the side chain R of the major glucosinolates occurring in the Brassicaceae (from Bjerg and Sorensen, 1987). Chemical name
Trivial name
CH2=CH-CH2 -
2-propenyl- or allyl glucosinolate
Sinigrin
CH2=CH-CH2-CH2 -
but-3-enyl glucosinolate
Gluconapin
CH2=CH-CH2-CH2-CH2 -
pent-4-enyl glucosinolate
Glucobrassicanapin
CH2=CH-CH-CH2 -
2-hydroxybut-3-enyl glucosinolate
Progoitrin
CH2=CH-CH2-CH-CH2 I OH
2-hydroxypent-4-enyl glucosinolate
Gluconapoleiferin
CH3-SO-CH2-CH2-CH 2-
3-methylsulfinylpropyl glucosinolate
Glucoiberin
CH3-SO-CH2-CH2-CH2-CH2-
4-methylsulfinylbutyl glucosinolate
Glucoraphanin
2-phenethyl glucosinolate
Gluconasturtiin
indol-3-ylmethyl glucosinolate (R1 = R4 = H)
Glucobrassicin
1-methoxyindol-3-ylmethyl glucosinolate (R1 = OCH3 ;R4 = H)
Neoglucobrassicin
4-hydroxyindol-3-ylmethyl glucosinolate (R1 = H ; R4 = OH)
4-Hydroxyglucobrassicin
4-methoxyindol-3-ylmethyl glucosinolate (R1 = H ; R4 = OCH3)
4-Methoxyglucobrassicin
Structure of R
Aliphatic glucosinolates
i
OH
Aromatic glucosinolates
Indole glucosinolates
326
I
~-z
o I
o o I
o
0
~
0
_~
_~ ~ I-i
.=~
0
rm
I
~--Z
o-z+
II o Z
r~ I
~
o
I
r
u=
o
2.5
2.23.2
,
m
t~ (o ~
100 %
C
. . m
43.5
Cr Jde fat !
i
100 % Figure 13.1 Chemical composition of the seed of a 00-cultivar of oilseed rape, Brassica napus (after Thies, 1994).
423 and B. rapa (Downey, 1964), followed by "zero erucic" B. j u n c e a (Kirk and Oram, 1981) and B. carinata (Alonso et al., 1991). This change in erucic acid content led to c o n c o m i t a n t shifts of all other fatty acids c o m p o n e n t s for purely calculatory r e a s o n s (see Table 13.3). While the relative increase in linoleic acid (vitamin F) was welcomed by h u m a n nutritionists, the technological disadvantage of the relatively high linolenic acid cont ent induced extensive breeding efforts (R6bbelen and Thies, 1980a; Scarth et al., 1995). These yielded promising lines with no more t h a n 3% linolenic acid (Rficker a n d R6bbelen, 1996), b u t none of these h a s yet been established at larger scales in agricultural productions. F u r t h e r d e m a n d s of the food i n d u s t r y were met by the breeders after m ut ageni c seed t r e a t m e n t a n d extensive screening, with B. n a p u s forms containing high oleic acid a n d others (B. rapa) with elevated palmitic acid c o n t e n t s (Table 13.3). For oleochemical purposes, where rapeseed oils with high erucic acid contents are desired, B. n a p u s lines with up to 60% erucic acid have been selected from interspecific crosses with preselected parents, i . e . B , oleracea cony. botrytis x B. rapa subsp, trilocularis 'Yellow Sarson' (Lfihs and Friedt, 1994). But m u c h beyond any n a t u r a l variability, molecular t r ans f or m at i on today offers the potential to produce transgenic varieties with u n u s u a l fatty acid profiles or even principally new c o n s t i t u e n t s not synthesized in the conventional B r a s s i c a seed till now (Table 13.4; for review see Murphy, 1994; Voelker, 1997). Evidently, the composition of storage c o m p o u n d s in the B r a s s i c a oilseeds is r e m a r k a b l y open to change, a n d breeding is highly promising provided efforts are sufficiently c o n t i n u o u s an d intense. Meal: The reduction of glucosinolates in the traditional rapeseed to less t h a n 10 % of their original contents (see reviews cited above) was a n o t h e r dramatic d e m o n s t r a t i o n of the potential of conventional breeding (R6bbelen and Thies, 1980 b). Oilseed meal from the new 00-rapeseed cultivars now allows full exploitation of the valuable protein in animal feed mixtures. However, the net protein utilization of rapeseed meal is still m u c h below soybean meal and the total of u n d e s i r e d c o m p o n e n t s still a m o u n t s to more t h a n 25% of the dry m a t t e r (Figure 13.1; for review see Bell, 1993; Uppstr6m, 1995). Hemicellulose with a b o u t 13 % of the dry m a t t e r const i t ut es a major share of the detrimental meal constituents. Youngs (1967; see also Downey et al., 1975) was the first to draw attention to the thin seed coat of yellowseeded B r a s s i c a forms. Since t hen breeders have wished to reduce the total fiber co n te n t of the meal by introducing this easy-to-screen c h a r a c t e r into their cultivars. However, the a p p r o a c h was more complicated t h a n expected and the first phenotypically stable lines of B. n a p u s were obtained only recently (Stringham et al., 1974; Chen and Heneen, 1992; Rashid et al., 1994; Tang et al., 1997). In the dehulled meal fraction, on the other h a n d , soluble oligosaccharides are the major u n d e s i r e d carbohydrates, i.e. raffinose (0,3 %, Gal-Glu-Fru) and stachyose (2,5 %, Gal-Gal-Glu-Fru), which are undigestible for swine (and h u m a n s ; see also Slominski et al., 1995); they a c c u m u -
424 T a b l e 13.3 Fatty acid c o m p o s i t i o n o f seed oils from B r a s s i c a species (abridged after U p p s t r 6 m , 1995); ~A u l d e t a l . , 1992; 2 including 4% 16" 1. Cultivar/type (a)
Fatty acid content (%) (b) 16:0
18:0
18:1
18:2
18:3
20:1
22:1
Traditional types
n
Victor (winter)
3.0
0.8
9.9
13.5
9.8
6.3
52.3
n
Target (spring)
3.0
1.5
20.9
13.9
9.1
12.2
38.6
r
Duro (winter)
2.0
1.0
12.9
13.4
9.1
8.9
49.0
r
Echo (spring)
4.5
1.3
33.3
20.4
7.6
9.4
23.0
r
Yellow Sarson
1.8
0.9
13.1
12.0
8.2
6.2
55.5
j
Indian origin
2.5
1.2
8.0
16.4
11.4
6.4
46.2
c
Ethiopian mustard
3.2
0.9
9.8
16.2
13.9
7.5
41.6
Zero erucic types
n
Low 22:1
4
2
62
20
9
2
z l~,
1
0 04
.
0 co
_~
