Plant cell culture protocols

VOLUME 111

M E T H 0 D S I N M 0 L E C U L A R B I 0 L 0 G Y'M John M. Walker, SERIES EDITOR 132 131 130 129 128 127 I 26 125 124 123 122 121 120 119 I I8 117 116 115 114 113 112

Biomformatics Methods and Protocols, ed1ted by Stephen M1sene1 and Stephen A Kwwetz, 1999 Flavoprotein Protocols, ed1ted by S K Chapman and G A Re1d /999 Transmption Factor Protocols, edited by Martm J T!•mm< /999 NMDA Protocols, edt ted by Mm L1, /999 Molecular Methods In Developmental Biology· Xenopus Developmental Biology Protocols, Vol. II, edited by Rocky S Tuan, /999 Developmental Biology Protocols, Vol. I, edited by Rody S Tuan, /999 Protein Kmase Protocols, ed1ted by Ala 1 h) IS recommended, followed by a thorough nnse m running tap water. Both mutagens are unstable in aqueous solution, with the advantage that the low concentratiOns used here do not need to be washed from the cells. The rate of breakdown is highly pH-dependent, and it IS important that mutagen solu-

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tions should be prepared using culture medium at tts normal p H (5.6). Further information about these mutagens can be found in ref. (9). It ts also posstble to select spontaneous mutants without mutagenic treatment, but a larger number of cultures may be requtred. Shoot regeneration from the resistant cell hnes ts a critical phase of the proce­ dure. The efficiency with which regeneration can be mduced by mampulating the hormonal composition of the medium (i.e., usmg RMOP or RMB) ts variable, and in the case of cell suspension cultures, may be quite low. Regeneration can often be stimulated by the mcluswn in the medmm of silver ions, m the form of silver nitrate at I 0 or 50 mg/L (10) . Resistance to the antimetabolite can also b e checked i n seedling progeny and callus derived from them. Mutagens can also be supplied at higher concentrations for short time penods to freshly isolated protoplasts or cell suspension cultures. The method ts fairly gen­ eral for a range of mutagens, mcluding NEU, NMU, and EMS In all cases, pre­ liminary expenments are needed to establish a suitable concentration and duration of treatment, one that gives a reduction to 1 0-50% colony formatwn m plated protoplasts, or cell suspension cultures compared to untreated controls. Recom­ mended concentration ranges to investigate are 0.3-1 0 rnM for NEU or NMU, or 0. 1-3% v/v EMS The duratiOn of treatment should be about 60 mm, after whtch the cells should be washed twice with fresh culture medium, before contmumg to culture and plating m the usual way. EMS is a volatile Iiqmd that needs to be dispensed into the liquid medmm m a fume hood. Prior to dtsposal, solutions contammg EMS should be inactivated by gradual addttion to a large excess of 3 M KOH in 95% ethanol, heated under reflux, repeatedly stirred for 2 h before dis­ posal down the drain, chased by a large volume of tap water UV mutagenesis t s best appl ied to protoplasts 24 h after tsol ation. D tshes o f protoplasts are placed under the UV source and the l ids removed before it ts turned on. A dose givmg 1 0-5 0% subsequent colony formatiOn com­ pared to nonmutagemzed contro ls should be used (typ tcally in the range 2 200-2000 erg/mm ).

References 1 . Dix, P. J. ( 1 986) Cell hne selection, in Plant Cell Culture Technology (Yeoman, M. M , ed.), B lackwell Scientific, Oxford, pp. 143-20 1 2. Dix, P. J. ( 1 994) IsolatiOn and charactensation of mutant cell lines, in Plant Tis­ sue Culture-Theory and Applications (Vasil, I . K. and Thorpe, T. A., eds.), Kluwer Academic, Dordrecht, pp. 1 19-138. 3 Dix, P. J. (ed.) ( 1 990) Plant Cell Line Selection. VCH, Weinhetm. 4. Negrutm I. (1 990) In vitro mutagenesis, in Plant Cell Line Selection (Dix, P. J , ed), VCH, Weinhetm, pp. 1 9-3 8. 5. Dix, P. J. ( 1 993) Use of chemical and physical mutagens zn vitro, m Plant Tissue Culture Manual · Fundamentals and Applicatwns (Lmdsey, K., ed. ), Kluwer Aca­ demtc, Dordrecht, F 1 , pp. 1-1 7.

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6. Maliga, P. ( 1 984) Cell culture procedures for mutant selection and characterisation

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m Nzcotzana plumbaginifo/za, in Cell Culture and Somatzc Cell Genetzcs ofPlants, vol. 1 , Laboratory Procedures and Thezr Applzcations (Vasil, I. K., ed.), Aca­ demic, New York, pp. 552-5562 Kmg, P. J. ( 1 9 84) InductiOn and mamtenance of cell suspensiOn cultures, m Cell Culture and Somatic Cell Genetzcs ofPlants, vol. 1 , Laboratory Procedures and Thezr applzcatzons (Vasil, I. K , ed ), Academic, New York, pp 1 30-1 3 8 Gihs sen, L. W J., Hamsch ten Cate, C. H , and Keen, B . ( 1 983) A rapid method of determinmg growth characteristics of plant cell populations in batch suspen­ sion culture. Plant Cell Rep 2, 232-23 5. Hagemann, R. ( 1 982) Induction ofplastome mutatiOns by nitrosourea compounds, in Methods zn Chloroplast Molecular Bwlogy (Edelman, M., Hallick, R. B . , and Chua, N H., eds ), Elsevier/North Holland, BIOmedical, Amsterdam, pp. 1 1 9-- 1 27 Pumhauser, L., Medgyesy, P., Czako, M., Dix, P. J., and Marton, L. ( 1 987) Sttmu­ latwn of shoot regeneration m Trztzcum aestzvum and Nzcotzana plumbagznifolza VIv tissue cultures usmg the ethylene mhibitor AgN03 • Plant Cell Rep. 6, 1 -4

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The Ge neration of Plastid Mutants In Vitro Philip J . Dix

1 . Introduction The plastid genome (or "plastome") encodes a number of proteins associ­ ated with the structure and function of chloroplasts, as well as tRNAs and rRNAs associated wtth the plastid translational machinery (1,2). Although there have been numerous studtes on the genetics of algal chloroplasts, similar stud­ Ies with higher plants have been hampered by the uniparental (maternal) pat­ tern of transmissiOn of chloroplasts observed in most species and also the shortage of suitable genetic markers (3). Two developments have added impetus to studies on htgher plant plastid genetics. First, procedures have been developed (4, 5) for the more efficient generation of plastome mutations. Second, breakthroughs m the genetic trans­ formation of plastids (6, 7) have led to an appreciation of the great biotechno­ logical potential of expressing foreign genes in the plastids (8). DNA delivery mto plastids has been achieved through biolistics or PEG-mediated uptake mto protoplasts, and selection of transformants IS based on one of two alternate strategies (9). Of these, the more attractive IS the use of plastid mutations con­ fernng msensitivity to antibiotics (10). As well as rapidly generatmg homo­ plasmic transplastomic hnes, these avoid the controversial use of bactenal antibiOtic resistance genes (9) and indeed can restrict the foreign DNA intro­ duced to solely the gene of interest. Thus, plastid mutations are now of interest both for the fundamental infor­ mation they can provtde on plastid function and as a means for selecting gene­ tically transformed plastJds. The protocols given here describe two reliable procedures to select for mutations m plastid ribosomal RNA or protem genes conferring insensitivity to several antibiOtics (streptomycm, spectinomycin, lmcomycin), m Solanaceous species. They use the mutagen mtroso-methylurea From Methods m Molecular Btology, Vol 111 Plant Cell Culture Protocols Ed1ted by R D Hall © Humana Press Inc , Totowa, NJ

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Table 1 Components of Modified K3 Medium Used for Isolation and Culture of N. plumbaginifolia Protoplasts8 Ingredient CaC12 2H20 KN03 NaH2P04 2H20 NH4N03 (NH4hS04 CoC12 6H20 CuS04 7H2 0 FeS04 7H20 Na2 EDTA H3B03 •

·

•

•

mg/L 900 2400 1 20 240 1 30 0.025 0.025 27.85 37.3 3.0

Ingredient

mg/L

KI MnS04 4H20 Na2 Mo04 2H20 ZnS04 7H2 0 myo-mosJtwl Nicotinic acid Pyridoxine HCl Thiamine HCl Xylose pH

0.75 6.7 0.24 2.3 1 00 1 .0 1 .0 10 250 5.6

·

·

"Sugar 1s not mcluded m the above table Sucrose or glucose 1s added as descnbed m the hst under Subheading 2. It 1s convement to make up 5 L of the mediUm double-strength, om1ttmg only the sucrose or glucose, and stormg at -20°C. It is generally recommended to filter-stenhze the mediUm, but for N plumbagmifolla protoplasts, good results can be obtamed w1th autoclaved mediUm

(NMU), which has been shown to be efficient in targeting the plastome (11). The procedure usmg protoplasts IS effective for Nlcotwna plumbagmifolza (4) and with modifications (see Note 4) can also be used to obtam herbicide resis­ tant mutants. The leaf stnp procedure (5) has been used successfully for sev­ eral species (N1cotiana tabacum, N. plumbaginifolia, Nicotiana sylvestns, Solanum mgrum, and Lycopers1con peruvwnum) and can also generate chloro­ phyll-deficient mutants on nonselective plates. 2 . Materials I . Shoot cultures of the species to be used (see above)· The procedures for obtain­ ing and maintaining these cultures, starting from seed, are as descnbed m Chap­ ter 27, this volume. 2. Protopl ast enzyme solutiOn. Modified K3 medmm {12; Table 1) containing 0.4 M sucrose and 0.5% Dnselase (Kyowa Hakko Kogyo Co., Tokyo, Japan) (see Note 1). K3 can be filter-sterilized or autoclaved m a pressure cooker, and stored for up to 4 wk, preferably m the cold room Driselase must be added immediately before use, and the resultmg enzyme solutiOn filter-stenhzed. 3. Protoplast wash solution, W5 (12): 1 5 0 rnM NaCl, 1 25 rnM CaC12, 5 mM KCi, 5 mM glucose, pH 5.6 W5 may be autoclaved and stored m the cold room for up to 2 mo 4 Protoplast culture medmm Modified K3 supplemented w1th 0 4 M glucose, 0 1 mg/L 2,4-dlchlorophenoxyacetic ac1d (2,4-D, from a 0. 1 mg/mL stock pre-

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pared w ith the dropw ise addition of 0 . 1 M NaOH to dissolve the hormone), 0.2 mg/L 6-benzylammopunne (BA, from a 0.5 mg/mL stock, prepared in 0. 1 M HCl), and 1 mg/L naphthaleneacetlc acid (NAA, from a l mg/mL stock prepared as for 2,4-D, above). Also, the same medmm w1th glucose levels reduced to 0.3 and 0.2 M Regeneration medmm (RMOP): Contams (per liter)· 4 6 g MS salts (see Appen­ dix), 20 g sucrose, 1 00 mg mesoinositol, 1 mg th1amine-HCl, 1 mg BA, 0 1 mg NAA (see item 4 above for stock solutions of hormones), pH 5.6, solidified with 0.65% D1fco Bacto-agar. Leaf strip medium for L. peruvzanum: Contains (per litre): 4.6 g MS salts, 20 g sucrose, l 00 mg mesoinositol, l mg thiamme-HCl, 0 5 mg mcotimc acid, 0.5 mg pyridoxine-HCl, 0.2 mg mdoleacetic ac1d (IAA), and 2 mg zeatin (Sigma) Stock solutiOns ( l mg/mL) of these hormones should be freshly prepared m 0. 1 M NaOH (IAA) or HCl (zeatm), filter-sterilized, and added to molten, autoclaved medium (pH 5 6, 0.65% D1fco Bacto-agar) before pounng Leaf stnp medmm for S mgrum: As for L. peruvzanum, except zeatm is replaced by l mg/L BA RM medium: Contains (per liter)· 4 6 mg MS salts and 20 g sucrose, pH 5.6, solidified with 0.65% Difco Bacto-agar. RM solution: as for RM medmm, but Without the agar. 60-Jlm mesh nylon boltmg cloth. Mutagen: N-mtroso-N-methylurea (NMU) (see Note 2). Protective clothmg. Plastic or rubber apron and disposable gloves, mdustnal organic vapor cartndge respirator with cartndges, and prefilters (obtamable from "Sa-fir," East Hoathly, East Sussex, England) Large sheets of absorbent paper backed w1th fml . 5 M NaOH. 5% (w/v) Streptomycm sulfate in H20, filter-stenhzed. 5% (w/v) Lmcomycm hydrochloride in H20, filter-sterilized. Culture room facilities: Preferred conditiOns: 25° C, 1 500 lx, 1 6-h d. 0.4 M glucose.

3. Methods Mutant isolation is described below, using two different starting materials, leaf strips and mesophyll protoplasts. The use of leaf strips is technically more straightforward and is described first. Healthy leaves from shoot cultures of any of the species mentioned in Subheading 1 . can be used in exactly the same way, the only differences being the culture media required. In our experience, S. nigrum gives substantially higher yields of streptomycin-, spectmomycin-, or lincomycin-insensitive mutants than the other species. Albmo mutants can readily be obtained in the same experiments, as described in Note 3 . The rest of this sectiOn deals wtth the isolatiOn of streptomycin- and hnco­ mycin-insensitive mutants of N. plumbagznifolia from cultures initiated from

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mesophyll protoplasts. ModificatiOns of this procedure, necessary to obtam mutants resistant to terbutryn and other photosynthetic herbicides, are described in Note 4 . 3. 1 . Isolation o f Antibiotic-Insensitive Mutants from Leaf Strips

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3 4 5.

6.

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Prepare mutagen solutwns after carefully readmg Note 2 Prepare a stock solu­ tion contammg 80 mg NMU m 20 mL of RM solutiOn, and filter-stenlize. For a 1 -mM solutwn, add 2.6 mL of stock to 97.4 mL of autoclaved RM solutiOn m a wide-necked 250-mL Erlenmeyer flask For a 5-rnM solutiOn, add 1 2 9 mL-8 7 1 mL of RM solutton. It ts Important that the RM solutiOns used have pH values of 5 5---{) 0 to enhance the stability of the mutagen Remove leaves from shoot cultures and cut into small stnps (2- to 3-mm wtde by 5- to 1 5-mm long, depending on leaf size). Add 2 5 0 stnps to 1 00 mL of each ( l and 5 mM) NMU solution and to 1 00 mL of RM solution (nonmutagenized control). Incubate the flasks on a rotary shaker (about 50 rpm) at 25°C for 90 min Decant mutagen solutiOn, and wash the leaf stnps four to five times m a large excess of RM solution or sterile distilled water (pH adjusted to 5 5-6 .0) For each treatment, place 200 of the stnps on the surface of selective medium m 40 plastic Petn dtshes (5 stnps/dish) The selective medta are RMOP for Nzcotz­ ana spectes, and the media descnbed in Subheading 2. for S mgrum and L peruvwnum, m each case supplemented with 500 mg/L streptomycm sulfate ( 1 000 mg/L can also be used for Nzcotzana spp and S mgrum ), I 00 mg/L spectmomycm, or lmcomycm hydrochlonde. The medmm is prepared by addt­ tton of a small aliquot from the concentrated, filter-stenhzed stock solutiOn of antibiotic to the autoclaved culture medmm, cooled to about 50°C pnor to pour­ mg mto dtshes. Place the remammg 50 stnps on the same medmm Without strep­ tomycin. Seal all dishes with parafilm, and mcubate m the culture room After about 40 d, the leaf strips on control dishes wtll show prolific shoot regen­ eratiOn, whereas those on antibiotic-containing medium are bleached and show little morphogenesis Green nodules wtll appear at the edges of some of the bleached leaf strips (from one or both mutagen treatments), and most of these will develop mto shoots. When shoots have at least two leaves that are normal, not "vitreous" (glassy and translucent) m appearance, remove cleanly with a scal­ pel, and transfer to RM medium (embedding cut stem m the medium) for rootmg Contmue to mcubate m the culture room After 4-8 wk, p lantlets with vtgorous roots are obtamed. Remove a smgle leaf, cut mto stnps, and test for msensttiVIty to the antJbtotlc on the same medium as that tmttally used for mutant selectiOn. Typically, msenstttvtty ts expressed m the mutants by the retentiOn of chlorophyll and the appearance of numerous green advent1t1ous shoots Rooted plants can either be propagated m vitro by nodal cuttmgs, or transferred to sotl for growth to matunty and genetic analysts. Both these procedures are descnbed m Chapter 28, thts volume.

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3.2. Isolation of Streptomycin or Lincomycin-Insensitive Mutants from Protoplast Cultures of N. plumbagin ifolia I . Remove healthy, fully expanded leaves from shoot cultures, sltce finely usmg a sterile razor blade (m a holder) or scalpel and forceps, and transfer to stenle protoplast enzyme solutiOn m 9-cm Petn dishes. Typically, 10 mL of solutiOn m a dish should be sufficient for three to four leaves, and three such dishes should provide enough protoplasts for one expenment. Protoplast yields are vanable, however, so It may be necessary to start with more material 2 Incubate overnight ( 1 2-1 6 h) at 25°C m the dark 3 . Swul the dishes several times to liberate protoplasts from leaf debns, remove the solutiOn with a Pasteur pipet, and filter through 60-�.tm nylon mesh mto a 1 00-mL Erlenmeyer flask. 4. Transfer the protoplast preparation to 1 0-mL capped glass centnfuge tubes (ster­ Ile), and spin at about 300g for 3 min. 5 Intact protoplasts float and form a tight green band at the surface of the medmm Remove this carefully with a Pasteur pipet, and transfer to a clean tube (not more than 1 mL/tube) Fill the tubes with W5 solutiOn, cap, invert to ensure thorough m1xmg, and spm at about 50g for 2 mm 6. Pipet off the supernatant, and resuspend the protoplast pellet m a small volume of protoplast culture mediUm (K3) supplemented with 0.4 M glucose Mix the con­ tents of the tube, count the mtact (spherical, with an umnterrupted plasma mem­ brane) protoplasts, and dilute to 1 05 /mL with the culture medmm 7 Transfer to 5 em Petn dishes, 5 mL/dish, usmg either previOusly calibrated Pas­ teur pipets or automatic pipets with wide-bore tips 8 To individual dishes, add (to a final concentration of 0. 1 or 0.3 mM ) NMU from a concentrated stock prepared m the culture medium and filter-stenltzed. (Impor­ tant: read Note 2 carefully before usmg the mutagen ) Wrap all dishes With parafilm and mcubate in the culture room at low light mtensity ( ca I 00 lx) 9 After 7-1 0 d, providmg divisions can be observed in the protoplasts, dilute the protoplasts 2x with fresh K3 mediUm with 0 4 M glucose. To do this, pipet the contents of each dish mto a 9-cm dish, and add 5 mL of medmm Seal and incu­ bate as before There is no need to wash out the mutagen, smce It IS unstable and breaks down within 48 h at the pH used 1 0. Monitor the development of the protoplast-derived cell aggregates, and make dilutions at suitable mtervals (see Note 5) In a good preparatiOn, these intervals should be of 7-1 0 d Each dilutiOn should be 2x, and lead to a doubling of the number of dishes contammg 1 0 mL culture. For the first dilutiOn, use K3 medmm with the glucose reduced to 0.3 M, and for the second, glucose reduced to 0 2 M W1thm 1 0--1 4 d of the latter di lutiOn, numerous microcolomes (about 1 -mm diameter) should be visible, and the cultures are then ready for platmg mto soltd medium 1 1 . For every four (more If there is a low colony density) dishes, prepare 500 mL of RMOP mediUm w1th 0.2 M glucose (instead of 2% sucrose), 0.65% agar, and 1 000 mg/L streptomycm sulfate or lincomycm hydrochlonde The antibiOtics are

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added from the concentrated filter-stenhzed stock solutwn to autoclaved medmm, held at 45°C m a water bath. Usmg a fine-tipped Pasteur pipet, remove the excess K3 medmm from the cul­ tures to be plated. This Is best done by tilting the dishes slightly and allowing the microcolonies to settle, so that the medtum can be removed from above them Then, using a broad-tipped pipet, wash the contents of four dishes mto the 500-mL molten medmm by repeatedly transferrmg small amounts of the medium to the dishes and suckmg up agam, together w1th the colomes. Mix well, and pour mto 9 e m Petri dishes (about 2 0 mLidish) Allow the agar to set, wrap the dishes with parafilm, and incubate in the culture room at I 000- to 1 500-lx dlummatwn Numerous white colomes should appear after 1-2 mo. Streptomycm- or hncomycm­ msensitive colomes are green and are easily selected visually. P1ck them off when large enough and transfer to the same medmm, but With sucrose reduced to 0. I M Resistant colonies will contmue to grow and remam green. Subculture them onto RMOP medmm, Without the antibiOtic, for shoot regeneratiOn Regenerated shoots are handled as described for mutants isolated from leaf stnps.

4 . Notes 1 . Dnselase powder 1s a crude preparation containmg much msoluble material that can quickly block the milhpore filter. This can be prevented by either spmning for a few minutes m a bench centnfuge, or filtenng, to obtam a clean solutiOn pnor to filter sterilization. 2 NMU Is a dangerous carcmogen and must be used wtth great care We recom­ mend the use of a respirator, and protective gloves and apron during all mampu­ lations mvolving the mutagen. It 1s Important to avmd skm contact. All working surfaces, balances, and the hke, where spillage m1ght have occurred, should be washed down immedtate1y after use. Placing large sheets of absorbent paper backed w1th fm1 on the Iammar flow work surface helps to contam any spillage. It IS a good Idea to exclude other workers from the work area wh1le manipula­ tiOns with mutagen are m progress In the event of skm contact, and routmely after use, wash hands m soap and water gently, avmdmg excess we rubbmg of the skin. Do not use NMU m alkahne solutiOns because 1t is very unstable. After filter-steriltzmg mutagen solutions, do not remove the syrmge from the mtlltpore umt tmmedtately, smce the pressure that has butlt up m the synnge can result m the release of an aerosol of the mutagen Mutagen solutwns should be macttvated before disposal. Add an excess of 5% NaOH in the fume hood, and leave open overnight, before pouring down the smk and chasmg with a large volume of tap water. Contammated apparatus should also be treated wtth 5% NaOH, but in th1s case, after an overmght soakmg, a second wash (> I h) is recommended, followed by a thorough nnse m runnmg tap water. Additional advtce on the use of these mutagens ts given m ref (11). 3 . For all the species mentiOned, albmo mutants have also been obtained from leaf stnps. These anse m response to the mutagenesis treatment on the dishes from

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which the selective agent (antibiOtic or herbicide) has been excluded. Among the nonnal green shoots differentiatmg from the leaf strips, some albino or vane­ gated shoots are frequently obtamed. Albmo shoots can be rooted and mamtained on RM medium m the same way as other shoot cultures. Albmo shoots can be obtained from vanegated ones by dissecting out white sectors and cultunng on the appropriate regeneration medium. 4 The procedure for Isolating antibwtic-res1stant mutants from protoplast cultures of N. plumbagzmfolia can be applted to the selectwn of mutants res1stant to her­ bicides that inh1b1t photosynthesis, providing a selective medtum perm1ttmg photomixotrophic growth IS used. Th1s is achieved by lowermg to 0.3% the glu­ cose level m the RMOP medium m which the m1crocolomes are plated. In order to reduce the osmotic stress resulting from plating m such a low-sugar medmm, an additional dilution step (with K3 medmm plus 0. 1 M glucose) 1s introduced into the protoplast culture procedure. For tnazme herb1c1des (e.g., terbutryn, atra­ 4 zine, stmazine ), a suttable selective level 1s I 0- M, and selection ts based on the greenmg of colomes, exactly as m the case of antlbtottc-resistant mutants. After retesting for resistance on selective medium, shoots are regenerated by transfer of small callus pieces to RMOP medium wtthout the herb1c1de Mutants reststant to metobromuron and bromoxynil have also been obtamed m thts way 5 The instructions for the gradual dilutiOn of protoplast cultures w1th fresh medium of decreasmg glucose concentration are given as accurately as poss1ble. The 7- to 1 0-d mterva1 should work, but careful monitonng of the cultures ts desirable If the growth rate of the colomes seems to be slow, a longer interval must be used On the other hand, raptd growth rates, especially tf accompamed by brownmg or the appearance of dead cells, mdtcates a requtrement for more raptd dtlutwn

References 1 . Dyer, T. A ( 1 985) The chloroplast genome and its products, in Oxford Surveys of Plant Molecular and Cell Bwlogy, vol 2 (Mifhn, B. J., ed.), Oxford Umverstty Press, New York, pp 147-1 77. 2. Borner, T and Sears, B. B. ( 1 986) Plastome mutants. Plant Mol Bzol 4, 69--92. 3. Medgyesy, P. ( 1 990) Selection and analysis of cytoplasmic hybrids, in Plant Cell Lzne Selection (Dtx, P J., ed ), VCH, Weinhetm, pp. 287-3 1 6 4. Cseplo, A and Ma!iga, P ( 1 984) Large scale isolation of maternally inhented hncomycin rest stance mutatwns m dtplotd Nzcotzana plumbagznzfolza protoplast cultures Mol Gen Genet 196, 407-4 1 2 5 McCabe, P F , Ttmmons, A. M., and Dtx, P. J. ( 1 989) A stmple procedure for the tsolatwn of streptomycm resistant plants in Solanaceae. Mol Gen Genet 216, 1 32-1 3 7. 6. Svab, Z., HaJduklewitz, P , and Maltga, P. ( 1 990) Stable transformation of plas­ tids in htgher plants Proc Nat!. Acad. Sci USA 87, 8526-8530 7. O'Neill, C M., Horvath, G. V , Horvath, E , Dtx, P J., and Medgyesy, P ( 1 993) Chloroplast transfonnatwn m plants: polyethylene glycol (PEG) treatment of pro­

toplasts IS an alternative to bwltsttc deltvery systems. Plant J. 3, 729-738

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8. McBnde, K. E., Svab, Z., Schaaf, D J., Hogan, P S. Stalker, D. M., and Maliga, P ( 1995) Ampltfication of a chtmenc Baczllus gene m chloroplasts leads to an extra­ ordinary level of an msecttctdal protein m tobacco. Rio/Technology 13, 362-365. 9. Dtx, P J. and Kavanagh, T A. ( 1 995) Transformmg the plastome genettc mark­ ers and DNA dehvery systems. Euphytzca 85, 29-34. 1 0. Kavanagh, T A., O'Driscoll, K. M , McCabe, P F., and Dix, P. J ( 1 994) Muta­ tions confernng hncomycm, spectmomycin, and streptomycin resistance m Solanum nigrum are located m three dtfferent chloroplast genes Mol Gen Genet 242, 675-680 1 1 . Hagemann, R ( 1 982) Induction of plastome mutations by nttroso-urea-com­ pounds, m Methods zn Chloroplast Molecular Bzology (Edelman, M., Hallick, R B., and Chua, N. H , eds.), Elsevter Btomedtcal, Amsterdam, pp. 1 1 9-1 27. 12. Maltga, P. ( 1 984) Cell culture procedures for mutant selection and charactenza­ twn in Nicotzana plumbagmifolza, m Cell Culture and Somatzc Cell Genetzcs of Plants, vol 1 (Vasil, I. K., ed.), Academic, New York, pp. 552-562.

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Agrobacterium-Med iated Tra nsformation of Petunia Leaf Disks Ingrid M. van der Meer

1 . Introduction Agrobacterzum-medtated transformation of plants is now applicable to many dicotyledonous and also several monocotyledonous plant species. It can be used to transform many dtfferent species based on various factors: the broad host range of Agrobacterium (1), the regeneration responsiveness of many dif­ ferent explant tissues (2), and the utility of a wide range of selectable marker genes (3) . In addition to tobacco, one of the first species that was routinely transformed usmg this method was Petunia hybrida. P. hybrida is a very good model plant for the analysis of gene function and promoter activity. It 1s readily transformed, the culture conditions are easy ful­ filled, generation time is 3--4 mo and one can grow up to 1 00 plants/m2 . Fur­ thermore, its genetic map is well developed, and it contams active transposable elements (4). The protocol presented here 1s a simplified vers10n of that of Horsch et al. (5) . The basic protocol involves the inoculation of surface-sterilized leaf disks with the appropriate disarmed strain of Agrobacterium tumefaciens carrying the vector of choice, which in this protocol confers kanamycin resistance. The plant tissue and Agrobacterzum are then coculttvated on regenerat10n medmm for a period of 2 or 3 d. During this tlme, the virulence genes m the bacteria are induced, the bacteria bind to the plant cells around the wounded edge of the explant, and the gene-transfer process occurs (6). Using a nurse culture of tobacco or Petunia cells during the coculture period may increase the transfor­ mation frequency. This is probably owing to a more efficient induction of the vtrulence genes. After the cocultivat10n period, the growth of the bactenal population is inhibited by bacteriostatic antib10tics ( cefotaxim or carbenicilFrom· Methods m Molecular Biology, Vol 1 1 1 Plant Cell Culture Protocols Ed1ted by A D Hall © Humana Press Inc , Totowa, NJ

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lm), and the leaf ttssue IS mduced to regenerate. The induction and develop­ ment of shoots on leaf explants occur m the presence of a selective agent agamst untransformed plant cells, usually kanamycin. Dunng the next 2-3 wk, the transformed cells grow mto callus or differentiate into shoots vta organogen­ esis. After 4-6 wk, the shoots have developed enough to be removed from the explant and induce rooting m preparation for transfer to sml. To speed up the rooting period, the shoots can be rooted without selection on kanamycin. In total, it takes about 2 mo, after inoculation of the leaf disks with Agrobacterium to obtain rooted plantlets that can be transferred to soil. 2. Materials 2. 1. Bacteria Media I . For the growth of Agrobactenum, use Luna broth (LB) medmm· I % Bacto-peptone (Dtfco, DetrOit, Ml). 0.5% Bacto-yeast extract (D1fco) 1 % NaCI. Autoclave, and cool medmm to at least 60°C Add appropnate ant1b10t1cs to select for plasm1ds (50 mg/L kanamycin for pBin l 9 {7}) 2. LB agar: LB medmm w1th 1 5 g/L agar (Difco) Autoclave, and cool medium to at least 60°C Add appropnate antibiotics to select for plasm1ds (50 mg/L kanamy­ cm for pBm l 9). Pour mto stenle 20-mm Petn d1shes 3 Agrobactenum inoculatiOn dliutton medmm Murashige and Skoog (8, and see Appendix) salts and v1tamms (4.4 g/L) (S1gma, Amsterdam). Autoclave.

2.2. Stock Solutions For convemence, most stock solutiOns are prepared at 1 000 times the con­ centration needed for the final media. The antibiotics are added to the medta after autoclaving when the temperature of the medta has cooled to 60°C. Cefotax1me. 250 mg/mL (Duchefa, Haarlem, The Netherlands, or S1gma), filter­ stenhze, keep at -20°C 2 Kanamycm: 250 mg/mL (Duchefa or Sigma), filter-stenhze, keep at -20°C 3 6-Benzylammopunne (6-BAP), (Sigma)· 2 mg/mL Dissolve 200 mg BAP in 4 mL 0 5 N HCl Add, while stirring, drop by drop H20 at 80-90°C and make up to I 00 mL Fi lter-sterihze. 4 1 -Naphtalene acetic acid (NAA) (Sigma)· I mg/mL, dissolved m DMSO No need to stenhze. Keep at -20°C DMSO should be handled under a fume hood

2.3. Plant Culture Media I . Cocult1vatton medmm. Murashtge and Skoog (8, and see Appendtx) salts and v1tamms (4.4 g/L) (Stgma), 30 g/L sucrose, 2 mg/L 6-BAP, 0 0 1 mg/L NAA, adjust pH to 5 . 8 w1th I M KOH, add 8 g/L agar (Bacto Difco) and autoclave. Pour mto stenle plastic dishes that are 20 mm h1gh (Greiner, Kremsmunster, Austna)

Petunia Leaf Disks

329

2. Regeneration and selection medium: Murashige and Skoog salts and vttamins (4.4 g/L) (Sigma), 30 g/L sucrose, 2 mg/L 6-BAP, 0.0 1 mg/L NAA, adJUSt pH to 5.8 with 1 M KOH, add 8 g/L agar (Bacto Difco ). Autoclave and cool medta to 60°C, add 250 mg/L cefotaxtme to kill off Agrobactenum, and the appropnate selective agent to select for transformed cells depending on the vector used ( 1 00 mg/L kanamycin for pBin 1 9) . Pour 25 mL mto each sterile 20-mm htgh Petri dish (Gremer) 3 . Rooting medium: Murashige and Skoog salts and vttamms (4.4 g/L) ( Stgma), 30 g/L sucrose, adjust pH to 5 . 8 with 1 M KOH, and add 7 g/L agar (Bacto Difco) Autoclave and cool media to 60°C, add 250 mg/L cefotaxtme to ktll off Agrobactenum, and add the appropnate selective agent to select for transformed shoots dependmg on the vector used ( 1 00 mg/L kanamycm for pBm 1 9). To speed up the rootmg process, kanamycin may be omttted from the rootmg medmm Pour m Magenta GA7 boxes (S tgma, 80 mL per box).

2.4. Plant Material, Sterilization, and Transformation P hybnda c.v W 1 1 5 (Mitchell), grown under standard greenhouse conditions 2. A tumejac1ens stram LBA 4404 and A tumefaczens LBA 4404 contaming pBm 1 9 (m which the gene of interest t s mserted) 3. 1 0% So Iutton of household bleach contammg 0. 1 % Tween or other surfactant 4 Stenle H20 5 Stenle filter paper (Whatman) and sterile round filters (Whatman, dtameter 90 mm)

2.5. General Equipment Stenle transfer factlities

2. Rotary shaker at 28°C 3 . Cork borer (or a paper punch) 4 Magenta GA 7 boxes (Stgma) and 20-mm htgh Petn dtshes (Gremer). 3. Methods 3. 1. Plant Material Young leaves are used as the explant source for transformation. These explants can be obtained from aseptically germinated seedlings or mtcro­ propagated shoots, but m this protocol, they are obtamed from greenhouse­ grown material. The genotype of the source material is important in order to obtain high transformation rates. P. hybnda cv.W l 1 5 gtves the best results and is most often used (see Notes 1 and 2). To obtain plant maten al suitable for transformation, seedlmgs should be germmated 4-6 wk pnor to transformation. Sow P. hybrzda (W 1 1 5) seeds in sotl 4--6 wk pnor to transformatiOn, and grow under standard greenhouse conditions in 1 0 x 1 0 x 1 0 em plastic pots. Use commerctally avatlable nutnttve solutiOn for house plants.

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van der Meer

3.2. Leaf Disk Inoculation I . Grow A tumefaczens culture overntght in LB at 28°C on a rotary shaker ( 1 3 0 rpm) with appropnate antibiotics to select for the vector (50 mg/L of kana­ mycm for pBin 1 9) (see Note 3). The Agrobacterzum hqutd culture should be started by inoculating 2 mL of liqutd LB with several bacterial colomes obtamed from an Agrobactenum streak culture grown on an LB agar plate at 28°C for 2-3 d. The streaked plate 1tself can be moculated from the ongmal -80°C frozen stock of the Agrobacterium stram (see Note 4). Th1s stock IS composed of a bac­ terial solutwn made from a 1 . 1 mixture of stenle glycerol (99%) and an over­ night LB culture of the Agrobacterzum 2 Prepare the culture for moculation of explants by taking 0.5 mL of the overmght culture and dilutmg 1 to 200 with Murashige and Skoog salts and v1tamms medmm (4 4 g/L) (Sigma) to a final volume of 1 00 mL The A tumefaciens mocu­ lum should be vortexed well pnor to use Pour the Agrobacterzum inoculum into four Petn d1shes 3. When the plants are I 0- 1 5 em h1gh, harvest the top leaves to prov1de ex plants (leaves 3-8 from the top) Lower leaves and leaves from flowermg plants should not be used, smce they usually have a lower transformatiOn and regeneratiOn response (see Note 1 ) 4 . Prepare harvested leaves for moculatwn by surface stenhzatwn for 1 5 m m m 1 0% solutiOn of household bleach contammg 0 I% Tween or other surfactant Wash the leaves thoroughly three t1mes with stenle H20 Keep them m stenle H20 until needed (see Note 5) All procedures followmg the bleach treatment are conducted m a stenle transfer hood to maintam t1ssue stenhty 5. Punch out leaf d1sks w1th a stenle ( 1 -cm dmmeter) cork borer (or cut mto small squares to produce a wounded edge) in one of the Petn d1shes contaimng the Agrobacterium moculum (20-25 disks/Petri d1sh) (see Note 6) Avmd the m1dnb of the leaf or any necrotic areas Cut 80-100 d1sks/construct (see Note 7). 6. Leave the d1sks m the inoculum for 20 mm After moculatwn, the explants are gently sandw1ched between two layers of stenle filter paper (Whatman) to remove excess moculum 7 (Optional) Prepare nurse culture plates by addmg 3 mL of cell suspenswn culture (e g , P. hybrzda cv Coomanche or Nzcotzana tabacum cv SR I ) to Petn d1shes contammg cocult1vatwn medmm Sw1rl the suspensiOn to spread the cells over the surface of the medium and cover w1th a stenle Whatman filter paper (diam­ eter 90 mm) (see Note 8). 8 Place 20 explants w1th the adax1al surface downward on each plate w1th coculttvation medmm (either with or without nurse cells), and mcubate for 2-3 d (see Note 9). Seal the plates w1th Nescofilm The culture cond1t10ns for the leaf d1sks are as follows a temperature of 25°C and a photopenod of 14 h hght (hght mtens1ty· 25--40 11ffi OI!m2/s)l l O h dark The controls to check the transformatiOn protocol are Leaf d1sks maculated with "empty" Agrobacterzum (w1thout pBm 1 9 vector) on regeneratiOn medmm w1thout selective agent (to check the regeneratwn).

Petunia Leaf Disks

33 1

Leaf disks inoculated With "empty" Agrobacterzum (without pBin 1 9 vector) on regeneration medium with selective agent (to check the efficiency of antibiotic selection) 9. After cocultlvation, transfer the disks to regeneration and selection medmm (seal the plates with Nescofilm), and continue incubation until shoots regenerate Transfer the explants every 2-3 wk to fresh regeneration and selection medium (see Note 1 0).

3.3. Recovery of Transformed Shoots 1 . After 2-3 wk, the first shoots will develop Cut off the shoots cleanly from the explant/ callus when they are 1-1 .5 em long, and place them upright in rootmg medium in Magenta boxes The shoots should be excised at the base without taking any callus tissue (see Note 1 1) . Take only one shoot from each callus on the explant to ensure no siblmgs are propagated representmg the same transfor­ mation event (see Note 12). Shoots from distinctly different calli on the same explant are, however, likely to be derived from different transformatiOn events and should be transferred separately. Give each shoot a code that allows it to be traced back to specific explants Cefotaxime is kept in the medtum to avoid Agrobacterzum regrowth. The anti­ biotic used to select for the transgenic shoots can be added to the medmm to select against escapes, although the rooting process is sped up when it is omitted. 2. (Optional) Before removing rooted shoots from sterile culture, transfer a leaf to selection medium to test for resistance to kanamycin Ifthe leaf 1s obtamed from a tranformed plantlet 1t should stay green and form callus on selectiOn med1um. If It ongmates from an untransformed plantlet, 1t should become brown/white and die within a few weeks. 3. After 3-4 wk the shoots will have formed roots. Remove plantlets, wash agar from the base under a running tap, plant the transformants m soli, and transfer them to the greenhouse (see Note 13). To retain humidity, cover the pots w1th Magenta boxes or place them m a plastic propagatiOn dome. The plants should then be allowed to come to ambient humidity slowly by gradually openmg the dome or Magenta box over a penod of 7 d (see Note 14). 4. Fertilize and grow under standard plant growth conditiOns.

3.4. Analysis of Transformants Tissue culture can be used to confirm that the putative transgenic shoots produced are expressmg the selectable marker gene (see Subheading 3.3., step 2). However, DNA analysis using Southern blotting or PCR w il l con­ firm whether regenerants have integrated the antibiOtic resistance gene (and also a gene of mterest if this was linked to it withm the T-DNA). For DNA analysis using Southern blotting (9), leaf DNA can be isolated accord m g to the protocol described by Dellaporte et al. (1 0) . A more rapid method can be used to Isolate genomic DNA as descnbed by Wang et al. (1 1) when PCR Is

332

van der Meer

going to be used to analyze the presence of foretgn DNA in the transformed plants (see Note 1 5) Usually, one to five coptes of the foretgn DNA is mtegrated m the plant genome usi n g the Agrobacterzum-mediated transformation method. However, position effects may silence the expression of the introduced gene (see Note 7) 4 . Notes

2.

3

4 5.

6.

7.

8

9. 10

Thts transformatiOn protocol works very well for the often-used P hybnda vane­ ties W l l 5 (Mitchell) and V26 However, some Petuma ltnes show poor regen­ eration from leaf dtsk explants, and consequently, few or no trans formants can be obtained from these plants. Umform, clean, and young plants will perform best It is important not to take leaf matenal from old, flowenng plants Instead of greenhouse matenal, aseptically germmated seedlings or micro­ propagated shoots could also be used as explant source. Then, of course, there IS no need to surface-stenhze the leaves. The Agrobactenum stram that ts most often used is LBA4404 (Clontech {12}). Also the more Virulent strams C58 or AGLO (13) can be used, but these can be more dtfficult to ehmmate after cocultlvatwn. Dunng growth, the Agrobacterzum culture will aggregate. The streaked plate can be reused for approx 3 wk if kept at 4 oc after growth Be very gentle wtth the plant matenal dunng stenhzatwn, smce the bleach will easily damage the leaves, especially wounded or weak, etiolated leaves Damaged tissue should not be used. Dtsks provide a very uniform explant and are conveniently generated with a cork borer or paper punch However, square explants or strips can also be used Av01d excessive woundmg during the process. The cork borer should be allowed to cool before use after flammg. Thts transformatiOn protocol will yteld approx 20 transgenic plants from I 00 tmttal explants The expressiOn level of the construct of interest can be greatly mfluenced by a positiOn effect owmg to Its stte of mtegratton mto the host plant genome. Thts stlencmg of expression owing to position effects can occur in 2�0% ofthe transgenic plants, especially tf weak promoters are used. Therefore, at least 20 transformants should be generated/construct The nurse culture ts not essential for transformatiOn, but can facilitate the process by increasing frequency and reducmg damage to the explant by the bactenum Any healthy suspension of tobacco or Petuma should work The suspensiOn cul­ tures can be maintamed by weekly transfer of 1 0 mL mto 50 mL of fresh suspen­ siOn culture medtum The cocultivation time may have to be optimized for different Agrobactenum strains carrying different vectors I f Agrobacterium contmues to grow on the regeneratiOn and selection medtum (formmg shmy gray-whtte bactenal colonies), 1 5 0 mg/L ofvancomycm can also be added to the medium already contammg cefotaxtme

Petunia Leaf Disks 11 1 2.

13.

14

15.

333

Care should be taken that only the stem and none of the associated callus IS moved to the rootmg medium Otherwise, no roots will develop. It is common for multiple shoots to arise from a single transformed cell. There­ fore, it is Important to separate mdependent transformatiOn events carefully so that siblmg shoots are not excised It is important to wash away all of the agar medium from the roots and to trans­ plant before the roots become too long When there is still agar left, Jt could enhance fungal growth. Gradual reduction m the humidity I S necessary to harden off the plantlets m soil. The roots must grow mto the sml, and the leaves must develop a protective wax cuticle. If the plantlets start to turn yellow and die from fungal contammatwn, the hd should be opened faster If the plantlets begm to wilt, the hd should be opened slower Confirmation of transformatiOn by PCR may not always be reliable, owmg to possible carryover of the Agrobacterium mto the whole plant. DNA analysis usmg Southern blot hybnd1zation Is a better way to confirm whether regenerants are true trans formants Furthermore, the number of mserts can be determmed at the same time using thts method

References I . Richie, S W. and Hodeges, T. K. ( 1 993) Cell culture and regeneratiOn of trans­ gemc plants, m Transgenzc Plants, vol I (Kung, S. and Wu, R., eds.), Academic, San D1ego, pp. 147-1 7 8 2 Jenes, B., Morre, H , Cao, J . , Zhang, W., and Wu, R (1 993). Techmques for gene transfer, m Transgenzc Plants, vo1 1 (Kung, S and Wu, R., eds) , Academic, San Diego, pp. 1 25-146 3 Bowen, B. A. ( 1 993) Markers for gene transfer, m Transgenzc Plants, vol l (Kung, S. and Wu, R., eds.), Academic, San Diego, pp. 89-124 4 Gerats, A G M , Hmts, H , VriJlandt, E , Marana, C , Souer, E , and Beld, M ( 1 990) Molecular characterization of a nonautonomous transposable element (dTph l ) of petuma Plant Cel/ 2, 1 1 2 1-1 1 2 8 5 Horsch, R B , Fry, J E., Hoffman, N. L., Eichho1tz, D , Rogers, S C , and Fraley, R. T ( 1 985) A simple and general method for transfernng genes mto plants Sczence 227, 1 229-! 23 ! 6. Hooykaas, P J. J. ( 1 989) Transformation of plant cells via Agrobactenum. Plant Mol Bwl 13, 327-3 36 7. Bevan, M. ( 1 984) Bmary Agrobactenum vectors for plant transformatiOn. Nuclezc Aczds Res 12, 87 1 1-872 1 8. Murash1ge, T. and Skoog, F. ( 1 962) A revised medmm for rapid growth and bw­ assays with tobacco tissue cultures. Plant Physwl 15, 473-497. 9 Mamahs, T , Fritsch, E. F , and Sambrook, J ( 1 982) Molecular clonzng A Labo­ ratory Manual Cold Spring Harbor Laboratory Press, Cold Spnng Harbor, NY 1 0. Dellaporte, S. L., Wood, J., and Hicks, J. B. ( 1 983) A plant DNA mm1preparat10n: versiOn II. Plant Mol. Bwl Rep. 1, 1 9--2 1

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1 1 . Wang, H., Qi, M , and Cutler, A J ( 1 993) A simple method of preparing plant samples for PCR. Nucleic Aczds Res 21, 4 1 53-41 54 12 Hoekema, A., HirSch, P. R , Hooykaas, P J 1., and Schilperoort, R. A ( 1 983) A bmary plant vector strategy based on separation of vzr and T region of the Agrobactenum tumefaczens TI-plasmid Nature 303, 1 79-180 13. Lazo, G R., Stein, P. A , and Ludwig, R. A. ( 1 99 1 ) A DNA transformation-compe­ tent Arabidopsts genomic hbrary m Agrobacterzum Bzo/Technology 9, 963-967

30

____________________ __

Transformation of Rice via PEG-Medi ated DNA Uptake i nto Protoplasts Karabi Datta and Swapan K. Datta

1 . Introduction For stable transformation of cereals through PEG-mediated DNA uptake into protoplasts, the two most critical requisites are the ability to Isolate and culture protoplasts m large numbers, and the development of an efficient and rehable system for routine plant regeneration from protoplasts (1-3). Based on early suc­ cess with mesophyll protoplasts of some dicotyledonous species, extensive efforts were made to induce sustained divisiOn m protoplasts Isolated from leaves or young shoots of different cereal plants. However, there is still no convmcmg evtdence of sustained dtvtsions in protoplasts isolated from leaves or shoots of any cereal In contrast, protoplasts isolated from embryogenic suspension cul­ tures could be induced to divide m culture (4). Obtainmg a fast-growmg and highly embryogemc suspensiOn culture is the most important factor for cereal plant regeneration from protoplasts (4-8). Microspore cultures, and immature or mature embryos may be used to obtain embryogenic call i which can eventual ly be used to establish embryogenic cell suspensiOns ECS (2, 7) . PEG-mediated gene transfer to rice protoplasts appears to be the most effi­ cient, reliable, inexpensive, and simplest method, when it works (2, 8, 9). In this system, from a suspension culture, a large population of protoplasts can be readily obtamed for transformation enabhng many chances of obtaimng inde­ pendent transformation events. RegeneratiOn of transgenic plants is possible under suitable in vitro conditiOns through selectiOn at an early stage of develop­ ment. However, the tissue-culture response may vary dependmg on the plant genotype, handling, and the condttion of the suspension cells. We have success­ fully used the procedure descnbed below for gene transfer to rice for at least I 0 Indica and several Japomca cultivars. From Methods

Edited

m

Molecular B1ology, Vol 1 1 1 Plant Cell Culture Protocols by R D Hall © Humana Press Inc , Totowa, NJ

335

Datta and Datta

336 2 . Materials 2. 1. Suspension Culture Establishment

2. 1 . 1 Plant Material Plant material is Oryza sativa-Indtca-type rice cultivar, Chinsurah Boro II, IR 72, Japomca type rice culttvar-Yamabiko: Immature mflorescence (panicles collected pnor to the emergence from the flag leaf sheath), immature embryos (pamcles collected 7-1 0 d after anthesis) and healthy mature seeds from the mentioned cultlvars.

2. 1 .2. General Equipment Laminar flow cabmet, mverted mtcroscope, hght microscope, mcubator, gyrat­ mg shaker, autoclave, and disttlled water plant 2 Nescofilm, flasks, beaker, measunng cylmder, medta bottles, forceps, scalpel, sctssors, sptrit lamp, and ptpet bulb 3 Plastic Petn dtshes (50 mm), 24-well COST A plates, disposable ptpets, 2- and I 0-mL capacity. 4 Greenhouse facihttes

2. 1 . 3. Sterilizing Solutions I . 70% Ethanol. 2 Sodium hypochlonte solut10n (e.g , 1 00 mL 1 .8% sodmm hypochlonte w1th two drops Tween 20) 3. Sterile dtsttlled water.

2. 1 .4. Media Mtcrospore culture medmm (R I ) Quanttty ( mg/L) Component NH4N03 1 650 KN03 1 900 CaCI2 2H20 440 MgS04 7H20 370 KH2P04 1 70 KI 0.83 6.3 H3 B03 MnS04 4H20 22.3 ZnS04 7H20 86 0 25 Na2Mo04 2H20 0 025 CuS04 5H20 CoCI2 6H20 0.025 37 3 Na2EDTA 27.8 FeS04 7H20 •

•

•

•

Transformation of Rice 10 Thiamine HCI 500 Glutamme 300 Casem hydrolysate 1 00 Myo-mosttol NAA 2 1 2,4-D Sucrose 6% w/v Fico II 400 1 0% w/v pH should be 5.6. Based on MS medium (10) . Steri lize by filtration 2. AA medmm (11) Quanttty (mg/L) Component 1 50 CaC1 2 2H20 250 MgS04 7H20 1 50 NaHzP04 H20 2950 KCI 0 75 KI 3.0 H3B03 10 0 MnS04 H20 ZnS04 7Hz0 2.0 0.25 Na2Mo04 2H20 0.025 CuS04 5H20 CoCI2 6H20 0 025 I 0 Ntcotme actd 1 .0 Pyndoxme-HCI 10 0 Thtamme-HCl 1 00 Inosttol 37.3 Na2EDTA 27 8 FeS04 7H20 876 L-G1utamine 266 Aspartic acid 1 74 Arginine Glycine 75 2,4-D 1 .0 Kinetin 0.2 GA3 0 1 20 g/L Sucrose/maltose pH should be 5 6 Sterilize by filterat10n. 3. Modtfied MS medtum Quanttty (mg/L) Component 1 650 NH4N03 1 900 KN03 440 CaCI2 2H20 370 MgS04 7H20 ·

•

•

•

•

•

337

338

Datta and Datta

1 70 KH2P04 KI 0.83 H3B03 6.3 22 3 MnS04 4H20 8.6 ZnS04 7H20 Na2Mo04, 2H20 0 25 CuS04 5 H20 0 025 CoCI2 6H20 0 025 27.8 FeS04 7H20 37.3 Na2 EDTA 05 Nicotmtc actd Pyridoxtne-HCl 0.5 1 0 Thmmine-HCl Glycine 20 300 Casein hydrolysate 1 00 Myo-mosttol 1 5 2 ,4-D Kmetm 0.5 Sucrose/maltose 30 g/L Agar 8 g/L pH should be 5 . 8 . This is based on MS medium (10) Steriltze by autoclavmg 4. R2 medmm (11) Quantity (mg/L) Component NaH2P04 2H20 240 KN03 4040 330 (NH4)2S04 247 MgS04 7H20 1 47 CaC12 2H20 0.50 MnS04 H20 ZnS04 7H20 0 50 H3B03 0 50 CuS04 5H20 0 05 0 05 Na2 Mo04 37.3 Na2 EDTA 27 8 FeS04 7Hz0 0.5 Ntcottmc actd 0.5 Pyndoxine-HCI 1 Thtamme-HCI 2 Glycine 1 00 Inositol 1 2,4-D 20 g Sucrose/maltose pH should be 5 8. S teriltze by autoclaving. •

·

·

·

•

•

·

•

Transformation of Rice

339

2.2. Protoplast Isolation, Transformation, and Regeneration

2.2. 1. General Equipment I. 2 3 4 5 6.

Centrifuge. Temperature-controlled shaker 1 2-mL round-bottom screw-cap centnfuge tubes. Petn dishes, 1 0 (deep model) and 3 5 em (Falcon) Nylon Sieves-50 and 25 J..U1l (Saulas, F93 1 00 Montreutl, France) Counting chamber (hemocytometer)

2.2.2. Solutions and Medium 1 . Enzyme so Iutton. 4% w/v cellulase onozuka RS, I % w/v macerozyme R l 0 (both Yakult Honsha Co., Japan), 0.02% pectolyase-Y23 (Se1shin Pharmaceu­ tical Co , Japan), 0.4 M manmtol, 6.8 mM CaCI2, pH 5 . 6; filter-sten hze, and store at -20°C 2 Wash solution 0 4 M mannitol, 0 1 6 M CaC12; autoclave 3. MaMg solutiOn (transformatiOn buffer). 0 4 M manmtol, 15 rnM MgCI 2 , I% (w/v) MES, pH 5.8, autoclave. 4. PEG solutiOn (40% w/v): Dt ssolve 80 g PEG 6000 (Merck, Art 1 2 033, 1 000) in 1 00 mL distilled water contammg 4 72 g Ca (N03h · 2H20 and 14.57 g manmtol. The PEG is dissolved by heatmg carefully m a microwave. Make the total volume 200 mL w1th distilled water. Divide the solution into two parts. In one part, the pH is adJusted to 1 0 .0 using I M KOH and left over­ night to stabilize Then, adJUSt the final pH to 8.2 usmg the second part of the solution Filter-stenlize the solutiOn and store as 5-mL ahquots at -20°C (see Note 1 ). 5 P I medmm· R2 medmm w1th 2 mg/L 2,4-D and 0 4 M maltose, pH 5 6, filter­ stenlize (Table 1). 6 Agarose-protoplast medtum 600 mg (dry autoclaved) Sea plaque agarose melted in 30 mL P I medmm 7. P2 medium. Soft agarose N 6 medium (13) with 6% maltose or sucrose, 2 mg/L 2,4-D, and 0.3 g Sea plaque agarose; autoclave (Table 1 ). 8. DNA for transformation - Plasmid DNA is used, 1 0 �Jg/sample of protoplast sus­ pensiOn ( 1 .5 x 1 06 protoplasts) is used for transformation. D1ssolve 1 0 mg calf thymus DNA (used as earner DNA) in 5 mL distilled water. Shear by passage through an I S -gauge needle to give an average fragment s1ze of 5- 1 0 kb (check by running gel) Carner and plasmid DNA is sterihzed by precipitation and washing in 96% ethanol (also possible with 70% ethanol), and dried m a laminar flow hood. DNA 1s dissolved m sterile double-distilled water (2 j..ig/fll) and stored at 4°C (or at -20°C for longer period). 9. Nurse cells--OC cell hne derived from seedlmgs of 0. sativa L. C5924, pro­ Vided by K. Syono of Umversity of Tokyo (see Note 2).

Datta and Datta

340 Table 1 Composition of the Media Used Component (NH4)2 S04 KH2 P04 KN03 NH4N03 CaCI2 2H2 0 MgS04 7H2 0 Na2 EDTA FeSO 7H2 0 NaH2P04 2H2 0 2H2 0 MnS04 4H20 H3B03 ZnS04 H 2 0 Kl CoC12 6H2 0 CuS04 5 H2 0 Na2 Mo04 2H 20 Thmmine-HCl Nicotinic acid Pyridox me-HCI Glycme Myo-mositol Kinetin NAA 2,4-D Sucrose g/U Maltose g/U Agar g/L Agarose g/L ·

·

·

P 1 mg/L

P2 mg/L

330 0

463 0 400 0 2830 0

4044 0 1 47.0 247 0 37 3 27.8 240 240 05 0.5 05

0.05 0 05 I 0 0.5 05 2.0 1 00 0

1 66 0 I 85 0 37.3 27 8

44 1 6 1 .5 08

I 0 05 05 20 1 00.0

P3 mg/L 1 70 0 1 900 0 1 650 0 440 0 370 0 37.3 27 8

22.3 6.3 86 0 83 0.025 0.025 0 25 10 05 0.5 2.0 I OO.O

2.0 1 .0

20 1 3 6.92 144. 1 2

20 60.0 60.0

30.0 8.0

pH 5 6 F i Iter-sterilized

3 or 6 pH 5 8 Autoclaved

pH 5 8 Autoclaved

0Either sucrose or maltose to be used

3. Method 3. 1. Growth of Donor Plants I . Break the dormancy of clean and pure seeds of denved lines m the oven at 50°C for 3-5 d. 2. Sow the seeds in seed boxes and water sufficiently to wet the soil

Transformation of Rice

34 1

3 . At 2 1 d after sowing, transplant the seedlings to 6-m. diameter pots (one seedling per pot) contammg soil and fertilizer (2.5 g [NH4h S04, 1 .25 g P205 and 0. 75 g K2 0/pot). 4. Grow the plants m the glass house, and keep them well watered. The glass house should have full sunshme and sufficient ventilation to mamtam daytime tempera­ tures of 27-29°C With a 90% humidity level.

3.2. Establishment of Embryogenic Suspension Culture

3.2. 1 . From Microspores I . Collect the tillers from the donor plants when most of the microspores are at the m1duninucleate stage In all cereals mvestigated, the early or midunmucleate stage of microspore development was found to give the best results (14). 2. Stenlize the selected spikes wtth 70% ethanol for 30 s, and then treat wtth sodmm hypochlorite solution for 7 min followed by washing three times with sterile deiomzed water (15) . 3. Float 30 anthers, each containing microspores a t the miduninucleate stage on the sur­ face of 10 mL of liquid microspore culture media (R 1) m 50-mm sterile Petri dishes. 4. Culture in the dark at 25°C. These floating anthers shed microspores mto the medium within 3-7 d. 5. After 4-6 wk, transfer the developing embryogemc calli (about 0 5 g) to 7 mL of AA medium (11) to establish the embryogemc cell suspension (Fig. lA). At th1s stage, subculturing t s necessary at least twice a week until a fine cell suspenswn IS obtamed. This cell suspension can then be subcultured every 7 d for long-time maintenance (at least 1 yr).

3.2.2. From Immature or Mature Embryos 1 . Stenlize dehulled Immature ( 1 2 d after anthesis) or mature seeds m 70% ethanol for 1 min, and then sodium hypochlorite ( 1 .8%) solution for 30 min followed by washing three times with sterile dewnized water. 2. Isolate the embryos from the stenle seeds, and place mdividually in 24-well COSTA plates with each well contammg 1 .5 mL of modified MS medmm. The scutellar tissue should be upward, since callus growth occurs only With th 1s onentatwn. 3. Incubate the embryos in the dark at 25°C After 3-4 d, cut off the emerging shoots and roots, and subculture the remaining tissues onto fresh medium. At least two more subcultures are necessary at 2-wk intervals until yellowish-white soft embryogenic calli develop on the surfaces of the scutella 4. Transfer this callus to 6 mL of liquid AA medium (11) m 50-mm Petri dishes, and 2 incubate on a gyratmg shaker at low speed (80 rpm) m diffuse light (3 ,.unol/m /s) at 25°C. 5. Subculture the callus every 7 d with continued manual selectiOn of small and densely cytoplasmic cells, which are transferred to 20 mL ofR2 medium in 100-mL Erlenmeyer flasks (see Note 3) These suspensiOns can be maintained for a long

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Transformation of Rice

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time by subcultunng every 7 d, and incubatmg with gyratory shakmg (80 rpm) at 25°C in dark or diffuse light.

3.3. Protoplast Isolation I . Transfer (approx) 3-5 g of suspension cells 3-4 d after subculture mto a deep 1 0-

2. 3. 4. 5 6.

cm Falcon Petri dish. Allow the suspension cells to settle and remove the medium by pipetmg off (see Note 4). Add 20 mL of enzyme solutwn, seal with Nescofilm, and mcubate Without shakmg at 30°C in the dark for 3-4 h (dependmg on protoplast release) (see Note 5) Add an equal volume of wash solutiOn to each Petn dish contammg the proto­ plast enzyme mixture Remove the protoplast suspension with a 1 0-mL pipet, and pour through 50- and 25-fllll steves into a sterile glass beaker ( I OO mL) Transfer the filtrate to l 0-mL round-bottom screw-cap centrifuge tubes, and cen­ trifuge for 1 0 min at 70g to separate off the enzyme solutiOn Di scard the supernatant, and resuspend the protoplast pellet m washmg solutiOn. Centrifuge and repeat once. Resuspend the pellet m l 0 mL of washing solutiOn. Count the density of protoplasts/mL usmg a hemocytometer Freshly Isolated protoplasts from ECS should appear densely cytoplasmic (Fig. lB; see Note 6)

3.4. Direct Gene Transfer to Protoplasts Using PEG I . Centnfuge the protoplasts

2. 3

4.

5. 6

m wash solutiOn at 70g for I 0 mm (I.e., third washing). Remove the supernatant, and resuspend the protoplast pellet m transformation buffer (i.e., MaMg solution). Adjust the protoplast density to 1 .5-2 0 x I 06/0.4 mL Distribute the protoplast suspension (0.4 mL contaimng 1 .5 x 1 0 6 protoplasts) into different centrifuge tubes using a 2 mL-pipet. Add 6-1 0 j.Ig of stenle plasmid DNA (preferably hneanzed) and 20 j.Ig of calf thymus carrier DNA (transformation can be performed without calf thymus) to each 0.4-mL ahquot of the protoplast suspension (see Note 7) Add 0.5 mL of the PEG solution dropw1se, and mix gently. Incubate at room temperature (or preferably at zooq for 1 0 mm. Add slowly 1 0 mL of wash solutiOn, mix, and centrifuge at 70g for 10 mm to remove the PEG. Discard the supernatant. Resuspend the protoplast pellet in 0.4 mL P 1 medium

Fig. I . (prevwus page) (A) Fertile transgenic nee plants from transformed proto­ plasts obtained usmg the PEG method. ECS developed from microspore culture (B) Freshly 1solated, densely cytoplasmic, protoplasts from ECS. (C) Early div1s1on in protoplast culture. (D) Protoplasts m selection medium (left: Petn dish nontransformed cell, right: Petri dtsh wtth transformed calli developing from transformed protoplasts). (E) Protoplast-derived calli m bead-type culture without selectiOn. (F) Putative transgenic rice plants in a plastic tray containing Yosh1da solutiOn after transfer to the transgenic greenhouse. (G) Transgenic fertile rice plants in the greenhouse.

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3.5. Culture of Protoplasts I . Melt agarose-protoplast medium m a microwave oven, and cool to 40°C 2 M1x 0.6 mL of the agarose-protoplast medmm with 0.4 mL of P I medmm con­ tammg protoplasts, and transfer mto a 3.5-cm Petn d1sh. Incubate at 20°C for I h The final density of the protoplasts should be around 1 5 x I 0 6/mL 3. Cut the solidified agarose gel mto four segments, and transfer each to a 5-cm Petn dish contaming 5 mL of P I medium (bead-type culture) (16) with or With­ out adding nurse culture (6,17) Incubate cultures in the dark at 28 5°C with slow shaking (40 rpm) 4. After 7 d, replace 2 mL medium w1th fresh P l medmm 5. At the l Oth d after protoplast isolation, remove (all) the nurse cells by transfer­ ring the segments to a fresh Petn d1sh con taming 5 mL of P 1 medi urn. Keep m dark at 28.5°C w1th slow shaking (30 rpm)

3. 6. Selection 1 . FirSt selectiOn. 1 4 d after transformatiOn, add the selective agent to the medmm (e.g., hygromycm B 25 mg/L, or kanamycm 50 mg/mL, G-4 1 8 25 mg/L, or phosphinotricm 20 mg/L, depending on the selectable marker gene used) Incu­ bate as above, but wtthout shakmg Early dtviswns of protoplasts are already present at this stage (Fig. 1 C). 2 Second selection· after 2 wk, the selection pressure should be mamtained in the same way by replacing 4 mL of medium with fresh medmm supplemented w1th the same concentratiOn of selective agent. Culture for another 2 wk Putatively transformed, protoplast-derived colomes are vts1ble at thts stage (Fig. 1 D) Nontransformed colonies without selectiOn are shown in Fig. IE. 3. Replace 4 mL of P l medmm with 2 mL of fresh P l medium and 2 mL of suspen­ sion culture medium (R2 medium) Without selective agent Culture for 2 wk. 4 Th1rd selectiOn: Transfer the visible calli (putative transformed colomes) onto soft agarose medium (P2 medmm) contaming 0.3% agarose and the same con­ centration of selective agent. Incubate m the dark at 25°C for 2 wk (see Note 8) 3. 7. Regeneration I . Transfer the vtsJble colonies to P2 medmm contaming 0.6% Sea plaque agarose without the selecttve agent (Table 1 ) Culture for another 2 wk under the same condttions 2. Transfer the selected embryogemc colonies with a size of ca. 1 .5--3 .0 mm d1ameter with developing somatic embryos to P3 medium (Table 1). Incubate in darkness at 25°C until the development ofembryos or embryogemc calh 1s observed (see Note 9). 3. Transfer the developmg embryos onto the same medium, and culture m the hght 2 (24 f..UTIOI!m /s), with a 1 6-h photoperiod at 25°C to obtain plantlets 4 Transfer the plantlets to MS medmm wtthout hormones unt1l a well-developed root system IS obtained. These plants can be transferred to the transgemc green­ house tf mamtamed under h1gh hum1dity for the first 2 wk (see Note 10) Alter­ natively, plants can be transferred to a culture solutton (18) m a plastic trays,

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which may be placed m a phytotron or greenhouse {wtth 29°C light penod, 2 1 °C dark period), 1 4 h/day, hght 1 60 ).lll10l/m2 /s, daylight supplemented with Philips HPL N400W mercury lamps if necessary and 70--95% relative humtdity (Fig. lF; Note 1 1 ). The plants when transferred to soil (Fig. lG) set seed after 3-4 mo.

4 . Notes 1 . For each transformation expenment ( 1 0 samples), use a new tube (5 mL) of PEG from the -20°C freezer to avoid posstble toxic effects and changes of pH 2. For nurse cells: any actively dividing nee cell suspension, even the same suspen­ sion culture used for protoplast isolation can be used. In our case, we use an OC cell line that Is not regenerable, but still actively dtvides 3 . Some suspension hnes (IR72) grow better m N 6LP medium, I.e., N6/P l basal medmm (13) supplemented with 1 g/L prolme, 860 mg/L glutamme, 1 . 5 mg/L 2,4-D, and 3% w/v sucrose or maltose (1, 5) 4 Regenerable embryogemc cell suspensiOn cultures (ECS) should be used for protoplast transformation. ECS can be obtamed for all genotypes of japonica, mdiCa, and IRRI-New plant-type nee. Usually, a 3- to 6-mo old suspensiOn culture shows a better effictency for protoplast tsolatwn, transformatiOn, and sub­ sequent regeneratiOn (1 9). 5 . Enzymatic digestion of suspension cells should not exceed 5 h; if sufficient pro­ toplasts are not released, the experiment for that day should be terminated. 6. The handling of protoplasts should be very gentle at all stages of the protocol 7. For the selectiOn, we use the selectable marker genes, such as the hygromycin resistance gene, kanamycm resistance gene, or a herbicide resistance gene Some­ times a gene of interest may be linked to the selectable marker gene. For cotransformabon, we add 1 0 J,tg ofplasm1d DNA contammg the gene of mterest, l 0 J.tg of selectable marker gene, and 20 J.tg of calf thymus DNA Supercotled plasmtd DNA (50 J,tg/mL/1 0 x 1 06 protoplasts even Without calf thymus DNA) is suitable for transient gene expression, as examined 24 h after transformation. 8. A total period of selectiOn pressure of 2 em in length) to root elongation medium in tubes. The smaller (< 1 em) shoots can be subcultured for one more cycle of shoot elongatiOn m Petn dishes. Calh w1th no shoots or shoots smaller than 1 em after two cycles on shoot elongation medium can be discarded (see Note 12). 5 Plants reachmg to the top of the culture tubes and With well-developed roots can be tested for PAT activity and transferred to sml in 56-66 d (see Fig. 1 ) They are grown to maturity m growth chambers under conditions similar to those used for donor plants.

3.4. Analysis of Transgene Expression

3.4. 1. Histochemical GUS Assay for Trans1ent Express1on Remove a sample of embryos (2--4 from 4 dishes each of control and +DNA) 2 d after bombardment, and soak separately m X-gluc solution (50--100 !JL!well of a mJCrotJter plate) 2 Seal the plate w1th Parafilm, and mcubate overnight at 37°C 3 Examme embryos under a stereo d1ssectmg miCroscope to v1suahze the blue, GUS-expressing umts

3.4.2. PA T Assay This assay is based on the detection of 1 4C-labeled acetylated PPT (nonra­ dioactive PPT used as substrate) after separation by TLC. Collect leaf samples on Ice m 1 5-mL Eppendorftubes (pnor to transferring puta­ tive trans formants to soil). Be sure to include ± control samples (optimum sample we1ght 20--3 0 mg; freeze m liqmd mtrogen and store at -70°C 1f not to be used the same day) 2. Add about 2 mg Po1yvmyi-Pyrrolidone (Sigma PVP40), some Silicone powder and 1 00 !JL of EB buffer to each tube Grind for 30 s with a polytron and keep samples on 1ce. 3. Centnfuge 1 0 m at 4°C, and collect supernatant m a fresh tube Repeat centnfugatwn

Particle Bombardment

355 Plant Seeds - 60 d

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Anthes1s - 12 d

,j,

Embryo Culture (dark) 4-6 d Callus lmt1at1on

,j,

Prebombardment Treatment 4-6 h Osmot1curr.

,j,

Bombardment

,j,

Postbombardment Treatment 1 6-20 h Osmot1cum � Culture 1n the Dark No Selection or 3 mg/1 B1alaphos 14 d Callus Formation � Culture 1n the Light 3 mg/1 B1alaphos B-1 0 d Formation of Shoots

,j,

Culture 1n the L1ght 5 mg/1 81alaphos 14 d Elongation of Shoots 1n Petn dishes � Culture 1n the Light 4-5 mg/1 81alaphos 1 4-20 d Elongation of Shoots 1n Tubes � Transfer of Plants to Soil - 60 d � Anthes1s - 30 d � Mature Seeds

Ftg. 1 Time frame for the production of transgenic wheat (cv Bobwhite) plants Times shown are averages for experiments performed in 1 995, those destgnated by ­ are approximate, and vary etther with the batch of donor plants or the individual callus I me or plant. Transgenic plants were transferred to soil 56-66 d after the mit1at10n of cultures (6)

4. Measure protem concentratiOn in the crude leaf extracts followmg the Bw-Rad protem assay relative to BSA as standard. 5. Adjust protein concentratiOn to 2.5 (�Jg/f!L for each sample wtth EB buffer Assay can be stopped here overnight (store at -20°C). The background acttvtty can be ehmmated by prectpttating the extracts wtth saturated (NH4hS04• 6. Use I 0 flL of extract for each sample for assay. 7. Aliquot 3 � ofbulk reactwn mtxture to 1 0 � of each sample extract. Mix well, and mcubate at 37°C for 1 h. Stop reactiOn on 1ce.

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Spot 1 3 � of each sample on a TLC plate (can be done by first spottmg 6.5 �. drymg wtth a han dryer, and spotting the rest to avmd spreading of mtxture). Up to 1 5 samples can be spotted on one plate. 9 Transfer the TLC plate (after all the sample extracts have dned) to a saturated chromatography tank for 1-1 5 h. 1 0. Remove plate and let dry completely (at least 1 5 mm) 4 I I Vtsuahze the 1 C-labeled acetylated PPT by overnight exposure to X-ray film 1 2 . Plants testing posthve for PAT are regarded as transformed (as the assay tests for expresston of the enzyme encoded by the transgene bar) and can be transferred to sotl. Characterizatwn of the trans genes can be accomphshed by DNA isolatwn and Southern hybridizatiOn using standard protocols 8

4. Notes Because the quality of donor plants directly affects the capac tty of the tmmature embryos to produce embryogemc callus, tt ts tmportant that the plants be grown under opttmal conditwns. Pestictde applicatiOn from pollination to harvest ttme should be avoided 2 For optimum response (espectally when using field-grown matenal, which is often contaminated), use within 2 d after collection 3 The stze of the caryopsts ts variable along the length of the sptke Therefore, a more umform sample ts obtained by collectmg caryopses only from the mtddle half of each sptke 4 The duration of sterilization treatment depends on the quality of the donor plants. Caryopses from growth chamber grown plants tend to be clean and can be stenl­ ized effectively m 1 0-- 1 5 mm. Matenal from field-grown plants needs a longer (30-mm) Clorox treatment 5 Although embryos rangmg m stze from 0.5-1 .5 mm are capable of producmg embryogemc callus, the best postbombardment response ts obtamed from 1 - to 1 2-mm embryos. Embryos at thts stage of development are the eastest to dtssect, since younger embryos are nearly transparent and difficult to locate, whereas the older ones are whtte owmg to stored starch m the scutellum Holdmg the cary­ opsts with forceps and making an mcision at the base easily exposes the embryo (the outline of the embryo is vtstble before dtssectwn) 6. Fifty to 60 tmmature embryos can be cultured in the same dtsh t f the donor plants appear healthy and free of infectton Nonetheless, tt ts better to culture an average of 20 embryos/dtsh from field matenal owmg to the probabthty of infectwn. The cultures should be exammed on a datly basis to detect early stgns of contammatwn. The contammated embryos should be removed along wtth the medmm around them to avotd infectmg the adjacent embryos. 7 The htghest frequenctes of transformatiOn and regeneration are obtamed wtth 4-6 h of pre- and 1 6 h of postosmottc treatment. 8. The concentration of gold particles and DNA m gold/DNA mixture can vary. We recommend 30--5 0 J.tg gold/shot for a uniform spread on the macrocarner disk, to obtam a more even and finer stze of blue GUS umts, and a conststent effictency of transformatiOn

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9. Rupture disks in a range of 650-1 550 are used, with the 1 1 00 psi dtsk bemg most common. 1 0. Among Basta, btalaphos, and PPT, btalaphos was found to be most reliable for selectiOn of wheat transformants in the accelerated protocol (6) . The time when selectiOn 1s imposed, the number of explants/d1sh, and the concentration of the selective agent are critical variables that depend on the quality of the embryos cultured. Generally, early selection is recommended for htgh-quahty embryos, and delayed selectiOn for inferior embryos. TransformatiOn frequencies of 0. 1-2.5% were obtamed with the protocol shown m Fig. 1 . 1 1 Cultures should be examined frequently to assess the concentration of the selec­ tive agent reqmred for the next step. For example, with high zeatin m the regen­ eratiOn medium ( 1 0 mg/L), too many shoots are formed. With such cultures, It is safe to use 5 or 6 mg/L b1alaphos during shoot elongation. With low ( 1 mg/L) or no zeatin, when only one or two green areas are seen in each explant, 4 or 5 mg/L bialaphos are sufficient 12. During the penod the explants and the regenerants are m Petn dtshes, bmlaphos can be used at 5 or 6 mg/L, since cross-protectiOn IS provided by the adJacent explants. However, tf very few shoots emerge after the first cycle of shoot elon­ gatwn, then 4-5 mg/L bmlaphos are sufficient, because only a smgle explant IS present and there is no crossprotectwn.

References 1 Oerke, E-C., Dehne, H-W , Schonbeck, F., and Weber, A ( 1 994) Crop Produc­ twn and Crop Protectwn Estzmated Losses m Ma;or Food and Cash Crops Elsevier, Amsterdam 2 Bialy, H. ( 1 992) Transgemc wheat finally produced Bw/Technology 10, 675 . 3 . Vast!, V , Castillo, A. M., Fromm, M E , and Vast!, I K ( 1 992) Herbtctde rests­ taut ferttle transgemc wheat plants obtamed by microproJecttle bombardment of regenerable embryogemc callus. Bw/Technology 10, 667-674 4. Vasil, V., Snvastava, V., Castillo, A. M., Fromm, M. E., and Vasil, I. K ( 1 993) Rapid productwn of transgenic wheat plants by direct bombardment of cultured tmmature embryos Rio/Technology 1 1 , 1 5 53-1 558. 5 Weeks, J. T., Anderson 0 D , and Blechl, A. E ( 1 993) Raptd productiOn of mul­ tiple mdependent lines of ferttle transgenic wheat (Trztzcum aestzvum) Plant Physiol 1 02, 1 077-1084 6. Altpeter, F., Vasil, V., Srivastava, V., Stoger, E., and Vast!, I K. ( 1 996) Acceler­ ated production of transgemc wheat ( Tntzcum aestivum L.) plants. Plant Cell Rep 16, 1 2-1 7 7. Ortiz, J P A , Regg1ardo, M I , Ravizzm1, R A., Altabe, S. G., Cervtgm, G. D. L , Spitteler, M. A., et a!. ( 1 996) Hygromycin rest stance as an effictent selectable marker for wheat stable transformation Plant Cell Rep 15, 877-8 8 1 8. Nehra, N. S , Chibbar, R. N., Leung, N . , Caswell, K., Mallard, C., Stemhauer, L , et a! ( 1 994) Self-fertile transgenic wheat plants regenerated from Isolated scutellar t1ssues followmg m1croprojectile bombardment w1th two dtstmct gene constructs. Plant J 5, 285-297

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9 Zhou, H., Arrowsmtth, J W., Fromm, M. E., Htronaka, C M., Taylor, M. L , Rodriguez, D., et al ( 1 995) Glyphosate-tolerant CP4 and GOX genes as a select­ able marker m wheat transformation Plant Cell Rep. 15, 1 59-1 63 1 0 . Becker, D., Brettschneider, R., and Lorz, H. ( 1 994) Ferti le transgenic wheat from microproJ ecttle bombardment of scutellar tissue. Plant J. 5, 299-307. 1 1 Takumt, S and Shimada, T ( 1 996) Production of transgenic wheat through par­ ticle bombardment of scutellar tissues: frequency ts mfluenced by culture dura­ tion J Plant Physwl 14 9, 4 1 8--423 1 2 Vasil, I. K. ( 1 996) Phosphmothncm-resistant crops, tn Herb1c1de-ReS1Stant Crops (Duke, S 0 , ed. ), Lewis Publishers, Boca Raton, FL, pp. 85-9 1 1 3 . Christensen, A. H. and Quat!, P. H. ( 1 996) UbtqUtttn promoter-based vectors for high-level expresston of selectable and/or screenable marker genes in monocoty­ ledonous plants. Transg Res 5, 2 1 3-2 1 8. 1 4. Altpeter, F . , Vast!, V , Srtvastava, V., and Vast!, I. K. ( 1 996) Integratton and expression of the htgh-molecular-wetght glutenin subumt l Ax 1 gene tnto wheat Nature Biotechno/ 14, 1 1 5 5-1 1 5 9 1 5 Taylor, M. G , Vast!, V., and Vast!, I K. ( 1 993) Enhanced GUS gene expresswn tn cereal/grass cell suspensions and immature embryos usng the matze ubtquittn­ based plasmtd pAHC25 Plant Cell Rep 1 2, 49 1--495 16 Snvastava, V., Vast!, V , and Vast!, I K. ( 1 996) Molecular charactenzation of the fate of transgenes tn transformed wheat ( Tnt1cum aest1vum L ) Theor Appl Genet. 92, 1 03 1-1 037. 1 7. Blechl, A. E. and Anderson, 0 D ( 1 996) ExpressiOn of a novel htgh-molecular­ wetght glutemn subumt gene in transgemc wheat Nature Bwtechnol 14, 875-879 1 8 . Murashige, T. and Skoog, F. ( 1 962) A revised medmm for rapid growth and bio­ assays wtth tobacco ttssue cultures. Physwl Plant 15, 4 73--497.

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Pl ant Transformation via Protoplast Electropo ration Georg e W. Bate s

1 . Introduction In electroporatwn, cells are permeabihzed by the application of very short, high-voltage electric pulses. Molecules ranging in size from small orgamc metabolites and reporter dyes to large macromolecules-including antibodies and plasmids--can be mtroduced into cells by electroporation. Electroporation Is effective on virtually any type of cell, and ts now the method of chOice for the genetic transformation of bactena and certain animal cell hnes. The pn­ mary application of electroporatiOn to plants has been for DNA uptake for studies of transient gene expressiOn and for stable transformation. However, electroporation has also been used to mtroduce RNAs (1,2), antibodies (3), and small molecules (4) into plant cells and Isolated organelles (5). Because the thick plant cell wall restricts macromolecule movement, most work on plant cell electroporation utilizes protoplasts. This has limited the use of electroporation for stable transformation to species whose protoplasts are regenerable. As protoplast regeneration systems are Improved, reports of new species of plants transformed by electroporat10n contmue to appear (for example, ref. 6). One advantage of electroporation over particle bombardment for stable transformation IS that electroporation results predominantly m single­ copy plasmid msertions (Bates, unpublished observations), whereas particle bombardment tends to introduce large plasmid concatemers. However, the main use of protoplast electroporation 1s in transient express1on assays for studies of transcriptional regulation (for example, 7, 8). These stud1es do not require protoplast regeneration. A growing number of recent reports indicate that electroporation can be used to introduce DNA into walled plant cells and plant tissues (9, 10). Tissue electroporation does not work in all cases, and the parameters for successful plant tissue electroporation are not yet clear. From Methods Ed1ted

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Molecular Biology, Vol 1 1 1 Plant Cell Culture Protocols

by R D Hall © Humana Press Inc , Totowa, NJ

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However, the success of tissue electroporatton in crops, such as maize and soybean, reopens the use of electroporation for stable transformation in major crops. This chapter provides a protocol for protoplast electroporation, a protocol for the selection of stable, kanamycin-resistant transformants, and notes on how to optimize these protocols for both stable transformation and transient expresswn. These protocols have been used for many years in the author's laboratory for the transformation of tobacco protoplasts, but they can be readily modified for use with protoplasts of other species and for the uptake of mole­ cules other than DNA. 2. Materials 2. 1. Protoplast Electroporation InstrumentatiOn· Electroporatwn equipment IS available from a variety of com­ mereta! manufacturers and can also be homemade Lists of commercial manufac­ turers and instrument specificatiOns, as well as a general dtscusston of homemade eqmpment can be found in Chassy et a!. (1 1). Two types of oc electrical pulses, square-wave pulses and capacitive dts­ charges, may be used for electroporahon. However, because the eqUipment IS less expenstve, most iaboratories use capaclilve-dtscharge electroporation sys­ tems The equipment presently used m the author' s laboratory ts the Cell-Porator® Electroporatwn System I manufactured by Gtbco BRL Life Technologies Inc. (Gaithersburg, MD). Thts capacttlve-dtscharge mstrument allows the pulse volt­ age to be adJUSted from 0 to 400 V, pulse length can be varied by selectmg one of etght dtfferent-stzed capacitors (from 1 0-1 980 )lF) (see Note 1) The Gtbco BRL Celi-Porator utilizes presterilized, disposable electroporatwn chambers to hold the cells during electroporatiOn For work wtth plant protoplasts, electroporation chambers should be selected that have a 0.4-cm electrode gap Chambers with 0 1 -cm electrode gaps can also be purchased, but are designed for electroporation of bacteria 2. Protoplasts: Protoplasts Isolated by standard procedures are suitable for electro­ poratwn. Protoplasts from a wtde range of spectes, organs, and cell cultures have been successfully electroporated However, tt ts important that the protoplasts be ofhtgh quality. Even m htgh-quahty protoplast preparatiOns, electroporatwn ktlls a substantial fractwn of the protoplasts PreparatiOns of margmal or low-quality protoplasts are likely to be completely killed by the electric shocks 3 DNA" The plasmid DNA used m electroporatwn does not have to be highly pun­ fied, but should be free of RNA and protems (such as RNase and restnctwn enzymes) that m1ght affect the vtabthty of the protoplasts The author's labora­ tory routmely uses plasmids isolated by alkaline lysts (12). RNA is removed from the plasmtd preparatiOns by RNaseA dtgestton followed by phenol extractiOn and ethanol precipitatiOn (see Note 2) Before electroporatton, the DNA must be sierihzed. DNA that has been etha­ nol-prectpttated and redtssolved m autoclaved water or TE ts probably suffi-

Protoplast Electroporatlon

36 1

ciently stenle. However, to be certam the DNA is sterile it can be passed through a 0.2-f..Ull pore, low-bindmg, cellulose acetate syrmge filter (e.g., Nalgene # 1 902520). It is convenient to fi lter-sterilize the DNA after d1lutmg 1t mto electro­ poration medium at the start of an experiment For transient expressiOn studies, linear and superc01led plasm1ds are equally effective (13). However, for stable transformation, lmear DNA IS 3- to 1 0-fold more efficient than superc01led (14). 4. Electroporation medmm: Hepes-buffered sahne (HBS): 1 50 mM KCI, 4 mM CaCI 2, I 0 mM HE PES (pH 7 .2), and enough mannitol to balance osmotically the protoplasts For tobacco mesophyll protoplasts, 0 2 1 M mannitol is used. The HBS can be prepared in advance, stenlized by autoclavmg, and stored at 4°C.

2.2. Selection of Stable Transformants 1 . Media for protoplast culture and selection of stable transformants: Tobacco mesophyll protoplasts may be cultured m K3G medium (K3 salts, v1tamms, and hormones {15] containing 0.4 M glucose as the carbon source and osmotic stabi­ lizer). K3G may be sterilized by autoclaving, and stored at 4°C 2 Callus medmm (CM)· Murash1ge and Skoog salts and v1tamms (1 6) plus 1 00 mg/L inositol, 3% sucrose, 1 mg/L benzyladenme, and I mg/L a-naph­ thaleneacetic acid For selection of kanamycm-res1stant stable trans formants, CM is amended by addition of vanous amounts of mannitol, Sea plaque agarose (FMC Corp , Rockland, ME), and kanamycin as descnbed below in the Subheading 3. CM and CM amended w1th mannitol and Sea plaque agarose can be prepared m advance, sterilized by autoclaving, and stored at 4°C Kana­ mycm should not be autoclaved. A I OOOX stock of kanamycin can be prepared by d1ssolvmg 1 00 mg/mL kanamycin sulfate m water; the stock should be fil­ ter-sterilized and stored at -20°C.

3. Methods The following procedures must be carried out under sterile conditiOns, prefer­ ably in a lammar flow hood. All solutions should be at room temperature (see Note 3). 3. 1. Protoplast Electroporation Freshly isolated protoplasts should be used for electroporation: After washing the protoplasts free of the enzymes used for cell-wall d1gestwn, pellet the proto­ plasts and resuspend them in 1 0 mL HBS + manmtol (see Note 4). 2 Determine the number of protoplasts/mL usmg a hemacytometer. 3 . Place aliquots of I x I 0 6 protoplasts into 1 5-mL conical centrifuge tubes, and pellet the protoplasts by centrifugation (50g for 5 mm). 4. Discard the supernatant. Usmg a disposable plastic transfer p1pet, gently resus­ pend each protoplast sample in 0.5 mL HBS + manmtol + DNA. The plasmid DNA concentration m the electroporatwn medmm should be 1 0- 1 00 �Jg/mL (see Note 5).

362 5 6

7.

8

9 l 0.

Bates Use a dtsposable plasttc transfer ptpet to transfer the protoplast samples to electroporation chambers Let stand for 5 mm. Resuspend the protoplasts by gentle agttatwn of the electroporatwn chamber, and then Immediately apply a smgle electric pulse (325 1!f, 300 V; see Note 6) [f you are using the Gtbco BRL Ceii-Porator, make sure the mstrument is on the low .n setting. Watt 2-3 mm, and then resuspend the protoplasts by agttation of the electro­ poratwn chamber. Transfer the protoplasts to a conical centrifuge containing 5 mL of K3G Rinse the electroporatwn chamber wtth 0. 5 mL HBS + manmtol, and combme the rinse wtth the protoplast sample (see Note 7) When all the protoplast samples have been electroporated, pellet the protoplasts, and resuspend each sample in 5 mL of culture medtum. Transfer the protoplast samples to a 60 x 1 5 mm Petn dish, and culture the proto­ plasts at 27°C. Protop lasts may be sampled after 24 h for transtent gene expresswn or roam­ tamed in culture for later selectiOn of stable trans formants

3.2. Selection of Stable Transformants The following procedure for selectiOn of stable transformants ts based on the agarose-bead culture technique of Shill ito et al. (1 7) . In this procedure, the protoplast culture is diluted and solidified by additiOn of low-melting-pomt agarose. When properly diluted, the embedded protoplasts are well separated from each other and grow into mdividual calh. Selection for transformants I S carried out after embeddmg the cells. This procedure permits calculation of plating efficiency and transformation efficiency, and allows isolation of indi­ vidual transformed clones. The method outlined here describes selection of kanamycm-resistant clones, but it can easily be adapted for use with other selectable markers or reporter genes. Dunng selectwn, the culture is progres­ sively diluted and the osmotic strength of the medium ts reduced. The sequence of medium changes described here has been optimized for work with proto­ plasts of Nicotiana tabacum, but can be modified for work With other species. l . Choose a plasmtd containmg a functiOnal neomycm phosphotransferase II gene such as pMON200 (18) or pBI 1 2 l (Clonetech, Palo Alto, CA). Lineanze the plasmtd by digestion wtth a suitable restnctwn enzyme. It is convenient to cut enough of the plasmid for several expenments. Then phenol-extract and ethanol­ precipitate the DNA, and resuspend tt m water at a concentration of l �JgiJ..!L . 2 Electroporate the protoplasts m the presence of 1 0-100 J..!g/mL of lmeanzed DNA, and culture them m 60 x 1 5 mm Petri plates m 5 mL of K3G (see Note 8) 3. After l wk of culture, the protoplasts wi ll have grown mto small-cell clusters (see Note 9) At this pomt, the protoplast-denved colomes are tmmoblltzed by a l : l dilution of the culture wtth medium containmg agarose Prepare in advance CM medtum + 0.23 M manmtol + 2 4% Sea plaque agarose, and stenhze It by

Protoplast Electroporation

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5 6.

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autoclavmg. Just before the transfer, melt this medium, and let it cool to JUSt above its gellmg temperature. While the agarose Is cooling, scrape any adhering protoplast colonies off of the Petn plate with a micropipet tip Transfer half of the culture (2.5 mL) to a new 60 x 1 5 mm Petn plate Now add 2 5 mL of the agarose-contaming medium to both Petn plates of protoplasts Mix thoroughly by swirling the plates. Place the Petn plates in the refngerator for 1 5 min to solidify the agarose. Culture the protoplasts at 27°C At the end of the second week of culture, divide the solidified culture mto wedges using a spatula, transfer the wedges to 100 x 1 5 mm Petn plates, and add 5 mL of liquid CM + 0. 1 3 M mannitol supplemented with 1 00 j.J.g/mL kanamycm (see Note 10). Culture at 27°C. At the end of the third week, add 5 mL CM supplemented with 1 00 j.J.g/mL kana­ mycm to each Petri plate. Thereafter, at weekly mterval s, remove 5 mL of hqmd from each Petn plate, and replace It With 5 mL of fresh CM supplemented With 1 00 j.J.g/mL kanamycm Transformed colomes should be visible after 4-5 wk of culture. When the transformed colomes are 2-3 mm in diameter (4-6 wk of culture), they can be picked out of the agarose using a spatula, and cultured on CM + 0.8% agar + 1 00 j.J.g/mL kanamycm. After another 2 wk of growth, they should be large enough to be transferred to regeneration medmm.

4 . Notes 1 . Pulse voltage, or more precisely electric field strength, and pulse length are two critical parameters m electroporati on, and must be optimized for each species and cell type Field strength depends on the voltage applied to the electroporatton chamber and the distance between the electrodes in the chamber. The appropnate units for field strength are V/em. Application of a 1 00 V pulse to a chamber with a 0.4-cm electrode gap results in a field strength of 250 V/em. Pulse length is determined by the size of the capacitor and the resistance of the electroporation medium. Di scharging a capacitor produces an exponentially decaymg pulse. The length of such pulses is best described by their RC time constant, which is the time reqmred for the pulse voltage to drop to 3 7% of its Initial value 2. Both RNA and protems present in a crude plasmid preparatton can be introduced mto the protoplasts by electroporation along with the DNA and may affect the experimental outcome. For example, in some early experiments in the author ' s laboratory, protoplasts were electroporated in the presence of a plasmid that had been treated with RNase, but not phenol-extracted. No transient gene expressiOn was observed until the RNase was removed from the plasmid preparations 3. Some electroporatlon protocols cail for chilling the cells m an ice bath during and Immediately after electroporation. The pores that form in the plasma membrane owing to electnc shocks have been shown to stay open longer if the cells are maintained at a temperature below the membrane's phase transition temperature. Chilling the cells would be expected to improve the efficiency of transformatiOn

364

4.

5.

6

7.

Bates by electroporatwn, because tt would allow more time for DNA uptake How­ ever, several studtes of transient and stable gene expressiOn have shown that chilling does not Improve electroporation effictency It turns out that the uptake of DNA dunng electroporatwn is electrophoretic (and not diffustve) and occurs dunng the electric pulse itself Thus, chillmg the cells ts unneces­ sary tf transformatiOn ts the goal of electroporation Chilhng may be useful, however, when el ectroporatwn ts used to mduce the uptake of molecules other than nuc letc acids. Because HBS is a nonphyswlogtcal, htgh-salt medmm, it ts advtsabie to hmlt the exposure of the protoplasts to thts medmm to 30 min. If a large number of samples are gomg to be electroporated, divide the protoplast preparation mto two batches. Leave one batch in the protoplast wash or in culture medmm, while the other batch ts resuspended m HBS, divided mto samples, and electroporated. Then when the first batch of protoplasts has been electroporated and dtluted mto cul­ ture medium (Subheading 3.1., step 7), pellet the second batch of protoplasts resuspend them m HBS and process them for electroporat10n. The effictency of both transtent expressiOn and stable transformation mcreases l inearly with DNA concentration from l 0 to l OO f.!g/mL. Transient expressiOn and stable transformatiOn are also increased by addition of "carner DNA," such as salmon sperm DNA. For transient gene expressiOn, the author's laboratory uses 1 0 f.!glmL supercoiled plasmid DNA + 50 f.!g/mL salmon sperm DNA (sheared by sonication). Because the earner DNA has been found to integrate along with the plasmtd, carrier DNA should be avotded for stable transformatiOn When the goal t s stable transformatiOn, thts laboratory uses 50 f.lg/mL of linear­ tzed plasmid DNA and no earner The efficiency of electroporation depends on pulse length and voltage A 325-!lf capactttve dtscharge mto 0.5 mL of HBS gtves a pulse of about l 0 ms (RC ttme constant) For tobacco mesophyll protoplasts, a l O-ms pulse of 300 V (750 V/em field strength m the electroporatton chamber) usually gives optimal results. The optimal setting can vary wtth species and cell type, and should be determmed empirically m prelimmary expenments. A quick way to begin to look for effec­ tive electroporat10n parameters ts to find pulse settings that result in 50% proto­ plast death by 24 h after the shocks Electroporatwn effictency can also vary between batches ofprotoplasts because ofbatch-to-batch dtfferences m protoplast viabthty. Batch-to-batch vanabthty com­ phcates transtent expressiOn studtes. To handle thts vanability, rephcates and appropnate controls must be mcluded m every expenment. Some transient expres­ sion studtes also mclude an mternal control. The approach t s to add a second plas­ mid carrying a reporter gene to each sample before electroporatwn as an internal control. ExpressiOn of the second plasmtd can be used to normalize both batch-to­ batch and sample-to-sample differences in transient gene expression Manufacturers of electroporation eqmpment mtend for the electroporatwn cham­ bers to be dtscarded after each use. However, the author finds that the chambers can be reused two or three times wtthout affectmg electroporation efficiency or

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365

protoplast viability. Rmsmg the chamber after electroporatwn with 0.5 mL HBS + manmtol not only helps remove all the electroporated protoplasts from the chamber, but also readies the chamber for the next sample. 8. High initial protoplast densities improve surviVal of the electric shocks. Up to 500 transformed clones can be recovered from a sample of I x I 0 6 protoplasts This would give an absolute transformation efficiency of 1 transformant for every 2000 electroporated protoplasts. However, the transformatiOn efficiency IS actu­ ally severalfold higher, because 50% of the protoplasts are killed directly by the electnc shock and no more than half of the surviVing protoplasts grow into calli This laboratory uses 50 J.tg/mL hnear DNA for stable transformatiOn South­ em blotting shows that 50-75% of the transformants have a single copy of the plasmid integrated. 9. The timing of the media changes described here should not be adhered too rigidly, but should be modified depending on how fast the protoplasts grow after electroporation. For tobacco protoplasts, the first dilution of the cultures IS done when the protoplasts have grown into m1crocolomes of 5- l 0 cells, which IS sually after 6-7 d of culture This first dilution may have to be delayed if the protoplasts are growmg more slowly-as will happen if electroporatwn kills more than about 75% of the protoplasts Too rapid a dilutiOn of the culture results m death of the protoplast-derived colomes within 24 h of the dilution I 0. As descnbed here, the selectiOn pressure starts out at 50 llg/mL of kanamycin and mcreases to about 1 00 llg/mL over a penod of a few weeks. Selection can be started earlier. For example, It can be convemently started I wk after e lectroporation by adding kanamycin at the first dilution of the culture This lab obtains the highest number of trans formants when addition of kanamycm IS delayed until the end of the second week. This may be because piating efficiency depends on cell density, so startmg selection early reduces the cell density in the culture, and this in turn, inhibits the growth of some trans­ formed clones SelectiOn for kanamycm resistance is very clean in tobacco. The author never observes untransformed clones growmg m the presence of kanamycin.

References I . Bailey-Serres, J. and Dawe, R. K. ( 1 996) Both 5' and 3' sequences of maize adh l mRNA are reqmred for enhanced translatiOn under low-oxygen conditiOns. Plant Physwl. 1 12, 685--695 2. Wintz, H. and Dietrich, A. ( 1996) Electroporation of small RNAs into plant proto­ plasts. mitochondrial uptake of transfer RNAs. Bwchem Bwphys Res Commun 223, 204--2 1 0. 3 Maccarrone, M., Veldmk, G A., Finazzi Agro, A., and Vhegenthart, J. F. ( 1 995) Lentil root protoplasts. a transient expression system suitable for coelectroporatwn of monocolonal ant1bodtes and plasmid molecules Bwchzm Bwphys. Acta 1 243, 1 3 6-142. 4. Jang, J.-C. and Sheen, J. ( 1 994) Sugar sensmg in higher plants. Plant Cell 6, 1 665--1679.

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5 To, K. Y , Cheng, M C , Chen, L F., and Chen, S C (1 996) Introductton and expressiOn of foretgn DNA m t solated spmach chloroplasts by electroporation Plant J 10, 737-743 . 6 Lee, L , Larmore, C. L., Day, P. R , and Turner, N. E ( 1 996) Transformation and regeneratiOn of creeping bentgrass (Agrostzs palustrzs Huds) protoplasts. Crop Scz 36, 40 1-406 7. Kao, C. Y., Cocciolone, S. M , Vastl, I. K., and McCarty, D. R. ( 1 996) Localiza­ tion and interactiOn of the cts-actmg elements for absctsic acid VIVIPAROUS 1 , and !tght achvahon o f the C 1 gene of matze. Plant Cel/ 8, i i 7 1-i 1 79. 8 Snowden, K. C., Buchholz, W. G., and Hall, T. C. ( 1996) Intron position affects expressiOn from the tpt promoter m nee. Plant Mol Bwl 3 1 , 689-692 9 Laursen, C M., Krzyzek, R. A , Fhck, C E., Anderson, P. C , and Spencer, T M ( 1 994) Productton of ferttle transgemc matze by electroporatwn of suspension culture cells Plant Mol Bwl. 24, 5 1-6 1 . 10. Chownra, G . M , Akella, V., Fuerst, P. E., and Lurqum, P F ( 1 996) Transgemc gram legumes obtamed by m planta electroporatton-medtated gene transfer. Mol Bwtechnol. 5, 85-96. 1 1 Chassy, B. M., Saunders, J A., and Sowers, A. E. (1 992) Pulse generators for electrofusion and electroporation, in Guzde to Electroporatwn and Electrofuswn (Chang, D. C , Chassy, B. M., Suanders J A , and Sowers, A. E., eds.), Academic, San Diego, pp. 555-569. 12 Sambrook, J , Fntsch, E F , and Mamatis, T . ( 1 989) Molecular Clonmg A Labora­ tory Manual, 2d ed. Cold Spnng Harbor Laboratory Press, Cold Spring Harbor, NY 1 3 . Bates, G W , Carle, S A., and Piastuch, W C ( 1 990) Lmear DNA mtroduced into carrot protoplasts by electroporatton undergoes ltgatwn and recirculanzatton Plant Mol. Bioi 14, 899-908 1 4. Bates, G. W ( 1 994) Genetic transformatiOn of plants by protoplast electro­ poratwn. Mol Bwtechnol. 2, 1 3 5-145. 15. Nagy, J. I. and Maitga, P ( 1 976) Callus mductton and plant regeneration from mesophyll protoplasts of Nzcotzana sylvestrzs. Z Pflanzenphyswl 78, 453-455 16 Murashtge, T and Skoog, F ( 1962) Revtsed medium for raptd growth and bioas­ says wtth tobacco tissue cultures Physwl. Plant 15, 4 73-497 1 7 Shilhto, R D , Paszkowskt, J , and Potrykus, I ( 1 983) Agarose platmg and a bead type culture technique enable and stimulate development of protoplast-denved colonies m a number of plant spectes Plant Cell Rep 2, 244-247. 18 Rogers, S G , Horsch, R. B., and Fraley, R. T ( 1 986) Gene transfer m plants· production of transformed plants usmg T1 plasmid vectors Methods Enzymol 1 18, 627-64 1 .

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Transformation of Maize via Tissue Electroporation Kathleen D 'Halluin, Els Bon ne, Martl e n Bossut, and Rosita Le Page

1 . Introduction Introduction of DNA into plant protoplasts via electroporation is a well­ known procedure. By givmg electrical impulses of high field strength, the cell membrane is reversibly permeabihzed, so that DNA molecules can be mtro­ duced into the cell {1) . Originally, maize transformatiOn efforts focused on electroporation of proto­ plasts (2). However, transient transformation by tissue electroporation was first demonstrated by Dekeyser et al. (3). Leaf bases of several monocot species, mclud­ ing maize, were shown to be amenable to electroporatiOn-mediated DNA uptake based on the transient expression of reporter genes. Stable transformants via tissue electroporation of matze have been described for enzymatically treated or mecham­ cally wounded immature embryos and type I callus (4), for mechanically wounded immature zygotic embryos (5), for enzymatically treated embryogenic maize sus­ pension cells (6), and for type II callus (7). Furthermore, transient expressiOn was observed after electroporation of intact immature maize embryos (8). Transient and stable electrotransformations were obtamed after electroporation of preplas­ molyzed, intact black Mexican sweet maize suspension cells (9). The followmg schedule outlines the steps for the stable transformation of enzymatically treated, Immature embryos and mechamcally wounded type I callus of maize. 2. Materials 2. 1. Electroporation 1 . Embryo culture medmm, Mah l VII· N6 basic medium (10) supplemented with

1 00 mg/L casem hydrolysate, 6 rnM L-prohne, 0.5 g/L MES, and 1 mg/L 2,4-D sohddied wtth 2.5 g/L Gelrite (Duchefa), pH 5 . 8 (see Notes 1 and 3). From Methods m Molecular Btology, Vol 1 1 1 Plant Cell Culture Protocols Edited by R D Hall © Humana Press Inc Totowa, NJ

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2.

3.

4

5 6. 7.

Mah l VII substrate. M: N6 basic medium; a: 1 00 mg/L casem hydrolysate and 6 mM L-proline; h: 0.5 giL MES; I · I mg/L 2,4-D; VII· 2.5 g/L Gelnte (Duchefa) Enzyme solution 0.3% macerozyme (Kmki Yakult, Nishinomiya, Japan) in CPW salts (11) supplemented with 1 0% manmtol and 5 mM MES, pH 5.6, filter-steril­ Ized (see Note 2) Washing medmm: N6 salt solution supplemented w1th 6 mM asparagme, 12 mM L-proline, I mg/L thiamin-HCl, 0 5 mg/L nicotinic ac1d, 1 00 mg/L casein hydroly­ sate, 1 00 mg/L mos1tol, 30 g/L sucrose, and 54 g/L manmtol, filter-stenhzed EPM buffer. 80 mM KCI, 5 mM CaCI2 , i 0 mM HEPES, and 0 425 M manmtol, pH 7 2 The same buffer without KCI 1s also needed. Agarose substrate· water with 0 45% BRL agarose Electroporatwn apparatus, electrodes, and cuvets (see Notes 4 and 6). DNA ofan appropriate plasmid beanng the bar selectable marker gene (see Note 5)

2.2. Selection and Regeneration of Transformants I . SelectiOn medmm Mah1 l VII Mah I VII substrate supplemented With 0 2 M man­ mtol and 2-5 mg/L filter-stenhzed phosphmothricm (PPT) (see Notes l and 3) Mahi l VII substrate. M. N6 basic medmm, a. 1 00 mg/L casem hydrolysate and 6 mM L-prohne; h. 0.5 g/L MES, , . 0 2 M manmtol, l . l mg/L 2,4-D, VII 2 5 g/L Gelnte (Duchefa) 2 SelectiOn medmm Mhi l VII: Mah l VII substrate from which casem hydrolysate and L-proline are omitted, and supplemented With 0 2 M manmtol and 2-5 mg/L filter-stenlized PPT Mh! l VII substrate M N6 basic medmm, h 0.5 g/L MES; , . 0 2 M manmtol, I 1 mg/L 2,4-D, VII 2 5 g/L Gelnte (Duchefa) (see Notes 1 and 3). 3 SelectiOn medmm Mh 1 VII Mah 1 VII substrate from which casem hydrolysate and L-prolme are omitted, and supplemented With 2-5 mg/L filter-stenhzed PPT (see Notes l and 3) Mh l VII substrate M: N6 basic medmm, h· 0 5 g/L MES; I . I mg/L 2,4-D, VII: 2.5 g/L Gelnte (Duchefa) 4 Selection medium Ahi i .5VII · correspondmg substrate, ofMh1 l VII substrate but MS (12, and see Appendix) basic medmm ("A") mstead of N6 basic medium ("M") supplemented with 0 2 M mannitol, 0 5 giL MES, I 5 mg/L 2 4-D, and 5 mg/L filter­ stenhzed PPT, solidified with 2.5 g/L Gelnte (Duchefa), pH 5.8 (see Notes l and 3) Ahi l .5 VII substrate A MS medmm; h· 0 5 g/L MES; 1 . 0 2 M manmtol, 1 5 1 5 mg/L 2,4-D, VII 2 5 g/L Gelnte (Duchefa) 5 RegeneratiOn medmm· MS medmm supplemented with 5 mg/L 6-benzylammo­ punne and 2 mg/L filter-stenhzed PPT (see Notes 1 and 3). 6. Shoot development media (see Notes 1 and 3): MS6%: MS medmm with 6% sucrose and supplemented With 2 mg/L fi lter­ stenhzed PPT MS1h: half-concentratiOn of MS medmm 7 Basta 0 5%· 0 5% (v/v) of a Basta stock solutiOn contaming 200 g/L giufosinate­ ammomum

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3. Methods 3. 1. Electroporation

3. 1 . 1 . Immature Embryos

2 3. 4

5 6. 7. 8

9.

Maize plants of the public inbred line H99 are grown in the greenhouse tn 20-L pots containing slow-release fertilizer. Growth conditions are at 25°C and 1 6-h hght of -20,000 lx (daylight supplemented by sodium vapor and mercury halide lamps); at night, temperature is reduced to 1 5-20°C. Kernels from ears 9-1 4 d after pollination are surface-sterilized m 3. 6% sodium hypochlorite for 1 5 mm. Immature zygotic embryos of the mbred lme H99 ( 1 -1 5 mm m length) are exc1sed and plated on Mah l VII substrate (see Note 1) Freshly isolated Immature embryos are collected from the Mah 1 VII substrate and enzymatically treated for 1-3 mm at room temperature with the enzyme solution (see Note 2) The embryos are then carefully, washed with filter-stenhzed washmg medmm (see Note 3). After washmg, approx 50 embryos are transferred mto a dtsposable mtcrocuvet contammg I 00 !JL matze electroporation buffer (EPM) (see Note 4). Ten m1crograms of hneanzed plasm1d DNA are added per cuvet and coincubated with the enzyme-treated embryos (see Note 5). After 1 h, the electroporatwn is carried out by dischargmg one pulse w1th a field strength of 375 V/cm from a 900-� capac1tor (see Note 6). The onginally descnbed (4) pre- or postelectroporat1on incubatiOn on ice can be omitted After a few minutes, the embryos are transferred onto culture substrate.

3. 1 .2. Type I Callus I . Immature embryos ( 1-1 5 mm m length) of the backcross (Pa9 1 x H99) x H99 are exc1sed from ears 9-1 2 d after pollination and plated w1th the1r embryonic axts m contact wtth the Mah l VII substrate (see Note 7). 2. Type I callus 1s imttated from tmmature embryos m the dark at 25°C 3 . Yellow, not too far differentiated, embryogenic t1ssue IS microscopically dts­ sected from developing, type I callus that has been cultured on Mah 1 VII sub­ strate for a period of - 1 .5--6 mo with subculture intervals of approx 3--4 wk Tissues older than 6 mo are discarded, smce the quahty of the callus declmes 4. The embryogenic tissue is cut through the menstems, using a mtcroscope, mto pteces of approx 1-1 5 mm and collected m EPM buffer without KCI. 5 The finely chopped tissue is washed several times wtth EPM buffer wtthout KCI to remove nucleases. 6. After 3 h of preplasmolysis in th1s buffer, the callus pieces are transferred to cuvets containmg 1 00 ,L of EPM supplemented with 80 mM KCl. 7. Ten mtcrograms of hnearized plasmid DNA are added per cuvet, and subsequent conditiOns are as for electroporatwn of Immature embryos

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3.2. 1 . Immature Embryos I

2 3. 4.

5 6 7. 8

The embryos are transferred either Immediately after electroporatwn to selective substrate MahJ I VII or to nonselective substrate for a few days (3-7 d) before transferring to selective substrate Mh1 l VII (see Note 8). The quality of the embryos IS the determmmg factor for the duect or nondirect transfer to selective substrate immediately after transformatiOn. The omisswn of L-proline and casein hydroly­ sate from the selective substrate makes the selectton more stnngent. Good-qual­ ity embryos from maize plants grown under more optimal conditions are plated Immediately on selective Mhi 1 VII substrate. After 2-3 wk, the embryos are transferred to selectiVe Mh l VII substrate. After 3-4 wk, the embryogemc tissue is selected and subcultured for another 3-4 wk on selective Mh l VII substrate w1th 2-5 mg/L PPT. Afterwards, the developmg embryogemc tissue IS Isolated and transferred to regeneratiOn medmm at 2 5 °C w1th a daylength of 1 6 h. Fluorescent lamps ("lumJiux wh1te" and "natural"; Osram, Mumch, Germany) are used with a hght mtensity of 2000 lx After 2 wk, the embryogenic tissue 1s subcultured onto fresh substrate with the same composition. Developmg shoots are transferred to MS6% medium With 2 mg/L PPT from which hormones are omitted Further developing shoots are transferred to nonselective MSI!z medium for fur­ ther development mto plantlets Plantlets are transferred to soil and grown to maturity m the greenhouse.

3.2.2. Type I Callus 1 . Immediately after electroporation, the callus p1eces are transferred to seiect1ve Ahi l .5VII substrate with 5 mg/L PPT. 2. After 3-4 wk, the prohferatmg callus pteces are subcultured onto Mh 1 VII sub­ strate with 5 mg/L PPT for a period of approx 2 mo with a subculture mterval of approx 3-4 wk. 3 Subsequent conditiOns for regeneratiOn are as for immature embryos of H99, except that 5 mg/L zeatm are used mstead of 6-benzylaminopurine for the mduc­ tlon of regeneratiOn from the selected embryogenic tissue.

3.3. Analysis of Putative Transgenic Plants

3.3 1 Phosphinothricin Acetyltransferase (PA T) Activity PAT activtty is detected by an enzymatic assay usmg 14C-Iabeled CoA. 1 4C-labeled acetylated PPT IS detected after separation by thin-layer chroma­ tography (13).

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3.3.2. Dot Assay or Basta Spraying Leaves of in vivo-grown plants are brushed With a 0.5% Basta® solution or 2 sprayed with 1 00 mL/m of a 0.5% Basta solution. Plants are assessed 8 d after Basta application. Plants expressing the bar gene have no symptoms, whereas leaves of nontransformed plants show necrosis.

3. 3. 3. Inheritance of the bar Gene Most plants should develop normally and form a normal tassel and ear. Seed set is obtained by selfing or crosspollination. Transgenic lines can be used either as male or female parent m the crossing or as both. The segregation of the Basta resistance gene is then determined by using a Basta dot assay or by a Basta spraying, and scored for segregation of resistant and sensitive pheno­ types. The maJority of the hnes obtained via electroporation show a Mendelian mhentance pattern for the bar gene locus (see Note 9). 4 . Notes

2. 3

4

5

6. 7.

All solid substrates are prepared m the following way· the sugar solutiOn and the salt solution with MES and the solidifying agent are autoclaved separately All other components are filter-sterilized and added after autoclavmg. The filter-sterilized enzyme solution is stored in the freezer at -20°C All liquid substrates and buffers are filter-stenlized and stored at room temperature in the dark, solutwns of hormones and L-prohne are stored m the freezer at -20°C, and v1tamm and casein hydrolysate solutions are stored m the refrigerator Microcuvets ( 1 93 8 PS; Kartell, Binasco, Italy) are stenlized m 70% ethanol for several minutes. Afterward microcuvets are filled with 1 mL agarose substrate (water with 0 45% BRL agarose), and after solidification I 00 J.!L of the electroporatwn buffer (EPM) IS p1peted on top of th1s agarose substrate. Parallel stainless-steel electrodes, 30 mm long, 2 mm thick, and 6 mm apart are mserted in such a way that by pushing the electrodes on the agarose substrate, the plant tissue I S collected between the electrodes in the EPM buffer. Plasmid DNA was purified on Qiagen (Q1agen Inc.) columns and resuspended m 1 0 rnM Tns-HCI, pH 7.9, and 0. 1 rnM EDTA at a concentration of I mg/mL. The plasmid DNA, carrying a chimeric cauliflower mosaic virus (CaMV) 35 S-bar3'nos gene, was linearized pnor to electroporation, usmg an appropriate enzyme A homemade electroporation umt consisting of a power supplier connected with an array ofcapacitors arranged m a circuit, as descnbed by Fromm et a!. (1), was used Since we observed a higher transformatiOn frequency from backcross matenal (Pa9 1 x H99) x H99 compared with the inbred lines H99 and Pa9 1 , and since It was easier, under our climatic conditions, to produce good donor material from the backcross compared with the inbred lmes, we continue to use the backcross m transformatiOn expenments

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8. The manmtol IS added m the first cu lture substrate immediately after electro­ poratJon to prevent an osmotic shock m the tissue that has been enzymatically treated or preplasmolyzed for 3 h in the EPM buffer with 0 425 M mannitol. 9 The maJority of the lines obtained with electroporatJon show a Mendelian inheri­ tance pattern for the bar gene locus m contrast with lines produced by particle bombardment. TransformatiOn of maize based on electroporation or micropro­ Jectile bombardment of type I callus and immature embryos have been further compared The level of transient expressiOn as duected by anthocyanin bwsyn­ thetJc regulatory genes revealed a sigmficantiy higher number of red spots on particle bombardment compared with electroporatwn. B oth transformatiOn procedures lead to the Iso latiOn of stab le trans­ formants denved from Immature embryos and type I callus as targets for transformatiOn usmg the bar gene as selectable marker Upon e lectroporahon, a clear selection was observed m ti ssue culture, but Basta-resistant pl ants could only be regenerated from a mmor fraction of the selected calli. With particle bombardment, a sigmfJcantly higher number of regenerants express­ ing the bar gene (up to ca. 40-fold) was obtamed However, segregatiOn analysis of progemes from transgemc lmes obtained from both transforma­ tiOn procedures revealed that m the maJ ority of the electroporatwn-denved hnes, the gene is inherited m normal Mendelian fashwn, whereas a lower fractiOn (about 50%) of the bar lines obtamed by microproJectile bombard­ ment show a normal Mendelian segregatiOn About 30% of the hnes produced by particle bombardment do not show an inhen tance of the Basta resi stance phenotype at all and yield only sensitive progemes. In reciprocal crosses of transformants produced by particle bombardment, segregation could be w1thm the expected range m one directiOn and abnormal, even all-sensitive, m the other duectwn Transformants should correctly express the mtroduced genes, 1f they are to be of practical value A drawback of d1rect gene delivery IS the unpredictable and often complex pattern of mtegration On ei ectroporation, about half of the transformants have a rather Simple IntegratiOn pattern, whereas the other half have a more complex mtegrahon pattern. With bwhstJc transformatiOn, the majority of the transformants have mtegrated multiple copies of the transgene, often fragmented and rearranged. This could explam the high proportion oflines obtamed with biol istics havmg an aberrant segre­ gation, since the presence of multiple copies of transgenes can lead to msta­ bil ity of their expression (14). In conclusiOn, stably transformed maize hnes using type I callus and Imma­ ture embryos can be produced by both direct gene transfer methods. The effi­ Ciency of transformatiOn IS significantly higher with particle bombardment compared with electroporatwn. However, qualitatively better transformants are produced by e l ectroporation, since a sig nific ant lower number of transformants have to be generated by elec troporatwn compared W ith biolistJcs m order to obtam a transgenic line d1splaymg a simple and "cor­ rect'' mtegration pattern

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References I . Fromm, M. E., Mornsh, F , Annstrong, C. W1lhams, R , Thomas, J , and Klein, T M. ( 1 990) Inheritance and expression of chimenc genes m the progeny of transgenic maize plants. Rio/Technology 8, 833-839 2. Rhodes, C. A., P1erce, D A , Mettler, I. J., Mascarenhas, D., and Detmer, J. J ( 1 988) Genetically transfonned maize plants from protoplasts Scrence 240, 204-207. 3 Dekeyser, R A , Claes, B., De RIJCke, R M U , Habets, M. E., Van Montagu, M C., and Caplan, A. B. ( 1 990) Transient gene expression m mtact and orgamzed rice tissues. Plant Cel/ 2, 59 1--{;02. 4. D ' Hallum, K., Bonne, E., Bossut, M , De Beuckeleer, M. and Leemans, J ( 1 992) Transgenic ma1ze plants by tissue electroporatwn Plant Cell 4, 1 495-1 505 5. Xiay1, K., Xmwen, Z., Heping, S., and BaoJian, L. ( 1 996) Electroporatwn of immature maize zygotic embryos and regeneration of transgemc plants. Transgemc Res. 5, 2 1 9-22 1 . 6. Laursen, C M , Krzyzek, R. A , Flick, C E , Anderson, P C , and Spencer, T M . ( 1 994) ProductiOn of fertile transgemc maize by electroporation o f suspensiOn culture cells Plant Mol Bzol 24, 5 1-{i I 7 Pescitelli, S. M. and Sukhapmda, K ( 1 995) Stable transformatiOn v1a electro­ poration mto maize Type II callus and regeneratiOn of ferti le transgemc plants. Plant Cell Rep. 14, 7 1 2-7 1 6 8 . Songstad, D . D., Halaka, F . G . , DeBoer, D . L , Annstrong, C. L., Hinchee, M. A. W., Ford-Santmo, C. G., et a! ( 1 993) Transient expressiOn of GUS and anthocya­ nin constructs in intact maize Immature embryos followmg electroporation. Plant Cell, Tissue Organ Cult 33, 1 95-20 1 . 9. Sabn, N., Pelissier, B , and TeJSSJe, J. ( 1 996) Transient and stable electrotrans­ formations of mtact black Mexican sweet maize cells are obtamed after preplasmolysJs Plant Cell Rep 15, 924-928. 10 Chu, C. C., Wang, C C , Sun, C. S., Hsu, C., Ym, K C., Chu, C. Y , et a! ( 1 975) Establishment of an efficient medmm for anther culture of rice through compara­ tive expenments on the mtrogen sources Scz Szn 18, 659-ti68 1 1 . Frearson, E. M., Power, J B., and Cocking, E. C. ( 1 973) The Isolation, culture and regeneration of Petuma leafprotoplasts Dev Bzol 33, 1 30-1 37. 1 2. Murash1ge, T. and Skoog, F ( 1 962) A revised medmm for rapid growth and bw­ assays with tobacco tissue cultures. Physzol Plant 15, 473-497 1 3 . De Block, M., Bottennan, J , Vandew�ele, M., Dockx, J , Thoen, C , Gosse1e, V., et a!. ( 1 987) Engmeenng herbiCide resi stance m plants by expressiOn of a detoxi­ fying enzyme. EMBO J 6, 25 1 3-25 1 8 14. Matzke, M A and Matzke, A J M ( 1 995) How and why do plants machvate homologous (trans)genes? Plant Physzol 107, 679--{;85

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Transformation of Maize Using Silicon Carbide Whiskers Jim M. Dunwell 1 . Introduction

As the most commercially valuable cereal grown worldwide and the best­ characterized in genetic terms, maize was predictably the first target for transfor­ mation among the important crops. Indeed, the first attempt at transformatiOn of any plant was conducted on maize (1). These early efforts, however, were inevi­ tably unsuccessful, since at that time, there were no rehable methods to permit the introduction of DNA into a cell, the expression of that DNA, and the Iden­ tification of progeny derived from such a "transgenic" cell (2). Almost 20 years later, these technologies were finally combined, and the first transgemc cereals were produced. In the last few years, methods have become mcreasingly effi­ cient, and transgenic maize has now been produced from protoplasts as well as from Agrobacterium-mediated or "Biolistic" delivery to embryogenic tissue (for a general comparison of methods used for maize, the reader is referred to a recent review-ref. 3). The present chapter will describe probably the simplest of the available procedures, namely the delivery of DNA to the recipient cells by vortexing them in the presence of silicon carbide (SiC) whiskers (this name will be used in preference to the term "fiber," smce it more correctly descnbes the single crystal nature of the material). This needle-shaped matenal was selected as a means of perforating cells (thereby allowing the entry of DNA), since it was known to be one of the hard­ est of"man-made" products; it is used commercially as an abrasive and a com­ ponent of saw blades. In fact, it is not a completely synthetic material, but is produced by carbonizmg (at high temperature in carbon monoxide) the s1licate found within the cells of rice husks. The chemically transformed product of one cereal is therefore being used to transform another cereal genetically. From Methods 1n Molecular B1ology, Vol 1 1 1 Plant Cell Culture Protocols Ed1ted by R D Hall © Humana Press Inc , Totowa, NJ

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Fig. l . (A) Scanning electronmicrograph of silicon carbide whiskers of the type used for transformation. (B) Group of maize cells after vortexing with silicon car­ bide whiskers, showing dark staining of a single-cell expressing the introduced GUS gene. In essence. the transfonnation method described below involves the agita­ tion ofsuspension-culture cells, with SiC whiskers and plasmid DNA

(Fig. 1).

The resultant transgenic cells are selected by growth on bialaphos and plants subsequently regenerated. Details of the procedure as developed for maize can

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be found in a series of recent papers and reviews (4-8). The scope of the method, however, is not restricted to matze, or indeed to cells of htgher plants, as shown by successful dehvery of DNA to cells of various algae (9), mcluding Chlamydomonas (10), tobacco (11, 12), Agrostis (13), and Lolium (Dalton, per­ sonal communication). Most recently, transient expression of the GUS marker was detected when dry wheat embryos were vortexed with DNA and whiskers for 1 0--30 min (14). It was also demonstrated that callus ttssue induced from the treated embryos contamed GUS-expressing sectors more than 1 mo after treatment. This very promising finding suggests that cells in culture are not necessanly the only possible target. AddttJOnally, recent results obtamed from node slices of Ameri can chestnut showed that treatment with whiskers increased the proportion of wounded tissue and thereby enhanced subsequent Agro ba cterium mediated gene delivery (15). It should be noted that the method as developed for the production of transgemc maize is the subject of a granted US patent (16) and that other pat­ ents are pending. These do not of course prevent the use of the technique for research purposes. -

2. Materials 2. 1. Source of Whiskers This ts one of the most important factors in the process. The preferred type of whiskers is that designated "Silar SC-9" and obtained from Advanced Com­ posites ( 1 525 South Buncombe Road, Greer, SC 2965 1 -9208). Other available types are TWS I OO (Tokat Carbon Company, Tokyo, Japan) (13) and Alfa Aesar (Johnson Mathey, Ward Hill, MA). Most whisker preparations are highly heterogeneous, rangmg m length from 5-500 J..U11 They can also dtffer in diameter (usual mean of 1 J..U11) and m the degree of hydrophobicity, both fac­ tors that are hkely to affect their efficiency (8). SiC whiskers are known to be a respiratory hazard (1 7, 18) and should therefore be treated with caution at all times, but especially when bemg weighed m the dry state. They should be dis­ posed of as hazardous waste. 2.2. Donor Plants Glass house-grown plants of the matze hybrid A l 88 x B73 are used as a source of embryos (see Note 1). 2.3. Callus Maintenance Medium For 1 L of N6 medmm (19), dissolve in distilled water: 4 g powdered N6 salts (Sigma), 30 g sucrose, 1 00 mg myo-inositol, 2 mg glycine, I mg thia­ mine, 0.5 mg pyridoxine HCl, 0.5 mg nicotmic acid, and 2 mg 2,4-D (from

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stock solutiOn [ 1 mglmL] made by dissolving 2,4-D m dilute KOH). Adjust to pH 6.0 with 1 M KOH, add 3 g Gelnte, and autoclave. 2.4. Callus Initiation Medium Medium N61 is N6 (20) modified by adding 0.69 g/L proline (6 mM) and reducmg the sucrose content to 20 g/L. Adjust pH to 6.0, add gelling agent as above, and autoclave. 2.5. Suspension Initiation Medium For 1 L MS medium (21), dissolve in distilled water: 4.3 g powdered salts (Sigma), 2 mg glycine, 0.5 mg thiamme, 0.5 mg pyridoxine HCl, 0.05 mg nico­ tinic acid, and 0 . 0 3 73 mg N a2EDTA. Adjust the pH to 6 .0 and autoclave . 2.6. Suspension Maintenance Medium For 1 L ofH9CP+, add to liquid MS· 30 g sucrose, 1 00 mg myo-inositol, 2 mg 2,4-D, 2 mg NAA, 0.69 g proline, 200 mg casein hydrolysate (both phytohor­ mones added from predissolved stocks in dilute KOH). Adjust to pH 6.0, and autoclave. (Add 5% sterile coconut water [Gibco] before subculture.) 2. 7. Pretreatment of Cells Medium N6(S/M) used for osmotic treatment ts liqmd N6 wtth 45 giL o-sorbitol, 45 giL o-mannitol, and 30 giL sucrose. Adjust pH to 6.0 and autoclave. 2.8. Selection of Transgenic Cells 1 . Bialaphos is obtamed by defonnulatiOn of the herbicide Herbiace (Metj i Seika, Japan). 2. Prepare N6( 1 B) from N6 and 1 mg/L btalaphos and N6(0.5B) with 0 5 mg/L bialaphos Solidify with 0.3% gelnte, or for embedding, with 0.6% Sea plaque agarose (FMC Bioproducts). Add bialaphos by filter-stenhzation after autoclav­ ing the media

2.9. Regeneration Media 1 . Regen 1 medium 1s MS with (per L) 60 g sucrose, 1 mg NAA, and 1 g myo-inosi­ tol Solidify with 0 3% Gelrite, pH 6.0 2. Regen 2 medium Is as above with 0.25 mg NAA and 30 g sucrose. 3 Regen 3 Is half-strength MS with 30 g sucrose, sohd1fied, and w1th pH as above.

2. 10. DNA Preparation Use a QIAGEN plasmid Maxi kit to purify plasmid DNA, and resuspend the DNA pellet in sterile Millipore-purified water at a final concentration of 1 f..lg/)!L.

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2. 1 1. Additional Requirements Bleach for sterilization, sterile water, forceps (curved tip), scalpel, Erlenm­ eyer flasks (250 mL), disposable pipets ( 1 0-mL w1de-bore), and 20-mesh stain­ less-steel s1eve are needed. 3. Methods 3. 1. Establishment of Suspension Cultures 1 . 1 0- 1 2 d after pollinatiOn, sterilize husked ears wtth 50% commercial bleach (final concentration 2.6% available chlorine), rinse three ttmes m water, remove Imma­ ture zygotic embryos under aseptic conditions, and place scutellum surface upward on N6I medmm 2 Add 3 g of proliferatmg type II callus from a selected smgle embryo to 20 mL of H9CP+ medium 3. Mamtain m 1 25-mL Erlenmeyer flask at 28°C in darkness on a rotary shaker ( 1 25 rpm) 4. Subculture every 3 . 5 d by addmg 3 mL packed cell volume (see Subheading 3.2.) and 7 mL old culture medmm to 20 mL fresh medmm. 5. If reqmred, cell suspensions can be cryopreserved (22). To thaw, submerge tubes in a water bath at 45°C, and immediately use a 5-mL dtsposable p1pet to plate cells onto a sterile filter paper m a 60 x 20 mm Petn dtsh. Remove excess cryoprotectant by repeated blotting wtth filter paper, place cells on solid N6 medtum, and subculture weekly for 3 wk, after which the suspensiOn can be reimtiated as above.

3.2. Pretreatment of Cells Prior to Transfo;mation l 2 3. 4. 5. 6.

7

One day after subculture, sieve suspensiOn cells through a 20-mesh stainless steve, and return to ongmal Erlenmeyer flask In used medmm. Measure packed cell volume (PCV) by using 1 0 mL wtde-bore dtsposable ptpet and allowmg cells to settle in lower half of ptpet. Replace H9CP+ medium with liqUid N6(S/M) at rate of3 mL to every I mL PCV. Return flasks to rotary shaker for 45 min. Resuspend cells evenly by pipetmg cells and medmm up and down repeatedly Usmg 1 0-mL disposable pipet, dispense 1 -mL aliquots mto 1 . 5-mL Eppendorf tubes. When cells have settled to bottom of tube, remove 0.5 mL medmm, leaving 1 · 1 proportions of PCV:medium.

3.3. Preparation of Whiskers I . Transfer about I 0-50 mg dry whiskers to preweighed 1 .5-mL Eppendorftubes in a fume hood. 2. Rewetg h tubes, and calculate the weight of whiskers. 3 Pterce the lid to avoid tts bemg displaced during heating, cover tt in aluminium fml, and autoclave the tubes.

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4. Store at room temperature prior to use Whiskers should be prepared fresh m an aqueous suspenston (5% w/v m stertle water) pnor to use (8).

3.4. Transformation Procedure I . Add 40 � of 5% whisker suspenston and 2 5 � plasmid DNA ( I j..tg/�) to the pretreated cells m an Eppendorf; gently premtx by tapping. 2. Place tube in multtsample holder of Vortex Geme II, and mtx at full speed for 60 s, or m the holder of a Mtxomat dental amalgam mtxer shaken at fixed speed for 1 s (See Notes 2, 3, and 4)

3.5. Selection of Transgenic Tissue 1 Add 0.5 mL fresh N6(S/M) medmm to the mtxture m the tube 2. Use a 200-� wtde-bore Ptpetman tip to dtspense the dtluted contents onto a 55-mm Whatman no. 4 filter paper overlaymg solid N6 medmm m a 60 x 20 mm plastic Petri dish. 3. Wrap dtshes m gas-permeable Urgopore tape (Stenlco, Brussels), and incubate at 28°C in darkness. 4. After 7 d, transfer the filter paper and cel ls to the surface of N6(1B) selectwn medium. Repeat after a further 7 d. (see Note 5) 5 Use a spatula to transfer cells from the filter paper mto 5 mL N6(0.5B) medium contammg 0 6% Sea plaque low-gellmg-pomt agarose m a stenle test tube kept at 3 7°C. 6. 01Vide the suspension into two altquots, and p late each evenly over 20 mL N6(0.5B) medmm soltdtfied with 0 3% Gelrite m a 1 00 x 25 mm Petri dtsh. 7 Seal with Nescofilm, and incubate at 28°C. 8 After 4--5 wk ptck offpotenttally transformed cal It, and transfer to the surface of fresh N6(1B) medmm 9. Subculture onto same medium every 2 wk.

3.6. Regeneration of Plants 1 . Transfer type II callus onto Regen I medium, and incubate at 2 5°C m darkness. 2. After 2-3 wk, transfer opaque, mature somatic embryos onto Regen 2 medmm. 2 Incubate in light ( -200 J..lO lll l/m /s with 1 6-h photoperiod) 3 . After a further 2-3 wk, shoots and roots emerge, and small plantlets can subse­ quently be transferred to glass tubes contaimng 1 5 mL Regen 2 medium. 4. After 7 d, transfer to sot! m a glass house for analysts (see Note 6). Grow to maturity in 3-gallon pots contammg equal parts of peat:perltte:s01l. 4. Notes 1.

The genotype recommended m this study produces a form of dispersed suspen­ sion very suitable for the method descnbed. Any other genotype with this charac­ teristic Is likely to be equally suttable. As ment10ned m Subheading 1., mature embryos have also been used successfully m wheat (14) .

Silicon Carbide Whiskers

38 1

2 Although these results were obtained usmg two particular forms of apparatus for mixmg, it can be assumed that any other eqUivalent form of mixmg device would give similar results. For example, the results on wheat (14) were achieved wtth a Micro tube mixer MT-360 (Tomy Seiko Co., Ltd., Japan) 3. To date, tnals on a range of material have gtven the best results with StC whis­ kers. There may, however, be other, as yet untested crystalline material of equal efficiency 4. As an alternative to the use of a random-mixing process for cells and whiskers, a fixed array of microneedles has been developed (23) 5 . It can be expected that other selective agents could be used in place of bialaphos 6. In an interesting comparative study (24) , It has been shown with Chlamydomo­ nas reinhardtu that transformants produced by a glass bead method (25) gtve a htgher frequency of random to homologous ( 1 000: 1 ) mtegratton events than those produced by using particle bombardment (24 1 ) (ref. 26). It may be that the form of the DNA (degree of condensatton) is an Important determmant of the type of integration pattern. If so, then the SiC method, m which the DNA IS not precipi­ tated, may be advantageous m those systems where random patterns are reqmred.

References 1 Coe, E H. and Sarkar, K. R ( 1 966) Preparatton of nucletc actds and a genetic transformatton attempt m matze. Crop Scz 6, 432-435 2. Dunwell, J M. ( 1 995) Transgenic cereals Chern Ind Sept 1 8 , 730-73 3 3 . Wilson, H M . , Bullock, W P., Dunwell, J M., Ellis, J. R , Frame, B , Register, J., et a!. ( 1 995) Maize, m Transformation ofPlants and Sml Mzcroorgamsms (Wang, K., Herrera-Estrella, A., and Van Montagu, M , eds.), Cambridge Umverstty Press, pp. 65-80 4. Frame, B. R., Drayton, P. R., Bagnell, S. V., Lewnau, C. J., Bullock, W. P., Wtlson, H M., et a!. ( 1 994) ProductiOn of ferttle transgemc matze plants by si licon carbtde whtsker-mediated transformatiOn. Plant J 6, 94 1-94 8. 5. Kaeppler, H. F. and Somers, D. A. ( 1 994) DNA dehvery to maize cell cultures using st hcon carbide fibers, m The Mmze Handbook (Freeltng, M., and Walbot, V , eds. ), Springer-Verlag, New York, pp 6 1 0--6 1 3 6. Thompson, J. A., Drayton, P. R., Frame, B . R., Wang, K , and Dunwell, J . M. ( 1 995) Maize transformatiOn utilizmg silicon carbide whiskers: a review. Euphytzca 85, 75-80. 7. Wang, K., Frame, B . R , Drayton, P. R , and Thompson, J. A. ( 1 995) Silicon­ carbide whtsker-mediated transformation: regeneration oftransgemc matze plants, in Gene Transfer to Plants (Potrykus, I. and Spangenberg, G., eds.), Springer, Berlm, pp 1 86-1 92. 8. Wang, K., Drayton, P. R., Dunwell, J. M., and Thompson, J. T ( 1 995) Whtsker­ medtated plant transformatiOn' an alternative technology. In Vztro Cell Dev Bzol 31, 10 1-104 9. Dunahay, T. G . , Adler, S A , and Jarvik, J W ( 1 997) Transformation of microalgae using sthcon carbide whiskers. Methods Mol Bzol. 62, 503-509

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1 0 Dunahay, T. G. ( 1 993) Transformation of Chlamydomonas reznhardtii with sib­ con carbide wh1skers. BwTechmques 15, 452--460. 1 1 Kaeppler, H F Gu, W . , Somers, D A , Rmes, H W , and Cockburn, A. F ( 1 990) Silicon carbide fiber-mediated DNA delivery into plant cells. Plant Cell Rep 9, 4 1 5--4 1 8. 1 2 . Kaepp1er, H. F , Somers, D. A., Rmes, H. W., and Cockburn, A F. ( 1 992) Silicon carbide fiber-mediated stable transformation of plant cells Theor Appl Genet 84, 560--566. 1 3 Asano, Y., Otsuki, Y , and Ugaki, M ( 1 99 1 ) Electroporation-medtated and sib­ con carbide whisker-medtated DNA dehvery m Agrostzs alba L. (Redtop) Plant Sci 9, 247-252. 14. Senk, 0 , Amur, I., Murat, K., Tetsuo, M , and Masaki, I. ( 1 996) Sihcon carb1de fiber-mediated DNA deli very mto cells of wheat (Tntzcum aestzvum L.) mature embryos. Plant Cell Rep 1 6, 1 3 3-1 36 Zmg, Z., Powell, W. A., and Maynard, C. A. ( 1 997) Usmg stlicon carbtde fibers 15 to enhance Agrobacterium-mediated transformatiOn of Amencan chestnut In Vztro Cell Dev Bwl 33, 63A 16 US Patent 5 302523, TransformatiOn of Plant Cells. April 1 2, 1 994 1 7 Vaughan, G L., Jordan, J , and Karr, S ( 1 99 1 ) The toxiCity, in vitro, of sthcon carbide whiskers. Envzron Res 56, 57-67. 1 8 Vaughan, G. L., Trendy, S A., and Wtlson, R B. ( 1 993) Pulmonary response, m vzvo, to sihcon carbtde whiskers. Envzron. Res 63, 1 9 1-201 19 Chu, C C., Wang, C. C., Sun, C S., Hsu, C., Yin, K C., Chu, C Y , et a!. ( 1 975) Establishment of an efficient mediUm for anther culture of rice through compara­ tive experiments on the mtrogen sources. Scz Sm 1 8, 659-668 20 Armstrong, C L. and Green, C. E ( 1 985) Estabhshment and maintenance of fnab1e, embryogemc matze callus and the involvement of L-proline. Planta 1 64, 207-2 1 4. 2 1 . Murashige, T. and Skoog, F. ( 1 962) A revised medium for rapid growth and bio­ assays wtth tobacco t1ssue cultures Physiol Plant 15, 473--497. 22 Shill ito, R. D., Carswell, G. K., Johnson, C. M., D1 Maio, J J., and Harms, C. T. ( 1 989) RegeneratiOn of ferti le plants from protop1asts of ehte mbred maize. Bzol Technology 7, 58 1-587 23 US Patent 545704 1 , Needle Array and Method of Introducmg Btolog1cal Sub­ stances mto Ltving Cells usmg the Needle Array. October 1 0, 1 995 24. Sodeinde, 0. A. and Kindle, K. L ( 1 993) Homologous recombmatwn in the nuclear genome of Chlamydomonas remhardtu. Proc. Nat/ Acad Scz USA 90, 9 1 99-9203. 25. Kindle, K L ( 1 990) Htgh-frequency nuclear transformatiOn of Chlamydomonas reznhardtu. Proc. Nat/. Acad. Scz USA 87, 1 228- 1232. 26 Kindle, K. L., Schnell, R. A., Fernandez, E., and Lefebvre, P. A. ( 1 990) Stable nuclear transformation of Chlamydomonas using the Chlamydomonas gene for mtrate reductase. J Cell Bwl 109, 2589-260 1 ,

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Directing Anthraq ui none Accumulat ion via Man ipulation of Morinda Suspension Cultures Marc J. M. Hagendoorn, Diaan C. L. Jamar, and Linus H. W. van der Plas

1 . Introduction Secondary metabolite accumulation in cell suspensions is the result of an interplay between primary metabolism, supplying the machinery, the energy, and the precursors, and the secondary metabolic pathways. For this reason, a successful strategy for producing considerable amounts of secondary plant products can hardly be obtained without the knowledge of the interactiOns between primary and secondary metabolism. These interactions are best studied m cell suspensions that produce significant amounts of secondary metabolites. In Morinda citrifolia cell suspensions, secondary metabolic pathways can easily be switched on, resulting m a considerable accumulatiOn (more than 1 0% of the dry weight) of anthraquinones (AQs, [1}). Anthraqumones are dyes that can be treated to give a variety of colors, e.g., yellow, red, purple, brown, or black (2). Rubiaceae AQs are all derivatives from the basic structure as shown in Fig. 1. M citrifolia is a tropical tree, inhabiting the islands on the edge of the Pacific Ocean (2). In M. Cltrifolia plants, AQs are only accumulated m the roots, and then mostly in the bark. Most of the AQs of M. citrifolia cell suspensions are glycosylated, especially as 0-glucosylxylosyl (therefore called primverosides [3}) and subsequently are stored in the vacuole. In the Rubiaceae, anthraquinones are produced via the shiktmate-mevalonate pathway. Intermediates from glycolysis and the pentose phosphate pathway are combmed to form shikimate and subsequently chorismate. This compound can, via several steps, be converted into phenylalanine, tyrosine, tryptophan, and via isochorismate, into anthraquinones. Thus, the b10synthes1s of anthraquinones From Methods

m

Molecular Brology, Vol 1 1 1 Plant Cell Culture Protocols Hall © Humana Press Inc , Totowa, NJ

Edited by R. D.

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Hagendoorn, Jamar, and van der Plas

Ftg. I Baste structure of anthraqumones found m the Rubtaceae. SubstJtuents mclude methyl, hydroxyl, and methoxyl groups. Numbered atoms ( 1-14) are C-atoms Os indtcate the posttlon of the 2-oxygen atoms.

shows mteractions with protein synthesis, other secondary pathways (phenyl­ propanmd pathway via phenylalanine), and hormone synthesis (auxm synthe­ SIS via tryptophan). We have stud1ed the mfluence of several auxm analogs on anthraqumone production. For 2,4-dlchlorophenoxy acetic ac1d (2,4-D) and ! -naphthalene acetic ac1d (NAA), we also have determmed the effects on cell d1vision. It appears that auxin analogs that are able to maintam cell d1vision have a nega­ tive influence on AQ production (4) . The auxin analog 2,4-D already stimu­ lates cell division at a much lower concentration than NAA. In Morinda cells growing in the presence of 4.5 � NAA, the AQ production can be Immedi­ ately blocked by the addition of 2,4-D (final concentration 4.5 � (5) . There­ fore, Mormda cell suspensions can be used as a model system, m wh1ch AQ production can be easily switched on and offby changing the auxm concentrations. Although there often is a negative correlatiOn between cell division and anthraquinone accumulation (see Fig. 2), affecting specifically the cell divi­ sion rate, th1s does not result in a change in AQ productwn. Poss1bly two dis­ tinct programs can be sw1tched on under regulation of auxins· a program focused on fast cell div1s1on and one in which productiOn prevails (4). These separate programs are also reflected m the cell compositiOn. The slowly diVid­ ing cells show a lower respiration rate, a h1gher soluble sugar content, and contam less protein (5, 6) . M. citrifolza cell suspenswns can also be grown m fermenters, as so-called contmuous cultures. After several weeks, a steady state is reached in wh1ch growth rate, cell composttton and AQ content (4, 7) are constant. Therefore, interactions between primary and secondary metabolism can be studied quan­ titatively over prolonged periods. Another advantage of the Morinda system is that changes m AQ accumula­ tion can easily be followed visually. A simple extraction and subsequent spec-

Directing Anthraquinone Accumulation

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Methods in Molecular BiologrM • 1 1 1 PLANT CELL CULTURE PROTOCOLS ISBN: 0-89603-549-2

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