Olive Propagation Manual (Landlinks Press)

Olive Propagation Manual

This page intentionally left blank

Olive Propagation Manual

Andrea Fabbri Dipartimento di Biologia Evolutiva e Funzionale Università degli Studi di Parma, Italy

Giorgio Bartolini CNR, Istituto per la Valorizzazione del Legno e delle Specie Arboree, Polo Scientifico, Sesto Fiorentino (Firenze), Italy

Maurizio Lambardi CNR, Istituto per la Valorizzazione del Legno e delle Specie Arboree, Polo Scientifico, Sesto Fiorentino (Firenze), Italy

Stanley George Kailis School of Plant Biology, Faculty of Natural and Agricultural Sciences, Crawley, WA Australia

© CSIRO 2004 All rights reserved. Except under the conditions described in the Australian Copyright Act 1968 and subsequent amendments, no part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, duplicating or otherwise, without the prior permission of the copyright owner. Contact CSIRO PUBLISHING for all permission requests. National Library of Australia Cataloguing-in-Publication entry Olive propagation manual. Bibliography. Includes index. ISBN 0 643 06676 4. 1. Olive – Propagation – Handbooks, manuals, etc. I. Fabbri, Andrea, 1948– . II. CSIRO Publishing. 634.6353 Available from Landlinks Press 150 Oxford Street (PO Box 1139) Collingwood VIC 3066 Australia Telephone: Local call: Fax: Email: Web site:

+61 3 9662 7666 1300 788 000 (Australia only) +61 3 9662 7555 [email protected] www.landlinks.com

Front cover Photos by the authors Figures and Plates: Figures 1.3, 3.7, 3.16, 3.17, 4.1, 4.4, 4.5, 6.3, 6.4, 6.7, 6.8, 6.12, 6.13, 6.18, 6.23. Plates VI, VII, IX, XI, XII, XIII, XVI, XVII, XVIII, XIX by Andrea Fabbri Figures 3.2, 3.4, 3.5, 3.6, 3.15, 3.18, 3.19, 4.8, 6.1, 6.2, 6.5, 6.6, 6.9, 6.10, 6.11, 6.14, 6.15, 6.16, 6.17, 6.19, 6.20, 6.21, 6.22. Plates II, III, IV, V, XIV, XV by Giorgio Bartolini Figures 3.8, 3.12, 3.13, 3.14, 4.2, 5.2, 5.3, 5.4, 5.5, 5.6. Plates I, VIII, X, XX, XXI, XXII, XXIII, XXIV, XXV, XXVI, XXVII, XXVIII, XXIX, XXX, XXXI, XXXII by Maurizio Lambardi Figure 1.2 by Stan Kailis Set in 10.5/13pt Minion Cover and text design by James Kelly Typeset by J&M Typesetting Printed in Australia by Ligare Disclaimer While the authors, publisher and others responsible for this publication have taken all appropriate care to ensure the accuracy of its contents, no liability if accepted for any loss or damage arising from or incurred as a result of any reliance on the information provided in this publication.

Dedicated to Gabriella, Pia Lucia, Marina and Lefki

This page intentionally left blank

Authors Andrea Fabbri is Full Professor of Arboriculture and Pomology at the University of Parma, Faculty of Agriculture. A large part of his professional activity has been focused on research and teaching on the olive, in various laboratories and universities in Italy and California. His main olive research subjects have been physiology and anatomy of adventitious rooting in cuttings, flower biology, freezing injury, systematic pomology (biomolecular characterisation), organic cultivation, ecophysiology. His research papers have been published on the most important horticultural scientific journals. He is presently involved in the development of olive cultivation in cold areas of Northern Italy.

Giorgio Bartolini is Head Researcher of the National Research Council (CNR) at the Istituto per la Valorizzazione del Legno e delle Specie Arboree (Trees and Timber Institute) of Florence, Italy. His activity has been focused on research concerning woody plants propagation by cuttings of olive, peach, grapevine, etc.; management of an international data bank (world olive germplasm); individuation of morphological and molecular markers for cultivar characterisation in O. europaea; gene expression induced by low temperature. He participates in joint research projects with research institutes and universities of Italy, California and Spain. He is author or co-author of many research papers published in international journals, books and in proceedings of international congresses and symposia.

viii

Olive Propa ga tion

Maurizio Lambardi is a Researcher of the National Research Council (CNR) at the Istituto per la Valorizzazione del Legno e delle Specie Arboree (Trees and Timber Institute) of Florence, Italy. He is also Lecturer of Woody Plant Biotechnology and Micropropagation at the University of Modena and Reggio Emilia, Faculty of Agriculture. He has wide-ranging expertise on woody plant micropropagation and biotechnology. His main areas of research on the olive have concerned seed germination, tissue culture, genetic transformation, germplasm cryopreservation. He is the author or co-author of over 90 scientific papers and reviews, published in leading international journals, books, proceedings of international congresses and symposia.

Stanley George Kailis is Professorial Fellow at The University of Western Australia in the School of Plant Biology. His antecendents came from the Dodecanese Island, Megisti. His interests focus on quality aspects of the olive. He is particularly interested in the propagation of olive varieties such as Kalamata, Konservolia, Leccino and Manzanilla. Stanley has made presentations on the olive at national and international forums. He has published numerous research papers in national and international journals. He has conducted numerous schools and workshops in Australia on olive growing, olive oil and table olive production, organoleptic evaluation of olive products and olive propagation.

Contact email [email protected] [email protected] [email protected] [email protected]

Foreword This publication deals with all issues concerning olive propagation. After an historical and thoroughly technical overview of the several available traditional techniques, the text focuses on the more modern, and more extensively employed, nursery procedures. The major recent scientific acquisitions, and the development of technological innovation that is a result of this knowledge, are also illustrated. The authors have interpretated the several subjects in a way that is both synthetical and exhaustive, and in a form that is accessible to all readers, be they students, technicians or farmers. This work represents a stimulus to scientific research in the sector, yet is also of universal interest. Their well-documented work is rich with related issues that invite the reader to go further in the field of plant propagation. Franco Scaramuzzi President ‘Accademia Economico-Agraria dei Georgofili’ of Florence, Italy

This page intentionally left blank

Contents

Authors

vii

Foreword

ix

1

Introduction

1

1.1 1.2 1.3

2 3 7

2

3

Fundamentals of plant propagation The importance of propagation for olive cultivation Olive propagation today

Flower and fruit biology in the olive

8

2.1

Olive cycles 2.1.1 Life or biological cycle 2.1.2 Annual cycle 2.1.3 Fruiting cycle

8 8 11 11

2.2

Stages of olive reproduction 2.2.1 Flower induction 2.2.2 Flower differentiation 2.2.3 Reproductive structures Inflorescence Flowers and flowering 2.2.4 Anthesis and pollination Sterility Fruit set 2.2.5 Fruit growth and seed development

12 12 13 13 13 14 16 18 18 19

Propagation by cutting

22

3.1

22 22 26

Biology of adventitious root formation in cuttings 3.1.1 Morphology and anatomy 3.1.2 Physiology

xii

Olive Propa ga tion

3.2

Techniques of propagation by cutting 3.2.1 Plant material

27 27

Branches

28

Shoots

30

Ovules

30

Suckers (or pollards)

33

3.2.2 Shoot collection and cutting preparation

33

3.2.3 Auxin treatments to promote rooting of cuttings

35

Using hydro-alcoholic solutions for quick-dip treatments

35

Using talcum powder formulations

37

Commercial preparations

39

Dilute auxin solutions

39

3.2.4 Techniques to improve the effectiveness of auxin treatments

39

Soaking the cuttings

39

Wounding

40

3.2.5 Root promoting compounds, used in combination

40

with auxin treatments

4

Growth regulators

41

Fungicides

41

Propagation by grafting

43

4.1

The purposes of grafting

43

4.2

Production of olive seedlings

46

4.2.1 Stone collection and quality of olive seeds

46

Olive seed dormancy

46

Sources of stones and quality of seeds

47

Harvesting time

49

Extraction, cleaning and storage of stones

49

4.2.2 Technique of propagation by seed

51

Pre-sowing treatments

51

Sowing

52

Germinating small quantities of valuable seed

54

Germinating seeds of other Olea species

54

Transplant and growth of seedlings

55

xiii

Contents

4.3

Theoretical and practical aspects of grafting 4.3.1 Histology of graft union 4.3.2 Graft incompatibility 4.3.3 Grafting on seedling rootstocks Collection and conservation of scionwood Grafting time Bark grafting technique Care of the grafted plants 4.3.4 Grafting on adult trees Topworking Grafting on suckers Grafting on wild olive trees 4.3.5 Grafted cuttings

55 55 58 58 59 59 59 62 62 62 63 64 73

4.4

Production of clonal rootstocks in the olive

73

4.5

Grafted plants or self-rooted plants?

74

5. In vitro propagation of the olive 5.1

Micropropagation 5.1.1 Stage 0: Collection of explants Explants from in field growing stock plants Explants from greenhouse growing stock plants 5.1.2 Stage I: Initiation of cultures Disinfection of explants End of Stage I 5.1.3 Stage II: Shoot proliferation Medium formulation Growth regulators Culture conditions and subculturing Shoot elongation 5.1.4 Stage III: Shoot rooting Rooting on IBA or NAA-containing medium Additional root-promoting methods Root induction by means of dipping method 5.1.5 Stage IV: Acclimatisation of plantlets Aims of acclimatisation How to acclimatise olive microplants 5.1.6 Field performance of micropropagated plants

77 77 81 81 81 82 82 83 83 84 84 87 87 87 87 87 88 88 88 89 89

xiv

Olive Propa ga tion

6

5.2

Somatic embryogenesis 5.2.1 Somatic embryogenesis from mature tissue explants

90 90

5.3

Synthetic seeds and micrografting

91

5.4

Considerations on olive micropropagation

92

5.5

In vitro conservation of olive 5.5.1 Slow growth storage 5.5.2 Cryopreservation

92 92 93

The olive nursery (stock plants, structures, equipment and operations) 96 6.1

Stock plants 6.1.1 Phase change 6.1.2 Training systems for stock plants

97 98 98

6.2

Greenhouses 6.2.1 Glass greenhouses 6.2.2 Plastic film greenhouses 6.2.3 Rigid-panel greenhouses PVC (polyvinylchloride) FRP (fibreglass-reinforced plastic) Polycarbonate, methacrylate, and other materials 6.2.4 Location of the greenhouse 6.2.5 Greenhouse heating 6.2.6 Greenhouse cooling 6.2.7 Computers for ambient control 6.2.8 Beds 6.2.9 Propagation greenhouses Control zone (instrumentation) Rooting zone Hardening zone 6.2.10 Shelter and storage

99 100 101 101 101 102 102 102 103 104 106 106 106 106 107 110 110

6.3

Substrates 6.3.1 Rooting substrates 6.3.2 Substrates for growing plants

111 111 113

6.4

Environmental conditions for rooting 6.4.1 Temperature 6.4.2 Humidity 6.4.3 Light

113 113 114 118

Contents

7

6.5

Hardening (1st transplant) 6.5.1 Containers and substrates 6.5.2 Transplanting environment

119 120 121

6.6

Plant growing (2nd transplant) 6.6.1 Containers and location of plants 6.6.2 Irrigation and fertilisation

122 123 123

6.7

Plant training

125

6.8

Plant certification 6.8.1 Genetic origin 6.8.2 Good sanitary conditions

126 127 128

Conservation of olive germplasm

129

Appendices Appendix 1 Rooting ability of all olive cultivars of which scientific literature is available

131

Olive germplasm collections throughout the world

133

Appendix 2

xv

This page intentionally left blank

1 Introduction

The European olive (Olea europaea L.) is a major source of edible oil and processed edible olives. Today, the olive is cultivated over a total world surface area of almost 10 million hectares, on 60% of which it represents the main crop. The traditional area of olive cultivation is the Mediterranean basin, which includes 95% of the olive orchards of the world, and where more than the 95% of the olive oil and the 75% of the table olives are produced. A rough estimate of the global number of olive trees is over 800 million. The annual yield of olives is estimated at 10 million tonnes, most of which is used for oil production and less than 10% consumed as table olives. Over the last 30 years, the production and the consumption of olive oil have increased together. It is unlikely that this trend will change in the near future, considering the recent introduction or increase of olive cultivation and olive-oil consumption in countries such as Japan, Australia, China and South Africa. Remarkable increases (up to 10 fold) have been observed in several countries, including Australia, where olive-oil consumption has passed the level of 1 kg/person/year. Hence, the volume of olive oil consumed worldwide during the next 10 years is expected to exceed three million tonnes annually. Such volumes of olive oil will require active farming programs and olive trees for both new olive orchards and replacement in existing olive groves. Furthermore, as the olive industry moves from traditional manual methods to mechanised operations, planting stock will need to be developed to meet future challenges. Varietal selection will need to be directed to clones that are early bearing, disease resistant and able to be mechanically harvested, and which will produce quality fruit and oil. Although these developments are the province of plant breeders, the follow-through will fall to the olive propagators and associated nurseries.

2

Olive Propa ga tion

1.1 Fundamentals of plant propagation Plant propagation consists of the application of specific biological principles and of particular techniques for the multiplication of plants. The plants obtained in the process should be identical, or as similar as possible, to the plants from which they are derived. All living organisms can be described by genotype and phenotype. Genotype is the whole set of genetic characteristics of the organism, which are controlled by a very high number of genes Any living cell of the organism has the availability of the whole set of genes, although only a minor part of these is activated in that given cell, according to its specialisation (i.e. the differentiation process it went through). Phenotype is the sum total of visible or measurable characters of the organism that characterise it as individual (such as, in fruit trees, fruit size, shape and quality, tolerances to stress and disease, phenological stages). A fundamental principle of biology is that the phenotype is the result of an interaction, between genotype and environment. As a result, if we want to obtain plants that are as similar as possible to their parent plants, we must produce individuals with the same genotype, and place them in identical environments. This latter condition is almost impossible to achieve, and at any rate is not a concern for propagators. Propagators should instead be aware of the implications of the genetic characteristics of propagules (any plant parts used for propagation), which are fundamental in defining the qualitative results of the propagation process. In this respect we can divide the propagation systems for horticultural plants in two main groups: reproduction and multiplication. In reproduction, or gamic or sexual propagation, the propagule is the seed (in higher plants), and particularly the embryo, derived from a fertilisation process. The seed carries the genes of two parents, and these can be arranged in a countless number of combinations. Even when it derives from self-pollination, the genotype of a seed is never identical to that of any of the parent plants, as there are several mechanisms that may cause variation in gene presence and position. Therefore the progeny are always to some extent different from any of the parents, and variability also occurs within the seedling population, just as happens with humans. This means that sexual propagation cannot be used for those species in which uniformity of the plantation and true-to-typeness for a series of biological and technical requirements are essential, as is the case of fruit trees. In species like the olive, seed propagation is therefore confined to the production of seedlings, to be used as rootstocks. In multiplication, or agamic or asexual propagation, the propagule is any other part of the tree, and therefore only carries identical somatic cells. This includes shoots, roots, buds and leaves, and even cells from the ovary tissues, which belong to the stock plant genotype. These tissues, being genetically identical to the stock plant, are able to generate individuals with the same genotype, which in turn will be able to be propagated indefinitely in the same way, thus generating a ‘clone’. Olive cultivars are clones, often very ancient clones, derived from countless asexual propagations, made with several techniques since ancient times. The success of propagation, whatever the technique adopted, depends on knowledge of the many aspects controlling the formation of a new individual:

Introduction

3

• genetic characteristics of species and cultivar which regulate their ability to be propagated • anatomical structure and physiology of the whole plant and of the organs to be propagated • methods most suited to propagate the various cultivars and rootstocks • techniques and structures, and their optimisation to obtain the best technical and economical results

1.2 The importance of propagation for olive cultivation The olive tree, Olea europaea (L.), is one of the most ancient cultivated fruit trees of the Old World, and its importance for the Mediterranean civilisations is witnessed by all classical sources. A remnant of the tropical flora of the mid-Tertiary, the olive is so typical of the Mediterranean that its presence qualifies a climate as Mediterranean, even in other areas of the world. The earliest signs of olive cultivation – the first wave – can be traced back to the 4th millennium BC and, before that, to areas of the eastern Mediterranean coasts and islands, although the ancestors of currently grown olive cultivars are still believed to have been domesticated in the mountainous territory south of the Caucasus, covering today’s eastern Turkey, western Iran, Lebanon, northern Israel, Syria and northern Iraq. From the eastern Mediterranean the olive moved westwards – the second wave – to Greece and the Aegean archipelago, although Crete and Cyprus probably belonged to the oldest olivegrowing centre. In these areas, collectively considered a secondary centre of diversity, the olive grew in importance, and possibly underwent deliberate selection by humans between the 3rd and the 2nd millennia BC. In Crete, in the 16th century BC, there existed in Knossos a huge deposit of clay jars, able to store five times the amount of oil the local population could consume in one year, thus indicating a strong possibility of a developed trade in olive oil. Around the beginning of the first millennium BC a third migration appears to have taken place. Again it was westwards, to Sicily and Tunisia, an area regarded as the olive tertiary centre of diversity. From there, around 600 BC, probably through Etruria (today’s Tuscany),

Figure 1.1 Distribution of the wild olive (Olea europaea oleaster), the progenitor of the cultivated olive. It is still present in most coastal areas of the Mediterranean basin. (Zohary & Spiegel-Roy 1975)

4

Olive Propa ga tion

the crop is reported by the classical historians to have reached the Romans. Up to this point the olive had moved slowly westwards, first on the ships of Phoenician merchants, and later on those of Greek colonists; these peoples had spread the crop in many other places along the Mediterranean, including Spain, France and northern Africa, with varied results. But the conquest of the whole area by the Roman legions, and its transformation into a vast united empire, made trade and communications far more intense, and the olive benefited from this situation. In addition, when Italy appeared unable to provide the required supply of olive oil, the Romans spread its cultivation in new areas, or favoured it in places where olive groves had already been established but had stagnated. The crop achieved its maximum economic importance in the 2nd to 3rd centuries AD, particularly in northern Africa, but also in Spain, Dalmatia, and French Provence. With the fall of the Roman empire, information about the olive becomes scarce. Its cultivation dropped dramatically, with the reduction in population and the abandonment of large areas that took place during the early middle ages. This was not the case in the territories under Arab rule, where the crop remained important, to the point that its cultivation was forbidden in Sicily in order to protect production in North Africa, probably the main producer at the time. In Europe, olive oil acquired new importance only in the 16th–17th centuries, when it became a significant trading commodity for Venetians, who imported it from their Aegean possessions such as Crete and Cyprus. It must be remembered that oil was not only used as food; it also had great importance as a medicine, and for illumination, massaging, soap production and wool processing. Thus, olive plantations slowly began to spread in the Mediterranean areas where they can still be found today, with the exception of most of northern Africa, where they were reintroduced on a large scale much more recently. The arrival of the olive in the western and southern hemisphere is also recent history. Argentina, California, Australia and South Africa – where the enthusiasm of Mediterranean migrants for the crop had ensured its introduction – all proved to have suitable environments for commercial olive cultivation. Both the early domestication of the olive and its diffusion in the Mediterranean region have been favoured, or should we more correctly say permitted, by the ability of the species to be propagated with simple techniques. It is certain that the earliest domesticated fruit crops could be transferred from the bush or the forest (where they were browsed by animals and man) via the most rudimentary forms of cultivation, thanks to the possibility of stabilising positive and superior characters that the early gatherers noticed in the wild plants, when transition from hunting/gathering to agriculture was taking place. This suitability to asexual propagation was true for all ancient fruit crops, such as pomegranate, grapevine, date and fig.

Introduction

5

Figure 1.2. Young olive orchard in Western Australia. New plantations in southern hemisphere countries follow the most modern technical guidelines.

Figure 1.3. Exceptionally grown olive tree in Greenough, Western Australia.

6

Olive Propa ga tion

The olive, from the origins of its cultivation until the second half of the 19th century, was only propagated agamically, by using either large cuttings, ovules or rooted suckers. The slowness of its diffusion, which was a general feature of fruit crops until recently, made it a normal practice to resort to on-farm propagation, which meant the production of small numbers of trees each year. This also meant the selection and stabilisation of local cultivars, which, given the antiquity of the crop and its spread in the region, account for the high number of genotypes found in the different Mediterranean countries. Caruso (1883) questioned these direct multiplication methods, instead advocating the advantages of indirect multiplication, i.e. grafting on seedlings. In reality, the main real advantage of the grafting technique was the possibility of mass propagating the olive, and therefore dropping the prices of the individual plants. This would produce several further advantages: cultivars with superior characteristics could easily be introduced in new areas of cultivation, planting density could be increased, and rootstocks with positive characteristics could be used. Therefore, although several researchers (Vivenza 1926; Casella 1934; Morettini 1942) in the 20th century demonstrated that direct multiplication was just as good as grafting with respect to olive-tree life and performance, the new technique spread due to the need to provide large numbers of plants for the expanding olive industry. The old systems of propagation survived until recently in many areas including Southern Italy and Andalusia (Spain), but grafting certainly became by far the most important propagation technique. The supremacy of grafting over direct rooting, apart from never-ending disputes over the field performance of the trees (see 4.5), was granted by the simple fact that sufficiently good rooting could only be obtained by cuttings 4 or more years old, and by relatively large ovules and suckers, which made the availability of propagation material quite scarce. It is not surprising then that research into the possibility of using 1- or 2-year-old olive cuttings for direct propagation started as early as 1940. Research centred on cutting characteristics, rooting substances and greenhouse environment. In less than two decades, direct propagation of olive semi-hardwood cuttings became a technical reality. Although the technique would undergo several improvements to increase its efficiency, the fundamental acquisition was available by the mid-1950s. This did not mean that grafting practices with olives were suddenly stopped. On the contrary, in Italy, which is the main producing country for olive nursery trees, cutting propagation slowly conquered a share of the market, which remained around 50% until the 1980s, increasing its share in the following decade to over 70%. More recently, olive tree production by micropropagation techniques is also gaining favour, after decades of research in numerous research stations. This reconquered supremacy of direct multiplication over grafting does not mean that this issue is settled forever; on the contrary, grafting is a technique that will most likely accompany the olive industry into the foreseeable future. The reasons for this are numerous, but some are particularly important. In the first place, not all cultivars are easily (i.e. economically) propagated from cuttings or in vitro, such as many table olive cultivars. Secondly, direct multiplication involves the use of more or less complex structures, which require money and training, and in many situations (such as new areas in developing countries) one or both of them may not be available. Thirdly, although the availability of clonal rootstocks is at present quite limited, research is involved in selecting rootstock genotypes which can improve the industry through effects on tree size, yield efficiency and stress tolerance.

Introduction

7

The main lesson to draw from this short history of the olive and its propagation is that all available techniques of propagation have had their importance in different historical times, although one technique has at times prevailed over others. On the other hand, the economic success of a commercial olive grove also depends on the choice of planting material and on the characteristics it bears due to the propagation technique, as described in depth in the following chapters. Besides, there are situations in which olive farmers may choose to propagate their own trees, and select the most suitable technique for their needs and conditions. Understanding the basics of olive propagation appears, therefore, to be one of the foundations for the technical training of the modern olive grower.

1.3 Olive propagation today The annual production of olive trees in the main olive-growing countries of the world is around 40 million, with 32 million in the Mediterranean basin and 8 million in the rest of the world (IOOC 2000). From a technical point of view, about 28 million trees are obtained by means of mist propagation, 7 million by grafting and 5 million by traditional techniques (ovule, cuttings from branches, etc.). The misting technique, which spread widely between 1950 and 1960, is the most common propagation method where there are no financial or technical constraints. Under such circumstances, and at any rate wherever the nurseries are medium to large in size, they can afford fairly high investment costs in terms of buildings (greenhouses), instruments (for the management and control of humidity levels, temperature, light, etc.) and skilled personnel. The material used for propagation comes from the nursery itself (stock plants), from neighbouring nurseries and from plants in production, generally from areas not more than 50 to 200 kilometres from the nursery. The result of all this is that, as a rule, the material is indigenous in origin; only where stock plants of exotic cultivars are collected and grown by nurseries or institutions can genotypes of other regions or countries be made available to local producers.

References Caruso, G. 1883. Monografia dell’olivo. Enciclopedia Agraria Italiana, vol. 3. UTET (ed), Torino (Italy), pp. 501–533. Casella, D. 1934. La propagazione dell’olivo nell’Italia meridionale. Proceedings ‘Convegno dell’olivicoltura meridionale’. Bari (Italy). IOOC (International Olive Oil Council) 2000. Catalogo mondiale delle varietà di olivo. Madrid. Morettini, A. 1942. Ricerche sul sistema radicale dell’olivo. Proceedings ‘Convegno di studi olivicoli’. Firenze (Italy), pp. 281–321. Vivenza, A. 1926. L’olivicoltura in Italia: l’Umbria. In VIII Congresso Internazionale di Olivicoltura, Rome, pp. 5–21. Zohary, D. & Spiegel-Roy, P. 1975. Beginnings of Fruit Growing in the Old World. Science, 187 (4174): 319–327.

2 Flower and fruit biology in the olive

Olive propagation may involve several aspects of plant biology. Therefore an introductory level of information on olive flower biology, which ultimately leads to seed formation, is instrumental in achieving a comprehensive knowledge of the object of propagation.

2.1 Olive cycles In the olive, as in all fruit trees, several biological and physiological cycles can be considered, according to the period and the events that are taken into consideration. Here, a life or biological cycle, an annual cycle and a fruiting cycle are synthetically described.

2.1.1 Life or biological cycle The life or biological cycle comprises the whole life-span of the tree. Trees from rooted cuttings and from grafted seedlings will be taken into consideration here (plants from micropropagation can be assimilated to self-rooted trees). The life of a cultivated olive tree is usually divided into four phases or stages. 1. Unproductive stage. During this stage the tree grows at high rates and is characterised by the absence of flowering and fruiting. Lack of production is due only to the absence of a sufficient equilibrium between the canopy and the root system. This stage should not be mistaken for the juvenile stage, which is only typical of seedlings. The very appearance of the first flowers marks its end. 2. Stage of increasing production. Flowering means production, and the tree increases its productive capacity as time passes. Its canopy grows, and with it the number of buds that are susceptible to flower induction. 3. Maturity stage, during which plant size and production have attained a maximum. Productivity can be considered constant, although it may fluctuate greatly from year to year. In this stage, the olive grove produces at its best.

Flo wer and fruit biolog y in the olive

9

Figure 2.1 Olive phenological stages according to Colbrant and Fabre (in Loussert & Brousse 1978). A, winter stage; B, bud break and elongation; C, inflorescence development; D, flower enlargement (flowers become spherical, on a short pedicel, and bracts diverge); E, corolla differentiation (pedicels elongate, distancing the flowers from the inflorescence axis); F, onset of anthesis, the first flowers open (corollas from green to white); F1, full bloom; G, petal fall (petals darken and separate from the calix); H, fruit set; I, fruit growth (1st stage, the size of a caryopsis); I1, fruit growth (2nd stage, the largest fruits are 8-10 mm in diameter; endocarp sclerification begins); L (not shown), véraison, the fruit colour turns from green to a dark colour (red and then black).

10

Olive Propa ga tion

4. Senescence stage. All processes typical of ageing (low vegetative activity, reduction of expansion of the root system, abundant flowering followed by poor fruit set, susceptibility to diseases, etc.) appear and indicate a tendency of the tree to weaken and die. The important stages in cultivated olive trees are the first three, as current practice usually involves uprooting long before senescence begins. The duration of these stages has, on average, changed with time, as olive cultivation technology has evolved. Table 2.1 shows how the durations of the different stages have been evaluated at different times. The dramatic changes in the stages of the olive life cycle are due to a number of technological inputs the industry has received in just half a century, such as soil management technology, fertilisation, pruning, training systems and planting density. Table 2.1 Duration in years of the four stages of cultivated olive trees, according to past and current reports Morettini (1950)

Maillard (1975)

Morettini (1972)

Present day

Unproductive stage

1–12

1–7

1–4

1–3

Production increase stage

12–50

7–35

4–15

3–12

15 onward

12 onward

Maturity stage

50–150

35–150

Senescence stage

150–200

Over 150

Figure 2.2 Yearly biological and cultivation cycles of olive in the Mediterranean, according to Pansiot and Rebour (1960); months indicated by roman numerals refer to northern hemisphere environments. A, rest period; B, period of active vegetative growth; B1, period of slow vegetative growth; C, flower bud differentiation; D, anthesis-fruit set (inflorescence emergence is usually four weeks before anthesis); E, fruit growth; F, pit hardening (endocarp sclerification); G, véraison; H, ripening; I, vernalisation; J, pruning; K, harvest; L, critical period for nitrogen; M, critical period for water.

Flo wer and fruit biolog y in the olive

11

2.1.2 Annual cycle Productive olive trees go through an annual cycle, which is well described by the succession of phenological stages (Fig. 2.1). Such stages are closely related to a series of physiological events, which in turn determine the timing of cultivation operations (Fig. 2.2). The most important phenological stages are: onset of vegetative activity (bud break), emission of inflorescences, anthesis, fruit set, and véraison (drupes turning black). All these events occur over a number of days, and both the onset of a given stage and its duration may strongly differ from year to year and among locations, owing to a number of environmental variables.

2.1.3 Fruiting cycle The fruiting cycle begins when the uncommitted bud receives the first stimuli leading to its induction to flower, and ends with the full ripening of the fruits derived from those flowers, and their eventual abscission or harvest. Unlike the annual cycle, then, the fruiting cycle extends over two, sometimes three solar years. This means that, in any given moment, two fruiting cycles are taking place on the same tree, and that they cannot fail to heavily influence each other (Fig. 2.3).

Figure 2.3 Biennial cycle of vegetative and reproductive processes in the olive (Rallo et al. 1994).

12

Olive Propa ga tion

2.2 Stages of olive reproduction 2.2.1 Flower induction The formation of flower-bearing buds is a process requiring the passage of the meristematic apex of the bud, undifferentiated in its early stages of growth, to a structure carrying flowers. Such a process is commonly divided into two stages: during the first one, the induction phase, the bud undergoes a series of conditionings, both internal and external to the plant, following which, in the meristematic apex, such biochemical modifications occur as ‘to commit’ it to the formation of reproductive structures. This is also termed the ‘irreversibility stage’ to indicate a condition of the bud in which there is

Figure 2.4 Schematic representation of flower bud differentiation in olive (Loussert & Brousse 1978; redrawn from Hartmann 1951). A, undifferentiated bud; B, elongation of the inflorescence axis and appearance of the terminal flower with flower primordia (S); C, well developed sepal (S) and petal (P) primordia; D, sepal and petal primordia at a more advanced stage, and appearance of stamen primordia (E).

Flo wer and fruit biolog y in the olive

13

only one ontogenic possibility left, i.e. of developing into a fruit bud; otherwise it will not undergo any further development. When the irreversibility stage is overcome, the bud is to be considered ‘induced’ to flower, and the next phase – differentiation – begins. The exact time of flower induction is difficult to determine, mostly because the modifications it involves are of a physiological nature, and therefore non-detectable at the microscopic level. In addition, the duration of this phase does not depend so much on the length of the process in the individual bud, but rather on the fact that it is not simultaneous in all parts of the canopy, and that it therefore takes place over a period of time, which is markedly influenced by environment and genotype. This graduality is present in all cultivars, although to different extents. Scientific evidence seems to indicate, for the olive bud, a two-step process, with a first wave of induction at the end of spring. These buds will undergo a later confirmation of their orientation to form flowers in late autumn, in accordance with the prevailing environmental and nutritional conditions that have occurred in the meantime (Fabbri & Benelli 2000).

2.2.2 Flower differentiation The differentiation stage involves a series of anatomical changes. During this stage the typical tissues of flower structures are formed, reaching completion immediately before anthesis. Flower differentiation takes place in winter (from late February to mid-March in the northern hemisphere) although in some areas it may last longer. The timing and extent of flower differentiation seem to depend on the achievement of specific chilling requirements. This means that low winter temperatures influence not so much floral evocation as the expression of a flowering potential already determined in warmer periods. As a rule, floral differentiation occurs during the 40–60 days before anthesis; therefore the process is completed as the inflorescence emerges and develops. The first signs of differentiation are a broadening of the bud apex towards a more or less flattened conical mass, with the four sepal primordia appearing as minute protuberances on the sides of the apex itself. At the axils of the leaflets enclosing the apex, more meristems (decussate couples) appear and develop, forming a branching that will form the inflorescence, which will comprise a large number of flowers. In the meantime, in each individual flower primordium, the various floral organs (petals, stamens and ovary) are formed centripetally (Fig. 2.4).

2.2.3 Reproductive structures Inflorescence Flower bud inflorescences are borne at leaf axils (a maximum of two per node). Usually flower buds are formed on the shoots developing the year before anthesis. Buds may remain dormant for more than a year and then develop into inflorescences, but in most cases undeveloped buds abscise. The inflorescence axis in the spring grows slowly, and finally emerges from the leaflets that protected it in the winter. Inserted on the main axis, at the axil of small bracts, are the secondary axils, decussate, of decreasing length from base to tip. These can be further branched, and this constitutes an important taxonomical character in cultivar description. Inflorescence colour is green to white or white-yellowish.

14

Olive Propa ga tion

Figure 2.5 Olive inflorescences (courtesy H. Rapoport).

Inflorescences (Fig. 2.5) can be described by their shape and size (10 to 70 mm), flower clustering, pedicel length, flower size, number of flowers (5 to 60). Some cultivars may bear mixed buds, i.e. developing a shoot that bears, in the same year’s growth, inflorescences on the basal nodes; the phenomenon is strongly influenced by the environment and is more common in warm climates. Inflorescence development begins in early spring (e.g. about mid-April in Central Italy), roughly one month after the start of differentiation, usually beginning on the south side of the tree (in the northern hemisphere). It is gradual, and does not follow a regular scheme like anthesis in the inflorescence; it may last 6–8 weeks, even more in warmer climates, in separate flushes. As a rule, the earlier the emission of inflorescences, the higher the expected production, as fruit set may take place in less dry conditions, but subsequent environmental events may markedly alter the forecast. Flowers are usually borne on 1-year-old shoots, but reports exist of inflorescences developed on 2- and sometimes 3-year-old branches. Flowers and flowering The olive flower is made of four verticils (whorls): calix, corolla, androecium and gynoecium (4 sepals, 4 petals, 2 stamens and 2 carpels; Fig. 2.6). The sepals are light green, short and rounded. The corolla is gamopetalous (tubed) with four white-yellowish lobes. The androecium consists of two opposite stamens inserted on the corolla, and each stamen consists of a filament topped by a large anther that is hemispherical, introverted and longitudinally dehiscent. Anthers contain bicellular pollen grains; the external walls of pollen grains have characteristic structures (Fig. 2.7). The gynoecium is a superior two-loculed ovary, made of two carpels. Each locule contains two anatropous ovules; the style is short and sturdy, and ends with a well-developed, bifid, papillate, plumose stigma. Calix, corolla, stigma and pollen exhibit characteristics that vary with the cultivar, but

Flo wer and fruit biolog y in the olive

15

Figure 2.6 Typical olive flower (courtesy H. Rapoport).

Figure 2.7 SEM (scanning electron microscope) photograph of olive pollen grains (Roselli 1977).

differences also exist among flowers on the same tree, and therefore such characteristics cannot be used for taxonomical purposes. Two types of flowers are present each season: perfect flowers, containing stamen and pistil, and staminate flowers, containing aborted pistils and functional stamens. The absence of stamens has only been observed in one cultivar, and is therefore very rare. The most important anomalies concern the ovary. ‘Ovary abortion’ refers to the absence of an ovary, or to a small, imperfect, non-persistent ovary. The perfect flower is evidenced by its

16

Olive Propa ga tion

large pistil, which nearly fills the space within the floral tube. The pistil is green when immature and deep green when open at full bloom. Staminate flower pistils are tiny, barely rising above the floral tube base. The style is small and brown, greenish-white, or white, and the stigma is large and plumose as it is in a functioning pistil. All olive trees display ovary abortion, although at different extents, depending on cultivar, year, environment, inflorescence and type of shoot. Its incidence is variable, from less than 10% to 30–40% of the flowers in the most common oil cultivars, up to 50–60% in table cultivars (Table 2.2). In some Italian cultivars, such as ‘Morchiaio’ in Tuscany, up to 70–90% of ovary abortion is reached. In spite of this, production is usually not depressed as normal harvests require no more than 1–4% of fruit set. One hundred per cent abortion cannot exist in a commercial cultivar, but ‘Swan Hill’, an ornamental cultivar selected by Hartmann in Australia (Hartmann 1967), displays that character, which is advantageous when olive trees are used in urban forestry or as ornamentals. Table 2.2 Percentages of ovary abortion for several olive cultivars. Data were collected from trees located in the Chianti region of Italy (Magherini 1971). Cultivar

1961

1962

1963

1964

Average

Frantoio

8.20

9.40

6.20

6.60

7.40

Moraiolo

29.30

18.00

7.20

12.95

16.08

Pendolino

26.20

22.50

31.40

18.45

23.40

Ascolana tenera

49.70

40.40

56.10

49.65

49.10

Uovo di Piccione

57.60

46.40

56.40

46.45

50.66

Morellona di Grecia

32.60

32.00

45.10

32.95

35.12

Grossa di Spagna

34.80

49.60

46.80

86.60

60.88

Sant’ Agostino

36.80

45.40

45.60

64.85

51.50

Gordales Sevillana

49.10

56.60

59.20

69.15

60.64

Yearly averages

36.03

35.57

39.33

43.07

2.2.4 Anthesis and pollination Full bloom occurs in full spring (e.g. May in warm areas such as California, Southern Italy, Greece and Spain; at higher latitudes and elevations full bloom is delayed into the first weeks of June). Differences can be observed among cultivars (Table 2.3), which are to be taken into account when selecting pollinators. Anthesis normally lasts 2–3 days on individual inflorescences, and 5–6 days on the individual tree, or up to 10–15 days if temperatures are relatively low. A flower is fully opened when both anthers and petals are separated. During the hottest part of the day anther dehiscence takes place, and an abundant amount of pollen is shed. The amount of pollen produced appears to be a varietal characteristic; for example ‘Leccino’ and ‘Frantoio’ produce small amounts of pollen, but larger quantities are produced by ‘Ascolana’, ‘Manzanilla’ and ‘Pendolino’. More important is the pollen’s ability to germinate: this characteristic appears to fluctuate (in vitro) between 12% and 60% (Zito & Spina 1956; Fernandez-Escobar et al. 1983).

17

Flo wer and fruit biolog y in the olive

Table 2.3 Flowering period of olive cultivars in Umbria, hills of Central Italy (Antognozzi et al. 1975). CULTIVAR Uovo di piccione Nocellara etnea Picholine Sant’ Agostino Bouteillan Carolea Santa Caterina Tanche Bella di Spagna Dolce di Andria Dritta di Moscufo Grossa di Spagna Morchiaio Moresca Nebba Rosciola Santagatese Ascolana tenera Bosana Cucca Fecciaro Gentile di Chieti Gordales Grossanne Itrana Manzanilla Passalunara Raja Sabina Casaliva Cellina Carmelitana Cerignola Corniolo Maurino Mignolo Moraiolo Corsini Morellona di Grecia Raza Sargano Taggiasca Ascolana Semitenera Coratina Dritta di Loreto Frantoio Frantoio Corsini Moraiolo Carboncella Nocellara Messinese Ottobratica Piangente Razzola Caninese Correggiolo Pendolino Rastellina Grappolo Ogliarola Olivago Savino Laurina Leccio del Corno Leccino

June 5

10

15

20

25

30

18

Olive Propa ga tion

Pollination is influenced by several factors, the most important being: • temperature – which has the effect of enhancing tube growth, although when it is too high the stigma may get dry. For anther dehiscence the optimum is 30°C with 50% RH (relative humidity). A good value of relative humidity also enhances pollen germination; • rain – which is always negative. Indeed, it may determine pollen grain plasmolysis, dilute stigma secretions, and hinder pollen transport; • wind – which is fundamental for this anemophilous species. When too strong it may transport masses of pollen away from the grove. Although olive pollen can be found as far as 12 kilometres from the originating tree, the effective range is up to 20–30 m. Among nutritional factors, good nitrogen fertilisation has been proven to lengthen the effective pollination period, and therefore to improve fruit set. Sterility Sterility may be due to factors different from those affecting ovary abortion, such as anomalies during meiosis producing (i) imperfect gametophytes (cytological sterility), quite rare in olive, and (ii) incompatibility (factorial sterility). Incompatibility occurs when a perfect pollen grain fails to germinate on the stigma, or germinates, but its tube growth is insufficient for fertilisation; this incompatibility may be between two cultivars (inter-incompatibility) or, as is often the case with the olive, a cultivar is genetically programmed not to be fertilised by its own pollen (self-incompatibility). Most olive cultivars are self-incompatible, or self-sterile, and care must be taken to establish orchards with more than one cultivar. It is therefore important to know which cultivars are best suited to fertilise the one we want to be the principal one. In any case, growing three or four cultivars in the same plot guarantees good set, even if the cultivar we are interested in is considered self-fertile, as is the case for ‘Frantoio’. Fruit set In the absence of sterility barriers, the pollen germinates on the stigma, and develops through the style a tube that reaches the ovary and fertilises the egg (‘double fertilisation’). The viable pistil has two carpels, each containing two ovules, but only one ovule is fertilised and develops. Thus, as a rule, in the fruit only one carpel containing one seed is present; occasionally, there may be none (parthenocarpy) or two. Following fertilisation, the flowers shed petals and stamens, the ovary grows in size, and at this point many pistils shrink and drop. Within a month from full bloom only 7–8% of flowers are still on the tree as fruitlets; of them, only 25% are present by late summer. The average fruit set is therefore around 2%, although yearly fluctuations can be wide. Fruit drop is used by the plant to adapt production to its elaborating surface; other factors may influence fruit drop, such as nutritional and water deficiencies, weather conditions during bloom, sterility, lack of pollinators and pests. Fruitlets compete for survival, starting about 10–15 days after full bloom.

Flo wer and fruit biolog y in the olive

19

2.2.5 Fruit growth and seed development The olive fruit is a drupe, which means it is made of two main parts: pericarp and seed. The pericarp is made of (i) the skin (exocarp), smooth and with stomata, (ii) the flesh (mesocarp), the tissue containing oil, and (iii) the pit, a lignified shell enclosing the seed. The ‘true seed’ (Fig. 2.8) consists in a seedcoat and a thick endosperm that both sheathe a large embryo made of flat cotyledons with a short radicle and plumule. As a rule there is only one seed, rarely two. In some Spanish cultivars the occurrence of nucellar polyembryony has been reported. Fruit shape and size, pit size and surface morphology vary greatly among cultivars, and are the most reliable morphological features to distinguish between cultivars.

Figure 2.8 Longitudinal section of an olive stone (seed and endocarp) (Krugman 1974).

Fruit growth is represented by the double sigmoid curve, typical of drupes (Fig. 2.9). Three stages can be separated: 1. Exponential growth, which is characterised by abundant cellular multiplication. The pit achieves a quasi-final size. 2. Growth slows down, or stops for a short period. During this stage the embryo completes its development and the pit hardens (sclerification). 3. Growth resumes due to cell enlargement, and gradually diminishes with time.

20

Olive Propa ga tion

Figure 2.9 Fruit growth of the cv. Lucques, expressed as a growth index (fruit length x diameter). Plants sprinkler (1) and drip (2) irrigated (Villemur et al. 1976).

This growth model is less evident if growth is represented by weight increases, rather than diameter or volume. The curve can even turn into a single sigmoid if dry weight is taken into account, thus showing that stage two is the one requiring most dry weight accumulation. In the course of fruit formation, and tightly connected to it, the vital ovule develops to form the seed. The embryo makes up most of the seed volume. The seedcoat, derived from the integuments which represented the main ovular tissues, is thin and leathery, and rich in vascular ridges. Between the seedcoat and the embryo is a layer of endosperm, rich in starch (King 1938). The embryo has two quite evident large cotyledons, the embryonic leaves (Fig. 2.10). A short radicle, located at the lowest end of the embryonic axis, will give rise to the root system. Between the cotyledons is a small plumule, from which will develop the future epigeic system, the plant parts that will be exposed to the open atmosphere. The embryo starts growing within three to four weeks after bloom, and reaches the globular stage at week six; after eight weeks the cotyledons appear to be well developed. The embryo is usually completely formed after five months from full bloom. No further morphological or anatomical changes appear to occur in the embryo, although dormancy is imposed on the seed late in the season (see 4.2.1). Seed growth means a gradual embryo enlargement, which at the end occupies most of the space inside the endocarp, at the expense of the endosperm.

Flo wer and fruit biolog y in the olive

21

Usually pollination and fecundation are essential for fruit set and early seed development. The presence of a vital seed in a growing drupe is not essential, as many fruits have their embryos aborted. As a consequence, many apparently normal fruits have no seed. The fruit can also develop without the presence of a fertilised ovule (parthenocarpy), but in this case the fruit remains distinctly smaller.

References Antognozzi, E., Cartechini, A. & Preziosi, P. 1975. Indagine sulla individuazione dei migliori impollinatori per olive da mensa della cultivar ‘Ascolana Tenera’. Proceedings ‘2nd Sem. Oleic. Int.’. Cordoba, 6–17 October. Fabbri, A. & Benelli, C. 2000. Flower bud induction and differentiation in olive. J. Hort. Sci. Biotech., 75 (2): 131–41. Fernandez-Escobar, R., Gomez-Valledor, G. & Rallo, L. 1983. Influence of pistil extract and temperature on in vitro pollen germination and pollen tube growth of olive cultivars. J. Hortic. Sci., 58 (2): 219–227. Hartmann, H.T. 1951. Time of floral differentiation of the olive in California. Botanical Gazette, 112: 323–327. Hartmann, H.T. 1967. ‘Swan Hill’ a new ornamental fruitless olive for California. California Agriculture, 21: 4–5. King, J.R. 1938. Morphological developmentof the fruit of the olive. Hilgardia, 1: 437–458. Krugman, S.L. 1974. Olea europaea L., Olive. In C.S. Schopmeyer (coord.) Seeds of woody plants in the United States. Agriculture Handbook 450. USDA, Washington, pp. 558–559. Loussert, R. & Brousse, G. 1978. L’olivier. G. P. Maisonneuve et Larose, Paris, pp. 465. Magherini, R. 1971. Osservazioni sull’aborto dell’ovario nell’olivo. L’Agricoltura Italiana, LXXI (5): 291–301. Maillard, F. 1975. In Loussert & Brousse, 1978. Morettini, A. 1950. Olivicoltura (1st edn). REDA, Rome. Figure 2.10 Morettini, A. 1972. Olivicoltura (2nd edn). REDA, Rome. Section of a mature embryo Pansiot, F.P. & Rebour, H. 1960. Amélioration de la culture de l’olivier. (courtesy H. Rapoport). FAO, Rome. Rallo, L., Torreño, P., Vargas, A. & Alvarado, J. 1994. Dormancy and alternate bearing in olive. Acta Hortic. 356: 127–136. Roselli, G. 1977. Osservazioni sulla scultura dell’esina del polline di alcune specie da frutto. 1. Olivo. Riv. Ortoflorofrutt. It., 61(2): 157–163. Villemur, P., Gonzales A. & Delmas, J.M. 1976. A propos de la floraison et de la fructification de quelques varieties d’olivier. L’olivier, 16 (3): 45–47. Zito, F. & Spina, P. 1956. Come germina il polline dell’olivo. Italia Agricola, 93(5): 413–425.

3 Propagation by cutting

Plant propagation by cuttings involves all techniques of asexual propagation in which the whole plant originates from a plant part that is put in the conditions of regenerating a root system and a canopy. This definition excludes grafting, in which the final plant derives from two different parent plants. Micropropagation can be considered a particular type of cutting propagation, but due to its technical peculiarity it is dealt with in chapter 5.

3.1 Biology of adventitious root formation in cuttings 3.1.1 Morphology and anatomy With the exception of the root developed by the embryo during germination, all roots formed in a plant are adventitious, as structures similar to buds do not exist in the root system. Thus, all lateral roots are adventitious, as their induction and differentiation in the root pericycle, and their occurrence along the older roots, depend on external factors. Here, attention will be focused only on roots arising from tissues originally destined for other functions, namely branches and shoots. The process of rhizogenesis, and the factors determining the formation of adventitious roots in aerial organs, are fundamentally the same in all the propagation systems adopted for the olive. As multiplication by semihardwood cuttings is the most common system of obtaining new olive plants by asexual propagation, the rooting process as it takes place in such material (that is, one-year-old or younger shoots) will be taken into consideration (Fig. 3.1). Any difference occurring in other systems will be mentioned when dealing with them in the appropriate sections. It is known that adventitious roots forming in cuttings are of two types: preformed roots and wound-induced roots. The presence of preformed roots has never been demonstrated in the olive, although latent meristems occur in old wood which can turn, under appropriate conditions, into roots. This is the case with ovules and sphaeroblasts

Propa ga tion by cutting

Epidermis Cork Phellogen Phelloderm Cortical parenchyma Fibres Sclereids

23

Periderm or Cortex

Primary phloem

Metaphloem Phloem parenchyma Secondary phloem

Sieve cells Cambium

Xylem rays

Xylem

Xylem parenchyma Vessel

Pith cells

Pith

Figure 3.1 Section of a 1-year-old shoot (Troncoso et al. 1975).

on branches. The roots obtained by propagating semi-hardwood cuttings therefore originate after the cutting is made by excising it from the stock plant. These are called wound-induced roots, as the first stimulus the rhizogenetic tissue receives is that of the wound, but roots also form through a series of modifications in the cutting environment. The term de novo-formed adventitious roots would therefore be more accurate; but, due to the nature and purpose of this book, the use of the term ‘root’, unless otherwise specified, will indicate such roots. The first events occurring in an olive cutting are merely a response to wounding, and are aimed at isolating the organ from the environment in order to avoid water loss through the cut area, which would lead the excised plant part to

24

Olive Propa ga tion

desiccation and death. Those outer cells that are suddenly in contact with the atmosphere die, and their remnants, often in the form of a layer of mucilages, are a first shield for the inner cell layers. Vessels, which can cause abundant water loss, are eventually plugged with gums (tyloses). Finally, living cells belonging to several tissues deeper below the cut surface start dividing to produce, within the first two weeks, large lumps of specialised parenchyma cells (callus), which in the end constitute a sort of thick cap on the lower end of the cutting. The completion of the process is indicated by a swelling of the cutting base. Its epidermis gets lighter in colour and shows occasional cracks and callus emergence, while the bottom is completely covered by the callus cap (Fig. 3.2). The top end of the cutting is usually sealed more internally, after a dieback of tissues that goes as deep as the loss of water determines. This can at times determine the loss of the most distal leaves and buds, an event that can negatively affect the survival of the cutting.

Figure 3.2 Bottom end of a cutting prior to root emergence.

The process leading to the formation of new roots in cuttings begins within a few days after excision. Specific differentiated cells abandon their physiological and cytological commitment, and regain the ability to divide (dedifferentiation). A lot of research has been devoted to the location of these early cells, although the task is not an easy one; indeed, a number of tissues and positions in the cuttings (phloem, medullary rays, callus, cortex, xylem, even pith) have been indicated in time. Most authors, though, indicate young, differentiating secondary phloem cells, located in the vicinity of

Propa ga tion by cutting

25

medullary rays, as those most sensitive to the stimuli leading to dedifferentiation. These cells, now meristematic (i.e. able to divide), start dividing and form, within the cutting, lumps of small cells called root initials. In the beginning these do not differentiate into specialised cells, but rather continue dividing until the size of the root initial is such as to exert a pressure on the outer cell layers, the tissues between the initial and the outside –usually the outer phloem with its schlerenchyma ring, and the cortex. At this point the structure has assumed a conical shape, with its base towards the central cylinder, and is more properly called a primordium. The primordium keeps growing towards the outside, and in the process its cells differentiate into the various tissues of a root; a strong rootcap makes it possible to push and crush the external cutting tissues, and allows the emergence of the primordium. By the time it emerges, the primordium is well structured (Fig. 3.3), and a connection between it and the central cylinder of the cutting is established. Still, the primordium is not yet functional, that is, it is not yet able to absorb water from the substrate. As can be seen in Fig. 3.1, the olive shoot (and therefore the semi-hardwood cutting) develops a ring of fibre bundles in the primary phloem, which becomes uninterrupted by the differentiation of sclereids in the gaps between one bundle and another. This creates a rigid sclerenchyma ring that may appear to be an insurmountable barrier for the primordium, and indeed this has been suggested as the reason for the failure in rooting of some cultivars (Ciampi & Gellini 1958; 1963). Further research, though, has demonstrated that the barrier can be crushed by the pressure of the growing primordium, or that the primordium can skip it by emerging from the bottom end

Figure 3.3 Root primordium (Ciampi & Gellini 1963).

26

Olive Propa ga tion

(Fig. 3.4), opening its way out through the softer callus tissue. At any rate, the presence of a thick sclerenchyma ring does not account for the varietal differences in rooting percentages (Troncoso et al. 1975; Fabbri 1980).

Figure 3.4 Young adventitious roots a few days after emergence.

3.1.2 Physiology It has been postulated that a substance called rhizocaline, present in leaves, buds and cotyledons, is able to move to the roots and stimulate rooting (Bouillenne & Went 1933) but, in spite of long years of research, rhizocaline still remains a hypothetical compound. However, different classes of plant growth regulators have been demonstrated to influence, positively or negatively, root initiation, including auxins, cytokinins, gibberellins, ethylene, polyamines, abscisic acid, growth retardants and phenolics. To date, auxins have been shown to have the greatest effect on rooting. Numerous reports have indicated the involvement of auxin in the initiation of adventitious roots, and that division of root initials depends on exogenous and endogenous auxin. Synthetic auxins, such as indole-3-butyric acid (IBA) and naphthaleneacetic acid (NAA), have been shown to be more effective than the naturally occurring indole-3-acetic acid (IAA) for rooting. IBA is actually commonly used in propagating olives by cuttings. The physiological role of cytokinins in root initiation and development is an ambiguous one: kinetin (a common type of cytokinin) has shown either stimulatory or inhibitory effects, depending on its concentration in the tissue. At any rate, in tissue culture the cytokinin levels must be lowered when rooting is desired. Roots are an important site for

Propa ga tion by cutting

27

gibberellin (GAs) metabolism, but these growth regulators do not appear to have a positive role in adventitious root formation. A similar conclusion has been reached for abscisic acid (ABA), although it seems to play an important role in enhancing root growth. A significant amount of research has been devoted to studying the effects of endogenous ethylene in adventitious root formation (Bartolini & Fabbri 1982; Bartolini et al. 1986a; 1986b). Ethylene certainly affects root initiation and development, although it might play different roles in the two stages. It has been suggested that auxin action takes place by enhancing ethylene synthesis. At present, however, the role of ethylene in the rooting process is not completely understood. Many studies have hypothesised a role for polyamines in the rooting process, and their relationship with auxins and peroxidases. According to Gaspar et al. (1997) IAA and putrescine, an important polyamine, might be required to initiate cell division at the end of the rooting inductive phase. Polyamines induce rooting in the olive (Rugini & Wang 1986; Rugini et al. 1997), possibly at the very early stages of rooting. It has also been suggested that polyamines might be considered precocious markers of rooting. At any rate, as other species are not affected by polyamine treatment, it is highly possible that their effects on the olive are due to the low endogenous polyamine content in olive tissues. Other naturally occurring substances that have been shown to exert an effect on rooting are phenolic compounds, which, according to their chemical structure, may be either stimulatory or inhibitory (Bartolini et al. 1988). In the olive, as in most species, juvenile tissues and organs root better than mature ones, and this peculiarity has been exploited for a series of propagation techniques, particularly to produce clonal rootstocks. Such behaviour has been attributed to larger amounts of stimulators and to a reduced presence of inhibitors, a condition that is gradually modified as tissues age. At any rate, although it is exploited when obtaining plants from rooted suckers, the commercial importance of this feature is negligible for the virtual absence of clonal rootstocks in the olive; on the other hand, propagating cultivars from juvenile or rejuvenated material might delay the onset of fruit bearing for an unacceptable number of years. What results in the end from physiological studies on the olive and other species is that a number of substances are needed to trigger the process, and that such substances are for the most part produced by leaves and buds. The process in the olive is particularly time consuming, requiring relatively high amounts of energy, i.e. carbohydrates, possibly in an appropriate C/N (carbon/nitrogen) ratio. This explains the need, when propagating cuttings made from young shoots, to retain as many leaves as possible, regardless of the technique used.

3.2 Techniques of propagation by cutting 3.2.1 Plant material With varying results, every part of the olive tree has been used for propagation. Shoots and branches are, as a rule, excellent propagation material when the proper techniques are applied. The olive tree is also very efficient at differentiating new meristems at the collar, where the transition from trunk to root system takes place, and on old wood (limbs) where latent unopened buds are maintained. This peculiarity explains the relative

28

Olive Propa ga tion

ease in propagating the olive from large plant parts, which enabled ancient growers to asexually propagate this tree. Root cuttings are the only organs of the olive not able to produce new plants; indeed, roots do not possess adventitious buds, and are incapable of producing such structures when separated from the parent plant. Branches The use of branches more than 4 or 5 years old – originally by the Phoenicians, Romans and Arabs – is a very ancient method of olive propagation. This method makes use of the possibility of producing shoots (from latent or adventitious buds) and roots from branches. Cuttings (20 to 50 cm long, with a diameter of 5 to 10 mm or more) are obtained from branches during the cold season (autumn–winter). The cuttings are kept until spring in a cool and not excessively damp place, in layers of sand. They are then positioned in an outdoors plot in various ways: • horizontally at the bottom of furrows (Fig. 3.5), covered with a few centimetres of soil. In spring, among the new shoots, the most vigorous are kept for growth and the others are eliminated, unless the cutting is of such a size to support more than one plantlet. After 6 to 8 months the plantlets have reached a height varying between 60 and 80 cm, and at their bottom end, near the insertion on old wood, a small root system should be present. The rooted shoots are then separated from the branch, after which they receive normal nursery care; • vertical or inclined. In this case the new shoots do not always produce roots, which can only be present on the old cutting wood. The new plant is therefore made of a conspicuous piece of the original wood (Fig. 3.6). With this method there can be a serious risk of root rot.

Figure 3.5 Branch cuttings positioned horizontally, before covering with soil.

Propa ga tion by cutting

29

Figure 3.6 Vertically positioned rooted branch cuttings.

Branch material is easily obtained from pruning operations, which in this case should be made in winter. As a rule, the cuttings should not be left in the open after collection; they should either be put to root or stratified in sand. The soil should be carefully pressed around the cuttings. In present day routine work, treatments with root-promoting chemicals are useful, and rooting performance is directly proportional to stem diameter. Rooting is also obtained from very old trunks, when uprooted and cut into sections 20–30 cm long; the new shoots and roots are produced from the living tissues that are in the cortex when the piece of wood is buried for a few centimetres (Fig. 3.7). Examples of traditional propagation by branch cutting are: • the ‘Cormoni’ (Apulia and Southern Italy in general) – 40 to 60 cm long branches with their foliage removed; they are planted directly in the definitive planting site in late autumn–early winter, at a depth of 35 to 50 cm, with the terminal 5–6 cm emerging; • the ‘Estacas’ (Spain) – big cuttings, as long as 2 to 3 m, more than 6 cm thick, obtained during pruning. About a third of their length is put into the ground (a hole 1 x 1 x 1 m, enriched with manure) in autumn and the rest is covered by a cone of soil, except the upper 20–30 cm. At the beginning of the following summer, when the apical shoots are 5 to 10 cm long, the mound is removed from around the cutting; • the ‘Garrotes’ (Spain) – cuttings 50 to 100 cm in length, and 3 to 5 cm in diameter. For rooting, they are directly planted out (3 or 4 per hole, at the corners of a triangle or square), or are placed in plastic containers with a volume of 20 to 30 litres of soil.

30

Olive Propa ga tion

Good results are obtained with these methods but they are not sufficient to justify nursery use. On a commercial scale, it is almost impossible to find the large quantities of material necessary for this kind of propagation. However, this technique can be useful for the small grower who needs to replant a small percentage of his orchard each year and is not willing to invest in propagation equipment. Shoots The introduction to the olive industry of the misting technique (Hartmann 1952) for shoot rooting has provided and still provides a valid alternative to the techniques used until then in olive-growing countries, namely grafting, ovules and severalyears-old woody cuttings (as described above). The technique uses shoots developed in the same year, or Figure 3.7 Section of old trunk wood, with a few rooted shoots, 1-year-old shoots which have not before excision. produced fruit (see 3.2.2). Ovules Ovules are characteristic swellings (tissue hyperplasia) which appear as protuberances and are found at the level of the collar (Fig. 3.8). At the ovule site, a build-up of sap due to a slowing down of its circulation occurs. This is most often found at the base of the trunk where the root structure joins the trunk, producing torsions in the vessels and thus slowing sap circulation. This determines hypernutrition of cambium cells, which actively proliferate and produce the extroflection of tissues, particularly parenchyma tissues that constitute most of the ovule. In some cultivars these structures can also be found in the lower trunk parts of 5- to 6-year-old trees. Accumulated starchy substances may cause the formation and emission of adventitious shoots and roots, the type of structure depending on whether or not the ovule is exposed to light. This property has been exploited by growers since ancient times to obtain new cloned plants (Fig. 3.9). The use of ovules for olive propagation is a method that was used mainly in the past and in areas at the fringes of olive cultivation. Francolini (1934) observed that olive trees derived from ovules and grown in poor, shallow and sub-arid soils gave better results than trees from grafting. This method has been more recently used in north-African countries such as Tunisia and Libya (Pansiot & Rebour 1960). The ovules can be removed with special cutting tools, and great care needs to be taken of the exposed tissues, which must be disinfected and protected. Planted at the

Propa ga tion by cutting

Figure 3.8 Ovules on an old olive trunk (* = ovule).

Figure 3.9 Detail of an ovule-propagated olive plant (Morettini 1972).

31

32

Olive Propa ga tion

beginning of spring, ovules are able to produce new trees as they contain both root primordia and dormant buds. Ovules are generally taken from the enlarged conical base of the trunk, the collar, which is particularly developed in old olive trees (Baldini 1986; Hartmann et al. 1990). Their removal from trunks is not common as it damages the stock plant. Ovules are taken from healthy trees at the beginning of spring or autumn, and they are then kept in slightly damp sand until the time comes to put them under earth in the ovulary, or ovule bed. The weight of an ovule varies, on average, from 100 g to 3 kg. If they are rooted in nurseries their weight is usually 500 to 800 g, but when they are to be planted directly in field their weight depends on the expected rainfall and on the possibility of irrigating. If water is expected to be lacking, very large ovules are used (e.g. up to 5 kg in Sfax, Tunisia). The bark of the ovule must be smooth and light in colour, with slight wrinkling which reveals the presence of latent buds, and the wood must have a healthy appearance. Those that are rotten, damaged or already rooted must be discarded. The ovulary (i.e. the site where the ovule is to be planted) is prepared first by turning over the soil to a depth of 80 cm, then the ovules are placed in furrows (20 cm deep) with the cut surface facing down (Fig. 3.10). Planting distances are 60 cm between rows and 30 cm between ovules along the row. Transplanting is done after 3–5 years; if the new plant has developed its own roots, it can be separated from the ovule. In this case the ovulary can be used as a continual source of plants, as in the stoolbed technique (Battaglini 1967).

Figure 3.10 Details of ovule propagation in the nursery; distances are in metres (Loussert & Brousse 1978).

Propa ga tion by cutting

33

Suckers (or pollards) Suckers develop at the base of the trunk and have their origin in ovules. Generally they are taken from the tree when they have acquired their own ovule masses, and have produced sufficiently developed roots (they cannot therefore be considered cuttings in a strict sense). To help these processes, the base of the tree is covered with a thin layer of soil which encourages the initiation of roots. Shoot girdling can further enhance adventitious root formation. In the spring, the rooted suckers are removed from the stock plant with some old, collar wood, to be grown in the nursery before being planted out. Although this method of multiplication can be used for the replacement of small numbers of trees, it cannot be used at nursery level because it is slow and costly. The use of suckers is part of common practice after a frost, to make up for losses in the most convenient way. Once the death of the upper part of the tree is confirmed, it is cut down and removed to avoid the presence of wood that can easily be attacked by parasites (fungi, insects, etc.), while the stump is left so that suckers can develop freely (the stump usually survives the cold because it is covered by a few centimetres of soil). After about one year, the less favourably positioned suckers are removed so that in the second or third year there is one, or at most two or three suckers, from which to grow a new canopy.

3.2.2 Shoot collection and cutting preparation For the preparation of cuttings, scions can be taken all the year round. In practice this is done in relation to the production cycle of self-rooted plants, which generally coincides

Figure 3.11 Time course of adventitious rooting (% of rooted cuttings) versus monthly shoot growth (mm) in the olive. Values are averages obtained by the authors over several years from cultivars of Central Italy, characterised by intermediate rooting ability; months refer to the northern hemisphere.

34

Olive Propa ga tion

Figure 3.12 Good quality shoot for cutting production.

Figure 3.13 Two cuttings were obtained from the shoot in Fig. 3.12. Figure 3.14 Olive semi-hardwood cuttings ready for planting in the rooting bed.

with two annual peak points of concentration of rooting compounds within tissues, i.e. April and September–October in Central Italy (Fig. 3.11). In temperate climates, at the borderline of the growing area for olives (such as Central Italy), it is possible to get shoots from stock plants that are long enough to make more than one cutting, but not more than once a year. From these shoots (Fig. 3.12), cuttings are obtained by dividing them into 10–15 cm long pieces (Fig. 3.13) of 4–6 mm in diameter, with 4 to 6 nodes. The 4 to 6 leaves at the distal end are retained, while the 2 to 3 basal nodes are left without leaves (Fig. 3.14). The basal cut must be made just under a node, and care must be taken to avoid leaving a stub of the internode, which can be a point of necrosis. Generally, new roots do not grow from internode tissues; they usually develop within node tissues. Once

Propa ga tion by cutting

35

the cuttings are prepared, basal treatment with a rooting hormone (see below) must be carried out within 20 to 40 minutes.

3.2.3 Auxin treatments to promote rooting of cuttings Auxins are root-promoting chemical agents. In spite of its belonging to the group of the naturally occurring plant hormones, IAA is not commonly used as a rooting promoter in commercial olive propagation. Indeed, in comparison with IAA, the two synthetic growth regulators below have shown to be stronger root promoters, light- and temperaturestable, and more resistant to microbial decomposition. • IBA is the best growth regulator for general use because it is not toxic to hardwood and semi-hardwood cuttings over a wide concentration range, and is very effective in the root promotion of a large number of plant species. • NAA is stronger than IBA in terms of stimulation of olive adventitious rooting. However, because uniform results are difficult to obtain with NAA, its use is mainly restricted to species or cultivars which respond unsatisfactorily to IBA. Some commercial preparations are composed of a mixture of IBA and NAA. As for the majority of woody plants, IBA is therefore the best auxin to promote rooting of olive cuttings. Hydro-alcoholic solutions or talc formulations are most commonly used in practising nurseries, as the nursery workers can easily prepare their own rooting treatment at a low cost by buying the IBA as pure product. Alternatively, commercial powder or gel preparations can be used. Using hydro-alcoholic solutions for quick-dip treatments In this method, the cuttings are treated by quick-dipping (5 seconds) their basal parts (0.5–1 cm) in an IBA hydro-alcoholic solution (Fig. 3.15), previously prepared from a

Figure 3.15 Quick-dip hydro-alcoholic IBA treatment. Prior to treatment, cuttings are tied in bunches for easier handling.

36

Olive Propa ga tion

concentrated stock solution (see box ‘Preparing a stock solution and a hydro-alcoholic solution of IBA for quick-dip treatments of olive cuttings’). The cuttings are then inserted in the rooting medium under misting (Fig. 3.16). Cuttings are most efficiently dipped as a bundle, rather than one by one. The cuttings should be inserted into the rooting medium immediately after treatment.

Figure 3.16 Insertion of cuttings into a perlite substrate.

PREPARING A STOCK SOLUTION AND A HYDRO-ALCOHOLIC SOLUTION OF IBA FOR QUICK-DIP TREATMENTS OF OLIVE CUTTINGS Stock solution (e.g. 100 mL at 50 000 ppm in absolute ethanol) • Weigh 5 g of IBA (acid form) and place in a small transparent glass jar with a 100-mL mark. • Add absolute ethanol and stir until the IBA is completely dissolved. • Bring to 100 mL final volume with ethanol. • Put in a dark glass bottle and store at 4°C (stable up to one year). Hydro-alcoholic solution for quick-dip treatment (e.g. 100 mL at 4000 ppm IBA in 30% ethanol) • Take 8 mL of stock solution and place in a beaker. • Add 22 mL of absolute ethanol and 70 mL of water (to avoid a partial auxin precipitation, use distilled or deionised water instead of tap water). • Pour a small amount of the solution into a flat container, to a depth of 1 cm. • Use immediately after preparation then discard the used solution; 100 mL is sufficient for a maximum of 600 cuttings.

In rooting treatments, the concentration of auxin is usually expressed in parts per million (ppm). In hydro-alcoholic treatments, it refers to the weight of the auxin compound per volume of the solution (w/v), e.g. 1000 ppm = 0.1 g of auxin in 100 mL of solution. However, to test the effectiveness of different growth regulators (e.g. IBA vs. NAA) in rooting promotion, equimolar concentrations (M) of the compounds, and not ppm, should be compared. With reference to the period of cutting collection, effective

Propa ga tion by cutting

37

concentrations of IBA in hydro-alcoholic solutions are in the range of 2000 to 4000 ppm: 2000 ppm when natural rooting hormone levels are high (cuttings collected from vigorous vegetative shoots of the stock plant), 4000 ppm when natural rooting hormone levels are low (cuttings collected during the rest period or during bloom). With respect to NAA, treatments with this growth regulator are generally in the range of 1000–2000 ppm. In general, ethanol concentrations in the rooting solution should be not higher than 30%, in order to limit the risks of strong dehydration and injury to the basal cutting tissue. As the quantity of IBA that can be dissolved is related to ethanol concentration, 30% ethanol may not dissolve the large amounts of auxin required to produce concentrated treatment solutions (e.g. over 4000 ppm). Salts of some auxin are available, and they can be simply dissolved in distilled or deionised water. Therefore, when high IBA concentrations are required, the potassium (K+) salt formulation can be used, enabling the preparation of 100% water solutions. However, because of the high cost of the K+-IBA compound, this practice is not common in commercial olive nurseries and talc formulations of auxins are preferred (see below). Using talcum powder formulations In root-promoting talc formulations, auxin is dispersed in the inert talcum powder. Here, concentration of auxin refers to weight per weight (w/w), that is grams of auxin per grams of talc (e.g. 1000 ppm = 0.1 g of auxin in 100 g of talc). Auxin–talcum powder mixtures can be purchased commercially (see p. 39), or prepared by the nursery workers using reagent grade auxin and talcum powder. To get a homogeneous dispersion, the auxin is previously dissolved in a solvent, which is then used to wet the talc (see box ‘Talcum powder formulation’). As with the hydro-alcoholic solution, bundles of cuttings are dipped in the powder with their 1–2 cm basal parts (Fig. 3.17). It may be beneficial to pre-wet cutting bases with water so that the powder adheres better. The cuttings are then lightly tapped to remove excess powder. To avoid brushing off the powder during insertion of cuttings in the rooting medium, a stick can be used to make a 5 mm hole in the medium before each cutting is inserted.

Figure 3.17 IBA treatment of cuttings in talcum powder.

38

Olive Propa ga tion

Talc formulations are advantageous in allowing treatments with high auxin concentrations (10 000 ppm or more), which are very useful when propagation of very difficult-to-root cultivars is pursued (Table 3.1). Another advantage is that talc formulations are easy to use. On the other hand, rooting results are less uniform with talc formulations than with hydro-alcoholic treatments, due to the variability in the amount of powder adhering to the cuttings. TALCUM POWDER FORMULATIONS Preparing 200 g of a 7500 ppm IBA stock formulation • Weigh 1.5 g of IBA (acid form) and put it in a small beaker. • Add diethyl ether and stir until the IBA is completely dissolved (alternatively, absolute ethanol can be used). • Weigh 200 g of pure talc and put it in a large container, so that the powder layer is about 1 cm thick. • Pour the IBA solution into the talc and add more solvent while stirring until all the talcum is wet. • Allow the solvent to evaporate completely (ethanol will require more time to evaporate than diethyl ether). • Stir the formulation until the talc again becomes a free-flowing powder.

Table 3.1 Rooting ability of the most commonly cultivated olive cultivars* High (100–66%)

Medium (66–33%)

Low (33–0%)

Cultivar (country)

Cultivar (country)

Cultivar (country)

Aglandau (France)

Aggezi Shami (Egypt)

Azéradj (Algeria)

Arbequina (Spain)

Azapa (Chile)

Bella di Spagna (Italy)

Ascolana tenera (Italy)

Bardhe i Tirane (Albania)

Bianchera (Italy)

Barnea (Israel)

Bella di Cerignola (Italy)

Biancolilla (Italy)

Bouteillan (France)

Bical Castelo Branco (Portugal)

Büyük Topak Ulak (Turkey)

Coratina (Italy)

Bidh el Hammam (Tunisia)

Carrasquenha (Portugal)

Cailletier (France)

Chemlal (Algeria)

Frantoio (Italy)

Çakir (Turkey)

Chemlali de Sfax (Tunisia)

Gordal de Granada (Spain)

Carrasqueño (Spain)

Domat (Turkey)

Leccino (Italy)

Chalkidiki (Greece)

Empeltre (Spain)

Lechin de Sevilla (Spain)

Chemchali (Tunisia)

Farga (Spain)

Cordovil Castelo Branco (Portugal)

Lucques (France)

Erkence (Turkey)

Gordal Sevillana (Spain)

Manzanilla de Sevilla (Spain)

Galega Vulgar (Portugal)

Leccio del Corno (Italy)

Mission (USA)

Kalamata (Greece)

Lianolia kerkyras (Greece)

Mixan (Albania)

Picholine (France)

Nabali Baladi (Israel)

Moraiolo (Italy)

Picholine marocaine (Morocco)

Nocellara Etnea (Italy)

Nocellara Messinese (Italy)

Picual (Spain)

Ogliarola Messinese (Italy)

Oblica (Croatia)

Sigoise (Algeria)

Oueslati (Tunisia)

Pendolino (Italy)

Taggiasca (Italy)

Salonenque (France)

Verdal (Spain)

Verdale de L’hérault (Spain)

Verdeal Alentejana (Portugal)

*The reported percentages are the average of results obtained by different authors, after IBA treatments (for a more detailed information, see Appendix 1).

Propa ga tion by cutting

39

Commercial preparations A number of commercial rooting preparations are available, differing in formulations and concentrations. Essentially, they contain one or more auxins in an inert matrix (talc or, less frequently, gel or paste, often mixed with lanolin to increase adherence), plus some minor additives (such as fungicides and microelements). These products are formulated to be effective for a large number of species. Usually, complete directions and a list of plants tested come with each product. However, there is very poor information on the activity of commercial preparations in inducing adventitious root formation in olive stem cuttings, as the olive is rarely among the tested species. The only guideline that can be suggested is to carefully check the composition of preparations, in order to choose the one in which the active ingredient concentration (auxin) is in the range indicated above. Dilute auxin solutions Low-concentration treatments have been occasionally used in olive propagation by putting the hormonal solution into the misting equipment, e.g. twice a week in the evening, after the last watering, for at least one month. With this technique, proper IBA concentrations are in the range of 50–200 ppm (Bartolini & Fiorino 1975).

3.2.4 Techniques to improve the effectiveness of auxin treatments Propagation of cuttings from many woody species is positively influenced by the application of some techniques that, in combination with auxin treatments, are able to improve rooting rates, the quality of adventitious roots, or both. Although they have never become routine in commercial olive propagation, worthy of note are the practices of soaking and wounding cuttings before auxin treatments. Soaking the cuttings With immersion of their bases in water (Fig. 3.18), cuttings lose by diffusion a number of natural substances, some of which have an inhibiting effect on the rooting process. At the same time, mechanisms which enable the cutting to root occur more readily. Soaking

Figure 3.18 Trays in which cuttings are left to soak overnight.

40

Olive Propa ga tion

does not alter the annual pattern of rooting response of cuttings, it enhances and stimulates an already existing ability. This effect is shown especially in hard-to-root olive cultivars. The control of water pH during soaking can further improve rooting ability. In the olive, a positive effect on both rooting percentage, and quality of roots has been reported when cuttings were soaked for 24 hours at a pH of 8.5 before treatment with 4000 ppm IBA (Fig. 3.19; Bartolini et al. 1977).

Figure 3.19 Comparison of rooting performance of olive cuttings following soaking in water at pH 8.5 (left) and 7.0 (right).

Wounding Basal wounding is often reported as beneficial to stimulate rooting of difficult-to-root species or cultivars. It consists of making thin longitudinal incisions at the base of cuttings with a sharp knife, before auxin treatment. Wounded cuttings display greater absorption of growth regulators at the base of the cuttings, especially when the quick-dip method is used. Following wounding and auxin treatment, callus and adventitious root production are greater, particularly along the edges of the incisions. With regard to the olive, there is no specific information on the subject. Considering the increase of propagation cost due to this practice, its application is justified in the commercial olive nursery only when rooting of very difficult-to-root cultivars is attempted.

3.2.5 Root promoting compounds, used in combination with auxin treatments Numerous compounds, other than auxins, have been tested over time for their ability to enhance adventitious root formation in olive, mainly in combination with IBA treatments. Although to date none of these substances has entered into the common nursery practice, some promising results are mentioned below.

Propa ga tion by cutting

41

Growth regulators Cytokinins: treatments with 75–150 ppm benzyladenine (BA) to the leaves of ‘Leccino’ and ‘Frantoio’ cuttings, previously quick-dipped in a 4000 ppm IBA solution, led to the increase of both rooting, and bud bursting (Bartolini & Del Ministro 1981). Gibberellins: unlike BA, the application of gibberellic acid (GA3, at 125–250 ppm concentrations) to the leaves of ‘Leccino’ cuttings, previously quick-dipped in a 4000 ppm IBA solution, produced a strong inhibition of both rooting, and bud bursting (Bartolini & Del Ministro 1981). Ethylene: this is a gas which, in plants, acts as a hormone in many physiological processes. In rooting, it has been reported to affect the induction of adventitious roots, as well as the elongation of preformed or latent root initials (Mudge 1988). In the olive (cv Maurino), basal treatments of cuttings with 10 mM 1-aminocyclopropane-1-carboxylic acid (ACC), the direct precursor in ethylene biosynthesis, in combination with IBA (4000 ppm), showed a positive effect on rooting only when cuttings were treated 1 to 3 hours after their preparation (Bartolini et al. 1986a.) Polyamines: it has been reported that polyamines can interfere in different ways with adventitious rooting of several species (Hausman et al. 1997). In particular, putrescine exerts a stimulatory effect on rooting, unlike spermidine and spermine, which seem to inhibit the process. As regards the olive, putrescine was shown to promote adventitious rooting of cuttings, in synergy with IBA treatments (Rugini et al. 1997). Therefore, putrescine could be considered for use with cuttings of olive cultivars which respond poorly to auxin treatments alone. Fungicides With many woody species, treatment of cuttings with fungicides, whether incorporated into the auxin-talcum powder or used alone, has been shown to protect newly formed roots from fungal attack, as well as increase survival and overall quality of the rooted cuttings. It should be noted that the use of cuttings affected by fungi and insects should always be avoided to limit the spread of diseases, and also because cuttings suffering biotic stresses have low rooting capacity.

References Baldini, E. 1986. Arboricoltura generale. CLUEB, Bologna (Italy), pp. 396. Bartolini, G. & Del Ministro, M. 1981. Influenze ed interazioni di fitoregolatori diversi sulla radicazione e sull’accrescimento dell’olivo in vivaio. Riv. Ortoflorofrutt. Ital., 6: 451–462. Bartolini, G. & Fabbri, A. 1982. Effetto dell’ACC (Ciclopropano-ammino-1-carbossilato) sulla radicazione di talee di olivo. Riv. Ortoflorofrutt. Ital., 66 (5): 377–384. Bartolini, G. & Fiorino, P. 1975. La multiplication par bouture de l’olivier avec la technique du brouillard: 1, Influence du nombre de feuilles, de bourgeons et des traitements foliaires sur l’émission des racines. Ann. Ist. Sperim. Olivicultura, vol. 3: 1–16. Bartolini, G., Fabbri, A. & Tattini, M. 1986a. The effects of regulators of ethylene synthesis on rooting of Olea europaea L. cuttings. Acta Hortic. 179: 841–846. Bartolini, G., Fabbri, A. & Tattini, M. 1986b. The effect of some exogenous growth regulators on rhizogenesis in Olea europaea L. cuttings. Olea, 17: 19–21. Bartolini, G., Fabbri, A. & Tattini, M. 1988. Phenolic acids and rhizogenesis in cuttings of ‘Frangivento’ olive. Olea, 19: 73–77. Bartolini, G., Fiorino, P. & Bouzar, M. 1977. Ricerche sull’influenza dell’immersione in acqua delle talee. 3, Effetto della bagnatura a pH diversi sulla capacità rizogena in talee di olivo, cv ‘Frantoio’. Riv. Ortoflorofruttic. Ital., 6: 409–417.

42

Olive Propa ga tion

Battaglini, M. 1967. Les méthodes traditionelles de propagation de l’olivier. Proc. ‘Sem. Oléic. Int. ‘. Spoleto (Italy), November, pp. 28. Bouillenne, R. & Went, F.W. 1933. Recherches experimentales sur la neoformation des racines dans les plantules et les boutures des plantes superieures. Annuel Jardin Botanique Buitenzorg, 43: 25–202. Ciampi, C. & Gellini, R. 1958. Studio anatomico sui rapporti tra struttura e capacità di radicazione in talee di olivo. Nuovo Giorn. Bot. Ital., 65: 417–424. Ciampi, C. & Gellini, R. 1963. Insorgenza e sviluppo delle radici avventizie in Olea europaea L. ; importanza della struttura anatomica agli effetti dello sviluppo delle radichette. Giorn. Bot. Ital., 70: 62–74. Fabbri, A. 1980. Influenza di alcuni caratteri anatomici sulla radicazione di talee di olivo cv ‘Frangivento’. Riv. Ortoflorofrutt. Ital., 64, (4): 325–335. Francolini, F. 1934. Contributo allo studio sulla moltiplicazione degli olivi. L’Italia Agricola, 71(1). Gaspar, T., Kevers, C. & Hausman, J.F. 1997. Indissociable chief factors in the inductive phase of adventitious rooting. In A. Altman & Y. Waisel (eds) Biology of Root Formation. Plenum Press, New York, 376 pp. Hartmann, H.T. 1952. Further studies on the propagation of the olive by cuttings. Proc. Amer. Soc. Hort. Sci., 59: 155–160. Hartmann, H.T., Kester, D.E. & Davies, F.T. 1990. Plant Propagation, Principles and Practices (5th edn) Prentice Hall, New Jersey, pp. 647. Hausman, J.F., Kevers, C., Evers, D. & Gaspar, T. 1997. Conversion of putrescine to 1-aminobutyric acid, an essential pathway for rooot formation by poplar shoots in vitro. In A. Altman & Y. Waisel (eds) Biology of Root Formation. Plenum Press, New York, pp. 133–139. Loussert, R. & Brousse, G. 1978. L’olivier. G. P. Maisonneuve et Larose, Paris, pp. 465. Morettini A. 1972. Olivicoltura (2nd edn). REDA, Rome, pp. 515. Mudge, K.W. 1988. Effect of ethylene on rooting. In T.D. Davis, B.E. Haissig & N. Sankhla (eds) Adventitious Root Formation in Cuttings. Dioscorides Press, Portland (Oregon), pp. 150–161. Pansiot, F. & Rebour, H. 1960. Amélioration de la culture de l’olivier. FAO, Rome, pp. 250. Rugini, E. & Wang, X.S. 1986. Effect of polyamines, 5-azacytidine and growth regulators on rooting in vitro of fruit trees, treated and untreated with Agrobacterium rhizogenes. Proc. Int. Congress of Plant Tissue and Cell Culture. Minnesota, p. 374. Rugini, E., Di Francesco, G., Muganu, M., Astolfi, S. & Caricato, G. 1997. The effect of polyamines and hydrogen peroxide on root formation in olive and the role of polyamines as an early marker for rooting ability. In A. Altman & Y. Waisel (eds) Biology of Root Formation. Plenum Press, New York, pp. 65–73. Troncoso, A., Valderrey, L., Prieto, J. & Linan, J. 1975. Algunas observaciones sobre la capacidad de enraizamiento de variedades de Olea europaea L. bajo tecnicas de nebulizacion I. Anales de Edafologia y Agrobiologia, XXXIV, (7–8): 461–471.

4 Propagation by grafting

4.1 The purposes of grafting In the olive, as well as in many fruit species, the purposes of grafting can be multiple. • Cloning (asexually propagating) genotypes (cultivars, selections, elite trees) that cannot be propagated by cutting or other methods, or that can only be propagated with such means at exceedingly high costs. Rooting ability differs markedly among olive cultivars (see Table 3.1 and Appendix 1): e.g. several cultivars for table olives are very hard to root or they do not root at all, while other techniques (trench layering, stoolbed layering, etc.) are little suited to the olive. This makes grafting the only viable technique for such cultivars. • Using the properties of certain rootstocks. Several fruit species have available a series of rootstocks able to hasten growth, anticipate onset of production, control plant vigour and improve fruit quality, but research has not produced anything comparable for the olive. • Using the properties of certain interstocks. However, this possibility has not yet been exploited with the olive. • Changing cultivar on established plants (topworking). Differently from other fruit species, the olive does not suffer from sudden changes in the consumer taste that can make a cultivar obsolete in a few years. But topworking can sometimes be necessary to replace a cultivar, at least in part, e.g. when a pollinator appears necessary in a grove where the planting of a pollinating cultivar has not been made, or the pollinating cultivar proves to be unsuitable to the task (because of a lack of overlapping at flowering times, poor interfertility, etc.). • Repairing damaged parts of trees. Occasionally the roots, the trunk, or large limbs of trees are severely damaged by winter injury, cultivation implements, rodents or

44

Olive Propa ga tion

diseases. Bridge grafting and inarching (Fig. 4.1) are techniques that can repair such damage and save the tree. • Indexing of virus diseases. These diseases are transmitted by grafting, i.e. an infected scion may infect healthy rootstock. Some cultivars display evident and peculiar symptoms of the infection, and are called ‘indicators’. It is therefore possible to test a genotype for the presence of a given virus by grafting it on a seedling of an indicator plant. As the first virus diseases of the olive have been recently described, it is possible that this technique will also gain importance in the near future for this species. • Research purposes. Grafting is a useful tool for studying a series of physiological aspects of olive biology, but they cannot even be outlined here.

Figure 4.1 Multiple inarching of a damaged olive tree: situation after five years (Wuhan, China).

Propa ga tion by grafting

45

On the other hand, grafting has some disadvantages: • The ‘seed to orchard’ procedure is far more lengthy and laborious than the ‘cutting to orchard’ one; hence, grafting propagation is far more expensive than cutting propagation; • Seedlings used as rootstocks are a genetically varied population of individuals, and their effects on the grafted cultivar may likewise be varied. This drawback can be overcome to some extent in the nursery by sorting out the seedlings. It can also be remedied after planting by inducing the scions to root and therefore obtaining trees on their own roots. This procedure requires planting by placing the grafting point below the soil level. However, this practice is uncommon in modern olive culture. • In many olive cultivars the germination of healthy and perfect seeds may be heavily delayed, as they must overcome a strong double dormancy (see 4.2.1); in addition to this, the germination percentages of several cultivars are very low. • Grafting is a complex operation which requires long practice. As a consequence, the production of grafted trees is usually restricted to specialised nurseries, where skilled labour is present. Grafting of fruit species can be carried out in many ways (see box ‘Terminology of grafting propagation’), and therefore the number of grafting procedures described in the scientific literature is countless. Not all of them have been adopted internationally, and only the most common procedure used in Mediterranean olive nurseries is described here.

TERMINOLOGY OF GRAFTING PROPAGATION Grafting means to put in contact two portions of tissue or two organs coming from two different trees, in such a way that they will heal and subsequently grow as one composite woody plant.The upper or distal part of this union (the scion) is a short (even just one node) piece of shoot carrying a number of dormant buds. After the healing of the graft union, it will develop into the canopy of the new plant. The plant from which the scion is obtained (the stock plant) should be tested for both its genetic response to the desired cultivar, and its good sanitary condition.The lower or basal part of the graft (the rootstock) will develop into the root system of the new tree. In the olive, it may be a seedling, a rooted cutting, a rooted sucker or ovule, or a micropropagated plant. With the exception of the seedling, propagation techniques give rise to clonal rootstocks, uniform and constant in their characteristics. A particular form of grafting is topworking, in which the rootstock may extend to the whole trunk and to the main scaffold branches (see 4.3.4). Budding is a form of grafting, where the scion is a small portion of tissue carrying one bud. Budding can be undertaken on olives, but it is not the favourite method for propagation.The callus is a mass of parenchyma cells, produced by the meristematic cells of the cambium (the regenerative tissue located between the bark and the wood) or by dedifferentiated cells, usually around wounds. In grafts, callus tissue proliferates to fill all empty spaces between rootstock and scion, determining the healing of the graft point.

46

Olive Propa ga tion

4.2 Production of olive seedlings Seed propagation in the olive is undertaken mainly for the purpose of producing seedlings to be used as rootstocks for olive trees. The advantage of seed propagation lies mostly in the possibility of producing a very large number of high-quality virus-free rootstocks. Moreover, the seeds are cheap and the production of seedlings can be carried out with little skill and equipment. One important drawback of rootstock production by seed is that the seedlings are heterogeneous in terms of vigour and root development, hence influencing growth characteristics of grafted plants which can differ quite markedly. A proper handling of olive seeds – from fruit collection up to seed germination and seedling development – is fundamental to produce fast-growing homogeneous rootstocks to be used in grafting propagation. The method of grafting scions on seedlings is still largely used in Italy where, in the highly specialised nurseries of Pescia (Pistoia province, Italy), the large numbers of plants required for establishing the new industrial olive plantations have been produced over the last half century. This would not have been possible if only branch cuttings and ovules had been available. The method is also used widely in Argentina, where it has allowed a rapid diffusion of the species. On the other hand, nurseries of other important olive-growing countries such as Spain almost exclusively use stem cuttings for olive propagation.

4.2.1 Stone collection and quality of olive seeds The use of high-quality seed is of prime importance for rootstock production, whether growers collect or produce the seed themselves or obtain it from others. As described above (see 2.2.5), what is commonly called ‘olive seed’ is actually a stone, i.e. the ‘true’ olive seed is enclosed in the fruit stony endocarp (the pit). Olive stones can be considered of good quality when (i) they are genetically true-to-type, and represent the desired olive cultivar or provenance, (ii) they are clean and free from disease and insects, and (iii) the embryos that they contain are viable and capable of high germination. Both wild olive trees (oleasters) and cultivars have been used in the past as sources of stones. A greater percentage of germination and more vigorous seedlings are obtained from the former. On the other hand, seedlings from wild olive stones are often non-cold-resistant, highly heterogeneous in growth, and have few tap roots which are invariably damaged during transplantation, so that rapid recovery is not possible. Moreover, derived seedling rootstocks have very thin bark and very short internodes, making grafting difficult. For these reasons, stones of cultivars are now preferred for propagation in olive nurseries. Olive seeds, both from wild trees and cultivars, are always affected by seed dormancy, for which embryo germinability immediately after fruit ripening is almost nil. Olive seed dormancy In the olive, when the fruit are ready for harvesting, embryo germinability is impeded by a double form of primary dormancy (see box ‘Seed dormancy’): a ‘mechanical dormancy’ due to the stony endocarp of the fruit which does not allow embryo imbibition and expansion (seed-coat dormancy), and a ‘chemical dormancy’ induced by some

Propa ga tion by grafting

47

germination inhibitors, i.e. naturally occurring chemicals which are presumably located in the endosperm and in its cover (testa). Experimental trials have shown that embryos excised from their seed coats and cultivated in vitro initiate promptly to germinate (Lagarda et al. 1983a; Acebedo et al. 1997), showing that the embryo by itself is not affected by any forms of dormancy. The seeds of many cultivars require up to 4 years for a complete removal of seed dormancy (Voyatzis & Pritsa 1994). Indeed, the seeds from just harvested fruits show nil or very low germinability (see box ‘Germinability’). During their storage, there is a progressive, although slow, natural overcoming of dormancy, so that the germinability increases and reaches a maximum, depending on the cultivar, between the 2nd and 4th year. Then, it gradually decreases in the following years. Further significant, though not generalisable, information on olive seed dormancy and germinability is available, among which (i) a 100% germinability of ‘Arbequina’ is obtained after the seeds are removed from the stony endocarps and maintained in tap water for 30 days (Sotomayor León & Durant Altisent 1994), (ii) the overcoming of dormancy can be promoted by the exposure of seeds to constant temperatures, e.g. 13°C for ‘Picholine’ (Instanbouli & Neville 1977), 15°C for ‘Manzanilla’ seeds (Lagarda et al. 1983b), 10°C (3–4 weeks) followed by 20°C for ‘Chondrolia Chalkidikis’ (Voyatzis 1995). Sources of stones and quality of seeds The best sources of stones for seed propagation are trees of specific cultivars, selected from olive orchards with plants in good conditions of productivity. When possible, it is advisable to select plants of the same cultivar in the inner part of the orchard, in order to reduce cross-pollination as much as possible and, therefore, heterogeneity of seedlings. Once trees producing good quality seeds have been located, it is recommended that the stones are collected every year from the same plants. The choice of cultivars used as sources of stones for seedling rootstock production generally results from the personal

SEED DORMANCY A seed separated from the plant may display primary dormancy, which prevents immediate germination and to various extent delays germination, until given environmental events have occurred. In nature, different kinds of primary dormancy have evolved, two of them concerning olive seeds: • ‘mechanical dormancy’ (or ‘seed coat dormancy’) depends on the presence of seed coats which exclude water and air penetration, and are too strong to allow embryo expansion during germination. Note that in the olive the term ‘seed coats’ refers to both the coats which cover the embryo (testa and endosperm), and the seed enclosing structure (the pit, formed by the stony fruit endocarp). Impermeability and hardiness of seed coats are due to a layer of macrosclereid cells, especially thick-walled on their outer surfaces and coated with a layer of waxy, cuticular substances. Hard or impermeable seed coats, which in nature are softened by the action of microorganisms in the soil, can be mechanically abraded or broken, or softened by means of treatments with acid or basic compounds. • ‘chemical dormancy’, due to chemical compounds which accumulate in embryo coverings (endosperm, inner seed coats) during the development of the seed and act as germination inhibitors, even for a long time after harvesting. Excising the embryo from the seed covering can overcome chemical dormancy.

48

Olive Propa ga tion

experience of nursery operators, who have acquired, with time, information on seed germinability and superior characteristics of derived seedlings in terms of vigour, quality of root apparatus, diameter reached at grafting time, and tolerance to biotic and abiotic stresses. Examples of cultivars used as stone sources are ‘Canino’, ‘Mignolo’, ‘Maurino’, ‘Moraiolo’, ‘Frangivento’ and ‘Americano’ in Italy; ‘Arbequina’ and ‘Verdal’ in Spain; ‘Allegra’ and ‘Oblonga’ in the USA; and ‘Frantoio’ in Australia. A limited use of stones from wild olive trees is still sporadically made in Italy and Spain. Olive nursery practice has repeatedly shown that different results, in terms of seedling production, are obtained using seeds from small- or large-fruited cultivars. As a rule, there is a strict correlation between fruit and stone size (Table 4.1). Seedlings derived from ‘small’ stones have a greater proportion of taproots than those grown from ‘large’ stones, whose root systems are more reduced and branched. As it is considered that seedlings derived from ‘large’ stones are more precocious and can be grafted 10 to 15 days earlier, it should follow that these seeds have generally better characteristics and their use should be preferable. On the other hand, small stones, although of lower productivity, display a higher and more rapid germination. As a consequence, nursery operators prefer to use small-fruited cultivars (such as those listed in Table 4.1) as donor plants. This orientation is also favoured by the lower costs of small stones. GERMINABILITY Germinability refers to the quickness and the vigour with which embryos start to germinate when the seeds are placed in proper environmental conditions. Several parameters have been proposed to express seed germinability, among which the following are the most used: • the germination percentage (or germination capacity), which indicates the percentage of seeds which are able to germinate and to produce seedlings within a specified length of time. • the germination rate (or average time of germination), which expresses the average number of days required for seed germination. This can be calculated as follows: GR (days) =

N1T1 + N2T2 + … + NnTn total number of seeds germinating

where N values are the numbers of seeds germinating within consecutive intervals of time, and T values indicate the days between the beginning of the test and the end of each subsequent interval of time. Although these two parameters give a good indication of seed quality, growth rate and the morphological appearance of seedlings must also be taken into consideration.

Germination percentages are quite variable, according to cultivar, technique, environment and year. As regards this aspect, research is quite scarce and, in addition, individual experiences (e.g. Scaramuzzi 1957, 1958; Guerrero 1997) are seldom comparable, just like nursery operators’ claims. As a rule, olive seeds are credited with poor germinability, although it varies greatly among cultivars (ranging, with a large approximation, between 5% to 60%). Low germination percentages are mainly due to embryo abortion and mechanical/chemical dormancy of embryos; these latter problems can be overcome by means of techniques such as soaking, scarification and stratification of stones (see 4.2.2).

Propa ga tion by grafting

49

Table 4.1 Classification of fruit and stones of olive by their size Fruit/stone size

Fruit weight (g)

Fruits per kg of fruits (n°)

Stone weight (g)

Stones per kg of stones (n°)

Small *

500

2000

Medium **

2–4

250–500

0. 5–1. 0

1000–2000

Large ***

>4

1. 0