Somatic Embryogenesis for Micropropagation of Coconut (Cocos nucifera L.)

By I.D. Anton, PhD

This is a publication on somatic embryogenesis of coconut. Somatic embryogenesis is a fascinating natural phenomenon where the embryo is formed from a single vegetative (somatic) cell instead of gametic fusion. Under specific conditions this process can occur in vitro and it can be used for cloning purposes. Coconut cloning or micropropagation has been explored for 40+ years with little success, as coconut is very recalcitrant to somatic embryogenesis. This book (PhD thesis) represents exciting research on the issue, which achieved more than three times higher overall efficiency than the previous protocol.

• Language: English
• Publisher: I.D. Anton, PhD
• Release date: Dec 24, 2014
• ISBN: 9781311467294

Book preview

Somatic embryogenesis for micropropagation of coconut (Cocos nucifera L.)

By I. D. Anton, PhD

Copyright 2014 I.D. Anton

Smashwords Edition

Statements by the author

This book is a PhD thesis (ebook version) and consists of my original work, both in research and writing, undertaken during my Doctor of Phylosophy candidature at The University of Queensland, Australia. This thesis does not have any content previously published by another person, except where stated otherwise and referenced accordingly here. To fulfill format requirements for ebook publishing some formatting adjustments were applied to my original PhD thesis manuscript as submitted to The University of Queensland.

Copyright of all material presented in my thesis resides with the copyright holder (author) of the material (real name: Irina D. Antonova, pen name: I.D. Anton).

For this work I acknowledge and express gratitude in appreciation of the expertise, contribution, guidance, patience and encouragement, as well as of the financial and logistic support of the following people and institutions:

Prof. Stephen Adkins (Principal Advisor, The University of Queensland /UQ)

Dr. Yohannes Samosir (Associate Advisor, UQ)

Dr. Sisunandar (Coconut Consultant to this project, Indonesia)

Dr. Erlinda Rillo (Coconut Consultant to this project, The Philippines)

Mr. Michael Foale (Coconut Consultant to this project, Australia)

Integrated Seed Research Unit /ISRU, SLCAFS, UQ

School of Land, Crop and Food Sciences / SLCAFS, UQ

University of Queensland Joint Research Scholarship Scheme

Australian Centre for International Agricultural Research /ACIAR

Indonesian Coconut and Other Palmae Research Institute /ICOPRI

Philippine Coconut Authority (PCA)

Family and acquaintances

Smashwords Edition, License Notes

This ebook is licensed for your personal enjoyment only. This ebook may not be re-sold or given away to other people. If you would like to share this book with another person, please purchase an additional copy for each recipient. If you’re reading this book and did not purchase it, or it was not purchased for your use only, then please return to your favorite ebook retailer and purchase your own copy. Thank you for respecting the hard work of this author.

TABLE OF CONTENTS

Somatic embryogenesis for micropropagation of coconut (Cocos nucifera L.)

Statements by the author

Abstract

Chapter 1: INTRODUCTION

Chapter 2: LITERATURE REVIEW

Chapter 3: GENERAL MATERIALS AND METHODS

Chapter 4: STARTING PROTOCOL

Chapter 5: OPTIMIZING THE PRODUCTION OF EMBRYOGENIC CALLUS

Chapter 6: INCREASING THE RATE OF SOMATIC EMBRYO FORMATION

Chapter 7: IMPROVING EMBRYO MATURATION AND GERMINATION

Chapter 8: INCREASING THE REGENERATION RATE OF PLANTLETS

Chapter 9: GENERAL DISCUSSION

BIBLIOGRAPHY

APPENDICES

About the author

Abstract

Coconut (Cocos nucifera L.) is native to the regions between 20 degrees North and 20 degrees South of the Equator, where it plays a significant socioeconomic role in the local communities. There it is referred to as ’The Tree of Life’, a eulogistic epithet describing its versatile use - more than 100 edible and non-edible products can be produced from it. Therefore, the coconut palm is grown in about 90 tropical countries on more than 10 million ha of land (Hamon et al., 1999).

Although coconut has a high local socioeconomic reputation, its production is experiencing many problems and consequently the area planted with this crop is declining. The conventional breeding approach using seed to replant land is very expensive due to the low production of seed for planting, and even when elite germplasm is available it takes decades to multiply up enough planting material for new areas (Adkins et al., 1999).

Hence over the past 40 years research has been directed towards developing a new technique for the micropropagation of coconut using somatic embryogenic approach. Throughout this time However, one conclusion is repeatedly made – coconut is very recalcitrant to somatic embryogenesis. And although the many obstacles to this are slowly being reduced, in order to successfully micropropagate coconut on a large scale bottlenecks in the protocol still exist, and those include inconsistency of the embryogenic response by explanted tissues, poor somatic embryo maturation and germination, low regeneration rate of the new plantlets and long time required to produce plants (1.5 years) (Samosir et al., 1998).

These bottlenecks and other problems were researched in the present study with the aim of trying to speed up the efficiency of coconut somatic embryogenesis process. Hence this thesis had the objectives to identify a starting protocol for coconut somatic embryogenesis; to select an appropriate for that aim explant; to optimize the production of embryogenic callus; to increase the rate of initiating coconut somatic embryos; to improve the maturation of somatic embryos and their germination efficiency; and to optimize the regeneration rate of the new plantlets. In order to identify a starting protocol, preliminary work was conducted, where existing protocols for coconut somatic embryogenesis were compared in their efficiency to induce somatic embryos. The protocol that stood out as the best in producing most embryogenic callus and subsequently embryos, as well as having the least dead (in culture) explants, was that of Nikmatullah (2001). Therefore,, the latter was chosen to be used as a starting protocol for this study.

New sources of explants were investigated during the current work as well, using tissues from different parts of in vitro derived 8 months old coconut plantlets. Those However, have shown to be unsuitable for somatic embryogenesis, since only non-embryogenic callus was developed by some of the inoculated tissues. The immature inflorescence explants were superior in producing embryogenic callus and somatic embryos; Therefore, they were selected as the preferred explant source to use in the next steps of the current study.

Optimizing the production of embryogenic callus was the first issue to address during the core work of this project. As a result of that the culture conditions were considerably improved by using vessels with larger headspace-medium ratio (3:1), as well as by selecting younger immature inflorescences and transversely segmenting the top half of the inflorescence spikes into smaller size (1 mm) sections. Further improvement was possible by studying the make up of the callus growth media. Amongst the administered for that purpose substances the applied together polyamines spermine (0.10 µM) and putrescine (7.5 mM) have proven to play a notably positive role in the induction of callus from coconut immature inflorescence explants. Thidiazuron (TDZ, 10 µM) too has shown a potential to improve the efficiency of the initial stage of coconut somatic embryogenesis, but only when applied in conjunction with other cytokinins (eg. BAP and 2iP). Smoke-saturated-water (SSW, 10 %) could only slightly diminish the amount of necrotising cultured explants, and high 2,4-D concentrations could not support the induction of callus from immature coconut inflorescences. Collectively taken, as a result of this current study the production of callus was improved by 300 %.

The rate of coconut somatic embryos formation was as well significantly increased (over 300 %), by the simultaneous application of suspension culture step, spermine (0.01 µM), SSW (10 %) and high auxin concentration (500 µM). Nevertheless the presence of TDZ and other cytokinins in the medium, as well as the absence of activated charcoal, were found to be unable to positively influence the somatic embryogenesis process.

Despite the considerable improvements made in the efficiency of inducing callus and initiating embryos, the poor maturation and germination (eg. 5 %, Verdeil et. al., 1999) of somatic embryos still remained a bottleneck to the whole somatic embryogenesis procedure. Therefore, further work was conducted in that direction and discovered that embryo maturation and germination rate can be elevated to 55 % by administering ancymidol (30 µM) to the somatic embryo maturation medium. This plant retardant has exhibited here three potential modes of action towards the cultured coconut somatic embryos: a) as a promoter of somatic embryo maturation and germination; b) as a preventor of pre-germination death of the somatic embryos; and c) as a preserver of non-germinating somatic embryos, that still can possess the potential to germinate in the future.

The work during the next step of the process – regeneration of the new plantlets – has shown that the omission of plant growth regulators from the media was crucial for the development of germinated embryos into new plantlets, where otherwise no plant regeneration occurred at all. The achieved here plantlet regeneration rate in the PGR-free medim was 56 %, which is higher than the previously reported 20 % regeneration rate (Verdeil et al., 1994) for coconut plantlets produced from immature inflorescences explants.

As a result of this current work a new method was developed for somatic embryogenesis of coconut from immature inflorescences explants (Fig. 9.2). The overall efficiency of this protocol is over three times higher than that of the starting protocol (Nikmatullah, 2001) selected during the preliminary work. Furthermore, when using this new method the entire duration for regenerating clonal coconut plantlets (up to the stage of first root and shoot emerging) takes up to 8 months, which is the shortest reported time for producing coconut plantlets via somatic embryogenesis (eg. 36 months from inflorescences explants (Verdeil et. al., 1999) and 18 months from sliced zygotic explants (Samosir, 1999, Fig. 9.2), presenting an additional valuable advantage of this newly developed method, from the perspective of the potential to micropropagate coconut on a commercial scale.

Keywords

coconut, somatic embryogenesis, micropropagation, polyamines, spermine, putrescine, ancymidol, smoke-saturated-water, thidiazuron

Chapter 1: INTRODUCTION

The coconut palm (Cocos nucifera L.) has high significance as a crop in the tropical regions, where it is grown in about 90 countries, on more than 10 million ha of land, providing income for more than 5 million smallholder farmers, while supplementing the food, drink and shelter supplies for millions more inhabitants (Hamon et al., 1999). Beside this, the coconut palm is regarded as an environmentally friendly tree, since it is able to grow in places where many other forms of plant will not (ie. by the sea), acting as a buffer to high winds, and a binder of unstable land on the coastal strand. In addition, the mechanical strength and resilience of the coconut palm trunk has given it the capacity to survive and recover from the assault of hurricanes and tidal waves (Persley, 1992).

Although the coconut palm has a high socioeconomic reputation, it is known to experience many problems leading to production decline, which has taken effect over the last 30 years (Hocher et al., 1999). This is due not only to the many pests and diseases that are found to attack the coconut palm in many regions, but also because many plantations are more than 70 years old. The natural yield decline with age is not being arrested with sufficient new plantings. Furthermore, the coconut palm is propagated only by seed, and its notorious heterozygosity (due to cross-pollination) makes genetic improvement through conventional breeding very slow and expensive (Blake et al., 1995). On the other hand, implementing a conventional breeding approach is very difficult, time consuming and expensive, due of its long juvenile phase (5 to 8 years) and low rate of seed production (50 to 100 seeds per year). Even when elite germplasm is available, the low multiplication index dictates a delay of many years in order to multiply up new planting materials on a commercial scale (Adkins et al., 1999).

Therefore,, traditional coconut breeding approach needs to be integrated with a biotechnological approach, such as in vitro micropropagation, in order to rapidly produce elite planting materials. Many scientists in various research institutes have explored clonal propagation via somatic embryogenesis in coconut, because of its advantage in offering the production of true-to-type plants in a rapid fashion. In addition this approach can lay a route for future genetic engineering studies on coconut. From the two major explant tissue types commonly used for somatic embryogenesis (zygotic and somatic tissues), zygotic tissue has been more successful. However,, the disadvantage of using this tissue type is that the explants coming from it are heterozygous in nature, which means true-to-type plants are very hard to produce (Hornung et al., 1999).

Somatic tissues do not exhibit the above described problem and Therefore, tissues from coconut palm inflorescences, leaves, stems or roots have been examined for their ability to form somatic embryos. Out of all these tissues the inflorescence often proves to be the most useful (Branton et al., 1983a; Cueto et al., 1997; Dussert et al., 1995b; Eeuwens et al., 1978; Hornung et al., 1999; Magnaval et al., 1997; Nikmatullah, 2001; Sugimura et al., 1989; Verdeil et al., 2001). These authors state the following reasons for this: the performance of the mother tree is already known; the removal of the explant allows the tree to survive (Rillo, 1989); the protocols are simpler and shorter to undertake than those for zygotic tissues (Hocher et al., 1999); the inflorescence explants were the explant tissue from which somatic embryogenesis of coconut was first achieved (Branton et al., 1983b); and most importantly, the regenerated plants from inflorescences explants are likely to be true-to-type. It is for these reasons that immature inflorescence explants were used in this current study.

The research issues that were addressed in this project are related to the improved clonal propagation of coconut by further optimising the process of coconut somatic embryogenesis. Although it is possible to undertake somatic embryogenesis, in order to successfully micropropagate coconut at a larger scale, practical application of the approach required that all stages of the process be improved. These stages and their associated problems include:

Stage 1 (callus induction and proliferation):

- Slow in vitro response

- Low caulogenic ability

- Intensive browning of the in vitro tissues

Stage 2 (somatic embryo initiation, maturation and germination):

- Low embryogenic capacity

- Poor em bryo maturation and germination

Stage 3 (plantlet regeneration and acclimatization):

- Low regeneration rate of the new plantlets

Therefore, the overall goal of this research was to ameliorate the efficiency of coconut somatic embryogenesis to a level (or as close as possible) that could allow for the commercial micropropagation of coconut. That goal was to be achieved by adopting the following objectives:

a) to identify a starting protocol and sources of explant for this work (Chapter 4)

b) to optimize callus production (Chapter 5);

e) to increase the rate of initiation of somatic embryos (Chapter 6);

f) to improve the maturation and germination of the somatic embryos (Chapter 7); and

g) to increase the regeneration rate of the new plantlets (Chapter 8)

h) to shorten the time for the entire process as much as possible (Chapters 4 to 8).

Chapter 2: LITERATURE REVIEW

2.1. COCONUT (Cocos nucifera L.)

It is likely that people have been making use of the coconut palm for more than half a million years, although it is very difficult to establish precisely, since there is very little archaeological evidence, much of which is likely to have been lost through sea level rises (Foale, 2003).

Existing evidence that coconuts were being used as early as 1200 years ago comes from 8th Century AD engraved slab, carrying the words of King Jayanasa of Indonesia: All the trees to be planted in this park – coconuts, arecanuts, sugar palm, sago palm – their fruits can be had by the people. At about the same time as this King Aggabodhi of Sri Lanka was also setting up a coconut garden according to the local literary evidence (Punchihewa, 1999).

Today the story of coconut and its presence around the world – from Asia to the Americas - is one in which evolution, immigration, trade, other cultural practices and the forces of nature all have played a part (Foale, 2003).

’The Tree of Life’, as the coconut palm is often referred to in its native tropical regions (between 20 degrees North and 20 degrees South of the Equator), is a very descriptive and well deserved eulogistic epithet, describing the palms very versatile use - almost every part of the tree is useful with more than 100 edible and non-edible products being made, directly or indirectly, from the coconut palm (Persley, 1992).

In the tropical regions the Tree of Life provides food, drink and work for many, and for the people of the temperate zone the coconut palm symbolises dreamy tropical relaxation on magnificent beaches. The palm is Therefore, often used to promote romantic, indulgent holidays with enjoyable tropical sun and azure sea waters (Foale, 2003).

2.1.1. Biology

Cocos nucifera L. or the coconut palm, belongs to the family Arecaceae, and it is included under the group of flowering plants of the monocotyledons. It occupies a conspicuous position in the plant kingdom having the typical features that are characteristics of most palms, such as a comparatively slow growth rate; absence of a tap root; an unusual stem thickening at the base of the trunk (known as the ‘bole’); an unbranched pillar-like stem; a continually growing terminal bud (known as a ‘cabbage’); a branched inflorescence which is enclosed in a sheath (known as a spadix); and a compact, crown of feathery, thick-cuticuled leaves, rendering natural beauty and elegance (Menon et al., 1958).

The roots. The amount and spread of the coconut palm root system depends on variety and the adequacy of soil drainage. Having no cambium, the roots reach a maximum diameter of 1 cm and are usually uniform in thickness. All bear many slender branches but there are no fine root hairs. The main roots are situated mostly in the top 90 cm of the soil, but some may go as deep as 9 m. The roots grow horizontally out of the bole of the tree, a feature that is most conspicuous in the Tall varieties. The coconut palm has no tap root, but adventitious roots are continually produced from an inverted dome within the soil that forms the basal 30-60 cm of the stem (Child, 1964).

The stem. Coconut palms can reach 30 to 40 m in height (Foale, 2003). The distinctively unbranched trunk develops from a single vegetative bud. This bud, situated at the apex of the stem, is the only vegetative bud formed on the whole tree (Sampson, 1923). The trunk begins to form usually after 5 years of growth (less in the Dwarf palm). Once formed, the stem of the coconut reduces very slightly in thickness as it ages (Child, 1964).

The stem has no designated area where the sap ascends or descends. It contains no cambial tissue, hence it is unable to repair damage to the stem. However,, due to the large number of stout conducting strands embedded in soft tissue running from one side of the trunk to the other, the tree can thrive even after receiving serious injury to its stem. The coconut stem is incapable of forming branches and Therefore, the leaves are attached directly to the stem (Sampson, 1923).

The crown. In adult trees the crown comprises of up to 40 open leaves (fronds). The same number, or up to one and a half times more, are present as unopened leaves inside the vegetative ’cabbage’ bud, which produces new leaves in succession. The young leaves appear in the centre of the crown as upward pointing arrows. As the leaves get older they bend down, and