Fernando, S.C.

Coconut Research Institute Repository

Clonal Propagation of Coconut: Prospects and Limitations S. C. Fernando Tissue Culture Division, Coconut Research Institute, Lunuwila, Sri Lanka ABSTRACT Coconut (Cocos nucifera L.) is the most important palm in the tropics. Introduction o f new varieties and hybrids with superior qualities is essential for crop improvement. However, achieving this goal by conventional means is very slow. Tissue culture is the only tool available for speeding up the process. This paper summarizes the current status, limiting factors in developing a viable clonal propagation method for coconut, possible measures to overcome limitations and future directions. The research shows that clonal propagation of coconut is possible through somatic embryogenesis. However, some major constraints that need to be overcome are heterogeneous responses due to the influence o f explant maturity and genotype, undefined culture conditions possibly caused by activated charcoal influencing the hormonal composition of the culture medium, production o f highly heterogeneous and compact calloids due to partial de-differentiation o f cells o f explants, poor efficiency in plant regeneration due to marked influence o f genotype and variable culture conditions and slow growth o f tissue-cultured plants under in vitro and early ex vitro conditions. Selection o f the most responsive explant, standardization of activated charcoal used and repeated multiplication o f embryogenic cell masses are some of the measures currently applied to overcome the limitations. Research towards selection of mother palms amenable to tissue culture, use o f AGPs to increase the number o f responsive mother palms and genetic manipulation of elite palms with regenerable genes are seen as the future directions in clonal propagation o f coconut. Key words: Coconut, clona propagation, AGP (Arabinogalatan proteeins) INTRODUCTION Coconut palm, the "Tree o f L i f e" , is an important oil crop in the tropics. There is a gradual decline in coconut production over the years due to constraints such as, maintaining senile plantations with low productivity, declined soil fertility, use o f unimproved planting material, marginal cultural practices, spread o f pests and diseases and increasing competit ion from other oil crops. However , coconut is still e c o n o m i c a l l y important for Sr i L a n k a and the industrial demand for lauric oil is high. Therefore, introduction o f new variet ies and hybrids with superior qualities is essential for the improvement o f the industry. Conventional breeding programmes for genetic improvement o f coconut are slow due to limitations attributed to the bulkiness, perennial nature o f the pa lm and its b io logy . T h u s the development o f a reliable clonal propagation method o f coconut to assist breeder is excit ing. Although research leading to a method o f clonal propagation in coconut was initiated in many countries in the early 1 9 7 0 ' s , an economica l ly viable protocol has not yet been developed. This paper summarizes the Sri Lankan experience on the current progress, limiting factors and possible measures to overcome the limitations and future directions towards the development o f a viable clonal propagation method for coconut . 322 Current status of coconut clonal propagation Since the inception o f research in coconut tissue culture at Wye Col lege , U K in the early 1 9 7 0 ' s , the response o f various somat ic tissues (tender leaf, immature inflorescence, root and shoot) and zygotic tissues (immature and mature zygotic embryos and plumule) to in vitro culture has been reported in many other countries worldwide (summarized in Femando , ,2001) . These indicated the possibility o f c l o n i n g c o c o n u t t h r o u g h s e v e r a l s t e p s i . e . , ca l logene.s is , s o m a t i c e m b r y o g e n e s i s , e m b r y o maturation and germination. The regenerated plants, though low in number , were sui table for field establishment. Up to now, hundred coconut palms regenerated in the Tissue Culture Divis ion o f the Coconut Research Institute o f Sri Lanka have been field established. T h e s e palms at different stages o f development including some o f which are already bearing nuts. A study using microsatel l i te markers showed tnat these c lones are genetically stable with field pe r fo rmance c o m p a r a b l e to seed-der ived palms (Fernando et al., 2 0 0 4 ) . Factors limiting the development of a clonal propagation protocol for coconut Callogenesis Heterogeneous response of explants The embryogenic potential o f coconut l ea f explants is very low ( < 10 % ) . Only explants derived from young leaves o f 10 -20 cm length from 12-24 month-o l d s e e d l i n g s r e s p o n d e d . F u r t h e r m o r e , the embryogenic potential o f these explants was o f short duration. T h i s l imi ts the use o f coconu t l e a f for c lonal propagat ion (Karunaratne et al., 1991) . Immature inf lorescence is a promising explant as it b e a r s n u m e r o u s m e r i s t e m a t i c p o i n t s . T h e embryogenic potential^ depends on the maturity s tage o f the exp l an t . H o w e v e r , the l a ck o f a marker greatly limits the ability to select the correct maturity s tage . T h e h ighes t ca l lu s ing reported so far i'is 3 0 % (Vidhanaa rachch i and Weerako on, 1997) . CLONAL PROPAGATION OF COCONUT Zygot ic tissues such as immature zygotic embryo and plumule (excised from mature zygotic embryo) hold potential for micropropagation. Unlike many somatic tissues ( inf lorescence and lea f tissues), the response o f zygo t ic t issues is more consis tent . H o w e v e r , the c a l l u s i n g f r e q u e n c y d e p e n d s on embryo developmental stage. Under optimum condi t ions , ca l lus ing is usual ly above 75 % in i m m a t u r e z y g o t i c e m b r y o ( D i y a s e n a , 1 9 9 8 ) and 6 0 % in plumule explants (Weerakoon, 2 0 0 7 ) . R e c e n t r e s e a r c h u n v e i l e d the high po ten t i a l o f unfer t i l i zed ova ry as an initial explant for c l o n i n g . T h e h i g h e s t f r e q u e n c y o f c a l l u s i n g reported from unfertilized ovary is 7 6 % (Perera et al, 2 0 0 7 ) . Somat ic tissues are the ideal explants for clonal propagation as the performance o f mother palm is known. However , the response o f these t issues ( excep t unfer t i l ized ovar i e s ) is genera l ly poor. Moreover , there is a marked difference among explants co l lec ted from different mother palms in terms o f cal lus initiation. This might be due to differences in genotype, explant maturity or mother palm age. Effect of activated charcoal Activated charcoal is an essential component in c o c o n u t t i s sue cu l tu re m e d i u m . It has s t rong adsorptive properties and its beneficial effects are attributed to the adsorption o f phenols and other growth inhibitory substances. However, a major disadvantage o f using activated charcoal is that it also can adsorb plant growth regulators (hormones and vi tamins) and some micro elements (Cu and Zn) (Pan and Staden, 1 9 9 8 ) . This creates undefined culture conditions which could lead to variable tissue response and non-reproducible results. Different types/ brands o f charcoal have different adsorptive c a p a c i t i e s b a s e d on thei r or ig in , age , s to rage condit ions and method o f activation. This has a significant effect on callus development even from zygotic tissues that give high yields. A charcoal-323 SECOND SYMPOSIUM ON PLANTATION CROP RESEARCH free medium was developed with the addition o f polyvinyl pyralidon ( 2 0 g L 1 ) or ascorbic acid (1100 juM) to culture medium in place o f charcoal that gave rise to 6 0 % callus in immature zygotic embryo explants. However, the embryogenic potential o f these calli was low (Diyasena , 1 9 9 8 ) . T h e low embryogenic potential o f coconut calli developed in charcoal-free medium was further confirmed by Adkins et al. ( 1 9 9 9 ) . Quality and quantity of callus initiated C o c o n u t t i s s u e s g e n e r a l l y p r o d u c e h i g h l y heterogeneous compact callus in relatively small quantities. Establ ishment o f cell suspensions is a useful tool for rapid multiplication o f callus but it requires friable callus. However, despite the various treatments applied, no friable callus was obtained (Fernando, 2 0 0 1 ) . Somatic embryogenesis and plant regeneration Generally, lowering in auxin levels and addition o f c y t o k i n i n to the m e d i u m i n d u c e d s o m a t i c embryogenes i s in coconu t (Chan et al., 1 9 9 8 ) . However, we found that application o f abscisic acid ( A B A ) induced somatic embryogenesis (Fernando and G a m a g e , 2 0 0 0 ) . T h e p lan t r e g e n e r a t i o n efficiency was further improved by increasing agar and addition o f polyethyleneglycol and A g N 0 3 in combination with A B A . But, the plant regeneration was low at 10 % (Weerakoon, 2 0 0 4 ) . However, calli derived from unfertilized ovary showed an increased somat ic embryo induction when they were transferred to a medium with lower level o f auxin and then to a hormone-free medium (Perera et al., 2 0 0 7 ) . Comparison o f different treatments revealed that callus from certain plumules/ immature zygotic e m b r y o s r e g e n e r a t e d p l a n t s i r r e s p e c t i v e o f the treatment. Thus it is necessary to study the geno type ef fec t further to dec ide on the vital f a c t o r for o b t a i n i n g p o s i t i v e r e s u l t s . T h i s phenomenon compl ica tes the improvement o f the medium that is essential for induction and expression o f somatic embryogenesis at a higher frequency. Slow growth of regenerated plants Growth o f clonal plants in vitro and early ex vitro is very slow. W h e n the growth o f regenerated clones was compared with embryo-cultured plants, clonal plants were seen to be smaller at the time o f transplanting and took a longer time to reach the stage required for field planting (Fernando et al., 2 0 0 4 ) . Possible ways to overcome limitations Selection of the most suitable explant Based on our studies, the unfertilized ovary seems to be the most suitable explant for clonal propagation o f e l i t e c o c o n u t p a l m s . F u r t h e r r e s e a r c h to determine the effect o f genotype and plant maturity stage needs to be carried out. Source of activated charcoal A c t i v a t e d c o c o n u t s h e l l c h a r c o a l ( A C S C ) , m a n u f a c t u r e d l o c a l l y b y H a y c a r b P L C w a s identified for incorporation in the coconut tissue culture medium. Its use is economical ly better over imported charcoal . Its suitability was confirmed by culturing immature zygotic embryos and plumules and also by testing free 2 ,4-dichlorophenoxyacet ic acid available in the medium by H P L C analysis. T h e use o f this s o u r c e o f ac t iva t ed c h a r c o a l continuously under controlled conditions o f moisture level is important to achieve consistent results in callogenesis and somatic embryogenesis (Fernando, 2 0 0 8 ) . Multiplication of embryogenic callus, somatic embryos and clonal plants T h e repor t by P e r e z - N u n e z et al ( 2 0 0 6 ) on embryogenic callus multiplication by subdividing cal lus and repeated subculturing is an efficient method for increasing callus quantity. Application o f this method for callus multiplication showed an average mul t ip l ica t ion rate o f f ive-fold and a variation o f the rate with genotype (Weerakoon, 2 0 0 7 ) . S e c o n d a r y s o m a t i c e m b r y o g e n e s i s is a phenomenon where new soma t i c e m b r y o s are 324 CLONAL PROPAGATION OF COCONUT initiated from primary somat ic embryos . Induction o f secondary somatic embryogenes is will also lead to highenmultiplication rates o f regenerated plants. U s e o f explants from in v/Yro-raised plants (re-culturing) may also be useful in obtaining higher m u l t i p l i c a t i o n r a t e s . T h i s wi l l o v e r c o m e the difficulties related to repeated collection o f explants from mother plants. Use of arabinogalactan proteins (AGPs) in culture medium Cultured ce l l s secre t metabo l i t es to the culture medium. T h e sec re t i ons can s t imula te in vitro development o f cul tured t i ssues . A m o n g these secretions, A G P s are important as they are reported to induce/ promote somat ic embryogenes is in non-responsive/ low-responsive t issues (Egertsdotter and Arnold, 1 9 9 5 ; Letarte et al., 2 0 0 6 ) . The type and quant i ty o f A G P s are c e l l type s p e c i f i c . Furthermore, the presence o f spec i f ic A G P s in spec i f i c quan t i t i e s is e s s en t i a l for succe s s fu l somatic, embryogenes is . I f A G P s are not secreted, an external supply to the medium is an alternative (Kreuger and van Hoist, 1 9 9 3 ) . It is thus possible that A G P s may have potential to induce somatic embryogenesis in highly recalcitrant species like coconut. This has not been applied in any palm. Studies are in p rogress to assess the effec t o f externally applied A G P s on initiation o f embryogenic callus and somatic embryos. Conditioned media i.e. cu l turemedia in which tissues have been cultured for s o m e t i m e s , c a r r o t s e e d e x t r a c t , c o c o n u t endosperm and Arabic gum are among the sources o f A G P s under investigation. Selection of mother palms more amenable to tissue culture Many tissue culture protocols have shown strong genotype dependence. Work done over the years in coconut tissue culture conf i rms that it applies to coconut as well (Weerakoon , 2 0 0 7 ) . It has been observed that ca l lus originated from individual embryos/ embryos col lec ted from open pollinated nuts o f individual palms could regenerate plants at a high frequency. The re fo r e , se lec t ion o f most responsive such mother palms among elite palms to be c loned would increase the eff ic iency and consis tency o f clonal propagation protocol. Presently, select ion o f such palms is only possible by culturing o f explants co l lec ted from a large number o f elite mother palms and selecting the most responsive palms. As this is a long-term, laborious and c o s t l y p r o c e s s , deve lopment o f mo lecu l a r markers is an alternative. Molecu la r markers in oil palm for select ion o f responsive palms for clonal propagation have been developed (Ong-Abdullah and Eng, 2 0 0 7 ) . Studies on gene transformation are a new avenue as genes related to in vitro regeneration in certain crops have been identified (Bout i l ier et al., 2 0 0 2 ) . Genetic transformation o f coconut using such genes c o u l d lead to a m o r e e f f i c i en t in vitro p lan t regeneration system. CONCLUSIONS The importance o f developing a clonal propagation method for coconu t is now well accepted . T h e difficulties encountered in realizing it have also been identified. At present, the unfertilized ovary appears to be the most suitable explant due to a relatively high regeneration potential and application in clonal p r o p a g a t i o n o f adul t e l i t e p a l m s . E f f o r t s to standardize callusing medium and multiplication o f embryogenic callus have given encouraging results. Further research towards selection o f mother palms amenable to tissue culture, use o f A G P s to increase the number o f responsive mother palms and genetic manipulation o f elite palms with genes related to in vitro regeneration will ultimately pave the way to a successful clonal propagation o f coconut. ACKNOWLEDGEMENTS The contribution o f the past and present members o f the staff o f the Tissue Culture Division, Coconut Resea rch Institute to the work presented in this paper is gratefully acknowledged. 325 SECOND SYMPOSIUM ON PLANTATION CROP RESEARCH REFERENCES Adkins, S .W, Samosir, Y.M.S. and Godwin, I.D. (1999). Control o f environment conditions and the use of polyamines can optimize the conditions for the initiation and proliferation of coconut somatic embryogenesis. pp. 321-340 . In: Oropeza, C , Verdeil, J .L . , Ashburner, G.R., Cardena, R. and Santamaria, J .M. (Eds). Current Advances in Coconut Biotechnology, Kluwer Academic Publishers, Dordrecht. Boutilier, K , Offringa, R., Sharma, V.K., Kieft Ft, Ouellet, T., Zhang, L., Hattori, J. , Liu, CM. , van Lammeren, A.A.M., Miki, B.L.A. , Custers, J .B .M. and van Lookeren, C.M.M. (2002) . Ectopic expression of B A B Y B O O M triggers a conversion from vegetative to embryonic growth. The Plant Cell 14:1737-1749. Chan, J .L. , Saenz, L., Talavera, C , Hornung, R., Robert, M. and Oropeza, C. (1998 ) . Regeneration o f coconut {Cocos nucifera L.) from plumule explants through somatic embryogenesis. Plant Cell Report 17: 515-521. Diyasena, R.A.C.N.M. ( 1 9 9 8 ) . Development o f a charcoal-free protocol for callogenesis and somatic embryogenesis in immature embryo explants of coconut (Cocos nucifera L.), M. Sc thesis, University of Colombo, Sri Lanka. Egertsdotter, U., Arnold, V. S. (1995) . Importance of arabinogalactan proteins for the development of somatic embryos of Norway spruce (Picea abies). Physiologia Plantarum 93: 334 - 345. Fernando, S.C. (2001) . Studies on in vitro somatic embryogenesis of Cocos nucifera L. (coconut), PhD thesis, University o f Colombo, Sri Lanka. Fernando, S.C. and Gamage, C.K.A. (2000) . Abscisic acid-induced somatic embryogenesis in immature embryo explants o f coconut (Cocos nucifera L.). Plant Science 151: 193-198. Fernando, S.C. (2008) . Potential o f activated coconut shell charcoal in in vitro culture of Cocos nucifera L. (coconut). Paper submitted for 6 4 , h Annual Sessions of S L A A S . Fernando, S . C , Weerakoon, L . K . , Perera, P.I.P., Bandupriya, H.DD., Ambagala, I.A., Gamage, C.K.A., Santha, E.S., Gunathilake, T.R. and Perera, L. (2004). Genetic fidelity and ex vitro performance of tissue-cultured coconut plants. Proceedings II o f the International Conference on 75" 1 Anniversary o f Coconut Research Institute o f Sri Lanka, p.47-57. Karunaratne, S., Gamage, C. and Kovoor, A. (1991). Leaf maturity, a critical factor in embryogenesis. Journal Plant Physiolog)' 139: 27-31 . Kreuger, M. and van Hoist, G.J. (1993). Arabinogalactan proteins are essential in somatic embryogenesis ofDaucus carota L. Planta 189: 243-248. Letarte, J . , Simion, E., Miner, M., and Kasha, K. J. (2006). Arabinogalactan proteins induce embryogenesis in wheat (Triticum aestivum L.) microspore culture. Plant Cell Report 24: 691-698. Ong-Abdullah, M. and Eng, O.S. (2007) . Biomarkers: Finding a niche in oil palm tissue culture. Part 2-Targeting the transcriptome.^O/7 Palm Bulletin 54:68-74. Pan, M.J. and Staden, J.V. (1998) . The use of charcoal in in vitro culture. Plant Growth Regulation 26: 155-163. Perera, P.I.P., Hocher, V , Verdeil, J .L . , Yakandawala, D.M.D. and Weerakoon, L.K. (2007). Enhancing effect of TDZ on callogenesis o f coconut (Cocos nucifera L.) ovary culture. Book o f Abstracts, Asia Pacific Conference on Plant Tissue Culture and Agribiotechnology, 17-21 June, 2007, Kuala Lumpur, Malaysia. P. 128. Perez-Nunez, M.T., Chan, J .L . , Saenz, L., Gonzalez, T , Verdeil, J .L . and Oropeza, C. (2006) . Improved somatic embryogenesis from coconut (Cocos nucifera L.) plumule explants cultured in vitro. In Vitro Cellular and Developmental Biology of Plants 42: 37-43. Vidhanaarachchi, V.R.M. and Weerakoon, L.K. (1997) . Callus induction and direct shoot formation in in vitro cultured immature inflorescence tissues of coconut. Cocos 12: 39-43. Weerakoon, L.K. (2004) . Coconut tissue and embryo culture research in Sri Lanka: Current developments and future cha l l enges . Proceedings I o f the International Conference on 75" 1 Anniversary o f Coconut Research Institute of Sri Lanka, p.41-61. Weerakoon, L.K. (2007) . Report o f the Tissue Culture Division, Annual Report 2007, Coconut Research Institute. 326 

Clonal propagation of coconut : prospectus and limitations

http://cri.nsf.ac.lk/bitstream/handle/1/3856/PR6154-322.pdf?sequence=2&isAllowed=y

Clonal propagation of coconut : prospectus and limitations

Abstract

Similar works

Full text

Available Versions

Coconut Research Institute Repository