Vidhanaarachchi, V.R.M.

Weerakoon, L.K.

Coconut Research Institute Repository

COCOS (1997), 12,39 - 43 Printed in Sri Lanka C A L L U S I N D U C T I O N A N D D I R E C T S H O O T F O R M A T I O N I N IN VITRO C U L T U R E D I M M A T U R E I N F L O R E S C E N C E T I S S U E S O F C O C O N U T V R M V i d h a n a A r a c h c h i a n d L K W e e r a k o o n Coconut Research Institute, Lunuwila, Sri Lanka A B S T R A C T Clonal propagation of elite palms is a promising possibility for pro­ducing uniform planting material and improving productivity in coconut lands. In the absence of a natural vegetative propagation method, in vitro culture remains the only approach for vegetative multiplication of coconut. Immature inflorescence material has shown to be a promising source of explants for coconut tissue culture. In the present study, four different culture media (medium 72; modified Eeuwens Y3 medium; coconut anther culture med im; modified Blake ' s medium) were evaluated for callus induc­tion in floral explants : Immature inflorescences were collected non-destruc-tively from 12 year old Sri Lanka-tall coconut palms and rachilla segments obtained from these inflorescences were cultured on above media . Out of the four culture media tested, callus production was observed only in the explants cultured on medium 72. In this med ium (which con­tained 24 U .M 2,4-dichlorophenoxyacetic acid and 0 . 2 5 % activated char­coal) , about 3 0 % of the floral explants produced compact , highly embryo-genie calli . Direct regeneration of shoots from floral pr imordia occurred at & very low frequency in the modified Eeuwens Y 3 medium which contained 200 u M 2,4-dichlorophenoxyacetic acid and 0 .2% activated charcoal . N o cal logenesis or direct organogenesis were observed in the floral explants cultured on the other two culture media tested. I N T R O D U C T I O N Coconut pa lms raised from seeds show a wide variation in the field due to their highly heterozygous nature. Vegetative multiplication of elite pa lms is a promis ing poss ib i l i ty for producing uniform planting material of 3 9 high quality. This would not only improve the productivity in coconut lands but a lso p roduce uniform parent material for breeding programmes . In vitro cul ture is the only channel open for intensive vegetat ive propagat ion of co­conu t and the development of a reliable in vitro c loning technique would a lso pave the way to genetic transformation of coconut by transfer of foreign genes that confer desirable characters. Recen t studies on immature inflorescence culture o f coconut have indicated that plant regenerat ion through somatic embryogenesis is possible bu t the technique has not yet been mastered (Verdeil et. al. 1994; Hornung , 1995). A t the Coconut Research Institute, of Sri Lanka, work on clonal propa­g a t i o n h a s been done ma in ly wi th immatu re e m b r y o (Karunara tne and Per iyapperuma, 1989) and leaf explants (Karunaratne et at 1991) and very little work has been done on inflorescence culture. Therefore, the present s tudy was under taken to assess the suitability of four different culture media for cal lus induct ion in immature inflorescence explants with a v iew to de ­velop a rel iable protocol for clonal propagat ion of coconut . M a t e r i a l s a n d m e t h o d s Immatu re inflorescences of 8 -10 c m outer spathe length were o b ­ta ined non-destruct ively (Rillo, 1989) from 12 year old Sri Lanka-tal l coco­nut pa lms . T h e inflorescences were surface sterilized for 15 minutes in 3 % ca lc ium hypochlor i te solution followed by thorough rinsing with sterile dis­tilled water. Then the outer a n d inner spathes were removed under aseptic condi t ions . T h e top and basal portions of the inflorescence were discarded and only the middle port ion of the inflorescence ( a b o u t 3 c m in l e n g t h ) was used for culturing. Rachil la was cut into 1-1.5 m m long sections and cul­tured into sc rew capped vials containing 15 ml of culture med ium. The fol­lowing cul ture media were used in this study. 1. M e d i u m 72 formulated by Karunaratne and Per iyapperuma (1989) containing 24 u M 2,4-dichlorophenoxyacetic acid ( 2 , 4 - D ) a n d o . 2 5 % activated charcoal . 2. Modif ied Eeuwens Y3 med ium described by Verdeil et. al. (1994) containing 200 u M 2,4-D and 0 .2% activated charcoal . 3. Modif ied Blake ' s med ium described by Ebert and Taylor (1990) conta in ing 200 u M 2,4-D and 0 .25% activated charcoal . 40 4. Coconut anther culture medium formulated by Tuyen and Guzman (1982) containing 10 uM 2,4-D, 10 um naphthaleneacetic acid and 0.25° activated charcoal. All the cultures were incubated in dark at 30 1 C. RESULTS The first sign of callogenesis (indicated by floral bud swelling) was observed in explants cultured on medium 72 (a medium developed for callus induction in immature embryo explants of coconut) after about 8 weeks of culturing. This was followed by the initiation of small, whitish calli from floral areas (Fig. 1). The callus tissues continued to grow into larger masses (without any subculturing) within 12-14 weeks of culturing (Fig. 2). About 30% of the cultured explants produced embryogenic calli (Fig. 3) on the above medium. In modified Eeuwens Y3 medium, transluscent outgrowths developed directly from the floral buds within 6 weeks of culturing. Some of these structures developed into shoots upon subculturing into a medium contain­ing 8 um 6-benzylaminopurine and 10 uM 2,4-D. However, the frequency of shoot regeneration was very low. Roots were induced on these shoots (Fig. 4) by dipping in 100 uM indoleacetic acid solution for 3 days followed by culturing them in an auxin-free medium as described by Fernando and Gamage (1995). DISCUSSION In the present study, emphasis was placed on callogenesis in flower meristem explants as it is the first stage in the process of somatic embryo-genesis. The results indicate that the culture medium developed for callogenesis in immature embryo explants of coconut is also suitable for callogenesis in floral explants. Work in progress is directed towards the in­duction of somatic embryogenesis in the calli obtained. The direct shoot regeneration observed in modified Eeuwens Y3 medium is also of importance as it could lead to the development of a regen­eration protocol devoid of a callus phase. Thus, attempts will be made to refine the culture conditions to increase the frequency of direct shoot forma­tion. 4 1 Fig. I ; Callus initiation on floral explants. (x 3) -ig. 2 ; Nodular, embryogcnic callus, (x 3) A C K N O W L E D G E M E N T S W e are mos t grateful to Mr. E. S. Shantha for his assistance in histo­logical work. We also gidtefully acknowledge tne assis tance ot Mr. Pemsir i Si lva and Mr. H.P. Asoka Kumara the photographers . Thanks are also due to the staff of Tissue Culture Division, Coconut Research Institute for their ass is tance in performing this investigation. References Eber t , A . and Taylor, H.F. (1990) . Assessment o f the changes of 2 ,4-dichlorophenoxyacet ic acid concentrat ions in plant t issue culture media in the presence of activated charcoal . Plant Cell Tissue and Organ cul ture 20 : 165-172. Fe rnando , S.C. and Gamage , C.K. (1995) . Clonal propagat ion of coconut : Improved cul ture condit ions for rhyzogenesis . C O C O S 10: 20-25 . Hornung , H . (1995) . Initiation of cal logenesis in coconut pa lm (Cocos nuciferaL.). In Lethal Yellowing: Research and Practical Aspects. 203 -215 . (Eds . C. Oropeza et. a l . ) . K luwer Academic Publishers . T h e Nether lands . Karunara tne , S. and Per iyapperuma, K. (1989) . Culture o f immature em­bryos of coconut (Cocos nucifera L.) callus proliferation and somatic e m -bryogenes is . Plant Sci. 62: 247-253 . Karunara tne , S. , Gamage , C. and Kovoor , A., (1991) . Leaf maturity, a crit ical factor in embryogenes is . J. Plant Physiol . 139: 2 7 - 3 1 . Ri l lo , E.P. (1989) . A nondestruct ive technique for collecting immature c o conut inflorescence for tissue culture. Phil. J. Coconut Studies 14(2): 16-17. Thanh-Tuen, T.N. and D e Guzman, E.V. (1983) . Format ion o f pol len e m ­bryos in cultured anthers of coconut (Cocos nucifera L . ) . Plant Science Let ters 29 : 81-88 . ' Verdeil , J . L . , Huet , C . ; G&sdemange , F. and Buffard-Morel, J. (1994) . Plant r egenera t ion from cul tured immature inf lorescences of coconu t (Cocos nucifera L . ) : Evidence for somatic embryogenesis . Plant Cell Repor ts 13: 2 1 8 - 2 2 1 . 4 3 

Callus induction and direct shoot formation in in vitro cultured immature inflorescence tissues of coconut

Vidhana Arachchi, V.R.M.