Coconut Research Institute Repository

Everard, J.M.D.T.

The application of bio technology to plant breeding and crop protection in coconut - Sri Lanka's efforts in coconut breeding

Peiris, T.S.G.

Mathes, D.T.

The analysis of long-term rainfall variability of selected locations in Sri Lanka

Vidhana Arachchi, L.P.

Yapa, P.A.J.

Mapa, R.B.

Somapala, H.

Effect of soil physical properties on ATPase activity, proline and carbohydrate levels of coconut (Cocos nucifera L) seedlings

Vidhana Arachchi, V.R.M.

Weerakoon, L.K.

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 pro­
ducing 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 induc­
tion 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 con­
tained 24 U .M 2,4-dichlorophenoxyacetic acid and 0 . 2 5 % activated char­
coal) , 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 co­
conu 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 propa­
g 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 coco­
nut 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 dis­
tilled 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 cul­
tured into sc rew capped vials containing 15 ml of culture med ium. The fol­
lowing 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 contain­
ing 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 in­
duction 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 regen­
eration protocol devoid of a callus phase. Thus, attempts will be made to 
refine the culture conditions to increase the frequency of direct shoot forma­
tion. 
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 histo­
logical 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 em­
bryos 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

Thattil, R.O.

Climate analysis-the effect of climate on coconut

Perera, L.

Russell, J.R.

Provan, J.

McNicol, J.W.

Powel, W.

Evaluation of genetic relationships between indigenous coconut(Cocos nucifera L.) accessions from Sri Lanka by means of AFLPprofiling

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Fernando, W.M.U.

Peries, R.R.A.

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Gajanayake, G.

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Hayward, M.D.

Kearsey, M.J.

Isozyme and quantitative traits polymorphisms in European provinces of perennial rye grass (Lolium perenne L)

National Science Foundation of Sri Lanka

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