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 o f 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 o f activated charcoal used and repeated
multiplication o f embryogenic cell masses are some o f the measures currently applied to overcome
the limitations. Research towards selection o f mother palms amenable to tissue culture, use o f
AGPs to increase the number o f responsive mother palms and genetic manipulation o f 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
for genetic i m p r o v e m e n t o f c o c o n u t are s l o w due
to limitations attributed to the bulkiness, perennial
C o c o n u t palm, the " T r e e o f L i f e " , is an important
n a t u r e o f t h e p a l m a n d its b i o l o g y . T h u s t h e
oil crop in the tropics. T h e r e is a gradual decline in
development o f a reliable clonal propagation method
coconut production o v e r the years due to constraints
o f c o c o n u t to assist breeder is e x c i t i n g . Although
such as, maintaining senile plantations with l o w
research leading to a m e t h o d o f clonal propagation
productivity, declined soil fertility, use o f unimproved
in c o c o n u t w a s initiated in many countries in the
planting material, marginal cultural practices, spread
early 1 9 7 0 ' s , an e c o n o m i c a l l y viable protocol has
o f pests and diseases and i n c r e a s i n g competition
not yet been developed.
from other oil c r o p s . H o w e v e r , c o c o n u t is still
e c o n o m i c a l l y i m p o r t a n t for S r i L a n k a and t h e
T h i s paper s u m m a r i z e s the S r i Lankan e x p e r i e n c e
industrial demand for lauric oil is high. T h e r e f o r e ,
on the current progress, limiting factors and possible
introduction o f n e w v a r i e t i e s and hybrids with
measures to o v e r c o m e the limitations and future
superior qualities is essential for the improvement
directions towards the d e v e l o p m e n t o f a v i a b l e
o f the industry. Conventional breeding programmes
clonal propagation m e t h o d for c o c o n u t .
322
CLONAL PROPAGATION OF COCONUT
Current status of coconut clonal propagation
Z y g o t i c tissues such as i m m a t u r e z y g o t i c e m b r y o
and plumule ( e x c i s e d from mature zygotic e m b r y o )
S i n c e the inception o f r e s e a r c h in c o c o n u t tissue
hold potential for m i c r o p r o p a g a t i o n . U n l i k e m a n y
culture at W y e C o l l e g e , U K in the early 1 9 7 0 ' s , the
somatic tissues ( i n f l o r e s c e n c e and l e a f tissues), the
response o f various s o m a t i c tissues (tender leaf,
r e s p o n s e o f z y g o t i c t i s s u e s is m o r e c o n s i s t e n t .
immature inflorescence, root and shoot) and zygotic
However,
tissues (immature and m a t u r e z y g o t i c e m b r y o s and
on e m b r y o d e v e l o p m e n t a l stage. Under optimum
plumule) to in vitro culture has b e e n reported in
c o n d i t i o n s , c a l l u s i n g is u s u a l l y a b o v e 7 5 % in
m a n y other c o u n t r i e s w o r l d w i d e ( s u m m a r i z e d in
immature
F e m a n d o , , 2 0 0 1 ) . T h e s e indicated the possibility o f
cloning coconut through
several steps i.e.,
the callusing frequency
zygotic
embryo
depends
(Diyasena,
1 9 9 8 ) and 6 0 % in plumule explants ( W e e r a k o o n ,
2007).
callogene.sis, s o m a t i c e m b r y o g e n e s i s , embryo
maturation and germination. T h e regenerated plants,
though l o w in n u m b e r , w e r e s u i t a b l e for
field
establishment. U p to now, hundred c o c o n u t p a l m s
regenerated in the T i s s u e Culture D i v i s i o n o f the
C o c o n u t R e s e a r c h Institute o f Sri L a n k a have been
R e c e n t r e s e a r c h u n v e i l e d the high
potential
o f u n f e r t i l i z e d o v a r y as an initial e x p l a n t for
cloning. T h e highest frequency o f callusing
reported from unfertilized ovary is 7 6 % (Perera
et al,
2007).
field established. T h e s e p a l m s at different stages
o f development including s o m e o f which are already
bearing nuts. A study using m i c r o s a t e l l i t e m a r k e r s
showed tnat these c l o n e s are g e n e t i c a l l y stable with
field p e r f o r m a n c e c o m p a r a b l e to s e e d - d e r i v e d
p a l m s ( F e r n a n d o et al., 2 0 0 4 ) .
S o m a t i c tissues are the ideal explants for clonal
propagation as the p e r f o r m a n c e o f m o t h e r palm is
k n o w n . H o w e v e r , the r e s p o n s e o f t h e s e t i s s u e s
( e x c e p t u n f e r t i l i z e d o v a r i e s ) is g e n e r a l l y poor.
M o r e o v e r , t h e r e is a m a r k e d d i f f e r e n c e a m o n g
Factors limiting the development of a clonal
propagation protocol for coconut
explants c o l l e c t e d from different mother p a l m s in
terms o f c a l l u s initiation. T h i s m i g h t b e due to
differences in g e n o t y p e , explant maturity or mother
Callogenesis
palm a g e .
Heterogeneous response of explants
Effect of activated charcoal
T h e e m b r y o g e n i c potential o f c o c o n u t l e a f explants
A c t i v a t e d c h a r c o a l is an essential c o m p o n e n t in
is very low ( < 1 0 % ) . O n l y e x p l a n t s derived from
c o c o n u t t i s s u e c u l t u r e m e d i u m . It h a s s t r o n g
young leaves o f 1 0 - 2 0 c m length from 1 2 - 2 4 month-
adsorptive properties and its b e n e f i c i a l effects are
old
F u r t h e r m o r e , the
attributed to the adsorption o f phenols and other
e m b r y o g e n i c potential o f these e x p l a n t s w a s o f
growth inhibitory s u b s t a n c e s . H o w e v e r , a m a j o r
short d u r a t i o n . T h i s l i m i t s t h e u s e o f c o c o n u t
disadvantage o f using activated c h a r c o a l is that it
l e a f for c l o n a l p r o p a g a t i o n ( K a r u n a r a t n e et al.,
also c a n adsorb plant growth regulators ( h o r m o n e s
1991).
and v i t a m i n s ) and s o m e m i c r o e l e m e n t s ( C u and
seedlings responded.
Immature i n f l o r e s c e n c e is a p r o m i s i n g explant as it
bears numerous
meristematic
points. The
e m b r y o g e n i c potential^ d e p e n d s on the maturity
stage o f the explant. However, the lack o f a
Zn) (Pan and Staden, 1 9 9 8 ) . T h i s creates undefined
culture conditions which could lead to variable tissue
r e s p o n s e and n o n - r e p r o d u c i b l e results. Different
types/ brands o f c h a r c o a l have different adsorptive
marker greatly limits the ability to select the correct
c a p a c i t i e s b a s e d on t h e i r o r i g i n , a g e , s t o r a g e
maturity s t a g e . T h e h i g h e s t c a l l u s i n g r e p o r t e d
c o n d i t i o n s and m e t h o d o f activation. T h i s has a
so far i'is 3 0 % ( V i d h a n a a r a c h c h i a n d W e e r a k o
significant effect on callus d e v e l o p m e n t even from
on, 1 9 9 7 ) .
z y g o t i c tissues that g i v e high yields. A c h a r c o a l -
323
SECOND SYMPOSIUM ON PLANTATION CROP RESEARCH
free m e d i u m was d e v e l o p e d with the addition o f
Slow growth of regenerated plants
1
polyvinyl pyralidon ( 2 0 g L ) or ascorbic acid ( 1 1 0 0
juM) to culture m e d i u m in p l a c e o f charcoal that
gave rise to 6 0 % callus in immature zygotic embryo
explants. H o w e v e r , the e m b r y o g e n i c potential o f
these calli w a s l o w ( D i y a s e n a , 1 9 9 8 ) . T h e low
e m b r y o g e n i c potential o f c o c o n u t calli developed
in charcoal-free m e d i u m w a s further c o n f i r m e d by
Growth o f clonal plants in vitro and early ex vitro
is very slow.
W h e n the growth o f r e g e n e r a t e d
clones w a s compared with embryo-cultured plants,
clonal plants w e r e seen to b e s m a l l e r at the time o f
transplanting and took a l o n g e r time to reach the
stage required for field planting ( F e r n a n d o et al.,
2004).
Adkins et al. ( 1 9 9 9 ) .
Possible ways to overcome limitations
Quality and quantity of callus initiated
Selection of the most suitable explant
Coconut
tissues
generally
produce
highly
h e t e r o g e n e o u s c o m p a c t callus in relatively small
B a s e d on our studies, the unfertilized ovary s e e m s
quantities. E s t a b l i s h m e n t o f cell suspensions is a
to be the most suitable explant for clonal propagation
useful tool for rapid multiplication o f c a l l u s but it
o f elite c o c o n u t p a l m s . Further research to
requires friable callus. However, despite the various
determine the effect o f g e n o t y p e and plant maturity
treatments applied, no friable callus w a s obtained
stage needs to b e carried out.
(Fernando, 2 0 0 1 ) .
Source of activated charcoal
Somatic
embryogenesis
regeneration
and
plant
Activated
coconut shell charcoal
manufactured
locally by Haycarb
(ACSC),
P L C was
Generally, lowering in auxin l e v e l s and addition o f
identified for incorporation in the c o c o n u t tissue
cytokinin
culture medium. Its use is e c o n o m i c a l l y better over
to t h e m e d i u m
induced
somatic
1998).
imported c h a r c o a l . Its suitability w a s confirmed by
However, w e found that application o f abscisic acid
culturing immature z y g o t i c e m b r y o s and plumules
( A B A ) induced s o m a t i c e m b r y o g e n e s i s (Fernando
and also by testing free 2 , 4 - d i c h l o r o p h e n o x y a c e t i c
and G a m a g e , 2 0 0 0 ) . T h e plant
acid available in the m e d i u m b y H P L C analysis.
e m b r y o g e n e s i s in c o c o n u t ( C h a n et al.,
regeneration
efficiency was further improved b y increasing agar
The
and addition o f p o l y e t h y l e n e g l y c o l and A g N 0 in
continuously under controlled conditions o f moisture
combination with A B A . B u t , the plant regeneration
level is important to a c h i e v e consistent results in
was low at 10 % ( W e e r a k o o n , 2 0 0 4 ) .
callogenesis and somatic embryogenesis (Fernando,
3
However,
calli derived from unfertilized o v a r y showed an
use o f this s o u r c e o f activated
charcoal
2008).
i n c r e a s e d s o m a t i c e m b r y o induction when they
were transferred to a m e d i u m with l o w e r level o f
auxin and then to a h o r m o n e - f r e e m e d i u m (Perera
et al., 2 0 0 7 ) .
Multiplication of embryogenic callus, somatic
embryos and clonal plants
The
report by P e r e z - N u n e z
et al ( 2 0 0 6 )
on
e m b r y o g e n i c callus multiplication b y subdividing
C o m p a r i s o n o f different treatments revealed that
callus from certain p l u m u l e s / i m m a t u r e z y g o t i c
embryos
regenerated
plants
irrespective o f
the treatment. T h u s it is n e c e s s a r y to study the
g e n o t y p e e f f e c t further to d e c i d e on t h e vital
factor
for o b t a i n i n g
positive
results.
This
c a l l u s and repeated s u b c u l t u r i n g
is an efficient
method for increasing c a l l u s quantity. Application
o f this method for c a l l u s multiplication showed an
a v e r a g e m u l t i p l i c a t i o n rate o f f i v e - f o l d and a
variation o f the rate with g e n o t y p e ( W e e r a k o o n ,
2007).
p h e n o m e n o n c o m p l i c a t e s the i m p r o v e m e n t o f the
medium that is essential for induction and expression
Secondary
somatic
o f somatic e m b r y o g e n e s i s at a higher
phenomenon
where new somatic e m b r y o s are
324
frequency.
embryogenesis
is
a
CLONAL PROPAGATION OF COCONUT
initiated from primary s o m a t i c e m b r y o s . Induction
r e s p o n s i v e such m o t h e r p a l m s a m o n g elite palms
o f secondary s o m a t i c e m b r y o g e n e s i s will also lead
to b e c l o n e d w o u l d i n c r e a s e the e f f i c i e n c y and
to highenmultiplication rates o f regenerated plants.
c o n s i s t e n c y o f c l o n a l propagation protocol.
U s e o f e x p l a n t s from in v/Yro-raised plants ( r e culturing) m a y also b e useful in o b t a i n i n g higher
Presently, s e l e c t i o n o f such p a l m s is only p o s s i b l e
multiplication rates. T h i s will o v e r c o m e the
by c u l t u r i n g o f e x p l a n t s c o l l e c t e d from a large
difficulties related to repeated collection o f explants
number o f elite mother palms and selecting the most
r e s p o n s i v e p a l m s . A s this is a long-term, laborious
from m o t h e r plants.
and c o s t l y p r o c e s s , d e v e l o p m e n t o f m o l e c u l a r
Use of arabinogalactan proteins (AGPs) in
m a r k e r s is an alternative. M o l e c u l a r markers in oil
culture medium
palm for s e l e c t i o n o f r e s p o n s i v e palms for clonal
propagation h a v e b e e n d e v e l o p e d ( O n g - A b d u l l a h
Cultured c e l l s s e c r e t m e t a b o l i t e s to the culture
and E n g , 2 0 0 7 ) .
m e d i u m . T h e s e c r e t i o n s c a n s t i m u l a t e in vitro
development o f cultured tissues. A m o n g these
Studies on g e n e transformation are a n e w avenue
secretions, A G P s are important as they are reported
as g e n e s related to in vitro regeneration in certain
to induce/ p r o m o t e s o m a t i c e m b r y o g e n e s i s in n o n -
crops h a v e b e e n identified ( B o u t i l i e r et al., 2 0 0 2 ) .
responsive/ l o w - r e s p o n s i v e tissues ( E g e r t s d o t t e r
G e n e t i c transformation o f coconut using such genes
and Arnold, 1 9 9 5 ; L e t a r t e et al., 2 0 0 6 ) . T h e type
and q u a n t i t y o f A G P s a r e c e l l t y p e
specific.
c o u l d l e a d to a m o r e e f f i c i e n t in vitro
plant
regeneration s y s t e m .
F u r t h e r m o r e , the p r e s e n c e o f s p e c i f i c A G P s in
s p e c i f i c q u a n t i t i e s is e s s e n t i a l f o r s u c c e s s f u l
CONCLUSIONS
somatic, e m b r y o g e n e s i s . I f A G P s are not secreted,
an external supply to the m e d i u m is an alternative
( K r e u g e r and v a n Hoist, 1 9 9 3 ) . It is thus p o s s i b l e
T h e i m p o r t a n c e o f d e v e l o p i n g a clonal propagation
m e t h o d for c o c o n u t is n o w w e l l a c c e p t e d . T h e
that A G P s m a y h a v e potential to induce s o m a t i c
e m b r y o g e n e s i s in h i g h l y r e c a l c i t r a n t s p e c i e s like
coconut. T h i s has not b e e n applied in any palm.
S t u d i e s are in p r o g r e s s to a s s e s s t h e e f f e c t o f
externally applied A G P s on initiation o f embryogenic
callus and somatic e m b r y o s . Conditioned media i.e.
c u l t u r e m e d i a in w h i c h tissues h a v e b e e n 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
difficulties encountered in realizing it have also been
identified. A t present, the unfertilized ovary appears
to b e the m o s t suitable explant due to a relatively
high regeneration potential and application in clonal
propagation
o f adult e l i t e p a l m s . E f f o r t s to
standardize callusing m e d i u m and multiplication o f
e m b r y o g e n i c callus have given encouraging results.
endosperm and A r a b i c g u m a r e a m o n g the s o u r c e s
Further research towards selection o f mother p a l m s
o f A G P s under investigation.
a m e n a b l e to tissue culture, u s e o f A G P s to increase
Selection of mother palms more amenable to
tissue culture
the n u m b e r o f responsive mother palms and genetic
manipulation o f elite p a l m s with genes related to in
vitro regeneration will ultimately pave the w a y to a
M a n y tissue culture p r o t o c o l s h a v e shown strong
successful c l o n a l propagation o f c o c o n u t .
genotype d e p e n d e n c e . W o r k done o v e r the years
in c o c o n u t tissue culture c o n f i r m s that it applies to
ACKNOWLEDGEMENTS
c o c o n u t as well ( W e e r a k o o n , 2 0 0 7 ) . It has b e e n
o b s e r v e d that c a l l u s o r i g i n a t e d from
individual
T h e contribution o f the past and present m e m b e r s
e m b r y o s / e m b r y o s c o l l e c t e d from open pollinated
o f the staff o f the T i s s u e Culture D i v i s i o n , C o c o n u t
nuts o f individual p a l m s c o u l d r e g e n e r a t e plants at
R e s e a r c h Institute to the w o r k presented in this
a high f r e q u e n c y . T h e r e f o r e , s e l e c t i o n o f m o s t
paper is gratefully a c k n o w l e d g e d .
325
SECOND SYMPOSIUM ON PLANTATION CROP RESEARCH
Anniversary o f Coconut Research Institute o f
REFERENCES
Sri Lanka, p.47-57.
Adkins, S . W , Samosir, Y.M.S. and Godwin, I.D. (1999).
Karunaratne, S., Gamage, C. and Kovoor, A. (1991). Leaf
Control o f environment conditions and the use
maturity, a critical factor in embryogenesis.
of polyamines can optimize the conditions for
Journal Plant Physiolog)' 139: 27-31.
the initiation and proliferation of coconut somatic
Kreuger, M. and van Hoist, G.J. (1993). Arabinogalactan
embryogenesis. pp. 3 2 1 - 3 4 0 . In: Oropeza, C ,
proteins are essential in somatic embryogenesis
Verdeil, J . L . , Ashburner, G.R., Cardena, R. and
ofDaucus carota L. Planta 189: 243-248.
Santamaria, J . M . (Eds). Current Advances in
Coconut Biotechnology, Kluwer Academic
Letarte, J., Simion, E., Miner, M., and Kasha, K. J. (2006).
Publishers, Dordrecht.
Arabinogalactan proteins induce embryogenesis
in wheat (Triticum aestivum L.) microspore
Boutilier, K , Offringa, R., Sharma, V.K., Kieft Ft, Ouellet,
culture. Plant Cell Report 24: 691-698.
T., Zhang, L., Hattori, J., Liu, C M . , van Lammeren,
A.A.M., Miki, B . L . A . , Custers, J . B . M . and van
Ong-Abdullah, M. and Eng, O.S. (2007). Biomarkers:
Lookeren, C.M.M. (2002). Ectopic expression o f
Finding a niche in oil palm tissue culture. Part 2 -
B A B Y B O O M triggers a c o n v e r s i o n from
Targeting the transcriptome.^O/7 Palm Bulletin
vegetative to embryonic growth. The Plant Cell
54:68-74.
14:1737-1749.
Pan, M.J. and Staden, J.V. (1998). The use o f charcoal in
in vitro culture. Plant Growth Regulation 26:
Chan, J.L., Saenz, L., Talavera, C , Hornung, R., Robert,
M. and Oropeza, C. ( 1 9 9 8 ) . Regeneration o f
155-163.
coconut {Cocos nucifera L . ) from plumule
Perera, P.I.P., Hocher, V , Verdeil, J . L . , Yakandawala,
explants through somatic embryogenesis. Plant
D.M.D. and Weerakoon, L.K. (2007). Enhancing
Cell Report 17: 515-521.
effect of T D Z on callogenesis o f coconut (Cocos
Diyasena, R . A . C . N . M . ( 1 9 9 8 ) . Development o f a
nucifera L.) ovary culture. Book o f Abstracts,
charcoal-free protocol for callogenesis and
Asia Pacific Conference on Plant Tissue Culture
somatic embryogenesis in immature embryo
and Agribiotechnology, 17-21 June, 2007, Kuala
explants o f coconut (Cocos nucifera L.), M. Sc
Lumpur, Malaysia. P. 128.
thesis, University o f Colombo, Sri Lanka.
Perez-Nunez, M.T., Chan, J . L . , Saenz, L., Gonzalez, T ,
Egertsdotter, U., Arnold, V. S. ( 1 9 9 5 ) . Importance o f
Verdeil, J . L . and Oropeza, C. (2006). Improved
arabinogalactan proteins for the development o f
somatic embryogenesis from coconut (Cocos
somatic embryos of Norway spruce (Picea abies).
nucifera L.) plumule explants cultured in vitro.
Physiologia Plantarum 93: 334 - 345.
In Vitro Cellular and Developmental Biology of
Plants 42: 37-43.
Fernando, S.C. ( 2 0 0 1 ) . Studies on in vitro somatic
embryogenesis o f Cocos nucifera L. (coconut),
Vidhanaarachchi, V.R.M. and Weerakoon, L.K. (1997).
PhD thesis, University of Colombo, Sri Lanka.
Callus induction and direct shoot formation in in
vitro cultured immature inflorescence tissues o f
Fernando, S.C. and Gamage, C.K.A. (2000). Abscisic
coconut. Cocos 12: 39-43.
acid-induced somatic embryogenesis in immature
embryo explants o f coconut (Cocos nucifera L.).
Weerakoon, L.K. ( 2 0 0 4 ) . Coconut tissue and embryo
Plant Science 151: 193-198.
culture
Fernando, S.C. ( 2 0 0 8 ) . Potential o f activated coconut
shell charcoal in in vitro culture of Cocos nucifera
, h
L. (coconut). Paper submitted for 6 4 Annual
future
Current
challenges.
Proceedings I o f the International Conference on
1
75" Anniversary o f Coconut Research Institute
Weerakoon, L.K. ( 2 0 0 7 ) . Report o f the Tissue Culture
Fernando, S . C , W e e r a k o o n , L . K . , Perera, P.I.P.,
Division, Annual Report 2007, Coconut Research
Bandupriya, H.DD., Ambagala, I.A., Gamage,
Institute.
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
326
in S r i L a n k a :
and
of Sri Lanka, p.41-61.
Sessions o f S L A A S .
II o f the International C o n f e r e n c e on 7 5 "
research
developments
1