Assy-Bah, B.

Engelmann, Florent

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!k*' Plant Cell, Tissue and Organ Culture 33: 19-24, 1993. 
--Q 1993 Kluwer Academic Publishers. Printed in the Netherlands. 
Medium-term conservation of mature embryos of coconut 
Béatrice Assy-Bah1'2 & Florent Engelmann2'* 
IDEFORIDPO (Institut des Forêts, Département Plantes Oléagineuses), La Mk, 13 BP 989 Abidjan 
13, Côte d'Ivoire; 20RSTOM (Institut Français de Recherche Scientifque pour le Développement en 
Coopération), Laboratoire de Ressources Génétiques et Amélioration des Plantes Tropicales, 911 av. 
Agropolis, BP 5045, 34032 Montpellier Cedex 01, France (* requests for offprints) 
1 
Received 6 April 1992; accepted in revised form 29 September 1992 
Key words: activated charcoal, disease control, germplasm storage, sucrose, zygotic embryos 
Abstract 
Optimal conditions for the medium-term conservation of zygotic embryos of coconut were developed. 
After 6 months of storage on a medium devoid of sucrose and containing 2gl- l  activated charcoal, 
100% of the embryos developed into whole plantlets within 5 months after transfer to the recovery 
medium. After a 12-month storage period on medium containing 15 g I-' sucrose and devoid of 
activated charcoal, 51% of the embryos germinated within 2 months after transfer to the recovery 
medium. 
Introduction 
The use of in vitro culture for the conservation of 
plant genetic resources calls for different tech- 
niques, according to the required time scale of 
storage. For medium-term conservation (several 
months to 1-2 years), the aim is to reduce the 
growth rate so as to increase the interval be- 
tween subcultures. This is achieved by modifying 
culture conditions, principally by lowering the 
temperature to 15-20°C in the case of tropical 
plants (Banerjee & De Langhe 1985), which are 
often cold sensitive. These slow growth tech- 
niques are now routinely employed for the 
storage of shoot cultures in many industrial 
laboratories as well as in regional and interna- 
tional germplasm conservation centers (Withers 
1991). 
Research on in vitro medium-term conserva- 
tion mainly concerns shoot cultures but experi- 
ments have also been conducted with cell sus- 
pensions, calluses and embryos (Engelmann 
1991). In this latter case, to our knowledge, only 
somatic embryos have been used and no work 
has been carried out yet with zygotic embryos. 
With somatic embryos, various techniques have 
been tried: encapsulation in alginate beads 
(Bapat & Rao 1988; Arumugam & Bhojwani 
1990), partial desiccatioi (Gray 1989), use of 
controlled atmosphere in which the oxygen con- 
centration is reduced (Engelmann 1990). 
Research on the conservation of coconut gen- 
etic resources started eight years ago, in the 
framework of a joint program involving IBPGR 
(International Board for Plant Genetic Re- 
sources, Rome), IRHO (Institut de Recherche 
pour les Huiles et Oléagineux, Département 
Oléagineux du CIRAD) and ORSTOM. Routine 
techniques for the collection (Assy-Bah et al. 
1987) and in vitro culture of zygotic embryos 
(Assy-Bah 1986; Assy-Bah et al. 1989) have 
been developed. Efficient cryopreservation tech- 
niques have been developed recently for imma- 
ture (Assy-Bah & Engelmann 1992a) and mature 
embryos (Assy-Bah & Engelmann 1992b). These 
should allow long-term conservation of coconut 
genetic resources in the form of zygotic embryos. 
However, there is a need for a medium-term 
~~S~~~ Fonds 
20 
conservation technique for embryos, because of 
problems and constraints linked to the occur- 
rence of diseases of coconut in different coconut 
growing areas (Frison & Putter 1992). For exam- 
ple, for material originating from areas where 
lethal yellowing occurs, parent palms that have 
not shown disease symptoms should be observed 
for one year (the period of incubation of the 
disease), before embryos may be released and 
exported to a country free of coconut lethal 
yellowing. In the meantime, excised embryos 
need to be maintained in vitro, preferably under 
slow growth conditions. 
In this paper, we describe the conservation of 
mature embryos of coconut under slow growth 
conditions for 6 and 12 months. 
Materials and methods 
Plant material 
The plant material used in this study consisted of 
mature embryos (10-12 months after pollina- 
tion) from seednuts of the variety Ghanaian 
Yellow Dwarf. Nuts were harvested from adult 
palms at the Port Bouet Research Station 
(IDEFOR, Iiory Coast). 
Methods 
Zygotic embryos were extracted from the nuts 
and inoculated in vitro according to the method 
of Assy-Bah et al. (1987). Embryos were cul- 
tured on the medium defined by Assy-Bah et al. 
(1989), containing Murashige & Skoog's (1962) 
macro- and microelements, Morel & Wetmore's 
(1951) vitamins, 41 mg 1-l NaFeEDTA, 
lOOmgl-' sodium ascorbate and 8gl- '  agar 
(Labosi AL 540). For storage experiments, treat- 
ments consisted of media with different sucrose 
concentrations (O, 5, 10, 15, 20, 60 g 1-') and the 
presence or absence of activated charcoal 
(2gl-l,  Sigma C 5386). For recovery after the 
storage period, embryos were transferred to the 
same basal medium containing 60 g 1-' sucrose 
and 2 g 1-1 activat'ed charcoal. Transfers were 
then performed monthly. 
The embryos were cultured in 24 X 150 mm 
test tubes containing 20ml of medium, sealed 
with plastic cling film. For storage experiments 
(6 and 12 months in storage), embryos were 
placed on the various media at 27 2 1°C in the 
dark. For recovery after the storage period, the 
embryos on recovery medium were kept in the 
same conditions until the gemmule emerged. 
They were then exposed to a photoperiod of 12 h 
light/l2 h dark at 35 pmol m-2 s-'. Fourteen to 
49 embryos per treatment were used. 
During storage, all germinated embryos were 
eliminated. The number of living, nongermi- 
nated embryos was recorded at the end of each 
storage period and their fresh weight increase 
measured. The number of dead embryos was 
also recorded at the end of each storage period. 
Embryos which died during storage turned 
brown and necrosed progressively. The number 
of embryos that had developed a gemmule after 
2 months on the recovery medium was noted. 
The number of plantlets sufficiently developed to 
be transferred in vivo after 5 months in culture 
on the recovery medium was recorded only in 
the case of embryos subjected to 6 months of 
storage. 
Results 
Embryos stored fol: 6 months 
The frequency of germination during storage fell 
gradually from 94 to 0% in line with the reduc- 
tion in sugar concentration on media containing 
charcoal (Table 1). All other treatments led to 
almost total inhibition of germination (only O to 
6% germination) on media lacking charcoal, 
except with 60gl-I of sugar where half of the 
embryos germinated. Embryos that had not 
germinated increased in weight in line with the 
increase in sugar concentration and reached a 
large size at the end of the storage period only in 
the presence of charcoal (Fig. 1). Without 
charcoal, the increase in fresh weight was less 
marked (Fig. 2) and there were fewer differences 
between sucrose treatments. The death rate 
during storage was low (O to 2%), except on a 
medium containing neither sucrose nor charcoal, 
where over 20% of the embryos died in storage. 
Germination of embryos stored on a medium 
containing charcoal was satisfactory for the low- 
3 3  
-'i) 21 
Table 1. The effect of the presence of activated charcoal and concentration of sucrose in the medium on the germination, death, 
survival and fresh weight increase of embryos during a 6-month storage period and on their further development. 
With 2 g 1-I charcoal (sucrose g l-') Without charcoal (sucrose g 1-') 
O 5 10 15 20 60 O 5 10 15 20 60 
Number of embryos 44 38 40 46 49 49 46 45 39 48 49 49 
Germinated embryos (%) O 10 40 73 65 93 O O 2 4 4 40 
Non germinated living 100 89 57 24 33 6 78 100 97 94 96 59 
Fresh weight increase 155 221 278 311 290 331 126 93 70 107 110 170 
Embryos with gemmule 77 60 42 42 20 4 30 44 36 44 47 49 
Plantlets (5 months) (%) 63 63 50 24 26 4 4 49 23 29 35 51 
cultured 
Dead embryos (%) O O 2 2 2 o 21 O O 2 O O 
embryos (%) 
of living embryos (%) 
(2 months) (%) 
Fig. 2. Development of an embryo after a 6-month storage 
period on a medium containing 5 gl-' sucrose, devoid of 
activated charcoal. 
Fig. 1. Development of an embryo after a 6-month storage 
period on a medium containing 5gl- '  sucrose and 2gl- l  
activated charcoal (Bar = 1 cm). 
Embryos stored for 12 months 
est sugar concentrations (60% with 5 g 1-l suc- 
rose, 100% without sucrose) two months after 
transfer to the recovery medium. However, 
storage in the absence of charcoal led to a much 
lower germination rate (30 to 49%), except in 
the case of embryos stored on 60 g 1-1 of sugar. 
Five months after transfer to the recovery 
medium, 63% of the embryos stored on medium 
with charcoal and O or 5 g 1-1 sucrose gave rise to 
plantlets which were sufficiently developed to be 
transferred in vivo (Fig. 3). Results were lower 
for embryos stored under all other conditions. 
The presence of activated charcoal during stor- 
age increased the germination rate of embryos 
subsequently transferred to the recovery medium 
(Table 2). Only 33 and lo%, respectively of 
living embryos failed to germinate after 12 
months under the conditions optimal for 6 
months of storage (O and 5 g 1-1 sucrose). With- 
out charcoal, the number of embryos that germi- 
nated during storage was comparatively lower 
and decreased with decrease in the sugar concen- 
tration, from 82% with 60 g 1-1 sucrose down to 
0% with 5 g 1-1 sucrose. Without sugar, no em- 
bryos could survive this storage period. 
22 
Fig. 3. Plantlet obtained from an embryo stored for 6 
months on a medium devoid of sucrose and containing 2 g 1-’ 
activated charcoal, after 5 months of culture on the recovery 
medium. 
The percentage of germinated embryos was 
lower when storage had been performed in the 
presence of activated charcoal after 2 months in 
culture on the recovery medium. The highest 
percentages of germination (51 and 41%) were 
obtained with embryos kept on media containing 
15 and 5gl-I  sucrose, respectively, but no 
charcoal. Germination was delayed in compari- 
son with control, non-stored embryos when 
embryos were kept on a charcoal-free medium. 
Y 
Y 
However , fully developed plantlets could finally 
be obtained from almost all embryos at the end 
of the storage period (data not shown). 
Discussion 
In this study, we have shown that mature em- 
bryos of one variety of coconut can germinate 
and give rise to fully developed plantlets after up 
to 12 months of storage under slow growth 
conditions. 
Optimization of sucrose concentration and use 
of activated charcoal were shown to be critical to 
success. Sucrose is the carbon source in the 
medium. A previous report has shown that the 
optimal concentration of sucrose is 60 g I-’ for 
normal embryo development (Assy-Bah et al. 
1989). When the sucrose concentration in the 
medium was lowered, growth decreased, leading 
to the death of embryos after 12 months in 
storage. Germination of the embryos and their 
further development depended greatly on the 
presence of activated charcoal. Growth of the 
embryos was increased and they germinated 
progressively, even with a low sucrose content 
and with charcoal in the storage medium. With 
charcoal, the results were optimal for a 6-month 
storage duration, but too much growth occurred 
over 12 months since all embryos germinated 
before the end of this period. On the contrary, 
without charcoal, growth was lowered and 
germination delayed for low sucrose concentra- 
tions. A large proportion of embryos remained 
ungerminated and alive, even during one year in 
storage. 
Various authors have shown that activated 
charcoal is an essential medium additive for the 
germination and development of coconut em- 
bryos (De Guzman et al. 1971; Karunaratne et 
al. 1985; Assy-Bah et al. 1989; Rill0 & Paloma 
1990). Activated charcoal is widely used in tissue 
culture. Its beneficial effects are attributed to its 
property of adsorbing toxic substances produced 
by the tissues (Pierik 1987) as well as com- 
ponents of the medium, such as growth reg- 
ulators (Ebert & Taylor 1990) and to the fact 
that it may contain elements which are necessary 
to the growth of the cultures (Pierik 1987). With 
coconut embryos, activated charcoal may adsorb 
23 
Table 2. The effect of the presence of activated charcoal and the concentration of sucrose in the culture medium on the 
germination, death, survival and fresh weight increase of embryos during a 12-month storage period and on their further 
development. 
With 2 g I-' charcoal (sucrose g 1-I) Without charcoal (sucrose g I-') 
O 5 10 15 20 60 O 5 10 15 20 60 
Number of embryos 48 48 39 40 45 45 37 44 14 47 18 47 
Germinated embryos (%) 29 72 87 95 84 88 O O 14 19 38 82 
Non germinated living 33 10 13 O 4 11 O 100 86 81 61 17 
Fresh weight increase 99 204 190 O 288 245 O 126 131 137 124 121 
cultured 
Dead embryos (%) 37 16 O 5 11 o 100 O O O O O 
embryos (%) 
of living embryos (%) 
(2 months) (%) 
Embryos with gemmule 19 10 13 O 4 11 O 41 14 51 39 4 
a toxic substance produced by the embryos, 
which impedes their germination. 
A delay in embryo development was not 
observed in comparing those stored in the pres- 
ence of activated charcoal with non-stored con- 
trol embryos. Indeed, all embryos living after 6 
months in storage in the presence of charcoal 
developed into a whole plant within an average 
of 5 months after placement on the recovery 
medium (Assy-Bah et al. 1989) or developed a 
gemmule after 2 months, for embryos stored for 
12 months. Without charcoal in the storage 
medium, germination and development were 
delayed. However, almost all living embryos 
gave rise to fully developed plantlets, with a 
delay of 2 to 3 months in comparison with non 
stored embryos. 
Additional work is needed to confirm these 
preliminary results. From a more fundamental 
point of view, the exact role of activated char- 
coal should be more extensively studied. For 
wider application, the technique should be tested 
with other varieties of coconut. Also a two-year 
storage duration should be tested, which would 
have useful applications in indexing for diseases 
of the coconut, such as those induced by mycop- 
lasma-like organisms (Frison & Putter 1992), 
since they have a two-year incubation period. 
Finally, palms originating from control and 
stored embryos should be observed for their 
genetic stability. 
In conclusion, this study has shown that the 
medium-term storage of coconut embryos is 
feasible. The safe preservation and international 
movement of coconut germplasm may therefore 
be foreseen in the coming years. 
Acknowledgements 
The authors wish to express their thanks to the 
personnel of the Port Bouët Coconut Research 
Station (IDEFOR, Ivory Coast) for kindly supp- 
lying plant material. This experiment was con- 
ducted in the tissue culture laboratory of the La 
Mé Research Station (IDEFOR, Ivory Coast) 
under the supervision of Dr. T. Durand-Gas- 
selin, head of the laboratory. The help of Dr. 
L.A. Withers (IBPGR, Rome) is kindly ac- 
knowledged for her comments on the original 
manuscript. This work is supported by IBPGR. 
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Medium-term conservation of mature embryos of coconut

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