Réponse d'embryons immatures de cocotiers (7 à 8 mois après la pollinisation) à la congélation par azote liquide (-196¼C) : survie et germinatio

Assy-Bah, B.

Engelmann, Florent

Horizon / Pleins textes

o 
I 
CRYOPRESERVATION OF IMMATURE EMBRYOS 
OF COCONUT (Cocos nucìfera L.) 
B. Assy-Bah1 and F. Engelmann2* 
1 : IRMO, Institut de Recherche pour les Huiles et OlCaginem, Station de Recherche de 
La MC, 13 BP 969 Abidjan 13, Côte d'Ivoire. 
2 : ORSTOM (Institut Français de Recherche Scientifique pour le Dkveloppement en 
CoopCration), Laboratoire de Ressources Genktiques et d'hklioration des Plantes 
Tropicales, 911 Av. Agropolis, BP 5045,34032 Montpellier Ckdex 01, France. 
* 
to whom correspondence should be addressed 
SUMMARY : Immature embryos of coconut (7 to 8 months after pollination) can 
withstand rapid freezing in liquid nitrogen after 4 hours of pregrowth on a semi-solid 
medium containing 600 g.1-l glucose and 10 or 15% glycerol or sorbitol. In these 
conditions, survival ranged from 10 to 43% and one embryo developed into a rooted 
plantlet, 2.5 months after freezing. 
KEY-WORDS : coconut, Cocos nucifera L., immature embryos, cryopreservation, genetic 
resources conservation. 
INTRODUCTION 
The preservation of genetic resources of coconut is a prioritary objective for the 
development of present and future breeding programs (1). This task is made difficult 
due to the characteristics of the nut, which is recalcitrant and is very large (2). Efficient 
in vitro techniques have been developed tor collecting (3) and culturing coconut embryos 
(4, 5). The conservation of coconut genetic resources is presently performed through 
field collections. However, the safe conservation of coconut germplasm is impossible to 
consider without a long-term storage technique. Only cryopreservation in liquid nitrogen 
(-196°C) presently offers a long-term conservation option (6). 
Cryopreservation has already been applied to more than 70 different plant 
species (7). Freezing of zygotic embryos has been mentioned for around 10 tropical 
species (8). Among tropical oleaginous crops, oil palm embryos can survive freezing (9). 
Preliminary work has been carried out recently on the cryopreservation of coconut using 
mature embryos (10,ll). Bajaj (10) observed initial recovery through callusing followed 
by death on halves of transversely cut embryos. .With whole embryos, Chin et al. (11) 
obtained callusing and the normal gemination of one embryo, 15 months after freezing. 
In this paper, we present the first report of the successful cryopreservation of 
immature embryos of coconut. 
MATERIALS AND METHODS 
Plant material 
The planting material used consisted of immature embryos (7 to 8 months after 
pollination) from PB 121 seednuts (Malayan Yellow Dwarf x West African Tall hybrid). 
The embryos were sampled according to the method of Assy-Bah et al. (3). The embryos 
weighed 5 to 15 mg and measured 0.1 to 0.3 mm in diameter (Fig. 1). 
Methods 
In vitro culture 
The embryos were cultured on a medium containing the macro- and 
microelements of Monnier ( 2), the vitamins of Morel and Wetmore (13), 1 mg.1-l Fe 
EDTA, 34 g.1-l sucrose and 45 g.1-l glucose, 0.01 mg.1" biotin, 200 mg.1" glutamine and 
Caseine, 100 mg.1-I inositol and sodium ascorbate, 2 g.1-I activated charcoal, 7 g.1-l agar. 
The pH was adjusted to 4.6 before autoclaving. 
The embryos were cultured in 24 x 150 mm test tubes containing 20 ml medium. 
They were placed at 27 f 1°C in the dark, until the gemmule emerged. They were then 
exposed to a photoperiod of 12 hours on 24 at 35 pE.m-2.s-1. 
Cryopreservation 
The embryos were placed for 4 hours in Petri dishes on standard medium from 
which the mixture of sucrose and glucose was replaced with 600 g.1-I glucose and the 
68 
.ff which the mixture of sucrose and glucose was replaced with 600 g.1-l glucose and the 
activated charcoal was omitted. Regrowth on this medium was compared with 
pregrowth on medium supplemented with the cryoprotectants glycerol, sorbitol or 
polyethyleneglycol (PEG) 6000, at 5, 10 or 15 %. The embryos were then placed in 
sterile 2 Ilfl qotubes and immersed rapidly in liquid nitrogen. M'ter 24 hours at 
-196"C, they were thawed rapidly by immersion of the ampoules for 30 s in a 40°C 
water-bath and transferred on the standard medim. Eighteen to twenty embryos were 
used per treatment. Contro1 treatments involved recovery on standard medium with 
md without pregrowth/cryoprotection and with and Without exposure to liquid 
nitrogen. 
SULTS 
During the cryoprotective treatments, the water content of. the embryos, 
expressed in % of their fresh weight, dropped from 82 to an average of 12%. 
after one month, the survival of the control embryos was satisfactory for all the 
gregrowth conditions (Table l), ranging from 73 to 100%. No difference was noted 
with control embryos which were not submitted to the cryoprotective treatment (-prg). 
after freezing in liquid nitrogen, survival was obtained in three conditions only : 
pregrowth with 10 and 15% glycerol (25 and 10% survival respectively) and 10% 
sorbitol (43% survival). PEG showed no cryoprotective effect at the concentrations 
employed. 
After 2.5 months in culture, germination of up to 100% of the control embryos 
could be observed. The embryos first showed some browning (Fig. 2), then increased in 
size (Fig. 3) and eventually germinated (Fig. 4). Some of them showed abnormal 
morphology including hypertrophy of the gemmule (Fig. 5) or malformation of the 
haustorium (Fig. 6). 
In the case of frozen embryos, only those treated with 15% glycerol have 
developed to date and one rooted plantlet has been obtained 2.5 months after freezing 
and thawing (Fig. 7). The germination of embryos treated and frozen in the same 
conditions was observed in two additional independent experiments. In the other 
conditions where survival was noted, no germination has yet been obtained, although 
the embryos increased in size. 
DISCUSSION/CONCLUSION 
During this work, the survival and germination of immature embryos were 
obtained after a 4-hour pregrowth on a medium containing a very high concentration of 
69 
$ 
? 
The technique described differs from that generally employed for zygotic 6 ,  
embryos of several tropical species. Indeed, in the case of oil palm (9), rubber (141, 
cassava (15), Howea and Veitckia (16) and tea (17), embryos were simply dehydrated 
for up to 5 hours in the air current of a laminar flow cabinet before freezing. With 
coconut, Bajaj (10) and Chin et al. (11) submitted the embryos to pregrowth in liquid 
medium containing cryoprotective substances (7% sucrose and dimethylsulfoxide in the 
first case, 10% dimethylsulfoxide in the second). Chin et al. (11) utilized a technique 
more similar to that usually empbyed in standard cryopreservation protocols, since 
after the cryoprotective treatment, the embryos were parfially dehydrated for one hour 
in the laminar flow before freezing. Inbur work, the embryos were too small for a 
precise control of their dehydration in air. Therefore we used pregrowth on semi-solid 
medium enriched with cryoprotectants. The very high concentrations of cryoprotectants 
employed were close to those used during the vitrification process (18,19) and induced 
a drastic dehydration of the embryos. It would be interesting to conduct thermal 
analysis during the freezing of the embryos in order to know if a vitrous transition or 
crystallization is taking place. 
The advantage of using immature embryos instead of mature ones is that they 
are much smaller, less differentiated and may be more likely to survive as a whole. 
However the drawback of utilizing such young embryos is that the conditions of their in 
vitro culture are not yet perfectly mastered. Unlike mature embryos, not all immature 
ones will survive, develop and germinate in vitro. 
In conclusion, this work demonstrated that it is possible to obtain the survival 
and the germination of immature embryos of coconut after freezing in liquid nitrogen. 
Trials are under way in order to improve the efficiency of the process and to try to 
adapt it to mature embryos. The long-term conservation of the genetic resources of 
coconut should thus be possible in the near future. 
ACKNOWLEDGEMENTS 
The authors wish to express their thanks to the personnel of the Marc Delorme 
Research Station (IRHO, @&e d‘Ivoire) for kindly supplying the plant material and to 
Dr. L.A. Withers for helpful comments on the manuscript. This work is supported by 
BPGR. 
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71 
Table 1 : Survival of control (-LN) and frozen (+LN) embryos after one month in culture. -pgr : no cryoprotective treatment ; 
+pgr : pregrowth with 600 g.1-* glucose only ; * : contaminated. 
I -PIF I +wl GLYCEROL (%) I SORBITOL (%) 1 PEG (9%) I 
l 1 2 1  5 1  n o l i s ]  2 I 5 I l O I 1 5 1 2 1  5 I 10 I 15 I -LN I 17/20 I 20/20 I 18/19 I 19/20 I 17/18 I 11/15 I 19/19 ] 19/19 I 17/18 ] * I 19/19 I 14/18 I 17/20 I * 
85% 100% 95% 95% 94% 73% 100% 100% 78% 85% 100% 94% 
0% 0% 0% 0% 29% 10% 0% 0% 43% 0% 0% 0% 0% 
+LN 0/2Q 0120 0119 0120 5/20 2/20 0120 0120 317 0120 0120 0120 0120 * 
5 mm 5 mm 
Figure 1 : Freshly dissected embryo. 
Figure 2 : Browning of a control embryo after 15 days in culture. 
5 mm 5 mm 
.Figure 3 : Increase in size of a control embryo after 3 weeks in culture. 
Figure 4 : Germination of a control embryo, after 8 weeks in culture. 
73 
Figure 5 : Abnormal development of a control embryo : hypertrophy of the gemmule. 
Figure 6 : Abnormal development of a control embryo ; hypertrophy of the haustorium. 
Figure 7 : Plantlet obtained from a frozen embryo after 2.5 months in culture. 


Cryopreservation of immature embryos of coconut (Cocos nucifera L.)

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