Assy-Bah, B.

Engelmann, Florent

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$ . “ r  C r y o - L e t t e r s  13, 117-126(1992) ,  
p u b l i s h e d  b y  C r y o - L e t t e r s ,  7 ,  Wootton Way, Cambridge CB3 9LX, U.K. 
CRYOPRESERVATION OF MA= EMBRYOS OF COCONUT 
(COCOS NUCIFERA L) 
AND SUBSEQUENT REGENERATION OF PLANTLETS 
B. Assy-BahlJ and F. Engelmann2* 
1: IRHO, Institut de Recherches pour les Huiles et OlCagineux, Station de La MC, 13 
BP 969 Abidjan 13, e6te d’Ivoire. 
2 ORSTOM (Institut Français de Recherche Scientifique pour le Dkveloppement en 
CoopCration) 911 av. Agropolis, BP 5045,34032 Montpellier CCdex, France. 
* to whom correspondence should be addressed 
SUMMARY: Mature embryos (10 to 12 months after pollination) of four varieties of 
coconut (hybrid PB 121, Cameroon Red Dwarf, Indian Tall, Rene11 Tall) could 
withstand cryopreservation in liquid nitrogen and develop into plants. Pretreatment 
consisted of a 4-hour desiccation in the air current of a laminar flow cabinet followed by 
a 11 to 20-hour culture on a medium containing 600 g.1-1 glucose and 15% glycerol. 
Freezing and thawing were carried out rapidly. Recovery rates varied between 33 and 
93% of frozen embryos, depending on the variety. 
KEY WORDS: Coconut, Cocos nucifera, mature embryos, cryopreservation, 
pretreatment, genetic resources conservation. 
INTRODUCXION 
The safe preservation of the genetic resources of coconut, which is a species with 
recalcitrant seeds (l), has been defined as a priority objective for the present and future 
development of coconut breeding programs (2). For eight years, ORSTOM and IRHO, 
ORSTOM Fonds, Documentaire 
in connexion with IBPGR (International Board for Plant Genetic Resources, Rome) 
have developed a research programme which led to the establishment of routine 
techniques for field collecting (3) and in vitro culture of coconut embryos (49 5). For 
long-term storage, a cryopreservation process has been developed recently using 
immature embryos (6). However, the drawback of the utilization of immature embryos 
is that, unlike mature ones, the conditions for their in vitro culture are not yet perfectly 
mastered (7). As a result, only a limited proportion of h a t u r e  embryos germinate and 
give rise to plants. Therefore, we have sought to develop a cryopreservation technique 
for mature embryos adapted from that set up for immature ones. This article presents 
the first results obtained in the cryopreservation of mature embryos from four different 
varieties of coconut. 
MATERIALS AND METHODS 
Plant material 
Tbe plant material used consisted of mature embryos (10-12 months after 
pollination) from seednuts of four varieties: the hybrid PB 121 (Malayan Yellow Dwarf 
x West African Tall), Indian Tall (IT7), Cameroon Red Dwarf (CRD) and Rene11 Tall 
(RT). The embryos were sampled according to the method of Assy-Bah et al. (3). The 
average initial weight of the embryos was 98.6 2 13.8 mg for PB 121, $1.6 2 12.4 mg for 
CRD, 112.3 k22.2 mg for IT7 and 146.5 235.4 mg for RT. 
Methods 
Jn vitro culture 
The embryos were cultured on the medium defined by +-Bah et al. (5) 
containing Murashige and Skoog's macro and microelements (8), Morel and Wetmore's 
vitamins (9), 41 mg.1-1 FeEDTA, 100 mg.1-1 sodium ascorbate, 60 g.1-1 sucrose, 2 g.1-1 
activated charcoal. The pH was adjusted to 5.5 before autoclaving. Transfer to new 
medium was carried out monthly. 
The embryos were cultured in 24 x 150 mm test tubes containing 20 ml of 
medium. They were placed at 27 21°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-l. 
Crvomeservation 
For pretreatment, the embryos were placed on open Petri dishes without culture 
medium and dehydrated for 4 hours in the air current of a laminar flow cabinet at room 
temperature. They were then transferred in open Petri dishes on the medium developed 
118 
I 
/ 
i. for the pretreatment of immature embryos of coconut ( 5 )  containing 600 g.1-1 glucose 
and 15% glycerol, and dehydrated for an additional period of 11 to 20 hours. Thus, the 
total duration of the pretreatment ranged from 15 to 24 hours. The embryos were then 
placed in 2 ml sterile cryotubes and immersed rapidly in liquid nitrogen. After 24 hours 
at -196°C they were thawed rapidly by plunging the ampoules into a 40°C water-bath 
for 2 min and then transferred on the standard medium. 13 to 25 embryos were used per 
treatment. Control treatments involved recovery on standard medium with and without 
desiccation and with and without exposure to liquid nitrogen. 
Water content measurement 
The water content of the embryos (expressed in % of their initial fresh weight) 
was monitored during pretreatment. Fresh weight measurement was performed on 
batches of 10 embryos before pretreatment and after dehydration for different periods 
of time. Dry weights were determined after 48 hours desiccation in an oven at 1CnOC. 
sessment of recovery 
SuMval of embryos was recorded after 1 month of culture. Embryos were 
considered alive when regrowth had been observed. After 3 months in culture, the 
development of the surviving embryos was examined by assessing the number of 
embryos having developed a gemmule (the length of which was measured) and at least 
one root. 
RESULTS 
The initial water content of the embryos was very similar in all 4 varieties 
(Table 1). It decreased rapidly during the first 15 hours of pretreatment, from an 
average of 78.4% to an average of 11.4%. The largest embryos (RT and IT) dehydrated 
more slowly and the smallest ones (CRD) more rapidly. Between 15 and 24 hours of 
pretreatment, the rate of dehydration slowed down. Over this period, the water content 
was lowered by an average of only 1.8 (average water content of 6.4 after 24 hours). 
Survival of unfrozen embryos was 100% without pretreatment (Table 1) and only 
slightly decreased after 24 hours of pretreatment. With frozen embryos, no suMval was 
observed without pretreatment. SuMval increased with increasing pretreatment 
durations for PB 121, RT and IT7 embryos. For 15 hours of Pretreatment, survival 
ranged between 6.6% (RT and IT7) and 35.3% (PB 121) and reached 85.7 (RT) to 
92.8% (PB 121) after 24 hours of pretrsatment. In the m e  of CRD embryos, suMval 
was greatest after 17 hours of pretreatment (73 ) and decreased for longer 
pretreatment durations. The haustorium of frozen embryos browned rapidly after 
thawing and did not develop further, contrarily to unfrozen controls (Fig.1). 
TaMe 1 : Water content (+ S.D.) and survival after 1 month culturing of control (-IN) and 
frozen (+IN) embryos of coconut as a function of the pretreatment duration. 
Fig. 1 : Development of control (C) and frozen (F) embryos after 1 month culturing. H: haustorium. 
Fig. 2 Development of plantlets originating from control (C) and frozen (F) embryos 
after 3 months culturing. 
Fig. 3: Development of a plantlet originating from a cryopreserved embryo after 4.5 
months culturing. 
121 
Table 2: Development-of control embryos after 3 months culturing as a function of the pretreatment 
duration. 
Table 3: Development of cryopreserved embryos after 3 months culturing as a function QI 
pretreatment duration. *: contamlnated. 
Rootedembryos I 
422 
'the 
Without pretreatment treatment, the development of unfrozen embryos was 
generally satisfactory (Table 2). 100% rooted plantlets were obtained with PB 121 and 
RT, 93% with CRD, but only 69% with IT7 embryos. With unfrozen pretreated 
embryos, the delay in their development was different among the varieties. With PB 121 
and RT, the number of developing embryos was slightly lowered only after a 24-hour 
pretreatment (73% for PB121,78% for RT). Rooting was also lowered and the average 
size of the gemmule drastically reduced. With CRD and IT7, the percentage of 
developing embryos was reduced much more for the same pretreatment duration (33% 
for CRD and 29% for IT7). The percentage of rooted embryos was not modified 
compared to untreated controls. However, the development of the gemmule was 
different between those two varieties: a similar decrease was observed with PB 121, RT 
and RT, whereas no change was observed with IT7 compared with untreated controls. 
4 
With frozen embryos (Table 3) the number of developing plants increased with 
the pretreatment duration in the case of PB 121 and IT7. With CRD, the optimal 
pretreatment duration was 17 hours. The highest percentage of developed embryos 
varied greatly between varieties, ranging from 33% with RT to 93% with PB 121. Under 
optimal pretreatment conditions, no decrease was observed in the recovery rate 
between unfrozen and frozen embryos of all varieties except RT. The average size of 
the gemmule of frozen embryos was lower than that of control embryos for non optimal 
pretreatment durations. It increased progressively with the pretreatment duration. The 
number of rooted plantlets was higher than that of unfrozen controls with PB 121, RT, 
IT7 and identical with CRD. The further development of the plantlets originating from 
cryopreserved embryos was comparable to that of the unfrozen controls (Fig. 2) and 
plantlets with fully expended leaves could be obtained after 4.5 months culturing 
(Fig. 3). 
DISCUSSION/CONCLUSION 
The technique established gave high survival rates and rapid regrowth and 
development in a large number of mature cryopreserved embryos of four varieties of 
coconut. Considerable improvements have been made in comparison with previous 
work on the same material. Bajaj (10) observed only transient callusing on halves of 
transversally cut embryos. Chin et al. (11) could regenerate only one whole coconut 
palm, 15 months after the cryopreservation of the embryo. More recently, Assy-Bah and 
Engelmann (6) obtained up to 43% survival after freezing of immature embryos, but 
only a limited number of plants were regenerated. 
This technique is original since pretreatment combines desiccation of the 
embryos in the air current of a laminar flow cabinet and the culture on a medium with 
high levels of cryoprotective substances. Such a combination has been employed up to 
now only with embryonic axes of pea (12). Indeed, pretreatment of zygotic embryos 
generally uses desiccation in an air flow (13) and pregrowth in the presence of 
123 
4' 
ì, 
P cryoprotective mediùm only in a limited number of cases, such as Capsella bumapastorii 
(14), JugZans (U), Pnuzus (16) and immature embryos of coconut (6). 
The pretreatment conditions set up for mature embryos of coconut were very 
drastic compared with those used for all other species: dehydration was very intense 
since the water conteht of the embryos was lowered from a high initial level (78.4%) to 
only 6.496, which gave maximal survival in three .of the varieties after cryopreservation. 
The duration of the pretreatment was very long (15 to 24 hours) certainly necessitated 
by the large size of the embryos. A recent review on cryopreservation of embryos (13) 
indicated that the initial water content of embryos is generally between 50 and 60% and 
maximal survival is observed with a higher water content (10 to 16%) reached after only 
2 to 4 hours dehydration. Further desiccation generally leads to a dramatic loss of 
viability probably caused by severe dehydration injury. However, coconut embryos are 
formed of two distinct parts, the haustorium and the meristematic pole, which probably 
have different water contents. Indeed, the tissues of the haustorium comprize mainly 
highly vacuolated cells. It would be interesting to measure their dehydration separately. 
Indeed, the fial water level of the meristematic pole may be closer to that generally 
observed with embryos of other species. The optimal dehydration duration varied 
between the coconut varieties tested in relation with the initial weight of the embryos: 
for the smallest ones (CRD) the maximal survival was obtained after 17 hours 
pretreatment, whereas it was observed after 24 hours with the other varieties which 
have larger embryos. 
The pretreatment and freezing conditions used in this study (high levels of 
cryoprotective substances causing a low water content compatible with freezing at a 
rapid rate) were similar to that used during a vitrification process (17,18). Vitrification 
may take place during freezing of coconut embryos and this could explain the high 
suMval rates obtained. Thermal analysis conducted during the freezing of the embryos 
would show if a vitrous transition or crystallization is taking place. 
The detrimental effects of the preculture were more apparent after 3 months in 
culture th= 1 month. With control embryos, the rate of development was mainly 
affected and not the survival. The effects of preculture were different between varieties: 
the delay was more pronounced with rapidly growing varieties like PB 121. On the 
contrary, with IT7, which grows very slowly, no difference was observed between 
untreated controls and pretreated embryos. 
With frozen embryos, similar delays were observed in comparison with 
pretreated controls, particularly with embryos which had not been pretreated in the 
optimal conditions, i.e. not enough or too much dehydrated. Differences appeared also 
between varieties, in relation with the size of the embryos The main difference in the 
development of frozen embryos compared with unfrozen controls was that growth of the 
haustorium was suppressed. Its tissues which are formed of highly vacuolated cells are 
completely destroyed during freezing. This is in accordance with the previous 
124 
observations of Chin et al (19) in the case of Howea and Veitchia zygotic embryos. 
The technique described in this paper is efficient since the percentage of 
embryos which are capable of developing into whole plants after cryopreservation is 
high with the 4 varieties tested. Moreover the development of cryopreserved embryos is 
normal. It is only delayed of one to two months, compared with control untreated 
embryos. 
However, this process has to be improved in several ways. The pretreatment 
duration has to be adapted to the different varieties and its conditions standardized. 
More research is needed to check to which extent the successive use of desiccation and 
pretreatment on cryoprotective medium is necessary. Finally, additional trials should be 
performed with larger numbers and new varieties of coconut. 
In conclusion, these preliminary experiments indicate that the safe long-term 
conservation of the genetic resources of coconut should be possible in the near future 
using cryopreservation. 
ACKNOWLEDGEMENTS 
The authors wish to express their thanks to the personnel of the Marc Delorme 
Resedrch Station (IRHO, CGte d'Ivoire) for kindly supplying the plant material. These 
experiments were performed in the tissue culture laboratory of the La M6 Research 
Station (IRHO, a t e  d'Ivoire). The in vitro culture of the embryos was supervised by 
Dr. T. Durand-Gasselin, head of the laboratory. The help of Dr. LA. Withers for her 
&m"mnts on the manuscript is gratefully acknowledged. This work is supported by 
IBPGR. 
REFERENCES 
1 M.W. King and E.H. Roberts, in "Recalcitrant Crop Seeds", H.F. Chin and E.H. 
Roberts Eds., Tropical Press Sdn. Bhd., Kuala Lumpur, Malaysia, 53-89 (1980). 
2 IBPGR , IBPGR Advisory Committee on In Wro Storage, Report of the Second 
Meeting, IBPGR Secretariat, Rome, 15 p (1985). 
3 B. Assy-Bah, T. Durand-Gasselin and C. Pametier, FAO/IBPGR Plant Genetic Res. 
Newslett., 71,4-10 (1987). 
4 B. Assy-Bah, 016agineux, 41,321-328 (1986). 
5 B. Assy-Bah, T. Durand-Gasselin, F,Engelmam and C. Pannetier, OEagineux, 44, 
6 B. Assy-Bah and F. Engelmann, Cryo-Lett., 13, (in press). 
7 F. Engelmann and B. Assy-Bah, in Proc. Intl. Workshop on Coconut Genetic 
Resources, Cipanas, Indonesia, 8-11 October 1991, (in press). 
515-523 (1989). 
125 
8 T. Murashige and F. Skoog, Physiol. Plant., 15,473-497 (1962). 
9 G. Morel and R.M. Wetmore, Am. J. Bot., 38,141-143 (1951). 
10 Y.P.S. Bajaj, Curr. Sci., 53,1215-1216 (1984). 
11 H.F. Chin, B. Krishnapillay and Y.L Hor, Pertanika, 9,299-303 (1989). 
12 DJ. Mycock, P. Berjak, F.C. Blakeway and P. Watt, in Proc. Intl. Conf. 'The Impact 
of Biotechnology in ~gridture", Amiens, France, 10-12 july 1989, CPl(l989). 
13 F. Engelmann, in Proc. XIIth Eucarpia Cong., "Reproductive Biology and Plant 
Breeding", Angers, France, 6-10 july 1992, (in press). 
14 M. Monnier and C. Leddet, Bull. Soc. Bot. Fr., 127,257-264 (1980). 
15 M.T. de Boucaud, M. Brison, C. Ledoux, C. Germain and A. Lutz, Cryo-Lett., 12, 
16 M.T. de Boucaud and M. Brison, in Proc. Cry0 91, Leuven, Belgium, 7-12 july 1991, 
17 A. Sakai, S. Kobayashi and I. Oiyama, Plant Cell Rep., 9,30-33 (1990). 
18 R. h g i s  and P. Steponkus, Plant Physiol., 92,666-671 (1990). 
19 H.F. Chin, B. Krishnapillay and .C. Almg, wo-Lett., 12,31-36 (1988). 
163-166 (1991). 
102 (1991). 
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Cryopreservation of mature embryos of coconut (Cocos nucifera L.) and subsequent regeneration of plantlets

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