Coconut Research Institute Repository

Ramanayaka, R.A.M.K.

Wijerathne, V.

Yalegam, L.L.W.C.

Biochemical study on germinated coconuts

Perera, P.I.P.

Enhancing effect of TDZ on callogenesis of coconut  (Cocos nucifera L) ovary culture

Fernando, L.C.P.

Manoj, P.

Hapuarachchi, D.C.L.

Edgington, S.

Evaluation of four isolates of Hirsutella thompsonii against coconut mite (Aceria guerreronis) in Sri Lanka

Jayasekara, C.

Gunathilake, K.D.P.P.

Processing technologies for virgin coconut oil and coconut based confectionaries and beverages

Peiris, T.S.G.

Hensen, J.W.

Lareef, Z.

Use of seasonal climate information to predict coconut production in Sri Lanka

Senarathne, S.H.S.

Vermicompost production in coconut lands

Fernando, M.T.N.

Zubair, L.

Ranasinghe, J.

Economic value of climate variability impacts on coconut production in Sri Lanka

Bandupriya, H.D.D.

Fernando, S.C.

Verdeil, J-L.

Malaurie, B.

COCOS (2007), 18  58 - 66 
Printed in Sri Lanka 
EFFECT OF ABSCISIC ACID ON SURVIVAL AND RECOVERY OF 
CRYOPRESERVED PLUMULE EXPLANTS OF COCONUT (COCOS 
NUCIFERA L.) 
 
H D D Bandupriya1, S C Fernando1, J-L Verdeil 2, B Malaurie3
 
1Coconut Research Institute, Lunuwila, Sri Lanka; 2CIRAD, TA40/02 Avenue 
Agropolis, 34398 Montpellier Cedex 5 - France;  3UMR 1098 BEPC, 
Institute for Research and Development (IRD), BP 64501, 911 Av Agropolis 
34394, Montpellier, France. 
  
ABSRACT 
 
Cryopreservation has great potential for long-term storage of coconut genetic 
resources. The recent development of the encapsulation-dehydration 
cryogenic technique dramatically facilitated progress with cryopreservation. 
The present study tested the effect of abscisic acid in the encapsulation-
dehydration method for cryopreservation of coconut plumules. Plumules 
were excised from mature zygotic embryos of Coconut var. Sri Lanka Tall 
and encapsulated in calcium alginate. The alginate beads were used to test 
effect of different cryoprotection pretreatments (mixtures of 0.75 M sucrose 
and abscisic acid (ABA) at concentrations of 0, 10, 20 and 40 µM), and 
exposure to silica gel for 16 hours for further dehydration, prior to freezing 
in liquid nitrogen. Effect of the treatments without freezing was also tested. 
The water loss from alginate beads following sucrose incubation and 
dehydration was monitored, and the survival and growth of plumules 
assessed at different stages.  
 
Addition of ABA to the sucrose pretreatment medium increased the survival 
rate of unfrozen plumules from 60% to over 80%. Pretreatment with 40 µM 
ABA gave the highest survival rate (84%) of frozen plumules. The recovery 
rate of plumules (frozen and unfrozen) pretreated without ABA was very low 
(6-8%). The addition of ABA increased the recovery rate significantly; 
frozen plumules that had been treated with 40 µM ABA recorded a 39% 
recovery rate. The improved recovery could be ascribed to increased water 
loss (69%) in plumules treated with 40 µM ABA.  
 
Key words: Coconut, plumule, cryopreservation, abscisic acid 
 
 
 
 58
INTRODUCTION 
 
The coconut palm (Cocos nucifera L.) is generally cultivated in the tropical 
regions both as large-scale plantations and in home gardens. The coconut 
palm yields a wide range of products and has been extensively cultivated 
over a long period of time. Consequently, many ecotypes have emerged 
through selection both by man and nature. Conservation of this genetic 
diversity is crucial for the development of future breeding programs before 
the ongoing genetic erosion takes its toll.  
 
Coconut genetic resources are presently maintained ex-situ in the field at 
great cost and subject to adverse weather conditions, pests and diseases. 
Cryopreservation is an important alternative method for long-term 
conservation of coconut germplasm. Cryopreservation reduces cell and tissue 
deterioration by virtually halting metabolism when they are stored at ultra 
low temperatures, usually that of liquid nitrogen (-196ºC).  
 
Coconut is propagated only by seeds. Coconut seed is recalcitrant in nature 
and large in size. Recalcitrant seeds cannot tolerate dehydration to moisture 
levels that would permit exposure to very low temperatures (Engelmann, 
2000). This is no doubt a constraint to using cryopreservation for conserving 
coconut genetic resources. 
 
Successful cryopreservation of mature zygotic embryos of coconut, using the 
pre-growth desiccation method, was reported by Assy-Bah and Engelmann 
(1992). However, only a few genotypes were tested. The large size of the 
embryo limits experimentation with other techniques such as those that 
require encapsulation. Coconut plumule i.e. the embryo meristem and first 
leaves, which is known to be free of viral diseases, is an interesting starting 
material for cryopreservation (Malaurie et al., 2002). Cryopreservation of 
plumules is vital because they are small in size (around 1.0 mm) and abound 
in meristematic cells. Using the encapsulation-dehydration technique, 
coconut plumules have been successfully cryopreserved but the percentage 
recovery is low (N’Nan, 2004). 
 
Artificial cryoprotection by incubation in sugar is generally required for 
recalcitrant species like coconut. Dehydration tolerance can be increased by 
adding abscisic acid (ABA) to the culture medium (Anandarajah and 
McKersie, 1990; Senaratna et al., 1991). ABA is also known to induce 
freezing tolerance in various species (Chen and Gusta, 1983). In the present 
study, the effect of ABA on the survival and recovery of coconut plumules, 
cryopreserved by the encapsulation-dehydration technique, was tested. 
 59
METHODOLOGY 
 
Mature zygotic embryos were extracted from 12 month-old seed nuts of the 
coconut variety Sri Lanka Tall. Plumules were excised from the embryos in a 
laminar airflow cabinet under a stereo microscope and precultured, for 4-7 
days, in Eeuwens Y3 growth medium (Eeuwens, 1976). The precultured 
plumules were encapsulated in 3% (w/v) sodium alginate and pretreated for 
three days in liquid Eeuwens medium, enriched with 0.75 M sucrose and 
ABA (at concentrations of 10, 20 and 40 µM), on a rotary shaker (90 rpm), at 
27ºC in the dark. After sucrose pretreatment, the beads were blotted on filter 
paper to remove surface moisture, weighed, and then dehydrated for 16 hours 
in glass jars with 20 plumules placed on top of 40 g silica gel separated by 
filter paper. The dehydrated beads were weighed, and half were transferred 
to sterile polypropylene cryotubes and directly plunged into liquid nitrogen. 
After at least 2 hours storage in liquid nitrogen,the tubes were warmed in a 
water bath at 40 ºC for 3 min. These frozen beads, and the unfrozen beads 
(subjected to pretreatment and dehydration only) were cultured in a recovery 
medium (Eeuwens Y3 growth medium).   
 
Encapsulated plumules (without any pretreatment, dehydration or freezing) 
were also cultured in Eeuwens Y3 growth medium, soon after encapsulation, 
as an untreated control. 
 
 The survival of plumules was assessed after two months in culture. A 
plumule manifesting new tissue growth, indicated by any sign of regrowth 
such as swelling, development of new leaf primordia and/or callus formation, 
was counted as a survivor. The recovery of plumules was assessed after 4 
months in culture. A plumule that grew and developed into a plantlet was 
counted as having recovered. The experimental design was a complete 
randomized design with three replicates, and 10 plumules per treatment. 
Arcsine transformed data was analysed using the GLM procedure (SAS 
Institute, USA) 
 
RESULTS AND DISCUSSION 
 
A critical parameter in cryopreservation is the removal of intracellular water, 
to prevent ice crystals forming during freezing or thawing and consequent 
cell damage (Fang et al, 2004). This necessitates the pretreatment of 
encapsulated plumules with cryoprotectants such as  sugars, sugar alcohols, 
DMSO etc.  
 
 60
Irrespective of ABA treatments, all the plumules that were not subjected to 
dehydration and cryostorage survived and developed into normal plantlets; 
and none of the plumules not subjected to dehydration, survived cryostorage 
(Table 1 and 2). The survival rate of dehydrated but unfrozen plumules 
varied from 60% to 84%. Very high survival rates (>80%) were observed in 
dehydrated, unfrozen plumules pretreated with 10-40 µM ABA, clearly 
indicating the positive effect of ABA on their survival. Similarly, 
pretreatment with ABA increased the survival rate of dehydrated, frozen 
plumules from 57% without ABA to 84% with 40 µM ABA.   
 
With both frozen and unfrozen, dehydrated embryos, the positive effect of 
ABA on recovery was quite clear. When they were pretreated only with 
sucrose (zero ABA) the recovery rate was very low but increased remarkably 
when ABA was added to sucrose (Table 2). Frozen plumules recorded the 
highest rate of recovery (39%) when pretreated with sucrose containing ABA 
at a concentration of 40 µM.  
 
Table 1: Effect of ABA concentration on survival of encapsulated plumules 
following dehydration and cryopreservation 
 
 
Treatment 
 
 Dehydration in hours 
 
 
 
Sucrose (M) + ABA 
(µM) 
                 No 
dehydration 
 
     Dehydration for 16 
hours 
 -LN +LN -LN +LN 
0.75 +   0 100 ± 0 0 ± 0 60 ± 12 57 ± 14 
0.75 + 10 100 ± 0 0 ± 0 82 ± 6 68 ± 15 
0.75 + 20 100 ± 0 0 ± 0 85 ± 8 39 ± 20 
0.75 + 40 100 ± 0 0 ± 0 84 ± 8 84 ± 5 
 
Values represent survival percentage ± SE.  
P (survival) < 0.0001; treatment differences were evaluated at 5% 
probability.  
LN:liquid nitrogen  
 
In this experiment, dehydration of plumules was initially by the sucrose 
pretreatment and then by exposure to silica gel for 16 hours. Water loss in 
plumules was lowest (62%, fresh weight basis) when pretreated with 0.75M 
sucrose without ABA, and highest (69%, fresh weight basis) when pretreated 
 61
with 0.75M sucrose containing ABA at a concentration of 40 µM. The 
increase in recovery rate of plumules with the latter pretreatment appears to 
be a result of increased dehydration. 
 
The influence of ABA and sugars on the tolerance of tissues to dehydration 
and cryopreservation has received much attention (Dumet et al., 1993; 
Nieves et al., 2001). Sucrose infiltrating into tissues helps to maintain their 
viability during dehydration and cryopreservation by stabilizing cell 
membranes. Sucrose, by replacing water, maintains membrane phospholipids 
in the liquid crystalline phase (Hoekstra et al., 1991), and prevents structural 
changes in soluble proteins (Carpenter et al., 1987). Sucrose, due to its solute 
properties, also promotes glass phase formation in the cytoplasm (Koster, 
1991).  ABA plays an important role in desiccation tolerance of several plant 
species (Senaratna et al., 1989; Shiota et al., 1999). ABA is associated with 
synthesis of proteins (Seijo, 2000) and compatible solute components, which 
are important in plant stress tolerance (Fang et al., 2004). A combination of 
ABA and sucrose treatments improved the survival of cryopreserved cocoa 
somatic embryos (Fang et al., 2004).  
 
Table 2: Effect of ABA concentration on recovery of encapsulated plumules 
following dehydration and cryopreservation 
 
Treatment 
 
 
Dehydration in hours 
 
 
Sucrose(M)+ ABA 
(µM) 
No dehydration           
 
Dehydration for 16 
hours 
 -LN +LN -LN +LN 
0.75 +   0   93 ± 4 0 ± 0   6 ± 6   8 ± 4 
0.75 + 10 100 ± 0 0 ± 0 45 ± 15 17 ± 9 
0.75 + 20 100 ± 0 0 ± 0 43 ± 2 23 ± 12 
0.75 + 40 100 ± 0 0 ± 0 45 ± 9 39 ± 6 
 
Values represent recovery percentage ± SE.  
P (recovery) < 0.0001; treatment difference were evaluated at 5% 
probability.  
LN:liquid nitrogen  
 
 
 
 
 62
Table 3: Mean percent water loss from plumules pretreated in sucrose and 
dehydrated with silica gel. 
  
Concentration of ABA (µM) in 
the sucrose solution 0 10 20 40 
Mean percentage of water loss 
(fresh weight basis) 62 67 68 69 
 
                                                                                                                                                    
N’Nan (2004) and Malaurie et al. (2002) using the encapsulation-
dehydration method with different sucrose concentrations and dehydration 
periods, achieved about 40-70% survival and about 20% recovery of coconut 
plumules after cryopreservation. In the present study, the optimum sucrose 
concentration and dehydration duration reported by N’Nan (2004) were used 
to investigate the influence of ABA on the survival and recovery of 
cryopreserved plumules. The preliminary results are encouraging and 
indicates the feasibility of using ABA for effective cryopreservation of 
coconut germplasm. It is likely that the recovery rate could be further 
improved by refining the protocol. 
 
ACKNOWLEDGEMENT 
 
Authors gratefully acknowledge the financial assistance provided by the 
IRD, through a research project funded by Bureau des Ressources 
Genetiques, Paris, France (BRG). Thanks are also due to all staff members of 
Lab Genetrop, IRD, France and Tissue Culture Division, CRI, Sri Lanka for 
their assistance in carrying out the experiments. We acknowledge the 
valuable comments of Dr L K Weerakoon, Head Tissue Culture Division on 
the manuscript. Thanks are also extended to Dr. T S G Peiris and Mr. 
J.D.J.S. Kularathne of the Biometry Division, CRI for their assistance in the 
statistical analysis of data.  
 
 
 
 
 
 
 
 
 
 
 63
REFERENCES 
 
Anandarajah, K. and McKersie, B. D. (1990). Manipulating the desiccation 
tolerance and vigor of dry somatic embryos of Medicago sativa L. 
with sucrose, heat shock and abscisic acid, Plant Cell Reports 9:451-
455. 
 
Assy-Bah and Engelmann, F. (1992). Cryopreservation of mature embryos of 
coconut (Cocos nucifera L.) and subsequent regeneration of plantlets, 
Cryo-letters 13,117-126. 
 
Blake, J. (1995). A brief history of coconut tissue culture, In: Oropeza, V., 
Howard, F. W., and Ashburner, G. R., (eds.) Lethal yellowing: 
research and practical aspects. Dordrecht, The Netherlands: Kluwer 
Academic Publishers, 217–223. 
Carpenter, J. F., Crowe, L. M. and Crowe, J. H. (1987). Stabilization of 
phosphofructokinase with sugars during freeze-drying: 
characterization of enhanced protection in the presence of divalent 
cations, Biochim. Biophys. Acta 923:109-115.  
Chen, T. H. H. and Gusta, L. V. (1983). Abscisic acid induced freezing 
tolerance in cultured plant cells, Plant Physiology 73: 71-75 
 
Dumet, D., Engelmann, F., Chabrillange, N., Duval,Y. and Dereuddre, J. 
(1993). Importance of sucrose for the acquisition of tolerance to 
desiccation and cryopreservation of oil palm somatic embryos, Cryo-
Letters 14:243-250 
 
Eeuwens, C. J. (1976). Mineral requirements for growth and callus initiation 
of tissue explants excised from mature coconut palm (Cocos 
nucifera) and cultured in vitro, Physiol. Plant. 36:23-28 
 
Engelmann, F. (2000). Importance of cryopreservation for the conservation 
of palnt genetic resources, In: Engelmann, F. and Takagi, (eds.) 
Cryopreservation of tropical plant germplasm. Current research 
progress and application, Japan International Center for Agricultural 
Sciences, Japan and International Plant Genetic Resources Institute; 
Rome, Italy,8-20 
 
 64
Fang, J. Y., Wetten, A. and Hadley, P. (2004). Cryopreservation of cocoa 
(Theobroma cocoa L.) somatic embryos for long term germplasm 
storage, Plant Science 166:669-675. 
 
Hoekstra, F.A., Crowe, J.H. and Crowe, L.M. (1991). Effect of sucrose on 
phase behaviour of membranes in intact pollen of Typha latifolia L. 
as measured with Fourier transform infrared spectroscopy, Plant 
Physiol. 97:1073-1079. 
Koster, K.L. (1991). Glass formation and desiccation tolerance in seeds, 
Plant Physiol. 96:302-304.  
Malaurie B.,  N’ Nan, O.,  Hocher, V.,  Ilbert, P.,  Grosdemang, F., Konan, J-
L.,  Zakra, N. and Verdeil, J-L. (2002). State of zygotic coconut 
embryo culture and cryopreservation research at IRD/CIRAD 
(France), Coconut embryo in vitro culture part П, 146-156. 
 
N’Nan, O. (2004). Utilisation des biotechnologies comme seconde voie pour 
les echanges et la conservation des ressources genetiques du cocotier 
(Cocos nucifera L.), PhD thesis, Angers University. 
 
Nieves, N., Martinez, M. E., Catillo, R., Blanco, M. A. and Gonzalez-
Olmedo, J. L. (2001). Effect of abscisic acid and jasmonic acid on 
partial desiccation of encapsulated somatic embryos of sugarcane, 
Plant Cell Tissue and Organ Culture 65:15-21 
 
Roberston, A.J., Ishikawa, M. and Gusta, L.V. (1995). The effect of 
prolonged abscisic acid treatment on the growth, freezing tolerance 
and protein patterns of Bromus inermis (Leyss) cell suspensions 
cultured at either 3º or 25ºC, J. Plant Physiology 145:137-142 
 
Seijo, G. (2000). Effect of preculture with sucrose and ABA on cell 
suspension  water status and its relation with vitrification 
resistance, R. Bras.Fisiol Veg, 12  (2):166-180. 
 
Senaratna T., Kott, L., Beversdorf, W. D. and McKersie, B. D. (1991). 
Desiccation of microspore derived embryos of oilseed rape (Brassica 
napus L.), Plant Cell Reports 10:342-344   
 
 65
Senaratna, T., McKersie, B.D. and Bowley, S.R. (1989). Desiccation 
tolerance of alfalfa (Medicago sativa L.) somatic embryos. Influence 
of abscisic acid, stress pretreatments and drying rates, Plant Sci. 
65:253-259.  
Shiota, H., Tachibana, K., Watabe, K. and Kamada, H. (1999). Successful 
long term preservation of abscisic acid-treated and desiccated carrot 
somatic embryos, Plant Cell reports 18:749-753. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 66


Effect of abscisic acid on survival and recovery of cryopreserved Plumule explants of coconut (Cocos nucifera L.)

Jayakody, J.A.D.S.S.

Tennakoon, N.A.

Fernandopulle, M.N.D.

Available soil phosphorus in coconut plantations in Boralu and Pallama soil series under different fertilizer regimes in Gampaha District of Sri Lanka

Verdeil, J.L.

COCOS (2007), 18 45 - 51 
Printed in Sri Lanka 
EFFECT OF ABSCISIC ACID ON SURVIVAL AND RECOVERY OF 
CRYOPRESERVED PLUMULE EXPLANTS OF COCONUT 
{COCOS NUCIFERA L.) 
H D D Bandupriya1, S C Fernando1, J-L Verdeil2, B Malaurie3 
'Coconut Research Institute, Lunuwila, Sri Lanka; CIRAD, TA40/02 Avenue 
Agropolis, 34398 Montpellier Cedex 5 - France; 3UMR 1098 BEPC, Institute for 
Research and Development (IRD), BP 64501, 911 Av Agropolis 34394, Montpellier, 
France. 
ABSRACT 
Cryopreservation has great potential for long-term storage of coconut genetic resources. 
The recent development of the encapsulation-dehydration cryogenic technique 
dramatically facilitated progress with cryopreservation. The present study tested the 
effect of abscisic acid in the encapsulation-dehydration method for cryopreservation of 
coconut plumules. Plumules were excised from mature zygotic embryos of coconut var. 
Sri Lanka Tall and-encapsulated in calcium alginate. The alginate beads were used to test 
the effect of different cryoprotection pretreatments (mixtures of 0.75 M sucrose and 
abscisic acid (ABA) at concentrations of 0,10,20 and 40 uM), and exposure to silica gel 
for 16 hours for further dehydration, prior to freezing in liquid nitrogen. Effect of the 
treatments without freezing was also tested. The water loss from alginate beads following 
sucrose incubation and dehydration was monitored, and the survival and growth of 
plumules assessed at different stages. 
Addition of ABA to the sucrose pretreatment medium increased the survival rate of 
unfrozen plumules from 60% to over 80%. Pretreatment with 40 pM ABA gave the 
highest survival rate (84%) of frozen plumules. The recovery rate of plumules (frozen 
and unfrozen) pretsreated without ABA was very low (6-8%). The addition of ABA 
increased the recovery rate significantly; frozen plumules that had been treated with 40 
u,M ABA recorded a 39% recovery rate. The improved recovery could be ascribed to 
increased water loss (69%) in plumules treated with 40 uM ABA. 
Key words: Coconut, plumule, cryopreservation, abscisic acid 
INTRODUCTION 
The coconut palm (Cocos nucifera L.) is generally cultivated in the tropical regions both 
as large-scale plantations and in home gardens. The coconut palm yields a wide range of 
products and has been extensively cultivated over a long period of time. Consequently, 
many ecotypes have emerged through selection both by man and nature. Conservation of 
this genetic diversity is crucial for the development of future breeding programs before 
the ongoing genetic erosion takes its toll. 
45 
Coconut genetic resources are presently maintained exsitu in the field at great cost and 
subject to adverse weather conditions, pests and diseases. Cryopreservation is an 
important alternative method for long-term conservation of coconut germplasm. 
Cryopreservation reduces cell and tissue deterioration by virtually halting metabolism 
when they are stored at ultra low temperatures, usually that of liquid nitrogen (- 196°C). 
Coconut is propagated only by seeds. Coconut seed is recalcitrant in nature and large in 
size. Recalcitrant seeds cannot tolerate dehydration to moisture levels that would permit 
exposure to very low temperatures (Engelmann, 2000). This is no doubt a constraint to 
using cryopreservation for conserving coconut genetic resources. 
Successful cryopreservation of mature zygotic embryos of coconut, using the pre-growth 
desiccation method, was reported by Assy-Bah and Engelmann (1992). However, only a 
few genotypes were tested. The large size of the embryo limits experimentation with 
other techniques such as those that require encapsulation. Coconut plumule i.e. the 
embryo meristem and first leaves, which is known to be free of viral diseases, is an 
interesting starting material for cryopreservation (Malaurie et al., 2002). 
Cryopreservation of plumules is vital because they are small in size (around 1.0 mm) and 
abound in meristematic cells. Using the encapsulation-dehydration technique, coconut 
plumules have been successfully cryopreserved but the percentage recovery is low 
(NlNan,2004). 
Artificial cryoprotection by incubation in sugar is generally required for recalcitrant 
species like coconut. Dehydration tolerance can be increased by adding abscisic acid 
(ABA) to the culture medium (Anandarajah and McKersie, 1990; Senaratna*?*tf/., 1991). 
ABA is also known to induce freezing tolerance in various species (Chen and Gusta, 
1983) . In the present study, the effect of ABA on the survival and recovery of coconut 
plumules, cryopreserved by the encapsulation-dehydration technique, was tested. 
METHODOLOGY 
Mature zygotic embryos were extracted from 12 month-old seed nuts of the coconut 
variety Sri Lanka Tall. Plumules were excised from the embryos in a laminar airflow 
cabinet under a stereo microscope and precultured, for 4-7 days, in Eeuwens Y 3 growth 
medium (Eeuwens, 1976). The precultured plumules were encapsulated in 3 % (w/v) 
sodium alginate and pretreated for three days in liquid Eeuwens medium, enriched with 
0,75 M sucrose and ABA (at concentrations of 10,20 and 4 0 uM), on a rotary shaker (90 
rpm), at 27°C in the dark. After sucrose pretreatment. the beads were blotted on filter 
paper to remove surface moisture, weighed, and then dehydrated for 16 hours in glassjars 
with 20 plumules placed on top of 40 g silica gel separated by filter paper. The dehydrated 
beads were weighed, and half were transferred to sterile polypropylene cryotubes and 
directly plunged into liquid nitrogen. After at least 2 hours storage in liquid nitrogen,the 
tubes were warmed in a water bath at 4 0 °C for 3 min. These frozen beads, and the 
unfrozen beads (subjected to pretreatment and dehydration only) were cultured in a 
recovery medium (Eeuwens Y 3 growth medium). 
46 
Encapsulated plumules (without any pretreatment, dehydration or freezing) were also 
cultured in Eeuwens Y3 growth medium, soon after encapsulation, as an untreated 
control. 
The survival of plumules was assessed after two months in culture. A plumule 
manifesting new tissue growth, indicated by any sign of regrowth such as swelling, 
development of new leaf primordia and/or callus formation, was counted as a survivor. 
The recovery of plumules was assessed after 4 months in culture. A plumule that grew 
and developed into a plantlet was counted as having recovered. The experimental design 
was a complete randomized design with three replicates, and 10 plumules per treatment. 
Arcsine transformed data was analysed using the GLM procedure (S AS Institute, USA) 
RESULTS AND DISCUSSION 
A critical parameter in cryopreservation is the removal of intracellular water, to prevent 
ice crystals forming during freezing or thawing and consequent cell damage (Fang et aly 
2004). This necessitates the pretreatment of encapsulated plumules with cryoprotectants 
such as sugars, sugar alcohols, DMSO etc. 
Irrespective of ABA treatments, all the plumules that were not subjected to dehydration 
and cryostorage survived and developed into normal plantlets; and none of the plumules 
not subjected to dehydration, survived cryostorage (Table 1 and 2). The survival rate of 
dehydrated but unfrozen plumules varied from 60% to 84%. Very high survival rates 
(>80%) were observed in dehydrated, unfrozen plumules pretreated with 10-40 uM 
ABA, clearly indicating the positive effect of ABA on their survival. Similarly, 
pretreatment with ABA increased the survival rate of dehydrated, frozen plumules from 
57% without ABA to 84% with 40 uM ABA. 
Table 1: Effect of ABA concentration on survival of encapsulated plumules 
following dehydration and cryopreservation 
Treatment Dehydration in hours 
Sucrose (M) + ABA 
(uM) 
No dehydration Dehydration for 16 
hours 
-LN +LN -LN +LN 
0 . 7 5 + 0 100 + 0 0 + 0 6 0 + 1 2 5 7 + 1 4 
0.75 + 10 100 + 0 0 + 0 82 + 6 6 8 + 1 5 
0.75 + 20 100 + 0 0 + 0 85 + 8 39 + 20 
0 . 7 5 + 4 0 100 ± 0 0 + 0 84 + 8 84 + 5 
Values represent survival percentage ± SE. 
P (survival) < 0.0001; treatment differences were evaluated at 5% probability. 
LN:liquid nitrogen 
4 7 
With both frozen and unfrozen, dehydrated embryos, the positive effect of ABA on 
recovery was quite clear. When they were pretreated only with sucrose (zero ABA) the 
recovery rate was very low but increased remarkably when ABA was added to sucrose 
(Table 2). Frozen plumules recorded the highest rate of recovery (39%) when pretreated 
with sucrose containing ABA at a concentration of 40 u.M. 
In this experiment, dehydration of plumules was initially by the sucrose pretreatment and 
then by exposure to silica gel for 16 hours. Water loss in plumules was lowest (62%, fresh 
weight basis) when pretreated with 0.75M sucrose without ABA, and highest (69%, fresh 
weight basis) when pretreated with 0.75M sucrose containing ABA at a concentration of 
40 uM. The increase in recovery rate of plumules with the latter pretreatment appears to 
be a result of increased dehydration. 
The influence of ABA and sugars on the tolerance of tissues to dehydration and 
cryopreservation has received much attention (Dumet et a l , 1993; Nieves et a l . 2001). 
Sucrose infiltrating into tissues helps to maintain their viability during dehydration and 
cryopreservation by stabilizing cell membranes. Sucrose, by replacing water, maintains 
membrane phospholipids in the liquid crystalline phase (Hoekstra et a l , 1991), and 
prevents structural changes in soluble proteins (Carpenter et al., 1987). Sucrose, due to 
its solute properties, also promotes glass phase formation in the cytoplasm (Koster, 
1991). ABA plays an important role in desiccation tolerance of several plant species 
(Senaratna et a l , 1989; Shiota et a l , 1999). ABA is associated with synthesis of proteins 
(Seijo, 2000) and compatible solute components, which are important in plant stress 
tolerance (Fang et a l , 2004). A combination of ABA and sucrose treatments improved the 
survival of cryopreserved cocoa somatic embryos (Fang et a l , 2004). 
Table 2: Effect of ABA concentration on recovery of encapsulated plumules following 
dehydration and cryopreservation 
Treatment Dehydration in hours 
Sucrose(M)+ ABA No dehydration Dehydration for 16 
(uM) hours 
-LN +LN -LN +LN 
0.75+ 0 93 + 4 0 + 0 6 + 6 8 + 4 
0.75 + 10 100 + 0 0 + 0 45+ 15 17 + 9 
0.75 + 20 100 + 0 0 + 0 43 + 2 23+12 
0.75+40 100 + 0 0 + 0 45 + 9 39 + 6 
Values represent recovery percentage ± SE. 
P (recovery) < 0.0001; treatment difference were evaluated at 5% probability. 
LN:liquid nitrogen 
48 
Table 3: Mean percent water loss from plumules pretreated in sucrose and dehydrated 
with silica gel. 
Concentration of ABA (^M) in 
the sucrose solution 0 10 20 40 
Mean percentage of water loss 
(fresh weight basis) 62 67 68 69 
N'Nan (2004) and Malaurie et al. (2002) using the encapsulation-dehydration method 
with different sucrose concentrations and dehydration periods, achieved about 40-70% 
survival and about 20% recovery of coconut plumules after cryopreservation. In the 
present study, the optimum sucrose concentration and dehydration duration reported by 
N'Nan (2004) were used to investigate the influence of ABA on the survival and recovery 
of cryopreserved plumules. The preliminary results are encouraging and indicates the 
feasibility of using ABA for effective cryopreservation of coconut germplasm. It is likely 
that the recovery rate could be further improved by refining the protocol. 
ACKNOWLEDGEMENT 
Authors gratefully acknowledge the financial assistance provided by the IRD, through a 
research project funded by Bureau des Ressources Genetiques, Paris, France (BRG). 
Thanks are also due to all staff members of Lab Genetrop, IRD, France and Tissue 
Culture Division, CRI, Sri Lanka for their assistance in carrying out the experiments. We 
acknowledge the valuable comments of Dr L K Weerakoon, Head Tissue Culture 
Division on the manuscript. Thanks are also extended to Dr.T S G Peiris and Mr. J D J S 
Kularathne of the Biometry Division, CRI for their assistance in the statistical analysis of 
data. 
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51 


Effect of abscisic acid on survival and recovery of cryopreserved plumule explants of coconut (Cocos nucifera L.)

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