INTRODUCTION


 

IN VITRO CONSERVATION OF COCONUT (Cocos nucifera L.) EMBRYOS 

BIOTROPIA NO. 25, 2005 : 11 – 21 

IN CULTURE MEDIA 
 

SUKENDAH1 and MA L. O. CEDO2 

 
1Dept. of Agronomy, Faculty of Agriculture,  Pembangunan Nasional “Veteran” University, 

Surabaya, Indonesia 
2 Assistant Professor, Dept. of  Horticulture, UPLB, Laguna 4031, 

Los Baños, Philippines 
 
 

The possibility of delaying the germination of mature coconut embryos in vitro for medium-term 
conservation of coconut was investigated.  Embryos of coconut was stored in sugar free, full-strength and 
half-strength MS media for 3 months with minimal loss in viability.  Germinability of embryos decreased 
with prolonged storage.  At the end of 9-month storage period, embryos kept in full-strength media were 
all non-germinable, while those stored in half-strength media still gave 15% germination.  Addition of 
mannitol up to 0.3M did show any significant effect on germination of embryos stored for 3 months.  
However, longer period of storage helps to a certain extent in the preservation of embryo viability.  The 
presence of mannitol in the storage medium also induced morphological abnormalities in seedlings 
derived subsequently from the stored embryos.  Mannitol at 0.4M was totally deleterious to the embryo.  
Significance of these findings on coconut genetic resources conservation are discussed. 

 
Keywords:  In vitro conservation/Coconut embryo. 
 
 

INTRODUCTION 
 

The safe conservation of genetic resources of coconut is faced with various 
problems. Coconut is a species with a truly recalcitrant seed which has no dormancy 
(Engelmann et al. 1995). Because of this characteristic, there is no possibility of 
storing it ex situ even for short periods of time. Genetic resource collections 
(germplasm) are conserved as special field genebanks  (living plants) in national or 
regional centers. 

Field genebanking, however, run the risk of being damaged by natural 
disasters, pests and diseases.  It also requires more space. Moreover, many sources 
of plant genetic materials (Indonesia, the Pacific, and Philippines) are still almost 
unexplored, and the small proportion of coconut germplasm currently available in 
existing field collections and certain accessions are now in danger of dying. The in 
vitro technique addresses all these concerns. It offers the possibility of providing 
duplicate, healthy collections for storage and use with less space. Since vegetative 
propagation of coconut is as yet not feasible, embryo culture technique is a 
promising alternative.  

Coconut embryo culture protocols are now available in the literature (Batugal 
and Engelmann 1998). In all these protocols, embryo germination commences soon 
after the embryos are placed in the culture medium. Subsequently, fully developed 
seedlings ready for acclimatization may be obtained within 5-7 months. For in vitro 
conservation, such quick growth response to the medium is not desired. Thus, efforts 

 11



 

are now focussed on modifying the medium and other culture conditions in order to 
delay the germination while at the same time preserving the germination potential of 
the embryos during storage.  

BIOTROPIA NO. 25, 2005 

Techniques which can be used to maintain collection in delaying the embryo 
germination are: the use of reduced temperature, minimized light, and modifications 
in media. Ashmore (1997) was able to store sugarcane shoots at 18ºC on reduced 
mineral salts, and banana shoots at 16ºC in the presence of plant growth subtances. 
Chatty and Tissouni (1999) showed that the suitable temperature for major palm 
species is about 25ºC, whereas lack of germination was observed at the extreme 
temperature of 5ºC.  The American palm germinated better at temperatures ranging 
from 25ºC to 35ºC, but did not germinate at 15ºC.  Meanwhile, Ng and Ng (1991) 
reported to slow down the growth rate, cultures are basically incubated under 
reduced light intensity or total darkness in conjunction with low incubation 
temperature. However, the modification of temperature and/or light to preserve 
cultures required a high cost because of electricity and equipment. 

Ng and Ng (1991) suggested that an alternative approach to retard growth was 
to use a reduced-growth medium. Recent publications on slow-growth media 
indicate that a variety of techniques are still being utilized, such as the reducing 
concentration of mineral salt, increasing, decreasing or eliminating sucrose, adding 
mannitol or sorbitol and growth retardant to the culture medium (Ng and Ng 1991).  
One of the advantages of this method is that the modification of the culture medium 
requires no special equipment. 

Preliminary studies on storage media conducted by Assy-Bah and Engelmann 
(1993) have indicated that embryos of coconut can be stored for 6 months on a 
medium devoid of sucrose and containing 2 g/l activated charcoal with eventual 
germination of stored embryos at 100%. Related study done by Damasco (2000) on 
Tagnanan Tall showed that ABA and mannitol at low concentrations (0.05 to 0.1M) 
might not be effective to inhibit growth in culture. However, high concentration of 
mannitol (0.2 and 0.3M) significantly reduced percent germination and subsequent 
growth of germinated embryos.  In this report, results on the use of reduced-nutrient 
media and mannitol for coconut embryo storage are presented. 
 
 
 

MATERIALS AND METHODS 
 
Collection, Sterilization, and Excision of Embryos 
 

Mature (11-12 months) coconut palm variety ‘Tagnanan Tall’ were collected 
from the Philippine Coconut Authority Zamboanga Research Center.  A cylinder of 
endosperm embedding the embryo was extracted from each nut using a 2-cm-
diameter cork borer. The endosperm cylinders were washed in 95% ethanol (1-2 
minutes) then sterilized in 100 % commercial bleach (5-6 % sodium hypochlorite) 
for 20 minutes.  

 12



 

Under aseptic condition, the embryos were excised from the solid endosperm 
cylinders, then sterilized in two changes of 10% commercial bleach for 5 minutes at 
each time and finally rinsed three times with sterile distilled water. Sterilized 
embryos were transferred to a sterile flask containing sterile distilled water for size 
selection.  The same size embryos (± 1 cm) were then inoculated singly onto liquid 
storage medium according to the treatments. 

Control of culture media for in vitro conservation of coconut – Sukendah and Ma L.O. Cedo 

 
Culture Media and Arrangement of Treatments 

 
The composition of the storage medium used in the present experiment was 

that of Assy-Bah (1986), at full-strength or half-strength. The medium contained 
activated charcoal and had no sucrose. After the required period of storage (3, 6, and 
9 months), embryos were germinated following the embryo culture protocol of the 
University of the Philippines at Los Baňos or UPLB (Batugal and Engelmann 1998). 

The experiment was conducted by culturing the embryos in test tubes 
containing 10 ml of full-strength or half-strength MS media without or with 
mannitol at 0.10 M, 0.2 M, 0.30 M, or 0.4 M.  Each treatment was replicated three 
times, each consisting of 35 embryos.  The cultures were randomized and incubated 
in a dark room with temperature maintained at 25°C.   
 
Observation of Embryos 
 
 Viability of embryos was evaluated based on the following parameters: Length 
of embryos, measured every 2 months since embryos have been incubated; Percent 
germination, it was done after the embryos have been preserved for 3, 6, and 9 
months of storage. The stored embryos from the experiment were transferred to the 
germination medium described in the UPLB protocol; Normal and abnormal 
seedlings, observed 3 months after embryos have been transferred to the germination 
medium.  Seedlings which showed well-developed roots & shoots were considered 
normal, and seedlings with no shoot, no root or stunted seedlings as abnormal 
seedling. Data were collected based on: a) Percent normal seedlings and abnormal 
seedlings, and b) Morphological features, observed by taken their photograph. 
 Observations of shoot length, number of leaves, leaf width, leaf length, length 
of primary root, and  number of adventitious roots for normal seedlings were done 5 
months after the embryos have been transferred to the germination medium. 
 

 
RESULTS AND DISCUSSIONS 

 
Coconut embryos that preserved in full-strength or half-strength MS basal 

medium significantly increased in length even up to 6th month of storage.  In overall, 
embryos maintained in half-strength media were significantly longer than embryos 
in the full-strength media (Table 1). Previous studies on reducing the mineral salt 
concentration showed that the cultures in general grow slowly or not at all (Ashmore 

 13



 

1997). Apparently the coconut embryos did not have the same response. No further 
study has reported on the response of coconut embryos treated in low-salt medium. 
Interestingly, at the end of each storage period, it was noted that the final volumes of 
the spent half-strength media were lower than those of the full-strength media (data 
not presented). This supports the view that greater growth (in length) of embryos in 
the half-strength media could have resulted from greater nutrient and water uptake 
by embryos in those media.  

BIOTROPIA NO. 25, 2005 

 By contrast, addition of mannitol to the storage medium tended to inhibit 
elongation of embryos during storage. Increasing amounts of mannitol (0.1M to 0.4 
M) increasingly reduced the final lengths of the embryos, particularly during the 
first two months of storage (Table 1). Beyond 6 months of storage, no significant 
difference on final length among embryos was noted. 
 
 
Table 1. Effects of level of nutrients and concentration of mannitol on elongation growth of coconut 

embryo during  storage  

Period of Storage (month) 
Treatments 

2 4 6 

 Final length of embryo (mm) 

Level of nutrients: 
Full-strength 
Half-strength 

 
9.76b* 
10.37a  

 
11.01ns 
11.56 

 
11.33ns 
11.71 

Concentration of 
Mannitol (M): 
0.0 
0.1 
0.2 
0.3 
0.4 

 
 
10.55 a 
10.65 a 
10.17 a 
  9.58 ab 
  9.38 b 

 
 
11.85ns 
11.83 
11.28 
11.37 
10.12 

 
 
12.27 ns  
12.23 
11.50 
11.45 
10.15 

* Means followed by the same letter in a column for each factor are not significantly different from each 
 other using LSD at 5% level of significance 
 
 

When the stored coconut embryos are placed in a germination medium such as 
Y3 (Eeuwens medium), they would start germinating or may exhibit a slight 
increase in size but still remain ungerminated or the embryos may take a 
deteriorative path and eventually dies. Table 2 shows that storing coconut embryos 
for 3 months in the modified MS medium, either at full-strength or half-strength, 
resulted in significant 10-30% reduction in percent germination. Prolonging the 
storage period to 6 months reduced even further the germination to 10-30% in five 
of the treatments and to zero (no germination) in the other five treatments. With 9 
months of storage all embryos kept in full-strength media become non-germinable, 
while those kept in half-strength media containing low amounts of mannitol showed 
13-20% germination. Mannitol when added to half-strength  (but not to full strength) 

 14



 

Table 2. Percent germination of stored embryos observed 2 months after transfer to the modified Y3 
 germination medium 

Control of culture media for in vitro conservation of coconut – Sukendah and Ma L.O. Cedo 

Period of Storage (month) Level of  
Nutrients 

Mannitol 
Concentration

(M) 3 6 9 

Full-strength 

 

0.0 
0.1 
0.2 
0.3 
0.4 

80.00 bc* 
70.00 c 
80.00 bc 
90.00 ab 
20.00 e 

30.00 b 
00.00 d 
20.00 cb 
00.00 d 
00.00 d 

00.00 c 
00.00 c 
00.00 c 
00.06 c 
00.00  c 

Half-strength: 0.0 
0.1 
0.2 
0.3 
0.4 

100.00 a 
  80.00 bc 
100.00 a 
  90.00 ab 
  40.00 d 

00.00 d 
20.00 cb 
30.00 b 
10.00 cd 
00.00 d 

00.00 c 
20.00 b 
20.00 b 
13.33 b 
00.00 c 

Unstored embryos  100.00 a 100.00 a 100.00 a 
* Means followed by  the same letter in a column are not significantly different from each other using 
 DMRT at 5% level of significance. 
 
 
media up to 0.3M appeared to help preserve the viability of the stored embryos. It is 
possible that the hypertonic condition created by the addition of mannitol at this 
level could have helped maintain membrane integrity, thus prevent rapid cell death 
which otherwise occurs in untreated coconut embryos during prolonged storage. 
Gomez et al. (1995) reported that mannitol significantly prevented EP-catalyzed 
hydrolysis of 40 and 50 kDa polypeptides. EP (endopeptidase) is a monomeric 
enzyme (Gomez et al. 1994) that has an important role in the digestion of cell wall 
protein. However, addition of high mannitol concentration (0.4M) significantly 
inhibited the germination of embryos. 
 After the required storage period, embryos were transferred to the Y3 
germination medium. Although high percentage of the stored embryos exhibited the 
early signs of germination and they were considered germinated (Table 2), many of 
these embryos did not develop into normal seedlings. In fact, some 10-70% of them 
showed abnormal growth such as produced well-developed roots but without shoots 
or well-developed shoot but little elongation of roots or stunted seedling even up to 
the third month after transfer to the Y3 medium (Table 3 & Fig. 1). 

Of the remaining embryos, normal seedlings were obtained from those stored 
in media without mannitol or with mannitol at 0.1M (Fig. 2). The presence of higher 
levels of mannitol (0.2 and 0.3M) in the storage medium seemed to have inhibited 
seedling development. Seedlings tended to produce shorter shoots whether stored in 
full-strength or half-strength media. With 0.4M mannitol, development of shoot and 
root totally failed (Table 4). Several authors have reported similar inhibitory effect 
of mannitol on growth of several crops (Ashmore 1997; Babu et al. 1999; and 
Damasco 2000). 

 15



 

Table 3. Percentage of normal and abnormal seedlings developed  from embryos stored for 3 months in  
full and half-strength media with or without mannitol 

BIOTROPIA NO. 25, 2005 

Abnormal Seedlings Mannitol 
Concen-
tration 

(M) 

Total 
Number 

of 
Embryos 
cultured 

Un- 
germinated
Embryos 

Normal 
Seedling No shoot 

(a) 
No root 

(b) 
Stunted 

(c) 

Total 
Abnormal 
Seedlings 
(a +b+ c) 

Full-
strength 
Medium: 

0.0 
0.1 
0.2 
0.3 
0.4 

 
 
 

10 
10 
10 
10 
10 

 
 
 

20 
30 
20 
10 
80 

 
 
 

80 
70 
50 
30 
0.0 

 
 
 

0.0 
0.0 
20 
30 
20 

 
 
 

0.0 
0.0 
0.0 
10 
0.0 

 
 
 

0.0 
0.0 
10 
20 
0.0 

 
 
 

0.0 
0.0 
30 
60 
20 

 
Half-

strength 
Medium: 

 
0.0 
0.1 
0.2 
0.3 
0.4 

 
 
 
 
 

10 
10 
10 
10 
10 

 
 
 
 
 

0 
20 
0 

10 
60 

 
 
 
 
 

80 
70 
60 
20 
0.0 

 
 
 
 
 

0.0 
0.0 
20 
50 
30 

 
 
 
 
 

10 
0.0 
0.0 
0.0 
0.0 

 
 
 
 
 

10 
10 
20 
20 
10 

 
 
 
 
 

20 
10 
40 
70 
40 

 

 
 
 
 

 
CBA 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 1. A. Abnormal seedling with stunted growth; B. Abnormal seedling with extensive root system 

but without shoot; C. Abnormal seedling with two sword leaves and an enlarged base but 
without fully formed root  

 16



 

Table 4.  Shoot and root development of seedling grown from embryos stored for 3 months in half- and 
full-strength MS medium with various concentration of mannitol 

Control of culture media for in vitro conservation of coconut – Sukendah and Ma L.O. Cedo 

Concentrations
mannitol 

(M) 

Shoot 
length 
(cm) 

Total no. of 
leaves/plant 

Average 
leaf width

(cm) 

Average 
leaf 

length 
(cm) 

Length of 
primary 

root 
(cm) 

No. of 
adventitious 

root 

 

Full-strength 
medium: 
 

0.0 
0.1 
0.2 
0.3 
0.4 

 

Half-strength 
medium: 
 

0.0 
0.1 
0.2 
0.3 
0.4 

 

Unstored 
embryos 
 

 

 
 
 

11.23 ns 
10.58 
8.05 
8.15 
0.00# 
 

 
 
 

11.48ns 
10.02 
10.90 
6.80 
0.00# 
 

17.66* 

 

 
 
 

2.50ns 
2.17 
2.00 
3.00 
0.00# 
 

 
 
 

2.50ns 
2.33 
2.50 
1.00 
0.00# 
 

3.00 

 

 
 
 

1.47ns 
1.43 
1.13 
1.20 
0.00# 
 

 
 
 

1.40ns 
1.50 
2.08 
0.40 
0.00# 
 

3.03** 

 

 
 
 

7.20ns 
8.08 
6.18 
6.92 
0.00# 
 

 
 
 

8.33ns 
6.93 
6.68 
2.40 
0.00# 
 

10.06 

 

 
 
 

8.93ns 
8.10 
5.88 
6.60 
0.00# 
 

 
 
 

6.08ns 
5.13 
6.25 
2.40 
0.00# 
 

10.57 

 

 
 
 

2.00ns 
1.17 
1.25 
2.00 
0.00# 
 

 
 
 

1.00ns 
1.17 
1.50 
1.00 
0.00# 
 

1.20 

ns = not significantly different among the treatments  in the same column 
* = significantly  different from stored embryos in the same column 
**  = highly significantly different  from stored embryos in the same column 
# = embryos remained germinated 
 
 

 
 

Figure 2. Normal five-month-old seedlings in full-strength medium with various concentrations of 
 mannitol 

 17



 

Inhibitory effect of osmotically active agents such as mannitol at high level has 
appeared during the storing period of embryos (Table 1). These embryos, then were 
not able to germinate even though supported by enough nutrient. Mannitol is a 
metabolically inactive sugar alcohol (Dodd et al. 1991). It is one of compatible 
solutes that can decrease plant water potential by osmotic adjustment (the 
accumulation of compatible solutes that promotes acclimation to dry) (Buchanan et 
al. 2005).  The large amount of mannitol in the cell could result in the increase of 
the osmotic pressure of the medium drastically. A high osmotic potential in the 
external medium can induce the protoplast to lose water, causing the cells to become 
plasmolyzed. If kept in this medium for extended period, it can cause cells to 
become permanently damaged. Study on leaf disks of maize under hyperosmotic 
condition by Del Longo (1993) revealed the cellular damage in both the sensitive 
and the resistant maize line.  Damage as indicated by, for example, the extension  of 
lipid peroxidation, the destruction of chlorophyll and carotenoids, the decrease in 
levels of protein sulfhydryl groups and the leakage of electrolytes from cells was 
apparent in leaf disks as a consequence of the applied stresses (0.5 M mannitol). 

BIOTROPIA NO. 25, 2005 

It should be mentioned that the stored embryos were not washed off before 
they were transferred to the germination media. So that the mannitol in the storage 
media was carried over to the new media. This could have caused the abnormal 
growth of the seedling. The washing step was not done, however to avoid the 
removal of important metabolites from possibly already leaky cells as suggested by 
Withers (1986; cited by Benson 1990). 

Microscopic observations of the paraffin sections of the embryos showed that 
high mannitol concentration caused plasmolysis of cells in the region of the apical 
meristem, which could have led to damage of shoot or root development (Fig. 3). 
Evidence from some researches showed that  the damage occurred on the hypocotyls 

 
 

 
 

Figure 3.   Plasmolyzed cells in  0.3 M mannitol medium 

 18



 

or in a critical number of cells of the axis would produce abnormal seedling or the 
germination would be retarded. Thus, the symptoms of slow growth, abnormal 
growth, or no growth could result from fundamental changes in membranes and 
macromolecules.  

Control of culture media for in vitro conservation of coconut – Sukendah and Ma L.O. Cedo 

The results showed that delaying the germination of coconut embryos of the 
variety ‘Tagnanan” was effective for up to 3-month-storage period.  After 3 months, 
the deterioration of embryos occurred very fast.  By the end of 9 months, most of the 
embryos showed significant loss in viability.  However, some embryos still showed 
viability potential after 9 months of storage (indicated by percent germination of 
about 20-30%).  Clearly, there is still a need for further studies on  the culture media 
of coconut embryos to increase the viability and germinability of embryos longer 
than 3 months period of storage. 

Addition of mannitol at a low concentration, lower than 0.2 M, gave a high 
germination rate (70-100%) and relatively high percentage of normal seedlings (50-
60%), both in the full-strength and half-strength media free of sucrose. Damasco 
(2000) reported that addition of mannitol at 0.05 to 0.1 M to the medium containing 
high level of sucrose (60 g/l) induced germination and growth of coconut embryos 
of the variety ‘Tagnanan’ with the same germination rate as the control treatment. It 
appeared that mannitol was not effective in delaying germination.  Since the present 
study was able to delay the germination up to 3 months and maintain high viability, 
further studies using other substances which could delay germination should be 
done. Sucrose analysis showed that coconut embryo contains very low amount of 
sucrose, only 42.49 mg per 100 g dry weight. Apparently, addition of some amount 
of sugar in the storage medium may still be important. 

The study indicated that it is possible to prolong the inactive metabolic phase 
of  coconut  embryos  before  the  active  germination  phase for at least 3 months by  
controlling the culture medium.Technique of delaying the germination may be used 
as a complementary conservation strategy of coconut germplasm. This technique 
may have a role in one or more activities in the conservation-use cycle such as in  in 
vitro collecting, in vitro exchange and distribution of coconut germplasm, and in 
vitro conservation.  
 
 
 

CONCLUSIONS  
 

The overall results indicate that it is possible to delay the germination of 
coconut embryo in vitro for up to 3 months without loss in viability.   

MS medium at half-strength was found to be a better medium for embryo 
storage than the full-strength medium. Addition of mannitol at low concentration 
(0.2M or lower) did not have any significant effect on storage. However, with 0.3 M 
or higher concentration of mannitol, the germination fell drastically to 30%. 
Furthermore, high concentration of mannitol induced abnormality in the resulting 
seedlings (formation of shoot without root, root without shoot, and stunting).   

 19



 

ACKNOWLEDGMENTS 

BIOTROPIA NO. 25, 2005 

 
The author would like to express her sincere gratitude to SEARCA and IPGRI 

for the financial support to endure this study.  She is also deeply grateful to the 
Director and staff of Department of Horticulture, UPLB, Philippines for the 
assistance and facilities and also to Mr. Carlos B. Carpio, Deputy Administrator, 
ARDB, PCA for the supply of  the coconut embryos. 
 
 

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ASSY-BAH, B. and F. ENGELMANN.  1993. Medium-term conservation of mature embryos of coconut 
(Cocos nucifera L.). Plant, Cell, Tissue and Organ Cult. 33:19-24. 

BABU, K.N., S.P. GEETHA, D. MINOO, P.N. RAVINDRAN and K.V. PETER. 1999. In vitro 
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BATUGAL, P.A and F. ENGELMANN. 1998. Coconut Embryo In Vitro Culture. Proceedings of the 
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CHATTY, Y. and T. TISSOUNI. 1999. Efect of temperature on germination of ornamental palm tees in 
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DAMASCO, O.P. 2000. Utilization of embryo culture technology for gemplasm conservation: 
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ENGELMANN, F., B. ASSAY-BAH, S. BAGNIOL, D. DUMET, and N. MICHAUX-FERRIERE. 1995. 
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GOMEZ, L.D., L.M. CASANO, M.ROUBY, M.S. BUCKERIDGE, and V.S. TRIPPI.  1994. Proteolytic 
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GOMEZ, L.D., L.M. CASANO, and V.S. TRIPPI. 1995. Effect of hydrogen peroxide on degradation of 
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 21


	INTRODUCTION 
	Observation of Embryos