MICROPROPAGATION OF COCONUT THROUGH PLUMULE CULTURE

Plumule tissues have found to be more responsive explants for clonal propagation of coconut. In the present study, the feasibility of using plumule explants for clonal propagation of a local coconut variety (Sri Lanka Tall) was a s s e s s e d . Plumules were cultured in three basal media (MS, Y3 and 72) supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) (24 400 \M) and activated charcoal. Somatic embryogenesis and plant regeneration were induced by incorporating of abscisic acid (ABA) into the medium. Attempts were made to improve plant regeneration by applying high agar concentration-induced water stress, cytokinin or silver nitrate in combination with ABA. The results revealed that callus can be initiated from plumule explants cultured in all three basal media tested. The callus formation depended on the concentration of 2,4-D in the media and the best effect was observed with the lower level (24 jiM) tested. The plant regeneration frequency observed with ABA treatment was 4.4 %. The combination of ABA with high agar concentration-induced water stress doubled the plant regeneration frequency. About 50 % of the regenerated plants could be successfully acclimatized and field planted. The results of the study indicate the possibility of regenerating plants from plumule explants of coconut. The method can be used for multiplication of improved seed material. However, the plant regeneration efficiency should be further improved for mass scale propagation of coconut using plumule explants.


INTRODUCTION
The coconut palm is a major plantation crop and the most important palm of the tropics.Due to its economic importance, breeding for crop improvement is of high priority.Coconut is a cross-pollinated and heterozygous plant, which is so far, propagated exclusively through seeds.Clonal propagation of coconut is of prime importance for rapid multiplication and distribution of the best genotypes obtained through conventional breeding.Furthermore, it would also allow rapid m< Implication of selected parental palms for establishing seed gardens and selected individuals, exhibiting resistance to biotic and/ or abiotic stresses, for increasing the yield.
Initial attempts have been made to micropropagate coconut through different explants such as immature inflorescence (Branton and Blake, 1983), tender leaf (Raju et al., 1984) and immature zygotic embryo (Karunaratne and Periyapperuma, 1989).Later studies by Hornung (1995) and Chan et al. (1998) have shown that the response of plumules (embryo meristem and first leaves) to in vitro culture conditions is better than that of other tissues.Therefore, plumules could be used as a model for clonal propagation of coconut.As plumules are excised from zygotic embryos, they can only produce clones of palms with unknown performance.However, if the embryos are obtained by controlled pollination of selected parents, the clones from these embryos are likely to show many of the desired characteristics of the parent palms (Chan er al., 1998).
This paper reports the progress of clonal plant regeneration of a local coconut variety (Sri Lanka Tall) using plumule explant.

Plant material
Mature nuts (12-14 month-old) of the variety Sri Lanka Tall coconut were used as the source of mature zygotic embryos.

Statistical analysis
Analysis of Variance was carried out to test the differences among treatments.

Callogenesis
Coconut somatic embryogenesis usually occurs through a callus phase.Therefore, production of embryogenic callus is a prerequisite for successful plant regeneration.Among several other factors, the presence of an auxin in the medium is critical for successful callogenesis (Auge, 1995) The results revealed that the callusing frequencies were higher and comparable in all the three basal media supplemented with lower (24 uM) concentration of 2,4-D.Increase in the concentration of 2,4-D up to 100 or 400 u.M resulted in a decrease in callusing (Table 1).Despite the statistically non-significant decrease in callusing frequency in Y3 supplemented with 100 uM 2,4-D, callus initiated in high 2,4-D containing media showed a poor growth compared to that of lower 2,4-D media.In this study, embryogenic callus (Fig. 1b) treated with 5 uM ABA, became more compact.After 5 weeks in the medium with ABA, whitish globular structures were visible.Upon transfer to the medium devoid of hormones for four weeks, some of the embryogenic structures elongated whereas the others developed into haustorial tissues or fused shoot-like structures.On repeated subculture to the Y 3 medium, the somatic embryos produced shoots (Fig. 1c).
Incorporating ABA to the culture medium resulted in normal development of somatic embryos that can develop into complete plants.However, the plant regeneration frequency was low (4.4 %).Among the treatments tested, only the application of high agar-induced water stress in combination with ABA had a positive effect and it doubled (9.0 %) the plant regeneration frequency (Table 2)
tested.The study was extended to assess the effect of medium 72 supplemented with higher level (100 uM) of 2,4-D and media MS and Y3 supplemented with lower level (24 \iM) of 2,4-D.
activated charcoal, which had been used by the relevant research groups.Different types of charcoal have different adsorption capacities, which results in variations in active auxin concentration in the medium.Therefore, the use of high levels of 2,4-D in the presence of unspecific type of charcoal could be the reason for the low callusing frequencies observed.This was further confirmed by the increase of callus production in those media by reducing the concentration of 2,4-D.With agreement of the results of this study, Saenz et al. (1999) reported that callusing in plumules cultured in medium described by Chan et al. (1998) could be increased up to 100 % by using a different type of charcoal.Furthermore, the use of different coconut genotypes for studies of different research groups might also have contributed to the variable results.Somatic embryogenesis and plant regenerationGenerally, somatic embryogenesis in coconut callus has been induced by gradual reduction of auxin concentration with a corresponding increase in the cytokinin level in the medium(Buffard-Morel et al., 1995).In contrast, somatic embryogenesis in coconut has also been induced by an early increase in 2,4-D concentration followed by a gradual reduction(Verdeil et al., 1994) or a rapid decrease in 2,4-D concentration and application of high concentration of cytokinin (Chan era/., 1998).However, research conducted at the CRISL revealed that an alternate treatment, removal of auxin and incorporating ABA to the culture medium, give rise to more consistent plant regeneration from coconut callus(Fernando and Gamage, 2000).Similarly,Samosir et al. (1999) reported improved somatic embryogenesis in coconut with the use of ABA.

.
The use of ABA in combination with osmotically active substances has resulted in improved somatic embryogenesis and plant regeneration in coconut(Samosir et al., 1999), rubber(Linossier et al., 1997) and several other crops.The moisture stress, induced by application of high agar concentration in the medium has also shown encouraging results in plant regeneration of rice(Jain et al., 1995).The effect of such a stress has not been tested for coconut previously.The enhanced results observed in this study might be due to promotion of the expression of embryo-specific genes, improved embryo development and increased production of storage reserves caused by moisture stress.and a good root system.When shoots did not produce roots spontaneously, the application of an IAA pulse resulted in successful rhyzogenesis in more than 90 % of the treated shoots.When the complete plants were transferred to the potting medium for acclimatization, about 50 % of plants survived and they could be transferred to field successfully (fig.Id).The results of.thisstudy indicated the possibility of regenerating clonal plants of the local variety Sri Lanka Tall using plumule explants.The concentration of 2,4-D in the callusing medium was found to be the critical factor determining success in callogenesis.Application of ABA in combination with high agar level-induced water stress improved plant regeneration frequency.The resulting plants could be acclimatized and field planted successfully.This method can be applied for multiplication of improved seed material.However, the plant regeneration efficiency of the current protocol should be further improved for mass scale propagation of coconut using plumule explants.