Plantlet regeneration through somatic embryogenesis in cocoa (Theobroma cacao L.)

Abstract

Investigations on 'Plantlet regeneration through somatic embryogenesis in cocoa' were undertaken in the Department of Agricultural Botany, College of Horticulture, Kerala Agricultural University, Vellanikkara during 1992-94. Studies were made to identify the most suitable medium, the most responsive genotype and most favourable conditions for embryogenesis in cocoa. Conditions for germination of embryoids to plantlet were also standardised. Among the different media tested for embryogenesis namely, MS, WPM and B5, MS medium was found to be the most ideal. Embryoids could be induced only from the tender cotyledon and embryonal axis of immature embryos of 100 days old pods. Other vegetative tissues like leaf, stem, petal, gynoecium, integumant etc. Yielded only non-embryogenic calli in media for somatic embryogenesis. An important finding in the present study was the standardisation of an ideal medium which favoured maximum embryogenesis from embryonic tissues. This medium was MS+ NAA 1.8 + thiamine 1 mg 1-1 + CW 15 per cent + sucrose 4 per cent. This is a modification of medium proposed for cocoa somatic embryogenesis by Adu-Ampomah et al. (1988). As already reported by other workers, the maximum embryogenesis occurred under dark incubation. The ideal incubation temperature was 30±20C. The embryoids originated singly or in clusters from the cotyledon explants. Most of the embryoids lacked a suspensor but some of them did have a suspensor. A typical embryoids had an embryonic axis and two cotyledons. However, aberrant forms were not uncommon with excessive proliferation of cotyledons as well as with disproportionate axes and cotyledons. The study helped to identify some genotype which showed maximum degree of embryogenesis. The Series I hybrid H 6.5 was found to be the ideal source of explant giving high frequency and intensity of embryogenesis as well as with larger sized embryoids having lesser percentage of abnormalities. The selfed progeny (S1) of this out-breeding crop exhibited minimum degree of embryogenesis. This indicates that the degree of embryogenesis may be associated with the vigour of the explant. Germination of embryoid to plantlet was a difficult process. Liquid media with 1/2 MS salts and 5 per cent sucrose was found to favour germination. A pretreatment was required to remove the inhibitors by washing and desiccation. Only embryoids of larger size (>4 mm) germinated properly to plantlet. The recovered plantlets were too small for field establishment. The most significant achievement in the present study was the plantlet regeneration from somatic embryoids and its planting out in the nursery. This was achieved by micrografting the embryoid derived plantlet to a three week old seedling rootstock. The presence of cotyledons was found to be inhibitory and at least a small leaflet in the embryoid derived plantlet was essential for success in micrografting.