Somatic embryogenesis in cocoa (Theobroma cacao L.)

Abstract

Investigation on somatic embryogenesis in cocoa (Theobroma cacao L.) were carried out at the college of Horticulture, Kerala Agricultural University, Vellanikkara during the period 1989-91, with the objective of studying the developmental potential of somatic embryos and its differentiation into plantlet by means of in vitro techniques. Stem and leaf segments, cotyledons and enbryonic axes of embryos collected from four typical genotypes of cocoa namely criollo, Amelonado, Amazon-forastero and Trinitario were used as explant. Cotyledons and embryonic axes of immature embryos (100 days pot anthesis) when incubated on MS basal semisolid medium supplemented with NAA 2 mg/1, thiamine 1 mg/1, casein hydrolysate 0.2 per cent (W/V) and coconut water 15 per cent (v/v) under dark for seven weeks resulted in high frequency and intensity of embryogenesis. Stem segements remined recalcitrant without embryoid regeneration, while leaf segments had a little potential. Auxins conditioned the culture for embryogenic competence while cytokinins had an inhibitory effect. The effect of NAA 2ppm was not replaceable by other auxins such as IAA, 2, 4-D. 2,4-D was a poor quantitative and qualitative stimulant of embryogenesis. Studies on auxincytokinin interaction revealed the counteracting effect of cytokinin on auxin. Fully mature embryoids germinated in hormone-free liquid medium consisting of half the salt concentration of MS and 5 per cent sucrose when incubated at 3000 lux (16 hours) for two weeks. De-cotyledonisation of embryoids and its rinsing with sterile distilled water and dessication, each for three minutes, enhanced the differentiation into plantlet. Shoot growth was stimulated by exogenous supply of NAA, GA3 and coconut water. In vitro rooting was promoted by reducing the salt concentration of MS medium to half strength and supplementing with IBA and activated charcoal. Germination and regeneration of embryoid into plantlet was dependent on its size. Sizes ranging from 0-4 mm were sub-optimal for germination and differentiation. Larger embroids (4-6 mm) had greater potential for differentiation. Quantitative and qualitative differnces were expressed by cocoa genotypes. Amelonado was found to be superior to Criollo and Amazon types for the induction of embryogenesis from cotyledons. Trinitario was the least efficient. Embryogenic potential of Amazon embryonic axes was superior to Criollo and Amelonado types. Trinitario embryonic axes remained recalcitrant. Plantlets were derived from embryoids within a time span of 13 weeks in Amelonado, Criollo and Amazon types.