Somatic embryogenesis, embryo conversion, micropropagation and factors affecting genetic transformation of Theobroma cacao L.

摘要

The effects of 5 carbon sources (glucose, fructose, maltose, sorbitol, sucrose) and 2 explant types (staminode and petal) on cacao somatic embryogenesis were evaluated. Glucose, fructose and sucrose were found to support cacao somatic embryo production, while no embryos were produced with sorbitol or maltose in the media. Staminode explants were more responsive to somatic embryogenesis than petals, producing up to 99% embryogenic explants and over 20 embryos per responsive explant. In contrast, petal explants produced a maximum of 34% embryogenic explants and a maximum of 8 embryos per responsive explant. Four different media (MS, WP, DKW, and DKW supplemented with potassium nitrate and amino acids) were tested for their ability to support and promote shoot development during embryo conversion. DKW medium supplemented with potassium nitrate and amino acids yielded the highest percentage of normal and total shoot production (59% and 92% respectively). DKW and WPM media supported a higher percentage of conversion (77% each) than did MS medium (59%). The ivory colored type II somatic embryos more frequently produced taproots than did the translucent type I embryos (65% and 35% respectively). In vitro pruning of plantlets with abnormal shoots resulted in axillary meristem growth and conversion into normal plantlets with a rate of 79%. A micropropagation system, including shoot proliferation, was developed for cacao by pruning in vitro somatic embryo derived plantlets. The addition of TDZ to the medium did not affect shoot production from internodal and apical explants. Higher rooting efficiencies of shoot explants were achieved using IBA. Micropropagated and somatic embryo derived plants were acclimated to greenhouse conditions with a success rate averaging 90%. Genetic transformation of cacao staminode tissue was attempted using Agrobacterium or biolistic techniques. Transgenic callus and proembryonic tissue expressing GFP were recovered but no embryo development was obtained. Agrobacterium and the antibiotic cefotaxime at 400mg/L were found to negatively influence staminode explant growth and somatic embryo production.