Somatic embryogenesis and transformation of cassava for enhanced starch production.

摘要

Cassava (Manihot esculenta Crantz), a member of the family Euphorbiaceae, is one of the most important food crops of sub-Saharan Africa. One of the requirements for the generation of genetically engineered cassava is an efficient and reproducible plant regeneration and transformation system. We describe here improved methods for plant regeneration of recalcitrant African cassava cultivars. This technology will allow us to increase the range of cultivars that can potentially be engineered using recombinant DNA technologies.; One of the constraints for cassava starch production is that it typically takes 9--12 months to yield a good harvest. This is longer than other major starch-producing crops such as corn and potatoes. We report here the generation of transgenic cassava with increased starch biosynthesis capacity, achieved by enhancing the activity of ADP-glucose pyrophosphorylase (AGPase), the rate-limiting enzyme in the starch biosynthesis. To do this, we transformed cassava with a modified E. coli glgC gene encoding AGPase. The glgC gene was modified by site-directed mutagenesis (K296E/G336D) to remove the allosteric regulation (enhancement by fructose1,6-P and inhibition by AMP) sites and to increase the velocity of the enzyme. Root-specific expression of the glgC gene product was achieved using the tuber-specific patatin promoter of potato. We obtained antibiotic-resistant transformed plants which have been shown to have integrated and expressed the transgene by PCR, Southern blot, RT-PCR and enzyme activity analyses. AGPase enzyme activity in transformed plants was increased by more than 65%. Significantly, transgenic plants expressing the bacterial glgC gene had two-fold greater top (leaf and stem) and root biomass than wild-type plants grown in the greenhouse.; Cassava has a high efficiency of photosynthesis. Therefore, we postulated that we could also enhance starch biosynthesis by increasing sucrose biosynthesis in cassava leaves. This was done by transforming cassava with maize sps gene that encodes sucrose phosphate synthase, the enzyme that catalyzes sucrose synthesis in leaves. Expression of the sps gene in leaves was driven by the leaf specific CAB1 promoter. Southern blotting, RT-PCR and sucrose phosphate synthase enzyme assays were used to demonstrate enhanced expression of the maize sps gene and increased SPS enzyme activity (58%--82%).