2003. Somatic embryogenesis and transformation of cassava for enhanced starch production

2003. Somatic embryogenesis and transformation of cassava for enhanced starch production

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ABSTRACT

Cassava (Manihot esculenta C'rantz), a member of the family Euphorbiaceae, is one of the most important food crops of sub-Saharan Africa. It is grown throughout the tropics including Asia and Latin America. The five main producers are; Nigeria, Brazil, Thailand, Zaire and Indonesia, which together account for 63% of the total world production of 120-130 million tons of fresh roots per year. The roots are the primary plant part consumed and store abundant amounts of starch. They provide a valuable source of cheap calories for about 500 million people, many of whom are subsistence farmers. In addition, cassava leaves and tender shoots are eaten as a vegetable in many parts of Africa and are an excellent source of vitamins, minerals and protein. Although most cassava is consumed by humans, it is also used in the production of ethanol for fuel, for animal feed, and as a raw material for the starch industry.

Cassava’s high photosynthctic rate, ability to grow on poor soils and its resistance to many pests and herbivores due to the presence of cyanogens make it an ideal crop for subsistence farmers. Furthermore, cassava is largely propagated clonally making it an ideal plant for improvement through genetic engineering.

One of the requirements for the generation of genetically engineered cassava is an efficient and reproducible plant regeneration and transformation system. One of my research objectives was to improve upon current technologies used for cassava transformation. We describe here improved methods for plant regeneration of recalcitrant African cassava cullivars. 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 the long growing season. Cassava typically takes 9-12 months to yield a good harvest. This is longer than other major starch-producing crops such as com and potatoes. The longer growing season of cassava also means that it may need more maintenance in the field than other crops. One objective of my research program was to increase the starch biosynthesis capacity of cassava by enhancing the enzyme activity of ADP-glucose pyrophosphorylase (AGPase), the rate-limiting enzyme in starch biosynthesis. To do this, we transformed cassava with a modified E. coli glgC gene that encodes AGPase. The glgC gene was modified by site-directional mutagenesis (K296E/G336D) to remove the allosteric regulation (enhancement by fructose-1, 6-P and inhibition by adenine monophosphate (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 putative 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%. Transgenic plants expressing the bacterial glgC gene had two-fold greater stem and root biomass than wild-type plants.

Cassava has a high efficiency of photosynthetic conversion of carbon dioxide into assimilates. We also 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 CAB1 promoter which is leaf specific. 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% to 82%). Given the long-term nature of field trials it is estimated that growth analysis studies will extend an additional
year from the current time  .