Genetic modification of cassava for enhanced starch production

Abstract

SummaryTo date, transgenic approaches to biofortify subsistence crops have been rather limited. This is particularly true for the starchy root crop cassava ( Manihot esculenta Crantz).Cassava has one of the highest rates of CO 2 fixation and sucrose synthesis for any C3 plant, but rarely reaches its yield potentials in the field. It was our hypothesis that starch production in cassava tuberous roots could be increased substantially by increasing the sink strength for carbohydrate. To test this hypothesis, we generated transgenic plants with enhanced tuberous root ADP-glucose pyrophosphorylase (AGPase) activity. This was achieved by expressing a modified form of the bacterial glgC gene under the control of a Class I patatin promoter. AGPase catalyses the rate-limiting step in starch biosynthesis, and therefore the expression of a more active bacterial form of the enzyme was expected to lead to increased starch production. To facilitate maximal AGPase activity, we modified the Escherichia coli glgC gene (encoding AGPase) by site-directed mutagenesis (G336D) to reduce allosteric feedback regulation by fructose-1,6-bisphosphate. Transgenic plants (three) expressing the glgC gene had up to 70% higher AGPase activity than control plants when assayed under conditions optimal for plant and not bacterial AGPase activity. Plants having the highest AGPase activities had up to a 2.6-fold increase in total tuberous root biomass when grown under glasshouse conditions. In addition, plants with the highest tuberous root AGPase activity had significant increases in above-ground biomass, consistent with a possible reduction in feedback inhibition on photosynthetic carbon fixation. These results demonstrate that targeted modification of enzymes regulating source-sink relationships in crop plants having high carbohydrate source strengths is an effective strategy for increasing carbohydrate yields in sink tissues.

Extending Cassava Root Shelf Life via Reduction of Reactive Oxygen Species Production

“…A, Plasmid construct of AOX. The codonoptimized Arabidopsis AOX, AtAox1A, was cloned into a pBI121-based three-dimensional vector in which the cauliflower mosaic virus 35S promoter was replaced by the root-specific patatin promoter followed by the NOS terminator (Siritunga and Sayre, 2003;Ihemere et al, 2006). B, AOX expression in roots of transgenic lines as determined by RT-PCR.…”

“…Somatic embryogenesis was induced using the method described by Ihemere et al (2006). Leaf lobes from 3-to 4-week-old in vitro cassava plants, variety TMS 60444, were cultured on MS medium containing 8 mg L 21 2,4-dichlorophenoxyacetic acid (2,4-D) for induction of embryogenesis.…”

“…The primers used for the site-directed mutagenesis reaction were AtAox1SDMF (59-CGTGATGTTGTGATGGTTG-TTCGTGCTGACGAGGCTCATCACC-39) and AtAox1SDMR (59-GGTGA-TGAGCCTCGTCAGCACGAACAACCATCACAACATCACG-39). The corrected codon-optimized AtAOX1A was removed from pUC19 using SmaI and SstI and placed under the control of a root-specific patatin promoter in a pBI121-based binary vector to generate 3D-AtAox1A CO (Siritunga and Sayre, 2003;Ihemere et al, 2006;Fig. 3A).…”

Influence of Marker Genes on Physicochemical Properties of Starch Produced by Transgenic Cassava (Manihot esculenta Crantz) Plants

“…Several transgenic cassava varieties have been developed to address numerous abiotic and biotic constraints militating against the crop production. For examples, cyanogenic glucosides-free, mosaic resistant and enhanced starch quantity and protein-rich cassava varieties have been produced by transgenic approach (Zhang et al, 2003, Jorgensen et al, 2005, Zhang et al, 2005, Ihemere et al, 2006. However, modification of cassava starch properties by transgenic approach has attracted limited research attention.…”

Starch meets biotechnology : in planta modification of starch composition and functionalities

“…A mutant variant of the AGPase gene from E. coli (GLGC16) was overexpressed in various plant species, such as cassava (Ihemere et al, 2006), maize and potato (Sweetlove et al, 1996), and the starch content is slightly increased. Surprisingly, Stark et al (1992) targeted this gene to plastids in transgenic potatoes resulting in significantly increased starch accumulation, and in some lines there was almost a 60% increase in starch content relative to control tubers.…”