Genetic modification of cassava for enhanced starch production.

To 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.

Cassava (Manihot esculenta Crantz).

During the last three years the generation of stably transformed cassava plants having value-added traits has become a reality. Currently, two Agrobacterium-mediated transformation systems are routinely used to engineer cassava. These systems use either somatic embryos or friable embryogenic calli. This paper presents detailed protocols for the transformation of cassava using primary somatic embryos. The effects of explant types, tissue culture conditions, and bacterial and plasmid related factors on transformation efficiency are discussed.

Iron Biofortification and Homeostasis in Transgenic Cassava Roots Expressing the Algal Iron Assimilatory Gene, FEA1.

We have engineered the tropical root crop cassava (Manihot esculenta) to express the Chlamydomonas reinhardtii iron assimilatory gene, FEA1, in its storage roots with the objective of enhancing the root nutritional qualities. Iron levels in mature cassava storage roots were increased from 10 to 36 ppm in the highest iron accumulating transgenic lines. These iron levels are sufficient to meet the minimum daily requirement for iron in a 500 g meal. Significantly, the expression of the FEA1 gene in storage roots did not alter iron levels in leaves. Transgenic plants also had normal levels of zinc in leaves and roots consistent with the specific uptake of ferrous iron mediated by the FEA1 protein. Relative to wild-type plants, fibrous roots of FEA1 expressing plants had reduced Fe (III) chelate reductase activity consistent with the more efficient uptake of iron in the transgenic plants. We also show that multiple cassava genes involved in iron homeostasis have altered tissue-specific patterns of expression in leaves, stems, and roots of transgenic plants consistent with increased iron sink strength in transgenic roots. These results are discussed in terms of strategies for the iron biofortification of plants.

Iron and protein biofortification of cassava: lessons learned.

Over two hundred and fifty million Africans rely on the starchy root crop cassava (Manihot esculenta) as their primary source of calories. Cassava roots, however, have the lowest protein:energy ratio of all the world's major staple crops. Furthermore, a typical cassava-based diet provides less than 10-20% of the required amounts of iron, zinc, vitamin A and vitamin E. The BioCassava Plus program employed modern biotechnologies to improve the health of Africans through development and delivery of novel cassava germplasm with increased nutrient levels. Here we describe the development of molecular strategies and their outcomes to meet minimum daily allowances for protein and iron in cassava based diets. We demonstrate that cyanogens play a central role in cassava nitrogen metabolism and that strategies employed to increase root protein levels result in reduced cyanogen levels in roots. We also demonstrate that enhancing root iron uptake has an impact on the expression of genes that regulate iron homeostasis in multiple tissues. These observations demonstrate the complex metabolic interactions involved in enhancing targeted nutrient levels in plants and identify potential new strategies for further enhancing nutrient levels in cassava.

Overexpression of hydroxynitrile lyase in cassava roots elevates protein and free amino acids while reducing residual cyanogen levels.

Cassava is the major source of calories for more than 250 million Sub-Saharan Africans, however, it has the lowest protein-to-energy ratio of any major staple food crop in the world. A cassava-based diet provides less than 30% of the minimum daily requirement for protein. Moreover, both leaves and roots contain potentially toxic levels of cyanogenic glucosides. The major cyanogen in cassava is linamarin which is stored in the vacuole. Upon tissue disruption linamarin is deglycosylated by the apolplastic enzyme, linamarase, producing acetone cyanohydrin. Acetone cyanohydrin can spontaneously decompose at pHs >5.0 or temperatures >35°C, or is enzymatically broken down by hydroxynitrile lyase (HNL) to produce acetone and free cyanide which is then volatilized. Unlike leaves, cassava roots have little HNL activity. The lack of HNL activity in roots is associated with the accumulation of potentially toxic levels of acetone cyanohydrin in poorly processed roots. We hypothesized that the over-expression of HNL in cassava roots under the control of a root-specific, patatin promoter would not only accelerate cyanogenesis during food processing, resulting in a safer food product, but lead to increased root protein levels since HNL is sequestered in the cell wall. Transgenic lines expressing a patatin-driven HNL gene construct exhibited a 2-20 fold increase in relative HNL mRNA levels in roots when compared with wild type resulting in a threefold increase in total root protein in 7 month old plants. After food processing, HNL overexpressing lines had substantially reduced acetone cyanohydrin and cyanide levels in roots relative to wild-type roots. Furthermore, steady state linamarin levels in intact tissues were reduced by 80% in transgenic cassava roots. These results suggest that enhanced linamarin metabolism contributed to the elevated root protein levels.

The Iron Assimilatory Protein, FEA1, from Chlamydomonas reinhardtii Facilitates Iron-Specific Metal Uptake in Yeast and Plants.

We demonstrate that the unique green algal iron assimilatory protein, FEA1, is able to complement the Arabidopsis iron-transporter mutant, irt1, as well as enhance iron accumulation in FEA1 expressing wild-type plants. Expression of the FEA1 protein reduced iron-deficient growth phenotypes when plants were grown under iron limiting conditions and enhanced iron accumulation up to fivefold relative to wild-type plants when grown in iron sufficient media. Using yeast iron-uptake mutants, we demonstrate that the FEA1 protein specifically facilitates the uptake of the ferrous form of iron. Significantly, the FEA1 protein does not increase sensitivity to toxic concentrations of competing, non-ferrous metals nor facilitate their (cadmium) accumulation. These results indicate that the FEA1 protein is iron specific consistent with the observation the FEA1 protein is overexpressed in cadmium stressed algae presumably to facilitate iron uptake. We propose that the FEA1 iron assimilatory protein has ideal characteristics for the iron biofortification of crops and/or for facilitated iron uptake in plants when they are grown in low iron, high pH soils, or soils that may be contaminated with heavy metals.