Progress 07/01/05 to 04/28/07
Outputs
Progress Report Objectives (from AD-416) To produce genetically enhanced cassava varieties with storage roots that are biofortified with nutritionally significant levels of vitamin E and vitamin A. Approach (from AD-416) Genes encoding enzymes for rate-limiting steps in the carotenoid and tocopherol biosynthetic pathways will be linked to storage root-specific promoters and inserted into appropriate binary vectors. The resulting gene constructs will be stably introduced into cassava via agrobacterium- mediated transformation. Storage roots from transformed plants will be analyzed for increases in carotenoid and tocopherol content. The agronomic performance and phenotypic stability of plants with the highest levels of carotenoids and tocopherols in storage roots will be tested under greenhouse conditions and ultimately in field trials. Significant Activities that Support Special Target Populations This report serves to document research conducted under a reimbursable agreement between ARS and The Ohio State University. Additional details of this research can be found in the report for the parent project 3622- 21000-028-00D, "Genetic Enhancement of Soybean Seed Value by Biotechnology." Cassava is a staple crop for large portions of the population of sub- Saharan Africa, Central America, South America, and southern Asia. Storage roots of cassava are rich in calories because of their high content of starch, but are deficient in many essential micronutrients. As a component of the BioCassava Plus program, we are conducting research to increase the vitamin E and provitamin A (beta-carotene) content of cassava storage roots by ten-fold through biotechnology. To date, nine transgenes have been introduced into cassava. Preliminary analyses of transgenic lines expressing the crtB gene for phytoene synthase has revealed increases in the pro-vitamin A or beta-carotene levels in storage roots from greenhouse grown cassava plants. In the limited sampling that has been conducted to date, roots from the top-performing lines have a light yellow appearance, and carotenoids are visually evident upon organic solvent extraction of the roots. Up to a ten-fold enhancement of beta-carotene levels in these roots has been confirmed by HPLC and spectrometric analyses. Vitamin E target levels have also been achieved in transgenic roots. To date, constitutive expression of homogentisate geranylgeranyl transferase (HGGT) has resulted in an approximately eight-fold increase in vitamin E-type compounds, principally in the form of gamma- and alpha-tocotrienol, in fibrous roots from tissue culture-maintained plants. These results point to HGGT expression as a viable approach for meeting the vitamin E target. Experiments are underway to co-express HGGT and gamma- tocopherol/tocotrienol methyltransferase genes under control of root- specific promoters to generate higher levels of the more nutritionally significant alpha-tocotrienol form of vitamin E. The project was monitored by the submission of mid-year and annual progress reports to the Gates Foundation through the project principal investigator and by an annual meeting and oral report to project participants.
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Progress 10/01/05 to 09/30/06
Outputs
Progress Report 4d Progress report. This report serves to document research conducted under a reimbursable agreement between ARS and Ohio State University. Additional details of this research can be found in the report for the parent project 3622- 21000-028-00D, "Genetic Enhancement of Soybean Seed Value by Biotechnology." The goal of this project is to enhance the nutritional value of cassava storage roots by increasing the content of vitamin E and pro-vitamin A through biotechnology. Cassava is the major source of calories for over 600 million people in sub-Saharan Africa and portions of Asia, but is deficient in many essential micronutrients. Constitutive and root-specific gene expression constructs were generated for phytoene synthase (crtB) and geranylgeranyl reductase (GGR). These constructs were transformed into cassava embryogenic calli for whole plant production and into a cassava composite root system for gene testing. Stable transformation of embryogenic
calli with root-specific constructs was confirmed by genetic analyses. Embryos have been obtained from these lines and are currently being regenerated. Calli transformed with the crtB gene under control of a constitutive promoter were visually observed to accumulate carotenoids (pro-vitamin A). These calli are currently forming embryos, which will be regenerated to obtain whole plants. A double gene expression construct that contains genes for GGR and homogentisate phytyltransferase (HPT) under control of root-specific promoters was prepared and will be used for upcoming transformation experiments. Detailed chemical analyses revealed the presence of tocotrienols, rather than the more nutritious tocopherols, as the nearly exclusive form of vitamin E in cassava storage roots. It is expected that introduction of the GGR and HPT constructs will result in the redirection of metabolic flux from the production of tocotrienols to the tocopherol class of vitamin E in cassava storage
roots. These findings will impact our design of strategies to improve the nutritional quality of Cassava and other crops.
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