Progress 10/01/12 to 09/30/13
Outputs
Progress Report Objectives (from AD-416): (1) Develop lines of potato cultivar Atlantic in which the genes for aparagine synthetase (ASy) and vacuolar acid invertase (VInv) are suppressed. (2) Quantify asparagine and reducing sugars in cold-stored tubers of these lines. (3) Measure acrylamide in potato chips made from these lines and relate to asparagine and reducing sugar content in raw tubers. (4) Evaluate the growth of suppressed lines and identify potential agronomic problems. Approach (from AD-416): Develop silenced lines: The RNA interference (RNAi) approach will be used for potato gene silencing. We will transform our existing lines of Atlantic that have strong suppression of the VInv gene with silencing constructs that target the two potato ASy genes (2). These lines will be characterized with regard to expression of the three silenced genes. Individuals with a range of ASy gene silencing will be selected for detailed analysis. Biochemical characterization of tubers: Tuber sucrose, glucose and fructose contents will be quantified by HPLC. Tuber asparagine content will be measured with the L-asparagine/L-glutamine assay kit (Megazyme). Acrylamide content of fried chips will be measured in the Department of Bacteriology, UW Madison. Evaluation of plant growth: Transformed lines will be grown in greenhouses using standard procedures to produce tubers for biochemical analysis. Growth characteristics of plants and tuber number, weight and shape will be documented for each line and for Atlantic controls. Differences between controls and silenced lines may indicate potential limits on agronomic performance that will be investigated further in subsequent years. This project was renumbered from 3655-21000-049-25S to 3655-21220-002-08S. This is the final report, project terminated 7/31/2013. Food safety is a primary concern for US agriculture. In 2002, research scientists discovered that trace amounts of acrylamide, a potential human carcinogen, are formed in many food products that are cooked at high temperatures. These products include potato chips and French fries. Acrylamide is produced during cooking when the simple sugars glucose and fructose react with the amino acid asparagine. Decreasing the content of glucose, fructose, and asparagine in raw potato tubers may be one way to minimize concerns related to acrylamide in processed potato products. Tissue culture plantlets in which the genes for asparagine synthetase and vacuolar acid invertase are suppressed were generated and planted in the greenhouse. Asparagine synthetase and vacuolar acid invertase are the enzymes primarily responsible for producing asparagine, glucose and fructose in potato tubers. Decreasing the expression of the genes needed to make these enzymes is hypothesized to produce tubers with less glucose, fructose and asparagine than that found in control tubers. Plants were evaluated for healthy appearance and for relative expression of the gene- silencing constructs. Tubers were harvested from pots after plants had senesced. Multiple lines that together covered a broad range of gene- silencing, from very effective to ineffective for each enzyme, were selected for further analysis. Tubers of gene-silencing lines derived from cultivar Russet Burbank were planted in research field plots in 2013 and plant emergence, canopy development, and overall health are being evaluated. Tubers based on cultivar Atlantic were planted in the greenhouse and are being evaluated for plant health and normal development. Tubers will be harvested from all lines in the fall and used for detailed molecular and biochemical analysis. The acrylamide-forming potential of each line will be evaluated. Targets for tuber asparagine, glucose, and fructose will be developed that can be used to guide future potato breeding efforts focused on acrylamide mitigation. This research completes Objective 1), Develop lines of potato cultivar Atlantic in which the genes for asparagine synthetase (ASy) and vacuolar acid invertase (VInv) are suppressed. It also make substantial progress toward Objective 2), Quantify asparagine and reducing sugars in cold- stored tubers of these lines, and Objective 3), Measure acrylamide in potato chips made from these lines and relate to asparagine and reducing sugar content in raw tubers, in that tubers are currently being produced for post-harvest analysis. This research partially completes Objective 4), Evaluate the growth of suppressed lines and identify potential agronomic problems in that above ground growth of plants is being assessed. Completion of this objective will occur after harvest when tuber size and yield will be evaluated.
Impacts
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Progress 10/01/11 to 09/30/12
Outputs
Progress Report Objectives (from AD-416): (1) Develop lines of potato cultivar Atlantic in which the genes for aparagine synthetase (ASy) and vacuolar acid invertase (VInv) are suppressed. (2) Quantify asparagine and reducing sugars in cold-stored tubers of these lines. (3) Measure acrylamide in potato chips made from these lines and relate to asparagine and reducing sugar content in raw tubers. (4) Evaluate the growth of suppressed lines and identify potential agronomic problems. Approach (from AD-416): Develop silenced lines: The RNA interference (RNAi) approach will be used for potato gene silencing. We will transform our existing lines of Atlantic that have strong suppression of the VInv gene with silencing constructs that target the two potato ASy genes (2). These lines will be characterized with regard to expression of the three silenced genes. Individuals with a range of ASy gene silencing will be selected for detailed analysis. Biochemical characterization of tubers: Tuber sucrose, glucose and fructose contents will be quantified by HPLC. Tuber asparagine content will be measured with the L-asparagine/L-glutamine assay kit (Megazyme). Acrylamide content of fried chips will be measured in the Department of Bacteriology, UW Madison. Evaluation of plant growth: Transformed lines will be grown in greenhouses using standard procedures to produce tubers for biochemical analysis. Growth characteristics of plants and tuber number, weight and shape will be documented for each line and for Atlantic controls. Differences between controls and silenced lines may indicate potential limits on agronomic performance that will be investigated further in subsequent years. Two approaches were used to develop lines of potato cultivar Atlantic in which the genes for asparagine synthetase (ASy) and vacuolar acid invertase (VInv) are suppressed. In the first approach, separate ribonucleic acids (RNA) interference silencing constructs were used for VInv and for ASy, with the ASy construct targeting a conserved region found in the two potato asparagine synthetase genes. A total of 70 plants were generated with this approach and a minimum of 24 tested positive for reduced expression of VInv and ASy. The second approach was to target the two ASy genes separately using two different silencing sequences. A total of 120 plants were generated using this approach, and a minimum of 41 tested positive for reduced expression of VInv and ASy. Tissue culture plantlets were generated for each line and transplanted to soil. We are evaluating the growth of suppressed lines in the greenhouse to identify potential agronomic problems that might develop from VInv suppression or ASy suppression. For most plants, growth and development appear to be normal. Some plants are showing abnormal growth, but to date we have not been able to correlate these differences with the degree of silencing of either the ASy genes or the VInv gene. We anticipate that the first set of tubers will be available in late 2012. We will quantify asparagine and reducing sugars in cold-stored tubers of these lines, and acrylamide content in chips made from those tubers. This research relates to Objectives 1), Develop lines of potato cultivar Atlantic in which the genes for asparagine synthetase (ASy) and vacuolar acid invertase (VInv) are suppressed; 2), Quantify asparagine and reducing sugars in cold-stored tubers of these lines; 3), Measure acrylamide in potato chips made from these lines and relate to asparagine and reducing sugar content in raw tubers; and 4), Evaluate the growth of suppressed lines and identify potential agronomic problems.
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