Progress 10/01/10 to 09/30/11
Outputs
Progress Report Objectives (from AD-416) Produce multiple independent transgenic cell lines and plants of peanut by microprojectile bombardment of embryogenic tissues cultured in vitro. Test for tissue specific expression of genes driven by tissue specific gene promoters in transformed peanut. Test peanut tissues transformed with fungal resistance genes for resistance to Aspergillus flavus/parasiticus. Approach (from AD-416) Embryogenic plant cell lines will be bombarded with DNA (containing genes for kanamycin or hygromycin resistance as selective markers) and stably transformed peanut tissues selected for antibiotic resistance. The tissue/organ specific expression of a beta-glucuronidase reporter gene driven by a soybean vegetative storage protein gene promoter will be tested in transgenic Arachis hypogaea progeny of the T1 and T2 generations. Other promoter/reporter or promoter/antifungal gene combinations will be similarly tested. As fungal resistance genes become available from cooperators, these genes will be tested for their effectiveness in inhibiting growth of Aspergillus flavus/parasiticus in peanut tissues transformed with these genes. Antifungal genes, namely a human anti-cell death gene Bcl-xl, and a chloroperoxidase gene (CPO-P) from a bacterium, Pseudomonas pyrrocinia, were tested for their effectiveness in peanuts. The CPO-P gene under the control of the gene expression trigger, called the CaMV35S promoter, was introduced into peanut tissue by a procedure called microprojectile bombardment, and multiple lines of peanuts containing the gene, were tested for the presence and functionality of the introduced gene. The presence of the gene expression products, ribonucleic acid (RNA), and the corresponding protein ensures that the gene was indeed transferred into the peanut tissue. One peanut line has been advanced for several generations, indicating that the gene insertion is stable and will be inherited in subsequent generations of peanut. This line also has shown antifungal property and a consistent 50-80 percent reduction in growth of the aflatoxin producing fungus, Aspergillus flavus, when seeds are inoculated in laboratory assays. Evaluations of the CPO-P line for aflatoxin contamination from field trials that were subjected to drought stress are yet to be performed. The second protein gene introduced into peanut through genetic transformation was Bcl-xL, a human anti-cell death gene. Bcl-xL has been shown to provide tolerance to biological and environmental stresses in other plants. Our Bcl-xL transgenic peanut also demonstrated resistance to stress induced by paraquat, a chloroplast- targeted herbicide. In addition to the generation and evaluation of transgenic lines containing putative anti-fungal genes, we have more thoroughly characterized the lipoxygenase (LOX) gene family in peanut. The lipoxygenase gene family is complex and many questions remain regarding the response of seed-expressed LOX genes to stress induced by environment and fungi, both of which contribute to aflatoxin contamination. Targeted induced mutations (TILLING) of LOX genes and other genes that may impact Aspergillus/aflatoxin contamination is being explored in peanut. Finally, we have also shown that altering the allergen proteins in peanuts (Ara h2 and Ara h6) did not lead to an increased risk of aflatoxin contamination. Research progress was monitored through teleconferencing, and scientific discussions at professional society meetings.
Impacts
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Progress 10/01/09 to 09/30/10
Outputs
Progress Report Objectives (from AD-416) Produce multiple independent transgenic cell lines and plants of peanut by microprojectile bombardment of embryogenic tissues cultured in vitro. Test for tissue specific expression of genes driven by tissue specific gene promoters in transformed peanut. Test peanut tissues transformed with fungal resistance genes for resistance to Aspergillus flavus/parasiticus. Approach (from AD-416) Embryogenic plant cell lines will be bombarded with DNA (containing genes for kanamycin or hygromycin resistance as selective markers) and stably transformed peanut tissues selected for antibiotic resistance. The tissue/organ specific expression of a beta-glucuronidase reporter gene driven by a soybean vegetative storage protein gene promoter will be tested in transgenic Arachis hypogaea progeny of the T1 and T2 generations. Other promoter/reporter or promoter/antifungal gene combinations will be similarly tested. As fungal resistance genes become available from cooperators, these genes will be tested for their effectiveness in inhibiting growth of Aspergillus flavus/parasiticus in peanut tissues transformed with these genes. Aflatoxin is a toxin and carcinogenic compound produced by this fungus on certain crops. Multiple genetic approaches to reduce aflatoxin contamination of peanut are being explored. Assumed antifungal genes being tested include a human anti-cell death gene Bcl-xl, and a chloroperoxidase gene (CPO-P) from a bacterium, Pseudomonas pyrrocinia. The CPO-P gene under the control of the gene expression trigger called the CaMV35S promoter, which was introduced into peanut by microprojectile bombardment, and multiple lines containing the gene, were tested for expression of the introduced gene. Expression was detected for both the direct gene expression product, ribonucleic acid (RNA), and protein. One line has been advanced for several generations, indicating that the gene insertion is stable and will be inherited in subsequent generations of peanut. This line also has shown a consistent 50-80 percent reduction in growth of the aflatoxin producing fungus, Aspergillus flavus, when seeds are inoculated in laboratory assays. Results from the CPO-P transgenic peanut lines have been published in "Peanut Science." Evaluations of the CPO-P line for aflatoxin contamination from field trials that were subjected to drought stress are yet to be performed. The second protein introduced into peanut through genetic transformation was Bcl-xL, a human anti-cell death gene. Bcl-xL transgenic plants of other species show less negative responses to a wide range of biological and environmental stresses. Our Bcl-xL transgenic peanut also demonstrated resistance to paraquat, a chloroplast-targeted herbicide. Research progress was monitored through teleconferencing, visits to cooperators' labs, and scientific discussions at professional society meetings.
Impacts
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Publications
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Progress 10/01/08 to 09/30/09
Outputs
Progress Report Objectives (from AD-416) Produce multiple independent transgenic cell lines and plants of peanut by microprojectile bombardment of embryogenic tissues cultured in vitro. Test for tissue specific expression of genes driven by tissue specific gene promoters in transformed peanut. Test peanut tissues transformed with fungal resistance genes for resistance to Aspergillus flavus/parasiticus. Approach (from AD-416) Embryogenic plant cell lines will be bombarded with DNA (containing genes for kanamycin or hygromycin resistance as selective markers) and stably transformed peanut tissues selected for antibiotic resistance. The tissue/organ specific expression of a beta-glucuronidase reporter gene driven by a soybean vegetative storage protein gene promoter will be tested in transgenic Arachis hypogaea progeny of the T1 and T2 generations. Other promoter/reporter or promoter/antifungal gene combinations will be similarly tested. As fungal resistance genes become available from cooperators, these genes will be tested for their effectiveness in inhibiting growth of Aspergillus flavus/parasiticus in peanut tissues transformed with these genes. Significant Activities that Support Special Target Populations Multiple genetic approaches to reduce aflatoxin contamination of peanut are being explored. Putative antifungal genes being tested include an anti-apoptotic gene Bcl-xl from mammals, and a chloroperoxidase gene (CPO- P) from a bacterium, Pseudomonas pyrrocinia. The CPO-P gene, under the control of the gene expression trigger called the CaMV35S promoter, was introduced into peanut by a process microprojectile bombardment, and multiple lines containing the gene were tested for expression of the requiring tranferred gene. Expression was detected for both the direct gene expression product, ribonucleic acid (RNA), and protein. One line has been advanced for several generations, indicating that the gene insertion is stable and will be inherited in subsequent generations. This line also has shown a consistent 50 percent reduction in growth of the aflatoxin producing fungus, Aspergillus flavus, when seeds are inoculated in laboratory assays. The CPO-P line was planted in two aflatoxin tests in 2006. Each test contained 10 plots of the CPO-P line that were subjected to drought stress and will be assayed for aflatoxin production. A manuscript has been accepted for publication on the antifungal activity of transgenic peanut expressing CPO-P. The second protein introduced into peanut through genetic transformation was Bcl-xL, an anti-apoptotic gene. Bcl-xL transgenic plants of other species show less negative responses to a wide range biotic and abiotic stresses. Our Bcl-xL transgenic peanut also demonstrated resistance to paraquat, a chloroplast (essential plant cell organelle) -targeted herbicide. Research progress was monitored through teleconferencing, emails, and reports.
Impacts
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Progress 10/01/07 to 09/30/08
Outputs
Progress Report Objectives (from AD-416) Produce multiple independent transgenic cell lines and plants of peanut by microprojectile bombardment of embryogenic tissues cultured in vitro. Test for tissue specific expression of genes driven by tissue specific gene promoters in transformed peanut. Test peanut tissues transformed with fungal resistance genes for resistance to Aspergillus flavus/parasiticus. Approach (from AD-416) Embryogenic plant cell lines will be bombarded with DNA (containing genes for kanamycin or hygromycin resistance as selective markers) and stably transformed peanut tissues selected for antibiotic resistance. The tissue/organ specific expression of a beta-glucuronidase reporter gene driven by a soybean vegetative storage protein gene promoter will be tested in transgenic Arachis hypogaea progeny of the T1 and T2 generations. Other promoter/reporter or promoter/antifungal gene combinations will be similarly tested. As fungal resistance genes become available from cooperators, these genes will be tested for their effectiveness in inhibiting growth of Aspergillus flavus/parasiticus in peanut tissues transformed with these genes. Significant Activities that Support Special Target Populations Multiple genetic approaches to reduce aflatoxin contamination of peanut are being explored. Putative antifungal genes being tested include a human anti-apoptotic gene Bcl-xl, and a chloroperoxidase gene (CPO-P) from a bacterium, Pseudomonas pyrrocinia. The CPO-P gene under the control of the gene expression trigger called the CaMV35S promoter, which was introduced into peanut by a microprojectile bombardment technique (called genetic transformation), and multiple lines containing the gene, were tested for expression of the gene that was transferred (transgene). Expression was detected for both the direct gene expression product, ribonucleic acid (RNA), and protein. One line has been advanced for several generations, indicating that the gene insertion is stable and will be inherited in subsequent generations. This line also has shown a consistent 50 percent reduction in growth of the aflatoxin producing fungus, Aspergillus flavus, when seeds are inoculated in laboratory assays. The CPO-P line was planted in two aflatoxin tests in 2006. Each test contained 10 plots of the CPO-P line that were subjected to drought stress and will be assayed for aflatoxin production. The second protein introduced into peanut through genetic transformation was Bcl-xL, a human anti-apoptotic gene. Bcl-xL transgenic plants of other species show less negative responses to a wide range biotic and abiotic stresses. Our Bcl- xL transgenic peanut also demonstrated resistance to paraquat, a chloroplast-targeted herbicide. Research progress was monitored through teleconferencing, visits to cooperators labs, and scientific discussions at professional society meetings and the Aflatoxin Elimination Workshop sponsored by industry stakeholders.
Impacts
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Publications
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Progress 10/01/06 to 09/30/07
Outputs
Progress Report Objectives (from AD-416) Produce multiple independent transgenic cell lines and plants of peanut by microprojectile bombardment of embryogenic tissues cultured in vitro. Test for tissue specific expression of genes driven by tissue specific gene promoters in transformed peanut. Test peanut tissues transformed with fungal resistance genes for resistance to Aspergillus flavus/parasiticus. Approach (from AD-416) Embryogenic plant cell lines will be bombarded with DNA (containing genes for kanamycin or hygromycin resistance as selective markers) and stably transformed peanut tissues selected for antibiotic resistance. The tissue/organ specific expression of a beta-glucuronidase reporter gene driven by a soybean vegetative storage protein gene promoter will be tested in transgenic Arachis hypogaea progeny of the T1 and T2 generations. Other promoter/reporter or promoter/antifungal gene combinations will be similarly tested. As fungal resistance genes become available from cooperators, these genes will be tested for their effectiveness in inhibiting growth of Aspergillus flavus/parasiticus in peanut tissues transformed with these genes. Significant Activities that Support Special Target Populations This report serves to document research conducted under a Specific Cooperative Agreement between the Agricultural Research Service (ARS) and the University of Georgia, Tifton. Additional details of research can be found in the report for the in-house project 6435-42000-019-00D, "Identification and Enhancement of Seed-Based Biochemical Resistance in Crops to Aflatoxin Producing Pathogens." Multiple genetic approaches to reduce aflatoxin contamination of peanut are being explored. Putative antifungal genes being tested include a chloroperoxidase gene (CPO-P) from a bacterium, Pseudomonas pyrrocinia. The CPO-P gene under the control of the gene expression trigger called the CaMV35S promoter, which was introduced into peanut by microprojectile bombardment, and multiple lines containing the gene, were tested for expression of the transgene. Expression was detected for both the direct gene expression product, ribonucleic acid (RNA), and protein. One line has been advanced for several generations, indicating that the gene insertion is stable and will be inherited in subsequent generations. This line also has shown a consistent 50 percent reduction in growth of the aflatoxin producing fungus, Aspergillus flavus, when seeds are inoculated in laboratory assays. The CPO-P line was planted in two aflatoxin tests in 2006. Each test contained 10 plots of the CPO-P line that were subjected to drought stress and will be assayed for aflatoxin production. Progress by cooperators was monitored through requiring routine teleconferencing, meetings, and scientific presentations of information relating to the project at professional society meetings, conferences and the Annual Aflatoxin Elimination Workshop sponsored by industry stakeholders.
Impacts
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