Progress 09/01/06 to 08/31/09
Outputs
OUTPUTS: Approximately 300 lines were produced from the mutant, antisense, and two of the Rep gene constructs. These lines have been grown into seedlings and have been evaluated for BBTV resistance using viruliferous aphids to challenge these plants. One of 32 plants that contained the mutant Rep gene construct was resistant; 4 of 74 plants that contained the antisense Rep gene construct were resistant; 5 of 38 plants that contained the partial Rep gene construct were resistant; and 10 of 126 plants that contained the partial Rep gene and the full-length Rep gene as inverted repeats were resistant to BBTV challenge. These lines have not developed bunchy top symptoms more than one year after challenge, while all of the 63 control plants had developed symptoms within three months after challenge. These putatively BBTV-resistant lines have been further characterized by molecular methods, including quantitative PCR and Southern hybridization analyses. These analyses indicate that the number of transgenes incorporated into these plants varies from 3 to more than 9 copies per plant. We are in the process of returning all of the putatively BBTV-resistant lines to tissue culture for multiplication by inducing the formation of vegetative propagules from the mother plant stocks. Currently, five resistant lines consisting of 270 individual plants with from 23 to 100 plants per line have been multiplied in tissue culture. Samples from these multiplied lines have been confirmed to contain the transgenes by PCR analyses . These multiplied lines will be retested for BBTV resistance by bioassay with viruliferous aphids before being eventually tested for BBTV resistance in field conditions. The multiplied lines that are BBTV-resistant will be grown to a size suitable for transplating to the field at the Waimanalo Field Station on Oahu, where field testing for BBTV and fungal resistance will be conducted. A provisional patent has been filed with the United States Patent Office for bananas with transgenic resistance to BBTV. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Banana bunchy top is the most devastating viral-caused disease of bananas in Hawaii and many areas of Asia, Africa and the Pacific. Banana bunchy top virus (BBTV) has the potential to destroy the banana industry in Hawaii and the Pacific Basin. Eradication efforts by the Hawaii Department of Agriculture have not been successful on any of the Hawaiian Islands. Control of the aphid vector using insecticides is expensive, inefficient, and poses environmental and health risks. Removal of infected plants is required to limit spread of the virus but requires identification of early symptoms as well as increased labor and chemical costs. New control strategies for the efficient control of BBTV are needed. The goal of this project is to develop environmentally sound approaches to control BBTV, which will directly benefit the commercial growers of the banana industry.
Publications
- Wayne Borth, Eden Perez, Kheng Cheah and John Hu. 2007. Transgenic Banana Resistant to Banana bunchy top virus. Proceedings of the 38th Annual Hawaii Banana Industry Association Conference. Held on Oahu, HI. August 24, 2007.
- Wayne Borth, Eden Perez, Kheng Cheah, Yan Chen, Wen-shuang Xie, Doug Gaskill, Said Khalil, John Hu 2009. Development of banana bunchy top virus-resistant transgenic banana plants. Presentation in the Plant Biology 2009, the Annul Meeting of the American Society of Plant Biologists. Held in Honolulu, HI. July 18-22, 2009.
- Wayne Borth, Eden Perez, Kheng Cheah, Yan Chen, Wen-shuang Xie, Doug Gaskill, Said Khalil, Dennis Gonsalves, John Hu 2009. Transgenic Banana Plants Resistant to Banana Bunchy Top Virus Infection. Acta. Hort. (in press)
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Progress 09/01/07 to 08/31/08
Outputs
OUTPUTS: The goal of this project is to develop environmentally sound approaches to control Banana bunchy top virus (BBTV), which will directly benefit the commercial growers of the banana industry. Embryogenic calli that have been initiated from immature male banana flowers have been used to produce embryogenic cell suspensions (ECS) of banana cultivar Dwarf Brazilian. These cell suspensions were used as the source of explants for Agrobacterium transformations using 5 constructs. Mutant and antisense constructs of the BBTV coat protein gene, and three Rep gene constructs of BBTV were used to produce more transgenic lines of Dwarf Brazilian. Embryos formed from transformed ECS were germinated on media containing antibiotics to select transformed lines. Approximately 300 lines were produced from the mutant, antisense, and two of the Rep constructs. These lines have been grown into seedlings and have been evaluated for BBTV resistance using viruliferous aphids to challenge these plants. Twenty lines have not developed bunchy top symptoms 6 months after challenge , while all of the control plants had developed symptoms within three months after challenge. These putatively BBTV resistant lines have been further characterized by molecular methods, including quantitative PCR and Southern hybridization analyses These analyses indicate that the number of transgenes incorporated into these plants varies from 3 to more than 9 copies per plant. The resistant lines will be multiplied in tissue culture and eventually tested in field conditions. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Banana bunchy top is the most devastating viral-caused disease of bananas in Hawaii and many areas of Asia, Africa and the Pacific. Banana bunchy top virus (BBTV) has the potential to destroy the banana industry in Hawaii and the Pacific Basin. The development of BBTV resistant banana plants through the use of the powerful tools of genetic engineering offer the quickest way to develop banana plants with long-lasting and broad-spectrum resistance to BBTV. The development of such cultivars will directly benefit the commercial banana growers of in Hawaii and the world.
Publications
- No publications reported this period
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Progress 09/01/06 to 08/31/07
Outputs
OUTPUTS: Embryogenic calli that have been initiated from immature flowers have been used to produce embryogenic cell suspensions (ECS) of banana cultivar Dwarf Brazilian. These cell suspensions were used as the source of explants for Agrobacterium transformations using 5 constructs. Mutant and antisense constructs of the Banana bunchy top virus (BBTV) coat protein gene, and three Rep gene constructs of BBTV were used to produce more transgenic lines of Dwarf Brazilian. Embryos formed from transformed ECS were germinated on media containing antibiotics to select transformed lines. These lines have been grown into seedlings and are currently being evaluated for BBTV resistance using viruliferous aphids to challenge these plants. The table below provides current details of these tests. Each plant in the table is considered an individual, independently transformed line. All challenged lines are currently being screened for the development of BBTV symptoms and any undesirable agronomic
characteristics. Construct Rep?2/5Rep was apparently lethal in banana since putatively transgenic lines containing this construct were produced but the development of these lines was arrested at an early stage and no mature plants survived. We have initiated more embryogenic calli from male inflorescences of Dwarf Brazilian and have produced ECS from these calli. We will produce more embryogenic calli and ECS from Dwarf Brazilian in September and December of 2007 and will transform these ECS with Agrobacterium using new constructs incorporating gene-silencing techniques that have recently been developed and that may afford very high resistance to BBTV infection.
PARTICIPANTS: Wayne Borth, Kheng Chaeh
TARGET AUDIENCES: banana farmers in Hawaii, plant virologists
Impacts
The goal of this project is to develop environmentally sound approaches to control Banana bunchy top virus (BBTV). The development of BBTV resistant banana plants through the use of the powerful tools of genetic engineering, offer the quickest way to develop banana plants with long-lasting and broad-spectrum resistance to BBTV. The development of such cultivars will directly benefit the commercial banana growers of Hawaii and the world.
Publications
- Wayne Borth, Eden Perez, Kheng Cheah and John Hu. 2007. Transgenic Banana Resistant to Banana bunchy top virus. Proceedings of the 38th Annual Hawaii Banana Industry Association Conference. Held on Oahu, HI. August 24, 2007.
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