Progress 09/15/04 to 09/14/07
Outputs
OUTPUTS: 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 Kauai or on the island of Hawaii. 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 Banana bunchy top virus (BBTV), which will directly benefit the commercial growers of the banana industry. Embryogenic calli that have been initiated from immature 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 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. More than 100 lines have been grown into seedlings and are currently being evaluated for BBTV resistance using viruliferous aphids to challenge these plants. All challenged lines are currently being screened for the development of BBTV symptoms and any undesirable agronomic characteristics. Preliminary results showed that several lines are resistant to BBTV infection. These promising lines will be furthered characterized by molecular methods. The best lines will be eventually tested in field
conditions. 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 growers in Hawaii and plant virologists
Impacts
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
- 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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Progress 10/01/05 to 09/30/06
Outputs
New embryogenic calli were initiated from immature flowers of two varieties of banana, Dwarf Brazilian and Williams. Embryogenic cell suspensions (ECS) of Dwarf Brazilian and Williams were produced and maintained as source of explants for transformation. The mutated Rep gene sequences of Banana bunchy top virus (BBTV) that was used previously in our lab to produce BBTV-resistant banana plants were used to produce more transgenic lines of Dwarf Brazilian and Williams. New BBTV Rep gene constructs have been developed to generate additional engineered BBTV resistant banana lines using genesilencing approaches. Selected embryos formed from transformed ECS were allowed to germinate and grow into seedlings and will be evaluated for the presence of the target transgene using Polymerase Chain Reaction (PCR). More transformation experiments will be conducted to evaluate GUS expression in the various stages of the developing seedlings and to optimize the transformation system.
Transgenic lines will be evaluated with molecular techniques including Real-Time PCR and Southern hybridization, and tested in the greenhouse for BBTV resistance using viruliferous aphids to transmit the virus.
Impacts
The goal of this project is to develop environmentally sound approaches to control Banana bunchy top virus (BBTV), which will directly benefit the commercial banana growers of in Hawaii. Banana production in Hawaii is severely limited by banana bunchy top disease caused by BBTV, which results in lost revenue, increased costs, and environmental degradation due to pesticide use. The disease can best be managed by the development of resistant cultivars
Publications
- No publications reported this period
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Progress 10/01/04 to 09/30/05
Outputs
We are still working on the MTA with the Farmacule Bioindustries Pty Ltd and the Queensland University of Technology in Australia, and working on the permits with the US federal and state regulatory agents to bring transgenic banana plants from Australia to Hawaii to conduct the field experiment. In the mean time, we have focused on production of BBTV-resistant transgenic banana plants in HI. We have initiated and established six lines each of ECS on both Williams and Dwarf Brazilian. These lines were able to regenerate somatic embryos after three months in solid M2 media. Maturation and germination of somatic embryos were made possible using a system developed in our lab previously. This regeneration system will be used to produce transgenic banana plants. Agro-transformation of ECS for both cultivars was tested using the protocol established by Dr. Dale. Transient expression of GUS gene was done using the ECS . We will transform the ECS with the mutant BBTV Rep gene
to produce BBTV resistant lines. We are also working on new gene constructs using gene silencing mechanism approaches.
Impacts
The goal of this project is to produce transgenic BBTV-resistant banana varieties Williams and Dwarf Brazilian. BBTV is a devastating disease of banana that affects the commercial growing areas here in Hawaii and in all other major banana-producing regions except South America. The development of transgenic banana plants with resistance to BBTV will help the growers to alleviate their profits and the consumers to get good quality banana. A reliable and robust system to transform and regenerate transgenic banana plants is the key to accomplish our goal.
Publications
- No publications reported this period
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Progress 10/01/03 to 09/30/04
Outputs
We have been working with federal and sate regulatory agents to bring transgenic banana plants from Australia to Hawaii to conduct the field experiment. In the mean time, we have had consistent success producing proembryotic and embryotic formations from callous formed in vitro. However, due to genomic differences between varieties and difficulties of forming embryogenic cell suspensions from the calloused explants, we have not yet formed a stable cell suspension. Current protocols for developing cell (especially embryogenic) suspensions vary among banana varieties. We also try to develop protocols useful for creating embryonic cell suspensions from a broad range of banana types. Once a suitable suspension protocol has been developed it will be used to transform bananas using Agro-bacterium to produce BBTV resistant lines of dwarf Brazilian and williams varieties.
Impacts
The goal of our banana bunchy top virus project is to produce transgenic virus-resistant bananas varieties dwarf Brazilian and williams. BBTV is a fast-spreading virus that prevents infected plants from producing fruits. Currently BBTV has been reported in every major banana-producing region except South America. It is paramount that research into BBTV continues, because bananas are extremely important as a food crop worldwide. The support of our projects will ensure that those whose livelihoods depend upon bananas will not be ruined by BBTV in the future.
Publications
- No publications reported this period
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