2010 Annual Report
The goal of this project is to develop virus-resistant plants using transgenic plant technology, which contributes directly to the objective of the in-house project.
Developing lettuce with resistance to tomato spotted wilt virus: Plants with virus resistance can be developed using transgenic plant technology, by transferring a viral gene or gene fragment to a host plant. When successful, this results in crops with highly specific and heritable resistance. Tomato spotted wilt virus (TSWV) is the most important virus affecting lettuce in Hawaii, and we have transformed lettuce plants with the TSWV nucleocapsid (N) gene in various configurations. In some lines, the nucleocapsid protein is expected to be produced, and in others only the nucleocapsid RNA will be produced. Previous work has shown that resistance can be conferred by expressing viral RNA, and viral protein expression is not always required for resistance.
The lettuce plants that were directly transformed (the primary transformants) were allowed to set seed, and a PCR test confirmed that the majority of those progeny were transgenic. A small number of seeds from the primary transformants were produced, so in order to produce plants for virus challenge experiments, several of the progeny plants were allowed to set seed. This resulted in hundreds of seeds of those transgenic lines. These seeds are being propagated for segregation analysis, and for evaluation of virus resistance.
Developing orchids with resistance to cymbidium mosaic virus, and evaluating orchid virus detection kits: The most important orchid viruses in Hawaii and worldwide are cymbidium mosaic virus (CymMV) and odontoglossum ringspot virus (ORSV). CymMV is more prevalent, but both viruses are serious challenges. We are working to develop transgenic orchids with resistance to CymMV, by transferring the CymMV coat protein gene to orchids. Our initial work is with Dendrobium varieties UH800 and UH306, which are very popular cut-flower varieties. We hope to provide virus resistant plants for Hawaiian cut-flower farms, which can have CymMV infection rates of over 95%.
We have cloned the CymMV coat protein (CP) gene and have developed vectors that will express this gene in both sense and antisense orientations. We are also in the process of transferring our constructs into a plant transformation vector for subsequent transfer to Agrobacterium. Protocorm-like bodies of the Dendrobium cultivars Jacquelyn Thomas UH800 and UH306 are being maintained in shaking culture, and these will be cocultivated with Agrobacterium to achieve gene transfer.
We are also using PCR as a sensitive virus detection method. We are doing this to resolve some serious discrepancies with the commercial antibody-based, orchid virus test kits. Some orchid growers use these kits, and these discrepancies are arising with recent large shipments of flasked orchids from overseas facilities.
The project is monitored via progress reports, meetings, site-visits, and telephone and email communications.