2009 Annual Report
Tomato spotted wilt virus and lettuce: Tomato spotted wilt virus (TSWV) is the most important virus affecting lettuce (Lactuca sativa) in Hawaii, and we are working to develop lettuce with TSWV-resistance. Earlier work has shown that virus-resistant plants can be produced by transferring a gene from the pathogenic virus into the host plant. This resistance is specific, thus a gene from TSWV might confer resistance to a similar strain of TSWV.
Using Agrobacterium-mediated transformation, we transformed lettuce plants with the TSWV nucleocapsid (N) gene, in either the sense or antisense orientation, using both the cauliflower mosaic virus (CaMV) 35S and tomato ubiquitin promoters, and followed by either the nos terminator or another plant promoter. After collecting and germinating seeds from mature primary transformants, R1 plants were evaluated for the presence of the transgenes. PCR assays for the TSWV N gene in leaf punches thus far indicate that most of the R1 lettuce plants carry the N gene. After identifying which R1 lines contain the transgene, we will proceed with inoculation trials using TSWV. We likewise transformed N. benthamiana plants with the above constructs, in order to assess their competency in inducing gene silencing.
Cymbidium mosaic virus and orchids: Orchids in Hawaii are also greatly affected by viruses. The most important of these are cymbidium mosaic virus (CymMV) and odontoglossum ringspot virus (ORSV), with CymMV being more prevalent. Much of the state’s orchids are produced on farms in East Hawaii. We recently conducted a virus infection survey of several East Hawaii orchid farms and found that cut-flower farms have very high (>85%) infection rates, while potted plant farms have much lower infection rates, at <5%. This is not surprising, as cut-flower farms have much older plants and flower-harvesting activity facilitates virus spread.
We are working to develop orchids with resistance to CymMV using transgenic technology. The effort is aimed at mitigating orchid industry losses to viruses, especially on cut-flower farms, and especially to CymMV. We are working with Dendrobium varieties UH800 and UH306; very popular on these farms.
In developing CymMV-resistant Dendrobiums, subsequent propagation of these plants will be by tissue culture. This is less efficient than the current method of crossing two inbred Dendrobiums to produce pods with thousands of seeds. However, virus resistance over the life of the plant greatly outweighs the added tissue culture costs.
We cloned the coat protein (CP) gene of CymMV and put it into the proper context for expression in plants. We are using vectors we developed that contain both the CaMV 35S and tomato ubiquitin promoters and the nos terminator.
We are in the process of transferring our constructs into a plant transformation vector for subsequent transfer to Agrobacterium. We have obtained protocorm-like bodies (plb’s), of UH800 and UH306 from a local germplasm producer. These plb’s will be cocultivated with Agrobacterium to achieve gene transfer.
ADODR monitored this project through meetings with cooperator, progress reports, email/telephone communications.