1a. Objectives (from AD-416)
The objectives of this cooperative research project are to: -Develop virus-resistant plants using transgenic plant technology. -Evaluate lines of transgenic lettuce and tomato for tomato spotted wilt virus (TSWV) resistance. -Develop orchid plants with resistance to cymbidium mosaic virus (CymMV).
1b. Approach (from AD-416)
We will work towards developing virus-resistant plants, using transgenic plant technology. Tomato spotted wilt virus (TSWV) has worldwide impact, and particularly in the tropics and subtropics. We have cloned and sequenced several TSWV genes, and transferred them to lettuce plants. We will evaluate these lines for TSWV resistance. In addition, we will evaluate lines of transgenic tomato for TSWV resistance. These tomato plants were developed by the PBARC research group, and contain a gene for TSWV resistance bred in using conventional means, in addition to a TSWV transgene. After initial greenhouse evaluations, we will move the most promising lines on to field trials. TSWV is an extremely important plant virus in Hawaii and elsewhere, and the development of TSWV-resistant lettuce and tomato will greatly benefit agriculture in Hawaii and abroad. Work will also begin towards developing orchid plants with resistance to cymbidium mosaic virus (CymMV), the most important orchid virus worldwide. Most Hawaiian orchid growers that sell cut flowers specialize in a just a few Dendrobium varieties, and experience CymMV infection rates of over 90%. These growers have selected cultivars with good tolerance to virus infection, but they are now severely limited with regard to bringing in new cultivars, as well as selling potted plants. In addition, shipments of orchids from overseas are often infected with CymMV, so there is an influx of new CymMV strains to Hawaii. Recent techniques for engineering virus resistance in plants can provide resistance to multiple strains of a plant virus, and we have cloned and sequenced several of the Hawaiian isolates of CymMV. The availability of CymMV-resistant dendrobiums will benefit the orchid cut-flower growers directly, and will also aid the entire orchid industry by reducing the overall numbers of virus-infected plants. Documents SCA with U of HI -Hilo.
3. Progress 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.