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.
3. Progress Report
Developing lettuce with resistance to tomato spotted wilt virus: Tomato Spotted Wilt Virus (TSWV) is one of the most serious diseases affecting Hawaii lettuce growers, causing significant losses every year. TSWV is persistently vectored by thrips and infection in lettuce results in the progressive appearance of numerous necrotic lesions approximately 0.5-1 cm in diameter. While these lesions approach coalescence, plants will generally wilt and die within 2 weeks of initial symptom appearance. Previous studies have shown that the introduction of the TSWV nucleocapsid protein gene (N-gene) into plant DNA can confer RNA-mediated protection for lettuce plants. We have transformed lettuce cv. Grand Rapids plants with the TSWV nucleocapsid (N) gene in various configurations. Twenty-four plants grown from seeds from the progeny of primary transformants (R2 plants) were challenged with virus freshly-collected from field-infected tomato. This initial challenge showed promising results with about a third of plants showing good resistance with no lesions, about a third with moderate resistance as determined by having significantly fewer lesion numbers and much better survival than controls. Plants with good resistance were allowed to set seed for further characterization. Other transgenic line challenges are underway, and the remaining transgene constructs are being used to transform both Romaine and leaf lettuce. Developing orchids with resistance to cymbidium mosaic virus, and evaluating orchid virus detection kits: Cymbidium mosaic virus (CymMV) and odontoglossum ringspot virus (ORSV) are the most important and common viruses infecting orchid plants in Hawaii. CymMV is more prevalent, and some cut-flower orchid farms on the Big Island have over 95% infection. We are working to develop transgenic Dendrobium orchid varieties Jacquelyn Thomas UH800 and UH306 with resistance to CymMV, by transferring the CymMV coat protein gene to these orchids. We cloned the CymMV coat protein (CymMVcp) gene and developed vectors in one construction. We have additionally developed vectors in another construction that will express the gene in both sense and antisense orientations. We are transferring these constructs into a plant transformation vector for subsequent transfer to Agrobacterium. Orchid virus detection: We are also using PCR as a sensitive virus detection method in conjunction with local growers’ in vitro propagation programs. There are serious discrepancies with commercial antibody-based orchid virus test kits which can be resolved with PCR. In addition, our PCR tests indicate an approximately 400 base pair discrepancy in the plants that have conflicting antibody-based results when compared to other positive plants. The project is monitored through site visits, meetings, and telephone and email communications.