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. Formerly 5320-21000-011-13S (11/10). Formerly 5320-21000-013-05S (05/13).
3. Progress Report:
The objectives of this cooperative research project to develop virus-resistant plants, evaluate lines of transgenic lettuce and tomato, and develop orchid plants resistant to virus, directly contributes to objective 1 of the inhouse parent project. This is the final report for this project. We developed lettuce (Lactuca sativa) cv. Grand Rapids with resistance to tomato spotted wilt virus (TSWV). As a first step, we isolated TSWV from a lettuce farm in Lalamilo, Hawaii Island. We then cloned and analyzed the nucleocapsid (N) protein gene from this isolate, and transferred it to lettuce plants using an Agrobacterium-mediated transformation protocol. The R2 generation of transformed plants was used for determining resistance, because the primary transformants exhibited poor seed set. This was primarily due to conditions in our laboratory, as increasing light intensity and some flower-handling modifications have resulted in better seed set in subsequent generations. We obtained a TSWV isolate from Lalamilo again to conduct inoculations of our transgenic plants and found that most of these plants were quite resistant. Lettuce plants develop numerous (hundreds, depending on plant size) necrotic lesions 4-5 days following inoculation. These lesions develop on all parts of the plant end eventually coalesce, with plant death resulting shortly thereafter. Most of the transgenic plants containing the TSWV N gene typically developed less than 10 lesions and went on to recover and set seed. Since the R2 generation of plants was used for these challenges, sibling plants not carrying the transgene were used for controls. All nontransgenic control plants died as a result of TSWV infection. TSWV affects many crops in Hawaii, and affects Romaine as well as green leaf lettuce. We initiated an effort to develop Romaine lettuce with TSWV resistance, using the same constructs we used for Grand Rapids. Three transformation protocols were used, but none were efficient at transforming Romaine lettuce. A single plant survived antibiotic selection and we determined that that plant was transgenic. However, that plant developed poorly and exhibited poor seed set. We recently planted some of those seeds and are currently determining whether they carry the transgene. If so, we will conduct TSWV challenges on these plants, in hopes that they exhibit resistance. We are also interested in developing orchid plants with virus resistance. We assayed >200 orchid plants from 4 large orchid farms (2 selling potted plants and 2 selling cut flowers) for the presence of 6 viruses: cymbidium mosaic virus (CymMV), odontoglossum ringspot virus (ORSV), impatiens necrotic spot virus (INSV), tomato spotted wilt virus (TSWV), and cucumber mosaic virus (CMV). CymMV was by far the most prevalent and we are now working to develop orchid plants with resistance to CymMV. Potted plant orchid farms have very low rates of virus infection, including CymMV. Cut-flower farms, however, sell flowers for arrangements and leis, so they generally have older plants which are maintained for as many years as possible. Flowers are harvested from these plants periodically, so harvesting shears efficiently move virus from plant to plant, and the cut-flower farms we surveyed have CymMV infection rates greater than 80%. These growers use plants with tolerance to CymMV, but cooler weather and stress can result in symptoms on flowers, and the production life of these plants is shortened due to virus infection. Movement of plants and tools can also serve to spread inoculum between farms. We plan to transform the dendrobium cultivars UH 306, UH 232 and UH 800, which are very popular plants on cut flower farms in Hawaii. We have cloned the CymMV coat protein gene from a local isolate, placed it in a plant transformation vector with the appropriate plant controlling elements (35S promoter and nos terminator). We have also placed it into a plant transformation vector.