1a. Objectives (from AD-416)
Objective 1: Develop the genomics of papaya for producing new knowledge about the regulation of transgenic disease resistance. [NP 301, C4, PS 4B] Objective 2: Develop methods for improved manipulation and expression of transgenes in key tropical/subtropical ornamental and fruit crop species. [NP 301, C4, PS 4A] Objective 3: Evaluate biotechnology risk and develop methods for practical adoption of selected transgenic crops. [NP 301, C4, PS 4C]
1b. Approach (from AD-416)
(1) Fingerprint and end-sequence approximately 40,000 clones from our existing bacteria artificial chromosome (BAC) library for anchoring the whole genome shotgun (WGS) sequence data that will be produced from two WGS libraries of the papaya genome, (2) mine the papaya BAC end and genomic sequences to develop 4,000 microsatellite markers (simple sequence repeats or SSRs) for constructing a high density genetic map of the papaya genome of at least 1,000 SSRs for combining with our amplified fragment length polymorphism (AFLP) map, (3) assemble and annotate the papaya genome sequences, (4) select a core set of evenly distributed SSRs to map major genes controlling fruit size and disease reactions, (5) develop a transient gene silencing system for functional genomic analysis in papaya, (6) characterize novel papaya disease resistance genes with the functional genomic tool, (7) determine the relationship between transgene copy number and gene silencing, (8) characterize the activity of SCYLV P0 and other viral suppressors of post-transcriptional gene silencing (PTGS) in Nicotiana benthamiana as a model system for application to sugarcane, (9) identify papaya genes with tissue-specific expression patterns for developing tissue-specific promoters, (10) use segmented and synthetic gene technology to develop and subsequently characterize transgenic papaya with resistance to wide range of papaya ringspot virus (PRSV) strains, (11) measure the extent, if any, of gene flow from commercial transgenic papaya to adjacent nontransgenic papaya fields, (12) develop and commercialize a transgenic Kapoho with segmented coat protein genes for the Hawaiian papaya industry, (13) develop data that are necessary to have the Rainbow transgenic papaya deregulated in Japan, and (14) develop, transfer, and commercialize transgenic papaya for developing countries with focus on Bangladesh. Replacing 5320-21000-011-00D (08/2010).
3. Progress Report
Deep genomic sequencing of genetically engineered (GE), virus resistant SunUp papaya has resulted in greater genome sequence coverage and improved assembly over the previous draft genome sequence. Description of gene content is currently underway through a computer program pipeline. Genome size distribution in the genus Anthurium, Anthurium genes of agronomic importance and the chloroplast genome of commercial Anthurium are being investigated with the aim of obtaining basic molecular information lacking in this group of plants to support development of improved cultivars. Research to characterize floral pigments of Anthurium cultivars has been initiated as an alternative and complementary approach to genetic classification, an area which is not completely understood among extant cultivars. This information should be useful for improving breeding programs and production of new cultivars for one of Hawaii’s most important commercial tropical flower industries. Promising lines were discovered during the evaluation of transgenic anthurium for resistance to burrowing nematodes in a shade-house potted plant bioassay. Hot water drenches were conducted on potted dracaena to eradicate reniform nematodes. Insect parasitic nematodes were found to infect and kill coffee berry borer larvae when sprayed on coffee cherries in laboratory conditions. Bacterial blight of anthurium has adversely affected the profitability of the industry since the 1980s. Screening methods for large quantities of transgenic anthuriums were developed and are being used to determine the practicality and economic feasibility of using transgenic lines as a method of control. Bed trials are being set up to mimic field practices and monitor the horticultural aspects of the plants. Coffee berry borer (CBB), one of the most devastating coffee pests worldwide, was recently discovered in the Kona coffee growing region of Hawaii. The industry is currently facing its toughest challenge and is desperately seeking ways to control this problem. Research is evaluating the potential for using the commercially available B. bassiana GHA strain as a control method for CBB in Hawaii. Persistence study results indicate that the GHA strain can survive longer than expected in the field. This information provides growers with a potential spray strategy for managing CBB disease on coffee. In collaboration with the Hawaiian pineapple industry we are conducting research to study flowering manipulation using genetic engineering. We are currently in the process of regenerating plants following transformation of non-flowering traits to the Hawaiian pineapple varieties. Non-synchronized coffee flowering results in higher high operational costs for both plantations that utilize mechanical harvesting as well as operations that harvest by hand. In addition, the introduction of the Coffee Berry Borer to Kona increases the detriment of non-synchronous flowering and fruit formation by having a constant source of berries for CBB reproduction in the fields. We are using a plant growth regulator and foliar fertilizer approach to provide more synchronous flowering in coffee at locations in Kona, Oahu, and Kauai.
1. Deregulation of genetically engineered (GE) Rainbow papaya in Japan. To date, Japan has not marketed U.S. fresh products derived from genetic engineering. Meanwhile, the U.S. is losing its major market share of Hawaiian papayas there due to the difficulty in supplying nonGE papaya economically, a situation caused by the prevalence of the devastating papaya ringspot virus (PRSV). In a cooperative effort led by ARS researchers at Hilo, HI, biosafety and other formal regulatory requirements of the Japanese government were completed, paving the way for the import for human consumption of virus resistant, (GE) Rainbow (line 55-1 derivatives) papaya fruit into Japan. The import and marketing of GE Rainbow papaya in Japan will aid in increasing U.S. market share of papayas there, support the U.S. Hawaiian Papaya industry and represent one of the first fresh GE products from the U.S. accepted and marketed in that country.
Gonsalves, D., Tripathi, S., Carr, J.B., Suzuki, J.Y. 2010. Papaya Ringspot Virus. The Plant Health Instructor. DOI: 10.1094/PHI-I-2010-1004-01. Available: http://www.apsnet.org/edcenter/intropp/lessons/viruses/Pages/PapayaRingspotvirus.aspx.