2004 Annual Report 1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Floral crop sales comprise one of the fastest growing segments of US agriculture, forming a $5.07 billion industry in 2003. Growth of the floral industry requires the introduction of new types of plants, while market share is maintained by introduction of genetically improved varieties. The floral industry is composed of many small growers which cannot internally support new crop development or germplasm enhancement. In 2003, 60% of the growers had sales less than $100,000. In addition, commercial breeding programs for floral crops typically do not breed for disease or stress tolerance or use biochemical procedures in selection. Most companies do not have the facilities for species evaluation, tissue culture, growth chamber studies, biochemical analysis, nor can they afford the time or space to perform the basic research studies. These facilities are required in new crop development, as well as for introgressing 'wild' and trans-genes into commercial breeding lines. This research will result in the release of new germplasm which can be used to develop new cultivars. The floral industry relies upon the USDA as one of their major sources of enhanced germplasm. This project involves multiple approaches to improve floral plants. A combination of basic and applied research will be used to develop new technology to create plants with improved pest, disease, and stress tolerance, as well as novel flower, leaf and fruit pigmentation. In Objective 1 of this project, Petunia and Capsicum will be utilized as model plants to study the genetic regulation of anthocyanin biosynthesis in flowers, fruits and leaves. The anthocyanin biosynthetic pathway is extremely well characterized and should be a useful model to study gene regulation. In addition, viruses known to effect anthocyanin expression will also be used to study gene regulation. The basic information obtained from Objective 1 will be used in Objective 2 to developed enhanced Petunia and Capsicum germplasm expressing novel pigmentation patterns. In Objective 3, technology developed in several other CRIS Projects within the Floral and Nursery Plants Unit will be used to create virus resistant plants. In this objective, new transformation technology will be developed which will overcome many of the licensing problems associated with the current technology. New promoters and gene constructs will be tested. The research to be undertaken falls under National Program 301, Component 1 - Genetic Resource Management and Component 2 - Genomic Characterization and addresses goals 1.1, 1.2, 1.3, 1.4, 1.5, 2.1 and 2.2 as described in the National Program Action Plan. In addition, this research contributes to National Program 302, Component 1- Analysis and Modification of Plant Genomes and Component 2 - Biological Processes that Determine Plant Productivity and Quality and addresses goals 1.1, 1.2, 2.2, and 2.3 as described in the National Program Action Plan. Specifically, these goals are: 301.1.1 Safeguarding Threatened Genetic Resources and Associated Information), 301.1.2 Conserving Genetic Resources and Associated Information Efficiently and Effectively, 301.1.3 Documenting and Characterizing Genetic Resources, 301.1.4 Expanding Germplasm Evaluations and Characterizations, 301.1.5 Technology Transfer of Genetic Resources and Associated Information, 301.2.1 Genome Characterization, 301.2.2 Genetic Improvement, 302.1.1 Molecular Characterization of Plant Genetic Systems, 302.1.2 Plant Transformation Systems and Influence of Transgenes on Genome Structure and Function, 302.2.2 Plant Tolerance to Environmental Stresses, and 302.2.3 Biological Basis for Expression of Value-Added Traits. 2.List the milestones (indicators of progress) from your Project Plan. Year 1 (FY 2004) Create virus infected Petunia lines expressing color break. Screen GenBank sequences for homology between regulatory genes and viruses. Develop PCR primers for anthocyanin genes. Determine the inheritance of Petunia leaf trichome number. Determine strong promoter region(s)for transformation of ornamentals. Year 2 (FY 2005) Release Capsicum germplasm. Determine the inheritance of pigmentation in Capsicum and Petunia. Develop an evolutionary tree of Petunia species in the integrifolia complex. Examine tissue specificity and level of virus and anthocyanin expression. Determine in color-break tissue which regulatory gene is affected. Determine if the pattern of viral distribution and anthocyanin pigmentation coincide. Examine mechanisms of resistance utilizing viral vectors. Year 3 (FY 2006) Transform model plants to evaluate scFv-based resistance. Evaluate transgenic model plants for viral resistance. Develop/adapt methods for transformation of ornamental hosts. Create scFv constructs to combine resistance to multiple viruses and/or genes. Determine gene(s) responsible for tissue specific pigmentation. Determine the expression pattern of regulatory genes in different tissues. Release shrub-like Petunia germplasm. Year 4 (FY 2007) Release cold tolerant Petunia germplasm. Create constructs to combine resistance to multiple viruses. Transform model plants to evaluate constructs. Initiate transformation of ornamental hosts with effective scFv constructs. Determine the methylation pattern of anthocyanin genes in virus infected tissue. Release new Capsicum germplasm with improved foliar and fruit pigmentation. Year 5 (FY 2008) Develop Petunia lines with leaves expressing different anthocyanins. Transform crop plants with effective constructs. Begin transformation of plants with HC-Pro. Determine resistance to multiple viruses and evaluate for possible recombination, synergism, etc. Determine resistance to multiple viruses and/or multiple virus genes. 3.Milestones: A. The milestones listed below were scheduled to be completed under Year 1. All milestones were completed. Create virus infected Petunia lines expressing color break. Screen GenBank sequences for homology between regulatory genes and viruses. Develop PCR primers for anthocyanin genes. Determine the inheritance of Petunia leaf trichome number. Determine strong promoter region(s)for transformation of ornamentals. B. The Year 2, 3, 4 and 5 milestones are listed below with a description of the anticipated outcomes. Year 2 (FY 2005) Capsicum germplasm with novel foliage color will be released. The inheritance of pigmentation in Petunia will be published. Because a CBD (Convention on Biology Diversity) Agreement with Brazil could not be obtained, Petunia leaf anthocyanin research is no longer being conducted. A new method for measuring evolution distance was developed based upon the sequence of introns. This research was completed and a paper was submitted for publication. The level of tobacco etch virus expression in infected Star Petunias will be compared to the phenotype to determine if the concentration of virus in a section of tissue corresponds to its phenotype. The level of tobacco etch virus expression in infected Star Petunias will be compared to the level of anthocyanin accumulation to determine if the concentration of virus in a section of tissue corresponds to concentration of anthocyanin. The level of tobacco etch virus expression in infected Star Petunias will be compared to the level of expression of different anthocyanin genes expression to determine which anthocyanin gene is affected by the virus. Model plants will be transformed with constructs designed to induce effective RNA silencing of Ornithogalum mosaic virus (OrMV) prior to transformation of Ornithogalum. The effectiveness of RNA silencing will be determined by challenge with potexviral vectors carrying OrMV sequences. In following years constructs designed to confer resistance to multiple viruses will be tested. Year 3 (FY 2006) Information on the expression of genes responsible for tissue specific pigmentation in the Petunia Star mutation will be published. Information on the expression of regulatory genes responsible for organ specific pigmentation in Capsicum will be published. A shrub-like Petunia derived from P. exserta will be released. Key "model" ornamental plants will be transformed with various anti-viral scFv constructs to evaluate this novel antibody-based form of engineered anti-viral resistance. Other scFv antibody constructs will be developed to combine resistance to multiple viruses and/or genes. Ornithogalum actin promoters have been isolated, and marker gene constructs will be used to compare biolistic, agrobacterium, and electrophoretic transformation methods for transient and stable transformation of Ornithogalum. When methods are established, constructs designed to confer multiple virus resistance will be used for transformation. Year 4 (FY 2007) Because a CBD (Convention on Biology Diversity) Agreement with Brazil could not be obtained, Petunia cold tolerant research is no longer being conducted. The methylation pattern of anthocyanin genes control by virus expression in the Petunia Star mutation will be determined. Novel Capsicum germplasm with improved foliar and fruit pigmentation will be released. Transgenic plants expressing chimeric or multiple scFv antibody constructs will be developed to examine the potential for a combined resistance to multiple viruses. Year 5 (FY 2008) Because a CBD (Convention on Biology Diversity) Agreement with Brazil could not be obtained, Petunia leaf anthocyanin research is no longer being conducted. In cooperation with CRADA partner, transformation systems for ornamental crops will be developed to allow the expression of anthocyanin regulatory genes. Begin transformation of plants with various potyviral HC-Pro genes; the expression of HC-Pro has the potential to alter existing patterns of RNA silencing, affecting plant phenotype, including anthocyanin gene expression, as well as potential virus resistance. Analysis of resistance to multiple viruses will be assessed in model plants, and later in ornamental plants transformed with appropriate constructs. 4.What were the most significant accomplishments this past year? A. Three near isogenic lines of Petunia that express the Star mutation at different levels (high, low and intermediate expressing lines) were created. The Petunia Star mutation is due to anthocyanin regulatory gene silencing and results in a white star pattern in colored flowers. These lines were infected with three different viruses (pepper mottle virus, tobacco etch virus and potato virus Y) that released the silencing, resulting in a near solid colored flower. The most effective virus for inhibiting silencing was tobacco etch. This research has resulted in a model system for studying gene silencing. Gene silencing is a very important method used by both plants and animals for controlling gene expression. Differences in gene expression are responsible for a wide range of responses from human cancer to patterned flowers. B. The Floral and Nursery Plants Research Unit in collaboration with University of New Hampshire determined the biochemical basis of an unusual Anagallis anthocyanin regulatory gene mutation that results in different anthocyanins in the upper (malvidin) and lower (delphinidin) epidermis of the petal. This information is being used by the University of New Hampshire to create new flower colors. Classical breeding can be used to modify flower color; however knowledge of the biochemical basis of the color is required. To date, two new colors (white and magenta) have been developed. Anagallis is used as a potted plant and only blue- or orange-flowered cultivars were previously available. This research has resulted in an expanded range of colors. Primers were developed that PCR amplified the dihydroflavonol reductase, anthocyanin synthase, and chalcone synthase genes in the anthocyanin pathway in Capsicum. These primers were used to screen mRNA expression in purple leaves of plants grown in different environments. Under high temperature and/or high daily light integral (total amount of light), anthocyanin accumulated due to an increase in chalcone synthase gene expression. This knowledge is an important first step in understanding how regulatory genes control tissue specific, structural gene expression. One of the recalcitrant goals in commercial plant breeding is to create plants expressing anthocyanins in the leaves. In addition, this information will be used to screen Capsicum germplasm for commercial potential as purple-leaved ornamental plants. This research is done in cooperation with the ARS Vegetable Laboratory in Beltsville, Maryland. Primers were developed that PCR amplified the dihydroflavonol reductase, anthocyanin synthase, and chalcone synthase genes in the anthocyanin pathway in Petunia. These primers were used to screen mRNA expression in white and colored tissue within the Petunia Star mutation. The Petunia Star mutation is due to anthocyanin regulatory gene silencing and results in a white star pattern in colored flowers. The white mutant tissue was due to the lack of chalcone synthase expression. Therefore the regulatory gene mutation responsible for the star phenotype, affects the expression of the chalcone synthase gene. This knowledge is an important first step in developing a model system to study how regulatory genes control structural gene expression. Differences in regulatory gene expression are responsible for a wide range of responses from human cancer to patterned flowers. Research has continued on the production and evaluation of disease resistant transgenic gladiolus through the incorporation of various viral genes. In collaboration with K. Kamo (also of the Floral and Nursery Plants Research Unit), transgenic gladioli have been produced which express either Cucumber mosaic virus (CMV) replicase or coat protein transgenes. Studies are underway to determine the degree of protection when transgenic plants are challenge inoculated with CMV. This work will facilitate the evaluation of virus resistance in transgenic gladiolus plants to yield improved floral quality and productivity. The helper component-proteinase (HC-Pro)genes of tobacco etch virus, pepper mottle virus, and potato virus Y were cloned into an expression vector for transformation of petunia and tobacco, in order to examine effects on gene regulation affecting flower color and viral replication. C. Cooperator on a funded USDA/1890 grant proposal 'Strengthening teachers' and students' knowledge of agricultural biotechnology through hands-on training workshops' with Tennessee State University. D. This report serves to document research conducted under a Cooperative Research and Development Agreement (Project #1230-21000-040-07T) between the Floral and Nursery Plants Research Unit, Vegetable Laboratory, and McCorkle Nurseries to develop Capsicum germplasm with ornamental/culinary applications. This project has just recently started. Germplasm with novel foliage and/or fruit colors was sent for commercial evaluation. From this evaluation, specific research goals will be developed for creating commercially acceptable lines. This report serves to document research conducted under a specific cooperative agreement 1230-21000-030-06S between the Floral and Nursery Plants Research Unit and Agricultural Research Council of South Africa. Most of the sales of potted-plants within the US are not just the standard crops (i.e., African violet, chrysanthemum, etc.) but also include unique new crops such as Ornithogalum and orchids. In this collaborative project many South African species were collected and evaluated in South Africa for horticultural potential. Production methods which can be used by rural South African farmers were then developed for selected species. FNPRU-ARS provided advice on evaluation and production. It is hoped that new South African species could be ultimately exported to US growers for final production. This report serves to document research conducted under a specific cooperative agreement 1230-21000-030-03S between the Floral and Nursery Plants Research Unit (FNPRU-ARS) and the Agricultural Research Council (ARC) of South Africa. The US floral industry has recognized the potential of Erica as a new crop, but a lack of germplasm for improved flower colors, heat tolerance and winter hardiness prevents further commercial development. South Africa is the center of origin for Erica and has a wide diversity of species. Collaborators at ARC collected many wild species from the Drakensberg Mountains of South Africa. Specific selections from these diverse collections are being vegetatively propagated for evaluation and breeding by the Floral and Nursery Plants Research Unit. FNPRU-ARS also obtained commercially available cultivars derived from the European species Erica carnea, E. cinerea, and E. tetralix. All of these cultivars were evaluated for heat and cold tolerance. Several cultivars were found to be both winter hardy and heat tolerant at Beltsville, Maryland. 5.Describe the major accomplishments over the life of the project, including their predicted or actual impact. This project was started in FY04. In the past project, germplasm was developed that was released as patented cultivars. In addition, the biochemical basis of flower color was determined for several crop species. This data was used to created germplasm with improved flower colors. 6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Ornithogalum `Chesapeake Snowflake' and Capsicum `Tangerine Dream' are now available for retail sales. A CRADA was signed for the development of Capsicum germplasm for commercial ornamental and/or culinary applications. Other CRADA agreements are currently being developed with two nurseries to transfer anthocyanin science genetics. An MTA agreement was signed to test the commercial potential of a weeping form of a purple-leaved Cercis. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. R. J. Griesbach presented a seminar on 'Genetics' to the Orlando, FL Orchid Society, November 10, 2003. Set-up a display at the Mid-Atlantic Nursery Trade Show in Baltimore, January 7-9, 2004. J. Hammond presented a talk 'Current status of genetically modified ornamentals' at the 11th International Symposium on Virus Diseases of Ornamental Plants, Tai-Chung, Taiwan, March 11, 2004. R.J. Griesbach presented a talk 'Biochemistry and Genetics of Flavonoid-based flower color' at the 8th Asia Pacific Orchid Conference in Tainan, Taiwan on March, 6-8, 2004. R.J. Griesbach presented a talk 'Molecular Heterogeneity of the Chalcone Synthase Intron in Petunia Species' at the Annual Meeting of the American Society for Horticultural Science in Austin Texas on July 17-20, 2004. R.J. Griesbach presented a seminar on 'Orchid Biology' to the Beltsville, MD Garden Club, May 26, 2004. Set-up a display at the Beltsville Agricultural Research Center Field Days, June 5, 2004. Review Publications Farzad, M., Griesbach, R., Hammond, J. Weiss, M., Elmendorf, H. 2003. Differential Expression of Three Key Anthocyanin Biosynthetic Genes in a Color-Changing Flower, Viola Cornuta cv. Yesterday, Today and Tomorrow. Plant Science. 165:1333-1342. Griesbach, R. 2004. Hemerocallis L. 'Chesapeake Belle'. HortScience. 39:190-191. Stommel, J.R., Griesbach, R.J. 2004. Capsicum annuum l. 'Tangerine Dream'. Hortscience. 39(2):448-449.