2007 Annual Report 1a.Objectives (from AD-416) The primary goal for this project is floral plant improvement, focusing on anthocyanin pigmentation and virus resistance. The specific goals are to elucidate the genetic control for anthocyanin pathways and production, using the Solanaceous crops Petunia and Capsicum as model plants; develop genetically improve Petunia and Capsicum germplasm with novel ornamental traits and/or improved pest, disease and stress tolerance; develop transgenic floral crops with induced resistance to viral diseases; and determine how viruses may regulate anthocyanin biosynthesis in the phenomenon known as color-break. 1b.Approach (from AD-416) Determine the expression and control of structural and regulatory genes for anthocyanin coloration of leaves, flowers, and fruit, including viral effects on flower-break. Develop Petunia and Capsicum germplasm with novel characteristics and/or pest, disease and stress tolerance. Initiate development of transgenic floral crops exhibiting induced resistance to selected major viral diseases. 3.Progress Report Additional research has been performed under a CRADA Agreement (58-3K95-5-1074; see #1230-21000-040-08T) with McCorkle Nurseries and Kerry’s Bromeliad Nursery. 4.Accomplishments Genetics of Flower Color Patterning in Petunia. The Petunia Star mutation is due to tissue specific gene silencing and results in a white star pattern in red flowers. The white silenced tissue was the result of an absence of chalcone synthase gene (Chs) expression. Tobacco etch virus (TEV) infection reversed Chs silencing and resulted in solid red flowers. The environment greatly influenced the effect of TEV on Chs silencing. The greatest effect was seen under high light and low temperature. As TEV-infected plants aged, regulatory gene expression increased and structural gene expression decreased. This phenomenon was not seen in virus-free plants. Gene silencing is a very important process used by both plants and animals to control gene expression. Differences in gene expression are responsible for a wide range of responses from human cancer to patterned flowers. This research was one of the FY 2007 milestones (Determine the methylation pattern of anthocyanin genes in virus infected tissue). This research is under National Program 301, Component I, Problem Area Ic and Id and Component II, Problem Area IIb. This research is under ARS Strategic Plan Goal 1, Performance measure 1.2.8. Genetics of Flower Color Patterning in Phalaenopsis. This research was done in cooperation with the Shrub Breeding Project with the Unit (1230-21000-041-00D). Anthocyanins pigments are responsible for red through blue flower, fruit and leaf colors. Anthocyanin pigmentation requires the coordinated expression of two classes of regulatory genes (Myb and Myc). The petals of Phalaenopsis stuartiana are white with small red spots. Through biolistic bombardment, it was demonstrated the spotting pattern in P. stuartiana was the result of a lack of Myb and/or Myb expression in the white tissue. This information is important in understand the genetics of differential pigmentation within the same tissue. Differential gene expression is responsible for a wide range of responses from patterned flowers to human cancer. This research was one of the FY 2007 milestones (Determine the genetics of anthocyanin gene regulation). This research is under National Program 301, Component I, Problem Area Ic and Id and Component II, Problem Area IIb. This research is under ARS Strategic Plan Goal 1, Performance measure 1.2.8. Identification of viral suppressors of RNA silencing. A transient expression system in floral tissue of a mutant Phalaeonopsis orchid (in which genes in the anthocyanin pigmentation pathway are silenced) has been utilized to examine the effects of viral suppressors of RNA silencing. Transient expression of the Tobacco etch virus (TEV) HC-Pro gene, a known suppressor of RNA silencing, causes anthocyanin pigmentation in cells bombarded with TEV HC-Pro constructs. Deletion mutants construct of HC-Pro have been produced to evaluate the effects of different regions of HC-Pro on release of RNA silencing in this system. The orchid transient expression system was also used to demonstrate that the ORF 6 of the carlavirus Phlox virus S is a suppressor of RNA silencing. This research was one of the FY 2007 milestones (Determine the genetics of anthocyanin gene regulation). This research is under National Program 301, Component I, Problem Area Ic and Id and Component II, Problem Area IIb. This research is under ARS Strategic Plan Goal 1, Performance measure 1.2.8. Transformation of Gladiolus with anti-viral genes. Research has continued on the production and evaluation of disease resistant transgenic gladiolus through the incorporation of various viral and antiviral antibody genes. In collaboration with the Floral and Nursery Plants Research Unit, Projects 1230-21000-037-00D and 1230-22000-022-00D, transgenic gladioli have been produced which express Cucumber mosaic virus (CMV) replicase, coat protein, or antiviral antibody transgenes. Studies include two varieties of gladiolus, different types of gene constructions and different promoters. Using the biolistic inoculation method developed in the laboratory, studies are underway to determine the degree of protection when transgenic plants are challenge-inoculated with CMV, as determined by symptom development, ELISA and quantitative Real Time RT-PCR. Initial challenge inoculation of the 60 transgenic lines containing coat protein or replicase gene or both showed that 9 lines were resistant to CMV. Initial challenge screening of transgenic gladioli containing antibody genes was been completed and the evaluation is in progress. This work will facilitate the evaluation of virus resistance in transgenic gladiolus plants to yield improved floral quality and productivity. This research contributes to two of the FY 2007 milestones (Transform model plants to evaluate constructs; and, Initiate transformation of ornamental hosts with effective scFv constructs). This research in this accomplishment contributes to ARS National Program 303, Component 3, Problem Statement 3B and ARS Strategic Plan Performance Measure 3.2.5. 5.Significant Activities that Support Special Target Populations 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. Cooperator on USDA/1890 grant proposal to increase small farmers’ awareness and adoption of biotechnology-based products. 6.Technology Transfer Number of active CRADAs and MTAs 2 Number of patent granted 4 Number of new commercial licenses granted 1 Number of web sites managed 1 Number of non-peer reviewed presentations and proceedings 3 Number of newspaper articles and other presentations for non-science audiences 7 Review Publications Hammond, J., Hsu, H.T., Huang, Q., Jordan, R.L., Kamo, K.K., Pooler, M.R. 2006. Transgenic approaches to disease resistance in ornamental crops. Journal of Crop Improvement. 17:155-210.