Progress 09/15/05 to 08/31/10

Outputs
OUTPUTS: North American native grape species, such as Vitis aestivalis, Vitis munsoniana and Vitis (Muscadinia) rotundifolia are tolerant to most pests and diseases. However, most of these species do not meet the expectations of consumers as fresh fruit grapes because the berry has large seeds and tough skin. "Fry Seedless" (patented by Ison's Nursery & Vineyards, GA) is the only seedless muscadine variety released until now. It appears to be fully parthenocarpic, has extremely small berries, insignificant commercial value and is of almost no use for breeding purposes. During the life of the project we developed a pioneer system for genetic transformation of muscadine grapes in liquid embryogenic cultures and generated large population of the mutagenic lines from Darlene and Surpeme premium fresh fruit muscadine varieties. We selected seedless large fruit mutation Supreme SM 14 -@8 which has been documented and evaluated since 2008. We established new set of mutagenic lines by treating muscadine somatic embryos with Y-irradiation which are very promising but will require further experiments for stabilization and multiplication.Graduate research under this project was presented and published as proceeding papers in 2 International Meetings including the 9th International Conference of Grape Genetics and Breeding, Udine, Italy and 5 National Meetings and Conferences. Undergraduate students presented their work as seminars in the respective program areas. PARTICIPANTS: 3.0 Research Associate, 3.0 Graduate Students 6 x 0.5 Undergraduate Students. TARGET AUDIENCES: The project was directed towards the grape researchers, commercial grape growers in Florida and southeastern states as well as small farmers who are growing grapes and those contemplating on growing grapes. In addition to that, year around the PI's lab was providing experiential learning in the area of DNA recombinant technology for FAMU/ College of Agriculture recruitment programs: 4-H, RATLER, Ag Discovery and for USDA Cochran Fellowship Program. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
1/With our research we strengthened the research linkages between FAMU/ College of Agriculture and USDA/ARS National Horticulture Research Lab, Forth Pierce. 2/In addition we developed new co-operations with the UF/Citrus Research Center, Lake Alfred and FSU, Gene Sequencing Facility. 2/Gene transformation and gene expression facility was established at FAMU/ Center for Viticulture and used as an effective hands on training platform in recombinant DNA technology for students, high school apprentices and various recruitment and outreach activities. 3/Altogether 3 graduate and 6 undergraduate students completed research work studies supported by this project. 4/ Our results are the real break through in seedless breeding of American native grapes.

Publications

• Colova-Tsolova, V., Perl, A., Krastanova, S., Samuelian and I., Atanassov, A. 2009: Genetic transformation of grape for disease and stress tolerance, In K. A. Roubelakis-Angelakis (ed.) Molecular Physiology & Biotechnology of Grapevine, Kluwer Academic Publishers , 509-531. A. Perl, V. Colova-Tsolova, Y. Esdat, 2004: Agrobacterium mediated transformation of grape embryogenic calli, In : Curtis, I. (ed.) Transgenic crop of the World, Essential Protocols, Kluwer Academic Publishers, 229-242. V.Colova, P. Bordallo, B. Phills, M. Bausher, 2007: Synchronized Somatic Embryo Development in Embryogenic Suspensions of Grapevine (Muscadinia rotundifolia Small and Vitis vinifera, L.), VITIS, (46) 1: 15-18. V. Colova, P.Bordallo, L.Parker, S. Leong, 2007: Evaluation Study of Fruit Quality and Photosynthesis of Two Training/ Trellis Systems and Canopy Management Practices for Carlos and Noble Muscadine Grapes in Florida, Journal International des Sciences de la Vigne et du Vin , 41(1) :43-49. V. Colova-Tsolova, J. Lu and A. Perl, 2005: Genetically Customized Seedless Grapes for Fresh Market Industry. Acta Horticulturae, 481-483.

Progress 09/15/09 to 08/31/10

Outputs
OUTPUTS: Development of triploids by interploid hybridization:To induce polyploidy and to excel the mutagenic experiments we treated in vitro plantlets and somatic embryogenic lines of a diploid seeded muscadine variety with the chemical and physical mutagenic agents.One set of inducible mutagenesis was done by treating young new growth of diploid seeded muscadine variety directly on 2 years old grapevines, grown on the field with chemical mutagenic substances in order to allow interploidy crosses and fruit set aiming triploid muscadine genotype at the same growing season. Another set of experiments was conducted with irradiation of one(1) and two (2) years old potted muscadine plants performed with the assistance of specialized private lab facility here in Florida.Seedless mutations SM14-ch8 and SM 14 -ch12 in Supreme 2 year's old vines in fruit set was obtained. The mutation forms are with the excellent texture, firmness, color and berry size superior to the one of Fry seedless. Numerous other seedless mutations has been registered and are under evaluation. PARTICIPANTS: 2.0 Graduate Students 2x 0.5 Undergraduate Students TARGET AUDIENCES: The project is directed towards the commercial grape growers in Florida and southeastern states as well as small farmers who are growing grapes and those contemplating on growing grapes. In addition to that, year around the PI's lab is the experiential learning platform for 3 academic courses in plant biotechnology and DNA research , majority of FAMU/ CESTA summer recruitment programs: 4-H, RATLER, Ag Discovery and USDA Cochran Fellowship Program, Capstone Program for High School Teachers in Food Safety PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
This research is designed to produce a new generation of genetically tailored seedless muscadine grapes that will provide biosafety guarantees and meet certain legal criteria for field release and commercialization. The results generated had a direct, positive impact on the marketability of the fresh fruit grape production in the southeastern United States and will add significant value to the muscadine industry

Publications

• No publications reported this period

Progress 09/15/08 to 09/14/09

Outputs
OUTPUTS: For the first time inducible seedless mutations SM14-ch8 and SM 14-ch12 of the top shelf fresh fruit muscadine variety Supreme was obtained. The mutation forms are with the excellent texture, firmness, color and berry size superior to the Fry seedless(only one existing until now seedless muscadine cultivar). Numerous other seedless mutations has been generated in 2008 and will be evaluated during the oncoming growing seasons and eventually developed to new varietes. The additionally secured modest financial support from Florida's Viticulture Advisory Counsel for continuation endorsed the significance of our study for the grape industry. The presented research is a seed grant effort to build up research and educational capacity in sustainable agriculture/viticulture, biotechnology and genetic engineering using "cutting-edge technology" achievements and employing technology transfer systems to address industry needs. Graduate research under this project was presented and published as proceeding papers in 6 national and 2 International Meetings including the 9th International Conference of Grape Genetics and Breeding, Udine, Italy and the 9th International Meeting of Grape Physiology and Biotechnology, Adelaide, Australia. The graduate student poster under this project was the 1st place winner at the 15th Biennial Research Symposium of the Association of Research Directors, Atlanta, GA March 28 - April 1, 2009. PARTICIPANTS: 1.0 Research Associate, 2.0 Graduate Students 2x 0.5 Undergraduate Students TARGET AUDIENCES: The project is directed towards the commercial grape growers in Florida and southeastern states as well as small farmers who are growing grapes and those contemplating on growing grapes. In addition to that, year around the PI's lab is the experiential learning platform for 3 academic courses in plant biotechnology and DNA research , majority of FAMU/ CESTA summer recruitment programs: 4-H, RATLER, Ag Discovery and USDA Cochran Fellowship Program, Capstone Program for High School Teachers in Food Safety. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Our research was designed to produce a new generation of genetically tailored seedless muscadine grapes that will provide biosafety guarantees and meet certain legal criteria for field release and commercialization. The success of our project has a direct, positive impact on the marketability of the fresh fruit grape production in the southeastern United States and adds significant value to the muscadine industry. Our results are the real break through in seedless breeding of the perennial fruit crops with extended juvenility and delayed fruit set. Capacity was build for establishment of Transformation Core Facility for Small Fruits at CESTA, FAMU to serve as an expert service center for other research users primarily in Southeastern United States.

Publications

• Violeta Colova, Tania Krastanova, Avi Perl, Suren Sanmuelian, Atanas Atanassov, 2009:Progress in genetic engineering of grape for disease and stress tolerance, In K. A. Roubelakis-Angelakis (ed.) Molecular Physiology & Biotechnology of Grapevine, Kluwer Academic Publishers,509-532.
• Tresia Walters, Julian Bourne, Suren Samuelian, Stephen Leong and Violeta Colova, 2009: DNA Profiling of Aestivales (Planshon) American Native Grapes, Proceeding of the 9th International Meeting of Grape Physiology and Biotechnology, November 22-27, 2008, Adelaide, Australia (in press).
• Suren K. Samuelian, Christian Kappel, Celine Camps, Ekaterina P. Simova, Serge Delrot Violeta M. Colova (Tsolova), 2009: Comparative analyses of differentially expressed genes involved in flavonoid biosynthesis in North American native grapes: Noble and Ison muscadinia vars. and Cynthiana aestivalis, Proceeding of the 9th International Meeting of Grape Physiology and Biotechnology, November 22-27, 2008, Adelaide, Australia(in press).

Progress 09/15/07 to 09/14/08

Outputs
OUTPUTS: The ongoing project made it possible and set us in a position to be the only group in the world that has developed and maintained stable somatic embryogenic lines from muscadine grapes. What is more important, our cell lines are from Supreme and Darlene, the two most attractive (high value) fresh fruit muscadine varieties on the market and we are holding the key technology for their genetic modification. We selected seedless large fruit mutation Supreme SM 14 -@8 which has been documented and evaluated and in 2007 we generated another set of mutagenic seedless lines which are very promising but will require further experiments for stabilization and multiplication. Synchronized embryogenic cell suspension: We use two parallel protocols for establishment of synchronized embryogenic lines in cell suspensions of Muscadinia rotundifolia varieties Supreme, Darlene and Ison. We have been able to achieve relatively high percent of embryogenic response for two types of explants (anthers and in vitro immature leaves) in muscadines (Colova et al., 2007). Cell suspension are developed based on synchronized somatic embryos arrest at PEMs developmental stage, with high ability to multiply. The initial cell inoculum at 1% (w/v) was used for the maintenance of the suspensions. Agrobacterium -mediated gene transformation. The research was comprised of 2 complete random blocks of simultaneously going experiments aiming genetically transforming of the existing muscadine grape varieties and conferring seedlesness as desirable quality trait. Each step of the transformation research was set up as serial of 3 independent randomized block experiments with 2 treatments (semisolid culture conditions and liquid culture conditions) and 20 replications. In vitro plates 100 X 10mm for semisolid culture conditions as sampling units, 50 ml Erlenmeyer vessels for liquid culture conditions as sampling units and magenta box for plant regeneration were used with unified fresh weight of the initial explants. Inducible Mutagenesis. In this year mutagenic experiments we treated in vitro plantlets and somatic embryogenic lines of a diploid seeded muscadine variety with physical mutagenic agent γ irradiation at the service industrial facility. For the first time in grape such a treatment is conducted on in vitro material. More then 300 micro propagated radiated explants are on selective stage. Altogether 20 tentative chemical mutants from Supreme varieties are under greenhouse evaluation. Additional 4 seedless mutants grown under field conditions were evaluated by cytoflowrometry. PARTICIPANTS: 1.0 Research Associate, 2.0 Graduate Students 2x 0.5 Undergraduate Students TARGET AUDIENCES: The project is directed towards the commercial grape growers in Florida and southeastern states as well as small farmers who are growing grapes and those contemplating on growing grapes. In addition to that, year around the PI's lab is the one providing experiential learning in the area of DNA research for CESTA summer recruitment programs: 4-H, RATLER, Ag Discovery and for USDA Cochran Fellowship Program, Capstone Program for High School Teachers in Food Safety and etc. Graduate research under this project was presented and published as proceeding papers in 2 International Meetings including the 9th International Conference of Grape Genetics and Breeding, Udine, Italy and 5 National Meetings and Conferences. Undergraduate students presented their work as seminars in the respective program areas and as a short presentations for AP Biology Class from Rickards High School PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Our project has strengthened the research linkages between CESTA and USDA/ARS National Horticulture Research Lab, Forth Pierce and developed new co-operations with the UF/Citrus Research Center, Lake Alfred and FSU, Gene Sequencing Facility. Gene transformation and gene expression facility was set up and it is used as an effective hands on training platform in recombinant DNA technology for students, high school apprentices and various College recruitment and outreach activities. Altogether 2 graduate and 6 undergraduate students completed or continue research work studies supported by this project. The proposed research is designed to produce a generation of seedless muscadine grapes by employing the recombinant DNA technology and inducible mutagenesis. The success of the project will have direct, positive impact on the marketability of the fresh fruit grape production in the southeastern United States and will add significant value to the muscadine industry. Our results will be the real break through in seedless breeding of the perennial fruit crops with extended juvenility and delayed fruit set. The capacity will be build for establishment of Transformation Facility for Small Fruits at CESTA, FAMU to serve as an expert service center for other research users primarily in Southeastern United States and international wide. Generation of carrier ready Ag Science professionals will be trained up to the advance of present day developmental biology and DNA research.

Publications

• S. K. Samuelian, Ch. Kappel, C. Camps, E. P. Simova, S. Delrot and V M. Colova (Tsolova), 2008: Differential screening of overexpressed genes involved in flavonoid biosynthesis in North American native grapes: muscadinia Noble var. and aestivalis Cynthiana var. Journal of Plant Science (submited).
• S. K. Samuelian, Ch. Kappel, C. Camps, E. P. Simova, S. Delrot, S. Leong and V M. Colova (Tsolova), 2008: Comparative analyses of differentially expressed genes involved in flavonoid biosynthesis in North American native grapes Noble and Ison muscadinia vars and Cynthiana aestivalis var.Australian Journal of Viticulture and Enology (submitted).
• Tresia Walters, Julian Bourne, Suren Samuelian, Stephen Leong and Violeta Colova, 2008: DNA Profiling of Aestivalis (Planshon) American Native Grape. Australian Journal of Viticulture and Enology (submitted).

Progress 09/15/06 to 09/14/07

Outputs
The experimental work for developing seedless muscadine varieties via recombinant DNA technology and inducible mutagenesisis progresses satisfactory and our results and accomplishments are according the timetable of the project. Recombinant DNA technology approach: Somatic embryogenic lines in suspension as a high frequency regeneration system for alien gene delivery have been established for the 'Supreme', 'Darlene' and 'Ison' muscadine varieties. Stable efficient Agrobacterium-mediated gene transformation system was developed for all targeted genotypes. The transformation experiments with 3 GFP reporter gene constructs and two genes of interest are ongoing and at the stage of in vitro genetic selection. Inducible mutagenesis approach: Protocols for chemical mutagenesis and γ irradiation of various in vitro and in vivo grape explants have been developed. Series of mutagenic experiments with the complex of chemical mutagenic agents for inducing polyploidy with 1year potted plants and 2 years vines of diploid seeded muscadine varieties 'Supreme' and 'Darlene' were successfully accomplished. Seedless 'Supreme' mutation SM 14 -@8 was registered and evaluated at fruitset during growing 2006 season. Further experiments to stabilize the mutation and multiply the result are ongoing. Accomplishments: Established synchronized embryogenic lines in suspension for Darlene muscadine variety; Induction of somatic embryogenesis (SE) in 'Darlene', 'Supreme' and 'Ison' muscadine varieties; Initial in vitro chemical mutagenic trials with 'Darlene' and 'Supeme' vars; Androgenesis trial in 'Supreme' var.; Initiation of SE in endosperm cell cultures; Graduate student recruitment; Graduate student appointment and curriculum development.

Impacts
The proposed research is designed to produce a new generation of genetically tailored seedless muscadine grapes that will provide biosafety guarantees and meet certain legal criteria for field release and commercialization. The success will be the real break through in seedless breeding of the perennial fruit crops with extended juvenility and delayed fruit set. The accomplishments of the project will have a direct, positive impact on the marketability of the fresh fruit grape production in the southeastern United States and will add significant value to the muscadine industry. The capacity will be build for establishment of Transformation Core Facility for Small Fruits at CESTA, FAMU to serve as an expert service center for other research users primarily in Southeastern United States, national and international wide.

Publications

• V. Colova, P. Bordallo, B. Phills, M. Bausher, 2007: Synchronized Somatic Embryo Development in Embryogenic Suspensions of Grapevine (Muscadinia rotundifolia Small and Vitis vinifera, L.), VITIS, 1, 36-41
• V. Colova, P.Bordallo, L.Parker, S. Leong, 2007: Fruit Quality and Photosynthesis of Two Training/ Trellis Systems and Canopy Management Practices for Carlos and Noble Muscadine Grapes in Florida, Journal International des Sciences de la Vigne et du Vin , 28 (1) 56-62.
• V. Colova, 2007: Current Trends in Florida Muscadine Grape Industry, South Regional Extension Conference, April 27- April 28, Sarasota, Florida
• V. Colova (Tsolova), 2006: Muscadine Grape:All Ameican Good Medicine, Annual Conference of North Caroline Nature Products Association, ASU, Boone, NC, November 17-18, 2006.

Progress 09/15/05 to 09/14/06

Outputs
The research for developing seedless muscadine varieties via recombinant DNA technology and inducible mutagenesisis progresses satisfactory and our results and accomplishments are according the timetable of the project. Recombinant DNA technology approach: Muscadine varieties Supreme, Darlene and Ison have been successfully introduced in vitro. Numbers of in vitro plants have been established via clonal micropropagation to serve as a donor plants for leaf explants for somatic embryogenesis initiation. Stable embryogenic lines have been established from young in vitro leaf explants of Darlene muscadine variety. The experimental work with Supreme and Ison varieties in regards of somatic embryogenesis is ongoing. Inducible Mutagenesis approach: To induce polyploidy and to excel the mutagenic experiments for the first year of the project we treated in vitro plantlests and somatic embryogenic lines of a diploid seeded muscadine variety with the complex of chemical mutagenic agents. In collaboration with Dr. Michael Bausher, Plant physiologist at USDA/ARS Horticulture Lab at Fort Pierce the methodology for karyotype identification by flowCytometry and genomic in situ hybridization (GISH) has been tested and will be used for analyzing the developed mutants. Accomplishments and Progress: Established synchronized embryogenic lines in suspension for Darlene muscadine variety; Induction of somatic embryogenesis (SE) in Darlene, Supreme and Ison muscadine varieties; Initial in vitro chemical mutagenic trial with Darlene var.; Androgenesis trial in Supreme var.; Initiation of SE in endosperm cell cultures; Graduate student recruitment; Graduate student appointment and curriculum development. We would like to underline our accomplishment to establish synchronous grapevine (muscadinia) somatic embryogenic cell lines in liquid culture, with steady high conversion rate and regeneration capacity. Only a high-frequency, synchronous embryogenic system in liquid culture is allowed to be explored the full advantage of the somatic embryogenesis (NOVAK, 1992) as it is essential in plant genetic improvement, cryopreservation, and for performing basic studies in plant physiology and plant genetics. Our results shows that Muscadinia rotundifolia SE can be produced successfully in suspension culture with high efficiency by cell fractionation trough Ficoll density gradient centrifugation and by manipulation of plant growth regulators (PGR) and cell density in the liquid fraction. Taking into consideration, the high regeneration rate, the present study is instrumental for the various genetic enhancements experiments (genetic transformation and inducible mutagenesis) with high scale in vitro propagation and lays out the steps for large-scale muscadinia somatic embryo production in bioreactors and further utilization in molecular farming. The fact that two cell populations can be distinguished with various potential in terms of somatic embryo development and differentiation could prove also very useful for functional genomics studies of embryogenesis in woody perennial plants.

Impacts
The proposed research is designed to produce a new generation of genetically tailored seedless muscadine grapes that will provide biosafety guarantees and meet certain legal criteria for field release and commercialization. The success will be the real break through in seedless breeding of the perennial fruit crops with extended juvenility and delayed fruit set. The accomplishments of the project will have a direct, positive impact on the marketability of the fresh fruit grape production in the southeastern United States and will add significant value to the muscadine industry. The capacity will be build for establishment of Transformation Core Facility for Small Fruits at CESTA, FAMU to serve as an expert service center for other research users primarily in Southeastern United States, national and international wide.

Publications

• V. Colova, P. Bordallo, B. Phills, M. Bausher, 2006: Synchronized Somatic Embryo Development in Embryogenic Suspensions of Grapevine (Muscadinia rotundifolia Small and Vitis vinifera, L.), Vitis, (submitted)
• V. Colova, L. Parker, P.Bordallo, 2005: The Identity Study of Norton/Cynthiana Grape Variety via DNA Fingerprinting. In: W.Qui and L.Kovacs ( eds) Proc Intern Grape Genom Symposium, St. Luis, MO, 20-27
• V. Colova-Tsolova, J. Lu and A. Perl, 2005: Genetically Customized Seedless Grapes for Fresh Market Industry. Acta Horticulturae, 481-483.
• V. Colova (Tsolova) and B. R. Phills, 2006: Genetic Enhancement for Seedlessness of Large Fruit Muscadine Grapes, In Proc 9th International Conference on Grape Genetics & Breeding, July 2-6, Udine, Italy
• L. Parker, P. Bordallo and V. Colova, 2006: Phylogenetics Analysis of American Native Cynthiana/Norton Grape using DNA Microsatellite Markers, In Proc. 9th International Conference on Grape genetics & Breeding, July 2-6, Udine, Italy