Source: UNIVERSITY OF FLORIDA submitted to
DEVELOPMENT OF IMPROVED GRAPE CULTIVARS VIA GENETIC ENGINEERING BIOTECHNOLOGY
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0210364
Grant No.
(N/A)
Project No.
FLA-APO-04626
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2007
Project End Date
Sep 30, 2013
Grant Year
(N/A)
Project Director
Gray, D. J.
Recipient Organization
UNIVERSITY OF FLORIDA
BOX 100494, JHMHC
GAINESVILLE,FL 32610
Performing Department
AGRI RES & ED CENTER, APOPKA
Non Technical Summary
Although grape is the world's most important fruit crop, relatively little progress in its genetic improvement has occurred. Florida is the nation's second-third largest consumer of grape products in the US, but almost all is imported because there are no local adapted varieties with sufficient quality. Breakthroughs in genetic engineering of grape that now have been achieved in Florida. This project continues the genetic eengineering research at MREC aimed at producing disease resistant varieties. The present project emphasizes development of disease resistant V. vinifera cultivars, particularly heat-tolerant varieties that may be of use in Florida. Development of improved varieties with other desirable traits, such as seedlessness, may be oursued in the future as well.
Animal Health Component
30%
Research Effort Categories
Basic
30%
Applied
30%
Developmental
40%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2011139104030%
2011139105030%
2032499105010%
2121139108030%
Goals / Objectives
1. To develop totipotent cell cultures of grape, which are amenable to genetic manipulation. 2. To develop useful genetic elements for use in crop improvement, including genes, promoters and other sequences with regulatory functions. 3. To genetically engineer target varieties and select plants with putatively improved phenotypic changes. 4. To utilize successful implementation of objectives 1-3 to develop populations of putative disease-resistant varieties. 5.To conduct screening in the greenhouse and field to select elite individual varieties with desirable phenotypic traits.
Project Methods
A. Embryogenic culture development. Embryogenic culture systems, and potentially, other types of culture systems, which allow plants to be regenerated from single cells, will be developed by experimentally testing the following: 1) Explant source; 2) Culture media; 3) Culture growth conditions. Germplasm to be emphasized include major varieties of the following: 1) Vitis vinifera; regionally-adapted vinifera hybrids; Vitis rootstock varieties; Vitis rotundifolia varieties. B. Development of genetically transformed plants. Methods for the repeatable transformation of target varieties will be developed through experimentation with genetic vectors and cell cultures. Initially, marker genes will be utilized to test performance of various constructs so that those with optimum traits, such as high transformation efficiencies, low gene silencing, etc., can be identified. Optimized transformation systems will utilize genes obtained through in-house discovery research and from outside sources to produce putatively improved plants. For example, the corresponding DNA sequence of target proteins will be deduced and putative genes will be utilized for producing transgenic plants, as described below. The primary transformation system utilizes Agrobacterium. Following insertion, the embryogenic cultures will be grown on media to select transgenic cells and, ultimately, plants. Plants that are shown to produce the marker genes will be selected for further testing to determine whether they also express target genes of interest. Such plants then will be tested for presence of transproteins produced by these target genes. C. Screening of selected plants. Unique phenotypes obtained through implementation will be analyzed for the presence of unique proteins, which will be correlated with desirable phenotypic traits. For example, disease resistance may be evaluated by direct inoculation with the pathogen. Plants successfully challenged in the greenhouse will be entered into field trials.

Progress 10/01/07 to 09/30/13

Outputs
Target Audience: The target udiences are 1) The grape growers of Florida, who have supported the research through the Florida Department of AQgriculture and Consummers Services, Viticultural Trust Fund. 2) The global grape genetic research community, fo which we have developed many of the fundamental techniques to genetically-improve grape. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? We have trained three successful postdocs and one Ph.D. student under this project. Numerous high school and college studendts have volunteered to conduct science projects and gain basic training in the lab. How have the results been disseminated to communities of interest? The results have been disseminated via refereed journal articles, presentations at state, federal and International scientific meetings. Reqular meetings with Florida grape growers occur to discuss progress. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We have successfully completed all of the major project goals.

Publications

  • Type: Journal Articles Status: Published Year Published: 2012 Citation: Maziah M, S. Shirani Bidabadi, C. Ghobadi and D.J. Gray. 2012. Effect of methyl jasmonate treatments on alleviation of polyethylene glycol -mediated water stress in banana (Musa acuminata cv. ‘Berangan’, AAA) shoot tip cultures. Plant Growth Regulation, 68: 161–169.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Dreyer, E., C. Sims, R. Rouseff, D. Gray and M. Sipowicz. 2013. Sensory and compositional characteristics of Blanc du Bois wine. Amer. J. Enol. Vitic. 64: 118-125.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Zhao, J., Z. T. Li, J Chen, R. J. Henney, D. J. Gray and J. Chen. 2013. Purple-leaved Ficus lyrata plants produced by overexpressing a grapevine MybA1 gene. Plant Cell Rep., Online Aug, 2013
  • Type: Book Chapters Status: Awaiting Publication Year Published: 2014 Citation: Trigiano, R. N., J. A. Franklin and D. J. Gray. A brief introduction to plant anatomy and morphology. In: Beyl, C. and Trigiano, R. N., (eds.), Plant Propagation Concepts and Laboratory Exercises, 2014


Progress 10/01/11 to 09/30/12

Outputs
OUTPUTS: Sequence analysis of the grape genome uncovered a large number of antibacterial and antifungal genes and associated promoters. These genetic elements have been isolated from various grape varieties and hybrids that display variations in promoter functionality and disease resistance performances. Using the novel anthocyanin-based promoter analysis technology that we developed, we demonstrated the strong transcription activity of several constitutive promoters from grape. These promoters are being tested and incorporated in transformation experiments in order to replace foreign promoters for driving selectable and functional genes for obtainment of transformed cells-plants and trait development. For disease resistance genes, their expression units covering regions of promoter, protein coding sequences and terminator have been isolated and cloned into transformation vectors using our proprietary high efficiency DNA cloning system. Genes from different varieties may contain functionally important sequence alterations-mutations associated with gain-loss of function and resistance performance. Also, DNA amplification during gene isolation often introduces mismatch mutations. Hence, we chose to test all gene variants that we have isolated to determine their true functionality. So far, over 100 vectors containing different variant genes from same-different varieties have been and/or are being evaluated via a rapid functional assay system using transgenic tobacco. A number of promising genes identified thus far include those encoding VvTL-1, 2S Albumin, Snakin-Defencin, Antimicorbial Peptide-1 (AMP-1), Antimicrobial Peptide-2 (AMP-2), and PR-1 proteins. Semi-cisgenic plants of 'Thompson Seedless' and 'Seyval Blanc' containing viral promoter and grape genes have been obtained and are being tested in the transgenic test field. These plants showed elevated resistance performance as compared to non-transformed control plants. Meanwhile, somatic embryos of 'Chenin Blanc' and other important varieties have been transformed with cisgenic constructs. However, efforts with 'Chenin Blanc' have been unsuccessful so far, and we are attempting various changes in the transformation protocol. Efforts are being made to select cisgenic plants that express various re-engineered grape genes. Extensive functional analysis of these grape genes and their incorporation into the transformation program will catapult engineering efforts for achieving enhanced disease resistance, including PD resistance, using cisgenic approach. Once obtained, these plants will be more readily available to growers with less stringent regulatory restrictions. A new state (UF) and federal (USDA/APHIS) approved field permit was obtained and plants containing these genes were planted in May 2012 for crucial field evaluation. Work to create fully cisgenic plants is actively underway. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: The target audiences include the following: 1) Scientists who are using information generated by this project to conduct research concerning the genetic improvement of grape, and 2) Grape growers and others involved in viticulture who await the availability of improved cultivars to be developed through this project. Efforts to deliver scientific-based information include the following: 1) Publication in refereed journals and book chapters, 2) Making presentations at scientific meetings and grower-based meetings, and 3) Providing interviews to reporters who publish our information in grower-trade magazines. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
A refereed publication was released as listed below plus two book chapters. A great deal of interest has been generated by utilizing a "cisgenic" or "intragenic" approach to genetic engineering. This approach is unique in that it uses only genetic elements from grape. More commonly termed "precision breeding", this approach is more consumer friendly and should be able to bypass many federal regulations that are currently keeping GM plants from the marketplace.

Publications

  • S.A. Dhekney, Z. T. Li, M. Dutt, and D. J. Gray. 2012. Initiation and Transformation of Grapevine Embryogenic Cultures. Chap. 18 In: Jim M. Dunwell and Andy C. Wetten (eds.), Transgenic Plants: Methods and Protocols, Methods in Molecular Biology, vol. 847:215-225. Humana Press/Springer Science+Business Media, LLC.
  • Li, Z.T., K-H Kim, J.R. Jasinski, M.R. Creech and D.J. Gray. 2012. Large-scale characterization of promoters from grapevine (Vitis spp.) using quantitative anthocyanin and GUS assay systems. Plant Sci. 196: 132-142.
  • M. Dutt, Z. T. Li, S.A. Dhekney, and D. J. Gray. 2012. Chap. 17 In: Co-transformation of Grapevine Somatic Embryos to Produce Transgenic Plants Free of Marker Genes. Jim M. Dunwell and Andy C. Wetten (eds.), Transgenic Plants: Methods and Protocols, Methods in Molecular Biology, vol. 847:201-213. Humana Press/Springer Science+Business Media, LLC. 2012.


Progress 10/01/10 to 09/30/11

Outputs
OUTPUTS: Several endogenous genes were isolated, including those to be tested for seedlessness and rot resistance. These genes were packaged into vectors and transformed into grape. Small grape plants are at various stages in the lab and greenhouse awaiting further evaluation. Field evaluations for Pierce's disease resistance in transgenic plants continue to show promise. We have one transgenic line of Freedom rootstock that is resisting disease, while nontransformed controls have died. Field evaluation for fungal disease resistance have identified two transgenic lines that show delayed black rot development. The ripe fruit from thses vines exhibit drammatic rot resistance. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Several refereed publicatiosn were released as listed below. A great deal of interest has been generated by utilizing a "cicgenic" or "intrageni" approach to genetic engineering. Both of thse approaches are unique in that they use only genetic elements from grape and thus more likely to be approved by APHIS BRS and EPA as well as being more palatable to the public.

Publications

  • Sandhu, A.K., D.J. Gray, J. Lu and L. Gu. 2011. Effects of exogenous abscisic Acid on antioxidant capacities, anthocyanins, and flavonol contents of Vitis rotundifolia (muscadine grapes). Food Chemistry, 126, 982-988.
  • Li, Z.T., S.A. Dhekney and D.J. Gray. 2011. Use of the VvMybA1 gene for non-destructive quantification of promoter activity via color histogram analysis in grapevine (Vitis vinifera) and tobacco. Transgenic Res.,20,1087-1097.
  • Stringer, S.J, S.A. Marshall and D.J. Gray. 2011. Eudora muscadine grape. HortScience, 46,143-144.
  • Dhekney, S.A., Z.T. Li and D.J. Gray 2011. Grapevines engineered to express cisgenic Vitis vinifera thaumatin-like protein exhibit fungal disease resistance. In Vitro Dev. Biol. Plant, 47,458-466.


Progress 10/01/09 to 09/30/10

Outputs
OUTPUTS: Field Testing Transgenic Grapevines for Fungal Disease and Pierce's Disease Resistance. In 2006, we obtained USDA APHIS approval for field testing via the "notification system". In January and April 2007, respectively, transgenic vines were established in the field at the Mid-Florida Research & Education Center. The vines either contained a gene called VVTL-1 that was recovered originally from grape and then re-engineered for increased expression before insertion back into Vitis vinifera or a proprietary antibacterial lytic peptide genes that will be evaluated for Pierce's disease resistance. In 2008, we began to evaluate plants containing VVTL-1 for fungal diseases. We obtained very high rot resistance in Thompson Seedless fruit from plants containing the VVTL-1 gene. Plants containing an additional proprietary gene recovered from grapevine, which shows powdery mildew resistance in fruit have been identified from greenhouse screening and are being readied for field tests. Most of the transgenic plants are from the variety Thompson Seedless. However, transgenic Merlot, Seyval Blanc and Syrah (Shiraz) are either in the field or greenhouse testing phases. In addition, a number of other genes and genetic elements are under development for grape and should have application in other crops as well. In 2009 - 2010, we began shifting emphasis in the laboratory program towards "cisgenic engineering", i.e., the use of grape genes to re-engineer grape. In this, we are developing a grape anthocyanin reporter marker gene; an anti-bacterial PR1 gene and the aforementioned VVTL-1 PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: The release of two new muscadine grape cultivars are of immediate use by growers. Additional development of cisgenic engineered grapevines will provide new varieties with enhanced disease resistance and quality traits. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
IMPACT: 2008/10 TO 2009/09 The outcome of high fruit rot resistance will be used to expedite the insertion of the VVTL-1 gene into additional species. The anthocyanin reporter marker gene is very convenient and should have application to other plants. Vitis rotundifolia (muscadine grape)has now been engineered with both VVTL-1 and a seedless construct. New muscadine cultivars provide increased choices for growers and consumers.

Publications

  • Dhekney, S.A., Z.T. Li and D.J. Gray. 2010. Factors influencing induction and maintenance of Vitis rotundifolia Michx. embryogenic cultures. Plant Cell Tissue and Organ Culture, Online First, DOI: 10.1007/s11240-010-9849-7.
  • Li, Z.T., S.A. Dhekney and D.J. Gray. 2010. PR-1 gene family of grapevine: a uniquely duplicated PR-1 gene from a Vitis interspecific hybrid confers high level resistance to bacterial disease in transgenic tobacco. Plant Cell Reports, Online First, DOI: 10.1007/s00299-010-0934-5
  • Li, Z.T., S.A. Dhekney and D.J. Gray. 2010. Molecular characterization of a SCAR marker linked to seedlessness in grapevine (Vitis vinifera) reveals a lack of sequence fidelity. Molecular Breed., 25:637-644.


Progress 10/01/08 to 09/30/09

Outputs
OUTPUTS: Plants containing an inserted version of the VVTL-1 gene were evaluated for resistance to various fungal diseases in field test plots. Two of five transgenic lines tested showed reistance to black rot and anthracnose diseases. The resistance persisted for two weeks longer than control lines. In 2009, fruit harvested from Thompson Seedless was evaluated for post-harvest diseases. The same two lines exhibited significant resistance to fruit rots after storage for three weeks at 24C. Studies of Pierce's disease resistance are ongoing using plants that contain two synthetic lytic peptide genes (Lima A and Lima B). Pierce;'s disease was detected in the vineyard in 2009 and a control vine died. We should obtain definitive results in 2010-2011. Two new muscadine grape varieties (Delicious and Southern Jewel) were released in 2009 and are sought-after by growers. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: The release of two new muscadine grape cultivars are of immediate use by growers. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The outcome of high fruit rot resistance will be used to expedite the insertion of the VVTL-1 gene into additional species, such as Vitis rotundifolia (muscadine grape). New muscadine cultivars provide increased choices for growers and consumers.

Publications

  • Gray, D.J., Z.T. Li, S.A. Dhekney, D.L. Hopkins and S.A. Sims, 2009. Delicious: An early-ripening, self-fertile, multi-purpose black-fruited muscadine grape. HortScience, 44(1), 200-201.
  • Gray, D.J., Z.T. Li, S.A. Dhekney, D.L. Hopkins and S.A. Sims, 2009. Southern Jewel: A self-fertile, black muscadine grape with fruit produced on bunches. HortScience, 44(5), 1476- 1477.
  • Dhekney, S. A., Z. T. Li, M. E. Compton, and D. J. Gray, 2009. Optimizing initiation and maintenance of Vitis embryogenic cultures. HortScience 44(5), 1400-1406.
  • Dhekney, S.A., Z.T. Li, M. Dutt and D.J. Gray, 2009. Factors Influencing Genetic Transformation and Plant Regeneration of Vitis. Amer. J. Enol. Vitic. 60, 285-292.
  • Dhekney, S. A., Z. T. Li., T. W. Zimmerman, and D. J. Gray, 2009. Using endogenous Vitis genes to produce disease resistant grapevines. 2009 Soc. In Vitro Biol. Ann. Meeting, Charleston SC, In Vitro Cell. Dev. Biol. 45, S38.
  • Gray, D. J., Z. T. Li, S. A. Dhekney, D. L. Hopkins, T. W. Zimmerman, 2009. Green genetic engineering technology: Rearrangement of endogenous functional genetic elements to create improved grapevines. In Vitro Biol. Ann. Meeting, Charleston SC, In Vitro Cell. Dev. Biol. 45, S39.


Progress 10/01/07 to 09/30/08

Outputs
OUTPUTS: Field Testing Transgenic Grapevines for Fungal Disease and Pierce's Disease Resistance. In 2006, we obtained USDA APHIS approval for field testing via the "notification system". In January and April 2007, respectively, transgenic vines were established in the field at the Mid-Florida Research & Education Center. The vines either contained a gene called VVTL-1 that was recovered originally from grape and then re-engineered for increased expression before insertion back into Vitis vinifera or a proprietary antibacterial lytic peptide genes that will be evaluated for Pierce's disease resistance. In 2008, we began to evaluate plants containing VVTL-1 for fungal diseases. The plants received no chemical control, except for one Ridomil application to control downy mildew, caused by an oomycetous fungus, which does not respond to VVTL-1. Plants containing an additional proprietary gene recovered from grapevine, which show powdery mildew resistance in fruit have been identified from greenhouse screen and are being readied for field tests. Most of the transgenic plants are from the variety Thompson Seedless. However, transgenic Merlot, Seyval Blanc and Syrah (Shiraz) are either in the field or greenhouse testing phases. In addition, a number of other genes and genetic elements are under development for grape and should have application in other crops as well. We continue to publish and seek patent protection for new inventions. Patents are needed to establish value of the technology and might generate the amount of financial investment needed to develop and commercialize transgenic grapevines. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
In early season comparisons with non-transgenic controls, two lines showed remarkable resistance to both black rot and anthracnose. However, by late July, anthracnose was noted in all plant lines and a broad spectrum chemical control program was instituted. Our hope is to "reset" the test so that all plants are nearly disease-free and then determine if there remains a difference in resistance. While we have the aforementioned results for fungal disease control now, evaluation for PD evaluation will take several years.

Publications

  • Gray, D. J., Z.T. Li, S. A. Dhekney, D. L. Hopkins, T. W. Zimmerman, 2008. Field tests of transgenic grapevine for disease resistance. In Vitro Biol. Ann. Meeting, Tucson AZ, In Vitro Cell. Dev. Biol. 44, S40, P-1013.
  • Li, Z. T., S.A. Dhekney, D.J. Gray, 2008. Probing the Vitis genome. Opportunities and pitfalls. 2008 Annual Meeting of the American Society for Horticultural Science, Orlando, FL, HortScience 43, 1080-1081.
  • Gray, D. J., S. A. Dhekney, Z.T. Li, T. W. Zimmerman, 2008. Green genetic engineering technology: The use of endogenous genes to create fungal disease-resistant grapevines. Caribbean Food Crops Society 44th Annual Meeting, Miami, FL Program Book, 59.
  • Li, Z. T. and D. J. Gray, 2007. Nucleotide sequences of 2S albumen gene and its promoter from grape and uses thereof, US Patent No. 7,250,296.
  • Gray, D. J., J. Subramanian and R. E. Litz. 2008. Selection of fungal resistant grape somatic embryos, US Patent No. 7,326,826 B2.
  • Gray, D. J., Z.T. Li, S. A. Dhekney, T. W. Zimmerman, 2008. Tracking pollen-mediated gene flow in transgenic grapevine. 2008 Annual Meeting of the American Society for Horticultural Science, Orlando, FL, HortScience 43, 1156.
  • Jayasankar, S. and D. J. Gray, 2008. In vitro plant pathology, Chap. 41, In: R. N. Trigiano, M. T Windham and A. S. Windham (Eds.), Plant Pathology: Concepts and Laboratory Exercises, 2nd Edition, CRC Press, Boca Raton, pp. 475-485, 2008.
  • Dhekney, S.A., Z.T. Li., M. Van Aman, M. Dutt, J. Tattersall, and D.J. Gray, 2007. Genetic transformation of embryogenic cultures and recovery of transgenic plants in Vitis vinifera, Vitis rotundifolia and Vitis hybrids, Proc. 2005 Int. Symp. Biotechnol. Temperate Fruit Crops & Tropical Species, ACTA Hort. 738, 743-748.
  • Dutt, M., Z.T. Li, K. Kelley, S.A. Dhekney, M. Van Aman, J. Tattersall, and D.J. Gray, 2007. Transgenic rootstock protein transmission in grapevines, Proc. 2005 Int. Symp. Biotechnol. Temperate Fruit Crops & Tropical Species, ACTA Hort. 738, 749-753.
  • Li, Z.T., S.A. Dhekney, M. Dutt, M. Van Aman, J. Tattersall, K. Kelley and D.J. Gray, 2007. Isolation and characterization of the 2S albumin gene and promoter from grapevine. Proc. 2005 Int. Symp. Biotechnol. Temperate Fruit Crops & Tropical Species, ACTA Hort. 738, 759-765.
  • Dutt, M., Z.T. Li, S.A. Dhekney and D.J. Gray, 2007. Transgenic plants from shoot apical meristems of Vitis vinifera Thompson Seedless via Agrobacterium-mediated transformation. Plant Cell Rep. 26, 101-2110.
  • Dhekney, S.A., Z.T. Li, M. Dutt and D.J. Gray, 2008. Agrobacterium-mediated transformation of embryogenic cultures and regeneration of transgenic plants in Vitis rotundifolia Michx. (muscadine grape). Plant Cell Rep. 77, 865-872.
  • Li, Z.T., S.A. Dhekney, M. Dutt, and D.J. Gray, 2008. An Improved Protocol for Agrobacterium-Mediated Transformation of Grapevine. Plant Cell Tiss. Organ Cult. 93, 311-321.
  • Dutt, M., Z.T. Li, S.A. Dhekney and D.J. Gray, 2008. A co-transformation system to produce transgenic grapevines free of marker genes. Plant Science 175, 423-430.
  • Gray, D. J., Z.T. Li, S. A. Dhekney, M. Dutt, D. L. Hopkins, T. W. Zimmerman, 2007. Field testing of transgenic grapevine for bacterial and fungal disease resistance. 2007 Annual Meeting of the American Society for Horticultural Science, HortScience 42:858, 2007.
  • Dhekney, S.A., Z.T. Li., M. Dutt, T. W. Zimmerman, and D.J. Gray Greenhouse Screening and Field Testing of Transgenic Grapevine for Fungal Resistance. 2007 Annual Meeting Society for In Vitro Biology, In Vitro, 43, S40.
  • Dhekney, S.A., Z.T. Li., M. Dutt, T. W. Zimmerman, and D.J. Gray, 2008. Overcoming obstacles to genetic transformation in Vitis. 2008 Soc. In Vitro Biol. Ann. Meeting, Tucson AZ, In Vitro Cell. Dev. Biol. 44, S40, P-1013.
  • Dhekney, S.A., Z.T. Li., D. L. Hopkins, T. W. Zimmerman, and D.J. Gray, 2008. Transgenic grapevine rootstocks for Pierces disease resistance. 2008 Annual Meeting of the American Society for Horticultural Science, Orlando, FL, HortScience 43, 1156.