D J Gray, Z T Li, S A Dhekney, M Dutt, D L Hopkins Mid-Florida Research & Education Center University of Florida/IFA

1. Genetic Engineering of Grapevine and Field Testing for Bacterial & Fungal Disease Resistance D J Gray, Z T Li, S A Dhekney,M Dutt, D L Hopkins Mid-Florida Research & Education Center University of Florida/IFAS T W Zimmerman Biotechnology & Agroforestry University of the Virgin Islands www.mrec.ifas.ufl.edu/grapes

2. Field Testing Transgenic Grapevine for Bacterial and Fungal Disease Resistance Objectives To test GM grapevines in Florida and the US Virgin Islands under USDA/APHIS approved conditions Evaluate for Pierce’s disease & fungal disease resistance Evaluate for commercially-useful qualities Begin to assess environmental risks of GM grape Determine extent of gene flow via pollen Determine if weedy hybrids occur www.mrec.ifas.ufl.edu/grapes

3. Grapevine & Genetic Improvement The Opportunity: • World’s most valuable fruit crop • 10 – 12th most valuable agricultural crop in US • But - Grape is particularly difficult to improve genetically • Genetic self-incompatibility makes breeding new varieties difficult • Long grape lifecycle – years to make recurrent crosses • Fine discrimination of wine type makes breeding a new “Cabernet” impossible • These obstacles have led to little improvement of desirable varieties, such as adding disease resistance to established wine varieties • An exception has been for table grape breeding where improved fruit types have been created by laborious breeding

4. Why Grape Research in the Subtropics?The Virgin Islands The Virgin Islands must import all grape products Therefore, like Florida, a large untapped local market exists Grape is a high-value crop suited for small farm production A new source of agricultural income BUT - Disease-resistant cultivars are needed www.mrec.ifas.ufl.edu/grapes

5. Why Grape Research in the Subtropics?Florida Florida is the US’s # 2 consumer of wine & grape products Existing muscadine-based industry satisfies less than 1% of market Therefore a large untapped local market exists Conventional varieties are needed for wine & seedless fruit But - All such varieties will die from Pierce’s disease & various fungal diseases if grown in Florida Conventional breeding cannot be used to create resistant versions of desirable varieties www.mrec.ifas.ufl.edu/grapes

6. Genetic Engineering of Grapevine • Insertion of genes for disease resistance into otherwise desirable varieties might result in grapevines that can be grown in the Virgin Islands and Florida and address the existing market • Agrobacterium-mediated genetic transformation of certain Vitis vinifera varieties and rootstocks is now routine & efficient • Li et al. 2006 In Vitro Cell. Dev. Biol. Plant 42:220-227 • Dhekney et al. 2007 ACTA Hort 738:749-753 • Therefore all needed technology is in place to evaluate use of genetic transformation in grapevine improvement www.mrec.ifas.ufl.edu/grapes

7. Diseases of Grapevine • Bacterial Disease • Pierce’s Disease – Florida & California • Prohibits production of Vitis vinifera in • the Southeastern US & is increasing in Ca • Fungal Diseases – Florida, Virgin Islands & Worldwide • Powdery Mildew – Most important disease of grape worldwide • Anthracnose • Downy Mildew • Ripe Rot • Many others • Viral Diseases – Worldwide • Grape fan leaf virus – most severe & widespread virus • Leaf roll, corky bark, many others

8. Pierce’s Disease • Endemic in Florida (the southern coastal plain) & California • Caused by Xylella fastidiosa • Xylem limited bacterium that infects a wide range of • vascular plants • Transmitted by xylem-feeding insects • Primarily leaf hoppers & sharpshooters – Glassy Winged Sharpshooter new in CA • Lethal to all Vitis vinifera & other non-native varieties

9. Pierce’s Disease Total loss of vineyard results

10. Pierce’s Disease Tolerant Vines

11. Anthracnose of Grapevine

12. Powdery Mildew of Grapevine Typical chlorotic lesions Powdery Mildew infects leaves, stems & fruit Aerial hyphae

13. Genetic Engineering of Grape • Direct insertion of genes (DNA) • Allows integration of single traits without disturbing desirable characteristics • Important for grape where varieties are highly prized and change is resisted (e.g. wine varieties) • Adds traits that normally are not found in grape • Such as novel types of disease resistance

14. Genetic Engineering of Grape • Modern techniques of molecular biology allow DNA to be analyzed and manipulated • Functional DNA, including genes, literally can be cut and pasted together into new and useful combinations • Potentially, unlimited possibilities result

15. Understanding Genetic Control ElementsBasic definitions: A segment of DNA that causes genes to become active. Promoters serve as a binding site for the enzyme RNA polymerase, which performs the first step of transcribing DNA in the adjacent gene. Promoter A segment of DNA that influences the activity of promoters by serving as a binding site for specific proteins. Enhancers may be physically separated from the promoter, but still influence it. Duplicating enhancers or modifying their DNA sequences can dramatically alter activity. Enhancer Element A heritable sequence of DNA that determines a particular characteristic in an organism. A gene may code for a wide range of functions. Most commonly, proteins are produced . Gene A segment of DNA that signals the end of a gene. Terminators serve to stop transcription by detaching RNA polymerase. Gene Terminator Also known as “transcription” and “translation” . RNA polymerase binds at the promoter and moves along the DNA strand, transcribing the sequence into Messenger RNA. When the terminator is reached, mRNA detaches and is translated into a protein, the structure of which is dictated by the original DNA sequence. RNA to Protein

16. A Conventional Genetic Control Element RNA Protein Gene Terminator Enhancer Element Core Promoter Gene Located on Chromosome * Chromosome (Double-Stranded DNA)

17. Bidirectional Dual PromoterComplexUS Patent # 7,129,343 2006 Protein RNA RNA Protein The patented BDPC is a new wayto arrange Genetic Control Elements. The BDPC provides more efficient control of genes and results in better protein production. Gene Terminator Core Promoter Core Promoter Gene Terminator Enhancer Element Enhancer Element Transgene - 1 Transgene - 2 Duplicated Elements in Divergent Orientation Duplicated Enhancer Conventional Arrangement of Gene Controlling Elements

18. Antibiotic Resistance GFP Functional Gene (To select GE cells) (To see GE cells) (Improved Trait) Source Genes Molecular Biology Antibiotic Resistance GFP Functional Gene Unique DNA Sequence for Genetic Engineering Assembly of DNA for Insertion into Grape

19. Burk et al. 2001 Yang et al. 1996 Bi-Functional Fusion Gene + EGFP NPTII Reporter Selectable A bi-functional marker gene

20. Functional Genes Tested in Grape at MREC • Vitis vinifera thaumatin-like protein gene • Tested for fungal resistance • Grape albumen protein gene • Tested for fungal resistance and seedlessness • Lytic peptide genes • Tested for PD and fungal resistance • Hybrid resistance genes • Tested for PD and fungal resistance

21. CsVMV-BDPC Transgene - 1 Transgene - 2 Lyt Pep EGFP/NPTII Term Term. Core Promoter Core Promoter Modified Enhancer Example of Transformation Vector: A Lytic Peptide-Containing Transformation Vector Based on a Bidirectional Promoter Complex (BDPC)

22. Grape Transformation System • Embryogenic cultures • Totipotent cells of somatic embryos are the target • Agrobacterium mediated transformation • Kanamycin selection of transgenic cells • Uses NPTII Gene

23. Selection of Transgenic Grape • Transient GFP expression • First visualized at 2 days and faded by 20 days • Stable GFP expression • Apparent within 20 days • GFP-positive embryogenic cultures • Isolated within 6 weeks • Plants produced from embryos are acclimated to ex vitro conditions

24. A B C E D Green Fluorescent Protein Expression in Vitis vinifera & Vitis rotundifolia • Embryos • B. Leaf/stem/tendril • C. Flowers • E. Anther/stigma • A-C = V. vinifera ‘Thompson • Seedless’ • D-E = V. rotundifolia ‘Alachua’ www.mrec.ifas.ufl.edu/grapes

25. Preparing Transgenic Grape Lines for Greenhouse Testing Plants from tissue culture are propagated to produce multiple clones Plants are arranged into populations to study disease resistance

26. Screening for Powdery Mildew-Resistant Transgenic Grapevines Transgenic vines producing “Thaumatin-like” protein are grown with non-transgenic controls in an area of high powdery mildew incidence and without chemical control. Vines are rated 3x’s per week for disease development throughout the growing season. Experimental population of transgenic and control vines Powdery Mildew Test Site in UF/IFAS Greenhouse

27. Powdery Mildew-Resistant Transgenic Grapevines (January 2005) Certain transgenic vines grown in an area of high powdery mildew pressure remained vigorous and show resistance to PM throughout the growing season. Initial Powdery Mildew Test Site in Greenhouse Resistant Transgenic Vines Susceptible Control Vines

28. Susceptible ‘Thompson Seedless’ Transgenic ‘Thompson Seedless’ Powdery Mildew Resistant Transgenic Grapevines Selected in Greenhouse at MREC Selected transgenic vines that express Vitis vinifera thaumatin-like protein (VVTL-1) exhibit an 8 day delay in visible lesion development compared to control vines VVTL-1 is an endogenous gene from grapevine 5 resistant lines have been selected for field trials www.mrec.ifas.ufl.edu/grapes

29. Testing Transgenic Plants for Resistance to Pierce’s Disease The Xylella bacterium, which causes Pierce’s disease, is injected directly into the xylem tissue of transgenic grape plants and control plants. The plants are then evaluated for resistance.

30. CK Test Samples Purified Protein Testing Xylem Sap for Lytic Peptide The presence of lytic peptide is determined by ELISA Pure xylem sap is exuded from decapitated plants due to root pressure Transgenic Vines

31. Tracking Bacterial Concentrations in Test Plants • After stem inoculation, the xylem sap from leaf petioles is placed on Xylella-specific culture media • The presence and number of bacterial colonies provides an early estimate of plant resistance • Periodic sampling provides information on internal spread of bacteria over time Dead Dead Seasonal dormancy

33. Susceptible Control Vines Transgenic Vines w Lytic Peptide Resistant Control ‘Tampa’ ‘Thompson Seedless’ PD Resistant Transgenic Grapevines Selected in Greenhouse at MREC These vines were inoculated in July 2004 Since even resistant controls developed symptoms, the PD test is considered to be stringent Lack of symptoms in transgenic plants that contain proprietary lytic peptide genes suggest high level of resistance Approximately 100 highly resistant lines have since been selected, 15 of which were propagated for field trials www.mrec.ifas.ufl.edu/grapes

34. Production of Transgenic Grapevines at MREC • Over 2,200 unique transgenic plants have been produced • 90%+ have been Thompson Seedless (the most widely planted variety in the US) • Nine different functional genes have been tested • Most have been discarded due to poor response • The hybrid LIMA gene appears to provide PD resistance • The native thaumatin gene appears to provide fungal resistance • Field tests must now be established

35. Selected Transgenic Plants Propagated For Field Trial www.mrec.ifas.ufl.edu/grapes

36. The Virgin Islands UVI Field Site • A protected site used for evaluation of GM plants • 5 lines containing VVTL-1, replicated 5-7 times • 29 transgenic vines plus 9 controls planted 1/2007 Approved through USDA APHIS notification process in October 2006 www.mrec.ifas.ufl.edu/grapes

37. Two ‘Thompson Seedless’ lines containing VVTL-1 gene (June 2007) The Virgin Islands Field SiteUniversity of the Virgin Islands, St. CroixEstablished January 2007 www.mrec.ifas.ufl.edu/grapes

38. The Florida Field Site • Isolated from cross-fertile wild & cultivated vines • 15 lines containing either of 2 experimental lytic peptide, replicated 4-5 times • 5 lines containing VVTL-1, replicated 8 times • Transgenic varieties used (180 plants = 60%) • 30% ‘Thompson Seedless’, 10% ‘Merlot’, 10% ‘Seyval Blanc’ • 10% ‘Freedom’ rootstock • Non-transgenic controls (120 plants = 40%) • Same scions and rootstocks as above (20%) • PD-resistant hybrids ‘Tampa’ and BN5-4 (20%) Approved through USDA APHIS notification process in October 2006 www.mrec.ifas.ufl.edu/grapes

39. The Florida Field SiteUF/IFAS Mid-Florida Research & Education Center www.mrec.ifas.ufl.edu/grapes

40. Trellises constructed in March 2007 The Florida Field Site www.mrec.ifas.ufl.edu/grapes

41. Planting April 2007 The Florida Field Site www.mrec.ifas.ufl.edu/grapes

42. The Florida Field Site ‘Thompson Seedless’, June 2007 www.mrec.ifas.ufl.edu/grapes

43. July 6, 2007 The Florida Field Site www.mrec.ifas.ufl.edu/grapes

44. What’s Next? Evaluation for disease resistance & clonal fidelity are ongoing Environmental risk assessment studies planned Greenhouse screening of endogenous genes and varieties will lead to new field planting in 2008-09 www.mrec.ifas.ufl.edu/grapes

45. Non-transgenic Transgenic Screening Endogenous Genes for use in Powdery Mildew Resistance ‘Syrah’ before veraision www.mrec.ifas.ufl.edu/grapes

46. Non-transgenic Transgenic Screening Endogenous Genes for use in Powdery Mildew Resistance ‘Syrah’ ripe www.mrec.ifas.ufl.edu/grapes

47. Acknowledgments • Florida Department of Agriculture & Consumer Services Viticulture Trust Fund • Long-term support of grape biotech research • USDA Tropical, Sub-Tropical Agricultural Research Grants Program • Support for endogenous gene discovery and field tests • Florida Genetics LLC (www.flgenetics.net) • Support for patent costs and commercialization efforts www.mrec.ifas.ufl.edu/grapes