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

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

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

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

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 In Vitro Cell. Dev. Biol. Plant 42:
Dhekney et al ACTA Hort 738:
Therefore all needed technology is in place to evaluate use of genetic transformation in grapevine improvement

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 Elements Basic 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
Terminator
Enhancer
Element
Core
Promoter
RNA
Protein
Located on
Chromosome *
Chromosome
(Double-Stranded DNA)

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

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

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

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

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. Green Fluorescent Protein Expression
in Vitis vinifera & Vitis rotundifolia C A E D B
Embryos
B. Leaf/stem/tendril
C. Flowers
E. Anther/stigma
A-C = V. vinifera ‘Thompson
Seedless’
D-E = V. rotundifolia ‘Alachua’

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
Susceptible Control Vines
Resistant Transgenic Vines

28. Powdery Mildew Resistant Transgenic Grapevines Selected in Greenhouse at MREC
Susceptible ‘Thompson Seedless’
Transgenic ‘Thompson Seedless’
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

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. Pure xylem sap is exuded from decapitated plants due to root pressure
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 CK
Purified Protein
Test Samples
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. PD Resistant Transgenic Grapevines Selected in Greenhouse at MREC
Susceptible
Control Vines
Transgenic Vines
w Lytic Peptide
Resistant Control
‘Tampa’
‘Thompson Seedless’
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

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

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

37. The Virgin Islands Field Site University of the Virgin Islands, St
The Virgin Islands Field Site University of the Virgin Islands, St. Croix Established January 2007
Two ‘Thompson Seedless’ lines
containing VVTL-1 gene (June 2007)

38. Approved through USDA APHIS notification
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

39. The Florida Field Site UF/IFAS Mid-Florida Research & Education Center

40. Trellises constructed in March 2007
The Florida Field Site
Trellises constructed in March 2007

41. The Florida Field Site Planting April 2007

42. The Florida Field Site ‘Thompson Seedless’, June 2007

43. The Florida Field Site
July 6, 2007

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

45. Screening Endogenous Genes for use in Powdery Mildew Resistance
Non-transgenic
Transgenic
‘Syrah’
before
veraision

46. Screening Endogenous Genes for use in Powdery Mildew Resistance
Non-transgenic
Transgenic
‘Syrah’
ripe

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 (
Support for patent costs and commercialization efforts