Genetic Transformation and Transgenic Plant Recovery from Vitis Species

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1 Genetic Transformation and Transgenic Plant Recovery from Vitis Species Sadanand Dhekney, Zhijian T. Li & Dennis J. Gray Mid Florida Research & Education Center Apopka, FL 32703

2 Rationale for Genetic Transformation of Vitis Limitations of Conventional Breeding Extreme heterozygosity and pronounced inbreeding depression F1 hybrid produced is of intermediate quality Long juvenile period of vines makes screening of new selections tedious and time consuming

3 Genetic Transformation Allows incorporation of single or few traits in popular varieties Potentially no change in existing desirable characteristics of a variety Circumvents problems encountered in conventional breeding Rapid screening of engineered trait

4 Potential Applications of Genetic Transformation in Vitis Disease Resistance Bacterial, Fungal and Viral Disease Resistance Abiotic Stress Tolerance Cold, Salt, Drought, Heavy Metals Quality Traits Seedlessness, Flavor, Reduced Browning, Enological Characteristics

5 Limitations Encountered in Genetic Transformation of Vitis Diverse Response of Species and Varieties Response to Cell Culture Rapid loss of embryogenic potential Loss of germination potential after prolonged culture Response to Transformation Low transient and stable transgene expression Good transient and stable transgene expression but poor secondary embryogenesis from stable callus Varying levels of Necrosis / Browning observed following co-cultivation with Agrobacterium

6 Differential Necrogenesis / Browning of Embryogenic Cultures Following Co-cultivation With Agrobacterium Orange Muscat Sauvignon Blanc Thompson Seedless Riparia Gloire

7 Resources Developed for Vitis Genetic Transformation Use of GFP as a reporter gene in grape transformation Development of a reporter-marker fusion gene (GFP/NPTII) Testing different promoters and enhancers for improving grape transformation Bi-directional duplex promoter complex development for enhanced transgene expression in grape (Li et al., 1999; 2001; 2004)

8 Embryogenic Culture System for Vitis Induction of embryogenic cultures leaves or floral tissues (Gray and Mortensen, 1987; Gray, 1989; 1992; 1995) Maintenance of embryogenic cultures on X6 medium (Li et al., 2001) Rapid proliferation using suspension cultures (Jayasankar et al., 1999) Germination of somatic embryos on MS1B medium

9 Genetic Transformation System SE at cotyledonary stage + Overnight Agro culture (EHA 105) 48 h. Co-cultivation Transfer to solid DM medium (30d) 3d wash in liquid DM medium Harden & transfer plants to greenhouse for testing Germinate transgenic embryo lines and regenerate plants Transfer to X6 medium & screen for transgenic embryo lines

10 Use of Preculture and Dithiothreitol (DTT) to Control Browning / Necrosis Treatments Preculture Control DTT- 0.5 g L -1 DTT- 1.0 g L -1 DTT- 1.0 g L -1 * No Preculture Control DTT- 0.5 g L -1 DTT- 1.0 g L -1 DTT- 1.0 g L -1 * Preculture = Transferring SE to fresh X6 medium for 7d prior to co- cultivation DTT- added to post co-cultivation wash medium * DTT - also added to Agrobacterium co-cultivation medium Thompson Seedless cultures were used for the experiments

11 Effect of Preculture and DTT Treatments on Culture Browning and Agrobacterium-mediated Transformation (30 embryos per Petri dish) Treatment Browning of Wash Medium Transient Expression Stable Callus Lines Stable Embryo Lines No Preculture 0 DTT g/l DTT wash g/l DTT wash g/l DTT co-cultivation & wash Preculture 0 DTT g/l DTT wash g/l DTT wash g/l DTT co-cultivation & wash

12 Effect of Preculture and DTT (1g l -1 ) on Browning of SE Following Co-cultivation Before co-cultivation No preculture - Control after co-cultivation Preculture - Control after co-cultivation No preculture - DTT after co-cultivation

13 Genetic Transformation of Vitis vinifera Transient GFP expression in SE GFP stable callus formation GFP stable SE formation GFP stable embryo proliferation Germinated SE GFP expression in leaf

14 Genetic Transformation of Vitis. rotundifolia Transient expression in PEM Transient expression in SE Callus formation PEM formation

15 Vitis rotundifolia (continued) Stably transformed PEM PEM proliferation Somatic Embryos Stably transformed somatic embryos

16 Vitis rotundifolia (continued) A B C D A- Germinating transgenic embryos C- GFP stable transgenic leaf B- GFP stable germinating embryo D- GFP stable transgenic root

17 Transient and Stable GFP expression, and Number of Plant Lines Recovered in Vitis Variety % Transient Expression No. of embryo lines No. of plant lines Vitis vinifera Thompson Seedless Merlot Sauvignon Blanc Pinot Noir Shiraz Chardonnay Cabernet Franc Superior Seedless Zinfandel Vitis rupestris 'St. George' Vitis rotundifolia 'Alachua' Vitis hybrids Conquistador Freedom Seyval Blanc

18 Current Efficiency of Transgenic Grapevine Production Procedure Time Frame Induction and maintenance of embryogenic cultures Genetic transformation and * regeneration of plants 7 months 4 months Cumulative time period from induction of cultures to transgenic plant regeneration 11 months * 1g embryogenic culture can give up to a 150 independent transgenic lines

19 Ongoing and Future Work Study responses of species and varieties to embryogenesis Evaluate more species and varieties for response to transformation Improve overall transformation efficiency Improve plant regeneration efficiency from somatic embryos

20 UF / IFAS MREC Grape Biotechnology Core Laboratory Research Personnel Dr. Dennis J. Gray Dr. Zijian T. Li Dr. Sadanand A. Dhekney Ms Marilyn V. Aman Manjul Dutt Jeremiah Tattersall Karen T. Kelley