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
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
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
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
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
Differential Necrogenesis / Browning of Embryogenic Cultures Following Co-cultivation With Agrobacterium Orange Muscat Sauvignon Blanc Thompson Seedless Riparia Gloire
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)
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
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
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
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 0.249 22 10 7 0.5 g/l DTT wash 0.065 23 14 9 1.0 g/l DTT wash 0.054 22 14 12 1.0 g/l DTT co-cultivation & wash 0.059 17 7 1.3 Preculture 0 DTT 0.058 25 17 10 0.5 g/l DTT wash 0.065 23 17 8 1.0 g/l DTT wash 0.042 22 19 11 1.0 g/l DTT co-cultivation & wash 0.068 18 14 4
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
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
Genetic Transformation of Vitis. rotundifolia Transient expression in PEM Transient expression in SE Callus formation PEM formation
Vitis rotundifolia (continued) Stably transformed PEM PEM proliferation Somatic Embryos Stably transformed somatic embryos
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
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 81.11 1400 795 Merlot 68.80 101 39 Sauvignon Blanc 24.66 44 4 Pinot Noir 10.00 19 5 Shiraz 72.20 7 5 Chardonnay 44.40 8 4 Cabernet Franc 72.20 1 1 Superior Seedless 76.60 28 1 Zinfandel 52.20 5 1 Vitis rupestris 'St. George' 28.13 74 3 Vitis rotundifolia 'Alachua' 58.00 31 4 Vitis hybrids Conquistador 68.80 20 5 Freedom 21.78 7 1 Seyval Blanc 67.80 44 1
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
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
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