Genetic Transformation of Embryogenic Cultures and Recovery of Transgenic Plants in Vitis vinifera, Vitis rotundifolia and Vitis Hybrids

Factors influencing Agrobacterium-mediated transformation of 15 Vitis rotundifolia, Vitis rupestris, Vitis vinifera and Vitis hybrids were explored. A green fluorescent protein/neomycin phosphotransferase II (GFP/NPT II) fusion gene was used to measure transient and stable transgene expression levels. Embryogenic cell cultures at different developmental stages were co-cultivated with Agrobacterium to determine the best stage for transformation. The effect of antioxidant treatment on recovery of transgenic embryo and plant lines was also studied. Transient and stable GFP expression varied widely among the species. For example, 1-81% of V. vinifera somatic embryos exhibited transient GFP expression, depending on the cultivar. Similarly, 060% of co-cultivated embryos produced stable transgenic lines. Proembryonal masses and cotyledonary stage somatic embryos were best for transformation of V. rotundifolia, V. rupestris and certain Vitis hybrids, whereas only cotyledonary stage somatic embryos were best for transformation of V. vinifera. Agrobacterium-induced browning/ necrosis was reduced by adding 1 g L dithiothreitol (DTT) antioxidant to the post cocultivation wash medium. Transgenic plants have been recovered from V. rupestris ‘St. George’, V. rotundifolia ‘Alachua’ and ‘Carlos’, V. vinifera ‘Cabernet Franc’, ‘Chardonnay’, ‘Merlot’, ‘Pinot Noir’, ‘Sauvignon Blanc’, ‘Shiraz’, ‘Superior Seedless’, ‘Thompson Seedless’ and ‘Zinfandel’, and Vitis hybrids ‘Conquistador’, ‘Seyval Blanc’ and ‘Freedom’. Research to optimize transformation conditions of additional Vitis species and hybrids is ongoing. INTRODUCTION Genetic improvement of Vitis has been carried out by hybridization and clonal selection (Olmo, 1942; Rantz, 1995). However, breeding in Vitis is limited by extreme heterozygosity and inbreeding depression (Winkler et al., 1974), which makes back crossing and recurrent selection difficult. F1 hybrids are of intermediate quality compared to parental lines and the long juvenile period of vines makes screening of new selections tedious and time consuming (Alleweldt and Possingham, 1988). As an alternative, genetic transformation might allow modification of popular grape cultivars by adding single traits without changing desirable characteristics (Gray et al., 2005). Transformation of embryogenic cultures has become routine only for a few cultivars and rootstocks, mostly using embryogenic cell cultures and Agrobacterium tumefaciens to effect gene transfer (Gray et al., 2005). Roadblocks to genetic transformation include poor embryogenic potential and low transformation efficiency due to severe browning/necrosis of cultures following co-cultivation with Agrobacterium (Perl et al., 1996). Initiation and maintenance of embryogenic cultures for a range of Vitis species and cultivars have been well documented (Gray and Mortensen, 1987; Gray, 1989, 1992, 1995). In the present study, the effects of developmental stage of embryogenic tissue and antioxidant treatment on Agrobacterium-mediated transformation were compared for two Vitis rotundifolia cultivars, Vitis rupestris ‘St. George’, nine Vitis vinifera cultivars and three Vitis hybrids. Proc. IS on Biotechnol. Temp. Fruit Crops & Trop. Species Eds. R.E. Litz and R. Scorza Acta Hort. 738, ISHS 2007 744 MATERIALS AND METHODS Induction of Embryogenic Cultures Embryogenic cultures of V. rupestris ‘St. George’, V. vinifera cultivars ‘Cabernet Franc’, ‘Chardonnay’, ‘Merlot’, ‘Pinot Noir’, ‘Sauvignon Blanc’, ‘Shiraz’, and ‘Zinfandel’, and Vitis hybrids ‘Conquistador’ and ‘Freedom’ were induced from anthers and ovaries as previously described (Gray and Mortensen, 1987). Embryogenic cultures of V. rotundifolia ‘Alachua’ and ‘Carlos’ (Robacher, 1993), V. vinifera ‘Thompson Seedless’ and ‘Superior Seedless’, and Vitis hybrid ‘Seyval Blanc’ (Gray, 1995) were initiated from in vitro grown leaves as previously described. Cultures were then maintained on growth regulator-free X6 medium (Li et al., 2001) for somatic embryo (SE) development and proliferation. Embryogenic liquid suspensions (Jayasankar et al., 1999) were produced from nine of the cultivars (see Table 3) in order to increase culture mass. Liquid suspension culture-derived PEMs and SEs were transferred to X6 medium and maintained as described above before use in transformation experiments. Transformation Vector A reporter-marker fusion gene consisting of an enhanced green fluorescent protein (GFP) and the neomycin phosphotransferase (NPTII) gene, driven by a double Cassava Vein Mosaic Virus (CsVMV) promoter was introduced in the T-DNA region of a pBIN119 derived binary vector (Li et al., 2001). This construct was introduced into Agrobacterium tumefaciens strain EHA 105 using the freeze-thaw method (Burrow et al., 1990). Preparation of Agrobacterium Cultures for Transformation Agrobacterium strain EHA 105 was cultured overnight at 26°C on an orbital shaker at 185 rpm in liquid MG/L medium (Garfinkle and Nester, 1980) containing 100 mg L kanamycin and 20 mg L rifampicin. Prior to transformation, the bacterial culture was pelleted by centrifugation at 6000 rpm for 8 min, resuspended in 25 ml liquid X2 medium (the same composition as X6 medium with 20 g L sucrose) and adjusted to an OD value of 0.6-0.8. The resuspended Agrobacterium was further cultured for an additional 3 h before use in transformation experiments. Transformation of Embryogenic Cultures PEMs and SEs were used for Agrobacterium co-cultivation as described earlier (Li et al., 2005). Embryogenic cultures were incubated in the bacterial solution for a period of ten minutes. Excess bacterial solution then was removed and the cultures were transferred to DM medium. This medium consisted of DKW basal salts (Driver and Kuniyuki, 1984), supplemented with 0.3 g L KNO3, 1.0 g L myo-inositol, 2.0 mg L each of thiamineHCl and glycine, 1.0 mg L nicotinic acid, 30.0 g L sucrose, 7.0 g L TC agar, 5.0 μM BA, 2.5 μM each of NOA and 2,4-D, at pH 5.7. Cultures were co-cultivated in the dark for two days and then washed for 3 days in liquid DMcck15 medium (DM medium containing 200 mg L each of carbenicillin and cefotaxime, and 15 mg L kanamycin). Cultures were then transferred to solid DMcck medium for callus formation and PEM proliferation. For selection of transgenic cells, V. rupestris, V. vinifera and Vitis hybrid cultures were maintained on DM medium for a period of one month, whereas V. rotundifolia cultures were maintained for a period of 3 months. Cultures were then transferred to X6cck70 medium for development of somatic embryos. Antioxidant Treatment The effect of the antioxidant dithiothreitol (DTT) on culture browning following co-cultivation with Agrobacterium was studied. SEs of different species and cultivars were co-cultivated with Agrobacterium for two days and then transferred to liquid DMcck15 medium supplemented with 0 or 1.0 g L DTT. SEs were then cultured for