We report an Agrobacterium-mediated transformation procedure optimized for 'Kyoho' that is a major table grapevine cultivar in Korea, and its transgenic plants with antifreeze protein gene of carrot (DcAFP). The full length of DcAFP coding region in accordance with the previous report was isolated from young leaves of carrot and recombined into a plant transformation vector. Ethylene inhibitors such as silver nitrate and aminoethoxyvinylglycin (AVG) supplemented in a co-cultivation medium distinctly increased frequency of shoot regeneration when explants were sub-cultured in a selection medium: particularly ten-fold higher in treatment with 0.1 mg/L AVG than one without ethylene inhibitor. Among various antibiotics and their concentrations, the combination of 150 mg/L cefotaxime plus 150 mg/L Clavamox TM was selected for elimination of Agrobacterium cells in addition to minimization of adverse effect on shoot regeneration, while 50 mg/L kanamycin monosulfate effectively suppressed regeneration of nontransgenic shoots. Applying the elucidated culture condition, we finally obtained a total of 5 transgenic 'Kyoho' plantlets with DcAFP, of which integration with the grapevine genome and transcription was confirmed by nucleic acid analyses.Keywords Genotype, Agrobacterium overgrowth, Antibiotics concentration, Vitis vinifera, Cold-tolerance
IntroductionGrapevine (Vitis spp.) is considered to be one of the major fruit crops in the world based on hectares cultivated and economic value (Torregrosa et al. 2015). Recently, various approaches to improve economically important characteristics in this crop has comprised genetic engineering, by which transgenic grapevines have been obtained in a stream (Iocco et al. 2001;Kim et al. 2013;Li et al. 2006). However, few efforts were involved in developing the event with enhanced cold-tolerance that is a primary characteristics in grapevine cultivation (Jin et al. 2009;Sun et al. 2016). Coldness like other abiotic stresses has adverse effects on the growth and development of grapevine plants (Wang and Nick 2017). A table grapevine 'Kyoho' is generally less cold-tolerant than the American grapevine cultivars derived from an interspecific hybridization (Sun et al. 2016), resulting in being cultivated in a greenhouse or buried during winter season in Korea. The former leads to increase cost of production while the latter make the scions be vulnerable to soil-borne diseases, especially to crown gall. Since the characterization of antifreeze protein (AFP) from Antarctic fishes (DeVries et al. 1970), many other AFPs were isolated from Arctic fishes, insects, and carrot at last (Jang et al. 1999;Meyer et al. 1999;Worrall et al. 1998). Functional studies on the AFP of carrot (DcAFP) further revealed the inhibitory effects of its protein on ice growth and recrystallization, which suggests the potential of this gene for crop improvement. Although genetic transformation technology encourages to express DcAFP for cold-tolerance in grapevines, each grapevine genotype crucially demands...
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