Optimization of Agrobacterium-mediated transformation procedure for grapevine ‘Kyoho’ with carrot antifreeze protein gene

• Published in: Journal of plant biotechnology Vol. 44; no. 4; pp. 388 - 393
• Main Authors: Shin, Hye Young, Kim, Gi Hoon, Kang, Sang Jae, Han, Jeung-Sul, Choi, Cheol
• Format: Journal Article
• Language: Korean
• Published: Korean Society for Plant Biotechnology 31.12.2017; (사)한국식물생명공학회
• Subjects: Genotype; Agrobacterium overgrowth; Antibiotics concentration; Cold-tolerance; Vitis vinifera
• ISSN: 1229-2818; 2384-1397

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 non-transgenic 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.