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Optimization of Agrobacterium-mediated transformation procedure for grapevine ‘Kyoho’ with carrot antifreeze protein gene
J Plant Biotechnol 2017;44:388-393
Published online December 31, 2017
© 2017 The Korean Society for Plant Biotechnology.

Hye Young Shin1・Gi Hoon Kim1・Sang Jae Kang2・Jeung-Sul Han1,3,4・Cheol Choi1,4

1Department of Horticultural Science, College of Agriculture & Life Sciences, Kyungpook National University, Daegu 41566, Republic of Korea
2School of Applied Life Science, College of Agriculture & Life Sciences, Kyungpook National University, Daegu 41566, Republic of Korea
3Department of Ecological Environment, College of Ecology & Environmental Science, Kyungpook National University, Sangju 37224, Republic of Korea
4Institute of Agricultural Science & Technology, Kyungpook National University, Daegu 41566, Republic of Korea
Correspondence to: C. Choi (⊠)
Department of Horticultural Science, College of Agriculture & Life Sciences, Kyungpook National University, Daegu 41566, Republic of Korea
e-mail: cc31@knu.ac.kr
Received August 22, 2017; Revised October 14, 2017; Accepted October 20, 2017.
cc This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
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 ClavamoxTM 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.
Keywords : Genotype, Agrobacterium overgrowth, Antibiotics concentration, Vitis vinifera, Cold-tolerance


December 2017, 44 (4)
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