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KMID : 0903620000410050450
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Journal of the Korean Society for Horticultural Science
2000 Volume.41 No. 5 p.450 ~ p.454
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Embryogenic Callus Induction and Agrobacterium - mediated Transformation in Bentgrass (Agrostis spp.)
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박미혜/Park, Mi Hyea
신정섭/김두환/조성현/정영수/Chai, Ming Liang/Senthil, Kalaiselvi/Shin, Jeong Sheop/Kim, Doo Hwan/Cho, Sung Hyun/Chung, Young Soo
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Abstract
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Embryogenic calli were induced from the seeds of creeping bentgrass (Agrostis palustris Huds.) cv. Regent, Cato, Mariner and colonial bentgrass (Agrostis tennis sibth. F₁. Oxcen.) cv. Tiger on MS medium supplemented with 2,4-D (2 ㎎·L^(-1) ). The effects of proline, glutamine, aspartic acid and the analogue of proline-thioproline on callus induction and formation of embryogenic callus were estimated systematically. For improving transformation efficiency, the embryogenic calli were preconditioned on fresh medium for 4-7 days and then cocultivated with Agrobacterium tumefaciens LBA 4404, which contains pCAMBIA 1300-SmGFP binary vector, harboring hygromycin phosphotransferase (HPT) and green fluorescent protein (GFP) genes. After 3 days of co-cultivation, the calli were washed thoroughly and transferred to MS medium containing 2 ㎎·L^(-1) of 2,4-D, 1.14 g·L^(-1) proline, 50 ㎎·L^(-1) hygromycin and 200 ㎎·L^(-1) of cefotaxime. After 2 months of selection, the actively growing calli of ´Regent´ were transferred to MS medium with 25 ㎎·L^(-1) hygromycin and 200 ㎎·L^(-1) cefotaxycine for regeneration. Two weeks later, some small plantlets formed. GFP expression was detected both in callus, shoot and roots, and PCR analysis revealed the presence of HPT fragment of the expected size (590 bp) in the transformed plants and its absence in control plant. Southern hybridization confirmed all the transformants contain HPT gene (1094 bp). This is the first report on Agrobacterium-mediated transformation of bentgrass.
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