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KMID : 1098420030110040289
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Korean Journal of Medicinal Crop Science
2003 Volume.11 No. 4 p.289 ~ p.297
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Agrobacterium Mediated Transformation of Rehmannia glutinosa L. with Glutathione S-Transferase Gene (Gh-5)
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Lim, Jung Dae
Sung, Eun Soo/Yang, Deok Chun/Yun, Song Joong/Chung, Ill Min/Kim, Myong Jo/Yu, Chang Yeon
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Abstract
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Using Agrobacterium-mediated transformation method the auxin-regulated cotton GST (Gh-5) constructs were used to transform Rehmannla glutinosa L. The PCR analysis was conducted to verify transgenicity. Based on the PCR analysis, there was verified that the 988 bp DNA band had showed in transgenic plant genomes in PCR analysis using Gh5-1 and Gh5-2 primers. The effects of cocultivation with Agrobacterium tumefaciens, regeneration and selection conditions on the transformation efficiency of Chinese foxglove (Rehmannia glutinosa L.) were investigated. Factors such as cocultivation period, use of acetosyringone, postcultivation in darkness, and different kanamycin concentrations for selection were assessed. In vitro regeneration, the number of leaves, shoot lengths and numbers on MS medium were superior to on B5 and WPM medium, and the shoot formation rate was highest level of 95% in cultured base part containing leaf stalk. Addition of acetosyringone at concentration of 200 μM to cocultivation medium and 3-day of cocultivation improved transformation frequencies. Exposure of explants to darkness for 4 weeks on selection medium resulted m further increased the regeneration frequency of transgenic shoots. In PCR analysis, the amplified fragments of Gh5 gene were detected (988 bp), and GST-expressing transgenic R, glutinosa L, plants had approximately three-fold higher activity in leaf extracts compared with control plant.
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