Team:UESTC-China/Protocol

From 2014.igem.org

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<h1 class="textEditingTitle" style="width:1100px">Agrobacterium-mediated Genetic Transformation to Tobacco </br></h1>
<h1 class="textEditingTitle" style="width:1100px">Agrobacterium-mediated Genetic Transformation to Tobacco </br></h1>
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         <p class="textEditingstyle">Tobacco was transformed essentially by following the leaf disk co-cultivation protocol of Horsch et al <i>(McCormick, Niedermeyer et al. 1986)</i>. Co-cultivation was initiated by dipping leaf disks in an Agrobacterium suspension, blotting them on sterile tissue paper, and incubating them for 2 d on MS medium <i>(Murashige and Skoog 1962 )</i>  containing naphthalene acetic acid (NAA 0.1mg/L), 6-Benzylaminopurine (6-BA,2.0mg/L). Cefotaxime sodium (Cef) was included in the medium (500mg/L) to inhibit Agrobacterium growth. The leaf disks were then transferred onto a medium containing antibiotics for transgenic plant selection(kanamycin, 50 mg/L), and NAA (0.1 mg/L), 6-BA (2.0 mg/L), Cef (500mg/L). And incubate them for 1 month on the medium above. At last, cut off the bud from the callus, put the buds into the mudium containing NAA (0.1mg/L), Cef (500mg/L) and kanamycin (25 mg/L).
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         <p class="textEditingstyle">Tobacco was transformed essentially by following the leaf disk co-cultivation protocol of Horsch et al <i>(McCormick, Niedermeyer et al. 1986)</i>. Co-cultivation was initiated by dipping leaf disks in an Agrobacterium suspension, blotting them on sterile tissue paper, and incubating them for 2 d on MS medium <i>(Murashige and Skoog 1962)</i>  containing naphthalene acetic acid (NAA 0.1mg/L), 6-Benzylaminopurine (6-BA,2.0mg/L). Cefotaxime sodium (Cef) was included in the medium (500mg/L) to inhibit Agrobacterium growth. The leaf disks were then transferred onto a medium containing antibiotics for transgenic plant selection(kanamycin, 50 mg/L), and NAA (0.1 mg/L), 6-BA (2.0 mg/L), Cef (500mg/L). And incubate them for 1 month on the medium above. At last, cut off the bud from the callus, put the buds into the mudium containing NAA (0.1mg/L), Cef (500mg/L) and kanamycin (25 mg/L).
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             <td class="lastmid">Qualitative detection</td>
             <td class="lastmid">Qualitative detection</td>
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             <td class="lastmid">Have 6 positive seedlings from every transgenic line (about 8 leaves age) , and 6 wild-type seedings with the same growth equally distributed into 3 650ml culture bottles. Treat with 10µl 37% formaldehyde for a week. </td>
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             <td class="lastmid">Have 6 positive seedlings from every transgenic line (about 8 leaves age) , and 6 wild-type seedings with the same growth equally distributed into three 650ml culture bottles. Treat with 10µl 37% formaldehyde for a week. </td>
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Revision as of 11:51, 17 October 2014

UESTC-China