Agrobacterium-mediated transformation of Eruca sativa |
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Authors: | Susan M H Slater W A Keller G Scoles |
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Institution: | (1) Department of Plant Sciences, College of Agriculture and Bioresources, University of Saskatchewan, 51 Campus Drive, Room 4D36 Agriculture Building, Saskatoon, SK, S7N 5A8, Canada;(2) Plant Biotechnology Institute, National Research Council of Canada, Saskatoon, SK, S7N 0W9, Canada |
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Abstract: | An Agrobacterium tumefaciens—mediated transformation system was developed for Eruca sativa (eruca). Hypocotyl explants were co-cultivated with bacterial cells carrying a plasmid harboring a uidA:nptII fusion gene along a phosphinothricin acetyl transferase (PAT) gene cassette, for a period of 2 days. These were grown on a high cytokinin/auxin medium containing 5.0 mg l?1 6-benzyladenine (BA), 1.0 mg l?1 indole-3-acetic acid (IAA), and 0.1 mg l?1 α-naphthaleneacetic acid (NAA). Explants were then transferred to a lower cytokinin/auxin medium containing 2.0 mg l?1 BA and 0.1 mg l?1 NAA along with 5.0 mg l?1 silver nitrate and 300 mg l?1 Timentin®. Upon transfer to a selection medium containing either 20 mg l?1 kanamycin or 2 mg l?1 L-phosphinothricin (L-ppt), shoot regenerants were observed. Expression of the transgenes in putative transformants was confirmed using a histochemical GUS assay. Presence of the PAT transgene in GUS-positive T0 plants was confirmed by Southern blot analysis. Moreover, spot tests of T1 seedlings were conducted using the L-ppt herbicide. A transformation frequency of 1.1% was obtained with more than 60% of transgenic lines containing single copies of the transgenes. |
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