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Agrobacterium-mediated transformation of Eruca sativa

Agrobacterium-mediated transformation of Eruca sativa 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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Plant Cell, Tissue and Organ Culture Springer Journals

Agrobacterium-mediated transformation of Eruca sativa

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References (40)

Publisher
Springer Journals
Copyright
Copyright © 2011 by Springer Science+Business Media B.V.
Subject
Life Sciences; Plant Pathology; Plant Physiology; Plant Sciences; Plant Genetics & Genomics
ISSN
0167-6857
eISSN
1573-5044
DOI
10.1007/s11240-010-9915-1
Publisher site
See Article on Publisher Site

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.

Journal

Plant Cell, Tissue and Organ Culture – Springer Journals

Published: Jan 5, 2011

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