Volume 18, Issue 57 (No.1&2-صفحات انگلیسی از 74 تا 82 2004)
2004, 18(57): 167-180 |
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Thidiazuron- induced Shoot Regeneration and Gene Transformation by Agrobacterium in Brassica napus L.. Journal title 2004; 18 (57) :167-180
URL: http://jsci.khu.ac.ir/article-1-1142-en.html
URL: http://jsci.khu.ac.ir/article-1-1142-en.html
Abstract: (4201 Views)
An in vitro method was developed for high frequency shoot regeneration and Agrob-acterium-mediated transformation of Brassica napus hypocotyl explants. The different concentrations of benzyladenine (BA), 1.5, 3.0 and 4.5 mg l-1, and thidiazuron (TDZ), 0, 0.15 and 0.30 mg l-1 were evaluated for shoot regeneration using hypocotyl explants 7, 14 and 21- day- old. Treatments were arranged in a randomized complete block design with three replications. Significant differences were found in the interactions of BA and TDZ concentrations along with the ages of the explants. Maximum shoots regeneration was obtained using 4.5 mg l-1 BA and 0.30 mg l-1 TDZ. Shoot regeneration was highly affected by the age of the explants. We found that 21-day-old explants were more likely to undergo shoot development than the others. Under these culture conditions, the highest percentage of shoot regeneration was 174.0% for hypocotyl explants. Regenera-ted shoots rooted when cultured on a root induction medium supplemented with 2 mg l-1 of indolebutyric acid(IBA).The rooted plantlets were successfully established in the soil and developed normal fertile flowers and viable seeds. In light of its efficiency, this hypocotyl regeneration method could be a suitable tool for genetic transformation. A. Fumefaciens strain LBA4404 carring disarmed binary vector pBI121 was used for plant transformation. After cocultivation for 48h, the hypocotyl explants were placed on shoot induction media containing 10 mgl-1 kanamycine. The green shoots were transferred to root induction medium. Rooted plantlets were transferred to green house. Six kanamycine resistance plants from 75 hypocotyl and grown for seed formation. GUS assay and PCR confirmed the introduction of the T-DNA into the transgenic rapeseed genome. The gene transfere system described in this method has a great potential for genetic improvement of Brassica napus by introducing genes responsible for agronomically important traits such as herbicide tolerance.
Published: 2004/01/15
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