biologia plantarum

International journal on Plant Life established by Bohumil Němec in 1959

Biologia plantarum 49:175-180, 2005 | DOI: 10.1007/s10535-005-5180-2

Efficient regeneration of Brassica napus L. hypocotyls and genetic transformation by Agrobacterium tumefaciens

P. Jonoubi1,2, A. Mousavi1,*, A. Majd2, A.H. Salmanian1, M. Jalali Javaran3, J. Daneshian4
1 National Research Center for Genetic Engineering and Biotechnology, Tehran, Iran
2 Teacher Training University, Tehran, Iran
3 Faculty of Agriculture, Tarbiat Modarres University, Tehran, Iran
4 Oilseeds Research Department, Seed and Plant Improvement Institute, Karaj, Iran

An efficient system for shoot regeneration and Agrobacterium-mediated gene transfer into Brassica napus was developed through the modification of the culture conditions. Different concentrations of benzyladenine (1.5, 3.0 and 4.5 mg dm-3) and thidiazuron (0.0, 0.15 and 0.30 mg dm-3) were evaluated for shoot regeneration of 7, 14 and 21-d-old hypocotyl explants. Maximum shoot regeneration frequency was obtained in 21-d-old explants using 4.5 mg dm-3 benzyladenine and 0.3 mg dm-3 thidiazuron. Under above culture condition, the highest percentage of shoot regeneration frequency was 200 %. Agrobacterium-infected explants grown on the selection medium gave rise to transgenic shoots at a frequency of 11.8 %. Transformed shoots rooted when cultured on a medium supplemented with 2 mg dm-3 of indolebutyric acid and 10 mg dm-3 kanamycin. The rooted plantlets were successfully established in the soil and developed fertile flowers and viable seeds. Evidences for transformation were confirmed by GUS assay and PCR analysis.

Keywords: age of explant; benzyladenine; gene transfer; thidiazuron; tissue culture
Subjects: Agrobacterium tumefaciens; auxins; Brassica napus; cytokinins; gene transfer; β-glucuronidase; hypocotyl; in vitro culture, regeneration, organogenesis, morphogenesis; nutrient medium B5 (Gamborg); nutrient medium, induction medium; nutrient medium MS (Murashige and Skoog); polymerase chain reaction (PCR); thidiazuron (TDZ)

Received: January 14, 2004; Accepted: October 18, 2004; Published: June 1, 2005  Show citation

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Jonoubi, P., Mousavi, A., Majd, A., Salmanian, A.H., Jalali Javaran, M., & Daneshian, J. (2005). Efficient regeneration of Brassica napus L. hypocotyls and genetic transformation by Agrobacterium tumefaciens. Biologia plantarum, 49(2), 175-180. doi: 10.1007/s10535-005-5180-2
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