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24 September 2008 Efficient Agrobacterium-mediated genetic transformation of oilseed mustard [Brassica juncea (L.) Czern.] using leaf piece explants
Indrajit Dutta, Prasenjit Saha, Sampa Das
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Leaf piece explants of five Brassica juncea (L.) Czern. cultivars were transformed with an Agrobacterium tumefaciens strain EHA105 harboring the plasmid pCAM-BIA1301, which contains the β-glucuronidase (uidA) and hygromycin phosphotransferase (hpt) genes under the control of cauliflower mosaic virus 35S (CaMV35S) promoter. Transgenic plants were regenerated on Murashige and Skoog (MS) medium fortified with 8.87 μM 6-benzylaminopurine, 0.22 μM 2,4-dichlorophenoxyacetic acid, and 20 μM silver nitrate in the presence of 30 mg/l hygromycin. When co-culture took place in the presence of 100 μM acetosyringone, the efficiency of stable transformation was found to be approximately 19% in the T0 generation, with the transgenic plants and their progeny showing constitutive GUS expression in different plant organs. Southern blot hybridization of uidA and hpt genes confirmed transgene integration within the genome of transformed plants of each cultivar. Inheritance of hpt gene for single copy T-DNA inserts showed a 3:1 pattern of Mendelian segregation in progeny plants through germination of T1 seeds on MS medium containing 30 mg/l hygromycin. The protocol described here reports superior transformation efficiency over previously published protocols and should contribute to enhanced biotechnology applications in B. juncea.

Indrajit Dutta, Prasenjit Saha, and Sampa Das "Efficient Agrobacterium-mediated genetic transformation of oilseed mustard [Brassica juncea (L.) Czern.] using leaf piece explants," In Vitro Cellular and Developmental Biology - Plant 44(5), 401-411, (24 September 2008).
Received: 6 March 2008; Accepted: 20 August 2008; Published: 24 September 2008

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Agrobacterium-mediated transformation
Brassica juncea
Leaf explants
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