Mehrotra, M. and Sanyal, I. and Amla , D.V. (2011) High-efficiency Agrobacterium-mediated transformation of chickpea (Cicer arietinum L.) and regeneration of insect-resistant transgenic plants. Plant Cell Reports, 30 (9). pp. 1603-1616.
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Abstract
To develop an efficient genetic transformation system of chickpea (Cicer arietinum L.), callus derived from mature embryonic axes of variety P-362 was transformed with Agrobacterium tumefaciens strain LBA4404 harboring p35SGUS-INT plasmid containing the uidA gene encoding β-glucuronidase (GUS) and the nptII gene for kanamycin selection. Various factors affecting transformation efficiency were optimized; as Agrobacterium suspension at OD600 0.3 with 48 h of co-cultivation period at 20°C was found optimal for transforming 10-day-old MEA-derived callus. Inclusion of 200 µM acetosyringone, sonication for 4 s with vacuum infiltration for 6 min improved the number of GUS foci per responding explant from 1.0 to 38.6, as determined by histochemical GUS assay. For introducing the insect-resistant trait into chickpea, binary vector pRD400-cry1Ac was also transformed under optimized conditions and 18 T0 transgenic plants were generated, representing 3.6% transformation frequency. T0 transgenic plants reflected Mendelian inheritance pattern of transgene segregation in T1 progeny. PCR, RT-PCR, and Southern hybridization analysis of T0 and T1 transgenic plants confirmed stable integration of transgenes into the chickpea genome. The expression level of Bt-Cry protein in T0 and T1 transgenic chickpea plants was achieved maximum up to 116 ng mg-1 of soluble protein, which efficiently causes 100% mortality to second instar larvae of Helicoverpa armigera as analyzed by an insect mortality bioassay. Our results demonstrate an efficient and rapid transformation system of chickpea for producing non-chimeric transgenic plants with high frequency. These findings will certainly accelerate the development of chickpea plants with novel traits.
| Item Type: | Article |
|---|---|
| Author Affiliation: | Plant Transgenic Lab, National Botanical Research Institute, Rana Pratap Marg, Lucknow 226 001, India |
| Subjects: | Plant Protection Crop Improvement |
| Divisions: | Chickpea |
| Depositing User: | Mr B Krishnamurthy |
| Date Deposited: | 03 Feb 2013 11:26 |
| Last Modified: | 03 Feb 2013 11:26 |
| Official URL: | http://dx.doi.org/10.1007/s00299-011-1071-5 |
| URI: | http://eprints.icrisat.ac.in/id/eprint/9414 |
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