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An efficient Agrobacterium tumefaciens -mediated genetic transformation of bitter melon (Momordica charantia L.)
| Content Provider | Semantic Scholar |
|---|---|
| Author | Thiruvengadam, Muthu Praveen, N. |
| Copyright Year | 2012 |
| Abstract | A simple and efficient protocol for Agrobacterium tumefaciens-mediated genetic transformation of bitter melon (Momordica charantia L.) has been developed. Pre-cultured leaf explants were transformed by co-cultivation with Agrobacterium tumefaciens strain LBA4404 harbouring a binary vector pBAL2 carrying the reporter gene beta-glucuronidase intron (gus) and the marker gene neomycin phosphotransferase (nptII). After co-cultivation, explants were transferred in to a callus induction medium containing 7.7 muM naphthaleneacetic acid (NAA) with 2.2 muM thidiazuron (TDZ), 100 mg L-1 kanamycin and 300 mg L-1 carbenicillin. Regeneration of adventitious shoots from callus was achieved on MS medium containing 5.5 muM TDZ, 2.2 muM NAA, 3.3 muM silver nitrate (AgNO3), 100 mg L-1 kanamycin and 300 mg L-1 carbenicillin. Transgenic shoots were excised from callus and elongated in MS medium fortified with 3.5 muM, gibberellic acid (GA3), 100 mg L-1 kanamycin and 300 mg L-1 carbenicillin. The transgenic elongated shoots were rooted in MS medium supplemented with 4.0 muM indole 3-butyric acid (IBA) and 100 mg L-1 kanamycin. The putative transgenic plants were acclimatized in the greenhouse and seeds were subsequently collected from mature fruits. Further, the presence of acetosyringone (300 muM) in the co-cultivation medium, infection of explants for 30 min and 3 days of co-cultivation proved to be critical factors for greatly improving the transformation efficiency. Histochemical GUS assay and polymerase chain reaction of field-established transgenic plants and their offspring confirmed the presence of the gus and nptII genes, respectively. Integration of T-DNA into the genome of putative transgenics was further confirmed by Southern blot analysis. The nptII gene expression in transgenic plants was confirmed by RT-PCR. A transformation efficiency of 7% was obtained. |
| Starting Page | 1094 |
| Ending Page | 1100 |
| Page Count | 7 |
| File Format | PDF HTM / HTML |
| Volume Number | 6 |
| Alternate Webpage(s) | http://www.cropj.com/tiruvengadam_6_6_2012_1094_1100.pdf |
| Language | English |
| Access Restriction | Open |
| Content Type | Text |
| Resource Type | Article |
