Transformation of Gossypium arboreum shoot apical meristem by Agrobacterium tumefaciens and regeneration of transgenic plants

Two Gossypium arboreum genotypes LD 327 and LD 694 were screened for plant regeneration from shoot apical meristem on half strength MS medium. The shoot apical meristems were excised from 5-day old in vitro germinated seedlings and co-cultivated for 2 days with Agrobacterium strain GV3101 having plasmid pPZP20010xnosnptll::35SdeOmega containing Cry 1Ac coding region under the control of CaMV 35S promoter double enhancer with an objective to introduce Cry 1Ac gene cassette into G. arboreum genotypes. The transformed shoot apical merlstems were allowed to regenerate and proliferate on half strength MS medium supplemented with carbenicillin 100 mg l⁻¹, cefotaxime 250 mg l⁻¹ for 42 days. The average shoot regeneration recorded after 42 days of co-cultivation was 51.82 and 67.58% for LD 327 and LD 694, respectively. Further, the regenerated shoots were transferred for selection on MS medium supplemented with carbenicillin 100 mg l⁻¹, cefotaxlme 250 mg l⁻¹ and kanamycin 20 mg l⁻¹. After 3 sub-culture cycles (30 + 30 + 20 days) on selection medium survival rate in LD 327 and LD 694 was 9.5 and 29.4% respectively. The shoots that survived selection were transferred to half strength MS medium leading to spontaneous root induction. These putative transformed plantlets were analyzed through polymerase chain reaction for the presence of Cry 1Ac transgene in the genomic DNA isolated from leaf, root, and stem. Amplification of 1845 bp DNA fragment corresponding to Cry 1Ac coding region was observed in all tissues analyzed, thus demonstrating introduction of Cry 1Ac transgene in G. arboreum genotype. This is the first report on transformation of G. arboreum shoort apical meristems genotype by Agrobacterium tumefaciens.