Agrobacterium-Mediated gene transfer using binary vector in lettuce (Lactuca sativa L.)

Lettuce cv. Sol-Let-1 was transformed by disarmed Agrobacterium tumefaciens LBA 4404 strain containing a reporter β-glucuronidase (gus) gene in binary vector (pBI 121) system along with kanamycin resistance gene (npt-II) under the control of CaMV35S and NOS promoters. Optimized high frequency plant regeneration protocol is a prerequisite to carry out genetic transformation studies. It was efficiently standardized using leaf (70.10%) on Murashige and Skoog (MS) medium supplemented with 1.00 mg L⁻¹ BAP (6-Benzyl Amino Purine) + 0.10 mg L⁻¹ NAA (Naphthalene acetic acid) and petiole (70.73%) on MS medium containing 0.25 mg L⁻¹ BAP+0.10 mg L⁻¹ NAA, respectively. Putative transgenic shoots from leaf and petiole explants were regenerated on MS selective medium containing MS + 1.0 mg L⁻¹ BAP and 0.10 mg L⁻¹ NAA+ 25 mg L⁻¹ kanamycin + 400 mg L⁻¹ cefotaxime and MS + 0.25 mg L⁻¹ BAP + 0.10 mg L⁻¹ NAA+ 25 mg L⁻¹ kanamycin + 400 mg L⁻¹ cefotaxime, respectively. MS medium supplemented with 0.50 mg L⁻¹ IBA was found better for root regeneration (68.00%) from in vitro developed shoots. Pre incubation of 48 hrs and co-cultivation of 48 hrs was found optimum as it produced maximum transgenic shoot regeneration from leaf (9.33%) and petiole explants (4.87%) on the selective medium. The putative transformants were randomly taken for the amplification of npt-ll and gus genes with specific primers and out of five random putative transgenic plantlets, three showed amplification of npt-ll and gus thereby indicating the presence/integration of npt-II and gus genes into the genome of transgenic lettuce. The expression of gus gene was analyzed by using biochemical and histochemical techniques of GUS assay. The gus gene was expressed in the PCR positive transgenic plantlets of lettuce. Thus, a protocol for in vitro plant regeneration and genetic transformation in lettuce with npt-ll and gus genes was optimized.