Re-evaluation of the effects of growth regulators on callus induction and shoot regeneration in Agrobacterium-mediated transformation of lettuce

Lettuce (Lactuca sativa) transformation varies by genotype. Various culture parameters have been studied in order to improve the transformation efficiency of lettuce cultivars. However, no improved transformation procedure for recalcitrant lettuce cultivars has yet been established. Here, we demonstrate the effects of varying concentrations and distinct combinations of growth regulators on recalcitrant lettuce transformation efficiency. More precisely, we assessed differences in the effects of several growth regulator combinations, including N-6(2-isopentenyl)-adenine (2ip), on induction of callus and regeneration of shoots after co-cultivation with Agrobacterium. When two commercial recalcitrant cultivars, Red Romaine and Bibb, were cultured on a medium with 2ip 1 mg l(-1), IAA 0.1 mg l(-1), and subsequently transferred to a second medium with BA 0.4 mg l(-1), NAA 0.05 mg l(-1) for selection and shoot regeneration, transformation efficiencies reached 8 and 9%, respectively. Stable integration and transmission of the transgene in T1 generation plants were confirmed by molecular analysis. This procedure represents a simple, efficient, and general means of transforming various lettuce cultivars, including recalcitrant commercial cultivars.