Optimization of Agrobacterium tumefaciens-mediated Transformation in Oxalis corniculata (L.)

Abstract

Background: Optimization of co-cultivation parameters during Agrobacterium-mediated transformation to Oxalis corniculata evaluated were bacterial density, infection period, acetosyringone (AS) concentration and co-cultivation temperature. Five fold transformation efficiency is achieved in transient gus gene expression after optimizing various parameters. The optimized conditions were Agrobacterium tumefaciens growth phase of A600nm 0.17, infection period of 30 min, addition of acetosyringone (AS) in co-cultivation medium (400 μM) and cocultivation temperature of 22°C. Methods: Oxalis corniculata (L.) were used for transformation. Bacterial culture was added to 50 ml of liquid YEP medium with kanamycin and rifampicin and grown until reaching the growth phase (A600nm). Bacterial density ranged from A600nm 0.17, 0.56,0.80 and 1.34 OD were used in the present study. The co-cultivation medium made of solid MS medium consisted of BAP 2 mg L-1 and NAA 0.5 mg L-1 and various concentrations of AS at 0, 50, 100, 200, 400,600 and 800 μM. Histochemical analysis of gene expression was carried out. Result: Higher bacterial density resulted in more transformation efficiency, but also higher necrosis in the explants. Dilution of bacterial suspension reduced necrosis in explants and resulted in higher transformation. The transformation efficiency is 64% when the infection process was carried out with acetosyringone in co-cultivation medium (400 μM). Our studies proved that among the optimized conditions, cocultivation temperature and acetosyringone were the critical parameters during Agrobacterium mediated transformation.