An optimized Agrobacterium-mediated transformation procedure for Phaseolus acutifolius A.!Gray

To improve Agrobacterium tumefaciens-mediated transformation of Phaseolus acutifolius, we examined the effect of different factors on T-DNA transfer by measuring transient expression levels of an intron-containing beta-glucuronidase gene. Improved transformation frequencies were obtained with an A. tumefaciens strain carrying nopaline-type virulence genes and when calli were infected with Agrobacterium cells in the early-log growth phase. Optimized co-cultivation was performed at 22degreesC under a 16/8-h (day/night) photoperiod in an acidic medium (pH 5.5) in the presence of 200 muM acetosyringone. By combining the best treatments, an efficient and reproducible transformation procedure was established for the R acutifolius genotype N1576. Southern and immunoblot analyses confirmed the stable integration and expression of the transgenes in the primary transgenic plants and their progeny.