Optimization of the conditions for Casuarina cunninghamiana Miq. genetic transformation mediated by Agrobacterium tumefaciens

Abstract

Using epicotyl fragments of the actinorhizal tree Casuarinacunninghamiana and the disarmed strain of Agrobacterium tumefaciens C58C1 (pGV2260) containing the pBIN19-35S-GUSINT binary vector, a method for the genetic transformation of C. cunninghamiana was established. Transformed cells were initially selected for 2 weeks on nutrient medium supplemented with kanamycin 20 mg L−1 during callus induction, and then subcultured with 50 mg L−1 until adventitious bud and shoot differentiation. Different factors involved in the early stages of the T-DNA transfer process were studied. Agrobacterium-mediated DNA delivery was most successful when epicotyl fragments excised from 45-day-old seedlings were co-cultivated with an exponentially growing culture of A. tumefaciens at an OD600nm of 0.3, for 5 days in the presence of 50 μM of acetosyringone. Kanamycin resistant calli were observed on 88.89 % of the explants and transgenic rooted C. cunninghamiana plants were obtained in 6 months. Evidence of genetic transformation was demonstrated by s-glucuronidase histochemical assays and polymerase chain reaction analyses. The possibility to obtain transgenic nitrogen-fixing nodules after inoculation by the soil actinomycete Frankia was established.