Abstract
Using epicotyl fragments of the actinorhizal tree Casuarina cunninghamiana and the disarmed strain ofAgrobacterium 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 shootdifferentiation. Different factors involved in the earlystages of the T-DNA transfer process were studied. Agrobacterium-mediated DNA delivery was most successfulwhen epicotyl fragments excised from 45-day-old seedlings were co-cultivated with an exponentially growingculture of A. tumefaciens at an OD600nm of 0.3, for 5 daysin the presence of 50 lM of acetosyringone. Kanamycinresistant calli were observed on 88.89 % of the explantsand transgenic rooted C. cunninghamiana plants wereobtained in 6 months. Evidence of genetic transformationwas demonstrated by ß-glucuronidase histochemical assaysand polymerase chain reaction analyses. The possibility toobtain transgenic nitrogen-fixing nodules after inoculationby the soil actinomycete Frankia was established.