Optimization of the conditions for Casuarina cunninghamiana Miq. genetic transformation mediated by Agrobacterium tumefaciens
Résumé
Using epicotyl fragments of the actinorhizal tree Casuarina cunninghamiana 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 lM 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 ß-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.