Agrobacterium-mediated genetic transformation of the desiccation tolerant resurrection plant Ramonda myconi (L.) Rchb.

In this paper we describe the first procedure for Agrobacterium tumefaciens-mediated genetic transformation of the desiccation tolerant plant Ramonda myconi (L.) Rchb. Previously, we reported the establishment of a reliable and effective tissue culture system based on the integrated optimisation of antioxidant and growth regulator composition and the stabilisation of the pH of the culture media by means of a potassium phosphate buffer. This efficient plant regeneration via callus phase provided a basis for the optimisation of the genetic transformation in R. myconi. For gene delivery, both a standard (method A) and a modified protocol (method B) have been applied. Since the latter has previously resulted in successful transformation of another resurrection plant, Craterostigma plantagineum, an identical protocol was utilized in transformation of R. myconi, as this method may prove general for dicotyledonous resurrection plants. On this basis, physical and biochemical key variables in transformation were evaluated such as mechanical microwounding of plant explants and in vitro preinduction of vir genes. While the physical enhancement of bacterial penetration was proved to be essential for successful genetic transformation of R. myconi, an additional two-fold increase in the transformation frequency was obtained when the above physical and biochemical treatments were applied in combination. All R0 and R1 transgenic plants were fertile, and no morphological abnormalities were observed on the whole-plant level.