An effective and reproducible transformation protocol for the model resurrection plant Craterostigma plantagineum Hochst.

Procedures previously established for plant regeneration and Agrobacterium tumefaciens-mediated genetic transformation of the desiccation-tolerant plant, Craterostigma plantagineum, have been further developed. A highly effective tissue culture system was established 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. The undesirable hyperhydricity of Craterostigma tissue in tissue culture was also circumvented by these modifications, which serve as an alternative to the previously described procedures. The high efficiency of plant regeneration from the callus phase provided the basis for optimising genetic transformation in Craterostigma. For gene delivery, both a standard (method A) and a modified protocol (method B), the latter having previously resulted in successful Agrobacterium-mediated transformation of monocot cereals, were applied. Physical and biochemical key variables in transformation were evaluated, such as gene gun-mediated microwounding of plant explants, infiltration of Agrobacterium suspension cultures into target tissues and the influence of in vitro pre-induction of vir genes. While the physical enhancement of Agrobacterium infection (microwounding, infiltration) had no positive effect, the in vitro pre-induction of vir genes (biochemical enhancement) resulted in a twofold increase in the transformation frequency as compared to the conventional protocol (method A).