Effect of exogenous calcium on Agrobacterium tumefaciens-mediated gene transfer in Hevea brasiliensis (rubber tree) friable calli

The influence of CaCl2 was investigated on Agrobacterium tumefaciens-mediat ed gene transfer in Hevea brasiliensis friable calli which are usually prol iferated on maintenance medium (MM) containing 9 mM CaCl2.Five A. tumefacie ns strains (C58pMP90, C58pGV2260, AGL1, LBA4404 and EHA 105) and two binary vectors (pGIN and pCAMBIA2301) were tested and the strain EHA105pC2301 was selected to conduct further experiments. The calli were precul tured on MM containing a range of CaCl2 concentrations, then inoculated with Agrobacte rium suspension. Transfer of friable calli from MM containing 9 mM CaCl2 to calcium-free medium significantly enhanced the transient beta-glucuronidas e activity. Interestingly, the use of calcium-free Agrobacterium resuspensi on medium to inoculate friable calli again dramatically increased the trans formation efficiency. Induction of Agrobacterium's virulence with acetosyri ngone remained an important factor to stimulate transformation.