Efficient Agrobacterium tumefaciens-mediated transformation of embryogenic calli and regeneration of Hevea brasiliensis Mull Arg. plants

An efficient procedure for producing transgenic Hevea brasiliensis callus and plant lines from clone PB 260 was established with Agrobacterium tumefaciens using strain EHA105 harbouring the vector pCAMBIA2301. Transformation capacity and competence of the embryogenic calli were improved after two cycles of cryopreservation. When the cocultivation temperature was reduced from 27 to 20 degrees C, the duration of this phase could be increased up to 7 days, promoting an increase in GUS activity. These transformation conditions led to the isolation of 24 callus lines resistant to paromomycin, which is used as a selection agent. Nineteen of these lines revealed the existence of one to four copies of T-DNA by Southern-blot analysis. Nine of them were transferred for regeneration by somatic embryogenesis. Three hundred seventy-four transgenic plants have thus been generated from six independent lines bearing 1, 2 or 3 copies of T-DNA. The efficiency and reproducibility of this method means that functional characterization of genes involved in natural rubber production can be envisaged.