Agrobacterium tumefaciens-mediated transformation of Hevea brasiliensis and production of transgenic callus lines

An efficient genetic transformation procedure has been developed via Agrobacterium tumefaciens on Hevea brasiliensis friable calli, and transgenic callus lines were established and characterised at the molecular level. Embryogenic friable calli precultured a fortnight on a CaC12-free maintenance medium were cocultivated with agrobacteria, which were induced with acetosyringone and resuspended in CaC12-free medium These conditions enhanced dramatically the transient activity of the reporter gene encoding ß-glucuronidase (GUS). Cocultivated friable calli were directly transferred as tissue aggregates of 1-2 mm on a maintenance medium supplemented with a bacterial static agent, the ticarciIIin, in order to favour plant tissue proliferation and to inhibit the Agrobacterium overgrowth. After two phases of proliferation for 3 weeks, GUS-positive cell clusters were observed, indicating that transgenic cells could proliferate. The medium was then supplemented with a selective agent, the paromomycin, at a concentration increasing at each subculture, until complete growth inhibition of non-transformed cells. DNA amplification of the transgenes was performed on the paromomycin-resistant and GUS-positive callus lines. Four transgenic callus lines have thus been established and characterised. Rubber tree friable tissue appeared to be particularly susceptible to various culture stresses. Strengthening of defence against oxidative stress of clone PB 260 by using DNA recombinant technology has been proposed in order to improve tissue culture and exploitation potential of this clone. Moreover, the use of transgenic callus lines can be a useful tool to study rubber tree cell metabolism. Finally, the availability of this transgenic material represents a way for clonal propagation of genetically engineered rubber trees. (Résumé d'auteur)