Agrobacterium-Mediated Genetic Transformation and Somatic Embryogenesis from Leaf Callus of Hevea brasiliensis: Effect of Silver Nitrate

Abstract

Somatic embryogenesis and plant regeneration systems developed in Hevea brasiliensis are being used in Hevea crop improvement through genetic approaches. In the present study, the effect of silver nitrate in improving the efficiency of the somatic embryogenesis pathway from leaf explants as well as Agrobacterium-mediated genetic transformation was studied. Experiments were done to find the effect of silver nitrate in callus proliferation and embryogenic callus initiation. Silver nitrate (0–30.0 mg/l) was added in the medium used for second and third subcultures for callus proliferation and in the embryo induction medium. Experiments were also carried out to find the effect of silver nitrate in controlling bacterial overgrowth and improve callus texture when freshly proliferated callus induced from leaf explants in the above medium was used as target tissue for Agrobacterium infection. Agrobacterium tumefaciens strain EHA 101 carrying a gene-encoding isopentenyltransferase (ipt) from the Ti plasmid of Agrobacterium tumefaciens was used for the study. Silver nitrate (0–30 mg/l) was added to the Agrobacterium infection as well as the cocultivation and selection medium. Addition of 20.0 mg/l silver nitrate in the callus proliferation medium used for the second and third subcultures and 10 mg/l in the embryo induction medium helped in the texture improvement of the callus which in turn increased the efficiency of the system by aiding embryogenic callus initiation. It was also observed that inclusion of 10.0 mg/l silver nitrate in the infection and cocultivation medium and 20 mg/l in the selection medium significantly controlled bacterial overgrowth, reduced tissue damage and improved the texture of callus in newly emerged putatively transgenic cell lines. Proliferation of the transgenic callus and somatic embryogenesis were also achieved. From the results, it has become evident that silver nitrate could improve somatic embryogenesis as well as Agrobacterium-mediated genetic transformation when included in the respective media used in the plant regeneration system developed from leaf explants.