Direct shoot organogenesis and Agrobacterium tumefaciens mediated transformation of Solanum trilobatum L.

Authors : Jayabalan SHILPHA, Manoharan JAYASHRE, Muthiah Joe Virgin LARGIA, Manikandan RAMESH

Pages : 866-877

View : 16 | Download : 10

Publication Date : 2016-12-01

Article Type : Research Paper

Abstract :Solanum trilobatum L. İs one of the most valued medicinal plants of Indian Ayurveda and Siddha medicine. An efficient protocol for direct shoot organogenesis and Agrobacterium tumefaciens mediated transformation using in vitro derived leaf explants of S. Trilobatum was established for the first time. High frequency direct shoot regeneration insert ignore into journalissuearticles values(89%); was achieved in MS medium containing 2 mg/L 6-benzylaminopurine and 0.25 mg/L indole-3-acetic acid. The Agrobacterium strain EHA 105 harboring the binary vector pCAMBIA 1301, which contained hygromycin phosphotransferase insert ignore into journalissuearticles values(hpt); as a selectable marker gene and β-glucuronidase insert ignore into journalissuearticles values(gus); as a reporter gene, was used for the transformation. The factors that influence the transformation frequency were optimized as follows: age of explants insert ignore into journalissuearticles values(40 days old);, acetosyringone concentration insert ignore into journalissuearticles values(150 μM);, duration of preculture insert ignore into journalissuearticles values(2 days);, bacterial optical density insert ignore into journalissuearticles values(O.D. 0.4);, Tween 20 concentration insert ignore into journalissuearticles values(0.01%);, infection time insert ignore into journalissuearticles values(15 min);, pH of cocultivation medium insert ignore into journalissuearticles values(5.4);, and cocultivation duration insert ignore into journalissuearticles values(3 days);. The putative transformants were screened on selection medium containing 15 mg/L hygromycin. The transgenic plants were confirmed by histochemical gus analysis, PCR, and RT-PCR analysis. A stable transformation efficiency of 64% was achieved under optimized transformation conditions. Thus, the protocol can be used for genetic improvement of this plant in the future.
Keywords : Acetosyringone, Agrobacterium tumefaciens, direct shoot organogenesis, genetic transformation, Solanum trilobatum