Development of an efficient -mediated transformation method and its application in tryptophan pathway modification in .

The biosynthetic pathway of vinca alkaloids has a long research history, including not only identification of metabolic intermediates but also the mechanisms of inter-cellular transport and accumulation of biosynthesized components. Vinca alkaloids pathway begins with strictosidine, which is biosynthesized by condensing tryptamine from the tryptophan pathway and secologanin from the isoprenoid pathway. Therefore, increasing the supply of precursor tryptophan may enhance vinca alkaloid content or their metabolic intermediates. Many reports on the genetic modification of use cultured cells or hairy roots, but few reports cover the production of transgenic plants. In this study, we first investigated a method for stably producing transgenic plants of , then, using this technique, we modified the tryptophan metabolism system to produce transgenic plants with increased tryptophan content. Transformed plants were obtained by infecting cotyledons two weeks after sowing with strain A13 containing a plant expression vector, then selecting with 1/2 B5 medium supplemented with 50 mg l kanamycin and 20 mg l meropenem. Sixty-eight regenerated plants were obtained from 4,200 cotyledons infected with , after which genomic PCR analysis using -specific primers confirmed gene presence in 24 plants with a transformation rate of 0.6%. Furthermore, we performed transformation into using an expression vector to join and genes, which are feedback-resistant mutant genes derived from . The resulting transformed plants showed exactly the same morphology as the wild-type, albeit with a marked increase in tryptophan and alkaloids content, especially catharanthine in leaves.