Genetic transformation of buckwheat (Fagopyrum esculentum M.) with Agrobacterium rhizogenes and production of rutin in transformed root cultures

Abstract

We developed an efficient protocol for transformation of buckwheat (Fagopyrum esculentum Moench.) root cultures by using stem explants that were infected by Agrobacterium rhizogenes strain 15834, a strain with the binary vector pBI121. Four weeks after infection, kanamycin-resistant roots appeared on 90% of explants that were maintained on hormone-free medium. PCR analysis of the neomycin phosphotransferase (NTPII) gene confirmed transformation in 17 of 20 kanamycin-resistant hairy root cultures. Detection of high levels of -glucuronidase (GUS) transcripts and enzyme activity, and GUS histochemical localization also confirmed the stable genetic transformation. We propagated isolated hairy roots in liquid medium to promote rapid growth and production of rutin, an important flavonol glycoside. The amount of hairy root clone 2 (12.6 g dry weight L-1) was around 2.4 times more than that of wild type root (5.3 g dry weight L-1). The content of rutin was found in hairy root clone 2 (1.3 mg/g dry weight) which was 2.6 times more than that of wild type root where the amount of rutin was 0.5 mg/g dry weight. Transgenic root cultures of F. esculentum will allow investigation of the molecular and metabolic regulation of rutin biosynthesis and evaluation of the genetic engineering potential of this species.