Genetic transformation of buckwheat (Fagopyrum esculentum M.) with Agrobacterium rhizogenes

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 ß-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. Among the six transformed root cultures and the wild type root culture, the growth rate ranged from 233 to 312 (mg/flask) and the rutin content ranged from 0.8 to 1.2 (mg/g D.W.). 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.