Transformation of Brassica napus with Agrobacterium tumefaciens based vectors

Abstract

A reproducible system to produce transgenic Brassica napus plants has been developed using stem segments. Stem segments from 6-7 week old plants were inoculated with an Agrobacterium tumefaciens strain containing a disarmed tumor-inducing plasmid pTiT37-SE carrying a chimeric bacterial gene encoding kanamycin resistance (pMON200). Stem explants were cocultured for 2 days before transfer to kanamycin selection medium. Shoots regenerated directly from the explant in 3-6 weeks and were excised, dipped in Rootone®, and rooted in soil. Transformation was confirmed by opine production, kanamycin resistance, and DNA blot hybridization in the primary transformants. Final proof of transformation was demonstrated by the co-transfer of opine production and kanamycin resistance to progeny in a Mendelian fashion. Over 200 transgenic Brassica napus plants have been produced using this system.