A highly efficient in vitro plant regeneration andAgrobacterium-mediated transformation ofBrassica oleraceavar.botrytis

Abstract

A highly efficient in vitro plant regeneration and Agrobacterium-mediated transformation of Brassica oleracea var. botrytis A reliable and efficient regeneration protocol from the cotyledons and hypocotyls is successfully established using three cauliflower genotypes. Thidiazuron (TDZ) is found to be an effective inducer of shoot formation. Both the hypocotyl and cotyledon segments are highly responsive (100%) to shoot formation and produce a maximum of 15.8 shoots per hypocotyl explant on MS salts with B 5 vitamins medium containing TDZ at 0.2 mg L (1 and a-naphthaleneacetic acid at 0.01 mg L (1 . Using this efficient regeneration protocol, the reporter b-glucuronidase (GUS) under the constitutive cauliflower mosaic virus (CaMV) 35S promoter is introduced into cauliflower cells of hypocotyls and cotyledons with Agrobacterium tumefaciens strain EHA105. By optimizing the parameters on the procedure of Agrobacterium-mediated transformation, a high transformation efficiency is demonstrated. The transformation efficiencies are 21.6% and 18.3% for cotyledons and hypocotyls, respectively. Integration of the transgenes in the plant genome is demonstrated by PCR and Southern hybridization analysis. Expression of the transgenes is confirmed by RT-PCR analysis and GUS staining. Compared with previous works, the present study provides higher regeneration and transformation efficiencies and establishes a highly efficient transformation system in cauliflower.

High efficiency transformation of Brassica oleracea var. botrytis plants by Rhizobium rhizogenes

“…Chakrabarty et al (2002) reported maximum transformation frequency (evidenced by GUS staining) of 22.6%. A similar range of transformation efficiencies was reported by Yu and co-workers in 2010 (21.6% and 18.3% for cotyledons and hypocotyls). In such context our protocol assures at least 2.5 fold increase in frequency of genetic modification (and more than sixfold increase when compared only to R. rhizogenes -mediated transformation).…”