An efficient regeneration and genetic transformation of maize through Agrobacterium and particle bombardment in immature embryos

Abstract

An efficient Agrobacterium mediated transformation system, from which transgenic tropical maize plants was directly generated without previous crosses with laboratory or temperate lines was established in present study. Experimental evaluations are focused on two main issues: i) Establishment of appropriate tissue culture conditions, which induced somatic embryogenesis from the immature embryo cells, and ii) The delivery of T-DNA toward these cells. A high rate of embryogenic calli, generated from immature embryo was obtained when 2-4 D (4mgL -1 ) was added to the MS based induction medium. Regeneration rate was observed upto 19 plants per gram in the calli by using BAP and KIN at 0.5mgL -1 . Regarding the Agrobacterium strains evaluated for their transformation capability on the tropical maize line MU-2092 used, best results are obtained from the strain when applied at OD 550 nm = 0.5-1.0. Physical micro-wounds before the Agroinfection proved to be an excellent way to promote both the T-DNA transfer toward the immature embryo and the acceleration in rates of transient GUS expression when compared with particle bombardment. The highest frequencies of transient GUS expression corresponding to the embryos as well as the regeneration of whole transgenic plants emerged from them, was obtained using immature embryos wounded by bombarding at 80 lb/in 2 . Analysis of the progenies confirmed the sexual transmission of the introduced genes and their stable expression.