A passage through in vitro culture leads to efficient production of marker-free transgenic plants in Brassica juncea using the Cre-loxP system

Abstract

The Cre-loxP site-specific recombination system was deployed for removal of marker genes from Brassica juncea (Indian mustard). Excision frequencies, monitored by removal of nptII or gfp genes in F₁ plants of crosses between LOX and CRE lines, were high in quiescent, differentiated somatic tissues but extremely poor in the meristematic regions (and consequently the germinal cells) thus preventing identification and selection of marker-free transgenic events which are devoid of both the marker gene as well as the cre gene, in F₂ progeny. We show that a passage through in vitro culture of F₁ leaf explants allows efficient development of marker-free transgenics in the F₂ generation addressing current limitations associated with efficient use of the Cre/loxP technology for marker gene removal.