Transformation of Arabidopsis thaliana Using Root Explants

Abstract

The small, rapidly cycling crucifer Arabidopsis thaliana has been generally accepted as an experimental model to study plant molecular biology and developmental genetics (National Science Foundation 1990). One of the important contributions to turn this weed into a model has been the elaboration of an efficient transformation system. We developed a simple and highly reproducible tissue-culture procedure for transformation of Arabidopsis using Agrobacterium tumefaciens as the gene delivery system and the roots of axenically grown plants as the explant source (Valvekens et al. 1988). Arabidopsis root explants have a high potential for rapid shoot regeneration. Critical for roots to form shoots over their entire length, is a short preculture of maximum 7 days on 2,4-dichlorophenoxyacetic acid (2,4-D)-containing medium prior to culture on medium with high concentration of the cytokinin 6(y,ydimethylallylamino)purine (2-ip). We incorporated this efficient regeneration method with Agrobacterium infection and developed a selection procedure for the regeneration of transgenic Ti shoots, transformed with the neomycin phosphotransferase II (nptll) gene conferring resistance to the antibiotic, kanamycin. With this method, initially developed for the Arabidopsis ecotype C24, transgenic T2 seeds are routinely obtained within 3 months of tissue culture at high frequency.