Design and Construction of a Transformation Vector to Induce Crown Gall Disease Resistance

摘要

Crown gall is a disease caused by Agrobacterium tumefaciens, a soil pathogen that greatly affects many fruit tree species and ornamental plants, producing large losses to growers and nurseries every year. Traditional breeding strategies to produce resistant plants have been successful only in those species where resistant germplasm exists. The objective of this work is to simultaneously silence both ipt and iaaM oncogenes of Agrobacterium tumefaciens by using a new hairpin RNAs transformation vector. To obtain this molecular construction only one chimerical transgene was made by the fusion of two 250 bp fragments, each one designed from a highly conserved sequence of ipt and iaaM genes. Transcription of the cassette, consisting of the sense and antisense sequences of the chimerical gene separated by intron, will produce mRNA with a hairpin estructure (hpRNA) and both Agrobacterium oncogenes should be silenced when transformed plants are infected. The construction was cloned in the binary plasmid pART harbouring the nptII gene. The new vector (pSilenceCG) has been used to transform the EHA105 strain of A. tumefaciens. To check the effectivity of this construction different transgenic tobacco lines have been produced after transforming disc leaves with the pSilenceCG The presence of nptll and ipt-iaaM trangenes was demonstrated by PCR and the nptII integration in the genome by Southern blot. The resistance to a strain of A. tumefaciens of some transgenic lines has been evaluated in vitro. Stem explants were evaluated for the presence of callus and weighed one month after infection. Transgenic lines showed lower callus production and weight than the infected non transformed control. Additionally, in vivo evaluation of the T_1 seedlings confirmed resistance to crown gall in the transgenic lines.