Transformation of wild Solanum species resistant to late blight by using reporter gene gfp and msh2 genes

Lenuta Rakosy-Tican

lrakosy@hasdeu.ubbcluj.ro
Babes-Bolyai University, Faculty of Biology and Geology, Plant Genetic Engineering Group, Clinicilor str. 5-7, 3400 Cluj-Napoca, Romania (Romania)

Adriana Aurori

Babes-Bolyai University, Faculty of Biology and Geology, Plant Genetic Engineering Group, Clinicilor str. 5-7, 3400 Cluj-Napoca, Romania (Romania)

Cristian M. Aurori

Babes-Bolyai University, Faculty of Biology and Geology, Plant Genetic Engineering Group, Clinicilor str. 5-7, 3400 Cluj-Napoca, Romania (Romania)

Gabriela Ispas

Babes-Bolyai University, Faculty of Biology and Geology, Plant Genetic Engineering Group, Clinicilor str. 5-7, 3400 Cluj-Napoca, Romania (Romania)

Ivan Famelaer

Vrije Universiteit, Brussel, Belgium (Belgium)

Abstract

Green fluorescent protein (gfp) reporter gene and nptII marker gene were used to optimize Agrobacterium tumefaciens (agro) mediated transformation of wild Solanum genotypes resistant to late blight. Different genotypes of Solanum bulbocastanum, S. chacoense, S. microdontum and S. verrucosum were assessed for their regeneration ability on MS based media and for agro-mediated transformation. As the first step reporter genes were used to optimize transformation protocol for each species and then the transfer of genes involved in mismatch repair of DNA were attempted in Solanum chacoense. For transformation, either leaf or stem fragments were used. It was shown that gfp is a valuable and elegant tool for monitoring the efficiency of transformation or the occurrence of chimera in all genotypes. Transformation efficiency was dependent on a plant genotype. A number of genotypes have been successfully transformed and they expressed constitutively the bright green fluorescence of gfp without any side effects. The most recalcitrant species proved to be S. microdontum, which did not regenerate plants although different media and phytohormones had been used. The best protocol for S. chacoense transformation was also found to work in the transfer of msh2 genes. Msh2 isolated from Arabidopsis was used and transferred either as mutated (Apa) or antisense (As) gene. The integration of msh2-mutated gene into S. chacoense genome was demonstrated by PCR amplification and confirmed by RT-PCR for some of the putative transgenic clones. The implications of mismatch repair in homologous recombination and its importance for potato improvement are discussed. 

Keywords:

Agrobacterium tumefaciens – mediated transformation, DNA mismatch repair, gfp, nptII marker gene