Method for obtaining transgenic plants expressing a protein with activity producing hydrogen peroxide by transformation by agrobacterium rhizogenes

A method for obtaining transgenic plants, comprising

• (a1) transforming dicotyledonous plant cells or dicotyledonous plant explants with Agrobacterium rhizogenes containing two vectors:
• a vector (i) carrying a T-DNA comprising a DNA sequence encoding an H2O2 producing protein, and a vector (ii) being the pRi of Agrobacterium rhizogenes, in order to obtain transformants, wherein said DNA sequence encoding an H2O2 producing protein is flanked by elements necessary for expression of said DNA sequence;
• and wherein said transformation with Agrobacterium rhizogenes induces the formation of roots on the transformants;
• or
• (a2) transforming dicotyledonous plant cells or dicotyledonous plant explants with Agrobacterium rhizogenes containing two vectors:
• a vector (i) containing a recombinant DNA comprising both a DNA sequence encoding an H2O2 producing protein and a gene encoding a protein of interest, and a vector (ii) being the pRi of Agrobacterium rhizogenes, in order to obtain transformants, wherein said DNA sequence encoding the H2O2 producing protein and said gene encoding a protein of interest are flanked by elements necessary for expression of said genes;
• and wherein said transformation with Agrobacterium rhizogenes induces the formation of roots on the transformants;
• and
• (b) visually selecting the transformants which express said H2O2 producing protein by the use of a peroxidase-based colorimetric test, wherein said test is carried out on said transformants in the presence of a substrate for said protein with H2O2 producing activity and said peroxidase for revealing the formation of H2O2, and wherein said formation of H2O2 is revealed by coloration of said transformants, thus leading to selected transformants;
• and
• (c) regenerating transgenic-plantlets out of said selected transformants and monitoring the expression of said H2O2 producing protein within said plantlets obtained, wherein said expression is monitored by the use of a peroxidase-based colorimetric test wherein expression of said H2O2 producing protein is monitored by the presence of H2O2, revealed by a peroxidase-based colorimetric test;
• and
• (d) sorting the transgenic plantlets which do not contain said pRi of Agrobacterium rhizogenes and optionally carrying out a molecular analysis of the progeny of said sorted plantlets, allowing the selection or the confirmation of the obtainment of transgenic plantlets containing only the DNA sequence encoding said H2O2 producing protein or the DNA sequence encoding said H2O2 producing protein and the gene encoding said protein of interest and not the pRi of Agrobacterium rhizogenes, wherein said transgenic plantlets containing the pRi of Agrobacterium rhizogenes exhibit phenotypic characteristics, such as crinkled leaves and shorter internodes, and transgenic plantlets which do not contain the pRi of Agrobacterium rhizogenes, do not exhibit said phenotypic characteristics;
• and
• (e) generating transgenic plants from said plantlets.