Establishment of transgenic hairy root cultures through a successive transformation method

TALANO, M., SOSA ALDERETE L. WEVAR OLLER A.L. ERIC I., AGOSTINI E., MEDINA M.I. ,

Villa Giardino Córdoba

Congreso; VX Jornadas Científicas de la Soc. de Biol. de Cba.; 2005

Soc. de Biol. de Cba.

Hairy root (HR) cultures have received special attention in the last years because of their multiple applications. They constitute a plant system with rapid in vitro growth without hormones and biochemical and genetic stability. Genetic engineering has allowed to transform plants with foreign genes to give or to improve special metabolic features. The establishment of transgenic HR can be obtained through a unique step of transformation, using genetically modified A.rhizogenes, or by an unusual method with two steps using A. tumefaciens and A. rhizogenes. The latter technique is called successive transformation. The aim of this work was to obtain transgenic HR cultures with peroxidases genes (tpx1, tpx2 and both) and with a gene involved in the ascorbic acid synthesis (GalUR) through successive transformation, to use them for future biotechnological applications. Stable transgenic HR clones of tobacco which expressed the foreign genes tpx1, tpx2 and both genes and tomato HR which expressed GalUR gene were established. As it could be analysed by PCR, the clones contained not only the rol C gene, which is characteristic of HR but also the foreign genes integrated in the transgenic plants used in the first step. Growth index, enzyme activities and the isoelectric focusing zymograms of the enzymes were analysed for transgenic and wild type clones. The results showed the efficiency of the successive transformation technique to obtain stable transgenic HR clones. This method constitutes a useful tool to give or to improve HR cultures features for biotechnological applications. Hairy root (HR) cultures have received special attention in the last years because of their multiple applications. They constitute a plant system with rapid in vitro growth without hormones and biochemical and genetic stability. Genetic engineering has allowed to transform plants with foreign genes to give or to improve special metabolic features. The establishment of transgenic HR can be obtained through a unique step of transformation, using genetically modified A.rhizogenes, or by an unusual method with two steps using A. tumefaciens and A. rhizogenes. The latter technique is called successive transformation. The aim of this work was to obtain transgenic HR cultures with peroxidases genes (tpx1, tpx2 and both) and with a gene involved in the ascorbic acid synthesis (GalUR) through successive transformation, to use them for future biotechnological applications. Stable transgenic HR clones of tobacco which expressed the foreign genes tpx1, tpx2 and both genes and tomato HR which expressed GalUR gene were established. As it could be analysed by PCR, the clones contained not only the rol C gene, which is characteristic of HR but also the foreign genes integrated in the transgenic plants used in the first step. Growth index, enzyme activities and the isoelectric focusing zymograms of the enzymes were analysed for transgenic and wild type clones. The results showed the efficiency of the successive transformation technique to obtain stable transgenic HR clones. This method constitutes a useful tool to give or to improve HR cultures features for biotechnological applications. Hairy root (HR) cultures have received special attention in the last years because of their multiple applications. They constitute a plant system with rapid in vitro growth without hormones and biochemical and genetic stability. Genetic engineering has allowed to transform plants with foreign genes to give or to improve special metabolic features. The establishment of transgenic HR can be obtained through a unique step of transformation, using genetically modified A.rhizogenes, or by an unusual method with two steps using A. tumefaciens and A. rhizogenes. The latter technique is called successive transformation. The aim of this work was to obtain transgenic HR cultures with peroxidases genes (tpx1, tpx2 and both) and with a gene involved in the ascorbic acid synthesis (GalUR) through successive transformation, to use them for future biotechnological applications. Stable transgenic HR clones of tobacco which expressed the foreign genes tpx1, tpx2 and both genes and tomato HR which expressed GalUR gene were established. As it could be analysed by PCR, the clones contained not only the rol C gene, which is characteristic of HR but also the foreign genes integrated in the transgenic plants used in the first step. Growth index, enzyme activities and the isoelectric focusing zymograms of the enzymes were analysed for transgenic and wild type clones. The results showed the efficiency of the successive transformation technique to obtain stable transgenic HR clones. This method constitutes a useful tool to give or to improve HR cultures features for biotechnological applications.