SANSBERRO pedro Alfonso
Agrobacterium tumefaciens-mediated transformation of Lotus tenuis and regeneration of transgenic lines
ESPASANDÍN, F; COLLAVINO, M; LUNA, C.; PAZ, R.; TARRAGÓ, J; RUIZ, O; MROGINSKI, L.; SANSBERRO, P.
PLANT CELL TISSUE AND ORGAN CULTURE
Lugar: The Netherlands; Año: 2010 vol. 102 p. 181 - 189
A protocol for the production of transgenic plants was developed for Lotus tenuis via Agrobacterium-mediated transformation of leaf segments. The explants were co-cultivated (for three days) with an A. tumefaciens strain harbouring either the binary vector pBi RD29A:oat arginine decarboxylase (ADC) or pBi RD29A:GUS, which carries the neomycin phosphotransferase II (nptII) gene in the T-DNA region. Following co-cultivation, the explants were cultured in Murashige and Skoog (MS) medium plus naphthalenacetic acid (NAA) and benzyladenine (BA) containing kanamycin (30 ìg ml-1) and cefotaxime (400 ìg ml-1) for 45 days. The explants were subcultured several times (for 2 weeks each time) to maintain the selection pressure during the entire period. About 40% of the explants inoculated with the pBiRD29:ADC strain showed 8-10 adventitious shoots per responsive explant through a direct system of regeneration, whereas 69% of the explants inoculated with the pBi RD29A:GUS strain produced 13-15 adventitious shoots per responsive explant. The selected transgenic lines were identified by PCR and Southern blot analysis. Three ADC transgenic lines were obtained from 30 infected explants, whereas 29 GUS transgenic lines were obtained from 160 explants. These results correspond to a transformation efficiency of 10 and 18.1%, respectively. More than 90% of the in vitro plantlets were successfully transferred to the soil. The increase in the activity of arginine decarboxylase from stressed ADC- Lt19 lines was accompanied by a significant rise in the putrescine level. The GUS transgenic line driven by the RD29A promoter showed that strong signals of osmotic stress were detected in leaves and stem tissues. All the transgenic plants obtained in the present study exhibited the same phenotype as the untransformed controls under non-stress conditions, and the stability of the gene introduced into the cloned materials was established.