Agrobacterium tumefaciens-mediated transformation of Lotus tenuis and regeneration of transgenic lines

ESPASANDIN FABIANA D; COLLAVINO M.M.; LUNA C.V.; PAZ R.C.; TARRAGÓ J.R.; RUIZ O.A.; MROGINSKI L.A.; SANSBERRO P.A.

PLANT CELL TISSUE AND ORGAN CULTURE

SPRINGER

Lugar: NEW YORK; Año: 2010 vol. 102 p. 181 - 189

0167-6857

A protocol for the production of transgenic plants has been developed for Lotus tenuis glaber Mill. via Agrobacterium mediated transformation of leaf segments. The explants were co-cultivated (for three days) with A. tumefaciens strain harboured the binary vector either pBi RD29A: oat arginine decarboxylase (ADC) or pBi RD29A:GUS, carrying the neomycin phosphotransferase II (nptII) gene in the T-DNA region. Following co-cultivation, the explants were cultured in the regeneration medium (MS plus naphtalenacetic acid and benzyladenine) containing kanamycin (30 μg mL-1) and cefotaxime (400 μg mL-1) for 45 days. The explants were subcultured several times (each 2 weeks) in order to maintain the selection pressure during the entire period. Forty percent of the inoculated explants with pBiRD29:ADC strain showed 8-10 adventitious shoots per responsive explants through a direct system of regeneration. Likewise, sixty nine percent of inoculated explants with pBi RD29A:GUS strain produced 13-15 adventitious shoots per responsive explants. PCR analysis using the primer sets for npt II yielded the expected products. All the transformed lines tested showed the predicted band 654 pb for the npt II gene. Three ADC transgenic lines were obtained from 30 infected explants. In the case of GUS, 29 transgenic lines were obtained from 160 analyzed explants. These results correspond to a transformation efficiency of 10 and 18.1 %, respectively. More than 90% of the in vitro plantlets could be successfully transferred to soil. All of the transgenic plants obtained in the present study exhibited the same phenotype as the non-transformed control and the stability of the introduced gene in the cloned materials was established.