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

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

PLANT CELL TISSUE AND ORGAN CULTURE

SPRINGER

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

0167-6857

Abstract A protocol for the production of transgenic plants was developed for Lotus tenuis via Agrobacteriummediated transformation of leaf segments. The explants were co-cultivated (for 3 days) with an A. tumefaciens strain harbouring either the binary vector pBi RD29A:oat arginine decarboxylase (ADC) or pBi RD29A:glucuronidase (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 medium supplemented with naphthalenacetic acid (NAA) and benzyladenine (BA) and containing kanamycin (30 lg ml-1) and cefotaxime (400 lg ml-1) for 45 days. The explants were subcultured several times (at 2-week intervals) to maintain the selection pressure during the entire period. About 40% of the explants inoculated with the pBiRD29:ADC strain produced eight to ten 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, corresponding to a transformation efficiency of 10 and 18.1%, respectively.