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

Año: 2010 vol. 102 p. 181 - 189

0167-6857

A protocol for the production of transgenicplants was developed for Lotus tenuis via Agrobacteriummediatedtransformation of leaf segments. The explantswere co-cultivated (for 3 days) with an A. tumefaciensstrain harbouring either the binary vector pBi RD29A:oatarginine decarboxylase (ADC) or pBi RD29A:glucuronidase(GUS), which carries the neomycin phosphotransferaseII (nptII) gene in the T-DNA region. Followingco-cultivation, the explants were cultured in Murashige andSkoog 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-weekintervals) to maintain the selection pressure during theentire period. About 40% of the explants inoculated withthe pBiRD29:ADC strain produced eight to ten adventitiousshoots per responsive explant through a direct systemof regeneration, whereas 69% of the explants inoculatedwith the pBi RD29A:GUS strain produced 13–15 adventitiousshoots per responsive explant. The selected transgeniclines were identified by PCR and Southern blotanalysis. Three ADC transgenic lines were obtained from30 infected explants, whereas 29 GUS transgenic lineswere obtained from 160 explants, corresponding to atransformation efficiency of 10 and 18.1%, respectively.More than 90% of the in vitro plantlets were successfullytransferred to the soil. The increase in the activity ofarginine decarboxylase from stressed ADC- Lt19 lines wasaccompanied by a significant rise in the putrescine level.The GUS transgenic line driven by the RD29A promotershowed strong signals of osmotic stress in the leaves andstem tissues. All of the transgenic plants obtained exhibitedthe same phenotype as the untransformed controls undernon-stress conditions, and the stability of the gene introducedinto the cloned materials was established.