INVESTIGADORES
LUNA claudia veronica
artículos
Título:
Agrobacterium tumefaciens-mediated transformation of Lotus tenuis and regeneration of transgenic lines
Autor/es:
F. D. ESPASANDIN; M. M. COLLAVINO; C. V. LUNA; R. C. PAZ; J. R. TARRAGO; O. A. RUIZ; L. A. MROGINSKI; P. A. SANSBERRO
Revista:
PLANT CELL TISSUE AND ORGAN CULTURE
Editorial:
SPRINGER
Referencias:
Lugar: The Netherlands; Año: 2010 vol. 102 p. 181 - 189
ISSN:
0167-6857
Resumen:
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. tumefaciensLotus tenuis via Agrobacteriummediated
transformation of leaf segments. The explants
were co-cultivated (for 3 days) with an A. tumefaciensA. 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 1315 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.
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 strong signals of osmotic stress in the leaves and
stem tissues. All of the transgenic plants obtained 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.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 1315 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.
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 strong signals of osmotic stress in the leaves and
stem tissues. All of the transgenic plants obtained 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.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 1315 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.
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 strong signals of osmotic stress in the leaves and
stem tissues. All of the transgenic plants obtained 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.Lt19 lines was
accompanied by a significant rise in the putrescine level.
The GUS transgenic line driven by the RD29A promoter
showed strong signals of osmotic stress in the leaves and
stem tissues. All of the transgenic plants obtained 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.