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Publication
Susceptibility of embryogenic and organogenic tissues of maritime pine (Pinus pinaster) to antibiotics used in Agrobacterium-mediated genetic transformation
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Advisor(s)
Abstract(s)
The effects of antibiotics commonly
used in Agrobacterium-mediated transformation
were studied on Pinus pinaster tissues. Embryogenic
tissue growth from three embryogenic lines
and adventitious bud induction from cotyledons
from three open-pollinated seed families were
analysed. Cefotaxizme, carbenicillin and timentin
commonly used for Agrobacterium elimination, at
concentrations of 200–400 mg l –1 did not inhibit
the embryogenic tissue growth on filter paper nor
as clumps. Adventitious bud induction and bud
number were significantly reduced for one of the
tested families when using 400 mg l–1 cefotaxime
or timentin. The selection agent kanamycin
significantly inhibited growth of embryogenic
tissue on filter paper in all the embryogenic
lines and concentrations tested (20–50 mg l–1).
Kanamycin also inhibited growth of embryogenic
clumps after two subcultures at 5–50 mg l–1.
In cotyledons, kanamycin inhibited adventitious
bud formation in the three seed families used,
regardless of the concentrations tested (5–
25 mg l–1). There was a significant effect of the
seed family on the bud induction and the number
of adventitious buds produced. From the results
obtained, we propose the use of timentin to
eliminate Agrobacterium in transformation
experiments, at concentrations of 400 mg l–1 for
embryogenic tissues and of 300 mg l–1 for cotyledons.
For selection of transformed tissues carrying
the kanamycin resistance gene, kanamycin
should be used at 20 mg l–1 for embryogenic tissues
on filter paper, at 5 mg l–1 when clumps are
in direct contact with the selection medium, and
bellow 5 mg l–1 for adventitious bud induction.
Description
Keywords
Adventitious bud induction Carbenicillin Cefotaxime Kanamycin Somatic embryogenesis Timentin
Citation
Plant Cell , Tissue and Organ Culture, 87, 33-40