Abstract
Protoplasts of three carrot cultivars were isolated from in vitro-grown plantlets by overnight incubation in an enzyme mixture composed of 1% (w/v) cellulase Onozuka R-10 and 0.1% (w/v) pectolyase Y-23. After cell immobilization in modified thin alginate layers, three types of β-lactam antibiotics (cefotaxime, carbenicillin, or timentin) at five different concentrations (100, 200, 300, 400, or 500 mg L−1) were added to the culture medium. In 20-d-old cultures, a different number of cell colonies had formed and varied on average from 27 to 56% in carbenicillin- and cefotaxime-containing media, respectively. Supplementation of the culture media with antibiotics at concentrations higher than 100 mg L−1 resulted in a decrease in plating efficiency in comparison with the controls. However, from all antibiotic treatments, except carbenicillin at concentrations of 400–500 mg L−1, efficient plant regeneration occurred. For this reason, we believe that cefotaxime and timentin in the concentrations analyzed here may be used in complex in vitro procedures or valuable carrot cultures as a prophylactic agent for prevention against occasional contaminations.
Highlights
Plant protoplast technology can be widely used in various aspects of plant science to study such fundamental processes as differentiation, dedifferentiation, and pluripotency of cellsE
Systems based on protoplast culture contain many steps and include time-consuming procedures as follows: (1) induction of in vitro-grown donor material; (2) source tissue slicing, conditioning, and digestion; (3) protoplast isolation and purification; (4) for some species, embedding of the cells before culture; and (5) plant regeneration preceded with stepwise reduction of the osmotic pressure by diluting the medium (Davey et al 2005b)
Our results demonstrated that in early cultures, carbenicillin at concentrations of 400–500 mg L−1 reduced the mitotic activity of carrot protoplast-derived cells gradually leading to complete arrest of cell divisions in older cultures, and as a result, a lack of plant regeneration was observed
Summary
Plant protoplast technology can be widely used in various aspects of plant science to study such fundamental processes as differentiation, dedifferentiation, and pluripotency of cellsE. Systems based on protoplast culture contain many steps and include time-consuming procedures as follows: (1) induction of in vitro-grown donor material; (2) source tissue slicing, conditioning, and digestion; (3) protoplast isolation and purification; (4) for some species, embedding of the cells before culture; and (5) plant regeneration preceded with stepwise reduction of the osmotic pressure by diluting the medium (Davey et al 2005b). A reduced growth rate is observed usually leading to cell/tissue/plant death, which eliminate the cultures completely from the program, and the long procedure of donor material establishment, protoplast isolation, or their regeneration into plants must be started again (Leifert and Waites 1992; Shehata et al 2010)
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