In vitro selection of transgenic sugarcane callus utilizing a plant gene encoding a mutant form of acetolactate synthase
Date
2013-02-05
Journal Title
Journal ISSN
Volume Title
Publisher
Springer
Abstract
Selection genes are routinely used in plant genetic
transformation protocols to ensure the survival of transformed
cells by limiting the regeneration of non-transgenic
cells. In order to find alternatives to the use of antibiotics as
selection agents, we followed a targeted approach utilizing a
plant gene, encoding a mutant form of the enzyme acetolactate
synthase, to convey resistance to herbicides. The
sensitivity of sugarcane callus (Saccharum spp. hybrids,
cv. NCo310) to a number of herbicides from the sulfonylurea
and imidazolinone classes was tested. Callus growth
was most affected by sulfonylurea herbicides, particularly
3.6 μg/l chlorsulfuron. Herbicide-resistant transgenic sugarcane
plants containing mutant forms of a tobacco acetolactate
synthase (als) gene were obtained following biolistic
transformation. Post-bombardment, putative transgenic callus
was selectively proliferated on MS medium containing
3 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 20 g/l sucrose,
0.5 g/l casein, and 3.6 μg/l chlorsulfuron. Plant regeneration
and rooting was done on MS medium lacking
2,4-D under similar selection conditions. Thirty vigorously
growing putative transgenic plants were successfully ex
vitro-acclimatized and established under glasshouse conditions.
Glasshouse spraying of putative transgenic plants
with 100 mg/l chlorsulfuron dramatically decreased the
amount of non-transgenic plants that had escaped the in
vitro selection regime. PCR analysis showed that six
surviving plants were als-positive and that five of these
expressed the mutant als gene. This report is the first to
describe a selection system for sugarcane transformation
that uses a selectable marker gene of plant origin targeted
by a sulfonylurea herbicide.
Description
CITATION: Van Der Vyver, C., et al. 2013. In vitro selection of transgenic sugarcane callus utilizing a plant gene encoding a mutant form of acetolactate synthase. In Vitro Cellular and Developmental Biology - Plant, 49(2):198–206, doi:10.1007/s11627-013-9493-0.
The original publication is available at http://www.springer.com/life+sciences/plant+sciences/journal/11627
The original publication is available at http://www.springer.com/life+sciences/plant+sciences/journal/11627
Keywords
Acetolactate synthase, Acetolactate synthase mutation, Sugarcane transformation, Sulfonylurea herbicide
Citation
Van Der Vyver, C., et al. 2013. In vitro selection of transgenic sugarcane callus utilizing a plant gene encoding a mutant form of acetolactate synthase. In Vitro Cellular and Developmental Biology - Plant, 49(2):198–206, doi:10.1007/s11627-013-9493-0