title: In vitroselection of transgenic sugarcane callus utilizing a plant gene encoding a mutant form of acetolactate synthase.
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 (Saccharumspp. 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 successfullyex 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 thein vitroselection regime. PCR analysis showed that six surviving plants wereals-positive and that five of these expressed the mutantalsgene. 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.
authors.affiliation: Institute for Plant Biotechnology, Department of Genetics, University of Stellenbosch, Stellenbosch, 7602 South Africa.
authors.forename: Christell
authors.initials: C
authors.lastname: van der Vyver
authors.forename: Tobie
authors.initials: T
authors.lastname: Conradie
authors.forename: Jens
authors.initials: J
authors.lastname: Kossmann
authors.forename: James
authors.initials: J
authors.lastname: Lloyd
keywords: Acetolactate synthase; Acetolactate synthase mutation; Sugarcane transformation; Sulfonylurea herbicide
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