Biolistic mediated sorghum (Sorghum bicolor L. Moench) transformation via mannose and bialaphos based selection systems

Abstract

Sorghum (Sorghum bicolor (L.) Moench) is recalcitrant to transformation and besides several reported cases of success, transformation is far from routine. The choice of the selectable markers is key factor influencing success and efficiency of transformation. In sorghum, concerns about flow of herbicide and antibiotic resistance gene into genetically related wild and weedy species have a direct bearing on the choice of suitable selectable markers in many tropical and subtropical regions. The authors studied the utility of bialaphos and phosphomannose isomerase selectable markers in microprojectile mediated transformation of P898012, a sorghum inbred line generally considered amenable to in vitro manipulation. Two plasmids containing bar gene, encoding bialaphos resistance, and manA gene, for phosphomannose isomerase, both under the control of maize ubiquitin promoter and nopaline synthase terminator were used to transform immature zygotic embryos (IZE) of sorghum via particle bombardment using a particle inflow gun. Transgene integration in putatively transgenic plants was confirmed by PCR and Southern blot analysis. The expression of the bar gene in transgenic plants was demonstrated by the BASTA leaf painting assay. Our results indicated a transformation efficiency of 0.11% and 75% escapes in the apparent bialaphos resistant plants. A transformation efficiency of 0.77% was observed on manA selection and all plants recovered contained the manA gene. Besides an improved transformation efficiency, mannose selection offers a more environmentally sound system as sugar metabolism is considered ecologically neutral should it inadvertently be transferred to wild sorghum species