Efficient induction of embryogenic callus and biolistic-mediated transient transformation in the CP۵۷-۶۱۴ sugarcane cultivar (Saccharum officinarum L.)

This study aimed to optimize embryogenic callus formation and transformation of the CP۵۷-۶۱۴ sugarcane variety using particle bombardment. Young leaf explants were cultured on modified Murashige and Skoog (MS) medium containing different concentrations of ۲,۴-dichlorophenoxyacetic acid (۲,۴-D; auxin) (۱, ۲, and ۴ mg L-۱) and two Dicamba concentrations (۴ and ۶ mg L-۱). The highest callus induction rate was achieved with ۲ mg L-۱ of ۲,۴-D. Subsequently, particle bombardment was used to introduce genetic constructs containing the uidA reporter gene driven by three promoters (CaMV۳۵S, ubiquitin, and actin۱) into the embryogenic calli. We assessed the impact of various biological, physical, and DNA parameters, including bombardment pressure, target distance, particle size, DNA amount, and osmotic pretreatment, through histochemical GUS assay to quantify blue spots. Results showed that the most effective combination involved bombardment at ۱۱۰۰ psi, ۶ cm target distance, ۱ μm gold particles coated with ۱ μg DNA, under a vacuum of ۲۵ inHg. Additionally, the best osmotic pretreatment utilized a solution containing ۰.۲ M sorbitol and ۰.۲ M mannitol. Notably, the construct driven by the ubiquitin promoter resulted in the highest transient expression of the uidA gene, suggesting its superiority for gene expression in this system.

کلیدواژه ها:

calli ، Gene expression ، particle bombardment ، Sugarcane