The effects of heat shock and phytohormons on Agrobacterium mediated transformation of maize (Zea mays L.)

he high efficiency of gene transfer to maize (Zea mays L.) using a standard binary vector is used widely in studies of genetic engineering and functional Genomics. The objective of the present study was to enhance efficiency of gene transfer to maize varieties using heat shock and hormones. To do this, embryo induction and plant regeneration from immature embryos of B73 and Mo17 cultivars immature embryos (1.5- 2 mm) were infected with Agrobacterium tumefaciens strain AGLO1 harboring pCAMBIA3301 binary vector containing gus gene. The immature embryos were treated by T1 (46 °C for 3 min, on ice for 1 min, centrifugation in 20000 g at 4 °C for 30 min) and T2 (without T1 treatment) treatments before inoculation with Agrobacterium. Moreover, the effects of Dithiothreitol (DTT) and Pluronic acid F68 (F68) detergents were studied on transformation efficiency by adding them to culture media. The results showed that heat shock and centrifugation treatments improved slightly genetic transformation of B73 but not on Mo17. The addition of DTT or F68 to co-culture medium led to a dramatic increase in T-DNA transfer to the B73, while there was no effects on Mo17 transformation. In B73, depending on the type of treatment, 18-5% of the immature embryos were produced resistant calli to phosphinotricin (PPT). Histochemical GUS assay on 8 weeks calli resistant to PPT and PCR analysis confirmed the transferring and expression of gus gene. These analyses showed 18% transformation frequency in T1+F68 treatment in comparation with 0% in T2-Detergents. Based on our results, the treatment T1+ F68 was suggested an efficient methodology to transfer genes by Agrobacterium to maize cultivar B73.