Soybean transformation and analysis of transformed tissues using PCR

Abstract

Factors which affect the transformation efficiency of Agrobacterium tumefaciens-mediated soybean transformation were studied. It was found that pre-induction of A. tumefaciens A281 using acetosyringone significantly increased the efficiency of transformation and this could be boosted further by octopine. The application of these pre-induction conditions led to the improvement in recovery of putative transformed soybean shoots although no fertile plants were recovered.

The co-transformation of T-strands from a wild type Ti plasmid pTiBo542 and T-strands of a binary vector pZA-7 in a virulent A. tumefaciens strain A281 containing a binary vector pZA-7 was studied. When transformation with the wild type T-strands was selected for, 82% of the transformants also contained the T-strands from the binary vector. The frequency of co-expression of genes contained on these two different T-strands was found to be 50%. The expression of two genes in the T-region of pZA-7 was random, although both genes were driven by a 35S promotor and terminated by a polyadenylation signal of the nopaline synthase gene and closely linked in the same T-DNA. Differential DNA methylation might be the cause of this inactivation of one of the two closely linked genes. The high frequency of co-transformation led to the development of a simple system for the expression of genes of interest in soybean tissues.

A simple, rapid and reliable system, the miniprep-PCR system, was developed for PCR analysis using very small amounts of plant tissue (10-50mg). The reliability of this system is ensured by the inclusion of an internal control into the system.