Soybean transformation and analysis of transformed tissues using PCR
Luo, Guangbin
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https://hdl.handle.net/2142/23344
Description
Title
Soybean transformation and analysis of transformed tissues using PCR
Author(s)
Luo, Guangbin
Issue Date
1993
Doctoral Committee Chair(s)
Widholm, Jack M.
Department of Study
Agronomy
Discipline
Agronomy
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Genetics
Language
eng
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.
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