Doctoral Dissertations

Date of Award

8-1997

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Life Sciences

Major Professor

Peter M. Gresshoff

Committee Members

Fred Allen, Karen Hughes, Brad Reddick

Abstract

The goals of this work included examining the culture parameters for initiating soybean embryogenic suspension cultures of selected soybean genotypes; examining tissue culture induced variance in progeny of soybean regenerants; developing a biolistic gene gun for soybean transformation; and attempts to transform selected soybean genotypes with genes for herbicide and virus resistance. The experiments were divided into five parts examining induction of somatic embryogenesis in soybean cultivars; the inheritance of the embryogenic phenotype through successive generations of progeny from regenerants; plasmids and gene constructs used for transformation studies; biolistic gene gun construction and optimization; and biolistic transformation studies with soybean genotypes amenable to embryogenic culture. Experiments were done to examine some of the parameters involved in somatic embryo induction and culture for the soybean genotypes Bragg and its non- and super- nodulating mutants, TN4-86, TN90-03, TN90-09, and the control Fayette. The data suggested broad genotypic differences in the response of soybean genotypes to induction of somatic embryogenesis in immature cotyledons. Establishment of sustainable embryogenic suspension cultures was also genotype dependent and was not very successful for the non- and super-nodulating mutants. Genotypes Bragg and TN4-86 produced sustainable embryogenic suspension cultures but Bragg was non- regenerable. TN4-86 could be regenerated from suspension culture. Two TN4-86 plants were regenerated from embryogenic suspension culture and the progeny of these plants were examined for the embryogenic phenotype by placing immature cotyledons into the embryo induction system for successive generations. Analysis indicated that the embryogenic phenotype did not seem to be controlled by a single locus. Also, seeds produced from the successive generations through the R4 generation were planted in the field and assessed for somaclonal variation. Dwarfism, 'partial steriles', and differences in maturity dates were detected. Genomic DNA's from the R1 progeny were analyzed by single primer DNA Amplification Fingerprinting (DAF) but no polymorphisms were detected among the progeny. For later transformation studies, plasmids were obtained containing a hygromycin resistance gene, a b-glucuronidase gene, and a phosphinothricin resistance gene (pat: resistance to the herbicides Basra or Ignite and the compound bialaphos). A plasmid construct was made for soybean transformation that contained the soybean mosaic virus coat protein (smv-cp) that might confer viral resistance through viral coat protein- mediated resistance. Herbicide selection experiments were done in vitro and in vivo to determine the effective concentrations of the herbicidal compounds on soybean tissues. It was found that the plant absorption of the different compounds was very important in determining the amount of selection. It was found that bialaphos was superior to pure phosphinothricin for in vitro selection and that the commercial herbicide formulation was superior to pure phosphinothricin. For soybean biolistic transformation studies, initially an electric discharge particle gun was constructed but this was superseded by construction of a better helium discharge particle gun. Optimal gene gun parameters were tested with tobacco, cowpea, and soybean leaves. Optimal parameters for bombardment of soybean embryogenic suspension tissues were also determined. Expression of the soybean mosaic virus coat protein gene was demonstrated in bombarded tobacco leaves by enzyme-linked immunosorbent assay (ELISA). Transformation attempts of TN4-86 and Bragg embryogenic suspension cultures were made with the helium discharge particle gun. In one experiment, two cultures were established that were resistant to hygromycin but unfortunately these cultures were lost to contamination. Four TN4-86 plants were regenerated from one bombardment experiment and were tested by polymerase chain reaction (PGR) for the presence of the smv-cp and pat genes. The results were negative for their presence as were the results for a large number of bombarded embryos from the same experiment. Low transformation frequencies and low culture conversion frequency of embryos to plants contributed to the lack of success in these transformation experiments.

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