The transformation of South African soya bean cultivars with a synthetic Basta resistance gene.
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Date
1995
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Abstract
The development of a genetic engineering system for soya bean (Glycine max L.) is
described in this thesis. Routine tissue culture regeneration systems were developed
for South African cultivars of soya bean despite the recalcitrant nature of this plant to
in vitro manipulation. Regeneration of shoots was obtained when cotyledons were
excised from seeds germinated for two days and cultured on B5 BA 20 medium
containing 2 mg/I BA. The important problems of in vitro shoot elongation and rooting
were overcome by culturing cotyledons in the dark for four weeks to produce shoots
with unusually long stems. This was followed by one week of culture under conditions
of high light intensity to obtain healthy green shoots which could be rooted , either in
sorbarods or on solid Y2MS 30 medium. The use of a mist bed for the hardening off of
rooted soya bean regenerants was essential for the recovery of fertile soya bean plants.
Molecular techniques for the cloning of foreign genes into binary vectors suitable for
plant genetic engineering were also studied and are described in the thesis. The Basta
herbicide resistance gene, pat, was successfully cloned into the binary vector pBI121
which contains the [beta]-glucuronidase (GUS) reporter gene, uidA. The new construct,
pB1121/Ac, was conjugated into various disarmed Agrobacterium tumefaciens strains
and these strains, along with other binary vector-containing strains, were used to
transform soya bean plant material. Although a protocol for the routine transformation
of soya bean was not developed, transgenic soya bean material resistant to kanamycin
and showing GUS activity was obtained. Transformation of wound sites on cotyledons
was obtained in several experiments and transgenic shoots were regenerated from
inoculated cotyledons. Only the A. tumefaciens strain C58C1 (pGV2260)(pJIT119) was able to transform cotyledonary cells of soya bean and, therefore, only kanamycin
resistant soya bean shoots were produced. Transgenic soya bean plants resistant to
the herbicide Basta were not produced due to the recalcitrant nature of the crop to
genetic engineering. Transformation of the non-recalcitrant plant, tobacco, which is a
model system for plant genetic engineering was achieved. The binary pat gene
containing vector constructed in th is study, as well as vectors obtained from AgrEvo,
were tested. The transgenic Basta resistant tobacco plants obtained were used to
optimize assay systems for the analysis of transformed plant material containing the pat
gene. These assay systems included the use of the polymerase chain reaction as well
as digoxigenin-Iabelling of a DNA probe suitable for detection of the pat gene.
Description
Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1995.
Keywords
Soybean--South Africa., Soybean--Genetic engineering., Theses--Genetics.