Efficient plant regeneration and transformation procedures and the stability of the
transgene are important to the success of the cotton and soybean biotechnology
industry. Engineering herbicide resistance into plants will provide the potential
solution to effective weed control in agriculture and reduce loss in crop yields due
to weeds. It will also provide cheaper control and decrease environmental
hazards. The aims of this study were to develop efficient regeneration and
transformation protocols for commercially grown soybean and cotton in South
Africa and to use tobacco as a model plant to study the stability of the pat gene
through seed generations, successive generations and high temperature and
drought stress regimes.
Tobacco (Nicotiana tabacum cv. Samsun) leaf disks were successfully
transformed with the pat gene. PCR analysis confirmed the integration of the pat
gene in all nine transgenic plants and T1, T2 and T3 progeny. Successive
generations, high temperatures and drought stress had no adverse effect on the
stability and expression of the pat gene in the transgenic tobacco plants.
In view of the economic importance of soybean (Glycine max) and cotton
(Gossypium hirsutum) in South Africa and the potential to improve commerciallygrown
cultivars by genetic transformation, a regeneration and transformation
protocol using the shoot apical meristem and Agrobacterium-mediated DNA
transfer was successfully developed, to obtain herbicide (Basta) resistant
commercially-grown South African soybean and cotton plants for the first time.
The frequencies of regenerated plants per meristem were 66% for Talana, 52% for
Ibis, 90% for Sabie, 74.6% for LRCC 101, 69.5% for Palala and 70% for 107/1.
Prior to transformation experiments, Talana and Ibis were screened for
susceptibility to virulent Agrobacterium tumefaciens sis 43. Both cultivars
produced tumours in response to infection and were therefore compatible hosts for
Agrobacterium-mediated DNA transfer. Transformation of Talana, Sabie and
107/1 with the pat gene, was successfully achieved following wounding of the
shoot apical meristem and injecting with Agrobacterium in the presence of
acetosyringone. Transformed explants and shoots grew in the presence of
kanamycin and PPT, indicating that the integrated pat gene was producing the
enzyme PAT which was successfully detoxifying the herbicide PPT. Final
transformation frequencies from the initial transformed meristems to regenerated
plants were 1.06% for Talana, 2.3-3% for Sabie and 1.2-2.3% for 107/1. These
transformation frequencies were higher than those reported in the literature. PCR
analysis of the extracted DNA from transgenic soybean and cotton shoots
confirmed the presence of the 558 bp pat coding region in the transformed plants.
The success of this study on the regeneration and transformation of soybean and
cotton indicates that South African agriculture now has available techniques for
plant regeneration and recombinant DNA technology for crop improvement of
soybean and cotton.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/5880 |
| Date | 09 December 2008 |
| Creators | McNaughton-Pascoe, Caeleen |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
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