Studies on the genetic engineering of herbicide resistance into South African tobacco cultivars.

Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1994.

Tobacco is an important crop in South Africa. The genetic basis of tobacco breeding is very narrow and cultivars are closely related. The production of new tobacco hybrids with novel characteristics through classical breeding techniques is difficult. Genetic engineering could assist plant breeders to introduce new herbicide, disease and pest resistance traits into existing proven cultivars. Plant genetic engineering has not previously been applied to the improvement of South African commercial tobacco cultivars. Agrobacterium-mediated leaf disc transformation was used to create transgenic tobacco plants from South African commercial tobacco cultivars TL33, J6 and 20/19. The cultivar samsun was also used to create transgenic plants. The Agrobacterium tumefaciens helper strain C58C1 (pGV2260) containing the binary vector pJIT119 was used to carry out the transformation. As well as the leaf disc transformation method, other methods of obtaining transgenic tobacco plants were explored. These methods included the use of Agrobacterium-mediated transformation of tobacco cell cultures and direct DNA-mediated transformation of tobacco protoplasts. The vector pJIT119 encodes the uidA gene for the β-glucuronidase (GUS) enzyme, the nptl/ gene for neomycin phosphotransferase (NPTII) and the sul I gene for the dihydropteroate enzyme conferring asulam resistance. The presence and expression of these three foreign genes uidA , npt/l and sul I from pJIT119 in transgenic tobacco plants was confirmed by a variety of experimental approaches, including the culture of transgenic plants on medium containing kanamycin or asulam, the GUS histochemical assay, the neomycin phosphotransferase assay, DNA dot-blot analysis, in situ hybridization, computerized image analysis, polymerase chain reaction and progeny analysis. A detailed analysis of individual transgenic plants is necessary in order to select those plants which express the foreign genes maximally. Only these plants would be given to plant breeders for field trial assessment. A high level of foreign gene inactivation was observed in transgenic tobacco plants obtained from the Agrobacterium-mediated leaf disc transformation method. Approximately 20% of the original transgenic plants were discarded as "escapes" as they contained a defective npt/l gene. The remaining kanamycin resistant plants, however, had inactive copies of either the sul I or the uidA gene, or both. The use of in situ hybridization and the polymerase chain reaction (PCR) helped to explain the foreign gene inactivation. The lack of foreign gene expression in individual transgenic plants was not due to the physical loss of entire foreign genes, DNA methylation or the position effect. The lack of expression was due to possible T-DNA rearrangements or deletions which disabled certain genes carried on the T-DNA. Transcription and translation of these foreign genes occurred, but the final uidA and sul I gene products (β-glucuronidase and dihydropteroate synthase, respectively) were possibly defective and did not confer GUS activity or asulam resistance on the transgenic plants The tissue specific activity of the uidA gene under the control of the cauliflower mosaic virus (CaMV) 358 promoter was studied. In the vegetative structures of transgenic tobacco plants, the uidA gene activity was located within the cells surrounding the vascular traces and within the glandular hairs. The effects of stress on 358 promoter activity was also investigated. Chemical and nutrient stess in vitro did not have a significant effect to decrease uidA gene expression under 358 promoter control. Foreign gene expression (uidA) under CaMV 358 promoter control may be enhanced by in vitro stress. Oxygen stress (anaerobic culture under waterlogged conditions) induced uidA expression in areas of the plant which usually did not show usual tissue specific patterns of uidA expression. The stage of differentiation in tissue culture when compared to the mature hardened off transgenic plant, also had an effect on the amount of uidA gene expression. Mature hardened off plants expressed less GUS activity than immature in vitro plants. The tissue specific pattern of foreign gene expression under CaMV 358 promoter direction was conserved in the reproductive structures of transgenic tobacco plants. In floral organs, the pattern of uidA gene expression was essentially the same as that found in vegetative tissues. In all floral organs examined, uidA expression was found associated with the vascular system and within the glandular hairs. The uidA gene with a CaMV 358 promoter was not expressed in pollen. Because of the ease of transformation of tobacco, it is possible that genes for pharmaceutically valuable proteins and peptides could be expressed in tobacco, for agricultural scale fine chemical production("pharming"). This could be of economic advantage for the survival of tobacco as a commercial agricultural crop in the future when tobacco smoking is no longer popular.