No description available.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1935. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 34-37).

Thesis--Leyden. / In Periodical Room.

This project is focused on transformation of Silene vulgaris mediated by Agrobacterium tumefaciens and A. rhizogenes. S. vulgaris is a good model plant to study gynodioecy, an evolutionary step from bisexuality to dioecy. Gynodioecious plants form in some individuals bisexual flowers, while the others possess only female flowers. The aim of this research is do develop a technique to introduce foreign genes into this plant to study its developmental consequences. Using A. rhizogenes we successfuly prepared hairy root cultures, which unfortunately do not form shoot regenerants. We have prepared a protocol to induce plant regenerants from S. vulgaris leaf fragments. The first results do not confirm that A. tumefaciens infected plant regenerants harbor reporter transgenes. We used Nicotiana tabacum as a positive control.

published_or_final_version / Botany / Doctoral / Doctor of Philosophy

No description available.

No description available.

No description available.

Cauliflower (Brassica oleracea var. botrytis) is described as a recalcitrant plant to genetic transformation processes especially Agrobacterium-mediated and as an extremely low frequency event then it requires a large amount of explants for this procedure to succeed. This thesis describes the development and refinement of a mass propagation system for cauliflower micropropagation and its use for overcoming recalcitrance to genetic transformation. Shoot meristematic tissue was taken from the curd of cauliflower and used to establish in-vitro cultures in liquid medium. Explants were cultured in a Murashige and Skoog (MS) medium containing various plant growth regulators combinations to induce shoot regeneration and which were optimised to be 2 mg L-1 (9.29 μM) kinetin and 1 mg L-1(4.9 μM) IBA. Shoots were cultured for 4–6 weeks to obtain rooted plants, which were then suitable for weaning and subsequently produce fully- developed in-vivo plants in pots in soil with a 95%+ success rate. A procedure for detection of the presence of insert DNA in recombinant plasmids in individual Agrobacterium tumefaciens strains was refined. Cauliflower was transformed using the EHA105 strain of A. tumefaciens harboring the binary vector pPRTL2 plasmid carrying the antioxidant gene Ascorbate peroxidase (APX) for increased stress resistance coupled with neomycin phosphotransferase II (nptII) for resistance to kanamycin and β-glucuronidase (GUS) as a marker gene. Selection was carried out in MS medium containing kanamycin (50 mg L-1), and surviving tissues were then tested by histochemical GUS assay.Agrobacterium-mediated plant genetic transformation requires a two-step process for its success: selection and regeneration of transformed tissues, and the elimination of the transformation vector (Agrobacterium). This study used carbenicillin and cefotaxime in MS media to eliminate A. tumefaciens, at selection levels of 25 and 50 mg L-1 kanamycin. Kanamycin severely reduced explant growth and regeneration of control cultures at concentrations as low as 10 mg L-1 and completely inhibited shoot organogenesis at 50 mg L-1. The integration of APX gene into putative transformant lines was confirmed using GUS and leaf disc assays. Genomic integration of the gene cassette was optimised using PCR analysis with primers flanking npt II and CaMV promoter regions. The stable integration of the APX gene in the putative transgenic plants was detected using PCR at 478bp. The result confirmed the first report of transformation with APX gene in Brassica oleracea. Thus, a protocol for effective Agrobacterium-mediated genetic transformation of cauliflower was optimized. Transformed and control lines were sub-cultured many times on maintenance medium over 2 years without any loss of the transgene and then tested for salt resistance as in-vitro and in-vivo plants using a leaf disc assay. Control plants had little or no NaCI resistance whilst transformed plants showed varying degrees of resistance. Analysis of APX gene expression under salt treatment showed that putative transgenic cauliflower survived salinity stress compared with control plants. Non-acclimated and acclimated in-vivo plants were also assessed for resistance to frost. Both non-acclimated and acclimated APX transformed lines showed improved frost resistance compared to controls. The results clearly confirmed that NaCI and frost resistance were stable traits attributable to improved APX expression.

Agrobacterium tumefaciens and A. rhizogenes are the causal agents of gall or hairy root disease, but normally the bacteria do not cause disease in wheat. However, both bacteria grew without inhibition when exposed to intact or wounded wheat roots or embryos, and they colonized wheat root surfaces to levels similar to dicotyledonous plants. A. tumefaciens and A. rhizogenes induced 23% cell death after a 1-h exposure to wheat embryo cells grown in 7.4 mM O2, while the extent of cell death at 2.1 mM 02 was 8%. Contact with A. tumefaciens or A. rhizogenes caused cultured wheat embryo and root cells to rapidly produce H202, which decreased when embryos and roots were cultured at 2.1 mM O2. Browning and autofluorescence, and an increase in ferulic acid in cell walls, were observed in wheat embryo and root epidermal cells exposed to Agrobacterium, but . neither lignin nor callose was detected. Agrobacterium appeared to induce resistance-like responses in wheat that may limit transformation efficiency. The inability to regenerate wheat plants using tissue culture has been a limitation to high efficiency transformation. Regeneration via somatic embryogenesis was improved significantly by simulating the in ovulo environment to which the immature wheat embryos are exposed. Triticum embryo culture medium (TEC) improved callus formation, somatic embryo formation, and regeneration from somatic embryos while reducing precocious germination when compared to growth on Murashige and Skoog medium. Regeneration frequencies were improved when embryos were cultured at the O2 concentration found in the wheat ovule (2.1 mM O2) rather than atmospheric 02concentration (7.4 mM O2). Agrobacterium-mediated transformation of wheat was limited by tissue necrosis following co-cultivation, and by poor plant regeneration. Reduction of necrosis and increased plant regeneration were accomplished by amending the culture medium with antioxidant compounds and by reducing the O2 tension in which the wheat embryos were cu1tured. Twelve days past anthesis (DPA), wheat embryos were co-cultivated with Agrobacterium tumefaciens strains WAg 11 or EHA 101, incubated on TEC medium containing antioxidant compounds (catalase, cysteine and ascorbic acid), and cultured at 2.1 mM O2 concentrations. Transformation was documented in 6.0% ofregeneratedA. tumefaciens WAg 11 exposed wheat plants using the firefly luciferase (luc) reporter system.