No description available.

No description available.

Regeneration and transformation protocols for 'Comet' red raspberry were optimized with the purpose of making the Agrobacterium-mediated gene transfer system efficient for this crop. Adventitious shoot regeneration from leaf discs was improved using explants 10 mm in diameter and transferring to fresh medium at the fourth week of incubation. Additions of liquid medium to solid medium during incubation decreased regeneration and attempts to release the suppressive influence of larger shoots on initials (apical dominance) did not succeed. The presence of claforan did not affect shoot regeneration, but inoculations with Agrobacterium and the presence of kanamycin decreased regeneration moderately or considerably, respectively. The threshold for kanamycin concentration for screening for kanamycin resistant transformed raspberry tissue was 30 to 40 mg l$ sp{-1}.$ The best co-incubation interval between wild-type Agrobacterium and 'Comet' leaf discs ranged from 2 days for highly virulent strains to 3 or more days for moderate to low virulent strains. Among several wild-type strains, C58 was chosen as the most appropriate partially because a disarmed form was commercially available for use as a non-oncogenic vector for transformation of red raspberry. / The binary plasmid pBI121 containing the marker genes NPTII and GUS encoding kanamycin resistance and $ beta$-glucuronidase activity, respectively, was successfully introduced into the Agrobacterium strain LBA4404, which is a disarmed C58 derivative. Transformation of 'Comet' red raspberry was apparently achieved by inoculating leaf disc explants with LBA4404 containing pBI121. The probable integration and expression of the foreign genes into the plant cells were confirmed by screening for kanamycin resistance, GUS assays and Southern blot analyses. This transformation system appears to be effective and may be useful in further studies on red raspberry for both introduction of genes for desirable agronomic traits and basic studies of gene expression.

Schwanniomyces occidentalis (Debaryomyces occidentalis ) is able to grow rapidly with high cell mass on cheap starch as a carbon source, produce strong amylolytic enzymes extracellularly and secrete large proteins without hyper-glycosylation and measurable extracellular proteases. Schw. occidentalis thus has a high potential as, a useful alternative to Saccharomyces cerevisiae in the production of heterologous proteins. However, the molecular study of Schw. occidentalis has been very limited due to the insufficient transformation system and lack of gene expression information. / A new transformation system of Schw. occidentalis has been developed. This system was based on vector YEp13 ( LEU2) and a stable leu auxotrophic mutant, Schw. occidentalis DW88, obtained by treating the yeast with 1-methyl-3-nitro-1-nitrosoguanidine. The transformation efficiency of YEp13 by spheroplast-mediating method was 103 transformants/mug DNA. The 2-mum replicon is proposed to be responsible for YEp13 replication in Schw. occidentalis. The YEp13 stability in Schw. occidentalis was low, but it kept its structure in the yeast, suggesting that Schw. occidentalis DW88 does not modify foreign DNA. / After analysis of 14 cloned Schw. occidentalis genes and comparison of associated genes from both Schw. occidentalis and S. cerevisiae, 25 codons were arbitrarily chosen as putative preferred codons for Schw. occidentalis. They are similar to those of S. cerevisiae, except for TTA for leucine, and AAA for lysine. Codon Bias Index (CBI), a criterion to evaluate gene expression, is calculated from preferred codons. A computer program (PCBI) which reads a gene containing introns was developed to quickly calculate CBI. / Schw. occidentalis DWSS should be a good host to produce and secrete heterologous proteins and the putative preferred codons and program PCBI can facilitate molecular study of Schw. occidentalis. (Abstract shortened by UMI.)

Dissertation (PhD)--University of Stellenbosch, 2006. / Bibliography. Also available via the Internet.

An Agrobacterium-mediated transformation system of purple raspberry ‘Amethyst’ was established after a series of experiments that determined the effect of genotype, inoculum density, and co-cultivation time on transformation. In this study, a plant regeneration protocol was established for ‘Joan J’ and ‘Polana’ (the regeneration protocol of ‘Amethyst’ was previously developed). Agrobacterium-mediated transformation was conducted for all three cultivars. The minimum killing level of hygromycin B and kanamycin was determined. Inoculum density and co-cultivation time were optimized. Polymerase chain reaction (PCR) verified a successful transformation of ‘Amethyst’ with the frequency of 3.3 ~ 4.4 % when leaves were infected with Agrobacterium EHA105 at the cell density of OD600 0.3 and co-cultivated for 3 days in the medium with 25.0 mg∙l-1 kanamycin. Transgenic lines with the PtFIT gene were hydroponically grown under iron sufficiency or deficiency. The real-time quantitative PCR verified the gene expression in response to iron sufficiency and deficiency conditions.

No description available.

Regeneration and shoot multiplication of common bean (Phaseolus vulgaris L. 'ICA Pijao') from half-cotyledonary nodes was achieved on modified Murashige and Skoog (1962) basal medium amended with 5 $ mu$M 6-benzylaminopurine. Histological studies confirmed the adventitious origin of the regenerated buds. Shoots were rooted ex vitro and developed into morphologically normal plants compared with seed-grown controls. The relative susceptibility of bean tissues to infection by a collection of wild-type Agrobacterium strains was tested. Positive transformation events were evaluated based on morphological and biochemical changes observed following Agrobacterium infection. The A. tumefaciens strain C58 was particularly virulent on greenhouse-grown plants, in vitro-derived stem sections, half-cotyledonary nodes and seedlings. A sensitive and rapid method was developed to detect opines using thin layer chromatography. Transient $ beta$-glucuronidase (GUS) gene expression was detected in 'ICA Pijao' bean buds regenerated from half-cotyledonary nodes following Agrobacterium-mediated gene transfer with the binary vector pGV1040 or p35SGUSINT. Four out of eight putative transformants contained the chimeric GUSINT gene following polymerase chain reaction (PCR) analysis. This was confirmed by Southern analysis of blotted PCR gels. However, there was no stable integration of the GUSINT gene as none of the R1 progeny showed an amplified GUSINT fragment with PCR.

No description available.

Thesis (M. Sc.)--University of Canterbury, 2008. / Typescript (photocopy). Includes bibliographical references (leaves 62-79). Also available via the World Wide Web.