Thesis (Ph. D.)--Murdoch University, 2006. / Thesis submitted to the Division of Science and Engineering. Includes bibliographical references (p. 191-207).

Thesis (M.S.)--Virginia Polytechnic Institute and State University. / Vita. Abstract. Includes bibliographical references (leaves 55-60). Also available via the Internet.

Thesis (Ph. D.)--University of Wisconsin-Madison, 1944. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

M.Sc. (Botany) / Please refer to full text to view abstract

M.Sc. (Botany) / Please refer to full text to view abstract

M.Sc. / Ophiopogon planiscapus var. nigrescens belongs to the family Convallariaceae. This plant exhibits unique black pigmented leaves and is, therefore. used in growing arrangements. The natural rate of multiplication. however. is slow and a reversion to green leaves in some plants necessitates the replacement of these plants, which is a time consuming activity. In vitro techniques allow the establishment of black clones and also speed up the multiplication process. A cultivation medium for optimal growth was formulated. The influence of other parameters on growth. multiplication and rooting was investigated and final hardening was done to yield plants suitable for the greenhouse.

The work on Spiraea in vitro shoot cultures was done to determine the feasibility of using light quality to modify endogenous phytohormone balances to decrease apical dominance. Such an effect would enable a reduction in the high levels of exogenous cytokinin benzyladenine (BA) applied in culture and thus reduce potential side-effects. The Spiraea in vitro light quality response was characterized by examining the effects of different light wavelengths on growth. A mixture of red/FR induced rates of shoot proliferation with 0.25 mg/1 BA that were as high as rates obtained under white light with 0.5 mg/1 BA. Shoot quality, as determined by the proportion of shoots 1 cm or longer (useful shoots), was highest under red/FR light. The lowest shoot proliferation rate was observed under blue light. When light wavelengths intermediate between blue and red light (green, yellow, and orange) were applied to explants only minor growth modifications occurred. Green light did not inhibit shoot initiation but inhibited shoot elongation at the 0.5 mg/1 BA level. The efficacy of the light source-filter combinations in the first experiment was studied in two further experiments. With the three light sources (tungsten filament, fluorescent, and metal halide) together with a blue filter, results supported the putative blue light inhibitory effect suggested in the first light quality experiment. Under the red filter, the tungsten filament source induced the highest shoot number means at both BA levels used (0.25 and 0.5 mg/1). Two factors may have contributed to the red/FR effect observed in the first experiment; the time under an incubation light regime before transfer to the treatment regime, and the photon fluence rate of each regime. In the subsequent study to examine these factors, shoot initiation was optimized at the lower BA levels of 0.25 and 0.4 mg/1 when cultures under low fluence red/FR were transferred after four weeks to white light of a higher fluence for one more week. Glyphosate, a known promoter of IAA oxidation, was used to investigate the presumed effect of lowered IAA-cytokinin interactions. Two types of responses to glyphosate occurred, each one dependent on the glyphosate concentration. At the lower glyphosate level (0.087 mg/1), cultures under both light regimes with 0.25 mg/1 of BA, showed a strong inhibition of shoot initiation. This inhibitory effect was overcome in cultures with 0.5 mg/1 of BA and an overall stimulatory response occurred as shoot initiation rates were as much as four-fold higher than in the previous experiments. For both BA levels, changes in shoot number were greater under white light than under red/FR. At the higher glyphosate level (0.2 67 mg/1), the shoot initiation rates were greater than glyphosate-free controls for both BA levels under white light although under red/FR the rates were virtually unchanged from controls. The glyphosate effect investigated for Spiraea cultures appears to be influenced by the levels of the cytokinin BA resulting in pleiotropic effects which depend on the specific concentrations of each component. / Land and Food Systems, Faculty of / Graduate

The propagation of wild-type virus and a transgenic viral vector was examined in cultured plant cells to identify factors affecting viral infection of and accumulation in cultured cells and to determine if viral vectors could be used to facilitate the expression of heterologous proteins in vitro. Tobacco mosaic virus (TMV) accumulation was examined in Nicotiana tabacum and N. benthamiana suspension and hairy root cultures. TMV accumulation was superior in N. benthamiana hairy roots. Hairy roots were infected by adding TMV to the liquid culture medium at the same time as root inoculation. Hairy root growth was unaffected by virus infection. The distribution of virus within root mats from shake-flask grown cultures was non-uniform and the concentration of virus accumulated in replicate cultures was variable. When N. benthamiana hairy roots were infected using 1.5 μg mL-1 TMV, the average maximum concentration of virus accumulated in the biomass was 1.6 ?? 0.25 mg g-1 dry weight, or 15-fold lower than in TMV infected N. tabacum leaves. Virus coat protein accumulated to a level of (26 ?? 10)% total soluble protein in the hairy roots. Inoculum virus concentration and the medium in which infection was performed affected the virus yield and the percentage of inoculated cultures that accumulated virus. When cultures were inoculated using 9.0 μg mL-1 TMV, virus accumulated in the biomass to 4.2 ?? 0.60 mg g-1 dry weight. Proportional scale-up of hairy root infection in shake flasks did not result in constant virus concentrations in the scaled cultures. TMV accumulation in bioreactor-infected and -grown hairy roots was poor. N. benthamiana hairy roots were infected with a TMV-based vector (30B-GFPC3) that encoded Cycle 3 green fluorescent protein (GFP). TMV-GFPC3 was (260 ?? 140)-fold less infectious than TMV as measured by local lesion assays. Propagation of TMV-GFPC3 could not be confirmed using mass balance. GFP was not detected in the infected hairy roots or the culture medium. Hairy roots represent a potentially viable culture-based system for the in vitro production of virus and virus products when field-grown agricultural systems do not adequately address issues of containment or product safety.

No description available.

Tissue-cultured shoots and plantlets usually have leaves with non-functional, open stomata and little epicuticular and cuticular wax, resulting in excess evapotranspiration after transplantation. Various strategies were evaluated to decrease ex vitro acclimatization difficulties for 'Silvan' blackberry, including transplanting unrooted shoots, increasing the medium agar concentration from 6 to 9 or 12 g/l and diluting the basal medium. Increased medium agar concentrations and medium dilution did not improve survival or growth. Stomatal function resumed sooner in new leaves of plantlets than shoots. High relative humidity ($>$95%) and low light intensity (90 $ mu$mol s$ sp{-1}$ m$ sp{-2}$) negatively affected stomatal closure both on acclimatizing transplants and greenhouse-grown plants. Guard cells developed on leaves in vitro were physiologically active but had apparent anatomical abnormalities that inhibited closure. A rapid clearing and staining method was developed for examination of foliar morphology using intact in vitro blackberry (Rubus sp. 'Silvan') and strawberry (Fragaria x ananassa Duch. 'Totem') plantlets and sections of greenhouse-grown 'Silvan' and 'Totem' leaves. This method involved three steps: (1) removing the chlorophyll by autoclaving in 80% ethanol; (2) dissolution of the protoplasm using 5% NaOH at 80$ sp circ$C; (3) post-alkali treatment with 75% bleach (4.5% NaClO) at room temperature for tissue-cultured plantlets and at 55$ sp circ$C for greenhouse-grown leaves. Aqueous safranin (10 mg/l) was used for staining.