Leaf size and shape are strongly influenced by the growth patterns of the epidermal tissue. Pavement cells are the prevalent cell type in the epidermis and during cell expansion they undergo a drastic shape change from a simple polyhedral cells to puzzled-shaped cell. The role of these cell protrusions, more commonly referred to as lobes, remains unknown but their formation has been proposed to help increase the structural integrity of the epidermal tissue. How the symmetry breaking event that initiates a lobe is controlled remains unknown, however pharmacological and genetic disruption of the microtubule system has been shown to interfere not only with lobe initiation but also with lobe expansion. Additionally, the role of microtubules in the pattering of microfibril deposition, the load-bearing structure of the cell wall, makes the microtubule system a good candidate to evaluate its dynamics as a function of shape change. Two main mechanical models for lobe initiation are evaluated here, one where microtubules serve as stable features suppressing local expansion and one where microtubules, similarly to the anisotropic expansion patterning in hypocotyl cells, pro-mote the local anisotropic expansion of the cell resulting in lobe formation. The main method to evaluate these models was through the use of long-term time-lapse image analysis using a plasma-membrane marker for accurate shape change quantification and a microtubule marker to quantify their location, persistence, and density as a function of cell shape change. Using the junctions where three cells come together,cells were sub-divided into segments and the shape of these segments were tracked using a new coordinate system that allowed the detection of new lobes as which can arise from ∼300 deflections. By mapping sub-cellular processes, such as microtubule persistence, to this coordinate system, correlations of microtubule organization and shape change was possible. Additionally, a subset of microtubules bundles that splay across the anticlinal and periclinal walls, perpendicular and parallel to the leaf surface respectively, were identified as marking the location and direction of lobe formation.Disrupting the cell boundary by partially digesting pectin, a main component in the middle lamella, revealed the cell-autonomous morphogenesis mechanism in pavementcells. Under pectinase treatment, cell invaginations were produced and similarly to lobes their initiation was microtubule and cellulose dependent. Lastly, stress prediction using finite-element models, based from live-cell images, co-localized regions of high cell wall stress with both microtubule persistence and shape shape locations in both lobing and invaginated segments. Together, a model of cellular shape change is presented where microtubules translate cell wall stresses to tissue morphogenesis.

Given the drawbacks associated with the use of antibiotics as feed additives and the imminent banning of its use in the European Union, the aim of this project was to develop an extract that could be used as an alternative feed additive in poultry production. The desired extract preferably had to be rich in antibacterial activity to control proliferation of undesired microorganisms, and antioxidant activity to boost the immune system of the poultry. A number of trial extraction procedures were employed on dried leaf material samples to identify the best extraction method. In preliminary extraction studies, direct extraction was performed on leaf samples from the Lowveld National Botanical Gardens (LNBG) and from University of Pretoria Botanical Garden (UP). The principle aim of preliminary studies was to identify the solvents that extracted high antibacterial and antioxidant activity while also extracting large quantities of material. The secondary objective was to test for differences in activities between samples collected from LNBG and UP. Five extractants of varying polarities; acetone, ethanol, ethylacetate, dichloromethane and hexane were used. Antibacterial activity of all extracts was quantified by a serial dilution microplate technique while bioautography was used in qualitative analysis of the antibacterial active compounds. ATCC strains of Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Enterococcus faecalis were used as test organisms. Qualitative antioxidant activity was determined by using a DPPH assay on TLC plates. Results from preliminary extraction studies showed larger quantities of material were present in extracts from the LNBG sample than in the UP sample. Two major antioxidant compounds (Rf values of 0.85 and 0.35 in EMW solvent system) were seen on DPPH sprayed TLC plates, while bioautography showed the presence of a number antibacterial active compounds in the acetone, ethanol and ethylacetate extracts with Rr values ranging between 0.85 and 0.56 on TLC plates developed in the EMW solvent system. MIC values of the extracts tallied with the results from bioautography. The acetone, ethanol and ethyl acetate extracts had the highest antibacterial activity while the hexane extracts had the lowest activity with average MIC value of 0.55 mg/ml for both the LNBG and UP samples. MIC values as low as 0.04 mg/ml were measured in the acetone and ethylacetate extracts of the LNBG sample against S. aureus and E. faecalis. Based on results from preliminary extraction studies, hexane was identified as a possible pretreatment solvent for application in enrichment procedures, acetone and ethanol were chosen as the main extractants and only the LNBG sample was used for future work. Enrichment procedures were employed along two pathways; the first pathway involved the use of hexane "wash" as a pretreatment procedure prior to extraction with acetone or ethanol. The second pathway involved the use of various mixtures of acetone in water and ethanol in water as extractants. The rationale of using these various ratios was an attempt to identify solvent mixtures that would selectively extract the bioactive components or otherwise selectively remove inactive material. A serial dilution microplate method was used to determine Minimum Inhibitory Concentrations (MICs) and the Trolox Equivalent Antioxidant Capacity (TEAC) assay was used to quantify antioxidant activity of all extracts. The optimal extract was the one developed by pretreatment with a single direct extraction with hexane prior to extraction with acetone. It had a TEAC value of 2.3, an increase in TEAC value of 283% compared to that of the crude acetone extract. The average MIC of the crude acetone extract against ATCC stains of S. aureus, Ps. aeruginosa, E. coli and E. faecalis had dropped from 0.15 mg/ml to 0.08 mg/ml in the optimal extract (an improvement in antibacterial activity of 87.5%). Since the optimal extract is intended for commercial application in poultry production, its antibacterial activity was tested against Campylobacter jejuni, Clostridium perfringens, Salmonella enteritidis, E. coli and multi drug resistant E. coli isolated from chickens. Its in vitro toxicity was ascertained using the brine shrimp assay and the MTT cytotoxicity assay on monkey kidney cells. The optimal extract was effective against Campylobacter jejuni and Clostridium perfringens with MIC values ranging from 40 µ/ml to 80 µ/ml. It was also active against multi-resistant strains of E. coli and Salmonella enteritidis (MIC values of 125 µ/ml for both strains). LC50 results from the brine shrimp assay and the MTT cytotoxicity assay on monkey kidney cells gave values of 863 µ/ml and 226 µ/ml respectively indicating low toxicity. These results meant that though in some cases the MICs of the optimal extract were higher than befitting of typical antibiotics, due to its relatively low toxicity, large quantities of the extract may possibly be feed to achieve the desired activity without causing any toxicity in the poultry. The major antioxidant compound was isolated by silica gel column chromatography. The isolated compound was identified by nuclear magnetic resonance and mass spectroscopy as combretastatin BS (2', 3', 4-trihydroxyl, 3, S, 4'-trimethoxybibenzyl), previously isolated from the seeds of C. kraussii and also from C. woodii leaves. Famakin (2002) showed this compound to be the major antibacterial compound in C. woodii leaves. Combretastatin BS (CBS) demonstrated in vitro cytotoxicity in the MTT assay on monkey kidney cells with an LC50 value of 1 0 µ/ml. In vitro cytotoxicity of CBS could be due to its antimitotic activity. The TEAC value of 7.9 found in this study means that combretastatin BS has about 8 times the antioxidant capacity of vitamin E. This is the first report of the antioxidant activity of any of the combretastatins. Tolerance of broiler chickens to the optimal extract was assessed at clinically inferred doses of 2 mg/kg, Smg/kg and 10 mg/kg . After 21 days of infeed-dosing with the optimal extract, none of the chickens died or showed any behavioral signs of toxicity. There were no statistically significant differences in weight gain between broilers fed the optimal extract and the positive and negative control. There was also no positive correlation between weight gain and amount of the optimal extract incorporated in feed. Although the optimal extract did not result in significant growth promotion relative to the positive and negative control, 2 mg/kg dose regimens showed the best Feed Conversion Ratio (FCR), with a 6.2% improvement compared to the negative control. The positive control was the only other feed regimen to provide a positive FCR with an improvement of 1.73% compared to the negative control. Because purchase of feed could represent up to 80% of costs of broiler production, this is an important finding. If these results can be confirmed, the product may therefore have commercial value. Repetition of the experiment with lower doses of the optimal extract on poultry challenged with bacterial infections is required to confirm the commercial applicability of this product. Copyright / Dissertation (MSc (Paraclinical Science))--University of Pretoria, 2004. / Paraclinical Sciences / unrestricted

Continental scale analysis of the savanna biome indicated that fire did not spread at tree canopy cover above 40%. This study investigates this relationship in a field study. It is possible that the type of tree (forest vs. savanna) may influence the amount of shade experienced by the understory and therefore this study also explores differences in LAI between congeneric pairs of forest and savanna tree species. Data were collected in two major South African savanna parks. Plots were set out to measure grass biomass in reference to canopy cover in both Kruger National Park (n=60) and the Hluhluwe-iMfolozi Game Reserve (n=82). Seven congeneric pairs were selected to compare leaf area and LAI between forest and savanna tree species using a destructive method. Against expectations, it was only when canopy cover reached 80% that grass fuel load was too low to support fire spread in all Kruger National Park plots (Pr=O) and 89% of the Hluhluwe-iMfolozi Game Reserve plots (Pr=0.11). No consistent, general relationships were evident with leaf area or LAI in comparisons between forest-savanna congeneric pairs. The significance of these findings and future direction is discussed.

Species are experiencing shifts in their phenology (i.e., seasonal timing of recurring biological events) due to climate change, leading to disruptions in the relative timing of interacting species. These shifts can be detrimental to the fitness of the consumer (e.g., herbivore) in the interaction. In its larval form, the monarch butterfly (Danaus plexippus) is a specialist herbivore that feeds on milkweed plants (Asclepias spp.). Given that plants generally experience seasonal declines in quality, it is hypothesized that if climate change disrupts the timing of the larval stage relative to the availability of younger milkweed plants, monarch performance will be negatively affected. In this thesis, I explore the potential for negative consequences for the eastern monarch population due to potential shifts in the timing of their interaction with milkweed—due to phenological shifts in either species. I used field surveys around Ottawa, ON to determine monarch oviposition preference on common milkweed (Asclepias syriaca) plants and the seasonal availability of their preferred plants. To determine the potential consequences for monarch fitness where females oviposit on non-preferred plants, I conducted a field experiment to assess the effect of milkweed size on monarch larval performance. Based on field surveys, females preferentially oviposited on smaller milkweed plants in earlier developmental stages with low levels of discolouration. Plants in early developmental stages were consistently available in large proportion over the summer season. These results suggest that even if the relative timing of the monarch-milkweed interaction in the eastern population is shifted due to climate change, there will likely be suitable milkweed plants available for oviposition throughout the breeding season, which could act as a buffer to disruptions in the relative timing of the interaction. I found that bigger plants exuded more latex and had thicker leaves than smaller plants. However, larval performance was unaffected by these plant quality differences. While it is unclear how the relative timing of the monarch-milkweed interaction will change in the future, my results suggest that shifts in the relative timing of their interaction within the breeding season are unlikely to have negative consequences for larval performance in eastern Ontario. Future studies should determine how the relative timing of the interaction will change in the region and explore how climate change will affect the quality of milkweed plants.

Medicinal plants have played an important role in drug discovery, with many pharmaceutical products originating from plants. Isolation and characterization of antibacterial compounds is still relevant today because of continuing development of resistance of bacteria to antibiotics. The aim of the study was to evaluate the antibacterial activity of leaf extracts of nine tree species (Acalypha sonderiana, Androstachys johnsonii, Dracaena mannii, Drypetes natalensis, Funtumia africana, Necepsia casteneifolia, Oncinotus tenuiloba, Turraea floribunda, and Xylia torreana) selected from the Phytomedicine Programme Database based on good antimicrobial activities. The next step was to select the most active plant species and to isolate and characterize the antibacterial compounds. A serial microplate dilution method was used to determine the minimal inhibitory concentration and bioautography was used to determine the number of antibacterial compounds in the extract and their Rf values. Four nosocomial infection pathogens (Escherichia coli, Enterococcus faecalis, Pseudomonas aeruginosa, and Staphylococcus aureus) were used as test organisms. Extracts of all the plant species were active with average MIC values ranging from 0.13 to 2.0 mg/ml against the four bacteria. MIC values as low as 0.08 mg/ml was obtained with F. africana and O. tenuiloba extracts against S. aureus. In bioautography seven of the nine leaf extracts had activity with clear zones of inhibition on bioautograms against the red background. F. africana was active against all four bacteria while O. tenuiloba had selective activity against P. aeruginosa with clear bands on the bioautogram. F. africana was chosen for further investigation because (a) it had good antibacterial activity against the four tested bacteria with MIC value as low as 0.08 mg/ml, (b) there were several active compounds against all the tested bacteria based on bioautography, (c) it is common in nature, and (d) as far as our literature survey could ascertain there was no published information on the antimicrobial activity of this plant species. The bulk powdered leaves of F. africana were extracted with acetone. The acetone extract was fractionated into five fractions (hexane, chloroform, butanol, H2O and 30% H2O in methanol) using solvent-solvent fractionation, to group the phytochemicals based on their polarity. Hexane and chloroform fractions were the most active with MIC values as low as 0.02 mg/ml for the chloroform fraction. One of the traditional uses of F. africana is to treat burns. As a result, the crude extract and its five fractions were also tested for anti-inflammatory activity using both the COX-1 and COX-2 assays. The crude extract and the hexane and chloroform fraction had moderate activity against both cyclooxygenase 1 and 2. The chloroform fraction was more active than the crude extract (59.7 ± 1.4%)with an inhibition of 68.2 ± 6.6%. Because there was no activity in the aqueous extracts and traditional healers usually use water as extractant, the pain relief experiences traditionally must be due to another anti-inflammatory mechanism. One antibacterial compound was isolated from the hexane fraction using column chromatography with silica gel as the stationary phase and a hexane ethyl acetate gradient as the mobile phase from low to high polarity. The isolated compound was identified as methyl ursolate using nuclear magnetic resonance (NMR) and mass spectrometry. Methyl ursolate has been isolated from a number of plant species. However, this is the first report on the isolation from Funtumia genus and the first report of its antimicrobial activity. Previous phytochemical investigation from the stem bark of F. africana led to the isolation of steroidal alkaloids of the conanine group. Methyl ursolate had a low activity with MIC values of >250 μg/ml against the four tested bacteria, but had better activity against five fungal (Candida albicans, Cryptococcus neomeforms, Fusarium oxysporum, Penicillium janthinellium, and Rhizoctonia solani) species with an MIC value of 63 μg/ml against F. oxysporum. The chloroform fraction had excellent activity with an MIC of 20 μg/ml and may be developed to become a useful complex drug. The more than one hundred fold lower activity of the isolated methyl ursolate compared to the activity of the chloroform fraction from which it was isolated, provides strong evidence of synergism. This may be good model system for studying synergism in antimicrobial preparations. / Dissertation (MSc)--University of Pretoria, 2010. / Paraclinical Sciences / unrestricted

Quercus praedelavayi Xing Y.W. et Zhou Z.K. sp. nov. is reported from the upper Miocene of the Xianfeng flora in central Yunnan, southwestern China. The fossil species is identified based on the detailed leaf morphological and cuticular examinations. The primary venation is pinnate and the major secondary venation is craspedodromous with regular spacing. Stomata are anomocytic and occur on abaxial epidermis. Trichome bases are unicellular and multicellular. The new fossil species shows the closest affinity with Quercus delavayi, an extant species distributing in southwestern China. The responses of the functional leaf traits to the climate change were studied by comparing the leaf characters of fossil species and its nearest living relative. The stomatal density of Q. praedelavayi is higher than Q. delavayi, which suggests a lower palaeoatmospheric CO2 concentration during the late Miocene. The trichome base density of Q. praedelavayi is higher than the extant Q. delavayi. Considering the palaeoclimatic reconstruction of Xianfeng flora, it rejected the hypothesis that increase in trichome density is an adaptation to the drier environment.

Quercus praedelavayi Xing Y.W. et Zhou Z.K. sp. nov. is reported from the upper Miocene of the Xianfeng flora in central Yunnan, southwestern China. The fossil species is identified based on the detailed leaf morphological and cuticular examinations. The primary venation is pinnate and the major secondary venation is craspedodromous with regular spacing. Stomata are anomocytic and occur on abaxial epidermis. Trichome bases are unicellular and multicellular. The new fossil species shows the closest affinity with Quercus delavayi, an extant species distributing in southwestern China. The responses of the functional leaf traits to the climate change were studied by comparing the leaf characters of fossil species and its nearest living relative. The stomatal density of Q. praedelavayi is higher than Q. delavayi, which suggests a lower palaeoatmospheric CO2 concentration during the late Miocene. The trichome base density of Q. praedelavayi is higher than the extant Q. delavayi. Considering the palaeoclimatic reconstruction of Xianfeng flora, it rejected the hypothesis that increase in trichome density is an adaptation to the drier environment.

In Europe, fossil fruits and seeds of Rhodoleia (Hamamelidaceae) have been described from the Upper Cretaceous to the Miocene, whereas no fossil record of Rhodoleia has been reported in Asia, where the modern species occur. Herein, 21 fossil leaves identified as Rhodoleia tengchongensis sp. nov. are described from the Upper Pliocene of Tengchong County, Yunnan Province, Southwest China. The fossils exhibit elliptic lamina with entire margins, simple brochidodromous major secondary veins, mixed percurrent intercostal tertiary veins, and looped exterior tertiaries. The leaf cuticle is characterized by pentagonal or hexagonal cells, stellate multicellular trichomes, and paracytic stomata. The combination of leaf architecture and cuticular characteristics suggests that the fossil leaves should be classified into the genus Rhodoleia. The fossil distributions indicate that the genus Rhodoleia might originate from Central Europe, and that migrated to Asia prior to the Late Pliocene. Additionally, insect damage is investigated, and different types of damage, such as hole feeding, margin feeding, surface feeding, and galling, are observed on the thirteen fossil leaves. Based on the damage frequencies for the fossil and extant leaves, the specific feeding behavior of insects on Rhodoleia trees appears to have been established as early as the Late Pliocene. The high occurrence of Rhodoleia insect herbivory may attract the insect-foraging birds, thereby increasing the probability of pollination.

Twenty-one fossil foliage, identified as Fokienia shengxianensis sp. nov. (Cupressaceae), were collected from the upper Miocene Shengxian Formation in Tiantai and Ninghai counties, Zhejiang Province of eastern China. These fossils can likely be distinguished from other extant genera in the family Cupressaceae s.l. except Fokienia by leaf external morphology alone. Foliar cuticular comparison with the only modern species in this genus, Fokienia hodginsii Henry et Thomas, further suggests that the present fossil species closely resembles the modern counterpart. Paleoecologically, the occurrence of Fokienia from the Shengxian Formation appears to support that the fossil site was covered by subtropical evergreen broad-leaved forest and under warm and humid conditions during the late Miocene, consistent with evidence from previously published fossil plants.

Eight fossil leaves identified as Exbucklandia tengchongensis sp. nov. (Hamamelidaceae) were collected from the Pliocene Mangbang Formation in Tengchong, Yunnan Province, Southwest China. The fossil leaves are characterized by the overall rounded lamina with entire margin, actinodromous venation, and cyclocytic stomata, which suggest the affinity within the genus Exbucklandia, particularly with E. populnea. A survey on the cuticles of the sun and shade leaves of modern E. populnea indicates that the shade leaves generally possess more pronounced undulate anticlinal cell walls and a much lower stomatal density than the sun leaves. Two morphotypes, i.e. sun vs. shade types, of the fossil leaves were therefore recognized. The distribution of the modern Exbucklandia suggests that the genus lives under a warm climate with a mean annual temperature (MAT) from 13 °C to 27 °C and a mean annual precipitation (MAP) from 800 mm to 2500 mm. Hence, E. tengchongensis might also live under a similar climatic condition in the Pliocene. Leaf margin analysis on the Tengchong flora supports this result. The little change of Neogene MAT in Southwest China is therefore supported.