Botany

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Resprouting and multi-stemming and the role of the persistence niche in the structure and dynamics of subtropical coastal dune forest in KwaZulu-Natal province, South Africa.
(2008) Nzunda, Emmanuel F.; Lawes, Michael John.; Griffiths, Megan Elizabeth.
Resprouting is an important means of plant regeneration especially under conditions that do not favour regeneration through seeding such as frequent disturbances, low productivity, unfavourable soil conditions, extreme cold and limited understorey light availability. Sprouts may be advantageous over seedlings because they have higher survival and growth rates than seedlings, since they use resources from parent plants unlike seedlings that have to acquire their own resources. Resprouting is well documented for ecosystems that experience severe disturbances that damage aboveground biomass. For example, resprouting is important for plant persistence against fire in fire-prone savannas and Mediterranean shrub-lands, and hurricanes and cyclones in tropical forests. In these ecosystems, resprouting often results in multi-stemming, because this dilutes the risk of damage among many stems, improving the chances of individual survival. This study was conducted at coastal dune forest at Cape Vidal in north-eastern South Africa, where there is a high incidence of multi-stemmed trees due to resprouting in response to chronic disturbances of low severity. This study examines (1) the importance of resprouting to tree survival and dynamics in an environment where disturbance severity is low but pervasive, and (2) how this resprouting strategy differs from the more familiar sprouting response to severe disturbances such as fire and hurricanes. Analysis of the relationship between multi-stemming and a number of disturbances potentially causing multi-stemming revealed that stem leaning and substrate erosion were the most important disturbances associated with multi-stemming. There were fewer multistemmed trees on dune slacks that had a stable substrate and were protected from sea winds than on dune crests and slopes that had unstable substrate and were exposed to sea winds. Trees resprouted and became multi-stemmed from an early stage to increase their chances of survival against leaning caused by strong sea winds and erosion, and occasional slumping of the unstable dune sand substrate. These low severity disturbances are persistent and are referred to as chronic disturbances in this thesis. As a result of these chronic disturbances, both single and multi-stemmed trees had short stature because taller individuals that emerged above the tree canopy would be exposed to wind damage. Under chronic disturbances plants may manifest a phylogenetically determined sprouting response. However, in this study resprouting and multi-stemming were the results of the tree-disturbance interaction and not a property of a plant or species and were not phylogenetically constrained. Because the disturbances are predominantly of low severity, leaning trees were able to regain the vertical orientation of the growing section by turning upward (a process referred to as ‘turning up’ in this study) and hence survive without resprouting. Species that were prone to turning upward had a low incidence and degree of leaning of their individuals, low frequency of loss of primary stems and high abundance of individuals. Although turning up is less costly to the individual than resprouting, it could only be used by leaning trees that had small angles of inclination and were not eroded. High intensities of the latter require that individuals resprout to survive. The form and function of resprouting varied between seedlings and juvenile and mature trees. Resprouting in seedlings resulted in a single replacement shoot, unlike sprouting in juvenile and mature trees that resulted in multi-stemmed trees. Like sprouting in juvenile and mature trees, sprouting in seedlings was not phylogenetically constrained. Resprouting in seedlings increased seedling persistence; hence species with more sprout seedlings had larger individual seedlings and seedling banks. Resprouting in seedlings increased the chances of seedling recruitment, whereas resprouting in juvenile and mature trees increased the chances of an established plant maintaining its position in the habitat. After disturbances of high severity, which destroy the photosynthesizing parts, plants resprout using carbohydrates stored below- or above ground. In this study, good resprouters stored more carbohydrates both below- and above ground than poor resprouters. The carbohydrates were mobilized for resprouting after disturbance. More carbohydrates were stored in stems than in roots because the prevailing disturbances were mostly of low severity and hence above ground resources were readily available. Similar to storage by plants in severely disturbed habitats, carbohydrates were stored by reserve formation, which competes for carbohydrates with growth and maintenance and forms permanent storage, rather than accumulation, which temporarily stores carbohydrates in excess of demands for growth and maintenance. Stored carbohydrates are not necessary for resprouting of plants after disturbances of low severity because they can resprout using resources remobilized directly from the disturbed photosynthesizing parts. However, in this study, stored carbohydrates served as a bet-hedge against occasional severe disturbances that occurred in addition to chronic disturbances. Allocation of carbohydrates to permanent storage diverts them from growth and reproduction and hence good resprouters had lower growth rates, seed output, seed size and seedling recruitment than poor resprouters. However, the costs of these traits that resulted in low recruitment from seed by good resprouters, were compensated for by high persistence of established individuals of good resprouters through recruitment of sprout stems. This study demonstrates that resprouting is not only advantageous in severely disturbed environments, but also in environments where disturbances are of low severity but nevertheless confer an advantage on individuals that persist. Thus in forest environments where aboveground biomass is seldom destroyed and individuals are relatively long-lived, resprouting can confer significant fitness and selective advantage on individuals.
The physiology of pinus patula seedlings in response to water stress and the implications for plantation regeneration in South Africa.
(2008) Rolando, Carol Ann.; Little, Keith MacMillan.; Pammenter, Norman William.
Pinus patula Schiede ex Schlect. & Cham. is the most widely planted softwood species for both pulpwood and saw timber in the South African forestry industry. High mortality of this species, often in excess of 20%, following planting is currently of major concern and has the potential to limit future deployment for commercial timber. Water stress is often reported to be a cause of mortality during regeneration in commercial forestry plantations yet, prior to 2007, there was no published research on the water relations of P. patula during regeneration in South Africa. This, together with questions raised by the industry as to the role of using water in the planting operation, initiated the series of studies conducted for this thesis. Water planting (application of water into the planting hole at the time of planting) of P. patula seedlings has been used commercially to reduce post-planting water stress and buffer against potentially extreme weather conditions immediately after planting. However, the primary role of the water, as well as its success in increasing survival following planting, has never been critically assessed. Since the use of water in the planting operation is expensive, it was essential that the benefits to using water were quantified, in terms of survival and growth, and justified, in terms of any monetary investment. In addition, there was a lack of local studies investigating the physiological characteristics of P. patula seedlings, particularly their tolerance to low soil water availability. To understand the role of water during the regeneration of P. patula in terms of plantation management and seedling physiology, a variety of research methodologies were used that included: applied field trials, multivariate methods (a retrospective investigation), pot trials and the development of a simple financial model. Four field trials were implemented to test the response in P. patula survival to water applied at planting. Two trials each were situated in the KwaZulu-Natal (KZN) Midlands and Mpumalanga Escarpment. The first trial at each site was planted in spring (October) and the second in summer (February). Watering treatments consisted of different quantities of water used in the planting operation and included 0.5 litres, 2 litres, 4 litres and no water (dry plant). Only at the spring planted trial in the KZN Midlands was survival of the dry planted seedlings significantly lower than that of the seedlings planted with water, at 90 days after planting. This may have been due to low rainfall during the week before and two weeks after planting, or the small size of the seedlings used in the trial. Application of 0.5 litres of water to the planting pit was sufficient to increase survival to a level equivalent to that where 2 or 4 litres of water was used, yet only increased soil moisture in the area immediately surrounding the seedling. This suggested that the role of the water applied during planting was increased root to soil contact. Overall, these four trials indicated that planting with water had the potential to increase survival only when soil water availability was low and rainfall sporadic. There was no effect of water applied at planting on early tree growth. While the results of the four field trials provided an indication of the effect of planting with water on subsequent survival of P. patula seedlings, there was concern that the results of the four trials may not be a true reflection of a dynamic situation. Survival in response to water applied at planting may vary from year to year and across forestry regions due to the unpredictable nature of rainfall and high air temperatures during the planting season, as well as the wide range of forestry sites across which P. patula seedlings are planted. To improve our understanding, a database of 58 trials was compiled where water and dry planting had been carried out. In this way it was possible to investigate whether the results from the four field trials were reflected in a range of previously conducted field trials implemented across time and space. The trials incorporated into the dataset were all planted to P. patula between 1990 and 2005 in the summer rainfall region of southern Africa. Data related to the climate, local weather, physiography and site management at each trial were also included. Summary statistics, linear correlation and multiple regression were used to determine if site-associated variables were related to an increase in survival in the water relative to the dry planted treatments. The analyses indicated that for all 58 trials, survival was lowest during the summer months, regardless of planting treatment. Planting with water was most likely to increase survival when used during spring, autumn and winter planting, although (as with the four applied field trials) there was no overall significant relationship between water planting and survival. Based on these results it was anticipated that an understanding of the water stress physiology of P. patula seedlings was required to explain the observed trends from a more fundamental perspective; if planting with water did not always increase survival, why not? Three pot trials were conducted to increase the understanding of the water relations of P. patula seedlings. These trials were also used to provide benchmark physiological data related to stressed (water) and unstressed seedlings. The first pot trial highlighted the importance of root plug moisture at the time of planting for increasing subsequent survival. The subsequent two pot trials were aimed at investigating the interaction between planting stock quality (as determined by measures of size) and soil water availability and the effect on survival, growth and physiology of P. patula seedlings. These results indicated that P. patula seedlings were not as sensitive to high air and soil temperatures (above 30°C) and low soil water availability (below -1.5 MPa) as previously thought. The seedlings were able to tolerate low soil water availability for several weeks and, following rewatering, were able to recover from moderate and severe water stress (a shoot water potential of below -1.5 MPa). This data supported the results from the four applied field trials and retrospective study of 58 trials, where the application of water to the seedlings at planting did not substantially increase survival. In the pot trials, stomatal conductance started to decrease when shoot water potential approached -0.8 to -0.9 MPa. Stomatal closure occurred at a shoot water potential between -1.2 MPa to -1.5 MPa. Mortality due to water stress occurred only in response to extended periods of low soil water and was associated with a shoot water potential of below -3.0 MPa. There was variability between seedlings in their potential for survival and growth. Inherently bigger seedlings had a greater capacity for new root growth following planting. New root growth, as well as a greater mass of new roots, was associated with higher shoot water potentials and higher rates of transpiration under conditions of low soil water availability. This indicated that seedling quality, as determined by size, may play a role in sensitivity to water stress. The field trials, retrospective study and pot trials indicated that the practice of planting with water was not always critical to the survival of P. patula seedlings. A simple financial model was developed to estimate whether planting with water represented a cost that could be used as a decision criterion, given certain growth parameters and management scenarios. The data projected by the model were also compared to actual research data for water versus dry planting (and the inclusion of an insecticide in the water). While these comparisons were specific to the parameters included in the model for this study, as well as the results of the research trials used in the benchmarking exercises, the model indicated that; 1) costs for planting with water were likely to be recovered only when no blanking (replacing of dead trees) was carried out, with capital invested at a low return rate (3%), 2) including an insecticide in the water increased the likelihood of cost recovery, and 3) site quality had an impact on the increase in survival required to recover planting method costs, with a greater percentage increase in survival required on lower quality sites. Lower quality sites often have a lower mean annual precipitation (associated with higher rainfall variability), or shallow soils (associated with lower soil water availability) and therefore are also likely to be sites where foresters may want to use water to reduce (drought related) mortality. The impact of site quality is thus also an important factor to include in any decisions regarding planting methods (i.e. using water) and their costs. Further investigations should be aimed at examining; 1) the interaction of root plug size (as determined by container type) and soil water availability on growth and physiology of P. patula seedlings, 2) the methods of grading seedlings within a population to select those that have a high potential for survival and growth, and 3) the effects of soil water availability on the physiology, survival and growth of P. patula cuttings, as well as other pine species and hybrids grown in South Africa, such as P. elliottii, P. elliottii x P. caribaea and P. patula x P. tecunumanii. It is likely that the proportion of forestry regions planted to these hybrids will increase in the future.
Sink regulation of photosynthesis in sugarcane
(2007) McCormick, Alistair James.; Watt, Derek Alexander.; Cramer, Michael D.
The C4 plant, sugarcane (Saccharum spp. hybrids), accumulates sucrose to high concentrations and, as a result, has been the focus of extensive research into the biochemistry and physiology of sucrose accumulation. Despite this, the relationship between source leaf photosynthetic rates and sucrose accumulation in the culm has not been well documented. The observations that photosynthetic activity declines during culm maturation in commercial cultivars and that high-sucrose accumulating ancestral genoptypes photosynthesize at rates two-thirds of those of low-sucrose ancestral Saccharum species indicate that source-sink communication may play a pivotal role in determining sucrose yield. The relationship between source and sink tissues in sugarcane was investigated using a supply-demand paradigm, an approach novel in the study of the crop. The demand for photosynthate from the primary culm growth sink was shown to be closely linked to photosynthetic rates, sucrose export and the eventual physiological decline of source leaves. Results from initial field experiments revealed that leaf assimilation rates were negatively correlated with leaf hexose concentrations, but not those of sucrose. Further manipulation of leaf sugar status, through sugar-feeding and cold-girdling techniques, demonstrated the regulatory role of leaf sugar concentrations on photosynthetic activity, thus revealing sucrose, and particularly hexose, as key signal molecules in the modulation of the amount of photosynthate available for export to the sink. Gene expression profiling, by means of array technologies, indicated that changes in leaf sugar status and photosynthetic rates result in concurrent modifications in the expression of several genes involved in fundamental metabolic pathways, including photosynthesis, carbohydrate metabolism, stress response and sugar-signaling. Notable amongst these, was the identification of a potential trehalose 6-phosphate (T6P) sugar-signaling mechanism, thus implicating the trehalose pathway as a central regulatory system in the communication of sink carbon requirements to the source leaf. This study demonstrated that maturation of the culm results in a decreased demand for sucrose, which invokes a sugar-mediated feedback signal to decrease leaf photosynthetic supply processes. However, sugarcane leaves appear to retain the capacity to increase the supply of assimilate to culm tissues under conditions of increased assimilate demand. Uncoupling of the signaling pathways that mediate negative feedback between source and sink tissues may result in improved leaf assimilation rates and, consequently, lead to increased sugarcane sucrose yields.
The effect of elevated atmospheric CO2 on the growth and physiology of Chromolaena odorata.
(2008) Lalla, Reshnee.
Rising atmospheric CO2 (Ca) concentrations have generated concern among scientists, mainly because of CO2’s role as a greenhouse gas and its influence on plant growth and development. Previous research has suggested that future CO2 enriched atmospheres may enhance the success of invasive aliens. Chromolaena odorata is an example of an invasive alien proving to be a serious threat to indigenous vegetation in South Africa, and effective control measures are desperately needed to curb infestations in the future. The current study aimed at assessing the response of C. odorata to elevated Ca and interactive factors, and was divided into two trials. During PART A, C. odorata was grown in competition with 2 grass species: Eragrostis curvula and Themeda triandra (selected for their differential preferences to nutrient availability). All three species were potted in a greenhouse at the University of KwaZulu-Natal (Howard College). There were 16 pots in total, and each pot contained four C. odorata plants, four T. triandra seedlings, and four E. curvula seedlings. Eight pots were exposed to elevated Ca (~700ppm), and eight pots were exposed to ambient Ca (~370ppm). The pots at each Ca treatment were further divided: four received high nutrient treatments (3L per addition), while the other four received low nutrient treatments (300 ml per addition). Studies on growth (e.g. plant height, dry weight, etc.), as well as physiology (e.g. Jmax), were undertaken. Results showed that generally, plants responded positively to high nutrient treatments. In contrast, elevated Ca did not affect growth or any of photosynthetic parameters of C. odorata significantly, but did reduce stomatal limitations. During PART B, C. odorata plants were grown monospecifically to assess whether there was a “chamber effect” associated with planting density. Pots at both Ca treatments contained either four C. odorata or two C. odorata seedlings. Growth and physiology were assessed. The fact that elevated Ca did not affect any of the photosynthetic parameters studied, suggests that photosynthetic down-regulation did not occur. This, together with the fact that no increase in stomatal limitations were observed in elevated Ca, implies that enhancement of photosynthetic assimilation could have occurred in C. odorata plants exposed to CO2 enrichment. Results from this study (PART A and PART B), when compared to previous research on this species, suggests that CO2 enrichment may enhance the success of monoculture populations of C. odorata. However, other species may gain competitive advantages over C. odorata occurring in mixed communites, under CO2 enriched environments. In addition, results of this study support the prediction that increasing Ca will reduce the importance of carbon as an external limiting resource, and that the extent of a plant’s response to Ca enrichment will depend on resources other than CO2. If increases in temperature caused by elevated Ca increases nutrient availability in the soil, then Ca could indirectly enhance the success of C. odorata occurring in mixed communities.
The performance and rooting of eucalyptus grandis x nitens cuttings.
(2007) Murugan, Nelisha.; Watt, Maria Paula Mousaco Deoliveira.; Mycock, David John.; Mokotedi, Mompe Edward Oscar.
Hybrid clones of Eucalyptus grandis and E. nitens (GN) have consistently been shown to be suitable for planting in cold, dry, marginal plantation sites, where they exhibit high yields and superior pulp properties. However, their clonal propagation is hindered by the very poor rooting success of cuttings. The present study aimed at assessing the effect of cutting type, time of year of setting cuttings and Seradix 2 application on rooting and development of cuttings of a commercially important Eucalyptus grandis x Eucalyptus nitens clone (GN107). Cuttings were prepared from clonal hedge coppice at the Mondi Business Paper, Trahar Technology Centre, Hilton. Three cutting types were used (cut at different distances from the node) for each terminal (situated below the apical bud) and non-terminal cuttings. The leaves were trimmed and, for half the cuttings, the base of the stem of cuttings were dipped in Seradix 2 rooting powder (3 g kg-1 4-(indole-3-yl)-butyric acid (IBA). They were then placed into rooting trays (128 inserts/ tray arranged as 8 rows x 16 columns). Seradix 2-treated and Seradix 2-untreated terminal and non-terminal cuttings, cut at, above and below the node (twelve treatments in total) were set in trays with one treatment per column of eight replicates, per tray. There were nineteen trays overall. The trays were filled with peat, perlite and vermiculite (3:3:1) and were maintained in a Mondi greenhouse, with air temperature at 25°C to 27°C (thermostatically activated fans), root zone temperature at 28°C (bed heaters) and 20 second misting at 10 minute intervals (automatic misters). The study was carried out in November 2005, April 2006 and June 2006. In the first experiment, both terminal and non-terminal cuttings were used; thereafter only non-terminal cuttings were used. The plantlet yield was very low, regardless of cutting type, Seradix 2 treatment and the time of year the cuttings were set. The highest plantlet production (12.5%) and rooting frequencies (13.8%) were achieved with non-terminal cuttings treated with Seradix 2. Although not statistically significant, Seradix 2 inhibited shoot production (31.4% for Seradix 2-untreated and 24.2% for treated cuttings). The position at which inserts were cut in relation to the node did not significantly affect the number of plantlets produced and non-terminal cuttings appeared hardier and performed better than terminal cuttings. The time of year of setting cuttings did not have any significant effect on plantlet yield, nonetheless, plantlet yield was highest in cuttings set in November (9.2%) and lowest in April (0.4%). In addition, cuttings set in November (spring), had superior shoot development in terms of the number of cuttings that produced shoots (regardless of root production), shoot length and the mass of shoots relative to root mass. The highest percentages of cuttings that produced roots (regardless of shoot growth) (10%) and the highest number of roots per cutting (2) were part of the June trial. Therefore, cuttings set in June (winter) had superior root development as compared with cuttings set in November (spring) or April (autumn). In all of the studies, three rooting patterns were observed in cuttings: roots produced only from the cut area only (type 1), only from the sides of the stem (type 2) and from both sites (type 3). Non-terminal cuttings treated with Seradix 2 showed a higher incidence of types 2 and 3 rooting patterns than the terminal cuttings. Seradix 2 application increased the prevalence of types 2 and 3 rooting patterns. Although not statistically different, cuttings dipped 2.5 cm into Seradix 2 produced more types 2 and 3 rooting patterns than cuttings dipped at the abaxial end only. Light microscopy of stem sections of cuttings indicated that roots appeared to originate from the xylem archs as well as from the cambium. The collected data indicate that it is necessary to continue research towards improving the efficiency of plantlet production of GN107 via cuttings. It appears that cuttings of this clone may be set throughout the year and that terminal cuttings should be avoided. In addition, the present practice at the Mondi Hilton nursery of treating cuttings with Seradix 2 needs to be reconsidered as although it increases rooting, it does not increase plantlet production due to its apparent inhibitory effect on shoot development.
Seed germination and medicinal properties of Alepidea species.
(2009) Mulaudzi, Rofhiwa Bridgeht.; Van Staden, Johannes.; Finnie, Jeffrey Franklin.
The rhizomes of Alepidea amatymbica and Alepidea natalensis are used for medicinal purposes. Because of the increase in demand for these plants the species is becoming scarce. As the seed biology of neither species is well defined, conditions as well as treatments required for optimum germination and vigour were studied. Seeds were exposed to various physical factors such as varying light and temperature conditions and cold stratification, sowing depth and seed storage. The effects of smoke-water, butenolide (3-methyl-2H-furo [2, 3-c] pyran-2-one) a novel smoke compound and chemical substances (gibberellins, kinetin and KNO3) were also tested in order to improve seed germination. Alepidea amatymbica and A. natalensis achieved the highest seed germination (72.5% and 80%, respectively) at 25 °C under a 16 h photoperiod with a mean germination time (MGT) of 18 and 12 days, respectively. Phytochrome studies showed that A. natalensis requires light for germination. Cold stratification (5 °C) for 14-28 days significantly improved the percentage germination of both species (> 90%) compared to non-stratified seeds (control) at 25 °C under a 16 h photoperiod. Sowing A. amatymbica and A. natalensis seeds at a depth of 0.5 cm resulted in higher percentage germination compared to 2.5 cm. The highest emergence rate for A. amatymbica was 40% at a sowing depth of 0.5 cm and the lowest emergence rate was 3% at 2.5 cm. Six months storage of A. natalensis seeds at room temperature (25 ± 2 °C) showed maximum germination (99%) with a MGT of 9 days. Smoke-water treatment of A. amatymbica seeds significantly enhanced germination from 72% to 91%. Smoke and butenolide at 10 °C and 25 °C promoted germination of A. natalensis seeds in a 16 h photoperiod. Smokewater application significantly improved both germination and seedling vigour of A. natalensis. GA3 (10-8 M) was the best treatment for achieving maximum percentage germination of A. natalensis seeds. Antibacterial (two Gram-positive bacteria: Bacillus subtilis, Staphylococcus aureus and two Gram-negative bacteria: Escherichia coli, Klebsiella pneumoniae), antifungal (Candida albicans), anti-inflammatory (COX-1 and -2) and genotoxicity tests (Ames test) were carried out on petroleum ether (PE), dichloromethane (DCM), 80% ethanol (EtOH) and water extracts of the two Alepidea species. Water extracts of A. natalensis rhizomes exhibited high activity (MIC values of 0.78 mg/ml) against the four bacterial strains. High activity was also observed in the PE and DCM leaf extracts of the same plant against the Gram-positive bacteria. The PE and DCM extracts of A. amatymbica rhizomes exhibited the best activity (MIC values of 0.39 mg/ml) against Bacillus subtilis. The rest of the extracts showed low activity (MIC values >1 mg/ml). All the extracts showed activity against Candida albicans, with A. natalensis leaf extracts exhibiting the highest antifungal activity with MIC values of 0.88, 0.20 and 0.78 mg/ml for PE, DCM and EtOH, respectively. EtOH extracts had inhibition less than 40% for both A. natalensis and A. amatymbica. All the PE extracts showed higher inhibitory activity for COX-2 than for COX-1. PE and DCM extracts had percentage inhibitions above 70% in both COX-1 and COX-2 assays. The Ames test for genotoxicity revealed that none of the plant extracts were genotoxic to the Salmonella TA98 tester strain.
Pharmacology and phytochemistry of South African plants used as anthelmintics.
(2009) Aremu, Adeyemi Oladapo.
Traditional medicine in South Africa is part of the culture of the people and has been in existence for a long-time. Although animal components form part of the ingredients used, plant material constitutes the major component. South Africa is endowed with vast resources of medicinal and aromatic plants which have been employed for treatment against various diseases for decades. A large number of South Africans still depend on traditional medicine for their healthcare needs due to its affordability, accessibility and cultural importance. Helminth infections are among the variety of diseases treated by traditional healers. These infections are regarded as neglected tropical diseases (NTDs) due to their high prevalence among the economically disadvantaged living in rural areas in different regions of the world.
Pharmacological, phytochemical and safety evaluation of commercial herbal preparations common in South Africa.
(2009) Ndhlala, Ashwell Rungano.
Herbal formulations claimed to cure several medical conditions including skin eruptions, chest pains, wounds, gout, menstrual pains, stress, nervous disorders, microbial and viral infections as well as stomach ailments have recently appeared as part of South African traditional medicine. The formulations consist of mixtures of extracts of different plant parts from several different plant species packaged in labelled bottles or boxes. The mixtures are available for sale in herbal shops and public places. While there has been widespread use of these herbal mixtures, there has been no scientific evidence to support their use. This project was aimed at documenting, validating claimed efficacy and testing the safety of fourteen unregistered commercial herbal preparations commonly sold in Pietermaritzburg. A detailed investigation of the pharmacological effects and safety of the plant components of one of the mixtures, Imbiza ephuzwato was also carried out.
Micropropagation and medicinal properties of Barleria greenii and Huernia hystrix.
(2009) Amoo, Stephen Oluwaseun.; Van Staden, Johannes.; Finnie, Jeffrey Franklin.
The crisis of newly emerging diseases and the resistance of many pathogens to currently used drugs, coupled with the adverse side-effects of many of these drugs have necessitated the continuous search for new drugs that are potent and efficacious with minimal or no adverse side-effects. The plant kingdom is known to contain many novel biologically active compounds, many of which could potentially have a higher medicinal value when compared to some of the current medications. Indeed, the use of plants in traditional medicine, especially in African communities, is gaining more importance due to their affordability and accessibility as well as their effectiveness. Exponential population growth rates in many developing countries has resulted in heavy exploitation of our plant resources for their medicinal values. In addition, plant habitat destruction arising from human developmental activities has contributed to the fragmentation or loss of many plant populations. Owing to these factors, many plant species with horticultural and/or medicinal potential have become either extinct or are threatened with extinction. These threatened species cut across different taxonomic categories including shrubs, trees and succulents. Without the application of effective conservation strategies, the medicinal and/or horticultural potential of such threatened species may be totally lost with time. The extinction of such species could lead to the loss of potential therapeutic compounds and/or genes capable of being exploited in the biosynthesis of new potent pharmaceutical compounds.
Systematic studies in Gnidia L. (Thymelaeaceae)
(2010) Beaumont, Angela Jane.; Edwards, Trevor John.; Van Staden, Johannes.
Gnidia L., variously estimated to contain 100–160 species, is the largest genus in the sub-cosmopolitan family Thymelaeaceae. Most species are shrubby, and occur in tropical and southern Africa, with one species reaching southern India and Sri Lanka, and 14 species endemic to Madagascar. Assorted segregate genera have been established using characters considered by some as too few, too trivial or unreliable. Generic limits have been contentious with authors following either a narrower concept of Gnidia or a broader circumscription within which segregate genera are placed in synonymy under Gnidia. Regional treatments for African and Madagascan floras have been published over the last century until very recently, but the genus was last revised in its entirety 153 years ago. Today, a broad-based concept of Gnidia is generally recognised, but there is no modern infrageneric classification, and species relationships are poorly understood. Homogenous groups of species are identified by their similarities of leaf length and width or bract length and width ratios. Species comprising the homogenous groups for leaf ratios differ to those comprising the homogenous groups based on bract ratios, and there is no correlation between leaf and bract length and width ratios. This suggests that the factors influencing leaf diversity differ from those influencing bract diversity. Bracts differ most from leaves in species with capitate inflorescences, and involucres of several layers of bracts likely protect reproductive organs (flowers) in heads. Previously overlooked morphological and micromorphological details, and morphometric analyses of leaf, bract and floral dimension data help define individual species, and clades of species derived from phylogenetic analyses of molecular data. Evidence from a phylogenetic analysis of nuclear ribosomal and plastid DNA sequence data confirms the polyphyly of Gnidia. Three lineages contain Gnidia species and species of genera from southern Africa, southern South America or Australia, while another lineage corresponds largely to the previously recognized genus Lasiosiphon. The genus Lasiosiphon is reinstated characterised by flowers mostly in heads, bracts different from the leaves, and the presence of smooth hairs; it now includes species with tetramerous flowers as well as ones with pentamerous flowers. Gynodioecy is recorded for the first time in a single species and represents the first documented example of sexual polymorphism involving unisexual flowers in Gnidia and sub- Saharan Thymelaeaceae. The findings of this thesis are discussed in terms of their phylogenetic value and contribution to our better understanding of the generic limits of Gnidia and its relationships with other southern hemisphere Thymelaeoideae. The circumscription and generic affinities of Gnidia as suggested by results presented in this thesis are compared to previous classification systems for congruence and dissimilarity.
Regulation of hyperhydricity in Aloe polyphylla propagated in vitro.
(2009) Ivanova, Mariyana Vasileva.; Finnie, Jeffrey Franklin.; Van Staden, Johannes.
Micropropagation of Aloe polyphylla, an endangered species with a high ornamental and medicinal value, is an important part of its conservation. However, the in vitro culture was hindered by the phenomenon of hyperhydricity. The research reported in this thesis was undertaken for two reasons. Firstly, to understand the role of various culture factors involved in the process of hyperhydricity in A. polyphylla and to identify the in vitro conditions, under which this disorder can be prevented. Secondly, we conducted an investigation into the underlying mechanisms of this phenomenon by probing if it was mediated through internal cytokinins. Ammonium (NH4 +) ions, applied cytokinins (CKs) and CK concentrations were tested in multifactorial combinations and significantly influenced the regeneration rate and occurrence of hyperhydricity. Shoots were grown on media with different NH4 + concentrations (10.3, 20.6 and 61.8 mM) and supplemented with BA, zeatin or TDZ at 0, 5 or 15 ìM. Elevating the levels of NH4 +, in the absence of CKs, could not induce hyperhydricity. Similarly, very low hyperhydricity was observed when CKs were added to media containing low NH4 + (10.3 mM). However, in the presence of higher NH4 + concentrations, CKs increased hyperhydricity in a concentrationdependant manner, suggesting that they were capable of inducing this syndrome only when other factors in the culture system were not optimised. High numbers of healthy looking shoots were produced on media with low NH4 + and low BA or zeatin (5 ìM). The use of TDZ resulted in the formation of buds, which did not develop into shoots. In view of the fact that NH4 + was supplied in the form of NH4NO3, it was difficult to determine if NH4 + or nitrate (NO3 -) ions were associated with the increase in hyperhydricity. We further examined the role of nitrogen (N) supplied as inorganic NH4 + or NO3 -, or organic glutamine. The omission of total N from the culture medium resulted in low multiplication and hindered shoot growth. Ammonium as the sole source of N depressed shoot regeneration and growth and escalated the frequency of hyperhydricity to ca. 50%. When NO3 - was used as the sole N source, shoots of fine quality were produced and hyperhydricity was completely eliminated. Overall, the MS N mix was superior to any single N source for multiplication and growth of shoots, suggesting a synergistic effect between NH4 + and NO3 - on shoot regeneration. Furthermore, not only the absolute amount of N, but also the relative amounts of NH4 + and NO3 - influenced the multiplication rate, frequency of hyperhydricity and shoot quality. The highest regeneration was obtained with NH4 + : NO3 - ratios (mM) of 20 : 40, 30 : 30 and 40 : 20. Decreasing the ratio of NH4 + : NO3 - lowered the occurrence of hyperhydricity. The potential of glutamine as the sole source of N was also demonstrated, since its application resulted in the production of good quality shoots and almost no hyperhydricity. Shoot explants grown in static liquid media became hyperhydric and lost their ability to regenerate. The type of gelling agent used to solidify the medium affected greatly hyperhydricity and shoot multiplication. Gelrite resulted in a significantly lower multiplication rate and four times higher hyperhydricity (64.7%) compared to when agar was used. Gelrite was further selected to test the hypothesis if hyperhydricity can be overcome by decreasing the relative matric potential of the media, and respectively the availability of water, as represented by increasing gelrite concentrations. Satisfactory reduction in hyperhydricity was achieved only at 16 g l-1 gelrite, however the regeneration also decreased. The nature of the gelling agent is therefore essential for the successful control of this phenomenon. It appears that a crucial prerequisite for the reduction of hyperhydricity in tissue cultures of A. polyphylla is the gaseous exchange between the in vitro atmosphere and the outside environment. In ventilated cultures, achieved by using a modified lid with a hole (d = 7 mm) covered with polyester or cotton mesh, hyperhydricity was completely eliminated, irrespectively of the type of gelling agent. Ventilation was further advantageous for the in vitro regenerants by increasing their leaf chlorophyll content as well as epicuticular wax deposition, the last one being indicative of the development of the water loss regulation mechanisms of explants. The increased culture ventilation, however, was negatively correlated with the regeneration rate and shoot growth. Endogenous CKs were measured in in vitro regenerants after an eight-week cycle to examine whether the hyperhydricity-inducing effect of exogenous CKs and gelling agents is associated with changes in the endogenous CK content. The content of endogenous CKs, determined by HPLC-mass spectrometry, in the shoots grown on CK-free media comprised isopentenyladenine-, trans-zeatin- and cis-zeatin-type CKs. The application of exogenous CKs resulted in an increase in the CK content of the shoots. Following application of zeatin, dihydrozeatin-type CKs were also detected in the newly-formed shoots. Application of BA to the media led to a transition from isoprenoid CKs to aromatic CKs in the shoots. Shoots grown on gelrite media contained higher levels of endogenous CKs compared to those on agar media. Total CK content of hyperhydric shoots was higher than that of normal shoots grown on the same medium. We suggest that the ability of exogenous CKs and gelrite to induce hyperhydricity in shoots of Aloe polyphylla is at least partially due to up-regulation of endogenous CK levels. However, hyperhydricity is a multifactor process in which different factors intervene.
Heavy metals in South African medicinal plants.
(2008) Street, Renée Anne.; Van Staden, Johannes.; Stirk, Wendy Ann.; Southway, Colin.
Plants are able to take up and accumulate certain environmental contaminants such as heavy metals. When the plants are ingested by man, these contaminants are transferred along the food chain. Due to the poorly regulated medicinal plant trade in South Africa, many opportunities exist for heavy metal contamination of medicinal plants namely contaminated harvest sites as well as poor drying, processing, storage, transport and manufacturing conditions. The concentrations of five heavy metals (As, Cd, Co, Ni, Pb) and six microelements (B, Cu, Fe, Mn, Mo, Zn) were determined in some commonly used South African medicinal plants obtained from street markets. Elemental content was determined using inductively coupled plasma optical emission spectrophotometry (ICP-OES). Some of the medicinal plant samples investigated contained As and Cd at levels exceeding the World Health Organization limits of 1 and 0.3 mg kg-1 respectively. Lead and Ni were detected in all the samples. Elevated Fe and Mn levels were recorded in certain plant species. The results revealed multiple metal contamination in some medicinal plant parts sold in local markets and is thus grounds for concern. The effects of Cd application on growth parameters of some medicinal plant species belonging to the Hyacinthaceae (Albuca setosa, Eucomis autumnalis, Eucomis humilis, Merwilla plumbea) gave insight into heavy metal accumulation and distribution in these species. Application of Cd at 5 mg l-1 over a 12 week period reduced growth in A. setosa. The medicinally used A. setosa bulbs accumulated 37 mg kg-1 Cd after 12 weeks. Cadmium application at 2 mg l-1 over a six week period had no effect on growth parameters of E. autumnalis or E. humilis. However, a substantial difference in total Cd accumulation was detected in the plants (40.2 and 15.3 mg kg-1 respectively). Cadmium application at 2 mg l-1 significantly reduced the fresh weight of leaves, bulbs and roots of M. plumbea. Although most of the Cd was stored in the roots, the medicinally used bulbs accumulated up to 11.6 mg kg-1 when applied at 10 mg l-1. The antagonistic effect between Cd and Zn treatments and their effect on micronutrient distribution in M. plumbea were investigated. Five treatments were evaluated: (1) Hoagland’s nutrient solution (HS) (control) (2) HS + Cd 2 mg l-1 (single) (3) HS + Cd 2 mg l-1 + Zn 50 mg l-1 (combination) (4) HS + Cd 2 mg l-1 + Zn 100 mg l-1 (combination) (5) HS + Cd 2 mg l-1 + Zn 150 mg l-1 (combination). Cadmium readily accumulated in leaves, bulbs and roots of M. plumbea when supplied at 2 mg l-1. Zinc at 50 mg l-1 led to increased Cd accumulation. However, further increases in Zn concentration showed an antagonistic effect of Zn on Cd uptake and accumulation. Thus, increasing Zn levels in soils may be favourable for reducing toxic Cd accumulation in M. plumbea plants. Boron was not significantly affected by the addition of Cd to the media. However, with an increase in Zn, leaf B content increased while the B content in the bulbs and roots decreased. Copper and Mo levels were not significantly affected by treatments with Cd or Cd/Zn combinations. Compared to the control, Cd and Cd/Zn applications caused an increase in Mn content in leaves, bulbs and roots. Iron levels of M. plumbea were not significantly affected by Cd in the media. However, with an increase of Zn in the Cd-containing media, Fe content in the leaves, bulbs and roots increased. Tulbaghia violacea is one of the few medicinal plants that is also frequently used as a leafy vegetable. Application of Cd at 2 and 5 mg l-1 to T. violacea of varying sizes (small 8 - 10 g, medium 16 - 20 g, large 80 – 95 g) elicited a difference in growth response, Cd accumulation and micronutrient distribution. Leaf length and fresh weight of leaves of the medium-size plants decreased with application of Cd at 2 mg l-1 whilst 5 mg l-1 Cd significantly decreased the number of leaves in small-sized plants. Small plants accumulated more Cd in the leaves than medium- or large-sized plants. Application of Cd at 2 mg l-1 and 5 mg l-1 lowered the leaf Cu, Fe, Mo and Zn contents in small- and medium-size plants. This study indicated that T. violacea has the ability to accumulate Cd. In addition, plant size plays an important role with regards to Cd accumulation and elemental distribution. The effect of various nutrient applications (10%, 50% and 100% Hoagland’s nutrient solutions (HS); and HS deficient in N, P or K) on growth parameters and micronutrient distribution in Dioscorea dregeana were investigated. Irrigating plants with 50% HS resulted in better growth performance, whereas a deficiency of either N, P or K negatively affected seedling growth. Plants grown in 10% HS contained higher total B, Fe and Mo levels compared to seedlings grown in 50% and 100% HS. Compared to the control, P deficiency resulted in a Fe increase in the leaves, tuber and roots while a lack of P and K significantly increased total Mn content in D. dregeana. The effect of excess Zn (100, 200 and 300 mg l-1) on growth performance, chlorophyll content and microelemental distribution on Dioscorea sylvatica was investigated. Growth parameters showed a significant decrease when supplied with Zn at 100 mg l-1. Zinc phytotoxicity was evident by the reduction in chlorophyll content. Highest Zn concentrations were detected in the roots. Certain micronutrients appear to be redistributed due to Zn toxicity. The effect of microelements (Cu, Zn) and heavy metals (Cd, Pb, Hg) on germination and seedling development of Bowiea volubilis, Eucomis autumnalis and Merwilla plumbea was investigated. Copper and Zn applied at 1 mg l.1 significantly reduced the percentage germination of E. autumnalis. Low concentrations (. 1 mg l.1) of Cu and Zn negatively affected the root growth of all three species. Mercury concentrations of 0.5 and 1 mg l.1 significantly decreased the percentage germination of B. volubilis and E. autumnalis respectively. Cadmium and Hg at 2 mg l.1 showed a negative effect on the root growth of B. volubilis. Concentrations of 0.5 mg l.1 of all heavy metals tested significantly decreased shoot length of M. plumbea. The effect of Cd on biological activity (anti-inflammatory, antibacterial and antifungal) of medicinal plants with previously confirmed activity was evaluated. When supplied with Cd at 2 mg l-1, Eucomis humilis bulbous extracts showed lower anti-inflammatory activity than the control for both COX-1 and COX-2 activity. Eucomis autumnalis bulbous extracts had greater COX-1 activity compared to the control. However, Cd suppressed the activity of COX-2. Compared with non-Cd-treated Merwilla plumbea plants (control), those supplied with Cd at 10 mg l-1 showed increased antibacterial activity against Bacillus subtilis, Klebsiella pneumoniae and Staphylococcus aureus. However, no change in activity against Escherichia coli was observed. Cadmium accumulation in the bulbs had no effect on antifungal activity of Tulbaghia violacea. Thus, optimized agricultural practices are essential for quality control of cultivated medicinal plants. The studies presented in this thesis collectively answer several questions related to heavy metal involvement in South African medicinal plants. The findings substantiate the need to regulate and monitor the South African medicinal plant trade against heavy metal contamination which will in turn provide a product of safety and quality to the consumer.
In vitro bulb induction in Eucomis zambesiaca Baker.
(2009) Cheesman, Lee.; Finnie, Jeffrey Franklin.; Van Staden, Johannes.
Eucomis L’ Hér. is a genus of 10 species that fall within the Hyacinthaceae family. Eucomis zambesiaca Baker is a summer-blooming bulbous geophyte occurring from northern South Africa to Malawi. Eucomis species are used in southern African traditional medicine for the treatment of various ailments, in particular, pain and inflammation. As a result, the bulbs are heavily harvested for trade in South Africa’s traditional ‘muthi’ markets. Over-collection of Eucomis species has seriously depleted natural populations and now Eucomis plants are among the 15 scarcest medicinal species to be traded. Micropropagation is a useful technique for rapid clonal multiplication of plant material which could potentially yield useful secondary metabolites as well as alleviate the pressure on the wild plant populations. The in vitro induction of storage organs is especially beneficial as it can limit the loss of plants during acclimatization as bulblets are hardier than shoots or plantlets. The aim of this research was to determine optimal growth conditions for bulblet induction of Eucomis zambesiaca. The effect of environmental and physiological parameters on the initiation and growth of bulblets was investigated. These included the effect of temperature, photoperiod, various carbohydrates at different concentrations and combinations as well as various plant growth regulators. Maximum number of bulblets per explant was obtained at 20 °C, with an average of three bulbs p er leaf explant. The average bulblet mass was 57 mg, which was significantly higher than bulblets formed at other tested temperatures. An 8 h light regime was the optimum photoperiod. The highest mean number of bulblets (1.4 per leaf explant) developed under the 8 h photoperiod and the bulblets that formed were large in size. They had a mean bulb diameter of 3.4 mm and a mean bulb weight of 42 mg. Different carbohydrates such as fructose, sucrose and glucose were tested at concentrations of; 1, 3, 6, 9 and 12%. Fructose at a concentration of 3% was found to produce the best results. An average of 1.2 bulbs formed per explant. The mean bulb diameter was 3.4 mm and mean bulb weight was 56.6 mg. Plant growth regulators (GA3, IAA, IBA, NAA, BA, zeatin, iP and others) were tested at concentrations of 1, 2 and 5 mg/L. 1 mg/L IBA was found to be the optimum hormone treatment for bulblet induction. Bulblets were large, had good leaves and well established roots. Medium supplemented with 1 mg/L IBA produced bulblets that had an average bulb diameter of 4.36 mm and a mean bulblet weight of 79.1 mg. Bulblets grown in vitro were transferred to vermiculite and placed in a misthouse to acclimatize. After 2 months the plantlets were transferred to pots containing a sand:soil mixture of 1:1 and placed in a greenhouse. There was a 80 to 90% survival rate.
Some aspects of megagametophyte development and post-shedding seed behaviour of Encephalartos natalensis (Zamiaceae)
(2009) Woodenberg, Wynston R.; Pammenter, Norman William.; Berjak, Patricia.
Very little is known about the post-shedding seed behaviour and megagametophyte development of the cycads, the most primitive extant seed-bearing plants, which pre-date the dinosaurs. In the present investigation, seeds of Encephalartos natalensis Dyer and Verdoorn were shed with relatively high mean embryo (3.33 g g-1) and megagametophyte (1.25 } 0.16 g g-1) WCs, when the developing embryo consisted primarily of the coiled, elongated suspensor bearing a rudimentary sporophyte at its tip. It was not surprising that these seeds were revealed as desiccation sensitive in the present investigation, as the embryos continued to develop after seed-shed, reaching a germinable size (.15 mm) only 4 . 6 months after seed abscission from the strobilus. Maintenance of the seeds in hydrated storage conditions was precluded by the proliferation of fungi, despite the application of the fungicide: BenlateR. Some seeds were also found to germinate in hydrated storage, despite the hard physical barrier to germination imposed by the enclosing sclerotesta. Seeds dusted with BenlateR and placed in eopen f storage in loosely closed paper bags had a longer life-span than those placed in hydrated storage; however, seeds stored in open storage were also overcome by fungi, but only around 18 months after seed-shed. Therefore, while the vigour and viability of the seeds appeared to decline slowly in the months after the embryos reached a germinable size, the life-span of stored E. natalensis seeds devoid of fungi is yet to be determined and will be the subject of further research. The current investigation also combined ultrastructural and viability retention studies to observe the post-shedding behaviour of the storage tissue, the megagametophyte. The cells of the megagametophyte became progressively packed with starch and protein as the two main storage reserves, a limited number of discrete lipid bodies, and occasional mitochondria all of which appeared to be embedded in an homogeneous matrix. When the development of the megagametophyte cells was analysed ultrastructurally, it was found that the unusual matrix was present from the inception of megagametophyte cellularisation, and contained microtubules and numerous very faintly-visible vesicles. Newly-formed megagametophyte cells were thus not highly vacuolated as previously thought, but dominated by an homogeneous matrix. Enzyme-gold localisation was employed in an attempt to determine the organelles responsible for the deposition of cell wall components during cellularisation of the megagametophyte. It appeared that ER-derived vesicles (and not Golgi-derived vesicles) were the principal contributors of the primary cell wall components, pectin and xylan. While cellularisation took place over approximately 1 - 2 weeks, subsequent development of the megagametophyte cells involved the accumulation of storage reserves, this phase lasting approximately 8 months -when the seeds were shed whether pollination/fertilisation had recently occurred, or not. At seed-shed, the cells of the megagametophyte were nucleated and contained a few mitochondria of a metabolically-active appearance. The occurrence of aerobic metabolism in these cells was confirmed by the tetrazolium (TTZ) test. Judging from the TTZ reactivity, the viability of the megagametophyte cells of fertilised seed appeared to decline slowly in the months after seed-shed, in parallel with extension growth of the embryo. The cell layer comprising the external surface of the megagametophyte showed marked ultrastructural differences from the inner cells, and may emerge as having an ‘aleurone-like’ function. It is, however, possible that the cells of the body of the gametophyte participate actively – at least in the earlier stages of post-shedding seed development – in mobilisation of stored reserves, which must support the development of the embryonic sporophyte.
The influence of root chilling on the hydraulic characteristics of selected Eucalyptus taxa.
(2008) Cheek, Michael.
The hydraulic conductance of a plant is a significant factor in determining the
Genetic analysis of Chaerephon pumilus (Chiroptera: Molossidae) from southern Africa.
(2008) Reddy, Devendran.
Chaerephon pumilus, the little free-tailed bat, (family: Molossidae) has a distribution throughout most of sub-Saharan Africa and the eastern region of Madagascar. The vast geographical distribution of this species is accompanied by considerable phenotypic variation, which may conceal cryptic species. The cytochrome b (845 nucleotides) and D-loop (314 nucleotides) regions of the mitochondrial DNA were sequenced to assess phylogenetic relationships within C. pumilus (southern Africa) and in relation to Chaerephon species from Madagascar (C. pumilus, C. leucogaster). Samples were obtained from KwaZulu-Natal, South Africa, and localities in Swaziland. The cytochrome b sample (n = 11) comprised four haplotypes, with a haplotype diversity of 0.6727, whilst the D-loop (n = 34) dataset comprised 13 haplotypes with a haplotype diversity of 0.8342. Neighbour joining, maximum parsimony and Bayesian analyses revealed congruent tree structures for both mtDNA regions. All Chaerephon taxa in this study formed a monophyletic clade with respect to the outgroup Mops midas. Chaerephon pumilus from the eastern side of Madagascar formed a well-supported monophyletic group, sister to a clade comprising C. pumilus (southern Africa) and C. leucogaster, and is suggested to comprise a separate species. Southern African C. pumilus formed two paraphyletic clades, A and B, separated by a genetic distance of 0.9 %. Chaerephon leucogaster formed a monophyletic group nested within southern African C. pumilus, suggesting conspecificity. However, the well-characterized morphology of C. leucogaster lends support to its specific status, and suggests the possible existence of cryptic species among southern African C. pumilus. Population genetic analysis suggests that two C. pumilus (southern African) clades have been expanding, one for between 2432 and 4639 years, and the other for the 11156 to 21280 years. A combined cytochrome b analysis, trimmed to 343 nucleotides, was carried out on the data from this study and that of Jacobs et al. (2004), also on southern African C. pumilus. Haplotypes from the Jacobs et al. (2004) study, which also identified two 0.9 % divergent clades (light- and dark-winged) were found to be identical or very similar to haplotypes from this study and were interspersed among southern African C. pumilus haplotypes in phylogenetic analyses. Chaerephon pumilus haplotypes from Zambia and Tanzania were found to be more closely related to those from southern Africa and to C. leucogaster than to C. pumilus (Madagascar), further indicating that this may be a separate species. Haplotypes from the light-winged clade of Jacobs et al. (2004) were identical to those of dark-winged samples from this study, suggesting that wing shade may not be diagnostic of the two clades.
Some investigations towards the cryopreservation of sugarcane germplasm.
(2009) Jaimangal, Ashika.; Pammenter, Norman William.; Berjak, Patricia.
Sugarcane has become an increasingly important crop in recent years, with South Africa featuring as one of the prominent producers. This has led to a significant growth in the South African sugarcane industry, translating into an increased demand for planting material. Although this demand is now satisfied by recent biotechnological advancements such as protocols for somatic embryogenesis to increase the production of planting material, such techniques are limited as a result of the progressive loss of the embryogenic potential of calli over time. In order to facilitate management of this material, it is desirable to develop a protocol for the long-term storage of the germplasm. This study reports on investigations of the different parameters that influenced the cryoprocess in attempts to develop a protocol for the successful cryopreservation of sugarcane somatic embryos of the 88H0019 variety. Experiments were carried out to determine in vitro culture conditions for successful induction of somatic embryos via both the direct and indirect routes of micropropagation. A suitable regeneration medium for plantlet establishment pre- and post-cooling was established (Chapter 2). Investigations were also carried out to ascertain the responses of somatic embryos to both rapid and slow dehydration techniques (Chapter 3). Finally, several cooling techniques (both slow and rapid), were applied, on partially dehydrated somatic embryos, either without, or after cryoprotection, in an attempt to achieve survival after cryopreservation of the somatic embryos (Chapter 4). Both directly- and indirectly-derived somatic embryos were converted, most successfully, on full strength Murashige and Skoog medium without addition of plant growth regulators. The initial mean water contents of directly- and indirectly-derived somatic embryos were not significantly different from each other (8.38±0.19 g g-1 and 8.45±0.33 g g-1 [dry mass basis], respectively). The percentage conversion at these water contents was also not significantly different; 97% for directly- and 98% for indirectlyinduced embryos. Slow dehydration by culture on a series of media with increasing concentrations of sucrose (from 0.2 M to 1.2 M) for a period of 48 h each was the most effective technique, with water content being reduced to 0.94±0.03 g g-1 and 0.95±0.02 g g-1 after dehydration on media containing 1.0 M sucrose, while maintaining between 98% and 100% conversion, respectively. Of the various cryoprotectants tested, proline and casamino acid had the least adverse effects on the somatic embryos. The encapsulation-vitrification cooling technique was the most efficient of all techniques employed. The best conditions involved encapsulation of embryo clumps in a solution of MS medium with 3% (w/v) Na-alginate and loading solution containing 2 M glycerol plus 0.4 M sucrose, followed by infiltration and dehydration at 0°C for various time intervals (0, 5, 10, 15, 20, 25, 30 min) with 1 ml PVS2 solution and thereafter, rapid immersion in liquid nitrogen. Under such conditions, 30% of the cryopreserved somatic embryos retained viability, going on to form callus from which shoots and roots were produced. Although somatic embryos of sugarcane of the local variety 88H0019 have proved to be recalcitrant to cryopreservation, the results obtained with explants that had been processed by encapsulation-vitrification suggest that this approach may be worth pursuing and refining.
Leaf ultrastructural studies of Avicennia marina in response to salinity under natural conditions.
(2007) Hiralal, Omitha.; Naidoo, Gonasageran.; Naidoo, Yougasphree.
In Richards Bay Harbour, the mangrove Avicennia marina exhibits a distinct natural productivity gradient. The fringe site, which is regularly inundated twice daily by tides, supports luxuriant adult A. marina trees that are 6-10 m tall and which form a dense, well-developed canopy. The landward site which is only inundated during high spring tides, supports diminutive or dwarf A. marina that are less than 1.5 m in height. In this study we compared leaves from fringe and dwarf sites with respect to morphology, ultrastructure and ecophysiology. Alterations in leaf morphology, ultrastructure and physiology of A. marina were compared at the fringe site (35 ‰) and dwarf site (60 ‰) using morphometric measurements, light (LM), transmission (TEM) and scanning microscopy (SEM). SEM and light microscopy revealed that multicellular salt glands were located on the thick, cutinised adaxial surface from leaves of both sites. The glands appeared to be scattered and protruding from individual crypts in fringe mangrove leaves whilst they appeared sunken and occluded by cuticular material in dwarf mangrove leaves. The salt glands on the abaxial surface were not sunken but obscured by the indumentum of peltate trichomes. Ultrastructural changes observed in dwarf mangrove leaves were associated with cuticle, cell walls, chloroplasts, mitochondria of mesophyll tissue and salt glands. Fringe mangrove leaves had chloroplasts with typical well-developed grana and stroma. Ultrastructural changes of chloroplasts were evident in dwarf mangrove leaves and included swelling and separation of thylakoids, disintegration of granal stacking and integranal lamellae, as well as loss of the integrity of the chloroplast envelope. Multivesicular structures were commonly found in vacuoles and associated with chloroplasts and mitochondria in both leaf types. In fringe mangrove leaves, mitochondria appeared spherical to tubular with a relatively smooth outer membrane and a highly convoluted inner membrane. Swelling and vacuolation of mitochondrial membranes, cristae and mitochondrial clustering in the cytoplasm around the chloroplasts were evident in dwarf mangrove leaves. Extensive lipid accumulation in the form of large, dense plastoglobuli occurred in the chloroplasts of dwarf mangrove leaves. There were characteristic differences in salt gland morphology of fringe and dwarf mangrove leaves, namely in the cell walls, vacuoles, and vesicle formation. In salt glands of dwarf mangrove leaves, a distinct withdrawal of the cytoplasm from the cell wall was observed. This feature was not observed in salt glands of fringe mangrove leaves. Numerous large vacuoles were observed in the secretory cells of glands of dwarf mangrove leaves compared to those of fringe plants. Multivesicular structures, vesicles and mitochondria were common features in both leaf types. Physiological studies involved a comparison of osmotic and ionic relations as well as whole plant responses in fringe and dwarf mangrove leaves. Relative leaf water content decreased by 7.8 % and specific leaf area by 17 % in dwarf compared to those of fringe mangroves. Dwarf mangrove leaves were 27.6 % thicker and leaf cuticle thickness 37.4 % higher than those from fringe mangroves. Fringe mangrove leaves displayed higher total chlorophyll contents by 27 %, with chlorophylls a and b being 22 % and 39.6 % higher, respectively than those of dwarf mangroves. Salt gland frequencies were higher in the apex, mid-lamina and base of fringe than dwarf mangrove leaves by 36 %, 45 % and 51 %, respectively. The concentration of glycinebetaine, a compatible, N-containing osmolyte was significantly higher by 40 % in dwarf than in fringe mangrove leaves. Concentrations of proline were 27 % lower in dwarf than in fringe mangrove leaves. The predominant inorganic ion detected in mature leaves was Na+, which was 19 % higher in dwarf than fringe mangrove leaves. Phosphorus was an element that appeared deficient in dwarf mangrove leaves, being 50 % lower compared to fringe mangrove leaves. The results of this investigation indicated that there were cytomorphological alterations as well as differences in physiological responses in leaves of A. marina at fringe and dwarf sites.
The effect of developmental status and excision injury on the success of cryopreservation of germplasm from non-orthodox seeds.
(2007) Goveia, Meagan Jayne Theresa.; Kioko, Joseph Ivala.; Berjak, Patricia.; Pammenter, Norman William.
The zygotic germplasm of plant species producing desiccation-sensitive seeds can be conserved in the long-term only by cryopreservation. Usually the embryonic axis is excised from the cotyledons and is used as the explant for cryopreservation as it is small and provides a large surface area:volume ratio. However the shoot of the axis of most species studied does not develop after excision, with the result that survival after cryopreservation is often recorded as callus production or simply explant enlargement and/or greening. Thus, besides explant size, factors such as in vitro regeneration techniques, physical injury induced upon excision and developmental status of the seed could compromise the success of cryopreservation. This study investigated the effect of the factors mentioned above, with particular attention to the developmental status of the seeds on explant in vitro development (section 3.1), response to dehydration (section 3.2) and cryopreservation of the desiccation-sensitive embryonic axes (section 3.3) of two species: Trichilia dregeana, T. emetica and embryos of a third, Strychnos gerrardii. For all three species, investigations were conducted on the embryonic axes/embryos excised from mature seeds immediately after fruit harvesting and from mature seeds stored under hydrated conditions for different periods (in order to achieve different degrees of development). In addition, preliminary studies were carried out on axes of T. dregeana to assess whether generation of reactive oxygen species (ROS) occurs in response to wounding upon axis excision (section 3.4). Excised embryonic axes of T. dregeana and T. emetica did not develop shoots in vitro unless the explants included attached cotyledonary segments. Following the development associated with short-term storage, however, the excised axes could develop shoots after complete cotyledon excision. The embryos from the (endospermous) seeds of S. gerrardii which included the paper-thin cotyledons, developed normally in vitro, with percentage germination increasing with seed storage time. For all three species, in vitro axis germination was promoted when activated charcoal was included in the germination medium, regardless of the developmental stage of the seeds. When dehydrated to approximately 0.3 g H2O g-1 dry mass (g g-1), embryonic axes from all three species failed to develop shoots even though a minimum of 50% produced roots in all cases. Hence, shoot production was shown to be more sensitive to desiccation than was root production. Furthermore, the sensitivity of the shoot apical meristem to desiccation was not ameliorated with seed storage for T. dregeana and T. emetica, but did decrease for S. gerrardii when seeds were stored for 6 – 8 weeks. The application of certain cryoprotectants did facilitate production of shoots after dehydration by a few axes of both Trichilia spp. For T. dregeana explants, combination of glycerol and sucrose allowed for 10% of the axes to retain the ability for shoot production after dehydration while for T. emetica explants, the combination of DMSO and glycerol (10 - 20% shoot production after dehydration) was best. The efficacy of the cryoprotectants was not influenced by storage period. The provision of cryoprotectants still needs to be tested for S. gerrardii. Survival of subsequent cryopreservation of T. dregeana and S. gerrardii explants was best achieved with rapid cooling in nitrogen slush, with the cooling procedure for T. emetica explants still to be optimized. The highest post-cryopreservation survival of T. dregeana axes was achieved when seeds had been stored for three months, while the seed storage period did not affect post-thaw survival of the axes of T. emetica or S. gerrardii. A small proportion of S. gerrardii explants only, produced shoots after cryopreservation, whereas the surviving embryonic axes of T. dregeana and T. emetica regenerated only as non-embryogenic callus. Although callus production is less desirable than successful seedling establishment, it has the potential for micropropagation if embryogenicity can be induced. Ultrastructural examination of the shoot apical meristem of T. dregeana after a 3-d recovery period, following excision, revealed considerable cellular derangement, although damage of individual organelles could not be resolved microscopically. Preliminary studies on T. dregeana involving a colorimetric assay using epinephrine, confirmed the generation of ROS in response to wounding associated with axis excision. Reactive oxygen species generated appeared to persist over prolonged periods rather than occurring only as a single oxidative burst. Hence, ROS production at the wound site could be the primary factor contributing to lack of shoot development. Axes immersed in the anti-oxidant, ascorbic acid (AsA) immediately after excision, showed lower ROS production and 10% shoot development when cultured in vitro, indicating that the oxidative burst coincident with, and after excision might be counteracted if immediate ROS production can be adequately quenched. Future investigations should aim to identify the specific ROS associated with wounding and optimize an anti-oxidant treatment(s) that will facilitate shoot development. Thus, the successful cryopreservation of the germplasm of the species tested, and others producing recalcitrant seeds, depends on a spectrum of species-specific factors, some still to be elucidated.
Southern African plants used to treat central nervous system related disorders.
(2009) Stafford, Gary Ivan.; Jäger, Anna Katharina.; Van Staden, Johannes.
The majority of the population in South Africa use traditional health care to treat various mental conditions. This thesis has two main objectives; to bring together a comprehensive and detailed record of psychotropic plants used in southern Africa by indigenous peoples for medicinal or cultural purposes. Secondly, this research attempts to investigate the validity and rationale of the use of these plants by screening them in various biological assays for psychotropic activity. Plants were selected, based on their traditional use and availability, and were screened in four assays, which detect biological activity of a useful nature. A number of in vitro enzymatic and neuronal signal transduction assays were employed in this thesis, the inhibition of the serotonin reuptake transporter protein (SERT); inhibition of catabolic enzymes (e.g. acetylcholinesterase, monoamine oxidase); GABAA- benzodiazepine receptor binding. The influence of legislation, past and present, on the state of traditional medicine is highlighted. Aspects of the philosophies and practises of the various practitioners of South African traditional medicine will be discussed. An annotated list compiled from available ethnobotanical literature of plants traditionally used for central nervous system-related purposes is provided. It contains more than 330 species, from 94 families, which are currently used or have been used for cultural, medicinal and recreational purposes related to the central nervous system (CNS). Where available, information pertaining to plant part used, preparation method, dosage, route of administration, known and potentially active constituents are included. Seventy five extracts from 34 indigenous plant species used in South African traditional medicine or taxonomically related to these were investigated for their affinity to the serotonin reuptake transport protein, making use of an in vitro [3H]-citalopram serotonin reuptake transport protein binding assay. Aqueous and 70% ethanolic extracts of various plant parts were screened and 45 extracts derived from 15 plant species showed affinity. The affinity of 12 extracts from four plants was characterized as high (more than 50% inhibition at 5, 1, and 0.5 mg/ml). Plant species with high affinity to the serotonin reuptake transport protein included Agapanthus campanulatus, Boophone disticha, Datura ferox and Xysmalobium undulatum. Agapanthus campanulatus yielded high activity in aqueous extracts from leaves and flowers. B. disticha showed high activity both in aqueous and ethanolic extracts of leaves and bulbs. D. ferox showed high activity in aqueous extracts from the seeds and X. undulatum showed high activity in the ethanolic extract of the whole plant. Two compounds, buphanadrine and buphanamine, were isolated by bioassay-guided fractionation on vacuum-liquid-chromatography (VLC) and preparative thin-layer-chromatography (TLC) from B. disticha. The structures of the compounds were determined by 1H and 13C NMR. Fractions were tested for affinity to the serotonin transporter in a binding assay using [3H]-citalopram as a ligand. The IC50 values of buphanidrine and buphanamine were 274 ìM (Ki = 132 ìM) and 1799 ìM (Ki = 868 ìM), respectively. The two alkaloids were also tested for affinity to the 5HT1A receptor, but only showed slight affinity. Aqueous and ethanol extracts of 43 plants that are traditionally used to treat against epilepsy and convulsions were initially tested in the GABAA-benzodiazepine receptor binding assay, where the binding of 3H-Ro 15-1788 (flumazenil) to the benzodiazepine site is measured. The GABAA-benzodiazepine receptor complex is involved in epilepsy and convulsions. Out of the 118 extracts tested, one aqueous and 18 ethanol extracts showed activity. The most active extracts were the ethanolic leaf extracts of Searsia tridentata, Searsia rehmanniana and Hoslundia opposita and the ethanolic corm extract of Hypoxis colchicifolia, which all showed good dose-dependent activity. A further forty-six ethanol extracts from another 35 species, both indigenous and exotic that are traditionally used predominantly as sedatives or to treat various CNS-related ailments were tested in the GABAA-benzodiazepine receptor-binding assay. Out of the 46 extracts tested, seven showed good activity and 10 showed moderate activity. The most active extracts were the ethanolic leaf extracts of Arctopus echinatus, Artemisa afra, four Helichrysum species and Mentha aquatica which all showed good dose-dependent activity. Two biflavonoids with activity in the 3H-Ro 15-1788 (flumazenil) binding assay were isolated by high pressure liquid chromatography (HPLC) fractionation of the ethanol extract of the leaves from Searsia pyroides. The structures of the two biflavonoids were elucidated by nuclear magnetic resonance spectroscopy (NMR) to be agathisflavone and amentoflavone. Agathisflavone and amentoflavone competitively inhibited the binding of 3H-Ro 15-1788 with a Ki of 28 and 37 nM, respectively. Extracts of Searsia dentata and Searsia pentheri were not as active as the extract from Searsia pyroides; both were found to contain apigenin and agathisflavone. The monomer apigenin, agathisflavone and amentoflavone were fitted into a pharmacophore model for ligands binding to the GABAA receptor benzodiazepine site. This reflected the affinities of the compounds in the [3H]-flumazenil binding assay. Mentha aquatica, a mint that is found in Europe and Africa, is used in Zulu traditional medicine for spiritual purposes. The ethanolic leaf extract showed a strong affinity to the GABA-benzodiazepine receptor. Viridiflorol from the essential oil and (S)-naringenin from an ethanolic extract was isolated by bioassay-guided fractionation using binding to the GABA-benzodiazepine site. Viridiflorol had an IC50 of 0.19 M and (S)-naringenin of 0.0026 M. Twenty plants used in Zulu traditional medicine for several CNS-related ailments were screened for MAO inhibition and specific MAO-B inhibition activity. MAO-B inhibitors are currently employed in the treatment of neurodegenerative related illnesses such as Parkinson's and Alzheimer's diseases. A photometric peroxidase linked assay was used to determine the inhibition of the oxidative deamination of tyramine by MAO isolated from rat liver. Ruta graveolens exhibited the best MAO inhibitory activity (ethyl acetate leaf extract = IC50 5 ± 1 ìg/ml, petroleum ether extract = 3 ± 1 ìg/ml) and specific MAO-B inhibition (ethyl acetate leaf extract = IC50 7 ± 6 ìg/ml petroleum ether extract = 3 ± 1 ìg/ml). Schotia brachypetala, Mentha aquatica and Gasteria croucheri also exhibited good MAO-B inhibition activity. Six extracts of varying polarity of Mentha aquatica were tested in a photometric peroxidase linked MAO bioassay. The 70% ethanol extract had highest inhibitory activity. (S)-Naringenin was isolated from the extract by bioassay guided fractionation on VLC and preparative TLC. The structure of the compound was determined by 1H, 13C and 13C-DEPT NMR and optical rotation. The IC50 values for MAO inhibition by naringenin were 342 ± 33 ìM for the rat liver mitochondrial fraction, 955 ± 129 ìM for MAO-A and 288 ± 18 ìM for MAO-B respectively. South African traditional medicine clearly utilizes many botanical species with CNS-related activity. Only a small number of the more than 330 southern African plant species reported to treat or alter the CNS have been scientifically evaluated. To date very few of the active compounds have been isolated and identified.

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