Doctoral Degrees (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.
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.
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.
Assessment of a process-based model to predict the growth and yield of Eucalyptus grandis plantations in South Africa.
(2005) Esprey, Luke John.; Smith, Colin William.; Pammenter, Norman William.; Sands, Peter.
It is believed that the process-based model 3-PG (Physiological Principles Predicting Growth; Landsberg and Waring, 1997) can potentially play a useful role within South African forestry, both as an operational and a strategic tool. Strategic applications may include the prediction of potential productivity on a site-by-site basis; broadscale productivity estimates based on remote sensing and the spatial application of 3-PG; identification of production constraints; and estimation of carbon fluxes to help address sustainability issues. Operationally, 3-PG could complement empiricallybased models or be used in conjunction with them as a hybridised product. The challenges of this study were therefore to see whether it is possible to adapt 3-PG to predict the growth and yield of E. grandis under South African conditions, test that model predictions are consistent with observed growth data and are biologically reasonable, and to assess the practicality of using 3-PG as either a strategic or operational tool. The main emphasis of this study was to understand the internal logic of 3-PG and how physiological processes are represented, and to develop methods to objectively parameterise and initialise the model. Thereafter a detailed model validation study was performed, ending off with selected potential applications of 3-PG within the South African context. The sensitivity of predicted stand volume (SV) and leaf area index (LAI) to the values of the species-specific parameters in 3-PG was examined. These analyses enabled the development of three distinct parameter sensitivity classes: insensitive parameters (i.e. those that can be varied widely without affecting the outputs studied), sensitive parameters (i.e. those whose value strongly affects the outputs, and non-linear parameters (i.e. those for which the outputs depend in a non-linear manner on the parameter value). Minimum data requirements for the parameterisation and initialisation of 3-PG are considered in detail. Conventional methods used for the parameterisation of models, specifically 3-PG, are reflected upon. An automated parameter estimation technique was examined and used for the estimation of selected parameters. Species-specific parameters were categorised according to data source estimation and sensitivity classes. Parameters describing allometric and age-dependent relationships were assigned values using observed data from biomass harvests. Critical parameters that could not be directly assigned using observed data were the ratio of foliage to stem allocation (i.e. P2 and p2o), allocation of net primary production (NPP) to roots (TJRX and T]Rn), optimum temperature for growth (7^,) and maximum canopy quantum efficiency (acx)- These were estimated using Parameter ESTimation, by fitting model output to corresponding observed growth data. As well as species-specific parameter values, mandatory inputs required by 3-PG include weather data, site-specific factors such as site fertility (FR) and physical properties of the soils, and stand initialisation data. Objective techniques to determine these site-specific factors and the initial values for the biomass pools were proposed. Most of these data are readily available for sites where experimental trials exist, or where monitoring networks are in place. However, this is the exception rather than the rule, so alternative data and information sources are required. These, together with the need for accurate weather inputs (especially monthly rainfall) and physical properties (especially soil texture, maximum available soil water and FR) of the sites being modelled were explored. 3-PG was validated using four simple tests by comparing predicted versus observed SV. Results showed that 3-PG predictions are relatively consistent with observed stand data. Analyses performed using time-series data showed model predictions accurately tracked observed growth in response to erratic and fluctuating weather conditions. Results from the initial model validation showed production on high and low productivity sites was under- and over-predicted, respectively. Further results presented here show a similar, but less marked trend (i.e. over- and under-predictions are not as extreme), and individual biases are less than those from predictions made using another locally developed parameter set. The application of 3-PG showed that the model is able to make estimates of tree growth that are consistent with those used within the forestry site classification. This showed the considerable potential 3-PG has for strategic planning by the forest industry (i.e. projected wood supplies etc) and in research planning (refining existing site classifications). The model could be useful in predicting growth in various areas where E. grandis is not grown, assisting in future decision making. 3-PG was able to identify growth constraints on a site-by-site basis and distinguish among them, and was able to identify environmental and site limitations to plantation growth, and how they vary in space and time. These results together with predictions of site productivity demonstrate the potential for 3-PG to be used to improve existing forest site classifications. The model comparison study between empirically-based models and 3-PG showed that although the empirical models made accurate predictions of volume under static climatic conditions, under fluctuating weather conditions empirical estimates of volume were less accurate than those made with 3-PG. 3-PG can therefore be used operationally with minimum input data to predict growth using enumeration data. This is useful in saving time and cutting costs. The use of process-based models (PBMs) in general, and 3-PG in particular, needs to be "championed'' to the South African forest industry. This is necessary for two reasons. Firstly, the model and the manner with which it describes physiological processes of growth need to be explained in layman's terms. This will demonstrate how and why a process-based model can work better in a fluctuating environment than empirically based models. Secondly the comparison between 3-PG and the local empirical models needs to be presented as an example of how 3-PG can be applied on an operational basis. It is accepted that much convincing is still required.
Some implications of associated mycoflora during hydrated storage of recalcitrant seeds of Avicennia marina (Forssk.) Vierh.
(2004) Calistru, Claudia.; Berjak, Patricia.; Pammenter, Norman William.; McClean, Michelle.
Three questions are considered in the context of the possible effects of seedassociated mycoflora, typified by Fusarium moniliforme, during hydrated storage of recalcitrant seeds of the tropical species, Avicennia marina. These are: 1) whether fungal infection reduces storage lifespan; 2) whether seeds become more susceptible to fungal attack during storage and whether they posses defence mechanisms that might suppress fungal proliferation in hydrated storage (production of antifungal compounds and 13-1,3-glucanase (EC 3.2.1.39) and chitinase (EC 3.2.1.14)] and 3) whether it is possible to discriminate ultrastructurally between inherent deteriorative changes and those that are fungally-induced. 1) The data indicate unequivocally that if fungal activity is curtailed, then the hydrated storage lifespan of A. marina seeds can be considerably extended. 2) When inoculated immediately with F. moniliforme, newly harvested seeds were extremely susceptible to the adverse effects of the fungus, while seeds that had been wet-stored for 4 days showed a considerably heightened resilience to the effects of the fungus prior to inoculation. The enhanced resilience, although declining, persisted in seeds stored hydrated for up to 10 days prior to inoculation, being lost after 12 days. This finding was supported by significant increase in 13-1,3-glucanase and chitinase and in antifungal compound production during 10 days of wet storage. After 14 days of wetstorage, seeds become more susceptible to the effects of fungusthanthose in the newly harvested condition. 3) The resilience of seeds that had been stored in the short-term was associated with ultrastructural changes indicative of enhanced metabolic activity associated with the onset of germination (e.g. increase in vacuolation, well-developed mitochondria and endomembrane system [ER and Golgi bodies]). However, with sustained stress associated with wet-storage IV conditions, the seeds became increasingly badly affected by the fungus, showing some ultrastructural fungally-induced abnormalities (e.g. nuclear lobing, presence of lipid bodies and prevalence of Golgi bodies that had many associated vesicles) and a decrease in 13-1,3-glucanase and chitinase activity. It is suggested that the decreased susceptibility of A. marina seeds during short-term storage relies on the ability to create an antifungal environment prior to infection (through synthesis and accumulation of pre-formed and induced antifungal compounds and antifungal enzymes), which would also be an effective strategy during germination in the natural environment.
The influence of the monocarpic herb, Isoglossa woodii, on subtropical forest tree dynamics and diversity.
(2009) Tsvuura, Zivanai.
Dominant understorey species, such as herbs, ferns, palms and shrubs may influence forest tree species diversity and dynamics. Their influence may be through shading the forest floor, thereby affecting regeneration of shade-intolerant species and reducing species diversity, or it may be through competition with seedlings for space and belowground resources, thus modifying or changing the structure of the forest. These effects may be compounded if the life cycle of the understorey species consists of synchronized reproductive and mortality events. This study examines the influence of a dominant understorey species, Isoglossa woodii (Acanthaceae), on regeneration of trees in Indian Ocean subtropical coastal dune forest in southern Africa. The species is a semiwoody herb and has population-wide synchronous reproduction at 4-7 year cycles after which it dies and regenerates from seed. In this thesis I examine three aspects of the ecology of this suppressive herb: (i) the ecological and environmental correlates of the distribution of I. woodii; (ii) the evolutionary advantages of synchronous monocarpy; and (iii) the ecological effects of the extensive cover and putative recruitment window caused by I. woodii on forest tree seedling dynamics and diversity. Isoglossa woodii covered 65–95 % of the understorey, while gaps in this understorey cover occupied the remaining 5–35 % of the area. The spatial distribution of I. woodii was strongly related to tree canopy structure, with the species excluded from sites with dense canopy cover. Woody seedling establishment was inhibited by low light availability (
Pollination, floral deception and evolutionary processes in Eulophia (Orchidaceae) and its allies.
(2009) Peter, Craig Ingram.
Orchids provide a model system for addressing evolutionary and ecological questions both because of their species diversity, and because the packaging of their pollen into pollinia facilitates the estimation of male and female pollination success. This thesis focuses on the ecology and evolution of pollination systems in the African orchid genus Eulophia, with an emphasis on deceptive pollination, mechanisms promoting cross-pollination, and pollinatordriven speciation. Pollination in the deceptive species E. zeyheriana is shown to depend on flower colour and proximity to the rewarding model species, Wahlenbergia cuspidata (Campanulacae). This study demonstrates the functional importance of colour matching between model and mimic in a floral Batesian mimicry system, as well as the importance of facilitation by the rewarding model [chapter 2]. The pollinaria of the vast majority of Eulophia and Acrolophia species undergo reconfiguration following removal by pollinators, similar to the phenomena first described by Darwin in some European orchids and which he hypothesised to be adaptations to limit pollinator mediated self-pollination. In chapter 3, a less common mechanism – anther cap retention – is described for E. foliosa. Observations of reconfiguration times were compared to the respective visit times by pollinators in a number of orchids (including Eulophia and Acrolophia) and asclepiads. In 18 of 19 species, pollinarium reconfiguration times exceed the average visit times, providing empirical support for Darwin’s cross-pollination hypothesis [chapter 4]. All of the 25 species of Eulophia examined are deceptive, but two of the three species in the small, closely related Cape genus Acrolophia examined in chapter 5 are rewarding. This translates into very high levels of pollen transfer efficiency in the rewarding A. cochlearis relative to the deceptive A. capensis and species of Eulophia. In addition, A. cochlearis exhibits high rates of pollinator-mediated self-pollination, as quantified using a novel method based on levels of inbreeding depression during embryo development. In chapter 6 the evolutionary divergence of long- and short-spurred forms of E. parviflora in response to different pollinators is investigated. This shows that divergence has occurred in floral morphology, scent chemistry and flowering phenology and that this can be attributed to adaptations to the respective bee and beetle pollinators of each form. This thesis also includes case histories of bee pollination in an additional five Eulophia species, and beetle-pollination in two other species of Eulophia with dense inflorescences and slow pollinarium reconfiguration [chapter 7]. In addition, four taxa were found to undergo auto-pollination [chapter 8]. The main conclusions of this thesis are that pollination of food-deceptive species can be enhanced by spatial proximity to, and floral colour matching with, sympatric rewarding species; that selection strongly favours traits that promote cross-pollination; that pollinatorshifts can drive speciation; and that floral adaptations for bee-, beetle-, and auto-pollination are found in South African representatives of Eulophia.
The potential of bulk segregant analysis and RAPD technology for identification of molecular markers linked to traits in sugarcane.
(1998) Msomi, Nhlanhla Sobantu.; Botha, Frikkie Coenraad.
The objective of the present study was to investigate the potential use of bulk segregant analysis (Michelmore et al., 1991) as a method to rapidly identify genetic markers linked to traits in sugarcane. Four bulked DNA samples were prepared from progeny of a sugarcane cross, AA157, based on segregation for the fibre trait. The bulks comprised five and ten individuals on either side of the fibre phenotypic extreme. The random amplified polymorphic DNA (RAPD) technique (Williams et ai., 1990) was used to screen for differences between the low and high fibre bulks. A total of 749 fragments were amplified in the bulks, eight of which were polymorphic. The segregation of the bulk specific polymorphism was analysed in 80 progeny of the AA157 cross; and seven were found to reproducibly segregate on a 1: 1 basis. This indicates that they are single dose fragments. A total of 79 polymorphisms were detected between the parents of the cross, indicating 10.5% variation in the genomic region sampled. Twenty two of the parental polymorphisms segregated as single dose fragments in the progeny of the cross AA157. Analyses of variance (ANOVAs), and multiple regression analyses, were used to ascertain linkage of the putative RAPD markers to fibre, and if linked, to determine the fibre variation ascribed respectively. Three RAPD fragments were found linked to the fibre trait. Fragments OPA17438 and OPC16889 (at the 99% significance level), and OPB1l464 (at the 95% significance level). These putative markers ascribed a total of 28.6% fibre variation in the 1993 season. The association of the RAPD markers with fibre in the different seasons (1992, 1993 and 1994) was investigated. Three RAPD markers were found linked to the fibre trait in each season, with a total of 5.5% and 31,4% fibre variation ascribed in the 1992 and 1994 seasons respectively. Marker OPA17438 was found to be linked to the fibre trait in all three seasons investigated, and marker OPC16889, was found linked to the fibre trait in the 1992 and 1993 seasons. Cross validation of the linkages of the RAPD markers to the fibre trait was carried out by a modified form of 'jacknifing' where the sample size was reduced to N-l0, and RAPD marker-fibre trait associations investigated as before. RAPD markers OPA17438 and OPC16889 were still consistent across the seasons, however marker OPA17438 was no longer linked to the fibre trait in the 1992 season. To investigate the genetic behaviour of RAPD based markers in sugarcane and the potential for their application in marker-assisted selection (MAS), two putative RAPD markers were converted to sequence characterised amplified regions (SCARs) (Paran and Michelmore, 1993). The RAPD fragments OPA17438, OPBl1464, and OPC16889 were excised from agarose gels, re-amplified and cloned into the pCR-Script SK (+) phagemid for sequencing. RAPD markers OPA17438 and OPB11 464 were converted to SCARs by using their sequences to design longer specific primers. A third SCAR marker, SAl1640, originally derived from sugarcane cDNA as a potential stem preferential expressed sequence tag, was included in the analysis to increase the sample size. All three SCAR markers segregated in a monomorphic fashion in the parents and progeny of the cross AA157. In addition, monomorphic length variants for markers, OPA17438 and OPB11 464 were detected with the SCAR amplification. All three SCARs segregated in a monomorphic fashion in different commercial varieties and bulks of S. officinarum and S. spontaneum, the progenitors of modern commercial varieties. The segregation analyses of the SCAR markers indicate that the RAPD polymorphism of marker SAl1640 was probably due to a point mutation or mismatch in the priming site. The segregation analyses of SCARs for the markers OPA17438 and OPB11464 indicate that their segregation in the RAPD analyses was due to an insertion mutation in the genetic locus. The combined results of the SCAR and RAPD segregation of markers OPA17438 and OPB11464 are indicative of preferential pairing in the cross AA157. Finally, to investigate the extent of linkage disequilibrium in a modern commercial variety, twenty two single dose RAPD fragments were investigated for their association with four traits in 53 progeny of cross AA157. The four traits investigated were fibre %cane, brix %cane, pol %cane and ers %cane over three seasons (1992, 1993 and 1994), at different ages of harvest (12, 8, and 9 months respectively). Seventeen linkages of RAPD markers to the four traits, over the three seasons, were detected. The phenotypic variation ascribed by the RAPD markers ranged from 7.6% fibre %cane variation explained by one marker in 1992, 29.6% fibre %cane (three markers) in the 1993 season to 10% (three markers) in 1994. A total of 14.1% brix %cane variation was ascribed by two markers in 1992, 9.6% (one marker) in 1993 and 16.3% (two markers) in the 1994 season. A total of 13.5% estimated recoverable sucrose %cane was ascribed by one marker in 1992, 12% (two markers) in 1993 and 15.3% (two markers) in the 1994 season. Two markers explained 17.2% pol %cane variation in 1992 and 25.4% in the 1994 season. Only four markers were detected across different environments, three of which were linked to fibre. These were OPA17438, OPB16618 and OPC16889, each linked to fibre in two seasons. RAPD marker OPB11 464 was linked to estimated recoverable sucrose %cane in two seasons. Two markers were found associated with different traits in a single season. RAPD marker OPB11 464 was found associated with brix %cane and estimated recoverable sucrose %cane in the 1993 season, and RAPD marker OPA17438 was found associated with all four traits in the 1994 season.
Some physiological and growth responses of three eucalyptus clones to soil water supply.
(2002) Manoharan, Printhan.; Pammenter, Norman William.
The response of three Eucalyptus spp. clones (GC550, GU210 and TAG14) to water availability was assessed in terms of growth, plant water status, leaf gas exchange, whole plant hydraulic characteristics (both root and shoot), stem xylem vulnerability. Furthermore, to experimentally assess the influence of hydraulic conductance on leaf physiology and plant growth, specimens of two of the clones were subjected to long-term root chilling. Prior to harvesting data were collected on the diurnal variation in leaf water potential (ΨL), transpiration rate (E), stomatal conductance (gs) and net CO2 assimilation rate (A). Main stem xylem vulnerability was assessed using ultrasonic acoustic emissions (UAE). Vulnerability of the main stem was assessed as the leaf water potential corresponding to the maximum rate of acoustic emissions (ΨL, EPHmax), and as the critical water potential triggering cavitation events, calculated as the mean of the water potentials of data points lying between 5 and 10% of the total accumulated emissions (ΨCAV,cUAE,%). Hydraulic conductance was measured on roots and shoots using the high-pressure flow meter (HPFM). Root data were expressed per unit root dry mass (Kr/trdw) and per unit leaf areas (Kr/LA), shoot data expressed per unit shoot dry mass (Ks/tsdw) and per unit leaf area (Ks/LA), and whole plant conductance was expressed per unit leaf area (KP/LA). Soil-to-leaf hydraulic conductance was also assessed as the inverse of the slope of the relationship between leaf water potential and transpiration rate (the evaporative flux, EF, method). A field study was undertaken on three month old TAGl4 and GU210 plants. Diurnal values of leaf water potential ΨL, E and gs were consistently higher in TAG14 than GU210, but A did not differ among the clones. Main stem xylem vulnerability (ΨCAV, cUAE,%) was higher in TAG14 than GU210. In both clones midday ΨL fell below ΨcAv,cUAE,%, suggesting lack of stomatal control of xylem cavitation. Kr/LA was higher in TAGl4 than GU210, whereas, Ks/LA and Ks/tsdw was higher in GU210 than TAGI4. A greater proportion of hydraulic resistances resided in the roots, particularly in GU210. Kp/LA was higher in TAGl4 than GU210 clone, although the significance was marginal (P=0.089). However, all the physiological measurements, were consistent with the concept of higher hydraulic conductances in TAGl4 leading to lower leaf level water stress. Above ground biomass was higher in TAG14 than GU210, in agreement with this concept, although this clone was more vulnerable than GU210. Material grown for 14 months in 25 l pots clones showed no differences in ΨSoil between the high and low watering supply, indicating that even the 'high' supply was inadequate to prevent water stress. In accordance with this, diurnal values of ΨL, gs, E and A did not differ significantly between treatments and clones. Early stomatal closure was apparent, maintaining ΨL constant during the middle of the day. Stem xylem vulnerability, assessed as both ΨL,EPHrnax and ΨCAV,cUAE,% showed that the main stem of GC550 was more vulnerable than other two clones, and that low watered plants were more resistant to xylem cavitation than those receiving high water. Midday ΨL fell below the vulnerability values assessed by both measures across treatments and clones, suggesting lack of stomatal control preventing stem xylem cavitation. There was no relationship between stem xylem cavitation and the shoot hydraulic conductances. Root pressures did not differ between either treatment or clones. Kr/LA was marginally higher in high watered plants, and Ks/LA and Ks/tsdw were higher in low watered plants, possibly by adjustment of leaf hydraulic architecture, and there were no clonal differences. Kr/LA was much lower than Ks/LA. Kp/LA did not differ between the watering treatment, but there was a clonal effect. Growth in dry mass was higher in high watered than low watered plants, but there were no differences among clones. As KP/LA was not affected by watering treatment there was no relationship between KP/LA and growth in total biomass. In plants grown for 21 months in 85 l pots low water treatment decreased midday ΨL, gs, E and A relative to high watered plants. Interclonal differences occurred at midday. Stem xylem vulnerability assessed as ΨCAV,cUAE,% and as ΨL,EPHrnax show similar trends as in the 14 months saplings, clonal differences being significant in ΨL, EPHmax. There was a 1:1 relationship between minimum leaf water potential and ΨL, EPHmax, suggesting that the water potential developed was limited by stem vulnerability. This implies stomatal control to reduce transpiration rates to prevent extensive cavitation occurring. These plants did not develop positive root pressures, indicating that recovery from xylem cavitations occurred through some other process. Kr/LA was higher in high watered plants than those receiving low water, and clonal differences were observed in Kr/trdw. There was no treatment effect in KS/LA and KS/tsdw, but a clonal effect was apparent. KP/LA was significantly different between treatment, and was reduced by low water in two clones, and increased by this in TAGI4. Reduced water availability reduced biomass production, with a greater effect on roots than shoots, such that low watering reduced root:shoot ratios. There was a weak but significant relationship between whole plant hydraulic conductance and maximum stomatal conductance, and between plant conductance and total biomass produced; these data are consistent with the concept of some hydraulic limitation to growth. Root chilling (achieved through chilling the soil) of two of the clones was used to experimentally manipulate hydraulic conductance to test the hydraulic limitation hypothesis. Short-term root chilling decreased both Kr/LA and KP/LA in both clones, but had marginal effects on leaf gas exchange. With long-term chilling the decrease in Kr/LA was observed only in GU210, with TAGl4 showing some adjustment to the treatment. As the roots constitute the major hydraulic resistance, KP/LA largely reflected those of the roots. Long-term root chilling significantly affected leaf physiological characteristics, despite the lack of effect on hydraulic conductance in TAGI4. Long term chilling decreased the whole plant dry mass, but the effect was smaller in TAGI4, and this clone also showed morphological adjustment, in that root growth was less adversely affected than shoot growth. The data from GU210 support the hydraulic limitation hypothesis; because of the morphological and physiological adjustment to long-term root chilling in TAGI4, the data are unsuitable to directly assess the hypothesis.
Establishing genetic diversity of Rwanda highland banana using random amplified polymorphic DNA markers.
(2006) Nsabimana, Antoine.; Van Staden, Johannes.
The characterization of the banana germplasm collection from Rubona - Rwanda was investigated using morphological and cytological characteristics of the genomic groups. Genetic diversity was assessed using Random Amplified Polymorphic DNA analysis. The survey was conducted to evaluate the distribution of banana cultivars in the four major growing regions of Rwanda. A total of 90 accessions from the National Banana Germplasm Collection at Rubona Rwanda were characterized and six characters of the fingers (length, width, weight, green life, post green life and length/width ratio) were subjected to principal component analysis (PCA). The cooking and beer clones were separated. The cooking clones were further grouped into three clone sets: Musakala, Nakabululu, and one that constitutes Nakitembe and Nfuuka clone sets. The AAB genomic group was separated from AAA, AB and ABB genomic groups. The results from the survey showed that East African Highland bananas are the most important genotype group in the four major banana growing regions of Rwanda ranging between 60 - 90% of banana mats counted. Several new Highland banana cultivars were recorded, such as 'Intokatoke', 'Igihuna', 'Ingenge', 'Ingaju', 'Icyerwa', 'Mitoki', 'Madamu', 'Inkokobora', 'Intokekazi', 'Bugoyi', 'Ishoki'. Amongst these cultivars, some were classified as cooking and others as brewing bananas. However, in the National Banana Germplasm Collection at Rubona - Rwanda, the uses of these cultivars are recorded differently therefore increasing the need for agro-morphological characterization. The assessment of ploidy level of accessions from the National Banana Germplasm Collection at Rubona - Rwanda, by flow cytometry showed misclassification of some accessions such as 'Pomme', 'Kamaramasenge', 'Gisubi kayinja', 'Gisubi kagongo', and 'Dibis' which were classified as diploid, diploid, triploid, and tetraploid respectively. They IV were found to be triploid, triploid, triploid, diploid and triploid. All these bananas were recently introduced into Rwanda, while the endemic Highland bananas were triploid. The genomic group and genetic similarities of 49 accessions were investigated using Random Amplified Polymorphic DNA markers. The genomic group of bananas assessed were established using OPA-18 (PILLAY et al., 2000) and OPG-17 primers. These primers showed bands 441 and 443 base pairs (bp) respectively for the accessions having only the B genome. Whilst they were absent for the accessions " having an A genome. The genetic similarity was estimated via a Simple Matching coefficient which showed the lowest value 0.46 measured between 'Ingumba' and 'Ishika 'and the highest value of 0.85 between 'Kirayenda' and 'Inyabukuwe'. The data of matrix of coefficient of similarity was subjected to cluster analysis with unweighted pair group method with arithmetic average (UPGMA). Each accession was clearly separated demonstrating the usefulness of RAPDs in analysis of genetic diversity. The results of this study are very important to the Curator of the banana germplasm collection in Eastern Central Africa and for the future breeding of this crop.
Characterisation and role of sugarcane invertase with special reference to neutral invertase.
(2000) Vorster, Darren James.; Botha, Frikkie Coenraad.; Huckett, Barbara Isobel.
The relationship between extractable invertase activities and sucrose accumulation in the sugarcane (Saccharum spp. hybrids) culm and in vivo invertase mediated sucrose hydrolysis was investigated to determine the significance of invertases in sucrose utilisation and turnover. In vitro activities were determined by assaying the soluble acid invertase (SAI), cell wall bound acid invertase (CWA) and neutral invertase (NI) from internodes three to ten in mature sugarcane plants of cultivar NCo376. Extractable activities were verified by immunoblotting. In vivo invertase mediated sucrose hydrolysis was investigated in tissue discs prepared from mature culm tissue of the same cultivar. Sugarcane NI had a higher specific activity than SAI (apoplastic and vacuolar) in the sucrose accumulating region of the sugarcane culm. CWA was also present in significant quantities in both immature and mature tissue. Sugarcane NI was partially purified from mature sugarcane culm tissue to remove any potential competing activity. The enzyme is non-glycosylated and exhibits catalytic activity as a monomer, dimer and tetramer. Most of the activity elutes as a monomer of native Mr ca 60 kDa. The enzyme displays typical hyperbolic saturation kinetics for sucrose hydrolysis. It has a Km of 9.8 mM for sucrose and a pH optimum of 7.2. An Arrhenius plot shows the energy of activation of the enzyme for sucrose to be 62.5 kJ.mol-1 below 30°C and -11.6 kJ.mol-1 above 30°C. Sugarcane NI is inhibited by its products, with fructose being a more effective inhibitor than glucose. Sugarcane NI is significantly inhibited by HgCI2, AgNO-3, ZnCI2, CuSO4 and CoCI2 but not by CaCI2, MgCI2 or MnCI2. Sugarcane NI showed no significant hydrolysis of cellobiose or trehalose. When radiolabelled fructose was fed to sugarcane internodal tissue, label appeared in glucose which demonstrates that invertase mediated hydrolysis of sucrose occurs. A combination of continuous feeding and pulse chase experiments was used to investigate the in vivo contribution of the invertases and the compartmentation of sugars. Sucrose is synthesised at a rate greater than the rate of breakdown at all stages of maturity in sugarcane culm tissue. The turnover time of the total cytosolic label pool is longer for internode three than internode six. A higher vacuolar:cytosolic sugar molar ratio than previously assumed is indicated. Developmentally, the greatest change in carbon allocation occurs from internodes three to six. The main competing pools are the insoluble and neutral fractions. As the tissue matures, less carbon is allocated to the insoluble and more to the neutral fraction. The neutral fraction consists mainly of sucrose, glucose and fructose. The compartmented nature of sugarcane storage parenchyma carbohydrate metabolism results in a system that is complex and difficult to investigate. A computer based metabolic flux model was developed to aid in the interpretation of timecourse labelling studies. A significant obstacle was the global optimization of the model, while maintaining physiologically meaningful flux parameters. Once the vacuolar:cytosolic molar ratio was increased, the model was able to describe the internode three and six labelling profiles. The model results were in agreement with experimental observation. An increase in the rate of sucrose accumulation was observed with tissue maturation. Only the internode three glucokinase activity was greater than the experimentally determined limit. The rate was however physiologically feasible and may reflect the underestimation of the in vivo rate. SAI and NI contributed to sucrose hydrolysis in internode three but not in internode six. The rates in internode six were set to fixed low values to enable the model to fit the experimental data. This does not however preclude low levels of in vivo SAI and NI activity, which would prove significant over a longer time period. The flow of label through the individual pools, which comprise the experimentally measured composite pools could be observed. This provides insight into the sucrose moiety label ratio, SPS:SuSy sucrose synthesis ratio, and the rate of 14CO2 release. The model provides a framework for the investigation and interpretation of timecourse labelling studies of sugarcane storage parenchyma.
Desiccation tolerance and sensitivity of vegetative plant tissue.
(1995) Sherwin, Heather Wendy.; Pammenter, Norman William.; Berjak, Patricia.
There is a great deal of work currently being done in the field of desiccation tolerance. Generally workers studying desiccation-tolerant plant tissues have concentrated on the mechanisms of desiccation tolerance without concomitant studies on why most plants cannot survive desiccation. The present study considers both a desiccation-tolerant plant as well as a range of desiccation-sensitive plants. The work incorporates physiological, biophysical, biochemical and ultrastructural studies in an attempt to get a holistic picture of vegetative material as it dries and then rehydrates. The plant species used in this study are: Craterostigma nanum, a so-called resurrection plant; Garcinia livingstonei, a drought-tolerant small tree; Isoglossa woodii, an understorey shrub which shows a remarkable ability to recover from wilting; Pisum sativum seedlings, which have a very high water content at full turgor; and finally, Adiantum raddianum, the maiden hair fern, which wilts very quickly and does not recover from wilting. The desiccation-tolerant plant, C. nanum, had an unusual pressure-volume (PV) curve which indicated that while large volume changes were taking place there was little concomitant change in pressure or water potential. The unusual nature of this PV curve made it difficult to assess the relative water content (RWC) at which turgor was lost. The desiccation-sensitive plants exhibited standard curvi-linear PV curves. The amount of nonfreezable water in the five species was studied and found to show no correlation with the ability to withstand dehydration or with the lethal water content. There were no differences in the melting enthalpy of tissue water between the tolerant and most of the sensitive plants. Isoglossa woodii had a lower melting enthalpy than the tolerant and the other sensitive species. Survival studies showed that the desiccation-sensitive plants all had similar lethal RWCs. The tolerant plant survived dehydration to as low as 1% RWC, recovering on rehydration within 24 hours. Membrane leakage studies showed that the sensitive plants all exhibited membrane damage at different absolute water contents, but very similar RWCs and water potentials. The increase in leakage corresponded to the lethal RWC for all the sensitive species. The desiccation-tolerant plant recovered from dehydration to very low water contents and did not show an increase in membrane leakage if prior rehydration had taken place. Without prior rehydration this tolerant plant exhibited an increase in leakage at similar RWCs and water potentials to that of the sensitive species. There did not appear to be much difference in the RWC at which damage to membranes occurred whether the material was dried rapidly or slowly. Respiration and chlorophyll fluorescence were studied to determine what effect drying and rehydration have on the electron transport· processes of the leaf. The chlorophyll fluorescence studies gave an indication of damage to the photosynthetic apparatus. Both qualitative changes as well as quantitative changes in fluorescence parameters were assessed. Characteristics like quantum efficiency (Fv/Fm)remained fairly constant for a wide range of RWCs until a critical RWC was reached where there was a sharp decline in Fv/Fm. Upon rehydration, C. nanum recovered to pre-stress levels, I. woodii showed no recovery and no further damage on rehydration, whilst the other species exhibited even more damage on rehydration than they had on dehydration. Respiration remained fairly constant or increased slightly during drying until a critical RWC was reached at which it suddenly declined. The RWC at which this decline occurred ranged from 15% and 20% in P. sativum and C. nanum respectively, to 50% for G. livingstonei. On rehydration respiration exceeded the levels measured in dehydrated material for the sensitive species. Unsuccessful attempts were made to fix material anhydrously for ultrastructural studies so standard fIxation was used. The ultrastructural studies revealed that changes had occurred in the ultrastructure of leaves of the sensitive species dried to 30% RWC particularly in A. raddianum and P. sativum. Drying to 5% RWC revealed extensive ultrastructural degradation which was worsened on rehydration in the sensitive species. The tolerant species showed ultrastructural changes on drying but these were not as severe as occurred in the sensitive species. The cell walls of the tolerant species folded in on drying. This folding was possibly responsible for the unusual PV curves found in this species. At 5% RWC the cells were closely packed and very irregular in shape. The cell contents were clearly resolved and evenly spread throughout the cell. The large central vacuole appeared to have subdivided into a number of smaller vacuoles. On rehydration the cells regained their shape and the cell contents had moved towards the periphery as the large central vacuole was reformed. Beading of membranes, which was common in the sensitive species, was not found in the tolerant species suggesting that membrane damage was not as severe in the tolerant species. Western Blot analysis of the proteins present during drying was performed to determine whether a class of desiccation-induced proteins, called dehydrins, were present. These proteins have been suggested to play a protective role in desiccation-tolerant tissue. It was found that C. nanum did, in fact, possess dehydrins, but so did P. sativum. The other three sensitive species did not show any appreciable levels of dehydrin proteins. The presence of dehydrins alone is, therefore, not sufficient to confer desiccation tolerance. While physiologically the damage occurring in the sensitive plants was similar to that of the tolerant plant, at an ultrastructural level the damage appeared less in the tolerant plant. On rehydration from low RWCs damage appeared to become exacerbated in the sensitive plants. This was in contrast to the tolerant plant where damage was apparently repaired. There appears, therefore, to be a combination of protection and repair mechanisms responsible for the ability of C. nanum to tolerate desiccation. The lethal RWC of the sensitive species was higher than that at which protective mechanisms, such as water replacement, might come into play. So it is not just the possible ability to replace tightly bound water that set the tolerant plant aside. It must also have mechanisms to tolerate damage at the higher RWCs which were damaging and lethal to the sensitive plants. The lethal damage to sensitive species appeared to be related to a critical volume, thus it is concluded that the tolerant plant had the ability to tolerate or avoid this mechanical damage during drying as well as the ability to remain viable in the dry state. It is hypothesised that the ability of the walls to fold in and the unusual nature of the PV curve may provide some answers to the enigma of desiccation tolerance.
The application of the heat pulse velocity technique to the study of transpiration from Eucalyptus grandis.
(1994) Olbrich, Bernard Wolfgang.; Pammenter, Norman William.
This thesis examines the application of the heat pulse velocity technique (HPV) to plantation-grown Eucalyptus grandis in the Eastern Transvaal, South Africa. The work addresses the application of the technique per se and is ultimately focused on improving the prediction of the hydrological impact of afforestation, to assist in the equitable management of South Africa's limited water resources. The verification of the HPV technique on E. grandis against the cut-tree method showed that the technique accurately reflected the water uptake in four three-year-old trees and a sixteen-year-old tree. It was found that accurate measurement of wound size and probe separation was essential for accurate water use estimates. The optimal probe allocation strategy for accurate measurements of transpiration in individual trees and stands of trees was examined. Stratifying the depths of implanted probes resulted in greater precision and repeatability in the HPV-derived estimates of sap flow in E. grandis. Given a limitation in the number of probes available to estimate stand transpiration, the results showed that sampling many individuals with a low sampling intensity (few probes per tree), rather than sampling few individuals intensively, improved the estimate of stand transpiration. An examination of the influence of tree age and season on transpiration rates showed that the transpiration rate per unit leaf area of E. grandis declined with age. Also, transpiration rates were higher in summer than under equivalent conditions of evaporative demand in winter. A seasonal change in the response of transpiration to VPD was implicated as the primary cause of this shift. A number of models were derived to predict transpiration from E. grandis. The variables vapour pressure deficit (VPO) and photosynthetically active radiation (PAR) were found to account for a large proportion of the observed variation in transpiration from the age sequence of trees studied. The models developed are applicable to trees of varying age, but are valid only for conditions where minimal soil water stress is experienced. The derived models were tested against two sets of independent data. This confirmed that a simple linear multiple regression adequately describes the relationship between transpiration and the two driving meteorological variables, PAR and VPO, in E. grandis. The application of a selection of the developed models on a sample data set from Sabie showed that transpiration from a three-year-old stand of E. grandis in summer may be more than double that for a sixteen-year-old stand under the same conditions. Simulated results also showed that transpiration in summer was about 25 to 50% higher than that from the same stand during winter conditions. Simulated transpiration rates from the young E. grandis stands were high, suggesting that further validation of the estimated rates is required before the models are applied. It is concluded that the HPV method is an ideal technique to estimate water use in E. grandis trees. The models developed represent a major advancement on previous models used to predict the hydrological impact of afforestation on mountain catchments.
Investigations into the responses of axes of recalcitrant seeds to dehydration and cryopreservation.
(2002) Wesley-Smith, James.; Berjak, Patricia.; Walters, Christina.; Pammenter, Norman William.
Achieving long-term storage of germplasm is critical for the conservation of plant biodiversity. Seed storage practices require that degradative reactions causing ageing be limited. By reducing the water content, cytoplasmic viscosity is increased to levels that minimise deteriorative reactions. Reducing the storage temperature additionally increases the storage lifespan by further reducing the rate at which such deleterious processes occur. Two broad categories of seeds can be distinguished based on their storage behaviour. Orthodox seeds are desiccation-tolerant; generally shed in the dry state and are metabolically quiescent. Such seeds are usually stored at low water contents (e.g. 5%), and their high cytoplasmic viscosity prevents freezing damage during cooling to subzero temperatures. On the other hand, desiccation-sensitive (recalcitrant) seeds do not undergo a maturation-drying phase, they are metabolically active at shedding, and sensitive to extreme or prolonged drying. Accordingly, recalcitrant seeds cannot be stored under conventional conditions because they do not survive drying to low water contents and are damaged by sub-zero temperatures, even when dried to the lowest water content tolerated. Therefore, procedures that facilitate harmless drying and cooling to low temperatures are required to achieve long-term storage of recalcitrant germplasm. Recalcitrant seeds that are dried rapidly can attain relatively lower water contents without injury. However, these seeds are usually large and this limits the drying rates that can be achieved even under favourable conditions. Isolating embryonic axes from the rest of the seed facilitates faster drying, and a consequent reduction in the water content at which damage occurs. In axes of many species, the level of drying attained before lethal desiccation damage occurs is sufficient to limit freeZing damage during cryogenic exposure and facilitate survival in vitro. However, many others are damaged when dried to water contents that preclude freezing, and also are killed if cooled to sub-zero temperatures at higher water contents. In such instances, the window of permissible water contents leading to survival may be small or nonexistent. A basic premise explored in this thesis is that by restricting the growth of intracellular ice crystals using increasingly rapid cooling rates, the range of permissible water contents can be widened, facilitating survival of axes at higher water contents. An overview of the problems associated with the long-term storage of recalcitrant germplasm, and the rationale behind such rapid cooling approach are presented in Chapter 1 of the present thesis. Subsequent chapters report investigations on the effects of variables required to dry and cryopreserve embryonic axes with minimum damage, in keeping with this approach. Collectively, those studies aimed at establishing a robust cryopreservation procedure for the conservation of recalcitrant germplasm with broad applicability across species. The approach presently adopted entailed manipulating the water content of excised axes using rapid drying to discrete water content ranges, and also using different methods to cool axes to cryogenic temperatures at various rates. The calorimetric properties of water in axes were investigated for Camellia sinensis (L.) O. Kuntze using differential scanning calorimetry (DSC). For all species, the effect of any drying or cooling treatment tested was determined by assessing the survival of axes in vitro, which provided the most reliable indicator of cellular damage. Additionally, the effects of different treatments upon the structural and functional integrity of axes were assessed using light and electron microscopy as well as measurement of electrolyte leakage. The studies undertaken are presented in a similar sequence to that in which they took place during the course of the experimental phase of this work. These are summarised below. Partial drying plays a pivotal role in the approach developed, and microscopy has contributed towards increasing present understanding of desiccation damage. Microscopy was used to determine the effects of drying rate upon the ultrastructure of recalcitrant axes. It was necessary to find reliable protocols to prepare specimens for light and electron microscopy that did not alter the architecture of the cells in the dry state. Freeze-substitution and conventional aqueous fixation were compared in Chapter 2 using variously dried material from three species. The results obtained revealed that an unacceptably high extent of artefactual rehydration occurs during aqueous fixation, and highlight the need for anhydrous processing of dehydrated samples. Significantly, that study also revealed that many cellular events commonly associated with desiccation damage (e.g. withdrawal, tearing and/or vesiculation of the plasmalemma) are not seen in freeze-substituted preparations, and are likely artefacts of aqueous fixation. Freeze-substitution was used subsequently (Chapter 3) to assess the effects of slow drying (2 - 3 days) or rapid drying (min) upon the survival of embryonic axes of jackfruit (Artocarpus heterophyllus Lamk.) Results confirmed the beneficial effects of rapid drying, and also provided insights into ultrastructural changes and probable causes underlying cellular damage that occur during a drying/rehydration cycle. Efforts subsequently focused on determining the effect of cooling rate upon survival of recalcitrant axes at various water contents. The study on embryonic axes of recalcitrant camellia sinensis (tea; Chapter 4) tested the hypothesis that rapid cooling facilitates survival of axes at higher water content by restricting the growth of ice crystals to within harmless dimensions. The presence of sharp peaks in DSC melting thermograms was indicative of decreased survival in vitro. These peaks were attributed to the melting of ice crystals sufficiently large to be detected by DSC as well as to cause lethal damage to axes. Increasing the cooling rate from 10°C min-1 to that attained by forcibly plunging naked axes into sub-cooled nitrogen increased the upper limit of water content facilitating survival in vitro from c. 0.4 to 1.1 - 1.6 g H20 g-1 (dry mass [dmb]). Subsequent studies tested whether a similar trend occurred in other recalcitrant species cooled under similar conditions. In order to investigate further the relationship between water content, cooling rate and survival it was necessary to achieve cooling rates reproducibly, and to quantify these reliably. The plunging device required to achieve rapid cooling, and instruments required to measure the cooling rates attained, are described in Chapter 5. That study investigated the effects of cryogen type, depth of plunge and plunging velocity on the cooling rates measured by thermocouples either bare or within tissues of similar size and water content as encountered in cryopreservation experiments. This plunger was used in subsequent studies to achieve the fastest cooling conditions tested. Favourable cooling conditions were selected, and the efficacy of this procedure to cryopreserve relatively large axes was tested (Chapter 6) using embryonic axes of horse chestnut (Aesculus hippocastanum L.) Axes at water contents above c. 0.75 g g-1 could not be cooled faster than c. 60°C S-1, but cooling rates of axes below this water content increased markedly with isopentane, and to a lesser extent with subcooled nitrogen. Axes were killed when cooled at water contents above 1.0 g g-1 but survived fully (albeit abnormally) when cooled in isopentane between 1.0 and 0.75 g g-1. Complete survival and increasingly normal development was attained at water contents below 0.75 g g-1, especially if isopentane was used. The study on horse chestnut axes emphasised that water content and cooling rate are co-dependent during non-equilibrium cooling. Accordingly, that study could not determine whether survival at lower water contents increased because of the corresponding increase in cooling rates measured, or because of the higher cytoplasmic viscosity that resulted from drying. That uncertainty was addressed by the study discussed in Chapter 7, using axes of the trifoliate orange (Poncirus trifoliata [L.] RAF.) That study investigated the effect of cytoplasmic viscosity upon survival of axes cooled and warmed at different rates. Survival and normal development was high at lower water contents, and seemingly independent of cooling rate at about 0.26 g g-1. At higher water contents the range of cooling rates facilitating survival became narrower and displaced towards higher cooling rates. This study revealed two conspicuous inconsistencies that questioned the beneficial effect of rapid cooling. Firstly, the fastest cooling rates did not necessarily facilitate the highest survival. Secondly, survival of fully hydrated axes was higher when cooled under conditions that encouraged - rather than restricted - the growth of intracellular ice crystals. These inconsistencies were explored further using embryonic axes of silver maple (Acer saccharinum L.). Freeze-fracture replicas and freeze-substitution techniques provided valuable insights into the way in which ice crystals were distributed in cells cooled using different methods at rates ranging between 3.3 and 97°C S-1. Extensive intracellular freezing was common to all treatments. Unexpectedly, fully hydrated axes not only survived cryogenic exposure, but many axes developed normally when cooled using the relatively slower methods (77 and 3.3°C S-1) if warming was rapid. The most conspicuous ultrastructural difference between plunge cooling and the relatively slower methods was the exclusion of ice from many intracellular compartments in the latter. It is possible that even the fastest warming cannot prevent serious cellular damage if ice crystals form within such 'critical' compartments. It is proposed that the intracellular location of ice is a stronger determinant of survival that the size attained by ice crystals. The study of A. saccharinum also investigated the recovery of axes cooled fully hydrated either rapidly (97°C S-1) or slowly (3.3°C S-1). This facet of the study showed that cell lysis became apparent immediately after warming only where damage was most extensive. In other cells damage became apparent only after 2.5 to 6 h had elapsed, thus cautioning against inferring survival from the ultrastructural appearance of cells immediately after warming. Microscopy enabled cell repair as well as the pattern of growth of cryopreserved tissues to be appraised at the cellular, tissue and organ levels. Similar studies are required to understand further the nature of freezing damage, and how those events affect cell function. The salient trends observed in previous chapters are brought together in Chapter 9.

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