Extracts of gelling agents have been shown to stimulate rooting and this study was initiated to investigate the presence of root stimulating substances in gelling agents. After screening a number of gelling agents, four were selected, namely; Agar Bacteriological, Agar Commercial Gel, Difco Bacto Agar and Gelrite were selected and examined for the presence of root-stimulating substances using mungbean bioassay. Water extracts of Agar Bacteriological, Agar Commercial Gel and Difco Bactol Agar stimulated rooting of mungbean cuttings. Addition of Charcoal neither reduced nor increased rooting produced by the water extract of the first two agars but when added in conjunction with Difco Bacto Agar rooting was reduced. Autoclaving, however reduced rooting in extracts of the gelling agents. The possibility that root-stimulating substances may not be the same in all the gelling agents can not be excluded. Extraction of Gelrite with water was problematical and was therefore excluded. IBA solution and water extracts of the gelling agents separately promoted good rooting in mungbeans cuttings. Rooting in extracts of autoclaved frozen-thawed gelling agents was poor, however, IBA + gelling agents gave high rooting at the 100% concentration and this could possibly be due to an additive effect of the IBA. Addition of charcoal reduced rooting significantly in extracts of IBA + gelling agents. Using 80% acidic methanol, reasonable levels of rooting substances were obtained from the residue extract of this complex (IBA + gelling agent+charcoal) of all the gelling agents except Gelrite indicating that root-promoting substances were adsorbed by charcoal. The low rooting in the presence of the Gelrite extract was attributed to the matrix of the polymer of the Gelrite. Ethyl acetate fractionated extracts (EA-pH 8.0; EA-pH 3.0; and Aqueous) obtained from the four gelling agents stimulated rooting indicating the presence of numerous root promoting substances. Gelrite gave good rooting with both the 50 and 100% concentrations of all the fractions. Purified water and ethanol extracts of the gelling agents exhibited auxin-like activity when separated by paper chromatography and compared with IBA and IAA standards. Using HPLC, IAA was quantified in all the gelling agents with Difco Bacto Agar and Agar Commercial Gel having the highest IAA concentration and Gelrite the lowest IAA concentration. IAA concentration in Agar Bacteriological was a third of the level detected in Difco Bacto Agar. The information from this work may enable researchers to consider gelling agents as sources of auxin-like compounds and other plant hormones as well as support media for use in tissue culture procedures and also increase the enthuse for further research into the nutrient types and levels in gelling agents. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2003.

No abstract available. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2003.

When imbibed at their optimum germination temperature of 25°C, achenes of Tagetes minuta L. germinate over a period of approximately 48 h. At temperatures of between 35°C and 39°C, the achenes do not germinate but enter into a state of thermoinhibition. These supra-optimal conditions do not harm the achenes, however, and when the temperature is reduced below 35°C radicle emergence may be observed within 4 h. Achenes which have been thermoinhibited for periods of 24 h or more show "accelerated germination" which takes only 24 h, although the actual germination curve is identical to that of normally germinated achenes. This suggests that the achenes are metabolically active at thermoinhibitory temperatures and undergo most of the processes of normal germination, but that at some point any further development is halted, preventing radicle emergence. When the temperature is reduced, this block on germination is removed and since the achenes are already primed for germination, this occurs within a short time. An analysis of the proteins produced by germinating and thermoinhibited achenes was conducted using SDS-polyacrylamide gel electrophoresis (SDS-PAGE). This procedure was able to resolve approximately 40 different protein bands, but no differences were observed between thermoinhibited and germinating achenes. Two dimensional polyacrylamide gel electrophoresis (20-PAGE) was able to resolve approximately 200 individual polypeptides. The vast majority of polypeptides in T. minuta achenes are acidic, although the number of neutral to basic polypeptides increases as germination progresses. Ten polypeptides were identified which were specific to thermoinhibited achenes. These formed two distinct groups on the twodimensional gels. The larger group contained seven proteins, ranging in size from 22 kDa to 26.7 kDa and with isoelectric points of between 3.0 and 4.0. The smaller group contained three polypeptides with molecular weights of about 14 kDA and a pi of approximately 3.0. These polypeptides were all extremely specific to thermoinhibited achenes and declined rapidly when the incubation temperature was reduced, in a manner which correlated with an increase in the germinability of the achenes. Several characteristics of the expression of these polypeptides were similar to characteristics of embryo-dormancy in seeds where dormancy is thought to be actively imposed by the expression of specific dormancy-associated genes. This, along with the very tightly-regulated nature of these 10 polypeptides, suggests that thermoinhibition in T. minuta may be regulated through gene expression and that these ten polypeptides may represent the products of genes responsible for preventing radicle emergence at unfavourable temperatures. Since these polypeptides were only resolved using silver-staining and could not therefore be used for amino acid sequence analysis, this hypothesis was further investigated using differential display of mRNA to isolate genes which are expressed specifically in thermoinhibited achenes. A large number of cDNA fragments which were specific to either germinating or thermoinhibited achenes were identified and extracted from the differential display gels. Those cDNAs specific to the thermoinhibited achenes were taken for further analysis. Of the 62 fragments excised from the gels, 25 could be reamplified to generate single bands of the correct size on agarose gels. A further 22 cDNAs produced multiple bands, where one band was much brighter than the others and correlated with the size of the original fragment. Thirteen of the cDNAs which' generated single bands were cloned into the plasmid vector pGEM®-T Easy and transformed into either Escherichia coli JM109 or E. coli XL1-Blue. Recombinant colonies were identified using blue-white colour selection and the presence of the insert confirmed by colony blotting and restriction analysis. Three clones were chosen for each of the cDNAs. Reverse northern analysis confirmed that all 39 clones were specific to the mRNA pool of thermoinhibited achenes. High quality sequence data were obtained for 27 of the cDNA samples, the remainder appeared to have been degraded in transit. Alignment of the various sequences revealed that a total of 14 different sequences had been cloned, indicating that several of the bands isolated from the differential display gels contained multiple sequences. Electronic homology searches tentatively identified three of the sequences, whilst the remainder did not show significant homology to any known sequences. Of the cDNAs identified in this way, one may encode a plant transcription factor-like or nuclear RNA-binding protein whilst the other two may encode an RNase-L Inhibitor-like protein and a miraculin homologue. The potential roles of such genes in the imposition or maintenance of the thermoinhibited state are discussed. Although further research needs to be conducted to isolate full length cDNA sequences and to determine their exact expression patterns in germinating and thermoinhibited achenes, these results are consistent with the hypothesis that thermoinhibition in T. minuta achenes is under positive genetic control in a manner analogous to embryo dormancy. This thesis represents the first molecular study of thermoinhibition as well as the first report of active control over this phenomenon in any species. Since thermoinhibition, unlike dormancy, can be rapidly imposed and released under strictly controlled conditions without the need for any dormancy breaking treatment, T. minuta achenes represent an excellent model system for studies on the molecular control of seed germination. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2003.

Environmental stresses such as drought, salinity and low temperature have been major selective forces throughout plant evolution and are important factors which limit crop plant distribution and agricultural productivity. An understanding of how crops adapt to adverse conditions is not only of theoretical interest, but also has considerable practical value . Low-temperature stress subtraction libraries were constructed in a pBluescript vector with the two-step-PCR amplified cDNAs using subtractive hybridization. One insert cs18 was obtained and the sequence analysis of insert cs18 revealed that the insert cDNA had a 76% homology with the sequences of the corresponding portion of glucose dehydrogenase from Thermoplasma acidophilum and 62.0% homology with a genomic DNA of Arabidopsis. Four clones, cs18-13, cs18-14, cs18-15, and cs18-16 from low-temperature stress soybean root conventional cDNA library have been confirmed to have inserts that could hybridize to the cs18 insert. One cDNA with a Xba I and Xho I fragment of approximately 3,500 bp in length corresponded to the insert cs18 , which probably encodes for glucose dehydrogenase, was obtained. Northern blot analysis indicated that cs18 mRNA was highly expressed in soybean root but moderately expressed in leaves under low temperature. Changes in the nuclei of meristematic root cells in response to severe salinity were studied. Roots are in direct contact with the surrounding solution . Thus, they are the first to encounter the saline medium and are potentially the first site of damage or line of defence under salt stress. Nuclear deformation or degradation in the soybean root meristem with 150 mM or higher NaCI led to sequential cell degradation, cell death and cessation of plant growth . However, this study indicates that an increase in CaCI[2] concentration up to 5 mM could partially prevent salt injury to the cells. Tissue culture is an excellent tool for elucidat ing the correlation between plant organizational levels and salt tolerance because of the possibility it offers for studying the physiology of intact plantlets together with that of organs and single cells using homogenous plant material under uniform environmental conditions. One NaCI-tolerant cell line (R100) was isolated during this study. The R100 callus cell line was significantly more tolerant to salt than the salt-sensitive line (S100) during exposure to salt stress. Salt tolerance in this culture was characterized by an altered growth behaviour, reduced cell volume and relative water content, and accumulation of Na+, Cl ¯, K+, proline and sugars when grown under salt stress and with its subsequent relief. The selection of this salt tolerant cell line has potential for contributing new genetic variability to plant breeders. Sugars are not only important energy sources and structural components in plants , they are also central regulatory molecules controlling physiology, metabolism, cell cycle , development, and gene expression in plants. The concentrations of glucose and fructose increased during salt stress and after relieving salt stress, at a rate closely corresponding to the increase in relative water content. Their accumulation was the earliest response detected during the removing of salt stress indicating that glucose and fructose may play important roles during salt-stress. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2000.

A replacement for silver thiosulphate as a commercial post-harvest treatment needs to be found. The longevity of cut carnation flowers is extended by all concentrations of ethanol tested. Compared to a water control, the vase-life of ethanol-treated flowers is between 150 and 250% longer. The greatest longevity increases are recorded with 3% ethanol. The use of ethanol as a post-harvest treatment was tested. The longevity increase as a result of ethanol application only occurs if the ethanol is applied as a holding solution. Pulse treatments are not effective at delaying the senescence of the flowers. The sooner the ethanol is applied, the greater the increase in vase life. If ethanol treatment is halted at any point during the experiment, the longevity of the flowers is reduced. It was observed that the longer the stems of ethanol-treated flowers, the greater the longevity increases. The ethanol holding solution does not prevent the action of external ethylene, thereby restricting the potential of ethanol as a commercial post-harvest treatment. Physiologically, flowers treated with ethanol exhibit a different senescence process to control flowers. The typical in-rolling of the petals of carnation flowers is not seen, instead the petals appear burnt. The ovaries are also notably effected by ethanol, being smaller and more yellow in colour. Ethanol treatment results in longevity increases by inhibiting the formation of ethylene, the plant hormone responsible for senescence. The concentration of the direct precursor to ethylene, ACC, as well as the activity of the enzyme that converts ACC to ethylene, ACC oxidase, is reduced to almost zero in the tissues of treated flowers. Another physiological factor affected by ethanol treatment is the carbohydrate status of the flowers. The normal sink activity of the ovary is inhibited by ethanol treatment. Although the carbohydrate content of the petals is found to decrease sharply in ethanol-treated flowers, these carbohydrates are not relocated to the ovary. The ovary does not increase in dry matter or chlorophyll content. The carbohydrate content decreases as a result of ethanol treatment, and when ¹⁴C sucrose was applied to petals, no radioactivity was recovered in the ovary. The petals and ovary are the organs most effect by ethanol activity, as when ¹⁴C ethanol was applied to cut carnation flowers as a pulse, the majority of the radioactivity was discovered here. The protein content of cells of both organs decreases significantly compared to control flowers. This is a total protein loss, rather than the destruction of specific systems. If the activity of alcohol dehydrogenase is prevented in ethanol-treated flowers, inhibiting the conversion of ethanol to acetaldehyde, no longevity increases are seen. The airspace surrounding treated flowers was found to contain ethanol and small amounts of acetaldehyde. The tissues of flowers treated with ethanol show an increase in the acetaldehyde content, as well as the ethanol content, especially in the ovary. The application of acetaldehyde directly to cut carnation flowers as a holding solution resulted in the vase life of the flowers increased by 150%. To determine the effectiveness of acetaldehyde as a post-harvest treatment, various concentrations of acetaldehyde were applied to cut carnation ftowers as a pulse treatment and a holding solution. Pulse treatments did not increase the vase life of flowers, and resulted in a number of negative effects in the flower. A holding solution of acetaldehyde does increase the longevity of cut carnation flowers, provided it is above a certain concentration. Treatments at concentrations below 1% acetaldehyde appear to promote flower senescence. The use of acetaldehyde as a post-harvest treatment has many of the same disadvantages as ethanol treatment. Acetaldehyde must also be applied as a holding solution for as long as possible. If removed from this solution, death of the organ occurred quickly. Acetaldehyde is also ineffective against external ethylene. A negative effect of acetaldehyde not found in ethanol-treated flowers, is that the longer the stem of cut carnation flowers, the shorter the resultant vase life. Physiologically the responses in cut carnation flowers were very similar to those seen in ethanol-treated flowers. Acetaldehyde inhibited the formation of ethylene completely. Almost no ACC can be found in treated tissues, and the action of ACC oxidase is completely reduced. The petals of acetaldehyde-treated flowers suffer from severe petal browning, rather than in rolling. The ovaries are particularly badly effected by treatment. There are large scale losses in fresh weight and chlorophyll content. The latter results in the ovaries appearing yellow in colour. They also show a loss in structure. The sink activity of these ovaries is destroyed. Like ethanol-treated flowers, the carbohydrate content of both the petals and ovaries are dramatically reduced. When ¹⁴C sucrose was applied to one of the. petals, almost no radioactivity was recorded in the ovary. There. is also a major loss in general protein content, slightly more severe than in ethanol-treated flowers. The conversion of ethanol to acetaldehyde is necessary in order to achieve longevity increases in ethanol-treated flowers. If the conversion of this acetaldehyde to ethanol is prevented in acetaldehyde-treated flower there is once again no longevity increase. Both ethanol and acetaldehyde are required within the system to result in increased longevity. Although ethanol and acetaldehyde treatments result in decreases in the total protein content of the flowers, certain enzymes remain active. Alcohol dehydrogenase is a bi-directional enzyme, capable of converting ethanol to acetaldehyde and then back to ethanol again. The activity of this enzyme, in both orientations, is increased in ethanol and acetaldehyde-treated flowers. The activity of pyruvate decarboxylase, which converts pyruvate to acetaldehyde, is also increased as a result of both treatments. The similarities of the physiological response of cut carnation flowers to ethanol and acetaldehyde post-harvest treatments, and the increased activity of these enzymes, indicate that the effect of both compounds on longevity is closely linked. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2000.

Sugarcane is an economically important crop and its high demand has necessitated the use of biotechnology methods to produce and accelerate the production of desirable genotypes. One such method is genetic transformation. However, as sugarcane is a highly polyploid crop, which originated from interspecific crosses between Saccharum spontaneum and S. officinarum, efforts to transform it are inhibited by transgene promoter silencing. As ancestral lines have a simpler genetic makeup than modern varieties, they may be useful to test promoter function. Intrinsic to the generation of transgenic plants is the ability to produce plants from specific species and varieties, for which an indirect method of regeneration is needed. Consequently, the first objective of this study was to determine a high yielding protocol for somatic embryogenic calli. The second was to transform such calli and produce regenerated plants to assess transgene expression. A preliminary study was conducted using eight ancestral varieties to determine which were the most responsive in culture. Leaf roll disks were cultured on 5 mg.1¯¹ 2, 4-D and callus production was assessed. Based on these results and the availability of plant material, S. spontaneum Nigeria 1, S. spontaneum Nigeria 2, S. spontaneum Coimbatore, S. officinarum NG 77-69, and S. officinarum Black Cheribon and the commercial polyploid variety NCo376 were selected and tested on 11 different callus induction media. The S. spontaneum variety that generated the highest percentage of leaf disks that produced callus and plant yield was Nigeria 1 (61 % and 259 plants/10 disks, respectively), whilst the S. officinarum variety was Black Cheribon (75 % and 90 plants/10 disks, respectively). The best media for both comprised of MS salts and vitamins, 20 g.1¯¹ sucrose, 0.5 g.1¯¹ casein hydrolysate 5 mg.1¯¹ 2, 4-D and 8 g.1¯¹ agar. NCo376 produced the most amount of callus (93 %) when cultured on media containing 3 mg.1¯¹ 2, 4-D and gave a final yield of 450 plants/10 disks. Based on the yields obtained above and the availability of plant material, the varieties S. spontaneum Nigeria 1 and S. officinarum NG77-69 were selected for genetic transformation studies. Calli of these varieties as well as that of NCo376 were microprojectile bombarded with either pEmuKN + pAHC27 or pEmuKN + pR₁₁F¯. Following bombardment, the calli were cultured onto paromomycin-containing (1 ml.1¯¹) selection media and regenerated plants were obtained after 8-12 weeks. Transgene integration into the plant genome was assessed using PCR and qPCR techniques, and indicated that all NCo376 plantlets contained the GUS and npt II transgenes. However, only 4 out of 5 and 2 out of 3 S. officinarum NG77-69 plants transformed with pAHC27 and pR₁₁F¯- respectively, and 6 out of 10 S. spontaneum Nigeria 1 plants transformed with pR₁₁F¯- contained these transgenes. The transformation efficiencies achieved for NCo376, for the constructs pAHC27 and pR₁₁F¯- was 0.27 and 0.33 transgenic plants/blast, respectively. For NG77-69 it was 0.27 and 0.13 transgenic plants/blast, whilst that of Nigeria 1 was 0.20 and 0.40 transgenic plants/blast. Stable transgene expression in acclimatized plants was then assessed using a histochemical GUS assay and none of the plants expressed the GUS gene. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2013.

The levels of the psychoactive alkaloids S-(-)-cathinone and its primary metabolite cathine, consisting of the diastereomers (+) -norpseudoephedrine and (-)- norephedrine were determined in Catha edulis (Vahl) Forssk. ex Endl., Catha transvaalensis Codd and Catha abbottii Van Wyk & Prins. Alkaloid levels were investigated in C. edulis plants collected from three different localities in South Africa, and one from a Nairobi khat market. The efficiency of three different methods for the extraction and isolation of cathinone and cathine were investigated, viz. an aqueous acid extraction, an organic solvent extraction and an aqueous acid extraction using the commercially available Extrelutᴿ procedure. The aqueous acid extraction resulted in the rapid loss of cathinone and yielded variable alkaloid levels in replicate studies. This was also observed when this method was coupled with the Extrelutᴿ procedure. In contrast, the organic solvent extraction did not result in a loss of cathinone and provided consistent results over a number of replicates; it also proved to be a simple and rapid method for extracting and isolating cathinone and cathine. A trifluoroacetic acid (TFA) derivatization procedure which has been suggested to produce characteristic diagnostic fragments for gas chromatography / mass spectrometry (GC/MS) identification, was investigated, but failed to produce consistent TFA derivatives of cathinone and cathine. However, underivatized cathinone and cathine were easily identified by GCMS due to their unambiguous mass spectra. All subsequent studies were undertaken using the organic solvent extraction and isolation method, coupled with GC analysis and GC/MS identification of underivatized cathinone and cathine. Leaves of C. edulis were found to contain cathinone and cathine at levels 100 times higher than those of C. transvaalensis. The alkaloids were undetectable in C. abbottii. Plants grown from cuttings of C. edulis collected from the Durban Botanical Gardens were found to contain cathinone and cathine at levels of 0.410 mg per gram fresh weight and 0.157 mg per gram fresh weight in leaves, respectively, while these levels in plants derived from different localities decreased in the order: Eastern Cape (0.319 mg/g f.w cathinone and 0.029 mg/g f.w cathine), Mpumalanga (0.139 mg/g f.w. cathinone and 0.171 mg/g f.w. cathine) and Nairobi (0.032 mg/g f.w. cathinone and 0.025 mg/g f. w. cathine). In an investigation of the cathinone levels in the different plant parts it was found that the highest levels were found in leaves of the shoot tip (0.243 mg/g f.w.) but decreased with the age of the leaf and developmental stage of the plant in the order: juvenile leaves (0.124 mg/g f.w.), mature leaves (0.035 mg/g f.w.), young stem (0.033 mg/g f.w.) and mature stem (0.004 mg/g f.w.). Concomitantly, cathine levels increased with the age of the leaf: leaves of the shoot tip (0.006 mg/g f.w.), juvenile leaves (0.011 mg/g f.w.), mature leaves (0.019 mg/g f.w.). The cathine level in the young stem material was found to be the highest in the entire plant (0.270 mg/g f.w.) but decreased markedly in the mature stem (0.052 mg/g f.w.). Both cathinone and cathine levels in the mature root were greater than levels in the mature stern, being 0.012 mg cathinone per gram fresh weight, and 0.063 mg cathine per gram fresh weight. Neither cathinone nor cathine were detectable in young root material. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2001.

An investigation was undertaken to determine whether Erythrina species indigenous to South Africa contained the same type of compounds as Erythrina species not found in South Africa and to determine whether they displayed any anti-inflammatory and antibacterial activity. Phytochemical analysis was conducted using thin layer chromatography. A great similarity was found in the leaf profiles of the species being studied. The leaf and bark extracts of E. caffra and E. lysistemon appear to have similar profiles when viewed under normal light and ultraviolet light, (254 and 366 nm). These two species have similar banding patterns when stained with fast blue reagent for flavonoids and potassium hydroxide reagent for coumarins. The five species that were tested appear to contain alkaloids, flavonoids, coumarins and triterpenes just like the species not found in South Africa from this genus. Dried bark and leaves from E. caffra, E. humeana, E. latissima, E. lysistemon and E. zeyheri were screened for anti-inflammatory and anti-bacterial activity. Ethanol, ethyl acetate and water extracts were screened for both anti-inflammatory and anti-bacterial activity. The cyclooxygenase bioassay was used to test for anti-inflammatory activity. The ethanol and ethyl acetate extracts generally displayed activity while the water extracts displayed no activity for both the bark and the leaves. The bark generally displayed more cyclooxygenase inhibitory activity than the leaves. The bark of E. caffra and E. lysistemon displayed the highest cyclooxygenase inhibitory activity. The disc diffussion bioassay was used to screen for anti-bacterial activity. Anti-bacterial activity was only detected in the water extracts of the leaves. The water extracts of the bark showed very little or no activity. The bark yielded more anti-bacterial activity than the leaves. Anti-bacterial activity was mainly displayed against Gram positive bacteria. The bark of E. caffra and E. lysistemon displayed the highest anti-bacterial activity. On the basis of the screening results it was decided to use bioasssay guided fractionation in an attempt to isolate putative anti-inflammatory and anti-bacterial compounds. A hexane extract from the bark of E. lysistemon was prepared and purified using a range of chromatographic methods. Vacuum liquid chromatography, separation using a chromatotron, thin layer chromatography and high performance liquid chromatography were used to isolate anti-inflammatory compound(s). The isolation proved to be unsuccessful as the pure compound had no cyclooxygenase inhibitory activity. It was subsequently determined that the compounds were lost during the HPLC procedure. An ethanolic extract of the bark of E. Iysistemon was purified in an attempt to isolate an anti-bacterial compound(s). Vacuum liquid chromatography and separation using the chromatotron was used to purify the crude extract. The more sensitive microtitre bioassay was used to test for anti-bacterial activity against S. aureus. The isoflavone, Wighteone was isolated. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2000.

Delairea odorata Lem., an asteraceous perennial vine indigenous to southern Africa, has become naturalised and invasive in many subtropical regions including California, South Australia and Hawaii. Biological control offers a potential long term solution to the management of this species in exotic locations. This study analysed aspects of the biology of D. odorata in its native environment to determine its suitability to classical biological control. To this end an examination of the reproductive biology and natural enemies of D. odorata was made. A study of the pyrrolizidine alkaloid profile was also conducted. Reproductive biology: Delairea odorata reproduces both sexually by seeds and asexually by stolons. The flowering season occurs over the autumn months from April to June. Results of the pollination trials indicate that D. odorata is a cross compatible species and an obligate outbreeder. There is no specialised pollination system and the predominant pollinators belong to the families Apidae, Syrphidae and Calliphoridae. Following pollination, numerous small achenes are produced. Laboratory trials indicate that these achenes germinate readily between 10 and 25 °C and, although germination occurs in both the light and dark, light clearly stimulates seed germination. Greenhouse trials conducted to determine the effect of light on growth and reproduction indicate that D. odorata is a shade tolerant species which shows plasticity in terms of growth form and deployment of biomass in response to changes in light intensity. Growth rate and allocation of biomass to vegetative and sexual reproduction are highest at an intermediate light level. However, greatest allocation of biomass is to stem growth regardless of light level. Natural enemies: Surveys for potential biological control agents against Delairea odorata were conducted in KwaZulu-Natal and several phytophagous species were associated with the plant. However, only one potentially suitable control agent was identified, a stem galling tephritid fly, Parafreutreta regalis Munro. Preliminary studies indicate this species to be fairly host-specific, a valuable asset if it is to be considered as a control agent. Furthermore, as D. odorata proliferates extensively by means of stem regeneration and elongation, galling of these growing points by P. regalis may limit stolon spread in exotic locations. Two species of parasitic wasp (Braconidae) were found to parasitise P. regalis pupae. If P. regalis is to be used as a control agent the likelihood of parasitisation in the new environment must be determined. Pyrrolizidine alkaloids: Host-specificity in insects is often dependent on host-plant chemistry (e.g. alkaloids or essential oils). Thus prior to any biological control programme it is important to determine if there are ecotypes of the host plant present. An investigation to determine the specificity of the pyrrolizidine alkaloid profile of D. odorata, occurring across KwaZulu-Natal, was made. The results indicate the presence of nine retronecine based pyrrolizidine alkaloids which occur in similar proportions in locally distributed plants. However, these alkaloid profiles differ considerably from those published for D. odorata occurring in California. This is an interesting and important result which indicates that chemotypes of D. odorata may exist, a factor which must be considered in the initiation of any biocontrol. If chemotypes of D. odorata are present this may affect the behaviour of natural control agents on the exotic plant populations. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2000.

Embryogenic tissue of Pinus patula Scheide et Deppe was initiated from immature green female cones during the months of November 1996 to February 1997 and December 1997 to January 1998. Tissue was maintained on MSG3 medium (BECWAR, NAGMANI and WANN 1990) supplemented with maltose. A comparison of various sugars as a carbohydrate source for maintaining embryogenic tissue showed that maltose was far superior to sucrose and the other sugars tested. Embryogenic tissue was successfully cryopreserved for up to 8 weeks using 0.3 M sorbitol and 5 % DMSO. Recovered tissue initially underwent a lag phase in tissue regrowth, but by the end of 5 weeks post-thaw, tissue proliferation was as vigorous as the unfrozen, untreated control. Fluoresceine diacetate (FDA) staining revealed that the embryonal head survived cryopreservation, but the highly vacuolated suspensor tissue had ruptured and died. Embryogenic tissue from two different families and four genotypes were successfully cryopreserved using this protocol. A comparison of commonly used cryopreservation techniques was conducted. It was found that the slow addition of the cryoprotectants over two days slowed the recovery rate of the tissue and increased the chances of contamination. Embryogenic tissue did not respond well to cryopreservation using a combination of the cryoprotectants PEG, glucose and DMSO (10-8-10%). Only a small proportion of the tissue survived, and initial tissue regrowth took up to 5 weeks. Embryogenic tissue was also set in gel and immersed directly in liquid nitrogen in an effort to cryopreserve tissue using the process of vitrification. However, none of the tissue survived, possibly due to insufficient dehydration prior to immersion in liquid nitrogen. Tissue recovery was highest when the tissue was precooled to -70°C in a container filled with isopropyl alcohol placed in a static freezer prior to immersion in liquid nitrogen. Recovery of tissue was improved by suspending the tissue on polyester grids and removing the liquid medium prior to placing onto MSG3 medium. Recovered tissue was bulked up using suspension cultures, and then paced onto MSG5 (BECWAR, NAGMANI and WANN 1990) or 240 medium (PULLMAN and WEBB 1994) to mature. Mature embryos were isolated from both media and germinated. Somatic plantlets were successfully hardened-off under greenhouse conditions. The successful cryopreservation of a number of genotypes and lines, and the maturation of recovered tissue has been achieved. This technique is now being actively incorporated into P. patula somatic embryo research, enabling the long-term storage of juvenile reference tissue while field trials are carried out and evaluated. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1999.