Doctoral Degrees (Research Centre for Plant Growth and Development)

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Micropropagation and pharmacological evaluation of Boophone disticha.
(2013) Cheesman, Lee.; Van Staden, Johannes.; Finnie, Jeffrey Franklin.; Light, Marnie Elizabeth.
Boophone disticha (L.f.) Herb is one of the most widely distributed bulbous species in southern Africa. Of Africa’s many bulbous plants, it is widely known for its poisonous and medicinal properties. It is of considerable ethnobotanical interest in traditional medicine because of its hallucinogenic alkaloids and it has great potential as an ornamental due to its fan-shaped foliage and large umbel of bright pink to deep red flowers. In South Africa, many bulbous plants are used in traditional medicine which are collected from wild populations. The high demand for trade and use of such plants, that are destructively harvested, places an enormous pressure on natural populations. According to the Red List of South African Plants, the conservation status of B. disticha has been listed as ‘declining’. It is, therefore, important to develop conservation strategies for these medicinal plants, such as the development of alternative propagation methods. Micropropagation is a useful technique for rapid clonal multiplication of plant material which could alleviate the pressure on the wild plant populations, as well as potentially producing useful secondary metabolites. The in vitro induction of storage organs is especially beneficial as it can limit the loss of plants during acclimatization since bulblets are generally hardier than shoots or plantlets. Thus, the main aim of this research was to establish a micropropagation protocol which could be a valuable tool for conservation of this plant species. In addition, B. disticha plants were assessed in various ethnopharmacological assays to evaluate their medicinal properties, and a preliminary study on the population genetics was also conducted. As part of the development of a suitable micropropagation protocol, the effect of environmental and physiological factors on the initiation and growth of bulblets were investigated. These factors included the effect of various plant growth regulators, carbohydrates, temperature, photoperiod and liquid culture. Different explants (i.e. ovaries, anthers, filaments, pedicels, embryos, seeds and bulb twin-scales) were tested to determine which explants were the most suitable for subsequent experiments. Although success was limited, twin-scales proved to be the most suitable explant and it was demonstrated that activated charcoal, ascorbic acid and N6- benzyladenine were required as media supplements. Antimicrobial activity was tested between different plant parts and seasons. The plant parts (roots, leaves, outer and inner bulb scales) were extracted with a range of differing polarity solvents. These were screened for antibacterial activity against Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae, and for antifungal activity against Candida albicans. Extracts from roots of plants collected in spring and summer showed the best antimicrobial activity against B. subtilis, E. coli and K. pneumoniae, indicating that plant part and collection time do affect activity. In vitro grown bulblets also showed antimicrobial activity, demonstrating that antibacterial properties were maintained in cultured plantlets. Extracts from plants collected in summer were tested for mutagenicity using the Ames test (Salmonella/microsome assay; plate incorporation method, with or without metabolic activation). None of the extracts tested were found to induce mutations and also did not modify the effect of the mutagenic compounds (2AA with S9 and 4NQO without S9). Although the results do not indicate a mutagenic response, this does not necessarily confirm that it is not mutagenic nor carcinogenic to other bacterial strains, however, B. disticha must be used with caution, especially considering the levels of alkaloids in the plant. The two major constituent alkaloids of B. disticha were identified as buphanidrine and distichamine. In the antibacterial assay, both compounds exhibited broad-spectrum micromolarlevel activity against the two Gram-positive and two Gram-negative bacteria tested. The best MIC value, of 0.063 mg/ml, was found for bupanidrine/distichamine against S. aureus, E. coli and K. pneumonia. The isolated compounds were tested and found to be neither mutagenic nor toxic at the concentrations tested. Thus, buphanidrine and distichamine are thought to be the constituents likely responsible for the medicinal properties of the plant. To determine the level of genetic variation between different populations of B. disticha, plants were collected from six wild populations in KwaZulu-Natal, South Africa. DNA was isolated and tested for genetic variation using ten Inter Simple Sequence Repeat (ISSR) primers. The level of inter-population polymorphism ranged between 23% and 39%, showing that the populations had low genetic polymorphism. From the genetic distance results, it was found that the Midmar and Umgeni Valley populations are closely related, and these populations are similar to two sister populations. The Amatikulu and Lions River populations were similar but slightly different to the other populations. Antimicrobial assays showed minor difference in activity from the six wild populations. Although the micropropagation of B. disticha had limited success, this study did develop a successful decontamination protocol as well as determine the most useful explant and supplements. This information provides an important starting point for the development of a successful micropropagation protocol for the conservation of B. disticha. Since, B. disticha is an important medicinal plant in South Africa, this study has also deepened our understanding of the constituents that could be responsible for the medicinal properties of B. disticha and, in so doing, confirmed the value of this plant for use in traditional medicine in South Africa.
Flower abscission in potted Plectranthus.
(2013) Rice, Laura Jane.; Van Staden, Johannes.; Finnie, Jeffrey Franklin.; Ascough, Glendon D.
Transport and post-harvest handling of flowers both cut and potted is one of the greatest challenges in the horticulture industry (REDMAN et al., 2002). Ethylene-induced flower abscission is responsible for the loss of crops (KIM et al., 2007). Flower abscission is greater when plants are transported (ABEBIE et al., 2005). This limits the sale of flowers and potted plants to areas close to the site of production and prevents export opportunities. South Africa is home to many spectacular species with great horticultural potential (RICE et al., 2011). Unfortunately however, development of a number of these species for export is difficult due to transport-induced flower abscission. Transport-induced flower abscission is a problem experienced by Dr Gert Brits, a breeder of Plectranthus in Stellenbosch in South Africa. In this study a number of Dr Brits’s Plectranthus varieties were used as model plants to understand the process of transport-induced flower abscission and develop a protocol for the prevention of such abscission. Flow cytometry was used to determine the ploidy levels of each of the varieties. It was important to be aware of this during the experiments as varieties with different ploidy levels have been reported to behave differently under stressful environmental conditions. Of the eight varieties examined, three were diploid (2n), one was triploid (3n), three were tetraploid (4n) and one was a mixopliod (2n/4n) variety. To determine the effects of packaging plants during transport and the effects of darkness on flower abscission, plants were packaged into perspex chambers and kept either in a 16 h photoperiod or in darkness for 96 h. Every 24 h the number of open and unopened flowers that had abscised was recorded. Both packaging and darkness increased flower abscission of open and unopened flowers in all eight varieties. Four varieties preferentially abscised open flowers; while the remaining four preferentially abscised unopened flowers. All eight varieties were exposed to different concentrations of ethylene (0, 0.1, 0.25 0.5, 1 and 2 μll-1) to determine their level of ethylene sensitivity. All of the Plectranthus varieties were determined to be extremely sensitive to ethylene. With 100% flower abscission occurring within 24 h at 1 and 2 μll-1 in all varieties. In order to determine what internal changes were causing this increase in flower abscission under these conditions, the changes in the expression of key ethylene biosynthetic enzymes, cytokinin content and carbohydrates in the flowers were examined. ACS and ACO are the two key enzymes in the ethylene biosynthetic pathway (JOHNSON & ECKER, 1998). Changes in the levels of mRNAs coding for these two enzymes were examined when plants were packaged and put into the dark. In general there was an upregulation of the ethylene biosynthetic pathway and in turn this may have increased ethylene production by the plants under simulated transport conditions. However, the changes were not large enough to be solely responsible for the increased flower abscission observed under simulated transport conditions. The concentrations of 43 cytokinins were measured in pedicle tissue from plants which had been kept in the dark for 0, 24, 48, 72 and 96 h. Of the 43 cytokinins measured 21 were below the level of detection. Concentrations for the remaining 22 cytokinins at each of the time points were examined and it was found that in general cytokinin concentrations increase when plants are packaged and put into the dark. DHZ-type cytokinins remained stable during the 96 h continuous dark monitoring period, with most of the changes observed in the tZ and iP types. Peaks in cytokinin concentrations are often followed by an increase in flower abscission, indicating that an increase in cytokinin concentrations may be one of the factors causing the increase in transport-induced flower abscission. Only glucose and fructose were detected in peduncle tissue. Changes in glucose and fructose over 24 h in the greenhouse and over 0, 24, 48, 72 and 96 h in simulated transport conditions were measured. During the day, glucose and fructose levels increased towards the afternoon and evening and decreased in the early morning. This is consistent with studies conducted on other species (ALONI et al., 1996). When plants were put into the dark, glucose and fructose levels increased slightly at 24 h and then decreased to levels similar to those measured in control plants. Although there were changes in glucose and fructose level in simulated transport conditions, they were very slight and it is unlikely that these changes are not responsible for the transport-induced flower abscission. These results suggest that the observed transport-induced flower abscission is the result of increased cytokinin concentrations and expression of ACO and ACS genes when plants are packaged and put into the dark. These changes in turn cause an increase in ethylene production by the plants, and the build-up of ethylene in the transport container causes flowers to abscise. Ethylene perception by the plant is the step which could be targeted to prevent flower abscission. A number of ethylene antagonists block the ethylene receptors in the plant and in so doing prevent the receptors from binding ethylene and transducing the abscission signal. 1-MCP isone such ethylene antagonist. To test whether 1-MCP could be used for the prevention of flower abscission in Plectranthus, plants were placed in sealed perspex chambers in the light and in the dark and treated with 100 nll-1 1-MCP for a single 6 h treatment, or for 6 h every day prior to continuous exposure to ethylene. 1-MCP treatment greatly reduced ethylene- and transport-induced flower abscission when plants were treated continuously, but reduced flower abscission for the first 24 h when pre-treated with a single 6 h exposure to 1-MCP.Transport-induced flower abscission in Plectranthus is the result of exposure to ethylene. The increase in ethylene production by the plants in transport conditions is likely due to an upregulation of the ethylene biosynthetic pathway and an increase in cytokinin concentrations or movement in the pedicle tissue. This transport-induced flower abscission can be prevented by continuous treatment with 100 nll-1 1-MCP during the transport period. By using 1-MCP plants can be transported for up to 4 d and the opportunity for export is made possible.
Medicinal properties and in vitro responses of Mayenus senegalensis (Lam.) exell.
(2003) Matu, Esther Ng'endo.; Van Staden, Johannes.
No abstract available.
Genetic modification in Pinus patula using transgenic technology.
(2006) Nigro, Sara Anna.; Van Staden, Johannes.; Makunga, Nokwanda P.; Jones, Nicky B.
Progress in tree biotechnology initially trailed behind agricultural crops due to their long life cycle, difficult tissue culture and regeneration protocols, and their abundance in natural forests. However, rapid global deforestation rates, together with an increased world demand for pulp, paper and timber products, have prompted scientific and commercial focus to improve genetic timber stocks. South Africa, a tree-poor country (where indigenous forests are protected), has relied almost solely on exotic plantations to meet its demand for timber. A pioneer study investigating the feasibility of using direct (biolistic) and indirect (Agrobacterium-mediated) methods for gene transfer was undertaken in Pinus patula Schiede et Deppe, a Mexican softwood and a forerunner for saw timber, pulpwood and paper in the South African forest industries. The aim of the transformation methods was to impart herbicide resistance to the trees. This was achieved via the introduction of a bar-GUS pAHC25 cassette under the control of the ubiquitin promoter. To provide target material for transformation, two in vitro micropropagation pathways were used: somatic embryogenesis and organogenesis. Both embryonal suspensor masses (ESM) and somatic embryos at various stages of development were initially used as target explants for the biolistic study using an established in vitro protocol. A stepwise selection was implemented in order to allow transformed (particularly bombarded) cultures the opportunity to regenerate under selection pressure using MSG3 maintenance medium supplemented with BASTA® herbicide at 1 mg l ¯¹ followed by 3 mg l¯¹ active ingredient at the next subculture. Biolistic transgene delivery was more efficient when sorbitol was included in the pre-bombardment medium enabling use of higher vacuum and shooting pressures, without lowering the regeneration potential of ESM significantly. Bombarded material from two genotypes (Lines 2 and 3) was regenerated to produce mature somatic embryos using an optimized regeneration regimen. The indirect study with Agrobacterium tumefaciens (LBA4404), transformed with the pAHC25 vector via triparental mating or heat shock, used a variety of target tissues including: mature somatic embryos, ESM and mature zygotic embryos (MZE's) - a novel in vitro system for P. patula. The Agrobacterium-mediated method resulted in optimized decontamination conditions using a combination of liquid MSG3 (or sterile dH₂O for mature embryos) supplemented with 500 mg l ¯¹ cefotaxime, with rotation, and sterile 65 mm Whatman No. 3 filter paper stacks, which avoided excess filtering and stress to transformation material. Further efforts to aid regeneration during the indirect study included L-proline post-transformation, though no mature somatic embryos were regenerated at the conclusion of the Agrobacterium-mediated study. Recovery of transformed ESM in both studies was best during the active growth phase 4-6 d after subculture. Regeneration with good somatic embryo potential was an exigent aspect in both transformation studies. Expression of positive histochemical GUS activity in all transformed material was confirmed by polymerase chain reaction (PCR) analysis indicating that Pinus patula tissue was amenable to transformation. A new bar PCR regime was implemented in P. patula. In the biolistic study, a higher transformation efficiency of bar amplicons (53%) than GUS amplicons (45%) was observed, reflecting their non-linked status on the pAHC25 transformation vector. This is the first report of biolistic transformation of P. patula that will allow for the production of transgenic ESM. The production of transgenic P. patula holds great promise for commercial development in the South African forestry industry. The application of transgenic trees in the timber industry is numerous but the aims most relevant to P. patula include wood modification and disease resistance to pathogens like pitch canker fungus.
Root-stimulating activity from various gelling agents used in tissue culture.
(2003) Arthur, Georgina Dede.; Van Staden, Johannes.; Stirk, Wendy Ann.
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.
Genetic transformation and micropropagation of Thapsia garganica L. - a medicinal plant.
(2003) Makunga, Nokwanda P.; Van Staden, Johannes.; Jäger, Anna Katharina.
No abstract available.
The role of smoke as a germination cue.
(2006) Light, Marnie Elizabeth.; Van Staden, Johannes.
No abstract available.
Differential gene expression in germinating and thermoinhibited achenes of Tagetes minuta L.
(2003) Hills, Paul Norman.; Van Staden, Johannes.
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.
Micropropagation of Hypoxis colchicifolia Baker, a valuable medicinal plant.
(2004) Appleton, Margaret Rae.; Van Staden, Johannes.
The large geophytic monocotyledon, Hypoxis colchicifolia Baker, has been identified for the importance of its corm extracts in the development of a potential non-toxic prodrug for the treatment of inflammation, certain malignancies and HIV-infection. The underground corms of this plant are also commonly used for therapeutic applications in traditional medicine in Kwazulu-Natal where it primarily occurs. A review of published literature revealed, however, that H. colchicifolia plants are currently harvested in an unsustainable manner from traditional collecting sites due largely to population growth, increased land use for urban development and agriculture, and the popularisation of Hypoxis plants for herbal remedies. A further search of historical records established that H. colchicifolia plants were dominant in grassland vegetation prior to 1950, but had rapidly declined since then. Quantitative data subsequently gathered in this study from comparative surveys of both H. colchicifolia and H. hemerocallidea populations from sites with near-pristine, disturbed, burnt and mown grassland vegetation showed for the first time that exposure to human activity and the grassland management practices of mowing and burning incurred not only a 75% reduction in plant density of both these Hypoxis species, but also the total destruction of mature plants of H. colchicifolia in frequently mown and burnt areas. Flowering data recorded in these surveys, and confirmed by monitoring field performance of cultivated H. colchicifolia plants, showed that a contributing factor to the plant's inability to withstand these pressures was that juvenile forms only reached flowering maturity after three to four years growth, thus adversely affecting seedling recruitment. It was concluded therefore that, since Hypoxis species responded differently to mowing and burning, geophytic plants should be considered individually and not as "forbs" during the planning of grassland management programmes for natural conservation areas. The need to cultivate H. colchicifolia to ensure its survival was also established using the new field data gathered in this study. Methods to propagate this species have, however, not been established. Data gathered on all the plants comprising a single population confirmed that mature plants survive to an estimated 20 years and longer in natural areas. Greatest hypoxoside yields were also obtained from corms with a fresh mass of 350g to 400g. Since these corms were estimated to be 10-years-old and older, propagation and cultivation methods that could sustain plant production and survival for long periods, and therefore increased hypoxoside yields, would have to be developed. Several micropropagation systems suitable for the mass production of H. colchicifolia and from which phenotypically normal plantlets were recovered, were therefore established via organogenesis, embryo culture and somatic embryogenesis. The latter cultures have not been reported previously for Hypoxis. In the former culture the toxic effects of phenolic leachates and browning were controlled, and improved plantlet regeneration achieved, by adding polyvinyl pyrrolidone to the medium and introducing distinct sequential aseptic steps into the micropropagation procedure developed. Defined protocols for the different phases of in vitro somatic embryogenesis are not readily available for monocotyledons, however, neither are the factors controlling embryogenesis and organ regeneration known. In this study the process of somatic embryogenesis from excised zygotic embryos of H. colchicifolia was shown to be complex and the resultant cultures very heterogeneous. Although the stage of development of the zygotic embryo explants was important at the time of inoculation, data showed that the induction and regulation of the processes of embryo culture and somatic embryogenesis were ultimately determined by the exogenously applied plant growth regulators. By comparing the different pathways leading to plantlet regeneration, and the morphological stages of development of the structures produced both on solid and in liquid media, not only photographically, but also quantitatively and schematically, the repeated formation of pseudoembryonic structures and neomorphs confirmed that they form an integral part in the in vitro somatic embryogenic pathway of H. colchicifolia. Evidence suggested not only that two types of somatic embryos are produced in the embryogenic cultures of H. colchicifolia, but that the pseudoembryonic structures produced resemble the pseudobulbils produced in polyembryonic cultures of Citrus. The success of the somatic embryogenic cultures was confirmed by the estimation that 28 112 somatic embryos and embryo clusters of H. colchicifolia could be obtained from 16 ml of somatic embryogenic liquid culture. Furthermore phenotypically normal plantlets regenerated from all of the micropropagation procedures developed were successfully transplanted from the laboratory, acclimatized under greenhouse conditions and their horticultural and field performances evaluated.
In vitro propagation of enset (Ensete ventricosum (Welw.) Cheesman)
(2003) Chimsa, Mulugeta Diro.; Van Staden, Johannes.
Enset (Ensete ventricosum) is an important food crop that is cultivated in Ethiopia. In vitro propagation: zygotic embryo culture, shoot tip culture, callus culture and somatic embryogenesis were investigated for this crop. Forty four percent germination of excised embryos of stored seeds of enset genotype Oniya was obtained when the embryos were placed horizontally on the medium that was supplemented with 0.5 mg l ̄¹BA and 0.2 mg lˉ¹ lAA, after germination of intact seeds could not be achieved. Over 85% embryos, excised from seeds of two wild enset genotypes shortly after seed harvest, were germinated on MS medium with and without plant growth regulators (PGRs). Addition of 5 g lˉ¹ activated charcoal (AC) prevented blackening of germinating zygotic embryos and improved in vitro growth of the seedlings. Contamination of culture was reduced to a tolerable level (below 7%) when eight to ten mm long shoot tips from greenhouse-grown suckers were decontaminated for 15 min in 3.5% sodium hypochlorite and rinsed three times with sterile distilled water. However, this contamination method was not sufficient to decontaminate shoot tips from field-grown suckers. Avoiding injury to the apical domes of the shoot tips at the initiation stage, addition of 7 g lˉ¹ AC to the medium and initiation of the shoot tips for two months before splitting for multiplication considerably decreased blackening and formation of callus for genotype Keberia and Mazia. Three to five normal shoots per shoot tip were produced when halved shoot tips from in vitro germinated seedlings of enset genotype Oniya was cultured on gelled and in liquid medium and when halved shoot tips of greenhouse-grown genotype Mazia were cultured in a liquid medium. One to two shoots/buds per shoot tip were regenerated from halved shoot tips of greenhouse-grown suckers on gelled medium for genotypes Keberia, Oniya and Mazia. The presence of BA did not result in a significant increase in the number of shoots per shoot tip both with intact and halved shoot tips. Therefore, wounding the apical dome by splitting appears necessary to release lateral buds. Both blackening of explants in the presence of AC and contamination of culture in vitro were not observed with in vitro grown plant material. Callus was produced on MS medium supplemented with 0.5 mg lˉ¹ BA + 0.2 mg lˉ¹l lAA from zygotic embryos of stored seeds of enset. Adventitious shoots from the callus were regenerated in the light on MS medium lacking PGRs. Embryogenic callus was obtained from shoot tips of genotype Mazia on MS medium with 0.5 mg lˉ¹ BA + 0.2 mg lˉ¹ lAA + 0.2 mg l ˉ¹2, 4-D. A large number of somatic embryos were produced from the embryogenic callus. The results of these studies can be used in enset clonal multiplication, conservation of germplasm and breeding of the crop.
Anti-bacterial and anti-inflammatory activity of medicinal plants used traditionally in Lesotho.
(2003) Shale, Thato Lucy.; Van Staden, Johannes.; Stirk, Wendy Ann.
A significant potion of the population in Lesotho relies on traditional medicine to meet its health care requirements. Traditional healers and herbalists were interviewed from Qacha's Nek (Highlands) and Mohale's Hoek (Lowlands) districts in Lesotho on plants used by the Basotho in traditional remedies. Fifteen plants were reported to be used for bacterial infections while thirteen plants were used for diseases associated with inflammation . Plant roots were most often used to make water extracts. Mainly high altitude plants are used with lowland healers obtaining most of their plant material from the highlands, either by collecting them or buying them from highland gatherers. Leaves and roots of plants used to treat bacterial infections were extracted with hexane, methanol and water and the respective extracts screened at 100 mg ml¯¹ for anti-bacterial activity using the disc diffusion bioassay. Seven species displayed very high anti-bacterial activity against both Gram-positive and Gram-negative bacteria. A number of plant extracts had medium inhibitory activity, mostly against Gram-positive bacteria. This activity was mainly found in the root extracts. Six of the thirteen plants screened for anti-inflammatory activity using the cyclooxygenase-1 (COX-1) bioassay had activity above 90%. Hexane and methanol extracts were the most active while water extracts usually had lower activity. Malva parviflora, Eriocephalus punctulatus and Asparagus microraphis exhibited high anti-inflammatory activity from hexane, methanol and water extracts made from leaf and root material. High anti-bacterial activity was also recorded from M. parviflora and E. punctulatus hexane, methanol and water extracts. An investigation on seasonal variation and plant part substitution in medicinal activities for these plants was carried out. Extracts of M. parviflora collected between June 1999 and July 2001 showed variation in anti-bacterial activity. Extracts made from leaves and roots inhibited the growth of both Gram-positive and Gram-negative bacteria. More bacterial strains were inhibited by extracts made from roots collected in cooler months. However, a trend in seasonal activity was not evident for either the roots or leaves because there was no detection of activity in some of the extracts made within the same months or seasons of the adjacent years. Variation in anti-inflammatory was detected for M. parviflora extracts. E. punctulatus leaf extracts did not exhibit any seasonal variation in anti-bacterial activity. Anti-inflammatory activity of E. punctulatus showed seasonal variation with the highest activity noted when material was collected during the cooler months and a decline in activity when collections were made during the warmer months. Hexane, methanol and water extracts made from leaves and roots of A. microraphis did not show any seasonal variation in anti-inflammatory activity. Thus, M. parviflora and E. punctulatus should be collected during the cooler months while A. microraphis can be collected throughout the year. Traditional healers, herbalists and vendors need to be encouraged to use aerial parts in substitution of ground parts which are reported to be highly utilized. Effect of storage on anti-bacterial and anti-inflammatory activities of M. parviflora, E. punctulatus and A. microraphis were monitored. Dried, ground leaf and root material of the three plants was stored in a cold room, at room temperature and in the Botanical Garden where the material was exposed to high and large changes in temperature. Dried hexane and methanol extracts made from leaves and roots of these plants were stored in a cold room and at room temperature. Initially, storage of the plant material under the three storage conditions caused an increase in antibacterial activity of the hexane, methanol and water extracts made from leaf and root material of M. parviflora and E. punctulatus. Storage for a longer period resulted in a decrease in inhibitory activity. TLC fingerprints developed from hexane and methanol extracts made from M. parviflora and E. punctulatus stored in a cold room and at room temperature showed a consistent number and colour of spots during the initial storage period. Prolonged storage resulted in a decline in the number and colour of detected spots. The stored hexane and methanol extracts made from leaves and roots showed a similar trend of increases and decreases in anti-bacterial activity as well as changes in spots with the storage of the extracts. Testing of the effect on anti-inflammatory activity of hexane, methanol and water extracts made from leaves and roots of M. parviflora, E. punctulatus and A. microraphis showed no change in inhibitory activity of hexane extracts obtained from the material and the extracts stored at the three storage conditions. Methanol and water extracts made from leaves exhibited an increase in activity with prolonged storage. Generally, the stability of the inhibitory activity was longer for the stored dried material than the plant extracts. Isolation of biological active compounds from M. parviflora was not successful due to loss in anti-bacterial activity as a result of collection of plant material from a different locality. Anti-inflammatory compounds could not be isolated due to insufficient amount and the synergistic effect of the active compounds . The purified compounds exhibited loss of activity following HPLC purification which then re-appeared upon recombining the fractions. A number of compounds were detected from essential oils of E. punctulatus using GC. Fractions containing these compounds gave positive anti-bacterial activity in the disc-diffusion , bioautographic and MIC bioassays as well as high anti-inflammatory activity with COX-1 and COX-2 anti-inflammatory bioassays. No anti-inflammatory compounds were isolated from A. microraphis.
A pharmacological study of some Nigerian medicinal plants.
(2005) Chukwujekwu, Jude Chinedu.; Van Staden, Johannes.
Petroleum ether, dichloromethane, and 80% ethanol extracts of 15 plant species collected in Nigeria were screened for in vitro antibacterial, anti-inflammatory and antimalarial activities. Antibacterial activity was tested using the agar diffusion method, while the minimum inhibitory concentrations (MIC) of the active extracts were determined using the microtitre serial dilution method. Most antibacterial activity detected was against Gram-positive bacteria with Staphylococcus aureus being the most susceptible. The highest activity was found in petroleum ether and dichloromethane leaf extracts of Mallotus oppositifolius; petroleum ether, dichloromethane and ethanolic root extracts of Newbouldia laevis; and ethanolic root extracts of Morinda lucida and Canthium subcordatum. Against the Gram-negative bacterium Escherichia coli, the highest activity was found in dichloromethane leaf extracts of Newbouldia laevis, ethanolic root extracts of Phyllanthus amarus, Mallotus oppositifolius, and Canthium subcordatum. A total of 60 plant extracts were screened for antiplasmodial activity. A chloroquine sensitive strain of Plasmodium falciparum (D10) was used. In the assay, the parasite lactate dehydrogenase (pLDH) activity was used to measure parasite viability. About 11 extracts showed promising activity with an IC₅₀ ranging from 2.5 to 13.4 µg/ml. The petroleum ether leaf extract of Hyptis suaveolens had the highest activity (IC₅₀ = 2.5 µg/ml). The cyclooxygenase (COX-1 and COX-2) assays were used to test for anti-inflammatory activity. All the plant species, with the exception of Hedranthera barteri and Picralima nitida showed anti-inflammatory activity. Apart for a few ethanolic extracts, all the activities were recorded with petroleum ether and dichloromethane extracts. Employing bioassay-guided activity fractionation, an antibacterial anthraquinone identified as emodin was isolated from ethanolic root extract of Senna occidentalis. Although this compound had been isolated from other sources, this was the first report of isolation from Senna occidentalis. Using a similar approach a novel antimalarial diterpenoid was isolated from the petroleum ether leaves extract of Hyptis suaveolens. It had IC₅₀ of 0.1 µg/ml. This new compound is worthy of further investigation and may act as an important lead compound for future antimalarial drugs.
Evaluation of anthelmintic, antiamoebic and antibacterial activity in traditional South African medicinal plants.
(2001) McGaw, Lyndy Joy.; Van Staden, Johannes.; Jäger, Anna Katharina.
Traditional medicine in southern Africa draws upon a vast selection of plants to treat gastrointestinal disorders such as diarrhoea and intestinal parasites. The evaluation of these plants for biological activity is necessary, both to substantiate the use of these plants by healers, and also a possible lead for new drugs or herbal preparations. After a survey of the existing ethnobotanical literature, plants used to treat stomach ailments such as diarrhoea, dysentery or intestinal worm infestations were selected and submitted to bioassays according to their traditional uses. Extracts of the chosen plants were made using the solvents hexane, ethanol and water, to ensure the extraction of compounds with a wide range of polarity. In total, 138 extracts were tested for antibacterial activity, 72 for anthelmintic activity, and 42 for antiamoebic activity. Antibacterial activity was evaluated using the disc-diffusion assay, and Minimal Inhibitory Concentration (MIC) values were determined using a microdilution assay. The extracts were tested against the Gram-positive bacteria Bacillus subtilis and Staphylococcus aureus, and the Gram-negative bacteria Escherichia coli and Klebsiella pneumoniae. Ethanolic extracts showed the greatest activity and Gram-positive bacteria were the most susceptible microorganisms. The free-living nematode Caenorhabditis elegans, which is morphologically similar to parasitic nematodes, was used in two different assays to evaluate anthelmintic activity. A microdilution technique was employed to investigate antiamoebic activity against the enteropathogenic Entamoeba histolytica, the causal organism of amoebic dysentery. These assays were suitable for the screening of a large number of extracts at one time. Several plants exhibited significant activity against these test organisms. Many species of plants belonging to the family Combretaceae are used in southern African traditional medicine against a variety of ailments, including abdominal complaints, bilharzia and diarrhoea. Extracts of powdered leaf material of 24 species belonging to the Combretaceae were prepared using the solvents ethyl acetate, acetone, methanol and water. These extracts were screened for anthelmintic activity. Significant activity was exhibited by C. apiculatum, C. hereroense and C. mossambicense. The most anthelmintic activity was shown by acetone extracts, followed by ethyl acetate, water and then methanol extracts. The aromatic rhizomes of Acarus calamus L. are used extensively in traditional medicine worldwide. They reportedly relieve stomach cramps and dysentery, and are used as anthelmintics. Rhizome extracts of A. calamus growing in KwaZulu-Natal, South Africa, exhibited anthelmintic and antibacterial activity in the initial general screening. Using bioassay-guided fractionation, the phenylpropanoid β-asarone was isolated from the rhizome. This compound possessed both anthelmintic and antibacterial activity. It has previously been isolated from A. calamus, and a related species, A. gramineus. Different varieties of A. calamus exhibit different levels of β-asarone, with the diploid variety containing none of the compound. Mammalian toxicity and carcinogenicity of asarones has been demonstrated by other researchers, supporting the discouragement of the medicinal use of Acarus calamus by traditional healers in South Africa. Schotia brachypetala was another plant to show good antibacterial activity in the initial screening. The roots and bark of S. brachypetala are used in South African traditional medicine as a remedy for dysentery and diarrhoea. The lack of pharmacological and chemical data on this plant prompted a further investigation into its antibacterial activity. The differences in activity of ethanol and water extracts with respect to plant part, season and geographical position were analysed. No extreme fluctuations in activity were noted. Two other Schotia species, S. afra and S. capitata, were included in the study, and both displayed good antibacterial activity. The storage of the plant, either as dried, ground plant material at room temperature, or as an extract residue at -15°C, had little effect on the antibacterial activity. Preparing the extracts from fresh or dry material also did not notably affect the activity. In general, the ethanolic extracts were more active than the aqueous extracts. The chemical profiles on TLC chromatograms were compared and found to be very similar in the case of ethanol extracts prepared in different months of the year, and from different trees. The extracts of the three species, and of the leaves stored under various conditions, as well as extracts prepared from fresh or dry material, also showed similar TLC fingerprints. However, various plant parts of S. brachypetala showed distinctly different chemical compositions. The leaves of S. brachypetala showed slightly higher antibacterial activity than the roots. Fractionation of the ethanol extract of the dried leaves using liquid-liquid partitioning and chromatographic techniques yielded 9,12,15-octadecatrienoic (linolenic) acid and methyl-5, 11,14,17-eicosatetraenoate. These fatty acids displayed antibacterial activity against the Gram-positive bacteria Bacillus subtilis and Staphylococcus aureus, and activity to a lesser extent against the Gram-negative Escherichia coli and Klebsiella pneumoniae. Linolenic acid is known to have antibacterial activity. The screening of plants for biological activity yielded valuable preliminary information about the plants used by traditional healers to treat gastrointestinal illnesses. The isolation of biologically active compounds from two highly active plants was achieved.
Cultivation of Combretum bracteosum (Hochst.) Brandis.
(2001) Koen, Kerry Jacqueline.; Van Staden, Johannes.
In maximizing South Africa's floral diversity, plant propagators have begun exploiting the rich array of indigenous plants, especially those with horticultural potential. Plants previously unavailable to the professional and amateur gardeners alike, are legally becoming common-place in nurseries. However, in promoting the trade of indigenous plants to nursery-owners, rapid, easy and cost effective methods of propagating these plants need to be established. Combretum bracteosum is one such indigenous plant, the aesthetic appeal thereof exhibits great potential for ornamentation, especially when flowering. In facilitating the introduction of Combretum bracteosum into nurseries, small gardens or even pots, investigations carried out aimed to determine and analyse quick and easy methods of propagating this plant. Of the various propagation techniques considered, only one, micropropagation, required specialized skill and training prior to carrying out the relevant procedures and protocols. The two other techniques used, which are accessible to most plant propagators, were seed germination and propagation from cuttings. Propagation by seed germination yielded less than optimal results from a commercial perspective. Although the hard pericarp surrounding the embryo did not impose any dormancy inducing mechanisms, such as the restriction of water uptake or the leaching of an inhibitory compounds, it did act as a mechanical barrier to the emerging radicle and roots. Recommendations for optimal Combretum bracteosum seed germination would be to remove the protective pericarp completely, incubate imbibed embryos in complete darkness at 25°C. After radicle emergence the germinating embryos could be moved into an alternating light: dark cycle. A more viable and simpler alternative to seed germination, was propagation by stem cuttings. Treating the cuttings with 10% and 50% or 75% of the commercially available Kelpak concentrate (using the Soak Method and Quick-dip Methods respectively), provided the most promising results, with the rapid development of roots and subsequent vegetative growth. Synthetic hormones such as IBA and NAA were also applied to the cuttings both alone or in combination however, although callus growth was profuse, root development was slow and unsubstantial, if any at all. Therefore, in recommending a protocol for the successful rooting of Combretum bracteosum cuttings taken during spring, summer or early autumn, the application of Kelpak at either 10% (Soak Method) or 50% (Quick-dip Method) of the full strength solution, is advised. Subsequent to hormone treatment, the cuttings still required attention with regard to nutrient supplementation as well as atmospheric moisture and temperature regulation. Success in generating Combretum bracteosum plantlets was obtained by germinating the seed in vitro as well as stimulating axillary shoot elongation from nodal explants. Placing the sterilized Combretum bracteosum embryo onto a nutrient rich basal medium (containing no hormones) was sufficient to stimulate 100% germination. The frequent poor availability of the seed may hamper the use of in vitro seed germination for commercial propagation purposes. The use of nodal explants from in vitro germinated stock plants, is a rapid and reliable means of generating a large seedling stock. Nodal explants excised from the newly developed shoot were subsequently placed onto 0.5 mg.ℓ ¯¹ BA which encouraged axillary bud elongation. After elongation, the lateral shoots were removed and placed onto a rooting medium (1.0 mg.ℓ ¯¹ IBA). The more mature nodal explants, collected from parent plants growing in vivo, required either a BA: NAA hormone combination or Kelpak to stimulate axillary shoot elongation, with the latter being most successful. Root initiation followed the protocol described above. Once rooted plantlets were hardened off they displayed a strong and vigorous growth, which is desirable from a commercial perspective. Upon maturity, the habit of many indigenous trees and shrubs could become too big for confined spaces such as the urban garden. Therefore, determining a means of modifying the plants' habit in order to maintain its suitability as a smaller garden plant was important. Treating the Combretum bracteosum plants with a 50 mg.ℓ ¯¹ paclobutrazol soil drench proved most successful, with the desired effects being visible within a few weeks of initial application. No negative morphological or developmental effects were noted on plants treated with the dwarfing agent, conversely however, the treated Combretum bracteosum plants were compact and bushy, with considerable visual appeal and aesthetic attractiveness.
Crinum moorei : propagation and secondary metabolite production in vitro.
(2002) Fennell, Catherine W.; Van Staden, Johannes.
As an alternative to conventional methods of vegetative propagation, micropropagation attracts much attention, because the levels of multiplication are increased, somaclonal variation is limited and disease-free material can be obtained. The technique is invaluable to the conservation of Crinum species belonging to the Amaryllidaceae which, as a group, possesses several biological features that make them particularly vulnerable. This is in addition to other problems relating to their value as horticultural material, traditional medicines and sources of phytochemicals of interest to medical science. Two in vitro systems are widely used for the propagation of amaryllidaceous species; regeneration from young floral stem explants and from twin-scales excised from bulbs. Although plantlet regeneration could be obtained from peduncle explants of Crinum moorei, a complex of factors including: the age of the floral stem; explant position and; hormonal factors, limited growth. Callus production was poor and indirect organogenesis could not be achieved. Twin-scales were used for the induction of somatic embryos. Morphologically these were different depending on the concentrations of 2,4-D and BA used in the induction medium. Although some of them went on to germinate, the use of somatic embryos for large-scale culture is not an efficient micropropagation route, owing to the low frequency of both embryo production and germination and to the long culture times. Regeneration of shoots and bulblets could, however, be readily induced from twin-scales using a series of modified MS media, and this despite the fact that explants from the bulb were more difficult to decontaminate than the above ground parts. Shoots arose in the axes of the twin-scales close to the basal plate. Initiation was greatest on a basic Murashige and Skoog medium, containing 4 g ℓ ¯¹ sucrose, and in the dark. No hormones were required. At high concentrations, the hormones stimulated abnormal organogenesis. Bulbing of the shoots was further enhanced using higher than normal levels of sucrose i.e. 6% and 5 g ℓ ¯¹ activated charcoal. The response was also influenced by the size of the twin-scale and its position in the parent bulb. Greater numbers of bulblets with larger diameters developed in large twin-scales from an intermediate position between the inner and outer scales. Furthermore, light, and a temperature of 25°C were required for normal bulblet development. Bulblets grown in this manner were used as a source of secondary explants by splitting them vertically in half. The addition of 10 mg ℓ ¯¹ BA resulted in multiple shoot development. In a liquid-shake culture system, this same multiplication medium induced the formation of meristemoid clusters whose rates of proliferation were higher than that achieved for shoot multiplication on either solid or static liquid media. The advantage of using meristematic clusters is that shoot hyperhydricity is avoided. Furthermore, the clusters can be mechanically separated; making the system ideal for automated plant production. Shoot morphogenesis, followed by the formation of bulblets occurred on solid MS media containing activated charcoal or high concentrations (6%) of sucrose. The induction of bulblets by sucrose was, however, slower, which may be beneficial for long-term storage and conservation ex situ. Compared to smaller bulblets, bulblets with a diameter of approximately 9 mm, acclimatized readily and grew rapidly after transferring them to the soil in greenhouse conditions. Biotechnological processes such as cell and tissue culture provide an ideal system for producing secondary metabolites, especially where their production in situ is hampered by poor resource availability or when chemical synthesis is difficult. In vitro produced Crinum moorei bulblets were found to contain nine alkaloids of the Amaryllidaceae type; three of which were released into the culture medium. Light was essential for alkaloid biosynthesis while the inclusion of BA and activated charcoal stimulated the production of specific alkaloids.
An evaluation of plants used in eastern Nigeria in the treatment of epilepsy and convulsion.
(2002) Ogbonnia, Steve Okwudili.; Van Staden, Johannes.
Schumanniophyton magnificum and Glypheae brevis are important medicinal plants growing wild in the West African rain forest. They are used in folkloric medicine in the treatment of epilepsy and convulsion as well as for some other diseases. The purpose of this work was to investigate the aspect of folkloric use in order to support folkloric claims and document the findings. The extracts were prepared from ground plant material by a continuous extraction method. Five hundred grams of ground plant material were continuously de-fatted with 2 L petroleum ether (60°- 80°) in a Soxhlet apparatus for about 5 h. The resulting marc was dried and the chemical constituents extracted hot in a Soxhlet apparatus for about 8 to 10 h with 2 L aqueous ethanol (70%). The efficacy of the extraction method was confirmed using standard bioassays and phytochemical analyses. The anti-convulsant activity of the crude extracts was evaluated in vivo against chemically induced convulsions using three different animal models, namely the strychnine, the picrotoxin and the pentylenetetrazole tests. The acute and delayed toxicity test results showed that in all the animal models investigated very high doses, about four times higher than the protective doses of the extracts, were required to kill 50% of the population of animal used. Phytochemical assays of the extracts indicated the presence of alkaloids only in S. magnificum root extract and glycosides in extracts from both species. The glycosides were positive to Baljet, Xanthydrol and Keller-Kiliani tests for cardiac glycosides. S. magnificum and G. brevis chemical constituents were initially isolated with a sequential fractionation method starting with a highly non-polar solvent and gradually increasing to a more polar solvent. The fractions were pooled on the basis of TLC similarity profiles when viewed under the UV light at 254 and 366 nm and were found to have two and four major UV absorbing fractions for S. magnificum and G. brevis respectively. Radio-receptor binding tests were used to assess the anti-convulsant activities of the hydro-alcoholic crude extracts, the organic and aqueous fractions of the crude extracts, partially purified components and pure components in in vitro tests against some standard GABA[A] receptor antagonists, muscimol and isoguvacine respectively. The anti-convulsant activities resided in the aqueous fractions of the hydro-alcoholic crude extracts of both plants. The purely organic fractions of G. brevis demonstrated no activity while all the fractions of the aqueous component demonstrated some degree of activity. The anti-convulsant activity of S. magnificum was found only in one fraction-Fraction 1. This Fraction was further investigated and one of the components appear to be responsible for the activity. The structure of the active constituent was 5,7dihdroxy-2 methylbenzopyran-4-one, a noreugenin. A second bioactive compound, schumanniofoside, was identified from Fraction M[5.2] from S. magnificum.
The effect of charcoal on tissue morphogenesis in vitro.
(2000) Pan, Manjing.; Van Staden, Johannes.
The effect of activated charcoal, autoclaving and culture media on sucrose hydrolysis in tissue culture media was investigated. Activated charcoal acidified an aqueous sucrose (5%) solution and culture media by about 1 to 2 units after autoclaving . Sucrose hydrolysis in tissue culture media and/or aqueous sucrose (5%) solutions containing activated charcoal (buffered to pH 5.8) was dependent on both the hydrogen ion concentration (pH) and autoclaving. After autoclaving, 70%, 56% and 53% sucrose hydrolysis were respectively recorded in a 5.0% sucrose solution, Murashige and Skoog (MS) and Gamborg B5 (B5) liquid media in the presence of 1.0% activated charcoal, added before autoclaving . In the absence of activated charcoal, autoclaving resulted in about 20% of the sucrose being hydrolysed The adsorption of 2, 4-dichlorophenoxyacetic acid (2,4-D) by activated charcoal from methanol and aqueous solutions was determinated using HPLC. The amount of the added 2,4-D decreased in both methanol and aqueous solutions in the presence of activated charcoal, compared with those in the absence of activated charcoal. In methanol and aqueous solutions, activated charcoal used at the level of 0.1% significantly reduced 2,4-D. About 68.4% and 60.9% respectively of the added 2,4-D was adsorbed by activated charcoal (1.0%) from these solutions. The changes of inorganic elements in MS-salt solutions, in the presence of activated charcoal, were analysed by SEM-EDX. The concentrations of magnesium (Mg), calcium (Ca), iron (Fe) and zinc (Zn) deceased in the presence of activated charcoal, while the concentrations of potassium (K), copper (Cu), manganese (Mn), phosphorus (P), and sulphur (S) increased in the MS salt solution in the presence of activated charcoal compared with no activated charcoal in the medium. This suggests that activated charcoal adsorbed calcium, magnesium, iron and zinc and released copper, manganese, phosphorus and sulphur. Rooting occurred when 7-day-old seedling hypocotyls of Daucus carota L. Cape Market were placed on MS medium supplemented with 2,4-D, and IAN/NAA in the presence of activated charcoal. Hypocotyls did not produce roots on the 2,4-D containing media in the absence of activated charcoal. The roots were produced polarly on the NAA/IAA-containing media in the presence of activated charcoal. No-polarity of root formation was observed on media supplemented with NAA/IAA without activated charcoal. Different responses of hypocotyls to a series of 2,4-D concentrations (0.5, 1.0, 3.05.0, 8.0, and 10.0 mg l ¯¹) were observed on media supplemented with 0.02, 0.1 and 0.5% activated charcoal. In the NAA/IAA containing media in the presence of activated charcoal, root number per hypocotyl decreased. Root number perhypocotyl, on the media supplemented with NAA and IAA, increased when hypocotyls were pre-cultured on MS medium supplemented with 2,4-D (1.0 mg l ¯¹) for 2-3 days. When hypocotyls were pre-cultured on a 2,4-D containing MS medium for 5 days, embryos emerged from the hypocotyls directly on the medium supplemented with 2,4-D in the presence of activated charcoal. Addition of activated charcoal to MS medium supplemented with 2,4-D resulted in somatic embryogenesis of Daucus carota. Somatic embryos were not formed on the medium in the absence of activated charcoal. In suspension culture, the incorporation of 0.01 to 1.0% concentrations of activated charcoal to the MS medium, irrespective of 2,4-D, increased the number of somatic embryos produced. The maximum number of somatic embryos were produced with 1.0% activated charcoal. Further development of embryos of Daucus carota occurred on the media in the presence of activated charcoal, and the embryos subsequently regenerated normal plantlets. Abnormal somatic embryos followed the addition of 3.0% activated charcoal to the medium.
Monocarpic senescence in Bidens pilosa L.
(2000) Zobolo, Alpheus Mpilo.; Van Staden, Johannes.
Senescence was examined in the economic weed Bidens pilosa, with the objectives to a) determine the effects of deflowering and defruiting on growth, chlorophyll content, photosynthesis and transpiration; b) to identify the stage of development of the head at which the flowers, seeds/fruit produce senescence signals; and c) to test for senescence activity in plant extracts made from the receptacles and leaves of Bidens pilosa. Total chlorophyll content in the controls, in association with the development of fruit, was lower in the final harvests when compared with earlier harvests in both pot and field-grown plant experiments. Deflowered Bidens pilosa plants had a higher chlorophyll concentration than both defruited and control plants in both pot and field-grown plants. Stem death of the control plants was higher than that of deflowered plants in both field and pot experiments. The present results suggest that deflowering is essential if the leaves are to be harvested commercially because it retards senescence and maintains growth. Fruit and flower heads were responsible for the reduction in leaf and stem growth after flowering in Bidens pilosa. Removing these organs slowed plant decline, suggesting that the flower head and especially the fruit are responsible for senescence. In contrast, the fruit were the main organs responsible for the decline in leaf chlorophyll concentration. In pot-grown plants in full sunlight, photosynthesis and transpiration were low in deflowered plants compared with the control and defruited plants 45 days after treatment, and it coincided with a low stomatal conductance. These results suggest that stomatal conductance played a role in lowering photosynthesis in deflowered plants. In contrast, the control plants had a higher stomatal conductance than deflowered plants 75 days after treatment, yet photosynthesis and transpiration rates were the same in both treatments. Thus stomatal conductance alone does not successfully explain differences in photosynthesis in these treatments. The dry weight of head with mature dry fruit was higher in plants grown at high light intensities than at medium or low light intensities. It coincided with a greater decline in chlorophyll concentration in the leaf nearest to the head and fruit. In contrast, photosynthesis was the same at all light intensities in the leaf nearest to the head and fruit. This suggests that high light accelerated the process of fruit maturation of the fruit which then influenced senescence in the leaf nearest to the flower head. Ethanolic and water extracts of senescent receptacles purified using paper chromatography, induced senescence of leaves in light but not in the dark. In ethanolic extracts, activity was detected in R[f]s 0.1, 0.2 and 0.3. In water extracts, activity was detected in R[f] 0.1. Senescent leaf extracts purified using column chromatography also induced senescence in light under greenhouse conditions. At high concentrations, activity was detected in fraction 10 eluted with ethyl acetate: methanol (55:45); fraction 11 eluted with ethyl acetate: methanol (50:50); fraction 12 eluted with methanol (100%) and in fraction 13 eluted with ethylacetate : isopropanol: water: acetic acid (52:28:28:4). Under growth room conditions, activity was detected in fractions 12, eluted with methanol (100%) and 13, eluted with ethyl acetate: isopropanol: water: acetic acid (52:28:28:4) in the presence of light. Fraction 1 (R[f] 0.00-0.10) from senescent receptacles, non-senescent and senescent leaves, obtained following thin layer chromatography of ethanolic extracts induced senescence under light. Fraction 1 was eluted with methanol. This fraction lacked activity when eluted with ethyl acetate. Fraction 4 (R[f] 0.25 - 0.35) from non-senescent leaf extracts, which co-chromatographed with 4-chloroindole acetic acid, gave activity in bean cuttings kept under continuous low light. Senescent leaf extracts showed no activity. Fraction 7 (R[f] 0.9 - 1.0) from non-senescent leaf extracts, also induced senescence in bean cuttings under light. The same Fraction from senescent leaf extracts lacked activity.
Alkaloids from three South African Crinum species.
(2000) Elgorashi, Esameldin Elzein.; Van Staden, Johannes.; Drewes, Siegfried Ernst.
The alkaloid content of three Crinum species namely C. bulbispermum, C. macowanii and C. moorei was investigated. The ethanolic extracts of C. bulbispermum yielded seven compounds. The new alkaloids 8α-ethoxyprecriwelline, N-desmethyl-8α-ethoxypretazettine and N-desmethyl-8β-ethoxypretazettine were isolated for the first time from a natural source. In addition, the known alkaloids bulbispermine, crinamine, 6-hydroxycrinamine and 3-O-acetylhamayne were isolated in this study. The ethanolic extracts of C. moorei were found to contain Iycorine, 1-O-acetyllycorine, crinine, 3-O-acetyllycrinine, epibuphanisine, powelline, crinamidine, undulatine, epivittatine, 1-epideacetylbowdensine, cherylline and the new alkaloids mooreine and 3-[4'-(2'-aminoethyl)phenoxy]bulbispermine. The alkaloids crinine, lycorine, bulbispermine, cherylline and hamayne were obtained from the ethanolic extracts of C. macowanii. In addition, the amine tyramine was identified during the isolation process. Dilute HCl solution extraction followed by GC analysis was used to investigate organ-to-organ and seasonal variation of alkaloids in each Crinum species, as well as the interspecific variation in these alkaloids over two consecutive years. Twelve alkaloids were identified, including crinine, epibuphanisine, powelline, crinamine, crinamidine, 6-hydroxycrinamine, 1-epideacetylbowdensine, 3-O-acetylhamayne, undulatine, Iycorine, 1-O-acetyllycorine and cherylline. Alkaloids were detected in all organs of C. moorei and C. macowanii. However, alkaloids were not detected in the leaves of C. bulbispermum. Organ-to-organ and seasonal variations in the total yield and total ring types of these alkaloids were noticed. Organ-to-organ and seasonal statistical variations were also detected for some of the individual alkaloids detected in each of these species. The results also showed that C. moorei had the highest levels of all individual alkaloids except crinamine when compared to C. bulbispermum and C. macowanii. Quantitatively, the detected alkaloids chemotaxonomically separated C. moorei from C. bulbispermum and C. macowanii. The results also indicated that C. macowanii is more closely related to C. bulbispermum. Qualitatively, Iycorine, 1-O-acetyllycorine, cherylline, crinamidine, 1-epideacetylbowdensine, crinine, crinamine and 3-O-acetylhamayne were detected in both C. moorei and C. macowanii, indicating the close relationship of these species.
Stress related responses in soybean.
(2000) Liu, Tao.; Van Staden, Johannes.
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.

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