Research Centre for Plant Growth and Development

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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.
Ameliorative effects of botanicals and rhizobacteria on the growth of Pelargonium sidoides and Solanum lycopersicum infested with Meloidogyne incognita.
(2021) Sithole, Nokuthula Thulisile.; Van Staden, Johannes.; Finnie, Jeffrey Franklin.
Abstract available in PDF.
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.
Antidiabetic and phytochemical properties of four selected medicinal plants.
(2019) Ratsoma, Manchela Francinah.; Van Staden, Johannes.; Finnie, Jeffrey Franklin.
Abstract available in pdf.
Breeding systems of some cold tolerant eucalyptus species.
(2002) Jones, Wayne Russell.; Van Staden, Johannes.
Seasonal flowering times for Eucalyptus nitens, E. dunnii, E. smithii, E. macarthurii and E. grandis were evaluated in clonal grafted orchards located at the Shaw Research Centre (SRC) in KwaZulu-Natal, South Africa. The orchards are situated at 29° 29 'South, 30° 11 'East at 1100 m above sea level. The climate is cool (MAT 16.7° C) with a January mean monthly maximum of 25.8° C and July minimum of 4.4° C. An estimated mean annual rainfall of 998 mm and median annual rainfall of 899 mm has been reported (PALLETT and MITCHELL 1993). It is evident that the different species flower consistently from one year to the next during the same period with similar mean flowering peaks. Long reproductive sequences where identified for all species relative to E. grandis, particularly E. smithii and E. dunnii. Paclobutrazol was used to initiate flowering to facilitate the study of the breeding systems of the different species. When applied as a soil drench during early summer an increase in the flower bud production in E. nitens, E. smithii and E. grandis was achieved. The use of various cytochemical methods to test pollen viability, were shown to be mere indicators of potential viability and lack the reliability for adequate testing of stored pollen. From the range of in vitro, pollen viability studies the most successful media for all species tested was 30 % sucrose with 150 mg r¯¹ boric acid. Without boric acid in the media, the response after 24 h was significantly poorer (p<0.001). Significant differences (p<0.05) in the area of pollen grains were found between and within species. There was no significant difference between E. dunnii and E. macarthurii at the species level. Pollen of E. smithii, E. grandis and E. nitens were significantly smaller than that of both E. dunnii and E. macarthurii. From isolation experiments which limited potential pollinators it is apparent that a reduction of pollinators not only leads to poorer capsule survival but also poorer seed set. Following an initial survey of pollinators of E. grandis, very few insects were recorded relative to surveys conducted in the natural habitats with indications that an association does exist between the presence of active pollinators and temperature. The potential of flowers to set seed is clearly demonstrated by the difference between open pollinated flowers and controlled pollinated flowers following intraspecific crosses where differences in seed yield per capsule are very often more than double for species such as E. nitens and E. macarthurii. Similarly with interspecific crosses, higher seed yields are extracted from crosses between closely related species. An extensive survey of orchards clearly demonstrates that E. nitens has the lowest clean seed recovery (13.8 %) significantly less than that of E. smithii (18.0 %) and both E. macarthurii and E. dunnii at 26.1 % and 26.0 % respectively.
Cadmium induces hypodermal periderm formation in the roots of the monocotyledonous medicinal plant Merwilla plumbea.
(Annals of Botany Company., 2010) Lux, Alexander.; Vaculık, Marek.; Martinka, Michal.; Liskova, Desana.; Kulkarni, Manoj G.; Stirk, Wendy Ann.; Van Staden, Johannes.
Background and Aims. Merwilla plumbea is an important African medicinal plant. As the plants grow in soils contaminated with metals from mining activities, the danger of human intoxication exists. An experiment with plants exposed to cadmium (Cd) was performed to investigate the response of M. plumbea to this heavy metal, its uptake and translocation to plant organs and reaction of root tissues. Methods. Plants grown from seeds were cultivated in controlled conditions. Hydroponic cultivation is not suitable for this species as roots do not tolerate aquatic conditions, and additional stress by Cd treatment results in total root growth inhibition and death. After cultivation in perlite the plants exposed to 1 and 5 mg Cd L-1 in half-strength Hoagland’s solution were compared with control plants. Growth parameters were evaluated, Cd content was determined by inductively coupled plasma mass spectroscopy (ICP-MS) and root structure was investigated using various staining procedures, including the fluorescent stain Fluorol yellow 088 to detect suberin deposition in cell walls. Key Results. The plants exposed to Cd were significantly reduced in growth. Most of the Cd taken up by plants after 4 weeks cultivation was retained in roots, and only a small amount was translocated to bulbs and leaves. In reaction to higher Cd concentrations, roots developed a hypodermal periderm close to the root tip. Cells produced by cork cambium impregnate their cell walls by suberin. Conclusions. It is suggested that the hypodermal periderm is developed in young root parts in reaction to Cd toxicity to protect the root from radial uptake of Cd ions. Secondary meristems are usually not present in monocotyledonous species. Another interpretation explaining formation of protective suberized layers as a result of periclinal divisions of the hypodermis is discussed. This process may represent an as yet unknown defence reaction of roots when exposed to elemental stress.
A comparative evaluation of the biological activities and phytochemical properties in Ehretia obtusifolia and Ehretia rigida.
(2021) Mnikathi, Mzamo Mpendulo Ntethelelo.; Finnie, Jeffrey Franklin.; Van Staden, Johannes.
Ehretia from the Boraginaceae family is predominantly found in parts of Asia and North America, with fewer species found in Africa, Europe, and Australia. The genus consists of more than 150 species, and species such as E. microphylla, E. accuminata, E. laevis have been reported on their medicinal prowess. Distribution of Ehretia in Southern Africa is found among a variety of habitats such as the lush forests of the Eastern Cape and Arid parts of Namibia. In South Africa, two species have been identified, namely E. rigida and E. obtusifolia and are used in traditional medicine. African and Asian countries traditional medicine is highly recommended because of its affordability. The aim of the study was to establish a baseline and compare different biological activities and phytochemical properties exhibited by the two South African species. In the study, phenolics, saponins, flavonoids, and tannins were detected in bark, roots, and leaves, of both species, but no detection of alkaloidsf. E. obtusifolia had a higher quantity of flavonoids than E. rigida. Both species exhibited high phenolic quantities in leaves with E. rigida having the highest quantity. Condensed tannins were found with a higher content in leaves than roots and bark for both species, with E. rigida containing higher quantities. E. rigida had the lower MIC’s compared to E. obtusifolia (0.195 mg/ml against M. luteus from ethyl-acetate root extracts). E. rigida had more samples with a MIC lower than 1 mg/ml than E. obtusifolia. The lowest MIC for leaves was 0.39 from ethyl-acetate extracts against S. aureus while methanol bark extracts also achieved 0.39 mg/ml against M. luteus. E. obtusifolia’s lowest MIC was 0.195 mg/ml from methanol leaf extracts against K. pneumoniae. Activity against C. albican was not as good as against the bacterial strains, as the lowest MIC was 0.78 mg/ml for both species. E. rigida and E. obtusifolia had dose-dependent antioxidant activity, with methanol and ethyl-acetate bark, leaf, and root extracts having the highest activities for both species. This study revealed that in comparison to literature, the activity achieved was similar or better when compared to the likes of E. laevis extracts. The α-glucosidase inhibitory activity reported in this study was dose-dependent. The relationship between antioxidant activity and antidiabetic activity is well documented and this study found that extracts with high antioxidant activity also had similarly high α-glucosidase inhibitory activity. It was E. rigida methanol extracts from bark and roots which exhibited the highest activities compared to E. obtusifolia. However, based on the dosedependent activity, E. obtusifolia is more potent because of the higher activity observed at the lowest concentrations. The study demonstrated that both species have good ethnopharmacological properties and were rich in phytochemicals, particularly phenolics and flavonoids. With E. rigida being the least studied of the two species with only one reported study, it was important to carry out this investigation as it has yielded further evidence that the genus Ehretia has multiple species with medicinal potential.
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.
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.
Cytokinins and the germination of Tagetes minuta L.
(2003) Gold, John David.; Van Staden, Johannes.; Stirk, Wendy Ann.
Tagetes minuta L. is a weedy herb that has been a rich source of fragrant oils, used as in the perfume and flavour industry. T. minuta achenes germinate erratically under field conditions. However, at the optimal germination temperature of 25 °C, 100 % germination is attained within 48 h of imbibition. The achenes are thermoinhibited at 35 °C. The aims of this project were to assess the role of cytokinins (CKs) in normal germination at 25 °C, and to investigate the factors that regulate thermoinhibition at 35 °C. CKs were extracted from achenes germinating at 25 °C at 0, 24; 48; 96 and 144 h after imbibition. Two different purification techniques were used, namely Dowex cation exchange resin followed by paper chromatography, or high performance liquid chromatography (HPLC). CK-like activity was tested with the soybean callus bioassay. With both techniques, a peak in CK-like activity appeared 24 h after imbibition, which coincides with the period during which most of the achenes germinated. For quantitative analysis, HPLC\mass spectrometry (MS) techniques were used. The isoprenoid CKs were far more abundant in T. minuta achenes than the aromatic CKs. cis-Zeatin (cZ) and its derivatives were the most abundant CKs. In total, 19 CK compounds were detected, including 4 free bases and a number of corresponding conjugates. Benzyladenine (BA) was the only aromatic CK detected. There was no common time at which active free base maximal concentrations were detected, suggesting that different CKs may have specific roles in the germination process, and thus peak at different times. This in turn suggests that germination is not a single process, but rather a correlative process involving a number of events, with specific CKs having specific roles relating to these correlative events. There is sufficient evidence obtained from both the soybean callus bioassay and HPLC/MS analysis to suggest that CKs have an active role in T. minuta germination. A decline in free BA during germination without corresponding conjugation, suggests that BA is actively used in early germination processes, possibly in the stimulation of DNA synthesis. Secondly, there was a distinct dihydrozeatin (DHZ) peak obtained at 24 h. Roughly 75 % of the achenes germinate between 16 and 26 h, thus it is likely that DHZ has an active role during the germination of T. minuta. Although CKs are probably not involved in the breaking of dormancy per se, the distinct peak in CK-like activity obtained in the bioassays, 24 h after imbibition, suggests that CKs have an active role in the germination of T. minuta. With respect to the regulation of thermoinhibition, a number of exogenous treatments were applied, including hormones [gibberellins (GA₄₊₇), abscisic acid (ABA), ethylene and a number of CKs], adenosine triphosphate (ATP) and incubation in 100 % oxygen. ABA was extracted from thermoinhibited and germinating achenes to assess the role of ABA in thermoinhibition and germination. While exogenous 0.1 mg L¯¹ GA₄₊₇ application slightly improved normal germination at 25°C, no treatments were effective in alleviating thermoinhbibition in T. minuta achenes. Thermoinhibition in T. minuta achenes may be under hormonal regulation, as there is strong evidence for the role of ABA in the maintenance of dormancy and thermoinhbition. High ABA levels were found in dry control samples. Additionally, exogenous ABA application inhibited normal germination, and the commencement of germination was accompanied by a decrease in endogenous ABA levels. A number of experiments relating to the imposition of thermoinhibition were carried out. Thermoinhibition appears to be very rapidly imposed. Germination is rapidly inhibited following shifting to higher thermoinhibitory temperatures, even after prolonged exposure to optimal germination temperatures. Results suggest active de novo biosynthesis of ABA in thermoinhibited achenes. Active biosynthesis of ABA during thermoinhibition suggests that this phytohormone is essential in the maintenance of thermoinhibition of T. minuta achenes. It thus appears that ABA is synthesized in the achenes in response to elevated temperatures that are unfavourable for germination to proceed. Unfavourable environmental conditions result in an achene-mediated inhibition of germination, which appears to be initiated and maintained by elevated levels of endogenous ABA.
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.
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.
Effect of nutrients and smoke solutions on seed germination and seedling growth of Tropical Soda Apple (Solanum viarum).
(Weed Science Society of America., 2011) Kandari, Laxman S.; Kulkarni, Manoj G.; Van Staden, Johannes.
Solanum viarum, commonly known as tropical soda apple (TSA), is native to Brazil and Argentina but has become a harmful weed in many countries with tropical climates. This study was conducted to reassess the seed biology of TSA found in South Africa. Cold stratification (14 d), acid scarification (20% H2SO4 for 5 min), and sandpaper scarification (30 s) significantly improved percentage germination when compared to the control. The highest germination (99.5%) was achieved when seeds were germinated in 50% Hoagland’s nutrient solution (HS). The lowest germination (66%) was recorded in the absence of phosphorus (P) under alternating light conditions. HS without nitrogen (N) completely inhibited seed germination of TSA under constant light conditions. These findings are useful in controlling TSA by amending the levels of N and P in soils. Seed germination of TSA was significantly enhanced by different concentrations of smoke-water and butenolide solution. Smoke-water dilution of 1:500 v/v and butenolide concentration of 10 -8M showed the highest seedling vigor indices (6,688 and 6,666, respectively) in comparison to the control (1,251) and gibberellic acid (GA3) concentrations (< 5,327). These findings suggest that germination of seeds or seedbanks of TSA might be successfully stimulated using smoke solutions. Subsequently, patches of seedlings emerging after treatment can be mechanically uprooted to reduce the infestation of TSA. However, justifying this with field trials is essential.
The effect of organic biostimulants and the mode of application on the growth and biochemical composition of Amaranthus hybridus L.
(2020) Ngoroyemoto, Nelson.; Van Staden, Johannes.; Finnie, Jeffrey Franklin.; Kulkarni, Manoj G.
Abstract available in PDF.
Elicitation, metabolomic analysis, and identification of antidiabetic compounds from selected indigenous plants = Ukuvuselelwa, Uhlaziyo Lokugayeka, nokuhlonzwa Kwezingxube Zesinqindasifo Sikashukela Ethathwe Ezihlahleni Zendabuko Eziqokiwe.
(2022) Ogbe, Abdulazeez Adeola.; Van Staden, Johannes.; Finnie, Jeffrey Franklin.
Diabetes mellitus (DM) is an endocrine disorder associated with high blood glucose levels accompanied by disruptions in the metabolism of fat, proteins and carbohydrates. DM is a chronic, non-communicable and medically incurable disease affecting millions of people globally, resulting in high morbidity and mortality rates, especially with the lingering coronavirus disease of 2019 (Covid-19). The use of western antidiabetic medicine has posed many challenges due to their perceived overall safety, treatment failure and cost. Many African communities rely on medicinal plants and their bioactive compounds as sources of medicine as a consequence of the poor state of health facilities, shortage of medical doctors and unaffordability of treatments. For this reason, this study partly evaluated the phytochemical contents, in vitro antioxidant and hypoglycaemic potentials of eleven indigenous plants using five different solvents. Putative hypoglycaemic agents from one of the most promising and readily available species were also identified using in silico molecular modelling. Secondary metabolites and their pharmacological activity have been reported as the basis for the wide use of plants in traditional medicine. However, due to the indiscriminate harvesting and environmental pressure, many valuable indigenous plant species have gone into extinction or are at least threatened. Moreover, plants' bioactive compounds are often produced in minute quantities, and prevailing environmental conditions further influence their concentrations in plants. Thus, due to indigenous plants' industrial and medicinal value, deliberate cultivation and elicitation strategies have been adopted for the en masse production of uniform indigenous plants and to influence the quality and quantity of their active principles. Thus, this study also assessed the effects of individual and co-inoculation of two isolated drought-resistant and growth promoting endophytes on the growth, drought tolerance, medicinal efficacy and metabolome changes in the leaves of Endostemon obtusifolius. In this research, the eleven plants were selected based on the traditional uses of the plants (or their related available species) for treating various ailments, including DM. The preliminary phytochemical quantification results revealed that the highest concentrations of phenolics, flavonoids and tannins were found in the crude extracts of Combretum krausssii, Lippia javanica, Psidium guajava, Pentanassia prenulloides, E. obtusifolius, Syzgium cordatum, Pachira aquatic and Catha edulis. The inhibitory effects of the crude extracts against the digestive enzymes α-amylase and α-glucosidase also showed that the crude extracts of C. edulis, C. krausssii, L. javanica, P. aquatica, P. guajava, P. prenulloides, E. obtusifolius and S. cordatum displayed excellent in vitro antioxidant and antidiabetic properties. These results validate the extensive use of these plants in the treatment of DM in many African communities. Furthermore, the 80% ethanol (v/v) leaf extract of S. cordatum (one of the most active and readily accessible specie from the previous study) was fractionated into four sub-extracts [petroleum ether (PE), dichloromethane (DCM), ethyl acetate (EtOAc) and water], and their phytochemical content, in vitro antioxidant and antidiabetic capacities were evaluated. Although the EtOAc extract was the richest of the sub-extracts in total phenolics, all four sub-extracts of S. cordatum showed good in vitro free radical scavenging and hypoglycaemic activities. In silico modelling evaluation of some (34) bioactive principles found in the Gas Chromatography-Mass Spectroscopy (GC-MS) analysis of the PE, DCM and EtOAc sub-extracts revealed that 21 compounds including andrographolide, benzylidene-iditol, cubenol and deoxyspergualin and bis[3,3,4,7-tetramethyl-1,3-2H-benzofuran-1-yl]-ether returned binding energy scores ≤ -7.5 kcal/mol against α-amylase and α-glucosidase enzymes indicative of their hypoglycaemic potentials. The physicochemical and toxicological properties of andrographolide, benzylidene-iditol, bis[3,3,4,7-tetramethyl-1,3-2H-benzofuran-1-yl]-ether and cubenol were predicted to be soluble with high gastrointestinal solubility and non-toxic following Lipinski's rule of five and Veber's rule. Thus, these results indicate that these compounds are potential candidates for oral drugs. The drought tolerance and in vitro plant growth-promoting properties of some endophytes isolated from E. obtusifolius (another active antidiabetic plant identified from the previous experiment) was evaluated. A total of 26 culturable endophytes (twelve fungi and fourteen bacteria) were isolated from the organs (leaf and root) of E. obtusifolius. These endophytic species displayed varying in vitro drought stress tolerance and plant-growth-promoting capacities. Two promising drought stress-tolerant and plant-growth-enhancing endophytic species (Fusarium oxysporum and Paenibacillus polymyxa) were subsequently identified using molecular tools. The identified bacterium (P. polymyxa) and fungus (F. oxysporum) exhibited a symbiotic relationship in an in vitro dual culture experiment. Paenibacillus polymyxa and F. oxysporum individual and co-inoculation differential effects on their host under varying water regimes was further evaluated. The plants were raised with or without endophyte infection under three watering regimes for two months, and their therapeutic efficacy, physiological, biochemical and metabolic responses were assessed. In this study, drought stress markedly affected the growth and hypoglycaemic potentials of E. obtusifolius. On the other hand, endophyte inoculation generally enhanced the dry shoot and root biomass, chlorophyll contents and fluorescence, total soluble sugar, relative water content, proline contents and superoxide dismutase activities in the leaves of E. obtusifolius, whereas their electrolyte leakage and malondialdehyde contents were lowered. As for phytochemical accumulation, while the total phenolic contents were slightly enhanced by the inoculation of endophytes in the leaves of E. obtusifolius, the flavonoid contents of the plant increased as the water deficit worsened. The EtOAc crude extracts' free radical scavenging capacity across the treatments remained unchanged; their in vitro α-glucosidase activity was negatively affected under moderate and severe drought stress but improved with endophyte inoculation. The metabolome difference between the twelve treatments was evaluated using GC-MS based metabolomics. The bi-plot PCA result revealed that the metabolome of fungal inoculated moderately stressed E. obtusifolius correlated less with the other E. obtusifolius plants under different treatments. Additionally, a heatmap of eight differential metabolites showed that the most responsive treatment (the co-inoculated severely drought-stressed plants) produced the highest quantities of non-protein amino acids and organic acids known to protect plant cells during abiotic stress. The leaf extracts of S. cordatum and E. obtusifolius showed remarkable antioxidant and antidiabetic potentials in this study. Although the putative active principles of these plants were identified using GC-MS analysis, proper isolation and quantification of these compounds can be explored by future studies. Moreover, some culturable endophytic species were isolated from the E. obtusifolius organs. Paenibacillus polymyxa and F. oxysporum showed their drought stress mitigating capacity in E. obtusifolius under varying water regimes. Although the concentration of some identified antidiabetic compounds in E. obtusifolius were up regulated, the mechanism involved in this observation requires further investigations. IQOQA Isifo sikashukela (Diabetes mellitus - DM) siwukungasebenzi kwezitho zangaphakathi okuhambisana nobuphezulu kwamazinga eglukhosi egazini, kuhambisana nokuphazamiseka kokugayeka kwamafutha, izakhamzimba ezingamaphrotheni nezinikimandla. IDM iyisifo esingelapheki, esingathelelani nesingelapheki ngokwemithi, esiphethe izigidi zabantu emhlabeni jikelele, kuphethe ngamazinga okugula aphezulu nokufa imbala, ikakhulu esimeni sokungakhawuki kwesifo ukhuvethe (coronavirus disease of 2019 - Covid-19). Ukusetshenziswa kwemithi eyizinqindimandla zesifo sikashukela yasentshonalanga sekudale izinselelo eziningi ngenxa yokucabangeka ukuthi kuthinta ukuphepha kwayo jikelele, ukwehluleka ukwelapha nezindleko. Iningi lemiphakathi yase-Afrika yethembele ezihlahleni eziyimithi (yokwelapha) nezingxube zayo ezinokuphilayo njengesizinda semithi ngokomphumela wesimo esingenele sezinsiza zezempilo, ukwesweleka kodokotela abelaphayo nokungameleki kwezindleko zokwelashwa. Ngalesi sizathu-ke, ingxenye yalolu cwaningo yahlola okuqukethwe ngamakhemikhali ezihlahla, ngezivikelizinhlayiya ezifakwe eshubhini lokuhlola nama-ejenti anezinga eliphezulu likashukela egazini avela kolunye uhlobo olwethembisayo nolutholakala kalula olwatholwa kusetshenziswa ukumodela kwemolekhula ngekhompiyutha. Kwabikwa ukuthi ukugayeka kokudla kwezinga lesibili, nomnyakazo wakho wezokwelapha ngemithi kuyisisekelo sokusetshenziswa kabanzi kwezihlahla emithini yendabuko. Nokho-ke, ngenxa yokuvunwa okunga okungakhethi nengcindezi yezokuvikelwa kwezemvelo, iningi lezinhlobo zezihlahla zendabuko zenani eliphezulu seziphelile kungenjalo-ke zisengcupheni. Naphezu-ke, izingxube ezinokuphilayo zivama ukukhiqizwa ngezamba ezincanyana, nezimo zokuvikelwa kwezemvelo ezikhona ziphinda zibe nomthelela wazo ogxile ezihlahleni. Ngakho-ke, ngenxa yenani lezihlahla zendabuko ngokwezimboni nakwezokwelapha, sekufakwe ngokwenhloso amasu okuzitshala nokuzivuselela ukuze zikhiqizele ngobuningi bazo nangokwefana kwezihlahla zendabuko nokuba nomthelela kohlonze nasebuningini kwemithetho esebenzayo. Ngalokho-ke lolu cwaningo lwahlola imithelela yokunye nokugonywa okuhlanganisiwe kokukhethiwe kokuhlala ezinhlayiyeni eziphilayo zokukhula, okumelana nesomiso nokukuthaza ukukhula, ukumelana nesomiso, ukwenza kahle ekwelapheni nezinguquko zokugayeka emaqabungeni e-Endostemon obtusifolius. Naphezu-ke, kwahlolwa i-ethanol (v/v) emuncwe emaqabungeni engama-80%, ye- S. cordatum (engenye yezinhlobo ezitholakala kalula nesebenzayo esukela ocwaningweni lwaphambilini) yacozululwa yaba ngokumunciwe okuncanyana [petroleum ether (PE), dichloromethane (DCM), ethyl acetate (EtOAc) namanzi], nokwamakhemikhali ezihlahla kwawo, nezivikelizinhlayiya ezifakwe eshubhini lokuhlola, nokukwazi ukulwa nesifo sikashukela. Nakuba isimuncwa i-EtOAc kwakuyiyo ecebe kunazo zonke izimuncwa ezincane zamafenolikhi aphelele, zonke izimucwa ezincane zozine ze- S. cordatum zakhombisa ukukwazi ukuthungatha okukhululekile nokunamandla nokulwa nezinga eliphezulu likashukela egazini. Ukuhlolwa kokumodelwa ngokwekhompiyutha, kwalokho okunokuphilayo okungama (34) ngokwemitheshwana yokunokuphilayo okwatholwa ohlaziyweni lwe-Gas Chromatography-Mass Spectroscopy (GC-MS) kwezimuncwa ezincane ze- PE, DCM ne-EtOAc zakhombisa ukuthi izingxube ezingama-21 kubalwa kuzo ne- andrographolide, benzylidene-iditol, cubenol ne-deoxyspergualin nebis[3,3,4,7-tetramethyl-1,3-2H-benzofuran-1-yl]-ether kwabuyisa imiphumela yamandla okubophezela ≤ -7.5 kcal/mol aphikisana nama-enzayimu e-α-amylase ne-a-glucosidase akhombisa ukuthi angakwazi ukulwa nezinga eliphezulu lesifo sikashukela. Okuqukethwe yiphysicochemical netoxicological andrographolide, benzylidene-iditol, bis[3,3,4,7-tetramethyl-1,3-2H-benzofuran-1-yl]-ether necubenol kwabikezelwa ngokukwazi ukugayeka ngokomgudusisu-mathumbu wokugayeka nokungabi nasihlungu kulandela umthetho kaLipinski wokuhlanu nomthetho weVeber. Ngalokho-ke, le miphumela ikhombisa ukuthi lezi zingxube zinganethuba lokungenela ukuba yimithi ephuzwayo. Kwahlolwa ukukwazi ukumela isomiso nokukwazi ukukhuthaza ukukhuliswa kwezinhlaka zezihlahla eshubhini lokho okuhlala kwezinye izinhlayiya zokuphilayo okwakhishwa ku- E. obtusifolius (esinye sezihlahla esilwa nesifo sikashukela esahlonzwa elingeni laphambilini). Isamba salokho okuhlala ezinhlayiyeni zokuphilayo sokukhulisekayo okungama-26 (ishuminambili lesikhunta namagciwane ayishumi nane) kwehluswa ezithweni (amaqabunga nezimpande) ze- E. obtusifolius. Lezi zinhlobo ezinokuhlala ezinhlayiyeni zalokho okuphilayo zakhombisa ukumelana nesomiso okwahlukene uma zifakwa eshubhini lokuhlola nokukwazi ukuba ngokukhuthazwa kokukhula kwezihlahla. Kwagcina sekutholwe izihlahla ezimbili ezikwazi ukumelana nokuhlukunyezwa yisomiso nokukwazi ukukhuthaza ukukhuliswa kwezihlahla kohlobo okuhlala kulo okuphilayo (Fusarium oxysporum nePaenibacillus polymyxa) kusetshenziswa amathuluzi anobumokhula. Ubugciwane obatholakala (P. polymyxa) nobukhunta (F. oxysporum) bakhombisa ubudlelwano obunokwencikana elingeni leshubhu lokuhlola elikhulisa ngakubili. Kwaphinda kwahlolwa iPaenibacillus polymyxa ne-F. oxysporum okungakunye nomgomo ohambisana nemithelela ehlusayo kokungumgcini wakho ezinhlotsheni zamanzi ezahlukene. Kwahlolwa izihlahla ezakhuliswa ngaphandle kokufakwa okuhlala kokuphilayo, ngaphansi kwezinhlobo ezintathu zokuchelela ngezinyanga ezimbili, nokusebenza kahle kokukwazi ukwelapha kwazo, ngokomzimba, ngokwamakhemikhali aphilayo nokwenza kwakho ekugayweni kokudla. Kulolu cwaningo, ukuhlukumeza kwesomiso kwaba nomthelela omkhulu ekukhuleni nokukwazi ukuvimba izinga eliphezulu likashukela kwe- E. obtusifolius. Ngakolunye uhlangothi, ukugonywa kwalokho okuhlala kokuphilayo kwavama ukukhuthaza ibhayomasi yezithombo nezimpande, okuqukethwe yiklorofili nokukhanyayo, ushukela oncibilika ngokuphelele, isikalo samanzi esinokuhambisana, okuqukethwe ngokwenziwa yiproline nesuperoxide okwenzeka ekuhlakazekeni kabili emaqabungeni e-E. obtusifolius, kanti kwehliswa ukuvuza kwe-elekthrolaythi yakho nokuqukethwe yimalondialdehyde. Ngokokunqwabelana kwamakhemikhali ezihlahla, ngenkathi okuqukethwe kwefenolikkhi kwakukhuthazwa ngukugonywa kwalokho okuhlala kokuphilayo emaqabungeni e-E. obtusifolius, okuqukethwe yiflavanoydi kwesihlahla kwakhula ngenkathi ukuncipha kwamanzi kudlanga. Okwe-EtOAc okungagayiwe okumuncwe ngomthamo okhululekile nokokuzingela okumawala kuzo zonke izinhlobo zokwelapha kwahlala kungaguqukile; ukusebenza kwe- α-glucosidase eshubhini lokuhlola kwatheleleka kabi ngaphansi kokuhlukunyezwa yisomiso esiphakathi nendawo nesadlulele, kodwa kwathuthuka ngokugonywa ngokuhlala kokuphilayo. Umehluko ekugayweni phakathi kokwelaphayo okuyishumi nambili wahlolwa kusetshenziswa i- GC-MS egxile kwezokugaya. Umphumela we bi-plot PCA waveza ukuthi ukugayeka kwesikhutha esigonyiwe kwahlukumeza ngokuphakathi kwendawo i- E. obtusifolius ehambisana kancane nezinye izihlahla ze- E. obtusifolius ngaphansi kokwelashwa okwahlukile. Ngokwengeziwe-ke, ibalazwe lokushisa kokugaya okunomehluko okuyisishiyagalombili kwakhombisa ukuthi ukwelapha okuzwela kakhulu (kwezihlahla ezigonywe ngokuhambisana ezihlukunyezwe kakhulu yisomiso) kwakhiqiza izibalo eziphezulu zezakhimaphrotheyni nama-esidi emvelo aziwa ngokuvikela izinhlayiya zezihlahla ngenkathi yokuhlukunyezwa ngokungenampilo. Okumuncwe emaqabungeni e- cordatum ne-E. obtusifolius kwakhombisa ubukhona kwezivikelizihlahla nokulwa nesifo sikashukela kulolu cwaningo. Nakuba imitheshwana esebenzayo ehlawumbiselwayo kwalezi zihlahla yabukwa kusetshenziswa uhlaziyo iGC-MS, ukwahluswa okufanele nokubalwa kwalezi zingxube kungahlolwa ezifundweni zangomuso. Naphezu-ke ezinye izinhlobo ezikhulisekayo zalokho okuhlala kokuphilayo zehluswa ezingxenyeni ze-E. obtusifolius. IPaenibacillus polymyxa ne-F. oxysporum kwakhombisa ukukwazi ukunciphisa ukukhandlwa yisomiso kwe- in E. obtusifolius okwalawulwa ngokuphakama, ukusebenza okwafakwa ekwethameleni kudinga okunye ukuhlolwa.
Enhancing phenolic compound production in medicinal plants.
(2017) Paine, Christine Susan.; Finnie, Jeffrey Franklin.; Van Staden, Johannes.
Abstract available in PDF file.
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.
Evaluation of plants used in African traditional medicine for asthma and related conditions.
(2014) Motlhatlego, Katlego Ellena.; Van Staden, Johannes.; Finnie, Jeffrey Franklin.
Traditional medicine is a form of discipline that has been applied within most South African societies with the objective of enhancing the physical and psychological health system in the country. Asthma is a complex inflammatory disease that involves the narrowing of the airways. The prevalence of asthma is increasing worldwide, and this chronic disease has been identified as a significant cause of morbidity and mortality. Asthma poses a major threat to health across the population of South Africa. The adverse effects of current treatments have encouraged the use of traditional medicine. The primary aim of the research study was to evaluate the efficacy of plants used in African traditional medicine against asthma and chest infections. This was achieved by screening Adansonia digitata, Ballota africana, Catha edulis, Datura stramonium, Pelargonium sidoides, Siphonochilus aethiopicus, Xerophyta retinervis and Zantedeschia aethiopica for their pharmacological properties against key bacteria; Staphylococcus aureus (ATCC 12600), Klebsiella pneumonia (ATCC 13883), Streptococcus pyogenes (ATCC 12344) and Haemophilus parainfluenzae (ATCC 7901) as well as the fungus Candida albicans (ATCC 10231) these microorganisms are known to cause chest infections. In the microdilution antibacterial assay, the crude extracts of the screened medicinal plants showed activity at minimal inhibitory concentrations (MICs) ranging from 0.098 to >12.5 mg/ml. In the disc-diffusion assay, only the ethanol extract of stems from Siphonochilus aethiopicus and water extract of leaves from Zantedeschia aethiopica showed zones of inhibition of 13.24 and 21.10 mm. All the other screened extracts showed no zones of inhibition, which may possibly indicate that plants were ineffective against Haemophilus parainfluenzae. One or more extracts from the tested plants were effective against one or both Gram-positive bacteria investigated in the study. There was no good antifungal activity shown in the study as the MIC and minimal fungicidal concentrations (MFCs) values were higher than 1 mg/ml. Genotoxicity of medicinal plant extracts that showed good antibacterial activity ≤ 0.5 mg/ml was evaluated using the Salmonella microsome assay without S9 metabolic activation. Two strains of Salmonella TA98 and TA102 were used in the Ames test. Most tested extracts were non-mutagenic in the Ames test except for the Siphonochilus aethiopicus roots which showed a dose dependent increase. The ethanolic crude extracts were screened in an immunological assay to determine the level of competitive binding to the receptors for the treatment of asthma and related conditions. Histamine is intimately associated with allergies. Datura stramonium flowers and fruits experienced remarkable histamine binding of approximately 97% at both concentrations (400 and 800 μg/ml). The immunological activity may be attributed to the various phytochemical constituents in the crude extracts. Ballota africana leaves and stems, Datura stramonium flowers and fruits, roots and stems as well as Zantedeschia aethiopica leaves showed excellent affinity with histamine ranging between 88 and 97% and these medicinal plants could potentially serve as a new effective antihistamine when compared to the currently available pharmaceuticals. Most of the medicinal plants tested may potentially provide remedies for asthma and related conditions such as eczema, rhinitis (hayfever), anaphylaxis, sinusitis, chronic obstructive pulmonary disease (COPD), emphysema, bronchiectasis and bronchitis.
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.
Factors influencing controlled pollination of Pinus patula.
(2002) Nel, André.; Van Staden, Johannes.
A study of factors contributing to successful controlled pollinations of Pinus patula Scheide et Deppe was undertaken. The pollen morphology of P. patula, P. oocarpa, P. greggii, P. elliottii, P. tecunumanii, P. caribaea and P. radiata was studied and the mean size of pollen grains was determined for these species. Clonal differences in pollen size within P. patula were also determined. The impact of pollen management practices on pollen viability was highlighted and a protocol for in vitro pollen viability testing of P. patula and other pine species was determined. A one percent agar solidified distilled water medium gave the best germination results after 72 hours incubation at 30 °C for a number of different Pinus species and P. patula clones. The addition of boric acid increase germination, although not significantly. The addition of sucrose to the pollen germination medium had a negative effect on pollen germination of P. patula, P. greggii and P. caribaea. Re-hydration of pollen for two hours prior to in vitro germination testing improved germination significantly. Incubation temperatures of above 38 °C were detrimental to germinating pollen grains. Stored pollen with low humidity (less than 10 %) of P. patula, P. greggii and P. caribaea could tolerate temperatures of up to 70 °C while still retaining some level of viability. The initiation and growth of the pollen tube was also studied and differences in pollen tube-lengths germinated at 30 °C for 72 hours were found between species studied. Flowering of different P. patula clones was monitored over seven seasons. Flowering periods varied in length between 4 and 14 days amongst five clones over the different seasons. The best cone-survival after controlled pollination was achieved with breathable micro-fibre material. Seed yields were also highest when breathable material was used for controlled pollination. The role of pollen viability in controlled pollination was also determined in pollination studies with low viability resulting in low cone survival and low seed yields. The temperature and relative humidity inside isolation bags were monitored and temperatures above 40 °C were reached inside bags constructed of nonbreathable material. These temperatures were lethal to pollen germinating in vitro. Relative humidity of between 80 and 100 % was maintained in non-breathable bagging material, constituting a risk of diseases causing cone-mortality. The application of fungicide before, during and after controlled pollination was ineffective in improving cone survival.

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