Browsing by Author "Van Staden, Johannes."

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Abscisic acid and other hormonal effects on growth in Spirodela.
(1969) Van Staden, Johannes.; Bornman, Chris H.
The effects of abscisic acid In particular, as well well as gibberellic acid and the cytoklnlns, 6-benzyladenine, kinetin, and 6-dlmethylal lalylamlnopurine, on the growth of Spirodela oligorrhiza were investigated. Abscisic acid effectively arrested growth permanently at concentrations down to 10¯¹ mg/I. Normal growth tended to be resumed at concentrations of 10¯² and 10¯³ mg/l between nine and twelve days after treatment. A concentration of 10[-8] mg/l, however, resulted in a significant increase in dry weight at both eight, nine and twelve days after introduction into the culture medium. It is suggested that the resumption of growth twelve days after treatment at those concentrations which inhibit growth up to nine days, was due to a possible progressive inactivation of abscisic acid resulting in a lowering of its concentration to a level that is promotive. It was furthermore found that the growth response of Spirodela in terms of dry weight production over a period of eight days is proportional to the log[10] concentration of abscisic acid. It is suggested that this curve can be used as a relatively reliable and easily performed bioassay to detect amounts of abscisic acid as low as 10[-5] μg. The assay is more reliable over the range 0.01 to 10,000 μg and appears not to be affected by gibberellin, benzyladenine and kinetin. The inhibitory effect of abscisic acid on growth in Spirodela was shown to be reversed by benzyladenine, kinetin and dimethylallalylaminopurine, although they were not equally effective in doing so. Benzyladenine at 1.0 mg/l was the most effective In overcoming growth inhibition by abscisic acid. Gibberellic acid, however, proved ineffective in reversing the inhibitory effect of abscisic acid on Spirodela oligorrhiza. The apparent Increases in growth obtained in some cases may have resulted more directly from gibberellic acid stimulation than from the Interaction of gibberel lie acid with abscisic acid.
Achene biology and the chemical control of Chromolaena odorata.
(1985) Erasmus, Daniël Jacobus.; Van Staden, Johannes.
Abstract viewable in PDF document.
Adventitious rooting in stem cuttings of Eucalyptus grandis Hill ex Maid.
(1988) Wilson, Philip John.; Van Staden, Johannes.
Adventitious rooting in stem cuttings of Eucalyptus grandis Hill ex Maid. was thought to be influenced by a putative inhibitor. In previous studies it has been usual to infer the presence of putative rooting inhibitors and promoters from the mung bean bioassay, but the possibility was raised that treatment responses in this assay could be mediated more by the concentration of the treatment solution than by the chemical identity of the solute. This appeared to be so: several solutes, including hydrochloric acid and common salt, were found to promote the rooting of mung bean cuttings when present in the treatment solution at an apparently injurous concentration. The concept of promoters and inhibitors of adventitious rooting, as constituted at present, was considered to be an unfavourable approach for further studies. Stem cuttings must contain a morphogen, broadly defined, which operates the 'switch' from stem to adventitious root. The leaves and buds of E.grandis stem cuttings did not appear to be sole sources of a morphogen (as is often assumed), but nevertheless the activity of the leaves and buds was good for rooting. This activity was reflected in the pattern of root emergence. A slight preponderance emerged from the leaf trace sectors of the stem, suggesting that the leaves and buds cause a morphogen (of unknown origin) to circulate in the cutting. The existence of a vascular morphogen was confirmed and it proved to be very mobile in the stem, suggesting that it is well distributed circumferentially at the base of the cutting rather than confined to the leaf trace sectors. It appeared to be super-abundant at the base of easy-to-root cuttings, but it was not possible to tell to what extent the morphogen was rendered accessible to the sites where roots initiate. In general, the rate of efflux from the transporting tissues, the rate of attenuation of the morphogen after efflux, and the number of potential sites for root initiation must interact on a small scale to determine rooting ability. The relative prominence of these groups of factors would be expected to vary with circumstances, for example at different locations within a single stem cutting, so the traditional concept of a limiting morphogen ('rhizocaline') is unhelpful in its simplest form. Nevertheless, the rhizocaline concept provides a starting point towards a more comprehensive view of adventitious rooting, which is required in order to predict and improve rooting ability. This view remains a remote objective because many of the factors which could be important have recieved very little attention and will be difficult to elucidate.
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.
Another culture of Solanum genotypes.
(1995) Liebenberg, Denise.; Van Staden, Johannes.
Being the third most cultivated crop in South Africa, potatoes are of great economic importance. As potatoes originated from cooler areas in the world, they do not easily adapt to South African conditions. The main objective of potato breeding is, therefore, to extend the crop's limited genetic base. Progress in crop improvement is slow due to dominance, segregation and other factors caused by the tetraploid character of cultivated potatoes. A new breeding program for rapid progress has been initiated at the Vegetable and Ornamental Plant Institute, Roodeplaat, South Africa, which comprises the combination of conventional and unconventional breeding techniques. The program is based on the reduction of the ploidy level from the tetraploid to the dihaploid level to facilitate crossings with diploid wild species. Anther culture is the preferred technique for the rapid reduction of the ploidy level and has been successfully applied on different members of the Solanaceae. Cultivated potato, Solanum tuberosum is, however, an important exception. In this study various potato genotypes (tetraploid cultivars, dihaploid breeding lines and a diploid wild species) were used in experiments concerning microtechniques, alternative culture methods and medium manipulation. The main objectives were to evaluate and compare the androgenetic ability of the various genotypes used and to try and identify the factors limiting their in vitro response. Regarding microtechnique, the study focussed on the investigation of the frequency of androgenesis - as a function of plant age - and the determination of defined flower bud lenqths representative of the correct microspore developmental stage for optimal androgenetic response. Combined with an extensive histological study on the microspore development within anthers, from the time of flower selection, after a cold-pretreatment and at various time-intervals during the culture period of 42 days, the following conclusions were reached: In vitro androgenetic response proved optimal when flowers of responsive genotypes were selected during the first seven to 21 days of the flowering period. Both microspore derived embryoid- and callus development were visible within responsive anthers after a culture period of only seven days. The flower bud length required for anthers to be in the optimal stage of microspore development, e.g. the uninucleate stage, varied between the different genotypes but could readily be determined with the DAPI (4,6-diamidino-2- phenylindole) technique. It was also concluded that anthers of the tetraploid cultivar Atzimba should be selected later, between the late-uninucleate and the early-binucleate developmental stages. This suggested a limited selection period for Atzimba anthers, as starch depositioning - which prevent embryogenesis - occurs within anthers during the binucleate stage. Histologically, Atzimba showed limited embryoid development with no embryoid release, while the diploid wild species, S. canasense, proved androgenetically unresponsive. Alternative culture methods were applied to study the effect of different culture phases (liquid, double layered and agar solidified) and anther orientations (lateral, dorsal and ventral) on the androgenetic response of the potato genotypes used. Liquid cultures, based on the so-called shed-pollen technique, enhanced the androgenetic response of the tetraploid cultivar Atzimba. Optimal embryogenesis was obtained for responsive breeding line 87.2002/3 with the utilization of agar solidified media, with maximal response when anthers were cultured in the lateral orientation. No response was observed from S. canasense. The effect of medium manipulation on the androgenetic response of the three genotypes was investigated. The utilization of various combinations of different concentrations of indole-3-acetic acid (1M) and benzyladenine (BA), the alteration of the initial time of incubation of anthers on the initiation media and the use of media without growth regulators compared to that containing gibberellic acid (GA[3]), were investigated. BA had to be present in the initiation media and had a major, though not exclusive, effect on embryogenesis compared to 1M. The optimal BA concentration varied between the two trials. IAA also had an increasing effect on anther response, both in the absence of BA and, especially, in addition with relatively high BA concentrations. In this experiment, only breeding line 87.2002/3 responded. The initial culture of anthers, during the first seven to 21 days of the culture period, on media containing growth regulators proved essential for microspore derived embryoid production in the tetraploid cultivar Atzimba. As these growth regulators are metabolized in the culture media, the regular transfer at shorter, two-weekly intervals to media containing metabolically active substances, proved important. GA[3] had no enhancing-effect on embryogenesis in any of the three tetraploid cultivars. The results obtained in this study suggest that the first 21 days is the critical stage in the anther culture period in terms of the optimal time for flower selection, embryoid induction and the increase in embryogenetic response due to growth regulator influence. It is important to pre-determine the developmental stage when most microspores were in the uninucleate stage of development and to correlate this stage with a specific flower bud length. This would assure maximum response of those genotypes amenable to anther culture. It also implies a more practical and economical starting pOint to anther culture experiments. Following the determination of microspore developmental stage and pollen fertility, flowers should be selected from the donor plants only during the first three weeks of the flowering period. The composition of the nutrient media used for potato anther cultures were sufficient with respect to growth regulators. The growth regulators SA, IAA and the amines glutamine and asparagine had to be present in the initiation media, especially during the first three weeks of the culture period. As microspore development within anyone anther was found to be asynchronous, the regular transfer of anthers to fresh media is recommended to assure proper development of all microspores. The use of a slightly higher IAA concentration could be considered, but care should be taken as too-high concentrations would induce callus production. Microspore derived embryoid production is preferred, as the ploidy level of callus derived plantlets normally varies and somaclonal variation can occur. Liquid media should be considered for anther culture of tetraploid genotypes, while embryoid production can be increased by culturing the anthers of responsive genotypes on agar solidified media on the lateral orientation. Finally, the diploid wild species S. canasense seemed androgenetically unresponsive, or the media and culture conditions used did not satisfy the specific requirements of this genotype. Androgenetic amenability should first be transferred by means of interspecific crossings with a responsive dihaploid genotype, such as the breeding line 87.2002/3.
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.
Anti-inflammatory and anti-bacterial activity of South African Erythrina species.
(2000) Pillay, Candice Claudia Natasha.; Van Staden, Johannes.; Jäger, Anna Katharina.
An investigation was undertaken to determine whether Erythrina species indigenous to South Africa contained the same type of compounds as Erythrina species not found in South Africa and to determine whether they displayed any anti-inflammatory and antibacterial activity. Phytochemical analysis was conducted using thin layer chromatography. A great similarity was found in the leaf profiles of the species being studied. The leaf and bark extracts of E. caffra and E. lysistemon appear to have similar profiles when viewed under normal light and ultraviolet light, (254 and 366 nm). These two species have similar banding patterns when stained with fast blue reagent for flavonoids and potassium hydroxide reagent for coumarins. The five species that were tested appear to contain alkaloids, flavonoids, coumarins and triterpenes just like the species not found in South Africa from this genus. Dried bark and leaves from E. caffra, E. humeana, E. latissima, E. lysistemon and E. zeyheri were screened for anti-inflammatory and anti-bacterial activity. Ethanol, ethyl acetate and water extracts were screened for both anti-inflammatory and anti-bacterial activity. The cyclooxygenase bioassay was used to test for anti-inflammatory activity. The ethanol and ethyl acetate extracts generally displayed activity while the water extracts displayed no activity for both the bark and the leaves. The bark generally displayed more cyclooxygenase inhibitory activity than the leaves. The bark of E. caffra and E. lysistemon displayed the highest cyclooxygenase inhibitory activity. The disc diffussion bioassay was used to screen for anti-bacterial activity. Anti-bacterial activity was only detected in the water extracts of the leaves. The water extracts of the bark showed very little or no activity. The bark yielded more anti-bacterial activity than the leaves. Anti-bacterial activity was mainly displayed against Gram positive bacteria. The bark of E. caffra and E. lysistemon displayed the highest anti-bacterial activity. On the basis of the screening results it was decided to use bioasssay guided fractionation in an attempt to isolate putative anti-inflammatory and anti-bacterial compounds. A hexane extract from the bark of E. lysistemon was prepared and purified using a range of chromatographic methods. Vacuum liquid chromatography, separation using a chromatotron, thin layer chromatography and high performance liquid chromatography were used to isolate anti-inflammatory compound(s). The isolation proved to be unsuccessful as the pure compound had no cyclooxygenase inhibitory activity. It was subsequently determined that the compounds were lost during the HPLC procedure. An ethanolic extract of the bark of E. Iysistemon was purified in an attempt to isolate an anti-bacterial compound(s). Vacuum liquid chromatography and separation using the chromatotron was used to purify the crude extract. The more sensitive microtitre bioassay was used to test for anti-bacterial activity against S. aureus. The isoflavone, Wighteone was isolated.
Antidiabetic and phytochemical properties of four selected medicinal plants.
(2019) Ratsoma, Manchela Francinah.; Van Staden, Johannes.; Finnie, Jeffrey Franklin.
Abstract available in pdf.
Aspects of structure, growth and morphogenesis in a new filamentous red alga (Ceramiaceae, Rhodophyta)
(1993) Stirk, Wendy Ann.; Van Staden, Johannes.
Pteroceramium, a descriptive name given to an undescribed winged species closely related to Ceramium, has uniaxial filamentous thallus construction with pseudodichotomous branching. Alternate branches become dominant. This pattern of growth is referred to as cellulosympodial growth. All growth is from an apical cell which cuts off subapical cells. The subapical cells develop into axial cells. Each axial cell cuts off six pericentral cells in a ring around its apical pole. The pericentral cells divide further to form the cortical band. Pc1 always forms on the outer face of the thallus as determined by the preceding pseudodichotomy and gives rise to the larger outer wing which is a lateral expansion of the cortical band. The smaller inner wing forms from Pc6 on the inner face. The other pericentral cells give rise apically to uniseriate spines. The pericentral cells also give rise to rhizoids and adventitious lateral branches. Each axial cell has a large central vacuole with a few peripheral chloroplasts, mitochondria and floridean starch granules. The smaller wing cells have a much denser cytoplasm with fewer small vacuoles, many chloroplasts which are more closely packed together and more floridean starch granules than axial cells. Chloroplasts have a typical Rhodophyta ultrastructure with single, evenly spaced thylakoids with phycobilisomes. Pit connections have a plug core but no plug cap. Pteroceramium has a typical Polysiphonia-type triphasic life history. The carposporophyte is naked and tetraspores are produced in a characteristic decussate cruciate arrangement. The effect of a number of physical and chemical factors on growth and morphogenesis was investigated. Pteroceramium grew best at irradiance levels between 79 μmol m⁻² S¯¹ and 129 μmol m⁻² S¯¹ with growth being limited at 30 μmol m⁻² S-I. The largest axial cells and wings were obtained from the material grown at 79 μmol m⁻² S¯¹ and the smallest measurements for material grown at 129 μmol m⁻² S¯¹. Monochromatic light fields of red, green and blue caused reduced growth rates compared to the control replicates grown in a white light from both incandescent and fluorescent lights. Light quality had no effect on morphogenesis. The critical daylength for maximum rates of cell elongation was 10 hours or longer, although 16 hours light caused a decrease in final axial cell volume. Optimum temperatures for growth of Pteroceramium were between 20°C and 25°C with growth decreasing at 15°C and 30°C. Axial cell volume was reduced and wing size was stunted at these two extreme temperatures tested. Scouring by sand caused axial cells to decrease in volume although the wings were unaffected. Smothering by sand did not prevent growth although axial cells and wings were greatly decreased in size, with the wings consisting of only one or two other cells. Tumbling to disrupt gravity did not affect the angle of each pseudodichotomy. Decreased levels of nitrogen and phosphorus limited growth but had little effect on axial cell volume and wing development. Pteroceramium was stenohaline with maximum growth at 34°/[00] and reduced growth at 300/[00] and 40°/[00]. Pteroceramium grew best at pH 7.5 and pH 8.5 with decreased growth at pH 6.5 and pH 5.5. The various pHs tested had little effect on morphogenesis. The best photosynthetic responses were obtained from material preconditioned at 80 μmol m⁻² S¯¹ compared with that at 30 μmol m⁻² S¯¹ and 150 μmol m⁻² S¯¹. There was a decrease in pigment content with increasing irradiance at which the alga was grown. Phycoerythrin was the dominant pigment. Exposure to a high irradiance (3000 μmol m⁻² S¯¹) for 30 minutes or longer inhibited photosynthesis. Plants did not fully recover even 24 hours later, indicating that this damage was permanent. Pteroceramium was able to acclimatize slowly over a week to temperature changes within the range of 15°C to 25°C. Rapid increases of 5°C within this temperature range increased photosynthetic performance and a rapid drop of 5°C decreased photosynthetic performance. However, a 10°C increase or drop reduced Pteroceramium's photosynthetic performance. Photosynthetic rates were decreased in alkaline conditions and increased in acidic conditions. Pteroceramium has well defined developmental patterns with basal band growth of axial cells and tip growth in the rhizoids. The pericentral cells are formed in a set sequence similar to Ceramium species with Pcl forming on the outer face, Pc2 and Pc3 forming on the lower and upper surface nearest to Pel respectively, Pc4 and PcS forming on the lower and upper surface respectively farthest from Pel, and Pc6 forming on the inner face. This sequence is unaffected by the direction of illumination or gravity. Exogenous application of plant hormones (IAA, GA3 and kinetin) in the concentration range of 10[-9] M to 10[-5] M had no effect on growth and morphogenesis in Pteroceramium. Application of polyamines and their precursors caused a decrease in growth and a reduction in cell size at concentrations higher than 10[-4] M. Polyamine inhibitors caused a reduction in growth and cell size at concentrations higher than 10[-5] M. Arginine increased growth at concentrations 10[-5] M and 10[-6] M. High power liquid chromatography (HPLC) separation of Pteroceramium extracts indicated that spermidine was present in Pteroceramium at approximately 38 μg spermidine g¯¹ fresh weight. The apical tip exerts an apical dominance effect on the subordinate branches, suppressing their elongation. Removal of the dominant apical tip increased adventitious branch formation. This effect was not reversed by application of exogenous IAA at concentrations of 10[-9] M to 10[-4] M.
Aspects of the role of cytokinins in adventitious root formation.
(1996) Taylor, Joslyn Leanda Susan.; Van Staden, Johannes.
The initiation and development of adventitious roots in cuttings are highly complex processes, influenced by both endogenous and exogenous factors. These vary from the environmental factors prior to the striking of the cutting, to the anatomical and physiological factors, within the stem. Encompassed are the nutrient status, physiological age and degree of differentiation of the tissues, and the balance of endogenous rooting inhibitors and/or promoters (including hormones). The role of cytokinins in root initiation and development has been perceived as that of an inhibitor. This investigation considered several aspects of the role played by cytokinins in the process of root development. A qualitative/quantitative analysis of the cytokinin-like activity in stem cuttings of several plants, including both easy- and difficult-to-root species was conducted on a comparative basis. There was no clear correlation between the type / level of cytokinins detected in the cuttings and the relative ease of root formation. Both qualitative and quantitative changes in the compounds exhibiting activity in the soybean callus bioassay were observed over the period of root formation in Impatiens stem cuttings. The effects on root formation in cuttings of exogenously applied auxins and cytokinins were investigated. Auxins generally promoted root number and elongation at relatively high concentrations (10[-4] M), but showed less effect on lateral root initiation and development. At high concentrations, cytokinins strongly inhibited root development, but did promote lateral root growth. In suspension culture, the effect of these hormones differed slightly, with IAA and IBA having no significant effect on root development, but NAA strongly stimulating lateral root initiation. Zeatin (10¯¹¹ M) significantly increased root length and the number of lateral roots produced. The effect of treatment of the stem cuttings with potassium permanganate and centrifugation was examined. While both these treatments have been perceived to increase root production in cuttings, no significant improvement in rooting ability following centrifugation (relative to the control) was observed. Impatiens cuttings centrifuged in the presence of distilled water showed a significantly reduced rooting ability relative to those centrifuged in the dry state. Treatment with an 8-hour pulse in 0.05 % potassium permanganate significantly increased the average root length. These treatments had an effect on the cytokinin levels and distribution in the stem cuttings. Slightly higher levels of cytokinins were associated with the increase in root number and length in both experiments.
Aspects related to the germination of Themeda triandra seed.
(1996) Baxter, Brent J. M.; Van Staden, Johannes.
Themeda triandra is a grass species of economic importance in Southern and Eastern Africa, and Australia. The species is being lost from grasslands and savannas in these areas due to poor agricultural practice, rangeland degradation, opencast mining and increased afforestation. Based on the poor re-establishment of the species from seed in sub-climax grasslands, dogma holds that T. triandra can not be re-established from seed. Recent research has, however, highlighted the potential to establish this species from seed, but the use of seed of T. triandra in re-vegetation of disturbed areas is limited by poor understanding of the seed biology of the species and low seed availability. In this Thesis ways to maximise the use of available seed are reported. Areas investigated include optimisation of seed storage conditions, overcoming primary seed dormancy, promoting germination of available seed and pre-treatment of seed to improve germination. The Thesis closes with an investigation of the environmental limits of tolerance of seedlings from the T. triandra ecotypes studied, when grown under field conditions at reciprocal sites. Two altitudinally and geographically distinct populations of T. triandra were studied; a high altitude grassland population at Cathedral Peak (Drakensberg: 1800 m) and a low altitude savanna population from the Umfolozi Game Reserve (Zululand: 90 m). At seed shed T. triandra seed is dormant. The depth and duration of primary seed dormancy varies between populations, but appears to reflect severity of the winter period experienced. More than 95% of T. triandra seed from the Drakensberg population was dormant at seed shed, compared to 55% of seed from the Zululand population. In both populations dormancy is lost during dry after-ripening. At seed shed T. triandra seed displays a high level of seed viability (> 80%). Seed temperature range -15°C to 70°C, was achieved at 25°C (± 2°C), at which temperature seed was held for 40 months. During this period viability decreased from over 80% to 50% and dormancy was lost through dry after-ripening within four (Zululand) to eight (Drakensberg) months. Loss of dormancy can be accelerated at higher temperatures, but is accompanied by rapid loss of seed viability. In contrast, viability can be maintained in storage at sub zero temperatures, but loss of dormancy is retarded. Loss of dormancy coinsides with the onset of spring. Dormant seed is capable 'of germination at a narrow range of constant temperatures (25 ° C to 40 ° C). With after-ripening, the range of temperatures at which germination takes place increases (15 ° C to 40 ° C) and the optimum temperature for germination decreases from 30 ° C in both populations to 25 ° C. After-ripened seed is capable of germination at lower water potentials than dormant seed. Similarly, seed from the low altitude population is capable of germination at lower water potentials (-1.0 MPa dormant: -1.5 MPa after-ripened) than seed from the high altitude population (-0.5 MPa dormant: -1.0 MPa afterripened). Dormancy in seed from the high altitude population is overcome by prolonged stratification (30d). In contrast, seed from the low altitude population responds to short duration stratification (5d) with longer periods proving detrimental to seed germination. Germination of dormant and non-dormant seed of T. triandra does not differ significantly in the light or dark. Neither does photoperiod, or red / far-red light exposure significantly affect germination. Seed response to light and temperature, as characterised under controlled conditions, was verified in a field seed burial experiment undertaken at the high altitude Drakensberg site during winter. Burial in soil does not affect the response of T. triandra seed to light or temperature. Loss of dormancy is accelerated in buried seed. After-ripened seed germinates over a wider range of temperatures than dormant seed. The mechanisms governing T. triandra seed dormancy and germination appear to be universal between ecotypes. Dormancy is enforced, in part, by the seed covering structures (glumes) which impose a mechanical restraint to radicle emergence. Approximately 85% of dormant seed, however, contains a dormant embryo. Embryo dormancy is enforced at seed shed by compounds inhibitory to seed germination. The germination process in T. triandra appears to be governed by endogenous gibberellins. Bioassay results reveal that endogenous gibberellin synthesis commences up to six hours sooner in after-ripened seed than in dormant seed and that the level, or concentration, of gibberellin-like compounds is substantially lower in after-ripened seed than in dormant seed. Similarly, the concentration of applied gibberellic acid required to achieve maximum germination of T. triandra seed decreased from 500 mg.l ¯¹ (8 week old seed) to 50 mg.l ¯¹ (78 week old seed) as dormancy is lost during after-ripening. Contrary to previous reports, boron does not promote T. triandra seed germination. Plant-derived smoke significantly promotes T. triandra seed germination (5% to 43% for dormant seed from the Drakensberg population). The effectiveness of smoke in promoting germination increased with increasing seed imbibition suggesting smoke action at a metabolic level. This suggestion is reinforced by the ability of smoke to bring about the germination of seed which had failed to germinate in water. Moreover when smoke is applied in combination with gibberellic acid the final level of seed germination following combined treatment is significantly greater than the level of germination achieved in the presence of either smoke or gibberellic acid alone. A similar result is achieved with joint application of smoke and kinetin, although the results were not statistically significant. Furthermore, smoke treatment reversed ABA-induced inhibition of germination of non-dormant T. triandra, wheat, radish and sunflower seed to a level equal to or greater than that achieved using GA[3] or kinetin. The possibility that smoke promotes seed germination by mimicking, or promoting the synthesis of endogenous gibberellins was investigated. Bioassay results revealed that smoke had no effect on increasing the level of endogenous gibberellin-like activity in T. triandra caryopses. The mechanism by which smoke acts to promote seed germination remains elusive, however results presented suggest that smoke may act to remove an ABA-induced block to seed germination. Consequently, it is suggested that smoke plays a permissive role in promotion of T. triandra seed germination by removing a block to the seed germination process thereby allowing endogenous gibberellins to act. Treatments which significantly improved the level of T. triandra seed germination were evaluated as seed pre-treatments. Significant improvement in germination was obtained following smoke (aq) and gibberellic acid (100 mg.l ¯¹) pre-treatment of seed. The effects of pre-treatment were evident on germination of seed for up to 21 days after pre-treatment. Seed pre-treatment with smoke had no affect on subsequent seedling growth, but gibberellic acid pre-treated seedlings developed abnormally. In contrast, short duration exposure of dormant seed to high temperature (70 0 C for 7 days) increased germination, seedling height and tiller number. Priming of seed in polyethylene glycol (PEG 8000) for 7 days significantly improves the level of T. triandra seed germination. The use of seed pre-treatment to maximise germination of T. triandra seed is discussed. Reciprocal transplanting of seedlings from both the Drakensberg and Zululand populations confirmed that the T. triandra populations under investigation are distinct ecotypes. Field transplant gardens were established in the Drakensberg, Zululand and at an intermediate altitude in Pietermaritzburg (800m). Less than 10% of planted seedlings died at any site. With increasing altitude of the field site, tiller number increased, but tiller allocation to reproduction decreased. Similarly, for both Zulu land and Drakensberg seedling transplants the time taken to reach anthesis increased with increasing altitude and the proportion of transplants which flowered decreased. These data are consistent with the climate of the field sites where the high altitude site experiences a short growing season and harsh winter while the Zululand site experiences a prolonged growing season and mild winter period. These data indicate that T. triandra ecotypes are tolerant of a wide range of environmental variables. The application of the data presented in this Thesis, in maximising the use of available seed of T. triandra for use in re-vegetation, is discussed.
Aspects relating to the occurrence of an inhibitor of tissue plasminogen activator in Erythrina caffra thunb. plants and in vitro cultures.
(1990) Meyer, Hendrik Johannes.; Van Staden, Johannes.
A double sandwich enzyme-linked immunosorbent assay (ELISA) was developed to quantify the proteinaceous inhibitor of tissue plasminogen activator (t-PA) which occur in the tissue of Erythrina caffra Thunb. Using the ELISA the t-PA inhibitor could be detected in nanogramme quantities on the micro titer plate. The concentration of the t-PA inhibitor was determined in different tissues of Erythrina caffra. t-PA inhibitor concentrations in the order of 1 000 microgrammes per gramme protein were found in the seeds. Relatively small quantities of t - PA inhibitor, in the order of 10 to 50 microgrammes per gramme protein, occurred in root, shoot, leaf and living bark material. The t-PA inhibitor was found to accumulate in a similar way to the storage proteins in developing seeds. The accumulation of the inhibitor is at a relatively low level during the early period of seed development but increases exponentially just before the seeds reach their maximum size. The t-PA inhibitor content of the cotyledons decreased drastically during the process of germination and subsequent seedling development. The disappearance of the inhibitor be the result of total degradation of the molecule can or partial proteolysis with the modified molecule still being present in the tissue. An attempt was made to increase the t-PA inhibitor content of excised leaves of Erythrina caffra with protein inducing substances such as polyamines, precursors of ethylene and phytic acid. The protein inducing compounds included cell wall hydrolysates of Erythrina caffra, the marine alga Ecklonia maxima Osbeck (Papenfuss) as well as Lycopersicon esculentum Mill which induced the, synthesis of proteinase inhibitors suggested to be involved in the defense mechanism of plants. None of the substances used, increased the t-PA inhibitor content of excised leaves or in vitro cultures of Erythrina caffra. It is suggested that the t-PA inhibitor is probably not involved in a defense mechanism of Erythrina caffra. A callus and suspension culture derived from shoot tissue was developed to determine the occurrence of the t-PA inhibitor in vitro. The optimal nutrient medium for the growth of callus was the salts and vitamins of MURASHIGE and SKOOG (1962). The medium was supplemented with 3 % sucrose, 0. 1 gramme per litre meso - inositol, 10 micromoles per litre benzyl adenine and 5 micromoles per litre 2,4- dichlorophenoxyacetic acid . Different auxins and cytokinins had a similar growth stimulatory effect on the growth of callus derived from a number of organs of Erythrina caffra. The callus from different organs did however, grow at different rates on the same nutrient medium. Callus derived from leaf, shoot, and cotyledonary tissue grew at similar rates on the nutrient media of MURASHIGE and SKOOG (1962), SCHENK and HILDEBRANDT (1972) and B5 (GAMBORG, MILLER and OJIMA, 1968) despite large differences in the concentration of the nutrients in the three nutri.ent media. The source of nitrogen and ratio of nitrate to ammonium was critical to the growth of callus cultures . The optimal concentration of nitrate and ammonium was 30 millimoles per litre . The growth of callus from different organs was significantly affected by the concentration of sucrose in the nutrient medium. A concentration of 3% was optimal for callus growth. Temperature had a significant effect on the growth of callus. The optimal temperature for callus growth was 25 °C. A shoot cell suspension culture was established and maintained at the same temperature and on the same medium as the callus cultures but with a ten times lower concentration of growth regulators. A low shake speed was essential for the growth of the suspension culture. Maximum growth was obtained at a shake speed of 60 rpm. Relatively low quantities of t-PA inhibitor, in the order of 1 to 5 microgrammes per gramme protein, was detected in the suspension cultures. An attempt was made to increase the t-PA inhibitor content of the suspension cultures with the pro te in i nduc i ng compounds used on excised leaves, but without success. However, the t-PA inhibitor content of the suspension culture was significantly increased with a ten times increase in the sulphate content of the nutrient medium.
Bark in traditional healthcare in KwaZulu-Natal, South Africa : usage, authentication and sustainability.
(2002) Grace, Olwen Megan.; Jäger, Anna Katharina.; Prendergast, Hew D. V.; Van Staden, Johannes.
Healthcare in South Africa is polarised between western and traditional African systems of therapy. The latter is consulted by the majority of the population and therefore plays an integral role in the delivery of healthcare to South Africans. Traditional medicines are primarily plant products with long storage lives, among which the dominance of bark is typical of southern African traditional healthcare systems. Expansion of the traditional healthcare sector during the twentieth century, in response to rising consumer demands, stimulated a lucrative trade in medicinal plants that is centred in KwaZulu-Natal. Since herbal medicines are sourced almost exclusively from indigenous vegetation, harvesting pressures exerted on the indigenous flora to meet demands for traditional medicines have rendered such resources non-sustainable. Although trees comprise a small fraction of South African medicinal plant species, bark from them constitutes a substantial proportion of the plant products used medicinally. Trees are among the most threatened medicinal plants in South African due to their limited abundance, the ecological sensitivity of the vegetation in which they occur, and destructive methods of commercial bark harvesting that frequently take place within protected areas. In KwaZulu-Natal, bark is harvested primarily from forests that occupy an extent of only 0.1 % in the province. Conservation of economically valuable tree species is particularly problematic since data necessary for the establishment of sustainable usage systems are absent or inaccessible. Alternatives to in situ conservation for renewable bark resources include propagation, multi-use timber systems and reintroduction of locally extinct species. To facilitate appropriate management of bark resources, there is a need for specialist publications and consolidated data with which sustainable usage levels may be determined. The importance of bark in South African traditional healthcare is poorly reflected by the ethno botanical literature. In this study, 180 bark species used in traditional healthcare in KwaZulu-Natal were inventoried from thorough literature surveys, but this number is anticipated to be a conservative reflection of actual statistics. Where trade data were recorded in the literature, they indicated intensive exploitation of bark resources in KwaZulu-Natal and throughout South Africa, but conservation and management data were lacking for 72 % of the species inventoried. A number of problems were encountered in the literature, of which vague information and the documentation of local vernacular nomenclature were the most troublesome. Despite the importance of traditional medicine, the country's political history led to the prevailing situation, where the traditional healthcare sector is largely unregulated. Coupled with increasingly limited availability of medicinal plants, the quality and appropriate use of traditional medicines is negatively affected by growing numbers of inadequately trained practitioners, herbalist retailers and plant gatherers. Possibilities of misidentification or purposeful adulteration of medicinal bark products therefore lead to concerns for patient safety, since dried bark is difficult or impossible to identify. Whilst bark characters are useful for field identifications, many useful diagnostic characters are lost through desiccation, and anatomy and morphology of bark are variable. Additionally, medicinal bark products used in KwaZulu-Natal, and their identification, are largely undocumented. This study focussed on eight bark species used medicinally in the province, elected by an esteemed traditional medical practitioner as having problematic identity. Monograph-type characterisation profiles were drawn up for reference specimens collected from various localities, and their medicinal bark products traded under vernacular names recorded in the literature. In the absence of standardised traditional medicines, there is a need for reliable and affordable methods for their authentication. Phytochemical bark characters identified by Thin Layer Chromatography (TLC) have proved useful in chemotaxonomic studies, and the technique is widely used for herbal drug authentication. TLC was tested here for authentication of medicinal bark products from the aforementioned study species. Three reference samples of each species were collected, and TLC-generated fingerprints compared. At the intraspecific level, TLC was useful in confirming the relationship of ethanol and hexane bark extracts, but was less meaningful in distinguishing between fingerprints of different species. Three medicinal bark products of each study species were purchased and fingerprints compared to a reference. The technique proved useful in confirming the identity of several medicinal bark products. Authentication of medicinal bark products may be useful in toxicology cases and in the accurate documentation of their trade. This research identified a complexity of issues surrounding the use of bark in traditional healthcare in KwaZulu-Natal, and indeed South Africa. A multi-faceted approach is required to secure their sustainability. Critical, however, to factors such as effective conservation and regulation of the traditional healthcare sector, is recognition of the importance, and documentation, of traditional bark medicines. The integrity of traditional healthcare, and the future of the South African flora, hinge upon the sustainable use of medicinal products such as bark.
The biochemical and cytokinin changes in the developing and germinating seeds of Podocarpus henkelli stapf.
(1981) Dodd, Malcolm Caulton.; Van Staden, Johannes.
A review of the literature revealed that there is a lack of depth in our knowledge of gymnospermous seeds with regard to the development and germination processes. The phytohormones, particularly the cytokinins have been implicated in these processes. The seeds of Podocarpus henkelii were thus selected as experimental material for studying the biochemical and cytokinin-like changes associated with development and germination. The development of these seeds was also followed at the ultrastructural level. These studies revealed that cellular detail within the female gametophyte only began .to form in December (early summer), approximately six weeks after fertilization had taken place. At this time some reserve protein was evident and the embryo sporophyte consisted of only a few pro-suspensor and pro-embryo cells. Concurrently, the cytokinin levels were fairly high in the female gametophyte but low in the epimatium. In both seed components two cytokinin- like compounds predominated which co-chromatographed with the free base cytokinin zeatin and its ribonucleoside. The second sample was taken in late January (mid-summer) by which time the embryo sporophyte had developed rapidly into a readily distinguishable seed component. The cellular detail indicated that much cell division had recently taken place and that the cells were currently increasing in size and accumulating starch and lipid. In the female gametophyte the soluble sugars were at the maximum level recorded during these experiments and the level of starch was increasing. The extractable cytokinin content of the seed was high at this time, particularly in the embryo sporophyte. In all three seed components cytokinin-like compounds which co-chromatographed with zeatin and ribosylzeatin were present. These high levels of cytokinin coincided with the rapid increase in both fresh and dry mass of the embryo sporophyte and female gametophyte. Ultrastructural studies of the third sample collected in mid-March (early autumn) showed that cellular changes were associated mainly with increases in cell size and the accumulation of food reserves, particularly starch. The cytokinin levels had decreased in all three seed components at this time. There was an increase in the cytokinin which co-chromatographed with glucosylzeatin in the female gametophyte. The seeds matured in late April (autumn) and had the unusual features of not drying out during maturation. Fresh seeds collected from the ground had a moisture content of ca. 62 per cent. The main food reserve was starch with relatively small amounts of protein and lipid also present. The seeds of Podocarpus henkelii germinated readily after scarification in the absence of water provided that their moisture content remained ca. 60 per cent. Seeds in which the moisture content had fallen below ca. 54 per cent required additional water for germination. The moisture content of the seeds fell rapidly under natural conditions and viability was lost below a moisture content of ca. 34 per cent. Unscarified seeds of 52 per cent moisture content placed under moist conditions at a constant 25°C took 23 weeks to achieve 68 per cent germination. These experiments showed that although the epimatium limited water uptake by the seeds it did not prevent moisture loss to the atmosphere. This appears to be the main factor contributing to the seed's inefficiency as a propagule. A small degree of after-ripening was recorded with the embryo sporophyte increasing in size with storage. This appeared to contribute to the increased rate of germination of the scarified seeds. An interesting feature of the seeds of Podocarpus henkelii is that they have the ability to fix atmospheric carbon, which is subsequently translocated from the epimatium to the female gametophyte and embryo sporophyte. The mature seeds were stored at 4°C for six weeks during which time little change had occurred at the ultrastructural level. Protein vacuoles in the embryo sporophyte had disappeared and in all three seed components cytokinin levels were low. Three days after scarification and the start of incubation, little change in cellular detail was apparent as limited rehydration was necessary due to the high moisture content. The cytokinin levels in the embryo sporophyte and epimatium had increased, whilst the levels in the female gametophyte had decreased at this time. In the embryo sporophyte lipid mobilization had commenced with these reserves apparently being metabolized within vacuoles. The rate of respiration measured in terms of increases in CO[2] evolution, increased 60 hours after the start of the incubation period, just 12 hours before ten per cent germination was recorded. Germination was accompanied by a large increase in the levels of cytokinins in the female gametophyte and embryo sporophyte. The cytokinins detected co-chromatographed with the free base cytokinin zeatin and its riboside, ribosylzeatin. Concurrently, marked ultrastructural changes were recorded with increases in the amounts of dictyosomes, endoplasmic reticulum and the formation of polyribosomes, all of which are indicative of increased metabolic activity. Similar increases in the female gametophyte were ofa lower order and occurred only after nine days of incubation. By this time the levels of cytokinins had decreased considerably. After 12 days of incubation 65 per cent of the seeds had germinated. As much of the food reserves in the female gametophyte and embryo sporophyte remained, it is suggested that these reserves are utilized for subsequent seedling establishment rather than for germination. The actual role that cytokinins play in the development and germination of these seeds is not clear. High levels of this phytohormone coincide with periods of food deposition and mobilization suggesting that they play an important part in these processes. The results of the biochemical, cytokinin and ultrastructural studies are discussed in relation to the developmental and germination processes and are compared to the data of other seeds.
Biochemical, physiological and agronomic response of various sweet potato cultivars/varieties to drought stress in rainout shelters and field conditions.
(2014) Laurie, Robert Naylor.; Van Staden, Johannes.; Finnie, Jeffrey Franklin.
Drought is and will always be an issue in the cultivation of plants. Some plants have the ability to withstand a drought conditions to a certain degree while others, with other useful attributes, fail dismally. The value of testing genotypes for the ability to tolerate drought cannot be underestimated and will enhance the progress in the selection of drought tolerant genotypes. Thus, the objective of this study was to investigate the physiological, biochemical and agronomical reaction of sweet potato plants to drought and the procedures which could be used to test for sweet potato drought tolerance in the field. This was made possible through the creation of an environment at ARC-Roodeplaat in which sweet potato plants could be subjected to drought stress conditions. Thirty five sweet potato genotypes were planted in three trials in rainout shelters and open fields to analyze their physiological, biochemical and agronomical responses to drought stress. The majority of the genotypes were selected breeding lines with some cultivars from America, Peru and South Africa. These genotypes were chosen due to their range of traits for incorporation in crosses in the sweet potato breeding programme of the Agricultural Research Council (ARC). Drought stress conditions on the plants were induced through selective irrigation practices. In Trial 1 control plants were cultivated at field capacity while drought stressed plants received 60% and 30% of the amount of water of the control, respectively. In Trial 2, genotypes were planted in the field and under rainout shelters respectively. The field plantings acted as the control and received normal rain and irrigation while the rainout shelter planting received irrigation corresponding to 30% of field capacity. The plants in Trial 3 were subjected to control and drought conditions with the drought stressed plants receiving 30% of the water of the control. Leaf harvesting and phenotypical measurements were conducted twice during the trial period i.e. 60 and 120 days after planting. The drought stress impacted the growth of the sweet potato plants significantly. Canopy cover and stem length were severely influenced by the drought stress and resulted in huge declines of the respective values in all trails. Canopy cover values declined by more than twice compared to the control while stem length values were reduced by up to 10 times compared to the control. Antioxidant systems with particular reference to ascorbate peroxidase (AP), super oxide dismutase (SOD) and glutathione reductase (GR) reacted to the stress imposed and increased significantly. It was observed that values of the respective antioxidant enzyme systems increased sharply in the latter part of the trial and that the increase was also more intense at severe stress. The analysis of the antioxidant system made it possible to distinguish between the genotypes regarding their reaction to the stress. Results for carbon discrimination experiments in all the trials indicated that a significant decline in values took place as the drought stress increased. The decline appeared to be slightly more pronounced as the stress progressed. Also, as in the case of the antioxidant systems, it was possible to distinguish between genotypes even in the control treatments. The plants responded to the drought stress to the effect that a similar trend, (compared to the antioxidants), was observed with regards to stomatal conductance although genotypical differentiation was not possible in any of the stress conditions. It was demonstrated in the trials that the relative water content (RWC) values in the leaves of plants subjected to drought stress declined significantly between water treatments. Drought stress in the three trials had a severe impact on the nitrate reductase (NR) activity in the leaves of the plants. The decline in values were substantial but no significant differences could be detected between the genotypes except for the breeding line 2005-1-16 and cultivars Purple Sunset, Beauregard and Zapallo. Slight non-significant differences were observed between the genotypes at mild stress conditions but the severe stress conditions proved too harsh. Significant increases in the proline content of the sweet potato plants subjected to drought stress resulted in differentiation between the genotypes in Trial 1 and Trial 2, especially during the latter stages of the trials and at severe stress. Large reductions, up to 97%, of root yield were detected in the three trials. It appeared that the severe stress treatment proved too harsh to accomplish significant differences between the genotypes in all the trials. In Trial 1 the genotype Resisto differed significantly from the other genotypes and seemed to tolerate the drought the best in the mild stress conditions. Water use efficiency (WUE) values did allow for discrimination between the genotypes in Trial 1. A large decline in WUE values were observed in Trial 2 in general, although a few breeding lines 2005-7-4, 2006-4-4 and ix 2006-7-7 were prominent with high WUE values and could be recommended for use in a breeding programme. In Trial 3 the cultivar Bophelo and 199062.1 also exhibited higher WUE values which correlate well with yield data obtained from the same Trial. This could also prove valuable in the selection process. Due to the fact that multiple traits make a valuable contribution to the decision-making process in the selection for possible screening methods, statistical correlation was undertaken to establish possible relationships between traits. Good correlation was found between yield, stomatal conductance and WUE in Trial 1. This confirmed the assumption that a drop in stomatal conductance will result in lower root yield. Proline correlated also very well with the antioxidant enzyme levels of GR and AP which indicates that while the antioxidant enzymes play a role in combatting oxidants proline aid in possible prevention of moisture loss and stabilization of cell membrane structures. In Trial 2 good correlation was observed between yield, LAI, NR and CCI and to a lesser extent carbon-13 discrimination. This confirmed the belief that a decrease in LAI and CCI should have a negative effect on the yield due to less canopy cover and less chlorophyll for the capture of sunlight for photosynthesis. Results from Trial 3 also indicated good relationships between proline, GR and AP, as well as good relationships between yield, WUE, carbon discrimination and stomatal conductance (gs). It can hereby be concluded that the reaction of sweet potatoes to drought stress revealed results that can be of help for use in the future to successfully establish a protocol whereby successful selection of genotypes can be made in a biochemical, physiological and agronomical way. The study also provided proof that some of the approaches and procedures used in these trials can be successfully implemented in the drought screening of sweet potato.
Biological activity of traditional medicinal plants used against venereal diseases in South Africa.
(2006) Buwa, Lisa Valencia.; Van Staden, Johannes.
Throughout the history of mankind, many infectious diseases have been treated with plant extracts. Venereal infections are one such group and are regarded as conditions that are highly responsive to traditional treatment. Aqueous, ethanol and ethyl acetate extracts of 13 plants used in South Africa for the treatment of venereal diseases were screened for in vitro antibacterial, antifungal, mutagenic and antimutagenic activities. Antibacterial activity was evaluated using the disc-diffusion and microdilution assays to determine the minimal inhibitory concentration (MIC) values of the extracts. The extracts were tested against the Gram-positive bacteria Bacillus subtilis and Staphylococcus aureus, and the Gram-negative bacteria Escherichia coli and Klebsiella pneumoniae. Among the plants tested, Gunnera perpensa, Harpephyllum caffrum, Hypoxis latifolia and Ledebouria ovatifolia showed the best antibacterial activity. The aqueous rhizome extract of Gunnera perpensa displayed good activity against Gram-negative bacteria with an MIC value of 0.78 mg/ml, and against S. aureus (0.78 mg/ml). Aqueous and ethanol extracts of H. caffrum bark were active against both Gram-positive and Gram-negative bacteria. Hypoxis latifolia aqueous corm extracts exhibited very good MIC values against K. pneumoniae (0.78 mg/ml), E. coli and S. aureus (1.56 mg/ml). Ethanol and ethyl acetate bulb extracts of Ledebouria ovatifolia displayed good activity against Bacillus subtilis bacteria with MIC values of 0.78 mg/ml and 0.39 mg/ml respectively. Antifungal activity was evaluated using the microdilution bioassay. Good activity was shown by the ethanolic bark extracts of Bersama lucens and Harpephyllum caffrum against Candida albicans. Only in the case of Harpephyllum caffrum did aqueous extracts have activity against Candida albicans. In the Ames test, all plant extracts showed a negative genotoxic response except for ethanol and ethyl acetate bulb extracts of Cyrtanthus obliquus which induced mutations in TA98. Moderate antimutagenic activity was observed with the ethyl acetate extract of G. perpensa and the ethanolic extract of H. latifolia. High antibacterial and antifungal activity detected with Harpephyllum caffrum bark extracts resulted in an investigation on seasonal and geographical variation of this inhibitory activity. Seasonal variation in antibacterial and antifungal activities was investigated in order to determine the best collection time to ensure potential high medicinal activity in plant preparations. The highest inhibitory activity was detected with plant material collected in June and December 2003, with a decline in activity when collections were made in September 2004. The chemical profiles of TLC chromatograms were compared and little variation was found, particularly in the case of plant material obtained from the Botanic Garden of the University of KwaZulu-Natal and a 'Muthi' Shop in Pietermaritzburg. Identification of active compounds from G. perpensa and H. caffrum was not successful due to insufficient amounts of isolated fractions.
The biosynthesis and production of hypoxoside in Hypoxis hemerocallidea Fisch. and Mey. in vivo and in vitro.
(1989) Bayley, Arlene Diane.; Van Staden, Johannes.
Hypoxoside, a phenolic diglucoside, with a diarylpentane-type structure, is thought to be the medicinally active constituent of corm extracts of Hypoxis hemerocallidea Fisch. & Mey. which are reputed to alleviate the symptoms of prostate hypertrophy and urinary infections. The biosynthes is and production of this unique phytochemical were investigated in H. hemerocallidea using both in vivo and in vitro systems. It was found, in root-producing callus, that [l4]C-phenylalanine and [14]C-t-cinnamic acid were efficient precursors for hypoxoside in comparison to [14]C-sodium acetate and [14]C-acetyl coenzyme-A, which were not incorporated into the phenolic compound. Thus, at least one aryl moiety of hypoxoside was derived, via phenylalanine and t-cinnamic acid, from the shikimate pathway. The acetate pathway did not appear to be involved in the biosynthetic process. The data supports the hypothesis that the molecule is formed from two cinnamate units with the loss of a carbon atom, in opposition to the proposal that the molecule is derived from head-to-tail condensation of acetate units onto a propenylic moiety. Despite the structural similarities between hypoxoside and caffeic and p-coumaric acids, these two hydroxycinnamic acids were not efficient precursors for hypoxoside in vivo or in vitro. A number of reasons are put forward to explain this finding. It was found that the greatest concentration of hypoxoside was located in the corms of intact plants. The major biosynthetic site of the molecule was also found to be located in this organ. Since the roots did accumulate the phytochemical to a small extent, the biosynthetic potential of these organs has not been disregarded. That of the leaves has been, however. The report by PAGE (1984) that the upper region of the corm contained a greater con cent ration of hypoxoside than the lower portion, is substantiated in this study, where this region was found to be more biosynthetically active than the lower half. Light microscopic and electron microscopic studies revealed that starch storing cells, which accumulated phenolics in their vacuoles, contained seemingly synthetically active tubular endoplasmic reticulum in their cytoplasm. A greater number of these cells were concentrated in the upper region as opposed to the lower half of the corm. It is postulated that these cells are the site for biosynthesis and accumulation of hypoxoside. The shikimate pathway, from which the precursors for hypoxoside are derived, was found, through the exposure of intact plants to [14]C-carbon dioxide, to be located mainly in the leaves. It is postulated from the above study and one in which [14]C-phenylalanine, [14]C-t-cinnamic acid, [14]C-p-coumaric acid and [14]C-caffeic acid were applied to intact plants, that phenylalanine and/or cinnamic acid are the transported form of the shi kimate derivatives. p-Coumaric and caffeic acids, which are metabolically more stable, are envisaged to be the sequestering forms. The investigation of the seasonal production of hypoxoside revealed that most of the synthesis and accumulation occurred after the corms had broken winter dormancy and after the flush of leaf growth had slowed down. During dormancy the production of hypoxoside appeared to cease. The in vjtro studies, where the effects of light, temperature, nutrients, plant growth regulators and supply of potential precursors, on hypoxoside production by root-producing callus were investigated, indicate that this metabolite is not simply a "shunt" metabolite. A number of factors other than precursor availability enhanced, or reduced the jn vjtro production of this phytochemical. Furthermore, production of the phytochemical and growth were not always antagonistic. Hypoxoside, the biosynthesis of which requires a more thorough investigation, is, however, according to this investigation, a typical secondary metabolite in many respects.
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.