Submitted in complete fulfillment for the Degree of Master of Technology: Biotechnology, Durban University of Technology, 2012. / Many secondary metabolites that have been extracted from medicinal plants have been used as source of clinical drugs. However, the concentration of the active metabolites in plants is generally low. An attractive alternative for producing these important secondary metabolites is via plant tissue culture technology. More particularly, the genetic transformation of a plant tissue by Agrobaterium rhizogenes has been employed for producing high yields of secondary metabolites. In a previous study, three structurally similar anthraquinones: 9,10-Anthracenedione, 1-Hydroxy-4-methylanthraquinone and 5,8-Dimethoxy-2,3,10,10a-tetrahydro-1H,4aH-phenanthrene-4,9-dione, and one steroid; Androst-5-ene-3, 17, 19-triol were isolated from the root extracts of C. triloba. The anthraquinones have shown to exhibit the anticancer mechanism which involves the inhibition of the activity of the human topoisomerase II enzyme that transforms supercoiled DNA to linear DNA. However, these anthraquinones were found in very low concentrations. Therefore, in this study we used plant cell and tissue culture systems (cell suspension, shoot and hairy root cultures) of C. triloba to increase the production of anthraquinones. Since the establishment of C. triloba in vitro plant systems required a source sterile explants, a protocol that involved the use of NaCIO was optimized for the sterilization and subsequent germination of C. triloba seeds which were micro-propagated into shoot cultures. These cultures provided a source explants for the induction of callus and hairy root cultures. The biomass of these plant cell and tissue cultures were subsequently bulked up for the extraction for anthraquinones and the yields were compared followed by fractionation and identification of the major compounds. The bioactivity of the fractions was evaluated by testing their cytotoxicity on cancer cells and anti-topoisomerase activity. The sterilization protocol that provided sterile seeds was found to be a solution of 30% NaCIO at an exposure time of 10 minutes. From the sterilized seeds shoot cultures were established on MS medium. The leaf explants of the shoot cultures were then used to induce callus cultures which subsequently were transferred to liquid medium whereby the total biomass of suspension cultures increased from 4 g to 134.18 g (wet weight). Also hairy roots cultures were established from stem explants with a low cell density inoculum of A. rhizogenes at a transformation efficiency of 73%. The growth of these hairy roots was slow in hormone free medium. This was overcomed with the use NAA and IAA which increased the xvii biomass from 1.03 g in the control culture (without hormone) to 23.91 g and 46.13 g respectively. An evaluation of the anthraquinones in the field root and hairy root, cell suspension and shoot culture extracts was carried out by using their Thin Layer Chromatography profiles and the High Performance Liquid Chromatography profiles as well as the standards, 9,10-Anthracenedione and 1-Hydroxy-4-methylanthaquinone. TLC analysis showed that the RF values of the fractions CT01 and CT02 matched the RF values of anthraquinones standards while HPLC analysis revealed that hairy root cultures supplemented with IAA (125.03 μg.mg-1) or NAA (98.25 μg. mg-1) produced a higher concentration of anthraquinones than the control culture (without hormone) (13.33 μg.mg-1), the field roots (33.51 μg. mg-1) and the shoot (3.23 μg.mg-1) and cell suspension cultures (13.17 μg.mg-1). Due to co-elution of the compounds in HPLC analysis, six fractions were isolated by Preparative Thin Layer Chromatography from the hairy root extract (obtained from the culture supplemented with NAA) and were coded as CT01, CT02, CT03, CT04, CT05 and CT06. The compounds in these fractions were identified by Electron Ionization-Liquid chromatography-Mass Spectroscopy and it was found that the hairy roots produced one acridone derivative; 5-Methoxy-2-nitro-10H-acridin-9-one, one naphthoquinone derivative; 2H-Naphto[2,3-b]pyran-5,10-dione,3,4-dihydro-2,2-dimethyl- and seven anthracenedione derivatives. These were: i) 5,8-Dimethoxy-2,3,10,10a-tetrahydro-1H,4aH-phenanthrene-4,9-dione, ii) 9,10-Anthracenedione, 2-methyl-, iii) 1-Hydroxy-4-methylanthraquinone, iv) 9,10-Anthracenedione, 2-ethyl-, v) 1,5-Diaminoanthraquinone, vi) Phenanthrene, 3,6-dimethoxy-9-methyl-, vii) 9,10-Anthracenedione, 1,4-dimethyl-. Fractions CT01 (5,8-Dimethoxy-2,3,10,10a-tetrahydro-1H,4aH-phenanthrene-4,9-dione, 9,10-Anthracenedione, 2-methyl- and 1-Hydroxy-4-methylanthraquinone) and CT02 (9,10- Anthracenedione, 2-ethyl-) were cytotoxic to the DU-145 cancer cell line at concentrations of 125 μg.mg-1 to 1000 μg.mg-1. These fractions also showed anti-topoisomerase activity as they inhibited the conversion of supercoiled DNA into linear DNA. In conclusion this is the first study that describes the transformation of C. triloba by A. rhizogenes mediated transformation and compares the production of anthraquinones in C. triloba hairy roots to the field roots, shoot and cell suspension cultures. This study has xviii indicated that hairy root cultures is a high-yielding production system for anthraquinones (5,8-Dimethoxy-2,3,10,10a-tetrahydro-1H,4aH-phenanthrene-4,9-dione, 1-Hydroxy-4-methylanthraquinone, 9,10-Anthracenedione, 2-methyl- and 9,10- Anthracenedione, 2-ethyl-) which could have the potential to be used in cancer therapy. In addition the discovery of C. triloba hairy roots having the biosynthetic capacity to synthesize five valuable anthraquinone derivatives that are not found the field roots has also been revealed. / National Research Foundation.

Thesis (PhDAgric)--Stellenbosch University, 1971. / ENGLISH ABSTRACT: The object of this anatomical study was to collect scientific data on the effect of Metoidogyne hapta Chitwood, 1949, on the roots of the following grapevine cultivars viz: Vitis vinifePa L. cvs. Steen and White French and the root-stocks, Jacquez, 1202 C, 99 R, Salt.Creek and Dogridge. These cultivars differed widely in their resistance to M. hapta attacks. In the roots of Steen, White French, Jacquez and 1202 C the formation of multinucleate syncytia by the destruction of the walls of groups of cells often occurred. In Salt Creek, Dogridge and 99 R roots, syncytia were observed in the stele only. The formation o,f abnormal xylem as a result of nematodal activities was a common occurrence. In the roots of these latter three cultivars, M. hapta could not complete its life cycle. Salt Creek, Dogridge, 99 R and often Steen formed a wound periderm which prevented the nematodes from reaching the xylem. Histological changes were often induced in advance of the invading nematodes. / AFRIKAANSE OPSOMMING: Die doel met hierdie anatomiese studie was om wetenskaplike inligting te versamel aangaande die uitwerking van Metoidogyne hapta Chitwood, 1949, op enkele cultivars van wingerdstokke,te wete Steen en Fransdruif van Vitis vinifera L. en die onderstokke Jacquez, 1202 C, 99 R, Salt Creek en Dogridge. Hierdie cultivars het onderling baie verskil in hul weerstandvermoe teen M. hapta. Ten gevolge van die vernietiging van die wande van selgroepe, in die wortels van Steen, Fransdruif, Jacquez en 1202 C, is veelkernige sinsiete (Eng. syncytia) gevorm. In die wortels van Salt Creek, Dogridge en 99 R, is sinsiete net in die sentrale silinder waargeneem. Die vorming van abnormale xileem weens nematodiese bedrywighede was 'n baie algemene verskynsel. M. hapta kon in laasgenoemde drie cultivars nie sy lewenskringloop voltooi nie. In Salt Creek, Dogridge, 99 R en dikwels ook in Steen, is wondperiderm gevorm, waardeur die nematodes verhinder was om die xileem te bereik. Die indringende nematodes het voor hulle uit dikwels histologiese veranderinge in die wortels teweeggebring.

Thesis (MSc)--Stellenbosch University, 1968. / Please refer to full text for abstract

Tissue culture and radicle excision techniques were employed to evaluate salt tolerance in alfalfa (Medicago sativa L.). Plant suspension cultures of either seedling root or shoot origin were studied in media with or without supplemental NaCl (3.54 g liter⁻¹). In most cases, the growth rates of root-derived cultures were stimulated by this low level of supplemental NaCl while most shoot-derived cultures were not stimulated by NaCl. Excised radicles of three populations of alfalfa which possessed widely differing ranges of germination salt tolerance were screened in four salts (NaCl, KCl, Na₂SO₄, and K₂SO₄) at six varying concentrations. As was observed in the tissue culture experiments, low levels of NaCl (7.09 g liter⁻¹) stimulated radicle elongation of all populations as compared to the elongation levels of the control solutions (no supplemental salts). In general, for NaCl, the population that posessed the highest degree of germination salt tolerance (Az-St 1982) also displayed the greatest rates of radicle elongation especially in the highest salt concentrations. Additionally, this population along with the moderately germination salt tolerant population (Az-ST 1979) maintained higher rates of elongation in KCl, K₂SO₄ and Na₂SO₄ than did the control germplasm which has little germination salt tolerance (Mesa Sirsa Control). Examinations of each individual population in all four salts simultaneously, indicated that the sulfate salts reduced radicle elongation to a greater extent than did the chloride salts. Evaluation of both osmotic effects and specific ion effects showed that the specific ion effects attributed to the anions were more detrimental to radicle elongation than were the osmotic effects.

No description available.

Kelpak®, a seaweed concentrate (SWC) prepared from the brown seaweed Ecklonia maxima (Osbeck) Papenfuss, improves overall plant mass and fruit yield in a variety of crops. The main active principals isolated from Kelpak® are cytokinins and auxins. Although these compounds are partly responsible for the growth promoting effect observed with Kelpak® application, they do not fully account for the complete effect of Kelpak® treatment. For this reason the focus has turned to polyamines (PAs) which are found in all cells of plants, animals and microorganisms, including eukaryotic algae. Polyamines also have growth promoting effects in plants. A study was carried out to investigate the PA levels in E. maxima and Kelpak® through a biennial cycle and to investigate if the PAs present in Kelpak® may have an effect on root growth, alleviating nutrient deficiency and the transport and accumulation of PAs in plants. To determine the amount of PA in the stipes, fronds and SWC prepared from E. maxima, samples were collected monthly over a two-year period (June 2009-June 2011). Extracts were benzoylated and quantified using a Varian HPLC. Putrescine concentrations ranged from 15.98-54.46 μg.g⁻¹, 6.01-40.46 μg.g⁻¹ and 50.66-220.49 μg.g⁻¹ DW in the stipe, fronds and SWC, respectively. Spermine concentrations ranged from 1.02-35.44 μg.g⁻¹, 1.05-26.92 μg.g⁻¹ and 7.28-118.52 μg.g⁻¹ DW in the stipe, fronds and SWC, respectively. Spermidine concentrations fell below the detection threshold. This is the first report of PAs being detected in a SWC. The seasonal pattern established for the stipe, frond and SWC followed the same trend over a biennial cycle. Polyamines accumulated in the seaweed tissue during periods of active growth and as a stress response elicited by rough wave action. This PA trend was similar to the cytokinin trend reported by MOONEY and VAN STADEN (1984b) for Sargassum heterophyllum which suggests that PAs play an important role in the hormone cascade during active growth. Routine monthly screening of Kelpak® carried out in the Research Centre for Plant Growth and Development indicated that Kelpak® consistently resulted in more rooting in the mung bean bioassay than the IBA control. The potential root promoting effect of PAs were investigated. Individually applied PAs did not increase rooting in the mung bean bioassay, but a synergistic relationship was observed between Put (10⁻³ M) and IBA (10⁻⁴ M). When applied together, rooting increased significantly above Put (10⁻³ M) and IBA (10⁻⁴ M) applied separately. The Put-auxin combination produced a similar number of roots to those treated with Kelpak®. It is possible that the PAs present in Kelpak® have a synergistic effect with auxins present in Kelpak® to promote root development and growth. Several physiological effects of Kelpak® and PAs on plant growth were investigated in a series of pot trials. Kelpak® significantly improved the growth of P- and K-deficient okra seedlings and masked the detrimental effects exerted by P- and K-deficiency. The application of PAs (10⁻⁴ M) significantly improved the seedling vigour index (SVI) of okra seedlings subjected to N-deficiency. The statistical difference was attributed to the N-containing growth regulators and polyamines being degraded and metabolized by the okra seedlings. Polyamine application did not alleviate P- and K-deficiency but increased root growth significantly in seedlings receiving an adequate supply of nutrients. It is likely that the additional PAs supported auxin-mediated root growth. A pot trial with okra plants was conducted to establish if the PAs in Kelpak®, applied as a soil drench or foliar application, are absorbed and translocated in a plant. Plants were also treated with Put, Spm, Spd to establish if PAs can be absorbed and translocated. Once the fruit had matured, plants were harvested and the endogenous PA content quantified by HPLC in the roots, stems and fruits. Applying PAs as a soil drench was not as effective as a foliar spray at increasing the PA content in the different plant parts. Kelpak® treatment (0.4%) did not contribute more PAs in any plant part. Spermidine concentrations were higher, in the various plant parts, than Put or Spm, irrespective of the mode of application. The application of Put, Spd and Spm increased Spd concentrations in the roots. Considering that Spd is the main PA produced in the roots and that exogenously applied PAs are readily converted to Spd, it seems evident that Spd is the preferred PA for long-distance transport in plants. The cytokinins and auxins in Kelpak® play an important role in stimulating growth in plants. It is, however, the totality of different compounds in Kelpak® that gives it its unique growth stimulating ability. Polyamines, occurring within the seaweed contribute to this activity, having an active role in root production and thus increased plant growth. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.

Abstract not available

Comprised of the author's previously published works. Includes bibliographical references.

Partial rootzone drying (PRD) is an irrigation management technique designed to reduce water use in grapevines without a decline in yield, thereby increasing water–use efficiency (measured as t/ML) (WUE). The principle of PRD is to keep part of the root system at a constant drying rate to produce soil-derived signals to above–ground plant organs to induce a physiological response. Major PRD effects include a reduced canopy size and greatly increased WUE with possible improvements in fruit quality. Although we have a good understanding of the hormonal physiology of PRD, little is known on the effect of PRD on partitioning of C, N and inorganic ions such as K. This thesis broadens our knowledge on the effects of PRD on grapevine field performance, growth and dry matter accumulation as well as its effects on physiology and biochemistry. In field experiments over 3 seasons, PRD reduced water use in grapevines without a significant decline in yield. PRD effects included reduced shoot growth and greatly increased WUE. Field–grown Cabernet Sauvignon, where the PRD grapevines were irrigated at half the control rate, and Shiraz where the PRD grapevines were irrigated at same rate as controls, confirmed that PRD is not simply an irrigation strategy that applies less water, rather it alters the way in which the plant responds to its environment, e.g. PRD alters the sensitivity of the stomatal response to atmospheric conditions and significantly influence enzymes that regulate nutrient accumulation and partitioning. PRD did not change the total amount of carbon and nitrogen on a whole plant basis. However, it caused a significant partitioning of carbon and nitrogen towards trunk, roots and fruit at the expense of shoot growth. This change in partitioning occurred as a result of altered activity of the enzymes controlling the assimilation of carbon and nitrogen. PRD significantly reduced nitrate reductase (NR) activity in grapevine leaves, which catalyses the first step in the assimilation of nitrate irrespective of the amount of water applied. The reduction in NR activity is correlated with the development of the PRD cycle and the associated reduction in stomatal conductance. PRD also significantly altered grapevine sucrolytic enzyme activity that regulate source:sink relationships. PRD showed transient increases in leaf sucrose phosphate synthase (SPS) activity (formation of sucrose) compared to control, but significantly reduced leaf neutral invertase (sucrose cleavage) and leaf starch content in both field and potted experiments. This may indicate an increased photosynthetic capacity and a reduction in its sink strength for sucrose in favor of organs such as fruit and roots. This hypothesis was reinforced by the fact that berries showed significantly higher levels in glucose and fructose early in the season. Berry sugar content and Brix at harvest however was unaffected. Although PRD had no significant effect on berry characteristics at harvest such as Brix and pH, it occasionally reduced per berry K+ content and increased total amino acid concentration that may lead to positive outcomes for wine quality. PRD–treated grapevine roots on the 'wet'– and 'drying'–sides differed greatly in enzyme activity and osmolality. PRD significantly increased osmolality in both wet and drying roots by increasing total osmolyte concentration that may facilitate the movement of water from wet to dry roots. The increases in osmolality were also associated with increased free polyamine production (spermidine and spermine) in PRD roots that may be related to increased root growth and density. / Thesis (Ph.D.)--School of Agriculture and Wine, 2005.

The increasing demand worldwide for Australian wine has driven the recent expansion in vineyard plantings which in turn, has increased the requirement for irrigation water in grape growing regions. Large areas of Australia's national vineyard are already irrigated with relatively poor quality water and many districts have a limited supply of water available for irrigation. Therefore, improving the efficiency of vineyard irrigation is essential for the long term sustainability of the Australian wine industry. Reducing the volume of irrigation applied to vineyards can improve water use efficiency (WUE) and reduce vine vigour. However, it can be difficult to accurately apply the required degree of water stress and this may result in a yield reduction. An irrigation technique known as partial rootzone drying (PRD) involves applying a continuous water deficit to alternate sides of the root system while ensuring the other half is well watered. This has been found to increase WUE, reduce vine vigour, improve fruit quality but not affect vine yield. Where the soil volume available for root growth is limited, so too is the resultant vine growth and yield, as access to water and nutrients is restricted. Shallow soil profiles present a major limitation to root development and grapevine vigour. In shallow soils, mounding topsoil from the vineyard mid row to form raised beds in the vine row has been found to improve vine growth and productivity. Soil mounds tend to have a higher moisture holding capacity than flat soil but the greater surface area of the mound can increase surface evaporation. Applying mulch to the mound surface has been shown to reduce evaporative soil moisture loss and conserve irrigation water. The general hypothesis tested in this experiment was that: 'Combining soil mounding, straw mulch and partial rootzone drying (PRD) irrigation will improve grapevine growth and production and reduce levels of sodium and chloride in the vine.' The experiment was established on Vitis vinifera cv. Shiraz in a mature vineyard at Padthaway, South Australia, where the soil profile consisted of a shallow loam over clay and limestone. Soils of the experimental site were classified as moderately saline because their electrical conductivity (ECse) was greater than 4 dS/m. Three main factors, irrigation method (standard or PRD), soil mounding (flat or mounded) and surface cover (bare or straw mulch) were combined into a 2X2X2 factorial experiment such that the randomised block experiment comprised three replicates of eight treatments. The irrigation treatments were control (the application of water to both sides of the vines) and PRD (the application of water to one side of the vines only at any time). In the PRD treatment the frequency of alternating the 'wet' and 'dry' sides was determined according to soil moisture measurements and was typically every 5-7 days. It was very difficult to accurately schedule the irrigation at this site to avoid applying a moisture deficit to the PRD treatment. The shallow soil profile dried very quickly following irrigation and there were problems with the accuracy of the soil moisture sensing equipment for the duration of the experiment. As a result, PRD vines experienced repeated, excessive soil moisture deficits such that vine growth and production were significantly reduced each season. Shoot length was measured weekly during the growing season, while photosynthetically- active radiation (PAR), leaf area and canopy volume were measured at full canopy. Shoot number and pruning weight were measured during dormancy. All measures of vegetative growth (with the exception of PAR) were reduced in response to PRD. The decrease in lateral shoot growth for PRD resulted in greater bunch exposure and PAR. As a direct result of the severe soil moisture deficits experienced by the PRD treatment, all components of yield were significantly reduced compared to the control treatment each season. In particular, bunch weight and berry weight were significantly lower in the PRD treatment compared to the control, which suggests a period(s) of severe soil moisture deficit was experienced. Despite the yield loss sustained by the PRD treatment, WUE was improved compared to the control treatment in the first two years of this experiment. Berry anthocyanin levels were higher for the PRD treatment than the control but this may be due to the reduction in berry size. Weekly volumetric soil moisture monitoring showed that mounded soil was wetter than flat soil each year at similar horizons. In addition, the larger soil volume of the mounded treatment enhanced vine root development. Vegetative growth was greater in the mounded treatment than the flat treatment. Mounded vines grew more shoots than non-mounded vines, although there was no effect of mounding treatment on shoot length. The difference in shoot number was significant only in year 2, possibly due to the time required for vine roots to establish in the mounds. Pruning weight and mean shoot weight were higher for the mounded treatment each year and mounded vines grew more shoots than non-mounded vines in years 1 and 2. The increase in shoot weight of mounded vines, relative to non-mounded, was most likely due to the increase in lateral shoot growth which is supported by the lower PAR values of the mounded treatment compared to the flat treatment. Each year soil mounding resulted in higher vine yields than in flat soil beds as a direct result of the increased vine capacity of mounded vines. The mounded treatment had more shoots per vine than the non-mounded treatment and thus more bunches per vine. In addition, bunch weights were higher in the mounded treatment each year, due mainly to improved fruit set and more berries per bunch. Despite the mounded treatment resulting in a denser canopy than the non-mounded treatment this did not affect fruit composition in years 1 and 2. WUE was higher for the mound treatment in years 2 and 3 only, due to the volume of irrigation water applied being reduced, yet mounded vines continued to produce higher yields than non-mounded vines. In year 3, berries from vines grown in mounded soil had significantly higher pH than berries from vines grown in flat soil beds. Mounding treatment did not consistently affect berry anthocyanin or phenolic levels. Soil moisture levels were higher in the mulch treatment than the bare treatment in all seasons. In contrast to the mounding treatment, wetter soil did not consistently lead to improved vine growth or yield. Mulched vines developed fewer roots than non-mulched vines which is likely to have limited vine access to water and nutrients. As a result, shoot growth was similar for both treatments each season. The only significant difference between treatments for pruning weight was found in year 3 and was due entirely to shoot weight. The mulched treatment had lower PAR than the bare treatment in year 3, probably the result of increased lateral shoot growth and thus increased shoot weight, although this was not significant. PAR was significantly higher for the mulch treatment, compared to the bare treatment, in year 1 only but this was not supported by significant increases in vegetative growth. The mulch treatment resulted in higher vine yield than the non-mulch treatment in years 1 and 3. This difference was significant in year 3 only when both bunch number and bunch weight were significantly higher for mulched vines. In year 1 only bunch weight was significantly higher for mulched vines. Differences between treatments occurred in year 2 for fruit composition, specifically juice TA and anthocyanin levels. The mulch treatment had significantly higher TA and a significantly lower anthocyanin concentration in berries than the non-mulch treatment in year 2. There was no evidence of increased shading in the mulched treatment relative to the bare treatment that year but the difference in anthocyanin concentration may be explained by the significantly smaller berries of the bare treatment. Analysis of samples taken regularly from the soil profile and vine rootzone showed that there was no treatment effect on soil salinity but that soil ECse increased with soil depth and time each year. Petiole samples were collected at flowering, veraison and pre-harvest and levels were deemed toxic by pre-harvest each year. The PRD treatment received approximately 60% of the salt applied to the control treatment. This did not reduce ECse but did result in lower measures of sodium and chloride in petioles and juice at harvest. Vines grown in soil mounds had access to a greater volume of soil water than the non-mounded vines. The mounded treatment had higher levels of pre-harvest petiole chloride in years 1 and 3 but this was significant only in year 3. There was no consistent trend in levels of sodium and chloride in the juice from either mound treatment, although in year 3 berry extract chloride levels were found to be significantly higher in the mounded treatment than the flat treatment. Similarly, a consistent trend in sodium and chloride levels of petioles and juice was not evident for the mulch treatment. Although, in year 3 petioles of vines grown in bare soil were found to contain significantly more petiole chloride than those which had straw mulch applied. The hypothesis that combining soil mounding, straw mulch and partial rootzone drying (PRD) irrigation will improve grapevine growth and production and reduce levels of sodium and chloride in the vine is rejected as there was not a consistent, cumulative effect of the three factors in this experiment. / Thesis (M.Ag.Sc.)--School of Agriculture and Wine, 2005.