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.

International Telemetering Conference Proceedings / October 26-29, 1998 / Town & Country Resort Hotel and Convention Center, San Diego, California / Dryden real-time flight simulators encompass the simulation of pulse code modulation (PCM) telemetry signals. This paper presents a new method whereby the calibration polynomial (from first to sixth order), representing the conversion from counts to engineering units (EU), is numerically inverted in real time. The result is less than onecount error for valid EU inputs. The Newton-Raphson method is used to numerically invert the polynomial. A reverse linear interpolation between the EU limits is used to obtain an initial value for the desired telemetry count. The method presented here is not new. What is new is how classical numerical techniques are optimized to take advantage of modern computer power to perform the desired calculations in real time. This technique makes the method simple to understand and implement. There are no interpolation tables to store in memory as in traditional methods. The NASA F-15 simulation converts and transmits over 1000 parameters at 80 times/sec. This paper presents algorithm development, FORTRAN code, and performance results.

Evolution of developmental genes is an important mechanism for plant morphological evolution. The LRL genes are an ancient group of bHLH transcription factors that positively regulate root hair development in angiosperms. Here I show that, in the moss Physcomitrella patens, two LRL genes are present and they positively regulate rhizoid and caulonema (a rhizoid-like cell type) development. GUS-transcriptional reporter plants show that both PpLRL1 and PpLRL2 are expressed in tissues giving rise to caulonemata. Loss-of-function mutants in either PpLRL1 or PpLRL2 led to defective rhizoid and caulonema development, and the Pplrl1 Pplrl2 double loss-of-function mutants completely lack rhizoids and caulonemata. Consistent with this, gain-of-function mutants show enhanced rhizoid and caulonema development. In addition, I show that the stimulatory effects of auxin and low phosphate on the development of rhizoids and/or caulonemata required PpLRL gene function. Together, these results show that LRL genes are conserved, positive regulators in tip-growing rooting cell development in land plants. To elucidate whether LRL genes belong to part of a conserved gene network, I use qRT-PCR to determine the transcriptional interaction between LRL genes and the Class I RSL genes, which is another group of conserved regulators of rhizoids and root hairs. Comparing the LRL-RSL network between P. patens and A. thaliana reveals that LRL and Class I RSL genes are transcriptionally independent of each other in P. patens but one LRL gene is transcriptionally downstream of Class I RSL genes in A. thaliana. This suggests that the gene network controlling tip-growing rooting cell development has changed since mosses and angiosperms last shared a common ancestor.

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, 2004. / ENGLISH ABSTRACT: This study investigated the activities of several of the enzymes involved in the alternative route of PEP metabolism via PEPc (EC 4.1.1.31). This reaction circumvents the adenyl ate-controlled PK (EC 2.7.1.40) reaction of the conventional glycolytic network under conditions of P stress. It was hypothesized that the synthesis of pyruvate under Pi stress would induce the PEPc alternative route and that C for pyruvate synthesis would primarily be imported via this route. This was assessed by looking at how total enzyme activities are perturbed under P stress and also by following the route of radioactive labelled 14C02 under sufficient (2 mM) and deficient P (2 JlM) conditions in either roots or nodules. The significance of the pathway under P stress, was further assessed by determining pool sizes of pyruvate that was synthesized from PEPc-derived C. The experiments were conducted under glasshouse conditions, as two separate studies: one to investigate the phenomenon of Pi stress and its consequences for PEPc-derived C metabolism, and the other one to study the enzymes involved. Seeds of Lupinus angustifolius (cv. Wonga) were inoculated with Rhizobium sp. (Lupinus) bacteria and grown in hydroponic culture. Tanks were supplied with either 2 JlM P04 (LP) or 2 mM P04 (control) and air containing 360 ppm CO2. Roots experienced pronounced P stress with a greater decline in Pi, compared to nodules. LP roots synthesized more pyruvate from malate than LP nodules, indicating the engagement of the PEPc route under Pi stress. In this regard, pyruvate is considered as a key metabolite under Pi stress. The role of pyruvate accumulation under Pi stress, was further highlighted by the metabolism of PEP via both the PK and PEPc routes. The enhanced PK activities supported these high pyruvate levels. Under P stress, PEPc activities increased in roots but not in nodules and these changes were not related to the expression of the enzyme. Root and nodular PEPc were not regulated by expression, but possibly by posttranslational control. The novelty of our results for symbiotic roots demonstrates that using metabolically available Pi is indeed a more sensitive indicator ofP stress. These results show that under Pi stress, nodules are able to maintain their Pi and adenylate levels, possibly at the expense of the root. It is suggested that nodules do not experience P stress to the same extent as roots or alternatively function optimally under conditions of low P availability. The increase in concentration of pyruvate synthesized from malate, indeed suggest that under LP conditions there is an increase requirement for pyruvate. It is clear from this data that the operation of bypass route in nodules should be investigated further. Nevertheless, this study provided incentives for understanding the role of C pathways in Ni-fixation, in particular under conditions ofP limitation. / AFRIKAANSE OPSOMMING: Die doel van hierdie studie was om die aktiwiteite van verskeie ensieme van die alternatiewe metaboliese roete via phosphoenolpirovaat karboksilase (pEPc, EC 4.1.1.31) te ondersoek. Dié reaksie omseil die adenilaat-beheerde pirovaatkinase (PK, EC 2.7.1.40) reaksie van die konvensionele glikolitiese weg onder toestande van fosfaat (P) stremming. Dit is gepostuleer dat die sintese van pirovaat onder toestande van Pstremming die alternatiewe roete via PEPc sou induseer en dat die koolstof (C) vir pirovaatsintese gevolglik hoofsaaklik vanaf hierdie roete sou kom. Dit is bepaal deur die veranderinge in die totale ensiemaktiwiteite wat sou plaasvind onder P-stremming te ondersoek. Daar is ook gekyk na die roete' wat radioaktiewe C C4C02) sou volg in wortles en wortelknoppies wat behandel is deur blootsteling aan eerder lae fosfaat (2 1lM) of genoegsame fosfaat (2 mM; kontrole), Die betekenis van die alternatiewe roete is ook ondersoek deur die poel-groottes van pirovaat, soos gesintetiseer via die PEPc reaksie, te bepaal. Twee eksperimente is in 'n glashuis uitgevoer. Eerstens is die verskynsel van Pstremming, asook die invloed daarvan op PEPc-afgeleide C-metabolisme, bepaal. Tweedens is die betrokke ensieme bestudeer. Sade van Lupinus angustifolius (cv. Wonga) is geïnokuleer met Rhizobium sp. (Lupinus) bakterieë en in 'n waterkultuur gekweek. Die houers is voorsien met óf2 IlM P04 (LP) óf 2 mM P04 (HP) en lug wat 360 ppm C02 bevat het. Wortels, anders as wortelknoppies, het 'n betekenisvolle afname in anorganiese P (Pi) ervaar. Onder P-stremming, het lae fosfaat wortels meer pirovaat vanaf malaat gesintetiseer as wortelknoppies, wat 'n definitiewe bydrae vanaf die PEPc roete impliseer. Hiervolgens is pirovaat 'n sleutel metaboliet onder P-stremming. Die belangrikheid van die akkumulering van pirovaat onder P-stremmende toestande is verder beklemtoon deur die toename in metabolisme van PEP via beide die PK- en die PEPcreaksies. Die toename in PK-aktiwiteite is goed gekorreleer met die verhoogde produksie van pirovaat. Onder toestande van P-stremming het die aktiwiteit van PEPc in wortels verhoog, maar nie in wortelknoppies nie. Dit was nie die gevolg van 'n verhoogde uitdrukking van die ensiem nie. Wortel- en wortelknoppie- uitdrukking van PEPc is derhalwe nie gereguleer deur die uitdrukking daarvan nie, maar eerder deur post-tranlasie kontrole. Hierdie resultate vir wortels met wortelknoppies demonstreer dat metaboliese Pi 'n beter maatstaf is om P-stres aan te dui. Hierdie resultate toon dat wortelknoppies beter daartoe instaat is om hul Pi-vlakke en adenilaatvlakke te reguleer, en dit mag ten koste van die gasheerwortel wees. Ons stel voor dat wortelknoppies nie P-stremming tot dieselfde mate ervaar as die gasheerwortel nie en dat dié knoppies optimaal funksioneer by lae Pi vlakke. Die verhoogde konsentrasie van pirovaat, wat vanaf malaat gesintetiseer is, impliseer dat daar 'n groter vereiste is vir dié metaboliet onder toestande van Pstremming. Hierdie studie het die rol van koolstofmetabolismein stikstofbindende organismes, spesifiek onder toestande van fosfaat-tekort, beklemtoon.

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

Thesis (MScAgric (Soil Science))--University of Stellenbosch, 2007. / This study forms part of an ARC Nietvoorbij research project on the long-term effect of different groundcover management practices in vineyards. This study concentrated on the root distribution of the vines after different groundcover practices were used for one decade. The vineyard on the research farm of the Agricultural Research Council at Robertson, South Africa, was divided into 56 equally distributed trail blocks, 14 treatments with four replicas each. For the study Chardonnay cultivar on 99 Richter rootstock was used. This study concentrated on only eight treatments. The eight different cover crop practices consisted of the mechanical treatment, chemical treatment, straw mulch treatment, permanent cover crop treatment and four annual cover crop treatments. The annual cover crop treatments consisted of triticale cover crop that was sprayed with an herbicide before bloom, a triticale cover crop that was left to die naturally, a grazing vetch cover crop that was sprayed with an herbicide before bloom and a grazing vetch cover crop that was left to die naturally. The vineyard was established in November 1992 while the different treatments started in April 1993. Root studies were conducted during the 2002/2003 season. In this study the auger method was used to determine root density (mm/cm³). The root density was correlated with data from the penetrometer studies, yield and shoot mass collected during the same season.

published_or_final_version / Dentistry / Doctoral / Doctor of Philosophy

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.

Controlled environment agriculture offers a solution to challenges including less available land, water deficits, and consumer demand for pesticide free produce. However, control of soil-borne diseases is a major limiting factor. The goal of this dissertation was to examine predictions of the hypothesis that border cells function to protect plant health by controlling microorganisms associated with plants grown in hydroponic culture. Border cells separate from root tips upon immersion in water, and appear to have important roles in the defense mechanisms of plant roots. The general objectives were (1) to study the delivery of border cells in hydroponics; (2) to evaluate interactions between border cells and microorganisms in hydroponics; and (3) to explore approaches to alter border cell production for improved root disease control. In this study it was confirmed that border cells can be released continuously into the solution of hydroponic culture suggesting that plants grown in this system may use extra energy in the production of new border cells. Free border cells interacted with microorganisms present in the hydroponic solution by secreting an extracellular capsule. Previous studies showed that proteins are a key component of this capsule, including lectins. The interaction of pea lectin and Nectria haematococca spores therefore was explored. Results demonstrated that pea lectin agglutinates fungal spores in a hapten-specific manner, and inhibits their germination. Lectin had no negative effect on root development suggesting that it could be used as a potential control for soil-borne diseases in hydroponics. To control the production of border cells, subsequent studies measured the impact of a transient exposure of root tips to different metabolites secreted by root caps and border cells. Exposure to specific metabolites altered the production of border cells without measurable effects on root growth and development. This is in contrast to results obtained with altered gene expression. For example, gene silencing of a border cell specific gene resulted in altered root growth.