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Thesis(M.S.)--Auburn University, 2006. / Abstract. Vita. Includes bibliographic references.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Plant growth promoting substances (PGPS) are emerging as useful tools in the investigation of important plant growth traits. Two PGPS, smoke-water derived from burning plant material and a synthetic strigolactone analogue, GR24, have been reported to regulate a wide variety of developmental and growth processes in plants. These PGPS are beginning to receive considerable attention in the area of improving plant biomass yield and production. Variation in growth between plants is a major impediment towards the complete understanding of the intrinsic processes that control biomass production. Callus cultures of the model plant Arabidopsis thaliana could overcome some of these hindrances. However, the suitability of these callus cultures as a model system for plant biomass production must be established first. This study aimed at using A. thaliana callus cultures as a platform to study the plant growth promoting activities of smoke-water and GR24. The first part of this study was conducted to develop an optimal protocol for inducing A. thaliana callus formation. Wild-type A. thaliana Col-O, as well as strigolactone deficient and insensitive mutants (max1-1, max2-1, max2-2, max3-9 and max4-1) were cultured for callus induction. Hypocotyl and leaf explants were cultured onto MS media supplemented with different hormone concentrations of 2,4-D and kinetin (2:2 mg/L 2,4-D:kinetin and 0.5:0.05 mg/L 2,4-D:kinetin). Both media proved suitable for callus induction of all genotypes, with max1-1 showing the highest efficiency (83.33% and 92.22%) of callus induction. Calli were then used as a platform for future investigations into the effects of smoke-water and GR24. Secondly, this study examined the effects of smoke-water and GR24 on wild-type A. thaliana Col-O callus. Basic physiological studies were conducted to determine if these two compounds would positively affect callus growth, as was shown in previous studies using whole plants. Calli cultivated on MS media containing the two different hormone concentrations were transferred onto the same fresh MS medium, supplemented with either smoke-water or GR24. Growth promotion by smoke-water and GR24 in calli was characterized by a significantly increased mass (biomass). Calli were additionally transferred onto MS medium containing either auxin only or kinetin only and supplemented with GR24 or smoke-water. In the auxin only system, increased mass was recorded for both GR24 and smoke-water treatments, while these two compounds seemed to reduce growth in the kinetin only system. The positive growth stimulatory effect observed for the auxin only system could be attributed to the synergistic relationship between auxin and strigolactones, whilst the reduced mass in the latter system could be due to the antagonistic interaction between strigolactones and cytokinins. Finally, this study has discovered a dual role of strigolactones in biomass accumulation and adventitious root formation for Arabidopsis thaliana callus. On an auxin- and cytokinin-free MS medium supplemented with GR24, calli of Arabidopsis thaliana strigolactone deficient mutants (max1-1 and max4-1) and the wild-type Col- O, but not the strigolactone response mutant (max2-2), showed enhanced biomass accumulation. In addition to this, the max4-1 mutant and wild-type Col-O demonstrated enhanced adventitious rooting, which was not apparent in max2-2. Together these data suggested that the biomass accumulation and the adventitious rooting activities of GR24 in Arabidopsis thaliana calli are controlled in a MAX2- dependent manner. The interaction between strigolactone, auxin and cytokinin signalling pathways in regulating these responses appears to be complex. Gene expression profiling showed regulation of stress-related genes such as B-box transcription factors, CALCINEURIN B-LIKE and RAP4.2 Genes encoding hormones associated with stress (ABA, ethylene) and defence mechanisms (JA) were upregulated. Expression of stress related genes indicated clues on some kind of stress mediation that might be involved during the regulation of the rhizogenic response. Conversely, smoke-water treatment could not enhance the biomass of the calli and nor could it induce adventitious rooting in the absence of auxin and cytokinin. This observation strongly emphasized the distinct roles of these two compounds, as well as the importance of the interaction and ratio of auxin and cytokinin in callus growth. This study has demonstrated a novel role of strigolactones in plant growth and development, i.e. enhancement of biomass production in callus cultures. Secondly the enhanced adventitious rooting ability is in agreement with recently published literature on the role of strigolactones in regulating root architecture. In vitro callus production is advantageous to plant sciences. It creates an opportunity for increasing plant material for cultivation and offers the use of cell cultures that accurately mimic specific growth responses. It could greatly contribute to the study of intricate regulatory and signalling pathways responsible for growth and development in plants. Because the regulation of plant biomass production is very complex and the molecular mechanisms underlying the process remain elusive, it is of paramount importance that further work be done in order to gain more in-depth insights and understanding of this aspect and subsequently improve efficiency and returns when applying biotechnology tools on commercially important crop plants. / AFRIKAANSE OPSOMMING: Verbindings wat plantgroei bevorder (PGBV) het as nuttige alternatief ontstaan om plant groei te ondersoek. Rook-water, afkomstig van verbrande plant material, en ‘n sintetiese strigolaktoon analoog, GR24, wat ‘n α, β-onversadigde furanoon funksionele groep in gemeen het, is vir die regulering van ‘n wye verskeidenheid ontwikkelings- en groei prosesse in plante verantwoordelik. Tans ontvang hierdie PGBVs aansienlik aandag in die area van die verbetering van plant biomassa opbrengs en -produksie. Die variasie in groei tussen plante is ‘n groot hindernis om die intrinsieke prosesse wat biomass produksie beheer, volledige te verstaan. Deur gebruik te maak van kallus kulture van die model plant Arabidopsis thaliana kan van hierdie hindernisse oorkom word. Tog moet die geskiktheid van kallus kulture as ‘n model sisteem vir plant groei biomass produksie eers gevestig word. Die doel van hierdie studie was om A. thaliana kallus kulture as ‘n platform vir die studie van die plantgroei bevorderingsaktiwiteite van rook-water en GR24 te gebruik. Die eerste deel van die studie is uitgevoer ten einde ‘n optimale protokol vir die induksie van A. thaliana kallus produksie te ontwikkel. Wilde tipe Col-0, asook strigolaktoon afwesige en onsensitiewe mutante (max1-1, max2-1, max2-2, max3-9 en max4-1) is vir kallus induksie gekultiveer. Hipokotiel en blaar eksplante is op MS medium wat verskillende hormoon konsentrasies van 2,4-D en kinetien (2:2 mg/L 2,4-D:kinetien en 0.5:0.05 mg/L 2,4-D:kinetien) bevat, oorgedra. Beide media was geskik vir kallus induksie van al die genotipes, met max1-1 wat die hoogste effektiwiteit (83.33% en 92.22%) van kallus induksie getoon het. Kalli is daarna as ‘n platform vir toekomstige navorsing i.v.m die effek van rook-water en GR24 gebruik. Tweedens ondersoek die studie die effek van rook-water en GR24 op wilde tipe Col-0 kallus. Basiese fisiologiese studies is uitgevoer om te bepaal of die twee verbindings ‘n positiewe effek op kallus groei toon soos aangedui in vorige studies waar intakte plante gebruik is. Kallus wat op MS medium wat die twee verskillende hormoon konsentrasies bevat gekultiveer was, is op dieselfde vars MS medium, wat addisioneel óf rook-water óf GR24 bevat, oorgedra. Die stimulering van groei van kalli deur rook-water en GR24 is deur ‘n merkwaardige toename in massa (biomassa) gekenmerk. Kallus is additioneel op MS medium wat slegs óf ouksien óf kinetin bevat (gekombineer met GR24 of rook-water behandeling), oorgedra. In die sisteem waar slegs ouksien toegedien is, is ‘n toename in massa waargeneem vir beide GR24 en rook-water behandelinge. In teenstelling hiermee, het die twee verbindings in die sisteem waar slegs kinetin toegedien is, ‘n vermindering in groei meegebring. Die positiewe groei stimulerende effek wat waargeneem is vir die sisteem waar slegs ouksien toegedien is, kan toegedra word aan die sinergistiese verhouding tussen die ouksien en strigolaktone; terwyl die verlaagde massa in die laasgenoemde sisteem aan die antagonistiese interaksie tussen strigolaktone en sitokiniene toegedra kan word. Laastens het hierdie studie het ‘n gelyktydige rol van strigolaktone vir biomassa akkumulasie en bywortelvorming in Arabidopsis thaliana kallus ontdek. Kallus van A. thaliana strigolaktoon afwesige mutante (max1-1 en max4-1) en die wilde tipe Col-0 (maar nie die strigolaktoon reagerende mutant (max2-2) het op ‘n ouksien en sitokinien vrye MS medium wat GR24 bevat ‘n verhoogde biomassa akkumulasie getoon. Die max4-1 mutant en wilde tipe Col-0 het verhoogde bywortelvorming getoon, wat nie so opmerklik by max2-2 was nie. Hierdie data het tesame voorgestel dat die biomassa akkumulasie en die bywortelvormingsaktiwiteite van GR24 in Arabidopsis thaliana kallus op ‘n MAX2-afhanklike wyse beheer word. Die interaksie tussen strigolaktoon, ouksien en sitokinien sein transduksie paaie vir die regulering van hierdie reaksies blyk kompleks te wees. Die geen uitdrukkingsprofiel het die regulering van stres verwante gene soos B-boks transkripsie faktore, CALCINEURIN B-LIKE en RAP4.2, getoon. Gene wat vir hormone wat aan stres (ABA, etileen) en verdedigingsmeganismes (JA) verwant is, is opgereguleer. Die uitdrukking van stress verwante gene dui op tekens van ‘n ander tipe stres bemiddeling wat dalk by die regulering van die risogeniese reaksie betrokke kan wees. In teenstelling, rook water behandeling kon nie die kallus biomassa verhoog nie en dit kon ook nie die bywortelingvorming in die afwesigheid van ouksien en sitokiniene induseer nie. Hierdie waarneming is ‘n sterk bevestiging vir die uitsonderlike rol van die twee verbindings, asook die belang van die interaksie en verhouding van ouksien en sitokinine vir die groei van kallus. Hierdie studie toon op ‘n nuwe rol van strigolaktoon in plant groei en ontwikkeling, d.w.s die verhoogde biomassa produksie in kallus kulture. Tweedens, die verhoogde bywortelvormingsvermoë is in ooreenstemming met literatuur wat onlangs gepubliseer is i.v.m die rol van strigolaktone in die regulering van wortel argitektuur. Die in vitro produksie van kallus is voordelig in plant wetenskappe. Dit skep ‘n geleentheid vir die vermeerdering van plant materiaal vir kultivering en bied die gebruik van selkulture wat spesifieke groei reaksies op ‘n merkwaardige wyse akkuraat namaak. Dit kan grootliks bydra tot die studie van die delikate regulatoriese en sein transduksie paaie wat vir groei en ontwikkeling van plante verantwoordelik is. Aangesien die regulering van plant biomassa produksie baie kompleks is en die molekulêre meganismes vir die proses onbekend bly is dit van grootskaalse belang dat meer werk gedoen word om ‘n meer in diepte insig en kennis van die aspekte en gevolglike verbetering van effektiwiteit en wins te kry deur die toepassing van biotegnologiese metodes op die gewas plante wat van kommersiêle belang is.

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Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Almost all processes during the life of a plant are affected by the environment. Changes in phytohormone, metabolite and protein levels follow in response to changes in the environment. Plant growth promoting substances can stimulate changes at these levels to facilitate increased plant growth and yields above what the plant would normally establish. In this study, the effects of two growth promoting substances, smoke water (SW) derived from bubbling smoke from the burning of plant material through water, and a synthetic strigolactone analogue, GR24, on plant growth and architecture, as well as the proteome and metabalome of salt stressed Nicotiana benthamiana seedlings were investigated. Physiological studies were conducted to identify the effects of the growth substances on salt stressed seedlings in a tissue culture system. Under non-stress conditions, SW treatment increased seedling fresh mass, root length and leaf area. Under salt stress conditions (100 mM and 150 mM NaCl), SW increased fresh mass, root length, leaf number and lateral root number significantly. Under non-stress conditions, GR24-treated seedlings showed increased fresh mass, leaf number and area and root length. When GR24-treated seedlings were placed under salt stress, the seedlings showed significant increases in fresh mass, leaf number and lateral root number, but only marginal increases in root length and leaf area. Despite these similarities, slight differences were observed in the metabolomes and proteomes of smoke water and GR24-treated seedlings, both with and without the addition of salt stress. Relatively few of the differentially expressed proteins could be identified with the instruments available. Changes in the metabolome indicated that photoassimilation and photosynthesis could be affected in response to smoke water and GR24 treatment. Our results suggest that smoke water and GR24 both promote growth under salt stress conditions in seedlings and we furthermore conclude that, although there are distinct overlaps between treatments, this is accomplished via slightly different mechanisms. / AFRIKAANSE OPSOMMING: Gedurende ‘n plant se lewe word omtrent alle prosesse deur die omgewing geaffekteer. Veranderinge in die omgewing word gevolg deur veranderinge in hormoon, metaboliet en protein vlakke. Plant groei stimulante affekteer hierdie vlakke om plant groei en -opbrengs na bo normalle vlakke te verhoog. In hierdie studie word die effek van twee groei stimulante, rook water verkry deur rook van plant materiaal deur water te borrel en ‘n sintetiese strigolaktoon, GR24, ondersoek op ‘n morfologiese, metaboliese en ‘n proteomiese vlak in Nicotiana benthamiana saailinge. ’n Studie is onderneem om die veranderinge as gevolg van die onderskeie groei stimulante te ondersoek in ‘n weefsel kultuur sisteem. Rook water het onder normale groei omstandighede vars en droeë massa, blaar aantal asook wortel en blaar lengte verhoog. Rook water het na sout behandeling (100 en 150 mM NaCl) steeds vars massa, wortel lengte, blaai aantal en laterale wortel aantal beduidend verhoog in vergelyking met die sout stres kontrole. Behandeling met GR24 het ook vars massa, wortel lengte, blaar aantal en grootte verhoog en onder sout stres met GR24 is ‘n beduidende vergroting opgemerk in vars massa, blaar grootte en laterale wortel aantal. Ongeag van die veranderinge in groei is klein verskille opgemerk in die metaboliet en protein studies. Net ‘n paar proteine kon positief geidentifiseer word met die apparaat beskikbaar. Verandering in die metaboloom wys na veranderinge in fotoassimilasie en fotosintese in reaksie tot rook water en GR24. Hierdie resultate lei tot die gevolgtrekking dat rook water en GR24 beide groei verbeter in saailing behandel met sout en ook dat alhoewel daar sekere ooreenkomste is tussen die reaksies as gevolg van die plant groei stimulante, dit wel geskiet deur geringe verskillende meganismes.

The stringent requirement for hemopoietic growth factors (HGF) in the induction of hemopoiesis in vitro has raised questions as to their possible role(s) in leukemogenesis. Several recent clinical studies have shown aberrant cell growth factor gene activation in patient derived leukemic cells. Assessment of growth factor activity is often based on in vitro bioactivity assays of conditioned media or body fluids. The specificity of this type of endpoint is, however, open to question due to the overlap in biological activities of many HGFs. In assessing the role of growth factor gene expression in a murine myeloid leukemia model I have used a sensitive RNA detection procedure coupled with a vector-probe system that enables the synthesis of uniformly labelled radioactive DNA probes to detect unambiguously the expression of particular growth factor genes. The Abelson murine leukemia virus (A-MuLV) derived myeloid transformants used in this study had previously been shown to produce a multi-lineage colony stimulating activity (CSA). While these A-MuLV transformants were shown to produce GM-CSF, it seemed likely that the multi-lineage CSA was due to another factor. In addition to confirming the expression of GM-CSF mRNA, I was able to show that the cells of all four A-MuLV transformed lines tested also expressed interleukin-3 mRNA. This finding was strongly corroborated by bio-activity data obtained using the CM from the A-MuLV myeloid transformants. Additional preliminary analysis by bioactivity assays have also shown the possible presence of interleukin-6 (IL-6) and a recently described pre-B cell factor suggesting perhaps a common mechanism underlying the activation of these various growth factor genes. / Medicine, Faculty of / Medical Genetics, Department of / Graduate