The effects of the synthetic strigolactone GR24 on Arabidopsis thaliana callus culture

Mdodana, Ntombizanele Thobela (2012-12)

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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