Enhancing drought and osmotic stress tolerance by overexpressing α-acetolactate decarboxylase and acetoin 2,3-butanediol dehydrogenase in planta

Thesis (MScAgric)--Stellenbosch University, 2020.ENGLISH ABSTRACT: The effect of certain rhizosphere colonising bacteria on plant growth has been extensively exploited in agriculture since the green revolution of 1950. The bacteria symbiotically colonise the rhizosphere, utilising exudates from the plant roots, while providing the plant with a number of beneficial actions such as increasing the nutrient availability to roots. These plant growth-promoting rhizobacteria (PGPR) have been well documented and researched since the green revolution began, however, many of the exact bacterial mechanisms responsible for plant growth promotion remain unknown. Previous research, aiming to elucidate such mechanisms, discovered that bacterial volatile organic compounds (VOCs) such as acetoin and 2,3-butanediol were able to increase growth of Arabidopsis thaliana. Subsequent research identified the genes responsible for the production of acetoin and 2,3-butanediol as a-acetolactate decarboxylase (ALDC) and 2,3-butanediol dehydrogenase (BDH1) respectively. These genes were previously successfully transformed into and expressed within Arabidopsis plants within our research group, leading to increased growth and disease tolerance. In this study, Arabidopsis thaliana plants were transformed with the ALDC and BDH1 genes using an Agrobacterium-mediated floral dip method in order to confirm the above-mentioned research, and to allow for the detection of the volatiles in the transgenic plants. However, due to a 240 base pair deletion within the ALDC gene, discovered in the T2 generation, further research could not be performed using Arabidopsis. Sugarcane (Saccharum officianarum) was transformed with the ALDC and BDH1 genes using a particle bombardment approach. Transgenic sugarcane plants were successfully genotyped, sequenced and assessed for transgene expression. The transgenic sugarcane was tested for increased growth, under both in vitro and ex vitro conditions, as well as for drought tolerance via an ex vitro pot trial. No significant differences were observed for the growth of the transgenic sugarcane in vitro compared to the untransformed control plants. The limited availability of transgenic material lead to difficulties in selecting plantlets that were of uniform size and root development for in vitro trials. This led to high variance in the data and inconsistent results within each transformed line. Larger quantities of transgenic material would have alleviated this issue by allowing for selection of plantlets at a uniform developmental stage. Neither were overall significant differences observed between the transformed and untransformed lines within the drought trial. Inconsistent conditions within the growth room where the drought trial was performed led to inconsistent drought pressures applied to the plants. In addition, a temperature spike during the trial led to the rapid onset of drought shock rather than the intended slower onset of drought stress. Untransformed sugarcane was also exposed to synthetic acetoin in vitro, with no significant differences in growth observed after the allowed growth period. In general, this study was inconclusive. However, various aspects of the research were identified which could lead to more conclusive and consistent results. In addition, a method for directly confirming the production of VOCs in planta still needs to be established.AFRIKAANSE OPSOMMING: Die effek van sekere rizosfeerkoloniserende bakterieë op plantgroei word sedert die groen rewolusie van 1950 op groot skaal in die landbou benut. Die bakterieë koloniseer op ‘n simbiotiese wyse die risosfeer deur om van die uitskeidings van die plantwortels gebruik te maak, terwyl die plant 'n aantal voordelige aksies aangebied word, soos die beskikbaarheid van voedingstowwe aan wortels. Hierdie plantgroei bevorderende risoobakterieë (PGPR) is goed gedokumenteer en nagevors sedert die groen rewolusie begin het, maar baie van die presiese bakteriële meganismes wat verantwoordelik is vir die bevordering van plantgroei bly onbekend. Vorige navorsing, met die doel om sulke meganismes te belig, het ontdek dat vlugtige organiese verbindings (VOC's) soos acetoïne en 2,3- butandiol die groei van Arabidopsis thaliana kon verhoog. Daaropvolgende navorsing het die gene wat onderskeidelik verantwoordelik is vir die produksie van asetoïen en 2,3-butaandiol as aasetolaktaat dekarboksilase (ALDC) en 2,3-butandiol dehidrogenase (BDH1) geïdentifiseer. Hierdie gene is voorheen suksesvol in Arabidopsis-plante getransfoormeer en tot uitdrukking gebring binne ons navorsingsgroep , wat gelei het tot groter groei en siektetoleransie. In hierdie studie was Arabidopsis thaliana-plante getransformeer met die ALDC en BDH1-gene met behulp van 'n Agrobacterium-gemedieerde blommedipmetode om die bogenoemde navorsings resultate te bevestig, en die opsporing van vlugtige verbindings in die transgene plante moontlik te maak. Weens 'n 240 basispaar verwydering in die ALDC geen, wat in die T2 generasie ontdek is, kon verdere navorsing nie met Arabidopsis uitgevoer word nie. Suikerriet (Saccharum officianarum) was getransformeer met die ALDC en BDH1 gene met behulp van 'n deeltjiebombardementbenadering. Transgeniese suikerrietplante is suksesvol genotipeer, gesekwenseer en beoordeel vir transgeenuitdrukking. Die transgeniese suikerriet was getoets vir verhoogde groei, onder in vitro en ex vitro toestande, sowel as vir droogteverdraagsaamheid deur middel van 'n ex vitro pot proef. Geen betekenisvolle verskille was waargeneem in die groei van die transgene suikerriet in vitro in vergelyking met die ontransformeerde kontroleplante nie. Die beperkte beskikbaarheid van transgene materiaal lei tot probleme met die seleksie van plantjies wat van eenvormige grootte en wortelontwikkeling was vir in vitro proewe. Dit het gelei tot 'n hoë variansie in die data en teenstrydige resultate binne elke getransformeerde lyn. Groter hoeveelhede transgene materiaal sou hierdie probleem verlig het deur die keuse van plantjies in 'n eenvormige ontwikkelingstadium. In die droogteverhoor was daar ook nie beduidende verskille tussen die getransformeerde en ongetransformeerde lyne gesien nie. Inkonsekwente toestande in die groeikamer waar die droogtetoets uitgevoer is, het gelei tot inkonsekwente droogtedruk op die plante. Daarbenewens het 'n temperatuurstyging tydens die proefneming tot die vinnige aanvang van droogteskok gelei, eerder as tot die beoogde stadiger aanvang van droogtestres. Ontransformeerde suikerriet is ook in vitro aan sintetiese asetoïne blootgestel, met geen noemenswaardige verskille in groei waargeneem na die toegelate groeiperiode nie. Oor die algemeen was hierdie studie onoortuigend. Verskeie aspekte van die navorsing is geïdentifiseer wat kan lei tot meer afdoende en konsekwente resultate. Daarbenewens moet 'n metode gevoer word om die produksie van VOC's in planta direk te bevestig.Master