Biotechnological approaches for sugarcane enhancement – Drought tolerance and Sucrose accumulation

Masoabi, Malira (2016-12)

Thesis (MScAgric)--Stellenbosch University, 2016.

Thesis

ENGLISH ABSTRACT: The application of biotechnological approaches can supplement traditional crop breeding strategies to combat the effects of adverse environmental conditions on plant production in order to bridge the gap between supply and demand worldwide. In Southern Africa adverse environmental conditions, such as drought, are a serious agronomic problem that threatens crop productivity, food security and eventually negatively impact the economy of the region. Urgent attention to overcome drought stress is therefore vital to eradicate or minimise its effects on crop productivity. Furthermore, the use of fossil fuels as the primary source of energy worldwide has led to demonstrable detrimental effects to the environment and its sustainability. Efforts have been made to seek other cost effective energy sources that counteract the disadvantages of fossil fuels towards the environment. In this regard, research has unravelled the potential of feedstock such as maize and sugarcane as fuel sources and tremendous advancements have been made to explore the possibility of using both sugarcane and maize for biofuel production. Sugarcane is an economically important crop that can be exploited both as food source and for production of biofuels. This study aimed to apply biotechnological methods to: firstly, produce drought tolerant sugarcane plants from three drought sensitive cultivars of South African origin using mutation breeding; secondly, manipulate the myo-inositol oxidation pathway (MIOP) in the model NCo310 sugarcane cultivar to alter sucrose and possibly cell wall composition which would enhance the plants viability for second-generation biofuel production. To accomplish this, optimal in vitro growth conditions for callus obtained from NCo376, N19 and N40 sugarcane cultivars were first established by testing different auxin concentrations for in vitro callus induction. NCo376, N19 and N40 callus grew optimally on 1, 0.5 and 2 mg/L 2,4-D, respectively. The NCo310 model cultivar was furthermore exposed to different ethylmethanesulfonate (EMS) concentrations to induce random mutations without compromising in vitro plant regeneration abilities of the model cultivar. Subsequently an optimal EMS concentration of 16 mM was selected and used for all further mutagenesis experiments across all cultivars. Additionally, calli from the NCo310 cultivar were exposed to polyethylene glycol (PEG) and mannitol at different concentrations to identify a suitable osmoticum, which simulates a drought stress environment in vitro. PEG at a 20% (w/v) concentration was determined as the most suitable osmoticum to induce negative osmotic pressure and allow selection of drought tolerant sugarcane plantlets in vitro. Sugarcane cultivars included in this study were consequently exposed to 16 mM EMS and in vitro selected on 20% PEG, which resulted in the survival of 7, 18 and 19 plantlets of NCo376, N19 and NCo310, respectively. These plantlets were multiplied to prepare for future ex vitro drought pot trials. NCo310 in vitro selected lines were subjected to ex vitro preliminary and follow-up pot trials to confirm drought tolerance. In the preliminary drought trials, single biological clones of selected lines were exposed to water deficit conditions. Of the 19 plantlets selected in vitro, only five outlived the wildtype NCo310 control plants. Plants were re-watered three days after plant to monitor possible plant recovery. Only one line, 310EP 4.1A, recovered from the drought stress and formed new shoots upon re-watering. To confirm these preliminary results, follow-up drought pot trials were executed using three biological repeats of each line together with NCo310 wildtype controls. . Preliminary results from this study indicate that biotechnological approaches, such as mutation breeding and in vitro selection for osmotic pressure, can potentially be successfully applied to develop crops to combat the negative effects of abiotic stresses, such as drought. Nucleotide sugars are activated monosaccharides that act as glycosyl donors in reactions that contribute to the production of cell wall biopolymers in plants. Cell wall components might play a role in biofuel potential of crops and are synthesised by various pathways. The nucleotide 5’-diphospho-sugar (NDP-sugar) interconversion pathway uses the sequential action of kinases and NDP-sugar phosphorylases to produce uridine 5’-diphosphate glucuronic acid (UDP-glucuronic acid), a precursor for cell wall sugars and lignocellulosic polymers. The NDP-sugar interconversion pathway is composed of the sugar nucleotide oxidation pathway (SNOP) and the myo-inositol oxidation pathway (MIOP). In this study one of the MIOP enzymes isolated from Arabidopsis thaliana, the glucuronokinase enzyme was overexpressed in NCo310 sugarcane, to evaluate changes in sucrose content. Morphological differences were observed between transgenic lines and the NCo310 wildtype, no significant sucrose content or glucoronokinase enzymatic activity differences were detected in leaf material isolated from these lines. Inconclusive results obtained from this study, together with the lack of published literature on the production of cell wall components and still remaining knowledge gaps regarding sucrose metabolism by the NDP-sugar interconversion pathways in sugarcane, demonstrate the need for further research into this particular field of cell wall biosynthesis and sucrose metabolism.

AFRIKAANS OPSOMMING: Tradisionele gewastelingstrategieë kan aangevul word deur die toepassing van biotegnologiese benaderings, om sodoende die gevolge van ongunstige omgewingstoestande op plantproduksie teë te werk en die wêreldwye gaping tussen vraag en aanbod te oorbrug. In Suidelike Afrika is ongunstige omgewingstoestande, soos droogte, ’n ernstige agronomiese probleem wat gewasproduktiwiteit en voedselsekerheid bedreig en uiteindelik ’n negatiewe uitwerking op die streek se ekonomie het. Dringende aandag aan die oorkoming van droogtestres is daarom noodsaaklik om die uitwerking daarvan op gewasproduktiwiteit te neutraliseer of te verminder. Boonop het die wêreldwye gebruik van fossielbrandstowwe as hoofenergiebron tot bewysbare skadelike gevolge vir die omgewing en die volhoubaarheid daarvan gelei. Daar word tans gepoog om ander kostedoeltreffende energiebronne te vind, wat die nadele van fossielbrandstowwe vir die omgewing sal teëwerk. In dié verband het navorsing die potensiaal van roumateriale soos mielies en suikerriet as moontlike brandstofhulpbronne blootgelê, en groot vooruitgang is reeds gemaak om die moontlikheid van hierdie gewasse vir biobrandstofvervaardiging te ondersoek. Suikerriet is ’n ekonomies belangrike gewas wat benut kan word met die oog op die verligting van voedselvoorraadtekorte en die vervaardiging van biobrandstowwe. Hierdie studie is gemik op die toepassing van biotegnologiese metodes om: in die eerste plek droogtebestande suikerrietplante uit drie droogte-sensitiewe kultivars van Suid-Afrikaanse oorsprong aan die hand van mutasieteling te vervaardig; en in die tweede plek die mioïnositol-oksidasieroete (MIOP) in die model NCo310-suikerrietkultivar te manipuleer ten einde sukrose, en moontlik selwandinhoud, te wysig en daardeur die plante se kiemkrag vir tweedegenerasie-biobrandstofvervaardiging te verhoog.Om dit te kon vermag, moes daar eers optimale in vitro-groeitoestande vir Saccharum-spesiehibriedkultivars NCo376, N19 en N40 bepaal word deur verskillende ouksienkonsentrasies vir in vitro-kallusinduksie te toets. NCo376, N19 en N40 het optimaal op onderskeidelik 1, 0.5 en 2 mg/L 2,4-D gegroei. Die NCo310-modelkultivar is verder aan verskillende konsentrasies etielmetaansulfonaat (EMS) blootgestel om willekeurige mutasies te induseer teen ’n koers wat waarskynlik ‘n hoë mutasiefrekwensies tot gevolg gehad het, sonder om in vitro-plantregenerasievermoëns te kompromitteer. Daarna is ’n optimale EMS-konsentrasie van 16 mM geselekteer en vir alle verdere mutagenese-eksperimente oor al die kultivars heen gebruik. Verder is kallusse van die NCo310-kultivar aan verskillende konsentrasies poliëtileenglikol (PEG) en mannitol blootgestel om ’n geskikte osmotikum te identifiseer wat ’n droogtestresomgewing in vitro simuleer. PEG, teen ’n konsentrasie van 20% (w/v), is bepaal as die mees geskikte osmotikum om negatiewe osmotiese druk te induseer en die seleksie van moontlike droogtebestande suikerrietplantjies in vitro moontlik te maak. Die suikerrietkultivars wat by hierdie studie ingesluit is, is daarna aan 16 mM EMS blootgestel en in vitro op 20% PEG geselekteer, wat tot die oorlewing van 7, 18 en 19 plantjies onderskeidelik van NCo376, N19 en NCo310 gelei het. Hierdie plantjies is verdeel om vir toekomstige ex vitro-droogteproewe in potte voor te berei. NCo310-, in vitro-geselekteerde lyne is aan voorlopige ex vitro- en opvolgproewe onderwerp om droogtebestandheid te bevestig. In die voorlopige droogteproewe is enkel- biologiese klone van geselekteerde lyne aan watertekorttoestande blootgestel. Van die 19 plantjies wat in vitro geselekteer is, het slegs 5 die wildetipe-NCo310 kontroleplante oorleef. Plante is weer drie dae ná plantsterfte nat gegooi om moontlike plantherstel te monitor. Slegs een lyn, naamlik 310EP 4.1A, het van die droogtestres herstel en nuwe lote uitgestoot nadat dit weer nat gegooi is. Om hierdie voorlopige resultate te bevestig, is opvolgdroogte-potproewe met behulp van drie biologiese herhalings van elke lyn uitgevoer, ingesluit 310EP 4.1A, tesame met die NCo310 wildetipe-kontroles. Alle lyne, behalwe 310EP 4.1A, het die kontroleplante oorleef. Nadat dit weer nat gegooi is, het slegs lyn 310EP 4.1A van die geïnduseerde droogtestrestoestande herstel. Die voorlopige resultate van hierdie studie dui gevolglik daarop dat biotegniese benaderings, soos mutasieteling en in vitro-seleksie vir osmotiese druk, moontlik suksesvol toegepas kan word om gewasse te ontwikkel wat die negatiewe gevolge van abiotiese strestoestande soos droogte kan weerstaan. Nukleotiedsuikers is geaktiveerde monosakkariede en vervul die rol van glikosielskenkers by reaksies wat tot die vervaardiging van selwandbiopolimere in plante bydra. Selwandkomponente speel ’n deurslaggewende rol by die biobrandstofpotensiaal van enige gewas en word via verskeie roetes gesintetiseer. Die onderlinge omsettingsroete van nukleotied 5’-difosfo(NDP)-suiker gebruik die opvolgaksie kinase en NDP-suikerfosforilase om uridien 5’-difosfaat(UDP)-glukuroniesuur te vervaardig, ’n voorloper vir selwandsuikers en lignosellulosepolimere. Die onderlinge omsettingsroete van NDP-suiker sluit die suikernukleotiedoksidasieroete (SNOP) en die mioïnositoloksidasieroete (MIOP) in. In hierdie studie is een van die MIOP-ensieme wat uit Arabidopsis thaliana geïsoleer is gevolglik ooruitgedruk in NCo310-suikerriet, ten einde die veranderings in sukrose-inhoud te evalueer. Alhoewel morfologiese verskille tussen transgeniese lyne waargeneem is toe die glukoronikase-geen in die NCo310-wildetipe ooruitgedruk is, is geen beduidende verskille in sukrose-inhoud of glukoronikase- ensimatiese aktiwiteit bespeur in blaarmateriaal wat uit hierdie lyne geïsoleer is nie. Die onbesliste resultate wat uit hierdie studie bekom is, die gebrek aan gepubliseerde literatuur oor die vervaardiging van selwandkomponente, asook die bestaande kennisgapings betreffende sukrosemetabolisme by die onderlinge omsettingroetes van NDP-suiker in suikerriet, dui op die behoefte aan verdere navorsing in hierdie spesifieke veld van selwandbiosintese en sukrosemetabolisme.

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