Attempting to enhance sugarcane growth through genetic modification

Van der Merwe, Suzane (2016-03)

Thesis (MSc)--Stellenbosch University, 2016.

Thesis

ENGLISH ABSTRACT: The current focus of plant breeding is to increase yield and meet the food and energy demands of a growing global population. Progress in this field of study is slow due to the complexity of yield, which is a complex trait governed by an array of different genes and various metabolic pathways. The progress falls drastically short of the increasing commercial demands. Biotechnological approaches can act as an alternative to traditional breeding, to help achieve yield increases that will meet the food and energy demands modern society faces. The purpose of this study was therefore to apply two separate biotechnological methods with the ultimate goal to promote growth in sugarcane. The well-studied plant phytohormone, cytokinin, has a pivotal role in plant development. Levels of the hormone are regulated by the cytokinin oxidase/dehydrogenase (CKX) gene family. The cytokinin oxidase/dehydrogenase enzyme carries the sole responsibility for cytokinin breakdown. This study, therefore, aimed at silencing the CKX genes present in sugarcane. The silencing attempt would ideally result in a higher cytokinin level being present in the transgenic plants. In turn, the higher cytokinin levels would then allow for increased plant growth, with the ultimate goal of increasing crop yield. It has been previously reported that silencing of only one of the genes, present in the CKX gene family, increases shoot and root weight. In order to silence one, or the majority of the CKX gene family, a novel RNA interference-based technology was employed. The method, referred to as the isothermal in vitro recombination system (IR-hpRNAi), allows for the assembly of a hairpin containing RNA vector in a single cloning step. It also allowed for the benefit of using the traditional sugarcane transformation vector, pUBI510+, containing an ubiquitin promoter and CamV terminator. Due to the novelty of the method in our laboratory, optimization was required. In addition to the IR-hpRNAi method, the traditional GATEWAY® cloning system was also successfully employed for the construction of a hairpin cassette. The second method employed was based on the introduction of the ALDC and BDH1 gene into Arabidopsis thaliana and subsequently into sugarcane. Certain rhizobacterial strains provide plants with enhanced plant health and increased growth. These beneficial rhizobacteria are known as plant growth-promoting rhizobacteria (PGPR). The PGPR are able to do provide the plant with beneficial effects via the release of specific volatile organic compounds (VOCs), amongst other mechanisms. Acetoin and 2,3-butanediol are two of the VOCs known to increase plant growth and disease resistance. To elicit ISR, the volatiles prime expression of specific ethylene- and jasmonic acid-response genes. The ALDC and BDH1 genes are responsible for the synthesis of the two respective VOCs. Increased disease resistance is caused by eliciting an induced systemic response (ISR) in the plant, which in turn allows the plant to respond quickly and efficiently to infection. An increase in disease resistance links closely to an increase in overall plant growth as it prevents any inhibition or limitation that would have been enforced through successful infection. Previous research demonstrated an increase in plant growth when transgenic lines, expressing ALDC and BDH1, were produced. During this study, transgenic Arabidopsis thaliana lines were created, expressing the ALDC and BDH1 genes, with the ultimate aim of producing plants with a more effective immune system. To test if the transgenic plants exhibit an increase in pathogen resistance infection trials were conducted by infecting the transgenic AB2 and A3 lines with the necrotrophic, fungal pathogen, Botrytis cinerea. Plant resistance was assessed quantitatively by assessing lesion development, and conducting sqRT-PCR analysis on several ISR-related genes. The A3 line, containing only the ALDC gene, exhibited a slight increase in disease resistance, while the ALDC and BDH1 containing AB2 line showed a decreased resistance. The study additionally aimed to produce ALDC and BDH1 expressing sugarcane lines that exhibit increased disease resistance, along with plant growth.

AFRKAANSE OPSOMMING: Tans is die fokus van plantteling om oes opbrengste van gewasse te verhoog. Daar is ‘n sterk toename in voedsel aanvraag as gevolg van die groeiende globale bevolking. Vordering in hierdie studieveld is egter stadig, omdat die genetiese eienskappe wat die metaboliese prosesse vir opbrengs en groei beheer, baie kompleks is. Die vordering val dus kort van die eise wat die moderne samelewing op die voedsel industrie plaas. Biotegnologie dien as 'n alternatiewe metode vir tradisionele teling. Dit kan moontlik help om voedsel opbrengste te verhoog tot op so ‘n punt dat dit voldoende sal wees vir die eise wat moderne samelewing stel. Die doel van hierdie studie was dus om twee afsonderlike biotegnologiese metodes te gebruik om plante groei te verbeter en uiteindelik oes opbrengste te verhoog. Sedert die ontdekking van sitokinien, is die planthormoon deeglik bestudeer en het dit bekend geraak vir sy sleutelrol in die ontwikkeling van plante. Dit is nou welbekend dat interne sitokinien hormoonvlakke gereguleer word deur die sitokinien oksidase/dehidrogenase (CKX) geen-familie. Die sitokinien oksidase/dehidrogenase ensieme wat geproduseer word, is alleenlik verantwoordelik vir die afbreek van sitokinien in plante. Ons studie was dus daarop gemik om CKX geenuitdrukking teenwoordig in suikerriet te stil. Dit is gedoen met die doel om 'n hoër vlak sitokiniene te bewerkstellig. Op sy beurt sou die hoër vlakke van sitokinien teenwoordig in die transgeniese suikerriet lei tot verbeterde groei en hoër gewas opbrengste. Dit is vroeër bewys dat wanneer een van die gene teenwoordig in die CKX geenfamilie verstil word, veroorsaak dit verhoging in plantmasse en wortel gewig. Verstilling van die geen was moontlik met ‘n biotegnologies-ontwerpte haarnaald (RNA-georiënteerde inmenging). Die metode, bekend as die isotermiese in vitro rekombinasie stelsel (IR-hpRNAi), maak voorsiening vir die bou van 'n haarnaald in ‘n toepaslike transformasie vektor. In hierdie spesifieke geval, vir ons studie, het die metode voorsiening gemaak vir die gebruik van die tradisionele suikerriet-transformasie vektor, pUBI510 +, wat 'n ubiquitine promotor en CamV termineringsgeen insluit. As gevolg van die metode se onbekendheid in ons laboratorium, was die optimalisering daarvan ‘n sleutelfaktor. Saam met die IR-hpRNAi metode, is die tradisionele GATEWAY® kloneringsisteem ook toegepas. Vir die tweede gedeelte het die studie gefokus op die bekenstelling van die ALDC en BDH1 gene in A. thaliana en daarna suikerrietplante. Sekere rhizobakterieë verskaf plante met ‘n verhoogde immuniteit. Hierdie voordelige rhizobakterieë staan bekend as plant groei-bevorderende rhizobakterieë (PGPR) en laat plante beter groei. Die bakterie verskaf ook ‘n versterkte immuunstelsel aan die plant. Een van die maniere waarop hierdie PGPR in staat is om die plante se verbeterde groei en versterkte immuunstelsel te fasiliteer, is deur middel van vlugtige organiese verbindings (VOVs). Acetoin en 2,3-butaandiol is twee van die VOVs wat PGPR vrystel. Verhoogde siekteweerstand is die gevolg van 'n geïnduseerde sistemiese immuunreaksie (ISR) in die plant. Vir ‘n ISR om in die plant plaas te vind moet ‘n spesifieke etileen (ET)- en jasmonsuur (JA)-reaksie ontlok word. Vorige studies het bepaal dat die VOVs verantwoordelik is vir die ISR deur die aktivering van spesifieke ET/JA verwante gene (Ryu et al., 2004a; Kwon et al., 2010; Rudrappa et al., 2010). Omdat ‘n vorige meestersstudies (Dempers, 2014) reeds ‘n toename in plantegroei vasgestel het, toe transgeniese lyne wat die ALDC and BDH1 gene uitdruk bestudeer is, het die huidige studie gefokus op patogeniese weerstand. Gedurende hierdie studie is transgeniese A. thaliana lyne geskep, wat die ALDC of BDH1 gene bevat. Hierdie twee gene is verantwoordelik vir acetoin (AC) en 2,3-butaandiol (2,3-BD) produksie. Die uiteindelike doel van die studie was om te toets of die transgeniese plante 'n toename in patogeenweerstand wys. Patogeenweerstand is getoets deur die transgeniese plante te infekteer met die nekrotrofiese swam, Botrytis cinerea. Plantweerstand is kwantitatief geassesseer deur die beoordeling van letsel ontwikkeling, asook deur semikwantitatiewe polymerasie-kettingreaksie (skRT-PKR) analise. Die skRT-PKR analise is uitgevoer om die uitdrukkingsvlakke van ISR-verwante gene te bepaal. Die A3 lyn, wat slegs die ALDC geen bevat het 'n effense toename in weerstand teen B. Cinerea getoon. Aan die ander kant het die AB2 transgeniese lyn, wat albei gene bevat, ‘n afname in weerstand getoon. Hierdie studie was ook daarop gemik om addisionele AB suikerriet lyne, wat beide die ALDC en BDH1 gene uitdruk, te produseer.

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