Browsing by Author "Van der Merwe, Suzane"
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- ItemAttempting to enhance sugarcane growth through genetic modification(Stellenbosch : Stellenbosch University, 2016-03) Van der Merwe, Suzane; Van der Vyver, Christell; Hills, Paul N.; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics. Institute for Plant Biotechnology.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.