Browsing by Author "Moyo, Tumelo"

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    Establishing grapevine hairy root cultures to study terpene production
    (Stellenbosch : Stellenbosch University, 2023-03) Moyo, Tumelo; Vivier, Melane; Young, Philip; Stellenbosch University. Faculty of Agrisciences. Dept. of Viticulture and Oenology.
    ENGLISH ABSTRACT: Terpenes are plant specialized or secondary metabolites that play a crucial role in plant growth, development, survival and interaction with the environment. Uses of terpenes in industry have grown over time and this demand has led to research regarding ways to improve the biosynthesis of terpenes in planta. Hairy roots are a promising experimental system to study terpene biosynthesis and accumulation. The aim of this study was to therefore establish hairy root cultures of several grapevine cultivars to study terpene production, profiles and quantification. In this study, two grapevine Rhizobium-mediated hairy root transformation methods were tested, differing in the starting material used for transformation (explants), as well as the species used; Rhizobium radiobacter C58C1 and Rhizobium rhizogenes A4T were tested for transformation efficiency. The grapevine scion cultivars used in the study were Vitis vinifera cvs Sultana, Redglobe, and Chardonnay, as well as the rootstocks Vitis Champini (Ramsey), and the interspecific hybrid Selection Openheim 4 (SO4). It was established that R. rhizogenes A4T was more effective in hairy root transformation because it had a moderate multiplication rate which enabled its elimination after co-cultivation with explants, unlike R. radiobacter C58C1 which was persistent in tissue cultures, thereby leading to its undesirable overgrowth on the explants. The results from the explants tested confirmed in vitro internodes of 6-week-old plants to yield higher numbers of hairy roots than the other method tested and was therefore adopted for the rest of the studies. The hairy roots were grown on half-strength Murashige and Skoog (MS) medium, supplemented with 1 g/l activated charcoal on both solid and liquid medium, after some optimisation to improve the sustained growth and integrity of the cultures. Sultana had the highest transformation success at 80% while SO4 had the least success at 13%. To confirm the transgenic state, the hairy roots were checked for the expression of the rolB genes: roots from Redglobe and Sultana tested negative, while those from SO4 and Ramsey tested positive. This was surprising and not considered conclusive in terms of the transgenic status of the Sultana and Redglobe roots, since they had the typical hairy root phenotypes; these cultures were therefore maintained for further analyses. Growth curve analysis in liquid medium confirmed that the cultures typically reach stationary phase after three weeks of culturing in liquid medium. Terpene profiling and quantification were conducted on the established hairy root cultures from the different cultivars using Gas Chromatography-Mass Spectrometry (GC-MS) after optimising some parameters of an existing volatile organic compound method. Hairy roots were shown to produce terpenes and the terpene profiles obtained from them differed from those of untransformed in vitro plantlet root and leaf profiles of the same genotypes. Increased accumulation of plant defence sesquiterpene α-humulene, allelopathic monoterpene α-pinene and fragrant linalool oxide, also implicated in plant defence, were seen enriched in the hairy root cultures. Compounds such as trans-b-ocimene, myrtenol, β-myrcene and geraniol, all implicated in insecticidal activity, showed reduced accumulation in the hairy roots. In a preliminary elicitation experiment, hairy root elicitation with MeJA led to the accumulation of trans-b- ocimene and linalool oxides which are involved in plant defence. Phytol was also detected in control hairy roots (not induced by the elicitor) which was an unusual occurrence since it is a chloroplast associated terpene. It would be interesting to study if transformation could have played a role in its accumulation in root tissue. The results obtained contribute to the knowledge of cultivar responses to Rhizobium- mediated transformation; provide a method for generating and propagating hairy roots in both solid and liquid medium; provide information on cultivar-specific grapevine root terpene profiles; and the ability of hairy roots to be induced to improve terpene accumulation in planta. Some of the problems experienced during the study also led to the identification of aspects that could be further optimised, such as the importance of testing alternative bacterial strains for transformation, or improvement of available strains to incorporate reporter genes or fluorescent markers in the transfer DNA to help in the transgenic root selection process. To further improve growth conditions of hairy root cultures, bioreactors may also be considered for use in order to enhance nutrient supply and aeration.