Masters Degrees (Viticulture and Oenology)

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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.
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    Sensory profiling, quality evaluation, and chemical profiling of South African de-alcoholised still wines
    (Stellenbosch : Stellenbosch University, 2023-03) Qwebani, Sinazo; Nieuwoudt, Helene; Brand, Jeanne; Pentz, Chris; Muller, Magdalena; Stellenbosch University. Faculty of Agrisciences. Dept. of Viticulture and Oenology.
    ENGLISH ABSTRACT: A relatively new category of wines with <0.5% v/v ethanol produced through physical removal of ethanol is in demand by many health-conscious consumers. However, this physical intervention has an impact on the wines’ sensory profiles. Accordingly, there is an interest in understanding this category’s sensory aspects and chemical composition. The aim of this study was to investigate the sensory profiles and quality of South African (SA) de-alcoholised still wines, and to do chemical profiling, with focus on volatile aroma compounds, of the wines. The sensory profiles of a set of commercial de-alcoholised still wines (n=16) were generated by the free listing (FL) method. Wine industry professionals (n=52) from seven different wine cellars participated were asked to freely list the sensory descriptors they associated with the wines upon tasting. For quality evaluation, the same panel was required to score the wines’ perceived quality using a 20-point scale. The sensory modalities evaluated for quality were appearance (max=3), nose (max=7), and palate (max=10) added up to a score of 20. In addition, to obtain a chemical profile, chemical compounds of a wine set (n=14) were quantified. However, data processing was required for the text generated from the free listing sensory evaluation method. For instance, free listing generated 11627 words which were standardised to 3846 descriptions after removal of non-sensory descriptions, repetitions of descriptions and comparisons. Thereafter, lemmatisation was done where descriptors were reduced to their roots and grouped into descriptor categories. The established sensory profile consisted of positive, neutral, and negative descriptor categories. The results of the study reveal that negative sensory descriptors were cited many times compared to positive characteristics: “off-odours”, “lack of aroma”, “lacks flavour”, and “watery”. However, it is important to note that there were positive descriptors: “good aroma” and “good palate”. Even though, “fruit” a neutral descriptor was the most cited which might have contributed positively to the sensory quality of these wines. In overall these results lack cultivar specific descriptions and thus wine authenticity which indicates that the complex aroma and flavour profile of full-strength white, rosé and red wines can be lowered drastically due to the process of de-alcoholisation. These sensory descriptors were then correlated with quality scores attained using the 20-point scale scoring system in which some of the wines received very low scores. As a result, the average of the overall scores was 13 out 20 which is explained as “borderline faulty”, “unbalanced wine”. In addition, the concentrations of the wines under study had 4 folds less of esters, 17 folds less of higher alcohols, and 3 folds less of fatty acids when compared to published ideal wines values, depicting poor wine quality. This pioneering study has established the sensory, quality, and chemical profile of this wine for the improvement of this category. As a result, major concerns both in sensory quality and the chemical profile of this new category have been successfully identified and insights into the way forward have been recommended to the wine industry and future studies.
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    Bunch quality and fertility of Vitis vinifera L. cv. Prime as affected by gibberellic acid (GA3) and s-abscisic acid (s-ABA).
    (Stellenbosch : Stellenbosch University, 2022-12) Masikane, Nonkululeko Nosipho; Avenant, Eunice; Avenant, J. H. ; Stellenbosch University. Faculty of Agrisciences. Dept. of Viticulture and Oenology.
    ENGLISH ABSTRACT: Table grapes are one of the most cultivated, economically important fruit crops in the world and is one of the major fruit industries in South Africa. There is an increase competition in the market that leads to an increase production costs for South African table grape industry, to produce quality grapes that meet market requirements nationally and internationally. Market requirements for grapes are consumer driven, and are based on bunch structure, berry size, colour uniformity, flavour, texture, seedlessness and firmness. Plant growth regulators (PGRs) have become an important tool in producing grapes of high export quality, and to meet these market requirements. Gibberellic acid (GA3) is one of the registered plant growth regulators that is used as a chemical thinner in the grapevine, it is has been found to play a role in berry set, thinning and development. The effectiveness of PGRs dependent on cultivar, application time and concentration. This study aimed at establishing the effect of gibberellic acid (GA3) and abscisic acid (s-ABA) for thinning, combined with GA3 for berry sizing (applied at the recommended dosages, using different application volumes obtained with commercial spray equipment), on fertility of a 5-year-old Vitis vinifera cv. Prime block, grafted onto Ramsey rootstock on the farm Newgro at Kanoneiland in the Orange River Region. Four thinning treatments were combined with four berry sizing treatments, resulting in 16 treatment combinations. The thinning treatments comprised of a Control (No thinning); 1 ppm GA3 at 10% set and 3 days later; 1 ppm of GA3 x 5, with the first application at 10% set, followed by four more applications at 3-day intervals; and 1 ppm GA3 plus 400 ppm s-ABA at 10% set, followed by 1 ppm GA3 3 days later. All thinning treatments were applied at a volume of 1000 L.ha-1 with a Nobili 2000 T mist blower spray pump. The berry sizing treatments, applied at 7 to 8 mm and 8 to 10 mm berry diameter, respectively, comprised of a Control (Dipping) and three treatments applied by spraying. The same active ingredient dosage (2 x 20g GA3 ha⁻¹) was used for all treatments, however different application volumes were used for the spraying treatments (90 L ha-1 with an electrostatic spray pump (ESS); 250 L.ha-1 and 500 L.ha- 1 respectively with a Cima A T50S2.15.L11 mist blower spray pump. Bunches of all treatments obtained commercially acceptable bunch lengths (>20 cm) and Extra-Large berry size (> 18 mm in diameter). The control had the most compact bunches, but there was no significant difference between the three chemical thinning treatments, regarding bunch length, bunch compactness, berry size and berry size distribution. Therefore, producers could use any of these three chemical thinning treatments for Prime. There was a tendency that the largest berry size was obtained with the Dipping and ESS berry sizing treatments. Although there were significant differences, the percentage of extra small berries of all sizing treatments was very low (< 1) in both seasons. Potential fertility was assessed through forced budding in a glasshouse and bud dissections. Actual fertility was assessed in the vineyard. None of the thinning or sizing treatments negatively affected fertility. All treatments obtained commercially acceptable levels (> 80%) of bud break (ranging from 92 % to 97%) and fertility (more than one bunch per sprouted bud). Fertility was not affected by the GA3 treatments applied over three consecutive seasons, probably due to the dosage range used (1 to 5 g.ha-1 for thinning and 40 g.ha-1 for berry sizing). When making the final decision of which chemical thinning treatment and which berry sizing application technique to use, the effect on berry size and bunch quality should also be considered. None of the treatments evaluated negatively affected berry size and bunch quality. Any of the application techniques evaluated could be used, if GA3 within the recommended dosage range is used and applied at the recommended phenological stages for Prime. This study also provided data on the efficacy of GA3+ABA as thinning agent for table grapes, to be used to extend the registration of s-ABA on table grapes to include its use as a thinning agent.
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    Characterization of cv. Cabernet Sauvignon (Vitis vinifera L.): the contribution of Ecklonia maxima seaweed extract
    (Stellenbosch : Stellenbosch University, 2022-12) Samuels, Liam Jay; Blancquaert, Erna; Setati, Mathabatha Evodia; Stellenbosch University. Faculty of Agrisciences. Dept. of Viticulture and Oenology.
    ENGLISH ABSTRACT: Conventionally grape cultivation makes use of chemical fertilizers and synthetic pesticides to sustain growth and ensure fruit production ultimately resulting in more resistant grapevines to pests and diseases. However, their application also imposes a negative impact on human health and the environment. Therefore, agricultural sustainability is increasingly encouraged to ensure food safety and the conservation of the environment. In this regard viticulture is urgently seeking alternative solutions to increase sustainability. Biostimulants is a novel and sustainable tool which may be a good alternative to reduce the chemical-dependency typical of conventional cultivation practices, thereby supporting eco-friendly methods to increase agricultural outputs, via fruit quality, and resistance to abiotic stress. Biostimulants is the term given to a range of products, of natural origin, known to promote vegetative and reproductive growth as well as increase tolerance against biotic and abiotic stress. Seaweed extracts, a sub-category of biostimulants, have been highlighted as one of the most promising groups of biostimulants for use in viticulture. Commercial seaweed extract products are often derived from Ascophyllum nodosum (often located on the coasts of the North Atlantic), with studies on its effects on plant physiology being very popular and well-researched. The South African species, Ecklonia maxima, and its effects on wine grapes, is however under researched. The aim of this study was to evaluate the influence of Ecklonia maxima (EM) seaweed extract on vine physiology, grape and wine quality within in the South African viticultural context on Vitis vinifera L. Cabernet Sauvignon. Foliar applications of a commercial Ecklonia maxima seaweed extract biostimulant, under the trade name Kelpak ®, were conducted before flowering, at fruit set and at véraison under two irrigation scenarios ((i) irrigated and (ii) non-irrigated). Grapevine photosynthetic capacity, chlorophyll content and water status were not impacted by the application of Ecklonia maxima. Leaf area and fruit composition were positively impacted by the treatments. The combination of EM applications and irrigation resulted in significantly higher leaf area. Furthermore, the leaf area continuously increased throughout the season which is the first study to report this finding. The latter resulted in increased concentrations of sugars, organic acids, amino acids and phenolic content in grape berries. Surprisingly, the increased concentrations of the metabolites at harvest were not reflected in the wine parameters (final alcohol content (%), wine pH and total acidity and phenolic content) made in this study. This suggests that fermentation kinetics altered the extraction of these metabolites. Our findings correspond with that of the use of Ascophylum nodosum which resulted in increased fruit quality. The study was only conducted in one season and subsequent seasons should be conducted to have a clear understanding of the photosynthetic, water use, and metabolic pathways induced when using Ecklonia maxima. Furthermore, Ecklonia maxima should be used in conjunction with conventional farming to enhance grapevine functioning and fruit quality.
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    Effect of post-harvest summer pruning on carbohydrate reserve status, bud break and fertility of Sultanina H5 in the Lower Orange River region.
    (Stellenbosch : Stellenbosch University, 2022-12) Toolo, Keboneilwe Boitumelo; Avenant, Eunice; Avenant, J. H.; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology.
    ENGLISH ABSTRACT: Carbohydrate reserves produced from photosynthesis are stored in perennial tissues of the grapevine in the form of starch and free-sugar fractions or soluble sugars, mostly sucrose, glucose, and fructose. These reserves are highly affected by viticultural practices altering the source-sink relationship in the grapevine. Post-harvest pruning is a practice widely applied by several table grape producers in South Africa aiming to channel carbohydrate reserve accumulation to the remaining shoots. Due to the high input costs of table grape production, any manipulation, including post-harvest summer pruning, should be applied only if it is scientifically proven to have practical and economic benefits. This study, comprising of two trials, aimed to determine whether post-harvest pruning results in increased carbohydrate reserve status, improved bud break and fertility, as well as to establish a base for quantifying and practically assessing the carbohydrate reserve status of grapevines. The first trial focused on establishing the seasonal dynamics of non-structural carbohydrate (NSC) reserves of Vitis vinifera L. cv. Sultanina H5 in the semi-arid Lower Orange River (LOR) and the Mediterranean Hex River Valley (HRV). Root, trunk, cane and/or shoot tissues were sampled monthly and analysed for NSC. The Anthrone method was used to analyse soluble sugars and starch, while enzymatic analysis was used to quantify specific sugars (sucrose, d-fructose and d- glucose). Starch and sucrose were the most abundant forms of NSC in all tissues in both regions. In both regions, soluble sugars in permanent tissues (roots, trunks, canes) reached their highest concentration during dormancy (June-July). The starch concentration was low in all tissues in winter (July), during grapevine dormancy, whereafter it increased to a peak occurring in August (before bud break). A steep decrease in starch concentration was recorded from dormancy to flowering in both regions, indicating a dependency of the vine on carbohydrate reserves during that period. Accumulation of NSC reserves began after flowering to the post-harvest period, reaching their second peaks in autumn. The overall higher soluble sugars and starch (roots and canes) concentrations in the tissues of the Mediterranean region is ascribed to the earlier accumulation of reserves, lower crop load and a shorter post-harvest period characteristic of this region. A basis was established for sampling grapevine tissues for qualitative assessment of grapevine NSC reserve status, linking sampling time to occurrence of peaks in soluble sugars and starch concentrations. It is recommended that sampling for qualitative assessment of soluble sugars should be done after leaf fall, during dormancy (June-July under the conditions of this study). Starch concentrations should be assessed before bud break (August under the conditions of this study). Based on significant positive correlations between NSC concentrations of different tissue types, tissue types that could be sampled for indication of the overall NSC status of the grapevine were identified. Canes and shoots could be sampled and analysed to indicate the overall NSC reserve status of the vine. These tissues are considered the most practical tissue types to sample for determination of grapevine NSC reserve status. The second trial investigated the effect of post-harvest summer pruning applied in the semi-arid Lower Orange River region on NSC reserve status, bud break and fertility of Sultanina H5 grapevines. Five post-harvest pruning treatments were applied, namely an early 33% and a 66% shoot removal pruning treatment one day after harvest (33_1dAH and 66_1dAH respectively), a late 33% and a 66% shoot removal pruning treatment 45 days after harvest (33_45dAH and 66_45dAH respectively) and a control (Ctr), in which no post-harvest summer pruning was applied. To quantify pruning severity, the number and length of removed shoots, as well as the number of leaves and leaf area removed were determined at the time of the post-harvest summer pruning treatment application. After winter pruning was applied, the removed canes and shoots were measured to calculate the overall shoot length and leaf area removed per vine. The day after pruning treatments were applied, photosynthetically active radiation (PAR), as well as photosynthetic activity and related physiological parameters were measured. Cane and/or shoot, stem and root tissue, were sampled on 4 dates for assessing the effect of pruning treatments on NSC reserve status. Bud break and fertility were assessed through forced bud break and bud dissection for potential bud break and fertility, while actual bud break and fertility were assessed in the vineyard. Post-harvest pruning proved to be beneficial for light penetration, but it did not improve the photosynthetic rate of the leaves. A few significant differences were recorded on the impact of the treatments on TNC. These however, do not show a clear trend. Post-harvest summer pruning did not have a significant effect on final bud break and potential fertility of grapevines in the season following the treatment. Based on this one season’s results, post-harvest pruning did not have overall practical benefits. Repeating the treatments for two more seasons on the same data vines, would indicate whether there is a carry-over effect of the practice on NSC, bud break and fertility. It is recommended that in a further phase of this project, available rapid and accurate methods to quantify carbohydrate reserves should be used and/or evaluated for use in grapevine studies, including Near-Infrared spectroscopy, as well as the starch iodine test (already commercially used in the apple and forestry industries).