Browsing by Author "Derman, Shannon Skye"

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    Protoplast-based biotechnology methods for Vitis vinifera
    (Stellenbosch : Stellenbosch University, 2020-12) Derman, Shannon Skye; Vivier, Melane A.; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
    ENGLISH ABSTRACT: As a major fruit crop worldwide, grapevine production yields the raw materials for the table grape, wine grape, raisin and grapeseed oil industries alike and mostly rely on commercial varieties of the European grape, Vitis vinifera L. As with most widely planted crops, the potential impacts of rapidly changing climatic conditions and associated biotic and abiotic stressors demand a renewed focus on plant improvement strategies. One of the techniques that has been prominent in the recent wave of novel crop improvement methods is that of protoplast biotechnology. Grapevines exhibit recalcitrance towards several biotechnological procedures, including protoplast methodologies. Although some successes have been reported, the potential benefits of protoplast-based methods are far from routine in grapevine science. This study aims to contribute to an existing body of grapevine protoplast research, by evaluating the standard methods of protoplast isolation from both Vitis embryogenic calli and other grapevine explants as productive sources of viable protoplasts and test their usefulness towards a number of applications. Somatic embryogenic cultures from three Vitis vinifera cultivars, namely Chardonnay, Pinotage and Muscat were established from immature influorescence-derived explants (anthers, ovaries and whole flower). Genotype-specific variability was obvious in the ability to form callus and specifically the extent of embryogenic callus recovery. Productive somatic embryogenic cultures were recovered from all cultivars, as well as regenerated plantlets, confirming the regeneration ability of the cultures. Chardonnay is known to respond well to culturing, which was confirmed in this study, and therefore was used for protoplast isolations, optimisations and application analysis. Two established enzymatic methods of protoplast isolation were first compared to identify the more superior of methods. The method using the higher concentration of enzymes (Cellulase (2%), Macerozyme (1%), Pectolyase (0.05%)) was higher yielding with a good viability of protoplasts recorded, and this method was then used to further evaluate and solve a number of technical issues during the isolation procedure. Adaptations were introduced to reduce the number of undigested cells remaining in the isolate after digestion, and to resolve aggregation of protoplasts to each other and to cell debris. The inclusion of a pre-isolation step of coating all plastics in Bovine Serum Albumin reduced protoplast aggregation but did not solve this problem. The optimised method, with a 12-hour digestion period, yielded an average of 9.4x10 cells per 100 mg of somatic embryo calli. The protoplasts were characterised using fluorescent microscopy to evaluate their integrity and viability, to assess the presence of cellulose in remaining cell walls after isolation, as well as to confirm that the sub-cellular structures of the protoplasts could be visualised using organelle-specific markers. Two other grapevine explants were also tested for potential use as efficient and viable protoplast sources, namely zygotic embryos, obtained from using embryo rescue techniques, and meristematic bulks, formed from shoot growth tips manipulated to form meristematic bulks in culture. An extension of the digestion period resulted in an increased yield of protoplasts from zygotic embryos, whilst a pre-plasmolysis treatment of the meristematic bulks increased the yield, but at a cost to the viability of the protoplasts. Our results showed that 1.36 times more protoplasts could be isolated from meristematic bulks compared to zygotic embryos. Despite explant-specific optimisations, 5 times and 3.7 times more protoplasts could be isolated from SEC than from zygotic embryos and meristematic bulks respectively. One of the advantages of meristematic bulks is the fact that it can be established relatively easily on demand, unlike embryogenic cultures (somatic and zygotic) whose explants are highly seasonal. The culturing of the protoplasts would need further time and experimentation and was beyond the scope of this study. Preliminary analyses of protoplasts from somatic embryogenic cultures and meristematic bulks, subjected to culturing confirmed that cell divisions occurred and the appearance of microcalli was evident, but no embryos formed yet. In line with the drive towards protoplast-based genome editing techniques in grapevine, somatic embryogenic protoplasts were subjected to transfection with the YFP reporter gene. Results showed positive transfection in protoplasts deriving from both Sultana and Garganega cultivars, at a transfection efficiency of <18% in both. Stable transformation of Chardonnay embryogenic calli using the GFP reporter gene was also conducted to be used as a resource for subsequent protoplast isolation experiments and as a control system for future transgene expressing protoplast systems. Multiple points of GFP expression were detected within the calli, but these calli tend to rapidly necrotise under selection and grow very slowly. Further transformations would be needed to secure the transgenic callus lines for future experiments. This study also attempted to use flow cytometric techniques to characterise and sort protoplast populations. The method was successful in characterising the protoplasts in solution and differentiating a sub-population with “ideal” characteristics from potentially less optimal subpopulations. However, when the sorted protoplasts were “harvested”, the recovery of viable protoplasts was not possible, and this aspect therefore needs further optimisations. This study was intended towards method validation, optimisations, as well as establishing resources and workflows to make protoplasting successful in our environment. Towards those aims, the study was successful and also expanded the current body of work on grapevine protoplasting by introducing results on two additional explants towards protoplast generation and potentially regeneration, as well as providing promising evidence that cell sorting of protoplasts could be a valuable addition in protoplasting workflows to characterise the populations, but hopefully also ultimately recover only the desired fractions. It is clear that the biggest challenge remains to make regeneration of protoplasts a routine technique to realise the full potential of protoplasts in grapevine biology and biotechnology.