Plant protoplasts are frequently used for transfection and have been applied in various plant species for the functional characterization of genes and the study of signaling pathways. Described systems for the use of protoplasts in grapevine (Vitis vinifera L.) are still rare, mainly due to the complexity of obtaining a sufficient quantity of viable protoplasts, transient transfection, and subsequent cultivation under in vitro conditions. The aim of the master's thesis was to optimize the isolation, cultivation, and transfection of protoplasts from the tissue culture of Vitis vinifera L. cv. Zweigelt, thereby contributing to the development of methods to enhance grapevine resistance to phytoplasmas. During enzymatic isolation of protoplasts, we examined the effects of leaf homogenization, leaf age, tissue culture age, enzyme solution composition and enzymatic digestion time. A trend was observed indicating better isolation from the first fully opened leaves, cut into 3 mm wide strips, from younger tissue cultures, with a five-hour enzymatic digestion using solution Z, the composition of which was adapted from Zhao et al. For the purpose of optimizing protoplast culture conditions, we also included shaking in some of the samples. We found out that shaking reduced viability, while protoplasts remained viable for up to three days when kept stationary. Protoplast transfection was performed in the presence of PEG and plasmid. The success of the transfection was confirmed using a plasmid carrying the eGFP gene, whose fluorescence was detected under a confocal microscope. With the LR reaction, which enables site-specific recombination between the attL and attR sites, we constructed a plasmid containing the sequences for the Cas9 protein, the guide RNA, and a fragment of the vvCWINV1B gene. The aim of this design was targeted inactivation of the vvCWINV1B gene, which, based on previous studies, shows increased expression upon phytoplasma infection. The use of such a transformed plant would allow for functional analysis of the gene's role during pathogenic interaction. Protoplasts from embryogenic callus of Zweigelt grapevine were used for transfection. The reaction was performed with 1.5 × 10$^5$ protoplasts and 10 μg of plasmid and was stopped after 5 minutes. We aimed to verify the success of the transfection using Sanger sequencing, but encountered difficulties in amplifying a portion of the genomic DNA. The results of our experiments revealed trends that serve as a foundation for further optimization studies. Based on these findings, it would be advisable to focus future experimental efforts on improving the protocols for isolation and cultivation of protoplasts from leaf tissue of the grapevine cultivar Zweigelt.
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