DEVELOPMENT OF A PROTOPLAST ISOLATION AND PEG-MEDIATED TRANSFECTION METHODS FOR PLASMIDS AND CAS9 PROTEIN IN CHILI (CAPSICUM ANNUUM L.) CV. INATA AGRIHORTI

• Main Author: Aji Sakti M P N, Hanggara
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/72684
• Institution: Institut Teknologi Bandung

Inata Agrihorti (Capsicum annuum L.) is a hybrid chilli cultivar with early maturation and high yield characteristics. Despite its many advantages, many chilli cultivars in Indonesia, including Inata Agrihorti, are still susceptible to various pathogens, such as infection by Potyvirus. The use of CRISPR/Cas9 genome editing technology can contribute to solving this problem by knocking out susceptibility genes. The CRISPR/Cas9 method can be performed transgenically or nontransgenically, with lower environmental risks. Therefore, an optimal method for transient transfection of Cas9 plasmids and/or proteins into Inata Agrihorti protoplasts is needed as an important step for non-transgenic genome editing applications. This study consisted of three stages of optimization: the optimization of chili mesophyll protoplast isolation, GFP:NLS plasmid (pK7WGF2) transfection, and Cas9:NLS:GFP protein transfection, both mediated by polyethylene glycol (PEG). Optimization of chili mesophyll protoplast isolation was carried out by the Tape Sandwich method combined with submersion using cellulase enzymes (1%–2%) and macerozyme (0.2%–0.4%) in three incubation time durations (1 hour, 2 hours, and 3 hours). The highest average yield of the protoplasts was 133,25 x 104 cells/mL from 0.1 g of chili leaves with a combination of 2% cellulase and 0.4% maserozyme within 3 hours of incubation. The plasmid transfection optimization aims to examine the effect of plasmid concentration (2.5 and 5 ?g/50?L) in 40% PEG 4000 and PEG 6000 on protoplast transfection efficiency. The best average efficiency of 48.71% was obtained from the 5 ?g/50?L plasmid and 40% PEG 4000 treatment. Optimization of Cas9 protein transfection was carried out to examine the effect of three protein concentration treatments (50, 100, and 1000 ?g/100?L) on the protoplast transfection efficiency mediated by 40% PEG 4000. The best Cas9 protein average efficiency obtained from the treatment of 1000 ?g of protein was 2.9%. These results indicate that the optimized methods can be further used to integrate the CRISPR/Cas9 system into the chilli protoplast in a non-transgenic manner.