PROTOPLAST-BASED TECHNOLOGY FOR IMPLEMENTATION OF NEW BREEDING TECHNIQUES IN CHICORY spp.

Chicory (Cichorium intybus L.) is a nutritionally and medicinally valuable plant with increasing significance in modern agriculture and biotechnology. This study aimed to optimize in vitro culture, protoplast isolation, and transfection protocols for 12 different chicory biotypes, laying the groundwork for future genome editing applications. Initially, in vitro cultures were established for all biotypes, ensuring consistent plant material for experimentation. Leaf characterization was performed to analyze morphological differences across biotypes. Following successful protoplast isolation, viability assays were conducted to determine the health and stability of the extracted protoplasts. Transfection was then carried out using two different methods: Polyethylene Glycol (PEG)-mediated transfection and electroporation, to evaluate their efficiency in DNA delivery. Following transfection, DNA extraction was performed from the protoplasts, followed by gel electrophoresis to confirm successful DNA uptake. To further validate the results, Sanger sequencing was conducted, focusing specifically on the Verona biotype, chosen for its agricultural and commercial importance. The findings from this study contribute valuable insights into protoplast-based transformation techniques in chicory, providing an efficient system for DNA-free genome editing and molecular studies. The optimized methodologies developed here offer a foundation for further genetic advancements in chicory, with potential applications in crop improvement and biotechnology.