CRISPR/Cas9 게놈 편집을 위한 Agrobacterium 매개 유전자 변형 및 식물 재생 시스템의 대마 (Cannabis sativa L.) 에서의 확립 = Agrobacterium-mediated genetic transformation and plant regeneration for CRISPR/Cas9 genome editing in hemp (Cannabis sativa L.) plant

다국어 초록 (Multilingual Abstract)

Cannabis (Cannabis sativa L.) is cultivated globally for its applications in medicine, fiber, oil, and seed production. With growing interest in cannabidiol (CBD) for its therapeutic potential and increasing regulation of tetrahydrocannabinol (THC) due to its psychoactive effects, the development of Cannabis cultivars with high CBD and low or null THC content has become a major breeding objective. Agrobacterium-mediated transformation is a well-established method for producing stably transformed plants, and recent advances in CRISPR/Cas9 gene-editing technology have further enabled precise genome modifications in a variety of plant species. In this study, I aimed to establish an efficient and reproducible in vitro regeneration and transformation system for Cannabis, in combination with CRISPR/Cas9 genome editing, to generate gene-edited lines with altered cannabinoid profiles.

Sterilization protocols were optimized using seeds of the Cannabis cultivar ‘Cheongsam’. Seeds treated with 1% hydrogen peroxide (H₂O₂) for 2 days in the dark, followed by husk and embryo membrane removal and a 0.2% sodium hypochlorite (NaClO) treatment for 15 minutes, showed a contamination rate reduced to 5%. Further contamination control was achieved by culturing on Murashige and Skoog (MS) medium containing 300 mg/L timentin, which effectively suppressed microbial growth without inhibiting seedling development. Cotyledon explants containing shoot apical meristem (SAM) regions and mature embryos were used for regeneration. Explants were co-cultivated with Agrobacterium tumefaciens strains LBA4404, GV3101, and AGL1 harboring CRISPR/Cas9 vectors pECO200 and pBAtC targeting the CsTHCAS gene, which encodes tetrahydrocannabinol acid synthase. Explants were cultured on MS medium supplemented with 0.4 mg/L thidiazuron (TDZ) and 0.2 mg/L 1-naphthaleneacetic acid (NAA), followed by transfer to rooting medium. After selection and molecular analysis, a total of six gene-edited Cannabis lines were obtained with insertions and deletions (In/Del) mutation rates of 0.6%, 0.2%, 0.3%, 0.3%, 0.2%, and 0.2%, respectively. This study successfully integrated sterile culture techniques, regeneration protocols, and CRISPR/Cas9-based genome editing to generate transgenic Cannabis lines with modified cannabinoid biosynthesis. These results provide a technical foundation for the development of stable, low-THC Cannabis cultivars through precise genetic modification.

목차 (Table of Contents)

• Table of contents i
• List of tables ⅳ
• List of figures ⅵ
• ABSTRACT viii
• 1.Introduction 1
• 2. Materials and methods 4
• 2.1 Plant materials and sample preparation 4
• 2.1.1 Establishment of sterile in vitro culture conditions 4
• 2.1.2 Protoplast culture 5
• 2.1.3 Explant culture 5
• 2.2 Evaluation of sgRNA efficiency using gene scissors technology 6
• 2.2.1 Isolation and Analysis of CsTHCAS Gene 6
• 2.2.2 Design of Single guide RNAs (sgRNAs) 7
• 2.2.3 Isolation of protoplasts 7
• 2.2.4 Polyethylene glycol (PEG) -mediated protoplast ribonucleoprotein (RNP) transfection 8
• 2.2.5 Next-generation sequencing (NGS) analysis 8
• 2.3 Gene editing vector cloning 8
• 2.3.1 Design of sgRNA oligonucleotides 8
• 2.3.2 Cloning into the pBAtC vector 9
• 2.3.3 Cloning into the pECO200 vector 10
• 2.4 Establishing plant regeneration conditions by evaluating Cannabis regeneration rates 10
• 2.4.1 Optimization of selection medium using phosphinothricin (PPT) 10
• 2.4.2 Agrobacterium-mediated transformation 11
• 2.5 Generation and analysis of gene-edited Cannabis plants 12
• 2.5.1 Genomic DNA (gDNA) extraction and T-DNA confirmation 12
• 2.5.2 Next-Generation Sequencing (NGS) for Indel detection 12
• 3. Results 35
• 3.1 Establishing of callus induction conditions via Cannabis cell differentiation rate assessment 35
• 3.1.1 Effect of 1% H2O2 on the germination rate of Cannabis seeds 35
• 3.1.2 Effect of NaClO concentration on the growth of Cannabis seedlings 39
• 3.1.3 Effect of antibiotic-based contamination suppression on the growth of Cannabis seedlings 42
• 3.2 Selection of Cannabis gene guide RNAs and evaluation of editing efficiency 45
• 3.2.1 Isolation and analysis of CsTHCAS gene 45
• 3.2.2 Design of single guide RNAs (sgRNAs) 49
• 3.2.3 Protoplast isolation 51
• 3.2.4 PEG-mediated RNP transfection in protoplasts 53
• 3.3 Agrobacterium-mediated transformation using three strains and plant regeneration from Cannabis sativa explants 56
• 3.3.1 Results of tissue culture using SAM explants 56
• 3.3.2 Results of tissue culture using mature embryos 59
• 3.4 Generation gene-edited Cannabis plants 61
• 3.4.1 T-DNA verification and mutation analysis 61
• 4. Discussion 66
• 5. References 68