CRISPR/Cas9-based targeted Lipoxygenase-2 gene editing in mungbean via transient transformation assays

Lipoxygenase 2 converts polyunsaturated fatty acids into volatile compounds, imparting an undesirable beany flavor to processed mungbean products. This experiment aims to assess the suitability of CRISPR/Cas9-based genetic modification of mungbean using protoplasts and agroinfiltration transient transformation assays. Robust and viable protoplasts were isolated using the tape sandwich method. Protoplast transfection was carried out using 40% PEG. A binary CRISPR/Cas9 construct targeting the Lipoxygenase 2 gene was agroinfiltrated into the abaxial leaf surface of two-week-old seedlings. Higher protoplast yield was obtained from fully grown, unifoliate leaves from 7-day-old seedlings compared to 12-day-old seedlings. The CaMV 35S promoter and CmYLCV are the best fits for transgene expression in mungbean and were selected for Cas9 and gRNA expression, respectively. Protoplast transformation with 5 minutes of PEG incubation achieved the highest transformation efficiency (~55%), compared to 15 and 30 minutes of PEG incubation. Mutations were detected by linear amplicon sequencing of the PCR-amplified target region. Transformed protoplasts showed a combination of mutation types, primarily insertion/deletions, with some substitutions on all gRNA target sites. Additionally, robust RUBY pigmentation and GUS expression were obtained, indicating successful transient expression of T-DNA. In the agroinfiltration assay, mutations were detected at target sites guided by gRNA1 and gRNA2. Hence, an efficient method for assessing CRISPR/Cas9 activity in mungbean using protoplast transformation and agroinfiltration was established. This study provides a practical foundation for future efforts towards CRISPR/Cas9-mediated targeted modification of the Lipoxygenase 2 gene via stable transformation to potentially reduce undesirable beany flavor in mungbean.