Tissue culture optimization and genome editing for yield improvement of an Indian rice landrace Chittimuthyalu

Chittimuthyalu, a rice landrace from Southern India, it known for its pleasant aroma, rich nutritive value, and excellent cooking qualities. However, it has poor plant type (tall and weak stem prone to lodging), and gives very less grain yield. The efforts to improve such precious rice accessions with existing cross breeding or random mutagenesis often results undesirable traits due to linkage drag or untargeted mutations in large numbers. Genome editing, the most precise breeding tool, offers a viable solution to address such issues. In this study, we developed an efficient tissue culture protocol for callus induction, transformation, and regeneration of Chittimuthyalu. The highest callus induction frequency was achieved on L3 basal media enriched with 2.5 mg/l 2,4-Dichlorophenoxyacetic acid (2,4-D) and 600 mg/l of both proline and glutamine. For regeneration, a combination of Thidiazuron (TDZ), 6-Benzylaminopurine (BAP), and kinetin yielded an optimal regeneration frequency. The optimized tissue culture protocol was utilized to transform a multiplex gene editing construct developed by combining the four guide RNAs designed from yield and disease resistance associated genes OsDEP1 , OsTB1 , OsCKX2 , and OsSWEET14 . The OsDEP1 genome-edited rice plants exhibit thicker culm, enhanced grain size, ~ 100% increase in the thousand grain weight, and ~ 50% increase total grain yield per plant. The optimized tissue culture protocol and developments of further edits in remaining genes will pave the way for improving agronomic traits of Chittimuthyalu. This study also highlights much needed efforts to develop efficient tissue culture and genome editing methods for wild rice species and landraces which will help bringing these hardy, climate resilient and nutrient rich accessions into mainstream cultivation.