In vitro gamma ray (60Co) mutagenesis is a powerful tool to achieve variability in commercial rice lines used by farmers, such as Lazarroz FL. We previously reported the optimized in vitro gamma mutagenesis system for Lazarroz FL Indica callus. As a continuation, in the present study, we targeted the ACC2 gene mutagenesis that provides tolerance to aryloxy-phenoxy-propionate (APP) fluazifop-P-butyl as a model to show the system's potential to create variability while providing a solution for weed management. The DL50 of fluazifop-P-butyl was calculated in calli as DL50 = 6,93 mg/L (0,425 mg/L − 15,743 mg/L, R2 = 0,402, 1000n) and regenerated vitroplants at an LD50 of 3.771 mg/L (R2 = 1, 290n). We used 5 mg/L fluazifop-P-butyl as a selection agent and the second round of selection of 10 mg/L (3000 vitroplants) resulted in one survivor plant when using calli as a starting material. The putative tolerant plant also tolerated 150 mg/L in the greenhouse. The ACC2 gene was sequenced, and a heteroecious mutation, T2222I/T2222M, was discovered that may be linked to tolerance. We improved the in vitro system by using seeds as a gamma irradiation starting point instead of embryogenic calli, followed by calli induction, regeneration, and exposure to the selection agent. The modification allowed higher gamma doses with an LD50 of 350 Gy and one to thirty-one putative tolerant plants. The in vitro model showed that gamma-ray mutants from recalcitrant indica rice materials are possible, and the use of selection agents such APP can help create variability useful for breeding a more resilient rice.

In vitro gamma ray (60Co) mutagenesis is a powerful tool to achieve variability in commercial rice lines used by farmers, such as Lazarroz FL. We previously reported the optimized in vitro gamma mutagenesis system for Lazarroz FL Indica callus. As a continuation, in the present study, we targeted the ACC2 gene mutagenesis that provides tolerance to aryloxy-phenoxy-propionate (APP) fluazifop-P-butyl as a model to show the system's potential to create variability while providing a solution for weed management. The DL50 of fluazifop-P-butyl was calculated in calli as DL50= 6,93 mg/L (0,425 mg/L - 15,743 mg/L, R2 = 0,402, 1000n) and regenerated vitroplants at an LD50 of 3.771 mg/L (R2 = 1, 290n). We used 5 mg/L fluazifop-P-butyl as a selection agent and the second round of selection of 10 mg/L (3000 vitroplants) resulted in one survivor plant when using calli as a starting material. The putative tolerant plant also tolerated 150 mg/L in the greenhouse. The ACC2gene was sequenced, and a heteroecious mutation, T2222I/T2222M, was discovered that may be linked to tolerance. We improved the in vitro system by using seeds as a gamma irradiation starting point instead of embryogenic calli, followed by calli induction, regeneration, and exposure to the selection agent. The modification allowed higher gamma doses with an LD50 of 350 Gy and one to thirty-one putative tolerant plants. The in vitro model showed that gamma-ray mutants from recalcitrant indica rice materials are possible, and the use of selection agents such APP can help create variability useful for breeding a more resilient rice.