Rice (Oryza sativa L.) is a crucial crop responsible for 20% of calories of half the human population and is facing constraints of yield related to biotic factors, such as pathogens, and abiotic stresses, such as salinity, drought, heat or cold [1–2]. Breeding can introduce desired traits based on genetic variability related to heat, salt, soil acidification stress, and plague tolerance to the few cultivars preferred by farmers in specific geographic areas [3–6]. Rice-induced genetic variability obtained by seed irradiation is well known and has been used since it was proven in 1928 on vegetables; however, it is slow and requires land, resources, and human capabilities to find desired traits [7–10]. Plant tissue culture provides a platform to produce gamma mutations from primitive embryogenic cells [11–13]. We previously reported a system to create variability using in vitro gamma radiation at an optimized dose of 60 Gy in embryogenic calli of Lazarroz indica rice [14]. The proposed model required a method to narrow the in vitro selection while correlating with desired mutations. In this paper, we suggest resistance to stressors such as the herbicide aryloxy-phenoxy-propionate (APP) to regenerate complete plants from tolerant mutated cells as a model for such challenges. The herbicide is commonly used to control weeds in rice by acting on the ACC2 enzyme, which is related to fatty acid synthesis, while mutations in the carboxyl transferase domain of ACC2 correlate with APP tolerance, as described below [15–21].
The herbicides acting on the ACC2 enzyme include at least two main groups, aryloxy-phenoxy-propionates (APP) and cyclohexanediones [15]. In grasses, there are two types of ACCases, cytosolic and plastidic. The second ACC2 is affected by APPs in rice. The APPs include clodinafop-propargyl, cyhalo-fop-butyl, fenoxapropethyl, metamifop, diclofop-meth fenthiaprop, quizalofop-ethyl, haloxyfop-R-methyl, and fluazifop-P-butyl. Fluazifop is a molecule of 327.25 g mol-1 that can control grasses at a dose of 210 g ai Ha− 1 and 420 g ai Ha− 1 preemergent with low residual effects [15–16]. Specific mutations in ACC2 enzymes correlate with APP tolerance, as described below.
The enzyme acetyl-CoA carboxylase 2 ACC2 (EC 6.4.1.2, UniProt: A2Y2U1) is located on chromosome 5 at amino acids 14,067,726 − 14,079,652 and is delivered to the plastid. Mutations in the carboxyl transferase domain of ACC2 between amino acids 1,781-2,078 and 2,027 − 2,096 are related to APPS tolerance, such as Ile1781-Leu, Trp-1999-Cys, Trp-2027-Cys, Ile-2041-Asn, Ile-2041-Val, Asp-2078-Gly, Cys-2088-Arg, and Gly-2096-Ala [16–19]. Mutations such as A2004 V provide wheat with tolerance to 10 g ai Ha-1 of quizalofop [16, 17]. Similarly, Oryza japonica ACCase 2 (LOC_Os05g22940), mutations on I1781 V, C2088R, and W2027C, also provide tolerance to APPs [21]. Finally, an indica rice with a mutation tolerant to APPs with the mutation I1781 L has been available in the USA market since 2018 [22].
This paper aims to improve our previous system and obtain more predictable traits that correlate with specific mutations. Here, we present a nonreported mutation, T2222I/T2222M, that may be linked to tolerance to APPs and present an improvement of our earlier in vitro system by using seeds instead of calli during gamma irradiation. We also show aryloxy-phenoxy-propionate (APP) fluazifop-P-butyl tolerance as a model to demonstrate the system's potential for incorporating features that correlate with mutations. The obtained mutant can be incorporated into the breeding program to look for functional variability.