Frequency and Spectrum of Chlorophyll Mutations in Rice (Oryza sativa L.)

Rice is one of the main staple food crops for almost half of the global human population. The generation of new variations is the first objective of the crop breeding programme for genetic improvement. Induced mutagenesis  is a universal technique (Bhat and Hakeem, 2023) to create novel alleles in rice (Jadhav et al., 2023) and forward (Gurunathan et al., 2019) and reverse (da Luz et al., 2021) genetic approaches are practiced to identify functional mutations. In rice breeding, many mutagenic methods have been applied to induce functional mutations, including ionizing radiation (e.g., fast neutrons, γ rays, X rays) and chemical substances (e.g., EMS, DEB, sodium azide) (Gulfishan et al., 2023) to generate varieties with enhanced genetic architecture (Kumawat et al., 2022). Among these, EMS is one of the majorly utilized mutagens to generate the mutant population in rice (Hameed et al., 2019; Jia et al., 2019) since the EMS possesses plasticity across the crop plants to generate novel genetic variations. Mutagenesis with EMS is an expensive method to induce high rates of point mutations in the crops (Gillmor and Lukowitz, 2020). Understanding on biological effects of mutagens is important for the successful plant breeding programmes (Jankowicz-Cieslak et al., 2021).
       
Moreover, the accuracy in following the mutagenesis protocol is also important to get the appropriate mutant population in crops with high rates of mutation to achieve the targets in the breeding programmes. The best indication of mutation occurrence in a mutagenesis protocol is the presence of chlorophyll mutants in the M₂ generation. In this study, the EMS mutagen was used to create the mutant population in a rice variety CO51, recorded the different types of chlorophyll mutants and analyzed the frequency of chlorophyll mutations at different doses of EMS.

Determination of lethal dose
 
A high-yielding variety, CO51, has been taken for the mutagenesis study. Ten batches of 100 healthy rice seeds each were pre-soaked in distilled water for 24 hours. Pre-soaked seed batches were treated with 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% and 1.0% of ethyl methanesulphonate (EMS) for 12 hours (Unan et al., 2022). Besides, the control batch without EMS treatment was also subjected. Immediately after the EMS treatment incubation period, after decanting the EMS liquid, 5% sodium thiosulfate solution was added to the treated seed batches to neutralize the EMS. Subsequently, seeds were thoroughly washed under running tap water for 6 hours to remove the residual EMS (Sagel et al., 2017). The seeds were sown in the raised bed nursery under field conditions. The observation on survival and lethality of the seedling for the different doses of EMS has been recorded at 14 DAS (Table 1). A kill curve analysis was carried out to express the lethality in a diagrammatically (Fig 1).




 
Generation of M₁ seeds and raising of the population
 
Since the LD value has resided between 0.5 to 0.6% of EMS dose, these two doses are used to generate M₁ generation along with a high dose of 0.7% of EMS. Hence, each batch containing 100 g of seeds was used to generate the M₁ generation with the aforementioned EMS doses. The seedlings were transplanted to the field 20 days after sowing. The seeds were harvested from 2462 healthy plants on a single plant basis. In these, seeds from 1253 (0.5% EMS), 842 (0.6% EMS) and 367 (0.7% EMS) plants were harvested.
 
Raising of M₂ families
 
1030, 710 and 360 M₂ families were raised from the seeds of M₁ plants harvested from the 0.5%, 0.6% and 0.7% EMS doses, respectively. The observations on chlorophyll mutants, viz., Albina, Striata, Xantha, Viridis, Aurea and Chlorina, were recorded at 10 DAS.

The mutagenic efficiency depends on the dose of the mutagen and it should be determined to get the optimum number of plant recovery with a higher number of mutations (Pratap and Kumar, 2011). The germination and survival rates progressively declined with increasing EMS concentrations (Table 1), which is consistent with prior findings (Hernández-Muñoz et al., 2019). Visible changes due to mutations induced by EMS could be expressed mostly in the M₂ generation (Jia et al., 2019) since most of the mutations are recessive. The presence of chlorophyll mutants in the M₂ progenies is an indicator for the occurrence of mutations in the mutant population (Prasannakumari et al., 2024). Our experiment generated different types of chlorophyll mutants, viz., Albina, Striata, Aurea, Xantha and Chlorina (Table 2; Fig 2). Lalitha et al. (2020) supported that the presence of mutations in the chlorophyll synthesis genes leads to the generation of different types of chlorophyll mutants.




       
In the 0.5 EMS dose, maximum and minimum frequencies of chlorophyll mutation were recorded as 66.6 and 3.57 in the M₂ families CO51-1973 M₁ and CO51-1285 M₁, respectively. In the 0.6% EMS dose, maximum and minimum frequency of chlorophyll mutation were recorded as 61.9 and 1.63 in the M₂ families CO51-778 M₁ and CO51-573 M₁. In the 0.7% EMS dose, maximum and minimum frequency of chlorophyll mutation were recorded as 39.39 and 1.58 in the M₂ families CO51-237M₁ and CO51-272M₁.
               
A total of 243, 200, 154 and chlorophyll mutants were found at the doses of 0.5% EMS, 0.6% EMS and 0.7% EMS, respectively. The chlorophyll mutation frequency from the observed plants is 16.99 (0.5% EMS), 11.35 (0.6% EMS) and 11.40 (0.7% EMS) (Table 3a). Besides, chlorophyll mutant frequency in terms of family volume numbers tested was 3.59 (0.5%), 6.47 (0.6%) and 9.44 (0.7%) (Table 3b). The increases in the EMS dose lead in increases in the chlorophyll mutation frequency in the M₂ families (Fig 3). Prasannakumari et al., (2024) reported that the frequency of chlorophyll mutation is increased when increasing the EMS and Gamma doses in rice.

The present study aimed to find out the mutagenic effects of ethyl-methane-sulfonate (EMS) on rice variety CO51 to generate genetic variation for crop improvement. The germination and survival rates progressively declined with increasing EMS concentrations. The LD₅₀ resided between the EMS doses of 0.5% and 0.6%, which indicates that the EMS doses between 0.5% and 0.6% could give enough plant recovery with mutations in the mutagenesis in rice. The occurrence of chlorophyll mutants in the M₂ generation confirms the accuracy level in the conduct of experiments in the mutation population generation. The frequency of mutation in the M₂ families recorded that 3.59, 6.47 and 9.44 for the EMS doses of 0.5%, 0.6% and 0.7% proves that an increase in the EMS dose leads to an increase in mutation frequency in rice. Conclusively, the mutant populations generated are highly reliable for screening for various traits of interest.