Barnyard millet (Echinochloa spp.) is a climate-tolerant crop with high drought and other abiotic stress tolerance. It is a major food staple in most arid and semi-arid areas, contributing to nutritional security and income support to smallholder farmers. Echinochloa frumentacea (Indian Barnyard Millet or Billion Dollar Grass) is most widely cultivated crop species in India, especially in arid and semi-arid areas. It is an annual grass that has fast growth rates and yields well even with limited agricultural inputs. Seeds are rich in proteins, dietary fibre and essential minerals, making them a valuable food source. It is known for its tolerance to drought conditions and poor soils (Bhat et al., 2018; Nautiyal and Bankoti, 2026). To overcome the effects of climate change-associated stresses and to improve the yield of millets, there is a need to develop stress-tolerant and high-yielding varieties. The rising incidence of drought caused by climate change creates the need for more climate-tolerant crop varieties.
       
There are several possible approaches to increase the stress tolerance and productivity of millets. Ever since the epoch-making discoveries of Muller and Stadler eighty years ago, the employment of mutation techniques using different agents of physical and chemical nature has generated an enormous amount of genetic diversity and has been of immense value in modern plant breeding and genetic studies. The use of induced mutations over the past half century has been highly significant in the development of crop varieties in every region of the world (Malekpoor et al., 2016). The widespread application of induced mutagenesis in plant breeding programs across the globe led to release of 3222 plant mutant varieties of 170 plant species in more than 60 countries aims across the globe. The developed varieties increase diversity and provide breeding material for conventional plant breeding thus contributing directly to the conservation and utilization of plant genetic resource (Ceccarelli, 2015). Mutation breeding has been used successfully for the improvement of crops during the past 80 years, as well as a complement to efforts exerted by traditional methods of plant breeding (Amin et al., 2015; Anne and Lim, 2020; Khan and Wani, 2005). Induced mutagenesis using chemical mutagen such as Ethyl Methane Sulphonate (EMS) has become an effective tool for plant improvement (Jankowicz-Cieslak et al., 2016; Lal et al., 2020; Priyadharshni et al., 2020). EMS induces high-frequency random point mutations across genome, enabling simultaneous modification of multiple loci associated with complex traits as drought tolerance (Chen et al., 2023; Lal et al., 2020). In millet crops, different approaches have been applied to mitigate the lodging stress and improve yield (Bizuayehu and Getachew, 2021; Jency et al., 2020) reported that mutation in Kodo millet CO3 variety by gamma radiation or ethyl methane sulfonate (EMS) could produce nonlodging mutants. They even developed mutants (named second mutants or M2) which showed higher lodging tolerance due to photosynthetic efficiency (Sharma, 1990). Mutation of alpha-tubulin-1 gene in tef by EMS produced the lodging-tolerant cultivar ‘Kegne’ (Jost et al., 2015). The present study aims to analyse the impact of EMS mutagenesis on increasing drought resistance in barnyard millet based on survival rates under water stress. The treatment involved pre-soaking and varying concentrations of EMS, followed by drought stress induction. The findings are useful in identifying the optimal EMS concentrations and treatment periods to enhance drought tolerance in barnyard millet (Pérez-Escamilla, 2017). Creating drought-resistant lines is vital in maintaining food security under changing climate scenarios (FAO, 2023). Induced mutations result from a gene interaction with mutagens or other environmental agents (Singh et al., 2021). Induced mutagenesis has been successfully used to generate more variability, facilitating for the isolation of mutants with useful characters of economic importance such as improved dwarf plant types for non-lodging, simultaneous maturity, heavy tillers, heavy grain yield, heavier seed size and useful seed color etc. in different crops (Ganapathy et al., 2008; Solanki and Sharma, 1999).  The optimal concentration of EMS for best shoot vigor and root development is 0.6%, which provides the best ratio of shoot vigour to root development. Higher doses of EMS (≥1.0%) promote a stress-adaptive response by increasing the allocation to root tissue while decreasing allocation to shoot tissue (i.e. reduced shoot growth and increased root allocation). The increased root: shoot ratio associated with greater concentrations of EMS may be due to a drought-stress response mechanism rather than increased productivity (Rubiales et al., 2018). The trends observed in this study are consistent with mechanisms of drought tolerance utilised by millet species that exhibit increased root growth and shifting of biomass partitioning towards increased root biomass in response to decreased water availability.

Experimental site and climatic conditions
 
The current study was carried out from 2022-2024 at two main sites- Research Laboratory, Department of Botany, School of Basic and Applied Sciences and School of Agricultural Sciences’ Agricultural field, Shri Guru Ram Rai University, Patel Nagar, Dehradun, Uttarakhand. The district of Dehradun is in the northern region of India and has geographical coordinates of latitude 29^o55' to 38^o31'N and longitude 77^o35' to 78^o20'E. The district lies at an average height of about 650 meters above sea level. The weather of Dehradun is typically categorised as sub-tropical, but it differs significantly with the season and elevation of the region. The topographical variation of the region is responsible for the change in temperature and weather patterns. There is a warm to hot climate during the summer season with temperatures varying from 27^oC to 40^oC. The warmer temperatures are typically observed in the summer months of May and June. In spite of the heat, the region sometimes experiences pre-monsoon rains, which bring temporary relief from the scorching heat. Dehradun experiences constant and intense rainfall during monsoons due to the effect of the Southwest Monsoon winds. The region is quite wet, with significant rainfall causing a humid climate and encouraging dense growth of vegetation. The region has an average rainfall of around 2200 mm annually, with most falling during the monsoon season. Weather in the winter months is cool to cold, with temperatures between 2^oC to 24^oC. The coldest months are typically December and January, where the temperature can occasionally drop below 2^oC. The weather during this period is mostly dry and crisp, with clear skies and lower humidity levels.
       
The climatic fluctuations of Dehradun make it an excellent place to conduct agricultural research, especially when it comes to the investigation of plant growth, physiology and adaptation to contrasting environmental conditions. The co-existence of warm summers, dense monsoon rainfall and chilly winters creates a wide variety of climatic conditions for investigating several crop species.
 
Plant material
 
Four varieties of barnyard were used in the experiment. Two varieties (PRJ-1 and VLM-207) were collected from the Uttarakhand State Tarai Development Corporation, Uttarakhand, India and other two local varieties were sourced from Maharashtra and Govind Ballabh Pant University of Agriculture and Technology, Pant Nagar, Uttarakhand, where they were originally developed. Seed purity was ensured prior to experimentation. For each treatment, 100 healthy, well-filled seeds were selected.

EMS mutagenesis treatment
 
Ethyl methane sulphonate (EMS) was used as a chemical mutagen. Seeds were pre-soaked in distilled water for 1, 4, 6 and 18 hours (maximum) at room temperature (~25^oC) to enhance metabolic activity and mutagen uptake. After pre-soaking, seeds were shade-dried for 1 hour. EMS solutions were freshly prepared in phosphate buffer (pH 7.0) at concentrations of 0.2%, 0.4%, 0.6%, 0.8%, 1.0% and 2.0% (v/v). A small quantity of dimethyl sulfoxide (DMSO) was added to improve solubility and penetration. Seeds were exposed to EMS solutions for a fixed duration i.e., 6 hrs (uniform across treatments) at room temperature with gentle agitation to ensure uniform treatment.
       
After treatment, seeds were thoroughly washed under running tap water and neutralized using 10% (w/v) sodium thiosulphate to inactivate residual EMS (Simon et al., 1999; Karthikeyan et al., 2022). Control seeds were treated with phosphate buffer alone. Each treatment consisted of three replicates, with 100 seeds per replicate. 12% was the soil moisture and the pot size was 12 × 12 inches.
       
Mutagenic effectiveness was measured in terms of mutations induced per unit dose of mutagen, while efficiency is the ratio of the frequency of mutations induced concerning the biological damage (Vinithashri et al., 2020).
 
Drought stress induction
 
A randomized experimental design was employed with three biological replicates for each EMS concentration and corresponding control. Screening was carried out in the M1 generation on seedling plates consisting of 12 × 12 inches pots under controlled conditions (temperature 25-30°C, relative humidity 50-60%).
       
Seedlings received minimal irrigation for the first four days after sowing to allow establishment, followed by complete withholding of water for eleven days, resulting in a total drought stress period of 15 days. Seedlings that survived the drought stress were transplanted to field conditions under low to minimal irrigation for preliminary validation. Selected M1 plants were advanced as individual progenies for subsequent M1‚ evaluation. Post-treatment seed viability was assessed using germination percentage, which served as the primary indicator of EMS toxicity. Initial drought-stress screening was conducted in the M1  generation at the seedling stage, while phenotypic evaluation was carried out in the M1‚  generation. For each EMS dose and control, 100 seeds were used per replication with three replications per genotype was maintained across generations. Seedlings were grown in 12 × 12 cm pots filled with soil along with FYM. Drought stress was imposed by withholding irrigation until soil moisture declined to approximately 12%, measured gravimetrically as the percentage of water relative to soil dry weight, which represents severe drought stress conditions (Malekpoor et al., 2016). Following a 15 - day stress period, surviving seedlings were allowed to recover and were subsequently transplanted to field conditions under low-input irrigation for further evaluation.
 
Parameters measured
 
The seed germination rate was calculated by counting the number of seeds germinated on daily basis.

 
Following 14 days of water deprivation, the survival rate which was the proportion of plants that survive under drought stress was determined.

Morphological parameters
 
At the end of the drought stress period, mean Plant height/shoot height (cm) was measured from the soil surface to the apical meristem and mean root length (cm) was measured from the collar region to the root tip after careful uprooting and washing. The root-to-shoot ratio was calculated to assess biomass allocation and adaptive responses to EMS-induced stress. Root- shoot ratios were calculated as root length divided by shoot length and showed a progressive increase under higher EMS-induced stress, indicating adaptive biomass reallocation toward root growth.
 
Ethical considerations
 
Because of the mutagenic and toxic characteristics of EMS all work involving EMS was performed under a fume hood with proper PPE, (personal protective equipment) (double layer chemical resistant gloves, laboratory coats, safety goggles, respiratory masks fitted with organic vapor cartridges and closed toe shoes). EMS residues and contaminated materials were deactivated by treating them with a 10% solution of sodium thiosulfate before discarding (Pérez-Escamilla, 2017). Some of the main criteria for evaluation of mutagenic effectiveness and efficiency were induction of mutations (Konzak et al., 1965; Nilan et al., 1965).
 
Statistical analysis
 
Data gathered were analysed using Jamovi statistics software. Tests were to establish statistical significance of effects of EMS concentrations and pre-soaking durations on barnyard millet survival rates in drought stress. The jamovi project (2024). Jamovi. (Version 2.6) [Computer Software).

As per the key findings, in this study, the extent of germination of barnyard millet was affected by time and concentration of EMS applied. All studied barnyard millet genotypes exhibited a similar trend towards decreasing germination percent with an increase in both concentration of EMS and the amount of exposure to EMS. This finding supports that the lethal as well as beneficial effects of EMS on seed viability were concentration dependent, as well as exposure time dependent. The findings revealed that for 1 hour of exposure, low to moderate concentrations (0.2% to 0.6%) produced a significant percent of germination. PRJ-1 and VLM-207 germinated at a high percent in response to low to moderate EMS concentrations after 1 hour, indicating that PRJ-1 and VLM-207 have a better ability to tolerate EMS induced stress when they were exposed for shorter periods of time. EMS concentrations greater than or equal to 0.8% resulted in a significant decrease in percent germination, with the Maharashtra genotype experiencing the most dramatic decrease, indicating that this genotype is particularly sensitive to EMS treatment. Additionally at the 2 hour exposure period, PRJ-1 maintained a high percentage of germination even at the moderate EMS level, while the M-D and Maharashtra genotypes exhibited a considerable decline in germination at EMS concentrations greater than 0.6%, indicating that different genotypes were able to tolerate different levels of stress from EMS treatment. All of the EMS treatment durations (4 and 6 hours) significantly increased the deleterious effects of EMS on germination; however, the extreme EMS concentration produced lower percent germination in all genotypes. PRJ-1 was the most tolerant of the EMS treatment, with VLM-207 and M-D as second and third most tolerant respectively and Maharashtra as the least tolerant. Ultimately, this study highlights the necessity to optimize both the concentration and period of exposure to EMS when inducing effective mutagenesis while ensuring the maintenance of seed viability. In all three varieties tested, EMS treatment at three months after sowing resulted in increases in growth, biomass allocation and seed yield for each of the three varieties (PRJ-1, M-D and Maharashtra) in a dose-dependent manner. From control conditions to 0.6% EMS, shoot length continuously increased but began to decline at higher concentrations of EMS (1% and 2%), indicating that EMS may inhibit growth if an excessive level of mutagenic pressure is applied to a plant. On the other hand, root length increased consistently as the concentration of EMS increased. As a result, the root: shoot ratio was steadily increased from approximately 0.23 at the control conditions to approximately 0.31-0.33 at 2% EMS (the highest ratios measured). Seed yield responded negatively to both high and low concentrations of EMS, with large increases in seed yield occurring only at moderate levels of EMS, specifically 0.6% EMS (560 g per 10 plants for PRJ-1; 345 g for M-D; and 245 g for Maharashtra). PRJ-1 exhibited the most productive potential for seed yield among the three varieties, while Maharashtra showed greater root allocation under elevated EMS conditions, indicating that the EMS treatment effects were not uniform across the three varieties tested. In summary, 0.6% EMS is the best treatment for  improving seed yield without compromising the balance between roots and shoots of the plant. The variety VLM-207 was not included in this evaluation due to field-related constraints during the experimental period. Identification of mutants was mainly noted with the PRJ-1 variety at the 0.6% EMS concentration, which produced stable and viable mutant lines across all 3 replicates. All mutant lines with the PRJ-1 (0.6%) EMS were selected and advanced through the M2 generation. Mutant lines produced at higher EMS levels showed decreased viability and inconsistent performance (Table 1 and 2).




       
Table 1 presents an effect of different EMS concentrations (0-2%) and duration of the EMS treatment applied to the seeds (1-6 hrs) affected the rates of germination (%) for 4 genotypes of barnyard millet. The study revealed that there was germination rates decreased with increased concentration of EMS and time of exposure. The highest germination rates were observed with the lowest concentrations of EMS and short exposure times (1-2 hrs). The two genotypes that had the greatest tolerance to EMS were PRJ-1 and VLM-207 because they had the highest rate of germination in most cases. M-D had a median sensitivity to EMS and was less sensitive than the Maharashtra genotype and more sensitive than the other two genotypes at the higher concentrations of EMS and at the longer times of exposure. Also, the highest concentration of EMS (2%) produced reductions in germination rates regardless of all the genotypes. Overall, there were distinct genotypic responses and the dose and length of time affect EMS mutagenesis and need for optimization.
       
The present study  demonstrated that EMS  treatment has created differences between barnyard millet genotypes with regards to both growth and yield traits, with the seed yield trait being the most reliably heritable and selectable trait (Table 2). Similar observations have been made with cereals and millets where vegetative traits are often poorly heritable due to the strong influence of environmental factors (Falconer and Mackay, 1996, Johnson et al., 1955). Therefore, it is unlikely that the improvement of these traits will be an efficient process through direct selection, under the same experimental conditions as were used in this study. On the other hand, the high heritability and genetic advance for the seed yield trait indicate that EMS treatment produced heritable variation for yield-related loci, particularly when the EMS was applied at moderate levels of mutagenic pressure, thus allowing the identification of heritable variation for yield traits (Panse and Sukhatme, 1967, Singh and Chaudhary, 1985). The elevated performance of other EMS-treated populations, especially at the moderate levels of exposure, is consistent with findings from earlier mutation breeding studies, which concluded that improved yields were produced by positive micro-mutations that have a role in the partitioning of assimilates and in the promotion of reproductive structures (Ankowiak-Cieslak and Till, 2016). The shoot-root correlation among MSH, RL indicates that shoot and root growth are regulated by correlated genes and are likely affected by pleiotropy or close linkage. A strong association between the R:S ratio with seed yield indicates that efficient biomass allocation plays an important role in obtaining high seed yields under stress induced by mutagens in plants. When roots are more developed, the plant can obtain increased water and nutrients for use during reproductive growth; therefore, this is extremely beneficial under less-than-ideal conditions, as has been demonstrated in stress-tolerant genotypes of cereal crops (Blum, 2011). In general, although early growth characteristics should not be relied upon for selection, both seed yield and root-shoot relationship are potential targets for mutation breeding with barnyard millet, particularly when utilizing optimal EMS concentration. Mean shoot height (MSH), root length (RL), the root-shoot (R:S) ratio and seed yield were evaluated for three varieties (PRJ-1, M-D, Maha) 90 days after sowing and the results are shown in the Table 2. Low to moderate levels of ethyl methane sulphonate (EMS) encouraged an increase in both MSH and seed yield; the maximum values for all three varieties were observed at a concentration of 0.6% EMS. Higher concentrations of EMS than 0.6% resulted in a reduction in both MSH and seed yield, while an increase in both RL and R:S ratio was noted. This indicates the shift of biomass allocation toward roots under conditions of increased mutagenic stress. Differences among the three varieties were apparent, with PRJ-1 producing the highest seed yield, while Maharastra had the greatest amount of root biomass allocation at the highest EMS levels, demonstrating genotype-specific responses to EMS treatment.
       
Seeds pre-soaked for 1 hour were found to have survival rates much lower than that of the seeds at 0.2% EMS, the survival rate was only 4-5% and at 0.8% EMS, it declined to 1%. This implies that shorter durations of pre-soaking (1 hour) were not sufficient to activate the embryos, hence the survival was poor under drought stress, especially at higher EMS concentrations. The survival rates were intermediate for seeds pre-soaked for 4 hours. The control group (0% EMS) survived at a rate of 60-70%, which remained the same at 0.4% EMS (60-70%) and 0.6% EMS (60-70%). But at 1% EMS, survival was marginally higher about 70-80% and at 2% EMS, it was again 60-70%. This indicated that pre-soaking for 4 hours gave a balance between embryo activation and EMS-induced stress, but higher concentrations (1%-2%) continue to present challenges to survival. Generally, these results suggest that lower EMS concentrations (0.2%-0.6%) in conjunction with extended pre-soaking times (18 hours) highly improve drought tolerance, as revealed by the significant survival rates (Fig 3). Conversely, higher EMS concentrations (1%-2%) and brief pre-soaking times (1 hour) decrease survival rates, which signifies the need for both EMS concentration and pre-soaking time to be optimized to enhance effective drought resistance in barnyard millet (Table 3, Fig 1, 2, 3 and 4).










       
The study’s findings showed that the concentration of ethyl methane sulphonate (EMS) and the length of pre-soaking significantly affected the survival rates. At lower EMS concentrations, the survival rates for seeds that had been pre-soaked for eighteen hours were noticeably high. In particular, the 90% survival rate for seeds treated with 0% EMS was maintained at 0.2% EMS (90%) and 0.4% EMS (90%). The survival rate, however, sharply decreased to 10-20% at a higher concentration of 0.8% EMS, suggesting that longer pre-soaking times and higher EMS concentrations have a detrimental effect on drought tolerance. The survival rates for seeds that had been pre-soaked for six hours remained comparatively constant across a range of EMS concentrations. The 70-80% survival rate for the control group (0% EMS) was constant across treatments. with 70-80% EMS 0.6%, 70-80% EMS 0.8%, 70-80% EMS 1% and 70-80% EMS 2%. Although the rates were lower than those of the 18-hour pre-soaking group, this indicates that moderate pre-soaking durations (6 hours) maintain survival rates even at higher EMS concentrations (Fig 2, 3,4).
       
It is utilized to present the correlation between a categorical variable (“CT Hours”) and a continuous variable (“Survived Percentage”). Analysis: CT Hours vs. Survived Percentage: CT Hours = 1: Average “Survived Percentage” is very low (close to 0). Error bar was also very small, reflecting low variability in data at this treatment time. CT Hours = 4: Average “Survived Percentage” increases significantly to around 60-70%. Error bar was still very small, reflecting less variability compared to longer treatment times. CT Hours = 6: Average “Survived Percentage” is still around 70-80%, as at 4 hours and error bar was very small. CT Hours = 18: Average “Survived Percentage” was around 70-80%, but error bar was very large. This reflects high variability in survival rates at this longer treatment time.     
 
Graphs and analysis of drought resistance
 
The continuous variable “Chemical Treatment Hours” is represented by the X-axis (CT-Hours). The percentage of plants that survived is shown on the Y-axis (Survived Percentage), which is likewise a continuous variable. Color (in PS-Hours): This represents the “Pre-Soaking Hours,” a categorical variable with values 0, 6, 7, 8, 10 and 12. In Fig  3, the graph looks to be a cross between violin plots with box plots integrated into them and scatter plots with a smooth line of best fit (probably an LOESS curve). Variables: The continuous variable “Chemical Treatment Hours” is represented by the X-axis (CT-Hour).
       
On the basis of data analysis on the correlation between the percentage of survivors and CT hours a distinct, non-linear relationship is depicted by the smooth line. The Survived Percentage first rises sharply as CT Hours rise from 1 to about 6. The Survived Percentage reaches a plateau after six hours and then starts to decline. This implies that there may be a CT-Hour threshold for survival, above which the treatment loses its effectiveness or even becomes harmful (Fig 4).

Impact of Pre-soaking (PS) hours on surviving percentage
Plots for violin
 
The distribution of Survived Percentage for each PS Hours category is displayed in violin plots on the right (Fig 4). The frequency of data points at various percentages is shown by the violin’s width. For every PS Hours category, the box plots inside the violins display the median, quartiles and possible outliers. Variability in survival rates is indicated by the broad distribution of the Survived Percentage in Hours 8 and 10. A greater concentration of data around the 75% mark is seen in PS Hours 6 and 12. PS Hours of 0 hours exhibit a high degree of fluctuation. A very high level of survival is demonstrated by PS hours of 7. Individual data points are displayed in a scatter plot that is coloured according to PS Hours. This makes it possible for us to observe the impact of PS Hours on the correlation between CT Hours and Survived Percentage. Variability is shown by the red dots, which stand for eight hours of PS, being dispersed over a large range of Survived Percentage values. The higher Survived Percentage values are typically where the green dots, which stand for seven hours of PS, are concentrated. Regardless of PS hours, the points at one hour of CT show a very low survival percentage. For maximizing survival, there seems to be a range of CT hours that work best. Survival is strongly influenced by PS Hours; higher and more stable survival rates are associated with certain PS Hours values. More research is necessary to fully understand the intricate relationship between CT and PS hours.
       
On the basis of present studies, the findings proves the efficacy of EMS mutagenesis in enhancing drought tolerance in barnyard millet. Lower EMS levels (0.2%-0.6%) were most effective in creating genetic diversity without affecting survival rates. Increased pre-soaking times (18 hours) also improved drought tolerance, probably because of enhanced embryo activation and nutrient mobilization. These findings were consistent with earlier studies involving EMS-induced mutagenesis in crops such as rice and wheat, in which lower dosages were successful in increasing stress resistance. The study also points out the necessity for optimizing the concentrations of mutagens and the durations of treatment in order to gain desirable characters in crop improvement programs (Choudhary et al., 2016).
       
Inducing drought resistance in barnyard millet varieties by using EMS mutagenesis is an environmentally friendly method for implementing agriculture in water-limited areas. Future studies need to concentrate on field tests and molecular profiling of EMS-induced mutants to detect certain genes that are related to drought tolerance. Mutations induced this way are called induced mutations. Induced mutations take place when a gene gets exposed to mutagens or other environmental factors (Singh et al., 2021).
       
In plant breeding, mutation induction is now a recognized technique for enhancing cultivars for particular traits and supplementing existing germplasm (Ramesh et al., 2019). According to Ganapathy et al., (2008), induced mutagenesis has been successfully used to increase variability and facilitating for the isolation of mutants with desirable characteristics of economic value, such as better dwarf plant types for non-lodging, simultaneous maturity, heavy tillers, heavy grain yield, heavier seed size and desirable seed color. In mutation breeding, a mutagen’s utility is determined by both its mutagenic efficacy (mutations per unit dose of mutagen) and mutagenic efficiency (mutation in relation to undesired changes/damage like sterility, lethality and injury) (Ramchander et al., 2014).

This study revealed effect of EMS mutagen and pre-soaking hours of seeds to increase barnyard millet’s resistance to drought. Longer pre-soaking times (18 hours) and lower EMS concentrations (0.2%-0.6%) were found to be the most effective ways to increase survival rates under drought stress. The results lay the groundwork for creating drought-tolerant barnyard millet cultivars that will support sustainable farming practices and ensure food security in arid areas.

This work was supported by Shri Guru Ram Rai University through the Seed Money Funded Grant Project. The authors express their sincere gratitude to the Department of Botany, School of Basic and Applied Sciences, Shri Guru Ram Rai University, Patel Nagar Campus, Dehradun, Uttarakhand, for their valuable support and facilities provided during the course of this research.
 
Disclaimers
 
The views and conclusions expressed in this research article are solely those of the authors and do not necessarily reflect the views of their affiliated institution. The authors are responsible for the accuracy and completeness of the information presented, but they do not accept any liability for any direct or indirect losses resulting from the use of this content.

Authors have declared that no competing interests exist.