Optimization of Mutagenic Doses in Bitter Gourd (Momordica charantia L.) for EMS and Gamma (γ) Rays

Bitter gourd, scientifically known as Momordica charantia L., is a tropical and subtropical vine belonging to the family cucurbitaceae. It is considered to be an old world species (Jadhav et al., 2009). People widely cultivate it for its bittertasting edible and medicinal fruit. It is monoecious in nature and native to South Asia (Pathak et al., 2014). Bitter gourd is low in calories and rich in essential nutrients, including vitamins A, C and K (Chaudhary, 1967). It also contains minerals like iron, potassium and dietary fibre. Chang et al., (1996) reported that oil contents of bitter gourd seed vary from 41 to 45% and the oils contain 22-27% stearic acid and 63-68% eleostearic acid. Fruit juice of bitter gourd has anti-diabetic properties which aid in retaining sugar levels in human body (Sreejayan and Rao, 1991). Cucurbitacin glucoside, which is present in bitter gourd, adds bitterness to the fruits. Among the cultivated cucurbits, it has been known as a prospective vegetable for foreign export by APEDA (Thangamani et al., 2011).
       
Proper resource use efficiency is a challenge for Indian agriculture, given the growing population and the need to mitigate food demand in a sustainable manner (Karthik et al., 2023). The productivity of bitter gourd in Bihar is reported to be lower than the national average. There is a noticeable difference in productivity, with Bihar struggling to achieve higher yields compared to the rest of the country. So, there is a need to develop high-yielding cultivars in Bihar to improve bitter gourd productivity. Developing cultivars with higher yield potential could potentially address the productivity gap and contribute to the region’s overall agricultural output.
 
In bitter gourd, gamma irradiation and EMS treatment led to an increased genetic variability in quantitative characters (Mallaiah and Zafar, 1986). Mutation breeding, involves the induction of mutations in plant propagules through chemical or physical mutagens. The resulting mutants are then selected for desirable changes and the process is repeated until the desired trait is stably expressed in subsequent generations. This method allows plant breeders to expand the genetic base of germplasm, creating new varieties or sources of variation for breeding programs. Mutation breeding depends on selfing mutants until the instigated character has a steady expression in the advanced mutant generations.
       
Mutation induction includes the treatment of plant propagules (seed or cutting or plant tissue) with chemical or physical mutagens. This is followed by selection for attractive (desirable) changes in the subsequent mutants. The plant breeders use mutation induction to expand the hereditary base of germplasm and utilize the mutant lines straightforwardly as new varieties or as sources of new variation. Mutation breeding has become an amazing strategy for creating novel plant genotypes (Penna et al., 2012). Mutation screening is the choice of individuals from a sizeable transformed population that meet explicit selection criteria and mutant affirmation is re-evaluating (Oladosu et al., 2016).
       
A bitter gourd landrace MC 013 was treated with gamma (g) rays and advanced another cultivar MDU 1 which has improvement for yield, long greenish white fruits, tolerant to pumpkin insects, fruit flies and leaf spot diseases (Rajasekharan and Shanmugavelu, 1984). Moreover, DBGy-201and DBGy-202: two gynoecious lines of bitter gourd (Momordica charantia L.) confined from native source as a spontaneous mutations (Behera et al., 2006). Overall, mutation breeding has proven to be an effective strategy for creating novel plant genotypes (Penna et al., 2012) and enhancing agricultural trait variability. Given these findings, the primary focus of the present study was to optimize mutagenic doses for gamma rays and EMS in bitter gourd.

For the pre sent investigation, Kahalgaon Local genotype was used which was collected from the farmer’s field of Kahalgaon region in Bhagalpur district of Bihar and through pure line selection, stable homozygous inbred line was developed by Department of Horticulture (Vegetable and Floriculture), BAC, BAU, Sabour in the year 2018. It produces green colour fruits with sharp tubercles. Present research was conducted to create various attributes for selection like macro and micro mutants with this genotype. In the present study, this genotype was treated with four concentrations of EMS, viz., 0.1, 0.2, 0.3 and 0.4% and three doses of gamma ray irradiation, viz., 0.25, 0.50 and 0.75 kGy. The details of treatments are given hereunder.
 
Chemical treatment by using EMS
 
Treatment was done in the laboratory at Department of Plant Breeding and Genetics, BAU, Sabour. Initially, sufficient seeds for chemical treatment were soaked in distilled water for 16 hours in a beaker. After 16 hours of pre-soaking treatment, the seeds were removed from beaker and added 60 seeds each in five conical flasks for seed treatment. To maintain 7.4 pH dilution of the combined stock solutions of 40.1 ml of dipotassium phosphate (K₂HPO₄) and 9.9 ml of monopotassium phosphate (KH₂PO₄) to 1000 ml with distilled H₂O was done. Then filled the conical flask 1 with 100 ml of distilled water because it acted as control and remaining conical flasks were filled with 100 ml of 0.05 M potassium phosphate buffer according to the experimental plan and then added required quantity of EMS solution i.e 100 µl, 200 µl, 300 µl and 400 µl to flask 2, flask 3, flask 4 and flask 5, respectively and all conical flasks were kept in mechanical shaker (140 rpm) for 8 hours. EMS was added to the conical flasks under laminar air flow chamber wearing mask, lab coat and gloves because it was a carcinogenic agent. Seed sowing was done immediately in pro-trays after treatment. For detoxifying EMS, we used 0.5 M NaOH solution. It was made by adding 20 g of NaOH in one litre of water. Solutions were decontaminated by disposal on to 2.5 times the volume of 0.5 M NaOH. They were left overnight and discarded in the hood on following days by rinsing the flasks for 30 minutes.
 
Physical treatment using gamma (y) rays
 
Treatment was done at Regional Nuclear Agriculture Research Centre (RNARC), Bidhan Chandra Krishi Viswavidyalaya (BCKV), Haringhata, West Bengal, India. 300 seeds per treatment were treated and replicated thrice in completely randomized design. The concentrations of various treatments were 0.25, 0.50 and 0.75 kGy. The capacity of gamma chamber at RNARC was 4983.38 Gy per hour and penetration was up to 8 mm thickness of seed. The time taken for seed treatment of various concentrations viz., 0.25, 0.50 and 0.75 kGy was 3.01 minutes, 6.02 minutes and 9.03 minutes, respectively. After seed treatment, seed sowing was done immediately on next day in the protrays.
 
Observations made in M₁ generation
 
Seed germination was calculated by per cent ratio of total number of seeds sown to number of seeds germinated. Similarly, survival rate was calculated by per cent ratio of seedlings survived to number of seeds germinated.
 
Estimation of Ld₅₀ value
 
LD₅₀ is the lethal dose of particular mutagen that will cause the death of 50% of the plant population being tested. LD50 value was calculated based on germination and survival plant percentage compared to control.
 

Germination and survival per centage
 
In the pre sent investigation, results regarding seed germination percentage revealed that per cent seed germination was decreased with an increase in concentration/dose of mutagens in M₁ generation. Higher concentrations of EMS and higher dose of gamma rays have put forth maximum inhibitory effect on seed germination percentage (Table 1). Among EMS treatments, the mean germination percentage was recorded highest in seeds treated with 0.1% EMS (65.00%) and lowest with 0.4% EMS (26.66%). In gamma irradiation treatments, the mean germination percentage was recorded highest in 0.25 kGy gamma irradiation (54.66%) and lowest in seeds treated with 0.75 kGy gamma irradiation (8.33%). Reduction in germination was due to inhibition of genetic and physiological processes by mutagens which leads to lethality. Belele et al., (2002) have reported reduction in seed germination percentage in french bean while increasing the mutagenic doses i.e. 200 Gy to 250 Gy. Reduction in seed germination due to increasing doses of mutagens was also recorded in other crops by Kumar et al., (2010), Girija and Dhanavel (2009) in cowpea; Mahla et al., (2010) in cluster bean; Chaudhari (2002) in lentil and Mejri et al., (2012) in faba bean.
       
A negative correlation was observed between dose of the mutagens and survival percentage. Increase in dose of mutagen reduced plant survival percentage. Plant survival is one of the most reliable parameters for evaluating the effects of any mutagen. Among various EMS treatments, the highest and lowest survival percentage was recorded in 0.1% EMS and 0.4% EMS respectively (Table 1). In case of gamma irradiation treatments, the highest and lowest survival percentages were recorded in 0.25 kGy and 0.75 kGy doses, respectively (Table 1). According to the experiments done by Shinde and More (2018) on cluster bean, it might happen due to point mutations or the injuries caused by the genetic material used, which eventually led to decrease the rate of respiration and energy production and finally a decrease in survival per centage. Similarly, an inverse relationship between dose of the mutagen and survival of plants has also been reported in cowpea by Kumar et al., (2010) and Mejri et al., (2012) in faba bean.


       
Kangarasu et al., (2014) opined that, LD₅₀ was the lethal dose of particular mutagen that will cause the death of 50% of the plant population being tested. In this experiment, the LD₅₀ value (the concentration at which 50% of the plant population survives) for EMS in bitter gourd was found between 0.2% and 0.3% EMS treatments. Specifically, the survival rate at 0.2% EMS suggested that the LD₅₀ is approximately 61.62%, meaning that a concentration of around 0.2% EMS was close to the lethal dose for 50% of the plant population as shown in Table 2. In case of 0.3% EMS and 0.4% EMS, LD₅₀ based on survival percentage was less than 50% i.e. 41.18% and 30.40% respectively. Concentrations of 0.3% and 0.4% EMS were more toxic, resulting in survival rates below 50% as shown in Table 2.



Among various EMS treatments tested, highest lethality was recorded in T₄ (0.4% EMS) and lowest lethality was recorded in T₁ (0.1% EMS). It showed linear increase with increasing concentration of EMS. The stimulatory effect of EMS at a lower dose is observed because at lower concentrations mutagens stimulate enzymes and growth hormones responsible for growth, yield and fruit quality, while, higher concentrations of mutagens had inhibitory effect (Sebastian et al., 2023). The similar trends were also obtained by Kavithamani et al., (2008), Tyagi and Khan (2010) in soya bean; Girija and Dhanavel (2009) in cowpea; Mahla et al., (2010) and Velu et al., (2012) in cluster bean.
       
The LD₅₀ value for gamma (g) rays in bitter gourd was observed at the concentration of 0.25 kGy (T₅) gamma rays where the survival plant population was 47.16% and LD₅₀ based on survival percentage was 63.51 % which was more than 50% survival percentage compared to treatment T₆ (0.50 kGy) and T7 (0.75 kGy) as indicated in Table 3. In case of 0.50 kGy and 0.75 kGy, LD₅₀ based on survival percentage was less than 50% i.e. 25.21% and 9.45% respectively. Higher doses of gamma rays lead to lower germination and survival rates, highlighting the detrimental effects of radiation on plant growth and viability as evident from Table 3. For practical applications, this information would be crucial in determining safe exposure levels for gamma rays in agricultural practices or genetic studies involving mutation breeding on bitter gourd.


       
Among various gamma irradiation treatments tested, highest lethality was recorded in T₇ (0.75 kGy) and lowest lethality was recorded in T₅ (0.25 kGy). It showed linear increase with increasing doses of gamma rays. Similar trend was also obtained by Tyagi and Khan (2010), Kavithamani et al., (2008) in soya bean; Girija and Dhanavel (2009) in cowpea; Mahla et al., (2010), Velu et al., (2012) and Patil and Rane (2015) in cluster bean.
       
The greater sensitivity at higher doses of mutagens has been attributed to various factors such as changes in the metabolic activity of the cells (Natarajan and Shivasankar, 1965), inhibitory effects of the mutagen (Ramulu, 1972) and to disturbances of balance between promoters and inhibitors of growth regulators (Meherchandani, 1975; Wani et al., 2012). The reduction in biological criteria (plant height and survival) might be attributed to a drop in the auxin level, inhibition of auxin synthesis, chromosomal aberrations or due to reduction of assimilation mechanism (Girija and Dhanavel, 2009).

Higher concentrations of EMS (0.4%) and Gamma (g) rays (0.75 kGy) recorded the highest lethality. This suggests that a higher concentration of EMS (0.4%) and Gamma (y) rays (0.75 kGy) resulted in a more significant impact, at inducing mutations but also cause more harm, leading to increased plant death in bitter gourd. Lower concentrations of EMS (0.1%) and Gamma (y) rays (0.25 kGy) recorded the lowest lethality. This indicates that a lower concentration of EMS and Gamma (y) rays had a milder effect, causing fewer mutations and less harm, leading to higher survival rates. There appears to be a linear relationship between the concentration of mutagens and lethality. As the concentration of EMS increased from 0.1% to 0.4%, lethality increased in a consistent and proportional manner. Similarly, as the concentration of Gamma (y) rays increased from 0.25 kGy to 0.75 kGy, lethality increased in a consistent and proportional manner. The LD₅₀ value for EMS and gamma (y) rays in bitter gourd were observed at the concentration of 0.2 % EMS and 0.25 kGy, respectively. Both EMS and gamma rays are effective mutagens, but their effectiveness is dose-dependent. Higher doses result in higher lethality, likely due to an increased number of mutations or a greater extent of damage.

The authors thankfully acknowledge the facilities received at BAU, Sabour and funding received under the State Non Plan Project (SNP/CI/Kharif/2016-5). We also acknowledge RNARC facility for treatment of seeds with gamma rays.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.