Introduction

Grasspea (Lathyrus sativus L.) are very resilient to drought, floods, and insect infestations, making them possibly the most ecologically robust legume crop1. In terms of both acreage and productivity, it is India’s third-most significant cool-season pulse crop. India leads the world in the cultivation of grasspea, while it is second in productivity after Bangladesh2. Because of its strong and hardy roots, it may be grown on a wide variety of soil types, even very poor soil and thick clays. The crop appears to be built to thrive in challenging environments because of its resilience and capacity to fix atmospheric nitrogen. It supplements nitrogen by fixing 67 Kg/ha in a single season3. Hence it is very popular among farmers4. The crop has a high nutritional content with 31% protein, 41% carbohydrate, 17% total dietary fibre and a rich source of vitamin B-complex in addition to its hardiness5.

In spite of such virtues, global area under its cultivation has decreased because of ban on its cultivation in many countries. The nutritious benefits of grasspea are reduced due to the presence of a harmful neurotoxin called β-ODAP (β- N-oxalyl-a, b-diaminopropionic acid), rendering its grain unfit for eating. The level of neurotoxin β-ODAP, (16.2 ± 0.5 g/Kg seed) is frequently found in the seeds of various grasspea varieties and causing Lathyrism, a permanent paralyzing condition6. This is a major obstacle in enhancing the nutritional value and increasing the production of this important crop until the development of low-toxin varieties. Only a few carefully chosen varieties, like Ratan, Prateek, and Mahateora in India and BARI Khesari-1 and BARI Khesari-2 in Bangladesh, have β-ODAP content below 0.10%, which is the acceptable limit in human food7,8. Moreover, grasspea seems to have relatively high amounts of trypsin inhibitor compared to many other food legumes (except soybeans), which poses another challenge for the crop’s improvement9. There are also reports available where reducing β-ODAP led to reduced yield of grasspea10.

Legume seeds contain trypsin and chemotrypsin antinutritional factor and it reduces the protein digestion that leads to enlargement of the pancreas11. Therefore, the goal of breeding grasspea is to develop varieties that are high in yield, produce a lot of biomasses, and have lower levels of neurotoxin and anti-nutritional factors, including trypsin inhibitors, to expand its cultivation across the country. The limited genetic diversity is the main obstacle for the improvement of grasspea by traditional breeding methods. Its narrow range of genetic variation because of its self-pollination and interspecific incompatibility. For these reasons, improvement strategies focusing on mutation breeding have been explored as a way of creating genetic variation12. It also offers a broader range for the identification of new genetic traits10. Over the past 75 years, mutation breeding has made a substantial impact by developing approximately 3200 mutant varieties for agricultural use globally. The largest contributions have been in cereals, followed by ornamental plants, legumes, and oilseeds. India released the highest number of varieties by mutation breeding after China8). Among all released mutant varieties worldwide, 776 mutants have been released for increased nutritional value.

The most common mutagen uses in mutation breeding are gamma radiation and EMS (Ethyl methane sulfonate). Numerous studies have been used on grasspea, a crop often overlooked, to enhance its genetic diversity through induced mutation. These efforts have primarily focused on identifying various plant traits, such as growth habits maturity stages13, seed and flower characteristics14), leaf mutations, and changes in chlorophyll levels and productivity15. Despite these efforts, there have been limited successes in modifying the nutritional and antinutritional content of the crop through induced mutation methods. Only two grasspea varieties—Poltavskaya from the former USSR and Bina Khesari-1 from Bangladesh16 have been created through mutation breeding methods that involve gamma rays (250 Gy) and electrical stimulation (0.1%), respectively17. Combining gamma rays with electrical stimulation can improve the plant’s antioxidant and β-ODAP content, leading to the creation of a mutant with a 0.1% neurotoxin content, significantly lower than the average toxin content in the non-mutant crop. A similar approach has been applied to other crops, including soybean, chickpea, and horse gram, for producing of mutants with lower trypsin inhibitor levels as a result of the mutagenic treatment18,19,20,21. In the course of evolution, as well as in crop improvement programmes, populations are constantly shifting towards superior types. The economic value of a plant depends upon the several characters. In the case of grasspea, the key features that matter are the number of seeds produced and neurotoxins content. Limited progress has been made previously in developing varieties that produce more seeds and have lower levels of β-ODAP through induced mutations. In this context, the present study was designed with the following objectives:

  1. 1.

    Understand the differential response of physical (γ-rays) and chemical (EMS) mutagens in three different plant varieties of grasspea.

  2. 2.

    Study of biochemical and yield related traits in M3 mutants’ lines of three different varieties of grasspea.

Materials and methods

Materials

For this investigation, three grasspea varieties—Nirmal, Biol-212 and Berhampur Local were employed. Nirmal variety was used for check of low β-ODAP content Biol-212 is a low β-ODAP + high yielding variety however, Berhampur Local is a variety that generally grown in West Bengal region of India. The germplasm kept at District Seed Farm, Bidhan Chandra Krishi Vigyan, Kalyani, WB (India) is the source of these varieties. The detail characteristics of these genotypes are presented in Supplementary Table 1.

Development of M1 and M2 generation by using gamma rays and ethyl methane sulphonate mutagens (EMS)

The development of M1, M2 and M3 populations is presented in Fig. 1. The gamma rays and EMS is the most commonly use physical and chemical. In the gamma ray chamber, three grasspea varieties (Nirmal, Biol-212, and Berhampur Local) with three bags containing three hundred healthy seeds were exposed to 400, 500, and 600 Gy radiation treatments. Irradiations were performed at the UGC- DAE consortium for scientific research, Kolkata center (South Campus of Jadavpur University, Salt Lake, Kolkata). The gamma-ray dosage rate was 7.12 Gy/min, and the irradiation source was 60Co. For treatments of EMS phosphate buffer was prepared. Sodium dihydrogen phosphate (NaH2PO4) and sodium hydrogen phosphate (Na2HPO4) were used to make the phosphate buffer. Total 27.80 g of NaH2PO4 were dissolved in 1000 cc of double-distilled water to made Solution “A”. Similarly, solution “B” was prepared by dissolving 53.65 g of Na2HPO4 in 1000 cc of double-distilled water. Total 100 cc (pH 7.0) combine solution prepared by the addition of the 39 cc solution “A” and the 61 cc solution “B”. Ultimately, this phosphate buffer was used to produce the EMS solution. After being treated with EMS solution, the seeds were shaken continuously in a shaker for 4 h and kept in an airtight container. All three varieties of grasspea were subjected to a mix of physical and chemical mutagens. The seeds of each variety were exposed to gamma rays at a dosage of 400 Gy, after which they were treated with two different concentrations of EMS-treated solution (0.5% and 1% V/V)22. This procedure was carried out step-by-step as previously described. The seeds were then carefully rinsed with water flowing from a tap for 1 h to eliminate any leftover impact from the mutagenic chemicals. Once the treatment was over, these seeds were considered as M0 seeds.

The M1 seeds of three different varieties and their corresponding non-treated (control) seeds were sown directly to the field at the Simanta farm in Kalyani, BCKV Nadia, WB (India), in 2012–2013. These seeds were planted in a randomized block design (RBD) in three replications containing 100 seeds of each variety with a spacing of 35 cm between rows and 20 cm between plants. These seeds of each genotype were considered as M1 plants. All standard practices were followed to produce a good crop. The reduction in the height of seedlings after 30 days after planting was recorded as effect of mutagens. The ratio of seed germination was used to determine the LD50 dose (the amount required for 50% plant lethal). During the maturity, each plant was collected separately22.

Fig. 1
figure 1

Flow chart for development of M3 mutants for increased biochemical and yield related traits in grasspea.

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The M2 generation (2013-14) was produced from a composite sample of 25 seeds from each M1 plant selected across the three different varieties during each experiment. A total of 300 seeds from each M1 generation were sown in RBD designed in three replications, maintaining 100 plants at a spacing of 35 × 20 cm to produced M2 seeds. Additionally, 600 seeds from each experimental group across all three varieties were cultivated using the plant-to-progeny method to enhance the population of mutated plants for selection and evaluation. The selection of mutant progenies was based on visual observation such as scoring of chlorophyll content and 628–662 M2 plants of each treatment were examined. The same agricultural practices and pest control strategies were applied consistently across all experimental groups. Both the experimental and control groups were evaluated for segregation.

Development and evaluation of M3 population

The M3 generation (2014-15) was developed from seeds that were harvested directly from the RBD trial of the M2 generation. A total of 300 seeds from each variety in each treatment were sown in the RBD experiment and the experiment was conducted in three replications containing 100 seeds. Data on the yield and specific quality indicators of grasspea like β-ODAP content (g/kg), protein content (%), and trypsin inhibitor activity (mg/g dry matter) from five random plants from each line and in each replication were recorded. Each selected line was observed closely for the specific traits for its selection. In M3 trials (2014-15), various economic and agronomic traits were recorded for 16 different traits such as germination, plant height (cm), days to 50% flowering, days to maturity, number of primary branches per plant, number of secondary branches per plant, number of pods per plant, pod length (cm), number of seeds per pod, number of seeds per plant, 100 seed weight (g), seed yield per plant (g), fresh weight per plant (g), ODAP content (%), protein content (%), trypsin Inhibitor Activity (TIU mg− 1 of dry matter). The detail of the procedure to record these data is presented in Supplementary Table 2.

Biochemical analysis

Grains for chemical examination were crushed in a grain mill to pass through an 80-mesh sieve, and the leftover seed powder was used for experiments. Bovine Serum Albumin (BSA) served as a standard protein in Lowry’s method23 to assess the protein content. The reactivity of the peptide nitrogen (s) with the copper particles in an antacid environment caused diminishment of the Folin-Ciocalteay phosphomolybdic phosphotungstic corrosive to heteropoly molybdenum blue by the copper-catalyzed oxidation of fragrant acids shape the premise of the Lowry strategy of protein concentration assurance. Since, the Lowry procedure is very sensitive to pH change, the test solution’s pH ought to be kept between 10 and 10.50.

To carry out the extraction, phosphate buffer (pH 7.00) was used. A mortar and pestle were used to thoroughly ground 40 mg of the sample (seed powder) in 10 ml of buffer after which it was centrifuged at 10,000 rpm, and the protein was estimated from the supernatant. Various concentrations of the working standard, ranging from 0.2 ml to 1 ml, were pipetted into a set of test tubes. In two more test tubes, 0.1 and 0.2 ml of the sample extract were pipetted. In every test tube, the volume was increased to 1 ml. A blank tube held 1 ml of water in it. Reagent C (50 ml of 2% Sodium carbonate + 50 ml of 0.1 N NaOH solutions + solution B 0.5 g Potassium sodium tartarate was mixed in 50 ml distilled water with 0.25 g of copper sulphate) was added to every tube, including the blank. These solutions were thoroughly mixed and incubated for 10 min. Then 0.5 ml of Folin reagent was added, mixed well and incubated at room temperature in the dark for 30 min and blue colour was developed. The final volume in each of the tube was 6.5 ml. The measurements were performed at 660 nm and the blank value was taken by taking zero. Protein concentration was plotted against the absorbance for standard calibration curve. The unknown sample’s absorbance was verified, and its concentration was calculated using the standard curve. The amount of protein in each 100 mg of dry seed was expressed in mg (%).

Trypsin inhibitor (TIA)

The TIA activity was measured indirectly. Trypsin hydrolyzes a synthetic substrate called BAPNA (N-ɑ-benzoyl-DL-arginine-p-nitroanilide) to yield yellow-coloured p-nitroanilide. The extracts were measured at 410 nm and yellow colour was recorded. Each sample’s 100 mg of dry plant material was extracted, homogenized, and centrifuged for 30 min@10,000 rpm at pH 8.10–8.20 in Tris-CaCl2 buffer for 5 min. Five different concentrations of the solution were taken in five tubes, and an aliquot of the supernatant was diluted with the buffer solution. The trypsin inhibitor activity was subsequently analyzed using the methodology suggested by Kakade et al. (1974)24. After adding 1 ml of trypsin solution in each tube, the tubes were placed in a water bath at 37 °C temperature to allow them to acclimate. Each tube received 2.5 ml of the substrate solution after ten minutes. The 40 mg of BAPNA hydrochloride that made up the substrate solution was dissolved in 0.5 ml of Dimethyl Sulfoxide (DMSO) and then topped off with 100 ml of Tris HCl buffer (pH 8.10). After an additional half-hour, the reaction was terminated by adding 0.5 ml of 30% (v/v) glacial acetic acid to each tube. In a spectrophotometer, the absorbance was measured at 410 nm against the substrate blank. Trypsin-free extracts in a range of 4–5 different concentrations made up the substrate blank. After that plot the absorbance against extract volume. It was established aliquot size of the extract was needed to inhibit 50% of the trypsin activity. One unit of trypsin inhibitor was thought to be represented by this aliquot size. Consequently, the quantity of inhibitor needed to prevent 50% of the hydrolysis of 0.02 mg of trypsin was known as a trypsin inhibitor unit. Trypsin inhibited units (TIU) per mg of sample dry matter were used to express TIA24.

Estimation of β-ODAP content

β-ODAP (β-N-oxalyl-L-α-β-diamino propionic acid) was estimated from grasspea seeds using the wet chemistry method suggested by Rao (1978)25. They have divided the protocol in the following steps.

Standard curve

Hygroscopic diammonium propionic acid (DAP) absorbs CO2 from the atmosphere and it is a stable salt 0.0054 g DAP hydrochloride was weighed and dissolved in 10 ml of 60% ethanol. Factor of conversion (β-ODAP) between DAP and BOAA is 1.691643. In each tube 4 ml of 3 N KOH, was added, sealed, and thoroughly vortexed. Followed by tubes were immersed in a hot water bath for 30 min. The solution was centrifuged for 15 min at 4500 rpm. After that colour reaction (discussed in coloured reaction step) done for the hydrolysed sample. Spectrophotometric readings at 420 nm were taken by setting absorbance of blank as zero. Finally, a graph of absorbance of standards was plotted against BOAA (β-ODAP).

Extraction procedure

Total 0.5 g powdered seed was weighed and placed into test tube after that 10 ml of 60% ethanol was added. The tubes were capped tightly and vortex after that tubes were shaken in shaker /mixer for 45 min. The solution centrifuged at 4500 rpm for 15 min. Decant the ethanol extract (unhydrolyzed fraction) into prelabelled test tubes.

Hydrolysis

In hydrolysis step conversion from β-ODAP in the sample to DAP was takes place. After that 2 ml of ethanol extract was pipette out into screw top test tube. Prepared a blank tube by 2 ml of 60% ethanol instead of ethanol extract. Added 4 ml of 3 N potassium hydroxide (KOH) in each tube including blank and capped tightly and vortex. After that placed all tubes in boiling water bath for 30 min. Then centrifuged at 4500 rpm for 15 min.

Colour reaction

For all samples/standard curve tubes/blank tube was done. Pipette the hydrolysed sample into screw top test tubes. Total 250 µl hydrolyzed sample/standard/blank was taken in another prelabelled test tube and 750 µl distilled water was added. Then 2000 µl of OPT solution was added. All the tubes were vortex and incubated for two hours at 40OC water bath. After 2 h spectrophotometric reading at 425 nm by setting absorbance of blank as zero was taken. Finally, % BOAA (β-ODAP) in the samples from the standard curve was determined by Lambert-Beer’s law.

\(A\,=\,C\varepsilon L\)

Where, (A) absorbance, (C) concentration, (ɛ) extinction co-efficient absorbance is constant and (L) is the path length which always be 1 cm.

Statistical analysis

ANOVA was performed for mutants of all three varieties for mean value of all fourteen traits. Fisher’s LSD test was used for post hoc comparisons. Fisher’s LSD was used to tested for normality where least significant difference between two mean of particular mutant and their respective parents were compared. All analysis was performed by using SPSS software.

Results

Effect of mutagen on M1 and M2 generations

There was a significant effect of gamma radiation, EMS, and their combination treatments on the M2 populations of all three varieties (Supplementary Table 6). In Nirmal variety except treatments 500 Gy and 400 Gy + 0.5% EMS no viable mutations were scored and rest of the treatment showed viable mutants. The results clearly showed that all of the treatments applied had potential to produce viable mutations in all three varieties. The variety Biol-212 had the highest frequency of viable mutations (0.64%), followed by Berhampur Local (0.22%) and Nirmal (0.26%). Impact of combined treatments of EMS and gamma radiation on the frequency of viable mutation of Nirmal and Biol-212 was varied 0.15 (600 Gy) to 0.61 (1% EMS), 0.16 (1% EMS) to 0.96 (400 Gy), while in Berhampur Local viable mutation frequencies was varied 0.15 (500 Gy and 400 Gy + 1% EMS) to 0.31 (600 Gy and 400 Gy + 0.5% EMS). In the Berhampur variety, the highest frequency was observed as a result of a combination of physical mutagenesis (600 Gy) and chemical mutagenesis (400 Gy + 0.5% EMS). In contrast, the Nirmal variety showed highest frequency by the use of chemical mutagen (1% EMS).

Screening of M3 population of morphological, biochemical and yield traits

A total of 51 mutants derived from the three varieties of grasspea were assessed in the M3 generation for biochemical and yield traits. The analysis of variance indicated significant differences for all traits examined except time to reach 50% flowering in the Berhampur Local variety (Table 1). The data also showed a considerable range of variation within the selected grasspea mutants.

Table 1 Analysis of variance for yield, its attributes and quality traits in M3 mutants of grasspea variety Nirmal, Biol-212 and Berhampur.
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Table 2 Nutrient quality in seeds of M3 mutants of grasspea variety Nirmal.
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In the majority of the Nirmal mutants showed variations, there were notable deviations in the average β-ODAP (%) and protein content (%). However, the average β-ODAP levels showed a clear downward trend compared to the standard, whereas the average protein content levels showed mix of increase and decrease trends. The mean β-ODAP levels ranged from 0.200% (Nm8) to 0.337% (Nm4). The mean β-ODAP value significantly lower than control (parent) in several mutants including Nm5 (0.261%), Nm6 (0.235%), Nm7 (0.212%), Nm8 (0.200%), and Nm11 (0.263%). While mean protein content levels varied between 22.75% (Nm4) and 31.08% (Nm9). The majority of the mutants showed an increase in mean protein content over the parent variety, except Nm2 (30.27%), Nm7 (30.27%), Nm9 (31.08%), and Nm12 (30.88%). The mean TIA (% dry matter) decreased from the standard in all the mutant lines except Nm3, Nm4, and Nm5, with the lowest values observed in Nm2 (16.58), Nm8 (15.15), Nm11 (15.88), and Nm12 (17.22) (Table 2).

Mean values for all the numerical traits also showed significant differences both upwards and downwards direction from the control in the majority of the Nirmal mutant lines. The average plant height at maturity was typically reduced in the mutants compared to the control. Among the mutants, the average plant height ranged from 53.45 cm in Nm12 to 118.45 cm in Nm2. Additionally, the average number of primary branches per plant varied from 4.47 in Nm12 to 9.17 in Nm6, with the overall average plant height for all mutants indicating an increase relative to their parents. The significant rise in average number of main branches per plant was recorded in mutants Nm1 (8.15), Nm3 (7.47), Nm6 (9.17), Nm9 (8.33), and Nm10 (7.67). For the average number of secondary branches per plant, the majority of mutants showed a decrease from their parents, with a range from 8.67 in Nm12 to 17.85 in Nm1. A significant increase in biomass weight was recorded in the mutants Nm1 (82.60 g), Nm2 (72.55 g), Nm3 (76.35 g), and Nm4 (92.55 g). The mean number of pods produced by each plant in the mutants was generally less than the control, except Nm1 (125.81 pods), Nm6 (105.20 pods), and Nm9 (102.85 pods), where the average value surpassed the control. The range for the average number of pods was from 67.73 (Nm12) to 125.81 (Nm1) (Supplementary Table 7).

The mean number of seeds per pod showed significant variation compared to control, with values ranged from 3.33 (Nm1) to 4.67 (Nm8). In the majority of the mutants, the mean number of seeds per plant fell below that of the parent, except Nm1 (401.10), Nm6 (411.92), Nm8 (409.05), and Nm9 (380.28). The range of mean number of seeds per plant varied from 289.85(Nm10) to 411.92 (Nm6). The average weight of 100 seeds was generally higher in the mutants compared to their parents, with the highest difference were observed between Nm8 and Nm9 (6.85 g to 9.05 g). There was also a significant increase was recorded in mean seed weight from the parent for mutants Nm1 (8.35 g), Nm7 (8.10 g), Nm9 (9.05 g), and Nm10 (8.63 g). The mean number of seeds produced by each plant in the mutants was higher than that of the parent, except Nm1 (33.85 g), Nm6 (31.25 g), and Nm9 (34.20 g). The range of mean seed production from the parent to the mutants varied 21.58 g (Nm4) to 34.20 g (Nm9). The mean time required for physiological maturity was varied from 107.33 days (Nm11) to 128.33 days (Nm9) significant decrease in mean time was recorded in Nm7 (111.33 days), Nm10 (109.67 days), and Nm11 (107.33 days). In contrast, significant increases in the average time required for physiological maturity were observed in mutants Nm3 (124.67 days) and Nm9 (128.33 days) (Table 3; Fig. 2).

Table 3 Nutrient quality in seeds of M3 mutants of grasspea variety Biol-212.
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Fig. 2
figure 2

Mutagenic effect on seed yield, fresh weight and 100 seed weight in mutants lines Nirmal grasspea variety.

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In the majority of the Biol-212 mutants, the mean values of β-ODAP (%) and protein content (%) show significant differences compared to parents and these differences were both positive and negative direction. However, there was only a significant decrease in mean values for β-ODAP content observed in mutants with TIA. The average β-ODAP content in these mutants generally decreased compared to the parents and it was ranged from 0.078% (Biom21) to 0.142% (Biom1). The mutant Biom10 (0.086%), Biom17 (0.085%), Biom19 (0.082%), Biom20 (0.085%), Biom21 (0.078%), and Biom22 (0.080%) showed a significant decrease mean value of β-ODAP content. Conversely, the average protein content in these mutants showed a positive shift compared to the parents except Biom9, Biom10, Biom11, Biom13, Biom24, Biom26, and Biom27. However, there were significant increases in average protein content in Biom1 (30.47%), Biom6 (30.06%), Biom7 (30.27%), Biom12 (30.06%), Biom15 (30.47%), Biom22 (29.86%), and Biom29 (29.45%). The mean concentration of TIA ranged from 14.16 (Biom27) to 17.88 (Biom27). In most of the mutant lines, the mean TIA concentration was lower than that of the parent lines. Notably, a significant decrease in mean TIA concentration compared to the parent was recorded in Biom3 (15.15), Biom6 (15.09), Biom9 (15.18), Biom11 (14.88%), Biom20 (14.68%), Biom26 (14.35%), Biom27 (14.16%), and Biom29 (15.00%) (Table 3).

In most of Biol-212 mutants, there was a significant decrease in mean values of plant height at maturity from the parental. However, there were some mutants, such as Biom8 (118.40 cm) and Biom16 (121.15 cm), did not show any decreased in plant height. While, the mean value for primary branches per plant ranged from 5.38 (Biom25) to 12.50 (Biom13). The mean number of primary branches per plant showed generally an increased from the parent with some exceptions. Among these, Biom1, Biom3, Biom6, Biom8, Biom9, Biom13, and Biom22 had significantly higher numbers of primary branches compared to their parents. In contrast, Biom2, Biom5, Biom12, and Biom29 had significantly lower numbers. Similarly, the number of secondary branches per plant tended to increase from the parental line, with several mutants showing a notably higher number of branches. The mean secondary branches per plant ranged from 10.38 (Biom2) to 26.67 (Biom22). Notably, Biom1, Biom3, Biom6, Biom8, Biom9, Biom13, Biom22, Biom29 had significantly more secondary branches than their parents. The weight of plant fresh matter also varied significantly from the control group, with mean values ranging from 47.75 g (Biom25) to 105.45 g (Biom22). A notable increase in weight from the parent was observed in Biom1, Biom3, Biom6, Biom7, Biom8, Biom9, Biom11, Biom12, Biom13, Biom22, Biom23, and Biom29.

Similarly, the number of pods per plant of Nirmal among most of the mutants of variety Biol-212 was reduced in parent and was distributed equally. There were notable significant positive and negative shifts in the standard deviations of the mean within the mutant population. The mean number of pods per plant among the mutants varied from 54.87 (Biom25) to 138.63 (Biom13). There was a marked increase in the average number of pods per plant from the parental variety, which was observed in mutants Biom1 (111.78), Biom7 (120.69), Biom8 (113.35), Biom11 (130.68), Biom13 (138.63), Biom14 (111.11), Biom17 (106.90), Biom19 (106.91), Biom20 (113.04), and Biom22 (123.71). The mean number of seeds per pod in most of the mutants was moved towards lower values compared to the parental variety, with only a few mutants, namely Biom1 (4.33), Biom5 (4.33), Biom11 (4.17), and Biom24 (4.17), significantly exceeding the average of the parental variety. The range of mean seeds per pod varied from 2.83 (Biom20) to 4.33 (Biom1 and Biom5). The mean number of seeds per plant shifted away from the parent and it was 212.92 (Biom25) to 533.37 (Biom11). The average yield of seeds per plant in the majority of the mutants was significantly reduced than the parent and exhibited a notable change in direction, both upwards and downwards. The average seed yield per plant ranged from 18.15 g (Biom25) to 47.25 g (Biom22), showing significant improvement in the average yield of Biom1 (38.86 g), Biom6 (38.30 g), Biom7 (39.10 g), Biom8 (40.25 g), Biom11 (36.75 g), Biom13 (42.85 g), Biom22 (47.25 g), and Biom23 (35.05 g) (Supplementary Tables 8 and Fig. 3).

Fig. 3
figure 3

Mutagenic effect on seed yield, fresh weight and 100 seed weight in mutants of grasspea variety Biol-212.

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The mean duration for achieving physiological maturity ranged from 112.00 days (Biom17) to 129.67 days (Biom11). This range also showed both negative and positive direction in the average number of days required for physiological maturity among the mutants. Notably, mutant Biom10, Biom17, Biom24, and Biom25 all showed a significant decrease in the average number of days required for physiological maturity from the parent, while Biom11 showed the highest duration of physiological maturity with 129.67 days (Supplementary Table 8).

The mean value of protein content (%) of Berhampur Local variety of M3 mutant showed a significant deviation from the parent and values were decreased for some mutants and also few mutants showed increased in mean value. Conversely, the mean protein content in all mutants was found lower than the parent, ranging from 0.156% (Blm8) to 0.533% (control). There were also significant decreased in the average protein content among specific mutants, such as Blm1 (0.479%), Blm3 (0.221%), Blm4 (0.226%), Blm6 (0.159%), Blm7 (0.427%), Blm8 (0.156%), Blm9 (0.339%), and Blm10 (0.275%). On the other hand, Blm2 and Blm8 showed slight increases in average protein content compared to the parent. The average protein content in a Blm3 was lower than the parent and did not show any significant rise in mean protein content. The highest protein content was observed in Blm8 (26.20%). The mean values of TIA (Trypsin inhibitor unit), varied from 23.69 (Blm5) to 27.83 (Blm7). Overall, TIA levels were lower in the mutant plants compared to the parent while, Blm5 showed the highest decrease in TIA, which was not significantly lower than in the control, while Blm3 showed the least change in TIA levels, indicating no significant increase over the parent. The range of TIA levels varied from 23.69 (Blm5) to 27.83 (Blm7) (Table 4).

Table 4 Nutrient quality in seeds of M3 mutants of grasspea variety Berhampur local.
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Similar to the two other varieties, the majority of Berhampur Local’s mutants experienced fluctuations in the average values from positive and negative directions compared to control of all their quantitative traits. Specifically, the mutations led to a decrease in mean height of mature plants compared to their parent except Blm1 (91.45 cm) and Blm5 (88.80 cm), which showed a notable increase in plant height. Plants also had fewer branches at the primary level, with values decreasing from 3.95 (Blm9) to 8.77 (Blm6). Nevertheless, the majority of the mutants exhibited a greater number of branches, with Blm1 (8.73), Blm2 (7.14), Blm3 (7.96), Blm4 (8.08), Blm5 (7.33), Blm6 (8.77), and Blm10 (8.09) demonstrating notable increases in comparison to their parental counterparts. The number of secondary branches varied from 7.14 (Blm9) to 23.75 (Blm1). The average weight of fresh foliage per plant was ranged 38.15 g (Blm9) to 82.75 g (Blm6) except, Blm7, Blm8, Blm9, and Blm10, the mean weight of fresh foliage increased significantly from the parent, with Blm1 (78.25 g), Blm3 (74.50 g), Blm5 (80.40 g), and Blm6 (82.75 g) were experiencing significantly increases. In a similar way, the average number of pods per plant decreased from 77.67 (Blm9) to 124.33 (Blm1) except Blm7, Blm8, Blm9, and Blm10, all other mutants exhibited a greater number of pods per plant compared to their parental lines. Notably, Blm1 (124.33), Blm2 (115.22), Blm3 (120.92), Blm4 (110.88), Blm5 (117.25), and Blm6 (108.44) showed highest significant increase. Conversely, the mean number of seeds per pod in most mutants showed a downward trend, markedly differing from that of their parent plants. The range of mean number of seeds per pod among the mutants varied 3.00 (Blm2) to 4.47 (Blm7). For most mutants, the mean number of seeds per pod decreased below than the parent, except Blm1, Blm3, and Blm6, which showed a significant increase in this value compared to the parent. Blm1 showed the highest average of seeds per pod (4.47). The average number of seeds per plant also showed a trend away from the parent, with Blm8 had the highest value at 473.27. The mean weight of 100 seeds in the mutants, excluding Blm7, show a positive shift from their parent, with a significant increase was observed in Blm2 (7.40 g), Blm5 (8.05 g), Blm6 (6.55 g), and Blm8 (8.43 g). However, Blm8 had highest weight (473.27 g). Similarly, the mean weight of 100 seeds per plant also shifted away from the parent in the mutant lines, with Blm2 showed the lowest mean value (5.65 g). The mean yield of seeds per plant increased in all the mutants except Blm4, Blm7, and Blm9. Significant positive shifts in average yield were observed in Blm2 (7.40 g), Blm5 (8.05 g), Blm6 (6.55 g), Blm8 (8.43 g). However, Blm5 showed the highest yield (31.65 g). The duration for the plants to attain physiological maturity ranged from 121.61 days (Blm9) to 134.00 days (Blm5). Although there were fluctuations in the mean values among the mutants, with some showing both increases and decreases, none of the mutants exhibited statistically significant changes in the average duration needed to reach physiological maturity (Supplementary Tables 9 and Fig. 4).

Fig. 4
figure 4

Mutagenic effect on seed yield, fresh weight and 100 seed weight in mutants of grasspea variety Berhampur local.

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In this study, after analyzing eleven traits contributing to yield and three biochemical characteristics and several grasspea mutants were identified as both economically and qualitatively valuable for both human and animal consumption. Among these mutants, five (Nm1, Nm2, Nm5, Nm6, and Nm8) from the Nirmal mutant, eleven (Biom6, Biom8, Biom9, Biom11, Biom13, Biom19, Biom20, Biom21, Biom22, and Biom23) from the Biol-212 mutant, and seven (Blm1, Blm2, Blm3, Blm4, Blm5, Blm6, and Blm8) from the Berhampur Local mutant, were outperformed from their parent plants in terms of yield (seed and/or fresh weight) as well as biochemical parameters such as low β-ODAP, Trypsin Inhibitor, and higher protein content (Supplementary Table 10).

Discussion

The primary challenge breeders face is to improve the crop quality with increasing yield. Grasspea is utilized as food, feed, and fodder, so selecting lines that maximize biomass, in addition to high-yielding. Grasspea, are crucial and affordable source of protein in human diets playing a key role in increasing protein levels. Grasspea cultivation is hindered by two main biochemical issues; β-ODAP and Trypsin Inhibitors8,10. Consequently, choosing lines characterized by low levels of β-ODAP and trypsin inhibitors with combinations of high protein content, can significantly enhance grasspea breeding initiatives26. Nevertheless, the limited genetic variation resulting from the prevalent self-pollination and interspecific incompatibility has impeded the conventional breeding efforts aimed at improving these traits in grasspea. Induced mutagenesis may serve as a viable alternative and a crucial method for introducing additional genetic variability, thereby expanding the opportunities for the isolation and characterization of novel genetic variants15. In the present study, two well-known varieties (Nirmal and Biol-212) with local variety Berhampur were used for the improvement of biochemical properties (reduced β-ODAP and Trypsin Inhibitors; increased protein content) by using gamma and EMS mutagens. These mutagens individually and combined effect showed significant effect. Previous studies have also documented seed mutations, including changes in color, size, and shape, in various crops, such as Seed mutations in chickpea, specifically concerning the size of the seeds. It has been observed that the frequency at which viable mutations are caused by physical and chemical mutagens varies according to the genotype types as well as the type of mutagen. The reason for this heterogeneity in the incidence of viable mutations might be because the mutagens operate differently on distinct base sequences in different genes27.

M3 lines of all three varieties with control were evaluated for biochemical and yield traits. The genetic diversity observed across all three varieties, subjected to eight mutagenic treatments along with a control for each variety, demonstrated significant variation in all fourteen traits among the treatments. The result showed effect of mutagens on M3 generation. Similarly, a high-yielding mutant of urdbean cultivar T9 was produced by using gamma irradiation and sodium azide28. Morphological and high yielding mutants were developed in black gram with more pod length, seeds per pod and 100 seed weight than the parent29. During this investigation, it was discovered that genetic variation for all characters increased more in the M3 generation than in the M2 generation. Additionally, the amount of variation varied for each character in each of the three varieties, indicating different varietal responses to gamma rays, EMS, and their combination treatments. The genetic modification procedures in the M3 generations typically resulted in a change in the mean values of several key characteristics, both beneficial and detrimental. However, the average values for the number of seeds per plant, weight of 100 seeds, weight of fresh seeds per plant, and yield per plant were generally more positive compared to the control group across all three varieties. Similarly, two varieties, “Poltavskaya” and “Bina Khesari-1,” were developed through mutation breeding in Russia and Bangladesh, respectively, using EMS (0.01%) and gamma rays (250 Gy)17. Asnake (2012)30 reported putative mutants with higher Methionine content than the parent line; in these altered putative mutant lines, the grass pea Methionine increased from 25% in the parent line to 50%. Similarly, our results also supported that mutation breeding enhance yield and related attributes in grasspea.

The focus of our study for development of high-yielding varieties with a low content of β-ODAP. The acceptable level of β-ODAP in human food is below 0.2%7. The M3 generation result showed mutant lines of all three varieties reduced β-ODAP, TIA and increased protein content. The mutant of all three varieties showed less than 0.2% β-ODAP. Mutant of Biol-212 showed less than 0.1% β-ODAP with more protein content and less TIA content which are very desirable for human consumption. The famous grasspea variety from India, “Pusa 24,” was chosen in a field in 1966 and was recognized as the first cultivar with low ODAP content (0.2%) in its seeds. Notably, a number of the other low-ODAP varieties in India and other nations can trace their ancestry back to the Pusa 24 variety. Rice fallow (LSD3, LSD6, Pusa-305, and Selection 1276) and upland (LSD1, LSD2) varieties with low (up to 0.2%) ODAP content have been developed31. The cultivar “Quila-blanco” was created in Chile in 1983 by selection from a heterogeneous population grown locally. This cultivar’s main traits are bold white seeds (weighing 28.7 g at 100 seeds) with a protein content of up to 24.0% and synchronous maturity32.

Biochemical traits β-ODAP, Trypsin Inhibitor, and protein content were improved in five out of 12 mutants of Nirmal, eleven out of 29 mutants of Biol-212, and seven out of ten mutants of Berhampur Local. But only Nm2 and Nm8 of Nirmal, Biom6, Biom8, Biom11, Biom13, Biom20, Biom22, and Biom23 of Biol-212, and Blm1, Blm3, Blm5, Blm6, and Blm8 of Berhampur Local exhibited non-segregating behavior in M3. The non-segregating populations are more stable and maintain traits generation to generation for low ODAP content (Tripathy and Lenka, 2010). These non-segregating is stable with improved quality of biochemical traits and further trial is going on to test the stability of these mutant lines. These mutants are commercially important for grasspea.

The agromorphological traits were also recorded for M3 mutants of all three varieties. There were significant variability was observed among the selected mutants in most of the yield attributing traits. The mutational change in each quantitative trait could micro or macro and caused significance in the improvement of the breeding programme depending on the characters involved. Similarly, mutagens produced early maturity grasspea genotypes13. The level of improvement varied mutants of all three varieties because all three varieties of present studies were diverse. Therefore, the performance for the different agromorphological traits varied33.

Conclusion

By considering all the results obtained from control and gamma irradiated and EMS and their combination in three varieties of grasspea plants, it can be concluded that the mutagen on grasspea seeds was effective in creating variability. The variability observed in present study was positive for biochemical traits (low β-ODAP and TIA high protein content). Similarly, the variability obtained after mutagen treatments is also found to be positive for yield and yield attribute traits. A considerable degree of genetic variability was recorded for the majority of traits, as indicated by significant variation resulting from mutagenic treatments in different varieties. This suggests that selecting progenies based on favorable means and higher variation in the early generations will be highly beneficial, ultimately contributing to the enhancement of yield and its components in the M3 generation. The mutants exhibiting improved yield and quality traits may play a crucial role in augmenting the grasspea germplasm. These mutants showed low β-ODAP and TIA high protein content that could be very beneficial in grasspea breeding and also be reduced the neurolathyrism.