Legume Research

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Studies on Effect of Mutagens on Quantitative Characters in M2 and M3 Generation of Horsegram [Macrotyloma uniflorum (Lam.) Verdc]

K. Geetha1,*, S. Divya1, S. Srividya1
1Regional Research Station, Paiyur, Krishnagiri-635 112, Tamil Nadu, India.
2Krishi Vigyan Kendra, Papparapatty, Dharmapuri-636 809, Tamil Nadu, India.
  • Submitted15-03-2021|

  • Accepted07-10-2021|

  • First Online 27-10-2021|

  • doi 10.18805/LR-4608

Background: Induction of mutation plays an important role in the breeding programme among modern methods of plant breeding. Genetic variability is prerequisite for crop improvement and through induced mutation it was found to be very effective for creating variability in the quantitative and qualitative characters. Hence, the present study was aimed to induce genetic variability within short time. In the present investigation positive as well as negative impact on quantitative traits were recorded.

Methods: The experiment material comprised of seeds of horsegram [Macrotyloma uniflorum (Lam.) Verdc] variety Paiyur 2 which was subjected to ethyl methane sulphonate (02, 0.3, 0.4 and 0.5%), gamma radiation (100, 200, 300 and 400 Gy) and combination treatments. The mutations affecting gross morphological changes in growth and yield characters such as plant habit, flowering, pod morphology, maturity and seed yield were scored as quantitative characters. The micro mutations at population level can be easily detected in the form of increased variations for quantitative traits in the segregation of mutagen treated populations. Micro mutations can alter morpho-physiological characters hence they are of a particular interest to the plant breeders. ANOVA test was performed to determine the significant differences and duncan’s multiple range test (p=0.05) to compare the differences among treatment means. 

Result: Among the twenty-five treatments studied both the mutagens, gamma radiations and Ethyl Methane Sulphonate proved to be very effective to induce variability in quantitative traits in M2 and M3 generations. The results of Duncan’s multiple range test analysis revealed that the treatment of mutagens in combinations induced more positive effects as compared to that of using alone. The combined treatments were found positive for improvement of primary branches per plant, no. of pods per plant, pod length, no. of seeds/pod and seed yield per plant in M2 and M3 generations whereas the treatment of gamma irradiation imparted its significant role for improvement of plant height, days required for 50% flowering, days to maturity and 1000 seed weight in both M2 and M3 generations whereas the treatment with Ethyl Methane Sulphonate is useful for improving no. of pods per plant and no. of seeds/pod in M2 generation alone.

Horsegram is a hardy drought resistant, annual, traditional and tropical grain legume. It derives its name from the fact that it is important as annual feed and as such fed to horses. It is the cheapest of pulses and is hence the poor man’s pulse. The bhusa of the crop after removal of pods is used as cattle feed. In Tamil Nadu the area under horsegram is about 18 per cent of the total area of 2.2 million hectares in India. The productivity of the crop is rather low with an average grain yield of 494 kg/ha as against 539 kg/ha of all the grain legumes as a whole. It ranks as the third among Indian pulses in area. The Southern States are the major horsegram growing States in the country.

        
Horsegram is cultivated in areas with annual rainfall of 300-600 mm and highly drought tolerant, but does not tolerate floodingor water logging. The favourable average temperature is 18-27°C. Horsegramis adapted to a wide range of well drained soils from sands and gravels to clay loams and heavy clays with neutral soils. It grows on soils having pH 5.5 to 8.
        
Genetic variability is the basic need in any of the crop to be improved. Mutation breeding is identified as the efficient method to enhance the genetic variability whenever the variation is not available in the natural resources. Knowing the limited variability in horsegram, present investigation has been carried out for the improvement in this crop by utilizing the variety Paiyur 2.
The authentic seeds of horsegram [Macrotyloma uniflorum (Lam.) Verdc] variety Paiyur 2 released from Regional Research Station, Paiyur were used. Ethyl Methane Sulphonate (EMS), Gamma rays and their combinations were employed in present study for the treatment of seeds of horsegram. EMS was used for the seed treatment of horsegram. Various concentrations of EMS (0.2% to 0.5%) were prepared in 0.1 m phosphate buffer pH -7.0. Selected seeds were soaked in distilled water for 10 hours and the wet seeds were treated with different concentrations of EMS (such as 0.2, 0.3, 0.4 and 0.5% v/v) for four hours. Healthy, dry and uniform seeds of horsegram with moisture content of 10-12% were treated with 100, 200, 300 and 400 Gy using gamma irradiation chamber at Bhabha Atomic Research Centre, Trombay. For combination treatments the gamma irradiated seeds were treated with different concentrations of EMS. The untreated seeds served as control. The seeds treated with various concentrations of EMS were washed thoroughly with tap water for 2 hours to terminate the reaction of chemical mutagen to leach out the residual chemicals. The treated seeds (675) from each treatment were used for raising M1 generation in field. Present investigation was carried out at Regional Research Station, Paiyur. All the experiments were carried out in triplicate following RBD design. The distance between two rows and two plants was 30 × 15 cm and the distance between two adjacent plots was one meter. The seeds of individually harvested M1 plants were sown in the experimental field to raise M2 generation in separate rows during Rabi season of the year 2018. The treated as well as control plants were screened for quantitative traits to study the induced variability. From each replication and treatment including control 20 plants were randomly selected for recording data on different quantitative characters in M2 generations. Data on nine quantitative traits such as plant height (cm), primary branches per plant, days required for 50% flowering, days for maturity, no. of pods per plant, pod length (cm), no. of seeds/pod, 1000 seed weight (g) and seed yield per plant (g) were recorded. All the surviving M2 plants were harvested individually and seeds of single plant from each treatment were kept separately for raising the M3 generation. Observations on quantitative characters in M3 generation were similar to that of M2 generation.
        
ANOVA test was performed to determine the significant differences due to various treatments while DMRT test (p=0.05) was employed to compare the differences among treatment means. The DMRT analysis was performed using SPSS Software.
Analysis of variance
 
M2 and M3 populations imparted significant differences for most of the traits studied.
 
Quantitative characters (Micro mutations) in M2 and M3 generations
 
EMS and Gamma radiations proved to be very effective to induce variability in quantitative traits in M2 and M3 generations (Table 1 and Table 2). Positive as well as negative impact on quantitative traits was well documented by Barshile et al., (2008) in chickpea, Bolbhat and Dhumal (2009), Dhumal and Bolbhat (2012) and Kanaka (2012) in horsegram.
 

Table 1: Duncan multiple range test (DMRT) of quantitative traits in M2 generation.


 

Table 2: Duncan multiple range test (DMRT) of quantitative traits in M3 generation.


 
Plant height
 
All the mutagens were effective for inducing variability in plant height in M2 generation (Table 1). The DMRT represented the similar group (Group h) for minimum plant height in 200 Gy+0.3% EMS (71.47 cm), 200 Gy+0.5% EMS (70.93 cm) and 300 Gy + 0.2% EMS (60.97 cm) as compared to control (92.13 cm). Gamma treatment of 100 Gy 98.43 cm) falls under Group a showed significant increase in plant height while other treatments showed -ve influence.
        
Similar trend in plant height was observed in M3 generation (Table 2). Maximum plant height 93.53 cm in M3 was noted in 100 Gy gamma rays (Group a). The minimum plant height of 72.23 cmwas noted in 300 Gy+0.4% EMS (Group f) through DMRT. The average height of control plants was 90.33 cm. All the doses/conc.ofgamma irradiation, EMS and their combinations caused reduction in plant height with few exceptions.Similar results werereported by Dalvi (1990). Gamma irradiation exhibited a stimulatory effect of significant increase in plantheight. Findings of Yaqoob and Abdur (2001) in mungbean and Barshile et al., (2008) in chickpea were in agreement with above results.
 
Number of primary branches per plant
 
Data obtained in M2 generation on number of primary branches per plant (Table 1) indicated that the mean values of this parameter showed positive and negative influence. 100 Gy and 0.4 % EMS (Group a) showed increased number of primary branches per plant. Maximum number of primary branches per plant was recorded in 100 Gy (9.23) and 0.4% EMS (8.87) over control (8.80). In combination treatments there was no definite pattern. The maximum decrease (6.63) was noted in 200 Gy + 0.5% EMS (Group j) compared to control (8.80). The highest increase (9.63) was noted in 100 Gy+0.5% EMS. In nine treatments the primary branches per plant were increased over control. Similar trend was obtained in M3 generation (Table 2). Six treatments showed increase in no. of primary branches per plant as compared to control and M2. DMRT revealed that maximum number of primary branches was recorded in 100 Gy+0.3% EMS (9.40) over control (8.93) and categorized as Group a. The treatment 200 Gy+0.5% EMS (Group j) recorded least number of primary branches as 7.10. Almost all the treatments of gamma irradiation and EMS showed positive as well as negative impact on primary branches per plant in horsegram. Dalvi (1990) and Singh et al., (2000) also noted similar trend with physical as well as chemical mutagen.
 
Number of days required for 50% flowering
 
The results of the gamma radiation in M2 generation in different treatments indicated that there was significant change in the number of days required for 50% flowering while in EMS and combination treatments there was no definite pattern. In all the gamma treatments, days to 50% flowering were less than control except 400 Gy. The minimum numbers of days required for 50% flowering were 66.87 DAS in 200 Gy (Group c) as compared to control (72.77 DAS). In the combination treatments, days to 50% flowering were less than control in 300 Gy with different concentrations of EMS viz., 0.2 and 0.3% and 400 Gy + 0.2% EMS. Similarpattern was noted for M3 generation (Table 2). The minimum days required for 50% flowering were 68.03 DAS in 300 Gy (Group b), 69.23 DAS in 200 Gy (Group b), 69.03 in 300 Gy+0.2% EMS (Group b), 70.13 in 300 Gy+0.3% EMS (Group b). The number of days required for 50% flowering was not muchas compared to control except few treatments. However, gamma radiation and combination with EMS treatments caused early flowering. Dalvi (1990) also noted similar results in horsegram with different mutagens. The results recorded by Gaikawad et al., (2005), Rudraswami et al., (2006), Manjaya and Nandavar (2007) and Ahire (2008) in different legumes were supportive to the present findings.
 
Number of days required for pod maturity
 
The data recorded in Table 1 revealed that GR had succeeded in reducing the number of days required for pod maturity as compared to control. The results of gamma irradiation were highly significant for reducing the number of days required for pod maturity. The minimum number of days (97.37 DAS) required for pod maturity was noted in 200 Gy (Group c). The data obtained for M3 generation was on par with M2 generation (Table 2) where the minimum days required for pod maturity were 99.03 DAS in 300 Gy. Gamma radiation (200 Gy) was successful to induce earlier pod maturity by about 3-4 days as compared to control. Singh et al., (2000) reported contradictory findings with reference to this parameter.
 
Number of pods per plant
 
Gamma radiation and EMS single and combination had induced variability in number of pods per plant in M2 generation. The data recorded in Table 1 revealed that the treatments had stimulatory as well as inhibitory effect. In M2 generation maximum number of pods per plant (59.23) was noted in 100 Gy+0.2% EMS (Group a), 59.13 in 300 Gy+0.3% EMS (Group a), 58.97 in 400 Gy+0.2% EMS (Group a), 58.66 in 200 Gy+0.2% EMS (Group a), 57.93 in 100 Gy (Group a) and 57.90 in 100 Gy (Group a) than control (57.00). The minimum number of pods per plant (48.40) were recorded at 200 Gy+0.4% EMS (Group f), (48.50) at 400 Gy+0.5% EMS (Group f), (49.40) at 0.5% EMS (Group f) and (49) 200 Gy+0.5% EMS (Group f) as compared to control. However, all the combination treatments have caused reduction in number of pods per plant except 100 Gy + 0.2% EMS, 200 Gy+0.2% EMS, 300 Gy + 0.3% EMS and 400 Gy +0.2% EMS.
        
The trend in variation of pod number observed in M3 generation was similar to that of M2 generation. M3 generation had shown slight increase in pod number as compared to M2 generation (Table 2). Maximum pods were recorded in 300 Gy+0.4% EMS (59.67- Group a) and minimum pods was recorded in 200 Gy+0.4% EMS (48.57- Group i) and 300 Gy+0.2% EMS (48.43- Group i). There was increase as well as decrease in number of pods per plant with different doses/concentrations used. The results of Dalvi (1990) for horsegram were in agreement with present study. Gaikawad et al., (2005) in lentil noted similar results. However, decrease in pod number was also recorded by Barshile et al., (2008) in chickpea.
 
Pod length
 
All the mutagenic treatments showed inhibitory effect on pod length except 300 Gy+0.2% EMS (5.67 cm- Group a) than control (5.2cm) in M2 generation. The treatments of GR, EMS and their combinations in M2 as well as in M3 did not show any significant change in pod length (Table 1). The results reported by Singh et al., (2000) in Vigna and Dalvi (1990) in horsegram were in conformity with present findings. M3 generation had shown slight increase in pod length as compared to M2 generation (Table 2). In M3 all the mutagenic treatments showed inhibitory effect on pod length except 200 Gy+0.2% EMS (5.80 cm- Group a) than control (5.60 cm).
 
Number of seeds per pod
 
All the treatments showed decreased number of seeds per pod except (0.3% EMS (5.93- Group a), 100 Gy+ 0.2% EMS (5.90- Group a), 300 Gy + 0.3% EMS and 400 Gy+0.2% EMS (6.00- Group a) compared to control (5.90). Data on total number of seeds per pod (Table 1) in M2 progeny showed non-significant changes as compared to control. M3 generation showed similar trend (Table 2). The results recorded in table indicated that all the treatments of mutagens of GR, EMS and GR+EMS exerted inhibitory effects on number of seeds per pod except 300 Gy + 0.2% EMS. Decrease in number of seeds per pod was recorded by Dalvi (1990), Dubey (1990), Gaikwad et al., (2005) and Barshile and Apparao (2006).
 
1000 seed weight
 
Results recorded on 1000 seed weight (Table 1) indicated that Gamma ray treatment of 100 Gy had exercised +ve effect on this parameter and showed increased 1000 seed weight (36.33g-Group a) compared to control (30.70 g). The results of M3 generation were on par with M2 generation (Table 2). The results with GR, EMS and GR+ EMS showed negative as well as positive impact in horsegram. Similar observations were also made by Singh et al., (2000) in urdbean, Gaikawad et al., (2005) in lentil Sagade (2008). Increase in 1000 seed weight was observed in 100Gy (34.53-Group a) and 400Gy+0.2% EMS (34.93- Group a) compared to control (32.23 g) in M3 generation.
 
Seed yield per plant
 
Mean values for seed yield per plant decreased in all treatments as compared to control (Table 1). All the mutagenic treatments of gamma radiation showed -ve effect except 300 Gy + 0.2% EMS (33.63 g- Group a). EMS (0.3%) (Group- abcde) caused maximum increase (31.03 g), while all other treatments showed reduction in seed yield per plant over control. The combination treatment 300 Gy+0.2% EMS had induced maximum increase (33.63 g) over control (30.67 g). But all other treatments had caused reduction as compared to control except 200 Gy+0.2% EMS (31.27g). In M3 generation seed yield per plant was increased in 300 Gy+0.2% EMS (33.90g- Group a) as compared to control (32.37 g). The results are in line with the findings of Priyanka et al., (2019). All the mutagenic treatments except few treatments showed inhibitory effect on seed yield per plant. Patil et al., (2004) in soybean, Auti (2005) in mung bean, Banu et al., (2005) in cowpea, Barshile et al., (2008) in chickpea and Senapati et al., 2008 in black gram also recorded adverse effect on seed yield per plant due to various types of mutagenic treatments. Hakande (1992) reported wider variability in yield due to mutagenic treatments in winged bean which was attributed to pollen sterility and genetical as well as physiological alterations caused by mutagens.
        
Yield is an important trait, as it governs the economic benefit. Its expression is inherited by many genes, which control the production, transport and storage of assimilates. Previous studies indicated that both additive and non-additive genes contribute to yield. The variability in yield was induced by mutagenic treatments. In the present study increased seed yield was attributed to increase in number of pods per plant, length of pods and 1000 seed weight per plant.
Both the mutagens proved to be very effective to induce variability in quantitative traits like plant height, primary branches per plant, number of days required for 50% flowering, days required for first pod maturity, number of pods per plant, pod length, number of seeds per pod, 1000 seed weight and yield per plant in M2 and M3 generations. Combination of EMS and gamma irradiation treatments showed better positive effects as compared to that of separate treatment of EMS and Gamma irradiation. The traits viz., primary branches per plant, no. of pods per plant, pod length, no. of seeds/pod and seed yield per plant in M2 and M3 generations can be improved through combined treatments. Gamma irradiation exerted its significant role for improvement of plant height, days required for 50% flowering, days to maturity and 1000 seed weight in both M2 and M3 generations. Within separate treatments gamma irradiation showed more positive effects than EMS.
All authors declared that there is no conflict of interest.

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