Effect of gamma radiation and EMS on mutation rate: their effectiveness and efficiency in faba bean (Vicia faba L.)

ABSTRACT

Faba bean is one of the most important grain legumes in the world because of its multiple uses and its ability to grow over a wide range of climatic conditions. The objective of this study was to evaluate the effectiveness and efficiency of single and combination doses of gamma rays and ethyl methanesulphonate (EMS) on two different varieties of faba bean. Studies on induced mutagenesis in faba bean involving EMS and gamma rays are sparse. Genetically pure, uniform, dry and dormant seeds of Vicia faba varieties Vikrant and PRT-12 were treated with four different doses of gamma rays (100, 200, 300 and 400 Gy) and of EMS (0.01, 0.02, 0.03 and 0.04%). However, the utility of a mutagenic agent is determined by its ability to induce a high frequency of desirable mutations as compared to undesirable ones; thus, it is necessary to assess the effectiveness and efficacy of mutagens. The results indicated that EMS is more effective in inducing mutations than gamma rays, with the following trend: EMS > gamma rays+EMS > gamma rays. However, with regard to mutagenic efficiency measured based on seedling injury, gamma rays were more efficient than EMS, with the following trend: gamma rays > gamma rays+EMS > EMS. Both mutagenic effectiveness and efficiency were found to be higher at lower doses of both the mutagens. The possible reason for decrease in mutagenic efficiency and effectiveness with increase in dose may be the higher dose of mutagens and less damaging effect of lower doses of mutagens.

KEYWORDS:

• Gamma rays
• ethyl methanesulphonate
• mutagenic effectiveness
• mutagenic efficiency

Introduction

Faba bean has gained increasing attention from the food industry and consumers, mainly due to the quality of its protein fraction, high nutritional value and easy availability round the year (Iqbal 2016). Induced mutagenesis has been recently recognized as the most efficient tool for improving nutritional properties. An optimum strategy for maintenance and utilization of locally available plant genetic resources is required for plant breeders to develop new varieties. Faba bean is a partially cross-pollinated crop. The out-crossing rate among different faba bean populations or different genotypes of the same population depends on various factors, such as floral and inflorescence characteristics of the genotype and pollination abundance. The lack of adequate pollination and reduced seed setting can be major constraint to yield. Faba bean seeds serve as a source of high content of proteins and starch along with good source of vitamins and minerals (Ofuya and Akhidue 2005). Induced mutagenesis has played a significant role in successful development of new varieties by upgrading a specific trait without altering the original genetic makeup of the cultivar (Raina et al. 2016). Physical and chemical mutagenesis has a number of advantages as mutagens induced random changes throughout the genomes, generating a wide range of mutations in all target genes, and a single plant can contain a large number of different mutations resulting a manageable population sizes (Raina et al. 2017). Mutations induced by physical and chemical mutagens increase the genetic variability for certain characters so that selection is more effective and the probability of getting the desired genotypes is considerably enhanced. Mutagenesis is a promising technology to increase variability in species in which natural genetic diversity is not large or, as often happens, where phenotypes desired by plant breeders are not available because they disappeared due to their poor competition ability in natural conditions. As per the reports of FAO, 2017 there are about 20 mutant varieties developed to date and the recently released mutant variety, Geca5, has improved attributes including good quality and early maturity. The list of mutant varieties developed along with their improved traits is presented in Table 1.

Table 1. Applications of induced mutagenesis for improvement in various traits of Vicia faba (IAEA 2017).

Mutant variety name	Mutagen treatment	Material	Registration date	Country	Highlight
Babylon	Gamma rays 30 Gy	–	1994	Iraq	Resistance to diseases, high yield and high protein content
Bronto	Gamma rays 60 Gy	Seed	1989	Poland	High yield
Chabanskii	–	Seed	1985	Russian Federation	Early maturity, grain yield 5 t ha^–1, green mass 47 t ha^–1 and dry matter 10.3 t ha^–1
Dino	Gamma rays 70 Gy	Seed	–	Poland	Short stem, early maturity (4 days), but smaller thousand grain weight (TGW)
Fritel	X-rays	–	1988	Germany	Improved plant architecture
Geca 5	Gamma rays 250 Gy	–	2008	Moldova, Republic of	Good quality and early maturity
Karna	Gamma rays	Seed	1983	Austria	High yield, erect, intermediate, suitable for grain and fodder
KYU 82	NMU, 0.01%	Seed	1987	Russian Federation	Early maturity, resistance to diseases and high yield
Martin	–	–	1994	Poland	Early and uniform maturity
Prikarpatskie 4	NEU+NMU+DMS+E	Seed	1986	Russian Federation	High yield
Rin-rei	Gamma and X-rays recurrent	–	1991	Japan	Dwarfness
Severinovskie 1	NMU, 0.01%	Seed	1992	Russian Federation	High yield and high protein content
Stego	Gamma rays 70 Gy	–	1987	Poland	Short stem, early maturity (4 days), but smaller TGW
Tifabo	X-rays	–	1990	Germany	Improved plant architecture
Tigo	X-rays	–	1988	Germany	Improved plant architecture
Tina	EMS	–	1988	Germany	Improved plant architecture
Tinos	–	–	1992	Poland	Terminal inflorescence plants, dwarfness, determinate growth and early maturity
Ti-Nova	X-rays 60 Gy	–	1986	Germany	Improved resistance to lodging, earlier and uniform maturity, reduced plant height, suitable for combine harvest
Tisesta	EMS	–	1991	Germany	Improved plant architecture
Tuwaitha	Gamma rays 40 Gy	–	1994	Iraq	Resistance to wilt, high yield and protein content

– Not specified

Mutagenic effectiveness and efficiency have much importance in mutation breeding experiments. The measure of frequency of mutations induced by unit dose of mutagen is considered as mutagenic effectiveness; whereas the mutagenic efficiency represents the mutations on the basis of biological damage i.e. sterility, injury and lethality. In mutation breeding programmes, the mutagenic effectiveness and efficiency are necessary for obtaining desirable mutations in plants (Smith 1972). Mutagenic effectiveness and efficiency depends upon the type of genotype used and the mutagen applied on it. Different workers have reported different effectiveness and efficiency of mutagens on different plants, e.g. grass pea (Waghmare and Mehra 2001), fenugreek (Koli and Ramkrishna 2002), lentil (Gaikward and Kothekar 2004; Laskar et al. 2018), clusterbean (Velu et al. 2007), chickpea (Laskar et al. 2015), blackcumin (Amin et al. 2016), cowpea (Girija and Dhanavel 2009), soybean (Khan and Tyagi 2010), garden pea (Sharma et al. 2009), and mungbean (Wani et al. 2017). Recently, laser irradiation has also been employed to meet agricultural demands and attain the goal of sustainable development (Abbas et al. 2018; Asghar et al. 2017). Pre-sowing laser of seeds has imposed beneficial effects on germination, seedling growth and yield of various crops (Asghar et al. 2016).

The present investigation was carried out to assess the efficiency and effectiveness of gamma ray mutagens in M₂ generation in faba bean. The genetic differences of different plant materials are responsible for the differential response of genotypes towards mutagens and thus the optimum dose for particular genotype varies for different mutagens (Goyal and Khan 2010; Raina et al. 2018).

Materials and methods

The plant material used are the two varieties of faba bean Vikrant and PRT-12, local varieties with better adaptability but lesser yield. Seeds of both varieties were procured from the Indian Agricultural Research Institute (IARI), New Delhi, India.

Raising M₁ generation

Uniform, healthy and dry seeds of each variety, with moisture content 12%, were irradiated with 100, 200, 300 and 400 Gy of gamma rays with a radioisotope ⁶⁰CO source (Gamma chamber Model-900 supplied by Bhabha Atomic Research Centre, Mumbai, India) at the National Botanical Research Institute, Lucknow, Uttar Pradesh, India. For the chemical mutagen, healthy seeds of uniform size of each variety were presoaked for 9 h in distilled water and treated with 0.01, 0.02, 0.03 and 0.04% of EMS for 6 h with intermittent shaking at room temperature of 22 ± 1°C. For combination treatments, dry seeds of each variety were first irradiated with gamma rays at 100, 200, 300 and 400 Gy doses and then treated with 0.01, 0.02, 0.03 and 0.04% EMS for about 6 h with intermittent shaking. On the next morning treated seeds of each dose and control were sown during the rabi season (mid-October) of 2012 for raising the M₁ generation. During the growing season, the average temperature recorded ranged from 30 to 35°C, the humidity was 50 to 57% and the day length was recorded as 12 h 10 min to 13 h 8 min. Three replications of 100 seeds each were sown for every treatment and control in each variety in a randomized complete block design (RCBD) at the Agriculture Farm, Aligarh Muslim University, Aligarh. The spacing was maintained at 30 cm (seed to seed in a row) and 60 cm (between the rows) in the field. Recommended agronomic practices were employed for preparation of field, sowing and subsequent management of the population of faba bean. The crop was harvested in March 2013 and seeds from each M₁ plants were stored and sown in the field during October 2013 to raise the M₂ generation.

Raising M₂ generation

Twenty-seven healthy seeds from each normal looking M₁ plant of all different treatments with their respective controls in both varieties were planted in plant progeny rows in M₂ generation during the rabi season of 2013. Different treatments and controls comprised of 50 progenies. The distance between seeds in a row and between the rows was kept at 30 × 60 cm, respectively. Three replications were maintained in each treatment.

Assessment of mutation frequency, mutagenic effectiveness and efficiency

Individual plants of the treatments along with the control were harvested separately for each treatment and desirable M₁ variants were selected and raised in M₂ generation; then mutation frequency, mutagenic effectiveness and efficiency were calculated (Konzak et al. 1965) and expressed as percentages.

Mutation frequency

On M₂ plant basis (% of mutated M₂ plants) mutation frequency was estimated as percentage of segregating M₁ plant progenies (Gaul 1964).

Mutagenic effectiveness

Mutagenic effectiveness is a measure of the frequency of mutations induced by unit dose of a mutagen. Formulae suggested by Konzak et al. (1965) were used to evaluate the mutagenic effectiveness of the mutagens used:

Mutagenic efficiency

The ratio of factor mutations to biological damage means desirable changes free from associated undesirable changes on mutagenesis. Mutagenic efficiency also represents the proportion of mutations in relation to biological damage. Mutagenic efficiency was calculated by the formula suggested by Konzak et al. (1965):

where “lethality” was calculated as percentage of plant survival reduction, “sterility” was calculated as percentage of seed fertility reduction and meiotic abnormality was calculated as percentage of meiotic abnormal cells.

Experimental results

Mutagenic effectiveness was calculated on the basis of number of mutations per unit dose of mutagen. Mutagenic effectiveness was found to be higher at lower doses/concentrations of both single and combination treatments of gamma rays and EMS. The estimates of effectiveness ranged from 0.04 to 0.06 in the variety Vikrant and 0.04 to 0.05 in the variety PRT-12 of gamma ray treatments, whereas the effectiveness of EMS treatments ranged from 58.33 to 83.33 and 33.33 to 66.66 in varieties Vikrant and PRT-12, respectively. The effectiveness of the combined gamma rays + EMS treatments ranged from 0.18 to 0.66 in the variety Vikrant and 0.16 to 1.00 in the variety PRT-12. In both varieties, EMS was found to be more effective than gamma rays and combination treatments. In both varieties, the order of effectiveness of mutagens was found to be EMS > gamma rays+EMS > gamma rays (Tables 2, 3; Figures 1, 2).

Table 2. Effectiveness and efficiency of gamma rays, EMS and their combination treatments in Vicia faba L. var. Vikrant in M₂ generation.

Treatment	% Seedling injury (I)	% Pollen sterility (S)	% Meiotic abnormalities (Me)	% Mutated plant progenies (Mp)	Mutagenic effectiveness	Mutagenic efficiency
						Mp/I	Mp/S	Mp/Me
100 Gy γ rays	2.75	4.25	9.20	6	0.06	2.18	1.41	0.65
200 Gy γ rays	4.13	12.76	10.70	12	0.06	2.90	0.94	1.12
300 Gy γ rays	10.34	17.55	12.91	14	0.04	1.35	0.79	1.08
400 Gy γ rays	13.09	19.14	17.29	16	0.04	1.22	0.83	0.92
0.01% EMS	3.44	4.14	8.40	4	66.66	1.16	0.96	0.47
0.02% EMS	5.17	7.25	8.89	10	83.33	1.93	1.37	1.12
0.03% EMS	12.75	10.36	9.28	12	66.66	0.94	1.15	1.29
0.04% EMS	13.75	14.50	12.93	14	58.33	1.01	0.96	1.08
100 Gy γ rays+0.01% EMS	4.13	7.97	11.15	8	0.66	1.93	1.00	0.71
200 Gy γ rays+0.02% EMS	7.58	15.42	13.80	14	0.58	1.84	0.90	1.01
300 Gy γ rays+0.03% EMS	13.79	24.46	17.39	16	0.29	1.16	0.65	0.92
400 Gy γ rays+0.04% EMS	17.58	26.06	15.58	18	0.18	1.02	0.69	1.15

Table 3. Effectiveness and efficiency of gamma rays, EMS and their combination treatments in Vicia faba L. var. PRT-12 in M₂ generation.

Treatment	% Seedling injury (I)	% Pollen sterility (S)	% Meiotic abnormalities (Me)	% Mutated plant progenies (Mp)	Mutagenic effectiveness	Mutagenic efficiency
						Mp/I	Mp/S	Mp/Me
100 Gy γ rays	1.39	5.69	6.32	4	0.04	2.87	0.70	0.63
200 Gy γ rays	2.78	10.88	9.36	10	0.05	3.59	0.91	1.06
300 Gy γ rays	8.68	17.09	8.89	12	0.04	1.38	0.70	1.34
400 Gy γ rays	10.77	18.13	10.38	16	0.04	1.48	0.88	1.54
0.01% EMS	2.43	2.12	4.82	2	33.33	0.82	0.94	0.41
0.02% EMS	3.47	3.72	5.55	8	66.66	2.30	2.15	1.44
0.03% EMS	11.11	6.91	8.78	10	55.55	0.90	1.44	1.13
0.04% EMS	11.45	13.82	10.41	14	58.33	1.22	1.01	1.34
100 Gy γ rays+0.01% EMS	2.43	7.77	10.43	6	1.00	2.46	0.77	0.57
200 Gy γ rays+0.02% EMS	6.60	15.54	13.5	12	0.50	1.96	0.77	0.88
300 Gy γ rays+0.03% EMS	12.85	24.27	13.30	14	0.25	1.08	0.57	1.05
400 Gy γ rays+0.04% EMS	18.06	23.31	14.34	16	0.16	0.88	0.68	1.11

Figure 1. Mutation rate of mutagens in relation to biological damage of Vicia faba L. var. Vikrant. Abbreviations: MRI, mutation rate based on seedling injury; MRP, mutation rate based on pollen sterility; MRMe, mutation rate based on meiotic abnormalities.

Figure 2. Mutation rate of mutagens in relation to biological damage of Vicia faba L. var. PRT-12. Abbreviations: MRI, mutation rate based on seedling injury; MRP, mutation rate based on pollen sterility; MRMe, mutation rate based on meiotic abnormalities.

Mutagenic efficiency was calculated on the basis of seedling injury (Mp/I), pollen sterility (Mp/S) and meiotic abnormalities (Mp/Me). On the basis of seedling injury, 200 Gy of gamma rays was most efficient among all mutagen treatments in both varieties. On the basis of pollen sterility, 100 Gy gamma rays was most efficient in the variety Vikrant, whereas 0.02% EMS was most efficient in the variety PRT-12 among all mutagen treatments.

On the basis of meiotic abnormalities, 0.03% EMS was most efficient in the variety Vikrant, whereas 400 Gy gamma rays was most efficient in the variety PRT-12 among all mutagen treatments. On the basis of seedling injury, the efficiency of mutagens in descending order was: gamma rays > gamma rays+EMS > EMS in both varieties. On the basis of pollen sterility the efficiency of mutagens in descending order was: gamma rays > EMS > gamma rays+EMS in the variety Vikrant, whereas it was EMS > gamma rays > gamma rays+EMS in the variety PRT-12.

On the basis of meiotic abnormalities, the efficiency of mutagens in descending order was: EMS > gamma rays+EMS > gamma rays in the variety Vikrant, whereas it was gamma rays > EMS > gamma rays+EMS in the variety PRT-12.

Discussion

Mutagenic effectiveness and efficiency have much importance in mutation breeding experiments. The measure of frequency of mutations induced by unit dose of mutagen is considered as mutagenic effectiveness; whereas the mutagenic efficiency represents the mutations on the basis of biological damage, i.e. sterility, injury and lethality. Thus, the response of genotype to the increasing dose of mutagen represents mutagenic effectiveness, and efficiency represents the proportion of mutations in relation to the undesirable biological effects. Mutagenic effectiveness and efficiency were observed in the M₂ generation. Results show that the effectiveness of EMS was higher than those of individual doses of gamma rays and combination treatments. Many workers have reported that EMS is more superior in causing useful mutations than gamma rays, as reported in rice (Kaul and Bhan 1977), lentil (Solanki and Sharma 1994), brinjal (Zeerak 1992), mungbean (Singh 2007), chickpea (Shah et al. 2008), urdbean (Thilagavathi and Mullainathan 2009), faba bean (Khursheed et al. 2016a), cowpea (Girija and Dhanavel 2009) blackcumin (Tantray et al. 2017) and soybean (Khan and Tyagi 2010). Decline in effectiveness at higher doses was observed, indicating that the increase in mutation rate was not proportional to the increase in the doses of mutagens. Some workers are of the view that the effectiveness and efficiency of some mutagens are influenced by pH and temperature (Veleminsky and Gichner 1970).

Three criteria, namely seedling injury (Mp/I), pollen sterility (Mp/S) and meiotic abnormalities (Mp/Me), were taken into consideration to determine the efficiency of gamma rays, EMS and their combination treatments. The present experiment shows that the efficiency of both single and combination treatments of gamma rays and EMS varies on the basis of criteria on which efficiency was calculated. In general, lower and moderate doses/concentrations of gamma rays and EMS were observed to be more efficient than higher ones. Many authors have reported higher efficiency at lower doses/concentrations of mutagens (Dhanavel et al. 2008 in Vigna unguiculata; Khan and Tyagi 2010 in Glycine max; Wani et al. 2011 in Vigna radiata; Khursheed et al. 2016b in Vicia faba). At higher doses/concentrations of mutagens the biological damage (seedling injury, pollen sterility and meiotic abnormalities) also increases (Konzak et al. 1965), resulting in decrease in efficiency. Similar results have already been reported by many workers (Dhanavel et al. 2008 in Vigna unguiculata; Shah et al. 2008 and Wani 2009 in Cicer arietinum; Bhosle and Kothekar 2010 and Dube et al. 2011 in Cyamopsis tetragonoloba; Kulkarni and Mogle 2013 in Macrotyloma uniflorum, Khursheed et al. 2015 in Vicia faba). Some workers have calculated mutagenic efficiency based on root length (Tripathy et al. 2012) because that helps us to plan our experiment even at seedling stage. Mutagenic efficiency based on seedling height and lethality has been considered more reliable by earlier workers. Efficient mutagens and their treatments are indispensable for the cost-effective use of the mutagen as a tool for the induction of mutations and their direct and indirect utilization in successful breeding programs. The success of mutation breeding programs depends on the choice of mutagen, dose and duration of mutagen and the target genotype. Therefore for successful mutation breeding in Vicia faba lower doses of EMS may be preferred to achieve higher frequency of mutations and less biological damage. The knowledge of optimal doses of mutagens is important in successful mutation breeding programs as it provides information on the balance between desirable and undesirable mutations.

Conclusions and future prospects

The present field experiment was conducted to study the response as mutagenic effectiveness and efficiency of two cultivars of faba bean, Vikrant and PRT-12, towards different doses/concentrations of both single and combination treatments of gamma rays and EMS. In both varieties, the order of effectiveness of mutagens was found to be EMS > gamma rays+EMS > gamma rays. Lower/moderate doses/concentrations of gamma rays and EMS were found to be more efficient than higher ones. On the basis of seedling injury, the efficiency of mutagens in descending order was: gamma rays > gamma rays+EMS > EMS in both varieties. On the basis of pollen sterility the efficiency of mutagens in descending order was: gamma rays > EMS > gamma rays+EMS in the variety Vikrant, whereas it was EMS > gamma rays > gamma rays+EMS in the variety PRT-12. On the basis of meiotic abnormalities, the efficiency of mutagens in descending order was: EMS > gamma rays+EMS > gamma rays in the variety Vikrant, whereas it was gamma rays > EMS > gamma rays+EMS in the variety PRT-12. Knowledge of relative biological effectiveness and efficiency of various mutagens and their selection is essential to recover high frequency of desirable mutations. Lower doses of both single and combination mutagen treatments may be employed for future mutation breeding programmes to gain the maximum desirable mutations.

Acknowledgments

The authors are grateful to the Chairman, Department of Botany, AMU, Aligarh, India for providing facilities for research work and UGC, India for financial support.

Disclosure statement

No potential conflict of interest was reported by the authors.