Qualitative Mutations Induced by Ethyl Methane Sulphonate in Phaseolus lunatus L.

Received 08 Sep, 2022

Accepted 02 Mar, 2023

Published 31 Mar, 2023

Background and Objective: Phaseolus lunatus commonly known as lima bean is an underutilized crop in Nigeria, which possess the potential to serve as an alternative source of protein supply but yet no considerable efforts have been made in time past towards the genetic improvement of this crop. This study was undertaken to assess the efficiency of ethyl methane sulphonate in inducing morphological and physiological mutations in lima bean, thus increasing the genetic variation available in the gene pool which can serve as an important source of variant genes for further breeding. Materials and Methods: Landraces of Phaseolus lunatus were obtained, presoaked in distilled water for 6 hrs and treated with freshly prepared concentrations of ethyl methane sulphonate (0.1, 0.2, 0.3 and 0.4%) for 6 hrs, a control experiment was also set up. Treated seeds and control were sown in polythene bags filled with top garden soil and arranged using a randomized complete block design to raise the M₁ and M₂ generation. Results: Morphological mutants: Invaginated leaf margin, bifurcated leaf apex, biapex leaf and enlarged leaf were observed in M₂ mutants treated with 0.2, 0.3 and 0.4% of the mutagen. Physiological mutants: A change in seed coat color from brown to black with off-white patches and a change in flower color from yellow to purple was observed in M₂ mutants treated with 0.4% ethyl methane sulphonate. The percentage frequency of M₂ progenies segregating for seed coat and flower color change was 92.31%. Conclusion: However, this research concludes that 0.3 and 0.4% ethyl methane sulphonate are more effective at inducing morphological and physiological mutations in Phaseolus lunatus.

INTRODUCTION

Pulses are the chief components of the agricultural system, effectively boosting food and nutrition, revenue and environment across the globe and hence assumed ideal for acquiring food security in the developing world including Nigeria. Phaseolus lunatus belong to the Family Fabaceae¹. It is commonly known as lima bean in English and Papala in Yoruba². Lima bean seeds are considered to be a good source of nutrients³, such as valuable proteins with content from 14.24 to 24.92%⁴ and rich in essential amino acids⁵. They provide complex carbohydrates, mainly starch and dietary fiber, vitamins (B complex) and minerals (zinc, iron and calcium)⁶. The most abundant proteins are storage proteins, primarily globulin and albumin⁷. In addition to their good macronutrient composition, lima bean is considered beneficial for health because of the low glycemic index due to the presence of slow-release carbohydrates⁸.

It is an underutilized crop in Nigeria, which possess the potential to serve as an alternative source of protein supply but yet no considerable efforts have been made in time past towards the genetic improvement of this crop. Plant scientists in Nigeria are currently using mutagens as a tool to broaden the genetic diversity of this crop to produce improved cultivars.

Seed coat colour is an important agronomic trait that determines the marketability of a dry bean variety. People in a location have a specific preference for certain colours of beans. A genetic understanding of lima bean seed coat colour inheritance is very important in cultivar development breeding and market acceptability. It is possible to induce a change in seed coat colour through mutation^9,10.

Mutation breeding is an alternative to conventional plant breeding and is a source of increasing variability, conferring specific improvement without significantly altering its acceptable phenotype¹¹.

Induced mutation breeding, which is recognized as a valuable supplement to conventional breeding in crop improvement, has been least applied in grain legumes¹². Among available mutagens, ethyl methyl sulphonate (EMS) is a potent and popular chemical mutagen that has been effectively used to induce a high density of random irreversible point mutations uniformly distributed in the genome¹³. Hence, this study was undertaken to assess the efficiency of ethyl methane sulphonate in inducing morphological and physiological mutations in lima bean, thus increasing the genetic variation available in the gene pool which can serve as an important source of variant genes for further breeding.

MATERIALS AND METHODS

Study area: The research was carried out in the research experimental field of the Department of Botany, Ahmadu Bello University, Zaria during the 2016 and 2017 planting seasons (June-November).

Research protocol: Landraces of Phaseolus lunatus were obtained from local farmers at Sabon Gari local government area of Kaduna State, Nigeria.

The seeds were presoaked in distilled water for 6 hrs and later transferred into freshly prepared concentrations of EMS (0.1, 0.2, 0.3 and 0.4) for 6 hrs. After treatment, the seeds were washed thoroughly under a running tap for 5 min to remove any residual effect of the mutagen. Treated seeds along with the control seeds were sown in triplicates in one hundred and fifty polythene bags (51.5×38.3 cm), each filled with sterilized top garden soil and arranged using a Randomized Complete Block Design (RCBD), to raise the first mutagenic (M₁) and second mutagenic (M₂) generation along with the control.

After the maturation of M₁ plants, pods were harvested from each treatment and threshed to obtain M₁ seeds which were sown to raise the M₂ generation. After flowering and maturation of the M₂ plants, pods were harvested and threshed and M₂ seeds were obtained. Leaves, flowers and seeds of M₂ plants were observed for morphological and physiological mutations.

Data collection: The number of plants showing morphological and physiological mutations was counted and percentages were calculated and presented in tables. The number of plant showing morphological variations in their leaves was presented in a bar chat.

RESULTS AND DISCUSSION

Morphological mutations such as invaginated leaf margin were observed in plants treated with 0.3% EMS, bifurcated leaf apex in plants treated with 0.2 and 0.3% EMS, biapex leaves were observed as a result of treatment with 0.3 and 0.4% ethyl methane sulphonate and enlarged leaf size was observed at the M₂ generation in plants treated with 0.3 and 0.4% EMS as represented on Fig. 1 and shown in Fig. 2. Morphological mutations that occurred may be a result of a change in the genes that code for the shape of the leaves due to treatment with the mutagens. This report corroborates findings in dolichos bean¹⁴ and tomato¹⁵.

Fig. 1: Leaf morphology mutant in M2 progenies

Fig. 2(a-d): Morphological mutants induced by ethyl methane sulphonate, (a) Invaginated leaf margin, (b) Bifurcated leaf apex, (c) Biapex leaf and (d) Enlarge leaf size

Fig. 3(a-d): Physiological mutants induced by ethyl methane sulphonate, (a) Control flower, (b) Mutant flower, (c) Control seed and (d) Mutant seed

Physiological mutants were observed in M₂ plants treated with 0.4% ethyl methane sulphonate (Table 1). The plants showed a purple and white colored flower which varied greatly from the control plants with a yellow and white flower. More so, the seed coat colour of M₂ seeds, treated with 0.4% ethyl methane sulphonate was black with off-white patches and larger in size compared to the control seed which had a brown seed coat colour and a smaller size (Fig. 3). The physiological spectrum of mutation which occurred in this study in the flower and seed coat colour could be a result of the occurrence of substitution mutation on the genes controlling these traits thereby leading to the replacement of the nucleotide of a triplet codon by another nucleotide resulting in the production of a protein with single amino acid and consequently altering the phenotype of the flower and seed. This conforms to the report of 0.1% EMS inducing a change in flower colour of grasspea¹⁶ and the change in the seed coat colour of cowpea exposed to 30KR of gamma rays¹⁷.

Table 1:

Seed coat and flower colour phenotype in M2 progenies

Concentration (%)	M2 progenies segregating seed coat colour	Mutant seed coat colour	Mutant flower colour	Number of plants
0	0	Nil	Nil	0
0.1	0	Nil	Nil	0
0.2	0	Nil	Nil	0
0.3	0	Nil	Nil	0
0.4	48	Black with off white patches	Purple	48

Table 2:

Frequency of M2 progenies segregating for seed coat/flower colour

Concentration (%)	Number of treated seeds	Number of plants observed	M2 segregating seed coat/flower colour	Frequency (%)
0	60	59	0	0
0.1	60	57	0	0
0.2	60	55	0	0
0.3	60	54	0	0
0.4	60	52	48	92.31

The highest percentage frequency (92.31%) of M₂ progenies segregating for seed coat colour and flower colour was observed at the 0.4% treatment as shown in Table 2. This is similar to the report on the effect of 15KR gamma irradiation on groundnut¹².

CONCLUSION

This research concludes that ethyl methane sulphonate is effective at inducing morphological and physiological mutations in Phaseolus lunatus at 0.3 and 0.4% concentrations. Morphological mutants such as invaginated leaf margin, bifurcated leaf apex and bi apex leaf were induced at 0.3% and enlarged leaves at 0.4% concentration at M₂ generation. An enlarged leaf increases the surface area which can as well increase the rate of photosynthesis and provide more foliage for animals. A change in seed coat colour from brown to black with off white patches increased seed size and a change in colour of flower from white to purple was also observed in mutants treated with 0.4% ethyl methane sulphonate at M₂ generation. The percentage frequency of M₂ progenies segregating for seed coat and flower colour change was 92.31%. The change in seed coat colour and increase in seed size has increased the genetic variability of the germplasm for further breeding and could also enhance its acceptability by farmers and final consumers.

SIGNIFICANCE STATEMENT

This study discovered that 0.3 and 0.4% EMS are efficient at inducing viable morphological and physiological mutations in Phaseolus lunatus that can be beneficial to breeders who hope to improve the available germplasm thus providing better varieties. This study will help researchers uncover critical areas in mutation breeding of Phaseolus lunatus that many researchers are yet to explore especially in Nigeria. Thus, a new theory on creating genetic variability and broadening the gene pool for further breeding of Phaseolus lunatus can be arrived at.

REFERENCES

1. Sandrine, M.A.D., K.K. Kevin and K.L. Clémence, 2020. Preliminary diversity assessment of lima beans (Phaseolus lunatus) cultivated in Côte D’Ivoire. Am. J. Plant Sci., 11: 2059-2065.
2. Adegbehingbe, K.T., 2013. Microbialogical and nutrient studies of fermented cooked lima bean (Phaseolus lunatus L.) seeds. Global J. Biol. Agric. Health Sci., 2: 94-101.
   
3. Jayalaxmi, B., D. Vijayalakshmi, R. Usha, M.L. Revanna, R. Chandru and P.H.R. Gowa, 2015. Effect of different processing methods on proximate, mineral and antinutrient content of lima bean (Phaseolus lunatus) seeds. Legume Res., 39: 543-549.
   
4. Ibeabuchi, J.C., D.C. Okafor, N.N. Ahaotu, C.N. Eluchie, I.M. Agunwah, M.N. Chukwu and C. Amandikwa, 2019. Effect of dehulling on proximate composition and functional properties of lima bean (Phaseolus lunatus) grown in Enugu State. J. Food Res., 8: 116-121.
   
5. Palupi, H.T., T. Estiasih, Yunianta and A. Sutrisno, 2021. Characterization of nutritional and functional properties of lima bean flour (Phaseolus lunatus L.). IOP Conf. Ser.: Earth Environ. Sci., 924: 012033.
   
6. Campos-Vega, R., G. Loarca-Piña and B.D. Oomah, 2010. Minor components of pulses and their potential impact on human health. Food Res. Int., 43: 461-482.
   
7. Agarwal, A., 2017. Proteins in pulses. J. Nutr. Disord. Ther., 7: 1000e129.
   
8. Bello-Pérez, L.A., S.G. Sáyago-Ayerdi, C.E. Chávez-Murillo and E. Agama-Acevedo, 2007. Proximal composition and in vitro digestibility of starch in lima bean (Phaseolus lunatus) varieties. J. Sci. Food Agric., 87: 2570-2575.
   
9. Moh, C.C., 1971. Mutation breeding in seed-coat colors of beans (Phaseolus vulgaris L.). Euphytica, 20: 119-125.
   
10. Satpute, R.A. and S.W. Suradkar, 2011. Effect of mutation on seed coat colour in groundnut (Arachis hypogaea L.). J. Exp. Sci., 2: 24-25.
    
11. Udage, A.C., 2021. Introduction to plant mutation breeding: Different approaches and mutagenic agents. J. Agric. Sci., 16: 466-483.
    
12. Dhanavel, D., P. Pavadai, L. Mullainathan, D. Mohana, G. Raju, M. Girija and C. Thilagavathi, 2008. Effectiveness and efficiency of chemical mutagens in cowpea (Vigna unguiculata (L.) Walp). Afr. J. Biotechnol., 7: 4116-4117.
    
13. Greene, E.A., C.A. Codomo, N.E. Taylor, J.G. Henikoff and B.J. Till et al., 2003. Spectrum of chemically induced mutations from a large-scale reverse-genetic screen in Arabidopsis. Genetics, 164: 731-740.
    
14. More, A.D. and S.S. Jagtap, 2016. Induction of morphological leaf mutations in Lablab purpureus (L.) sweet through chemical and physical mutagens. Int. J. Curr. Microbiol. Appl. Sci., 5: 592-597.
    
15. Kessler, S., M. Kim, T. Pham, N. Weber and N. Sinha, 2001. Mutations altering leaf morphology in tomato. Int. J. Plant Sci., 162: 475-492.
    
16. Ramezani, P., M. Siavoshi, A.D. More, M. Ebrahimi and S. Dastan, 2017. Gamma rays and EMS induced flower color mutation in grasspea (Lathyrus sativus Linn.). J. Agric. Sci., 23: 423-427.
    
17. Gaafar, R.M., M. Hamouda and A. Badr, 2016. Seed coat color, weight and eye pattern inheritance in gamma-rays induced cowpea M2-mutant line. J. Genet. Eng. Biotechnol., 14: 61-68.