Register      Login
Crop and Pasture Science Crop and Pasture Science Society
Plant sciences, sustainable farming systems and food quality
RESEARCH ARTICLE

Phenotypic variations in M2 generation by ethyl methanesulfonate mutagenesis in lucerne (Medicago sativa L.)

S. S. Jade A , V. N. Gaikwad A , S. P. Jadhav A , P. S. Takawale A * and R. A. Bahulikar https://orcid.org/0000-0002-0442-4607 A *
+ Author Affiliations
- Author Affiliations

A BAIF Development Research Foundation, Central Research Station, Urulikanchan, Dist. Pune 412 202, Maharashtra, India.


Handling Editor: Marta Santalla

Crop & Pasture Science 75, CP23047 https://doi.org/10.1071/CP23047
Submitted: 21 February 2023  Accepted: 9 October 2023  Published: 30 October 2023

© 2024 The Author(s) (or their employer(s)). Published by CSIRO Publishing

Abstract

Context

Mutagenesis is an effective method to create genetic variation in an available accession.

Aim

This study aimed to generate variability in a genetically distinct genotype of lucerne (Medicago sativa L.) by using a chemical mutagen.

Methods

Eight different ethyl methanesulfonate (EMS) concentrations were used to determine lethal dose for 50% of the population (LD50). Among them, two EMS concentrations were used to treat 20 000 seeds of BAL-08-01 accession. A total of 8301 individual plants from 1225 families in the second generation (M2) were established under field conditions. Phenotypic observations were recorded as per Distinctness, Uniformity and Stability (DUS) testing guidelines.

Key results

Wide variations were observed for all phenotypic characters. Among these, green fodder yield, plant height and number of tillers varied with a range of 1–372.5 g, 8.1–120.8 cm and 0–77 per plant, respectively. Variations were also recorded for other characters such as leaf shape, flower colour, primary branching, etc. A total of 86.76% of plants showed purple colour flowers. Principal component analysis was performed for fodder characters from each EMS concentration. First, two principal components contributed an overall 97.06% variability. M2 plants outside 95% ellipses show the highest phenotypic variations.

Conclusion

EMS is effective in inducing mutation in lucerne. Phenotypic mutations were recognised in the M2 generation for the quantitative and qualitative characters and green fodder yield.

Implications

The resulting high level of variability will serve as source material for the improvement of lucerne for various traits.

Keywords: biomass estimation, ethyl methanesulfonate, genetic variability lucerne, green fodder yield, heritability, mutagenesis.

References

Ariraman M, Gnanamurthy S, Dhanavel D, Bharathi T, Murugan S (2014) Mutagenic effect on seed germination, seedling growth and seedling survival of Pigeon pea (Cajanus cajan (L.) Millsp). International Letters of Natural Sciences 16, 41-49.
| Crossref | Google Scholar |

Arisha MH, Shah SNM, Gong Z-H, Jing H, Li C, Zhang H-X (2015) Ethyl methane sulfonate induced mutations in M2 generation and physiological variations in M1 generation of peppers (Capsicum annuum L.). Frontiers in Plant Science 6, 399.
| Crossref | Google Scholar | PubMed |

Aung B, Gruber MY, Amyot L, Omari K, Bertrand A, Hannoufa A (2015a) Ectopic expression of LjmiR156 delays flowering, enhances shoot branching, and improves forage quality in alfalfa. Plant Biotechnology Reports 9, 379-393.
| Crossref | Google Scholar |

Aung B, Gruber MY, Amyot L, Omari K, Bertrand A, Hannoufa A (2015b) MicroRNA156 as a promising tool for alfalfa improvement. Plant Biotechnology Journal 13, 779-790.
| Crossref | Google Scholar | PubMed |

Biazzi E, Nazzicari N, Pecetti L, Brummer EC, Palmonari A, Tava A, Annicchiarico P (2017) Genome-wide association mapping and genomic selection for alfalfa (Medicago sativa) forage quality traits. PLoS ONE 12, e0169234.
| Crossref | Google Scholar | PubMed |

Burton GW (1952) Quantitative inheritance of grasses. In ‘Proceedings of 6th International Grassland Congress. Vol. 1’, Pennsylvania State College 17–23 August 1952. pp. 277–283.

Burton GW, DeVane EH (1953) Estimating heritability in tall fescue (Festuca arundinacea) from replicated clonal material. Agronomy Journal 45, 478-481.
| Crossref | Google Scholar |

Chen YL, Liang HL, Ma XL, Lou SL, Xie YY, Liu ZL, Chen LT, Liu YG (2013) An efficient rice mutagenesis system based on suspension-cultured cells. Journal of Integrative Plant Biology 55, 122-130.
| Crossref | Google Scholar | PubMed |

Dhakshanamoorthy D, Selvaraj R, Chidambaram A (2010) Physical and chemical mutagenesis in Jatropha curcas L. to induce variability in seed germination, growth and yield traits. Romanian Journal of Biology – Plant Biology 55, 113-125.
| Google Scholar |

Duan H-R, Wang L-R, Cui G-X, Zhou X-H, Duan X-R, Yang H-S (2020) Identification of the regulatory networks and hub genes controlling alfalfa floral pigmentation variation using RNA-sequencing analysis. BMC Plant Biology 20, 1-17.
| Crossref | Google Scholar |

Espina MJ, Ahmed CMS, Bernardini A, Adeleke E, Yadegari Z, Arelli P, Pantalone V, Taheri A (2018) Development and phenotypic screening of an ethyl methane sulfonate mutant population in soybean. Frontiers in Plant Science 9, 394.
| Crossref | Google Scholar |

Gaikwad BS (2021) Analysis of genetic variability in M2 generation of cowpea [Vigna Unguiculata (L.) Walp]. International Journal of Researches in Biosciences, Agriculture and Technology II, 211-215.
| Google Scholar |

Galpaz N, Burger Y, Lavee T, Tzuri G, Sherman A, Melamed T, Eshed R, Meir A, Portnoy V, Bar E (2013) Genetic and chemical characterization of an EMS induced mutation in Cucumis melo CRTISO gene. Archives of Biochemistry and Biophysics 539, 117-125.
| Crossref | Google Scholar | PubMed |

Greene EA, Codomo CA, Taylor NE, Henikoff JG, Till BJ, Reynolds SH, Enns LC, Burtner C, Johnson JE, Odden AR, Comai L, Henikoff S (2003) Spectrum of chemically induced mutations from a large-scale reverse-genetic screen in Arabidopsis. Genetics 164, 731-740.
| Crossref | Google Scholar | PubMed |

Gyamfi MO, Eleblu JSY, Sarfoa LG, Gyamfi L, Asante IK, Opoku-Agyemang F, Danquah EY (2022) Induced variations of ethyl methane sulfonate mutagenized cowpea (Vigna unguiculata L. Walp) plants. Frontiers in Plant Science 13, 952247.
| Crossref | Google Scholar |

Hamd Alla WA, Bakheit BR, Abo-Elwafa A, El-Nahrawy MA (2013) Evaluate of some varieties of alfalfa for forage yield and its components under the New Valley conditions. Journal of Agroalimentary Processes and Technologies 19, 413-418.
| Google Scholar |

Hammer Ø, Harper DAT, Ryan PD (2001) Past: paleontological statistics software package for education and data analysis. Palaeontologia Electronica 4(1), 9.
| Google Scholar |

Henry IM, Nagalakshmi U, Lieberman MC, Ngo KJ, Krasileva KV, Vasquez-Gross H, Akhunova A, Akhunov E, Dubcovsky J, Tai TH (2014) Efficient genome-wide detection and cataloging of EMS-induced mutations using exome capture and next-generation sequencing. The Plant Cell 26, 1382-1397.
| Crossref | Google Scholar | PubMed |

Johnson HW, Robinson HF, Comstock RE (1955) Estimates of genetic and environmental variability in soybeans. Agronomy Journal 47, 314-318.
| Crossref | Google Scholar |

Koli P, Bhardwaj NR (2018) Status and use of pesticides in forage crops in India. Journal of Pesticide Science 43, 225-232.
| Crossref | Google Scholar | PubMed |

Li Z, Jiang L, Ma Y, Wei Z, Hong H, Liu Z, Lei J, Liu Y, Guan R, Guo Y, Jin L, Zhang L, Li Y, Ren Y, He W, Liu M, Htwe NMPS, Liu L, Guo B, Song J, Tan B, Liu G, Li M, Zhang X, Liu B, Shi X, Han S, Hua S, Zhou F, Yu L, Li Y, Wang S, Wang J, Chang R, Qiu L (2017) Development and utilization of a new chemically-induced soybean library with a high mutation density. Journal of Integrative Plant Biology 59, 60-74.
| Crossref | Google Scholar | PubMed |

Mba C, Afza R, Bado S, Jain SM (2010) Induced mutagenesis in plants using physical and chemical agents. In ‘Plant cell culture: essential methods’. Vol. 20. (Eds MR Davey, P Anthony) pp. 111–130. (John Wiley & Sons)

Menda N, Semel Y, Peled D, Eshed Y, Zamir D (2004) In silico screening of a saturated mutation library of tomato. The Plant Journal 38, 861-872.
| Crossref | Google Scholar | PubMed |

Min X, Luo K, Liu W, Zhou K, Li J, Wei Z (2022) Molecular characterization of the miR156/MsSPL model in regulating the compound leaf development and abiotic stress response in alfalfa. Genes & Development 13, 331.
| Crossref | Google Scholar |

Mofokeng MA, Bello Z, Mashingaidze K (2022) Phenotypic analysis of pigeon pea reveal genotypic variability under different environmental interaction. In ‘Legumes Research-Volume 1.’ (Eds JC Jimenez-Lopez, A Clemente) (IntechOpen: London)

Nair AS, Gayathri G (2022) Optimization of doses for Ethyl Methane Sulphonate (EMS) and analysis of M1 generation of fodder cowpea [Vigna unguiculata (L.) Walp]. The Pharma Innovation 11, 593-598.
| Google Scholar |

Nasri F, Zakizadeh H, Vafaee Y, Mozafari AA (2022) In vitro mutagenesis of Chrysanthemum morifolium cultivars using thylmethanesulphonate (EMS) and mutation assessment by ISSR and IRAP markers. Plant Cell, Tissue and Organ Culture 149, 657-673.
| Crossref | Google Scholar |

Raval V, Patel A, Rathod S, Sumita Z, Vashi JM, Chaudhari B (2018) Genetic variability, heritability and genetic advance studies in okra (Abelmoschus esculentus (L.) Moench). International Journal of Chemical Studies 6, 3319-3321.
| Google Scholar |

Rychowdhury R, Tah J (2011) Chemical mutagenic action on seed germination and related agro-metrical traits in M1 Dianthus generation. Current Botany 2, 19-23.
| Google Scholar |

Sayed MRI, Alshallash KS, Safhi FA, Alatawi A, ALshamrani SM, Dessoky ES, Althobaiti AT, Althaqafi MM, Gharib HS, Shafie WW, Allah MMA, Sultan FM (2022) Genetic diversity, analysis of some agro-morphological and quality traits and utilization of plant resources of alfalfa. Genes 13, 1521.
| Crossref | Google Scholar | PubMed |

Scholtz GDJ (2008) A grading system for Medicago sativa hay in South Africa. PhD Thesis, University of the Free State, Bloemfontein, South Africa.

Shah SNM, Gong ZH, Arisha MH, Khan A, Tian SL (2015) Effect of ethyl methyl sulfonate concentration and different treatment conditions on germination and seedling growth of the cucumber cultivar Chinese long (9930). Genetics and Molecular Research 14, 2440-2449.
| Crossref | Google Scholar | PubMed |

Shen XH, Jiang C, Zheng W, Feng P, Wang Q, Lai YC (2020) Mutagen effect on the growth, physiology and microstructure of alfalfa (Medicago sativa L). Applied Ecology and Environmental Research 18, 3497-3510.
| Crossref | Google Scholar |

Singh T, Ramakrishnan S, Mahanta SK, Tyagi VC, Roy AK (2018) Tropical forage legumes in India: status and scope for sustaining livestock production. In ‘Forage groups’. (Eds RL Edvan, EM Santos) (IntechOpen)

Takawale PS, Jade SS, Bahulikar RA, Desale JS (2019) Diversity in Lucerne (Medicago sativa L.) germplasm for morphology, yield and molecular markers and their correlations. Indian Journal of Genetics and Plant Breeding 79, 453-459.
| Google Scholar |

Talebi AB, Talebi AB, Shahrokhifar B (2012) Ethyl methane sulphonate (EMS) induced mutagenesis in Malaysian rice (cv. MR219) for lethal dose determination. American Journal of Plant Sciences 3, 1661-1665.
| Crossref | Google Scholar |

Tsuda M, Kaga A, Anai T, Shimizu T, Sayama T, Takagi K, Machita K, Watanabe S, Nishimura M, Yamada N, Mori S, Sasaki H, Kanamori H, Katayose Y, Ishimoto M (2015) Construction of a high-density mutant library in soybean and development of a mutant retrieval method using amplicon sequencing. BMC Genomics 16, 1014.
| Crossref | Google Scholar |

Vandemark GJ, Ariss JJ, Bauchan GA, Larsen RC, Hughes TJ (2006) Estimating genetic relationships among historical sources of alfalfa germplasm and selected cultivars with sequence related amplified polymorphisms. Euphytica 152, 9-16.
| Crossref | Google Scholar |

Wijekoon CP, Singer SD, Weselake RJ, Petrie JR, Singh S, Jayawardhane KN, Shah S, Chen G, Eastmond PJ, Acharya SN (2020) Enhancement of total lipid production in vegetative tissues of alfalfa and sainfoin using chemical mutagenesis. Crop Science 60, 2990-3003.
| Crossref | Google Scholar |

Yang H, Chang G, Bao W, Chai X, Zhou X (2012) Morphological variation of space mutation in alfalfas. Acta Prataculturae Sinica 21, 222-228.
| Google Scholar |