Skip to main navigation menu Skip to main content Skip to site footer
Plant Science Today (PST)

Research Articles

Early Access

Optimisation of in vitro ethyl methanesulfonate- induced mutagenesis for future application in development of mutants in aromatic rice (Oryza sativa L.)

DOI
https://doi.org/10.14719/pst.16318
Submitted
24 June 2026
Published
10-11-2026

Abstract

This study optimised in vitro culture conditions and ethyl methanesulfonate (EMS)-induced mutagenesis in the aromatic rice landrace Tulasikanti for optimising the regeneration protocol and determining the optimal EMS treatment. Mature seeds were cultured on Murashige and Skoog (MS) medium with different plant growth regulator combinations to optimise callus induction and regeneration. The resulting calli were treated with EMS at 0.1 %, 0.2 % and 0.3 % along with an untreated control (0 % EMS), for exposure durations of 2, 4 and 6 hr to determine the median lethal dose (50 % lethality (LD50)) and evaluate callus survival and regeneration efficiency. Plant growth regulators (PGRs) significantly influenced callus induction, with the highest embryogenic callus formation (85 %) recorded on T4 (MS + 2.0 mg L-1 2,4-dichlorophenoxyacetic acid (2,4-D) + 0.1 mg L-1 6-benzylaminopurine (BAP) + 0.25 mg L-1 naphthaleneacetic acid (NAA)) medium. Further, EMS treatment caused a concentration- and exposure time-dependent decline in callus survival, with 0.2 % EMS for 6 hr identified as the LD50. The highest shoot regeneration (48.2 %) occurred on R2 (MS + 0.01 mg L-1 NAA + 0.25 mg L-1 Kn + 2.0 mg L-1 BAP) medium following 0.2 % EMS treatment, whereas higher EMS concentrations exerted pronounced cytotoxic effects. Rooting was highly efficient, with approximately 90 % of regenerated shoots forming healthy roots. Thus, the study provided a reproducible in vitro EMS mutagenesis framework that can efficiently balance mutagenic intensity and regeneration, thereby providing a foundation for future mutant development for aromatic rice improvement and the future identification of semi-dwarf mutants.

References

  1. 1. Bautista RC, Counce PA. An overview of rice and rice quality. Cereal Foods World. 2020;65(5):52. https://doi.org/10.1094/CFW-65-5-0052
  2. 2. Kumar A, Raman A, Yadav S, Verulkar SB, Mandal NP, Singh ON, et al. Genetic gain for rice yield in rainfed environments in India. Field Crops Res. 2021;260:107977. https://doi.org/10.1016/j.fcr.2020.107977
  3. 3. Roy S, Banerjee A, Basak N, Kumar J, Mandal NP. Aromatic rice. In: Oliveira AC, Pegoraro C, Viana VE, editors. The future of rice demand: quality beyond productivity. Cham: Springer International Publishing; 2020. p. 251–82. https://doi.org/10.1007/978-3-030-37510-2_11
  4. 4. Keerthana U, Senapati AK, Bag MK, Prabhukarthikeyan SR, Naveenkumar R, Yadav MK, et al. Leveraging genetic diversity of aromatic rice landraces in Odisha for sustainable rice blast resistance. Physiol Mol Plant Pathol. 2025;138:102682. https://doi.org/10.1016/j.pmpp.2025.102682
  5. 5. Sao R, Sahu PK, Patel RS, Das BK, Jankuloski L, Sharma D. Genetic improvement in plant architecture, maturity duration and agronomic traits of three traditional rice landraces through gamma ray-based induced mutagenesis. Plants (Basel). 2022;11(24):3448. https://doi.org/10.3390/plants11243448
  6. 6. Xu Y, Zhang X, Li H, Zheng H, Zhang J, Olsen MS, et al. Smart breeding driven by big data, artificial intelligence and integrated genomic-enviromic prediction. Mol Plant. 2022;15(11):1664–95. https://doi.org/10.1016/j.molp.2022.09.001
  7. 7. Rime J, Dinesh MR, Sankaran M, Shivashankara KS, Rekha A, Ravishankar KV. Evaluation and characterization of EMS derived mutant populations in mango. Sci Hortic. 2019;254:55–60. https://doi.org/10.1016/j.scienta.2019.04.015
  8. 8. Sharma V, Thakur M, Maan SS, Verma K, Thakur A, Penna S. In vitro mutagenesis: a non-invasive technology for effective crop improvement to assure food and nutritional security-current trends, advancements and future perspectives. J Plant Growth Regul. 2025;44(2):484–507. https://doi.org/10.1007/s00344-024-11484-8
  9. 9. Sun L, Lai M, Ghouri F, Nawaz MA, Ali F, Baloch FS, et al. Modern plant breeding techniques in crop improvement and genetic diversity: from molecular markers and gene editing to artificial intelligence a critical review. Plants (Basel). 2024;13(19):2676. https://doi.org/10.3390/plants13192676
  10. 10. Maluszynski M, Ahloowalia B, Ashri A, Nichterlein K, Van Zanten L. Induced mutations in rice breeding and germplasm enhancement: assessment and orientation towards the 21st century. Proc 19th Sess Int Rice Comm, Cairo, Egypt. 1998;9:7–9.
  11. 11. Zhang J, Liu X, Li S, Cheng Z, Li C. The rice semi-dwarf mutant sd37, caused by a mutation in CYP96B4, plays an important role in the fine-tuning of plant growth. PLoS One. 2014;9(2). https://doi.org/10.1371/journal.pone.0088068
  12. 12. Salgotra RK, Chauhan BS. Genetic diversity, conservation and utilization of plant genetic resources. Genes (Basel). 2023;14(1):174. https://doi.org/10.3390/genes14010174
  13. 13. Dar SA, Khan IS, Majeed G, Tyub S, Nawchoo IA, Kamili AN. Chemical-induced variations in callus regenerated Atropa acuminata plants: a study on ethyl methanesulfonate effects. Biotechnol Rep. 2025;46. https://doi.org/10.1016/j.btre.2025.e00888
  14. 14. Sevanthi AM, Kandwal P, Kale PB, Prakash C, Ramkumar MK, Yadav N, et al. Whole genome characterization of a few EMS-induced mutants of upland rice variety Nagina 22 reveals a staggeringly high frequency of SNPs which show high phenotypic plasticity towards the wild-type. Front Plant Sci. 2018;9:1179. https://doi.org/10.3389/fpls.2018.01179
  15. 15. Desai S, Jadhav A, Ramteke A, Dhole V, Bapat V, Gaikwad N. Genetic improvement of two Indian non-basmati aromatic rice landraces through physical and chemical mutagenesis. Int J Radiat Biol. 2022;98(1):82–9. https://doi.org/10.1080/09553002.2021.1987567
  16. 16. Türkoğlu A, Haliloğlu K, Tosun M, Bujak H, Eren B, Demirel F, et al. Ethyl methanesulfonate (EMS) mutagen toxicity-induced DNA damage, cytosine methylation alteration and iPBS-retrotransposon polymorphisms in wheat (Triticum aestivum L.). Agronomy. 2023;13(7):1767. https://doi.org/10.3390/agronomy13071767
  17. 17. Chakraborty S, Mahapatra S, Hooi A, Ali MN, Satdive R. Determination of median lethal (LD50) and growth reduction (GR50) dose of gamma irradiation for induced mutation in wheat. Braz Arch Biol Technol. 2023;66. https://doi.org/10.1590/1678-4324-2023220294
  18. 18. Singh S, Kumar A, Rana V, Kumar A, Sharma VR. In vitro callus induction and plant regeneration in basmati rice (Oryza sativa L.) varieties. J Pharmacogn Phytochem. 2018;7(5S):65-9.
  19. 19. Murashige T, Skoog F. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant. 1962;15(3):473–97. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
  20. 20. Das S, Pradhan B, Parida S, Samantaray S. Callus mutagenesis using EMS and mutagenic response in aromatic rice (Oryza sativa L.) landraces of India. Agric Mech Asia Afr Lat Am. 2022;53(5):7665–74.
  21. 21. Li R, Fan L, Lin J, Li M, Liu D, Sui S. In vitro mutagenesis followed by polymorphism detection using start codon targeted markers to engineer brown spot resistance in Kalanchoe. J Am Soc Hortic Sci. 2019;144(3):193–200. https://doi.org/10.21273/JASHS04571-18
  22. 22. Murugan T, Awasthi OP, Singh B, Rudra SG. In vitro mutagenesis using habituation and PBR autotrophy based indirect somatic embryogenesis (ISE) system in Kinnow mandarin. Physiol Mol Biol Plants. 2024;30(8):1297–1312. https://doi.org/10.1007/s12298-024-01498-7
  23. 23. Mutanda M, Figlan S, Shargie NG, Gwata ET. Mutation breeding: an underutilized strategy for improving finger millet productivity and nutritional quality. Front Sustain Food Syst. 2025;9:1679819. https://doi.org/10.3389/fsufs.2025.1679819
  24. 24. Penna S, Bhagwat SG. Mutagenesis and selection: reflections on the in vivo and in vitro approaches for mutant development. In: Mutation breeding for sustainable food production and climate resilience. Singapore: Springer Nature Singapore; 2023. p. 99–127. https://doi.org/10.1007/978-981-16-9720-3_4
  25. 25. Khan MN. In vitro callus induction of aromatic rice depends on the concentration of 2,4-D. Malays J Halal Res. 2019;2(2):9–13. https://doi.org/10.2478/mjhr-2019-0007
  26. 26. Trunjaruen A, Sayam RA, Maneerattanarungroj P, Taratima W. Effects of cultivation media on in vitro callus induction and regeneration capabilities of pakaumpuel rice (Oryza sativa L.), Thai rice landrace. Walailak J Sci Technol. 2020;17(1):37–46. https://doi.org/10.48048/wjst.2020.5349
  27. 27. Wafai AH, Mokhdomi TA, Amin A, Husaini AM, Bukhari S, Wani ZA, et al. An efficient method for development of callus and suspension culture in Crocus sativus L. (saffron) using 6-BAP, 2-4 D and NAA enriched media. Int J Curr Trends Sci Technol. 2017;7(12):20416–9.
  28. 28. Hasan MN, Bhuiyan FH, Hoque H, Jewel NA, Ashrafuzzaman M, Prodhan SH. The effect of plant growth regulators (PGRs) on efficient regeneration of 12 recalcitrant indica rice (Oryza sativa L.) genotypes. Am J Biochem Biotechnol. 2021;17:148–59. https://doi.org/10.3844/ajbbsp.2021.148.159
  29. 29. Das P, Tanti B, Borthakur SK. In vitro callus induction and indirect organogenesis of Brucea mollis Wall. ex-Kurz - a potential medicinal plant of Northeast India. S Afr J Bot. 2018;119:203–11. https://doi.org/10.1016/j.sajb.2018.09.012
  30. 30. Kumar S, El-Hawaz R, Li Z, Lawson J, Parris S, Kangben F, et al. Optimized auxin and cytokinin interactions enable direct somatic embryogenesis in the peach rootstock 'Guardian®' from immature cotyledons. Int J Mol Sci. 2025;26(17):8698. https://doi.org/10.3390/ijms26178698
  31. 31. Ming NJ, Binte Mostafiz S, Johon NS, Abdullah Zulkifli NS, Wagiran A. Combination of plant growth regulators, maltose and partial desiccation treatment enhance somatic embryogenesis in selected Malaysian rice cultivar. Plants (Basel). 2019;8(6):144. https://doi.org/10.3390/plants8060144
  32. 32. Chitphet P, Sanevas N, Vuttipongchaikij S, Wongkantrakorn N. An effective protocol for callus induction and plant regeneration in an indica rice cultivar RD43. Int J Plant Biol. 2025;16(2):48. https://doi.org/10.3390/ijpb16020048
  33. 33. Mostafiz SB, Wagiran A, Johan NS, Zulkifli NS, Ming NJ. The effects of temperature on callus induction and regeneration in selected Malaysian rice cultivar indica. Sains Malays. 2018;47:2647–55. https://doi.org/10.17576/jsm-2018-4711-07
  34. 34. Alam Khan N, Uddin I, Rana MS, Jahan N, Sumi MA, Rashid HO. Callus induction, regeneration and establishment of rice plant from mature embryo. J Adv Biol Biotechnol. 2021;24(9):10–8. https://doi.org/10.9734/jabb/2021/v24i930238
  35. 35. Chen T, Huang L, Wang M, Huang Y, Zeng R, Wang X, et al. Ethyl methyl sulfonate-induced mutagenesis and its effects on peanut agronomic, yield and quality traits. Agronomy. 2020;10(5):655. https://doi.org/10.3390/agronomy10050655
  36. 36. Raina A, Laskar RA, Wani MR, Jan BL, Ali S, Khan S. Comparative mutagenic effectiveness and efficiency of gamma rays and sodium azide in inducing chlorophyll and morphological mutants of cowpea. Plants (Basel). 2022;11(10):1322. https://doi.org/10.3390/plants11101322
  37. 37. Dhange PR, Gowda APM, Kandpal K, Ramegowda HV. Determination of lethal dose (LD50) and mutagenic effects of ethyl methane sulfonate (EMS) on germination and survival of seedlings in Stevia rebaudiana Bertoni. Mysore J Agric Sci. 2024;58(2):214–20.
  38. 38. Thakur G, Paul S, Kumar A. Mutagenic effectiveness and efficiency of ethyl methane sulphonate (EMS) mutagen in linseed (Linum usitatissimum L.). J Oilseeds Res. 2020;37(4):260–6. https://doi.org/10.56739/jor.v37i4.136741
  39. 39. Snyman M, Huynh TV, Smith MT, Xu S. The genome-wide rate and spectrum of EMS-induced heritable mutations in the microcrustacean Daphnia: on the prospect of forward genetics. Heredity. 2021;127(6):535–45. https://doi.org/10.1038/s41437-021-00478-x
  40. 40. Warren JJ, Forsberg LJ, Beese LS. The structural basis for the mutagenicity of O6-methylguanine lesions. Proc Natl Acad Sci U S A. 2006;103:19701–6. https://doi.org/10.1073/pnas.0609580103
  41. 41. Loewe L, Hill WG. The population genetics of mutations: good, bad and indifferent. Philos Trans R Soc Lond B Biol Sci. 2010;365(1544):1153–67. https://doi.org/10.1098/rstb.2009.0317
  42. 42. Kashtwari M, Wani AA, Dhar MK, Jan S, Kamili AN. Development of an efficient in vitro mutagenesis protocol for genetic improvement of saffron (Crocus sativus L.). Physiol Mol Biol Plants. 2018;24(5):951–62. https://doi.org/10.1007/s12298-018-0576-6
  43. 43. Di Pauli V, Fontana PD, Lewi DM, et al. Ethyl methanesulfonate-induced mutagenesis generates genetic and morphological variations in an elite Argentine sugarcane genotype. Discov Plants. 2025;2:230. https://doi.org/10.1007/s44372-025-00298-x
  44. 44. Ahmed AI, Tayyab M, Shakeel A, Rehman HM, Shaheen N, Khan SHU, et al. Optimization of LD50 and induction of mutation through EMS in Dalbergia sissoo. J Genet Appl Biotechnol. 2025;1(1). https://doi.org/10.66432/3sj9s175
  45. 45. Altındal N, Karadoğan T, Altındal D. Genetic variation in EMS-treated potato calli assessed by ISSR. Plant Cell Tissue Organ Cult. 2025;163(2):79. https://doi.org/10.1007/s11240-025-03265-2
  46. 46. Chen L, Duan L, Sun M, Yang Z, Li H, Hu K, et al. Current trends and insights on EMS mutagenesis application to studies on plant abiotic stress tolerance and development. Front Plant Sci. 2023;13:1052569. https://doi.org/10.3389/fpls.2022.1052569
  47. 47. Rashid M, Shamshad A, Jankuloski L. Effects of ethyl methanesulfonate (EMS) on seedling and yield contributing traits in Basmati rice. Preprints. 2021. https://doi.org/10.20944/preprints202103.0453.v1
  48. 48. Rafi S, Kamili AN, Ganai BA. In vitro mutagenesis via EMS and gamma irradiations. In: Biotechnologies and genetics in plant mutation breeding. Oakville: Apple Academic Press; 2023. p. 181–203. https://doi.org/10.1201/9781003305064-9
  49. 49. Sabagh AE, Mbarki S, Hossain A, Iqbal MA, Islam MS, Raza A, et al. Potential role of plant growth regulators in administering crucial processes against abiotic stresses. Front Agron. 2021;3:648694. https://doi.org/10.3389/fagro.2021.648694
  50. 50. Zahid NA, Sinniah UR, Md Hatta MA, Zulperi D, Papenbrock J, Hakiman M. In vitro regeneration and biotechnological advances in tissue culture of Zingiber officinale Roscoe: a comprehensive review. Plant Cell Tissue Organ Cult. 2026;166(1):12. https://doi.org/10.1007/s11240-026-03514-y

Downloads

Download data is not yet available.