Highly efficient mutagenesis using N-methyl-N-nitrosourea on male germ cells of maize

Authors

  • Huaidong Tian 1. Laboratory for Plant Germplasm and Genetic Resources of Crop, School of Life Science, Shanxi University, Taiyuan 030002, China
  • Zhen Li 1. Laboratory for Plant Germplasm and Genetic Resources of Crop, School of Life Science, Shanxi University, Taiyuan 030002, China
  • Pengfei Niu 1. Laboratory for Plant Germplasm and Genetic Resources of Crop, School of Life Science, Shanxi University, Taiyuan 030002, China
  • Chenghua Tian 2. Sorghum Research Institute, Shanxi Agricultural University, Jinzhong 030600, Shanxi, China
  • Nan Li 1. Laboratory for Plant Germplasm and Genetic Resources of Crop, School of Life Science, Shanxi University, Taiyuan 030002, China
  • Feng Yan 1. Laboratory for Plant Germplasm and Genetic Resources of Crop, School of Life Science, Shanxi University, Taiyuan 030002, China
  • Xiaohui Shen 1. Laboratory for Plant Germplasm and Genetic Resources of Crop, School of Life Science, Shanxi University, Taiyuan 030002, China
  • Hailin Ma 3. Shanxi Dafeng Seed Industry, Taiyuan 030031, China
  • Guimei Cui 3. Shanxi Dafeng Seed Industry, Taiyuan 030031, China

DOI:

https://doi.org/10.25165/ijabe.v18i6.9610

Keywords:

Zea maysL., inbred line, in vitro pollens, MNU, mutagenization

Abstract

The mutagenesis with ethyl methanesulfonate (EMS) mixed in paraffin oil on maize (Zea mays L.) pollens has been the main means for inducing genic mutants of maize. However, it is necessary to develop a new technique for improving efficiency of random mutagenesis of genes in maize, and to promote mutant creation for functional-genomic research and breeding of maize. In this study, the technique of N-methyl-N-nitrosourea (MNU) mutagenesis on maize male germ cells was developed by establishing the method of MNU treatment of invitro maize pollens and screening the derived progeny populations. The phosphate-buffered protective-solution-1 (PS1) containing 30% glycerol, 15% ethylene glycol, 15% dimethyl sulfoxide, and 0.4% sucrose was determined as the optimum formula for protecting invitro pollens at 20°C; the effect of MNU concentration, treatment time, pH, and PO_4^(3-) concentration on the survival rate of pollens treated in the MNU solutions containing PS1 formula was clarified, performing the single-factor and orthogonal experiments for pollen viability; the reproductivity of treated pollens was confirmed, examining their germination and insemination; the MNU treatments under each group condition all increased the mutant-character occurrence in the M1-M2 populations derived by insemination of mutagenized male germ cells, in contrast to the EMS treatment; when the pollens were treated for 40 min in 1.0 mM MNU solution (pH: 4.8) containing 20 mM PO_4^(3-), the mutant-character incidence most-significantly reached the maximum value of 10.5%, which was 6.2 times higher than that based on the EMS treatment; the mutant population was obtained by further screening the M3 populations. The MNU-mutagenic technique remarkably improves the efficiency of broad-spectral induction of maize mutants, which can provide support for the novel functional-gene identification and diversified breeding of maize. Key words: Zea maysL.; inbred line; in vitro pollens; MNU; mutagenization DOI: 10.25165/j.ijabe.20251806.9610 Citation: Tian H D, Li Z, Niu P F, Tian C H, Li N, Yan F, et al. Highly efficient mutagenesis using N-methyl-N-nitrosourea on male germ cells of maize. Int J Agric & Biol Eng, 2025; 18(6): 48–55.

References

Krishnan A, Guiderdoni E, An G, Hsing Y I C, Han C D, Lee M C, et al. Mutant resources in rice for functional genomics of the grasses. Plant Physiology, 2009; 149(1): 165–170.

Alonso J M, Stepanova A N, Leisse T J, Kim C J, Chen H, Shinn P, et al. Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science, 2003; 301(5633): 653–657.

Kuromori T, Hirayama T, Kiyosue Y, Takabe H, Mizukado S, Sakurai T, et al. A collection of 11, 800 single-copy Ds transposon insertion lines in Arabidopsis. Plant Journal, 2004; 37(6): 897–905.

Mao Y F, Yang X X, Zhou Y T, Zhang Z J, Botella J R, Zhu J K. Manipulating plant RNA-silencing pathways to improve the gene editing efficiency of CRISPR/Cas9 systems. Genome Biology, 2018; 19(1): 149.

Byrne M, Taylor W C. Analysis of Mutator-induced mutations in the iojap gene of maize. Molecular and General Genetics, 1996; 252: 216–220.

May B P, Liu H, Vollbrecht E, Senior L, Rabinowicz P D, Roh D, et al. Maize-targeted mutagenesis: a knockout resource for maize. Proceedings of the National Academy of Sciences, 2003; 100(20): 11541–11546.

McCarty D R, Settles A M, Suzuki M, Tan B C, Latshaw S, Porch T, et al. Steady-state transposon mutagenesis in inbred maize. Plant Journal, 2005; 44(1): 52–61.

Liu L, Gallagher J, Arevalo E D, Chen R, Skopelitis T, Wu Q Y, et al. Enhancing grain-yield-related traits by CRISPR–Cas9 promoter editing of maize CLE genes. Nature Plants, 2021; 7(3): 287–294.

Wang N L, Long T, Yao W, Xiong L Z, Zhang Q F, Wu C Y. Mutant resources for the functional analysis of the rice genome. Molecular Plant, 2013; 6(3): 596–604.

Neuffer M G, Ficsor G. Mutagenic action of ethyl methanesulfonate in maize. Science, 1963; 139(3561): 1296–1297.

Neuffer M G, Sheridan W F. Defective Kernel Mutants of Maize. I. Genetic and lethality studies. Genetics, 1980; 95(4): 929–944.

Tran Q H, Bui N H, Kappel C, Dau N T N, Nguyen L T, Tran T T, et al. Mapping-by-sequencing via mutMap identifies a mutation in ZmCLE7 underlying fasciation in a newly developed EMS mutant population in an elite tropical maize inbred. Genes, 2020; 11(3): 281.

Nie S J, Wang B, Ding H P, Lin H J, Zhang L, Li Q G, et al. Genome assembly of the Chinese maize elite inbred line RP125 and its EMS mutant collection provide new resources for maize genetics research and crop improvement. Plant Journal, 2021; 108(1): 40–54.

Messing J, Bharti A K, Karlowski W M, Gundlach H, Kim H R, Yu Y, et al. Sequence composition and genome organization of maize. Proceedings of the National Academy of Academy of Sciences, 2004; 101(40): 14349–14354.

Schnable P S, Ware D, Fulton R S, Stein J C, Wei F, Pasternak S, et al. The B73 maize genome: complexity, diversity, and dynamics. Science, 2009; 326(5956): 1112–1115.

Jiao Y, Peluso P, Shi J, Liang T, Stitzer M C, Wang B, et al. Improved maize reference genome with single-molecule technologies. Nature, 2017; 546(7659): 524–527.

Lu X D, Liu J S, Ren W, Yang Q, Chai Z G, Chen R M, et al. Gene-indexed mutations in maize. Molecular Plant, 2018; 11(3): 496–504.

Wang B B, Hou M, Shi J P, Ku L X, Song W, Li C H, et al. De novo genome assembly and analyses of 12 founder inbred lines provide insights into maize heterosis. Nature Genetics, 2023; 55(2): 312–323.

Tian H D, Li J, Tian B H, Niu P F, Li Z, Yue Z X, et al. Method for N-methyl-N-nitrosourea mutagenesis on hermaphroditic germ cells of rice. Chinese Bulletin of Botany, 2019; 54(5): 625–633. (in Chinese)

Tian H D, Li Y, Guo Y P, Qu Y J, Zhang X Y, Zhao X X, et al. Involvement of a rice mutation in storage protein biogenesis in endosperm and its genomic location. Planta, 2024; 260(1): 19.

Viana V E, Pegoraro C, Busanello C, de Oliveira A C. Mutagenesis in rice: The basis for breeding a new super plant. Frontiers in Plant Science, 2019; 10: 1326.

Yan H, Zhang W, Wang Y, Jin J, Xu H, Fu Y, et al. Rice LIKE EARLY STARVATION1 cooperates with FLOURY ENDOSPERM6 to modulate starch biosynthesis and endosperm development. Plant Cell, 2024; 36(5): 1892–1912.

Volk G M, Harris J L, Rotindo K E. Survival of mint shoot tips after exposure to cryoprotectant solution components. Cryobiology, 2006; 52(2): 305–308.

Eto T, Takahashi R, Kamisako T, Hioki K, Sotomaru Y. A study on cryoprotectant solution suitable for vitrification of rat two-cell stage embryos. Cryobiology, 2014; 68(1): 147–151.

Abdelgadir H A, Johnson S D, Van Staden J. Pollen viability, pollen germination and pollen tube growth in the biofuel seed crop Jatropha curcas (Euphorbiaceae). South African Journal of Botany, 2012; 79: 132–139.

Cui G, Sun Y, Hao Y, Du J, Wang Y. The improvement of maize pollen in vitro germination method and its role in pollen-mediated plant genetic transformation. Chinese Bulletin of Botany, 2012; 47(2): 155–161.

Till B J, Cooper J, Tai T H, Colowit P, Greene E A, Heniko V S, et al. Discovery of chemically induced mutations in rice by TILLING. BMC Plant Biology, 2007; 7(1): 19.

Suzuki T, Eiguchi M, Kumamaru T, Satoh H, Matsusaka H, Moriguchi K, et al. MNU-induced mutant pools and high performance TILLING enable finding of any gene mutation in rice. Molecular Genetics and Genomics, 2008; 279(3): 213–223.

Swain C G, Scott C B. Quantitative correlation of relative rates. Comparison of hydroxide ion with other nucleophilic reagents toward alkyl halides, esters, epoxides and acyl halides. Journal of the American Chemical Society, 1953; 75(1): 141–147.

Neale S. Mutagenicity of nitrosamides and nitro-samidines in micro-organisms and plants. Mutation Research, 1976; 32: 229–266.

Ohgaki H, Ludeke B I, Meier I, Kleihues P, Lutz W K, Schlatter C. DNA methylation in the digestive tract of F344 rats during chronic exposure to N-methyl-N-nitrosourea. Journal of Cancer Research and Clinical Oncology, 1991; 117(1): 13–18.

Engelbergs J, Thomale J, Rajewsky M F. Role of DNA repair in carcinogen-induced ras mutation. Mutation Research, 2000; 450: 139–153.

Cooke M S, Evans M D, Dizdaroglu M, Lunec J. Oxidative DNA damage: mechanisms, mutation, and disease. FASEB Journal, 2003; 17(10): 1195–1214.

Downloads

Published

2025-12-26

How to Cite

Tian, H., Li, Z., Niu, P., Tian, C., Li, N., Yan, F., … Cui, G. (2025). Highly efficient mutagenesis using N-methyl-N-nitrosourea on male germ cells of maize. International Journal of Agricultural and Biological Engineering, 18(6), 48–55. https://doi.org/10.25165/ijabe.v18i6.9610

Issue

Vol. 18 No. 6 (2025): IJABE

Section

Animal, Plant and Facility Systems

License

IJABE is an international peer reviewed open access journal, adopting Creative Commons Copyright Notices as follows.


Authors who publish with this journal agree to the following terms:


  1. Authors retain copyright and grant the journal right of first publication with the work simultaneously licensed under a Creative Commons Attribution License that allows others to share the work with an acknowledgement of the work's authorship and initial publication in this journal.

  2. Authors are able to enter into separate, additional contractual arrangements for the non-exclusive distribution of the journal's published version of the work (e.g., post it to an institutional repository or publish it in a book), with an acknowledgement of its initial publication in this journal.

  3. Authors are permitted and encouraged to post their work online (e.g., in institutional repositories or on their website) prior to and during the submission process, as it can lead to productive exchanges, as well as earlier and greater citation of published work (See The Effect of Open Access).