Generation of Rice Mutants by Chemical Mutagenesis
Chemical mutagenesis of rice has been used extensively to generate useful genetic variation for the purpose of breeding improved varieties. More recently, advances in high-throughput genotyping platforms have enabled the efficient detection of point mutations generated by chemical agents. This in turn has renewed interest in using traditional chemical mutagenesis to generate mutant populations for gene discovery and functional characterization. Targeting of Induced Local Lesions in Genomes (TILLING) is a powerful reverse genetics method which combines chemical mutagenesis with the high-throughput discovery of point mutations. Numerous chemical mutagens have been shown to be effective in generating point mutations and small deletions in rice. This chapter describes the use of a combination of sodium azide (NaN3) and N-nitroso-N-methylurea to generate populations that are suitable for TILLING as well as forward genetics and mutation breeding.
Key wordsChemical mutagenesis Reverse genetics Forward genetics TILLING Mutation breeding
This protocol was developed with the technical assistance of P.M. Colowit and supported by the USDA Agricultural Research Service CRIS Project 5306-21000-016/17-00D and grant 2004-353604-14265 from the USDA Cooperative State Research, Education, and Extension Service, NRI Plant Genome Program.
- 1.Rutger JN (1983) Applications of induced and spontaneous mutation in rice breeding and genetics. In: Brady NC (ed) Advances in agronomy. Academic, New York, NY, pp 383–413Google Scholar
- 7.Koornneef M (2002) Classical mutagenesis in higher plants. In: Gilmartin PM, Bowler C (eds) Molecular plant biology: a practical approach, vol 1. Oxford University, Oxford, UK, pp 1–11Google Scholar
- 12.Maple J, Møller SG (2007) Mutagenesis in Arabidopsis. In: Rosato E (ed) Methods in Molecular Biology: Circadian Rhythmns: Methods and Protocols. Humana, Totowa, NJ, pp 197–206Google Scholar
- 14.Wu JL, Wu C, Lei C, Baraoidan M, Bordeos A, Madamba MR, Ramos-Pamplona M, Mauleon R, Portugal A, Ulat VJ, Bruskiewich R, Wang G, Jan Leach J, Khush G, Leung H (2005) Chemical-and irradiation-induced mutants of indica rice IR64 for forward and reverse genetics. Plant Mol Biol 59:85–97PubMedCrossRefGoogle Scholar
- 17.Owais WM, Kleinhofs A (1988) Metabolic activation of the mutagen azide in biological systems. Mutat Res 197:31–323Google Scholar
- 20.Suzuki T, Moriguchi K, Tsuda K, Eiguchi M, Kumamaru T, Satoh H, Kurata N (2010) Neighboring nucleotide bias around MNU-induced mutations in rice. Rice Genet Newsl 25:90–91Google Scholar
- 21.Szarejko I, Maluszynski M (1999) High frequency of mutations after mutagenic treatment of barley seeds with NaN3 and MNH with application of inter-incubation germination period. Mutat Breed Newsl 44:28–30Google Scholar