Reducing the content of nornicotine in tobacco via targeted mutation breeding

Cultivated tobacco produces secondary alkaloids involved in the formation of nitrosamines with health concerns. The recent identification of target genes in nicotine and nornicotine biosynthetic pathways now allows biotechnological approaches for their control. We demonstrate here that mutation breeding can be used as an alternative to genetically modified (GM) plants for generating nornicotine-free tobacco. Ten alleles of the NtabCYP82E4 gene (nicotine N-demethylase) were identified by screening 1,311 M2 families of tobacco ethylmethane sulphonate (EMS) mutants. Alkaloid analysis indicated that the nornicotine contents of homozygous M2 plants carrying nonsense or missense alleles of NtabCYP82E4 were very low or near-null. Backcrossing with tobacco elite varieties yielded BC1 plants phenotypically undistinguishable from parental lines. This major objective of tobacco breeders in the last few decades could be reached in a period of less than 1.5 years, including the creation of highly mutagenised tobacco mutant collections and the detection of mutated alleles using a simple and versatile detection technology (capillary electrophoresis-single strand conformation polymorphism, CE-SSCP) accessible to most breeding companies and crop species.