Development of a nornicotine-reduced flue-cured tobacco line via EMS mutagenesis of nicotine N-demethylase genes

Substantial progress had been made in reducing nornicotine accumulation in burley tobacco, as nornicotine is a precursor of the carcinogen N-nitrosonornicotine (NNN). Three members of the CYP82E2 family encoding nicotine N-demethylase (NND) have been reported to be responsible for the majority of nicotine demethylation that forms nornicotine in burley tobacco. We had obtained a nonsense mutant of each NND member in flue-cured tobacco from an ethyl methanesulfonate (EMS)-mutagenized population. In this study, we developed dCAPS markers for each nonsense mutation. Using marker-assisted selection, NND mutants were crossed with each other to generate a triple mutant GP449. In line with previous reports, the triple knockout caused significantly decreased levels of nornicotine and NNN in flue-cured tobacco. With the decreased nornicotine, the nicotine level was expected to accumulate. However, the nicotine level in GP449 was significantly decreased to 72.80% of wild type. Realtime RT-PCR analysis showed that the nicotine reduction was correlated with inhibited expression of nicotine biosynthetic pathway genes. The triple mutant and dCAPS markers can be utilized to develop new flue-cured tobacco varieties with lower levels of nornicotine and NNN.