Abstract
Due to potential regulations that could affect nicotine levels in some tobacco products, there is interest in using genetic modification to reduce levels of this pyridine alkaloid in tobacco leaves. Enzymes coded by A622 genes have previously been indicated to be involved in one of the latter steps of tobacco alkaloid biosynthesis. Whole tobacco plants with reduced A622 activity have never been evaluated, however. We utilized CRISPR/Cas9-based editing to introduce deleterious mutations into the two A622 genes present in the Nicotiana tabacum genome. Double homozygous A622 mutant genotypes established in four recipient genotypes varying for the presence/absence of mutations in other alkaloid biosynthetic genes exhibited severely reduced nicotine accumulation in field and greenhouse experiments. A622 knockout lines exhibited lower nicotine levels than previously created genotypes with deleterious mutations in BBL genes also associated with one of the latter steps in tobacco alkaloid biosynthesis. Reduced A622 activity resulted in plants with drastically reduced growth and development, however. A622 mutant lines were later flowering and produced green leaf yields that were 60.6% lower, on average, than those for non-A622-mutated control lines. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11032-022-01293-w.