Tobacco plants having reduced nicotine demethylase activity

Abstract

The present invention generally relates to methods and materials involved in producing tobacco plants having reduced levels of conversion of nicotine to nornicotine. In certain embodiments, the invention is directed to mutations in a nicotine demethylase gene, tobacco plants comprising mutations in a nicotine demethylase gene, and tobacco compositions and products thereof. In other embodiments, the invention is directed toward nicotine demethylase RNA interference, tobacco plants comprising a nicotine demethylase RNA interference transgene, and tobacco compositions and products thereof.

What is claimed is: 
     
         1 . A tobacco hybrid, variety, or line comprising plants having a mutation in a nicotine demethylase gene, wherein the mutation is a stop codon at residue 329 relative to SEQ ID NO:2, said plants exhibiting a non-converter phenotype, and whose homozygous progeny have a reversion rate that is reduced at least 2× compared to the reversion rate of the corresponding tobacco hybrid, variety, or line comprising plants having a wild type nicotine demethylase gene. 
     
     
         2 . The tobacco hybrid, variety, or line of  claim 1 , wherein said hybrid, variety, or line comprises Burley type tobacco plants. 
     
     
         3 . The tobacco hybrid, variety, or line of  claim 2 , wherein said progeny have a reversion rate that is reduced 10× to 1000× compared to the reversion rate of the corresponding tobacco hybrid, variety, or line comprising plants comprising a wild type nicotine demethylase gene. 
     
     
         4 . The tobacco hybrid, variety, or line of  claim 1 , wherein said hybrid, variety, or line comprises dark type tobacco plants. 
     
     
         5 . The tobacco hybrid, variety, or line of  claim 4 , wherein said progeny have a reversion rate that is reduced 2× to 100× compared the reversion rate of the corresponding tobacco hybrid, variety, or line comprising plants comprising a wild type nicotine demethylase gene. 
     
     
         6 . The tobacco hybrid, variety, or line of  claim 1 , wherein said hybrid, variety, or line comprises flue-cured type tobacco plants. 
     
     
         7 . The tobacco hybrid, variety, or line of  claim 6 , wherein said progeny revert to a converter phenotype at a rate per generation that is reduced 2× to 100× compared to the reversion rate of a corresponding tobacco hybrid, variety, or line comprising plants comprising a wild type nicotine demethylase gene. 
     
     
         8 . The tobacco hybrid, variety, or line of  claim 1 , wherein said hybrid, variety, or line comprises Oriental type tobacco plants. 
     
     
         9 . The tobacco hybrid, variety, or line of  claim 8 , wherein said progeny revert to a converter phenotype at a rate per generation that is reduced 2× to 100× compared the reversion rate of the corresponding tobacco hybrid, variety, or line comprising plants comprising a wild type nicotine demethylase gene. 
     
     
         10 . The hybrid, variety, or line of  claim 1 , wherein said hybrid, variety, or line is a variety. 
     
     
         11 . An M1 tobacco plant that is heterozygous for a mutant allele at a nicotine demethylase locus encoding an amino acid sequence of SEQ ID NO:2, said allele comprising a stop codon mutation at residue 329 relative to SEQ ID NO:2, wherein the tryptophan at amino acid 329 is replaced with a stop codon, wherein at least a portion of first generation self-pollinated progeny of said M1 tobacco plant exhibit a non-converter phenotype, and wherein at least a portion of first generation self-pollinated progeny of said M1 tobacco plant revert to a converter phenotype at a rate that is statistically significantly less than the reversion rate of the progeny of a corresponding tobacco plant that comprises a wild type allele at said nicotine demethylase locus. 
     
     
         12 . Progeny of the tobacco plant of  claim 11 , said progeny having said mutant allele at said nicotine demethylase locus and having said non-converter phenotype. 
     
     
         13 . An M1 tobacco plant carrying in its genome a mutant allele at a nicotine demethylase locus encoding an amino acid sequence of SEQ ID NO:2, said mutant allele comprising a stop codon at residue 329 relative to SEQ ID NO:2, said plant exhibiting a non-converter phenotype and whose progeny revert to a converter phenotype at a rate that is statistically significantly less than the reversion rate of the progeny of a corresponding tobacco plant that comprises a wild type allele at said nicotine demethylase locus. 
     
     
         14 . Progeny of the tobacco plant of  claim 13 , said progeny having said mutant allele at said nicotine demethylase locus and having said non-converter phenotype. 
     
     
         15 . The plant of  claim 13 , wherein said M1 plant is a plant of a variety selected from the group consisting of BU 64, CC 101, CC 200, CC 27, CC 301, CC 400, CC 500, CC 600, CC 700, CC 800, CC 900, Coker 176, Coker 319, Coker 371 Gold, Coker 48, CU 263, DF911, Galpao tobacco, GL 26H, GL 350, GL 600, GL 737, GL 939, GL 973, HB 04P, K 149, K 326, K 346, K 358, K394, K 399, K 730, KDH 959, KT 200, KT204LC, KY 10, KY 14, KY 160, KY 17, KY 171, KY 907, KY907LC, KTY14 x L8 LC, Little Crittenden, McNair 373, McNair 944, msKY 14xL8, Narrow Leaf Madole, NC 100, NC 102, NC 2000, NC 291, NC 297, NC 299, NC 3, NC 4, NC 5, NC 6, NC7, NC 606, NC 71, NC 72, NC 810, NC BH 129, NC 2002, Neal Smith Madole, OXFORD 207, ‘Perique’ tobacco, PVH03, PVH09, PVH19, PVHSO, PVH5 1, R 610, R 630, R 7-11, R 7-12, RG 17, RG 81, RG H51, RGH 4, RGH 51, RS 1410, Speight 168, Speight 172, Speight 179, Speight 210, Speight 220, Speight 225, Speight 227, Speight 234, Speight G-28, Speight G-70, Speight H-6, Speight H20, Speight NF3, TI 1406, TI 1269, TN 86, TN86LC, TN 90, TN 97, TN97LC, TN D94, TN D950, TR (Tom Rosson) Madole, VA 309, VA359 and a variety essentially derived from any of the above varieties.