Tobacco having altered leaf properties and methods of making and using

Abstract

This disclosure provides tobacco plants containing a PMT RNAi and tobacco plants having a mutation in PMT, and methods of making and using such plants.

What is claimed is: 
     
         1 . A method of making a  Nicotiana tabacum  plant, comprising:
 inducing mutagenesis in  N. tabacum  cells to produce mutagenized  N. tabacum  cells;   obtaining one or more  N. tabacum  plants from the mutagenized  N. tabacum  cells; and   identifying at least one of the  N. tabacum  plants that comprises a mutated PMT sequence.   
     
     
         2 . The method of  claim 1 , further comprising identifying at least one of the  N. tabacum  plants that exhibits reduced amounts of nicotine relative to a  N. tabacum  plant lacking a mutated PMT. 
     
     
         3 . The method of  claim 1 , further comprising identifying at least one of the  N. tabacum  plants that, when material from the at least one plant is cured, exhibits a reduced amount of at least one TSNA relative to cured material from a  N. tabacum  plant lacking a mutated PMT. 
     
     
         4 . The method of  claim 1 , wherein leaf from the mutant  N. tabacum  plant exhibits comparable or better quality than leaf from the plant lacking a mutated PMT sequence. 
     
     
         5 . The method of  claim 1 , wherein the  N. tabacum  plant is a Burley type, a dark type, a flue-cured type, or an Oriental type. 
     
     
         6 . A variety of  Nicotiana tabacum , the variety comprising plants having a mutation in an endogenous nucleic acid, the wild type endogenous nucleic acid encoding the PMT sequence shown in SEQ ID NO:2, 4, 6, 8 or 10. 
     
     
         7 . The variety of  claim 6 , wherein leaf from the mutant plants exhibits a reduced amount of nicotine relative to leaf from a plant lacking the mutation. 
     
     
         8 . The variety of  claim 6 , wherein material from the mutant plants, when cured, exhibits a reduced amount of at least one TSNA relative to cured material from a plant lacking the mutation. 
     
     
         9 . The variety of  claim 6 , wherein leaf from the mutant  N. tabacum  plant exhibits comparable or better quality than leaf from the plant lacking a mutated PMT sequence. 
     
     
         10 . Cured leaf from the  N. tabacum  variety of  claim 6 . 
     
     
         11 . A tobacco product comprising the cured leaf of  claim 10 . 
     
     
         12 . The tobacco product of  claim 11 , selected from the group consisting of cigarettes, smokeless tobacco products, tobacco-derived nicotine products, cigarillos, non-ventilated recess filter cigarettes, vented recess filter cigarettes, cigars, snuff, electronic cigarettes, e-vapor products, pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, and cut tobacco. 
     
     
         13 . A RNA nucleic acid molecule comprising a first nucleic acid between 15 and 500 nucleotides in length and a second nucleic acid between 15 and 500 nucleotides in length, wherein the first nucleic acid has a region of complementarity to the second nucleic acid, wherein the first nucleic acid comprises at least 15 contiguous nucleotides of the sequence shown in SEQ ID NO:1, 3, 5, 7 or 9. 
     
     
         14 . The nucleic acid molecule of  claim 13 , further comprising a spacer nucleic acid between the first nucleic acid and the second nucleic acid. 
     
     
         15 . A method of making a  Nicotiana tabacum  plant, comprising:
 transforming  N. tabacum  cells with the nucleic acid molecule of  claim 13  to produce transgenic  N. tabacum  cells;   regenerating transgenic  N. tabacum  plants from the transgenic  N. tabacum  cells; and   selecting at least one transgenic  N. tabacum  plant that comprises the nucleic acid molecule or the construct.   
     
     
         16 . The method of  claim 15 , further comprising identifying at least one transgenic  N. tabacum  plant having reduced amount of nicotine relative to a  N. tabacum  plant not transformed with the nucleic acid molecule. 
     
     
         17 . The method of  claim 15 , further comprising identifying at least one transgenic  N. tabacum  plant that, when material from the at least one transgenic  N. tabacum  plant is cured, exhibits a reduced amount of at least one TSNA relative to cured material from a  N. tabacum  plant not transformed with the nucleic acid molecule. 
     
     
         18 . The method of  claim 15 , wherein leaf from the selected transgenic  N. tabacum  plant exhibits comparable or better quality than leaf from the non-transformed  N. tabacum  plant. 
     
     
         19 . A transgenic  Nicotiana tabacum  plant comprising a vector, the vector comprising a RNA nucleic acid molecule having a length of 15 to 500 nucleotides and having at least 95% sequence identity to a PMT nucleic acid shown in SEQ ID NO:1, 3, 5, 7 or 9. 
     
     
         20 . The transgenic  N. tabacum  plant of  claim 19 , wherein the plant exhibits reduced amount of nicotine in the leaf relative to leaf from a  N. tabacum  plant lacking the nucleic acid molecule. 
     
     
         21 . The transgenic  N. tabacum  plant of  claim 19 , wherein, when material from the at least one transgenic  N. tabacum  plant is cured, exhibits a reduced amount of at least one TSNA relative to cured material from a  N. tabacum  plant lacking the nucleic acid molecule. 
     
     
         22 . The transgenic  N. tabacum  plant of  claim 19 , wherein leaf from the plant exhibits comparable or better quality than leaf from a  N. tabacum  plant lacking the nucleic acid molecule. 
     
     
         23 . Cured leaf from the transgenic  N. tabacum  plant of  claim 19 . 
     
     
         24 . A tobacco product comprising the cured leaf of  claim 23 . 
     
     
         25 . The tobacco product of  claim 24 , selected from the group consisting of smokeless tobacco products, tobacco-derived nicotine products, cigarillos, non-ventilated recess filter cigarettes, vented recess filter cigarettes, cigars, snuff, pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, and cut tobacco.