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.

1 .- 25 . (canceled) 
     
     
         26 . A tobacco plant comprising:
 (a) a first induced mutation in a first endogenous nucleic acid molecule encoding a putrescine N-methyltransferase 1a (PMT1a) protein;   (b) a second induced mutation in a second endogenous nucleic acid molecule encoding a PMT1b protein;   (c) a third induced mutation in a third endogenous nucleic acid molecule encoding a PMT2 protein;   (d) a fourth induced mutation in a fourth endogenous nucleic acid molecule encoding a PMT3 protein; and   (e) a fifth induced mutation in a fifth endogenous nucleic acid molecule encoding a PMT4 protein,   wherein the tobacco plant is a  Nicotiana tabacum  plant.   
     
     
         27 . The tobacco plant of  claim 26 , wherein:
 (a) one wild-type allele of the first endogenous nucleic acid molecule encodes an amino acid sequence comprising SEQ ID NO: 2;   (b) one wild-type allele of the second endogenous nucleic acid molecule encodes an amino acid sequence comprising SEQ ID NO: 4;   (c) one wild-type allele of the third endogenous nucleic acid molecule encodes an amino acid sequence comprising SEQ ID NO: 6;   (d) one wild-type allele of the fourth endogenous nucleic acid molecule encodes an amino acid sequence comprising SEQ ID NO: 8; and   (e) one wild-type allele of the fifth endogenous nucleic acid molecule encodes an amino acid sequence comprising SEQ ID NO: 10.   
     
     
         28 . The tobacco plant of  claim 26 , wherein:
 (a) the first endogenous nucleic acid molecule comprises a nucleic acid sequence at least 95% identical to SEQ ID NO: 1;   (b) the second endogenous nucleic acid molecule comprises a nucleic acid sequence at least 95% identical to SEQ ID NO: 3;   (c) the third endogenous nucleic acid molecule comprises a nucleic acid sequence at least 95% identical to SEQ ID NO: 5;   (d) the fourth endogenous nucleic acid molecule comprises a nucleic acid sequence at least 95% identical to SEQ ID NO: 7; and   (e) the fifth endogenous nucleic acid molecule comprises a nucleic acid sequence at least 95% identical to SEQ ID NO: 9.   
     
     
         29 . The tobacco plant of  claim 26 , wherein:
 (a) the first endogenous nucleic acid molecule comprises a nucleic acid sequence 100% identical to SEQ ID NO: 1;   (b) the second endogenous nucleic acid molecule comprises a nucleic acid sequence 100% identical to SEQ ID NO: 3;   (c) the third endogenous nucleic acid molecule comprises a nucleic acid sequence at 100% identical to SEQ ID NO: 5;   (d) the fourth endogenous nucleic acid molecule comprises a nucleic acid sequence at 100% identical to SEQ ID NO: 7; and   (e) the fifth endogenous nucleic acid molecule comprises a nucleic acid sequence at least 95% identical to SEQ ID NO: 9.   
     
     
         30 . The tobacco plant of  claim 26 , wherein each of the first induced mutation, the second induced mutation, the third induced mutation, the fourth induced mutation, and the fifth induced mutation comprise a mutation individually selected from the group consisting of an insertion, a deletion, and a substitution. 
     
     
         31 . The tobacco plant of  claim 26 , wherein the tobacco plant comprises reduced expression or activity of each of the PMT1a, PMT1b, PMT2, PMT3, and PMT4 proteins as compared to a control tobacco plant lacking the first induced mutation, the second induced mutation, the third induced mutation, the fourth induced mutation, and the fifth induced mutation. 
     
     
         32 . The tobacco plant of  claim 26 , wherein the tobacco plant comprises a reduced amount of N-methylputrescine as compared to a control tobacco plant lacking the first induced mutation, the second induced mutation, the third induced mutation, the fourth induced mutation, and the fifth induced mutation. 
     
     
         33 . The tobacco plant of  claim 26 , wherein at least one of the first induced mutation, the second induced mutation, the third induced mutation, the fourth induced mutation, and the fifth induced mutation is within a methyltransferase domain. 
     
     
         34 . The tobacco plant of  claim 26 , wherein at least one of the first induced mutation, the second induced mutation, the third induced mutation, the fourth induced mutation, and the fifth induced mutation results in a truncated protein. 
     
     
         35 . The tobacco plant of  claim 26 , wherein at least one oft the first induced mutation, the second induced mutation, the third induced mutation, the fourth induced mutation, and the fifth induced mutation results in a premature stop codon. 
     
     
         36 . The tobacco plant of  claim 26 , wherein at least one of the first induced mutation, the second induced mutation, the third induced mutation, the fourth induced mutation, and the fifth induced mutation results in a non-conserved amino acid substitution. 
     
     
         37 . The tobacco plant of  claim 26 , wherein the tobacco plant comprises a reduced amount of nicotine as compared to a control tobacco plant lacking t the first induced mutation, the second induced mutation, the third induced mutation, the fourth induced mutation, and the fifth induced mutation. 
     
     
         38 . The tobacco plant of  claim 26 , wherein the tobacco plant is of a type selected from the group consisting of a Burley type, a dark type, a flue-cured type, and an Oriental type. 
     
     
         39 . The tobacco plant of  claim 26 , wherein the tobacco plant is of a tobacco variety selected from the group consisting of BU 64, CC 101, CC 200, CC 13, CC 27, CC 33, CC 35, CC 37, CC 65, CC 67, CC 301, CC 400, CC 500, CC 600, CC 700, CC 800, CC 900, CC 1063, Coker 176, Coker 319, Coker 371 Gold, Coker 48, CU 263, DF911, GL 26H, GL 338, GL 350, GL 395, GL 600, GL 737, GL 939, GL 973, GF 157, GF 318, RJR 901, HB 04P, K 149, K 326, K 346, K 358, K394, K 399, K 730, NC 196, NC 37NF, NC 471, NC 55, NC 92, NC2326, NC 95, NC 925, PVH 1118, PVH 1452, PVH 2110, PVH 2254, PVH 2275, VA 116, VA 119, KDH 959, KT 200, KT204LC, KY 10, KY 14, KY 160, KY 17, KY 171, KY 907, KY907LC, KTY14×L8 LC, Little Crittenden, McNair 373, McNair 944, msKY 14×L8, 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, PVH50, PVH51, 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, TN90LC, TN 97, TN97LC, TN D94, TN 5 D950, TR (Tom Rosson) Madole, VA 309, and VA359. 
     
     
         40 . A tobacco product comprising cured tobacco material from the tobacco plant of  claim 26 . 
     
     
         41 . The tobacco product of  claim 40 , wherein the tobacco product is selected from the group consisting of a cigarette, a cigarillo, a non-ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, cut tobacco, chewing tobacco, and snus. 
     
     
         42 . The tobacco product of  claim 40 , wherein the cured tobacco material comprises cured leaf material. 
     
     
         43 . The tobacco product of  claim 40 , wherein the tobacco product further comprises aged, conditioned, or fermented tobacco material. 
     
     
         44 . The tobacco product of  claim 40 , wherein the tobacco product comprises an ingredient selected from the group consisting of a binder, a plasticizer, a stabilizer, and a flavoring. 
     
     
         45 . A seed of the tobacco plant of  claim 26 , wherein the seed comprises the first induced mutation, the second induced mutation, the third induced mutation, the fourth induced mutation, and the fifth induced mutation.