Compositions and methods based on pmt engineering for producing tobacco plants and products having altered alkaloid levels

Abstract

The present disclosure provides compositions and methods related to tobacco plants with altered total alkaloid and nicotine levels and commercially acceptable leaf grade, their development via breeding or transgenic approaches, and production of tobacco products from these tobacco plants.

1 .- 32 . (canceled) 
     
     
         33 . Cured tobacco material from a tobacco plant comprising:
 (a) a first knockout mutant allele in a putrescine N-methyltransferase (PMT)1a gene (PMT1a), wherein a wildtype allele of the PMT1a gene encodes the polypeptide of SEQ ID NO: 12, and wherein the first knockout mutant allele in the PMT1a gene comprises the sequence of SEQ ID NO: 432; and   (b) a second knockout mutant allele in a PMT1b gene, wherein a wildtype allele of the PMT1b gene encodes the polypeptide of SEQ ID NO: 11; and   (c) a third knockout mutant allele in a PMT2 gene, wherein a wildtype allele of the PMT2 gene encodes the polypeptide of SEQ ID NO: 13; and   (d) a fourth knockout mutant allele in a PMT3 gene, wherein a wildtype allele of the PMT3 gene encodes the polypeptide of SEQ ID NO: 14; and   (e) a fifth knockout mutant allele in a PMT4 gene, wherein a wildtype allele of the PMT4 gene encodes the polypeptide of SEQ ID NO: 15;   
       wherein the tobacco plant produces a leaf comprising a nicotine level less than the nicotine level of a leaf from a control tobacco plant not having each of the first, second, third, fourth, and fifth knockout mutant alleles when grown and processed under comparable conditions. 
     
     
         34 . The cured tobacco material of  claim 33 , wherein the cured tobacco material comprises cured tobacco leaf. 
     
     
         35 . The cured tobacco material of  claim 33 , wherein the tobacco plant is of a tobacco type selected from the group consisting of flue-cured tobacco, air-cured tobacco, dark air-cured tobacco, dark fire-cured tobacco, Galpão tobacco, and Oriental tobacco. 
     
     
         36 . The cured tobacco material of  claim 33 , wherein the tobacco plant is of a tobacco type selected from the group consisting of Burley tobacco, Maryland tobacco, and dark tobacco. 
     
     
         37 . The cured tobacco material of  claim 34 , wherein the cured tobacco material exhibits reduced mold infection as compared to control cured tobacco material from the variety LA Burley 21. 
     
     
         38 . The cured tobacco material of  claim 37 , wherein the mold is from a genus selected from the group consisting of  Cladosporium, Penicillium, Alternaria, Aspergillus , and  Mucor.    
     
     
         39 . The cured tobacco material of  claim 33 , wherein the tobacco plant is derived from or in the genetic background of a tobacco 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×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, TN 97, TN97LC, TN D94, TN D950, TR (Tom Rosson) Madole, VA 309, or VA359, Maryland 609, HB3307PLC, HB4488PLC, KT206LC, KT209LC, KT210LC, KT212LC, R610LC, PVH2310, NC196, KTD14LC, KTD6LC, KTD8LC, PD7302LC, PD7305LC, PD7309LC, PD7318LC, PD7319LC, PD7312LC, and ShireyLC. 
     
     
         40 . The cured tobacco material of  claim 33 , wherein the cured tobacco material is selected from the group consisting of sun-cured tobacco material, air-cured tobacco material, and fire-cured tobacco material. 
     
     
         41 . The cured tobacco material of  claim 34 , wherein the cured tobacco leaf comprises a higher leaf grade than a control cured tobacco leaf from the tobacco variety LA BU 21. 
     
     
         42 . A tobacco blend comprising the cured tobacco material of  claim 33 . 
     
     
         43 . The tobacco blend of  claim 42 , wherein the cured tobacco material constitutes at least 10% of the tobacco blend by volume. 
     
     
         44 . A tobacco product comprising cured tobacco leaf from a tobacco plant comprising:
 (a) a first knockout mutant allele in a putrescine N-methyltransferase (PMT)1a gene (PMT1a), wherein a wildtype allele of the PMT1a gene encodes the polypeptide of SEQ ID NO: 12, and wherein the first knockout mutant allele in the PMT1a gene comprises the sequence of SEQ ID NO: 432; and   (b) a second knockout mutant allele in a PMT1b gene, wherein a wildtype allele of the PMT1b gene encodes the polypeptide of SEQ ID NO: 11; and   (c) a third knockout mutant allele in a PMT2 gene, wherein a wildtype allele of the PMT2 gene encodes the polypeptide of SEQ ID NO: 13; and   (d) a fourth knockout mutant allele in a PMT3 gene, wherein a wildtype allele of the PMT3 gene encodes the polypeptide of SEQ ID NO: 14; and   (e) a fifth knockout mutant allele in a PMT4 gene, wherein a wildtype allele of the PMT4 gene encodes the polypeptide of SEQ ID NO: 15;   
       wherein the tobacco plant produces a leaf comprising a nicotine level less than the nicotine level of a leaf from a control tobacco plant not having each of the first, second, third, fourth, and fifth knockout mutant alleles when grown and processed under comparable conditions 
     
     
         45 . The tobacco product of  claim 43 , 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, and cut tobacco. 
     
     
         46 . The tobacco product of  claim 43 , wherein the tobacco product is a smokeless tobacco product. 
     
     
         47 . The tobacco product of  claim 45 , wherein the smokeless tobacco product is selected from the group consisting of loose leaf chewing tobacco, plug chewing tobacco, moist snuff, snus, and nasal snuff. 
     
     
         48 . The tobacco product of  claim 43 , wherein the tobacco product further comprises a flavoring. 
     
     
         49 . The tobacco product of  claim 43 , wherein the tobacco product further comprises a filler. 
     
     
         50 . The tobacco product of  claim 48 , wherein the filler comprises reconstituted tobacco. 
     
     
         51 . The tobacco product of  claim 48 , wherein the filler comprises expanded tobacco. 
     
     
         52 . The tobacco product of  claim 48 , wherein the filler comprises non-tobacco plant material.