# US 11,304,440 B2 · Tobacco having altered leaf properties and methods of making and using > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,304,440 B2 - Document ID: US11304440B2 - Status: Granted - Kind: B2 - Application number: 16/674,838 - Filed: 2019-11-05 - Published: 2020-03-05 - Granted: 2022-04-19 - Earliest claimed: 2014-04-08 - Assignees: Altria Client Services LLC - Applicants: Altria Client Services LLC - Inventors: Chengalrayan Kudithipudi; Alec J. Hayes; Marcos Fernando de Godoy Lusso; Jerry Whit Morris - Web page: https://patentgrep.com/patent/US11304440B2 ## CPC - A24D 1/00: Cigars; Cigarettes - A24B 13/00: Tobacco for pipes, for cigars, e.g. cigar inserts, or for cigarettes; Chewing tobacco; Snuff (reconstituted tobacco products A24B3/14) - A24D 3/043: {with ventilation means, e.g. air dilution (A24D3/041 takes precedence)} - C12N 15/8218: {Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]} - C12N 15/8243: {involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine} - C12N 15/8261: {with agronomic (input) traits, e.g. crop yield} - A24F 40/20: Devices using solid inhalable precursors - A24D: CIGARS; CIGARETTES; TOBACCO SMOKE FILTERS; MOUTHPIECES OF CIGARS OR CIGARETTES; MANUFACTURE OF TOBACCO SMOKE FILTERS OR MOUTHPIECES - A24B: MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - A24F: SMOKERS' REQUISITES; MATCH BOXES; SIMULATED SMOKING DEVICES (inhaling appliances for medical purposes, shaped like cigars, cigarettes or pipes A61M15/06) ## 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. ## Claims ### Claim 1 A method of generating a transgenic Nicotiana tabacum plant comprising: (a) introducing an RNAi nucleic acid molecule to a N. tabacum cell, wherein said RNAi nucleic acid molecule comprises a first nucleic acid sequence at least 95% identical to SEQ ID NO: 11, and a second nucleic acid sequence at least 95% identical to SEQ ID NO: 12, and a spacer nucleic acid comprising a sequence at least 95% identical to SEQ ID NO: 13 between said first nucleic acid sequence and said second nucleic acid sequence; (b) generating at least one transgenic N. tabacum plant from said N. tabacum cell; and (c) selecting at least one transgenic N. tabacum plant generated in step (b) comprising said RNAi construct, wherein said at least one transgenic N. tabacum plant comprises a reduced amount of nicotine as compared to a control plant lacking said RNAi nucleic acid molecule. ### Claim 2 (depends on 1) The method of claim 1, wherein the first nucleic acid sequence is 100% identical to SEQ ID NO: 11. ### Claim 3 (depends on 1) The method of claim 1, wherein the second nucleic acid is 100% identical to SEQ ID NO: 12. ### Claim 4 (depends on 1) The method of claim 1, wherein said RNAi nucleic acid molecule comprises a sequence encoding a promoter that is operable in a plant cell operably linked to said first and second nucleic acid sequences. ### Claim 5 (depends on 4) The method of claim 4, wherein said promoter is selected from the group consisting of a constitutive promoter and a tissue-specific promoter. ### Claim 6 (depends on 5) The method of claim 5, wherein said constitutive promoter is selected from the group consisting of a cassava mosaic virus promoter, a cauliflower mosaic virus 35S promoter, an actin promoter, and a glyceraldehyde-3-phosphate dehydrogenase promoter. ### Claim 7 (depends on 5) The method of claim 5, wherein the tissue-specific promoter is selected from the group consisting of a putrescine N-methyl transferase (PMT) promoter and a quinolinate phosphoribosyltransferase promoter. ### Claim 8 (depends on 1) The method of claim 1, wherein said transgenic N. tabacum plant is of a tobacco type selected from the group consisting of a Burley type, a dark type, a flue-cured type, and an Oriental type. ### Claim 9 (depends on 1) The method of claim 1, wherein expression of said RNAi nucleic acid molecule generates a small hairpin RNA molecule. ### Claim 10 (depends on 1) The method of claim 1, wherein said introducing comprises a method selected from the group consisting of electroporation, calcium phosphate precipitation, polyethylene glycol transformation, heat shock, lipofection, microinjection, and viral-mediated nucleic acid transfer. ### Claim 11 (depends on 1) The method of claim 1, wherein the transgenic N. tabacum plant comprises a reduction in the amount of a PMT polypeptide comprising the amino acid sequence of SEQ ID NO: 8 as compared to a control N. tabacum plant lacking the RNAi nucleic acid molecule. ### Claim 12 Cured tobacco material comprising leaf from a Nicotiana tabacum plant comprising an RNAi nucleic acid molecule, wherein said RNAi nucleic acid molecule comprises a first nucleic acid sequence at least 95% identical to SEQ ID NO: 11, and a second nucleic acid sequence at least 95% identical to SEQ ID NO: 12, and a spacer nucleic acid comprising a sequence at least 95% identical to SEQ ID NO: 13 between said first nucleic acid sequence and said second nucleic acid sequence, and wherein said cured tobacco material comprises a reduced amount of nicotine as compared to cured tobacco material from a control plant lacking said RNAi nucleic acid molecule. ### Claim 13 (depends on 12) The cured tobacco material of claim 12, wherein the cured tobacco material is selected from the group consisting of air cured tobacco material, fire cured tobacco material, flue cured tobacco material, and sun cured tobacco material. ### Claim 14 (depends on 12) The cured tobacco material of claim 12, wherein said leaf comprises a reduced amount of nicotine as compared to leaf from a control plant lacking said RNAi nucleic acid molecule. ### Claim 15 (depends on 12) A tobacco product comprising the cured tobacco material of claim 12. ### Claim 16 (depends on 15) The tobacco product of claim 15 wherein the tobacco product is selected from the group consisting of cigarettes, cigarillos, non-ventilated recess filter cigarettes, vented recess filter cigarettes, cigars, snuff, pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, cut tobacco, and snus. ### Claim 17 (depends on 12) The cured tobacco material of claim 12, wherein the first nucleic acid sequence is 100% identical to SEQ ID NO: 11. ### Claim 18 (depends on 12) The cured tobacco material of claim 12, wherein the second nucleic acid is 100% identical to SEQ ID NO: 12. ### Claim 19 (depends on 1) The method of claim 1, wherein the spacer nucleic acid comprises a sequence 100% identical to SEQ ID NO: 13. ### Claim 20 (depends on 12) The cured tobacco material of claim 12, wherein the spacer nucleic acid comprises a sequence 100% identical to SEQ ID NO: 13.