REDUCED-EXPOSURE TOBACCO PRODUCTS
The present invention provides methodologies, including the utilization of genetically modified (GM) tobaccos, for the development and production of consumer acceptable PREPs encompassing the following: 1) production of low tar-to-nicotine ratio cigarettes, which allow smokers to obtain satisfying, amounts of nicotine more efficiently than conventional cigarettes while reducing whole smoke deliveries; thereby reducing smoker ‘compensation’ that exists with conventional low-yield cigarettes; 2) reduction of harmful tobacco-specific nitrosamines in tobacco products by genetic means and by extracting nicotine from tobacco and combining such with genetically modified reduced-nicotine tobacco; 3) production of improved expanded tobacco which utilizes genetically modified increased-nicotine tobacco; and 4) production of reconstituted tobacco which includes any combinations of the following: genetically modified increased-nicotine tobacco, genetically modified reduced-nicotine tobacco, tobacco leaf fractions, and freshly harvested freeze-dried tobacco.
1 .- 148 . (canceled)
149 . A method of producing a nicotine buffer, comprising:
a) crossing an increased-nicotine transgenic Nicotiana tabacum plant with an increased-sugar Nicotiana tabacum plant;
b) selecting an increased-nicotine and increased-sugar Nicotiana tabacum progeny plant; and
c) producing a cigarette comprising a portion of the increased-nicotine and increased-sugar Nicotiana tabacum plant, wherein the sugar acts as the nicotine buffer.
150 . The method according to claim 149 , wherein the increased-nicotine transgenic Nicotiana tabacum plant is produced by:
a) transforming a Nicotiana tabacum plant with a construct comprising, in the 5′ to 3′ direction, a promoter operably linked to a heterologous nucleic acid encoding an enzyme or transcription factor that up-regulates nicotine synthesis;
b) regenerating transgenic Nicotiana tabacum plants from the transformed plant; and
c) selecting a transgenic Nicotiana tabacum plant having increased-nicotine content relative to a non-transformed control plant.
151 . A method for producing a nicotine buffer, comprising
a) producing a transgenic Nicotiana tabacum plant that expresses a heterologous nucleic acid sequence that confers elevated nicotine and fatty acid levels, as compared to a non-transformed control plant, and
b) producing a cigarette that comprises a portion of the plant, wherein the fatty acids act as the nicotine buffer.
152 . The method according to claim 151 , wherein step a) comprises:
1) transforming a Nicotiana tabacum plant with (i) a first construct comprising, in the 5′ to 3′ direction, a promoter operably linked to a heterologous nucleic acid encoding an enzyme or transcription factor that up-regulates nicotine synthesis; and (ii) a second construct comprising, in the 5′ to 3′ direction, a promoter operably linked to a heterologous nucleic acid encoding an enzyme or transcription factor that up-regulates fatty acid synthesis;
2) regenerating transgenic Nicotiana tabacum plants from the transformed plant; and
3) selecting a transgenic Nicotiana tabacum plant having increased-nicotine content and increased fatty acids as compared to a non-transformed control plant.
153 . A method according to claim 151 , wherein step a) comprises:
1) crossing an increased-nicotine transgenic Nicotiana tabacum plant with an increased fatty acid Nicotiana transgenic plant;
2) selecting an increased-nicotine and increased fatty acid Nicotiana tabacum progeny plant; and
3) producing a cigarette comprising at least a portion of the progeny plant.
154 . A method for increasing nicotine and cured reducing sugar content in a Nicotiana tabacum plant, comprising:
a) transforming a Nicotiana plant having elevated cured reducing sugar content with a nucleic acid conferring an increased-nicotine phenotype;
b) regenerating transgenic Nicotiana tabacum plants from the transformed plant; and
c) selecting a progeny plant having increased cured reducing sugar content and increased-nicotine content as compared to a non-transformed control plant.
155 . A method of increasing alkaloid biosynthesis in a Nicotiana tabacum plant or part thereof, comprising:
a) transforming a Nicotiana tabacum plant with at least one nucleic acid sequence, wherein the nucleic acid increases quinolate phosphoribosyl transferase and putrescine N-methyltransferase expression;
b) regenerating transgenic Nicotiana tabacum plants from the transformed plant; and
c) selecting a plant having increased alkaloid biosynthesis;
wherein the plant has increased quinolate phosphoribosyl transferase and putrescine N-methyltransferase expression relative to a non-transformed control plant.
156 . A tobacco plant characterized by increased nicotine levels and high cured reducing sugar content, wherein at least one transgene confers the increased nicotine levels.
157 . The tobacco plant according to claim 156 , wherein the plant has a nicotine content greater than 3.5 percent and a cured reducing sugar content from between about 12% and about 30%.
158 . A method of making a tobacco product having lower levels of tobacco-specific nitrosamines, comprising:
a) producing a reduced nicotine transgenic Nicotiana tabacum plant or plant portion, wherein the plant or plant portion expresses at least one heterologous nucleic acid that down-regulates nicotine production;
b) producing tobacco from the transgenic plant or plant portion;
c) adding nicotine to the tobacco; and
d) producing a product comprising the tobacco, wherein the product has a lower level of tobacco-specific nitrosamines as compared to a control product comprising a non-transformed control plant or plant portion.
159 . A product produced by the method of claim 158 , wherein the product is cigarette tobacco.
160 . The tobacco product of claim 159 , wherein the cigarette tobacco comprises filler having a level of tobacco-specific nitrosamines below about 0.5 microgram per gram of tobacco.
161 . The tobacco product of claim 160 , wherein the level of tobacco-specific nitrosamines is less than 0.05 microgram (50 ppb) per gram of tobacco.
162 . A method of making reconstituted tobacco, comprising:
a) providing plant material selected from the group consisting of:
(i) an increased-nicotine transgenic Nicotiana tabacum plant or plant portion, wherein the plant or plant portion expresses at least one heterologous nucleic acid that up-regulates the production of nicotine in the transgenic plant or plant portion;
(ii) a reduced-nicotine transgenic Nicotiana tabacum plant or plant portion, wherein the plant or plant portion expresses at least one heterologous nucleic acid that down-regulates the production of nicotine in the transgenic plant or plant portion;
(iii) deproteinized tobacco fiber or tobacco fiber portion; and
(iv) freeze-dried tobacco or tobacco portion; and
b) combining and reconstituting any combination of the plant material.
163 . A cigarette comprising the reconstituted tobacco of claim 162 , wherein the cigarette has a reduced yield of compounds selected from the group consisting of polycyclic aromatic hydrocarbons, tobacco-specific nitrosamines, benzo(a)pyrene, phenol, and catechols as compared to a cigarette made from a control plant.