Classifications

• • A—HUMAN NECESSITIES
  • A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
  • A24B—MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
  • A24B3/00—Preparing tobacco in the factory
  • A24B3/12—Steaming, curing, or flavouring tobacco
• • A—HUMAN NECESSITIES
  • A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
  • A24B—MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
  • A24B3/00—Preparing tobacco in the factory
  • A24B3/04—Humidifying or drying tobacco bunches or cut tobacco
• • A—HUMAN NECESSITIES
  • A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
  • A24B—MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
  • A24B15/00—Chemical features or treatment of tobacco; Tobacco substitutes, e.g. in liquid form
  • A24B15/10—Chemical features of tobacco products or tobacco substitutes

Definitions

• the present specification includes methods used to improve leaf quality during curing.
• the present specification also includes air-curing, fire-curing, and a novel hybrid curing process.
• the present specification further includes the reduction of polyaromatic hydrocarbons and tobacco specific nitrosamines (TSNAs) using the provided hybrid curing methods.
• TSNAs tobacco specific nitrosamines
• Cured tobacco is the result of many physical and chemical changes that transform tobacco from a green, high-moisture leaf obtained at harvest to an aromatic, low-moisture leaf that is sold and used in tobacco products. Physical and chemical changes begin before tobacco is harvested. As leaves ripen and begin the process of leaf senescence, chemical changes begin and continue even after the tobacco is cut and hung in a bam to cure. Therefore, there are many environmental conditions, before and after harvesting, that can influence the properties of cured tobacco.
• Fire curing practice typically includes three to six fires over a period of about twenty-one to thirty days and takes place at a process point that can lead to the formation of tobacco specific nitrosamines (TSNAs). Furthermore, fire-curing can vary from grower to grower and region to region adding variability.
• TSNAs tobacco specific nitrosamines
• HPHCs Harmful and Potentially Harmful Constituents
• the present specification provides for, and includes, a method for curing tobacco leaves comprising subjecting a tobacco leaf to air curing and applying one or more smoke finish attributes to the air cured leaf to generate an air/fire combination cured tobacco leaf.
• the present specification provides for, and includes, an air / fire combination cured tobacco leaf comprising one or more smoke finish attributes and further comprising a lower amount of NNN, NNK, or both, as compared to a control cured leaf of a tobacco plant cured using a conventional fire-curing process.
• the present specification provides for, and includes, an air/ fire combination cured tobacco leaf comprising one or more smoke finish attributes and lower amounts of one or more polyaromatic hydrocarbons as compared to a control cured leaf of a tobacco plant cured using a conventional fire-curing process.
• the present specification provides for, and includes, a tobacco product comprising tobacco cured using an air/fire combination curing method.
• Figure l is a graph showing NOx (nitrogen oxides) emissions over time in parts per billion during the fire-curing of tobacco when using a smoldering fire.
• the top trend line on the graph represents NO levels and the bottom trend line on the graph represents NO2 levels.
• Figure 2 is a graph showing NOx (nitrogen oxides) emissions over time in parts per billion during the fire-curing of tobacco when using an open flame fire.
• the top trend line on the graph represents NO levels and the bottom trend line on the graph represents NO2 levels.
• a plant or “at least one plant” may include a plurality of plants.
• phrases such as “between X and Y” and “between about X and Y” should be interpreted to include X and Y.
• phrases such as “between about X and Y” mean “between about X and about Y” and phrases such as “from about X to Y” mean “from about X to about Y.”
• the transitional phrase “consisting essentially of’ means that the scope of a claim is to be interpreted to encompass the specified materials or steps recited in the claim and those that do not materially affect the basic and novel characteristic(s) of the claimed specification.
• the term “consisting essentially of’ when used in a claim of this specification is not intended to be interpreted to be equivalent to “comprising.”
• plant refers to a whole plant or a cell or tissue culture derived from a plant, comprising any of: whole plants, plant components or organs (e.g ., leaves, flowers, stems, roots, fruits, vasculature, anthers, pistils, buds, tillers, and branches), plant tissues, seeds, plant cells, and/or progeny of the same.
• a progeny plant can be from any filial generation, e.g., Fi, F2, F3, F4, F5, F 6 , F7 , and so on.
• a plant cell is a biological cell of a plant, taken from a plant or derived through culture from a cell taken from a plant.
• a plant cell can be any plant cell taken for culture.
• a plant cell can be a non-photosynthetic cell.
• a plant cell can also be a non-reproductive, or somatic, plant cell.
• plant or part thereof includes plant cells, plant protoplasts, plant cells of tissue culture from which tobacco plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as leaves, flowers, stems, roots, fruits, vasculature, anthers, pistils, buds, tillers, branches, and pollen.
• a “population of plants” or a “population of seeds” means a set comprising any number, at least two, of individuals, objects, or data from which samples are taken for evaluation. Most commonly, the terms relate to a breeding population of plants from which members are selected and crossed to produce progeny in a breeding program.
• a population of plants can include the progeny of a single breeding cross or a plurality of breeding crosses, and can be either actual plants or plant derived material, or in silico representations of the plants or seeds.
• the population members need not be identical to the population members selected for use in subsequent cycles of analyses or those ultimately selected to obtain final progeny plants or seeds.
• a population of plants or seeds is derived from a single biparental cross, but can also derive from two or more crosses between the same or different parents.
• a population of plants or seeds can comprise any number of individuals, those of skill in the art will recognize that plant breeders commonly use population sizes between 20 to more than several thousand, between several hundred and several thousand, and between several thousand and tens of thousands of individuals. The highest performing 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of a population is what is commonly selected to be used in subsequent crosses in order to improve the performance of subsequent generations of the population.
• a tobacco plant can be from any plant from the Nicotiana tabacum genus including, but not limited to Nicotiana tabacum tabacum; Nicotiana tabacum amplexicaulis PI 271989; Nicotiana tabacum benthamiana PI 555478; Nicotiana tabacum bigelovii PI 555485; Nicotiana tabacum debneyi ; Nicotiana tabacum excelsior PI 224063; Nicotiana tabacum glutinosa PI 555507; Nicotiana tabacum goodspeedii PI 241012; Nicotiana tabacum gossei PI 230953; Nicotiana tabacum hesperis PI 271991; Nicotiana tabacum knightiana PI 555527; Nicotiana tabacum maritima PI 555535; Nicotiana tabacum megalosiphon PI 555536; Nicotiana tabacum nudicaulis PI 555540; Nicotiana tabacum panic
• TSNAs Tobacco-specific nitrosamines
• NNN N-nitrosonomicotine
• NNK 4- (methylnitrosamino)-l-(3-pyridyl)-l-butanone
• TSNAs are not present in significant quantities in growing tobacco plants or fresh cut tobacco (green tobacco), but are formed during the curing process specifically during the late yellowing and early browning stages. Without being limited by any theory, bacterial populations which reside on tobacco leaves can cause the formation of nitrites from nitrate during curing and possibly affect the direct catalysis of the nitrosation of secondary amines at physiological pH values.
• TSNAs are formed as a result of the nitrosation of tobacco alkaloids in the presence of nitrogen oxides (NOx). For example, NNN is formed by the nitrosation of the alkaloid, nomicotine.
• the nitrosating agent in air-cured tobacco is usually nitrite, derived from the reduction of leaf nitrate by the action of microbes during curing, referred to as nitrate-reducing microorganisms.
• nitrate-reducing microorganisms derived from the reduction of leaf nitrate by the action of microbes during curing.
• the nitrosating agents are both nitrite and any of several nitrogen oxides formed during the fire-curing process.
• HPHCs harmful and potentially harmful constituents
• an HPHC is selected from the group consisting of Acetaldehyde, Acetamide, Acetone, Acrolein, Acrylamide, Acrylonitrile, Aflatoxin Bl, 4-Aminobiphenyl, 1-Aminonaphthalene, 2-Aminonaphthalene, Ammonia, Anabasine, o- Anisidine, Arsenic, A-a-C (2-Amino-9H-pyrido[2,3-b]indole), Benz[a]anthracene, Benz[j]aceanthrylene, Benzene, Benzo[b]fluoranthene, Benzo[k]fluoranthene, Benzo[b]furan, Benzo[a]pyrene, Benzo[c]phenanthrene, Beryllium, 1,3 -Butadiene, Cadmium, Caffeic acid, Carbon monoxide, Catechol, Chlorinated dioxins/furans, Chromium, Chr
• an HPHC is selected from the group consisting of Acetaldehyde, Arsenic, Benz[a]anthracene, Benzene, Benzo[a]pyrene, Cadmium, Crotonaldehyde, Dibenz[a,h]anthracene, Dibenzo[a,e]pyrene, Dibenzo[a,h]pyrene, Ethylbenzene, Indeno[l,2,3-cd]pyrene, and Toluene.
• Curing methods allow for the slow oxidation and degradation of carotenoids in the tobacco leaf. This produces various compounds in the tobacco leaves that give cured tobacco its flavor profile that contributes to the organoleptic properties of the end product consumed by adult tobacco consumers. Curing methods vary with the type of tobacco, but generally include air-curing, fire-curing, and flue-curing. The following are meant to be representative examples of curing methods and are not meant to limit the methods described herein for reducing TSNAs and HPHCs [0029] Air-cured tobaccos include Burley, Maryland, and dark tobaccos. The common factor is that air-curing is primarily without artificial sources of heat and humidity. Burley tobaccos are light to dark brown in color, high in oil, and low in sugar.
• Burley tobaccos are air- cured in barns.
• Major Burley tobacco growing countries include Argentina, Brazil, Italy, Malawi, and the United States of America.
• Maryland tobaccos are extremely fluffy, have good burning properties, low nicotine, and a neutral aroma.
• Major Maryland tobacco growing countries include the United States of America and Italy.
• tobacco plants or seeds provided herein are in a Burley tobacco background selected from the group consisting of Clay 402, Clay 403, Clay 502, Ky 14, Ky 907, Ky 910, Ky 8959, NC 2, NC 3, NC 4, NC 5, NC 2000, TN 86, TN 90, TN 97, R 610, R 630, R 711, R 712, NCBH 129, HB4488PLC, PD 7319LC, Bu 21 xKy 10, HB04P, Ky 14> ⁇ L 8, Kt 200, Newton 98, Pedigo 561, Pf561 and Va 509.
• a Burley tobacco background selected from the group consisting of Clay 402, Clay 403, Clay 502, Ky 14, Ky 907, Ky 910, Ky 8959, NC 2, NC 3, NC 4, NC 5, NC 2000, TN 86, TN 90, TN 97, R 610, R 630, R 711, R 712, NCBH 129, HB4488PLC, PD 7319LC, Bu 21 xKy 10, HB04
• modified or non-modified tobacco plants, seeds, hybrids, varieties, or lines provided herein are in any Burley background selected from the group consisting of TN 90, KT 209, KT 206, KT212, and HB 4488.
• modified or non-modified tobacco plants or seeds provided herein are in a Maryland tobacco background selected from the group consisting of Md 10, Md 40, Md 201, Md 609, Md 872 and Md 341.
• Dark air-cured tobaccos are distinguished from other types primarily by its curing process which gives dark air-cured tobacco its medium- to dark-brown color and distinct aroma. Dark air-cured tobaccos are mainly used in the production of smokeless tobacco products including chewing tobacco and snuff.
• modified or non-modified tobacco plants or seeds provided herein are in a dark air-cured tobacco background selected from the group consisting of Sumatra, Jatim, Dominican Cubano, Besuki, One sucker, Green River, Virginia sun-cured, and Paraguan Passado.
• Dark fire-cured tobaccos are generally cured with low-burning wood fires on the floors of closed curing bams. Dark fire-cured tobaccos are used for making pipe blends, cigarettes, chewing tobacco, snuff and strong-tasting cigars. Major growing regions for dark fire-cured tobaccos are Tennessee, Kentucky, and Virginia.
• tobacco plants or seeds provided herein are in a dark fire-cured tobacco background selected from the group consisting of Narrow Leaf Madole, Improved Madole, Tom Rosson Madole, Newton's VH Madole, Little Crittenden, Green Wood, Little Wood, Small Stalk Black Mammoth, DT 508, DT 518, DT 592, KY 171, DF 911, DF 485, TN D94, TN D950, VA 309, and VA 359.
• a dark fire-cured tobacco background selected from the group consisting of Narrow Leaf Madole, Improved Madole, Tom Rosson Madole, Newton's VH Madole, Little Crittenden, Green Wood, Little Wood, Small Stalk Black Mammoth, DT 508, DT 518, DT 592, KY 171, DF 911, DF 485, TN D94, TN D950, VA 309, and VA 359.
• a practice for harvesting dark tobacco is to cut the plants late in the afternoon and allow them to wilt on the ground overnight before spiking.
• “spiking” is a term of art that refers to hanging tobacco to be cured upside down on sticks designed for placement in a curing barn. This practice is used to avoid sunburn, which occurs when dark tobacco is exposed to high sunlight intensity during the hot period of the day and results in an undesirable crude green color in the cured leaf.
• Sufficient wilting is important to minimize leaf breakage and to facilitate handling of the plants between spiking and housing; sufficiently wilted tobacco also is less likely to sweat and house burn, and will yellow and cure better. Sufficient wilting before housing also reduces the moisture that is brought into the barn, which ultimately restricts the growth of nitrate-reducing microorganisms.
• Growers begin housing dark tobacco (e.g., air-curing or fire-curing) when it is as far along in the yellowing phase as possible. Delaying the curing process to wait for tobacco to finish yellowing, however, can result in an increase in the nitrate-reducing microorganisms, yet curing the tobacco before yellowing is completed can cause “bluing” of the tobacco, which results in an undesirable color. In addition, improperly curing dark tobacco can result in “green” tobacco. While several pre-harvesting factors can lead to “green” tobacco, critical factors occur in the barn during curing. For example, if not managed correctly, relative humidity, temperature, and/or airflow, all of which affect the rate of leaf drying, can lead to “green” tobacco and also can affect TSNA levels.
• dark tobacco e.g., air-curing or fire-curing
• certain management practices can be used during the curing process to lower or reduce TSNAs in tobacco.
• growers can increase the temperature (e.g., starting the first fire) within 48 hours, within 36 hours, within 24, within 18 hours, within 12 hours, within 10 hours, within 8 hours, within 6 hours, within 5 hours, within 4 hours, within 3 hours, within 2 hours, within 1 hour, or immediately after housing the tobacco.
• growers can reduce the relative humidity in the barn to 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, or 45% or less.
• growers can reduce the relative water activity (a w .) in the plants to, for example, 0.90 or less (e.g., 0.89, 0.88, 0.87, 0.86, 0.85, 0.84, 0.83, 0.82, 0.81, 0.80,
• conventional curing methods As used herein “conventional curing methods,” “conventional air-curing methods,” and “conventional fire-curing methods” refer to curing methods and practices as described in the 2017-2018 Burley and Dark Tobacco Production Guide (B. Pearce ed., (2017)). Unless otherwise stated, conventional methods are used as controls for comparison to dislcosed curing conditions or methods.
• curing season refers to the time period during which tobacco is cured after the growing season.
• the growing season is typically between June and August with the curing season immediately following typically including the months of September through November.
• the duration of air curing is more variable than fire curing due to temperature and moisture fluctuations from year to year.
• Leaf quality of air cured tobacco is influenced by moisture and temperature conditions inside the curing facility during the curing period. Control of the curing process is affected mainly by spacing of the tobacco in the curing facility and management of the drying rate. The drying rate is controlled primarily by operating the ventilators, plastic covering, or other air control means to regulate the ventilation rates.
• a “smoke finish attribute” refers to any aroma, flavor, or chemical characteristic derived from a conventional fire curing process.
• a smoke finish attribute refers to an increase in polyaromatic hydrocarbons in a fire-cured leaf compared to tobacco leaf cured by other curing methods.
• a smoke finish attribute refers to an increase in phenolic acid compounds in a fire-cured leaf compared to tobacco leaf cured by other curing methods.
• a smoke finish attribute refers to an increase in HPHCs in a fire-cured leaf compared to tobacco leaf cured by other curing methods.
• a smoke finish attribute refers to an increase in NNN in a fire-cured leaf compared to tobacco leaf cured by other curing methods. In a further aspect, a smoke finish attribute refers to an increase in NNK in a fire-cured leaf compared to tobacco leaf cured by other curing methods. In a further aspect, a smoke finish attribute refers to an increase in NNN, NNK, or both, in a fire-cured leaf compared to tobacco leaf cured by other curing methods.
• flavor and aroma attributes refer to the concentration of phenols present in a tobacco leaf after curing
• the phenol concentration in a cured tobacco leaf is between 400 and 4000 parts per million (ppm), between 450 and 3500 ppm, between 500 and 3000 ppm, between 750 and 2500 ppm, or between 1000 and 2000 ppm.
• the phenol concentration in a cured tobacco leaf is at least 400 ppm, at least 450 ppm, at least 500 ppm, at least 750 ppm, at least 1000 ppm, at least 2000 ppm, at least 2500 ppm, at least 3000 ppm, at least 3500 ppm, at least 4000 ppm, or more.
• the phenol concentration in a cured tobacco leaf is between 400 and 4000 ppm.
• the phenol concentration in a cured tobacco leaf is between 450 and 3500 ppm.
• the phenol concentration in a cured tobacco leaf is between 500 and 3000 ppm.
• the phenol concentration in a cured tobacco leaf is between 750 and 2500 ppm. In a further aspect, the phenol concentration in a cured tobacco leaf is between 1000 and 2000 ppm. In a further aspect, the phenol concentration in a cured tobacco leaf is at least 400 ppm In a further aspect, the phenol concentration in a cured tobacco leaf is at least 450 ppm In a further aspect, the phenol concentration in a cured tobacco leaf is at least 500 ppm In a further aspect, the phenol concentration in a cured tobacco leaf is at least 750 ppm In a further aspect, the phenol concentration in a cured tobacco leaf is at least 1000 ppm In a further aspect, the phenol concentration in a cured tobacco leaf is at least 2000 ppm In a further aspect, the phenol concentration in a cured tobacco leaf is at least 2500 ppm In a further aspect, the phenol concentration in a cured tobacco leaf is at least 3000 ppm In a further aspect,
• a smoldering fire refers to a slow, low-temperature, flameless form of combustion. Smoldering occurs on the surface rather than in the gas phase for a flaming fire. Smoldering fire temperatures typically less than half that of flaming combustion. Smoldering fires are created by covering few hardwood slabs with about 12 to about 18 inches of hardwood sawdust. The moisture content of the hardwood sawdust will impact the burn rate and temperature of the combustion. Reducing the layer and amount of hardwood slabs and thoroughly covering the slabs with sufficient sawdust better enables the maintenance and control of the smoldering fire.
• smoldering smoke chemistry refers to a chemical fingerprint of a tobacco leaf cured using a smoldering fire.
• the chemical fingerprint of a tobacco leaf cured using a smoldering fire comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 50, at least 75, or at least 100 identifiable chemical constituents.
• an open flame fire refers to a fire where a flame is present.
• An open flame fire is faster burning and has a higher temperature of combustion compared to a smoldering fire.
• Open flame fire combustion occurs in the gas phase for a flaming fire but on the surface of the solid fuel for smoldering fire.
• Open flame fires can be created using more hardwood slabs that can be stacked and layered in the barn floor with little or no hardwood sawdust covering. The moisture content of the hardwood sawdust will impact the burn rate and temperature of the combustion. Increasing the layer and amount of hardwood slabs, lightly covering the slabs with hardwood sawdust or removing the hardwood sawdust completely better enables the combustion to burn with an open flame.
• flame combustion chemistry refers to a chemical fingerprint of a tobacco leaf cured using an open flame fire.
• the chemical fingerprint of a tobacco leaf cured using an open flame fire comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 50, at least 75, or at least 100 identifiable chemical constituents.
• air/fire combination cured refers to a tobacco leaf cured using conventional air curing techniques until the finishing stage when the tobacco leaf is subjected to a controlled smoke process in order to apply smoke finish attributes.
• a controlled smoke process is used to apply smoke finish attributes before the finishing stage of a curing process.
• a controlled smoke process is used to apply smoke finish attributes during the finishing stage of a curing process.
• a controlled smoke process is used to apply smoke finish attributes after the finishing stage of a curing process.
• PAH polyaromatic hydrocarbons
• Most PAH compounds have boiling point temperatures well in excess of the smoldering temperatures achieved in a commercial wood smoke generator.
• Most phenols have boiling points between 200°C to 300°C (392°F to 572°F) and escape the water filter as gases. Phenols are more typically associated with smoke and flavor characteristics of the fire-curing process.
• a tobacco plant is of a tobacco variety selected from the group consisting of a flue cured variety, a bright variety, a Burley variety, a Virginia variety, a Maryland variety, a dark variety, a Galpao variety, an Oriental variety, and a Vietnamese variety.
• a modified tobacco plant provided herein is of a tobacco variety selected from the group consisting of a flue cured variety, a bright variety, a Burley variety, a Virginia variety, a Maryland variety, a dark variety, a Galpao variety, an Oriental variety, and a Turkish variety.
• a tobacco cell is of a tobacco variety selected from the group consisting of a flue cured variety, a bright variety, a Burley variety, a Virginia variety, a Maryland variety, a dark variety, a Galpao variety, an Oriental variety, and a Vietnamese variety.
• a modified tobacco cell is of a tobacco variety selected from the group consisting of a flue cured variety, a bright variety, a Burley variety, a Virginia variety, a Maryland variety, a dark variety, a Galpao variety, an Oriental variety, and a Turkish variety.
• a tobacco leaf is of a tobacco variety selected from the group consisting of a flue cured variety, a bright variety, a Burley variety, a Virginia variety, a Maryland variety, a dark variety, a Galpao variety, an Oriental variety, and a Vietnamese variety.
• a cured tobacco leaf or plant part is of a tobacco variety selected from the group consisting of a flue cured variety, a bright variety, a Burley variety, a Virginia variety, a Maryland variety, a dark variety, a Galpao variety, an Oriental variety, and a Turkish variety. Skilled artisans further understand that cured tobacco does not constitute a living organism and is not capable of growth or reproduction
• Flue-cured tobaccos (also called “Virginia” or “bright” tobaccos) amount to approximately 40% of world tobacco production. Flue-cured tobaccos are often also referred to as “bright tobacco” because of the golden-yellow to deep-orange color it reaches during curing. Flue- cured tobaccos have a light, bright aroma and taste. Flue-cured tobaccos are generally high in sugar and low in oils. Major flue-cured tobacco growing countries are Argentina, Brazil, China, India, Africa and the United States of America.
• tobacco plants or seeds or modified tobacco plants or seeds provided herein are of a flue-cured tobacco variety selected from the group consisting of the varieties listed in Table 2, and any variety essentially derived from any one of the foregoing varieties. See WO 2004/041006 Al.
• modified tobacco plants or seeds provided herein are in a flue-cured variety selected from the group consisting of K326, K346, and NCI 96.
• Air-cured tobaccos include “Burley,” “Maryland,” and “dark” tobaccos.
• the common factor linking air-cured tobaccos is that curing occurs primarily without artificial sources of heat and humidity.
• Burley tobaccos are light to dark brown in color, high in oil, and low in sugar. Burley tobaccos are typically air-cured in barns.
• Major Burley growing countries include Argentina, Brazil, Italy, Malawi, and the United States of America.
• Maryland tobaccos are extremely fluffy, have good burning properties, low nicotine and a neutral aroma.
• Major Maryland growing countries include the United States of America and Italy.
• tobacco plants or seeds or modified tobacco plants or seeds provided herein are of a Burley tobacco variety selected from the group consisting of the tobacco varieties listed in Table 3, and any variety essentially derived from any one of the foregoing varieties..
• modified tobacco plants or seeds provided herein are in a Burley variety selected from the group consisting of TN 90, KT 209, KT 206, KT212, and HB 4488.
• tobacco plants or seeds or modified tobacco plants or seeds provided herein are of a Maryland tobacco variety selected from the group consisting of the tobacco varieties listed in Table 4, and any variety essentially derived from any one of the foregoing varieties. Table 4. Maryland Tobacco Varieties
• Dark air-cured tobaccos are distinguished from other tobacco types primarily by its curing process, which gives dark air-cured tobacco its medium-brown to dark-brown color and a distinct aroma. Dark air-cured tobaccos are mainly used in the production of chewing tobacco and snuff.
• modified tobacco plants or seeds provided herein are of a dark air-cured tobacco variety selected from the group consisting of Sumatra, Jatim, Dominican Cubano, Besuki, One sucker, Green River, Virginia sun-cured, and Paraguan Passado, and any variety essentially derived from any one of the foregoing varieties..
• Dark fire-cured tobaccos are generally cured with low-burning wood fires on the floors of closed curing barns. Dark fire-cured tobaccos are typically used for making pipe blends, cigarettes, chewing tobacco, snuff, and strong-tasting cigars. Major growing regions for dark fire- cured tobaccos are Tennessee, Kentucky, and Virginia in the United States of America.
• tobacco plants or seeds or modified tobacco plants or seeds provided herein are of a dark fire-cured tobacco variety selected from the group consisting of the tobacco varieties listed in Table 5, and any variety essentially derived from any one of the foregoing varieties.
• Oriental tobaccos are also referred to as Greek, aroma and Turkish tobaccos due to the fact that they are typically grown in eastern Mediterranean regions such as Turkey, Greece, Bulgaria, Ardia, Iran, Lebanon, Italy, and Bulgaria.
• the small plant size, small leaf size, and unique aroma properties of Oriental tobacco varieties are a result of their adaptation to the poor soil and stressful climatic conditions in which they have been developed.
• tobacco plants or seeds or modified tobacco plants or seeds provided herein are of an Oriental tobacco variety selected from the group consisting of the tobacco varieties listed in Table 6, and any variety essentially derived from any one of the foregoing varieties. Table 6.
• tobacco plants or seeds or modified tobacco plants or seeds provided herein are of an cigar tobacco variety selected from the group consisting of the tobacco varieties listed in Table 7, and any variety essentially derived from any one of the foregoing varieties. Table 7. Cigar Tobacco Varieties
• tobacco plants or seeds or modified tobacco plants or seeds provided herein are of a tobacco variety selected from the group consisting of the tobacco varieties listed in Table 8, and any variety essentially derived from any one of the foregoing varieties.
• a tobacco plant, seed, or cell is from a variety selected from the group consisting of the tobacco varieties listed in Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, and Table 8.
• a modified or non-modified plant, seed, plant component, plant cell, or plant genome provided herein comprises a recombinant DNA construct provided herein.
• cured tobacco material or tobacco products provided herein comprise modified or non-modified tobacco plants, plant components, plant cells, or plant genomes provided herein.
• modified refers to plants, seeds, plant components, plant cells, and plant genomes that have been subjected to mutagenesis, genome editing, genetic transformation, or a combination thereof.
• a plant, seed, plant component, plant cell, or plant genome provided herein is a modified plant, modified seed, modified plant component, modified plant part, modified plant cell, or modified plant genome.
• a plant provided herein is a modified plant.
• a seed provided herein is a modified seed
• a plant component provided herein is a modified plant component.
• a plant cell provided herein is a modified plant cell.
• a modified plant, seed, plant component, plant cell, or plant genome provided herein comprises one or more transgenes.
• a transgene refers to a polynucleotide that has been transferred into a genome by any method known in the art.
• a transgene is an exogenous polynucleotide.
• a transgene is an endogenous polynucleotide that is integrated into a new genomic locus where it is not normally found.
• a modified plant, seed, plant component, plant cell, or plant genome provided herein comprises one or more cisgenes.
• cisgenesis or “cisgenic” refers to genetic modification of a plant, plant cell, or plant genome in which all components (e.g promoter, donor nucleic acid, selection gene) have only plant origins (/.£ ⁇ ., no non-plant origin components are used).
• a modified plant, plant cell, or plant genome provided herein is cisgenic.
• a modified tobacco plant provided herein comprises no non-tobacco genetic material or sequences.
• “comparable conditions” refers to similar environmental conditions, agronomic practices, and/or curing process for growing or curing tobacco and making meaningful comparisons between two or more plant genotypes so that neither environmental conditions nor agronomic practices (including curing process) would contribute to, or explain, any differences observed between the two or more plant genotypes.
• environmental conditions include, for example, light, temperature, water, humidity, and nutrition ( e.g ., nitrogen and phosphorus).
• agronomic practices include, for example, seeding, clipping, undercutting, transplanting, topping, suckering, and curing. See Chapters 4B and 4C of Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford (1999), pp. 70-103.
• a “reduced” or “increased” level refers to a statistically significant change (reduction or increase) from a reference point.
• “statistically significant” refers to a p-value of less than 0.05, a p-value of less than 0.025, a p-value of less than 0.01, or a p-value of less than 0.001 when using an appropriate measure of statistical significance (e.g., a one-tailed two sample t-test).
• similar or “statistically similar” refers to a p- value of greater than 0.05.
• control plant refers to a comparator plant that is an unmodified tobacco plant of the same variety or a tobacco plant having no transgene of interest, depending on the context or the purpose of the control plant.
• a control tobacco plant can be a tobacco plant cured using a curing practice standard in the field. Control tobacco plants and plants of interest are grown under comparable conditions.
• measurements of the level of total TSNAs, individual TSNA, total or individual alkaloid, or leaf grade index values mentioned herein for a cured leaf from a tobacco plant, variety, cultivar, or line refer to average measurements, including, for example, an average of multiple leaves (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more leaves) of a single plant or an average measurement from a population of tobacco plants from a single variety, cultivar, or line.
• a population of tobacco plants or a collection of tobacco leaves for determining an average measurement can be of any size, for example, 5, 10, 15, 20, 25, 30, 35, 40, or 50. Industry-accepted standard protocols are followed for determining average measurements or grade index values.
• a plant component provided herein includes, but is not limited to, a leaf, a stem, a root, a seed, a flower, pollen, an anther, an ovule, a pedicel, a fruit, a meristem, a cotyledon, a hypocotyl, a pod, an embryo, endosperm, an explant, a callus, a tissue culture, a shoot, a cell, and a protoplast.
• this specification provides tobacco plant cells, tissues, and organs that are not reproductive material and do not mediate the natural reproduction of the plant.
• this specification also provides tobacco plant cells, tissues, and organs that are reproductive material and mediate the natural reproduction of the plant.
• this specification provides tobacco plant cells, tissues, and organs that cannot maintain themselves via photosynthesis.
• this specification provides somatic tobacco plant cells. Somatic cells, contrary to germline cells, do not mediate plant reproduction.
• this specification provides tobacco endosperm. In another aspect, this specification provides a tobacco endosperm cell. In a further aspect, this specification provides a male or female sterile tobacco plant, which cannot reproduce without human intervention.
• a modified plant, seed, plant part, or plant cell provided herein comprises one or more non-naturally occurring mutations.
• a mutation provided herein suppresses TSNA levels in a cured leaf from a tobacco plant.
• Types of mutations provided herein include, for example, substitutions (point mutations), deletions, insertions, duplications, and inversions. Such mutations are desirably present in the coding region of a gene; however, mutations in a promoter or other regulatory region, an intron, an intron-exon boundary, or an untranslated region of a gene may also be desirable.
• a modified tobacco plant provided herein further comprises one or more mutations in one or more loci encoding a nicotine demethylase (e.g CYP82E4 , CYP82E5, CYP82E10) that confer reduced amounts of nomicotine ( See U.S. Patent Nos. 8,319,011; 8,124,851; 9,187,759; 9,228,194; 9,228,195; 9,247,706) compared to control plant lacking one or more mutations in one or more loci encoding a nicotine demethylase.
• a nicotine demethylase e.g CYP82E4 , CYP82E5, CYP82E10
• a tobacco plant provided herein further comprises one or more mutations in a Nicl locus, aNic2 locus, or both, which confer reduced amounts of nicotine compared to a control plant lacking one or more mutations in a Nicl locus, aNic2 locus, or both (See U.S. Patent Application Publication number US2016/0374387).
• tobacco plants provided herein comprise one or more non-naturally existing mutant alleles at Nicl or Nic2 locus which reduce or eliminate one or more gene activity from Nicl or Nic2 locus. In an aspect, these mutant alleles result in lower nicotine levels (See U.S. Publication number US2016/0374387).
• a tobacco plant provided herein further comprises a mutation in a Nicl Deletion Gene 1 (Ndgl) or Nicl Deletion Gene 1 (Ndg2) or both (See U.S. Patent Application Publication number US2016/0374387).
• Tobacco material obtained from tobacco lines, varieties or hybrids of the present specification can be used to make tobacco products.
• tobacco product is defined as any product made or derived from tobacco that is intended for human use or consumption.
• a tobacco product provided herein comprises cured components from a tobacco plant provided herein.
• a tobacco product provided herein comprises cured tobacco leaves from a tobacco plant provided herein.
• Tobacco products provided herein include, without limitation, cigarette products (e.g ., cigarettes, bidi cigarettes, kreteks), cigar products (e.g., cigars, cigar wrapping tobacco, cigarillos), pipe tobacco products, products derived from tobacco, tobacco-derived nicotine products, smokeless tobacco products (e.g., moist snuff, dry snuff, snus, chewing tobacco, moist smokeless tobacco, fine cut chewing tobacco, long cut chewing tobacco, pouched chewing tobacco), films, chewables (e.g., gum), lozenges, dissolving strips, tabs, tablets, shaped parts, gels, consumable units, insoluble matrices, hollow shapes, reconstituted tobacco, expanded tobacco, and the like. See, for example, U.S. Patent Publication No. US 2006/0191548.
• dark air cured, dark fire cured, and dark air-fire combination cured tobacco provided for and included in the current specification may be used in smokeless tobacco products.
• cigarette refers a tobacco product having a “rod” and “filler”.
• the cigarette “rod” includes the cigarette paper, filter, plug wrap (used to contain filtration materials), tipping paper that holds the cigarette paper (including the filler) to the filter, and all glues that hold these components together.
• the “filler” includes (1) all tobaccos, including but not limited to reconstituted and expanded tobacco, (2) non-tobacco substitutes (including but not limited to herbs, non-tobacco plant materials and other spices that may accompany tobaccos rolled within the cigarette paper), (3) casings, (4) flavorings, and (5) all other additives (that are mixed into tobaccos and substitutes and rolled into the cigarette).
• a method provided herein comprises preparing a tobacco product using a cured tobacco leaf from a tobacco plant provided herein. In a further aspect, a method provided herein comprises preparing a tobacco product using a cured tobacco leaf from a modified or non- modified tobacco plant provided herein.
• Tobacco products derived from plants of the present specification also include cigarettes and other smoking articles, particularly those smoking articles including filter elements, where the rod of smokeable material includes cured tobacco within a tobacco blend.
• a tobacco product of the present specification is selected from the group consisting of a cigarillo, a non-ventilated recess filter cigarette, a vented recess filter cigarette, a bidi cigarette, a cigar, snuff, pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, hookah tobacco, shredded tobacco, and cut tobacco.
• a tobacco product of the present specification is a smokeless tobacco product.
• Smokeless tobacco products are not combusted and include, but not limited to, chewing tobacco, moist smokeless tobacco, snus, and dry snuff.
• Chewing tobacco is coarsely divided tobacco leaf that is typically packaged in a large pouch-like package and used in a plug or twist.
• Moist smokeless tobacco is a moist, more finely divided tobacco that is provided in loose form or in pouch form and is typically packaged in round cans and used as a pinch or in a pouch placed between an adult tobacco consumer’s cheek and gum.
• Snus is a heat treated smokeless tobacco.
• Dry snuff is finely ground tobacco that is placed in the mouth or used nasally.
• a tobacco product of the present specification is selected from the group consisting of loose leaf chewing tobacco, plug chewing tobacco, moist snuff, and nasal snuff.
• a tobacco product of the present specification is selected from the group consisting of an electronically heated cigarette, an e- cigarette, an electronic vaporing device.
• the present specification further provides a method for manufacturing a tobacco product comprising tobacco material from tobacco plants provided herein.
• methods provided herein comprise conditioning aged tobacco material made from tobacco plants provided herein to increase its moisture content from between about 12.5% and about 13.5% to about 21%, blending the conditioned tobacco material to produce a desirable blend.
• the method of manufacturing a tobacco product provided herein further comprises casing or flavoring the blend. Generally, during the casing process, casing or sauce materials are added to blends to enhance their quality by balancing the chemical composition and to develop certain desired flavor characteristics. Further details for the casing process can be found in Tobacco Production, Chemistry and Technology, Edited by L. Davis and M. Nielsen, Blackwell Science, 1999.
• Tobacco material provided herein can be also processed using methods including, but not limited to, heat treatment (e.g ., cooking, toasting), flavoring, enzyme treatment, expansion and/or curing. Both fermented and non-fermented tobaccos can be processed using these techniques. Examples of suitable processed tobaccos include dark air-cured, dark fire cured, burley, flue cured, and cigar filler or wrapper, as well as the products from the whole leaf stemming operation. In one aspect, tobacco fibers include up to 70% dark tobacco on a fresh weight basis. For example, tobacco can be conditioned by heating, sweating and/or pasteurizing steps as described in U S. Publication Nos. 2004/0118422 or 2005/0178398.
• Tobacco material provided herein can be subject to fermentation. Fermenting typically is characterized by high initial moisture content, heat generation, and a 10 to 20% loss of dry weight. See, e.g, U.S. Patent Nos. 4,528,993; 4,660,577; 4,848,373; and 5,372,149.
• fermentation can change either or both the color and texture of a leaf.
• evolution gases can be produced, oxygen can be taken up, the pH can change, and the amount of water retained can change. See, for example, U.S. Publication No.
• Cured, or cured and fermented tobacco can be further processed (e.g., cut, expanded, blended, milled or comminuted) prior to incorporation into the oral product.
• the tobacco in some cases, is long cut fermented cured moist tobacco having an oven volatiles content of between 48 and 50 weight percent prior to mixing with a copolymer and, optionally, flavorants and other additives.
• tobacco material provided herein can be processed to a desired size.
• tobacco fibers can be processed to have an average fiber size of less than 200 micrometers. In one aspect, tobacco fibers are between 75 and 125 micrometers. In another aspect, tobacco fibers are processed to have a size of 75 micrometers or less. In one aspect, tobacco fibers include long cut tobacco, which can be cut or shredded into widths of about 10 cuts/inch up to about 110 cuts/inch and lengths of about 2.54 mm (0.1 inches) up to about 25.4 mm (1 inch). Double cut tobacco fibers can have a range of particle sizes such that about 70% of the double cut tobacco fibers falls between the mesh sizes of -20 mesh and 80 mesh.
• oven volatiles are determined by calculating the percentage of weight loss for a sample after drying the sample in a pre- warmed forced draft oven at 110°C for 3.25 hours.
• An oral product can have a different overall oven volatiles content than the oven volatiles content of the tobacco fibers used to make the oral product. The processing steps described herein can reduce or increase the oven volatiles content.
• Tobacco material provided herein can be processed to have a total oven volatiles content of about 10% by weight or greater; about 20% by weight or greater; about 40% by weight or greater; about 15% by weight to about 25% by weight; about 20% by weight to about 30% by weight; about 30% by weight to about 50% by weight; about 45% by weight to about 65% by weight; or about 50% by weight to about 60% by weight.
• Those of skill in the art will appreciate that “moist” tobacco typically refers to tobacco that has an oven volatiles content of between about 40% by weight and about 60% by weight (e.g., about 45% by weight to about 55% by weight, or about 50% by weight).
• the present specification provides for, and includes, a method for curing tobacco leaves comprising subjecting a tobacco leaf to air curing and applying one or more smoke finish attributes to the air cured leaf to generate an air/fire combination cured tobacco leaf.
• air curing is for a duration of at least 30 days.
• air curing is for a duration of between 5 and 150 days, of between 10 and 150 days, of between 20 and 150 days, of between 30 and 150 days, of between 40 and 150 days, of between 50 and 150 days, of between 60 and 150 days, of between 70 and 150 days, of between 80 and 150 days, of between 90 and 150 days, of between 100 and 150 days, of between 110 and 150 days, of between 120 and 150 days, of between 130 and 150 days, or of between 140 and 150 days.
• air curing is for a duration of between 20 to 60 days, of between 40 to 80 days, of between 60 to 100 days, of between 80 to 120 days, or of between 100 to 140 days.
• air curing is a for a duration of between 30 to 60 days, of between 40 to 70 days, of between 50 to 80 days, of between 60 to 90 days, of between 70 to 100 days, of between 80 to 110 days, of between 90 to 120 days, of between 100 to 130 days, of between 110 to 140 days, or of between 120 to 150 days.
• air curing is for a duration of at least 5 days, at least 10 days, at least 20 days, at least 30 days, at least 40 days, at least 50 days, at least 60 days, at least 70 days, at least 80 days, at least 90 days, at least 100 days, at least 110 days, at least 120 days, at least 130 days, at least 140 days, or at least 150 days.
• the present specification provides for, and includes, an air/fire combination curing method wherein one or more smoke finish attributes are applied.
• one or more smoke finish attributes are flavor or aroma attributes.
• a flavor attribute is selected from the group consisting of constituents derived from the pyrolysis of lignin, Syringol and related compounds; constituents derived from the pyrolysis of cellulose, maltol, furfural, and various cyclopentenolones.
• an aroma attribute is selected from the group consisting of constituents derived from the pyrolysis of lignin, Syringol and related compounds; constituents derived from the pyrolysis of cellulose, maltol, furfural, and various cyclopentenolones.
• the present specification provides for, and includes, an air/fire combination curing method wherein a tobacco leaf cured using this method comprises a similar amount of phenolic compounds compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• an air/fire combination curing method provides a tobacco leaf that comprises a similar amount of Catechol, Cresol, Phenol, or a combination thereof, compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• an air/fire combination curing method provides a tobacco leaf that comprises a similar amount of Catechol, Phenol, or a combination thereof, compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• an air/fire combination curing method provides a tobacco leaf that comprises a similar amount of Catechol, Cresol, or a combination thereof, compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• an air/fire combination curing method provides a tobacco leaf that comprises a similar amount of Catechol compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• an air/fire combination curing method provides a tobacco leaf that comprises a similar amount of Cresol compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process. In a further aspect, an air/fire combination curing method provides a tobacco leaf that comprises a similar amount of Phenol compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• the present specification provides for, and includes, an air/fire combination curing method wherein a tobacco leaf cured using this method comprises a significant reduction of at least one harmful and potentially harmful constituent (HPHC) compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• HPHC harmful and potentially harmful constituent
• an air/fire combination curing method provides a tobacco leaf that comprises a significant reduction of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen, at least twenty, at least twenty-one, at least twenty -two, at least twenty -three, at least twenty -four, or at least twenty-five HPHCs compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire curing process.
• an air/fire combination curing method provides a tobacco leaf that comprises a significant reduction in Acetaldehyde, Acetamide, Acetone, Acrolein, Acrylamide, Acrylonitrile, Aflatoxin Bl, 4-Aminobiphenyl, 1 -Aminonaphthalene, 2-Aminonaphthalene, Ammonia, Anabasine, o-Anisidine, Arsenic, A-a-C (2-Amino-9H-pyrido[2,3-b]indole), Benz[a]anthracene, Benz[j]aceanthrylene, Benzene, Benzo[b] fluoranthene, Benzo[k]fluoranthene, Benzo[b]furan, Benzo[a]pyrene, Benzo[c]phenanthrene, Beryllium, 1,3- Butadiene, Cadmium, Caffeic acid, Carbon monoxide, Catechol, Chlorinated dioxin Bl
• an HPHC is selected from the group consisting of Acetaldehyde, Acrolein, Arsenic, Benz[a]anthracene, Benzene, Benzo[a]pyrene, Cadmium, Catechol, Chlorinated dioxins, Chlorinated furans, Chromium, Chrysene, Cresols, Crotonaldehyde, Dibenz[a,h]anthracene, Dibenzo[a,e]pyrene, Dibenzo[a,h]pyrene, Ethylbenzene, Formaldehyde, Indeno[l,2,3-cd]pyrene, Lead, Nickle, Phenol, Propionaldehyde, Selenium, Toluene, and a combination thereof, as compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• an air/fire combination curing method provides a tobacco leaf that comprises a significant reduction of Acetaldehyde, Acrolein, Arsenic, Benz[a]anthracene, Benzene, Benzo[a]pyrene, Cadmium, Catechol, Chlorinated dioxins, Chlorinated furans, Chromium, Chrysene, Cresols, Crotonaldehyde, Dibenz[a,h]anthracene, Dibenzo[a,e]pyrene, Dibenzo[a,h]pyrene, Ethylbenzene, Formaldehyde, Indeno[l,2,3-cd]pyrene, Lead, Nickle, Phenol, Propionaldehyde, Selenium, and Toluene, or a combination thereof, as compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• the present specification provides for, and includes, an air/fire combination curing method wherein a tobacco leaf cured using this method comprises an amount of at least one HPHC that is reduced by at least 25 % as compared to a tobacco leaf grown under similar growth conditions and cured under a conventional fire-curing process.
• the present specification provides for, and includes, an air/fire combination curing method that provides a tobacco leaf comprising an amount of at least one HPHC that is reduced by between 1% and 100%, between 1% and 90%, between 1% and 80%, between 1% and 70%, between 1% and 60%, between 1% and 50%, between 1% and 45%, between 1% and 40%, between 1% and 35%, between 1% and 30%, between 1% and 25%, between 1% and 20%, between 1% and 15%, between 1% and 10%, or between 1% and 5% compared to a tobacco leaf grown under similar growth conditions and cured under a conventional fire-curing process.
• the present specification provides for, and includes, an air/fire combination curing method that provides a tobacco leaf comprising an amount of at least one HPHC that is reduced by at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% compared to a tobacco leaf grown under similar growth conditions and cured under a conventional fire-curing process.
• the present specification provides for, and includes, an air/fire combination curing method wherein a tobacco leaf cured using this method comprises an amount of at least one HPHC that is reduced by at least 0.05 parts per million as compared to a tobacco leaf grown under similar growth conditions and cured under a conventional fire-curing process.
• the present specification provides for, and includes, an air/fire combination curing method that provides a tobacco leaf comprising an amount of at least one HPHC that is reduced by between 0.001 and 1000 parts per million, between 0.005 and 1000 parts per million, between 0.01 and 1000 parts per million, between 0.05 and 1000 parts per million, between 0.1 and 1000 parts per million, between 0.5 and 1000 parts per million, between 1.0 and 1000 parts per million, between 1.0 and 100 parts per million, between 1.0 and 10 parts per million between 2.0 and 1000 parts per million, between 3.0 and 1000 parts per million, between 4.0 and 1000 parts per million, between 5.0 and 1000 parts per million, between 10 and 1000 parts per million, between 50 and 1000 parts per million, between 100 and 1000 parts per million, between 200 and 1000 parts per million, between 300 and 1000 parts per million, between 400 and 1000 parts per million, between 500 and 1000 parts per million, between 600 and 1000 parts per million, between 700 and 1000 parts per million, between 800 and 1000 parts per million, or between 900 and 1000 parts per million as compared to a
• the present specification provides for, and includes, an air/fire combination curing method that provides a tobacco leaf comprising an amount of at least one HPHC that is reduced by at least 0.001 parts per million, at least 0.005 parts per million, at least 0.01 parts per million, at least 0.05 parts per million, at least 0.1 parts per million, at least 0.5 parts per million, at least 1.0 part per million, at least 2.0 parts per million, at least 3.0 parts per million, at least 4.0 parts per million, at least 5.0 parts per million, at least 10 parts per million, at least 50 parts per million, at least 100 parts per million, at least 200 parts per million, at least 300 parts per million, at least 400 parts per million, at least 500 parts per million, at least 600 parts per million, at least 700 parts per million, at least 800 parts per million, at least 900 parts per million, or at least 1000 parts per million, as compared to a tobacco leaf grown under similar growth conditions and cured under a conventional fire-curing process.
• the present specification provides for, and includes, an air/fire combination curing method wherein a tobacco leaf cured using this method comprises a reduced amount of N'-nitrosonomicotine (NNN), an amount of 4-methylnitrosoamino-l-(3-pyridyl)-l- butanone (NNK), or both compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• NNN N'-nitrosonomicotine
• NNK 4-methylnitrosoamino-l-(3-pyridyl)-l- butanone
• an air/fire combination curing method provides a tobacco leaf comprising an amount of NNN that is reduced by at least 50% as compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• an air/fire combination curing method provides a tobacco leaf comprising an amount of NNN that is reduced by between 1% and 100%, between 5% and 100%, between 10% and 100%, between 15% and 100%, between 20% and 100%, between 25% and 100%, between 30% and 100%, between 35% and 100%, between 40% and 100%, between 45% and 100%, between 50% and 100%, between 55% and 100%, between 60% and 100%, between 65% and 100%, between 70% and 100%, between 75% and 100%, between 80% and 100%, between 85% and 100%, between 90% and 100%, or between 95% and 100% as compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• an air/fire combination curing method provides a tobacco leaf comprising an amount of NNN that is reduced by at least 5%, at least
• an air/fire combination curing method provides a tobacco leaf comprising an amount of NNN that is reduced by at least 1 part per million as compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• an air/fire combination curing method provides a tobacco leaf comprising an amount of NNN that is reduced by between 0.001 and 10 parts per million, between 0.005 and 10 parts per million, between 0.01 and 10 parts per million, between 0.05 and 10 parts per million, between 0.1 and 10 parts per million, between 0.5 and 10 parts per million, between 1.0 and 10 parts per million, between 2.0 and 10 parts per million, between 3.0 and 10 parts per million, between 4.0 and 10 parts per million, between 5.0 and 10 parts per million, between 6.0 and 10 parts per million, between 7.0 and 10 parts per million, between 8.0 and 10 parts per million, or between 9.0 and 10 parts per million as compared to a tobacco leaf grown under similar growth conditions and cured under a conventional fire-curing process.
• an air/fire combination curing method provides a tobacco leaf comprising an amount of NNN that is reduced by at least 0.001 parts per million, at least 0.005 parts per million, at least 0.01 parts per million, at least 0.05 parts per million, at least 0.1 parts per million, at least 0.5 parts per million, at least 1.0 parts per million, at least 2.0 parts per million, at least 3.0 parts per million, at least 4.0 parts per million, at least 5.0 parts per million, at least 6.0 parts per million, at least 7.0 parts per million, at least 8.0 parts per million, at least 9.0 parts per million, or at least 10 parts per million as compared to a tobacco leaf grown under similar growth conditions and cured under a conventional fire-curing process.
• the present specification provides for, and includes, an air/fire combination curing method that provides a tobacco leaf comprising an amount of NNK that is reduced by at least 50% as compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• an air/fire combination curing method provides a tobacco leaf comprising an amount of NNK that is reduced by between 1% and 100%, between 5% and 100%, between 10% and 100%, between 15% and 100%, between 20% and 100%, between 25% and 100%, between 30% and 100%, between 35% and 100%, between 40% and 100%, between 45% and 100%, between 50% and 100%, between 55% and 100%, between 60% and 100%, between 65% and 100%, between 70% and 100%, between 75% and 100%, between 80% and 100%, between 85% and 100%, between 90% and 100%, or between 95% and 100% as compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• an air/fire combination curing method provides a tobacco leaf comprising an amount of NNK that is reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% as compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• an air/fire combination curing method provides a tobacco leaf comprising an amount of NNK that is reduced by at least 0.2 parts per million as compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• an air/fire combination curing method provides a tobacco leaf comprising an amount of NNK that is reduced by between 0.001 and 5 parts per million, between 0.005 and 5 parts per million, between 0.01 and 5 parts per million, between 0.05 and 5 parts per million, between 0.1 and 5 parts per million, between 0.2 and 5 parts per million, between 0.3 and 5 parts per million, between 0.4 and 5 parts per million, between 0.5 and 5 parts per million, between 0.6 and 5 parts per million, between 0.7 and 5 parts per million, between 0.8 and 5 parts per million, between 0.9 and 5 parts per million, between 1.0 and 5 parts per million, between 1.0 and 10 parts per million, between 2.0 and 5 parts per million, between 3.0 and 5 parts per million, or between 4.0 and 5 parts per million as compared to a tobacco leaf grown under similar growth conditions and cured under a conventional fire-curing process.
• an air/fire combination curing method provides a tobacco leaf comprising an amount of NNK that is reduced by at least 0.001 parts per million, at least 0.005 parts per million, at least 0.01 parts per million, at least 0.05 parts per million, at least 0.1 parts per million, at least 0.3 parts per million, at least 0.4 parts per million, at least 0.5 parts per million, at least 0.6 parts per million, at least 0.7 parts per million, at least 0.8 parts per million, at least 0.9 parts per million at least 1.0 part per million, at least 2.0 parts per million, at least 3.0 parts per million, at least 4.0 parts per million, or at least 5.0 parts per million as compared to a tobacco leaf grown under similar growth conditions and cured under a conventional fire-curing process.
• the present specification provides for, and includes, an air/fire combination curing method wherein one or more smoke finish attributes are applied using a controlled smoke process.
• a controlled smoke process is selected from the group consisting of open flame combustion, smoldering combustion, cold smoke generation, combustion using a wood smoke generator, and any method known in the art that produces smoke without combustion.
• the present specification provides for, and includes, an air/fire combination curing method used to cure a tobacco leaf wherein that leaf does not comprise a transgene.
• a tobacco leaf cured using an air/fire combination curing method is not modified.
• a tobacco leaf cured using an air/fire combination curing method comprises at least one mutation in a nicotine demethylase gene.
• a tobacco leaf cured using an air/fire combination curing method is a modified tobacco leaf.
• a tobacco leaf cured using an air/fire combination curing method is a modified tobacco leaf comprising increased levels of one or more antioxidants as compared to a control leaf of an unmodified tobacco plant of the same variety when grown under comparable conditions.
• the present specification provides for, and includes, an air/fire combination curing method used to cure a tobacco leaf wherein that leaf is a leaf of a dark tobacco plant.
• the present specification provides for, and includes, an air/fire combination curing method used to cure a tobacco leaf wherein that leaf is from a tobacco plant selected from the group consisting of a burley tobacco leaf, an oriental tobacco leaf, or a bright tobacco leaf.
• an air/fire combination cured tobacco leaf comprising one or more smoke finish attributes and wherein the amount of N'-nitrosonomicotine (NNN), the amount of 4-methylnitrosoamino-l-(3-pyridyl)-l- butanone (NNK), or the amount of both are reduced in comparison to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• NNN N'-nitrosonomicotine
• NNK 4-methylnitrosoamino-l-(3-pyridyl)-l- butanone
• an air/fire combination cured tobacco leaf comprises an amount of NNN that is reduced by at least 50% as compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• an air/fire combination cured tobacco leaf comprises an amount of NNN that is reduced by between 1% and 100%, between 5% and 100%, between 10% and 100%, between 15% and 100%, between 20% and 100%, between 25% and 100%, between 30% and 100%, between 35% and 100%, between 40% and 100%, between 45% and 100%, between 50% and 100%, between 55% and 100%, between 60% and 100%, between 65% and 100%, between 70% and 100%, between 75% and 100%, between 80% and 100%, between 85% and 100%, between 90% and 100%, or between 95% and 100% as compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• an air/fire combination cured tobacco leaf comprises an amount of NNN that is reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% as compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• the present specification provides for, and includes, an air/fire combination cured tobacco leaf comprising one or more smoke finish attributes and wherein the amount of NNN is reduced by at least 1 part per million as compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• an air/fire combination cured tobacco leaf comprises an amount of NNN that is reduced by between 0.001 and 10 parts per million, between 0.005 and 10 parts per million, between 0.01 and 10 parts per million, between 0.05 and 10 parts per million, between 0.1 and 10 parts per million, between 0.5 and 10 parts per million, between 1.0 and 10 parts per million, between 2.0 and 10 parts per million, between 3.0 and 10 parts per million, between 4.0 and 10 parts per million, between 5.0 and 10 parts per million, between 6.0 and 10 parts per million, between 7.0 and 10 parts per million, between 8.0 and 10 parts per million, or between 9.0 and 10 parts per million as compared to a tobacco leaf grown under similar growth conditions and cured under a conventional fire-curing process.
• an air/fire combination cured tobacco leaf comprises an amount of NNN that is reduced by at least 0.001 parts per million, at least 0.005 parts per million, at least 0.01 parts per million, at least 0.05 parts per million, at least 0.1 parts per million, at least 0.5 parts per million, at least 1.0 parts per million, at least 2.0 parts per million, at least 3.0 parts per million, at least 4.0 parts per million, at least 5.0 parts per million, at least 6.0 parts per million, at least 7.0 parts per million, at least 8.0 parts per million, at least 9.0 parts per million, or at least 10 parts per million as compared to a tobacco leaf grown under similar growth conditions and cured under a conventional fire-curing process.
• the present specification provides for, and includes, an air/fire combination cured tobacco leaf comprising one or more smoke finish attributes and wherein the amount of NNK is reduced by at least 50% as compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• an air/fire combination cured tobacco leaf comprises an amount of NNK that is reduced by between 1% and 100%, between 5% and 100%, between 10% and 100%, between 15% and 100%, between 20% and 100%, between 25% and 100%, between 30% and 100%, between 35% and 100%, between 40% and 100%, between 45% and 100%, between 50% and 100%, between 55% and 100%, between 60% and 100%, between 65% and 100%, between 70% and 100%, between 75% and 100%, between 80% and 100%, between 85% and 100%, between 90% and 100%, or between 95% and 100% as compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• an air/fire combination cured tobacco leaf comprises an amount of NNK that is reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% as compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• the present specification provides for, and includes, an air/fire combination cured tobacco leaf comprising one or more smoke finish attributes and wherein the amount of NNK is reduced by at least 0.2 part per million as compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• an air/fire combination cured tobacco leaf comprises an amount of NNK that is reduced by between 0.001 and 5 parts per million, between 0.005 and 5 parts per million, between 0.01 and 5 parts per million, between 0.05 and 5 parts per million, between 0 1 and 5 parts per million, between 0.2 and 5 parts per million, between 0.3 and 5 parts per million, between 0.4 and 5 parts per million, between 0.5 and 5 parts per million, between 0.6 and 5 parts per million, between 0.7 and 5 parts per million, between 0.8 and 5 parts per million, between 0.9 and 5 parts per million, between 1.0 and 5 parts per million, between 2.0 and 5 parts per million, between 3.0 and 5 parts per million, or between 4.0 and 5 parts per million as compared to a tobacco leaf grown under similar growth conditions and cured under a conventional fire-curing process.
• an air/fire combination cured tobacco leaf comprises an amount of NNK that is reduced by at least 0.001 parts per million, at least 0.005 parts per million, at least 0.01 parts per million, at least 0.05 parts per million, at least 0.1 parts per million, at least 0.3 parts per million, at least 0.4 parts per million, at least 0.5 parts per million, at least 0.6 parts per million, at least 0.7 parts per million, at least 0.8 parts per million, at least 0.9 parts per million at least 1.0 part per million, at least 2.0 parts per million, at least 3.0 parts per million, at least 4.0 parts per million, or at least 5.0 parts per million as compared to a tobacco leaf grown under similar growth conditions and cured under a conventional fire-curing process.
• the present specification provides for, and includes, an air/fire combination cured tobacco leaf comprising one or more smoke finish attributes and wherein the cured tobacco leaf comprises a similar amount of phenolic compounds compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• an air/fire combination cured tobacco leaf comprises a similar amount of Catechol, Cresol, Phenol, or a combination thereof, compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• an air/fire combination cured tobacco leaf comprises a similar amount of Catechol, Phenol, or a combination thereof, compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• an air/fire combination cured tobacco leaf comprises a similar amount of Catechol, Cresol, or a combination thereof, compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• an air/fire combination cured tobacco leaf comprises a similar amount of Catechol compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• an air/fire combination cured tobacco leaf comprises a similar amount of Cresol compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process. In a further aspect, an air/fire combination cured tobacco leaf comprises a similar amount of Phenol compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• the present specification provides for, and includes, an air/fire combination cured tobacco leaf comprising one or more smoke finish attributes wherein the tobacco leaf comprises a significant reduction of at least one HPHC compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• an air/fire combination cured tobacco leaf comprises a significant reduction of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen, at least twenty, at least twenty-one, at least twenty -two, at least twenty -three, at least twenty-four , or at least twenty -five HPHCs compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• an air/fire combination cured tobacco leaf comprises a significant reduction of an HPHC selected from the group consisting of Acetaldehyde, Acetamide, Acetone, Acrolein, Acrylamide, Acrylonitrile, Aflatoxin Bl, 4- Aminobiphenyl, 1-Aminonaphthalene, 2-Aminonaphthalene, Ammonia, Anabasine, o-Anisidine, Arsenic, A-a-C (2-Amino-9H-pyrido[2,3-b]indole), Benz[a]anthracene, Benz[j]aceanthrylene, Benzene, Benzo[b]fluoranthene, Benzo[k]fluoranthene, Benzo[b]furan, Benzo[a]pyrene, Benzo[c]phenanthrene, Beryllium, 1,3-Butadiene, Cadmium, Caffeic acid, Carbon monoxide, Catechol, Chlorin
• an HPHC is selected from the group consisting of Acetaldehyde, Acrolein, Arsenic, Benz[a]anthracene, Benzene, Benzo[a]pyrene, Cadmium, Catechol, Chlorinated dioxins, Chlorinated furans, Chromium, Chrysene, Cresols, Crotonaldehyde, Dibenz[a,h]anthracene, Dibenzo[a,e]pyrene, Dibenzo[a,h]pyrene, Ethylbenzene, Formaldehyde, Indeno[l,2,3-cd]pyrene, Lead, Nickle, Phenol, Propionaldehyde, Selenium, Toluene, and a combination thereof, as compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• an air/fire combination cured tobacco leaf comprises a significant reduction of an HPHC selected from the group consisting of Acetaldehyde, Acrolein, Arsenic, Benz[a]anthracene, Benzene, Benzo [a] pyrene, Cadmium, Catechol, Chlorinated dioxins, Chlorinated furans, Chromium, Chrysene, Cresols, Crotonaldehyde, Dibenz[a,h]anthracene, Dibenzo[a,e]pyrene, Dibenzo[a,h]pyrene, Ethylbenzene, Formaldehyde, Indeno[l,2,3-cd]pyrene, Lead, Nickle, Phenol, Propionaldehyde, Selenium, and Toluene, or a combination thereof, as compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• HPHC selected from the group consisting of
• the present specification provides for, and includes, an air/fire combination cured tobacco leaf comprising one or more smoke finish attributes and wherein the amount of at least one HPHC is reduced by at least 25 % as compared to a tobacco leaf grown under similar growth conditions and cured under a conventional fire-curing process.
• the present specification provides for, and includes, an air/fire combination cured tobacco leaf wherein the amount of at least one HPHC is reduced by between 1% and 100%, between 1% and 90%, between 1% and 80%, between 1% and 70%, between 1% and 60%, between 1% and 50%, between 1% and 45%, between 1% and 40%, between 1% and 35%, between 1% and 30%, between 1% and 25%, between 1% and 20%, between 1% and 15%, between 1% and 10%, or between 1% and 5% compared to a tobacco leaf grown under similar growth conditions and cured under a conventional fire-curing process.
• the present specification provides for, and includes, an air/fire combination cured tobacco leaf wherein the amount of at least one HPHC is reduced by at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% compared to a tobacco leaf grown under similar growth conditions and cured under a conventional fire-curing process.
• the present specification provides for, and includes, an air/fire combination cured tobacco leaf comprising one or more smoke finish attributes and wherein the amount of at least one HPHC is reduced by at least 0.05 parts per million as compared to a tobacco leaf grown under similar growth conditions and cured under a conventional fire-curing process.
• the present specification provides for, and includes, an air/fire combination cured tobacco leaf wherein the amount of at least one HPHC is reduced by between 0.001 and 1000 parts per million, between 0.005 and 1000 parts per million, between 0.01 and 1000 parts per million, between 0.05 and 1000 parts per million, between 0.1 and 1000 parts per million, between 0.5 and 1000 parts per million, between 1.0 and 1000 parts per million, between 2.0 and 1000 parts per million, between 3.0 and 1000 parts per million, between 4.0 and 1000 parts per million, between 5.0 and 1000 parts per million, between 10 and 1000 parts per million, between 50 and 1000 parts per million, between 100 and 1000 parts per million, between 200 and 1000 parts per million, between 300 and 1000 parts per million, between 400 and 1000 parts per million, between 500 and 1000 parts per million, between 600 and 1000 parts per million, between 700 and 1000 parts per million, between 800 and 1000 parts per million, or between 900 and 1000 parts per million as compared to a tobacco leaf grown under similar growth conditions and cured under a conventional fire-curing process.
• the present specification provides for, and includes, an air/fire combination cured tobacco leaf wherein the amount of at least one HPHC is reduced by at least 0.001 parts per million, at least 0.005 parts per million, at least 0.01 parts per million, at least 0.05 parts per million, at least 0.1 parts per million, at least 0.5 parts per million, at least 1.0 parts per million, at least 2.0 parts per million, at least 3.0 parts per million, at least 4.0 parts per million, at least 5.0 parts per million, at least 10 parts per million, at least 50 parts per million, at least 100 parts per million, at least 200 parts per million, at least 300 parts per million, at least 400 parts per million, at least 500 parts per million, at least 600 parts per million, at least 700 parts per million, at least 800 parts per million, at least 900 parts per million, or at least 1000 parts per million, as compared to a tobacco leaf grown under similar growth conditions and cured under a conventional fire-curing process.
• the present specification provides for, and includes, an air/fire combination cured tobacco leaf comprising one or more smoke finish attributes and wherein the tobacco leaf is a leaf of a tobacco plant comprising a nicl or nic2 mutant allele.
• an air/fire combination cured tobacco leaf further comprises at least one mutation in a nicotine demethylase gene.
• an air/fire combination cured tobacco leaf further comprises a ndgl or ndg2 mutant allele.
• an air/fire combination cured tobacco leaf is a modified tobacco leaf.
• a modified tobacco leaf comprises increased levels of one or more antioxidants as compared to a control leaf of an unmodified tobacco plant of the same variety when grown under comparable conditions.
• an air/fire combination cured tobacco leaf comprising one or more smoke finish attributes does not comprise a transgene.
• the present specification provides for, and includes, a cured tobacco leaf comprising one or more smoke finish attributes and lower amounts of one or more polyaromatic hydrocarbons as compared to a control cured leaf of a tobacco plant cured using a conventional fire-curing process.
• a cured tobacco leaf comprises a significant reduction of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, or more than thirteen polyaromatic hydrocarbons compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• a tobacco leaf cured using an air/fire combination curing method wherein the tobacco leaf comprises a significant reduction of an HPHC selected from the group consisting of Acetaldehyde, Acetamide, Acetone, Acrolein, Acrylamide, Acrylonitrile, Aflatoxin Bl, 4-Aminobiphenyl, 1- Aminonaphthalene, 2-Aminonaphthalene, Ammonia, Anabasine, o-Anisidine, Arsenic, A-a-C (2-Amino-9H-pyrido[2,3-b]indole), Benz [a] anthracene, Benz[j]aceanthrylene, Benzene, Benzo[b]fluoranthene, Benzofk] fluoranthene, Benzo[b]furan, Benzo[a]pyrene, Benzo[c]phenanthrene, Beryllium, 1,3-Butadiene, Cadmium, Caffeic acid, Carbon mon
• a tobacco leaf cured using an air/fire combination curing method wherein the tobacco leaf comprises a significant reduction of an HPHC selected from the group consisting of Acetaldehyde, Arsenic, Benz[a]anthracene, Benzene, Benzo[a]pyrene, Cadmium, Crotonaldehyde, Dibenz[a,h]anthracene, Dibenzo[a,e]pyrene, Dibenzo[a,h]pyrene,
• the present specification provides for, and includes, an air/fire combination cured tobacco leaf comprising one or more smoke finish attributes wherein the amount of at least one polyaromatic hydrocarbon is reduced by at least 25 % as compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• the present specification provides for, and includes, an air/fire combination cured tobacco leaf wherein the amount of at least one polyaromatic hydrocarbon is reduced by between 1% and 100%, between 1% and 90%, between 1% and 80%, between 1% and 70%, between 1% and 60%, between 1% and 50%, between 1% and 45%, between 1% and 40%, between 1% and 35%, between 1% and 30%, between 1% and 25%, between 1% and 20%, between 1% and 15%, between 1% and 10%, or between 1% and 5% compared to a tobacco leaf grown under similar growth conditions and cured under a conventional fire-curing process.
• the present specification provides for, and includes, an air/fire combination cured tobacco leaf wherein the amount of at least one polyaromatic hydrocarbon is reduced by at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• the present specification provides for, and includes, an air/fire combination cured tobacco leaf comprising one or more smoke finish attributes wherein the amount of at least one polyaromatic hydrocarbon is reduced by at least 0.05 parts per million as compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• the present specification provides for, and includes, an air/fire combination cured tobacco leaf wherein the amount of at least one polyaromatic hydrocarbon is reduced by between 0.001 and 1000 parts per million, between 0.005 and 1000 parts per million, between 0.01 and 1000 parts per million, between 0.05 and 1000 parts per million, between 0.1 and 1000 parts per million, between 0.5 and 1000 parts per million, between 1.0 and 1000 parts per million, between 2.0 and 1000 parts per million, between 3.0 and 1000 parts per million, between 4.0 and 1000 parts per million, between 5.0 and 1000 parts per million, between 10 and 1000 parts per million, between 50 and 1000 parts per million, between 100 and 1000 parts per million, between 200 and 1000 parts per million, between 300 and 1000 parts per million, between 400 and 1000 parts per million, between 500 and 1000 parts per million, between 600 and 1000 parts per million, between 700 and 1000 parts per million, between 800 and 1000 parts per million, or between 900 and 1000 parts per million as compared to a tobacco leaf grown under similar growth conditions and cured under a conventional fire-curing process
• the present specification provides for, and includes, an air/fire combination cured tobacco leaf wherein the amount of at least one polyaromatic hydrocarbon is reduced by at least 0.001 parts per million, at least 0.005 parts per million, at least 0.01 parts per million, at least 0.05 parts per million, at least 0.1 parts per million, at least 0.5 parts per million, at least 1.0 parts per million, at least 2.0 parts per million, at least 3.0 parts per million, at least 4.0 parts per million, at least 5.0 parts per million, at least 10 parts per million, at least 50 parts per million, at least 100 parts per million, at least 200 parts per million, at least 300 parts per million, at least 400 parts per million, at least 500 parts per million, at least 600 parts per million, at least 700 parts per million, at least 800 parts per million, at least 900 parts per million, or at least 1000 parts per million, as compared to a tobacco leaf grown under similar growth conditions and cured under a conventional fire-curing process.
• the present specification provides for, and includes, an air/fire combination cured tobacco leaf comprising one or more smoke finish attributes wherein the amount of Benzo[a]pyrene is reduced by at least 0.05 parts per million as compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• the present specification provides for, and includes, an air/fire combination cured tobacco leaf wherein the amount of Benzo[a]pyrene is reduced by between 0.001 and 1000 parts per million, between 0.005 and 1000 parts per million, between 0.01 and 1000 parts per million, between 0.05 and 1000 parts per million, between 0.1 and 1000 parts per million, between 0.5 and 1000 parts per million, between 1.0 and 1000 parts per million, between 2.0 and 1000 parts per million, between 3.0 and 1000 parts per million, between 4.0 and 1000 parts per million, between 5.0 and 1000 parts per million, between 10 and 1000 parts per million, between 50 and 1000 parts per million, between 100 and 1000 parts per million, between 200 and 1000 parts per million, between 300 and 1000 parts per million, between 400 and 1000 parts per million, between 500 and 1000 parts per million, between 600 and 1000 parts per million, between 700 and 1000 parts per million, between 800 and 1000 parts per million, or between 900 and 1000 parts per million as compared to a tobacco leaf grown under similar growth conditions and cured under a conventional fire-curing process
• the present specification provides for, and includes, an air/fire combination cured tobacco leaf wherein the amount of Benzo[a]pyrene is reduced by at least 0.001 parts per million, at least 0.005 parts per million, at least 0.01 parts per million, at least 0.05 parts per million, at least 0.1 parts per million, at least 0.5 parts per million, at least 1.0 parts per million, at least 2.0 parts per million, at least 3.0 parts per million, at least 4.0 parts per million, at least 5.0 parts per million, at least 10 parts per million, at least 50 parts per million, at least 100 parts per million, at least 200 parts per million, at least 300 parts per million, at least 400 parts per million, at least 500 parts per million, at least 600 parts per million, at least 700 parts per million, at least 800 parts per million, at least 900 parts per million, or at least 1000 parts per million, as compared to a tobacco leaf grown under similar growth conditions and cured under a conventional fire-curing process.
• the present specification provides for, and includes, an air/fire combination cured tobacco leaf comprising one or more smoke finish attributes and lower amounts of one or more polyaromatic hydrocarbons further comprising an amount of N'- nitrosonomicotine (NNN), an amount of 4-methylnitrosoamino-l-(3-pyridyl)-l-butanone (NNK), or an amount of both individually that are reduced in comparison to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• NNN N'- nitrosonomicotine
• NNK 4-methylnitrosoamino-l-(3-pyridyl)-l-butanone
• an air/fire combination cured tobacco leaf comprising lower amounts of one or more polyaromatic hydrocarbons further comprises an amount of NNN that is reduced by at least 50% as compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire curing process.
• an air/fire combination cured tobacco leaf comprising lower amounts of one or more polyaromatic hydrocarbons further comprises an amount of NNN that is reduced by between 1% and 100%, between 5% and 100%, between 10% and 100%, between 15% and 100%, between 20% and 100%, between 25% and 100%, between 30% and 100%, between 35% and 100%, between 40% and 100%, between 45% and 100%, between 50% and 100%, between 55% and 100%, between 60% and 100%, between 65% and 100%, between 70% and 100%, between 75% and 100%, between 80% and 100%, between 85% and 100%, between 90% and 100%, or between 95% and 100% as compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• an air/fire combination cured tobacco leaf comprising lower amounts of one or more polyaromatic hydrocarbons further comprises an amount of NNN that is reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% as compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• the present specification provides for, and includes, an air/fire combination cured tobacco leaf comprising one or more smoke finish attributes and lower amounts of one or more polyaromatic hydrocarbons that further comprises an amount of NNN that is reduced by at least 1 part per million as compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• an air/fire combination cured tobacco leaf comprising lower amounts of one or more polyaromatic hydrocarbons further comprises an amount of NNN that is reduced by between 0.001 and 10 parts per million, between 0.005 and 10 parts per million, between 0.01 and 10 parts per million, between 0.05 and 10 parts per million, between 0.1 and 10 parts per million, between 0.5 and 10 parts per million, between 1.0 and 10 parts per million, between 2.0 and 10 parts per million, between 3.0 and 10 parts per million, between 4.0 and 10 parts per million, between 5.0 and 10 parts per million, between 6.0 and 10 parts per million, between 7.0 and 10 parts per million, between 8.0 and 10 parts per million, or between 9.0 and 10 parts per million as compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• an air/fire combination cured tobacco leaf comprising lower amounts of one or more polyaromatic hydrocarbons further comprises an amount of NNN that is reduced by at least 0.001 parts per million, at least 0.005 parts per million, at least 0.01 parts per million, at least 0.05 parts per million, at least 0.1 parts per million, at least 0.5 parts per million, at least 1.0 part per million, at least 2.0 parts per million, at least 3.0 parts per million, at least 4.0 parts per million, at least 5.0 parts per million, at least 6.0 parts per million, at least 7.0 parts per million, at least 8.0 parts per million, at least 9.0 parts per million, or at least 10 parts per million as compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• the present specification provides for, and includes, an air/fire combination cured tobacco leaf comprising one or more smoke finish attributes and lower amounts of one or more polyaromatic hydrocarbons that further comprises an amount of NNK that is reduced by at least 50% as compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• an air/fire combination cured tobacco leaf comprising lower amounts of one or more polyaromatic hydrocarbons further comprises an amount of NNK that is reduced by between 1% and 100%, between 5% and 100%, between 10% and 100%, between 15% and 100%, between 20% and 100%, between 25% and 100%, between 30% and 100%, between 35% and 100%, between 40% and 100%, between 45% and 100%, between 50% and 100%, between 55% and 100%, between 60% and 100%, between 65% and 100%, between 70% and 100%, between 75% and 100%, between 80% and 100%, between 85% and 100%, between 90% and 100%, or between 95% and 100% as compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• an air/fire combination cured tobacco leaf comprising lower amounts of one or more polyaromatic hydrocarbons further comprises an amount of NNN that is reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% as compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• the present specification provides for, and includes, an air/fire combination cured tobacco leaf comprising one or more smoke finish attributes and lower amounts of one or more polyaromatic hydrocarbons that further comprises an amount of NNK that is reduced by at least 0.2 part per million as compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• an air/fire combination cured tobacco leaf comprising lower amounts of one or more polyaromatic hydrocarbons further comprises an amount of NNK that is reduced by between 0.001 and 5 parts per million, between 0.005 and 5 parts per million, between 0.01 and 5 parts per million, between 0.05 and 5 parts per million, between 0.1 and 5 parts per million, between 0.2 and 5 parts per million, between 0.3 and 5 parts per million, between 0.4 and 5 parts per million, between 0.5 and 5 parts per million, between 0.6 and 5 parts per million, between 0.7 and 5 parts per million, between 0.8 and 5 parts per million, between 0.9 and 5 parts per million, between 1.0 and 5 parts per million, between 2.0 and 5 parts per million, between 3.0 and 5 parts per million, or between 4.0 and 5 parts per million as compared to a tobacco leaf grown under similar growth conditions and cured under a conventional fire-curing process.
• an air/fire combination cured tobacco leaf comprising lower amounts of one or more polyaromatic hydrocarbons further comprises an amount of N K that is reduced by at least 0.001 parts per million, at least 0.005 parts per million, at least 0.01 parts per million, at least 0.05 parts per million, at least 0.1 parts per million, at least 0.3 parts per million, at least 0.4 parts per million, at least 0.5 parts per million, at least 0.6 parts per million, at least 0.7 parts per million, at least 0.8 parts per million, at least 0.9 parts per million at least 1.0 parts per million, at least 2.0 parts per million, at least 3.0 parts per million, at least 4.0 parts per million, or at least 5.0 parts per million as compared to a tobacco leaf grown under similar growth conditions and cured under a conventional fire-curing process.
• the present specification provides for, and includes, an air/fire combination cured tobacco leaf comprising one or more smoke finish attributes and lower amounts of one or more polyaromatic hydrocarbons that further comprises a similar amount of phenolic compounds compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• an air/fire combination cured tobacco leaf comprising lower amounts of one or more polyaromatic hydrocarbons further comprises a similar amount of Catechol, Cresol, Phenol, or a combination thereof, compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• an air/fire combination cured tobacco leaf comprising lower amounts of one or more polyaromatic hydrocarbons further comprises a similar amount of Catechol, Phenol, or a combination thereof, compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• an air/fire combination cured tobacco leaf comprising lower amounts of one or more polyaromatic hydrocarbons further comprises a similar amount of Catechol, Cresol, or a combination thereof, compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• an air/fire combination cured tobacco leaf comprising lower amounts of one or more polyaromatic hydrocarbons further comprises a similar amount of Catechol compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• an air/fire combination cured tobacco leaf comprising lower amounts of one or more polyaromatic hydrocarbons further comprises a similar amount of Cresol compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• an air/fire combination cured tobacco leaf comprising lower amounts of one or more polyaromatic hydrocarbons further comprises a similar amount of Phenol compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• an air/fire combination cured tobacco leaf comprising one or more smoke finish attributes and lower amounts of one or more polyaromatic hydrocarbons that further comprises a nicl or nic2 mutant allele.
• an air/fire combination cured tobacco leaf comprising lower amounts of one or more polyaromatic hydrocarbons comprises at least one mutation in a nicotine demethylase gene.
• an air/fire combination cured tobacco leaf comprising lower amounts of one or more polyaromatic hydrocarbons further comprises a ndgl or ndg2 mutant allele.
• an air/fire combination cured tobacco leaf comprising lower amounts of one or more polyaromatic hydrocarbons is a modified tobacco leaf.
• a modified tobacco leaf comprises increased levels of one or more antioxidants as compared to a control leaf of an unmodified tobacco plant of the same variety when grown under comparable conditions.
• an antioxidant is selected from the group consisting of anthocyanidin, flavanone, flavanol, flavone, flavonol, isoflavone, hyhroxybenzoic acid, hydroxy cinnamic acid, ellagitannin, stibene, lignan, carotenoids, and glycyrrhzin.
• an antioxidant is selected from the group consisting of Delphnidin, Cyanidin, Procyanidin, Prodelphinidin, Hesperetin, Naringenin, Catechin, Epicatechin, Apigenin, Luteonin, Quercetin, Myricetin, Genistein,Daidzein, Gallic acid, Ferunic acid, Cinamic acid, Coumeric acid, Chlorogenic acid, Coffeic acid, Sanguiin, Resveratrol, Sesamin, Caretonoids, and Vitamin C.
• a modified tobacco leaf does not comprise a transgene.
• a tobacco product comprising air/fire combination cured tobacco.
• a tobacco product is selected from the group consisting of a cigarette, a kretek, a bidi cigarette, a cigar, a cigarillo, a non- ventilated cigarette, a vented recess filter cigarette, pipe tobacco, snuff, snus, chewing tobacco, moist smokeless tobacco, fine cut chewing tobacco, long cut chewing tobacco, pouched chewing tobacco product, gum, a tablet, a lozenge, and a dissolving strip.
• Embodiment 1 A method for curing tobacco leaves comprising: a. subjecting a tobacco leaf to air curing; and b. applying one or more smoke finish attributes to the air cured leaf to generate an air/fire combination cured tobacco leaf.
• Embodiment 2 The method of Embodiment 1, wherein said air curing is for a duration of at least 30 days.
• Embodiment 3 The method of Embodiment 1 or 2, wherein said air curing is for a duration of at least 40 days.
• Embodiment 4 The method of any one of Embodiments 1 to 3, wherein said air curing is for a duration of between 30 days and 100 days.
• Embodiment 5 The method of any one of Embodiments 1 to 4, wherein said air curing is for a duration of between 30 days and 80 days.
• Embodiment 6 The method of any one of Embodiments 1 to 5, wherein said air curing is for a duration of between 30 days and 60 days.
• Embodiment 7 The method of any one of Embodiments 1 to 6, wherein said air curing is for a duration of between 40 days and 60 days.
• Embodiment 8 The method of any one of Embodiments 1 to 7, wherein said one or more smoke finish attributes comprise a compound selected from the group consisting of Syringol, Maltol, and Furfural.
• Embodiment 9 The method of any one of Embodiments 1 to 8, wherein said one or more smoke finish attributes comprise flavor or aroma attributes similar to a tobacco leaf grown under similar growth conditions and cured using a conventional fire curing process.
• Embodiment 10 The method of any one of Embodiments 1 to 9, wherein said air/fire combination cured tobacco leaf comprises a similar amount of phenolic acid compounds compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• Embodiment 11 The method of any one of Embodiments 1 to 10, wherein said phenolic acid compounds are selected from the group consisting of Catechol, Cresol, Phenol, and a combination thereof.
• Embodiment 12 The method of any one of Embodiments 1 to 11, wherein said air/fire combination cured tobacco leaf comprises a significant reduction of at least one harmful and potentially harmful constituent (HPHC) compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• HPHC harmful and potentially harmful constituent
• Embodiment 13 The method of any one of Embodiments 1 to 12, wherein said air/fire combination cured tobacco leaf comprises a significant reduction of at least two HPHCs compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• Embodiment 14 The method of any one of Embodiments 1 to 13, wherein said one or more smoke finish attributes comprise a significant reduction of a HPHC selected from the group consisting of Acetaldehyde, Acrolein, Arsenic, Benz[a]anthracene, Benzene, Benzo [a] pyrene, Cadmium, Chlorinated dioxins, Chlorinated furans, Chromium, Chrysene, Crotonaldehyde, Dibenz[a,h]anthracene, Dibenzo[a,e]pyrene, Dibenzo[a,h]pyrene, Ethylbenzene, Formaldehyde, Indeno[l,2,3- cd]pyrene, Lead, Nickle, Propionaldehyde, Selenium, and Toluene, and a combination thereof, as compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• Embodiment 15 The method of any one of Embodiments 1 to 14, wherein the amount of N'-nitrosonomicotine (NNN), the amount of 4-methylnitrosoamino-l-(3- pyridyl)-l-butanone (NNK), or the amount of both are reduced in comparison to a tobacco leaf grown under similar growth conditions and cured using a conventional fire curing process.
• NNN N'-nitrosonomicotine
• NNK 4-methylnitrosoamino-l-(3- pyridyl)-l-butanone
• Embodiment 16 The method of any one of Embodiments 1 to 15, wherein the amount of NNN is reduced by at least 20%, at least 30%, at least 40%, or at least 50% as compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• Embodiment 17 The method of any one of Embodiments 1 to 16, wherein the amount of NNN is reduced by at least 1 parts per million (ppm), at least 2 ppm, at least 5 ppm, at least 10 ppm, at least 20 ppm, or greater than 20 ppm as compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire curing process.
• ppm parts per million
• Embodiment 18 The method of any one of Embodiments 1 to 17, wherein the amount of NNK is reduced by at least 20%, at least 30%, at least 40%, or at least 50% as compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• Embodiment 19 The method of any one of Embodiments 1 to 18, wherein the amount of NNK is reduced by at least 0.2 ppm, at least 1 ppm, at least 2 ppm, at least 5 ppm, at least 10 ppm, at least 20 ppm, or greater than 20 ppm as compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• Embodiment 20 The method of any one of Embodiments 1 to 19, wherein said one or more smoke finish attributes are applied using a controlled smoke process.
• Embodiment 21 The method of any one of Embodiments 1 to 20, wherein said controlled smoke process is selected from the group consisting of open flame combustion, smoldering combustion, cold smoke generation, wood smoke generator, and any method known in the art that produces smoke without combustion.
• Embodiment 22 The method of any one of Embodiments 1 to 21, wherein said tobacco leaf is selected from the group consisting of a burley tobacco leaf, an oriental tobacco leaf, or a bright tobacco leaf.
• Embodiment 23 The method of any one of Embodiments 1 to 22, wherein said tobacco leaf is a dark tobacco leaf.
• Embodiment 24 The method of any one of Embodiments 1 to 23, wherein said tobacco leaf is leaf of a tobacco plant that does not comprise a transgene.
• Embodiment 25 The method of any one of Embodiments 1 to 24, wherein said tobacco leaf comprises at least one mutation in a nicotine demethylase gene.
• Embodiment 26 The method of any one of Embodiments 1 to 25, wherein said tobacco leaf comprises a nicl or nic2 mutant allele.
• Embodiment 27 The method of any one of Embodiments 1 to 26, wherein said tobacco leaf comprises a ndgl or ndg2 mutant allele.
• Embodiment 28 The method of any one of Embodiments 1 to 27, wherein said tobacco leaf is a modified tobacco leaf comprising increased levels of one or more antioxidants as compared to a control leaf of an unmodified tobacco plant of the same variety when grown under comparable conditions.
• Embodiment 29 An air/fire combination cured tobacco leaf comprising one or more smoke finish attributes and a lower amount of N, NNK, or both, as compared to a control cured leaf of a tobacco plant cured using a conventional fire-curing process.
• Embodiment 30 The cured tobacco leaf of Embodiment 29, wherein said cured tobacco leaf further comprises a significant reduction of at least one HPHC.
• Embodiment 31 The cured tobacco leaf of Embodiment 29 or 30, wherein said cured tobacco leaf further comprises a significant reduction of a HPHC selected from the group consisting of Acetaldehyde, Acrolein, Arsenic, Benz[a]anthracene, Benzene, Benzo[a]pyrene, Cadmium, Chlorinated dioxins, Chlorinated furans, Chromium, Chrysene, Crotonaldehyde, Dibenz[a,h]anthracene, Dibenzo[a,e]pyrene, Dibenzo[a,h]pyrene, Ethylbenzene, Formaldehyde, Indeno[l,2,3-cd]pyrene, Lead,
• a HPHC selected from the group consisting of Acetaldehyde, Acrolein, Arsenic, Benz[a]anthracene, Benzene, Benzo[a]pyrene, Cadmium,
• Embodiment 32 The cured tobacco leaf of any one of Embodiments 29 to
• cured tobacco leaf further comprises a similar amount of phenolic acid compounds compared to a traditional fire curing process.
• Embodiment 33 The cured tobacco leaf of any one of Embodiments 29 to
• phenolic acids are selected from the group consisting of Catechol, Cresol, Phenol, or a combination thereof.
• Embodiment 34 The cured tobacco leaf of any one of Embodiments 29 to
• said cured tobacco leaf further comprises an amount of NNN reduced by at least 50% in comparison to said cured leaf of a tobacco plant cured using a conventional fire-curing process.
• Embodiment 35 The cured tobacco leaf of any one of Embodiments 29 to
• said cured tobacco leaf further comprises an amount of NNN reduced by at least 1 part per million in comparison to said cured leaf of a tobacco plant cured using a conventional fire-curing process.
• Embodiment 36 The cured tobacco leaf of any one of Embodiments 29 to
• said cured tobacco leaf further comprises an amount of NNK reduced by at least 50% in comparison to said cured leaf of a tobacco plant cured using a conventional fire-curing process.
• Embodiment 37 The cured tobacco leaf of any one of Embodiments 29 to
• said cured tobacco leaf further comprises an amount of NNK reduced by at least 0.2 parts per million in comparison to said cured leaf of a tobacco plant cured using a conventional fire-curing process.
• Embodiment 38 The cured tobacco leaf of any one of Embodiments 29 to
• said tobacco leaf is leaf of a tobacco plant that does not comprise a transgene.
• Embodiment 39 The cured tobacco leaf of any one of Embodiments 29 to
• said cured tobacco leaf further comprises a nicl or nic2 mutant allele.
• Embodiment 40 The cured tobacco leaf of any one of Embodiments 29 to
• said cured tobacco leaf further comprises at least one mutation in a nicotine demethylase gene.
• Embodiment 41 The cured tobacco leaf of any one of Embodiments 29 to
• said cured tobacco leaf further comprises a ndgl or ndg2 mutant allele.
• Embodiment 42 The cured tobacco leaf of any one of Embodiments 29 to
• said cured tobacco leaf further comprises a modification conferring an increased amount of one or more antioxidants wherein said increased amount is compared to a control cured leaf of an unmodified tobacco plant of the same variety when grown and cured using comparable conditions.
• Embodiment 43 An air/fire combination cured tobacco leaf comprising one or more smoke finish attributes and lower amounts of one or more polyaromatic hydrocarbons as compared to a control cured leaf of a tobacco plant cured using a conventional fire-curing process.
• Embodiment 44 The cured tobacco leaf of Embodiment 43, wherein said polyaromatic hydrocarbons are selected from the group consisting of Acetaldehyde, Arsenic, Benz[a]anthracene, Benzene, Benzo[a]pyrene, Cadmium, Crotonaldehyde, Dibenz[a,h]anthracene, Dibenzo[a,e]pyrene, Dibenzo[a,h]pyrene, Ethylbenzene, Indeno[l,2,3-cd]pyrene, Toluene, or a combination thereof, as compared to a tobacco leaf grown under similar growth conditions and cured using a conventional fire-curing process.
• said polyaromatic hydrocarbons are selected from the group consisting of Acetaldehyde, Arsenic, Benz[a]anthracene, Benzene, Benzo[a]pyrene, Cadmium, Crotonaldehyde, Dibenz
• Embodiment 45 The cured tobacco leaf of Embodiment 43 or 44, wherein the amount of Benzo[a]pyrene is reduced by 90% in comparison to said cured leaf of a tobacco plant cured under a conventional fire-curing process.
• Embodiment 46 The cured tobacco leaf of any one of Embodiments 43 to
• Embodiment 47 The cured tobacco leaf of any one of Embodiments 43 to
• said cured tobacco leaf further comprises an amount of NNN reduced by at least 50% as compared to said cured leaf of a tobacco plant cured using a conventional fire-curing process.
• Embodiment 48 The cured tobacco leaf of any one of Embodiments 43 to
• said cured tobacco leaf further comprises an amount of NNN reduced by at least 1 part per million as compared to said cured leaf of a tobacco plant cured using a conventional fire-curing process.
• Embodiment 49 The cured tobacco leaf of any one of Embodiments 43 to
• said cured tobacco leaf further comprises an amount of NNK reduced by at least 50% as compared to said cured leaf of a tobacco plant cured using a conventional fire-curing process.
• Embodiment 50 The cured tobacco leaf of any one of Embodiments 43 to
• Embodiment 51 The cured tobacco leaf of any one of Embodiments 43 to
• said cured tobacco leaf further comprises a similar amount of phenolic acid compounds compared to a traditional fire curing process.
• Embodiment 52 The cured tobacco leaf of any one of Embodiments 43 to
• phenolic acids are selected from the group consisting of Catechol, Cresol, Phenol, or a combination thereof.
• Embodiment 53 The cured tobacco leaf of any one of Embodiments 43 to
• Embodiment 54 The cured tobacco leaf of any one of Embodiments 43 to
• said cured leaf further comprises a nicl or nic2 mutant allele.
• Embodiment 55 The cured tobacco leaf of any one of Embodiments 43 to
• said cured leaf further comprises at least one mutation in a nicotine demethylase gene.
• Embodiment 56 The cured tobacco leaf of any one of Embodiments 43 to
• said cured leaf further comprises a ndgl or ndg2 mutant allele.
• Embodiment 57 The cured tobacco leaf of any one of Embodiments 43 to
• said cured leaf further comprises a modification conferring an increased amount of one or more antioxidants wherein said increased amount is compared to a control cured leaf of an unmodified tobacco plant of the same variety when grown and cured using comparable conditions.
• Embodiment 58 A tobacco product comprising cured tobacco leaf cured using an air/fire combination curing method.
• Embodiment 59 The tobacco product of Embodiment 58, wherein said product is selected from the group consisting of a cigarette, a kretek, a bidi cigarette, a cigar, a cigarillo, a non-ventilated cigarette, a vented recess filter cigarette, pipe tobacco, snuff, snus, chewing tobacco, moist smokeless tobacco, fine cut chewing tobacco, long cut chewing tobacco, pouched chewing tobacco product, gum, a tablet, a lozenge, and a dissolving strip.
• Tobacco seeds are germinated in a greenhouse before being transplanted to a field during the summer months. Tobacco plants are grown using standard dark tobacco production practices as described in the 2017-2018 Burley and Dark Tobacco Production Guide (B. Pearce ed., (2017)).
• Table 1 Five experimental dark tobacco varieties cured under experimental curing conditions in the 2017 field season
• Amounts of TSNAs are determined by adding deuterated internal standards to approximately 0.75 gram of cured tobacco leaf followed by extraction with ammonium acetate. The sample is rotated and the extract is cleaned by solid phase extraction. The sample is analyzed by liquid chromatography with tandem mass spectrometry (LC/MS/MS). HPHC content is determined based on the protocol presented in ISO 21766:2018, similar to Coresta Recommended Method No. 72, Determination of TSNA in Tobacco and Tobacco Products by LC-MS/MS.
• Amounts of Benzo[a]pyrene are determined by adding deuterated internal standard Benzo[a]pyrene-dl2 to approximately 1.0 gram of cured tobacco leaf followed by extraction with methanol. The extract is cleaned with solid phase extraction, concentrated, and analyzed by gas chromatography- mass spectrometry (GC/MS). Benzo[a]pyrene content is determined based on the protocol presented in “Determination of Benzo[a]pyrene in Tobacco and Tobacco Products by GC-MS, Similar to Coresta Recommended method No. 82.
• Amounts of formaldehyde, acetaldehyde, and crotonaldehyde are determined after extraction from approximately 1.0 gram of cured tobacco using lOOmM ammonium formate extraction solution.
• the extract is derivatized with 2,4-dinitrophenylhydrazine.
• the resulting hydrazines are analyzed using ultra-performance LC/MS/MS at Enthalpy Analytical in Durham, NC.
• Amounts of cadmium and arsenic are determined by subjecting approximately 0.5 grams of cured tobacco leaf to closed vessel microwave digestion using nitric acid. The resulting sample is analyzed by inductively-coupled plasma mass spectrometry using a dynamic reaction cell cadmium and arsenic content is determined based on the protocol presented in Determination of Cadmium and Arsenic in Tobacco Products by ICP-MS.
• the content of NO and NO2 are determined using a NO x emissions analyzer (CT2000-00033, Cascade Technologies, Herndon, VA), at the SPAREC facility in Blackstone, Virginia.
• CT2000-00033 Cascade Technologies, Herndon, VA
• a tobacco leaf grade is assigned by a trained USDA grader.
• Leaf grade is determined using an Official Standard Grade published by the Agricultural Marketing Service of the US Department of Agriculture (7 U.S.C. ⁇ 511). See, e.g., Official Standard Grades for Kentucky and Tennessee Fire-Cured and Foreign-Grown Fire-Cured Tobacco (U.S. Type 22, 23 and Foreign Type 96), effective November 7, 1986 (51 F.R.40405) .
• Temperature and humidity are monitored and recorded throughout curing. Temperature is measured both inside and outside of the curing barn. Inside the curing bam, temperature is measured at Tier level in the middle of the bam using conventional thermometers. Humidity is measured inside and outside of the bam. Humidity is measured using a hygrometer and can be recorded as either water content (mg per cm 3 ) or as percent relative humidity. IMonnit Wireless Sensors (U14-002, Monnit Corporation, South Salt Lake, UT) are placed inside the curing bam to record inside temperature and RH.
• Example 2 Dark tobacco cured by standard fire curing
• the standard fire curing method used in this study is based on the 2017-2018 Burley and Dark Tobacco production guide (B. Pearce ed., (2017)). After the pre-curing methods described in Example 1 above, 15 sticks of tobacco from each of the five experimental varieties are placed in a barn. Each experimental bam is filled with all varieties at the same time. The first phase is the yellowing phase. Tobacco is allowed to yellow and the first firing is performed when yellowing is nearly complete. The first firing is performed between five and eight days after housing and the initial fires are about 100T. barn top ventilators are left open during this phase.
• the second phase is color setting.
• Color setting begins when yellowing is completed which is indicated by a solid yellow leaf lamina with little or no brown color.
• temperature is increased to between 37.7°C and 46.1°C (100°F and 115°F) with additional fires.
• Bam top ventilators are closed during this phase. Color setting conditions are maintained until the leaf lamina is a solid brown color.
• This phase lasts between 7 and 14 days and involves multiple firings. Ventilators are opened between firings. Brown color appearing one-half to two-thirds up the leaf indicates the end of the color setting phase.
• the third phase is drying. During drying, ventilators are opened and heat is increase to no greater than 54.4°C (130°F). Drying is complete when little to no green color is present and when the tobacco lamina shatters when touched.
• the final phase is finishing phase. After drying, bam temperatures are maintained at no greater than 48.9°C (120°F).
• the barn is ventilated for several days before two slow firings over a 10 to 14 day period to impart a smoke finish. Smoke volume is maximized to impart smoke finish characteristics to the leaf surface.
• the firing phase uses little to no ventilation. All tobacco should be cured in barns that have adequate ventilation that avoids high relative humidity and prevents house bum. Sticks should be housed such that spacing is a minimum of 8” in a non-lapped barn and a minimum of 12” in a lapped barn. Tobacco must not be taken down before the tobacco has finished curing or stripped when too high in case.
• Example 3 Dark tobacco cured by air curing with a smoke finish
• Heat sources include gas burners or coke stoves. Heat sources are typically not required when air-curing dark tobacco.
• the tobacco is sampled for chemistry analysis. After samples are removed, two or more slow firings (smoldering fires) over a 10 to 21 day period are conducted to impart a smoke finish.
• Smoke volume is maximized by using a single layer of hard wood slabs completely covered by 12 inches to 18 inches of moist (about 20% Oven Volatiles OV to about 50%OV) hardwood sawdust.
• the leaf should be in case order (about 15%OV to 22%OV) at the time of the fires.
• Tobacco leaf is allowed to come back into order prior to subsequent fires. Each fire can last for about 1 to about 3 days.
• Smoldering fire temperatures should be about 90°F and not exceed 100°F and the RH inside the bam should remain above 60.
• Example 4 Dark tobacco cured by air curing with a cold smoke finish
• Tobacco is grown and pre-cured as described in Example 1. After the pre-curing methods described above, 15 sticks of tobacco from each of the five experimental varieties are placed in a barn. Each experimental barn is filled with all varieties at the same time. Air-curing is performed as described in Example 3.
• Example 5 Dark tobacco cured by an accelerated Kentucky style fire curing method
• Tobacco is grown and pre-cured as described in Example 1. After the pre-curing methods described above, 15 sticks of tobacco from each of the five experimental varieties are placed in a barn. Each experimental barn is filled with all varieties at the same time. Fire curing is performed as described in Example 2 with the modifications described below. Accelerated curing is the same process as traditional curing but starts a few days earlier. The tobacco is housed and it stays in the barn for about 5 days to allow it to yellow. In an accelerated curing situation, the fire starts on the same day or the following day after the tobacco is housed.
• Example 6 Dark tobacco cured by a Virginia style fire curing method
• Tobacco is grown and pre-cured as described in Example 1. After the pre-curing methods described above, 15 sticks of tobacco from each of the five experimental varieties are placed in a barn Each experimental barn is filled with all varieties at the same time Fire curing is performed as described in Example 2 with the modifications described below. Virginia style firing uses lower heat ( ⁇ 115 °F) due to smaller scattered fires that last only about 1 day to about 3 days. Virginia Style is used to remove moisture from the barn during periods of high relative humidity.
• Example 7 Evaluation of HPHCs in Tobacco cured with Experimental curing methods
• the five dark tobacco varieties are grown and cured as described in Examples 1 to 6. Tobacco leaves are sampled and HPHCs are determined using methods described in Example 1. All five dark tobacco varieties have significantly reduced amounts of Acetaldehyde, Arsenic, Benz [a] anthracene, Benzene, Benzo[a]pyrene, Cadmium, Crotonaldehyde, Dibenz[a,h]anthracene, Dibenzo[a,e]pyrene, Dibenzo[a,h]pyrene, Ethylbenzene, Indeno[l,2,3- cd]pyrene, and Toluene, when cured using an air-cure with a smoke finish or a cold smoke finish as compared to standard fire-curing, accelerated fire-curing, and a Virginia style fire-cure.
• All five dark tobacco varieties have significantly reduced amounts of NNN and NNK when cured using an air-cure with a smoke finish or a cold smoke finish as compared to standard fire-curing, accelerated fire-curing, and a Virginia style fire-cure. All five dark tobacco varieties show a similar amount of the phenolic acid compounds Catechol, Cresol, and Phenol under all five curing methods.
• Example 8 Evaluation of NO and NO2 during Experimental curing methods [0200] The five dark tobacco varieties are grown and cured as described in Examples 1 to 6. Tobacco leaves are sampled and NO and NO2 are determined using methods described in Example 1. The levels of NO and NO2 correlate with the temperature inside the experimental curing bam. The levels are highest in the accelerated fire-curing bam and lowest in the air-cure with smoke finish barn and the cold smoke finish barn.
• Example 9 Evaluation of grade by stalk position after experimental curing methods [0201]
• Leaf quality is measured using a numerical grade index, based on the USDA official standard grades for dark tobacco, as described in Example 1, for all five dark tobacco varieties cured under the five different experimental curing conditions.
• the numerical leaf grade index is consistent for each variety across each different experimental barn. While variation is seen when comparing different varieties, the different curing processes do not significantly change the E!SDA numerical grade index.

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