TOBACCO PLANTS HAVING ALTERED AMOUNTS OF ONE OR MORE ALKALOIDS IN LEAF AND METHODS OF USING SUCH PLANTS

20250064011

18/943128

November 11, 2024

This disclosure provides a number of sequences involved in the transport of alkaloids from the root to the leaf in tobacco, methods of using such sequences, tobacco plants carrying modifications to such sequences, and tobacco products made from such plants.

Altria Client Services LLC (Richmond, VA, US)

1. A tobacco hybrid, variety, line, or cultivar comprising plants having a mutation in one or more endogenous nucleic acids having a sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, and 90.

2. The tobacco hybrid, variety, line, or cultivar of claim 1, wherein leaf from the plants exhibit a reduced amount of at least one alkaloid relative to leaf from a plant lacking the mutation.

3. The tobacco hybrid, variety, line, or cultivar of claim 2, wherein cured leaf from the plants exhibit a reduced amount of at least one tobacco specific nitrosamine (TSNA) relative to cured leaf from a plant lacking the mutation.

4. Seed produced by the tobacco hybrid, variety, line, or cultivar of claim 1, the seed comprising the mutation in one or more endogenous nucleic acids having a sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, and 90.

5. A method of making a tobacco plant, comprising the steps of: inducing mutagenesis in Nicotiana tabacum cells to produce mutagenized cells; obtaining one or more plants from the mutagenized cells; and identifying at least one of the plants that comprises a mutation in one or more endogenous nucleic acids having a sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, and 90.

6. The method of claim 5, further comprising identifying at least one of the plants that comprises leaf exhibiting a reduced amount of at least one alkaloid relative to leaf from a plant lacking the mutation.

7. The method of claim 6, further comprising identifying at least one of the plants that comprises leaf that, when cured, exhibit a reduced amount of at least one TSNA relative to cured leaf from a plant lacking the mutation.

8. The method of claim 5, wherein mutagenesis is induced using a chemical mutagen or ionizing radiation.

9. The method of claim 8, wherein the chemical mutagen is selected from the group consisting of nitrous acid, sodium azide, acridine orange, ethidium bromide, and ethyl methane sulfonate (EMS).

10. The method of claim 8, wherein the ionizing radiation is selected from the group consisting of x-rays, gamma rays, fast neutron irradiation, and UV irradiation.

11. The method of claim 5, wherein mutagenesis is induced using TALEN.

12. The method of claim 5, wherein mutagenesis is induced using zinc-finger technology.

13. A method for producing a tobacco plant, said method comprising the steps of: crossing at least one plant of a first tobacco line with at least one plant of a second tobacco line, the plant of the first tobacco line having a mutation in one or more endogenous nucleic acids having a sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, and 90; and selecting for progeny tobacco plants that have the mutation.

14. The method of claim 13, further comprising selecting for progeny tobacco plants that comprise leaf exhibiting a reduced amount of at least one alkaloid relative to leaf from a plant lacking the mutation.

15. The method of claim 14, further comprising selecting for progeny tobacco plants that comprise leaf that, when cured, exhibit a reduced amount of at least one TSNA relative to cured leaf from a plant lacking the mutation.

16. A tobacco product comprising cured leaf from a tobacco plant having a mutation in one or more endogenous nucleic acids having a sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, and 90.

17. The tobacco product of claim 16, wherein the leaf exhibits a reduced amount of at least one alkaloid relative to leaf from a plant lacking the mutation.

18. The tobacco product of claim 17, wherein the cured leaf exhibits a reduced amount of at least one TSNA relative to cured leaf from a plant lacking the mutation.

19. A method of producing a tobacco product, the method comprising: providing cured leaf from a tobacco plant having a mutation in one or more endogenous nucleic acids having a sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, and 90; and manufacturing a tobacco product using the cured leaves.

20. The method of claim 19, wherein the cured leaf exhibits a reduced amount of at least one alkaloid relative to cured leaf from a plant lacking the mutation.

21. The method of claim 19, wherein the cured leaf exhibits a reduced amount of at least one TSNA relative to cured leaf from a plant lacking the mutation.

22. The method of claim 19, wherein the mutation is selected from the group consisting of a point mutation, an insertion, a deletion, and a substitution.

23. A method of screening plants, comprising: providing plant material from the plant of claim 1; and determining the amount of one or more alkaloids in plant tissue.

24. The method of claim 23, wherein the plant tissue is leaf.

25. The method of claim 23, wherein the plant tissue is root.

01; 01; 01; 01; 24; 07; 12; 12; 01

edspap.20250064011