Tobacco plants having altered amounts of one or more alkaloids in leaf and methods of using such plants

1. A Nicotiana tabacum plant having an induced mutation in an endogenous gene, wherein a naturally occurring sequence of said endogenous gene has the sequence shown in SEQ ID NO:15, wherein the mutation results in reduced expression of the endogenous gene relative to a corresponding plant lacking the mutation, wherein leaf from the plant exhibits a reduced amount of at least one alkaloid relative to leaf from a corresponding plant lacking the mutation.

2. The N. tabacum plant of claim 1 , wherein cured leaf from the plant exhibits a reduced amount of at least one tobacco specific nitrosamine (TSNA) relative to cured leaf from a corresponding plant lacking the mutation.

3. Seed produced by the N. tabacum plant of claim 1 , wherein the seed comprises the induced mutation.

4. A method of making a tobacco plant, comprising the steps of:

(a) inducing mutagenesis in Nicotiana tabacum cells to produce mutagenized cells;

(b) obtaining one or more plants from the mutagenized cells;

(c) identifying at least one of the plants that comprises a mutation in an endogenous gene, wherein said endogenous gene has the sequence shown in SEQ ID NO:15, wherein the mutation results in reduced expression of the endogenous gene relative to a corresponding plant lacking the mutation; and

(d) identifying at least one of the plants that comprises leaf that exhibits a reduced amount of at least one alkaloid relative to leaf from a corresponding plant lacking the mutation.

5. The method of claim 4 , further comprising identifying at least one of the plants that comprises leaf that, when cured, exhibits a reduced amount of at least one tobacco-specific nitrosamine (TSNA) relative to cured leaf from a plant lacking the mutation.

6. The method of claim 4 , wherein mutagenesis is induced using a chemical mutagen or ionizing radiation.

7. The method of claim 6 , wherein the chemical mutagen is selected from the group consisting of nitrous acid, sodium azide, acridine orange, ethidium bromide, and ethyl methane sulfonate (EMS).

8. The method of claim 6 , wherein the ionizing radiation is selected from the group consisting of x-rays, gamma rays, fast neutron irradiation, and UV irradiation.

9. The method of claim 4 , wherein mutagenesis is induced using TALEN.

10. The method of claim 4 , wherein mutagenesis is induced using zinc-finger technology.

11. A method for producing a tobacco plant, said method comprising the steps of:

(a) 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 an endogenous gene, wherein a naturally occurring sequence of said endogenous gene has the sequence shown in SEQ ID NO:15, wherein the mutation results in reduced expression of the endogenous gene relative to a corresponding plant lacking the mutation, wherein leaf from the plant of the first tobacco line exhibits a reduced amount of at least one alkaloid relative to leaf from a corresponding plant lacking the mutation;

(b) selecting for progeny tobacco plants that have the mutation and exhibit reduced expression of the endogenous gene relative to a corresponding plant lacking the mutation; and

(c) selecting for progeny tobacco plants that comprise leaf exhibiting a reduced amount of at least one alkaloid relative to leaf from a corresponding plant lacking the mutation.

12. The method of claim 11 , further comprising selecting for progeny tobacco plants that comprise leaf that, when cured, exhibit a reduced amount of at least one tobacco-specific nitrosamine (TSNA) relative to cured leaf from a plant lacking the mutation.

13. A tobacco product comprising cured leaf from a tobacco plant having a mutation in an endogenous gene, wherein said endogenous gene has the sequence shown in SEQ ID NO:15, wherein the mutation results in reduced expression of the endogenous gene relative to a corresponding plant lacking the mutation, wherein the leaf exhibits a reduced amount of at least one alkaloid relative to leaf from a corresponding plant lacking the mutation.

14. The tobacco product of claim 13 , wherein the cured leaf exhibits a reduced amount of at least one tobacco-specific nitrosamine (TSNA) relative to cured leaf from a plant lacking the mutation.

15. A method of producing a tobacco product, the method comprising:

(a) providing cured leaf from a tobacco plant having a mutation in an endogenous gene, wherein said endogenous gene has having the sequence shown in SEQ ID NO:15, wherein the mutation results in reduced expression of the endogenous gene relative to a corresponding plant lacking the mutation, wherein the leaf exhibits a reduced amount of at least one alkaloid relative to leaf from a corresponding plant lacking the mutation; and

(b) manufacturing a tobacco product using the cured leaves.

16. The method of claim 15 , wherein the cured leaf exhibits a reduced amount of at least one tobacco-specific nitrosamine (TSNA) relative to cured leaf from a plant lacking the mutation.

17. The method of claim 15 , wherein the mutation is selected from the group consisting of a point mutation, an insertion, a deletion, and a substitution.