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

Abstract

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.

1 .- 25 . (canceled) 
     
     
         26 . A tobacco plant comprising an induced mutation in an endogenous gene, wherein a naturally occurring sequence of the endogenous gene comprises SEQ ID NO: 39, wherein the induced mutation results in reduced expression of the endogenous gene relative to a corresponding tobacco plant lacking the mutation. 
     
     
         27 . The tobacco plant of  claim 26 , wherein the tobacco plant exhibits reduced transport of nicotine from root tissue to leaf tissue relative to the corresponding tobacco plant lacking the mutation. 
     
     
         28 . The tobacco plant of  claim 26 , wherein the induced mutation is a point mutation. 
     
     
         29 . The tobacco plant of  claim 26 , wherein the induced mutation is selected from the group consisting of an insertion or a deletion. 
     
     
         30 . The tobacco plant of  claim 26 , wherein the tobacco plant is of a tobacco type selected from the group consisting of a Burley type, a dark type, a flue-cured type, a Maryland type, and an Oriental type. 
     
     
         31 . The tobacco plant of  claim 26 , wherein the tobacco plant is of a variety selected from the group consisting of BU 64, CC 101, CC 200, CC 13, CC 27, CC 33, CC 35, CC 37, CC 65, CC 67, CC 301, CC 400, CC 500, CC 600, CC 700, CC 800, CC 900, CC 1063, Coker 176, Coker 319, Coker 371 Gold, Coker 48, CU 263, DF911, GL 26H, GL 338, GL 350, GL 395, GL 600, GL 737, GL 939, GL 973, GF 157, GF 318, RJR 901, HB 04P, K 149, K 326, K 346, K 358, K394, K 399, K 730, NC 196, NC 37NF, NC 471, NC 55, NC 92, NC2326, NC 95, NC 925, PVH 1118, PVH 1452, PVH 2110, PVH 2254, PVH 2275, VA 116, VA 119, KDH 959, KT 200, KT204LC, KY 10, KY 14, KY 160, KY 17, KY 171, KY 907, KY907LC, KTY14×L8 LC, Little Crittenden, McNair 373, McNair 944, msKY 14×L8, Narrow Leaf Madole, NC 100, NC 102, NC 2000, NC 291, NC 297, NC 299, NC 3, NC 4, NC 5, NC 6, NC7, NC 606, NC 71, NC 72, NC 810, NC BH 129, NC 2002, Neal Smith Madole, OXFORD 207, ‘Perique’ tobacco, PVH03, PVH09, PVH19, PVH50, PVH51, R 610, R 630, R 7-11, R 7-12, RG 17, RG 81, RG H51, RGH 4, RGH 51, RS 1410, Speight 168, Speight 172, Speight 179, Speight 210, Speight 220, Speight 225, Speight 227, Speight 234, Speight G-28, Speight G-70, Speight H-6, Speight H20, Speight NF3, TI 1406, TI 1269, TN 86, TN86LC, TN 90, TN90LC, TN 97, TN97LC, TN D94, TN D950, TR (Tom Rosson) Madole, VA 309, and VA359. 
     
     
         32 . Seed produced by the tobacco plant of  claim 26 , wherein the seed comprises the induced mutation. 
     
     
         33 . A method of making a tobacco plant, the method comprising:
 (a) inducing mutagenesis in tobacco cells to produce mutagenized tobacco cells;   (b) obtaining one or more tobacco plants from the mutagenized tobacco cells;   (c) identifying at least one tobacco plant obtained in step (b) that comprises a mutation in the endogenous gene, wherein a naturally occurring sequence of the endogenous gene comprises SEQ ID NO: 39, wherein the mutation results in reduced expression of the endogenous gene relative to a corresponding tobacco plant lacking the mutation, and wherein the tobacco plant exhibits reduced transport of nicotine from root tissue to leaf tissue relative to leaf from the corresponding tobacco plant lacking the mutation.   
     
     
         34 . The method of  claim 33 , wherein mutagenesis is induced using a chemical mutagen or ionizing radiation. 
     
     
         35 . The method of  claim 34 , wherein the chemical mutagen is selected from the group consisting of nitrous acid, sodium azide, acridine orange, ethidium bromide, and ethyl methane sulfonate. 
     
     
         36 . The method of  claim 34 , wherein the ionizing radiation is selected from the group consisting of X-rays, gamma rays, fast neutron irradiation, and ultraviolet radiation. 
     
     
         37 . The method of  claim 34 , wherein mutagenesis is induced using a TALEN. 
     
     
         38 . The method of  claim 34 , wherein mutagenesis is induced using zinc-finger technology. 
     
     
         39 . The method of  claim 33 , wherein the induced mutation is a point mutation. 
     
     
         40 . The method of  claim 33 , wherein the induced mutation is selected from the group consisting of an insertion or a deletion. 
     
     
         41 . The method of  claim 33 , wherein the tobacco plant is of a tobacco type selected from the group consisting of a Burley type, a dark type, a flue-cured type, a Maryland type, and an Oriental type. 
     
     
         42 . A method of producing a tobacco plant, the method comprising:
 (a) crossing a plant of a first tobacco line with a plant of a second tobacco line, wherein the plant of the first tobacco line comprises an induced mutation in an endogenous gene, wherein a naturally occurring sequence of the endogenous gene comprises SEQ ID NO: 39, wherein the induced mutation results in reduced expression of the endogenous gene relative to a corresponding tobacco plant lacking the mutation, and wherein the plant of the first tobacco line exhibits reduced transport of nicotine from root tissue to leaf tissue relative to the corresponding tobacco plant lacking the mutation;   (b) selecting a first group of progeny tobacco plants that comprise the induced mutation and exhibit reduced expression of the endogenous gene relative to the corresponding tobacco plant lacking the mutation; and   (c) selecting a second group of progeny tobacco plants from the first group of progeny tobacco plants selected in step (b) that comprise a leaf exhibiting reduced transport of nicotine from root tissue to leaf tissue relative to the corresponding tobacco plant lacking the mutation.   
     
     
         43 . The method of  claim 42 , wherein a plant of the second tobacco line exhibits a phenotypic trait selected from the group consisting of: disease resistance, high yield, high grade index, early maturing, medium maturing, late maturing, 5-10 leaves per plant, 11-leaves per plant, and 16-21 leaves per plant. 
     
     
         44 . The method of  claim 42 , wherein the second plant of the second tobacco line comprises the induced mutation. 
     
     
         45 . The method of  claim 42 , wherein the first tobacco line, the second tobacco line, or both, is of a tobacco type selected from the group consisting of a Burley type, a dark type, a flue-cured type, a Maryland type, and an Oriental type.