	US 12,171,177 B2
	Genetic approach for achieving ultra low nicotine content in tobacco
Ramsey S. Lewis, Apex, NC (US); Ralph E. Dewey, Apex, NC (US); and Juan Sanchez Tamburrino, Williamsville, NY (US)
Assigned to North Carolina State University, Raleigh, NC (US); and 22ND CENTURY LIMITED, LLC, Mocksville, NC (US)
Appl. No. 17/785,153
Filed by North Carolina State University, Raleigh, NC (US); and 22ND CENTURY LIMITED, LLC, Willamsville, NY (US)
PCT Filed Jan. 8, 2021, PCT No. PCT/US2021/012742 § 371(c)(1), (2) Date Jun. 14, 2022, PCT Pub. No. WO2021/142296, PCT Pub. Date Jul. 15, 2021.
Claims priority of provisional application 62/958,505, filed on Jan. 8, 2020.
Prior Publication US 2023/0029171 A1, Jan. 26, 2023
Int. Cl. A24B 3/12 (2006.01); A01C 21/00 (2006.01); A01G 22/45 (2018.01); A01H 1/00 (2006.01); A01H 6/82 (2018.01); A24B 15/10 (2006.01); C12N 15/82 (2006.01); A01C 23/04 (2006.01)

CPC A01H 1/101 (2021.01) [A01C 21/00 (2013.01); A01G 22/45 (2018.02); A01H 6/823 (2018.05); A24B 3/12 (2013.01); A24B 15/10 (2013.01); A01C 23/042 (2013.01)]

9 Claims

1. A method of curing one or more leaves of a Nicotiana plant comprising:

(A) a modification that reduces the activity of BBLa, BBLb, and BBLc or reduces expression of a nucleic acid encoding BBLa, a nucleic acid encoding BBLb, and a nucleic acid encoding BBLc; and

(B) a homozygous recessive allele of nic1 or a homozygous recessive allele of nic1 and a homozygous recessive allele of nic2,

the method comprising:

(a) a yellowing process comprising heating the leaf at a temperature starting at 92° F.-96° F. and increasing to a temperature of 104° F.-108° F. at a rate of about 1° F. per hour until reaching a maximum upper temperature and holding at the upper temperature for a period of about 52-58 hours;

(b) a leaf drying process comprising drying at a temperature of about 120° F. for about 22 hours; and

(c) a stem drying process comprising drying at a temperature of about 132° F.-138° F. for about 50 hours to about 65 hours,

thereby curing the one or more leaves of the Nicotiana plant and producing one or more cured leaves of the Nicotiana plant.