# US 8,999,713 B2 · Methods of multiplying conifer embryogenic tissue > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 8,999,713 B2 - Document ID: US8999713B2 - Status: Granted - Kind: B2 - Application number: 13/602,983 - Filed: 2012-09-04 - Published: 2013-03-28 - Granted: 2015-04-07 - Earliest claimed: 2011-09-28 - Assignees: Weyerhaeuser NR Company - Applicants: Pramod K. Gupta; Diane G. Holmstrom; Pamela L. Carlson - Inventors: Pramod K. Gupta; Diane G. Holmstrom; Pamela L. Carlson - Web page: https://patentgrep.com/patent/US8999713B2 ## CPC - A01H 4/005: {Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques} - C12N 5/04: Plant cells or tissues {(culture media C12N5/0025)} - A01H: NEW PLANTS OR {NON-TRANSGENIC} PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract The invention provides methods for multiplying conifer embryogenic tissue. The methods of the invention comprise the steps of: (a) culturing conifer embryogenic tissue in or on a solid multiplication medium comprising one or more growth hormones; and (b) culturing the conifer embryogenic tissue multiplied in step (a) in or on a liquid multiplication medium comprising one or more growth hormones, wherein the initial concentration of growth hormones in the solid multiplication medium of step (a) is greater than the initial concentration of growth hormones in the liquid multiplication medium of step (b). ## Claims ### Claim 1 A method of multiplying conifer embryogenic tissue comprising the steps of: (a) culturing conifer embryogenic tissue in or on a first multiplication medium, wherein the first multiplication medium is a solid multiplication medium comprising one or more growth hormones, to multiply the conifer embryogenic tissue; and (b) culturing the conifer embryogenic tissue multiplied in step (a) in or on a second multiplication medium, wherein the second multiplication medium is a liquid multiplication medium comprising one or more growth hormones, to further multiply the conifer embryogenic tissue, wherein the initial concentration of the growth hormones in the solid multiplication medium of step (a) is about two to about three times the initial concentration of the growth hormones in the liquid multiplication medium of step (b). ### Claim 2 (depends on 1) The method of claim 1, wherein the initial concentration of the growth hormones in the solid multiplication medium of step (a) is about two to about two and one-half times the initial concentration of the growth hormones in the liquid multiplication medium of step (b). ### Claim 3 (depends on 1) The method of claim 1, wherein the growth hormones in the multiplication medium of steps (a) and (b) are at least one of an auxin and a cytokinin. ### Claim 4 (depends on 3) The method of claim 3, wherein the initial concentration of auxin in the solid multiplication medium of step (a) is from about 2.0 mg/L to about 5.0 mg/L. ### Claim 5 (depends on 3) The method of claim 3, wherein the initial concentration of auxin in the solid multiplication medium of step (a) is from about 2.0 mg/L to about 3.0 mg/L. ### Claim 6 (depends on 3) The method of claim 3, wherein the initial concentration of cytokinin in the solid multiplication medium of step (a) is from about 0.2 mg/L to about 0.25 mg/L. ### Claim 7 (depends on 3) The method of claim 3, wherein the initial concentration of auxin in the liquid multiplication medium of step (b) is from about 0.25 mg/L to about 1.1 mg/L. ### Claim 8 (depends on 3) The method of claim 3, wherein the initial concentration of cytokinin in the liquid multiplication medium of step (b) is from about 0.025 mg/L to about 0.1 mg/L. ### Claim 9 (depends on 3) The method of claim 3, wherein the auxin is 2,4-dichlorophenoxyacetic acid. ### Claim 10 (depends on 3) The method of claim 3, wherein the cytokinin is at least one of 6-benzylaminopurine and 6-furfurylaminopurine. ### Claim 11 (depends on 3) The method of claim 3, wherein the multiplication medium of step (a) and step (b) each comprises the growth hormones of 2,4-dichlorophenoxyacetic acid, 6-benzylaminopurine, and 6-furfurylaminopurine. ### Claim 12 (depends on 9) The method of claim 9, wherein the initial concentration of 2,4-dichlorophenoxyacetic acid in the solid multiplication medium of step (a) is about three times the initial concentration of 2,4-dichlorophenoxyacetic acid in the liquid multiplication medium of step (b). ### Claim 13 (depends on 10) The method of claim 10, wherein the initial concentration of 6-benzylaminopurine in the solid multiplication medium of step (a) is about two and one-half times the initial concentration of 6-benzylaminopurine in the liquid multiplication medium of step (b). ### Claim 14 (depends on 10) The method of claim 10, wherein the initial concentration of 6-furfurylaminopurine in the solid multiplication medium of step (a) is about two and one-half times the initial concentration of 6-furfurylaminopurine in the liquid multiplication medium of step (b). ### Claim 15 (depends on 11) The method of claim 11, wherein the initial concentration of 2,4-dichlorophenoxyacetic acid in the solid multiplication medium of step (a) is about three times the initial concentration of 2,4-dichlorophenoxyacetic acid in the liquid multiplication medium of step (b); the initial concentration of 6-furfurylaminopurine in the solid multiplication medium of step (a) is about two and one-half times the initial concentration of 6-furfurylaminopurine in the liquid multiplication medium of step (b); and the initial concentration of 6-benzylaminopurine in the solid multiplication medium of step (a) is about two and one-half times the initial concentration of 6-benzylaminopurine in the liquid multiplication medium of step (b). ### Claim 16 (depends on 1) The method of claim 1, further comprising culturing the conifer embryogenic tissue in or on the solid multiplication medium of step (a) for a period of time to produce about 50 mg to about 250 mg fresh weight biomass. ### Claim 17 (depends on 1) The method of claim 1, further comprising culturing the conifer embryogenic tissue multiplied in step (b) in or on development medium to form conifer cotyledonary embryos. ### Claim 18 (depends on 1) The method of claim 1, wherein the conifer embryogenic tissue is pine embryogenic tissue. ### Claim 19 (depends on 18) The method of claim 18, wherein the pine embryogenic tissue is Loblolly pine embryogenic tissue.