IP Library › Granted Patent US 12,201,075
Granted Patent B2
US 12,201,075 · App. 17/500,864 · Granted Jan 21, 2025

Methods and compositions for axillary shoot micropropagation of

Inventors: David S. Hart (Lenexa, KS); Jonathan McGiveron (Sparks, NV); Michael Kane (Gainesville, FL)
Assignees: Jushi IP, LLC; Phytotech Labs, Inc.
A01H4/008A01H4/005A01H6/28
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Quick Facts
Patent No.
US 12,201,075
App. No.
17/500,864
Granted
Jan 21, 2025
Kind
B2
Abstract

A plant medium composition and methodology of application of the medium as it pertains to the micropropagation of Cannabis and related plant species is described that yields two to four-fold increase in the number of biologically functional nodes from axillary shoots thereby allowing for exponential amplification of such plant species. The compositions comprise basal plant media supplemented with: (i) one or more cytokinins, and (ii) one or more gibberellins and/or brassinolides. This achievement in exponential shoot growth of these plant species represents a marked step forward in the Cannabis industry as it pertains to commercial micropropagation of a recreationally and medicinally important plant whose extracts are the subject of numerous clinical trials.

Claims (51)

1. A composition comprising a plant tissue from a Cannabaceae plant and a sterile culture medium comprising:

(i) a basal plant medium, wherein the basal plant medium is Driver Kuniyaki Walnut (DKW) medium,

(ii) one or more ribosides selected from 6-(3,3-dimethylallylamino)-9-0-D-ribofuranosylpurine (iPR), 6-((E)-4-hydroxy-3-methylbut-2-enylamino)-9-0-D-ribofuranosylpurine (tZR), and 6-(4-hydroxy-3-methylbutylamino)-9-O-D-ribofuranosylpurine (DHZR), and

(iii) gibberellic acid A3,

wherein the culture medium is essentially free of auxins,

wherein the culture medium stimulates axillary shoot growth of Cannabaceae grown in a sterile tissue culture to a greater extent than Cannabaceae grown in the sterile tissue culture without the culture medium, and

wherein the culture medium stimulates axillary shoot growth of Cannabaceae grown in the sterile tissue culture to a greater extent than DKW medium.

2. The composition of claim 1 , wherein the composition further comprises one or more brassinolides.

3. The composition of claim 1 , wherein the culture medium stimulates axillary shoot growth of Cannabaceae grown in sterile tissue culture at a rate of approximately 1.5 nodes or more per month more than Cannabaceae grown in sterile tissue culture without the culture medium.

4. The composition of claim 1 , wherein the one or more ribosides are present in a concentration of from 0.1 μM to 6.0 μM.

5. The composition of claim 1 , wherein the one or more ribosides is iPR or tZR.

6. The composition of claim 1 , wherein the culture medium stimulates axillary shoot growth of Cannabaceae grown in sterile tissue culture at a rate of approximately 2.8 nodes or more per month more than Cannabaceae grown in sterile tissue culture without the culture medium.

7. The composition of claim 1 , wherein the one or more ribosides are present in a concentration of from 0.1 μM to 30 μM.

8. The composition of claim 1 , wherein the gibberellic acid A3 is present in a concentration of from 0.1 μM to 9.0 μM.

9. The composition of claim 2 , wherein the one or more brassinolides are one or more of 2,4-epi-brassinolide, 2,8-homo-brassionolide, and 2,4-epi-castasterone.

10. The composition of claim 1 , wherein the culture medium further comprises one or more of: vitamins, carbohydrates, buffers, amino acids, complex organic ingredients, salts, and gelling agents.

11. The composition of claim 1 , wherein the culture medium further comprises a gelling agent that is one or more of agar, gellan gum, xantham gum, guar gum, gum arabic, agargellan, and carrageenan.

12. A plant tissue culture comprising a plant tissue excised from a Cannabaceae plant and a culture medium;

wherein the culture medium is a sterile composition comprising:

(i) a basal plant medium, wherein the basal plant medium is Driver Kuniyaki Walnut (DKW) medium,

(ii) one or more ribosides, wherein the one or more ribosides is selected from 6-(3,3-dimethylallylamino)-9-β-D-ribofuranosylpurine (iPR), 6-((E)-4-hydroxy-3-methylbut-2-enylamino)-9-β-D-ribofuranosylpurine (tZR), and 6-(4-hydroxy-3-methylbutylamino)-9-β-D-ribofuranosylpurine (DHZR), and

(iii) gibberellic acid A 3 ,

wherein the gibberellic acid A 3 is present in the composition in a concentration of from 0.5 μM to about 7.0 μM or from about 1.0 μM to about 7.5 μM

wherein the composition stimulates axillary shoot growth of Cannabaceae grown in the tissue culture to a greater extent than Cannabaceae grown in sterile tissue culture without the composition.

13. The plant tissue culture of claim 12 ,

wherein the basal plant medium further comprises one or more of the following components:

Mg 2+ at a concentration of from about 0.10 mM to about 10 mM;

SO 4 2− at a concentration of from about 0.1 mM to about 15 mM;

Ca 2+ at a concentration of from about 0.1 mM to about 10 mM;

K + at a concentration of from about 1 mM to about 40 mM;

PO 4 3− at a concentration of from about 0.1 mM to about 10 mM;

iron at a concentration of from about 1×10 −3 mM to about 1.0 mM; and

EDTA at a concentration of from about 1×10 −3 mM to about 1 mM.

14. The plant tissue culture of claim 12 , further comprising:

(iv) one or more brassinolides.

15. The plant tissue culture of claim 12 , wherein the riboside is iPR or tZR.

16. The plant tissue culture of claim 12 , wherein the gibberellic acid A 3 is present in the composition in a concentration of from about 1.0 μM to about 7.5 μM.

17. The composition of claim 1 , wherein the culture medium is a gelled composition comprising DKW, tZR, water, and gibberellic acid A 3 .

18. A plant tissue culture comprising a plant tissue from a Cannabaceae plant and a sterile gelled Cannabaceae growth composition comprising Driver Kuniyaki Walnut (DKW) medium, and the following:

(a)-6-(3,3-dimethylallylamino)-9-(3-D-ribofuranosylpurine (iPR),

(b) 6-(4-hydroxy-3-methylbutylamino)-9-(3-D-ribofuranosylpurine (DHZR),

(c) gibberellic acid A 3 , and (d) 2,4-epi-brassinolide,

wherein the gelled composition is essentially free of auxins; and

wherein the gelled composition stimulates axillary shoot growth of the plant tissue to a greater extent than the Cannabaceae plant grown in the tissue culture without the composition.

19. A composition, comprising a plant tissue from a Cannabaceae plant and a gelled sterile culture medium comprising:

(a) Driver Kuniyaki Walnut (DKW) medium and at least one of 6-(3,3-dimethylallylamino)-9-(3-D-ribofuranosylpurine (iPR), and 6-(4-hydroxy-3-methylbutylamino)-9-(3-D-ribofuranosylpurine (DHZR),

(b) one or more of vitamins, carbohydrates, buffers, amino acids, salts, and gelling agents,

(c) gibberellic acid, and

(d) water,

wherein the gelled sterile culture medium has a pH of about 5.6 to 5.8 and comprises no auxins, and

wherein the gelled sterile culture medium stimulates axillary shoot growth of Cannabaceae grown in a tissue culture to a greater extent than Cannabaceae grown in the sterile tissue culture without the culture medium.

Assignments (4)
SECURITY INTEREST Recorded Mar 27, 2026
From: JUSHI IP, LLC
To: FG AGENCY LENDING LLC
Reel/Frame 074212/0363 →
CORRECTIVE ASSIGNMENT TO CORRECT THE PRIORITY U. S. PROVISIONAL APPLICATION NO. FROM 60/091,181 TO U. S. PROVISIONAL APPLICATION NO. 63/091,181 PREVIOUSLY RECORDED ON REEL 69398 FRAME 952. ASSIGNOR(S) HEREBY CONFIRMS THE ASSIGNMENT. Recorded Dec 3, 2024
From: NULEAF SPARKS CULTIVATION, LLC
To: JUSHI IP, LLC
Reel/Frame 069503/0675 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Nov 25, 2024
From: NULEAF SPARKS CULTIVATION, LLC
To: JUSHI IP, LLC
Reel/Frame 069398/0952 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jan 20, 2022
From: HART, DAVID; MCGIVERON, JONATHAN; KANE, MICHAEL
To: NULEAF SPARKS CULTIVATION, LLC; PHYTOTECH LABS, INC.
Reel/Frame 058716/0287 →
Continuity (2)
Provisional Application 63091181 · Oct 13, 2020
Related Publication 20220110278A1 · Apr 14, 2022
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