Cacao cell suspension protocol

Patent metadata

Field	Value
Publication number	US-11925166-B2
Application number	US-202017617215-A
Country	US
Kind code	B2
Filing date	Jun 5, 2020
Priority date	Jun 7, 2019
Publication date	Mar 12, 2024
Grant date	Mar 12, 2024

Abstract

Provided herein are methods for establishing Theobroma cell suspension cultures using young leaves as a source of explants. Also provided are induction, proliferation, and suspension media used for producing Theobroma cell suspension cultures. These methods and media may be useful for producing secondary metabolites in Theobroma , as well as for isolating virus particles associated with Theobroma diseases.

First claim

What is claimed is:

1. A method for preparing a Theobroma cell suspension culture, comprising:
obtaining an explant from a young Theobroma leaf, wherein the Theobroma is Theobroma cacao;
sterilizing the explant;
inducing friable callus from the explant on a callus induction medium, wherein the callus induction medium comprises:
a base plant medium;
1-naphthaleneacetic acid (NAA) at a concentration of 1 mg/L; and
6-benzylaminopurine (BAP) at a concentration from 0.5 mg/L to 1 mg/L;

proliferating the induced callus on a callus proliferation medium, wherein the callus proliferation medium comprises: the base plant medium and tidiazuron, and
wherein the base plant medium comprises:
woody plant medium salts;
vitamins;
L-glutamine;
myo-inositol; and
sucrose; and

suspending the proliferated callus in a suspension medium, wherein the suspension medium comprises:
salts;
vitamins;
L-glycine;
sucrose;
penicillin;
2,4 -dichlorophenoxyacetic acid (2,4-D); and
kinetin.

2. The method of claim 1 , wherein the sterilization comprises contacting the explant in a hypochlorite solution under vacuum condition.

3. The method of any one of claim 1 , wherein the induction of friable callus is obtained after a photoperiod of 16 hours of light and 8 hours of dark.

4. A Theobroma cell suspension culture produced by the method of any one of claim 1 .

5. A method for producing a Theobroma secondary metabolite, comprising:
obtaining an explant from a young Theobroma leaf, wherein the Theobroma is Theobroma cacao;
sterilizing the explant;
inducing friable callus from the explant on a callus induction medium, wherein the callus induction medium comprises:
a base plant medium;
1-naphthalene acetic acid (NAA) at a concentration of 1 mg/L; and
6-benzylaminopurine (BAP) at a concentration from 0.5 mg/L to 1 mg/L;

proliferating the transformed friable callus on a callus proliferation medium,
wherein the callus proliferation medium comprises: the base plant medium and tidiazuron, and
wherein the base plant medium comprises:
woody plant medium salts;
vitamins;
L-glutamine;
myo-inositol; and
sucrose;

suspending the proliferated callus in a suspension medium, wherein the suspension medium comprises:
salts;
vitamins;
L-glycine;
sucrose;
penicillin;
2,4-dichlorophenoxyacetic acid (2,4-D); and
kinetin (KIN); and
recovering a secondary metabolite from the suspension medium.

6. The method of claim 5 , further comprising transforming the induced callus with an expressible transgene that encodes a product in the biosynthetic pathway of the secondary metabolite.

7. The method of claim 6 , wherein the transformation is achieved by particle bombardment or by Agrobacterium mediation.

8. The method of any one of claim 5 , wherein the secondary metabolite is a phenol, a flavonoid, a methylxanthine, or a fatty acid.

9. The method of any one of claim 5 , wherein the sterilization comprises contacting the explant in a hypochlorite solution under vacuum.

10. The method of any one of claim 5 , wherein the induction of friable callus is obtained after a photoperiod of 16 hours of light and 8 hours of dark.

11. A Theobroma secondary metabolite produced by the method of any one of claim 5 .

12. A callus induction medium for inducing friable callus from Theobroma leaf explants, wherein the Theobromao is Theobroma cacao comprising:
a base plant medium;
1-naphthaleneacetic acid (NAA) at a concentration of 1 mg/L, and
6-benzylaminopurine (BAP) at a concentration from 0.5 mg/L to 5 mg/L.

13. A callus proliferation medium for proliferating friable callus induced from Theobroma leaf explants, wherein the Theobroma is Theobroma cacao , comprising:
a base plant medium wherein the base plant medium comprises:
woody plant medium salts;
vitamins;
L-glutamine;
myo-inositol; and
sucrose; and
optionally one or more vitamins; and
tidiazuron (TDZ) at a concentration from 1 mg/L to 5 mg/L.

Assignees

• Mars Inc

Inventors

• Garcia Claudia Yanet
• Britto Dahyana
• Marelli Jean-Philippe
• Silva Lidiane Dos Santos

Classifications

• A61K36/5777

  Theobroma, e.g. cocao or cocoa · CPC title

• A01H6/60 Primary

  Malvaceae, e.g. cotton or hibiscus · CPC title

• A01H4/002

  Culture media for tissue culture · CPC title

• A01H4/005

  Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques · CPC title

• A01H5/10

  Seeds · CPC title