Scalable bioreactor culture process and system for the maturation of conifer somatic embryos

• US 7,235,402 B2
• Filed: 03/21/2005
• Issued: 06/26/2007
• Est. Priority Date: 05/06/1999
• Status: Expired due to Fees

• Current Assignee: Universite Laval
• Original Assignee: Universite Laval
• Inventors: Tremblay, Francine M., Aubry, Dany, Archambault, Jean
• Primary Examiner(s): BELL, KENT L
• Application Number:
  US11/085,930
• Publication Number:
  US 20050287660A1
• Time in Patent Office: 827 Days
• Field of Search: 435/422, 435/261, 435/431, 435/420, 435/410
• US Class Current: 435/422
• CPC Class Codes: C12M 21/06 for in vitro fertilization
• CPC Class Codes: C12M 25/14 Scaffolds; Matrices in general C12N5/0068
• CPC Class Codes: C12M 29/06 Nozzles; Sprayers; Spargers; Diffusers per se B01F23/231, B01J19/26
• CPC Class Codes: C12M 41/34 of gas

Abstract

A bioreactor culture system and process for producing conifer somatic embryos comprise a closed vessel, a biomass immobilization matrix, a liquid culture medium recirculating equipment, and a gas control equipment. The biomass immobilization matrix is installed in a closed vessel, a liquid culture medium is introduced in the closed vessel, and the level of liquid culture medium in the closed vessel is kept lower than the biomass immobilization matrix. The liquid culture medium recirculating equipment sprays liquid culture medium from the closed vessel onto the biomass immobilization matrix to thereby irrigate the maturing immobilized biomass.

Claims

1. A process for the mass production of mature conifer somatic embryos in a bioreactor comprising:

(a) inoculating a suitable culture medium in said bioreactor with conifer embryogenic tissues;

(b) immobilizing said embryogenic tissues onto a biomass immobilizing matrix contained in said bioreactor, under initial flooding conditions by mixing said culture medium and said embryogenic tissues under low shear conditions until said embryogenic tissues attach to said biomass immobilization matrix;

(c) reducing the level of said culture medium in said bioreactor to a level such that only a lower end of said matrix or less, remains immersed in said medium; and

(d) subjecting said attached embryogenic tissues to a maturation step under controlled minimal humidified conditions, thereby enabling mass production of mature conifer somatic embryos.

2. The process of claim 1, wherein said level of medium in (c) is reduced to a level below that of the biomass immobilizing matrix.

3. The process of claim 1, which further comprises:

(a) installing a biomass immobilization matrix in a closed vessel of said bioreactor; and

(b) sterilizing said biomass immobilization matrix and said closed vessel, prior to the inoculating step.

4. The process of claim 1, wherein the maturation step comprises:

(a) maintaining an immobilized, maturing biomass under sterile conditions; and

(b) spraying of a liquid medium over said immobilization matrix.

5. The process of claim 1, further comprising removing most of said culture medium between said immobilizing of said tissues and said maturation step.

7. The process of claim 1, further comprising periodical nutrient refreshment or replacement.

9. The process of claim 1, further comprising gassing of said bioreactor at a controlled flow rate to maximize somatic conifer embryo production.

11. The process of claim 1, further comprising harvesting and germinating the mature somatic embryos.

13. The process of claim 1, wherein said immobilizing matrix has a vertical spiral configuration.

14. The culture of conifer somatic embryos of claim 1, wherein at least 60% of said mature somatic embryos are mature cotyledonary somatic embryos.

15. The culture of conifer somatic embryos of claim 1, wherein at least 70% of said mature somatic embryos are mature cotyledonary somatic embryos.

16. The process of claim 1, further comprising the step of partially drying said immobilization matrix to reduce humidity level following step c).

6. A process for the mass production of mature conifer somatic embryos in a bioreactor comprising:

(a) installing a biomass immobilization matrix in a closed vessel of said bioreactor;

(b) sterilizing said matrix and said closed vessel;

(c) introducing a suitable liquid culture medium in said closed vessel to immerse said matrix;

(d) adding an inoculum suspension of conifer embryogenic tissues to said liquid culture medium;

(e) mixing said liquid culture medium and said conifer embryogenic tissues under low shear conditions until said embryonic tissues attach to said immobilizing matrix and form an immobilized, maturing biomass;

(f) removing most of said liquid culture medium;

(g) maintaining said immobilized, maturing biomass under sterile conditions; and

(h) spraying of a residual or replacement liquid medium over said immobilization matrix to provide minimum relative humidity, thereby enabling mass production of mature conifer somatic embryos.

8. The process of claim 6, further comprising periodical nutrient refreshment or replacement.

10. The process of claim 6, further comprising gassing of said bioreactor at a controlled flow rate to maximize somatic conifer embryo production.

12. The process of claim 6, further comprising harvesting and germinating the mature somatic embryos.