Method for propagating microorganisms within plant bioreactors and stably storing microorganisms within agricultural seeds

• US 10,362,787 B2
• Filed: 12/24/2014
• Issued: 07/30/2019
• Est. Priority Date: 12/24/2013
• Status: Active Grant

• Current Assignee: AIT Austrian Institute of Technology GmbH (Government of Austria)
• Original Assignee: AIT Austrian Institute of Technology GmbH (Government of Austria)
• Inventors: Mitter, Birgit, Naveed, Muhammad, Berninger, Teresa, Compant, Stephane, Sessitsch, Angela, Von Maltzahn, Geoffrey, Flavell, Richard Bailey, Toledo, Gerardo V., Djonovic, Slavica, Marquez, Luis Miguel, Johnston, David Morris, Millet, Yves Alain, Lyford, Jeffrey, Leff, Jonathan W., Samayoa, Phillip, Sadowski, Craig
• Primary Examiner(s): Ibrahim, Medina A
• Application Number:
  US15/107,965
• Publication Number:
  US 20160330976A1
• Time in Patent Office: 1,679 Days
• Field of Search: None
• CPC Class Codes: A01N 63/25 Paenibacillus
• CPC Class Codes: C12N 1/20 Bacteria; Culture media therefor
• CPC Class Codes: G01N 33/0098 Plants or trees wood G01N33/46

Abstract

The present invention relates to methods of scalably producing microorganisms by propagating them within plant tissues and introducing them into agricultural seeds to improve their shelf-life during long-term storage, to produce substances of interest, and to create libraries of microbes.

Claims

1. A method of producing a indol-3 carboxylic acid within a bacterial colonized cereal plant bioreactor, the method comprising:

a. germinating a cereal seed into which at least one inoculant bacterial endophyte has been introduced, wherein the bacterial endophyte is from the genus Burkholderia, comprises a 16S rRNA nucleic acid sequence comprising SEQ ID NO;

1446 and can grow on nitrogen free media, to produce the bacterial colonized cereal plant bioreactor; and

b. growing the bacterial colonized cereal plant bioreactor under conditions such that the bacterial endophyte proliferates and the indol-3 carboxylic acid is produced in the bacterial colonized cereal plant bioreactor, wherein the indol-3 carboxylic acid is produced at a higher concentration in the bacterial colonized cereal plant bioreactor compared to an isoline plant grown from a non-inoculated cereal seed.

2. The method of claim 1, wherein the at least one inoculant bacterial endophyte is introduced into the cereal seed by the method of:

contacting at least one flower of a cereal plant in the course of a flowering phase of the cereal plant with a preparation comprising a population of the at least one inoculant bacterial endophyte, wherein the preparation comprises 10⁶ to 10⁹ CFU per ml, whereby the inoculant bacterial endophytes enter the flowering plant via at least one flower and are conveyed to an interior of at least one seed produced by the flowering plant.

4. The method of claim 1, wherein the cereal plant is corn.

5. The method of claim 1, wherein the cereal plant is wheat.

6. The method of claim 1, wherein the indol-3 carboxylic acid improves root length.

3. A method of producing an ACC-deaminase within a bacterial colonized cereal plant bioreactor, the method comprising:

a. germinating a cereal seed into which at least one inoculant bacterial endophyte has been introduced, wherein the bacterial endophyte is from the genus Burkholderia, comprises a 16S rRNA nucleic acid sequence comprising SEQ ID NO;

1446 and can grow on nitrogen free media, to produce the bacterial colonized cereal plant bioreactor; and

b. growing the bacterial colonized cereal plant bioreactor under conditions such that the bacterial endophyte proliferates and the ACC-deaminase is produced in the bacterial colonized cereal plant bioreactor, wherein the ACC-deaminase is produced at a higher concentration in the bacterial colonized cereal plant bioreactor compared to an isoline plant grown from a non-inoculated cereal seed.

7. The method of claim 3, wherein the bacterial endophyte is introduced into the cereal seed by contacting at least one flower of a cereal plant in the course of a flowering phase of the cereal plant with a preparation comprising a population of the bacterial endophyte, wherein the preparation comprises 10⁶ to 10⁹ CFU of the bacterial endophyte—

per ml, whereby the inoculant bacterial endophyte enter the flowering plant via at least one flower and is conveyed to an interior of at least one cereal seed produced by the flowering cereal plant.

9. The method of claim 3, wherein the cereal plant is corn.

10. The method of claim 3, wherein the cereal plant is wheat.

11. The method of claim 3, wherein the ACC-deaminase improves root length.

12. The method of claim 7, wherein the cereal plant is wheat and the ACC-deaminase improves root length.

8. A method of producing a ribonucleoside diphosphate reductase within a bacterial colonized cereal plant bioreactor, the method comprising:

a. germinating a cereal seed into which at least one inoculant bacterial endophyte has been introduced, wherein the bacterial endophyte is from the genus Burkholderia, comprises a 16S rRNA nucleic acid sequence comprising SEQ ID NO;

1446 and can grow on nitrogen free media, to produce the bacterial colonized cereal plant bioreactor; and

b. growing the bacterial colonized cereal plant bioreactor under conditions such that the bacterial endophyte proliferates and the ribonucleoside diphosphate reductase is produced in the bacterial colonized cereal plant bioreactor, wherein the ribonucleoside diphosphate reductase is produced at a higher concentration in the bacterial colonized cereal plant bioreactor compared to an isoline plant grown from a non-inoculated cereal seed.

13. The method of claim 8, wherein the at least one inoculant bacterial endophyte is introduced into the cereal seed by the method of:

contacting at least one flower of a cereal plant in the course of a flowering phase of the cereal plant with a preparation comprising a population of the at least one inoculant bacterial endophyte, wherein the preparation comprises 10⁶ to 10⁹ CFU per ml, whereby the inoculant bacterial endophytes enter the flowering plant via at least one flower and are conveyed to an interior of at least one seed produced by the flowering plant.

14. The method of claim 8, wherein the cereal plant is corn.

15. The method of claim 8, wherein the cereal plant is wheat.

16. The method of claim 8, wherein the ribonucleoside diphosphate reductase improves root length.

17. The method of claim 13, wherein the cereal plant is wheat and the ribonucleoside diphosphate reductase improves root length.