Methods and devices for creating doubled haploid embryos using oil matrices

Patent metadata

Field	Value
Publication number	US-10278345-B2
Application number	US-201615248010-A
Country	US
Kind code	B2
Filing date	Aug 26, 2016
Priority date	Aug 29, 2014
Publication date	May 7, 2019
Grant date	May 7, 2019

Abstract

Methods for preserving viability of plant tissues such as plant embryos are provided herein. Also included are methods for storing genomic DNA and/or molecular marker assay materials in an oil bilayer as part of a high-throughput molecular characterization system. Moreover, plant embryos may be treated while in an oil matrix. The treatment may include chromosome doubling, Agrobacterium -mediated transformation, or herbicide selection as part of an embryo rescue process.

First claim

We claim:

1. A method of treating one or more plant embryos with a doubling agent, said method comprising:
a. placing doubling media between two oils, wherein one of the oils is more dense than water and the other is less dense than water;
b. placing one or more plant embryos in the doubling media;
c. selecting one or more plant embryos; and
d. transferring the selected plant embryos to media for germination or storage.

2. The method of claim 1 , wherein said one or more plant embryos are haploid embryos.

3. The method of claim 1 , wherein between steps (c) and (d) cellular material is collected from the one or more plant embryos; DNA is obtained from the cellular material; and genotypic information is obtained from the one or more plant embryos.

4. The method of claim 3 , wherein said plant embryos are selected based on genotypic information.

5. A method of selecting one or more plant embryos during doubled haploid production, said method comprising:
a. placing doubling media between two oils, wherein one of the oils is more dense than water and the other is less dense than water;
b. placing one or more plant embryos in the doubling media and
c. selecting one or more plant embryos for germination or storage.

6. The method of claim 5 , wherein between steps (b) and (c) cellular material is collected from the one or more plant embryos; DNA is obtained from the cellular material; and genotypic information is obtained from the one or more plant embryos.

7. The method of claim 6 , wherein said one or more plant embryos are selected based on genotypic information.

8. The method of claim 7 , further comprising transferring the selected plant embryos to media for germination or storage.

9. The method of claim 5 , wherein said doubling media comprises an anti-microtubule agent.

10. The method of claim 5 , wherein said doubling media comprises colchicine, pronamide, dithipyr, am iprophosmethyl or trifluralin.

11. The method of claim 5 , wherein said one or more plant embryos are haploid.

12. The method of claim 5 , wherein said one or more plant embryos in step (c) are haploid maize embryos produced by a cross between a male inducer line and a female line of interest.

13. The method of claim 12 , wherein said male inducer line contains a marker gene that is expressed in embryo tissue.

14. The method of claim 13 , wherein said marker gene expresses anthocyanin pigments.

15. The method of claim 14 , wherein plant embryos that are white are selected to be transferred to media for germination or storage.

16. The method of claim 15 , wherein selection of the white plant embryos is performed using a camera or other imaging device.

17. The method of claim 15 , wherein expression of anthocyanin is enhanced by aeration of the doubling media.

18. The method of claim 15 , wherein expression of anthocyanin is enhanced by placing the plant embryos in a hypotonic doubling media comprising perfluorodecalin.

19. The method of claim 15 , further comprising germinating or storing the selected plant embryos.

Assignees

• Pioneer Hi Bred Int

Inventors

• Arnold Randal
• Barreiro Roberto
• Cope Matthew Paul
• Hunter Clifford P
• Schares Justin Andrew
• Wu Xinli Emily
• Yun Yue

Classifications

• C12N15/8205

  Agrobacterium mediated transformation · CPC title

• A01H4/006 Primary

  Encapsulated embryos for plant reproduction, e.g. artificial seeds · CPC title

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Q2600/13

  Plant traits · CPC title

• A01C1/025

  Testing seeds for determining their viability or germination capacity · CPC title