# US 10,477,859 B2 · Plant embryo storage and manipulation

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- Number: US 10,477,859 B2
- Document ID: US10477859B2
- Status: Granted
- Kind: B2
- Application number: 15/507,032
- Filed: 2015-06-04
- Published: 2017-09-28
- Granted: 2019-11-19
- Earliest claimed: 2014-08-29
- Assignees: Pioneer Hi-Bred International, Inc.
- Applicants: Pioneer Hi-Bred International, Inc.
- Inventors: Randal Arnold; Matthew Paul Cope; Justin Andrew Schares; Yue Yun
- Web page: https://patentgrep.com/patent/US10477859B2

## CPC

- A01N 3/00: Preservation of plants or parts thereof, e.g. inhibiting evaporation, improvement of the appearance of leaves {or protection against physical influences such as UV radiation using chemical compositions} (preservation or chemical ripening of fruit or vegetables A23B7/00); Grafting wax
- A01H 4/00: Plant reproduction by tissue culture techniques {; Tissue culture techniques therefor}
- A01N: PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF (preservation of food or foodstuff A23); BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES (preparations for medical, dental or toiletry purposes which kill or prevent the growth or proliferation of unwanted organisms A61K); PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01H: NEW PLANTS OR {NON-TRANSGENIC} PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES

## Abstract

Methods for obtaining genetic material from plant embryos while preserving their viability are provided. In the methods, preservation of viability may be maintained by suspending the embryos in an aqueous solution surrounded by an oil matrix. Genetic material may be obtained from an aliquot of the aqueous solution and may be used directly for molecular analysis, or whole genome amplification may be performed prior to molecular analysis.

## Claims

### Claim 1

A method for performing molecular analysis of one or more plant embryos comprising agitating the plant embryos to remove cellular material for molecular analysis, storing the one or more plant embryos in an aqueous solution surrounded by a matrix of at least two oils, wherein one of the at least two oils is more dense than the aqueous solution and one of the at least two oils is less dense than the aqueous solution, obtaining genetic material from the cellular material removed from the plant embryos, and performing molecular analysis of the genetic material.

### Claim 2 (depends on 1)

The method of claim 1 wherein the one or more plant embryos comprise one or more immature plant embryos.

### Claim 3 (depends on 1)

The method of claim 1 wherein antimicrobial agents and/or minimal growth media are added to the aqueous solution.

### Claim 4 (depends on 1)

The method of claim 1 wherein the one or more plant embryos are stored under dark conditions.

### Claim 5 (depends on 1)

The method of claim 1 wherein the one or more plant embryos are stored at approximately 4 degrees Celsius.

### Claim 6 (depends on 1)

The method of claim 1 further comprising transferring the one or more plant embryos for continued storage.

### Claim 7 (depends on 1)

The method of claim 1 further comprising transferring the one or more plant embryos to a germination medium.

### Claim 8 (depends on 1)

The method of claim 1 further comprising germinating at least one of the one or more plant embryos.

### Claim 9 (depends on 1)

The method of claim 1 wherein the method of storing is automated.

### Claim 10 (depends on 1)

The method of claim 1 wherein said molecular analysis is genotyping.

### Claim 11 (depends on 1)

The method of claim 1, further comprising the step of selecting or discarding the plant embryo based on the results of the molecular analysis.

### Claim 12 (depends on 1)

The method of claim 1, wherein the plant embryo is washed in the aqueous solution prior to agitation.