# US 11,111,548 B2 · Systems and methods for genotyping seed components

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- Number: US 11,111,548 B2
- Document ID: US11111548B2
- Status: Granted
- Kind: B2
- Application number: 14/473,114
- Filed: 2014-08-29
- Published: 2016-03-03
- Granted: 2021-09-07
- Earliest claimed: 2014-08-29
- Assignees: Pioneer Hi-Bred International, Inc.
- Applicants: Pioneer Hi Bred International Inc
- Inventors: Randal Arnold; Matthew Paul Cope; Justin Schares; Yue Yun
- Web page: https://patentgrep.com/patent/US11111548B2

## CPC

- C12Q 1/6895: for plants, fungi or algae
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 2600/13: Plant traits
- C12Q 2600/156: Polymorphic or mutational markers
- C12Q 2600/16: Primer sets for multiplex assays
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

The invention provides methods for obtaining genetic material from plant embryos while preserving their viability as well as methods for performing a molecular analysis of plant embryos, particularly with small quantities of genetic material. The methods may include the steps of collecting shed cellular material from one or more embryos; obtaining DNA from the shed cellular material; performing a molecular analysis of the DNA; and germinating at least one of the one or more embryos. A further extension of this method includes determining whether to germinate and grow the embryo or to discard the embryo based on its genotype as part of a breeding process.

## Claims

### Claim 1

A method of selecting maize embryonic tissue to culture into a plant with a known genotype comprising:
(a) collecting shed cellular material from maize embryonic tissue in a container comprising the maize embryonic tissue and a non-destructive medium consisting essentially of an aqueous solution, wherein the shed cellular material was obtained by agitating the container without the addition of chemicals that would induce cell lysis;
(b) removing the maize embryonic tissue from the container comprising the shed cellular material and the non-destructive medium;
(c) obtaining DNA from the shed cellular material in the non-destructive medium consisting essentially of an aqueous solution;
(d) genotyping the DNA from the shed cellular material;
(e) selecting maize embryonic tissue to culture into a plant based on the genotype of the DNA from the shed cellular material; and
(f) culturing the maize embryonic tissue to produce a maize plant with a known genotype.

### Claim 2 (depends on 1)

The method of claim 1, wherein the maize embryonic tissue comprises diploid tissue.

### Claim 3 (depends on 1)

The method of claim 1, wherein the maize embryonic tissue comprises haploid tissue.

### Claim 4 (depends on 1)

The method of claim 1, wherein the maize embryonic tissue comprises doubled haploid tissue.

### Claim 5 (depends on 1)

The method of claim 1, wherein the DNA is obtained by exposing the shed cellular material to cold and then heat followed by agitation.

### Claim 6 (depends on 5)

The method of claim 5, wherein said cold, heat, and agitation steps are repeated.

### Claim 7 (depends on 1)

The method of claim 1 wherein the DNA is obtained by heating of the shed cellular material and agitation.

### Claim 8 (depends on 7)

The method of claim 7 wherein said heat and agitation steps are repeated.

### Claim 9 (depends on 1)

The method of claim 1, wherein obtaining DNA from the shed cellular material comprises extracting the DNA by adding magnetic particles to the shed cellular material.

### Claim 10 (depends on 1)

The method of claim 1, wherein said genotyping comprises one or more of single nucleotide polymorphism detection, restriction fragment length polymorphism identification, random amplified polymorphic detection, amplified fragment length polymorphism detection, DNA sequencing, whole genome sequencing, allele specific oligonucleotide probes, and DNA hybridization to DNA microarrays or beads.

### Claim 11 (depends on 1)

The method of claim 1, further comprising performing whole genome amplification on the DNA from the shed cellular material.

### Claim 12 (depends on 1)

The method of claim 1, wherein the maize embryonic tissue has been removed from the container using an automated method.

### Claim 13 (depends on 1)

The method of claim 1, wherein the non-destructive medium consisting essentially of an aqueous solution also comprises a chromosomal doubling agent.

### Claim 14 (depends on 13)

The method of claim 13, wherein the chromosomal doubling agent is colchicine.

### Claim 15 (depends on 1)

The method of claim 1, wherein the maize embryonic tissue is stored at 4° C.

### Claim 16 (depends on 1)

The method of claim 1, wherein the maize embryonic tissue is stored to prevent germination until after the genotype of the DNA from the shed cellular material has been obtained.