# US 10,945,390 B2 · Method of selecting corn embryos

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- Number: US 10,945,390 B2
- Document ID: US10945390B2
- Status: Granted
- Kind: B2
- Application number: 15/743,191
- Filed: 2016-07-06
- Published: 2019-04-25
- Granted: 2021-03-16
- Earliest claimed: 2015-07-09
- Assignees: Syngenta Participations AG
- Applicants: Syngenta Participations AG
- Inventors: William Paul Bullock; Ulrich Stephan Hannappel; Katie Marie Hunter
- Web page: https://patentgrep.com/patent/US10945390B2

## CPC

- A01H 1/045: {using molecular markers}
- C12N 15/1003: {Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor}
- C12Q 1/6895: for plants, fungi or algae
- C12Q 2600/13: Plant traits
- A01H: NEW PLANTS OR {NON-TRANSGENIC} PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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 present invention relates to a viability-retaining method of selecting corn embryos having a desired trait, said method comprising extracting embryos, removing associated non-embryogenic tissue or DNA from the exterior of the embryo; and extracting DNA directly from the embryo. The invention surprisingly shows that germinating embryos sampled directly for their DNA content are capable of continuing the germination process to form normal seedlings.

## Claims

### Claim 1

A viability-retaining method of selecting corn embryos having a desired trait, said method comprising:
a) extracting embryos;
b) removing associated non-embryogenic tissue or DNA from the exterior of the embryo; and
c) extracting DNA directly from the embryo by contacting the embryos with a cell lysis solution which disrupts cells but does not reduce embryo viability.

### Claim 2 (depends on 1)

The method according to claim 1, wherein steps a) and b) are performed simultaneously using a liquid stream of solution.

### Claim 3 (depends on 1)

The method according to claim 1, wherein step b) comprises contacting the embryos with a solution comprising nutritional salt and optionally a carbohydrate source before DNA extraction.

### Claim 4 (depends on 1)

The method according to claim 1, wherein step c) comprises contacting the embryos with a soaking solution comprising nutritional salt and optionally a carbohydrate source after DNA extraction.

### Claim 5 (depends on 1)

The method according to claim 1, wherein step b) comprises contacting the embryos with a solution comprising a nutritional salt and optionally a carbohydrate source before DNA extraction and step c) comprises contacting the embryos with a soaking solution comprising a nutritional salt and optionally a carbohydrate source after DNA extraction.

### Claim 6 (depends on 1)

The method according to claim 1, wherein step b) comprises washing the embryos post extraction to remove associated non-embryogenic tissue or DNA before DNA extraction directly from the embryo.

### Claim 7 (depends on 6)

The method according to claim 6, wherein the washing step comprises contacting the embryos with a liquid.

### Claim 8 (depends on 6)

The method according to claim 6, wherein the washing step further comprises the use of an abrasive.

### Claim 9 (depends on 1)

The method according to claim 1, wherein the embryos are mechanically opened to further facilitate removal of DNA from ruptured embryonic cells.

### Claim 10 (depends on 1)

The method according to claim 1, wherein step b) further comprises removing contaminants enzymatically.

### Claim 11 (depends on 1)

The method according to claim 1, wherein molecular marker analysis is performed using DNA extracted from embryos.

### Claim 12 (depends on 11)

The method according to claim 11, wherein the DNA is additionally cleaned or concentrated via alcohol precipitation.

### Claim 13 (depends on 1)

The method according to claim 1, wherein the embryo is an immature embryo.

### Claim 14 (depends on 1)

The method according to claim 1, wherein the method is automated or semi-automated.