# US 12,180,466 B2 · Plant tissue collection, RNA extraction and genetic analysis

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- Number: US 12,180,466 B2
- Document ID: US12180466B2
- Status: Granted
- Kind: B2
- Application number: 17/556,710
- Filed: 2021-12-20
- Published: 2022-04-14
- Granted: 2024-12-31
- Earliest claimed: 2018-02-27
- Assignees: WinField Solutions, LLC
- Applicants: Winfield Solutions, LLC
- Inventors: Jonathan Zuk; Stephanie Wedryk
- Web page: https://patentgrep.com/patent/US12180466B2

## CPC

- C12N 15/1003: {Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor}
- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- C12Q 1/6895: for plants, fungi or algae
- C12Q 2600/158: Expression markers
- 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

Methods of extracting ribonucleic acid from plant tissue involve treating multiple groups of plant seeds with different plant growth regulators. Each group of plant seeds can be treated with a different plant growth regulator. Methods further involve germinating the plant seeds to form seedlings, harvesting tissue samples from subgroups within each group of plant seeds and seedlings at different timepoints, and extracting total RNA from each harvested tissue sample. Methods can also involve sequencing the extracted RNA, determining an expression level of a plurality of genes relative to a negative control, and identifying a subset of differentially expressed genes at each distinct timepoint.

## Claims

### Claim 1

A method of treating plant tissue, the method comprising:
treating plant seeds with a plant growth regulator composition;
harvesting tissue samples from seedlings derived from the plant seeds after the plant seeds have germinated; and
extracting RNA from each harvested tissue sample,
wherein the plant growth regulator composition comprises about 0.03 wt % to about 0.2 wt % kinetin, about 0.005 wt % to about 0.15 wt % gibberellic acid, and about 0.008 wt % to about 0.17 wt % indole-3-butyric acid.

### Claim 2 (depends on 1)

The method of claim 1, wherein harvesting tissue samples comprises harvesting at least a portion of an embryo, an endosperm, a radicle, a coleoptile, or a combination thereof.

### Claim 3 (depends on 1)

The method of claim 1, wherein extracting RNA comprises extracting total RNA.

### Claim 4 (depends on 3)

The method of claim 3, further comprising performing whole-genome sequencing of the total RNA.

### Claim 5 (depends on 1)

The method of claim 1, wherein harvesting tissue samples comprises harvesting the tissue samples at multiple timepoints.

### Claim 6 (depends on 5)

The method of claim 5, wherein the timepoints comprise about 12 hours after planting, about 24 hours after planting, about 96 hours after planting, about 120 hours after planting, and/or about 144 hours after planting.

### Claim 7 (depends on 5)

The method of claim 5, wherein the multiple timepoints are each separated by at least 12 hours.

### Claim 8 (depends on 5)

The method of claim 5, wherein a unique tissue sample is harvested at each of the timepoints.

### Claim 9 (depends on 5)

The method of claim 5, wherein each of the multiple timepoints corresponds to a different developmental stage of the seedlings.

### Claim 10 (depends on 1)

The method of claim 1, wherein the plant seeds comprise one or more of corn seeds, bean seeds, wheat seeds, barley seeds, or alfalfa seeds.

### Claim 11 (depends on 1)

The method of claim 1, wherein treating plant seeds with a plant growth regulator composition comprises applying the plant growth regulator composition to the seeds and incubating the seeds for a period thereafter.

### Claim 12 (depends on 11)

The method of claim 11, wherein the period is at least about 12 hours.

### Claim 13

A method of treating plant tissue, the method comprising:
treating plant seeds with a first plant growth regulator composition at a first timepoint; and
treating seedlings derived from the plant seeds with a second growth regulator composition, different than the first plant growth regulator composition, at a second timepoint,
wherein the first and second plant growth regulator compositions comprise about 0.03 wt % to about 0.2 wt % kinetin, about 0.005 wt % to about 0.15 wt % gibberellic acid, and about 0.008 wt % to about 0.17 wt % indole-3-butyric acid.

### Claim 14 (depends on 13)

The method of claim 13, further comprising treating the seedlings with a third plant growth regulator composition, different than the first and second growth regulator compositions, at a third timepoint.

### Claim 15 (depends on 13)

The method of claim 13, wherein the first and second timepoints are separated by at least about 12 hours.

### Claim 16 (depends on 13)

The method of claim 13, wherein the plant seeds comprise one or more of corn seeds, bean seeds, wheat seeds, barley seeds, and alfalfa seeds.

### Claim 17 (depends on 16)

The method of claim 16, wherein the first plant growth regulator composition is different for each of the one or more corn seeds, bean seeds, wheat seeds, barley seeds, and/or alfalfa seeds.

### Claim 18 (depends on 16)

The method claim 16, wherein the second plant growth regulator composition is different for each of the one or more corn seeds, bean seeds, wheat seeds, barley seeds, and/or alfalfa seeds.

### Claim 19 (depends on 16)

The method of claim 16, further comprising harvesting plant tissue derived from the one or more corn seeds, bean seeds, wheat seeds, barley seeds, and alfalfa seeds at different timepoints.

### Claim 20 (depends on 16)

The method of claim 16, wherein the first timepoint is different for each of the one or more corn seeds, bean seeds, wheat seeds, barley seeds, and/or alfalfa seeds.