# US 10,480,001 B2 · Method for the generation of a monoclonal plant cell line

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- Number: US 10,480,001 B2
- Document ID: US10480001B2
- Status: Granted
- Kind: B2
- Application number: 14/115,629
- Filed: 2011-06-07
- Published: 2014-02-20
- Granted: 2019-11-19
- Earliest claimed: 2011-06-07
- Assignees: Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V.
- Applicants: Janina Kirchhoff; Stefan Schillberg; Andreas Schiermeyer; Helga Schinkel; Rainer Fischer
- Inventors: Janina Kirchhoff; Stefan Schillberg; Andreas Schiermeyer; Helga Schinkel; Rainer Fischer
- Web page: https://patentgrep.com/patent/US10480001B2

## CPC

- C12N 15/8241: {Phenotypically and genetically modified plants via recombinant DNA technology}
- A01H 4/005: {Methods for micropropagation; Vegetative plant propagation using cell or 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)
- A01H: NEW PLANTS OR {NON-TRANSGENIC} PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES

## Abstract

The invention provides a method for the generation of a monoclonal plant cell line from a heterologous population of plant cells, comprising the following steps: (a) provision of a heterologous population of plant cells; (b) preparation of protoplasts from said heterologous population of plant cells; (c) separation of single protoplasts by subjecting the preparation of protoplasts to flow cytometric sorting; (d) regeneration of a separated single transformed protoplast until the formation of a microcolony by co-cultivation in the presence of feeder cell material; (e) removal of the microcolony from the feeder cell material and cultivation of the microcolony until the formation of a monoclonal plant cell line.

## Claims

### Claim 1

A method for the generation of a monoclonal plant cell line from a heterogeneous population of plant cells, comprising the following steps:
(a) providing a heterogeneous population of plant cells;
(b) preparing a heterogeneous population of protoplasts from the heterogeneous population of plant cells, wherein at least one of the protoplasts from the prepared heterogeneous population of protoplasts is transformed with at least one expression vector encoding at least one fluorescent protein and a resistance against at least one selection agent;
(c) initiating cell wall regeneration in said heterogeneous population of protoplasts for fluorescence activated cell sorting (FACS);
(d) providing a cell deposition device containing feeder cells in liquid medium containing wells;
(e) sorting by FACS the prepared at least one protoplast from the heterogeneous population of protoplasts in which the cell wall regeneration has been initiated, wherein said at least one protoplast is highly fluorescent;
(f) separating the sorted at least one protoplast into at least one different liquid medium-containing well containing the feeder cells for monoclonal plant cell generation such that said at least one well for monoclonal plant cell line generation contains a single prepared protoplast permitted to directly contact the feeder cells;
(g) regenerating the single separated protoplast from said at least one well into a microcolony by co-cultivating the separated protoplast in the same well as the feeder cells; and
(h) removing the microcolony from the feeder cells and cultivating the microcolony in the presence of said at least one selection agent against which said at least one protoplast is transformed with said at least one expression vector encoding a resistance until a monoclonal plant cell line from said at least one protoplast is established.

### Claim 2 (depends on 1)

The method according to claim 1, further comprising regenerating the monoclonal plant cell line established in step (h) into whole fertile plants.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the at least one protoplast of step (b) is additionally transformed to produce one or more desired products selected from the group consisting of heterologous proteins or polypeptides, secondary metabolites, and markers for diagnostic or analytic purposes and wherein the monoclonal plant cell line of step (h) produces said one or more desired products.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the heterogeneous population of plant cells is provided from an already transgenic heterogeneous plant suspension culture comprising transgenic cells.

### Claim 5 (depends on 1)

The method according to claim 1, wherein the at least one protoplast is transformed with a resistance against at least one antibiotic or herbicide selection agent.

### Claim 6 (depends on 1)

The method according to claim 1, wherein said monoclonal plant cell line is stable for at least 2 months.

### Claim 7 (depends on 3)

The method according to claim 3, wherein said monoclonal plant cell line produces stable amounts of said one or more desired products for at least 2 months.

### Claim 8 (depends on 1)

The method according to claim 1, wherein said monoclonal plant cell line is stable for at least 12 months.

### Claim 9 (depends on 3)

The method according to claim 3, wherein said monoclonal plant cell line produces stable amounts of said one or more desired products for at least 12 months.