Methods for producing transformed plants

Patent metadata

Field	Value
Publication number	US-12559762-B2
Application number	US-202118026102-A
Country	US
Kind code	B2
Filing date	Sep 15, 2021
Priority date	Sep 16, 2020
Publication date	Feb 24, 2026
Grant date	Feb 24, 2026

Abstract

Methods for highly efficient and fast generation of transformed plants, highly efficient, mild and fast generation methods for introducing a biomolecule into an intact plant cell by a laser-assisted transfection method, and the regeneration of cell lines, whole tissues or organisms thereof.

First claim

What is claimed is:

1 . A method of producing a transformed plant by introducing an expression vector into an intact plant cell with a laser-assisted transfection method comprising the steps of:
(i) providing an intact plant or a plant explant isolated from any organ and or tissue of a plant and comprising intact plant cells or an isolated plant cell with a cell wall; (ii) providing an expression vector and a selectable marker; (iii) delivering said expression vector and the selectable marker into the plant cell by a laser-assisted transfection method, wherein the cell or tissue is surrounded by a liquid, and the laser focal point is within the liquid adjacent to the cell with a cell wall and not directly on the cell with a cell wall; (iv) identifying the cells transfected with the expression vector; and (v) selecting the cells transfected with the expression vector and regenerating the cells to intact plantlets by growing in and/or on a growth media.

2 . The method according to claim 1 , further comprising the step of screening the transformed plants, optionally by genetic characterization.

3 . The method according to claim 1 , wherein the selectable marker is a visual marker, a gene encoding a visual marker, a gene that confers resistance to antibiotic or any other marker that can be used to select and/or identify transformed plants and/or plant tissue or is a combination of more than one selectable markers, and wherein the cells transfected with the expression vector and the selectable marker are identified by microscopy and/or by cultivation on solid medium containing appropriate selective agents.

4 . The method according to claim 1 , wherein the expression vector comprises a gene of interest and a selectable marker gene, and wherein the expression vector is delivered into the plant cell in combination with a separate visual marker molecule.

5 . The method according to claim 1 , wherein the laser irradiation conditions are adjusted for delivering of the expression vector by the laser-assisted transfection method with a cell membrane impermeable fluorescent substance, which is not passively or actively taken up by the intact plant cell.

6 . A method for introducing a biomolecule into an intact plant cell by a laser-assisted transfection method comprising the steps of:
(i) providing a plant explant isolated from any organ and or tissue of a plant and comprising intact plant cells or an isolated plant cell with a cell wall and a selectable marker; (ii) providing a biomolecule and a selectable marker; and (iii) delivering said biomolecule and the selectable marker into the plant cell by a laser-assisted transfection method, wherein the cell or tissue is surrounded by a liquid and the laser focal point is within the liquid adjacent to the cell with a cell wall and not directly on the cell with a cell wall.

7 . The method according to claim 6 , wherein the biomolecule is selected from the group consisting of DNA, RNA, a polypeptide, a protein and a combination thereof.

8 . The method according to claim 6 , wherein the method further comprises the step of screening of the transfected plant cells, optionally by genetic characterization.

9 . The method according to claim 6 , wherein the selectable marker is a visual marker, a gene encoding a visual marker, a gene that confers resistance to antibiotic or any other marker that can be used to select and/or identify transformed plants and/or plant tissue or is a combination of selectable markers, and wherein the cells are transfected with an expression vector and the selectable marker is identified by microscopy, and/or by cultivation on solid medium containing appropriate selective agents.

10 . The method according to claim 9 , wherein the expression vector comprises a gene of interest and a selectable marker gene, and wherein the expression vector is delivered into the plant cell in addition with a separate visual marker molecule.

11 . The method according to claim 6 , wherein the method further comprises the step of selecting the cells transfected with the biomolecule and optionally regenerating the cells to intact plantlets by growing in and/or on a growth media.

12 . The method according to claim 6 , wherein a plurality of different biomolecules are introduced into an intact plant.

13 . The method according to claim 6 , wherein the laser irradiation conditions are adjusted for delivering the biomolecule by the laser-assisted transfection method.

14 . The method according to claim 1 , wherein the laser is a multiphoton laser operating under pulsing conditions and wherein the laser power is between 0.5 watts and 3 watts and the wavelength is between 700 nm and 900 nm.

15 . The method according to claim 1 , wherein said plant is selected from the group consisting of bean, pea, soybean, sunflower, cereal species including maize, rice, wheat and trees including Gymnosperms and hardwood.

16 . The method according to claim 3 , wherein the microscopy is fluorescence microscopy.

17 . The method according to claim 9 , wherein the microscopy is fluorescence microscopy.

Assignees

• Fraunhofer Ges Forschung

Inventors

• Augustine Sruthy Maria
• Vadakan Cherian Anoop
• Seiling Kerstin
• Raven Nicole
• Di Fiore Stefano
• Schillberg Stefan
• Commandeur Ulrich

Classifications

• C12N15/8206 Primary

  by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated · CPC title