Method for producting a cereal plant with foreign DNA

• US 5,767,368 A
• Filed: 04/03/1995
• Issued: 06/16/1998
• Est. Priority Date: 04/03/1995
• Status: Expired due to Fees

• Current Assignee: Michigan State University Board of Trustees Operating
• Original Assignee: The Board of Trustees of Michigan State University (Michigan State University)
• Inventors: Zhong, Heng, Sticklen, Masomeh B.
• Primary Examiner(s): Robinson, Douglas W.
• Assistant Examiner(s): HAAS, THOMAS M
• Application Number:
  US08/415,426
• Time in Patent Office: 1,170 Days
• Field of Search: 435/172.3, 435/240.4, 435/320.1, 435/240.45, 435/240.5, 435/413, 435/418, 435/419, 435/424, 435/430, 435/431, 800/205, 800/DIG. 56, 536/23.6, 536/23.7, 536/24.1
• US Class Current: 800/279
• CPC Class Codes: C12N 15/8207 by mechanical means, e.g. microinjection, particle bombardment, silicon whiskers
• CPC Class Codes: C12N 15/8274 for herbicide resistance
• CPC Class Codes: C12N 15/8286 for insect resistance
• CPC Class Codes: Y02A 40/146 Genetically Modified [GMO] plants, e.g. transgenic plants

Abstract

A method for isolating a cereal plant with foreign DNA by bombarding meristem primordia tissue with particles coated with the foreign DNA in a culture media is described. The foreign DNA in the transformed plants can provide proteins which impart disease and/or insect resistance or other desirable properties.

Claims

1. A method for producing a maize plant with foreign DNA encoding a herbicide or insecticide resistance which comprises:

(a) providing isolated shoot apex meristem primordia of germinated embryos of the maize which have been separated from a coleoptile and exposed by removal of leaf primordia of the plant in a first culture medium comprising a cytokinin selected from the group consisting of N₆ -benzyladenine, 6-furfurylamino-purine and 6-(4-hydroxy-3-methylbut-2-enylamino)purine at a concentration between about 0.001 to 10 μ

M and optionally an auxin selected from the group consisting of 3,6-dichloro-o-anisic acid, 2,4-dichlorophenoxyacetic acid, indole-3-acetic acid, indole-3-butyric acid and α

-naphthaleneacetic acid at a concentration of between about 0.001 to 10 μ

M with frequent subculturing over time to enable the meristem primordia to be transformed with the foreign DNA;

(b) bombarding the subcultured meristem primordia with micron sized particles coated with the foreign DNA to transform the meristem primordia with the DNA;

(c) growing the bombarded meristem primordia in a second culture medium containing the auxin and optionally the cytokinin and selecting for transformed meristem primordia using the herbicide; and

(d) regenerating the meristem primordia containing the foreign DNA in a culture medium containing the auxin to produce the transformed maize plant which has the herbicide or insect resistance.

2. The method of claim 1 wherein in addition the foreign DNA encodes an enzyme which is used as a visual selection marker.

3. The method of claim 2 wherein the enzyme is β

-glucuronidase.

4. The method of claim 1 wherein the first and second culture medium contains casein hydrolysate and N₆ -benzyladenine and optionally 2,4-dichlorophenoxyacetic acid and the meristem primordia in step (a) are cultured in darkness for four weeks and then in the light for four weeks.

5. The method of claim 4 wherein the first culture medium and second culture medium contain MS medium.

6. The method of claim 5 wherein the first culture medium and second culture medium contain 0.5 to 1 g/liter casein hydrolysate and the 2,4-dichlorophenoxyacetic acid.

7. The method of claim 1 wherein the foreign DNA includes a promoter.

8. The method of claim 7 wherein the promoter is rice actin 1 promoter.

9. The method of claim 8 wherein the foreign DNA includes a 5'"'"' intron and a 5'"'"' non-coding exon.

10. The method of claim 1 wherein the foreign DNA encodes a bar gene for herbicide resistance.

11. The method of claim 1 wherein the foreign DNA encodes a pin 2 gene for insect resistance.

12. The method of claim 1 wherein the foreign DNA encodes a bar gene for resistance to glufosinate ammonium and a pin-2-gene for insect resistance.

13. The method of claim 1 wherein the foreign DNA is contained in plasmid pTW-a.

14. The method of claim 1 wherein the auxin in step (c) is indole-3-butyric acid (IBA).

15. The method of claim 14 wherein the indole-3-butyric acid is present in an amount between 0.001 and 10 μ

M.