Transformation of soybeans

• US 20030046733A1
• Filed: 09/06/2001
• Published: 03/06/2003
• Est. Priority Date: 09/06/2001
• Status: Abandoned Application

• Current Assignee: Kalyani Mallika Dias
• Original Assignee: Kalyani Mallika Dias
• Inventors: Dias, Kalyani Mallika
• Application Number:
  US09/948,292
• Publication Number:
  US 20030046733A1
• Time in Patent Office: Days
• US Class Current: 800/294
• CPC Class Codes: C12N 15/8205 Agrobacterium mediated transformation

Abstract

Soybean are transformed by inserting a functional gene into an explant of a soybean (particularly after being pre-treated with high doses of cytokinin (hormone)), transferring embryonic axes explants of the mature soybean seeds incubated on wet filter papers in the presence of at least one phenol compound naturally produced when plant cells have been wounded, to induce vir genes, and incubated in the dark in such presence at 20° C.-25° C. for at least 24 hr. After incubation, the explants are transferred to a media to develop shoots from explants, control Agrobaterium growth, and after shoot elongation, separated shoots, with or without roots, are either transferred to soil, or contacted with at least 1 mg/l IBA before transplant.

Claims

1. A method for transforming soybean comprising:

a) inserting a functional gene into a cell of a soybean explant of a soybean seed;

b) explants of the soybean seeds developed from step a) are then transferred to a porous substrate for incubation;

c) after incubation, the explants are transferred to a media of macro and micro salts supplemented with 1×

B5 vitamins; and

d) after shoot elongation, separated shoots, with or without roots, are either

i. transferred to soil;

or

ii. contacted with at least 1 mg/l IBA before transplant.

2. The method of claim 1 wherein a soybean is first pretreated with hormone before step a).

3. The method of claim 2 wherein the hormone is a cytokinin preferably 6-benzyleaminopurine.

4. The method of claim 2 wherein the hormone comprises at least about 40 mg/L of 6-benzyleadinopurine for at least 1.5 hours.

5. The method of claim 1 wherein Agrobacterium is used for inserting the functional gene into the cell.

6. The method of claim 5 wherein embryonic axes explants of the soybean seeds are developed in claim 1 step a) and are transferred to the porous substrate.

7. The method of claim 6 wherein the transfer to the porous substrate is carried out in the presence of at least one phenol compound naturally produced when plant cells have been wounded, to induce vir genes.

8. The method of claim 7 wherein incubation is in the dark in the presence of the at least one phenol compound at 20°

C.-25°

C. for at least 24 hours.

9. The method of claim 1 step c) wherein the shoot regeneration is performed in the presence of an antibiotic to control Agrobacterium growth.

10. The method of claim 9 wherein the shoot regeneration is performed in the presence of a selection agent.

11. The method of claim 10 wherein the selection agent comprises an antibiotic or a herbicide or a growth inhibitor.

12. The method of claim 11 wherein the selection agent is chosen from the group consisting of kanamycin, bialophos, hygromycin, and glyphosate.

13. The method of claim 1 wherein the porous medium contains a signal compound.

14. The method of claim 5 wherein the porous medium contains a signal compound.

15. The method of claim 7 wherein the porous medium contains a signal compound.

16. The process of claim 14 wherein the signal compound is selected from the group consisting of acetosyringone, alpha-hydroxyacetosyringone, acetovillone, syringaldehyde, syringic acid, sinapinic acid, and mixtures thereof.

17. The process of claim 1 wherein the porous medium comprises paper.

18. The process of claim 5 wherein the porous medium comprises paper.

30. The process of claim 2 wherein the pretreatment is effected with an effective amount of a cytokinin.

31. The process of claim 30 wherein the cytokinin is present in a concentration of at least 20 mg/L.

32. The method of claim 1 wherein the insertion of the functional gene into the cell is done by a mechanical or physical DNA delivery method.

33. The method of claim 32 wherein the mechanical or physical DNA delivery method is selected from the group consisting of biolistic delivery, whisker delivery, or electroporation.

19. A method of pretreating soybean tissue for development into a transgenic soybean plant comprising:

a) Pre-treating sterile seeds with a relatively high concentration of at least about 40 mg/L of 6-benzyleaminopurine for at least 1.5 hours.

20. The method of claim 19 wherein the relatively high concentration of a solution of 6-bezyleaminopurine between about 40-100 mg/L.

21. The method of claim 20 wherein the pretreatment is performed for at least 2.5 hours.

22. The method of claim 20 wherein after the pretreatment, an explant derived from embryonic axes of a mature soybean seed is co-cultivated with Agrobacterium species containing a chimeric gene in the presence of liquid plant tissue culture media.

23. The method of claim 20 wherein after the pretreatment, an explant derived from embryonic axes of a mature soybean seed is co-cultivated with Agrobacterium species containing a chimeric gene in the presence of plant media.

24. The method of claim 22 wherein virulence is induced in the Agrobacterium by incubating the tissues at a temperature between 20°

C.-25°

C.

25. The method of claim 22 wherein virulence is induced in the Agrobacterium by incubating the tissues at a temperature between 22°

C.-24°

C.

26. The process of claim 22 wherein co-cultivation is performed on porous support media.

27. The process of claim 26 wherein the porous support medium comprises paper.

28. The process of claim 26 wherein the porous support medium contains a signal compound.

29. The process of claim 28 wherein the porous support medium contains a signal compound selected from the group consisting of acetosyringone, alpha-hydroxyacetosyringone, acetovanillone. syringaldehyde, syringic acid, sinapinic acid, and mixtures thereof.