Preparation and use of plant embryo explants for transformation
The present invention relates to excision of explant material comprising meristematic tissue from seeds, and storage of such material prior to subsequent use in plant tissue culture and genetic transformation. Methods for tissue preparation, storage, and transformation are disclosed, as is transformable meristem tissue produced by such methods, and apparati for tissue preparation.
1. A method for obtaining transformable soybean plant tissue comprising:
(a) obtaining a soybean plant seed;
(b) preparing an explant from the soybean plant seed under conditions wherein the explant does not germinate and remains viable and competent for genetic transformation, thereby producing transformable plant tissue; and
(c) storing said explant, wherein storing the explant is carried out for from about 1 hour to about 20 months;
wherein the soybean plant seed from which the explant is obtained has an internal moisture content of about 3-8%; and the explant has an internal moisture content of from about 3% to about 11%.
2. The method of claim 1 , wherein the explant comprises a substantially intact seed modified to expose at least a first transformable cell of the seed.
3. The method of claim 2 , wherein preparing the explant comprises drilling at least a first aperture in the seed.
4. The method of claim 2 , wherein preparing the explant comprises nicking the seed.
5. The method of claim 2 , wherein the substantially intact seed comprises a meristem of the seed.
6. The method of claim 1 , further comprising the step of:
(d) transforming at least a first cell of the explant with a selected DNA.
7. The method of claim 6 , further comprising the step of:
(e) regenerating a transgenic plant from said cell, wherein the plant is stably transformed with the selected DNA.
8. The method of claim 7 , wherein the explant is stored prior to step (e) under conditions wherein the explant does not germinate.
9. The method of claim 1 , further defined as comprising dehydrating said seed and/or explant prior to or after preparing the explant.
10. The method of claim 7 , further defined as comprising increasing the moisture content of the explant prior to or concurrently with step (e).
11. The method of claim 1 , wherein preparing the explant is carried out in the absence of a liquid.
12. The method of claim 8 , wherein the explant is stored at a temperature of between about −80° C. and about 60° C.
13. The method of claim 8 , wherein the explant is dehydrated prior to storing.
14. The method of claim 1 , further defined as comprising preparing a plurality of explants from a plurality of seeds and selecting one or more explant exhibiting at least a first characteristic indicative of the ability to transform the explant and/or regenerate a plant from the explant.
15. The method of claim 14 , wherein the characteristic is selected from the group consisting of color, size, shape, density, proximate analysis, carbohydrate content, protein content, fat content, fibre (ash) content, moisture content, viability, germplasm, intactness, pathogen presence, and optical characteristics.
16. The method of claim 6 , further defined as comprising priming the plant seed and/or the explant prior to or concurrently with step (d).
17. The method of claim 16 , wherein priming the seed comprises contacting the seed with an aqueous solution.
18. The method of claim 17 , wherein the aqueous solution comprises a disinfectant.
19. The method of claim 18 , wherein the aqueous solution comprises bleach or alcohol.
20. The method of claim 1 , wherein preparing the explant is automated.
21. The method of claim 20 , wherein preparing the explant comprises an automated process in which the plant seed is oriented as it passes through a mechanical separator to provide a substantially uniform output of regenerable meristematic plant tissue.
22. The method of claim 6 , wherein step (d) is carried out by bacterially-mediated transformation or microprojectile bombardment.
23. The method of claim 7 , wherein the transgenic plant is regenerated without producing a callus tissue culture.
24. The method of claim 7 , further defined as comprising disinfecting the plant seed and/or the explant prior to step (e).
25. The method of claim 24 , comprising disinfecting the explant.
26. The method of claim 24 , wherein disinfecting comprises application of a disinfectant selected from the group consisting of bleach, alcohol, ozone, chlorine gas, ultraviolet light, temperatures of −20° C. or lower, and exposure to a temperature higher than 50° C.
27. The method of claim 26 , wherein disinfecting comprises application of a nystatin and/or thiabendazole.
28. The method of claim 8 , wherein storing the explant is carried out for from about 1 hour to about 24 hours.
29. The method of claim 7 , wherein the method is carried out without manipulating the development of the explant with a plant growth regulator.
30. The method of claim 7 , wherein step (e) comprises culturing the explant in the presence of a selective agent at about 35° C.
31. The method of claim 7 , wherein step (e) comprises culturing the explant under lighting conditions that allow for normal plastid development.
32. The method of claim 7 , wherein at least a portion of the regenerating is performed at a temperature of up to about 40° C.
33. The method of claim 1 , wherein preparing an explant comprises:
a) subjecting a singulated seed that comprises a seed coat, cotyledon tissue, and meristematic tissue to a force sufficient to fracture the seed; and
b) separating the meristematic tissue from the seed coat and optionally the cotyledon tissue, wherein the method is mechanized.