Soybean transformation and regeneration methods

• US 5,968,830 A
• Filed: 03/28/1997
• Issued: 10/19/1999
• Est. Priority Date: 03/28/1997
• Status: Expired due to Term

• Current Assignee: Mississippi State University
• Original Assignee: Mississippi State University
• Inventors: Dan, Yinghui, Reichert, Nancy A.
• Primary Examiner(s): Robinson, D. W.
• Assistant Examiner(s): FOX, DAVID T
• Application Number:
  US08/825,469
• Time in Patent Office: 935 Days
• Field of Search: 435/172-3, 435/419, 435/426, 435/430, 800/DIG. 26
• US Class Current: 435/469
• CPC Class Codes: A01H 4/005 Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
• CPC Class Codes: C12N 15/8201 Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
• CPC Class Codes: C12N 15/8205 Agrobacterium mediated transformation
• CPC Class Codes: C12N 15/8207 by mechanical means, e.g. microinjection, particle bombardment, silicon whiskers

Abstract

A method of transforming and regenerating soybean plants relies on selection of hypocotyl explants as the target material. Hypocotyl explants can be transformed either by microparticle bombardment with DNA-coated microparticles of inert metals, or by co-culturing with an Agrobacterium strain. The transformed explants can be successfully regenerated, using a protocol including culturing on a shoot induction medium, followed by transfer to a shoot elongation medium to form rooted plantlets, which are transplanted to soil.

Claims

1. A method of regenerating soybeans (G. max) via organogenesis, comprising:

obtaining hypocotyl explants from germinated seedlings of soybean plants whose regeneration is desired,

maintaining said hypocotyl explants on a shoot induction medium comprising a cytokinin until shoots form at the acropetal end of said hypocotyl explant,

excising shoots from said hypocotyl explant and maintaining said shoots on a shoot elongation medium until said shoots are competent on a rooting medium, and maintaining said shoots on a rooting medium until rooted plantlets are formed, and

transplanting said plantlets to soil.

2. The process of claim 1, wherein said hypocotyl explant is split longitudinally, and said explant is placed, split-side down, on said shoot induction medium.

3. The method of claim 1, wherein said hypocotyl explant is maintained intact, and laid on its side or placed upright on said shoot induction medium such that the basipetal end of said explant is submerged in said medium.

4. The method of claim 1, wherein said explant is maintained under continuous darkness while on said shoot induction medium.

5. The method of claim 1, wherein said explant is maintained on a 16 hr photoperiod while on said shoot initiation/elongation medium.

6. The method of claim 1, wherein said shoot induction medium comprises benzyladenine.

8. The process of claim 7, wherein said plantlets are obtained by regeneration according to the process of claim 1.

11. The process of the claim 10, wherein said regeneration is effected pursuant to the process of claim 1.

7. A method for transforming soybean plants to express exogenous DNA, comprising:

obtaining a hypocotyl explant from germinated seedlings of a soybean plant, maintaining said explant on a shoot induction medium comprising a cytokinin for 16-32 hours, bombarding said explant, with the acropetal end facing up, with microparticles of an inert metal coated with exogenous DNA comprising a plasmid which comprises an expression gene, said expression gene encoding the expression of a protein exogenous to said soybean plant, maintaining said bombarded hypocotyl explant on a medium selective for growth of transformed tissues, followed by maintenance on a shoot elongation medium and preparing plantlets from shoots so obtained.

9. The process of claim 7, wherein said plasmid comprises a gene whose expression product may be detected by a flourometric or histochemical assay or procedure for detecting foreign DNA, and said bombarded hypocotyl plants, following growth on a medium selected for transformed tissues, are assayed for expression of said gene by said assay or procedure.

10. A method of transforming soybeans, comprising obtaining a hypocotyl soybean explant from germinated seedlings, and maintaining on its side or upright on a shoot induction medium comprising a cytokinin with a basipetal end thereof submerged in said medium,

pre-culturing and/or co-culturing an Agrobacterium strain with a virulence enhancing substance, and adding said pre- or co-cultured bacteria to the acropetal end of said upright hypocotyl explant or by complete submersion,

co-incubating said hypocotyl explant for a time sufficient to permit transfection of said hypocotyl explant by said bacteria,

disinfecting said hypocotyl explant, maintaining said hypocotyl explant on a shoot induction medium and assaying said explant for expression of introduced foreign DNA, and regenerating hypocotyl explants positive for expression of said DNA.