Method for producing transgenic cucumber that produces high levels of superoxide dismutase

• US 6,084,152 A
• Filed: 04/14/1999
• Issued: 07/04/2000
• Est. Priority Date: 04/14/1998
• Status: Expired due to Fees

• Current Assignee: Korea Advanced Institute of Science and Technology
• Original Assignee: Korea Advanced Institute of Science and Technology
• Inventors: Kim, Jae-Whune, Kwon, Suk Yoon, Kwak, Sang Soo, Lee, Haeng-Soon
• Primary Examiner(s): Fox, David T.
• Assistant Examiner(s): IBRAHIM, MEDINA AHMED
• Application Number:
  US09/291,562
• Time in Patent Office: 447 Days
• Field of Search: 800/278, 800/287, 800/289, 800/294, 800/307, 800/309, 800/317.1, 800/317.4, 800/313, 514/783, 435/430.1, 435/431, 435/252.3, 435/469, 435/320.1, 435/252.2, 536/23.2, 536/23.6, 536/23.1
• US Class Current: 800/287
• CPC Class Codes: A01H 4/005 Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: C12N 15/8205 Agrobacterium mediated transformation
• CPC Class Codes: C12N 15/8235 Fruit-specific
• CPC Class Codes: C12N 15/8257 for the production of primary gene products, e.g. pharmaceutical products, interferon
• CPC Class Codes: C12N 15/8277 Phosphinotricin
• CPC Class Codes: C12N 9/0089 acting on superoxide as acceptor (1.15)

Abstract

The invention relates to a transgenic plant that produces high levels of superoxide dismutase (SOD) and to a method for producing the transgenic plant. The hypocotyl section of seedlings is co-cultured with Agrobacterium transformant and regenerated by adventitious shoot induction and by root induction, where the Agrobacterium transformant contains an expression vector that comprises the promoter of a fruit-dominant ascorbate oxidase gene, an SOD gene isolated from cassava, and an herbicide-resistant bar gene. The present invention also relates to a method for inducing adventitious shoot from hypocotyl sections in plant tissue culture, thus providing a method for the efficient production of transgenic plants maintaining higher SOD activity in fruits. Therefore, the SOD transgenic cucumber of the present invention can be used for cosmetics, additives in functional foods, and medicines as well as having tolerance to herbicides and environmental stresses.

Claims

1. A method for producing a transgenic Cucurbitaceae plant comprising:

(a) constructing an expression vector for plant transformation that comprises a cucumber fruit-dominant promoter, a superoxide dismutase (SOD) gene derived from cassava, and a selectable marker gene;

(b) transferring the vector constructed in step (a) to a transformant;

(c) co-culturing the transformant of step (b) with Cucurbitaceae plant tissue; and

(d) regenerating the transformed tissue into a mature transgenic plant.

2. The method of claim 1, wherein the Cucurbitaceae plant tissue is selected from the group consisting of cucumber (Cucumis sativus L.), pumpkin (Cucurbita spp.), watermelon (Citrullus vulgaris ), and melon (Cucumis melo L.).

3. The method of claim 1, wherein the cucumber fruit-dominant promoter is an ascorbate oxidase (ASO) promoter.

4. The method of claim 1, wherein the SOD gene comprises the nucleotide sequence shown in SEQ ID NO:

1.

5. The method of claim 1, wherein the expression vector has a restriction map as shown in FIG. 1 and designated ASOp+mSOD1/pGPTV-Bar.

6. The method of claim 1, wherein the selectable marker gene comprises an herbicide-resistant gene.

7. The method of claim 6, wherein the herbicide-resistant gene comprises a bar gene.

8. The method of claim 1, wherein the transformant is Agrobacterium tumefaciens.

9. The method of claim 8, wherein the Agrobacterium tumefaciens is Agrobacterium tumefaciens LBA 4404 having KCTC Accession No. 0585BP.

10. The method of claim 1, wherein the co-culturing of step (c) comprises:

(i) excising hypocotyl segments bearing cotyledons of a germinated seedling to eliminate the root portion of the seedling;

(ii) infecting the excised hypocotyl segments bearing cotyledons with the transformant of step (b); and

(iii) culturing the infected hypocotyl segments bearing cotyledons in medium under conditions sufficient for adventitious shoot induction.

11. The method of claim 10, wherein the regenerating of step (d) comprises:

(i) inducing adventitious shoot from the infected hypocotyl segments bearing cotyledons in selective medium;

(ii) rooting the induced adventitious shoot in medium under conditions sufficient for root induction; and

(iii) acclimatizing the rooted plantlet to soil.

12. The method of claim 10, wherein the excised hypocotyl segments bearing cotyledons are approximately 2 to approximately 3 mm in length.

13. The method of claim 10, wherein the hypocotyl segments bearing cotyledons are excised at about 3 to about 5 days after germination.

14. The method of claim 10, wherein the hypocotyl segments bearing cotyledons are excised at about 3 to about 5 days after the germinated seedling has one intact cotyledon or two half-cotyledons.

15. A transgenic Cucurbitaceae plant produced by the method of claim 1.

16. A composition comprising a transgenic Cucurbitaceae plant of claim 15 or a derivative thereof, and a carrier.

17. The composition of claim 16 which is a cosmetic composition, a nutriceutical composition or a pharmaceutical composition.