WO1998048613B1 - Compositions and methods for plant transformation and regeneration - Google Patents
Compositions and methods for plant transformation and regenerationInfo
- Publication number
- WO1998048613B1 WO1998048613B1 PCT/US1998/007264 US9807264W WO9848613B1 WO 1998048613 B1 WO1998048613 B1 WO 1998048613B1 US 9807264 W US9807264 W US 9807264W WO 9848613 B1 WO9848613 B1 WO 9848613B1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- concentration
- callus
- plant
- transformed
- auxin
- Prior art date
Links
- 230000008929 regeneration Effects 0.000 title claims abstract 9
- 238000011069 regeneration method Methods 0.000 title claims abstract 9
- 230000001131 transforming Effects 0.000 title claims abstract 8
- 239000000203 mixture Substances 0.000 title claims abstract 4
- 206010020649 Hyperkeratosis Diseases 0.000 claims abstract 49
- RYGMFSIKBFXOCR-UHFFFAOYSA-N copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 claims abstract 20
- 229910052802 copper Inorganic materials 0.000 claims abstract 20
- 239000010949 copper Substances 0.000 claims abstract 20
- 230000001939 inductive effect Effects 0.000 claims abstract 13
- 210000001519 tissues Anatomy 0.000 claims abstract 12
- 230000000408 embryogenic Effects 0.000 claims abstract 4
- 241000196324 Embryophyta Species 0.000 claims 39
- GUBGYTABKSRVRQ-YOLKTULGSA-N Maltose Natural products O([C@@H]1[C@H](O)[C@@H](O)[C@H](O)O[C@H]1CO)[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 GUBGYTABKSRVRQ-YOLKTULGSA-N 0.000 claims 24
- 239000002363 auxin Substances 0.000 claims 24
- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 claims 24
- 239000004062 cytokinin Substances 0.000 claims 19
- 239000000463 material Substances 0.000 claims 18
- 230000001172 regenerating Effects 0.000 claims 13
- 210000001161 Embryo, Mammalian Anatomy 0.000 claims 11
- 150000007523 nucleic acids Chemical class 0.000 claims 11
- 108020004707 nucleic acids Proteins 0.000 claims 11
- 241000209219 Hordeum Species 0.000 claims 6
- 241000209140 Triticum Species 0.000 claims 6
- 238000004519 manufacturing process Methods 0.000 claims 5
- 244000075850 Avena orientalis Species 0.000 claims 4
- 235000007319 Avena orientalis Nutrition 0.000 claims 4
- 235000007558 Avena sp Nutrition 0.000 claims 4
- 239000004459 forage Substances 0.000 claims 4
- 235000007340 Hordeum vulgare Nutrition 0.000 claims 3
- 240000000218 Cannabis sativa Species 0.000 claims 2
- 239000002253 acid Substances 0.000 claims 2
- 230000035755 proliferation Effects 0.000 claims 2
- 230000001737 promoting Effects 0.000 claims 2
- 235000021307 wheat Nutrition 0.000 claims 2
- 239000005631 2,4-D Substances 0.000 claims 1
- OVSKIKFHRZPJSS-UHFFFAOYSA-N 2,4-Dichlorophenoxyacetic acid Chemical compound OC(=O)COC1=CC=C(Cl)C=C1Cl OVSKIKFHRZPJSS-UHFFFAOYSA-N 0.000 claims 1
- GOSWTRUMMSCNCW-HNNGNKQASA-N 9-ribosyl-trans-zeatin Chemical compound C1=NC=2C(NC\C=C(CO)/C)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O GOSWTRUMMSCNCW-HNNGNKQASA-N 0.000 claims 1
- 241000287937 Colinus Species 0.000 claims 1
- IWEDIXLBFLAXBO-UHFFFAOYSA-N Dicamba Chemical compound COC1=C(Cl)C=CC(Cl)=C1C(O)=O IWEDIXLBFLAXBO-UHFFFAOYSA-N 0.000 claims 1
- 239000005504 Dicamba Substances 0.000 claims 1
- 229960001669 Kinetin Drugs 0.000 claims 1
- QANMHLXAZMSUEX-UHFFFAOYSA-N Kinetin Natural products N=1C=NC=2N=CNC=2C=1NCC1=CC=CO1 QANMHLXAZMSUEX-UHFFFAOYSA-N 0.000 claims 1
- NWBJYWHLCVSVIJ-UHFFFAOYSA-N N-benzyladenine Chemical compound N=1C=NC=2NC=NC=2C=1NCC1=CC=CC=C1 NWBJYWHLCVSVIJ-UHFFFAOYSA-N 0.000 claims 1
- 240000007594 Oryza sativa Species 0.000 claims 1
- 235000007164 Oryza sativa Nutrition 0.000 claims 1
- 241000209504 Poaceae Species 0.000 claims 1
- 241001464837 Viridiplantae Species 0.000 claims 1
- 240000008042 Zea mays Species 0.000 claims 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims 1
- 229940023877 Zeatin Drugs 0.000 claims 1
- 230000015572 biosynthetic process Effects 0.000 claims 1
- 235000005822 corn Nutrition 0.000 claims 1
- 235000005824 corn Nutrition 0.000 claims 1
- 238000005755 formation reaction Methods 0.000 claims 1
- 235000009566 rice Nutrition 0.000 claims 1
- UZKQTCBAMSWPJD-FARCUNLSSA-N trans-zeatin Chemical compound OCC(/C)=C/CNC1=NC=NC2=C1N=CN2 UZKQTCBAMSWPJD-FARCUNLSSA-N 0.000 claims 1
Abstract
Improved compositions and methods for transformation and regeneration of plants from embryogenic callus are disclosed that include, for example: use of an intermediate-incubation medium after callus induction to increase the competence of the transformed cells for regeneration; dim light conditions during early phases of selection; use of green callus tissue as a target for microprojectile bombardment; and media with optimized levels of phytohormones and copper concentrations.
Claims
AMENDED CLAIMS
[received by the International Bureau on 24 November 1998 (24.11.98); original claims 5, 8, 9 and 11-26 amended; new claims 27-41 added; remaining claims unchanged (4 pages)]
1. A method for producing a transformed plant, comprising the steps of: introducing a nucleic acid into a cell of an embryogenic callus to produce a transformed plant cell; culturing the transformed plant cell on a callus-induction medium comprising an auxin, thereby promoting proliferation of the transformed plant cell to produce a transformed callus; culturing the transformed callus on an intermediate-incubation medium comprising an auxin and a cytokinin, thereby promoting continued proliferation and formation of a transfbπned structure that is competent to regenerate; and culturing the transformed structure on a regeneration medium to produce the transformed plant.
2. The method of claim 1 wherein the auxin is selected from the group consisting of 2,4- dichlorophenoxyacetic acid, dicamba, na hthaleneacetic acid, indoleaceύc acid, and mixtures thereof.
3. The method of claim 1 wherein the cytokinin is selected from the group consisting of 6- benzylaminopurine, zeatin, zeatin riboside, kinetin, 2iP, and mixtures thereof.
4. The method of claim 1 wherein die callus-induction medium comprises the auxin at a concentration of about 0.1 mg/L to about 5 mg/L.
5. The method of claim 1 wherein the callus-induction medium further comprises a cytokinin at a concentration of from about 0.01 mg/L to about 2.0 mg/L.
6. The method of claim 1 wherein the intermediate-incubation medium comprises d e auxin at a concentration of about 0.1 mg/L to about 5 mg/L.
7. The method of claim 1 wherein the intermediate-incubation medium comprises a cytokinin at a concentration of about 0.1 mg/L to about 5 mg/L.
8. The method of claim 1 wherein: the callus-induction medium comprises an auxin at a concentration of about 0.1 mg/L to about 5 mg/L; and the intermediate-incubation medium comprises an auxin at a concentration of about 0.1 mg/L to about 5 mg/L and a cytokinin at a concentration of about 0.1 mg/L to about 5 mg L.
9. The method of claim 8 wherein ύ e callus-induction medium comprises copper at a concentration of about 0.1 μ.M to about 50 μM.
10. The method of claim 1 wherein die intermediate -incubation medium comprises copper at a concentration of about 0.1 μM to about 50 μM.
11. The method of claim 1 wherein the callus-induction and intermediate-incubation media comprise maltose as a sugar source.
12. The method of claim 11 wherein the maltose is present at a concentration of about 30 g/L.
13. The method of claim 1 wherein culturing the transformed plant cell on a callus-induction medium comprises culturing the transformed plant cell under dim light conditions, thereby producing a green transformed callus.
14. The mediod of claim 1 wherein introducing the nucleic acid comprises bombardment of the embryogenic callus with microprojectiles coated with the nucleic acid at a rupture pressure of about 600 psi to about 900 psi.
15. The method of claim 1 wherein:
60
(a) the callus induction medium comprises an auxin at a concentration of about 0.1 mg/L to about 5 mg/L, copper at a concentration of about 0.1 μM to about 50 μM and maltose at a concentration of about 30 g/L, and
(b) the intermediate incubation medium comprises an auxin at a concentration of about 0.1 mg/L to about 5 mg/L, a cytokinin at a concentration of about 0.1 mg/L to about 5 mg/L, copper at a concentration of about 0.1 μM to about 50 μM and maltose at a concentration of about 30 g/L.
16. The method according to claims 1 or 15 wherein uie plant is selected from the group consisting of barley, wheat, oat, corn, rice, and turf and forage grasses.
17. The method according to claim 16 wherein die plant is a barley plant selected from die genotypes Golden Promise, Galena, Harrington, Morex, Moravian IE, and Salome.
18. The me od of according to claim 16 wherein the plant is a wheat plant selected from the genotypes Bobwhite, Anza, Karl and Yecora Rojo.
19. A me od of producing a transformed barley plant, comprising:
(a) introducing a nucleic acid molecule into cells of an immature zygotic barley embryo by microprojectile bombardment so as to transform at least some of the cells of the embryo;
(b) culturing the embryo in the dark for about 10-14 days;
(c) transferring the embryo to a callus induction medium comprising a sugar source, an auxin at a concentration of about 1 mg L to about 2.5 mg/L, and copper at a concentration of about 0.1 μM to about 50 μM, and incubating the embryo in the dark so that callus material is obtained;
(d) transferring the callus material to an intermediate induction medium comprising a sugar source, an auxin at a concentration of about 0.1 mg/L to about 5 mg/L, a cytokinin at a concentration of about 0.1 mg/L to about 5 mg/L and copper at a concentration of about 0.1 μM to about 50 μM, and incubating such that green structures are observed on the callus material;
(e) transferring the callus material comprising the green structures to a regeneration medium and incubating such that shoots are obtained; and
(f) transferring the shoots to rooting medium to obtain transformed barley plants.
20. The method of claim 19 wherein die sugar source comprises maltose
21. A method for producing a transformed bailey plant, comprising:
(a) placing an immature zygotic barley embryo on a callus induction medium comprising a sugar source, an auxin at a concentration of about 0.1 mg/L to about 5 mg/L, a cytokinin at a concentration of 0 mg/L to about 5 mg/L and copper at a concentration of about 0.1 μM to about 50 μM, and incubating in dim light conditions so as to form green regenerative callus material;
(b) introducing a nucleic acid molecule into the callus by microprojectile bombardment to produce transformed callus;
(c) transferring the callus to an intermediate incubation medium comprising a sugar source, an auxin at a concentration of about 0.1 mg/L to about 5 mg/L, a cytokinin at a concentration of about 0.1 mg/L to about 5 mg/L and copper at a concentration of about 0.1 μM to about 50 μM, and incubating such ttiat green structures are observed on me callus material;
(d) transferring die callus material comprising the green structures to a regeneration medium and incubatmg such that shoots are obtained; and
(e) transferring the shoots to rooting medium to obtain transformed bailey plants.
22 The method of claim 21 wherein me sugar source comprises maltose.
23. A method for producing a transformed wheat plant, comprising:
(a) placing an immature zygotic wheat embryo on a callus induction medium comprising , sugar source, an auxin at a concentration of about 0.1 mg/L to about 5 mg/L, a cytokinin at a concentration of 0 mg/L to about 5 mg/L and copper at a concentration of about 0.1 μM to about 50 μM, and incubating in dim light conditions so as to form green regenerative callus material;
6 1
(b) introducing a nucleic acid molecule into the callus by microprojectile bombardment to produce transformed callus;
(c) transferring the callus to an intermediate incubation medium comprising a sugar source, an auxin at a concentration of about 0.1 mg/L to about 5 mg/L, a cytokinin at a concentration of about 0.1 mg/L to about 5 mg/L and copper at a concentration of about 0.1 μM to about 50 μM, and incubating such that green structures are observed on die callus material;
(d) transferring the callus material comprising the green structures to a regeneration medium and incubating such that shoots are obtained; and
(e) transferring the shoots to rooting medium to obtain transformed wheat plants.
24. The method of claim 23 wherein me sugar source comprises maltose.
25. A method for producing a transformed oat plant, comprising:
(a) placing an oat seed on a callus induction medium comprising a sugar source, an auxin at a concentration of about 0.1 mg/L to about 5 mg/L, a cytokinin at a concentration of 0 mg/L to about 5 mg/L and copper at a concentration of about 0.1 μM to about 50 μM, and incubating in dim light conditions so as to form green regenerative callus material;
(b) introducing a nucleic acid molecule into the callus by microprojectile bombaidmeπt to produce transformed callus;
(c) transferring die callus to an intermediate incubation medium comprising a sugar source, an auxin at a concentration of about 0.1 mg/L to about 5 mg/L, a cytokinin at a concentration of about 0.1 mg/L to about 5 mg/L and copper at a concentration of about 0.1 μM to about 50 μM, and incubating such that green structures are observed on die callus material;
(d) transferring the callus material comprising the green structures to a regeneration medium and incubating such that shoots are obtained; and
(e) transferring the shoots to rooting medium to obtain transformed oat plants.
26. The method of claim 25 wherein the sugar source comprises maltose.
27. A method for producing a transformed turf or forage plant, comprising:
(a) placing an turf or forage grass seed on a callus induction medium comprising a sugar source, an auxin at a concentration of about 0.1 mg/L to about 5 mg/L, a cytokinin at a concentration of 0 mg/L to about 5 mg/L and copper at a concentration of about 0.1 μM to about 50 μM, and incubating in dim light conditions so as to form green regenerative callus material;
(b) introducing a nucleic acid molecule into the callus by microprojectile bombardment to produce transformed callus;
(c) transferring the callus to an intermediate incubation medium comprising a sugar source, an auxin at a concentration of about 0.1 mg/L to about 5 mg/L, a cytoltiiiin at a concentration of about 0.1 mg/L to about 5 mg/L and copper at a concentration of about 0.1 μM to about 50 μM, and incubating such that green structures are observed on die callus material;
(d) transferring the callus material comprising the green structures to a regeneration medium and incubating such that shoots are obtained; and
(e) transferring the shoots to rooting medium to obtain transformed turf or forage grass plants.
28. The method of claim 27 wherein the sugar source comprises maltose.
29. A memod of producing green regenerative plant tissue suitable for transformation from a monocotyledenous plant, comprising:
(a) removing an immature embryo from the plant;
(b) incubating the immature zygotic embryo in dim light conditions on a callus induction medium comprising a sugar, an auxin at a concentration of about 0.1 mg/L to about 5 mg/L, a cytokinin at a - concentration of about 0 mg/L to about 5 mg/L and copper at a concentration of about 0.1 μM to about 50 μM, so as to form callus material
(c) transferring the callus to an intermediate induction medium comprising an auxin at a
concentration of about 0.1 mg/L to about 5 mg/L, a cytokinin at a concentration of about 0.1 mg/L to about 5 mg/L, a sugar source, and copper at a concentration of about 0.1 μM to about 50 μM, and incubating in dim light conditions so as to form green regenerative plant tissue suitable for transformation.
30. The method of claim 29 wherein the sugar source comprises maltose.
31. A method of producing a transformed plant, comprising introducing a nucleic acid molecule into green regenerative plant tissue produced according to claim 29.
32. A metiiod of generating regenerative green plant tissue suitable for transformation from a monocotyledenous plant, comprising:
(a) incubating a seed of the plant in dim light conditions on a callus induction medium comprising a sugar source, an auxin at a concentration of about 0.1 mg/L to about 5 mg/L, a cytokinin at a concentration of about 0 mg/L to about 5 mg/L and copper at a concentration of about 0.1 μM to about 50 μM, so as to form callus material
(b) transferring the callus to an intermediate induction medium comprising an auxin at a concentration of about 0.1 mg/L to about 5 mg/L, a cytokinin at a concentration of about 0.1 mg/L to about 5 mg/L, a sugar source, and copper at a concentration of about 0.1 μM to about 50 μM, and incubating in dim light conditions so as to form green regenerative plant tissue suitable for transformation.
33. The method of claim 32 wherein the sugar source comprises maltose.
34. A metiiod of producing a transformed plant, comprising introducing a nucleic acid molecule into green regenerative plant tissue produced according to claim 32.
35. A metiiod of producing green regenerative plant tissue suitable for transformation from a wheat plant, comprising:
(a) removing an immature embryo from the plant;
(b) incubating the immature zygotic embryo in dim light conditions on a callus-induction medium comprising a sugar source, an auxin at a concentration of about 0.1 mg/L to about 5 mg/L, a cytokinin at a concentration of about 0 mg/L to about 5 mg/L and copper at a concentration of about 0.1 μM to about 50 μM, so as to form highly embryogenic daughter tissue structures
(c) transferring die daughter tissue structures to fresh callus induction medium and incubating under dim light conditions so as to form green regenerative plant tissue suitable for transformation.
36. The method of claim 35 wherein die sugar source comprises maltose.
37. A method of producing a transformed plant, comprising introducing a nucleic acid molecule into green regenerative plant tissue produced according to claim 35.
38 A transgenic plant produced by the method of claims 1, 8, 15, 16, 17, 18, 19, 21, 23, 25, 27, 31, 34 or
37.
39 A transgenic plant that is a progeny of a transformed plant according to claim 30.
40. Seed of a transgenic plant according to claim 38 or 39.
41. A method of producing a transgenic plant comprising.
(a) producing a transformed plant by the method of claims 1, 8, 15, 16, 17, 18, 19, 21, 23, 25, 27, 31, 34 or 37
(b) producing a transgenic plant that is a progeny of die transformed plant.
Priority Applications (6)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AT98915484T ATE243410T1 (en) | 1997-04-29 | 1998-04-13 | METHODS OF PLANT TRANSFORMATION AND REGENERATION |
| AU69658/98A AU727873B2 (en) | 1997-04-29 | 1998-04-13 | Compositions and methods for plant transformation and regeneration |
| DE69815837T DE69815837T2 (en) | 1997-04-29 | 1998-04-13 | METHODS FOR PLANT TRANSFORMATION AND REGENERATION |
| JP54450198A JP2001521391A (en) | 1997-04-29 | 1998-04-13 | Compositions and methods for transforming and regenerating plants |
| EP98915484A EP0979031B1 (en) | 1997-04-29 | 1998-04-13 | Methods for plant transformation and regeneration |
| CA002287942A CA2287942A1 (en) | 1997-04-29 | 1998-04-13 | Compositions and methods for plant transformation and regeneration |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US08/845,939 US6235529B1 (en) | 1997-04-29 | 1997-04-29 | Compositions and methods for plant transformation and regeneration |
| US08/845,939 | 1997-04-29 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| WO1998048613A1 WO1998048613A1 (en) | 1998-11-05 |
| WO1998048613B1 true WO1998048613B1 (en) | 1998-12-30 |
Family
ID=25296482
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/US1998/007264 WO1998048613A1 (en) | 1997-04-29 | 1998-04-13 | Compositions and methods for plant transformation and regeneration |
Country Status (8)
| Country | Link |
|---|---|
| US (2) | US6235529B1 (en) |
| EP (1) | EP0979031B1 (en) |
| JP (1) | JP2001521391A (en) |
| AT (1) | ATE243410T1 (en) |
| AU (1) | AU727873B2 (en) |
| CA (1) | CA2287942A1 (en) |
| DE (1) | DE69815837T2 (en) |
| WO (1) | WO1998048613A1 (en) |
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| US5320961A (en) | 1992-11-16 | 1994-06-14 | Board Of Trustees Operating Michigan State University | Method for asexual in vitro propagation of fertile corn plants |
| US5281529A (en) | 1992-11-16 | 1994-01-25 | Board Of Trustees Operating Michigan State University | Method for in vitro sexual reproduction of corn plants |
| WO1994019930A1 (en) | 1993-03-11 | 1994-09-15 | National Research Council Of Canada | Enhanced regeneration system for cereals |
| JP3424971B2 (en) | 1994-01-20 | 2003-07-07 | 松下電器産業株式会社 | Surface acoustic wave filter |
| JPH07213183A (en) * | 1994-02-04 | 1995-08-15 | Sapporo Breweries Ltd | Method for regenerating plant body of barley |
| JPH07255304A (en) | 1994-03-17 | 1995-10-09 | New Oji Paper Co Ltd | Method for large amount of multiplication of plant of genus acacia |
| WO1996004392A2 (en) | 1994-07-29 | 1996-02-15 | Pioneer Hi-Bred International, Inc. | Transgenic cereal plants |
-
1997
- 1997-04-29 US US08/845,939 patent/US6235529B1/en not_active Expired - Fee Related
-
1998
- 1998-04-13 EP EP98915484A patent/EP0979031B1/en not_active Revoked
- 1998-04-13 CA CA002287942A patent/CA2287942A1/en not_active Abandoned
- 1998-04-13 WO PCT/US1998/007264 patent/WO1998048613A1/en not_active Application Discontinuation
- 1998-04-13 AU AU69658/98A patent/AU727873B2/en not_active Ceased
- 1998-04-13 JP JP54450198A patent/JP2001521391A/en active Pending
- 1998-04-13 AT AT98915484T patent/ATE243410T1/en not_active IP Right Cessation
- 1998-04-13 DE DE69815837T patent/DE69815837T2/en not_active Expired - Fee Related
-
2001
- 2001-04-03 US US09/825,217 patent/US6541257B2/en not_active Expired - Fee Related
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