Title:
TRANSGENIC PLANTS COMPRISING A CONDITIONALLY LETHAL GENE
Document Type and Number:
WIPO Patent Application WO/2000/037060
Kind Code:
A2
Abstract:
Genetic constructs, transformation vectors and methods are taught for production of transgenic plants which can be selectively removed from a growing site by application of a chemical agent or physiological stress. The invention links a target gene for the trait of commercial interest to a conditionally lethal gene, which can be selectively expressed to cause plant death. By use of the genetic constructs, transformation vectors and methods of the present invention, invasion of environments and contamination of commercial non-engineered productions by transgenic plants can be avoided. Methods are also taught for transformation of $i(Brassica) species.
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Inventors:
KELLER W A (CA)
FABIJANSKI S F (CA)
ARNISON P G (CA)
FABIJANSKI S F (CA)
ARNISON P G (CA)
Application Number:
PCT/CA1999/001223
Publication Date:
June 29, 2000
Filing Date:
December 22, 1999
Export Citation:
Assignee:
CA NAT RESEARCH COUNCIL (CA)
KELLER W A (CA)
FABIJANSKI S F (CA)
ARNISON P G (CA)
KELLER W A (CA)
FABIJANSKI S F (CA)
ARNISON P G (CA)
International Classes:
C12N5/10; A01H5/00; C12N15/09; C12N15/12; C12N15/31; C12N15/82; (IPC1-7): A61K31/00
Domestic Patent References:
| WO1997040179A1 | 1997-10-30 |
Foreign References:
| EP0198288A2 | 1986-10-22 | |||
| EP0636310A1 | 1995-02-01 | |||
| US5254801A | 1993-10-19 | |||
| US5777201A | 1998-07-07 |
Other References:
DATABASE WPI Week 8625 Derwent Publications Ltd., London, GB; AN 1986-157860 XP002149218 & JP 61 088874 A (MITSUBISHI COPR; MITSUBISHI MONSANTO KK), 7 May 1986 (1986-05-07)
DATABASE WPI Week 8847 Derwent Publications Ltd., London, GB; AN 1988-333483 XP002149219 & JP 63 245668 A (SEO S), 12 October 1988 (1988-10-12)
ZOU J ET AL: "Modification of seed oil content and acyl composition in the Brassicacea by expression of a yeast sn-2 acyltransferase gene" PLANT CELL,US,AMERICAN SOCIETY OF PLANT PHYSIOLOGISTS, ROCKVILLE, MD, vol. 9, no. 9, June 1997 (1997-06), pages 909-923, XP002122743 ISSN: 1040-4651
DATABASE WPI Week 8847 Derwent Publications Ltd., London, GB; AN 1988-333483 XP002149219 & JP 63 245668 A (SEO S), 12 October 1988 (1988-10-12)
ZOU J ET AL: "Modification of seed oil content and acyl composition in the Brassicacea by expression of a yeast sn-2 acyltransferase gene" PLANT CELL,US,AMERICAN SOCIETY OF PLANT PHYSIOLOGISTS, ROCKVILLE, MD, vol. 9, no. 9, June 1997 (1997-06), pages 909-923, XP002122743 ISSN: 1040-4651
Attorney, Agent or Firm:
Morrow, Joy D. (Ontario K1P 5Y6, CA)
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Claims:
CLAIMS:
| 1. | A genetic construct comprising a conditionally lethal gene operably associated with a promoter functional in a plant cell, said gene being used to select, identify or selectively kill a plant expressing said gene. |
| 2. | A genetic construct comprising: a conditionally lethal first gene adapted for expression in a plant cell; and, a second gene adapted for expression in a plant cell; wherein either or both gene is operably associated with a promoter functional in a plant cell. |
| 3. | The construct of claim 2, wherein the second gene is heterologous. |
| 4. | The construct of claim 3, wherein the heterologous gene codes for a pharmaceutical product. |
| 5. | The construct of claim 3, wherein the heterologous gene codes for an industrially useful enzyme. |
| 6. | The construct of claim 3, wherein the heterologous gene codes for rennin and/or hirudin. |
| 7. | The construct of claim 2, wherein the second gene, when expressed, changes the phenotype of the plant. |
| 8. | The construct of claim 2, wherein the second gene codes for a protein, peptide or antisense RNA. |
| 9. | The construct of claim 2, wherein the second gene codes for an input or output trait. |
| 10. | The construct of claim 1 or claim 2, wherein the conditionally lethal gene is an oncogene. |
| 11. | The construct of claim 1 or claim 2, wherein the conditionally lethal gene is oncogene 2 from Agrobacterium tumefaciens. |
| 12. | The construct of claim 1 or claim 2, wherein the conditionally lethal gene is expressed in response to chemical or stress. |
| 13. | The construct of claim 1 or claim 2, wherein the conditionally lethal gene is lethal only when an exogenous substance is applied. |
| 14. | The construct of claim 1 or claim 2, wherein the conditionally lethal gene is lethal when it is expressed and no exogenous substance need be applied. |
| 15. | The construct of claim 1 or claim 2, wherein the conditionallyly lethal gene is oncogene 4, a low temperature inducible gene from Arabidopsis, the gene coding for methoxinine dehydrogenase, the gene coding for rhizobitoxine synthase, or the gene coding for phosphonate monoester hydrolase. |
| 16. | The construct of claim 13, wherein the conditionally lethal gene is the gene coding for methoxinine dehydrogenase, the gene coding for rhizobitoxine synthase, or the gene coding for phosphonate monoester hydrolase. |
| 17. | The construct of claim 1 or claim 2, wherein the promoter is inducible. |
| 18. | The construct of claim 1 or claim 2, wherein the promoter is tissuespecific. |
| 19. | The construct of claim 1 or claim 2, wherein the promoter is constitutive. |
| 20. | A plant transformation vector comprising the genetic construct of any one of claims 1 to 19. |
| 21. | A plant comprising the genetic construct of any one of claims 1 to 20. |
| 22. | A plant comprising the genetic construct of any one of claims 11,12,13 and 15. |
| 23. | A plant transformed with the vector of claim 20. |
| 24. | The plant of any of claims 21 to 23 which is Brassica. |
| 25. | The Brassica plant of claim 24 which has altered oil composition. |
| 26. | The Brassica plant of claim 25 which has high oleic, low linoleic acid genotype. |
| 27. | The Brassica plant of claim 26 which is variety AG 019 or derivatives thereof. |
| 28. | A method for producing a transgenic plant which can be removed from a growing environment, comprising: transforming a plant cell with the genetic construct or vector of any one of claims 1 to 20; and, regenerating the plant cell to a whole plant. |
| 29. | A method for removing the plant of claim 22 from a growing environment, comprising application of a chemical agent which is converted to a phytotoxic agent by a product of a conditionally lethal gene, wherein the agent is applied at a level which, upon conversion by the gene product, results in a sublethal level of converted substrate. |
| 30. | A method for visual identification of the plant of claim 22, comprising: application of a chemical agent which is a substrate of the product of the conditionally lethal gene, wherein the agent is applied at a level which, upon conversion by the gene product, results in a sublethal level of converted substrate; visually identifying the plants which manifest the sublethal phenotype. |
| 31. | The method of claim 29 or claim 30 wherein the genetic construct or vector comprises oncogene 2 as the conditionally lethal gene, and wherein the chemical agent is an indoleamide or a related derivative. |
| 32. | The method of claim 31 wherein the indoleamide is naphthalene acetamide. |
| 33. | A method for selecting a transgenic plant of claim 22, comprising: application of a chemical agent which is a substrate for the product of the conditionally lethal gene, wherein the agent is applied at a level which, upon conversion by the gene product, results in a sublethal level of converted substrate; visually identifying the plants which manifest the sublethal phenotype; and, allowing the identified plants to recover into normal plants in the absence of the chemical agent. |
| 34. | The method of claim 33 wherein the genetic construct or vector comprises oncogene 2 as the conditionally lethal gene, and wherein the chemical agent is an indoleamide or a related derivative. |
| 35. | The method of claim 34 wherein the indoleamide is naphthalene acetamide. |
| 36. | The method of any one of claims 29 to 35 wherein the plant is Brassica. |
| 37. | The method of claim 36 wherein the Brassica plant has altered oil composition. |
| 38. | The method of claim 37 wherein the Brassica plant has high oleic acid, low linoleic acid content. |
| 39. | The method of claim 38 wherein the Brassica plant is variety AG019 or derivatives thereof. |
| 40. | A method for visual identification of a germinating seed or plant embryo comprising oncogene 2 as a transgene, comprising: culturing the seed or embryo on a medium containing an indoleamide or a related derivative; and visually identifying the germinated seed or embryo which manifests the phenotype. |
| 41. | A method for selecting a germinating seed or plant embryo comprising oncogene 2 as a transgene, comprising: culturing the seed or embryo on a medium containing an indoleamide or a related derivative; visually identifying the germinated seed or embryo which manifest the phenotype; and, transferring the identified seed or embryo to a medium without indoleamide; thereby obtaining the germinating seed or plant embryo comprising oncogene 2 as a transgene. |
| 42. | The method of claim 40 or 41 wherein the medium of step (a) contains an auxin transport inhibitor and the medium of step (b) does not contain an auxin transport inhibitor. |
| 43. | The method of any one of claims 40 to 42, wherein the inhibitor is N (lnaphthyl) phthalamic acid; 2,3,5 triiodobenzoic acid; 9hydroxyfluorene9carboxylic acid; erythrosine; eosine; fluorescein; semicarbazone; or ethanphon. |
| 44. | The method of any one of claims 40 to 43, wherein the indoleamide is naphthalene acetamide and the inhibitor is naphthylphthalamic acid. |
| 45. | The method of any one of claims 40 to 44, wherein the seed or embryo is Brassica. |
| 46. | The method of claim 45, wherein the Brassica seed or embryo has altered oil composition. |
| 47. | The method of claim 46, wherein the Brassica seed or embryo has high oleic acid, low linoleic acid content. |
| 48. | The method of claim 47, wherein the Brassica seed or embryo is variety AG019 or derivatives thereof. |
| 49. | A method for selecting a transgenic plant cell during transformation, comprising: transforming a plant cell with a genetic construct or vector comprising an oncogene adapted for expression in a plant cell; exposing said plant cell to a formula comprising a benign auxin derivative of a plant hormone, which is converted into an active hormone by the product of the oncogene, and an auxin transport inhibitor; culturing the cell to form a group of cells; visually identifying the group of cells which manifests the phenotype associated with the active hormone; and, allowing the identified group of cells to recover in the absence of the derivative. |
| 50. | The method of claim 49, wherein the oncogene is oncogene 2. |
| 51. | The method of claim 49, wherein the benign derivative is naphthalene acetamide and the inhibitor is naphthylphthalamic acid. |
| 52. | The method of any one of claims 49 to 51, wherein the plant cell is Brassica. |
| 53. | The method of claim 52 wherein the Brassica plant cell has altered oil composition. |
| 54. | The method of claim 53 wherein the Brassica plant cell has high oleic acid, low linoleic acid content. |
| 55. | The method of claim 54 wherein the Brassica plant cell is variety AG019 or derivatives thereof. |
| 56. | A method for transforming Brassica napus, comprising inclusion of naphthalene acetic acid in the media at the callusing and recovery step. |
| 57. | The method of claim 56 wherein the Brassica napus has altered oil profile. |
| 58. | The method of claim 56 wherein the Brassica napus is variety AG019. |
| 59. | A plasmid selected from the group consisting of: pJH121, pJH122, pJH123, pJH125, pJH126, and pJH130. |
Description:
INTERNATIONALSEARCHREPORT internealApplicationNo PCT/CA99/01223 C.(Conttnuat) on) DOCUMENTS CONS) DEREDTOBERELEVANT CategoryCitabonofdocument,withindication, where appropnate,oftherelevantpassagesRelevanttodaimNo. XEP0636310A(SANDOZLTD;1,10-14, SANDOZ-PATENT-GMBH;SANDOZ-ERFINDUNGEN20-23, VGBH)1February1995(1995-02-01)28, 30-35, 40,41 page1,line33-page2,line10;claims 1,2,4,5;example1 XUS5254801A(DOTSONSTANTONBETAL)1-3,5, 19October1993(1993-10-19)8-16, 18-24,28 column5,line26-line13;example1 column2,line8-line15 XUS5777201A(POUTRECANDACEGLORIAET56-58 AL)7July1998(1998-07-07) examples4-8 XDATABASEWPI56-58 Week8625 DerwentPublicationsLtd.,London,GB; AN1986-157860 XP002149218 &JP61088874A(MITSUBISHICOPR; MITSUBISHIMONSANTOKK), 7May1986(1986-05-07) abstract ADATABASEWPI56-58 Week8847 DerwentPublicationsLtd.,London,GB; AN1988-333483 XP002149219 &JP63245668A(SEOS), 12October1988(1988-10-12) abstract AZOUJETAL:"Modificationofseedoil56-58 contentandacylcompositioninthe Brassicaceabyexpressionofayeastsn-2 acyltransferasegene" PLANTCELL,US,AMERICANSOCIETYOFPLANT PHYSIOLOGISTS,ROCKVILLE,MD, vol.9,no.9,June1997(1997-06),pages 909-923,XP002122743 ISSN:1040-4651 thewholedocument 3 InttionalapplicationNo. INTERNATIONALSEARCHREPORTPCT/CA99/01223 BoxIObservationswherecertain claimswerefoundunsearchable(ConUnuatlon of Item toffirstsheet) ThisIntemationalSearchReporthasnotbeenestablished inrespectofcertainclaimsunderArticle17(2)(a)forthefollowingr
easons: 1.1-1 ClaimsNos.: becausetheyrelatetosubjectmatternotrequiredtobesearchedbythi
sAuthority,namely: 2.QClaims Nos.: becausetheyrelatetopartsoftheInternationalApplicationthatdon
otcomplywiththeprescribedrequirementstosuch anextentthatnomeaningfulInternationalSearchcanbecarriedout,s
pecifically: 3.jClaimsNos.: becausetheyaredependentdaimsandarenotdraftedinaccordancewith
thesecondandthirdsentencesofRule6.4(a). Box11ObservationswhereunityofinventionIslacking(Continuation
ofItem2offirstsheet) ThisIntemationalSearchingAuthorityfoundmultipleinventionsint
hisinternationalapplication,asfollows: seeadditionalsheet 1.2 Asallrequiredadditionalsearchfeesweretimelypaidbytheapplican
t,thisIntemationalSearchReportcoversall searchableclaims. 2.As ati searchableclaimscouldbesearchedwithouteffortjustifyinganaddi
tionalfee,thisAuthoritydidnotinvitepayment ofanyadditionalfee. 3.7j As onlysomeoftherequiredadditionalsearchfeesweretimelypaidbythe
applicant,thisInternationalSearchReport coversonlythoseclaimsforwhichfeeswerepaid,specificallyclaims
Nos.: 4.Norequiredadditionalsearchfeesweretimelypaidbytheapplicant
.Consequently,thisIntemationalSearchReportis restrictedtotheinventionfirstmentionedintheclaims; itiscoveredbydaimsNos.: RemarkonProtestThe additionalsearchfeeswereaccompaniedbytheapplicant'sprotest. !Noprotestaccompaniedthepaymentofadditionalsearchfees. u IntemationalApplicationNo. PCTtA 99 t1223 FURTHERINFORMATIONCONTINUEFROMPCT/ISA/210 ThisInternationalSearchingAuthorityfoundmultiple(groupsof) inventionsinthisinternationalapplication,asfollows: 1.Claims:1-55,59 Ageneticconstructcomprisingaconditionallylethalgene operablyassociatedwithapromotorfunctionalinaplant cell,saidgenebeingusedtoselect,identifyor selectivelykillaplantexpressingsaidgene;agenetic constructcomprisingaconditionallylethalfirstgene adaptedforexpressioninaplantcellandasecondgene adaptedforexpressioninaplantcell,whereineitheror bothgeneisoperablyassociatedwithapromotorfunctional inaplantcell;aplanttransformationvectorcomprising anyofsaidgeneticconstructs;aplantcomprisinganyof saidgeneticconstructs;aplanttransformedwith aforementionedvector;amethodforproducingatransgenic plantwhichcanberemovedfromagrowingenvironment;a methodforremovingsaidplantfromagrowingenvironment;a methodforvisualidentificationofsaidplant;amethodfor selectingsaidtransgenicplant;methodsforvisual identificationofagerminatingseedorplantembryoanda methodforselectingatransgenicplantcell. 2.Claims:56-58 AmethodfortransformingBrassicanapus,comprising inclusionofnaphthaleneaceticacidinthemediaatthe callusingandrecovery. INTERNATIONALSEARCHREPORT Internr ial ApplicatbnNo ., rmatbn on patenttamily msnbsr PCT/CA99/01223 PatentdocumentPublicationPatent family Publication citedinsearchreportdatemember (s)date EP0198288A22-10-1986US5180873A 19-01-1993 AU602306B 11-10-1990 AU5615586A 23-10-1986 US5278057A 11-01-1994 WO9740179A30-10-1997US5773697A 30-06-1998 AU2739297A 12-11-1997 BR9708742A 03-08-1999 CN1219201A 09-06-1999 EP 0910649 A 28-04-1999EP 0910649 A 28-04-1999 EP0636310A01-02-1995AU684076B 04-12-1997 AU6613494A 19-01-1995 CA2127298A 07-01-1995 CZ9401611A 12-07-1995 HU68797A 28-07-1995 JP7143829A 06-06-1995 PL304110A 09-01-1995 SK79294A 05-01-1995 US5639951A 17-06-1997 ZA 9404890 A 06-01-1996ZA9404890 A 06-01-1996 US5254801A19-10-1993NONE US5777201A07-07-1998AU3516793A 16-09-1993 CA2092661A 14-09-1993 EP0561569A 22-09-1993 JP6014667A 25-01-1994 JP61088874A07-05-1986JP1845276C 25-05-1994 JP5055106B 16-08-1993 JP63245668A12-10-1988NONE