[go: up one dir, main page]

WO1999061597A2 - Methodes et materiels ameliores de transformation - Google Patents

Methodes et materiels ameliores de transformation Download PDF

Info

Publication number
WO1999061597A2
WO1999061597A2 PCT/US1999/011250 US9911250W WO9961597A2 WO 1999061597 A2 WO1999061597 A2 WO 1999061597A2 US 9911250 W US9911250 W US 9911250W WO 9961597 A2 WO9961597 A2 WO 9961597A2
Authority
WO
WIPO (PCT)
Prior art keywords
dna
rna
launching platform
plant
trans
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US1999/011250
Other languages
English (en)
Other versions
WO1999061597A3 (fr
WO1999061597A9 (fr
Inventor
Lada Rasochova
Thomas L. German
Paul G. Ahlquist
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Wisconsin Alumni Research Foundation
Original Assignee
Wisconsin Alumni Research Foundation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Wisconsin Alumni Research Foundation filed Critical Wisconsin Alumni Research Foundation
Priority to BR9911065-2A priority Critical patent/BR9911065A/pt
Priority to EP99953356A priority patent/EP1086237A2/fr
Priority to AU43101/99A priority patent/AU763096B2/en
Priority to JP2000550982A priority patent/JP3959965B2/ja
Priority to CA002329509A priority patent/CA2329509C/fr
Publication of WO1999061597A2 publication Critical patent/WO1999061597A2/fr
Publication of WO1999061597A9 publication Critical patent/WO1999061597A9/fr
Publication of WO1999061597A3 publication Critical patent/WO1999061597A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8203—Virus mediated transformation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8205—Agrobacterium mediated transformation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells

Definitions

  • RNA virus-based vectors can spread to non-target plants by mechanical means and/or by insects. Such spread can be prevented by using vectors that can replicate and/or move only in target plants expressing the appropriate trans-acting factors.
  • Another aspect of the subject invention pertains to a method of genotypically or phenotypically modifying a host cell, comprising introducing a DNA-launching platform which encodes a viral RNA molecule and an exogenous RNA segment in a location which does not disrupt the replication of said viral RNA segment or said exogenous RNA segment, whereby the exogenous RNA segment confers a detectable trait in the host cell.
  • the subject invention applies to a wide array of plant cells.
  • Figure 7 depicts DNA-launching platforms which can be used in accord with the teachings contained herein.
  • Figure 14 shows the efficient replication of launched BMV RNA3 in (la + 2a)- transgenic plants.
  • Figure 18 shows the successful GFP expression from the launched BMV RNA3 in protoplasts.
  • SEQ DD NO. 7 pB12LR8 — partial nucleotide sequence includes BMV la and 2a expression cassettes.
  • SEQ DD NO. 8 pB12LR9 — partial nucleotide sequence includes BMV la and 2a expression cassettes.
  • the subject invention is directed towards a method of transfection employing a DNA-launching platform which encodes a modified viral RNA molecule comprising an RNA viral component attached to an exogenous RNA component and a DNA-dependent RNA pol promoter.
  • the DNA-dependent RNA pol promoter is preferably but not necessarily fused within up to 10 nucleotides of the 5 ' transcriptional start site of the modified viral RNA molecule, and more preferably within up to 5 nucleotides of the 5' transcriptional start site.
  • RNA-launching platform comprising the RNA3 viral replication segment, as well as the exogenous RNA of interest
  • RNA3 viral replication segment as well as the exogenous RNA of interest
  • DNA-launching platforms could be also derived from either RNA1 or RNA2.
  • sequence encoding the la protein could be replaced with an exogenous RNA; replication would require the expression of la (e.g., separate expression plasmid).
  • the DNA- launching platform also comprises a ribozyme situated proximate to the 3' end of the modified RNA3, wherein said ribozyme cleaves the RNA3 at the 3' end.
  • viral segments from other known viruses, and/or subviral agents can be used to formulate DNA-launching platforms of the subject invention.
  • BMV is merely one representative example of the many viruses suitable for practicing the subject invention. It is widely accepted that principles on which the subject invention is based are broadly applicable to a myriad of viruses.
  • RNA viruses are seed transmitted from one generation to the next. This property can be exploited to effect genotypic transformation of a plant. That is to say, the modified RNA remains transmissible from one generation to the next, just as seed-borne virus infections are transmitted from one generation to the next.
  • Binary vectors for expressing the BMV la and 2a proteins in plants were constructed. Starting with the pBI101.2 construct (Clontech, Palo Alto, CA), the GUS gene was removed by first cutting the construct with EcoRI and SnaBI. The overhanging restriction fragment ends were filled in by treatment with Klenow fragments and dNTPs. The restriction fragment ends were religated forming the pB 101.2LR1.
  • a DNA-launching platform having a BMV RNA3 with a GUS gene insertion wherein the GUS is downstream of an additional BMV subgenomic promoter was constructed.
  • the pB3LRl 5 construct was cut with Aval and the restriction fragment ends were filled in with Klenow fragment and dNTPs. Construct was then cut with Clal and dephosphorylated.
  • the pB3MI22 was cut with Clal and Stul and a B3GUS fragment was isolated. The isolated B3GUS fragment was then ligated to the cut pB3LR15 construct to form a new construct of pB3GUSCPLRl 9 ( Figure 5).
  • a DNA-launching platform wherein the BMV RNA3 coat protein was replaced with the SHMV (Sunn hemp mosaic virus) coat protein and the GUS gene was inserted downstream of an additional BMV subgenomic promoter was constructed.
  • the pB3RS4 (Sacher et al, 1988) was cut with Aval, blunted with Klenow fragment and dNTPs, and cut with Kpnl.
  • the SHMV coat protein fragment was isolated using a low-melting agarose gel.
  • the pB3GUSLRl 7 was cut with Stul and Kpnl and dephosphorylated.
  • the SHMV coat protein fragment was ligated to the cut pB3GUSLRl 7 thereby forming pB3GUSCPLR24 ( Figure 7).
  • NTl Plating Medium (1 liter) was made with NTl medium and 72.86 g mannitol, the pH was adjusted to 5.5-5.7, and the resulting mixture was autoclaved.
  • the cells were centrifuged at 800 rpm for 5 min. The supernatant was discarded. About 40 ml of wash solution was added, cells were resuspended and were centrifuged at 800 rpm for 5 min. The supernatant was discarded. The cells were then resuspended in three volumes of enzyme digestion solution, and incubated for 60 min. at room temperature.
  • the cells were resuspended in 1 ml of ice-cold 20 mM CaCl 2 solution. 0.1 -ml aliquots were dispensed into prechilled eppendorf tubes. About 1 ⁇ g of plasmid DNA was added to the cells. The cells were frozen in liquid nitrogen. The cells were thawed by incubating the test tube in a 37°C water bath for 5 min. 1 ml of YEP medium was added to the tube and incubated at 28°C for 2-4 h with gentle shaking to allow the bacteria to express the antibiotic resistance genes. The tubes were centrifuged for 30 s and the supernatant solution was discarded. The cells were resuspended in 0.1 ml YEP medium. The cells were plated on a YEP agar plate containing selection antibiotic(s) and incubated at 28 °C. Transformed colonies appeared in 2-3 days.
  • Transgenic N. tabacum and N. benthaniana plants were produced according to the procedures discussed above.
  • the plants were transfected with a DNA-launching platform containing a GUS gene (Figure 5a) by particle bombardment as described in Examples 5 and 7.
  • the plants were incubated for 3-5 days and then assayed for ⁇ -glucuronidase (GUS) activity using 1 mg/ml X-Gluc (5-bromo-4-chloro-3-indolyl glucucuronide) as substrate in 0.1M potassium phosphate buffer, pH 7.0, 50 ⁇ M potassium ferrocyanide, and 2% Triton® X-100.
  • GUS ⁇ -glucuronidase
  • Example 9 Transfection of Transgenic Plants Expressing BMV la. 2a. 3 a. and CP
  • BMV RNA3 derivatives contained the GUS gene in place of the coat protein ORF ( Figure 5a) (these were inoculated with or without coat protein expression plasmid, Figure 5b), or had the BMVCP gene translated from an additional subgenomic RNA driven from BMV or CCMV subgenomic promoter ( Figures 5c and 5d), or had the SHMV coat protein translated from an additional BMV subgenomic RNA ( Figure 7b).
  • N. benthamiana plants were transfected using a particle bombardment as described above with a DNA-launching platform for BMV RNA3 having the GFP gene in place of BMV coat protein (Figure 6e).
  • the GFP expression was determined 24 hrs post inoculation using a fluorescent microscope.
  • Figure 17 shows the successful expression of GFP in (la + 2a)- transgenic N. benthamiana.
  • Example 16 Transfection of Progeny From ( 1 a+2a -Transgenic N. benthamiana With BMV R ⁇ A3 DNA-Launching Platform
  • Transgenic plants are obtained having one or more trans-acting factors fused to the DNA-dependent RNA polymerase promoter and terminator.
  • Such factors may include the viral replicase (126K/183K), movement protein (30K), or coat protein (17.6K).
  • At least one cis- acting sequence necessary for TMV RNA replication is removed from transgenes.
  • the transacting factors are stably expressed in the plant cell or their expression may be induced if an inducible promoter is used.
  • PVX Potato virus X
  • the 5 ' end has an m7Gppp cap and the 3' end is polyadenylated.
  • a full-length cDNA clone of PVX has been constructed and infectious RNA transcripts obtained (Hemenway et al, 1990).
  • a DNA-launching plasmid is constructed based on PVX RNA containing PVX cDNA precisely fused at its 5' end to a DNA-dependent RNA polymerase promoter and having a polyadenylation site at its 3 ' end.
  • a convenient restriction site may also be included at the 3 ' end.
  • a foreign gene may be expressed from an additional subgenomic RNA.
  • Transgenic plants are obtained having one or more trans-acting factors fused to the
  • a DNA-launching plasmid is constructed having a DNA-dependent RNA polymerase promoter, polyadenylation site, and the PVX cDNA sequence in which the ORF2
  • Transgenic plants are obtained having one or more trans-acting factors fused to the DNA-dependent RNA polymerase promoter and terminator.
  • Such factors may include protein A or capsid protein precursor alpha, and preferably will also include a movement protein from a plant virus, such as 30K of TMV or 35K of RCNMV.
  • Trans-acting factors are stably expressed in the plant cell or their expression may be induced if an inducible promoter is used.
  • Transgenically expressed trans-acting factors preferably lack at least one cis-acting factor which is necessary for their replication, such as the 5 ' and/or 3 ' end.
  • a DNA-launching plasmid is constructed based on TSWV RNA M in which the Gl and G2 coding sequences are replaced with at least one foreign gene or sequence.
  • Such DNA- launching plasmid contains a DNA-dependent RNA polymerase promoter and TSWV RNA M cDNA fused to the self-cleaving ribozymes at the 5' and 3' ends.
  • a DNA- launching plasmid is constructed based on TSWV RNA S in which the N coding region is replaced with a foreign gene or sequence.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Virology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne de nouvelles méthodes et de nouveaux matériels destinés à la transformation d'une cellule hôte et à l'expression dans celle-ci d'ARN exogène. Spécifiquement, l'invention concerne des plates-formes de lancement d'ADN utilisées pour introduire un segment viral réplicant fixé à un polynucléotide exogène dans une cellule, de manière que le polynucléotide exogène soit exprimé dans ladite cellule et confère un trait détectable.
PCT/US1999/011250 1998-05-22 1999-05-21 Methodes et materiels ameliores de transformation Ceased WO1999061597A2 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
BR9911065-2A BR9911065A (pt) 1998-05-22 1999-05-21 Processos melhorados e materiais para transformação
EP99953356A EP1086237A2 (fr) 1998-05-22 1999-05-21 Methodes et materiels ameliores de transformation
AU43101/99A AU763096B2 (en) 1998-05-22 1999-05-21 Improved methods and materials for transformation
JP2000550982A JP3959965B2 (ja) 1998-05-22 1999-05-21 形質転換のための改良された方法および材料
CA002329509A CA2329509C (fr) 1998-05-22 1999-05-21 Methodes et materiels ameliores de transformation

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US8652698P 1998-05-22 1998-05-22
US60/086,526 1998-05-22

Publications (3)

Publication Number Publication Date
WO1999061597A2 true WO1999061597A2 (fr) 1999-12-02
WO1999061597A9 WO1999061597A9 (fr) 2000-02-24
WO1999061597A3 WO1999061597A3 (fr) 2000-03-30

Family

ID=22199163

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1999/011250 Ceased WO1999061597A2 (fr) 1998-05-22 1999-05-21 Methodes et materiels ameliores de transformation

Country Status (7)

Country Link
EP (1) EP1086237A2 (fr)
JP (1) JP3959965B2 (fr)
AR (1) AR020324A1 (fr)
AU (1) AU763096B2 (fr)
BR (1) BR9911065A (fr)
CA (1) CA2329509C (fr)
WO (1) WO1999061597A2 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001071017A3 (fr) * 2000-03-24 2002-04-04 Chiron Spa Arn de nodavirus modifie pour apport de gene
WO2002059336A3 (fr) * 2001-01-25 2003-09-04 Large Scale Biology Corp Inhibition cytoplasmique de l'expression genique et expression d'une proteine etrangere dans une plante de type monocotyledone par un vecteur viral de plante
US9765349B2 (en) 2003-02-03 2017-09-19 Ibio, Inc. System for expression of genes in plants

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2540668C (fr) 2003-10-01 2010-12-21 Japan Science And Technology Agency Fragment d'adn, methode de production de transformant servant a la production de proteines et utilisation connexe

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA1288073C (fr) * 1985-03-07 1991-08-27 Paul G. Ahlquist Vecteur de transformation de l'arn
WO1990012107A1 (fr) * 1989-03-31 1990-10-18 The Salk Institute Biotechnology/Industrial Associates, Inc. Systeme d'expression par recombinaison base sur le virus de la mosaique du tabac satellitaire
JPH04121200A (ja) * 1990-09-07 1992-04-22 Nippon Nohyaku Co Ltd 植物細胞におけるポリペプチドの製造法
EP0694070B1 (fr) * 1993-09-15 2002-04-10 Chiron Corporation Vecteurs composes d'alphavirus recombinants
GB9703146D0 (en) * 1997-02-14 1997-04-02 Innes John Centre Innov Ltd Methods and means for gene silencing in transgenic plants

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001071017A3 (fr) * 2000-03-24 2002-04-04 Chiron Spa Arn de nodavirus modifie pour apport de gene
JP2003527863A (ja) * 2000-03-24 2003-09-24 カイロン エセ．ピー．アー． 遺伝子送達のための改変ノダウイルスｒｎａ
US7179457B2 (en) 2000-03-24 2007-02-20 Chiron S.R.L. Modified nodavirus RNA for gene delivery
JP4815091B2 (ja) * 2000-03-24 2011-11-16 ノバルティス ヴァクシンズ アンド ダイアグノスティクス エスアールエル 遺伝子送達のための改変ノダウイルスｒｎａ
WO2002059336A3 (fr) * 2001-01-25 2003-09-04 Large Scale Biology Corp Inhibition cytoplasmique de l'expression genique et expression d'une proteine etrangere dans une plante de type monocotyledone par un vecteur viral de plante
US6800748B2 (en) 2001-01-25 2004-10-05 Large Scale Biology Corporation Cytoplasmic inhibition of gene expression and expression of a foreign protein in a monocot plant by a plant viral vector
US9765349B2 (en) 2003-02-03 2017-09-19 Ibio, Inc. System for expression of genes in plants

Also Published As

Publication number Publication date
CA2329509A1 (fr) 1999-12-02
CA2329509C (fr) 2009-10-06
AR020324A1 (es) 2002-05-08
AU763096B2 (en) 2003-07-10
EP1086237A2 (fr) 2001-03-28
JP3959965B2 (ja) 2007-08-15
WO1999061597A3 (fr) 2000-03-30
WO1999061597A9 (fr) 2000-02-24
JP2002516089A (ja) 2002-06-04
BR9911065A (pt) 2001-02-06
AU4310199A (en) 1999-12-13

Similar Documents

Publication Publication Date Title
US8148608B2 (en) Systems and methods for clonal expression in plants
US5491076A (en) Expression of foreign genes using a replicating polyprotein producing virus vector
US6531647B1 (en) Gene silencing methods
US20190048358A1 (en) Expression of Foreign Sequences in Plants Using Trans-Activation System
US20240218385A1 (en) Negative-strand rna viral vector and plant genome editing method without transformation
Ambrós et al. Agroinoculation of Citrus tristeza virus causes systemic infection and symptoms in the presumed nonhost Nicotiana benthamiana
CN101166827A (zh) 在植物中表达蛋白质
US20190211345A1 (en) Citrus tristeza virus based vectors for foreign gene/s expression
US20150067918A1 (en) Citrus plants resistant to citrus huanglongbing (ex greening) caused by candidatus liberibacter asiaticus (las) and bacterial canker caused by (xanthomonas axonopodis pv. citri) (xac)
Beernink et al. Direct agroinoculation of maize seedlings by injection with recombinant foxtail mosaic virus and sugarcane mosaic virus infectious clones
US20100269220A1 (en) Enhancing expression of value-added genes by transgenic expression of tombusvirus-based p19 gene mutants
US20120084884A1 (en) Stably transformed ferns and related methods
CA2404471C (fr) Construction capable de liberation en forme circulaire fermee a partir d'une sequence nucleotidique plus grande permettant l'expression specifique du milieu et/ou l'expression a regulation de developpement de sequences genetiques selectionnees
US10308946B2 (en) Expression cassette for transformation comprising a modified viral sequence driven by a suitable promoter
WO1999061597A2 (fr) Methodes et materiels ameliores de transformation
US7005559B1 (en) Expression silencing system and different uses thereof
WO2008119136A1 (fr) Procédés et constructions perfectionnés pour transformation à médiation par agrobacterium sans marqueur
WO2017011815A2 (fr) Agrumes résistants au citrus huanglongbing (anciennement greening) provoqué par candidatus liberibacter asiaticus (las) et au chancre bactérien provoqué par (xanthomonas axonopodis pv. citri) (xac) au moyen de gènes de la défensine de l'épinard dans des vecteurs du ctv
Birch et al. Regulation of transgene expression: progress towards practical development in sugarcane, and implications for other plant species
Zhang Characterization of grapevine vein clearing virus expression strategy and development of caulimovirus infectious clones
El Mohtar Exploring Citrus tristeza virus-based vector limits for heterologous gene/s expression
Klöti Genetic transformation of rice (Oryza sativa L.) to confer resistance to rice tungro bacilliform virus (RTBV)
Rose Molecular and biological investigations for the description and taxonomic classification of celery latent virus and a German Celery mosaic virus isolate from Quedlinburg
PrimaryTransformants Resistance to Tospoviruses in Nicotiana benthamiana Transformed with the N Gene of Tomato Spotted Wilt Virus: Correlation Between Transgene Expression and Protection In PrimaryTransformants
AU2001243939A1 (en) A construct capable of release in closed circular form from a larger nucleotide sequence permitting site specific expression and/or developmentally regulated expression of selected genetic sequences

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AL AU BA BB BG BR CA CN CU CZ EE GD GE HR HU ID IL IN IS JP KP KR LC LK LR LT LV MG MK MN MX NO NZ PL RO SG SI SK SL TR TT UA UZ VN YU ZA

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW SD SL SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
AK Designated states

Kind code of ref document: C2

Designated state(s): AE AL AU BA BB BG BR CA CN CU CZ EE GD GE HR HU ID IL IN IS JP KP KR LC LK LR LT LV MG MK MN MX NO NZ PL RO SG SI SK SL TR TT UA UZ VN YU ZA

AL Designated countries for regional patents

Kind code of ref document: C2

Designated state(s): GH GM KE LS MW SD SL SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

COP Corrected version of pamphlet

Free format text: PAGES 1/22-22/22, DRAWINGS, REPLACED BY NEW PAGES 1/20-20/20; DUE TO LATE TRANSMITTAL BY THE RECEIVING OFFICE

AK Designated states

Kind code of ref document: A3

Designated state(s): AE AL AU BA BB BG BR CA CN CU CZ EE GD GE HR HU ID IL IN IS JP KP KR LC LK LR LT LV MG MK MN MX NO NZ PL RO SG SI SK SL TR TT UA UZ VN YU ZA

AL Designated countries for regional patents

Kind code of ref document: A3

Designated state(s): GH GM KE LS MW SD SL SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

WWE Wipo information: entry into national phase

Ref document number: 1999953356

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 43101/99

Country of ref document: AU

ENP Entry into the national phase

Ref document number: 2000 550982

Country of ref document: JP

Kind code of ref document: A

Ref document number: 2329509

Country of ref document: CA

NENP Non-entry into the national phase

Ref country code: KR

WWP Wipo information: published in national office

Ref document number: 1999953356

Country of ref document: EP

WWG Wipo information: grant in national office

Ref document number: 43101/99

Country of ref document: AU