WO2001064023B1 - Marker free transgenic plants: engineering the chloroplast genome without the use of antibiotic selection - Google Patents

Marker free transgenic plants: engineering the chloroplast genome without the use of antibiotic selection

Info

Publication number
WO2001064023B1
WO2001064023B1 PCT/US2001/006275 US0106275W WO0164023B1 WO 2001064023 B1 WO2001064023 B1 WO 2001064023B1 US 0106275 W US0106275 W US 0106275W WO 0164023 B1 WO0164023 B1 WO 0164023B1
Authority
WO
WIPO (PCT)
Prior art keywords
plant
dna sequence
plastid
phytotoxic
aldehyde
Prior art date
Application number
PCT/US2001/006275
Other languages
French (fr)
Other versions
WO2001064023A1 (en
Inventor
Henry Daniell
Original Assignee
Univ Auburn
Univ Central Florida
Henry Daniell
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 Univ Auburn, Univ Central Florida, Henry Daniell filed Critical Univ Auburn
Priority to EP01918263A priority Critical patent/EP1294221A4/en
Priority to CA002401954A priority patent/CA2401954A1/en
Priority to NZ521707A priority patent/NZ521707A/en
Priority to AU2001245359A priority patent/AU2001245359A1/en
Publication of WO2001064023A1 publication Critical patent/WO2001064023A1/en
Publication of WO2001064023B1 publication Critical patent/WO2001064023B1/en
Priority to US10/741,379 priority patent/US7795497B2/en
Priority to US12/850,733 priority patent/US20110072541A1/en

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    • 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
    • C12N15/8209—Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers
    • 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
    • C12N15/8214—Plastid 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)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance

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

Abstract

The present invention provides for a method to circumvent the problem of using antibiotic resistant selectable markers. In particular, target plants are transformed using a plastid vector which contains heterologous DNA sequences coding for a phytotoxin detoxifying enzyme or protein. The selection process involves converting an antibiotic-free phytotoxic agent by the expressed phytotoxin detoxifying enzyme or protein to yield a nontoxic compound. The invention provides for various methods to use antibiotic-free selection in chloroplast transformation.

Claims

AMENDED CLAIMS[received by the International Bureau on 13 August 2001 (13.08.01); original claims 2-20, 25-26 and 28-29 amended; new claims 30-38 added; remaining claims unchanged (5 pages)]
1. An integration and expression plastid vector competent for stably transforming the plastid genome, where growth is inhibited by an antibiotic-free phytotoxic agent, which comprises an expression cassette, which expression cassette comprises as operably joined components, a 5' part of the plastid DNA sequence inclusive of a spacer sequence, a promoter operative in said plastid, a DNA sequence encoding a detoxifying enzyme or protein acting as a selectable marker which is capable of detoxifying said antibiotic-free phytotoxic agent in the cells to the corresponding nontoxic compound, at least one restriction site for the insertion of a heterologous target gene, a transcription termination region functional in said plastid, and the 3' part of a plastid DNA sequence inclusive of the spacer sequence.
2. The vector of claim 1 wherein said vector further comprises a ribosome binding site and a 5' untranslated region (5' UTR).
3. The vector of claim 1 wherein a heterologous DNA sequence coding for a molecule of interest is inserted in one of the restriction sites.
4. A chloroplast vector of claim 2 wherein the molecule of interest is a polypeptide.
5. A vector of claim 1, wherein the antibiotic-free phytotoxic agent is a phytotoxic aldehyde and the detoxifying enzyme or protein is an aldehyde dehydrogenase capable of detoxifying said phytotoxic aldehyde.
6. A vector of claim 5 competent for stably transforming the chloroplast genome where growth is inhibited by a phytotoxic aldehyde, wherein the phytotoxic aldehyde is selected from the group consisting of acetaldehyde, formaldehyde, propronaldehyde, utyraldehyde and betaine aldehyde.
7. A vector of claim 6, wherein plastid is tobacco chloroplast.
8. An integration and expression plastid vector competent for stably transforming the plastid genome where growth is inhibited by a phytotoxic aldehyde, which vector comprises an expression cassette which comprises as operably joined components, a 5' part of the plastid DNA sequence inclusive of a spacer sequence, a promoter operative in said plastid, a DNA sequence encoding betaine aldehyde dehydrogenase (BADH) as a selectable marker which is capable of detoxifying said phytotoxic aldehyde in the cells to glycine betaine, a heterologous DNA sequence which codes for a molecule of interest, a transcription termination region functional in said plastid, and a 3' part of a plastid DNA sequence inclusive of the spacer sequence.
9. A vector of claim 8, wherein said promoter is a Prrn promoter, wherein said expression cassette further comprises a DNA sequence coding for a selectable marker, a transcription termination region of the psbA gene, and wherein said expression cassette is inserted between the tral and trnA genes of the chloroplast genome.
10. A stably transformed plant which comprises a chloroplast which has been stably transformed with a vector of claim 1 or claim 8.
11. The stably transformed plant of claim 10, wherein the plant is a solanaceoυs plant edible for a mammal,
12. The stably transformed plant of claim 10, wherein the plant is a crop plant edible for a mammal.
13. A stably transformed plant of claim 10, wherein the plant is a monocotyledonous plant, selected from the group of rice, wheat, grass, rye, barley, oat, or maize.
14. A stably transformed plant of claim 10, wherein the plant is a dicotyledonous plant, selected from the group of soybean, peanut, grape, sweet potato, pea, canola, tobacco, tomato or cotton.
15. A stable transformed plant of claim 10, wherein the plant is a tobacco, tomato, potato, rice, brassica, cotton, maize or soybean.
16. A stable transformed plant of claim 10, wherein the plant is a homoplasmic plant.
17. A vector of claim 1 or 8, wherein the selectable marker is driven by a promoter operative in green and non-green tissues selected from the group consisting of the 16SrRNA promoter, the psbA promoter, the atpB promoter, or the accD promoter.
18. A method for transforming the plastid genome of a plant cell, said method comprising introducing into cells of a plant species whose growth is inhibited by an antibiotic- free phytotoxic agent, an expression cassette which comprises as operably linked components, a 5' part of a plastid DNA sequence inclusive of a spacer sequence, a promoter operative in said plastid, a DNA sequence encoding a detoxifying enzyme or protein acting as a selectable marker for transgenic plant cells and capable of detoxifying said phytotoxic agent in the cells to the corresponding nontoxic compound, a heterologous target DNA sequence, a transcription termination region functional in said plant chloroplast cells, and the 3' part of the plastid DNA sequence inclusive of a spacer sequence.
19. The method of claim 18 wherein the heterologous target DNA sequence codes for a molecule of interest.
20. The method of claim 18 wherein the antibiotic-free phytotoxic agent is a phytotoxic aldehyde, and the DNA sequence encoding a detoxifying enzyme or protein codes for an aldehyde dehydrogenase capable of detoxifying said phytotoxic aldehyde.
21. The method of claim 20 wherein the phytotoxic aldehyde is selected from the group consisting of acetaldehyde, formaldehyde, propronaldehyde, utyraldehyde and betaine aldehyde.
22. A method of claim 18, wherein said method further comprises culturing said plant in a plant growth medium comprising said phytotoxic aldehyde, and selecting transformed plant cells capable of growth in the presence of said phytotoxic aldehyde.
23. A method of claim 22, wherein said method further comprises regenerating a transformed plant from said transformed plant cells.
24. A method of claim 20 wherein said phytotoxic aldehyde is betaine aldehyde and the aldehyde dehydrogenase is betaine aldehyde dehydrogenase (BADH).
25. A method of claim 24, wherein said DNA sequence is derived from at least one of a plant and a microorganism.
26. A method of claim 25, wherein said plant is selected from a sugar beet or a spinach.
27. A method of claim 18, wherein the promoter is selected from a group consisting of 16SrRNA, psbA, accD and atpB promoters.
28. A vector of claim 1 or 8, wherein the phytotoxic agent is selected from a group consisting of triazines, sulfonylureas, imidazolinones, aryloxyphenoxypropionates, cyclohexanediones, glyphosate, bromoxynil, phenoxycarboxylic acids, glufosinate, cyanamide, dalapon, betaine aldehyde and polyethylene glycol, and wherein the detoxifying agent is selected from a group consisting of an enzyme or protein capable of detoxifying triazines, sulfonylureas, imidazolinones, aryloxyphenoxypropionates, cyclohexanediones, glyphosate, bromoxynil, phenoxycarboxylic acids, glufosinate, cyanamide, dalapon, and the chlB gene, and betaine aldehyde dehydrogenase, or the TSP1 gene.
29. A method of claim 18, where the expression cassette further comprises a ribosome binding site (rbs) and a 5' untranslated region (5TJTR) to enhance expression.
31
30. A method for transforming the plastid genome of a plant cell in which selection is antibiotic- free and based upon chlorophyll synthesis in the dark, said method comprising: introducing into cells of a higher plant species whose ability to synthesize chlorophyll is inhibited by lack of light, an expression cassette which comprises as operably linked components, a 5 ' part of a plastid DNA sequence inclusive of a spacer sequence, a promoter operative in said plastid, a DNA sequence encoding a pigment biosynthesis gene acting as a selectable marker for transgenic plant cells, a heterologous DNA sequence coding for a gene of interest, a transcription termination region functional in said plant chloroplast cells, and the 3' part of the plastid DNA sequence inclusive of a spacer sequence, and allowing said plant ceils to grow in the dark, and selecting transgenic green shoots.
31. The method of claim 30, further comprising the step: Regenerating the transgenic green shoots.
32. The method of claim 30, wherein the pigment biosynthesis gene is the prorochlorophyllide reductase (chIB) gene.
33. A method for transforming the plastid genome of a plant cell in which selection is antibiotic-free, and based upon feed back inhibition, said method comprising: introducing into cells of a higher plant species, an expression cassette which comprises as operably linked components, a 5' part of a plastid DNA sequence inclusive of a spacer sequence, a promoter operative in said plastid, a DNA sequence encoding a mutant gene coding for an enzyme which is insensitive to feed back inhibition, a heterologous DNA sequence coding for a gene of interest, a transcription termination region functional in said plant chloroplast cells, and the 3' part of the plastid DNA sequence inclusive of a spacer sequence, and allowing said plant cells to grow in a medium lacking specific araino acids, and selecting transgenic shoots.
34. The method of claim 33, further comprising the step: Regenerating the transgenic shoots.
35. The method of claim 18, wherein the antibiotic-free phytotoxic agent is an herbicide, and the DNA sequence encoding a detoxifying enzyme or protein codes for an enzyme or protein capable of detoxifying said herbicide.
36. The method of claim 35, wherein the herbicide is selected from a group
32 consisting of triazines, sulfonylureas. imidazolinones, aryloxyphenoxypropionates, cyclohexanediones, glyphosate, bromoxynil, phenoxycarboxylic acids, glufosinate, cyanamide, and dalapon, and wherein the detoxifying enzyme or protein is selected from a group consisting of an enzyme or protein capable of detoxifying triazines, sulfonylureas, imidazolinones, aryloxyphenoxypropionates, cyclohexanediones, glyphosate, bromoxynil, phenoxycarboxylic acids, glufosinate, cyanamide, and dalapon.
37. A plant according to claim 10, wherein said plant is the progeny thereof.
38. A method of claim 25, wherein said microorganism is E. coli.
33
PCT/US2001/006275 2000-03-02 2001-02-28 Marker free transgenic plants: engineering the chloroplast genome without the use of antibiotic selection WO2001064023A1 (en)

Priority Applications (6)

Application Number Priority Date Filing Date Title
EP01918263A EP1294221A4 (en) 2000-03-02 2001-02-28 Marker free transgenic plants: engineering the chloroplast genome without the use of antibiotic selection
CA002401954A CA2401954A1 (en) 2000-03-02 2001-02-28 Marker free transgenic plants: engineering the chloroplast genome without the use of antibiotic selection
NZ521707A NZ521707A (en) 2000-03-02 2001-02-28 Marker free transgenic plants: engineering the chloroplast genome without the use of antibiotic selection
AU2001245359A AU2001245359A1 (en) 2000-03-02 2001-02-28 Marker free transgenic plants: engineering the chloroplast genome without the use of antibiotic selection
US10/741,379 US7795497B2 (en) 2000-03-02 2003-12-19 Marker free transgenic plants: engineering the chloroplast genome without the use of antibiotic selection
US12/850,733 US20110072541A1 (en) 2000-03-02 2010-08-05 Marker free transgenic plants engineering the chloroplast genome without the use of antibiotic selection

Applications Claiming Priority (8)

Application Number Priority Date Filing Date Title
US18630800P 2000-03-02 2000-03-02
US60/186,308 2000-03-02
US20876300P 2000-06-02 2000-06-02
US60/208,763 2000-06-04
US25740600P 2000-12-22 2000-12-22
US60/257,406 2000-12-22
US25915400P 2000-12-28 2000-12-28
US60/259,154 2000-12-29

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US09/807,722 A-371-Of-International US20020137214A1 (en) 2000-03-02 2001-02-28 Marker free transgenic plants engineering the chloroplast genome without the use of antibiotic selection
US10/741,379 Continuation US7795497B2 (en) 2000-03-02 2003-12-19 Marker free transgenic plants: engineering the chloroplast genome without the use of antibiotic selection

Publications (2)

Publication Number Publication Date
WO2001064023A1 WO2001064023A1 (en) 2001-09-07
WO2001064023B1 true WO2001064023B1 (en) 2001-12-13

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EP (1) EP1294221A4 (en)
AU (1) AU2001245359A1 (en)
CA (1) CA2401954A1 (en)
WO (1) WO2001064023A1 (en)

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US8581035B2 (en) 2006-08-31 2013-11-12 Monsanto Technology Llc Plant transformation without selection

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WO2001072959A2 (en) 2000-03-01 2001-10-04 Auburn University Plastid transformation vectors for expressing human proteins in plants
US20100251425A9 (en) 1998-05-15 2010-09-30 University Of Central Florida Expression of human interferon in transgenic chloroplasts
CA2471737C (en) 2001-12-26 2016-01-26 University Of Central Florida Expression of protective antigens in transgenic chloroplasts and the production of improved vaccines
WO2004005480A2 (en) * 2002-07-03 2004-01-15 University Of Central Florida Plastid genetic engineering via somatic embryogenesis
CA2493364A1 (en) 2002-07-26 2004-02-12 Basf Plant Science Gmbh Inversion of the negative-selective effect of negative marker proteins using selection methods
AU2003285818A1 (en) * 2002-11-28 2004-06-18 Universiteit Leiden A method for marker-less integration of a sequence of interest into the genome of a cell
US20040210961A1 (en) 2003-03-07 2004-10-21 Palys Joseph Michael Markerless transformation
US20090007294A1 (en) * 2004-09-01 2009-01-01 Henry Daniell Genetic Engineering of Male Sterility in Plants
CA2608671C (en) 2005-05-27 2018-05-15 University Of Central Florida Chloroplasts engineered to express pharmaceutical proteins
AU2006290733B8 (en) 2005-09-16 2012-07-05 Bayer Intellectual Property Gmbh Transplastomic plants expressing lumen-targeted protein
GB2447416B (en) * 2006-12-15 2010-08-25 Uni I Stavanger Methods and vectors for transformation of plastids and marker excision using site-specific recombination
AU2008232543B2 (en) 2007-03-30 2013-01-17 University Of Central Florida Research Foundation, Inc. Chloroplasts engineered to express pharmaceutical proteins in edible plants
WO2015058214A1 (en) 2013-10-18 2015-04-23 Trustees Of The University Of Pennsylvania Oral delivery of angiotensin converting enzyme 2 (ace2) or angiotensin-(1-7) bioencapsulated in plant cells
PL3068869T3 (en) 2013-11-15 2021-03-08 The Trustees Of The University Of Pennsylvania Compositions for suppression of inhibitor formation against factor viii in hemophilia a patients
MA41180A (en) 2014-12-17 2017-10-24 Bayer Cropscience Nv PLANTS CHARACTERIZED BY IMPROVED PHOTOSYNTHETIC CARBON BINDING CAPACITY
WO2017038835A2 (en) * 2015-08-28 2017-03-09 Honda Motor Co., Ltd. Method of producing plastid transformants
JP6696806B2 (en) * 2015-08-28 2020-05-20 本田技研工業株式会社 Method for producing plastid transformant
CN108699548A (en) 2015-11-16 2018-10-23 宾夕法尼亚州立大学托管会 The therapeutic protein targeted delivery that biology is encapsulated in plant cell is to target cell type to treat disease
CN109554445B (en) * 2019-01-23 2021-09-21 山东省花生研究所（山东省农业科学院花生工程技术研究中心） Effective and simple method for analyzing genetic relationship between peanut species

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US5877402A (en) * 1990-05-01 1999-03-02 Rutgers, The State University Of New Jersey DNA constructs and methods for stably transforming plastids of multicellular plants and expressing recombinant proteins therein
US5633153A (en) * 1994-10-14 1997-05-27 Calgene, Inc. Aldehyde dehydrogenase selectable markers for plant transformation
EP1002115B9 (en) * 1997-08-07 2008-12-03 Auburn University Universal chloroplast integration and expression vectors and transformed plants
JP2002520024A (en) * 1998-07-10 2002-07-09 カルジーン エルエルシー Expression of herbicide resistance gene in plant plastids

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8581035B2 (en) 2006-08-31 2013-11-12 Monsanto Technology Llc Plant transformation without selection
US8847009B2 (en) 2006-08-31 2014-09-30 Monsanto Technology Llc Plant transformation without selection
US9617552B2 (en) 2006-08-31 2017-04-11 Monsanto Technology Llc Plant transformation without selection

Also Published As

Publication number Publication date
EP1294221A4 (en) 2003-06-25
WO2001064023A1 (en) 2001-09-07
CA2401954A1 (en) 2001-09-07
AU2001245359A1 (en) 2001-09-12
EP1294221A1 (en) 2003-03-26

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