WO1992006205A1 - A process for the gene manipulation of plant cells, recombinant plasmids, recombinant bacteria, plants - Google Patents
A process for the gene manipulation of plant cells, recombinant plasmids, recombinant bacteria, plants Download PDFInfo
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- WO1992006205A1 WO1992006205A1 PCT/EP1991/001883 EP9101883W WO9206205A1 WO 1992006205 A1 WO1992006205 A1 WO 1992006205A1 EP 9101883 W EP9101883 W EP 9101883W WO 9206205 A1 WO9206205 A1 WO 9206205A1
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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
- C12N15/8202—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
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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
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/52—Genes encoding for enzymes or proenzymes
Definitions
- This invention relates to a process for the gene manipulation of plant cells, which comprises introducing foreign DNA into plant cells by infecting the plant cells with one or more recombinant Agrobacterium tumefaciens strains, which contain foreign DNA to be transferred to the plant cells between the left- and right-hand ends of T-DNA necessary for such a transfer and are capable of transferring this foreign DNA to the. plant cells.
- the invention further relates to a recombinant plasmid consisting of a bacterial vector plasmid and at least one DNA insert, and to a recombinant Agrobacterium tumefaciens strain, which contains foreign DNA to be transferred to plant cells between the left- and right-hand ends of T-DNA necessary for such a transfer and is capable of transferring this foreign DNA to the plant cells.
- the invention also relates to the plants deriving from the transformed cells and to the parts
- substantially dicotyl plant species such as
- Transposons are, in fact, genetic elements which can move within the genome of the host cells in which they are normally present. They have virtual ends, repeated border sequences which are in general invertedly repeated and may be rather different in length. At the location of their insertion into the genome a piece of host sequence is found back directly repeated to the left and the right of the transposon sequence. There is generally no specific selection of the location of insertion. In case of excision either the original sequence is restored or a deletion occurs or part of the sequence remains at the relevant location (insertion).
- a transposon consists of a transposase gene located between terminal sequences which contain transposase binding sites and optional binding sites for other host enzymes and terminate with the repeated border sequence. These terminal sequences, together with the directly repeated sequences of the target DNA to the left- and right-hand of the border sequence, will also be referred to hereinbelow as left- and right-hand transposon ends.
- the present invention provides a process which removes the main objections of the known methods and gives a very efficient and stable transformation of cells of monocotyl plants, which has not been found possible so far. For this purpose use is made according to the invention of the
- the invention relates in a first aspect to a process for the gene manipulation of cells of plants which, in essence, cannot be transformed by Agrobacterium tumefaciens with integration by T-DNA, with foreign DNA being introduced into the plant cells by infecting the plant cells with one or more recombinant Agrobacterium tumefaciens strains which contain foreign DNA to be transferred to the plant cells between the left- and right-hand ends of T-DNA necessary for such a transfer and are capable of transferring this foreign DNA to the plant cells, the foreign DNA located between the left- and right-hand ends of T-DNA comprising:
- transposase gene located in an expression cassette active in the plant cells but not between the left- and right- hand transposon ends necessary for integration in the DNA of the plant cells
- T-DNA are meant all those plant species in which
- Agrobacterium tumefaciens is not capable of effecting an efficient integration of the T-DNA in the plant genome, which roughly means almost all the non-dicotyl plants.
- the invention is therefore eminently suited for the gene manipulatin of monocotyl plants and other non-dicotyl plants, preference being given according to the invention to gene manipulation of plant cells of cereals such as maize, wheat, barley, rye, oat, rice etc., grasses such as ryegrass and fescue, leguminous plants such as leek, onion and garlic, ornamental plants such as tulipe, hyacinth etc. and other plant species such as conifer etc.
- transposase gene which is not located between the left- and right-hand transposon ends necessary for integration in the DNA of the plant cells.
- transposase gene which is not located between the left- and right-hand transposon ends necessary for integration in the DNA of the plant cells.
- transposase gene which is not located between the left- and right-hand transposon ends necessary for integration in the DNA of the plant cells.
- transposase gene which is not located between the left- and right-hand transposon ends necessary for integration in the DNA of the plant cells.
- recombinant transposon containing no DNA fragment which can lead in the plant cells to expression of an active
- transposase This means that the recombinant transposon is an inactive transposon. Consequently, a desirable high stability of the transformed plants can be obtained.
- the drawback of instability normally connected with the use of transposons is, in fact, the result of the presence of a complete (or active) transposon, i.e. a transposon with a transposase gene located between the transposon ends. This gene is then co-incorporated with the transposon in the plant genome and may therefore give rise for several generations to the undesirable instability.
- an active transposase gene namely in order to ensure efficient
- transposase gene leads to the formation of transposase which, in cooperation with nuclear factors of the plane, is capable of effecting efficient integration of the transposon in the genome but if this transposase gene is not within a transposon, in
- the transposase gene preferably consists of a genomic clone of the transposase gene, i.e. of genomic DNA which comprises not only the encoding exons but also the introns normally present.
- genomic DNA which comprises not only the encoding exons but also the introns normally present.
- a promoter suitable for this purpose is the known CaMV 35S promoter.
- the use of the pTR1' or pTR2' promoter is also posible and favorable if, as is also preferably done according to the invention, transfer occurs by microinjection with the apical meristem being mechanically damaged.
- the wound-inducible regulators of gene expression for these promoters are present in the meristematic cells.
- the use of such a construct of a genomic transposase gene under control of another promoter such as the CaMV 35S, the pTR1' or the pTR2' promoter therefore constitutes a highly
- the recombinant transposon comprises at least one foreign gene located in an expression cassette active in the plant cells.
- an expression cassette active in the plant cells By this is meant any gene that is to be incorporated in the genome of the plant for whatever reason. It may be a gene coding for a desirable property of the plant, such as
- the gene to be introduced into the plant may therefore be a gene within the scope of plant breeding or a gene within the scope of biological production of specific products (proteins and enzymatic products, DNA, RNA etc.).
- the recombinant transposon contains a promoter active in plants, instead of a foreign gene in an expression cassette active in plants. Consequently, it becomes possible to alter the expression behavior of specific genes and thus the phenotype.
- one or more selectable marker genes suitable for selection of transformed bacteria and located in an expression cassette active in the bacteria are located between the left- and right-hand ends of T-DNA.
- Marker genes suitable for this purpose are known to those skilled in the art. These are often genes the expression of which leads to a resistance to specific antibiotics, such as resistance to kanamycin,
- one or more selectable marker genes suitable for selection of transformed bacteria and located in an expression cassette active in bacteria form part of the recombinant transposon.
- the recombinant transposon in a gene of the host the transformed plant shows another phenotype (which is not due to the introduced genetic information as such) or that the foreign gene introduced proves to function differently in later generations than in the first generations (e.g. as a result of mutation) the recombinant transposon can be cloned for further examination from the plant genome into bacteria again by means of selection with the marker gene active in bacteria.
- one or more selectable marker genes suitable for selection of transformed plant cells and located in an expression cassette active in the plant cells are located between the left- and right-hand ends of T-DNA.
- selectable marker genes suitable for this purpose are known to those skilled in the art.
- resistance to, e.g., antibiotics or herbicides can be mentioned as a property which, as is known to those skilled in the art, can be used for a selection of successfully
- one or more selectable marker genes suitable for selection of transformed plant cells and located in an expression cassette active in the plant cells form part of the recombinant transposon.
- This preferred embodiment has the advantage that the marker genes are co-incorporated in the genome of the plant so that also later generations can be selected on the basis of the
- tumefaciens strains one of which comprises between the left- and right-hand ends of T-DNA the transposase gene located in an expression cassette active in the plant cells and the other the recombinant transposon between the left- and right-hand ends of T-DNA.
- This variant has the advantage of having a wide range of applications, i.e. there can always be used the same Agrobacterium strain supplying the transposase gene and only the second strain containing the recombinant transposon must be constructed for each new gene. In this second strain there is used a construct in which a multiple cloning site is located between the transposon ends so as to have a great freedom of choice with respect to the DNA fragments to be incorporated in the transposon.
- both the first and the second A. tumefaciens strain will comprise between the left- and right-hand ends of T-DNA at least one marker gene for selection of transformed bacteria and at least one marker gene for selection of transformed plant cells. It is preferred here that at least the marker genes for selection of transformed plant cells which carry the two bacterial strains within the T-DNA ends are different from each other so as to recognize plant cells successfully transformed by the two strains.
- An alternative embodiment of the process according to the invention is characterized in that the plant cells are infected with one recombinant A. tumefaciens strain which comprises between the left- and right-hand ends of T-DNA both the transposase gene located in an expression cassette active in the plant cells and the recombinant transposon.
- This variant has the advantage of more chance of success of the transformation, i.e. the desired DNA transfer and integration will have taken place in a larger part of the treated plant cells.
- the A. tumefaciens strain will comprise between the left- and right-hand ends of T-DNA at least one marker gene for selection of transformed bacteria and at least one marker gene for selection of transformed plant cells.
- the process according to the invention is of special importance to the transformation of plant cells of a monocotyl plant species such as maize, wheat, barley, rye, oat, rice etc. An efficient transformation of such crops has not been found possible so far.
- the invention can also be used, however, for the purpose of altering phenotypic characteristics of plants resulting from the presence of inactive transposons in one or more genes of the plant by removing such a transposon from the gene in which it is contained.
- the invention provides a process for the gene manipulation of cells of plants which, in essence, cannot be transformed by Agrobacterium tumefaciens with integration by T-DNA, which comprises introducing foreign DNA into the plant cells by infecting the plant cells with a recombinant Agrobacterium tumefaciens strain which contains foreign DNA to be transferred to the plant cells between the left- and right-hand ends of T-DNA necessary for such a transfer and is capable of transferring this foreign DNA to the plant cells, which process is characterized in that plant cells are infected which contain one or more inactive
- transposons but no active transposase gene and that the foreign DNA located between the left- and right-hand ends of T-DNA comprises a transposase gene located in an expression cassette active in the plant cells, which cassette is not located between the left- and right-hand transposon ends necessary for integration in the DNA of the plant cells .
- the present invention is not subject to special
- cells of the apical meristem in germinating seeds are infected with the bacteria.
- the infection is carried out by means of needleless high-pressure injection.
- needleless high-pressure injection By this is meant a
- the invention is not limited to such a method and comprises the possibility of carrying out the infection by means of microinjection.
- other meristematic or embryogenic tissues or cells can be used for introducing a foreign gene.
- the invention in another aspect relates to a recombinant plasmid consisting of a bacterial vector plasmid and at least one DNA insert, which recombinant plasmid is characterized in that at least one DNA insert comprises a transposase gene located in an expression cassette active in plant cells, which expression cassette active in plant cells and filled with a transposase gene is not located between the left- and right- hand transposon ends necessary for integration in the DNA of plant cells.
- the invention further relates to a recombinant plasmid consisting of a bacterial vector plasmid and at least one DNA insert, which recombinant plasmid is characterized in that at least one DNA insert comprises a recombinant transposon which contains the left- and right-hand transposon ends necessary for integration in the DNA of plant cells but no DNA fragment which can lead in plant cells to expression of an active transposase.
- a preferred embodiment of such a recombinant plasmid is characterized in that between the left- and right-hand transposon ends the recombinant transposon comprises one or more restriction endonuclease recognition sequences suitable as cloning site.
- Such a plasmid has a wide range of
- a special preferred embodiment of such plasmids is characterized in that the recombinant transposon comprises between the left- and right-hand transposon ends at least one foreign gene located in an expression cassette active in plant cells.
- the recombinant transposon comprises betwe n the left- and right-hand transposon ends one or more selectable marker genes suitable for selection of transformed plant cells and located in an expression cassette active in plant cells, and that the recombinant transposon comprises between the left- and right-hand transposon ends one or more selectable marker genes suitable for selection of transformed bacteria and located in an expression cassette active in bacteria.
- the invention provides a
- Agrobacterium tumefaciens strain which contains foreign DNA to be transferred to plant cells between the left- and right-hand ends of T-DNA necessary for such a transfer and is capable of transferring this foreign DNA to the plant cells, which strain is characterized in that a transposase gene located in an expression cassette active in plant cells is located between the left- and right-hand ends of T-DNA, which expression cassette active in plant cells and filled with a transposase gene is not located between the left- and right-hand transposon ends necessary for integration in the DNA of plant cells.
- the invention also provides a recombinant Agrobacterium tumefaciens strain which contains foreign DNA to be transferred to plant cells between the left- and right-hand ends of T-DNA necessary for such a transfer and is capable of transferring this foreign DNA to the plant cells, which strain is characterized in that between the left- and right-hand ends of T-DNA a recombinant transposon is located, which comprises the left- and right-hand transposon ends necessary for integration in the DNA of plant cells but no DNA fragment which can lead in plant cells to expression of an active transposase.
- the recombinant transposon may comprise between the left- and right-hand transposon ends at least one foreign gene located in an expression cassette active in plant cells and/or one or more selectable marker genes suitable for selection of transformed plant cells and located in an expression cassette active in plant cells, and/or one or more selectable marker genes suitable for selection of transformed bacteria and located in an expression cassette active in bacteria.
- a special embodiment of such a recombinant Agrobacterium tumefaciens strain is characterized in that also a transposase gene located in an expression cassette active in plant cells is located between the left- and right-hand ends of T-DNA, which expression cassette active in plant cells and filled with a transposase gene is not located between the left- and right-hand transposon ends necessary for integration in the DNA of plant cells.
- the invention is finally also contained in plants and parts consisting of one or more cells or products thereof, which plants derive from plant cells transformed by the process according to the invention.
- parts and products of plants is meant, e.g., the bulb, the tuber or the root, the flowers, the seed and the meal made therefrom etc.
- FIG. 1 diagrammetically show in Fig. 1 a T-DNA construct comprising both a foreign gene X included in an inactive transposon and a transposase gene not located in an active transposon.
- Fig. 2a a T-DNA construct comprising a foreign gene X included in an inactive transposon.
- Fig. 2b a T-DNA construct which can be used in
- Fig. 2a combination with that of Fig. 2a and comprises a transposase gene not located in an active transposon.
- Fig. 3a a T-DNA construct comprising a foreign gene X included in an inactive transposon which comprises a marker gene interrupted by a second inactive transposon for selecting transformed plant cells.
- Fig. 3b a T-DNA construct which can be used in
- Fig. 4a a T-DNA construct comprising a foreign gene X included in an inactive transposon, which foreign gene X is interrupted by a second inactive transposon,
- Fig. 4b a T-DNA construct which can be used in
- Fig. 5a the structure of the recombinant plasmid pMH10, pMH10-LTS and pMH10-DsB, respectively a plasmid having a complete transposon, a transposase gene with only the left transposon end and a transposon without transposase gene,
- Fig. 5b an example of a T-DNA construct with plasmid pMH10-LTS according to the invention
- Fig. 6a the structure of the recombinant plasmid
- Fig. 6b an example of a T-DNA construct with this plasmid according to the invention
- Fig. 7 the structure of the recombinant plasmid
- Fig. 8 the structure of the recombinant plasmid
- Fig. 10 the structure of the recombinant plasmid
- pUCSSAc9Ds19 suitable for cloning foreign genes between the transposon ends of Ac9
- Fig. 11a the structure of the recombinant plasmid pUCSS19tpnAc9P35S,
- Fig. 11b the structure of the recombinant plasmid pUCSS19tpnAc9PTR1,
- Fig. 11c a T-DNA construct with the recombinant plasmid pUCSS19tpnAc9P35S according to the invention.
- Fig. 12a the structure of the recombinant plasmid pUCSSDs19BTKA
- Fig. 12b a T-DNA construct with this plasmid according to the invention
- Fig. 13a a cointegrate plasmid with the constructs pMH11Ds4IPTkan and pUCtpnAc9P35S, and
- Fig. 13b an example of a T-DNA construct with the cointegrate plasmid pMH11Ds4IPTkan/pUCtpnAc9P35S.
- X represents a foreign gene located in an expression cassette, not separately shown, active in plant cells; if the gene is interrupted by an insert, the component parts of the gene are indicated by X' and X", respectively;
- S1 represents a marker gene which can be used for the selection of transformed plant cells; here, too, the component parts are indicated by S1' and S1", respectively, when the gene is interrupted by an insert; S2 and S3 also represent marker genes suitable for the selection of successfully transformed plant cells, further referred to as plant selection genes;
- s1 and s2 represent marker genes suitable for the selection of transformed bacteria, further referred to as bacteria selection genes;
- tpn represents a genomic clone of a transposase gene located in an expression cassette, not separately shown, which can function in plant cells;
- LTA and RTA represent respectively the left end of T-DNA and the right end of T-DNA, which ends are necessary for transferring the T-DNA by the Agrobacterium tumefaciens
- LTS and RTS represent respectively the left end of a transposon and the right end of an active or inactive
- Wx represents the sequence of the waxy gene of maize from which Ac9 was cloned
- Fig. 1 shows a T-DNA construct according to the
- transposase gene tpn not located on an active transposon.
- the selection genes shown in Fig. 1 are facultative, for that matter, in particular one of the bacteria selection genes and one of the plant selection genes being dispensable, if
- transposase gene it is important that it is not located within the transposon ends LTS and RTS. This does not mean that the transposon ends must be completely absent: often removal of (a significant portion of) one of the transposon ends will be sufficient to prevent efficient incorporation of the transposase gene in the genome of the transformed plant cells. Thus, for instance, one transposon end could be retained in Fig. 1 beside the transposase gene (see Fig. 5a).
- Figs . 2a and 2b show a preferred embodiment consisting of an analogous construct, the elements being distributed over two different plasmids.
- the inactive transposon having a foreign gene X and a plant selection gene Sl contained therein is located on a first plasmid the T-DNA of which is shown in Fig. 2a.
- Fig. 2b shows the T-DNA of a second plasmid which contains a transposase gene and a plant selection gene S2.
- each of the two T-DNA constructs also contains a bacteria selection gene (si and s2,
- T-DNA constructs according to the invention may contain additional or other elements than are shown in the drawings, e.g. one or more additional foreign genes, one or more additional bacteria or plant selection genes etc.
- Figs . 3a and 3b show a preferred embodiment resembling those of Figs. 2a and 2b.
- An inactive transposon having a foreign gene X and a plant selection gene Sl contained therein is located on a first plasmid, the T-DNA of which is shown in Fig. 3a.
- the plant selection gene Sl is interrupted by a second inactive transposon which, if
- Fig. 3b shows the T-DNA of a second plasmid
- This plasmid is identical with the plasmid shown in Fig. 2b.
- Figs . 4a and 4b show a preferred embodiment
- FIGs. 5 through 13 show DNA constructs which will be further illustrated by the Examples .
- Ds mutations from genes of maize.
- Agrobacterium is used herein to introduce the transposase gene into cells of the apical meristem of maize.
- the plasmid pMHIO (Fig. 5a) contains the PstI clone of transposon Ac9 in the waxy gene of maize (Pohlman et al, 1984, Muller-Neumann et al, 1984), a Pstl-BamHI fragment of plasmid pBR322 (Bolivar et al, 1977) with a functional ori sequence for replication in gram-negative bacteria, but a nonfunctional amp and tet resistance gene and moreover a BamHI-
- the plasmid pGV3850 was obtained from Laboratorium voor Genetika RUG, Ledeganckstraat 35, B9000 Ghent, Belgium.
- Seeds of ten maize lines were disinfected with NaClO 2% and then laid on sterile wet sand to germinate on sterile sand at 25°C in the dark.
- Three days after germination 50 nl induced A. tum. culture were integrated with the construction pMH10 LTS by genetic recombinantion between the borders of the T-DNA (Fig. 5b), injected into the apical meristem by
- the induction medium has been described by Vernade et al (1988). After one week at 20°C the young plants were cultured together with untreated plants in large pots in an air- conditioned greenhouse in a mixture of compost and sandy clay in their normal growing season (May to October). Each plant was fertilized manually, both the plume and the ear being kept in small bags. Each plant was individually watered in the pot. The ears were harvested in September and October and after drying the seed was stored in paper bags during winter at 6°C. The next year the plants were sown on ear lines in the soil in the greenhouse and again fertilized manually and evaluated by corn growers for their characteristics both during growth and in surmaturity.
- the first contains the ⁇ -glucuronidase gene of
- Escherichia coli located between the left- and right-hand transposon ends of Ac9 (wx), which, in turn, is located between the Left and Right Border (LB and RB) of the T-DNA on a non- oncogenic Ti plasmid.
- the second contains a complete
- transposon Ac9(wx) located between LB and RB of the T-DNA.
- the A.tum. strain containing the transposon Ac9 in the T-DNA is shown in Fig. 5a.
- the other A.tum. strain was obtained in the following manner:
- Plasmid pBI 221 contains the ⁇ -glucuronidase gene of E. coli (uidA) under the control of the CaMV 35S promoter (Cauliflower Mosaic Virus 35S RNA promoter) and the mRNA distal end of the nopaline synthase gene of the Ti plasmid of A. tum. T37. This plasmid was available from Dr R. Jefferson, c/o AFRC
- the plasmid pMH10 shown in Fig. 5a was cut with EcoRI and partially with Hindlll and the 10.1 kb fragment was ligated with the 3 kb EcoRI-Hindlll fragment of pBI 221 containing the sequence of the chimeric uidA gene of this last plasmid.
- the resulting plasmid pMH10Ds15GUS contains the chimeric uidA gene in a transcription orientation contrary to that of the (partially) deleted transposase gene.
- Example 2 The same maize lines described in Example 1 were used for the transformation.
- the apical meristem was infected by microinjection with 50 nl of a mixture of the two above-described A. tum. strains induced for transfer.
- a first phase some hundreds of germs were tested after 1 or 2 weeks for ⁇ -glucuronidase activity by incubating a piece of tissue containing the apical meristem with X-gluc (5-bromo-4-chloro-3-indolyl-D-glucuronide). In about 7.5% of the tissues blue staining occurred.
- X-gluc (5-bromo-4-chloro-3-indolyl-D-glucuronide
- Beta- glucuronidase activity was detected by covering the gel with a thin agarose gel containing the substrate methyl-umbelliferyl- D-glucuronide which fluoresces in UV light if the ⁇ - glucuronidase enzyme causes release of methyl umbelliferone.
- a fluorescent band was found in about 1% of the tested plants.
- DNA was prepared from leaves and pieces of roots of these plants and, as in RFLP mapping, hybridized with a 2.9 kb Hindlll-EcoRI fragment containing the chimeric uidA gene.
- Hindlll-EcoRI fragment containing the chimeric uidA gene.
- the mutagenic properties of a transposon are used by integrating a chimeric transposon containing a plant selection gene but no transposase gene in the genome of barley by means of a transposase gene that is not located between transposon ends.
- the first strain contains the chimeric transposon between the T-DNA ends and the second strain contains the transposase gene between the T-DNA ends.
- the employed plasmids are pMH10Ds15GUS and pMH10LTS. As described before, they are conjugated from Escherichia coli to Agrobacterium and integration in pGV3850 selected by also adding spectinomycin to the medium.
- Seeds of 12 ear lines were disinfected with 6% NaCIO and the seeds laid on moist sand to germinate at 4°C. Germinating seeds were further incubated at room temperature under sterile conditions.
- tobacco ⁇ -glucuronidase activity was found when the gene was present in the infection, with or without additional infection with the transposase gene.
- the chloramphenicol acetyl transferase gene in an expression cassette consisting of the 35S promoter of CaMV and the nos-terminator of the T-DNA of Agrobacterium tumefaciens is available as plasmid pCAMVCN from Pharmacia-LKB, Bromma, Sweden.
- the complete chimeric gene was cut as Xbal fragment.
- the plasmid pMH10 was partially cut with Bell (position 1530 in Ac9Wx) and BamHI (position 4498 in Ac9Wx) and the 8.1 kb fragment was ligated back.
- This plasmid contains unique restriction sites in the remaining Ac9 sequence: Nsil (five sites at position 571, 972, 1003, 1183, and 1251 of Ac9Wx), Xhol (at position 1125 of Ac9Wx) and Xbal (at position 1257 of Ac9Wx) (Fig. 5a).
- the cat expression cassette was cloned into the unique Xbal site of Ac9.
- the resulting plasmid was called pMH10Ds34CAT (Fig. 7), conjugated to Agrobacterium and integrated in the plasmid pGV3850. By transformation of tobacco the expression of the cat gene was proved.
- the genes iaaM and iaaH from the T-DNA of Agrobacterium tumefaciens Ach5 were cloned from the plasmids pGV814 (EcoRl-Clal clone of iaaM) (Budar et al, 1986) and pGV824 (Hindlll clone of iaaH) (Budar et al, 1986).
- pMH11 is a derivative of pMH10 obtained by cutting in the vector plasmid of pMH10 the 0.8 kb Sphl fragment located between the str/spc resistance gene and the (partially deleted) tet resistance gene, blunt ending the ends with S1 nuclease and ligating back the vector.
- the PstI clone of Ac9Wx was again incorporated in the resulting vector in the unique PstI site. The orientation, however, is
- plasmid pMH11Ds4IPTkan (Fig. 9) was conjugated to Agrobacterium and integrated in pGV3850. by additional selection with spectinomycin and then with
- the Sphl-Kpnl fragment from mini-Sa a derivative of pR702 (Leemans et al, 1983) containing the sm-sp adenosyl transferase gene encoding resistance to streptomycin and spectinomycin was blunt ended with T4 DNA polymerase and cloned into the plasmid pUC19 (Yanisch-Perron et al, 1985), blunt end cut with Sspl.
- This plasmid was 4330bp and will be referred to hereinbelow as pUCSS19.
- the PstI clone of Wx-Ac9 was cut from pMH10 (Fig. 5a) and blunt ended with T4 DNA polymerase. This fragment was cloned into the 4030bp PvuII fragment of pUSS19. This plasmid was 8840bp and will be referred to hereinbelow as pUCSSAc9.
- pUCSSAc9 was cut with Nsil and MscI and the Nsil end was blunt ended with T4 DNA polymerase. This fragment was 4980bp and contained the ends of transposon Ac9Wx. Into this was cloned the 300bp PvuII fragment of pUC19.
- the plasmid was 5280 bp and will be referred to as pUCSSAcDs19. It can be used for cloning numerous genes with their expression cassette. It can be conjugated to Agrobacterium with plasmid pGV3850 as described in the first example by additional selection with
- Bacillus thuringiensis var. Hopkins 1715 (Klier et al, 1982), the sequence of which was published by H ⁇ fte et al, 1986, was obtained from S.A. Solvay, Brussels, Belgium, as plasmid pBT424. The strain is available from the Institut Pasteur in Paris, France.
- the complete double expression cassette pTR1 '-BTtox terminator gene 4/pTR2'-nptll terminator ocs was cut again with Kpnl and partially with PstI as a 5.7 kb fragment and cloned into the corresponding restriction sites of pUCSSAcDsl9.
- the terminator of gene 4 contains different stop codons in the three reading frames upstream of the polyadenylation signal.
- pUCSSDs19BTKA The resulting plasmid (pUCSSDs19BTKA) (Fig. 12a) was conjugated to Agrobacterium and integrated in pGV3850 by additional selection with spectinomycin as described before (Fig. 12b).
- the construct was used for making maize lines resistant to Pyralidae, in the manner as described in Comparative
- Example 2 with the difference that use was made of a second Agrobacterium strain containing a transposase gene without transposon ends (pMH10LTS).
- the plasmid pMH10 (Fig. 5a) was cut with PstI and the 4810bp Ac9 wx fragment, cloned into pUC19, cut wit PstI. This plasmid was 7500bp and will be referred to hereinbelow as pUC19Ac9wx. The work was further done with the orientation 1, namely the one in which the BamHI sites are remotest from each other.
- the Ac9 wx fragment was cut again, as BamHI fragment. This fragment was partially cut with Cfr10I and two fragments were stored: the 3.4 kb Cfr10I fragment and the 4.2 kb BamHI- Cfr10I fragment. The first contained a transposase gene without a promoter and the second a transposase gene with a promoter. The 4.2 kb fragment was cloned into pUCSS19, cut with Xmal and BamHI. This plasmid (8.5kb) contained the normal transposase gene of Ac but no transposon ends
- pUCSS19tpnAc9PAc9 pUCSS19tpnAc9PAc9
- the 3.4 kb fragment was cloned into pUC19, cut with Xmal, and the orientation was determined using the Xbal site in the MCS and in the transposase gene (position 1260 of Ac9Wx).
- This plasmid (7.7 kb) is called pUCSS19tpnAc9cds) and may serve for cloning new promoters upstream of the transposase gene.
- This plasmid was cut with PstI and BamHI and the CaMV 35S promoter fragment from pBl221, cut with PstI and BamHI, was cloned into it.
- the resulting plasmid is called pUCSS19tpnAc9P35S
- Agrobacterium Ach5 (Velten et al, 1984) was obtained as a
- pUCS119tpnAc9PTR1' contains the transposase gene under control of the pTR1' promoter (Fig. 11b).
- Both plasmids can be conjugated, as described before, to Agrobacterium and integrated in pGV3850 by additional
- pTR1' promoter has the advantage that in case of microinjection or bombardment of Agrobacterium under high pressure this promoter strongly expresses in the wounded tissue.
- the employed pMH10 derivatives were: pMH10Ds15GUS, pMH10Ds34CAT, pMH10Ds121AA and pMH11Ds4IPTkan and are all carriers of a spectinomycin
- the cointegrate of the recombinant plasmids pMH11Ds4IPTkan and pUCSS19Ac9P35S is shown in Fig. 13 by way of example. These cointegrate plasmids were transmitted by transformation to the strain HB101 and after selection for ampicillin and spectinomycin they were characterized by plasmid preparation and restriction enzyme analysis. Then the plasmids were transmitted by conjugation to Agrobacterium. as described before.
- the resulting Agrobacterium strains are an example of the use shown in Fig. 1.
- the resulting recombinant Agrobacterium strains were tested on tobacco for expression of respectively nopaline synthase (pGV3850 marker), uidA, cat, root formation and shoot formation and finally for transposition, before they were used on cereals.
Abstract
Description
Claims
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NL9002116 | 1990-09-27 | ||
NL9002116A NL9002116A (en) | 1990-09-27 | 1990-09-27 | METHOD FOR GENETICALLY MANIPULATING PLANT CELLS, RECOMBINANT PLASMID, RECOMBINANT BACTERIA, PLANTS. |
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WO1992006205A1 true WO1992006205A1 (en) | 1992-04-16 |
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PCT/EP1991/001883 WO1992006205A1 (en) | 1990-09-27 | 1991-09-26 | A process for the gene manipulation of plant cells, recombinant plasmids, recombinant bacteria, plants |
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EP (1) | EP0554273A1 (en) |
JP (1) | JPH06501155A (en) |
CA (1) | CA2089072A1 (en) |
IE (1) | IE913359A1 (en) |
NL (1) | NL9002116A (en) |
WO (1) | WO1992006205A1 (en) |
Cited By (16)
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US5489520A (en) * | 1990-04-17 | 1996-02-06 | Dekalb Genetics Corporation | Process of producing fertile transgenic zea mays plants and progeny comprising a gene encoding phosphinothricin acetyl transferase |
WO1999020776A1 (en) * | 1997-10-20 | 1999-04-29 | Cotton Incorporated | In planta method for the production of transgenic plants |
US6051430A (en) * | 1996-02-09 | 2000-04-18 | Het Nederlands Kanker Instituut | Vectors and methods for providing cells with additional nucleic acid material integrated in the genome of said cells |
WO2000044919A1 (en) * | 1999-01-29 | 2000-08-03 | New Zealand Institute For Crop & Food Research Limited | Transformation and regeneration of allium plants |
WO2001081600A2 (en) * | 2000-04-20 | 2001-11-01 | Btg International Limited | Transgenic plants |
US6822144B1 (en) | 1997-01-24 | 2004-11-23 | Pioneer Hi-Bred International, Inc. | Methods for Agrobacterium-mediated transformation |
US7026528B2 (en) | 1996-06-21 | 2006-04-11 | Monsanto Technology Llc | Methods for the production of stably-transformed, fertile wheat employing agrobacterium-mediated transformation and compositions derived therefrom |
US7067719B1 (en) | 1999-05-05 | 2006-06-27 | Seminis Vegetable Seeds, Inc. | Transformation of Allium sp. with Agrobacterium using embryogenic callus cultures |
US7250554B2 (en) | 2002-02-20 | 2007-07-31 | J.R. Simplot Company | Precise breeding |
EP2105446A2 (en) | 1998-09-23 | 2009-09-30 | ZymoGenetics, Inc. | Cytokine receptor zalpha11 |
WO2010037209A1 (en) | 2008-10-03 | 2010-04-08 | Agrisoma Biosciences Inc. | Production of modified fatty acids in plants |
US8273949B2 (en) | 2002-02-20 | 2012-09-25 | J.R. Simplot Company | Precise breeding |
WO2013104026A1 (en) | 2012-01-11 | 2013-07-18 | The Australian National University | Method for modulating plant root architecture |
US9290777B2 (en) | 2007-02-05 | 2016-03-22 | National University Of Singapore | Putative cytokinin receptor and methods for use thereof |
US10428336B2 (en) | 2013-10-16 | 2019-10-01 | The Australian National University | Method for modulating plant growth |
WO2021019394A1 (en) | 2019-07-26 | 2021-02-04 | Oxford University Innovation Limited | Modified plants |
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US4732856A (en) * | 1984-04-03 | 1988-03-22 | Carnegie Institution Of Washington | Transposable elements and process for using same |
EP0267159A2 (en) * | 1986-11-07 | 1988-05-11 | Ciba-Geigy Ag | Process for the genetic modification of monocotyledonous plants |
WO1989005859A1 (en) * | 1987-12-21 | 1989-06-29 | The Upjohn Company | Agrobacterium mediated transformation of germinating plant seeds |
NL8801444A (en) * | 1988-06-06 | 1990-01-02 | Solvay | Genetic transformation of eukaryotic cells - esp. plant cells, by treating dried cells with DNA soln. |
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1990
- 1990-09-27 NL NL9002116A patent/NL9002116A/en not_active Application Discontinuation
-
1991
- 1991-09-25 IE IE335991A patent/IE913359A1/en unknown
- 1991-09-26 JP JP51593891A patent/JPH06501155A/en active Pending
- 1991-09-26 CA CA 2089072 patent/CA2089072A1/en not_active Abandoned
- 1991-09-26 EP EP19910917384 patent/EP0554273A1/en not_active Withdrawn
- 1991-09-26 WO PCT/EP1991/001883 patent/WO1992006205A1/en not_active Application Discontinuation
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EP0267159A2 (en) * | 1986-11-07 | 1988-05-11 | Ciba-Geigy Ag | Process for the genetic modification of monocotyledonous plants |
WO1989005859A1 (en) * | 1987-12-21 | 1989-06-29 | The Upjohn Company | Agrobacterium mediated transformation of germinating plant seeds |
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US5550318A (en) * | 1990-04-17 | 1996-08-27 | Dekalb Genetics Corporation | Methods and compositions for the production of stably transformed, fertile monocot plants and cells thereof |
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US6051430A (en) * | 1996-02-09 | 2000-04-18 | Het Nederlands Kanker Instituut | Vectors and methods for providing cells with additional nucleic acid material integrated in the genome of said cells |
US7026528B2 (en) | 1996-06-21 | 2006-04-11 | Monsanto Technology Llc | Methods for the production of stably-transformed, fertile wheat employing agrobacterium-mediated transformation and compositions derived therefrom |
US6822144B1 (en) | 1997-01-24 | 2004-11-23 | Pioneer Hi-Bred International, Inc. | Methods for Agrobacterium-mediated transformation |
AU752717B2 (en) * | 1997-10-20 | 2002-09-26 | Cotton Incorporated | In planta method for the production of transgenic plants |
US5994624A (en) * | 1997-10-20 | 1999-11-30 | Cotton Incorporated | In planta method for the production of transgenic plants |
WO1999020776A1 (en) * | 1997-10-20 | 1999-04-29 | Cotton Incorporated | In planta method for the production of transgenic plants |
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US7737328B2 (en) | 1999-01-29 | 2010-06-15 | New Zealand Institute For Crop & Food Research Limited | Transformation and regeneration of Allium plants |
US7714191B2 (en) | 1999-05-05 | 2010-05-11 | Seminis Vegetable Seeds, Inc. | Transformation of allium sp. with agrobacterium using embryogenic callus cultures |
US7067719B1 (en) | 1999-05-05 | 2006-06-27 | Seminis Vegetable Seeds, Inc. | Transformation of Allium sp. with Agrobacterium using embryogenic callus cultures |
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US8252974B2 (en) | 2002-02-20 | 2012-08-28 | J.R. Simplot Comany | Precise breeding—low acrylamide foods |
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US9290777B2 (en) | 2007-02-05 | 2016-03-22 | National University Of Singapore | Putative cytokinin receptor and methods for use thereof |
US8546645B2 (en) | 2008-10-03 | 2013-10-01 | Agrisoma Biosciences Inc. | Production of modified fatty acids in plants through rDNA targeted integration of heterologous genes |
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Also Published As
Publication number | Publication date |
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JPH06501155A (en) | 1994-02-10 |
CA2089072A1 (en) | 1992-03-28 |
IE913359A1 (en) | 1992-04-08 |
NL9002116A (en) | 1992-04-16 |
EP0554273A1 (en) | 1993-08-11 |
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