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WO2001073084A2 - Procede pour produire rapidement des plantes monocotyledones transgeniques - Google Patents

Procede pour produire rapidement des plantes monocotyledones transgeniques Download PDF

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WO2001073084A2
WO2001073084A2 PCT/DE2001/001209 DE0101209W WO0173084A2 WO 2001073084 A2 WO2001073084 A2 WO 2001073084A2 DE 0101209 W DE0101209 W DE 0101209W WO 0173084 A2 WO0173084 A2 WO 0173084A2
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plants
tissue
transgenic
dna
medium
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WO2001073084A3 (fr
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Fredy Altpeter
Juan-Carlos Popelka
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Institut fuer Pflanzengenetik und Kulturpflanzenforschung
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Institut fuer Pflanzengenetik und Kulturpflanzenforschung
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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
    • C12N15/8206—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated
    • C12N15/8207—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated by mechanical means, e.g. microinjection, particle bombardment, silicon whiskers
    • 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)
    • C12N15/8201—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

Definitions

  • the invention relates to a method for producing transgenic, fertile, monocotyledonous plants.
  • Protoplast methods have been widely used for rice, with DNA being transferred into protoplasts by liposomes, PEG and electroporation.
  • a large number of transgenic plants have been grown in various laboratories (Shimamoto et al. (1989); Datta et al. Bio / Technology 8, 736-740 (1990)), but the protoplast method requires the long-term establishment of embryogenic suspension cultures.
  • Some regenerations of protoplast cultures are sterile and phenotypically abnormal due to long-term suspension culture (Davey et al. J Exp. Bot. 42, 1129-1169 (1991); Rhodes et al. Science 240, 204-207 (1988)).
  • the particle bombardment or _4grob ⁇ 2ctertwm-mediated transmission methods have used calli induced by immature explants as targets.
  • calli induced by immature explants After particle bombardment are transgenic Plants of rice, barley, wheat and rye have been described (Christou et al. Bio / Technology 9, 957-962 (1991); Gordon-Kamm et al. Plant Cell 2, 603-6128 (1990); Somers et al. Bio / Technology 10, 1589-1594 (1992), Wan et al. Plant Physiol. 104, 37-48 (1994), Vasil et al. (1992), Castillo et al.
  • transgenic rye plants In addition, the fact that only two independent transgenic plants were created from a single experiment means that it must be an initial report on transgenic rye plants and not a reproducible process. In fact, no further reports on transgenic rye plants have been published since 1994. This level of reproducibility is certainly too low for genetic studies and for economic applications. The efficiency of the described method was also very low, since 1383 explants were required to produce 2 transgenic rye plants.
  • Selectable marker genes are known to be used to identify transgenic events during the in-vitro process of plant transformation, but are unrelated to crop improvement and are therefore not required once the transgenic plant has been produced.
  • Selectable group I marker genes encode, for example, herbicide or antibiotic resistance and therefore allow selection pressure to be exerted by means of an appropriate cytotoxic selective agent in order to suppress cell division and plant regeneration in non-transformed tissue (Veiten and Schell Nucleic Acid Res. 13, 6981-6987 (1985)).
  • a second group of selectable markers supports the growth and regeneration of transformed cells under special culture conditions, for example phosphomannose isomerase or IPT (Joersbo et al. Physiol. Plant. 105, 109 - 1 1 (1999); Ebinuma et al. 94, 2117-2121 (1997)). If selectable markers remain in transgenic plants, their gene products must be analyzed for their effects on biosafety and the environment.
  • transformation systems In order to reduce the risks and problems associated with selectable markers, transformation systems have been developed which allow the marker genes to be eliminated after the transformation.
  • marker genes and useful transgenes are inserted into the plant genome as unlinked fragments, and if they get into uncoupled gene loci, they can be separated into cleaving sexual offspring.
  • the overall efficiency for the generation of the desired marker gene-free but useful transgene-carrying events is reduced compared to the generation of useful transgene-carrying events while maintaining the marker genes. This reduced efficiency is the result of low cotransformation rates and frequent integration of different plasmids at coupled gene loci without their segregation in the subsequent generation (McKnight et al. Plant Mol. Biol.
  • Agrobacterium-mediated transformation methods are also known. Such processes have been used principally in dicotyledonous plants. .4grob ⁇ cterz ⁇ .w -mediated transformation in dicotyledons facilitates the transfer of larger pieces of heterologous nucleic acids compared to other transformation methods, such as particle bombardment, electroporation, polyethylene glycol-mediated transformation processes and the like. In addition, it seems Transformation rarely generates genre combination and rather results in the integration of a small number of gene copies in the plant chromosome.
  • Monocots are not a natural host of Agrobacterium. Although roughly mediated transformation for asparagus (Bytebier B., et al. Proc. Ntl. Acad. Sei. USA 84: 5354-5349, 1987) and for Dioscora bublifera (Schafer et al. Nature 327: 529-532, 1987) was published, it was generally accepted that plants of the Gramineae family cannot be transformed with Agrobacterium (Potrykus I. Biotechnology 8: 545-543, 1990).
  • Monocot plants are generally less receptive than dicotyledons in terms of Agrobacterium-mediated transformation, and thus have become largely direct DNA transfer method used for the transformation of monocots.
  • Direct DNA transfer methods include the uptake of pure DNA stimulated by polyethylene glycol or electroporation and the transformation using the particle gun.
  • Agrobacterium-mediated gene transfer usually results in the insertion of a discrete, non-recombinant DNA segment into the host genome, and thus it would be desirable to develop methods for Agrobacterium-mediated transformation of monocotyledons.
  • monocotyledons are considered to be relatively difficult to transform with Agrobacterium, there are various reports of gene transfer in monocotyledons with Agrobacterium -mediated transformation (Boulton et al. (1989) Plant Mol. Biol. 12:31; Chan et al. (1992) Plant Cell Physiol. 33: 577, Gould et al. (1991) Plant Physiol. 95: 426; Graves et al. (1986) Plant Mol. Biol.
  • Tissue culture is obtained by culturing an explant for more than seven days on a dedifferentiation medium.
  • the prolonged cultivation process in this method has the disadvantage that the risk of undesirable properties is increased.
  • the present invention is therefore based on the object of developing an efficient, rapid and simple method for producing transgenic plants.
  • the occurrence of somaclonal variation during tissue culture should be reduced in order to obtain as many regenerated plants as possible per explant.
  • the immature embryos were cocultivated with Agrobacterium in one variant.
  • the phase of undifferentiated tissue growth after gene transfer was minimized.
  • a marker-gene-free and selection-agent-free process was developed for plant species which are "unruly" with regard to tissue culture, such as Monocotyledons, preferred for rye, barley and wheat, since avoiding the use of selection agents allows the transgenic tissue cultures to grow and regenerate.
  • transgenic plants can be regenerated in addition to non-transgenic plants from tissue cultures after gene transfer, and these transgenic plants can be used during the in vitro phase Have the DNA analysis separated from the non-transgenic regenerated plants.
  • the method is intended to preserve other important features such as, in particular, the ability of the tissues to regenerate into plants and the reproductive and productivity of the transformation events.
  • the method should have a reproducibly high transformation efficiency and be applicable to a wide range of genotypes.
  • the main variant 1 is characterized by a significant reduction in the mutagenic tissue culture phase, which is necessary for the production of transgenic monocotyledonous plants. This is achieved by introducing the foreign DNA by coculturing a freshly explanted immature embryo with Agrobacterium, which contains a plasmid with a heterologous nucleic acid, without prior exploration of the explants on a callus-inducing culture medium.
  • the main variant 2 aims at a tissue culture phase that is as short as possible, which follows the biolistic or Agrobacterium vQvmittQhen gene transfer into immature explants or the calli induced therefrom and is required for the production of transgenic monocotyledonous plants. This is achieved in that no selection is used during this phase and only the formation of the first somatic embryos is awaited, which takes place in a few days to weeks after the gene transfer.
  • the successful transformation events are preferably selected during the regeneration phase of the monocotyledonous plants, specifically during the development of shoot primordia into shoots.
  • the main variant 3 consists in the introduction of foreign DNA without marker genes into plant tissue, culture of the plant tissue without the addition of selection agents, regeneration of plants from the aforementioned plant tissue without addition of selection agents, DNA analysis of the regenerated plants formed and selection of the transgenic plants.
  • foreign DNA is introduced into regenerable plant tissue, and any type of foreign DNA can be introduced into plant species.
  • foreign DNA is any type of DNA that is introduced into plant cells from the outside. Methods to introduce this DNA into plant cells are known to those skilled in the art, such as particle bombardment using a device described in US Pat. 5,179,022.
  • the type of DNA included in foreign DNA includes DNA that is already present in the plant cell, DNA from another plant, DNA from another organism, or externally generated DNA, such as DNA sequences, that have an antisense message from a plant gene contain, or DNA sequences containing a synthetic version of a gene in which the nucleotide sequence has been modified.
  • the first step of the present invention is to isolate regenerable tissue from a plant.
  • Any regenerable plant tissue can be used in accordance with the present invention.
  • a tissue that can be regenerated into a differentiated plant after the introduction of foreign DNA is usually referred to as regenerable plant tissue.
  • regenerable plant tissue is usually immature or mature zygotic embryos or calli induced from these tissues (Altpeter et al. Plant Cell Rep. 16, 12-17 (1996)).
  • an immature plant embryo is used as the target tissue for the gene transfer without prior culture.
  • Immature embryos can be created using known methods in the art. For example, the production of immature wheat embryos by Vasil et al. (1993) and the production of immature rye embryos by Castillo et al. (1994).
  • calli are used as regenerable target tissues for gene transfer.
  • the preferred calli are embryogenic calli, which are produced by immature embryos through a short, maximum seven-day culture phase. Such calli can be obtained by isolating and cultivating immature embryos on a nutrient medium Carbohydrates and plant growth regulators are produced. Callus generating media are well known in the art and any culture medium or preparation method can be used.
  • the second step of the process is the introduction of foreign DNA into the plant tissue.
  • This process is referred to here as transformation.
  • Any method can be used to introduce foreign DNA into regenerable plant tissue. Such methods include particle bombardment (Weeks et al, (2993)); Vasil et al., (1992)), -4grob ⁇ cterz ' w / w transformation (Chan et al., Plant Mol. Biol. 22, 491-506 (1993)), electroporation of regenerable tissue (Shillito et al., Bio / Technology 3, 1099-1103 (1985)), silicon carbide fiber-mediated gene transmission (Dalton et al, Plant Sei. 132, 31 - 43 (1999)) and protoplast-mediated gene transmission (Shimamoto et al, (1989), Datta et al . (1990)) a.
  • the regenerable tissue is preferably transformed using Agrobacterium or the particle bombardment method. Immature embryos without preculture are preferred. After the gene transfer, callus is induced by the explant.
  • the regenerable tissue is kept for a short period after the introduction of the foreign DNA, e.g. the particle bombardment or the coarse ⁇ cte ⁇ ww-mediated gene transfer.
  • the nutrient medium used for this growth period contains no selection agent and this nutrient medium leads to undifferentiated cell growth and prepares the subsequent plant regeneration.
  • This cultivation period is kept short and is up to six weeks, preferably it does not last longer than about four weeks and ideally not longer than 7-17 days. A longer period of undifferentiated growth is not required in the present invention.
  • the regenerable plant tissue is transferred into a shoot induction medium which induces the regeneration of plants from the tissue, the shoot induction medium used for the regeneration preferably containing no selection agent.
  • the shoot induction medium used for the regeneration preferably containing no selection agent.
  • the shoot induction medium used for this step can be any medium that enables the formation of shoots from regenerable tissue.
  • a preferred shoot induction medium is free from plant growth regulators for shoot and root formation from regenerable tissue (for example a medium according to Weeks et al, 1993).
  • the regenerable plant tissue is transferred into this medium as quickly as possible after the transformation.
  • the regeneration of the shoot is preferably carried out via somatic embryogenesis. This is preferably done between one day and 6 weeks, preferably not later than 4 weeks, ideally not later than 7 to 17 days after the introduction of the foreign DNA.
  • the polymerase chain reaction was preferably used to identify transgenic plants which were transformed via the biolistic gene transfer. Plants over Gene transfer were preferably identified using ELISA (Enzyme Linked Immunosorbent Assay). Transgenic plants can be identified before or after the plants are transferred to soil culture. DNA analysis is preferably carried out during the in v / tro culture of the transgenic plants, which avoids the laborious transfer of numerous non-transgenic plants into soil.
  • the plants can then be transferred into earth culture and further cultivated according to the prior art until seed formation.
  • the main variant 1 of the invention relates to the transformation of rye (secale cereale L.) with stable expression of the transgene according to the invention with Agrobacterium as a gene transfer system consisting of the cocultivation of a freshly obtained rye explant or a callus induced by a rye exploration with Agrobacterium containing a plasmid a heterologous nucleic acid. Immature embryos are preferably used as explants.
  • the method consists of contacting at least one immature embryo or an explant-induced callus of a rye plant with Agrobacterium, which is suitable for transferring at least one gene into cells of the embryo or cells of the explant-induced callus.
  • the embryo or the callus induced by the explant is then cocultivated with Agrobacterium.
  • the embryos or explant-induced callus are then cultured in a medium that promotes callus formation with the addition of an antibiotic in concentrations that are suitable for stopping the growth of Agrobacterium.
  • the plants that express the selectable marker transgene are regenerated on a medium with selective agent to select plants that express the gene.
  • a preferred embodiment comprises the stable transformation of the regenerated plants. Immature embryos with a length of 0.8 mm to 2.0 mm are preferably used. The concentration of Agrobacterium is preferably between approximately 0.5 OD and 4.0 OD, particularly preferably between approximately 1.6 OD to 2.4 OD. Contacting is usually carried out in a suspension. The cocultivation can be carried out on a solid, liquid or medium containing MS salt.
  • Another preferred form relates to the regeneration of the embryos in medium with an antibiotic which is suitable for stopping the growth of Agrobacterium.
  • Timentin a mixture of 1500 mg of ticarcillin sodium and 100 mg of potassium clavunate, is preferably used as the antibiotic. Timentin concentrations between approximately 100 mg / 1 and 300 mg / 1 are particularly suitable, in particular a concentration of 150 mg / 1.
  • the embryos or alternative explants, such as inflorescences, armpit buds, meristems, are usually cultivated for about 0 to 15 days before cocultivation with Agrobacterium.
  • P refers the embryos or alternative explants, such as inflorescences, armpit buds, meristems, are cultivated with Agrobacterium for about 0 to 5 days before co-cultivation.
  • the length of the cocultivation step is usually between one and four days.
  • the culture phase before the transfer of the tissues under conditions of plant regeneration is usually 1 to 60 days after cocultivation, with seven to 17 days being preferred.
  • the selection for transgenic plants which express the selectable marker transgene can be carried out during the entire tissue culture phase after cocultivation or during the regeneration phase.
  • the selection of the marker gene expression is preferably carried out during the stretching growth of the shoot primordia to shoots.
  • Another preferred variant (main variant III) completely dispenses with the use of selection marker genes and the use of corresponding selection agents.
  • the transformed plants are found using biochemical detection methods (ELISA).
  • the main variants I to III of the present method are characterized by numerous advantages.
  • the process is not limited to certain plant species. It only depends on the use of regenerable plant tissue and thus on tissue culture conditions, processes and gene transfer methods that support the regenerative capacity of transformed tissue and at the same time promote the efficiency of the process.
  • Another advantage according to the invention is that the previous transformation methods require 8 to 9 months in order to, for example, transgenic rye plants obtained (Castilo et al, 1994).
  • the bombarded regenerable tissues were subcultured for 3 to 4 weeks on selection medium according to the prior art in order to allow callus multiplication.
  • the rapid method according to the invention requires less than 2 to at most 3 months to produce, for example, transgenic rye, barley or wheat plants. Long-term culture before and after the transformation treatment and the use of selection agents are not necessary according to the main variant III with the method according to the invention.
  • the main variant III of the present invention thus represents a fast, reproducible and efficient system for producing transgenic plants which are free from selectable or screenable marker genes.
  • it provides a fast and efficient marker-free transformation system for monocot crops using immature embryos as the starting tissue.
  • the method can be used for the transformation and regeneration of transgenic rye, barley and wheat plants.
  • the plants regenerated according to the invention are phenotypically normal and fully fertile.
  • Transgenic plants that are free of selectable and screenable marker genes can be transferred into soil culture within less than two to a maximum of three months after the preparation of the primary explants.
  • the transgenes are stably passed on to the Fl progeny.
  • transgenic plants namely to improve the public acceptance of transgenic crop plants by reducing the adverse environmental effects that the use of selectable markers entails. It can be done with the simple and quick procedure regulatory requirements of the tissues in question are met, and transgenic crop plants are generated with value-enhancing gene sequences that are free of selectable and screenable marker genes.
  • selectable and screenable markers are not required, various selectable marker systems, including herbicides such as bialophos and also antibiotics such as paramomycin or hygromycin, or other screenable markers such as GUS or GFP, can also (if necessary) together with the described method of the main variants I and II can be used.
  • the rapid marker-free transformation method described according to the invention usually also produces uniform, non-chimeric transformants. Regeneration is preferably carried out via somatic embryogenesis. Due to the short tissue culture time, embryogenic callus sectors are small at the regeneration stage. Therefore, only one branch is regenerated from each callus sector. As the PCR analysis for stable transgene integration showed, leaf segments of different parts of the transgenic plants are uniform in the transgene integration. Progeny analyzes also showed that most of the transgenic plants after self-splitting split 3: 1 between transgenic and non-transgenic plants, as was to be expected for a single dominant gene.
  • the present invention offers advantages to plant species that are inconsistent in tissue culture. It is particularly useful for monocot species. It is even more particularly useful for plants that cannot be kept on the callus stage for a long time without losing their ability to regenerate. Three particularly useful species are rye, barley and wheat in the present invention.
  • transgenic rye in sufficient numbers has become possible for genetic studies and commercial applications.
  • rye and wheat for example, it was found that the process is genotype-independent. This means that this method can be used with any type of rye and wheat, including both winter and summer wheat and rye. It can be used to transgenic plants from summer rye inbred lines such as L22 or winter rye inbred lines such as L20, summer wheat varieties such as Bobwhite and winter wheat breeding lines such as W08. This means that the method can even be used for homozygous inbred lines of cross-pollinating species such as rye and elite genotypes of wheat.
  • Agrobacterium -mediated transformation advantages are that, as a rule, simpler transgene insertion patterns occur than with biolistic gene transfer or the direct uptake of DNA in protoplasts.
  • transgenes are usually inserted in actively transcribed regions using Agrobacterium, in contrast to the alternative gene transfer methods. Simple transgene integration patterns and insertion in actively transcribed regions leads to stable transgene expression in generative offspring.
  • larger DNA fragments can be transferred using Agrobacterium than with other gene transfer methods, which is particularly important for complementation studies and for changing entire metabolic pathways.
  • Immature rye embryos which had not been ripened, or up to 5 days on a MS medium (Murashige and Skoog, Physiol. Plant. 15: 473-497 (1962) modified according to Casillo et al (1994)) were used Calli were infected with Agrobacterium precultivated on LB medium, and with the aid of the Agrobacterium strain AGLO (Lazo et al., Bio / Technology 9: 963-967 (1991)), a T-DNA with a constitutive expression cassette was controlled under the control of the ubiquitin promoter and NOS terminators (Christensen and Quail, Transgenic Res. 4: 44-51 (1996) were transferred with the selectable nptgen marker gene.
  • Leaf samples were taken from the shoot tips of regenerated plants, the proteins extracted and an NPTII expression analysis carried out by means of ELISA and Western blot. In the preferred treatment, 31 independent transgenic rye plants were identified from 500 explants. These plants were selected and planted in soil.
  • Figure 2a shows ELISA using 20 ⁇ g total protein extract compared to 150 and 20pg NPTII standards.
  • Figure 2b shows NPTII Western blot of selected plants using 10 ⁇ g total protein and NPTII-specific antibody.
  • transgenic, marker gene-free rye plants by means of biolistic gene transfer. Immature rye embryos were grown on a Casillo et al. (1994) modified MS medium (Murashige and Skoog, Physiol. Plant. 15: 473-497 (1962). These tissues were coated with microparticles with two constitutive expression cassettes of useful genes under the control of the ubiquitin promoter and NOS terminators (Christensen and Quail, Transgenic Res.
  • cDNA's of different fragment lengths were integrated into the rye genome under control of the sequences mentioned.
  • the cultivation of the calli after the particle bombardment was carried out for 7 to 17 days in callus induction medium (composition as in Castillo et al. 1994) and then for 4-5 weeks in a shoot induction medium (composition as in Castillo et al. 1994).
  • Rooted shoots had formed 4-5 weeks after the start of culture on the shoot induction medium. Samples were taken from the shoot tips, DNA extracted and DNA analysis carried out by means of PCR. 20 of the regenerated rye plants from Kalli, which resulted from 1400 different explants, had the transformed foreign DNA. These plants were selected and planted in soil.
  • Figure 3 shows PCR analyzes using primer pairs that initiate in the control sequences flanking the cDNA (ubiquitin promoter and nos terminator) compared to the plasmid control and fragment size markers (Fig.
  • PCR analyzes using primer pairs that are in the ubiquitin promoter and the respective cDNA initiate compared to the plasmid control and fragment size markers (Fig.3b) as well as transgenic markergen-free rye plants after transfer to soil cultures (Fig. 3c).
  • the transgene was transferred to the generative progeny in a stable manner, as confirmed by PCR and Southern blot analyzes (Fig. 3d).
  • Immature wheat embryos were placed on a Casillo et al. (1994) modified MS medium (Murashige and Skoog, Physiol. Plant. 15: 473-497 (1962). These tissues were coated with microparticles with a constitutive expression cassette of a useful gene under the control of the ubiquitin promoter and NOS- Terminators (Christensen and Quail, Transgenic Res. 4: 44-51 (1996).
  • a gamma-tocopherol methyltransferase from Arabidopsis was integrated into the wheat genome under control of the sequences mentioned.
  • the cultivation of the calli after the particle bombardment was carried out for 7 to 17 days in Callus induction medium (composition as in Castillo et al. 1994) and then for 4-5 weeks in a shoot induction medium (composition as in Castillo et al. 1994).
  • Rooted shoots had formed 4-5 weeks after the start of culture on the shoot induction medium. Samples were taken from the shoot tips, DNA extracted and DNA analysis carried out by means of PCR. 10 of the regenerated wheat plants from Kalli, which originated from 500 different explants, had the transformed foreign DNA. These plants were selected and planted in soil.
  • Figure 4a shows PCR analyzes using primer pairs which initiate in the ubiquitin promoter and the cDNA of the gamma-tocopherol methyltransferase, compared to the plasmid control and fragment size markers and transgene expression detected with Northern blot (Fig. 4b).
  • Fig. 1 Coarse ⁇ cterz ' mediated gene transfer to freshly explored immature barley embryos (main variant I).
  • Co-culture of immature barley embryos that were not pre-cultivated prior to contact with Agrobacterium leads to expression of the transgene (in this case the reporter gene gfp (green fluorescent protein) in the embryo tissue (Fig. A), the regenerating callus (Fig. B) and the generative ones Descendants (Fig. D) of fertile plants
  • the expression of the transgene (gfp) is also biochemical via Western blot analysis using a specific antibody in the protein extracts, obtained from leaf samples from various transgenic barley plants (Fig. C; 1-7), The specificity of the antibody is demonstrated by reaction with gfp protein (Fig.
  • Fig. 2 NPTII expression of regenerated transgenic rye plants [Secale cereale L.) after Agrobacterium-mediated gene transfer.
  • NPTII ELISA with 20 ⁇ g crude protein extract per well (1A: 150pg NPTII protein; 1B: 20pg NPTII protein; IC: 20 ⁇ g crude protein extract from an nptll transgenic rye plant; 1D: 20 ⁇ g crude protein extract from a wild-type rye plant). Arrows indicate protein extracts from transgenic rye plants with NPTII expression.
  • Fig. 3a PCR analysis of genomic DNA of transgenic, marker gene-free rye plants for the cointegration of two useful gene expression cassettes (y- Tocopherol methyltransferase and ferretin).
  • M 1Kb ladder
  • Kl transgenic rye with
  • 183, 206, 212 independent transgenic wheat lines.
  • 1-5 DNA extracted from five different shoots of the transgenic wheat plants was used as a template.
  • 4B Northern blot detection of the expression of the gammatocopherol methyltransferase (arrow) in transgenic wheat plants 183 and 212, which were produced without the use of selection marker genes and without the use of selection agents.
  • NC RNA isolated from a non-transgenic wheat plant. A radioactively labeled probe from the coding region of the gammatocopherol methyltransferase from Arabidopsis was used for the hybridization.

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Abstract

L'invention concerne un procédé permettant de produire des plantes monocotylédones transgéniques fertiles, notamment pour produire du froment, de l'orge et du seigle transgéniques, par introduction d'ADN étranger, afin d'obtenir une hérédité stable de l'expression transgénique dans la lignée sexuelle. L'invention comprend trois variantes permettant de produire rapidement des plantes monocotylédones transgéniques fertiles et normales. L'invention permet de minimiser les phénomènes de variation somaclonale. Cette mutagenèse qui intervient fréquemment pendant la culture tissulaire et la sélection de phénomènes de transformation génétiques dans des plantes cultivées monocotylédones, induit fréquemment des propriétés indésirables telles que des troubles de la fertilité ou une aptitude réduite à la croissance et un rendement réduit, jusqu'à la perte de pouvoir de régénération des cultures tissulaires de plantes. Le froment et l'orge constituent des exemples de plantes qu'il est difficile de régénérer à partir de cultures tissulaires pour former des plantes et par conséquent de transformer par voie génétique, en raison de la mutagenèse qui intervient fréquemment du fait de la culture tissulaire. Le seigle constitue un exemple de plante qu'il est difficile de régénérer à partir de cultures tissulaires pour former des plantes et par conséquent à transformer par voie génétique, en raison de la mutagenèse qui intervient fréquemment du fait de la culture tissulaire. Les procédés présentés contiennent des exemples de production de plantes transgéniques fertiles de seigle, de froment et d'orge par transfert de gènes induit par voie biolistique et par agrobacterium, ainsi que leurs lignées génératives à intégration transgénique et expression stables. Lesdits procédés nécessitent moins de deux à trois mois entre le moment de l'extraction de l'explant et le transfert des plantes transgéniques en terre.
PCT/DE2001/001209 2000-03-29 2001-03-23 Procede pour produire rapidement des plantes monocotyledones transgeniques Ceased WO2001073084A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU73821/01A AU7382101A (en) 2000-03-29 2001-03-23 Methods for rapidly producing transgenic monocotyledonous plants

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE2000115458 DE10015458A1 (de) 2000-03-29 2000-03-29 Verfahren zur raschen Herstellung von transgenen, markergen-freien Pflanzen
DE10015458.1 2000-03-29

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WO2001073084A2 true WO2001073084A2 (fr) 2001-10-04
WO2001073084A3 WO2001073084A3 (fr) 2002-04-04

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1502955A1 (fr) * 2003-08-01 2005-02-02 Institut für Pflanzengenetik und Kulturpflanzenforschung Procédé de production de plantes gramineae stablement transformées par transformation des zygotes isolées induite par agrobacterium
US8049067B2 (en) 2002-12-06 2011-11-01 Del Monte Fresh Produce Company Organogenic transformation and regeneration
US8507758B2 (en) 2003-03-07 2013-08-13 Seminis Vegetable Seeds, Inc. Markerless transformation
DE102015016445A1 (de) 2015-12-21 2017-06-22 Kws Saat Se Restorer-Pflanze
DE102015017161A1 (de) 2015-12-21 2017-06-22 Kws Saat Se Restorer-Pflanze
CN116042694A (zh) * 2022-11-24 2023-05-02 中国科学院南京土壤研究所 禾本科狼尾草属植物非组培遗传转化方法
US11840693B2 (en) 2015-12-21 2023-12-12 KWS SAAT SE & Co. KGaA Restorer plants

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990001551A1 (fr) * 1988-07-29 1990-02-22 Washington University School Of Medicine Production de polypeptides pour usage commercial au moyen d'un tissu d'endosperme transforme genetiquement
DE4309203C1 (de) * 1993-03-22 1994-04-21 Holt Claus Von Prof Dr Verfahren zur Produktion von transgenischen Pflanzen
US5981840A (en) * 1997-01-24 1999-11-09 Pioneer Hi-Bred International, Inc. Methods for agrobacterium-mediated transformation
AU7479298A (en) * 1997-05-16 1998-12-08 Pioneer Hi-Bred International, Inc. Recovery of transformed plants without selectable markers by nodal culture and enrichment of transgenic sectors
US6037522A (en) * 1998-06-23 2000-03-14 Rhone-Poulenc Agro Agrobacterium-mediated transformation of monocots

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8049067B2 (en) 2002-12-06 2011-11-01 Del Monte Fresh Produce Company Organogenic transformation and regeneration
US8507758B2 (en) 2003-03-07 2013-08-13 Seminis Vegetable Seeds, Inc. Markerless transformation
EP1502955A1 (fr) * 2003-08-01 2005-02-02 Institut für Pflanzengenetik und Kulturpflanzenforschung Procédé de production de plantes gramineae stablement transformées par transformation des zygotes isolées induite par agrobacterium
WO2005014827A1 (fr) * 2003-08-01 2005-02-17 Sungene Gmbh Procede de production de graminacees fertiles, transformees de maniere stable, mettant en oeuvre une transformation a mediation d'agrobacteries de zygotes de graminacees isoles
DE102015016445A1 (de) 2015-12-21 2017-06-22 Kws Saat Se Restorer-Pflanze
DE102015017161A1 (de) 2015-12-21 2017-06-22 Kws Saat Se Restorer-Pflanze
US11312967B2 (en) 2015-12-21 2022-04-26 KWS SAAT SE & Co. KGaA Restorer plants
EP4008176A1 (fr) 2015-12-21 2022-06-08 KWS SAAT SE & Co. KGaA Plante restauratrice
US11840693B2 (en) 2015-12-21 2023-12-12 KWS SAAT SE & Co. KGaA Restorer plants
CN116042694A (zh) * 2022-11-24 2023-05-02 中国科学院南京土壤研究所 禾本科狼尾草属植物非组培遗传转化方法

Also Published As

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DE10115761A1 (de) 2001-12-20
WO2001073084A3 (fr) 2002-04-04
DE10015458A1 (de) 2001-10-11
AU7382101A (en) 2001-10-08

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