WO2001073084A2

WO2001073084A2 - Methods for rapidly producing transgenic monocotyledonous plants

Info

Publication number: WO2001073084A2
Application number: PCT/DE2001/001209
Authority: WO
Inventors: Fredy Altpeter; Juan-Carlos Popelka
Original assignee: Institut Für Pflanzengenetik Und Kulturpflanzenforschung (Ipk)
Priority date: 2000-03-29
Filing date: 2001-03-23
Publication date: 2001-10-04
Also published as: DE10115761A1; AU7382101A; DE10015458A1; WO2001073084A3

Abstract

The invention relates to a method for producing transgenic, fertile, monocotyledonous plants, in particular, for producing transgenic wheat, barley and rye by introducing foreign DNA while achieving stable heredity of the transgenic expression in sexual offspring. The invention consists of three variants which lead to the rapid production of fertile and normal transgenic monocotyledonous plants. The invention results in minimizing the occurrence of somaclonal variation. This mutagenesis, which often occurs during the tissue culture and selection of genetic transformation events in monocotyledonous cultivated plants, often leads to undesired features such as fertility disorders or reduced vigorousness and yield potential up to a loss in ability of the tissue cultures to regenerate into plants. Wheat and barley are examples of plants which are difficult to regenerate out of tissue cultures and into plants due to frequently occurring tissue culture-related mutagenesis and are thus difficult to be able to genetically transform. Rye is an example of a plant that, due to particularly frequent tissue culture-related mutagenesis, is especially difficult to regenerate out of tissue cultures and into plants and is thus particularly difficult to be able to genetically transform. The depicted methods contain examples of producing fertile transgenic plants of rye, wheat and barley using biolistic-mediated and Agrobacterium-mediated gene transfer as well as their generative offspring with stable transgenic integration and expression. The inventive methods require a period of only less than two to a maximum of three months from the moment of explant extraction up to the transfer of the transgenic plants into soil.

Description

Process for the rapid production of transgenic, monocotyledonous plants

description

The invention relates to a method for producing transgenic, fertile, monocotyledonous plants.

In the past 15 years, it has become possible to transfer genes from a large number of organisms to useful plants using recombinant genetic engineering. This progress has opened up enormous opportunities to improve the resistance of plants to pests, diseases and herbicides, and to modify biosynthesis processes to change the quality of plant products and to generate new valuable substances in crops (Knutson et al. PNAS USA 89 , 1992, 2624-2628; Piorier et al., Science 256, 520-523 (1992); Vasil et al., Bio / Technologie 10, 667-674 (1992)).

Three alternative transformation methods are currently used for monocot species: direct DNA transfer to isolated protoplasts (Shimamoto et al., Nature 338, 274-276 (1989)), particle bombardment-mediated DNA transfer (Fromm et al. Bio / Technology 8, 833) - 839 (1990)), and / fgrobαcter / wra-mediated transformation (Cheng et al. Plant Physiol. 115, 971-980 (1997)).

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. 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. Bio / Technology 12, 1366-1371 (1994) ) and after Agrobacterium-mediated gene transfer, transgenic plants of rice, barley and wheat have been described (Hiei et al. Plant J. 6, 271-282 (1994); Ishida et al. Nature Biotechnol. 14, 745 - 750 (1996) ; Tingay et al. Plant J. 11, 1369-1376 (1997); Cheng et al. Plant Physiol. 115, 971-980 (1997)).

So far there is only one report that describes the production of transgenic rye plants. The biolistic gene transfer was followed by callus selection using an appropriate selective agent and the regeneration of two transgenic plants (Castillo et al. Bio / Technology 12, 1366-1371 (1994)). The method is time consuming and takes 8 to 9 months from harvesting the explants to obtaining transgenic plants. This method also suffers from a loss of regenerability of the transgenic callus. Only 33% of the transgenic calli could still be regenerated after the selection for expression of the selectable marker gene bar, which codes for the phosphinotricin acetyl transferase, was carried out. 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)). The addition of herbicide or antibiotic components as selective agents to plant cells often has a negative effect not only on the non-transformed cells but also on transgenic cells and can therefore interfere with the regeneration process and in particular the efficiency and reproducibility of the production of transgenic plants, especially in " unruly "like rye, barley and wheat. 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. Such safety investigations can delay or even block the commercialization of transgenic plants. The public's reaction to the problem of selectable markers of bacterial origin can have a negative effect on the commercial potential of transgenic crops, even if the safety of the selectable markers has been scientifically proven (Malik and Saroha J. Plant Biochem. Biotechnol. 8, 1 - 13 (1999)). If multiple transgenes have to be inserted into the same genetic background, several different selectable marker genes are required.

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. In cotransformation systems, 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. 8, 439-445 (1987), Deblock and Debrouwer Theor. Appl. Genet 82, 257-263 (1991), Komari et al. Plant J. 10, 165-174 (1996)). Directed cutting out of the selectable marker genes by means of site-specific recombination (Rüssel et al. Mol. Gen. Genet. 234, 49-59 (1992)) or transposition-mediated repositioning (Goldsbrough et al. Bio / Technology 11, 1286-1292 (1993)), followed by cleavage into marker genes and useful transgenes in the sexual offspring, have been successfully used for the production of marker gene-free transgenic plants. However, the construction of corresponding relevant transformation vectors is quite complicated and the activation of the enzyme-assisted excision either requires quite time-consuming sexual crossings followed by cleavage analyzes (Goldsbrough et al. Bio / Technology 11, 1286-1292 (1993)) or the transient expression of the excitation-mediating enzymes , The transient expression of excision-mediating enzymes is often accompanied by the generation of undesired stable expression of such enzymes (Gleave et al. Plant Mol. Biol. 40, 223-235 (1999)), which reduces the efficiency of the generation of the desired genetic events free of marker genes and further reduced without excision-mediating enzymes. The reproducibility in these systems therefore depends heavily on the reproducible and precise expression excision by Indian enzymes. The overall efficiency of generating the desired genetic events is reduced compared to transgenic plants with marker genes.

Agrobacterium-mediated transformation methods are also known. Such processes have been used principally in dicotyledonous plants. .4grobαcterzι.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 (monocots) 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. For an overview, see Gene Transfer to Plants, Potrykus et al., Eds., Springer-Verlag, Berlin, 1995.

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. Although 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. 7:43; Grimsley et al. (1987) Nature 325: 177; Raineri et al. (1990) Bio / Technology 8:33; U.S. Pat. No. 5,177,010 to Goldman et al. and U.S. Pat. No. 5,187,073 to Goldman et al.).

However, many of these early studies on Agrobacterium -mediated transformation of monocots were the subject of controversy (Potrykus (1990) Bio / Technology 8: 5350) and were not independently confirmed.

More recent studies report successful coarse αctertwm-mediated transformation of barley (Tingay et al. Plant J. 11, 1369-1376 (1997), Weizen Cheng et al. Plant Physiol. 1 15, 971-980 (1997) and rice (Hiei et Plant J. 6: 271 (1994), Dong and others (1996) Molecular Breeding 2: 267. Cultivated immature scutella were bombarded with particles and then co-cultivated with Agrobacterium for 2 to 3 days, followed by callus culture with selective agent and regeneration of transgenic barley plants (Tingay et al. Plant J. 11, 1369-1376 (1997). Cheng et al. Plant Physiol. 115, 971-980 (1997) cocultivated either three to four hours of precultivated immature embryos (are considered as freshly isolated), one to six days of pre-cultivated immature embryos or ten to 25 days of pre-cultivated embryogenic callus with Agrobacterium for 2 to 3 days, followed by a callus culture phase with selective agent and the regeneration of fertile wheat plants al. (Plant J. 6: 271 (1994)), Dong and others (Molecular Breeding 2: 267 (1996)) cultivated various tissues of rice, including shoot tips, scutella, immature embryos, calli, from young roots and Scutella were induced, and cells in suspension cultures induced by Scutella were cocultured with Agrobacterium tumefaciens, resulting in various degrees of expression of the reporter transgene in these tissues. Transgenic plants were only regenerated from Scutellurn-induced calli, which were co-cultivated with Agrobacterium. Stable integration, expression and inheritance of the transgenes have also been reported. US Pat. No. 5,591,616 to Hiei et al. discloses a method for transforming a monocot (rice) consisting of transforming a tissue culture during the dedifferentiation process or dedifferentiated tissue culture with Agrobacterium. 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 advanced methods of coarse-grained transformation of rice involve the use of long-term in vitro selection methods in tissue cultures after the transformation process. Tissue culture manipulations have been associated with the induction of somaclonal variation (Kaeppler et al. (1993) Proc. Natl. Acad. Sei. USA 90: 8773; Phillips et al. (1994) Proc. Natl. Acad. Sei. USA 91: 5222) , U.S. Pat. No. 6,037,522 claims an Agrobacterium-shrouded rye transformation protocol using immature inflorescences as the target tissue for gene transfer. However, the examples only include transgenic rice plants. Rye is considerably more "stubborn" in terms of tissue culture than rice. According to our investigations, immature inflorescences are not a suitable target tissue for rye transformation.

The present invention is therefore based on the object of developing an efficient, rapid and simple method for producing transgenic plants.

In particular, the occurrence of somaclonal variation during tissue culture should be reduced in order to obtain as many regenerated plants as possible per explant. In order to develop a method with the shortest possible tissue culture phase, the immature embryos were cocultivated with Agrobacterium in one variant. In another variant, the phase of undifferentiated tissue growth after gene transfer was minimized. In a third variant, 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. An important aspect of this variant is that, with the right timing of gene transfer, tissue culture and the start of regeneration, a high proportion of 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 task is accomplished according to the claims. Three main variants are used.

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. According to the invention, foreign DNA is introduced into regenerable plant tissue, and any type of foreign DNA can be introduced into plant species. In general, 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. Such tissue is preferably immature or mature zygotic embryos or calli induced from these tissues (Altpeter et al. Plant Cell Rep. 16, 12-17 (1996)).

In a preferred embodiment of the present invention, 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).

In an alternative embodiment, 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.

Once the regenerable plant tissue is isolated, 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)), -4grobαcterz ^' 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.

In the main variants of the invention, 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.

In a preferred embodiment of the invention, the regenerable tissue is kept for a short period after the introduction of the foreign DNA, e.g. the particle bombardment or the coarse αcteπww-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.

After the transformation and a short phase with undifferentiated tissue growth, 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. This is in contrast to the previous state of the art, according to which regenerable plant tissue initially have to go through an extended selection period before the regenerable tissues are transferred into the shoot induction medium (Vasil et al., 1993; Castillo et al., 1994), or after which regenerable plant tissues are exposed to a selection period during exposure in a shoot induction medium (EP 0709462).

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).

According to the invention, 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.

After at least one shoot has formed, a small part of the shoot is removed for DNA extraction or protein extraction. Any method for DNA analysis or protein analysis that allows the determination of small amounts of DNA or protein is compatible with the present method. According to the invention, 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. Subsequently, the plants that express the selectable marker transgene are regenerated on a medium with selective agent to select plants that express the gene. In a preferred embodiment, the use of a marker gene and a selection medium is not necessary to identify the plants that express a potentially useful transgene. 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. Prefers 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.

1. 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.

2. With the marker-free transformation method described, a higher transformation frequency is achieved than in previously published cases (Castillo et al., 1994). This statement about the transformation frequency applies to the frequency that was observed in the marker-free method, with complicated, efficiency-reducing marker elimination experiments being unnecessary. As a result of the rapid generation of transgenic plants which are free from selectable and screenable marker genes, risk identification and commercialization of the transgenic plants can be accelerated and simplified. The applicability of this marker-free transformation method to e.g. Rye inbred lines increase reproducibility in this "recalcitrant" and cross-pollinated plant. Plants created using this method are generally fertile.

3. 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. By reducing the time for callus multiplication according to main variant II and, or by using explants without preculture according to main variant I, 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. It is surprising that even after the marker-free transformation method has been carried out, the transformed cells regenerate just as quickly as non-transformed cells and therefore the proportion of transgenic and non-transgenic plants in the total regenerated plants does not require automated molecular analysis, although this can increase the efficiency of the method. 4. The regenerates were stronger and healthier both in vz ^' tro culture and in earth culture than those derived from selection media.

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. In a preferred embodiment, it provides a fast and efficient marker-free transformation system for monocot crops using immature embryos as the starting tissue. In particular, 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.

The process has achieved an important goal in the production of 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.

In addition, although 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.

It is particularly noteworthy that, for the first time, the reproducible production of transgenic rye in sufficient numbers has become possible for genetic studies and commercial applications. For 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.

Advantages of the Agrobacterium -mediated transformation are that, as a rule, simpler transgene insertion patterns occur than with biolistic gene transfer or the direct uptake of DNA in protoplasts. In addition, 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. In addition, 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.

The invention is explained in more detail with the following examples. The examples are in no way intended to limit the present invention.

1. Main variant I

Production of transgenic barley plants using Agrobacterium - mediated gene transfer.

GrobαcteπwmstammAGLl (Lazo et al. 1991) with two consuming expression cassettes in the T-DNA (hygromycin B resistance gene hph under the control of the 55S promoter and nos terminators and the reporter gene gfp under the control of the ubiquitin promoter and nos terminators) was not previously explored precultivated barley embryos for 2-3 days on callus induction medium according to Hagio et al. (Plant Cell Rep 14: 329-334 (1995)) cocultivated. Then the explants were cultivated on callus induction medium containing 150 mg / L timentin and after a further five days cultivated on selective callus induction medium with 10-30 mg / L hygromycin B and 150 mg / L timentin. After one to three two-week selection phases, the explants were transferred to selective regeneration medium (Hagio et al.) And after a further 5 weeks, regenerated plants could be planted in soil. Figure la-d shows expression of the gfp gene in different stages of tissue culture and transgene progeny, as well as the molecular evidence for transgene integration (E: Southern blot) and transgene expression (F: Western blot).

2. Main variant II

Production of transgenic rye plants using Agrobacterium -mediated gene transfer.

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. Calli were cocultivated with Agrobacterium culture medium containing 150mM acetosyringone for 2 to 3 days, then for 3 weeks on callus induction medium containing 150mg / L timentin of the composition according to Castillo et al. (1994) cultivated without a selective agent and on 150 mg / L timentin-containing shoot induction medium of the composition according to Castillo et al. (1994) with 30 mg / L paramomycin sulfate regenerated as a selective agent.

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 20μg total protein extract compared to 150 and 20pg NPTII standards. Figure 2b shows NPTII Western blot of selected plants using 10 μg total protein and NPTII-specific antibody.

3. Main variant III

Production of 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. 4: 44-51 (1996) As examples, cDNA's of different fragment lengths (gamma-tocopherol methyltransferase from Arabidopsis and ferret gene from pea) 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. 3a) and 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).

4. Main variant III

Production of transgenic, marker gene-free wheat plants by means of biolistic gene transfer.

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). As examples, 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).

Legends for the illustrations

Fig 1) Coarse αcterz ^' 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. F; PC) and no reaction with protein extracts from non-transgenic barley plants (Fig. F; NC). The stable integration of the gfp transgene into the barley genome is by Southern blot analysis using a radioactively labeled sample from the coding region of the gfp transgene in various barley plants detected by hybridization signals (Fig. E 1-11). Identically treated DNA extracts of non-transgenic barley (Fig. E; NC) show no hybridization signal.

Fig. 2) NPTII expression of regenerated transgenic rye plants [Secale cereale L.) after Agrobacterium-mediated gene transfer.

(A) NPTII ELISA with 20μg crude protein extract per well (1A: 150pg NPTII protein; 1B: 20pg NPTII protein; IC: 20μg crude protein extract from an nptll transgenic rye plant; 1D: 20μg crude protein extract from a wild-type rye plant). Arrows indicate protein extracts from transgenic rye plants with NPTII expression.

(B) NPTII Western blot with 10 μg crude protein extract per lane (M: marker, + K: 20pg NPTII, -K: 20pg crude protein extract of a wild-type rye plant).

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

Cointegration of both expression cassettes, K2: ferritin plasmid control, K3: y-

Tocopherol-methyltransferase plasmid control, wt: wild type, 1-10 = transgenic

Rye plants. Primers in the control sequences flanking the respective cDNA

(ubiquitin promoter, nos terminator) were used for DNA amplification.

The comparison with the 1Kb ladder demonstrates the integration of both more completely

Expression cassettes in transgenic rye plants 1-10.

3b) PCR analysis of genomic DNA of transgenic, marker-gene-free rye plants for integration of the ferretin (left) and the γ-tocopherol methyltransferase

Transgens (right). M: 100bp ladder, + P: respective plasmid controls, WT: wild type, 1-

10 = transgenic rye plants. Primer pairs which initiate in the ubiquitin promoter and the respective cDNA were used for DNA amplification.

3c) Transgenic, marker-gene-free rye plants after transfer to soil.

3d) Southern blot analysis for integration of marker gene-free rye plants with the y-

Tocopherol methyltransferase transgene.

Plasmid DNA (+ P), wild-type (WT) genomic DNA and transgenic rye plants

(1-8) were digested with the restriction enzyme BglII and separated on 0.8% agarose gel, transferred to the nitrocellulose membrane and hybridized with a radioactively labeled probe (approx. 500 bp from the coding sequence of the γ-tocopherol methyltransferase transgene) and visualized with a phophore imager ,

Fig. 4) Marker gene-free transgenic wheat plants

A) PCR detection of the integration of the expression cassette ubiquitin promoter - coding sequence Gammatocopherolmethyltransferase from Arabidopsis - Nos terminator in genomic DNA from winter wheat. The primers advertise in the promoter area: Ubi-F2 Universe: 5'- GTC TGG TTG GGC GGT CGT TCT AG - 3 'or in the coding area of the gammatocopherol methyltransferase: gTMT Reverse: 5 ^' - CGC CTC AGT GGA TGT AGC A - 3 ' an expected amplification product of 900 bp. PC: plasmid control, NC: DNA of a non-transgenic wheat variety was used as a PCR template used. 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.

Claims

claims

1) Process for the production of transgenic, monocotyledonous plants, in particular those types of culture which are considered to be very "recalcitrant" in tissue culture, characterized by the introduction of foreign DNA characterized by a) isolation of regenerable tissue from said plants and, if appropriate, cultivation in a known per se Tissue culture medium, b) introduction of the foreign DNA into the plant tissue according to techniques known per se, the foreign DNA comprising those DNA sequences which serve in the plants to improve their utility or to produce additional valuable substances, and c) regeneration of fertile ones transgenic plants

2) Method according to claim 1 for the production of transgenic marker gene-free plants, in particular those cultivars which, like monocotyledonous plants, are considered to be very "recalcitrant" in tissue culture, by introducing foreign DANN, characterized by a) isolating regenerable tissue from said plants and optionally cultivation in a tissue culture medium known per se, b) introduction of the foreign DNA according to techniques known per se, the foreign DNA comprising those DNA sequences which serve in the plants to improve their utility or to produce additional valuable substances, without that said DNA encodes a selectable or screenable marker, c) if necessary after a brief cultivation for a period of from several hours to four weeks in the tissue culture medium mentioned under a) or another tissue culture medium which contains no selection agent and the shoot induction by adding growth regulators suppressed, placement the tissue in a shoot induction medium known per se or the preferred shoot induction medium which allows shoots and roots to be formed from said tissue, said shoot induction medium containing no compounds for the selection of regenerable tissue; d) after formation of at least one shoot, removal of shoot tissue for DNA extraction and DNA analysis by means of molecular methods or protein extraction and biochemical analysis for the detection of transgenic plants and e) transfer of the transgenic plants verified after gene transfer has taken place in earth culture and f) further cultivation of the transgenic plants to achieve stable inheritance of transgene integration and expression in sexual progeny of the transgenic plants.

3) Method according to claim 1 for transforming rye and barley with Agrobacterium, comprising the following steps: a) contacting at least one immature embryo immediately after explantation or a callus induced by an explant with Agrobacterium, which is suitable for at least one gene in the embryo tissue or to transfer the callus induced by the explant, b) co-cultivating the embryo or the callus induced by the explant with Agrobacterium, c) cultivating the embryo or the callus induced by the explant in a medium which promotes callus formation, with the addition of an antibiotic in concentrations, which are suitable for stopping the growth of Agrobacterium, d) regeneration of the plants which express the selectable marker transgene either on a medium with selective agent, in order to select plants which express the gene or by regeneration of all plants, with one being selectable Marker gene and an ent the selection medium is omitted, shoot induction with subsequent selection by ELISA using proteins extracted from shoot parts and transferring the transgenic plants verified after gene transfer to earth culture until stable inheritance has been achieved.

4) Method according to claim 1 and 2, characterized in that as regenerable tissue anthers, microspores, inflorescences, immature or mature embryos, seeds, leaf tissue, meristematic tissue or calli, which have been induced from these tissues, which regenerate by somatic embryogenesis or organogenesis , are used, preferably immature plant embryos or calli induced from them, which are generated in a nutrient medium which contains no selection agent. 5) Method according to claims 1-3, characterized in that immature embryos or calli induced therefrom by self-fertilized homozygous donor plants are briefly cultivated in a tissue culture medium as regenerable tissue and the introduction of the foreign DNA into these tissues takes place and these tissues preferably via somatic embryogenesis or regenerated into plants via organogenesis.

6) Method according to claims 1-3, characterized in that meristematic tissue is isolated as regenerable tissue and the introduction of the foreign DNA into this tissue or calli induced therefrom takes place and these tissues are regenerated to plants via somatic embryogenesis or organogenesis.

7) Method according to claim 1,2 and 4-6, characterized in that the regenerable tissue is obtained from a wide range of genotypes and cultures.

8) Method according to claim 1-7, characterized in that regenerable tissue from homozygous inbred lines is used in cross-pollinated crops, such as rye.

9) Method according to claims 1-8, characterized in that a medium is used for tissue culture, the nutrient salts as described in Murashige and Skoog 1962, carbohydrates such as e.g. Contains sucrose and possibly growth substances such as auxins or cytokinins and is preferably solidified with phytagel or agarose.

10) Method according to claims 1-9, characterized in that the introduction of the foreign DNA into the regenerable tissue takes place several hours up to four weeks after culturing the regenerable tissue, preferably 3 to 7 days after culturing the explants.

11) Method according to claims 1-10, characterized in that the introduction of the foreign DNA into the regenerable tissue takes place immediately after the explantation and without prior preculture of the regenerable tissue. 12) Method according to claim 1-11, characterized in that the foreign DNA used: 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 contain an antisense message from a plant gene or DNA sequences that contain a synthetic version of a gene in which the nucleotide sequence has been modified.

13) Method according to claim 1, 2 and 4 to 12, characterized in that the introduction of the foreign DNA by any gene transfer method such as e.g. Particle bombardment, Agrobacterium transformation, electroporation of regenerable tissue, silicon carbide fiber-mediated gene transfer and protoplast-mediated gene transfer is carried out, preferably by particle bombardment or Agrobacterium transformation.

14) Method according to claims 1-13, characterized in that the placement in the shoot induction medium takes place after several hours up to four weeks after the introduction of the foreign DNA, preferably 7 to 17 days after the introduction of the foreign DNA.

15) Method according to claim 1-14, characterized in that a medium is used for sprout induction, the nutrient salts as described in Murashige and Skoog 1962, carbohydrates such as sucrose and preferably contains no growth agents and is preferably solidified with phytagel or agarose.

16) Method according to claims 1, 2 and 4-15, characterized in that selection and screening marker-free transgenic monocot plants, preferably cereal plants, are produced, in particular wheat and rye plants.

17) The method of claims 1-3, wherein the regenerated plants are stably transformed.

18) The method of claims 1-3, wherein the immature embryos are preferably about 0.8 mm to 2.0 mm in length at the time of explantation. 19) The method of claim 1 and 3, wherein the concentration of Agrobacterium is between about 0.5 OD and 4.0 OD.

20) The method of claim 19, wherein the concentration of Agrobacterium is preferably between about 1.6 OD to 2.4 OD.

21) Method according to claim 1 and 3 and 11 and 12, wherein for the step of contacting Agrobacterium and the plant tissues are present in a suspension in a liquid

22) The method of claims 1 and 3, wherein the cocultivation takes place on a solid medium.

23) Method according to claim 1 and 3, wherein the step of co-cultivation takes place on a liquid medium.

24) Method according to claim 1 and 3, wherein a medium containing MS salt is used for the cocultivation.

25) Method according to claim 1 and 3, wherein a medium containing MS salt is used for the regeneration step.

26) Method according to claims 1 and 3, additionally comprising the step of regenerating the embryos, by culturing the tissue in medium with an antibiotic which is suitable for stopping the growth of Agrobacterium.

27) Method according to claims 1, 3 and 26, wherein timentin, a mixture of 1500 mg of ticarcillin sodium and 100 mg of potassium clavunate, is used as the antibiotic.

28) The method of claim 27, wherein the concentration of the timentin is between about 100 mg / 1 and 300 mg / 1. 29) The method of claim 27, wherein the concentration of the timentin is 150 mg / 1.

30) Method according to claim 1 and 3, wherein the length of the step of co-cultivation is between one and four days.

31) Method according to claim 1 and 3, wherein the length of the Kultuφhase before the transfer of the tissues under conditions of plant regeneration is between 1 and 20 days after cocultivation.

32) Method according to claim 1 and 3, wherein the length of the Kultuφhase before the transfer of the tissues under plant regeneration conditions is preferably between 7 and 17 days after cocultivation.

33) The method of claim 1 and 3, wherein the selection for transgenic events that express the selectable marker transgene is applied during the whole tissue culture phase after cocultivation.

34) Method according to claim 1 and 3, wherein the selection for transgenic plants which express the selectable marker transgene is applied during the regeneration phase.

35) Method according to claim 1 and 3, wherein a transgene is transmitted without a selectable marker transgene and no selection is applied.

36) Method according to claims 1, 3 and 35, wherein the transformed plants are found by a known molecular or biochemical detection method.