DD283646A5 - PROCESS FOR PRODUCING TRANSGENIC PLANTS - Google Patents

Abstract

The invention relates to a method for introducing genetic material into plant proembryos or embryos using microinjection technology. The novel, reproducible method according to the invention is used for the production of transgenic plants. Material genetically; Proembryonic plants; embryos; Microinjection technology; Transgenic plants}

Description

Method of introducing genetic material into plants

Scope of the invention

The present invention is a novel ₉ reproducible method for introducing genetic material into zygotic embryos of plants using the microinjection _technology? which is characterized by its universal applicability and is used for the production of transgenic plants *

Char akteristik the known prior art

The genetic manipulation of hereditary genetic material using recombinant DBA technology is now available in a variety of procedures and techniques that are already routinely used in many laboratories

Undoubtedly the best studied and most widely used method is the A ^ robacterium transformation system®

A ^ robaoterium cells have on their Ti plasmid a large ШA fragment _9, the so-called T-DUA region _f, which is integrated into the plant genome during the natural transformation of plant cells *

This natural gene transfer system can be used after the implementation of various modifications as a gene-vector system for the targeted transformation of plants (Chilton _s MD ₉ 1983) ·

The Ag; However, it has the decisive advantage that the host range of the Agrobacteria can be applied to certain dicotyledonous plants as well as to a few representatives of the monocotyledons (hernialis).

steens et al, 1984; Hookas-Van-Slagtoen et al. , 1984), but they are insignificant from an agri-economic point of view. This means that the most important crops are not accessible for effective gene transfer.

In addition, the Agrobacteria used are pathogens that induce characteristic disease symptoms in the form of cancerous tissue growths in their host plants and can therefore only be handled in strict compliance with strict safety requirements in the laboratory.

Alternative transformation systems designed to overcome these drawbacks of the Agrobacterium transformation system and aimed at introducing exogenous DNA into plant protoplasts, such as the direct gene transfer of vector-free DNA into protoplasts (Paszkowski et al. , 1984, Potrykus et al. , 1986). and microinjection of vector-free DNA into protoplasts (Spangenberg et al. , 1986, Steinbiss and Stabel, 1983, Morikawa and Yamada, 1985) or cells (Nomura and Komamine, 1986), or callus (Melnikov PV et al., GB 2,200,367). must be regarded as problematic insofar as the regeneration of whole plants from plant protoplasts of a variety of plant species, especially from the group of Gramineae, still represents a problem.

Investigations by Yamada et al. , 1986, and Toriyma et al. , 1986, among others, for the regeneration of rice protoplasts and Harris et al. , 1988 (wheat) and Rhodes et al (corn) have made some progress here.

Another disadvantage of these alternative transformation systems relates to the still relatively low transformation rates, which currently lie at a maximum at values between 1% and 5%.

These low transformation rates make it still needed ^ the DNA to be inserted with markers (eg _e B ₈ anti "biotics resistance genes) to provide ^ that enable rapid selectioning the transformants from the large number not" transformed cells "

But this does * that at the time no really satisfactory transformation process is available * which allows also a commercial point of efficient and cost-effective production of transgenic plants with new and Nütz = "lent properties, particularly in terms of the plants from the group of monocotyledoneae _#

It must, therefore, be regarded as an urgent task to develop procedures for a rapid, efficient and reproducible transformation of all plants, irrespective of their taxonomic position and the peculiarities associated with it _. and thus ensure a commercially effective and economical production of transgenic plants »

This applies especially to plants of the group Monocotyledoneae "in particular to those of the family Gramineae " in which the agri-economic most important crops such as wheat ^ barley _s rye oats, corn ₉ rice _s millet and _{e e} m® and are therefore of very particular economic interest, especially since no satisfactory methods have yet been available for the production of transgenic monocotyledonous plants ©

The first attempts in this direction provide two only recently developed transformation "process _e These are firstly, the injection of exogenous DUA in

the young blooms of rye plants (de la Pena et al., 1987) and the Agrobacterium- mediated viral infection of maize stalks with maize streak virus (Grimsley et al. 1987) Disadvantages, for example, the first-mentioned method has proved to be non-reproducible,

Object of the invention

In the context of the present invention, it has now surprisingly been possible to develop a highly efficient transformation system for plants, irrespective of their taxonomic affiliation and the peculiarities associated therewith, which is free from the abovementioned limitations of the previously known methods and thus thus characterized by its universal applicability and ensures effective and economical production of transgenic plants®

Presentation of the ffesens of the invention

It is an object of the present invention to develop methods which permit a rapid, efficient and reproducible transformation of all plants, regardless of their economic position and the peculiarities associated therewith *

According to the invention the object is achieved by a method for introduction of genetic material into plant ₉ which is characterized _s that mikroinjiziertj a ША-containing solution in young zygotic embryos of plants that have a high regeneration potential in the form of a natural embryogenesis ₉ indicates the microinjected embryos microcultivated into suitable culture media which promote embryogenesis and subsequently regenerate them into whole, complete and viable plants

Another object of the present invention relates to the preparation of transgenic whole plants, which have been transformed using the method according to the invention with a recombinant DHA molecule ₉ which leads to new and desirable properties in the transformed plant and the transgenic plants produced in this way and their nachkommen®

The present invention further relates to plants, which from transformed primary or secondary embryos that said recombinant DIA molecule ₉ are regenerated and the semen _s, moreover, the offspring of plants ₉ which have been regenerated from said transgenic plant material, as well as mutants and variants thereof ©

In the context of this invention, the origin of transgenic plants means derivatives of the transformed mother plant produced both sexually and asexually

The present invention further encompasses hybrid genetic constructions containing one or more genes or DNA sequences which result in novel and desirable properties in the transformed plant, cloning vehicles and host organisms.

Another object of the present invention relates to the use of the inventive transformation method for producing transgenic plants with new and desirable properties.

In the following description, a number of terms are used which are common in recombinant DNA technology as well as in plant genetics. In order to assure a clear and consistent understanding of the description and claims as well as the scope to which the expressions are deemed to set forth, the following definitions are set forth.

Embryo: Early stage of development of a plant consisting of shoot and root vegetation point and cotyledons.

Proembryo: 2 to multicellular stage of embryonic development, prior to the formation of shoot and root vegetation meristem and cotyledons (see Fig. 1).

Embryogenesis: development of an embryo, starting from a cell that has the potency for embryo formation.

Zygotic embryos: Embryos that are formed from a fertilized egg.

Somatic embryos: Embryos formed from somatic cells with embryogenic potency.

Chimera: cell, group of cells, tissue or plant composed of cells with different genetic information.

Plant material: In culture or as such viable plant parts such as protoplasts, cells, callus, tissues, embryos, plant organs, buds, seeds, etc., and whole plants.

Plant Cell : Structural and physiological unit of the plant consisting of a protoplast and a cell wall.

Protoplast: Regenerate plant cell or tissue isolated, cell wall-less "bare" plant cell with the potential to regenerate a cell clone or whole plant.

Cell clone: a population of genetically identical cells arising from a cell through ongoing mitoses.

Plant tissue: group of plant cells organized in the form of a structural and functional unit.

Plant organ: Structural and functional unit of several tissues, such as root, stem, leaf or embryo.

Transgenic chimera: plants or tissues that do not contain a foreign DNA stably integrated into the genome in all cells but only in tissue sectors.

Transgenic plant: Plant that contains a foreign DNA stably integrated into the genome in all cells.

Heterologous gene (s) or DNA: A DNA sequence encoding a specific product or products or performing a biological function derived from a species other than that into which said gene is introduced; said DNA sequence is also referred to as foreign gene or foreign DNA.

Homologous gene (s) or DNA: A DNA sequence encoding a specific product or products or performing a biological function derived from the same species into which said gene is introduced.

Synthetic gene (s) or DNA: A DNA sequence that encodes a specific product or products or that performs a biological function and that is synthetically produced.

Plant promoter: A DNA expression control sequence that ensures the transcription of any homologous or heterologous DNA gene sequence in a plant, if said gene sequence is operably linked to such a promoter.

Termination sequence: DNA sequence at the end of a transcription unit that signals the end of the transcription process.

Overproducing Plant Promoter (OPP): A plant promoter capable of effecting , in a transgenic plant cell, the expression of any operably linked functional gene sequence (s) to an extent (as measured in terms of RNA or polypeptide amount) which is significantly higher than is naturally observed in host cells that are not transformed with said OPP.

3 '/ 5' untranslated region: DNA segments located downstream / upstream of the coding region which, while transcribed into mRNA but not translated into a polypeptide. This region contains regulatory sequences, such as the ribosome binding site (5 ') or the polyadenylation signal (3').

DNA expression vector: A cloning vehicle, such as a plasmid or a bacteriophage, containing all the signal sequences necessary for the expression of an inserted DNA in a suitable host cell.

DNA transfer vector: Transfer vehicle, such as a Ti plasmid or a virus, which allows the introduction of genetic material into a suitable host cell.

Figure 1 (after Randolph et al. , 1936) shows the different phases of the development of a cereal embryo using the example of maize ( Zea mays ). The development of other cereals such as wheat ( Triticum aestivum ), rice ( Oryza sativa ), barley ( Hordeum vulgare) and other gramines proceeds in an analogous manner:

Fertilization of the egg in the embryo bag (1) leads to the formation of the zygote (2). This develops via ongoing mitoses to the various phases of the pro-embryo (3-25). The transition from the proembryo to the embryo (25-26) is characterized by the formation of shoot meristem and root meristems and scutellum (modified cotyledon).

The pictures are not drawn on the same scale; The proembryo (17) has a length of about 0.05 mm, the proembryo (25) about 1 mm, and the embryo (35) about 8-10 mm.

The arrows indicate where the preferred areas for microinjection of the DNA-containing solution are.

Figure 2 (after Randolph et al. , 1936) shows the different phases of the development of a dicotyledemal embryo using the example of the shepherd's purse ( Capsella bursa pastoris ). The fundamentals of development are the same in different species; Details can be very different.

Fertilization of the egg in the embryo bag leads to the formation of a zygote (A). This develops as a result of ongoing mitoses in the proembryo (P) and suspensor (S). The transition from the proembryo (globular) to the embryo (E) lies between the phases Q and R and begins with the formation of the plants of the two cotyledons.

The arrows indicate the areas preferred for microinjection of the DNA-containing solution.

The transformation process according to the invention for introducing genetic material into plants is based on the microinjection of a DNA-containing solution into young zygotic embryos of plants which naturally have a high regeneration potential in the form of natural embryogenesis.

Preferred in the context of this invention is the microinjection of a solution containing DNA into zygotic proembryos or embryos of monocotyledonous plants, in particular into zygotic embryos of cereals and other grasses for which no transformation system is currently available.

Up to the time of the present invention, it has not been possible to locate, isolate and in the form of an in vitro culture very young zygotic embryos, which are at a very early stage of development and have cell numbers between 4 and <1000 cells cultivate.

Rather, it has previously been assumed that such very young zygotic embryos (proembryos) are not capable of growth and reproduction in culture. The present reports on the isolation and cultivation of zygotic embryos are limited to embryos that have a minimum size of a few thousand cells and thus appear less suitable for a possible microinjection due to the much too high cell number.

In the context of the present invention, surprisingly, for the first time now it has been possible by means of specific process measures to isolate zygotic embryos from the plant structures surrounding them at an early proembryonic stage (see Figures 1 and 2) and to cultivate them in vitro .

The microinjection of the DNA-containing solution into the embryos derived from fertilized oocytes is carried out according to the invention with the aid of a microinjection apparatus equipped with manually or motor-controllable micromanipulators.

The specific structure and the equipment of said micro-injection devices are widely described in the literature. Microinjection equipment is now also offered by various companies and can be obtained directly from them as complete equipment (for example ZEISS, Narashige Nikon Instr., Leitz etc.).

The thus transformed by microinjection Proembryonen and embryos can then be stimulated by the inventive method by microculture for direct embryogenesis and then very easily regenerated to whole plants.

To achieve this goal, the microinjected proembryos and embryos are first introduced individually or in small groups, preferably in wells of commercially available microculturing plates in suitable culture media and microcultivated until the formation of embryo-specific structures.

Of course, in the context of this invention, other, suitable for the microcultivation of zygotic pro / embryos suitable cultivation methods.

Following differentiation into embryos, they are finally transferred to a vitamin- and hormone-free or low-hormone medium in which the further differentiation to mature embryos and finally their germination to the plantlet takes place.

The plantlets thus obtained can then be transferred to soil and cultivated further in the same way as normal seedlings.

An essential advantage of the inventive transformation method over the previously known methods is based on the extraordinarily high transformation rates, which can be achieved with this method and which are higher by values of between 20% and 60% by a power of ten than the maximum previously described in the literature achievable transformation rates.

Moreover, the direct regeneration of plants from embryos via direct embryogenesis completely or at least partially avoids the negative effects of 'somaclonal variation' which usually occur in other alternative processes, especially those whose regeneration step is via a callus phase become. However, this means that the genetically modified plant - with the exception of the introduced gene - is identical to the parent plant, which is of crucial importance for the purposes of plant breeding.

A further decisive advantage of the method according to the invention is its universal applicability, irrespective of the taxonomic affiliation of the target plant in question and the peculiarities and procedural difficulties associated with the previously available transformation methods, eg in the form of a restriction to certain dicotyledons Use of the Agrobacterium Transfoрmationssystems or yet insufficient regenerability of monocotyledonous protoplasts using direct gene transfer methods.

This universality of the method according to the invention is based on transferring genetic material by means of a physical method (microinjection) into a structure (young sexual embryos) available from each plant, regardless of their taxonomic affiliation, which is the formation of normal, fertile, whole plants guaranteed during the subsequent regeneration.

In addition to the already mentioned advantages of the inventive transformation method over the previously known methods are to be mentioned as further process advantages:

a) a greatly reduced regeneration time, since the regeneration takes place via a direct embryogenesis and eliminates the usual callus phase and the morphogenetic induction phase of the in vitro cultures

b) if the microinjected structures are susceptible to secondary embryogenesis or secondary adventitious growth, it is possible to induce in vitro segregation of the resulting chimeric transformants as well

c) make an in vitro propagation of the transformants obtained.

d) Up to 100% survival rate of the transformed, microinjected structures due to a gentle injection technique.

Thus, it is within the scope of the present invention for the first time possible to generate transgenic plants, especially those from the group of Monocotyledoneae , with new and desirable properties, independently of the aforementioned limitations, with high efficiency, by adding a DNA-containing solution into zygotic plant Microembryinated proembryos or embryos with high regeneration potential, the microinjected zygotic embryos or embryos microcultured in suitable embryoider structures promoting culture media and produced from the thus obtained, usually chimeric Regeneraten optionally non-chimeric transgenic plants.

In detail, the method according to the invention comprises the following specific method steps:

a) Isolation of individual, for microinjection suitable multi- to multicellular zygotic pro / embryos from the ovules of donor plants;

b) selection of the isolated zygotic pro / embryos for the subsequent microinjection and introduction of the selected pro / embryos into a suitable culture medium;

c) microinjection of a DNA-containing solution containing one or more DNA fragments into the zygotic pro / embryos isolated under a);

di) microculturing the microinjected zygotic pro / embryos in suitable culture media promoting direct embryogenesis;

СІ2) Alternatively: cultivating the injected pro-embryos under conditions that allow the development of a morphogenic cell culture or the formation of secondary embryos; and

f) regeneration of said transformed zygotic pro / embryos to complete usually chimeric plants.

g) Preparation of non-chimeric transgenic plants with new and desirable properties.

Donor plants, which can be used as starting material for the isolation of zygotic embryos and embryos, can be used both under field conditions and in the greenhouse. When using plants which require vernalization to induce flower formation, such as e.g. Wheat, barley, oats, rye, maize, etc. as donor plants, care must be taken that these varieties are expediently exposed to a cold period, which may be between 1 to 5 weeks, to obtain an optimal budding and, as a consequence, optimal development of the ovules to ensure. The time of harvest of the donor plants depends on the plant species used and on the climatic conditions under which the plants are grown. In general, the donor plants are harvested 2 to 20 days after the pollination has taken place, but especially when the zygotic embryos have reached a suitable stage of development between the early proembryonic phase and the embryo phase, preferably between the 2-cell stage and the 5000 cell stage, extends (see Figures 1 and 2).

The isolation of the zygotic proembryos and embryos is preferably carried out with the aid of fine scalpels and fine needles or finely pulled tweezers under microscopic control.

The isolation can be done, for example, by excising the Nucellusgewebe containing the embryo bag and subsequent preparation of the embryo sack, wherein the pro- / embryo is prepared from the isolated embryo sack. In addition, of course, any other suitable isolation method can be used.

The zygotic proembryos and embryos obtained in this way are subsequently transferred into a drop of a liquid culture medium. The subsequent selection of suitable for a microinjection embryonic stages is preferably also under optical control, usually a stereomicroscope or an inverted microscope and for a microselection of cellular structures suitable, known in the art selection systems are used, as described for example in Koop and Schweiger (1985 ), Spangenberg (1986) or in Schweiger et al (1987).

Preferred for subsequent microinjection are embryonic stages ranging from early globular proembryonic stages to early embryonic stages. In particular, embryo stages are to be mentioned at this point, which comprise 2 to about 5000 cells.

Since good microinjectivity is often accompanied by a low regeneration frequency of the microinjected structures due to only small numbers of cells in early proembryonic stages, further criteria should be taken into account when selecting the optimal stages, as e.g. a good isolation of the embryonic or embryonic stages and a good regeneration capacity.

In the case of gramineae, therefore, in addition to early golbular proembryonic stages with scutellum (see Fig. 1, phase 17 to 25), in particular early embryonic stages (see Fig. 1, phases 25 to 30) are particularly suitable for use in the inventive method to look at, since the latter are distinguished not only good insulating but also by a high Regeneratiosfähigkeit. In addition, a good Mikroinizizierbarkeit is guaranteed because the intended and intended for microinjection area (Sprossmeristemanlage) from relatively few

Cells exists. Very particularly preferred in the context of this invention

therefore early embryonic stages, which except the scutellum already a good

have recognizable shoot and root meristem (see Fig. 1, phase 26-27).

Also, for the introduction of genetic material into plant from the group of Dicotyledoneae via microinjection early globular Proembryonalstadien are in particular (see. Fig. 2, stage 0, P, Q) as well as early embryonic stages (early to late-Kotelydonar stages) (see FIG. Fig. 2, phase R, S, T) are particularly suitable.

Finding or selecting the optimal development stage for the respective target plant as starting material for the process according to the invention is possible for the person skilled in the art on the basis of the present, detailed description without unreasonable experimental effort.

Microinjection of the DNA-containing solution into zygotic proembryos or embryos can be carried out by means of methods known per se, which are described, inter alia, by Steinbiss and Stabel, 1983; Neuhaus et al. , 1984, 1986; Lawrence and Davies, 1985; as described in Morikawa and Jamada, 1985.

Specifically, the microinjection of the plant pro / embryos can thereby be carried out using a specific microinjection apparatus, the core of which is micropipettes controllable by micromanipulators.

On the one hand, this is the actual injection pipette whose outer diameter is between 1.00 mm and 2.00 mm. Preferred is a diameter of 1.2 mm to 1.5 mm. The tip diameter of the injection capillary, on the other hand, is between ί 1 and 10 μπι. Especially suitable for use in the process according to the invention are injection capillaries with tip diameters between 0.5 μm and 1 μm. In the context of the present invention, the microinjection capillary used is preferably microelectrode

capillaries with fused glass filaments of borosilicate glass. Of course, all other glass types or other materials suitable for carrying out the microinjection may also be used. This microinjection pipette is usually connected via a pressure regulating unit, the so-called microinjector, to a gas source, in particular a nitrogen source or a compressed air source, which ensures a very finely metered transfer of the injection solution from the micropipette into the plant tissue. A second pipette, the so-called holding pipette, serves to fix the zygotic embryos during the microinjection process.

These holding pipettes usually have a bowl-shaped tip with a diameter between 0.02 mm and 1.00 mm, preferably between 0.1 mm and 0.5 mm. By applying a weak negative or positive pressure, the plant pro / embryos can be sucked in and thus fixed or released again after the microinjection of the DNA-containing solution. A third micropipette, the so-called collection pipette, is used to collect the microinjected embryos and transfer them to suitable microculture plates.

Suitable collection pipettes usually have an inner diameter between 0.1 mm and 1.0 mm, so that a trouble-free suction of the micro-injected embryos is guaranteed.

The actual microinjection process usually begins with the introduction of previously isolated and selected proembryos or embryos in 10 μΐ to 200 μΐ of a suitable culture medium on specially prepared coverslips and the aspiration of a suitable pro- / embryo using the holding pipette, while this by releasing and then re-aspirating in its orientation with respect to the injection pipette can be varied until an optimal for microinjection alignment of the pro- / embryo is reached.

After penetration of the micropipette tip into the embryonic tissue of the proembryos or into the tissue of the embryos, the release of the DNA-containing injection solution takes place.

When using embryos, injection of the DNA-containing solution into the tissue of the shoot meristem is preferred.

The injection volume varies depending on the cell size. It is usually between 1 pl and 100 pl per cell injected.

Preferred within the scope of this invention is an injection volume of 20 μl to 40 μl per cell injected.

In order to ensure a uniform transformation of all cells involved in the construction of the multicellular structure to be injected, these are injected depending on the existing cell number between 1 to 1000 times, but preferably between 4 to 60 times.

According to the invention, this can be achieved by rotating the zygotic proembryos and embryos to be injected between the different injection processes so that the largest possible number of different cells are taken.

As DNA solutions which are suitable for a microinjection into plant cells, for example buffer solutions are possible which contain the transforming DNA in a suitable concentration.

The preferred within the context of this invention DNA concentration is 0.1 μg / μl to 10 ug / ul, but especially at 0.5 ug / ul to 5 ug / ul. Most particularly preferred is a DNA concentration of 0.5 ug / ul to 1.5 _μ ε / μ1

The pH of the buffer solution may vary between values of pH 7 to pH 8; According to the invention, a pH range between pH 7.5 and pH 7.8 is preferred.

For the integration of the foreign gene into the genomic DNA of the plant cell, it is advantageous if the transforming DNA is flanked by neutral DNA sequences (carrier DNA). Under neutral DNA sequences

In the context of this invention, it is intended in particular to understand those sequences which are not related to the function of the transforming DNA to be transferred but are only of importance for the integration of the introduced DNA into the plant genome. The neutral DNA sequences flanking the transforming DNA may be of synthetic origin as well as obtained by treatment with suitable restriction enzymes from naturally occurring DNA sequences. For example, as a carrier DNA naturally occurring plasmids are suitable, which have been opened with a selective restriction enzyme. However, the DNA sequence produced for gene transfer can also have an annular structure (plasmid structure). Such plasmids consist of a DNA strand into which the foreign gene is integrated with the expression signals.

An example of such a plasmid is the freely available plasmid pUC8 (described in Messing, J. and J. Vieira, Gene J_9, 269-276, 1982). Furthermore, fragments of naturally occurring plasmids can also be used as carrier DNA. For example, as the carrier DNA, the deletion mutant can be used for gene VI of Caulifield Mosaic Virus.

According to the invention, the transforming DNA can be in the form of either open or closed rings (plasmid structure); it can have an over-branched or, preferably, a linearized form.

Preferred in the context of this invention is the use of a mixture of linearized and spirally wound form.

The zygotic proembryos and embryos can then be sucked up after microinjection with the aid of the collecting pipette and transferred into a suitable culture medium.

The optical control of the entire microinjection process is usually carried out under microscopic observation. Inverse microscopes or stereomicroscopes with a combination of incident and transmitted light are preferred.

The magnification chosen depends on the size of the material to be injected and fluctuates between 10 and 800 times the magnification.

The in vitro cultivation of the transformed proembryos and embryos following the microinjection is preferably carried out in culturing media which are suitable for the further development of the embryos up to the fully formed plant.

The composition of these media is characterized primarily by the fact that they mainly contain the essential for the growth and development of the plant cell mineral substances or elements in their compounds (macroelements), such as. Nitrogen, phosphorus, sulfur, potassium, calcium, magnesium and iron, besides the so-called trace elements (microelements) such as e.g. Sodium, zinc, copper, manganese, boron, vanadium, selenium and molybdenum. While the macroelements are generally present in amounts between 10 mg / l and a few hundred mg / l, the concentration of the microelements is generally at most a few mg / l.

Other ingredients of said media include a number of essential vitamins such as nicotinic acid, pyridoxine, thiamine, inositol, biotin, lipoic acid, riboflavin, Ca-pantothenate, etc., a suitable readily assimilable carbon source such as sucrose or glucose, as well as various growth regulators in the form of natural or synthetic Phytohormones from the class of auxins and cytokinins in the concentration range of 0.01 mg / 1 to 20 mg / 1.

Particular care should be taken in accordance with the invention that the composition of the media and the concentration ratios of their individual components can change depending on the stage of development of the embryos. This applies in the first place to the growth regulators used, which must be used in a balanced relationship, so that, if desired, a controlled course of direct embryogenesis is ensured.

Within the scope of this invention, a concentration range of the phytohormones from the class of the auxins and the cytokinines is preferably between 0.01 mg / l and 20 mg / l, but in particular between 0.8 mg / l and 1.2 mg / l.

The culture media are moreover osmotically stabilized by addition of sugar alcohols (for example mannitol), sugars (for example glucose) or salt ions and are adjusted to pH values from 4.5 to 7.5, preferably from 5.2 to 6.5.

In addition to the use of synthetic media having a well-defined composition of various individual components of known concentration, it is also possible according to the invention to use complex nutrient media which are completely or at least partially composed of natural components.

In this connection, reference is made, by way of example, to potato extract, coconut milk and liquid endosperm, which can be obtained by aspiration from the ovules of plants.

While the first cultivation steps, which are directly followed by microinjection, are preferably carried out in liquid culture media, which may optionally overlay a solidified culture medium, in the further course of embryonic development, either liquid or solid culture media, which may be obtained by adding one of the Usually, solidifying agents used in microbiological technology can be produced, or a combination of solid and liquid media can be used.

Suitable solidifying agents include, for example, agar, agarose, alginate, pectinate, Gelrite®, gelatin, and the like. Preference is given to agar and agarose, in particular agarose and low-melting-type agarose (LMT).

When using liquid media, it may be expedient to add an osmotically neutral thickening medium to the medium so that the density of the medium increases, without thereby increasing the osmotic value.

This results in the embryos floating on the medium, thus ensuring optimal gas exchange with the surrounding atmosphere.

Preferred within the scope of this invention are synthetic polymers, e.g. Co-polymers of sucrose and epichlorohydrin having a molecular weight between 70,000 and 100,000, but are not limited thereto.

The microinjected proembryos or embryos in the manner described above are preferably transferred directly to commercially available microculturing plates immediately after microinjection, in media having a composition suitable for the in vitro cultivation of zygotic embryos and embryos.

The microculturing of the microinjected zygotic embryos or embryos is preferably carried out in a liquid culture medium which, depending on the specific requirements of the plant material used, can be treated with an osmotically neutral thickener and which may optionally be superimposed on a solid medium.

The culturing density is preferably between 1 proembryo or embryo / μΐ and 50 proembryos or embryos / μΐ culture medium, very particularly preferably between 1 proembryo or embryo / μΐ and 5 probembryos or embryos / μΐ culture medium.

The selected cultivation density depends on the size and development status of the pro / embryos used.

It proves to be advantageous in the context of this invention, if the cultivation of the microinjected proembryos or embryos is at least temporarily carried out in a 2 compartment system, which acts as a moist chamber, the outer compartment, for example

is formed by water, by culture medium or by an aqueous mannitol solution while the embryos are in the inner compartment.

It is also advantageous to transfer the microinjected proembryos or embryos every 1 to about 12 days, but preferably every 2-3 days, into fresh culture medium or to coat with fresh culture medium, the volume of the medium depending on the size of the embryos and in The rule is between 0.5 μΐ and 10 μΐ and thereby, a ratio embryo: medium of 1: 0.2 μΐ to 1:10 μΐ is present.

When the embryos microcultivated in the manner described above have reached a certain developmental stage, usually after 10 to 30 days, the embryos are transferred to solid media for differentiation of the shoot and root apex, which may optionally be superimposed by a liquid medium. In general, these are the culture media commonly used for the regeneration of plants, which cause a differentiation of shoot and / or root growth due to their balanced content of phytohormones, in particular from the class of auxins and cytokinins.

After a cultivation period of about 4 to 8 weeks, depending on the plant material used, but at the latest after germination of the embryos, they can be transferred to solid media, which have no or only a low hormone concentration and here until the formation of small plantlets (seedlings ) are further cultivated.

Depending on the plant material used, it may be advantageous to induce induction of rooting, e.g. by transferring the embryos to known root-inducing media [cf. e.g. Medium S4 (Table 9); H4 (Table 10)].

The thus formed transgenic, usually chimeric plantlets can then be planted in soil and treated in the same way as normal seedlings.

Non-chimeric transgenic plants can then be easily prepared from the transgenic chimeras obtained in the manner described above using methods known per se.

There are basically two alternative ways of producing non-chimeric transgenic plants.

a) In plants susceptible to secondary embryogenesis or secondary adventitious shoot formation, it is possible to separate and separate the secondary structures derived from single cells in vitro from the primary, usually chimeric, regenerants, and thus obtaining pure transgenic plants. This is particularly easy if the or one of the transmitted foreign genes allows the application of selective culture conditions so that only the transgenic cells survive.

The transgenic regenerates can then be cultured in the manner described above and regenerated to whole plants.

b) Homogeneously transformed (non-chimeric) plants, on the other hand, can also be obtained by optionally repeated self-pollination of primary transgenic chimeras. Since the sexual offspring comes from a single cell, namely the fertilized egg cell, a part of these offspring will be complete. _; Transgenic plants, and indeed when the transgenic part of the chimera in the formation of pollen or egg cells is involved.

The method for the production of transgenic plants with the aid of microinjection technology described above, with all its sub-steps, is part of the present invention.

The inventive method is practically universally applicable, since virtually all plants form zygotic embryos. It encompasses both the microinjection of natural DNA sequences and of hybrid gene constructs artificially generated using recombinant DNA technology.

Primarily encompassed by the broad scope of the present invention are recombinant DNA molecules containing DNA sequences which confer useful and desirable properties on the transformants.

These are preferably recombinant DNA molecules that contain one or more gene sequences that encode a useful and desirable characteristic and that are under the regulatory control of and are operably linked to plant-active expression signals, such that expression thereof Gene sequences in the transformed plant cell is ensured.

Thus, for use in the method of the invention, all those genes which are expressed in the plant cell and which confer on the plant a useful and / or desired property, such as e.g. an increased resistance to pathogens (for example to phytophathogenic fungi, bacteria, viruses etc.), a resistance to chemicals [for example to herbicides (such as triazines, sulfonylureas, imidazolinones, triazolopyrimidines, bialaphos, glyphosates, etc.), insecticides or other biocides] ; Resistance to adverse (endaphic or atmospheric) environmental influences (for example to heat, cold, wind, special, extreme soil conditions, humidity, dryness, osmotic stress, etc.); or lead to an increased or better quality formation of reserve or storage materials in leaves, seeds, tubers, root, stems, etc. Among the desirable substances that can be produced by transgenic plants include, for example, proteins, starches, sugars, amino acids, alkaloids, fragrances, colors, fats, etc.

Also encompassed by the present invention are genes which contain pharmaceutically acceptable active substances, e.g. Alkaloids, steroids, hormones, immunomodulators, and other physiologically active substances.

Resistance to cytotoxins can be achieved, for example, by transfer of a gene capable of providing, upon expression in the plant cell, an enzyme which detoxifies the cytotoxin, e.g. neomycin phosphotransferase type II or aminoglycoside phosphotransferase type IV, which contribute to the detoxification of kanamycin, hygromycin and other aminoglycoside antibiotics or a glutathione S-transferase, cytochrome P-450 or other catabolic enzymes known to be triazines, sulfonylureas or detoxify other herbicides. Resistance to cytotoxins may also be mediated by a gene expressing in a plant a particular form of a 'target enzyme' (cytotoxin action site) which is resistant to the activity of the cytotoxin, e.g. a variant of acetohydroxy acid synthase that is insensitive to the inhibitory effect of sulfonylureas, imidazolinones or other herbicides that interact with this specific metabolic step; or a variant of EPSP synthase, which proves to be insensitive to the inhibitory effect of glyphosate. It may prove advantageous to express these altered target enzymes in a form which facilitates their transport into the correct cellular compartment, e.g. in the above case in the chloroplasts, allowed.

In certain cases it may be advantageous to direct the gene products into the mitochondria, the vacuoles, the endoplasmic reticulum or other cell regions, possibly even into the intercellular spaces (apoplasts).

Resistance to certain fungal classes can be achieved, for example, by the introduction of a gene that expresses chitinase in the plant tissues. Numerous phytopathogenic fungi contain chitin as an integral part of their hyphae and spore structures, such as the Basidiomycetes (fire and rust fungi), Asomycetes and Fungi Imperfecti (including Alternaria and Bipolaris, Exerophilum turcicum, Colletotricum, Gleocercospora and Cercospora) . Chitinase is capable of mycelial growth of certain pathogens

inhibit in vitro . A plant leaf or root expressing chitinase constructively or in response to the ingress of a pathogen is protected against attack by a large number of different fungi. Depending on the situation, constitutive expression may be advantageous compared to inducible expression, which occurs in many plants as a normal response to pathogen attack, since chitinase is immediately present in high concentration without having to wait for a lag phase for the new synthesis.

For example, resistance to insects may be conferred by a gene encoding a polypeptide that is toxic to insects and / or their larvae, such as the crystalline protein of Bacillus thuringiensis [Barton et al, 1987; Vaeck et al, 1987]. A second class of proteins that confer insect resistance are the protease inhibitors. Protease inhibitors form an ordinary part of plant storage structures (Ryan, 1973). Bowman-Birk protease inhibitor isolated and purified from soybeans has been shown to inhibit the intestinal protease of Tenebrio larvae (Birk et al, 1963). The gene encoding the trypsin inhibitor from cowbud is described in Hilder et al. (1987).

A gene encoding a protease inhibitor may be placed in a suitable vector under the control of a plant promoter, particularly a constructive promoter, such as the CaMV 35S promoter [described in Odell et al, (1985)]. The gene, for example the coding sequence of the Bowman-Birk protease inhibitor from soybean, can be obtained by the cDNA cloning method described by Hammond et al. (1984). Another possibility for the preparation of a protease inhibitor is its synthetic preparation, provided that it has less than 100 amino acids, such as the trypsin inhibitor of lima bean. The coding sequence can be predicted by back-translating the amino acid sequence. In addition, restriction sites are built at both ends, which are suitable for the respective desired vector. The synthetic gene is prepared by synthesis of overlapping oligonucleotide fragments of 30 to 60 base pairs

prepared by first subjected to a kinase reaction, then linked together (Maniatis et al, 1982) and finally cloned in an appropriate vector. Using DNA sequencing, a clone having the insert in a correct orientation can then be identified. For the discharge into the embryos isolated plasmid DNA is used.

Also included in the present invention are genes that encode pharmaceutically active ingredients, eg, alkaloids, steroids, hormones, and other physiologically active substances, as well as flavins, vitamins, and dyes. Genes contemplated within the scope of this invention therefore include, but are not limited to, plant-specific genes such as, for example, Zeingen (Wienand et al, 1981), mammalian genes such as insulin gene, somatostatin gene Interleucingeins, the t-PA gene (Pennica et al, 1983), etc., or genes of microbial origin such as the NPT II gene as well as synthetic genes such as insulin gene (Itakura et al, 1975).

In addition to naturally occurring genes which code for a useful and desirable property, it is also possible in the context of this invention to use genes which have been previously modified with the aid of chemical or genetic engineering methods.

Furthermore, the broad concept of the present invention also encompasses those genes which are completely prepared by chemical synthesis.

Also suitable for use in the method according to the invention are other useful DNA sequences, in particular non-coding DNA sequences with regulatory functions. For example, these noncoding DNA sequences are capable of regulating the transcription of an associated DNA sequence in plant tissues.

In this context, for example, the promoter of the hps70 gene may be mentioned, which can be induced by a heat shock (Spena et al , 1985); the 5 'flanking sequence of the nuclear-encoded sequence of the small

Subunit of the ribulose-1,5-biphosphate carboxylase (RUBISCO) gene [Morelli et al , 1985] or the chlorophyll a / b binding protein which are light-inducible; or the 5'-flanking region of the maize alcohol dehydrogenase (adhl-s) gene which induces transcription of an associated reporter gene [eg chloramphenicol acetyltransferase gene (CAT)] under anaerobic conditions (Howard et al, 1977).

Another group of regulatable DNA sequences relates to chemically-regulated sequences, e.g. are present in the PR ("pathogenesis related proteins") protein genes of tobacco and are inducible by means of chemical regulators.

The regulatable DNA sequences mentioned above by way of example may be of natural or synthetic origin or may consist of a mixture of natural and synthetic DNA sequences.

Suitable genes or DNA sequences which can be used in the context of the present invention are both homologous and heterologous gene (s) or DNA as well as synthetic gene (s) or DNA according to the definition made in the context of the present invention.

The DNA sequence can be constructed exclusively from genomic, from cDNA or synthetic DNA. Another possibility is to construct a hybrid DNA sequence consisting of both cDNA and genomic DNA and / or synthetic DNA.

In this case, the cDNA may be from the same gene as the genomic DNA or else both the cDNA and the genomic DNA may be from different genes. In any case, however, both the genomic DNA and / or the cDNA, each by itself, can be made from the same or different genes.

If the DNA sequence includes portions of more than one gene, these genes may be from either one and the same organism, from several organisms, the different strains or varieties of the same species or

belong to different species of the same genus, or from organisms that belong to more than one genus of the same or another taxonomic entity (Reich), descend.

In order to ensure the expression of said structural genes in the plant cell, it is advantageous if the coding gene sequences are first operably linked to expression sequences which are functional in plant cells.

The hybrid gene constructions in the context of the present invention thus contain, in addition to the structural gene (s), expression signals which include both promoter and terminator sequences as well as further regulatory sequences of the 3 'and 5' untranslated regions.

Any promoter and terminator capable of inducing induction of expression of a coding DNA sequence (structural gene) may be used as part of the hybrid gene sequence. Particularly suitable are expression signals derived from genes of plants or plant viruses. Examples of suitable promoters and terminators are e.g. those of the nopaline synthase genes (nos), the octopine synthase genes (ocs) as well as the cauliflower mosaic virus genes (CaMV) or homologous DNA sequences, which still have the characteristic properties of said expression signals.

Preferred within the scope of this invention are the 35S and 19S expression signals of the CaMV genome or their homologs obtained by molecular biological methods, e.g. in Maniatis et al., 1982, can be isolated from said genome and linked to the coding DNA sequence.

For the purposes of the present invention, homologs of the 35S and 19S expression signals are to be understood as meaning sequences which are essentially homologous to the starting sequences despite slight sequence differences and which still fulfill the same function as these.

The starting material for the ßSS transcription control sequences according to the invention, for example, the seal fragment of the CaMV strain "S", which includes the nucleotides 6808-7632 of the gene map (Frank G et al. , 1980) are used.

The 19S promoter and 5 'untranslated region is located on a genomic fragment between the PstI site (position 5386) and the HindIII site (position 5850) of the CaMV gene map (Hohn et al. , 1982). The corresponding terminator and 3 'untranslated region is located on an EcoRV / BglII fragment between position 7342 and 7643 of the CaMV genome.

Further preferred in the context of this invention are the expression signals of the CaMV strain CM 1841, the complete nucleotide sequence of which is described in Gardner RC et al. , 1981.

Another effective representative of a plant promoter that can be used is an overproducing plant promoter. This type of promoter should, if operably linked to the gene sequence encoding a desired gene product, be able to mediate the expression of said gene sequence.

Overproducing plant promoters which may be used in the present invention include the promoter of the small subunit (ss) of soybean ribulose-1,5-bisphosphate carboxylase [Berry-Lowe et al. , J. Molecular and App. Gene. , U 483-498 (1982)] and the promoter of the chlorophyll a / b binding protein. These two promoters are known to be induced by light in eukaryotic plant cells [see, eg, Genetic Engineering of Plants, an Agricultural Perspective ,

A. Cashmore, Plenum, New York 1983, pages 29-38; Coruzzi G. et al. , The Journal of Biological Chemistry , 258 : 1399 (1983) and Dunsmuir,

P. et al., Journal of Molecular and Applied Genetics, 2 \ 285 (1983)].

The various DNA sequence sections can be linked to one another by means of methods known per se to form a complete coding DNA sequence. Suitable methods include, for example, the in vivo recombination of DNA sequences having homologous portions as well as the in vitro linking of restriction fragments.

As cloning vectors, one generally uses plasmid or virus (bacteriophage) vectors with replication and control sequences derived from species compatible with the host cell.

The cloning vector usually carries an origin of replication, as well as specific genes that lead to phenotypic selection characteristics in the transformed host cell, in particular to resistance to antibiotics or to certain herbicides. The transformed vectors can be selected from these phenotypic markers after transformation in a host cell.

Selectable phenotypic markers that can be used in the context of this invention include, for example, without this being a limitation of the subject invention, resistance to ampicillin, tetracycline, hygromycin, kanamycin, metotrexate, G418 and neomycin.

Suitable host cells in the context of this invention are prokaryotes, including bacterial hosts such as, for example, A.tumefaciens , E. coli , S. typhimurium and Serratia marcescens , as well as cyanobacteria. Furthermore, eukaryotic hosts such as yeasts, mycelium-forming fungi and plant cells may also be used in the invention.

The splicing of the hybrid gene construction according to the invention into a suitable cloning vector takes place by means of standard methods as described, for example, in Maniatis et al., 1982.

As a rule, the vector and the DNA sequence to be spliced are first cut with suitable restriction enzymes. Suitable restriction enzymes are e.g. those which provide blunt-ended fragments, such as e.g. SmaI, Hpal and EcoRV, or enzymes which form cohesive ends, e.g. EcoRI, Sacl and BamHI.

Both blunt-ended and cohesive-end fragments that are complementary to each other can be recombined into a single, consistent DNA molecule using appropriate DNA ligases.

Smooth ends may also be prepared by treating DNA fragments having overhanging cohesive ends with the Klenow fragment of E. coli DNA polymerase by filling in the gaps with the corresponding complementary nucleotides.

On the other hand, cohesive ends can also be made artificially, e.g. by adding complementary homopolymeric tails to the ends of a desired DNA sequence and the cut vector molecule using a terminal deoxynucleotidyl transferase or by adding synthetic oligonucleotide sequences (linkers) which carry a restriction site and subsequent cutting with the corresponding enzyme.

The cloning vector and the host cell transformed with this vector are used according to the invention to increase the copy number of the vector. With an increased copy number, it is possible to isolate the vector carrying the hybrid gene construction of the invention, and e.g. for the introduction of the chimeric gene sequence into the plant cell.

In a further process step, these plasmids are used to introduce the desired gene product encoding structural genes or non-coding DNA sequences with regulatory function in the plant cell and to integrate into the plant genome.

Another object of the present invention therefore relates to the production of plant recipient cells which contain said structural gene or other natural DNA sequences incorporated into their genome.

Also encompassed by the broad concept of this invention are transgenic plants that have been transformed by the method of the invention described above, and their asexual and / or sexual progeny, which still have the new (and desirable) effects of transformation of the parent plant. n) feature (s), as well as general plant reproductive material.

By definition, the term asexual and / or sexual progeny of transgenic plants therefore also includes within the scope of this invention all mutants and variants which still have the characteristic properties of the transformed starting plant, as well as all cross and fusion products with the transformed plant material.

In the context of this invention, by the term of the plant propagation material, by definition, e.g. herbal protoplasts, cells, cell clones, cell agglomerates, callus tissue and / or organ cultures, as well as seeds, pollen, ova, zygotes, embryos or other reproductive material resulting from germ line cells are understood, this exemplary listing means no limitation of the subject invention.

Plant parts, e.g. Blossoms, stems, fruits, leaves, roots derived from transgenic plants or their progeny previously transformed by the method of the invention and thus at least partially constructed from transgenic cells are also subject of this invention.

The inventive method is suitable for the transformation of all plants, in particular those of the systematic groups Angiospermae and Gymnospermae.

Among the gymnospermae the plants of the class Coniferae are of special interest.

Among the angiosperms , besides the deciduous trees and shrubs of particular interest are plants of the families Solanaceae , Cruciferae , Compositae , Liliaceae , Vitaceae , Chenopodiaceae , Rutaceae , Alliaceae , Amaryllidaceae , Asparagaceae , Orchidaceae , Palmae , Bromeliaceae , Rubiaceae , Theaceae , Musaceae , Malvaceae or Gramineae and the order Leguminosae and here especially the family Papilionaceae . Preferred are representatives of Solanaceae , Cruciferae , Leguminosae , Malvaceae and Gramineae .

Among the target crops in the present invention include, for example, those selected from the series: Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis, Kajorana, Cichorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Hemerocallis, Nemesia, Pelargonium, Panicum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browallia, Glycine, Lolium, Zea, Triticum and Sorghum, Gossypium, Ipomoea, Passiflora, Cyclamen, Malus, Prunus, Rosa, Rubus, Populus, Santalum, Allium, Lilium, Narcissus, Pineapple, Arachis, Phaseolus and Pisum .

Particularly favored are representatives of the family of the Gramineae , such as plants, which are cultivated over large areas and deliver high crop yields. Examples include corn, rice, wheat, barley, rye, oats, millet or sorghum and pasture grasses.

Further target crops which are particularly preferred for use in the process according to the invention are, for example, plants of the genera Allium , Avena , Hordeum , Oryza , Panicum , Saccharum , Seeale , Setaria, Sorghum , Triticum , Zea , Musa , Cocos , Phoenix and Elaeis

The detection of a successful Tranaformat ion can be done in a conventional manner, for example, by molecular biology studies, which particularly belonged to the "Southern blot" analysis

The extracted DUA is first treated with restriction enzymes, then subjected to electrophoresis in a 0.8% to 1% agarose gel, on a nitrocellulose membrane (Southern, Ε _β M _o , J _e Mol _e Biol _e 98, 503- 517 (1975)) and with the DNA to be detected which previously had undergone translation (Rigby, W ₁₉ J ,, Dieckmann, M ₀₉ Rhodes, G _e and P _a Berg, J. Moto Biol. "237-251 )) was subjected to (DNA-specific activities of 5 χ Ю ⁸ to 10 χ 10 Cepeine / ^ ug), hybridized the filters are long times per hour with an aqueous solution of 0.03M sodium citrate and 0.3M sodium chloride at 65 ⁰ C washed. The hybridized DNA is visualized by blackening an X-ray film for 24 to 48 hours. "

To illustrate the more general description and to better understand the vorliege iden invention will now be made to specific embodiments, which have no limiting character _s , unless it is specifically pointed out. The same applies to all exemplary lists included in the preceding description *

Auaführtuigabeispiele Example 1s Construction of the plasmid pABDI

We cut into the freely accessible plasmids pkm 21 and pkm 244 (Beck, E _9, et al * _s Gene J ^ _9327-336 (1982)) with the restriction endonuclease PstI · The fragments of the plasmids

which are used for recombination are purified by electrophoresis in 0 ₈ 8% agarose gel. The plasmid pkm 21244 obtained from the compound of the fragments is a combination of the 5 ⁸ and 3 'Ba I 31 deletions of the HPT Ii gene as described by Beck et al., Gene J ^ _9, 327-336 (1982). The coupling plasmid pJPAX is constructed to link the promoter signal of the cauliflower mosaic virus to the HindIII fragment of plasmid pkm _21244. The coupling plasmid pJPAX becomes from the Pias-

pUC8 and pUC9 [Messing, J. and J. Vieira, Gene 1_9, 269-276 (1982)]. Ten base pairs of the coupling sequence of plasmid pUC9 are cleaved at the HindIII and SalI positions and then filled with the adhesive ends with the polymerase I Klenow fragment [Jacobsen, H., et al., Eur. J. Biochem , 4_5, 623, (1974)] and joining the polynucleotide chain to restore the HindIII position. A synthetic coupling member of 8 base pairs (Xhol) is inserted at the SmaI position of this separated coupling sequence. Recombination of the appropriate Xor1 and HindIII fragments of the pUC8 plasmid and the modified pUC9 plasmid yields the plasmid pJPAX with a partially asymmetric coupling sequence with the contiguous restriction sites: EcoRI, SmaI, BamHI, Sail, PstI, HindIII, BamHI, Xhol and EcoRI , The CaMV gene VI promoter region, which is linked to the NPT II structural gene, is derived from the genome of the CaMV strain CM 4-184, a variant of the CaMV strain CM 1841, whose complete nucleotide sequence is described in Gardner RC et al , 1981 is. The CaMV promoter region is cloned into the 6 kbp (kbp: 1000 base pair) cosmid pHC 79, a derivative of the E. coli plasmid pBR322 [Hohn B and Collins J, 1980], with the CaMV genome and the cosmid pHC79 Bstll cut and the resulting fragments are linked together. The connection of the 5 'expression signal of the CaulifIower Mosaic Virus gene VI and the HindIII fragment of the NPT II gene is inserted in the plasmid pJPAX by inserting the promoter region of the CaulifIower Mosaic Virus gene VI between the PstI and the Hind HI position causes. The resulting plasmid is cut open at the unique HindIII position and the HindIII fragment of plasmid pkm 21244 is incorporated into this cleavage site in both orientational directions to yield plasmids pJPAX Cakm and pJPAX Cakm. To create an EcoRV sequence in the vicinity of the 3 'termination signal of the NPT-II hybrid gene, a BamHI fragment of the plasmid pJPAX Cakm is inserted into the BamHI position of the plasmid pBR327 [Soberon, X. et al. , Gene 9_, 287-305 (1980)]. The plasmid pBR327 Cakm is obtained. The EcoRV fragment of plasmid pBR327 Cakm, containing the novel DNA construction, is used to replace the EcoRV region of CaulifIower mosaic virus gene VI, which is cloned at the Sall position in plasmid pUC8, thereby the protein-encoding DNA sequence of the NPT II

Gens under the control of the 5 'and 3' expression signals of the CaulifIower Mosaic Virus gene VI. The plasmids thus prepared are designated pABDI and pABDII.

Example 2: Construction of the plasmid pDH51

The plasmid pDH 51 is described in Pietrzak et al. , 1986. This plasmid contains the 35S promoter region and terminator region of CaMV separated by an inserted polylinker region containing numerous cloning sites.

The starting material for the 35S promoter-Z terminator region is a seal fragment of CaMV "S" which comprises the nucleotides 6808-7632 of the gene map (Frank G et al. , Cell , 2J ₁ : 285-294, 1980), and US Pat into the SmaI site of pUCl8 (Norrander J. et al. , Gene , 2b: 101-106, 1983), to give plasmid pDB21. This is then digested with the restriction enzyme Hphl. The cohesive ends are removed by treatment with T4 DNA polymerase.

A fragment containing the 35S promoter region [pUCl8 pos. 21 (Hphl) -412 (Smal) plus CaMV pos. 6808-7437 (Hphl)] is linked to Kpnl linkers, cut with NcoI (position 6909 CaMV), the cohesive ends filled in using the Klenow fragment of the DNA polymerase, linked to EcoRI linkers and finally digested with EcoRI and Kpnl ,

The resulting EcoRI / KpnI fragment containing the CaMV promoter sequence is then spliced into the plasmid pUCl8 between the KpnI and EcoRI restriction sites. This gives the plasmid pDG2.

A second fragment carrying the termination signals [CaMV pos. 7439 (HphI) -7632 plus pUCl8 pos. 413-1550 (Hphl)], is cut with EcoRI, the cohesive ends are filled in with Klenow, provided with HindIII linkers and digested with HindIII and SphI.

The resulting SphI-HindIII fragment containing the CaMV terminator sequence is then spliced into the plasmid pDG2 into the HindIII and SphI restriction sites.

Example 3: Construction of the plasmid pHP23

The construction of plasmid pHP23 is carried out by insertion of an EcoRV fragment of the plasmid pABD1, which contains the APH (3 ') II structural gene and a part of the 19S RNA promoter sequence of CaMV, into the SmaI site of the plasmid pDH51.

Example 4: Isolation and injection of zygotic maize embryos

a) Isolation and selection of maize embryos Young zygotic proembryos and embryos are isolated from Zea mays cv. Alpine, cv. Issar, G-4083 or cv. Tokano, 3 to 22 days after pollination isolated (see Fig. 1 Phase 17 to 30).

The corncobs are surface sterilized by a 70% ethanol treatment for 30 seconds, and the singled young corn kernels are kept in Petri dishes wet with sterile distilled water until the zygotic embryos are isolated.

The zygotic embryos are isolated under microscopic control (10-5x magnifications) using fine scalpels and fine forceps. The size and developmental stage of the isolated embryos ranges between phase 17 and 30 of Figure 1.

The isolation is conveniently carried out by

a) excising a median longitudinal incision in the area of the nucellus,

b) subsequent preparation of the embryo sack and

c) Removing the embryo from the isolated embryo sac.

For the selection of the embryos one usually uses a stereomicroscope (50x - 200x) or an inversion microscope (6Ox-2OOx) as well as a hand drawn microcapillary, which is equipped with a silicone tube

or for a specific micro-selection system as described by Koop and Schweiger, 1985; Spangenberg, 1986 and Schweiger et al. , 1987.

Immediately after isolation, the zygotic proembryos and embryos thus selected are transferred for subsequent microinjection into 50 μΐ to 100 μΐ droplets of culture medium (ml) without Ficoll previously applied to coverslips of 24 mm to 40 mm. These microinjection plates are described in Section [b]; C)]. Until injection, the microinjection plates are placed in a two-compartment system as described in section (c).

b) Microinjection of pHP23 plasmid DNA into maize embryos

A) Apparatus

Microinjection of the maize embryos and embryos is performed under microscopic observation on an inverted microscope or a stereomicroscope with a combination of incident and transmitted light. The enlargement depends on the size of the embryos and varies between 10-800 times magnification.

The manipulation of the zygotic embryos or embryos is carried out with the help of two micromanipulators, which can be attached to the microscope stage (motor-driven manipulators) or laterally next to the microscope stand (mechanical manipulators). Both micromanipulators are equipped with capillary holders.

As a microinjection capillary microelectrode capillaries are used with molten glass filaments of borosilicate, which have an outer diameter of 1.00 mm to 1.50 mm. For fixation of the embryos during the injection process, specially prepared holding capillaries are used for this purpose. These are glass tube sections from Sodaglass with an outside diameter of 1.00 mm to 1.50 mm. Both capillary types are drawn out on a commercially available capillary pulling device, so that suitable injection capillaries (tip diameter about <1 μπι) and Haltekapillaren arise.

To complete the holding capillaries, use a micro-forge on which the solid holding capillary is broken and polished vertically at the required opening diameter of 0.02 mm to 1.00 mm (rounded end with small opening).

The injection of the DNA solution is carried out via a microinjector. It is a pneumatically operated pressure device that allows a very finely dosed injection into the cells of the embryos. The injection volume varies depending on the cell size between 1 pl and 100 pl. Suitable microinjectors can be obtained, for example, from Eppendorf, FRG or Bachhofer, FRG.

B) DNA solution

The microinjected DNA solution (0.01 μg / μl to 2.0 μg / μl) is a spirally-spun or linearized pHP23 DNA or a mixture of spirally-spun and linearized pHP23 DNA in a Tris-HCl buffer consisting of 50 mM Tris-HCl, pH 7-7.8 and 50 mM NaCl.

C) microinjection

The microinjection of the previously described DNA-containing solution into the cells of maize embryos or proembryons takes place analogously to that described in Neuhaus et al. , 1984; 1986 described method. The areas preferred for microinjection are marked by arrows in FIG.

First a cover glass (24x40 mm) is covered half-way with another cover glass (5 χ 30 mm to 35 mm) or a transparent Tesafilmstreifen of 1-4 superimposed layers of the same size on the long side, so that an edge is formed. On the cover glass prepared in this way, the embryos are transferred into 10 .mu.l to 200 .mu.M media drops Ml culture medium without Ficoll. With the help of the holding capillary small embryos are held during the injection, larger embryos are fixed with the help of the holding capillary and the cover glass edge and then injected. Each embryo is injected 4x to 60x depending on the number of cells and rotated between each injection, so that

if possible all cells are hit. Subsequently, the injected zygotic embryos are further cultured in the appropriate medium.

c) Microcultivation of microinjected proembryos and embryos of

Immediately after injection, the embryos are transferred to the wells of a polycarbonate microcultivation plate (Spangenberg, 1986, Spangenberg et al., 1986) with a hand drawn microcapillary connected to a silicone tube or with the bent tip of a needle, respectively 0.5 μΐ to 2 μΐ culture medium (ml) containing Ficoll. Alternatively, about 1 μΐ of an Ml medium solidified with low-melting agarose (eg Sea Plaque®) can be introduced into each well and the proembryos or, respectively,. Embryos are cultured in about 1 μM of a liquid Ml medium layered over the solidified medium.

As a rule, the microinjected embryos are individually microcultivated. However, up to 5 embryos may also be placed together in one well of the polycarbonate microculture plate.

The microcultured embryos loaded microculture plates are placed in a two compartment system (Falcon organ tissue culture dish, manufactured by Falcon, USA), which acts as a humid chamber and usually in the outer compartment, from 2 ml to 4 ml of a 0 , 2 M mannitol solution (Koop and Schweiger, 1985; Spangenberg et al., 1986) contains water or Ml culture medium.

Of course, any other two-compartment system suitable for embryo culture may also be used.

Every 2 to 3 days, the injected embryos are transferred to fresh culture medium (M1) with Ficoll, depending on their stage of development, whether in the same volume or in a larger volume of 4 μΐ to 10 μΐ on Terasaki plates (microwave plate; 60 χ 10 μΐ , Nunc, Roskilde, Denmark).

The Petri dishes (two-compartment system and Terasakiplatten) are then sealed with Parafilm and incubated at a temperature of 25 ° to 28 ⁰ C in the dark or a light (dim light, about 500 lux) / dark rhythm of 16/8 hours ,

d) Cultivation of the embryos and regeneration of whole maize plants After this time (10 to 30 days after injection) the germinated plantlets (length 1 cm to 3 cm), which consist of 10% to 60 % of the Proembryonen (see stages 17 to 24 of Fig. 1) and more than 90% of the injected embryos (see stages 17 to 24 of Fig. 1), transferred to larger culture vessels (eg 330 ml culture cans 68x110 mm, Greiner, FRG), the 30 ml to 50 ml of solid culture medium (M2) or Gelrite-solidified MS medium without added hormone. The corn plants are cultivated at 25 ° to 28 ° C and a light / dark rhythm of 16/8 hours (4000 lux, cold white light) until rooting begins and the plantlets have reached a size of 5 cm to 15 cm. This is usually done after 15 to 20 days, after which the plants are brought into the greenhouse, transferred to soil and cultivated further as normal corn seedlings to the maturity of the plants.

Table 1: Culture medium (ml) for the cultivation of zygotic maize

embryos Concentration (mg / 1) Culture medium Ml 1650 component 1900 NH ₁₁ NO ₃ 440 KNO ₃ 370 CaCl ₂ .2H ₂ O 170 MgSO 2 .2H ₂ O 40 NH ₂ PO ₁ * 6.2 PeNa ₂ -EDTA 22.3 H ₃ BO ₃ 8.6 MnSOu'4 H ₂ O 0.83 ZnSOu-4 H ₂ O 0.25 KI 0,025 Na ₂ MoO _2, -2H ₂ O 1 CuSOu-5 H ₂ O 750 BAP ¹ 100 glutamine 0.4 Myo-inositol 60000 Thiamine HCl 200000 sucrose 6000 Ficoll 400® ² agarose pH 5.6

BAP Benzylaminopurine

Ficoll 400® (Pharmacia: Synthetic polymer of a co-polymerisation of sucrose and epichlorohydrin with a molecular weight of 400,000.

Table 2: Culture medium (M2) for the cultivation of zygotic maize embryos

Culture medium M2 Concentration (mg / 1) component 300 KH ₂ POu 1000 KNO ₃ 1000 NH ₃ NO ₃ 500 Ca (NO ₃ ) u'4H ₂ O 72 MgSOu'7 H ₂ O 65 KCl 5.0 MnSOu-H ₂ O 0.25 Na ₂ MoOu * 2H ₂ O 0.17 ZnSOu-H ₂ O 0.16 H ₃ BO ₃ 0.08 KI 37.25 Na ₂ -EDTA 27.85 FeSOu'7 H ₂ O 5.0 niacin 5.0 Pyridoxine HCl 1.0 Thiamine HCl 100 inositol 2.0 glycine 0.5 IAA ³ 20000 sucrose 8000 agar 500 activated carbon

pH 5.9

IAA indole acetic acid

Example 5: Isolation and injection of zygotic rapeseed embryos

a) Isolation and selection of oilseed rape embryos

Immature zygotic embryos are isolated from Brassica napus L. (genotype CrGC 5 "rapid cycling") and cv. Bronowski Ag903 isolated, which are grown in the greenhouse at a temperature of 16 ° C and a light / dark rhythm of 16/8 hours.

Pods 3 cm to 6 cm in length are collected for 5 to 20 days, preferably 8 to 15 days after pollination of the rape plants, surface-sterilized for 30 minutes in 1% calcium hypochlorite solution and washed three times in sterile distilled water. Subsequently, 5 to 8 pods are transferred together into a Petri dish (10 cm diameter) with 4 ml of distilled water. The Petri dishes are sealed with Parafilm and stored at 4 ° C or at room temperature (25 ° to 28 ° C) until isolation of the immature zygotic embryos. With the help of fine tweezers and scalpels, the pods are opened under optical control at a magnification of 10 to 20 times under a stereomicroscope. 8 to 15 ovules are taken from the pods, transferred into 1 ml to 3 ml of hormone-free culture medium (R3) in Petri dishes of 6 cm in diameter and stored at 4 ° C or 25 ⁰ C until the isolation of the zygotic embryos. Then, with the help of two dissecting needles, the ovules are opened and the zygotic embryos in developmental stages and sizes, the early globular proembryonic stages (see Fig. 2, Phase 0, P, Q) to early or late cotyledonous stages approximately 2000-row ( See Figure 2, phase R, S, T), isolated under microscopic control. Immediately after isolation, the embryos are read out analogously to the method described under point (la), in which case hormone-free medium (R3) is used instead of the medium (III) without Ficoll mentioned under point la).

b) Microinjection of pHP2 3 DNA into oilseed rape embryos

The introduction of pHP23 DNA into oilseed rape embryos via microinjection is carried out in a manner analogous to that described under point 4b).

The areas preferred for microinjection are marked with arrows in FIG.

c) Microcultivation of microinjected oilseed rape embryos

The microculturing of the microinjected oilseed rape embryos is carried out analogously to the method described under item 4c) for the cultivation of maize embryos. In this case, however, the wells of the polycarbonate microculture plates are filled with different culture media depending on the size and developmental stage of the microinjected oilseed rape embryos. Young globular embryos to early heart-shaped embryos are transferred to 0.5 μΐ to 1.0 μΐ of liquid endosperm, which is isolated by aspirating the contents of an unripe rape seed plant which is in a similar developmental stage.

Rapeseed embryos in the late heart-to-early torpedo stage are microcultured in 0.5 μΐ to 1.0 μΐ mixture of liquid rapeseed endosperm (obtained above) and synthetic culture medium (R3) (usually a 1: 1 mixture).

Microinjected oilseed rape embryos in the late torpedo stage to early cotyledonar stage are microcultured directly on 0.5 μΐ to 1.0 μΐ of synthetic culture medium (R3).

The microinjected embryos are cultured at a density of 1 to 5 embryos / well on the microculture plates and incubated in the humid chamber two-compartment system (as described under 4c) at 25 ° C in the dark or in the light (dim light, about 500 lux) / dark rhythm of 16/8 hours incubated.

d) Cultivation of microinjected oilseed rape embryos and regeneration of whole plants

Every 2nd and 4th day, starting 1 to 4 days after microinjection and depending on the size and stage of development of the microinjected oilseed rape embryos, they are tested on wells of poly-

carbonate microculture plates with 0.5 μΐ to 2.0 μΐ liquid culture medium (ИЗ) or transferred in 4 μΐ to 10 μΐ of the culture medium (R4) in wells of Terasaki plates.

The small volume range (0.5 μΐ to 2.0 μΐ) is used for young oilseed rape embryos that have reached stages of development between the early heart-shaped and early torpedo stages. In contrast, the large volume range (4 μΐ to 10 μΐ) is used for microinjected embryos in the development stage between the late torpedo stage and the late cotyledonar stage.

This culture phase takes place at a temperature of 25 ° to 28 ° C and in a light / dark rhythm of 16/8 hours (4000 lux, cold white light).

The regeneration of whole plants from the micro-injected rapeseed embryos, as well as the induction of secondary embryos from the stem of the primary regenerates is done by transferring the microinjected oilseed rape embryos to solid culture medium (R5). Normally, 5 embryos are always transferred together to 7 ml culture medium (R5) in 6 cm diameter petri dishes. The Petri dishes are then sealed with parafilm and incubated at 25 ° to 28 ° C in a light / dark rhythm of 16/8 hours (4000 lux, cold white light) for 1 to 3 weeks to plant regeneration or to induce secondary embryogenesis.

After regeneration of green plantlets - by direct germination of the microinjected primary embryos or by differentiation of the induced secondary embryoids - these are transferred for further development in 50 ml plastic containers with 25 ml culture medium (R6) and at a temperature of 23 ° C to 28 ⁰ C and a lighting period of 10 to 16 hours (4000 lux, cold white light) cultivated until they have reached a size of 1 cm to 3 cm.

After the shoot and roots are formed, the rape seedlings are transferred into 330 ml culture cans containing 30 ml to 50 ml solid culture medium (R6) until they reach a size of 5-10 cm. This usually happens after another 1 to 3 weeks, after which the plants are brought into the greenhouse and continue to be grown like normal rape seedlings to the maturity of the plants.

Table 3: Culture media (R3 ', Ri,) for the cultivation of zygotic

Canola embryos component concentration (Mg / 1) - 100 R ₃ Ru 120000 Ca (NO ₃ ) ₂ '4 H ₂ O 500 500 0.2 KNO ₃ 125 125 0.3 MgSOu-7 H ₂ O 125 125 0.5 KH ₂ PO ₁ , 125 125 pH 6.0 MnSO 4, · 4H ₂ O 25 25 H3BO3 10 10 ZnSO 4, · 4H ₂ O 10 10 Na ₂ MoOi ₁ ^ H ₂ O. 0.25 0.25 CuSO 4, 5H ₂ O 0,025 0,025 CoCl ₂ -6H ₂ O 0,025 0,025 Na ₂ -EDTA 37.3 37.3 FeSOi ₄ -? H ₂ O 27.8 27.8 glycine 2 2 niacin 5 5 Pyridoxine HCl 0.5 0.5 Thiamine HCl 0.5 0.5 folic acid 0.5 0.5 biotin 0.05 0.05 inositol 100 100 glutamine 1000 1000 asparagine 1000 serine - sucrose 20000 BAP 0.1 IAA ⁵ 0.5 NAA ⁶ - pH 5.8

BAP: benzylaminopurine IAA: indole acetic acid NAA: naphthyl-1-acetic acid

Table 4: Culture media (R ₅ ;

Canola embryos component concentration - (Mg / 1) - 100 - 7000 Rs pH 5.8 Rs - 1000 KNO ₃ 2500 2500 - pH 5.8 CaCl ₂ -2H ₂ O 150 150 MgSO 4 H ₂ O 250 250 (NHOS (H 134 134 NaH ₂ POu-H ₂ O 150 150 KI 0.75 0.75 H ₃ BO ₃ 3.0 3.0 MnSO ₄ -H ₂ O 10.0 25 ZnS0i »-4 H ₂ O 2.0 2.0 Na ₂ MoO ₁ , -2H ₂ O 0.25 0.25 CuSOu-5 H ₂ O 0,025 0,025 CoCl ₂ -6H ₂ O 0,025 0,025 Na ₂ -EDTA 37.3 37.3 FeS0i, -7 H ₂ O 27.8 27.8 inositol 100 100 niacin 1.0 Pyridoxine HCl 1.0 Thiamine HCl 10.0 sucrose 30000 BAP 0.05 Difco-Bacto® agar 8000 activated carbon

for the cultivation of zygotic

BAP: Benzylaminopurine

Example ба: Isolation and injection of zygotic wheat proembryos and embryos followed by direct germination

a) Isolation and selection of zygotic wheat proembryos and embryos

The immature zygotic embryos are isolated 4 to 12 days after pollination from the immature ovules of wheat.

The preparation and selection of the zygotic embryos is carried out in a manner analogous to that described under point 4a) for maize or for the method described under point 5a) for oilseed rape.

After surface sterilization of the ovules and introduction into distilled water, the zygotic embryos are isolated using a stereomicroscope, fine scalpels and fine forceps and then selected for subsequent microinjection. Most suitable for microinjection are embryos located between the 16 and 500 cell stages.

The isolated and selected zygotic embryos are first transferred to square plastic dishes, each having 4 wells (Multidish 4, Nuncton Delta SI, Nunc, Denmark), in 0.5 ml of culture medium WI (Table 5). There they are usually left in the dark for one day at a temperature of 4 ° C. The preferred culture density is 8 to 10 embryos per well.

b) Microinjection of pHP23 DNA into Wheat Proembryos and Embryos The introduction of pHP23 DNA into wheat embryos via microinjection is carried out in a manner analogous to that described under point 4b). The injection of the solution containing DNA preferably takes place in the region of the embryonic axis, a region of meristemoid cells from which the plumula and the radicle later emerge.

c) Microcultivation of microinjected wheat proembryos and embryos After microinjection, the injected embryos are further cultured for another day under the same conditions and on the same culture plates.

One day after injection, the embryos are cultured at 25 ° C under diffuse light for a further 3 to 5 days. After a total culture time of 5 to 7 days in liquid culture medium, the embryos are transferred to Terasaki plates containing 2 layers of different media.

The first layer consists of 10 μΐ medium W2, which is solidified with Sea Plaque® agarose, the second layer is 10 μΐ liquid medium Wl, which is covered over the solid medium. Of course, for the solidification of medium W2, other, suitable for the cultivation of plant embryos, solidification media can be used, preferably agarose from 'Low melting type ¹ (LMP agarose). Cultivation also takes place in this case under the same conditions, ie at a temperature of 25 ° C and under diffuse light.

After a cultivation period of 7 to 15 days, depending on the size of the embryos, they begin to germinate. After 10 to 14 days, the microinjected embryos are therefore transferred in multiwell culture dishes to 1 ml to 5 ml of culture medium W2, which is solidified by Sea Plaque® agarose, and at a temperature of 25 ° C in a light / dark rhythm of 16 h / 8 h further cultivated for differentiation.

d) Cultivation of embryos and regeneration of whole wheat plants . A week after the microinjection, the germinated plantlets are transferred to larger culture vessels. These are culture cans with a volume of 60 ml containing 15 ml of the culture medium W3, these are later exchanged for larger culture cans with a volume of 330 ml containing 30 ml to 50 ml of W4 medium.

When the plantlets have reached a certain size, they are transferred to soil and taken to the greenhouse, where they are treated like normal wheat seedlings.

Table 5a Culture media (W1, W2, W3, W4) for the cultivation of zygotic wheat embryos

Wl [mg / 1] _ W2 [mg / 1] W ₃ [mg / 1] - W4 [mg / 1] - KNO ₃ 1900 1900 1900 100 1900 100 NHi ₄ NO ₃ 165 165 165 750 165 7 50 CaCl ₂ -2H ₂ O 440 440 440 440 MgS0i »« 7 H ₂ O 370 370 370 7000 370 7000 KH ₂ POu 170 170 170 170 FeNa ₂ -EDTA 40 40 40 40 MnSOu-4 H ₂ O 22.3 22.3 22.3 22.3 H ₃ BO ₃ 6.2 6.2 6.2 6.2 ZnSOu-4 H ₂ O 8.6 8.6 8.6 8.6 KI 0.83 0.83 0.83 0.83 Na ₂ MoO ₁ , ^ H ₂ O. 0.25 0.25 0.25 0.25 CuSO ₄ -SH ₂ O 0,025 0,025 0,025 0,025 kinetin - - 1.0 2.0 BAP 0.5 0.5 - - NAA ⁹ - - 0.5 1.0 1 O IAA 0.1 0.2 - - Thiamine, HCl 1.0 1.0 0.4 0.4 sucrose 20000 20000 30000 20000 glucose 5000 5000 Myo-inositol 100 100 glutamine 500 500 Sea Plaque® , 11 agarose 6000

BAP: Benzylaminopurine

NAA: naphthyl-1-acetic acid

IAA: indole acetic acid

Sea Plaque® Agarose: FMC Corporation, Marine Colloids Div. Rockland,

Example 6b: Isolation and Injection of Wheat Pro-ZEmbryonen with subsequent induction of adventitious embryoids

a) Isolation and selection of zygotic pro-embryos of wheat

The immature zygotic embryos are isolated 4 to 12 days after pollination from the immature ovules of wheat.

The preparation and selection of the zygotic embryos is carried out in a manner analogous to that described under point 4a) for maize or for the method described under point 5a) for rapeseed.

After surface sterilization of the ovules (15 minutes in 1.8% (v / v) sodium hypochlorite solution followed by a triple wash with sterile distilled water) and placing in distilled water, the zygotic pro / embryos are incubated under microscopic control ( 10 χ - 50 χ magnification) with the help of a scalpel and dissecting needles. The large and developmental stage of the isolated pro-embryos ranges between approximately 20-cell globular proembryos with suspensor and embryos with skutellum and coleoptyl.

Immediately after isolation, the selected zygotic pro-embryos are then transferred for subsequent microinjection into 50 μΐ to 100 μΐ droplets of culture medium WZ 1, which were previously applied to coverslips of 24 × 40 mm. These microinjection plates are described in Section [4b) C)]. Until microinjection, the microinjection plates are placed in a two-compartment system as described in Section 4c).

b) Microinjection of pHP23 DNA into wheat zygotic pro-embryos The introduction of pHP23 DNA into proEM embryos of wheat via microinjection is carried out in exactly the same way as described in point 4b).

c) Microculturing of inocropri-pro-embryos of wheat Immediately following injection, the embryos are transferred to the wells of a polycarbonate microcultivation plate with a hand drawn microcapillary connected to a silicone tube or with the bent tip of a needle (Spangenberg, 1986 , et al., 1986), each containing 0.5 μΐ to 1 μΐ of culture medium WZ 2. In addition, this solid culture medium WZ 2 with 0.5 μΐ to 1 μΐ liquid endosperm - by sucking the contents of an immature wheat ovule similar developmental stage - overlaid.

As a rule, the microinjected pro / embryos are individually microcultivated. However, up to 10 pro / embryos may also be transferred together into one well of the polycarbonate microculture plate. The microcultured embryos loaded microculture plates are placed in a two compartment system ("Falcon organ tissue culture dish" No. 3037, Fa. Falcon, USA), which acts as a humidity chamber and usually in the outer compartment 2 ml to 3 ml a 0.2 M mannitol solution (Koop and Schweiger, 1985, Spangenberg, 1986, e_t al 1986), this sealed with parafilm and incubated at a temperature of 25-28 ° C in the dark.

d) Cultivation of the microinjected wheat embryos, induction of secondary embryoids and regeneration of whole plants Every 2 to 3 days - starting 2 days after the injection of the wheat pro- / embryos - 0.5 μΐ to 2 μΐ of culture medium WZ 3 is added to the individual cultures. - Loaded with injected Pro-ZEmbryonen - wells of the microculture plates until the pro- / embryos have reached a size of about 2-5 mm.

Subsequently and according to their stage of development, the injected embryos are transferred individually or up to 5 embryos together to 1 ml of culture medium WZ 4 in wells of square plastic dishes (4 well multidish, Nunc, Denmark).

After a culture period of 4 to 6 weeks on culture medium WZ 4, the induction of callus with potential for secondary embryogenesis takes place. These embryogenic calli are split and transferred to 10 ml of WZ 5 culture medium in Sterilin 125 AP plastic cans (Sterilin Ltd., Feltham, England) for differentiation of sprouts.

The further development of these shoots and their rooting is carried out after repeated transfer to 30 ml to 80 ml culture medium WZ 5 in larger culture vessels (330 ml culture doses, 68x110 mm, Fa. Greiner, FRG). When the differentiated plantlets reach a certain size (5 cm to 10 cm) and have developed a sufficient root system, they are transferred to soil and taken to the greenhouse, where they are treated like normal wheat seedlings.

Table 5b Culture media (W1, W2, W3, W4) for the cultivation of zygotic wheat embryos

Wl [mg / 1] W2 [mg / 1] W3 [mg / 1] W4 [mg / 1] NH ₁₄ NO ₃ 1650 1650 1650 3300 KNO ₃ 1900 1900 1900 3800 CaCl ₂ -2H ₂ O 440 440 440 880 MgSO ₁₁ -? H ₂ O 370 370 370 740 KH ₂ PO ₁ , 170 170 170 340 KI 0.83 0.83 0.83 1.66 H ₃ BO ₃ 6.2 6.2 6.2 12.40 MnSOi ₁ -4 H ₂ O 22.3 22.3 22.3 44,60 ZnSCH · 4H ₂ O 8.6 8.6 8.6 17,20 Na ₂ Mo0i4'2 H ₂ O 0.25 0.25 0.25 0.5 CuSO 4 .5H ₂ O 0,025 0,025 0,025 0.5 CoCl ₂ '6 H ₂ O 0,025 0,025 0,025 0.5 Na ₂ -EDTA 37.3 37.3 37.3 74.6 FeS0i, -7 H ₂ O 27.8 27.8 27.8 55.6 nicotinic acid 0.5 0.5 0.5 0.5 Pyridoxine HCl 0.5 0.5 0.5 0.5 Thiamine HCl 0.1 0.1 0.1 0.1 glycine 2.0 2.0 2.0 2.0 cefotaxime - - - 0.1 LMP agarose - 6000 - 6000 sucrose 30000 30000 30000 20000 inositol 200 200 200 200 casein hydrolyzate 200 200 200 200 glutamine - - - 500 2,4-D ¹² 1.0 1.0 - 2.0 kinetin 1.0 1.0 - - ABA ¹³ GA ₃ ¹⁴ 0.5 0.1 0.5 0.1 - -

, 2,4-D: 2,4-Dichlorophenoxyacetic acid, ABA Abszisinsäure GA ₃ Giberellinsäure

Table 5c: culture medium WZ 5

WZ 5 [mg / 1] KH ₂ PO ₁ , 300 KNO ₃ 1000 NHi ₁ NO ₃ 1000 Са (Ж) з) і * «4 H ₂ O 500 MgS0i "-7 H ₂ O 72 KCl 65 MnSO ₁₁ H ₂ O 5.0 ZnSO ₁₁ H ₂ O 0.17 H ₃ BO ₃ 0.16 KI 0.08 Na ₂ -EDTA 37.25 FeSOu-7 H ₂ O 27.85 nicotinic acid 5.0 Pyridoxine HCl 5.0 Thiamine HCl 1.0 inositol 100 Glycine - 15 IAA 2,0 0,5 sucrose 20000 agar 8000 activated carbon 500 pH 5.9 -

IAA indole acetic acid

Example 7: Isolation and Injection of Zygotic Rice Embryos

a) Isolation and selection of zygotic rice pro-embryos and embryos The isolation of immature zygotic proembryos or embryos takes place from the immature ovules of rice until 12 days after pollination.

The preparation and selection of the zygotic embryos or embryos is carried out in a manner analogous to that described under Example 4a) for maize or for the method described under Example 5a) for rapeseed.

After surface sterilization of the ovules and introduction into distilled water, the zygotic embryos or embryos are isolated with the aid of a stereomicroscope (Zeiss), fine scalpels and fine forceps and then selected for subsequent microinjection. Best suited for microinjection are proembryos or embryos located between phase 20 and phase 25 (see Fig. 1).

The isolated and selected zygotic proembryos or embryos are first in square plastic dishes, each having 4 wells (Multidish 4, Nuncton Delta SI, Fa. Nunc, Denmark), transferred to 0.5 ml of culture medium RSI (Table 6). The preferred culture density is 4 to 6 proembryos or embryos per well.

b) Microinjection of pHP 23 DNA into Rice Proembryos or Embryos The introduction of pHP 23 DNA into rice proembryos or embryos via microinjection is carried out in exactly the same way as described in Example 4b). The injection of the DNA-containing solution is preferably carried out in the embryonic cells designated by an arrow (cf Fig. I).

c) Microcultivation of microinjected rice embryos

After the microinjection, the injected proembryos or embryos are further cultured for another day under the same conditions and on the same culture plates.

2 days after the injection, the pro / embryos are cultured at 25 ° C and under diffused light for a further 5 to 7 days. After a total culture time of 7 to 10 days in liquid culture medium, the embryos are transferred to Terasaki plates containing 2 layers of different media. The first layer consists of 5 μΐ medium RS2 solidified with a low-melting agarose, such as Sea Plaque® agarose (0.6% v / w); the second layer forms 5 μΐ liquid medium RS1, which is covered over the solid medium. Of course, for the solidification of medium RS2 other, suitable for the cultivation of plant embryos, solidification media can be used, preferably agarose of the 'Low Melting Type ¹ (LMP agarose). Cultivation also takes place in this case under the same conditions, ie at a temperature of 25 C and under diffused light.

After a cultivation period of 7 to 15 days, depending on the size of the pro / embryos used, they begin to germinate. After 15 days, the microinjected pro / embryos are therefore transferred in multiwell culture dishes to 1 to 2 ml culture medium RS2, which is e.g. is solidified by Sea Plaque® agarose and further cultivated at a temperature of 25 ° C in a light / dark rhythm of 16/8 hours for differentiation.

d) Cultivation of embryos and regeneration of whole rice plants . 4-5 weeks after the microinjection, the sprouted plantlets are transferred to larger culture vessels. These are commercial culture cans with a volume of 60 ml containing 15 ml of the culture medium RS3. These are later exchanged for larger culture cans with a volume of 330 ml containing 30 to 40 ml of RS4 medium.

When the plantlets reach a certain size, they are transferred to soil and taken to the greenhouse, where they are treated like normal rice seedlings.

Table б Media for the cultivation of zygotic rice pro-embryos

RS1 [mg / 1] - RS2 [mg / 1] - 160 RS 3 [mg / 1] - RS 4 [mg / 1] - - 160 KNO ₃ 1900 1900 Sea plaque® 0.6% 1900 100 1900 100 Agar 0.75% NH ₁ ^ NO ₃ 165 1650 1650 - 1650 CaCl ₂ '2 H ₂ O 440 440 440 160 440 MgSO 4 -7H ₂ O 370 370 370 Agar 0.75% 370 KH ₂ PO ^ 170 170 170 170 FeNa ₂ -EDTA 40 40 40 40 MnSOit'4 H ₂ O 22.3 22.3 22.3 22.3 H ₃ BO ₃ 6.2 6.2 6.2 6.2 ZnSO 4, · 4H ₂ O 8.6 8.6 8.6 8.6 KI 0.83 0.83 0.83 0.83 Na ₂ Mo0i, «2 H ₂ O 0.25 0.25 0.25 0.25 CuSO 4 .5H ₂ O 0,025 0,025 0,025 0,025 kinetin - - 1.0 1.0 BAP ¹⁶ 0.5 0.5 - - IAA ¹⁷ 0.2 0.2 0.5 0.5 NAA ¹⁸ - - 0.5 0.5 Thiamine, HCl 1.0 0.4 0.4 0.4 sucrose 20000 20000 25000 20000 glucose 5000 5000 Myo-inositol 100 100 Casein hydrolyzate 300 glutamine 500 Agarose / Agar

._ BAP: Benzylaminopurine ._ IAA: Indole Acetic Acid

NAA: naphthyl-1-acetic acid

Example 8: Isolation and Injection of Zygotic Barley Proembryos and Embryos

a) Isolation and selection of zygotic barley proembryos and embryos

Immature zygotic proembryos and embryos are found in Hordeum vulgare cv. Igri, isolated 6 to 14 days after pollination.

The ovules obtained from the barley ears are sterilized for 15 minutes in a 1.8% (v / v) sodium hypochlorite solution surface and then washed three times in sterile distilled water.

The preparation and selection of the zygotic Рго- / ЕтЪгуопеп is carried out in a manner analogous to that described under point 4a) for corn or under point 5a) for oilseed rape.

After surface sterilization of the ovules and introduction into distilled water, the zygotic pro / embryos are isolated under microscopic control (10χ-50χ magnification) with the aid of a scalpel and fine forceps.

The major and developmental stages of isolated pro / embryos are in an area that includes early globular proembryos with suspensor and immature embryos with skutellum and coleoptyl.

The isolated and selected zygotic embryos are first transferred to square plastic dishes, each having 4 wells (Multidish 4, Nunclon Delta SI, Nunc, Denmark), in 0.5 ml of culture medium Gl, at a preferred density of 8 to 10 Pro -ZEmbryonen per well and stored there until microinjection at 25 ° C.

b) Microinjection of pHP23 DNA into Peristyle Zygotic Pro-Embryos The introduction of pHP23 DNA into barley-pro-embryos via microinjection is carried out in exactly the same way as described in point 4b). The injection of the solution containing DNA preferably takes place in the region of the embryonic axis, a region of meristemoid cells from which the plumula and the radicle later emerge. The preferred injection site is indicated by an arrow in Fig. 1.

c) Microcultivation of microinjected barley pro-embryos

The microculturing of the microinjected Рго- / ЕтЪгуопеп of barley is carried out analogously to the method described in section 4c) for the cultivation of maize embryos.

In this case, however, the wells of the polycarbonate microculture plates are filled with 0.5 μΐ to 1.0 μΐ liquid culture medium Gl. Subsequently, the microinjected pro-embryos are transferred to the microculture plates at a density of 1 to 20 pro / embryos per well, and the well is filled up with 0.5 .mu.l to 1 .mu.l of liquid culture medium Gl.

d) Cultivation of barley pro-embryos and regeneration of whole plants

The microinjected pro-embryos are regularly transferred to fresh culture medium G1 every 3 to 12 days and transferred to new microculture chambers, the wells containing 1 .mu.l to 10 .mu.l of culture medium G1, depending on the size and number of the microcultured proEM embryos. When the pro-embryos reach a size of 2 mm to 5 mm, they are transferred to medium G2 at a density of 16 embryos per 10 ml of culture medium and Petri dish. These are sealed with parafilm and then incubated in the dark for a period of 5 days at a temperature of 25 ° C. Subsequently, the cultures are kept at a temperature of 23 ° C and a lighting period of 10 hours day (4000 lux, cold white light), until the green.

After the regeneration of green plantlets they are transferred for further development in commercial 60 ml plastic containers with 25 ml medium G3 and at a temperature of 23 ° C to 26 ° C and a lighting time of 10 to 16 hours (4000 lux, cold white light) cultivated until they reach a size of 1 cm to 3 cm. Root formation is achieved by re-transferring the regenerated plantlets into culture medium G3. After the formation of several shoots and roots, the winter barley can be vernalisiert by transferring the plant pots in 240 ml containers and at 4 ° C and a lighting period of 10 to 16 hours at 2000 lux (Phillips, warm white light) for a period of 6 to Cultivated for 8 weeks.

Table 7 Media for the cultivation of zygotic barley pro-embryos

component Gl [mg / 1] - G2 [mg / 1] G ₃ [mg / 1] NH ₁₁ NO ₃ 165 750 165 165 KNO ₃ 1900 100 1900 1900 CaCl ₂ -2H ₂ O 440 0.4 440 440 MgSO 4 H ₂ O 370 20000 370 370 NH ₂ PO ₁ , 170 - 170 170 FeNa ₂ -EDTA 40 5.6 40 40 H ₃ BO ₃ 6.2 6.2 6.2 MnSO 4, · 4H ₂ O 22.3 22.3 22.3 ZnSO 4, · 4H ₂ O 8.6 8.6 8.6 KI 0.83 0.83 0.83 Na ₂ MoOu · 2H ₂ O 0.25 0.25 0.25 CuSOu'5 H ₂ O 0,025 0,025 0,025 BAP ¹⁹ 0.4 - glutamine 7 50 7 50 Myo-inositol 100 100 Thiamine HCl 0.4 0.4 sucrose 20000 20000 "Sea Plaque" ® agarose 6000 6000 pH 5.6 5.6

BAP Benzylaminopurine

Example 9 : Isolation and Injection of Zygotic Cotton Proembryos and Embryos

a) Isolation and selection of pro- / embryos from cotton

Young zygotic proembryos and embryos are isolated from immature 1 cm to 2 cm fruits of Gossypium hirsutum about 4 to 20 days after pollination.

The fruit stands are first pre-cleaned under running tap water, possibly with the addition of a commercially available kitchen cleaner (Lux, Pril, etc.). After thorough cleaning, the fruit stand is sprayed with 70% ethanol and then washed with distilled water. The unripe fruits are now sterilized with a 3.6% sodium hypochlorite solution containing 0.1% Tween 20 as an additive for 45 minutes. Subsequently, the fruits are sufficiently washed with sterile water and stored in a moist Petri dish (room temperature). With the help of fine tweezers and scalpels, the fruits are opened under the stereomicroscope with a 10 to 20-fold magnification. From the fruits, the embryo sacs are removed and transferred to square plastic dishes, each with 4 wells (Multi dish 4, Nunclon Delta SI, Fa. Nunc, Denmark) in about 0.5 ml of hormone-free medium (Cl). Then, with the help of two dissecting needles from the embryo sacs, the embryos and the embryos in different sizes, ranging from globular 20-cell stages (see Fig. 2, phase 0, P, Q) to early to late cotyledonous stages (2000) see Fig. 2, phase R, S, T), isolated under microscopic observation. After isolation, the embryos are read out analogously to the method described under Example 4a), in which case hormone-free medium (Cl) is used.

b) Microinjection of pHP23 DNA into Cotton Pro / Embryos The introduction of pHP23 into these pro / embryos via microinjection is performed in a manner analogous to that described in Example 4b). The preferred injection site is marked in Fig. 2 by an arrow.

Microcultivation of Microinjected Cotton Pro TEM Embryos The cultivation of the microinjected cotton Pro TEM embryos is carried out analogously to the method described under Example 4c) for the cultivation of maize embryos. The first microculture phase in Cl medium takes about 7 days. Subsequently, the pro-embryos are cultured for 4 to 6 weeks in C2 medium in suitable culture vessels (see Example 4c and 4d). The subsequent regeneration to small plantlets takes place in C3 medium. When these plantlets have reached a size of about 6 to 10 cm, they can be transferred to the greenhouse, where they can be cultivated to maturity like normal cotton plants.

Table 7 Media for the cultivation of zygotic cotton embryos

component Cl [mg / 1] C2 [mg / 1] C3 [mg / 1] NH ^ NO ₃ 240.12 1650 1650 KNO ₃ 505.56 1900 1900 CaCl ₂ 2 2 H ₂ O 176.42 440 440 MgSO 4 .7H ₂ O 492.96 370 370 KH ₂ POI, 27.20 170 170 H ₃ BO ₃ 1.85 6.3 6.3 MnS0i, '4 H ₂ O 5.07 22.3 22.3 ZnS0i, «7 H ₂ O 2.59 8.6 8.6 KI 0.25 0.83 0.83 Na ₂ Mo0i, ^o 2 H ₂ O. 0.22 0.25 0.25 CuS0i + "5 H ₂ O 0.0075 0,025 0,025 CoCl ₂ -6H ₂ O 0.0071 0,025 0,025 Na ₂ FeEDTA 20 40 40 inositol 100 100 100 nicotinic acid 0.5 1.0 1.0 Pyridoxine HCl 0.5 1.0 1.0 Thiamine HCl 10 10 10 glycine 2.0 - - MgCl ₂ ABA ²⁰ GA ²¹ 7 50 0.1 0.1 0.1 glucose 30000 30000 - sucrose - - 20000 Gelrite® _ _ 1600

ABA abscisic acid GA gibberellic acid

Example 10: Isolation and Injection of Zygotic Soybean Proembryos and Embryos

a) Isolation and selection of Рго- / ЕілЪгуопеп from soybeans

Young zygotic proembryos and embryos are isolated from 10 mm to 50 mm long pods (about 3 to 20 days after pollination) of Glycine max cv Marple Arrow. The plants are grown in the greenhouse at a temperature of 19 ° C to 21 ° C and a light / dark rhythm of 16/8 hours.

Prior to isolation, the pods are sterilized by treatment with a 1.5% calcium hypochlorite solution containing 0.1% Tween-20 for 10 minutes followed by sufficient rinsing with sterile distilled water. The thus treated pods are stored in humidified Petri dishes at about 4 ⁰ C in the dark until further treatment. With the help of fine tweezers and scalpels, the pods under the stereomicroscope are opened at a 10 to 20-fold magnification. The ovules are removed from the pods and transferred into about 1 ml of hormone-free medium (S3) in Petri dishes of 3 cm diameter. From the ovules, the embryo sacs are removed and transferred into about 1 ml of hormone-free medium (S3) in Petri dishes of 3 cm diameter. Then, with the help of two dissecting needles from the embryo sacs, the proembryos and embryos of various sizes, ranging from early globular stages of the proembryonic stage (see Fig. 2, Phases 0, P, Q) to early to late cotyledonous stages of approximately 2000 cells (cf. Fig. 2, phase R, S, T), isolated under microscopic observation. After isolation, the embryos are read out analogously to the method described under Example 4a), in which case hormone-free medium (S3) without Ficoll is used.

b) Microinjection of pHP23 DNA in soybean pro / embrvons

The introduction of pHP23 into these pro / embryos via microinjection is carried out in an exactly analogous manner to the method described under Example 4b).

c) Microcultivation of microinjected soybean pro-embryos

The cultivation of the microinjected soybean pro-ZE embryos is carried out analogously to the method described in Example 4c) for the cultivation of maize embryos. In this case, however, the wells of the polycarbonate microculture plates are filled with different amounts of culture medium, depending on the size and developmental stages of the microinjected pro-embryos. Young globular proembryons are placed on a 0.1 μΐ to 0.5 μΐ layer of Sl medium and overlaid with 0.1 μΐ to 0.5 μΐ S3 medium. In this way, 1-10 proembryos are cultured in one well of the microculture plate. Heart-shaped and later cotyledonary stages are placed on a 0.2 μΐ to 0.5 μΐ layer of medium Sl and covered with 0.2 μΐ to 0.5 μΐ S3 medium [cf. Example 7c)]. These larger stages of development are always preferably cultivated individually. The microculture plates are incubated in the two-compartment system serving as a humid chamber [as described in example 4c] at 21 ° C. in the dark or in bright (dim light about 500 lux) for a dark rhythm of 16/8 hours.

d) Cultivation of the mikroin.j ized Pro-ZEmbryonen and regeneration of whole plants

Every 2 and 4 days starting 1 to 4 days after microinjection and depending on the size and developmental stage of the microinjected embryos they are provided with new culture medium by addition of 0.2 .mu.ΐ to 0.5 .mu.M S3 medium. After about 6 to 10 days, depending on the size of the cultured embryos, they are transferred to 4 μΐ to 10 μΐ of the culture medium S2 in wells of Terrasaki plates. Here, too, the cultured pro-embryos are covered with 0.2 μΐ to 1 μΐ of S3 culture medium after transfer into the wells [cf. Example 7c)]. In order to ensure sufficient moisture, about 1 ml to 2 ml of sterile water are deposited on the edge of the wells of the Terrasaki plates. The embryos are again incubated at 25 ° C and a HellZDukelrhythmus of 16/8 hours (4000 lux, cold white light).

Every 3 to 4 days, the developing embryos are humidified by adding 0.2 μΐ to 1.0 μΐ S3 medium. After regeneration of small seedlings, they are grown in Costar plates ("Costar wells";

24 wells; 16 mm diameter; # 3524; Tissue Culture Cluster, Cambridge, Mass.) On S3 medium (with 0.8% agarose) and cultured further under the culture conditions described above. It may be necessary to re-induce root induction by adding hormones into the medium. This can be achieved, for example, by keeping the plantlets on root-inducing medium (S4) for 3 to 6 days. After forming several leaflets and roots, these plantlets are transferred in 300 ml culture cans with 20 ml to 50 ml of S3 medium containing 0.8% agarose. When these plantlets have reached a size of about 6 cm to 10 cm, they can be brought into the greenhouse, where they can be cultivated to maturity like normal soybean plants.

Table 9 Media for the cultivation of zygotic soybean embryos

sl S2 S3 S4 component [Mg / 1] [Mg / 1] [Mg / 1] [Mg / 1] NH ₁₊ NO ₃ 1650 1650 1650 - KNO ₃ 1900 1900 1900 1900 CaCl ₂ -2H ₂ O 440 440 440 440 MgSO 2 .2H ₂ O 370 370 370 370 NH ₂ POi ₁ 170 170 170 170 FeNa ₂ -EDTA 40 40 40 40 H ₃ BO ₃ 6.2 6.2 6.2 6.2 MnSO 4 -4H ₂ O 22.3 22.3 22.3 22.3 ZnSOu'4 H ₂ O 8.6 8.6 8.6 8.6 KI 0.83 0.83 0.83 0.83 Na ₂ Mo0i »-2 H ₂ O 0.25 0.25 0.25 0.25 CuSOu-5 H ₂ O 0,025 0,025 0,025 0,025 inositol 10000 10000 10000 10000 Thiarain-HCl 40 40 40 40 sucrose 30000 20000 10000 10000 BAP ²² 0.6 0.4 - - zeatin 0.1 0.05 - - IBA ²³ - - - 1 Sea Plaque® Agarose 6000 6000 - - Difco agar Bacto® - - - 7000 pH 5.8 5.8 5.8 5.8

.- BAP: benzylammopurine

IBA: indolebutyric acid

Example 11: Isolation and Injection of Zygotic Sunflower Proembryos and Embryos

a) Isolation and selection of pro-embryos from sunflowers

Young zygotic proembryos and embryos are isolated from immature 6 mm to 8 mm long fruits (light gray to white pericarp) of Helianthus annum about 4-20 days after pollination. The fruit stands are first pre-cleaned under running tap water, possibly with the addition of a commercially available kitchen cleaner (Lux, Pril, etc.). After thorough cleaning, the fruit stand is sprayed with 70% ethanol and then washed with distilled water. The immature fruits with gray to white fruit wall are then removed from the fruit stand with the help of tweezers and collected in 330 ml plastic cans. Each of these cans should be about 1/4 full. The immature fruits are now sterilized with a 1.5% calcium hypochlorite solution containing 0.1% Tween 20 as an additive for 15 minutes. Subsequently, the fruits are washed sufficiently with sterile water and stored in a humidified Petri dish (4 ° C). With the help of fine tweezers and scalpels, the fruits under the stereomicroscope are opened at a 10-20 fold magnification. From the fruits, the embryo sacs are removed and transferred into about 1 ml of hormone-free medium (H3) in Petri dishes of 3 cm diameter. Subsequently, with the help of two dissecting needles from the embryo sacs, the embryos of different sizes are examined, which are from early globular stages of the proembryonic stage (see Fig. 2, Phases 0, P, Q) to early to late cotyledonous stages of about 2,000 teeth (see Fig. 2, phase R, S, T), isolated under microscopic observation. After isolation, the embryos are read out analogously to the method described under Example 4a), in which case hormone-free medium (H3) without Ficoll is used.

b) Microinjection of pHP23 DNA into sunflower pro / embryos

The introduction of pHP23 into these pro / embryos via microinjection is carried out in an exactly analogous manner to the method described under Example 4b).

c) Microcultivation of microinjected sunflower embryos

The cultivation of the microinjected sunflower embryos is carried out analogously to the method described under Example 4c) for the cultivation of maize embryos. In this case, however, the wells of the polycarbonate microculture plates are filled with different amounts of culture medium depending on the size and developmental stages of the microinjected embryos. Young globular proembryons are placed on a 0.1 μΐ to 0.5 μΐ layer of medium Hl and overlaid with 0.1 μΐ to 0.5 μΐ H3 [cf. Example 7c)]. In this way 1-10 proembryos can be cultured in one well of the microculture plate. Heart-shaped and later cotyledonary stages are placed on a 0.2 μΐ to 0.5 μΐ layer of Hl and overlaid with 0.2 μΐ to 0.5 μΐ H3 medium. These larger stages of development are preferably cultivated individually. The microculture plates are incubated in the two-compartment system serving as humidity chamber [as described under example 4c] at 25 ^° C. in the dark or in light (dim light, about 500 lux) / dark rhythm of 16/8 hours.

d) Cultivation of microinjected pro / embryos and regeneration of whole plants

Every 2 and 4 days starting 1 to 4 days after the microinjection and depending on the size and the stage of development of the microinjected embryos, they are provided with new culture medium by addition of 0.2 μΐ to 0.5 μΐ H3 medium. After about 6-10 days, depending on the size of the cultured embryos, they are transferred to 4 μΐ to 10 μΐ of the culture medium H2 in wells of Terrasaki plates. Here, too, the cultured embryos are covered after transfer into the wells with 0.2 μΐ to 1 μΐ H3 culture medium. To ensure sufficient moisture, about 1 ml to 2 ml of sterile water are deposited on the edge of the wells of the Terrasaki plates. The embryos are again incubated at 25 ° C and a Hell / Dukelrhythmus of 16/8 hours (4000 lux, cold white light).

Every 3-4 days, the developing embryos are humidified by adding 0.2 μΐ to 1.0 μΐ H3 medium. After regeneration of small seedlings, they are grown in Costar plates on H3 medium (at 0.8%

Agarose) and cultured under the culture conditions described above. It may be necessary to re-induce root induction by adding hormones into the medium. This can be achieved, for example, by keeping the plantlets on a root-inducing medium (H4) for 3-6 days. After forming several leaflets and roots, these plantlets are transferred into 330 ml culture cans containing 30 ml to 50 ml H3 medium containing 0.8% agarose. When these plants have reached a size of about 6 to 10 cm, they can be brought into the greenhouse, where they can be cultivated to maturity like normal sunflower plants.

Table 10 Media for the cultivation of zygotic sunflower embryos

component Hl [mg / 1] H2 [mg / 1] H3 [mg / 1] H4 [mg / 1] NH ₃ NO ₃ 1650 1650 1650 - KNO ₃ 1900 1900 1900 1900 CaCl ₂ -2H ₂ O 440 440 440 440 MgSOu'2 H ₂ O 370 370 370 370 NH ₂ POu 170 170 170 170 FeNa ₂ -EDTA 40 40 40 40 H ₃ BO ₃ 6.2 6.2 6.2 6.2 MnSOu-4 H ₂ O 22.3 22.3 22.3 22.3 ZnSOu-4 H ₂ O 8.6 8.6 8.6 8.6 KI 0.83 0.83 0.83 0.83 Na ₂ Mo0i »-2 H ₂ O 0.25 0.25 0.25 0.25 CuSOu'5 H ₂ O 0,025 0,025 0,025 0,025 inositol 100 100 100 100 niacin 0.5 0.5 0.5 0.5 Pyridoxine HCl 0.5 0.5 0.5 0.5 Thiamine HCl 0.1 0.1 0.1 0.1 casein hydrolyzate 500 500 500 500 Sucrose BAP ²⁴ NAA ²⁵ 30000 0.6 20000 0.4 10000 10000 0.1 Sea plaque® agarose 6000 6000 - - Difco agar Bacto® - - - 7000

BAP: Benzylaminopurine

NAA: naphthyl-1-acetic acid

Example 12: Isolation and Injection of Zygotic Arabidopsis Proembryos and Embryos

a) Isolation and Selection of Arabidopsis Pro-ZE Embryos Young zygotic proembryos and embryos are isolated from 5-15 mm long pods (about 3-20 days after pollination) of Arabidopsis thaliana cv. Columbia isolated. The plants are grown in the greenhouse at a temperature of 16 ° C to 18 ° C and a light / dark rhythm of 16/8 hours. Prior to isolation, the pods are sterilized by treatment with 1.5% calcium hypochlorite for 10 minutes and then rinsed sufficiently with sterile distilled water. The pods thus treated are stored in humidified Petri dishes at about 4 ° C in the dark until further treatment. With the help of fine tweezers and scalpels, the pods under the stereomicroscope are opened at a 10-20 fold magnification. The ovules are removed from the pods and transferred into about 1 ml of hormone-free medium (A3) in Petri dishes of 3 cm diameter. Subsequently, with the aid of two dissecting needles from the ovules, the proembryos and embryos of different sizes are obtained, which are from early globular stages of the proembryonic stage (see Fig. 2, phases 0, P, Q) to early to late cotyledonous stages of about 2000 rows (cf. Fig. 2, phase R, S, T), isolated under microscopic observation. After isolation, the embryos are read out analogously to the method described under Example 4a), in which case hormone-free medium (A3) without Ficoll is used.

b) Microinjection of pHP23 DNA into Arabidopsis pro / embryos

The introduction of pHP23 into these pro / embryos via microinjection is carried out in an exactly analogous manner to the method described under Example 4b).

c) Microcultivation of microinjected Arabidopsis pro / embryos

The cultivation of the microinjected Arabidopsis pro / embryos is carried out analogously to the method described in Example 4c) for the cultivation of maize embryos. In this case, however, the pits of the polycarbonate microculture plates become involved depending on the size and developmental stages of the microinjected embryos

filled different amounts of culture medium. Young globular proembryons are placed on a 0.1 μΐ to 0.5 μΐ layer of Al and overlaid with 0.1 μΐ to 0.5 μΐ A3 medium [cf. Example 7c)]. In this way 1-10 proembryos can be cultured in one well of the microculture plate. Heart-shaped and later cotyledonary stages are placed on a 0.2 μΐ to 0.5 μΐ layer of Medium Al and overlaid with 0.2 μΐ to 0.5 μΐ A3 medium. These larger stages of development are preferably cultivated individually. The microculture plates are incubated in the two-compartment system serving as humidity chamber [as described under example 4c] at 21 ° C. in the dark or in continuous light (about 500 lux).

d) Cultivation of the microinjected pro-embryos and regeneration of whole plants

Every 2 and 4 days starting 1 to 4 days after the microinjection and depending on the size and the stage of development of the microinjected embryos they are provided with new culture medium by addition of 0.2 .mu.ΐ to 0.5 .mu.A A3 medium. After about 6-10 days, depending on the size of the cultured embryos, they are transferred to 4 μΐ to 10 μΐ of the culture medium A2 in wells of Terrasaki plates. Also in this case, the cultured embryos are overlaid with 0.2 μΐ to 1 μΐ A3 culture medium after transfer to the wells. In order to ensure sufficient moisture, about 1 ml to 2 ml of sterile water are deposited on the edge of the wells of the Terrasaki plates. The embryos are again incubated at 21 ⁰ C and steady light (about 500 lux).

Every 3-4 days, the developing embryos are humidified by adding 0.2 μΐ to 1.0 μΐ A3 medium. Small plantlets (10-15 days) develop in this culture phase, which are then transferred to Costar culture plates with 1 ml to 1.3 ml of A3 medium containing 0.8% agarose. After the formation of several leaves and roots, these plantlets are transferred into commercially available 330 ml culture cans containing 30 ml to 50 ml of A3 medium containing 0.8% agarose. When these plantlets have reached a size of about 5 cm to 6 cm, they can be brought into the greenhouse, where they can be cultivated to maturity like normal Arabidopsis plants.

Table 11 Media for the cultivation of Arabidopis embryos

component Al [mg / 1] A2 [mg / 1] A3 [mg / 1] KNO ₃ 2500 2500 2500 CaCl ₂ -2H ₂ O 150 150 150 KgSOk'l H ₂ O 250 250 250 (NHU) SO * 134 134 134 NaH ₂ PO ₄ -H ₂ O 150 150 150 KI 0.75 0.75 0.75 H3BO3 3.0 3.0 3.0 MnSOu'4 H ₂ O 10.0 10.0 10.0 ZnSO 4 -4H ₂ O 2.0 2.0 2.0 Na ₂ MoO _2, -2H ₂ O 0.25 0.25 0.25 CuS0i, -5 H ₂ O 0,025 0,025 0,025 CoCl ₂ -6H ₂ O 0,025 0,025 0,025 Na ₂ -EDTA 37.3 37.3 37.3 FeS0i "-7 H ₂ O 27.8 27.8 27.8 inositol 100 100 100 niacin 1.0 1.0 1.0 Pyridoxine HCl 1.0 1.0 1.0 Thiamine HCl 10.0 10.0 10.0 Sucrose BAP ²⁶ 30000 0.6 20000 0.1 4000 Sea plaque® agarose 6000 6000 - kinetin 0.05 0.05 - ph 5.8 ph 5.8 ph 5.8

BAP: Benzylaminopurine

Example 13: Detection of the NPT-II gene in the plant heritage

Leaf tissues of the regenerated and transformed plants are dissolved at ⁰ ° C in 15% sucrose solution containing 50 mmol / l ethylenediamine-N, N, N ', N'-tetraacetic acid, EDTA), 0.25 mol / l sodium chloride and 50 mmol / 1 ctjCc.a-tris-ihydroxymethyl-methylamine hydrochloride (TRIS-HCl) homogenized at pH 8.0. By centrifuging the homogenate for 5 minutes at 1000 g, the nuclei are roughly separated. The cell nuclei are resuspended in 15% sucrose solution containing 50 mM EDTA and 50 mM TRIS-HCl at pH 8.0, treated with sodium dodecyl sulfate (SDS) to a final concentration of 0.2%, and Heated to 70 ⁰ C for 10 minutes. After cooling to 2O-25 ° C the mixture potassium acetate is added to a concentration of 0.5 mol / l. This mixture is incubated for 1 hour at 0 ⁰ C. The precipitate is centrifuged (centrifuged for 15 minutes at 4 ° C in a microcentrifuge). From the supernatant liquid, the DNA is precipitated by adding 2.5 times the volume of ethanol at 20-25 ° C. The separated DNA is dissolved in a solution of 10 mM TRIS-HCl containing 10 pg / ml ribonuclease A, incubated for 10 minutes at 37 ° C, added with proteinase K to a concentration of 250 ug / ml and for an additional hour Incubated at 37 ° C. Proteinase K is removed by phenol and chloroform / isoamyl alcohol extractions. From the aqueous phase, the DNA is precipitated by addition of 0.6 volume of 0.6 M isopropanolic sodium acetate solution and in 50 .mu.l of a solution containing 10 mmol / 1 TRIS-HCl and 5 mmol / 1 EDTA, at pH 7.5 solved. This preparation yields DNA sequences predominantly containing more than 5ΟΌ0Ο base pairs. Restriction of this DNA with EcoRV endonuclease, hybridization of the fragments with radioactively labeled HindIII fragments of the NPT-II gene and comparison with the plasmid pABDI shows in a "Southern Blot" analysis the presence of the NPT-II gene in the nuclear DNA of the transformed plant cells.

Example 14: Dot-blot analysis

10 μg of genomic DNA are digested overnight with EcoRI and then applied directly to a nitrocellulose membrane without previous electrophoretic separation (see method of Schleicher and Schull GmbH, Dassel, FRG).

The hybridization reaction and the subsequent autoradiography are carried out analogously to the Southern blot method.

Example 15: Southern blot analysis:

The Southern blot analysis of the plant DNA is carried out according to a method described by Paszkowski and Saul, 1983.

Approximately 5 μg of genomic DNA are optionally cut with EcoRV or HindIII restriction enzymes and the resulting fragments are separated by electrophoresis in a 0.8% to 1% standard agarose gel. (Maniatis T et al. , 1982).

The nick translation is replaced by the "random primer" method described by Feinberg and Vogelstein, 1983.

Example 16 Investigation of the Aminoglycoside Phosphotransferase Enzyme

activity of transformed plants

Leaf pieces (100 to 200 mg) are homogenized in 20 μΐ extraction buffer in an Eppendorf centrifuge tube. The buffer is a modification of the procedure described by Herrera-Estrella, L., DeBlock, M., Messens, E., Hernalsteens, J.-P., Van Montagu, M. and J. Schell, EMBO J. 2, 987-995 (1983) used buffer in which albumin from bovine blood is replaced by 0.1 M sucrose. The extracts are centrifuged for 5 minutes at 12,000 g and the supernatant phase is treated with bromophenol blue to a final concentration of 0.004%. By electrophoresis in a 10%, non-denaturing polyacrylamide gel, the proteins are separated from 35 μΐ of the supernatant phase. The gel is overlaid with a kanamycin and у ^{ѣ 2} P-labeled ATP-containing agarose gel, incubated, and the phosphorylated reaction products are transferred to Whatmanp81 phosphocellulose paper. The paper is washed six times with deionized water at 90 ^° C. and then autoradiographed.

Results:

The regeneration of whole, transgenic plants from microinjected zygotic embryos via direct embrogenesis could be carried out in rapeseed as well as corn, barley and rice within 8 weeks, whereby the achieved regeneration rates are very high. In secondary embryogenesis plants, the secondary regenerants were used to determine the transformation frequency. For all other plants, the following method is used: after fertilization, the seeds of these plants are germinated and this plant generation used to provide evidence for the transformation and expression of the gene. The transformation frequencies are in a range of 5% - 35%.

The stable integration of the complete microinjected genes into the high-molecular Pi generation DNA, derived from the primary regenerant, could be demonstrated by Southern blot analysis of the genomic DNA of said Fl generation plants. Expression of the integrated genes was detected using the enzyme assay described in Example 11 (assay of aminoglycoside phosphotransferase enzyme activity).

Deposit:

The plasmid pHP23 used within the scope of the present invention has been assigned to the German Collection of Microorganisms (DSM), in Braunschweig, Federal Republic of Germany, in accordance with the requirements of the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the Purpose of Patenting , deposited. A statement on the viability of the deposited samples was made by the said International Depository.

microorganism Deposit date Deposit number * Date of the ability certificate pHP23 (Escherichia coli DHI 21.12.1987 DSM 4323 12/21/1987 transformed with pHP2 3 plasmid DNA

* Deposit number, issued by the International Depository, as described above.

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Claims (49)

claims: 1. A method for introducing genetic material into plants, characterized in that one microinjected a DNA-containing solution into young zygotic embryos or embryos of plants that have a high regeneration potential in the form of a natural embryogenesis, the microinjected pro- / embryos in suitable microculturing the embryogenesis-promoting culture media and optionally non-chimeric, transgenic plants produced from the thus obtained, usually chimeric regenerants. 2. The method according to claim 1, which is characterized by the following specific process steps: a) Isolation of individual, for microinjection suitable multi- to multicellular zygotic pro / embryos from the ovules of donor plants; b) selection of the isolated zygotic pro / embryos for the subsequent microinjection and introduction of the selected pro / embryos into a suitable culture medium; c) microinjection of a DNA-containing solution containing one or more DNA fragments into the zygotic pro / embryos isolated under a); d) microculturing the microinjected zygotic pro / embryos in suitable culture media promoting direct embryogenesis; f) regeneration of said transformed zygotic pro / embryos to complete, usually chimeric, plants; and g) Preparation of non-chimeric, transgenic plants with new and desirable properties. 3. The method according to claim 2, characterized in that the cultivation of the microinjected pro- / embryos is carried out under conditions that allows the development of a morphogenic cell culture or the formation of secondary embryos. 4. The method according to claim 1, characterized in that one carries out the microinjection using a microinjection apparatus, which consists essentially of the following parts: a) a microscope for optical control of the injection process b) motorized or mechanically operated micromanipulators with capillary holders c) microinjection capillaries with an outer diameter of 1.0 mm to 2.0 mm and a tip diameter of ΐΐ 1 μΐΐι to 10 μπι d) Holding capillaries with a tip diameter of 0.02 mm to 1.0 mm e) a pneumatically controlled pressure device for injecting the DNA-containing solution. 5. A process according to claim 4, characterized in that the microinjection capillaries are microelectrodes with fused glass filaments of borosilicate. 6. The method according to claim 1, characterized in that the microinjection process proceeds in the following substeps: a) introducing the Proembryonen or embryos in 10 μΐ to 200 μΐ media drops on coverslips b) Fix the embryos using the holding capillary c) injection of the DNA-containing solution into the individual cells of the pro / embryo with the aid of the microinjection capillary d) rotation of the pro / embryos after each injection and re-injection into single cells e) Cultivating the injected pro / embryos in suitable culture media. 7. The method according to claim 6, characterized in that injecting the DNA-containing solution in plant Pro / embryos, which are in an optimal developmental stage, i. which are characterized by good microinjectibility, good insulating properties and good regeneration capacity. 8. A method according to claim 6, characterized in that one uses embryos that are in a former globular prostembryonic stage to early embryonic stage. 9. The method according to claim 6, characterized in that early embryonic stages are used, which already have a well recognizable shoot and Wurzelmeristemanlage in addition to the scutellum. 10. The method according to claim 6, characterized in that microinjected the DNA-containing solution into plant proembryos or embryos, which are in 2 to about 5000 cell stage. 11. The method according to claim 10, characterized in that the plant pro / embryos are in the 4 to 100 cell stage. 12. The method according to claim 6, characterized in that one carries out the microinjection process 1 to 1000 times per embryo or proembryo. 13. The method according to claim 12, characterized in that microinjected 4 to 60 times per embryo or proembryo. 14. The method according to claim 12, characterized in that the DNA-containing solution is injected into the tissue of Sprossmeristemanlage. 15. The method according to claim 6, characterized in that the injection volume is 1 pl to 100 pl per cell injected. 16. The method according to claim 6, characterized in that the DNA concentration of the injected DNA-containing solution is between 0.1 pg / μΐ and 10 ug / ul. 17. The method according to claim 6, characterized in that said DNA-containing solution is a buffer solution. 18. The method according to claim 17, characterized in that the pH of the buffer solution is in a range between pH 7.0 and pH 8.0. 19. The method according to claim 6, characterized in that said DNA-containing injection solution containing the transforming DNA a) in linearized form; b) in spirals form; or c) in the form of a mixture of linearized and spirally-spun DNA. 20. Method according to claim 19, characterized in that said transforming DNA is flanked by neutral DNA sequences. 21. The method according to claim 6, characterized in that the microinjection-specific DNA-containing solution contains a rekobminantes DNA molecule, which leads in the transformed plant to useful and desirable properties. A method according to claim 21, characterized in that said recombinant DNA molecule consists essentially of one or more structural genes encoding a useful and desirable characteristic (s) and (s) in the plant cell active expression signals are operably linked (are), so that an expression in the plant cell is ensured. 23. A method according to claim 21, characterized in that said recombinant DNA molecule consists of either genomic DNA, a cDNA or synthetic DNA or a combination of said DNA. 24. The method according to claim 22, characterized in that said structural gene a) confers increased resistance or tolerance to pathogens, herbicides, insecticides or other biocides to the transformed plant; b) conferring on the transformed plant increased resistance or tolerance to adverse environmental factors selected from the group: heat, wind, cold, dryness, moisture, extreme soil conditions, osmotic stress; (c) improve the formation and quality of reserve and storage materials in leaves, seeds, tubers, roots and stems; or d) encodes pharmaceutically acceptable active substances or other physiologically active substances. 25. The method according to claim 2, characterized in that the DNA-containing solution contains a non-coding DNA having a regulatory function. 26. The method according to claim 22, characterized in that said expression signals derived from genes of plants or plant viruses. 27. The method according to claim 26, characterized in that said expression signals are derived from genes of Cauliflower mosaic virus (CaMV) or are homologous thereto. 28. The method according to claim 27, characterized in that it is a) the 35 S expression signals of the CaMV genome or its homologs; or b) the 19 S expression signals of the CaMV genome or their homologs. 29. Process according to claim 3, characterized in that the microculturing of the microinjected proeembryos and embryos is carried out in a liquid culture medium which stimulates direct embryogenesis. 30. The method according to claim 29, characterized in that said culture medium is mixed with an osmotically neutral thickener. 31. The method according to claim 29, characterized in that said liquid culture medium is coated over a solid medium. 32. The method according to claim 30, characterized in that said thickening agent is co-polymers of sucrose and epichlorohydrin having a molecular weight of between 70,000 and 400,000. 33. The method according to claim 31, characterized in that said solid medium contains a solidifying agent selected from the group consisting of agar, agarose, alginate, pectinate, gelatin or Gelrite®. A method according to claim 29, characterized in that said culture medium has the following composition: a) for growth and development of the plant cell essential mineral substances or elements in their compounds selected from the group consisting of nitrogen, phosphorus, sulfur, potassium, calcium, magnesium and iron, among others. (Macroelements) and sodium, zinc, copper, manganese, boron, vanadium, selenium and molybdenum u.a. (Microelements) b) essential vitamins selected from the group consisting of nicotinic acid, pyridoxine, thiamine, inositol, biotin, lipoic acid, riboflavin, Ca-pantothenate and the like. c) an easily assimilable carbon source selected from the group consisting of sucrose, glucose and the like. d) various growth regulators in the form of natural or synthetic phytohormones selected from the class of auxins and cytokinins. e) osmotic stabilizers selected from the group consisting of sugar alcohols, sugar or salt ions and the like. 35. The method according to claim 34, characterized in that said culture media containing the various growth regulators from the group of auxins and cytokinins in a balanced ratio to each other, so that a controlled process of direct embryogenesis is guaranteed. 36. The method according to claim 35, characterized in that the phytohormones from the group of the auxins and the cytokinins in a concentration ratio between 0.01 mg / 1 and 20.0 mg / 1 are present, depending on the respective stage of development of the embryos. 37. The method according to claim 34, characterized in that the pH of the culture medium is between pH 4.5 and pH 7.5. 38. The method according to claim 34, characterized in that complex culture media are used for the cultivation of the embryos, which are wholly or at least partially composed of natural components. 39. A method according to claim 38, characterized in that said natural components are those selected from the group consisting of potato extract, coconut milk or liquid endosperm and the like. 40. A method according to claim 29, characterized in that the cultivation density of the pro / embryos is between 1 pro / embryo per μΐ culture medium and 50 pro / embryos per μΐ culture medium, depending on the size and the development status of the pro / embryos , 41. The method according to claim 29, characterized in that the microcultivation of the pro / embryos is carried out at least temporarily in a two-compartment system. 42. The method according to claim 29, characterized in that the microinjected pro- / embryos transferred every 1 to 12 days in fresh culture medium or overlaid with fresh culture medium. 43. A method according to claim 2, characterized in that the pro / embryos, when they have reached a certain stage of development, usually after 10 to 30 days, for the differentiation of the shoot and root vertexes on solid culture media commonly used for the regeneration of plants and which may optionally be over-coated by a liquid medium. 44. The method according to claim 2, characterized in that the pro / embryos after a cultivation period of 4 to 8 weeks, but at the latest after the germination of the embryos transmits to solid media that have no or only low hormone concentration. 45. The method according to claim 44, characterized in that planting the regenerated plantlets in soil and treated in the same manner as normal seedlings. 46. A process for the preparation of pure homozygous transformants according to claim 1, characterized in that a) in plants susceptible to secondary embryogenesis or secondary adventitious shoot formation, in vitro separate the secondary structures derived from single cells from the primary, mostly chimeric regenerants, isolated and regenerated to whole plants or b) Transgenic chimeric plants optionally repeatedly crossed with themselves. 47. Method according to claim 1, characterized in that proembryos or embryos of plants selected from the group consisting of the families Solanaceae , Cruciferae , Compositae , Liliaceae , Vitaceae , Chenopodiaceae , Rutaceae , Alliaceae , Amaryllidaceae , Asparagaceae , Orchidaceae , Palmae , Bromeliaceae , Rubiaceae , Theaceae , Musaceae , Gramineae or the order Leguminosae . 48. The method according to claim 1, characterized in that one uses proembryos or embryos of plants of the family Malvaceae . 49. Method according to claim 48, characterized in that proembryos or embryos of plants selected from the group consisting of the genera Allium , Avena , Hordeum , Oryzae , Panicum , Saccharum , Seeale , Setaria , Sorghum , Triticum , Zea , Musa , Cocos , Phoenix , Elaeis used. Listen I Sttfcui