DD265911A5 - Process for the transformation of hereditary genetic material - Google Patents

Abstract

The present invention relates to a method for the transformation of hereditary genetic material with simple purely chemical process measures as well as the plant material obtainable by this process. The transfer of the genetic material into the plant cell is carried out directly without the use of a natural Pflanzeninfektioesen system such as a plant bacterium or plant virus and without transmission by insects or phytopathogenic fungi, but by co-incubation of the transforming DNA and plant protoplasts in a suitable incubation medium. In this way, desired genes can be easily and efficiently transferred to plant material, resulting in plants with improved properties.

Description

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Field of application of the invention

The present invention relates to an improved process for the transformation of plant protoplasts with simple purely chemical process measures as well as the herbal material obtainable by this process. By introducing new genetic information into plant material, plants with new and / or improved properties can be produced. Considering, for example, the rapid growth of the world's population and the concomitant increase in food and commodity needs, increasing the yield of crops and increasing the production of herbal ingredients, in particular food and medicine advances, are among the most urgent tasks of biological research. In this context, z. To mention the following essential aspects *, an increase in the resistance of crops to unfavorable soil conditions or climatic conditions, to diseases and pests, increased resistance to pesticides such as insecticides, herbicides, fungicides and bactericides, and a favorable change in nutrient content or crop yield Plants. Such desirable effects could generally be brought about by induction or increased formation of protective substances, valuable proteins or toxins. A corresponding influence of plant genetic material can occur for example by reacting a certain ^l: remdgen specifically in plant cells infiltrates without making this conventional breeding methods use.

Characteristic of the known state of the art

The transfer of DNA sequences into plant cells using genetically engineered plant bacteria is known from publications in the literature, such as 111 Nature, Vol. 303, 209-213 (1983); / 3 / Nature, Vol. 304, 184-187 (1983); IAI Scientific American 248 (6), 50-69 (1983); / 6 / EMBO Journal 2 (6), 987-995 (1983); / 6 / Science 222,476-482 (1983); / 71 Science 223, 498-498 (1984); or / 8 / proc. Natl. Acad. Be. USA 80,4803-4807 (1983). The methods described therein exploit the natural plant-infecting properties of these bacteria to introduce new genetic into plant cells. Heretofore, primarily plant pathogens have been used as vectors for this purpose, for example Agrobacterium tumefaclens or its Ti-plosmide or cauliflower mosaic virus. Recently developed methods now also allow the direct introduction of a gene into plant cells without the ingestion of biological vectors. (/ 9 / Potrykus, I. et al., Plant Molec BIoI Rep 3,117-128 (1985); 10 / Shillito, RD et al., Bio / Technology 3,1099-1103,11985]).

These methods, which have become known under the heading "direct gene transfer", permit a vector-free transformation of plant cells without the use of plant-infectious systems, such as plant-pathogenic bacteria, viruses, insects or fungi.

This also eliminates all restrictions that are given by the host specificity of the pathogens. The development of the plants from the transformed cells is not affected by the application of the novel methods for the transformation of plant ΖβΙΙβη.

A major problem in the application of gene transformation stems from the difficulty of identifying the transformed cells or tissues.

The lower the transformation frequency in a gon transformation, the more difficult and expensive it is to find the few cell clones resulting from the transformed cells under the enormous number of non-transformed clones. The application of conventional screening methods is therefore almost or completely impossible with low transformation frequency, unless it is a gene with selective marker function (B. resistance to a specific substance). Low Transformaiionshäufigkelt thus requires a huge effort in genes without selectable marker Funktlon. Therefore, the usual screening methods for the sanctioning of transformed cell clones can only be meaningfully and successfully used in transformations with genes without marker function, if the transformation frequency is in the order of 10 "to 10" ^{2. At} present, these high target transformation rates can be achieved only with the use of electroporation, even in conjunction with other methods used in microbiological gene transfer research, such as poly-L-ornithine or poly-L-lysine treatment, liposome fuelone, DNA proteic complexing, charge distraction protoplasts membrane, fusion with microwave protoplasts or calclum phosphate coprecipitate, and especially polyethylene glycol treatment and heat shock (heat shock) method (/ 10 / Shillito, et al., Bio / Technology 3,1099-1103 (1985)) so far only reproducible transformation rates when using purely chemical process measures in an order of magnitude of up to 10 "'can be achieved. In electroporation (/ 11 / Neumann, "et al., The EMBO Journal 7, 841-845 (1982), / 10 / Shillito et al., Bio / Technology 3, (1986)), protopiasts in mannitol / calcium are or a mannitol / magnesium solution by discharging a capacitor over the SuspenslonsflOsslgkeit briefly applied to a voltage pulse of high intensity.

This causes a polarization of the protoplast membrane and a reversible opening of pores in the membrane,

whereby the transfer of DNA into the cell is facilitated.

However, this method has many disadvantages and is subject to certain limitations. On the one hand, a relatively large amount of equipment is required to carry out the transformation with the aid of electroporation

necessary, which is associated with a corresponding cost and work attachment. On the other hand, the application of this

Method due to the high sensitivity of plant protoplasias only within a very narrow Gre.izbereiches

possible. Therefore, in order to ensure the viability of the transformed cells, certain parameters such as voltage, capacitance and field strength values may be varied only within these narrow limits (e.g.

Voltage range 1400-1700V (/ 10 / Shillito, R.D. et al., Bio / Technology 3, [1985]). Aim of the invention These disadvantages can surprisingly in the context of the process according to the invention with simple purely chemical Procedural measures are overcome. The investigations carried out in the context of the present invention on the principles for recording and Inteoration of DNA in plant cells have made it clear that the transformation process of

a multiplicity of mutually influencing parameters is dependent. Building on these investigations, the transformation frequency can now be determined by varying and optimally coordinating the parameters relevant to the transformation, in the

Compared to conventional Verfahrer, so significantly increased that the aforementioned aspired Transformation rates of 10 ~ ³ to 10 ~ ^{2 and} above are easily achieved. With the help of this on purely chemical measures lending process of the invention next to the Transformation frequency now surprisingly, the reproducibility and survival of the treated Protoplasts are increased very much. While hitherto with reu chemical processes maximum transformation frequencies in the range of 10 ~ * are achieved

Now, when using the method according to the invention, it is now possible to produce reproducible

Tran8formationefrequ <zen to achieve an order of magnitude up to a few percent, comparable to the Electroporation. In contrast to electroporation, the method improved according to the invention is a method which without

special equipment and therefore cost and less labor intensive can be performed. Another

Vortoil, compared to methods that require electroporation, consists in the novel way, now large To be able to transform amounts of plant protoplasts at the same time. Comparable limitations, as previously discussed for the electroporation method in relation to the survival rate of the

treated cells are not given when using the method according to the invention.

The parameters determining the transformation according to the invention are variable over a wide range and

do not affect the transformation efficiency and viability of the treated protoplasts within this range.

In addition, the entire protoplast population after transformation using the

according to the invention as far less heterogeneous as in the case of a transformation by means of F.! oporporatlon, both concerning the life and the Tellungsfähigkeit the treated ProtopU iten. Thus, when using the

according to the invention, for example, without further survival rates of the treated protoplasts of 80% and more, while maintaining their potency and thus the possibility of forming new colonies.

By contrast, the comparable survival rates for electroporation use only about 10%. It has now been shown, contrary to expectations, that of a large number of parameters, which individually or with each other, the Transformation frequency in direct gene transfer into protoplasts can influence, by the right choice and Combining only a few of these parameters or measures with little technical and time effort optimal Transformat'onsrate can be achieved. The following parameters crystallized as essential:

1) DNA sample: shape, size, concentration, time of application

2) Carrier DNA: shape, concentration, size

3) Mg ² *: concentration, time of application 4} platemembrane

modifying agent: concentration, time of application.

Among the factors essential for the process according to the invention are primarily the Mg ¹ * concentration and the Concentration oes the plasma membrane modifying agent of crucial importance for the increase of Transformation efficiency. One observes a synergistic interaction between these two mentioned factors,

resulting in a strong increase in the transformation frequency.

Also of importance are the time and order of application of the Mg '* ions, the modifying agent

and the transforming DNA.

This crucial improvement and simplification of the method for direct gene transfer into plant protoplasts and

ultimately for the production of genetically modified plants can be achieved by the following essential steps of the invention:

Isolation of the protoplasts from plant material and optionally incubation of the isolated protoplasts in a suitable nutrient medium,

Resuspending the isolated protoplasts in a standardized saline solution,

Transfer of the inolated protoplasts from the saline solution to a transformation medium optimized for the transformation, which contains Mg'mon,

Addition of the DNA to be injected and of a plasma membrane modifying agent,

Incubation of protoplasts and DNA in the presence of a plasma membrane modifying substance for a time sufficient to allow penetration of the DNA into the protoplasts,

- separation of the treated protoplasts from represent incubation and optionally Derun resuspension in an aqueous CaCl _r solution

Separation of the protoplasts from the CcClj solution,

Incubation In a culture medium suitable for wide protoplate development and

- if desired, regeneration of complete transformed plantone.

Dwtogung dM essence of the invention The present invention is based on the object, an improved method for the transformation of plant protoplasts

to provide.

In the opening of the present invention, the following definitions apply: Plant material: In culture or as such viable plant parts, such as protoplasts, cells, callus, tissue,

Embryos, plant organs, buds, seeds and the like as well as whole plants. Gene: structures with flanking expression signals

Structural gene: Proteln-encoding DNA sequence

Expression signals: Promoter signal and Terminatlonsslgnal plant effective

Expression signal: expression signal that is functional in plants. Promoter signal: the transcription-initiating signal

Terminatlonsslgnal: the transcription-terminating signal Enhancerslgnal: the Trnscriptfon amplifying signal

Replication! Gnal: the DKA replication-enabling signal

Integration signal: DNA sequence which promotes integration of the gene into genomic DNA Hybrid gene: from heterologous DNA sequences, i. H. DNA sequences of different origin constructed gene,

which are natural, c-DNA as well as synthetic DNA sequences

can

Trager DNA: the gene flanking neutral, d. H. DNA sequence not involved in the function of the gene Carrier DNA: Neutral DNA without transforming gene, which is added to the transforming gene

this against nucleases

Isolated gene: DNA sequence separated from the original DNA which encodes a single protein NPT-II gene: neomycin S'-phosphotransferase gene, type II of transposon Tn 6 (/ 1 / Rothstdin, S.J. W.S. Reznikoff, Cell 23, 191-199, (1981)) Genomic DNA: DNA of a plant genome (total or part thereof)

c-DNA: a copy of an m-DNA produced by reverse transcriptase.

The present invention thus relates in essence to an improved process for the transformation of plant protoplasts and, if desired, the production of whole, genetically-modified plants by regeneration from said transformed protoplasts, which comprises

- Plant protoplasts isolated from any plant tissues and optionally cultured in one of the nutrients usually used for the cultivation of plant protoplasts;

Said protoplasts before the actual transformation for 20 minutes to β hours in a preincubation medium containing alkaline earth and / or alkali cations, preferably Ca ¹⁺ , K * and / or Na * ions and a suitable C source, at a temperature of 4 to 10 ⁹ C ahead;

- The protoplasts then separated from the Vorinkubatlonamedium and> n the actual transformation medium, containing as essential ingredient 0.1 to 6OmM, preferably 10 to 3 ^Λ M Mg '* - ions, in the presence or absence of Ca'Monen resuspended;

- Immediately thereafter, a DNA sample containing one or more genes u . . ' Z * \ nrrll of expression signals active in pianos, as well as a carrier DNA which lays out transformant solution;

- a few seconds to 20 minutes, preferably 0.1 to 10 minutes, later added a plasma membrane modifying agent;

Incubate the protoplasts and DNA sample in said transformation solution for a cell compartment which ensures the uptake of the DNA in the protoplasts; and

If desired, whole plants are regenerated from the transformed protoplasts.

From this description it is clear that the inventive transfer of new genes into the plant cells

direct route · without using a ·· natural phytopathogenic system, such as a plant bacterium, one

Plant virus or transmission by insects or phytopathogenic fungi takes place. For this purpose, the

Treating plant protoplasts treated directly with the gene to be transferred, introducing them initially into a solution suitable for solution, there for a certain time, then passing them together with the foreign gene into the actual donor-forming medium and there for a period of time sufficient for the uptake of the foreign gene

Protoplasts sufficient, bellet. As plant protoplasts preferably each of a single plant species or one of the species are subordinate

used systematic unit.

Isolated · Herbal protoplasts, which are also suitable as starting material for isolated cells and tissues, can be obtained from

Any part of the plant, such as, for example, leaves, seedlings, stems, flowers, roots, pollen or embryos, may be obtained, and leaf protoplasts are preferably used.The isolated protoplasts may also be obtained from cell cultures

be won. Methods for the isolation of protoplasts can be found, for example, in / 12 / Potrykus, I. and Shlllito,

R.D., Methods in Enzymology 118, 499-678 (1986). As solutions in which dio protoplasts are cultured, preferably osmotically stabilized culture media, such as

they are commonly used for protoplast cultures into consideration.

There are already numerous cultural media available, which can be found in individual media components or groups of such Differentiate components. However, all media are generally constructed according to the following principle: they contain one Group of inorganic ions in the concentration range of about 10 mg / 1 to some. One hundred mg / 1 to a few hundred

mg / 1 (so-called macroelements such as nitrate, phosphate, sulfate, potassium, magnesium, iron), another group of inorganic ions in amounts of a maximum of a few mg / i (the so-called microelements such as cobalt, zinc,

Copper, manganese), as well as a number of vitamins (for example, inositol, folic acid, thiamine), an energy and Carbon source such as sucrose or glucose, and growth regulators in the form of natural or

synthetic phytohormones from the classes of auxins and cytokines in the concentration range of 0.01 to 10mfl / l. The

Culture media are also osmotically stabilized by the addition of sugar alcohols (for example mannitol) or sugar

(For example, glucose) or salt ions (for example, CaCli) and are adjusted to a pH of 6.6 to 6.5.

A more detailed description of common culture media can be found, for example, in / 13 / Koblitz, H., Methodological Aspects of the Zeil ·

and Tissue Breeding in Gramineae with Special Consideration of Cereals, Crop XXII, 1974, 93-11 <7.

Before the protoplasts are transferred into the actual transformation medium, a preliminary incubation takes place in advance

a medium that optimally prepares the protoplasts for subsequent transformation, which in one

marked increase in transformation and survival rates of the treated protoplasts.

Under certain conditions, it is also possible to transform directly into said preincubation medium

perform.

Said medium is a standardized saline solution which, in addition to a suitable C source, e.g. one Sugar or sugar alcohol such as glucose or mannitol, various salts, e.g. As NaCl, CaCl ₂ , KCl, in a concentration of

1 mM to 200 mM. The pH of this salt solution is advantageously at pH values of pH 6 to pH 8.

Shortly before the intended transformation, the protoplasts from the preincubation solution become the actual Transformation medium transferred. It is a mannitol solution containing Mg ^n- ions in a concentration of

0.1 mM to 6OmM, preferably in a concentration of 1OmM to 3OmM. The pH of the incubation solution is at pH 6.6 to pH 12, especially at pH 7 to pH 10.

Immediately after introduction of the isolated protoplasts into the incubation medium, the addition of the DNA sample takes place in one Concentration from 2 μg / ml to 20 μl / μl, preferably in a concentration of μg / ml to μμρ / αιΙ. The DNA, consisting of a structural gene and plant-active expression signals, is advantageously neutralized len Flanking DNA sequences (carrier DNA), which allow the integration of the gene into the genomic DNA of the plant cell. It is advantageous to use the gene in linearized form. Proved particularly suitable in the course of durchgeführton Experiments a TrAger DNA concentration from 60 μg / ml to 70 pg / ml with an average size between 4 "j and ml It is also advantageous to use neutral DNA such as animal or plant DNA, lambda DNA, plasmin 'JNA or

any other DNA suitable for carrying out the process of the invention is used in excess as a carrier

Use DNA to protect the gene against degradation by nucleases. A few seconds to 20 minutes, preferably 0.1 minutes to 10 minutes after the addition of the DNA to the incubation solution

the application of polyethylene glycol is carried out to a final concentration of 10% to 30%. High transformation rates are achieved> nan at a PEG concentration of 20% to 28%.

In addition to polyethylene glycol but other higher alcohols or alcohol-like substances, which also the Modify protoplast membrane and used for example in the field of Zellfuslon, in the

present inventive method can be used. These are, for example, long-chain, polyhydric alcohols, such as polypropylene glycol (426 to 4000 g / mol), polyvinyl alcohol or polyhydric alcohols, their

Hydroxy groups are partially or completely etherified, and plant-compatible, common in the agricultural area Detergents as described, for example, in the following publications:

/ 14 / "McDeckcheon's Detergents and Emulsifiers Annual" MC Publishing Corp., Rldgewood New Jersey, 1981; Stäche, H., "Tensld Diving Book", Carl Hanser Verlag, Munich / Vienna, 1981

If polyethylene glycol itself is used (as in Examples 1 and 2), it is preferable to use those having a molecular weight between 10 000 g / mol and 10000 g / mol, in particular between 3000 g / mol and 8000 g / mol. Among the above-mentioned agents, the substance polyethylene glycol itself is preferable, particularly a CMS type polyethylene glycol solution which has a relatively high proportion of Ca ²⁺ ions. (/ 15 / Negmtiu, I. et al., Theor, Appl. Gene. "72, 279-286, [1986a]).

Particularly advantageous for the course of the transformation was an incubation period with polyethylene glycol between 20 minutes and 6 hours.

After the carried out in the manner indicated above treatment v></, the protoplasts resuspended in fresh culture medium, the cell density advantageously to values between i 'χ 10 ⁴ and 8 χ 10 ⁴ protoplasts per ml of culture medium is adjusted.

When using PEG In a concentration of> 20%, it is expedient to gradually dilute the protoplast after the transformation with a 2- to 10-fold volume of a solution containing Ca'Mon and thus PEG residues present by subsequent sedimentation and resuspension in fresh culture medium wash out. To be particularly advantageous for the transformation results thus an aqueous CaCli solution with a Ca ion concentration of ²⁴ 0.1 M to I ₁ OM. Proved

The method according to the invention is practically unlimited applicable

 In first? In its entirety, the present invention relates to chimeric genetic constructions which constitute a central component

have several structural genes coding for novel useful and desirable properties and operably linked to expression signals functional in plant cells.

For use in the method according to the invention, in the first place, all the genes which are used in the plant Cell are expressed and give the plant a useful β and / or desired property, such as. B. an increased Resistance or tolerance to pathogens (eg, biophytogenic insects, fungi, bacteria, viruses, etc.) Herbicides, insecticides or other biots, to climatic influences and local peculiarities (eg heat, Cold, wind, dryness, humidity, extreme soil conditions, osmotic stress, etc.) or increased Formation of reserve and storage materials in butternut, seeds, tubers, roots, stems, etc. Likewise, the present invention encompasses genes which are suitable for pharmaceutically acceptable active substances, such as

e.g. Alkaloids, steroids, hormones, immunomodulators, and other physiologically active substances.

So you can any genetic genes of plant origin, such as the zein gene (/ 18 / Wienand, U., et al., Mol. Genet. 1 * 2,440-444 [1981p, of animal origin, such as the TPA gene (Tissua-type plasminogen actlvator gene; / 17 / Pennlca, D., et al., Nature MI, 214-221, [1983]), of microbial origin, such as the NPT-II gene, or also

of synthetic origin, such as the insulin gene (/ 18 / Stepien, P., et al., Gene 24, 289-297 [1983]) into the genome of

Plants transferred, provided that the structure of Gone expression signals are flanked in plants

are active, wherein the expression signals of plant, animal, microbial or synthetic origin may be.

In the context of this invention, expression signals are primarily promoter and termination sequences,

but also other regulatory sequences of the 5 'and 3' untranslated regions upstream or downstream. downstream of the structural gene sequences.

The promoter sequences include i.a. a recognition site for the RNA polymerase to which this enzyme is specific

binds and thus initiates the transcription.

Responsible for the binding affinity are certain DNA sequences that accumulated in prokaryotic promoters

occurrence. These so-called "consensus" sequences are generally found in the sequence range -10 to -30, to which

ATG start codon of the structural gene. These are two hexanucleotide sequences, called "Pribnov-Schaller-Box"

and their nucleotide sequence within these sequences and their distance from one another the affinity of the DNA

Decisively influence polymerase to the promoter. In eukaryotic cells, in this context, the so-called "TATA" box, an adenine and thymiule, is produced Sequence portion, which is located 20-30 nucleotides upstream of the Transkrlptlons initiation site, of particular importance As genes that can be used in the context of the present invention, both homologous and come

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 coding DNA sequence may be constructed exclusively from genomic, cDNA or synthetic DNA. A

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 both the cDNA and the cDNA

Genomic DNA can come from different genes. In either case, however, both the genomic DNA and / or the cDNA may be made individually, from the same or different genes.

If the DNA sequence contains portions of more than one gene, these genes can either be of one and the same Organism, of several organisms, more than one strain, one variety or species of the same genus

or from organisms belonging to more than one genus of the same or to any other taxon monies (Reich).

In order to ensure the expression of said structural genes in plant cells, the coding oensequenzen

Initially operably linked to expression sequences functional in plant cells.

The transferred genes, consisting of structural genes and flanking expression signals, can be both natural

occurring genes as well as hybrid genes. In the process according to the invention, preference is given to using those genes whose expression signals of animal or, in particular, plant or synthetic origin »Ind. Examples of such genes are:

a) complete genes from plants, consisting of the structural gene with "a natural expression signals;

b) fully synthetic genes, consisting of a structural gene of synthetic origin, flanked by expression signals of synthetic origin;

c) structural genes of plant origin, flanked by plant-active expression signals, whereby structural genes and Expressionsslgnale originate from different plant species;

d) structural genes of plant origin, flanked by Expresslonssigneie synthetic origin;

e) structural genes of animal, microbial or synthetic origin, flanked by expression symbals of plant origin; or

f) structural genes of animal or microbial origin, flanked by expression signals of synthetic origin.

The Hybrid Gene Constructions In the context of the present invention, therefore, in addition to the structural gene (s), expression signals which include both promoter and terminator sequences as well as further regulative ORPs of the 3 'and 5' non-translatable regions are included.

Very particular preference is given to structural genes of bacterial origin, flanked by expression signals of plant origin, in particular those of plant virus origin.

In this case, any promoter and terminator capable of inducing the expression of a coding DNA sequence (structural gene) can be used as part of the hybrid gene sequence. Examples of suitable promoters and terminators are e.g. These include, for example, the nopaline synthase genes (nos), the octoplone synthase genes (ocs) and the cauliflower mosaic virus gene (CaMV).

Preferred within the scope of this invention are the 35S and 195 expression signals of the CaMV genome, which are determined by molecular biology methods such as ale ζ. B. in / 19 / Maniatis et al., 1982, from said genome IsgIImI and can be linked to the coding DNA sequence.

As starting material for the 35S Tran8kriptionskontroll sequences Jrfindungsgemöß z. B. the Sea Ι · fragment of CaMV Strain "S" comprising nucleotides 6808-7632 of the gene map (/ 20 / Frank G et al., 1980). The 19S promoter and 5'-transcribed regions are located on a genomic fragment between the Pst) site (position

6386) and the Hind HI site (position 6860) of the CaMV gene map (/ 21 / Hohn et al., 1982).

The corresponding terminator and 3 'translated region is located on an EcoRV / 8g Ill-Fragmont between position V 342

and 7643 of the CaMV genome.

In the 196 promoter region, it is a typical eukaryotic / promoter, the de ι CaMV gene Vl Vl upstream and is responsible for the expression of the gene Vl product (Virue envelope protein). Another effective member of a plant cell functional promoter that can be used is one

overproducing plant promoter. These types of promoters should be as long as they provide in an operable manner with the gene sequence is linked coding for a desired gene product capable tein, the expression of said gene sequence m.

Overproducing plant promoters that can be used in the present invention,

exclude the promoter of the small subunit (email subunit; se) of ribuloso-1,6-bis-phosphate carboxyl.ise

Soybean / 22 / (Berry-Lowe et al., J. Mol. And Appl. Gen., 1: 483-498 (1892)) and the chlorophyll a / b promoter. Binding protein. These two promoters are known to be light-emissive in eukaryotic plant cells

(See, for example., 23 / Genetic Human Mering of Plant, Agricultural Perspective, A. Cashmore, Plenum, New York 1883, pages 29-38, / 24 / Coriini G. at al., The Journal of Biological Chemistry , 288: 1399 (1983) and / 26 / Dunsnvuir, P. at til.,

Journal of Molecular and Applied Genetic ·, 2: 285 (1983)). For an application according to the present invention, the exprssion signals of the gen Vl of the Cauliflower moi Virus proved to be particularly advantageous. Particularly preferred in the context of this invention are the expression signals of the CaMV strain CM1841, the complete DNA sequence at / 26 / Gardner et al., 1981. The hybrid genes are prepared by microbiological methods known per se, the reading frame of the coding

is maintained for the proteins to be produced by the plant cell. Current methods are known and have been described, for example, in the following publications: / 27 / "Molecular Cloning", Maniatis, T., Fritsnh, E.F. and J. Sambrook,

Cold Spring Harbor Laboratory, 1982, and / 20 / Recombinant DNA Techniques, Rodriguez, R.L. and R.C. TaIt, Addlson-Wetley Publishing Comp., London, Amsterdam, Don Mills. Ontario, Sydney, Tokyo, 1983. For the integration of the foreign gene into the genomic DNA of the plant cell is t advantageous if the gene consisting of Structural and plant expression expressions flanked by neutral DNA sequences (carrier DNA). The Carrier DNA can consist of two linear DNA strands, so that the insert into the plant cell Construction is a linear DNA molecule. However, the iDNA sequence produced for gene transfer can search for an annular Have structure (plasmid structure). Such plasmids consist of a DNA strain in which the foreign gene with the ExnresBionsslgnalen is integrated. The carrier DNA may be of synthetic origin or by treatment with appropriate Reotriction enzymes are obtained from naturally occurring DNA sequences. For example, as a carrier DMA

naturally occurring plasmas opened with a selective restriction enzyme.

A bolsplel for a dendritic plasmid is the freely available plaemid pUC8 (described in / 28 / Mussing, J. and J. Vlera, Gere

19.269 to 276.1982). Wolter can also be used as carrier DNA fragments of naturally occurring plasmids.

For example, the Deletlons mutant for gene Vl of the Coullflower mosaic virus can be used as Tragor DNA. The likelihood of genetic transformation of a plant cell can be increased by several factors. Thus, as we know from experiments in yeast, the number of successful stable gene transformations increases DmIi: increasing number of copies of the new genes per cell,

2) when combining a replication signal with the new gene, as well

3) when combining integration sequences with the new gene.

Thus, the erfindungggemäße method is particularly advantageous to apply when the gene transferred in with plant cells

effective replication and / or integration sequences is combined.

The expression of a gene in a plant cell depends on the transcription frequency of the gene in a messenger RNA Sequence. It is therefore also advantageous if the new gene with an enhancer which enhances this transcription

combined. In particular, those methods are to be emphasized, to which a gene is transferred that in

Plant cells is combined with effective replication, integration and enhancement events. Furthermore, it is of great procedural advantage if the transferred gene has a selective marker function,

that is, the transformed plant cells can be separated from the untransformed plant cells by selective self-assembly. Such a marker function allows a rational operation in that only those

Plant cells must be regenerated by conventional microbiological measures to KaIIi or complete plants, in

the genome of which is a gene for expression, which allows marker-specific selection measures.

While protoplasts, as plant cells, which are to be grown as exterior materials for transformation into butter, Cell culture cells, cells in tissue tissues, pollen, pollen tubes, oocytes, embryo sacs or zygotes and embryos

Different stages of development are protoplasts because of the direct application without further

Pre-treatments preferred. Another object of the present invention therefore relates to the resulting from the erfindiingsgemäßen process

transformed protoplasts and the resulting plant cells, cell aggregates, embryos, plants and

Seeds, as well as their progeny, which possess the new genes obtained by the transformation and those derived therefrom

have resulting advantageous properties.

Also included are all cross and fusion products of plant material transformed according to the present invention containing the

have the new genes obtained by transformation and have the advantageous properties resulting therefrom.

The inventive method is suitable for the transformation of all plants, in particular those of the systematic groups Angiosperm «· and Gymnosperm ··.

Among the gymnosperms the plants of the class Conifer ·· are of particular interest.

Among the Angiosperm ·· are in addition to the deciduous trees and shrubs of particular interest plants of the families

Solanacoae, CrucHwM, Composite, UH * cm, VHaceae, Chenopodiacea, Rutaceae, Alllecea, Amaryllidac, ..., Atparagaceae,

Orchldaea ·, palm ··, bromeliac ···, Rublaceae, Theacea ·, Musaceae or Gramineae and the order Legumlnosae and especially the family Papllionaceae. Preference is given to representatives of the families Solaneceae, Cruclferae and Gramlnaae. Particularly noteworthy are plants of the genera Nieotinna, Petunia, Hyoscyamus, Brasslca and Lollum, such as Nicotians tabacum, Nteotiana plumbagenifolla, Petunia hybride, Hyoscyamus mutlcus, Brasslca napus, Brassica rapa and Lollum niurtiflorum.

The crops with high crop yields such as corn, rice, wheat, barley, rye, oats or millet are primarily objects of efforts in the field of plant cell transformation.

All plants that can be produced by regeneration from protoplasts can also be transformed using the errindtinpsgemäßen method. Representatives of the Gramineae family (grasses), which also includes cereals, have not yet been genetically manipulated. It has now been demonstrated that with the method of direct gene transformation as described above, gramineous protoplasts, that is to say cereal cells, can also be genetically transformed. In the same way elna is Trensformation of the crops of the genera Solanum, Nlcotiana, Brastiica, Beta, Pisum, Phasaolus, Glycine ·, Hellanthus, Alllum. Avena, Hordeum, Oryzoe, Setarla, Seeal, Sorghum, Trrtlcum, Zea, Muua, Cocos, Cydonla, Pyru ·, Malus, Phoenix. Elaals, Rubus, Fragariu, Prunus, Arachls, Seeale, Panlcum, Sacchnrum, Coffee, Canaille, Muse, Pineapple, VWe or Citrus possible and desired, although the total yields and acreage for this worldwide geiinger

Regeneration of cultured protoplasts to whole plants is described in / 29 / Evans, et al., "Protoplast Isolation and Culture" in Handbook of Plant (Ml Culture, 1: 124-176 (MacMillan Publishing Co. New York 1983 / 30 / MR Davey, "Recent Developments in the Culture and Regeneration of Plant Protoplasts", Protoplasts, 1983 Lecture Proceedings, pages 19-29, (BirkMuser, Basel 1982 "; / 31 / PJ Dale," Protoplast Culture and Plant Regeneration of Cereals and Other Recalcitrant Crops ", in Protoplasts 1983 Lecture Proceedings, pages 31-41, (Blrkhäuser, Basel 1983) and / 32 / H Binding," Regeneration of Plants ", in Plant Pn.toplaste, page 21 -37, (CRC Press, Boca Raton 1985) and / 32 / Potrykus I and Shillto RD, MVhods In Enzymology, Vol.118, Plant Molecular Biology, Eds. A. and H. Weissbach, Academic Press, Orlando, 1986, described.

The regeneration processes differ from plant species to plant species. In general, however, a suspension of transformed protoplasts, cells or tissues containing numerous copies of the introduced genes is first produced. On the basis of these suspensions, the induction of embryo formation can subsequently take place. The development of embryos is allowed to proceed to the stage of maturity and germination, as is the case with naturally occurring embryos. As a rule, however, the protoplasts are stimulated in one of the known culture media for division and cell wall formation. There are finally callus cultures, which by treatment with certain drugs such. B. Auxinen and Cytokinlnen for root · or Sproßbildung can be induced. In addition to these growth substances, the culture media usually contain different amino acids. It proves to be advantageous to add glutamic acid and proline to the medium, especially in the case of non-specific compounds such as maize and alfalfa. Sprouting and rooting occur at the same time.

The iliese obtained Pfiänzchen can then be transferred into soil and cultivated in the same way as normal seedlings.

Efficient regeneration depends primarily on the medium, the genotype and the history of the culture. If these drill variables are adequately controlled, the regeneration is completely reproducible and repeatable.

Due to new developments in the field of in vitro cultivation of plants, primarily in the field of plant regeneration, it is now possible to regenerate whole plants even from representatives of the Gramin family ···, starting from plant prompt residues. Examples of successfully performed regenerator experiments on gramini are i.a. in / 33 / Abdullah, R., et al. Bto / Technology, 4: 1087-1090. / 34 / Fujimura, T., et al., Plant Tissue Culture Lett, 2: 74-76, 1985/35 / Torlyama, K., "T al., Tm" r Appi Qmurt, 73: 16-19, 188, / 36 / Yamada, Y., et al. Plant Cell Rep, 8: 85-88, 1986 (Rice) and in co-pending U.S. Patent Application Serial No. 056,506, filed June 24, 1987, entitled "Regeneration of · ··· plants from protoplast".

The following plants can therefore also be used in the context of the present invention: Lolium, Z-M, Trrtlcum, 8orghurn, 8aceharum, Bromus, Oryza, Avena, Hordeum, S cal and Seiaria.

The detection of transformed genes can be carried out in a manner known per se, for example by means of cross-analysis and molecular-biological investigations, which include, in particular, the "Southern blot" analysis and enzyme activity tests.

As part of the cross-breeding analysis, the mature plants, which were used from transformed plant cell, are first crossed with themselves for semen production. Some of the yarns contain the introduced genes that code for a new and desirable trait, in proportion, that obeys the established inheritance of inheritance.

These seeds can be used to produce plants with new and desired characteristics.

Homozygous lines can be obtained by repetitive self-sterilization and production of inbroth. This' ./ inbreeding! These can then be used to develop hybrids. In this method, an inbred line is crossed with a further inbred line.

Parts, ie can be obtained from regenerated plants, such. B. flowers, seeds, leaves, branches, fruits u. a. are also part of the present invention, provided that these parts include transformed cells. Progeny (including hybrid progeny), varieties and mutants of the regenerated plants are also part of this invention

The "Southern blot" analysis can be carried out, for example, as follows: The DNA taken from the transformed cells or protoplasts, after treatment with restriction enzymes, is subjected to electrophoresis in 1% agarose gel, onto a nitrocellulose membrane (/ 37 / Southern, EM, J. Biol. 98, 503-517 [1975]) and with the DNA to be detected, which is a Nlck translation (/ 38 / Rigby, W..I., Dieckmann, M., Rhodes, C. and P. Berg, J Mol. Biol.

113,237-261, (1977)) (DNA-specific activity of 6χ10 * to 10χχαρ.Γη./μρ), hybridized. The filters

are washed three times for one hour each with an aqueous solution of 0.03 M sodium citrate and 0.3 M sodium chloride at 65 ⁰ C>. The hybridized DNA is visualized by blackening an X-ray film for 24 to 48 hours. An assay for enzyme activity can be explained in more detail in the test for aminoglycoside phosphotransferase (enzyme for kanamycin-specific phosphorylation), for example as follows: callus or leaf pieces (100 to 200 mg) are homogenized in 20 μl extraction buffer in an Eppendorf centrifuge tube. The buffer is a modification of the method described by / 5 / 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 was omitted from bovine blood and 0.1 M sucroas added. Di « ^c ^ .i« Kit. Centrifuge at 12000g for 5 minutes and add bromophenol blue to the supernatant phase to a final concentration of 0.004%. By electrophoresis in a 10%, non-denaturing rOlyacrylamid-GeI proteins are separated from 35μΙ of the supernatant phase. The gel is overlaid with a kanamycin and Y-labeled ATP-containing agarose gel, incubated, and the phosphorylated reaction products are transferred to Whaimanp81 phosphocellulose paper. The paper is washed six times with deionized water at 90 ^° C. and then autoradiographed.

The following examples serve to illustrate the present invention without, however, limiting it. They describe the construction of a hybrid gene and its incorporation into carrier DNA sequences of cyclic character, the transfer of this hybrid gene into plant cell cells, the selection of the transformed plant cells and the regeneration of complete plants from the transformed plant cells, and their cross-linking and molecular biological analysis.

Ausfuhranflstoisptole

In the following examples, the following abbreviations are used:

Media:

HeNa / F (10mMHepes, pH 7.1, SmMCaCI ₂ , 16OmM NaCl, 0.2M mannitol, / 39 / Fromm, M, et al., I1985J)

K ₃ (0.1mg / l2.4D, 1.0mg / INAA, 0.2mg / IBAP; / 40 / Shillito, RDetal. (1981); MI / Nagy.JlandMaliga.P., (19761)

KA ₁ AO (/ 42 / Installe, P. et al., [1985J]

LS (/ 43 / Linsmaler, EM, S ¹ IOOk, F., Physiology Plantarum 18, 1-12, 127, 1965 1)

MaCa (0.4 M mannitol, 15 mM CaCl ₂ × 2 H ₂ O, pH 6.6)

MaMg (0.4-0.5 M mannitol, 16 mM MgCl ₂ , 0.1% MES, pH 5.6)

M (/ 42 / Installe, P. et al., (1985))

MAP ₁ AO (/ 42 / Installe, P. et al., (1985))

MDs (/44/Negrutiu,l.etal.,(1983))

RP (/ 42 / Installe, P. et al., (1985))

R'SA (/ 42 / Inetal, P. et al., (1985))

T (/ 45 / Nltsch, J.P. and C. Nitsch, (1969))

W ₆ (154mM NaCl, 125mM CaCl ₂ x 2H ₂ O, 5mMKCl, 5mM glucose, pH 5.6-6, U / 46 / Menczeletal., (1981l)

chemicals:

BAP Benzylaminopurine

2.4 D (2.4-dichlorophenoxy) acetic acid

NAA Napthylselg'iäure

EDTA ethylenediamine n-N, N, N ', N'-tetraacetic acid

PEG CMS CMS Type Polyethylene Glycol (/ 15 / Nogrutiu, I. el el. (1986a))

PEG 6R R-Type Polyethylene Glycol (/ 10 / Shillito, R.D. et al., (1985))

Tris-HCl a.a.a-tris-fhydroxymethyl-methylamine hydrochloride

NPT-Il gene neomycin 3'-phosphotransferase gene, type II

tables:

ATF Absolute Transformation Frequency

GPPL Gas Office ProtoplaetenzaM

RTF Relative Tranetormatlonsf tquenz

UE Surviving protoplasts after transformation treatment

ZT Zahldei Trensformanten

The percentages given in the following Ausführungsbelspielen are weight / volume information (w / v, volumetric weight)

KuntMSChreibung of the pictures

Figure 1 shows the construction of the plasmid vectors pABD I and pABD II containing the NPT II structures. Figure 2 demonstrates the influence of MgCl ₂ concentration (A) and MgCl ₂ PEC interaction (B) on the transformation rates in Nicotiana taoacum ov Petit Havanna SR ₁ (A and B) and Nlcotlana plubaglntfolla (A). Fig.A Curvea: N.tabaeumSRt

Curve b: N. plumbaginrfolia

Note the semilogarithmic scale of the MgCl ₂ concentration (abscissa). The PEG concentration in this case is 20% (w / v)

Fig. B Curve a: IQmMMgCI ₂

Curve b: 3OmMMgCI ₂

-11- 265

Example 1 Construction of the Plasmid pABDI

The freely accessible pla3mids pkm21 and pkm24 * (/ 47 / Beck, E., et al., Gene 19, 323-336 (1982)) are cut with the restriction endonuclease Psti. The fragment of the plasmids used for recombination is cut are purified by electrophoresis in 0.8% agarose gel., The plasmid pkm21244 obtained from the junction of fragments contains a combination of the 6 'and 3' Bal31 deletions of the NPT-II gene, as from / 47 / Beck In order to link the promoter signal of the cauliflower mosaic virus to the HindIII fragment of the plasmid pkm21244, the coupling plasmid pJPAX is constructed and the coupling plasmid p JPAX is prepared from the plasmids pUCO and pUC9 (/ 28 / Messing, J, and J. Vieira, Gene 19, 266-276 [1982]) 10 base pairs of the coupling sequence of plasmid pUC9 are cleaved at the HindIII and SalI positions and then filled in adhesive ends with the polymerase I Klenow fragment ( / 48 / Jacobsun, K., et al., Eu- J. Blechern. 46,623, [1974]) and connecting *, the polynucleotide chain, thus restoring 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 plasmid pUC8 and the modified plasmid pUC9 yields the plasmid pJPAX with a partially asymmetric coupling sequence with the consecutive restriction sites: EcORI, SMaI, BamHI, Sail, PstI, HindIII, BamHI, X »· ..! and EcoRI. The CaMV gene VI promoter pagion linked to the NPT-II structural gene is derived from the genome d. JaMV strain CM4184, a variant of the CaMV strain CM1841, whose complete nucleotide sequence is / 26 / Gardner KC et al., 1981. The CaMV promoter rogion is cloned into the 6kb cosmid pHC79, a derivative of E. coli plasmid pBR322 (/ 49 / Hohns B. and Collins J., 1980), with the CaMV genome and Cosid pHC79 cleaved with B3t II and the resulting fragments are linked together. The connection of the b'-expression signal of Cauliflower mosaic virus gene Vl and the HindIII fragment of the NPT-II gene is carried out in the plasmid pJPAX (insertion of the promoter region of the cauliflower mosaic virus gene Vl between the Pstl and The resulting plasmid is cut at the unique HindIII position and the HindIII fragment of plasmid pkm21244 is inserted into this cleavage site in both orientational directions to give the plasmids pJPAX Cakm ⁺ and pJPAX Cakm " To create an EcoRV sequence near the 3 'termination signal of the NPT-II hybrid gene, a BamHI fragment of the plasmid pJPAX Cakm ^{4 is} inserted into the BamHI position of the plasmid pBR327 (/ 60 / Soberon, X. et al , Gene 9, 287-306 [1980]). The plasmid pBR327 Cakm is obtained. The EcoRV fragment of the plasmid pBR327 Cakm containing the new DNA construction is used to construct the EcoRV region of the cauliflower mosaic virus -Gen Vl to replace which en de r Rope Position In the plasmid pUC8 is cloned, which places the protein-encoding DNA sequence of the NPT-II gene under the control of the 6 'and 3' expression signals of the cauliflower mosaic god Vl. The plasmids thus prepared are designated pABDI and pABDII (see Figure 1).

Example 2: Transformation of Protoplates of Nkotiana tebacum cv Petit havanna SRI by transfer of the NPT-II gene as a Tall de plasmid pABDI: nH may allow Mg ² VPEG treatment.

Tobacco protoplasts of Nicotians tabacum c.v. Petit Havana are prepared by conventional procedures starting from a tobacco suspension culture (/ 12 / Potrykus I and Shillito RD, Methods in Enzymology, Vo1118, Plant Molecular Biology, ed. A and H., Weissbach, Academic Press, Orlando, 1986) unfolded leaves of β-week old shoot cultures are removed under sterile conditions and thoroughly wetted with an enzyme solution of the following composition:

Enzyme solution: H ₂ O 70ml

Sucrose 13 g

Macerozyme RIO Ig

Cellulaee 2 g

'Onozuka' R10 (Yakult Co. Ltd. .. Japan) Drisallase (Chemical Factory Schweizerholle, Switzerland 0.13g

2 (n-morpholine) -ethanesulfonic acid (MES) 0.5 ml

pH 6.0

The leaves are then cut into squares of 1-2 cm and allowed to float on the abovementioned enzyme solution. The incubation takes place overnight at a temperature of 26 C in the dark. This approach is then easily panned and for further? Incubated for 30 minutes until complete digestion.

This suspension is then filtered through a 100 mm sieve sieve, rinsed with 0.6 M sucrose (MES, pH 5.6) and then centrifuged for 10 minutes at 4 COO-5000 rpm. The protoplasts gather on the surface of the medium, which then peeled off wildly under the protoplasts, ». B. using e 'wr sterilized syringe. The protoplasts graze in a K3A medium (sucrose [102.96 g / l; xyioa ?!10, 25 g / l); 2,4-dichlorophenoxyacetic acid D [0.10 mg / l | I-Naphtylessigsflure [1.00mg / 1]; 6-benzylsminopurine [0.20 mg / l]; pH 6.8 »(/ 12 / Potrykus I and Shillito, R.D. et al., 1981) containing 0.4 M sucrose.

To carry out the transformation experiments, the protoplasts are first washed, counted and then in a We-medium (154 mM NaCl, 126 mM CaCl ₂ 2H ₂ 0.5 mM KCl, SmM glucose, pH 5.6, / 46 / Menciel, L. et al [1981]), which ensures a high survival rate of isolated protoplasts, resuspended at a cell density of 1-2.5 χ 10 * cells per ml. Pie protoplasts are then subjected to an incubation period of 30 minutes at 6 to 8 ° C for dia transformation experiments.

Ku. 7 Before the actual transformation of the isolated protoplasts, the W ₆ medium is replaced by the actual transformation medium. It is a mannitol / magnesium solution (MaMg solution: 0.4-0.5 mM Mannitol, 0.1% MES [morpholineethanesulfonic acid], pH 5.6) with a Mg ²⁺ concentration of 12 to 16 mM , For this purpose, the protoplasts are first separated from the W _s solution by centrifugation at 100 g for 5 minutes and resuspended in the MaMg-Medinm (0.3 ml). Then 66 .mu.l of an aqueous DNA solution containing 5-1 μg of the plasmid pABDI and 50 .mu.C calf thymus carrier DNA are added to this suspension. The latter is a neutral carrier DNA without transforming site, added to this approach in excess to protect against nuclease prediltration of the DNA to be transformed

becomes. After a period of about 0.1 to 10 minutes after the addition of DNA, the application of an aqueous 40% polyethylene glycol solution (w / v) until a final concentration of 24 to 28% (w / v) is reached. Particularly advantageous is the use of PEG before .. CMS type. It is a Ca ^2> containing (0.1 M Ca [NO ₃ I ₂ 4 H ₃ O) 0.4N Mannitlö '<ng, the PEG molecular weight from about 1000 to about 6000 (to a final concentration of 40% w / v). The pH of this solution is at pH 7 to 9 (/ 16 / Neprutiu, I. et al., P986aj)

In the case of Nicotian® tabacum cv Petit Havana SRI, preferably PEG CMS 4000 is used. The pH of the transformation medium is then adjusted to a value of pH 7. This mixture is incubated with occasional panning for 30 minutes at 26 ⁰ C.

When using high PEG concentrations of> 20%, a stepwise dilution with 3 x to 5 times the volume of a 0.2 M CaCl 2 solution is advantageous. Subsequently, the protoplasts treated in the manner indicated are centrifuged off (5 minutes at 100 g) and in fresh K ₃ medium resuspension (0.3 ml protoplast solution in 10 ml fresh K ₃ -M9dium). The further incubation is carried out in 10 ml diameter Petri dishes at 24 ° C. and darkness, the population density being 4 to 8 × 1 f / protoplasts per ml After 3 days, the culture medium per dish is divided into 0.3 parts by volume fresh K diluted ₃ Medlum and incubated for farther 4 days at 24 ⁰ C and 3000 lux artificial light. Nacli total of 7 Togen developed from the protoplasts clones are embedded containing culture medium in 1% agarose solidified, 50 mg / l kanamycin, and after the Cultivar culture method (/ 51 / Shillito, RD et al., Plant Cell Reports, 2,244-247 (1983)) is grown under dunking at 24 ° C. The nutrient medium is replaced every 5 days with fresh, similar nutrient solution ,

Example 3: Regeneration of Kanamydn-resirtent N. tabecum c. v. Petit Havana SR1

After 3 to 4 weeks of continued culture in culture medium containing ktinamycin, the resistant cells of 2 to 3 mm diameter are grown on agar-solidified LS culture medium (/ 43/ünsmaier, EM and Skook, F., Physiol. Plant 18, 1-127 [1965]). containing 0.05 mg / l 2,4-DlChIOrPhOnOXyCSsIgSaUr ?, 2 mg / l l-naphthylacetic acid, 0.1 mg / l 6-benzylaminopurine, 0.1 mg / l kinetin and 76 mg / l kanamycin. By shoot induction on LS medium containing 150 mg / l kanamycin and 0.2 mg / l 6-benzylaminopurine and subsequent rooting on T medium (/ 45 / Nitsch, YP and C. Nitsch, Science 163,85-87 ( 196dl) one obtains Kunemycin-resistant Nicotiana tabacum plants of the Petit Havana SRI variety.

Example 4: Transformation of Nicotiana plumbaginHolia protoplasts by transfer of the NPT-ti gene into the pABDI pasmin with HlH * Mg / PEQ treatment.

The isolation of the protoplastone and the pre-incubation provided for the actual transformation experiments Protoplasts are orally analogous to the method described for Nicotiana tabacum 8R1. The actual transformation of the nested in the manner specified N. plumbaginHolla protoplasts is in a Mannitol / Magneslum solution (MaMgUg: 0.4 to O ₁ SmM mannitol, 0.1% MES (morpholineethanesulfonic acid, pH 5.6) with a Mg ¹⁺ ion concentration from 22 to 27 mM. After the addition of an aqueous DNA solution (65 μΙ) containing 5

up to 10 μg of the pla * mlds pABDI and 50 mg calf thymus carrler DNA, the application of an aqueous 40% iyen

Polyethylene glycol solution (w / v) until a final concentration of 18 to 22% (w / v) is reached. The time between DNA addition and PEG application is usually only about 0.1 to 10 minutes. Also in the hall of Nicotiana plumbaglnrfolia proves the use of PEG CMS 4000 to be particularly advantageous for achieving high Trnnsforr.iationsraten. The pH of the transformation medium is then adjusted to a value of pH 7. It

followed by a about 30-minutlge incubation of these Tran "formation solution boi a temperature of 26 ⁰ C with occasional

Swirling the mixture i. Dia Isolated protoplasts were initially high in medium for a period of 4 to 12 days Hormone concentration ((ΚΑ, ΑΟ medium, / 42 / In parts, P. et al., J. Plant, Phyeiol., 119, 433-454, (1985)) in a ZeH density of 2 to

3 χ 10 * protoplasts / ml cultured.

After the formation of cell aggregates, the surviving colonies are read, counted and in high dilution (0.6

to 2χ10 'colonies / ml in an MDs medium) / 44 / Negrutiu, I. et al., Theor. Appl. Genet. 6, 341-347, (1983)) or a ΜΑΡ-medlum, '42 / Instelfe, P. et al., (19S5J) with low hormone content, containing 20 to 40 mg / l kanamycin sulphate. Subsequently, the colonies are embedded in agar (1% agarose) and cultured according to the, bead-type '/ 61 / Shillito, RD et al., 11983]) culturing method bsi a temperature of 24 ⁰ C and in the dark. The nutrient medium is doing every 5

Days replaced by fresh similar culture solution. Example B: Regeneration Kiinamycin-relted UHr N. plumbsgintfollu plants After a cultivation period of 3 to 4 weeks, the resistant cells are reached in a diameter of 2-5 mm

have read, and cultured on solid media containing 60mg / l kanamycin, another 3 to 5 weeks.

The regeneration of entire plants is carried out according to the information in / 42 / Insole, P. et al. (1985) by transfer of

Reslstenten KaIIi from an RP medium to a R'SA and / or M medium.

Example β: Detection of the NfT-H gene in the plant heritage

0.5 g of callus of the transformed cell culture or leaf tissue of the plants regenerated therefrom are dissolved at ⁰ ° C. in 15% sucrose solution containing 60 mMol / II-butyldiamine-N, N, N, N'-tetra-essig-acid, EDTA, O. , 25mol / l sodium chloride and 60mMol / l aaa-trls-lhydroxyrnethyO-methylamln hydrochloride (TRIS-HCl) homogenized at pH 8.0. Centrifuging the homogenate for 6 minutes at 1000g roughly separates the cell nuclei. Nuclei are dissolved in 16% sucrose solution containing 50 mmol / L EDTA and 50 mmol / l TRIS-HCl, pH 8.0, resuspended at, with sodium dodecyl sulfate to a final concentration of 0.2% \ replaced and for 10 minutes at 7O ⁰ C heated. After cooling to 20 to 25 ⁰ C of potassium acetate is added to a concentration of 0.5 mol / l. This mixture is incubated for 1 hour at ⁰ ° C. The precipitate is separated by centrifugation (16 minutes at 4 ⁰ C in a microcentrifuge). From the supernatant liquid, the DNA is precipitated by adding 2.6 times the volume of ethanol at 20 to 25X. The separated DNA is dissolved in a solution of 10 mM Tris-HCl containing 10 μg / ml ribonuclease A, incubated for 10 minutes at 37 ° C., added with ProMlnase K to a concentration of 260 μg / ml and incubated for a further hour at 37 ° C. ⁰ C incubated. Protainae K is removed by phenol and chloroform / isoamyl alcohol extractions. From the aqueous phase is the

DNA was precipitated by addition of 0.6 volume of 0.6 molar isopropanolic sodium acetate solution and dissolved in 50 .mu.l of a solution containing 10 .mu.MiIZI TRIS-HCl and 5 mmol / l EDTA at pH 7.5. This preparation yields DNA sequences predominantly containing more than 50,000 base pairs. Restriction of the DNA with EcoRV endonuclease, hybridization of the fragments with radiolabelled HindIII fragments of the NPT-Il-Qens and comparison with the p'asmid pABDI shows in a Southern Blof analysis the species-like nature of the NPT-II gene in the cell nucleus DNA of the transformed Nicotiana tabacum and Nicotiana ptumbaginifolia cells

Ereebnlsteil

In the following, the obtained transformants were discussed in connection with various transformation parameters.

The transformation rates achieved are reproduced in the form of "relative transformation frequencies" (RTF) or "absolute transformation frequencies" (ATF).

The relative tranformational frequencies are the ratio between the number of transformants and the surviving fraction (capable of forming colonies) of an unselected cultured protoplast population, whereas the absolute transformation frequency is defined as the ratio between the same number Transformants and the original number of protoplasts that had been characterized as living before transformation. A comparison between the ATF and RTF values provides a good picture of the protoplast survival after performing the individual transformation and selection steps.

Influence of Structure and Concentration of the DNA Used on the TransformatlonsrtteTabitlla 1: Influence of the DNA structure and the Konzcntrationbverhältnisses between plasmid DNA (pABDI) and carrier DNA on the Tiansformationsraten (RTF and ATF) in Nicotiana plumbagtnrfotla protoplasts.

The transformation experiments were performed in a MaCa solution at a PEG CMS4 concentration of 13%.

DNA concentration GPPL UE ZT RTF ATF [Ug / ml) · x10 x10 ⁴ XiO " ⁴ XiO " ⁴ Linear pABDI 10 + 20 2 45 4 0.085 0.02 10 + 50 2 49.6 23 0.464 0.12 50 + 10 2 46.4 1 0,022 0.005 Circular pABDI 10 + 50 2 45.4 0 <00:01 <0.005 50 + 10 2 46.1 0 <0.01 <0.005

The influence of the DNA structure on the transformation rates can be demonstrated by the example of Nicotiana plumbaglnrfolia

It can be seen in the course of the transformation experiments carried out that it is possible to achieve significantly higher transformation frequencies when using linear DNA compared to a circular DNA. In the case of linear DNA, the transformation rates achieved are up to two orders of magnitude higher than the corresponding values when using circular DNA.

Table 1 makes it clear that not only the DNA structure, but also the relationship between plasmid DNA and added carrier DNA play a role for the achievable transformation frequencies.

The best rosultato is achieved when the carrier DNA is significantly in excess relative to the plasmid DNA, with a ratio of 10:60 (plasmid DNA: carrier DNA) being particularly advantageous.

Results obtained with the hens confirm results that could previously be determined for the tobacco system / 10 / Shilllto, R.D. et al., Bio / Technology 3, [1985]).

2. Influence of PEG Concentration on the Transformation Rate

Table 2: Effect of PEG concentration on the transformation rates (RTF and ATF) of Nlcotlana plumbaginrfolia and N. tabacum c. v. Pettt Havana SR1 (C.S.) Protoplasts. The transformer experiments were performed in a Ws-Selz solution at a DNA concentration ratio between plasmid and carrier DNA of 1: S.

PEG Concentrate%, w / v) GPPL S ZT RTF ATF PEG CMS 4 N. plumbaginifolia

8% 0.72 - 2 0.13 0.03

(± 0.02) 13% 1.05 - 7 0.26 0.063

(± 0.035) 20% 0.90 - 22 1.45 0.25

«(± 0.06)

24% 1.0 - 32 2.0 0.32

(± 0:07)

27% 1.0 - 46 3.1 0.46 (± 0.05)

N. tabacum, SRi PEG CMS β 13% 0.33-14-0.28

(± 0.16) 20% 0.33 - 30 - 0.90

(± 0.05) 26% 0.33 - 40 - 1.2

(± 0.08)

Table 2 illustrates the influence of different PEG concentrations on the achievable transformation frequencies in N. tabacum 8ft 1 and N. plumbaginrfolia. In both cases a clear correlation between the two parameters can be established. The increase in PEG concentration from 8% to 27% in the case of N. plumbaginrfolia and from 13% to 26% in N. tabteum SRI is accompanied by an increase in the transformation frequencies achieved, which range from 0.03 χ 10 ⁴ 0.28 χ 10 ~ ⁴ , resp. from 0.46 x 1 (T ⁴ to 1.2 χ 10 ~ ⁴ anteigen.

3. BnfluederMgCtrKoniemrationaufdteTranafotmatiorisrate

Table 3: Influence of the MgCl 2 concentration, the PEG solution used, at high PEG concentrations (> 25%), the wash conditions on the transformation rate of N. tabaeum 8R1 protopiasts. For the transformation experiments, both PEG CMS4 and PEG CMS6 solutions with a final concentration of 20% are used (exceptions are given separately in the table).

Transformation GPPL χ 10 »ZT ATFx 10 '» I.Kontrollen

W ^ kOInMg ²⁺ 0.69 77 0.13 ± 0.03

He Na / F, no Mg ¹⁺ 0.26 265 1.0 ± 0.38 MaMg12mM, 8% PSG6R,

1600V, 3 pulses, 1 Ki) JOnF 0.28 324 1.3 ± 0.26

MaMgSmM, no PEG 0.33 0 As above, + 0.1 M MgCl,

after addition of DNA 0.33 0

2. MgClr addition of non-sticks. before transformation

W, + MaMg (1.1, MgCMmM) 0.37 1033 2.7 ± 0.46 W ₅ + MnMg (1: 1, MgCl ₁ SmM) 0.33 1087 3.3 ± 0.48

MaMgSmM 0.50 2045 4.1 ± 0.51

MaMg 12m M 0.50 2056 4.1 ± 0.86

3. MgClr added 2 h before transformation

MaMgCmM 0.33 382 1.2 ± 0.30

4. PEG assembly (MaMg 12mM)

PEOCMS4 (24%) 0.32 693 2.2 ± 0.56

PEGR6 (24%) 0.32 474 1.6 ± 0 34

6. Watching Requirements (MaMg 12mM; 25% PEG)

CaCl ₂ -2H ₂ O 0.2M 0.32 1127 3.6 ± 0.66

Vj OM βββ 2.1 ± 0.42

* MaMg ... mM indicates the MgCIrKonztotration in a MaMq solution.

a) Applmurtlomzeitpunkt: As can be seen from Table 3, let the Betten result lists with a transformation rate of 4.1 χ 10 "'achieve, if the addition of MgCl * in transformation medium immediately before the actual transformation takes place.

In contrast, if the transformation is carried out only 2 hours after the MgCIr application has taken place, the Transformation rates our still at values of 1.2 χ 10 "'.

Also, a combination of a Mannlt / MgClj solution (MaMg solution) with a Wg salt solution In a mixing ratio of 1: 1 leads to a more or less strong decrease in the transformation frequency as a function of the molar MgClj concentration. Halving, for example, the MgCl 2 concentration of the MaMg solution leads to a decrease in the transformation rate by approximately 20%.

On the other hand, if one compares the results of the control experiments, which are only in the presence of suitable buffer solutions (Weodsr

HeNa / F solution) ("> Fromm, M., et al., Proc. Natl. Acad. ScI. U8A 82, 5842-5848, (1985)), but in the absence of Mg ² 'ions In these cases, the transformation frequencies are lower by an order of magnitude greater than 10. Even with the use of electroparatlon, the high transformations discussed above can not be achieved.

PEG / Mo "Synergism: The synergistic effect of simultaneous administration of Mg ^n- ions and PEQ is very clear in a comparison of the transformation results of the control experiments, which are carried out in the presence of MgCl ₁ (6 mM) but without the addition of PEQ, and the actual Exemplary embodiments in which MgCl ₁ and PEG are used together (Table 3).

Oil control experiments in the presence of MgCl ₂ at a concentration of 6 mM but without the addition of PEG in no case lead to detectable transformation events.

In contrast, when applying PEG (at a final concentration of 20%, immediately after (0.1 to 10 min) of the addition of DNA into the MgCI-containing transformation medium, the above-mentioned high transformation rates of 4.1 x 10 " 'to reach.

These transformation rates are therefore more than a power of ten higher than the comparable results, results when using PEG alone without MgCl ₂ addition (see Table 2).

In contrast to tobacco, where maximum transformation rates are achieved in a concentration range of 12 to 15 mM MgCl ₂ at a concomitant PEG CMS concentration of 28% (Figure 2 B), the opthnum hyperelium in N. plumbagln Kolia is higher than MgCI ₂ values shifted (· Fig. 2A).

Maximum transformation rates are achieved in this coat with MgClj concentrate ions in a range between 20 to 30 mM and a PEG concentration of 20%. At the same time, it is possible to achieve transformer conversion rates of 3.9 χ 10 ~ ⁴ .

PEQ composition: In addition to the parameters mentioned above, the composition of the PEG solution used has an influence on the achievable transformation frequencies. Using PEG CMS / 16 / Negrutiu, I. et al., (1986a) and PEG 6R / 10 / Shillito, RD et al., (1985) under otherwise identical experimental conditions (PEG concentration 24%, MgCl 2 concentration 12 mM), in the latter case significantly higher transformation rates can be achieved, which are higher by a factor of 1.4.

When using high PEG concentrations of 24% (20% in the case of N. plumbaginffol) ·) it is advantageous to wash the treated protoplasts with a 0.2 M CaCl ₂ .2HjOhaitigen solution.

By this measure, the transformation rates can be increased by a factor of 1.7 compared to sinei treatment with a simple saline solution (We solution).

4. Evidence of the transmission of the transformed gene to the sexual progeny and its establishment as normal Pflaniwngen

Extensive genetic cross-linking analysis and detailed molecular biology studies (for example, Southern blot analysis of the DNA of the plant body; study of the enzymatic activity of the ΑΓηΙηοςΙν ^ βΙορηοβρη <ΜΓβηβίθΓββθ, ie "the enzyme for kenamycin-specific phosphorylation) with the genetically modified < Plants (first generation and progeny) have given the following results:

1. The bacterial gene is stably incorporated into the plant genome;

2. It is usually transferred unchanged and regularly to cross-breeding offspring;

3. Its inheritance corresponds to that of a natural, simple, dominant plant gene;

4. Molecular analysis by DNA hybridization and enzyme assay confirms the results of genetic cross-analysis:

6. The genetically modified plants have retained their normal, natural phenotype during treatment; So no undesirable changes have been detected.

These results show that the best way for the targeted genetic modification of plant material has been found with the method according to the invention of direct gene transfer into protoplasts. The genetic change is stable and undesirable changes in the genotype of the plant do not occur.

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Claims (41)

1. A process for the transformation of hereditary genetic material, characterized in that - Plant protoplasts isolated from any plant tissues and cultured in one of the nutrient media commonly used for the cultivation of plant protoplasts; - said protoplasts prior to the actual transformation of 20 minutes to 6 hours in a pre-incubation medium containing Ca ²⁺ -, K ⁺ - and nations itowie a suitable carbon source, at a temperature of 4 bit) 1O ⁰ C preincubated; - The protoplasts then separated from the Vorinkubationsmedium and in the ₄ , actual transformation medium containing, as an essential component, 0.1 to 60 mM Mg ²⁺ ions in the presence or absence of Ca ²⁺ ions, resuspended; Immediately thereafter a DNA sample containing one or more genes under the control of plant-active expression signals and a carrier DNA into which transformation solution enters; Add 0.1 to 10 minutes later a plasma membrane modifying agent and - Protoplasts and DNA sample of said Transformaticnslösung incubated for a period sufficient for the uptake of the DNA into the protoplasts. 2. The method according to claim 1, characterized in that said transformation solution contains Mg ²⁺ ions in a concentration of 10 to 3OmM. 3. The method according to claim 1, characterized in that it is the plasma membrane modifying agent is polythylene glycol. 4. The method according to claim 3, characterized in that the final concentration of polyethylene glycol in said incubation medium is 10% to 30%. 5. The method according to claim 4, characterized in that the final concentration for polyethylene glycol is 20% to 28%. 6. The method according to claim 1, characterized in that the pH value of the incubation solution at pH 5.6 to pH 12 Hegt. 7. The method according to claim 6, characterized in that the pH of the incubation solution is at pH 7 to pH 10. 8. The method according to claim 1, characterized in that the concentration of the DNA sample containing one or more allergens of any origin under the control of plant-active expression signals and a carrier DNA 2 to 20pg / ml. 9. The method according to claim 8, characterized in that the concentration of said DNA sample 5 to 10 mg / ml. 10. The method according to claim 1, characterized in that the transformation solution is additionally added neutral DNA without transforming gene as the carrier DNA in excess. A method according to claim 10, characterized in that said neutral DNA is animal DNA, plant DNA, A-DNA, plasmid DNA or any DNA suitable for carrying out the method. 12. The method according to any one of claims 10 or 11, characterized in that said neutral DNA is calf thymus carrier DNA. 13. The method according to claim 10, characterized in that the concentration of the carrier DNA is 50 to 70 mg / ml. 14. The method according to claim 1, daduceh characterized in that the preincubation of the protoplasts over a period of 20 minutes to 1 hour. 15. The method according to claim 1, characterized in that the co-incubation of protoplasts and DNA in the presence of a plasma membrane modifying agent in the actual Transformationerm / dlum containing as essential constituents 0.1 to 6OmM Mg ²⁺ ions in the presence or absence of Ca ²⁺ ions, over a period of 10 minutes 6 hours. 16. The method according to claim 1, characterized in that one gene ah transforming gene used, Dossen structural part of plant, animal, microbial, viral or synthetic origin lot and whose Expiresionssignale of plant, animal or synthetic origin. 17. The method according to claim 16, characterized in that said transforming gene consists either of genomic ONA from a cDNA or from synthetic DNA. 18. The method according to claim 16, characterized in that said transforming gene is composed of both genomic DNA and cDNA and / or synthetic DNA. 19. The method according to claim 16, characterized in that the transforming gene from gene fragments of several organisms, the various genera listeners, is composed. 20. The method according to claim 16, characterized in that the transforming gene from gene fragments of more than one strain, a variety or a species of the same organism is composed. 21. The method according to claim 16, characterized in that the transforming gene is composed of portions of more than one gene of the same organism. A method according to claim 16, characterized in that said transforming gene has a structural portion conferring a useful and desirable property on the transforming plant. A method according to claim 22, characterized in that said structural gene imparts increased resistance or tolerance to pathogens, herbicides, insecticides and / or other biocides to the plant. A method according to claim 22, characterized in that said structural gene improves the formation and quality of reserve and storage materials in leaves, seeds, tubers, roots and stems. 25. The method according to claim 22, characterized in that said structural gene encodes pharmaceutically acceptable active ingredients. 26. The method according to claim 16, characterized in that said expression signals are derived from genes of plants or plant viruses. 27. The method according to claim 16, characterized in that said expression signals derived from a plant gene encoding the small subunit of Ribulosebisphosphatcarboxylase or the chlorophyll a / b binding protein. 28. The method according to claim 16, characterized in that said expression signals derived from genes of plant viruses. 29. Method according to claim 28, characterized in that it is Cauliflower Mosaic Virus (CaMV). 30. The method according to claim 28, characterized in that it is the 35S expression signals of the CaMV genome. 31. The method according to claim 29, characterized in that it is the 19S expression signals of the CaMV genome. 32. The method according to claim 1, characterized in that are used as plant protoplasts such a single plant species or one of the type subordinate systematic unit. 33. The method according to claim 32, characterized in that said protoplasts are obtained from leaves, seedlings, stems, flowers, roots, pollen or embryos. 34. The method nacrt claim 33, characterized in that they are leaf protoplasts. 35. The method according to claim 32, characterized in that said protoplasts are obtained from cell cultures. 36. The method according to claim 1, characterized in that it is protoplasts of plants of the families Solanaceae, Crucifarae, Composttae, Liliaceae, Vrtaceae, Chenopodiaceae, Rutaceae, Alliaceae, Aaparagaceae, Orchidaceae, Palmae, Bromeliaceae, Rubiaceae, Theaceae, Musaceae or Gramineae or the order Legumlnosae acts. 37. The method according to claim 1, characterized in that it is protoplasts of the families Solanaceae, Cructferae and Qramlneae. 38. The method according to claim 1, characterized in that it is protoplasts of corn, rice, wheat, barley, rye, oats or millet. 39. The method according to claim 1, characterized in that it is protoplasts of the genera Solanum, Nicotiana, Brsssica. Beta, Pisum, Phaseolum, Glycine, Hellanthus, Allium, Avena, Hordeum, Oryzae, Setarla, Seeal, Sorghum, Triticum, Zea, Musa, Cocos, Cydonia, Pyrus, Malus, Phoenix, Elauite, Rubus, Fragaria, Prunu », Arachis, Seeale, Panlcum, Saccharum, Coffee, Camellia, , Musa, pineapple, lemon or citrus. 40. The method according to claim 1 for the transformation of plants, characterized in that they are regenerable from protoplasts. 41. The method of claim 1 for the transformation of plants of the group Nicotlana tabacum, Nicotiana plumbagenifolia, Petunia hybrida, Hyosyamus muticus and Brassica napus by transfer of the NPT-II gene, characterized in that the NPT-II gene with promoter and Terminationssitjnalen the CaMV gene Vl provides this incorporates into the pCU8 plasmid and transfers the resulting chimeric plasmid by Mg ³ VPolyethylenglykolbehandlung in isolated Promptesten of plants of the above group.