Process for controlling plant pests.

The present invention relates to a method for controlling pests on plants. In particular, it relates to a method for controlling pests on plants by exposing the pests to proteinase inhibitors which are biologically synthesized by the plants. The invention also relates to plants which, owing to their content of proteinase inhibitors, inhibit pest infestation or are toxic to pests.

A. Proteinases and their inhibitors

An enzyme is usually referred to as a proteinase which hydrolyzes peptide bonds, thereby destroying molecules which contain such bonds, for example proteins. Proteinases that separate the peptide bond of a terminal amino acid are called "exoproteinases". Enzymes that hydrolyze a non-terminal peptide bond are called "endoproteinases". However, in the context of the present invention, the term is also considered applicable to substances that cleave peptide bonds by mechanisms other than hydrolysis.

Both classes of proteinase show different modes of action. Exoproteinases can cut at either the N-terminal or the C-terminal end of the peptide. Aminopeptidases act at the N-terminal end, carboxypeptidases attack at the C-terminal end.

Endopeptidases are generally more specific in their effects. By convention, these enzymes are divided into four main classes, which reflect the type of hydrolyzed bonds and / or the well surrounding the site of the hydrolysis activity and / or the requirement for a characteristic part in the proteinase.

Serine endoproteinases are characterized by the participation of a serine hydroxyl group of the protein to be degraded in the hydrolysis reaction. As a group, these proteinases are probably best characterized. However, they appear to be far more common in microbial and animal tissues than in plant tissues.

Thiol proteinases, also known as sulfhydryl proteinases, are apparently the most common group of proteinases in plant tissues. They are characterized by the obvious involvement of sulfur in some form in the hydrolysis reaction. A free sulfhydryl group on the cysteine ​​has been identified in the active center of a number of these proteinases.

The acidic proteinases, also known as carboxyl proteinases, are widespread in the plant and animal kingdom. The active sites appear to contain two aspartic acid side chains that share a common proton and a hydroxyl group from a tyrosine residue.

The metal proteinases are generically defined in the context of the present invention in such a way that they include those proteinases which do not fall into one of the above three groups and which need at least one metal ion for their hydrolytic activities. Calcium, zinc and iron are among the metals commonly found in such proteases.

Proteinase inhibitors are substances that act as antagonists by preventing or restricting the activity of proteinases. A large number of classification schemes have been developed to correlate the activities of certain inhibitors with the respective target proteinases and / or other inhibitors. The most common classification correlates the activity of the inhibitor with that of the protein nose. Therefore, the four main classes of proteinase inhibitors are serine proteinase inhibitors, thiol proteinase inhibitors, acid proteinase inhibitors and metal proteinase inhibitors.

Serine proteinase inhibitors occur naturally in various tissue types in plants. They are apparently effective against a very large number of serine proteinases, which can originate from both insects and microorganisms. There is evidence that the target proteinase is inactivated by the formation of a stable proteinase-inhibitor complex that is unable to hydrolyze peptides.

From a mechanistic point of view, the inhibitors of acidic proteinases and metal proteinases are less well understood. They are found throughout the plant kingdom, although they are apparently less ubiquitous than serine proteinase inhibitors. It is believed that thiol proteinase inhibitors act primarily through a mechanism that blocks the characteristic sulfur residue.

Proteinase inhibitors can also be classified based on structural features. A large number of low molecular weight proteinase inhibitors are known, mostly of non-natural, synthetic origin and are suitable as laboratory reagents. A number of naturally occurring, low molecular weight inhibitors have been characterized that come from bacterial and fungal sources. This group includes inhibitors such as the leupeptins, antipaine and pepstatins. Many naturally occurring proteinase inhibitors are actually proteins that are produced directly by gene expression in the cell or that are synthesized in a sequence of chemical, enzymatically controlled reactions, under the control of those enzymes that are produced directly by gene expression in the cell.

B. Proteinase inhibitors in plants

Plant proteinase inhibitors are common in plant tissues, apparently as part of a biological control mechanism to protect the plant from attack by a variety of pests, particularly insects or microorganisms. In response to such an attack, the plant is believed to release one or more proteinase inhibitors which inactivate the proteinases in the attacker's body when the attacker is ingested. Such inactivation very likely interferes with the attacker's metabolic processes, particularly his digestive processes, and thus slows down or inhibits his metabolism.

The addition of proteinase inhibitors to the diet of certain insects has been shown to inhibit their growth. Gatehouse and Boulter (1983) have shown, for example, that the trypsin inhibitor from the bean (Vigna unguiculata) inhibits the development of the larva of Callosobruchus maculatus (spotted bean beetle); the inhibitors from the soybean (Glycine max) and from the lima bean (Phaseolus lunatus) are much less effective. Murdock et al. (1987) have shown that some insects of the order Coleoptera (beetles) experience growth retardation when exposed to thiol proteinase inhibitors.

These results indicate that naturally occurring proteinase inhibitors are suitable means of controlling insects or microbial pests on plants. However, the use of naturally occurring inhibitors on a large scale suffers from a number of disadvantages. Such compounds are typically proteins, and it is known that it is expensive to isolate (or otherwise produce), purify and formulate them in large quantities. Since many of them are rapidly degraded in the environment, they are only active for a short time, which makes repeated applications necessary. In addition to rapid degradation, many proteinase inhibitors are water soluble and washed away after the first rain storm or after the first watering.

The non-specificity of many proteinase inhibitors may be even more disadvantageous. Many inhibitors show activity against very different insects and microorganisms. Accordingly, the use of a single inhibitor against a single pest can very well have a tremendous, unfavorable effect on useful insects and / or microorganisms.

C. In Vivo Synthesis of Proteinase Inhibitors in Plants

As a result of developments in the field of molecular biology, the control of genetic processes that lead to the biological production of proteins in a wide variety of biological organisms is now routine in many cases. Of particular importance in the development of genetic engineering or The recombinant DNA technology is the transplantation of genes from one organism to another, which is quite different in its properties from the original organism, so as to endow the recipient organism with a phenotype that is not characteristic of it.

The transformation of plants has developed more slowly than the transformation of other eukaryotic organisms. Only in recent years have the techniques for transforming plant tissue and regenerating adult fertile plants become more numerous and reliable, so that it has now become possible to transform plants in such a way that they express individual proteins. Quite powerful methods are available today for this purpose.

Given the advances in recombinant DNA technology and the disadvantages associated with the use of proteinase inhibitors in a pure or in a substantially pure form, it is now possible to develop transgenic plants that are capable of biological proteinase -Inhibitors to synthesize the plants with a new or Provide an additional defense system to protect yourself from attack by insects and / or microorganisms, so that no more pesticide has to be applied from the outside.

The disadvantages that have been shown above for the topical application of proteinase inhibitors are completely or at least largely eliminated by the creation of resistant plants. The inhibitor is produced in the plant in a pure form and in sufficient quantity and does not need to be applied or formulated. Degradation in the environment and losses are therefore minimal, if not insignificant. The biological synthesis of a particular proteinase inhibitor also allows some control over its specificity in the target plant.

The ability to provide plants at the genetic level with the ability to express a foreign proteinase inhibitor, that is, a proteinase inhibitor that they do not normally express, has recently been demonstrated (Hilder et al., 1987). In this work, tobacco plants were transformed with the gene encoding the trypsin inhibitor from Vigna unguiculata (eye bean), and in this way the tobacco was conferred resistance to Heliothis virescens.

It is very clear that on the one hand this demonstration allows certain conclusions, but on the other hand it should be borne in mind that tobacco is only a model system that can be easily transformed and is therefore generally used for introductory experiments on plant transformation. However, the present invention is directed to transforming a variety of plants with genes for a variety of proteinase inhibitors, not only for the purpose of model studies but rather for commercial utility.

The range of variation with regard to the plants and the inhibitors in the context of the present invention is huge. The transformation of tobacco with genes encoding non-trypsin proteinase inhibitors is of interest because of its special model character. The transformation of a variety of mono- and dicotyledons with widespread usefulness and significant commercial use represents a significant advance, the use of which is of great importance for the whole field of agriculture. The provision of plants with increased resistance to pests has significant advantages not only for the growers but also for the consumers of these plants, which are suitable as a source of food, feed, ornamental, fiber, energy and pharmaceuticals.

Summary of the invention

The present invention relates to a method for controlling plant pests which is essentially based on exposing the pest to a pesticidally effective amount of a proteinase inhibitor, the inhibitor itself being biologically synthesized as a result of genetic manipulation in the plant, the plant being a mono- or is dicotyledons. The invention also relates to the transgenic plants which contain genes which encode proteinase inhibitors or genes which encode protein precursors of the desired proteinase inhibitors, and preferably to those plants which also express these genes.

The focus of this invention is on transgenic plants which, as a phenotype, have the ability to in vivo synthesize a proteinase inhibitor which is foreign to these plants, the inhibitor preferably being produced in an amount sufficient to control a particular pest and in this way giving the plants a new or improved resistance to this pest is conferred.

Another object is the process for the production of new transgenic monocotyledonous plants (monocotyledons) which have the above-mentioned phenotype.

Another object is the development of new transgenic dicotyledonous plants (dicotyledons) with said phenotype.

Another important object of this invention is a method for controlling plant pests, which he^fexposed to transgenic plants according to the invention.

Brief description of the pictures

<ul id="ul0001" list-style="none"><li>Fig. 1: Construction of pRK252 / Tn903 / Bglll.</li><li>Fig. 2: Construction of pCIB5.</li><li>3 and 4: Construction of pCIB4.</li><li>Fig. 5: Construction of pCIB2.</li><li>Figure 6: Construction of pCIB10, a broad host spectrum plasmid containing the T-DNA boundaries and a plant selection gene.</li><li>Fig. 7: Construction of pCIB710.</li><li>Fig. 8: Construction of pCIB10 / 710.</li><li>9: Sequence of a cystatin gene which is incorporated into a transgenic plant according to the present invention</li><li>FIG. 10: Synthetic gene fragments that are prepared and then combined to form the cystatin gene from FIG. 9.</li><li>Fig. 11: Synthetic gene fragments which are combined with the soybean Kunitz trypsin inhibitor gene.</li></ul>

Brief description of tables A and B

Table A contains a list of plants, ordered by their uses. It was taken from Christie (1987).

Table B lists typical representatives of transgenic plants which contain genes for proteinase inhibitors or for precursors of proteinase inhibitors which are produced according to the present invention and lists typical representatives of insects and other pests to which these plants are resistant or that can be kept under control by these plants. The scope of the invention as described here is in no way limited by this exemplary listing.

DETAILED DESCRIPTION OF THE INVENTION

A. General aspects of the invention

Generally speaking, the present invention is directed to a method of controlling a plant pest that attacks a particular target plant, the method essentially based on exposing that pest to a pesticidally effective amount of a proteinase inhibitor in or on a plant that<ul id="ul0002" list-style="none"><li>(a) said inhibitor is biologically synthesized in the plant as a result of expression of a foreign gene encoding the proteinase inhibitor or as a result of expression of one or more foreign genes encoding one or more precursors of the proteinase inhibitor; in which</li><li>(b) said plant either</li><li>(i) is a monocot, typically selected from the group consisting of cereals, vegetable and tuber-producing plants, oil plants, sugar plants, forage and lawn grasses, fiber and wood-producing plants, and aromatic and spice plants; or</li><li>(ii) is a dicotyledon, typically selected from the group consisting of cereals, protein-producing plants, fruit-supplying plants, vegetable and tuber-supplying plants, nut-supplying plants, oil plants, sugar plants, feed legumes, fiber and wood supplying plants as well as aromatic and aromatic plants.</li></ul>

A second general aspect of the present invention is directed to a method for controlling a plant pest which attacks a particular target plant, which second method is characterized in that the pest is exposed to a pesticidally effective amount of a proteinase inhibitor in or on the plant that inhibitor:<ul id="ul0003" list-style="none"><li>(a) is biologically synthesized in the plant as a result of the expression of a foreign gene encoding the proteinase inhibitor or as a result of the expression of one or more genes encoding one or more precursors of the proteinase inhibitor; and</li><li>(b) is to be selected from the group of non-trypsin proteinase inhibitors consisting of inhibitors of thiol proteinases, metal proteinases, acid proteinases and serine proteinases.</li></ul>

A special aspect of the present invention is directed to a method for controlling a plant pest which attacks a specific target plant, the method essentially being based on exposing the pest to a pesticidally effective amount of a proteinase inhibitor in or on a plant, characterized in that that said inhibitor:<ul id="ul0004" list-style="none"><li>(a) is biologically synthesized in the plant as a result of expression of a foreign gene encoding the proteinase inhibitor or as a result of expression of one or more foreign genes encoding one or more precursors of the proteinase inhibitor; in which</li><li>(b) the inhibitor is selected from the group of proteinase inhibitors consisting of inhibitors of thiol proteinases, metal proteinases, acid proteinases and non-trypsin serine proteinases.</li></ul>

Another aspect of the present invention relates to a transgenic plant that has a foreign gene capable of expressing a proteinase inhibitor, or a foreign gene or group of genes capable of expressing one or more precursors of a proteinase inhibitor , contains, characterized in that<ul id="ul0005" list-style="none"><li>(a) said gene or said gene group comprises at least one coding section which codes for a proteinase inhibitor or one or more precursors of a proteinase inhibitor, the proteinase inhibitor being selected from the group consisting of inhibitors of serine proteinases, thiol proteinases, metal proteinases and acidic proteinases is selected; in which</li><li>(b) said plant either<ul id="ul0006" list-style="none"><li>(i) is a monocot, typically selected from the group consisting of cereals, vegetable and tuber-producing plants, oil plants, sugar plants, forage and lawn grasses, fiber and wood-producing plants, and aromatic and spice plants; or</li><li>(ii) is a dicotyledon, typically selected from the group consisting of cereals, protein-producing plants, fruit-supplying plants, vegetables and tuber-supplying plants, nut-supplying plants, oil plants, sugar plants, feed legumes, fiber and wood supplying plants as well as aromatic and aromatic plants.</li></ul></li></ul>

Furthermore, the present invention also relates to a transgenic plant which has a foreign gene which is capable of expressing a proteinase inhibitor or a foreign gene or a foreign gene group which is capable of expressing one or more precursors of a proteinase inhibitor, contains, characterized in that<ul id="ul0007" list-style="none"><li>(a) said gene or said gene group comprises at least one coding segment which codes for a proteinase inhibitor or one or more precursors of a proteinase inhibitor, the proteinase inhibitor being selected from the group consisting of inhibitors of thiol proteinases, metal proteinases, acidic proteinases and not Trypsin serine proteinases is selected; in which</li><li>(b) said plant either<ul id="ul0008" list-style="none"><li>(i) is a monocot, typically selected from the group consisting of cereals, vegetable and tuber-producing plants, oil plants, sugar plants, forage and lawn grasses, fiber and wood-supplying plants, and aromatic and spice plants; or</li><li>(ii) is a dicotyledon, typically selected from the group consisting of cereals, protein-producing plants, fruit-producing plants, vegetables and tubers-producing plants, nut-producing plants, oil plants, sugar plants, feed legumes, fiber and wood supplying plants, plants containing drugs as well as aromatic and aromatic plants.</li></ul></li></ul>

The present invention also relates to transgenic plants which express a proteinase inhibitor which is suitable for the above-mentioned method for controlling a pest which attacks a target plant.

B. Definitions

In order to ensure a clear and precise linguistic understanding of the description, the claims and the scope of protection, the following terms are defined within the scope of this invention as follows:

Biological synthesis of a proteinase inhibitor:

Synthesis of a proteinase inhibitor in a host cell. This term includes: (1) processes in which an active proteinase inhibitor is produced as a protein as a result of the expression of a gene encoding this proteinase inhibitor; (2) processes in which a translation product is changed post-translationally by enzymatic action; and (3) processes in which a protein or proteins are produced as a result of the expression of a gene or genes, the protein or proteins participating in reactions that convert one or more protein or non-protein precursors into an active proteinase. Form inhibitor. This latter process also includes cases in which exogenous or endogenously occurring substances in the plant are converted into active proteinase inhibitors in subsequent reactions.

Biological synthesis as a result of gene expression:

Synthesis of a product in a host cell that results directly from gene expression (ie as a product of translation) or synthesis of a product that results indirectly from gene expression (ie as a product of chemical reactions carried out by translation products).

Pest control:

Killing a pest or inhibiting its activity so that the host is spared.

Foreign gene or foreign gene group:

A gene or group of genes derived from a host that is different from the one that ultimately shows the desired genotype; or a gene or group of genes derived from the same host, with the proviso that this host is genetically modified in some sense. Changes in this latter situation specifically include, but are not limited to, any exchange in the DNA sequence of the gene or any addition of one or more coding segments encoding an additional phenotype (e.g. an antibiotic marker).

Gene:

A DNA sequence that contains all the sections necessary to translate a protein. The necessary sections include at least a promoter sequence, a coding sequence and a termination signal. These sections can, but need not in any way come from the same source; that is, the term "gene" as used herein includes stretches of DNA derived from the same or from different sources.

Non-trypsin proteinase:

A proteinase, the mode of action of which differs significantly from that of trypsin according to agreed classification schemes or which the person skilled in the art considers to be different. Illustrative, non-limiting examples of this term are proteinases that have two or more modes of action, the most important of which is significantly different from that of a trypsin, although one may be less significant or the least significant as that of a trypsin. A non-trypsin proteinase inhibitor according to the invention at least interferes with the primary mode of action, regardless of any interference with the trypsin mode of action.

Pesticide effective amount:

A lot enough to fight a pest.

Plant:

A plant in the traditional sense, but including plant tissue, whether in a plant or as a cultivated tissue, for example as part of tissue cultures in nutrient media.

Precursors of a proteinase inhibitor:

A substance, optionally from protein, that is enzymatically converted into an active proteinase inhibitor in the host cell or that participates (either as a reactant or as an enzyme) in the synthesis of a proteinase inhibitor. This expression includes, for example, individual compounds or groups of compounds that occur endogenously or exogenously for a host cell. The term also includes enzymes that participate in the synthesis of a proteinase inhibitor from cellular substrates; in this case both the substrate and the enzyme are considered to be a proteinase inhibitor.

Proteinase:

A substance, either a single compound or a group of co-acting compounds, that inactivates a protein. Although the term "proteinase" is interchangeable with "protease" here, the term "proteinase" should preferably be used here and below.

Proteinase inhibitor:

A substance, either a single compound or a group of co-acting compounds, that is capable of inactivating a protein.

Transgenic plant:

A plant that contains at least one DNA sequence, which can be a gene and differs from the corresponding DNA sequence in the analog wild-type plant. Here and below, the expression includes any plant in which any DNA is changed relative to the corresponding DNA of the wild type plant. However, the changed DNA does not necessarily have to give the wild type plant a new phenotype. Transgenic plants in this sense accordingly also include plants with an increased number of gene copies compared to the wild type and plants with modified DNA sequences, which are not necessarily restricted to those which code for proteins.

Functional similarity of inhibitors:

Inhibitors are functionally similar if they can be exchanged for one another without significant deterioration in activity, ie if they have a similar effect and their level of action is of the same order of magnitude.

C. Transgenic Plants and Pest Control Procedures

(1) Transgenic plants containing proteinase inhibitors of all four classes and the use of these inhibitors

The present invention offers the possibility of protecting a number of transgenic monocotyledonous and dicotyledonous plants from plant pests. This protection is based on proteinase inhibitors which are representative of all four common classes of such inhibitors and which are biologically synthesized in these plants. The pests that can be controlled in this way include above all insects, mites, fungi and bacteria.

Target plants which are of particular interest in the context of the present invention are in particular (a) monocots, typically selected from the group of ornamental plants and those plants which are listed in Table A as cereals, vegetables and bulbs-producing plants, oil plants, sugar plants, feed crops and lawn grasses, fiber and wood supplying plants as well as aromatic and aromatic plants are listed; and (b) dicotyledons, typically selected from the group of ornamental plants and those plants which are listed in Table A as cereals, protein-producing plants, fruit-producing plants, vegetable and tuber-supplying plants, nut-producing plants, oil plants, sugar plants , Feed legumes, fiber and wood-supplying plants as well as aromatic and aromatic plants are listed. In contrast, dicotyledons containing drugs are not included in this aspect of the invention.

Preferred monocotyledonous plant types include cereals, plants producing vegetables and tubers, sugar plants, and forage and lawn grasses. Plants of the genera Avena (oats), Hordeum (barley), Oryza (rice), Sorghum (millet), Triticum (wheat), Dactylis (ball grass) and Saccharum (sugar cane) and Zea mays (corn) are particularly preferred.

Plants of the genus Dactylis and Zea mays, particularly Zea mays, are particularly preferred.

Preferred dicotyledonous plant types include fruit-supplying plants, vegetables and tubers-supplying plants, oil plants, sugar plants, feed legumes and fiber and wood-supplying plants. Plants of the genera Lycopersicon (tomato), Solanum (potato), Pisum (pea), Beta (turnip), Glycine (soybean), Brassica (rapeseed and cabbage) and Gossypium (cotton) are particularly preferred. Most preferred are plants of the genera Lycopersicon, Solanum and Gossypium. Another group of preferred plants consists of potatoes, rapeseed, tomatoes, soybeans, peas and cotton. Inhibition can be observed, for example, in transgenic plants which contain at least one foreign gene or a foreign gene group which codes for an inhibitor of a serine proteinase. Inhibitors of at least one serine proteinase are preferably selected from the group consisting of thrombin, plasmin, elastase, kallikrein, subtilisin, cathepsin G, chymase, acrosine, plasminogen activator, Ci-esterase, enterokinase, tryptase, post-proline-cutting enzyme ( Prolyl endoproteinase), ATP-dependent protease, thermitase, mast cell proteinase I and II, Streptomyces griseus proteinase A, Staphylococcus aureus V8 proteinase, Tenebrio a proteinase, urokinase, blood coagulation factors, Complement-activating factors and the serine carboxypeptidases as well as a proteinase, which has a substantial structural or functional similarity to one of the aforementioned.

In addition, the inhibitor can be a trypsin or a chymotrypsin inhibitor. If the proteinase is chymotrypsin, the inhibitor is preferably a potato 1, potato II, tomato I or tomato 11 inhibitor. The inhibition also manifests itself as a result of the in vivo synthesis of other inhibitors of serine proteinases if one or more of these inhibitors is a member of the Bowman-Birk inhibitor family, the soybean-Kunitz inhibitor family, the bovine pancreatic trypsin (Kunitz ) Inhibitor family, the Kazal-Trypsin inhibitor family, the Streptomyces subtilisin inhibitor family, the potato inhibitor I family, the potato inhibitor II family, the ai-proteinase inhibitor family, the Hirudin family, the Bdellin family, the Eglin family, the Inter-ai-Trypsin inhibitor family, the Serpin superfamily, the Ci inhibitor family, the Ascaris inhibitor family, the Leupeptine, the Antipaine, Elastinal and is chymostatin. Inhibitors are included which have a substantial structural or functional similarity to one of the aforementioned inhibitors.

Particularly preferred serine proteinase inhibitors are the soybean Kunitz trypsin inhibitor, ai-antitrypsin (an example of the α1-proteinase inhibitor family), Eglin C and Eglin C mutants, especially Eglin C (Arg 45).

A preferred group of inhibitors that are biologically synthesized in a transgenic plant and that are useful in pest control, particularly insect control, consists of the thiol proteinase inhibitors. Preferred examples of this are the inhibitors of papain, bromelain, ficin, calpain, cathepsin B, cathepsin C, cathepsin L, cathepsin H, cathepsin S, chymopapain, clostripain, asclepain, prolyl endopeptidase, pyroglutamyl peptidase, dipeptyl proteinase -Proteinase B, Streptococcus Proteinase, Staphylococcus Thiolproteinase and Actinidin as well as proteinases, which have a substantial structural or functional similarity to one of the aforementioned.

Particularly preferred inhibitors are: cystatin, calpastatin, bromelain inhibitor, antipain, leupeptin, chymostatin and E64 or a descendant thereof, and inhibitors which have a substantial structural or functional similarity to the aforementioned. E64 is the common name for [N- (L-3-transcarboxyoxiran-2-carbonyl) -L-leucyl] -amido (4-guanido) butane. For descendants of E64, the group -NH- (CH₂)₄-NH-C (= NH) -NH₂ by different alkyl groups or by groups such as -NH- (CH₂)₄-NH₂, -NH- (CH₂)₂-CHs, -NH- (CH₂)₇-NH₂ or -0₂CCH = CH-CO-NHCH (i-propyl) -CO-NH- (CH₂)₂-CH- (CHs) 2 replaced. The preferred inhibitors of the cystatin type include, for example, those from the group: chicken egg white cystatin, human cystatin A, human cystatin B, human cystatin C, human cystatin S, rat cystatin a, rat cystatin β and kininogen, for example L-kininogen and H-kininogen.

A particularly preferred thiol proteinase inhibitor is egg white cystatin.

In addition, transgenic plants can be resistant to plant pests if they produce a metal proteinase inhibitor by in vivo synthesis. The preferred inhibitors of this type include, in particular, those which inhibit carboxypeptidase A, carboxypeptidase B, aminopeptidase, collagenase, calcium-dependent neutral proteinase, thermolysin, angiotensin-converting enzyme, renal dipeptidase, enkephalinase, gelatinase and keratinase, or inhibitors of proteinases, which have an essential structural or functional similarity to the aforementioned proteinases.

Particularly preferred inhibitors are: potato carboxypeptidase inhibitor, mammalian collagenase inhibitor, &lt;X.2 macroglobulin, tissue bradykinin-enhancing peptide, phosphoramidon, bestatin and amastatin, and inhibitors which have a substantial structural or functional similarity to the above-mentioned inhibitors.

Finally, inhibition is also observed when the inhibitor inhibits an acidic proteinase. Preferred inhibitors of this type inhibit pepsin, renin, cathepsin D, chymosin, penicillinopepsin and Scytalidium acidic protease B as well as inhibitors of proteinase which are structurally or functionally similar to one of the aforementioned.

Within this group, particular preference is given to: pepstatin, Ascaris carboxyl proteinase inhibitor, Bauhinia pepsin inhibitor, Scopolia japonica proteinase inhibitor and potato cathepsin D inhibitor and an inhibitor which has a substantial structural or functional similarity to the abovementioned inhibitors.

Typical plant pests include insects, mites, fungi and bacteria; insects are of particular importance. Table B lists some preferred representatives of target plants. They are genetically manipulated, produce the specified proteinase inhibitors and are therefore able to fight the specified target insects. This exemplary listing is in no way intended to limit the invention.

Primary target pests of monocots, for example maize, are representatives of the orders Coleoptera (beetle) and Lepidoptera (butterflies), in particular of the genera Diabrotica, Diatraea, Ostrinia and Heliothis. Primary target pests of dicotyledons, such as potatoes, rapeseed, tomatoes, soybeans, peas or cotton, are also representatives of the Coleoptera and Lepidoptera orders, in particular of the genera Diabrotica, Diatraea, Ostrinia, Heliothis, Spodoptera and Anthonomus.

The proteinase inhibitors can be expressed in any part of the plant, for example in the roots, stems, leaves, seeds or pollen of the plant. The proteinase inhibitor is preferably expressed in the part of the plant which is the primary point of attack of the pest to be controlled.

(2) Transgenic plants containing non-trypsin proteinase inhibitors and the use of these inhibitors

The present invention also provides means for protecting transgenic single and double cotyledon target plants from plant pests, or for controlling the pests by means of transgenic plants. This protection is based on proteinase inhibitors that are representative of all four common classes and that are biologically synthesized in these plants. In particular, this protection is based on inhibitors of non-trypsin proteinases of all four classes, preferably on inhibitors of non-trypsin serine proteinases. Pests that can be controlled in this way are predominantly insects, mites, fungi and bacteria. Plants of particular interest in this aspect of the present invention, as already mentioned, include mono- and dicotyledons from Table A and are typically selected from the group consisting of cereals, protein-producing plants, fruit-producing plants, vegetables and tubers. supplying plants, nut-supplying plants, oil plants, sugar plants, forage and lawn grasses, forage legumes, fiber and wood-supplying plants, as well as aromatic and aromatic plants and, in addition, drug-containing plants.

Preferred monocotyledon plant types include cereals, vegetable and tuber-producing plants, sugar plants and forage and lawn grasses. Plants of the genera Avena (oats), Hordeum (barley), Oryza (rice), Sorghum (millet), Triticum (wheat), Dactylis (ball grass) and Saccharum (sugar cane) and Zea mays (corn) are particularly preferred. Most preferred are plants of the genus Dactylis and Zea mays.

Preferred dicotyledonous plant types include fruit-supplying plants, vegetables and tubers-supplying plants, oil plants, sugar plants, feed legumes, and fiber and wood-supplying plants and drug-containing plants. Plants of the genera Lycopersicon (tomato), Solanum (potato), Pisum (pea), Beta (turnip), Glycine (soybean), Brassica (rape and cabbage), Gossypium (cotton) and Nicotiana (tobacco) are particularly preferred. Plants of the genera Lycopersicon, Solanum, Gossypium and Nicotiana are most preferred.

Inhibition can be observed, for example, in transgenic plants which contain at least one foreign gene or a foreign gene group which codes for an inhibitor of a non-trypsin serine proteinase. Inhibitors of at least one non-trypsin serine proteinase, selected from the group consisting of thrombin, plasmin, elastase, kallikrein, subtilisin, cathepsin G, chymase, acrosine, plasminogen activator, CI esterase, enterokinase, tryptase, post-proline, are preferred cutting enzyme (prolyl endoproteinase), ATP-dependent protease, thermmitase, mast cell proteinase and II, Streptomyces griseus proteinase A, Staphylococcus aureus V8 proteinase, Tenebrio a proteinase, urokinase, Blood coagulation factors, complement-activating factors and the serine carboxypeptidases as well as a proteinase, which has a substantial structural or functional similarity to one of the aforementioned. In addition, the inhibitor can be a chymotrypsin inhibitor. If the proteinase is chymotrypsin, the inhibitor is preferably a potato 1, potato II, tomato 1 or tomato 11 inhibitor.

The inhibition also manifests itself as a result of the in vivo synthesis of other inhibitors of non-trypsin serine proteinases when one or more such inhibitors is a member of the Bowman-Birk inhibitor family, the Streptomyces subtilisin inhibitor family, the potato inhibitor I family, the potato inhibitor 11 family, the ai proteinase inhibitor family, the hirudin family, the Bdellin family, the Eglin family, the inter-u1-trypsin inhibitor family, the serpin superfamily, the α-inhibitor family, the Ascaris inhibitor family, the Leupeptine, the Antipaine, Elastinal or Chymostatin. Inhibitors are included which have a substantial structural or functional similarity to one of the aforementioned inhibitors.

Particularly preferred non-trypsin serine proteinase inhibitors from this aspect of the invention are Eglin C and Eglin C mutants, especially Eglin C (Arg 45).

A group of inhibitors preferred in this particular aspect of the invention, which are biologically synthesized in a transgenic plant and which are useful in pest control, particularly of insects, contains, as already mentioned, an inhibitor of a thiol proteinase. Preferred and particularly preferred examples of such inhibitors are listed in the preceding sections and are also preferred from this point of view of the invention.

In addition, transgenic plants can be resistant to pests if an inhibitor of a metal proteinase is produced by the in vivo synthesis. Preferred and particularly preferred inhibitors from this aspect of the invention are those mentioned as preferred in the preceding paragraphs. Finally, inhibition is observed when the inhibitor is an acid proteinase inhibitor. In this aspect of the invention, preferred and particularly preferred inhibitors are also those which are mentioned as preferred in the preceding sections.

Typical plant pests include insects, mites, fungi and bacteria; insects are of particular importance; Table B lists some preferred representatives of target plants. They are genetically manipulated, produce the specified proteinase inhibitors and are therefore able to fight the specified target insects. This exemplary listing is in no way intended to limit the invention. Typical pests to be controlled are mentioned in the previous sections.

D. DNA sequences

In the transformation of plants which is necessary to produce the transgenic plants according to the invention and to use the method according to the invention, DNA sequences which code for the proteinase inhibitors or precursors of the proteinase inhibitors are used for the manipulation. Accordingly, this invention also relates to the DNA sequences used according to the invention. The transformations are described in detail below.

Generically speaking, the invention relates to an essentially pure DNA sequence which comprises a coding sequence for a proteinase inhibitor and which is selected from the group consisting of inhibitors of thiol proteinases, metal proteinases, acid proteinases and non-trypsin serine proteinases, or a coding sequence for one or more precursors which participate in the biological synthesis of a proteinase inhibitor, preferably a non-trypsin proteinase inhibitor, selected from the group consisting of thiol proteinases, metal proteinases, acidic proteinases and serine proteinases. In addition, the invention relates to an essentially pure DNA sequence which encodes a proteinase inhibitor, selected from the group consisting of inhibitors of thiol proteinases, metal proteinases, acid proteinases and non-trypsin serine proteinases.

The invention is also to be understood to include vectors bearing sequences encoding proteinase inhibitors or precursors of proteinase inhibitors and those sequences which have been isolated in substantially pure form.

Preference is given to sequences for inhibitors which are active against plant pests, in particular insects, mites, fungi and bacteria. Of particular importance are those sequences that code inhibitors or precursors of inhibitors that are effective against insects.

These are typically sequences which either originate from an animal, a bacterium, a fungus or a plant which belongs to a species other than the target plant or which have an essential sequence homology with proteinase inhibitor genes which originate from one of the abovementioned organisms .

In cases where the invention is directed to an inhibitor of a non-trypsin serine proteinase, it is an inhibitor selected from the group consisting of inhibitors of chymotrypsin, thrombin, plasmin, elastase, kallikrein, subtilisin, cathepsin G, chymase, acrosine, plasminogen activator, CI esterase, enterokinase, tryptase, post-proline cutting enzyme (prolyl endoproteinase), ATP-dependent protease, thermitase, mast cell proteinase I and II, Streptomyces griseus proteinase A, Staphylococcus aureus V8 proteinase, Tenebrio a proteinase, urokinase, blood coagulation factors, complement-activating factors and the serine carboxypeptidases as well as an inhibitor of a proteinase that has a substantial structural or functional similarity to one of the above.

As previously mentioned, the invention also encompasses DNA sequences encoding one or more precursors that participate in the biological synthesis of a proteinase inhibitor, said inhibitor being selected from the group consisting of thiol proteinases, metal proteinases, acidic proteinases and serine proteinases . In this case, the serine proteinase is selected from the group consisting of trypsin, chymotrypsin, thrombin, plasmin, elastase, kallikrein, subtilisin, cathepsin G, chymase, acrosine, plasminogen activator, CI esterase, enterokinase, tryptase, post-proline cutting enzyme (prolyl endoproteinase), ATP-dependent protease, thermmitase, mast cell proteinase and 11, Streptomyces griseus proteinase A, Staphylococcus aureus V8 proteinase, Tenebrio a proteinase, urokinase, blood coagulation factors, Complement activating factors and the serine carboxypeptidases and a proteinase that has a substantial structural or functional similarity to one of the above.

More specifically, it can be one or more sequences that encode precursors that result in an inhibitor that is a member of the Bowman-Birk inhibitor family, the soybean Kunitz inhibitor family, the bovine pancreatic trypsin (Kunitz) Inhibitor family, the Kazal-Trypsin inhibitor family, the Streptomyces subtilisin inhibitor family, the potato inhibitor I family, the potato inhibitor II family, the ai-proteinase inhibitor family, the Hirudin family, the Bdellin family, the Eglin family, the Inter-ai-Trypsin inhibitor family, the Serpin superfamily, the CI inhibitor family, the Ascaris inhibitor family, the Leupeptine, the Antipaine, the Is elastinal or chymostatin, or an inhibitor of chymotrypsin, or an inhibitor that is essentially structurally or functionally similar to a chymotrypsin inhibitor. In the latter case, it can be a sequence which codes the potato I, potato II, tomato I or tomato 11 inhibitor.

Another object of the invention is that the DNA sequence encodes a proteinase inhibitor or one or more precursors of a proteinase inhibitor that belongs to the class of thiol proteinase inhibitors. Preferred examples of this are the inhibitors of papain, bromelain, ficin, calpain, cathepsin B, cathepsin C, cathepsin L, cathepsin H, cathepsin S, chymopapain, clostripain, asclepain, prolyl endopeptidase, pyroglutamyl peptidase, dipeptyl proteinase -Proteinase B, Streptococcus Proteinase, Staphylococcus Thiolproteinase and Actinidin as well as proteinases, which have a substantial structural or functional similarity to one of the aforementioned.

From this point of view, an inhibitor is particularly preferred which is either a cystatin, calpastatin, bromelain inhibitor, antipain, leupeptin, chymostatin or E64 or a descendant thereof, or one which has a substantial structural or functional similarity to it. In cases where the inhibitor is a cystatin, it can be selected from the group consisting of egg white cystatin, human cystatin A, human cystatin B, human cystatin C, human cystatin S, rat cystatin a, Rat cystatin β and kininogen, for example L-kininogen and H-kininogen.

Furthermore, the DNA sequence can be a sequence which encodes a proteinase inhibitor or one or more precursors of a proteinase inhibitor, the proteinase being a metal proteinase. For example, it can be the inhibitor of carboxypeptidase A, carboxypeptidase B, aminopeptidase, collagenase, calcium-dependent neutral proteinase, thermolysin, the angiotension-converting enzyme, renal dipeptidase, enkephalinase, gelatinase or keratinase or an inhibitor of a proteinase which has an essential structural or functional similarity to one of the aforementioned.

From this point of view, particular preference is given to DNA sequences encoding inhibitors or one or more precursors of inhibitors, and the inhibitors preferably from the group consisting of potato carboxypeptidase inhibitor, mammalian collagenase inhibitor, a₂-Macroglobulin, tissue bradykinin-enhancing peptide, phosphoramidon, bestatin and amastatin as well as inhibitors which have a substantial structural or functional similarity to the above.

Finally, the invention also relates to DNA sequences which encode a proteinase inhibitor or one or more precursors of a proteinase inhibitor which is an inhibitor of an acidic proteinase. Of particular interest from this point of view are those cases in which it is an inhibitor of pepsin, renin, cathepsin D, chymosin, penicillinopepsin or scytalidium acidic protease B or an inhibitor of a proteinase that is essentially structurally or functionally similar to a the aforementioned has. Particularly preferred are those cases in which the inhibitor is a pepstatin, Ascaris carboxyl proteinase inhibitor, Bauhinia pepsin inhibitor, Scopolia japonica proteinase inhibitor or potato cathepsin D inhibitor or has a substantial structural or functional similarity to one of these inhibitors.

E. Vectors

Vectors which are produced by standard methods and which contain the DNA sequences described above represent an additional subject of the invention. Vectors are recombinant DNA sequences which are used for the isolation and propagation purposes of the DNA sequences mentioned and for the transformation of suitable hosts can be used with these sequences. Preferred vectors for isolation and propagation are plasmids which can be propagated in a suitable host microorganism, for example E. coli. Preferred vectors for the transformation are those which are suitable for the transformation of plant cells or of Agrobacterium. In particular, in cases where plant cells, not protoplasts, are to be transformed, the preferred vector is a vector derived from the Ti plasmid. Any of the vectors mentioned can be used for direct gene transfer in protoplasts. Suitable vectors that can be used as starting material are state of the art. Suitable vectors for the transformation of plant tissue and protoplasts are described in de Framond et al. (1983), An et al. (1985), Potrykus et al. (1985) and Rothstein et al. (1987). In addition, many other vectors are known which are suitable as a starting material in the context of the present invention.

The vectors can be constructed and propagated in a suitable host, for example in E. coli. Suitable E. coli strains include HB101, JM83, DH1, DH5a, LE392, etc. The vectors according to the invention can be used as such in direct gene transfer or in microinjection technology. In certain cases it may be advantageous to linearize the vector before use. Alternatively, the vectors can be transferred to an Agrobacterium host. This transfer is accomplished by conventional techniques such as "biparental mating" (Simon et al., 1983b), "triparental mating" (Ditta et al., 1980) or transformation (Holsters et al., 1978). Suitable Agrobacterium strains include, but are not limited to, A. tumefaciens LBA4404, CIB542 and C58Z707.

Preferred vectors are those comprising the preferred DNA sequences mentioned above. Vectors which function in plant cells or in Agrobacterium are also preferred. The vectors described in the examples are particularly preferred.

F. Manufacturing process

The invention also encompasses a method for producing the DNA sequences mentioned, in which the DNA sequence is isolated from a naturally occurring source, optionally mutated, or synthesized chemically or enzymatically.

The invention also encompasses a process for the preparation of the vectors mentioned which contain a DNA sequence as defined above, characterized in that the said DNA sequence is inserted into a vector which functions in plant cells or in Agrobacterium.

Furthermore, the invention comprises a method for the production of transgenic plants as defined above, characterized in that a plant cell is transformed with a vector which contains a DNA sequence according to the invention or is cocultured with Agrobacterium which contains such a vector, and in that the plant cell is regenerated into a plant.

Preferred methods are those which lead to the preferred DNA sequences, vectors and transgenic plants according to the invention. The methods described in the examples are particularly preferred.

EXAMPLES

The following examples serve to illustrate both general and specific aspects of the invention. They only serve to explain the invention and have no limiting character. The scope of the disclosure or claims is in no way restricted thereby.

<ul id="ul0009" list-style="none"><li>ABBREVIATIONS</li><li>Bp: base pairs.</li><li>2,4-D: 2,4-dichlorophenoxyacetic acid.</li><li>Dicamba: 3,6-dichloro-2-methoxybenzoic acid.</li><li>EDTA: 1-ethylenediamine N, N, N ', N'-tetraacetic acid.</li><li>kbp: kilobase pairs.</li><li>MES: 2- (N-morpholino) ethanesulfonic acid.</li><li>MW: molecular weight.</li><li>NAA: a-naphthalene acetic acid.</li><li>PEG: polyethylene glycol.</li><li>SDS: sodium dodecyl sulfate.</li><li>Tris-HCl: Tris (hydroxymethyl) methylamine hydrochloride.</li><li>Rpm: revolutions per minute.</li></ul>

MEDIA

SH-O medium:

Medium by Schenk and Hildebrandt (1972); without hormones. SH medium can be liquid or solid, solidified by 0.8% agar or 0.5% GeIRite®. The medium is usually heat sterilized by autoclaving at about 110 to 121 ° C for 15 to 20 minutes.

SH-30 medium:

Sh-O medium containing 30 µM dicamba.

SH-45 medium:

SH-O medium containing 45 µM dicamba.

MS medium and OMS medium:

Media from Murashige and Skoog (1962). The media can be solidified with 0.8% agar or agarose or with 0.5% GeIRite®.

Beasley and Ting Medium (germination medium for embryos):

Medium according to Beasley and Ting (1973).

KM-8p medium:

This medium includes macro elements, micro elements and Fe-EDTA as described by Kao and Michayluk (1975) as well as the following organic compounds: Biotin (0.01 mg / liter), pyridoxine-HCl (1 mg / liter), thiamine-HCl (10 mg / liter), nicotinamide (1 mg / liter), nicotinic acid (0.1 mg / liter), folic acid ( 0.4 mg / liter), D-Ca pantothenate (1 mg / liter), p-aminobenzoic acid (0.02 mg / liter), choline chloride (1 mg / liter), riboflavin (0.2 mg / liter), Vitamin B12 (0.02 mg / liter), glycine (0.1 mg / liter), sucrose (0.25 g / liter), glucose (68.4 g / liter), mannitol (0.25 g / liter) , Sorbitol (0.25 g / liter), cellobiose (0.25 g / liter), fructose (0.25 g / liter), Mannose (0.25 g / liter), rhamnose (0.25 g / liter), ribose (0.25 g / liter), xylose (0.25 g / liter), myo-inositol (0.1 g / liter) ), Citric acid (40 mg / liter), fumaric acid (40 mg / liter), malic acid (40 mg / liter), sodium pyruvate (20 mg / liter), adenine (0.1 mg / liter), guanine (0, 03 mg / liter), thymidine (0.03 mg / liter), uracil (0.03 mg / liter), hypoxanthine (0.03 mg / liter), cytosine (0.03 mg / liter) glutamine (5.6 mg / liter), alanine (0.6 mg / liter), glutamic acid (0.6 mg / liter), cysteine ​​(0.2 mg / liter), asparagine, Aspartic acid, cystine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine (each 0.1 mg / liter).

The solution is filter sterilized. The final pH 5.8. The macro elements are prepared as a 10-fold concentrated, the micro-elements as a 1000-fold concentrated stock solution. Citric, fumaric and malic acid as well as sodium pyruvate are prepared as a 100-fold concentrated stock solution and mixed with NH₄0H adjusted to pH 6.5. Adenine, guanine, thymidine, uracil, hypoxanthine and cytosine are prepared as a 1000-fold concentrated stock solution and mixed with NH₄0H adjusted to pH 6.5. The amino acids are made up as a 10-fold concentrated stock solution (pH 6.5 with NH₄0H) added to get the final concentrations. The vitamin stock solution is usually prepared as a 100-fold concentrated solution.

N6 medium:

This medium includes macro elements, micro elements and Fe-EDTA as described by Chu et al. (1975) and the following organic compounds: pyridoxine-HCl (0.5 mg / liter), thiamine-HCl (0.1 mg / liter), nicotinic acid (0.5 mg / liter), glycine (2.0 mg / liter), and sucrose (30 g / liter). The solution is autoclaved. The final pH is 5.6. Macro elements are prepared as a 10-fold concentrated stock solution and micro-elements as a 1000-fold concentrated stock solution. The vitamin stock solution is usually prepared as a 100-fold concentrated solution.

YEB Medium:

5 g / liter beef extract, 1 g / liter yeast extract, 5 g / liter peptone, 5 g / liter sucrose, adjusted to pH 7.2 with NaOH, 2 mM MgCl₂-Addition after autoclaving.

Agarose:

The production and cleaning are described for example by Guiseley and Renn (1975). Agarose is one of the components of agar. Commercially available agar usually consists of a mixture of neutral agarose and ionic agaropectin with a large number of side groups. Usually a certain number of side chains remain intact and determine the physicochemical properties of agarose, such as gel formation and melting temperature. Agarose that melts at low temperatures, especially SeaPlaque * agarose, is a preferred strengthening agent in the process described here.

Casein hydrolyzate:

Casein Hydrolyzate - Enzymatic hydrolyzate of bovine milk, Type 1, Sigma Co., St. Louis, MO, USA.

Cellulase RS and R-10:

Yakult Honsha Co. Ltd., Tokyo, Japan.

GeIRite®:

GeiRite Gellan Gum, Scott Laboratories Inc., Fiskersville, RI, USA.

Nalgene® filters:

Nalge Co., division of Sybron Corp., Rochester, New York, USA.

Pectolyase Y-23®

Seishin Pharmaceutical Co. Ltd., Tokyo, Japan.

Parafilm®:

Parafilm⁰ Labor Film - American Can Co., Greenwich, CT, USA.

GENERAL PROCEDURE RECOMBINANT DNA TECHNOLOGY

Since many of the techniques used in the context of this invention are routine for the person skilled in the field of recombinant DNA technology, a brief description of the generally used methods is given below so that they do not have to be specified again and again in the following specific exemplary embodiments. All of these routine procedures are described in Maniatis et al. (1982) unless otherwise noted.

A. Cutting with restriction endonucleases.

Typically, about 50 to 500 µg / ml DNA is contained in the reaction mixture in the buffer solution recommended by the manufacturer, New England Biolabs, Beverly, MA. 2 to 5 units of restriction endonuclease are added for each µg DNA and the reaction mixture is incubated at the temperature recommended by the manufacturer for one to three hours. The reaction is stopped by heating at 65 ° C. for 10 minutes or by extraction with phenol; precipitation of the DNA with ethanol follows. This technique is also described on pages 104-106 of the Maniatis et al. Reference.

B. Treatment of DNA with polymerase to produce blunt ends.

DNA fragments are added to a reaction mixture in the buffer recommended by the manufacturer, New England Biolabs, in a concentration of 50 to 500 µg / ml. The reaction mixture contains all four deoxynucleotide triphosphates in a concentration of 0.2 mM. The reaction takes place at 15 ° C. for 30 minutes and is then ended by heating to 65 ° C. for 10 minutes. For fragments obtained by cutting with restriction endonucleases that produce 5 'protruding ends, such as EcoRI and BamHI, the large fragment, or Klenow fragment, of the DNA polymerase is used. T4 DNA polymerase is used for fragments obtained from endonucleases that produce 3 'protruding ends, such as Pstl and Sacl. The use of these two enzymes is described on pages 113 to 121 of the Maniatis et al. Reference.

C. Agarose gel electrophoresis and purification of DNA fragments from the gels.

Agarose gel electrophoresis is carried out in a horizontal apparatus as described on pages 150-163 of the Maniatis et al. Reference. The Tris-borate buffer described there is used as a buffer. The DNA fragments are stained with 0.5 µg / ml ethidium bromide, which is either already present in the gel or tank buffer during the electrophoresis or is only added after the electrophoresis. The DNA is made visible by illumination with short or boring ultraviolet light. If the fragments are to be separated from the gel, agarose is used which can be gelled at low temperature and obtained from Sigma Chemical, St. Louis, Missouri. After electrophoresis, the desired fragment is cut out, placed in a plastic tube, heated to 65 ° C. for about 15 minutes, extracted three times with phenol and precipitated twice with ethanol. This method is compared to that of Maniatis et al. described on page 170 changed slightly.

D. Attaching synthetic linker fragments to DNA ends.

If a new endonuclease interface is to be added to the end of a DNA molecule, the molecule may first be treated with DNA polymerase to create blunt ends, as described in Section B. About 0.1 to 1.0 µg of this fragment is added to about 100 ng of phosphorylated linker DNA (New England Biolabs), which is in a volume of 20 11.1 to 30 µl of one recommended by the manufacturer, together with 2 µl of T4 DNA ligase (New England Biolabs) and 1 mM ATP. After overnight incubation at 15 ° C, the reaction is terminated by heating at 65 ° C for 10 minutes. The reaction mixture is diluted to about 100 μl in a buffer suitable for the restriction endonuclease which cuts the synthetic linker sequence. Approximately 50 to 200 units of this endonuclease are added to this approach. The mixture is incubated for 2 to 6 hours at the appropriate temperature, then the fragment is subjected to agarose gel electrophoresis and purified as described in Section C. The resulting fragment should now have endings as generated by cutting with the respective restriction endonuclease. These ends are usually cohesive so that the resulting fragment can now be easily linked to other fragments that have the same cohesive ends.

E. Removal of 5'-terminal phosphates from DNA fragments.

Recircularization of a vector during plasmid cloning can be reduced by treating the vector plasmid with phosphatase (discussed on page 13 of the Maniatis et al. Reference). After digesting the DNA with the correct restriction endonuclease, a unit of alkaline phosphatase from the intestine of calves is added, which can be obtained from Boehringer-Mannheim, Indianapolis, IN. The DNA is incubated at 37 ° C for one hour and then extracted twice with phenol and precipitated with ethanol.

F. Linking the DNA fragments.

If fragments with complementary cohesive ends are to be linked to one another, approximately 100 ng of each fragment are incubated in a reaction mixture of 20 11.1 to 40 μl with approximately 0.2 units of T4 DNA ligase (New England Biolabs) in the buffer recommended by the manufacturer. The incubation period is between 1 to 20 hours at a temperature of 15 ° C. If DNA fragments with blunt ends are to be linked, they are incubated in the manner indicated above, the amount of T4 DNA ligase being increased to 2 to 4 units in this case.

G. Transformation of E. coli.

The E. coli strain HB101 is used for most experiments. The DNA is introduced into E. coli using the calcium chloride method described by Maniatis et al., Pages 250 to 251. Transformed bacteria are capable of selective growth on media containing a suitable antibiotic. This selective growth ability makes it possible to distinguish the desired bacteria from the host bacteria that do not receive transforming DNA. The determination of suitable antibiotics for the selection of host bacteria is routine and is based on the knowledge of the resistance genes that are on the inserted DNA and the sensitivity of the host bacteria to certain active substances. If, for example, it is known that a certain host bacterium is sensitive to the antibiotic ampicillin and there is a corresponding resistance gene against ampicillin on the introduced transforming DNA, then ampicillin is a suitable antibiotic for the selection of the transformants.

H. Screening of E. coli for plasmids.

After transformation, the resulting colonies of E. coli are checked for the presence of the desired plasmid using a rapid plasmid isolation procedure. Two common methods are described on pages 366 to 369 of the Maniatis et al. Reference.

I. Large-scale isolation of plasmid DNA.

Methods for isolating plasmids from E. coli on a large scale are described on pages 88 to 94 of the Maniatis et al. Reference.

J. Cloning in M13 phage vectors.

For the following description, it goes without saying that the double-stranded replicative form of the phage M13 derivatives is used for routine procedures, such as cutting with restriction endonuclease, linking, etc.

I. Identification of proteinase inhibitors

Example 1: In vitro test for the selection of proteinase inhibitors which are active against target insects

A. Test procedure.

Proteinase inhibitors are initially tested for activity against the desired target insect by measuring the ability of individual inhibitors to inhibit proteolysis by homogenates from the intestine of the insect. The intestines are made with C0₂ cut out anesthetized or frozen larvae in the second or third molting stage; the intestines are frozen on dry ice immediately after being cut out. The intestines are homogenized in 100 mM Tris, 10 mM EDTA, pH 8.5 (Wolfson and Murdock, 1987) using 5 to 10 ul buffer per intestine. The homogenate is centrifuged at 5000 rpm for 5 minutes at 4 ° C to separate particles and the supernatant is stored in 1 ml aliquots at -20 ° C. Protease activity is measured essentially according to the method of Wolfson and Murdock (1987), in which ¹⁴C-BSA (New England Nuclear) was used as the substrate and the rate of solubilization of the radioactivity was measured over time over 32 minutes. The pH optimum of protease activity is determined by determining the extent of hydrolysis in each of the following buffers:<tables id="tabl0001" num="0001"><img file="EP0348348A2_D0001.tif" /></tables>

Subsequent experiments are carried out at the pH of the maximum activity. The inhibitor is incubated for 5 minutes at 25 ° C. with the homogenate and the test buffer. The substrate, 14C-methylated bovine serum albumin (NEN Products, Boston), adjusted to a specific activity of - 0.5 µCi / mg with unlabelled BSA, is added and 20 µl aliquots are added after 0, 2, 4, 8, 16 and 32 Minutes removed. These aliquots are added to 200 µl ice cold 10% trichloroacetic acid and kept in ice for 30 minutes. Samples are centrifuged at 5000 rpm for 10 minutes at 4 ° C to precipitate insoluble protein. Two 75 µl aliquots of each supernatant are placed in 8 to 10 ml Scintiverse 11 scintillation cocktail and the radioactive decay is counted. The rate of solubilization of the radioactivity is determined and the rate for each inhibitor is compared to that of the control.

B. Screening with Diabrotica undecimpunctata intestinal homogenates.

In a typical experiment in which intestinal homogenates from D. undecimpunctata are used, the optimal pH for the hydrolysis is pH 4. A number of inhibitors are examined in a standard test which uses 10 μl homogenate, 80,000 cpm ¹⁴Contains C-BSA in a final volume of 200 ul 0.2 M ß-alanine / HCl, pH 4.0. Pepstatin activity is measured in 0.2 M glycine / HCl, pH 2.0. Typical results are shown in Table 1.<tables id="tabl0002" num="0002"><img file="EP0348348A2_D0002.tif" /></tables>

Proteolysis by intestinal homogenates from D. virgifera and D. balteata shows a similarly strong inhibition by inhibitors of the thiol proteinases.

C. Inhibition of proteolysis in intestinal homogenates of Diabrotica virgifera and Ostrinia nubilalis.

Proteinase inhibitors are tested for their ability to inhibit proteolytic activity in D. virgifera and 0. nubilalis intestinal homogenates using the methods described above. The results shown in Table 2 are consistent with data obtained with other inhibitors showing that intestinal proteinases in Ostrinia, but not in Diabrotica, are inhibited by serine proteinase inhibitors. In particular, Eglin C (Arg45) is shown to very effectively inhibit proteolysis by O. nubilalis intestinal homogenates; Eglin C (Arg45) is significantly more potent than Eglin C and another trypsin inhibitor, Vigna unguiculata trypsin inhibitor (Hilder et al., 1987).<tables id="tabl0003" num="0003"><img file="EP0348348A2_D0003.tif" /></tables><tables id="tabl0004" num="0004"><img file="EP0348348A2_D0004.tif" /></tables>

Example 2: Feeding trial for the selection of proteinase inhibitors which are active against target insects

Another method of determining the effectiveness of inhibitors against a single target insect is to add the inhibitor to the diet. The inhibitor solution is applied to the surface of normal food (eg young maize leaves for Ostrinia nubilalis and Heliothis zea, tobacco leaf disks for H. virescens, maize roots or cultivated maize tissue for Diabrotica spec.) And dried. This food is fed to newly hatched larvae and the survival rate and weight are measured after 5 to 6 days. Alternatively, larvae in the 2nd or 3rd molting stage can be weighed before and 3 days after feeding with the treated food.

A. Influence of proteinase inhibitors on the growth of Heliothis zea.

In a typical experiment, corn leaf strips (1 cm²) wetted with 12 gl aliquots of solutions of 10 mg / ml test material and placed in individual food pots, each with a newly hatched H. zea larva. The weight of the larvae is measured for 5 days. Control proteins such as bovine serum albumin or thaumatin cause a significant (P &lt;0.05) increase in weight compared to untreated leaves. Leaves with serine proteinase inhibitors such as Kunitz trypsin inhibitor from soya or lima bean trypsin inhibitor cause a significant decrease in larval weight compared to the control (Table 4).<tables id="tabl0005" num="0005"><img file="EP0348348A2_D0005.tif" /></tables>

B. Influence of proteinase inhibitors on the growth of diabrotics.

Feeding trials with Diabrotica spec. confirm the in vitro test described in Example 1. In a representative experiment, cells from a maize cell suspension are soaked with 2.5% sucrose solution, which contains the desired amounts of inhibitors, and fed to D. undecimpunctata larvae in the second molting stage. The weight gain after 5 days is significantly lower (t-test p &lt;0.05) than in controls when the cells are treated with leupeptin, antipain or pepstatin in concentrations exceeding 0.3 mg / ml. In similar experiments, larval growth of D. virgifera is inhibited by doses of leupeptine as low as 0.1 mg / ml (Table 5).<tables id="tabl0006" num="0006"><img file="EP0348348A2_D0006.tif" /></tables>

C. Influence of proteinase inhibitors on the growth of Ostrinia.

The influence of Eglin C (Arg45) on the growth of newly hatched O. nubilalis larvae is determined using the test described above. Aliquots of the inhibitor solution are at 1 cm² given large corn leaf slices; each corn leaf is placed in a separate feeding pot together with a freshly hatched larva. 20 larvae are tested for each dose. The pots are incubated for 6 days at 29 ° C. and then the survival rate, the final insect weight and the eaten leaf area are determined. The application of 125 µg Eglin C (Arg45) to each leaf piece causes a significant drop in the final weight of the larvae (p&gt; 0.05). Typically, contact with Eglin C (Arg45) results in a decrease of approximately 30% in the average final weight of the larvae compared to untreated controls.

11. Genes and vectors

A proteinase inhibitor gene is isolated from the source in which it occurs naturally or artificially and, if necessary, characterized by conventional methods. If the gene is inducible, it is activated by the associated regulator. The RNA resulting from this activation is isolated and used to create a cDNA library. This library is used for discriminatory screening using radioactively labeled cDNA generated from (1) RNA isolated from the activated system and (2) RNA isolated from a second unactivated system. cDNA clones that correspond to the induced clones are then isolated and sequenced.

If the desired gene is not known to be inducible, it can be isolated using immunological methods. The protease inhibitor protein is used as an antigen to produce an antibody, which is then used to screen a recombinant phage cDNA library in λ-GT1 according to the method of Young and Davis (1983). Positive clones are screened with radiolabelled mixed oligonucleotides (Wood et al., 1985) that were generated according to the known amino acid sequence of the protein.

Example 3: Proteinase inhibitor genes

A. Purification of proteinase inhibitors.

Many proteinase inhibitors are commercially available in purified or partially purified form and the cleaning instructions for many others are known [see Barrett and Salvesen (1986) for extensive references]. Material can be further purified by affinity chromatography on columns with the associated inactivated enzymes.

In a typical purification with the pineapple bromelain inhibitor, the enzyme column with bromelain is essentially as described by Anastasi et al. (1983) for carboxymethyl papain. The bromelain is treated with iodoacetamide to inactivate it. The carboxymethylbromelain is then coupled to CNBr-activated Sepharose (Pharmacia) according to the method recommended by the manufacturer. After intensive washing, the column is in 0.05 M NaP0₄, pH 4.0, 0.5 M NaCl, 0.1% Brij 35 equilibrated and is treated with a column volume of 50 mg / ml bovine serum albumin in the same buffer in order to block non-specific binding sites. The column is washed carefully and the partially purified bromelain (Sigma) dissolved in pH 4.0 buffer containing 10% glycerol instead of Brij 35 is applied to the column. After careful washing with pH 4.0 buffer, the column is washed with 0.05 M NaP0₄, pH 11.5, 0.5 M NaCl, 10% glycerol eluted. The activity is checked by the ability to breakdown¹⁴Inhibiting C-BSA by bromelain is being tested.

When isolating inhibitors that have a high level of activity against the protease of a single insect are isolated, the efficiency of purification can be checked by the test described in Example 1. To increase the selectivity, the insect enzyme can be purified by affinity chromatography on an inhibitor column and then used to prepare an affinity column for the purification of new inhibitors. If necessary, further purifications of inhibitors for protein sequencing can be achieved using the reverse phase HPLC.

B. Amino acid sequencing of proteinase inhibitors.

In the case of proteins whose amino acid sequence is not yet known from the literature, the amino acid sequence is determined by automatic Edman degradation, using a Model 470A protein sequencer (Applied Biosystems, Foster City, CA) equipped with an on-line reverse phase HPLC for Analysis of the phenylthiohydantoin derivatives of the amino acids and a Model 900 data analysis system. Peptides are produced by enzymatic degradation with trypsin, Lys-C (lysine endopeptidase), Arg-C (arginine endopeptidase) or Glu-C (Staphylococcus aureus protease V8) and separated by reverse phase HPLC before sequencing.

C. Synthesis of a proteinase inhibitor-encoding gene: egg white cystatin.

For proteins with less than 150 amino acids, the amino acid sequence of which is fully known, a gene can be constructed by DNA synthesis. In the case of chicken egg white cystatin, the amino acid sequence (Schwabe et al., 1984; Turk et al., 1983) is retranslated using the genetic code with the codon frequency derived from all available maize proteins of the GenBank database using the computer programs of genetics Computer group from the University of Wisconsin was calculated. Translation stop and start signals are added together with BamHI linkers to facilitate subsequent manipulations at both ends. This process results in the sequence shown in FIG. 9.

Oligonucleotides which correspond to sections A to K (FIG. 10) are synthesized with the aid of a model 380A DNA synthesizer (Applied Biosystems, Foster City CA) with β-cyanoethyl chemistry.

The gene is assembled in three steps:<ul id="ul0010" list-style="none"><li>(1) Add 5'-phosphate:<ul id="ul0011" list-style="none"><li>A 5'-phosphate is added to the 5 'ends of all fragments except fragments A and K by adding 40 pmol of each fragment B, C, D, E, F, G, H, I and J and the T4 polynucleotide Kinase are mixed, according to the method described by Maniatis et al. (1982).</li></ul></li><li>(2) Hybridization:<ul id="ul0012" list-style="none"><li>After removing excess reagent by phenol / chloroform extraction, chloroform extraction and ethanol precipitation, the precipitate containing the phosphorylated fragments is dissolved in T4 ligase buffer. 40 pmol of each fragment A and K are added, the mixture is heated to 85 ° C., then slowly cooled to 15 ° C. and incubated at this temperature for at least 4 hours so that the fragments can hybridize.</li></ul></li><li>(3) Link:<ul id="ul0013" list-style="none"><li>ATP is added to a concentration of 1 mM along with T4 ligase and the incubation is continued for 4 hours. The reagents are removed by extraction and precipitation as in step (1). To check the effectiveness of the reaction, an aliquot of the products is analyzed in a 10 to 15% acrylamide gel. A band corresponding to 363 bp is visible. If necessary, the corresponding fragment can be purified by preparative gel electrophoresis before proceeding to link it to the vector.</li></ul></li></ul>

D. Synthesis of a gene encoding a proteinase inhibitor: Eglin C and Eglin C mutants.

(1) Eglin C gene:

The preparation of the plasmid pML147, which contains the Eglin C gene, is described in EP 146 785. The E. coli strain HB101 / pML147, which contains this plasmid, was deposited on January 28, 1988 with the German Collection of Microorganisms (DSM), Mascheroder Weg 1b, D-3300 Braunschweig, Federal Republic of Germany under the accession number DSM 4380. The 230 bp EcoRI-BamHl fragment, which contains the complete Eglin C gene, is isolated from 10 μg plasmid pML147 by cutting with the restriction endonucleases EcoR1 and BamHl and subsequent electrophoresis in 1.5% agarose with a low melting point. The EcoRI end is converted to a BamHI end by linking to a linker which is chosen such that the coding sequence is in the correct reading frame. After isolation, the modified fragment is inserted into the BamHI site of the CaMV35S promoter in the pCIB710 vector

inserted as described in Example 5.

(2) M13 cloning of the Eglin C gene:

About 0.5 ug of the 230 bp EcoRI-BamHI fragment, which contains the complete Eglin C gene, is isolated from 10 ug plasmid pML147 as described above. This DNA fragment (10 ng) is mixed with 40 ng M13mp8, which had previously been cut with EcoRI and BamHl, and in 50 mM Tris-HCl pH 7.4, 10 mM MgCl₂, 10 mM ATP, 10 mM dithiothreitol in the presence of 0.125 units of T4 DNA ligase in a volume of 15 ul (Zoller et al., 1983). The resulting solution is used to transform E. coli strain JM101 (Zoller et al., 1983). The transformation mixture is applied to X-Gal (IPTG indicator agar) plates (Zoller et al., 1983). 40 blue (wild type) and 650 colorless plaques are obtained.

(3) Production of M13mp8 single-stranded DNA:

2nd ml E. coliJM101 culture in L medium (10 g / liter bactotrypton, 5 g / liter Bacto yeast extract, 5 g / liter NaCI, 5 g / liter glucose, 0.1 g / liter ampicillin) to an optical density ( OD₆₂₃) of about 0.5, are inoculated with a colorless plaque taken from the agar plate (see above) and agitated at 180 rpm for about 4 to 5 hours at 37 ° C. The grown culture is then centrifuged for 5 minutes in an Eppendorf centrifuge. The supernatant is placed in a fresh centrifuge tube and centrifuged again. 200 µl 20% PEG and 1.5 M NaCl are added and the mixture is incubated for 20 minutes at room temperature and centrifuged again. The supernatant is discarded and the pellet is dissolved in 100 μl 50 mM Tris-HCl, pH 7.8, 1 mM EDTA (TE buffer). The mixture is mixed with 50 µl phenol / TE buffer, incubated for 15 minutes at room temperature and then centrifuged for 5 minutes in an Eppendorf centrifuge. 10 0 Na acetate buffer, pH 6, and 250 µl absolute alcohol are added to 100 µl supernatant. The mixture is incubated at -20 ° C. overnight and then centrifuged for 10 minutes as described above. The pellet is washed with 1 ml of 80% ethanol and centrifuged again. The pellet is dried for 10 minutes at room temperature and then dissolved in 50 ul TE buffer. The solution contains about 5 µg M13mp8 single-stranded DNA.

(4) Preparation of the gene encoding Eglin C (Arg45):

A method is used which is known as "site directed mutagenesis" (Zoller et al., 1983). The following nucleotide is produced by chemical synthesis for the mutagenesis of the Eglin C gene: 5'-CT CCT GTT ACT CG GAC C-3 '

The base marked with an asterisk differs from the corresponding base in the coding strand of the Eglin C gene (a T) and ultimately ensures the mutation of Leu45 to Arg45. 10 ul of the oligonucleotide (1 OD / ml = 500 ng) are in 20 ul 0.07 M Tris-HCl pH 7.6, 0.01 M MgCl₂, 50 mM dithiothreitol with y-³²P ATP and T4 polynucleotide kinase (Boehringer) according to the method of Maniatis et al. (1982) (p. 125) activated. The activated oligonucleotide is dissolved in 10 µl TE buffer (50 ng / µl).

1 µg M13mp8 single-stranded DNA and 50 ng of the activated oligonucleotide primer are in 10 µl 50 mM Tris-HCl pH 7.8 and 100 mM MgCl₂ Incubated for 30 minutes at 45 ° C and then at room temperature for 5 minutes for mating. 1 µl of 10 mM dATP, dGTP, dCTP and dTTP, 1 µl of T4 DNA ligase, 2 µl of 50 mM dithiothreitol, 1 µl of 10 mM ATP, µl of gelatin (5 mg / ml), µl of 10-fold concentrated Klenow buffer (0, 66 M Tris-HCl pH 7.6.50 mM MgCl₂, 50 mM dithiothreitol) and 1 µl (2.5 units) DNA polymerase (Klenow fragment) are added. The mixture is incubated for 5 minutes at 22 ° C. and then for 16 hours at 15 ° C. and then separated electrophoretically in 1% agarose. The resulting circular, double-stranded DNA is stained with ethidium bromide and released from the gel by electroelution (about 10 ng in 15 μl TE buffer). With 5 µl (about 3.5 ng) of the DNA obtained in this way, E. coli strain JM101 transformed and spread on X-Gal / IPTG indicator plates (see above). About 100 colorless plaques are obtained.

40 these plaques are used to inoculate 2 ml each of E. coliJM101 culture (see section (3)). After cultivation, the supernatants, which contain phages and single-stranded DNA, are separated from the E. coli cells by centrifugation. The cell pellets already contain the corresponding mutated double-stranded DNA. 50 μl each of the 40 phage supernatants are filtered through nitrocellulose, washed twice with TE buffer, incubated in vacuo for 2 hours at 80 ° C. and checked for the presence of the mutated DNA sequence according to Southern (1975) by using the oligonucleotide primer as a radioactive probe hybridization is used. 12 phage supernatants that may contain the Eglin C (Arg45) gene are identified. 4 of these positive phage supernatants become about 1:10⁵ diluted, mixed with E. coli JM101 and applied to indicator agar. Phages from 3 of the resulting plaques each are isolated. The single-stranded DNA is isolated therefrom in the manner described above. These 12 single-stranded DNAs are sequenced according to Sanger (1981 and 1977). All 12 single-stranded DNAs contain the desired mutated Eglin C sequence. The corresponding double-stranded DNA (Eglin C (Arg45) gene in plasmid M13mp8) from the corresponding E. coli cell pellets is then prepared in a mini-preparation (see above). By cutting with the restriction endonucleases EcoRI and BamHl, the EcoRI-BamHI insert which contains the mutated gene is cut out of the vector, isolated and cloned in the vector pHRi148 / EcoRI / BamHI (EP 146 785). The resulting plasmid pJPG18 is isolated and thus E. coli strain HB101 transformed.

The 230 bp EcoRI fragment, which contains the mutated Eglin C (Arg45) gene, is again cut from the vector pJPG18 and adapted as described for the non-mutated Eglin C gene in section (1).

(5) Production of the gene encoding Eglin C (Pro44):

The mutation from Thr44 to Pro44 is carried out in a manner analogous to that described in section (4). The mutagenic oligonucleotide used has the following structure: 5'-CT CCT GTT<img file="EP0348348A2_D0007.tif" /> CT CTG GAC-3 '

The base marked with an asterisk differs from the corresponding base in the coding strand of the Eglin C gene (an A) and ultimately ensures the mutation from Thr44 to Pro44. When the mutation mixture is processed, 18 putative Eglin C (Pro44) mutants are obtained. By cloning the Eglin C (Pro44) DNA in the vector pHRI148 / EcoRI / BamHI, the plasmid pJB591 is obtained and used as described.

Example 4: Construction of a vector derived from the Ti plasmid

The vector pCIB10 (Rothstein et al., 1987) is a vector derived from the Ti plasmid which can be used for the transfer of chimeric genes to plants via Agrobacterium tumefaciens. The vector is derived from the plasmid pRK252, which has a wide host range and which was developed by Dr. W. Barnes, Washington University, St. Louis, Mo. The vector also contains a gene which mediates kanamycin resistance in Agrobacterium and which is derived from the transposon Tn903, and left and right T-DNA border sequences from the Ti plasmid pTiT37. Between the border sequences there is a polylinker region from the plasmid pUC18 and a chimeric gene which causes kanamycin resistance in plants.

In a first process step, the plasmid pRK252 is modified in such a way that the tetracycline resistance gene is replaced by the kanamycin resistance gene from the transposon Tn903 (Oka et al., 1981). Another modification relates to the replacement of the only EcoR1 interface in pRK252 with a BG111 interface (see FIG. 6, which provides a summary overview of the modifications given above). The plasmid pRK252 is first cut with the endonucleases Sall and Smal and then treated with the large subunit of the DNA polymerase to produce blunt ends. The large vector fragment is purified using agarose gel electrophoresis. Next, the plasmid p368 is cut with the BamHI endonuclease and treated with the large fragment of the DNA polymerase. The approximately 1050 bp fragment is then isolated after performing agarose gel electrophoresis. This fragment contains the gene from the transposon Tn903, which confers resistance to the antibiotic kanamycin (Oka et al., 1981). Plasmid p368 has been deposited with the ATCC under the accession number 67700. To create smooth ends, both fragments are treated with the large subunit of the DNA polymerase. The two fragments are then mixed and incubated overnight at a temperature of 15 ° C. with T4 DNA ligase. After transforming the E. coli strain HB101 and selecting kanamycin-resistant colonies, the plasmid pRK252 / Tn903 is obtained.

The plasmid pRK252 / Tn903 thus obtained is cut at its only EcoR1 interface and then treated with the large subunit of the E. coli DNA polymerase to produce smooth ends. This fragment is mixed with synthetic linkers containing BglII restriction sites and incubated overnight with T4 DNA ligase. The DNA resulting from a treatment is cut with an excess of Bg111 restriction endonuclease and the larger vector fragment is purified with the aid of agarose gel electrophoresis. The resulting fragment is again incubated with T4 DNA ligase to recircularize the fragment via its newly added Bg111 cohesive ends. After transformation of the E. coli strain HB101, the plasmid pRK252 / Tn903 / Bglll is obtained (FIG. 1).

In a further process step, a derivative of the plasmid pBR322 is constructed which, in addition to the T-DNA border sequences from the Ti plasmid and the polylinker region from the plasmid pUC19, contains a selectable gene for kanamycin resistance in plants (FIG. 2). The plasmid pBR325 / Eco29 contains the 1.5 kBp EcoRI fragment from the Nopalin Ti plasmid pTiT37. This fragment contains the left T-DNA border sequence (Yadav et al., 1982). To replace the EcoR1 ends of this fragment with HindIII ends, the plasmid pBR325 / Eco29 is cut with EcoRI and then incubated with nuclease S1. This is followed by an incubation with the large fragment of the DNA polymerase to produce blunt ends. This reaction mixture is mixed with synthetic HindIII linkers and incubated with T4 DNA ligase. The resulting DNA is cut with the endonucleases Clal and an excess of HindIII; the resulting 1.1 kbp fragment, which contains the left T-DNA border sequence, is purified using gel electrophoresis. Next, the polylinker region of plasmid pUC19 is isolated by cutting the plasmid DNA with the endonucleases EcoR1 and Hindlll and isolating the smaller fragment (approximately 53 bp) by agarose gel electrophoresis. The plasmid pBR322 is then cut with the endonucleases EcoR1 and Clal, mixed with the other two fragments previously isolated, incubated with T4 DNA ligase and the E coli strain HB101 is thus transformed. The resulting plasmid pCIB5 contains the polylinker region and the left T-DNA border sequence integrated into a derivative of the plasmid pBR322 (FIG. 2).

In a further process step, a plasmid is constructed which contains a gene which mediates the expression of kanamycin resistance in plants (FIG. 4). The plasmid Bin 6 is available from Dr. M. Bevan, Plant Breeding Institute, Cambridge, UK. This plasmid is also described in Bevan (1984). The plasmid Bin 6 is cut with EcoR1 and HindIII. The approximately 1.5 kbp fragment, which contains the chimeric neomycin phosphotransferase (NPT) gene, is isolated and then purified by agarose gel electrophoresis. The fragment is mixed with pUC18 plasmid DNA, which was previously cut with the endonucleases EcoRI and HindIII. After incubation with T4 DNA ligase, the E coli strain HB101 is transformed with the resulting DNA. The resulting plasmid is called pUC18 / neo. This plasmid contains an undesirable BamHI site between the neomycin phosphotransferase gene and the terminator sequence of the nopaline synthase gene (see Bevan, 1984). To remove this recognition sequence, the plasmid pUC18 / neo is cut with the BamHI endonuclease, followed by treatment with the large subunit of DNA polymerase to produce blunt ends. In order to recircularize the fragment, it is then incubated with T4 DNA ligase. The E. coli strain HB101 is transformed with this fragment. The resulting plasmid, pUC18 / neo (Bam) no longer has a BamHI recognition sequence.

In a further process step, the right T-DNA border sequence is inserted immediately next to the chimeric NPT gene (FIG. 4). The plasmid pBR325 / Hind23 contains the 3.4 kBp HindIII fragment of the plasmid pTiT37. This fragment has the right T-DNA border sequence (Bevan et al., 1983). The plasmid pBR325 / Hind23 is cut with the endonucleases Sacll and Hindlll and a 1.9 kbp fragment, which contains the right border sequence, is isolated in purified form following agarose gel electrophoresis. The plasmid pUC18 / neo (Bam) is cut with the endonucleases Sacll and Hindlll and the 4.0 kBp vector fragment isolated using agarose gel electrophoresis. The two fragments are mixed together, incubated with T4 DNA ligase and the E. coli strain HB101 is thus transformed. The resulting plasmid pCIB4 (Fig. 4) contains the right T-DNA border sequence and the selectable marker for kanamycin resistance in plants in a derivative of the plasmid pUC18.

In a last process step, a plasmid is constructed which contains both the left and the right T-DNA border sequence and between these border sequences the kanamycin resistance gene which can be selected in plants and the polylinker of the plasmid pUC18 (FIG. 5). First, the plasmid pCIB4 is cut with the HindIII endonuclease, followed by treatment with the large subunit of the DNA polymerase to produce blunt ends and fragmentation with the endonuclease EcoRI. The 2.6 kbp fragment, which contains the chimeric kanamycin resistance gene and the right T-DNA border sequence, is isolated using agarose gel electrophoresis. The plasmid pCIB5 is then cut with the endonuclease AatII, treated with T4 DNA polymerase to produce blunt ends and then cut with the endonuclease EcoRI. The larger vector fragment is purified using agarose gel electrophoresis, mixed with the pCIB4 fragment and incubated with T4 DNA ligase. The E. coli strain HB101 is transformed with this fragment. The resulting plasmid pCIB2 (Fig. 5) is a derivative of plasmid pBR322, which contains the desired sequences between the two T-DNA border sequences.

The following steps complete the construction of the vector pCIB10. They are shown in Fig. 6. The plasmid pCIB2 is cut with the endonuclease EcoRV and, as described above, provided with synthetic linkers which have a BglII recognition site. After cutting with an excess of BglII, the approximately 2.6 kbp fragment is isolated using agarose gel electrophoresis. The plasmid pRK252 / Tn903 / Bglll previously described (FIG. 1) is cut with the endonuclease BglII and then treated with phosphatase in order to prevent recircularization. These two DNA fragments are mixed together and incubated with T4 DNA ligase. E. coli strain HB101 is then transformed. The resulting plasmid is the completed vector pCIB10.

Example 5: Construction of a chimeric gene with the CaMV 35S promoter

A. Construction of a CaMV 35S promoter-cassette plasmid:<ul id="ul0014" list-style="none"><li>pCIB710 is constructed as shown in FIG. 7. This plasmid contains CaMV promoter and transcription termination sequences for the 35S RNA transcript (Covey et al., 1981). An 1149 bp BglII restriction fragment of the CaMV DNA (bp 6494 to 7643; Hohn et al., 1982) is derived from the plasmid pLW111, which was released on 14. May 1986 with the American Type Culture Collection under the accession number ATCC 40235, isolated; alternatively, the fragment can be isolated directly by preparative agarose gel electrophoresis. It is mixed with BamHI-cut pUC19 plasmid DNA, treated with T4 DNA ligase and thus E. coli transformed (note that the BamHI restriction site in the resulting plasmid is destroyed by linking the BglII cohesive ends to the BamHI cohesive ends). The resulting plasmid, called pUC19 / 35S, is then used in an oligonucleotide-mediated in vitro mutagenesis to insert the BamHI recognition sequence GGATCC directly after the CaMV nucleotide 7483 (as described in Hohn et al., 1982). The resulting plasmid pCIB710 contains the CaMV 35S promoter and transcription termination region, separated by a BamHI restriction site. DNA sequences inserted into this BamHI site are expressed in plants by these CaMV transcription regulatory sequences (also note that pCIB710 contains no ATG translation initiation codons between the start of transcription and the BamHI site).</li></ul>

B. Insert the CaMV 35S promoter / terminator cassette into CIB10.

The following steps are shown in Fig. 8. The pCIB10 and pCIB710 plasmid DNA is cut, mixed and linked with EcoRI and SalI. The resulting plasmid pCIB10 / 710 contains the CaMV 35S promoter / terminator cassette inserted into the plant transformation vector pCIB10. The CaMV 35S sequences are located between the T-DNA border sequences in pCIB10 and are thus inserted into the plant genome during plant transformation experiments.

C. Transfer to Agrobacterium.

An intermediate E. coli host, the E. coli strain S17-1, is used for the transfer of the binary plasmid derived from pCIB10 from E. coliHB101 to Agrobacterium. This E. coli strain, available from Agrigenetics Research Co., Boulder Co., is described in Simon et al. (1983a). It contains mobilization functions that allow a direct transfer of the plasmid pCIB10 to Agrobacterium via a conjugation. This avoids the need to transfer naked plasmid DNA directly to Agrobacterium. First, pCIB10 plasmid DNA is introduced into S17-1 cells treated with calcium chloride. Cultures of transformed S17-1 cells are then mixed with A. tumefaciens LBA4404 (Ooms et al., 1981) and paired on N agar (Difco) plates overnight at room temperature. A sample is taken from the resulting bacteria and inoculated on AB minimal medium containing 50 μg / ml kanamycin (Ditta et al., 1980) and plated out. Incubation takes place at 28 ° C. The grown colonies are spread on the same medium a second time and then again on N agar plates. Slowly insert the BamHI recognition sequence GGATCC directly after the CaMV nucleotide 7483 (as described in Hohn et al., 1982). The resulting plasmid pCIB710 contains the CaMV 35S promoter and transcription termination region, separated by a BamHI restriction site. DNA sequences inserted into this BamHI site are expressed in plants by these CaMV transcription regulatory sequences (also note that pCIB710 contains no ATG translation initiation codons between the start of the transcription and the BamHI site).

B. Insert the CaVM 35S promoter / terminator cassette into pCIB10.

The following steps are shown in Fig. 8. The pCIB10 and pCIB710 plasmid DNA is cut, mixed and linked with EcoR1 and Sall. The resulting plasmid pCIB10 / 710 contains the CaMV 35S promoter / terminator cassette inserted into the plant transformation vector pCIB10. The CaMV 35S sequences are located between the T-DNA border sequences in pCIB10 and thus two plant transformation experiments are inserted into the plant genome.

C. Transfer to Agrobacterium.

An intermediate E. coli host, the E. coli strain S17-1, is used for the transfer of the binary plasmid derived from pCIB10 from E. coli HB101 to Agrobacterium. This E. coli strain, available from Agrigenetics Research Co., Boulder, Co., is described in Simon et al. (1983a). It contains mobilization functions that allow a direct transfer of the plasmid pCIB10 to Agrobacterium via a conjugation. This avoids the need to transfer naked plasmid DNA directly to Agrobacterium. First, pCIB10 plasmid DNA is introduced into S17-1 cells treated with calcium chloride. Cultures of transformed S17-1 cells are then mixed with A. tumefaciens LBA4404 (Ooms et al., 1981) and paired on N agar (Difco) plates overnight at room temperature. A sample is taken from the resulting bacteria and inoculated on AB minimal medium containing 50 μg / ml kanamycin (Ditta et al., 1980) and plated out. Incubation takes place at 28 ° C. The washed colonies are spread on the same medium a second time and then again on N agar plates. Slowly growing colonies are spread on an AB minimal medium with kanamycin and isolated colonies. This method isolates agrobacteria which contain the plasmid pCIB10.

111. Transformation and regeneration

Example 6: Transformation of tobacco

Protoplasts from Nicotiana tabacum cv. "Coker 176" are produced as follows: Four to five week old shoot cultures are grown under aseptic conditions in an MS medium without hormones at a temperature of 16 ° C and a photoperiod of 16 hours light / 8 hours dark. Approx. 1.5 g of leaf tissue are removed from the plant and distributed evenly into 8 to 10 petri dishes (100 x 25 mm, Lab-Tek), each containing 10 ml of an enzyme solution. The enzyme solution contains 1 0 /₀ Cellulase R-10, 0.25% Macerase, from Calbiochem Ca., 1% Pectolyase Y-23, from Seishin Pharmaceuticals Co., 0.45 M mannitol and 0.1 x K3 salts (Nagy and Maliga, 1976). The tobacco leaves are cut into thin strips using a scalpel. The petri dishes are then sealed and incubated with the enzymes on a rotary shaker at a rotation speed of 35 rpm and room temperature for a period of 4 to 5 hours.

The contents of the petri dishes are then filtered through a funnel covered with a fine-meshed fabric (gauze) and collected in a collecting vessel. The filtrate is then pipetted into "Babcock" bottles, each containing 35 ml of a washing solution (the washing solution contains: 0.45 M sucrose, 0.1% MES and 0.1 x K3 salts). The bottles are centrifuged at 80 xg for 10 minutes, which enriches the protoplasts on the surface of the bottles. The protoplasts are removed using a 1 ml pipette, collected in a bottle and washed two more times. The resulting protoplasts are suspended in K3 medium in a 15 ml single-use centrifuge tube. The concentration of the protoplasts is determined by counting in a Fuchs-Rosenthal hemocytometer. The protoplasts are then plated in petri dishes (100 x 20 mm, Corning) containing 6 ml of a liquid K3 medium at a density of 100,000 / ml. The petri dishes with the protoplasts are then incubated for two days at a temperature of 26 ° C in the dark. The cell wall is regenerated during this time.

After the two-day incubation has ended, 5 μl of a stationary A. tumefaciens culture (washed in a YEB medium), the 50th _I.Ig / ml Kanamycin as an additive, at a temperature of 28 ° C until the stationary phase is reached), which contain the desired plasmids, added to the protoplasts. After an incubation period of three more days at 26 ° C, cefotaxime (Calbiochem) is added until a final concentration of 500 μg / ml is reached in order to kill the agrobacteria. The following day, the cells are diluted with 3 ml of fresh K3 medium per petri dish. Then cefotaxime is again used up to a final concentration of 500 _I.lg / ml added. The cells are then cultured at a temperature of 26 ° C. for 2 to 3 weeks and then screened for selective media as in de Block et al. (1984).

Example 7: Regeneration of whole cotton plants from cotyledon explants

A. Cotton variety Acala SJ2 in a medium that induces an even callus.

Seeds of Gossypium hirsutum (cotton) variety Acala SJ2 are sterilized for three minutes with 95% alcohol, washed twice with sterile water, then placed in a 15% sodium hypochlorite solution for 15 minutes and washed again in sterile water. To produce seedlings, the seeds sterilized in this way are germinated in the dark for about 14 days on one of the customary agar media. The cotyledons of these seedlings are cut into segments, which are then transferred under sterile conditions to a callus-inducing medium which is composed of the macro and micro salts of Murashige and Skoog Medium (MS) and additionally thiamine HCI (0.4 mg / Liters), glucose (30 mg / liter), NAA (2 mg / liter), kinetin (1 mg / liter), myo-inositol (100 mg / liter) and agar (0.8%). The cultures are incubated at about 30 ° C with a photoperiod of 16 hours light / 8 hours dark in a "Percival" incubator. The lighting takes place with the help of fluorescent light (cold daylight), with an illuminance of approximately 2000 to 4000 1x. Calli that develop a white to gray-green color develop on the cultivated tissue fragments within three to four weeks. The calli formed are inoculated every three to four weeks on a callus growth medium containing myo-inositol (100 mg / liter), sucrose (2.0 g / liter), NAA (2 mg / liter) and agar, and there cultivated again. Four to six months after the transfer of the tissue explants to the callus-inducing medium, somatic embryos are formed. The callus and embryos are kept alive by inoculating for three to four weeks on fresh callus growth medium and re-culturing.

Somatic embryos that develop on pieces of tissue are either inoculated on fresh callus growth medium or transferred to a specific germination medium for embryos (Beasley and Ting, 1973). The plants that develop from the somatic embryos are transferred to the Beasley and Ting medium, which contains ammonium nitrate (15 mg / liter) and casein hydrolyzate (15 mg / liter) as an organic nitrogen source. The medium is stabilized by a solidifying agent (Gelrite®) and the plantlets are transferred to pots. The somatic embryos develop into little plants within about three months. The plants are rooted when they reach the 6 to 8 leaf stage (the size of the plants is then between 7.5 and 10 cm) and are transferred to soil. The cultivation takes place for a period of three to four weeks in an incubator at high humidity. The plantlets are then transferred to the greenhouse. When the plants have hardened, they are planted out in open, processed soil.

B. Cotton variety Acala S72 in half diluted callus inducing medium.

The process of A is repeated, in which case a semi-concentrated MS medium is used in which all of the media components are reduced to half in their stated concentration. Essentially the same results are obtained as when using the fully concentrated MS medium.

C. Different cotton varieties.

The process measures described in A and B are carried out using the Acala cotton varieties SJ4, SJ2C-1, GC510, B1644, B2724, B1810, the "picker" variety Siokra and the "stripper" variety FC2017. All of the varieties listed can be successfully regenerated.

D. Regeneration of cotton plants starting from cotyledon explants with the interposition of a cell suspension culture.

The process measures described in A are repeated until a callus is produced which is capable of forming somatic embryos. Pieces of 750 mg to 1000 mg of the actively growing embryogenic callus are in 8 ml aliquots of a liquid suspension culture medium, which is composed of the macro and micro salts of the MS medium, and also thiamine HCI (0.4 mg / liter) Contains sucrose (20 g / liter), myo-inositol (100 mg / liter) and NAA (2 mg / liter) as additives, suspended in dishes and then incubated on a rotating drum at a rotation speed of 1.5 rpm and a light / dark rhythm of 16 hours / 8 hours. The light comes from fluorescent lamps (cold daylight) and has a strength between 2000 and 4500 1x. After four weeks, the suspension culture is through a nylon mesh with a mesh size of 840₁Filtered 1m to remove larger clumps of cells. The fraction, which only comprises particles with a size of less than 840 11 m, is allowed to settle and then washed once with 20 to 25 ml of a fresh suspension culture medium. This cell suspension is transferred to dishes (2 ml per dish) and each dish is diluted with 6 ml of fresh suspension culture medium. The cultures are kept alive by repeating the process steps described above at intervals of 10 to 12 days. With each repetition, the suspension culture is repeated by the 840₁Filtered in the network and only that fraction, the cell aggregates smaller than 840 ₁₁m contains, is transferred to fresh suspension culture medium. In all cases, the fraction containing cell clumps larger than 840 µm is placed on a callus growth medium to obtain somatic embryos. The somatic embryos that develop on the callus growth medium are removed and transferred to an embryo germination medium. Using the experimental description described in A, the embryos first germinate, which then develop into small plants and then into plants growing outdoors.

E. Alternative intermediate suspension culture.

The process steps described in D are repeated except that in this case 750 to 1000 mg of embryogenic callus tissue are transferred to a DeLong bottle containing 15 to 20 ml of liquid MS medium with 2 mg / liter of NAA. The bottle with the culture is incubated on a rotary shaker at a rotation speed of 100 to 110 rpm. After three weeks, the suspension culture is filtered through a nylon mesh with a mesh size of 840 μm in order to obtain the large cell clumps for the development of whole plants, as described in D. The fraction that has cell aggregates with a size of less than 840 μm is first allowed to settle, then washed once in a liquid MS medium and then resuspended in 2 ml to 5 ml of a liquid MS medium. The cell suspension is cultured again by transferring 1 ml to 2 ml of the suspension into a DeLong bottle containing 15 ml of fresh MS medium. The cultures are kept alive by repeating the process steps described above at seven to ten day intervals. For each new cultivation, only those suspensions are used that have cell aggregates with a size of less than 840 11m. The larger cell clumps (840 µm and larger) are used for the development of whole plants.

F. Production of Plants from Large Clumps of Cells Cultivated in Suspensions.

After repeating the cultivation three to four times using the experimental descriptions described in D and E, 1.5 ml to 2 ml of the cell suspension are removed from the dishes or DeLong bottles and placed on agar-solidified MS medium containing 2 mg / Liters of NAA and Beasley and Ting Medium containing 500 mg / liter of casein hydrolyzate. After three to four weeks, embryogenic calli with developing embryos become visible. In this case too, cell clumps with a size of 840 μm and more are transferred to a callus growth medium. From these clumps of cells, one obtains embryogenic clumps with developing embryos that ultimately grow into plants.

Example 8: Transformation of cotton suspension culture cells to a tumor phenotype using Agrobacterium LBA 4434

A. Growth of the vegetable suspension culture.

An Acala cotton suspension culture is cultivated in dishes, the medium (MS medium with 2 g / liter NAA) being changed every seven to ten days. After changing the medium, the dish is turned 90 ° and the cells are allowed to settle. Before the transformation, the supernatant is removed using a pipette and the resulting cells are treated as described above.

B. Description of the Agrobacterium vector.

The Agrobacterium strain LBA 4434 (Hoekema et al., 1983) contains a binary plant transformation system based on the Ti plasmid. In such a binary transformation system, one of the plasmids contains the T-DNA region of the Ti plasmid, while the second plasmid has the vir region of the Ti plasmid. The transformation of the plant is guaranteed by the interaction of both plasmids. In the Agrobacterium strain LBA 4434, the T-DNA plasmid pAL1050 contains the T_L Sequence of pTiAch5, an octopine-Ti plasmid. The vir plasmid in strain LBA 4434, pAL4404, on the other hand, contains the intact virulence region of pTiAch5 (Ooms et al., 1982). The Agrobacterium strain 4434 can be obtained from Dr. Robert Schilperoort, Department of Biochemistry, Leiden University, The Netherlands.

C. Cultivation of Agrobacteria; Growing conditions.

The transforming Agrobacterium strain is taken from a glycerol strain culture and cultivated. The next day, an aliquot is taken from this overnight culture and inoculated with a 50 ml culture. The agrobacteria are cultivated on YEB medium to which antibiotics have been appropriately added. The optical density of the 50 ml overnight culture at 600 nm is determined, the culture is centrifuged and the pellet is resuspended in a growth medium for plant cells (MS medium which contains 2 mg / ml NAA) until an OD₆₀₀ of 0.5 is reached. 8 ml of this bacterial suspension is added to each dish containing the plant cells according to section A.

D. infection.

The dishes with the plant and bacterial cells are first shaken to resuspend all cells, and then incubated again for 3 hours on a shaker to give the agrobacteria the opportunity to attach to the plant cells. The cells are then allowed to settle and the remaining supernatant is removed. An aliquot of fresh growth medium is then added to the dishes and incubated on a shaker for 18 to 20 hours in the presence of any agrobacteria that may still be present. The cells are then allowed to settle again, the supernatant is removed and the cells are washed twice with a solution of growth medium and cefotaxime (200 μg / ml). After washing, the cells from the individual dishes are resuspended in 10 ml growth medium with cefotaxime (200 mg / ml each) and 1 ml aliquots thereof are plated onto Petri dishes.

E. Culturing the transformed tissue.

The cells infected with agrobacteria grow on the growth medium, which contains no hormone additives, and thereby show that the tissue has the wild-type phytohormone genes in the T-DNA. The corresponding cells develop into tumors and thus offer further evidence of the successful transformation of the cultures.

Example 9: Transformation of cotton suspension culture cells to a kanamycin-resistant, non-tumorous phenotype

The same procedural measures as previously described in Example 8 are used, except that other transforming Agrobacteria are used and that the plant selection medium contains an antibiotic for the selection of transformed plant tissues.

A. Cultivation of the vegetable tissue.

The plant tissue is cultivated analogously to the process measures given in Example 8, Section A.

B. Description of the Agrobacterium vector.

The transforming agrobacteria have the T-DNA-containing binary vector pCIB10 (Rothstein et al., 1987) and the vir plasmid pAL4404. The pCIB10 T-DNA contains a chimeric gene composed of the promoter of the nopaline synthase gene, the coding region of Tn5 (which encodes the enzyme neomycin phosphotransferase) and the terminator of the nopaline synthase gene.

C. Cultivation of Agrobacteria, Growth Conditions.

Agrobacteria with the plasmid pCIB10 are cultivated on YEB medium with 50 μg / ml kanamycin. The other cultivation conditions correspond to those described in Example 8, Section C.

D. infection.

The transformation of plant cells is carried out according to the process measures described in Example 8. In this case, however, the aliquots (1 ml) resulting from section C are plated directly onto media enriched with selective antibiotics. The selection medium contains either kanamycin (50 µg / ml) or G418 (25 µg / ml). The expression of the chimeric nos / neo / nos gene in the transformed plant tissue allows the selection of this tissue for one of the aforementioned antibiotics.

E. Culturing the transformed tissue.

In this and in all the following examples, the plant growth media contain phytohormones according to the information given in Example 7.

After 2 to 4 weeks, the transformed tissue becomes visible on the selection plates. Uninfected and control tissues show no signs of growth. Eventually it turns brown and dies. In contrast, transformed tissue grows very well in the presence of kanamycin and G418. At this point, well-growing pieces of tissue are inoculated onto fresh selection medium and further cultivated there.

F. Cultivation of Somatic Embryos.

Form from the fabric pieces. somatic embryos. They are inoculated on fresh medium (non-selective medium).

G. Germination.

When the embryos begin to differentiate and germinate, that is, when they begin to develop roots and have 2 to 3 leaves, they are transferred to growth medium containers. The plants are cultivated to the 6 to 8 leaf stage before being removed from the agar medium.

H. Plant cultivation.

The plants are planted in potting soil and covered with a beaker to maintain high humidity and cultured in an incubator for 4 to 8 weeks. At this point the beaker is removed and the plants are brought into the greenhouse. The plants grow in the greenhouse, develop flowers and finally form seeds.

Example 10: Transformation of cells from a cotton suspension culture to a hygromycin-resistant, non-tumorous phenotype

Unless otherwise stated below, the experiments are carried out according to the information in Example 9. In contrast to Example 8, other transforming agrobacteria are used in this case. In addition, the plant selection medium contains an antibiotic that is suitable for the selection of transformed plant tissue.

A. Cultivation of the vegetable tissue.

The plant tissue is cultivated analogously to the process measures given in Example 8, Section A.

B. Description of the Agrobacterium vector.

The transforming agrobacteria contain the binary T-DNA vector pCIB715 (Rothstein et al., 1987) and the vir plasmid. The T-DNA of pCIB715 contains a chimeric gene composed of the promoter and terminator of the CaMV 35S transcript (Odell et al., 1985) and the coding sequence for hygromycin B phosphotransferase (Gritz and Davies, 1983) is.

C. Cultivation of Agrobacteria.

The agrobacteria, which contain the plasmid pCIB715, are on YEB medium, the 50th _I.Lg / ml contains kanamycin, cultured.

D. infection.

The transformation is carried out in accordance with the method steps detailed in Example 8. In this case, however, the aliquots (1 ml each) resulting in section (c) are plated directly onto the media with the selective antibiotics. The selection medium contains 50 ILg / ml hygromycin. The expression of the chimeric hygromycin gene in transformed plant tissue allows the selection of this tissue on hygromycin-containing media.

E. Culturing the transformed tissue.

The cultivation is carried out analogously to the process measures described in Example 9, Section E. The antibiotic used in the plant selection medium in this case is hygromycin.

Example 11: Transformation of plant cells by bombardment with microprojectiles

Another method of introducing foreign DNA sequences into plant cells involves attaching said DNA to particles, which are then attached to the plant cells with the aid of an apparatus as described by Klein et al. (1988). Any plant tissue or organ can be used as the target of this procedure, including, but not limited to, embryos, apical and other meristems, buds, somatic and sexual tissues in vivo and in vitro. Transgenic cells and callus are selected according to established procedures known in the art. Affected tissues are induced to form somatic embryos or to regenerate shoots in order to obtain transgenic plants according to established procedures known to the expert. The appropriate methods can be chosen according to the type of plant used.

The regenerated plant can be chimeric with regard to the foreign DNA that is incorporated. If the cells that contain the foreign DNA develop in either micro or macro spores, the integrated foreign DNA will be inherited by the sexual offspring. If the cells containing the foreign DNA are somatic cells of the plant, non-chimeric transgenic plants are grown by conventional methods of vegetative propagation either in vivo, ie made from buds or stem sections, or in vitro according to established methods known in the art. Such methods can be chosen according to the type of plant used.

Example 12: Transformation of plant cells by injection

The transfer of DNA into plant cells is also accomplished by injection into isolated protoplasts, cultured cells and tissues, as described by Reich et al. (1986 a and b), and achieved by injection into meristematic tissue from seedlings and plants, as described by de la Pena et al. (1987), Graves and Goldman (1986), Hooykaas-Van Slogteren et al. (1984) and Grimsley et al. (1987 and 1988). Transgenic plants and their progeny are obtained by methods known to those skilled in the art.

Example 13: Production of a special type of callus from Zea mays, Elite inbred line Funk 2717

Zea mays plants from the Elite inbred line Funk 2717 are cultivated until flowering in the greenhouse and self-pollinated. Immature flasks with about 2 to 2.5 mm long embryos are removed from the plants and in 10% Clorox^9-Solution disinfected for 20 minutes. The embryos are sterile removed from the corn kernels and placed with the embryonic axis down on OMS medium containing 0.1 mg / liter 2,4-D, 6% (w / v) sucrose and 25 mM L-proline and with 0.24% (w / v) gelrite⁰ is solidified (initiation medium). After culturing in the dark at 27 ° C. for two weeks, the callus which has formed on the scutellum is removed from the embryo and transferred to B5 medium (Gamborg et al., 1968) which contains 0.5 mg / liter of 2,4-D contains and is previously solidified with 0.24% (w / v) Gelrite®. The callus is transferred to fresh medium every 2 weeks. A total of 8 weeks after the embryos have been placed on the initiation medium, the callus special type can be identified on the basis of its characteristic morphology. This callus is further cultivated on the same medium. After a further 2 months, the callus is transferred to N6 medium, which contains 2 mg / liter 2,4-D and is solidified by adding Gelrite °, and is vaccinated periodically.

Example 14: Preparation of a suspension culture from Zea mays-Elite inbred line Funk 2717

The callus described in this example is cultivated for at least 6 months. The type of callus that is selected for further cultivation is comparatively not slimy, is granular and very crumbly, so that it disintegrates into small individual cell aggregates when placed in liquid medium. Cultures containing aggregates of highly vacuolated cells are discarded. Approximately 500 mg aliquots of the above described special callus from Zea mays of the elite inbred line Funk 2717 are placed in 125 ml Delong flasks in 30 ml N6 medium with 2 mg / liter 2,4-D. After culturing for one week at 26 ° C in the dark on a rotary shaker (130 rpm, 2.5 cm turning radius), the medium is replaced by fresh one. The suspensions are cultivated again under the same conditions for a further week. The cultures are then examined and those that show a small number of large, vacuolated cells are retained. Suspension cultures containing aggregates with large, vacuolated cells are discarded. The preferred tissue consists of cytoplasmic, dividing cell aggregates, which characteristically have a smoother surface than the usual type of cell aggregate. In the retained cultures, at least 50% of the cells are present in these small aggregates. This is the desired morphology. These suspensions also have a large growth rate with a doubling time of less than a week. The suspension cultures are inoculated weekly by placing 0.5 ml PCV (packed cell volume: volume of sedimenting cells in a pipette) in 25 ml fresh medium. After 4 to 6 weeks in which the further cultivation is carried out in the manner described above, the cultures increase by 2 to 3 times per week inoculation. Cultures in which more than 75% of the cells have the desired morphology are used for further cultivation. The lines are maintained by always selecting the flask with the best morphology for vaccination. In some cases, the dispersion of the cultures is increased by regular filtration every two weeks through stainless steel sieves with a pore size of 630 µm, without this procedure being absolutely necessary.

A Zea mays suspension culture has been deposited with the American Type Culture Collection (ATCC), deposit number 40326. This deposit was made in accordance with the Budapest Treaty (filing date: May 20, 1987).

Example 15: Production of protoplasts from cell cultures of Zea mays

1 up to 1.5 ml of PCV from cells of the suspension culture, which was prepared according to the preceding example, are incubated in 10 to 15 ml of a filter-sterilized solution containing 4% (w / v) cellulase RS and 1% (w / v) Rhozymes in KMC saline solution (8.65 g / liter KCI, 16.47 g / liter MgC1₂.6H₂0 and 12.5 g / liter CaCl₂-2H₂0.5 g / liter MES pH 5.6) contains. The reaction takes place at 30 ° C for 3 to 4 hours on a slow shaker table. The formation of the protoplasts is monitored with an inverted microscope (plankton microscope). The protoplasts released are collected as follows: The reaction mixture is successively filtered through sieves with a mesh size of 100 µm and 50 µm. The protoplasts are flushed through the sieves with the volume corresponding to the original enzyme solution. 10th The protoplast suspension is placed in a single-use plastic centrifuge tube and underlaid with 1.5 to 2 ml of a 0.6 M sucrose solution (buffered at pH 5.6 with 0.1% (w / v) MES and KOH). The tubes are centrifuged at 60 to 100 xg for 10 minutes and the protoplasts, which have accumulated in the intermediate layer, are picked up with a pipette and placed in a new tube. The protoplasts are resuspended in 10 ml of new KMC saline solution and 5 min. centrifuged at 60 to 100 xg. The supernatant is removed and then discarded. The protoplasts are carefully resuspended in the remaining drop. Then 10 ml of a 13/14 concentrated KMC solution are gradually added. After another five minutes of centrifugation, the supernatant is removed and the protoplasts are resuspended in a 6/7 concentrated KMC solution. An aliquot is removed for counting and the protoplasts are sedimented again by centrifugation. The protoplasts are in as much KM-8p medium or a 0.5 M mannitol solution with 6 mM MgCl₂ or in any other medium suitable for the transformation described in the examples below, resuspends that 10⁷ Protoplasts are contained in one ml.

Example 16: Transformation of Zea mays protoplasts by electroporation

A. All steps except for the heat shock treatment are performed at room temperature (22 ° to 28 ° C). The protoplasts from the previous example are in 0.5 M mannitol with 0.1% (w / v) MES and 6 mM MgCl₂ resuspended. The resistance of this suspension is measured in the chamber of a Dialog Electroporator® (DIA-LOG, GmbH, D-4000 Düsseldorf 13, Federal Republic of Germany) and with a 300 mM MgCl₂-Solution set to 1 to 1.2 kQ. The protoplasts are subjected to a heat shock treatment by immersing the beaker with the sample in a water bath at 45 ° C. for 5 minutes, followed by cooling to room temperature in ice. 4 µg of linearized plasmid containing a plant-selectable hygromycin resistance gene as described by Rothstein et al. (1987) or contains chimeric gene constructs, which are described in Examples 32, 36, 41 and 46, and 20 µg calf thymus carrier DNA were given. 0.125 ml 24% (w / v) PEG (MW: 8000) in 0.5 M mannitol with 30 mM MgCl₂ are added to the protoplasts. The mixture is mixed well but carefully and incubated for 10 minutes. The sample is placed in the electroporator chamber and three times for 10 seconds with initial voltages of 1500, 1800, 2300 or 2800 Vcm.¹ pulsed, with an exponential decay time of 10 µs.

The protoplasts are cultivated as follows: The samples are placed in 6 cm petri dishes at room temperature. After a further 5 to 15 minutes, 3 ml of KM-8p medium with 1.2% (w / v) SeaPlaque® agarose and 1 mg / liter of 2,4-D are added. The agarose and protoplasts are mixed well, then the medium is allowed to gel.

B. The procedure of A is repeated with one or more of the following modifications:<ul id="ul0015" list-style="none"><li>(1) The resistance of the protoplast preparation is set to 0.5 to 0.7 kΩ.</li><li>(2) The PEG used is a PEG with a molecular weight of 4000.</li><li>(3) No PEG or half a volume of 12% (w / v) PEG is added.</li><li>(4) It is pulsed at 3 second intervals.</li><li>(5) After electroporation, the protoplasts are placed in dishes placed on a plate which is cooled to 16 ° C.</li><li>(6) After electroporation, the protoplasts are placed in tubes containing 10 ml of a 6/7 concentrated KMC solution or W5 solution containing 380 mg / liter KCI; 18.375 g / liter CaCl₂· 2H₂O; 9 g / liter NaCl; 9 g / liter glucose; pH 6.0 contains, washed, sedimented by centrifugation at 60 × g for 10 minutes, resuspended in 0.3 ml of KM medium and plated out as indicated in A.</li><li>(7) No calf thymus carrier DNA is added.</li></ul>

Example 17: Transformation of Zea mays protoplasts by treatment with polyethylene glycol

A. The protoplasts are in the last step of Example 15 in a 0.5 M mannitol solution with 12 to 30 mM MgC1₂ resuspended. The protoplasts are subjected to a 5 minute heat shock at 45 ° C as described in Example 16. The suspended protoplasts are distributed in 0.3 ml aliquots in centrifuge tubes for transformation. Over the next 10 minutes, DNA (as in Example 16A and PEG solution (MW 6000.40% (w / v); Ca (N0₃)₂ 0.1 M; Mannitol 0.4 M; pH 8 to 9 with KOH) so that the final concentration of PEG is 20%. The aliquots are incubated for 30 minutes with occasional gentle shaking, the protoplasts are placed in petri dishes (0.3 ml original protoplast suspension per 6 cm diameter dish) and cultured as described in Example 16A.

B. The procedure of A is repeated. However, after 30 minutes of incubation, the protoplasts are washed in the PEG solution by adding 0.3 ml of W5 solution five times in 2 to 3 minute intervals. The protoplast suspension is then centrifuged and cultivated after removing the supernatant, as described in Example 16 (A).

C. The procedures of A and B are repeated with the modification that the final concentration of PEG is between 13 and 25% (w / v).

Example 18: Regeneration of callus from protoplasts

The plates containing the protoplasts in agarose are kept in the dark at 26 ° C. After 14 days, the protoplasts become colonies. Colon agarose is placed on the surface of a 9 cm diameter petri dish containing 30 ml of N6 medium containing 2 mg / liter 2,4-D solidified with 0.24% (w / v) gelrite^@ contains. This medium is referred to as 2N6 medium. The callus is cultivated further in the dark at 26 ° C, callus pieces are inoculated onto fresh, solid 2N6 medium every 2 weeks.

Example 19: Selection of transformed callus from Zea mays

Example 18 is repeated with the modification that 100 mg / liter or 200 mg / liter hygromycin B is added to the 2N6 medium in order to be able to select transformed material.

Example 20: Regeneration of maize plants

A. Callus is placed on 2N6 maintenance medium and on ON6 (N6 medium without 2,4-D) and N61 (0.25 mg / liter 2,4-D and 10 mg / liter kinetin) medium in order to achieve this Initiate regeneration. Callus, which grows on ON6 and N61 media, is cultivated in the light (16 hours / day, 800 to 8000 Ix, white fluorescent lamps). Callus grown on N61 is transferred to ON6 medium after 2 weeks because prolonged cultivation on N61 medium is harmful to the callus. The callus is vaccinated every 2 weeks, even if it is to be transferred back to the same medium formulation. Little plants appear in about 4 to 8 weeks. When the plants have reached a height of at least 2 cm, they are transferred to 0N6 medium in GA7 containers. Roots form in 2 to 4 weeks. If the roots look as if they could support growth, the plants are transferred to peat pots in soil and shaded for 4 to 7 days. It is often helpful to put a planted-out plastic pot on the planted plant for 2 to 3 days to harden it. When the plants are fully developed, they are treated like normal maize plants and grown to maturity in the greenhouse. To obtain offspring, the plants are self-pollinated or crossed with wild types.

B. The procedure of A is repeated with the modification that 100 mg / liter or 200 mg / liter hygromycin B is added to the callus maintenance medium.

Example 21: Preparation of embryogenic suspensions from tissue of Dactylis glomerata L. (knotty grass)

A. Embryogenic callus is initiated from basal sections of the youngest leaves of D. glomerata L. grown in the greenhouse, as described by Hanning and Conger (1982). The leaves are immersed in a 1:10 diluted Clorox ° solution [a solution of 5.25% (w / v) sodium hypochlorite; The Clorox Company, Oakland, CA 94623, USA] surface-sterilized and then aseptically cut into small sections of 1 to 5 mm in length or diameter. These sections are placed on sterile SH-30 medium containing 0.8% (w / v) agarose as a gelling agent. Callus and / or embryogenic structures appear within 2 to 6 weeks after application during cultivation at about 25 ° C. Embryogenic callus is maintained by inoculating fresh SH-3 medium at intervals of 2 to 4 weeks and culturing in the dark at 25 ° C.

B. Embryogenic suspension cultures are initiated by introducing about 0.5 g embryogenic callus (fresh weight) into 50 ml liquid medium, which is described by Gray and Conger (1985) and contains 45 μM dicamba and 4 g / liter casein hydrolyzate. The suspension cultures are cultivated at 27 ° C. and a photoperiod of 16 hours of light (3300 lx), 8 hours of dark on a rotary shaker at about 130 rpm in 125 ml Delong vessels, which are sealed with a metal cap and Parafilm °. After approximately 4 weeks, the large lumps are allowed to settle for approximately 30 seconds and 10 ml aliquots of the supernatant medium containing small cell clusters are removed and transferred to 50 ml fresh medium. This process is repeated every 3 to 4 weeks using the most successful cultures, which are determined by their smaller pile size and better quality due to the presence of small, cytoplasmic cells. After 5 to 8 transfers, the suspensions are essentially free of embryogenic cells and the majority of the embryogenic cell clusters are quite small (150 to 2000 µm).

Example 22: Isolation and purification of Dactylis glomerata L. protoplasts

Protoplasts are prepared from the embryogenic suspension cultures of the previous example by aseptically filtering the cells through a Nalgene® 0.2 µm filter unit and then adding 0.5 g of cells (fresh weight) to a 12.5 ml mixture of protoplasts and enzyme mixture in a petri dish. The filter-sterilized enzyme mixture consists of 2% (w / v) Cellulase.RS, 7 mM CaCl2 H20, 0.7 mM NaH2P04^* H₂0.3 mM MES (pH 5.6), glucose (550 mOs / kg H₂0 of pH 5.6). The mixture is agitated on a orbital shaker at about 50 rpm in dim light (&lt;400 Ix) for about 4 to 5 hours. All of this is then sieved through a stainless steel sieve (100 µm mesh size), distributed in 12 ml centrifuge tubes and centrifuged at about 60 to 100 xg for about 5 minutes. The protoplast-containing sediment is then three times with KM-8p protoplast culture medium, which on 550 mOs / kg H₂0 adjusted with glucose, washed. At this point, a further step for cleaning the protoplasts can be inserted. In this case, the washed protoplasts on 10 ml KM-8p culture medium, which on 700 mOs / kg H₂0 is set with sucrose, layered. After centrifugation at 60 to 100 xg for about ten minutes, the protoplasts, which form a band at the interface, are collected using a fine pipette. Finally, the protoplasts are resuspended in 1 to 2 ml of KM-8p culture medium and sieved through a stainless mesh screen (20 µm mesh size). The protoplasts released are collected, washed and resuspended for culture in KM-8p medium or in osmotically adjusted medium which is suitable for the transformation.

Example 23: Dactylis glomerata L. Protoplast culture and callus growth

A. The cleaned protoplasts are in a density of about 5 x 10⁵ Protoplasts / ml plated in KM-8p culture medium that conditioned 1.3% (w / v) SeaPlaque® agarose (FMC Corp., Marine Colloids Division, Rockland, Maine, USA) and 30 to 40% (v / v) Medium (obtained from 3 to 4 week old D. glomerata L. embryogenic suspension cultures by filtering the medium through a sterile Nalgene® 0.2 µm filter, the medium by adding glucose to 550 mOs / kg H₂0 adjusted and sterile filtered again) contains. The plates are then set up in the dark at a constant temperature of 28 ° C. After 10 to 14 days, the agarose is cut into wedges and placed in a "bead" culture as described by Shillito et al. (1983) is given using 20 ml of SH-45 suspension culture medium with 3% (w / v) sucrose per 3 ml of original culture embedded in agarose. The plates are placed on a shaker and shaken at about 50 rpm in the light of 670 Ix. New suspension cultures form as the colonies grow out of the agarose and release cells into the liquid medium. The resulting cells cultivated in the suspension are plated on SH-30 medium solidified by agar and placed in the dark until callus forms.

B. Protoplasts are cultivated as described in A except that the culture medium contains no conditioned medium.

Example 24: Transformation of Dactylis glomerata L. protoplasts using electroporation

A. Immediately after cleaning the protoplasts, electroporation according to Shillito et al. (1985) by using a linearized plasmid as described in Example 16. The protoplasts are washed to a density of about 7 x 10 after the last wash⁶ Protoplasts / ml in electroporation buffer (0.4 M mannitol, 6 mM MgC1₂) resuspended. The protoplasts are placed in 0.7 ml aliquots in 10 ml plastic centrifuge tubes. Plasmid DNA in a final concentration of 10 µg / ml and sonicated calf thymus DNA in a concentration of 50 µg / ml are added to the tubes. Then 0.38 ml of PEG solution [24% (w / v) PEG 6000 in 0.4 M mannitol, 30 mM MgCl₂, 0.1% (w / v) MES (pH 5.6)] was added and the solution was mixed slightly. The protoplast suspension is transferred to the chamber of a Dialog® electroporator and 10 pulses with an initial voltage of 3250 Vcm.¹ and an exponential decay constant of 10 µs are given at 30 s intervals. The sample is removed from the chamber and placed in a 10 cm diameter petri dish. 10 ml of KM-8p medium containing 1.2% (w / v) SeaPlaque® agarose is added, the protoplasts are evenly distributed throughout the medium and the agarose gels.

B. Example 24 A is repeated until the initial voltage used is 2500 Vcm-¹, 3000 Vcm-¹, 3500 Vcm-¹, 4000 Vcm-¹ or 5000 Vcm-¹ is.

C. Examples 24 A and B are repeated, except that a PEG with MW 4000 or a PEG with MW 8000 is used.

D. Examples 24A through C are repeated except that the final concentration of PEG is between 10% and 30% (w / v).

Example 25: Transformation of Dactylis glomerata L. protoplasts by treatment with polyethylene glycol (PEG)

A. The direct gene transfer mediated by PEG is carried out according to Negrutiu et al. (1987). After the last wash, the protoplasts in 0.5 M mannitol with 15 mM MgCl₂ at a density of approximately 2 x 10⁶ resuspended per ml. The protoplast suspension is distributed in 1 ml aliquots in 10 ml plastic centrifuge tubes. The DNA is added as described in Example 24 above and then 0.5 ml of the PEG solution is added [40% (w / v) PEG 4000 in 0.4 M mannitol, 0.1 M Ca (N0₃)₂, pH 7.0]. The solutions are mixed gently and incubated for 30 minutes at room temperature (about 24 ° C) with occasional shaking. Then 1.4 ml of the washing solution are added and the contents of the tube are mixed gently. The washing solution consists of 87 mM mannitol, 115 mM CaC1₂, 27 mM MgCl₂, 39 mM KCI, 7 mM Tris / HCl and 1.7 g / liter myo-inositol, pH 9.0. Four additional 1.4 ml aliquots of wash solution are added at 4 minute intervals, with mixing after each addition. The tube is then centrifuged at 60 xg for about 10 minutes and the supernatant is discarded. The sedimented protoplasts are taken up in 1 ml of KM-8p culture medium and placed in a 10 cm petri dish. 10 ml of KM-8p medium containing 1.2% (w / v) Sea-Plaque® agarose is added. The protoplasts are evenly distributed in the medium and the agarose gelled.

B. The procedure of A is repeated with one or more of the following modifications:<ul id="ul0016" list-style="none"><li>(1) The pH of the washing solution is adjusted to 5.6 or 7.0.</li><li>(2) The PEG used has a molecular weight of 2000, 6000 or 8000.</li><li>(3) The washing medium consists of 154 mM NaCl, 125 mM CaCl₂, 5 mM KCI, 5 mM glucose, adjusted to pH 6.0 with KOH, or from 0.2 M CaC1₂, 0.1% (w / v) MES, adjusted to pH 6.0 with KOH, or from 0.2 M CaCl₂, 7 mM Tris / HCl, adjusted to pH 9.0 with KOH.</li></ul>

Example 26: Transformation of Dactylis glomerata L. protoplasts after heat shock treatment

The transformation is carried out as described in Examples 24 or 25, except that the protoplasts are incubated at 45 ° C. for 5 minutes before the distribution of the aliquots into the tubes for transformation or after the distribution of the aliquots and before the addition of PEG .

Example 27: Selection of transformed colonies

A. The culture plates (petri dishes), which contain the protoplasts from Examples 24 to 26, are incubated for 10 days in the dark at about 25 ° C. and then in 5 equal slices for the "bead" culture (Shillito et al., 1983 ) cut. 4 of the slices are placed in 20 ml SH-45 culture medium with 4 g / liter casein hydrolyzate and 20 µg / ml hygromycin B. The fifth disc is placed in 20 ml of the same medium, but without hygromycin B, as an unselected control. After 4 to 5 weeks, the putatively transformed protoplast-derived cell colonies, which grow in hygromycin B, are cut out of the agarose and placed in a 19 mm petri dish with 2 ml of liquid SH-45 medium containing 20 μg / ml hygromycin B, given, which is shaken at about 50 rpm on a rotating shaker. After a further 4 to 5 weeks, all colonies that grow into new suspensions are transferred to 125 ml Erlenmeyer flasks and drawn in a similar manner to the parental suspension culture, except that 20 μg / ml hygromycin B is contained in the medium.

The new suspensions are inoculated into new medium every 1 to 3 weeks using SH-45 medium containing 4 g / liter casein hydrolyzate and 20 µg / ml hygromycin B. Cells with these suspensions are also plated on solid SH-30 medium containing 20 µg / ml hygromycin B and incubated at about 25 ° C in the dark. Calli growing from the plated cells are inoculated into fresh medium every 2 weeks. The cells that grow in the presence of hygromycin B are very likely to be transformants.

B. The selection is carried out as described in Example 27 (A), except that the protoplast-derived cell colonies, which grow in medium containing hygromycin B, on SH-30 medium agar plates containing 20 μg / ml hygromycin B included, placed and incubated at about 25 ° C in the dark.

Example 28: Regeneration of transformed Dactylis glomerata L. plants

AD glomerata L. callus (obtained as described in Example 27) derived from protoplasts is grown on solid SH-30 medium and vaccinated every 2 weeks. All embryos that form are removed, plated on a germination medium (SH-O) and brought into the light (3700 to 4600 Ix). These embryos germinate in 1 to 4 weeks and the resulting plantlets are placed on SH-O medium in the light to form root systems. They are brought into the greenhouse at the 6- to 12-leaf stage and hardened gradually.

B. Callus (obtained as described in Example 27) derived from protoplasts is placed on SH-O medium containing 0.24% (w / v) GelRite⁰ is solidified, drawn in the light (3700 to 4600 Ix) and vaccinated every two weeks. The resulting plantlets are placed on a 1: 1 mixture of SH-O and OMS medium containing a combination of 0.12% (w / v) GelRite^@ and 0.4% (w / v) agar is solidified and illuminated to form root systems. They are brought into the greenhouse at the 6- to 12-leaf stage and hardened gradually.

C. Small plantlets are obtained as described in A and B, placed on OMS medium which is solidified with 0.8% (w / v) agar, and illuminated so that root systems can develop. They are brought into the greenhouse at the 6 to 12-leaf stage and hardened gradually.

D. Small plants are obtained as described in A, on a 1: 1 mixture of SH-O and OMS medium, solidified with a combination of 0.12% (w / v) GeIRite® and 0.4% (w / v) Agar, laid and illuminated to form root systems. They are brought into the greenhouse at the 6 to 12 leaf stage and hardened gradually.

Example 29: Transformation and regeneration of tomatoes

Tomatoes are transformed with manipulated A tumefaciens (Nelson et al., 1988) which contains a marker which can be selected in plants (for example kanamycin resistance) and the chimeric gene which codes for the protease inhibitor of interest. The leaf disc transformation of tomatoes with A. tumefaciens is carried out according to McCormick et al. (1986).

Example 30: Transformation and regeneration of potatoes

Potatoes are transformed with manipulated A. tumefaciens (Stockhaus et al., 1987) which contains a marker which can be selected in plants (for example kanamycin resistance) and the chimeric gene which codes for the protease inhibitor of interest.

IV. Results

Example 31: Bioassay of transformed cotton

Heliothis virescens eggs are obtained from the Tobacco Insect Control Laboratory at North Carolina State University, Raleigh, North Carolina. The eggs are on a gauze layer, which is transferred to large covered beakers. The beaker is lined with damp filter paper to guarantee constant moisture. The Heliothis eggs are incubated at a temperature of 29 ° C. Under these conditions, the larvae hatch within three days. After hatching, the larvae are transferred as soon as possible into small covered plastic containers (1 larva / container), each containing a cotton leaf disc. The larvae are transferred using a fine-bristled brush.

The leaf disks, which have a diameter of one centimeter, are previously punched out of the leaves of cotton plants and applied to a round, moistened filter paper in the plastic container. At least 6 to 10 leaf disks from both young and old leaves are tested from each plant. The leaf disks are replaced at 2 daily intervals or depending on the food intake as required. The growth rates (the size or the total weight of all larvae within a test group) and the mortality rate of the larvae feeding on leaves of transformed plants are compared with those of the control animals feeding on untransformed cotton leaves.

Larvae feeding on leaf disks derived from cotton plants transformed with the protease inhibitor gene show a greatly reduced growth rate and a 100% increase in mortality compared to the controls.

Example 32: Corn Resistant to Diabrotica Damage by Expression of Egg White Cystatin

A. Construction of the pCIB715 / 710 cystatin vector.

The cystatin gene is synthesized as described in Example 3C. The 363 bp DNA sequence is purified and inserted into the BamHI site of the pCIB710 vector as described in Example 5. The resulting vector is called pCIB710 cystatin. This vector is cut with EcoRI and the resulting DNA is treated with alkaline phosphatase. Plasmid pCIB715 (Rothstein et al., 1987) is cut with EcoRI and linked with pCIB710-cystatin from above. The resulting vector is called pCIB715 / 710 cystatin.

B. Maize transformation and regeneration.

Corn tissue is transformed with the pCIB710 vector, or preferably the pCI B715 / 710 vector, which carries the cystatin gene scub, and plants are regenerated as described in Examples 16 to 20. As a control, plants transformed only with the pCIB710 vector or the pCIB715 vector without a gene insert are produced in the same way. Transformed plant tissue is selected by sensitivity to the corresponding antibiotic. The original plants are self-fertilized and seeds are obtained (T1 seeds).

C. Test of plants for cystatin expression.

Plants grown from the T1 seeds are examined for the presence and expression of the cystatin gene using various tests.<ul id="ul0017" list-style="none"><li>(1) DNA is isolated and cut with BamHI; the degradation products are separated electrophoretically on a 1.5% agarose gel. The DNA fragments are transferred to nitrocellulose and with the cystatin gene that with^32p is marked by nick translation, hybridizes [see Maniatis et al. (1982) because of the techniques]. The presence of the cystatin gene is identified by a band that corresponds to approximately 363 base pairs and hybridizes with the sample.</li><li>(2) RNA is found by the Northern blot method (Maniatis et al., 1982) as a band corresponding to about 380 bases and with the ³²P-cystatin gene hybridized, which is described above.</li><li>(3) Cystatin protein is detected using standard immunological techniques with a rabbit monoclonal antibody produced against commercially available (Sigma) cystatin.</li><li>(4) Cystatin activity is found by purifying material from the plant extracts using immunological methods, using the rabbit polyclonal anti-cystatin antibody and Protein A Sepharose, and testing the isolated material for the ability to inhibit the proteolysis of ¹⁴Inhibit C-BSA by papain and by intestinal homogenates from Diabrotica as described in Example 1.</li></ul>

D. Resistance of transformed maize plants to damage from Diabrotica.

Seedlings obtained from T1 seeds are planted in coarse vermiculite in 100 mm petri dishes (5 per dish, 5 dishes). When the second leaves appear on the seedling, each dish is infected with 20 second-molting diabrotica larvae. After 7 days, the number, the weight of the survivors and the weight of the washed roots of the maize plant are determined. The resistance of the transformed plants is determined by a statistically significant (Student's Test p &lt;0.05) decrease in the weight gain of the larvae, decrease in the survival rate of the larvae or in the decrease in the loss of root weight compared to insect-free plants, in that plants, which express the cystatin gene, are compared with control plants which have only been transformed with the vector without a gene insert, or with non-transformed plants.

Example 33: Potatoes and tomatoes which are resistant to damage from Leptinotarsa ​​decemlineata (Colorado beetles) by the expression of cystatin

A. Construction of the pCIB10-cystatin vector.

The cystatin gene is synthesized and linked to the vector pCIB710 as described in Example 32A. The cystatin gene and the 35S CaMV promoter continue to be cloned out of the pCIB710 vector into the pCIB10 vector. For this, pCIB710 is cut with Xbal and EcoRI, the 1593 bp fragment is isolated and this fragment is linked with Xbal and EcoR1 cut pCIB10 in order to obtain the pClB10-cystatin vector.

B. Transformation and regeneration of plants.

The pCIB10 cystatin vector, or preferably the pCIB715 / 710 cystatin vector, is introduced into A. tumefaciens carrying a virulence plasmid such as LBA 4404 (Example 5) or pCIB542. pCIB542 is a manipulated A. tumefaciens vir plasmid derived from pTiBo542 (Hood et al., 1986). In pCIB542, the bacterial kanamycin resistance gene is replaced by a bacterial streptomycin / spectinomycin resistance gene. The strain, which carries both pCIB710 cystatin or pCIB715 / 710 cystatin and pCIB542, is used to transform transformed tomato plants according to the method of Fischhoff et al. (1987) or as in

To make example 29.

Potato plants containing pCIB10 cystatin or pCIB715 / 710 cystatin are grown according to the method of Stockhaus et al. (1987), Example 30.

C. Test of plants for cystatin expression.

Tests of the transformants for cystatin expression are carried out as described above for corn.

D. Resistance of transformed plants to damage by L. decemlineata.

10th four week old plants are infected with 5 larvae of L. decemlineata in the 2nd molting stage. The larvae are allowed to eat for 4 days and then the insect mortality, the weight gain of the insect and the extent of the damage done to the plants are determined. The resistance of the transformed plants is determined by a statistically significant (Student's t test p &lt;0.05) decrease in the weight gain and survival rate of the larvae or a decrease in plant damage by using plants that express the cystatin gene with control plants that only transformed with the vector without gene insertion, or with non-transformed plants.

Example 34: Coiled grass (Dactylis glomerata), which is resistant to damage by the expression of cystatin

through Coleoptera (beetle)

A. Construction of the vector.

pCIB710 cystatin and pCIB715 / 710 cystatin are prepared as described in Example 32A.

B. Transformation and regeneration of plants.

Transformed plants are obtained as described in Examples 24 and 28 using pCIB710 cystatin and pCIB715 / 710 cystatin.

C. Test of plants for cystatin expression.

Tests of the transformants for cystatin expression are carried out as described in Example 32C.

D. Resistance of transformed plants to damage from Diabrotica undecimpunctata.

Seedlings obtained from T1 seeds are planted in fine soil in 100 mm petri dishes (10 / dish, 5 dishes). When the second leaves appear on the seedling, each dish is infected with 20 second-molting diabrotica larvae. After 7 days, the number and weight of survivors are determined, along with the weight of the plant's washed roots. The resistance of the transformed plants is determined by a statistically significant (Student's t test p &lt;0.05) reduction in the weight gain of the larvae, reduction in the survival rate of the larvae or reduction in the loss of root weight compared to insect-free plants, in that plants, which express the cystatin gene can be compared with control plants which are only transformed with the vector without a gene insert or with non-transformed plants.

Example 35: Cotton that is resistant to damage by Anthonomus grandis (American cotton beetle) by expression of cystatin

A. Construction of the vector.

pCIB715 / 710 cystatin and pCIB10 cystatin are prepared as described in Examples 32A and 33A.

B. Transformation and regeneration of plants.

Transformed plants are obtained as described in Examples 7 to 10 using pCIB 715/710 cystatin or pCIB10 cystatin in Agrobacterium.

C. Test of plants for cystatin expression.

Tests of the transformants for cystatin expression are carried out as described in Example 32C.

D. Resistance of transformed plants to A. grandis damage.

10th transformed plants are grown until capsules begin to form. Each plant is infected with 3 adult female A. grandis. The damage done to the plants is assessed after a week and surviving adults are removed. The damage from larvae, the number of larvae and the weight per plant are measured at weekly intervals for 4 weeks. The resistance of the transformed plants is determined by a statistically significant (Student's t test p &lt;0.05) decrease in the weight gain of the larvae, decrease in the survival rate of the larvae or decrease in the loss of root weight compared to insect-free plants by plants which express the cystatin gene, with control plants that are only transformed with the vector without a gene insert, or with non-transformed plants.

Example 36: Maize which is resistant to damage by Lepidoptera (butterflies) larvae by expression of the soybean Kunitz trypsin inhibitor

A. Construction of the pCIB715 / 710-CTI vector.

The soybean trypsin inhibitor cDNA is determined by the method of Hoffman et al. (1984). The 652 bp fragment is changed so that the sequence begins at the methionine start codon (44 in the original fragment). Codons for the missing three amino acids plus a stop codon are added to the 3 'end. BamHl linkers are attached to both ends. This construction is achieved by first cutting the 652 bp fragment with Eco571 and BstXI and isolating the resulting 517 bp fragment after agarose gel electrophoresis. The oligonucleotides shown in Figure 11 are synthesized. Phosphates are attached to the 5 'ends of the 517 bp fragment and the oligonucleotides 3M and 5C and all fragments are linked as described above. The resulting fragment is isolated and linked to the BamHI site of the pCIB710 vector as described above. The resulting vector is called pCIB710-KTI.

B. Maize transformation and regeneration.

Corn tissue is transformed with the pCIB710 vector or, preferably, with the pCIB715 / 710 vector, which contains the soybean Kunitz trypsin inhibitor gene insert, and plants are regenerated as described in Examples 16 to 20. For control purposes, plants are produced in the same way, but with the pCIB710 vector or the pCIB715 vector without gene insertion. Transformed plant tissue is selected based on its sensitivity to the corresponding antibiotic. The initial plants are self-pollinated and seeds (T1 seeds) are obtained.

C. Test of plants for the expression of the Kunitz trypsin inhibitor from soybeans.

Plants grown from T1 seeds are tested for the presence and expression of the Kunitz trypsin inhibitor gene from soybeans using the following test procedures.<ul id="ul0018" list-style="none"><li>(1) DNA is isolated and cut with BamHI; the degradation products are separated electrophoretically on a 1.5% agarose gel. The DNA fragments are transferred to nitrocellulose and hybridized with the Kunitz trypsin inhibitor gene from soybeans, which by nick translation with³²P is marked [see Maniatis et al. (1982) because of the techniques]. The presence of the Kunitz trypsin inhibitor gene is identified by a band that corresponds to approximately 630 bp and hybridizes with the sample.</li><li>(2) RNA is found by the Northern blot method as a band which corresponds to about 650 bases and with which ³²P-Kunitz soybean hybridized trypsin inhibitor gene described above.</li><li>(3) Kunitz trypsin inhibitor protein from soybeans is detected using standard immunological tests with a rabbit polyclonal antibody which was produced against commercially available (Sigma) Kunitz trypsin inhibitor after purification by reverse phase HPLC on a Vydac phenyl column.</li><li>(4) The activity of the Kunitz trypsin inhibitor from soybeans is determined by using immunological methods to purify material from plant extracts using the polyclonal anti-soybean Kunitz trypsin inhibitor antibody from rabbit and protein A Sepharose and then isolating the isolated material on the Ability to test the proteolysis of ¹⁴Inhibit C-BSA by trypsin using the test described in Example 1.</li></ul>

D. Resistance of transformed plants to damage from Lepidopteran larvae.

T1 seeds are germinated and leaf pieces obtained from seedlings in the 4-leaf stage are fed to newly hatched larvae of Ostrinia nubilalis or Heliothis zea. Freshly hatched larvae are placed in individual feeding pots with a 1 cm² Leaf piece set. 50 insects are tested per group. After 5 days, the insect weight, insect survival and the amount of eaten leaves are determined. The resistance of the transformed plants is determined by a statistically significant (Student's t Test p &lt;0.05) decrease in insect survival, insect weight or in the amount of leaves eaten by leaves of plants that contain the Kunitz trypsin inhibitor gene from soybeans express with those of control plants transformed with the vector without gene insertion or with those of untransformed plants.

Example 37: Cotton which is resistant to damage by Le. By expression of the Kunitz trypsin inhibitor from soybeans

p

I do

p

is larvae

A. Construction of the pClB10-CTI vector.

The Kunitz trypsin inhibitor gene from soybeans is removed together with the 35S CaMV promoter from pCiB710-KTi (Example 36A) using suitable restriction enzyme and linked to pCIB10. This vector is called pCIB10-KTI.

B. Transformation and regeneration of plants.

Transformed plants are obtained as described in Examples 7 to 10 using pCIB10-KTI or pCIB715 / 710 KTI (Example 36A) in Agrobacterium.

C. Test of plants for the expression of the trypsin inhibitor.

Tests of transformants for the expression of the Kunitz trypsin inhibitor from soybeans are carried out as described in Example 36C.

D. Resistance of transformed plants to Lepidoptera.

Leaf discs from 4-week-old transformed plants are fed to newly hatched Heliothis virescens, H. zea or Pectinophora gossypiella. Freshly hatched larvae are placed in individual feeding cups with a 1 cm² Leaf piece set. 50 insects are tested per group. After 5 days, the insect weight, insect survival and the amount of leaves eaten are determined. The resistance of the transformed plants is determined by a statistically significant (Student's t test p &lt;0.05) decrease in larval weight, larval survival and in the amount of leaves eaten by the leaves of plants in which the Kunitz trypsin inhibitor gene expresses is compared with that of control plants transformed with the vector without gene insertion, or with those of non-transformed plants.

Example 38: Tomatoes which are resistant to damage from lepidopteran larvae by the expression of the Kunitz trypsin inhibitor from soybeans

A. Construction of the vector.

pCIB715 / 710-KTI and pCIB10-KTI are prepared as described in Examples 36A and 37A.

B. Transformation and regeneration of plants.

Transformed plants are prepared as described in Example 33B using the pCIB10-KTI vector or the pCIB715 / 710-KTI vector.

C. Test of plants for the expression of the trypsin inhibitor.

The transformants are tested for the expression of the Kunitz trypsin inhibitor from soybeans as described in Example 36C.

D. Resistance of Transformed Plants to Lepidopteran Damage.

Leaf discs from 4-week-old transformed plants are fed to newly hatched Heliothis zea or Manduca sexta. Freshly hatched larvae are placed in individual feeding cups with a 1 cm² Leaf piece set. 50 insects are tested per group. After 5 days, the insect weight, insect survival and the amount of leaves eaten are determined. The resistance of the transformed plants is determined by a statistically significant (Student's t test p &lt;0.05) decrease in larval weight, larval survival and in the amount of leaves eaten by the leaves of plants in which the Kunitz trypsin inhibitor gene expresses is compared with that of control plants transformed with the vector without gene insertion, or with those of non-transformed plants.

Example 39: Tobacco which is resistant to damage from lepidopteran larvae by the expression of the Kunitz trypsin inhibitor from soybeans

A. Construction of the vector.

The pCIB10-KTI vector is prepared as described in Example 37A.

B. Transformation and regeneration of plants.

Transformed plants are prepared as described in Example 6 using the pCIB10-KTI vector or the pCIB715 / 710-KTI vector (Example 36A) in Agrobacterium.

C. Test of plants for the expression of the trypsin inhibitor.

The transformants are tested for the expression of the Kunitz trypsin inhibitor from soybeans as described in Example 36C.

(D) Resistance of transformed plants to damage by lepidopters.

Leaf discs from 4-week-old transformed plants are fed to newly hatched Heliothis zea or Manduca sexta. Freshly hatched larvae are placed in individual feeding cups with a 1 cm² Leaf piece set. 50 insects are tested per group. After 5 days, the insect weight, insect survival and the amount of leaves eaten are determined. The resistance of the transformed plants is determined by a statistically significant (Student's t test p &lt;0.05) decrease in larval weight, larval survival and in the amount of leaves eaten by the leaves of plants in which the Kunitz trypsin inhibitor gene expresses is compared with that of control plants transformed with the vector without gene insertion, or with those of non-transformed plants.

Example 40: Coiled grass (Dactylis glomerata) which is resistant to damage by Lepidopteran larvae due to the expression of the Kunitz trypsin inhibitor from soybeans

A. Construction of the vector.

pCIB710-KTI and pCIB715 / 710-KTI are prepared as described in Example 36A.

B. Transformation and regeneration of plants.

Transformed plants are obtained as described in Examples 24 to 28 using pCIB710-KTl and pCIB715 / 710-KTI.

C. Test of plants for the expression of the trypsin inhibitor.

The test of transformants for the expression of the Kunitz trypsin inhibitor from soybeans is carried out as described in Example 36C.

D. Resistance of transformed plants to damage from Lepidopteran larvae.

Seedlings obtained from T1 seeds are planted in fine soil in 100 mm petri dishes (10 per dish, 5 dishes). When the second leaves appear on the seedling, each shell is infected with 20 second-molting Crambus caliginosellus larvae. After 7 days, the number and weight of the survivors are determined together with the weight of the washed grass roots. The resistance of transformed plants is determined by a statistically significant (Student's t Test p &lt;0.05) drop in larval weight gain, drop in larval survival rate, or drop in root weight loss, relative to insect-free plants, by the plants that express the Kunitz trypsin inhibitor gene from soybeans with control plants transformed with the vector without gene insertion, or compared to non-transformed plants.

Example 41: Maize which is resistant to damage by lepidopteran larvae by the expression of α1-antitrypsin

(A) Construction of the pCIB715 / 710-AATI vector.

The DNA sequences encoding ai-antitrypsin are made according to the method of Rosenberg et al. (1984). For insertion into the BamHI site in the pCIB710 vector, the DNA fragment is cut with appropriate enzymes and the oligonucleotides necessary to create compatible ends are linked to pCIB710 cut with BamHI by the above described Procedure is applied. The resulting vector is called pCIB710-AATI. This vector is cut with EcoRI and the resulting DNA is treated with alkaline phosphatase. Plas mid pCIB715 (Rothstein et al., 1987) is cut with EcoRI and linked to the pCIB710-AATI described above. The resulting vector is named pCIB715 / 710-AATI.

(B) Transformation and regeneration of corn.

Corn tissue is transformed with the pCIB710 vector, or preferably with the pCIB715 / 710 vector, which contains the ai-antitrypsin gene insert, and plants are regenerated as described in Examples 16 to 20. For control purposes, plants are produced in the same way, but with the pCIB710 vector or the pCIB715 vector without gene insertion. Transformed plant tissue is selected based on its sensitivity to the corresponding antibiotic. The initial plants are self-pollinated and seeds (T1 seeds) are obtained.

(C) Test of plants for ai-antitrypsin expression.

Plants grown from T1 seeds are analyzed for the presence and expression of the a1-antitrypsin gene using the following test procedures.<ul id="ul0019" list-style="none"><li>(1) DNA is isolated and cut with BamHI; the degradation products are separated electrophoretically on a 1.5% agarose gel. The DNA fragments are transferred to nitrocellulose and hybridized with the ai-antitrypsin gene, which by nick translation with³²P is marked. The presence of the ai-antitrypsin gene is detected by a band that corresponds to approximately 1200 bp and hybridizes with the sample.</li><li>(2) RNA is found by the Northern blot method as a band corresponding to about 1200 bases and hybridizing with the 32P-a1 antitrypsin gene described above.</li><li>(3) ai-antitrypsin protein is detected using standard immunological tests by a rabbit polyclonal antibody produced against commercially available (Sigma) ai-antitrypsin.</li><li>(4) The activity of ai-antitrypsin is determined by immunologically purifying plant extract material using rabbit polyclonal anti-ai antitrypsin and protein A Sepharose and testing the isolated material for its ability to prevent proteolysis of ¹⁴Inhibit C-BSA by trypsin using the test described above.</li></ul>

D. Resistance of transformed plants to damage from Lepidopteran larvae.

T1 seeds are germinated and leaf pieces obtained from seedlings in the 4-leaf stage are fed to newly hatched larvae of Ostrinia nubilalis or Heliothis zea. Freshly hatched larvae are placed in individual feeding pots with a 1 cm² Leaf piece set. 50 insects are tested per group. After 5 days, the insect weight, insect survival and the amount of eaten leaves are determined. The resistance of the transformed plants is determined by a statistically significant (Student's t test p &lt;0.05) decrease in insect survival, insect weight or amount of leaves eaten, by leaves of plants expressing the ai-antitrypsin gene with those of control plants which have been transformed with the vector without gene insertion or with those of untransformed plants.

Example 42: Cotton Produced by Expression of a

i

-Antitrypsin is resistant to damage from lepidopteran larvae

A. Construction of the pCIB10-AATI vector.

The ai-antitrypsin gene is removed together with the 35S CaMV promoter from pCIB710-AATI (Example 41A) using suitable restriction enzymes and linked to pCIB10. This vector is called.pCIB10-AATI.

B. Transformation and regeneration of plants.

Transformed plants are obtained as described in Examples 7 to 10 using pCIB10-AATI or pCIB715 / 710-AATI (Example 41A) in Agrobacterium.

C. Test of plants for expression of ai-antitrypsin.

Tests of transformants for the expression of ai-antitrypsin are carried out as described in Example 41C.

D. Resistance of transformed plants to Lepidoptera.

Leaf discs from 4-week-old transformed plants are fed to newly hatched Heliothis virescens, H. zea or Pectinophora gossypiella. Freshly hatched larvae are placed in individual feeding cups with a 1 cm² Leaf piece set. 50 insects are tested per group. After 5 days, the insect weight, insect survival and the amount of leaves eaten are determined. The resistance of the transformed plants is determined by a statistically significant (Student's t test p &lt;0.05) decrease in larval weight, larval survival and in the amount of leaves eaten by the leaves of plants in which the ai-antitrypsin gene is expressed with those of control plants transformed with the vector without gene insertion or with those of non-transformed plants.

Example 43: Tomatoes which are resistant to damage by Lepidopteran larvae by the expression of ai-antitrypsin

A. Construction of the vector.

pCIB715 / 710-AATI and pCIB10-AATI are prepared as described in Examples 41A and 42A.

B. Transformation and regeneration of plants.

Transformed plants are prepared as described in Example 33B using the pCIB10-AATI vector and the pCIB715 / 710-AATI vector.

C. Test of plants for expression of ai-antitrypsin.

The transformants are tested for expression of ai-antitrypsin as described in Example 41C.

D. Resistance of Transformed Plants to Lepidopteran Damage.

Leaf discs from 4-week-old transformed plants are fed to newly hatched Heliothis zea or Manduca sexta. Freshly hatched larvae are placed in individual feeding cups with a 1 cm² Leaf piece set. 50 insects are tested per group. After 5 days, the insect weight, insect survival and the amount of leaves eaten are determined. The resistance of the transformed plants is determined by a statistically significant (Student's t test p &lt;0.05) decrease in larval weight, larval survival and in the amount of leaves eaten by the leaves of plants in which the ai-antitrypsin gene is expressed with those of control plants transformed with the vector without gene insertion or with those of non-transformed plants.

Example 44: Tobacco which is resistant to damage by Lepidopteran larvae by the expression of ai-antitrypsin.

A. Construction of the vector.

pCIB715 / 710-AATI and pCIB10-AATI are prepared as described in Examples 41A and 42A.

B. Transformation and regeneration of plants.

Transformed plants are prepared as described in Example 6 using the pCIB10-AATI vector or the pCIB715 / 710-AATI vector (Example 41A) in Agrobacterium.

C. Test of plants for expression of ai-antitrypsin.

The test of the transformants for the expression of ai-antitrypsin is carried out as described in Example 41C.

Resistance of transformed plants to damage from lepidopters.

Leaf discs from 4-week-old transformed plants are fed to newly hatched Heliothis zea or Manduca sexta. Freshly hatched larvae are placed in individual feeding cups with a 1 cm² Leaf piece set. 50 insects are tested per group. After 5 days, the insect weight, insect survival and the amount of leaves eaten are determined. The resistance of transformed plants is determined by a statistically significant (Student's t test p &lt;0.05) decrease in larval weight, larval survival and in the amount of leaves eaten, by the leaves of plants in which the ai-antitrypsin gene is expressed, with those of control plants transformed with the vector without gene insertion, or with those of non-transformed plants.

Example 45: Coiled grass (Dactylis glomerata), which is produced by the expression of α

1

-Antitrypsin is resistant to damage from lepidopteran larvae.

A. Construction of the vector.

pCIB710-AATI and pCIB715 / 710-AATI are prepared as in Example 41A.

B. Transformation and regeneration of plants.

Transformed plants are obtained as described in Examples 24 to 28 using pCIB710-AATI and pCIB715 / 710-AATI.

C. Test of plants for expression of ai-antitrypsin.

The test of transformants for the expression of ai-antitrypsin is carried out as described in Example 41C.

D. Resistance of transformed plants to damage from Lepidopteran larvae.

Seedlings obtained from T1 seeds are planted in fine soil in 100 mm petri dishes (10 dishes, 5 dishes). When the second leaves appear on the seedling, each shell is infected with 20 second-molting Crambus caliginosellus larvae. After 7 days, the number and weight of the survivors are determined together with the weight of the washed grass roots. The resistance of transformed plants is determined by a statistically significant (Student's t Test p &lt;0.05) drop in larval weight gain, drop in larval survival rate or drop in root weight loss, relative to insect-free plants, by the plants that express the ai-antitrypsin gene, be compared with control plants transformed with the vector without a gene insert, or with non-transformed plants.

Example 46: Maize resistant to damage from Lepidopteran larvae by expression of Eglin

A. Construction of the pCIB715 / 710 Eglin vector.

The DNA sequences encoding Eglin C and Eglin C mutants are obtained as described in Example 3D. For insertion into the BamHI site of the pCIB710 vector, the DNA fragment is cut with suitable enzymes and the synthetic oligonucleotides necessary to generate compatible ends are linked together with BamHI cut pCIB710 using the method described above . The resulting vector derived from the Eglin C (Arg45) mutant is called pCIB710-Eglin. This vector is cut with EcoR1 and the resulting DNA is treated with alkaline phosphatase. The plasmid pCIB715 (Rothstein et al., 1987) is cut with EcoRI and linked to the CIB710-Eglin from above. The resulting vector is called pCIB715 / 710-Eglin.

B. Maize transformation and regeneration.

Corn tissue is transformed with the pCIB710 vector or preferably with the pCIB715 / 710 vector containing the Eglin C (Arg45) gene insert, and plants are regenerated as described in Examples 16 to 20. For control purposes, plants are produced in the same way, but with the pCIB710 vector or the pCIB715 vector without gene insertion. Transformed plant tissue is selected based on its sensitivity to the corresponding antibiotic. The initial plants are self-pollinated and seeds (T1 seeds) are obtained.

C. Test of plants for Eglin expression.

Plants grown from T1 seeds are tested for the presence and expression of the Eglin C (Arg45) gene using the following test methods.<ul id="ul0020" list-style="none"><li>(1) DNA is isolated and cut with BamHI; the degradation products are separated electrophoretically on a 1.5% agarose gel. The DNA fragments are transferred to nitrocellulose and hybridized with the Eglin C (Arg45) gene, which by nick translation with^32p is marked. The presence of the Eglin gene is identified by a band that corresponds to approximately 230 bp and hybridizes with the sample.</li><li>(2) RNA is found by the Northern blot method as a band which corresponds to about 230 bases and with which ³²P-Eglin C (Arg45) gene hybridized, which is described above.</li><li>(3) Mutated Eglin C (Arg45) protein is detected using standard immunological tests with a rabbit polyclonal antibody produced against Eglin C (Arg45).</li><li>(4) Eglin activity is determined by using immunological methods to purify material from plant extracts with the aid of the polyclonal anti-Eglin C (Arg45) antibody from rabbit and protein A Sepharose and then to test the isolated material for the ability to perform proteolysis from ¹⁴Inhibit C-BSA by trypsin using the test described above.</li></ul>

D. Resistance of transformed plants to damage from Lepidopteran larvae.

T1 seeds are germinated and leaf pieces obtained from seedlings in the 4-leaf stage are fed to newly hatched larvae of Ostrinia nubilalis or Heliothis zea. Freshly hatched larvae are placed in individual feeding pots with a 1 cm² Leaf piece set. 50 insects are tested per group. After 5 days, the insect weight, insect survival and the amount of eaten leaves are determined. The resistance of the transformed plants is determined by a statistically significant (Student's t Test p &lt;0.05) decrease in insect survival, insect weight or in the amount of leaves eaten by leaves of plants that contain the Eglin C- (Arg45) gene express with those of control plants transformed with the vector without gene insertion or with those of untransformed plants.

Example 47: Cotton which is resistant to damage by lepidopteran larvae by expression of Eglin

A. Construction of the pCIB10-Eglin vector.

The Eglin C (Arg45) gene is removed together with the 35S CaMV promoter from pCIB710-Eglin (Example 46A) using suitable restriction enzyme and linked to pCIB10. This vector is called pCIB10-Eglin.

B. Transformation and regeneration of plants.

Transformed plants are obtained as described in Examples 7 to 10 using pCIB10-Eglin or pCIB715 / 710 Eglin (Example 46A) in Agrobacterium.

C. Test of plants for Eglin expression.

Tests of transformants for Eglin expression are carried out as described in Example 46C.

D. Resistance of transformed plants to Lepidoptera.

Leaf discs from 4-week-old transformed plants are fed to newly hatched Heliothis virescens, H. zea or Pectinophora gossypiella. Freshly hatched larvae are placed in individual feeding cups with a 1 cm² Leaf piece set. 50 insects are tested per group. After 5 days, the insect weight, insect survival and the amount of leaves eaten are determined. The resistance of the transformed plants is determined by a statistically significant (Student's t test p &lt;0.05) decrease in larval weight, larval survival and in the amount of leaves eaten by the leaves of plants in which the Eglin C (Arg45) gene is expressed with that of control plants which are transformed with the vector without gene insertion, or with those of non-transformed plants.

Example 48: Tomatoes which are resistant to damage by Lepidopteran larvae by Eglin expression

A. Construction of the vector.

pCIB715 / 710-Eglin and pCIB10-Eglin are prepared as described in Examples 46A and 47A.

B. Transformation and regeneration of plants.

Transformed plants are prepared as described in Example 33B using the pCIB10-Eglin vector or the pCIB715 / 710-Eglin vector.

C. Test of plants for Eglin expression.

The transformants are tested for Eglin expression as described in Example 46C.

D. Resistance of Transformed Plants to Lepidopteran Damage.

Leaf discs from 4-week-old transformed plants are fed to newly hatched Heliothis zea or Manduca sexta. Freshly hatched larvae are placed in individual feeding cups with a 1 cm² Leaf piece set. 50 insects are tested per group. After 5 days, the insect weight, insect survival and the amount of leaves eaten are determined. The resistance of the transformed plants is determined by a statistically significant (Student's t test p &lt;0.05) decrease in larval weight, larval survival and in the amount of leaves eaten by the leaves of plants in which the Eglin C (Arg45) gene is expressed with that of control plants transformed with the vector without gene insertion or with those of non-transformed plants.

Example 49: Tobacco that is resistant to damage from lepidopteran larvae by Eglin expression

A. Construction of the vector.

pCIB715 / 710-Eglin and pCIB10-Eglin are prepared as described in Examples 46A and 47A.

B. Transformation and regeneration of plants.

Transformed plants are prepared as described in Example 6 using the pCIB10-Eglin vector or the pCIB715 / 710-Eglin vector (Example 46A) in Agrobacterium.

C. Test of plants for Eglin expression.

The transformants are tested for expression as described in Example 46C.

D. Resistance of Transformed Plants to Lepidopteran Damage.

Leaf discs from 4-week-old transformed plants are fed to newly hatched Heliothis zea or Manduca sexta. Freshly hatched larvae are placed in individual feeding cups with a 1 cm² Leaf piece set. 50 insects are tested per group. After 5 days, the insect weight, insect survival and the amount of leaves eaten are determined. The resistance of the transformed plants is determined by a statistically significant (Student's t Test; &lt;0.05) decrease in larval weight, larval survival and in the amount of leaves eaten by the leaves of plants in which the Eglin C (Arg45) gene is expressed with that of control plants which are transformed with the vector without gene insertion, or with those of non-transformed plants.

Example 50: Ball grass (Dactylis glomerata) which is resistant to damage by Lepidopteran larvae by the expression of Eglin.

A. Construction of the vector.

pCIB710-Eglin and pCIB715 / 710-Eglin are prepared as described in Example 46A.

B. Transformation and regeneration of plants.

Transformed plants are obtained as described in Examples 24 to 28 using pCIB710-Elgin and pCIB715 / 710-Eglin.

C. Test of plants for the expression of Eglin.

The test of transformants for the expression of Eglin is carried out as described in Example 46C.

D. Resistance of transformed plants to damage from Lepidopteran larvae.

Seedlings obtained from T1 seeds are planted in fine soil in 100 mm petri dishes (10 per dish, 5 dishes). When the second leaves appear on the seedling, each shell is infected with 20 second-molting Crambus caliginosellus larvae. After 7 days, the number and weight of the survivors are determined together with the weight of the washed grass roots. The resistance of transformed plants is determined by a statistically significant (Student's t Test p &lt;0.05) drop in larval weight gain, drop in larval survival rate, or drop in root weight loss, relative to insect-free plants, by the plants that express the Eglin C (Arg45) gene, be compared with control plants transformed with the vector without gene insertion, or with non-transformed plants.<tables id="tabl0007" num="0007"><img file="EP0348348A2_D0008.tif" /></tables><tables id="tabl0008" num="0008"><img file="EP0348348A2_D0009.tif" /></tables><tables id="tabl0009" num="0009"><img file="EP0348348A2_D0010.tif" /></tables><tables id="tabl0010" num="0010"><img file="EP0348348A2_D0011.tif" /></tables><tables id="tabl0011" num="0011"><img file="EP0348348A2_D0012.tif" /></tables><tables id="tabl0012" num="0012"><img file="EP0348348A2_D0013.tif" /></tables><tables id="tabl0013" num="0013"><img file="EP0348348A2_D0014.tif" /></tables><tables id="tabl0014" num="0014"><img file="EP0348348A2_D0015.tif" /></tables><tables id="tabl0015" num="0015"><img file="EP0348348A2_D0016.tif" /></tables><tables id="tabl0016" num="0016"><img file="EP0348348A2_D0017.tif" /></tables><tables id="tabl0017" num="0017"><img file="EP0348348A2_D0018.tif" /></tables><tables id="tabl0018" num="0018"><img file="EP0348348A2_D0019.tif" /></tables><tables id="tabl0019" num="0019"><img file="EP0348348A2_D0020.tif" /></tables>

CREDENTIALS

<ul id="ul0021" list-style="none"><li>An, G., Watson, BD, Stachel, S., Gordon, MP, Nester, EW, Embo J. 4, 277, 1985 Anastasi, A., Brown, MA, Kembhavi, AA, Nicklin, MHJ, Sayers, CA. , Sunter, DC, Barrett, AJ, Biochem. J. 211, 129, 1983</li><li>Barrett, AJ, Salvesen, G. (ed.), Proteinase inhibitors, Research monographs in cell and tissue physiology 12, Elsevier, Amsterdam, 1986</li><li>Beasley, CA, Ting, JP, Amer. J. Bot. 60, 130, 1973</li><li>Bevan, M., Barnes, WM, Chilton, M.-D., Nucl. Acids Res. 11, 369, 1983</li><li>Bevan, M., Nucl. Acids Res. 12, 8711, 1984</li><li>de Block, M., Herrera-Estrella, L., van Montagu, M., Schnell, J., Zambryski, P., EMBO J. 3, 1681, 1984 Christie, BR (ed.), CRC Series in Agriculture, CRC Handbook of Plant Science in Agriculture 1, CRC Press,</li><li>Boca Raton, Florida, 1987</li><li>Chu, C., Wang, C., Sun, C., Hsü, C., Yin, K. Chu, C., Bi, Fi., Scientia Sinica 18, 659, 1975</li><li>Covey, SN, Lomonossoff, GP, Hull, R., Nucl. Acids Res. 9, 6735, 1981</li><li>Ditta, G., Stansfield, S., Corbin, D., Helinski, DR, Proc. Natl. Acad. Sci. USA 77, 7347, 1980</li><li>Fischhoff, DA, Bowdish, KS, Perlak, FJ, Marrone, PG, McCormick, SM, Niedermeyer, JG, Dean, DA, Kusano-Kretzmer, K., Mayer, EJ, Rochester, DE, Rogers, SG, Fraley, RT , Biotechnology 5, 807, 1987 de Framond, AJ, Barton, KA, Chilton, M.-D., Biotechnology 1, 262, 1983</li><li>Gamborg, OL, Miller, RA, Ojima, K., Exptl. Cell Res. 50, 151, 1968</li><li>Gatehouse, AMR, Boulter, D., J. Sci. Food Agric. 34, 345, 1983</li><li>Graves, ACF, Goldman, SL, Plant Mol. Biol. 7, 43, 1986</li><li>Gray, DJ, Conger, BV, Plant Cell Tissue Organ Culture 4, 123, 1985</li><li>Grimsley, N., Hohn, T., Davies, JW, Hohn, B., Nature 325, 177, 1987</li><li>Grimsley, NH, Ramos, C., Hein, T., Hohn, B., Biotechnology 6, 185, 1988</li><li>Gritz, L., Davies, J., Gene 25, 179, 1983</li><li>Guiseley, KB, Renn, DW, Agarose, Marine Colloids Division, FMC Corporation, Rockland, Maine, 1975 Hanning, GE, Conger, BV, Theor. Appl. Genet. 63, 155, 1982</li><li>Hilder, VA, Gatehouse, AMR, Sheerman, SE, Barker, RF, Boulter, D., Nature 330, 160, 1987 Hoekema, A., Hirsch, PR, Hooykaws, PJJ, Schilperoort, RA, Nature 303, 179, 1983</li><li>Hoffman, LM, Sengupta-Gopalan, C., Paaren, HE, Plant Mol. Biol. 3, 111, 1984</li><li>Hohn, T., Richards, K., Lebeurier, G., In: Gene cloning in organisms other than E. coli, Current Topics in Microbiology and Immunology 96, 193, 1982</li><li>Holsters, M., de Waele, D., Depicker, A., Messens, E., van Montagu, M., Schell, J., Mol. Gen. Genet. 163, 181, 1978</li><li>Hood, EE, Helmer, GL, Fraley, RT, Chilton, M.-D., J. Bacteriol. 168, 1291, 1986</li><li>Hooykaas-Van Slogteren, GMS, Hooykaas, PJJ, Schilperoort, RA, Nature 311, 763, 1984</li><li>Kao, KN, Michayluk, MR, Planta 126, 105, 1975</li><li>Klein, TM, Gradziel, T., Fromm, ME, Sanford, JC, Biotechnology 6, 559, 1988</li><li>Maniatis, T., Fritsch. EF, Sambrook, J., Molecular cloning, a laboratory manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, 1982</li><li>McCormick, S., Niedermeyer, J., Fry, J., Barnason, A., Horsch, R., Fraley, R., Plant Cell Reports 5, 81, 1986 Murashige, T., Skoog, F., Physiol. Plans. 15, 473, 1962</li><li>Murdock, LL, Brookhart, G., Dunn, PE, Foard, DE, Kelley, S., Kitch, L., Shade, RE, Shucle, RH, Wolfson, JL, Comp. Biochem. Physiol. 87B, 783, 1987</li><li>Nagy, JI, Maliga, P., Z. Plant Physiol. 78, 453, 1976</li><li>Negrutiu, 1st, Shillito, R., Potrykus, 1st, Biasini, G., Sala, F., Plant Mol. Biol. 8, 363, 1987</li><li>Nelson, RS, McCormick, SM, Delannay, X., Dube, P., Layton, J., Anderson, EJ, Kaniewska, M., Proksch, RK, Horsch, RB, Rogers, SG, Fraley, RT, Beachy, RN, Biotechnology 6, 403, 1988</li><li>Odell, JT, Nagy, F., Chua, N.-H., Nature 313, 810, 1985</li><li>Oka, A., Sugisaki, H., Takanami, M., J. Mol. Biol. 147, 217, 1981</li><li>Ooms, G., Hooykaas, PJJ, Moolenaar, G., Schilperoort, RA, Gene 14, 33, 1981</li><li>Ooms, G., Hooykaas, PJJ, van Veen, RJM, van Beelen, P., Regensburg-Tuink, TJG, Schilperoort, RA, Plasmid 7, 15, 1982</li><li>de la Pena, A., Lörz, H., Schell, J., Nature 325, 274, 1987</li><li>Potrykus, J., Saul., MW, Petruska, J., Paszkowski, J., Shillito, RD, Mol. Gen. Gene. Genet. 199, 183, 1985 Reich, TJ, lyer, VN, Haffner, M., Holbrook, LA, Miki, BL, Can. J. Bot. 64, 1259, 1986a</li><li>Reich, TJ, lyer, VN, Miki, BL, Biotechnology 4, 1001, 1986b</li><li>Rothstein, SJ, Lahners, KN, Lotstein, RJ, Carozzi, NB, Jayne, SM, Rice, DA, Gene 53,153,1987 Rosenberg, S., Barr, PJ, Najarian, RC, Hallewell, RA, Nature 312, 77, 1984</li><li>Sanger, F., Nicklen, S., Coulson, AR, Proc. Natl. Acad. Sci. USA 74, 5463, 1977</li><li>Sanger, F., Science 214, 1205, 1981</li><li>Schenk, RU, Hildebrandt, AC, Can. J. Bot., 50, 199, 1972</li><li>Schwabe, C., Anastasi, A., Crow, H., McDonald, JK, Barrett, AJ, Biochem. J. 217, 813, 1984</li><li>Shillito, RD, Paszkowski, J., Potrykus, I., Plant Cell Reports 2, 244, 1983</li><li>Shillito, RD, Saul MW, Paszkowski, J., Müller, M., Potrykus, 1., Biotechnology 3, 1099, 1985</li><li>Simon, R., Priefer, U., Pühler, A., in: Pühler, A. (ed.), Molecular Genetics of the Bacteria-Plant Interaction, Springer Verlag, Berlin, 98, 1983a</li><li>Simon et al., Biotechnology 1, 784, 1983b</li><li>Southern, EM, J. Mol. Biol. 98, 503, 1975</li><li>Stockhaus, J., Eckes, P., Blau, A., Schell, J., Willmitzer, L., Nucl. Acids Res. 15, 3479, 1987 Turk, V., Brzin, J., Longer, M., Ritonja, A., Eropkin, M., Borchart, U., Machleidt, W., Hoppe-Seyler's Z. Physiol. Chem 364, 1487, 1983</li><li>Wolfson, JL, Murdock, LL, Entomol. Exp. Appl. 44, 235, 1987</li><li>Wood, WI, Gitschier, J., Lasky, LA, Lawn, RM, Proc. Natl. Acad. Sci. USA 82, 1585, 1985 Yadav, NS, Vanderleyden, J., Bennett, DR Barnes, WM, Chilton, MD, Proc. Natl. Acad. Sci. USA 79, 6322, 1982</li><li>Young, RA, Davis, RW, Proc. Natl. Acad. Sci. USA 80, 1194, 1983</li><li>Zoller, MJ, Smith, M., in: Wu, R., Grossmann, L., Moldave, K., Methods in Enzymology 100, 468, 1983</li></ul>

Filing:

<ul id="ul0022" list-style="none"><li>(1) E. coli HB101 / pML147 was deposited on January 28, 1988 under the deposit number DMS 4380 at the German Collection of Microorganisms and Cell Cultures in Braunschweig, Federal Republic of Germany. The following microorganisms have been deposited with the American Type Culture Collection (ATCC) in Rockville, Maryland, USA:</li><li>(2) Plasmid pLW111 (ATCC 40235) on May 14, 1986.</li><li>(3) Zea mays cell culture (ATCC 40326) on May 20, 1987.</li><li>(4) Plasmid p368 (ATCC 67700) on May 19, 1988.</li></ul>