RU2126047C1 - Method of preparing zea mays l plants exhibiting resistance to damages caused by insects - Google Patents

Abstract

FIELD: agriculture, selection. SUBSTANCE: method is designated for construction of transgenic plants exhibiting resistance to damages caused by insects and can be used in transformation of totipotent protoplasts by vector containing structural gene that encodes polypeptide of crystalline protein δ-endotoxin in Bacillus thuringiensis. Before transformation tissue of nonmatured embryos is isolated, cultured to formation of special callus type. Callus is treated with enzyme preparation to obtain totipotent protoplasts and the latter is subjected for transformation for fertile plants generating. Regeneration of plants from protoplast cells ensures to use somatic hybridization for producing the improved species of Zea mays L. EFFECT: improved method of plants preparing. 31 cl, 4 dwg, 6 tbl, 34 ex

Description

The present invention relates to a method for producing cereal plants (Zea mays) resistant to damage caused by insects that regenerate from protoplasts or from cells derived from protoplasts
BACKGROUND OF THE INVENTION
(Part A) Regeneration of Zea mays plants from protoplasts
The description of the regeneration of Zea mays from callus dates mainly from the mid-70s (for example, Green and Phillips, Crop. Se. 15 (1975) 417-421; Harms et al., Z. Pflanzenzuechtg; 77 (1976) 437-352; European Patent Applications EP-0160390, Zowe and Smith (2985), EP-0176162, Cheng (1985), EP-0177738, Close (1985)). Attempts have been made to obtain division and subsequent regeneration of plants from Zea mays protoplasts for more than 12 years (Potrykus et al., Jn. Cell Genetics in Higher Plants, Dudits et al., (Eds), Akademiai Kiado, Budapest (1976) 129- 140, and references therein, Harm "Maize and Cereal Protoplasts - Facte and Perspectives" Maize for Biological Research, WF Sheridan, ed. (1982); Dale in: Protoplasts (1983); Potrykus et al. (Eds) Zecture Proceedings, Experientia Supplementum 46, Potrykys et al., Eds Birkhauser, Basel (1983) 31-41 and references therein). However, Zea mays protoplasts always yielded growth only in cultures that were not able to regenerate fertile plants (Potrykus et al., Mol. Gen. Genet 156 (1977) 347-350; Potrykus et al., Theor. Appl. Genet., 54 (1979) 209-214; Choury and Zurawski, Yheor. Appl. Genet. 59 (1981) 341-344; Vasil, in: Proc 5th Int. Cong, Plant Tissue and Cell Culture, Fujiwara Tokyo (1982) 101-104; Imbric-Milligan and Hodges, Planta, 168 (1986) 395-401; Vasil and Vasil, Theor. Appl. Genet. 73 (1987) 793-798). However, none of these references describes the procedure for producing protoplasts capable of regenerating fertile plants.

 The ability to produce such protoplasts will enable genetic manipulation of this important cereal crop, using, for example, transformation or fusion of protoplasts or using protoplasts as starting material for the selection of valuable new phenotypes.

 Although there was great interest in the genetic transformation of Zea mays, there is no description of a successful method, in vitro, which can lead to a regenerated transformed plant, although there were descriptions of the transformation of the Zea mays protoplast, which led to non-regenerated callus (Fromm et al., Nature, 319 (1986) 791-793). One method for producing transgenic Zea mays plants can be found by stably transforming protoplasts capable of regenerating whole fertile plants. However, no method has been described for the preparation of Zea mays protoplasts capable of differentiating with the appearance of whole fertile plants.

 This and other objectives were achieved in accordance with the present invention by disclosing a method for producing Zea mays protoplasts that can divide to form callus colonies. Protoplasts can be transformed, and calli can regenerate Zea mays fertile plants. The process for preparing Zea mays protoplasts capable of dividing and forming callus, which is then induced to regenerate into whole fertile plants, requires a special type of callus. Such calluses and methods for their preparation and identification are described hereinafter.

 For the initial cell suspension, a special type of callus is used, when subcultured, they lead to suspensions that can be used to isolate protoplasts. These protoplasts are capable of division and the formation of callus, which can be induced on plant regeneration. Cell division or callus formation may be promoted by certain agents, such as O-lower alkanoylsalicylic acid (O-acetylsalicylic acid and its homologs), plant growth regulators or DMSO, or combinations thereof.

 The regeneration of plants from cells originating from Zea mays protoplasts in culture is essential for applying somatic hybridization, for obtaining improved Zea mays varieties through somaclonal variability, and for using genetic engineering to obtain new Zea mays plants cultivated or inbred plant varieties with new phenotypic traits .

) является штаммом бактерий, который продуцирует кристаллический белок, также упоминаемый как дельта-эндотоксин. Технически, этот кристаллический белок является протоксином, который превращается в токсин при проглатывании личинками чешуекрылых жесткокрылых и двукрылых насекомых.(Part B) Zea mays insecticidal corn plants
Bacillus thuringiensis (hereinafter

) is a strain of bacteria that produces crystalline protein, also referred to as delta endotoxin. Technically, this crystalline protein is a protoxin that turns into a toxin when swallowed by Lepidoptera Lepidoptera and Diptera.

является потенциально важным инсектицидом, не обладающим известными вредными воздействиями на людей, других теплокровных, птиц, рыб или насекомых, других чем личинки чешуекрылых, жесткокрылых и двукрылых насекомых. Активность

токсина является крайней высокой, так что требуются только нанограммовые количества для успешного уничтожения личинок насекомых. Другие преимущества использования кристаллического белка

в качестве инсектицида включает его широкий спектр действия против личинок чешуекрылых, жесткокрылых и двукрылых насекомых, и для таких личинок возникают затруднения развития устойчивости к кристаллическому белку, даже когда кристаллический белок используют в широких масштабах. Указанные личинки являются основной проблемой в сельском и лесном хозяйствах и особенно при выращивании кукурузы.Crystalline protein

is a potentially important insecticide that does not have known harmful effects on humans, other warm-blooded animals, birds, fish or insects, other than the larvae of Lepidoptera, Cole-winged and dipteran insects. Activity

the toxin is extremely high, so only nanogram quantities are required for the successful destruction of insect larvae. Other benefits of using crystalline protein

as an insecticide, it includes its wide spectrum of action against Lepidoptera, Coleoptera and Diptera insects, and for such larvae it is difficult to develop resistance to a crystalline protein, even when a crystalline protein is used on a large scale. These larvae are a major problem in agriculture and forestry, and especially when growing corn.

 Crystalline protein is effective as an insecticide when it is applied to plants infected with larvae of Lepidoptera, Coleoptera or Diptera.

кристаллический белок (протоксин) выделяют из Bacillus и наносят на растения с помощью стандартных методов, таких как опудривание дустом или распыление. Препараты, содержащие

, кристаллический белок, коммерчески используют в качестве биологических инсектицидов. Например, Бастоспейн, выпускаемый Биохем Продактс Лтд., Дипель, выпускаемый Эббот Лабораториз, и Турцид, выпускаемый Сандоз АГ. Тот факт, что

продуцирует кристаллический белок только во время споруляции, является значительным недостатком для производства и применения этого биологического инсектицида. Такое ограничение фазы роста, особенно в промышленном процессе, может привести в результате к неудобствам и излишнему времени, требующемуся для производства.Still

crystalline protein (protoxin) is isolated from Bacillus and applied to plants using standard techniques such as dusting with dust or spraying. Preparations containing

, a crystalline protein, is commercially used as biological insecticides. For example, Bastospain manufactured by Biochem Products Ltd., Dipel manufactured by Abbot Laboratories, and Turcid manufactured by Sandoz AG. The fact that

produces crystalline protein only during sporulation, is a significant disadvantage for the production and use of this biological insecticide. Such a limitation of the growth phase, especially in an industrial process, can result in inconvenience and excessive time required for production.

 In addition, the costs associated with this production makes it difficult for such a biological insecticide to compete effectively with other commercially available chemical-based products, such as, for example, pyrethroid derivatives.

токсина является, например, тот факт, что белок обычно остается на поверхности обработанных растений, где он является эффективным только против поверхностью питающихся личинок и где он инактивируется при продолжительном воздействии ультрафиолетового излучения. Такая инактивация может быть по крайней мере одной из причин общей потери персистентности кристаллического белка в окружающей среде. Следовательно, необходимо частое и дорогостоящее нанесение кристаллического белка.Another disadvantage regarding application

toxin is, for example, the fact that the protein usually remains on the surface of the treated plants, where it is effective only against the surface of feeding larvae and where it is inactivated by prolonged exposure to ultraviolet radiation. Such inactivation may be at least one of the reasons for the overall loss of persistence of crystalline protein in the environment. Therefore, frequent and costly application of crystalline protein is necessary.

кристаллический белок или белок, обладающий по существу токсичностью по отношению к насекомым

кристаллического белка, в растениях.These and other disadvantages can be eliminated by the introduction and expression of a gene encoding

crystalline protein or a protein that is substantially toxic to insects

crystalline protein in plants.

кристаллический белок или белок, обладающий по существу токсичностью по отношению к насекомым

кристаллического белка, в протопластах кукурузы, и регенерации фертильных трансгенных растений кукурузы из трансформированных протопластов и выращивания этих устойчивых к насекомым растений кукурузы.The present invention describes how these disadvantages can be addressed by introducing and expressing a gene encoding

crystalline protein or a protein that is substantially toxic to insects

crystalline protein in maize protoplasts; and regenerating fertile transgenic maize plants from transformed protoplasts and growing these insect-resistant maize plants.

 Taking advantage of genetic engineering, the gene responsible for producing a useful polypeptide can also be transformed into a donor cell, in which the gene is usually found, in a host cell, in which the gene is not found in nature; Cohen and Boyer, US Pat. Nos. 4,237,224 and 4,468,464. Indeed, there are few restrictions for such transfers. Genes can be transferred between viruses, bacteria, plants and animals. In some cases, the transferred gene is functional or can be made functional in the host cell. When the host cell is a plant cell, the whole plant can be made functional in the host cell. When the host cell is a plant cell, the whole plant can sometimes be regenerated from the cell.

 Genes typically contain regions of DNA sequences, including a promoter and a transcription region. The transcription region usually contains a 5'-untranslated region encoding a sequence and a 3'-untranslated region.

 The promoter contains the DNA sequence necessary to initiate transcription, during which the transcribing region is converted into mRNA. In eukaryotic cells, it is believed that the promoter includes a region recognized by RNA polymerase and a region that places RNA polymerase on DNA to initiate transcription. This last region is referred to as the TATA box, which is typically found about 30 nucleotides higher from the transcription initiation site.

 The region after the promoter is a sequence that is transcribed in mRNA but not translated into a polypeptide. This sequence consists of the so-called 5'-untranslated region and is believed to contain sequences that are responsible for initiating transcription, such as a ribosome binding site.

имеет ген с кодирующей последовательностью, которая транслируется в аминокислотную последовательность инсектицидного протоксина кристаллического белка.The coding region is a sequence that is immediately below the 5'-untranslated region in the DNA or corresponding RNA. It is a coding region that is translated into polypeptides in accordance with the genetic code. For instance,

has a gene with a coding sequence that translates into the amino acid sequence of an insecticidal protoxin of a crystalline protein.

 The coding region is located below the sequence that is transcribed into mRNA but not translated into a polypeptide. This sequence is called the 3'-untranslated region and is believed to contain a signal that leads to the end of transcription, and in eukaryotic mRNA, a signal that causes polyadenylation of the transcribed strand. It is believed that mRNA polyadenylation has processing and transport functions.

 Natural genes can be transferred entirely from donated blood to the host cell. However, it is often preferable to construct a gene containing the desired coding sequence with a promoter and, in some cases, 5 ′ and 3 ′ untranslated regions that do not exist in nature in the same gene as the coding region. Such constructs are known as chimeric genes.

. Эти методы позволяют продуцировать кристаллический белок, но не устраняют недостатков применения кристаллического белка в качестве коммерческого инсектицида.Genetic engineering methods have already been described for improved ways to obtain crystalline protein. For example, Schnepf et al., US Pat. Nos. 4,448,885 and 4,467,036, describe plasmids for producing crystalline protein in bacterial strains other than

. These methods allow the production of crystalline protein, but do not eliminate the disadvantages of using crystalline protein as a commercial insecticide.

токсина непосредственно в растения, чтобы позволить растениям защищать самих себя:, (Klausner A., Biotechnology, 2 (1984) 409 - 413). Adang et al. Европейская заявка на патент EP-0142924 (Агригенетикс) и De Greve H.M.J. et al. EP-0193269 (Плант Генетик Системс Н.В.) заявили способ клонирования генов токсина из Bt в табак. Ни в одной из этих публикаций не приводится примеров трансформации однодольных растений.Proposals have been made to clone genes

toxin directly into plants to allow plants to protect themselves :, (Klausner A., Biotechnology, 2 (1984) 409 - 413). Adang et al. European Patent Application EP-0142924 (Agrigenetics) and De Greve HMJ et al. EP-0193269 (Plant Genetic Systems N.V.) claimed a method for cloning toxin genes from Bt into tobacco. None of these publications provide examples of transformation of monocotyledonous plants.

в клетках растений кукурузы и новых способов контроля личинок насекомых, которые питаются на растениях кукурузы, содержащих

кристаллический белок или подобный полипептид, обладающий по существу токсичностью по отношению к насекомым (инсектицидной активностью)

кристаллического белка. Под "контролем" как здесь упоминается, понимают или умерщвление личинок или по крайней мере снижение их питания. Кроме того, существует необходимость создания способа защиты растений кукурузы против повреждений, вызываемых паразитами или патогенами, заключающегося в продуцировании количества пестицидного или антипатогенного белка в клетке растения и растении соответственно, это количество является достаточно эффективным для уничтожения или контроля паразита или патогена. Кроме того, существует необходимость в предпочтительном создании способа защиты растений кукурузы против повреждений насекомыми, заключающегося в продуцировании инсектицидно эффективного количества

кристаллического белка или белка, обладающего по существу токсичностью против насекомых

кристаллического белка, в клетках растений. Далее, существует необходимость в создании способа защиты растений кукурузы от повреждений химическими агентами, такими как, например, гербициды, чтобы такие химикаты могли быть безопасно нанесены на растения кукурузы.There is a need to develop new methods for producing crystalline protein

in the cells of corn plants and new ways of controlling insect larvae that feed on corn plants containing

crystalline protein or similar polypeptide having substantially toxicity to insects (insecticidal activity)

crystalline protein. By "control" as mentioned here, they mean either the killing of the larvae, or at least a decrease in their nutrition. In addition, there is a need to create a method for protecting corn plants against damage caused by parasites or pathogens by producing an amount of a pesticidal or antipathogenic protein in a plant cell and a plant, respectively, this amount is sufficiently effective to kill or control the parasite or pathogen. In addition, there is a need for the preferred creation of a method of protecting corn plants against insect damage, which consists in producing an insecticidally effective amount

crystalline protein or a protein that is substantially toxic to insects

crystalline protein in plant cells. Further, there is a need for a method of protecting corn plants from damage by chemical agents, such as, for example, herbicides, so that these chemicals can be safely applied to corn plants.

 From the prior art, at the time of the invention, it was not possible to predict that cereal plants (Zea mays), in particular fertile plants, can be regenerated from protoplasts, from cells derived from protoplasts or callus originating from protoplasts. Even less could be assumed that Zea mays protoplasts containing stable incorporated exogenous DNA can regenerate transgenic plants, in particular Zea mays fertile transgenic plants.

 The objects of the present invention are described in detail below.

 A method for producing insect resistant Zea mays L. plants, comprising isolating tissue from immature embryos, culturing said tissue in a medium for initiating callus and / or in a medium for maintaining callus proliferation, until a special type of callus is formed which is non-sticky, granular and loose structures and containing dense cytoplasmic, dividing cells, the selection of the above type of callus and its subsequent treatment with an enzyme preparation to remove cell walls, collection, purification y and transformation of the obtained totipotent protoplasts with a vector containing a structural gene encoding a polypeptide of the crystalline protein of δ-endotoxin of Bacillus thuringiensis toxic to insects and flanked by transcriptional control sequences containing a promoter and 5'- and 3'-untranslated sequences that are functional in cereal , further sowing of transformed totipotent protoplasts on a nutrient medium for cell division and formation, subculturing them on a regenerator medium tion and subsequent production of fertile plants Zea mays L., resistant to damage caused by insects.

 The invention is further directed to the method according to claim 1, characterized in that after the selection of a particular type of callus, it is transferred into a liquid nutrient medium to form a suspension of cells or cell aggregates, subculturing the resulting suspension over time and with a frequency sufficient to maintain cells and cell aggregates in a viable state, followed by selection and preservation of cultures containing aggregates of dividing cells with a dense cytoplasm, which are characterized by a smoother surface than others cell aggregates.

 In FIG. 1 shows callus that is suitable for use in Zea mays initial embryogenic suspensions (approximately 7X magnification). (Stage a).

 In FIG. Figure 2 shows the dense cytoplasmic, dividing cells that are retained in the cultures of stages b - d (magnification of about 20X).

In FIG. Figure 3 shows the nucleotide sequence of the endotoxin gene of Bacillus thuringiensis var. Kurstaki: HD1
In FIG. Figure 4 shows the sequence of the B. thuringiensis δ-endotoxin crystalline protein polypeptide encoded by the structural gene.

 A preferred nucleotide sequence that colorants a crystalline protein is one that is shown by nucleotides 156-3623 in the sequence, or a shorter sequence that encodes an insecticidal fragment of such a crystalline protein; Geiser et al., Gene, 48 (1986) 109-118.

Definitions
Zea mays / corn: In the present description or in the claims, these terms refer to any plant or part of a plant (protoplasts, cells, tissues, organs, organelles, embryos, calli, shoots, etc.) belonging to the species Zea mays. The terms, as used here, include variety, race, species, inbred lines and Zea mays hybrids. The present invention includes, but is not limited to such inbred maize lines, for example Funr 5N984, Funk 5N986, Funk 2717, Funk 211D, Funk 2N217A, B73, A632, CM105, B37, B84, B14, Vc17, R 186, MS 71, A188, F A91, A641 and W 117. All elite genotypes (varieties, varieties, inbred lines, hybrids, etc.) are preferred, including the elite inbred lines Funk 5 N 984, Funk 5N 986, Funk 2717, Funk 211D and Funk 2N217A , more preferred elite line inbred Funk 2717.

 BMS: "Black Mexican Sweet Corn", an old small-grained variety of corn with a dark blue grain pericarp, giving the grain a "black" tint.

Plant cell
Structural and physiological unit of plants, consisting of protoplast and cell wall.

 The term "plant cell" refers to any cell that is either part of or separated from a plant.

 Some examples of cells covered by the present invention include differentiated cells that are part of a living plant; differentiated cells in culture; undifferentiated cells in culture; undifferentiated tissue cells such as callus or tumors, seeds, embryos (embryos); shoots and pollen.

Plant tissue
A group of plant cells organized into a structural and functional unit. Any plant tissue in a plant or culture. This term includes, but is not limited to, the whole plant, plant cells, plant organs, plant seeds, protoplasts, cell culture and any group of plant cells organized into structural and / or functional units. The use of this term in combination with or in the absence of any specific type of plant tissue, as listed above, or in other words covered by this definition, is not intended to exclude any other type of plant tissue.

Plant organ
A separate and apparently structured and differentiated part of plants, such as a root, trunk, leaf, flower bud or embryo. A plant organ may consist of various types of plant cells or plant tissues.

Proplast
An isolated plant cell without a cell wall.

 Transgenic plant. A plant that has stably introduced exogenous DNA in its genetic material. The term includes exogenous DNA, which can be introduced into protoplasts in various forms, including, for example, naked DNA in a circular, linear or supercoiled form, DNA contained in nucleosomes or nuclei or in their parts, DNA complexed or associated with other molecules, DNA enclosed in liposomes, spheroplasts, cells or interlayers. Various methods for introducing exogenous DNA into interlayers are known in the art and can be used to produce transgenic plants of the present invention.

Culture
The proliferating mass of cells in an undifferentiated or partially differentiated state. The term "cell culture" includes callus cultures, consisting of a mass of cells growing on a cured culture medium, and suspension cell cultures growing in a dispersed state in a mixed liquid culture medium.

Embryo (fetus)
The earliest stage of plant development, or originating from the zygote (then called the sexual embryo), or from the embryogenic somatic cell (then called the somatic embryo), with stages of recognizable morphology, structure and cellular organization, including cell to the globular stage, to the cotyledon stage. (Corn germ development is described, for example, in Randolph, LF, J. Agril. Research, 53 (1936) 881-916; cereal germ development is generally described, for example, Brown, WV, Phytomorphology, 10 (1960) 215-223 )

The escape
A multicellular structure consisting of a sprout and a root in the shape of a small plant.

 Chimeric sequence or chimeric gene.

 A DNA sequence containing at least two heterologous parts, for example, parts originating from or having essentially a sequence homologous to pre-existing DNA sequences that are not associated in previous steps. Pre-existing DNA sequences can be of natural or synthetic origin.

 The coding sequence of DNA.

 A DNA sequence that, when transcribed and translated, results in the formation of a cell polypeptide.

Gene
A discrete chromosomal region consisting of regulatory DNA sequences that are responsible for controlling expression, namely, translation and transcription, and from a colony sequence that is transcribed and translated to produce a specific polypeptide.

Originating from:
in the context of this description, genes, parts of genes or other DNA sequences "derived from" other DNA sources encompass genes, parts of genes or other DNA sequences that are identical or substantially homologous to the material of the DNA source.

Phenotypic trait
An observed property resulting from the expression of one or more genes.

 Pre-existing DNA sequence.

 The DNA sequence that exists before its use, in whole or in part, in the product or method according to the present invention. Although this pre-existence usually reflects natural origin. Previously existing sequences can be of natural or synthetic origin.

Essential sequence homology
Substantial functional and / or structural equivalence between nucleotide or amino acid sequences. Functional and / or structural differences between sequences having substantial sequence homology will be minimal.

Dicamba
3,6-Dichloro-2-methoxybenzoic acid;
MES 2- (N-morpholino) ethanesulfonic acid;
2,4-D: 2,4-dichlorophenoxyacetic acid.

 Picloram: 4-amino-3,6,6-trichloropicolinic acid.

Tris-HCl: alpha, alpha, alpha-tris- (hydroxymethyl) methylamine hydrochloride;
EDTA: 1-ethylenediamine-N, N, N ', N'-tetraacetic acid;
PEG: polyethylene glycol.

Agarose: The preparation and purification of agarose are described, for example, by Guiseley and Renn, ("The Agarose Monograph"; Marine Colloids Division FMC Corp. (1975)). Agarose is one of the components of agar. Commercially available agar usually consists of a mixture of neutral agarose and ion agaropectin with a large number of side groups. Commercial agarose is usually obtained from agar by conventional methods. Usually a certain amount of side chains remains intact and determines the physicochemical properties of agarose, such as gelation and melting point. Low melting agarose, especially Sea Plague ^R agarose is the preferred curing agent in the process described hereinafter.

SH Wednesday: Wednesday, Schenka R.Yu., et al. Can. H. Bot., 50 (1972) 199-204; without growth regulators (SH medium may be liquid or solidified 0.8% (w / v) agar or 0.5% (w / v) GelRite ^R ) The medium is usually sterilized using techniques known in the art, for example , by heat or sterilized by autoclaving at 121 ^o C and 15 lb / ⁱⁿ² (1.2 kg / ^cm2) pressure for about 15-20 minutes.

MS Environment: Murashige T.and Shoog F., Physiol Plant., 15 (1962) 473
B5 Environment: Gambord, O.et al., Exp. Cell Res., 50 (1968) 151 N6 medium: Chu et al. Scientia sinica, 18 (1975) 659.

 The composition is given in table. 1.

KM medium: KaO KNet.al., Planta, 126 (1975) 105-110. This medium can be liquid or solidified agar, agarose or Gel Rite ^R and can also be prepared and used without ascorbic acid, vitamin D and vitamin A The components of the medium, with the exception of the curing agent, are usually sterilized by filtration through a 0.2 μm filter.

 The composition of the medium used is given in table. 1.

 (Macronutrients and trace elements of the medium correspond to those given in the literature).

Notes to the table. 1:
a: macronutrients are usually prepared as a 10x concentrated storage solution, and macronutrients as 1000x concentrated storage solutions.

b: citric, fumaric and maleic acids (each at a final concentration of 40 mg / l) and sodium pyruvate (final concentration of 20 mg / l) are prepared as 100x concentrated storage solutions, pH 6.5 is adjusted with NH ₄ OH and added to this environment.

 c: Adenine (0.1 mg / l final concentration) and guanine, thymine, uracil, hypoxanthine and cytosine (final concentration of each 0.03 mg / l) are prepared in the form of 1000x concentrated storage solutions, pH 6.5 is adjusted as described above, and added to this medium.

d: The following amino acids are added to this medium using 10x storage solutions (pH 6.5 with NH ₄ OH) to obtain the desired final concentrations: glutamine (5.6 mg / L), alanine, glutamic acid (each 0, 6 mg / l), cysteine (0.2 mg / l), aspargin, aspartic acid, cystine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine (each 0, 1 mg / l).

 e: Vitamin storage solution is usually prepared 100x concentrated.

Cellulose RS: Cellulose RS Yakult Hongsha Co., Ltd., 1.1.19 Higashi-Shinbashi, Minato-ku, Tokyo 105, Japan
Pectoliasis Y-23: Seishin Pharmaceutical Co., Ltd. 4-13 Koamijo, Nihonbashi, Tokyo, Japan.

фильтр: Нальге Кол., Отделение Сирбон Корп. Рочестер, Нью-Йорк, 14602, США.

filter: Nalge Kol., Branch Sirbon Corp. Rochester, New York, 14602, USA.

 Bgl I [Restriction enzyme Bgl I]; New England Biolabs, 32 Tozer Road, Beverly, MA, 01915, USA, or any other commercial supplier.

 Bam HI Restriction enzyme Bam HI; New England Biolabs, 32 Tozer Road, Beverly, MA, 01915, USA, or any other commercial supplier.

 Hydromycin B: cytotoxin, hydromycin B, purified; cat. N 400050 Calbiochem Bering Diagnostics, La Yolla, CA 92037, USA, Group N 702296.

: ГельРайтГеллан Гум, Скотт Лабораториз Инк., Gel Rite Фискерсвилл, РИ 02823.Gol

: GelRightGellan Gum, Scott Laboratories Inc., Gel Rite Fiskersville, RI 02823.

Prime Rit randon primer Rit of IBI Random primer Rit:
International Biotechnology Inc., BO Box 1565, New Haven, CT 07606, USA (Catalog No. 77800; group NF 630-01).

контейнер. Маленький этерильный контейнер
(примерно 7 х 7 х 10 см) изготовлен из полупрозрачного пластика с полупрозрачной крышкой: используют для выращивания побегов, культур побегов и т. д. Поставщик: Магента Корп., 3800 Н.Милуокси Авеню, Чикаго, ИЛ, 60641, США. Может быть заменен любым подобным стерильным полупрозрачным контейнером (например, стеклянным или пластиковым).

container. Small ether container
(approximately 7 x 7 x 10 cm) is made of translucent plastic with a translucent lid: used for growing shoots, shoot crops, etc. Supplier: Magenta Corp., 3800 N. Miluoxy Avenue, Chicago, IL, 60641, USA. It can be replaced by any similar sterile translucent container (e.g. glass or plastic).

Convert microEinsteins to suites
mkE - luxury
0.1 - 200 - 8 - 16700
0.1 - 100 - 8 - 8350
30 - 90 - 2500 - 7500
10 - 40 - 830 - 3330 \\\ 30 - 80 - 2500 - 6670
Deposition of plasmids in microorganisms or in plant cells
In accordance with the present invention, the following listed plasmids (in microorganisms) and plant cells were deposited in accordance with the requirements of the Budapest Treaty:
(1) Zea mays suspension cell culture 6-2717 CG Funk 2717 was deposited in the American Type Culture Collection (ATCC), Rockville, USA, and has ATCC storage number 40326 (deposited date: May 20, 1987).

 (2) Plasmid pCIB712 (in E. coli strain MC1061) was deposited in ATCC and has ATCC storage number 67407 (date of deposit: May 18, 1987).

 (3) Plasmid pSCHI (in E. coli strain K12) was deposited at the German Microorganism Collection (DMS) in Göttingen, Germany, and has DMS storage number 4129 (date of deposit: May 29, 1987).

 (4) Plasmid pCIB10 (35 SBt (in E. coli strain MC1061) was deposited in ATCC and has ATCC storage number 67329 (date of deposit: February 27, 1987).

 (5) Plasmid pE16 (8-C4) in E. coli strain HB101) was deposited in ATCC and has ATCC storage number 67420 (date of deposit: May 29, 1987).

 (6) Plasmid pCIB10 (19 SBt (in E. coli strain HB 101) was deposited in ATCC and has ATCC storage number 67330 (date of deposit: May 27, 1987).

Detailed Description of the Present Invention (Part A) Regeneration of Zea mays Maize Plants from Protoplasts
Calluses used in the present invention can be derived from any tissue of Zea mays plants. Preferably, this tissue is an immature tissue, such as an immature panicle, an immature embryo, the basal part of a young leaf and, in principle, any other Zea mays tissue capable of forming callus. A particularly useful tissue is the shield of the immature Zea mays embryo.

 In principle, all Zea mays genotypes (varieties, varieties, inbred lines, hybrids, etc.) are useful in the present invention. The most valuable are commercially interesting genotypes.

 Preferred are all elite genotypes, especially all inbred lines, most preferred elite inbred lines. Suitable inbred lines are, for example, Funk 2717, Funk 211D, Funk 2N 217A, B 73, A632, CM105, B37, B84, B14, Mo 17, R 168, MS71, A188, FA91, A641, and W117. Additional examples of useful Zea mays genotypes are Funk 5 N 984 and Funk 5N986. Especially preferred are the elite inbred lines Funk 2717, Funk 211D, Funk 5N984 or Funk 2N217A, and the elite genotype Funk 2717 is most preferred.

 In the description below, a method for producing embryogenic suspensions of Funk 2717 will be disclosed, from which protoplasts that can regenerate into fertile plants can be isolated. It should be understood that other maize genotypes may be used in the described method using in most cases routine techniques known to those skilled in the art.

 Stage (a). Callus formation.

 Zea mays plants of the Funk 2717 genotype are grown before flowering and pollen formation. Immature embryos are removed from the cobs and placed on medium for initiation. The embryos typically have a length between 1 and 4 mm, preferably between 1.5 and 3 mm, and more preferably between 2 and 2.5 mm. The initiation medium may be liquid or solidified. Any initiation medium capable of inducing callus may be used. Some suitable examples include, but are not limited to, those based on the MS medium (Murashige T. and SRoog F., Physiol. Plant. 15 (1962) 473), medium No. 6 (Chu et al., Scientia Sinica 18, (1975) 659), B5 medium (Gamborg o et al., Exp. Cell. Res. 50 (1968) 151), SH medium or KM medium with the corresponding concentrations of sugars and plant growth regulators. Other suitable combinations are described in Plant Culture Media "George E.E. et al / eds /, Exegetics Ltd, Edington, Westbury, Wiltshire, England / 1987 /.

Callus is removed from cultured immature embryos and placed on a support medium. Any supportive medium capable of proliferating callus may be used. Some suitable examples include, but are not limited to, MS-based, N 6, B5, or KM-based media, with appropriate concentrations of sugars and plant growth regulators. The material is subcultured on fresh support medium at an appropriate time interval, for example, every 1-120 days, preferably 3-21 days, more preferably 5-14 days. Initiation and maintenance can be carried out in the light or in the dark, preferably in the dark. The temperature may be between 0 and 50 ^o C, preferably between 20 and 32 ^o C, more preferably between 25 and 28 ^o C.

 Subculturing is continued under the same conditions until the callus is a special type of callus. A special type of callus suitable for use in initiating suspensions in accordance with the present invention is relatively non-sticky, granular and loose. In FIG. 1 shows callus that is suitable for use in initiating embryogenic suspensions in accordance with the present invention. Suitable callus is subcultured at appropriate time intervals, for example, every 1-120 days, preferably 3-21 days, more preferably 5-14 days. The specified special type of callus, which is able to regenerate Zea mays fertile plants and whose protoplasts can be regenerated into fertile plants, can be isolated and is one of the preferred variants of the present invention.

Stage (b) - (d) suspension culture
A special type of callus from step (a) is placed on a liquid medium to form a suspension of cells and cell aggregates. Some suitable examples of liquid media include, but are not limited to, media based on M-medium, N 6 medium, B5 medium, and KM medium with corresponding concentrations of sugars and plant growth regulators. The preferred medium is N6 medium.

 The cultures are subcultured with a frequency sufficient to maintain the cells and cell aggregates in a viable and proliferating state, for example, every 1-120 days, preferably 3-21 days, more preferably 5-10 days. Cultures that contain aggregates of dense cytoplasmic, dividing cells are retained. Cultures with a high content of expanded cells are discarded. The number of cells that are tightly cytoplasmic may increase over time.

 A desirable type of suspension culture is a culture containing preferably at least 50%, and more preferably at least 70% of the cells in aggregates of dense, cytoplasmic dividing cells. The desired type of suspension culture is obtained after a suitable period of time, usually after about 7 days, preferably after about 30 days. The temperature and light conditions are the same as in step (a). Suspension culture was deposited in ATCC, Rockville, Maryland, USA, under storage number 40326 (date of deposit: May 20, 19687).

Therefore, the present invention describes a method for producing suspension cultures of Zea mays cells and cell aggregates from which protoplasts can be isolated, and protoplasts are able to regenerate fertile plants, consisting of the stages:
(a) receiving callus, which is relatively non-sticky, granular and loose, on induction and support media.

 (b) transferring callus to a liquid medium to form a suspension of cells or cell aggregates.

(c) subculturing the suspension (i) for a period of time (preferably about 7 days) sufficient to produce protoplasts in a viable, dividing stage capable of regeneration into fertile plants, and (ii) with a frequency sufficient to maintain any cells and cell aggregates in a viable state, and
(d) selection and preservation of those cultures from the product of stage (c) that contain aggregates from dense, cytoplasmic, dividing cells.

Stage (e1) - obtaining protoplasts
Protoplast isolation is carried out by incubating calli or a suspension culture of cells or cell aggregates (obtained in accordance with steps (a) - (d) above) with an enzyme preparation that removes cell walls. Suitable enzyme preparations are known in the art. A particularly suitable enzyme preparation is, for example, a preparation containing 4% w / v RS pulp (Yakult Pharmacyutical Ind., Co., Ltd., Shingikan Sho, Nishinomiya, Japan) with 1% w / v Rosima (Rum and Haas, Philadelphia , PA, USA) in CMC saline solution (8.65 g / l KCl, 12.5 g / l CaCl ₂ • 2H ₂ O, 16.47 g / l MgSO ₄ • 7H ₂ O, 5 g / l MES pH 5.6). Digestion is carried out at a temperature between 0 and 50 ^o C, preferably between 10 and 35 ^o C, more preferably between 26 and 32 ^o C. Digestion is continued until protoplasts are released. The time required for digestion is usually from 30 minutes to 5 days, preferably between 1 hour and 1 day, more preferably between 3 and 5 hours.

Therefore, in addition, the present invention provides a method for producing Zea mays protoplasts capable of regenerating fertile plants, said method comprising the steps of:
(a) obtaining callus, which is relatively non-sticky, granular and loose, in callus-inducing and callus-supporting environments,
(b) transferring callus to a liquid medium to obtain a suspension of cells or cell aggregates,
(c) subculturing the suspension (i) for a period of time (preferably about 7 days) sufficient to produce protoplasts in a viable, dividing stage capable of regeneration into fertile plants, and (ii) with a frequency sufficient to maintain any cells and cell aggregates in a viable state,
(d) selecting and maintaining those cultures from the product of step (c) that contain aggregates of dense, cytoplasmic, dividing cells,
(e1) removing cell walls using suitable enzymes and isolating the resulting protoplasts.

 Released protoplasts are collected and purified using standard techniques such as filtration and centrifugation.

 Stage (e2). Treatment of protoplasts of exogenous DNA.

 Protoplasts can be treated with exogenous DNA in such a way as to obtain cells that contain all or part of exogenous DNA stably introduced into their genetic material. Exogenous DNA is any DNA added to a protoplast. Exogenous DNA may contain a promoter active in Zea mays or may use a promoter already present in the Zea mays genome.

Exogenous DNA may be a chimeric gene or part of it. Processing of protoplasts of exogenous DNA can be carried out in accordance with the methods described in the following publications:
(Paszkowski J. et al. The EMBO Journal 3, 12 (1984) 2717-2732; European Patent Application EP-0 164 575 (Paszkowski, J. et al);
Shillito RD et al. Bio (Techonology, 3 (1985) 1099-1103; Potrykus I et al., Mol. Gen. Genet 199 (1985) 183-188; Zoers H., et al., Mol. Gen. Genet., 199 (1985) 178-182; Fromm ME et al., Nature, 319 (1986) 79) 1-793; British Patent Application GB-2 140,822 (Mettler IJ): and Negrutiu I. et al., Plant Mol. Biology 8 (1987) 363-373). These publications are provided here as a prior art.

 Exogenous DNA can be added in any form, such as, for example, unprotected linear or circular DNA, DNA encapsulated in liposomes, DNA in charoplasts, DNA and other plant protoplasts, DNA complexed with salts. DNA in the form of nucleosomes, chromosomes or nuclei or parts thereof, and the like. The introduction of foreign DNA can be stimulated using any known suitable method in the art, including the techniques described in the above links.

 First of all, the chimeric genes considered in the present invention are those that provide transformed plant protoplasts originating from protoplasts to tissues and, finally, originating from protoplasts to plants, the appearance of new phenotypic properties (valuable properties), such as increased resistance to parasites ( for example, Arthropods, including insects and ticks, nematodes, etc.); increased resistance to pathogens (for example, to phytopathogenic fungi, bacteria, viruses, etc.); increased resistance to chemicals (e.g. herbicides (such as triazines, carbamates, ureas, dinitroanilides, sulfonylureas, imidazolinones, triazolo-pyrimidines, bialaphos, glyphosate, etc.), insecticides or other biocides), increased resistance to cytotoxins (e.g. to hydromycin, kanamycin, chloramphenicol, etc.); increased resistance to harmful environmental influences (soil or atmospheric) (for example, heat, cold, wind, soil conditions, moisture, drought, etc.); with increased formation of the desired substances (for example, in leaves, seeds, tubers, roots, stems, etc.). Desirable substances produced by a genetically modified plant include proteins, starches, sugars, amino acids, alkaloids, aromatic substances, coloring agents, fats, etc.); reduced formation of undesirable substances (for example, toxins, protease inhibitors, undesirable aromatic substances, undesirable proteins, fats, carbohydrates or secondary metabolites, etc.); or modified morphological or evolutionary phenotypic properties (for example, changes in height, nature of growth, shape, color, strength, flowering time, etc.).

 Cytotoxin resistance can be imparted by a gene expressing an enzyme in plant cells that detoxifies a cytotoxin, for example, neomycin phosphotransferase type II or aminoglycoside phosphotransferase type IV for detoxification of kanamycin, hydromycin and other aminoglycoside P or trans-glutathione S-transferase another catabolic enzyme known for the detoxification of triazine, sulfonylurea or other herbicides. Cytotoxin resistance can also be imparted using a gene that expresses a “target enzyme” form (cytotoxin action site) in a plant that is cytotoxin resistant, for example, an acetohydroxy acid synthase that is insensitive and inhibited by sulfonylureas or imidazolinones or other herbicides acting at this metabolic stage, or a form of 5-enolpyruvyl shikimate-3-phosphate (EPSP) synthase, which is insensitive to glyphosate inhibition. It may be appropriate to express these altered target enzymes in a form that allows their transport in the plant cell to the desired cell division, namely to the chloroplast, in the above examples.

 In some cases, it is advisable to target the gene product in mitochondria, vacuoles, into endoplasmic vesicles or other cell particles, or even into the intercellular (apoplastic) space.

 Resistance to certain classes of fungi can be imparted by introducing a gene that expresses chitinase in plant tissues. Many plant pathogenic fungi contain chitin as an integral part of the hyphae or spore structure, including basidiomycetes (smut and rust) and ascomycetes and imperfect fungi (including Alternaria and Bipolaris, Exerohilum turcicum, Colletotricum, Gleocercospora and Cercospora). Chitinase can inhibit the growth of mycelium of certain pathogen in vintro. The leaves and roots of a plant expressing chitinase are protected against many types of fungal attacks.

 Resistance to parasites can be imparted by a gene encoding a pesticidal polypeptide.

 Resistance to Arthropods can be given, for example, by a gene encoding a polypeptide that is toxic to the stages of larvae and adults, or makes the plant unattractive to larvae, so that they eat less.

Вторым классом белков, которые будут придавать устойчивость растению, являются ингибиторы протеаз. Ингибиторы протеаз являются обычными составляющими запасных структур растения (Ryan C., Ann. Rev. Plant Physiol; 24 (1973) 173 - 196). Ингибирование развития насекомых было приписано наличию ингибитра трипсина в муке (Lipke H. , Fraenkel G. S. and Liener I.E., J. Agg. Foos chem. 2 (1954) 410 - 414). Очищенный ингибитор протеазы Баумана-Бирка, выделенный из соевой муки, как было показано, ингибирует протеазы кишечника личинок Tenebrio (Berk V, Gertler A. and Khalef S., Biochem. Biophys. Acta, 67 (1963) 326 - 328). Ген, кодирующий ингибитор трипсина вигны китайской, описан Hilder et al (Nature, 330 (1987) 160 - 163).A suitable gene in this context is crystalline protein.

The second class of proteins that will confer resistance to the plant are protease inhibitors. Protease inhibitors are common constituents of plant storage structures (Ryan C., Ann. Rev. Plant Physiol; 24 (1973) 173 - 196). Inhibition of insect development has been attributed to the presence of a trypsin inhibitor in flour (Lipke H., Fraenkel GS and Liener IE, J. Agg. Foos chem. 2 (1954) 410-414). A purified Bauman-Birka protease inhibitor isolated from soybean flour has been shown to inhibit intestinal proteases of Tenebrio larvae (Berk V, Gertler A. and Khalef S., Biochem. Biophys. Acta, 67 (1963) 326 - 328). The gene encoding the Chinese cowpea trypsin inhibitor is described by Hilder et al (Nature, 330 (1987) 160-163).

 The gene encoding the protease inhibitor can be placed under the control of a plant promoter, preferably a constitutive promoter, such as the 35S protector of the sunflower mosaic virus (CaM V), in a suitable vector such as the pCIB 710 vector described below.

For example, a gene encoding a sequence of a Bauman-Birka soybean protease inhibitor can be obtained using cDNA cloning methods described by Hammond et al. (J. Biol. Chem. 259 (1984) 9883 - 9890). The corresponding clone can be identified either by immunoprecipitation of a hybrid selected by mRNA, as described by Hammond et al., Or by probing at a low fiber with a synthetic oligonucleotide containing 50 or more bases from the 5'-end of the sequence encoding a predetermined amino acid sequence (amino acid sequences are shown in handbook of G. Fasman "Handbook of Biochemistry and Molecular Biology, 2 (1976) 611 - 620). For example, for probe cloning of a soybean trypsin inhibitor from soybeans, a probe was used en the following nucleotide:
AA Asp Phe Val Leu Asp Asn Glu Gly Asn Pro Leu Glu Asn Gly Probe 5'-GAC TTC GTT GTG GAC AAC GAA GGT AAC CCG GTG GAA AAC GGT Gly Thr Tyr Tyr Ile Leu Ser AGP Ile Thr Ala Phe Gly Gly GGT ACC TAC TAC ATC CTG TCC GAC ATC ACC GCT TTC GGT GGT-3 '
The cloned DNA fragment can be sequenced using, for example, the Maxim and Gilbert techniques (Methods Enzymol., 65 (1980) 499-560) and the starting translation site is identified. If the cloned DNA fragment is longer than the coding sequence, additional 5'- and 3'-sequences are resected by Bal 31. If the cloned DNA fragment is shorter than the coding sequence, the cloned DNA is used as a primer to stretch the mRNA primer (Maniatis et al., Molecular Cloning: A Laboratory Manual (1982), Cold Spring Harbor, NY) and reclaim expanded DNA.

 The appropriate linkers are added to the DNA encoding the protein and the fragment is inserted into the selected vector (cassette), for example pCIB 710, so that the gene is under the control of the desired promoter (CaMV 35S and pCIB 710).

 An alternative method for generating a gene for protease inhibitors with less than 100 amino acids, such as a trypsin inhibitor of lima beans, is to synthesize them. The coding sequence is predetermined by the corresponding reverse translation and restriction sites for the target vector, included at each end. A synthetic gene is obtained by synthesizing overlapping oligonucleotides from 30-60 bases. These fragments kinase, ligate (Maniatis et al., Supra) and clone into the corresponding vector.

 A clone whose insertion is in the desired orientation can be identified by sequencing. Plasmid DNA is isolated and used to introduce maize into protoplasts.

Therefore, the present invention describes a method in which the method described in steps (a) to (d) further comprises the step of:
(e2) treating protoplasts from step (e1) capable of regenerating exogenous DNA into fertile plants to obtain cells that contain all or part of exogenous DNA stably introduced into their genetic material.

Стадия (f). Посев протопластов.A process for the preparation of Zea mays having stably introduced exogenous DNA, preferably exogenous DNA expressed in Zea mays, is another preferred aspect of the present invention.
A particularly preferred method for producing transgenic Zea mays is where the stably introduced exogenous DNA is a chimeric gene that encodes a polypeptide that is substantially toxic to the insect (insecticidal activity) of the crystalline protein

Stage (f). Sowing protoplasts.

Protoplasts are seeded for cultivation with or without DNA treatment in a liquid or cured culture medium. Some examples of suitable culture media include, but are not limited to, media based on KM medium, N6 medium, B5 medium and MS medium, with appropriate concentrations of sugars and plant growth regulators. A preferred medium is a KM medium containing a curing agent. Agarose is a preferred curing agent, especially Sea Plague ^R agarose (European Patent Application EP-0 129 668, Shillito RD et al., (1984). When used, Sea Plague ^R agarose concentrations may be between 0.1% and 2, 5% w / v, preferably between 0.6% and 1.5% w / v The optimum concentration of other curing agents may vary, but can be easily determined.

 The medium in which protoplasts are usually cultured in the presence of suitable plant growth regulators (such as auxins and / or cytokinins, etc.) promotes protoplast division and colony formation. Suitable plant growth regulators include, but are not limited to, 2,4-dichlorophenoxyacetic acid (2,4-D), 3,6-dichloro-2-methoxybenzoic acid (dicamba), 4-amino-3,6,6-trichloropicolinic acid (picloram) and para-chlorophenoxyacetic acid (pCPA). The amount of plant growth regulator is effective for regulating plant growth, a non-toxic amount. The concentration of such plant growth regulators is usually in the range from 0.01 to 100 mg / L, preferably 0.1 to 100 mg / L, more preferably 0.1 to 10 mg / L, even more preferably 0.3 to 3 mg / l

 Unexpectedly, it was found that O-lower alkanoylsalicylic acids (o-acetylsalicylic acid and its homologs / alone or in combination with plant growth regulators and / or DMSO promote the division or formation of colonies from plant protoplasts, preferably Zea mays protoplasts. The term "lower alkanoyl" means alkanoyl groups having 1 to 7, preferably 1 to 4, more preferably 2 to 3 carbon atoms.

 Preferred O-lower alkanoylsalicylic acids are O-acetylsalicylic acid and O-propionylsalicylic acid, preferably O-acetylsalicylic acid.

 The amount of O-lower alkanoylsalicylic acid should be sufficient to promote cell division and colony formation. An effective growth-regulating, non-toxic amount is preferred. Suitable concentrations of O-lower alkenoyl-salicylic acids are in the range of 0.1 to 3000 mg / L, preferably in the range of 10 to 300 mg / L, more preferably 10 to 100 mg / L, and even more preferably about 100 mg / l

 The amount of DMSO is sufficient to promote cell division or colony formation. Suitable concentrations of DMSO are those in the range of 0 to 3%, preferably 0.01 to 2%, more preferably 0.1 to 1%.

 Therefore, the present invention describes a method for increasing cell division or colony formation from plant protoplasts, preferably Zea mays protoplasts, comprising cultivating protoplasts in the presence of an efficiently controlling plant growth of a non-toxic amount of O-lower alkanoylsalicylic acid sufficient to promote cell division or colony formation. 0.01 to 100 mg / d, preferably 0.1 to 100 mg / l, more preferably 0.1 to 10 mg / l, even more preferably 0.3 to 3 mg / l of a plant growth regulator, are used.

 Preferred is the presence of DMSO in an amount promoting cell or colony formation. Preferred amounts of DMSO are 0 to 3%, preferably 0.01 to 2%, more preferably 0.1 to 1%.

 This method of increasing the division or colony formation from protoplasts is suitable for all plant protoplasts and is not limited to Zea mays. It is particularly suitable for Angiospermae and Gymnospermae, for example, plants of the following families: Solanaceae, Crueiferae, Liliaceae, Yitaceae, Chenopodiaceae, Rutaceae, Bromoliaceae, Rubidiaceae, Theaceae, Musaceae or Graminaceae, and especially of the family Puminuminapae. Preferred plants are representatives of Solanaceae, Cruciferae and Gramineae.

 The medium in which protoplasts are cultured may contain a medium that was previously balanced for the growth of suitable cells, for example, Zea mays or Dactylis glomerafa.

 Protoplasts can be cultured on solid or liquid medium without subculturing for a period of time up to 12 weeks, preferably up to 6 weeks, more preferably in the range from 3 to 4 weeks. Alternatively, a solid medium may be placed in a liquid medium, as described in European patent application EP-0 129 668, Shillito R.D. et al., (1984), or processed in some other way to help fission and / or colony formation from protoplasts. One suitable method is culturing together with cultured cells as described by Shneyour et al. / Plant Scince Zett. 33 (1984) 293) and Smitn et al. (Plant Science Zett 36 (1984) 67) and in Example 8S.

Protoplasts are cultured in the light or, preferably, in the dark at a temperature between 0 and 50 ^o C, preferably between 20 and 32 ^o C, more preferably between 25 and 28 ^o C. The light intensity is usually 0.1 to 200 μE / (m ² • c) (microEstien) (m ² • s), preferably 0.1 to 100 μE / (m ² • s), more preferably 30 to 90 μE / (m ² • s), even more preferably 10 to 40 μE / (m ² • s). Callus develops from protoplasts cultured on a solid medium, and callus originating from protoplasts according to stage (f) can be subcultured on a support medium, as described for callus in stage (a), and then subcultured on a regenerating medium according to stage (g) ), or can be directly subcultured on a regenerating medium according to stage (g).

 Stage (g). Sprout regeneration.

 Callus, which is formed from protoplasts according to step (f), is subcultured one or more times in suitable media to regenerate a fertile plant. Some examples of suitable culture media include, but are not limited to, those based on N6 medium, B5 medium, MS medium, or KM medium, with appropriate concentrations of sugars, plant growth regulators, and in some cases DMSO. The preferred medium is N6 medium without growth regulators, or containing cytoxin and / or other plant growth regulators that stimulate the regeneration of sprouts and / or containing O-lower alkanoylsalicylic acid, optionally in additional combination with DMSO, as described above in step (1). Callus appears containing differentiated cells and cell aggregates (Fig. 6) after an appropriate period of time, usually 1 week to 6 months, more typically 1 to 3 months. Callus then differentiates to form sprouts after an appropriate additional time, usually from 1 week to 6 months, more typically 1-3 months.

Callus is cultured at this stage in the light. The light intensity is usually between 0.1 and 100 μE / (m ² • s), preferably between 30 and 80 μE / (m ² • s).

 Stage (h). Getting a fertile plant from a germ.

 Sprouts are transferred to a suitable medium, for example, N6 medium without plant growth regulators. Alternatively, growth regulators that stimulate root or shoot growth can be added. Sprouts are cultivated on this medium until they form roots. The time required for root formation is usually 1 to 6 weeks, more typically about 2 weeks. When the roots and sprouts have grown enough, the plants are transferred to the soil and carefully pressed. A sufficient root length at this stage is an interval from 1 to 10 cm, more typically 2 to 5 cm, and for shoots it is in the range from 1 to 15 cm, more typically 2 to 10 cm.

 Plants are grown before flowering and sexual reproduction or vegetative propagation is carried out in the usual way.

 Transgenic maize plants containing exogenous DNA can be maintained in culture or can be regenerated into plants. Corn plant cells into which exogenous DNA can be introduced include any corn plant cells capable of accepting, replicating, and expressing foreign genes at an appropriate level. Some suitable corn plant cells are, for example, cells isolated from the inbred lines Funk 57984, Funk 5H986, Funk 2717, 211D, Funk 2N217A, B73, A632, CM105, B37, B84, B14, Mo 17, R168, MS71, A188, FA91, A641, and W117. All elite genotypes are preferred. More preferred are all elite genotypes. More preferred are all inbred lines. Even more preferred are all elite inbred lines, especially the elite inbred lines Funk 5N984, Funk 5N986, Funk 2717, Funk 211D or Funk 2N217A, and even more preferred Funk 2717.

 A culture medium capable of supporting a particular plant cell in the culture may depend on the genotype of the corn plant used. For example, some suitable media include, but are not limited to, MS-based (Murashige and Skoog (1962)), N6 (Chu et al., (1975)), B5 (Gamborg et al. (1968)), and KM (Kao and Michayluk (1975)) media with appropriate concentrations of sugars and plant growth regulators.

Therefore, the present invention describes a method for the regeneration of Zea mays plants from protoplasts, comprising the steps of:
(e1) producing protoplasts capable of being regenerated in fertile plants from cells and cell aggregates of steps (d),
(f) plating the protoplasts on a suitable medium in which the protoplasts divide and form cells,
(g) subculturing the cells obtained in step (f) to regenerate the sprouts, and (h) cultivating the sprouts on a growth medium to obtain fertile plants.

 The invention further provides a method in which Zea mays protoplasts contain stably introduced exogenous DNA, preferably exogenous DNA expressed in Zea mays plants. More preferred is the cultivation of Zea mays protoplasts in the presence of 0.01-100 mg / L plant growth regulator (preferably 2,4-D, dicamba, piclorama or pCPA, most preferably 2,4-D) and in the presence of O-lower alkanoylsalicylic acid (preferably 2,4-D) and in the presence of O-lower alkanoylsalicylic acid (preferably O-acetylsalicylic acid or O-propionylsalicylic acid, most preferably O-acetylsalicylic acid) in an amount sufficient to promote cell division or the formation of olonium. Most preferred is a method that further comprises the presence of DMSO in an amount sufficient to promote cell division or colony formation.

 Additionally described is a method for producing transgenic Zea mays protoplasts capable of regenerating into fertile transgenic plants, including processing Zea mays protoplasts capable of regenerating in fertile plants with exogenous DNA to obtain cells that contain all or part of exogenous DNA included in their genetic material.

 Suitable exogenous DNAs are, for example, chimeric genes described above or subsequently. The term "sprouts" includes any plant material capable of sexual or non-sexual reproduction, or propagated in vivo or in vitro. Such sprouts preferably consist of protoplasts, cells, calluses, tissues, embryos or seeds of regenerated plants. The offspring of regenerated plants and regenerated transgenic plants include mutants and variants.

 Detailed description.

токсина. Рассматриваемые клетки растений кукурузы включают все генотипы (сорта, разновидности, инбредные линии, гибриды и т.п.) растений кукурузы, в которых может быть введена чужеродная ДНК, реплицирована и экспрессирована. Некоторые примеры подходящих генотипов растений кукурузы включают, но не ограничиваются такими инбредными линиями, как Funk 2717, Funk 211D, Funk 2N217A, B73, A632, CM105, B37, B84, В14, Mo17, 168, M71, A188, FA91, A641 и W117. Funk 5N984 и 5N986 являются дополнительными примерами полезных генотипов Zea mays. Предпочтительно генотипом является элитный генотип, более предпочтительно элитная инбредная линия, выбранная в группе, состоящей из Funk 5N984, Funk 5N986, Funk 2717, Funk 211D или Funk 2N217A, а еще более предпочтительно им является инбредная линия Funk 2717.(Part B) insecticidal corn plants (Zea mays)
The present invention provides a method for producing a chimeric gene

toxin. The considered cells of maize plants include all genotypes (varieties, varieties, inbred lines, hybrids, etc.) of maize plants in which foreign DNA can be introduced, replicated and expressed. Some examples of suitable maize plant genotypes include, but are not limited to, inbred lines such as Funk 2717, Funk 211D, Funk 2N217A, B73, A632, CM105, B37, B84, B14, Mo17, 168, M71, A188, FA91, A641 and W117 . Funk 5N984 and 5N986 are additional examples of useful Zea mays genotypes. Preferably, the genotype is an elite genotype, more preferably an elite inbred line selected in the group consisting of Funk 5N984, Funk 5N986, Funk 2717, Funk 211D or Funk 2N217A, and even more preferably it is the inbred line Funk 2717.

или полипептид, обладающий по существу инсектицидными свойствами кристаллического белка

Кодирующая последовательность химерического гена не известна в ассоциации с указанным промотором в природных генах.The chimeric gene described in the present invention contains a promoter region that functions efficiently in corn plants and a coding region that encodes a crystalline protein

or a polypeptide having substantially insecticidal properties of a crystalline protein

The coding sequence of the chimeric gene is not known in association with the specified promoter in natural genes.

 5'- and / or 3'-untranslated regions can independently be associated in nature with either the promoter or the coding region, or neither the promoter nor the coding region. Preferably, either the 5'- or 3'-nontranslire region is associated with the promoter in the natural genes, and even more preferably both 5'- and 3'-regions are associated with the promoter in the natural genes.

будет особенно трудно экспрессироваться в клетках растений.Based on the state of the art, it was currently not possible to predict that corn cells would express an insecticidal polypeptide at any level, and especially in sufficient quantities, to impart insecticidal properties to the cells. In particular, it was believed that the polypeptide as a large and insoluble and as a polypeptide having insecticidal properties of a crystalline protein

it will be especially difficult to express in plant cells.

Инсектицидным количеством является такое количество, которое при наличии в клетках растений, убивает личинок насекомых или по крайней мере снижает существенно их кормление.In order to be considered insecticidal, plant cells must contain an insecticidal amount of a toxin with insecticidal activity of a crystalline protein

An insecticidal amount is such an amount that, when present in the cells of plants, kills insect larvae or at least significantly reduces their feeding.

 Gene.

 Transcription of control sequences.

 The chimeric gene described in the present invention contains transcriptional control sequences including a promoter and 5'- 3'-untranslated sequences that are functional in corn plants. These sequences can independently come from any source, such as genes from a virus, plant, or bacteria.

 Viral promoters and 5'- and 3'-untranslated sequences suitable for use are functional in maize plants and are derived, for example, from plant viruses such as sunflower mosaic virus (CaMV). The sunflower virus has been characterized and described by Hon in the variety. in Current Topics in Microbiology and Immunology, 96 (1982) 194-220 and in Appendices A to G. This description is given here as a prior art.

 CaMV is an atypical plant virus that contains double-stranded DNA. At least two CaMV promoters are functional in plants, namely the 19S promoter, which is the result of transcription of the CaMV VI gene and the 35S transcript promoter, the 19S promoter and 356C promoter are the preferred viral plant promoters for use in the present invention.

 CaMV 19S promoters and 5'-untranslated regions can be obtained using a restriction map, such as the map described in FIG. 4 on page 199 of the article from Hon et al., Or from the sequence given in Appendix C to the Hon article with employees.

 In order to isolate the CaMV 19S promoter and, in some cases, the adjacent 5'-untranslated region, a restriction fragment of the CaMV genome containing the desired sequence is selected. A suitable restriction fragment that contains the 195 promoter and the 5'-untranslated region is the fragment between the Pst I site starting at position 5386 and the Hind III site starting at position 5850 of FIG. 4 and annex C of Hon's article with coworkers.

 By similar methods, the 35S CaMV promoter can be obtained as described in Example 6 below.

 Unwanted sequences in the restriction fragment in some cases can be removed using standard techniques. Some suitable techniques for deleting unwanted nucleotides include the use of exonucleases (Maniatis et al., Molecular Cloning, Cold Spring Harbor Laboratory. (1982) 135-139) and oligonucleotide directed mutagenesis (Loller and Smit, Meth. Enzymol., 100 (1983) 468-500).

 A detailed procedure can be used to obtain the desired 3'-untranslated region. For example, a suitable 3 ′ untranslated CaMV 19S gene sequence can be obtained by isolating the region between the EcoRV site at position 7342 and the Bgl II site at position 7643 of the CaMV genome as described in Appendix 4 of Hon's article with coworkers.

 Examples of promoters of plant genes and 5'- and 3'-untranslated regions suitable for use in the present invention also include promoters of a gene encoding the small subunit of ribolose bisphosphate carboxylase and chlorophyll a / b binding protein. These regions of the plant gene can be isolated from plant cells using methods comparable to those described above for isolating the corresponding regions from CaMV, see Morelli et al., Nanure, 315 (1985) 200-204.

Suitable promoters and 5'- and 3'-untranslated regions from bacterial genes include those that are present in the T-DNA region of Agrobacterium plasmids. Some examples of suitable Agrobacterium plasmids include the Ti plasmid A. turefaciens plasmid and the Ri plasmid
A. thizogenes. Agrobacterium promoters and the 5 ′ and 3 ′ untranslated regions useful in the present invention are in particular those that are present in the genes encoding octopin synthase and nopaline synthesis. These sequences can be obtained by methods similar to those described above for isolation of CsMV and plant promoters and untranslated sequences; see Bevan et al., Nabure, 304 (1983) 184-187.

дельта-эндотоксина кристаллического белка. Полипептид для целей настоящего изобретения обладает по существу токсичностью

дельта-эндотоксина кристаллического белка, если он является инсектицидным для подобного ряда личинок насекомых, как и кристаллический белок от штаммов

Некоторые подходящие штаммы включают, например,

var. Rurstaki,

var. berliner,

var. alesti;

var. tolworthi;

var. sotto;

var. dandrolimus;

var. tenebrionis;

var. san diego; и

var. aizawai.Coding region
The coding region of the chimeric gene contains a nucleotide sequence that encodes a polypeptide that is essentially toxic

delta endotoxin crystalline protein. The polypeptide for the purposes of the present invention has substantially toxicity.

delta-endotoxin crystalline protein, if it is insecticidal for a similar series of insect larvae, like crystalline protein from strains

Some suitable strains include, for example,

var. Rurstaki,

var. berliner,

var. alesti;

var. tolworthi;

var. sotto;

var. dandrolimus;

var. tenebrionis;

var. san diego; and

var. aizawai.

var. Rurstaki, а особенно

var. Rurstaki HDI.The preferred strain are

var. Rurstaki, and especially

var. Rurstaki HDI.

Альтернативно кодирующая область может содержать последовательность, которая отличается от последовательности, которая существует в

, но является эквивалентной из-за вырождения генетического кода.The coding region may exist in nature in

Alternatively, the coding region may comprise a sequence that is different from a sequence that exists in

but is equivalent due to degeneration of the genetic code.

 The coding sequence of a chimeric gene can also encode a polypeptide that is different from a suitable crystalline delta endotoxin protein, but which is still substantially toxic to the insect crystalline protein. Such a coding sequence will usually be a variant of the natural coding region. A “variant” of a natural DNA sequence is a modified form of a natural sequence that performs the same function. The variant may be a mutation or may be a synthetic DNA sequence and is essentially homologous to the corresponding natural sequence. "Essential sequence homology" is to be understood here as a DNA fragment having a nucleotide sequence sufficiently similar to another DNA fragment to obtain a protein having similar properties; or a polypeptide having an amino acid sequence sufficiently similar to another polypeptide to exhibit similar properties. Typically, the DNA sequence is substantially homologous to the second DNA sequence if at least 70%, preferably at least 80%, and even more preferably at least 90% of the active regions of the DNA sequence are homologous. Two different nucleotides are considered homologous in the coding region of the DNA sequence for the purpose of determining significant homology if substituting one for the other is a mute mutation.

 Preferably, the nucleotide sequence is essentially homologous to at least that part or those parts of the natural sequence that are responsible for insecticidal activity.

кристаллического белка, так как он имеет по крайней мере некоторые из одинаковых антигенных детерминат.The polypeptide expressed by the chimeric gene used in the present invention will usually also have at least some immunological properties of natural

crystalline protein, since it has at least some of the same antigenic determinants.

.

продуцирует кристаллический белок с субъединицей, которая является протоксином, имеющим Мг примерно 130000-140000. Эта субъединица может быть расщеплена протеазами или щелочами с образованием инсектицидных фрагментов, имеющих Мг 50000 и возможно даже ниже. Химерические гены, которые кодируют такие фрагменты протоксина или даже его меньшие части, согласно настоящему изобретению могут быть сконструированы, поскольку фрагмент или части фрагментов имеют требуемую инсектицидную активность. Протоксин, инсектицидные фрагменты протоксина и инсектицидные части этих фрагментов могут быть слиты с другими молекулами, такими как полипептиды.Therefore, in the present invention, the polypeptide encoded by the chimeric gene is preferably structurally related to the delta-endoxin of a crystalline protein produced

.

produces a crystalline protein with a subunit, which is a protoxin having Mg of about 130,000-140000. This subunit can be cleaved by proteases or alkalis to form insecticidal fragments having Mg 50,000 and possibly even lower. Chimeric genes that encode such protoxin fragments, or even smaller parts thereof, of the present invention can be constructed since the fragment or parts of the fragments have the desired insecticidal activity. Protoxin, insecticidal fragments of protoxin and insecticidal portions of these fragments can be fused with other molecules, such as polypeptides.

 Coding regions suitable for use in the present invention can be obtained from crystalline protein toxin genes isolated from Bt, for example, see Whiteley et al., PCT Application WO 86, 01536 and US Pat. Nos. 4,448,885 and 4,467,036. Preferred Nucleotide Sequence which encodes a crystalline protein is as shown from nucleotide 156 to 3623 and a sequence of formula (1) or a shorter sequence that encodes an insecticidal fragment of such a crystalline protein; Geiser et al., Gene 48 (1986) 109-118.

 Therefore, suitable for use in the present invention is also a gene having a nucleotide sequence of formula I, genes having a sequence essentially homologous to a gene of formula I, and genes encoding a polypeptide having a sequence of formula II, or having a sequence essentially homologous to a sequence of formula II, and preferably those of these genes that are expressed in plants, more preferably in Zea mays.

 The coding region defined by nucleotides 156 to 3623 of sequence I encodes a polypeptide of the sequence of formula II.

 The sequence of formula II, see the end of the description.

 Therefore, the present invention also describes a polypeptide having a sequence of formula II, or insecticidal parts of this polypeptide.

являются токсичными к другим, чем чешуекрылые насекомые. Токсичность

штамме tenebrionis по отношению к жесткокрылым насекомым и последовательность ассоциированного гена токсина обсуждается Herrustadt and Soares иEP-0 202 739 и E - 0 213 817.In addition, it was shown that some strains

are toxic to lepidopteran insects. Toxicity

the tenebrionis strain with respect to the winged insect and the sequence of the associated toxin gene is discussed by Herrustadt and Soares and EP-0 202 739 and E - 0 213 817.

 Vectors.

 In order to introduce a chimeric gene into plant cells, the gene is first inserted into the vector. If the gene is not available in an amount sufficient for transformation, the vector can be amplified by replication in the host cell. The most convenient host cells for amplification are bacterial or yeast cells. When a sufficient amount of the chimeric gene is available, it is introduced into the corn protoplasts of the present invention. The introduction of the gene into corn plants can be carried out using the vector used for replication or using another vector.

rhizogenes. Методы клонирования гетерологических генов в бактерии описаны Coher et al., патенты США 4237224 и 468464.Some examples of bacterial host cells suitable for replication of a chimeric gene include Escherihia bacteria, such as E. coli, and Agrobacterium, such as A. tumefaciens or

rhizogenes. Cloning methods for heterologous genes in bacteria are described by Coher et al., US Pat. Nos. 4,237,224 and 4,684,464.

в E.coli описана Wong et al., J. Biol. Chem. 258 (1983), 1960-1967.Replication of genes encoding a crystalline protein

in E. coli, described by Wong et al., J. Biol. Chem. 258 (1983), 1960-1967.

 Some examples of host yeast cells suitable for replicating the genes of the present invention include Saccharomyces yeast.

 Any vector into which a chimeric gene can be inserted and which replicates in a suitable host cell, such as a bacterium or yeast, can be used in the present invention to amplify genes. The vector may be, for example, derived from a phage or plasmid. Some examples of vectors derived from phages useful in the invention include vectors derived from M13 or lambda. Some suitable vectors originating from M13 include M13 mp18 and M13 mp19. Some suitable vectors originating from lambda include lambda-gt11, lambda-gt7 and lambda Sharon 4.

 Some vectors derived from plasmids that are particularly suitable for replication in bacteria include pBR322 (Bolivar et al., Gene 2 (1977) 95-113); pUC18 and pUC19 (Harrander et al., Gene, 26 (1983) 101-104); and Ti plasmids (Bevan et al., Nature, 304 (1983) 184-187). Preferred vectors for amplifying genes in bacteria are pBR322, pUC18 and pUC19.

 Design vectors for replication.

 To construct a chimeric gene suitable for replication in bacteria, a promoter sequence, a 5'-untranslated sequence, a coding sequence and a 3'-untranslated sequence are inserted into or combined in the desired order in a suitable vector, such as the vector described above. In order for it to be suitable, the vector must be capable of replication in the host cell.

 The promoter, the 5'-untranslated region, the coding region, and the 3'-untranslated region, which make up the chimeric gene, can first be combined into a single unit outside the vector, and then inserted into the vector. Alternatively, portions of the chimeric gene can be inserted separately into the vector. Preferably, the vector also contains a gene that confers a property to the host cell, allowing selection of cells containing the vector. A preferred property is antibiotic resistance. Some examples of useful antibiotics include ampicillin, tetracycline, hygromycin, G418, and neomycin.

 The insertion or combination of a gene with a vector is carried out according to standard methods, such as using recombinant DNA (Maniatis et al., (1982)) and recombinant homology (Hinnen et al., Proc. Natl. Acad. Sci 75 (1978) 1929-1933) .

 Using recombinant DNA techniques, the vector is cut, the desired DNA sequence is inserted between the cut portions of the vector, and the ends of the desired DNA sequence are ligated to the corresponding ends of the vector.

 It is most convenient to cut the vector using suitable restriction endonucleases. Some suitable endonucleases include those that form blunt ends, such as Smal, Hpal and EcoRV, and those that form sticky ends, such as EcoR1, Sac1, and BamH1.

 The desired DNA sequence usually exists as part of a large DNA molecule, such as a chromosome, plasmid, transposon, or phage. The desired DNA sequence is cut from its source and, in some cases, modified so that its ends can be connected to the ends of the cut vector. If the ends of the desired DNA sequence and the cut vector are blunt ends, they are joined by blunt end ligases, such as T4 DNA ligase.

 The ends of the desired DNA sequence can also be connected to the ends of the cut vector in the form of sticky ends, in this case using a ligase for sticky ends, which can also be T4 DNA ligase. Other suitable sticky end ligases include, for example, E. coli DNA ligase.

 Sticky ends are most conveniently formed by cutting the desired DNA and vector sequences with the same restriction endonuclease. In this case, the desired DNA sequence and the cut vector have sticky ends that are complementary to each other.

 The sticky ends can also be constructed by adding complementary homopolymer tails to the ends of the desired DNA sequence and to the cut vector, using the terminal deoxynucleotidyl transferase or by adding a synthetic oligonucleotide sequence recognized by a specific restriction endonuclease, known as a linker, and cleavage with an endonase, such as Manon etonase al., (1962).

 Construction of vectors for plant transformation.

токсин или

-подобный токсин, вектор предпочтительно также содержит последовательность ДНК, которая обеспечивает отбор или скрининг клеток растений кукурузы, содержащих вектор, в присутствии клеток, которые не содержат вектор. Такие отбираемые или скринируемые маркеры могут быть в векторе природно, в этот вектор введен химерический ген, или же маркер может быть введен в вектор до или после введения химерического гена. Альтернативно, отбираемый или скринируемый маркерный ген или его часть могут быть сначала соединены с желаемым химерическим геном или любой его частью, а рекомбинантные гены или сегменты генов могут быть введены в вектор как единое целое. Отбираемый или скринируемый маркер сам может быть химерическим.In addition to the chimeric gene encoding

toxin or

-like toxin, the vector preferably also contains a DNA sequence that allows selection or screening of corn plant cells containing the vector in the presence of cells that do not contain the vector. Such selectable or screenable markers can be naturally occurring in the vector, a chimeric gene is introduced into the vector, or the marker can be inserted into the vector before or after the introduction of the chimeric gene. Alternatively, a selectable or screenable marker gene, or part thereof, may first be coupled to the desired chimeric gene or any part thereof, and recombinant genes or gene segments may be integrated into the vector. The selectable or screenable marker itself may be chimeric.

 A preferred selectable marker is a gene encoding antibiotic resistance. The gene must be capable of expression in cells that must be transformed. Cells can be cultured in a medium containing an antibiotic, and those cells containing a vector that have an increased ability to survive in the medium are selected. Genes that confer resistance to corn toxins, such as hygromycin or G413, may be useful as a selectable marker.

 Some examples of genes that confer antibiotic resistance include, for example, neomycin phosphotransferase (resistance to kanamycin and G418, Velten et al., EMBO J. 3 (1984) 2723-2730), hygromycin phosphotransferase (hygromycin resistance, van den Elzen et al., Plant Molec. Biol., 5 (1985) 299-300).

-токсина, и CaMV 35S промотор, функционально соединенный с отбираемым маркерным геном, таким как G418 устойчивость или устойчивость к гигромицину. Примерами таких векторов являются pCIB710 (355 (G418 устойчивость) и pIRV (устойчивость к гигромицину), как описано ниже.Preferred vectors for transforming maize plants are the caMV 35S promoter operably linked to the coding region

-toxin, and CaMV 35S promoter functionally linked to a selectable marker gene, such as G418 resistance or hygromycin resistance. Examples of such vectors are pCIB710 (355 (G418 resistance) and pIRV (hygromycin resistance), as described below.

кристаллический токсин или полипептид, обладающий по существу инсектицидной активностью

кристаллического токсина.The invention also describes fertile corn plants whose cells contain a chimeric gene that expresses

crystalline toxin or polypeptide having substantially insecticidal activity

crystalline toxin.

 Insecticides.

кристаллического белка. Клетки растений сами могут содержать инсектицид. Клетки растений, используемых непосредственно в качестве инсектицидов, могут быть культивируемыми клетками растений или могут быть составляющими живого растения.The plant cells used in the present invention contain a chimeric gene and can be used to produce a polypeptide having substantially insecticidal activity.

crystalline protein. Plant cells themselves may contain an insecticide. Plant cells used directly as insecticides can be cultured plant cells or can be components of a living plant.

смотри, например, Deacon, Aspeots of Microbiology, 7 (1983) Cole et al., ed, American Society for Microbiology and Miller et al., Seience 219 (1983) 715.The toxin can also be isolated from plant cells using known techniques, for example, extraction or chromatography. The extract may be an extract of all plant cells, a partially purified extract, or a pure polypeptide preparation. Any such extract or chromatographic isolate can be used in the same way as crystalline protein.

see, for example, Deacon, Aspeots of Microbiology, 7 (1983) Cole et al., ed, American Society for Microbiology and Miller et al., Seience 219 (1983) 715.

var. tenebrionis являются токсичными для жесткокрылых насекомых-паразитов. Важными насекомыми паразитами, принадлежащими к порядку Coleoptera, являются, например, колорадский картофельный жук, долгоносик хлопковый и кукурузный корневой червь.Insecticidal cells are toxic to lepidopteran insects that attack maize cells and plants, such as the cotton scoop (Heliothis zea) corn moth (Ostrinia nubilalis). In addition, corn cells carrying the crystalline toxin gene

var. tenebrionis are toxic to parasitic beetles. Important insect parasites belonging to the Coleoptera order are, for example, the Colorado potato beetle, the cotton weevil and the corn root worm.

кристаллического белка, в котором промотор, 5'-нетранслируемая область, и необязательно 3'-нетранслируемая область гена происходят из гена растения или гена вируса растения, и
(b) экспрессию полипептида
В настоящем изобретении также описан способ уничтожения личинок насекомых, заключающийся в кормлении личинок инсектицидным количеством трансгенных клеток Zea mays, содержащих ген, который кодирует кристаллический токсин Bacillus thuringiensis или полипептид, обладающий по существу инсектицидной активностью кристаллического белка Bacillus thuringiensis.Therefore, the present invention describes a method for preparing a polypeptide in Zea mays having substantially insecticidal activity of a crystalline protein of Bacillus thuringiens, said method comprising:
(a) introducing into the Zea mays cells a gene encoding a polypeptide having substantially insecticidal activity

a crystalline protein in which a promoter, a 5'-untranslated region, and optionally a 3'-untranslated region of a gene are derived from a plant gene or a plant virus gene, and
(b) expression of the polypeptide
The present invention also describes a method for killing insect larvae by feeding the larvae with an insecticidal amount of Zea mays transgenic cells containing a gene that encodes a crystalline toxin of Bacillus thuringiensis or a polypeptide having substantially insecticidal activity of the crystalline protein Bacillus thuringiensis.

var tenebrionis или ее инсектицидные участки.In addition, a method for killing Lepidopteran larvae is described, which comprises feeding the larvae with an insecticidal amount of transgenic corn plant cells that contain a chimeric gene. Plant cells can be cultured plant cells or can be components of a living plant. In addition, the method includes the destruction of Coleopteran larvae by feeding them with an insecticidal number of cells containing a chimeric gene having a coding sequence of crystalline toxin

var tenebrionis or its insecticidal regions.

 Utility.

кристаллический белок) стабильно в геном таких растений, и изменения их фенотипа. Кроме того, протопласты могут быть слиты с протопластами того же или другого сорта, чтобы получить новые сочетания ядерной ДНК или новые сочетания ядерной и органелль ДНК. Эта последняя возможность обеспечивает, например, конструирование новых растений с мужской стерильностью для целей селекции. Кроме того, протопласты могут быть использованы в качестве источника материала для клонирования, на котором может быть проведен мутагенез и/или селекция на желаемый фенотип. Примерами желаемых фенотипов могут быть, например, повышенная устойчивость к фитотоксичным химикатам, к патогенам, к паразитам, к вредным воздействиям окружающей среды, модифицированные морфологические или эволюционные свойства и измененное содержание метаболитов.The method described in the present invention provides Zea mays protoplasts that can be regenerated into fertile plants. This possibility arises from the introduction of exogenous DNA (e.g., a gene encoding

crystalline protein) is stable in the genome of such plants, and changes in their phenotype. In addition, protoplasts can be fused with protoplasts of the same or a different kind to obtain new combinations of nuclear DNA or new combinations of nuclear and organelle DNA. This last opportunity provides, for example, the construction of new plants with male sterility for breeding purposes. In addition, protoplasts can be used as a source of material for cloning, on which mutagenesis and / or selection for the desired phenotype can be carried out. Examples of desired phenotypes include, for example, increased resistance to phytotoxic chemicals, pathogens, parasites, environmental hazards, modified morphological or evolutionary properties, and altered metabolite content.

 Example 1a Obtaining a special type of callus Zea mays genotype Funk 2717.

Zea mays plants of the Funk 2717 genotype are grown to flowering in a greenhouse and pollinated with pollen from the same genotype. Immature corn cobs containing embryos 1.5 to 2.5 mm long are removed from the plants and sterilized in a 10% Clorox ^R solution for 20 minutes. The embryos are separated from the grains and placed with the germ stem down on Muroshigi-Skoog medium (Physiol. Plant), 15 (1962) 473 - 497) containing 0.1 mg / L 2,4-D 6% w / v sucrose and 25 mM Z-proline, cured 0.24% w / v Gelrite ^R (initiating medium). After two weeks of culturing in the dark at 27 ^° C, the callus that developed on the scab was removed from the embryo and placed on B5 medium (0.5 mg / L 2,4-D), rejected Gelrite ^R. The material is subcultured (passivated) every 2 weeks on fresh medium. About 8 weeks after placing the embryo on the initiating medium, a special type of callus is identified by its characteristic morphology. This callus is further subcultured on the same medium. After an additional period of 2 months, the callus is transferred and sterilely subcultured on N6 medium containing 2 mg / L 2,4-D and cured Gelrite ^R.

 Example 1b Obtaining a special type of callus Zea mays genotype Funk 5N984.

Zea mays plants of the Funk 5N984 genotype are grown to flowering in a greenhouse and pollinated with pollen from the same genotype. Immature pieces of ears containing embryos 1.5 to 2.5 mm long are removed from the plants and sterilized in a 15% Clorox ^R solution for 15 minutes. The embryos are removed from the kernels and placed down with germinal stem onto Schenck and Hildebrandt medium (Can. J. Bot 50 (1972) 199 - 204) containing 1.0 mg / L 2,4-D, 2% w / v sucrose, 100 mg / l casein hydrolyzate, 100 mg / l myo-inositol and 25 mM Z-proline, cured 0.8% w / v Fitagar ^R (initiating medium).

After two weeks of culturing in the dark at 27 ^° C, the preferred callus is selected by the presence of a well-formed globular, somatic embryo that has developed on the scutellum of some explants. These calluses are removed and placed either on B5 medium (0.5 mg / l 2,4-D plus 2% w / v sucrose, 100 mg / l casein hydrolyzate, 100 mg / l myo-inositol and 25 mM L-proline / or on Schenck or Hildebrandt medium (described above). The material is subcultured every 2 weeks on fresh medium. After at least 8 weeks from placing the embryo on the initiating medium, a special type of callus is identified by its characteristic morphology. A special type of callus can then be subcultured then the same medium, but preferably it is transferred and subcultured with erialno on N6 -.. medium containing 2 mg / l 2,4-D and solidified with 0.24% w / v Gelrite ^R embryogenic suspension cultures can be derived from these callus cultures by the method of Example 2.

 Example 2. Preparation of a suspension of Zea mays genotype Funk 2717.

 The callus described in Example 1a is left in culture for at least 6 months. The type of callus selected for subcultivation is relatively non-sticky granular and very loose, so that it is divided into individual cell aggregates when placed in a liquid medium. Cultures containing aggregates with large, expanded cells do not leave.

Aliquots of approximately 500 mg of the Zea mays special callus described in step (a) above, of the Funk 2717 elite genotype, are placed in 30 ml of N 6 medium containing 2 mg / L 2,4-D in 125 ml short-duration flasks. After 1 week of cultivation at 26 ^o C in the dark on a rotating rocking chair (130 rpm, 2.5 cm stroke), the medium is replaced with fresh medium. Suspensions are subcultured in the same way again every week.

 During this time, cultures inspect and leave those cultures in which there is not a large number of expanded cells. Suspension cultures containing aggregates with large expanded cells are discarded. A preferred tissue consists of dense cytoplasmic fissile cell aggregates that have a characteristic smoother surface than most cell aggregates. These abandoned cultures contain at least 50% of the cells found in these small aggregates. This is the desired morphology.

 These suspensions also have a high growth rate with a doubling time of less than 1 week. Suspension cultures are subcultured weekly by transferring 0.5 ml of densified cell volume (CSF: precipitated cell volume in a pipette) to 25 ml of fresh medium. After 4–6 weeks of subcultivation in this way, cultures increase 2–3 times in a week of subculture. For further subculture, cultures are left in which more than 75% of the cells possess the desired morphology. They support cultures, always choosing for subculture flasks, the contents of which have the best morphology. In some cases, periodic filtering through a stainless steel mesh with a pore size of 630 μm (every 2 weeks) is used to increase the dispersion of the cultures, but this is not necessary.

 Susa-core culture Zea mays 6-277-CG (Funk 2717) was deposited at ATCC under storage number 40326. This deposit was made in accordance with the Budapest Treaty (date of deposit: May 20, 1987).

 Example 3. The preparation of protoplasts from suspension cultures Zea mays.

Incubate 1-1.5 ml of PCV cell suspension culture prepared as described in Example 2 in 10-16 ml of a mixture consisting of 4% w / v RS cellulase with 1% w / v Rosima in KMS saline (8.65 g / L KCl, 16.47 g / L MgCl ₂ 6H ₂ O and 12.5 g / L CaCl ₂ , 2H ₂ O, 5 g / L MES pH 5.6).

Digestion is carried out at 30 ^o C on a shaking table for 3 to 4 hours. Obtaining is controlled under a reverse microscope for released protoplasts.

 Isolated protoplasts are harvested as follows. The drug is filtered through a 100 μm mesh sieve, then through a 50 μm sieve. Protoplasts are washed through a sieve with a volume of KMS saline equal to the initial volume of the enzyme solution. Place 10 ml of protoplast preparation in each of several plastic free centrifuge tubes and 1.5 to 2 ml of a 0.6 M sucrose solution, buffered to pH 5.6 with 0.1% w / v morpholinoethanesulfonic acid (MES) and KOH layered down. The tubes are centrifuged at 60-100 g for 10 minutes and protoplasts are collected from the interface and placed in a fresh tube.

The protoplast preparation is resuspended in 10 ml of fresh KMS saline and centrifuged for 5 minutes at 60-100 g. The supernatant is removed and discarded, and the protoplasts are carefully resuspended in the remaining drop and then 10 ml of KMS solution with a strength of 13/14 are gradually added. After 5 minutes of centrifugation, the supernatant is again removed and the protoplasts are resuspended in a 6/7 strength KMS solution. An aliquot was taken for counting and protoplasts were again pelleted by centrifugation. Protoplasts are resuspended at a concentration of 10 ⁷ (ml in KM medium (table 1) or in a mannitol solution having the same osmotic properties as KM medium and containing 6 mM MgCl ₂ or in any other suitable medium used for transformation as described in the following examples: This protoplast suspension is used for cultivation, as described in example 8a, or where it is performed, for transformation of protoplasts, as described in examples 8 and 9.

 Example 4. General methods of recombinant DNA.

 Since many recombinant DNA techniques used in the present invention are routine for those skilled in the art, a brief description of these commonly used techniques is provided here, and not in every case where they arise below. Except as noted here, all of these routine procedures are described in Maniatis et al. (1982).

 A. Digestion with restriction endonucleases.

Typically, the DNA is in the reaction mixture in an amount of about 50-500 μg / ml in a buffer solution recommended by the manufacturer, New Inglade Biolabs, Beverly, MA, 2-5 units of restriction endonuclease are added for every 1 μg of DRC and the reaction mixture is incubated at the temperature recommended manufacturer, for 1 to 3 hours. The reaction is completed by heating at 65 ^° for 10 minutes or by phenol extraction, followed by DNA precipitation with ethanol. This technique is also described on pages 104 - 106 by Maniatis et al.

 B. DNA polymerase processing to create flush ends.

DNA fragments were added to the reaction mixture at 50-500 mg / ml in the buffer recommended by the manufacturer, New England Biolabo. The reaction mixture contains all four deoxynucleotide triphosphates at a concentration of 0.2 mm. The reaction mixture was incubated at 15 ^° C. for 30 minutes, and then terminated by heating to 65 ^° C. for 10 minutes. For fragments obtained by digestion with restriction endonucleases that create 5'-protruding ends, such as EcoRI and Bam HI, use a large fragment or fragment of the Maple DNA polymerase.

 For fragments produced by endonucleases that produce 3'-protruding ends, such as PstI and SacI, use T4 DAY polymerase. The use of these two enzymes is described on pages 113 -121 of the work of Maniatis et al.

 C. Electrophoresis on agarose gel and purification of DNA fragments from gels.

Agarose gel electrophoresis is carried out in a horizontal device, as described on pages 150-163 of the work of Maniatis with employees. The buffers used are the Trisborate buffer described herein. DNA fragments are visualized by staining with 0.5 μg / ml ethidinium bromide, which is either present in the gel and tank buffer or added after electrophoresis. DNA is visualized by short-wave or long-wave ultraviolet light. When fragments are to be isolated from the gel, low temperature gel agarose obtained from Sigma Chemical, St. Louis, Missouri is used. After electrophoresis, the desired fragment is excised, placed in a plastic tube, heated to 65 ^° C. for about 15 minutes, then extracted three times with phenol and ethanol precipitated twice. This procedure is slightly modified compared to that described in the work of Maniatis with employees on page 170.

 D. Attachment of synthetic linker fragments to the ends of DNA.

When it is desired to add a new endonuclease restriction site to the end of the DNA molecule, such a molecule is first treated with DNA polymerase to create flush ends, if necessary, as described in the above section. Approximately 0.1 to 1.0 μg of this fragment is added to about 10 ng of the phosphorylated DNA linker obtained from New Ingled in a volume of 20 to 30 μl containing 2 μl of T4 DNA ligase, from New Ingled Biolabs and 1 mM ATP in buffer recommended by the manufacturer. After overnight incubation at 15 ^° C., the reactions are terminated by heating the mixture at 65 ^° C. for 10 minutes. The reaction mixture is then diluted to about 100 μl in a buffer suitable for restriction endonucleases that cleave the synthetic linker sequence, and approximately 50-200 units of this endonuclease are added. The mixture was incubated at the appropriate temperature for 2-6 hours, then the fragment was subjected to agarose gel electrophoresis and the fragment was purified as described above. The resulting fragment will now have ends with the ends obtained by digestion with restriction endonuclease.

 These ends are usually sticky, so the resulting fragment is now easily ligated with other fragments having the same sticky ends.

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

During the cloning step of the plasmid, treatment of the plasmid vector with phosphatase reduces the recirculation of the vector (discussed on page 13 of Maniatis et al.). After digesting the DNA with the appropriate restriction endonuclease, one unit of cow gastric alkaline phosphatase is added, obtained from Boehringer-Mannheim, Indianapolis, IN, DNA is incubated at 37 ^° C for one hour, then extracted twice with phenol and precipitated with ethanol.

 F. Ligation of DNA fragments.

When combining fragments with complementary sticky ends, approximately 100 ng of each fragment is incubated in a 20-40 μl reaction mixture containing approximately 0.2 T4 units of New Biolabs DNA ligase in a buffer recommended by the manufacturer. Incubation is continued for 1 hour 20 minutes at 15 ^o C. When connecting DNA fragments having blunt ends, they are incubated as described above, except that the amount of T4 DNA ligase is increased by 2-4 times.

G. Transformation of DNA in E. coli
For most experiments, E. coli strain HB101 is used. DNA is introduced into E. coli using the calcium chloride procedure described by Maniatis and coworkers on pages 250-251.

 H. Screening of E. coli for plasmids.

 After transformation, the resulting E. coli colonies are screened for the presence of the target plasmid using the quick plasmid isolation procedure. Two convenient procedures are described on pages 366-369 in Maniatis et al. (1982).

 1. Large-scale isolation of plasmid DNA.

 Procedures for the isolation of large quantities of plasmids in E. coli are described on pages 86-94 of Maniatis et al. (1982).

 f. Cloning in phage N13 vector.

 In the following description, it should be understood that the double-stranded replicative form of derivatives of phage M13 is used for routine procedures, such as restriction endonuclease digestion, ligation, etc.

 Example 5. Construction of a chimeric gene in plasmid pBR322.

 To fuse the CaMV gene VI promoter and the protoxin coding sequences, a phage derivative of the mp 19 vector is constructed (Janiscn - Perron et al., (1985)).

First, a DNA fragment containing approximately 155 nucleotides 5 ′ to the protoxin coding region and adjacent approximately 1346 nucleotides of the coding sequence is inserted into mp 19. Phage mp 19 ds rf is digested in a double-stranded replicative form) of DNA with restriction endonucleases Sac I and Sma I and the vector fragment of approximately 7.2 kpu is purified after electrophoresis via low gel temperature agarose using standard procedures. Plasmid pK U 25/4, containing approximately 10 kpo (kilopar bases) of Baccillus thuringiensis DNA, including the protoxin gene, was obtained from Dr. J. Nues, Tsiba-Geigi Ltd., Basel, Switzerland. The nucleotide sequence of the protoxin gene present in plasmid pK U 25/43 is shown in sequence (1). Plasmid pK U 25/4 DNA is digested with HpaI and SacI endonuclases and the 1503 po fragment (containing nucleotides 2 to 1505) in sequence (1) is purified as described above). This fragment contains sequences of approximately 155 at the bacterial promoter and approximately 1346 at the start of the protoxin coding sequence). About 100 ng of each fragment was then mixed, T4 DNA ligase was added and incubated overnight at 15 ^° C. The resulting mixture was transformed into E. coli strain HB 101, mixed with the bacterial indicator E. coli JM101 and plated as described (Messing J. Meth Enzymol 101 (1983) 20). Below, for further construction, a single phage is used, called mp 19 (b)
Then the DNA fragment containing the CaMV gene VI promoter and some coding sequences of the VI gene are inserted into mp 19 / bt. Phage mp 19 / bt ds rf DNA is digested with Ba HI, treated with a large DNA fragment by polymerase to create blunt ends and re-digested with endonuclease Pst I. The large vector fragment is purified by electrophoresis as described above. Plasmid pABDI described Pashkovsky with employees, EMBO J. 3 (1984) 2717-2722. Plasmid pABDI DNA is digested with Pst I and Hind III. A fragment of approximately 465 in length containing the CaMV gene VI promoter and approximately 75 in the coding sequence of gene VI is purified.

Two fragments are ligated and plated as described above. One of the resulting recombinant phages, called mp 19 / bt ca, is used in the following experiment:
Phage mp 19 / bt ca contains CaMV gene VI promoter sequences part of the VI gene coding sequence, approximately 155 for the Baccillus thuringiensis DNA sequence above the protoxin coding sequence, and approximately 1346 for the protoxin coding sequence. To fuse CaMV promoter sequences exactly to protoxin encoding sequences, the introduced DNA is deleted using directed mutagenesis of mp19 / bt ca DNA oligonucleotides. DNA oligonucleotide with the sequence
(5 ') TTCGGATTGTTATCCATGGTTGGAGGTCTGA
(3 ') are synthesized using routine procedures using the DNA Biosystems DNA synthesizer. This oligonucleotide is complementary to these sequences in the mp 19 / bt ca DNA phage at the 3'-end of the CaMV promoter nucleotides 5762 to 5778 from Hon (1982)) and the start of the protoxin encoding sequence (nucleotides 156 to 172) in formula 1 above. The general procedure for mutagenesis is as described by Zoller and Smith (1983). Approximately 5 μg of single stranded DNA of phage mp 19 / bt ca are mixed with 0.3 μg of phosphorylated oligonucleotide in a volume of 40 μl. The mixture is heated to 65 ^° C. for 5 minutes, cooled to 50 ^° C. and slowly cooled to 4 ^° C. Then, buffer, nucleotide triphosphatase, ATP, T4 DNA ligase and a large fragment of DNA polymerase are added and incubated overnight at 15 ^° C. as described (see Zoller and Smith (1983)). After agarose gel electrophoresis, circular double-stranded DNA is purified and transfected into E. coli strain JM101. The resulting plates are screened for sequences that hybridize with a 32P-labeled oligonucleotide and phage are analyzed by restriction endonuclease DNA analysis. Among the resulting phage clones, there will be clones that have correctly deleted unexpected sequences between the CaMV gene VI promoter and the protoxin coding sequence. This phage is called mp19 (bt ca) del.

 A plasmid is then constructed in which the 3'-coding region of the protoxin gene is fused to CaMV transcription end signals. First, plasmid pABDI DNA is digested with Bam HI endonucleases and BglII isolate a 0.5 kbp fragment containing CaMV transcription terminator sequences. Then the plasmid pU C19, Yanish - Perronn et al., Gene 33 (1985) 103-119, digested with Bam HI, mixed with a fragment of 0.5 kpo and incubated with T4 DNA ligase. After transformation of NK into E. coli strain HB101, one of the resulting clones, called plasmid p702, is obtained.

 Plasmid p702 DNA is then digested with SacI and SmaI endonucleases and a larger fragment of approximately 3.2 kpo is isolated by electrophoresis. Plasmid pK U 25/4 DNA is digested with Aha III and Sai I endonucleases and a 2.3 kpo fragment (nucleotides 1502 according to 3773 in formula I above) is isolated after gel electrophoresis containing the 3'-section of the sequence encoding protoxin / nt 1504 according to 3773 the sequence shown in formula I. These two DNA fragments are mixed, incubated with T4 DNA ligase and transformed into E. coli strain HB101. The resulting plasmid is p702 / bt.

кристаллический белок, затем с терминатором транскрипции CaMV.Finally, mp 19 / bt ca / del ds rf DNA phage particles and plasmid p702 / bt are combined to create a plasmid containing a protoxin coding sequence flanked by the CaMV promoter and terminator sequences. Phage mp 19 / bt del DNA is digested with Sea I and Spn I endonucleases and a fragment of about 1.75 kp is purified by agarose gel electrophoresis and plasmid p702 / bt is similarly digested
DNA with ScaI and SalI endonucleases isolate a fragment of approximately 2.5 kpo. Finally, the plasmid pBk 322 DNA (Bolivar et al., (19677) was digested with SalI and SphI endonucleases and the larger 4.2 kpo fragment was isolated. All three DNA fragments were mixed and incubated with T4 DNA ligase and transformed into E. coli, strain HB 101. The resulting plasmid, pBK 322 / bt 14 is a derivative of pBK 322 containing the CaMV gene VI promoter, and translation start signals fused to the coding sequence

crystalline protein, then with CaMV transcription terminator.

 Example 6a Construction of pTOX containing a chimeric gene encoding the insecticidal toxin gene of Bacillus thuringiensis var. tenebrionis.

 A gene encoding the insecticidal crystalline protein gene of Bacillus thuringiensis var. benebrionia has been characterized and sequenced (Sekar V et al., Proc. Natl. Acad. Sci. USA 84 (1987) 7036-7040). This coding sequence is isolated on a suitable restriction fragment, such as Hind III. A fragment of approximately 3 ko and inserted into a suitable plant expression vector, such as plasmid pCIB 770 (Rothstein 5 et al., Gene, 63 (1987) 153-161). Plasmid pCIB 770 contains the chemical kanamycin gene for expression in plants, as well as the promoter and terminator of the 35S RNA of the CaMV transcript (sunflower mosaic virus), separated by single Bam HI sites. The restriction fragment carrying the toxin coding sequence is made compatible with the single Bam HI site of pCIB 770 using a suitable molecular adapter and ligated together.

Example 6b Construction of pSAH containing a chimeric gene encoding an insecticidal toxin gene
Baccillus thuringiensis strain San diego.

 The gene encoding the insecticidal protein Baccillus thuringiensis strain san diego has been characterized and sequenced by Herrnstadt et al., EP-0 202 739 and EP-0 213 818. This coding sequence is isolated on a suitable restriction fragment and inserted into a suitable plant expression vector such as pCIB 770. Plasmid pCIB770 contains the chimeric kanamycin gene for expression in plants, as well as the promoter and terminator of the 35 S CaMV (Sunflower Mosaic Virus) transcript of RNA, separated by a single Bam HI site. A restriction fragment carrying a toxin coding sequence is made compatible with a single Bam HI site of pCIB 770 using a suitable molecular adapter and ligated together.

 Example 7A Construction of the plasmid pCIB 710.

 Plasmid pZW III (available from S. Howell University of California, San Diego) consists of three small EcoRI fragments of the BYI strain of sunflower mosaic virus (CaMV) (Franck et al. (1980) Cell 21: 285-294) cloned into pMB9. p ZW III digest and isolate the BgelII fragment 1150 po / base pair number 6494-7643, Hohn et al., Current Topics in Microbiology and Immunology 96 (1982) 193-216). This fragment is ligated to the Bam HI site of p UC 19. This restriction fragment codes for both the promoter for the 35 S pHK CaMV and the polyA addition signal (i.e., terminator) for this transcript.

Mutagenesis in vitro
A single Bam HI site is inserted between this promoter and the terminator using in vitro mutagenesis. A 36-base oligonucleotide is synthesized which is identical to the CaMV sequence into which the region is inserted with the exception of such a Bam HI restriction site in base pair N 7463 in the sequence.

 Clone BglII fragment 1150 from the above in M13 mp 19 and isolate single-stranded phage DNA.

 This single-stranded DNA is annealed with a synthetic oligonucleotide, a new strand is synthesized using the oligonucleotide as a primer and the DNA is transfected into JM101 strains (Zoller and Smit, DHK, 3 (1984) 479-488), then isolated as described by Zoller and Smith (above).

Selection of the target mutant phage
An oligonucleotide of 36 bases was labeled by Kinasing with 32-P-ATP.

 A group of dies of transfected M13 phage is localized on a nitrocellulose filter. This filter is hybridized with a 36-mer labeled filter and washed at elevated temperature. Mutated phages are stable when washed at elevated temperatures.

 One of these phages stable at elevated temperatures is sequenced to confirm the presence of a Bam HI site.

Final assembly of the pCIB 710
The double-stranded DNA isolated from this phage was digested with Hind III and EcoRI pUC19 was digested with Hind III and EcoRI. These two digested DNAs are ligated. Transformants are wiped away by ampicillin resistance. One transformant from this ligation is pCIB710.

 Plasmid pCIB710 does not have ATC start codons between this Bam HI site that is inserted and the transcription start point.

Example 76. Construction of pCIB712
The hygromycin resistance gene (Gritz, Z. and Davies J. Gene 25 (1983) 179-188) is located on approximately 1,150 base pairs of the BamHI fragment from plasmid p LG90. Plasmid pLG90 was obtained from Dr. Lida Grits. Applied Biotechnology, 80 Rogers St., Cambridge, Mass. 02142. This fragment is linked to BglII linkers, and then inserted into a single BamHI site of pCIB 710, destroying the BamHI site and constructing plasmid pCIB 712. Plasmid pCIB 712 was deposited in ATCC, Pc, Maryland, ATS, , USA, under storage number 67407. This deposit was made in accordance with the Budapest Treaty (date of deposit: May 18, 1987).

 Plasmid pCIB712 is linearized using the appropriate enzyme, for example, SmaI.

Example 7c Construction of pCIB10 / 34 Sbt
Plasmid pCIB710 described above.

Construction of pCIB10
1. A T-DNA fragment containing the left edge of pTi 737 is isolated from pB R 325 (EcoR 129) Yadav et al., Proc. Natl. Acad Sci. USA 79 (1982) 6322-6326, pBR 325 (EcoR 129 cut with EcoRI and link 1.1 kb fragment with Hind III linkers (New England Biolabs).

2. Plasmid pBR322 (Bolivar et al., Gene, 2 (1977) 95-103) was cut with Hind III
3. The fragment containing the left edge described in step 1% is ligated into a Hind digest of pBR 322.

 4. A plasmid containing the left edge constructed in step 3 is digested with ClaI and Hind III and the 1.1 ko Hind III / ClaI fragment is isolated (Hepburn et al., J. Mol. Appl. Genet., 2, (1983) 315 -329).

 5. Plasmid p UC18 (Horrander et al., Genl 26 (1983) 101-106) was cut with Hind III and Eco RI; polylinker 60 will be labeled at the end using T4 polynucleotide kinase and gamma-32p-ATP and isolated from the acrylamide gene.

 6. Plasmid pBR 322 was cut with EcoRI and ClaI and a large fragment was isolated.

 7. The Hind III / EcoRI 60 polylinker and the Hind III / ClaI 1.1 fragment of EcoR 129 are ligated into pBR 322, cut with ClaI and EcoRI, constructing pCIB5.

8. The chimeric gene conferring resistance to kanamycin (nos-neo) is taken from Binb (Bevan, Nuc. Acid Res. 12 (1984) 8711-8721) as a SalI / EcoRI fragment
9. Plasmid pUC18 cut with EcoRI and SalI
10. The SalI / EcoRI fragment containing the chimeric stage 8 gene is ligated into pUC18, cut with EcoRI and SalI
11. BanHI recognition site in the terminal sequence of this chimeric gene is destroyed by cutting Bam HI, filled using T4 DNA polymerase and ligated.

 12. The resulting plasmid was cut with Sst II (Bethesda Research Laboratories) and Hind III.

 13. The fragment containing the 5'-part of the nos promoter and the right edge of the pTi T37 is isolated by cutting pBR 325 (Hind 23) with HindIII and Sst II and the 1.0 kb fragment is isolated.

 14. This Hind III / SstII fragment 1.0 is ligated into pUC18 vector of step 12, pCIB4 is constructed.

 15. pCIB5, containing the left edge of T-DNA, was cut into Aat II, creating a blunt end by treatment with T4 DNA polymerase, and then cut with EcoRI.

 16. pCIB4 cut Hind III creating a blunt end by processing using the Klenov fragment of E. coli DNA polymerase 1, and cut EcoRI.

17. The vector of step 29 is ligated with a large fragment of step 16, constructing a pCIB2, CoIEI replicon containing the left and right edges of T-DNA flanking the chimeric Km ^r gene and polylinker.

 18. Plasmid pR 102 (Jorgensen et al Mol. Gen. Gemet 177 (1979) 65-72) was digested with Bam HI and filled using the Klenov fragment.

 19. A partial Alul digest of plasmid pAO3 is prepared (Oka et al, Nature 276, (1978) 845-847 and Oka et al, J. Mal. Biol., 147 (1981) 217-226).

 20. AluI digest ligated into the restricted pRZ 102 from step 18, selecting the desired transformants for kanamycin resistance.

 21. The resulting plasmid has the coding sequence Tn 903 present on the Bam HI fragment of 1.05 ko; this fragment is isolated after BamHI digestion and filling with the Klenov fragment.

 22. Plasmid pRK 252, a derivative of a number of patients with host plasmid pRK 2, was obtained from Dr. Don Helinsky of the University of California, San Diego. This plasmid lost the BglII site present in the related plasmid pRK 290 (Ditta et al., Proc. Nat. Acad. Sci., USA, 77 (1980) 262-269). p RK 252 is digested with SmaI and SalI is filled using a Klenov fragment and the large fragment obtained from this digest is isolated.

 23. The fragment containing TN903 isolated in step 21 is ligated into a large fragment from pRK 252, constructing pRK252 Km.

 24. Plasmid pPK252 Km cut EcoRI get blunt ends using a maple fragment and link BglII linkers (New England Biolabs).

 25. Plasmid pCI82 was cut with EcoRV and linked with BglI linkers (New England Biolbs).

 26. Bgl II linked pCIB2 from step 25 is ligated into the vector of step 24, constructing pC1810.

Example 7. Construction pCIBIOa
Example 7c can be repeated with the replacement of pRK252 with pRK290.

 1. Plasmid pRZ102 (Jorgensen et al., (1979), supra) was digested with BamHI and filled using Klenov.

 2. Prepare a partial digest of AluI plasmid pA03 (Oka et al., (1978) above).

 3. AluI digestion is ligated to the restriction enzyme from step 18, Example 7c, and target transformants are selected using kanamycin resistance.

 4. The resulting plasmid has a Tn903 coding sequence present on a 1.05 kb BamHI fragment; this fragment was isolated after BamHI digestion and filled with a Klenov fragment.

 5. The plasmid pRK290, which is a derivative of the common host plasmid RK2, was obtained from Dr. Don Helinsky of the University of California, San Diego, pRK290 was digested with SmaI and SaII was filled using the Klenov fragment and a large fragment obtained in the result of such digestion.

 6. Tn908 containing the fragment isolated in step 35 was ligated into a large fragment from pRK290. Constructing pRKK290Km.

 7. The plasmid from step 6 was digested with BgIII, filled using the Klenov fragment and ligated, destroying its BgIII site, in order to construct pRK290Km.

 8. The plasmid pRK290Km was cut using EcoRI, connected with blunt ends using a Klenov fragment, and connected using BglII linkers (New England Biolabs).

 9. Plasmid pCIB2 was cut using EcoRV and connected to BglII linkers (New England Biolabs).

 10. The linked BglII pCIB2 from step 9 was ligated to the vector from step 7, constructing pCIB10a.

 Example 7e Construction of the chimeric protoxin gene of Bacillus thuringiensis with the CaMV35S promoter.

A. Construction of CaMV35S promoter cassette
Plasmid pCIB710 was constructed as shown in FIG. 107. Such a plasmid contains the CaMY promoter and transcriptional terminator sequences of the 35S RNA matrix (Covey S. N. Lomonosoff G. P., Hull R., Nucleic Acids Research (1981) 6735-6747), 1149 bp BgIII restriction fragment CaMV DNA (bp 7643-6494 in Hon et al. (1982)) was isolated from plasmid pLVIII (obtained from Dr. S. Howell, University of California, San Diego; or such a fragment can be isolated directly from CaMV DNA) by preparative agarose gel electrophoresis described above and mixed with BamHI-digested plasmid pUC19 DNA, treated with T ₄ DNA ligase and and trans formed in E. coli (Note: the BamHI restriction site in the resulting plasmid was destroyed by ligation of BglII sticky ends with BamHi sticky ends).

 The resulting plasmid, named pUC19 / 35S, was then used in oligonucleotide-directed in vitro mutagenesis to introduce the BamHI recognition sequence GGATCC immediately after CaMV nucleotide 7483 in the Hon link. The resulting plasmid, pCIB710, contains the CaMV35S promoter region and the transcriptional termination region, separated by a BamHI restriction site. DNA sequences inserted at such a BamHI site can be expressed in plants using such CaMV transcriptional regulatory sequences. (It should also be noted that pCIB710 does not contain any ATG translation initiation codons between the start of transcription and the site).

B. Insertion of CaMV35S Promoter / Terminator Cassette into pCIB10
The following steps are reflected in FIG. eleven
Plasmids pCIB10 and pCIB70 were digested with EcoRI and SalI, mixed and ligated. The resulting plasmid, pCIB10 710 has a CaMV35S promoter / terminator cassette inserted into the plant transformation vector pCIB10. CaMV35S sequences are located between the T-DNA boundaries in PCIB10 and thus, they can be introduced into the plant genome during plant transformation experiments.

 C. Introduction of the Bacillus thuringiensis protoxin gene into pCB10 / 710.

показанная на фиг. 12, содержит протоксиновый ген между CaMV 35S промоторной и транскрипционной терминаторной последовательностями.The following steps are shown in FIG. 12. As a source of the protoxin gene, plasmid pCIB10 / 19B was digested with BamHI and NcoI, and a fragment of 3.6 kb containing the protoxin gene was isolated by preparative gel electrophoresis. This fragment was then mixed with a synthetic NcoI-BamHI adapter having the sequence 5'-CATGGCCGGATCCGGC-3 ', and then digested with BamHI. At this stage, BamHI sticky ends are formed at both ends of the protoxin fragment. Then, the resulting fragment was inserted into a BamHI-digested pCIB10 / 710. The resulting plasmid, pCIB10 / 35S

shown in FIG. 12, contains a protoxin gene between the CaMV 35S promoter and transcriptional terminator sequences.

 Plasmid pCIB10 (19SBt) [in E. coli strain HB101] was deposited with ATCC and assigned ATCC catalog number 67330 (date of deposit: February 27, 1987).

 pCIB10 / 35SBt in strain MC1061 was deposited with ATCC, Rockville, Maryland, USA under catalog number 67329. Such deposit was carried out in accordance with the requirements of the Budapest Treaty (date of deposit: February 27, 1987).

Example 8. Transformation of Zea mays protoplasts by electroporation
(a) All steps except heat shock were carried out at room temperature. The protoplasts were resuspended in the last step of Example 3 in a 0.5 M mannitol solution containing 0.1% w / v. MES and 6 mM MgCl _{2 the} resistance of such a suspension was measured in the cassette of the interactive electroporator ^R and maintained at 1-1.2 Kom using 300 mM MgCl ₂ solution for this purpose. Protoplasts were subjected to heat shock by immersing the tube containing the sample for 5 minutes in a water bath at a temperature of 45 ^o C, after which it was cooled with ice to room temperature. To aliquots of such a 0.25 ml suspension, 4 μg of linearized pCIB712 plasmid containing the hydromycin resistant gene and 20 μg of calf thymus DNA carrier were added.

0.125 ml of 24% w / v was added to the protoplasts. a solution of polyethylene glycol (PEG) (mol.m. PEG 8000) in 0.5 M mannitol containing 30 mm MgCl ₂ . The resulting mixture was well but gently mixed and incubated for 10 minutes. The sample was transferred to the electroporator chamber and, after 10 second intervals, was exposed to pulses three times with initial voltages of 1000, 1200, 1500, 1800, 2300, and 2800 cm ⁻¹ , the time of exponential decay and pulse being 10 μs).

The cultivation of protoplasts was carried out as follows:
Samples were placed in Petri dishes with a diameter of 6 cm at room temperature. Over the next 5-15 minutes was added 3 ml of KM medium containing 1.2% wt. /about. Sea Plague ^R agarose, 1 mg / L 2,4-D and 100 mg / L O-acyl salicylic acid. Agarose in protoplasts was well mixed and the medium was allowed to gel.

 (b) The procedure of step (a) was repeated, except that the protoplast drug resistance was maintained at 0.5-0.7 kOhm.

 (c) The procedure of step (a) was repeated, except that PEG with mol.m was used as PEG. 4000.

 (d) The procedure of step (c) was repeated, except that the protoplast drug resistance was maintained at 0.5-0.7 kOhm.

 (e) The procedure of step (a) was repeated, except that PEG was not added.

 (f) The procedure of step (b) was repeated, except that PEG was not added.

 (g) The procedure of step (a) was repeated, except that half the volume was 12% w / v. PEG.

 (h) Repeated procedure (b), except that half the volume of 12% w / v was added. PEG.

 (i) The procedure of step (c) was repeated, except that half the volume of 12% w / v was added. PEG.

 (j) The procedure of step (d) was repeated, except that half the volume of 12% w / v was added. PEG.

 (k) The procedure of steps (a) to (j) was repeated, except that pulses were applied at 3 s intervals.

(l) Repeated steps (a) - (k) with the difference that after electroporation, the protoplasts were placed in cups located on a plate cooled to 16 ^o C.

 (m) Repeated steps (a) - (l) with the difference that after the electroporation stage the protoplasts were placed in tubes, washed with 10 ml of 6/7 KMS solution, collected by centrifugation at 60g for 10 min, resuspended in 0.3 ml of KM medium and were sown according to the method of stage (a).

(n) The procedure of step (m) was repeated, except that the medium used for washing the protoplasts was a W5 solution (380 mg / L KCl; 18.375 g / d CaCl ₂ • 2HO; 9 g / L NaCl; 9 g / l glucose; pH 6.0).

(o) Repeat steps (a) to (n) with the exception that the medium used for plating the protoplasts was a KM medium containing 30% vol. Schenk and Hildebrandt medium (1972), which contained 30 μM Dicamba ^R , in which a cell suspension of Dactylis glomcrata was previously grown.

(p) Repeat steps (a) to (n) with the exception that KM medium containing 15% vol. was used as the medium used for plating the protoplasts. Schenk and Hildebrandt medium (Can. J. Bot. 50 (1972), 199 - 204) containing 30 μM Dicamba ^R in which cell suspensions of Dactylis glomerata were pre-grown.

 (q) Steps (a) - (n) were repeated with the exception that KM medium containing 30% vol. was used as a medium for plating protoplasts. medium N6, in which the cell suspensions described in Example 3 were pre-grown.

 (r) Repeated steps (a) to (n) with the exception that KM medium containing 15% vol. was used as a medium for plating the protoplasts. medium N6, in which the cell suspensions described in Example 3 were pre-grown.

(s) Steps (a) - (r) were repeated with the exception that the protoplasts were transferred to the culture medium (KM medium containing 0.5 mg / L 2,4-D and 100 mg / L O-acetylsalicylic acid, and 1.2% w / v Sea Plagul ^R agarose) per crop filter, at a density of 0.5 million protoplasts / ml.

In this embodiment, Durapore filters (catalog number CVW PO4700, pore size 0.22 μm, diameter 47 mm) were numbered with a lead pencil along the edge of the outer surface as they were removed from the tank. Such an operation was carried out in order to be sure that the upper side is facing plant crops and that only one filter is used for each crop. Such filters were autoclaved in distilled, deionized water in a translucent container (GA 7 ^R or any similar container) for 20 minutes. After cooling, the water was replaced with liquid KM medium described above, but not containing gelling agents, at least 3 hours before placing the filters on the crop.

 Inoculum cultures were prepared from a suspension culture of black Mexican sweet corn (BMS) (Green Hort. Sci 12 (1977) 131, Smith et al. Plant Sci Lett. 36 (1984) 67 / or from the embryogenic suspensions described in Example 3.

In the case when BMS sterile KM medium containing 0.5 mg / l 2,4-D and 100 mg / l O-acetylsalicylic acid (2 mg / ml in 2 vol./vol. DMCO containing 0.1% w / v MES pH 6.0) was added to sterile Sea Plague ^R agarose to obtain a final agarose concentration of 1.2% w / v. After heating to melt the agarose, the medium was cooled to 44 ^° C in a water bath, 1 ml of PCV suspension of BMS was added per 10 ml of medium, and 5 aliquots were distributed in 60 mm diameter Petri dishes. After the medium had solidified, sterile Durapore filters were placed on the surface, numbered on the upper side.

In the case when embryogenic corn suspension cultures were used as a plant culture, the osmolarity of N6 medium was increased with glucose to 530-540 mOS / kg H ₂ O, the pH of the system was again set to 6.0 and the medium was sterilized through filter 0, 2 microns. Added 0.5 mg / l 2,4-D and 100 mg / l O-acetylsalicylic acid (2 mg / ml in 2% vol./about. DMCO containing 0.1% wt./about. MES, pH 6 , 0). This medium was added to sterile Sea Plagul ^R agarose to a final agarose concentration of 1.2% by weight. /about. After heating to melt the agarose, the medium was cooled to 44 ^° C in a water bath, 1 ml of PCV embryogenic suspension culture was added per 10 ml of medium, and 5 ml aliquots were distributed in 60 mm diameter Petri dishes. After the medium had solidified, sterile Durapore filters were placed on its surface with the numbered side up.

Protoplast preparations were diluted with chilled (44 ^o C) KM medium with 1.2% wt./about. Sea Plague ^R agarose containing 0.5 mg / L 2,4-d and 100 mg-L O-acetylsalicylic acid to a concentration of 0.5 million protoplasts / ml. 0.5 ml of such a preparation was slowly pipetted onto the surface of each filter.

 (t) Steps (a) - (s) were repeated with the exception that no carrier veal thymus DNA was added.

 (u) Repeated steps (a) to (t) with the exception that 50 μg of linearized pC1B712 plasmid was added.

 (v) Repeated steps (a) - (u) with the exception that the plasmid pCIB 712 was not linearized.

Example 9. Transformation of Zea mays protoplasts by treatment with polyethylene glycol
(a) Protoplasts were resuspended in the last step of Example 3 in a 0.5 M mannitol solution containing 12-30 mM MgCl ₂ . Heat shock was carried out for 5 minutes at 45 ^° C. as described in Example 8a. Protoplasts were distributed in aliquots for transformation in centrifuge tubes, 0.3 ml of suspended protoplasts per tube. Over the next 10 minutes, the following substances were added; DNA (as described in examples 8a - 8V) into a PEG solution (PEG 6000 40% w / v; Ca (NO ₃ ) ₂ 0.1M; mannitol 0.4M; pH 8 - 9 with KOH) as a result final concentration of 20% wt./about. Aliquots were incubated for 30 minutes with occasional gentle shaking, after which the protoplasts were placed in Petri dishes (0.3 ml of the initial suspension of protoplasts per cup with a diameter of 6 cm) and cultured as described in examples 8a or 8o - 8r.

(b) The protoplasts were resuspended in the last step of Example 3 in a 0.5 M mannitol solution containing 12-30 mM MgCl ₂ at a density of 20 million protoplasts / ml. Heat shock was carried out for 4 min at 45 ^o C, as described in example 8a. Protoplasts were distributed in aliquots for transformation in centrifuge tubes, 0.5 ml protoplasts per tube. Over the next 10 minutes, the following components were added: DNA (as described in Example 5a) and a PEG solution (PEG 8000 (sigma or other equivalent variety), 36% w / v, 0.1 M Ca (NO ₃ ) ₂ 0 , 4 M mannitol, 0.1% MES maintaining pH 7-8 for 3 hours by exposure to KOH sterilized through a 0.2 μm filter to a final concentration of 18% w / v. Aliquots were incubated for 30 min with gentle shaking and then the protoplasts were placed in Petri dishes and cultured as described in examples 8a - 8s.

(c) Steps (a) and (b) were repeated and the protoplasts were washed after 30 minutes of incubation in PEG by adding 0.3 ml of the W5 solution of Example 8n five times at 2/3 minute intervals, then the system was centrifuged, the supernatant was removed and protoplasts were cultured as described in examples 8a - 8s
(d) According to another procedure, the protoplasts were washed at 5-minute intervals by sequentially adding 1 ml, 2 ml and 5 ml of the W5 solution of Example 8n. The system was then centrifuged and the protoplasts were cultured as described in examples 8a - 8s.

 (c) Repeated steps (a) - (d) with the exception that the final concentration of PEG was 15% wt./about.

 (f) Steps (a) - (e) were repeated with the exception that the final concentration of PEG was 25% w / v.

 (g) Steps (a) - (d) were repeated with the exception that the final concentration of PEG was 12% w / v.

 (h) Repeated steps (b) - (f) with the exception that the protoplasts were washed with brine KMC according to example 8n.

Example 10a Protoplast callus regeneration
Plates containing protoplasts in agarose were placed in the dark at 26 ^° C. Cell colonies developed from protoplasts within 14 days. Colar-containing agarose was transferred onto the surface of 9 cm diameter Petri dishes containing 30 ml of N6 medium with 2 mg / L 2,4-D, which solidified during processing with 0.24% weight. /about. Gelright (2N6). The colonies were cultured to produce callus: Callus was further cultured in the dark at 26 ^° C and pieces of callus were subcultured every 2 weeks on fresh Nb medium containing 2 mg / L 2,4-D (which solidified during processing 0.24% w / vol. Gelright ^R ).

Example 10b Callus regeneration of their protoplasts
Plates containing seed culture protoplasts were placed in the dark at 26 ^° C. Colonies appeared for about 2 weeks. The complete filters were then transferred onto the surface of 9 cm diameter Petri dishes containing 2 mg / L 2,4-D medium, which solidified upon exposure to 0.24% w / v, Gelright ^R (2N 6), or the colonies were removed and individually transferred to such an environment. The developed callus was additionally cultured in the dark at 26 ^° C and pieces of callus were subcultured every 2 weeks on fresh N6 medium containing 2 mg / L 2,4-D (which solidified upon processing 0.24% w / v Gelright ^R ).

 Example 11a Selection of transformed callus Zea mays.

Examples 10a and 10b were repeated with the exception that 50 mg / L hygromycin was added to 2N6 in order to select the transformed cells
Example 11B Selection of transformed callus Zea mays.

 Examples 10a and 10b were repeated with the exception that 100 mg / L hygromycin was added to 2N6 in order to select the transformed cells.

 Example 11c Selection of transformed callus Zea mays.

 Examples 10a and 10b were repeated with the exception that 200 mg / L hygromycin was added to 2N6 so as to select the transformed material.

Example 12a Plant regeneration
Callus was placed on 2N6 medium to preserve both 0N6 (N6 hormone-free medium) and N61 (0.25 mg / L 2,4-D + 10 mg / L kinetin) to initiate regeneration. The materials were grown on 0N6 and N61 medium in the light (16 hours of daylight with 10-30 μE / m ² • s from a fluorescent lamp). Callus expressed on N61 medium was transferred after 2 weeks to 0N6 medium since a longer residence time on N61 plates is harmful. Callus was subcultured every 2 weeks, even if it was again transferred to the original medium.

After 4-8 weeks, shoots appeared
Once the shoots reached a height of at least 2 cm, they were transferred to 0N6 medium in G47 containers or other suitable culture vessels. Within 2-4 weeks, roots appeared. If the roots looked sufficiently formed to support growth, the shoots were transferred to peat pots, which were placed in the shade for the first 4-7 days. Sometimes it can be helpful to place an inverted plastic cup over the grafts for several days.

 Immediately after the adaptation of the plants, they were treated like ordinary Zea mays plants and grown in a greenhouse until ripening in order to test the effect of fertilizers and inheritance of introduced genes.

 Example 12b Plant regeneration.

 Example 12a was repeated with the exception that 50 mg / L hygromycin was added to the medium in order to maintain callus.

Example 12c Plant regeneration
The procedure of Example 12a was repeated with the exception that 100 mg / L hygromycin was added to the medium used to preserve callus.

 Example 12d Plant regeneration.

 The procedure of Example 12a was repeated with the exception that 200 mg / L hygromycin was added to the medium used to preserve callus.

 Example 13. Transformation and regeneration.

 Examples 8, 9, 10, and 11 were repeated except that the plasmid pCIB10 / 35 SBt was used as the DNA plasmid and hygromycin was replaced with the antibiotic C418 at the selection stages.

ген, pIRV линеаризовывали на соответствующем сайте, дистальном по отношению к химерическим генам, перед электропорацией.Example 14. Construction of a pIRV containing both a chimeric resistance gene for hygromycin and a chimeric BT gene
Plasmid pCIB was digested with SmaI; plasmid pCIB10 / 35 Sb + was digested with SphI was treated with a large fragment of DNA polymerase (Klenov enzyme) to develop shoot ends, then SmaI was digested. A fragment of approximately 4.8 kO containing a chimeric BT gene was purified by agarose gel electrophoresis and ligated with SmaI digested pCIB712DNA. The resulting pIRV plasmid contains both the chimeric hygromycin resistance gene and the chimeric

gene, pIRV was linearized at the corresponding site distal to the chimeric genes before electroporation.

 Example 15. Transformation and regeneration.

 Examples 8, 9, 10 and 11 were repeated with the exception that the plasmid pIRV was used as the DNA plasmid and hygromycin was used for selection.

 Example 16. Construction of a plasmid carrying the chimeric chitinase and chimeric hygromycin resistance gene.

 The cDNA clone, PSCHI, containing the coding region of the tobacco chitinase gene was identified in the library of tobacco DNA cloned into the PstI site of pBR322. Hybrid selective translation yielded a protein product that hybridizes with an anti-bean chitinase antibody (Chinshi et al: Proc.Nate. Acad. Sci USA 84 (1987) 89-93). From the lambda library of tobacco DNA, the corresponding genomic clone was identified using a cDNA clone as a probe (Maniatis cit. Reference). To identify the Pst I site common with the cDNA clone and the genomic clone, hybridization and restriction mapping methods were used.

Establishing the sequence of genomic DNA above such a common PstI site made it possible to detect the beginning of an open reading frame for the chitinase-structural gene, as well as the SphI recognition site at position 35, starting from the starting site. The steps described below were used to combine this upper genomic DNA fragment with a downstream cDNA fragment, forming the full length of the chitinase coding region:
a) The full-length gene was constructed in the pGVI plasmid vector, which contains the CaMV 35S promoter fragment, the polycenylation termination signal from CaMV and the polylinker between them. The polarity of the polylinker forces orientates the upper fragment; the exact orientation of the bottom fragment is determined by sequence analysis.

 The upper SphI / PstI fragment of the genomic clone is isolated and purified; then it is ligated into the plasmid pGYI, which was digested with SphI and PstI.

 b) The resulting intermediate plasmid was digested with PstI. The lower PstI fragment was isolated from the pSCHI clone and ligated into such a digested intermediate vector.

 c) A clone with the correct orientation and fusion in the frame of the cDNA fragment with the upper genomic DNA fragment was confirmed by DNA sequence analysis. The resulting plasmid was called the pSCHI plasmid. Plasmid pSCHI (in E. coli strain K12 / was deposited with Deutsche Sammlung von Mikroorganismen. Göttingen, Germany, catalog number DSM 4129. Such a deposit was made in accordance with the requirements of the Budapest Treaty (date of deposit: May 29, 1987).

 The complete chitinase gene flanked by the CaMV 35S promoter and terminator regions was excised from pSCHI by EcoRI digestion. The fragment was created with blunt ends using a large fragment of DNA polymerase I (Maniatis, cited above). The receptor plasmid pCIB712, which contains the chimeric gene for hygromycin resistance that is expressed in plant cells, was digested with SmaI • SmaI - digested pCIB 712 and a blunt-ended fragment containing the chimeric chitinase gene was ligated together using T4 ligase (Maniatis, cited) . The resulting plasmid was named pCIBcht. Prior to protoplast transformation, pCIBcht was linearized at the site distal to chimeric genes.

 Example 17. The construction of a vector carrying a chimeric AHAS gene, which confers resistance to sulfonylurea.

 a. Isolation of sulfonylurea resistant (SU-R) Arabidopsis plants.

Seeds of M2 Arabidopsis thaliana (Colombian race) mutagenized by ethyl methanesulfonate (EMS) were obtained according to the method of Houna and Somerville (Mol. Gen. Genet. 204 (1986) 430-434), as well as the method of Somerville and Ogrin (Methods of chloroplast molecular biology, ed Edelman et al. (1982) 129-138). EMS was used at a concentration of 0.3% for 14-16 hours and M1 seeds were sown and grown with a density of 2-3 plants per cm ² .

 Sulfonylurea resistant (SU-R) Arabidopsis plants were selected and grown from M2 seeds according to the method of Haun and Somerville (see link above).

 b. Construction of the recombinant lambda phage library SU-R Arabidopsis DNA.

 DNA was isolated from SU-R plants of the species Arabidorsis according to the method of Jen and Hilton (J. Bacteriol. 166 (1986) 491-499). Arabidopsis DNA was completely digested with XlaI (New England Biolabs).

 4-8 kho DNA fragments were isolated from the agarose gel using a NA45 DEAE membrane (Schleitzer and Schuell, Kim, N.G. Catalog No. 03431) and cloned into a lambda-ongC-X vector (Strategin, 3770, Tancy, S. San Diego, CA 92121 according to the methods recommended by the manufacturers Recombinant phage DNA was encapsulated using the Gigipak-plus kit obtained from Strategen.

 with. Cloning of the SU-R acetoxyhydroxy acid synthase (AHAS) gene of plants of the Arabidopsis variety SU-R.

Plasmid pE16 / 8-c4 containing the IZV2 locus of Saccharomgeces cerevisiol (Zh. Polina, Carlsberg Res. Comn. 49 (1984 (577-584), obtained from J. Poline, was transformed into E. coli HB101 to receive a large-scale plasmid preparation. Plasmid pE16 / 8-c4 was deposited in the American Type Culture Collection under catalog number 67420. Such deposition was performed according to the requirements of the Budapest Treaty (deposit date: May 29, 1987). Plasmid pE16 / 8-04 DNA was digested with EcoRI and a 2.1 kho fragment (Eco 2.1) containing the AHAS coding sequence (Falx et al. Nucl. Acid. Res. 13 (1985) 4011-4027) you elyali from an agarose gel. Fragment Eco 2,1 subjected to radiation-label isotope phosphorus 32 to a specific activity of 5-8 • October ⁸ cpm / .mu.g using Prime Time kit (International Bioteknolodzhiz, INC).

Recombinant phages containing Arabidopsis Xba I fragments were plated on E. coli VCS 257 according to Maniatis et al. (1982) (Cold Spring Harbor, New York) and Strategen Protocols. Duplicate lambda plaque elevators were obtained from each phage plate using a Colony / Plague Screen ^R membrane (New England Newclair Reservation Products) according to manufacturers instructions. Plaque elevators were processed as follows:
i) washed for 5 hours with 5X SSC, 2% SDS at 65 ^° C (all prescriptions are given by Maniatis et al. (see link above);
ii) were hybridized in a hybridization mixture (5 x SSC, 1% SDS, 5 x Denhardt's solution, 200 μg / ml denatured salmon DNA, 25% formamide, 10% dextanesulfate; 10 ml of mixture per 150 mm membrane) for 16-20 hours at 42 ^o C;
iii) subjected to hybridization in a hybridization mixture (10 ml per 150 mm membrane) containing 2 ng / ml of denatured radiolabeled Eco 2.1 fragment for 18-20 hours at 42 ^o C;
iv) washed with 2 x SSC, 1% SDS at room temperature for 30 minutes;
v) washed with 5 x SSC, 1% SDS, 25% formamide for 6-8 hours at 42 ^° C. with a four-fold change of the wash solution.

 Membranes were exposed to x-ray film (Kodak X-Omat AR). Phage plaques exhibiting appropriate hybridization on duplicate membranes were purified using a second batch of lambda plaque elevators and hybridized using an Eco 2.1 probe.

 Recombinant phage plaque lysates containing an insert homologous to the yeast AHAS sequence were prepared according to the Maniatis et al. (see above). Phage DNA was prepared from plaque lysates in the presence of Lambda Sorb (Promega. 2800. S. Fish Hatchery Rd., Madison 53711 in accordance with the methods recommended by the manufacturers.

d. Construction and confirmation of the transformation vector pC1B808 containing the SU-R Arabidopsis AHAS gene
Recombinant phage DNA was digested with Xbal, and a 5.8 kho insert fragment was cloned b Xba-I-digested pC1B10 transformation vector to give pC1B803. Plasmid pC1B803 was transferred from the host E.coli HE101 to Agrobacterium tumefaciens C1B542 by tripartite cross-breeding (Ditt et al. Proc. Acad. Sci USA 77 (1980 (262-269). A tumefaciens pC1B803 / C1B542 was used for transformation of Arabian leaves with the method of Loyd et al. (Science 234 (1980 (464 / -466) and Sheikholeslam and Wicks (Plant. Mol. Biol, 8, (1986 / 291-298). Transformed tissues were regenerated in plants according to the method of Lloyd et al. ( see link above) and Feldman and Marx (Plant Sei 47, (1987 / 63-69). It has been shown that the growth of transformed tissues in a culture medium is tolerant ten to 3-10 ng / ml of chlorosulfuron The germination and growth of transgenic plants of Arabidopsis on agar was shown to be tolerant to 10-30 ng / ml of chlorosulfuron.

 e. Construction of the transformation vector pC1B804 containing the chimeric SU-R AHAS gene.

 The location of the AHAS coding sequence within the 5.8 Xba fragment was determined by nuclease-S1 mapping (Maniatis et al., See above link), by primer extension method (McKnight et al., Cell, 25 (1981) 385-398) and confirmed by establishing the DNA sequence (Sanger et al. Proc. Nat. Acad. Sci, USA 74 (1977) 5463-5467) and by comparing the protein sequence with bacterial and yeast AHAS sequences with bacterial and yeast AHAS sequences (Falco et al. Nucl. Acid. Res 13 (1985) (4011-4027) Xbal fragment size 5.8 kb was cloned into Bluescript (Strategin) and sequences above the AHAS coding sequence were deleted within 10-20 bp of the upstream AHAS initiation codon using the Exo / Mung deletion kit. The corresponding linker was attached to the end point of the deletion. the gene was cut at the site of the corresponding restriction site, at least 10 bp above the AHAS of the termination codon, and the same linker that was used at the 5'-end of the gene was attached to the 3'-end of the gene. A fragment containing the AHAS coding sequence was cut into vectors by cutting at linker sites and cloned into the Bam HI site pC1B710.

 The BamHI site pC1B710 was adapted or converted to a site compatible with the linker used above, if necessary, in order to obtain the linker SU-R AHAS gene. The orientation of the AHAS gene insert in pC1B710 was determined using appropriate restriction enzymes. A clone containing the AHAS coding sequence correctly oriented relative to the transcriptional direction of the CaMV 35S promoter was designated as pC1B04.

Example 18. Promotion of callus formation from Zea mays protoplasts under the influence of 2,4-D acetylsalicylic acid
Protoplasts were resuspended in the last step of Example 3 in a 6/7 KMS solution in an amount of 7.5 million per ml. O-Acetylsalicylic acid was dissolved in an amount of 2.5 mg / ml in KM medium. The resulting solution was sterilized on a filter. Then, immediately before use, the culture medium was added O-acetylsalicylic acid. Aliquots of the protoplast suspension were placed in Petri dishes with a diameter of 6 cm at room temperature. 3 ml of KM medium containing 1.2% w / v. Sea Plague ^R agaroses, 2,4-D and O-acetylsalicylic acid were added so as to obtain the correct concentration of 2,4-D and O-acetylsalicylic acid and a protoplast density of 0.5 million per ml. Agarose and protoplasts were thoroughly mixed and the medium allowed to gel.

 The dishes were cultured as described in Example 7. After 3 weeks, the number of macroscopic colonies appearing in the dishes was counted. In the table. Figures 2 and 3 show the results obtained for protoplasts from two different independently obtained embryogenic suspension cultures.

In the table. 2 shows the formation of colonies of Zea maus protoplasts in the presence of O-acetylsalicylic acid and 2,4-D (numbers indicate the numbers of the formed colonies)
Table 3 shows the formation of Zea mays protoplast colonies in the presence of O-acetylsalicylic acid and 2,4-D. (The numbers indicate the number of colonies formed)
Example 19. Enhanced colony formation from protoplasts as a result of the addition of O-acetylsalicylic acid and 2,4-D.

 Protoplasts were prepared and plated for cultivation as described in Example 18, using 2 ml of medium and a Petri dish 6 cm in diameter for this purpose.

 The dishes were cultured as described in Example 10. After 3 weeks, the number of macroscopic colonies appearing in the dishes was counted. The results are presented in table. 4. Again, it was found that O-acetylsalicylic acid stimulates the formation of colonies. However, at concentrations of 300 mg / L and higher, a toxic effect was observed. It was suggested that this effect is associated with the problem of establishing pH and it is possible that the effect can be eliminated by introducing the appropriate buffer into the medium.

And tab. 4 shows the formation of colonies (calculated per cup) of protoplasts in the presence of O-acetylsalicylic acid and 2,4-D
(The numbers indicate the number of colonies formed)
Example 20. Promotion of colony formation from Zea mays protoplasts under the influence of DMSO and O-acetylsalicylic acid and in the presence of 2,4-D.

 Protoplasts were prepared and plated in an amount of 0.75 million per ml as described in Example 18 using 1 ml of medium and Petri dishes with a diameter of 3.5 cm. O-acetylsalicylic acid was introduced into the medium. O-acetylsalicylic acid was dissolved in DMSO and then added to the medium before plating the protoplasts. To obtain a solution in DMSO, 1 g of O-acetylsalicylic acid was dissolved in 10 ml of DMSO and added to 90 ml of distilled water. Then the resulting solution before using sterilization on the filter.

 The dishes were cultured as described in Example 10. After 3 weeks, the number of macroscopic colonies formed in the dishes was counted. The results obtained are presented in table 5. Using only DMSO (1% v / v) does not significantly increase or decrease the efficiency of colony formation of Zea mays antiplasts and clearly enhances the stimulatory activity of O-acetylsalicylic acid.

In the table. Figure 5 shows the effect of O-acetylsalicylic acid on the formation of Zea mays protoplast colonies, which demonstrates the stimulatory effect of the dissolution of O-acetylsalicylic acid in DMSO (the number of formed colonies)
Example 21. Enhanced formation of protoplast colonies as a result of the addition of 2,4-D and related plant growth regulators (auxins).

 Protoplasts were resuspended in the last step of Example 3 in a 6/7 KMS solution in an amount of 5 million per ml.

Aliquots of the protoplast suspension were placed in Petri dishes with a diameter of 6 cm at room temperature. 2 ml of KM medium containing 1.2% w / v were added. Sea Plague ^R agarose, as well as 2,4-D, Pikloram, Dikamba or para-chlorophenoxyacetic acid (pCPA) in order to achieve the correct concentrations of plant growth regulator. A final protoplast density of 0.54 million per ml was used. Agarose and protoplasts were mixed well and the medium was allowed to gel.

 The dishes were cultured as described in Example 10. After 3 weeks, the number of macroscopic colonies appearing in the dishes was counted. The results are presented in table. 6.

In the table. 6 shows the formation of colonies from cultured Zea mays protoplasts in the presence of various plant growth regulators (auxins) / number of colonies formed)
Example 22 Construction of a Deleted Bt Protoxin Gene Containing About 725 Amino Acids and Construction of a Chimeric Gene Containing Such a Deleted Gene with a CaMV 35s Promoter
A depleted protoxin gene containing approximately 725 amino acids was obtained by removing the COOH terminal portion of the gene by cleavage of the restriction endonuclease at the Kn pI site at position 2325 of the sequence. Plasmid pCIB10 / 35 sBr (Fig. 12) was digested with Bam HI and Kn pI and a Bam HI / Kn pI fragment of approximately 2.2 kbp containing the deletion protoxin gene was isolated by preparative agarose gel electrophoresis. To convert the KnpI site at the 3 ′ end to a BAm HI site, such a fragment was mixed with a KnpI / Bam HI adapter oligonucleotide and ligated. Then such a fragment was mixed with Bam HI-digested pCIB10 / 710. The resulting transformants, designated pCIB10 / 35SBt (Kn p1 and shown in Fig. 15, contain a deletion protoxin gene of about 725 amino acids in size. Such transformants were selected on kanamycin.

протоксинового гена, содержащего примерно 645 аминокислот и конструирование химерного гена, содержащего такой делецированный ген с CaMV 35S промотором.Example 23. Construction of the deleted

a protoxin gene containing approximately 645 amino acids; and constructing a chimeric gene containing such a deleted gene with the CaMV 35S promoter.

(фиг. 12) переваривали с Bam HI и BCl I и Bam HI/Bcl I, фрагмент размером 1,9 k b p, содержащий делецированный протоксиновый ген, выделяли методом препаративного электрофореза агарозном геле. Поскольку BCII создает липкий конец, совместимый с Bam HI, никакого дополнительного манипулирования не требуется перед лигированием такого фрагмента на BamHI - расщепленную pCIB10/710. Полученную в результате плазмиду, со структурой pCIB10/35 SBt (BcII), подвергали селекции не канамицине.A deduced protoxin type containing approximately 645 amino acids was obtained by removing the COOH terminal portion of the gene by cleavage of the restriction endonuclease at the Bcl I site at position 2090 of the sequence. Plasmid pCIB10 / 35S

(Fig. 12) were digested with Bam HI and BCl I and Bam HI / Bcl I, a 1.9 kbp fragment containing the deleted protoxin gene was isolated by preparative agarose gel electrophoresis. Since BCII creates a sticky end compatible with Bam HI, no additional manipulation is required before ligation of such a fragment on BamHI - cleaved pCIB10 / 710. The resulting plasmid, with pCIB10 / 35 SBt (BcII) structure, was subjected to non-kanamycin selection.

протоксинового гена, содержащего примерно 607 аминокислот и конструирование химерического гена, содержащего делецированный ген с CaMV 35S промотором.Example 24. The construction of the deleted

protoxin gene containing approximately 607 amino acids; and constructing a chimeric gene containing a deleted gene with the CaMV 35S promoter.

b mp18 и использованием стандартных методик олигонуклеотидного мутагенеза, описанных выше. После мутагенеза, двухнитевую репликационную ДНК готовили из М13 клона, который затем переваривали с Bam HI. Фрагмент размером около 1,9 k b p, содержащий делецированный протоксиновый ген, вставляли в Bam HI - расщепленную pCIB10/710. Полученную в результате плазмиду, имеющую структуру pCIB10/35S

(607), подвергали селекции на канамицине.The depleted protoxin gene was obtained by introducing the Bam HI cleavage site (GGATCC) after nucleotide 1976 in sequence. Such an operation was carried out by cloning a BamHI fragment containing a protoxin sequence from pCIB10 / 35 S

b mp18 and using standard oligonucleotide mutagenesis techniques described above. After mutagenesis, double-stranded replication DNA was prepared from the M13 clone, which was then digested with Bam HI. A fragment of about 1.9 kbp in size containing the deleted protoxin gene was inserted into the Bam HI-cleaved pCIB10 / 710. The resulting plasmid having the structure pCIB10 / 35S

(607) were subjected to selection on kanamycin.

Claims (31)

 1. A method of obtaining Zea maysL plants resistant to damage caused by insects, comprising isolating tissue from immature embryos, culturing said tissue in a medium for initiating callus and / or in a medium for maintaining callus proliferation, until a special type of callus is formed, characterized by non-sticky, granular and friable structures and containing dense cytoplasmic dividing cells, selection of the specified type of callus and subsequent processing of it with an enzyme preparation to remove cell walls, collection, purification, etc. transformation of the obtained totipotent protoplasts with a vector containing a structural gene encoding a polypeptide of the crystalline protein of the δ-endotoxin of Bacillus thuringiensis toxic to insects and flanked by transcriptional control sequences containing the promoter and 5'- and 3'-untranslated sequences that are functional in cereals, further sowing transformed totipotent protoplasts on a nutrient medium for cell division and formation, subculturing them on a medium for regeneration and subsequent preparation of Zea mays L. fertile plants resistant to insect damage.  2. The method according to claim 1, characterized in that after the selection of a particular type of callus, it is transferred into a liquid nutrient medium to form a suspension of cells or cell aggregates, subculturing the resulting suspension over time and with a frequency sufficient to maintain cells and cell aggregates in viable state with subsequent selection and preservation of cultures containing aggregates of dividing cells with a dense cytoplasm and characterized by a smoother surface than other cell aggregates.  3. The method according to claim 1 or 2, characterized in that the promoter and 5'- and 3'-untranslated regions are derived from plant genes, viruses or bacteria.  4. The method according to claim 3, characterized in that the promoter, 5'- or 3'-untranslated region are derived from DNA sequences that are present in Agrobacterium plasmids and cause expression in plants.  5. The method according to p. 4, characterized in that the promoter comes from the Ji plasmid Agrobacterium tumefaciens.  6. The method according to claim 4, characterized in that the DNA sequences are derived from a gene that encodes octopin synthase.  7. The method according to claim 4, characterized in that the DNA sequences are derived from a gene that encodes nopalin synthase.  8. The method according to claim 3, characterized in that the promoter, 5'- or 3'-untranslated region come from the DNA of the plant virus.  9. The method of claim 8. characterized in that the plant virus is a cauliflower mosaic virus.  10. The method according to claim 9, characterized in that the promoter of the cauliflower mosaic virus is the 19S promoter.  11. The method according to claim 9, characterized in that the promoter of the cauliflower mosaic virus is a 35S transcript promoter.  12. The method according to claim 3, wherein the promoter, 5 ′ or 3 ′ untranslated region of the gene is derived from a plant gene that encodes a small subunit of ribulose carboxylase bisphosphate or chlorophyll a / b binding protein.  13. The method according to claim 1 or 2, characterized in that Bacillus thuringiensis is selected from the group of subspecies consisting of Bt.var. Kurstaki, Bt.var. berliner, Bt. var. alesti, Bt.var. toluorti, Bt.var. sotto, Bt.var. dendrolimusi, Bt.var. tenebrionis, Bt.var. san diego, Bt.var. asawai.  14. The method according to item 13, wherein Bt.var. Kurstaki is Bt. var. Kurstaki ND1.  15. The method according to claim 1 or 2, characterized in that the structural gene is a nucleotide sequence of the formula I shown in the description (Fig. 3).  16. The method according to claim 1 or 2, characterized in that the polypeptide has a mol.m. ~ 130,000 - 140,000 Yes, or its insecticidal fragments.  17. The method according to claim 1 or 2, characterized in that the polypeptide is fused to another molecule.  18. The method according to claim 1 or 2, characterized in that the structural gene is essentially homologous to the nucleotide sequence that encodes the crystalline protein of δ-endotoxin Bacillus thuringiensis.  19. The method according to claim 1 or 2, characterized in that the structural gene is capable of hybridization with the coding region of a gene that encodes a crystalline δ-endotoxin protein of Bacillus thuringiensis.  20. The method according to claim 1 or 2, characterized in that the polypeptide essentially has the immunological properties of a crystalline protein of Bacillus thuringiensis.  21. The method according to claim 1 or 2, characterized in that the gene encodes a polypeptide with the sequence of formula II given in the description (Fig. 4).  or an insecticidal portion of said polypeptide.  22. The method according to p. 1 or 2, characterized in that the gene encodes an insecticidal polypeptide having sequence homology with the sequence shown in paragraph 21.  23. The method according to claim 1, characterized in that the gene is enclosed in a vector capable of replication in bacteria or yeast.  24. The method according to item 23, wherein the vector is derived from a plasmid.  25. The method according to paragraph 24, wherein the vector is derived from plasmid pBR 322.  26. The method according to item 23, wherein the vector comes from phage.  27. The method according to p, characterized in that the vector is capable of replication in Escherichia coli.  28. The method according to claim 1 or 2, characterized in that the genotype of Zea mays L. is an inbred line of Zea mays L.  29. The method according to p. 28, characterized in that the genotype Zea mays L. is an elite inbred line Zea mays L.  30. The method according to clause 29, wherein the elite Zea mays L. is Funk 2717, which is deposited in the American type culture collection under storage number ATCC 40326.  31. The method according to claim 1 or 2, characterized in that the protein of δ-endotoxin Bac. thuringiensis is produced in an amount sufficient to make the plant unattractive to insects.

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