DE60028751T2 - HERBICIDRESISTENT PLANTS - Google Patents

Description

The The present invention relates to recombinant DNA technology and in particular the production of transgenic plants, the substantial Resistance or substantial tolerance for herbicides compared with non-transgenic similar Have plants. The invention also relates to u.a. the nucleotide sequences (and expression products thereof) involved in the production of transgenic Plants used or produced by the transgenic plants become.

Plants, the substantially "tolerant" for a herbicide when exposed to it, provide a dose / response curve ready, which is shifted to the right in comparison with the one by the similar way subjected to non-tolerant similar Plants is provided. Such dose / response curves have the "dose" on the X-axis plotted and the "percentage Killing "," herbicidal action "etc. on the y-axis applied on. Tolerant plants will typically be at least require twice as much herbicide as non-tolerant similar plants, to produce a given herbicidal action. Plants that Substantially "resistant" to the herbicide are few, if any, necrotic, lytic, chlorotic or other lesions, when exposed to the herbicide in concentrations and amounts that typically used by the agricultural community be used to kill weeds on the field on the annexes for commercial Purposes are to be cultivated.

It is particularly preferred that the Plants are substantially resistant or substantially tolerant to herbicides (hereafter "glyphosate") are the 5-enolpyruvyl-shikimate phosphate synthetase hereinafter "EPSPS") as their place of action of which N-phosphonomethylglycine (and its various Salts) is the outstanding example.

The Herbicide can be used either before or after emergence as usual Techniques for herbicides are applied to fields, crops include those that are resistant to (against) the herbicide was made. The present invention provides et al Nucleotide sequences useful in the preparation of such herbicide tolerant or resistant plants.

According to the invention is a isolated polynucleotide having the sequence shown in SEQ ID NO: 43 illustrated sequence. The invention also provides a polynucleotide exclusive of cDNA encoding the rice and corn EPSPS ready containing an EPSPS coded and complementary for one is that when incubated at a temperature of 65 to 70 ° C in 0.1-concentrated citrate buffered saline, which contains 0.1% SDS followed of rinsing at the same temperature with 0.1-concentrated citrate buffered Saline, containing 0.1% SDS, still hybridized with the sequence shown in SEQ ID NO: 43. An inventive EPSPS-coding However, polynucleotide can be obtained by screening of plant genomic DNA libraries are obtained with a nucleotide which represents an intron within the sequence of SEQ ID NO: 43, and the invention concludes also such a sequence, which is available from the survey, a.

The Invention includes also an isolated polynucleotide comprising a region which a chloroplast transit peptide and a glyphosate-resistant 5-enolpyruvyl-shikimate phosphate synthase (EPSPS) 3 'of the peptide where the region under the expression control is a plant-functional promoter, with the provisos, that the Promoter is not heterologous with respect to the region and the chloroplast transit peptide is not heterologous with respect to the synthase.

By "heterologous" is meant from a different source, and accordingly "non-heterologous" means originating from the same source - but at the level of a gene instead of an organism or a tissue. For example, the CaMV35S promoter is clearly heterologous to a Petunia EPSPS coding sequence insofar as the promoter is from a virus and the sequence (the expression of which it controls) is from a plant. However, the term "heterologous" according to the present invention has an even closer meaning. For example, "heterologous" with respect to the present invention means that the Petunia EPSPS coding sequence is "heterologous" with respect to, for example, a promoter also derived from Petunia - unlike that expressing the EPSPS gene controlled. In this sense, the Petunia promoter derived from the Petunia EPSPS gene, which is then used to control the expression of an EPSPS coding sequence, as it were from Petunia, is "non-heterologous" to the coding sequence. However, "non-heterologous" does not mean that the promoter and coding sequence must necessarily have been obtained from one and the same (original or precursor) polynucleotide. Same with respect to transit peptides. For example, a sunflower-derived Rubisco chloroplast transit peptide is "heterologous" to the coding sequence of an EPSPS gene as it were from the sunflower (the same plant, the same tissue or the same cell). A Rubisco transit peptide coding sequence derived from sunflower is "non-heterologous" to a rubisco enzyme coding sequence also derived from the sunflower, even if the origins of both sequences are distinct polynucleotides present in different cells, Tissues or sunflower plants may have been present.

• (i) Wenigstens einen Transkriptionsverstärker, der die verstärkende Region ist, die stromaufwärts vom Transkriptionsstart der Sequenz ist, aus der der Verstärker erhalten wird, und wobei der Verstärker als solcher nicht als Promotor entweder in der Sequenz, in der er endogen umfaßt ist, oder dann, wenn er heterolog als Teil eines Konstrukts vorhanden ist, funktioniert;
• (ii) Den Promotor aus dem Reis-EPSPS-Gen;
• (iii) Die genomische Sequenz aus Reis, die das Reis-EPSPS-Chloroplastentransitpeptid codiert;
• (iv) Die genomische Sequenz, die das Reis-EPSPS codiert;
• (v) Einen Transkriptionsterminator;

worin die Reis-EPSPS-codierende Sequenz dahingehend modifiziert ist, daß eine erste Position mutiert ist, so daß der Rest an dieser Position Ile anstelle von Thr ist, und daß eine zweite Position mutiert ist, so daß der Rest an dieser Position Ser anstelle von Pro ist, wobei die Mutationen in EPSPS-Sequenzen eingeführt sind, die die folgende kondervierte Region GNAGTAMRPLTAAV im Enzym vom Wildtyp umfassen, so daß die modifizierte Sequenz GNAGIAMRSLTAAV lautet.A preferred form of the polynucleotide comprises the following components in the 5 'to 3' direction of transcription:

• (i) at least one transcriptional enhancer which is the amplifying region upstream of the transcriptional start of the sequence from which the enhancer is obtained, and wherein the enhancer as such is not included as a promoter either in the sequence in which it is endogenously encompassed, or then, when heterologous as part of a construct, works;
• (ii) the promoter from the rice EPSPS gene;
• (iii) the rice genomic sequence encoding the rice EPSPS chloroplast transit peptide;
• (iv) The genomic sequence encoding the rice EPSPS;
• (v) a transcription terminator;

wherein the rice EPSPS coding sequence is modified such that a first position is mutated so that the remainder at this position is Ile instead of Thr, and that a second position is mutated such that the remainder at this position is Ser instead of Pro is wherein the mutations are introduced into EPSPS sequences comprising the following constrained region GNAGTAMRPLTAAV in the wild-type enzyme such that the modified sequence is GNAGIAMRSLTAAV.

The reinforcing Region includes prefers a sequence whose 3'-end at least 40 nucleotides upstream of the next transcription start the sequence from which the amplifier is obtained. In another embodiment of the polynucleotide the reinforcing Region a region whose 3'-end at least 60 nucleotides upstream the next Starts, and in yet another embodiment of the polynucleotide comprises the enhancing region a sequence whose 3'-end at least 10 nucleotides upstream of the first nucleotide of the TATA consensus sequence the sequence from which the amplifier is obtained.

The polynucleotide according to the invention may comprise two or more transcriptional enhancers, wherein one particular embodiment of the polynucleotide may be present as a tandem.

in the polynucleotide according to the invention can the 3'-end of the amplifier or the first amplifier, if more than one is present, between 100 and 1000 nucleotides upstream of the Codons corresponding to the translation start of the EPSPS transit peptide, or the first nucleotide of an intron in the 5'-untranslated region Region for the case that the Region contains an intron, be. In a particularly preferred embodiment of the polynucleotide is the 3'-end of the amplifier or first amplifier between 150 and 1000 nucleotides upstream of the codon, the translation start of the EPSPS transit peptide, or the first nucleotide of an intron in the 5'-untranslated region Region, and in a more preferred embodiment, the 3 'end of the amplifier or first amplifier between about 300 and about 950 nucleotides upstream of the first codon corresponding to the translation start of the EPSPS transit peptide, or the first nucleotide of an intron in the 5'-untranslated region Be a region. In a still more preferred embodiment, the 3'-end of the enhancer or first amplifier between about 770 and about 790 nucleotides upstream of the Codons corresponding to the translation start of the EPSPS transit peptide, or the first nucleotide of an intron in the 5'-untranslated region Located in the region.

In an alternative polynucleotide of the invention can be the 3'-end of the amplifier or first amplifier between about 300 and about 380 nucleotides upstream of the Codons corresponding to the translation start of the EPSPS transit peptide, or the first nucleotide of an intron in the 5'-untranslated region Region, and in a preferred embodiment this alternative Polynucleotide is the 3 'end of the amplifier or first amplifier between about 320 and about 350 nucleotides upstream of the Codons corresponding to the translation start of the EPSPS transit peptide, or the first nucleotide of an intron in the 5 'untranslated region.

in the polynucleotide according to the invention can the region upstream the promoter from the rice EPSPS gene comprise at least one enhancer, which originates from a sequence upstream of the transcription start either the CaMV35S or FMV35S promoter.

Corresponding the polynucleotide in the 5 ' to 3'-direction one first amplifier, the one transcriptional enhancer Region includes, which originates from a sequence upstream of the transcription start of the GOS2 promoter, and a second enhancer comprising a transcriptional enhancement region comprises which originates from a sequence upstream of the transcription start either the CaMV35S or FMV35S promoter.

alternative the polynucleotide in the 5 ' to 3'-direction one first amplifier, the one transcriptional enhancer Region includes, which originates from a sequence upstream of the transcription start of the Rice actin promoter, and a second enhancer comprising a transcriptional enhancement region comprises which originates from a sequence upstream of the transcription start either the FMV435S or CaMV35S promoter.

alternative the polynucleotide in the 5 ' to 3'-direction one first amplifier, the one transcriptional enhancer Region includes, which originates from a sequence upstream of the transcription start of the Barley plastocyanine promoter, and comprise a second enhancer, the one transcriptional enhancer Region includes, which originates from a sequence upstream of the transcription start either the CaMV35S or FMV35S promoter.

alternative the polynucleotide in the 5 ' to 3'-direction one first amplifier, the one transcriptional enhancer Region includes, which originates from a sequence upstream of the transcription start of the Maize polyubiquitin protomotors, and comprise a second enhancer, the one transcriptional enhancer Region includes, which originates from a sequence upstream of the transcription start either the CaMV35S or FMV35S promoter.

alternative the polynucleotide in the 5 ' to 3'-direction one first amplifier, the one transcriptional enhancer Region includes, which originates from a sequence upstream of the transcription start of the FMV35S promoter, and a second enhancer comprising a transcriptional enhancement region comprises which originates from a sequence upstream of the transcription start of the FMV35S promoter.

Independent of the identity and juxtaposition of the various polynucleotides present in the polynucleotide amplifier can the nucleotides 5 'of the Codons, which initiate the translation of rice EPSPS chloroplast transit peptide represents Kozack-prefers. What is meant by that is One skilled in the art and will continue from the following examples become apparent.

Especially preferred embodiments of the polynucleotide according to the invention have a non-translated region that includes a sequence that functions as an intron located 5 'of the rice genomic sequence, which encodes the rice EPSPS chloroplast transit peptide. The untranslated one Region may include the sequence shown in SEQ ID NO: 55. In particular, the untranslated region may be the maize ADHI intron comprising the sequence SEQ ID NO: 55.

The Polynucleotide of the invention may comprise a virus-derived translation enhancer, located within the untranslated region 5 'of the genomic Rice sequence encoding the rice EPSPS chloroplast transit peptide. The skilled person is aware of the identity of such suitable translation enhancers - like the Omega and Omega sequences, that come from TMV, and those that come from the tobacco etch virus and how such translational enhancers can be introduced into the polynucleotide the wished To provide the result.

The polynucleotide of the invention may further comprise regions encoding proteins which may confer to plant material containing them at least one of the following agronomically desirable traits: insect resistance, fungi, viruses, bacteria, nematodes, stress, dehydration and herbicides. Although such a polynucleotide contemplates that the herbicide resistance-conferring gene is different from an EPSPS, such as glyphosate oxido-reductase (GOX), the herbicide may be different from glyphosate, in which case the resistance-conferring genes may be selected from the group consisting of phosphinothricin acetyltransferase (PAT), hydroxyphenylpyruvate dioxygenase (HPPD), glutathione S-transferase (GST), cytochrome P450, acetyl-CoA carboxylase (ACCase), acetolactate synthase (ALS), protoporphyrinogen oxidase (PPO), dihydropteroate synthase, polyamine transport proteins, superoxide dismutase (SOD), bromoxynilonitrilase, phytoene desaturase (PDS), the product of the tfdA gene, available from Alcaligenes eutrophus, and known mutagenized or otherwise modified variants of the proteins. In the event that the polynucleotide provides multiple herbicide resistance, sol herbicides are selected from the group consisting of dinitroaniline herbicide, triazolopyrimidines, uracil, a phenylurea, triketone, isoxazole, acetanilide, oxadiazole, triazinone, sulfonanilide, amide, anilide, RP201772, fluorochlorideone, norflurazon and triazolinone-type herbicide; Postemergent herbicide is selected from the group consisting of glyphosate and salts thereof, glufosinate, asulam, bentazone, bialaphos, bromacil, sethoxydim or other cyclohexanedione, dicamba, fosamine, flupoxam, phenoxypropionate, quizalofop or other aryloxyphenoxypropanoate, picloram, fluorormetron, atrazine or another triazine, metribuzin, chlorimuron, chlorosulfuron, flumetsulam, halosulfuron, sulfometrone, imazaquin, imazathapyr, isoxaben, imazamox, metosulam, pyrithrobac, rimsulfuron, bensulfuron, nicosulfuron, fomesafen, fluroglycofen, KIH9201, ET751, carfentrazone, ZA1296, sulcotrione, paraquat, Diquat, Bromoxynil and Fenoxaprop.

In the case, that this Polynucleotide sequences comprises which encode insecticidal proteins, these proteins can be derived from the Group selected are made from crystal toxins derived from Bt, including secreted ones Bt toxins; Protease inhibitors, lectins, Xenhorabdus / Photorhabdus toxins consists; the fungal resistance-conferring genes can be selected from the group consisting of those known AFPs, defensins, chitinases, Encode glucanases, Avf-Cf-9. Particularly preferred insecticides Proteins are cryIAy, cryIAb, cry3A, Vip1A, Vip1B, cysteine protease inhibitors and snowdrop lectin. For the Case, that Polynucleotide includes bacterial resistance conferring genes, these can selected from the group which consists of those who are Cecropine and Techyplesin and analogs thereof. Virus resistance conferring genes can be the group selected which consists of those containing viral envelope proteins, motor proteins, encode viral replicases and antisense and ribozyme sequences which for the Provision of virus resistance are known; whereas the stress, salt and and dehydration resistance lending genes can be selected from those that Glutathione-S-transferase encode the sequence containing the known CBF1 regulatory sequence represents, and genes for the provision of enrichment of trehalose are known.

The polynucleotide according to the invention can for reinforcement the expression of protein encoded by him Sequences are modified by mRNA instability motifs and / or random splice regions can be removed or attachment-preferred codons can be used so that expression of the thus modified polynucleotide in a plant is substantially similar Protein with a substantially similar activity / function provides to those by expression of the unmodified Polynucleotide is obtained in the organism in which the protein coding Regions of the unmodified polynucleotide are endogenous. The degree of identity between the modified polynucleotide and a polynucleotide that endogenously contained in the plant and essentially the same protein encoded, may be such that a Cosuppression between the modified and endogenous sequences is prevented. In this case, the degree of identity should be between preferably less than about 70% of the sequences.

The Invention includes furthermore, a biological or transformation vector incorporating the polynucleotide according to the invention includes. Accordingly, with "vector" among other things one the following: a plasmid, virus, cosmid or bacterium, transformed or transfected to contain the polynucleotide.

The Invention includes and plant material associated with the polynucleotide or vector was transformed, as well as such transformed plant material, which has been further transformed with a polynucleotide, or which includes regions encode the proteins that contain plant material that contains it, at least confer one of the following agronomically desirable characteristics can: resistance to insects, fungi, viruses, bacteria, nematodes, Stress, Dehydration and herbicides.

The Invention includes furthermore, morphologically normal, fertile whole plants, which consist of regenerates the material disclosed in the immediately preceding paragraph were, their progeny seeds and parts, with the offspring the polynucleotide or vector of the invention is stable in its genome recorded and inherited in Mendelian manner.

The Invention includes further morphologically normal fertile plants containing the polynucleotide of the invention contained and made from the crossing of plants that come out with the polynucleotide according to the invention or vector transformed material, and plants result, which were transformed with a polynucleotide, the Includes regions, encode the proteins that contain plant material that contains it, at least one of the following agronomically desirable features transmitted can: resistance against insects, fungi, viruses, bacteria, nematodes, stress, dehydration and herbicides, the progeny of the resulting plants, their seeds and parts.

plants of the invention selected from the group be made of crops, Fruits and vegetables like canola, sunflower, tobacco, sugarbeet, cotton, corn, wheat, Barley, Rice, Sorghum, Tomato, Mango, Peach, Apple, Pear, Strawberry, Banana, melon, potato, carrot, lettuce, cabbage, onion, soy spp., Cane, pea, field bean, poplar, grape, citrus, alfalfa, Rye, oats, turf and forage grasses, flax and oilseed rape and nußerzeugenden Plants, unless they are already specifically mentioned, their progeny, seeds and parts.

Such particularly preferred plants include corn, soybean, cotton, sugar beet and canola.

The Invention Furthermore, a method for the selective control of weeds in one Field, the field weeds and plants of the invention or their herbicidal resistant offspring, the method Application of a glyphosate-type herbicide to the field in one sufficient amount to combat of weeds without substantially affecting the plants. According to this Procedures can one or more of a herbicide, insecticide, fungicide, nematocide, Bactericidal and antiviral agents on the field (and thus on the contained therein) either before or after application of the glyphosate herbicide.

• (i) Transformieren von Pflanzenmaterial mit dem Polynukleotid oder Vektor der Erfindung;
• (ii) Selektieren des so transformierten Materials; und
• (iii) Regenerieren des so selektierten Materials zu morphologisch normalen, furchtbaren ganzen Pflanzen.

The invention further provides a method of producing plants that are substantially tolerant to or substantially resistant to glyphosate herbicide, comprising the following steps:

• (i) transforming plant material with the polynucleotide or vector of the invention;
• (ii) selecting the material so transformed; and
• (iii) regenerating the material so selected to morphologically normal, terrible whole plants.

The Transformation can be the introduction of the polynucleotide in the material by any known means, but in particular by: (i) biolistic bombardment of the material with particles coated with the polynucleotide; (Ii) impale of the material on silicon carbide fibers coated with a solution which comprises the polynucleotide; or (iii) introducing the Polynucleotide or vector in Agrobacterium and cocultivation of the thus transformed Agrobacterium with plant material, is transformed and then regenerated. plant transformation, selection and regeneration techniques requiring routine modification with respect to a particular species of crop may require Expert well known. The transformed plant material can be selected by its resistance to glyphosate.

The The invention further provides the use of the polynucleotide of the invention or vector in the production of plant tissues and / or morphologically normal fertile whole plants ready that are substantially tolerant for or are substantially resistant to glyphosate herbicide.

The Invention includes a method of selecting biological material, that has been transformed to express a gene of interest, wherein the transformed material is the polynucleotide or the vector the invention comprises and wherein the selection comprises contacting the transformed one Materials with glyphosate or a salt thereof and selecting of the survivor Material includes. The material may be of plant origin and may in particular from a monocotyledonous Come from the group selected from the group consisting of barley, wheat, Corn, rice, oats, rye, sorghum, pineapple, sugarcane, banana, Onion, asparagus and leek exists.

• (a) Erzeugen von regenerierbarem Gewebe aus der zu transformierenden Pflanze;
• (b) Transformieren des regenerierbaren Gewebes mit der Fremd-DNA, worin die Fremd-DNA eine selektierbare DNA-Sequenz umfaßt, worin die Sequenz in einem regenerierbaren Gewebe als Selektionsvorrichtung wirkt;
• (c) zwischen ca. 1 Tag und ca. 60 Tage nach Schritt (b), Plazieren des regenerierbaren Gewebes aus Schritt (b) in ein Medium, das Schößlinge aus dem Gewebe erzeugen kann, worin das Medium ferner eine Verbindung enthält, die zur Selektion von regenerierbarem Gewebe verwendet wird, das die selektierbare DNA-Sequenz enthält, um die Identifizierung oder Selektion des transformierten regenerierten Gewebes zu erlauben;
• (d) nachdem wenigstens ein Schößling aus dem selektierten Gewebe aus Schritt (c) gebildet wurde, Übertragen des Schößlings auf ein zweites Medium, das Wurzeln aus dem Schößling erzeugen kann, um ein Pflänzchen zu erzeugen, worin das zweite Medium gegebenenfalls die Verbindung enthält; und
• (e) Kultivieren des Pflänzchens zu einer fruchtbaren transgenen Pflanze, worin die Fremd-DNA auf Nachkommenschaftspflanzen in Mendelscher Weise weitergegeben wird, dadurch gekennzeichnet, daß die Fremd-DNA das erfindungsgemäße Polynukleotid ist oder die selektierbare DNA-Sequenz, die von der Fremd-DNA umfaßt ist, das erfindungsgemäße Polynukleotid umfaßt und die Verbindung Glyphosat oder ein Salz davon ist. Die Pflanze kann eine einkeimblättrige Pflanzen wie oben angegeben sein – besonders bevorzugt aus Banane, Weizen, Reis, Mais und Gerste ausgewählt, und das regenerierbare Gewebe kann aus embryogenen Kalli, somatischen Embryos, unreifen Embryos etc. bestehen.

The invention further includes a method of regenerating a fertile transformed plant to contain foreign DNA comprising the steps of:

• (a) generating regenerable tissue from the plant to be transformed;
• (b) transforming the regenerable tissue with the foreign DNA, wherein the foreign DNA comprises a selectable DNA sequence, wherein the sequence acts as a selection device in a regenerable tissue;
• (c) between about 1 day and about 60 days after step (b), placing the regenerable tissue of step (b) in a medium capable of producing shoots from the tissue, wherein the medium further contains a compound which binds to Selection of regenerable tissue containing the selectable DNA sequence to permit identification or selection of the transformed regenerated tissue;
• (d) after at least one shoot of the selected tissue of step (c) has been formed, transferring the shoot to a second medium capable of producing roots from the shoot to produce a plantlet wherein the second medium optionally contains the compound; and
• (e) cultivating the seedling to a fertile transgenic plant wherein the foreign DNA is propagated to progeny plants in a Mendelian manner, characterized in that the foreign DNA is the polynucleotide of the invention or the selectable DNA sequence derived from the foreign DNA comprising polynucleotide of the invention and the compound is glyphosate or a salt thereof. The plant may be a monocotyledonous plant as specified above - most preferably selected from banana, wheat, rice, maize and barley, and the regenerable tissue may consist of embryogenic calli, somatic embryos, immature embryos, etc.

The The present invention will become apparent from the following description with the associated drawings and sequence protocols further apparent become.

sequence Listings

• SEQ ID NO: 1-42 PCR primers.
• SEQ ID NO: 43 Genomic rice EPSPS sequence (from ATG).
• SEQ ID NO: 44 Genomic rice EPSPS sequence, the double mutation contains.
• SEQ ID NO: 45 FMV amplifier.
• SEQ ID NO: 46 CaMV35S enhancer 1.
• SEQ ID NO: 47 CaMV35S enhancer Second
• SEQ ID NO: 48 maize polyubiquitin enhancer.
• SEQ ID NO: 49 rice actin enhancer.
• SEQ ID NO: 50 rice GOS2 enhancer.
• SEQ ID NO: 51 Barley plastocyanine enhancer.
• SEQ ID NO: 52 G1 Rice EPSPS promoter + 5 'upstream region.
• SEQ ID NO: 53 G3 rice EPSPS promoter + 5 'upstream region.
• SEQ ID NO: 54 G23 Rice EPSPS promoter + Adh1 intron in 5 'upstream region.
• SEQ ID NO: 55 maize Adh1 intron.

List of figures

1 : Genomic schematic map of Rice EPSPS.

2 : Vector pCR4-OSEPSPS (rice dmEPSPS gene in vector pCR4-Blunt).

3 : Schematic representation of the strategy used to introduce the double mutation.

4 : Vector pTCV1001.

5 : Vector pTCV1001OSEPSPS (includes rice dmEPSPS gene in vector pTCV1001).

6 : Vector pTCV1001EPSPSPAC (includes rice dmEPSPS gene in vector pTCV1001).

7 : Vector pBluSk + EPSPS (includes rice dmEPSPS gene in pBluescript SK + vector).

8th : Vector pPAC1.

9 : Vector pTCVEPSPSPH.

10 : Vector pTCVEPSPSADH.

11 : Vector pBluSKEPSPSADH (includes rice dmEPSPS gene and Adh1 intron)

12 Photos: Vector pIGPD9.

13 : Vector Zen 9

14 : Vector Zen 12

15 : Vector Zen 18

16 : Introduction of Zen vectors into super binary vectors.

Production of plants, the tolerant of Glyphosate treatment is by overexpression of a mutant EPSPS under the control of a non-heterologous promoter

Of the Term "amplifier" as in this specification used refers to those sequences upstream of the Promoter, which does not include the promoter itself, but to reinforce or Regulation of transcription start from the promoter act. Of the Term "EPSPS promoter deletion" as used in this specification used refers to the EPSPS promoter together with nucleotides from the enhancer native to the EPSPS gene is, i. Nucleotides that are upstream (5 ') of the promoter.

In The transformation of plant material will be understood by those skilled in the art recognize that, although particular types of target material (e.g., embryogenic cell suspension culture or de-differentiating immature embryos) and special transformation techniques (e.g., using Agrobacterium or particle bombardment) are given in the following examples, the present Invention is not limited to these particular embodiments and such target materials and methods used interchangeably can be. Furthermore, the term "plant cells" as used in this description throughout the invention uses isolated cells, including suspension cultures, as well as cells in an intact or partially intact tissue such as Embryo, scutella, microspore, microspore-derived embryo or somatic Designate cells from plant organs. Although the specific examples limited to corn, wheat and rice are, the invention is more similar As it were applicable to any of a wide range of agricultural crops and ornamental plants using suitable methods of plant cell transformation transformed can be.

General Molecular biological methods are used according to Sambrook et al. (1989) "Molecular cloning: A Laboratory Manual ", 2nd edition, Cold Spring Harbor Lab. Press, performed.

Example 1. Generation a cDNA probe for Rice EPSPS

A partial length cDNA encoding rice EPSPS is obtained using reverse transcriptase PCR (RT-PCR). Complete RNA is isolated from two week old rice plants (Oryza sativa L. indica var. Koshihikari) using the TRI-ZOL ^™ method (Life Technologies). First strand cDNA synthesis is performed using Superscript II reverse transcriptase (Life Technologies) with 200 ng of EPSPS degenerate reverse primer 10 (SEQ ID NO: 1) and 2 μg of complete RNA according to the protocols provided. The synthesis and cDNA amplification of the second strand by PCR is performed using EPSPS degenerate primers 10 and 4 (SEQ ID NO: 1 and SEQ ID NO: 2) and PCR beads (Pharmacia) according to manufacturer's instructions. All letter encodings are standard abbreviations (Eur. J. Biochem. (1985) 150: 15).

SEQ ID NO: 1 EPSPS degenerates reverse 10

SEQ ID NO: 2 EPSPS degenerates forward 4

The products are cloned into vector pCR2.1 (Invitrogen) using a TA-Cloning Kit ^™ according to the manufacturer's recommendation. Plasmid is recovered from selected colonies and the sequence analyzed by a method involving computer-based homology searches (BLAST) to confirm high homology of the cloned RT-PCR product with known plant EPSPS sequences.

Example 2. Isolation of genomic EPSPS sequences from rice and cloning of the rice EPSPS gene

A region of genomic DNA containing the complete rice EPSPS gene and 5 'upstream region is isolated from a λEMBLSP6 / T7 genomic library consisting of 5 day old etiolated rice saplings (Oryza sativa L. Indica var. IR36) (Clontech). 1 x 10 ⁶ plaque forming units (pfu) are screened using the ³² P-labeled rice EPSPS cDNA probe (Example 1) using the protocols provided by the manufacturer. Positive plaques are subjected to subsequent rounds of hybridization screening until a plaque moiety of a cross-hybridizing plaque is obtained. λ DNA is prepared from the pure phage stock according to the method described by Sambrook et al., 1989. The obtained DNA is analyzed by restriction digestion and Southern blotting using the same ³² P-labeled rice EPSPS cDNA as a probe. Cross-hybridization restriction fragments, if applicable, are truncated using a method such as Perfectly Blunt ^™ (Novagen) and cloned into an appropriate vector such as pSTBlue (Novagen). The DNA is then sequenced using an ABI377A PRISM automated DNA sequencer. 1 Figure 4 is a schematic representation of the rice EPSPS gene with some of the restriction sites labeled.

A 3.86 kb fragment of the rice EPSPS gene containing the coding region, the EPSPS promoter, some of the 5 'upstream region and the terminator is obtained by PCR. Oligonucleotide primer OSGRA1 (SEQ ID NO: 3) is used in conjunction with OSEPSPS3 (SEQ ID NO: 4) to amplify the desired region. OSEPSPS3 contains additional SacI and SmaI restriction enzyme sites to facilitate subcloning of the gene during the later stages of vector construction. A schematic localization of these primers is in 1 specified.

Pfu Turbo ^™ polymerase with high accuracy (Stratagene) is used to perform the PCR reaction with DNA obtained from the λ preparation (described above) as amplification template. The PCR product of the expected size is cloned into pCRblunt 4-TOPO ^™ (Invitrogen) and sequenced for integrity checking.

Example 3. Mutation of T to I and P to S in rice EPSPS

The mutation from T to I and P to S is obtained by the introduction of two point mutations. These mutations are introduced into the genomic EPSPS gene from rice by PCR using oligonucleotide primers containing the desired mutation. A schematic diagram indicating the binding sites of the primers used is in FIG 3 shown.

Two separate PCR reactions are performed (both using the λDNA as a template).

The resulting PCR products are joined using equimolar concentrations of each PCR product as template to the two oligonucleotides SalIEnd and EcoRVEnd in a new PCR reaction. A subset of the reaction product is analyzed by agarose gel electrophoresis and stored in pCR-BluntII ^™ (Invitrogen). Plasmid DNA is recovered and sequenced to detect successful uptake of the double mutation.

The DNA fragment containing the double mutation is inserted into the genomic EPSPS clone from rice ( 1 ) is introduced as follows. The clone containing the double mutant is digested with EcoRV and SalI. The plasmid containing rice EPSPS DNA PCR product is similarly digested and the EcoRV / SalI fragment containing the double mutant into the rice EPSPS gene in pCR4Blunt-TOPO ^™ using standard cloning techniques , which are described in Sambrook et al., 1989, ligated and transformed into competent E. coli. Plasmid is recovered from resulting colonies and sequenced to confirm the presence of the double mutation without further changes. This plasmid, pCR4-OSEPSPS, is in 2 shown.

The rice genomic EPSPS gene containing the double mutant ( 2 ) is excised from pCR4-Blunt-TOPO ^™ using PstI and NotI and ligated into pTCV1001 ( 4 ) to pTCV1001SEPSPS ( 5 ) and this is transformed into E. coli for amplification. Next, the PacI / EcoRV restriction fragment from λ-DNA ( 1 ) and plated in pTCV10010SEPSPS ( 5 ) to pTCV1001EPSPSPAC ( 6 ) to create. The rice EPSPS gene, which now contains the sequence from PacI to SacI ( 6 ), from pTCV1001EPSPSPAC ( 6 ) were excised as EagI / SacI fragment and ligated into similarly digested pBluescript SK + to prepare pBluSK + EPSPS ( 7 ). Additional rice EPSPS upstream regions and desired enhancers are assembled (as described below) and ligated into the pBluescript SK + vector using XbaI / PacI.

Example 4. Generation of individually reinforced Rice EPSPS promoter fusions

1 Figure 4 shows the binding sites of primers G1 and G2 used to generate a series of deletions at the 5 'end of the rice EPSPS gene. The G1 and G2 primers are used in combination with the RQCR10 primer using the EPSPS lambda DNA template from Rice and Pfu Turbo ^™ polymerase (Stratagene) using the protocols provided by the manufacturer.

The resulting products are analyzed by agarose gel electrophoresis and cloned into pCR-BluntII-TOPO ^™ vector (Invitrogen). The sequence of the resulting products is determined to ensure that there is no change in the sequence of the EPSPS genomic clone from rice. Clones for the further procedure are selected based on their orientation within the vector by determining whether or not Xho I digestion removes only the polylinker sequence instead of the entire insert from the vector.

The sequences of the CaMV35S and FMV35S genes and their associated 5 'upstream regions are published in the EMBL database (for example, entry # v00141 and x06166). Primers are designed to amplify only the enhancer regions upstream of the genes. The CaMV35S enhancer 1 (SEQ ID NO: 36) is thus obtained by PCR using primers SEQ ID NO: 12 and SEQ ID NO: 13 in conjunction with Pfu Tubo ^™ polymerase and pMJB1 DNA (A59870) as template. Alternatively, a different region of the CaMV35S enhancer is obtained using similar methods (SEQ ID NO: 37). The FMV35S enhancer is chemically synthesized (SEQ ID NO: 35).

The The following oligonucleotide primers are used.

The Sequence of the amplified and cloned molecules after cloning in the PCR Blunt II TOPO vector (Invitrogen). The pCR Blunt II TOPO vector, the EPSPS 5'UTR deletion contains is digested with XbaI / XhoI. The amplifier will be out of its corresponding pCR-Blunt-22-TOPO vector also removed using XbaI / XhoI digestion and in the first Vector ligated containing the EPSPS promoter deletions, the were generated by PCR.

Example 5. Generation of double reinforced Rice EPSPS promoter fusions

to further increase Expression from the rice EPSPS promoter may be an additional amplifier to be used in conjunction with either the 35S or FMV amplifier. In one Example of a cloning strategy for this goal will be first Amplifier / EPSPS fusions made (similar Example 4) comprising a single (first) amplifier. Such first amplifier are from upstream regions 5 'of the promoters the rice GOS2, rice actin 1, corn polyubiquitin, CaMV35S, FMV35S and barley plastocyanin genes. The nucleotide sequences these regions are published in the EMBL database (entry numbers x51910, x15865, u29159, v00141, x06166 and z28347, respectively). Become a primer created just the desired ones Transcriptional enhancer regions 5 'to these genes to amplify (SEQ ID NO: 15 to 22).

DNA is isolated from greenhouse-grown plants (corn, barley or rice) using the DNAeasy protocol (Qiagen) and used as the starting template for PCR amplification. The PCR products are cloned into pCR-BluntII-TOPO and sequenced for verification of authenticity. The enhancer-EPSPS fusion is performed as previously described in Example 4 (using Xba / XhoI exchange) except that in the case of rice actin 1, where the enhancer is inserted as an XbaI / PstI fragment (an XhoI Site is replaced by a PstI site due to the presence of an XhoI site in the rice actin enhancer), and maize polyubiquitin wherein the enhancer is inserted as a SpeI / XhoI fragment (the XbaI site is replaced by a SpeI). Location due to the presence of an XbaI site in the maize polyubiquitin stronger replaced). It is noted that an internal XhoI site is used with respect to the maize polyubiquitin enhancer region. The second enhancer, which may be either the CaMV35S enhancer or the FMV enhancer, is amplified using primers 35SXho and 3553 (SEQ ID NO: 23 and SEQ ID NO: 13) (35S) and FMVXho and FMV3 (SEQ ID NO: 25 and SEQ ID NO: 26). These primers facilitate introduction of an XhoI site (or PstI site) at the 5 'and 3' ends of the enhancer. Alternatively, a PacI site is introduced at the 3 'end instead of the XhoI site using primers 35SPac (SEQ ID NO: 24) or FMVPAC3 (SEQ ID NO: 27).

To the sequencing will be the PCR product, either XhoI: XhoI, PstI / PacI (If rice actin is the first amplifier is), or XhoI / PacI, introduced into the construct that is the fusion first Amplifier: EPSPS gene either at the XhoI site or between the XhoI and PacI or PstI and PacI restriction sites as required. The introduction at the XhoI site will construct the duplicate enhancer fusion constructs used either the G1 or G2 EPSPS promoter deletions include. Both the XhoI / PacI and the PstI / PacI fragments are used to construct dual amplifier fusions that comprising the G3 EPSPS promoter deletion. If the second amplifier as XhoI fragment introduced is, the orientation of the amplifier is determined by PCR.

Example 6. Insertion of the Adh1 intron into the 5 'UTR of the rice EPSPS gene

Insertion of the maize Adh1 intron I into the desired rice EPSPS promoter deletion (eg, prepared as described in Example 4) is performed prior to generation of the fusion construct with the desired enhancer (enhancer). In this particular example, the Adh1 intron is introduced into the G2 EPSPS promoter deletion. One skilled in the art will appreciate that a similar methodology for introducing the Adh1 intron can be adapted to other EPSPS promoter deletions. The maize Adh1 intron is inserted into the constructs by PCR. The Adh1 intron is obtained from a suitable source such as genomic maize DNA or a vector such as pPAC1 ( 8th ) using primers Adh5 (SEQ ID NO: 28) and Adh3 (SEQ ID NO: 29):

The resulting PCR product is denatured and used as a primer in conjunction with Adh5Pac (SEQ ID NO: 30) to obtain the desired product using the vector pTCV1001EPSPSPAC (FIG. 6 ) as a template.

The resulting PCR product is cloned into PCR BluntII (Invitrogen). The PacI: HindIII fragment is derived from the genomic rice clone ( 1 ) and inserted into pTCV1001 to generate pTCVEPSPSPH ( 9 ). Next, the PacI / NcoI PCR product obtained above, comprising the Adh1 intron, is inserted into pTCVEPSPH as shown by the scheme ( 9 ). The PacI: EcoRV fragment present in the cloned EPSPS gene containing the double mutant ( 10 ) is excised and replaced with the PacI / EcoRV fragment from pTCVEPSPSPH comprising the Adh1 intron sequence ( 9 ). Finally, the complete EPSPS gene comprising the Adh1 sequence is prepared from pTCVEPSPSADH ( 10 ) was excised as an EagI / SacI fragment and cloned into pBluescript SK + to give pBlueSKEPSPSADH ( 11 ).

Example 7. Introduction of optimized pre-ATG consensus sequence (Kozak) over site-directed mutagenesis for Constructs containing the maize Adh1 intron include

Optionally, site-directed mutagenesis is performed on constructs containing the Adh1 intron using the QuickChange Site Directed Mutagenesis Kit (Stratagene). This is done on the PacI / SacI EPSPS fragment in pBluescript SK + ( 11 ) before fusion with the enhancer: EPSPS promoter fusions performed. The following oligonucleotides are used according to the protocols provided to optimize the KOZAK sequence.

Clones are recovered by restriction analysis using KpnI Plasmid analyzed. The correctly altered DNA is by a additional KpnI restriction site compared to the unaltered DNA. The sequence is then verified by automated DNA sequencing. The altered DNA sequence can on original Constructs using the particular restriction enzyme sites from SphI or PacI at the 5 'end and AvrII or EcoRV at the 3 'end as needed for transfer every vector become.

Example 8. Completion of EPSPS expression cassettes, in the 5 'to 3' direction, enhancer region (s), rice EPSPS promoter upstream region, EPSPS promoter, EPSPS 5'UTR + (optional) maize Adh1 intron 1, rice EPSPS plastid transit peptide encoding Region, mature EPSPS coding Region of rice and rice EPSPS gene terminator region

The single and double amplified rice EPSPS promoter fusions (Examples 4 and 5) contained in the pCR Blunt II TOPO vectors are excised using XbaI and PacI and inserted into the similarly digested pBluescript SK + clone containing the remainder of the rice EPSPS sequence ( 7 / 11 ). However, if the corn polyubiquitin enhancer: EPSPS fusion is to be used, it is first cloned into pBluescript using SpeI / PacI. The remainder of the rice EPSPS sequence is then inserted as PacI / SacI to complete the expression cassette. This last cloning step leads to the required gene constructs. Examples of constructs obtainable using the above strategies are given in Table 1 below. Schematic maps are in the 13 - 15 specified.

Example 9. Subcloning of EPSPS expression cassettes from Bluescript in pIGPD9

If desired and especially for the transformation of plants by direct DNA methods (whisker, bombardment and protoplasts), the EPSPS expression constructs are excised from pBluescript using XmaI and cloned into pIGPD9 ( 12 ). The use of this vector for transformation avoids the transfer of antibiotic resistance genes to the plant as the selection is based on the complementation of an auxotrophic his B E. coli mutant with the gene expressing IGPD (the his B product). The pIGPD9-derived plasmids derived from the insertion of the XmaI fragments of pZEN9, 11, 12, 14, 15, 16, 18, 20, 23, 24 and 25 are labeled with pZEN9i, pZEN11i, pZEN12i ... etc . designated.

Large DNA preparations for use in plant transformation using the Maxi-prep method (Qiagen) using the manufacturer's received delivered logs.

Example 10. Preparation of DNA for plant transformation

The The above method describes the assembly of "EPSPS expression cassettes" which are in a 5 'to 3' direction (one) amplifier sequence (s), an EPSPS promoter from rice, a region containing a rice EPSPS transit peptide a region encoding a mature rice EPSPS enzyme, the Glyphosate is resistant to T-to-I and P-to-S changes at the indicated positions, and include a rice EPSPS gene terminator.

Possibly include the desired Cassettes also include a drug selection marker gene (e.g., ampicillin resistance, Kanamycin resistance, etc.), a T-DNA left or right border region and (optionally) a framework attachment region, the 5 'and / or 3 'to the above Construct is added. The person skilled in the art will recognize that similar Methods such as those described above can be used to address these get added components and put them in the desired To clone positions.

Example 11. Transformation of maize lines using an Agrobacterium strain, the a super-binary Contains vector, an EPSPS expression cassette between the right and left edges containing T-DNA; Selection and regeneration of plant cells and plants that are resistant to glyphosate

Construction of Agrobacterium strain

Bluescript plasmid DNA (eg ZEN9, ZEN11, ZEN14, ZEN15, ZEN18, ZEN20, ZEN12, ZEN23, ZEN24 or ZEN25) is digested with either XmaI or with XbaI / SacI and the resulting (~ 5-6.5 kb) EPSPS-encoding fragment ligated in a position in the cloning site, located between the right and left T-DNA borders of similarly restricted pSB1. For example, in the case of using the XmaI fragment of pZEN18, this ligation generates the plasmid pZEN18BS11 ( 16 ). The construction of plasmid pSB11 and the construction of its parent pSB21 is described by Komari et al. (1996, Plant J. 10: 165-174). The T-DNA region of pZEN18 is inserted into the super binary pSB1 vector (Saito et al. EP 672 752 A1 ) is integrated by a method of homologous recombination ( 16 ) to generate the plasmid pSB1ZEN18. To achieve this, the plasmid pZEN18SB11 is transformed into the E. coli strain HB101, which is then transformed according to the triple crossing method of Ditta et al. (1980, Proc Natl. Acad Sci., USA 77: 7347-7351) paired with an Agrobacterium LBA4404 harboring pSB1 to produce the transformed strain of Agrobacterium, LBA4404 (pSB1ZEN18), in which the presence of the cointegrate plasmid pSB1ZEN18 is selected on the basis of resistance to spectinomycin. The identity of pSB1ZEN18 is also confirmed based on SalI restriction analysis ( 16 ). LBA4404 strains containing the directly analogous constructs pSB1ZEN9, pSB1ZEN11, pSB1ZEN12, pSB1ZEN14, etc. are similarly constructed starting from the XmaI fragments of pZEN9, ZEN11, ZEN12, ZEN14, etc.

Alternatively, using similar procedures to the one described above, a similar fragment of pZEN9, ZEN11, etc. is homologously recombined into a position between the right and left edges of the superbinary vector pTOK162 ( 1 in US 5591616 ) to produce a similar set of cointegrate plasmids selected in Agrobacterium based on combined resistance to kanamycin and spectinomycin.

Of the Agrobacterium strain LBA4404 containing a helper plasmid PAL4404 (with a complete vir region) is from the American Type Culture Collection available (ATCC 37349). An alternative useful strain is Agrobacterium EHA101 (1986, Hood et al., J. Bacteriol., 168 (3): 1283-1290), the helper plasmid with the vir region from the highly virulent Agrobacterium tumefaciens strain A281.

manufacturing of Agrobacterium suspensions

Agrobacterium strains LBA4404 (pSB1ZEN9), LBA4404 (pSB1ZEN11) etc. are each on plates coated, containing solid "PHI-L" medium, and at 28 ° C in the dark for Cultivated for 3 to 10 days.

PHI-L medium is as described on page 26 (Example 4) of WO 98/32326. PHI-L medium, which is prepared in double-distilled water, comprises 25 ml / l stock solution A, 25 ml / l stock solution B, 450.9 ml / l stock solution C and 50 mg / l spectinomycin. The stock solutions are sterilized by autoclaving or filtration. Stock solution A is 60 g / l K ₂ HPO ₄ and 20 g / l NaH ₂ PO ₄ adjusted to pH 7.0 with KOH; Stock solution B is 6 g / l MgSO ₄ .7H ₂ O, 3 g / l KCl, 20 g / l NH ₄ Cl, 0.2 g / l CaCl ₂ and 50 mg / l FeSO ₄ .7H ₂ O; Stock solution C is 5.56 g / L glucose and 16.67 g / L agar (A-7049, Sigma Chemicals, St. Louis, MO, USA).

Alternatively, the Agrobacteria are cultured for 3-10 days on a plate containing YP medium (5 g / L yeast extract, 10 g / L peptone, 5 g / L NaCl, 15 g / L agar at pH 6.8), as reported by Ishida et al. (1966, Nature Biotechnology, 14, 745-750) or, alternatively, as described by Hei et al. in US 5591616 described (AB medium (Drlica and Kado, 1974; Proc Natl Acad Sci., USA 71: 3677-3681)), but in each case modified to provide the appropriate antibiotic selection (for example, containing 50 mg / ml spectinomycin in U.S.P. Case of Agrobacterium strain LBA4404 (pSB1ZEN9), etc., or containing both 50 mg / ml spectinomycin and 50 mg / ml kanamycin in the case of using Agrobacterium containing a super-binary vector derived from pTOK 162).

plates from Agrobacterium prepared as described above at 4 ° C stored and used within one month of manufacture. to Production of suspensions becomes a single colony from the Original plate spread on a plate at pH 6.8 5 g / l yeast extract (Difco), 10 g / l peptone (Difco), 5 g / l NaCl, 15 g / l Agar (Difco) and 50 mg / L spectinomycin (or as needed for the particular strain from Agrobacterium). Plates are at 28 ° C in the dark for Incubated for 2 days.

Suspensions of Agrobacterium for transformation of plant material are prepared in a similar manner as in US 5591616 described prepared. (Using good microbiological practice to avoid contamination of aseptic cultures) 3x5 mm spoons of Agrobacterium are removed from the plates, transferred to 5 ml of sterile AA liquid medium in a 14 ml Falcon tube and suspended therein. As used herein, at pH 5.2, the AA liquid medium contains the major inorganic salts, amino acids, and vitamins, as defined by Toriyama and Hinata (1985) in Plant Science 41, 179-183, the minor inorganic salts of Murashige-Skoog medium (Murashige and Skoog, 1962, Physiol. Plant. 15, 473-497), 0.5 g / l casamino acids (casein hydrolyzate), 1 mg / l 2,4-dichlorophenoxyacetic acid (2,4-D), 0.2 mg / l kinetin, 0.1 mg / l gibberellin , 0.2 M glucose, 0.2 M sucrose and 0.1 mM acetosyringone.

alternative are suspensions of Agrobacterium for the transformation of plant material in a similar Manner as described in WO 98/32326. 3 × 5 mm spoon of Agrobacterium are removed from plates in 5 ml sterile PHI-A basal medium as described in Example 4 on page 26 of WO 98/32326 and suspended therein or alternatively combined in 5 ml of the sterile PHI-I Medium, also in Example 4 on page 26 of WO 98/32326 is described. In each case, 5 ml of 100 mM 3'-5'-Dimethoxy-4-hydroxyacetophenone also added. PHI-A base medium at pH 5.2 comprises 4 g / l CHU (N6) base salts (Sigma C-1416), 1.0 ml / l Eriksson vitamin mixture (1000X, Sigma E-1511), 0.5 mg / l thiamine · HCl, 1.5 mg / ml 2,4-D, 0.69 g / l L-proline, 68.5 g / l sucrose and 68.5 g / l glucose. PHI-I combination medium, also adjusted to pH 5.2 with KOH and filter sterilized, comprises 4.3 g / l MS salts (GIBCO-BRL), 0.5 mg / ml nicotinic acid, 0.5 mg / ml pyridoxine · HCl, 1.0 mg / ml thiamine · HCl, 100 mg / l myo-inositol, 1 g / l vitamin-casamino acids (Difco), 1.5 mg / ml 2,4-D, 0.69 g / l L-proline, 68.5 g / l sucrose and 36 g / l glucose.

alternative are suspensions of Agrobacterium for the transformation of plant material in a similar As described by Ishida et al. (1996), Nature Biotechnology, 14, 745-750. 3 × 5 mm spoon of Agrobacterium are removed from plates, transferred to 5 ml LS-inf medium and suspended therein. LS-inf medium (Linsmaier and Skoog, 1965, Physiol. Plant 18, 100-127), adjusted with KOH to pH 5.2, contained major and minor inorganic LS salts, 0.5 mg / ml nicotinic acid, 0.5 mg / ml pyridoxine · HCl, 1.0 mg / ml thiamine · HCl, 100 mg / l myo-inositol, 1 g / l vitamin test casamino acids (Difco), 1.5 mg / ml 2,4-D, 68.5 g / l sucrose and 36 g / l glucose.

As also generated, the suspension of Agrobacterium is vortexed to make a uniform suspension and the cell population is adjusted to 0.5 x 10 ⁹ to 2 x 10 ⁹ cfu / ml (preferably the lower limit). 1 x 10 ⁹ cfu / ml corresponds to an OD value (1 cm) of ~ 0.72 at 550 nm.

Agrobacterium suspensions are divided into 1 ml batches into sterile 2 ml microcentrifuge tubes and as soon as as possible used.

Corn lines for transformation

suitable Maize lines for transformation include without limitation A188, F1 P3732, F1 (A188 × B73Ht), F1 (B73Ht × A188), F1 (A188 × BMS) one. The varieties A188, BMS (Black Mexican Sweet) and B73Ht become from the Ministry of Agriculture, forestry and fisheries obtained by the specialist well known. P3732 is obtained from IWATA RAKUNOU KYODOKUMIAI. Close suitable corn lines also a variety of A188x inbred hybrids (e.g. PHJ90 × A188, PHN46 × A188, PHPP8 × A188 in Table 8 of WO 98/32326) and elite inbred lines of different heterotic groups (e.g., PHN46, PHP28 and PHJ90 in Table 9 from WO 98/32326).

To the For example, immature embryos are produced from "Hi-II" corn. "Hi-II" is a hybrid between Inzüchtungen (A188 × B73), generated by reciprocal hybrids between Hi-II parent A and Hi-II Parent B, available from the Maize Genetic Coorporation Stock Center, University of Illinois at Champaign, Urbana, Illinois. Seeds, referred to as "Hi-II" seeds, are obtained from these Intersections, are in a greenhouse or field planted out. The resulting Hi-II plants will be self- or cross-pollinated with sibling plants.

Production of immature Embryos, infections and cocultivation

The Transformation of immature embryos of maize is made by contacting of the immature embryo with the appropriate recombinant described above strains performed by Agrobacterium. An immature embryo means the embryo of an immature seed that at the stage of maturation after pollination is. Immature embryos are in an intact tissue that is responsible for cell division is capable to lead to callus cells, who can then differentiate to produce the tissues and organs of a whole plant. preferred Material for transformation also excludes the scutella of embryos one, the dedifferentiated calli with the ability to induce for the regeneration of normal fertile plants that originally transformed were able to. Preferred material for transformation thus also includes callus, which from such dedifferentiated immature zygotic embryos or Scutella is from.

immaturity Corn embryos are aseptically made from developing flasks like by Green and Phillips (1976, Crop. Sci. 15: 417-421) or alternatively by the methods of Neuffer et al. (1982, "Growing Maize for genetic purposes ", Maize for biological research, W.F. Sheridan, ed., University Press, University of North Dakota, Grand Forks, North Dakota, USA) isolated. For example, immature maize embryos are between 1 and 2 mm (preferably 1 and 1.2 mm) in length aseptic from female ears 9-12 (preferred 11) Days after pollination isolated using a sterile spatula. typically, For example, flasks with 2.63% sodium hypochlorite are allowed to soak for 20 minutes before washing sterilized deionized water and aseptic removal surface sterilized by immature embryos. Immature embryos (preferably about 100 in number) are directly in a 2 ml microcentrifuge tube dripped, which contains about 2 ml of the same medium as for the preparation the suspension of Agrobacterium is used (the alternatives as described above). The lid of the tube is closed and the content by swirling for mixed for a few seconds. The medium is decanted off, 2 ml fresh Medium is added and vortexing is repeated. The entire medium is then stripped to the washed immature Embryos at the bottom of the tube leave.

If the immature maize embryos are made, there is the next phase of the process, the infection step, in it, they with the transformed To bring strain of Agrobacterium into contact.

In An example of this procedure is infection in one liquid Medium, the main one inorganic salts and vitamins of N6 medium (1987, C.C. Chu, Proc. Symp. Plant Tissue Culture, Science Press Beijing, Pp. 43-50), as described in Example 4 of WO 98/32326. 1.0 ml suspension of Agrobacterium produced in PHI-A medium as described above becomes the embryos in the microcentrifuge tube given and for swirled for about 30 s. Alternatively, 1.0 ml suspension of Agrobacterium, also prepared as described above in either PHI-I medium or in LS-inf medium.

To Standing for 5 minutes, the suspension of Agrobacterium and embryos in a Petri dish, containing either 1) PHI-B medium or 2) PHI-J medium or 3) contains LS-AS medium, dependent of whether the original Suspension of Agrobacterium in PHI-A medium, PHI-I medium or LS-inf medium was produced. The Agrobacterium suspension is used peeled off a Pasteur pipette, the embryos manipulated so that they the axle side down on the medium, the bowl with Parafilm sealed and in the dark at 23-25 ° C for three days cocultivation incubated. PHI-B medium at pH 5.8 comprises 4 g / l CHU (N6) base salts (Sigma C-1416), 1.0 ml / l Eriksson vitamin mixture (1000X, Sigma E-1511), 0.5 mg / l thiamine · HCl, 1.5 mg / ml 2,4-D, 0.69 g / l L-proline, 0.85 mg / l silver nitrate, 30 g / l Sucrose, 100 μM Acetosyringone and 3 g / L gelrites (Sigma). PHI-J medium, also discontinued to pH 5.8 4.3 g / l MS salts (GIBCO-BRL), 0.5 mg / ml nicotinic acid, 0.5 mg / ml pyridoxine · HCl, 1.0 mg / ml thiamine · HCl, 100 mg / l myo-inositol, 1.5 mg / ml 2,4-D, 0.69 g / l L-proline, 20 g / l sucrose, 10 g / l glucose, 0.5 g / l MES (Sigma), 100 mM acetosyringone and 8 g / l purified Agar (Sigma A-7049). LS-AS medium (Linsmaier and Skoog, 1965, Physiol. Plant 18, 100-127), adjusted to pH 5.8 with KOH, contains inorganic LS main and Minor salts, 0.5 mg / ml nicotinic acid, 0.5 mg / ml pyridoxine · HCl, 1.0 mg / ml thiamine · HCl, 700 mg / l L-proline, 100 mg / l myo-inositol, 1.5 mg / ml 2,4-D, 20 g / l sucrose, 10 g / l glucose, 0.5 g / l MES, 100 mM acetosyringone and 8 g / l purified agar (Sigma A-7049).

After the production of immature embryos as described above, an alternative method of achieving the transformation is to use them during and after a period of dedifferentiation as in US 5591616 described to infect. Immature embryos are placed on solid LSD 1.5 medium containing inorganic LS salts and vitamins in addition to 100 mg / ml casamino acids, 700 mg / l L-proline, 100 mg / l myo-inositol, 1.5 mg / ml 2 , 4-D, 20 g / l sucrose and 2.3 g / l gelrites. After 3 weeks at 25 ° C, the scallella-derived calli are collected in a 2 ml microcentrifuge tube and submerged in 1 ml of Agrobacterium suspension prepared as described above in AA medium. After standing for 5 minutes, the resulting calli are transferred to solid 2N6 medium containing 100 mM acetosyringone and incubated in the dark at 25 ° C for a 3-day period of cocultivation. Solid 2N6 medium comprises the inorganic salts and vitamins of N6 medium (CC Chu, 1978, Proc. Symp Plant Tissue Culture, Science Press, Beijing, pp. 43-50) containing 1 g / L casamino acids, 2 mg / l 2,4-D, 30 g / l sucrose and 2 g / l gelrites.

Resting and Selection of transformants

After cocultivation, the embryos are optionally transferred to a plate containing PHI-C medium, sealed with parafilm and incubated in the dark for 3 days for a "rest" step prior to selection. PHI-C medium at pH 5.8 comprises 4 g / l CHU (N6) base salts (Sigma C-1416), 1.0 ml / l Eriks son vitamin mixture (1000X, Sigma E-1511), 0.5 mg / l thiamine · HCl, 1.5 mg / ml 2,4-D, 0.69 g / l L-proline, 0.85 mg / l Silver nitrate, 30 g / l sucrose, 0.5 g / l MES, 100 mg / l carbenicillin and 8 g / l purified agar (Sigma A-7049). As disclosed in WO 98/32326, the desire to include this retirement step varies throughout the corn line transformation process and is a matter of experiment.

For the selection step about 20 embryos are transferred to a number of fresh plates, contain the PHI-D selection medium or LSD-1.5 selection medium, sealed with parafilm and incubated in the dark at 28 ° C. PHI-D selection medium adjusted to pH 5.8 with KOH comprises 4 g / l CHU (N6) base salts (Sigma C-1416), 1.0 ml / l Eriksson vitamin mixture (1000X, Sigma E-1511), 0.5 mg / l thiamine · HCl, 1.5 mg / ml 2,4-D, 0.69 g / l L-proline, 0.85 mg / l silver nitrate, 30 g / l Sucrose, 0.5 g / l MES, 100 mg / l carbenicillin and 8 g / l purified Agar (Sigma A-7049) and 0.1 mM to 20 mM tissue culture grade N- (phosphonomethyl) -glycine (Sigma P-9556). LDS 1.5 selection medium, adjusted to pH 5.8 with KOH, includes inorganic LS main and minor salts (Linsmaier and Skoog, 1965, Physiol. Plant 18, 100-127), 0.5 mg / ml nicotinic acid, 0.5 mg / ml pyridoxine · HCl. 1.0 mg / ml thiamine · HCl, 700 mg / l L-proline, 100 mg / l myo-inositol, 1.5 mg / ml 2,4-D, 20 g / l sucrose, 0.5 g / l MES, 250 mg / l cefotaxime, 8 g / L purified agar (Sigma A-7049) and 0.1 mM to 20 mM N- (phosphonomethyl) glycine of tissue culture quality (Sigma P-9556).

Alternatively, for the Case, that Starting material for selection from immature embryos derived calli are as disclosed in WO 5591616, such calli are sterilized with Washed water containing 250 mg / l cefotaxime before switching to LSD 1.5 selection medium be cultivated.

The Embryos or clusters of cells resulting from the immature embryo multiply (if necessary using a sterile Scalpels) on plates containing fresh selection medium, at 2-week intervals over one Total period of about 2 months transferred. herbicide-resistant Calli are then due to continued growth on the same Medium increases until the diameter of the selected callus is about Exceeds 1.5 cm.

The Concentration of N- (phosphonomethyl) -glycine in the selection medium is suitably selected to a desirable one Number of original transformants, and is preferred in the range of 0.3-5 mM. The concentration used in the selection medium is preferred of N- (phosphonomethyl) glycine about 1 mM for the first two weeks of selection and about 3mM afterwards.

Regeneration of transformants / propagation and analysis of transformed plant material

The selected calli become normal fertile plants according to, for example regenerated by the methods described by Duncan et al. (1985, Planta, 165, 322-332), by Kamo et al. (1985, Bot Gaz. 146 (3), 327-334) and / or West et al. (1993, The Plant Cell, 5, 1361-1369) and / or Shillito et al. (1989) Bio / Technol. 7, 581-587.

For example, selected calli are transferred with a diameter of 1.5-2 cm to regeneration / maturation medium and incubated in the dark fo _r approximately 1-3 weeks to mature the somatic embryos. Ei _n suitable regeneration medium, PHI-E medium (WO 98/32326) is adjusted to pH 5.6 with KOH and comprises 4.3 g / l MS salts (GIBCO-BRL), 0.5 mg / ml nicotinic acid , 0.5 mg / ml pyridoxine · HCl, 0.1 mg / ml thiamine · HCl, 100 mg / l myo-inositol, 2 mg / l glycine, 0.5 mg / l zeatin, 1.0 mg / ml indoleacetic acid , 0.1 mM abscisic acid, 100 mg / l carbenicillin, 60 g / l sucrose, 8 g / l purified agar (Sigma A-7049) and optionally 0.02 to 1 mM tissue culture grade N- (phosphonomethyl) glycine (Sigma P -9556).

The calli are then transferred to rooting / regeneration medium and cultured at 25 ° C under a scheme of either 16 h daylight (270 mEm ^-2 s ^-1 ) and 8 h dark or under continuous illumination (~ 250 mEm ^-2 s ^-1 ) until saplings and roots develop. Suitable rooting / regeneration media are either LSZ medium as described in the following paragraph (optionally without phosphonomethylglycine) or PHI-F medium at pH 5.6, containing 4.3 g / l MS salts (GIBCO-BRL), 0.5 mg / ml nicotinic acid, 0.5 mg / ml pyridoxine · HCl, 0.1 mg / ml thiamine · HCl, 100 mg / l myo-inositol, 2 mg / l glycine, 40 g / l sucrose and 1.5 g / l Gelrite includes.

Alternatively, selected calli are transferred directly to LSZ regeneration medium adjusted to pH 5.8 with KOH and main and minor inorganic LS salts (Linsmaier and Skoog, 1965, Physiol. Plant 18, 100-127), 0.5 mg / ml nicotinic acid, 0.5 mg / ml pyridoxine · HCl, 1.0 mg / ml thiamine · HCl, 700 mg / l L-proline, 100 mg / l myo-inositol, 5 mg / ml zetain, 20 g / l Sucrose, 0.5 g / l MES, 250 mg / l cefotaxime, 8 g / l purified agar (Sigma A-7049) and optionally 0.02 to 1 mM N- (phosphonomethyl) glycine (Sigma P-9556) from turned includes high-quality cultured food. After an incubation period in the dark, the plates are exposed (continuous or light / day as above) and plantlets regenerated.

Small plantlets are transferred to individual glass tubes containing either PHI-F medium or half-concentrated LSF medium at pH 5.8, the main LS salt (Linsmaier and Skoog, 1965, Physiol. Plant 18, 100-127) in half concentration , LS minor salts, 0.5 mg / ml nicotinic acid, 0.5 mg / ml pyridoxine · HCl, 1.0 mg / ml thiamine · HCl, 100 mg / l myo-inositol, 20 g / l sucrose, 0.5 g / l MES, 8 g / l of purified agar (Sigma A-7049) and cultured for approximately one more week. The plantlets are then transferred to pots with soil, hardened in a growth chamber (85% relative humidity, 600 ppm CO ₂ and 250 mEm ^-2 s ^-1 ) and cultured to maturity in a soil mixture in the greenhouse.

The first (T0) generation of plants obtained as above are self-fertilized, to obtain seed of the second generation (T1). Alternatively (and preferred) is the first generation of plants reciprocal with a other non-transgenic maize inbred line crossed to seed the second generation. It is then expected that the offspring of these intersections (T1) 1: 1 for split the herbicide resistance feature. T1 seeds are sown in the greenhouse or field used and the degree of resistance, inheritance of Resistance and breakdown of resistance to the herbicide glyphosate through this and subsequent generations through observation of differential plant survival, the fertility and symptoms of necrosis in the tissue after a Spray treatment with glyphosate (suitably formulated and optionally as a salt) in a range of application rates between 25 and 2000 g / ha and in a series of growth stages between and including V2 and V8 (or alternatively 7-21 Days after germination). These reviews are relative too vulnerable Cleavage mutants and relative to similar, untransformed Lines made of corn that are not genes of the present invention or sequences of the prior art, the resistance to glyphosate to lend, include. Transgenic lines resistant to glyphosate are selected and selfed again or with a non-transgenic Inbred line back crossed.

In All stages in the above procedure are tissue samples of transformed Callus, plantlet, T0- and T1 plant material optionally taken and by 1) Southern analyzes and PCR to detect the presence, copy number and integrity of transgenes to show 2) Northern analyzes (or similar) to measure the expression of mRNA from transgenes, 3) quantitative Western analysis of SDS gels to measure expression levels of EPSPS and 4) Measurement of EPSPS enzyme activity levels in the presence and absence of glyphosate to more accurately evaluate how much EPSPS that expresses is derived from the transgene.

Such Analytical methods are generally known in the art. Suitable methods to study for the presence, integrity and expression of the transgene by PCR, to carry out Southern analysis, for cloning and expression of mature rice EPSPS in E. coli, for the purification of rice EPSPS, for the production of polyclonal antibodies against purified rice EPSPS, for Western analysis of EPSPS starches in callus and plant tissues and to measure EPSPS activity levels in plant-derived extracts at a concentration of Glyphosate, which is between the endogenous glyphosate-susceptible EPSPS and the glyphosate-resistant product of the EPSPS-encoding transgene different, will be in greater detail described below in Examples 17-20.

Table 2

Transgene expression in Calli, resulting from the transformation of immature embryos from A188 × B73 (Hi-II) corn come with Agrobacterium strain LBA4404, which is a super binary vector contains the EPSPS expression cassettes of Bluescript vectors (pZEN11, 12, 14 or 15) within the T-DNA borders

The table shows the results of the EPSPS enzyme assay (+/- 100 μM glyphosate at 100 μM PEP) based on enzyme assays of extracts of stably transformed callus from A188 × B73 regeneratable corn transformed using Agrobacterium containing a super binary vector carrying the EPSPS expression cassettes of the pZEN11, ZEN12, ZEN14 or ZEN15 pBluescript plasmids. Each callus line represents a single event that is double-tested. The ratio of true (allowed approximately 8% inhibition) tolerant enzyme activity expressed by transgenic to endogenous susceptible activity (> 98% inhibited + glyphosate) is calculated. The mutated EPSPS is relatively strongly expressed in some lines, especially lines 5 and 12, whereas, considering the reduced Vmax of the tolerant enzyme relative to the wild type (about 1/3), it can be estimated that the tolerant enzyme is 2-6 -fold the normal strength of the endogenous EPSPS is expressed (this calculation is ver ver complicates that in some selected calli expression of the transgene occurs in addition to an apparent approximately 2 to 2.5 fold increase in background expression of the susceptible endogenous enzyme).

Example 12. Transformation of corn lines by bombardment with particles coated with DNA which includes an EPSPS expression cassette; selection and regeneration of plant cells and plants that are resistant against glyphosate

In another example is crumbling embryogenic callus, which originates from immature maize embryos, induced on a solid medium and biolistically transformed. Similar the method described in Example 11 is transformed Callus then selected based on differential growth rate in medium, that is a range of glyphosate concentrations contains. Resistant callus is selected and regenerated to provide T0 plantlets which are transferred into pots, cultivated to maturity and in the greenhouse self- or cross-fertilized. The progeny seeds (T1) are then reared to other generations of plants to be tested for resistance to glyphosate and on transgene presence, integrity and expression as described in Example 1.

Inducing callus from immature embryos

Type II crumbling embryogenic callus suitable for transformation is from un mature embryos from, for example, A188 × B73 maize. Alternative inbred lines such as from B73 and hybrid lines of maize may also be used, for example, including those listed in Example 11. Immature embryos of corn between 1 and 2 mm in length are aseptically isolated from female ears, typically about 11 days after pollination, using the procedures given in Example 11.

immaturity For example, embryos are plated on an N6-based medium (Chu et al., 1975, Scientia Sinica, 18, 659-668), adjusted with KOH to pH 5.8, 1 mg / l 2,4-D, 2.9 g / l L-proline, 2 mg / l L-glycine, 100 mg / l casein hydrolyzate, N6 main salts, N6 minor salts, N6 vitamins, 2.5 g / L Gelrite (or 2 g / L "Gel") and 20 g / L sucrose contains. Alternative suitable media include, for example, a similar one Medium containing MS salts (Murashige and Skoog, 1962, Physiol. Plant, 15, 473-497) contains instead of N6 salts. Alternatively, the medium may contain about 10 mg / L dicamba instead of 2,4-D contain.

immaturity Embryos are incubated in the dark on the above medium at about 25 ° C, to induce the callus. Callus material type II is through visual selection of fast growing crumble embryogenic cells by methods known in the art and e.g. described in WO 98/44140 selected. For example, suitable recipient cells are selected manually by selecting more preferred Selecting cells that are at the surface of a cell cluster can and further by their lack of differentiation, a small size and a high core / cytoplasm volume ratio be identifiable can. A suspension culture is initiated from tissue within the callus, the least differentiated, softest and most crumbling appears. Tissue with this morphology will be on fresh plates from media approx. 8-16 Days after the original one Plating the immature embryo transfer. The tissue then becomes routinely all 14 to 21 days by takeover of about 10% of the pieces subcultured, which reach about 1 g. In each step will only Material with the desired Morphology type II or type III further subcultured.

manufacturing of cell suspension cultures

Preferably, within six months of the callus induction described above, dispersed suspension cultures are introduced into liquid media containing appropriate hormones such as 2,4-D and NAA, optionally delivered in the form of slow release hormone capsule treatments, such as in Examples 1 and 2 of US 5550318 described. Optionally, hormone levels within cultures are maintained by occasionally adding fresh hormone supplementation. Suspension cultures are introduced, for example, by adding about 0.5 g of callus tissue to a 100 ml flask containing 10 ml of suspension culture medium. Every 7 days the culture is further subcultured by transfer, using a sterile end-to-end pipette, 1 ml of settled cells and 4 ml of conditioned medium to a fresh flask containing fresh medium. Large aggregates of cells that can not pass through the pipette tip are excluded at each subculturing step. Optionally, suspension cultures are passed through a suitable sieve (eg, about 0.5-1.0 mm mesh size) in each subculturing step. After 6 to 12 weeks, the culture is dispersed. Suitable cell suspension culture media include, for example, a medium adjusted to pH 6.0 and main and minor salts according to Murashige and Skoog (1962) (optionally modified to contain a reduced amount, 1.55 g / l ammonium nitrate), 30 g / l sucrose, 0.25 mg / l thiamine, 10 mg / l dicamba, 25 mM L-proline, 200 mg / l casein hydrolyzate, 100 mg / l myo-inositol, 500 mg / l potassium sulfate and 400 mg / l potassium hydrogen phosphate contains. Alternatively, the cell suspension medium contains 2,4-D and / or NAA instead of dicamba.

antifreeze of cell suspension cultures

Optionally, suspension cultures obtained as described above are cryopreserved using antifreeze agents and procedures as exemplified in Example 2 US 5550318 to be discribed. Frozen preservation requires the addition of antifreeze at ice temperature to precooled cells, also at ice temperature, in a stepwise manner over a period of 1 to 2 hours. The mixture is stored at ice temperature and the final volume of antifreeze is equal to the volume of the cell suspension. The final concentrations of antifreeze are, for example, 10% dimethylsulfoxide, 10% polyethylene glycol (6000 Mw), 0.23 M L-proline and 0.23 M glucose. After an equilibration period of 30 minutes at ice temperature, the mixture is divided into approximately 0.5 ml aliquots, transferred to 2 ml microcentrifuge tubes and slowly cooled to a temperature of -8 ° C at a rate of 0.5 ° C / min , After a period of ice nucleation, the sample is further cooled to -35 ° C and then immersed in liquid nitrogen. When needed for use, frozen samples are thawed by first bathing in their containers in water at about 40 ° C for 2 min and then slowly filling be thawed constantly. The mixture of cells and antifreeze is then pipetted onto a filter placed over a layer of BMS "feed" cells at 25 ° C. After the thawed tissue begins to grow, it is transferred back to fresh solid culture medium and, once established (within 1 to 2 weeks), is further transferred to cell suspension culture medium. Once the growth in the liquid suspension culture has re-established, the cells are used for transformation.

Particle-mediated transformation

DNA derived from plasmid pIGPD9 ( 12 ) containing Xma1 EPSPS expression cassettes (ie, pZEN9i, ZEN11i, ZEN12i, ZEN14i, etc.) is purified, concentrated (eg by anion exchange chromatographic or CsCl ₂ gradient densitometric isolation of plasmid DNA from cells of a suitable HisB, RecA Host strain of E. coli (eg DH5α: hisB-) after growth to stationary phase in a 5 × A minimal medium (K ₂ HPO ₄ 52.5 g, KH ₂ PO ₄ 22.5 g, (NH ₄ ) ₂ SO ₄ 5 g and sodium citrate · 2H ₂ O 2.5 g per liter) and as a concentrated solution (preferably about 1 mg / ml) in sterile water provided DNA as ring-shaped plasmid DNA or alternatively restricted with XmaI to provide a linear fragment containing an EPSPS expression cassette, and used after purification by agarose gel electrophoresis and electroelution.

A suitable device for bombardment is, for example, the Biorad PDS1000 helium cannon. The plate is placed 5-6 cm below the brake screen used to stop the Capton macro projectile. The DNA construct is grown on tungsten or gold particles with an average diameter of ~ 1.0 μm in a similar manner as described by Klein et al. , 1987, Nature, 327, 70-73. For example, 1.25 mg tungsten or gold particles (prewashed in ethanol at 65 ° C. for 12 h) are successively admixed with approximately 20-30 mg DNA, 1.1 M CaCl ₂ and 8.7 mM spermidine to a final volume of approx 0.6 ml mixed. The mixture is vortexed for 10 min at 0-4 ° C, centrifuged at low speed (about 500 g) for 5 min and the bulk of the supernatant decanted off to leave the tungsten particles suspended in a final volume of about 30 ml. 1-10 μl aliquots are pipetted onto the macroprojectile of the particle gun.

Out Type II and / or Type III callus derived suspension cultures be in culture for 3-5 months maintained (or alternatively obtained from cryopreservation), fresh subcultivated and then through a sieve with ~ 0.5-1.0 mm Mesh size sieved from stainless steel. Approximately 0.5 ml packed Cell volume of cells that are recovered from the filtrate then pipetted on 5 cm paper filter and before transfer Vacuum dried on a Petri dish containing a stack of three containing 7 cm paper filters moistened with suspension culture medium. each Slabs of suspension cells are centered on the sample plate tray, the Petri dish lid removed and twice at a vacuum of 28 inHg bombed twice. 0.1 or 1.0 mm screens are approx. 2.5 cm below the brake plate placed to the injury of the to mitigate bombarded tissue. After the bombing will be remove the plant cells from the filter in cell suspension culture medium zurücksuspendiert and for 2-21 days cultured. Alternatively, the bombarded callus becomes plate by plate transferred to a plate, a similar one contains solid medium (For example, containing 8 g / L of purified agar), and the like Way at ~ 25 ° C cultivated in the dark.

selection of transformants

To The transformation will be cells that are unselected in liquid or solid culture, transferred to filters and overcoated on solid medium, the one row (0.1-20 mM) of selecting concentrations of N- (phosphonomethyl) glycine of tissue culture quality (Sigma) overlays. Suitable solid selection media include media adjusted with KOH to pH 5.8 or 6.0 containing either MS or N6 salts (such as the above for described the callus induction or, with appropriate addition from Agar, those above for the growth of cells in liquid suspension described) and N- (phosphonomethyl) glycine. suitable Close selection media also, for example, the selection media described in Example 11 but in this case modified so that they lack antibiotics. Transform calli expressing the resistant EPSP synthase enzyme are selected based on their growth at concentrations, the inhibitory for similar Preparations of untransformed cells are. Growing lumps are subcultured on fresh selection medium. Is preferred the concentration of N- (phosphonomethyl) glycine used in the selection medium about 1 mM for the first two weeks of selection and about 3mM afterwards. After 6-18 weeks become presumptive resistant calli identified and selected.

Regeneration of transformants / propagation and analysis of transformed plant material

The selected calli become normal fertile plants according to, for example regenerated by the methods described by Duncan et al. (1985, Planta, 165, 322-332), by Kamo et al. (1985, Bot Gaz. 146 (3), 327-334) and / or West et al. (1993, The Plant Cell, 5, 1361-1369) and / or Shillito et al. (1989) Bio / Technol. 7, 581-587.

To the For example, plants become efficient by transferring the embryogenic callus regenerated on Murashige and Skoog medium, adjusted to pH 6.0, the 0.25 mg / l 2,4-D, 10 mg / l 6-benzyl-aminopurine and optionally 0.02 to 1 mM N- (phosphonomethyl) glycine. After about 2 weeks will be the tissue on a similar Transfer medium, but the hormones are missing. If necessary, the hormone level gradually through multiple transmissions and over a longer one Reduced period of up to 6 to 8 weeks. shoots, which develop after 2 to 4 weeks are transferred to MS medium, containing 1% sucrose and solidified with 2 g / l of gel, into which they then rooted.

alternative Methods and media used for regeneration are as in Example 1, except that the media used contain no antibiotic.

method for cultivating plants to maturity, for further propagation of Plants through generations, for the analysis of inheritance of resistance against glyphosate and for analysis of the presence, integrity and expression of the EPSPS transgene are as described in Example 11.

Example 13. Transformation of maize lines with DNA including an EPSPS expression cassette on silicon carbide whiskers; Selection and regeneration of plant cells and plants that are resistant to glyphosate.

In another example is corn lines, for example hybrid lines with the genotype A188 × B73 lock in, prepared as cell suspensions and by contacting the Cells with silicon carbide wiskers, coated with DNA, transformed using methods, essentially as described by Frame et al. are described (1994, Plant J. 6, 941-948). As described in the previous examples, the so generated transformed callus based on the differential growth rate selected in medium containing a range of glyphosate concentrations contains to small plants Regenerated (T0), which are cultivated to maturity and either self- or cross-fertilized to progeny seeds (T1) for further breeding provide. Plant and plant material will be resistant across from Glyphosate and on transgene presence, integrity and expression analyzed as described in the previous examples.

Inducing callus from immature embryos, production of cell suspension cultures

Maize cell suspensions which are suitable for transformation are optionally cryopreserved and in the same manner as described in Example 12.

transformation

DNA derived from plasmid pIGPD9 ( 12 ) containing XmaI EPSPS expression cassettes (ie, pZEN9i, ZEN11i, ZEN12i, ZEN14i, etc.) is purified, concentrated (eg by anion exchange chromatography or CsCl ₂ gradient densitometric isolation of plasmid DNA from cells of a suitable HisB, RecA Host strain of E. coli (eg DH5α: hisB-) after growth to stationary phase in a 5 × A minimal medium (K ₂ HPO ₄ 52.5 g, KH ₂ PO ₄ 22.5 g, (NH ₄ ) ₂ SO ₄ 5 g and sodium citrate · 2H ₂ O 2.5 g per liter) and as a concentrated solution (preferably about 1 mg / m) in sterile water DNA is provided as circular plasmid DNA or alternatively restricted with XmaI to provide a linear fragment containing an EPSPS expression cassette, and used after purification by agarose gel electrophoresis and electroelution.

The Transformation is done exactly as described by Frame et al. (1994) carried out. Alternatively, the method becomes something as described below modified.

One leaves cells cultured in liquid culture in cell suspension medium one day after subculturing in a shake flask. Consumed medium is decanted off and drawn off, and 12 ml of N6 medium at pH 6.0 (Chu et al., 1975) modified to contain 6 mM L-proline, 20 g / l sucrose, 2 mg / l 2,4-D, 0.25 M sorbitol and 0.25 M mannitol are added per 4 ml of packed cell volume. The flask is returned to the shaker (rotationally shaken at 125 rpm and incubated at 26-28 ° C) for 45 minutes. At the end of this period, 1 ml aliquots of cell suspension are removed using a wide bore pipette into a series of sterile microcentrifuge tubes. After settling the cells in each tube, 0.6 ml of the supernatant from spent medium is removed to leave most of the remaining contents as settled cells. 50 mg of silicon carbide whiskers (Silar SC-9 Whisker, Advanced Composite Materials Corp., Greer, SC, USA) are suspended by vortexing in 1 ml of the modified N6 medium described above. 40 ml of these suspended whiskers and 25 mg of the plasmid or linear DNA which includes the EPSPS expression cassette are then added to each cell of seeded cells. The tubes are vortexed manually 2 to 3 times, mixed in a Mixomat (Mixomat Dental Amalgamator (Degussa, Ontario, Canada)) for 1 second and then with 0.3 ml of N6 medium (modified as described above) for each microcentrifuge tube is added. The suspended cells are then plated on a filter disc (200 μl / plate) overlaid on solid N6 medium (same as the modified N6 medium described above, but without sorbitol, without mannitol and with 30 g / l sucrose and 3 g / l gelrites). Each plate is then wrapped with Urgopore tape (Stelrico, Brussels) and incubated in the dark for 1 week at 26-28 ° C.

Selection of transformants

transformed Callus is as described in Example 12 or alternatively as in Frame et al. (1994), except that N- (phosphonomethyl) glycine is used, in a range of concentrations between 1 and 5 mM instead in the publication by Frame et al. specified Bialaphos.

Regeneration of transformants / propagation and analysis of transformed plant material

plants be regenerated as described in Example 12, multiplied and bred. Plants are analyzed for resistance to glyphosate, and plant material is based on transgene presence, integrity and expression as in example 12 described analyzed.

Example 14. Transformation of rice lines using an Agrobacterium strain, the a super-binary Contains vector, an EPSPS expression cassette between the right and left edges which includes T-DNA; Selection and regeneration of plant cells and plants that are resistant to glyphosate.

In another example, scutella from mature seeds are isolated from appropriate lines of rice (including, for example, the varieties Koshihikari, Tsukinohikari, and Asanohikari), dedifferentiated, and the resulting callus transformed by infection with Agrobacterium. After selection and regeneration, transgenic plantlets (T0) are obtained, which are cultured to maturity and either self-fertilized or cross-fertilized to provide progeny seeds (T1) for further breeding. Plants and plant material are evaluated for resistance to glyphosate and analyzed for transgene present, integrity and expression as described in the previous examples. As an alternative to the methods described below, the methods used in Example 1 are used US 5591616 suitably adapted so that glyphosate is used instead of hygromycin for selection.

Construction of Agrobacterium strain; Preparation of Agrobacterium suspension

A strain of Agrobacterium containing the superbinary vector with the desired EPSPS expression cassette between the right and left edges is constructed as described in Example 1 (using electroporation to transform Agrobacterium with plasmid DNA). Suspensions are prepared according to the procedures described in Example 1. Alternatively, the transformed strain of Agrobacterium is cultured for 3 days on AB medium (Chilton et al., 1974, Proc Natl. Acad Sci., USA, 71, 3672-3676) containing a suitable antibiotic selection (eg, 50 mg / ml). l spectinomycin in the case of LBA4404 (pSB1ZEN18 etc.)), and scraped from the plate to a suspension in AAM medium (Hiei et al., 1994, The Plant Journal, 6 (2), 271-282) at a density from 1-5 x 10 ⁹ cells / ml.

Rice varieties, production from Callus from Scutella

rice varieties are for example Oryza sativa L. Tsukinohikari, Asanohikari and Koshihikari.

Mature seeds are peeled, surface sterilized by washing in 70% ethanol and then Soaked in 1.5% NaOCl for 30 minutes. After rinsing in sterile water, they are cultured at 30 ° C in the dark for 3 weeks on 2N6 medium at pH 5.8 containing the major salts, minor salts and vitamins of N6 medium (Chu 1978, Proc. Symp. Plant Tissue Culture , Beijing: Science Press, p. 43-50), 30 g / l sucrose, 1 g / l casein hydrolyzate, 2 mg / l 2,4-D and 2 g / l gelrites. Grown callus derived from the seed cutella is subcultured on fresh 2N6 medium for 3-7 days. Grown callus (diameter 1-2 mm) is selected, suspended in 2N6 liquid medium (without Gelrite) and cultured in flasks in the dark on a 125 rpm shaker and at 25 ° C. The medium is changed every 7 days. Cells growing in the logarithmic phase after 3-4 transformations are used for transformation.

Infection, transformation and selection

suspended Reiskallus cells are allowed to settle out of the suspension and then resuspended in the suspension of Agrobacterium, for several Minutes left in contact and then let settle again and without rinsing on 2N6-AS medium (2N6 medium, adjusted to pH 5.2, 10 g / l D-glucose and 100 mM acetosyringone) and plated in the Dark at 25 ° C for 3-5 days incubated. Growing material is carefully mixed with 250 mg / l cefotaxime rinsed in sterile water and then to 2N6-CH medium (2N6 medium adjusted to pH 5.8 with KOH, 250 mg / l cefotaxime and 0.5-5 mM N- (phosphonomethyl) glycine of tissue culture quality contains) or alternatively on 2N6K-CH medium (2N6 medium, modified as by Hiei et al. 1994, but instead of hygromycin 0.5-5 mM N- (phosphonomethyl) glycine of tissue culture quality containing) and for 3 weeks in the dark at 25 ° C cultured. Growing colonies become on a second plate Selection medium for another period of 7-14 Subcultured for days.

regeneration and analysis of plants

growing Colonies are plated on a regeneration medium at pH 5.8, the semi-concentrated N6 major salt, N6 minor salts, N6 amino acids, vitamins from AA medium (Chilton et al., 1974), 1 g / l casein hydrolyzate, 20 g / l sucrose, 0.2 mg / l naphthaleneacetic acid, 1 mg / l kinetin, 3 g / l Gelrites and optionally 0.04-0.1 mM N- (phosphonomethyl) glycine. These plates are included Incubated at 25 ° C and kept under continuous illumination (~ 2000 lux). As in Example 11, regenerated plants are finally grown up Transfer soil into pots and in a greenhouse matured.

plants are propagated and substantially as described in Example 11 cultured (For example, the transgenic plants are selfed). plants are analyzed for resistance to glyphosate, and plant material is essentially based on transgene presence, integrity and expression analyzed as described in Example 11.

Example 15. Transformation of wheat lines with DNA including an EPSPS expression cassette Use of microprojectile bombardment; Selection and regeneration of plant cells and plants that are resistant to glyphosate

In another example, immature embryos are isolated from suitable lines of wheat (including, for example, spring wheat cultivar BobWhite or Jaggar), incubated on hormone-containing (2,4-D) medium for 2 days, and transformed by bombardment with DNA-coated particles. After a period of recovery and continued growth of callus, callus-forming embryos are subcultured by a series of media containing a fixed amount of glyphosate and (serially diluted) decreasing amounts of 2,4-D to induce somatic embryogenesis. The selected material is regenerated to form shoots on a medium also containing glyphosate, transferred to rooting medium, and regenerated into plantlets (T0) for maize, which are grown to maturity and either self-fertilized or cross-fertilized as in the previous examples. to provide progeny seeds (T1) for further breeding. Plants and plant material are evaluated for resistance to glyphosate and analyzed for transgene present, integrity and expression as described in the previous examples. As an alternative to the methods described below, those in Example 1 are described US 5631152 used described method.

Production of immature embryos

Wheat plant lines (for example, spring wheat Triticum aestivum variety Bob White) are cultivated to maturity in the greenhouse and caryopses are isolated 11-15 days after flowering. Caryopses are surface sterilized by treatment for 15 minutes in 5% NaOCl and then repeated in sterile Washed water. Immature embryos are aseptically isolated on 3 cm nylon mesh squares (1.5 mm mesh) on A2 medium. A2 medium, adjusted to pH 5.8, is 4.32 g / l of Murashige-Skoog salts, 20 g / l of sucrose, 0.5 g / l of L-glutamine, 2 mg / l of 2,4-D, 100 mg / l casein hydrolyzate, 2 mg / l glycine, 100 mg / l myo-inositol, 0.5 mg / l nicotinic acid, 0.1 mg / l thiamine · HCl and 2.5 g / l gelrites. Embryos are placed in a 2.5 cm solid disc containing a number of about 50. The plates are sealed with Leukopore tape and incubated at 25 ° C in the dark for 2 days. Four hours prior to bombardment, the embryos are transferred to plates containing fresh A2 medium supplemented with 36.44 g / L D-sorbitol and 36.44 g / L D-mannitol. The embryos are transmitted from plate to plate by means of the nylon net on which they sit. The embryos sit on this medium at elevated osmotic concentration for 4 h at 25 ° C in the dark before being bombarded.

Particle-mediated transformation

DNA derived from plasmid pIGPD9 ( 12 ) containing XmaI EPSPS expression cassettes (eg, pZEN9i, ZEN11i, ZEN12i, ZEN14i, etc.) is purified, concentrated (for example, by anion exchange chromatographic or CsCl ₂ gradient densitometric isolation of plasmid DNA from cells of a suitable HisB vector). , RecA host strain of E. coli (eg DH5α: hisB-) after growth to stationary phase in a 5 x A minimal medium (K ₂ HPO ₄ 52.5 g, KH ₂ PO ₄ 22.5 g, (NH ₄ ) ₂ SO ₄ 5 g and sodium citrate · 2H ₂ O 2.5 g per liter) and as concentrated solution (preferably about 1 mg / ml) in sterile water provided DNA as ring-shaped plasmid DNA or alternatively restricted with XmaI to to provide a fragment containing EPSPS expression cassette, and used after purification by agarose gel electrophoresis and electroelution.

Particles are prepared and coated with DNA in a manner similar to that described by Klein et al. 1987, Nature, 327, 70-73. The preparation of DNA-coated particles and the operation of the particle gun is as described in Example 2. Alternatively, the details are as follows. For example, 60 mg of gold or tungsten particles (~ 1.0 μm) in a microcentrifuge tube are repeatedly washed in HPLC grade ethanol and then repeatedly in sterile water. The particles are resuspended in 1 ml of sterile water and distributed in 50 μl aliquots in microcentrifuge tubes. Gold particles are stored at 4 ° C, tungsten particles at -20 ° C. 3 mg of DNA are added to each subset of (thawed) particles and the tubes are vortexed at full speed. While maintaining a near continuous vortex, 50 μl of 2.5 M CaCl ₂ and 20 μl of 0.1 M spermidine are added. After 10 minutes of further vortexing, the samples are centrifuged for 5 seconds in an Eppendorf microcentrifuge, the supernatant is removed, and the particles are washed in successive additions of HPLC grade ethanol. The particles are carefully resuspended in 60 μl ethanol and then distributed in 10 μl aliquots on the surface of each of the macrocarriers to be used in the PDS1000 particle gun.

components The PDS1000 particle gun is made by immersion in 70% ethanol and air drying sterilized. The target plates, made like described above, with about 50 embryos arranged in about 2.5 cm disc are placed 6 cm in front of the brake screen. 1100 psi bursting discs are then used for bombing. Each plate will be once or bombed twice.

bombed Panels are sealed with pore tape and stored at 25 ° C in the Darkness for stored for about 16 h. From the surface of the medium through the helium shockwave dissolved Embryos are obtained and also overnight on fresh plates from the same, supplemented with mannitol and sorbitol A2 medium incubated. The bombarded embryos are then transferred to fresh plates of A2 medium and for 1 week at 25 ° C incubated in the dark before selection.

Selection and regeneration for transformants

After this recovery period, callus-forming embryos are removed from the nets and transferred to A2 2P medium (A2 medium adjusted to pH 5.8 containing 2 mM N- (phosphonomethyl) glycine) at a density of 20 plates / plate. After one week on A2 2P medium, calli are removed on A1 2P medium (A2 medium containing only 1.0 mg / l 2,4-D and 2mM N- (phosphonomethyl) glycine) for 2 weeks and then on A 0.5 2P medium (A2 medium containing only 0.5 mg / L 2,4-D and 2 mM N- (phosphonomethyl) glycine) for another two weeks. If appropriate, the two-week incubation periods are reduced to 1 week and / or the middle step of incubation on A1 2P medium is omitted. Optionally, the selection concentration of N-phosphonomethylglycine is between 0.5 and 10 mM, although 2 mM is preferred. The total time for this period of callus induction with decreasing amounts of 2,4-D in the medium is 2-10 Weeks, preferably 3-6 weeks, and most preferably about 4 weeks.

to favoring a maximum shoot growth and to deter the development of roots, the calli then become transferred to Z-medium. Z medium is A2 medium, but 10 mg / l zeatin instead of 2,4-D contains and also contains 0.1 mM N- (phosphonomethyl) glycine. Optional is N- (phosphonomethyl) glycine in the range of 0.04-0.25 mM. Regenerating calli are used on this medium for one Period of 3 weeks before the subculture saved, which is well-developed shoots cut out become. There's probably just one event on a single callus is generated (which represents a single embryo), the entire Callus removed on a fresh plate and with the cut out Sapling (saplings) saved to ensure that multiple Clones that originate from the same callus, not as separate Events counted become. Calli with only partially developed shoots or without regenerating Sectors are for returned to the Z medium for another three weeks. At the end of this period non-regenerating calli are discarded.

Become saplings kept on Z medium until 4 or more well-developed leaves (the on a length of about 2 cm) have formed. The regenerating plant material is then carefully transferred to plastic tubs containing 0.5 MS medium. 0.5 MS medium at pH 5.8 is 2.16 g / l Murashige-Skoog salts, 15 g / l sucrose, 2.5 g activated charcoal, 2.5 g / l gelrites, 1 mg / l glycine, 50 mg / L myo-inositol, 0.25 mg / L nicotinic acid, 0.25 mg / L pyridoxine · HCl, 0.05 mg / l thiamine · HCl and 0.1mM N- (phosphonomethyl) glycine (optionally from 0.0 to 0.25 mM).

As soon as the plants have rooted can potted them in soil and weaned them or on individual glass tubes are removed, the 0.5 MS (without N- (phosphonomethyl) gycin) and 2.5 g / l of activated carbon. It is preferred that activated carbon present in the rooting medium to any remaining PGRs or to adsorb selection chemicals transferred to the plantlet and to create a dark rooting environment thereby being physiologically abnormal green To avoid roots.

The Callus induction and the first week of regeneration occur 25 ° C in the darkness. The second week of regeneration occurs at low Light at 25 ° C and then subsequent Weeks at about 2500 lux with a 16-hour light period.

Propagation, breeding and Analysis of transformed plant material

method for the production of T1 and other offspring are all professional well known and essentially as in the previous examples described. Method for analyzing the inheritance of resistance to Glyphosate and presence, integrity and expression of transgenes are as in previous examples.

Example 16. Transformation of wheat lines with DNA including an EPSPS expression cassette Electroporation of protoplasts; Selection and regeneration of Plant cells and plants that are resistant to glyphosate

In another example, plasmid or linear DNA comprising an EPSPS expression cassette identical to that used in Examples 12, 13 and 15 is used for direct transformation of protoplasts of a line of wheat that can be regenerated into fertile plants (see. US 5231019 ). Isolated protoplasts of wheat, preferably from leaf tissue or cells in culture (see OL Gamborg and LR Wetter, Plant Tissue Culture Methods, 1975, 11-21), are incubated with approximately 2 x 10 ⁶ protoplasts / ml in 0.4 M mannitol produced at pH 5.8. To this suspension are first added 0.5 ml of 40% w / v polyethylene glycol (PEG) having a molecular weight of 6000 in modified F medium (Nature (1982), 296, 72-74) at pH 5.8 and secondly 65 ml Water containing 15 mg of the desired plasmid or linear DNA and 50 mg calf thymus DNA. The mixture is incubated together at 26 ° C for 30 minutes, occasionally stirred and then diluted in F-Medium (Nature (1982), 296, 72-74). The protoplasts are isolated by low speed centrifugation, taken up in 4 ml of CC culture medium (Potrykus, Harms, Lorz (1979), Theor. Appl. Genet., 54, 209-214) and incubated in the dark at 24 ° C.

Alternatively, and in addition to treatment with PEG, transformation of cereal protoplasts is carried out using further steps of heat shock and / or electroporation (E. Neumann et al., (1982) EMBO J., 7, 841-845). For example, wheat protoplasts are incubated in an aqueous solution of DNA and mannitol, heated to 45 ° C for 5 minutes and then cooled to 0 ° C over a period of 10 seconds. Then polyethylene glycol is added (Mr 3K-8K) until the final concentration is about 8%. G / V is. After careful but careful mixing, the treatment is carried out in an electroporator. The chamber of a dialogue "Porator" (Dialog, Dusseldorf, Germany) is sterilized by rinsing with 70% ethanol and subsequent drying in sterile air. Suspensions of protoplasts (about 2 × 10 ⁶ protoplasts / ml in 0.4 M mannitol + DNA) are adjusted with manganese chloride to a measured electrical resistance of about 1.4 kΩ. 0.4 ml volumes are subjected to three pulses of applied voltages between 1000 and 2000 V at 10 second intervals. The protoplasts thus transformed are then collected and redissolved in CC culture medium.

The Professionals will recognize that many Permutations and variations of these transformation methods are possible and that to For example, a transformation also by increasing the pH to 9.5 and / or increasing the Concentration of calcium ions in the solution in which the transformation carried out will, can be improved.

To 3-14 days subsets of developing cell cultures are transferred to medium, the alternative selection levels of tissue culture grade N- (phosphonomethyl) glycine (Sigma) between 1 and 5 mM (preferably 2 mM). So identified resistant Cell colonies (which show growth at concentrations of glyphosate, at least 2 times higher than those tolerated by untransformed controls are transferred to fresh agar medium, which is also a series of selection concentrations of glyphosate, and as in Example 15 described subcultivated between plates, which are consecutive contain descending concentrations of 2,4-D. Growing resistant Colonies can (by PCR etc.) for the presence of the recombinant DNA become. It may be possible or not, to effect high selection in the callus step. In any case, all growing calli will be taken along.

growing Calli are then transferred to shoot regeneration medium, containing zeatin and N- (phosphonomethyl) glycine, and then rooting medium, exactly as described in Example 15. Fertile transgenic plants, then express the glyphosate-resistant EPSPS synthase regenerated, selected and tested as it is known in the art and in Example 15, using the example given in FIG 11 described analysis method.

Example 17. Procedure for testing EPSPS activity and determination of kinetic constants. Method of testing EPSPS activities in raw plant material and distinguishing the proportion of the totality, which is resistant to glyphosate

EPSPS enzyme assay

Assays are generally performed according to the radiochemical method of Padgette et al. 1987 (Archives of Biochemistry and Biophysics, 258 (2) 564-573) with K ⁺ ions as the main species of the cationic counterion. Assays in a total volume of 50 μl in 50 mM HEPES (KOH) pH 7.0 at 25 ° C contain purified enzyme or plant extract (see below), suitably diluted in HEPES, pH 7.0, containing 10% glycerol and 5 mM DTT , ¹⁴ C-PEP either as a variable substrate (for kinetic determinations) or fixed at 100 or 250 μM and shikimate-3-phosphate (K ⁺ salt) at 2 or 0.75 mM as indicated. Optionally, assays for studies on crude plant extracts may also contain 5 mM KF and / or 0.1 mM ammonium molybdate. Assays are started by addition of ¹⁴ C-phosphonoenolpyruvate (cyclohexylammonium salt) and stopped after 2-10 minutes (preferably 2 minutes) by addition of 50 μl of a solution of 1 part 1 M acetic acid and 9 parts ethanol. After arrest, 20 μl are loaded onto a Synchropak AX100 (25 cm x 4.6 mm) column and assayed using a mobile phase isocratic elution with 0.28 M potassium phosphate pH 6.5 at a flow of 0.5 ml / ml. chromatographed over 35 minutes. Under these conditions, the retention times for PEP and EPSP are approximately 19 and 25 minutes, respectively. A CP 525TR scintillation counter is connected to the end of the AX100 column. It is equipped with a 0.5 ml flow cell and the flow rate of the scintillant (Ultima Flo AP) is adjusted to 1 ml / min. Relative peak areas of PEP and EPSP are integrated to determine the percentage conversion of labeled PEP to EPSP. Apparent K _m and V _max values are determined by fitting least squares fit to a single weight hyperbola using Graphite 3.09b from Erithacus Software Ltd. customized. K _m values are generally ensured using 8-9 concentrations of variable substrate in the range of K _m / 2-10 K _m and triplicate points. Unless specifically stated, data points are included in the analysis only if there is a substrate to EPSPS conversion of <30%.

Shikimate-3-Pi (S3P) is prepared as follows: to 7 ml 0.3 M TAPS pH 8.5, the 0.05 M shikimate, 0.0665 M ATP (Na salt), 10 mM KF, 5 mM DTT, and 0.05 M MgCl ₂ .6H ₂ O, 75 .mu.l of a solution of shikimate kinase with 77 units (μmolmin ^-1) ml ^-1 are added. After 24 h at room temperature, the reaction is terminated by brief heating to 95 ° C. The reaction solution is diluted 50-fold in 0.01 M Tris.HCl pH 9 and chromatographed by anion exchange on Dowex 1 × 8-400 using a 0-0.34 M LiCl ₂ gradient. The S3P fractions are combined, freeze-dried and then redissolved in 7 ml of distilled H ₂ O. 28 ml of 0.1 M Ba (CH ₃ COOH) ₂ and 189 ml of absolute ethanol are then added. This solution is stirred overnight at 4 ° C. The resulting precipitate of tri-barium-S3P is collected and washed in 30 ml of 67% ethanol. The washed precipitate is then dissolved in about 30 ml of distilled H ₂ O. By adding either K ₂ SO ₄ , the K ⁺ or TMA ⁺ salt of S3P is generated as needed. Great care is taken to add a minimal excess of sulfate. The BaSO ₄ precipitate is removed and the supernatant containing the required salt of S3P is freeze-dried. Each salt is weighed and analyzed by ¹ H NMR. Thus produced S3P preparations are according to ¹ H-NMR> 90% pure and contain (according to their masses and integration of ³¹ P-NMR) only small residues of potassium sulfate.

Production of extracts of plant material for suitable for the EPSPS test are

callus or plantlet material (0.5-1.0 g) becomes a fine frozen powder in liquid nitrogen cooled mortar and pestle ground. This powder is in an equal volume a suitable chilled Extraction buffer (e.g., 50 mM HEPES / KOH buffer at pH 7.5, 1mM EDTA, 3mM DTT, 1.7mM "Pefabloc" (serine protease inhibitor), 1.5mM leupeptin, 1.5mM pepstatin A, 10% v / v glycerol and 1% polyvinylpyrrolidone contains) taken up, resuspended, mixed and centrifuged in a refrigerated centrifuge, around debris deposit. The supernatant is on a chilled PD10 column from Sephadex G25 in 25 mM HEPES-KOH buffer at pH 7.5, containing 1 mM EDTA, containing 3 mM DTT and 10% V / V glycerol. Protein becomes by the Bradford method, standardized using Bovine serum albumin, estimated. Part of the extract is in liquid Nitrogen frozen. A part is examined immediately.

Plant extract EPSPS assays are standardly performed as described above with 0.1 mM ¹⁴ C-PEP and 0.75 mM shikimate-3-Pi either in the absence or presence of 0.1 mM N- (phosphonomethyl) glycine. Under these test conditions, it is estimated that the resistant form is inhibited by EPSPS (see below) by <8.5%, while the wild type susceptible form is substantially completely inhibited (> 98%). Thus, the degree of activity (A) observed in the presence of glyphosate is taken as about 92% of the level of resistant enzyme derived from the expression of the transgene, while the degree of susceptible wild-type EPSPS as the total level of EPSPS activity that in the absence of glyphosate, minus the value of A × ca. 1.08. Because it is estimated that the Vmax value of the mutant enzyme is only about one third of the Vmax of the wild-type enzyme (and because the Km values for PEP of both wild-type and mutant forms are about 20 μM or less estimated), the level of expression of the polypeptide of the mutant enzyme relative to the level of expression of the wild-type endogenous EPSPS is estimated to be about 3 times higher than the ratio calculated on the basis of the ratio of their relative observed activities. The overall level of EPSPS polypeptide expression (mutant + wild type) is also estimated by Western (see below).

Example 18. Cloning and expression of wild-type and mutant cDNA, the mature rice EPSPS encoded in E. coli. Purification and Characterization of Wild-type and mutant rice EPSPS.

Expression, purification and characterization of wild-type mature rice EPSPS

Rice EPSPS cDNA is amplified from RNA using RT-PCR derived from the Rice variety Koshigari is isolated using Superscript RT from BRL in accordance with the Manufacturer supplied recommendation is used. PCR is under Use of Stratagene's Pfu turbopolymerase according to the procedures of Manufacturer supplied procedures. The following oligonucleotides be in the amplification reaction and in the reverse transcription steps used.

The PCR product is cloned into pCRBluntII using the Zero Blunt TOPO kit from Invitrogen ned. The sequence of the insert is confirmed by sequencing and it is verified that the predicted open reading frame corresponds to that of the predicted rice EPS rice mature protein with the exception of the presence of an initiating Met. The cloned and verified rice EPSPS sequence is excised using NdeI and XhoI and the purified fragment cloned into pET24a (Novagen) similarly digested. The recombinant clones are introduced into BL21 (DE3), a codon-optimized RP strain of E. coli supplied by Stratagene. The EPSPS protein is expressed in this strain after the addition of 1 mM IPTG to the fermentor medium (LB supplemented with 100 μg / ml kanamycin). The recombinant protein of the correct predicted mass is determined i) on the basis of Coomassie staining of SDS gels from cell extracts and side-by-side comparison with Coomassie-stained gels from extracts of similar E. coli cells which have an empty pET24a. Vector, and ii) identified by Western analysis using a polyclonal antibody raised against previously purified EPSPS plant protein. The mature rice EPSPS protein is purified at about 4 ° C as follows. 25 g of wet weight of cells are washed in 50 ml of 0.1 M HEPES / KOH buffer at pH 7.5 containing 5 mM DTT, 2 mM EDTA and 20% v / v glycerol. After low speed centrifugation, the cell pellet is resuspended in 50 ml of the same buffer but also containing 2 mM "Pefabloc", a serine protease inhibitor. The cells are evenly suspended using a glass homogenizer and then disrupted at 10,000 psi using a Constant Systems (Budbrooke Rd, Warwick, UK) Basic Z cell disruptor. The crude extract is centrifuged at about 30,000 g for 1 h and the pellet discarded. Protamine sulfate (saline) is added to a final concentration of 0.2%, mixed and the solution allowed to stand for 30 minutes. Precipitated material is removed by centrifugation for 30 min at about 30,000 g. Aristar grade ammonium sulfate is added to a final concentration of 40% saturation, stirred for 30 minutes and then centrifuged at -27,000 g for 30 minutes. The pellet is resuspended in ~ 10 ml of the same buffer as used for cell digestion, additional ammonium sulfate is added to bring the solution to about 70% saturation, the solution is stirred for 30 min and recentrifuged to give Pellet which is resuspended in about 15 ml of S200 buffer (10 mM HEPES / KOH (pH 7.8) containing 1 mM DTT, 1 mM EDTA and 20% v / v glycerol). This is filtered (0.45 μm), coated and chromatographed on a K26 / 60 column containing Superdex 200 equilibrated with S200 buffer. EPSPS-containing fractions detected based on EPSPS enzyme activity are pooled and applied to a xk16 column containing 20 ml of HP Q-Sepharose equilibrated with S200 buffer. The column is washed with S200 buffer and then the EPSPS eluted with a linear gradient developing from 0.0 M to 0.2 M KCl in the same buffer. EPSPS elutes within a single peak corresponding to a salt concentration of 0.1 M or below. EPSPS-containing fractions detected based on EPSPS enzyme activity are pooled and loaded on a HiLoad xk26 / 60 column from Superdex 75 equilibrated with Superdex 75 buffer (25 mM HEPES / KOH (pH 7.5) 2 mM DTT, 1 mM EDTA and 10% v / v glycerol). EPSPS later elutes from the column, as might have been expected based on the molecular weight of the putative dimer. This may be due to the interaction of the protein with the gel matrix at low ionic strength. EPSPS-containing fractions identified on the basis of enzyme activity are pooled and applied to a 1 ml column of MonoQ equilibrated with the same Superdex 75 buffer. The column is washed with starting buffer and EPSPS elutes as a single peak in the course of a 15 ml linear gradient developing between 0.0 and 0.2 M KCl. EPSPS is obtained almost pure (> 90%) in this purification step. Optionally, EPSPS is further purified by exchange in Superdex 75 buffer containing 1.0 M (Aristar) ammonium sulfate and applying to a 10 ml column of phenylsepharose equilibrated in the same buffer. EPSPS is eluted as a single early peak during the course of a linear gradient of decreasing ammonium sulfate developing between 1.0 and 0.0 M ammonium sulfate.

Cloning, expression, Purification and characterization of glyphosate-resistant mutant Rice EPSPS

The Rice EPSPS cDNA in pCRBlunt is used as a template for two more PCRs the following primer pairs used for the introduction of specific changes were developed:

The resulting products are gel purified and placed in a tube in equimolar Concentrations given to serve as template for another round of PCRs with the rice 5'- and 3'-oligonucleotides to serve. The resulting products are included in pCRBluntII Using the Zero Blunt TOPO kit from Invitrogen cloned. It will be confirmed, that the DNA sequence of the Inserts and their predicted open reading frame that of the predicted ripe EPSPS protein of rice chloroplast (with the exception of the presence of an introductory Met), and also that the desired changes (the specific mutation from T to I and P to S at specific Positions in the EPSPS sequence). The so cloned and verified rice EPSPS sequence is determined using NdeI and XhoI cut out and the purified fragment in similar Way digested pET24a (Novagen) cloned. The recombinant clones are introduced in BL21 (DE3), a codon-optimized RP strain of E. coli supplied by Stratagene. The EPSPS protein is in this strain after addition of 1 mM IPTG to the fermenter medium (LB, suppl at 100 μg / ml Kanamycin). The recombinant protein of the correct predicted Mass is i) based on Coomassie staining of SDS gels from cell extracts and side-by-side comparison with Coomassie-stained gels of extracts more similar E. coli cells transformed with an empty pET24a vector and ii) by Western analysis using a polyclonal antibody identified against previously purified plant EPSPS protein was generated. This mutated form of rice EPSPS will be in a similar Way to the top for Purified and characterized rice EPSPS wild-type method.

The resulting rice EPSPS mutant form, tested as above in the presence of 2 mM shikimate-3-Pi, has an apparent Vmax of about 10 μmol / min / mg and a Km value for PEP of 22 μM. At 40 μM PEP, the IC ₅₀ value for the potassium salt of glyphosate is about 0.6 mM. The estimated Ki value for potassium glyphosate of the mutant EPSPS is about 0.2 mM.

Example 19. Preparation of antibodies against purified rice EPSPS and western analysis methods

standard procedures for the production of polyclonal antisera in rabbits are used. Rabbits are young females of New Zealand whites. Immunization courses exist out of 4 doses each containing about 100 mg taken at monthly intervals be administered. Each dose in phosphate buffered saline will with complete Freund's adjuvant (dose 1) or incomplete adjuvant (2 ~ 4 doses) and injected at four subcutaneous sites. A pre-blood collection is taken before dose 1 is administered. A test blood sample will be taken 14 days after the second dose, to confirm the immune reaction. Deadline blood collection is taken 14 days after the fourth dose and for used the experiment. A fifth and last dose will be given if at least 6 weeks after the 4th dose and the last blood sample (also for the experiment used) will be 14 days later taken. antibody are tested against (i) purified native Ripe Rice EPSPS from Wild-type (Example 8) as well as (ii) generated against SDS-denatured rice EPSPS polypeptide, which is eluted from a band cut from a 12% SDS gel (the correct position of the protein being determined by side-by-side Coomassie staining) Band is marked exactly).

12% polyacrylamide gels are used for SDS gel electrophoresis and Western blotting. Electrophoresis is performed at a constant current of 100 V for 90 minutes. Gels are blotted overnight against nitrocellulose sheets at 4 ° C at constant 30V. Sheets are blocked in 5% Marvel Marvel buffered saline containing 0.1% Tween (PBS-Tween) for 1 or 2 hours, washed three times in PBS-Tween and in rice EPSPS-Rb1 primary antibody at approximately 1, 3 mg IgG / ml (usually equivalent to a 1: 4000 to 1:20 000 dilution of timed blood collection). These antibody dilutions are placed in PBS (phosphate buffered saline) containing 1% Marvel and 0.05% Tween 20 for 2 hours. Secondary antibody, goat anti-rabbit HRP (Sigma A6154) is used at 1:10 000 or 1:20 000 in PBS containing 0.05% Tween and 1% Marvel. Incubation with secondary antibody is continued for 1 hour, the blot is washed three times in PBS (0.05% Tween), ECL substrate is usually applied for 1 minute and the film is exposed for 10-60 seconds. Negative control blots are blots of (1) preimmune serum at a dilution calculated to give the same [IgG] as in test immunoserants (IgG is routinely purified and quantified from a subset of each serum so that these dilutions can be calculated directly). , and also with (2) immune serum produced against Freund's adjuvant alone. The IgG concentration in control immune sera is adjusted so that controls have a suitable concentration of IgG. To perform this calculation, IgG is determined from ro The antiserum was purified by filtration through a 0.45 μm syringe filter and passed through a 1 ml HiTrap Protein G column (Pharmacia, Cat. No .: 17-0404-01). The bound IgG is eluted from the column with 0.1 M glycine · HCl pH 2.9 and dialyzed against PBS overnight. The IgG concentration is estimated by UV determination (a light path of 1 cm of a 1 mg / ml solution of pure IgG has an absorbance at a wavelength of 280 nm of 1.35). From the IgG yield, a calculation of the IgG concentration in the crude antiserum can be performed and correct dilutions in Western blots calculated accordingly.

Plant tissue samples are prepared, for example, as described in Example 17. Alternatively, for The Western analysis used a much simpler procedure. 100-200 mg Plant tissues for analysis are rapidly (for example, using an Ultraturrax, a beading machine or a glass homogenizer) Homogenized in an equal volume of buffer (similar to Example 7), for 5 minutes in a chilled Eppendorf microfuge centrifuge and a) a small subset of the supernatant analyzed for protein using the Bradford method and b) the majority of the supernatant 1: 1 mixed with Laemli SDS "sample buffer", heated and then ready for the application stored on gels. Typically, SDS disc gels loaded with 10 protein samples in 10 wells. typically, these have 1 to 10 μg raw extracts of plant material for analysis next to a standard curve with 0 to 20 ng pure rice EPSPS. In some cases, Western analysis performed using antibodies that against purified wild-type EPSPS from Brassica napus (expressed and cleaned using similar ones Method as described above) were generated. In this case is the strenght the cross reaction of the antibodies less strong Rice EPSPS (or opposite Wheat or maize EPSPS from endogenous plants) than in the case but antibodies generated against rice EPSPS are used it nevertheless provides a sufficient response for useful quantitative information with respect to the standard curve to be derivable.

Example 20. Isolation genomic DNA from transgenic plant material. PCR analysis. DNA probe production and hybridization. Copy number and integrity of the transgene

genomic DNA is made from plants, plantlets and callus material using, for example, the method by Dellporta et al. (1983) isolated, Chromosome Structure and Function: Impact of New Concepts, 18'th Stadler Genetics Symposium. J.P. Gustafson and R. Appels, eds., New York: Plenary Press, pp. 263-282, or alternatively using a DNAeasy kit (Qiagen). Transgenic callus and Plant cleavage containing the mutated rice EPSPS transgene are prepared using fluorescence PCR using the Oligonucleotide primers SEQ ID NO: 39 and 40 identified specifically for the Mutations in the genomic EPSPS sequence from rice are. The fluorescent dye SYBR-Green, with double-stranded DNA is intercalated, is included in the PCR reaction, so that samples, containing the mutant rice EPSPS gene, by an increase in Fluorescence can be identified in the sample using an ABI3377 machine is detected. Alternatively, the professionals know that the Primers can be labeled fluorescently, and technologies such as "molecular signals" and "scorpions" are available.

A typical PCR reaction, prepared in 96-well optical plates and sealed with optical covers (PE Biosystems), consists of the following:
5.0 μl gDNA template (Qiagen DNeasy preparation)
12.5 μl 2 × SYBR Green premix
2.5 μl 5 pmol / μl stock forward primer
2.5 μl 5 pmol / μl stock reverprimer
2.5 μl of ddH ₂ O
25.0 μl total tvolumen

The following cyclization parameters are followed:
Stage 1: 50 ° C × 2 min
Stage 2: 95 ° C × 10 min
Step 3: 50 cycles of 95 ° C x 15s, 60 ° C x 45s

changes the fluorescence within the samples is removed every 7 seconds Level 3 of the reaction recorded.

For the Southern blotting be about 10 micrograms DNA for used any restriction digest. Genomic DNA is used with appropriate Restriction enzymes (e.g., HindIII) according to manufacturer's instructions (e.g. Promega) digested. Restriction enzymes are selected which both inside and outside cut the mutated EPSPS sequence. DNA is being used of TAE (0.04 M Tris-acetate, 1 mM EDTA) 0.8% agarose gels. Southern blotting is according to procedure carried out, which are disclosed by Sambrook et al., 1989, using of HyBond N + nitrocellulose blotting membrane (Amersham Pharmacia). The DNA is using the membrane by UV exposure networked.

For the generation DNA fragments used for specific probes are purified by purification isolated from gels from restriction digests of plasmid DNA or by PCR generated. For example, a 700 bp fragment, the intron 1 of the rice EPSPS gene, generated by PCR using the primers as shown below.

Such probes are labeled with ³² P using the random priming method, for example with Ready To Go DNA tag beads (Amersham Pharmacia), and purified using, for example, MicroSpin G-25 columns (Amersham Pharmacia).

blots of DNA gels are at 65 ° C in 5x SSC, 0.5% SDS, 2X Denhardt's solution, 0.15 mg / ml denatured salmon sperm DNA prehybridized for at least one hour. The blot is then freshened with denatured probe at 65 ° C for 16-24 h prehybridization solution hybridized. Membranes are blotted dry and used visualized by autoradiography.

If Southern blotting is a single integration event of the transgene at a single location indicated by the hybridization the probe with only a single restriction fragment of specific size, then the result is used by rehybridizing the blot confirmed an alternative probe. For controls Untransformed material is used. In addition, the blot can continue probed with hybridization probes specific for others Regions of the transgenic construct are (for example, the promoter, 5'-UTR intron or Upstream enhancer sequences) for integrity of the construct. In addition, in case that the Agrobacterium transformation used, specific probes used to the present or Absence of DNA display that extends across the right and left Edge of the super-binary Vector extends beyond.

SEQ ID NO: 43 rice EPSPS genomic (from ATG - WT sequence)

SEQ ID NO: 44 rice EPSPS genomic (from ATG including double mutant - shown)

SEQ ID NO: 45 FMV amplifier

SEQ ID NO: 46 CaMV35S enhancer 1

SEQ ID NO: 47 CaMVS35S enhancer 2

SEQ ID NO: 48 maize polyubiquitin enhancer

SEQ ID NO: 49 rice actin enhancer

SEQ ID NO: 50 rice GOS2 enhancer

SEQ ID NO: 51 Barley plastocyanine enhancer

SEQ ID NO: 52 rice genomic G1 EPSPS (to ATG)

SEQ ID NO: 53 rice genomic G3 EPSPS (to ATG)

SEQ ID NO: 54 rice genomic G2 EPSPS + maize Adh1 intron

SEQ ID NO: 55 maize Adh1 intron

sequence Listing

Claims (16)

A polynucleotide comprising the following components in the 5 'to 3' direction of transcription: (i) at least one transcriptional enhancer which is the amplifying region upstream of the transcriptional start of the sequence from which the enhancer is obtained; the amplifier as such not acting as a promoter either in the sequence in which it is endogenously encompassed, or when heterologous as part of a construct; (ii) the rice EPSPS gene promoter; (iii) the rice genomic sequence encoding the rice EPSPS chloroplast transit peptide; (iv) the genomic sequence encoding the rice EPSPS; (v) a transcription terminator; wherein the rice EPSPS coding sequence is modified such that a first position is mutated such that the remainder at this position is Ile instead of Thr, and that a second position is mutated such that the remainder at this position is Ser instead of Pro is wherein the mutations are introduced into EPSPS sequences comprising the following conserved region GNAGTAMRPLTAAV in the wild type enzyme such that the modified sequence is GNAGIAMRSLTAAV. Polynucleotide according to Claim 1, wherein the reinforcing Region comprises a sequence whose 3 'end is at least 40 nucleotides upstream of the next Transcriptional starts of the sequence from which the amplifier is obtained becomes. Polynucleotide according to Claim 1, wherein the reinforcing Region comprises a sequence whose 3 'end is at least 10 nucleotides upstream of the first nucleotide of the TATA consensus sequence of that sequence is out of the amplifier is obtained. Polynucleotide according to one of the claims 1 to 3, comprising first and second transcriptional enhancers. Polynucleotide according to one of the claims 1 to 4, wherein the 3'-end of the amplifier or first amplifier between 100 to 1,000 nucleotides upstream of the codon, which is the start of translation of the EPSPS transpeptide corresponds, or, if the polynucleotide according to claims 1 to 4 additionally a Intron in the 5'-untranslated Region of the first nucleotide of the intron in the 5 'untranslated region Region is. Polynucleotide according to one of the claims 1 to 5, wherein the region upstream of the promoter from the rice EPSPS gene at least one amplifier which originates from a sequence upstream of the Transcription launches of either the CaMV35S or FMV35S promoter is. Polynucleotide according to Claim 6, which is in the 5'- to 3'-direction one first amplifier, the one transcriptional enhancer Region originating from a sequence upstream of the Transcriptional starts of the FMV35S promoter, and a second amplifier comprising a transcriptional enhancement region which comes from a sequence upstream of the transcription start of the CaMV35S promoter. Polynucleotide according to one of the claims 1 to 7, wherein 5 'of rice genomic sequence containing the rice EPSPS chloroplast transit peptide encodes a non-translated region that contains a sequence which functions as an intron. Polynucleotide according to one of the claims 1 to 8, which further includes regions that encode proteins that Plant material that contains it, at least one of the following agronomically desirable characteristics can lend: Resistance to insects, fungi, viruses, bacteria, nematodes, stress, Dehydration and herbicides. Vector containing the polynucleotide according to each preceding claim. Plant material containing the polynucleotide according to a the claims 1 to 9 or the vector according to Claim 10 was transformed, or plant material with the polynucleotide according to one of the claims 1 to 9 or the vector according to Claim 10, and further comprising a polynucleotide transformed or transformed, comprising regions, encode the proteins, the plant material containing them, at least confer one of the following agronomically desirable characteristics can: Resistance to insects, fungi, viruses, bacteria, nematodes, stress, Dehydration and herbicides. Morphologically normal, fertile whole plants regenerated from the material according to claim 11, their progeny seeds and parts, or morphologically normal, fertile whole Plants comprising the polynucleotide according to any one of claims 1 to 9 and resulting from the crossing of plants regenerated from material transformed with the polynucleotide according to any one of claims 1 to 9 or the vector according to claim 10, and plants that have been transformed with a polynucleotide comprising regions that encode proteins that can confer plant material containing them at least one of the following agronomically desirable traits: resistance to insects, fungi, viruses, bacteria, nematodes, stress, dehydration, and herbicides , the progeny of the resulting plants, their seeds and parts. Method for the selective control of weeds in one Field, where the field is weeds and plants or offspring according to Claim 12, wherein the method comprises applying a glyphosate herbicide on the field in an amount sufficient to combat the Weeds without substantially affecting the plants. Process according to the preceding claim, further comprising the application to the Field either before or after application of the glyphosate herbicide of one or more of the following: a herbicide, insecticide, Fungicide, nematocide, bactericide and antiviral agent. Process for the production of plants that are substantial tolerant of or substantially resistant to glyphosate herbicide, comprising the following steps: (i) transforming plant material with the polynucleotide according to one of the claims 1 to 9 or the vector according to Claim 10; (ii) selecting the material so transformed; and (iii) regenerating the thus selected material morphologically normal, terrible whole plants. Method for regenerating a fertile transformed Plant to contain foreign DNA, including the following Steps: (a) generating regenerable tissue from the to transforming plant; (b) transforming the regenerable Tissue with the foreign DNA, wherein the foreign DNA comprises a selectable DNA sequence, wherein the sequence acts as a selection device in a regenerable tissue; (C) between about 1 day and about 60 days after step (b), placing of the regenerable tissue of step (b) into a medium containing shoots from the tissue, wherein the medium further comprises a compound contains which is used for the selection of regenerable tissue, the contains the selectable DNA sequence to identify or to allow selection of the transformed regenerated tissue; (D) after at least one sapling was formed from the selected tissue of step (c), transferring of the sapling on a second medium, which can produce roots from the sapling to one plantlets where the second medium is optionally the compound contains; and (e) cultivating the plantlet to a fertile transgenic plant, wherein the foreign DNA is on Progeny plants are passed on in Mendelian manner, wherein it is an optional step between step (b) and step (c) placing the transformed material on callus-inducing Medium, characterized in that the foreign DNA is the polynucleotide according to one of the claims 1 to 9 or the selectable DNA sequence that is derived from the foreign DNA comprising, the polynucleotide according to one of claims 1 to 9 and the compound is glyphosate or a salt thereof.