DD279269A5 - METHOD FOR PRODUCING A HERBICIDE TOLERANT PLANT - Google Patents

Abstract

The invention relates to a process for the preparation of a herbicide-tolerant plant. Using genetic engineering methods, the enzyme activity of glutathione-S-transferase in the plants is significantly increased. The process according to the invention consists in transforming a plant with a recombinant DNA molecule which is capable of transferring herbicide tolerance, which is based on the detoxification of the herbicide, to a plant.

Description

Process for the preparation of a herbicide-tolerant plant Field of application of the invention

The invention relates to a method of producing a herbicide-tolerant plant using recombinant DNA technology, said herbicide tolerance resulting from the detoxification of the corresponding herbicide.

Characteristic of the known state of the art

The glutathione S-transferases (EC 2.5.1 * 18) belong to a class of enzymes that play an important role in the xenobiotics detoxification process. These enzymes are found in most living organisms, including microorganisms, plants, insects and higher animals. Although each of the glutathione-S-transferase (GST) enzymes within this class differs from the other, "all of these enzymes have overlapping substrate specificity. See: Dakobi et al. , "Glutathione S-transferase J Binding and Physical Properties," in Glutathione: Metabolism and Function , ed. I. Arias and W Oakoby (Raven Press, New York, 1976); Reddy et al. , "Purification and Characterization of Individual Glutathione S-Transferase from Sheep Liver," Archives of Biochem. and biophys. , 224 : 87-101 (1983)

Of the many GST functions, catalysis of the conjugation of glutathione with electrophilic compounds is of particular interest. H. Rennenberg, "Glutathione

Metabolism and Possible Biological Roles in Higher Plants "" Phytochemlstry "21; 2771-2781 (1982); see: Meister and Tate," Glutathione and Related Gamma-Glutamyl Compounds: Biosynthesis and Utilization, "in Ann." Rev. "Biochem . 45 : 560-604 (1976) Numerous xenobiotics, including herbicides, pesticides and insecticides belong to the electrophilic compounds In the course of the conjugation of glutathione and an electrophilic compound, the sulfhydryl group of glutathione interacts with the electrophilic center of this compound Glutathione acts as a nucleophile, and this conjugation is catalyzed by a specific GST enzyme (Rennbeir> supra ).

For plants, this reaction is of particular importance as it offers a means of detoxifying xenobiotic agents. The conjugated, electrophilic xenobiotic compound becomes water-soluble and thus no longer toxic to the plant.

Object of the invention

The invention provides a process for producing plants which are tolerant of herbicides by significantly increasing the enzyme activity of glutathione-S-transferase in the plants using genetic engineering methods.

Explanation of the essence of the invention

The object of the invention is to provide "plants" which are tolerant to herbicides.

- 2a -

ΖΪ9Ζ19

The present invention relates to a method for producing herbicides of tolerant plants, wherein the herbicide tolerance is transferred by recombinant DNA molecules and the herbicides are datoxified by production of proteins.

Known detoxification mechanisms include the conjugation of glutathione to electrophilic compounds, a reaction catalyzed by glutathione S-transferase; the conjugation of D-amino acid to 2,4-D.i.chlorphenoxyacetic acid (2,4-D) as well as the hydroxylation and carbohydrate conjugation of sulfonylureas.

The recombinant DNA molecule consists of various sequence segments derived from genomic DNA and / or cDNA and assembled into a complete glutathione S-transferase-encoding DNA sequence.

The DNA sequence encoding a glutathione S-transferase polypeptide is derived from a mammal. The DNA sequence coding for a glutathione-S-transferase PoIypoptid the rat "has the following nucleotide sequence:

40 50 60

ATGCCTATGATACTGGGATACTGG MPMI LGYW

70 80 90 100 110 120

AACGTCCGCGGGCTGACACACCCGATCCGCCTGCTCCTGGAATACACAGACTCAAGCTAT NVRGLTHPIRLL LEYTDSSY

130 140 150 160 170 180

GAGGAGAAGAGATACGCCATGGGCGACGCTCCCGACTATGACAGMGCCAGTGGCTGAAT EEKRYAMGDAPDYDRSQWLN

- 2 B -

190 200 210 220 230 240 GAGAAGTTCAAACTGGGCCTGGACTTCCCCAATCTGCCCTACTTAATTGATGGATCGCGC EKFKLGLDFPNLPYLIDGSR

250 260 270 280 290 300 AAGATTACCCAGAGCAATGCCATAATGCGCTACCTTGCCCGCAAGCACCACCTGTGTGGA KITQSNAIMRYLARKHHLCG

310 320 330 340 350 360 GAGACAGARGAGGAGCGGATTCGTGCAGACATTGTGGAGAACCAGGTCATGGACAACCGC ETESERIRADTVENQVMDNR

370 380 390 400 410 420 ATGCAGCTCATCATGCTTTGTTACAACCCCGACTTTGAGAAGCAGAAGCCAGAGTTCTTG MQLIMLCYNPDFEKQKPEFL 4'iO 440 450 460 470 480

aagaccatccctgagaagatgaagctctactgagttcctgggcaagcgaccatggttt ktipekmklyseflgkrpwf

490 500 510 520 530 540

gcaggggacaaggtcacctatgtggatttccttgcttatgacattcttgaccagtaccac agdkvtyvdflaydildqyh

550 560 570 580 590 600

ATTTTTGAGCCCAAGTGCCTGGACGCCTTCCCAAACCTGAAGGACTTCCTGGCCCGCTTC IFEPKCLDAFPNLKDFLARF

610 620 630 640 650 660

gagggcctgaagaagatctctgcctacatgaagagcagccgctacctctcaacacctata eglkkisaymkssrylstpi

670 680 690 700 710 720

ttttcgaagttggcccaatggagtaacaagtag fsklaqwsnk

This DNA sequence may also have the following nucleotide sequence:

- 2c -

CTG TAC AGC ATG 1 10 GCT CCC GAC DID GAC 13 O CAG Leu Tyr Ser mead GGG GAT Ala Per Asp Tyr Asp AGA AGC gin 150 Gly Asp 1 70 bad Ser TAC AGT GAG AAG CTG GGC CTG GAC TTC CTG TGG Tyr Ser Glu Lys TTC AAA Leu Gly Leu Asp Phe CCC AAT Leu Trp 30 19 O Phe Lys 210 Per Asn CTG TTA ATT GAT CAC AAG ATC ACC CAG GCC CCC Leu Leu He Asp GGG TCA His Lye He Thr gin AGC AAT Ala Per Gly Ser 25 0 Ser Asn 270 2 GAG CGC TAC CTT AAG CAC AAC CTT TGT ACA ATC Glu bad Tyr Leu GGC CGG Lys His Asn Leu Cys GGG GAG Thr He Gly bad Gly Glu ACC GAG AGG ATT 2 90 GAC GTT TTG GAG AAC 31 O ATG GAG Thr Glu bad He CGT GTG Asp Val Leu Glu Asn CAG GCT mead Glu 330 bad Val 3 50 Gin Ala AGA CGC CTA CAG ATG GTC TGC TAC AGC TTT GAC bad bad Leu gin TTG GCC mead Val Cys Tyr Ser CCT GAC Phe Asp 10 37 O Leu Ala 390 Per Asp CTT AAG AAG CCA TTA GAG GGT CTC CCT ATG GAG Leu Lys Lys Per GAG TAC Leu Glu Gly Leu Per GAG AAG mead Glu Glu Tyr 43 0 Gly Lys 450 4 AAG TAC TCC GAA GGC AAG CAG CCA TGG GGG AAG Lys Tyr Ser Glu TTC CTG Gly Lys gin Per Trp TTT GCA Gly Lys Phe Leu Phe Ala CAC ATT ACG DID 4 70 TTT CTT GTT TAC GAT 49 O GAT AAC His He Thr Tyr GTG GAT Phe Leu Val Tyr Asp GTC CTT Asp Asn 510 Val Asp 5 30 Val Leu CGT ATA TTT AAG TGC CTG GAC GCC AAC CAA bad He Phe GAA CCC Lys Cys Leu Asp Ala TTC CCA Asn gin Glu Per Phe Per

55 O 570

CTG AAG GAC TTC GTG GCT CGG TTT GAG GGC CTG AAG AAG ATA TCT Leu Lys Asp Phe VaI Aia Arg Phe GIu GIy Leu Lys Lye He Ser

5 90 61 0 630

GAC TAC ATG AAG AGC GGC CGC TTC CTC TCC AAG CCA ATC TTT GCA Aep Tyr Met Lys Ser Gly Arg Phe Leu Ser Lye Pro He Phe AIa

6 50

AAG ATG GCC TTT TGG AAC CCA AAG TAG Lys Met Ala Phe Trp Asn Pro Lys End

This DNA sequence may also have the following nucleotide sequence:

10 30

ATG CCT ATG ACA CTG GGT TAC TGG GAC ATC CGT GGG CTG GCT CAC Met Pro Met Thr Leu Gly Tyr Trp Asp lie Arg Gly Leu Ala His

50 7 0 90

GCC ATT CGC CTG TTC CTG GAG ACT ACA GAC ACA AGC ACT GAG GAC Ala He Arg Leu Phe Leu Glu Tyr Thr Asp Thr Ser Tyr Glu Asp

1 10 13 O

AAG AAG TAC AGC ATG GGG GAT GCT CCC GAC TAT GAC AGA AGC CAG Lys Lys Tyr Ser Met Gly Asp Ala Pro Asp Tyr Asp Arg Ser Gin

150 1 70

TGG CTG AGT GAG AAG TTC AAA CTG GGC CTG GAC TTC CCC AAT CTG Trp Leu Ser Glu Lys Phe Lys Leu Gly Leu Asp Phe Pro Asn Leu

19 0 210

CCC TAC TTA ATT GAT GGG TCA CAC AAG ATC ACC CAG AGC AAT GCC Pro Tyr Leu Gly Asp Gly Ser His Lys He Thr Gin Ser Asn AIa

2 30 25 0 270

ATC CTG CGC TAC CTT GGC CGG AAG CAC AAC CTT TGGGGG GAG ACA lie Leu Arg Tyr Leu Gly Arg Lys His Asn Leu Cys Gly Glu Thr

- 2β -

2 90 31 0

GAG GAG GAG AGG ATT CGT GTG GAC GTT TTG GAG AAC CAG GCT ATG Glu GIu Glu Arg lie Arg VaI Asp VaI Leu Glu Asn Gin Ala Met

330 3 50

GAC ACC CGC CTA CAG TTG GCC ATG GTC TGC TAC AGC CCT GAC TTT Asp Thr Arg Leu Gin Leu Ala Met VaI Cys Tyr Ser Pro Asp Phe

37 O 390

GAG AGA AAG AAG CCA GAG TAC TTA GAQ GGT CTC CCT GAG AAG ATG Glu Arg Lye Lys Pro Glu Tyr Leu Glu GIy Leu Pro Glu Lye Met

4 10 43 0 450 AAG CTT TAC TCC GAA TTC CTG GGC AAG CAG CCA TGG TTT GCA GGG Lye Leu Tyr Ser Glu Phe Leu GIy Lys Gin Pro Trp Phe Ala Gly

4 70 49 0

AAC AAG ATT ACG TAT GTG GAT TTT CTT GTT TAC GAT GTC CTT GAT Asn Lys lie Thr Tyr VaI Asp Phe Leu VaI Tyr Asp VaI Leu Asp

510 5 30

CAA CAC CGT ATA TTT GAA CCC AAG TGC CTG GAC GCC TTC CCA AAC Gin His Arg Phe Glu Pro Lys Cys Leu Asp Ala Phe Pro Asn

55 0 570

CTG AAG GAC TTC GTG GCT CGG TTT GAG GGC CTG AAG AAG ATA TCT Leu Lys Asp Phe VaI Ala Arg Phe Glu Gly Leu Lys Lys lie Ser

5 90 61 O G30 GAC TAC ATG AAG AGC GGC CGC TTC CTC TCC AAG CCA ATC TTT GCA Asp Tyr Met Lys Ser Gly Arg Phe Leu Ser Lys Pro lie Phe Ala

6 50

AAG ATG GCC TTT TGG AAC CCA AAG TAG Lys Met Ala Phe Trp Asn Pro Lys End

The DNA sequence may also have the following nucleotide sequence:

A GAC CCC AGC ACC ATG CCC ATG ACA CTG GGT TAC TGG GAC ATC mead Per mead Thr Leu Gly Tyr Trp Asp He 5 0 70 CGT GGG CTA GCG CAT GCC ATC CGC CTG CTC CTG GAA TAC ACA GAC bad Gly Leu Ala His Ala He bad L.3U Leu Leu Glu Tyr Thr Asp 90 11 0 130 TCG AGC DID GAG GAG AAG AGA TAC ACC ATG GGA GAC GCT CCC GAC Ser Ser Tyr Glu Glu Lys bad Tyr Thr mead Gly Asp Ala Per Asp 1 50 17 O TTT GAC AGA AGC CA3 TGG CTG AAT GAG AAG TTC AAA CTG GGC CTG Phe Asp bad Ser gin Trp Leu Asn Glu Lys Phe Lys Leu Gly Leu 190 2 10 GAC TTC CCC AAT CTG CCC TAC TTA ATT GAT GGA TCA CAC AAG ATC Asp Phe Per Asn Leu Per Tyr Leu He Asp Gly Ser His Lys He 23 0 250 2 ACC CAG AGC AAT GCC ATC CTG CGC DID CTT GGC CGC AAG CAC AAC Thr gin Ser Asn Ala He Leu bad Tyr Leu Gly bad Lys His Asn 70 29 O 310 CTG TGT GGG GAG ACA GAA GAG GAG AGG ATT CGT GTG GAC ATT CTG Leu Cys Gly Glu Thr Glu Glu Glu bad He bad Val Asp He Leu 3 30 35 O GAG AAT CAG CTC ATG GAC AAC CGC ATG GTG CTG GCG AGA CTT TGC Glu Asn gin Leu mead Asp Asn bad mead Val Leu Ala bad Leu Cys 370 3 90 DID AAC CCT GAC TTT GAG AAG CTG AAG CCA GGG TAC CTG GAG CAA Tyr Asn Per Asp Phe Glu Lys Leu Lys Per Gly Tyr Leu Glu gin 41 0 430 4 CTG CCT GGA ATG ATG CGG CTT TAC TCC GAG TTC CTG GGC AAG CGG Leu Per Gly mead mead bad Leu Tyr Ser Glu Phe Leu Gly Lys bad

- 2g -

50 TGG TTT GCA GGG GAC 47 O ACC TTT GTG GAT TTC 490 GCT CCA Trp Phe Ala Gly Asp AAG ATC Thr Phe Val Asp Phe ATT Ala Per 5 10 Lys He 53 O He GAT GTT CTT GAG AGG GTG TTT GAG GCC ACG CTG TAC Asp Val Leu Glu bad AAC CAA Val Phe Glu Ala Thr TGC Leu Tyr 550 Asn gin 5 70 Cys GCG TTC CCA A /> C CTG TTC ATA GCG CGC TTT GGC GAC Ala Phe Per Asn Leu AAG GAT Phe He Ala bad Phe GAG Gly Asp 59 O Lys Asp 610 Glu 6 AAG AAG ATC TCC GAC AAG TCC AGC CGC TTC CCA CTG Lys Lys He Ser Asp TAC ATG Lys Ser Ser bad Phe CTC Per Leu Tyr mead Leu 30 CCT CTG TTC ACA AAG 65 O ATT TGG GGC AGC AAG 670 GAC AGA Per Leu Phe Thr Lys ATG GCT He Trp Gly Ser Lys DAY Asp bad 6 90 mead Ala 71 O End GAC AGG TGG GCT TTA AGA TAC CAA ATC TCC GTT CCT Asp bad Trp Ala Leu GGA GAA bad Tyr gin He Ser TGG Val Per 730 Gly Glu 7 50 Trp CAA GAG CCC TAA GGA AGG ATT CCT GAG CCC AGC TGC gin Glu Per End Gly GCG GGC bad He Per Glu Per CAG Ser Cys 77 O Ala Gly 790 gin 8th GTT TTC TTC CTT CCT CCA GTC CCC AAG CCT CAG CAT Val Phe Phe Leu Per TCC ATT Per Val Per Lys Per TAC Gin His Ser He Tyr 10 TCA TTT TTT GGT CAT 83 O CCT GCC AAA CAC AGG 850 TTA CTC Ser Phe Phe Gly His CAA ATT Per Ala Lye His bad CTC Leu Leu 8th 70 gin He 89 O Leu GCC CTA GCA ACT CCT DAY CAA AAT AGC CTT AAG AAA Ala Leu Ala Thr Per TTC CAT End gin Asn Ser Leu CTA Lys Lys Phe His Leu

- 2h - Z19ZC3

910 9 30

TTA AAG TGC CCC GCC CCC ACC CCT CGA GCT CAT GTG ATT GGA TAG Leu Lye Cys Pro AIa Pro Thr Pro Arg Ala His VaI He Gly End

95 0 970 9

TTG GCT CCC AAC ATG TGA TTT TGG GCA GGT CAG GCT CCC CGG Leu AIa Pro Asn Met End Leu Phe Trp AIa Gly Gin AIa Pro Arg

90 101 0 1 030

CAG ATG GGG TCT ATC TGG AGA CAG TAG ATT GCT AGC AGC TTT GAC Gin Met Gly Ser He Trp Arg Gin End He Ala Ser Ser Phe Asp

10 50 107 0

CAC CGT AGC CAA GCC CCT CTT CTT GCT GTT TCC CGA GAC TAG CTA HIs Arg Ser Gin AIa Pro Leu Leu Ala VaI Ser Arg Asp End Leu

1 090 11 10

TGA GCA AGG TGT GCT GTG TCC CCA GCA CTT GTC ACT GCC TCT GTA End Ala Arg Cys Ala VaI Ser Pro Ala Leu VaI Thr Ala Ser VaI

113 0 1 150 11

ACC CGC TCC TAC CGC TCT TTC TTC CTG CTG CTG TGA GCT GTA CCT Thr Arg Ser Tyr Arg Ser Phe Phe Leu Leu Leu End Ala VaI Pro

70 119 0 1 210

CCT GAC CAC AAA CCA GAA TAA ATC ATT CTC CCC TTA AAA AAA AAA Pro Asp His Lys Pro Glu End Lie Leu Pro Leu Lye Lye Lys

AAA AAA AAA A Lys Lys Lys

The herbicide-tolerant plants are transformed with a recombinant DNA molecule which codes for an enzyme which is responsible for the detoxification of herbicides

 The recombinant DNA molecules are through a gene sequence

which encodes a glutathione-S-transferase polypeptide, and herbicide-tolerant transformed plants having increased glutathione-S-transferase enzyme activity. In the context of this invention, the plant cell is transformed with a glutathione-S-transferase gene which confers herbicidal tolerance to the plant in the course of its expression or overexpression.

In addition, the present invention also relates to plants which have been regenerated from transformed plant cells and their seeds, moreover also to the progeny of plants regenerated from transgenic plant cells, and mutants and variants thereof.

The invention also relates to chimeric genetic constructs containing a glutathione S-tranferase gene on cloning vehicles and host organisms as well as methods for transferring herbicide tolerance to plants.

In the following, the enclosed figures are to be briefly characterized and explained:

Figure 1 sets forth the sequencing strategy for the pGTR200 cl? NA insert of Υ ^ 200, a rat liver GltJtathion S-transferase gene.

Figure 2 shows the plasmid pCIB710, an E. coli replicon comprising the promoter for the CaMV 35S SNA transcript and its terminator, and polyA accessory.

V y <J- ί? ') -j ι

Figure 3 shows the construction of plasmid pCIB12, a in E. coli replicon, containing the chimeric gene with the CaMV 35S promoter linked to the glutathione S-transferase cDNA gene from rat liver Y, 200, and the CaMV terminator is linked.

Figure 4A illustrates the construction of the plasmid pCIB14, a broad host range replicator, containing two chimeric genes within a T-DNA border sequence, the chimeric gene consisting of the CaMV βSS promoter labeled with the "glutathione-S" promoter.

Transfe.raae gene Y, 200, the CaMV terminator and kanamycin

Resistance gene, nos-neo-nos, linked iet.

Figure AB shows the completion of a partial clone using the in vivo recombination method.

^F igure 4C shows the completion of a part of clone using the in vitro linking method.

Figure 5 shows fluorescence induction patterns typical of non-transgenic tobacco leaves. The upper curve is obtained after the leaves have taken up 10 M atrazine over a period of 48 hours. The lower curve is obtained when pure buffer solution is taken up.

Figure 6 shows the fluorescence induction patterns obtained in leaves from transgenic pCIB14 tobacco plants after 48 hours of uptake of 10 N atrazine, revealing three classes of responses: (i) no detoxification of the atrazine, upper-grade; (ii) significant detoxification of atrazine, lower curve; (iii) a middle detoxification, middle curve.

Figure 7a shows the construction of plasmids pCIBS. Figure 7b shows the construction of the plasmid pClB4. Figure 7c shows the construction of plasmid pCIB2.

Figure 7d / e describes the construction of the plaamide pCIBlO. Figure 7f / g describes the construction of the plasmid pCID10a.

In the following description, a number of printouts are used which are common in recondite DNA technology as well as in plant genetics.

In order to ensure a clear and consistent understanding of the description and claims as well as the scope of the said terms, the following definitions are set forth:

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

Homologue, 3) gene (s) or DNA : A DNA sequence that encodes a specific product, product or biological function and that is derived from the same species into which said gene is introduced.

Plant promoter · A DNA expression control sequence which ensures transcription of any homologous or heterologous DNA gene sequence in a plant, if operably linked to such a promoter.

Overproducing Plant Promoter (OPP) : Plant promoter capable of expressing, in a transgenic plant cell, the expression of each functional sequence (in) in an auoma β υ (gcmosuei in l'Orni the KNA mim "the

Polypeptide level) which is significantly higher than is naturally observed in host cells not transformed with pm OIM '.

Glutathione-S-Trant; f erase : The definition of this enzyme occurs in a functional manner and includes any glutathione S-transferaae (GST) capable of conjugating glutathione and an electrophilic compound in a given and desired plant catalyze. Thus, this term includes not only the enzyme from the specific plant species involved in the genetic transformation, but also includes glutathione S-transferae (GSTs) from other plant species as well as from microorganisms or mammalian cells, if these GSTs are capable to fulfill their natural function in the transgenic plant cell.

The term GST includes amino acid sequences that are shorter or longer than the sequences of natural GSTs, e.g. functional hybrids or partial fragments of GSTs or their analogues.

Plant ; Any photosynthetic member from the Planta realm characterized by a merobran-coated nucleus, a chromosome-organized genetic material, membrane-coated cytoplasmic organelles, and the ability to perform meiosis.

Plant Cell : Structural and physiological unit of the plant consisting of a protoplast and a ze. Lwand.

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

Plant organ : A delimited and clearly visible differentiated part of a plant, such as root, stem, leaf or embryo.

-7

Herbicide-tolerant plants with increased GS ^T enzyme activity;

The present invention relates to methods for the production of herbicide-tolerant plants whose herbicide tolerance based on detoxification of the herbicide is conferred by recombinant DNA molecules.

Known detoxification mechanisms include the hydroxylation of Ui.i carbohydrate conjugation of sulfonylurea (Hutchison et al. , Pesticide et al. , 1979, PhysJol. , 22 ; 243-249 (1984)), D-araic acid conjugation ur, 2-4 -D (Davidonis et al. , Plant Phys. , 70: 357-360 (3352;) and the conjugation of glutathione to electrophilic compounds catalyzed by glutathione S-transforase.

The present invention relates to herbicide tolerant plants transformed with a rekoaibin DNA molecule which encodes an enzyme responsible for the detoxification of herbicides. The recombinant DNA molecules are characterized by a gene sequence encoding a glutathione S-transferase polypeptide, and herbicide tolerant transformed plants having increased glutathione S-transferase enzyme activity. The glutathione-containing plant cells and plants are transformed by a glutathione S-transferase gene, which upon expression in said plant cell or plant enhances GST enzyme activity and thus confers herbicide tolerance to the plant. In the context of this invention, gene manipulation techniques are used for the modification of these plants.

In the context of the present invention, a "herbicide-tolerant plant" is defined by a plant that survives in the presence of a normally effective herbicidal concentration and preferably exhibits normal growth.

Herbicide tolerance in plants, according to the invention, refers to a detoxification mechanism in the plant, even if the herbicide dungeon or target is still sensitive.

Resistance is the maximum »tolerance to be understood.

Detoxification must be distinguished from other mechanisms for transmitting pheromic herbicide tolerance in which the herbicidal binding site is altered rather than long-sensitive.

In the context of the present invention, the inhibitor inhibitor remains unchanged in the plant, but the herbicide is not bound, since it is e.g. previously detoxified by the GST enzyme.

The term "herbicidal tolerance" as used in the present invention is therefore intended to encompass both tolerance and resistance to herbicides, said tolerance or resistance to the detoxification of herbicides, for example due to increased GST enzyme activity. is due. A herbicide tolerant plant can survive without damage in the presence of certain herbicides which are lethal to herbicidal-sensitive plants or impair their growth or vitality.

In the context of the present invention, herbicides are to be understood as meaning all herbicides which can be detoxified, for example by the formation of conjugates of plant ulutathione or glutathione analogues or homologs, or of any electrophilic compound. Particularly preferred in the context of the present invention are herbicides which have a chlorine radical. Examples of such herbicides, which are not limiting, are in the context of the present invention: triazines, including the chloratrazines, acetamides, including the Chloracßtanilide, sulfonylureas, imidazolinones, thiocarbamates, chlorinated Nitrobem'.ole, diphenyl ether and others. Some specific examples of such herbicides include

- 9 - ZfS2Cf

the atrazine, alachlor, S-ethyl dipropyl thiocarbamate and diphenyl ether. See also: Herbicide Resistance in Plants (H. LeBaron and J. Gresael, ed., 1982).

Some of the herbicides mentioned are potent photosynthetic inhibitors in the plant.

Frear et al. , Phytochemistry , 9: 2123-2132 (1970) (chlorotriazines); Frear et al. , Pesticide Biochem. and Physiol. , 20: 299-310 (1983) (diphenyl ether); Lay et al. , Pesticide Biochem. and Physiol. , 6: 442-456 (1976) (thiocarbamates); and Frear et al. , Pesticide Biochem. and Physiol. , 23: 56-65 (1985) (Metribuzin). Other herbicides lead to the inactivation of enzymes involved in the biosynthesis of amino acids.

Although the term "herbicides" is used to describe these compounds, the use of this term in the context of this invention should not be considered as limiting. For example, there are numerous insecticides and pesticides (used to control diseases, parasites and predators) that have harmful effects on the plant's vitality when applied to the plant. The insecticidal or pesticidal agent may possibly be incorporated into the plant through the leaves, stem or soil via the root system.

In addition, there are numerous xenobiotics that are among the electrophilic compounds that can be conjugated to glutathione in a reaction catalyzed by the GST enzyme activity. These xenobiotic compounds are also included in the present invention.

A specific embodiment of the present invention comprises herbicides which are sensitizers, i. Inhibitors of GST enzyme activity. These sensitizers inhibit the endogenous detoxification mechanism by forming a GST sensitizer conjugate.

-ίο-

The simultaneous application of a herbicide and a sensitizer, eg tridiphrene, to a plant leads to an inhibition of the enzyme-catalyzed conjugation between glutathione and the herbicide. Ezra et al. , "Tridiphane as a Synergist for Herbicides in Corn ( Zea mays ) and Proso Millet (Panicum miliaceum), Weed Science , 33: 287-290 (1985).

In the context of the present invention, therefore, the use of gene manipulation techniques to transfer GST enzyme activity or GST enzyme activity to a plant results in a transgenic plant having increased tolerance to sensitizers such as tridiphane.

For example, a sensitizer and a herbicide may be applied to this catfish in combination with herbicide-sensitive and at the same time herbicide-tolerant plants according to the present invention. This combination of sensitizer and herbicide will usually be toxic to the herbicide-sensitive plants but not to transgenic plants.

Any plant containing glutathione or an analogous compound, such as homo-glutathione, which is susceptible to genetic manipulation using genetic manipulation techniques can be used in this invention. The transgenic plant must also be able to express the GST gene.

In the context of the present invention, plants are to be understood as meaning plant cells, plant protoplasts, plant tissue cultures which can be cultivated and induced to form whole plants, but also plant calli, plant clumps and plant cells as intact constituents of a plant and plant parts

- 11 -

The term "plant" also refers to pollen, which is transformable by means of gene manipulation techniques.

The highest concentrations of glutarium in plants are generally found in the subcellular compartments of plant tissue, such as in the plant plastids, in particular in the chloroplays (Rennberg, H., Phytochemiotry ., 21: 2771-2781 (1982) Glutathione has a gamma-L-glutarayl-L-cysteinyl-glycine structure, and a homologous form of glutathione, homo-glutathione, has been found in some plants and has the structure of gamma-L-glutamyl-L-glutathione. Cysteinyl-bi'ta-Alnnin B [Carnegie, P., Hiochoin J. , £ 9: 459-471 (1963) and Carnegie, P., Biochem J. , £ 9: 471-478 (1963)).

Plants have different amounts of glutathione or homo-glutathione.'So one finds, for example, in various legumes mainly Homo-glutathione, while other legumes preferably contain glutathione. Normally, in cases where one of the two components, either homo-glutathione or glutathione, predominates in the plant, the other component is present only in low concentration. (Race mountain, supra ).

The region encoding the glutathione S-transferase (GST) gene used in accordance with the present invention may be of homologous or heterologous origin relative to the plant cell or plant to be transformed. However, it is in any case necessary that the gene sequence encoded for GST be expressed and result in the production of a functional enzyme or polypeptides in the resulting plant cell. The present invention therefore includes plants which possess either homologous or heterologous GST genes and which express the GST enzyme.

Furthermore, the GST may be derived from other plant species or from organisms belonging to another taxonomic unit, e.g. from microbes or from mammals.

-12-

As previously described, the GST enzyme is a class of multifunctional enzymes. Therefore, it is necessary to select a GST gene which catalyzes the conjugation of glutathione and an electrophilic compound.

Because glutathione acts as a substrate of GST, prior to this invention, it was uncertain whether the GST enzyme, specific for glutathione conjugation, would accept homo-glutathione in transformed plants. Frear et al. , Phytochemistry , 9: 2123-2132 (1970) (shows that GlutatMon-S-Trnsferase is specific for induced glutathione int). The required GST enzyme, which is specific for glutathione, can be identified and selected by using an assay in which the substrate specificity is determined to differentiate the different glutathione S-Vansferases. In a typical assay, the glutathione-specific GST can be characterized by affinity chromatography [Tu et al. , Biochem. and biophys. Research Comm. , 108 ; 461-467 (1982); Tu et al. , J. Biol. Chem. , 258 : 4659-4662 (1983); and Jakoby et al. in Glutathione; Metabolism and Function , (Raven Press, New York, 1976)].

In a specific embodiment of this invention, the GST is a plant GST that is homologous to the plant to be transformed. In another embodiment of this invention, the GST is a plant GST heterologous to the plant to be transformed.

To the plants, the one. GST surplus include corn and sorghum. Another embodiment of the present invention includes a mammalian GST. Mammalian GSTs are known and described in Reddy et al. , Archives of Biochem. and biophys. , 224 : 87-101 (1963) (sheep liver); Tu et al. , J. Biol. Chem. , 258 : 4659-4662 (1983) (rat tissue from the heart, kidney, liver, lung, spleen and testis). The preferred G £ T gene is characterized by the coding region of the GS'f gene from rat liver, in particular by

- η - ZfSZCS

Y, 200 described in Example I and the completed Y, 187 described in Example IB. Another aspect of the present invention relates to the coding region of a GST gene from the rat brain, in particular the cDNA clone GlY. »Which is described in example IE. However, it is also known other genes that can be used in the present invention. See: Mannervik, B., Adv. Enzymol Relat. Areas Mol. Biol ., 52 ··· 357-417 (1985), incorporated by reference into this invention.

The DNA sequence coding for the glutathione-S-transferase can be constructed exclusively from genomic or from cDNA. Another possibility is to construct a hybrid DNA sequence consisting of both cDNA and genomic DNA. In this case, the cDNA may be from the same gene as the genomic DNA, or both the cDNA and the genomic DNA may be from different genes. In any case, however, both the genomic DNA and / or the cDNA, each by itself, can be made from the same or different genes.

If the DNA sequence includes portions of more than one gene, these genes may be from either one and the same organism, from multivariate organisms belonging to more than one strain, variety or species of the same genus, or from organisms containing more than belong to a genus of the same or another taxonomic unit (Reich).

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

Thus, a variety of embodiments are encompassed by the broad concept of this invention.

- 14 -

One embodiment of this invention is characterized by a chimeric gene sequence containing:

(a) a first gene sequence encoding a glutathione S-transferase polypeptide having glutathione S-tranferase activity upon expression of the gene in a given plant cell; and

(b) one or more additional gene sequences operably linked to both sides of the GST coding region. These additional gene sequences contain promoter and / or terminator regions. The plant regulatory sequences may be heterologous or homologous relative to the host cell.

Any promoter and terminator capable of inducing induction of expression of a GST coding region may be used as part of the chimeric gene sequence. Examples of suitable promoters and terminators are e.g. those of the nopaline synthase genes (nos), itr octopine synthase genes (ocs.) as well as the cauliflower mosaic virus genes (CaMV).

An effective representative of a plant promoter that can be used is an overproducing plant promoter. This type of promoter, if operably linked to the gene sequence for the GST, should be able to mediate the expression of said GST in a manner that enhances the transformed plant against a herbicide due to its presence GST enzyme activity is tolerant.

Overproducing plant promoters which can be used in the present invention include the promoter of the small subunit (email subunit) of the soybean ribulose-1,5-bisphosphate carboxylate (Berry-Lowe et al. , J ₁ Molecular and App. Gen. , U 483-498 (1982) 1 and the promoter of the chlorophyll a / b binding proteins These two promoters are

- is -

known to be induced by light in eukaryotic plant cells [see, eg, Genetic Engineering of Plants, to Agricultural Perspective , A. Cashmore, Plenum, New York 1983, pages 29-38; Coruzzi G. et al. , The Journal of Biological Chemistry , 2_58: 1399 (1983) and Dunsmuir, P. et al. , Journal of Molecular and Applied Genetics , 2: 285 (1983)].

The chimeric gene sequence consisting of a glutathione S tranferase gene operably linked to a plant promoter can be spliced into a suitable cloning vector. In general, raan uses plasmid or viral (bacteriophage) vectors with replication and control sequences derived from species compatible with the host cell.

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

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

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

- 16 -

The introduction of DNA into host cells can be carried out by means of methods known per se. For example, bacterial host cells can be transformed after treatment with calcium chloride. In plant cells, DNA can be introduced by direct contact with the protoplasts produced from the cells.

Another way of introducing DNA into plant cells is to place the cells in contact with Viron or me Agrobacteriuro . This can be accomplished by infecting sensitive plant cells or by co-culturing protoplasts derived from plant cells.

These procedures will be discussed in detail below.

For the direct incorporation of DNA into plant cells, a number of methods are available. For example, the genetic material contained in a vector can be micropiped directly into the plant cells using micropipettes for the mechanical transfer of recombinant DNA Material can also be introduced after treatment of the protoplasts with polyethylene glycol in them. (Paszkowski et al. , EMBO J. , 3: 2717-22 (1984)].

Another object of the present invention relates to the introduction of the GST gene into the plant cell by means of electroporation [Shillito et al. , Biotechnology , 3: 1099-1103 (1985); Fromm et al. , Proc. Natl. Acad. Be. USA , £ 2: 5824 (1985)].

In this technique, plant protoplasts are electroporated in the presence of plasmids containing the GST gene.

Electrical impulses of high field strength lead to a reversible increase in permeability of biomembranes and thus enable the introduction of the plasmids, electroporated plant protoplasts

-π-

renew their cell wall, divide and form callus tissue. The selection of the transformed plant cells with the expressed GST enzyme can be carried out with the aid of the previously described phenotypic markers.

The cauliflower mosaic virus (CaMV) can be used in the context of this invention as ale vector for the introduction of the GST gene into the plant (Hohn et al. , In "Molecular Biology of Plant Tumor", Arndomie ProsB, New York, 1982 , Seito 549- "JCiO; llowol I, US Patent Patent No. A, 407,956).

The entire viral DNA genome of CaMV is thereby integrated into a bacterial parent plasmid to form a recombinant DNA molecule that can be propagated in bacteria. After cloning, the recombinant plaemide is cleaved by restriction enzymes either randomly or at very specific nonessential sites within the viral portion of the recombinant plasmid, e.g. within the gene that encodes the transmissibility of the virus encoded by aphids to incorporate the GST gene sequence.

A small oligonucleotide, a so-called linker, which has a single, specific residue. ' The recombinant plasmid modified in this way is cloned again and further modified by splicing the GST gene sequence into a single-site re-repression site.

The modified viral portion of the recombinant plasmid is then excised from the parent bacterial plasmid and used for inoculation of the plant cells or the whole plants.

Another method for introducing the GST genes into the cell utilizes the infection of the plant cell with Agrobacterium tumefaciens , which is transformed with the GST gene. The transgenic plant cells are then under suitable, the

Cultivating cultivating conditions known to those skilled in the art, so that they form shoots and roots and ultimately whole plants result.

The GST gene sequences, for example, can be converted into suitable plant cells with the aid of the Ti plastid of Agrobacterium tumefacions [DeCleene et al. , Bot. Rev. , 47: 147-194 (1981); Bot. Rev., kl \ 389-466 (1976)], The Ti plasmid is transmitted in the course of infection by Agrobacterium tumefaciens to the plant and stably integrated us plant genome. [Horsch et al. , Science , 233 : 496-493 (1984); Fraley et al. , Proc. No t i. Acad. Be. USA , 80: 4803 (1983)].

For plants whose cells are not sensitive to infection with Agrobacterium , one can resort to the co-cultivation of Agrobacterium with the corresponding protoplasts.

Ti plasmids have two regions that are essential for the production of tranformed cells. One of them, the transfer DNA region, is transferred to the plant and leads to the induction of tumors. The other, the virulence-conferring (vir) region, is only for training, but not for maintaining the tumors eating Liull. The transfer DNA region can be increased in size by incorporation of the GST gene sequence without compromising its ability to transfer. By removing the tumor-causing genes whereby the transgenic plant cells remain non-tumorigenic and by incorporating an aelokiionierbaren marker, the modified Ti plasmid can be used as a vector for the transfer of gene design according to the invention into a suitable plant cell.

The vir region effects the transfer of the T-DHA region of Agrobacterium to the genome of the plant cell, regardless of whether the T-DNA region and the vir region are on the same vector or on

- 19 - J? 9It.J

different vectors within the same Agrobacterium cell. A vir region on a chromosome also induces the transfer of T-DNA from a vector: into a plant cell.

Preferred is a system for transferring a T-DNA region from an Agrobacterium to plant cells, characterized in that the vir region and the T-DNA region are on different vectors. Such a system is known by the term "binary vector system" and the vector containing the T-DNA is referred to as a "binary vector".

Any T-DNA containing vector that is transmissible in plant cells and that allows selection of the transformed cells is suitable for use in this invention. Preferred is a vector made from a promoter, a coding sequence and pCIB10.

Any vector having a vir region which effects the transfer of a T-DNA region of Agrobacterium to plant cells may be used in this invention. The preferred vector having a vir region is pCIB542.

Plant cells or plants transformed according to the present invention can be isolated by means of a suitable phenotypic marker, which is part of the DNA in addition to the GST gene. Examples of such phenotypic markers, but not to be considered limiting, include antibiotic resistance markers, such as kanamycin resistance and hygromycin resistance genes, or herbicide resistance markers. Other plipfiotypic markers are known to those skilled in the art and may also be used within the scope of this invention.

All plants whose cells can be transformed by direct introduction of DNA or by contact with Agrobacterium and can then be regenerated to complete plants, the erfindungagemässen process for the preparation of trangener whole

-20- Z79ZG *}

Plants containing the transferred GST gene. There is an ever-growing body of evidence suggesting that, in principle, all plants from regenerated cells or tissues are running, including, but not limited to, all major cereals, sugarcane, sugarbeet, cotton, fruit trees and other tree species, Legumes and vegetables.

Among the target cultures in the present invention include, for example, those selected from the series: Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vlgna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopaia, Brassica, Raphanus, Sina pis , Atropa, Capsicum, Datura, Hyoscyamus, lycopersion, Nicotiana, Solsnum. Petunia, Digitalis, Majorana , Cichorlum, Helianthus, Lactuca, Bromus, Aspaurus, Antirrhinum, Hemerocallis, Neme8Ja, Pelargonium, Panicum, Pennise ': um. Ranunculus, Senecio , Salpiglossis, Cucumia, Browallia. Glycine, LoHum, Zea, Triticum and Sorghum , including those selected from the series: Ipomoea, Paasiflora, Cyclamen, Malus, Prunus, Rosa, Rubua, Populu e, Santalum, Allium, Ix lium, Narcissus, Pineapple, Arachis, Phaseol u u id Pi sum .

The knowledge about the extent to which all these plants can be transformed with the help of Agrobacterium are still limited at present. For example, In'vitro can also be used to transform plant species that are not natural host plants for Agrobacteriuro . For example, monocotyledonous plants, especially the cereals and grasses, are not natural hosts for Agrobacterium .

There is now increasing evidence that monocotyledonone can also be transformed with Agrobacterium so that, using new sclerimantell approaches that are now becoming available, cereals and grassy species are susceptible to transformation [Griraeluy, N , et al. , Na ture , 325; 177-179 (1987)).

- 21 -

Regeneration of cultured protoplasts to whole plants is described in Evane, et al. , Protoplast Isolation and Culture, in Handbook of Plant Cell Culture , U 124-176 (MacMillan Publishing Co. New York 1983); MR Davey, "Recent Developments in Tht Culture and Regeneration of Plant Protoplasts," ProtoplaBts , 1983 - Lecture Proceedings, pages 19-29, (Birkhäuser, Basel 1983); PJ Dale, "Protoplast Culture and Plant Regeneration of Cereals and Other Recalcitrant Crops," in Protoplast s 1983 - Lecture Proceedings, page 31-Al, (Uirkhäuser, Basel 1983); and H. Binding, "Regeneration of Plants", in Plant Protoplasts , pages 21-37, (CRC Press, Boca Raton 1985).

The regeneration differs from plant species to plant species. In general, however, a suspension of transformed ·! Protoplasts, cells or tissues containing numerous copies of the GST genes. On the basis of these suspensions, the induction of embryo formation can subsequently take place. Development of embryos is allowed to proceed to maturity and germination, as is the case with naturally occurring embryos. The culture media usually contain various amino acids and hormones, such as e.g. Auxin and cytokinins. It also proves to be advantageous to add glutamic acid and proline to the medium, especially in species such as maize and alfalfa. The shoot and root formation generally occurs simultaneously. Efficient regeneration depends primarily on the medium, the genotype and the history of the culture. If these three variables are adequately controlled, the regeneration is completely reproducible and repeatable.

Plants which are suitable for the use according to the invention include, for example, species from the genera Lotus, Medicare /, Onobrychis, Trifolium, Trigonella, Citrus, Linum, Manihot, Daucus, Arabldopsis, Bra: sica, Raphanue, Sinapis, Atropa, Capsicum , Datura, Hyo8cyarou8, Lycopereicon, Nicotiana, Solanum, Petunia, Majorana, Cichorium, Hfeiianthus, Lactuca, Asparagus , Antirrhinum, Panicum,

- 22 -

Pennisetum, Ranunculua, Salpiglossis, Glycine, GosBypium, Malus, PrunuB, Rosa, Populua, Allium, Lilium, Narciasus, Pineapple, Arachio, Phaseolua and Pit> um .

After finding out that oryza (rice) can be regenerated from protoplasts into whole plants, it should now also be possible to regenerate other plants of the family Gramineae . The following plants can therefore also be used in the context of the present invention: LoIium , Zea, Triticum , Sorghum and Bromus .

Particularly preferred according to this invention are plants of the genera Nicotana spp. (eg tobacco), Glycine spp. (especially Glycine max , soybean) and Gossypium spp. (Daum wool).

The mature plants grown from transformed plant cells are crossed with themselves for semen production. Some of the seeds contain the genes for increased GST enzyme activity in a ratio that obeys the established principles of heredity. These seeds can be used to produce herbicide tolerant plants. The tolerance of these seeds can be determined, for example, by growth of the seeds in soil containing herbicides. Alternatively, the herbicidal tolerance of the transformed plant can be determined by applying the herbicide to the plant.

Homozygous lines can be obtained by repeated self-fertilization and inbred line production. These inbred lines can in turn be used to develop herbicide tolerant hybrids. In this process will one? Herbicide-tolerant In / .uchtlinie crossed with another inbred line for the production of herbicide-resistant hybrids.

Parts which can be obtained from regenerated plants, e.g. Pigs, seeds, leaves, twigs, fruits and the like are also part of the present invention, provided that

23 -

Contain cid-tolerant cells. Progeny (including hybrid progeny), varieties and mutants of the regenerated plants are also part of the present invention.

Use of GST Gene Constructions and Herbicide Tolerant plants

The previously described GST gene constructs can be used in vectors as intermediates in the production of herbicide tolerant plant cells, plant organs, plant tissues, and whole plants.

The importance of herbicidal plants according to the present invention is apparent. Namely, these plants allow farmers to first plant a herbicide-tolerant crop and then treat the field with the herbicide to control weeds without damaging the crop.

In addition, a herbicide tolerant plant allows farmers to grow crops on areas that have been previously treated with herbicides, for example, in a crop rotation, followed by a naturally tolerant plant following a naturally sensitive plant. These herbicidally treated cultivated areas may contain a "herbicide surplus" in the soil such that they are naturally derived from herbicidal sensitive crops as a result of crop rotation by this herbicide surplus. in the soil, if they did not prove tolerant (Sheets, T., Residue Reviews , 32: 287-310 (1970); Burnside et al. , W eed Science , lj>: 290-293 (1971)). ].

For example, farmers usually grow corn and soybeans in alternating successions. While the maize plants are inherently tolerant of certain herbicides, e.g. various

-24-

Nine triazines, such as atrazine, may damage the more sensitive soybean plants once they have been planted on an area previously treated with herbicides. Using a herbicide tolerant plant according to the present invention avoids damage due to herbicidal excess in the soil (Fink et al. , Weed Science , 17 , 35-36 (1969) (Soybeans); Khan et al. , Weed Research , 2J .: 9-12 (1981) (Oats and Timothy Stocks); Brink.nan et al. , Crop Science , 20: 185-189 (1980) (Oats); Eckert et al. , J. Ranp.e Mf .nit. , Tb: 219-224 (1972) (Whiskey)).

The present invention also includes a method of plant control characterized by comprising a mixed population consisting of a herbicide-sensitive plant, e.g. a weed, and a herbicide-tolerant plant according to the invention with an effective amount for controlling the herbicide-sensitive plant amount of a herbicide brings into contact. Therefore, the method of platelet treatment of herbicidal tolerant plants and herbicidal-sensitive weeds with herbicides on the field, whereby both plant types are simultaneously brought into contact with the herbicide in the course of the treatment operation, is also a graft of the present invention.

Another object of the present invention relates to a previously described method of plant control, which is characterized in that a herbicide and a sensitizer in a Miechpopulation are applied simultaneously to both herbicide-tolerant and h.Bizidsensitive plants.

The term "plant-controlling amount of a herbicide" functionally describes a rate of application of a herbicide which is capable of affecting the growth or development of a particular plant. The application rate can therefore either be kept as small as possible, so that they for a

- 25 -

Decrease or suppression of growth or development is sufficient or "it can be so large" that irreversible damage to the sensitive plant occurs.

The actual application rate depends, on the one hand, on the herbicide used, and on the other plant to be controlled. For dicotyledonous plants and weeds z. B. are usually required application rates between 0.5 and 1.5 kg / ha. For monocotyledonous plants, the required herbicidal concentrations are generally between 0.5 and about 2.0 kg / ha. Various herbicides, such as the sulfonylureas "are known" that they are effective even at very low rates.

The herbicides may be contacted with the respective plants using well-known spraying or spreading techniques. For example, the prepublished leaflet application may be used to control weeds by atrazine in the presence of atrazine-tolerant plants of the present invention.

In the general description of the present invention, reference will now be made, for convenience of understanding, to specific embodiments which are included in the specification for purposes of illustration and which are not limiting unless specifically noted.

exemplary The invention will be explained in more detail below with reference to some examples.

- 25a -

purifies.

The methods used in the following examples are generally described by Maniatis et al., Molecular Cloning, Cold Spring Harbor Laboratory, (1982). Enzymes can be purchased from New Englang Biolabs, unless otherwise noted. They will be used according to the manufacturer's instructions, unless otherwise specified.

-26-

Be IA ; Isolation of GST cDNA Clone Y , 200 Antibodies :

Antisera to Homogeneous Liver-Glutathione-S-Transferases (GSTs) (AFG) are prepared according to previously described procedures [Tu et al. , Nucleic Acids ReB. , K): 5407-5419 (1982)). The IgG fraction is purified on a Protein A-Sopharoae (Pharmacia) column and concentrated by ultrafiltration (Amicon, XM-50 membrane) as described by Kraus and Rosenberg [Kraus & Rosenberg, Proc. Natl. Acad. Be. USA , 9_, 4015-4019 (1982)]. It is then dissolved in 50% glycerol and stored 0.2 mg / ml heparin at -18 ⁰ C.

Isolation of polysomes The livers (about 26 g) of awei male Sprague-Dawley rats

(Body weight about 300 g) are extracted with a Potter Elvehjem homogenizer in 150 ml (final volume) of 50 mM Tris-HCl, pH 7.5, 25 mM MgCl ₂ , 0.25 M sucrose solution containing bentonite (1 mg / ml), tieparin (0.2 mg / ml) and cycloheximide (1 μβ / □) in different aliquots to give a 15% (w / v) homogenate. The Polysomenieolation is performed exactly according to the method described by Kraue and Rosenberg (see above). The yields before and after the

Dialysis amounts to 1389 A, "units or 1208 A ₀ , _n units.

Immobilization of polysome-antibody complexes and elution specific mRNA

1130 Α _ο , ~ polysome units are recovered for immunoinabsorption with anti-GST IgG (7.1 mg). Protein A-Sepharoae affinity chromatography and the elution of bound RNA molecules are performed according to the instructions of Kraus and Rosenberg aupra . The eluted RNA molecules are immediately adapted to 0.5 M NaCl and 0.5% dodecyl sulfate and further purified on an oligo (dT) cellulose column [Aviv & Leder, Proc. Natl. Acad. Be. USA , 69: 1408-1412 (1972); Bantle et al. Anal Biochem. , 72: 413-417 (1976)}. The purified poly (At) RNA molecules are isolated by in vitro translation and immunoprecipitation (Tu et al. , Nucleic Acids Res ., K): 5407-5419 (1982); Pelham and Jackson,

- 27 -

Eur. J. Biochem. , §] _ '. 247-256 (1976)], before being sliced for cDNA synthesis. The immunoprecipitated material is resolved on a dodecyl sulfate polyacrylaraid gel and visualized by fluorography (Laemmli, Nature , 227 ; 680-684 (1970); Swanstrom and Shenk, Anal. Biochem. , 86: 184-192 (1978)).

Isolation of GST cDNA clones

The synthesis of the cDNA is carried out analogously to the method of Okayama and Berg [Okayama and Berg, Mol. Cell Biol. , 2: 161-170 (1982)], except for minor deviations, according to a modification of Gublor and Hoffman [Gubler and Hoffman, Gene, 25: 263-269 (1983) J. For cDNA synthesis, approximately 100-500 ng of poly (A +) RNA from immunoprecipitated polysemes are used. The reverse transcription of the mRNA into the corresponding cDNA is carried out in a 40 μΐ reaction unit containing 50 mM Tris-HCl pH 8.3, 100 mM NaCl, 10 mM MgCl ₂ , 10 mM DTT, 4 mM sodium pyrophosphate, 1.25 mM four ilNTPs, 1800 U / ml RNAsin (Promega Biotec), 100 μg / ml oligo- (dT) 12-18 (Pharmacia / PL) and 3000 U / ml reverse transcriptase (Molecular Genetics).

The reaction mixture is incubated for a period of 25 minutes at a temperature of 43 ⁰ C. Anechlieseend the reaction is terminated by the addition of 2 μΐ 0.5 M EDTA. The reaction mixture is extracted with one part by volume of phenol / chloroform and the aqueous phase is then extracted with half a volume of chloroform. The organic phase is back-extracted again with TE buffer.

The aqueous phases are subsequently combined. After adding one volume of 4 M ammonium acetate, the nucleic acids are precipitated by addition of two volumes of ethanol and then chilled on dry ice for 20-30 minutes. The solution is then warmed to room temperature for 5 minutes and centrifuged for 15 minutes in a Kppenriorf zone ("Myrofugo") at a temperture of 4 ° C. The resulting pellet is dissolved in 25 μM TE buffer. The process of nucleic acid precipitation and recovery

- 28 -

The reaction is repeated as described above by adding ammonium acetate (25 μM, 4 M) and 100 μM ethanol. The pellet thus obtained is then washed with 70% ethanol, dried and dissolved in / 0 μΐ water.

The exchange of the mRNA strand of the mRNAicDMA hybrid is carried out in a 50 μΐ reaction unit containing 20 mM TripHCl pH 7.5, 5 mM MgCl ₂ , 10 mM ammonium sulfate, 100 mM KCl, 0.15 mM beta-NAD, 0.04 mM of the four dNTP's, 20 μΐ of the first strand product, 10-20 μCio "P-dATP, 10 U / ml of E. coli DNA ligase (New England Biolabs), 230 U / ml of DNA Polymeraee (Boehringer Mannheim). contains and 8.5 U / ml E.coli RflAse H (Pharmacia / PL) The incubation time is 90 minutes at a temperature of 12 ° to 14 ⁰ C and a further hour at room temperature The double stranded cDNA is phenol. chloroform. Purified and recovered by ethanol precipitation as described for the Eretstrang product, the final dried pellet is dissolved in 20 μΐ water.

10 μΐ of the double-stranded cDNA are amplified in a total volume of 20 μΐ containing 100 mM potassium cacodylate pH 7.0, 1 mM CoCl ₂ , 0.2 mM DTT, 0.1 mM dCTP and 500 U / ml terminal deoxynucleotide transferase (Pharmacia. PL) with a dCTP attachment. The incubation period is 1 to 2 minutes at a temperature of 37 ⁰ C. Anechliessend 20 μΐ 4 mM EDTA are added and the enzyme by Hicze-inactivated by incubation at 65 ⁰ C for 10 minutes.

PstI-digested and dG-tagged pBR322 (Betheeda Research Labs, Inc.) is added at a molar ratio of about 1: 1 to the dC-containing double-stranded cDNA (ie, about 5-10 fold excess in view of the molecular weight of the vector compared to the assumed amount of cDNA). The DNA-echoic solution (cDNA + vector) is amplified up to. a total DNA concentration of 0.5-2.0 ng / μΐ (0.5 ng is optimal) in the presence of 10 mM Tris-HCl pH 7.5, 1 mM SDTA and 150 mM NaCl. These

- 29 -

Mixture is first incubated for 5 minutes at 65 ⁰ C, followed by the actual renaturation reaction ("annealing") takes place at a temperature of 55-58 ⁰ C over a period of 90 minutes.

E. coli strain MM294 is transformed with the renatured cDNA: vector complex using 5 μΐ DNA per 200 μΐ aliquot of transformation competent cells [Hanahan, J. Mol. Biol. , 155 ; 557-580 (1983)]. The transfer frequency of the control is about 1-2 χ 10 ⁸ transformants per covalently closed circular pBR322 DNA. The transformed cells are plated on LM plates (without magnesium) containing 17 μg / ml tetracycline (Hanahan, supra ). The resulting colonies are assayed for ampicillin sensitivity. Tetracycline-resistant and ampicillin-senive (approximately 50%) colonies are picked for further analysis.

Hybrid-Selected In Vitro Translation of pGTR2G 0 Plaemide DNA molecules from 354 ampicillin-antigenic transformants are purified using an alkaline lysis method [Birnboim and Doyl, Nucleic Acid Research , U 1513-1523 (1979)]. The purified DNA molecules are then digested with PstI to determine the size of the integrated cDNA molecules. Of these, a total of 134 visible cDNA inserts were present after performing agarose gel electrophoresis. Inserts of greater than 800 nucleotides are subjected to further analysis by Southern blot hybridization (Southern, J. Mol. Biol. , 98: 5.03- 517 (1975)] using Y (pGTR261) and Y (pGTR262) as probe molecules (Lai et al. , J. Biol. Chem. , 259 : 5536-5542 (1984); Tu et al. , J. Biol. Chem. , 259 : 9434-9439 (1984)].

12 clones which do not hybridize with these probes are included by means of the hybrid-selected in vlf.ro Translation [Cleveland et al. , Cell , 20: 95-105 (1980)]. One of these negative clones is called pGTR200. Poly (A +) RNA molecules except rat liver, immobilized by pGTR200 DNA immobilized on activated aminophenyl thioether cellulose (APT paper).

- 30 -

are selected, are eluted at 75 ⁰ C and 100 ⁰ C and then used for in vitro translation in the rabbit reticulocyte lysate system. The in vitro tranolation products are immunoprecipitated with the aid of antisera to the total Kattenleber GCTs, followed by a dodecylsulfate-polyacrylamide gel electrophoresis. The immunoprecipitated product has the same mobility as Y,. By pGTR200, no other class of fist unrnroinhpites is oplaktierr. In the past, disruption of the Hvbrid-flakliorli »in vitro TrunnlutionBfxnurimoiiLo with Y and Y clones did not yield products with Y, subunits (Lai et al ., Supra ;

Tu et al. , supra ). Nucleotide sequence of the pGTR200 DNA InBert

The DNA sequence of the οDNA in pGTR200 is determined according to the strategy presented in Figure 1 using the Maxam and Gilbert chemical method [Maxam and Gilbert, Method *? Enzymol. , 65: 499-560 (1980)]. The DNA fragments obtained due to cleavage with various restriction enzymes are labeled at the 3 'end. Each determination was repeated at least once.

The nucleotide sequence is shown below. For the 218 residues of the open reading frame, the single-letter hippet code for amino acids was used [Old and Primrose, Principles of Gene Manipulation , (1985), B. Lacrow's Publications, London, p. 346]. The poly (A) addition, AATAAA is underlined.

10 20 30 40 50 60 CTGAAGCCAAATTGAGAAGACCACAGCGCCAGAACCATGCCTATGATACTGGGATACTGG

MPMILGYW

70 80 90 100 110 120 AACGTCCGCGGGCTGACACACCCGATCCGCCTGCTCCTGGAATACACAGACTCAAGCTAT NVRGLTHPIRLLLEYTDSSY

130 140 150 160 170 180 GAGGAGAAGAGATACGCCATGGGCGACGCTCCCGACTATGACAGAAGCCAGTGGCTGAAT EEKRYAMGDAPDYDRSQWLN

190 200 210 220 230 240 GAGAAGTTCAAACTGGGCCTGGACTTCCCCAATCTGCCCTACTTAATTGATGGATCGCGC EKFKLGI, U FPNLPYLIDGSR

- 31 -

250 260 270 280 290 300 AAGATTACCCAGAGCAATGCCATAATGCGCTACCTTGCCCGCAAGCACCACCTGTGTGGA KIT. QSNAIMRYLARK H HLCG

310 320 330 3A0 350 360 GAGACAGAGGAGGAGCGGATTCGTGCAGACATTGTGGAGAACCAGGTCATGGACAACCGC ETEEERIRADIVENQVMDNR

370 380 390 AOO AlO A20 ATGCAGCTCATCATGCTTTGTTACAACCCCGACTTTGAGAAGCAGAAGCCAGAGTTCTTG MQLIMLCYNPDFEKQKPEFL

A30 AAO A50 A60 A70 A80 AAGACCATCCCTGAGAAGATGAAGCTCTACTCTGAGTTCCTGGGCAAGCGACCATGGTTT KTIPEKMKLYSEFLGKRPWF

A90 500 510 520 530 5A0 GCAGGGGACAAGGTCACCTATGTGGATTTCCTTGCTTATGACATTCTTGACCAGTACCAC AGUKVTYVDFLAYDI LDQYH

550 560 570 580 590 600 ATTTTTGAGCCCAAGTGCCTGGACGCCTTCCCAAACCTGAAGGACTTCCTGGCCCGCTTC IFF. PKCLDAFPNLKDFLARF

610 620 630 6A0 650 660

gagggcctgaagaagatctctgcctacatgaagagcagccgctacctctcaacacctata eglkkisaymkssrylstpi

670 680 690 700 710 720

ttttcgaagttggcccaatggagtaacaagtaggcccttgctacactggcactcacagag fsklaqwsnk *

730 7A0 750 760 770 780 AGGACCTGTCCACATTGGATCCTGCAGGCACCCTGGCCTTCTGCACTGTGGTTCTCTCTC

790 800 8) 0 820 830 8A0 CTTCCTGCTCCCTTCTCCAGCTTTGTCAGCCCCATCTCCTCAACCTCACCCCAGTCATGC

850 860 870 880 890 900 CCACATAGTCTTCATTCTCCCCACTTTCTTTCATAGTGGTCCCCTTCTTTATTGACACCT

910 920 930 9A0 950 960 TAACACAACCTCACAGTCCTTTTCTGTGATTTGAGGTCTGCCCTGAACTCAGTCTCCCTA

970 980 990 1000 1010 1020 GACTTACCCCAAATGTAACACTGTCTCAGTGCCAGCCTGTTCCTGGTGGGGGAGCTGCCC

1030 10AO 1050 1060 1070 CAGGCCTGTCTCATCTTTAATAAAGCCTGAAACACAAAAAAAAAAAAAA

Example I B: Isolation of the Partial GST cDNA Clone Y 187 The cDNA clone Y is obtained using the same method in principle. 187. Y, 187 is a partial ciiNA clone, which is known as am 5'-end sequence encoding 96 nucleotides missing. The

-32- ZfS ZC 9

The nucleotide and amino acid sequence, as well as the sequence for the 96 missing nucleotides and for Yyl87 are shown below:

1 0 30

ATG CCT ATG ACA CTG GGT TAC TGG GAC ATC CGT GGG CTG GCT CAC Met Pro Met Thr Leu GIy Tyr Trp Aep He Arg GIy Leu Ala His

50 7 0 90

GCC ATT CCC CTG TTC CTG GAG ACT ACA GAC ACA AGC ACT GAG GAC Ala lie Arg Leu Phe Leu GIu Tyr Thr Asp Thr Ser Tyr GIu Asp

1 10 13 0

AAG AAG TAC AGC ATG GGG GAT GCT CCC GAC TAT GAC AGA AGC CAG Lyo Lys Tyr Ser Met GIy Asp Ala Pro Asp Tyr Asp Arg Ser GIn

150 1 70

TGG CTG AGT GAG AAG TTC AAA CTG GGC CTG GAC TTC CCC AAT CTG Trp Leu Ser Glu Lys Phe Lys Leu Glu Leu Aap Phe Pro Asn Leu

19 0 210

CCC TAC TTA ATT GAT GGG TCA CAC AAG ATC ACC CAG AGC AAT GCC Pro Tyr Leu He Asp GIy Ser His Lye He Thr Gin Ser Asn AIa

2 30 25 0 270

ATC CTG CGC TAC CTT GGC CGG AAG CAC AAC CTT TGT GGG GAG ACA Ιΐβ Leu Arg Tyr Leu GIy Arg Lye Hie Asn Leu Cys GIy GIu Thr

2 90 31 0

GAG GAG GAG AGG ATT CGT GTG GAC GTT TTG GAG AAC CAG GCT ATG GIu GIu GIu Arg He Arg VaI Asp VaI Leu GIu Aan Gin AIa Met

330 3 50

GAC ACC CGC CTA CAG TTG GCC ATG GTC TGC TAC AGC CCT GAC TTT Asp Thr Arg Leu GIn Leu AIa Met VaI Cye Tyr Ser Pro Asp Phe

37 0 390

GAG AGA AAG AAG CCA GAG TAC TTA GAG GGT CTC CCT GAG AAG ATG GIu Arg Lys Lys Pro GIu Tyr Leu GIu GIy Leu Pro GIu Lya Met

4 10 43 0 450 AAG CTT TAC TCC GAA TTC CTG GGC AAG CAG CCA TGG TTT GCA GGG Lys Leu Tyr Ser Glu Phe Leu Gly Lyo GIn Pro Trp Phe Ala GIy

4 70 4, 0

AAC AAG ATT ACG TAT GTG GAT TTT CTT GTT TAC GAT GTC CTT GAT Asn Lys He Thr Tyr VaI Asp Phe Leu VaI Tyr Asp VaI Leu Asp

510 5 30

CAA CAC CGT ATA TTT GAA CCC AAG TGC CTG GAC GCC TTC CCA AAC GIn Ηΐε Arg He Phe GIu Pro Lys Cys Leu Asp Ala Phe Pro Asn

55 0 570

CTG AAG GAC TTC GTG GCT CGG TTT GAG GGC CTG AAG AAG ATA TCT Inu Lys Ähtp Phe VaI AIa Arg Phe GIu GIy Leu Lys Lys He Ser

5 90 61 0 630 GAC TAC ATG AAG AGC GGC CGC TTC CTC TCC AAG CCA ATC TTT GCA Asp Tyr Met Lye Sev Giy Arg Phe Leu Ser Lys Pro He Phe AIa

- 33 - Z79ZC9

6 50

AAG ATG GCC TTT TGG AAC CCA AAG TAG Lys Met Ala Phe Trp Αβη Pro Lye End

Example IC : The complete one, Y. 187 corresponding clone dip missing nucleotides are added to the co-combination or in vitro by linking the corresponding restriction fragments to ^v ul87. These methods are shown in Figures 4B and 4C.

Recombination can be achieved by the introduction of a genomic DNA clone, as a component of the plasmid colEl (eg pUC8 or pBR322) and a cDNA Klqns, which is located on the plasmid inc Pl (eg pRK29O), in the E. coli strain SR43. (ATCC Accession No. 67217, filed September 22, 1986). The strain SR43 carries the mutations polAl supF183 [Tacon, W. & Sherratt, D., Mol. Gen. Genet. , 147 ; 331-335 (1976)) so that replication of the ColEl plasmid at a restrictive temperature (42 ⁰ C) is inhibited.

A Überfuhrung the SR43-3akterien containing the two plasmids, from a perraissive temperature of 28 ⁰ C to 42 ⁰ C while maintaining the Auswahlseinrbarkeit on antibiotic resistance., Which is on the Plasmid- ColEl (eg Ap -refeistenz of pBR322) allows the selection of bacteria containing a cointegrated form of the two plasmids.

Such cointegrated plasmids come about through recombination of homologous DNA regions.

The isolation of cointegrated plasmids allows the cloning of the entire cDNA sequence as well as the upstream genomic DNA in the form of a single Petl or BamHI fragment.

- 34 -

Restriction of the upstream DNA by BaI31, followed by the addition of a BamHI linker, allows the isolation of clones containing the entire coding region (confirmed by DNA sequence analysis).

The entire coding region is cloned in the form of a BamHI fragment into pCID710, as described in Example IA, and used for plant cell transcription.

The same fragment is cloned into pDR540 for expression in E. coil as described in Example TV, below.

Another possibility for the construction of a complete clone corresponding to Y, 187 'is the linking of appropriate restriction fragments of the cDNA and genomic clones (Figure I).

Plasmid pUC19 contains the 5 'end of the genomic clones spliced in as a PstI-HindIII fragment containing approximately the first 400 bases of the coding sequence.

The 3 'end of the coding sequence of the cDNA can be isolated as a 630 base HinfI fragment from the partial cDNA clone in BR325. The pUC plasmid cleaved at its single Hinfl site and the HinfI fragment carrying the 3 'end of the gene are joined together to restore the entire coding sequence of the GST gene. The complete gene is isolated as a Bgll-PstI fragment.

Isolation of the entire coding region, without the upstream DNA sequences, can be achieved by excising the upstream DNA using BaI31 followed by BamHI linker addition. This BamHI fragment is then inserted into pCIB710 or into pDR540, respectively, according to the information given above.

-35-

Example ID : Hybrid clones

Hybrid clones derived from coding sequences for different genes are in principle constructed in the same manner as before for the combination of the partial cDNA

Clones Y, 187 and the missing nucleotides were described (Beib

IC) by using heterologous genes as the Aiisgangamatorial for the two DNA fragments.

Example IE : Isolation of the GST-eDNA Clone GlY Using the phage expression vector λ gt11 [Young, RA and Davis, RW, Proc. Natl. Acad. Be. USA , 80: 1194 (1983)], a cDNA gene library is prepared starting from a polyCA) RNA previously isolated from rat brain by the method described above (see Example IA).

The cDNA is then isolated by antibody screening using antibodies directed against GST from the rat brain.

The isolation of the RNA as well as the preparation and isolation of the associated cDNA is carried out according to previously known methods, as described, for example, in Young and Davis [Young, RA and Davis, RW, Science , 222 : 778-782 (1983)).

The nucleotide sequence and the associated amino acid sequence of the resulting cDNA clone GlY are shown below:

10 30

A GAC CCC AGC ACC ATG CCC ATG ACA CTG GCT TAC TGG GAC ATC Met Pro Met Thr Leu GIy Tyr Trp Asp lie

5 0 70

CGT GGG CTA GCG CAT GCC ATC CGC CTG CTC CTC GAA TAC ACA GAC Arg GIy Leu Ala His Ala He Arg Lou Lt * u L.ou GIu Tyr Thr Aup

90 11 0 130

TCG AGC TAT GAG GAG AAG AGA TAC ACC ATG GGA GAC GCT CCC GAC Ser Ser Tyr GIu GIu Lye Arg Tyr Thr Met GIy Asp Ala Pro Asp

1 50 17 0

TTT GAC AGA AGC CAG TGG CTG AAT OAG AAG TTC AAA CTG GGC CTG Phe Asp Arg Ser GIn Trp Leu Asn GIu Lys? He Lye Leu GIy Leu

- 36 -

190 2 10

GAC TTC CCC AAT CTG CCC TAC TTA ATT GAT GGA TCA CAC AAG ATC Asp Phe Pro Αβη Leu Pro Tyr Leu I.le Αβρ Giy Ser Hie Lye He

23 0 250 2

ACC CAG AGC AAT GCC ATC CTG CGC ACT CTT GGC CGC AAG CAC AAC Thr GIn Ser Αβη Ala He Leu Arg Tyr Leu Giy Arg Lye Hie Aen

70 29 0 310

CTG TGT GGG GAG ACA GAA GAG GAG AGG ATT CGT GTG GAC ATT CTG Leu Cys GIu GIu Thr GIu GIu GIu Arg He Arg VaI Asp He Leu

3 30 35 0

GAG AAT CAG CTC ATG GAC AAC CGC ATG GTG CTG GCG AGA CTT TGC GIu Asn GIn Leu Met Aep Asn Arg Met VaI Leu AIa Arg Leu Cys

370 3 90

TAT AAC CCT GAC TTT GAG AAG CTG AAG CCA GGG TAC CTG GAG CAA Tyr Aen Pro Asp Phe GIu LyB Leu Lye Pro GIy Tyr Leu GIu GIn

41 0 ATG ATG CGG CTT TAC 430 GAG TTC CTG GGC AAG 4 CTG CCT GGA mead mead bad Leu Tyr TCC Glu Phe Leu Gly Lye CGG Leu Per Gly 47 0 Ser 490 bad 50 GCA GGG GAC AAG ATC TTT GTG GAT TTC ATT CCA TGG TTT Ala Gly Αβρ Lye lie ACC Phe Val Αβρ Phe He GCT Per Trp Phe 5 10 Thr 53 0 Ala CTT GAG AGG AAC CAA TTT GAG GCC ACG TGC TAC GAT GTT Leu Glu bad Αβη gin GTG Phe Glu Ala Thr CyB CTG Tyr Asp Val Val Leu

550 5 70

GAC GCG TTC CCA AAC CTG AAG GAT TTC ATA GCG CGC TTT GAG GGC Asp AXa Phe Pro Asn Leu Lys Asp Phe XIe AIa Arg Phe GXu GXy

59 0 610 6

CTG AAG AAG ATC TCC GAC TAC ATG AAG TCC AGC CGC TTC CTC CCA Leu Lys Lye He Sar Aep Tyr Met Lys Ser Ser Arg Phe Leu Pro

30 65 0 670.

AGA CCT CTG TTC ACA AAG ATG GCT ATT TGG GGC AGC AAG TAG GAC Arg Pro Leu Phe Thr Lye Met Ala He Trp GXy Ser Lye End Αβρ

6 90 71 0

CCT GAC AGG TGG GCT TTA GGA GAA AGA TAC CAA ATC TCC TGG GTT Pro Αρρ Arg Trp AXa Leu GXy GXu Arg Tyr GXn IXe Ser Trp VaX

730 7 50

TGC CAA GAG CCC TAA GGA GCG GGC AGG ATT CCT GAG CCC CAG AGC Cys GXn GXu Pro End GXy AXa GXy Arg IXe Pro GXu Pro GXn Ser

77 0 790 8

CAT GTT TTC TTC CTT CCT TCC ATT CCA GTC CCC AAG CCT TAC CAG Here VaX Phe Phe Leu Pro Ser He Pro VaX Pro Lys Pro Tyr GXn

10 83 0 850

CTC TCA TTT TTT GGT CAT CAA ATT CCT GCC AAA CAC AGG CTC TTA Leu Ser Phe Pha GXy His GXn He Pro AXa Lye Hie Arg Leu Leu

-37-

8 70 89 0

AAA GCC CTA GCA ACT CCT TTC CAT DAY CAA AAT AGC CTT CTA AAG Lye. Ala Leu Ala Thr Pro Phe His End Gin Asn Ser Lau Leu Lye

910 9 30

TTA AAG TGC CCC GCC CCC ACC CCT CGA GCT CAT GTG ATT GGA TAG Leu Lys Cys Pro Ala Pro Thr Pro Arg Ala His VaI lie Gily End

95 0 970 9

TTG GCT CCC AAC ATG TGA TTT TGG GCA GGT CAG GCT CCC CGG Leu Ala Pro Asn Met End Leu Phe Trp Ala GIy Gin Ala Pro Arg

90 101 0 1 030

CAG ATG GGG TCT ATC TGG AGA CAG TAG ATT GCT AGC AGC TTT GAC Gin Met Gly Ser He Trp Arg Gin End He Ala Ser Ser Phe Asp

10 50 107 0

CAC C3T AGC CAA GCC CCT CTT CTT GCT GTT TCC CGA GAC TAG CTA His Arg Ser Gin Ala Pro Leu Leu Ala VaI Ser Arg Αβρ End Leu

1 090 11 10

TGA GCA AGG TGT GCT GTG TCC CCA GCA CTT GTC ACT GCC TCT GTA End Ala Arg Cye Ala VaI Ser Pro Ala Leu VaI Thr Ala Ser VaI

113 0 1 150 11

ACC CGC TCC TAC CGC TCT TTC TTC CTG CTG CTG TGA GCT GTA CCT Thr Arg Ser Tyr Arg Set Phe Phe Leu Leu Leu End Ala VaI Pro

70 119 0 1 210

CCT GAC CAC AAA CCA GAA TAA ATC ATT CTC CCC TTA AAA AAA AAA Pro Asp His Lye Pro GIu End He He Leu Pro Leu Lye Lys Lys

AAA AAA AAA Lys Lye Lye

Example II : Construction of the Plaamld pCIB710

The plasmid pLW111, ATCC No. 40235, consists of the three smaller EcoRI fragments of the BJI strain of Cauliflower Mosaic Virus (CaMV) [Franck et al. , Cell , 21: 285-294 (1980)], which are cloned into pMB9. The plasmid pLWIII is digested with BglII and a 1149 bp fragment (base pairs H6494-7643) isolated. This fragment is spliced into the BamHI site of pUC19. This repression fragment encodes both the promoter of the CaMV 35S RNA and the polyA addition signal (ie the terminator) for this transcript.

- 38 -

In Vitro Mutagene8e

Between this promoter and terminator, a single BamHI restriction site is inserted via in vitro mutagenesis. A 36 Ease oligonucleotide is synthesized which is identical to the corresponding sequence of the CaMV region except for a BamHI restriction site incorporated into base sequence # 7464 in the sequence.

The above mentioned 1149 Pp IgIII fragment is first cloned into M19inp 19, followed by isolation of a single-stranded phage DNA, which is then annealed to the synthetic oligonucleotide.

Using this. Oligonucleotides as primers, a new DNA strand is then first synthesized and then the DNA introduced into the E. coli strain JMlOl IZoller & Smith, DNA 3: 479-488 (198O) J by transfection.

The isolation of the M13 phage with the incorporated BamHI site is performed as described by Zoller & Smith, supr a.

Selection of the desired mutant phage

The 36-base oligonucleotide is labeled in a kinase reaction with ^yl P-ATP. A panel of Ml3 phage plaques obtained by transfection is transferred to a nitrocellulose filter

 Diener Filter is hybridized with the labeled 36-base oligonucleotide and then washed at increasing temperatures. The labeled oligonucleotide attached to the mutant phage is still stable at higher wash temperatures

 One of these elevated temperature stable phages is isolated and sequenced to confirm the presence of the BamHI site.

Construction of pCIB710

Double-stranded DNA isolated from this phage is digested with HindIII and EcoRI. püC19 is also cleaved with HindIII and EcoRI. These two digested DNA molecules then become together

- 39 - 1 * 9161

connected. Traneformants are selected for their ampicillin resistance. One of the transformants resulting from this linkage reaction is pCI3710, a plasmid shown in Figure 2.

Example III ; Construction of plasmids pCIBII, pCIB12, pCIB13 and pCIBIA

1. The plasmid pGTR200 is digested with Haell, PstI and PvuII and the 678 bp Haell / PstI fragraent containing the GST coding sequence isolated from an agarose gel,

2. This Haell / PstI fragment is blunt-ended by treatment with TA DNA polymerase, to which are linked by TA DNA ligase BarnHI linker (dCGGATCCG-New England Biolabs).

3. The plasmid pCIB710 is cut with BamHI and then treated with alkaline phosphatase from Ka.berdarm (Boehringer Mannheim).

A. The BamHI-linked GST fragment described above is spliced into the BaraHI digested plasmid pCIB710, the ligation mixture transformed into the E. coli strain HB101, and the desired transformants selected for their ampicillin resistance. It is possible to characterize both transformants which contain the GST coding sequence with regard to the transcription starting from the CaMV promoter in the correct orientation (pCIB12), as well as those with an opposite orientation (pCIB11).

5. The plasmid pCIB10 (see Example IV) is digested with XbaI and EcoRI.

6. The plasmid pCIBl2 is also digested with the aid of XbaI and EcoRI and the smaller fragment isolated from an Agarote gel.

- 40 -

7. Dan isolated XbaI / EcoRI fragment carrying the chimeric gene is spliced into the previously digested plasmid pCIB10; the entire ligation mixture is subsequently transformed into E. coli HB101. The transformants are selected for their kanamycin resietence. These trun-formants, which contain the GST coding sequence in the appropriate orientation with respect to transcription from the CaMV prorotor, are designated pCIB14 (see Figure 3).

8. Plasmid pCIB11 is similarly manipulated to construct pCIB13. It is pCIB13 "in plasroid, which contains the GST coding region in an orientation that precludes a pirated transcription, starting from the CaMV promo to ·.

Introduction of pCIB13 and pCIB14 in Agrobacterium Purified plasraid DNA of the type pCIB13 or pCIB14 is analyzed with the aid of Tranformation in Agrobacterium tumefaciens Al 36 introduced I Watson et al. , J. Bacteriol. , 123 ; 255-264 (1975) J, which is the Plasmid pCIB542 (see also Example VII, unteji). The Transformants are on kanamycin (50 pg / ral) and on spectinomycin

(25 μg / ml).

Example IV : Injection of Y , 200 GST into the trp-lac expression vector

C yy C C

Plasmid pCIB12 is digested with BamHI to isolate a 7Q8 bp Barn-linked GST gene fragment. The Plaeraid pDR54O (Pharmacia PL Biochemical), a trp-lac Explo sion vector iRussel, DR and Bennett, GN, Gene , 20: 231-243 (1982) J is also digested with BamHI and the GST fragment in the spliced appropriate restriction site. The resulting recombinant plaemide is transformed into E. coli strain JM103. Cultures of the resulting strain are inducible at any time by the addition of 1.0 mM leopropyl-beta-D-thiogalactoside (IPTG).

- 41 -

After induction by IPTG and expression of the Y _b 200 GST gene, the bacterial host is pelleted. The pellet formed is then resuspended in 59 ml of 0.2 M Tris-HCl, pH 8.0, and 1 mM EDTA. To this suspension is added 0.5 ml of 100 mM phenylethylsulfonyl fluoride (PMSF) in ethanol and 5 ml of lysozyme (10 mg / ml) in buffer solution.

The suspension is incubated for about 15 minutes at a temperature of 37 ^U C until the bacterial cells are lysed. The lysed bacterial cells present in the suspension are pelleted again. The supernatant is collected and then dialyzed against 25 mM Trie-HCl, pH 8.0, with 1/100 volume PMSF.

The dialyzed supernatant is then assayed for GST activity. Thereafter, the dialyzed residue is packed on an S-hexylglutathione-agarose affinity column at a flow rate of 0.5 ml / minute. The column is washed with 25 mM Tris-HCl and 0.2 M KCl until the absorbance at 280 nm drops below a value of 0.005. After all unbound material is washed off the column, the bound material is eluted with 25 mM Tris-HCl, 0.2 M XCl (pH 8.0) containing 5 mM S-Hbxylglutathione and 2.5 mM glutathione. The eluted fractions are monitored by their absorbance at 280 nm.

The fractions suspected of containing the purified enzyme are individually challenged with 0.1 M NaPO 4 buffer (pH 6.5) containing PEG to concentrate the fractions, and then the same buffer without PEG, dialyzed.

The volumes in each of the dialyned fractions are registered. For each fraction the concentration and the amount of the sample are calculated and then diluted so that in the end the same proportion in each fraction is present in relation to the starting fraction. Each fraction is examined for enzyme activity.

The determination of enzyme activity is carried out using 1-chloro-2,4-dinitrobenzene (ClDNB) alle substrate. For each reaction, to 5 mM reduced glutathione (30.8 mg / ml in buffer, 0.1 M sodium phosphate, pH 6.5) and GST enzyme is added 1 mM ClDNB (20.2 mg / ml in ethanol).

ClDNB and reduced glutathione are made fresh daily. The reaction unit has a final volume of 1 ml. The reaction is carried out at room temperature and started by addition of ClDNB. The course of the reaction is monitored by means of a Gilford spectrophotonizer at a wavelength of 340 nm. A typical reaction procedure involves 10 to 50 units of GST, with 1 unit corresponding to the conversion of 1 mmol of substrate per minute and ml.

The protein concentration of the enzymatically active fractions in the starting material is determined (Biorad, BSA standard). Each of the enzymatically active fractions is analyzed on a 15% Läramli gel using dilution series of standard enzymes (1.0 μg , 0.3 μg and 0.1 μg) as quantitative standards. Detection of the purified enzyme is by immunoblotting using specific antibodies.

Example V : Construction of pCIB10 and pCIB10a The construction of plasmids pCIB10 and pCIB10a is in the pendent

U.S. Patent Application Serial No. 757,098, filed Jul. 19, 1985, entitled "Transformation of Plastids and Mitochondria", which is incorporated herein by reference.

The following individual steps, which have been described and numbered as follows in the above referenced patent application, are carried out for the construction of the plasrides (see Figure 7a-g):

Construction of pC IB10

15. A T-ONA fragment containing the left border sequence of the plasmid

pTiT37 is expressed by pBR325 (EcoRI29) 1Yadav et al. , Proc. Natl. Acad. Be. USA , | 9: 6322 (1982) J. pBR325 (EcoR129) is cut with EcoRI and the 1.1 kb fragment linked to HindIII linkers (New England Biolabs) ^

16. Plasmid pBR322 (Bolivar et al., Gene, 2:75) is cut with HindIII.

17. The fragment containing the left border sequence mentioned in step 15 is spliced into the HindIII digested plasmid pBR322,

18. The plasmid pBR322 with the left border sequence from step 17 is digested with CIaI and HindIII; The 1.1 kb HindIII / ClaI fragment is isolated (Hepburn et al .

J. Hol. Appl. Genet. , 2: 211 <1983) J.

. 19. The Piasmid pHCl8 (Norrander et al, Gene, U \ 101 (1983), J is cut with HindIII and EcoRI, a 60 Bb extensive polylinker is endraarkiert using T4 polynucleotide kinase and gamma ^ P-dATP and an acrylamide Gel isolated.

20. The plasmid pBR322 is cut with EcoRI and CIaI and the groüsa fragment isolated.

The 60 bp Hindlli / EcoRI polylinker and the 1.1 Kb HindIII / ClaI fragment from EcoRI29 are spliced into the pBR322 plasmid previously cut with CIaI and EccRI to give PCIB5 (step 15-21). see Figure 7a).

22. From Bin6 iBevan, Nucleic Acids Res , Jj: 8711 (1984) J, a chimeric gene coding for kanamycin resistance (noe-nao-nos) is recovered in the form of a Sall / EcoRI fragment.

-44-

23. Plaemid pUC18 is cut with EcoRI and Sail,

24. The Sall / EcoRI fragment containing the chimeric Ten mentioned in step 22 is spliced into the EcoRI and Sail-cut plasmid pUC18.

2 ^ς . The BamHI recognition site in the termination sequence of this chimeric gene is disrupted by cutting with DamHI, supplemented using T4 + DNA l'polymerase and finally recombined.

26. The resulting plasmid is cut with Sstll (Bethesda Research Laboratories) and HindIII.

27. A 1.0 Kb fragment carrying the 5 'part of the nos promoter and the right border sequence of plasmid pTiT37 is isolated by cutting pBR325 (Hind23) with HindIII and SstII,

28. This 1.0 Kb Hindll / Satll fragraent is spliced into the plasmid pUC18 from step 26 to give pCIB4 (step 22-28, see Figure 7b).

29. The plasmid pCIB5, which contains the left DNA border sequence, is cut with Aatll, blunt-ended by treatment with T4 DNA polymerase, and then cut again with EcoRI.

3C. pCIB4 is cut with HindIII, provided dur · ^ treatment with the Kl.inow fragment of E. coli DNA polymerase blunt-ended and then cut with EcoRI.

31. The restriction-derived plaemide pCIB5 from step 29 is linked to the truncated plaemide pCIB4 (step 30) to give pCIB2, a ColEI replicon containing the left and the left

- 45 - inches

contains a right T-DNA border sequence containing a chima'rea kanamycin resistance gene and a polylinker P v. ··· (step 29-31, see Figure 7c).

32. The plasmid pRZ102 iJorgenson et__al. , Mol. Gen. Genet ; 177 ; 65 (1979) J is digested with BamHI and supplemented using Klenow.

33. V.8 is a teilaeiae Alul digestion of the plasmid pA03 [Oka, J. MjI. Biol . , 1_7_4: 217 (198I) J.

34. The Alul digestion product of plasmid pA03 is spliced into plasmid pRZ102 from step 32, whereby the desired trana formants can be selected for their kanaroycin-resistance.

35. The resulting plasmid has the coding sequence of Tn9O3 on a 1.05 Kb BamHI fragment which is isolated after DamHI digestion. This fragment is then treated with Klenow DNA Poylymeraee.

36. The plasmid pRK252, a derivative of the plasmid RK2, which has a wide host range, can be described by Dr. med. Don Helinski at the University of California in San Diego (USA). This plasmid lacks the BglII site found in the star plasmid pRK29O [Ditta et al ., Proc. Natl. Acad. Be. USA , JJ '. 7347 (1980) j.

pRK252 is digested with Smal and Sail, and supplemented with the help of Klenow; the large fragment resulting from this digestion is isolated.

37. The Tn903-containing fragment isolated in step 35 is spliced into the large fragment of pRK252 to give pRK252Km (step 32-37, see Figure 7d).

- 46 -

38. The plaamid pRK252Km is cut with EcoRI, blunt-ended using Klenow and linked to BglII linkers (New England Biolabs).

39. The plasmid pCIB2 is cut with EcoRV and the smaller fragment ,. that contains the right border sequence and the nos-neo-nos sequence is isolated. This fragment is supplemented with Klenow polymerase and linked to Bglll-Linkorn (New England Biolabs).

40. The UrIII Frajjmont from step 39 becomes with the l'lasmid

from step 38 which carries a linker to form pCIB10 (step 38-40, see Figure 7e).

Construction of pCIBlOa i

41. The plasmid pRZ102 iJorgeneen, et al , Mo l gene. Genet ., 177 : 65 (1979) J is blocked with BamHI and filled in using Klenow.

42. partial digestion of the plasmids pA03 10ka et al. , Nature , 276 : 845 C1978) J.

43. The material digested with the aid of AIuI is spliced into the restricted plasmid pRZ102 from step 32, selecting the desired transformants for their kanamycin resietence.

44. The resulting plasmid has the coding sequence of Tn903 on a 1.05 kb BamHI fragment; this fragment is isolated after completion of BamHI digestion and after filling with the aid of Klenow.

45. The plasraid pRK290, a derivative of the plasmid RK2, which comprises a broad host range, can be described by Dr. med. Don Helinski at the University of California, San Diego (USA). pRK29O is digested with Smal and Sail, supplemented with Klenow, and the large fragment resulting from this digestion isolated.

46. The Tn9O3-containing fragment isolated in step 35 is spliced into the large fragment of pRK2 to give pRK29OKm.

47. The plaemide of step 46 is digested with DgIII, supplemented with the help of Klenow, and rejoined to destroy its Bglll site. The result is the plasmid pKR290Km (step 41-47, see Figure 7f).

48. Plaemid pRK290Km is cut with EcoRI, blunt-ended using Klenow and fitted with BglII-Link ™ (New England Biolabs).

49. Plaemid pCIB2 is cut with EcoRV and also annealed with BglII linkers (New England Biolabs).

50. The BglII-linkered plasmid pCIB2 from Schrit is spliced into the vector from step 47 to give pCIB10a (step 48-50, see Figure 7g).

The process steps described under point 41-50 can be carried out in the same way using the plasmid pRK252 instead of pRK290.

Example VI : Construction of pCIB23 and pCIB24, vectors that target the GST enzyme to the chloroplasts of the transformed plants

The plasmid pSRS2.1 containing the 5 "sequence of the small subunit (SSU) of ribulose-bis-phosphate-carboxylase (RuPUC) of soybeans iBerry-Lowe et al. , J. Mol. Appl. Genet. , U 483- 498 (1982)) is available from Dr. Richard Meagher of the Department of Genetics of the University of Georgia at Athens, Georgia 30602 (USA).

- 48 - i? 9 <! T ν

This plasmid is pre-digested with EcoRI. The 2.1 Kb fragment containing the SSU 5 'region from soybeans is isolated from an Agaroee gel. The 2.1 Kb EcoRI fragment is then digested with Ddel and a 471 bp Ddel fragment is isolated. This fragment contains the transit peptide and part of the second exon.

The 471-Db Ddel fragment is treated with Klenow (New England Diolabs DNA Polynurae). A BgIII linker Id treated with kinase (CAGATCTC New England Biolabs®) is linked to this fragment, this BglII fragment is then digested with TaqI, and the resulting TaqI fragments are treated with Klenow (Bethesda Research Labs DNA Polymerase) Fragments were linked to BamHI-Lir.kern previously treated with kinase Id (CGCGGATCCGCG) New England Biolabs J and then purified These BamHI fragments are then digested with Bgl II and Bam HI A Bam HI / Bgl II fragment of approximately 400 Bp containing the SSU 5 ¹ Ragion is binned.

The plasmid pCIBVIO is geochipped with BahaI and then treated with calf intestinal alkaline phosphatides. The 400 bp BamHI / BglII fragrent (see above) is spliced into this plasmid (pCIB710). The linker product is transferred to E. coli HBlOI and the transformants are selected on Ampilli.cin.

In this way one finds transformants carrying the BaraHI / BglII fragment in both orientations. In plasmid pSCR2, the 5 ¹ region of the transit polypeptide is immediately adjacent to the 35S promoter. In contrast, the BamHI / BglII fragment in the plasmid pSCR1 is in the opposite orientation.

The plasmid pCIB12 is digested with BamHI and the 708 bp BaraHI fragment carrying the GST gene is heolated.

The Plaomid pSCR2 is also cut with BamHI and subsequently treated with calf intestinal alkaline phocatase. The 708 Dp BamHI fragment from the plasmid pCIB12 is then inserted into the BamHI-treated plasmid pSCR2. The linkage product is converted into HB101 and the transformants are selected for ampicillin.

One finds transformants that have the GST gene in both directions with respect to the control regiocs at the 5 'end. The clone pCIB22 has the GST gene in a direction suitable for transcription starting from the CaMV promoter. In clone pCIB21, on the other hand, the GST gene has the opposite orientation. pCIB22 is digested with XbaI / EcoRI. The fragment carrying the chimeric gene is isolated from a gel and purified.

The plasmid pCIB10 is also digested with XbaI and EcoRI. The XbaI / EcoRI fragment carrying the chimeric gene is spliced into the previously digested plasmid pCIB10 and the transformants are selected for their kanamycin resistance. The resulting plasmid pCIB24 is a broad host range plasmid containing the chimeric gene in direct association with a chloroplast translate peptide sequence.

The plasmid pCIB23 is constructed starting from the clone pCIB21 instead of pCIB22 and using similar enzymatic steps. This plasimide contains the GST gene in the opposite orientation compared to pCIB24.

These plasmids are transferred into Agrobacterium strains in a manner similar to that previously described for pCIB13 and pCIBH.

Example VI : Construction of the plasmid pCIB542, an Agropin Vir Hider plasmid carrying a spectinomycin resistance gene in the region of the T-DNA

The Ti plasmid pTiBo542 ISciaky, Ώ., Montoya, AL & Chilton, MD, Plasmid , Ij 238-253 (1978) J is of particular interest because agrobacteria containing this plasmid are capable of producing the agronomically important legumes To infect Aifalfa and Soybeans | Hood, EF., Ot al. , Bio / Technolop.y, 2: 702-708 (1904) j.

The construction of a pTiBo542 derivative which is deletion in the region of its T-DNA is described in Hood, Elizabeth E., (1985) Ph.D. thesis; Washington University, St. Ljuis, Mo. (USA). In this construction, designated EHAlOl, the T-DNA is replaced by a kanamycin resistance gene. The starting plasmid of EHAlOl, A281, is ATCC under ATCC no. 53487 deposited.

Starting from EHAlOl, a derivative is constructed in which the kanamycin resistance gene is replaced by spectinomycin resistance. Hood, Washington University, Ph.D. thesis (1985) J, has a 1.7 Kb region homologous to the left side of Bam a of the plasmid pTiBo542 iHood, et al. , (1984) J, as well as an 8 kb region homologous to the right side of Bam2a of pTiBo542, separated by a single EcoRI site. Plasmid pMON30, ATCC No. 67113, contains the spectinomycin / streptomycin resistance gene (spc / str) of Tn7 [Hollingsheaci, S. and Vapnek, D., Plasmid , 1_3: 17-30 (1985)]. The t'lasmid pMON30 is digested with EcoRi; the 5.5 Kb fragment containing the spc / str genes is isolated from an agarose gel and spliced into the EcoRI truncated plasmid pi delta 307. The desired recombination product is selected in the form of a spectinomycin-resistant (50 μg / ml) and a tetracycline-resistant (10 μg / ml) transformant. This plasmid is introduced into Agrobacterium strain A136 / EHA1O1 and selected for its streptomycin resistance, tetracycline resistance and kanaraycin resistance phenotype. Merodiploid Cells ("homogenotes") iMatzke, AJM & Chilton, MD, J. Mol. Ap pl.

- 51 -

Genet ., U 39-49 (198I) J with the EHAIOI plasmid and the spectinomycin plasmid are incubated after introduction of the expulsion plasmid R751-pMG2 [Jacoby, G. et al. , J. Dacterio , '., 127 : 1278-1285 (1976) 1 and selection for gentamycin (50 μg / ml) and spectinomycin ".

The preferred merodiploid cell is characterized by a gentamicin-resistant, spectinoraycin-resistant, tetracycline-sensitive and kanamycin-sensitive phenotype. The structure of the resulting plasmid is confirmed by Southern blot studies.

Example VIII : Test of plants for atrazine tolerance Based on numerous studies, such as

1) the fluorescence induction; and

2) Seedlings' ability to grow at concentrations of atrazine toxic to control or wild-type plants has been shown to produce regenerated tobacco plants bearing the GST gene construction pCIB14 tols ^ ant Bind to atrazine.

The fluorescence induction gives information about the state of the photochemical apparatus of the plant iVoss et al , Weed Science , 32 : 675-680 (1984)]. In the course of this assay, leaf tissue is irradiated with light of a particular wavelength and the resulting fluorescence is registered at a second wavelength. Figure 5 shows a pattern of fluorescence induction typical of a punched out (untransformed) tobacco leaf infiltrated with buffer solution passing over the cut off. Petiolus (petiole) is recorded (lower curve). One first recognizes a large increase in fluorescence when the light is turned on, which then reaches a peak, followed by a gradual decrease in Hess signal over time.

This pattern demonstrates that the chloroplasts were excited to fluoresce by incident light at a wavelength characteristic of the system. At this l'okt

- 52 -

energy is removed from the photosystem in the form of electrons, which are transmitted via the electron transport pathways of Photoeystem II

and I drain this is reflected by the gradual

Drop of the curve for the fluorescence signal.

But when the leaf is infiltrated with a 10 * M atrazine solution, the result is an image as shown in the upper curve of Figure 5. In this case, too, the light energy is first absorbed ", which causes the chloroplasts to fluoresce, but there is no energy shift because the electron flow is blocked at the quinone-binding stage of the Photosysteir II Photosystem in the excited state solidifies, therefore, no drop is seen after the initial increase in fluorescence.

If these measurements were carried out in the presence of 10 × M atrazine in the presence of 10 × M atrazine genetically engineered according to the present invention, all the control plants show the same fluorescence induction patterns as the non-transgenic tobacco plants.

On the other hand, if the measurements were made on the test plants, three clusters of fluorescence induction meters can be distinguished (Figure 6).

Some of the transgenic plants show no evidence of atrazine detoxification (the uppermost curve), some show intermediate detoxification (third line), and some show significant (lower curve) atrazine ionization due to the normal electron abstraction the photosystem becomes obvious. Out of 27 plants characterized in this way, 5 showed clear evidence of the ability to detoxify atrazine.

- 53 -

Example IX:

Groba cte rium Infection of Plant Materials The different genotypes of Agrobacterium tumefaciens are grown on an AB minimal medium. I Watson, B. et al ., J. Bacteriol. , J_23: 255-264 (1975), added to mannitol xst, cultured for 48 hours at a temperature of 28 ^U C. The bacteria are pelleted in MSilN medium with a twofold dilution; resuspended and incubated for three hours at a temperature of 25 ° C. For the Hevöti? ϊUnless of the MSKN mode is used tine vcu '' otorl \ o 'pulwrl' ornate mixture, which has been purchased from KC Biological, and which contains the macro and micro elements of the .nirashige and Skoog medium in the form of theirv Contains salts. The final concentration of the constituents of the MSBN medium corresponds to that of the medium according to Murashige and Skocg. In addition, the MSBN medium (final concentrations) contains: benzyladenine (1 mg / l); Naphthylacetic acid (0.1 mg / l); Myo-inoaitol (i mg / l); Nicotinic acid (1 mg / l); Pyridoxine (1 mg / l); Thiamine KCl (10 mg); and sucrose (30 mg / l) J. The pH is adjusted to a value of pH 5.7 to 5.8.

Leaves of leaf discs of in vitro cultured Nicotiana tabacum cv. petite Havana SRI plants 10 minutes »float on a bacterial suspension in a modification of a method of Hursch, R. et al., Science , 27: 1229-1231 (1985). The leaf discs are then transferred to filter paper on an MSN medium without antibiotics. After 48 hours, the leaf pieces are dipped in liquid MSBN medium containing 500 mg / l carbenicillin, and then transferred to a solid selection medium containing 100 mg / l kanamycin and 500 mg / l carbenicillin.

Plant ripening and self-pollination

Saplings that develop on the dialectin from the calli are removed and transferred to OMS medium containing 100 mg / l kanamycin and 250 mg / l carbenicillin. The development is then allowed to proceed for three more weeks. Afterwards, the plants are planted in soil and transferred to the greenhouse. Flowering begins 4 to 8 weeks after

- 54 - Zf9ZCS

Spend the plants in a greenhouse. At the time when the flower opens and its anthurium separate, they are removed with the help of tweezers and rubbed on the scars for self-pollination of each individual flower. The seed capsules mature within AO days.

Seed progeny testing of control plants, transgenic control plants and transgenic test plants The seeds are first removed from the mature seed capsule under aseptic conditions and stored in sterile petri dishes. The seeds are then transferred to a medium soft seed germination medium (SKM) composed of all macro- and microelements in the form of their salts and in a concentration corresponding to the medium of Murashige & Skoog (KC Diologicals), of 1 mg / 1 thiamine hydrochloride as well 0.6% purified agar (Difco®). The medium atrazine is added in analytical quality, in a concentration of 10 ^-7 M, 3 χ 1O ^-7 M, 5 χ 1θ "Μ ^7, 8 χ 10" H ⁷ and 10 ^"6M.

Growth and survival rate of seedlings is determined at said concentration and at an atrazine level of zero, over a period of 16 days; the results are shown in Table 1, below. Although all control plants and transgenic control plants germinate, they only reach the cotyledon stage in their growth at atrazine concentrations of 5χ10 M or at higher concentrations. Among the seedlings resulting from the self-pollination of pCIB14 plants, approximately 75% green remains in an atrazine-concentrate uration of 5 χ 10 M and forms primary leaves, whereas those seedlings live at atrazine concentrations of 8 χ 10 M or higher concentrations only cotyledons form before bleach pie and die off.

-55-

Z? 9

Although the tolerant seedlings on atrazine-containing medium do not show the same good growth as on atrazine-free medium, they can still be easily distinguished from the sensitive controls on the same medium.

Atrazine concentrations (M)

Genotype of the seedlings 10 " ⁷ M 3 χ 10" ⁷ M 5 κ 10 ~ ⁷ M 8 χ 10 " ⁷ sts 10 " ⁶ m control + + ο O O LBA 4404 + + O O O I am 6 + + O O O pCIBH + + O O O pCIB14 + + + O O

The table clearly demonstrates the difference in growth and survival between the progeny of transgenic pCIB14 plants and the controls. Positive growth is indicated by "+", negative growth by an "o".

While the present invention has been described in detail herein by way of illustration and example for purposes of clarity and understanding, it is to be understood that certain changes and modifications can be practiced within the scope of this invention, the scope of which is indicated solely by the scope of the appended claims are predetermined.

In the context of the present invention, not only those DNA molecules which are specified with specific nucleotide sequence and encode a glutathione-S-transferaee polypeptide form part of the invention, but likewise their variants or mutants which are suitable for polypeptides with glutathione Encode -S-transferase activity.

Claims (20)

-56- claims A process for the preparation of a herbicide-tolerant plant, which comprises transforming a plant with a recombinant DNA molecule capable of conferring herbicide tolerance based on the detoxification of the herbicide to a plant. 2. The method according to claim 1, characterized in that said recombinant DNA molecule consists of different sequence sections, which are derived from genomic DNA and / or cDNA and which are composed to a / Final glutathione S-transferase-encoding DNA sequence. 3. The method according to claim 2, characterized in that said DNA sequence sections are composed of both genomic DNA and cDNA. 4. A method according to claim 2, characterized in that said DNA sequence portions of gene fragments of several organisms belonging to different genera are composed. 5. The method according to claim 2, characterized in that said DNA sequence segments are composed of gene fragments of more than one strain, a variety or a species of the same organism. 6. A method according to claim 2, characterized in that said DNA Sequenzabschnitle from portions of more than one gene of the same organism are composed. ?. A method according to any one of claims 1 to 6, characterized in that said DNA sequence encoding a glutathione S-transferase polypeptide is derived from a mammal. - 57 - 8. A method according to the fine of claims 1 to 7, characterized in that said DNA sequence encoding a glutathione S-transferase polypeptide derived from a rat. 9. Method according to claim 8, characterized in that said DNA sequence encoding a rat glutathione S-transferase polypeptide has the following nucleotide sequence: 40 50 60 ATGCCTATGATACTGGGATACTGG MPMILGYW 70 80 90 100 110 120 AACGTCCGCGGGCTGACACACCCGATCCGCCTGCTCCTGGAATACACAGACTCAAGCTAT NVRGLTHPIRLLLEYTDSSY 130 140 150 160 170 180 GAGGAGAAGAGATACGCCATGGGCGACGCTCCCGACTATGACAGAAGCCAGTGGCTGAAT EEKRYAMGDAPDYDtSQWLN 190 200 210 220 230 240 gagaagttcaaactgggcctggacttccccaatctgccclxttaattgatggatcgcgc ekfklgldfpnlpylidgsr 250 260 270 280 290 300 AAGATTACCCAGAGCAATGCCATAATGCGCTACCTi. JCCGCAAGCACCACCTGTGTGGA KITQSNAIMRYLARKHHLCG 310 320 330 340 350 360 GAGACAGAGGAGGAGCGGATTCGTGCAGACATTGTGGAGAACCAG & TCATGGACAACCGC ETEEERIRADIVENQVMDNR 370 380 390 400 410 420 ATGCAGCTCATCATGCTTTGTTACAACCCCGACTTTGAGAAGCAGAAGCCAGAGTTCTTG MQLIMLCYNPDFEKQKPEFL 430 440 450 460 470 480 AAGACCATCCCTGAGAAGATGAAGCTCTACTCTGAGTTCCTGGGCAAGCGACCATGGTTT KTIPEKMKLYSEFLGKRPWF 490 500 510 520 530 540 GCAGGGGACAAGGTCACCTATGTGGATTTCCTTGCTTATGACATTCTTGACCAGTACCAC AGDKVTYVDFLAYDILDQYH 550 560 570 580 590 600 ATTTTTGAGCCCAAGTGCCTGGACGCCTTCCCAAACCTGAAGGACTTCCTGGCCCGCTTC IFEPKCLDAFPNLKDFLARF 610 620 630 640 650 660 GAGGGCCTGAAGAAGATCTCTGCCTACATGAAGAGCAGCCGCTACCTCTCAACACCTATA EGLKKISAYMKSSRYLSTPI 670 680 690 700 710 720 TTTTCGAAGTTGGCCCAATGGAGTAACAAGTAG
FSKLAQWSNK - 58 - OSZG 10. The method according to claim 8, characterized in that said DNA sequence which codes for a glutathione S-transferase polypeptide of the rat üie following nucleotide sequence: 1 10 13 0 TAC AGC ATG GGG GAT GCT CCC GAC TAT GAC AGA AGC CAG Tyr Ser Met GIy Asp Ala Pro Asp Tyr Asp Arg Ser GIn 150 1 70 TGG CTG AGT GAG AAG TTC AAA CTG GGC CTG GAC TTC CCC AAT CTG 'irp Leu Ser Giu Lys Phe Lys Leu Gily Leu Asp Phe Pro Asn Leu 19 0 210 CCO TAC TTA ATT GAT GGG TCA CAC AAG ATC ACC CAG AGC AAT GCC Pro Tyr Leu lie Asp Gly Ser His Lys He Thr GIn Ser Asn AIa 2 30 25 0 270 ATC CTG CGC TAC CTT GGC CGG AAG CAC AAC CTT TGT GGG GAG ACA He Leu Arg Tyr Leu GIy Arg Lys His Asn Leu Cys GIy GIu Thr 2 90 31 0 GAG GAG GAG AGG ATT CGT GTG GAC GTT TTG GAG AAC CAG GCT ATG GIu GIu GIu Arg He Arg VaI Asp VaI Leu GIu Asn Gin AIa Met 330 3 50 GAC ACC CGC CTA CAG TTG GCC ATG GTC TGC TAC AGC CCT GAC TTT Asp Thr Arg Leu GIn Leu AIa Met VaI Cys Tyr Ser Pro Asp Phe 37 0 390 GAG AGA AAG AAG CCA GAG DAY TTA GAG GGT CTC CCT GAG AAG ATG GIu Arg Lys Lys Pro GIu Tyr Leu GIu GIy Leu Fro GIy Lys Met 4 10 A3 0 450 AAG CTT TAC TCC GAA TTC CTG GGC AAG CAG CCA TGG TTT GCA GGG Lys Leu Tyr Ser GIu Phe Leu GIy Lys Gin Pro Trp Phe Ala GIy 4 70 49 0 AAC AAG ATT ACG TAT GTG CAT TTT CTT GTT TAC GAT GTC CTT GAT Asn Lys He Thr Tyr VaI Asp Phe Leu VaI Tyr Asp VaI Leu Asp 510 5 30 CAA CAC CGT ATA TTT GAA CCC AAG TGC CTG GAC GCC TTC CCA AAC Gin His Arg He Phe GIu Pro Lys Cys Leu Asp Ala Phe Pro Asn 55 0 570 CTG AAG GAC TTC GTG GCT CGG TTT GAG GGC CTG AAG AAG ATA TCT Leu Lys Asp Phe VaI AIa Arg Phe GIu GIy Leu Lys Lys He Ser 5 90 61 0 630 GAC TAC ATG AAG AGC GGC CGC TTC CTC TCC AAG CCA ATC TTT GCA Asp Tyr Met Lys Ser Gly Arg Phe Leu Ser Lys Pro He Phe AIa 6 50 AAG ATG GCC TTT TGG AAC CCA AAG DAY
Lys Met AI Phe Trp Asn Pro Lys End - 59 - 11. The method according to claim 8, characterized in that said DNA sequence encoding a rat glutathione S-transferase polypeptide having the following nucleotide sequence: 1 0 30 ATG CCT ATG ACA CTG GGT TAC TGG GAC ATC CGT GGG CTG GCT CAC Met Pro Met Thr Leu GIy Tyr Trp Asp He Arg Gly Leu Ala His 50 7 0 90 GCC ATT CGC CTG TTC CTG GAG ACT ACA GAC ACA AGC ACT GAG GAC Ala He Arg Leu Phe Leu GIu Tyr Thr Asp Thr Ser Tyr GIu Asp 1 10 13 0 AAG AAG TAC AGC ATG GGG GAT GCT CCC GAC TAT GAC AGA AGC CAG Lys Lys Tyr Ser Met GIy Asp Ala Pro Asp Tyr Asp Arg Ser GIn 150 1 70 TGG CTG AGT GAG AAG TTC AAA CTG GGC CTG GAC TTC CCC AAT CTG Trp Leu Ser Glu Lys Phe Lys Leu Gily Leu Asp Phe Pro Asn Leu 19 0 210 CCC TAC TTA ATT GAT GGG TCA CAC AAG ATC ACC CAG AGC AAT GCC Pro Tyr Leu He Asp Gly Ser His Lys He Thr GIn Ser Asn AIa 2 30 25 0 270 ATC CTG CGC TAC CTT GGC CGG AAG CAC AAC CTT TGT GGG GAG ACA He Leu Arg Tyr Leu GIy Arg Lys His Asn Leu Cys GIy GIu Thr 2 90 31 0 GAG GAG GAG AGG ATT COT GTG GAC GTT TTG GAG AAC CAG GCT ATG GIu GIu GIu Arg He Arg VaI Asp VaI Leu GIu Asn Gin AIa Met 330 3 50 GAC ACC CGC CTA CAG TTG GCC ATG GTC TGC TAC AGC CCT GAC TTT Asp Thr Arg Leu GIn Leu AIa Met VaI Cys Tyr Ser Pro Asp Phe 37 0 390 GAG AGA AAG AAG CCA GAG TAC TTA GAG GGT CTC CCT GAG AAG ATG GIu Arg Lys Lys Pro Glu Tyr Leu GIu GIy Leu Pro GIu Lys Met 4 10 43 0 450 AAG CTT TAC TCC GAA TTC CTG GGC AAG CAG CCA TGG TTT GCA GGG Lys Leu Tyr Ser Glu Fhe Leu Gly Lys GIn Pro Trp Phe Ala GIy 4 70 49 0 AAC AAG ATT ACG TAT GTG GAT TTT CTT GTT TAC GAT GTC CTT GAT Asn Lys He Thr Tyr VaI Asp Phe Leu VaI Tyr Asp VaI Leu Asp 510 5 30 CAA CAC CGT ATA TTT GAA CCC AAG TGC CTG GAC GCC TTC CCA AAC Gin His Arg He Phe Glu Pro Lys Cys Leu Asp Ala Phe Pro Asn 55 0 570 CTG AAG GAC TTC GTG GCT CGG TTT GAG GGC CTG AAG AAG ATA TCT Leu Lys Asp Phe VaI AIa Arg Phe Giu GIy Leu Lys Lys He Ser 5 90 61 0 630 GAC TAC ATG AAG AGC GGC CGC TTC CTC TCC AAO CCA ATC VTT GCA Asp Tyr Met Lys Ser Gly Arg Phe Leu Ser Lys Pro He Phe AIa -60- 6 50 AAG ATG GCC TTT TCG AAC CCA MG DAY
Lys Met Ala Phe Trp Asn Pro Lys End 12. The method according to claim 8, characterized in that said DNA sequence encoding a rat glutathione S-transferase polypeptide having the following nucleotide sequence: 10 30 A GAC CCC AGC ACC ATG CCC ATG ACA CTG GGT TAC TGG GAC ATC Met Pro Met Thr Leu GIy Tyr Trp Asp He 5 0 70 CGT GGG CTA GCG CAT GCC ATC CGC CTG CTC CTG GAA TAC ACA GAC Arg GIy Leu Ala His Ala He Arg Leu Leu Leu GIu Tyr Thr Asp 90 11 0 130 TCG AGC TAT GAG GAG AAG AGA TAC ACC ATG GGA GAC GCT CCC GAC Ser Ser Tyr GIu GIu Lys Arg Tyr Thr Met GIy Asp Ala Pro Asp 1 50 17 0 TTT GAC AC-A AGC CAG TGG CTG AAT GAG MG TTC AM CTG GGC CTG Phe Asp Arg Ser GIn Trp Leu Asn GIu Lys Phe Lys Leu GIy Leu 190 2 10 GAC TTC CCC MT CTG CCC TAC TTA ATT GAT GGA TCA CAC MG ATC Asp Phe Pro Asn Leu Pro Tyr Leu He Asp GIy Ser His Lys He 23 0 250 2 ACC CAG AGC MT GCC ATC CTG CGC TAT CTT GGC CGC MG CAC MC Thr GIn Ser Asn Ala He Leu Arg Tyr Leu GIy Arg Lys His Asn 70 29 0 310 CTG TGT GGG GAG ACA GM GAG GAG AGG ATT CGT GTG GAC ATT CTG Leu Cys GIu GIu Thr GIu GIu GIu Arg lie Arg VaI Asp He Leu 3 30 35 0 GAG MT CAG CTC ATG GAC MC CGC ATG GTG CTG GCG AGA CTT TGC GIu Asn GIn Leu Met Asp Asn Arg Met VaI Leu AIa Arg Leu Cys 370 3 90 TAT MC CCT GAC TTT GAG MG CTG MG CCA GGG TAC CTG GAG CM Tyr Asn Pro Asp Phe GIu Lys Leu Lys Pro GIy Tyr Leu GIu GIn 41 0 430 4 CTG CCT GGA ATG ATG CGG CTT TAC TCC GAG TTC CTG GGC MG CGG Leu Pro GY Met Met Arg Leu Tyr Ser GIu Phe Leu GIy Lys Arg 50 47 0 490 CCA TGG TTT GCA GGG GAC MG ATC ACC TTT GTG GAT TTC ATT GCT Pro Trp Phe Ala GIy Asp Lys He Thr Phe VaI Asp Phe He AIa 5 10 53 0 TAC GAT GTT CTT GAG AGG MC CM GTG TTT GAG GCC ACG TGC CTG Tyr Asp VaI Leu GIu Arg Asn Gin VaI Phe GIu AIa Thr Cys Leu 550 5 70 GAC GCG TTC CCA MC CTG MG GAT TTC ATA GCG CGC TTT GAG GGC Asp Ala Phe Pro Asn Leu Lys Asp Phe He AIa Arg Phe GIu GIy - 61 - 59 o CTG AAG AAG
Leu Lys Lys AGA CCT CTG Arg Pro Leu CCT GAC AGG
Pro Asp Arg ATC TCC GAC Hey Her Asp TTC ACA AAG Phe Thr Lys 6 90 TGG GCT TTA Trp AIa Leu 610 TAC ATG AAG Tyr Met Lys 65 0 ATG GCT ATT Met Ala He GGA GAA AGA GIy GIu Arg TCC AGC CGC Ser Ser Arg TGG GGC AGC Trp GIy Ser 71 TAC CAA ATC Tyr Gin He TTC CTC CCA Phe Leu Pro 670 AAG TAG GAC Lys End Asp TCC TGG GTT Ser Trp VaI 730 7 50 TGC CAA GAG CCC TAA GGA GCG GGC AGG ATT CCT GAG CCC CAG AGC Cys Gin GIu Pro End GIy Ala GIy Arg He Pro GIu Pro Gin Ser 77 0 CAT GTT TTC TTC CTT CCT His VaI Phe Phe Leu Pro CTC TCA TTT TTT GGT CAT Leu Ser Phe Phe Giy His 8 70 AAA GCC CTA GCA ACT CCT Lys AIa Leu AIa Thr Pro 910 9 30 TTA AAG TGC CCC GCC CCC ACC CCT CGA GCT CAT GTG ATT GGA TAG Leu Lys Cys Pro AIa Pro Thr Pro Arg Ala His VaI He Giy End 95 0 970 9 TTG GCT CCC AAC ATG TGA TTA TTT TGG GCA GGT CAG GCT CCC CGG Leu Ala Pro Asn Met End Leu Phe Trp Ala Giy Gin AIa Pro Arg 90 101 0 1 030 CAG ATG GGG TCT ATC TGG AGA CAG TAG ATT GCT AGC AGC TTT GAC GIn Met Gly Ser He Trp Arg Gin End He Ala Ser Ser Phe Asp 10 50 107 0 CAC CGT AGC CAA GCC CCT CTT CTT GCT GTT TCC CGA GAC TAG CTA His Arg Ser GIr ₁ Ala Pro Leu Leu Ala VaI Ser Arg Asp Ent: Leu 1 090 11 10 TGA GCA AGG TGT GCT GTG TCC CCA GCA CTT GTC ACT GCC TCT GTA End Ala Arg Cys Ala VaI Ser Pro AIa Leu VaI Thr AIa Sor VaI 113 0 1 150 11 ACC CGC TCC TAC CGC TCT TTC TTC CTG CTG CTG TGA GCT GTA CCT Thr Arg Ser Tyr Arg Ser Phe Phe Leu Leu Leu End Ala VaI Pro 70 119 0 1? .1O CCT GAC CAC AAA CCA GAA TAA ATC ATT CTC CCC TTA AAA AAA AAA Pro Asp His Lys Pro Giu End He He Leu Pro Leu Lys Lys Lys AAA AAA AAA A
Lys Lys Lys - 62 - 13. The method according to any one of claims 1 to 12, characterized in that said DNA sequence encoding a glutathione S-transferase polypeptide is operably linked to a plant promoter and / or a terminator region and in the plant cell is expressible. IA. A method according to claim 13, characterized in that said DNA sequence is heterologous Urspruch in relation to the plant
Promoter. 15. The method according to claim 14, characterized in that it is in the plant promoter to a promoter selected from the
Group of nos, ocs and CaMV promoters. 16. The method according to claim 14, characterized in that said plant promoter is the promoter of the small subunit of the ribulose-1,5-bisphosphate carboxylase of the soybean or the promoter of the chlorophyll a / b binding protein. 17. The method according to claim 1, characterized in that said herbicides are a chloroacetamide, a sulfonylurea, a Triazi.n, a diphenyl ether, an imidazolinone or a thiocarbamate. 18. The method according to claim 1, characterized in that said recombinant DNA molecule by means of a suitable transformation method selected from the group consisting of direct transformation method, Agrobaktfcrium-mediated transformation method, virus-mediated transformation ancestor and Co-Transformatdonsverfahren in introduced the plant cell or plant and the
resulting transgenic plant cell optionally to a whole
Plant is regenerated. 19. Process according to claim 18, characterized in that said plant cells are those selected from the group consisting of tobacco, soybeans or cotton. 20. The method according to any one of claims 1 to 19, characterized in that said herbicide-tolerant plants also include mutants and variants. 21. The method according to any one of claims 1 to 20, characterized in that said herbicide-tolerant plants also include the progeny of said plants. 22. The method according to any one of claims 1 to 21, characterized in that said herbicide-tolerant plants also include parts of plants selected from the group consisting of flowers, seeds, leaves, twigs, fruits ^ .a, provided that these parts herbicidal include tolerant cells. FO 7.5 / WB / sm * / tr Ju -YS