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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
  • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
  • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
  • C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
  • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
  • C12N15/8263—Ablation; Apoptosis
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
  • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)

Definitions

• Instant invention relates to a method of controlling plant pathogens by pathogen inducible expression of a deacetylase (deac) gene encoding a protein exhibiting N-acetyl-PPT (e.g., N-acetyl-phosphinothricine) specific deacetylase activity in combination with the application of a composition containing one or more non-toxic compounds (e.g., N- acetyl-PPT), allowing - after deacetylation within the plant cell - inducible and selective destruction of cells within a living plant after pathogen infestation.
• a deacetylase deacetylase
• pathogen-resistant transgenic plants which are sensitized to respond more rapidly to infection and thus are protected against virulent pathogens.
• One possible way is to express a pathogen toxin under control of a constitutive promoter in order to control pathogen attack e.g., crystal protein derived from Bacillus thuringiensis (US 5,322,938), insect-specific peptidase inhibitors (WO 95/35031), sarcotoxin (EP- A-0 798 381), ribozymes (US 5,641,673), chitinase (US 4,940,840) etc.
• crystal protein derived from Bacillus thuringiensis US 5,322,938
• insect-specific peptidase inhibitors WO 95/35031
• sarcotoxin EP- A-0 798 381
• ribozymes US 5,641,673
• chitinase US 4,940,840
• a different approach is the expression of a cytotoxin under control of a pathogen inducible promoter to induce a hypersensitive response, for example, the barnase system (cf. US 5,750,386).
• tissue- or development-specific plant promoters are leaky", i.e., they are also expressed to a certain extent in other tissues or developmental stages or are also inducible by other environmental stress factors than pathogen infection.
• the transgenic plants may suffer from cell damage caused by undesired expression of the toxins and result in a loss in productivity.
• deac system for the selective ablation of plant tissue and cells is known from EP-A-0 531 716.
• specific cell ablation can be achieved for any plant tissue of interest, e.g., for the induction of transgenic male or female sterility by anther or pistil specific expression of a deacetylase gene.
• a deac gene By fusing a deac gene with a tapetum-specific promoter male sterility (MS) can be induced by external application of N-acetyl-phosphinothricine.
• MS tapetum-specific promoter male sterility
• N-acetyl-phosphinothricine-specific deacetylase protein the expression product of the deac gene, converts the non- toxic N-acetyl-derivative of phosphinothricine into the herbicidal active compound (WO 98/27201).
• nucleotide sequences coding for suitable deacetylase (deac) proteins (or deacetylases) with appropriate substrate specificities have been described previously: argE from Escherichia coli, encoding N-acetyl-ornithine deacetylase, dea from Streptomyces viridochromogenes (EP-A-0 531 716), dead from Stenotrophomonas sp. (DSM 9734) and dead from Comamonas acidovorans (DSM 11070), both encoding hippurate hydrolases (WO 98/27201). It is understood that other nucleotide sequences encoding proteins with deacetylase activity can be used in the context of the present invention.
• the use of the deac system i.e. the expression of a deac gene in a plant under control of a pathogen inducible promoter, optionally within a specific tissue (e.g., roots, leaves, fruits, seeds) in combination with the external application of N- acetyl PPT is suitable for pathogen control by selective destruction of the infested plant cells or tissues.
• a pathogen inducible promoter e.g., roots, leaves, fruits, seeds
• the formation of the plant-toxic compound i.e. deacetylated PPT
• the formation of the plant-toxic compound i.e. deacetylated PPT
• the formation of the plant-toxic compound is fast enough to trigger a hypersensitive' response causing rapid cell death and therefore, prevents the pathogen from further spreading throughout the plant or plant tissue.
• the use of the deac system for pathogen control by selective ablation of pathogen infested plant tissue and cells has a number of advantages, e.g., with respect to resistance development or the broad effectiveness against plant pathogens, compared to known sytems for plant pathogen control.
• cell death can only be induced by a combination of deac expression and application of N-acetyl PPT.
• the deac activity as such is not harmful and thus, plant cells are not affected by deac expression in case of use of a leaky' promoter.
• Another advantage of the deac system according to the invention is that the optimal time window for the application of N-acetyl-PPT, resp., PPT can be chosen by the farmer depending on pathogen attack.
• the present invention relates to a nucleic acid molecule comprising the coding region of, resp., a nucleotide sequence encoding a deacetylase, under control of a pathogen inducible regulatory element, such as a promoter or a promoter in combination with an enhancer.
• an object of the invention is the use of a deac gene under control of a pathogen inducible regulatory element, preferably a promoter, or a nucleic acid molecule of the invention for the preparation of a pathogen-tolerant plant cell or plant.
• the nucleic acid molecule according to the invention is preferably a DNA or RNA molecule, for instance genomic or cDNA.
• the nucleotide sequences coding for a deacetylase and/or a pathogen inducible promoter, which are part of the nucleic acid molecule of the invention, may be isolated from natural sources or synthesized by known methods (Sambrook et al., Molecular Cloning, A Laboratory Manual, 2. Aufl. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).
• the nucleic acid molecule according to the invention may also comprise a fragment, derivative or allelic variant of said nucleotide sequence encoding a deacetylase provided that the biological activity of a N-acetyl-PPT specific deacetylase is restored. Further, the nucleic acid molecule according to the invention may also comprise fragments, derivatives and allelic variants of a pathogen inducible promoter provided that the biological activity of a pathogen inducible promoter is restored.
• the fragments, derivatives and allelic variants of said nucleotide sequences coding for a N-acetyl-PPT specific deacetylase or a pathogen inducible promoter might have been caused by one or more mutations such as deletions, substitutions, insertions and/or recombinations, which may have naturally occurred, or which may have been induced by genetic engineering or molecular biological techniques (e.g., Sambrook et al., loc.cit.).
• nucleic acid molecule according to the invention is linked to one or more regulatory elements, which ensure transcription and synthesis of a translatable RNA in procaryotic or eucaryotic cells (e.g., enhancer, tissue-specific elements, 3'-poly A-tail, e.g., Gielen et al., EMBO J. 1989, 8:23-29).
• regulatory elements e.g., enhancer, tissue-specific elements, 3'-poly A-tail, e.g., Gielen et al., EMBO J. 1989, 8:23-29.
• the expression product i.e. the deacetylase protein
• the expression product can be localized in any compartment (e.g., cytosol, vacuole, apoplast, cell wall, plastid, mitochondrium etc.) of the transformed plant cell.
• cytosol a nucleotide sequence coding for a signal peptide, resp., targeting sequence.
• nucleotide coding for a signal peptide, resp., targeting sequence is well known in the art (e.g., Braun et al., EMBO J.l l (1992), 3219-3227; Wolter et al., Proc. Natl. Acad. Sci. USA 85 (1988), 846-850; Sonnewald et al., Plant J. 1 (1991), 95-106).
• the invention relates to vectors, preferably plasmids, cosmids, viruses, bacteriophages, and other vectors common in genetic engineering, which comprise a nucleic acid molecule according to invention.
• the invention relates to a host cell, preferably procaryotic or eucaryotic cell, comprising a nucleic acid molecule or a vector according to the invention and the progeny of said host cell.
• the host cell of the invention is characterized in that it has been transformed and/or genetically modified by a nucleic acid molecule according to the invention or by a vector according to the invention.
• the progeny of said host cell is further characterized by the presence of the nucleic acid molecule according to the invention.
• the host cell is a bacterial cell.
• the host cell of the invention is further characterized in that the introduced nucleic acid molecule is of heterologous origin, i.e. it does not occur naturally in these cells.
• the present invention relates to a process for the production of a transgenic plant cell comprising the step of introducing a nucleic acid molecule or a vector according to the invention into the genome of a plant cell by known transformation technologies, e.g., by stable integration into the genome of said plant cell, said transgenic plant cell comprising a deacetylase under control of a pathogen inducible regulatory element.
• the transformed plant cell obtainable by the process of the invention is a further embodiment of the invention.
• the plant cell according to the invention comprises a deacetylase under control of a pathogen inducible promoter and is further characterized in that the introduced nucleic acid molecule is of heterologous origin, i.e. it does not occur naturally in these cells.
• the transformed plant cell of the invention additionally comprises a nucleic acid molecule conferring PPT-resi stance (such as the pat or bar gene) as known, e.g., by EP-A-0 242 236, EP-A-0 242 246, EP-A-0 257 542 or EP-A-0 531 716, obtainable, e.g., by introducing the nucleic acid molecule of the invention into a known PPT-resistant or tolerant plant cell according to the process of the invention.
• a nucleic acid molecule conferring PPT-resi stance such as the pat or bar gene
• transgenic plant cell of the invention can be regenerated into a complete plant.
• a further object of the invention is a process for the production of a transgenic plant comprising the step of regenerating a complete plant from a plant cell according to invention, said plant exhibiting pathogen-resistance after treatment with N-acetyl-PPT.
• a plant is obtainable by applying to the transgenic plant cell of the invention a well known process for plant regeneration.
• a plant cell comprising a gene conferring PPT-resi stance e.g., a pat or bar gene
• a nucleic acid molecule or a vector according to the invention such a plant exhibits pathogen-resistance after treatment with either N-acetyl-PPT and/or PPT.
• a plant according to the invention exhibits additionally PPT-tolerance, the treatment of said plant with PPT results simultaneously in a herbicidal and pathogen- controlling effect.
• constitutive low-level expression of the pat or bar gene can act prophylactically to protect non-pathogen infected tissues.
• cloning vectors In order to prepare the integration of foreign nucleic acid molecules into higher plants a high number of cloning vectors are available, containing a replication signal for E.coli and a marker gene for the selection of transformed bacterial cells. Examples of such vectors are pBR322, pUC series, M13mp series, PACYC 1 84, pBinAR etc.
• the desired sequence may be integrated into the vector at a suitable restriction site.
• the obtained plasmid is used for the transformation of E.coli cells.
• Transformed E.coli cells are cultivated in a suitable medium and subsequently harvested and lysed. The plasmid is recovered.
• plasmid DNA As an analyzing method for the characterization of the obtained plasmid DNA, use is generally made of restriction analysis, gel electrophoresis and other biochemical or molecular biological methods. After each manipulation the plasmid DNA may be cleaved and the obtained DNA fragments may be linked to other DNA sequences. Each plasmid DNA may be cloned into the same or other plasmids. In order to integrate DNA into plant host cells, a wide range of techniques are available.
• These techniques comprise the transformation of plant cells with T-DNA by using Agrobacterium tumefaciens or Agrobacterium rhizogenes as transformation medium, by polyethylene glycol treatment, the fusion of protoplasts, the injection or electroporation of DNA, the integration of DNA by means of the biolistic method as well as further possibilities.
• plasmids In the case of electroporation and injection of DNA into plant cells, there are no special demands made on the plasmids used. Simple plasmids such as pUC derivatives may be used. However, in case whole plants are to be regenerated from cells transformed in such a way, a selectable marker gene should be present.
• Ti- or Ri-plasmid is used e.g. for the transformation of the plant cell, usually at least the right border, more frequently, however, the right and left border of the Ti- and Ri-plasmid T-DNA should be connected to the foreign gene to be integrated as a flanking region.
• the DNA which is to be integrated should be cloned into special plasmids, namely either into an intermediate vector or into a binary vector. Due to sequences homologous to the sequences within the T-DNA, the intermediate vectors may be integrated into the Ti- or Ri-plasmid of the Agrobacterium due to homologous recombination. This also contains the vir-region necessary for the transfer of the T-DNA. Intermediate vectors cannot replicate in Agrobacteria. By means of a helper plasmid the intermediate vector may be transferred to Agrobacterium tumefaciens (conjugation). Binary vectors may replicate in E.coli as well as in Agrobacteria.
• Agrobacterium contains a selectable marker gene as well as a linker or polylinker which is framed by the right and the left T-DNA border region. They may be transformed directly into Agrobacteria (Holsters et al, Mol. Gen. Genet. 163 (1978), 1 81 -1 87).
• the Agrobacterium acting as host cell should contain a plasmid carrying a vir-region. The vir-region is usually necessary for the transfer of the T-DNA into the plant cell. Additional T-DNA may be present.
• the Agrobacterium transformed in such a way is used for the transformation of plant cells.
• T-DNA for the transformation of plant cells was investigated intensively and described sufficiently in EP 120 516; Hoekema, In: The Binary Plant Vector System Offsetdrukkerij Kanters B.V., Alblasserdam (1985), Chapter V; Fraley et al., Crit. Rev. Plant. Sci., 4, 1-46 and An et al. EMBO J. 4 (1985), 277-287.
• plant explants may suitably be co-cultivated with Agrobacterium tumefaciens or Agrobacterium rhizogenes.
• Agrobacterium tumefaciens or Agrobacterium rhizogenes From the infected plant material (e.g. pieces of leaves, stem segments, roots, but also protoplasts or suspension- cultivated plant cells) whole plants may then be regenerated in a suitable medium which may contain antibiotics or biocides for the selection of transformed cells.
• the plants obtained in such a way may then be examined as to whether the integrated DNA is present or not.
• Other possibilities in order to integrate foreign DNA by using the biolistic method or by transforming protoplasts are known to the skilled person (cf. e.g. Willmitzer, L., 1993 Transgenic plants.
• the introduced DNA Once the introduced DNA has been integrated in the genome of the plant cell, it usually continues to be stable there and also remains in the genome of the descendants of the originally transformed cell. It usually contains a selectable marker which confers resistance against a biocide such as phosphinotricine or against an antibiotic such as kanamycin, G 418, bleomycin or hygromycin etc. to the transformed plant cells.
• the individually selected marker should therefore allow for a selection of transformed cells to cells lacking the integrated DNA.
• the transformed cells grow in the usual way within the plants (see also McCormick et al., Plant Cell Reports 5 (1986), 81-84).
• the resulting plants can be cultivated in the usual way and cross-bred with plants having the same transformed genetic heritage or another genetic heritage.
• the resulting hybrid individuals have the corresponding phenotypic properties.
• the resulting plants are viable and produce seeds.
• Two or more generations should be grown in order to ensure whether the phenotypic feature is kept stably and whether it is transferred. Furthermore, seeds should be harvested in order to ensure that the corresponding phenotype or other properties will remain.
• Another object of the invention is a plant obtainable by the process of the invention, said plant comprising a plant cell of the invention.
• the plant according to the invention displays pathogen resistance after treatment with N-acetyl PPT (and/or PPT).
• the plant of the invention is an angiosperm, gymnosperm, monocotyledonous or dicotyledonous plant, especially a crop plant, in particular a plant for the production of fruits, nuts, vegetables, fodder, cereals, ornamentals, herbs, etc., such as, Dilleniidae, Rosanae, Fabaceae, Anacardiaceae, Euphorbiaceae, Hamamelididae, Solananaceae, Pooideae, especially selected from the group consisting of rye, barley, oat, maize, wheat, millet, cassava, sago, rice, lens, pea, maniok, potato, tobacco, tomato, rape, canola, soya, sugar beet
• the progeny of a plant or plant cell comprises fruits, seeds, tubers, root-stocks, seedlings, cuttings, calli, cell cultures etc. which are characterized by the presence of the nucleic acid molecule according to the invention.
• the hypersensitive reaction induced by the deac system according to the invention does not attack the pathogen directly, but acts by inhibiting further spreading of the infection. Due to this mechanism the deac system according to the invention is not only highly effective against a large number of plant pathogens (viruses, fungi (particularly biotrophic fungi), bacteria, insects, acarina, nematodes and gastropoda), but also prevents or delays the development of resistance phenomena due to circumvention of the pathogen metabolism. Furthermore, the life cycle of the pathogen is considerably affected resulting in a decreased pathogen attack in the next generation. Therefore, the instant invention provides ecological advantages for man and environment. Furthermore, the invention is not restricted to a single specific pathogen or a specific pathogenic mechanism, but may cover a very broad range of very different pathogens simultaneously.
• another embodiment of the invention is a process for controlling one or more pathogens in plants comprising the nucleic acid molecule of the invention, whereby N- acetyl-PPT (and/or PPT) is applied to said plants or the area under cultivation.
• the deac system according to the invention offers an inducible solution for pathogen control (pathogen tolerance can be induced by application of N-acetyl-PPT, resp. PPT), it can be used to optimize agricultural productivity dependent from environmental conditions which influence pathogen stress. In the case that crop plants are not infested by pathogens, N-acetyl-PPT, need not be applied.
• the term 'deac system' shall have the meaning of one or more nucleotide sequence(s) coding for a protein exhibiting deacetylase activity under control of a pathogen-inducible promoter in combination with the application of one or more non-toxic compounds which act as substrate(s) of said deacetylase(s) or compositions comprising said non-toxic compounds, which - after deacetylation - lead to a limited toxic effect within a plant tissue, plant cell and/or developmental stage of the plant.
• N-acetyl-PPT N-acetylated PPT
• Specificity for N-acetyl PPT refers to its ability to deacetylate N-acetyl PPT, but does not limit the activity to N-acetyl PPT exclusively.
• T T' is not restricted to the herbicidal active compound phosphinothricine (2- amino-4-methylphosphinobutyric acid, glufosinate), but shall also comprise derivatives and related compounds such as desmethylphosphinothricine (2-amino-4- hydroxyphosphinobutyric acid), bialaphos (phosphinothricyi-alanyl-alanine), desmethylbialaphos and the like, their salts, racemic mixtures and active enantiomers.
• N- acetyl PPT relates to an acetylated form of PPT, or a composition containing one or more of such acetylated PPT compounds.
• PPT-tolerant plants which express, e.g., the phosphinothricin acetyltransferase (pat or bar) gene
• pathogen control or resistance is also achievable by herbicide treatment with PPT.
• N-acetyl-PPT is generated by the plant itself, and is selectively reconverted to the active herbicide only in the tissue expressing the deac gene.
• pathogen-inducible regulatory elements such as a pathogen inducible promoter
• a pathogen inducible promoter can be found in the art, and are understood to comprise regulatory elements inducible either by pathogen structures or by the reaction of the plant to pathogen infection (e.g. wounding or stress).
• pathogen infection e.g. wounding or stress.
• defence molecules such as phytoalexins, PR- proteins or protease inhibitors and by a reaction called hypersensitive cell death, which immobilizes the pathogen at the site of penetration.
• a number of defence genes have been cloned (e.g. pathogen-related (PR)-protein genes, Martins et al., 1993, Mol. Gen. Genet. 236: 1 79-186) and their pathogen or stress inducible promoters are available (cf. table 1).
• pathogen means any pathogen or pest with which plants can be infested, whether plant- specific or not, such as viruses, bacteria, fungi (especially biotrophic fungi), insects, acarina, nematodes, gastropoda, and also any other endo- or ectoparasite.
• Transgenic plants are generated, expressing a deac gene under control of a pathogen- inducible (e.g., PR-protein or wound-inducible) promoter. The development of the plants is not affected under normal and under stress conditions, since the deac protein is not toxic to the cells per se.
• the plants are monitored for pathogen infection and the chemical, e.g., N-acetyl-PPT or PPT is applied before or as soon as an infection becomes manifest.
• Table 1 provides examples for some pathogen- or wound-inducable promoters.
• N- acetyl-PPT may be combined with other transgenic strategies for pathogen resistance (e.g., antifungal protein, ribosomal inhibiting protein, chitinases, glucanases) or other molecular biological approaches including qualitative and/or quantitative modification/alteration of plant products, e.g., by cross breeding or transformation technology.
• pathogen resistance e.g., antifungal protein, ribosomal inhibiting protein, chitinases, glucanases
• other molecular biological approaches including qualitative and/or quantitative modification/alteration of plant products, e.g., by cross breeding or transformation technology.
• Figure 1 describes the structure of plasmid pHOE6AC, comprising the pat coding sequence flanked by CaMV 35S promoter and terminator sequences.
• the dead structural gene was fused with the pathogen inducible promoter prpl-1 from potato (Martini et al., 1993, Mol. Gen. Genet. 236: 179-186) and the promoter-gene cassette was subcloned in the binary vectors pPCV801 (selectable marker: Pnos-hpt, Koncz et al., 1989, Proc. Natl. Acad. Sci. USA 86: 8467-8471) and pHOE6AC (selectable marker: P35S-p t, cf. Fig. 1). These constructs were transformed into potato (Solanum tuberosum) by Agrobacterium mediated gene transfer (de Block, 1988, Theor. Appl. Genet. 76: 161-11 A).
• Transformed plants were selected on hygromycin or phosphinothricin medium, depending on the plasmid construct, and tested for the presence of the transgene by southern blot analysis.
• necrosis formation - was observed in most of the deac-positive plants (pPCV801 -transformants) down to 0.4 mg/ml.
• the effects of phosphinothricine treatment on the pHOE6AC-plants were less pronounced, probably due to the expression of the pat gene in those transformants.
• surface irritations and weak necrosis development were observed in many of the deac-positive plants at phosphinothricine concentrations of 2 and 1 mg/ml.
• Deac transformants without Na salicylate preincubation as well as induced and uninduced wild type and pat control plants did not exhibit any tox symptoms after N- acetyl-phosphinothricine and phosphinothricine treatment, respectively.
• transgenic plants with the highest inducible deac expression levels were chosen for infection experiments with Phytophtora infestans. Plants were sprayed two-times with 5 mg/ml N-acetyl-D,L-phosphinothricine (pPCV801 -transformants) or 2 mg/ml D,L- phosphinothricine (pHOE6AC-transformants) and infected with spore suspension of Phytophtora infestans 1 week after the last spraying.
• pPCV801 -transformants 5 mg/ml N-acetyl-D,L-phosphinothricine
• pHOE6AC-transformants 2 mg/ml D,L- phosphinothricine

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