Indian Patents. 208585:NOVEL SELECTION MARKER

Title of Invention	NOVEL SELECTION MARKER
Abstract	This invention is concerned with the use of cyanamid hydratase as a selection marker in plant transformation. Cyanamid acts as a herbicide and plants transformed with the gene coding for cyanamide hydratase are able to convert the cyanamide into urea which enables the selection of transformed plants by survival under cyanamide pressure.

Full Text	FIELD OF THE INVENTION This application is concerned with a novel selectable marker, especially with the use of cyanamide hydratase as a selection marker in transformation experiments, more specifically in plant transformation experiments. BACKGROUND ART Cyanamide (H2N-C=N) is a nitrile derivate which, like other nitrile derivates, is used in agriculture for stimulation of growth and for plant protection. Cyanamide in aqueous solution or in the form of its calcium salt is used as a fertilizer by providing ammonia to the soil by its metabolic conversion. It has, however, the additional advantage of acting as a herbicide. To use it as fertilizer it has to be applied before sowing. Chemically, cyanamide belongs to the class of nitriles. In spite of the relatively rare occurrence in nature of compounds containing the nitrile group, enzymes that hydrate this group have been found in bacteria and plants (e.g. Nagasawa T., et al. (1988) Biochem. Biophys. Res. Commun. 155 1008-1016; Endo T. and Watanabe I. (1989) FEES Lett. 243 61-64) . Also in the fungus Myrothecium verrucaria a nitrile hydrating enzyme was found (Stransky H. and Amberger A. (1973) Z. Pflanzenphysiol. 7 74-87), which hydrates the nitrile group of cyanamide with formation of urea: Maier-Greiner et al. have isolated the enzyme and cloned the gene coding for it {Proc. Natl. Acad. Sci. USA 8_8, 4260-4264, 1991). They have demonstrated that this enzyme shows an extremely narrow substrate specificity, where compounds chemically related to cyanamide are not recognized as substrates. Selectable markers have to confer a dominant phenotype on transformed cells which is able of being used as a selection criteria. These falls into two classes: one class of genes which confers either cell viability or lethality in the presence of a selective agent and a class of genes which has negligible effects on cell survival but which confers transformed cells with some distinguishing physical characteristic. In plant transformation the fraction of cells incorporating the novel DNA is generally low, so most stable transformation schemes use markers which ensure the survival of transformed cells in the presence of a selective agent. A number of selection markers of this first group has been known and used for plant transformation experiments for several years. Included are the enzyme neomycin phosphotransferase (npt) which confers resistance to a group of antibiotics including kanamycin, paromomycin, geneticin and neomycin, mutant forms of the enzyme acetolactate synthase (als) which confer resistance to imidazolinones, sulfonylureas, triazolopyrimidines and pyrimidyloxybenzoates and the enzyme hygromycin 3"-0-phosphotransferase (hpt) which confers resistance to hygromycin. Also available are chloramphenicol transferase (cat) which detoxifies chloramphenicol and dihydrofolate reductase (dhfr) which neutralizes the toxic effects of methotrexate. Another possibility is to use the bar gene for resistance to the herbicide bialaphos (WO 97/05829}. Although there already are a number of selectable markers available, there is still need for another marker. This is due to several reasons: when transgenic plants are being transformed for a second time with a new construct it is necessary to select for the newly formed transformants with the help of a second selectable marker. the above mentioned selection markers are not applicable on in all plant species. some of the compounds which have to be added to enable selection are antibiotics. Spreading of genes which give resistance to antibiotics or herbicides should be minimized as much as possible to avoid the risk of conferring resistance to pathogens. some of the compounds which have to be added to enable selection are relatively expensive. There is a need for cheaper selection agents. SUMMARY OF THE INVEHTION The invention now provides the use of a gene coding for cyanamide hydratase (CAH) as a new selection marker. Preferably this can be used for the transformation of plants. The gene comprises the Nucleotide sequence of SEQIDNO: 1 or muteins thereof having cyanamide hydratase function. The invention comprises a method for the selection of transformed plants which comprises constructing a vector carrying a coding sequence for CAH and a gene of interest, transforming the vector to plants or plant parts or plant cells or callus and growing the resulting transformants in a medium which comprises cyanamide. The invention is also directed to the use of cyanamide for the selection of plants transformed with a gene coding for CAH. Further part of the invention are expression cassettes comprising a nucleotide sequence coding for cyanamide-hydratase and a gene of interest. Also part of the invention are vectors with this expression cassette and hosts, including Agrobacterium, harboring such a vector. Further, plants transformed with such a vector and/or such an Agrobacterium form part of the invention. Accordingly the invention provides a method for selection of transformed plants comprising: (a) constructing a vector comprising a coding sequence for cyanamide hydratase and a gene of interest wherein said cyanamide hydratase is the selection marker, and, (b) transforming plants or plant parts or plant cells with said vector, and (c) growing the transformants in a medium comprising cyanamide wherein said coding sequence comprises the nucleotide sequence of SEQ ID NO.l. Description of the Figures of the Accompanying Drawings Fig.l. outilne of the T-DNA in pMOG874 Fig.2 outline of the T-DNA in pMOG1156 Fig .3 outKne of the T-DNA in PMOG22 Fig.4 outline of the T-DNA In pMOGlOOS Fig.5 outHne of the T-DNA in pMOG1278 Fig.6 outline of the T-DNA in pMOG1295 Fig7 outline of the T-DNA in pMOG1253 Fig .8 outline of the expression cassette In pMOG873 Fig .9 outline of the expression cassette in pMOG617 DETAILED DESCRIPTION OF THE INVENTION The invention is directed to the use of a gene coding for cyanamide hydratase as a selectable marker. The enzyme cyanamide hydratase (CAH) confers resistance to cyanamide which is a compound that has herbicidal activity. It has now been found that this property of the gene can be used in transformation technology to help in discerning transformed plants from non-transformed plants. However, the herbicidal activity alone is not sufficient to make a gene useful as a selectable marker. For that it is also needed that the gene is expressed in those cells which are submitted to selective conditions. This can be either by constitutive expression or expression in specific tissues like callus, seed, embryogenic tissues and meristematic tissues, Furthermore, it is needed that the gene converts susceptibility of a plant to a toxic compound into tolerance without any residual toxic activity. Also the presence of a large enough Window" between the concentration of toxic compound needed for selection and the concentration which in the presence of the selection gene at which still growth can be seen is of importance for the use of a selection marker gene. In addition, the system should preferably function sufficiently cell autonomously, such that in a chimaeric tissue (i.e. a tissue with a mosaic of transformed and untransformed cells) untransformed cells are not protected by neighboring transformed cells and therefore survive selection. Surprisingly, the combination of the gene coding for CAH and the toxic properties of cyanamide qualify for their use as selection marker system. This invention shows that it is possible to select transformants on basis of their tolerance to cyanamide. An additional advantage is that the cyanamide is converted into urea which is converted in various plants in NH3 and CO2. The NH3 can be used by the plant as source of nitrogen. This is an additional selection possibility to increase the ""window" between tolerance and selection. Normally, the culture media contain ammonia and nitrate (contained in the Murashige and Skoog media, see Table 2 and 4). If these are left out or their concentration is decreased the transformed plants containing the CAH gene will convert the cyanamide present in the medium as selection agent into urea and further into ammonia which can be used as nitrogen source. The non-transformed plants are unable to do so, thus in addition to the herbicidal effect of cyanamide they will also suffer from a competitive disadvantage in the area of nitrogen uptake. The nucleotide sequence coding for CAH is preferably the sequence as depicted in SEQIDNO:!. Also muteins of this sequence may be considered as being part of the invention. Muteins are nucleotide sequences which alter in their nucleotide sequence but still have similar functional and immunological characteristics as the sequence presented in SEQIDNO:!. These muteins are also called functional variants. In addition, the polynucleotides of the invention specifically include those sequences substantially identical (determined as described below) with the gene sequences of the invention and that encode proteins that retain the functional activity of the proteins of the invention. Thus, in the case of the CAH gene disclosed here, the above term includes variant polynucleotide sequences which have substantial identity with the sequences disclosed here and which encode proteins which still have cyanamide degrading activity. "Percentage of sequence identity" for polynucleotides and polypeptides is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions {i.e. gaps) as compared to the reference sequence (which does not comprise additions or deletions} for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of match positions, dividing the number of match positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Optimal alignment of sequences for comparison may be conducted by computerized implementations of known algorithms (e.g., GAP, BESTFIT, FASTA and TFAST in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI, or BlastN and BlastX available from the National Center for Biotechnology Information), or by inspection. The term "substantial identity" or "substantial similarity" means that a polypeptide comprises a sequence that is able to hybridize with the target polypeptide under stringent conditions. With stringent conditions a solution of 2 * SSC and a temperature of 65C is meant. Polypeptides which are "substantially similar" share sequences as noted above except that residue positions which are not identical may differ by conservative amino acid changes. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine and isoleucine: a group_ of amino acids having aliphatic-hydroxyl side chains is serine and threonine: a group of amino acids having amide-containing side chains is asparagine and glutamine: a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan: a group of amino acids having basic side chains is lysine,arginine and histidine: and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Substantial identity of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 70% sequence identity, preferably at least 80%, more preferably at least 90% and most preferably at least 95%. Another indication that nucleotide sequences are substantially identical is if two molecules specifically hybridize to each other under stringent conditions. Stringent conditions are sequence dependent and will be different in different circumstances. Generally, stringent conditions are selected to be about 10 °C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. The Tm of a hybrid, which is a function of both the length and the base composition of the probe, can be calculated using information in Sambrook, T. et al. (1989) Molecular Cloning - A Laboratory Manual {second edition), Volume 1-3, Cold Spring Harbor Laboratory, Cold Spring. Typically, stringent conditions for a Southern blot protocol involve washing at 65C with 0.2 X SSC. For preferred oligonucleotide probes, washing conditions are typically about 42"C in 6X SSC. The present invention provides a chimeric DNA sequence which comprises an open reading frame capable of encoding a protein having cyanamide hydratase activity. The term chimeric DNA sequence shall mean to comprise any DNA sequence which comprises DNA sequences not naturally found in nature. For instance, chimeric DNA shall mean to comprise DNA comprising the said open reading frame in a non-natural location of the plant genome, even if said plant genome would normally contain a copy of the said open reading frame in its natural chromosomal location. Similarly, the said open reading frame may be incorporated in the plant genome wherein it is not naturally found, or in a replicon or vector where it is not naturally found, such as a bacterial plasmid or a viral vector. Chimeric DNA shall not be limited to DNA molecules which are replicable in a host, but shall also mean to comprise DNA capable of being ligated into a replicon, for instance by virtue of specific adaptor sequences, physically linked to the open reading frame according to the invention. The open reading frame may or may not be linked to its natural upstream and downstream regulatory elements. The open reading frame may be derived from a genomic library. In this latter it may contain one or more introns separating the exons making up the open reading frame that encodes a protein according to the invention. The open reading frame may also be encoded by one uninterrupted exon, or by a cDNA to the mRNA encoding a protein according to the invention. Open reading frames according to the invention also comprise those in which one or more introns have been artificially removed or added. Each of these variants is embraced by the present invention. Preferably the open reading frame is derived from the soil fungus Myrothecium verrucaria (as described in Maier-Greiner, U.H. et al,, Proc. Natl. Acad. Sci. USA , 4260-4264, 1991), In order to be capable of being expressed in a host cell in a way that the expressed protein can confer resistance to the toxic selection agent, a chimeric DNA according to the invention will usually be provided in an expression cassette with regulatory elements enabling it to be recognized by the biochemical machinery of the host and allowing for the open reading frame to be transcribed and translated in the host. It will usually comprise a transcriptional initiation region which may be suitably derived from any gene capable of being expressed in the host cell of choice, as well as a translational initiation region for ribosome recognition and attachment. In eukaryotic plant cells, an expression cassette usually comprises in addition a transcriptional termination region located downstream of said open reading frame, allowing transcription to terminate and polyadenylation of the primary transcript to occur. In addition, the codon usage may be adapted to accepted codon usage of the host of choice. The principles governing the expression of a chimeric DNA construct in a chosen host cell are commonly understood by those of ordinary skill in the art and the construction of expressible chimeric DNA constructs is now routine for any sort of host cell, be it prokaryotic or eukaryotic. In order for the open reading frame to be maintained in a host cell it will usually be provided in the form of a replicon comprising said open reading frame according to the invention linked to DNA which is recognized and replicated by the chosen host cell. Accordingly the selection of the replicon is determined largely by the host cell of choice. Such principles as govern the selection of suitable replicons for a particular chosen host are well within the realm of the ordinary skilled person in the art. A special type of replicon is one capable of transferring itself, or a part thereof, to another host cell, such as a plant cell, thereby co-transferring the open reading frame according to the invention to said plant cell. Replicons with such capability are herein referred to as vectors. An example of such vector is a Ti-plasmid vector which, when present in a suitable host, such as Agrohacteriuw turnsfaciens, is capable of transferring part of itself, the so-called T-region, to a plant cell. Different types of Ti-plasmid vectors {vide: EP 0 116 718 Bl} are now routinely being used to transfer chimeric DNA sequences into plant cells, or protoplasts, from which new plants may be generated which stably incorporate said chimeric DNA in their genomes. A particularly preferred form of Ti-plasmid vectors are the so-called binary vectors as claimed in (EP 0 120 516 Bl and US 4,940,838) . Other suitable vectors, which may be used to introduce DNA according to the invention into a plant host, may be selected from the viral vectors, e.g. non~integrative plant viral vectors, such as derivable from the double stranded plant viruses (e.g. CaMV) and single stranded viruses, gemini viruses and the like. The use of such vectors may be advantageous, particularly when it is difficult to stably transform the plant host. Such may be the case with woody species, especially trees and vines. The expression "host cells incorporating a chimeric DNA sequence according to the invention in their genome" shall mean to comprise cells, as well as multicellular organisms comprising such cells, or essentially consisting of such cells, which stably incorporate said chimeric DNA into their genome thereby maintaining the chimeric DNA, and preferably transmitting a copy of such chimeric DNA to progeny cells, be it through mitosis or meiosis. Such host cells can be prokaryotic organisms such as bacteria, but also eukaryotic organisms such as yeast. Also cells from eukaryotes in tissue culture, such as cell cultures of plants or animals like mammals can be envisaged to stably incorporate the chimeric DNA. According to a preferred embodiment of the invention plants are provided, which essentially consist of cells which incorporate one or more copies of said chimeric DNA into their genome, and which are capable of transmitting a copy or copies to their progeny, preferably in a Mendelian fashion. By virtue of the transcription and translation of the chimeric DNA according to the invention those cells that produce the CAH will show enhanced resistance to cyanamide. Although the principles which govern transcription of DNA in plant cells are not always understood, the creation of chimeric DNA capable of being expressed in tissue which is subject to selection by cyanamide, such as callus, seed, embryogenetic tissues or meristematic tissues, or constitutive expression, is now routine. Transcription initiation regions routinely in use for expression of the transformed polynucleotide in a constitutive way are promoters obtainable from the cauliflower mosaic virus, notably the 35S RNA and 193 RNA transcript promoters and the so-called T-DNA promoters of Agrobacterium tumefaciens. In particular to be mentioned are the nopaline synthase promoter, octopine synthase promoter (as disclosed in EP 0 122 791 Bl) and the mannopine synthase promoter. In addition plant promoters may be used, which may be substantially constitutive, such as the rice actin gene promoter. The choice of the promoter is not essential, although it must be clear that constitutive high-level promoters should show expression in tissue on which the selection takes place. It is further known that duplication of certain elements, so-called enhancers, may considerably enhance the expression level of the DNA under its regime [vide for instance: Kay R. et al. (1987), Science Z36, 1299-1302: the duplication of the sequence between -343 and -90 of the CaMV 35S promoter increases the activity of that promoter). In addition to the 35S promoter, singly or doubly enhanced, examples of high-level promoters are the light-inducible ribulose bisphosphate carboxylase small subunit {ri?cSSU} promoter and the chlorophyll a/b binding protein (Cab) promoter. Also envisaged by the present invention are hybrid promoters, which comprise elements of different promoter regions physically linked. A well known example thereof is the so-called CaMV enhanced mannopine synthase promoter (US Patent 5,106,739), which comprises elements of the mannopine synthase promoter linked to the CaMV enhancer. Specifically with monocot transformation the use of introns between promoter and selectable marker gene enhances expression. The term "promoter" thus refers to a region of DNA upstream from the structural gene and involved in recognition and binding RNA polymerase and other proteins to initiate transcription. A "plant promoter" is a promoter capable of initiating transcription in plant cells. A "constitutive promoter" is a promoter which is active under most environmental conditions and states of development or cell differentiation. A constitutive promoter is preferable for this invention because selection for transformants may be made at various stages and with various tissues. Thus a constitutive promoter does not limit the selection possibilities. Choice of an appropriate constitutive promoter in this respect is of importance for the use of other promoters in the same transformation process. It is known that duplication of promoters is influential to the expression of the genes under control of said promoters. Since it is the goal of the expression of a selection marker only to be used for selection of plants which are simultaneously transformed with a gene of interest one should keep in mind that using the same promoter for the selectable marker gene and the gene of interest can cause problems. As regards the necessity or a transcriptional terminator region, it is generally believed that such a region enhances the reliability as well as the efficiency of transcription in plant cells. Use thereof is therefore strongly preferred in the context of the present invention. As regards the applicability of the invention in different plant species, it has to be mentioned that one particular embodiment of the invention is merely illustrated with transgenic tomato, potato, rice and Arabidopsis plants as an example, the actual applicability being in fact not limited to these plant species. Although some of the embodiments of the invention may not be practicable at present, e.g. because some plant species are as yet recalcitrant to genetic transformation, the practicing of the invention in such plant species is merely a matter of time and not a matter of principle, because the amenability to genetic transformation as such is of no relevance to the underlying embodiment of the invention. "Transformation of plants" is meant to be any method in which DNA is introduced into a plant. Such a transformation process should not necessarily contain a regeneration and/or tissue culture period. Transformation of plant species is now routine for an impressive number of plant species, including both the Dicotyledoneae as well as the Monocotyiedoneae. In principle any transformation method may be used to introduce chimeric DNA according to the invention into a suitable ancestor cell. Methods may suitably be selected from the calcium/polyethylene glycol method for protoplasts (Krens, F.A. et al., 1982, Nature £, 12-1 A; Negrutiu I. et al, June 1987, Plant Mol. Biol. 8, 363-373), electroporation of protoplasts (Shillito R.D. et al,, 1985 Bio/Technol. 3, 1099-1102), microinjection into plant material (Crossway A. et al., 1986, Mol. Gen. Genet. 2£2, 179-185), (DNA or RNA-coated) particle bombardment of various plant material (Klein T.M. et al., 1987, Nature 327, 70), infection with (non-integrative) viruses, in planta Agrobacterium tumefaciens mediated gene transfer by infiltration of plants or transformation of mature pollen or microspores (EP 0 301 316} and the like. A preferred method according to the invention comprises grobacterium-mediated DNA transfer. Especially preferred is the use of the so-called binary vector technology as disclosed in EP A 120 516 and U.S. Patent 4,940,838). Tomato transformation is preferably done essentially as described by Van Roekel et al. (Van Roekel, J.S.C., et ai. Plant Cell Rep. 1, 64 4-647) . Potato transformation is preferably done essentially as described by Hoekema et al. {Hoekema, A., et al. 1, 273-278 1989). Although considered somewhat more recalcitrant towards genetic transformation, monocotyledonous plants are amenable to transformation and fertile transgenic plants can be regenerated from transformed cells or embryos, or other plant material. Presently, preferred methods for transformation of monocots are microprojectile bombardment of embryos, explants or suspension cells, and direct DNA uptake or (tissue) electroporation (Shimamoto, et al., Nature 338, 274-276, 1989). Transgenic maize plants have been obtained by introducing the Streptomyces hygroscopicus jbar-gene, which encodes phosphinothricin acetyltransferase (an enzyme which inactivates the herbicide phosphinothricin), into embryogenic cells of a maize suspension culture by microprojectile bombardment (Gordon-Kamm, Plant Cell, 2, 603-618, 1990) . Wheat plants have been regenerated from embryogenic suspension culture by selecting embryogenic callus for the establishment of the embryogenic suspension cultures (Vasil Bio/Technol. 8, 429-434, 1990) . The combination with transformation systems for these crops enables the application of the present invention to monocots. Monocotyledonous plants, including commercially important crops such as rice and corn are also amenable to DNA transfer by Agrobacterium strains (vide WO 94/00977; EP 0 159 418 Bl; Gould J, Michael D, Hasegawa O, Ulian EC, Peterson G, Smith RH, Plant. Physiol. , 426-434, 1991). To obtain transgenic plants capable of expressing more than one chimeric gene, a number of alternatives are available including the following: A. The use of DNA, e.g a T-DNA on a binary plasmid, with a number of modified genes physically coupled to a second selectable marker gene. The advantage of this method is that the chimeric genes are physically coupled and therefore migrate as a single Mendelian locus. The invention is especially useful in this respect, since it enables for a second selectable marker which can be introduced next to an already existing selectable marker- gene of interest combination. Thus selection for retransformants can be performed irrespective of the nature of the first selectable marker. B. Cross-pollination of transgenic plants each already capable of expressing one or more chimeric genes, preferably coupled to a selectable marker gene, with pollen from a transgenic plant which contains one or more chimeric genes coupled to another selectable marker. Afterwards the seed, which is obtained by this crossing, maybe selected on the basis of the presence of the two selectable markers, or on the basis of the presence of the chimeric genes themselves. The plants obtained from the selected seeds can afterwards be used for further crossing. In principle the chimeric genes are not on a single locus and the genes may therefore segregate as independent loci. Also here the option to select for both selectable markers is one of the advantages of the present invention. C. The use of a number of a plurality of chimeric DNA molecules, e.g. plasmids, each having one or more chimeric genes and a selectable marker. If the frequency of co- transformation is high, then selection on the basis of only one marker is sufficient. In other cases, the selection on the basis of more than one marker is preferred. D. Consecutive transformation of transgenic plants already containing a first, second, (etc), chimeric gene with new chimeric DNA, optionally comprising a selectable marker gene. As in method B,the chimeric genes are in principle not on a single locus and the chimeric genes may therefore segregate as independent loci. E. Combinations of the above mentioned strategies. The actual strategy may depend on several considerations as maybe easily determined such as the purpose of the parental lines (direct growing, use in a breeding program, use to produce hybrids) but is not critical with respect to the described invention. Although not necessary for this invention, it is known that practically all plants can be regenerated from cultured cells or tissues. The means for regeneration vary from species to species of plants, but generally a suspension of transformed protoplasts or a petri plate containing transformed explants is first provided. Shoots may be induced directly, or indirectly (from callus) via organogenesis or embryogenesis and subsequently rooted. Next to the selective compound, the culture media will generally contain various amino acids and hormones, such as auxin and cytokinins. Efficient regeneration will depend on the medium, on the genotype and on the history of the culture. If these three variables are controlled regeneration is usually reproducable and repeatable. After stable incorporation of the transformed gene sequences into the transgenic plants, the traits conferred by them can be transferred to other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed. Example 1 Cloning the fungal gene encoding cyanamide hydratase (CAH) in a heterologous expression cassette a. Constructs for transformation to dicots Construct pMOG874 contains the coding region from the cyanamide hydratase gene from the soil fungus Myrothecium verrucarla which is operably linked to the CaMV 35S promoter and the CaMV 35S terminator. This chimeric gene is cloned in the binary vector pBIlOl (Jefferson et al. EMBO J. 6, 3901/ 1987) replacing the -glucuronidase coding region and the nopaline synthase terminator. The construct is obtained by adding an Xhol site at the 5" end and a SstI site at the 3" end of a 899 bp cDNA fragment of CAH (position 23 5-1197 of sequence published by Maier-Greiner et al. (1991) Proc. Natl. Acad. Sci. USA 88:4260-4 2 64} by PCR using the primers pi: 5"ACCGAGCTCGAATTCGGCACGAGGTTGACATGATACCTTCCTG 3" and p2: 5"GACCTCGAGAATTCGGCACGAGGTACGATCCTACTTCCTCGC 3" between the sites Xhol and SstI of the plant expression vector pRTlOl, both sites belonging to the polylinker which is inserted between the 35S promoter and the 35S termination signal of pRTlOl (Topfer et al. 1987, Nucl. Acids Res. 15: 5890). The chimeric gene is then cleaved with PstI, overhanging ends are polished with T4 DNA polymerase and the fragment is cloned blunt in the Smal site of pBIN19 (Bevan, M. Nucl. Acids Res. 12:8711-8721, 1984). In construct pMOG1156 an additional ii-glucuronidase gene operably linked to the 35S promoter and the 35S terminator is inserted as Xhol/Sall fragment in the Sail site of pMOG874. Both constructs contain in addition to the novel CAH selection marker the conventional NPTII selection marker linked to the nopaline synthase promoter and the nopaline synthase terminator as it is in pBINl9. b. Constructs for transformation to monocots In the same way as pMOG874 was made the expression cassette was cloned into a high copy vector (pRTlOl, Topfer, R. et al., Nucl. Acids Res. 15, 5890, 1987) resulting in pMOG873 (fig. 8) A derivative of pMOG22 (fig. 3, deposited at the Centraal Bureau voor Schimmelcultures, Baarn, The Netherlands on January 29, 1990, under no. CBS 101.90) was made by introducing a Kpn I restriction site in the polyl inker of pMOG22 between the EcoR I and Sma I site. The orientation of the polylinker was also reversed. This plasmid, denominated pMOGlOOS, contains a hygromycin resistance gene between the left and right T-DNA borders (fig. 4). The 1.7 kb expression cassette comprising the cah gene under control of the 35S promoter and the 35S terminator was cloned between the Hind III and BamH I restriction sites. This plasmid was denominated pMOG1278 (fig. 5}. Binary vector pMOG1295 (fig. 6) is a derivative of pMOG1278 and contains in the Sal I restriction site a GUS-expression cassette as described in Vancanneyt, G. et al. {Mol. Gen. Genet., , 245-250, 1990}. pMOG1253 was made departing from pMOGlS (Sijmons, P.C. et al,, Bio/Technol. 8, 217-221, 1990) which contains the double enhanced 35S promoter, the AlMV RNA4 leader sequence, the GUS-gene and the nos-terminator in an expression cassette as an EcoR I - Hind III fragment. The plasmid p35S GUS INT (Vancanneyt, 1990) was digested with SnaB I and Msc I; the resulting 426 bp fragment, containing part of the coding region for the GUS gene and ST-LSl intron, was isolated and cloned into pMOGlB linearized with SnaB I and Msc I. From the resulting plasmid a 3189 bp EcoR I - Hind III fragment was isolated and cloned into pMOG22, resulting in pMOG1253 (fig. 7). pMOG617 (fig. 9) was made by cloning the hygromycin expression cassette from pMOG22 in the Hind III site of high copy vector pMOGlS. Exaiig)le 2 Potato transformation Described below is the method used for transformation of stem segments of Solanum tuberosum cv. Kardal using Agrobacterium tumefaciens. Nodal explants from in vitro grown potato plants were used 3 to 8 weeks after transfer. The plants were grown on Multiplication Medium (MUM) under a 16h light period (1700 lux) at 24°C and a 8 h dark period at 21°C {The various media can be found in Table 2). Stem segments of approximately 5 mm were cut on sterile filter paper soaked with Washing Medium (WAM) and collected in a flask containing Washing Medium. For approximately 300 explants the Washing Medium was replaced by Pre cultivation Medium (PRM). The flasks were cultured at 80 rpm at the same culture conditions as described above for approximately 24 h. All binary vectors used in this study contained the nptll gene as a plant selectable marker and the nptlll as bacterial selectable marker. Plasmid pMOG410 additionally harbored a chimeric gus gene containing an intron (Vancanneyt et al. Mol. Gen. Genet., 220, 245-250, 1990). Plasmid pMOG1156 additionally harbored the gus gene and the chimeric cah gene encoding cyanamide hydratase. Plasmid pMOG874 additionally harbored the cah gene. Plasmids were maintained in E.coli and A. tumefaciens under kanamycin selection. The Agrojbacterium strain used in this study harbored a rifampicin selection marker in a C58 chromosomal background. The construction of the helper strain EHA105 is described by Hood et al. (1993), Transg. Res. 2, 208-218. Agrobacteria were grown overnight in LB medium with antibiotics (rifampicin 2D mg/1, kanamycin 100 mg/1). The overnight culture was diluted to OD6oo= 0 1 and grown to OD6oo= 0.3 in LB without antibiotics in approximately 2 h time. Bacterial suspensions were centrifuged at 1600xg for 15 minutes at room temperature. Bacteria were resuspended in Washing Medium and used for cocultivation experiments. The Pre cultivation Medium was removed from the flasks and replaced by the Agrobacterium suspension. The flasks were incubated for 20 minutes after which the explants were rinsed twice with Washing Medium. The explants were dried on sterile filter paper and incubated for 48 h on plates containing Cocultivation Medium (COM). Then, the explants were transferred to Post cultivation Medium (POM) and incubated for 72 h. The explants were then transferred to Shoot inducing Medium (SIM) containing several concentrations cyanamide or kanamycin. After two weeks the explants were subcultured on the same medium and approximately three weeks later the explants were placed on Shoot elongation Medium (SEM) containing cyanamide or kanamycin as mentioned above. When shoots were large enough to cut they were transferred to Root inducing Medium (RIM). Shoots that were able to root were then transferred to Root inducing Medium containing 50 mg/1 cyanamide or 30 mg/1 kanamycin. Simultaneously the transgenic nature of the shoots was determined by testing leaflets of the rooted shoots for expression of the gus gene using a histochemical GUS assay. It appeared that for pMOG1156 rooting of transgenic shoots on medium containing cyanamide was completely correlated with expression of the gus gene. Described below is the method used for transformation of cotyledons of Lycopersicon esculentum cv. Money Maker using Agrobacterium tumefaclens. The binary vectors and Agrobacteria strains for this transformation method are identical to those described above. Tomato seedlings were germinated on Germination Medium (GEM) under a 16 h light period (1700 lux) at 240 and a 8 h dark period at 21°C (The contents of the various media can be found in Table 4). Cotyledon explants of 5 to 7 day old seedlings were cut on sterile filter paper soaked with Washing Medium (WAM) and placed on plates containing Cocultivation Medium (COM)- The plates, each containing approximately 50 explants, were incubated overnight under the same conditions as described above. The pre incubated explants were carefully submerged in the Agrobacterium inoculi:mi for 20 minutes. The explants were then blotted dry on sterile filter paper and incubated for 4 8 h on the second set of Cocultivation plates. In procession the explants were incubated for 72 h on plates containing Postcultivation Medixjrn (POM) after which the explants were transferred to Shoot inducing Medium (SIM) containing several concentrations of cyanamide or kanamycin. Every three weeks the explants were subcultured on the same medium. After approximately 8-12 weeks shoots were excised and placed on Root inducing Medium (RIM). Shoots that were able to root were then transferred to Root inducing Medium containing 50 mg/1 cyanamide or 30 mg/1 kanamycin. Simultaneously leaflets of the rooted shoots were tested for expression of the gus gene in a histochemical GUS assay. E»ang>le 3 Tomato transforniation Described below is the method used for transformation of cotyledons of Lycopersicon esculentum cv. Money Maker using Agrobacterium tumefaciens. The binary vectors and Agrobacteria strains for this transformation method are identical to those described above. Tomato seedlings were germinated on Germination Medium (GEM) under a 16 h light period (1700 lux) at 24°C and a 8 h dark period at 21 C (The contents of the various media can be found in Table 4). Cotyledon explants of 5 to 7 day old seedlings were cut on sterile filter paper soaked with Washing Medium (WAM) and placed on plates containing Cocultivation Medium (COM). The plates, each containing approximately 50 explants, were incubated overnight under the same conditions as described above. The pre incubated explants were carefully submerged in the Agrobacterium inoculum for 20 minutes. The explants were then blotted dry on sterile filter paper and incubated for 48 h on the second set of Cocultivation plates. In procession the explants were incubated for 72 h on plates containing Postcultivation Medium (POM) after which the explants were transferred to Shoot inducing Medium (SIM) containing several concentrations of cyanamide or kanamycin. Every three weeks the explants were subcultured on the same medium. After approximately 8-12 weeks shoots were excised and placed on Root inducing Medium (RIM) . Shoots that were able to root were then transferred to Root inducing Medium containing 50 mg/1 cyanamide or 30 mg/1 kanamycin. Simultaneously leaflets of the rooted shoots were tested for expression of the gus gene in a histochemical GUS assay. Described below is the method used for transformation of root segments of Arabidopsis thaliana cv. C24 using Agrobacterium tumefaciens. The binary vectors for this transformation method are identical to those described above. Six mg of Arabidopsis seeds were germinated in a flask containing liquid Germination Medium (GM) under 16 h light period (1700 lux) at 24°C and a 8 h dark period at 21"C at 80 rpm. (The contents of various media can be found in Table 4). Roots of 9 days old seedlings were isolated in a sterile petridish and collected in a drop of Germination Medium (GM). Roots were cut in segments of approximately 3-5 mm and approximately 100 explants were spread evenly on a nylon membrane {0 8 cm) which was placed on plates containing Callus Inducing Medium (CIM). The plates were incubated 3 days under the same conditions as described above. The Agrobacterium strain used in this study harbored a rifampicin selection marker in a C 58 chromosomal background. The construction of the helper strain MOGlOl is described by Hood et al. (1993). Agrobacteria were grown overnight in LB medium with antibiotics (rifampicin 20 mg/1, kanamycin 100 mg/1) . The overnight culture was diluted 1:10 in LB without antibiotics and grown for approximately 3 hours. Bacterial suspensions were centrifuged at 1600xg for 15 minutes at room temperature. Bacteria were resuspended in GM and adjusted to OD600=0.1 and used for cocultivation. The membrane containing approximately 100 explants was incubated for 2 minutes with the Agrobacterium suspension and dried on sterile filter paper to remove excess of bacteria. The membrane with explants are cultured for 4 8 h on CIM plates. After rinsing the membrane and explants with liquid GM these were incubated on Shoot Induction Medium (SIM) plates containing several concentrations of cyanamide or kanamycin. After 5 days the membrane with the explants was transferred to the same medium (SIM) for subculture. The second subculture was after 2 weeks. Approximately four weeks after cocultivation 60 shoots per cyanamide concentration were excised and placed on plates with Shoot Elongation Medium (SEM) containing 30 mg/1 cyanamide. Shoots which were able to root are tested on their transgenic character by testing leaflets and flowers for expression of the gus gene using a histochemical GUS assay. Three experiments were performed. Shoots obtained from Exp. 98-8 and 98-11 were transferred to rooting medium (SEM) containing 30 mg/litre cyanamide. Shoots obtained from Exp. 98-13 were transferred to rooting medium containing the same concentration as the selection medium (SIM), for results see Table 4a. The shoots obtained from the kanamycin selection (50 mg/litre) were transferred to rooting medium containing 25 mg/litre Kanamycin. Root explants transformed with pMOG 410 were not able to regenerate on cyanamide containing medium. Even 20 mg/litre cyanamide was already enough to prevent regeneration of explants transformed with a construct without the cah gene. At 20 till 4 0 mg/litre cyanamide some callus development was observed, but at 50 rag/Iitre and higher explants were not viable and turned completely brown. On the other hand explants transformed with the cah gene (pMOG 1156) were able to regenerate at all cyanamide concentrations, even at 80 mg/litre. At lower concentrations the regeneration of shoots was faster than with kanamycin. Although more shoots were available 60-65 shoots were harvested per treatment and placed on rooting medium. At the lower cyanamide concentrations the same amount of shoots developed as with kanamycin selection (approx. 70-100 per petridish). There is a clear correlation between callus development and GUS expression on cyanamide selection with root explants transformed with pMOG 1156 (fig. 4b). GUS analysis of shoots obtained on cyanamide 0 mg/litre (NS) showed no staining, indicating that cyanamide is needed to obtain transgenic Described below is the method used for transformation of callus derived from scutellum of mature embryos of Oryza satlva cv. Taipei 309 using AgroJbacterliun tumefaciens strain LEAlll9-pMOGl295 (harboring the cah-gene) and strain LBA1119-pMOGl253 (control). Sterile dehusked rice seeds were germinated on plates containing Callus Induction Medium (CIM) in the dark at 28""C. (The contents of various media can be found in Table 5) . After 3 weeks embryogenic callus derived from the scutellum is isolated and subcultured on the same medium under the same conditions. After 2-3 weeks embryogenic calli were cut in segments of approximately 2-3 mm and cultured plates containing CIM for 4 days. The Agrobacterium strains used in this study harbored a rifampicin selection marker in a C 58 chromosomal background. The construction of the helper strain EHA105 is described by Hood et al. (1993). Agrobacteria were grown for 4 days on plates containing AB medium with antibiotics (rifampicin 20 mg/1, kanamycin 100 mg/1} . Agrohacteria were collected in LIM and the OD600 was adjusted till 1.0-1.5. This suspension was used for cocultivation. Calli were incubated for 10 minutes with the Agrobacterium suspension and dried on sterile filter paper to remove excess of bacteria. Calli were cultured for 48 h on Coculture Medium (COM) plates at 2b°C in the dark. 50 pMOG1295 calli and 20 pMOGl253 calli were cultured per concentration of cyanamid. The following concentrations of cyanamide were used: 0, 15, 30, 60, 100, 150, 200, 300 and 500 mg/1. Hygromycin was applied in a concentration of 50 mg/1. Calli were incubated on First Selection Medium (FSM) plates containing several concentrations of cyanamide or hygromycin at 28°C in the dark. After 3 weeks the calli were transferred to Embryo Induction Medium I (EIM I) containing the same concentration of cyanamide or hygromycin . After another 3 weeks the calli were subcultured on Embryo Induction Medium II (EIM II) containing the same concentration of cyanamide or an increased concentration of hygromycin (75 mg/1}. Calli were transferred to Shoot Induction Medium (SIM) containing the same concentration of cyanamide as during FSM, EIM I, EIM II and were cultured under 12 hours light period (2600 LUX) and 12 hours dark at 28 °C. Approximately 3 weeks after transferring calli to SIM, shoots were regenerating and excised and placed in jars containing Pre-Greenhouse Medium (PGM). No calli were formed at concentrations of 100 mg/1 or higher of cyanamid. At 15 mg/1 cyanamide the regeneration frequency of callus from both constructs was the same (pMOG1253 7 out of 16 calli were able to regenerate, pMOG1295 17 out of 44) . At 30 mg/1 cyanamide only 11 calli of pMOG1295 showed green callus development and 6 were able to be regenerated. Table 5. Media required for Oryza sativa Taipei 309 transformation Example 6 Rice transformation by particle gun Described below is the method used for transformation of non- morphogenic cell suspensions of Oryza sativa cv. IR 52 using a particle inflow gun (PIG) according to Finer et al. (Plant Cell Rep. 11, 323-328, 1992). A long-term, non-morphogenic suspension culture of Oryza sativa cv. IR 52 was subcultured in weekly intervals in liquid LS-4 (Linsmaier and Skoog, Physiol. Plant. 18, 100-127, 1962} medium and maintained on a rotary shaker (110 rpm) at 280 in the dark. (The contents of the LS-4 medium can be found in Table Z) . 3-4 days after the last subculture 1.5 ml of this cell suspension (appro. 1.5 x 10 cells) were evenly spread on a filter paper (Whatman no 4) which was subsequently placed on solidified LS-4 medium and cultivated in the dark at 28"C for 24 h and directly used for bombardment thereafter. For microprojectile bombardment a home-made particle inflow gun (PIG) according to Finer et al. (1992) was used. 300 \|ag tungsten particles coated with either pMOG 617 (35S-gus and 35S-hyg) or pMOG 873 {35S-cah) were loaded on a particle support. The particles were accelerated by a 2.5 bar helium pulse and had to pass a 500 \|jm metal stop screen, placed 2 cm under the particle support. The suspension cells were placed 15 cm under the particle support. The PIG was evacuated to 30 mbar before bombardment. After bombardment the cells were cultured at 28°C in the dark for 3 days. Then the filters with the cells were transferred to solid LS-4 medium containing various concentrations of cyanamide or 50 mg/1 hygromycinB (see table 6) . The subculture was repeated every 9 days. Resistant microcalli that were visible after 4-6 weeks were transferred to fresh LS-4 medium containing the respective selective agent. From two experiments 7 + 41 calli transformed with pMOG617 were found resistant to hygromycin, while for the transformation with pMOG873 7 calli survived cyanamide 20 mg/1 in the first experiment (results from the second experiment not yet available) and 0+4 calli remained viable on 40 mg/1 cyanamide. No calli were formed at concentrations of 50 mg/1 cyanamide or higher. The transgenic nature was confirmed by testing parts of the developing callus for the presence of the DNA in the callitransformed with pMOG873. One of the 4 surviving calli on 40 mg/1 cyanamide showed positive in a PCR experiment on the cah-gene. Table 6. Media required for Oryza sati-va cv transformation IR 52 Table 7. pMOG617 pMOG 873 Example 8 Maize killing curve Stock solutions of cyanamide were prepared in water at 10 and 100 mg/ml and filter sterilised. Aliquots were stored at -20°C. Media were prepared by adding MS medium (4.4g), sucrose (20g), 2,4-D C2.0mg) and agar (8g) to 1 litre of water. After autoclaving the appropriate amount of cyanamide (0, 10, 30, 50, 100, 150 mg/L cyanamide) was added and the media was poured into 9cm petri dishes. BMS liquid was prepared as above minus agar. BMS cells were added to media containing cyanamide in three ways; a. BMS cell suspension added to falcon tube and liquid removed. Then the BMS cells were arranged on the surface of the agar in clumps of approximately 5mm in diameter, 5 clumps per plate, 3 plates per concentration, while on the base of each petri dish the outline of each clump was marked; b. Approximately 0.5ml pack cell volume plus 1.5ml BMS liquid added to surface of agar and the cells were spread finely over surface of agar. Three plates per treatment were set up. c. Approximately 0,5ml pack cell volume plus 1.5ml BMS liquid were added to filter paper overlying agar. Cells were spread evenly over surface of filter. One plate per treatment was set up. The plates were sealed with micropore tape and incubated at 25°C in the dark. The growth of the cells was observed after 7 and 14 days. RESULTS Day 8 The growth of BMS cells on cyanamide was assessed after 8 days. The clumps of BMS cells arranged on the surface of the control media had increased in size and outgrown their original outline. Cells on 10 mg/1 had not outgrown their outline but the height of the clumps had increased forming an uneven surface. A slight reduction of growth with increased cyanamide concentration was apparent with the maximum effect on growth observed at 50 mg/1 cyanamid. The cells which were spread over the surface of the control media had grown well and densely covered the surface of the media. A significant reduction in growth was observed on the lowest level of cyanamide (10 mg/1), however, an increased cell density was clearly visible. A slight increase in cell density was evident on 30 mg/1 cyanamid, but it was difficult to distinguish different growth rates on higher concentrations. Cells on all levels of cyanamide remained a milky white colour, no browning of cells was observed. Day 15 {Table 9} The reduction in growth of BMS cells on lOmg/1 was still very clear after 15 days on cyanamide, however, the cells arranged in clumps had outgrown their original outline. Cells spread directly over the surface of the agar showed a similar response to those arranged in clumps with a notable reduction in growth observed on 30mg/l plus. Cells on 50mg/X plus showed no signs of growth and the surface of the clumps remained very flat but the cells were still milky white in colour. A similar response was observed with cell spread over a filter. However, small raised lumps were observed on the surface on all filters but these did not develop further into colonies and were evidently comprised of the larger cell aggregates from a mixed population of sizes typical in BMS suspensions. Samples were taken from the clumps of cells on all levels of cyanamide for observation under a light microscope. With increasing levels of cyanamide there were increasing numbers of dead cells where the cell contents had shrunk away from the cell wall, and an increase in the number of dark bodied starch grains. Cells were observed resuspended in water and with FDA stain under UV light. Table 9. The experiment was repeated with cyanamide concentrations of 0, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 mg/1 cyanamid. The results were similar to those described above, i.e. for the cells aggregated in clumps a slight reduction in growth was seen at 10 mg/1. From a concentration of 20 mg/1 cyanamide on the cell clumps showed no outgrowing from their original outline, but at the lower concentrations ( higher concentrations). Above 50 mg/1 the clumps showed a slight orange tinge. The results with cells spread over the surface of the agar or on the filters were similar in that at a concentration of 10 mg/1 showed a slight growth (approximately doubling of the number of original cells), while at concentrations of 20 mg/1 and higher exhibited limited signs of growth. Example 8 Kill curves in banana (Misa) To test the potential of cyanamide as a selection agent for transformation in banana two kill curves were set up with a regenerable embryogenic suspension of Grand nain 6 day old embryogenic suspension CEd6b) cultures, subbed routinely in M2 2,4D liquid, containing 4.32 g/1 MS salts, 45g/l sucrose, standard Ix concn. MS vitamins, lOOmg/1 glutamine, 100 mg/1 myo-inositol, 100 mg/1 biotin, lOOmg/1 malt extract at pH 5.3 and added after autoclaving 1.2mg/l 2,4-D and 0.8 mg/1 picloram. Cultures were sieved (>250n, Kill Curve Medium A : M2/MS/1.0 2,4-D (as M2/MS/2,4~D except only 1.0 mg/1 2,4-D, no picloram and +2.25 g/1 gelrite): this medium promotes the rapid division and growth of embryogenic callus, but not embryos. Kill Curve Medium B : M2/SH/0.5Pic, 0.5 2,4-D (as M2/MS/2,4-D except only 0.5 mg/1 2,4-D and 0.5 mg/1 picloram, SH salts (4.32g/l) instead of MS,. + 2.25 g/1 gelrite) : This medium promotes the early development of embryos which can be matured and germinated by transfer to alternative media. Cyanamide was added to both media types, after autoclaving, to concentrations of 0,20,30,50,75,100,150 mg/1. The results are depicted in Table 10, where the figures on cell growth are approximate visual estimates, not precise measurements of callus volume. There is no significant visual browning of cultures and release of phenolics until concentrations of >75 mg/1. Generally cultures just stop growing, with cell division being widely inhibited. Cyanamide inhibits the growth of embryogenic callus by 40-50% at even low concentrations of 20mg/l, without causing significant visual damage. Embryogenesis was totally inhibited at the lowest concentration tested here. We claim; 1. Method for selection of transformed plants comprising: (a) constructing a vector comprising a coding sequence for cyanamtde hydratase and a gene of interest wherein said cyanamide hydratase is the selection marker, and, (b) transforming plants or plant parts or plant cells with said vector, and (c) growing the transformants in a medium comprising cyanamide wherein said coding sequence comprises the nucleotide sequence of SEQ ID NO.l. 2. A method according to claim 1 wherein said plants are monocot plants. 3. A method according to claim 2 wherein said plants are selected from the group consisting of; rice; maize and banana plants. 4. A method according to claim 1 wherein said plants are dicot plants. 5. A method according to claim 4 wherein said plants are selected from the group consisting of: potato; tomato and arabldopsls plants. 6. Method for the selection of transformed plants, substantialy as herein described, with reference to the accompanying drawings.

Full Text

FIELD OF THE INVENTION
This application is concerned with a novel selectable marker, especially with the use of cyanamide hydratase as a selection marker in transformation experiments, more specifically in plant transformation experiments.
BACKGROUND ART
Cyanamide (H2N-C=N) is a nitrile derivate which, like other nitrile derivates, is used in agriculture for stimulation of
growth and for plant protection. Cyanamide in aqueous solution or in the form of its calcium salt is used as a fertilizer by providing ammonia to the soil by its metabolic conversion. It has, however, the additional advantage of
acting as a herbicide. To use it as fertilizer it has to be applied before sowing.
Chemically, cyanamide belongs to the class of nitriles. In spite of the relatively rare occurrence in nature of compounds containing the nitrile group, enzymes that hydrate this group have been found in bacteria and plants (e.g. Nagasawa T., et al. (1988) Biochem. Biophys. Res. Commun. 155 1008-1016; Endo T. and Watanabe I. (1989) FEES Lett. 243 61-64) . Also in the fungus Myrothecium verrucaria a nitrile hydrating enzyme was found (Stransky H. and Amberger A. (1973) Z. Pflanzenphysiol. 7 74-87), which hydrates the nitrile group of cyanamide with formation of urea:

Maier-Greiner et al. have isolated the enzyme and cloned the gene coding for it {Proc. Natl. Acad. Sci. USA 8_8, 4260-4264, 1991). They have demonstrated that this enzyme shows an extremely narrow substrate specificity, where compounds chemically related to cyanamide are not recognized as substrates.
Selectable markers have to confer a dominant phenotype on transformed cells which is able of being used as a selection criteria. These falls into two classes: one class of genes which confers either cell viability or lethality in the presence of a selective agent and a class of genes which has negligible effects on cell survival but which confers transformed cells with some distinguishing physical characteristic.

In plant transformation the fraction of cells incorporating the novel DNA is generally low, so most stable transformation schemes use markers which ensure the survival of transformed cells in the presence of a selective agent.
A number of selection markers of this first group has been known and used for plant transformation experiments for several years. Included are the enzyme neomycin phosphotransferase (npt) which confers resistance to a group of antibiotics including kanamycin, paromomycin, geneticin and neomycin, mutant forms of the enzyme acetolactate synthase (als) which confer resistance to imidazolinones, sulfonylureas, triazolopyrimidines and pyrimidyloxybenzoates and the enzyme hygromycin 3"-0-phosphotransferase (hpt) which confers resistance to hygromycin. Also available are chloramphenicol transferase (cat) which detoxifies chloramphenicol and dihydrofolate reductase (dhfr) which neutralizes the toxic effects of methotrexate.
Another possibility is to use the bar gene for resistance to the herbicide bialaphos (WO 97/05829}.
Although there already are a number of selectable markers available, there is still need for another marker. This is due to several reasons:
when transgenic plants are being transformed for a second time with a new construct it is necessary
to select for the newly formed transformants with the help of a second selectable marker.
the above mentioned selection markers are not applicable on in all plant species.
some of the compounds which have to be added to enable selection are antibiotics. Spreading of genes which give resistance to antibiotics or herbicides should be minimized as much as possible to avoid the risk of conferring resistance to pathogens.
some of the compounds which have to be added to enable selection are relatively expensive. There
is a need for cheaper selection agents.
SUMMARY OF THE INVEHTION
The invention now provides the use of a gene coding for cyanamide hydratase (CAH) as a new selection marker. Preferably this can be used for the transformation of plants. The gene comprises the

Nucleotide sequence of SEQIDNO: 1 or muteins thereof having cyanamide hydratase function.
The invention comprises a method for the selection of transformed plants which comprises constructing a vector carrying a coding sequence for CAH and a gene of interest, transforming the vector to plants or plant parts or plant cells or callus and growing the resulting transformants in a medium which comprises cyanamide.
The invention is also directed to the use of cyanamide for the selection of plants transformed with a gene coding for CAH.
Further part of the invention are expression cassettes comprising a nucleotide sequence coding for cyanamide-hydratase and a gene of interest. Also part of the invention are vectors with this expression cassette and hosts, including Agrobacterium, harboring such a vector. Further, plants transformed with such a vector and/or such an Agrobacterium form part of the invention.
Accordingly the invention provides a method for selection of transformed plants comprising:
(a) constructing a vector comprising a coding sequence for cyanamide hydratase and a gene of interest wherein said cyanamide hydratase is the selection marker, and,
(b) transforming plants or plant parts or plant cells with said vector, and
(c) growing the transformants in a medium comprising cyanamide wherein said coding sequence comprises the nucleotide sequence of SEQ ID NO.l.
Description of the Figures of the Accompanying Drawings Fig.l. outilne of the T-DNA in pMOG874 Fig.2 outline of the T-DNA in pMOG1156 Fig .3 outKne of the T-DNA in PMOG22 Fig.4 outline of the T-DNA In pMOGlOOS Fig.5 outHne of the T-DNA in pMOG1278 Fig.6 outline of the T-DNA in pMOG1295 Fig7 outline of the T-DNA in pMOG1253 Fig .8 outline of the expression cassette In pMOG873 Fig .9 outline of the expression cassette in pMOG617

DETAILED DESCRIPTION OF THE INVENTION
The invention is directed to the use of a gene coding for cyanamide hydratase as a selectable marker.
The enzyme cyanamide hydratase (CAH) confers resistance to cyanamide which is a compound that has herbicidal activity. It has now been found that this property of the gene can be used in transformation technology to help in discerning transformed plants from non-transformed plants. However, the herbicidal activity alone is not sufficient to make a gene useful as a selectable marker. For that it is also needed that the gene is expressed in those cells which are submitted to selective conditions. This can be either by constitutive expression or expression in specific tissues like callus, seed, embryogenic tissues and meristematic tissues, Furthermore, it is needed that the gene converts susceptibility of a plant to a toxic compound into tolerance without any residual toxic activity. Also the presence of a large enough Window" between the concentration of toxic compound needed for selection and the concentration which in the presence of the selection gene at which still growth can be seen is of importance for the use of a selection marker gene. In addition, the system should preferably function sufficiently cell autonomously, such that in a chimaeric tissue (i.e. a tissue with a mosaic of transformed and untransformed cells) untransformed cells are not protected by neighboring transformed cells and therefore survive selection.
Surprisingly, the combination of the gene coding for CAH and the toxic properties of cyanamide qualify for their use as selection marker system.
This invention shows that it is possible to select transformants on basis of their tolerance to cyanamide.
An additional advantage is that the cyanamide is converted into urea which is converted in various plants in NH3 and CO2. The NH3 can be used by the plant as source of nitrogen. This is an additional selection possibility to increase the ""window" between tolerance and selection. Normally, the culture media contain ammonia and nitrate (contained in the Murashige and Skoog media, see Table 2 and 4). If these are left out or their concentration is decreased the transformed

plants containing the CAH gene will convert the cyanamide present in the medium as selection agent into urea and further into ammonia which can be used as nitrogen source. The non-transformed plants are unable to do so, thus in addition to the herbicidal effect of cyanamide they will also suffer from a competitive disadvantage in the area of nitrogen uptake.
The nucleotide sequence coding for CAH is preferably the sequence as depicted in SEQIDNO:!. Also muteins of this sequence may be considered as being part of the invention. Muteins are nucleotide sequences which alter in their nucleotide sequence but still have similar functional and immunological characteristics as the sequence presented in SEQIDNO:!. These muteins are also called functional variants. In addition, the polynucleotides of the invention specifically include those sequences substantially identical (determined as described below) with the gene sequences of the invention and that encode proteins that retain the functional activity of the proteins of the invention. Thus, in the case of the CAH gene disclosed here, the above term includes variant polynucleotide sequences which have substantial identity with the sequences disclosed here and which encode proteins which still have cyanamide degrading activity.
"Percentage of sequence identity" for polynucleotides and polypeptides is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions {i.e. gaps) as compared to the reference sequence (which does not comprise additions or deletions} for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of match positions, dividing the number of match positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Optimal alignment of sequences for comparison may be conducted by computerized implementations of known algorithms (e.g., GAP, BESTFIT, FASTA and TFAST in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI, or BlastN and BlastX available from the National Center for Biotechnology Information), or by inspection.
The term "substantial identity" or "substantial similarity" means that a polypeptide comprises a sequence that is able to hybridize with the target polypeptide under stringent conditions. With stringent conditions a solution of 2 * SSC and a temperature of 65*C is meant.

Polypeptides which are "substantially similar" share sequences as noted above except that residue positions which are not identical may differ by conservative amino acid changes. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine and isoleucine: a group_ of amino acids having aliphatic-hydroxyl side chains is serine and threonine: a group of amino acids having amide-containing side chains is asparagine and glutamine: a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan: a group of amino acids having basic side chains is lysine,arginine and histidine: and a group of amino acids having sulfur-containing side chains is cysteine and methionine.
Substantial identity of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 70% sequence identity, preferably at least 80%, more preferably at least 90% and most preferably at least 95%. Another indication that nucleotide sequences are substantially identical is if two molecules specifically hybridize to each other under stringent conditions. Stringent conditions are sequence dependent and will be different in different circumstances. Generally, stringent conditions are selected to be about 10 °C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. The Tm of a hybrid, which is a function of both the length and the base composition of the probe, can be calculated using information in Sambrook, T. et al. (1989) Molecular Cloning - A Laboratory Manual {second edition), Volume 1-3, Cold Spring Harbor Laboratory, Cold Spring. Typically, stringent conditions for a Southern blot protocol involve washing at 65**C with 0.2 X SSC. For preferred oligonucleotide probes, washing conditions are typically about 42"C in 6X SSC.
The present invention provides a chimeric DNA sequence which comprises an open reading frame capable of encoding a protein having cyanamide hydratase activity. The term chimeric DNA sequence shall mean to comprise any DNA sequence which comprises DNA sequences not naturally found in nature. For instance, chimeric DNA shall mean to comprise DNA comprising the said open reading frame in a non-natural location of the plant genome, even if said plant genome would normally contain a copy of the said open reading frame in its natural chromosomal location. Similarly, the said open reading frame may be incorporated in the plant genome wherein it is not naturally found, or in a replicon or vector where it is not naturally found, such as a bacterial plasmid or a viral vector. Chimeric DNA shall not be limited to DNA molecules which are replicable in a host, but shall also mean to comprise DNA capable of being ligated into a replicon, for instance by virtue of specific adaptor

sequences, physically linked to the open reading frame according to the invention. The open reading frame may or may not be linked to its natural upstream and downstream regulatory elements.
The open reading frame may be derived from a genomic library. In this latter it may contain one or more introns separating the exons making up the open reading frame that encodes a protein according to the invention. The open reading frame may also be encoded by one uninterrupted exon, or by a cDNA to the mRNA encoding a protein according to the invention. Open reading frames according to the invention also comprise those in which one or more introns have been artificially removed or added. Each of these variants is embraced by the present invention.
Preferably the open reading frame is derived from the soil fungus Myrothecium verrucaria (as described in Maier-Greiner, U.H. et al,, Proc. Natl. Acad. Sci. USA , 4260-4264, 1991),
In order to be capable of being expressed in a host cell in a way that the expressed protein can confer resistance to the toxic selection agent, a chimeric DNA according to the invention will usually be provided in an expression cassette with regulatory elements enabling it to be recognized by the biochemical machinery of the host and allowing for the open reading frame to be transcribed and translated in the host. It will usually comprise a transcriptional initiation region which may be suitably derived from any gene capable of being expressed in the host cell of choice, as well as a translational initiation region for ribosome recognition and attachment. In eukaryotic plant cells, an expression cassette usually comprises in addition a transcriptional termination region located downstream of said open reading frame, allowing transcription to terminate and polyadenylation of the primary transcript to occur. In addition, the codon usage may be adapted to accepted codon usage of the host of choice. The principles governing the expression of a chimeric DNA construct in a chosen host cell are commonly understood by those of ordinary skill in the art and the construction of expressible chimeric DNA constructs is now routine for any sort of host cell, be it prokaryotic or eukaryotic.
In order for the open reading frame to be maintained in a host cell it will usually be provided in the form of a replicon comprising said open reading frame according to the invention linked to DNA which is recognized and replicated by the chosen host cell. Accordingly the selection of the replicon is determined largely by the host cell of choice. Such principles as govern the selection of suitable replicons for a particular chosen host are well within the realm of the ordinary skilled person in the art.

A special type of replicon is one capable of transferring itself, or a part thereof, to another host cell, such as a plant cell, thereby co-transferring the open reading frame according to the invention to said plant cell. Replicons with such capability are herein referred to as vectors. An example of such vector is a Ti-plasmid vector which, when present in a suitable host, such as Agrohacteriuw turnsfaciens, is capable of transferring part of itself, the so-called T-region, to a plant cell. Different types of Ti-plasmid vectors {vide: EP 0 116 718 Bl} are now routinely being used to transfer chimeric DNA sequences into plant cells, or protoplasts, from which new plants may be generated which stably incorporate said chimeric DNA in their genomes. A particularly preferred form of Ti-plasmid vectors are the so-called binary vectors as claimed in (EP 0 120 516 Bl and US 4,940,838) . Other suitable vectors, which may be used to introduce DNA according to the invention into a plant host, may be selected from the viral vectors, e.g. non~integrative plant viral vectors, such as derivable from the double stranded plant viruses (e.g. CaMV) and single stranded viruses, gemini viruses and the like. The use of such vectors may be advantageous, particularly when it is difficult to stably transform the plant host. Such may be the case with woody species, especially trees and vines.
The expression "host cells incorporating a chimeric DNA sequence according to the invention in their genome" shall mean to comprise cells, as well as multicellular organisms comprising such cells, or essentially consisting of such cells, which stably incorporate said chimeric DNA into their genome thereby maintaining the chimeric DNA, and preferably transmitting a copy of such chimeric DNA to progeny cells, be it through mitosis or meiosis. Such host cells can be prokaryotic organisms such as bacteria, but also eukaryotic organisms such as yeast. Also cells from eukaryotes in tissue culture, such as cell cultures of plants or animals like mammals can be envisaged to stably incorporate the chimeric DNA. According to a preferred embodiment of the invention plants are provided, which essentially consist of cells which incorporate one or more copies of said chimeric DNA into their genome, and which are capable of transmitting a copy or copies to their progeny, preferably in a Mendelian fashion. By virtue of the transcription and translation of the chimeric DNA according to the invention those cells that produce the CAH will show enhanced resistance to cyanamide. Although the principles which govern transcription of DNA in plant cells are not always understood, the creation of chimeric DNA capable of being expressed in tissue which is subject to selection by cyanamide, such as callus, seed, embryogenetic tissues or meristematic tissues, or constitutive expression, is now routine. Transcription initiation regions routinely in use for expression of the transformed polynucleotide in a constitutive way are promoters obtainable from the cauliflower mosaic virus, notably the 35S RNA and 193 RNA transcript promoters and the

so-called T-DNA promoters of Agrobacterium tumefaciens. In particular to be mentioned are the nopaline synthase promoter, octopine synthase promoter (as disclosed in EP 0 122 791 Bl) and the mannopine synthase promoter. In addition plant promoters may be used, which may be substantially constitutive, such as the rice actin gene promoter. The choice of the promoter is not essential, although it must be clear that constitutive high-level promoters should show expression in tissue on which the selection takes place. It is further known that duplication of certain elements, so-called enhancers, may considerably enhance the expression level of the DNA under its regime [vide for instance: Kay R. et al. (1987), Science Z36, 1299-1302: the duplication of the sequence between -343 and -90 of the CaMV 35S promoter increases the activity of that promoter). In addition to the 35S promoter, singly or doubly enhanced, examples of high-level promoters are the light-inducible ribulose bisphosphate carboxylase small subunit {ri?cSSU} promoter and the chlorophyll a/b binding protein (Cab) promoter. Also envisaged by the present invention are hybrid promoters, which comprise elements of different promoter regions physically linked. A well known example thereof is the so-called CaMV enhanced mannopine synthase promoter (US Patent 5,106,739), which comprises elements of the mannopine synthase promoter linked to the CaMV enhancer.
Specifically with monocot transformation the use of introns between promoter and selectable marker gene enhances expression.
The term "promoter" thus refers to a region of DNA upstream from the structural gene and involved in recognition and binding RNA polymerase and other proteins to initiate transcription. A "plant promoter" is a promoter capable of initiating transcription in plant cells. A "constitutive promoter" is a promoter which is active under most environmental conditions and states of development or cell differentiation.
A constitutive promoter is preferable for this invention because selection for transformants may be made at various stages and with various tissues. Thus a constitutive promoter does not limit the selection possibilities.
Choice of an appropriate constitutive promoter in this respect is of importance for the use of other promoters in the same transformation process. It is known that duplication of promoters is influential to the expression of the genes under control of said promoters. Since it is the goal of the expression of a selection marker only to be used for selection of plants which are simultaneously transformed with a gene of interest one should keep in mind that using the same promoter for the selectable marker gene and the gene of interest can cause problems.

As regards the necessity or a transcriptional terminator region, it is generally believed that such a region enhances the reliability as well as the efficiency of transcription in plant cells. Use thereof is therefore strongly preferred in the context of the present invention.
As regards the applicability of the invention in different plant species, it has to be mentioned that one particular embodiment of the invention is merely illustrated with transgenic tomato, potato, rice and Arabidopsis plants as an example, the actual applicability being in fact not limited to these plant species.
Although some of the embodiments of the invention may not be practicable at present, e.g. because some plant
species are as yet recalcitrant to genetic transformation, the practicing of the invention in such plant species is merely a matter of time and not a matter of principle, because the amenability to genetic transformation as such is of no relevance to the underlying embodiment of the invention.
"Transformation of plants" is meant to be any method in which DNA is introduced into a plant. Such a transformation
process should not necessarily contain a regeneration and/or tissue culture period.
Transformation of plant species is now routine for an impressive number of plant species, including both the Dicotyledoneae as well as the Monocotyiedoneae. In principle any transformation method may be used to introduce chimeric DNA according to the invention into a suitable ancestor cell. Methods may suitably be selected from the calcium/polyethylene glycol method for protoplasts (Krens, F.A. et al., 1982, Nature £, 12-1 A; Negrutiu I. et al, June 1987, Plant Mol. Biol. 8, 363-373), electroporation of protoplasts (Shillito R.D. et al,, 1985 Bio/Technol. 3, 1099-1102), microinjection into plant material (Crossway A. et al., 1986, Mol. Gen. Genet. 2£2, 179-185), (DNA or RNA-coated) particle bombardment of various plant material (Klein T.M. et al., 1987, Nature 327, 70), infection with (non-integrative) viruses, in planta Agrobacterium tumefaciens mediated gene transfer by infiltration of plants or transformation of mature pollen or microspores (EP 0 301 316} and the like. A preferred method according to the invention comprises grobacterium-mediated DNA transfer. Especially preferred is the use of the so-called binary vector technology as disclosed in EP A 120 516 and U.S. Patent 4,940,838).
Tomato transformation is preferably done essentially as described by Van Roekel et al. (Van Roekel, J.S.C., et ai. Plant Cell Rep. 1, 64 4-647) . Potato transformation is preferably done essentially as described by Hoekema et al.
{Hoekema, A., et al. 1, 273-278 1989).

Although considered somewhat more recalcitrant towards genetic transformation, monocotyledonous plants are amenable to transformation and fertile transgenic plants can be regenerated from transformed cells or embryos, or other plant material. Presently, preferred methods for transformation of monocots are microprojectile bombardment of embryos, explants or suspension cells, and direct DNA uptake or (tissue) electroporation (Shimamoto, et al., Nature 338, 274-276, 1989). Transgenic maize plants have been obtained by introducing the Streptomyces hygroscopicus jbar-gene, which encodes phosphinothricin acetyltransferase (an enzyme which inactivates the herbicide phosphinothricin), into embryogenic cells of a maize suspension culture by microprojectile bombardment (Gordon-Kamm, Plant Cell, 2, 603-618, 1990) . Wheat plants have been regenerated from embryogenic suspension culture by selecting embryogenic callus for the establishment of the embryogenic suspension cultures (Vasil Bio/Technol. 8, 429-434, 1990) . The combination with transformation systems for these crops enables the application of the present invention to monocots.
Monocotyledonous plants, including commercially important crops such as rice and corn are also amenable to DNA transfer by Agrobacterium strains (vide WO 94/00977; EP 0 159 418 Bl; Gould J, Michael D, Hasegawa O, Ulian EC, Peterson G, Smith RH, Plant. Physiol. , 426-434, 1991).
To obtain transgenic plants capable of expressing more than one chimeric gene, a number of alternatives are
available including the following:
A. The use of DNA, e.g a T-DNA on a binary plasmid, with a
number of modified genes physically coupled to a second
selectable marker gene. The advantage of this method is that
the chimeric genes are physically coupled and therefore
migrate as a single Mendelian locus. The invention is
especially useful in this respect, since it enables for a
second selectable marker which can be introduced next to an
already existing selectable marker- gene of interest
combination. Thus selection for retransformants can be
performed irrespective of the nature of the first selectable
marker.
B. Cross-pollination of transgenic plants each already
capable of expressing one or more chimeric genes, preferably
coupled to a selectable marker gene, with pollen from a
transgenic plant which contains one or more chimeric genes
coupled to another selectable marker. Afterwards the seed,
which is obtained by this crossing, maybe selected on the
basis of the presence of the two selectable markers, or on
the basis of the presence of the chimeric genes themselves.
The plants obtained from the selected seeds can afterwards
be used for further crossing. In principle the chimeric
genes are not on a single locus and the genes may therefore

segregate as independent loci. Also here the option to select for both selectable markers is one of the advantages
of the present invention.
C. The use of a number of a plurality of chimeric DNA
molecules, e.g. plasmids, each having one or more chimeric
genes and a selectable marker. If the frequency of co-
transformation is high, then selection on the basis of only
one marker is sufficient. In other cases, the selection on
the basis of more than one marker is preferred.
D. Consecutive transformation of transgenic plants already
containing a first, second, (etc), chimeric gene with new
chimeric DNA, optionally comprising a selectable marker
gene. As in method B,the chimeric genes are in principle not
on a single locus and the chimeric genes may therefore
segregate as independent loci.
E. Combinations of the above mentioned strategies.
The actual strategy may depend on several considerations as maybe easily determined such as the purpose of the parental lines (direct growing, use in a breeding program, use to produce hybrids) but is not critical with respect to the described invention.
Although not necessary for this invention, it is known that practically all plants can be regenerated from cultured cells or tissues. The means for regeneration vary from species to species of plants, but generally a suspension of transformed protoplasts or a petri plate containing transformed explants is first provided. Shoots may be induced directly, or indirectly (from callus) via organogenesis or embryogenesis and subsequently rooted. Next to the selective compound, the culture media will generally contain various amino acids and hormones, such as auxin and cytokinins. Efficient regeneration will depend on the medium, on the genotype and on the history of the culture. If these three variables are controlled regeneration is usually reproducable and repeatable.
After stable incorporation of the transformed gene sequences into the transgenic plants, the traits conferred by them can be transferred to other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.

Example 1
Cloning the fungal gene encoding cyanamide hydratase (CAH) in a heterologous expression cassette
a. Constructs for transformation to dicots
Construct pMOG874 contains the coding region from the cyanamide hydratase gene from the soil fungus Myrothecium verrucarla which is operably linked to the CaMV 35S promoter and the CaMV 35S terminator. This chimeric gene is cloned in the binary vector pBIlOl (Jefferson et al. EMBO J. 6, 3901/ 1987) replacing the -glucuronidase coding region and the nopaline synthase terminator.
The construct is obtained by adding an Xhol site at the 5" end and a SstI site at the 3" end of a 899 bp cDNA fragment of CAH (position 23 5-1197 of sequence published by Maier-Greiner et al. (1991) Proc. Natl. Acad. Sci. USA 88:4260-4 2 64} by PCR using the primers pi: 5"ACCGAGCTCGAATTCGGCACGAGGTTGACATGATACCTTCCTG 3" and
p2: 5"GACCTCGAGAATTCGGCACGAGGTACGATCCTACTTCCTCGC 3" between the sites Xhol and SstI of the plant expression vector pRTlOl, both sites belonging to the polylinker which is inserted between the 35S promoter and the 35S termination signal of pRTlOl (Topfer et al. 1987, Nucl. Acids Res. 15: 5890).
The chimeric gene is then cleaved with PstI, overhanging ends are polished with T4 DNA polymerase and the fragment is cloned blunt in the Smal site of pBIN19 (Bevan, M. Nucl. Acids Res. 12:8711-8721, 1984).
In construct pMOG1156 an additional ii-glucuronidase gene operably linked to the 35S promoter and the 35S terminator is inserted as Xhol/Sall fragment in the Sail site of pMOG874.
Both constructs contain in addition to the novel CAH selection marker the conventional NPTII selection marker linked to the nopaline synthase promoter and the nopaline synthase terminator as it is in pBINl9.
b. Constructs for transformation to monocots
In the same way as pMOG874 was made the expression cassette was cloned into a high copy vector (pRTlOl, Topfer, R. et al., Nucl. Acids Res. 15, 5890, 1987) resulting in pMOG873 (fig. 8)

A derivative of pMOG22 (fig. 3, deposited at the Centraal Bureau voor Schimmelcultures, Baarn, The Netherlands on January 29, 1990, under no. CBS 101.90) was made by introducing a Kpn I restriction site in the polyl inker of pMOG22 between the EcoR I and Sma I site. The orientation of the polylinker was also reversed. This plasmid, denominated pMOGlOOS, contains a hygromycin resistance gene between the left and right T-DNA borders (fig. 4). The 1.7 kb expression cassette comprising the cah gene under control of the 35S promoter and the 35S terminator was cloned between the Hind III and BamH I restriction sites. This plasmid was denominated pMOG1278 (fig. 5}.
Binary vector pMOG1295 (fig. 6) is a derivative of pMOG1278 and contains in the Sal I restriction site a GUS-expression
cassette as described in Vancanneyt, G. et al. {Mol. Gen. Genet., , 245-250, 1990}.
pMOG1253 was made departing from pMOGlS (Sijmons, P.C. et al,, Bio/Technol. 8, 217-221, 1990) which contains the double enhanced 35S promoter, the AlMV RNA4 leader sequence, the GUS-gene and the nos-terminator in an expression cassette as an EcoR I - Hind III fragment. The plasmid p35S GUS INT (Vancanneyt, 1990) was digested with SnaB I and Msc I; the resulting 426 bp fragment, containing part of the coding region for the GUS gene and ST-LSl intron, was isolated and cloned into pMOGlB linearized with SnaB I and Msc I. From the resulting plasmid a 3189 bp EcoR I - Hind III fragment was isolated and cloned into pMOG22, resulting in pMOG1253 (fig. 7).
pMOG617 (fig. 9) was made by cloning the hygromycin expression cassette from pMOG22 in the Hind III site of high copy vector pMOGlS.
Exaiig)le 2
Potato transformation
Described below is the method used for transformation of stem segments of Solanum tuberosum cv. Kardal using
Agrobacterium tumefaciens.
Nodal explants from in vitro grown potato plants were used 3 to 8 weeks after transfer. The plants were grown on Multiplication Medium (MUM) under a 16h light period (1700 lux) at 24°C and a 8 h dark period at 21°C {The various media can be found in Table 2). Stem segments of approximately 5 mm were cut on sterile filter paper soaked with Washing Medium (WAM) and collected in a flask containing Washing Medium. For approximately 300 explants the Washing Medium was replaced by Pre cultivation Medium (PRM). The flasks were cultured at 80 rpm at the same culture conditions as described above for approximately 24 h.

All binary vectors used in this study contained the nptll gene as a plant selectable marker and the nptlll as bacterial selectable marker. Plasmid pMOG410 additionally harbored a chimeric gus gene containing an intron (Vancanneyt et al. Mol. Gen. Genet., 220, 245-250, 1990). Plasmid pMOG1156 additionally harbored the gus gene and the chimeric cah gene encoding cyanamide hydratase. Plasmid pMOG874 additionally harbored the cah gene. Plasmids were maintained in E.coli and A. tumefaciens under kanamycin selection.
The Agrojbacterium strain used in this study harbored a rifampicin selection marker in a C58 chromosomal background. The construction of the helper strain EHA105 is described by Hood et al. (1993), Transg. Res. 2, 208-218.
Agrobacteria were grown overnight in LB medium with antibiotics (rifampicin 2D mg/1, kanamycin 100 mg/1). The overnight culture was diluted to OD6oo= 0 * 1 and grown to OD6oo= 0.3 in LB without antibiotics in approximately 2 h time. Bacterial suspensions were centrifuged at 1600xg for 15 minutes at room temperature. Bacteria were resuspended in Washing Medium and used for cocultivation experiments. The Pre cultivation Medium was removed from the flasks and replaced by the Agrobacterium suspension. The flasks were incubated for 20 minutes after which the explants were rinsed twice with Washing Medium. The explants were dried on sterile filter paper and incubated for 48 h on plates containing Cocultivation Medium (COM). Then, the explants were transferred to Post cultivation Medium (POM) and incubated for 72 h. The explants were then transferred to Shoot inducing Medium (SIM) containing several concentrations cyanamide or kanamycin. After two weeks the explants were subcultured on the same medium and approximately three weeks later the explants were placed on Shoot elongation Medium (SEM) containing cyanamide or kanamycin as mentioned above. When shoots were large enough to cut they were transferred to Root inducing Medium (RIM). Shoots that were able to root were then transferred to Root inducing Medium containing 50 mg/1 cyanamide or 30 mg/1 kanamycin. Simultaneously the transgenic nature of the shoots was determined by testing leaflets of the rooted shoots for expression of the gus gene using a histochemical GUS assay. It appeared that for pMOG1156 rooting of transgenic shoots on medium containing cyanamide was completely correlated with expression of the gus gene.

Described below is the method used for transformation of cotyledons of Lycopersicon esculentum cv. Money Maker using Agrobacterium tumefaclens. The binary vectors and Agrobacteria strains for this transformation method are identical to those described above.
Tomato seedlings were germinated on Germination Medium (GEM) under a 16 h light period (1700 lux) at 240 and a 8 h dark period at 21°C (The contents of the various media can be found in Table 4). Cotyledon explants of 5 to 7 day old seedlings were cut on sterile filter paper soaked with Washing Medium (WAM) and placed on plates containing Cocultivation Medium (COM)- The plates, each containing approximately 50 explants, were incubated overnight under the same conditions as described above.
The pre incubated explants were carefully submerged in the Agrobacterium inoculi:mi for 20 minutes.
The explants were then blotted dry on sterile filter paper and incubated for 4 8 h on the second set of Cocultivation plates. In procession the explants were incubated for 72 h

on plates containing Postcultivation Medixjrn (POM) after which the explants were transferred to Shoot inducing Medium (SIM) containing several concentrations of cyanamide or kanamycin. Every three weeks the explants were subcultured on the same medium. After approximately 8-12 weeks shoots were excised and placed on Root inducing Medium (RIM). Shoots that were able to root were then transferred to Root inducing Medium containing 50 mg/1 cyanamide or 30 mg/1 kanamycin. Simultaneously leaflets of the rooted shoots were tested for expression of the gus gene in a histochemical GUS assay.

E»ang>le 3
Tomato transforniation
Described below is the method used for transformation of cotyledons of Lycopersicon esculentum cv. Money Maker using Agrobacterium tumefaciens. The binary vectors and Agrobacteria strains for this transformation method are identical to those described above.
Tomato seedlings were germinated on Germination Medium (GEM)
under a 16 h light period (1700 lux) at 24°C and a 8 h dark period at 21 C (The contents of the various media can be found in Table 4). Cotyledon explants of 5 to 7 day old seedlings were cut on sterile filter paper soaked with Washing Medium (WAM) and placed on plates containing Cocultivation Medium (COM). The plates, each containing approximately 50 explants, were incubated overnight under the same conditions as described above.
The pre incubated explants were carefully submerged in the Agrobacterium inoculum for 20 minutes.
The explants were then blotted dry on sterile filter paper and incubated for 48 h on the second set of Cocultivation plates. In procession the explants were incubated for 72 h on plates containing Postcultivation Medium (POM) after which the explants were transferred to Shoot inducing Medium (SIM) containing several concentrations of cyanamide or kanamycin. Every three weeks the explants were subcultured on the same medium. After approximately 8-12 weeks shoots were excised and placed on Root inducing Medium (RIM) . Shoots that were able to root were then transferred to Root inducing Medium containing 50 mg/1 cyanamide or 30 mg/1 kanamycin. Simultaneously leaflets of the rooted shoots were tested for expression of the gus gene in a histochemical GUS assay.

Described below is the method used for transformation of root segments of Arabidopsis thaliana cv. C24 using Agrobacterium tumefaciens. The binary vectors for this transformation method are identical to those described above. Six mg of Arabidopsis seeds were germinated in a flask containing liquid Germination Medium (GM) under 16 h light period (1700 lux) at 24°C and a 8 h dark period at 21"C at 80 rpm. (The contents of various media can be found in Table 4).

Roots of 9 days old seedlings were isolated in a sterile petridish and collected in a drop of Germination Medium (GM). Roots were cut in segments of approximately 3-5 mm and approximately 100 explants were spread evenly on a nylon membrane {0 8 cm) which was placed on plates containing Callus Inducing Medium (CIM). The plates were incubated 3 days under the same conditions as described above. The Agrobacterium strain used in this study harbored a rifampicin selection marker in a C 58 chromosomal background. The construction of the helper strain MOGlOl is described by Hood et al. (1993). Agrobacteria were grown overnight in LB medium with antibiotics (rifampicin 20 mg/1, kanamycin 100 mg/1) . The overnight culture was diluted 1:10 in LB without antibiotics and grown for approximately 3 hours. Bacterial suspensions were centrifuged at 1600xg for 15 minutes at room temperature. Bacteria were resuspended in GM and adjusted to OD600=0.1 and used for cocultivation.
The membrane containing approximately 100 explants was incubated for 2 minutes with the Agrobacterium suspension and dried on sterile filter paper to remove excess of bacteria. The membrane with explants are cultured for 4 8 h on CIM plates. After rinsing the membrane and explants with liquid GM these were incubated on Shoot Induction Medium (SIM) plates containing several concentrations of cyanamide or kanamycin. After 5 days the membrane with the explants was transferred to the same medium (SIM) for subculture. The second subculture was after 2 weeks. Approximately four weeks after cocultivation 60 shoots per cyanamide concentration were excised and placed on plates with Shoot Elongation Medium (SEM) containing 30 mg/1 cyanamide. Shoots which were able to root are tested on their transgenic character by testing leaflets and flowers for expression of the gus gene using a histochemical GUS assay.

Three experiments were performed. Shoots obtained from Exp. 98-8 and 98-11 were transferred to rooting medium (SEM) containing 30 mg/litre cyanamide. Shoots obtained from Exp. 98-13 were transferred to rooting medium containing the same concentration as the selection medium (SIM), for results see Table 4a. The shoots obtained from the kanamycin selection (50 mg/litre) were transferred to rooting medium containing 25 mg/litre Kanamycin.

Root explants transformed with pMOG 410 were not able to regenerate on cyanamide containing medium. Even 20 mg/litre cyanamide was already enough to prevent regeneration of explants transformed with a construct without the cah gene. At 20 till 4 0 mg/litre cyanamide some callus development was observed, but at 50 rag/Iitre and higher explants were not viable and turned completely brown.

On the other hand explants transformed with the cah gene (pMOG 1156) were able to regenerate at all cyanamide concentrations, even at 80 mg/litre. At lower concentrations the regeneration of shoots was faster than with kanamycin.
Although more shoots were available 60-65 shoots were harvested per treatment and placed on rooting medium. At the lower cyanamide concentrations the same amount of shoots developed as with kanamycin selection (approx. 70-100 per petridish).
There is a clear correlation between callus development and GUS expression on cyanamide selection with root explants transformed with pMOG 1156 (fig. 4b). GUS analysis of shoots obtained on cyanamide 0 mg/litre (NS) showed no staining, indicating that cyanamide is needed to obtain transgenic

Described below is the method used for transformation of callus derived from scutellum of mature embryos of Oryza satlva cv. Taipei 309 using AgroJbacterliun tumefaciens strain LEAlll9-pMOGl295 (harboring the cah-gene) and strain LBA1119-pMOGl253 (control). Sterile dehusked rice seeds were germinated on plates containing Callus Induction Medium (CIM) in the dark at 28""C. (The contents of various media can be found in Table 5) . After 3 weeks embryogenic callus derived from the scutellum is isolated and subcultured on the same medium under the same conditions. After 2-3 weeks embryogenic calli were cut in segments of approximately 2-3 mm and cultured plates containing CIM for 4 days. The Agrobacterium

strains used in this study harbored a rifampicin selection marker in a C 58 chromosomal background. The construction of the helper strain EHA105 is described by Hood et al. (1993). Agrobacteria were grown for 4 days on plates containing AB medium with antibiotics (rifampicin 20 mg/1, kanamycin 100 mg/1} . Agrohacteria were collected in LIM and the OD600 was adjusted till 1.0-1.5. This suspension was used for cocultivation. Calli were incubated for 10 minutes with the Agrobacterium suspension and dried on sterile filter paper to remove excess of bacteria. Calli were cultured for 48 h on Coculture Medium (COM) plates at 2b°C in the dark. 50 pMOG1295 calli and 20 pMOGl253 calli were cultured per concentration of cyanamid. The following concentrations of cyanamide were used: 0, 15, 30, 60, 100, 150, 200, 300 and 500 mg/1. Hygromycin was applied in a concentration of 50 mg/1. Calli were incubated on First Selection Medium (FSM) plates containing several concentrations of cyanamide or hygromycin at 28°C in the dark. After 3 weeks the calli were transferred to Embryo Induction Medium I (EIM I) containing the same concentration of cyanamide or hygromycin . After another 3 weeks the calli were subcultured on Embryo Induction Medium II (EIM II) containing the same concentration of cyanamide or an increased concentration of hygromycin (75 mg/1}. Calli were transferred to Shoot Induction Medium (SIM) containing the same concentration of cyanamide as during FSM, EIM I, EIM II and were cultured under 12 hours light period (2600 LUX) and 12 hours dark at 28 °C. Approximately 3 weeks after transferring calli to SIM, shoots were regenerating and excised and placed in jars containing Pre-Greenhouse Medium (PGM). No calli were formed at concentrations of 100 mg/1 or higher of cyanamid. At 15 mg/1 cyanamide the regeneration frequency of callus from both constructs was the same (pMOG1253 7 out of 16 calli were able

to regenerate, pMOG1295 17 out of 44) . At 30 mg/1 cyanamide
only 11 calli of pMOG1295 showed green callus development and
6 were able to be regenerated.
Table 5. Media required for Oryza sativa Taipei 309
transformation

Example 6
Rice transformation by particle gun
Described below is the method used for transformation of non-
morphogenic cell suspensions of Oryza sativa cv. IR 52 using a particle inflow gun (PIG) according to Finer et al. (Plant Cell Rep. 11, 323-328, 1992).
A long-term, non-morphogenic suspension culture of Oryza sativa cv. IR 52 was subcultured in weekly intervals in liquid LS-4 (Linsmaier and Skoog, Physiol. Plant. 18, 100-127, 1962} medium and maintained on a rotary shaker (110 rpm) at 28*0 in the dark. (The contents of the LS-4 medium can be found in Table Z) . 3-4 days after the last subculture 1.5 ml of this cell suspension (appro. 1.5 x 10 cells) were evenly spread on a filter paper (Whatman no 4) which was subsequently placed on solidified LS-4 medium and cultivated in the dark at 28*"C for 24 h and directly used for bombardment thereafter. For microprojectile bombardment a home-made particle inflow gun (PIG) according to Finer et al. (1992) was used. 300 |ag tungsten particles coated with either pMOG 617 (35S-gus and 35S-hyg) or pMOG 873 {35S-cah) were loaded on a particle support. The particles were accelerated by a 2.5 bar helium pulse and had to pass a 500 |jm metal stop screen, placed 2 cm under the particle support. The suspension cells were placed 15 cm under the particle support. The PIG was evacuated to 30 mbar before bombardment. After bombardment the cells were cultured at 28°C in the dark for 3 days. Then the filters with the cells were transferred to solid LS-4 medium containing various concentrations of cyanamide or 50 mg/1 hygromycinB (see table 6) . The

subculture was repeated every 9 days. Resistant microcalli that were visible after 4-6 weeks were transferred to fresh LS-4 medium containing the respective selective agent. From two experiments 7 + 41 calli transformed with pMOG617 were found resistant to hygromycin, while for the transformation with pMOG873 7 calli survived cyanamide 20 mg/1 in the first experiment (results from the second experiment not yet available) and 0+4 calli remained viable on 40 mg/1 cyanamide. No calli were formed at concentrations of 50 mg/1 cyanamide or higher. The transgenic nature was confirmed by testing parts of the developing callus for the presence of the DNA in the callitransformed with pMOG873. One of the 4 surviving calli on 40 mg/1 cyanamide showed positive in a PCR experiment on the cah-gene.

Table 6. Media required for Oryza sati-va cv
transformation

IR 52

Table 7. pMOG617

pMOG 873

Example 8

Maize killing curve
Stock solutions of cyanamide were prepared in water at 10 and 100 mg/ml and filter sterilised. Aliquots were stored at -20°C.
Media were prepared by adding MS medium (4.4g), sucrose (20g), 2,4-D C2.0mg) and agar (8g) to 1 litre of water. After autoclaving the appropriate amount of cyanamide (0, 10, 30, 50, 100, 150 mg/L cyanamide) was added and the media was poured into 9cm petri dishes. BMS liquid was prepared as above minus agar.
BMS cells were added to media containing cyanamide in three ways;
a. BMS cell suspension added to falcon tube and liquid
removed. Then the BMS cells were arranged on the surface of
the agar in clumps of approximately 5mm in diameter, 5 clumps
per plate, 3 plates per concentration, while on the base of
each petri dish the outline of each clump was marked;
b. Approximately 0.5ml pack cell volume plus 1.5ml BMS liquid
added to surface of agar and the cells were spread finely
over surface of agar. Three plates per treatment were set up.
c. Approximately 0,5ml pack cell volume plus 1.5ml BMS liquid
were added to filter paper overlying agar. Cells were spread
evenly over surface of filter. One plate per treatment was
set up.
The plates were sealed with micropore tape and incubated at
25°C in the dark. The growth of the cells was observed after 7 and 14 days.

RESULTS
Day 8
The growth of BMS cells on cyanamide was assessed after 8
days.
The clumps of BMS cells arranged on the surface of the
control media had increased in size and outgrown their
original outline. Cells on 10 mg/1 had not outgrown their
outline but the height of the clumps had increased forming an
uneven surface. A slight reduction of growth with increased
cyanamide concentration was apparent with the maximum effect
on growth observed at 50 mg/1 cyanamid.
The cells which were spread over the surface of the control
media had grown well and densely covered the surface of the
media. A significant reduction in growth was observed on the
lowest level of cyanamide (10 mg/1), however, an increased
cell density was clearly visible. A slight increase in cell
density was evident on 30 mg/1 cyanamid, but it was difficult
to distinguish different growth rates on higher
concentrations.
Cells on all levels of cyanamide remained a milky white
colour, no browning of cells was observed.
Day 15 {Table 9}
The reduction in growth of BMS cells on lOmg/1 was still very clear after 15 days on cyanamide, however, the cells arranged in clumps had outgrown their original outline. Cells spread directly over the surface of the agar showed a similar response to those arranged in clumps with a notable reduction in growth observed on 30mg/l plus. Cells on 50mg/X

plus showed no signs of growth and the surface of the clumps remained very flat but the cells were still milky white in colour. A similar response was observed with cell spread over a filter. However, small raised lumps were observed on the surface on all filters but these did not develop further into colonies and were evidently comprised of the larger cell aggregates from a mixed population of sizes typical in BMS suspensions.
Samples were taken from the clumps of cells on all levels of cyanamide for observation under a light microscope. With increasing levels of cyanamide there were increasing numbers of dead cells where the cell contents had shrunk away from the cell wall, and an increase in the number of dark bodied starch grains. Cells were observed resuspended in water and with FDA stain under UV light.

Table 9.

The experiment was repeated with cyanamide concentrations of 0, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 mg/1 cyanamid. The results were similar to those described above, i.e. for the cells aggregated in clumps a slight reduction in growth was seen at 10 mg/1. From a concentration of 20 mg/1 cyanamide on the cell clumps showed no outgrowing from their original outline, but at the lower concentrations (
higher concentrations). Above 50 mg/1 the clumps showed a
slight orange tinge.
The results with cells spread over the surface of the agar or
on the filters were similar in that at a concentration of 10
mg/1 showed a slight growth (approximately doubling of the
number of original cells), while at concentrations of 20 mg/1
and higher exhibited limited signs of growth.
Example 8
Kill curves in banana (Misa)
To test the potential of cyanamide as a selection agent for
transformation in banana two kill curves were set up with a regenerable embryogenic suspension of Grand nain 6 day old embryogenic suspension CEd6b) cultures, subbed routinely in M2 2,4D liquid, containing 4.32 g/1 MS salts, 45g/l sucrose, standard Ix concn. MS vitamins, lOOmg/1 glutamine, 100 mg/1 myo-inositol, 100 mg/1 biotin, lOOmg/1 malt extract at pH 5.3 and added after autoclaving 1.2mg/l 2,4-D and 0.8 mg/1 picloram.
Cultures were sieved (>250n, Kill Curve Medium A : M2/MS/1.0 2,4-D (as M2/MS/2,4~D except only 1.0 mg/1 2,4-D, no picloram and +2.25 g/1 gelrite): this medium promotes the rapid division and growth of embryogenic
callus, but not embryos.

Kill Curve Medium B : M2/SH/0.5Pic, 0.5 2,4-D (as M2/MS/2,4-D except only 0.5 mg/1 2,4-D and 0.5 mg/1 picloram, SH salts (4.32g/l) instead of MS,. + 2.25 g/1 gelrite) : This medium promotes the early development of embryos which can be matured and germinated by transfer to alternative media. Cyanamide was added to both media types, after autoclaving, to concentrations of 0,20,30,50,75,100,150 mg/1. The results are depicted in Table 10, where the figures on cell growth are approximate visual estimates, not precise measurements of callus volume. There is no significant visual browning of cultures and release of phenolics until concentrations of >75 mg/1. Generally cultures just stop growing, with cell division being widely inhibited. Cyanamide inhibits the growth of embryogenic callus by 40-50% at even low concentrations of 20mg/l, without causing significant visual damage. Embryogenesis was totally inhibited at the lowest concentration tested here.

We claim;
1. Method for selection of transformed plants comprising:
(a) constructing a vector comprising a coding sequence for cyanamtde hydratase and a gene of interest wherein said cyanamide hydratase is the selection marker, and,
(b) transforming plants or plant parts or plant cells with said vector, and
(c) growing the transformants in a medium comprising cyanamide wherein said coding sequence comprises the nucleotide sequence of SEQ ID NO.l.

2. A method according to claim 1 wherein said plants are monocot plants.
3. A method according to claim 2 wherein said plants are selected from the group consisting of; rice; maize and banana plants.
4. A method according to claim 1 wherein said plants are dicot plants.
5. A method according to claim 4 wherein said plants are selected from the group consisting of: potato; tomato and arabldopsls plants.
6. Method for the selection of transformed plants, substantialy as herein described, with reference to the accompanying drawings.

Documents:

837-mas-1998 abstract.pdf

837-mas-1998 claims duplicate.pdf

837-mas-1998 claims.pdf

837-mas-1998 correspondence others.pdf

837-mas-1998 correspondence po.pdf

837-mas-1998 description (complete) duplicate.pdf

837-mas-1998 description (complete).pdf

837-mas-1998 drawings.pdf

837-mas-1998 form-1.pdf

837-mas-1998 form-13.pdf

837-mas-1998 form-19.pdf

837-mas-1998 form-26.pdf

837-mas-1998 form-4.pdf

837-mas-1998 others.pdf

837-mas-1998 pct search report.pdf

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Patent Number

208585

Indian Patent Application Number

837/MAS/1998

PG Journal Number

39/2007

Publication Date

28-Sep-2007

Grant Date

02-Aug-2007

Date of Filing

20-Apr-1998

Name of Patentee

SYNGENTA MOGEN BV

Applicant Address

OF EINSTEINWEG 97 2333 CB LEIDEN

Inventors:

#	Inventor's Name	Inventor's Address
1	BRIGITTE DAMM	OBER ESCHHACHERSTRASSE 141 61352 BAD HOMBURG

PCT International Classification Number

C 12 N 15/60

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA