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WO1995006127A1 - Method for the production of transgenic cereals - Google Patents

Method for the production of transgenic cereals Download PDF

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Publication number
WO1995006127A1
WO1995006127A1 PCT/FI1994/000365 FI9400365W WO9506127A1 WO 1995006127 A1 WO1995006127 A1 WO 1995006127A1 FI 9400365 W FI9400365 W FI 9400365W WO 9506127 A1 WO9506127 A1 WO 9506127A1
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Prior art keywords
plant
transgenic
transformed
plants
gene
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PCT/FI1994/000365
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French (fr)
Inventor
Marjatta Salmenkallio-Marttila
Kristian Aspegren
Anneli Ritala
Leena Mannonen
Ulrika KURTÉN
Riitta PUUPPONEN-PIMIÄ
Veli Kauppinen
Teemu H. Teeri
Original Assignee
Valtion Teknillinen Tutkimuskeskus
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Priority to AU74631/94A priority Critical patent/AU7463194A/en
Publication of WO1995006127A1 publication Critical patent/WO1995006127A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8206—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated
    • C12N15/8207—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated by mechanical means, e.g. microinjection, particle bombardment, silicon whiskers

Definitions

  • the present invention relates to plant biotechnology and specifically to a new method for obtaining stable transgenic monocotyledonous plants, especially cereals, and to a method for obtaining transgenic cereal seeds.
  • the present invention thus provides cereals which carry in their genome at least one foreign gene giving to the plant a desirable new trait, but which cereals do not carry any additional undesired selectable genes in their genome.
  • a new application of electroporation may overcome at least some of these prob ⁇ lems.
  • Dekeyser et al. (1990) have demonstrated transient expression of genes delivered into leaf segments of different cereal species by electroporation.
  • PCT- application WO 92/09696 the same method is applied to obtain transgenic maize plants from immature zygotic embryos or type I embryogenic callus. To prepare them to take up DNA, the callus or immature embryos are first wounded, either mechanically or chemically. Foreign DNA is then transferred to them by electro- poration. The electroporated embryos or callus are then cultured for several weeks on selective medium before resistant calli are grown into plants. The method is based on the ability to produce embryogenic callus from the scutellar tissue of immature embryos.
  • Particle bombardment is another gene transfer method which has been successfully used in transforming cereals.
  • PCT application WO 90/01551 transformation of monocot plants with exogenous DNA to produce foreign proteins is described.
  • the PCT-application WO 91/02071 discloses a system for genetic transformation of monocotyledonous plant cell suspension, e.g. maize cell suspension, by particle bombardment and the selection of genetic transformants from the suspensions. Fertile plants are then produced from the transformed cells by conventional methods.
  • Particle bombardment has also been used to produce several transgenic rice varieties by transforming the scutellar tissue of immature embryos (Christou et al. 1991).
  • In addition to the "traditional" biolistic apparatus there is a new micro- targeting device for particle bombardment of plant meristems (Sautter 1993), but so far tobacco is the only transgenic plant produced with this method.
  • a grass embryo there are several separately transformable meristematic regions.
  • a grass embryo also reaches a relatively high degree of differentiation (e.g. Esau, 1977).
  • Esau 1977
  • an embryo independent mutant sectors which can ⁇ not have a mutant cluster in common, are proven anatomically, if a shoot or shoot group exists as a separate shoot primordium or as a meristematic tissue area separ ⁇ ated from the apex of the shoot primordium by a leaf or leaf primordium.
  • the anatomical data from barley embryos proves that the embryo contains at least six separate shoot meristems or prospective shoot meristems which will constitute mutually exclusive mutant sectors in the plant (Jacobsen 1966). Similar results have been reported for rice in which the spikes on the primary side-shoots mutate independently of the apical spike.
  • the present invention is developed to utilize the whole potential of the numerous meristems of the grass embryo.
  • the present in- vention utilizes conventional gene transfer methods, the gist of the system lying in the treatment of the plant material.
  • the present inventors have now developed a new efficient method for transforming grasses and cereal plants.
  • the method of the invention is based on the fact that a grass embryo has several separately transformable meristematic regions. E.g. a barley grain has, depending on its size and stage of development, even 16 separ- ately transformable meristems.
  • the transformation is thus directed to such a region of the embryo in which the leaf primordia and the side shoot primordia or their initial cells are located.
  • the transformed embryos are germinated and cultivated directly without any selec- tion, and the rachises and awns of the ears produced are analyzed as regards the transferred genes.
  • a special advantage of the present method is thus the fact that no selection is used when growing the transformed embryo material into plants so as to exploit the whole growing capacity of the meristematic region. Consequently, it is not necessary to insert selectable genes into the genetic construct used for the transformation of the embryonic material.
  • Another special advantage of the method of the invention is the minimal requirement of tissue culture, and it helps to elim ⁇ inate culture induced mutations and somaclonal variation.
  • the direct germination of the transformed embryos also makes it possible to use var- ieties that are difficult to regenerate in tissue culture.
  • a further object of the invention is to provide a method for generating transgenic cereal seed comprising generating transgenic plants by the above method, cultivat ⁇ ing green plantlets and deriving seed from the resulting mature plants.
  • the method of the invention to produce a transgenic monocot plant is applicable to grasses and cer ⁇ eals, primarily barley, Hordeum vulgare L., but can be used also for e.g. wheat, oat, rye, rice or maize. Accordingly, the present invention provides a method for generating transformed cereal plants comprising transforming the embryo material and growing it directly into plants, wherein the growth medium does not comprise any selective substances for the transformed trait.
  • the material to be transformed must be an embryo, either zygotic or somatic, immature or mature and it preferably contains several separate meristematic regions to be transformed. Parts of an embryo, particularly the nodal region, or intact embryos can be transformed.
  • the term embryo or embryo material should be understood to include all stages of development from the globular stage to the mature stage of zygotic and somatic embryos.
  • the embryos may be transformed directly after isolation or after being germinated for 1 to 5 days prior to the trans- formation.
  • GUS ⁇ -glucuronidase enzyme
  • NPTII neomycin phosphotransferase enzyme
  • the cDNA coding for ⁇ -glucuronidase under control of the maize alcohol dehydrogenase I promoter was transferred by particle bombardment to the meristematic cells of immature barley embryos and mature embryos of barley, wheat and maize to dem ⁇ onstrate the general applicability of the method to different cereal species.
  • a histological assay for ⁇ -glucuronidase was performed for the transformed embryos. The blue stain indicating the transformation events was localized in the meristematic regions of the embryos by serial sectioning of the samples.
  • the cDNA coding for neomycin phosphotransferase was placed under control of the cauliflower mosaic virus 35S promoter and transferred by particle bombardment to the meristematic cells of barley embryo.
  • the transformed embryos were germinated and cultivated without selection so as to produce as many shoots as possible.
  • the rest of the spike after grain removal were then analyzed as regards the transferred genes, and grains of the positive ears were used for the production of several gen ⁇ erations of transgenic barley plants.
  • the method of this invention can also be used to produce transgenic cereal plants expressing any other desirable new trait.
  • enzymes of importance in brewing such as ⁇ -glucanases and amylolytic and proteolytic enzymes.
  • barley and wheat especially interesting are some agrono- mically important characteristics like preharvest sprouting.
  • Cereal grains differ in their nutritional characteristics.
  • the value of cereal protein is less than that from animal products mainly because of deficiencies of several essential amino acids.
  • the food and feed value of wheat and barley grains can be improved with genetic • engineering of their storage protein genes.
  • the method of this invention can also be used to produce transgenic plants that produce other foreign products, such as pharmaceutical peptides, proteins or compounds of secondary metabolism.
  • Direct gene transfer methods are used for the transformation. Suitable methods are e.g. particle bombardment and electroporation, electrophoresis may also be used.
  • the transformed embryos or embryo parts are germinated on modified MS medium without selection so as to allow all the potentially transformed meristems to form shoots.
  • the enhancement can be effected e.g. by cutting the main shoots of the plants after sprouting. Later the rooted plantlets are grown in soil for seed production. The rachises of the ears produced are then analyzed as regards the transferred genes, and grains of the positive ears are used for the production of transgenic cereals.
  • the seeds from the cultivated green plantlets may be derived by harvesting them directly from the resulting mature plants or by obtaining seeds from plants one or more generations remote from the original green plantlets.
  • T 0 primary transformant Tj first progeny of T 0
  • Fig. 1 Diagram of a transversely cut barley embryo.
  • Fig. 2A The vector pKAH24 containing the endo- ⁇ -glucanase gene egll.
  • Fig. 2B The vector pKAH21 containing the endo- ⁇ -glucanase gene egll.
  • Fig. 2C The vector pHTT303 containing the neomycin phosphotransferase gene nptll.
  • Fig. 2D The vector pAT13 containing the ⁇ -glucuronidase gene uidA.
  • Fig. 3 Histochemical localization of ⁇ -glucuronidase gene in trans ⁇ formed immature embryos of barley.
  • Fig. 4 Histochemical localization of ⁇ -glucuronidase gene in trans ⁇ formed mature embryos of barley.
  • Fig. 5 Histochemical localization of ⁇ -glucuronidase gene in trans ⁇ formed mature embryos of wheat.
  • Fig. 6 Histochemical localization of ⁇ -glucuronidase gene in trans- formed mature embryos of maize.
  • Fig. 8 NPTII gel assay of leaf extracts from T 0 and T j barley plants.
  • Lane PC represents a positive control from tobacco transgenic for
  • Samples I to IV represent four spikes of the T 0 plant Toivo and samples 1 to 25 their progeny (TJ, so that 1 to 7 are the offspring of I, 8 to 14 are offspring of II, 15 to 18 off- spring of III, and 19 to 25 offspring of IV. Samples are numbered so that they are equivalent in Figures 8, 9 and 10.
  • the intensity of the bands in the barley samples as well as the number of integration specific frag- ments indicate that about two copies of pHTT303 were integrated into the barley genome. Note that part of the T 0 spikes may be chimeric and part of the T j plants homozygous for the transferred DNA, resulting in some variation of band intensities between the samples.
  • Fig. 10 Analysis of methylation pattern of the transferred DNA in Tj plants.
  • BamHI, EcoRl and Hindlll digested total DNA samples (5 ⁇ g) were in addition restricted with either Dp ⁇ l (Dl) or Dpnll (D2) and probed with plasmid pHTT303.
  • the positive control (PC) is BamHI, EcoRl and Hindlll digested pHTT303 that was further restricted with either Dpnl (Dl) or Dpnll (D2).
  • 5 pg of digested plasmid was mixed with 5 ⁇ g.DNA from non-trans ⁇ formed barley, digested with same enzymes as the plasmid.
  • the S lane represents the negative control containing DNA isolated from non-transformed barley plant spikes.
  • the three T- plant samples 5 p ⁇ l (Dl) or Dpnll (D2) and probed with plasmid pHTT303.
  • the positive control (PC) is BamHI, EcoRl and Hind
  • Example 1 Construction of chimaeric genes for expression in plants.
  • the 5.2 kb plasmid consists of the barley ⁇ -amylase promoter and the gene egll of Trichoderma reesei coding for the endo- ⁇ -glucanase I in the E. coli cloning vector pUC18 (Norrander et al., 1983).
  • the plasmid pKAH21 contains the same egll gene under control of cauliflower mosaic virus 35S promoter.
  • the plasmid constructs pKAH24 and pKAH21 are shown in Figures 2A and 2B.
  • pHTT303 (expressing neomycin phosphotransferase II)
  • the gene nptll coding for neomycin phosphotransferase II was combined at its 5' end to the cauliflower mosaic virus 35S promoter and at its 3' end to a terminator of the gene 7 of T-DNA.
  • the recombinant gene resides in the E. coli cloning vector pUC18 (Norrander et al, 1983) as a Bglll-Sacl fragment between the BamHI and S ⁇ cl sites of the vector.
  • the plasmid construct pHTT303 is shown in Figure 2C.
  • the plasmid pAT13 was a gift from Carlsberg Research Institute.
  • the 6.5 kb plasmid consists of the alcohol dehydrogenase 1 promoter, a fragment from intron 1 of the A dh I gene, a ⁇ -glucuronidase coding region, and the nopaline synthase polyadenylation region inserted into pUC8 (Klein et al. 1988), as pAI j GusN.
  • the plasmid construct pAT13 is shown in Figure 2D.
  • Example 2 Transformation of immature embryos of barley by particle bom ⁇ bardment.
  • Immature embryos of the size of 0.5 to 1 mm were isolated from developing seeds of barley (Hordeum vulgare L. cv. Kymppi). The embryos were bombarded at the embryonic axis. For particle bombardment the isolated embryos were placed scutellum down on modified MS medium (Olsen, 1987) supplemented with 0.4 mg/1 BA and 35 g/1 maltose and solidified with 3 g/1 gellan gum. The principle of particle bombardment is described by Sanford et al. (1987) and Klein et al. (1987). The helium modification of the Biolistic R PDS-1000 device (Bio-Rad) was used for transformation.
  • sample chamber pressure 3.1 kPa pressure of helium shock wave 9.0 MPa; distance between macrocarrier and stopping screen 6 mm; distance between macrocarrier and rupture disk 9 mm.
  • Particles were prepared by coating gold particles (1.0 ⁇ m in diameter) with the plasmid pKAH24 ( ⁇ -glucanase), or with the plasmid pAT13 ( ⁇ -glucuronidase), or with a 1:1 mixture of plasmids pHTT303 (neomycine phosphotransferase) and pAT13. In each bombardment, 1.0-2.0 ⁇ g of DNA was used and each sample was bombarded once.
  • Mature seeds of barley (Hordeum vulgare L.), wheat (Triticum aestivum L.) and maize (Zea mays L.) were surface sterilized first with 70% ethanol for 5 min and then with NaOCl (4% active Cl) for 10 min, rinsed several times with sterile distilled water and germinated on moist filter paper for 1 day.
  • the embryos were separated from the seeds and part of the coleoptile and leaves covering the meristematic regions of the embryo were removed with a scalpel.
  • the embryos were transformed by particle bombardment with the plasmid pAT13 as described in Example 1.
  • Immature embryos of barley and mature embryos of barley, wheat and maize transformed with the plasmid pAT13 were stained with X-gluc (5-bromo-4- chloro-3-indolyl- ⁇ -D-glucuronic acid cyclohexylammonium salt) with the method of Jefferson (1987) with the modifications of Ritala et al. (1993) to detect the activity of the transferred uidA gene coding for ⁇ -glucuronidase.
  • the stained embryos were embedded in paraffine and sectioned with microtome to localize the transformed cells.
  • FIG. 3 The localization of blue stain indicating transformation in the meristematic regions is shown in Figure 3 (3A-3C) for immature embryos of barley, Figure 4 (4A-4D) for mature embryos of barley, Figure 5 (5A-5D) for mature embryos of wheat, and Figure 6 (6A-6C) for mature embryos of maize.
  • the embryos were transferred on modified MS medium (Olsen 1987) supplemented with 0.4 mg/1 BA and 35 g 1 maltose and solidified with 3 g/1 gellan gum for germination.
  • the plates were incubated at 23°C in light (50 ⁇ mol m ⁇ 2 s "1 ).
  • the embryos were germinated without any selective substances in the medium.
  • the shoots were transferred to modified MS rooting medium (Olsen 1987) without hormones and solidified with 3 g/1 gellan gum.
  • the plantlets were potted in soil and grown to maturity in the green ⁇ house. The embryos from all green grains were isolated for further cultivation, the rachises and/or awns were screened for the presence of the transferred gene.
  • Neomycin phosphotransferase II activity in the plants was assayed according to McDonnell et al. (1987) with the following modifica- tions: Extraction buffer with 0.13 g/1 leupeptine and a small amount of purified sea sand was added to the leaf material which was then ground thoroughly in an Ep- pendorf tube and centrifuged. The supernatant was collected and the protein con- centration determined (Bradford, 1976). The amount of extract used in the enzyme assay corresponded to 20 ⁇ g of total protein. The reaction mixture was blotted after 5 minutes centrifugation via a dot blot apparatus (Milliblot-D, Millipore) onto Whatman P81 paper. Alternatively, the NPTII gel assay (Reiss et al. 1984, mod- ified by Van den Broeck et al. 1985) was used.
  • the plant material was screened by the polymerase chain reaction (PCR) carried out in a Perkin Elmer cetus 9600 thermocycler.
  • the com- plete PCR mixture contained 100-500 ng of genomic or 0.5 pg of pKAH21 DNA, 12.5 pmol of each oligonucleotide primer, 200 ⁇ M dNTPs, 0.5 U Dynazyme and buffer supplied by the enzyme manufacturer (Finnzymes Oy) in a total volume of 100 ⁇ l. Thirty cycles were performed under following conditions: 75 s at 95°C, 2 min at 55°C, and 3 min at 72°C.
  • Primers were designed to amplify a 557 bp frag- ment of the cDNA for EGI.
  • the forward primer was 5'-AGGACACCTCGGTGG- TCCTT-3' and the reverse primer 5'-AGAGTGAGGGGTCAAGGCATT-3'.
  • the PCR performance was controlled by including a primer pair amplifying the promo ⁇ ter fragment of one of the ⁇ -amylase genes.
  • Total DNA isolated from a transgenic barley cell line (VTT-6-93002, transformed with pHTT303 and pKAH21) was used as a positive control.
  • the amplified samples were analyzed by electrophoresis in a 2% agarose gel.
  • Plants grown from immature embryos transformed with the plasmid pKAH24 were screened for the gene egll by PCR. About 1400 spikes were analyzed from 146 plants. Nine plants of these (12 spikes) were shown to contain the transferred gene (Fig. 7).
  • PCR polymerase chain reaction
  • the complete PCR mixture contained 100-500 ng total or 0.15 pg pHTT303 DNA, 50 pmol of each oligonucleotide primer, 200 ⁇ M dNTPs, 2 U DynazymeTM and buffer supplied by the enzyme manufacturer (Finnzymes Oy) in a total volume of 100 ⁇ l. Thirty cycles were performed under following conditions: 75 s at 94°C, 2 min at 55°C, and 3 min at 72°C. Primers were designed to amplify a 5' sequence of nptll in pHTT303.
  • the forward primer used was 5'-ACACGCTGAAATCACCAGTCTC-3' (+1 from start of transcription) and the reverse primer 5'-CTCGTCCTGCAGTTC- ATTC-3' or 5'-TCGCCCAATAGCAGCCAGTC-3' (+281 and +417 from start of transcription, respectively).
  • the PCR performance was controlled by including a primer pair amplifying the promoter fragment of one of the ⁇ -amylase genes. The amplified samples were analyzed by electrophoresis in a 2% agarose gel.
  • NPTII activity was analyzed from the 227 plants of the T 0 generation by the dot blot method.
  • One plantlet proved to be NPTII positive, and this plant was named and is later referred to as Toivo.
  • Toivo was transferred to potting soil and grown in the growth room. By now it has produced 98 fertile spikes. Embryos from green grains were isolated for further cultivation to Tj progeny. The rest of the spikes of Toivo after grain removal were screened for the presence of the nptll gene with the PCR technique. Of the 90 spikes screened, six were positive for the nptll gene. Four of them gave positive signals in the NPTII gel assay (Fig. 8) as well. The presence of the transferred nptll gene in these four was confirmed with Southern blot hybridization (Fig. 9).
  • Total DNA from the plant material was isolated according to Dellaporta et al. 1983.
  • isolated total DNA was restricted with enzymes BamHI, EcoRl, H dIII, Dp l and/or Dpnll in the buffers recommended by the enzyme manufacturers.
  • 5 ⁇ g digested DNA was separated by electrophoresis in 0.8 % agarose gels. The gels were blotted onto MagnaGraph (Micron Separation Inc.) nylon membranes (Southern 1975).
  • the 32 P-dCTP -labeled plasmid p ⁇ TT303 was used as a probe.
  • Hybridizations at 42°C overnight in 50 % formamide
  • wash ⁇ ing of the filters were carried out according to Sambrook et al. (1989). After washing the filter was exposed to X-ray film using intensifying screens at -70°C for three days.
  • the DNA samples from the four T 0 shoots and from fifteen T j progeny shoots contained copies of the nptll and promoter fragments, and in addition several extra fragments (Fig. 9).
  • the extra fragments do not corre- spond to partial digestion fragments of pHTT303. This indicates integration of the transferred pHTT303 into the genome of Toivo as well as passage of both the transferred sequences and the integration pattern to T- progeny. From this fact it is also apparent that all of the transgenic tillers originate from a single transformed cell and show integration at one locus.

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Abstract

The present invention relates to plant biotechnology and specifically to a new method for obtaining stable transgenic monocotyledonous plants, especially cereals, and to a method for obtaining transgenic cereal seeds. The present invention thus provides cereals which carry in their genome at least one foreign gene giving to the plant a desirable new trait, but which cereals do not carry any additional selectable genes in their genome. In the method of this invention a transgenic monocotyledonous plant is produced by providing a genetic construct comprising at least one gene heterologous to the plant to be transformed, whereby no additional selectable genes are inserted into the genetic construct, transforming the meristematic cells of the embryo of a monocot plant with the said genetic construct, and generating a whole plant by germinating and cultivating the transformed embryo without selection.

Description

METHOD FORTHEPRODUCTION OFTRANSGENIC CEREALS
Field of the Invention
The present invention relates to plant biotechnology and specifically to a new method for obtaining stable transgenic monocotyledonous plants, especially cereals, and to a method for obtaining transgenic cereal seeds. The present invention thus provides cereals which carry in their genome at least one foreign gene giving to the plant a desirable new trait, but which cereals do not carry any additional undesired selectable genes in their genome.
Background of the Invention
Recently there have been several reports of transformation of plant cells with genes of bacterial, plant or mammalian origin. It has been proposed to use whole plant tissues for production of heterologous protein material in plant hosts. Many of the transformation experiments have been made in tobacco plant, which can easily be transformed by Agrobacterium infection or electroporation of protoplasts (PCT application WO 91/02066). Tobacco plant can be regarded as a model system in plant transformation.
There is at present no satisfactory method available for routine transformation of barley or other cereals. Cereals belonging to the Monocotyledonae have proved to be very recalcitrant to genetic engineering. The Agrobacterium -mediated trans- formation routinely used for dicotyledonous plants has not been successful in transforming monocot plants. However, an application of Agrobacterium-mediaϊed transformation of monocots is disclosed in WO 92/14828, wherein a method for genetic transformation of cereals is described, in which a bacterial vector carrying a foreign gene is cocultivated with a microspore culture of the plant to be trans- formed. The microspore culture is then regenerated in a selective medium. The failure to achieve satisfactory Agrobacterium-mduced transformation in cereals has led to increased interest in other methods for the production of transgenic plants, such as the use of DNA-coated microprojectiles and direct uptake of DNA into cells or protoplasts stimulated either electrically or chemically. In addition to the problem of delivering foreign DNA into the target cell there is the problem of the competence of the cell to integrate the DNA into its genome, regenerate to a new plant and produce progeny with the transferred new trait. This means that one basic requirement for the use of any of these methods is an efficient culture and regeneration system that guarantees a large scale regeneration of fertile plants from single transformed cells.
At the moment plant regeneration remains the major limitation to the application of genetic engineering of plant cells to crop improvement. The currently used gene transfer techniques require the use of embryogenic cultures, which are rather diffi- cult to establish and maintain. The ability to produce such regenerable cultures is not only a species specific but also a cultivar specific feature.
A new application of electroporation may overcome at least some of these prob¬ lems. Dekeyser et al. (1990) have demonstrated transient expression of genes delivered into leaf segments of different cereal species by electroporation. In PCT- application WO 92/09696 the same method is applied to obtain transgenic maize plants from immature zygotic embryos or type I embryogenic callus. To prepare them to take up DNA, the callus or immature embryos are first wounded, either mechanically or chemically. Foreign DNA is then transferred to them by electro- poration. The electroporated embryos or callus are then cultured for several weeks on selective medium before resistant calli are grown into plants. The method is based on the ability to produce embryogenic callus from the scutellar tissue of immature embryos.
Particle bombardment is another gene transfer method which has been successfully used in transforming cereals. In the PCT application WO 90/01551 transformation of monocot plants with exogenous DNA to produce foreign proteins is described. The PCT-application WO 91/02071 discloses a system for genetic transformation of monocotyledonous plant cell suspension, e.g. maize cell suspension, by particle bombardment and the selection of genetic transformants from the suspensions. Fertile plants are then produced from the transformed cells by conventional methods. Particle bombardment has also been used to produce several transgenic rice varieties by transforming the scutellar tissue of immature embryos (Christou et al. 1991). In addition to the "traditional" biolistic apparatus there is a new micro- targeting device for particle bombardment of plant meristems (Sautter 1993), but so far tobacco is the only transgenic plant produced with this method.
Eventhough there has been considerable success lately in the field of cereal trans¬ formation, none of the published methods offer a general routinely applicable method for obtaining large numbers of transformed plants. In comparison to the earlier ones the present invention offers a relatively fast and simple method for producing transgenic cereal plants, which method furthermore is universally app¬ licable to all cereals independent of genotype or cultivar.
In a grass embryo there are several separately transformable meristematic regions. A grass embryo also reaches a relatively high degree of differentiation (e.g. Esau, 1977). For example in large barley seeds there are nine meristems leading to nine mutually exclusive mutant sectors, and up to seven additional meristems leading to mutually exclusive mutant sectors can be present in the embryo of the seed (Jacobsen, 1966, see Fig. 1). In an embryo independent mutant sectors which can¬ not have a mutant cluster in common, are proven anatomically, if a shoot or shoot group exists as a separate shoot primordium or as a meristematic tissue area separ¬ ated from the apex of the shoot primordium by a leaf or leaf primordium. The anatomical data from barley embryos proves that the embryo contains at least six separate shoot meristems or prospective shoot meristems which will constitute mutually exclusive mutant sectors in the plant (Jacobsen 1966). Similar results have been reported for rice in which the spikes on the primary side-shoots mutate independently of the apical spike. The present invention is developed to utilize the whole potential of the numerous meristems of the grass embryo. The present in- vention utilizes conventional gene transfer methods, the gist of the system lying in the treatment of the plant material.
Summary of the Invention
The present inventors have now developed a new efficient method for transforming grasses and cereal plants. The method of the invention is based on the fact that a grass embryo has several separately transformable meristematic regions. E.g. a barley grain has, depending on its size and stage of development, even 16 separ- ately transformable meristems. The transformation is thus directed to such a region of the embryo in which the leaf primordia and the side shoot primordia or their initial cells are located.
The transformed embryos are germinated and cultivated directly without any selec- tion, and the rachises and awns of the ears produced are analyzed as regards the transferred genes. A special advantage of the present method is thus the fact that no selection is used when growing the transformed embryo material into plants so as to exploit the whole growing capacity of the meristematic region. Consequently, it is not necessary to insert selectable genes into the genetic construct used for the transformation of the embryonic material. Another special advantage of the method of the invention is the minimal requirement of tissue culture, and it helps to elim¬ inate culture induced mutations and somaclonal variation. Thus the transformed embryos can be germinated directly without the laborious cell cultivation step. The direct germination of the transformed embryos also makes it possible to use var- ieties that are difficult to regenerate in tissue culture.
It is therefore an object of this invention to provide a method for producing a transgenic monocotyledonous plant by providing a genetic construct comprising at least one gene heterologous to the plant to be transformed, whereby no additional selectable genes are inserted into the genetic construct, transforming the meriste¬ matic cells of the embryo of a monocot plant with the said genetic construct with direct gene transfer methods, and generating a whole plant by germinating and cultivating the transformed embryo without selection.
A further object of the invention is to provide a method for generating transgenic cereal seed comprising generating transgenic plants by the above method, cultivat¬ ing green plantlets and deriving seed from the resulting mature plants.
The plants as well as seeds obtained by the above defined methods are also objects of the present invention.
Detailed Description of the Invention
It has now been found that certain modifications in relation to the selection of transformable material and the culture technique enable significantly enhanced efficiency to be achieved in grass and cereal transformation. The method of the invention to produce a transgenic monocot plant is applicable to grasses and cer¬ eals, primarily barley, Hordeum vulgare L., but can be used also for e.g. wheat, oat, rye, rice or maize. Accordingly, the present invention provides a method for generating transformed cereal plants comprising transforming the embryo material and growing it directly into plants, wherein the growth medium does not comprise any selective substances for the transformed trait.
The material to be transformed must be an embryo, either zygotic or somatic, immature or mature and it preferably contains several separate meristematic regions to be transformed. Parts of an embryo, particularly the nodal region, or intact embryos can be transformed. The term embryo or embryo material should be understood to include all stages of development from the globular stage to the mature stage of zygotic and somatic embryos. The embryos may be transformed directly after isolation or after being germinated for 1 to 5 days prior to the trans- formation. To demonstrate the method of the invention the β-glucuronidase enzyme (GUS) and the neomycin phosphotransferase enzyme (NPTII) were used as an example because there are suitable assays to detect their activity in plant tissues. The cDNA coding for β-glucuronidase under control of the maize alcohol dehydrogenase I promoter was transferred by particle bombardment to the meristematic cells of immature barley embryos and mature embryos of barley, wheat and maize to dem¬ onstrate the general applicability of the method to different cereal species. A histological assay for β-glucuronidase was performed for the transformed embryos. The blue stain indicating the transformation events was localized in the meristematic regions of the embryos by serial sectioning of the samples. The cDNA coding for neomycin phosphotransferase was placed under control of the cauliflower mosaic virus 35S promoter and transferred by particle bombardment to the meristematic cells of barley embryo. The transformed embryos were germinated and cultivated without selection so as to produce as many shoots as possible. The rest of the spike after grain removal were then analyzed as regards the transferred genes, and grains of the positive ears were used for the production of several gen¬ erations of transgenic barley plants.
To demonstrate the use of the method for the production of transgenic cereal plants using other gene constructions immature embryos of barley were transformed with the plasmid pKAH24 containing the egll gene coding for endo-β-glucanase I of Trichoderma reesei under control of the barley α-amylase promoter. The trans¬ formed embryos were germinated and cultivated without selection to produce as many shoots as possible. 9 of the 146 plants analyzed by the polymerase chain reaction contained the transferred gene.
The method of this invention can also be used to produce transgenic cereal plants expressing any other desirable new trait. Especially interesting for barley are enzymes of importance in brewing such as β-glucanases and amylolytic and proteolytic enzymes. For barley and wheat especially interesting are some agrono- mically important characteristics like preharvest sprouting. Cereal grains differ in their nutritional characteristics. The value of cereal protein is less than that from animal products mainly because of deficiencies of several essential amino acids. The food and feed value of wheat and barley grains can be improved with genetic • engineering of their storage protein genes. The method of this invention can also be used to produce transgenic plants that produce other foreign products, such as pharmaceutical peptides, proteins or compounds of secondary metabolism.
Direct gene transfer methods are used for the transformation. Suitable methods are e.g. particle bombardment and electroporation, electrophoresis may also be used. The transformed embryos or embryo parts are germinated on modified MS medium without selection so as to allow all the potentially transformed meristems to form shoots. At this stage the growth of the tillers can be enhanced, if desired. The enhancement can be effected e.g. by cutting the main shoots of the plants after sprouting. Later the rooted plantlets are grown in soil for seed production. The rachises of the ears produced are then analyzed as regards the transferred genes, and grains of the positive ears are used for the production of transgenic cereals.
The seeds from the cultivated green plantlets may be derived by harvesting them directly from the resulting mature plants or by obtaining seeds from plants one or more generations remote from the original green plantlets.
Abbreviations
T0 primary transformant Tj first progeny of T0
Brief Description of the Drawings
Fig. 1 Diagram of a transversely cut barley embryo.
Fig. 2A The vector pKAH24 containing the endo-β-glucanase gene egll.
Fig. 2B The vector pKAH21 containing the endo-β-glucanase gene egll. Fig. 2C The vector pHTT303 containing the neomycin phosphotransferase gene nptll. Fig. 2D The vector pAT13 containing the β-glucuronidase gene uidA.
Fig. 3 (A-C) Histochemical localization of β-glucuronidase gene in trans¬ formed immature embryos of barley.
Fig. 4 (A-D) Histochemical localization of β-glucuronidase gene in trans¬ formed mature embryos of barley.
Fig. 5 (A-D) Histochemical localization of β-glucuronidase gene in trans¬ formed mature embryos of wheat.
Fig. 6 (A-C) Histochemical localization of β-glucuronidase gene in trans- formed mature embryos of maize.
Fig. 7 Screening for egll by PCR technique.
Fig. 8 NPTII gel assay of leaf extracts from T0 and Tj barley plants. Lane PC represents a positive control from tobacco transgenic for
35S-nptII and lane NC a negative control from a non-trans¬ formed barley plant. Samples I to IV represent four spikes of the T0 plant Toivo and samples 1 to 25 their progeny (TJ, so that 1 to 7 are the offspring of I, 8 to 14 are offspring of II, 15 to 18 off- spring of III, and 19 to 25 offspring of IV. Samples are numbered so that they are equivalent in Figures 8, 9 and 10.
Fig. 9 Southern blot analysis of transgenic T0 and Tj plants. BamHI,
EcoRI and Hindlll digested total DNA samples (5 μg) were probed with plasmid pHTT303. The positive control (PC) is
Bam I, EcoRI and Hindlll digested pHTT303 (5 pg, representing approximately one copy per haploid genome), mixed with 5 μg BamHI, EcoRl and Hindlll digested DNA from a non-trans¬ formed barley. This digestion releases vector (2.6 kb), nptll gene (1.7 kb) and 35S promoter (1.0 kb) fragments. The S and L lanes represent negative controls containing DNA isolated from non- transformed barley spikes and leaves, respectively. Samples II to rv represent spikes of the T0 plant Toivo and samples 8 to 25 their progeny (Tj). Samples are numbered so that they are equi¬ valent in Figures 8, 9 and 10. The intensity of the bands in the barley samples as well as the number of integration specific frag- ments indicate that about two copies of pHTT303 were integrated into the barley genome. Note that part of the T0 spikes may be chimeric and part of the Tj plants homozygous for the transferred DNA, resulting in some variation of band intensities between the samples.
Fig. 10 Analysis of methylation pattern of the transferred DNA in Tj plants. BamHI, EcoRl and Hindlll digested total DNA samples (5 μg) were in addition restricted with either Dpήl (Dl) or Dpnll (D2) and probed with plasmid pHTT303. The positive control (PC) is BamHI, EcoRl and Hindlll digested pHTT303 that was further restricted with either Dpnl (Dl) or Dpnll (D2). 5 pg of digested plasmid was mixed with 5 μg.DNA from non-trans¬ formed barley, digested with same enzymes as the plasmid. The S lane represents the negative control containing DNA isolated from non-transformed barley plant spikes. The three T- plant samples
9, 15 and 22 are offspring of T0 spikes II, III and IV, respectively. See text for conclusions. Experimental
Unless otherwise stated all experiments described herein are effected according to conventional procedures used in genetic engineering.
Example 1. Construction of chimaeric genes for expression in plants.
pKAH24 and pKAH21 (expressing endo-/?-glucanase)
The 5.2 kb plasmid consists of the barley α-amylase promoter and the gene egll of Trichoderma reesei coding for the endo-β-glucanase I in the E. coli cloning vector pUC18 (Norrander et al., 1983). The plasmid pKAH21 contains the same egll gene under control of cauliflower mosaic virus 35S promoter. The plasmid constructs pKAH24 and pKAH21 are shown in Figures 2A and 2B.
pHTT303 (expressing neomycin phosphotransferase II)
The gene nptll coding for neomycin phosphotransferase II was combined at its 5' end to the cauliflower mosaic virus 35S promoter and at its 3' end to a terminator of the gene 7 of T-DNA. The recombinant gene resides in the E. coli cloning vector pUC18 (Norrander et al, 1983) as a Bglll-Sacl fragment between the BamHI and Sαcl sites of the vector. The plasmid construct pHTT303 is shown in Figure 2C.
pAT13 (expressing ?-glucuronidase)
The plasmid pAT13 was a gift from Carlsberg Research Institute. The 6.5 kb plasmid consists of the alcohol dehydrogenase 1 promoter, a fragment from intron 1 of the A dh I gene, a β-glucuronidase coding region, and the nopaline synthase polyadenylation region inserted into pUC8 (Klein et al. 1988), as pAIj GusN. The plasmid construct pAT13 is shown in Figure 2D. Example 2. Transformation of immature embryos of barley by particle bom¬ bardment.
Immature embryos of the size of 0.5 to 1 mm were isolated from developing seeds of barley (Hordeum vulgare L. cv. Kymppi). The embryos were bombarded at the embryonic axis. For particle bombardment the isolated embryos were placed scutellum down on modified MS medium (Olsen, 1987) supplemented with 0.4 mg/1 BA and 35 g/1 maltose and solidified with 3 g/1 gellan gum. The principle of particle bombardment is described by Sanford et al. (1987) and Klein et al. (1987). The helium modification of the BiolisticR PDS-1000 device (Bio-Rad) was used for transformation. The following conditions were used: sample chamber pressure 3.1 kPa; pressure of helium shock wave 9.0 MPa; distance between macrocarrier and stopping screen 6 mm; distance between macrocarrier and rupture disk 9 mm. Particles were prepared by coating gold particles (1.0 μm in diameter) with the plasmid pKAH24 (β-glucanase), or with the plasmid pAT13 (β-glucuronidase), or with a 1:1 mixture of plasmids pHTT303 (neomycine phosphotransferase) and pAT13. In each bombardment, 1.0-2.0 μg of DNA was used and each sample was bombarded once.
Example 3. Transformation of mature embryos of barley, wheat and maize by particle bombardment.
Mature seeds of barley (Hordeum vulgare L.), wheat (Triticum aestivum L.) and maize (Zea mays L.) were surface sterilized first with 70% ethanol for 5 min and then with NaOCl (4% active Cl) for 10 min, rinsed several times with sterile distilled water and germinated on moist filter paper for 1 day. The embryos were separated from the seeds and part of the coleoptile and leaves covering the meristematic regions of the embryo were removed with a scalpel. The embryos were transformed by particle bombardment with the plasmid pAT13 as described in Example 1. Example 4. Histochemical localization of the ^-glucuronidase gene.
Immature embryos of barley and mature embryos of barley, wheat and maize transformed with the plasmid pAT13 were stained with X-gluc (5-bromo-4- chloro-3-indolyl-β-D-glucuronic acid cyclohexylammonium salt) with the method of Jefferson (1987) with the modifications of Ritala et al. (1993) to detect the activity of the transferred uidA gene coding for β-glucuronidase. The stained embryos were embedded in paraffine and sectioned with microtome to localize the transformed cells. The localization of blue stain indicating transformation in the meristematic regions is shown in Figure 3 (3A-3C) for immature embryos of barley, Figure 4 (4A-4D) for mature embryos of barley, Figure 5 (5A-5D) for mature embryos of wheat, and Figure 6 (6A-6C) for mature embryos of maize.
Example 5. Growing the transformed plants.
After transformation the embryos were transferred on modified MS medium (Olsen 1987) supplemented with 0.4 mg/1 BA and 35 g 1 maltose and solidified with 3 g/1 gellan gum for germination. The plates were incubated at 23°C in light (50 μmol m~2 s"1). The embryos were germinated without any selective substances in the medium. After three weeks the shoots were transferred to modified MS rooting medium (Olsen 1987) without hormones and solidified with 3 g/1 gellan gum. After 2 to 3 weeks the plantlets were potted in soil and grown to maturity in the green¬ house. The embryos from all green grains were isolated for further cultivation, the rachises and/or awns were screened for the presence of the transferred gene.
Example 6. Screening for the transformed plants.
Assay for NPTTI activity. Neomycin phosphotransferase II activity in the plants was assayed according to McDonnell et al. (1987) with the following modifica- tions: Extraction buffer with 0.13 g/1 leupeptine and a small amount of purified sea sand was added to the leaf material which was then ground thoroughly in an Ep- pendorf tube and centrifuged. The supernatant was collected and the protein con- centration determined (Bradford, 1976). The amount of extract used in the enzyme assay corresponded to 20 μg of total protein. The reaction mixture was blotted after 5 minutes centrifugation via a dot blot apparatus (Milliblot-D, Millipore) onto Whatman P81 paper. Alternatively, the NPTII gel assay (Reiss et al. 1984, mod- ified by Van den Broeck et al. 1985) was used.
Screening of plants by PCR technique.
Screening for egll. The plant material was screened by the polymerase chain reaction (PCR) carried out in a Perkin Elmer cetus 9600 thermocycler. The com- plete PCR mixture contained 100-500 ng of genomic or 0.5 pg of pKAH21 DNA, 12.5 pmol of each oligonucleotide primer, 200 μM dNTPs, 0.5 U Dynazyme and buffer supplied by the enzyme manufacturer (Finnzymes Oy) in a total volume of 100 μl. Thirty cycles were performed under following conditions: 75 s at 95°C, 2 min at 55°C, and 3 min at 72°C. Primers were designed to amplify a 557 bp frag- ment of the cDNA for EGI. The forward primer was 5'-AGGACACCTCGGTGG- TCCTT-3' and the reverse primer 5'-AGAGTGAGGGGTCAAGGCATT-3'. The PCR performance was controlled by including a primer pair amplifying the promo¬ ter fragment of one of the α-amylase genes. Total DNA isolated from a transgenic barley cell line (VTT-6-93002, transformed with pHTT303 and pKAH21) was used as a positive control. The amplified samples were analyzed by electrophoresis in a 2% agarose gel.
Plants grown from immature embryos transformed with the plasmid pKAH24 were screened for the gene egll by PCR. About 1400 spikes were analyzed from 146 plants. Nine plants of these (12 spikes) were shown to contain the transferred gene (Fig. 7).
Screening for nptll. The plant material was screened by the polymerase chain reaction (PCR) carried out in a Techne PHC-2 thermocycler. The complete PCR mixture contained 100-500 ng total or 0.15 pg pHTT303 DNA, 50 pmol of each oligonucleotide primer, 200 μM dNTPs, 2 U Dynazyme™ and buffer supplied by the enzyme manufacturer (Finnzymes Oy) in a total volume of 100 μl. Thirty cycles were performed under following conditions: 75 s at 94°C, 2 min at 55°C, and 3 min at 72°C. Primers were designed to amplify a 5' sequence of nptll in pHTT303. The forward primer used was 5'-ACACGCTGAAATCACCAGTCTC-3' (+1 from start of transcription) and the reverse primer 5'-CTCGTCCTGCAGTTC- ATTC-3' or 5'-TCGCCCAATAGCAGCCAGTC-3' (+281 and +417 from start of transcription, respectively). The PCR performance was controlled by including a primer pair amplifying the promoter fragment of one of the α-amylase genes. The amplified samples were analyzed by electrophoresis in a 2% agarose gel.
During in vitro rooting, NPTII activity was analyzed from the 227 plants of the T0 generation by the dot blot method. One plantlet proved to be NPTII positive, and this plant was named and is later referred to as Toivo. Toivo was transferred to potting soil and grown in the growth room. By now it has produced 98 fertile spikes. Embryos from green grains were isolated for further cultivation to Tj progeny. The rest of the spikes of Toivo after grain removal were screened for the presence of the nptll gene with the PCR technique. Of the 90 spikes screened, six were positive for the nptll gene. Four of them gave positive signals in the NPTII gel assay (Fig. 8) as well. The presence of the transferred nptll gene in these four was confirmed with Southern blot hybridization (Fig. 9).
Screening of the Tj generation
Progeny of four shoots of Toivo carrying the nptll gene were studied further. They produced twenty five plantlets as T- progeny. PCR analysis indicated that among the sets of siblings five out of seven, two out of seven, four out of four and four out of seven, contained the nptll gene. The NPTII enzyme activity analysis revealed that fourteen of them also expressed the transferred gene (Fig. 8). The integration of the nptll gene in the genomes of T- progenies was confirmed by Southern blot hybridization (Fig. 9). Example 7. Southern blot analysis of T0 and T, plants as evidence for trans¬ formation and inheritance.
Total DNA from the plant material was isolated according to Dellaporta et al. 1983. For Southern blots, isolated total DNA was restricted with enzymes BamHI, EcoRl, H dIII, Dp l and/or Dpnll in the buffers recommended by the enzyme manufacturers. 5 μg digested DNA was separated by electrophoresis in 0.8 % agarose gels. The gels were blotted onto MagnaGraph (Micron Separation Inc.) nylon membranes (Southern 1975). The 32P-dCTP -labeled plasmid pΗTT303 was used as a probe. Hybridizations (at 42°C overnight in 50 % formamide) and wash¬ ing of the filters were carried out according to Sambrook et al. (1989). After washing the filter was exposed to X-ray film using intensifying screens at -70°C for three days.
Total DNAs isolated from the rest of the spikes of Toivo (T0 generation) after grain removal and from leaves of the T- progeny were analyzed for the presence of the nptll gene. The isolated DNAs were digested simultaneously with the enzymes BamHI, EcoRl and Hindlll. This releases vector (2.6 kb), nptll (1.7 kb) and 35S promoter (1.0 kb) fragments. When hybridized with the pHTT303 probe, the BamHl-EcoRl-Hindlll -digested total DNA sample of the non-transformed con¬ trol shows a 2.6 kb band as background. In addition to this unspecific band (that overlaps with the vector band), the DNA samples from the four T0 shoots and from fifteen Tj progeny shoots contained copies of the nptll and promoter fragments, and in addition several extra fragments (Fig. 9). The extra fragments do not corre- spond to partial digestion fragments of pHTT303. This indicates integration of the transferred pHTT303 into the genome of Toivo as well as passage of both the transferred sequences and the integration pattern to T- progeny. From this fact it is also apparent that all of the transgenic tillers originate from a single transformed cell and show integration at one locus. In order to further confirm that the DNA hybridizing to pHTT303 is not carry-over of the plasmid DNA used for transformation, we performed analysis with the restriction enzymes Dpnl and Dpnll. They both recognize and cleave the sequence GATC, but while Dpnll cleavage is inhibited by the methylation of the A residue, Dpnl requires methylation. Our E. coli host DH5α performs methylation of the A residue of the GATC sequence, as do all strains wild type for the dam locus. How¬ ever, the A residue is not methylated in eukaryotes (Torres et al. 1993). Figure 10 shows that the pHTT303 plasmid isolated from E. coli is cleaved by Dpnl but not Dpnll. On the other hand, the sequences homologous to pHTT303 in the barley DNA are cleaved by Dpnll but not Dpnl, indicating that their methylation status is different from that of the plasmid. We conclude that the pHTT303 homologous sequences in the barley samples have been replicated by the barley cells.
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Claims

Claims
1. A method for generating a transgenic monocotyledonous plant, comprising transforming the embryo material of the host plant and growing it directly into plant, wherein the growth medium does not comprise any selective substances for the transformed trait.
2. A method for producing a transgenic monocotyledonous plant, comprising a) providing a genetic construct comprising at least one gene heterologous to the plant to be transformed, whereby no selectable genes are inserted into the said genetic construct, b) transforming meristematic cells of the embryo of the host plant with the said genetic construct, and c) generating a whole plant by germinating and cultivating the transformed embryo without selection.
3. A method according to claim 1 or 2 wherein the monocotyledonous plant is barley.
4. A transgenic monocotyledonous plant or a progeny thereof comprising at least one gene heterologous thereto stably integrated into its genome and being obtain¬ able by the process of claim 1 or 2.
5. The transgenic monocotyledonous plant or a progeny thereof according to claim 4 wherein the plant is barley.
6. A method for generating transgenic cereal seed comprising generating transgenic plants by the method according to claim 1 or 2, cultivating green plantlets and deriving seed from the resulting mature plants.
7. A seed of a transgenic monocotyledonous plant, said seed comprising at least one gene heterologous to the said plant stably integrated into its genome and being obtainable by the method of claim 6.
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