WO1995006127A1 - Procede pour la production de cereales transgeniques - Google Patents

Procede pour la production de cereales transgeniques Download PDF

Info

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
Authority
WO
WIPO (PCT)
Prior art keywords
plant
transgenic
transformed
plants
gene
Prior art date
Application number
PCT/FI1994/000365
Other languages
English (en)
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
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Valtion Teknillinen Tutkimuskeskus filed Critical Valtion Teknillinen Tutkimuskeskus
Priority to AU74631/94A priority Critical patent/AU7463194A/en
Publication of WO1995006127A1 publication Critical patent/WO1995006127A1/fr

Links

Classifications

    • 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.

Abstract

L'invention se rapporte à la biotechnologie végétale et plus particulièrement à un nouveau procédé pour obtenir des plantes monocotylédones transgéniques stables, en particulier des céréales, ainsi qu'à un procédé pour obtenir des graines de céréales transgéniques. Cette invention concerne par conséquent des céréales qui portent dans leurs génomes au moins un gène étranger donnant à la plante un nouveau trait désirable mais qui ne portent pas de gènes sélectionnables additionnels dans leurs génomes. Dans ce procédé, on produit une telle plante monocotylédone transgénique en formant un produit de synthèse génétique comportant au moins un gène hétérologue à la plante à transformer, pour qu'aucun gène sélectionnable additionnel ne soit introduit dans le produit de synthèse génétique, en transformant les cellules méristématiques de l'embryon d'une plante monocotylédone avec ledit produit de synthèse génétique, et en produisant une plante entière en faisant germer et en cultivant l'embryon ainsi transformé sans sélection.
PCT/FI1994/000365 1993-08-20 1994-08-22 Procede pour la production de cereales transgeniques WO1995006127A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU74631/94A AU7463194A (en) 1993-08-20 1994-08-22 Method for the production of transgenic cereals

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
FI933682A FI933682A0 (fi) 1993-08-20 1993-08-20 Metod foer att producera transgena saedesvaexter
FI933682 1993-08-20

Publications (1)

Publication Number Publication Date
WO1995006127A1 true WO1995006127A1 (fr) 1995-03-02

Family

ID=8538451

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/FI1994/000365 WO1995006127A1 (fr) 1993-08-20 1994-08-22 Procede pour la production de cereales transgeniques

Country Status (3)

Country Link
AU (1) AU7463194A (fr)
FI (1) FI933682A0 (fr)
WO (1) WO1995006127A1 (fr)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996004392A2 (fr) * 1994-07-29 1996-02-15 Pioneer Hi-Bred International, Inc. Cereales transgeniques
WO1998004723A1 (fr) * 1996-07-26 1998-02-05 Regents Of The University Of Minnesota Avoine transgenique fertile
US5736369A (en) * 1994-07-29 1998-04-07 Pioneer Hi-Bred International, Inc. Method for producing transgenic cereal plants
WO2003007698A2 (fr) * 2001-07-19 2003-01-30 Monsanto Technology Llc Nouveau procede de production de plantes transgeniques
WO2008028121A1 (fr) * 2006-08-31 2008-03-06 Monsanto Technology Llc Transformation de plantes sans sélection
US10584350B2 (en) 2016-10-27 2020-03-10 Board Of Trustees Of Michigan State University Structurally modified COI1
EP3456825A4 (fr) * 2016-05-13 2020-03-18 Kaneka Corporation Procédé d'édition du génome d'une plante
US11499158B2 (en) 2016-05-13 2022-11-15 Kaneka Corporation Method for modifying plant
US11518998B2 (en) 2016-05-13 2022-12-06 Kaneka Corporation Method for creating transformed plant

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992020809A1 (fr) * 1991-05-15 1992-11-26 Agracetus, Inc. Procede de creation d'un riz transforme

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992020809A1 (fr) * 1991-05-15 1992-11-26 Agracetus, Inc. Procede de creation d'un riz transforme

Cited By (36)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996004392A3 (fr) * 1994-07-29 1996-03-28 Pioneer Hi Bred Int Cereales transgeniques
US5736369A (en) * 1994-07-29 1998-04-07 Pioneer Hi-Bred International, Inc. Method for producing transgenic cereal plants
AU697373B2 (en) * 1994-07-29 1998-10-01 Pioneer Hi-Bred International, Inc. Transgenic cereal plants
WO1996004392A2 (fr) * 1994-07-29 1996-02-15 Pioneer Hi-Bred International, Inc. Cereales transgeniques
WO1998004723A1 (fr) * 1996-07-26 1998-02-05 Regents Of The University Of Minnesota Avoine transgenique fertile
US5773269A (en) * 1996-07-26 1998-06-30 Regents Of The University Of Minnesota Fertile transgenic oat plants
US8212109B2 (en) 2001-07-19 2012-07-03 Monsanto Technology Llc Method for the production of transgenic plants
WO2003007698A2 (fr) * 2001-07-19 2003-01-30 Monsanto Technology Llc Nouveau procede de production de plantes transgeniques
WO2003007698A3 (fr) * 2001-07-19 2004-04-08 Monsanto Technology Llc Nouveau procede de production de plantes transgeniques
US10253322B2 (en) 2001-07-19 2019-04-09 Monsanto Technology Llc Method for the production of transgenic plants
US9353377B2 (en) 2001-07-19 2016-05-31 Monsanto Technology Llc Method for the production of transgenic plants
EP2390337A1 (fr) * 2006-08-31 2011-11-30 Monsanto Technology LLC Transformation de plantes sans sélection
EP3269818A1 (fr) * 2006-08-31 2018-01-17 Monsanto Technology LLC Transformation de plantes sans sélection
AU2007289111B2 (en) * 2006-08-31 2013-03-28 Monsanto Technology Llc Plant transformation without selection
US8513016B2 (en) 2006-08-31 2013-08-20 Monsanto Technology Llc Methods for producing transgenic plants
US8581035B2 (en) 2006-08-31 2013-11-12 Monsanto Technology Llc Plant transformation without selection
US8847009B2 (en) 2006-08-31 2014-09-30 Monsanto Technology Llc Plant transformation without selection
US8853488B2 (en) 2006-08-31 2014-10-07 Monsanto Technology Llc Methods for rapidly transforming monocots
CN101528932B (zh) * 2006-08-31 2014-12-10 孟山都技术有限公司 植物无选择转化
CN104357535A (zh) * 2006-08-31 2015-02-18 孟山都技术有限公司 植物无选择转化
US8962326B2 (en) 2006-08-31 2015-02-24 Monsanto Technology Llc Methods for producing transgenic plants
US8124411B2 (en) 2006-08-31 2012-02-28 Monsanto Technology Llc Methods for producing transgenic plants
US9617552B2 (en) 2006-08-31 2017-04-11 Monsanto Technology Llc Plant transformation without selection
US9783813B2 (en) 2006-08-31 2017-10-10 Monsanto Technology Llc Methods for rapidly transforming monocots
US8395020B2 (en) 2006-08-31 2013-03-12 Monsanto Technology Llc Methods for rapidly transforming monocots
US10006036B2 (en) 2006-08-31 2018-06-26 Monsanto Technology Llc Methods for producing transgenic plants
US10233455B2 (en) 2006-08-31 2019-03-19 Monsanto Technology Llc Plant transformation without selection
WO2008028121A1 (fr) * 2006-08-31 2008-03-06 Monsanto Technology Llc Transformation de plantes sans sélection
US11718855B2 (en) 2006-08-31 2023-08-08 Monsanto Technology, Llc Methods for producing transgenic plants
US11091766B2 (en) 2006-08-31 2021-08-17 Monsanto Technology Llc Methods for producing transgenic plants
US10941407B2 (en) 2006-08-31 2021-03-09 Monsanto Technology Llc Plant transformation without selection
EP3456825A4 (fr) * 2016-05-13 2020-03-18 Kaneka Corporation Procédé d'édition du génome d'une plante
US11499158B2 (en) 2016-05-13 2022-11-15 Kaneka Corporation Method for modifying plant
US11518998B2 (en) 2016-05-13 2022-12-06 Kaneka Corporation Method for creating transformed plant
US11591605B2 (en) 2016-05-13 2023-02-28 Kaneka Corporation Plant genome editing method
US10584350B2 (en) 2016-10-27 2020-03-10 Board Of Trustees Of Michigan State University Structurally modified COI1

Also Published As

Publication number Publication date
AU7463194A (en) 1995-03-21
FI933682A0 (fi) 1993-08-20

Similar Documents

Publication Publication Date Title
Ritala et al. Fertile transgenic barley by particle bombardment of immature embryos
US7803988B2 (en) Plant transformation method
AU643846B2 (en) Plant transformation method using agrobacterium species
Cheng et al. Production of fertile transgenic peanut (Arachis hypogaea L.) plants using Agrobacterium tumefaciens
AU738153C (en) Methods for the production of stably-transformed, fertile wheat employing agrobacterium-mediated transformation and compositions derived therefrom
Suzuki et al. Production of transgenic plants of the Liliaceous ornamental plant Agapanthus praecox ssp. orientalis (Leighton) Leighton via Agrobacterium-mediated transformation of embryogenic calli
Dong et al. Transgenic flax plants from Agrobacterium mediated transformation: incidence of chimeric regenerants and inheritance of transgenic plants
Sarria et al. Transgenic plants of cassava (Manihot esculenta) with resistance to Basta obtained by Agrobacterium-mediated transformation
Suzuki et al. Agrobacterium-mediated production of transgenic plants of Muscari armeniacum Leichtl. ex Bak.
Zuker et al. Wounding by bombardment yields highly efficient Agrobacterium-mediated transformation of carnation (Dianthus caryophyllus L.)
WO2002014520A2 (fr) Procede relatif a l'induction d'embryogenese somatique directe et d'embryogenese secondaire dans les cellules de plantes a monocotyledon et les plantes fertiles a monocotyledon a regenerescence rapide
EP1407000B1 (fr) Methode de transformation et plantes transgeniques ainsi produites
González et al. Production of transgenic Eucalyptus grandis x E. urophylla using the sonication-assisted Agrobacterium transformation (SAAT) system
Solleti et al. Additional virulence genes in conjunction with efficient selection scheme, and compatible culture regime enhance recovery of stable transgenic plants in cowpea via Agrobacterium tumefaciens-mediated transformation
US7022894B2 (en) Methods of transforming plants and identifying parental origin of a chromosome in those plants
Oneto et al. Biolistic maize transformation: Improving and simplifying the protocol efficiency
Sangwan et al. Effect of culture conditions on Agrobacterium-mediated transformation in datura
WO1995006127A1 (fr) Procede pour la production de cereales transgeniques
Sanyal et al. Agrobacterium tumefaciens-mediated transformation of chickpea (Cicer arietinum L.) using mature embryonic axes and cotyledonary nodes
Hewezi et al. Dehydrating immature embryo split apices and rehydrating with Agrobacterium tumefaciens: A new method for genetically transforming recalcitrant sunflower
Robledo-Paz et al. Genetic transformation of garlic (Allium sativum L.) by particle bombardment
Mendel et al. Gene transfer to barley
US20030233676A1 (en) Increasing host plant susceptibility to agrobacterium infection by overexpression of the arabidopsis VIP1 gene
Li et al. Recovery of transgenic asparagus plants by particle gun bombardment of somatic cells
Arokiaraj Genetic transformation of Hevea brasiliensis (rubber tree) and its applications towards crop improvement and production of recombinant proteins of commercial value

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU BR CA CN CZ FI JP LT LV NO NZ RU US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA