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WO1995011967A1 - Zygote viable - Google Patents

Zygote viable Download PDF

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Publication number
WO1995011967A1
WO1995011967A1 PCT/EP1994/003485 EP9403485W WO9511967A1 WO 1995011967 A1 WO1995011967 A1 WO 1995011967A1 EP 9403485 W EP9403485 W EP 9403485W WO 9511967 A1 WO9511967 A1 WO 9511967A1
Authority
WO
WIPO (PCT)
Prior art keywords
zygote
dna
gamete
treated
paternal
Prior art date
Application number
PCT/EP1994/003485
Other languages
English (en)
Inventor
Aksel Buchter-Larsen
Hans Christian Pedersen
Original Assignee
Sandoz Ltd.
Sandoz-Patent-Gmbh
Sandoz-Erfindungen Verwaltungsgesellschaft M.B.H.
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 Sandoz Ltd., Sandoz-Patent-Gmbh, Sandoz-Erfindungen Verwaltungsgesellschaft M.B.H. filed Critical Sandoz Ltd.
Priority to AU79394/94A priority Critical patent/AU7939494A/en
Publication of WO1995011967A1 publication Critical patent/WO1995011967A1/fr

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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)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • 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
    • 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
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/04—Plant cells or tissues

Definitions

  • the present invention relates to the production of novel organisms, and in particular to the production of transformed plants.
  • the present invention substantially overcomes this disadvantage.
  • viable zygote in which chromosomal DNA from one of the parental gametes has been substantially degraded.
  • viable zygote is meant the fusion product of a maternal and paternal gamete, which product is capable of undergoing mitotic cell division when exposed to a suitable environment (in vivo, in vitro, ex vivo, or in uter ⁇ ). It is preferred that at least some of the degraded DNA from one of the gametes is incorporated into the chromosomes of the other gamete.
  • the degradation preferably involves the introduction into the DNA of both double and single strand breaks.
  • the DNA may be degraded to the extent that chromosome fragments remain, or it may be somewhat more degraded, for example to the extent that transcribable gene fragments do not remain.
  • the zygote may optionally be treated with an agent such as benzamide or 3-amino benzamide which inhibits enzymes such as the nuclear polymerizing ADP-ribosyl transferase; DNA ligase II; deoxynucleotidyl terminal transferase and the repair DNA polymerases.
  • an agent such as benzamide or 3-amino benzamide which inhibits enzymes such as the nuclear polymerizing ADP-ribosyl transferase; DNA ligase II; deoxynucleotidyl terminal transferase and the repair DNA polymerases.
  • Such agents include ultra-violet light and alkylating agents (dimethylsulphate for example) as well as other agents such as l,3-Bis-(2-chloroethyl)-l-nitrosourea and even low levels of DNAase which are known to introduce strand-breaks.
  • the paternal gamete DNA has been degraded.
  • the gametes may comprise the combination of (i) an ovum and a sperm, or (ii) an egg cell or other ovule-derived cell capable of functioning thereas, and a pollen grain and/or sperm cell.
  • the paternal gamete Prior to fertilization, the paternal gamete may be treated with an agent capable of degrading or otherwise cleaving its chromosomal DNA in response to a specific stimulus provided to the zygote. It is particularly preferred that the paternal gamete has been treated substantially in the dark with a photo-excitable DNA intercalater, and that fertilization is also effected substantially in the dark.
  • “dark” is meant, the substantial absence of ultraviolet light and other electromagnetic radiations having a wavelength of from about 0.1 to about l ⁇ m. In particular “dark” means the substantial absence of electromagnetic radiations having a wavelength of from about 350 to 800 nm.
  • the DNA intercalater may be an acridine compound, including acridine orange or acridine yellow, or it may be any other suitable compound, for example: ethidium bromide, 6-mercapto purine or propidium iodide.
  • the specific stimulus may be visible light or ultraviolet light.
  • visible or ultraviolet light is meant electromagnetic radiations having a wavelength of from about 0.1 to about l ⁇ m, and more particularly those radiations having wavelengths within the range of about 100 to 800nm
  • the zygote may comprise a substantially haploid genome, or a genome contributed substantially by the maternal gamete.
  • the amount of degradation may be chosen by the skilled man, having regard to the amount of intercalater introduced into the DNA and amount of stimulus provided to the thus intercalated DNA.
  • Chromosome fragments, optionally comprising genes or gene fragments, originating from the paternal gamete are preferably integrated into chromosomal DNA originating from the maternal gamete. This integration may take place in the zygote in the nuclear part thereof which is derived from the maternal gamete, although the invention also includes the possibility that the integration takes place in the mitochondria or chloroplasts (if present). Such fragments may of course be stably present in the zygote absent such integration.
  • the zygote may be treated with any suitable known ploidy increasing agent, either subsequent to degradation of the DNA but prior to the first mitotic division of the zygote, or subsequent to the first mitotic division of the zygote but prior to degradation of the DNA.
  • suitable ploidy increasing agents include colchicine, dinitroaniline herbicides such as oryzalin and trifluralin, amiprophos methyl and nitrous oxide.
  • the paternal chromosomal DNA prior to its degradation, comprises a suitable marker gene.
  • marker genes include genes involved in the generation of a phenotypically identifiable trait, antibiotic resistance-inducing genes, herbicide resistance - inducing genes, and others such as those encoding beta glucuronidase and luciferase.
  • the invention also includes an organism, particularly a non-human animal or a plant, resulting from cell division of the zygote according to the present invention.
  • the invention still further includes the progeny of the organism, or the seeds of the organism and/or the progeny thereof.
  • the invention still further includes a method of producing a viable zygote according to the invention, which has an altered chromosome complement when compared with the wild type, comprising fertilizing a maternal gamete with a paternal gamete, characterized in that in the zygote, chromosomal DNA from one of the gametes is substantially degraded.
  • the paternal gamete is treated with a photo-excitable DNA intercalating agent in the dark, fertilization of the maternal gamete takes place in the dark, and that the zygote is subjected to visible or uv light before or after its first mitotic division.
  • the invention still further includes a zygote obtainable by the above disclosed method.
  • the invention will be further apparent from the following which describes the production of essentially maternal haploid transformed sugar beet plants in which the paternally originating GUS marker gene has been incorporated into the maternal genome as a consequence of the paternal DNA having been selectively destroyed post fertilization.
  • PGM-B 2 ml boron-free PGM
  • PGM used for sugar beet pollen is defined as: sucrose 300 g/1; CaCl 2 2H 2 O 150 mg 1; H 3 BO 3 100 mg/1; 50mM MES, (pH 6.0)).
  • the tube is foiled to prevent light from reaching the pollen. All subsequent operations are performed under conditions of reduced light intensity.
  • PGM-B is added so that the total volume is about 10 ml.
  • the thus treated pollen is centrifuged at about lOOOrpm for about 5 min (Hereaus Labofuge), the Acridine Orange- containing supernatant is removed and the precipitated pollen is washed with 10 ml PGM-B.
  • the thus washed pollen is centrifuged as above, and the precipitate is resuspended in about 0.5-1 ml PGM-B.
  • the Acridine Orange treatment is considered successful if thus treated pollen can germinate in the dark but not in the light. Moreover, under a UV-microscope orange colored nuclei are visible in the pollen tubes of the germinating pollen.
  • ovules are harvested (in the dark or in very dim light) from the pollinated flowers (normally sugarbeet pollen fertilize the egg cell about 17h post- pollination, after about 21h the zygote is formed and the first mitotic division is about 25h post-pollination) and placed on a suitable haploid embryo induction medium in a petri-dish which is subsequently sealed and covered with foil until exposure to light.
  • a suitable haploid embryo induction medium in a petri-dish which is subsequently sealed and covered with foil until exposure to light.
  • some of the ovules are exposed to visible light (2000 ⁇ Em "2 S _1 ) from a Nolpi light source at a distance of about 50cm for 15 or 30 min. Following such exposure the petri- dishes are re-sealed, placed in dark at 25°C and the thus exposed ovules are cultured for about 3-6 weeks until harvest of embryos.
  • Harvested embryos are transferred to regeneration medium. Developing shoots are cloned on standard sugar beet cloning medium and the thus developed shoots are rooted by transfer to medium containing Indole butyric acid (IBA). Rooted plants are transferred to a greenhouse. The plants are analysed for morphological abnormalities; chromosome number group (flow cytometry); isozyme production; transgenic status (X-Gluc GUS-assay); and glyphosate resistance.
  • IBA Indole butyric acid
  • Pollen is harvested from plants transgenic for the GUS-marker gene, pooled, treated with Acridine Orange for 60 minutes and washed as described above. This treated pollen is used to pollinate a single CMS-plant. Ovules are harvested 24 hours or four days post-pollination and treated with light as described above. The results demonstrate that acridine Orange loaded pollen can germinate on stigma and grow through the micropyle to the egg cell and fertilize the egg cell to form a viable zygote that can develop into an embryo. Moreover, certain chimeric embryos are found to be GUS-positive in cotyledons, but GUS-negative in root and root-hairs.
  • the egg cell has divided at least once meaning that the nucleic acid in the cell(s) of the zygote which are destined to form the root part of the embryo is selectively destabilised by the light treatment. From this particular chimeric embryo, shoots are regenerated and cloned. Some of the shoots consisted of mainly GUS-negative cells, other were mainly GUS-positive. The GUS-positive cells are regenerated into plants and rooted.
  • the thus rooted plants are GUS positive in all tissues (including roots) meaning that the observed chimerism is not due to integration of the transgene behind a tissue-specific (shoot) promoter or is otherwise under the control of a shoot specific region on the chromosome, but is due to the late treatment of light to the developing zygote.
  • the amount of Acridine Orange per cell of the zygote thus gradually diminishes a the zygote divides.
  • ovules are harvested and exposed to different amounts of visible light.
  • the amount of light used for degradation of the chemical chromosome inter ⁇ calating complex is regulated individually for each fertilization event so that the amount of light given to any fertilized egg correlates positively with the donor-DNA fragmentation.
  • Table 1 summarizes the results of the above treatment. Row A of the Table relates to ovules isolated from unpollinated flowers.
  • the present invention enables the production of transformed plants wherein gene transfer from one haploid genome to another (paternal to maternal) occurs thus enabling the production, with or without embryo rescue, of embryos or seeds having a near haploid [or near halved tetraploid] chromosome set.
  • the present invention has been exemplified with respect to the production of transformed sugar beet plants transgenic for the paternally originating GUS marker gene, it will be appreciated that it is not limited to plants, and that transformed versions of any organism which results from the mitotic division of a zygote may be produced. Moreover, if it can be determined where the egg cell is located in the ovule, it is possible using the method according to the invention to direct light specifically at the egg cell thus cleaving only DNA contained within this fertilized cell. This leaves the nucleic acids in the central nucleus - which gives rise to endosperm - relatively intact.
  • the ovule is loaded with acridine orange
  • precise direction of light to only the egg cell will result in a similar situation, but with the degradation of mainly maternally-derived DNA in the zygote.
  • the technique can equally well be applied to animals.
  • eggs may be collected treated with acridine orange and fertilized with non-treated sperm to facilitate degradation of the maternally originating genome and survival of the paternally originating genome in the zygote.
  • the present invention enables the introduction of agronomically important traits (such as nematode, insect or virus resistance) from wild species into cultured distantly related species, with the proviso that the pollen from the wild species is capable of inducing zygote formation in the cultured species.
  • agronomically important traits such as nematode, insect or virus resistance
  • Genes of interest may be introduced into hitherto difficult to transform crop species by a process involving the introduction of a gene (by known transformation techniques) into relatives distantly related to the difficult to transform species.
  • the gene is then introduced into the said crop species by the asymmetric chromosome transfer technique according to the invention. This protocol is especially useful for the introduction of desired traits into difficult to transform of otherwise recalcitrant crops.
  • the method according to the invention provides the possibility to transfer chromosomes or chromosome fragments containing the desired genes: (i) from maintainer or restorer lines to male sterile Unes without the loss of male sterile character; or (ii) between maintainer lines and restorer lines such that the desired genes are allelic.
  • Numerous medical applications of the present invention include the replacement of mutated or otherwise defective regions of recipient chromosomes at the zygotic level in gametes suspected of containing such defective chromosome regions.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Botany (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

La présente invention se rapporte à un zygote viable dans lequel l'ADN chromosomique provenant de l'un des gamètes parents a été sensiblement dégradé. L'invention se rapporte également à des organismes, en particulier des plantes, obtenus par division cellulaire du zygote; ainsi qu'à un procédé de production d'un zygote viable comprenant un complément chromosomique modifié par rapport au zygote de phénotype sauvage, ce procédé consistant à féconder un gamète femelle avec un gamète mâle et se caractérisant en ce que, dans le zygote, l'ADN chromosomique provenant de l'un des gamètes est sensiblement dégradé. Le gamète mâle est de préférence traité, dans l'obscurité, au moyen d'un agent d'intercalation d'ADN, la fécondation du gamète femelle s'effectuant également dans l'obscurité, et le zygote étant exposé à une lumière visible et/ou ultraviolette avant ou après sa première division mitotique.
PCT/EP1994/003485 1993-10-25 1994-10-24 Zygote viable WO1995011967A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU79394/94A AU7939494A (en) 1993-10-25 1994-10-24 Viable zygote

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB9321975.6 1993-10-25
GB939321975A GB9321975D0 (en) 1993-10-25 1993-10-25 Improvements in or relating to organic compounds

Publications (1)

Publication Number Publication Date
WO1995011967A1 true WO1995011967A1 (fr) 1995-05-04

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Application Number Title Priority Date Filing Date
PCT/EP1994/003485 WO1995011967A1 (fr) 1993-10-25 1994-10-24 Zygote viable

Country Status (3)

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AU (1) AU7939494A (fr)
GB (1) GB9321975D0 (fr)
WO (1) WO1995011967A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002098210A3 (fr) * 2001-06-05 2004-04-15 Univ Missouri Procede de doublement des chromosomes
CN113125227A (zh) * 2020-12-25 2021-07-16 上海乐辰生物科技有限公司 核型分析用制备高分辨染色体的秋水溶液及应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SU436645A1 (ru) * 1973-01-25 1974-07-25 Центральная Ордена Трудового Красного Знамени Генетическая Лаборатория Им.И.В.Мичурина Способ преодолени нескрещиваемости плодовых культур
RU2002406C1 (ru) * 1992-01-16 1993-11-15 Pashchenko Vasilij M Способ получени мутаций растений

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SU436645A1 (ru) * 1973-01-25 1974-07-25 Центральная Ордена Трудового Красного Знамени Генетическая Лаборатория Им.И.В.Мичурина Способ преодолени нескрещиваемости плодовых культур
RU2002406C1 (ru) * 1992-01-16 1993-11-15 Pashchenko Vasilij M Способ получени мутаций растений

Non-Patent Citations (13)

* Cited by examiner, † Cited by third party
Title
ACTA GENET. SIN., vol. 8, no. 1, 1981, pages 50 - 55 *
BANGA, S.S., ET AL.: "Gametic gene transfer in Indian mustard (Brassica juncea)", HEREDITY, vol. 53, no. 2, 1984, pages 293 - 298 *
BIOLOGICAL ABSTRACTS, vol. 73, Philadelphia, PA, US; abstract no. 45541, WU, C., ET AL.: "Carp Cyprinus-carpio gynogenesis with reference to establishing a pure line" *
BULETINUL ACADEMIEI DE STIINTE A REPUBLICII MOLDAVA, STIINTE BIOLOGICE SI CHIMICE, no. 5, 1992, pages 19 - 23 *
CHIN, S.-F., ET AL.: "Pollination with irradiated pollen in rice Oryza sativa L.: I. First (M1) generation", HEREDITY, vol. 63, no. 2, 1989, pages 163 - 170 *
DATABASE CAB CAB INTERNATIONAL, WALLINGFORD, OXON, GB; PASHCHENKO, V.M., ET AL.: "Effects of combined treatment with ultraviolet radiation and sensitizer dyes on germinating pollen" *
DATABASE WPI Section Ch Week 7525, Derwent World Patents Index; Class C03, AN 75-42189W *
DATABASE WPI Section PQ Week 9410, Derwent World Patents Index; Class P13, AN 94-080672 *
MORRISON, R.A., ET AL.: "Haploid plants from tissue culture: new plant varieties in a shortened time frame", BIOTECHNOLOGY, vol. 6, June 1988 (1988-06-01), pages 684 - 690, XP001344669 *
PANDEY, K.K., ET AL.: "Evidence for gene transfer by the use of sublethally irradiated pollen in Zea mays and theory of ocurrence by chromosome repair through somatic recombination and gene conversion", MOL. GEN. GENET., vol. 191, no. 3, 1983, pages 358 - 365 *
RU-C-2002406 (PASHCHENKO V M) 15 NOVEMBER 1993 *
SAUTON, A., ET AL.: "Induction of gynogenetic haploid plants in muskmelon Cucumis-melo L. by use of irradiated pollen", AGRONOMIE, vol. 7, no. 2, 1987, pages 141 - 147, XP008103758, DOI: doi:10.1051/agro:19870209 *
SHINTAKU, Y., ET AL.: "Chromosomal variation in hybrids between Nicotiana repanda Willd. and N.tabacum L. through pollen and egg-cell irradiation techniques", GENOME, vol. 32, no. 2, April 1989 (1989-04-01), pages 251 - 256 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002098210A3 (fr) * 2001-06-05 2004-04-15 Univ Missouri Procede de doublement des chromosomes
US7135615B2 (en) 2001-06-05 2006-11-14 The Curators Of The University Of Missouri Chromosome doubling method
CN113125227A (zh) * 2020-12-25 2021-07-16 上海乐辰生物科技有限公司 核型分析用制备高分辨染色体的秋水溶液及应用

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Publication number Publication date
GB9321975D0 (en) 1993-12-15
AU7939494A (en) 1995-05-22

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