WO1995011967A1

WO1995011967A1 - Viable zygote

Info

Publication number: WO1995011967A1
Application number: PCT/EP1994/003485
Authority: WO
Inventors: Aksel Buchter-Larsen; Hans Christian Pedersen
Original assignee: Sandoz Ltd.; Sandoz-Patent-Gmbh; Sandoz-Erfindungen Verwaltungsgesellschaft M.B.H.
Priority date: 1993-10-25
Filing date: 1994-10-24
Publication date: 1995-05-04
Also published as: GB9321975D0; AU7939494A

Abstract

The present invention provides a viable zygote in which chromosomal DNA from one of the parental gametes has been substantially degraded. The invention also provides: organisms, particularly plants, resulting from cell division of the zygote; and a method of producing a viable zygote having 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 preferably treated with a DNA intercalating agent in the dark, fertilization of the maternal gamete takes place in the dark, and the zygote is subjected to visible and/or ultra-violet light before or after its first mitotic division.

Description

VIABLE ZYGOTE

The present invention relates to the production of novel organisms, and in particular to the production of transformed plants.

The production of organisms which result from the cell division of zygotes has long been known. Such conventional production of organisms disadvantageously does not readily enable the DNA of substantially only one of the gametes originally comprised by the zygote to be expressed.

Inter alia, the present invention substantially overcomes this disadvantage.

According to the present invention there is provided a viable zygote in which chromosomal DNA from one of the parental gametes has been substantially degraded. By "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 uterό). 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.

In the case that it is desired to prevent repair of the degraded DNA, 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. It will be appreciated that the normal replication of DNA should not be substantially interfered with by the addition to the zygote of such agents, and thus their concentrations must be chosen accordingly. The determination of these concentrations is a routine matter for the skilled man. Moreover, in a further embodiment of the invention, the maternal gamete is subjected to DNA damaging agents prior to fertilization. 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.

It is preferred that 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.

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. By "dark" is meant, the substantial absence of ultraviolet light and other electromagnetic radiations having a wavelength of from about 0.1 to about lμm. 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. By visible or ultraviolet light is meant electromagnetic radiations having a wavelength of from about 0.1 to about lμm, and more particularly those radiations having wavelengths within the range of about 100 to 800nm

As a consequence of the paternal DNA having been substantially completely degraded 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.

It is preferred that the paternal chromosomal DNA, prior to its degradation, comprises a suitable marker gene. Such 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.

It is particularly preferred that 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.

Harvesting of pollen from transgenic plants

Flowers on a CMS-sugar beet plant are pollinated with Acridine orange loaded pollen from a transgenic plant comprising genes encoding beta-glucuronidase, NPTII, and genes conferring resistance to glyphosate. Such pollen is harvested from newly opened flowers in the morning by suction into a dry glass-tube. Alternatively, inflorescences with no opened flowers are removed and placed in water in a jar (100% RH). After a few days, when flowers have opened, the inflorescences are moved to conditions of low humidity after which the anthers dehisce enabling the collection of high quality pollen.

Treatment of pollen with Acridine Orange

Within 30 minutes from harvest, the pollen obtained as indicated above is poured directly into a centrifuge tube containing 2 ml boron-free PGM (PGM-B). (PGM used for sugar beet pollen is defined as: sucrose 300 g/1; CaCl₂2H₂O 150 mg 1; H₃BO₃ 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. About 0.5 ml of a Acridine Orange stock solution: 1 mg/ml PSM-B is added to the PGM-B treated pollen (final Acridine Orange concentration 0.2 mg/ml) and the suspension is mixed gently. After 1-4 h in the dark, 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. Pollination with the Acridine Orange-loaded pollen, light treatment and ovule/embryo culture. Using a thin martenhair brush unpollinated flowers of CMS sugarbeet plants are pollinated in the dark with the Acridine Orange-loaded pollen suspended in PGM-B. Thus treated flower stems are then covered with foil or black plastic and thus treated plants are then returned to a growth room. After at least 22 hours 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. About 22-24h post pollination, some of the ovules are exposed to visible light (2000 μEm ^"2S^_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.

Example 1.

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. This pronounced chimerism is thought to be due to the relatively late exposure of the ovule to light (4 days after pollination). At this time 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.

Example 2.

Flowers on a MS-sugar beet plant are pollinated with acridine orange loaded pollen as described above. By using a GUS-test on pollen (Pollen is germinated in PGM for 30 min, X-gluc (1 mg/ml in PGM) is added 1 : 1 to the germinating pollen population, incubation at room temperature overnight) the pollen was found to segregate 1 : 1 (481 +505-) in GUS expression. No differences are found between Acridine Orange-treated pollen and non-treated pollen with respect to GUS-expression during germination.

One, three or ten days after pollination, 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.

TABLE 1

Code Numbers Treatment Embryos of ovules hrs¹ AO, light (2000 μE), +/- lid harvested %

A 70 0 control, dark 11 15,7

B 50 22 AO-treated, dark 2 4 c, 10 - AO-treated, light 15', + lid 1 10

C 137 - AO-treated, light 15', - lid 4 2,9

D 116 - AO-treated, light 30', - lid 5 4,3

E 13 72 AO-treated, dark 0 0

F 22 - AO-treated, light 15', - lid 0 0

H 32 - AO-treated, light 30', -lid 4 12,5

J 4 10 days AO-treated, dark 0 0

G 16 - AO-treated, light 30', + lid 1 6,3

Total 470 28

': hrs/days from pollination to harvest of ovules, light treatment within 1 hr after harvest of ovules. Of the 28 harvested embryos, 18 survived to shoots.

From 400 "experimental" ovules harvested (Rows B-G in Table 1), 17 embryos are obtained, of which 11 survived to shoots. Table 2 shows that the percentage survival of embryos obtained from ovules which had been fertilized in the dark with acridine orange loaded pollen, is very similar to that obtained for control ovules.

Table 2:

embrvos survived % survival

Unpollinated control 11 7 64%

AO-treated 17 11 65% Two of the experimental embryos which survived to shoots are GUS-positive. Of the 11 clones derived after polhnation with acridine orange treated pollen 8 were transferred to the greenhouse. Chromosome counting using flow cytometry shows that some of the cloned plants have a chromosome number around the haploid level. Most of the cloned plants from both of the GUS-positive clones are scored as being on the haploid level, and isozyme analysis indicates that the transgene is coupled to the aconitase locus, since only the GUS- positive plants are heterozygous in the ACO-isozyme. Table 3 provides a summary of the analysis of such plants.

Table 3.

As the above results demonstrate, 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.

Although 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. In the case that 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. For example, in the case of fish, 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 skilled man will appreciate that 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. Thus known problems associated with post-zygotic incompatibility following pollination of cultured species with pollen from wild species are overcome.

In the case that pollen loaded with the DNA fragmenting agent is placed directly on the style, haploid seeds are produced directly without the need for ovule culture or embryo rescue.

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.

Certain problems presently encountered with regulatory affairs organizations in respect of the production of transformed plants may be overcome or otherwise circumvented by the method according to the invention.

Claims

1. A viable zygote in which chromosomal DNA from one of the parental gametes has been substantially degraded.

2. A zygote according to claim 1, wherein at least some of the degraded DNA is incorporated into the chromosomes of the other gamete.

3. A zygote according to either of claims 1 or 2, wherein the DNA has been degraded to the extent that chromosome fragments, optionally comprising transcribable genes or fragments thereof, remain.

4. A zygote according to either of claims 1 or 2, in which the DNA has been degraded to the extent that transcribable gene fragments do not remain.

5. A zygote according to any preceding claim which has been treated with an agent which substantially prevents DNA repair.

6. A zygote according to any preceding claim, wherein the paternal gamete DNA has been degraded.

7. A zygote according to any one of the preceding claims, wherein the gametes comprise (i) the combination of 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.

8. A zygote according to any preceding claim, wherein prior to fertilization, the paternal gamete is treated with an agent capable of degrading or otherwise cleaving its chromosomal DNA in response to a specific stimulus provided to the zygote.

9. A zygote according to the preceding claim, wherein the paternal gamete has been treated in the dark with a photo-excitable DNA intercalater, and optionally, fertilization has been effected substantially in the dark.

10. A zygote according to claim 9, wherein the DNA intercalater is selected from the group consisting of acridine orange, acridine yellow, ethidium bromide, 6-mercapto purine, and propidium iodide.

11. A zygote according to any one of claims 8-10, wherein the specific stimulus is visible light or ultraviolet light.

12. A zygote according to any preceding claim, wherein the genome is contributed substantially by the maternal gamete.

13. A zygote according to the preceding claim, wherein chromosome fragments, optionally comprising genes or gene fragments, originating from the paternal gamete are integrated into chromosomal DNA originating from the maternal gamete.

14. A zygote according to any preceding claim, which has been treated with a ploidy increasing agent.

15. A zygote according to the preceding claim, in which the ploidy is increased subsequent to degradation of the DNA but prior to the first mitotic division, or wherein the ploidy is increased subsequent to the first mitotic division of the zygote but prior to degradation of the DNA, or wherein the ploidy is increased prior to degradation of the DNA.

16. A zygote according to either of claims 14 or 15, wherein the ploidy increasing agent is selected from the group consisting of colchicine, dinitroaniline herbicides such as oryzalin and trifluralin, amiprophosmethyl and nitrous oxide.

17. A zygote according to any one of the preceding claims, wherein the chromosomal DNA, prior to its degradation, comprises a marker gene.

18. A zygote according to the preceding claim, wherein the marker gene is selected from the group consisting of genes involved in the generation of a phenotypically identifiable trait, antibiotic resistance inducing genes, herbicide resistance inducing genes, beta glucuronidase, and luciferase.

19. An organism resulting from cell division of the zygote of any one of the preceding claims.

20. An organism according to the preceding claim, in the form of a non-human animal, a plant, the progeny of the organism or plant, and the seeds of the plant or the progeny of the plant.

21. An organism according to either of claims 19 or 20, which has been treated with a ploidy increasing agent prior to the production of gametes thereby.

22. A method of producing a viable zygote according to any one of claims 1-18 or an organism according to any one of claims 19-21, 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.

23. A method according to the preceding claim, comprising the steps of: i. treating the paternal or maternal gamete with an agent capable of degrading or otherwise cleaving its chromosomal DNA in response to a specific stimulus provided to the zygote; ii. fertilizing the maternal gamete with the paternal gamete; iii. subjecting the zygote to the said stimulus.

24. A method according to claim 23, wherein 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 the zygote is subjected to white light before or after its first mitotic division.

25. A zygote according to any one of claims 1-18, obtainable by the method of any one of claims 22-24.

26. A method of producing an organism comprising obtaining a zygote by the method of any one of claims 22-24, and culturing the thus obtained zygote in vivo, in vitro, ex vivo or in utero so that it undergoes mitosis.