WO2006032504A2 - Procede permettant d'introduire des mutations genetiques dans des cellules vegetales - Google Patents

Procede permettant d'introduire des mutations genetiques dans des cellules vegetales Download PDF

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WO2006032504A2
WO2006032504A2 PCT/EP2005/010270 EP2005010270W WO2006032504A2 WO 2006032504 A2 WO2006032504 A2 WO 2006032504A2 EP 2005010270 W EP2005010270 W EP 2005010270W WO 2006032504 A2 WO2006032504 A2 WO 2006032504A2
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plant
msh2
dna
gene
mismatch repair
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PCT/EP2005/010270
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English (en)
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WO2006032504A3 (fr
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Gabriela Ispas
Ivan Famelaer
Geert Angenon
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Vrije Universiteit Brussel
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
    • 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
    • C12N15/01—Preparation of mutants without inserting foreign genetic material therein; Screening processes 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
    • 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

Definitions

  • the present invention relates to mutagenesis in plants.
  • the present invention relates to methods for efficiently introducing genetic mutations into the genomes of plant cells. More specifically, the present invention discloses a method that can be especially useful for generating collections of plant mutants with mutation-saturated genomes.
  • the present invention aims at providing an efficient method to introduce genetic mutations into the genomes of plant cells. In doing so, the present invention aspires to enable the generation of libraries of mutants of various plant species, wherein a greater proportion of the genes will be containing a mutation, and wherein a greater number of different phenotypes will be obtained than achieved using current methods. This effect of the present invention will help close the 'phenotypic gap' present in the existing libraries of plant mutants.
  • repetitive sequences are mostly found outside of gene regions that code for proteins and hence, the increased mutation frequency of said repetitive sequences in MSH2- deficient plants will most likely result in a limited number of discernible phenotypes. Therefore, said increased mutation frequency of repetitive sequences is not useful for production of libraries of mutant plants.
  • WO0188192 described a way to generate hypermutable mammalian cells by introducing into said cells a dominant negative variant of a gene involved in MMR. Such cells show increased efficiency of mutagenesis in response to mutagenic agents and can be screened for mutations in genes of interest or for novel phenotypes. Following mutagenesis, genetic stability of the cells was re-established by turning off the expression of the dominant negative MMR gene. WO0188192 did not extend its findings to plant cells. However, there exist important differences in DNA repair systems between plants and mammalian species. For example, and also relevant to alkylating mutagens, plants do not possess methylguanyl methyl transferases, whereas mammals do.
  • WO0188192 used an inducibly expressed dominant negative MMR gene, such that following mutagenesis the expression of the said dominant negative gene could be turned off. Presumably, this was found crucial for re-establishing the genetic stability of the mutagenised cells.
  • reliable inducible systems that would work at the whole plant level and/or in seeds, and/or in a wide range of plants, are not available. While some systems have been developed for the model plants Arabidopsis thaliana or Nicotiana tabacum, these only work with particular well-characterized lines expressing a critical level of a repressor or activator and it remains very difficult to work with other lines, or to develop such lines for other (crop) plant species.
  • the uptake and transport of an inducer of expression in plant tissues and/or seeds may be problematic, especially if MMR should be suppressed only in particular cells of a plant, such as, e.g., in a few meristematic cells that would give rise to progeny.
  • a skilled person would not have considered applying the teachings of WO0188192 to plants cells.
  • a skilled person would also consider other ways of MMR suppression, in particular ways which are in general not readily reversible, as unsuitable for preparation of plant mutagenesis libraries.
  • WO0224890 described a method for increasing the efficiency of targeted gene mutation and/or of homologous recombination in a plant cell, wherein the activity of the MLH1 gene involved in MMR has been suppressed by one of multiple approaches in said plant cell.
  • this document did not investigate the possibility to increase the efficiency of random mutagenesis with mutagenic agents in plant cells by suppressing the activity of MMR.
  • EP 1 333 095 described that recombination between partially homologous DNA sequences can be facilitated through inactivation of the mismatch repair system in cells.
  • EP 1 333 095 utilised this finding to achieve targeted replacement of an endogenous sequence by a foreign DNA sequence.
  • targeted recombination is mechanistically entirely distinct from random mutagenesis, such as, e.g., using alkylating agents. Therefore, EP 1 333 095 does not disclose nor suggest using MMR deficient cells in random mutagenesis.
  • WO 02/054856 described the possibility to suppress mismatch repair system in plant cells using chemical inhibitors, thereby generating spontaneously hypermutable plant cells, and optionally subject the said plant cells to further chemical mutagenesis. Genetic stability of the cells following mutagenesis was re-established through removal of the chemical inhibitor of MMR.
  • chemical MMR inhibitors entails several disadvantages. Many such inhibitors (e.g., anthracenes) lack specificity and may show a wide variety of other effects, such as alterations in mitotic recombination, cell cycle regulation, single stranded DNA cleavage, double stranded DNA cleavage, general cytotoxicity, etc.
  • inhibitors including ATPase, nuclease and polymerase inhibitors, influence numerous metabolic reactions apart from MMR.
  • chemical MMR inhibitors e.g., cell cycle arrest, chromosome breakage, cell death, etc.
  • chemical inhibitors may suffer from inadequate uptake and/or transport in plant tissues and/or seeds, which may be particularly troublesome if one desires to suppress MMR only in particular tissues or cells of a plant, such as, e.g., in a few meristematic cells that give rise to progeny.
  • the present invention provides a method for efficiently introducing genetic mutations into the genome of a plant cell.
  • the method utilizes the fact that suppressing the function of an endogenous system of DNA repair will cause increased resistance of plant cells to high doses of mutagenic agents, as well as increased mutagenic effect of these agents in said plant cells.
  • the method provided in the present invention comprises the steps of: (i.) suppressing the activity of at least one endogenous system of DNA repair in said at least one plant cell; and (ii.) exposing said at least one plant cell to at least one mutagenic agent.
  • the present invention demonstrates that suppressing the mismatch repair system dramatically increases the ability of plant cells to sustain DNA damage induced by mutagenic agents and to accumulate mutations.
  • the method of the present invention comprises suppressing the activity of the mismatch repair system.
  • the present invention also realises that advantageous ways of suppressing the mismatch repair system in plant cells may comprise the following: partial or complete inactivation of at least one copy of at least one gene involved in said mismatch repair system, down-regulation of the expression of at least one gene involved in said mismatch repair system, introducing a dominant negative variant of at least one gene involved in said mismatch repair system, introducing an antibody or an expression cassette encoding such antibody capable of binding to at least one protein involved in said mismatch repair system, introducing agents capable of saturating the activity of said mismatch repair system, and over-expression of at least one gene involved in said mismatch repair system or a biologically active variant thereof.
  • advantageous ways of suppressing the mismatch repair system in plant cells may comprise the following: partial or complete inactivation of at least one copy of at least one gene involved in said mismatch repair system, down-regulation of the expression of at least one gene involved in said mismatch repair system, introducing a dominant negative variant of at least one gene involved in said mismatch repair system, introducing an antibody or an expression cassette encoding
  • an advantageous way of suppressing the mismatch repair system in plant cells may comprise introducing a dominant negative variant of at least one gene involved in said mismatch repair system.
  • dominant negative MMR variant could not be efficiently turned off in plant cells after mutagenesis.
  • MMR deficient plants can be propagated through several generations without the need to re-establish MMR proficiency.
  • MMR deficient plants obtained by introducing a dominant negative variant of an MMR gene can be propagated through several generations without the need to reduce the expression of the said dominant negative MMR variant.
  • some mutant phenotypes may appear, this does not substantially interfere with growth, reproduction and phenotypic characterisation, which is surprising.
  • the present application discloses that plant cells with suppressed mismatch repair system (such as, e.g., by dominant negative MMR variants) can be efficiently mutagenised.
  • plant cells with suppressed mismatch repair system such as, e.g., by dominant negative MMR variants
  • mutagenised plant cells are suitable for preparation of plant libraries, since the inventors found that MMR deficient plants may be propagated without the need to re-establish MMR proficiency (such as, e.g., without the need to reduce the expression of the said dominant negative MMR variant). Together, this may help to close the 'phenotypic gap' in such libraries.
  • the above detailed ways of suppressing the MMR in plant cells are advantageous over chemical MMR inhibitors which, as detailed above, often show non-specific effects and suffer from inadequate uptake and/or transport in plant tissues and/or seeds.
  • the above methods allow for specifically targeting the MMR process, and MMR in any desired cell type of a plant.
  • the present invention preferably employs mutagenic agents capable of introducing a random mutation into the genome of plant cells.
  • the present invention also realizes the advantages of using DNA-alkylating agents to mutagenise plant cells. Because of the fact that DNA-alkylating agents induce basepair substitutions rather than deletions or insertions, they can yield a qualitatively richer spectrum of mutations than other methods, such as X-ray irradiation or insertion of transposable elements. Basepair substitutions induced by DNA-alkylating agents will often result in single amino acid changes in the affected proteins, which might lead to for example a complete loss of function of the protein, a partial loss of its function, or to gaining a novel function. In this way a large variety of alleles can be created. Accordingly, in several embodiments of the present invention said method uses DNA-alkylating agents for mutagenesis.
  • the present invention provides mutagenised plant cells, as well as plants and plant parts and derivatives derived from these plant cells.
  • the present invention also envisages collections comprising such plants.
  • the present invention relates in particular to efficiently introducing random genetic mutations into the genome of a plant cell.
  • nucleic acid DNA, RNA, or other related compositions of matter that may include substitution of similar moieties.
  • nucleic acids may include bases that are not found in DNA or RNA, including, but not limited to, xanthine, inosine, uracil in DNA, thymine in RNA, hypoxanthine, and so on.
  • Nucleic acids may also include chemical modifications of phosphate or sugar moieties, which can be introduced to improve stability, resistance to enzymatic degradation, or some other useful property.
  • the present invention provides a method for introducing at least one genetic mutation into at least one plant cell, comprising the steps of: (i.) suppressing the activity of at least one endogenous system of DNA repair in said at least one plant cell; and (ii.) exposing said at least one plant cell to at least one mutagenic agent.
  • the DNA damage caused by applying a mutagenic agent would be to a large extent removed by the DNA repair systems before the cell would undergo the next cell division. This would in turn by large prevent the occurrence of mutations in the progeny of said cell.
  • the present invention performs mutagenesis in a plant cell with suppressed action of one or more DNA repair systems.
  • Such cells will be unable to efficiently remove the DNA damage caused by the mutagenic agent and this DNA damage will be turned into mutations - i.e., deviations from the DNA sequence of the mother cell before the mutagenesis - in daughter cells originating from the mutagenised mother cell.
  • the present method should therefore amplify the effect of said mutagenic agent and help obtain plant cells with much wider spectrum of mutations than can be achieved with currently available methods.
  • DNA damage is defined as any deviation from the usual base pairing between A-T and G-C, such as by way of example and not limitation: apurinic/apyrimidinic sites, base adducts, alkylated bases, dimers, mismatches, and loopouts resulting from DNA polymerase slippage. Also under DNA damage belong any deviation from the usual structure of the phosphodeoxyribose backbone of DNA, such as double strand breaks and single-strand nicks. Depending on the nature of the DNA damage, a cell will utilize various endogenous systems of DNA repair to re-establish the normal DNA structure.
  • the present invention anticipates that said endogenous system of DNA repair will be chosen from the group comprising mismatch repair system, nucleotide excision repair system, base excision repair system, direct reversal system, double strand break repair system, and enzymatic photoreactivation system. Also, because the proofreading activity of DNA polymerases considerably reduces the occurrence of mismatches during DNA replication, said proofreading activity will too be considered to constitute a DNA repair system in the context of the present invention.
  • MMR mismatch repair system
  • mutS orthologues identified in plants are of particular importance. While these currently comprise mutS orthologs isolated from Arabidopsis (MSH1 , MSH2, MSH3, MSH4, MSH5, MSH6, MSH7, and MutS2), maize (Musi , Mus2, Mus3), wheat (MSH2, MSH3, MSH6, MSH7), canola (MSH2), rice (MSH6), tobacco (MSH2), and sugarcane (MSH2, MSH3, MSH5, MSH6, MSH7, MutS2), the present invention also anticipates that future identified plant mutS homologues can be used in the context of the present invention.
  • the mutL orthologues comprise for example MLH1 , MLH2, MLH3, PMS1 , and PMS2.
  • MLH1 , MLH3, PMS1 the mutL orthologues isolated from plants, such as from Arabidopsis (MLH1 , MLH3, PMS1), rice (MLH1), and from sugarcane (MLH1 , MLH3, PMS1 ).
  • NER nucleotide excision repair system
  • the NER system is used by the cell to repair various types of damage, and is often involved in repairing DNA injuries induced by UV radiation, comprising cyclobutane pyrimidine dimers and 6-4 photoproducts, and by chemical agents, such as bulky DNA adducts.
  • the NER system includes the "global genome NER", which repairs the lesions over the entire genome, as well as the “transcription coupled NER", that repairs transcription blocked lesions present in transcribed DNA strand.
  • direct reversal system is meant a mechanism that involves a single enzyme- catalysed reaction to remove certain types of DNA damage.
  • DNA damage may comprise base alkylation, especially methylation of guanine at position 06, yielding O6-methylguanine.
  • MGMT methylguanine methyltransferases
  • base excision repair (BER) is understood the system in which the damaged DNA base is recognized and eliminated from the DNA strand by the action of DNA glycosylases, followed by repair of the apurinic/apyhmidinic site in several steps, comprising incision, gap filling and sealing.
  • the BER system repairs mainly DNA damage that arises spontaneously in a cell from hydrolytic events, such as deamination, base loss, base fragmentation by ionizing radiation, and oxidative damage.
  • zymatic photoreactivation system is understood the removal of UV-induced damage, such as cyclobutane pyrimidine dimers and 6-4 photoproducts, from DNA by the action of photolyases.
  • the "double strand break repair system” (DSBR) is involved in repairing DNA lesions, wherein both DNA strands of a DNA double helix are broken. Such lesions are usually caused by ionizing radiation, chemical agents, or arise during recombination.
  • the two mechanisms of DSBR comprise (i) homologous recombination and (ii) non-homologous end- joining. In homologous recombination the double strand break is repaired using genetic information from a sister chromatid. Non-homologous end-joining involves the joining of two broken ends.
  • said endogenous system of DNA repair is the mismatch repair system (MMR).
  • MMR is also responsible for signaling the extent of DNA damage to cellular mechanisms that control cell division and programmed cell death. Accordingly, if the amount of DNA damage exceeds a certain threshold, MMR will play a role in instructing the cell to interrupt the progression of the cell cycle until the damage is repaired. Alternatively, with even higher load of DNA damage the cell will be instructed to initiate apoptosis. This may likely constitute the cytotoxic effect of the mutagenesis treatment, which effectively limits the frequency of mutations that can be introduced with classical mutagenesis.
  • the invention anticipates that suppressing the activity of said endogenous system of DNA repair will be accomplished by means chosen from the group comprising: (i) exposure to a chemical agent; (ii) partial or complete inactivation of at least one copy of at least one gene involved in said endogenous system of DNA repair, said inactivation being attributable to for example base substitutions, methylation, insertions and/or deletions within the sequence of said gene; (iii) down-regulation of expression of at least one gene involved in said endogenous system of DNA repair, said down-regulation may for example result from base substitutions, insertions, or deletions in the regulatory sequences of said gene, or alternatively, said down-regulation may be accomplished using anti-sense nucleic acids or RNA silencing, or RNA interference; (iv) introducing a dominant negative variant of at least one gene involved in said endogenous system of DNA repair; (v) introducing an antibody or an expression cassette encoding such antibody, capable of binding to at least one protein involved in said endogen
  • the invention anticipates that suppressing the activity of said endogenous system of DNA repair will be accomplished by means chosen from the group comprising: (i) partial or complete inactivation of at least one copy of at least one gene involved in said endogenous system of DNA repair, (ii) down-regulation of the expression of at least one gene involved in said endogenous system of DNA repair, (iii) introducing a dominant negative variant of at least one gene involved in said endogenous system of DNA repair, (iv) introducing an antibody or an expression cassette encoding such antibody, capable of binding to at least one protein involved in said endogenous system of DNA repair, (v) introducing agents capable of saturating the activity of said endogenous system of DNA repair, and (vi) over-expression of at least one gene involved in said endogenous system of DNA repair or a biologically active variant thereof.
  • the approach (vi) may lead to titrating out of other components of the endogenous system of DNA repair by the over-expressed member, whereby the DNA repair system will be compromised.
  • the above means may in general not be readily reversible and may result in substantially permanent (e.g., substantially maintained upon further propagation of the mutagenised cells) suppression of the endogenous DNA repair system.
  • reversible use of the above means is also envisioned, whereby proficiency of endogenous DNA repair system following mutagenesis would be re- established.
  • biologically active variant is preferably defined to mean a peptide which has at least about 80%, preferably at least about 90%, and more preferably at least about 95%, sequence identity (e.g., by BLAST sequence comparison algorithm) to a naturally occurring gene.
  • Biologically active variants of the gene may encode peptides that have at least about 10%, preferably at least about 50%, and more preferably at least about 90%, of the activity of the peptides encoded by naturally occurring gene.
  • Such variants also encompass fragments of the naturally occurring gene encoding biologically active polypeptide fragments. The activity may be measured by any method known in the art and may involve testing of the ability of the variant to suppress an endogenous system of DNA repair upon over-expression in cells.
  • the present invention can use one of the above methods to permanently or transiently suppress the activity of the mismatch repair system.
  • the invention anticipates that suppressing the activity of said mismatch repair system will be accomplished by means chosen from the group comprising: (i) partial or complete inactivation of at least one copy of at least one gene involved in said mismatch repair system, (ii) down-regulation of the expression of at least one gene involved in said mismatch repair system, (iii) introducing a dominant negative variant of at least one gene involved in said mismatch repair system, and (iv) introducing an antibody or an expression cassette encoding such antibody, capable of binding to at least one protein involved in said mismatch repair system, (v) introducing agents capable of saturating the activity of said mismatch repair system, and (vi) over-expression of at least one gene involved in said mismatch repair system or a biologically active variant thereof.
  • the approach (vi) may lead to titrating out of other components of the mismatch repair system by the over-expressed member, whereby the of the mismatch repair system will be compromised.
  • the above means may in general not be readily reversible and may result in substantially permanent (e.g., substantially maintained upon further propagation of the mutagenised cells) suppression of the mismatch repair system.
  • reversible use of the above means is also envisioned, whereby proficiency of the mismatch repair system following mutagenesis may be re ⁇ established.
  • said mismatch repair system is suppressed by introducing into said at least one plant cell a dominant negative variant of at least one gene involved in said mismatch repair system.
  • Dominant negative variants cause a mismatch repair defective phenotype even in the presence of a wild-type gene in the same cell.
  • This approach offers the advantage that often the knowledge of the nucleic acid and/or protein sequence of the particular protein to be inhibited by a dominant negative variant is not required, because the high conservation of many proteins involved in mismatch repair system allows the use of a dominant negative variant from another plant species.
  • the present invention demonstrates in one of the examples that a dominant negative G 671 D mutant of the Arabidopsis thaliana MSH2 protein can exert a dominant inhibitory effect on the MSH2 function in Nicotiana plumbaginifolia.
  • this embodiment combines the advantageous effect that the suppression of the mismatch repair system has on the ability of said plant cell to sustain high load of DNA damage with the easiness of suppression achieved by using a dominant negative variant of at least one gene involved in said mismatch repair system.
  • the use of a dominant negative MMR variant allows for specifically targeting the MMR process, and the dominant negative MMR variant can easily be expressed in any desired cell type of a plant.
  • the expression of the dominant negative MMR variant in said plant cells does not depend on provision of an external inducer or may even be constitutive, typically by using endogenous or exogenous constitutive promoters.
  • the use of non-inducible expression systems can be advantageous, because systems of inducible expression which would reliably work at whole plant level and/or in a wide range of plants are not available.
  • constitutive expression of the dominant negative MMR variant in mutagenised plant cells does not interfere with the use of such cells to obtain mutant plants and that plants comprising and/or consisting of MMR deficient cells can be propagated and characterised on phenotypic level.
  • the expression of the dominant negative MMR in said plant cells may be inducible.
  • said at least one gene involved in MMR is a eukaryotic orthologue of the MutS gene of the bacterium Escherichia coli.
  • said eukaryotic orthologue of the MutS gene of E. coli is chosen from the group comprising MSH1 , MSH2, MSH3, MSH4, MSH5, MSH6, MSH7, MutS2, Musi , Mus2, and Mus3 isolated from various plant species.
  • the present invention uses a dominant negative variant of the MSH2 gene isolated from various plant species to suppress the activity of the MMR system.
  • Said MSH2 gene can be for example the MSH2 gene isolated from Arabidopsis thaliana.
  • the normal MSH2 protein forms heterodimers with the MutS homologue MSH6, said heterodimer recognising mainly mismatches, or with the MutS homologue MSH3, said latter heterodimer recognising mainly insertions and deletions of 2-14 bases.
  • said heterodimers associate with other proteins involved in the MMR system and begin to thread along the DNA strand searching for a signal that helps to differentiate the original DNA strand from the newly synthesized one. Afterwards, the system repairs said defect in the newly synthesized DNA strand. Dominant negative variants of said MSH2 gene will interfere with this mismatch repair mechanism and cause a mismatch repair defective phenotype even in the presence of a wild-type MSH2 gene in the same cell.
  • said dominant negative variant of the MSH2 gene carries a mutation affecting the amino acid sequence of the ATP-binding domain of the corresponding MSH2 protein, and more specifically the conserved box within said ATP- binding domain, comprising the amino acid sequence TGPNMGGKSTFI.
  • the corresponding MSH2 protein carrying a mutation in said ATP-binding domain tends to recognise and associate with defects in DNA structure, but cannot perform the subsequent steps of the MMR response. In staying associated with said defects in DNA structure, it also prevents heterodimers comprising wild-type MSH2 to recognise and associate with the defects in DNA, thus exerting a dominant negative effect.
  • said mutation in the ATP-binding domain of MSH2 is chosen from a group comprising all possible mutations that change the G (GIy) residue at a position corresponding to position G 671 in the Msh2 protein sequence of Arabidopsis thaliana.
  • such mutation can represent a G to D (Asp) change.
  • said at least one gene involved in the mismatch repair is a eukaryotic orthologue of the MutL gene of the bacterium Escherichia coli. More specifically, in another embodiment, the present invention uses dominant negative variants of MLM , MLH2, MLH3, PMS1 and PMS2.
  • the method of the present invention suppresses the activity of the MMR system in at least one plant cell by introducing into said at least one plant cell a dominant negative variant of at least one gene chosen from the group comprising MSM ,
  • MLH3, PMS1 and PMS2 isolated from various plant species. If a dominant negative variant of a MutS or MutL orthologue from other species than plants would exert a dominant negative effect in a plant cell, this can also be used within the scope of the present invention.
  • Such orthologues may be isolated from for example bacteria, yeast, fungi, or animals.
  • the present invention uses as a mutagenic agent one capable of introducing random mutations into the genome of at least one plant cell.
  • the present invention uses as a mutagenic agent to introduce random mutations into the genome of at least one plant cell a step chosen from the group comprising: chemical mutagen, irradiation with ultraviolet light, irradiation with X-rays, irradiation with gamma rays, and a transposable genetic element.
  • said mutagenic agent will be a chemical agent and in particular a DNA-alkylating agent.
  • DNA-alkylating agents a more specific embodiment of the invention makes use of ethylmethane sulphonate (EMS) and N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG). Said agents are able to introduce a methyl group on the 06 position of guanine. O6-methylguanine lesions can give rise to G-C to A-T transitions in subsequent DNA synthesis steps in propagation of the cell. Hence, this type of mutagenesis in general introduces basepair substitutions.
  • EMS ethylmethane sulphonate
  • MNNG N-Methyl-N'-nitro-N-nitrosoguanidine
  • deletions resulting from X- ray irradiation or insertions resulting from transposable elements which tend to result in frameshift mutations and partial or complete loss-of-function of a gene
  • basepair substitutions introduced by DNA-alkylating agents will mainly lead to amino acid substitutions. Therefore, such mutations can reveal a much larger spectrum of phenotypes, among others partial loss- of function phenotypes and gain-of-function phenotypes, than other forms of random mutagenesis.
  • said chemical agent will induce frameshift mutations.
  • the method described in the present invention can be used to introduce genetic mutations into at least one somatic or germ plant cell, which is capable of propagation.
  • plant cell may be a meristematic cell that gives rise to progeny.
  • the present method can also be used to mutagenise seed of a plant.
  • said plant cell can be derived from various plant species, such as by way of example and not limitation: maize, wheat, rice, barley, sorghum, tobacco, tomato, potato, Brassica spp., soybean, pea, sunflower, cotton, peanut, Arabidopsis thaliana, Nicotiana, and Medicago.
  • the present invention provides a plant cell that is obtainable by the method of the invention.
  • an additional embodiment of the invention provides a plant that is derived by propagation from said mutagenised cell.
  • progeny of said plant obtained in generative or vegetative manner is provided by the invention.
  • the present invention also provides parts or derivatives of said plant, which are suitable for propagation, such as organ tissue, leaves, stems, roots, shoots, protoplasts, somatic embryos, anthers, petioles, pollen, cells in culture, and seeds.
  • parts or derivatives of said plant which are suitable for consumption, such as seeds, fruits, stems and leaves, are provided by the present invention.
  • parts or derivatives of a plant obtainable from said mutagenised plant cell are also provided by the invention, comprising organ tissue, leaves, stems, roots, shoots, protoplasts, somatic embryos, anthers, petioles, pollen, cells in culture, and seeds.
  • the present invention also provides the plant obtainable from said mutagenised plant cell and the progeny of said plant obtained in generative or vegetative manner, said plant and the progeny thereof having at least one mutant phenotype.
  • Such plants will be a valuable resource in identification of novel advantageous phenotypes in plants, in mutation breeding and in forward and reverse genetics approaches.
  • the present invention also provides the nucleic acid isolated from the plant obtainable from said mutagenised plant cell and the progeny of said plant obtained in generative or vegetative manner, said plant and the progeny thereof having at least one mutant phenotype, wherein said nucleic acid comprises at least one mutation responsible for said at least one mutant phenotype.
  • nucleic acids comprising at least one nucleic acid carrying at least one mutation responsible for said at least one mutant phenotype according to the previous embodiment is anticipated. Such collection will be a valuable resource for forward and reverse genetics approaches in plants.
  • the present invention also provides an assay of the presence of mutagenic agents in an environmental sample, comprising the steps of: (i) exposing at least one plant cell to an environmental sample; (ii) assessing the occurrence of mutations in plants or plant parts and derivatives obtainable by propagation of said at least one plant cells. Further description of additional aspects of the described method can be found in the accompanying examples.
  • Figure 1 illustrates RT-PCR (A) and radioactive probing of the RT-PCR membrane (B) for different lines of transgenic N. plumbaginifolia overexpressing the Msh2 G671D mutant and the two controls: wild type N. plumbaginifolia (P2) and A. thaliana.
  • A RT-PCR
  • B radioactive probing of the RT-PCR membrane
  • P2 wild type N. plumbaginifolia
  • P2 wild type N. plumbaginifolia
  • A. thaliana A. thaliana
  • Figure 2 illustrates A. Location of point mutation in the dominant negative Msh2 G671D mutant and position of T-DNA insertion in Msh2 protein for 002708 line. Abbreviations:
  • MutS_N and MutS_C MutS domains found in proteins of the MutS family; MutSd: DNA binding domain of MutS family; MutSac: ATPase domain of MutS family.
  • the mutation G671 D in the dominant negative mutant is located in ATPase domain of Msh2 protein.
  • the T-DNA insertion caused a deletion in MUTSac domain, from 645 to 924 aac (Leonard et al. (2003). Plant Physiology 133: 328-338).
  • DNA insertion lines (002707 and 002708).
  • CoIO and 002707 line the PCR amplification with the combination of primers a and c indicate the absence of T-DNA insertion in the region
  • C-D primers indicates the deletion of this region in T-DNA insertion mutant while the amplification with the combination b of primers (A-LBbI) indicates the localisation of T-DNA insertion downstream to A primer.
  • FIG. 3 Spontaneous albino phenotype in Nicotiana plumbaginifolia overexpressing Msh2 G671 D mutants (A) and Arabidopsis thaliana Msh2-T-DNA insertion mutant: 002708 (B). Spontaneous callusogenesis from plantlets derived from seeds.
  • C Nicotiana plumbaginifolia overexpressing Msh2 G671 D mutant
  • D Arabidopsis thaliana Msh2 T-DNA insertion mutant 002708.
  • FIG. 1 Dynamic of cell division in protoplast population isolated from wild type P2 and plants overexpressing Msh2 G671D mutant. After 14 days of culture (dark, at room temperature) were scored in successive microscopic fields the number of single cells, two cells and microcolonies formed. The mean of these values are represented in the graph.
  • Figure 5 A. Gradual reduction of total leaf surface for wild type N. plumbaginifolia germinated on medium containing increasing concentrations of MNNG (0-100 ⁇ M). The higher the mutagen concentration, the stronger the delay exerted on plant development.
  • B Graphic representation of reduction in total leaf surface in N. plumbaginifolia plantlets derived from seeds germinated on medium containing different concentrations of MNNG. The amplitude of leaf reduction in mutated N. plumbaginifolia was quantified with the ASSESS program and expressed in relative values, reported to the maximum recorded value.
  • Figure 7 A. The difference in total leaf surface observed for plantlets in Petri dishes are retrieved to a lesser extent in untreated adult plants grown in greenhouse conditions, regardless of their position on the plant (A1 - P2 and A2 - Msh2 G671 D/S16). Wild type and transgenic plants derived from EMS 0.3% - 9Oh treated seeds, have a normal development in greenhouse conditions (A3 - P2 and A4 - Msh2 G671D/S16).). Leaf position on longitudinal axe: A: basal; B: 1/3 inferior; C: 1/3 superior; D: apical leaf. B. The yield of seeds harvested per individual capsule from mutated plants was considerable reduced compared to the yield of seeds harvested per individual capsule from untreated plants (B1 - P2 and B2 - Msh2 G671D/S16).
  • Figure 8 Phenotype of Msh2 G671 D/S16 - M2 plantlets generated from seeds collected from EMS treated parental plants. None of these abnormal phenotypes were observed for M2 wild type plantlets obtained from mutated M1 parental wild type plants.
  • Figure 9. Figure 9 Induced variegated phenotype after EMS mutagenic treatments in Arabidopsis thaliana Msh2-T-DNA insertion mutant 002708 A. multiple spotted, B. half white
  • FIG. 10 A. Germination of CoIO and 002708-T4 mutated seeds treated with 0.3% EMS for 18h. Difference in germination efficiency is observed between CoIO and 002708 for long EMS treatment (18h), but not for short EMS treatment (3h). B. Percentage of seed germination after EMS treatment. According to the germination test the resistance to EMS treatment (0.3%, 18h) is higher in successive generations of Msh2 deficient A. thaliana (002708-T4 and 002708-T5) than in controls CoIO and 002820 line.
  • Table 1 Segregation of albino (white and light green) phenotype in successive generations of Msh2 T-DNA insertion mutant: 002708.
  • the observed ratio of 1 :15 for albino in T5-4 generation indicates the mendelian inheritance of a double recessive mutation, and may explain the low frequency of albino in T4 and the absence of this phenotype in other 002708 offspring lines.
  • the seed stocks used in our experiment were obtained starting from a limited number of seeds SALK 002708 and seeds SALK 002707, supplied from SIGnAL: SaIk Institute Genomic Analysis Laboratory (http://signal.salk.edu/tdna_protocols.html). DNA was extracted from plantlets of CoIO, 002708 and 002707 lines using Plant DNA Mini Kit (Qiagen).
  • T-DNA fragment in msh2 gene was confirmed by PCR, using the left border T- DNA anchored primer LBbI (gcgtggaccgcttgctgcaact) and Msh2 specific primers A (atggagaatacatgatatcttcaagc), D (ctttcagtgtcaatgtgagcgctgac) and C (gcggatgaaagtggacttatctccca).
  • the primers A, D and C span the ATP binding domain of the Msh2 protein and the exact location of A, D and C primers is illustrated in Figure 2A.
  • the PCR amplification of 1 kb A-D fragment and 0.4 kb C-D fragment indicate the absence of T- DNA insertion in this region, while the amplification obtained with LBbI- A primers combination indicates the disruption of msh2 gene in the ATP binding site, downstream to the primer A.
  • the Msh2 point mutant was generated starting from A.thaliana cDNA Msh2 -gi 3914056 (generous gift of dr. Culligan), introducing the G 671 D point mutation in the TGPNM conserved domain, by SOEing PCR technique (Splicing by Overlap Extension).
  • the bordering primers used are: primer A (atggagaatacatgatatcttcaagc) containing the internal EcoRV restriction site and primer D (ctttcagtgtcaatgtgagcgctgac) containing the msh2 internal Eco47lll site. These restriction sites were used for cloning of the mutated fragment into the wild type msh2 gene.
  • the internal point mutation was introduced using C-B primers: primer C (gcggatgaaagtggacttatctccca) in sense and primer B (tgggagataagtccactttcatccgc), complementary to primer C, in antisense orientation.
  • primer C gcggatgaaagtggacttatctccca
  • primer B tgggagataagtccactttcatccgc
  • Apal site used for direct screening of the introduced point mutation.
  • the principle of SOEing PCR is presented in the Annexes and the exact location of A-D primers is illustrated in Figure 2A.
  • the presence of desired mutations was confirmed by sequencing.
  • the Ncol-Sphl fragment containing the full mutated Msh2 cDNA was isolated using a triple digestion: Ncol, Sphl and Pvul.
  • the 2.9kb fragment corresponding to Msh2 G 671 D mutant, flanked by Ncol site at 5' end and Sphl site at 3' end was isolated from the gel, and the ends polished by treatment with T4 DNA polymerase. Consequently to the fill-in and polishing of ends the fragment was ready for blunt-end cloning in the backbone of the modified binary vector FRG8 (Timmermans et al. (1990) J. Biotech. 14: 333-344).
  • the sense orientation of mutated Msh2 G 671 D was confirmed by restriction mapping.
  • the binary vector was introduced into Agrobacterium tumefaciens LBA 4404 by heat shock. Agrobacterium leaf-disc transformation of N.
  • plumbaginifolia P2 was performed as described by Horsch and collaborators (Horsch et al. (1985) Science 227: 1229-1231 ). Transformed plants were generated and selected on MS medium supplemented with 50mg/l kanamycine. The number of T-DNA insertion loci was determined from the kanamycine resistance segregation ratio.
  • the Arabidopsis msh2-specific primers will allow amplification of transcribed Msh2 in transgenic plants overexpressing Msh2 mutant and in Arabidopsis, but not necessarily in WT N. plumbaginifolia.
  • msh2 - mRNA for amplification of endogenous msh2 - mRNA in Nicotiana, couples of degenerate primers were used and different RT-PCR amplification profiles, starting from two weeks young leaves, without success.
  • Protoplasts were isolated from WT and plants overexpressing Msh2 G671 D protein, following the protocol of Negrutiu (Negrutiu (1981) Z.Planen physiologie 104: 431-442).
  • Negrutiu Negrutiu (1981) Z.Planen physiologie 104: 431-442.
  • MNNG mutagenesis we followed basically the protocol of King (King (1983). Cell Culture and somatic cell genetics of plants , voll: Laboratory procedures and their applications. Editor: lndra K. Vasil, Accademic Press.lnc, ISBN: 0-12-7150011983) adapted to microwell plates. Particular attention was given to the adjustment of protoplast density during MNNG treatment since the higher the cell density, the smaller the effect of any one MNNG concentration.
  • the protoplast density was counted with a Thoma chamber and adjusted to 500000 protoplasts/ml.
  • the mutagen MNNG was obtained from Sigma and diluted in DMSO to a stock concentration of 1OmM. Protoplasts were treated with MNNG to a final concentration of 10 ⁇ M mutagen, for a period of 10 minutes. Because of the loss of activity, MNNG was not washed from the protoplasts in the further steps but diluted 10 times in protoplasts culture medium. After the mutagenic treatment protoplasts were cultured at 10 times dilution, in a final volume of 500 ⁇ (50 ⁇ l treated protoplasts plus 450 ⁇ K3M medium) in multiwell plates, in culture room at 25°C and absence of light.
  • the plating efficiency was counted after 2 weeks according to the protocol of Harris and Oparka (Harris and Oparka (1994) Plant Cell Biology-A practical approach; Fluorescent probes for studies of living plant cells. Oxford University Press: pp 45-461994).
  • To 40 ⁇ protoplast suspension 4 ⁇ of 1mg/ml FDA (fluorescein diacetate) was added. The single cells; two sister cells formed after the first division and microcolonies were scored under a UV-light Axiophot microscope (Zeiss).
  • a second negative control beside CoIO was used for the germination test.
  • the line is called 002820, is originated from the same SIGnAL collection and the T-DNA insertion was identified at the level of methionine synthase gene, based on in silico analysis. Since the germination efficiency may be affected by the conditions in the greenhouse, the moment of harvesting and the storage conditions, the seeds used in this experiment were obtained from 002708 line, CoIO and 002820 line grown, harvested and stored in the same conditions. The scoring of occurrence of white sectoring in leaves was assayed ( a similar method was used by Preuss and Britt (Preusee and Britt(2003) Genetics.
  • RTLS total leaf surface
  • a number of 100 seeds were sowed per Petri dish and two to four Petri dishes scored for each variant.
  • the ASSESS program American Society of Plant Pathology
  • the RTLS represents the % of green leaf area reported to the total surface of the Petri dish (constant). After 2-3 weeks , in the stage of 4-6 leaves the Petri dishes were pictured with a digital camera and the RTLS measured using the ASSESS program.
  • Nicotiana plumbaginifolia transgenic lines overexpress the Msh2 G 671 D dominant negative mutant
  • N. plumbaginifolia T 0 competitive inhibition (Cl) transformants were screened by RT-PCR and a total number of 12 transgenic lines overexpressing the Msh2 G 671 D dominant negative mutant were identified ( Figure 1 ). No Msh2 expression was observed whatsoever neither for A. thaliana 2 weeks old plantlets nor for the wild type N. plumbaginifolia young leaves.
  • Ade et al. (Ade et al. (1999) MoI. Gen. Genet. 262: 239- 249) mentioned that msh2 gene is very poorly expressed in plant tissues. They have observed expression of A. thaliana Msh2 mainly in cell suspension with Msh2 most strongly expressed in cells in exponential growing phase, 2 days after inoculation. The msh genes expression pattern with very poor level of expression in somatic tissues and increased level of expression in highly dividing mitotic tissues and anthers in meiotic division was confirmed in wheat by Dong et al. (Dong et al. (2002) Genome 45: 116-124), and in Arabidopsis by Ade et al. (Ade et al. (2001) Genome 44: 651-657).
  • CaMV 35S and Msh2 promoters Consequently between the activity pattern of CaMV 35S and Msh2 promoters seems to be a strong difference. While both promoters are active in mitotically dividing cells, in mature vegetative tissues (mature leaves) just CaMV-35S promoter is highly active. Differential activity is observed as well for meiotically dividing cells, with Msh2 promoter up regulated during meiosis (Dong et al. (2002) Genome 45: 116-124) and background level of activity for CaMV 35S promoter in pollen mother cells in meiosis I with a slight increased activity in meiosis II.
  • This differential pattern of activity between the CaMV 35S and Msh2 promoters makes the CaMV 35S promoter suitable to assess MMR deficiency in mitotic dividing cells, meristematic apex and vegetative tissues but less appropriate to analyze meiotic specific processes in the context of MMR deficiency.
  • MMR deficiency is associated with a spontaneous mutator phenotype in Arabidopsis thaliana and Nicotiana plumbaginifolia, phenotype absent in MMR proficient plants
  • the T-DNA insertion in SALK 002708 line was located in At3g18525 locus based on in silico analysis, locus corresponding to msh2 gene. More precisely, based on the information published by Leonard et al., 2003 and our results, the T-DNA insertion is situated in one of the exons located in 3' terminal part, in the ATP binding site (Fig. 2A). The absence of PCR amplification of C-D fragment confirms that the T-DNA insertion caused the deletion of this region in 002708 line, C-D fragment being a part of msh2 deletion: exons 7 to 13, caused by the single T-DNA insertion (Leonard et al. (2003) Plant Physiol. 133: 328-338).
  • T4 generation we identified an "albino" phenotype, encompassing the whole plant and this phenotype was retrieved in generation T5 in one single line called T5-4 (Fig.3B).
  • Fig.3 A One single event of variegated (sectorial albino development) was recovered for one of the 20 primary competitive inhibition N. plumbaginifolia transformants (Fig.3 A). From this variegated plant, albino auxotrophic plants as well as normal green autotrophic Nicotiana were obtained in a further step by dissection of the apical meristem and leaves, indicating the location of mutation in one of the cells located in central zone of the meristem. It may be that during organogenesis, in MMR deficient background a mutation occurred in one of the albino loci and further on through somatic recombination the homozygotation of this mutation took place, leading to the observed variegated phenotype. A slight reduction in germination frequency of 002708 line (78-85%) compared with colO control (approximate 98%) was observed constantly in different experiments.
  • the second spontaneous mutator phenotype observed in MMR deficient plants is the generation of callus from seeds sowed on MS medium, without hormone supplement and without selection. Just one single event was observed in 4.000 sowed seeds for msh2 deficient Arabidopsis and respectively one single event in approximate 2.000 sowed Nicotiana MMR deficient plants (Fig. 3, C and D). No spontaneous or EMS induced callusogenesis was observed whatsoever in a similar number of wild type Nicotiana and CoIO Arabidopsis.
  • Nicotiana overexpressing Msh2 dominant negative mutant presents increased resistance to high dose of MNNG, based on protoplast plating efficiency
  • protoplasts were isolated from wild type and transgenic Nicotiana overexpressing Msh2 G 671 D protein. The results were scored after 14 days of culture in optimum conditions and represented as number of single cells (no division), two cells (one single division) and microcolonies (more than one division) per microscopic field.
  • the MNNG treatment blocked cells to enter first division, with the blockage being stronger for the second division, with a ration of protoplasts dividing more than once compared to the untreated protoplasts of 15 microcolonies vs 76,5 microcolonies per microscopic field for treated vs untreated protoplasts, giving a comparative value of 19,6 % - 15 x 100/76,5- ( Figure 4).
  • the MNNG blockage of the cell division is more relaxed in the plants overexpressing the dominant negative Msh2 mutant.
  • An increase in number of cells undergoing first and more than one division is observed for all 7 MMR deficient lines, with notable results for S16, where no significant difference is observed between the number of microcolonies formed in absence: 79 microcolonies and/or presence of mutagenic treatment: 82,5 microcolonies per microscopic field (Figure 4), giving a comparativetric value of 95,75%.
  • the comparison with untreated controls shows that the blockage of division is not completely removed, but constantly for the protoplasts with MMR deficiency the frequency of the cells being able to pass over the first block of division, is higher than the frequency of wild type cells able to divide in the same conditions.
  • the profile of cell division in MNNG treated protoplasts is similar to one for untreated cells ( Figure 4). This line was chosen for further studies on tolerance to alkylating agents at plantlet level.
  • reaction to the treatment with alkylating agents in plants will depend on the moment of the treatment, dose of mutagenic agent, targeted organ and plant species.
  • RTLS indicates no significant difference between P2 and G 671 D Msh2/ S16 plantlets germinated from seeds treated with 0.3% EMS for prolonged period of time: 9Oh (Figure 6C).
  • MMR deficient Arabidopsis thaliana shows increased resistance to high dose of EMS and increased frequencies of mutations at reduced dozes of EMS
  • Nicotiana overexpressing Msh2 dominant-negative mutant is a plant system where
  • A. thaliana SALK 002708 is a clear situation where the MMR system is dysfunctional due to the disruption of Msh2 gene through T-DNA insertion.
  • EMS treatment we have used the germination test for seeds treated with mutagenic agent. When low EMS concentrations and reduced treatment durations were used: 0.6% EMS for 3h and respectively 0.3% EMS for 6h, no significant differences in percentage of germination have been observed for CoIO and Msh2 deficient Arabidopsis. Nevertheless the MMR deficient plants presented mutated phenotypes with an increased frequency, scored based on albino marker.
  • a stronger mutagenic treatment with 0.3%EMS for 18h will cause embryo lethality and consequently the germination test will be the most adequate to illustrate the resistance of plants to these type of mutagenic treatment. Since the germination efficiency may vary in function of the greenhouse conditions, the moment of harvesting and storage conditions, the 002708 line, CoIO as well as the second negative control: the SALK line 002820, were grown, harvested and treated with mutagen for the germination test, simultaneously, within one experiment.
  • T4 and T5 of MMR deficient Arabidopsis show an increased resistance to EMS treatment compared to MMR proficient plants ( Figure 10B).
  • a germination frequency of 40% was observed for T4 generation of 002708 line compared with 10% germination efficiency for CoIO mutated seeds ( Figure 10 A and B).
  • Similar results were obtained for MMR deficient plants in T5 generation, with 70% germination for MMR deficient plants compared to 30% germination for 002820 line used as a first control and 38% germination for CoIO plants used as second control (Figure 10B).
  • Example 3 Genotypic analysis of EMS-mutagenised wild-type and mismatch repair deficient N. plumbaginifolia
  • N. plumbaginifolia sequences were selected from Genbank. The total length of the amplicons is 9812 base pairs (bp). 32 M2 plants from a WT line and 32 M2 plants from the MMR deficient line S-16 were screened for SNPs. Therefore a total of 628000 bp was screened for each genotype.
  • Candidate mutant alleles identified by TILLING were reamplified by PCR and sequenced. One confirmed mutation was found in the 628 kb screened DNA of M2 WT plants, and four confirmed mutations were found in 628 kb DNA of M2 mismatch repair deficient plants (see Table 3).
  • the invention has demonstrated that increase in mutation frequency in response to chemical mutagens (here in particular alkylating agents) can be achieved in plant cells deficient in the mismatch repair system.
  • the mutation frequency in WT EMS treated N. plumbaginifoiia is in the same range as that detected by TILLING in EMS treated A. thaliana, i.e. 1 mutation per 300 kb screened (Greene et al. Spectrum of chemically induced mutations from a large-scale reverse-genetic screen in Arabidopsis. Genetics 164: 731-740, 2003).
  • mismatch repair deficient N. plumbaginifoiia a four-fold increase in mutation frequency was found at the DNA level. This confirms the increase in induced mutations in mismatch repair deficient plants observed at the phenotypic level.

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Abstract

La présente invention se rapporte à un procédé permettant d'introduire des mutations génétiques dans le génome de cellules végétales. Le procédé selon l'invention comprend les étapes consistant : (i) à supprimer l'activité d'au moins un système endogène de réparation de l'ADN dans au moins une cellule végétale ; (ii) à exposer ladite cellule végétale à au moins un agent mutagène. Certains modes de réalisation consistent à supprimer le système de réparation du mésappariement dans ladite cellule végétale, et à utiliser un agent d'alkylation de l'ADN pour la mutagenèse. L'invention a pour but de permettre la génération de banques de mutants de diverses espèces végétales, dans lesquelles une proportion plus importante de gènes présentera une mutation et un nombre plus important de phénotypes différents sera obtenu que ce qu'il est possible de réaliser à l'aide des procédés actuels. La présente invention aura pour effet de combler la 'lacune phénotypique' touchant les banques existantes de mutants végétaux.
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010074562A1 (fr) * 2008-12-22 2010-07-01 Keygene N.V. Utilisation de l'arn à double brin de manière à augmenter l'efficacité d'une altération génétique ciblée à l'intérieur de protoplastes végétaux
WO2011078665A1 (fr) * 2009-12-21 2011-06-30 Keygene N.V. Techniques améliorées de transfection de protoplastes
CN101597328B (zh) * 2008-06-06 2012-07-11 中国科学院遗传与发育生物学研究所 促进植物体细胞胚胎发生和脂肪酸合成的转录因子及其编码基因与应用
CN102812803A (zh) * 2012-07-02 2012-12-12 青岛市农业科学研究院 一种豌豆ems突变体库构建的方法
WO2015164805A1 (fr) * 2014-04-24 2015-10-29 Purdue Research Foundation Mutagenèse induite

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997005268A1 (fr) * 1995-07-26 1997-02-13 Setratech Recombinaison homologue dans des cellules eucaryotes a systeme de reparation des mesappariements inactive
WO1999019492A2 (fr) * 1997-10-10 1999-04-22 Aventis Cropscience S.A. Procedes pour obtenir des varietes de plantes
WO2001059092A2 (fr) * 2000-02-11 2001-08-16 The Johns Hopkins University Procedes d'obtention de microbes hypermutables
WO2001061012A1 (fr) * 2000-02-18 2001-08-23 Nicolaides Nicholas C Procede de generation de plantes tres sujettes aux mutations
WO2001088192A2 (fr) * 2000-05-12 2001-11-22 The Johns Hopkins University Procede de generation d'organismes a mutabilite tres elevee
WO2002054856A1 (fr) * 2001-01-15 2002-07-18 Morphotek Inc. Inhibiteurs chimiques de reparation de desappariements
WO2003054217A2 (fr) * 2001-12-20 2003-07-03 Boyce Thompson Institute For Plant Research Sequences msh2 de plantes et methodes d'utilisation correspondantes

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997005268A1 (fr) * 1995-07-26 1997-02-13 Setratech Recombinaison homologue dans des cellules eucaryotes a systeme de reparation des mesappariements inactive
WO1999019492A2 (fr) * 1997-10-10 1999-04-22 Aventis Cropscience S.A. Procedes pour obtenir des varietes de plantes
WO2001059092A2 (fr) * 2000-02-11 2001-08-16 The Johns Hopkins University Procedes d'obtention de microbes hypermutables
WO2001061012A1 (fr) * 2000-02-18 2001-08-23 Nicolaides Nicholas C Procede de generation de plantes tres sujettes aux mutations
WO2001088192A2 (fr) * 2000-05-12 2001-11-22 The Johns Hopkins University Procede de generation d'organismes a mutabilite tres elevee
WO2002054856A1 (fr) * 2001-01-15 2002-07-18 Morphotek Inc. Inhibiteurs chimiques de reparation de desappariements
WO2003054217A2 (fr) * 2001-12-20 2003-07-03 Boyce Thompson Institute For Plant Research Sequences msh2 de plantes et methodes d'utilisation correspondantes

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
ADE JULES ET AL: "Functional analysis of the Arabidopsis thaliana mismatch repair gene MSH2" GENOME, vol. 44, no. 4, August 2001 (2001-08), pages 651-657, XP001208017 ISSN: 0831-2796 *
ANDREW SUSAN E ET AL: "Tissues of MSH2-deficient mice demonstrate hypermutability on exposure to a DNA methylating agent" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 95, no. 3, 3 February 1998 (1998-02-03), pages 1126-1130, XP002375421 ISSN: 0027-8424 *
HOFFMAN PETER D ET AL: "Rapid accumulation of mutations during seed-to-seed propagation of mismatch-repair-defective Arabidopsis" GENES & DEVELOPMENT, vol. 18, no. 21, 1 November 2004 (2004-11-01), pages 2676-2685, XP002375419 ISSN: 0890-9369 *
ISPAS GABRIELA ET AL: "Interferance of Mismatch Repair System on genetic recombination in plants." MEDEDELINGEN FACULTEIT LANDBOUWKUNDIGE EN TOEGEPASTE BIOLOGISCHE WETENSCHAPPEN UNIVERSITEIT GENT, vol. 67, no. 4, 2002, pages 239-241, XP008062435 ISSN: 1373-7503 *
LEONARD JEFFREY M ET AL: "Reduction of stability of Arabidopsis genomic and transgenic dna-repeat sequences (Microsatellites) by inactivation of AtMSH2 mismatch-repair function." PLANT PHYSIOLOGY (ROCKVILLE), vol. 133, no. 1, September 2003 (2003-09), pages 328-338, XP002375417 ISSN: 0032-0889 cited in the application *
RAKOSY-TICAN LENUTA ET AL: "Transformation of wild Solanum species resistant to late blight by using reporter gene gfp and msh2 genes" PLANT BREEDING AND SEED SCIENCE, vol. 50, 2004, pages 119-127, XP008062469 ISSN: 1429-3862 *
STUDAMIRE BARBARA ET AL: "Separation-of-function mutations in Saccharomyces cerevisiae MSH2 that confer mismatch repair defects but do not affect nonhomologous-tail removal during recombination" MOLECULAR AND CELLULAR BIOLOGY, vol. 19, no. 11, November 1999 (1999-11), pages 7558-7567, XP002375528 ISSN: 0270-7306 *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101597328B (zh) * 2008-06-06 2012-07-11 中国科学院遗传与发育生物学研究所 促进植物体细胞胚胎发生和脂肪酸合成的转录因子及其编码基因与应用
WO2010074562A1 (fr) * 2008-12-22 2010-07-01 Keygene N.V. Utilisation de l'arn à double brin de manière à augmenter l'efficacité d'une altération génétique ciblée à l'intérieur de protoplastes végétaux
JP2012513199A (ja) * 2008-12-22 2012-06-14 キージーン・エン・フェー 植物プロトプラストにおける標的化遺伝子改変の効率を増大させるための二本鎖ｒｎａの使用
JP2013514764A (ja) * 2009-12-21 2013-05-02 キージーン・エン・フェー プロトプラストを形質移入するための改善された技術
CN102791865A (zh) * 2009-12-21 2012-11-21 凯津公司 用于转染原生质体的改进技术
WO2011078665A1 (fr) * 2009-12-21 2011-06-30 Keygene N.V. Techniques améliorées de transfection de protoplastes
AU2010335107B2 (en) * 2009-12-21 2014-07-03 Keygene N.V. Improved techniques for transfecting protoplasts
EP2813572A3 (fr) * 2009-12-21 2015-02-18 Keygene N.V. Techniques améliorées de transfection de protoplastes
CN104651394A (zh) * 2009-12-21 2015-05-27 凯津公司 用于转染原生质体的改进技术
CN102791865B (zh) * 2009-12-21 2015-07-01 凯津公司 用于转染原生质体的改进技术
JP2015165812A (ja) * 2009-12-21 2015-09-24 キージーン・エン・フェー プロトプラストを形質移入するための改善された技術
CN102812803A (zh) * 2012-07-02 2012-12-12 青岛市农业科学研究院 一种豌豆ems突变体库构建的方法
WO2015164805A1 (fr) * 2014-04-24 2015-10-29 Purdue Research Foundation Mutagenèse induite

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