EP0915984A1 - Production de graines apomictiques - Google Patents

Production de graines apomictiques

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Publication number
EP0915984A1
EP0915984A1 EP97923882A EP97923882A EP0915984A1 EP 0915984 A1 EP0915984 A1 EP 0915984A1 EP 97923882 A EP97923882 A EP 97923882A EP 97923882 A EP97923882 A EP 97923882A EP 0915984 A1 EP0915984 A1 EP 0915984A1
Authority
EP
European Patent Office
Prior art keywords
leu
sequence
asn
dna
val
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP97923882A
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German (de)
English (en)
Inventor
Sape Cornelis De Vries
Eduard Daniel Leendert Schmidt
Gerrit Jan Van Holst
Valerie France Gabrielle Hecht
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Syngenta Participations AG
Original Assignee
Novartis AG
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Filing date
Publication date
Application filed by Novartis AG filed Critical Novartis AG
Publication of EP0915984A1 publication Critical patent/EP0915984A1/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • 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
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • 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
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis

Definitions

  • the present invention relates to the production of genetically transformed plants.
  • the invention relates inter alia to a process for inducing apomixis, to the apomictic seeds which result from the process, and to the plants and progeny thereof which result from the germination of such seeds.
  • Apomixis which is vegetative (non-sexual) reproduction through seeds, is a genetically controlled reproductive mechanism found in some polyploid non-cultivated species. The process is classified as gametophytic or non-gametophytic. In gametophytic apomixis - of which there are two types (apospory and diplospory), multiple embryo sacs which typically lack antipodal nuclei are formed, or else megasporogenesis in the embryo sac takes place. In adventitious embryo ⁇ y (non-gametophytic apomixis), a somatic embryo develops directly from the cells of the embryo sac, ovary wall or integuments. In adventitious embryony, somatic embryos from surrounding cells invade the sexual ovary, one of the somatic embryos out-competes the other somatic embryos and the sexual embryo and utilizes the produced endosperm.
  • apomixis would provide for true-breeding, seed propagated hybrids. Moreover, apomixis could shorten and simplify the breeding process so that setting and progeny testing to produce and/or stabilize a desirable gene combination could be eliminated. Apomixis would provide for the use as cultivars of genotypes with unique gene combinations since apomictic genotypes breed true irrespective of heterozygosity. Genes or groups of genes could thus be "pyramided and "fixed" in super genotypes. Every superior apomictic genotype from a sexual- apomict j c cross would have the potential to be a cultivar. Apomixis would allow plant breeders to develop cultivars with specific stable traits for such characters as height, seed and forage quality and maturity.
  • Breeders would not be limited in their commercial production of hybrids by (i) a cytoplasmic-nuclear interaction to produce male sterile female parents or (ii) the fertility restoring capacity of a pollinator. Almost all cross-compatible germplasm could be a potential parent to produce apomictic hybrids.
  • apomixis would simplify commercial hybrid seed production.
  • the potential benefits to accrue from the production of seed via apomixis are presently unrealized, to a large extent because of the problem of engineering apomictic capacity into plants of interest.
  • the present invention provides a solution to that problem in that it provides the means for obtaining plants which exhibit the adventitious embryony type of apomixis.
  • a method of producing apomictic seeds comprising the steps of:
  • integuments in one or more of the following: carpel, integuments, ovuie, ovule premordium, ovary wall, chalaza, nucellus, funicle and placenta.
  • integuments also includes those tissues, such as endothelium, which are derived therefrom.
  • embryoogenic is meant the capability of cells to develop into an embryo under permissive conditions. It will be appreciated that the term “in an active form” includes proteins which are truncated or otherwise mutated with the proviso that they initiate or amplify embryogenesis whether or not in doing this they interact with the signal transduction components that they otherwise would in the tissues in which they are normally present
  • plant material includes protoplasts, isolated plant cells (such as stomatal guard cells) possessing a cell wall, pollen, whole tissues such as emerged radicle, stem, leaf, petal, hypocotyl section, apical meristem, ovaries, zygotic embryo per se, roots, vascular bundle, pericycle, anther filament, somatic embryos and the like.
  • a further embodiment of the invention relates to a DNA molecule comprising a nucleotide sequence encoding a protein the presence of which in an active form in a cell, or membrane thereof, renders said cell embryogenic.
  • the said nucleotide sequence may be introduced into the plant material, inter alia, via a bacterial or viral vector, by micro-injection, by co-incubation of the plant material and sequence in the presence of a high molecular weight glycol or by coating of the sequence onto the surface of a biologically inert particle which is then introduced into the material.
  • kinase capable of spanning a plant cell membrane.
  • the kinase may be a leucine rich repeat receptor like kinase which has the capacity to auto-phosphorylate.
  • leucine rich repeat receptor like kinase examples include Arabidopsis RLK5 (Walker, 1993), Arabidopsis RPS2 (Bent et al. 1994), Tomato CF-9 gene product (Jones ef al. 1994), Tomato N (Whitham et al. 1994), Petunia PRK1 (Mu er al. 1994), the product of the Dmsophila Toll gene (Hashimoto ef al.
  • the protein may comprise a ligand binding domain, a proline box, a transmembrane domain, a kinase domain and a protein binding domain.
  • the extracellular (ligand binding) domain serves as an inhibitor of the kinase domain in the ligand-f ree state. This arrest is removed after binding of the ligand.
  • the protein either lacks a ligand binding domain or the domain is functionally inactivated so that the kinase domain can be constftutiveiy active in the absence of an activating signal (ligand).
  • the protein binding domain is preferably located intra-cellulariy.
  • the said sequence further encodes a cell membrane targeting sequence.
  • the sequence may be that which is depicted in SEQ ID Nos. 1 , 2, 20, or 32, or it may be similar in that it is complementary to a sequence which hybridizes under stringent conditions with the said sequences and which encodes a membrane bound protein having kinase activity.
  • similar is meant a sequence which is complementary to a test sequence which is capable of hybridizing to the inventive sequence.
  • the nucleic acid constituting the test sequence preferably has a TM within 20°C of that of the inventive sequence.
  • the TM values of the sequences are preferably within 10°C of each other. More preferably the hybridization is performed under stringent conditions, with either the test or inventive DNA preferably being supported.
  • a denatured test or inventive sequence is preferably first bound to a support and hybridization is effected for a specified period of time at a temperature of between 50 and 70°C in double strength citrate buffered saline (SSC) containing 0.1%SDS followed by rinsing of the support at the same temperature but with a buffer having a reduced SSC concentration.
  • SSC double strength citrate buffered saline
  • a buffer having a reduced SSC concentration are typically single strength SSC containing 0.1%SDS, half strength SSC containing 0.1%SDS and one tenth strength SSC containing 0.1%SDS.
  • Sequences having the greatest degree of similarity are those the hybridization of which is least affected by washing in buffers of reduced concentration. It is most preferred that the test and inventive sequences are so similar that the hybridization between them is substantially unaffected by washing or incubation in one tenth strength sodium citrate buffer containing 0.1%SDS.
  • the sequence may be modified in that known mRNA instability motifs or polyadenylation signals may be removed and/or codons which are preferred by the plant into which the sequence is to be inserted may be used so that expression of the thus modified sequence in the said plant may yield substantially similar protein to that obtained by expression of the unmodified sequence in the organism in which the protein is endogenous.
  • the sequence is preferably under expression control of an inducible or developmentally regulated promoter, typically one of the following: a promoter which regulates expression of SERK genes in planta, the Arabidopsis ANT gene promoter, the promoter of the 0126 gene from Phalaenopsis, the carrot chitinase DcEP3-1 gene promoter, the Arabidopsis AtChitlV gene promoter, the Arabidopsis LTP-1 gene promoter, the Arabidopsis bel- 1 gene promoter, the petunia fbp-7 gene promoter, the Arabidopsis AtDMCI promoter, the pTA7001 inducible promoter.
  • a promoter which regulates expression of SERK genes in planta typically one of the following: a promoter which regulates expression of SERK genes in planta, the Arabidopsis ANT gene promoter, the promoter of the 0126 gene from Phalaenopsis, the carrot chitinase DcEP3-1 gene
  • the DcEP3-1 gene is expressed transiently during inner integument degradation and later in cells that line the inner part of the developing endosperm.
  • the AtChilV gene is transiently expressed in the micropylar endosperm up to cellularisation.
  • the LTP-1 promoter is active in the epidermis of the developing nucellus, both integuments, seed coat and early embryo.
  • the bel-1 gene is expressed in the developing inner integument and the fbb-7 promoter is active during embryo sac development.
  • the Arabidopsis ANT gene is expressed during integument development, and the 0126 gene from Phalaenopsis is expressed in the mature ovule.
  • sequence is expressed in the somatic cells of the embryo sac, ovary wall, nucellus, or integuments.
  • the endosperm within the apomictic seed results from fusion of polar nuclei within the embryo sac with a pollen-derived male gamete nucleus. It is preferred that the sequence encoding the protein is expressed prior to fusion of the polar nuclei with the male gamete nucleus.
  • the invention further includes a DNA, but preferably a recombinant DNA, comprising a sequence encoding a protein the presence of which in an active form in a cell, or membrane thereof, renders said cell embryogenic.
  • a DNA encoding a protein which is a leucine rich repeat receptor like kinase and comprises a ligand binding domain, a proline box, a transmembrane domain, a kinase domain and a protein binding domain, the ligand binding domain optionally being absent or functionally inactive.
  • the invention embodies a DNA comprising a DNA sequence encoding a N-terminal protein fragment having the following amino acid sequence: Gh Ser Trp Asp Pro Thr Leu Val Asn Pro Cys ThrTrp Phe HE Val Thr Cys Asn.
  • a specific embodiment of the invention relates to a DNA comprising a DNA sequence encoding a protein having the sequence depicted in SEQ ID Nos. 3 or 21 , or a protein substantially similar thereto which is capable of being membrane bound and which has kinase activity.
  • substantially similar is meant a pure protein having an amino acid sequence which is at least 90% similar to the sequence of the proteins depicted in SEQ ID No 3 below.
  • two amino acid sequences with at least 90% similarity to each other have at least 90% identical or conservatively replaced amino acid residues in a like position when aligned optimally allowing for up to 8 gaps with the pmviso that in respect of each gap a total not more than 4 amino acid residues is affected.
  • conservative replacements may be made between amino acids within the following groups:
  • the invention futher embodies a DNA comp ⁇ sing a DNA sequence encoding a N-terminal protein fragment having the following amino acid sequence: Val Xaa Gh Ser Trp Asp ProThrLeu Val Asn Pro CysThrTrp Phe His Val ThrCys Asn, with Xaa being a variable amhoacti,bLit preferably LeuorVai
  • a DNA comprising a DNA sequence encoding a N-terminal protein fragment having the following amino acid sequence: Val Xaa Gh Ser Trp Asp Pro Thr Leu Val Asn Pro Cys Thr Trp Phe He Val Thr Cys Asn XabXac XadXaeVaJXafArgVal A ⁇ Leu GVAsn Xag Xah Leu SerGry te Ljeu Xa FtoGkj Le ⁇ Xaa to
  • Xac Glu or Asp or H ⁇
  • the DNA further encodes a cell membrane targeting sequence, and that the protein encoding region is under expression control of a developmentally regulated or inducible promoter, such as, for example, a promoter which regulates expression of SERK genes in planta, the carrot chitinase DcEP3-1 gene promoter, the Arabidopsis AtChitlV gene promoter, the Arabidopsis LTP-1 gene promoter, the Arabidopsis bel-1 gene promoter, the petunia fbp-7 gene promoter, the Arabidopsis ANT gene promoter, or the promoter of the 0126 gene from Phalaenopsis; the Arabidopsis AtDMCI promoter, or the pTA7001 inducible promoter.
  • a developmentally regulated or inducible promoter such as, for example, a promoter which regulates expression of SERK genes in planta, the carrot chitinase DcEP3-1 gene promoter, the Arabidopsis AtChitlV
  • the said DNA include those depicted in SEQ ID Nos. 1 , 2, 20 or 32, or those which are complementary to one which hybridizes under st ⁇ ngent conditions with the said sequences and which encode a membrane bound protein having kinase activity.
  • the DNA may be modified in that known mRNA instability motifs or polyadenylation signals may be removed and/or codons which are preferred by the plant into which the DNA is to be inserted may be used so that expression of the thus modified DNA in the said plant may yield substantially similar protein to that obtained by expression of the unmodified DNA in the organism in which the protein is endogenous.
  • the invention still further includes a vector which contains DNA as indicated in the three immediately preceding paragraphs, plants transformed with the recombinant DNA or vector, and the progeny of such plants which contain the DNA stably inco ⁇ orated, and/or the apomictic seeds of such plants or such progeny.
  • the recombinant DNA molecules of the invention can be introduced into the plant cell in a number of art-recognized ways. Those skilled in the art will appreciate that the choice of method might depend on the type of plant, i.e. monocot or dicot, targeted for transformation. Suitable methods of transforming plant cells include microinjection (Crossway et al., BioTechniques 4:320-334 (1986)), electroporation (Riggs et al, Pmc. Natl. Acad. Sci.
  • transgenic plants in particular transgenic fertile plants transformed by means of the aforedescribed processes and their asexual and/or sexual progeny, which still contain the DNA stably inco ⁇ orated, and/or the apomictic seeds of such plants or such progeny.
  • the transgenic plant according to the invention may be a dicotyledonous or a monocotyledonous plant.
  • Such plants include field crops, vegetables and fruits including tomato, pepper, melon, lettuce, cauliflower, broccoli, cabbage, brussels sprout, sugar beet, com, sweetcorn, onion, carrot, leek, cucumber, tobacco, alfalfa, aubergine, beet, broad bean, celery, chicory, cow pea, endive, gourd, groundnut, papaya, pea, peanut, pineapple, potato, safflower, snap bean, soybean, spinach, squashes, sunflower, sorghum, water-melon, and the like; and ornamental crops including Impatiens, Begonia, Petunia, Pelargonium, Viola, Cyclamen, Ve ⁇ ena, Vinca, Tagetes, Primula, Saint Paulia, Ageratum, Amaranthus, Anthirrhinum, Aquilegia, Chrysanthemum, Cineraria, Clover, Cosmo
  • the DNA is expressed in "seed crops" such as com, sweet com and peas etc. in such a way that the apomictic seed which results from such expression is not physically mutated or otherwise damaged in comparison with seed from untransformed like crops.
  • seed crops such as com, sweet com and peas etc.
  • transgenic maize More preferred are transgenic maize, wheat, barley, sorghum, rye, oats, turf and forage grasses, millet and rice.
  • sorghum More preferred are maize, wheat, sorghum, rye, oats, turf grasses and rice.
  • soybean, cotton, sugar beet, sugar cane, oilseed rape, tobacco and sunflower are more preferred herein.
  • soybean, cotton, tobacco, sugar beet and oilseed rape are more preferred herein.
  • soybean, cotton, tobacco, sugar beet and oilseed rape are more preferred herein.
  • progeny' is understood to embrace both, “asexually” and “sexually” generated progeny of transgenic plants. This definition is also meant to include all mutants and variants obtainable by means of known processes, such as for example cell fusion or mutant selection and which still exhibit the characte ⁇ stic properties of the initial transformed plant, together with all crossing and fusion products of the transformed plant material. This also includes progeny plants that result from a backcrossing, as long as the said progeny plants still contain the DNA according to the invention
  • Another object of the invention concerns the proliferation material of transgenic plants.
  • the proliferation material of transgenic plants is defined relative to the invention as any plant material that may be propagated sexually or asexually in vivo or in vitm. Particularly preferred within the scope of the present invention are protoplasts, cells, calli, tissues, organs, seeds, embryos, pollen, egg cells, zygotes, together with any other propagating material obtained from transgenic plants.
  • Parts of plants such as for example flowers, stems, fruits, leaves, roots o ⁇ ginatmg in transgenic plants or their progeny previously transformed by means of the process of the invention and therefore consisting at least in part of transgenic cells, are also an object of the present invention.
  • a further object of the invention is a method of producing apomictic seeds, but preferably seeds that are of the adventitious embryony type, comprising the steps of:
  • the kinase protein being expressed by the DNA according to the invention is preferably a leucine rich repeat receptor like kinase and comprises a ligand binding domain, a proline box, a transmembrane domain, a kinase domain and a protein binding domain.
  • the said kinase protein may tack a functional ligand binding domain but comprises a proline box, a transmembrane domain, a kinase domain and a protein binding domain.
  • the genetic properties engineered into the transgenic seeds and plants described above are passed on by sexual reproduction or vegetative growth and can thus be maintained and propagated in progeny plants.
  • said maintenance and propagation make use of known agricultural methods developed to fit specific pu ⁇ oses such as tilling, sowing or harvesting.
  • Specialized processes such as hydroponics or greenhouse technologies can also be applied.
  • measures are undertaken to control weeds, plant diseases, insects, nematodes, and other adverse conditions to improve yield.
  • Use of the advantageous genetic properties of the transgenic plants and seeds according to the invention can further be made in plant breeding which aims at the development of plants with improved properties such as tolerance of pests, herbicides, or stress, improved nutritional value, increased yield, or improved structure causing less loss from lodging or shattering.
  • the various breeding steps are characterized by well-defined human intervention such as selecting the lines to be crossed, directing pollination of the parental lines, or selecting appropriate progeny plants. Depending on the desired properties different breeding measures are taken.
  • the relevant techniques are well known in the art and include but are not limited to hybridization, inbreeding, backcross breeding, multiline breeding, variety blend, interspecific hybridization, aneuploid techniques, etc.
  • Hybridization techniques also include the sterilization of plants to yield male or female sterile plants by mechanical, chemical or biochemical means.
  • Cross pollination of a male sterile plant with pollen of a different line assures that the genome of the male sterile but female fertile plant will uniformly obtain properties of both parental lines.
  • the transgenic seeds and plants according to the invention can be used for the breeding of improved plant lines which for example increase the effectiveness of conventional methods such as herbicide or pestidice treatment or allow to dispense with said methods due to their modified genetic properties.
  • new crops with improved stress tolerance can be obtained which, due to their optimized genetic "equipment" , yield harvested product of better quality than products which were not able to tolerate comparable adverse developmental conditions.
  • Propagation material to be used as seeds is customarily treated with a protectant coating comp ⁇ sing herbicides, insecticides, fungicides, bacte ⁇ cides, nematicides, molluscicides or mixtures thereof.
  • a protectant coating comp ⁇ sing herbicides, insecticides, fungicides, bacte ⁇ cides, nematicides, molluscicides or mixtures thereof.
  • Customarily used protectant coatings comp ⁇ se compounds such as captan, carboxin, thiram (TMTD * ), methalaxyl (Apron*), and pi ⁇ miphos-methyl (Actellic*). If desired these compounds are formulated together with further carriers, surfactants or application- promoting adjuvants customarily employed in the art of formulation to provide protection against damage caused by bacterial, fungal or animal pests.
  • the protectant coatings may be applied by impregnating propagation material with a liquid formulation or by coating with a combined wet
  • plants produced as described in the examples set forth below are grown in pots in a greenhouse or in soil, as is known in the art, and permitted to flower. Pollen is obtained from the mature stamens and used to pollinate the pistils of the same plant, sibling plants, or any desirable plant. Similarly, the pistils developing on the transformed plant may be pollinated by pollen obtained from the same plant, sibling plants, or any desirable plant.
  • Transformed progeny obtained by this method may be distinguished from non-transformed progeny by the presence of the introduced gene(s) and/or accompanying DNA (genotype), or the phenotype conferred.
  • the transformed progeny may similarly be selfed or crossed to other plants, as is normally done with any plant carrying a desirable trait.
  • tobacco or other transformed plants produced by this method may be selfed or crossed as is known in the art in order to produce progeny with desired characteristics.
  • other transgenic organisms produced by a combination of the methods known in the art and this invention may be bred as is known in the art in order to produce progeny with desired characteristics.
  • a method of obtaining embryogenic cells in plant material comprising transforming the material with a recombinant DNA sequence or a vector according to the invention, expressing the sequence in the material or derivatives thereof and subjecting the said material or derivatives to a compound which acts as a ligand for the gene product of the said sequence.
  • the invention further relates to a method of generating somatic embryos under in vitm conditions wherein the SERK protein is overexpressed ectopically.
  • the invention still further includes the use of the said DNA in the manufacture of apomictic seeds, in which use the sequence is expressed in the vicinity of the embryo sac.
  • the SERK gene may be expressed in transgenic plants such as, for example, an Arabidopsis plant, under the control of plant expression signals, particularly a promoter which regulates expression of SERK genes in planta, but preferably a developmentally regulated or inducible promoter such as, for example, the carrot chitinase DcEP3-1 gene promoter, the Arabidopsis AtChitlV gene promoter, the Arabidopsis LTP-1 gene promoter, the Arabidopsis bel-1 gene promoter, the petunia fbp-7 gene promoter, the Arabidopsis ANT gene promoter, or the promoter of the 0126 gene from Phalaenopsis; the Arabidopsis AtDMCI promoter, or the pTA7001 inducible promoter.
  • a promoter which regulates expression of SERK genes in planta but preferably a developmentally regulated or inducible promoter such as, for example, the carrot chitinase DcEP3-1 gene
  • the promoters of the DcEP3-1 and the AtChit IV genes may be cloned and characterized by standard procedures.
  • the DcSERK coding sequence (SEQ ID No. 2) is cloned behind the DcEP3-1 , the AtChit IV or the AtLTP-1 promoters and transformed into Arabidopsis. The ligation is performed in such a way that the promoter is operably linked to the sequence to be transc ⁇ bed.
  • This construct which also contains known marker genes providing for selection of transformed mate ⁇ al, is inserted into the T-DNA region of a binary vector such as pBIN19 and transformed into Arabidopsis. transformation into Arabidopsis is performed by the vacuum infiltration or root transformation procedures known to the skilled man.
  • Transformed seeds are selected and harvested and (where possible) transformed lines are established by normal selfing.
  • Parallel transformations with 35S promoter-SERK constructs and the entire SERK gene itself are used as controls to evaluate over-expression in many cells or only in the few cells that naturally express the SERK gene.
  • the 35S promoter-SERK construct may give embryo formation wherever the signal that activates the SERK-mediated transduction chain is present in the plant.
  • a testing system based on emasculation and the generation of donor plant lines for pollen carrying LTP1 promoter-GUS and SERK promoter-bamase is established.
  • the same constructs (35S, EP3-1 , AtChitlV, AtLTP-1 and SERK promoters fused to the SERK coding sequence) are employed for transformation into several Arabidopsis backgrounds. These backgrounds are wild type, male sterile, fis (allelic to emb 173) and p ⁇ mordia timing (pt)-1 lines, or a combination of two or several of these backgrounds.
  • the wt lines are used as a control to evaluate possible effects on normal zygotic embryogenesis, and to score for seed set without fertilization after emasculation.
  • the ms lines are used to score directiy for seed set without fertilization.
  • the fis lines exhibit a certain degree of seed and embryo development without fertilization, so may be expected to have a natural tendency for apomictic embryogenesis, which may be enhanced by the presence of the SERK constructs.
  • the pt-1 line has superior regenerative capabilities and has been used to initiate the first stably embryogenic Arabidopsis cell suspension cultures. Combinations of several of the above backgrounds are obtained by crossing with each other and with lines containing ectopic SERK expressing constructs. Except for the ms lines, propagation can proceed by normal self ing, and analysis of apomictic traits following emasculation. A similar strategy is followed in which the ATChilV, AtLTP-1 and SERK promoters are replaced by the bel-1 and fbp-7 promoters as well by other promoters specific for components of the female gametophyte.
  • Additional constructs are generated that have constitutive receptor kinase activity.
  • Most of the receptor kinases of the SERK type act as homodimeric receptors, requiring autophosphorylation before being able to activate downstream signal transduction cascades.
  • the extracellular domain serves as an inhibitor of the kinase domain in the ligand-free stage. This arrest is removed after binding of the ligand (Cadena and Gill, 1992).
  • mutant homodimeric (in cells that do not have a natural population of SERK proteins) or heterodimeric (in cells that also express the unmodified forms) proteins can be generated with a constitutively activated kinase domain.
  • This approach when coupled to one of the promoters active in the nucellar region, results in activation of the embryogenic pathway in the absence of the activating signal. This may be an important alternative in cases where it is necessary or desirable to have activation of the SERK pathway only dependant on specific promoter activity and independent of temporal regulation of an activating signal.
  • the present invention has been particularly described by way of the production of apomictic seed by heterologous expression of the SERK gene in the nucellar region of the ca ⁇ el, the skilled man will recognize that other genes, the products of which have a similar structure/function to the SERK gene product, may likewise be expressed with similar results.
  • the example illustrates apomictic seed production in Arabidopsis, the invention is, of course, not limited to the expression of apomictic seed-inducing genes solely in this plant.
  • the present disclosure also includes the possibility of expressing the SERK (or related) gene sequences in the transformed plant material in a constitutive - tissue non ⁇ specific manner (for example under transcriptional control of a CaMV35S or NOS promoter).
  • tissue specificity is assured by the localized presence within the vicinity of the embryo sac of the ligand of the product of the said gene.
  • the SERK (or related) gene products may interact with proteins such as transc ⁇ ption factors which are involved in regulating embryogenesis. This interaction withtn tissue which has been transformed according to the present disclosure is also part of the present invenbon.
  • the SERK gene (and others as indicated in the preceding paragraph) may be transformed into plant mate ⁇ al which may be propagated and/or differentiated and used as an explant from which somatic embryos can be obtained.
  • Expression of such sequences in the transformed tissue substantially increases the percentage of the cells in the tissue which are competent to form somatic embryos, in compa ⁇ son with the number present in non-transformed like tissue.
  • SEQ ID NO. 1 depicts the Daucus carota genomic clone of the putative receptor kinase (SERK) associated with the transition of competent to embryogenic cells;
  • SEQ ID NO. 2 depicts the cDNA of the said putative kinase
  • SEQ ID NOs. 3 depicts the predicted protein sequence of the SERK protein encoded by the DNA of SEQ ID NO:1.
  • SEQ ID NOs: 4-16 depict the sequences of va ⁇ ous PCR p ⁇ mers.
  • SEQ ID NOs. 17-19 depict specific peptides contained within the gene product of SEQ ID NO. 2.
  • SEQ ID NO: 20 depitcts the Arabidopsis thaliana partial genomic clone of the putative receptor kinase (SERK) associated with the transition of competent to embryogenic cells.
  • SERK putative receptor kinase
  • SEQ ID NO: 21 depicts the predicted protein sequence of the SERK protein encoded by the
  • SEQ ID NOs: 22, 24, 26, 28 and 30 depict the partial DNA sequences of 5 EST clones with high homology to the SERK LRR sequences .
  • SEQ ID Nos. 23, 25, 27, 29 and 31 depict the predicted protein sequence of the partial DNA sequences of the 5 EST clones of SEQ ID Nos: 22, 24, 26, 28 and 30.
  • SEQ ID NO: 32 depicts the nuclotide sequence of the SERK cDNA from Arabidopsis thaliana.
  • SEQ ID NO: 33 depicts the predicted amino acid sequence of the SERK protein from
  • FIG. 1 shows the results of an RT-PCR experiment performed on RNA extracted from the indicated tissues. 40 cycles followed by Southern blotting of the resulting bands is necessary to visualize SERK expression. Lanes include explants at day 7, treated for less (lane 1 ) or more (lane 2) then 3 days with 2,4-D. In the original a very faint signal is visible in lane 2, but not in lane 1. Established embryogenic cultures (lanes 4-6) but not a non-embryogenic control (lane 3) express the SERK gene. In carrot plants, no expression is detectable except for developing seeds after pollination (lane 7).
  • Figure 2A shows the results of a whole-mount in situ hybridization with the SERK cDNA on 7 day explants treated for 3 days with 2,4 D. Few cells on the surface of the explant express the SERK gene, and those cells that do are the ones that become embryogenic.
  • Figure 2B shows a whole mount in situ hybridization on a partially dissected seed containing a globular zygotic embryo. Hybridization is visualized by DIG staining.
  • Figure 3 shows SERK expression in embryogenic hypocotyl cells during hormone-induced activation, determined by whole mount in situ hybridization . Ban 50 mm
  • A-E Cell population generated by mechanical fragmentation of the activated hypocotyls. Only few of a certain type of cell, defined enlarged cell show SERK expression (asterisks). Small cytoplasmic cells (c), enlarging cells (eg) and large cells (I) never show SERK expression.
  • G-l Proliferating mass coming from the inner hypocotyl tissues 10 days after the beginning of the hormonal treatment (longitudinal section).
  • G a single enlarged cells showing SERK expression is detectable within a row of negative ceils showing the same mo ⁇ hology.
  • H a single enlarged cell showing serk expression is detaching from the surface of the proliferating mass.
  • I a cluster of enlarged cells showing SERK expression is detectable at the surface of proliferating tissue.
  • FIG. 4 shows the phenotype of Arabidopsis WS plants transformed with the 2200 bp SERK- lucrferase consturct at the seedling level. Pictures were taken at 28 days after germination of T2 seeds. In plant II and III no clear shoot me ⁇ stem is visible at the seedling stage, 7 days after ger ⁇ mination. The first two leaves, if they develop at all, are needleshaped as hown on the pictures taken 28 days after germination.
  • plant I which shows no clear phenotype, already starts flowe ⁇ ng.
  • Secondary shoot me ⁇ stems are already developing in plant no II and will also develop later from no III.
  • Shoot me ⁇ stems, influorescences and normal flowers eventually develop on all plants.
  • Figure 5 shows how the 2200 bp SERK luciferase construct affects the number of developing ovules in the siliques of transformed plants.
  • Figure 6 shows autophosphorylation of purified SERK fusion protein in vrtm.
  • Lane 1 purified SERK fusion protein
  • Lane 2 se ⁇ ne phosphate
  • Lane 3 threonine phosphate
  • Lane 4 thyrosine phosphate.
  • the following desc ⁇ ption illustrates the isolation and cloning of the SERK gene and the production of apomictic seed by heterologous expression of the said gene in the nucellar region of the ca ⁇ el so that somatic embryos form which penetrate the embryo sac and are encapsulated by the seed as it develops.
  • RT-PCR (Liang and Pardee, 1992) were used besides conventional differential screening of cDNA libraries.
  • Labeled probes for differential screening were obtained from RNA out of a ⁇ 30 mm sieved sub-population of cells from either embryogenic or non-embryogenic cell cultures. Employing these probes in a library screen of approximately 2000 plaques yielded 26 plaques that failed to show any hybridization to either probe. These so-called cold plaques were purified and used for further analysis. From the total number of plaques that did hybridize, about 30 did so only with the probe from embryogenic cells.
  • ddRT-PCR reactions using a combination of one anchor primer and one decamer primer were performed on mRNA isolated from three embryogenic, and three non-embryogenic suspension cultures. About 50 different ddRT-PCR fragments were obtained from each reaction.
  • LTP Lipid Transfer Protein
  • cDNA clone 31-50 encodes a leucine-rich repeat containing receptor-like kinase
  • the mRNA corresponding to the isolated clone 31-50 had an open reading frame of 1659 nucleotides encoding a protein with a calculated Mw of 55 kDa. Because clone 31-50 is mainly expressed in embryogenic cell cultures it was renamed Somatic Embryogenesis Receptor Kinase (SERK).
  • the SERK protein contains a N-terminal domain with a five-times repeated leucine-rich motif that is proposed to act as a protein-binding region in LRR receptor kinases (Kobe and Deisenhofer, 1994). Between the extracellular LRR domain of SERK and the membrane-spanning region is a 33 amino acid region rich in proiines (13), that is unique for the SERK protein.
  • SPPPP sequence conserved in extensins, a class of universal plant cell wall proteins (Varner and Lin, 1989).
  • the proposed intracellular domain of the protein contains the 11 subdomains characteristic of the catalytic core of protein kinases.
  • the core sequences HRDVKAAN and GTLGYIAPE in respectively the kinase subdomains VB and VIII suggest a function as a serine / threonine kinase (Hanks et al. 1988).
  • Another interesting feature of the intracellular part of the SERK protein is that the C-terminal 24 amino-acids resembles a single LRR.
  • the serine and threonine residues present within the intracellular LRR sequence are surrounded by acidic residues and might be targets for the autophosphorylation of SERK, thereby regulating the ability of other proteins to interact with this receptor-kinase in a similar fashion as described for the SH2 domain of the EGF family of tyrosine receptor kinases.
  • Hybridization of the SERK cDNA clone to the carrot genome revealed the presence of only a single main hybridizing band after digestion with EcoRI , probably reflecting a single SERK gene in the carrot genome. This was confirmed after digestion with Ddel, an enzyme that cuts three times within the SERK gene. No signal was observed after Northern blotting of mRNA from embryogenic cell cultures and hybridization with labeled SERK probes, reflecting the low levels of transcript present in these cultures. Detection of the SERK transcript on the original spot-dot Northerns was only possible after long exposure times compared with other probes.
  • the ability of the SERK protein to autophosphorylate was investigated in vitm, using a previously described autophosphorylation assay (Mu et al. 1994), with a bacterial fusion protein that contained the complete intracellular region of the SERK protein.
  • the bacterially expressed SERK fusion protein was able to autophosphorylate, indicating that the SERK protein is able to fulfill a role as a protein kinase in vivo (Heldin, 1995).
  • SERK gene corresponds with the first appearance of competent cells during hypocotyl activation
  • the shape of the enlarging and fully enlarged cells could change from oval to elongate or t ⁇ angular.
  • Cell tracking on a total of 24,722 cells released from seven days activated hypocotyls showed that only 20 single cells formed a somatic embryo. Because of their dependance on continued 2,4-D treatment, the embryo- forming single ceils are still in the competent cell stage. All of the embryo-forming single cells belonged to the category of 3,511 enlarged cells, that contained therefore competent cells in a frequency of 0.56%.
  • hypocotyls treated for ten days with 2,4-D the number of SERK-positive cells had increased to 3.04% and included at this stage also ceils present in small clusters. No SERK transcript was ever detected in small cytoplasm-rich cells or large vacuolated cells. Hypocotyls were also treated for only one day with 2,4-D and subsequently cultured in hormone-free medium for a total of seven or ten days. Under these conditions explant celts proliferated and gave rise to roots and non-embryogenic cell cultures, while SERK expression could never be detected.
  • SERK gene corresponds with the occurrence of competent cells in established embryogenic cell cultures
  • SERK expression is not restricted to competent single cells, but may persist in small clusters of embryogenic cells. No SERK expression was encountered during the late globular, heart and to ⁇ edo-stages of somatic embryogenesis.
  • the SERK gene is transiently expressed in zygotic embryogenesis
  • the expression of the SERK gene in carrot plants was determined by RT-PCR. The results indicate that no SERK mRNA accumulates in any of the adult plant organs nor in flowers prior to pollination.
  • the first occasion when SERK expression can be detected is in flowers at three days after pollination (DAP), at which stage fertilization has taken place and endosperm development has commenced.
  • DAP days after pollination
  • SERK mRNA remains present in flowers up to twenty DAP, corresponding with the eariy globular stage of the zygotic embryo (Yeung et al. 1996).
  • Whole mount in situ hybridization on partially dissected carrot seeds confirmed that the SERK gene was only expressed in eariy embryos up to the globular stage. Expression was observed in the entire embryo including the suspensor.
  • Cell cultures were derived from Daucus camta cv. Flakkese and maintained as previously described (De Vries ef a/. 1988a). Cell suspension cultures were maintained at high cell density in B5 medium (Gamborg et al. 1968) supplemented with 2 mM 2,4-D (B5-2 medium). Embryo cultures with globular, heart and to ⁇ edo-stage somatic embryos were derived from ⁇ 30 mm sieved cell cultures cultured at low cell density (100 000 cells / ml) in B5 medium without 2,4-D (B5-0). For hypocotyl explant induction experiments, plantlets were obtained from seed of Daucus camta cv.
  • BTL hybridot manifold
  • Genomic DNA was isolated according to Sterk et al. (1991). Samples of 10 mg genomic DNA were digested with different restriction enzymes and separated on agarose gel, and transferred to nytran-plus membrane (Schleicher & Schuell). Hybridization of RNA blots took place at 42°C in hybridization buffer containing 50% formamide, 6xSSC, 5xDenhardt, 0.5% SDS and 0.1 mg/ml salm sperm DNA. Hybridization of DNA blots was performed as previously described (Sterk et al. 1991). Following hybridization, filters were washed under stringent conditions (3x20 min in 0.1% SSC, 1% SDS, at 65°C).
  • RNA Filters were exposed to Kodak X-Omat AR film. The integrity and the amount of RNA on the blots was confirmed by hybridization with an 18S ribosomal RNA probe. Nucleotide sequence analysis was performed on an ABI 373A automated DNA sequencer (Applied Biosystem).
  • cDNA libraries Two independent cDNA libraries were constructed with equal amounts of poly(A) * -RNA from total established cell cultures grown for six days in B5-2 medium, sieved ⁇ 125 mm cell cultures grown for six days in B5-0 medium and sieved ⁇ 30 mm cell cultures grown for six days in B5-0 medium.
  • cDNA synthesis and cloning into the Uni-ZAPTM XR vector was performed according to the manufacturers protocol (Stratagene).
  • [ PJdATP labeled probes were prepared using random prime labeling on first strand cDNA. Pooled probes from embryogenic and non-embryogenic cell populations were hybridized to two pairs of nitrocellulose filters, each containing 1000 plaques from one cDNA library.
  • cDNA synthesis took place by annealing 1 mg of total RNA in 10 ml buffer containing 200 mM KCl, 10 mM Tris-HCl (pH 8.3), and 1 mM EDTA with 100 ng of one of the following anchor primers: (5'- l 1 1 I I I I I I I GC-3'), (5'- l I I I I I I I I CTG-3'), (5'- l I I I I t i l l I I CA-3'). Annealing took place by heating the mix for 3 min. at 83°C followed by incubation for 30 min at 42°C.
  • Annealing was followed by the addition of 15 ml pre-warmed cDNA buffer containing 16 mM MgCI 2 , 24 mM Tris-HCl (pH 8.3), 8 mM DTT, 400 mM dNTP, and 4 Units AMV reverse transcriptase (Gibco BRL).
  • cDNA synthesis took place at 42°C for 90 min.
  • First strand cDNA was phenol/chlorophorm extracted and precipitated with ethanol using glycogen as a carrier.
  • the PCR reaction was performed in a reaction volume of 20 ml containing 10% of the synthesized cDNA, 100 ng of anchor primer, 20 ng of one of the following 10-mer primers: (5- GGGATCTAAG-3') , (5'-TCAGCACAGG-3'), (5'-GACATCGTCC-3') ( ( ⁇ '-CCCTACTGGT-S'), (5'- ACACGTGGTC-3'), (5'-GGTGACTGTC-3'), 2 mM dNTP, 0.5 UnrtTag enzyme in PCR buffer (10 mM T ⁇ s-HCI (pH 9.0), 1.5 mM MgCI 2 , 50 mM KCl, 0.01% gelatin and 0.1% T ⁇ ton X100) and 6 nM [a- 32 ?] dATP (Amersham).
  • PCR parameters were 94°C for 30 sec, 40°C for 1 mm, and 72°C for 30 sec for 40 cycles using a Cetus 9600 (Perkin-Elmer).
  • Amplified and labeled cDNAs were separated on a 6% denatu ⁇ ng DNA sequencing gel. Gels were d ⁇ ed without fixation and bands were visualized by 16 hours of autoradiography using Kodak X-omatic film. Bands containing differentially expressed cDNA fragments of 150-450 nucleotides were cut out of the gel and DNA was extracted from the gel slices by eiectroelution onto DE-81 paper (Whatmann).
  • Amplified PCR products were blunt-ended using the Klenow fragment of E.coli DNA Polymerase I (Pharmacia), purified on Sephacryl-S200 columns (Pharmacia), ligated into a Smal linearized pBluesc ⁇ pt vector II SK " (Stratagene) and transformed into E coli using electroporation.
  • RNA from Daucus camta were obtained from S&G Seeds (Enkhuizen). Controlled pollination was performed by hand.
  • Flower tissue RNA was obtained from three compete umbels for each time-point and contained all flower organs including pollen grains. 2 mg of total RNA from adult plant tissue or cell cultures was annealed at 42°C with 50 ng oligo (5'- TCTTGGACCAGATAATTC-3') in 10 ml annealing buffer (250 mM KCl, 10 mM Tris-HCl pH 8.3, 1 mM EDTA). After 30 mm.
  • Optimal induction was achieved with longitudinal hypocotyl sections with a thickness of at least 90 mm.
  • hypocotyl sections were exposed to 2,4-D for onty 1 day, and subsequently transferred to B5-0 medium (Guzzo et al 1994).
  • Whole mount in situ hyb ⁇ dization on developing seeds was performed by removing the chalazal end of the seeds to allow easier probe penetration
  • hyb ⁇ dization the enveloping layers of integuments and endosperm were carefully removed to expose the developing embryos, tn situ hyb ⁇ dization on sections was performed as desc ⁇ bed previously (Sterk et al. 1991) except for the use of non- radioactive probes.
  • Hyb ⁇ dization solution consisted of PBS containing 0.1% Tween 20, 330 mM NaCl, 50 mg/ml hepa ⁇ n, and 50% deionized formamide. Hyb ⁇ dization took place for 16 hours at 42°C using digoxigenm-labeled sense or antisense ⁇ boprobes (Boeh ⁇ nger Mannheim).
  • a 1.4 kB Sspl cDNA fragment of the SERK cDNA encoding most of the open reading frame apart trom the N-terminal three LRRs was cloned into the pGEX expression vector (Pharmacia).
  • a fusion protein consisting of SERK and the glutathione S-transferase gene product was synthesized by a three hours induction of transformed E.coli with 2 mM IPTG. Fusion protein was isolated and purified as described previously (Horn and Walker, 1994). Purified fusion protein was coupled to glutathione agarose beads (Sigma) and incubated for 20 min.
  • the pAcHLT-B and pAcHLT-C baculovirus transfer vectors were used for the cloning of two cDNA fragments of the carrot SERK gene.
  • the Sspl 1.41 kB fragment of carrot DcSERK cDNA was cloned into the Smal site of pAcHLT-B and the Sspl / Pvull 1.07 kB fragment of carrot DcSERK cDNA was cloned into the Smal site of pAcHLT-C.
  • the first construct contains the complete C-terminal part of the DcSERK protein and from the putative extracellular region the proline-rich region and three of the lecuine-rich repeats.
  • the second construct contains only the putative intracellular region of the DcSERK gene product. Nucleotide sequence analysis was performed in order to confirm the presence and the orientation of the DcSERK cDNA within the vector.
  • the resulting transfer vectors were used to transfect (lipofect) insect cell culture Sf21 from Spodoptera frugiperda in combination with linearized AcMNPV baculovirus DNA.
  • Monolayers of SF21 ceils were transfected in 35 mm petridishes containing 2 ml of Hink's medium.
  • One microgram of linearized AcMNPV baculovirus DNA (Baculogold, Invitrogen) was added to 5 microgram of pAcHLT / SERK vector construct in 25 microliter of water.
  • Fifteen microliter of Lipofectin (BRL) was mixed with 10 microliter of water, after which the DNA solution was added.
  • Hink's medium After mixing 200 microliter of Hink's medium was added to the mix and the solution was transferred to the cell monolayer, from which the medium was removed. After one hour, 500 microliter of Hink's medium was added and the cells were incubated for anotehr 3 hours. Finally, 1 ml of Hink's medium with 20% foetal bovine serum (FBS) was added and the cells were incubated for 4 days. After transfection, the viral infection could be identified by the reduced growth of cells, the swollen shape and the enlarged nucleus. After four days, infected cells were harvested and the medium containing infectious budded virus was collected and used for plaque assays and amplification of recombinant virus stocks.
  • FBS foetal bovine serum
  • Single recombinant virus plaques were isolated from monolayers of cells infected with a titration range of the primairy virus stock. Infections was performed in 35 mm petridishes with monolayers of cells. Virus stocks were diluted in 600 microlieter of Graces medium and added to the cell monolayer, followed by a 90 minutes incubation period at in Graces medium with 20% FBS. Afterwards, 3% Sea Plaque agarose was autoclaved, mixed with an equal amount of 2x Graces medium with 20% FBS and from the resulting agarose overlay solution 2 ml. was spread over the cell monolayers after removal of the viral inoculum. After 4 days of incubation single plaques could be visulalized and purified for further analysis.
  • Cells were lysed for 45 min on ice in twenty volumes of 1x insect cell lysis buffer (10 mM Tris pH 7.5, 130 mM NaCl, 1% Triton, 100 mM NaF, 10 mM NaPi, 10 mM NaPPi, with proteinase inhibitors: 16 mg/l benzamidine, 10 mg/l phenanthroline, 10 mg/l aprotinin, 10 mg/l leupeptin, 10 mg/l pepstatin A, 1 mM PMSF).
  • 1x insect cell lysis buffer 10 mM Tris pH 7.5, 130 mM NaCl, 1% Triton, 100 mM NaF, 10 mM NaPi, 10 mM NaPPi, with proteinase inhibitors: 16 mg/l benzamidine, 10 mg/l phenanthroline, 10 mg/l aprotinin, 10 mg/l leupeptin, 10 mg/l pepstatin A, 1 mM PMSF).
  • the lysate was cleared by centrifugation at 10.000 g for 30 min and the supernatant was batchwise incubated in TALON resin (with high affinity for the 6xHIS tag of the recombinant fusion protein). Binding was performed by gentle agitation for 20 min. at room temp. The resin was washed three times with lysis buffer, followed by an elution step with lysis buffer with 200 mM imidazole. Purified fusion protein was collected and purifty and integrity was tested by SDS-PAGE.
  • Protein kinase activity was detemined by incubating 1 microgram of purified fusion protein for 30 min. at room temp, in a buffer containing 10 mM MgCI2, 10 mM MnCI2, 1 mM DTT and 10 ⁇ M [gamma-32]ATP (105 pm/pmol ATP).
  • the autophosphorylated fusion protein was purified after SDS-PAGE from the gel in a buffer containing 50 mM NH4C03, 0.1 % SDS, 0.25% beta-mercaptoethanol. Protein was precipitated with 20 ⁇ g/ml BSA and 20% (w/v) solid trichloroacetic acid.
  • the precipitate was collected after centrifugation, hydrolysed in 50 ⁇ l 6N HCI for 1 hour at 120 degrees Celcius. HCI was subsequently removed by lyophilization and the pellet was resuspended in a buffer consiting of 2.2% formic acid and 7.8% acetic acid. Hydrolysed protein was loaded onto cellulose thin layer chromatography plates together with control amino acid samples (phosphoserine, phosphothreonine, phosphotyrosine). Chromatography was performed in a buffer containing propionic acid: 1M ammonium hydroxide: isopropyl alcohol (90:35:35 v/v/v).
  • the separated amino-acids were visualized by spraying with 0.25% ninhydrin in aceton, followed by heating for 5 min. at 65 degrees Celcius. Plates were afterwards exposed to Phospho Imager casettes in order to detect the phospho-labeled aminoacids.
  • fusion proteins (10 ⁇ g) were mixed in complete Freund adjuvant and injected IP into BALBc mice. After 4 weeks booster antigen was injected (10 ⁇ g purified fusion protein in imcomplete Freund adjuvant). Two weeks later a final booster was injected. One week after the final booster, serum was collected from these mice. The specificity and the titer of the resulting sera was tested on Western blots using total insect cell extracts with or without the SERK fusion proteins. INTRODUCTION OF THE SERK GENE INTO PLANTA AND THE PRODUCTION OF APOMICTIC SEED
  • the binary vector pMT500 is based on the pBIN19 vector (Bevan, 1984) and contains the firefly luciferase gene downstream of a polylinker containing 5 unique restriction sites was created by uni-directional ligation of the firefly luciferase coding region followed by the polyadenylation sequence from the pea rbcS::E9 gene in the H/ ⁇ dlll-Xoal site of the binary vector pMOG ⁇ OO (kindly provided by Mogen N.V., Leiden, The Netherlands).
  • the binary vector pMOG ⁇ OO is based upon pBIN19 (Bevan, 1984) but while in pBIN19 the polylinker is flanked by the left border and the neomycin phosphotransferase (NPT II) expression cassette, the polylinker in pMOG800 is flanked by the right border and the NPT II expression cassette.
  • NPT II neomycin phosphotransferase
  • a Kpnl / Sstl fragment containing the SERK genomic DNA was isolated and cloned into the Kpnl / Sstl sites of the binary vector pMT500.
  • the resulting DNA construct, pMT531 contained the 2200 bp genomic SERK DNA fragment as promoter sequence, the luciferase gene as vital reporter, and the E9 transcription terminator sequence.
  • the binary vector pMT531 was transformed by electroporation into Agmbacterium tumefaciences strains MOG101 and MOG301 (for transformation into carrot cells) and into Agmbacterium tumefaciences strain C58C1 (for transformation into Arabidipsis thaliana plants). Transformed colonies were selected on LB plates with 100mg/l kanamycin.
  • the firefly luciferase coding sequence under control of the genomic carrot Hindlll / Dral 2200 bp DNA fragment was introduced into carrot cells by Agmbacterium tumefaciens mediated transformation of hypocotyl segments. Transformation of Daucus camta cv. 'Amsterdamse bak' was performed by slicing one week old dark grown seedlings into segments of 10 to 20 mm. Segments were incubated for 20 minutes in a freshly prepared 10 fold diluted overnight culture of Agmbacterium..
  • the segments were dried and transferred to a modified Gamborgs B5 medium (P1 medium; S&G seeds, Enkhuizen, The Netherlands) supplemented with 2 ⁇ M 2,4-D (P1-2) and solidified with agar (Difco, Detroit, Mi, USA ). After two days of culture in the dark at 25 ⁇ 0.5 _C, segments were transferred to solidified P1-2 medium supplemented with kanamycin (100 mg -I "1 ), carbenicillin (500 mg-l" 1 ; Duchefa) and vancomycin (100 mg l "1 ; Duchefa). After three weeks segments were transferred to fresh plates and transformed calli were selected after an additional three weeks.
  • Transformed calli were grown on P1-2 plates with antibiotics for 3 weeks at a 16 hour light/8 hour darkness regime.
  • Transformed embryogenic suspension cultures were initiated as described by transferring 0.2 g callus to 10 ml liquid P1-2 medium supplemented with 200 mg -l" 1 kanamycin, 250 mg-l" 1 carbenicillin and 50 mg-l "1 vancomycin.
  • 1 to 3 volumes of fresh medium were added to the culture at weekly intervals.
  • cultures were subcultured to a packed cell volume of 2 ml per 50 ml medium every two weeks and incubated at a 16 hour light / 8 hour darkness regime at 25 ⁇ 0.5 °C.
  • hypocotyl segments were sprayed with lucife ⁇ n to test whether luciferase expression could be detected in transformed callus shortly after transformation.
  • a large number of hypocotyl segments showed luciferase activity at the cut edges, but did not develop calli. Instead, growth of bacteria occurred, suggesting that the luciferase activity was of bactenal origin.
  • calli were obtained that showed luciferase activity in variable amounts, while no bacterial growth could be observed anymore.
  • calli measuring 5 to 10 mm in diameter were used to start suspension cultures. At this time no bactenal contamination was observed.
  • a control transformation experiment in which luciferase expression under influence of the CaMV 35S promoter was observed in single cells and cell clusters in the suspension culture demonstrating that the luciferase protein is active in Daucus camta suspension cultured cells.
  • the bottom layer consisted of 1 ml P1-0 medium with 5 mM Ca 2 + and 0.2 % phytagel. Two hundred thousand cells ( ⁇ 30 ⁇ m and ⁇ 50 ⁇ m populations) in B5-0 medium without Ca 2+ supplemented with 0.1 % phytagel were poured on top of the bottom layer. For this layer B5 was applied since, at room temperature, phytagel solidified in P1 medium without Ca 2+ . After 2 hours of solidification an additional P1-0 layer with 0.2 % phytagel was poured onto the cell layer preventing the B5 layer to move.
  • luciferin detection on single cells was determined with a CCD camera for a period of 5 times one hour (Schmidt et al. (1997) Development 124: 2049-2062). After 7 days of culture, luciferin was removed from the cultures by extensive washing with P1 -0 medium.
  • Wildtype WS plants were grown under standard long day conditions: 16 hours light and 8 hours dark.
  • the first emerging mfluorescense was removed in order to increase the number of mfluorescenses. Five days later, plants were ready for vacuum infiltration.
  • Agrobacterium strain C58C1 containing the transformation plasmid was grown on a LB plate with 50 mg/l kanamycin, 50 mg/l rifampicin and 25 mg/l gentamycin.
  • a single colony was used to inoculate 500 ml of LB medium containing 50 mg/l kanamycin, 50 mg/l rifampicin and 25 mg/l gentamycin.
  • the cultures were grown O/N at 28 degrees Celcius and the resulting log phase culture (OD6000.8) was centrifuged to pellet the cells and resuspended in 150 ml of infiltration medium (0.5x MS medium (pH 5.7) with 5% sucrose and 10 ⁇ l/l benzylaminopu ⁇ ne).
  • the inflorescenses of 6 Arabidopsis plants are submerged in the infiltration suspension while he remaining parts of the plants (which are still potted) are placed upside down on meshed wire to avoid contact with the infiltration suspension.
  • Vacuum is applied to the whole set-up for 10 min. at 50 kPa. Plants are directly afterwards placed under standard long day conditions. After completed seed setting the seeds were surface sterilized by a 1% sodium hypochlorite soak, then thoroughly washed with sterile water and plated onto petridishes with O. ⁇ xMS medium and 80 mg/l kanamycin in order to select for transformed seeds. After 5 days germination under long day conditions (10.000 lux), the transformed seedlings could be identified by their green color of their cotyledons (the untransformed seedlings turn yellow), and were further grown in soil under C1 lab conditions under long day conditions. This vacuum infiltration method resulted in approximately 0.1% transformed seeds.
  • a T2 generation could be obtained from the four plants I, II, III and V ( Figure 4). Within the siliques of the T2 generation of plants no. Ill and V, an early inhibition in development could be observed in appoximatetely 25-50 % of the seeds. The plants I and II did not show a reduction in the number of developing seeds.( Figure 5). Similar results were observed in a T3 generation, in which again approximately 25-50% of the seeds showed an early inhibition of normal seed development.
  • a lambda ZipLox genomic library made form Arabidopsis Landsberg erecta total genomic DNA is screened for the presence of homologous sequences.
  • Three different lambda clones with inserts of 14, 18 and 20 kb respectively are obtained.
  • the 14 kb clone is digested by EcoRI and the resulting fragments subcloned into pBluescript vectors. Fragments spanning the entire coding sequence of the AtSERK gene are isolated, sequenced and compared with the Daucus homologues. The resulting sequence is shown as SEQ ID NO: 20.
  • a lambda ZAPII cDNA library is screened tor the presence of homologous sequences.
  • Four lambda clones are obtained and their inserts subcloned into pBluescript vectors using the helper phage excision procedure. Fragments spanning the entire AtSERK cDNA coding sequence of the AtSERK gene are isolated, sequenced and compared with the Daucus homologues. The resulting sequence is shown as SEQ ID NO: 32. Plasmids containing promoter sequences
  • Arabidopsis thaliana LTP1 promoter fragment is obtained from the binary plasmid pUHIOOO (Thoma, S., Hecht, U., Kipper, A., Borella, J., De Vries, S.C., Sommerville, C. (1994) Plant Physiol. 105, 35-45) by digestion with BamHI and H/ndlll and cloning into pBluescript SK " (pMT121).
  • the CaMV 35S promoter enhanced by duplication of the -343 to -90 region is isolated from the pMON999 vector by digestion with W/ndlll and Sstl and cloned into the pBluescript SK " vector (pMT120).
  • Plasmid SLJ 9691 is a construct consisting of pBluescript SK+ in which the Arabidopsis thaliana DMC1 genomic clone (accession number U76670) is cloned into the EcoRV site.
  • SLJ 9691 carries EcoRV fragments of the 5' end of the AtDMCI gene with the following modification: a Bglll site instead of the second Hpal site, two ATG codons in the first exon and an Xhol site at the ATG codon of the second exon.
  • the promoter of the FBP7 gene is cloned by subcioning the 0.6 kb H/ndlll - Xbal genomic DNA fragment of FBP7 into the H/ ⁇ dlll - Xbal site of pBluescript KS-, resulting in the vector FBP201.
  • pAtSERK is constructed for transformation of the Arabidopsis thaliana SERK cDNA under the control of different promoters.
  • the full length Arabidopsis thaliana cDNA clone of SERK (Seq ID No. NEW) is obtained from a pBluescript SK- plasmid.
  • a Smal - Kpnl 2.1 kb fragment containing the AtSERK cDNA is cloned into pBIN19 Smal - Kpnl.
  • the polyadenylation sequence from the pea rbcS::E9 gene (Millar et al., 1992), Plant Cell 4: 1075-1087) is placed downstream from the AtSERK cDNA by cloning a Klenow-filled EcoRI - Hindlll E9 DNA fragment into the Klenow- filled Xmal site of the pBIN19:AtSERK vector in order to generate the binary vector pAtSERK. Construction of plant expression vectors
  • the pAtSERK binary vector is used to generate the following promoter-AtSERK constructs.
  • the AtLTPI promoter is cloned in the Smal site of the pAtSERK binary vector as a Klenow-filled Kpnl-Ss ⁇ * DNA fragment to give the pAtLTPI AtSERK vector.
  • the CaMV 35S promoter is cloned in the Smal site of the pAtSERK binary vector as a Klenow-filled Kp ⁇ l-Ssfl fragment to give the p35SAtSERK vector.
  • AtDMCI promoter consisting of the Bglll - Xhol 3.3kB fragment from the clone SLJ 9691 is filled in with Klenow and cloned into the Smal site of the pAtSERK binary vector to give the pAtDMCl AtSERK vector.
  • a Sacl-Kpnl fragment of FBP2101 is filled in with Klenow and cloned into the Smal site of the pAtSERK binary vector to give the pFBP2101 AtSERK vector.
  • Wild type Arabidopsis thaliana WS plants are grown under standard long day conditions: 16 hours light and 8 hours dark.
  • the first emerging inflorescence is removed in order to increase the number of influorescences. Five days later, plants are ready for vacuum infiltration.
  • Agrobacterium strain C58C1 containing the transformation plasmid (the pAtLTPI AtSERK vector or the p35SAtSERK or the pAtDMCl AtSERK vector or the pFBP2101 AtSERK vector) is grown on a LB plate with 50 mg/l kanamycin, 50 mg/l rifampicin and 25 mg/l gentamycin.
  • a single colony is used to inoculate 500 ml of LB medium containing 50 mg/l kanamycin, 50 mg/l rifampicin and 25 mg/l gentamycin.
  • the cultures are grown O/N at 28 degrees Celsius and the resulting log phase culture (OD6000.8) is centrifuged to pellet the cells and resuspended in 150 ml of infiltration medium (0.5x MS medium (pH 5.7) with 5% sucrose and 1 mg/l benzylaminopurine).
  • infiltration medium 0.5x MS medium (pH 5.7) with 5% sucrose and 1 mg/l benzylaminopurine.
  • the inflorescences of 6 Arabidopsis plants are submerged in the infiltration suspension while the remaining parts of the plants (which are still potted) are placed upside down on meshed wire to avoid contact with the infiltration suspension. Vacuum is applied to the whole set-up for 10 mm. at 50 kPa. Plants are directly afterwards placed under standard long day conditions.
  • the seeds are surface sterilized by a 1% sodium hypochlo ⁇ te soak, then thoroughly ished with ste ⁇ le water and plated onto petridishes with 0.5xMS medium and 80 mg/l kanamycin in order to select for transformed seeds.
  • the transformed seedlings could be identified by their green colour of their cotyledons (the untransformed seedlings turn yellow), and are further grown in soil under long day conditions. This vacuum infiltration method resulted in approximately 0.1% transformed seeds.
  • the inflorescences from transgenic and not transgenic Arabidopsis thaliana plants are analysed by Whole mount in situ hybridisation analysis with AtSERK cDNA as probe.
  • the inflorescences in different stages of development are fixed for 60 mm. in PBS containing 70 mM EGTA, 4% paraformaldehyde, 0.25% glutaraldehyde, 0.1% Tween 20, and 10% DMSO. Samples are then washed, treated with protemase K for 10 mm, again washed and fixed a second time.
  • Hyb ⁇ disation solution consisted of PBS containing 0 1% Tween 20, 330 mM NaCl, 50 mg/ml hepa ⁇ n, and 50% deionized formamide.
  • Hyb ⁇ disation took place for 16 hours at 42°C using digoxigenm-labeled sense or antisense ⁇ boprobes (Boeh ⁇ nger Mannheim). After washing, the cells are treated with RNaseA and incubated with anti-digoxigenin-alkalme phosphatase conjugate (Boeh ⁇ nger Mannheim) which had been preabsorbed with a plant protein extract. Excess antibody is removed by washing followed by ⁇ nsing in staining buffer (100 mM T ⁇ s-HCI pH 9.5, 100 mM NaCl, 5 mM MgCI 2 , 1 mM levamisole) and the staining reaction is performed for 16 hours in a buffer containing NBT and BCIP. Observations are performed using a Nikon Optiphot microscope equipped with Nomarski optics. The transformed plants show ectopic expression of SERK in the vicinity of the embryo sac.
  • GrrATGACATT GTTGGGGATC CCTATCACTG GATTCCTGTT TTGCTGACCC TCTGTTCAAT 3900
  • GCT GCA AAT ATA TTA TTG GAC GAA GAA TTT GAG GCT GTT GTA GGT GAT 1218 Ala Ala Asn He Leu Leu Asp Glu Glu Phe Glu Ala Val Val Gly Asp 360 365 370 375
  • GCT GTA AGG GGT ACC TTG GGC TAC ATA
  • GCT CCC GAG TAC CTC TCG ACT 1314 Ala Val Arg Gly Thr Leu Gly Tyr He Ala Pro Glu Tyr Leu Ser Thr 395 400 405
  • GAG AAA AAG TTG GAG ATG CTG GTC GAT CCT GAC CTG GAG AAC AAT TAC 1506 Glu Lys Lys Leu Glu Met Leu Val Asp Pro Asp Leu Glu Asn Asn Tyr 460 465 470
  • MOLECULE TYPE DNA (genomic)
  • AAAAAAATTC ACATTTTTGG TATGAGATTG CrCACATGAT AGTGAACCTC TTTAACATTT 480
  • GCTITGCATA CTTTGAGGGT TACTCTACTT GATCCAAACA ATGTCTTGCA GAGCTGGGAT 1860
  • CTGGTCTCAA TTGAGAACTT D3AGGTCTGT ATCTAAAATT TCTAAATGCG ATTTTCGCCT 967
  • CAC ATT CCT TTA CAG AAC TTT GAG AAC AAC CCG AGG TTG GAG GGA CCG 622 His He Pro Leu Gin Asn Phe Glu Asn Asn Pro Arg Leu Glu Gly Pro 195 200 205

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Abstract

La présente invention concerne, entre autres, un procédé pour produire des graines apomictiques. Ce procédé comprend les étapes consistant à (i) transformer le matériau de la plante à l'aide d'une séquence de nucléotides codant une protéine dont la présence dans une cellule, ou une membrane de celle-ci rend la cellule embryogène, (ii) regénérer le matériau ainsi transformé en plantes, ou en carpelle contenant des pièces de ces dernières, et (iii) exprimer la séquence au voisinage du sac de l'embryon. La protéine peut être une kinase de type récepteur à unité de répétiion riche en leucine, qui est, de préférence, modifiée dans la mesure ou le domaine de liaison au ligand est effacé ou fonctionnellement inactivé.
EP97923882A 1996-05-14 1997-05-13 Production de graines apomictiques Withdrawn EP0915984A1 (fr)

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GBGB9610044.1A GB9610044D0 (en) 1996-05-14 1996-05-14 Improvements in or relating to organic compounds
GB9610044 1996-05-14
PCT/EP1997/002443 WO1997043427A1 (fr) 1996-05-14 1997-05-13 Production de graines apomictiques

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JP (1) JP2000510342A (fr)
KR (1) KR20000011136A (fr)
CN (1) CN1218510A (fr)
AR (1) AR007130A1 (fr)
AU (1) AU713130B2 (fr)
BR (1) BR9709098A (fr)
CA (1) CA2254839A1 (fr)
GB (1) GB9610044D0 (fr)
HU (1) HUP9901477A3 (fr)
IL (1) IL126490A0 (fr)
PL (1) PL329872A1 (fr)
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WO (1) WO1997043427A1 (fr)

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WO1998008961A2 (fr) * 1996-08-30 1998-03-05 Olsen Odd Arne Genes specifiques de l'endosperme et du nucelle, leurs promoteurs et leurs utilisations
GB9626858D0 (en) * 1996-12-24 1997-02-12 Gatsby Plant Science Innovatio Transcriptional regulation in plants
US6452068B1 (en) 1998-01-28 2002-09-17 The Rockefeller University Chemical inducible promoters used to obtain transgenic plants with a silent marker
US6262228B1 (en) * 1998-08-17 2001-07-17 Tularik Inc. IRAK3 polypeptides and methods
GB9823098D0 (en) * 1998-10-22 1998-12-16 Novartis Ag Organic compounds
EP1057891A1 (fr) * 1999-06-02 2000-12-06 Centrum Voor Plantenveredelings- En Reproduktieonderzoek Utilisation de l'activateur transcriptionel BNM3 pour contrôler l'embryogenèse et les procédés de régénération de plantes
EP1094113A1 (fr) * 1999-10-22 2001-04-25 Genetwister Technologies B.V. Régénération
AU2001237421B2 (en) 2000-03-02 2006-06-22 Limagrain Europe Embryo sac-specific genes
WO2002083912A2 (fr) * 2001-04-10 2002-10-24 Syngenta Participations Ag Apomixie inductible
US7264964B2 (en) 2001-06-22 2007-09-04 Ceres, Inc. Chimeric histone acetyltransferase polypeptides
EP1382682A3 (fr) * 2002-07-17 2004-06-30 Expressive Research B.V. Modulation des voies de développement des plantes
JP4062356B2 (ja) * 2004-04-16 2008-03-19 治 北島 フリンジ咲きの花を有するニチニチソウおよびその育種方法
EP1621629A1 (fr) * 2004-07-28 2006-02-01 Expressive Research B.V. Procédé augmentant la résistance aux pathogènes dans les plantes
CN1939910A (zh) 2004-12-31 2007-04-04 孙飘扬 氨基嘧啶类化合物及其盐和其制备方法与药物用途
WO2006094774A2 (fr) * 2005-03-03 2006-09-14 Rijk Zwaan Zaadteelt En Zaadhandel B.V. Mise en correspondance de descendance inverse
AU2006222150B2 (en) 2005-03-03 2010-12-23 Rijk Zwaan Zaadteelt En Zaadhandel B.V. Near reverse breeding
US7550579B2 (en) 2005-04-29 2009-06-23 Pioneer Hi-Bred International, Inc. Pericarp-preferred regulatory element
EP2119786A1 (fr) 2008-05-13 2009-11-18 Expressive Research B.V. Production améliorée de composés améliorant la santé chez les plantes
NL2004624C2 (en) 2010-04-28 2011-11-01 Stichting Dienst Landbouwkundi A new glycosyltransferase protein and its role in the metabolism of phenylpropanoid volatiles in tomato.
CN103260394B (zh) * 2010-11-24 2016-08-24 纽海姆有限公司 双重目的的授粉西瓜
MX2014008243A (es) * 2012-01-06 2015-02-20 Pioner Hi Bred International Inc Un metodo para seleccionar plantas por elementos geneticos que inducen la partenogenesis en plantas.
CN103430831B (zh) * 2013-08-15 2014-08-13 黑龙江省农业科学院经济作物研究所 获得无融合生殖亚麻种子的方法
US20240018535A1 (en) * 2020-03-19 2024-01-18 Institute Of Genetics And Developmental Biology, Chinese Academy Of Sciences Method for improving plant genetic transformation and gene editing efficiency
WO2022186295A1 (fr) * 2021-03-02 2022-09-09 国立研究開発法人産業技術総合研究所 Procédé pour induire l'embryogenèse d'une plante de graine sans fécondation, protéine utilisée dans celui-ci, acide nucléique, vecteur et plante de graine recombinante dans laquelle un embryon peut être généré sans fécondation

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BR8807155A (pt) * 1987-07-31 1989-10-17 Maxell Hybrids Inc Inducao assexual de esterilidade masculina transmissivel e apomixia em plantas
US5811636A (en) * 1995-09-22 1998-09-22 The United States Of America As Represented By The Secretary Of Agriculture Apomixis for producing true-breeding plant progenies
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WO1997043427A1 (fr) 1997-11-20
CN1218510A (zh) 1999-06-02
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HUP9901477A3 (en) 2001-11-28
KR20000011136A (ko) 2000-02-25
PL329872A1 (en) 1999-04-12
GB9610044D0 (en) 1996-07-17
AU2953997A (en) 1997-12-05
HUP9901477A2 (hu) 1999-09-28
RU2223320C2 (ru) 2004-02-10
AU713130B2 (en) 1999-11-25
CA2254839A1 (fr) 1997-11-20
AR007130A1 (es) 1999-10-13
IL126490A0 (en) 1999-08-17
JP2000510342A (ja) 2000-08-15

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