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WO1994029465A1 - Procede de generation de plantes males steriles - Google Patents

Procede de generation de plantes males steriles Download PDF

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Publication number
WO1994029465A1
WO1994029465A1 PCT/EP1994/001840 EP9401840W WO9429465A1 WO 1994029465 A1 WO1994029465 A1 WO 1994029465A1 EP 9401840 W EP9401840 W EP 9401840W WO 9429465 A1 WO9429465 A1 WO 9429465A1
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WIPO (PCT)
Prior art keywords
plants
plant
gene
male
antisense
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PCT/EP1994/001840
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English (en)
Inventor
Robert Dirks
Klaus Trinks
Bert Uijtewaal
Klaus Bartsch
Roger Peeters
Rainer Höfgen
Hans-Dieter Pohlenz
Original Assignee
Nunhems Zaden B.V.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Priority claimed from EP93109226A external-priority patent/EP0628635A1/fr
Application filed by Nunhems Zaden B.V. filed Critical Nunhems Zaden B.V.
Priority to JP7501308A priority Critical patent/JPH08510916A/ja
Priority to AU69723/94A priority patent/AU701920B2/en
Priority to US08/556,944 priority patent/US6262339B1/en
Priority to EP94918390A priority patent/EP0701619A1/fr
Priority to PCT/EP1994/001840 priority patent/WO1994029465A1/fr
Priority to CA002164732A priority patent/CA2164732A1/fr
Publication of WO1994029465A1 publication Critical patent/WO1994029465A1/fr
Priority to NO954978A priority patent/NO954978L/no

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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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • 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
    • C12N15/8289—Male sterility

Definitions

  • Male sterility is a property that is highly recognized in plant breeding. Male sterility enables the combination of traits from two parental lines, with the ultimate goal to supress negative traits from either parent with the genes from the other parent, and to superimpose positive traits carried by both parents. This results in a vigour that equals or exceeds either parental line.
  • Several natural sources e.g. cytoplasmic sterility or systems based on nuclear male sterility are already being used for many years. In crops were natural sources of male sterility are not available or not suitable, e.g. in tomato, hand emasculation and manual hybridization is still being carried out.
  • the International Patent Application WO 90/08830 is directed to methods for the production of male sterile plants by the expression of either a gene encoding a protein inhibitor, or a so-called killer gene.
  • the expression of the genes in the male flowers leads to cell death of the anthers.
  • the International Patent Application WO 90/08831 teaches a method for the inhibition of cell-respiration by expression of a disrupter gene.
  • disrupter proteins are the mammalian uncoupling protein (UCP), a mutated form of the gene for the ⁇ -1 subunit of F ⁇ ATPase, and a mutated, synthetic form of the olil gene encoding subunit 9 of the F 0 -ATPase.
  • UCP mammalian uncoupling protein
  • a mutated form of the gene for the ⁇ -1 subunit of F ⁇ ATPase a mutated, synthetic form of the olil gene encoding subunit 9 of the F 0 -ATPase.
  • the International Patent Application WO 89/10396 discloses methods for the generation of male sterile plants.
  • the plant cells are transformed with a male-sterility DNA.
  • male-sterility DNA are those encoding DNAses, RNAses, proteases, or enzymes of phytohormone synthesis, such as cytokinin or antisense DNA coding for a strand of DNA complementary to a strand of DNA that is naturally transcribed in the plant cells.
  • EP-A-0 329 308 dicloses a method to produce male-sterile plants by using anti- sense DNA.
  • the development of functional pollen grains is blocked because fo anti-sense DNA directed to genes which are specifically expressed in the microspores, preferably in the premeiotic stage. All systems described above suffer from a serious drawback in the sense that plants are terminally male sterile because of the cell death of the tapetal cells and eventually the microspores and thus the pollengrains, or the microspores directly. Maintaining lines that carry the male sterility gene requires back crossing with a wild type isogenic line. This results in a segregation and consequent loss of 50 % of this backcross population due to the fact that 50 % of the lines will not carry the male sterility gene, and will be male fertile.
  • Auxotrophic mutants of plants are characterized by the inability to synthesise one or more enzymes involved in metabolic pathways. Such mutants have been recovered in plant cells via a variety of techniques (for a review, see Pythoud and King, 1990).
  • the invention is directed to a process for the generation of male sterility in plants comprising the steps of
  • the invention is further directed to a process for the generation of conditional refertible male sterility in plants by
  • inhibitory DNA encompasses any DNA that will cause that a certain metabolic pathway is blocked, e. g. an antisense DNA.
  • antisense is to be understood as a DNA sequence which is complementary to a target or substrate DNA sequence, the expression of the antisense leads to expression of an inhibitory RNA and the inhibition of the expression of the target (see also EP 140 308).
  • the principles of antisense RNA are described by Inouye (1988) and Izant and Weintraub (1984).
  • Inhibitory DNA might also code for ribozymes that selectively cuts and therefore inhibits target sequences (EP 321 201). But there exist also other ways to inhibit gene expression and translation.
  • the inhibitory DNA is transcribed in the anther cells into a RNA which selectively inhibits the expression of genes coding for enzymes or proteins which are involved in the biosynthesis of amino acids.
  • the invention relates to plants wherein the DNA sequence is expressed under the control of a tapetum-specific promotor. Plants regenerated from plant cells with an inhibitory DNA will be unable to produce functional tapetum cells and will produce non functional pollen or no pollen at all.
  • the invention is also related to transgenic plants containing gene constructs comprising a male organ specific promotor operably linked to inhibitory DNA sequences.
  • the inventions also pertains to male sterile plants, parts thereof and cells as well as its reproduction material.
  • the invention further pertains to the seeds produced by the transgenic plants and all progeny that exhibit the desirable trait herein described.
  • the promotor contains the DNA sequence which is necessary for the initation of transcription. Further downstream, i.e. following the promotor, is the so-called 5' non translated region which is also involved in the initiation of transcription. In most cases the promotor will be located at the 5' end of the gene, but it can also vary in its position.
  • the coding region is followed further downstream by the so-called 3' untranslated region. This region does contain signals which cause the termination of transcription and in eucaryotic cells an additional signal that causes the polyadenylation of the transcribed RNA. Termination sequences such as 3'polyadelylation signals are also well described and used in a routine fashion (see Lloyd et al. 1986).
  • the DNA constructs or chimeric genes may also include leader sequences and signal sequences.
  • the DNA sequences which regulate the expression may be derived from different sources, e.g. plant, virus or bacterial genes which are active in plants.
  • Inducible promotors as opposed to constitutive promotors enable directed and controlled expression.
  • Inducible promotors may be expressable depending upon the development of the cell or the type of tissue.
  • the invention relates to a process wherein the male sterility is induced by expression of inhibitory DNA solely in it's male organs, especially in anther cell layers, preferably in tapetum cells.
  • the antisense RNA is preferentially expressed in tapetum layer in order not to disturb the physiological processes taking place in non reproductive tissues and in the female plant organs.
  • tapetum specific promoters or controls of expression have been published (see f.i. Seurinck et al. 1990, and Wyatt et al. 1992).
  • said antisense genes are preferably expressed under the control of a pollenspecific promoter.
  • Constructs containing chimeric genes composed of a tapetum specific promotor and an antisense coding region against a well defined target at the mRNA or the HnRNA can be made according to well known procedures (see Maniatis et al. 1982).
  • Antisense gene expression preventing the formation of aspartokinase leads to starvation for lysine, threonine, isoleucine and methionine, and would therefore be very suitable for obtaining male sterility.
  • the spraying with these amino acids or with aspartyl- ⁇ -phosphate of aspartate- ⁇ - semialdehyde leads to resumption of the normal metabolism.
  • Acetolactate synthase inhibition leads to a combined valine, isoleucine, leucine auxotrophy and non functional EPSP synthase (5- enolpyruvylshikimic acid-3-phosphate synthase) would lead to phenylalanine, tryptophan and tyrosine deprivation.
  • Non functional dehydrodipicolinate synthase leads to lysine auxotrophy.
  • the invention is directed to a process in which another enzyme involved in the biosynthesis of amino acids, the glutamine synthetase (EP-A-0 290 987) is inhibited.
  • glutamine synthetase (EP-A-0 290 987) is inhibited.
  • Ammonium ions are incorporated into glutamine by the action of this enzym on glutamate.
  • the glutamine synthetase plays a critical role in nitrogen metabolism. Inhibition of this enzym leads to nitrogen starvation and consequently to male sterility.
  • the antisense can also be directed against introns in the heteronuclear mRNA (HnRNA) so that the splicing out of the introns would either not be possible or would lead to non sense coding information, resulting in a loss of the enzymatic function of the protein for which the corresponding RNA (mRNA or HnRNA) was targeted against by the antisense RNA.
  • HnRNA heteronuclear mRNA
  • the preparation of transformed plants comprises the following steps: ligating the coding region of the inhibiting DNA sequence to a promotor and a terminator which is selectively active in male organ plant cells, transferring and integrating said contructed DNA sequence in the genome of a plant cell, regenerating whole plants from transformed cells.
  • Agrobacterium tumefaciens as a vector, for example (An et al. 1986, EP 0 116 718) biolistic approaches (Klein et a. 1987) electroporation technologies (Shillito et al. 1985), liposome mediated gene delivery (Gad et al. 1990), direct gene transfer using polyethyleenglycol (Dewulf and Negrutiu, 1991) and protoplast transformation (EP 0 164 575).
  • plasmids which are used.
  • Commonly used plasmids as e. g. pUC-derivatives, bBR322, M13 mp plasmids, EMBL plasmids can be employed.
  • the selection of plant cells which have been transformed is enabled by the use of a selectable marker gene which is also transferred.
  • the expression of the marker gene confers a phenotypic trait that enables the selection. Examples for such genes are those coding for antibiotica or herbicide resistance, e.g. neomycin or phosphinothricin resistance.
  • Plants which can be protected may be either monocotyledons or dicotyledons.
  • Examples of families that are of special interest are Solanaceae and Brassicaceae. Examples of species of commercial interest that can be protected include:
  • Beta vulgaris (Chenopodiaceae)
  • male sterility is induced in plants such as potato, tomato, wheat, cabbage, chicore whitloof and Chinese cabbage.
  • constructs designed for testing antisense activity of the expressed sequences are preferentially introduced into dicotyledonous plants via a binary Ti vector system in Agrobacterium tumefaciens.
  • An example of a gene useful primarily as a screenable marker for identification of plant cells harbouring foreign genes is a gene that codes for an enzyme producing an chromogenic product, e. g. the beta- glucorinidase (GUS, Jefferson et al. (1987)).
  • the genetically created nuclear encoded tapetum specific amino acid auxotrophy in these plants leads to a conditional refertible male sterility.
  • the plants exhibit conditionally male infertile, but the fertility can be restored by supplying the substrate not produced due to the metabolic block, i. e. the missing amino acid(s).
  • the substrates may be added (exogenous supplementation) or produced in the cells (endogenous supplementation).
  • the internal synthesis may be dependend on the activity of another inducible promotor.
  • the substrate is added or applicated externally.
  • the invention is especially related to a process in which the fertility of the conditional unfertile plants is restored by spraying the amino acids onto the plants.
  • the culture medium is modified to contain the amino acid(s) or their precursors that are unable to be synthesised due to the metabolic block imposed by the expression of the transferred inhibitory DNA construct.
  • the concentration of the required metabolite in the culture medium may range from 0.001 to 10 mM, preferably from 0.01 to 1 mM, especially 0.05 mM to 0.5 mM, more preferred from 0.025 to 0.3 mM.
  • a piece of callus of approximately 1 to 2 mm, an internode, a leaf segment or another plant part is subcultured on callus induction and callus propagation medium (such as MS medium (Murashige and Skoog, 1962) containing 0.1 mg/l ⁇ -napthyl acetic acid and 1 mg/l Benzyladenine) or plant propagation medium (for internodes) (such as basic MS medium without growth regulators (Murashige and Skoog, 1962) without the amino acid or the amino acid precursor as mentioned above.
  • callus induction and callus propagation medium such as MS medium (Murashige and Skoog, 1962) containing 0.1 mg/l ⁇ -napthyl acetic acid and 1 mg/l Benzyladenine
  • plant propagation medium for internodes
  • the transferred plant material stops growing (max 1 week) and dies after transfer on the minimal medium (not supplemented with the required metabolite), and shows an auxotrophic requirement corresponding to the metabolite for which the biosynthesis was impaired due to the expression of the antisense construct.
  • the antisense construct is not effectively, expressed only at low levels or even absent. The distinguishment between these alternatives can be made by RNA hybridisation (northern blotting).
  • protoplast transformation direct gene transfer, see f. i. Dewulf and Negrutiu, 1991
  • the initial culture medium is supplemented with concentrations of 0.025 to 0.08 mM of the required compound.
  • the concentration is increased at levels between 0.05 and 0.3 mM in order to maintain viability and sufficient growth.
  • the same scheme for evaluation can be followd as above: first selection for a selectable marker and subsequently testing the transformants for an auxotrophic requirement on non-supplemented medium such as described above. Such an auxotrophic requirement will also lead to cell death if the starvation period will last too long.
  • transgenic plants expressing antisense construct(s) impairing an amino acid biosynthesis are brought back to fertility by spraying the required amino acid or precursors following the metabolic block.
  • Spraying is performed prior to the development of reproductive tissues especially tapetal cells. Spraying is performed at least once within 5 days, preferentially within 3 days, especially preferred within 2 days. The plants are sprayed preferentially under the leaf surface and around the flowers when these are developping. The spraying is applied until fertilisation has taken place or at least to the time that functional pollen is produced. The self pollination is then be performed by hand if necessary.
  • the amino acid may be sprayed in any suitable formulation (see e. g. "Pesticides Formulations” (1986) by Marcel Dekker, 2nd ED., New York or “Spray Drying Handbook", (1979) by G.Goodwin, London). They are sprayed preferrentially in a water solution.
  • the frequency of spraying as well as the concentration of applied amino acids can vary within a broad range. If the desired amino acid(s) is (are) not toxic to the plant it is no problem to spray higher concentrations than needed to restore fertility.
  • the concentrations required for obtaining reversion of fertility may range from 0.01 to 10 mM, preferrentially from 0.3 to 5 mM, more preferrentially from 0.5 mM to 3 mM, especially preferred from 1 mM to 2 mM.
  • the invention also relates to a process in which the restoration of the fertility of the F1 generation is accomplished by reinitiation of normal metabolism by suppression of the expression of antisense constructs.
  • the safener ethyl-1 -(2,4-dichlorphenyl)-5-trichloromethyl-(1 H)-1 ,2,4-triazole-3- carboxylate increases the tolerance level of wheat to fenoxaprop-ethyl, when applied in mixture with this postemergence graminicide.
  • the safener treatment accelerates the rate of hydrolysis of the herbicide by induction of an isoenzyme gene of the glutathion-S-transferase family, which efficiently inactivates the herbicide by conjugation with glutathione.
  • the enzyme is not present in untreated plants and is strictly dependent on spraying with the compound. This effect is apparent already few hours after treatment of the plants.
  • the induced state lasts for several days after one application of the chemical safener. It can be extended for a longer period by application of the safener as seed treatment or by several rounds of spraying.
  • Transformed plants harbouring this type of construct are restored to male fertility by spraying the plants with the chemical safener in concentrations around 100 gAI/ha.
  • conditional male fertility is important for maintaining the male sterile culture.
  • a DNA sequence exhibiting inducible enzymatic activity from wheat was isolated.
  • the F1 hybrid is to be made by crossing a male sterile motherplant obtained by the procedure as described above, with another selected male parent. This leads to a male sterile F1 hybrid; this is for such crops however not relevant because of the use of only the leaves.
  • fertility in the F1 hybrid has to be restored. This can be done by crossing the F1 hybrid which has to be restored.
  • the male sterile line can be crossed with a transgenic line that encodes a functional gene that when transcription takes place will result in a functional protein, restoring the deficiency introduced by the antisense construct.
  • the restorer, and parental line must produce a functional mRNA that will not be recognized by the antisense RNA from the female parent.
  • an antisense RNA against the introns is introduced in the line that has to be made male sterile and by introducing a cDNA copy of the gene in the paternal line.
  • the mRNA encoded by the cDNA will not be recognized by the antisense RNA and normal metabolic activities will be restored resulting in a fertile F1 hybrid plant.
  • the Arabidopsis thaliana A9 gene promoter and the Antirhinum promoter TAP1 were tested for their specific expression both in Arabidopsis and N. tabacum by means of the ⁇ -glucuronidase system basically as described by Martin et al., 1992 and Scott, 1993.
  • RNA and DNA preparations were performed as described by Sambrook et al., 1989 and Jackson et al., 1993.
  • ALS activity was determined essentially as described by Miflin (1971). Protein was extracted from about 100 mg plant material followed by a fractionated ammoniumsulfate precipitation (25-70 %) using 100-1000 ⁇ g of the precipitate for further assays with 40 mM sodium pyruvate as substrate and 0.32 mM thiaminpyrophosphate as coenzyme in presence of 0.5 mM MnS0 4 in 20 mM sodium-phosphate buffer, pH 7.5: incubation was for 1 h at 30°C, the enzyme reaction was stopped with 5 mM ZnS0 4 , the supernatant acidified with HCI to decarboxylate the reaction product acetolactate to acetoin. After addition of 1.7 % alphanaphtol and 0.17 % creatin and 1h incubation at roomtemperatur the absorbance of the samples was detected at 530 nm.
  • CIM callus induction medium
  • cefotaxim in the transformation of Arabidopsis is possible but known to interfere with the ability for regeneration.
  • Tissue culture and transformation of leaf discs was essentially performed as described by Rocha-Sosa et al., 1989.
  • Potato leaf discs of the commercial cultivar Desiree and the tobacco cultivars SR1 and Samsun NN were used as explants.
  • Agrobacterial growth after the cocultivation was inhibited by the addition of cefotaxim.
  • Selection of transformants was carried out by the addition of kanamycin in the culture medium.
  • the potato ALS gene was cloned by a nested PCR approach into the PCR 1000 vector system because the first set of primers only yielded faint and ambigious bands.
  • Nested PCR primers are ones that are internal to the first primer pair.
  • the larger fragment produced by the first round of PCR is used as the template for the second PCR.
  • Nested PCR can also be performed with one of the first primer pair and a single nested primer. The sensitivity and specificity of both DNA and RNA can be significantly increased by using this method.
  • the primers were homologous to the sequence of the tobacco SurB gene (database accession number X 07645, Lee et al., 1988) and the first pair comprised position 415-444 resp. 2376-2409 and the second more inward pair of primers 445- 477 res. 2341-2375.
  • the 5'oligo was provided with an EcoRI site and the 3'oligo with an additionally BamHI site for cloning purposes.
  • An Asp 718/Notl framgent of the ALS gene was cut from this vector, blunted and ligated into the Smal of pUC 19.
  • a Kpnl/Sall fragment with suitable orientation was out from pUC-ALS and cloned into the expression cassette of a plant binary vector, Bin AR (Hofgen and Willmitzer, 1990), resulting in a potato ALS gene in antisense orientation with respect to the CaMV 35S promoter of the expression cassette; plant termination sequences were provided by an octopine synthase 3'end.
  • the plasmid was designated pH29. 3.3 Isolation of a 355 bp anther/tapetum specific promoter
  • a 355 bp anther/tapetum specific promoter fragment from the A9 gene of Arabidopsis thaliana was generated by the construction of two primers, one at positionl 084-1106 containing a Hindlll site at position 1089-1094 and the other one at position 1425-1448 containing a Xbal site at position 1138- 1143, respectively, using the polymerase chain reaction.
  • the 355 bp promoter fragment was cloned into the Hindlll-Xbal cut pBI/101 vector (Jefferson et al., 1987) forming pNun3.
  • the potato ALS gene as described above was recloned from pH29 into BamHi/Sacl cut pNun3 forming pNun5 by using a primer pair with unique restriction sites.
  • the first primer at position 445-478 contained a Sad site at its 5'- end.
  • the second primer at position 2340-2375 contained a BamHI site at its 5'-end.
  • Another fusion plasmid of an antisense potato ALS gene behind an another/tapetum specific promoter was created by ligation of a BamHI cut pVDH187 with the BamHI cut potato ALS gene out of pH29 forming pVDH92 after control of proper orientation.
  • pVDH187 was originally derived from pBIN19 (Bevan, 1984) in which the Tapl promoter of Antirhinum (Nacken et al., 1991) was ligated after EcoRI/BamHI digestion.
  • This promoter was followed by a BamHI/Hindlll ligated polyadenylation signal from the nopaline synthase gene of the agrobacterium Ti plasmid.
  • a GUS-intron gene (Vancanneyt et al., 1990) in between the CaMV 35S promoter and polyadenylation signal was ligated in this plasmid after Hindlll digestion.
  • an antisense-tapetum promoter construct was created with an Arabidopsis ALS gene (database accession number X51514, Sathasvisan et al., 1990). After Dral/Bglll digestion, Bglll linkers were ligated to the Arabidopsis ALS gene fragment. The Bglll cut fragment was inserted into BamHI cut pVDH187 forming pVDH190 when antisense orientation was checked.
  • tobacco and potato explants were transformed with an ALS gene placed in reverse orientation, under the control of a constitutive promoter.
  • An antisense ALS gene (pH29) was constructed under control of the heterologous constitutive 35S CaMV promoter and transfered to Agrobacterium tumefaciens pGV2260. This Agrobacterium strain (H29) was applied for a standard leaf disc transformation procedure of potato plants. Rooted plantlets were replicated and one copy of each plant was transfered to MS medium (Murashige and Skoog, 1962) without casamino acids complementation, to screen for plants unable to grow without exogenous supply of amino acids.
  • Plant P.H. 29-2, -23, - 41 developed phenotypical symptoms, two plants only initially after potting but recovering later.
  • Several siblings of P.H. 29-41 usually even died soon after transfer from tissue culture to soil.
  • Genomic southern blots were performed to correlate phenotypical symptoms and the integration of ALS antisense genes.
  • ALS assays were performed with tissues of control plants of different development stages: very young tissue near to the vegetative bud, fully developed and expanded leaves and old but still green leaves at the stem base (Table 1).
  • Table 1 ALS acitivity in different tissues of control plants and of transgenic ALS antisense plants
  • middle leaf 16 0.2 +/-0.03 19.4 %
  • Desiree control 20 1.6 +/- 0.24 100 plants
  • ALS activity of P.H. 29-2 was reduced to 33 % of wild type activity and the phenotypically severely affected plant P.H 29-41 showed only 15 % of wildtype activity (Table 1).
  • ALS antisense plants showed a clear correlation of phenotype and the reduction of ALS activity when compared to control plants. 15 % remaining enzyme activity of ALS is just sufficient to sustain growth of the transgenic plant P.H 29-41.
  • the reduction in enzymatic acticity correlates to the results obtained in northern blot analysis.
  • ALS antisense plants were scored for transcriptional regulation of some other key regulatory enzymes within the affected pathway (see above). We found no alteration when compared to RNA of the control plants.
  • the ⁇ -glucuronidase gene was fused to the another specific promoters and Gus assays were performed basically as described by Martin et al., 1992 and Scott, 1993.
  • the expression of the antisense was clearly confined solely to anther tissue and the expression specificity and timing were comparable to the patterns as described by Scott, 1993.
  • N. tabacum and Arabidopsis thaliana were transformed with gene constructs expressing an antisense ALS gene under the control of anther specific promoters A9 and Tap1 respectively.
  • anther specific promoters A9 and Tap1 Of several hundered independend transformants, with the bacterial strains Nun 3, 5 and VDH 190 and 192 respectively, selected by means of kanamycin resistance and by the confirmation of the transgenic nature of the transformants by means of a ⁇ -glucuronidase assay, several sterile plants or plants with reduced fertility and without seed set were recovered. Molecular analysis is currently carried out to confirm the relation between the reduction of ALS in the anther tissue and the observed decrease of fertility.
  • Arabidopsis genotype C-24
  • 36 out of these 340 putative sterile plants produced pollen that was not stainable and in several cases the morphology of the pollen was clearly abnormal (broken pollen).
  • Hood EE Helmer GL, Fraley RT, Chiltron MD (1986) J Bact 168, 1291-1301.
  • GUS Protocols Using the GUS gene as a Reporter of Gene Expression. pp 23-43. Eds. Sean R. Gallagher. ACADEMIC PRESS, INC. San Diego, California.
  • Negrutiu I De Brouwer D, Dirks R, Jacobs M (1985) Mol Gen Genet 199, 330-337.
  • Negrutiu I. De Brouwer D., Dirks R. and Jacobs M. (1985). Amino acid auxotrophs from protoplast cultures of Nicotiana plumbaginifolia, Viviani. 1. BUdR enrichment selection, plant regeneration, and general characterization. Mol Gen Genet 199, 330-337.
  • Vergunst A et al. (lab of prof. Dr. P. Hooykaas: Clusius Laboratory, Institute of Molecular Plant Sciences, Wassenaarseweg 64, 2333 AL Leiden. The Netherlands (manuscript in preparation).

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Abstract

L'invention se rapporte à un procédé permettant d'induire la stérilité dans des plantes mâles, et consistant à (a) transformer une cellule de plante au moyen de séquences d'ADN qui inhibent sélectivement l'expression de composés métaboliques essentiels et (b) régénérer les plantes à partir desdites cellules de plantes. Les cellules endommagés au cours de la biosynthèse de composés métaboliques de base s'étiolent et finissent par mourir. De tels processus comprennent la biosynthèse d'acides aminés, la biosynthèse d'acide nucléique ainsi que d'autres processus de biosynthèse tels que le cycle d'acide citrique, le processus à base de phosphate de pentose, le métabolisme d'acide gras, et la biosynthèse de vitamines, qui vont rendre la cellule inactive par appauvrissement en substances nutritives, si au moins une enzyme ou protéine impliquée dans cette voie est inactivée par l'utilisation d'ADN de recombinaison inhibiteurs.
PCT/EP1994/001840 1993-06-08 1994-06-07 Procede de generation de plantes males steriles WO1994029465A1 (fr)

Priority Applications (7)

Application Number Priority Date Filing Date Title
JP7501308A JPH08510916A (ja) 1993-06-08 1994-06-07 雄不稔性植物を作り出す方法
AU69723/94A AU701920B2 (en) 1993-06-08 1994-06-07 Process for generating male sterile plants
US08/556,944 US6262339B1 (en) 1993-06-08 1994-06-07 Process for generating male sterile plants
EP94918390A EP0701619A1 (fr) 1993-06-08 1994-06-07 Procede de generation de plantes males steriles
PCT/EP1994/001840 WO1994029465A1 (fr) 1993-06-08 1994-06-07 Procede de generation de plantes males steriles
CA002164732A CA2164732A1 (fr) 1993-06-08 1994-06-07 Procede de production de plantes males steriles
NO954978A NO954978L (no) 1993-06-08 1995-12-07 Fremgangsmåte for generering av hannlige planter

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EP93109226A EP0628635A1 (fr) 1993-06-08 1993-06-08 Procédé de préparation de plantes mâles stériles
EP93109226.6 1993-06-08
PCT/EP1994/001840 WO1994029465A1 (fr) 1993-06-08 1994-06-07 Procede de generation de plantes males steriles

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WO2000060101A1 (fr) * 1999-03-31 2000-10-12 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Marqueurs de selection metaboliques destines aux plantes
WO2003078629A1 (fr) 2002-03-20 2003-09-25 Basf Plant Science Gmbh Produit de synthese et procede de regulation de l'expression genique
US7230168B2 (en) 2001-12-20 2007-06-12 The Curators Of The University Of Missouri Reversible male sterility in transgenic plants by expression of cytokinin oxidase
WO2006125678A3 (fr) * 2005-05-23 2007-07-26 Nunhems Netherlands Bv Plantes de sterilite male reversible
EP2267139A2 (fr) 1998-04-08 2010-12-29 Commonwealth Scientific and Industrial Research Organization Procédés ét moyens d'obtention de phénotypes modifies
EP2612918A1 (fr) 2012-01-06 2013-07-10 BASF Plant Science Company GmbH Recombinaison in planta
US8598332B1 (en) 1998-04-08 2013-12-03 Bayer Cropscience N.V. Methods and means for obtaining modified phenotypes
WO2013184768A1 (fr) 2012-06-05 2013-12-12 University Of Georgia Research Foundation, Inc. Compositions et méthodes d'inactivation génique dans les plantes
EP2781151A1 (fr) 2013-03-18 2014-09-24 Bayer CropScience AG Procédés de séparation de graines hybrides à partir d'un mélange d'agents de contrôle biologique de graines
US9029527B2 (en) 1998-03-20 2015-05-12 Commonwealth Scientific And Industrial Research Organisation Synthetic genes and genetic constructs
EP2980220A1 (fr) 2005-09-20 2016-02-03 BASF Plant Science GmbH Procédés améliorés de contrôle de l'expression de gènes
US9441239B2 (en) 1998-04-08 2016-09-13 Commonwealth Scientific & Industrial Research Organisation Methods and means for obtaining modified phenotypes
US9708621B2 (en) 1999-08-13 2017-07-18 Commonwealth Scientific And Industrial Research Organisation Methods and means for obtaining modified phenotypes
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US6291741B1 (en) 1994-01-31 2001-09-18 Gene Shears Pty. Limited Method for the production of modified plants
WO1995020668A1 (fr) * 1994-01-31 1995-08-03 Nickerson Biocem Limited Procedes de production de plantes modifiees
US9029527B2 (en) 1998-03-20 2015-05-12 Commonwealth Scientific And Industrial Research Organisation Synthetic genes and genetic constructs
US9963698B2 (en) 1998-03-20 2018-05-08 Commonwealth Scientific And Industrial Research Organisation Control of gene expression
EP3214177A2 (fr) 1998-04-08 2017-09-06 Commonwealth Scientific and Industrial Research Organisation Procédés et moyens pour obtenir des phénotypes modifiés
US9441239B2 (en) 1998-04-08 2016-09-13 Commonwealth Scientific & Industrial Research Organisation Methods and means for obtaining modified phenotypes
EP2267139A2 (fr) 1998-04-08 2010-12-29 Commonwealth Scientific and Industrial Research Organization Procédés ét moyens d'obtention de phénotypes modifies
EP2267138A2 (fr) 1998-04-08 2010-12-29 Commonwealth Scientific and Industrial Research Organization Procédés et moyens d'obtention de phénotypes modifiés
US8598332B1 (en) 1998-04-08 2013-12-03 Bayer Cropscience N.V. Methods and means for obtaining modified phenotypes
WO2000060101A1 (fr) * 1999-03-31 2000-10-12 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Marqueurs de selection metaboliques destines aux plantes
US9708621B2 (en) 1999-08-13 2017-07-18 Commonwealth Scientific And Industrial Research Organisation Methods and means for obtaining modified phenotypes
US10190127B2 (en) 1999-08-13 2019-01-29 Commonwealth Scientific And Industrial Research Organisation Methods and means for obtaining modified phenotypes
US7230168B2 (en) 2001-12-20 2007-06-12 The Curators Of The University Of Missouri Reversible male sterility in transgenic plants by expression of cytokinin oxidase
US7951997B2 (en) 2001-12-20 2011-05-31 Monsanto Technology Llc Reversible male sterility in transgenic plants by expression of cytokinin oxidase
WO2003078629A1 (fr) 2002-03-20 2003-09-25 Basf Plant Science Gmbh Produit de synthese et procede de regulation de l'expression genique
WO2006125678A3 (fr) * 2005-05-23 2007-07-26 Nunhems Netherlands Bv Plantes de sterilite male reversible
EP2980220A1 (fr) 2005-09-20 2016-02-03 BASF Plant Science GmbH Procédés améliorés de contrôle de l'expression de gènes
WO2013102875A1 (fr) 2012-01-06 2013-07-11 Basf Plant Science Company Gmbh Recombinaison in planta
EP2612918A1 (fr) 2012-01-06 2013-07-10 BASF Plant Science Company GmbH Recombinaison in planta
WO2013184768A1 (fr) 2012-06-05 2013-12-12 University Of Georgia Research Foundation, Inc. Compositions et méthodes d'inactivation génique dans les plantes
EP2781151A1 (fr) 2013-03-18 2014-09-24 Bayer CropScience AG Procédés de séparation de graines hybrides à partir d'un mélange d'agents de contrôle biologique de graines

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