EP1206559A2 - Inhibition conditionnelle de multiplication vegetative - Google Patents

Inhibition conditionnelle de multiplication vegetative

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Publication number
EP1206559A2
EP1206559A2 EP00961249A EP00961249A EP1206559A2 EP 1206559 A2 EP1206559 A2 EP 1206559A2 EP 00961249 A EP00961249 A EP 00961249A EP 00961249 A EP00961249 A EP 00961249A EP 1206559 A2 EP1206559 A2 EP 1206559A2
Authority
EP
European Patent Office
Prior art keywords
gene
rooting
inhibiting
product
codes
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Withdrawn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
EP00961249A
Other languages
German (de)
English (en)
Inventor
Wilhelmus Maria Van Der Krieken
Michiel Maria Van Lookeren Campagne
Jan Kodde
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.)
Plant Research International BV
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Plant Research International BV
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Plant Research International BV filed Critical Plant Research International BV
Publication of EP1206559A2 publication Critical patent/EP1206559A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8291—Hormone-influenced development
    • C12N15/8295—Cytokinins

Definitions

  • the present invention relates to a method for rooting plants which are protected against undesired vegetative propagation, in addition to a DNA construct and a transgenic plant which enable conditional inhibition of vegetative propagation.
  • Improvement stations invest a great deal of money in making new plant varieties. This money is recovered by selling the developed plants. A major problem arises here however in that the new varieties are often propagated illegally via cuttings or via grafting. An estimated 25 to 35% of all sold carnations is thus vegetatively propagated illegally (i.e. without payment of royalties) .
  • the present inventors have developed a technology for inhibiting root formation conditionally (under particular conditions) . This technique is described in the international application 099/06579, the content of which is incorporated herein by means of reference.
  • the action mechanism associated with this technology is based on the following principles.
  • the plant growth regulator auxin is involved in the induction of roots on a shoot (adventive root formation) and in side root formation (lateral root formation) .
  • Auxin occurs in some plants in a degree sufficient to induce roots on cuttings and in the case of some other plants it has to be administered additionally via a cutting powder (or via a solution) .
  • Antagonists for these auxin-induced processes are also present in the plant.
  • These antagonists are signal molecules such as for instance cytokinins and ethylene. Cytokinins not only inhibit rooting but can also inhibit graft union and lateral root formation.
  • cytokinins zeatin or benzylaminopurine administered for a short period of time (4 hours) is capable of completely blocking in vitro root induction with auxin.
  • the gene that codes for a protein which can play a key role in the formation of cytokinin is the isopentenyl transferase gene (ipt gene) from A ⁇ robacterium tumefaciens.
  • ipt gene isopentenyl transferase gene
  • the conversion of 5'- adenosine monophosphate + isopentenyl pyrophosphate in the cytokinin isopentenyl adenine is catalysed by the enzyme isopentenyl transferase. This enzyme is present after transcription and translation of the ipt gene.
  • Isopentenyl adenine is converted rapidly in the plant to the very active cytokinin zeatin.
  • Cytokinin formed via the ipt gene has a paracrine effect (at the location of synthesis) , so that pleiotropic effects (undesired side effects) will be limited.
  • genes with which rooting or graft union can be inhibited are genes which induce cell death at the location where and the moment at which rooting or graft union would normally speaking be desirable. Regulation of genes which control cell division (cyclins and cell cycle genes, actin genes, scarecrow genes etc.) and genes which are involved in induction, formation and development of adventive root formation can be used to inhibit root formation (for instance via inhibition of functional gene expression) . In addition, genes which play a part in auxin transport, such as the PIN genes, can be used to inhibit root formation and graft union. In addition to blocking adventive root formation, the above stated genes can also be used to stop root formation at the moment when the root primordium is being formed or has already been formed.
  • cytokinins formed after expression of the ipt gene are poorly transported in the plant, pleiotropic effects are limited. Expression of the ipt gene in a wounded leaf also has few adverse effects, because cytokinin is naturally involved in the recovery from wounds .
  • the above described system is in principle very useful in preventing undesired vegetative propagation of plants. However, it also prevents propagation when this is desired, such as by the developer or breeder of the plant itself or with the permission of the developer or breeder . It is therefore the object of the present invention to provide a method whereby, if so required or desired, the inhibition of the vegetative propagation can be discontinued.
  • a method for enabling rooting or grafting of a plant which contains in its genome a gene which is placed under the regulation of an inducible promoter and which codes for a rooting-inhibiting product or a product leading to rooting inhibition, comprising of treating the plant prior to production of a cutting or graft or treating the cutting or graft after production of the cutting or graft with a stimulus which: a) discontinues the effect of the rooting- inhibiting gene; and/or b) results in expression of a second gene likewise present in the plant which is under the regulation of an inducible promoter and the expression product of which discontinues the effect of the rooting-inhibiting gene.
  • a stimulus which: a) discontinues the effect of the rooting- inhibiting gene; and/or b) results in expression of a second gene likewise present in the plant which is under the regulation of an inducible promoter and the expression product of which discontinues the effect of the rooting-inhibiting gene.
  • the stimulus usually a substance which causes rooting inhibition to be discontinued directly or indirectly (via induction of the second gene)
  • the stimulus can be made available by the owner of the plant variety to those who are authorized for vegetative propagation of a plant which is patented or protected by plant breeders ' rights and provided with the rooting-inhibiting mechanism.
  • the stimulus which discontinues the effect of the rooting-inhibiting gene can be chosen from substances which inhibit the biosynthesis of the rooting-inhibiting product and structural analogs of the rooting-inhibiting product .
  • Particularly suitable rooting-inhibiting substances are the endogenous plant hormones cytokinins. These can be produced in a plant in which the rooting-inhibiting mechanism is active via expression of the ipt gene, the product of which plays a key part in the cytokinin biosynthesis .
  • the stimulus in particular the substance, which inhibits the biosynthesis of the cytokinin is a substance which is a structural analog of a precursor of a cytokinin, such as the substrates of IPT from which isopentenyl adenine and subsequently, via other enzymes, zeatin is made. Examples are shown in Table 1A.
  • CBP various 4-substituted 2-methylpyrrolo [2, 3-d]pyrimidines, including: 4-cyclobutylamino-2-methylpyrrolo [2, 3-d] pyrimidine (CBP)
  • CBP 4-cyclobutylamino-2-methylpyrrolo [2, 3-d] pyrimidine
  • the second gene can thus be a gene coding for the antisense version of the rooting-inhibiting gene.
  • the messenger transcribed from the antisense version of the gene captures the mRNA of the sense version whereby no translation to protein takes place.
  • the protein is the rooting inhibitor itself or is required in the biosynthesis thereof. Now the product is no longer formed, the rooting inhibition is discontinued.
  • the second gene preferably codes for an antisense version of the ipt gene.
  • the second gene can further be a gene which codes for a second sense version of the rooting-inhibiting gene. Co-suppression can hereby be effected.
  • the second gene preferably codes for a sense version of the ipt gene .
  • the second gene can also be one of the rolABC genes of Aqrobacterium rhizo enes. Expression of the rolABC genes makes the plant more insensitive to cytokinin.
  • the first gene is under the regulation of an inducible promoter which is modified, for instance a wound-inducible promoter which regulates expression of the ipt gene. The modification ensures that a repressor can interact with the promoter.
  • the second gene the expression of which is likewise inducible (for instance via a chemical substance or via another method) , codes for a repressor-protein which inhibits expression of the first gene.
  • An example hereof is the GAL4 repressor system.
  • the rooting-inhibiting gene is therefore preferably the ipt gene, which is under the regulation of for instance a modified wound- inducible promoter, wherein the modification is such that the repressor of the GAL4 regulation system can interact therewith.
  • the second gene can further code for a degradation enzyme of the rooting-inhibiting gene product.
  • the rooting-inhibiting gene product is preferably cytokinin and the second gene then codes for instance for cytokinin-oxidase .
  • the second gene can be a mutated cytokinin receptor gene, whereby the cytokinin pathway cannot take place and the rooting becomes conditional.
  • the expression of the second gene is inducible (for instance with a chemical compound) and can therefore be switched on at the moment rooting is required by administering the inducer. Induction proceeds via the promoter.
  • Examples hereof are gene-regulation systems which make use of promoters which are inducible with for instance ethanol, copper ions (or other metals) , tetracycline, dexamenthasone (and other glucocorticoid promoters) , benzothiadiazole, N- (aminocarbonyl) -2-chlorobenzene sulfonamide or tebufenozide .
  • the invention further relates to a transgenic plant which is reversibly protected against undesired vegetative propagation, which plant contains in its genome a first gene which is placed under the regulation of an inducible first promoter and which codes for a rooting-inhibiting product or a product leading to rooting inhibition, and a second gene which is placed under the regulation of a second promoter inducible by means of another stimulus and which codes for a product which deactivates the rooting-inhibiting expression product of the first gene.
  • the invention further relates to a DNA construct comprising transcription-initiation sequences, including an inducible promoter, and optionally transcription termination sequences operably linked to a second gene as defined above.
  • the same DNA construct can advantageously further contain a first gene which codes for a rooting- inhibiting product or a product leading to rooting inhibition provided with transcription-initiation sequences operably linked thereto, including an inducible promoter, and optionally transcription termination sequences.
  • the first gene is advantageously the ipt gene .
  • the second gene can in both the transgenic plant and the DNA construct be everything as defined above.
  • the invention further relates to a method for reversing rooting inhibition in a cutting or the growing together of a rootstock and upper stem (graft union) of a transgenic plant as described above, comprising of treating the cutting or graft with the stimulus of the second promoter and placing the cutting or graft on a rooting medium.
  • a stimulus usually a substance
  • this advantageously forms part of a composition, such as for instance a cutting powder or a graft promoter (substance which enhances the growing together of the graft with the lower stem) or a solution. Rooting or graft union is then only possible in the presence of this cutting powder, the graft promoter or a solution thereof.
  • the cutting powder or graft promoter (or solutions thereof) can be administered after making the cutting or graft.
  • the plants from which the graft or cutting is made can also be treated beforehand with the substance for inhibiting the effect of the ipt gene.
  • the substances which discontinue the rooting inhibition directly, i.e. not via the genetic route, are mentioned above.
  • other cytokinin inhibitors can be readily found among the arsenal of available substances. Finding these other substances can be done by testing a large number of substances in so-called high throughput screening (HTS) systems. Many thousands of compounds can be tested in the HTS systems.
  • the IPT protein or bacteria containing or producing the IPT-protein
  • cytokinin precursors (5 ' -adenosine monophosphate and isopentenylpyrophosphate) and potential inhibitors for testing.
  • IP isopentenyl adenine
  • zeatin is formed.
  • the effectiveness of the substances which inhibit IP or zeatin formation van be further optimized via so-called "combinatorial chemistry". All this may result in the identification of substances which are suitable for direct application to discontinue the ipt activity in the plant (conditional inhibition of rooting) .
  • the action of the genetic mechanism whereby rooting inhibition is discontinued is based on expression induction.
  • the second gene which discontinues the activity of the rooting-inhibiting product is switched on at the desired moment by induction via its promoter.
  • Inducible promoters are for instance the per se known systems inducible using alcohol and tetracycline.
  • Other examples of inducers are copper ions (or other metals), dierithasone (and other glucocorticoid promoters) benzothiadiazole, N- (aminocarbonyl) -2-chlorobenzene sulfonamide or tebufenozide .
  • DNA constructs containing the rooting-inhibiting gene and/or the gene which leads to discontinuation of the rooting inhibition with a marker gene, such as GUS or GFP. It is then possible throughout the whole chain - from producer tot consumer - to perform a very rapid test for the presence of the reporter, such as by means of a GUS assay or using a fluorescence meter.
  • the construct which is incorporated can be identified via a Southern blot or PCR reaction.
  • rooting-inhibiting gene is understood to mean a gene which codes for a rooting-inhibiting product or product leading to rooting inhibition, or a gene which codes for a product which can prevent graft union or which results in prevention of graft union.
  • the gene product of the rooting- inhibiting gene is referred to as "rooting-inhibiting gene product”.
  • Dit product is itself not necessarily rooting-inhibiting, but in any case results in inhibition of rooting or prevention of graft union.
  • ipt gene resulted in rolB-ipt SRI tobacco tissue in a very strong increase in the formation of axillary shoots (between ten and twenty relative to the control plants which formed no axillary shoots), which points to an increased cytokinin content via expression of the ipt gene.
  • transgenic rolB-ipt tobacco shoots Twenty of these transgenic rolB-ipt tobacco shoots were incubated for three weeks on Murashige and Skoog medium with 2% sucrose and the auxins indol-3-butyric acid (IBA) or naphthalene acetic acid (NAA) .
  • the auxins were present during the entire incubation period in the following concentrations: 0, 1, 3.2, 10 and 100 ⁇ M.
  • As control the same incubations were performed with wild type SRI tobacco shoots.
  • the transgenic tobacco plants formed no roots.
  • the wild type control shoots formed roots at all auxin concentrations. Strong callus formation was observed at 100 ⁇ M auxin.
  • the rolB-ipt SRI tobacco shoots were incubated with 10 ⁇ M IBA plus 10 or 100 ⁇ M of the anti-cytokinin 2- cyclobutylamino-4-ethylamino-6-chloro-l, 3, 5-triazine . These shoots (see figure) were able to form roots, in contrast to the shoots which were incubated without anti-cytokinin. This shows that the effect of expression of the ipt gene can be prevented by anti-cytokinins . Other anti-cytokinins are listed in Table IB.
  • Anti-cytokinin activity can also be obtained via introduction of a gene construct which prevents the effect of expression of the ipt gene, as explained in the description introduction.
  • the techniques used for the above stated experiments are described in the following references:
  • MS medium Murashige T, Skoog F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physioloqia Plantarum 15, 473-497.

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  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
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  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
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Abstract

La présente invention concerne un procédé permettant d'enraciner ou de greffer une plante contenant dans son génome un gène, placé sous la régulation d'un promoteur inductible, codant pour un produit inhibant l'enracinement ou pour un produit conduisant à une inhibition de l'enracinement. Ce procédé consiste à traiter la plante, avant production d'une bouture ou d'une greffe, ou de traiter la bouture ou le greffage, après leur production, au moyen d'une substance qui, ou bien stoppe l'effet du gène inhibant l'enracinement, ou/et permet l'expression d'un second gène du même genre, présent dans la plante, se trouvant sous la régulation d'un promoteur inductible, et dont le produit d'expression stoppe l'effet du gène inhibiteur d'enracinement. l'invention concerne, en outre, une plante transgénique, pourvue d'un mécanisme d'inhibition d'enracinement et d'un mécanisme qui stoppe l'inhibition d'enracinement, ainsi qu'un ADN chimère destiné à réaliser l'arrêt de l'inhibition d'enracinement.
EP00961249A 1999-08-26 2000-08-28 Inhibition conditionnelle de multiplication vegetative Withdrawn EP1206559A2 (fr)

Applications Claiming Priority (3)

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NL1012917 1999-08-26
NL1012917 1999-08-26
PCT/NL2000/000597 WO2001018170A2 (fr) 1999-08-26 2000-08-28 Inhibition conditionnelle de multiplication vegetative

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EP1206559A2 true EP1206559A2 (fr) 2002-05-22

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AU2001273454A1 (en) 2000-07-13 2002-01-30 Pioneer Hi-Bred International, Inc. ZmaXIG1 polynucleotides and methods of use
DE60333546D1 (de) * 2003-05-06 2010-09-09 Univ Palackeho PyrazoloÄ4,3-DÜpyrimidines, verfahren zur ihre herstellung und deren verwendung
EP1656378A4 (fr) * 2003-08-15 2011-05-11 Irm Llc Composes et compositions inhibiteurs de l'activite du recepteur tyrosine kinase
WO2005118588A1 (fr) * 2004-06-02 2005-12-15 Takeda Pharmaceutical Company Limited Composé heterocyclique fusionné
EP1792630A1 (fr) * 2005-12-02 2007-06-06 The Procter & Gamble Company Conjugués d'acides aminés et de la vitamine B3 pour l'administration transcutanée de la vitamine B3
CN107711499A (zh) * 2017-10-23 2018-02-23 西北农林科技大学 一种不同时间点6‑ba处理抑制培苗不定根发生的方法

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DE3810286A1 (de) * 1988-03-25 1989-10-12 Max Planck Gesellschaft Transgene pflanze mit modifizierter physiologie, morphologie und modifiziertem hormonmetabolismus, gewebekulturen dieser pflanze und verfahren zu ihrer herstellung
JP2000513937A (ja) * 1996-06-28 2000-10-24 ユニバーシティー オブ ブリストル 植物におけるｇタンパク質シグナル伝達
US6229066B1 (en) * 1997-07-30 2001-05-08 The Curators Of The University Of Missouri Cytokinin oxidase
IL134282A0 (en) * 1997-07-30 2001-04-30 Inst Voor Agrobiologisch En Bo Inhibition of unwanted hormone action in plants

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WO2001018170A2 (fr) 2001-03-15
WO2001018170A3 (fr) 2001-09-20

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