EP0866850A4 - Analogues auxiniques d'acide indole-3-acetique - Google Patents

Analogues auxiniques d'acide indole-3-acetique

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Publication number
EP0866850A4
EP0866850A4 EP96943547A EP96943547A EP0866850A4 EP 0866850 A4 EP0866850 A4 EP 0866850A4 EP 96943547 A EP96943547 A EP 96943547A EP 96943547 A EP96943547 A EP 96943547A EP 0866850 A4 EP0866850 A4 EP 0866850A4
Authority
EP
European Patent Office
Prior art keywords
group
plant
acetic acid
iaa
carbon atoms
Prior art date
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Ceased
Application number
EP96943547A
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German (de)
English (en)
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EP0866850A1 (fr
Inventor
Jhy-Jhu Lin
Jianqing Lan
Nacyra Assad-Garcia
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Life Technologies Corp
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Life Technologies Inc
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Publication of EP0866850A1 publication Critical patent/EP0866850A1/fr
Publication of EP0866850A4 publication Critical patent/EP0866850A4/fr
Ceased 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018—Culture media for cell or tissue culture
    • C12N5/0025—Culture media for plant cell or plant tissue culture
    • 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
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/008—Methods for regeneration to complete plants
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/34—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom
    • A01N43/36—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom five-membered rings
    • A01N43/38—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom five-membered rings condensed with carbocyclic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/10—Indoles; Hydrogenated indoles with substituted hydrocarbon radicals attached to carbon atoms of the hetero ring
    • C07D209/18—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8205—Agrobacterium mediated transformation

Definitions

  • This invention relates to the use of an indole-3-acetic acid (IAA) analogue as a plant hormone stimulatory to plant growth, to regeneration of plant cells and tissues, and to transformation of plant cells. It particularly relates to the use of mono- and multi-substituted IAA molecules.
  • the invention also relates to compositions comprising IAA analogues of the invention.
  • Plant growth is affected by a variety of physical and chemical factors. Physical factors include available light, day length, moisture and temperature. Chemical factors include minerals, nitrates, cofactors, nutrient substances and plant growth regulators or hormones, for example, auxins, cytokinins and gibberellins.
  • Indole-3-acetic acid is a naturally-occurring plant growth hormone identified in plants. IAA has been shown to be directly responsible for the increase in growth in plants in vivo and in vitro . The characteristics influenced by IAA include cell elongation, internodal distance (height) , leaf surface area and crop yield. IAA and other compounds exhibiting hormonal regulatory activity similar to that of IAA are included in a class of plant regulators called "auxins.”
  • cytokinins such as 6-furfurylamino purine (kinetin) and 6-benzylamino purine (BAP)
  • cytokinin-based preparations are most effective in combination with auxins. While the mechanism by which cytokinins affect the growth cycle of plants is far from being understood, it is apparent that they affect leaf growth and prevent aging in certain plants.
  • Major crop varieties of particular interest in this regard are agricultural crops such as maize, wheat, rice, soybeans and cotton.
  • Embryo culturing has been shown to be important in making difficult interspecies crosses, while shoot-tip culturing is important in rapid clonal multiplication, development of virus-free clones and genetic resource conservation work.
  • Callus, cell, and protoplast cultures have been shown to be important for cultures in which organization is lost but can be recovered.
  • Plant genetic engineering techniques have also been established. These techniques include gene transfer by transformation or by protoplast fusion. In gene transfer, the steps involved are: (a) identification of a specific gene; (b) isolation and cloning of the gene; (c) transfer of the gene to recipient plant host cells: (d) integration, transcription and translation of the DNA in the recipient cells; and (e) multiplication and use of the transgenic plant (T. Kosuge, CP. Meredith and A. Hollaender, eds (1983) Genetic Engineering of Plants. 26:5-25; Rogers et al. (1988) Methods for Plant Molecular Biology [A. Weissbach and H. Weissbach, eds.] Academic Press, Inc., San Diego, CA) .
  • plant cell protoplasts are fused by standard chemical (e.g., PEG) or electroporation techniques. After regeneration of the fused cells, interspecies amphidiploids have been obtained.
  • the technique may provide desired amphidiploids which cannot be made by conventional means, and presents possibilities for somatic recombination by some variant of it.
  • the foregoing techniques are widely in use (Chaleff, R.S. (1981) Genetics of Higher Plants. Applications of Cell Culture. Cambridge: Cambridge University Press) , and newly inserted foreign genes have been shown to be stably maintained during plant regeneration and are transmitted to progeny as typical Mendelian traits (Horsch et al. (1984) Science 223:496, and DeBlock et al. (1984) EMBO 3:1681). These foreign genes retain their normal tissue specific and developmental expression patterns.
  • the Agrojbacterium tume-aciens-mediated transformation system has proved to be efficient for many dicotyledonous plant species.
  • Barton et al. (1983, Cell 32:1033) reported the transformation and regeneration of tobacco plants
  • Chang et al. (1994, Planta 5:551-558) described stable genetic transformation of Arajbidopsi.? thaliana .
  • the Agrrojbacterium method for gene transfer was also applied to monocotyledonous plants, e.g., in plants in the Liliaceae and Amaryllidaceae families (Hooykaas-Van Slogteren et al., 1984, Nature 311:763-764) and in Dioscorea bulbifera (yam) (Schafer et al. , 1987, Nature 327:529-532); however, this method did not appear to be efficient for the transformation of graminaceous onocots, which include such food crops as wheat, rice and corn.
  • Transformation of food crops was obtained with alternative methods, e.g., by polyethylene glycol (PEG) - facilitated DNA uptake (Uchimiya et al. (1986) Mol. Gen. Genet. 204:204-207) and electroporation (Fromm et al. (1986) Nature 319:791-793), both of which used protoplasts as transfer targets.
  • Monocot and dicot tissues may be transformed by bombardment of tissues by DNA-coated particles (Wang et al. (1988) Plant Mol. Biol. 11:433-439; Wu, in Plant Biotechnology (1989), Kung and Arntzen, Eds., Butterworth Publishers, Stoneha , MA) .
  • Regeneration was described in rice (Abdullah et al. (1986) Bio/Technology 4:1087-1090) and maize (Rhodes et al. (1988) Bio/Technology 6:56-60 and (1988) Science 240:204-207).
  • the present invention satisfies these needs by providing compounds and compositions which stimulate plant growth, regeneration of plant cells and tissues, and transformation of plant cells and tissues.
  • the compounds of the invention comprise mono- or multi-substituted IAA (indole-3-acetic acid) or ester or salt derivatives thereof.
  • the invention also provides compositions comprising one or more of these IAA analogues and, optionally, a carrier.
  • the invention contemplates the use of such auxinic analogues to affect growth, regeneration and transformation in both monocotyledonous and dicotyledonous plants.
  • the invention provides monosubstituted IAA analogues having a substituent group at the 2, 4, 5, 6 or 7 position of the IAA structure, wherein said substituents are halo- or alkyl-, alkoxy-, acyl-, acylamido- and acyloxy-substituent groups having 1-10 carbon atoms.
  • the invention also provides multi- substituted IAA analogues having two to five, same or different, substituent groups at different positions selected from positions 2, 4, 5, 6 or 7 of the IAA structure wherein said substituents are halo- or alkyl-, alkoxy-, acyl-, acylamido- and acyloxy-substituent groups having 1-10 carbon atoms.
  • compositions of the invention may include, in addition to one or more of the mono- or multi-substituted compounds, one or more additional plant growth regulators.
  • plant growth regulators include, for example, a cytokinin, a gibberellin, etc., in definite proportions for wide application to various plants.
  • the invention is exemplified with compositions comprising mono- or multi-substituted IAA analogues having between one and five, same or different, substituent groups that are halo- , alkyl-, alkoxy-, acyl-, acylamido- or acyloxy-substituent groups at positions 2, 4, 5, 6 and/or 7 of the IAA structure, and a cytokinin to affect the growth of plants.
  • the invention further relates to media comprising the compounds and compositions of the invention.
  • Such media comprise one or more IAA analogues and optionally an IAA analogue and optionally a plant growth regulator, e.g. , cytokinin, to stimulate plant growth, to stimulate regeneration of plant cells and tissues, and to stimulate transformation of plant cells and tissues.
  • a plant growth regulator e.g. , cytokinin
  • the invention also relates to a method of stimulating plant growth comprising (a) applying to a plant, plant cell or tissue an effective amount of the compound or composition of the invention and optionally, applying one or more additional plant growth regulators, for example, a cytokinin, a gibberellin, etc. , and (b) incubating the plant cell or tissue under conditions sufficient to stimulate the regeneration of the plant cell or tissue.
  • additional plant growth regulators for example, a cytokinin, a gibberellin, etc.
  • the invention also provides a method for stimulating the regeneration of plant cells and/or tissues comprising (a) applying to a plant cell or tissue an effective amount of the compound or composition of the invention and applying one or more additional plant growth regulators, for example, a cytokinin, a gibberellin, etc. , and (b) incubating the plant cell or tissue under conditions sufficient to stimulate the regeneration of the plant cell or tissue.
  • a plant cell or tissue an effective amount of the compound or composition of the invention and applying one or more additional plant growth regulators, for example, a cytokinin, a gibberellin, etc.
  • the invention provides a method for stimulating the transformation of plant cells and/or tissues comprising (a) contacting the plant cell or tissue with a nucleic acid molecule (e.g., by transformation or protoplast fusion) , (b) applying to the plant cell or tissue an effective amount of a compound or composition of the invention and, optionally, one or more additional plant growth regulators, for example, a cytokinin, a gibberellin, etc., and (c) incubating the plant cell or tissue under conditions sufficient to stimulate transformation of the plant cell or tissue with the nucleic acid molecule.
  • the compounds and compositions of the invention also may be used to stimulate regeneration or growth of the transformed tissue or cells, thus providing a method to obtain a transgenic plant.
  • the invention also concerns a method of attenutating or alleviating environmental stress in a plant, plant cell or tissue comprising (a) contacting a plant, plant cell or tissue which has been exposed to an environmental stress (such as drought, excess temperature, diminished temperature, chemical toxicity [e.g., antibiotic, herbicides], pollution, excess light, and diminished light) with an effective amount of the compounds or compositions of the invention, and (b) incubating said plant, plant cell or tissue under conditions sufficient to attenuate or alleviate said stress.
  • an environmental stress such as drought, excess temperature, diminished temperature, chemical toxicity [e.g., antibiotic, herbicides], pollution, excess light, and diminished light
  • Figure 1 presents the chemical structure of IAA where R x - R 5 are hydrogen and the numbers (l)-(7) represent the numbering pattern for the IAA chemical structure.
  • Figure 2 presents the chemical structures of some halogenated IAA auxinic analogues.
  • Figure 3 presents the chemical structures of some mono ⁇ substituted, alkyl-IAA auxinic analogues having an alkyl group in the 4 position.
  • the present invention also contemplates alkyl-IAA compounds having the same alkyl substituent group at position 2, 5, 6 or 7.
  • Exemplified in Figure 3 are alkyl-IAA structures having an alkyl (R) group with 1-4 carbon atoms.
  • the instant invention provides IAA analogues with alkyl substituents with 1-10 carbon atoms.
  • Figure 4 presents the chemical structures of some mono- substituted, alkoxy-IAA auxinic analogues having an alkoxy group in the 4 position.
  • the present invention also contemplates alkoxy-IAA compounds having the same alkoxy substituent group at position 2, 5, 6 or 7.
  • Exemplified in Figure 4 are alkoxy-IAA structures having an alkoxy group with 1-4 carbon atoms. The instant invention, however, provides
  • IAA molecules with alkoxy substituents with 1-10 carbon atoms are provided.
  • Figure 5 presents the chemical structures of some mono- substituted, acyl-IAA auxinic analogues having an acyl group in the 4 position.
  • the present invention also contemplates acyl-IAA compounds having the same acyl substituent group at position 2, 5, 6 or 7.
  • Exemplified in Figure 5 are acyl-IAA structures having an acyl group with 1-4 carbon atoms.
  • the instant invention provides acyl-substituted IAA molecules having acyl groups with 1-10 carbon atoms.
  • Figure 6 presents the chemical structures of some mono ⁇ substituted, acylamido-IAA auxinic analogues having an acylamido group in the 4 position.
  • the present invention also contemplates acylamido-IAA compounds having the same acylamido substituent group at position 2, 5, 6 or 7.
  • Exemplified in Figure 6 are acylamido-IAA structures having an acylamido group with 1-4 carbon atoms.
  • the instant invention provides acylamido-substituted IAA molecules having acylamide groups with 1-10 carbon atoms.
  • Figure 7 presents the chemical structures of some mono- substituted, acyloxy-IAA auxinic analogues having an acyloxy group in the 4 position.
  • the present invention also contemplates acyloxy-IAA compounds having the same acyloxy substituent group at position 2, 5, 6 or 7.
  • Exemplified in Figure 7 are acyloxy-IAA structures having an acyloxy group with 1-4 carbon atoms.
  • the instant invention provides acyloxy substituted IAA molecules having acyloxy groups with 1-10 carbon atoms.
  • Figure 8 documents tomato plant growth with increasing concentrations of 6-BrIAA (0.5, 2.0 and 8.0 mg/l) in the absence (top row) and in the presence (bottom row) of BAP (0.5 mg/l) on the formation of roots (top row) and calli (bottom row) .
  • Figure 9 documents potato plant growth with increasing concentrations of 6-BrIAA (0.5, 2.0 and 8.0 mg/l) in the absence (top row) and in the presence (bottom row) of BAP (0.5 mg/l) on the formation of roots (top row) and calli (bottom row) .
  • Figure 10 documents tobacco plant growth with increasing concentrations of 6-BrIAA (0.5, 2.0 and 8.0 mg/l) in the absence (top row) and in the presence (bottom row) of BAP (0.5 mg/l) on the formation of roots and calli (top rcw) and shoots and calli (bottom row) .
  • Figure 11 documents cassava plant growth in the presence of 5-FIAA or 7-FIAA (2.0 mg/l) and BAP (0.5 mg/l) on shoot formation.
  • IAA indole-3-acetic acid
  • R,-R 5 hydrogen
  • This term refers not only to the free acid form but also to an amide, an ester or a salt form of IAA. Included in the meaning of IAA are, for example, such salt and ester derivatives as the sodium, potassium, ammonium, dimethylamine, ethanolamine, etc. salts and amides and the lower alkyl esters.
  • monosubstituted IAA refers to an IAA molecule of Figure 1 where one or the Rj-R 5 groups represents a halo-, an alkyl-, an alkoxy-, an acyl-, an acylamido- or an acyloxy- substituent group at the 2, 4, 5, 6 or 7 position in the IAA chemical structure.
  • multi-substituted IAA refers to an IAA molecule of Figure 1 where two or more of the Ri-Rj groups represent the same or different halo-, alkyl-, alkoxy-, acyl-, acylamido- or acyloxy-substituent group in at least two of the positions corresponding to the 2, 4, 5, 6 or 7 position in the IAA chemical structure.
  • auxinic analogue(s) of IAA or IAA analogue or IAA auxinic analogue as used herein refers to a mono- or multi-substituted IAA that comprises, for example, one or more of tha groups including a halo-, an alkyl-, an alkoxy-, an acyl-, an acylamido-, an acyloxy- and the like.
  • the analogues include not only the free acid form but also an amide, an ester or a salt form of the mono- or multi-substituted IAA analogues.
  • a halo-group refers to a halogen including, but not limited to, iodo-, bromo-, chloro- and fluoro-groups.
  • An alkyl-group includes, but is not limited to, an alkyl, R-, (linear, branched or cyclic; saturated or unsaturated) , wherein R has 1-10 carbon atoms.
  • An alkoxy-group includes, but is not limited to, an alkoxy, R-O- (linear, branched or cyclic; saturated or unsaturated) , wherein R has 1-10 carbon atoms.
  • An acyl-group includes, but is not limited to, an acyl, R-C(O)- (linear, branched or cyclic; saturated or unsaturated) , wherein R has 1-10 carbon atoms.
  • An acylamido-group includes, but is not limited to, an acylamido, R-C(0)-NH- (linear, branched or cyclic; saturated or unsaturated) , wherein R has 1-10 carbon atoms.
  • An acyloxy-group includes, but is not limited to, an acyloxy, R-C(0)-0- (linear, branched or cyclic; saturated or unsaturated) , wherein R has 1-10 carbon atoms.
  • plant growth regulator or hormone refers to a naturally occurring or synthetic compound that acts as a hormone in regulating plant growth.
  • Plant growth regulators are exemplified by auxins, cytokinins and gibberellins.
  • auxin or cytokinin as used herein refers to a plant growth regulator that affects the growth of plants.
  • An auxin is exemplified by a compound such as indole-3-acetic acid (IAA) , indole-3-butyric acid (IBA), 2,4- dichlorophenoxyacetic acid (2,4-D), naphthaleneacetic acid
  • a cytokinin is exemplified by a compound such as 6- benzyiamino purine (BAP) , N 6 - ( ⁇ 2 isopentenyl) adenine (2iP) , isopentenylpyrophosphate (ipp), 6-(4-hydroxy-3-methyl-2- transbetenylamino)purine (zeatin) , 6-furfurylaminopurine
  • a compound can be tested for auxin activity using a bioassay, e.g., the elongation of coleoptiles of Avena sativa (Bottger et al. (1978) Planta 140:89) or the root growth inhibition of Chinese cabbage (Maru o et al. (1974) in Plant Growth Substance, p. 419, Hirokawa Publishing Co., Inc., Tokyo) or the hypocotyl swelling of mung bean (Marumo et al. (1974) supra) .
  • a bioassay e.g., the elongation of coleoptiles of Avena sativa (Bottger et al. (1978) Planta 140:89) or the root growth inhibition of Chinese cabbage (Maru o et al. (1974) in Plant Growth Substance, p. 419, Hirokawa Publishing Co., Inc., Tokyo) or the hypocotyl swelling of mung bean (Marumo et al. (1974) supra
  • Cytokinin activity may be measured in assays designed to evaluate the promotion of growth in plants (e.g., tobacco bioassays, etc.) as is well known in the art (Skoog et al. 1967) Phytochem 6:1169-1192; Morris (1986) Ann. Rev. Plant Physiol. 37:509-538; Horgan (1984) in Advanced Plant Physiol (Wilkins, M.B., ed.) pp. 53- 75, Pitman Publishing, London; Letham and Palni (1983) Ann. Rev. Plant Physiol 34:163-197; and Chen (1981) in Metabolism and Molecular Activities of Cytokinins (Guern, J. and Peaud- Lenoel, C.
  • cytokinin/auxin concentration ratio cause the enhancement in plant growth to occur preferentially in certain tissues.
  • a high cytokinin/auxin ratio promotes growth of shoots
  • a low cytokinin to auxin ratio promotes the growth of roots (Depicker et al. (1983) in Genetic Engineering of Plants, T. Kosunge, CP. Meredith and A. Hollaender, eds., Plenum Press, New York, p. 154) .
  • medium refers to a solid or liquid comprising nutrient sufficient to support plant cell growth, the regeneration of plant cells and tissues, and the transformation of plant cells and tissues.
  • carrier refers to a chemically- or biologically- or physiologically-acceptable molecule that is hydrophobic of hydrophilic or amphoteric and that is useful in facilitating the eff ctiveness of an active ingredient (i.e., an IAA analogue of the invention) in a plant.
  • a plant as used herein refers to a whole plant or a part of a plant comprising, for example, a locus of a plant, a cell of a plant, a tissue of a plant, an explant, or seeds of a plant.
  • This term further contemplates a plant in the form of a suspension culture or a tissue culture including, but not limited to, a culture of calli, protoplasts, embryos, organs, organelles, etc.
  • transformed plant or transformed plant tissues refers to introduction of a nucleic acid molecule, e.g. , native of foreign DNA, into a plant or plant tissue by transformation or protoplast fusion.
  • transgenic plant or transgenic plant tissue refers to a plant or plant tissue stably transformed with a foreign gene.
  • transient expression refers to a plant or plant tissue transformed with a DNA, where that DNA is expressed only for a short period of time immediately after transformation.
  • genetic engineering refers to the introduction of foreign, often chimeric, genes into one or more plant cells which can be regenerated into whole, sexually competent, viable plants which can be self-pollinated or cross-pollinated with other plants of the same species so that the foreign gene, carried in the germ line, can be inserted into or bred into agriculturally useful plant varieties.
  • regeneration refers to the production of at least one newly developed or regenerated plant tissue, e.g., root, shoot, callus, etc., from a cultured plant tissue or unit, e.g., leaf disc, seed, etc.
  • percent regeneration. % regeneration or regeneration efficiency as used herein refer to the number of tissue cultured plant units producing at least one newly developed or regenerated tissue as a percentage of the total number of tissue cultured plant units, e.g., fnumber of leaf discs with shoots X 100) . total number of leaf discs
  • affecting plant growth or growth affecting or affector or affect refer to any one of a number of plant responses which improve or change, relative to what is observed in the absence of the growth regulator, some characteristic of overall plant growth, for example, stimulation of seed germination, inducing rooting, suppressing shooting, promoting cell proliferation, stimulating callus growth, etc.
  • effective amount refers to the amount or concentration of a compound that is a plant growth regulator or hormone administered to a plant such that the compound stimulates or invokes one or more of a variety of plant growth responses.
  • a plant growth response includes, among others, the induction of stem elongation, the promotion of root formation, the stimulation of callus formation, enhancement of leaf growth, stimulation of seed germination, increase in the dry weight content of a number of plants and plant parts, and the like.
  • the present invention relates to the discovery that mono and multi-substituted IAA analogues have utility as auxins in affecting plant growth.
  • IAA analogues have utility as auxins in affecting plant growth.
  • cytokinin For example, in combining 5-bromo-IAA with cytokinin, both callus and shco formation are observed [disclosed in copending U.S. application serial no. 08/430,209 filed April 27, 1995].
  • IAA auxinic analogues are compared to IAA in functioning as an auxin in both monocots and dicots. For example, it was found that 5-bromo-IAA was between two and four times more effective than IAA in stimulating the regeneration of green calli from Arabidopsis thaliana .
  • Growth affecting compositions of the present invention comprise an IAA analogue, or a mixture of an IAA analogue and one or more additional plant growth regulators, such as cytokinin, gibberellin or the like, mixed with a carrier or auxiliary nutrients.
  • additional plant growth regulators such as cytokinin, gibberellin or the like
  • BAP, 2iP and kinetin with an IAA analogue is also exemplified in particular embodiments of this invention. It is contemplated that other cytokinins or other plant growth regulators known to the art can be utilized with an IAA analogue to make a growth affecting composition of the invention.
  • cytokinin or a different plant growth regulator can be admixed with an IAA analogue to make a growth enhancing composition of the invention.
  • plant growth regulators are known to the art and include, but are not limited to, BAP, 2iP, ipp, zeatin, kinetin, gibberellin, and the like, as described in Skoog et al. (1967) Phytochemistry 6:1169-1192 and Theologis (1989) in Plant Biotechnology (Kung and Arntzen, eds.) Butterworth Publishers, Stoneham, MA.
  • various IAA analogues were screened for auxinic activity by incubating different plant tissues, e.g., tobacco and tomato leaf discs and potato stems in (a) MS complete medium (obtained from Life Technologies, Inc., Gaithersburg, MD) containing different concentrations of auxin only and (2) the MS complete auxin medium containing different ratios of cytokinin/auxin.
  • MS complete medium obtained from Life Technologies, Inc., Gaithersburg, MD
  • tomato leaf discs, tobacco leaf discs and potato stems were incubated in the MS complete medium comprising different amounts (i.e., 0.5, 2.0 and 8.0 mg/l) of 6-BrIAA in the absence and presence of a cytokinin, e.g., BAP (0.5 mg/l) .
  • a cytokinin e.g., BAP (0.5 mg/l)
  • Figure 9, upper row shows the direct correlation between root formation from tomato leaf discs and increasing concentration of 6- BrlAA.
  • IAA analogues were screened for auxinic activity.
  • IAA derivative structures such as 2-bromoindole-3-acetic acid (2-BrIAA) , 6-bromoindole- 3-acetic acid (6-BrIAA) , 7-bromoindole-3-acetic acid (7- BrlAA) , 5-chloroindole-3-acetic acid (5-C1IAA) , 5- fluoroindole-3-acetic acid (5-FIAA) , 7-fluoroindole-3-acetic acid (7-FIAA) , 5-iodoindole-3-acetic acid (5-IIAA) , 5- ethylindole-3-acetic acid (5-EtIAA) , 7-ethylindole-3-acetic acid (7-EtIAA) , and 5-methoxyindole-3-acetic acid (5-MeOIAA) were tested for the ability to function as an auxin in the regeneration of plant tissues in
  • Roots Callus Roots Callus Roots Callus Roots Callus
  • IAA derivatives were further evaluated for auxinic activity in the presence of a cytokinin.
  • Table 2 indicates the ability of the different IAA analogues (at a concentration of 2 mg/l) in the presence of BAP (at a concentration of 0.5 mg/l) to stimulate the regeneration of shoots and/or calli from tobacco, tomato and potato. Incubation of plant tissues TABLE 2. Regeneration of shoots or calli from different plant tissues using IAA analogues together with cytokinin
  • halogenated IAA analogues were comparable to IAA in inducing the regeneration of calli in tobacco, while alkyl-IAA and alkoxy-IAA analogues showed auxinic activity similar to that of IAA in tomato and potato under these conditions. These IAA analogues were further evaluated for their abilities to effect the regeneration of plant tissues comprising foreign DNA.
  • Tobacco and potato tissues were subjected to Agrobacterium-mediated transformation techniques, including cocultivation with Agrobacterium containing pBI121 and selecting with MS complete medium containing 0.5 mg/l BAP, 2 mg/l IAA derivative and 100 mg/l kano ycin.
  • Table 3 indicates that the IAA analogues tested (7-BrIAA, 5-C1IAA, 5- FIAA, 7-FIAA, 5-EtIAA, 7-EtIAA and 5-MeOIAA) , in the presence of BAP, stimulated the regeneration of kanamycin resistant calli in transformed tobacco and potato, and shoots in transformed tobacco.
  • halogenated-IAA, alkyl-IAA and alkoxy-IAA derivatives exhibited IAA-type auxinic activities in transgenic plant tissues.
  • examples of IAA analogues of the invention were evaluated for auxinic activity in the presence of a cytokinin.
  • Table 4 indicates the ability of 2-BrIAA, 6-BrIAA, 7-BrIAA, 5- FIAA, 5-EtIAA and 7-EtIAA (at a concentration of 2 mg/l) in the presence of 0.5 mg/l of BAP to stimulate the regeneration of shoots and callus from cassava leaves and stems. It was also shown that 5-FIAA promoted the formation of shoots in cassava stems and to a greater extent from cassava leaves.
  • IAA analogues of the present invention were applied to the regeneration of plant tissues known in the art to be difficult to regenerate, such as cassava, woody plants, and monocotyledonous crops [Vasil and Vasil (1994) in Plant Cell and Tissue Culture (Vasil and Thorpe, eds.) , Kluwer Academic Publishers, Dordrech, Netherlands; Chee (1995) Plant Cell Reports 14:753- 757; Burns and Schwartz (1996) Plant Cell Reports 15:405-408; Mihaljevic et al. (1996) Plant Cell Reports 15:610-614; Schopke et al. (1996) Nature Biotechnology 14:731].
  • Figure 11 documents the regeneration of shoots in cassava leaves and stems incubated in MS complete medium comprising 2 mg/l of 5- FIAA and 0.5 mg/l BAP. TABLE 3. Regeneration of Agrobacterium-mediated transformed plant tissues using IAA analogues
  • the cassava cissue is transferred from a medium containing a high amount of auxin to another medium containing auxin and cytokinin.
  • the regeneration of shoots from cassava tissue was obtained without tissue transfer from a high auxin medium to a medium containing auxin and cytokinin.
  • the regeneration of shoots in cassava tissue, according to the invention showed significant improvement in the number of shoots regenerated.
  • the IAA analogues of the present invention not only exhibit auxinic activity but also improve the yield of plant tissue regenerated, as exemplified in plant tissues traditionally described as being difficult to regenerate, e.g., cassava, woody plants, maize, soybean, wheat, etc.
  • the practice of the present invention contemplates a wide variety of plant growth responses, including stimulation of seed germination and breaking of dormancy; increasing yields; hastening ripening and color production in fruit; increasing flowering and fruiting; stimulating shoot formation; inducing callus development; inducing rooting and causing cell proliferation; increasing the hardiness of various plant species; and increasing the dry weight content of a number of plants and plant parts.
  • any other modification of a plant, seed, fruit or vegetable so long as the net result is to increase the growth or maximize any beneficial or desired property of the agricultural and horticultural crop or seed, is intended to be included within the scope of advantageous responses achieved by the practice of the present invention.
  • compositions of the present invention are further utilized for plant regeneration from transgenic plants.
  • Genetic engineering of plants generally involves two complementary processes.
  • the first process involves the genetic transformation of one or more plant cells of a specifically characterized type.
  • transformation it is meant that a foreign gene, typically a chimeric gene construct, is introduced into the genome of the individual plant cells, typically through the aid of a vector which has the ability to transfer the gene of interest into the genome of the plant cells in culture.
  • the second process then involves the regeneration of the transformed plant cells into whole sexually competent plants. Neither the transformation nor regeneration process need be 100% successful but must have a reasonable degree of reliability and reproducibility so that a reasonable percentage of the cells can be transformed and regenerated into whole plants.
  • the two processes, transformation and regeneration, must be complementary.
  • the complementarity of the two processes must be such that the tissues which are successfully genetically transformed by the transformation process must be of a type and character, and must be in sufficient health, competency and vitality, so that they can be successfully regenerated into whole plants.
  • the invention envisions the genetic transformation of tissues in culture derived from leaf discs or hypocotyl explants.
  • the transformed tissues can be induced to form plant tissue structures, which can be regenerated into whole plants.
  • the transformation technique of the present invention is one which makes use of the Ti plasmid of A . tumefaciens.
  • an A . tumefaciens culture it is most advantageous to use a non-oncogenic strain of the Agrobacterium as the vector carrier so that normal non-oncogenic differentiation of the transformed tissue is possible.
  • the chimeric construction including a foreign gene of interest must contain a promoter which is effective in plant cells to cause transcription of the gene of interest and a polyadenylation sequence or transcription control sequence also recognized in plant cells.
  • Promoters known to be effective in plant cells include the nopaline synthase promoter, isolated from the T-DNA of Agrobacterium, and the cauliflower mosaic virus 35S promoter. Other suitable promoters are known in the art. It is also preferred that the vector which harbors the foreign gene of interest also contain therein one or more selectable marker genes so that the transformed cells can be selected from non- transformed cells in culture. In many applications, preferred marker genes include antibiotic resistance genes so that the appropriate antibiotic can be used to segregate and select for transformed cells from among ceils which are not transformed.
  • the details of the construction of the vectors containing such foreign genes of interest are known to those skilled in the art of plant genetic engineering and do not differ in kind from those practices which have previously been demonstrated to be effective in tobacco, petunia and other model plant species.
  • the foreign gene should obviously be selected as a marker gene (Jefferson et al. (1987) EMBO J. 6:3901-3907) or to accomplish some desirable effect in plant cells. This effect may be growth promotion, disease resistance, a change in plant morphology or plant product quality, or any other change which can be accomplished by genetic manipulation.
  • the chimeric gene construction can code for the expression of one or more exogenous proteins, or can cause the transcription of negative strand RNAs to control or inhibit either a disease process or an undesirable endogenous plant function.
  • tissues are seeds, the seeds are then allowed to germinate on an appropriate germinating medium containing a fungicide.
  • the hypocotyl portion of the immature plant is removed and sectioned into small segments averaging approximately 0.5 centimeters apiece. The hypocotyl explants are allowed to stabilize and remain viable in a liquid or agar plant tissue culture medium.
  • the tissues can promptly be inoculated with a suspension culture of transformation competent non-oncogenic Agrobacterium.
  • the inoculation process is allowed to proceed for a short period, e.g., two days, at room temperature, i.e., 24°C.
  • the remaining treated tissues can be transferred to a selective agar medium, which contains one or more antibiotics toxic to Agrobacterium but not to plant tissues, at a concentration sufficient to kill any Agrobacterium remaining in the culture.
  • Suitable antibiotics for use in such a medium include carbenicillin, cefotaxi e, etc. as the bactericide for Agrobacterium and kanamycin as the selective antibiotic for transformed plant tissues.
  • tissue culture medium which, in addition to its normal components, contains a selection agent.
  • the selection agent exemplified herein by kanamycin, is toxic to non-transformed cells but not to transformed cells which have incorporated genetic resistance to the selection agent and are expressing that resistance.
  • a suitable tissue culture medium is the MS medium to which are added an auxinic analogue of the invention and a cytokinin, with or without antibiotics.
  • the surviving transformed tissues are transferred to a secondary medium to induce tissue regeneration.
  • the surviving transformed tissue will thus continue to be regenerated into a whole plant through the regeneration technique of the present invention or through any other alternative plant regeneration protocols.
  • compositions employed in the practice of the present invention will depend upon the type of response desired, the formulation used and the type of plant treated.
  • the invention contemplates the use of a ratio of cytokinin concentration to auxin concentration of between approximately 50.0 and 0.001, and preferably between approximately 5.0 and 0.05, and more preferably between approximately 2.0 and 0.25.
  • the chemical compounds employed as active components of the growth enhancing compositions of the present invention may be prepared in accordance with processes well known in the prior art or may be obtained commercially from readily available sources.
  • the IAA analogues of the invention are useful in making a plant less susceptible to the toxicities of antibiotics. Such IAA analogues are also useful in enabling plants to overcome stress, e.g., environmental stress, physical stress, chemical stress, pollution, contamination, drought, light, and the like.
  • compositions may be applied at any developmental stage of the plant species to obtain plant hormone or maintenance effects throughout maturity and to expedite regrowth in damaged tissues during early developmental stages, depending upon the concentration used, the formulation employed and the type of plant species treated.
  • compositions of the present invention are preferably used in conjunction with specific auxiliary nutrients or other plant growth regulators in precise proportions to achieve a particular synergistic, growth enhancing response in various type of plants.
  • the present compositions may additionally be used in association with fungicides to increase the disease resistance of various plants, making the plant tissue resistant to invasion by pathogens by influencing the enzyme and plant processes which regulate natural disease immunity.
  • the present compositions possess essentially no phytotoxic activity of their own, they may sometimes be used in conjunction with herbicides to stimulate the growth of unwanted plants in order to make such plants more susceptible to a herbicide.
  • results achieved in the practice of the present invention as growth enhancing responses in agricultural and horticultural crops, as well as perennial and annual household plants species.
  • substituted anilines are available and can be used as starting materials to synthesize corresponding substituted phenylhydrazines which, in turn, can be converted to substituted IAA compounds.
  • Substituted anilines can be converted to corresponding substituted phenylhydrazines by the method of Robinson (1957) Can. J.Che . 35:1570.
  • Both a 4-halo-IAA and a 6-halo-IAA compound can be synthesized individually by the method of Majima and Hoshino (1925) Ber 58:2042 as described by Fox and Bullock (1951) J. Am. chem. Soc. 73:2756.
  • the 4- and 6-chloroindolylmagnesium iodide complexes are condensed with chloroacetonitrile and the resulting nitriles are hydrolyzed to the corresponding indole-3- acetic acids.
  • the protocol for the synthesis of a monosubstituted halogen-IAA comprises the following steps.
  • a substituted phenylhydrazine HCl (0.05 mole) is dissolved in 30% acetic acid, pH 4.0, and is added to 0.1 mole 3-formylpropionic acid solution that is freshly-prepared (See below) .
  • the precipitate is collected and dissolved in 75 ml pyridine.
  • 100 ml of concentrated HCl and 25 ml of 85% H 3 P0 4 are added and the resultant solution is refluxed for 10 hours in the dark under N 2 .
  • the reaction mixture is diluted with 600 ml H 2 0, filtered and the filtrate is extracted with ether several times.
  • the ether fractions are pooled and washed with water.
  • the indole acetic acid is extracted back into 0.5 M NaOH (200 ml) , boiled and precipitated with concentrated HCl (pH 1.0). Soapy tars are decanted or filtered off.
  • the substituted IAA is recrystallized twice from H 2 0, H 2 0/ethanol, toluene or ethyl acetate/hexane.
  • 3-formylpropionic acid is freshly prepared by adding 200 ml of fresh 1 M NaOCl solution to 24.9 g (0.20 mole) of glutamic acid in 400 mi of 0.5 N NaOH solution, stirring until it gives a negative test with starch-iodide paper and is then acidified by adding 70 ml of 3N HCl.
  • Multi-halogenated IAA compounds can be synthesized according to the guidelines provided by the methods of Engvild (1977) Acta Chem. Scand. B3l:338 (e.g., 4,6-, 4,7-, 5,7-, 6,7- dichloro-IAAs) , or the method of Baldi et al. (1985) J. Label. Compd. Radiopharm. 22:279 (e.g., 5,6-, 4,5-dichloro-IAAs) or the method of Hatano et al. (1987) Experientia 43:1237-1239 (e.g., 5,6-, 6,7-, 4,5-, 4,6-, 5,7-, 4 , 7-dichloro-IAA) , etc.
  • Experientia 43:1237-1239 e.g., 5,6-, 6,7-, 4,5-, 4,6-, 5,7-, 4 , 7-dichloro-IAA
  • Multi-substituted IAA analogues have 2 or more different substituents selected from halo-, alkyl-, alkoxy-, acyl-, acylamido-, acyloxy-substituent groups can be synthesized using synthetic techniques well known in the art [Vasil and Vasil (1994) in Plant Cell and Tissue Culture (Vasil and Thorpe, eds.), Kluwer Academic Publishers, Dordrech, Netherlands; Chee (1995) Plant Cell Reports 14:753-757; Burns and Schwartz (1996) Plant Cell Reports 15:405-408; Mihaljevic et al. (1996) Plant Cell Reports 15:610-614; Schopke et al. (1996) Nature Biotechnology 14:731).
  • IAA compounds comprising combinations of alkyl-, halo- and acyl-substituent groups (e.g., 2-methyl-5,7-dichloro-IAA, 2-COOH-5-methyl-IAA, 2-COOH-7-chloro- IAA, etc.) can be prepared according to Fox and Bullock (1951) J. Am. Chem. Soc. 73:2756-2759; Hoffman et al. (1952) J. Biol. Chem. 196:437 and Engvild (1977) Acta Chem. Scand. B31:338-344, etc.
  • alkyl-, halo- and acyl-substituent groups e.g., 2-methyl-5,7-dichloro-IAA, 2-COOH-5-methyl-IAA, 2-COOH-7-chloro- IAA, etc.
  • Nicotiana tobaccum Xanthi Seeds of Nicotiana tobaccum Xanthi are provided by Dr. James Saunders (USDA, Beltsville, MD) . Young tobacco leaves are removed and cut into small pieces. The explants are incubated on (1) MS complete medium (obtained from Life Technologies, Inc., Gaithersburg, MD) containing different concentrations of auxin related compounds only or (2) MS complete medium containing different concentrations of auxin-related compounds and 0.5 mg/l benzylaminopurine (BAP) using an 18 h light/6 h dark cycle until the formation of green calli and shoots.
  • MS complete medium obtained from Life Technologies, Inc., Gaithersburg, MD
  • BAP benzylaminopurine
  • Tomato seeds of variety "Moneymaker” are provided by Dr. James Saunders (USDA, Beltsville, MD) . Young tomato leaves are removed and cut into small pieces. Explants are incubated in MS complete medium with auxin in the presence or absence of cytokinin as described in section (a) above for tobacco.
  • Seeds of Arabidopsis thaliana ecotypes Columbia and Landersberg ereta are provided by Dr. Keith Davis (The Ohio State University, Columbus) . Tissues of hypocotyl are removed from 10-day-old seedlings, transferred to MS complete medium containing (1) different concentrations of IAA cr IAA analogue with different concentrations of N b - ( ⁇ ,Isopentenyl) adenine (2iP) and (2) different concentrations of IAA analogue with different concentrations of 2iP, BAP, or kinetin. The explants are incubated at 23°C using an 18 h light/6 h dark cycle until the formation of green calli and shoots.
  • Seeds of Oryza sativa cv . Orion are kindly provided by Dr. James Saunders (USDA, Beltsville, MD) .
  • the surfaces of the seeds are sterilized as follows: mature seeds are soaked in 0.5% detergent with shaking for 1 h, transferred to a solution containing 20% bleach and 0.1% Tween20® and vacuumed with shaking. The seeds are then rinsed with sterilized distilled water three times. At this point the seeds are transferred onto a MS complete medium containing different concentrations of BAP and different concentrations of IAA analogue, incubated in the dark at 25°C for 1 month, and then incubated at 25°C using an 18 h light/6 h dark cycle until the formation of green calli and shoots.
  • the regeneration of plant tissues using tissue culture depends on plant hormones such as auxin and cytokinin. It is known that the presence of an auxin in plant tissue cultured on Murashige and Skoog (MS) medium (Murashige, T. and F. Skoog (1962) Physiol. Plant 15:473-497) stimulates the formation of root structure whereas the formation of callus is observed when not only the auxin but also a cytokinin complement the MS nutrient medium.
  • MS Murashige and Skoog
  • evaluation cf a test compound as a potential new auxin is performed by incubating tobacco leaf discs in (a) the MS complete medium containing different concentrations of auxin only and (b) the MS complete medium containing different ratios of cytokinin to auxin concentrations (cytokinin/-auxin) .
  • halogenated IAA compounds are chemically synthesized and tested for plant growth regulatory activity. As shown in Figure 2, the following compounds are tested for auxin activity: 2-iodo-IAA, 4-iodo-IAA, 5-iodo-IAA, 6-iodo-IAA, 7- iodo-IAA, 2-bromo-IAA, 4-bromo-IAA, 5-bromo-IAA, 6-bromo-IAA, 7- bromo-IAA, 2-fluoro-IAA, 4-fluoro-IAA, 5-fluoro-IAA, 6-fluoro- IAA and 7-fluoro-IAA.
  • each compound is compared to that of IAA. Different concentrations of each compound are tested for the ability to stimulate root, shoot and callus formation from tobacco and tomato leaf discs and potato stems. The ability to stimulate root formation is evaluated for each compound in the presence of different concentration ratios of cytokinin (e.g., BAP) to test compound.
  • cytokinin e.g., BAP
  • alkyl substituted IAA compounds are chemically synthesized and tested for plant growth regulatory activity. Shown in Figure 3 are nine monosubstituted IAA compounds having an alkyl substituent group at position 4.
  • the present invention also contemplates alkyl-IAA compounds having alkyl substituents with 1-10 carbon atoms and having the same or different alkyl substituent groups at position 2, 5, 6 or 7.
  • the biological activity of each compound is compared to that of IAA. Different concentrations of each compound are tested for the ability to stimulate root, shoot and callus formation from tobacco and tomato and tomato leaf discs and potato stems. The ability to stimulate root, shoot and callus formation is evaluated for each compound in the presence of different concentration ratios of cytokinin (e.g. , BAP) to test compound.
  • cytokinin e.g. , BAP
  • alkoxy-substituted IAA compounds are chemically synthesized and tested for plant growth regulatory activity. Shown in Figure 4 are nine monosubstituted IAA compounds having an alkoxy substituted group at position 4.
  • the present invention also contemplates alkoxy-IAA compounds having alkoxy substituent groups with 1-10 carbon atoms and having the same or different alkoxy substituent groups at position 2, 5, 6 or 7.
  • the biological activity of each compound is compared to that of IAA. Different concentrations of each compound are tested for the ability to stimulate root, shoot and callus formation from tobacco and tomato leaf discs and potato stems. The ability to stimulate root shoot and callus formation is evaluated for each compound in the presence of different concentration ratios of cytokinin (e.g., BAP) to test compound.
  • cytokinin e.g., BAP
  • acyl substituted IAA compounds are chemically synthesized and tested for plant growth regulatory activity. Shown in Figure 5 are nine monosubstituted IAA compounds having an acyl substituent group at position 4.
  • the present invention also contemplated acyl-IAA compounds having acyl substituents with 1- 10 carbon atoms and having the same or different acyl substituent groups at position 2, 5, 6 or 7.
  • the biological activity of each compound is compared to that of IAA. Different concentrations of each compound are tested for the ability to stimulate root, shoot and callus formation from tobacco and tomato leaf discs and potato stems. The ability to stimulate root formation is evaluated for each compound in the presence of different concentration ratios of cytokinin (e.g., BAP) to test compound.
  • cytokinin e.g., BAP
  • acylamido substituted IAA compounds are chemically synthesized and tested for plant growth regulatory activity. Shown in Figure 6 are nine monosubstituted IAA compounds having an acylamido substituent group at position 4.
  • the present invention also contemplated acylamido-IAA compounds having acylamido substituents with 1-10 carbon atoms and having the same acylamido substituent groups at position 2, 5, 6 or 7.
  • the biological activity of each compound is compared to that of IAA. Different concentrations of each compound are tested for the ability to stimulate root, shoot and callus formation from tobacco and tomato leaf discs and potato stems. The ability to stimulate root formation is evaluated for each compound in the presence of different concentration ratios of cytokinin (e.g., BAP) to test compound.
  • cytokinin e.g., BAP
  • acyloxy substituted IAA compounds are chemically synthesized and tested for plant growth regulatory activity. Shown in Figure 7 are nine monosubstituted IAA compounds having an acyloxy substituent group at position 4.
  • the present invention also contemplated acyloxy-IAA compounds having acyloxy substituents with 1-10 carbon atoms and have the same or different acyloxy substituent groups at position 2, 5, 6 or 7.
  • the biological activity of each compound is compared to that of IAA. Different concentrations of each compound are tested for the ability to stimulate root, shoot and callus formation from tobacco and tomato leaf discs and potato stems. The ability to stimulate root formation is evaluated for each compound in the presence of different concentration ratios of cytokinin (e.g., BAP) to test compound.
  • cytokinin e.g., BAP
  • multi-substituted IAA analogues di-, tri-, tetra- or penta-substituted IAA compounds, comprising between two and five substituent groups that are individually selected from the group consisting of a halo-, an alkyl-, an alkoxy-, an acyl-, an acylamido- and an acyloxy- substituent group in at least two of the positions corresponding to the 2, 4, 5, 6 or 7 position in the IAA chemical structure of Figure 1, are synthesized and tested for plant growth regulatory activity.
  • a halo- substituent group includes, but is not limited to, an iodo-, a bromo-, a fluoro- and a chloro-group;
  • an alkyl-substituent group includes, but is not limited to, a linear, branched or cyclic alkyl group, R, having 1-10 carbon atoms;
  • an alkcxy-substituent includes, but is not limited to, a linear, branched or cyclic alkoxy group, RO-, having 1-10 carbon atoms;
  • an acyl-substituent group includes, but is not limited to, a linear, branched or cyclic acyl group, R-C(O)-, having 1-10 carbon atoms;
  • an acylamido-substituent group includes, but is not limited to, a linear, branched or cyclic acylamido-group, R-C(0)-NH-, having 1-10 carbon atom
  • each multi-substituted IAA its auxinic activity is tested and compared to that of IAA.
  • Different concentrations of each compound are tested for the ability to stimulate root, shoot and callus formation from tobacco and tomato leaf discs and potato stems.
  • the ability to stimulate root formation is evaluated for each compound in the presence of different concentration ratios of cytokinin (e.g., BAP) to test compound.
  • cytokinin e.g., BAP
  • Example 3 The use of IAA auxinic analogues for stimulating the regeneration of transgenic plants. (a) Tobacco
  • Plant transformation is carried out according to the Agrobacterium-mediated transformation procedure essentially as described by Lin et al. [(1994) Focus 16:72-77)].
  • the leaf discs are incubated with 10 10 cells/ml Agrobacterium tumefaciens LBA4404 cells, containing pBI121 harboring the GUS reporter gene, in MS complete medium with 0.5 mg/l 2-(N-morpholino) ethanesulfonic acid (MES) for 10 min, transferred to solid MS complete medium, and incubated for 2 days at 25°C, using an 18 h light/6 h dark cycle for cocultivation.
  • MES 2-(N-morpholino) ethanesulfonic acid
  • the explants are transferred to MS madia containing different ratios of 3AP/IAA or BAP/IAA analogue, 100 mg/l kanamycin and 500 mg/l carbenicillin and incubated at 25°C using an 18 h light/6 h dark cycle for shoot formation.
  • Plant transformation is carried out according to the Agrobacterium-mediated transformation procedure essentially as described by Lin et al. (1994) Focus 16:72-77. Incubation conditions are as described above in section (a) for tobacco.
  • Plant transformation in potato ste s is carried out as described above in section (a) for tobacco.
  • Arabidopsis thaliana For Arabidopsis thaliana , tissues of hypocotyl are removed from 10-day-old seedlings and preincubated in MS complete medium containing 0.5 mg/l 2, 4-D and 0.5 mg/l kinetin for three days. The explants are immersed in 10 q cells/ml of Agrobacterium tumefaciens LBA4404 containing pBI121 for 20 min, and transferred to solid MS medium containing 500 mg/l carbenicillin, 50 mg/l kanamycin, and various ratios of IAA analogue/2iP or IAA/2iP for callus and shoot formation. Arabidopsis explants were incubated at 25°C, using an 18 h light/6 h dark cycle.
  • rice Oryza sativa
  • rice is transformed using the particle bombardment method of Wang et al. (supra) or the Agrobacterium- ediating technique of Hiel et al. (1994) Plant Journal 6:271 or, alternatively, using the electroporation method as described by Dekeyser et al. (1990) Plant Cell 2:591- 602.
  • Regeneration of transformed rice is performed according to Abdullah et al. (1986) Bio/Technology 4:1087-1909 or, alternatively, according to Raineri et al. (1990) Bio/Technology 8:33-38.
  • maize is transformed and regenerated according to the procedures of Rhodes et al. (1988) Bio/Technology 6:56-60 and (1988) Science 240:204-207.
  • an IAA auxinic analogue is used as the auxin to stimulate plant growth in accordance with the invention.
  • one or more additional plant growth regulators may be added to the IAA analogue-comprising plant growth compositions.
  • Non-Transgenic Plants as documented in Figures 8-10 and Tables 1. 2 and 4. Seeds of Nicotiana tobaccum Xanthi, potato and tomato were kindly provided by Dr. James Saunders (USDA, Beltsville) . Cassava plants were provided by Dr. Dick Sayer (The Ohio State University, Columbus, OH) .
  • the leaf discs were incubated with 10 9 cells/ml of Agrobacterium tumefaciens LBA4404 cells containing pBI121 in MS complete medium with 0.5 mg/l MES for 10 minutes, transferred to solid MS complete medium, and incubated for two days at 25°C, using an 13 hour light/6 hour dark cycle for cocultivation.
  • the explants were transferred to the MS medium containing a different ratio of BAP/IAA or BAP/IAA analogue, 100 mg/l kanamycin and 500 mg/l carbenicillin and incubated at 25°C using an 18 hour light/6 hour dark cycle for shoot formation.
  • potato leaf discs were transformed with the Agrobacterium-mediation technique as described above for tobacco.
  • the potato explants were then subjected to incubation with IAA analogues in the absence and presence of cytokinin to screen for auxinic activities.
  • Potato stems were excised as described above and incubated in the MS complete medium (a) with 25, 50 and 100 mg/l kanamycin and either with/without 2 mg NAA or auxin derivatives or with/without 0.5 mg/l BAP (a ctyokinin) . After one month incubation, potato stems incubated in 50 mg or 100 mg/l kanamycin with/without BAP only showed necrosis of stem which indicted the toxicity of kanamycin. However, potato stems incubated in the MS medium containing 2 mg/l NAA and 100 mg/kanamycin, became expanded ( Figure 12) . In some cases, the presence of auxin or auxin derivatives showed the formation of calli in some of the potato stems.
  • the stems incubated for one month with 50 mg/l kanamycin and 0.5 mg/l BAP were transferred into MS complete medium containing 2 mg/l NAA with/without 100 mg/l kanamycin, or MS medium containing 2 mg/l NAA, 0.5 mg/l BAP, and with/without 100 mg/l kanamycin.
  • the formation of calli on the potato stems was observed when the potato stems were transferred to the MS medium containing auxin or auxin derivative with/without BAP. However, all the potato stems were dead after continued incubation without the addition of auxin or auxin derivatives ( Figure 13) .
  • Tobacco leaves were removed from one month old plants and cut into small pieces (0.5 cm x 0.25 cm) . These tissues were incubated in the MS complete medium in 0.5 mg/l BAP overnight and bombarded with microprojectiles (1300 psi) (helium source, obtained from BioRad) . After bombardment, the tobacco explants were incubated in (1) MS complete medium with 0.5 mg/l BAP, and (2) MS complete medium with 0.5 mg/l BAP and 2 mg/l NAA for three days before transferring to MS complete medium with 0.5 mg/l BAP. Significant improvement of the regeneration of tobacco shoots were shown in those explants incubated in MS medium containing NAA and BAP compared to those shoots incubated in MS medium with BAP only.

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Abstract

Composés et compositions capables de stimuler la croissance des plantes, de régénérer des cellules et des tissus de plantes et de transformer des cellules et des tissus de plantes. Ces composés et compositions comprennent des analogues auxiniques monosubstitués et plurisubstitués d'acide indole-3-acétique (IAA) comprenant des groupes substituants tels que des groupes halo, alkyle, alcoxy, acyle, acylamido et acyloxy. Cette invention concerne un procédé d'utilisation de ces analogues auxiniques de IAA monosubstitués ou plurisubstitués pour modifier la croissance, la régénération ou la transformation dans des plantes monocotylédone et dicotylédone ainsi que dans des tissus de plantes transgéniques. Cette invention concerne également l'utilisation de ces analogues auxiniques d'IAA en présence d'autres régulateurs de croissance des plantes tels que la cytokinine, etc... pour améliorer la croissance des plantes.
EP96943547A 1995-11-30 1996-11-29 Analogues auxiniques d'acide indole-3-acetique Ceased EP0866850A4 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US777095P 1995-11-30 1995-11-30
US7770P 1995-11-30
PCT/US1996/019167 WO1997020034A1 (fr) 1995-11-30 1996-11-29 Analogues auxiniques d'acide indole-3-acetique

Publications (2)

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EP0866850A1 EP0866850A1 (fr) 1998-09-30
EP0866850A4 true EP0866850A4 (fr) 2000-11-22

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EP (1) EP0866850A4 (fr)
AU (1) AU1276497A (fr)
WO (1) WO1997020034A1 (fr)

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JP3943661B2 (ja) * 1996-07-11 2007-07-11 株式会社資生堂 インドール誘導体及びこれを有効成分とする発根誘導剤
AU2001245670A1 (en) * 2000-03-10 2001-09-24 Invitrogen Corporation Materials and methods for the regeneration of plants from cultured plant tissue
CN106256196B (zh) * 2015-11-20 2018-05-29 刘寒冬 高效诱导哥伦比亚型拟南芥愈伤组织的方法及诱导培养基
ES2636363B2 (es) * 2016-04-05 2018-05-07 Instituto Nacional De Investigación Y Tecnología Agraria Y Alimentaria (Inia) Uso de compuestos para la regulación del crecimiento vegetal

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WO1997020034A1 (fr) 1997-06-05
AU1276497A (en) 1997-06-19
EP0866850A1 (fr) 1998-09-30

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