JP2000501395A - Auxin analog of indole-3-acetic acid - Google Patents

Abstract

  (57) [Summary] The present invention provides compounds and compositions that can stimulate plant growth, regeneration of plant cells and tissues, and transformation of plant cells and tissues, including halo-, alkyl-, alkoxy-, acyl- Mono- or polysubstituted auxin analogs of indole-3-acetic acid (IAA), including substituents such as-, acylamide-, and acyloxy- groups. The present invention relates to methods of using such mono- and poly-substituted auxin analogs of IAA to affect the growth, regeneration, or transformation of monocot and dicot plants, as well as transgenic plant tissues. About. The present invention also contemplates the use of these auxin IAA analogs in the presence of other plant growth regulators (eg, cytokinins, etc.) to enhance plant growth.

Description

DETAILED DESCRIPTION OF THE INVENTION                   Auxin analog of indole-3-acetic acidCross-reference of related applications   This application is based on provisional application 60 / 007,770 filed on November 30, 1995, and April 27, 1995. Is a continuation-in-part of co-pending application Ser. Incorporated herein by reference.Field of the invention   The present invention relates to plant growth, regeneration of plant cells and tissues, and plant cell morphology. Indole-3-acetic acid (IAA) analogues as plant hormones stimulatory for quality inversion Regarding the use of The invention particularly relates to the use of mono- and polysubstituted IAA molecules. . The present invention also relates to compositions comprising the IAA analogs of the present invention.Background of the Invention   Plant growth is affected by various physical and chemical factors. physically Factors include available light, photoperiod, humidity, and temperature. Chemical Elements include minerals, nitrates, cofactors, nutrients, and plant growth regulators (Plant growth regulator) or hormones (eg, auxin, cytoca Inine, and gibberellin).   Indole-3-acetic acid (IAA) is a naturally occurring plant growth identified in plants Is a hormone. IAA increases plant growth in vivo and in vitro Has been shown to be a direct cause of Features affected by the IAA include: These include cell elongation, internode distance (height), leaf surface area, and crop yield. IAA And other compounds that exhibit hormone regulating activity similar to that of IAA, Included in the class of plant regulators called "auxins".   Cytokinins (eg, 6-furfurylaminopurine (kinetin) and 6-furfurylaminopurine (kinetin)) Preparations based on benzylaminopurine (BAP) are also growth stimulants (growth st imulator). However, preparations based on cytokinins Is most effective in combination with auxin. Cytokinins grow in plants The mechanisms that influence the cycle are far from understood, but they do affect leaf growth. And prevent aging of certain plants.   The general purpose of this field is to use methods such as tissue culture and genetic engineering. The successful operation and regeneration of plants of major crop species. About this Major crop species for specific purposes include corn, wheat, rice, soybean, and wheat. It is an agricultural product like ta.   In vitro culture techniques have been established for regenerating plants. (Reinert,  Edited by J. and Bajaj, Y.P.S., (1977)Plant Cell , Tissue and Organ Culture, Be rlin: Springer; Simmonds, N.W. (1979)Principles of Crop Improvement, Lon don: Longman; Vasil, I.K., Ahuja, M.K. and Vasil, V. (1979) Plant Tissue Culture In Plant Breeding "Adv.Genet. 20: 127-215). Regenerate plants Specific in vitro culture techniques include embryo culture, shoot apex culture, and Cultures of cells, cells, and protoplasts. Embryo culture is difficult interspecies Has been shown to be important in For clonal propagation, virus-free clone development, and conservation of genetic resources Is important. Culture of callus, cells, and protoplasts lose organization It has been shown to be important for cultures that can be recovered.   Genetic engineering techniques for plants have also been established. These techniques include traits Gene transfer by conversion or by protoplast fusion. gene The transfer involves the following steps: (a) identification of a particular gene; (b) isolation and And (c) transferring the gene into a recipient plant host cell; Integration, transcription, and translation of this DNA in transgenic cells; and (e) transgene Propagation and Use of Nick Plants (T. Kosuge, C.P. Meredith and A. Hollaender) (1983)Genetic Engineering of Plants, 26: 5-25; Rogers et al. (1988).Methods for Plant Molecular Biology [A. Weissbach and H.S. Weissbach edition] Academic  Press, Inc., San Diego, CA). In protoplast fusion, plant cell protoplasts Last uses standard chemical (e.g., PEG) or electroporation techniques. Fused by art. After regeneration of the fused cells, interdiploid diploids were obtained. This technique Surgery can provide the desired diploid that cannot be made by conventional means, and The possibility of somatic recombination by some of the mutants is presented. The above technology is , Widely used (Chaleff, R.S. (1981)Genetics of Higher Plants , Applications of Cell Culture , Cambridge: Cambridge University Press). So Therefore, the newly inserted foreign gene should be maintained stably during plant regeneration. And are transmitted to progeny as a typical Mendelian trait (Horsch (1984) Science 223: 496, and DeBlock et al. (1984) EMBO 3: 1681). this These foreign genes maintain their normal tissue-specific and developmental expression patterns. Carry.   Agrobacterium tumefaciens-mediated transformation systems are effective in many dicot species. Proven to be. For example, Barton et al. (1983, Cell 32: 1033) describe tobacco Plant transformation and regeneration have been reported and described in Chang et al. (1994, Planta 5: 551-558). ) Described stable genetic transformation of Arabidopsis thaliana.   The Agrobacterium method for gene transfer has also been applied to monocotyledonous plants (e.g., For example, plants of the families Liliaceae and Amaryllidaceae (Hooykaas-Van Slogteren et al., 1 984, Nature 311: 763-764) and Dioscorea bulbifera (yam) (Schafer Et al., 1987, Nature 327: 529-532). However, this method is a monocotyledonous plant of the grass family (This includes edible crops such as wheat, rice, and corn) The conversion did not seem to be effective.   Transformation of food crops can be performed, for example, by using polyethylene glycol (PEG) -enhanced DNA (Uchimiya et al. (1986) Mol. Gen. Genet. 204: 204-207) and electroporation Obtained by a different method by the authors (Fromm et al. (1986) Nature 319: 791-793). . Both methods used protoplasts as targets for transfer. Monocotyledonous and bibiotic Cotyledon plant tissue is transformed by bombardment of the tissue with DNA-coated particles (Wang et al. (1988) Plant Mol. Biol. 11: 433-439; Wu,Plant Biotechno logy (1989), edited by Kung and Arntzen, Butterworth Publishers, Stoneham, MA) . Regeneration is performed 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 2 Four 0: 204-207).   Thus, while regeneration and transformation protocols have been established, monocots and There remains a need to stimulate regeneration and transformation of dicotyledonous plants. In fact, Plants are difficult to regenerate and transform (Vasil and ~ asil (1994)Plant Cell and Tissue Culture (Vasil and Thorpe), Kluwer Academic Publishers, Do rdrech, Netherlands; Chee (1995) Plant Cell Peports 14: 753-757; Burns and And Schwartz (1996) Plant Cell Reports 15: 405-408; Mihaljevic et al. (1996) Pla. (nt Cell Reports 15: 610-614; Schopke et al. (1996) Nature Biotechnology 14: 731) . In addition, there is a need to stimulate plant growth, especially after transformation and regeneration.Summary of the Invention   The present invention relates to plant growth, regeneration of plant cells and tissues, and plant cells and tissues. By providing compounds and compositions that stimulate cell and tissue transformation. Meet these needs. The compounds of the present invention may be mono- or poly-substituted IAAs (indoles). -3-acetic acid), or an ester or salt derivative thereof. The present invention also provides one A composition comprising these IAA analogs and optionally a carrier is provided. You. The present invention relates to the growth, regeneration, and transformation of both monocots and dicots. The use of such auxin analogs to affect G.A. In particular, The present invention provides a monosubstituted IAA having a substituent at position 2, 4, 5, 6, or 7 of the IAA structure. Provide a nalog. Wherein the substitution is a halo- having 1 to 10 carbon atoms. Or alkyl-, alkoxy-, acyl-, acylamide-, and acyl Oxy-substituent. The invention also relates to positions 2, 4, 5, 6, or 7 of the IAA structure. Having from 2 to 5 identical or different substituents at different positions selected from Provides substituted IAA analogs. Here, the substituent has 1 to 10 carbon atoms. Halo- or alkyl-, alkoxy-, acyl-, acylamide-, And acyloxy-substituents.   The compositions of the present invention may comprise, in addition to one or more mono- or poly-substituted compounds, one or more Of additional plant growth regulators. Such plant growth regulators , For example, a clear ratio of cytokinins for widespread application to various plants, Gibberellin and the like. In certain embodiments, the present invention relates to growing a plant. At positions 2, 4, 5, 6, and / or 7 of the IAA structure to affect B-, alkyl-, alkoxy-, acyl-, acylamide-, or acyl- Monosubstitution with the same or different substituents between one and five being xy-substituents Or exemplified by compositions comprising polysubstituted IAA analogs, as well as cytokinins .   The present invention further relates to media containing the compounds and compositions of the present invention. like this Medium stimulates plant growth, stimulates plant cell and tissue regeneration, and One or more IAA analogs may be used to stimulate the transformation of plant cells and tissues. In addition, IAA analogs and plant growth regulators as needed (e.g., Cytokinin).   The invention also relates to a method of stimulating plant growth. The method comprises the steps of (a) Applying an effective amount of a compound or composition of the invention to the cells or tissues of the product, and Optionally, one or more additional plant growth regulators (e.g., cytokinin , Gibberellin, etc.), and (b) the plant cells or tissues Incubating under conditions sufficient to stimulate cell or tissue regeneration Include.   The present invention also provides a method for stimulating the regeneration of plant cells and / or tissues. provide. The method comprises the steps of: (a) providing an effective amount of a compound or composition of the present invention to plant cells. Or one or more additional plant growth regulators (eg, Tokainin, gibberellin, etc.) and (b) plant cells or tissues Incubated under conditions sufficient to stimulate plant cell or tissue regeneration The step of performing   The present invention also provides a method for stimulating the transformation of plant cells and / or tissues. Provide a way. The method comprises the steps of (a) transforming a plant cell or tissue (e.g., Contacting with a nucleic acid molecule (by protoplast fusion), (b) a compound of the invention or Or an effective amount of the composition, and optionally one or more additional plant growth regulators Quality (e.g., cytokinins, gibberellins, etc.) to plant cells or tissues. And (c) transforming a plant cell or tissue into a plant cell or tissue. Incubating with nucleic acid molecules under conditions sufficient to stimulate You. The compounds and compositions of the present invention can also be used to regenerate transformed tissues or cells. Or can be used to stimulate growth, thus obtaining a transgenic plant Provide a way to:   The present invention also provides for reducing the environmental stress of a plant, plant cells or tissues or How to mitigate. The method includes: (a) environmental stresses (e.g., drought, Temperature, too low temperature, chemical toxicity (eg antibiotics, herbicides), pollution, excessive light, Plants and cells or tissues of plants that have been exposed to Contacting with an effective amount of the compound or composition; and (b) the plant, plant cells Alternatively, incubate the tissue under conditions sufficient to reduce or alleviate this stress. In the process.Figure description   FIG.₁~ R_FiveShows the chemical structure of IAA where is hydrogen and the numbers (1)-( 7) shows the numbering pattern for the IAA chemical structure.   FIG. 2 shows the chemical structures of some halogenated IAA auxin analogs.   FIG. 3 shows some monosubstituted alkyl-IAA oximes having an alkyl group at the 4-position. 1 shows the chemical structure of an analog. The present invention also provides for the same alkyl substituent to be 2, 5, Alkyl-IAA compounds having a 6 or 7 position are contemplated. In FIG. Exemplified by an alkyl-IAA structure having an alkyl (R) group having three carbon atoms . However, the present invention is directed to IAA compounds having alkyl substituents having 1 to 10 carbon atoms. Provide a nalog.   FIG. 4 shows several mono-substituted alkoxy-IAA Au with an alkoxy group at the 4-position. 1 shows the chemical structure of a toxin analog. The present invention also provides the same alkoxy substituent with 2 Alkoxy-IAA compounds having positions 5, 6, or 7 are contemplated. In FIG. Exemplified by an alkoxy-IAA structure having an alkoxy group having 1 to 4 carbon atoms Is done. However, the present invention has alkoxy substituents having 1 to 10 carbon atoms. Provide IAA molecules.   FIG. 5 shows several mono-substituted acyl-IAA auxins having an acyl group at position 4. 1 shows the chemical structure of Nalog. The present invention also provides for the substitution of the same acyl substituent with 2, 5, 6, or Or acyl-IAA compounds having a 7-position. In FIG. 5, one to four carbons An acyl-IAA structure having an acyl group having an atom is exemplified. However, the present invention An acyl-substituted IAA molecule having an acyl group having 1 to 10 carbon atoms is provided. Offer.   FIG. 6 shows some monosubstituted acylamide-IAA having an acylamide group at position 4. 1 shows the chemical structure of an auxin analog. The invention also provides the same acylamide substituent Is intended at acyl positions 2, 5, 6, or 7. FIG. Wherein the acylamide-I having an acylamide group having 1 to 4 carbon atoms An AA structure is exemplified. However, the present invention relates to acylamido having 1 to 10 carbon atoms. An acylamide-substituted IAA molecule having a thiol group.   FIG. 7 shows some monosubstituted acyloxy-IAA having an acyloxy group at position 4. 1 shows the chemical structure of an auxin analog. The invention also relates to the same acyloxy substituent Are intended at 2, 5, 6, or 7 positions. FIG. Wherein acyloxy-I having an acyloxy group having 1 to 4 carbon atoms An AA structure is exemplified. However, the present invention relates to acyloxy having 1 to 10 carbon atoms. An acyloxy-substituted IAA molecule having a thio group is provided.   FIG. 8 shows BAP (0.5) based on root formation (top row) and callus formation (bottom row). mg / l) in the absence (top row) and in the presence (bottom row) of increasing concentrations of 6-BrIAA (0 Demonstrates the growth of tomato plants using .5, 2.0, and 8.0 mg / l).   FIG. 9 shows BAP (0.5) based on root formation (top row) and callus formation (bottom row). mg / l) in the absence (top row) and in the presence (bottom row) of increasing concentrations of 6-BrIAA (0 .5, 2.0, and 8.0 mg / l).   Figure 10 shows root and callus formation (top row) and shoot and callus formation Based on (bottom row), the absence (top row) and presence (bottom row) of BAP (0.5 mg / l) Of tobacco plants using increasing concentrations of 6-BrIAA (0.5, 2.0, and 8.0 mg / l) in Demonstrate growth.   FIG. 11 shows 5-FIAA or 7-FIAA (2.0 mg / l) and BAP based on shoot formation. (0.5 mg / l) demonstrates the growth of cassava plants in the presence.Detailed description of the invention   The following definitions are intended to describe their intent or scope in the specification and claims. Provided to clarify the enclosure.   the termIndole-3-acetic acidOrIAAIs, as used herein, R₁~ R_Five Is hydrogen. This term refers to the IAA as well as the free acid form. Also refers to the amide, ester, or salt form of IAA means, for example, Thorium, potassium, ammonium, dimethylamine, ethanolamine, etc. Salts and ester derivatives such as salts and amides and lower alkyl esters Is included.   the termMonosubstitute IAAIs, as used herein, R₁~ R_FiveOne of the groups is IAA Halo-, alkyl-, alkoxy-, at position 2, 4, 5, 6, or 7 in the chemical structure; Diagram showing acyl-, acylamide-, or acyloxy-substituent One IAA molecule.   the termPolysubstituted IAAIs, as used herein, R₁~ R_FiveTwo or more of the groups are I At least two positions corresponding to positions 2, 4, 5, 6, or 7 in the AA chemical structure Same or different halo-, alkyl-, alkoxy-, acyl-, acylamido Refers to the IAA molecule of FIG. 1 indicating a do- or acyloxy-substituent.   the termIAA Auxin analogOrIAA analogOrIAA Auxinanaro G Is used herein, for example, halo-, alkyl-, alkoxy -Including one or more groups, including-, acyl-, acylamide-, acyloxy-, etc. Refers to mono- or polysubstituted IAA.   As used herein, an analog is a mono- or polysubstituted IAA analog. It includes the amide, ester, or salt forms thereof as well as the free acid form.   Halo groups include iodo-, bromo-, chloro-, and fluoro- groups, but Halogen not limited to these.   Alkyl groups are alkyl, R- (linear, branched, or cyclic; saturated or unsaturated). Saturates). Here, R has 1 to 10 carbon atoms. I do.   Alkoxy groups include alkoxy, R-O- (linear, branched, or cyclic; saturated or Or unsaturated). Where R is 1-10 carbon sources Have children.   An acyl group can be acyl, R—C (O) — (linear, branched, or cyclic; saturated or Unsaturated). Here, R represents 1 to 10 carbon atoms Have.   Acylamide groups include acylamide, R—C (O) —NH— (linear, branched, Is cyclic; saturated or unsaturated). Where R is 1 Has ~ 10 carbon atoms.   Acyloxy groups include acyloxy, R—C (O) —O— (linear, branched, or Cyclic; saturated or unsaturated). Where R is 1 to Has 10 carbon atoms.   the termPlant growth regulator or plant growth hormoneIs the field used in this specification. Naturally occurring or acting as hormones in regulating plant growth Refers to a synthetic compound. Plant growth regulators include auxins, cytokinins, and And gibberellins.   the termauxinOrCytokininRefers to a plant as used herein A plant growth regulator that affects the growth of plants. Auxin, for example, India Toluene-3-acetic acid (IAA), indole-3-butyric acid (IBA), 2,4-dichlorophenoxyacetic acid (2,4-D), naphthaleneacetic acid (NAA), 5,6-dichloroindole-3-acetic acid (5,6-Cl_Two -IAA) and the like. Cytokinin is 6-benzyla Mino pudding (BAP), N⁶・ (Δ_TwoIsopentenyl) adenine (2iP), isopentenyl Lupirophosphate (ipp), 6- (4-hydroxy-3-methyl-2-transbethenyla) Mino) purine (zeatin), 6-furfurylaminopurine (kinetin), etc. Such compounds are exemplified. Compounds can be used in bioassays (eg, Avena sati Elongation of va cotyledon sheath (Bottger et al. (1978) Planta 140: 89) or root growth of Chinese cabbage Inhibition (Marumo et al. (1974)Plant Growth Substance, 419, Hirokawa Publishing  Co., Inc., Tokyo), or the swelling of the hypocotyl of the green crab (Marumo et al. (1974)Above)) Can be used to test for auxin activity. Cytokinin activity can be measured in the field Assays designed to assess growth promotion in plants, as is well known in (Eg, tobacco bioassays, etc.) ((Skoog et al., 196 7) Phytochem 6: 1169-1192; Morris (1986) Ann. Rev. Plant Physiol. 37: 5 09-538; Horgan (1984) Advanced Plant Physiol (Wilkins, M.B.), pp. 53-75, P. itman Publishing, London; Letham and Palni (1983) Ann. Rev. Plant Physio1  34: 163-197; and Chen (1981)Metabolism and Molecular Activities of C ytokinins (Guern, J. and Peaud-Lenoel, C. eds., Springer, New York, pp. 34-43) ). Changes in the cytokinin / auxin concentration ratio have been shown to enhance plant growth It occurs preferentially in the organization. For example, high cytokinin / auxin ratios Promote the growth of roots, while low cytokinin to auxin ratios (Depicker et al. (1983) Genetic Engineering of Plants, T. Kosunge , C.P. Meredith and A. Hollaender, eds., Plenum Press, New York, p. 154).   the termMedium (single)OrMedium (multiple)Refers to a plant as used herein Cell growth, regeneration of plant cells and tissues, and shape of plant cells and tissues A solid or liquid that contains sufficient nutrients to support transversion.   the termCareerRefers to hydrophobic or hydrophilic or both, as used herein Of the active ingredient in plants (ie, the IAA analog of the present invention) Chemically or biologically or physiologically useful to promote efficacy. Refers to an acceptable molecule.   the termplantRefers to an entire plant or a part of a plant as used herein . Plants include, for example, certain parts (locus) of plants, plant cells, plant tissues , Explants, or plant seeds. This term refers to a suspension culture or tissue Plants in the form of a culture are further contemplated. These include calli, protoplasts, embryos , Organs, organelles, etc., but are not limited thereto.   the termTransformed plantsOrTransformed plant tissueIs used herein Transformation of a nucleic acid molecule into a plant or plant tissue, for example, from foreign DNA Replacement or protoplast fusion.   the termTransgenic plantsOrTransgenic plant tissueIs used in this specification When used in plants, plants or plant tissues stably transformed with foreign genes Say.   the termTransient expressionIs transformed with DNA, where the DNA is A plant or plant tissue that is expressed for a period of time.   the termGenetic manipulationAs used herein, is referred to as one or more plant cells. Refers to the introduction of a subsequent (often chimeric) gene. This plant cell is completely sexually active It can be regenerated into a viable and viable plant that is retained in the germline So that the introduced foreign gene can be inserted or crossed into agriculturally useful plant species. It can be pollinated or cross-pollinated with other plants of the same species.   the termPlaybackIs a tissue or unit of a cultured plant, as used herein. (Eg, leaf disks, seeds, etc.) Or generation of regenerated plant tissue (eg, roots, shoots, calli, etc.).   the termPlay percentage,Playback%OrRegeneration efficiencyIs the field used in this specification. A tissue-cultured plant that produces at least one newly developed or regenerated tissue. The number of physical units is defined as a percentage of the total number of plant units in tissue culture (eg, Say:   the termAffect plant growthOrAffect growthOrAffector OrInfluenceAs used herein, is the number of whole plants growing Characteristics (eg, stimulation of seed germination, induction of roots, suppression of shoot formation, cell proliferation Promotion of reproduction, stimulation of callus growth, etc.) in the absence of growth regulators Refers to any one of a number of plant responses that improve or alter as compared to   the termEffective amountA compound, as used herein, is a component of one or more plants. Plant growth regulators, which are administered to plants to stimulate or produce a long response Or the amount or concentration of a compound that is a plant growth hormone. As plant growth response In particular, induction of stem elongation, promotion of root formation, stimulation of callus formation, enhancement of leaf growth, Stimulation of seed germination, increased dry weight content of many plants and plant parts, etc. No.   The present invention describes that mono- and multi-substituted IAA analogs can be used to affect plant growth. It relates to the finding that it has utility as a xin. For example, 5-bromo-IAA When combined with Itokainin, both callus and shoot formation are observed [ No. 08 / 430,209 (filed Apr. 27, 1995) ].   IAA auxin analogs are present in both monocots and dicots. Compared to IAA in functioning as a toxin. For example, 5-bromo-IAA IAA in stimulating the regeneration of green callus from Arabidopsis thaliana Has been found to be 2-4 times more effective.   The growth affecting composition of the present invention may be mixed with a carrier or supplemental nutrient. An IAA analog, or an IAA analog and one or more additional plant growth regulators ( For example, cytokinins and gibberellins). BAP, 2iP, And the use of kinetin and IAA analogs is also a particular embodiment of the invention. Is exemplified. Other cytokinins or other plant growth regulators known in the art The substance is combined with an IAA analog to produce a growth affecting composition of the invention. It is intended that it can be used for More than one cytokinin or different A plant growth regulator (eg, gibberellin etc.) comprising a growth enhancing composition of the invention It is also contemplated that it can be mixed with an IAA analog to produce a product. Also, The selection of plant growth regulators depends on the incubation that characterizes the growth of a particular plant Or it may change at different stages of the application cycle. Plant growth regulators are And Skoog et al. (1967) Phytochemistry 6: 1169-1192 and The ologis (1989)Plant Biotechnology(Edited by Kung and Arntzen) Butterworth Publ BAP, 2iP, ipp, zeatin, kine, as described in ishers, Stoneham, MA But not limited to, tin, gibberellin and the like.   Compounds and compositions of the invention affect the growth cycle of plants and plant tissues Such mechanisms are not currently fully understood, but will be demonstrated hereinbelow. As such, they elicit responses that affect many growths in various plant species. It is clear that it plays an important role in guiding.   In certain embodiments of the invention, the various IAA analogs are derived from various plant tissues (eg, For example, tobacco and tomato leaf disks and potato stalks) MS complete medium containing only various concentrations of auxin (Life Technologies, Inc., Gai (obtained from Thersburg, MD) and (2) various ratios of cytokinins / orchids By incubating in MS complete auxin medium containing syn Screened for auxin activity.   In specific embodiments, tomato leaf discs, tobacco leaf discs And potato stalks in the absence of cytokinin (eg, BAP (0.5 mg / l)) And various amounts of 6-BrI in the presence (ie, 0.5, 2.0, and 8.0 mg / l) Incubation was performed in MS complete medium containing AA. Tomato leaf disk If only 6-BrIAA is present in the incubation mixture, Only root formation was observed from the leaf discs. Fig. 9, upper row is tomato Correlation between Root Formation from Leaf Discs and Increasing Concentrations of 6-BrIAA Show. However, as shown in FIG. 9, bottom row, In MS complete medium containing 0.5 mg / l BAP and 0.5, 2.0, or 8.0 mg / ml 6-BrIAA When incubated, callus was formed in each case, but roots were formed. Did not.   Identical auxin and cytokinin conditions resulted in potato stem (FIG. 8) and taba When applied to coco leaf disks (FIG. 10), various growth responses were observed. In potatoes, as the concentration of 6-BrIAA increases from 0.5 mg / l to 2.0 mg / l, Formation increased. However, at a concentration of 8.0 mg / ml, root formation was reduced (FIG. 8, top row). ). If BAP (0.5 mg / l) was present in the incubation mixture, Callus formation was observed at all nin / auxin ratios (Figure 9, bottom row). Contrast Typically, leaf disks of tobacco were exposed to various concentrations of 6-BrIAA in the absence of BAP. (Fig. 10, upper row), both root formation and callus formation were observed. On the other hand, in the presence of both 6-BrIAA and BAP (FIG. 10, bottom row), shoot and cal The formation of both forms occurred. These results indicate that various plant tissues were treated with auxin. Auxin levels are important for plant tissue regeneration. It showed that both effects were quantitative and quantitative.   In another embodiment of the invention, the IAA analog is screened for auxin activity. It was leaned. IAA derivative structures (eg, 2-bromoindole-3-acetic acid (2-Br IAA), 6-bromoindole-3-acetic acid (6-BrIAA), 7-bromoindole-3-acetic acid ( 7-BrIAA), 5-chloroindole-3-acetic acid (5-CIIAA), 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-ethylin Dole-3-acetic acid (7-EtIAA) and 5-methoxyindole-3-acetic acid (5-MeOIAA) ) To auxin standards in the art (eg, IAA and NAA). Were tested for their ability to function as auxins in plant tissue regeneration. Table 1 shows that various IAA analogs were identified as roots or mosquitoes from tobacco, tomato, and potato. Demonstrate the ability to stimulate lupus regeneration. Generally, 2 or 6 bromine substitutions Is an IAA analog at position 7 or a fluorine substitution at position 5 or 7, Auxin activity equal to or better than the observed auxin activity It seems to show.NA = Not available. + (-) Means preliminary observation.   These IAA derivatives can also be treated with auxin in the presence of cytokinins. The activity was evaluated. Table 2 shows the species in the presence of BAP (at a concentration of 0.5 mg / l). Various IAA analogs (at a concentration of 2 mg / l) from tobacco, tomato, and potato Shows the ability to stimulate shoot and / or callus regeneration. Auxin and Incubation of plant tissue in the presence of both cytokinins was tested In all three plants (ie, tobacco, tomato, and potato) Shoots, whereas auxin and cytokinin Only occurs in tobacco incubated with both, auxin and Occurs in tomatoes and potatoes incubated with both cytokinins Did not. In general, halogenated IAA analogs are responsible for callus regeneration in tobacco. Comparable to IAA in deriving, while alkyl-IAA and alkoxy- The IAA analogs are responsible for the auxin of IAA in tomatoes and potatoes under these conditions. It showed auxin activity similar to the activity. NA = Not available. + (-) Means preliminary observation.   These IAA analogs also have the ability to effect regeneration of plant tissue, including foreign DNA. Power was evaluated. Tobacco and potato tissues contain Agrobacterium-mediated traits Provided with conversion technology. This technology can be used for co-culture with Agrobacterium containing pBI121. MS containing 0.5 mg / l BAP, 2 mg / l IAA derivative, and 100 mg / l kanamycin Includes selection using whole medium. Table 3 shows the IAA analogs tested (7-BrIAA, 5-CIIA A, 5-FIAA, 7-FIAA, 5-EtIAA, 7-EtIAA, and 5-MeOIAA) in the presence of BAP Kanamycin-resistant calli in transformed tobacco and potatoes, Stimulates regeneration of kanamycin-resistant shoots in rabies transformed tobacco Indicates that Generally, halogenated IAA, alkyl-IAA, and alkoxy-IAA Derivatives showed IAA-type auxin activity in transgenic plant tissues .   Further, examples of the IAA analogs of the present invention include those in the presence of cytokinins. It was evaluated for xin activity. Table 4 shows 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 BAP Ability to stimulate shoot and callus regeneration from cassava leaves and stems Show. 5-FIAA is bigger in cassava stems and from cassava leaves It was also shown that to some extent promoted shoot formation.   In another embodiment of the present invention, the IAA analogs of the present invention are difficult to reproduce Is known in the art to be (e.g., cassava, woody And monocotyledonous crops) [Vasil and Vasil (1994)Plant Cell and Tissue Cul ture (Edited by Vasil and Thorpe), Kluwer Academic Publishers, Dordrech, Nethe rlands; Chee (1995) Plant Cell Reports 14: 753-757; Burns and Schwartz ( 1996) Plant Cell Reports 15: 405-408; Mihaljevic et al., (1996) Plant Cell Report. ts 15: 610-614; applied to regeneration of Schopke et al. (1996) Nature Biotechnology 14: 731]. Was done. FIG. 11 shows MS complete medium containing 2 mg / l 5-FIAA and 0.5 mg / l BAP. Demonstrates shoot regeneration in incubated cassava leaves and stems You.+ (-) Means preliminary observation.   Traditionally, to achieve shoot regeneration from cassava leaves and stems, Cassava tissue is derived from media containing large amounts of auxin, Transfer to another medium containing kainin. In the present invention, the system from cassava tissue Regenerates auxin and cytokinin from high auxin media Obtained without transplantation of the tissue into the medium. Further, the cassava tissue according to the present invention The regeneration of the shoots in the test showed a significant improvement in the number of regenerated shoots. Obedience Thus, plant tissues that have traditionally been described as difficult to regenerate (eg, , Cassava, woody plants, corn, soybeans, wheat, etc.) Thus, the IAA analogs of the present invention not only exhibit auxin activity, but also It also improves the yield of tissue.   The practice of the present invention will stimulate seed germination and break dormancy; increase yield; Promote maturation and coloration; flowering and increased outcome; stimulation of shoot formation; callus Induction of root development; induction of root formation and cell proliferation; strength of various plant species Increased health; including increased dry weight content of many plants and plant parts Contemplate a wide variety of plant growth responses. In addition to these categories of responses , The end result is increased crop and horticultural crop or seed growth, or As long as it maximizes any of its beneficial or desired characteristics, Any other variation of the vegetables is within the advantageous response achieved by practicing the invention. Is intended to be included.   A suitable application of the growth enhancing composition of the present invention to the culture of plant tissue is to shoot Induces root, callus regeneration. This effect can be seen in monocot and dicot Occurs in both plant species and applies to a wide variety of plants.   The compositions of the present invention are further utilized for plant regeneration from transgenic plants. You.   Genetic engineering of plants generally involves two complementary processes. First process Involves the genetic transformation of one or more plant cells of a particularly characterized type. Trait The conversion allows the foreign gene (typically a chimeric gene construct) to be grown, typically Helping a vector with the ability to transfer a gene of interest into the genome of a growing plant cell To be introduced into the genome of individual plant cells. Then, The second process involves the regeneration of transformed plant cells into whole plants that can be crossed. Including. Neither the transformation process nor the regeneration process need be 100% successful, but % Of cells can be transformed and regenerated into whole plants Must have reliability and reproducibility.   The two processes (transformation and regeneration) must be complementary. Two Process complementation is successfully transformed by the transformation process The tissue must be of a certain type and characteristic, and be sufficiently healthy and competent They must be powerful and vital, so that they can It must be able to play well.   Successful transformation and regeneration techniques are described in the prior art as monocotyledonous plants and Demonstrated for dicotyledonous plants. For example, transformation and regeneration of tobacco plants Barton et al., Cell 32: 1033 (April 1983), whereas cotton regeneration was 1 As described in Umbeck, U.S. Pat. No. 5,004,863, issued Apr. 2, 991. Further In addition, rice transformation and regeneration is described in Abdullah et al. (1986) Bio / Technology 4: 1087. Corn, whereas corn is described in Rhodes et al. (1988) Blo / Tec. hnology 6: 56-60 and Science 240: 204-207. And played.   The most common methodology used to transform cells of dicotyledonous species is the phytopathogen Related to the use of Agrobacterium tumefaciens. Agrobacterium-mediated traits Conversion was achieved in some monocotyledonous plants, but WuPlant Biotechnology (1989) pp. 35-51, by Butterworth Publishers, Stoneham, MA. Other gene transfer methods (eg, polyethylene glycol method, electroporation, etc.) Solutions, direct injection, particle bombardment, etc.) Was. The present invention is useful for any transformation method including a plant regeneration step.   In certain embodiments, the present invention is directed to a leaf disk or hypocotyl explant. Suppose gene transformation of a tissue in culture. The transformed tissue is induced To form plant tissue structures. This can regenerate into a whole plant.   The transformation technique of the present invention is described in This technology uses the Ti plasmid of tumefaciens. You. A. as a transformation medium When using tumefaciens cultures, the transformed Non-tumorigenic Agrobacterium so that normal non-tumorigenic differentiation of It is most advantageous to use tumorigenic strains as vector carriers. Once planted In order to be effective when introduced into a target cell, a chimeric construct containing the desired foreign gene must be used. Architecture is a promoter that is effective in triggering the transcription of a gene of interest in plant cells. And polyadenylation sequences or transcriptional regulatory elements that are also recognized in plant cells. Must contain columns. Promoters known to be effective in plant cells Nopaline synthase promoter isolated from T-DNA of Agrobacterium And the cauliflower mosaic virus 35S promoter. other Suitable promoters are known in the art. Transformed cells are in culture Have the foreign gene of interest so that it can be selected from untransformed cells. That the vector also contains one or more selectable marker genes therein. preferable. In many applications, preferred marker genes include appropriate antibiotics The substance separates and selects transformed cells from non-transformed cells. Antibiotic resistance genes, such as may be used to select.   Details of the construction of a vector containing such a foreign gene of interest are described in Plant Genetics. It is known to those skilled in the art of child manipulation, and tobacco, petunia, and other models. It is substantially different from the practice previously demonstrated to be effective in Dell plant species. No. Foreign genes have been identified as marker genes (Jefferson et al. (1987) EMBO J. 6:39). 01-3907) or to achieve any desired effect in plant cells. Should be chosen easily. This effect has the effect of promoting growth, disease resistance, Changes in condition or quality of plant products, or any that can be achieved by genetic engineering Other changes may be made. Chimeric gene constructs are useful for disease processes or unwanted One or more exogenous proteins to control or inhibit any of the intrinsic plant functions. Can encode the expression of a protein, or can result in the transcription of negative strand RNA.   To begin the transformation and regeneration process on plant tissue, Sterilize the tissue surface to prevent inadvertent contamination of the resulting culture. Is necessary. If the tissue is seed, then the seed is It is germinated on a cut germination medium. Four to ten days after germination, the hypocotyl part of the immature plant is removed. And cut into small segments each averaging about 0.5 cm. Hypocotyl explant The pieces are stabilized in liquid or agar plant tissue culture medium and viable. Leave.   Once the tissues have stabilized, they are transformed into non-tumorigenic Aggrobact It can be quickly inoculated with a suspension culture of erium. The inoculation process can be performed for a short period of time (eg, 2 Days) at room temperature (ie, at 24 ° C.).   At the end of the inoculation period, the remaining treated tissues can be transplanted onto selective agar. This choice cold The culture medium contains one or more of the following: toxic to Agrobacterium but not to plant tissue. Use antibiotics enough to kill any remaining Agrobacterium in the culture. Contained in various concentrations. Suitable antibiotics for use in such media include Ag Carbenicillin, cefotaxime, etc. as robacterium fungicides, and their forms Kanamycin is a selective antibiotic for transformed plant tissue.   The tissue is then placed on a tissue culture medium containing the usual components of the tissue culture plus a selection agent. Cultured in The selection agent (exemplified herein by kanamycin) is Toxic to untransformed cells, but genetically resistant to selected drug Toxic to transformed cells that have integrated sex and express their resistance Not gender. Suitable tissue culture media are auxin analogs and cytokines of the invention. MS media with inin added with or without antibiotics. Raw Existing transformed tissue is transferred to a second medium to induce tissue regeneration. It is. Therefore, surviving transformed tissues can be obtained through the regeneration technique of the present invention. Or continue to regenerate through any other alternative plant regeneration protocol Be an object.   The exact amount of the composition that affects growth used in the practice of the present invention is: Depends on the type of response desired, the formulation used and the type of plant being treated . The present invention relates to the invention of about 50.0-0.001, and preferably about 5.0-0.05, and more preferably Use the ratio of cytokinin concentration to auxin concentration of about 2.0-0.25 Intended to be.   The chemical compound used as an active ingredient of the composition for promoting growth of the present invention includes: It can be prepared according to processes well known in the prior art or is readily available. It can be obtained commercially from sources.   The IAA analogs of the present invention make plants less susceptible to antibiotic toxicity. Useful for Such IAA analogs can also cause plant stress (circularity). Environmental stress, physical stress, chemical stress, pollution, pollution, drought, light, etc.) Useful in making it possible to overcome.   The composition of the present invention may be used at any stage of development of the plant Mon or for maintenance effects and on tissue damaged during the early stages of development Concentration, formulation used, and processing to promote regrowth in It may be applied depending on the type of plant species being treated.   The compositions of the present invention are preferably used to enhance specific synergistic growth in various types of plants. Certain supplemental nutrients or other plant growth in precise ratios to achieve a strong response Used in combination with modulators. The composition of the present invention is useful for disease resistance of various plants. Can be used in combination with further fungicides to increase the viability of the plant tissue. By controlling innate disease immune enzymes and plant processes Resistant to infestation by pathogens. The composition of the invention essentially comprises Although they themselves have no phytotoxic activity, they sometimes remove unwanted plants. grass In order to make these plants more susceptible to It can be used in combination with herbicides. However, agricultural and horticultural crops, Implementation of the invention, such as growth-enhancing responses in annual and annual home plant species It is preferred to consider the results achieved in.   The following examples are realized by applying the preferred compositions of the present invention to various plant species 9 is an illustration of a wide range of plant growth responses that can be performed. Nevertheless, the present invention is effective It is not intended to be limited to these optimal ratios of the components. Because those skilled in the art Do you find that the composition of the invention shown above in the description is an effective growth enhancer? It is. In addition, other plants, seeds, fruits, and other plants not specifically exemplified in this specification. Recognition of the improved results of using the compositions of the present invention with respect to vegetables and vegetables The person skilled in the art will readily think that it is easy in the ability of the person skilled in the art. . The following examples illustrate the utility of the present invention without limiting the scope of the invention Provided for.Example Embodiment 1 FIG. IAA Chemical synthesis of auxin analogs (A) Synthesis of halogenated IAA   The synthesis of monohalogenated IAA compounds is mainly based on J. March,Advanced Organic Chemi stry (1985) J. Wiley and Son, pages 1032-1033. Based on the Shah Indole synthesis method. This method uses the original 3-formylpropionic acid Depends on the Fisher ring closure of phenylhydrazone. Of substituted phenylhydrazine Due to the general utility of indole-3-acetic acids substituted on the benzene ring This method makes it attractive for preparation (Fox and Bullock (1951) J. Am. Chem.  Soc. 73: 2756 and Hatano et al., (1987) Experientia 43: 1237).   Alternatively, a wide variety of substituted anilines are available and the corresponding substituted An output for synthesizing phenylhydrazine (which can then be converted to a substituted IAA compound) Can be used as a source. Substituted anilines are described in Robinson (1957) Can. J. Chem. 35 : 1570 can be converted to the corresponding substituted phenylhydrazine.   2- and 4-halogen-substituted phenylhydrazines can be combined with 3-formylpropionic acid When used with 2- and 4-halogen substituted phenylhydrazones ( Cyclizing to the corresponding 7- and 5-halo-IAA, respectively). 3-halogen Preparations of substituted phenylhydrazones are cyclized to give C₁₈Using reverse phase HPLC, the water: Tanol: 4- and 6-halo-IAA isomers that can be separated by elution with acetic acid To form 4-halo-IAA is eluted prior to 6-halo-IAA. 2-halo-IAA compound The product was prepared according to the procedure described by Porter and Thimann (1965) 4: 229-243. Can be synthesized. Each isomer product is analyzed by thin-layer chromatography, mass spectrometry, and It can be identified using nuclear magnetic resonance.   Both 4-halo-IAA and 6-halo-IAA compounds were purchased from Fox and Bullock (1951) J. Am. A m. chem. Soc. 73: 2756, Majima and Hoshino (1925 ) Can be synthesized individually by the method of Ber 58: 2042. 4- and 6-chloroi iodide The magnesium complex, concentrated with chloroacetonitrile and obtained. The nitrile is hydrolyzed to the corresponding indole-3-acetic acid.   The protocol for the synthesis of monosubstituted halogen-IAA includes the following steps. Replacement Fe Nylhydrazine hydrochloride (0.05 mol) is dissolved in 30% acetic acid (PH 4.0) and Add to a 0.1 M solution of freshly prepared (see below) 3-formylpropionic acid Add. After cooling, collect the precipitate and dissolve in 75 ml pyridine. In this solution 100 ml concentrated hydrochloric acid and 25 ml 85% H_ThreePO_FourAnd the resulting solution is N_Twounder Reflux in the dark for 10 hours.   600 ml H of reaction mixture_TwoDilute with O, filter, and extract the filtrate several times with ether. Put out. The ether fractions are pooled and washed with water. Indole acetic acid, 0. Back-extract into 5M NaOH (200 ml), boil and precipitate with concentrated hydrochloric acid (pH 1.0). stone The soapy tar is decanted or removed by filtration. Replace IAA with H_TwoO, H_TwoO Recrystallize twice from / ethanol, toluene, or ethyl acetate / hexane.   Stir 3-formylpropionic acid with starch-iodide paper until negative result While adding 200 ml to 24.9 g (0.20 mol) of glutamic acid in 400 ml of 0.5N NaOH solution Freshly prepared by adding fresh 1 M NaOCl solution, then 70 ml of 3N  Acidify by adding HCl.   Polyhalogenated IAA compounds are described in Engvild (1977) Acta Chem. Scand. B31: 338 (example For example, the method of 4,6-, 4,7-, 5,7-, 6,7-dichloroIAA), or Baldi et al., (1985) J.  Label. Compd. Radiopharm. 22: 279 (eg, 5,6-, 4,5-dichloro-IAA) Or Hatano et al., (1987) Experientia 43: 1237-1239 (e.g., 5,6-, 6,7-, 4,5-, 4,6-, 5,7-, 4,7-dichloro-IAA). Can be synthesized.   (B) Alkyl, alkoxy, acyl, acylamide, and acyloxy IAA Synthesis of   Using the procedure outlined above (Example 1 (A)), 2, 4, 5, 6, or 7 Alkyl, alkoxy, acyl, acylamide, or acyloxy substituent at the Is prepared. Synthetic reaction to obtain desired substituted IAA compound Using an appropriately substituted phenylhydrazine. If necessary, reactive substituents (Eg, acyl, acylamide, acyloxy, etc.) And then deprotect it.   Halo, alkyl, alkoxy, acyl, acylamide, acyloxy substituent Multisubstituted IAA analogs having two or more different substituents selected from Synthetic techniques (Vasil and Vasil (1994), well known inPlant Cell and Tissse Cult ure (Edited by Vasil and Thorpe), Kluwer Academic Publishers, Dordrech, Netherla nds; 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 1 5: 610-614; synthesized using Schopke et al. (1996) Nature Biotechnology 14: 731). obtain. For example, an IAA compound containing a combination of alkyl, halo, and acyl substituents (For example, 2-methyl-5,7-dichloro-IAA, 2-COOH-5-methyl-IAA, 2-COOH-7-chloro B-IAA) and Fox and Bullock (1951) J. Am. Am. Chem. Soc. 73: 2756-2759; H offman et al., (1952) J.C. Biol. Chem. 196: 437, and Engvild (1977) Acta Chem. Scand. B31: 338-344 and the like.Embodiment 2. FIG. Evaluation of auxin activity in non-transgenic plants (A) Tobacco     Nicotiana tobaccum Xanthi seeds were obtained from Dr. James Saunders (USDA, Beltsvi lle, MD). The young tobacco leaves are collected and cut into small pieces. Explants were prepared using (1) MS complete medium (L) containing only various concentrations of auxin-related compounds. ife Technologies, Inc., Gaithersburg, MD) or (2) various Concentration of auxin-related compound and 0.5 mg / l of benzylaminopurine (BAP) Green callus on MS complete medium with 18 h light / 6 h dark cycle And incubate until shoots form.(B) Tomato   The seeds of the varieties "Moneymaker" tomato are provided by Dr. James Saunders (USDA, Beltsvil le, MD). The young tomato leaves are harvested and cut into small pieces. Explants were transferred to cytocaica as described above in section (a) for tobacco. Incubate in MS complete medium with auxin in the presence or absence of inin. Beate.   (C) potatoes   Potato seeds of the cultivar “Kent V.F.” were purchased from Dr. James Saunders (USDA, Beltsvill e, MD). The young potato stalks are collected and cut into small pieces. Explants As described above for tobacco in section (a) above, In MS complete medium with auxin in the presence or absence of I do.   (D) Arabidopsis thaliana   Seeds of the Arabidopsis thaliana ecotypes Columbia and Landersberg ereta are Ke Provided by Dr. ith Davis (The Ohio State University, Columbus). Hypocotyl Were harvested from day 10 seedlings, and (1) various concentrations of N⁶・ (Δ_TwoIsopentenyl) MS complete containing various concentrations of IAA or IAA analog with adenine (2iP) Medium, and (2) various concentrations with 2iP, BAP, or kinetin at various concentrations Transfer onto MS complete medium containing IAA analogs. Explants, 18 hours light / 6 hours Incubate at 23 ° C using a dark cycle until green calli and shoots are formed. Lab.   (E) Rice     Seeds from Oryza sativa cultivar Orion were purchased by Dr. James Saunders (USDA, Beltsville , (MD) in good faith. Seeds to form rice embryogenic calli Sterilize the surface of the seeds as follows: 0.5% surfactant for 1 hour while shaking the mature seeds Reduce pressure while shaking. The seeds are then rinsed three times with sterile distilled water. at this point, Seeds on MS complete medium containing various concentrations of BAP and various concentrations of IAA analogs And incubate in the dark at 25 ° C. for 1 month, then 18 hours light / 6 hours Use a dark cycle to incubate at 25 ° C until green calli and shoots are formed. Cubate.   (F) Cassava   Stems or third young leaves were harvested from January cassava seedlings and cut into small pieces . Explants were treated with various concentrations of auxin-related compounds and 0.5 mg / l benzylamino acid. Incubated on MS complete medium containing purine (BAP). Explants for 18 hours Form green calli and shoots at 23 ° C using a light / 6 hour dark cycle And incubated until the   Regeneration of plant tissue using tissue culture is similar to auxin and cytokinin. Depends on various plant hormones. Murashige and Skoog (MS) media (Murashige, T .; And F. Skoog (1962) Physiol. Plant 15: 473-497) The presence of auxin stimulates the formation of root structures, while Formation of cytokinin as well as auxin is replenished to the MS nutrient medium. It is known that if it is, it will be observed. Therefore, potential new auxies The evaluation of the test compound as a component was performed using leaf disks of tobacco (a) at various concentrations. MS complete medium containing auxin alone, and (b) different concentrations for auxin MS complete medium containing cytokinin (cytokinin / auxin) at different ratios And by incubating.   In certain embodiments of monosubstituted IAA analogs, 15 halogenated IAA compounds are It is chemically synthesized and tested for plant growth regulating activity. As shown in FIG. 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-I AA, 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. Biological of each compound The activity is compared to the biological activity of the IAA. Various concentrations of each compound can be used in tobacco recycling. Leaf and tomato leaf discs and roots and potatoes from potato stalks The ability to stimulate the formation of callus and callus is tested. Stimulates root formation Ability to test compounds at various concentration ratios of cytokinin (eg, BAP) Are evaluated for each compound in the presence of   In another embodiment of the monosubstituted IAA analog, the alkyl-substituted IAA compound is Synthesized and tested for plant growth regulating activity. In Fig. 3, Alki is fourth 9 shows nine monosubstituted IAA compounds having a thiol substituent. The present invention also provides 1-10 Alkyl-IAA compounds having an alkyl substituent having a hydrogen atom, and Are alkyl-IAA compounds having various alkyl substituents at positions 2, 5, 6, or 7. Intended thing. The biological activity of each compound is compared to the biological activity of the IAA. Various concentrations of each compound were added to tobacco and tomatoes and tomato leaf disks. Its ability to stimulate the formation of roots, shoots and callus from potato stalks And test. The ability to stimulate root, shoot, and callus formation was tested Each compound in the presence of different concentration ratios of cytokinin (eg, BAP) Evaluate things.   In another embodiment of the monosubstituted IAA analog, the alkoxy substituted IAA compound is Chemically synthesized and tested for plant growth regulating activity. In Figure 4, Al is fourth 9 shows nine monosubstituted IAA compounds having a Coxy substituent. The invention also provides 1 to 10 An alkoxy-IAA compound having an alkoxy substituent having the carbon atoms of Alcohols having the same or different alkoxy substituents at the 2, 5, 6, or 7 positions Xy-IAA is intended for compounds. The biological activity of each compound is determined by the biological activity of the IAA. Compare with Various concentrations of each compound were added to tobacco leaf disks and tomatoes. Stings root, shoot, and callus formation from leaf discs and potato stalks Test for vigorous ability. Ability to stimulate root, shoot, and callus formation The force is determined by the presence of various concentration ratios of cytokinin (eg, BAP) to the test compound. Each compound is evaluated in the presence.   In another embodiment of the monosubstituted IAA analog, the acyl-substituted IAA compound is chemically And tested for plant growth regulating activity. In FIG. 5, acyl is placed at the 4th position. 9 shows nine monosubstituted IAA compounds having a substituent. The present invention also relates to a carbon source comprising 1 to 10 carbon atoms. Acyl-IAA compounds having an acyl substituent having An acyl-IAA compound having an acyl substituent at the 2, 5, 6, or 7 position is intended. . The biological activity of each compound is compared to the biological activity of the IAA. Various concentrations of each Compounds are tobacco leaf disks and tomato leaf disks and potatoes Test their ability to stimulate the formation of roots, shoots, and callus from stalks You. The ability to stimulate root formation was determined by varying the concentration of Each compound is evaluated in the presence of a nin (eg, BAP).   In another embodiment of the monosubstituted IAA analog, the acylamide substituted IAA compound is It is chemically synthesized and tested for plant growth regulating activity. In FIG. 9 shows nine monosubstituted IAA compounds having an acylamide substituent at the position. The invention also provides Acylamide-IAA having an acylamide substituent having 1 to 10 carbon atoms Compounds and compounds having the same acylamide substituent at positions 2, 5, 6, or 7 Silamide-IAA compounds are contemplated. The biological activity of each compound is determined by the biological Compare with activity. Various concentrations of each compound were added to tobacco leaf disks and tomatoes. Roots, shoots, and callus shapes from leaf disks and potato stalks Test for ability to stimulate growth. Test compounds have the ability to stimulate root formation Each compound in the presence of different concentration ratios of cytokinins (eg, BAP) About   In another embodiment of the monosubstituted IAA analog, the acyloxy-substituted IAA compound is It was chemically synthesized and tested for plant growth regulating activity. In Figure 7, the fourth place 9 shows nine monosubstituted IAA compounds having a siloxy substituent. The present invention also provides Acyloxy IAA compounds having an acyloxy substituent having 0 carbon atoms, and And the same or various acyloxy substituents at the 2, 5, 6, or 7 positions An acyloxy-IAA compound is intended. The biological activity of each compound is And to compare the activity. Various concentrations of each compound were added to tobacco leaf disks and tobacco. Mato leaf discs and roots, shoots and callus from potato stalks Test for ability to stimulate formation. The ability of the test compound to stimulate root formation Each compound in the presence of different concentrations of cytokinins (eg, BAP) Is evaluated.   In certain embodiments of multi-substituted IAA analogs, 2, 3, 4, or 5-substituted IAA Compounds (which correspond to positions 2, 4, 5, 6, and 7 in the IAA chemical structure of FIG. 1) Halo, alkyl, alkoxy, acyl, acyl in at least two corresponding positions 2 and 5 individually selected from the group consisting of amide and acyloxy substituents (Including substituents between and) were synthesized and tested for plant growth regulating activity. these In the polysubstituted IAA compound of the above, the halo substituent is an iodo group, a bromo group, , And chloro groups; however, the alkyl substituents are from 1 to 10 A straight, branched, or cyclic alkyl group R having 5 carbon atoms, Without limitation, alkoxy substituents may be straight-chain, branched, having 1 to 10 carbon atoms. Or including, but not limited to, the cyclic alkoxy group RO; , Containing a linear, branched or cyclic acyl group R--C (O)-having 1 to 10 carbon atoms. But not limited to; acylamide groups have from 1 to 10 carbon atoms Including, but not limited to, linear, branched, or cyclic acylamide groups RC (O) -NH- And acyloxy substituents are straight-chain having 1 to 10 carbon atoms; Including, but not limited to, branched or cyclic acyloxy groups R—C (O) —O—. No.   For each polysubstituted IAA, test its auxin activity and compare it to that of the IAA I do. Various concentrations of each compound were applied to tobacco leaf disks and tomato leaves. Stimulates the formation of roots, shoots and calli from discs and potato stalks Test the ability to Ability to stimulate root, shoot and callus formation In the presence of various concentration ratios of cytokinin (eg, BAP) to test compound Then, each compound is evaluated.Embodiment 3 FIG. IAA auxinicity to stimulate regeneration of transgenic plants Use of analog a)tobacco   Plant transformation was performed using the Agrobacteri described in Lin et al. ((1994) Focus 16: 72-77). Performed essentially according to the um-mediated transformation procedure.   About tobacco, 10 leaf disks^TenAgrobacterium tumefacie in cells / ml ns LBA4404 cells (containing pBI121 with GUS reporter gene) together with 0.5 mg / l 10 minutes in MS complete medium containing 2- (N-morpholino) ethanesulfonic acid (MES) Incubate, transfer to MS complete solid media, and 18 hr light / Incubate at 25 ° C. for 2 days using a 6 hour dark cycle. After co-culture , Explants with various concentrations of BAP / IAA or BAP / IAA analog, 100 mg / l Transfer to MS medium containing Syn, and 500 mg / l carbenicillin, and Incubate at 25 ° C using an 18 hour light / 6 hour dark cycle to form To   (B) Tomato   Plant transformation was performed using the Agrobacteri described in Lin et al. ((1994) Focus 16: 72-77). Performed essentially according to the um-mediated transformation procedure. Incubation conditions are Same as described in section (a) above for tobacco.   (C) potatoes   Transformation of plants with potato stalks was performed as described in section (a) above for tobacco. To be implemented.   (D) Arabidopsis thaliana   For Arabidopsis thaliana, the hypocotyl tissue was collected from the seedlings on day 10 and 3% on MS complete medium containing 0.5 mg / l 2,4-D and 0.5 mg / l kinetin. Pre-incubate for days. Explants containing 10 pBI121⁹Agrobacter in cells / ml immersion in ium tumefaciens LBA4404 for 20 minutes and callus and shoot formation 500 mg / l carbenicillin, 50 mg / l kanamycin, and various ratios Transfer to IAA analog / 2iP or MS solid medium containing IAA / 2iP. Arabidopsis Explants are incubated at 25 ° C. using an 18 hour light / 6 hour dark cycle did.   (E) Rice   Transform monocotyledonous plants by Wu, "Methods for Transforming Plant Cells"Plant Biotechn ology (1989), edited by Kung and Arntzen, Butterworth Publishers, Stoneham, MA Performed according to methods known in the art as described more fully. Rice and rice Transformation of monocotyledonous plants such as wheat is described in Wang et al. (1988) Plant Mol. Biol. 11: 433- It is preferred to use the particle bombardment method as described in 439. New Regeneration of the transformed monocotyledonous plants is carried out by known methods as described in Example 5. It is performed according to the procedure (Vasil, Biotechnology (1988) 5: 387-402).   For example, use rice (Oryza sativa) as a particle bombard from Wang et al. Or the Agrobacterium-mediated technique of Hiel et al. (1994) Plant Journal 6: 271. Or as described by Dekeyser et al. (1990) Plant Cell 2: 591-602. Transform using the electroporation method. Regeneration of transformed rice, Ab (1986) Bio / Technology 4: 1087-1909, or Raineri et al. (1990 ) Perform according to Bio / Technology 8: 33-38.   In a further example, corn is harvested from Rhodes et al. (1988) Bio / Technology 6 : 56-60 and (1988) Science 240: 204-207. Live.   In all cases, IAA auxin analogs are used in accordance with the invention in plant growth. Used as an auxin to stimulate If required, one or more Additional plant growth regulators are added to the plant growth composition containing the IAA analog. obtain.   (F) Cassava   Stems or leaves from the cassava seedlings of January (preferably the first, second and The third leaf, and more preferably the lower quarter of the leaf closest to the petiole, into small pieces Cut. Exogenous DNA can be prepared using techniques known in the art (eg, A. tumifaciens). technique (Lin et al., (1994) supra) or micro bombardment technique (Schopke (1996) Nature Biotechnology 14: 731). Transgeni The cassava tissue is exposed to the IAA analog of the present invention and the cassava tissue is reconstituted. Raw was tested.Embodiment 4. FIG. Screening substituted IAA compounds for auxinic activity   (A)The non-transgenic plants shown in Figures 8-10 and Tables 1, 2 and 4 object   Nicotiana tobaccum Xanthi, potato and tomato seeds are available from James Saunder Courtesy of Dr. S (USDA, Beltsville). Cassava seedlings, Dick Sa Courtesy of Dr. yer (The Ohio State University, Columbus, OH).   Young tobacco leaves, potato stalks, tomato leaves and cassava from the January plant Leaves and stems were harvested and cut into small pieces. Explants were prepared by (1) MS complete medium containing only toxin-related compounds (2) Various concentrations of auxin-related On MS complete medium containing the serial compound and 0.5 mg / l benzylaminopurine (BAP) Green calli, shoots or roots using a 18 hour light / 6 hour dark cycle To form.   The results of root, shoot, and callus formation were compared with 6-BrIAA alone or BAP and 6-BrIA And potato (FIG. 9) incubated in MS complete medium containing ), And tobacco (FIG. 10), and cassava (FIG. 11). Auxin activity Screening results for various IAA analogs Table 1 shows the MS complete medium containing IAA analog and BAP. In Table 2, and in MS complete media with various IAA analogs and IAPs Table 4 shows the cassava regeneration.   (B)Transgenic plants shown in Table 3   Acrobacterium-mediated plant transformation was described by Lin et al., (1984) Focus 16: 72-77. Performed as listed. For example: (I)tobacco   In tobacco, a leaf disk is replaced with 10 containing pBI121.⁹Aglob of cells / ml acterium tumefaciens 1 in MS complete medium with 0.5 mg / l MES with LBA4404 cells Incubate for 0 min, transfer to solid MS complete media and 13 h for co-culture Incubated for 2 days at 25 ° C. using a light / 6 hour dark cycle. Both After culture, explants were harvested with various ratios of BAP / IAA or BAP / IAA analog, 100 mg / l Transfer to MS medium containing mycin, and 500 mg / l carbenicillin, and Incubate at 25 ° C using a 18 hour light / 6 hour dark cycle for plate formation. I did it.   (ii) potato   In another embodiment, a potato leaf disc is prepared as described above for tobacco. And transformed using Agrobacterium-mediated technology. The potato explants were then Incubation with IAA analogs in the absence and presence of cytokinins And screened for auxin activity.   (iii) Cassava   Stems or third young leaves are harvested from 1 month old cassava plants and Cut into pieces. Explants were grown in MS complete liquid medium with pBI121^TenA. tumefaci Incubate with ens cells for 20 minutes, transfer to MS complete solid medium and 2 days Incubated. After two days of incubation, explants were harvested at 500 mg / l Benicillin, 50 mg / l kanamycin, 0.5 mg / l BAP, and various concentrations of auxin Were transferred to complete MS media containing the thiol-related compounds. Explants 18 hours light / 6 hours dark Incubate at 23 ° C until green calli and shoots are formed I was vate.Embodiment 5 FIG. Reducing stress with auxins and auxin derivatives (A) Attenuation of chemical toxicity   Potato stalks are excised as above and 25, 50, and 100 mg / l kanamycin With and without 2 mg NAA or auxin derivative Or MS complete medium with / without 0.5 mg / l BAP (cytokinin) (a) Incubated in. After 1 month incubation / with BAP Potato stalks incubated in 50 mg / l or 100 mg / l kanamycin Only showed necrosis, which is toxic to kanamycin. However, 2 mg / l NAA and 100 Potato stems incubated in MS medium containing mg / kanamycin elongate (Figure 12). In some experiments, callus on some of the potato stems Was shown in the presence of auxin or an auxin derivative.   Auxin has the ability to restore the viability of antibiotic-treated potato stems 1 month with 50 mg / l kanamycin and 0.5 mg / l BAP 2 mg / l NAA with / without 100 mg / l kanamycin MS medium containing, or 2 mg / l NAA with / without 100 mg / l kanamycin, 0.5 m Transferred to MS medium containing g / l BAP. Auxin with / without BAP with potato stem Or callus formation on potato stems when transferred to auxin derivative-containing MS medium Was observed. However, continuous without auxin or auxin derivatives After a good incubation, all potato stems died (FIG. 13).   The interaction of a similar antibiotic with auxin was found to be 2,4-D, 5-bromo-IAA and IA A was observed in the medium containing A. In addition, various antibiotics (eg, hygro Mycin and chloramphenicol) are auxin-treated potato stems And tobacco leaves showed the same interaction.B. Reduction of physical damage using auxins and auxin derivatives   Tobacco leaves were collected from 1 month old plants and cut into small pieces (0.5 cm × 0.2 5cm). These tissues were incubated overnight in MS complete medium containing 0.5 mg / l BAP. And bombarded using a microprojectile (1300 psi) ( Helium source, obtained from Biorad). Has 0.5 mg / l BAP after bombardment Prior to transfer to MS complete medium, tobacco explants were (1) MS complete with 0.5 mg / l BAP Medium and (2) MS complete medium with 0.5 mg / l BAP and 2 mg / l NAA for 3 days Cubbed. Shoots incubated in MS medium with BAP only Significant improvement in tobacco shoot regeneration compared to contains NAA and BAP Shown in explants incubated in MS medium. In addition, pBI121 Bombard the tobacco explants using and with 0.5 mg / l BAP, and 2 Similar results were shown when incubated with / without mg / l NAA. Transformed tissues were purified with MS complete with 0.5 mg / l BAP and 100 mg / l kanamycin. Selection was made by incubation of explants in medium. All these results Auxin reduces physical stress (eg, bombardment), and This indicates that the regeneration of the plant tissue could be restored.   All publications, patent applications, and patents cited in this specification are as if Indicate that various publications, patent applications, or patents are to be incorporated by reference. It is incorporated by reference to the extent that it appears physically and individually.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 15/09 C12N 15/00 A (81) Designated country EP (AT, BE, CH, DE, DK, ES , FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN , TD, TG), AP (KE, LS, MW, SD, SZ, UG), UA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU , AZ, BB, BG, BR, BY, CA, CH, CN, CZ, DE, DK, EE, ES, FI, GB, GE, HU, IL, IS, JP, KE, KG KP, KR, KZ, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI SK, TJ, TM, TR, TT, UA, UG, UZ, VN (72) Inventor Lan, Jiankin United States Maryland 20876, Germantown, Red Admiral Way 12129 (72) Inventor Assad-Garcia, Nasila United States Mary Land 20877, Geysersburg, Towncrest Court 8405

Claims (1)

[Claims] 1. An auxin analog of IAA for stimulating plant growth, regeneration of plant cells or tissues, or transformation of plant cells or tissues, comprising the chemical structure Wherein R _{1 to} R ₅ are independently a halo group, an alkyl group having 1 to 10 carbon atoms, an alkoxy group having 1 to 10 carbon atoms, 1 to 10 carbon atoms. An acyl group having an atom, an acylamide group having 1 to 10 carbon atoms, an acyloxy group having 1 to 10 carbon atoms, or a salt, ester, or amide derivative thereof, provided that Auxin analogs of IAA, except for indole-3-acetic acid and 5-bromoindole-3-acetic acid. 2. The auxin analog according to claim 1, wherein R _{1 to} R ₅ are individually selected from the group consisting of a bromo group, a chloro group, a fluoro group, and an iodo group. 3. The auxin analog of claim 1, wherein R ₁ -R ₅ are individually selected from the group consisting of alkyl groups having 1-10 carbon atoms. 4. The auxin analog according to claim 1, wherein said R _{1 to} R ₅ are individually selected from the group consisting of alkoxy groups having 1 to 10 carbon atoms. 5. The auxin analog of claim 1, wherein R _{1 to} R ₅ are individually selected from the group consisting of acyl groups having 1 to 10 carbon atoms. 6. The auxin analog according to claim 1, wherein R _{1 to} R ₅ are individually selected from the group consisting of acylamide groups having 1 to 10 carbon atoms. 7. The auxin analog according to claim 1, wherein said R _{1 to} R ₅ are individually selected from the group consisting of acyloxy groups having 1 to 10 carbon atoms. 8. The auxin analog of IAA is 2-bromoindole-3-acetic acid, 6-bromoindole-3-acetic acid, 7-bromoindole-3-acetic acid, 5-chloroindole-3-acetic acid, 5-fluoroindole-3- 2. The method of claim 1, wherein the acetic acid is selected from the group comprising acetic acid, 7-fluoroindole-3-acetic acid, 5-ethylindole-3-acetic acid, 7-ethylindole-3-acetic acid, and 5-methoxyindole-3-acetic acid. Auxin analog as described. 9. 2. The auxin analog of claim 1, further comprising a growth stimulating effective amount of one or more plant growth regulators. 10. 2. The auxin analog of claim 1, further comprising a growth stimulating effective amount of a cytokinin. 11. Said cytokinin is benzylaminopurine ^{(BAP), N 6 · (} Δ 2 isopentenyl) adenine (2iP), and 6-furfuryl - is selected from the group consisting of amino purine (kinetin), auxin according to claim 10 analog. 12. The auxin analog of claim 10, wherein the concentration ratio of cytokinin to auxin is between about 16.0 and about 1.0. 13. 2. The auxin analog of claim 1, further comprising a molecule selected from the group consisting of fungicides, herbicides, and antibiotics. 14． 2. The auxin analog of claim 1, wherein said regeneration is related to root formation or shoot formation. 15. A composition for stimulating plant growth, regeneration of plant cells or tissues, or plant cell or tissue transformation, comprising an effective amount of an auxin analog of IAA capable of functioning as a plant hormone, and Chemical structure Wherein R _{1 to} R ₅ are independently a halo group, an alkyl group having 1 to 10 carbon atoms, an alkoxy group having 1 to 10 carbon atoms, 1 to 10 carbon atoms. An acyl group having an atom, an acylamide group having 1 to 10 carbon atoms, an acyloxy group having 1 to 10 carbon atoms, or a salt, ester, or amide derivative thereof, provided that , Except for indole-3-acetic acid and 5-bromoindole-3-acetic acid. 16. Said R ₁ to R _5, individually, bromo group, chloro group, selected from the group consisting of fluoro, and iodo, The composition of claim 15. 17． Wherein R ₁ to R ₅ are individually selected from the group consisting of alkyl groups having 1 to 10 carbon atoms The composition of claim 15. 18. Wherein R ₁ to R ₅ are individually selected from the group consisting of alkoxy radicals having 1 to 10 carbon atoms The composition of claim 15. 19. The composition according to claim 15, wherein R _{1 to} R ₅ are individually selected from the group consisting of acyl groups having 1 to 10 carbon atoms. 20. Wherein R ₁ to R ₅ are individually selected from the group consisting of acylamido group having 1 to 10 carbon atoms The composition of claim 15. 21. Wherein R ₁ to R ₅ are individually selected from the group consisting of acyloxy groups having 1 to 10 carbon atoms The composition of claim 15. 22. The auxin analog of IAA is 2-bromoindole-3-acetic acid, 6-bromoindole-3-acetic acid, 7-bromoindole-3-acetic acid, 5-chloroindole-3-acetic acid, 5-fluoroindole-3- 16. The method of claim 15, wherein the acetic acid is selected from the group comprising acetic acid, 7-fluoroindole-3-acetic acid, 5-ethylindole-3-acetic acid, 7-ethylindole-3-acetic acid, and 5-methoxyindole-3-acetic acid. A composition as described. 23. 16. The composition of claim 15, further comprising a growth stimulating effective amount of one or more plant growth regulators. 24. 16. The composition of claim 15, further comprising a growth stimulating effective amount of a cytokinin. 25. Said cytokinin is benzylaminopurine ^{(BAP), N 6 · (} Δ 2 isopentenyl) adenine (2iP), and 6-furfuryl - is selected from the group consisting of amino purine (kinetin), the composition of claim 24 object. 26. 25. The composition of claim 24, wherein the concentration ratio of cytokinin to auxin is between about 16.0 and about 1.0. 27. 16. The composition of claim 15, further comprising a compound selected from the group consisting of a fungicide, a herbicide, and an antibiotic. 28. 16. The composition of claim 15, wherein said regeneration relates to root formation or shoot formation. 29. A method for stimulating plant growth, regeneration of plant cells or tissues, or transformation of plant cells or tissues, comprising incubating the plant with an effective amount of an auxin analog of IAA, The auxin analog can function as a plant hormone in regulating plant growth and has the following chemical structure: Wherein R _{1 to} R ₅ are independently a halo group, an alkyl group having 1 to 10 carbon atoms, an alkoxy group having 1 to 10 carbon atoms, 1 to 10 carbon atoms. An acyl group having an atom, an acylamide group having 1 to 10 carbon atoms, and an acyloxy group having 1 to 10 carbon atoms, or a salt, ester, or amide derivative thereof, However, except for indole-3-acetic acid and 5-bromoindole-3-acetic acid, so that an improvement or change in the growth characteristics of the plant when compared to the corresponding plant in the absence of the auxin analog of the IAA. The method occurs in the plant in the presence of an auxin analog of the IAA. 30. Wherein R ₁ to R ₅ are, individually, bromo group, chloro group, selected from the group consisting of fluoro, and iodo, The method of claim 29. 31. Wherein R ₁ to R ₅ are individually selected from the group consisting of alkyl groups having 1 to 10 carbon atoms, The method of claim 29. 32. Wherein R ₁ to R ₅ are individually selected from the group consisting of alkoxy groups having 1 to 10 carbon atoms, The method of claim 29. 33. Wherein R ₁ to R ₅ are individually selected from the group consisting of an acyl group having 1 to 10 carbon atoms, The method of claim 29. 34. Wherein R ₁ to R ₅ are individually selected from the group consisting of acylamido group having 1 to 10 carbon atoms, The method of claim 29. 35. Wherein R ₁ to R ₅ are individually selected from the group consisting of acyloxy groups having 1 to 10 carbon atoms, The method of claim 29. 36. The auxin analog of IAA is 2-bromoindole-3-acetic acid, 6-bromoindole-3-acetic acid, 7-bromoindole-3-acetic acid, 5-chloroindole-3-acetic acid, 5-fluoroindole-3- 30. The method according to claim 29, wherein the acetic acid is selected from the group comprising acetic acid, 7-fluoroindole-3-acetic acid, 5-ethylindole-3-acetic acid, 7-ethylindole-3-acetic acid, and 5-methoxyindole-3-acetic acid. The described method. 37. 30. The method of claim 29, wherein the auxin analog of IAA is incubated with the plant in the presence of one or more growth regulators. 38. 30. The method of claim 29, wherein the auxin analog of IAA is applied to the plant in the presence of cytokinin. 39. 39. The method of claim 38, wherein said cytokinin and said auxin analog are present in a ratio between about 1.0 and about 16.0. 40. 30. The method of claim 29, further comprising a molecule selected from the group consisting of a fungicide, a herbicide, and an antibiotic. 41. 30. The method of claim 29, wherein the plant is selected from the group consisting of a part, cell, tissue, immature seed, and mature seed of the plant. 42. 30. The method of claim 29, wherein the plant is a plant tissue culture. 43. 30. The method of claim 29, wherein said plant is a monocotyledonous plant. 44. 30. The method of claim 29, wherein the plant is a dicotyledon. 45. 30. The method of claim 29, wherein said plant is a transgenic plant. 46. 30. The method of claim 29, wherein the plant is selected from the group consisting of woody, cassava, and monocot crops. 47. 30. The method of claim 29, wherein the plant growth characteristic is root formation or shoot formation.