JP2002528394A - Compositions containing compounds that modulate plant cell growth and methods of use - Google Patents

Abstract

  (57) [Summary] The present invention generally relates to compositions containing one or more compounds that regulate the growth of plants, plant cells, and plant tissues. More specifically, the present invention relates to one or more auxin-like compounds (monosubstituted, disubstituted-, or polysubstituted phenylacetic acid compounds, or ester or salt derivatives thereof (e.g., 2-, 3- And compounds such as 4-bromophenylacetic acid and derivatives thereof). The invention also relates to such compositions further comprising one or more additional compounds that regulate the growth of plants, plant cells, and plant tissues. Such compounds include cytokinins (eg, zeatin, kinetin, and 6-benzylaminopurine (BAP)) and gibberellins.

Description

DETAILED DESCRIPTION OF THE INVENTION

BACKGROUND OF THE INVENTION The present invention is in the field of plants and in the field of plant cell biology. The present invention relates generally to compositions containing one or more compounds that regulate the growth of plants and plant cells. More specifically, the present invention relates to one or more auxin-like compounds (monosubstituted, disubstituted-, or polysubstituted phenylacetic acid compounds, or ester or salt derivatives thereof (e.g., 2-, 3-, And compounds such as 4-bromophenylacetic acid and derivatives thereof). The present invention also provides for the regulation of plant and plant cell growth,
Such compositions further comprising one or more additional compounds. Such compounds include cytokinins (eg, zeatin, kinetin, and 6).
-Benzylaminopurine (BAP)) and gibberellin. The invention also relates to methods of regulating the growth of plants and plant cells. The method comprises contacting the plant cell with one or more of the above compositions. The present invention also relates to methods for producing and culturing genetically modified plants and plant cells.
The method includes obtaining a genetically modified plant or plant cell, and contacting the plant or plant cell with one or more of the above compositions. The invention also relates to the genetically modified plants and plant cells produced by such a method.

[0002] Plant growth is affected by various physical and chemical factors. Physical factors include available light, photoperiod, humidity, and temperature. Chemical factors include minerals, nitrates, cofactors, nutrients, and plant growth regulators, or hormones (eg, auxins, cytokinins, and gibberellins).

[0003] Indole-3-acetic acid (IAA) is a naturally occurring plant growth hormone identified in plants. IAA has been shown to be directly responsible for increased growth in plants in vivo and in vitro. Characteristics affected by the IAA include cell elongation, internodal distance (height), and leaf surface area. IAA and other compounds that exhibit hormone-modulating activity similar to that of IAA are included in a class of plant regulators called "auxins".

[0004] Cytokinin-based preparations such as 6-furfurylaminopurine (kinetin) and 6-benzylaminopurine (BAP) are also known as growth stimulating factors. However, preparations based on cytokinins are most effective in combination with auxins. The mechanisms by which cytokinins influence the growth cycle of plants are far from understood, but it is clear that they affect leaf growth and prevent senescence in certain plants.

A general goal in this field is to successfully manipulate and regenerate plants of major crop varieties using methods such as tissue culture and genetic engineering. Major crop varieties of particular interest in this interest are agricultural crops such as corn, wheat, rice, soybean, and cotton.

[0006] In vitro culture techniques have been established for plant regeneration. (Rei
nert, J .; And Bajaj, Y .; P. S. (1977) Plant C
cell Tissue and Organ Culture, Berlin;
Springer; Simmonds, N .; W. (1979) Principl
es of Crop Improvement, London; Longma
n; Vasil, I .; K. Ahuja, M .; K. And Vasil, V .; (1
979) "Plant tissue cultures in geneti
cs and plant bleeding ", Adv. Genet. 20:
127-215). Specific in vitro culture techniques for regenerating plants include culture of embryos, culture of shoot tips, and culture of callus, cells, and protoplasts. Embryo culture has been shown to be important in performing difficult interspecific crosses, but shoot tip cultivation requires rapid cloning, virus-free clone development, and It is important in the preservation work. Culture of callus, cells, and protoplasts has been shown to be important for cultures that have lost organization but are capable of recovery.

[0007] Genetic engineering techniques for plants have also been established. These techniques include gene transfer by transformation or by protoplast fusion. Gene transfer involves the following steps: (a) identification of specific genes; (b) isolation and cloning of genes; (c) transfer of genes into recipient plant host cells; (d). Integration, transcription and translation of DNA in recipient cells; and (e) propagation and use of transgenic plants (T. Kosuge, CP.
Meredith and A.M. Edited by Hollaender (1983) Genet
ic Engineering of Plants, 26: 5-25; Rog
ers et al. (1988) Methods for Plant Molecula.
r Biology, A. et al. Weissbach and H.W. Weidsbach, Ed., Academic Press, Inc. , San Diego, CA). In protoplast fusion, plant cell protoplasts are fused by standard chemical techniques (eg, PEG) or electroporation techniques.
After regeneration of the fused cells, diploids between species have been obtained. This technique can provide the desired diploid that cannot be made by conventional means, and demonstrates the potential for somatic recombination by some of its variants. The above technique is widely used (Chaleff, RS (1981) Genetics of Hig).
her Plants, Applications of Cell Cult
ure, Cambridge: Cambridge University P
and newly inserted foreign genes have been shown to remain stable during plant regeneration and to be transmitted to progeny as typical Mendelian traits (Horsch et al., Science 223: 496 ( 1984), and De
Block et al., EMBO 3: 1681 (1984)). These foreign genes retain their normal tissue-specific and developmental expression patterns.

[0008] Transformation systems mediated by Agrobacterium tumefaciens have proven to be effective for many dicot species.
For example, Barton et al. (Cell 32: 1033 (1983)) reported on the transformation and regeneration of tobacco plants, and Chang et al. (Plantant 5:
551-558 (1994)) is Arabidopsis thaliana.
Described a stable genetic transformation.

The Agrobacterium method for gene transfer has also been applied to monocotyledonous plants, eg, as follows: Liliaceae and Amaryllida
In plants of the ceae family (Hooykaas-Van Slogteren et al., 1984, Nature 311: 763-764), Dioscorea
bulbifera (Ya yam) (Schaffer et al., 1987, Na
cure 327: 529-532), and in rice and corn (Ishida et al., 1996, Nat. Biotechnology 14:
745-750; Hiei et al., 1997, Plant Mol. Biol. 35
: 205-218).

[0010] Transformation of food crops can be accomplished using other methods (eg, polyethylene glycol (PEG), to promote DNA uptake (Uchimiya et al., Mol. Gen.
Genet. 204: 204-207 (1986)), and electroporation (Fromm et al., Nature 319: 791-793 (1986)).
By). Both of these used protoplasts as targets for transfer. Monocot and dicot tissues can be transformed by bombardment of the tissue with DNA-coated particles (Wang et al. (1988) Plan.
t Mol. Biol. 11: 433-439; Wu, Plant Biote
chronology (1989), edited by Kung and Arntzen, Butte.
rworth Publishers, Stoneham, MA). Regeneration was performed using rice (Abdulah et al. (1986), Bio / Technology 4: 1).
087-1090) and corn (Rhodes et al., Bio / Techn.
6: 56-60 (1988) and Science 240: 20.
4-207 (1988)).

Thus, while regeneration and transformation protocols have been established, there remains a need to stimulate regeneration and transformation of monocots and dicots. In fact, some plants are difficult to regenerate and transform (Vasil and Vasil (1994), Plant Cell and Tissue).
Culture (edited by Vasil and Thorpe), Kluwer Acad
emic Publishers, Dordrech, Netherlands
Chee, Plant Cell Reports 14: 753-757 (
1995); Burns and Schwartz, Plant Cell Re.
ports 15: 405-408 (1996); Mihaljevic et al., P.
lant Cell Reports 15: 610-614 (1996); S
Chopke et al., Nature Biotechnology 14: 731 (
1996)). Furthermore, it is necessary to stimulate the growth of plants, especially after transformation and regeneration. Finally, promote in vitro cultivation of plants, plant tissues, and plant cells and protoplasts to allow plant growth and regeneration in vitro and to facilitate genetic manipulation and development of hybrid plants There remains a need for improved culture media.

The present invention meets these needs by providing a composition that stimulates plant growth, regeneration of plant cells and tissues, and transformation of plant cells and tissues. The compositions of the present invention comprise one or more mono- or poly-substituted phenylacetic acid (PAA) compounds, or ester or salt derivatives thereof, having auxin activity on plant cells and tissues. They have the following general formula:

[0013]

Embedded image Here, X represents one or more halo groups (particularly one or more fluoro-, chloro-, bromo-, or iodo-groups), one or more alkyl- (R-) groups, one or more alkoxy groups. -(RO-) group, one or more alkylamino- (RNH-) groups, one or more acyl- (RCO-) groups, one or more acylamido- (RCONH-) groups, and one or more acyloxy groups. -(RCOO-) groups selected from the group consisting of one or more substituents, which may be the same or different. According to a preferred aspect of the present invention, R can be CH ₃ (CH ₂ ) _n , where n is an integer ranging from 0 to 10. The present invention contemplates the use of such compounds to affect growth, regeneration, and transformation in both monocots and dicots. In particular, the present invention relates to 2, 4, 5, and / or PAA structures.
Alternatively, there is provided a composition containing one or more such compounds having a substituent at the 6-position. Here, the substituent is halo-, alkyl-, alkylamino-, alkoxy-, acyl-, acylamide- having 1 to 10 carbon atoms.
Or an acyloxy-substituent. The present invention also provides compositions containing one or more polysubstituted PAA compounds having 2 to 5 substituents.
At positions selected from the group consisting of positions 2, 4, 5, and 6 of the PAA structure, the substituents may be the same or different. Here, the above substituent is 1 to 10
Halo-, alkyl-, alkylamino-, alkoxy- having 4 carbon atoms
, Acyl-, acylamido-, or acyloxy- substituents. Preferred PAA compounds for use in compositions according to these aspects of the present invention include
-Bromophenylacetic acid, 3-bromophenylacetic acid, 4-bromophenylacetic acid, and derivatives thereof, but are not limited thereto.

The compositions of the present invention may comprise one or more of the above monosubstituted or polysubstituted PAAs
May contain one or more additional plant growth regulators. Such plant growth regulators include, for example, cytokinins (eg, zeatin, 6-furfurylaminopurine (kinetin), or 6-benzylaminopurine (BAP) in a fixed ratio for a wide range of applications to various plants.
)), Gibberellin and the like. In specific embodiments, the invention provides:
Exemplified using a composition comprising a mono- or polysubstituted PAA compound of the invention having between 1 and 5 substituents and a cytokinin to affect plant growth, These substituents can be the same or different, and at positions 2, 4, 5, and / or 6 of the PAA structure, halo-, alkyl-, alkylamino-, alkoxy-, acyl-, acylamide- , And acyloxy-substituents.

The invention further relates to a culture medium containing one or more compounds or compositions of the invention as described above. Such culture media include, for example, one or more of the above PAA's.
A compound, and optionally one or more of the above plant growth regulators, to stimulate plant growth, to stimulate plant cell and tissue regeneration, and /
Or to stimulate transformation of plant cells and tissues.

[0016] The present invention also relates to a method of stimulating the growth of a plant, plant cell, or plant tissue. The method comprises the steps of: (a) applying an effective amount of one or more compounds or compositions of the present invention to a plant, plant cell, or plant tissue; and (b) the plant, Incubating a plant cell or plant tissue under conditions sufficient to stimulate the growth of the plant, plant cell or plant tissue.

The present invention also provides a method for stimulating the regeneration of plant cells and / or tissues. The method comprises the steps of: (a) applying an effective amount of one or more compounds or compositions of the present invention to plant cells or tissues; and (b)
Incubating the plant cells or tissues under conditions sufficient to stimulate regeneration of the plant cells or tissues.

[0018] The invention also relates to a method for culturing a plant, plant cell, or plant tissue in vitro. The method includes the following steps: (a) obtaining a plant, plant cell, or plant tissue to be cultured; (b) converting the plant, plant cell, or plant tissue to one or more of the present invention. And c) incubating the plant, plant cell, or plant tissue under conditions suitable to support the cultivation of the plant, plant cell, or plant tissue.

The invention further provides a method for the transformation of plant cells and / or tissues. The method comprises the steps of: (a) contacting the plant cells or tissue with a nucleic acid molecule (eg, by transformation or protoplast fusion); (b) contacting the plant cells or tissue with Contacting an effective amount of one or more compounds or compositions of the present invention; and (c) contacting the cells or tissues of the plant with
Incubating with the nucleic acid molecule under conditions sufficient to induce transformation of a plant cell or tissue. The compounds and compositions of the present invention can also be used to stimulate the regeneration or growth of transformed tissues or cells, and therefore,
A method for obtaining a transgenic plant is provided. Thus, the present invention also provides
It also relates to transformed or transgenic plants, plant cells, and plant tissues produced by the methods described above.

[0020] The invention also relates to a method of reducing or reducing environmental stress in a plant, plant cell or tissue. The method includes the following steps: (a) environmental stress (eg, drought, excessive temperature, reduced temperature, chemical toxicity (eg,
Antibiotics, herbicides), contaminants, plants exposed to excessive and reduced light),
Contacting a plant cell or tissue with an effective amount of one or more compounds or compositions of the present invention; and Incubating under conditions sufficient for.

Other preferred embodiments of the present invention will be apparent to one of ordinary skill in light of the following drawings and description of the invention, and of the claims.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Unless defined otherwise, all technical and scientific terms used herein are commonly understood by one of ordinary skill in the art to which this invention pertains. It has the same meaning as the meaning that is generally understood. The following definitions are used in the description and claims.
Provided to provide clarity of intent or scope of their use.

As used herein, the term “phenylacetic acid” or “PAA” refers to X
Is hydrogen, referring to the chemical structure of FIG. The term refers not only to the free acid form of PAA, but also to the amide, ester, or salt form. PAA means:
For example, salts and amides such as sodium, potassium, lithium, magnesium, calcium, ammonium, dimethylamine, ethanolamine and the like, and salts and ester derivatives such as lower alkyl esters are included. Examples of ammonium salts include tetraalkylammonium salts (eg, tetramethylammonium halide) and trialkylbenzylammonium halide salts (eg, benzyltrimethylammonium chloride salt).

As used herein, the term “monosubstituted PAA” refers to the PAA of FIG.
A molecule wherein X is one of the positions 2, 4, 5, or 6 of PAA
Wherein the substituent is a halo-group (for example, fluoro-, chloro-, butyl-
Such as lomo- or iodo-), an alkyl-group (R-, especially where R is _Three (CH_Two)_nAnd n is an integer that can range from 0 to 10
), An alkoxy-group (RO-, especially where R is CH_Three(CH_Two)_nAnd that
And n is an integer that can range from 0 to 10), an alkylamino-group
(RNH-, especially where R is CH_Three(CH_Two)_nAnd n is from 0
An acyl-group (RCO-, especially where
R is CH_Three(CH_Two)_nAnd n can range from 0 to 10
Is an integer), an acylamide-group (RCONH-, especially where R is CH_Three(
CH_Two)_nAnd n is an integer that can range from 0 to 10),
Or an acyloxy-group (RCOO-, especially where R is CH_Three(CH_Two)_nso
And n is an integer that can range from 0 to 10)
Refers to a molecule.

As used herein, the term “polysubstituted PAA” refers to the PAA of FIG.
A molecule, wherein X represents two or more substituents at two or more positions corresponding to the 2, 4, 5, or 6 positions in the chemical structure of PAA, wherein the substituents are May be the same or different, and wherein these substituents may be selected from the halo-, alkyl-, alkoxy-, alkylamino-, acyl-, acylamido-, and acyloxy- substituents described above. Say.

As used herein, the term “auxinic analog (s) of PAA” or “PAA analog” or “PAA auxinic analog”
Or "PAA compounds" can be used interchangeably and include, for example, one or more of the above-described halo-, alkyl-, alkoxy-, alkylamino-, acyl-, acylamido-, and acyloxy-substituents. Refers to mono- or polysubstituted PAA.

As used herein, a PAA compound includes not only the free acid form, but also the amide, ester, or salt form of a mono- or polysubstituted PAA compound. .

Halo-group refers to halogen and includes, but is not limited to, iodo-, bromo-, chloro-, and fluoro-groups.

Alkyl-groups include, but are not limited to, R-groups (linear, branched, or cyclic; saturated or unsaturated). Here, R has 1 to 10 carbon atoms.

Alkoxy-groups include, but are not limited to, RO-groups (linear, branched, or cyclic; saturated or unsaturated). Here, R has 1 to 10 carbon atoms.

Alkylamino-groups include, but are not limited to, RNH-groups (linear, branched, or cyclic; saturated or unsaturated). Here, R has 1 to 10 carbon atoms.

Acyl-groups include, but are not limited to, RCO- (linear, branched, or cyclic; saturated or unsaturated). Here, R has 1 to 10 carbon atoms.

Acylamido-groups include, but are not limited to, acylamido, RCONH- (linear, branched or cyclic; saturated or unsaturated). Where R is
It has from 1 to 10 carbon atoms.

Acyloxy-groups include, but are not limited to, acyloxy, RCOO- (linear, branched or cyclic; saturated or unsaturated). Here, R is 1
From 10 to 10 carbon atoms.

Examples of groups R in the above compounds include methyl, ethyl, ethenyl, ethynyl, propyl, isopropyl, propenyl, propynyl, butyl, isobutyl, butenyl, butynyl, pentyl, 2-pentyl, 3-pentyl, hexyl, Hexenyl, heptyl, heptenyl, octyl, octenyl, nonyl, nonenyl, decyl,
Decenyl and the like, but are not limited thereto.

[0036] As used herein, the term "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 include auxin, cytokinin, gibberellin, ethylene, and abasic acid.
(ABA).

As used herein, the term “auxin” or “cytokinin”
Refers to a plant growth regulator that affects plant growth. Auxin is
Indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), 2,4-
Dichlorophenoxyacetic acid (2,4-D), naphthaleneacetic acid (NAA), 5,6-
It is exemplified by compounds such as dichloro indole-3-acetic acid (5,6-Cl ₂ -IAA). Cytokinin is 6-benzylaminopurine (BAP)
, N ⁶ · (Δ ₂ -isopentenyl) adenine (2iP), isopentenyl pyrophosphate (ipp), 6- (4-hydroxy-3-methyl-2-transbutenylamino) purine (zeatin), 6-furfuryl Exemplified by compounds such as aminopurine (kinetin) and the like. Compounds can be used in biological assays (eg, Ave).
Na sativa cotyledon sheath elongation (Bottger et al., Planta 140
: 89 (1978)) or inhibition of root growth of Chinese cabbage (Marumo et al. (197)
4), Plant Growth Substance, page 419, Hirok
awa Publishing Co. Inc. (Tokyo, Tokyo), or swelling of hypocotyls of P. japonicus (Marumo et al. (1974) supra)) can be tested for auxin activity. Cytokinin activity can be determined as is well known in the art (Skoog et al. (1967) Phytochem 6: 1169-1192;
Morris, Ann. Rev .. Plant Physiol. 37: 509-
538 (1986); Horgan (1984) Advanced Plant
Physiol (Wilkins, MB, ea.) Pitman Pub
lishing, London, pp. 53-75; Letham and Palni
, Ann. Rev .. Plant. Physiol, 34: 163-197 (19
83); and Chen (1981) Metabolism and Mole.
cultural Activities of Cytokinins (Guern
, J. et al. And Peaud-Lenoel, C .; Ed.) Springer, New
York, pp. 34-43), which can be measured in assays designed to assess growth promotion in plants, such as tobacco bioassays. Variations in the cytokinin / auxin concentration ratio cause enhanced plant growth to occur preferentially in certain tissues. For example, a high cytokinin / auxin ratio promotes shoot growth, while a low cytokinin / auxin ratio promotes root growth (Depicker et al. (1983) G
enetic Engineering of Plants (T. Kosun
ge, C.I. P. Meredith and A.M. Hollaender) Plen
um Press, New York, p. 154).

As used herein, the term “medium” or “medium (
media) "is a solid or liquid composition containing sufficient nutrients to support in vitro cultivation, growth, regeneration, and / or transformation of plants, plant cells, plant tissues, protoplasts, and the like. A thing.

A plant culture medium is composed of a number of components, and these components vary from culture medium to culture medium. A “1 × formulation” is mentioned to refer to any aqueous solution that contains some or all of the components found in the culture medium of a plant at working concentrations. "1 ×
"Formulation" can mean, for example, a plant culture medium or any subgroup of components of that medium. The concentration of a component in a 1 × solution is about the same as the concentration of that component found in plant culture formulations used to maintain or culture plant cells or tissues in vitro. The plant culture medium used for the cultivation of plants, plant cells, plant tissues, etc. in vitro is a 1 × formulation by definition. When multiple components are present, in a 1 × formulation, each component has a concentration approximately equal to the concentration of those components in the plant culture medium. For example, Gamborg's B
The -5 plant culture medium contains, among other ingredients, 0.134 g / L ammonium sulfate, 2.5 g / L potassium nitrate, and 20 g / L sucrose. A “1 × formulation” of the components of these media contains approximately the same concentration of these components in solution. Thus, when referring to a “1 × formulation”, it is intended that each component in the solution has the same or nearly the same concentration as found in the described plant culture medium. You. The concentration of the components in the 1 × formulation of the plant culture medium is
It is well known to those skilled in the art. Vasil and Thorpe, Plant Cell
and Tissue Culture, Dordrech, Netherlands
ands: Kluwer Academic Publishers (1995
)checking. It is incorporated herein by reference in its entirety.
However, osmolality and / or pH may differ in a 1 × formulation, especially when fewer components are included in the 1 × formulation, as compared to the culture medium.

A “10 × formulation” is meant to refer to a solution in which each component in the solution is 10 times more concentrated than the same component in the plant culture medium. For example, a 10 × formulation of Gamburg's B-5 plant culture medium, among other ingredients, has 1.34 g / L ammonium sulfate, 25 g / L potassium nitrate, and 20 g / L potassium nitrate.
It may contain 0 g / L sucrose (compared to the 1 × formulation above). A “10 × formulation” may include a number of additional components at about 10 times the concentration found in 1 × culture medium. As is readily apparent, "20x formulation", "25x formulation",
“50 × formulation” and “100 × formulation” refer to solutions containing components at about 20-fold, 25-fold, 50-fold, or 100-fold concentration, respectively, when compared to 1 × culture medium. Show. Again, the osmolality and pH of the media formulation and the concentrated solution can vary. See U.S. Patent No. 5,474,931. This relates to techniques for enriching culture media, the disclosure of which is hereby incorporated by reference in its entirety.

As used herein, the term “carrier” is hydrophobic or hydrophilic or amphiphilic and is useful in promoting the effectiveness of an active ingredient in a plant (ie, PAA analogs of the invention), refers to chemically, or biologically, or physiologically acceptable molecules.

As used herein, the term “plant” refers to an entire plant or a part of a plant, for example, plant parts, plant cells, plant tissues, plant embryos, explants Includes pieces or plant seeds. The term further refers to calli, protoplasts, embryos, organs,
A plant in the form of a suspension culture or tissue culture, including but not limited to cultures such as organelles, is contemplated.

As used herein, the term “transformed plant” or “transformed plant tissue” refers to a transformation or protoplast fusion of a nucleic acid molecule (eg, natural or foreign DNA). Into plants or plant tissues.

As used herein, the term “transgenic plant” or “transgenic plant tissue” refers to a plant or plant tissue that has been stably transformed with a foreign gene.

The term “transient expression” refers to a plant or plant tissue transformed with DNA, wherein the DNA is expressed only for a short period immediately after transformation.

As used herein, the term “genetically engineer” refers to the ability of foreign, often chimeric, genes to be regenerated into full, sexually competent, viable plants1 Refers to introduction into one or more plant cells. Can this plant be self-pollinated,
Or, it can be cross-pollinated with other plants of the same species, so that the foreign gene carried by the germline can be inserted or crossed into agriculturally useful plant varieties.

As used herein, the term “regeneration” refers to at least one newly generated or regenerated plant from a cultured plant tissue or unit (eg, leaf disks, seeds, etc.) Refers to the production of tissue (eg, roots, shoots, calli, etc.).

As used herein, the term “percent”, “% regeneration”, or “regeneration efficiency” refers to, for example, as a percentage of the total number of tissue-cultured plant units, Refers to the number of tissue cultured plant units that produce at least one newly generated or regenerated tissue:

[0049]

(Equation 1) As used herein, the term "affecting plant growth" or "affecting growth" or "influencing factors" or "affecting" is observed in the absence of growth regulators. Some characteristics of overall plant growth (eg, stimulation of seed germination, induction of root formation, suppression of shoot formation, promotion of cell growth, Stimulus) or any one of a number of plant responses that improve or alter stimuli. It is further understood that in the context of the present invention, these terms may also include toxic effects on plant tissues.

As used herein, the term “effective amount” is administered to a plant such that the compound stimulates or effects one or more of a variety of plant growth responses. Refers to the amount or concentration of a compound such as a plant growth regulator or hormone.
In response to plant growth, among others, induction of stem elongation, promotion of root formation, stimulation of callus formation, enhancement of leaf growth, stimulation of seed germination, dry weight content of many plants and parts of plants And the like.

Compositions The present invention generally relates to compositions that stimulate plant growth, regeneration of plant cells and tissues, and transformation of plant cells and tissues. The compositions of the present invention comprise one or more mono- or poly-substituted phenylacetic acid (PAA) compounds, or ester or salt derivatives thereof, having auxinic activity on plant cells and tissues. The present invention provides for growth,
The use of such PAA compounds to affect regeneration and transformation is contemplated.

In a particular embodiment of the invention, the various PAA compounds are (a) MS complete medium containing only different concentrations of auxin (LifeTechnolo).
gies, Inc. And (2) MS complete medium containing various ratios of cytokinins / auxins in various plant tissues (eg, leaf disks of tobacco and tomato, and potatoes). The stalks) were screened for auxin activity by incubating.

Traditionally, to achieve shoot regeneration from potato stalks, potato tissue was transferred from a medium containing large amounts of auxin to another medium containing auxin and cytokinin. It is. In the present invention, regeneration of shoots from potato tissue was obtained without transferring the tissue from a large amount of auxin medium to a medium containing auxin and cytokinin. Furthermore, according to the invention, the regeneration of shoots in potato tissue showed a significant improvement in the number of regenerated shoots. Thus, the compositions of the present invention not only exhibit auxin activity, but also plant tissues traditionally described as difficult to regenerate (eg, cassava, woody plants, corn, soybeans, wheat, etc.). Also improve the yield of regenerated plant tissue.

The practice of the present invention contemplates a wide variety of plant growth responses. This response includes stimulating seed germination and break dormancy; increasing yield; accelerating ripening and coloring in fruit; increasing flowering and fruit set; stimulating shoot formation; inducing callus growth Inducing roots and causing cell proliferation; increasing the strength of various plant species; and increasing the dry content of many plants and parts of plants. In addition to these categories of responses, any other modification of a plant, seed, fruit, or vegetable may result in increased net growth or any increase in agricultural crops or agricultural seeds and horticultural crops or horticultural seeds. As long as it maximizes the beneficial or desired properties of
It is intended to fall within the advantageous response achieved by practicing the invention.

Appropriate application of a growth enhancing composition of the present invention to a culture of plant tissue induces shoot, root, or callus regeneration. This effect occurs in both monocotyledonous and dicotyledonous species and applies to a wide variety of plants.

The composition of the present invention has auxin activity on plant cells and tissues.
Includes one or more mono- or polysubstituted phenylacetic acid (PAA) compounds, or ester or salt derivatives thereof. Compounds according to this aspect preferably have the following general formula:

[0057]

Embedded image Here, X is one or more substituents, which may be the same or different, and which may have one or more halo groups (especially one or more fluoro-, chloro-, bromo- -Or iodo- group), one or more alkyl- (R-) groups, one or more alkoxy- (RO-) groups, one or more alkylamino- (RNH-) groups, one or more acyl groups. -(RCO-) group, one or more acylamido- (RCONH
-) Group, and one or more acyloxy- (RCOO-) groups. According to a preferred aspect of the present invention, R can be CH ₃ (CH ₂ ) _n , where n is an integer ranging from 0 to 10. These compounds can be obtained commercially or synthesized by methods well known in the art.

In particular, the present invention comprises one or more monosubstituted PAA compounds having substituents at positions 2, 4, 5, and / or 6 of the PAA structure (FIG. 1). A composition is provided wherein the substituents are halo- having 1 to 10 carbon atoms.
, Alkyl-, alkylamino-, alkoxy-, acyl-, acylamide-,
Or an acyloxy-substituent. The present invention also provides compositions containing one or more polysubstituted PAA compounds having 2 to 5 substituents. P
At a position selected from the group consisting of positions 2, 4, 5, and 6 of the AA structure (FIG. 1);
The substituents can be the same or different. Here, the substituent is a halo-, alkyl-, alkylamino-, alkoxy-, acyl-, acylamido-, or acyloxy- substituent having 1 to 10 carbon atoms. Preferred PAA compounds for use in compositions according to these aspects of the invention include 2-bromophenylacetic acid, 3-bromophenylacetic acid, 4-bromophenylacetic acid, 4-chlorophenylacetic acid and derivatives thereof, It is not limited to these.

The PAA compound is preferably included in the composition of the present invention at a concentration that promotes optimal growth, culture, regeneration, or transformation of plant cells, tissues, and the like. Preferred concentration ranges for the PAA compound used in the compositions of the present invention include, but are not limited to, about 0.01 mg / ml to about 100 mg / ml, about 0.05 mg / ml to about 100 mg / ml, about 0 mg / ml to about 100 mg / ml. 0.1 mg / ml to about 50 mg / ml, about 0.5 mg
/ Ml to about 50 mg / ml, about 0.75 mg / ml to about 25 mg / ml, about 1 mg / ml to about 25 mg / ml, about 2 mg / ml to about 20 mg / ml, about 2.5 mg / ml to about 20 mg / ml. ml, about 2.5 mg / ml to about 15 mg / ml
ml, about 2.5 mg / ml to about 10 mg / ml, about 2.5 mg / ml to about 9
mg / ml, about 2.5 mg / ml to about 8 mg / ml, about 2.5 mg / ml to about 7.5 mg / ml, about 2.5 mg / ml to about 7 mg / ml, about 2.5 mg / ml
ml to about 6 mg / ml, about 2.5 mg / ml to about 5 mg / ml, about 3 mg / ml
ml to about 5 mg / ml, and about 3 mg / ml to about 4 mg / ml. As will be readily apparent to those skilled in the art, the concentration of a given PAA analog may be increased or decreased beyond the above ranges, and may be increased or decreased for plant growth, culture, regeneration, or transformation. The effect of a reduced concentration can be determined using routine experimentation.

According to another aspect of the present invention, the PAA compound is preferably included in the composition of the present invention at a concentration that produces a toxic effect on plant tissue. A more preferred concentration range of the PAA compound used in the composition of the present invention includes, but is not limited to,
About 0.5 mg / l to about 1000 mg / l, about 0.5 mg / l to about 850 mg
/ L, about 0.5 mg / l to about 750 mg / l, about 0.5 mg / l to about 650
mg / l, about 0.5 mg / ml to about 500 mg / ml, about 0.5 mg / l to about 350 mg / l, about 0.5 mg / ml to about 250 mg / l, about 0.5 mg / ml.
1 to about 150 mg / l, about 0.5 mg / l to about 100 mg / l, about 0.5 m
g / l to about 50 mg / l, and about 0.5 mg / l to about 20 mg / l.

The compositions of the present invention may comprise one or more of the above monosubstituted or polysubstituted PAAs
And one or more additional plant growth regulators. For example, it is contemplated that other cytokinins or other plant growth regulators known in the art may be utilized with one or more PAA compounds to make additional compositions that affect growth of the invention. Is done. Plant growth regulators that can be used to advantage in the compositions of the present invention are known in the art, and are described in Skoog et al., Phytoc at a certain ratio for a wide range of applications to various plants.
hemistry 6: 1169-1192 (1967) and Theolog.
is (1989) Plant Biotechnology (Kung and A
rntzen) Butterworth Publishers, Stone
ham, MA, including, but not limited to, BAP, 2iP, ipp, zeatin, kinetin, gibberellin, and the like. In certain embodiments, the present invention can be the same or different to affect plant growth, and at positions 2, 4, 5, and / or 6 of the structure of PAA,
One or more monosubstituted one or more of the present invention having between 1 and 5 substituents which may be halo-, alkyl-, alkylamino-, alkoxy-, acyl-, acylamido-, and acyloxy- substituents. Or using a composition containing a polysubstituted PAA compound and cytokinin.

According to the present invention, one or more cytokinins or other growth regulators are used in a range of concentrations that promote optimal growth, culture, regeneration, or transformation of plant cells, tissues, etc. May be used in the composition. Preferred ranges of concentrations of cytokinins or other growth regulators used in the compositions of the present invention include, but are not limited to, about 0.01 mg / ml to about 100 mg / ml, about 0.05 mg / ml to about 100 mg / ml. / Ml, about 0.1 mg / ml to about 50 mg / ml, about 0.5 mg
/ Ml to about 50 mg / ml, about 0.75 mg / ml to about 25 mg / ml, about 1 mg / ml to about 25 mg / ml, about 2 mg / ml to about 20 mg / ml, about 2.5 mg / ml to about 20 mg / ml. ml, about 2.5 mg / ml to about 15 mg / ml
ml, about 2.5 mg / ml to about 10 mg / ml, about 2.5 mg / ml to about 9
mg / ml, about 2.5 mg / ml to about 8 mg / ml, about 2.5 mg / ml to about 7.5 mg / ml, about 2.5 mg / ml to about 7 mg / ml, about 2.5 mg / ml
ml to about 6 mg / ml, about 2.5 mg / ml to about 5 mg / ml, about 3 mg / ml
ml to about 5 mg / ml, and about 3 mg / ml to about 4 mg / ml. As will be readily apparent to one of skill in the art, the concentration of a given cytokinin or other growth regulator may be increased or decreased beyond the above ranges, and may be dependent on the growth, culture, regeneration, or transformation of the plant. The effect of increased or decreased concentration can be determined using routine experimentation.

The exact amount of growth affecting composition used in the practice of the present invention will depend on the type of response desired, the formulation used, and the type of plant being treated.
The present invention relates to a method for preparing a cytokinin or other compound comprising between about 50.0 and 0.001, preferably between about 5.0 and 0.05, and more preferably between about 2.0 and 0.25. The use of the ratio of the concentration of the growth regulator to the concentration of the PAA analog is contemplated.

The mechanisms by which the compounds and compositions of the present invention affect the growth cycle of plants and plant tissues are not completely understood at this time. However, as shown in the examples below, it is clear that the compositions of the present invention play a significant role in inducing a number of growth affecting responses in various plant species.

Culture Medium The present invention further relates to a plant culture medium containing one or more of the above PAA compounds or compositions of the present invention. Such culture media can be used, for example, to stimulate one or more of the above-described PAA compounds, and optionally one or more of the above-described plant growth regulators, to stimulate plant growth, and to enhance plant cell and plant cells. To stimulate tissue regeneration,
And to stimulate transformation of plant cells and tissues.

A cell culture medium according to this aspect of the invention is aqueous-based or solid or semi-solid to form a “basal medium” and is deionized,
Contains many components in a solution of distilled water. As components that may be present in the basal medium of the present invention, amino acids, vitamins, inorganic salts, D-glucose, sucrose, MES,
Glycine, and coagulation reagents such as agar, agarose, gellite, charcoal, and coconut. Each of these components is, for example, S
igma (Saint Louis, Missouri).

As amino acid components that can be contained in the medium of the present invention, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine , L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline,
L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-
Valine is mentioned. These amino acids are, for example, Sigma (Saint
Louis, Missouri).

As the vitamin components that can be contained in the medium of the present invention, biotin, choline chloride, D
-Ca ⁺⁺ - pantothenate, folic acid, i- inositol, niacinamide, pyridoxine, riboflavin, thiamine, and vitamin B ₁₂ and the like. These vitamins are, for example, Sigma (Saint Louis, Missouri)
) Can be obtained commercially.

As the inorganic salt component that can be used in the medium of the present invention, a calcium salt (for example, C
aCl ₂ ), CuSO ₄ , FeSO ₄ , KCl, magnesium salt (eg, MgC
l ₂ ), manganese salts (eg, MnCl ₂ ), sodium acetate, NaCl, NaH
CO ₃ , Na ₂ HPO ₄ , Na ₂ SO ₄ , and ions of the trace elements selenium, silicon, molybdenum, vanadium, nickel, tin, and zinc. These trace elements may be in various forms, preferably Na ₂ SeO ₃ , Na ₂ Si
O ₃ , (NH ₄ ) 6 Mo ₇ O ₂₄ , NH ₄ VO ₃ , NiSO ₄ , SnCl, and ZnS
It may be provided in the form of a salt such as O. These inorganic salts and trace elements can be obtained commercially, for example, from Sigma (Saint Louis, Missouri).

[0070] Examples of plant cell culture media that can be used to prepare the culture media of the present invention include, but are not limited to, Anderson's Pla.
nt Culture Media, CLC Basal Media, Gam
burg's Media, Guilard's Marine Plant
Culture Media, Provasoli's Marine Me
dia, Kao and Michaeluk's Media, Murash
image and Skoog Media, McCown's Woody P
lant Media, Knudson Orchid Media, Lind
emann Orchid Media and Vacin and Went
Media. Formulations for these media that are commercially available, as well as for many other commonly used plant cell culture media, are well known in the art and are described, for example, in GIBCO BRL Products. a
nd Reference Guide (Life Technologies
, Inc. Rockville, Maryland), and Sigma P.
lant Cell Culture Catalog (Sigma; St
. Louis, Missouri).

As those skilled in the art will appreciate, any of the above media of the present invention may comprise an indicator or selection agent (eg, dyes, antibiotics, amino acids, enzymes, substrates, etc.), filters (eg, charcoal), salts , Such as polysaccharides, ions, surfactants, stabilizers, etc.
It is intended that one or more additional components may also be included.

In a particularly preferred embodiment of the invention, the culture medium described above may contain one or more buffer salts at a concentration sufficient to provide optimal buffering capacity for the culture medium. According to one aspect of the present invention, the buffer salt is added before, during, or during the addition of one or more PAA compounds and optionally one or more cytokinins or other growth regulators to the medium. Later, it can be added in powdered form to the powdered medium.

The components of the medium can be dissolved in a liquid carrier or can be maintained in a dry form. When dissolved in a liquid carrier at the preferred concentrations shown in Table 1 (ie, a “1 × formulation”), the pH of the medium is about 4 to 8, preferably
It should be adjusted to about 4.5 to 7, 5 to 6, or 5.5 to 6. Of course, the optimal p for a given culture medium to be used for a particular cell type
H can also be determined empirically by those skilled in the art using known methods.

The type and method of liquid carrier used to dissolve the components in the solution will vary and can be determined by one skilled in the art by merely performing routine experimentation. Typically, the components of the medium can be added in any order. As will be readily apparent to those skilled in the art, each of the components of the culture medium may react with one or more other components in solution. Accordingly, the present invention provides a culture medium supplemented as described above with one or more PAA compounds, and also optionally one or more cytokinins or other plant growth regulators, and components thereof. Include any reaction mixture formed after mixing.

Preferably, the solution containing a component is more concentrated than the concentration of the same component in a 1 × media formulation. The ingredients can be concentrated 10 times (10 × formulation)
Can be 20-fold concentrated (20 × formulation), 25-fold concentrated (25 × formulation), 50-fold concentrated (50 × formulation), or 100-fold concentrated (1
00 × formulation). Higher concentrated formulations can be made as long as the components remain soluble and stable. See U.S. Patent No. 5,474,931. This relates to a method for solubilizing culture medium components at high concentrations.

If the components of the medium are prepared as separate, concentrated solutions, a suitable (sufficient)
A quantity of each concentrate is mixed with a diluent to yield a 1 × media formulation. Typically, the diluent used is water, but other solutions, including aqueous buffers, aqueous saline solutions, or other aqueous solutions, may be used in accordance with the present invention.

Alternatively, the culture medium of the present invention can be prepared in the form of a dry powder and coagulated with one or more solvents, preferably aqueous solvents such as water or saline solution, and reconstituted. This results in an agglomerated dry powder plant culture medium that dissolves more easily in the makeup. See, for example, co-pending U.S. Patent Application Serial No. 09 / 023,790, filed February 13, 1998. This relates to a method for the production of an agglomerated dry powder culture medium, the disclosure of which is hereby incorporated by reference in its entirety.

[0078] The culture medium of the present invention is typically sterilized to prevent unwanted contamination. Sterilization can be achieved, for example, by mixing the concentrated components to produce a sterile culture medium, followed by a low protein binding membrane filter having a pore size of about 0.1 to 1.0 μm (eg, Millipore, Bedford, (Available commercially from Massachusetts). Or,
A subgroup of the concentrated components can be sterile filtered and stored as a sterile aqueous solution. These sterile concentrates can then be mixed under sterile conditions with sterile diluents to produce a concentrated 1 × sterile media formulation. Autoclaves or other sterilization methods based on increasing temperatures are not preferred. This is because certain components of the culture media of the present invention can be thermolabile and can be irreversibly degraded by temperatures such as those achieved during most heat sterilization methods. The dry powder culture medium of the present invention, including the agglomerated dry powder culture medium described above, is described, for example, in co-owned, co-pending U.S. Patent Application No. 09, filed February 13, 1998. / 023,790, can be sterilized by irradiation using gamma irradiation or ultraviolet irradiation.

Although the culture medium of the present invention is exemplified herein as suitable for the cultivation of rice, corn, wheat, tobacco, potato, and soybean, the culture medium of the present invention may be any plant Species-derived cells, tissues, embryos, protoplasts, etc.
It can be used to support in vitro growth, culture, regeneration, and substrate conversion. Suitable methods for the isolation and cultivation of plants, plant cells, plant tissues, etc., and in vitro are well known to those of skill in the art (see, eg, US Pat. No. 5,674,7731, the disclosure of which is hereby incorporated by reference). , Which are hereby incorporated by reference in their entirety).

Use of Compositions and Culture Media The compounds, compositions, and culture media of the present invention may be used to stimulate the growth, regeneration, transformation, or culture of plant cells, tissues, protoplasts, embryos, and the like. It can be used in the various ways used. Thus, in a further aspect, the invention relates to a method of stimulating the growth or regeneration of a plant, plant cell or plant tissue. The method comprises the steps of: (a) applying to a plant, plant cell, or plant tissue an effective amount of one or more compounds or compositions of the present invention; Incubating the plant, plant cell, or plant tissue under conditions sufficient to stimulate the growth or regeneration of the plant, plant cell, or plant tissue (eg, in one or more culture media of the present invention). The present invention also provides a method of stimulating the regeneration of plant cells and tissues. The method comprises the following steps:
(A) applying an effective amount of one or more compounds or compositions of the present invention to a plant cell or tissue, and (b) regenerating the plant cell or tissue from the plant cell or tissue. Incubating under conditions sufficient to stimulate.
The present invention also relates to a method of reducing or reducing environmental stress in a plant, plant cell, or plant tissue. The method includes the following steps: (a) environmental stresses (eg, drought, excessive temperature, reduced temperature, chemical toxicity (eg, antibiotics, herbicides), contamination, excessive light and reduction). Contacting a plant, plant cell, or tissue that has been exposed to light) with an effective amount of one or more compounds or compositions of the invention; and (b) contacting the plant, plant cell, or tissue, Incubating under conditions sufficient to reduce or alleviate the above stresses. The invention also relates to a method of culturing a plant, plant cell, or plant tissue in vitro. The method comprises the following steps: (a) the plant to be cultured, the plant cell,
Or obtaining a plant tissue; (b) contacting the plant, plant cell, or plant tissue with one or more compounds or compositions of the present invention; and (c) the plant, plant cell, or plant tissue. Incubating under appropriate conditions to support the culture of plants, plant cells, or plant tissues. Furthermore, the present invention provides a method for transformation of plant cells and / or tissues. The method comprises the steps of: (a) contacting a plant cell or tissue with a nucleic acid molecule (eg, by transformation or protoplast fusion); (b) plant cell or tissue according to the present invention. Contacting with an effective amount of one or more compounds or compositions of
And (c) incubating the plant cell or tissue with the nucleic acid molecule under conditions sufficient to induce transformation of the plant cell or tissue. The compounds and compositions of the present invention can also be used to stimulate the regeneration or growth of transformed tissues or cells, thus providing a method for obtaining transgenic plants. Accordingly, the present invention also relates to transformed or transgenic plants, plant cells, and plant tissues produced by the methods described above.

Suitable applications of the growth enhancing compounds and compositions of the present invention include shoots,
Contacting a culture of plant tissue with one or more compounds or compositions of the present invention to induce root or callus regeneration. This effect occurs in both monocotyledonous and dicotyledonous plant species, and applies to a wide variety of plants.

The compounds and compositions of the present invention may further be utilized for plant regeneration from transgenic plants.

Plant genetic engineering generally involves two complementary processes. The first process involves the genetic transformation of one or more plant cells of a specifically characterized type. By transformation, a foreign gene, typically a chimeric gene construct,
It is meant to be introduced into the genome of an individual plant cell, typically with the aid of a vector capable of transferring the gene of interest into the genome of the plant cell in culture. The second process then involves the regeneration of the transformed plant cells into whole plants with sexual potential. Neither the transformation nor the regeneration process requires 100% success, but a reasonable degree of reliability and confidence that a reasonable percentage of cells can be transformed and regenerated into whole plants Must have reproducibility.

[0084] The two processes of transformation and regeneration must be complementary. The complementarity of the two processes is that the tissue that has been successfully genetically transformed by the transformation process must be of a certain type and characteristic, and that it is sufficiently healthy, competent, and It must have vitality so that they can be successfully regenerated into whole plants.

[0085] Successful transformation and regeneration techniques have been demonstrated in the prior art for monocots and dicots. For example, transformation and regeneration of tobacco plants is reported in Barton et al., Cell 32: 1033 (April 1983), while regeneration of cotton is described in US Pat. No. 5,004,863, the disclosure of which is incorporated by reference. Which are incorporated herein by reference in their entirety). further,
Transformation and regeneration of rice is described in Abdulah et al., Bio / Technolo.
gy 4: 1087-1090 (1986), while corn is described in Rhodes et al., Bio / Technology 6: 56-60 (1988).
) Was transformed and regenerated.

The most common methodology used for the transformation of cells of dicotyledonous species involves the use of the plant pathogen, Agrobacterium tumefaciens. Other gene transfer methods (eg, the polyethylene glycol method, electroporation, direct injection, particle bombardment, etc.) are also available from Wu (P
lant Biotechnology, Stoneham, MA; Butte
rworth Publishers, pp. 35-51 (1989)). The present invention is useful for any transformation method including a plant regeneration step.

In certain embodiments, the invention contemplates genetic transformation of tissue in culture from leaf disks or hypocotyl explants. The transformed tissue can be induced to form a structure of a plant tissue. This structure s can be regenerated into a whole plant.

The transformation technique of the present invention Tumefaciens Ti plasmid is used. A. In using a culture of Tumefaciens as a transformation vehicle, Agrobacteri is used as a vector carrier.
It is most advantageous to use non-tumorigenic strains of um, so that non-tumorigenic differentiation of normal transformed tissues is possible. To be effective once introduced into plant cells, chimeric constructs containing the foreign gene of interest are:
It must contain a promoter that is effective in plant cells to cause transcription of the gene of interest, and a polyadenylation or transcription control sequence also recognized in plant cells. Promoters known to be effective in plant cells include Ag
Nopaline synthase isolated from T. DNA of R. bacterium, and the 35S promoter of cauliflower mosaic virus.
Other suitable promoters are known in the art. It is preferred that the vector carrying the foreign gene of interest contain one or more selectable marker genes therein, so that transformed cells can be selected from non-transformed cells in culture. Can be done. In many applications, preferred marker genes include antibiotic resistance genes, such that the appropriate antibiotic is used to separate and select transformed cells from non-transformed cells. Can be used.

Details of the construction of vectors containing such foreign genes of interest are known to those skilled in the genetic engineering of plants and are useful in tobacco, petunia, and other model plant species. Does not differ in essence from these implementations that have been previously demonstrated. Foreign genes should clearly be selected as marker genes (Jefferson et al., EMBO J. 6: 3901-3907).
(1987)) or should achieve some desirable effects in plant cells. The effect can be growth promotion, disease resistance, a change in the quality of the plant morphology or plant product, or any other change that can be achieved by genetic engineering. The chimeric gene construct can encode the expression of one or more foreign proteins or cause transcription of negative strand RNA to control or inhibit either the disease process or unwanted endogenous plant function. obtain.

To begin the process of transformation and regeneration on plant tissue, it is necessary to first sterilize the surface of the tissue to prevent inadvertent contamination of the resulting culture. If the tissue is a seed, the seed is germinated on a suitable germination medium containing an antifungal agent. Four to ten days after germination, the hypocotyl parts of the immature plant are harvested and cut into small segments, each averaging about 0.5 cm. Hypocotyl explants are stabilized and remain viable in liquid or agar plant tissue culture media.

Once the tissues have been stabilized, they can be quickly inoculated with a non-tumorigenic, non-tumorigenic Agrobacterium suspension culture capable of transformation. The inoculation process is performed at room temperature (ie, 24 ° C.) for a short period of time (eg, 2 days).
Let go.

At the end of the inoculation time, the remaining treated tissue may be transferred to a selective agar medium. This medium contains one or more antibiotics that are toxic to Agrobacterium, but not to the plant, with all A-
The bacterium is contained in a concentration sufficient to kill bacterium. Suitable antibiotics for use in such media include carbenicillin, cefotaxime, etc., as antibacterials for Agrobacterium, and kanamycin, as selective antibiotics for transformed plant tissue. . The tissue can then be cultured on a tissue culture medium containing one or more selective agents in addition to its usual components, preferably on a culture medium of the present invention. The selection agent, exemplified by kanamycin herein, is toxic to non-transformed cells, but incorporates the genetic resistance to the selection agent, and is transformed to express that resistance. Not toxic to damaged cells. The surviving transformed tissue is transferred to a secondary medium, preferably a culture medium of the present invention, containing a selection agent to induce tissue regeneration. Thus, the living transformed tissue is continued to regenerate into a complete plant by the regeneration technique of the present invention or by any other alternative plant regeneration protocol.

The PAA compounds and compositions of the present invention are also useful in producing plants that are less susceptible to antibiotic toxicity. Such PAA compounds and compositions are also useful in enabling plants to overcome stress (eg, environmental stress, physical stress, chemical stress, contamination, contamination, drought, light, etc.). .

The compounds and compositions of the present invention may be used at concentrations used to obtain phytohormones or maintenance effects throughout maturation and to promote regeneration in damaged tissue during early developmental stages. Depending on the formulation and the type of plant species to be treated, it can be applied at any stage of development of the plant species.

The compounds and compositions of the present invention are preferably used to achieve specific synergistic growth enhancing responses in various types of plants in the correct ratio of certain supplemental nutrients or other plants. Used in combination with growth regulators. The compounds and compositions of the present invention further enhance the disease resistance of various plants and thereby regulate the immunity of natural diseases and the processes of the plants by affecting the processes of the pathogens and thereby preventing invasion by pathogens. It can be used in combination with antifungal agents to make plant tissue resistant. Although the compounds and compositions of the present invention do not inherently have their own phytotoxin activity, they sometimes make unwanted plants more sensitive to herbicides. Can be used in combination with a herbicide to stimulate the growth of the plant. However, it is preferred to consider the results achieved in the practice of the present invention as a response that enhances growth in agricultural and horticultural crops, as well as perennial and annual plant species.

[0096] Other suitable modifications and adaptations to the methods and applications described herein will be apparent and can be made without departing from the scope of the invention or any of its embodiments. It will be apparent to those skilled in the relevant art. However, having now described the invention in detail, the invention will be more clearly understood with reference to the following examples. The following examples are included herein for illustrative purposes only, and are not intended to limit the invention.

Examples Materials and Methods Culture Medium. GIBCO BRL Murashige and Skoog (
MS) Complete medium-50x concentrate (Cat. # 10494-011) with 940 ml of sterile water for liquid growth format based on membrane or 940 ml with 0.8% agar for semi-solid growth format 20 from Salt I, Salt II, and Acid Soluble, along with any of the following sterile waters:
It was prepared by mixing ml of each component.

[0098] Plant material. Rice (Oryza sativa L.), corn (Zea
mays) and wheat (Triticum aestivam L.) seeds were sterilized by first placing them in sterile water for 30 minutes with gentle agitation. The seeds were then submerged in 95% alcohol for 1 minute.
The alcohol solution was removed and the seeds were removed from the flask with handle (Side-arm-
flash). 15% commercial bleach and 0.1% Tween (
A solution of ® 20 was prepared and added to the flask. The seeds were depressurized for 20 minutes with shaking. The vacuum was removed but shaking continued for another 25 minutes. Seeds are rinsed three times with sterile water in an uncontaminated hood and semi-solid MS
Planted in medium.

Tobacco (Nicotiana tabaccum cv. Xanti) and tomato (Lycopersicon esculentum L. Mill.)
For cultivation, young leaves were collected from 1 month old plants and punched with a punch to prepare a uniform leaf disk. Leaf disks were placed in MS complete medium in Petri dishes.

Potato (Solanum tuberosus cv. Atlanti)
For c) and cassava (Manihot esculenta) plant cultures, approximately 1 month old stems (0.8 cm internodes) were used. Stems were placed in MS complete medium in Petri dishes.

For soybean (Glycine max L.) culture, seeds were sterilized by placing the seeds in 95% ethanol for 3 minutes. The ethanol was then removed, 10% bleach was added, and the seeds were stirred for 15 minutes. The seeds were then transferred directly from the bleaching solution to sterile MS medium (7 seeds per plate). Plates were incubated at 28 ° C. for 4 days in the dark. The plates are then checked for fungi and bacteria, and the clean seeds are
Boxes and incubated for an additional 11 days. The resulting 15-day-old soybean stalks were cut into approximately 1 cm long segments and the MS in Petri dishes
Placed in medium.

All explants and seeds were incubated for 1 month at 25 ° C. using an 18 hour light / 6 hour dark cycle. Transformation of potato and cassava, A. a high concentration containing Km ^R gene tumefaciens LBA
4404 co-culture. Lin et al. (1995) Focus 16
: 72.

Results Auxin-like chemicals (PAA derivatives) were treated with tobacco leaf disks, tomato leaf disks, and potato stems with MS salt and various concentrations of auxin-like chemicals alone or with auxin-like chemicals. Auxin activity was assessed by incubating in culture media containing any of the combinations with cytokinins. Callus or root formation was observed in media containing only auxin-like chemicals, while shoot regeneration was also observed in addition to root and / or callus formation in media containing auxin-like chemicals and cytokinins. Was found. In general, more callus formation was observed with the combination of cytokinins and high concentrations of auxin-like chemicals (FIG. 2).

Nine different auxin-like chemicals were evaluated for auxin activity. Six of these compounds (2,4-dichlorophenoxyacetic acid (2,4-D),
2-bromophenylacetic acid, 3-bromophenylacetic acid, 4-bromophenylacetic acid,
4 (bromomethyl) phenylacetic acid, and 4-bromomandelic acid) showed auxin activity (Tables 1 and 2).

To demonstrate the auxin activity and possible application of these novel compounds in plant tissue culture, we have chosen to optimize the media formulation for potato shoot regeneration. Showed only minimal auxin activity 4
-Bromophenylacetic acid was selected. Interestingly, the regeneration efficiency was higher in potato cultivar Atlantic than in the medium containing 4-bromophenylacetic acid,
There was a two-fold increase when compared to media containing the well-characterized auxin, NAA. An increase of more than 11-fold was observed in shoot regeneration of potato variety Kent. This variety is recognized as a potato variety that is difficult to regenerate in tissue culture (Table 3).

Further evaluation of the effects of 4-bromophenylacetic acid was performed in combination with various cytokinins. Interestingly, the effects of cytokinin-like zeatin can improve regeneration efficiency up to 10-fold. In addition, shoot regeneration could be increased up to 57-fold using membrane-based liquid culture (Table 4). All of these demonstrated that 4-bromophenylacetic acid is a useful auxin and can be used to improve the regeneration of potato shoot formation.

Regeneration of transgenic plants was assessed by Agrobacterium-mediated transformation. A. with potato stem, pBII21
After co-culture with Tumefaciens LBA4404, the potato stem was
Different concentrations of cytokinin and kanamycin for selection of antibiotic resistant plants were incubated on media containing 4-bromophenylacetic acid.
Transformation efficiency is increased about 40-fold in agar-based cultures and
For tic varieties, a 95-fold increase in membrane-based liquid cultures. S
For nowden and Russet Burbank varieties, transformation efficiencies are improved from 0 to 10-25 fold in agar-based cultures and up to 57-920 fold in membrane-based liquid cultures. (Table 5).

Various crops such as soybean, cassava, corn, wheat, and rice were used to further evaluate the auxin activity of 2-bromophenylacetic acid, 3-bromophenylacetic acid, and 4-bromophenylacetic acid. . Either callus formation or root formation was seen when plant tissues were incubated in media containing these compounds (Table 6). All these results indicate that auxin activity was detected, and that regeneration of both untransformed and transformed shoots was once the concentration of the chemical was optimized using cytokininting. Demonstrate what was achieved after

Significant improvement in potato shoot regeneration was demonstrated by Snowden and Russ.
Various potato varieties, such as et Burbank, have been demonstrated using various cytokinins with 4-bromophenylacetic acid. In addition, callus and root formation was observed by treating cassava tissue with 4-bromophenylacetic acid. This was not shown in those treated with conventional auxins such as 2,4-D (Table 6).

[0110]

[Table 1]

[0111]

[Table 2]

[0112]

[Table 3]

[0113]

[Table 4]

[0114]

[Table 5]

[0115]

[Table 6] Although the invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be understood that
What can be done in a broad and equivalent range of conditions, formulations and other parameters without affecting the scope of the invention or any particular embodiment thereof;
It will be apparent to one skilled in the art that such modifications or changes are intended to be included within the scope of the appended claims.

All publications, patents, and patent applications mentioned in this specification are indicative of the level of those skilled in the art to which this invention pertains, and each individual publication, patent or patent application is referred to by reference. To the same extent as if specifically and individually indicated to be incorporated herein by reference.

[Brief description of the drawings]

FIG. 1 is a diagram of the chemical structure of phenylacetic acid (PAA) and shows preferred substituents used to produce the PAA compounds used in the compounds and compositions of the present invention.

FIG. 2 shows that, in the presence of various concentrations of 6-benzylaminopurine (BAP),
Tobacco (FIG. 2A), as determined by callus and root formation, and plant regeneration in response to bromophenylacetic acid ("new auxin").
Explant growth of tomatoes (FIG. 2B) and potatoes (FIG. 2C) are documented in detail.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 5/04 C12N 5/00 C 5/10 F (81) Designated country EP (AT, BE, CH, CY) , DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW) , ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, K, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR , LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, UZ, VN, YU, ZA, ZW (72) Inventor Lan, Junkin United States of America Maryland 20876, Germantown, Red Admiral Way 12129 F-term (reference) 2B022 AB13 AB15 AB20 EA01 2B030 AA02 AB03 AD04 CA10 CB02 CD03 CD06 CD09 CD10 CD13 CD17 4B065 AA88X AA89X AB01 BA01 BB08 BB35 CA53 4H006 AA01 AA03 AB06 BJ50 BM30 BM72 BM73 BS10 4H011 AB03 BA01 BA06 BB13 BB21 BC18

Claims (26)

[Claims] 1. A compound for regulating the growth of a plant cell, wherein the compound comprises a mono- or polysubstituted phenylacetic acid (PAA) compound, or an ester or salt derivative thereof; The compound has auxin activity on plant cells and tissues and has the following general formula: Wherein X is one or more halo-groups, one or more alkyl- (R-) groups, one or more alkoxy- (RO-) groups, one or more alkylamino- (RNH-) groups, 1
An acyl- (RCO-) group or more, an acylamide- (RCONH-) group or more,
And at least one substituent selected from the group consisting of at least one acyloxy- (RCOO-) group. 2. The compound according to claim 1, wherein R is a linear, branched or cyclic hydrocarbon. 3. The compound according to claim 2, wherein said hydrocarbon is a saturated hydrocarbon. 4. The compound according to claim 2, wherein said hydrocarbon is an unsaturated hydrocarbon. 5. The compound according to claim 1, wherein R is CH ₃ (CH ₂ ) _n , wherein n is an integer in the range of 0-10. 6. The compound according to claim 1, wherein said halo group is selected from the group consisting of a fluoro group, a chloro group, a bromo group and an iodo group. 7. The compound according to claim 1, wherein X is one or more halo groups. 8. The method of claim 7, wherein the one or more halo groups is one or more bromo groups.
The compound according to the above. 9. The method of claim 7, wherein said one or more halo groups is one or more chloro groups.
The compound according to the above. 10. The compound of claim 1, wherein said compound is a mono- or disubstituted phenylacetic acid compound or a derivative thereof. 11. The compound according to claim 1, wherein the compound is selected from the group consisting of 2-bromophenylacetic acid, 3-bromophenylacetic acid, 4-bromophenylacetic acid, 4- (bromomethyl) phenylacetic acid, and 4-bromomandelic acid, and derivatives thereof. Selected,
A compound according to claim 10. 12. A composition comprising one or more compounds according to claim 1. 13. The composition of claim 12, further comprising at least one cytokinin or gibberellin. 14. The composition of claim 13, wherein said cytokinin is selected from the group consisting of zeatin, 6-furfurylaminopurine (kinetin) and 6-benzylaminopurine (BAP). 15. A method of affecting the growth of a plant, plant cell or plant tissue, comprising: (a) providing the plant, plant cell or plant tissue with an effective amount of one or more.
And (b) incubating the plant, plant cell or plant tissue under conditions sufficient to affect the growth of the plant, plant cell or plant tissue. ,Method. 16. A method of affecting the regeneration of a plant, plant cell or plant tissue, comprising: (a) an effective amount of one or more plants, plant cells or plant tissue.
And (b) incubating the plant, plant cell or plant tissue under conditions sufficient to affect regeneration of the plant, plant cell or plant tissue. ,Method. 17. A plant culture medium comprising one or more compounds according to claim 1. 18. The culture medium according to claim 17, further comprising at least one cytokinin or gibberellin. 19. The culture medium according to claim 18, wherein said cytokine is selected from the group consisting of zeatin, 6-furfurylaminopurine (kinetin) and 6-benzylaminopurine (BAP). 20. A method for in vitro culturing a plant, plant cell or plant tissue, comprising: (a) obtaining a plant, plant cell or plant tissue to be cultured in vitro; Contacting the plant tissue with one or more of the plant culture media of claim 17; and (c) supporting said plant, plant cell or plant tissue in vitro culturing said plant, plant cell or plant tissue. And incubating under suitable conditions. 21. A method for producing a transformed plant, plant cell or plant tissue, comprising: (a) transforming said plant, plant cell or plant tissue into said plant, plant cell or plant tissue. Contacting with a nucleic acid molecule, (b) the plant,
Contacting a plant cell or plant tissue with an effective amount of one or more compounds of claim 1, and (c) transforming said plant, plant cell or plant tissue into said plant, plant cell or plant tissue. Incubating with the nucleic acid molecule under conditions sufficient to induce 22. A transformed plant, plant cell or plant tissue produced by the method of claim 21. 23. A method for producing a transgenic plant, plant cell or plant tissue, wherein (a) transforming the transformed plant, plant cell or plant tissue of claim 22 with one or more plants. Contacting with the compound described in
b) incubating the transformed plant, plant cell or plant tissue under conditions sufficient to affect the regeneration or growth of the transformed plant, plant cell or plant tissue. Method. 24. A transgenic plant, plant cell or plant tissue produced by the method of claim 23. 25. A method for reducing or alleviating environmental stress in a plant, plant cell or plant tissue, comprising: (a) removing an effective amount of one or more plants, plant cells or plant tissue from environmental stress. A method comprising: contacting with a compound according to claim 1; and (b) incubating said plant, plant cell or plant tissue under conditions sufficient to reduce or alleviate said stress. 26. The method of claim 26, wherein the environmental stress is drought, excessive temperature, reduced temperature,
26. The method of claim 25, wherein the method is selected from the group consisting of chemical toxicity, contamination, excess light and reduced light.