WO2000024249A2

WO2000024249A2 - Compositions comprising plant cell growth regulating compounds and methods of use thereof

Info

Publication number: WO2000024249A2
Application number: PCT/US1999/025263
Authority: WO
Inventors: Jhy-Jhu Lin; Nacyra Assad-Garcia; Jianqing Lan
Original assignee: Invitrogen Corporation
Priority date: 1998-10-28
Filing date: 1999-10-28
Publication date: 2000-05-04
Also published as: EP1124417A2; AU1327000A; WO2000024249A3; CA2348411A1; JP2002528394A

Abstract

The present invention relates generally to compositions comprising one or more compounds that regulate the growth of plants, plant cells and plant tissues. More particularly, the invention relates to compositions, such as culture media, comprising one or more auxin-like compounds, including mono-, di- or multi-substituted phenylacetic acid compounds or ester or salt derivatives thereof, including compounds such as 2-, 3-, and 4-bromophenylacetic acid and derivatives thereof and the like. 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, including cytokinins (such as zeatine, kinetin, and 6-benzylaminopurine (BAP)) and giberellins. The invention also relates to methods for regulating the growth of plants, plant cells and plant tissues, to methods for the production and cultivation of genetically modified (e.g., transformed and/or transgenic) plants, plant cells and plant tissues, and to genetically modified plants, plant cells and plant tissues produced by such methods.

Description

Compositions Comprising Plant Cell Growth Regulating Compounds and Methods of Use Thereof

BACKGROUND AND SUMMARY OF THE INVENTION

The present invention is in the fields of plant and plant cell biology. The invention relates generally to compositions comprising one or more compounds that regulate the growth of plants and plant cells. More particularly, the invention relates to compositions, such as culture media, comprising one or more auxin-like compounds, including mono-, di- or multi-substituted phenylacetic acid compounds or ester or salt derivatives thereof, including compounds such as 2-, 3-, and 4-bromophenylacetic acid and derivatives thereof and the like. The invention also relates to such compositions further comprising one or more additional compounds that regulate the growth of plants and plant cells, including cytokinins (such as zeatine, kinetin, and 6-benzylaminopurine (BAP)) and giberellins. The invention also relates to methods of regulating the growth of plants and plant cells comprising contacting a plant cell with one or more of the above- described compositions. The invention also relates to methods for the production and cultivation of genetically modified plants and plant cells comprising obtaining a genetically modified plant or plant cell and contacting the plant or plant cell with one or more of the above-described compositions. The invention also relates to genetically modified plants and plant cells produced by such methods.

Plant growth is affected by a variety of physical and chemical factors. Physical factors include available light, day length, moisture and temperature. Chemical factors include minerals, nitrates, co-factors, nutrient substances and plant growth regulators or hormones, for example, auxins, cytokinins and gibberellins.

Indole-3-acetic acid (IAA) is a naturally-occurring plant growth hormone identified in plants. IAA has been shown to be directly responsible for the increase in growth in plants in vivo and in vitro. The characteristics influenced by IAA include cell elongation, internodal distance (height) and leaf surface area. IAA and other compounds exhibiting hormonal regulatory activity similar to that of IAA are included in a class of plant regulators called "auxins." Preparations based on cytokinins, such as 6-furfurylaminopurine (kinetin) and 6- benzylaminopurine (BAP), are also known to be growth stimulators. However, cytokinin-based preparations are most effective in combination with auxins. While the mechanism by which cytokinins affect the growth cycle of plants is far from being understood, it is apparent that they affect leaf growth and prevent aging in certain plants. It is a general objective in the field to successfully engineer and regenerate plants of major crop varieties using methods such as tissue culture and genetic engineering. Major crop varieties of particular interest in this regard are agricultural crops such as maize, wheat, rice, soybean and cotton.

To regenerate plants, in vitro culture techniques have been established. (Reinert, J., and Bajaj, Y.P.S., eds. (1977) Plant Cell Tissue and Organ Culture, Berlin: Springer; Simmonds, N.W. (1979) Principles of Crop Improvement, London: Longman; Vasil, I.K., Ahuja, M.K. and Vasil, V. (1979) "Plant tissue cultures in genetics and plant breeding," Adv. Genet. 20: 127-215.) Specific in vitro culture techniques to regenerate plants include embryo culturing, shoot tip culturing and callus, cell and protoplast culturing. Embryo culturing has been shown to be important in making difficult interspecies crosses, while shoot-tip culturing is important in rapid clonal multiplication, development of virus-free clones and genetic resource conservation work. Callus, cell, and protoplast cultures have been shown to be important for cultures in which organization is lost but can be recovered. Plant genetic engineering techniques have also been established. These techniques include gene transfer by transformation or by protoplast fusion. In gene transfer, the steps involved are: (a) identification of a specific gene; (b) isolation and cloning of the gene; (c) transfer of the gene to recipient plant host cells: (d) integration, transcription and translation of the DNA in the recipient cells; and (e) multiplication and use of the transgenic plant (T. Kosuge, C.P. Meredith and A. Hollaender, eds. (1983) Genetic Engineering of Plants, 26:5-25; Rogers et al. (1988) Methods for Plant Molecular Biology, A. Weissbach and H. Weissbach, eds., Academic Press, Inc., San Diego, CA). In protoplast fusion, plant cell protoplasts are fused by standard chemical (e-g-, PEG) or electroporation techniques. After regeneration of the fused cells, interspecies amphidiploids have been obtained. The technique may provide desired amphidiploids which cannot be made by conventional means, and presents possibilities for somatic recombination by some variant of it. The foregoing techniques are widely in use (Chaleff, R.S. (1981) Genetics of Higher Plants, Applications of Cell Culture, Cambridge: Cambridge University Press), and newly inserted foreign genes have been shown to be stably maintained during plant regeneration and are transmitted to progeny as typical Mendelian traits (Horsch et al., Science 223:496 (1984), and DeBlock et al., EMBO 5: 1681 (1984)). These foreign genes retain their normal tissue specific and developmental expression patterns. The Agrobacterium tumefaciens mediated transformation system has proven to be efficient for many dicotyledonous plant species. For example, Barton et al. (Cell 52:1033(1983)) reported the transformation and regeneration of tobacco plants, and Chang et al. (Planta 5:551-558 (1994)) described stable genetic transformation of Arabidopsis thaliana. The Agrobacterium method for gene transfer was also applied to monocotyledonous plants, e.g., in plants in the Liliaceae and Amaryllidaceae families (Hooykaas-Van Slogteren et al, 1984, Nature 311:763-764), in Dioscorea bulbifera (yam) (Schafer et al, 1987, Nature 327:529-532) and in rice and maize (Ishida et al., 1996, Nat. Biotechnology 14:745-750; Hiei et al, 1997, Plant Mol Biol. 35:205-218). Transformation of food crops was obtained with alternative methods, e.g., by polyethylene glycol (PEG), facilitated DNA uptake (Uchimiya et al, Mol. Gen. Genet. 204:204-207 (1986)) and electroporation (Fromm et al, Nature 319:791-793 (1986)), both of which used protoplasts as transfer targets. Monocot and dicot tissues may be transformed by bombardment of tissues with DNA-coated particles (Wang et al. (1988) Plant Mol. Biol. 77:433-439; Wu, in Plant Biotechnology (1989), Kung and Arntzen, Eds., Butterworth Publishers, Stoneham, MA). Regeneration was described in rice (Abdullah et al. 1986) Bio/Technology 4: 1087-1090) and maize (Rhodes et al, Bio/Technology 5:56-60 (1988) and Science 240:204-207 (1988)). Thus, although regeneration and transformation protocols have been established, there remains a need to stimulate regeneration and transformation of monocotyledonous and dicotyledonous plants. Indeed, some plants have been difficult to regenerate and transform (Vasil and Vasil (1994) in Plant Cell and Tissue Culture (Vasil and Thorpe, eds.), Kluwer Academic Publishers, Dordrech, Netherlands; Chee, Plant Cell Reports 74:753-757 (1995); Burns and Schwartz, Plant Cell Reports 75:405-408 (1996); Mihaljevic et al, Plant Cell Reports 75:610-614 (1996); Schopke et al, Nature Biotechnology 14:731 (1996)). Moreover, there is a need to stimulate growth of the plants, particularly after transformation and regeneration. Finally, there remains a need for improved culture media facilitating the in vitro cultivation of plants, plant tissues, and plant cells and protoplasts, to permit in vitro growth and regeneration of plants as well as to facilitate genetic manipulation and development of hybrid plants.

The present invention satisfies these needs by providing compositions which stimulate plant growth, regeneration of plant cells and tissues, and transformation of plant cells and tissues. The compositions of the invention comprise one or more mono- or multi-substituted phenylacetic acid (PA A) compounds, or ester or salt derivatives thereof, which have auxinic activity on plant cells and tissues and which have the general formula of:

wherein X is one or more substitutent groups, which may be the same or different, selected from the group consisting of 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- (RO-) groups, one or more alkylamino- (RNH-) groups, one or more acyl- (RCO-) groups, one or more acylamido- (RCONH-) groups, and one or more acyloxy- (RCOO-) groups. According to a preferred aspect of the invention, R may be CH₃(CH₂)_n, wherein n is an integer ranging from 0 to 10. The invention contemplates the use of such compounds to affect growth, regeneration and transformation in both monocotyledonous and dicotyledonous plants. In particular, the invention provides compositions comprising one or more such compounds having a substituent group at the 2, 4, 5 and/or 6 position of the PAA structure, wherein said substituents are halo-, alkyl-, alkylamino-, alkoxy-, acyl-, acylamido- or acyloxy-substituent groups having 1-10 carbon atoms. The invention also provides compositions comprising one or more multi-substituted PAA compounds having two to five substituent groups, which may be the same or different, at positions selected from the group consisting of positions 2, 4, 5 and 6 of the IAA structure wherein said substituents are halo-, alkyl-, alkylamino-, alkoxy-, acyl-, acylamido- or acyloxy- substituent groups having 1-10 carbon atoms. Preferred PAA compounds for use in compositions according to these aspects of the invention include, but are not limited to, 2- bromophenylacetic acid, 3-bromophenylacetic acid, 4-bromophenylacetic acid, and derivatives thereof and the like.

The compositions of the invention may include, in addition to one or more of the above-described mono- or multi-substituted PAA-derived compounds, one or more additional plant growth regulators. Such plant growth regulators include, for example, a cytokinin (such as zeatine, 6-furfurylaminopurine (kinetin) or 6-benzylaminopurine (BAP)), a gibberellin, and the like, in definite proportions for wide application to various plants. In specific embodiments, the invention is exemplified with compositions comprising mono- or multi-substituted PAA compounds of the invention having between one and five substituent groups, which may be the same or different, that are halo-, alkyl-, alkylamino-, alkoxy-, acyl-, acylamido- and acyloxy-substituent groups at positions 2, 4, 5 and/or 6 of the PAA structure, and a cytokinin to affect the growth of plants. The invention further relates to culture media comprising one or more of the above-described compounds or compositions of the invention. Such culture media may, for example, comprise 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, to stimulate regeneration of plant cells and tissues, and/or to stimulate transformation of plant cells and tissues.

The invention also relates to a method of stimulating the growth of a plant, plant cell or plant tissue comprising (a) applying to a plant, plant cell or plant tissue an effective amount of one or more of the compounds or compositions of the invention, and (b) incubating the plant, plant cell or plant tissue under conditions sufficient to stimulate the growth of the plant, plant cell or plant tissue.

The invention also provides a method for stimulating the regeneration of plant cells and/or tissues comprising (a) applying to a plant cell or tissue an effective amount of one or more of the compounds or compositions of the invention, and (b) incubating the plant cell or tissue under conditions sufficient to stimulate the regeneration of the plant cell or tissue.

The invention also relates to methods for cultivating a plant, plant cell or plant tissue in vitro, comprising (a) obtaining a plant, plant cell or plant tissue to be cultivated,

(b) contacting the plant, plant cell or plant tissue with one or more of the compounds or compositions of the invention, and (c) incubating the plant, plant cell or plant tissue under conditions suitable to support cultivation of the plant, plant cell or plant tissue.

In addition, the invention provides a method for transformation of plant cells and/or tissues comprising (a) contacting the plant cell or tissue with a nucleic acid molecule (e.g., by transformation or protoplast fusion), (b) contacting the plant cell or tissue with an effective amount of one or more of the compounds or compositions of the invention and (c) incubating the plant cell or tissue under conditions sufficient to induce transformation of the plant cell or tissue with the nucleic acid molecule. The compounds and compositions of the invention also may be used to stimulate regeneration or growth of the transformed tissue or cells, thus providing a method to obtain a transgenic plant. The invention therefore also relates to transformed or transgenic plants, plant cells and plant tissues produced by the above-described methods.

The invention also concerns a method of attenuating or alleviating environmental stress in a plant, plant cell or tissue comprising (a) contacting a plant, plant cell or tissue which has been exposed to an environmental stress (such as drought, excess temperature, diminished temperature, chemical toxicity (e.g., antibiotic, herbicides), pollution, excess light, and diminished light) with an effective amount of one or more of the compounds or compositions of the invention, and (b) incubating said plant, plant cell or tissue under conditions sufficient to attenuate or alleviate said stress. Other preferred embodiments of the present invention will be apparent to one of ordinary skill in the art in light of the following drawings and description of the invention, and of the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

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

Figure 2 documents the growth of tobacco (Figure 2A), tomato (Figure 2B), and potato (Figure 2C) explants, as determined by callus and root formation and plant regeneration, in response to 4-bromophenylacetic acid ("New Auxin"), in the presence of different concentrations of 6-benzylaminopurine (BAP).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIEMENTS

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as would be commonly understood by one of ordinary skill in the art to which the present invention pertains. The following definitions are provided in order to provide clarity as to the intent or scope of their usage in the specification and claims.

The term "phenylacetic acid" or "PAA" as used herein refers to the chemical structure of Figure 1, wherein X = hydrogen. This term refers not only to the free acid form but also to an amide, an ester or a salt form of PAA. Included in the meaning of PAA are, for example, such salt and ester derivatives as the sodium, potassium, lithium, magnesium, calcium, ammonium, dimethylamine, ethanolamine, etc. salts and amides and the lower alkyl esters. Examples of ammonium salts include tetralkylammonium salts such as tetramethylammonium halide and trialkylbenzylammonum halide salts, such as benzyltrimethylammonium chloride salts. The term "monosubstituted PAA" as used herein refers to a PAA molecule of

Figure 1 wherein X represents one substitutent group present at the 2, 4, 5 or 6 position of PAA, which substitutent group may be a halo- group (e.g., fluoro-, chloro-, bromo-, iodo-, etc.), an alkyl- group (R-, particularly wherein R is CH₃(CH₂)_n and n is an integer which may range from 0 to 10), an alkoxy- group (RO-, particularly wherein R is CH₃(CH₂)_n and n is an integer which may range from 0 to 10), an alkylamino- group (RNH-, particularly wherein R is CH₃(CH₂)_n and n is an integer which may range from 0 to 10), an acyl- group (RCO-, particularly wherein R is CH₃(CH₂)_n and n is an integer which may range from 0 to 10), an acylamido- group (RCONH-, particularly wherein R is CH₃(CH₂)_n, and n is an integer which may range from 0 to 10), or an acyloxy- group (RCOO-, particularly wherein R is CH₃(CH₂)_n and n is an integer which may range from 0 to 10).

The term "multi-substituted PAA" as used herein refers to a PAA molecule of Figure 1 wherein X represents two or more substituent groups present at two or more of the positions corresponding to the 2, 4, 5 or 6 position in the PAA chemical structure, which substituent groups may be the same or different and which may be selected from the above-described halo-, alkyl-, alkoxy-, alkylamino-, acyl-, acylamido- and acyloxy- substitutent groups

The terms "auxinic analogue(s) of PAA" or "PAA analogue" or "PAA auxinic analogue" or "PAA compound" as used herein may be used interchangeably, and refer to a mono- or multi-substituted PAA that comprises, for example, one or more of the above- described halo-, alkyl-, alkoxy-, alkylamino-, acyl-, acylamido- and acyloxy- substitutent groups.

As used herein, the PAA compounds include not only the free acid forms but also amide, ester or salt forms of the mono- or multi-substituted PAA compounds.

A halo-group refers to a halogen including, but not limited to, iodo-, bromo-, chloro- and fluoro-groups.

An alkyl-group includes, but is not limited to, an R- group (linear, branched or cyclic; saturated or unsaturated), wherein R has 1-10 carbon atoms. An alkoxy-group includes, but is not limited to, an RO- group (linear, branched or cyclic; saturated or unsaturated), wherein R has 1-10 carbon atoms.

An alkylamino-group includes, but is not limited to, an RNH- group (linear, branched or cyclic; saturated or unsaturated), wherein R has 1-10 carbon atoms.

An acyl-group includes, but is not limited to, an RCO- (linear, branched or cyclic; saturated or unsaturated), wherein R has 1-10 carbon atoms.

An acylamido-group includes, but is not limited to, an acylamido, RCONH- (linear, branched or cyclic; saturated or unsaturated), wherein R has 1-10 carbon atoms.

An acyloxy-group includes, but is not limited to, an acyloxy, RCOO- (linear, branched or cyclic; saturated or unsaturated), wherein R has 1-10 carbon atoms. Examples of the group R in the above-described compounds include, but are not limited to, 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.

The term "plant growth regulator or hormone" as used herein refers to a naturally occurring or synthetic compound that acts as a hormone in regulating plant growth. Plant growth regulators are exemplified by auxins, cytokinins, gibberellins, ethylene and abasic acid (ABA).

The term "auxin" or "cytokinin" as used herein refers to a plant growth regulator that affects the growth of plants. An auxin is exemplified by a compound such as indole- 3-acetic acid (IAA), indole-3-butyric acid (IBA), 2,4-dichlorophenoxyacetic acid (2,4-D), naphthaleneacetic acid (NAA), 5,6-dichloroindole-3-acetic acid (5,6-Cl₂-IAA) and the like. A cytokinin is exemplified by a compound such as 6-benzylaminopurine (BAP), N⁶-(Δ₂-isopentenyl)adenine (2iP), isopentenylpyrophosphate (ipp), 6-(4-hydroxy-3- methyl-2transbutenylamino)purine (zeatin), 6-furfurylaminopurine (kinetin) and the like. A compound can be tested for auxin activity using a bioassay, e.g., the elongation of coleoptiles of Avena sativa (Bottger et al., Planta 140:89 (1978)) or the root growth inhibition of Chinese cabbage (Marumo et al. (1974) in Plant Growth Substance, p. 419, Hirokawa Publishing Co., Inc., Tokyo) or the hypocotyl swelling of mung bean (Marumo et al. (1974) supra). Cytokinin activity may be measured in assays designed to evaluate the promotion of growth in plants (e.g., tobacco bioassays, etc.) as is well known in the art (Skoog et al. (1967) Phytochem 6:1169-1192; Morris, Ann. Rev. Plant Physiol 37:509-538 (1986); Horgan (1984) Advanced Plant Physiol (Wilkins, M.B., ea.) Pitman Publishing, London, pp. 53-75; Letham and Palni, Ann. Rev. Plant Physiol 34:163-197 (1983); and Chen (1981) Metabolism and Molecular Activities of Cytokinins (Guern, J. and Peaud-Lenoel, C, eds.) Springer, New York, pp. 34-43). Variations of the cytokinin/auxin concentration ratio cause the enhancement in plant growth to occur preferentially in certain tissues. For example, a high cytokinin/auxin ratio promotes growth of shoots, whereas a low cytokinin/auxin ratio promotes the growth of roots (Depicker et al. (1983) Genetic Engineering of Plants (T. Kosunge, C. P. Meredith and A. Hollaender, eds.) Plenum Press, New York, p. 154).

The term "medium" or "media" as used herein refers to a solid or liquid composition comprising nutrients sufficient to support the in vitro cultivation, growth, regeneration and/or transformation of plants, plant cells, plant tissues, protoplasts and the like.

A plant culture medium is composed of a number of ingredients and these ingredients vary from one culture medium to another. A "IX formulation" is meant to refer to any aqueous solution that contains some or all ingredients found in a plant culture medium at working concentrations. The "IX formulation" can refer to, for example, the plant culture medium or to any subgroup of ingredients for that medium. The concentration of an ingredient in a IX solution is about the same as the concentration of that ingredient found in a plant culture formulation used for maintaining or cultivating plant cells or tissues in vitro. A plant culture medium used for the in vitro cultivation of plants, plant cells, plant tissues and the like, is a IX formulation by definition. When a number of ingredients are present, each ingredient in a IX formulation has a concentration about equal to the concentration of those ingredients in a plant culture medium. For example, Gamborg's B-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 " IX formulation" of these medium components contains about the same concentrations of these ingredients in solution. Thus, when referring to a "IX formulation," it is intended that each ingredient in solution has the same or about the same concentration as that found in the plant culture medium being described. The concentrations of ingredients in a IX formulation of plant culture medium are well known to those of ordinary skill in the art. See Vasil and Thorpe, eds., Plant Cell and Tissue Culture, Dordrech, Netherlands: Kluwer Academic Publishers (1995), which is incorporated by reference herein in its entirety. The osmolality and/or pH, however, may differ in a IX formulation compared to the culture medium, particularly when fewer ingredients are contained in the IX formulation. A "10X formulation" is meant to refer to a solution wherein each ingredient in that solution is about 10 times more concentrated than the same ingredient in the plant culture medium. For example, a 10X formulation of Gamborg's B-5 plant culture medium may contain, among other ingredients, 1.34 g/L ammonium sulfate, 25 g/L potassium nitrate, and 200 g/L sucrose (compare IX formulation, above). A "10X formulation" may contain a number of additional ingredients at a concentration about 10 times that found in the IX culture medium. As will be readily apparent, "20X formulation," "25X formulation," "50X formulation" and "100X formulation" designate solutions that contain ingredients at about 20-, 25-, 50- or 100- fold concentrations, respectively, as compared to a IX culture medium. Again, the osmolality and pH of the media formulation and concentrated solution may vary. See U.S. Patent No. 5,474,931, which is directed to culture media concentrate technology, the disclosure of which is incorporated by reference herein in its entirety.

The term "carrier" as used herein refers to a chemically- or biologically- or physiologically-acceptable molecule that is hydrophobic or hydrophilic or amphoteric and that is useful in facilitating the effectiveness of an active ingredient (i.e., a PAA analogue of the invention) in a plant.

The term "a plant" as used herein refers to a whole plant or a part of a plant comprising, for example, a locus of a plant, a cell of a plant, a tissue of a plant, an embryo of a plant, an explant, or seeds of a plant. This term further contemplates a plant in the form of a suspension culture or a tissue culture including, but not limited to, a culture of calli, protoplasts, embryos, organs, organelles, etc.

The term "transformed plant" or "transformed plant tissues" as used herein refers to introduction of a nucleic acid molecule, e.g., native or foreign DNA, into a plant or plant tissue by transformation or protoplast fusion.

The term "transgenic plant" or "transgenic plant tissue" as used herein refers to a plant or plant tissue stably transformed with a foreign gene.

The term "transient expression" refers to a plant or plant tissue transformed with a DNA, where that DNA is expressed only for a short period of time immediately after transformation.

The term "genetic engineering" as used herein refers to the introduction of foreign, often chimeric, genes into one or more plant cells which can be regenerated into whole, sexually competent, viable plants which can be self-pollinated or cross-pollinated with other plants of the same species so that the foreign gene, carried in the germ line, can be inserted into or bred into agriculturally useful plant varieties.

The term "regeneration" as used herein refers to the production of at least one newly developed or regenerated plant tissue, e.g., root, shoot, callus, etc., from a cultured plant tissue or unit, e.g., leaf disc, seed, etc. The terms "percent", "% regeneration" or "regeneration efficiency" as used herein refer to the number of tissue cultured plant units producing at least one newly developed or regenerated tissue as a percentage of the total number of tissue cultured plant units, e.g., (number of leaf discs with shoots X 100) total number of leaf discs

The terms "affecting plant growth" or "growth affecting" or "affector" or "affect" as used herein refer to any one of a number of plant responses which improve or change, relative to what is observed in the absence of the growth regulator, some characteristic of overall plant growth, for example, stimulation of seed germination, inducing rooting, suppressing shooting, promoting cell proliferation, stimulating callus growth, etc. It is further understood that within the context of the present invention, these terms may also encompass toxic effects on plant tissues.

The term "effective amount" as used herein refers to the amount or concentration of a compound such as a plant growth regulator or hormone, administered to a plant such that the compound stimulates or invokes one or more of a variety of plant growth responses. A plant growth response includes, among others, the induction of stem elongation, the promotion of root formation, the stimulation of callus formation, enhancement of leaf growth, stimulation of seed germination, increase in the dry weight content of a number of plants and plant parts, and the like.

Compositions

The present invention relates generally to compositions which stimulate plant growth, regeneration of plant cells and tissues, and transformation of plant cells and tissues. The compositions of the invention comprise one or more mono- or multi- substituted phenylacetic acid (PAA) compounds, or ester or salt derivatives thereof, which have auxinic activity on plant cells and tissues. The invention contemplates the use of such PAA compounds to affect growth, regeneration and transformation in both monocotyledonous and dicotyledonous plants. In particular embodiments of the invention, various PAA compounds were screened for auxinic activity by incubating different plant tissues, e.g., tobacco and tomato leaf discs, and potato stems in (a) MS complete medium (obtained from Life

Technologies, Inc., Gaithersburg, MD) containing different concentrations of auxin only and (2) MS complete medium containing different ratios of cytokinin/auxin.

Traditionally, in order to achieve shoot regeneration from potato stems, the potato tissue is transferred from a medium containing a high amount of auxin to another medium containing auxin and cytokinin. In the present invention, the regeneration of shoots from potato tissue was obtained without tissue transfer from a high auxin medium to a medium containing auxin and cytokinin. In addition, the regeneration of shoots in potato tissue, according to the invention, showed significant improvement in the number of shoots regenerated. Thus, the compositions of the present invention not only exhibit auxinic activity but also improve the yield of plant tissue regenerated, as exemplified in plant tissues traditionally described as being difficult to regenerate, e.g., cassava, woody plants, maize, soybean, wheat, etc.

The practice of the present invention contemplates a wide variety of plant growth responses, including stimulation of seed germination and breaking of dormancy; increasing yields; hastening ripening and color production in fruit; increasing flowering and fruiting; stimulating shoot formation; inducing callus development; inducing rooting and causing cell proliferation; increasing the hardiness of various plant species; and increasing the dry weight content of a number of plants and plant parts. In addition to these categories of responses, any other modification of a plant, seed, fruit or vegetable, so long as the net result is to increase the growth or maximize any beneficial or desired property of the agricultural and horticultural crop or seed, is intended to be included within the scope of advantageous responses achieved by the practice of the present invention.

Suitable applications of the growth enhancing compositions of the present invention to cultures of plant tissues induce the regeneration of shoots, roots or calli. This effect occurs in both monocotyledonous and dicotyledonous plant species and applies to a wide variety of plants.

The compositions of the invention comprise one or more mono- or multi- substituted phenylacetic acid (PAA) compounds, or ester or salt derivatives thereof, which have auxinic activity on plant cells and tissues. Compounds according to this aspect preferably have the general formula:

wherein X is one or more substitutent groups, which may be the same or different, and which is selected from the group consisting of 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- (RO-) groups, one or more alkylamino- (RNH-) groups, one or more acyl- (RCO-) groups, one or more acylamido- (RCONH-) groups and one or more acyloxy- (RCOO-) groups. According to a preferred aspect of the invention, R may be CH₃(CH₂)_n, wherein n is an integer ranging from 0 to 10. These compounds may be obtained commercially or synthesized by methods well-know in the art. In particular, the invention provides compositions comprising one or more mono- substituted PAA compounds having a substituent group at the 2, 4, 5 and/or 6 position of the PAA structure (Figure 1), wherein the substituent group is a halo-, alkyl-, alkylamino-, alkoxy-, acyl-, acylamido- or acyloxy-substituent group having 1-10 carbon atoms. The invention also provides compositions comprising one or more multi- substituted PAA compounds having two to five substituent groups, which may be the same or different, at positions selected from the group consisting of positions 2, 4, 5 and 6 of the PAA structure (Figure 1) wherein the substituents are halo-, alkyl-, alkylamino-, alkoxy-, acyl-, acylamido- or acyloxy-substituent groups having 1-10 carbon atoms. Preferred PAA compounds for use in compositions according to these aspects of the invention include, but are not limited to, 2-bromophenylacetic acid, 3-bromophenylacetic acid, 4-bromophenylacetic acid, 4-chlorophenylacetic acid, and derivatives thereof.

PAA compounds are preferably included in the compositions of the invention at concentrations that promote optimal growth, cultivation, regeneration or transformation of plant cells, tissues, etc. Preferred concentration ranges of PAA compounds for use in the present compositions include, without limitation, about 0.01 mg/ml to about 100 mg/ml, about 0.05 mg/ml to about 100 mg/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, about 2.5 mg/ml to about 15 mg/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 to about 6 mg ml, about 2.5 mg/ml to about 5 mg/ml, about 3 mg/ml to about 5 mg/ml, and about 3 mg/ml to about 4 mg/ml. As will be readily apparent to one of ordinary skill in the art, the concentration of a given PAA analogue can be increased or decreased beyond the ranges described above, and the effect of the increased or decreased concentration on plant growth, cultivation, regeneration or transformation can be determined using routine experimentation.

According to another aspect of the present invention, PAA compounds are preferably included in the compositions of the present invention at concentrations that cause toxic effects on plant tissues. Further preferred concentration ranges of PAA compounds for use in the present compositions include, without limitation, about 0.5 mg/1 to about 1000 mg/1, about 0.5 mg/1 to about 850 mg/1, about 0.5 mg/1 to about 750 mg/1, about 0.5 mg/1 to about 650 mg/1, about 0.5 mg/ml to about 500 mg/ml, about 0.5 mg/1 to about 350 mg/1, about 0.5 mg/ml to about 250 mg/1, about 0.5 mg/1 to about 150 mg/1, about 0.5 mg/1 to about 100 mg/1, about 0.5 mg/1 to about 50 mg/1, and about 0.5 mg/1 to about 20 mg/1.

The compositions of the invention may include, in addition to one or more of the above-described mono- or multi-substituted PAA-derived compounds, one or more additional plant growth regulators. For example, it is contemplated that other cytokinins or other plant growth regulators known in the art can be utilized with one or more PAA compounds to make additional growth-affecting compositions of the invention. Plant growth regulators that may be advantageously used in the compositions of the invention are known in the art and include, but are not limited to, BAP, 2iP, ipp, zeatin, kinetin, gibberellin, and the like, as described in Skoog et al, Phytochemistry 6: 1169-1192 (1967) and Theologis (1989) Plant Biotechnology (Kung and Arntzen, eds.) Butterworth Publishers, Stoneham, MA, in definite proportions for wide application to various plants. In specific embodiments, the invention is exemplified with compositions comprising one or more mono- or multi-substituted PAA compounds having between one and five substituent groups, which may be the same or different and which may be halo-, alkyl-, alkylamino-, alkoxy-, acyl-, acylamido- and acyloxy-substituent groups at positions 2, 4, 5 and/or 6 of the PAA structure, and one or more cytokinins to affect the growth of plants.

According to the invention, the one or more cytokinins or other growth regulators may be used in the compositions of the invention at concentration ranges that promote optimal growth, cultivation, regeneration, or transformation of plant cells, tissues, etc. Preferred concentration ranges of cytokinins or other growth regulators for use in the present compositions include, without limitation, about 0.01 mg/ml to about 100 mg/ml, about 0.05 mg/ml to about 100 mg/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, about 2.5 mg/ml to about 15 mg/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 to about 6 mg/ml, about 2.5 mg/ml to about 5 mg/ml, about 3 mg/ml to about 5 mg/ml, and about 3 mg/ml to about 4 mg/ml. As will be readily apparent to one of ordinary skill in the art, the concentration of a given cytokinin or other growth regulator can be increased or decreased beyond the ranges described above, and the effect of the increased or decreased concentration on plant growth, cultivation, regeneration or transformation can be determined using routine experimentation. The precise amount of growth affecting compositions employed in the practice of the present invention will depend upon the type of response desired, the formulation used, and the type of plant treated. The invention contemplates the use of a ratio of cytokinin or other growth regulator concentration to PAA analogue concentration of between approximately 50.0 and 0.001, preferably between approximately 5.0 and 0.05, and more preferably between approximately 2.0 and 0.25.

The mechanisms by which the compounds and compositions of the present invention affect the growth cycle of plants and plant tissues is not fully understood at present. However, as shown in the Examples below, it is apparent that the present compositions play a significant role in inducing a number of growth affecting responses in a variety of plant species.

Culture Media

The invention further relates to plant culture media comprising one or more of the above-described PAA compounds or compositions of the invention. Such culture media may, for example, comprise 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, to stimulate regeneration of plant cells and tissues and to stimulate transformation of plant cells and tissues.

The cell culture media according to this aspect of the invention are aqueous-based or solid or semi-solid, comprising a number of ingredients in a solution of deionized, distilled water to form "basal media." Ingredients which may be in the basal media of the present invention include amino acids, vitamins, inorganic salts, D-glucose, sucrose, MES, glycine and solidified reagents such as agar, agrose, gelrite, charcoal and coconut. Each of these ingredients may be obtained commercially, for example from Sigma (Saint Louis, Missouri).

Amino acid ingredients which may be included in the media of the present invention include 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. These amino acids may be obtained commercially, for example from Sigma (Saint Louis, Missouri).

Vitamin ingredients which may be included in the media of the present invention include biotin, choline chloride, D-Ca^-pantothenate, folic acid, z^'-inositol, niacinamide, pyridoxine, riboflavin, thiamine and vitamin B₁₂. These vitamins may be obtained commercially, for example from Sigma (Saint Louis, Missouri).

Inorganic salt ingredients which may be used in the media of the present invention include a calcium salt (e.g., CaCl₂), CuSO , FeSO₄, KC1, a magnesium salt (e.g., MgCl₂), a manganese salt (e.g., MnCl₂), sodium acetate, NaCl, NaHCO₃, Na₂HPO , Na₂SO₄, and ions of the trace elements selenium, silicon, molybdenum, vanadium, nickel, tin and zinc. These trace elements may be provided in a variety of forms, preferably in the form of salts such as Na₂SeO₃, Na₂SiO₃, (NH₄)6Mo₇O₂₄, NH₄VO₃, NiSO₄, SnCl and ZnSO. These inorganic salts and trace elements may be obtained commercially, for example from Sigma (Saint Louis, Missouri).

Examples of plant cell culture media that may be used to prepare the culture media of the present invention include, but are not limited to, Anderson's Plant Culture Media, CLC Basal Media, Gamborg's Media, Guillard's Marine Plant Culture Media, Provasoli's Marine Media, Kao and Michayluk's Media, Murashige and Skoog Media, McCown's Woody Plant Media, Knudson Orchid Media, Lindemann Orchid Media, and Vacin and Went Media. Formulations for these media, which are commercially available, as well as for many other commonly used plant cell culture media, are well-known in the art and may be found for example in the GIBCO BRL Products and Reference Guide (Life Technologies, Inc., Rockville, Maryland), and in the Sigma Plant Cell Culture Catalogue (Sigma; St. Louis, Missouri).

As the skilled artisan will appreciate, any of the above media of the invention may also include one or more additional components, such as indicating or selection agents (e.g., dyes, antibiotics, amino acids, enzymes, substrates and the like), filters (e.g., charcoal), salts, polysaccharides, ions, detergents, stabilizers, and the like. In a particularly preferred embodiment of the invention, the above-described culture media may comprise one or more buffer salts, at concentrations sufficient to provide optimal buffering capacity for the culture medium. According to one aspect of the invention, a buffer salt may be added in powdered form to the powdered medium prior to, during or following addition of the one or more PAA compounds and optionally the one or more cytokinins or other growth regulators to the medium.

The medium ingredients can be dissolved in a liquid carrier or maintained in dry form. If dissolved in a liquid carrier at the preferred concentrations shown in Table 1 (i.e., a "IX formulation"), the pH of the medium should be adjusted to about 4-8, preferably about 4.5-7, 5-6 or 5.5-6. Of course, optimal pH for a given culture medium to be used on a particular cell type may also be determined empirically by one of ordinary skill in the art using known methods.

The type of liquid carrier and the method used to dissolve the ingredients into solution vary and can be determined by one of ordinary skill in the art with no more than routine experimentation. Typically, the medium ingredients can be added in any order. As will be readily apparent to one of ordinary skill in the art, each of the components of the culture medium may react with one or more other components in the solution. Thus, the present invention encompasses the culture media supplemented as described above with one or more PAA compounds and optionally also with one or more cytokinins or other plant growth regulators, as well as any reaction mixture which forms after these ingredients are combined.

Preferably, the solutions comprising ingredients are more concentrated than the concentration of the same ingredients in a IX media formulation. The ingredients can be 10-fold more concentrated (10X formulation), 20-fold more concentrated (20X formulation), 25-fold more concentrated (25X formulation), 50-fold more concentrated (50X concentration), or 100-fold more concentrated (100X formulation). More highly concentrated formulations can be made, provided that the ingredients remain soluble and stable. See U.S. Patent No. 5,474,931, which is directed to methods of solubilizing culture media components at high concentrations. If the media ingredients are prepared as separate concentrated solutions, an appropriate (sufficient) amount of each concentrate is combined with a diluent to produce a IX medium formulation. Typically, the diluent used is water but other solutions including aqueous buffers, aqueous saline solution, or other aqueous solutions may be used according to the invention.

Alternatively, the culture media of the invention may be prepared in dry powder form and then agglomerated with one or more solvents, preferably an aqueous solvent such as water or a saline solution, to produce an agglomerated dry powder plant culture medium that will more readily dissolve upon reconstitution. See, e.g., commonly owned, co-pending U.S. Application No. 09/023,790, filed February 13, 1998, which relates to methods for production of agglomerated dry powder culture media, the disclosure of which is incorporated by reference herein in its entirety.

The culture media of the present invention is typically sterilized to prevent unwanted contamination. Sterilization may be accomplished, for example, by filtration through a low protein-binding membrane filter of about 0.1-1.0 μm pore size (available commercially, for example, from Millipore, Bedford, Massachusetts) after admixing the concentrated ingredients to produce a sterile culture medium. Alternatively, concentrated subgroups of ingredients may be filter-sterilized and stored as sterile solutions. These sterile concentrates can then be mixed under aseptic conditions with a sterile diluent to produce a concentrated IX sterile medium formulation. Autoclaving or other elevated temperature-based methods of sterilization are not favored, since certain of the components of the present culture media may be heat labile and irreversibly degraded by temperatures such as those achieved during most heat sterilization methods. Dry powder culture media of the invention, including the above-described agglomerated dry powder culture media, may be sterilized by irradiation, for example with γ irradiation or ultraviolet irradiation, as described in commonly owned, co-pending U.S. Application No. 09/023,790, filed February 13, 1998.

Although the culture media of the invention are exemplified herein as suitable for cultivation of rice, maize, wheat, tobacco, potato and soybean, the present culture media may be used to support the growth, cultivation, regeneration and transformation in vitro of cells, tissues, embryos, protoplasts, and the like from any plant species. Suitable methods for isolation and in vitro cultivation of plants, plant cells, plant tissues, and the like are well-known to those of ordinary skill in the art (see, e.g., U.S. Patent No. 5,674,731 , the disclosure of which is incorporated by reference herein in its entirety).

Uses of Compositions and Culture Media

The present compounds, compositions and culture media of the invention may be used in a variety of methods used for stimulating growth, regeneration, transformation, or cultivation of plant cells, tissues, protoplasts, embryos and the like. Thus, in additional aspects, the present invention relates to methods of stimulating the growth or regeneration of a plant, plant cell or plant tissue comprising (a) applying to a plant, plant cell or plant tissue an effective amount of one or more of the compounds or compositions of the invention, and (b) 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, for example in one or more of the culture media of the invention. The invention also provides methods for stimulating the regeneration of plant cells and/or tissues comprising (a) applying to a plant cell or tissue an effective amount of one or more of the compounds or compositions of the invention, and (b) incubating the plant cell or tissue under conditions sufficient to stimulate the regeneration of the plant cell or tissue. The invention also concerns methods of attenuating or alleviating environmental stress in a plant, plant cell or tissue comprising (a) contacting a plant, plant cell or tissue which has been exposed to an environmental stress (such as drought, excess temperature, diminished temperature, chemical toxicity (e.g., antibiotic, herbicides), pollution, excess light and diminished light) with an effective amount of one or more of the compounds or compositions of the invention, and (b) incubating said plant, plant cell or tissue under conditions sufficient to attenuate or alleviate said stress. The invention also relates to methods for cultivating a plant, plant cell or plant tissue in vitro, comprising (a) obtaining a plant, plant cell or plant tissue to be cultivated, (b) contacting the plant, plant cell or plant tissue with one or more of the compounds or compositions of the invention, and (c) incubating the plant, plant cell or plant tissue under conditions suitable to support cultivation of the plant, plant cell or plant tissue. In addition, the invention provides a method for transformation of plant cells and/or tissues comprising (a) contacting the plant cell or tissue with a nucleic acid molecule (e.g., by transformation or protoplast fusion), (b) contacting the plant cell or tissue with an effective amount of one or more of the compounds or compositions of the invention, and (c) incubating the plant cell or tissue under conditions sufficient to induce transformation of the plant cell or tissue with the nucleic acid molecule. The compounds and compositions of the invention may also be used to stimulate regeneration or growth of the transformed tissue or cells, thus providing a method to obtain a transgenic plant. The invention therefore also relates to transformed or transgenic plants, plant cells and plant tissues produced by the above-described methods.

Suitable applications of the growth-enhancing compounds and compositions of the present invention include contacting cultures of plant tissues with one or more of the compounds or compositions of the invention to induce the regeneration of shoots, roots or calli. 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.

Genetic engineering of plants generally involves two complementary processes. The first process involves the genetic transformation of one or more plant cells of a specifically characterized type. By transformation it is meant that a foreign gene, typically a chimeric gene construct, is introduced into the genome of the individual plant cells, typically through the aid of a vector which has the ability to transfer the gene of interest into the genome of the plant cells in culture. The second process then involves the regeneration of the transformed plant cells into whole sexually competent plants. Neither the transformation nor regeneration process need be 100% successful but must have a reasonable degree of reliability and reproducibility so that a reasonable percentage of the cells can be transformed and regenerated into whole plants.

The two processes, transformation and regeneration, must be complementary. The complementarity of the two processes must be such that the tissues which are successfully genetically transformed by the transformation process must be of a type and character, and must be in sufficient health, competency and vitality, so that they can be successfully regenerated into whole plants.

Successful transformation and regeneration techniques have been demonstrated for monocots and dicots in the prior art. For example, the transformation and regeneration of tobacco plants was reported in Barton et al, Cell 32:1033 (April 1983), whereas the regeneration of cotton is described in U.S. Patent No. 5,004,863, the disclosure of which is incorporated by reference herein in its entirety. Further, transformation and regeneration of rice was described by Abdullah et al, Bio/Technology 4: 1087-1090 (1986), whereas maize was transformed and regenerated as described in Rhodes et al, Bio/Technology (5:56-60 (1988). The most common methodology used for the transformation of cells of dicot plant species involves the use of the plant pathogen Agrobacterium tumefaciens. Other methods of gene transfer have also been described, e.g., the polyethylene glycol method, electroporation, direct injection, particle bombardment, etc., as described by Wu (in: Plant Biotechnology, Stoneham, MA: Butterworth Publishers, pp. 35-51 (1989)). The present invention will be useful with any method of transformation that includes plant regeneration steps.

In a specific embodiment, the invention envisions the genetic transformation of tissues in culture derived from leaf discs or hypocotyl explants. The transformed tissues can be induced to form plant tissue structures, which can be regenerated into whole plants.

The transformation technique of the present invention is one which makes use of the Ti plasmid of A. tumefaciens. In using an A. tumefaciens culture as a transformation vehicle, it is most advantageous to use a non-oncogenic strain of the Agrobacterium as the vector carrier so that normal non-oncogenic differentiation of the transformed tissue is possible. To be effective once introduced into plant cells, the chimeric construction including a foreign gene of interest must contain a promoter which is effective in plant cells to cause transcription of the gene of interest and a polyadenylation sequence or transcription control sequence also recognized in plant cells. Promoters known to be effective in plant cells include the nopaline synthase promoter, isolated from the T-DNA of Agrobacterium, and the cauliflower mosaic virus 35S promoter. Other suitable promoters are known in the art. It is also preferred that the vector which harbors the foreign gene of interest also contain therein one or more selectable marker genes so that the transformed cells can be selected from non-transformed cells in culture. In many applications, preferred marker genes include antibiotic resistance genes so that the appropriate antibiotic can be used to segregate and select for transformed cells from among cells which are not transformed.

The details of the construction of the vectors containing such foreign genes of interest are known to those skilled in the art of plant genetic engineering and do not differ in kind from those practices which have previously been demonstrated to be effective in tobacco, petunia and other model plant species. The foreign gene should obviously be selected as a marker gene (Jefferson et al, EMBO J. <5:3901-3907 (1987)) or to accomplish some desirable effect in plant cells. This effect may be growth promotion, disease resistance, a change in plant morphology or plant product quality, or any other change which can be accomplished by genetic manipulation. The chimeric gene construction can code for the expression of one or more exogenous proteins, or can cause the transcription of negative strand RNAs to control or inhibit either a disease process or an undesirable endogenous plant function.

To initiate the transformation and regeneration process for plant tissues, it is necessary to first surface sterilize tissues to prevent inadvertent contamination of the resulting culture. If the tissues are seeds, the seeds are then allowed to germinate on an appropriate germinating medium containing a fungicide. Four to ten days after germination the hypocotyl portion of the immature plant is removed and sectioned into small segments averaging approximately 0.5 centimeters apiece. The hypocotyl explants are allowed to stabilize and remain viable in a liquid or agar plant tissue culture medium.

Once the tissues have stabilized, they can promptly be inoculated with a suspension culture of transformation competent non-oncogenic Agrobacterium. The inoculation process is allowed to proceed for a short period, e.g., two days, at room temperature, i.e., 24°C.

At the end of the inoculation time period, the remaining treated tissues can be transferred to a selective agar medium, which contains one or more antibiotics toxic to Agrobacterium but not to plant tissues, at a concentration sufficient to kill any Agrobacterium remaining in the culture. Suitable antibiotics for use in such a medium include carbenicillin, cefotaxime, etc., as the bactericide for Agrobacterium and kanamycin as the selective antibiotic for transformed plant tissues. The tissues may then be cultivated on a tissue culture medium, preferably a culture medium of the invention, which, in addition to its normal components, contains one or more selection agents. The selection agent, exemplified herein by kanamycin, is toxic to non-transformed cells but not to transformed cells which have incorporated genetic resistance to the selection agent and are expressing that resistance. The surviving transformed tissues are transferred to a secondary medium, preferably a culture medium of the invention with the selection agent, to induce tissue regeneration. The surviving transformed tissue will thus continue to be regenerated into a whole plant through the regeneration technique of the present invention or through any other alternative plant regeneration protocols.

The PAA compounds and compositions of the invention are also useful in making a plant less susceptible to the toxicities of antibiotics. Such PAA compounds and compositions are also useful in enabling plants to overcome stress, e.g., environmental stress, physical stress, chemical stress, pollution, contamination, drought, light, and the like.

The present compounds and compositions may be applied at any developmental stage of the plant species to obtain plant hormone or maintenance effects throughout maturity and to expedite re-growth in damaged tissues during early developmental stages, depending upon the concentration used, the formulation employed and the type of plant species treated.

The compounds and compositions of the present invention are preferably used in conjunction with specific auxiliary nutrients or other plant growth regulators in precise proportions to achieve a particular synergistic, growth enhancing response in various type of plants. The present compounds and compositions may additionally be used in association with fungicides to increase the disease resistance of various plants, making the plant tissue resistant to invasion by pathogens by influencing the enzyme and plant processes which regulate natural disease immunity. While the present compounds and compositions possess essentially no phytotoxic activity of their own, they may sometimes be used in conjunction with herbicides to stimulate the growth of unwanted plants in order to make such plants more susceptible to a herbicide. However, it is preferred to regard the results achieved in the practice of the present invention as growth enhancing responses in agricultural and horticultural crops, as well as perennial and annual household plants species.

It will be readily apparent to one of ordinary skill in the relevant arts that other suitable modifications and adaptations to the methods and applications described herein are obvious and may be made without departing from the scope of the invention or any embodiment thereof. Having now described the present invention in detail, the same will be more clearly understood by reference to the following examples, which are included herewith for purposes of illustration only and are not intended to be limiting of the invention.

Examples

Materials and Methods

Culture Media. GIBCO BRL Murashige and Skoog (MS) Complete Medium- 50X Concentrate (Cat. # 10494-011) was prepared by mixing 20 ml of each component from Salt I, Salt II and Acid Soluble either with 940 ml of sterilized water for membrane based liquid growth format, or with 940 ml sterilized water with 0.8% agar for semi-solid growth format.

Plant Material. Seeds of Rice (Oryza sativa L.), Sweetcorn (Zea mays) and Wheat (Triticurn aestivam L.) were sterilized by first placing them in sterile water while gently stirring for 30 minutes. The seeds were then immersed in 95% alcohol for 1 min. The alcohol solution was remove and the seeds were placed into a Side-arm-flask. A solution of 15% commercial bleach plus 0.1% Tween^® 20 was prepared and added to the flask. A vacuum was applied to the seeds for 20 minutes while shaking. The vacuum was removed but shaking continued for an additional 25 min. The seeds were rinsed three times with sterile water in a clean hood, and planted in semi-solid MS medium.

For cultivation of Tobacco (Nicotiana tobaccum cv. Xanti) and Tomato (Lycopersicon esculentum L. Mill.), young leaves from one-month-old plants were removed and punched by hole puncher to prepare uniform leaf discs. The leaf discs were placed into MS complete media in petri dishes. For cultivation of Potato (Solanum tuberosus cv. Atlantic) and Cassava (Manihot esculenta) plants, stems (0.8 cm internodo) approximately one month old were used. The stems were placed into MS complete media in petri dishes.

For cultivation of Soybean (Glycine max L.), seeds were sterilized by placing them into 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 bleach solution into sterile MS medium (7 seeds per plate). The plates were incubated in the dark at 28°C for 4 days. The plates were then inspected for fungi and bacteria, and clean seeds were transferred to Magenta Boxes and incubated for an additional 11 days. The resulting 15 day old Soybean stems were cut into segments approximately 1 cm long and placed into MS medium in petri dishes.

All explants and seeds, were incubated for 1 month at 25°C, using an 18 hour light/6 hour dark cycle. Potato and Cassava transformation was generated by co- cultivating high concentration of A. tumefaciens LBA4404 containing Km^R gene. See Lin et al. (1995) Focus 16: 72. Results

Auxin-like chemicals (PAA derivatives) were evaluated for auxin activity by incubating the tobacco leaf discs, tomato leaf discs and potato stems in culture medium containing MS salts and different concentrations of either auxin-like chemicals only, or a combination of auxin-like chemicals and cytokinin. Callus or root formation was observed in the medium containing the auxin-like chemicals only, while the regeneration of shoots was also found in addition to root and/or callus formation in the medium containing auxin-like chemicals and cytokinin. In general, more callus formation was observed in the combination of cytokinin with high concentration of auxin-like chemicals (Figure 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). In order to demonstrate the auxin activity and possible application of these new compounds in plant tissue culture, we chose 4-bromophenylacetic acid which only showed marginal auxin activity, to optimize medium formulation for the potato shoot regeneration. Interestingly, the regeneration efficiency increased two fold in potato variety, Atlantic comparing the medium containing 4-bromophenyacetic acid with the medium containing well characterized auxin, NAA. More than 11 fold increased was observed in the shoot regeneration of potato variety, Kent, which is regarded as a difficult potato variety for regeneration in tissue culture (Table 3).

Further evaluation of effects of 4-bromophenylacetic acid was performed in combination with different cytokinines. Interestingly, the effect of cytokinine like zeatine is able to improve the regeneration efficiency up to ten fold. In addition, the shoot regeneration was able to increase up to 57 fold using the membrane-based liquid culture (Table 4). All these demonstrated that 4-bromophenylacetic acid is a useful auxin and can be used for improving the regeneration of potato shoot formation. Regeneration of transgenic plants was evaluated by Agrobactenum-mediated transformation. After co-cultivation of potato stems with A. tumefaciens LBA4404 with pBI121, the potato stems were incubated onto the medium containing 4- bromophenylacetic acid with different cytokinine and kanamycin for the selection of antibiotics resistant plants. The transformation efficiency was increased about 40 fold in agar based culture, and 95 fold in membrane based liquid culture for the Atlantic variety. For varieties of Snowden and Russet Burbank, the transformation efficiency were improved from 0 to 10-25 fold in agar based culture, and 0 to 57-920 fold in the membrane based liquid culture (Table 5). Different crops such as soybean, cassava, maize, wheat, and rice were used to evaluate further for the auxin activity of 2-bromophenylacetic acid, 3-bromophenylacetic acid and 4-bromophenylacetic acid. Either callus formation or root formation was shown when the plant tissues were incubated in the medium containing theses compounds (Table 6). All these results demonstrate that auxin activity was detected and the regeneration of both non-transformed shoots or transformed shoots will be improved once the optimization of the concentration of chemicals with cytokinine was achieved.

Significant improvement of potato shoot regeneration was demonstrated using different cytokinins with 4-bromophenylacetic acid in different varieties of potato such as Snowden and Russet Burbank. Furthermore, callus and root formation was observed by treating the cassava tissues with 4-bromophenylacetic acid, which didn't show in those treated by conventional auxin such as 2,4-D (Table 6).

00 IN.

Oύ

Having now fully described the present invention in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious to one of ordinary skill in the art that the same can be performed by modifying or changing the invention within a wide and equivalent range of conditions, formulations and other parameters without affecting the scope of the invention or any specific embodiment thereof, and that such modifications or changes are intended to be encompassed within the scope of the appended claims.

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

Claims

WHAT IS CLAIMED IS:

1. A compound for regulating the growth of a plant cell, wherein said compound comprises a mono- or multi-substituted phenylacetic acid (PAA) compound, or an ester or salt derivative thereof, which has auxinic activity on plant cells and tissues and has the general formula of:

wherein X is one or more substitutent groups selected from the group consisting of one or more halo- groups, one or more alkyl- (R-) groups, one or more alkoxy- (RO-) groups, one or more alkylamino- (RNH-) groups, one or more acyl- (RCO-) groups, one or more acylamido- (RCONH-) groups, and one or more acyloxy- (RCOO-) groups.

2. The compound of claim 1, wherein R is a linear, branched or cyclic hydrocarbon.

3. The compound of claim 2, wherein said hydrocarbon is a saturated hydrocarbon.

4. The compound of claim 2, wherein said hydrocarbon is an unsaturated hydrocarbon.

5. The compound of claim 1, wherein R is CH₃(CH₂)_n, wherein n is an integer which ranges from 0 to 10.

6. The compound of 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 of claim 1, wherein X is one or more halo groups.

8. The compound of claim 7, wherein said one or more halo groups are one or more bromo groups.

9. The compound of claim 7, wherein said one or more halo groups are one or more chloro groups.

10. The compound of claim 1, wherein said compound is a mono- or a di- substituted phenylacetic acid compound or a derivative thereof.

11. The compound of claim 10, wherein said 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.

12. A composition comprising one or more of the compounds of claim 1.

13. The composition of claim 12, further comprising at least one cytokinin or giberellin.

14. The composition of claim 13, wherein said cytokinin is selected from the group consisting of zeatine, 6-furfurylaminopurine (kinetin), and 6-benzylaminopurine

(BAP).

15. A method of affecting the growth of a plant, plant cell or plant tissue comprising (a) applying to a plant, plant cell or plant tissue an effective amount of one or more of the compounds of claim 1, 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.

16. A method of affecting the regeneration of a plant, plant cell or plant tissue comprising (a) applying to a plant, plant cell or plant tissue an effective amount one or more of the compounds of claim 1, and (b) incubating the plant, plant cell or plant tissue under conditions sufficient to affect the regeneration of the plant, plant cell or plant tissue.

17. A plant culture medium comprising one or more of the compounds of claim 1.

18. The culture medium of claim 17, further comprising at least one cytokinin or giberellin.

19. The culture medium of claim 18, wherein said cytokinin is selected from the group consisting of zeatine, 6-furfurylaminopurine (kinetin), and 6- benzylaminopurine (BAP).

20. A method of cultivating a plant, plant cell or plant tissue in vitro, comprising (a) obtaining a plant, plant cell or plant tissue to be cultivated in vitro, (b) contacting the plant, plant cell or plant tissue with one or more of the plant culture media of claim 17, and (c) incubating the plant, plant cell or plant tissue under conditions suitable to support cultivation of the plant, plant cell or plant tissue in vitro.

21. A method of producing a transformed plant, plant cell or plant tissue comprising (a) contacting the plant, plant cell or plant tissue with a nucleic acid molecule to be transformed into the plant, plant cell or plant tissue, (b) contacting the plant, plant cell or plant tissue with an effective amount of one or more of the compounds of claim 1, and (c) incubating the plant, plant cell or plant tissue under conditions sufficient to induce transformation of the plant, plant cell or plant tissue with the nucleic acid molecule.

22. A transformed plant, plant cell or plant tissue produced by the method of claim 21.

23. A method of producing a transgenic plant, plant cell or plant tissue comprising (a) contacting the transformed plant, plant cell or plant tissue of claim 22 with one or more of the compounds of claim 1, and (b) incubating the transformed plant, plant cell or plant tissue under conditions sufficient to affect regeneration or growth of the transformed plant, plant cell or plant.

24. A transgenic plant, plant cell or plant tissue produced by the method of claim 23.

25. A method of attenuating or alleviating environmental stress in a plant, plant cell or tissue comprising (a) contacting a plant, plant cell or plant tissue which has been exposed to an environmental stress with an effective amount of one or more of the compounds of claim 1, and (b) incubating said plant, plant cell or plant tissue under conditions sufficient to attenuate or alleviate said stress.

26. The method of claim 25, wherein said environmental stress is selected from the group consisting of drought, excess temperature, diminished temperature, chemical toxicity, pollution, excess light, and diminished light.