WO1990001058A1 - Method for improved somatic embryogenesis using synthetic auxin analogs - Google Patents

Abstract

A method for improved somatic embryogenesis is disclosed wherein callus tissue from a suitable plant source is induced to form somatic embryos in an induction medium containing selected synthetic auxin analogs.

Description

Description

Method for Improved Somatic Embryoσenesis Using Synthetic Auxin Analogs

Technical Field

The present invention relates generally to methods for regenerating whole plants from tissue cultures and more specifically to improved methods for somatic embryogenesis using compounds which promote the formation of somatic embryos.

Background of the Invention

Plant cells possess the capacity to differentiate into whole plants from single cells or small cell colonies, termed callus. Callus is typically composed of undifferentiated cellular aggregates which do not manifest the typical leaf, stem or root tissues found in an adult plant. During the differentiation process, callus cells grow into diverse tissue types which give rise to distinct plant organs. The process of differentiation and formation of plant parts which ultimately form whole plants is called regeneration.

Plant cell regeneration has numerous advantageous applications, such as the uses described in Handbook of Plant Cell Culture. D.A. Evans, et al. eds, Vols. 1-4, MacMillian Publishing Co., N.Y. (1986). For example, cells can be manipulated to effect cell and organelle fusion, cell selection and gene introduction. Plant cells can also be rapidly grown in masse for a number of purposes. Plant cell regeneration can be used to evaluate the effects of cellular manipulation at the whole plant level. Traditional genetic manipulation of plants, using a plant breeding process, requires the regeneration of plant cells to adult plants that are reproductively mature. Hence, the selections made from the above methods must be regenerated for the gene change to be incorporated into a breeding program.

The two most important methods for plant cell regeneration are somatic organogenesis and somatic embryogenesis. For a recent review of these two processes, see Evans, D.A. et al.. , "Growth and Behavior of Cell Cultures: Embryogenesis and Organogenesis" in Plant Cell Culture. T.A. Thorpe, ed. , Academic Press, pp. 45-113 (1981) . In general, organogenic regeneration occurs at low frequencies, requires a separate rooting process to give rise to whole plants and is an expensive undertaking for commercial plant propagation.

By comparison, the process of somatic embryogenesis generates numerous, bipolar, naked embryos containing the rudiments of both shoot and root axes, which are capable of direct development into an entire plant. Commercialization of this process is considered more desireable and economical than organogenic cloning due to its higher yield of plants and similarity to the development of the seed embryo.

Somatic embryos may also be used as a source of valuable natural products, such as oils, flavorings or other phytochemicals, which are synthesized in highly differentiated plant tissues. These products could be produced in fermentors, bioreactors or other scaled-up production systems capable of maintaining the embryonic cells.

One problem encountered with large scale use of somatic embryogenesis lies with normal plantlet formation after embryo germination. Lutz, J.D. et al.. "Somatic Embryogenesis for Mass Cloning of Crop Plants" in Tissue Culture in Forestry and Agriculture. R.R. Henke et al. eds., Plenum Press, NY, pp. 105-116 (1985). For example, studies using naked or encapsulated somatic embryos of carrot illustrate that an extremely low percentage of somatic embryos germinate and develop into plantlets even when the best conditions for germination are used. Drew, R.K.L., "The Development of Carrot (Daucus carota L.) Embryoids (Derived from Cell Suspension Culture) into Plantlets on a Sugar-free

Basal Medium," Hort. Res. .19:79-84 (1979); Kitto, S.L. and J. Janick, "Production of Synthetic Seed by Encapsulating Asexual Embryos of Carrot," J. American Hort. Society. 110:277-282 (1985). However, to be commercially useful for cloning purposes, somatic embryos must germinate rapidly, as do seeds, and produce vigorous shoots and roots. Such results have been achieved by utilizing chemicals known generally as plant auxins. The term auxin, originally applied to the naturally-occurring compound indole-3-acetic acid, is generally applied to structurally related, naturally occurring or chemically synthesized substances that possess the ability to promote cell elongation. Salisbury, F.B. and C. Ross, Plant Physiology.

Wadsworth Publishing, Belmont, CA, Chapter 21 (1969) . The auxins are compounds included within a larger class of chemicals, termed "plant growth regulators," having the ability to affect plant cell growth (elongation) and development. These chemicals include: gibberellin, abscisic acid (ABA) , ethylene and cytokinins such as kinetin.

Certain auxins, especially the arylphenoxy acids, have found broad commercial use as plant growth regulators and herbicides. The structures of selected arylphenoxy acids are presented in Figure 1, accompanied by a reference numeral (presented in brackets in the text of this portion of the disclosure) . One of the most widely used of such arylphenoxy acids is 2,4-dichlorophenoxyacetic acid (2,4-D) [I]. These che icals promote cell growth in stems and shoots but can also selectively kill plants. Loos, M.A., "Phenoxyalkanoic Acids," in Herbicides: Chemistry Degradation and Mode of Action. P.C. Kearney and D.D. Kaufmann eds. , M. Dekker, Inc., N.Y., pp. 1-128 (1975). The ability of phenoxypropanoic acids to promote plant cell elongation has been extensively investigated.^' It is known that if the propanate side chain is substituted with a phenoxy group at the third position, the resultant chemical lacks auxin cell elongation activity and fails to function as a herbicide. An example of such a chemical is 3-(2,4- dichlorophenoxy)propanoic acid [II]. Fawcett, CH. et al.. "The Degradation of Certain Phenoxy Acids, Amides, and Nitrites Within Plant Tissues," in The Chemistry and Mode of Action of Plant Growth Substances. R.L. Wain and F. Wightman, eds., Butterworths, London, pp. 187-194 (1965) . However, substitution of the phenoxy group at the second position of a side chain, as in a racemic mixture of 2-(2,4-dichlorophenoxy)propanoic acid [III] results in an active auxin structure.

This latter type of compound is optically active, having an asymmetric carbon at the 2 position of the propanoate side chain, and the activity and potency depends on the enantio er studied. For example, the (+)-d enantiomer of 2-(2,4 dichlorophenoxy)propanoic acid [IV] is an active auxin, but is not more potent than 2,4-D [I]. Smith, M.S. et al., "Studies on Plant Growth-regulating Substances," Ann. Appl. Biol 3_9.:295-307 (1952); Aberg, B., "Some New Aspects of the Growth Regulating Effects of Phenoxy Compounds," in Plant Growth Regulation. R. M. Klein ed. , Iowa State University Press, Ames, IA, pp. 219-232 (1959).

The (-)-enantiomer of 2-(2,4-dichlorophenoxy) propanoic acid [V] is not an active auxin and has been shown to antagonize auxin action. Smith, M.S. et al.. "Antagonistic Action of Certain Sterioisomers on the Plant-growth Regulating Activity of Their Enantiomorphs," Nature 169:833-834. (1952). The racemic mixture of 2-(2,4-dichlorophenoxy)propanoic acid [III] is slightly less growth active than 2,4-D [I]. The ability of 4-(2,4-dichlorophenoxy)butanoic acid (4-(2,4-DB) ) [VI] to enhance plant cell elongation varies with the plant treated and it is generally held that 4-(2,4-DB) lacks auxin activity. Fawcett, CH. et al.. "The Degradation of Certain Phenoxy Acids, Amides, and Nitrites Within Plant Tissues," in The Chemistry and Mode of Action of Plant Growth Substances. R.L. Wain and F. Wightman, eds. , Butterworths, London, pp. 187-194 (1965) ; Galston, A.W. and P.J. Davies, Control

Mechanisms in Plant Development. Prentice Hall, Inc., Englewood Cliffs, New Jersey, p. 82 (1970) . However, 4- (2,4-DB) can be metabolized, by /9-oxidation, to 2,4-D which is active. In Sinapis arvenis L. , 4-(2,4-DB) is active on whole plants as an herbicide since this plant possesses a strong ^-oxidation system to convert 4-(2,4- DB) to 2,4-D. On the other hand, no herbicidal activity is seen in whole plants of alfalfa or clover, as they lack an active ^-oxidation metabolic system. Thus, 4-(2,4-DB) would not be expected to be more active than 2,4-D in any plant, as it is only expected to be active when converted to 2,4-D. In alfalfa, 4-(2,4-DB) would be expected to be inactive when compared to 2,4-D. When 9-oxidation is active in plant cells, metabolism occurs in two-carbon units. Thus, active auxin structures occur as two carbon extensions of 2,4-DB. The auxins 6-(2,4 dichlorophenoxy)hexonic acid [VII] and 8-(2,4-dichlorophenoxy)octanoic acid [VIII] would therefore be reduced in two carbon units to the active 2,4-D in species capable of _/9-oxidation. In species lacking _/9-oxidation, these auxins would be expected to be as inactive as 2,4-DB.

The auxin analogs 3-(2,4 dichlorophenoxy) ropanoic acid [II] and 5-(2,4-dichlorophenoxy)pentanoic acid [IX] are inactive auxin structures for one of two reasons. If ^-oxidation is present, these structures are reduced to 2,4 dichlorophenol, which has no auxin activity. If ^-oxidation is not present, these structures show no inherent auxin activity on their own. Fawcett et al. , loc. cit.

Auxins are also used in plant cell culture to promote cell proliferation and growth (Evans, D.A. et al.. "Growth and Behavior of Cell Cultures: Embryogenesis and Organogenesis," in Plant Cell Culture, T.A. Thorpe ed. , Academic Press, pp. 45-113, (1981)) and for the initiation of somatic embryogenesis in cell culture (Sharp, W.R. et al.. , "The Physiology of in Vitro Asexual Embryogenesis," Hort. Reviews 2:368-310 (1980)). Thus the term "auxin activity" actually includes two components. One action is manifest at the whole plant level, which accounts for cell elongation and herbicide activity. The second component is auxin action on cell culture development, which promotes embryo formation in vitro. In somatic embryogenesis, plant cells are typically exposed to an auxin-containing induction medium in order to induce somatic embryo formation from callus. For subsequent expression of embryos, the callus is transferred to a regeneration medium which contains reduced auxin levels or no auxin at all.

Wetherell, D.F., "In Vitro Embryoid Formation in Cells Derived from Somatic Plant Tissues," in Propagation of Higher Plants Through Tissue Culture. K.W. Hughes ed. , U.S. Dept. Energy Publication CONF-7804111, UC-48, pp. 102-124 (1978) ; Kohlenbach, H.W. "Comparative Somatic Embryogenesis," in Frontiers of Plant Tissue Culture 1978. T.A. Thorpe, ed. , Univ. Calgary Offset Printing Services, pp. 59-65 (1978); Yamada, Y., "Tissue Culture Studies of Cereals," in Applied and Fundamental Aspects of Plant. Cell. Tissue, and Organ Culture. J.

Reinert and Y.P.S. Bajaj, eds. Springer Verlag, Berlin, pp. 144-159 (1977).

In a published review of over 300 cell culture research reports from dicotyledonous and monocotyledonous crop species capable of somatic embryogenesis, it was found that the majority of researchers used 2,4-D [I] as the auxin in the induction medium. D.A. Evans et aJL. , supra. (1981) . Cell cultures were then generally transferred to an embryo regeneration medium containing no hormones.

Further, an extensive survey, using carrot cell cultures, of a number of synthetic auxins found that 2,4-D [I], 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) [X], 2-methyl-4-chlorophenoxyacetic acid [XI] and 4-chlorophenoxyacetic acid [XII] were superior auxins when used to induce somatic embryogenesis. They also discovered that /9-naphthoxyacetic acid [XII] could induce embryogenesis in carrot. Kamada, H. and H. Harada, "Studies on Organogenesis in Carrot Tissue Cultures. I. Effects of Growth Regulators on Somatic

Embryogenesis and Root Formation," Z. Pflanzenphysiol. 1:255-266 (1979).

The effect of 2,4-D and α-naphthaleneacetic acid (α-NAA) [XIV] in the induction medium of alfalfa cell cultures with respect to embryo yields has also been investigated. Walker, K.A. et al., "Organogenesis in Callus Tissue of Medicago sativa. The Temporal Separation of induction Processes form Differentiation Processes," Plant Sci. Lett. 16:23-30 (1979). They found 2,4-D to be more potent and to stimulate greater embryo yields than α-NAA.

In other studies, additional plant growth regulators or herbicides have been used to stimulate somatic embryogenesis. For example, picloram

(4-amino-3,4,6-trichloropicolinic acid) has been used as an auxin source for various plant cell cultures (Collins, G.B. et al. , "Use of 4-amino-3,5,6- trichloropicolinic acid as an Auxin Source in Plant Tissue Culture," Crop Sci. 18.:286-288 (1978)) . DICAMBA (3,6-dichloro-o-anisic acid) [XV] has been used to induce callus and embryo formation in Dactylis glomerata (Gray, D. . et al. , "Somatic Embryogenesis in Suspension and Suspension-derived Callus Cultures of Dactylis glomerata." Protoplasma 122:196-202 (1984)). In both cases, these herbicides have auxin-like properties that promote cell elongation and embryo induction.

A recent report tested the effect of substituted benzoic acid derivatives on induction of somatic embryogenesis in maize (Zea mays L.) (Close, K.R. and L.A. Lude an, "The Effect of Auxin-like Plant Growth Regulators on Somatic Embryogenesis from Elite Maize Inbreds," Plant Sci. Lett. 52.:81-89 (1987)). They found that genotype effects, which often result in low regeneration in some inbred lines, can be overcome by using the appropriate substituted benzoic acid plant growth regulators. These growth regulators fall in a distinctly different class than the substituted phenoxy-acid growth regulators described above, however. In a study testing the abil-Ltyof certain auxins to block carrot somatic embryogenesis, it was found that the addition of 2-(4-chlorophenoxy)propanoic acid [XVI] to the regeneration medium blocked embryogenesis but to a lesser degree than the addition of 2,4-D to the regeneration medium. When 2-(4-chlorophenoxy)propanoic acid was applied as the (+)-d enantiomorph [XVII], it was active in inhibiting somatic embryogenesis, whereas the (-)-l enantiomorph [XVIII] was inactive (Caldas, L.S., "Effects of Various Growth Hormones on the

Production of Embryoids from Tissue Culture of the Wild Carrot, Daucus carota L.," Ph.D. Thesis, The Ohio State University, Columbus, Ohio, pg. 125 (1971)). These chemicals were not tested for their ability to induce embryogenesis, but only as inhibitors of embryo regeneration. The above study demonstrated that racemic 2-(4-chlorophenoxy)propanoic acid [XVI] and (+)- 2-(4-chlorophenoxy)propanoic acid [XVII] are excellent inhibitors of somatic embryo development. Neither the 2-phenoxypropanoic acids nor the 4- phenoxybutanoic acids have been tested to determine their ability to induce somatic embryogenesis. Both, however, have been found active in promoting plant cell elongation in callus culture. See, e.g., Sanchez de Jimenez, E. and E. Murillo, "Mecoprop as a Growth Factor in Wheat and Rye Tissue Cultures," Can. J. Bot. 57:1439-1483 (1979); McComb, A.J. and J.A. McComb, "Differences Between Plant Species in Their Ability to Utilize Substituted Phenoxybutanoic Acids as a Source of Auxin for Tissue Culture Growth," Plant Sci. Lett. 11:227-232, (1978).

A number of studies indicate that auxins which produce callus growth do not necessarily induce embryogenesis. In Antirrhinum_f for example, it has been observed that while indole-3-acetic acid, α-naphthaleneacetic acid and other auxins produce callus growth, they do not induce somatic embryo formation. Only ø-naphthoxyacetic acid [XIII] and 2,4-D were effective in this regard. Sangwan, R.S. and H. Harada, "Chemical Regulation of Callus Growth, Organogenesis, Plant Regeneration and Somatic Embryogenesis in Antirrhinum manus Tissue and Cell Cultures," J. Expt. Bot. 26:869-877 (1975). A similar result was obtained using orange cell cultures. Kochba, J. and P. Spiegel-Roy, "The Effects of Auxins,

Cytokinins and Inhibitors of Embryogenesis in Habituated Callus of the Shamouti Orange (Citrus sinesis) ." Z. Pflanzenphysiol 81:283-288 (1977).

This lack of correlation between auxin-stimulated plant cell elongation and the ability to induce somatic embryo formation in cell cultures has also been noted in other species. Sharp, W.R. et a . , "The Physiology of In Vitro Asexual Embryogenesis," Hort. Reviews 2.:268-310 (1980) . It is therefore impossible to predict the ability of a chemical compound to induce somatic embryogenesis in cell culture, based on its ability to stimulate cell elongation in whole plants, to stimulate cell elongation in culture or based on its herbicidal properties. This lack of predictability of auxin- induced somatic embryogenesis is a problem which hinders the commercial development of plant propagation by somatic embryogenesis.

Accordingly, it is an object of the present invention to provide a novel and improved method for producing numerous high quality somatic embryos from plant tissue.

It is a further object of the present invention to provide a novel and improved method whereby a high percentage of somatic embryos ultimately germinate and develop into plantlets.

It is a another object of the instant invention to provide a method of somatic embryogenesis capable of large scale plant production. Disclosure of the Invention

These and other objects are achieved in accordance with the present invention, wherein a method for induction of somatic embryo formation in plants is provided. In the practice of the instant invention, callus tissue is induced to form somatic embryos by growth of the callus in the presence of a medium containing mineral salts, a carbon source and at least one synthetic auxin analog having the general structure of a compound in accordance with the formula:

*2

R₄

wherein

X is 0, S, Se, NH or CH₂;

Rl is a carboxylic acid side chain; and

R2, R₃ and R₄ are each independently H, Cl, Br, 1, F, NO2 Cι_ιo straight chain alkyl or 0-Cι_ι₀ straight chain alkyl; with the proviso that when X is O, R]_ is CH2-COOH, and R₃ is Cl, then:

R2 cannot be Cl unless R4 is not H or Cl; or R2 cannot be H or CH₃ unless R4 is not H.

Typically, representative synthetic auxin analogs employed in the practice of the invention comprise a central phenoxy ring moiety, a C₃.-.1₀ carboxylic acid side chain substituted to the phenoxy group side chain and a halo, methyl, acetyl or higher order side chain independently substituted at the 2-, 4- or 5-positionε on the phenoxy ring. Alternatively, synthetic auxin analog growth regulators with the general structure of a phenoxy ring and a propanoic or butanoic acid side chain substituted to the phenoxy oxygen, may be used.

Following incubation in the induction medium, the induced tissue is regenerated and germinated under conditions sufficient to cause root and shoot formation.

The use of the above described compounds in the induction medium has the surprising and unexpected effect of inducing higher somatic embryo yields, producing improved embryo morphology, and improving germination of the somatic embryos so treated.

Further, the use of 2-phenoxypropanoic acid or 4- phenoxybutanoic acid and related compounds notably improves the subsequent morphological and physiological quality of somatic embryos without deleteriously affecting embryo yields.

Brief Description of the Drawings

Figure 1 is a diagrammatic representation of the generalized structure of various arylphenoxy acid compounds discussed in the present disclosure;

Figure 2 is a diagrammatic representation of a generalized structure of one class of synthetic auxin analogs used in the instant invention; Figure 3 is a diagrammatic representation of the generalized structure of an alternative class of synthetic auxin analogs used in the instant invention; and

Figure 4 is a diagrammatic representation of selected synthetic auxin analog structures in selected embodiments of the present invention. Detailed Description of the Invention

This invention provides a method for enhancing the quality of embryos produced from plant somatic tissue derived from species which are capable of somatic embryogenesis, such as alfalfa, celery and lettuce. The resultant somatic embryos have a number of uses including plant cloning and natural product synthesis.

In the practice of the invention, callus tissue is induced to form somatic embryos by contacting the callus with a medium containing mineral salts, a carbon source and at least one synthetic auxin analog having the general structure of a compound in accordance with the formula:

R₂

/

/

R₄

wherein X is o, s, Se, NH or CH₂;

Rl is a carboxylic acid side chain; and R2, R₃ and R4 are each independently H, Cl, Br, I, F, N0₂, Cι_ιo straight chain alkyl or O-Ci-io straight chain alkyl; with the proviso that when X is 0, Ri is CH2-COOH, and R₃ is Cl, then:

R2 cannot be Cl unless R4 is not H or Cl; or R2 cannot be H or CH3 unless R4 is not H. Such compounds will generally have an even number of carbon atoms in the portion of the Ri side chain extending between the phenoxy oxygen and the carboxyl carbon. Such compounds are generally recognized as displaying greater activity than the compounds having odd-ordered alkyl chains. Fawcett, CH. et ___)_. , "The Degradation of Certain Phenoxy Acids, Amides, and Nitrites Within Plant Tissues," in The Chemistry and Mode of Action of Plant Growth Substances. R.L. Wain and F. Wightman, eds. , Butterworths, London, pp. 187-194 (1965) .

Throughout the disclosure of the present invention, reference will be made to selected synthetic auxin analogs depicted in Figure 4 by including the reference numeral which accompanies the structural representation. Typically, representative synthetic auxin analogs employed in the practice of the invention comprise a central phenoxy ring moiety, a C₃---1₀ straight chain carboxylic acid substituted at the phenoxy group side chain and a halo, methyl, nitro, acetyl or higher order side chain independently substituted at the 2-, 4- or 5- positions phenoxy ring. Such compounds are generally in accordance with the formula:

R₂

/

R₄

wherein X is O, S, Se, NH or CH₂;

R_la is -(CH2)_n~^C00H wherein n is zero or any odd positive whole number; and R , R3 and R4 are as previously defined; with the proviso that when X is 0, n is 1, and R₃ is Cl, then:

R cannot be Cl unless R4 is not H or Cl; or R cannot be H or CH₃ unless R₄ is not H. Alternatively, synthetic auxin analog compounds with the general structure of a phenoxy ring and a branched chain carboxylic acid substituted to the phenoxy oxygen, may be used. These synthetic auxin analogs are generally in accordance with the formula:

*2

/

R₄

wherein

X is 0, S, Se, NH or CH₂;

R_lb is -CH-(CH₂) -COOH

I (CH2)m~CH₃ where m is zero or any positive whole number and p is zero or any even positive whole number; and

R2, R₃ and R4 are as previously defined.

Following incubation in the induction medium, the induced tissue is regenerated and germinated under conditions sufficient to cause root and shoot formation.

Numerous important crop and horticultural species, including alfalfa, celery and carrot, have been shown to be capable of propagation through tissue culture and somatic embryogenesis. For a recent list of such species, see Evans, D.A. et al. , "Growth and Behavior of Cell Cultures: Embryogenesis and Organogenesis," in Plant Tissue Culture: Methods and Applications in

Agriculture. Thorpe, ed. , Academic Press, pg. 45 et seq. (1981) . This list, however, is by no means exhaustive, and the practice of the present invention will be found to be beneficial in the propagation of species which are subsequently shown to be capable of undergoing somatic embryogenesis.

In one embodiment of the present invention, sterilized plant tissue source material, e.g., alfalfa petioles, are plated on a maintenance medium containing mineral salts, a carbon source, a plant growth regulator and at least one auxin.

The mineral salts used in the media of the invention are well-known materials in the art, and are comprised of macroelements and microelements. The mineral salt macroelements and microelements used in the induction medium are well known materials generally selected from the following compounds: ammonium sulfate, potassium nitrate, monopotassium phosphate, magnesium sulfate heptahydrate, manganese sulfate dihydrate, zinc sulfate heptahydrate, boric acid, potassium iodine, calcium chloride dihydrate, ferrous sulfate heptahydrate, ethylenediamine tetraacetic acid (disodium salt) . Other combinations of mineral salts may also be used. Representative culture media mineral salts include, but are not limited to: Schenk- Hildebrandt salts (SH) , Schenk, R.U. and A.C Hildebrandt, Can. J. Bot. .50:199-204 (1972), or Murashige-Skoog salts (MS), Murashige, T. and F.K. Skoog, Physiol. Plant. 15:473-497 (1962). For example, SH mineral salts comprise (in milligrams per liter) : potassium nitrate (2500) , calcium chloride dihydrate (200) , magnesium sulfate heptahydrate (400) , ammonium dihydrogen phosphate (300) , potassium iodide (1.0), boric acid (5.0), manganese sulfate monohydrate (10) , zinc sulfate heptahydrate (1.0), sodium olybdate dihydrate (0.1), cupric sulfate pentahydrate (0.2), cobalt chloride hexahydrate (0.1), ferrous sulfate heptahydrate (15) , disodium ethylenediamine tetraacetic acid (20) . As another example, MS mineral salts comprise (in milligrams per liter) : ammonium nitrate (1650) , potassium nitrate (1900) , calcium chloride dihydrate (440) , magnesium sulfate heptahydrate (370) , cupric sulfate pentahydrate (0.025) , manganese sulfate monohydrate (16.9), zinc sulfate heptahydrate (8.6), potassium phosphate (170) , boric acid (6.2), potassium iodine (0.83), sodium molybdate dihydrate (0.25), cobalt chloride hexahydrate (0.025), disodium ethylenediamine tetra^*a-c*etic acid (37.3), ferrous sulfate heptahydrate (27.8).

Vitamins are also generally included in the medium. Typical vitamins, such as inositol, nicotinic acid, pyridoxine hydrochloride, and thiamine hydrochloride, among others, are included in plant tissue culture medium, in accordance with known techniques.

A carbon source, generally consisting of readily metabolizable carbohydrate, is also included in the medium. The most commonly used carbon source is the disaccharide sucrose. Other saccharides can also be employed in plant tissue culture medium, however, with varying results.

An optional plant growth regulator such as a cytokinin is often included at a concentration sufficient to stimulate the growth of the plant tissue. Typically, kinetin (Sigma Chemical Co. ; Hoechst) can be employed as a plant growth regulator and will generally be used at a concentration of approximately 0.1 to 20μM, more usually at an optimum range of from approximately 5 to 15μM.

The auxin employed in the maintenance media used in the practice of the invention is preferably a known auxin such as α-naphthaleneacetic acid, at a concentration of approximately 5 to 50^M, with an optimum range of from approximately 15 to 30μM. Other auxins such as 2,4-D, DICAMBA (2,6-dichloro-o-anisic acid) , or IAA (indole-3-acetic acid) may also be used. Callus which is formed from the plant tissue source material is separated from the remaining uncallused tissue and repeatedly subcultured on a maintenance medium until a desired mass of tissue is formed. The callus tissue is then transferred to an induction medium containing mineral salts, a carbon source, optionally a plant growth regulator (as described above) , and one or more synthetic auxin analogs.

The carbon source employed in the induction medium of the invention is usually a carbohydrate source, desirably at least one carbohydrate, preferably sucrose, in the concentration of approximately 3g per 100ml media.

Routinely, the induction medium is solidified by the addition of a gelling agent, such as agar or the proprietary compound Gelrite™ (Kelco Commercial Development) .

The synthetic auxin analogs utilized in the practice of the present invention include those compounds of formulas XIX, XlXa and XlXb, disclosed above. Conveniently, the preferred synthetic auxin analogs, having the general structure of a compound in accordance with the formula:

*2

R₄

wherein X is O, S, Se, NH or CH₂;

Rl is a carboxylic acid side chain having zero or an odd number of carbon atoms between X and the carboxyl carbon; and R₂, R3 and R4 are each independently H, Cl, Br, I,

F, N0₂, Cι_ιo straight chain alkyl or -C^-IQ straight chain alkyl, with regard for the proviso stated above; can be subdivided into two classes. In the first sub-class, the preferred analogs are generally in accordance with the formula:

*2

/

R₄

wherein

X is 0, S, Se, NH or CH₂;

Ri_a is -(CH₂)_n-COOH where n is zero or any odd positive whole number; and

R2, R₃ and R4 are as previously defined; with the proviso that when X is 0, n is 1, and R₃ is Cl, then:

R₂ cannot be Cl unless R₄ is not H or Cl; or R₂ cannot be H or CH₃ unless R₄ is not H.

Such analog compounds will generally comprise a central phenoxy ring moiety, a C₃.--₁₀ straight chain carboxylic acid substituted at the phenoxy group side chain and a halo, methyl, acetyl or higher order side chain independently substituted at the 2-, 4- or 5-positions phenoxy ring.

The second sub-class of presently preferred synthetic auxin analogs are generally in accordance with the formula:

R2

R4

wherein

X is O, S, Se, NH or CH₂; Ri is -CH-(CH₂)_p-COOH

I ( ^H2)m~^CH ₃ where m is zero or any positive whole number and p is zero or any even positive whole number; and R₂, R₃ and R4 are as previously defined.

These analog compounds differ from the analogs in the first sub-class in that they possess the general structure of a phenoxy ring and a branched chain carboxylic acid substituted to the phenoxy oxygen. These synthetic auxin analogs will be capable of being resolved into optical isomers, due to the asymmetry at the phenoxy-bound carbon. As will be seen, desirably the (+) stereoisomer will be employed as the auxin analog, either in substantially pure form or as a major component of a mixture with the (-) isomer.

Representative synthetic auxin analogs of the invention include: racemic 2- 2,4-dichlorophenoxy)propanoic acid (XXI); (+) 2- 2,4-dichorophenoxy)propanoic acid (XXII); racemic 2- 2-methyl-4-chlorophenoxy)propanoic acid (XXIV); racemic 2- 2,4,5-trichlorophenoxy)propanoic acid (XXVI); (+) 2- 2,4,5-trichlorophenoxy)propanoic acid (XXVII) ;

(-) 2- 2,4,5-trichlorophenoxy)propanoic acid (XXVIII); racemic 2- 4-chlorophenoxy)propanoic acid (XXIX); (+) 2- 4-chlorophenoxy) ropanoic acid (XXX) ; racemic 2- 2-methyl-4-chlorophenoxy)butanoic acid (XXXII) ; (+) 2- 2-methyl-4-chlorophenoxy)butanoic acid (XXXIII) and 4- 2,4-dichlorophenoxy)butanoic acid (XXXV).

Numerous synthetic auxin analogs in accordance with the present invention can be obtained from commercial and other sources. For example: racemic 2-(2,4-dichlorophenoxy) ropanoic acid (XXI), racemic 2-(2,4,5-trichlorophenoxy)propanoic acid (XXVI) and racemic 2-(4-chlorophenoxy)propanoic acid (XXIX) are available from Sigma Chemical Co., St. Louis, MO;

4-(2,4-dichlorophenoxy)butanoic acid (XXXV) is available from Chem Service, Inc., P.O. Box 194, West Chester, PA;

(+) 2-(2,4-dichorophenoxy)propanoic acid (XXII) is available from K.V. Thimann, Thimann Laboratories, University of California, Santa Cruz, Santa Cruz, CA; racemic 2-(2-methyl-4-chlorophenoxy)propanoic acid (XXIV) , (+) 2-(2-methyl-4-chlorophenoxy)propanoic acid; (-) 2-(2-methyl-4-chlorophenoxy)propanoic acid, racemic 2-(2-methyl-4-chlorophenoxy)butanoic acid (XXXII);

(+) 2-(2-methyl-4-chlorophenoxy)butanoic acid (XXXIII) , (+) 2-(2,4,5-trichlorophenoxy)propanoic acid (XXVII) and (-) 2-(2,4,5-trichlorophenoxy)propanoic acid (XXVIII) are available from R.L. Wain, Crown Point, Scolton Street, Kent, TN255BZ England and

(+) 2-(4-chlorophenoxy)propanoic acid (XXX) is available from V. Tortorella, Dept. Farmico-Chemico, Universita delgi Studi de Bari, Bari, Italy. Alternatively, analog compounds within the scope of the invention can be produced in accordance with general principles of chemical synthesis well known in the art. For example, numerous phenoxy propanoates can be synthesized in accordance with the teachings of

Smith, M.S. et al. , "Studies on Plant Growth-Regulating Substances. V. Stearic Factors in Relation to Mode of Action of Certain Aryloxyalkyl-Carboxylic Acids," Ann. Appl. Biol. 19.(3) :295-307 (1952); Matell, M. and A. Fredga, "Studies on Synthetic Growth Substances. III.

The Steric Relations of the Optically Active α-Phenoxy- Propionic Acid," Arkiv Kemi 4.(20) :325-330 (1951); Matell, M. , "Stereochemical Studies on Plant Growth Regulators. I. Optically Active α-(3,4- Dichlorophenoxy)-Propionic Acid and α-(2,4- dichlorophenoxy)-Propionic Acid," Arkiv Kemi 4.(37) :473- 478 (1952); and Witiak, D.T. et al.. "Hypocholesterolemic Agents. Compounds Related to Ethyl α.-(4-Chlolophenoxy)-α-Methylpropionate," J. Medicinal Chem. 11:1086-1089 (1968).

In general, the synthesis of phenoxy ring moiety synthetic auxin analogs of the present invention will be effected by preparing the sodium phenoxide of the appropriate substituted phenol. The carboxylate side chain will be added to the sodium phenoxide as the ethyl-bromo carboxylic acid. From this intermediate, the acid or the salt of the acid will be readily prepared.

For those synthetic auxin analogs of the present invention having optical isomers, preparation in accordance with the disclosed protocols will generally provide the racemic mixture. The selected stereoisomer can generally be resolved, or partly purified, from the racemic mixture by recrystallization of the phenoxy acid with an optically-active base such as quinine, brucine, strychnine or yohimbine. As described in the above- cited references, the (+)-d acid/(-)-l base salt will ordinarily have a different solubility than the corresponding (-)-d acid/(-)-l base salt, providing a means of separation by recrystallization.

It will also be readily understood that other means known in the art will be found useful to separate desired stereoisomers from racemic mixtures, such as chiral column affinity purification as described in Wainer, I.W. and T.O. Doyle, "Stereoisomeric

Separations," Liquid Chromotography 2. (February 1984) or in Regis Lab Notes, Regis Chemical Co., Morton Grove, IL, pp. 6-7 (January 1984) . Alternatively, the desired stereoisomer can be synthesized directly by stereo- directed synthesis in accordance with principles generally known in the art.

These synthetic auxin analogs are found to be effective in inducing somatic embryogenesis. The synthetic auxin analogs are generally present in the medium at concentrations ranging from approximately 0.1 to SOOμM, more usually from approximately 0.5 to 300μM. It should be understood that other phenoxy-containing compounds, such as those containing a hexanoic acid, an octanoic acid, or other higher homologs, when substituted on the phenoxy oxygen, will also be effective in inducing somatic embryo formation in plants.

Cells are induced to form embryos by incubation in the induction medium of the invention for approximately four days after which time cells are transferred to a regeneration medium.

The regeneration medium employed in the invention is also a medium well-known in the art, consisting of mineral salts and other components, as described above, but generally without auxins. Alternatively, auxins may be present at concentrations much lower than for the induction medium.

Further, the regeneration medium may contain other beneficial substances desirable for plant cell growth and development, such as ammonium. In addition to ammonium, or as an alternative, one or more amino acids can also be advantageously included.

It is known that amino acids are grouped generally in accordance with certain characteristics of particular subclasses. Amino acid residues can be generally subclassified into four major subclasses as follows:

Acidic - i.e., the residue has a negative charge due to loss of H ion at physiological pH;

Basic - i.e., the residue has a positive charge due to association with H ion at physiological pH;

Neutral/non-polar, i.e., the residues are not charged at physiological pH and the residue is repelled by aqueous solution; and

Neutral/polar, i.e., the residues are not charged at physiological pH and the residue is attracted by aqueous solution .

It is understood, of course, that in a statistical collection of individual residue molecules some molecules will be charged, and some not. To fit the definition of charged, a significant percentage (at least approximately 25%) of the individual molecules are charged at physiological pH.

For the naturally occurring protein amino acids, subclassification according to the foregoing scheme is as follows:

Acidic: Aspartic acid and Glutamic acid;

Basic: Arginine, Histidine and Lysine;

Neutral/polar: Glycine, Serine, Cysteine,

Threonine, Asparagine, Glutamine, Tyrosine; Neutral/non-polar: Alanine, Valine, Isoleucine, Leucine, Methionine, Phenylalanine, Proline and Tryptophan. In the present application, the L-form of any amino acid residue having an optical isomer is intended unless otherwise expressly indicated.

In certain presently preferred embodiments of the present invention, the regeneration medium can contain an addition of one or more neutral/non-polar amino acids. Of such amino acids, particularly preferred are L-proline and L-alanine. Other amino acids which may prove beneficial for regeneration include arginine, glutamine, asparagine, serine, lysine, glycine, glutamic acid and aspartic acid. Plant cells are incubated for approximately three weeks in this medium after which the formed embryos are usually aseptically transferred to a solidified conversion medium consisting of half-strength mineral salts, a carbon source such as carbohydrate, preferably maltose at approximately 1.5% (w/v) , and with reduced concentration or no auxins. Somatic embryo quality is observed after approximately 30 days, by counting the percent of embryos which ultimately form plants with roots and shoots. In an alternative embodiment of the present invention, germinated seedlings from a plant capable of undergoing somatic embryogenesis, such as celery, are sterilized and cotyledons, hypocotyls, petioles or leaf discs are removed therefrom. Explants are placed in an induction medium containing mineral salts and a carbon source, as described above, a plant growth regulator, such as kinetin, in a concentration ranging from approximately 0.1 to l.OμM, more preferably 0.3 to 0.7_/..M, and one or more of the synthetic auxin analogs deεcribed above, in concentrations ranging from approximately 0.5μM to 3.0ιM.

After induction, callus tissue is transferred to a regeneration medium lacking in auxins but containing mineral salts, as previously described, and carbohydrate, preferably a disaccharide such as sucrose, in a concentration of approximately 3g per 100ml media. This medium may also contain other beneficial constituents such as but not limited to NH+ and amino acids such as L-alanine.

In still another embodiment of the present invention, cotyledons, hypocotyl or leaf disc explants from germinated seeds of a plant able to undergo somatic embryogenesis, such as lettuce, are transferred to a solidified induction medium containing mineral salts and a plant growth regulator, as described above, carbohydrate, preferably a disaccharide such as sucrose, in a concentration of approximately 3g per 100ml media, and synthetic auxin analog. The synthetic auxin analog used can be any one of the synthetic auxin analogs described above, including 4- chlorophenoxyacetic acid (pCPA; XXXV) . After growth and subculture at approximately three week intervals, callus is transferred to a solidified regeneration medium containing mineral salts and carbohydrate, such as the disaccharide maltose in the approximate concentration of 3g per 100ml. The regeneration medium may also contain NH4⁺ or any of the other beneficial components described previously. Appearance of embryos is determined by microscopic determination after approximately three weeks. Experimental

The following examples serve to illustrate certain preferred embodiments and aspects of "the present invention and are not to be construed as limiting the scope thereof.

In the experimental disclosure which follows, all weights are given in grams (g) or -milligrams (mg) , all concentrations are given as millimolar (mM) or micromolar (μM) and all volumes are given in liters (L) or milliliters (mL) unless otherwise indicated.

Example 1.

The synthetic auxin analog 2-(2,4,5- trichlorophenoxy) propanoic acid (2,4,5-TP) [XXVI] will ordinarily be prepared by first reacting 0.2 gram atoms of sodium with excess ethanol and adding 0.2 moles of 2,4,5-trichlorophenol.

Upon warming, a clear solution will be obtained and 0.2 moles of ethyl α-bromopropanoate will be added. The mixture will then be boiled under reflux for ten hours, 200mL of 10% NaOH will be added and the heating will be continued for one hour "to hydrolyze the ester. Thereafter, the alcohol will be distilled off and the acid will be precipitated with excess ice cold hydrochloric acid. The yield at this point will be approximately 89%. The 2,4,5-TP can then be recrystallized twice from 25% ethanol to give white needles with a melting point of 179-180.5^βC

Example 2.

The racemic 2-(2,4,5-trichlorophenoxy)propanoic acid [XXVI] produced in accordance with Example 1 can be resolved into its stereoisomer≤ in two stages. First, the salt of the (-)-acid isomer [XXVXII] can be formed with (-)-strychnine and second, the salt of the (+)-acid isomer [XXVII] can be formed with (+)-yohimbine. In each case, one equivalent of the racemic acid will be treated with 1/2 equivalent of the optically active base and 1/2 equivalent of sodium hydroxide. To prepare the salt of the (-)-acid stereoisomer [XXVIII], 16.7g of finely powdered strychnine will be suspended in 2L boiling 20% ethanol. To the vigorously stirred suspension, 27g of racemic 2,4,5,-TP, dissolved in 50mL IN NaOH and 50mL ethanol, will be added. The mixture will then be brought to boiling with continuous stirring.

The resultant, nearly clear solution will be filtered and the salt separated upon cooling. The yield at this point will be approximately 27g. This product can be recrystallized five times from 20% ethanol to yield approximately 8g of the (-)-acid salt.

To prepare the salt of the (+)-acid stereoisomer [XXVII], 19.5g of yohimbine hydrochloride will be dissolved in IL boiling water. The base will be precipitated with excess ammonium hydroxide, filtered and washed with hot water. The moist base will then be suspended in 2L 25% ethanol and a mixture of 27g of 2,4,5-TP in 50mL IN NaOH and 50mL ethanol will be added to the suspension with vigorous stirring. The salt will begin to crystallize from the completely clear solution as a mass of very fine needles. The yield at this point will be approximately 28g. Recrystallization three times from acetone will yield approximately 9.4g of the (+)-acid salt.

Example 3.

In a similar protocol to the synthesis described in Example 1, racemic 2-(4-chlorophenoxy)propanic acid [XXIX] will be synthesized by refluxing equal molar parts of sodium 4-chlorophenolate and ethyl α- bromopropanoate in ethyl methyl ketone. Then, 94g (0.7 mole) of 4-chlorophenol and 122.5g (0.7 mole) of ethyl 2-bromopropanoate will be refluxed for 48 hours with 276.4g of K₂C0₃ in 500mL of MeCOEt. The reaction mixture will be cooled and poured into

500mL of H₂0. The ketone layer will be separated and the H₂0 layer further extracted with Et₂0, dried over Na Sθ₄, and filtered. The combined ether and ketone layers will be concentrated under reduced pressure to produce 150g (89%) of ethyl α-(4-chlorophenoxy) propanoate, having a boiling point of 155-158°C

Then, DL-2-(4-chlorophenoxy)propanoic acid is prepared from 150g (0.65 mole) of ethyl α-(4- chlorophenoxy) ropanoate by refluxing in 10% aqueous NaOH with stirring for two hours, producing 121.9g (66%) of the DL acid. Recrystallization from petroleum ether (60-80°) produces white crystals with a melting point of approximately 113-114°C

Example 4.

The racemic mixture of DL-2-(4-chlorophenoxy) propanoic acid [XXIX] produced in accordance with Example 3 can be resolved into its stereoisomers generally in accordance with the protocol outlined in Example 2.

For example, L-(-)-2-(4-chlorophenoxy)propanoic acid [XXXI] is prepared by suspending 16.7g (0.042 mole) finely powdered (-)-brucine in 2L of boiling 20% EtOH in H₂0. Then, 20g (0.1 mole) of DL-2-(chlorophenoxy) propanoic acid in 50mL of IN NaOH and 50mL of EtOH will be added to the stirred suspension. The boiling mixture is stirred until a clear solution results. The solution is then filtered and the salt will crystallize on cooling. Five additional recrystallizations from 20% EtOH-H2θ will produce approximately 10.6g (44%) of brucine salt ([α]25 D -25.4 degrees).

Utilizing twelve times the above amount, 122g of optically pure (-)-acid salt will be obtained. The acid is liberated from 122g of salt by acidification with 5% H₂S0₄, and extraction with ether. The ether is dried over NaSθ₄, filtered and removed under reduced pressure. After recrystallization from petroleum ether (60-80°C), 28.8g (70%) of L-(-)-2-(chlorophenoxy) ropanoic acid [XXXI] will be obtained, having a melting point of approximately 104-105"C

Similarly, D-(+)-2-(4-chlorophenoxy)propanoic acid [XXX] will be obtained from DL-2-(chlorophenoxy) propanoic acid by following the protocol of Example 2 for resolving similar compounds.

The resolution will be accomplished by utilizing (+)-yohimbine obtained from the HCl salt. Three recrystallizations of the yohimbine salt of D-(+)-2- (chlorophenoxy)propanoic acid from Me₂CO will afford white crystals ([α]+64.8 degrees). The D-(+) acid [XXX] is liberated from the salt as in L-(-)-2-(chlorophenoxy) propanoic acid, above. Recrystallization from petroleum ether (60-80°C) will afford white crystals, having a melting point of approximately 104-105°C Following the protocols outlined in Examples 1 through 4 and the preceeding disclosure, with appropriate adjustments being made to produce the desired end product, one will be able to obtain the synthetic auxin analogs of the present invention.

Example 5. Alfalfa Embryogenesis

Embryogenesis can be routinely induced in alfalfa in the Regen S line (Saunders, J.W. and E.T. Bingha , "Production of Alfalfa Plants- from Callus Tissue," Crop Sci. 12.:804-808 (1972)). Plants of Medicago sativa cultivar Regen S, derived from the second cycle recurrent selection for regeneration from a cross of the varieties Vernal and Saranac, and identified as RA-3 were used. Callus formation was initiated by surface sterilizing petioles with 50% Clorox^® in water for five minutes, washing with water and plating on Schenk-Hildebrandt (SH) medium containing 25μM α-naphthaleneacetic acid, lOμM kinetin, 3% (w/v) sucrose and 0.8% washed agar (termed maintenance medium) . Callus which formed on the explant tissue was separated from the remaining uncallused tissue and repeatedly subcultured at three week intervals on maintenance medium under indirect light at 27°C Callus was collected at 18 to 24 days after subculture.

Somatic embryogenesis induction was performed by transferring the callus to 2,4-D-containing induction medium. One gram of callus was transferred to 30mL of agar-solidified induction medium in 100 x 15mm petri plates. The induction medium contained SH salts, 3% sucrose, an auxin source, as specified in each case, and kinetin. Cells were induced to form embryos by incubation on this medium for three or four days at 27°C in low light. Incubation on induction medium gave rise to callus which regenerated somatic embryos by the following method.

Induced cells were aseptically smashed on a petri dish using a stainless steel spatula. Seventy-five mg of cells were plated and spread onto approximately 30mL of agar-solidified medium in 60 x 15mm petri dishes. Regeneration medium in all cases was on SH medium containing 7.4mM to 9.4mM ammonium and 30mM L-proline. Each treatment was generally plated in five to ten replicates. Dishes were Parafilm^®-wrapped and incubated for 21 days at 27°C under 16 hour illumination from cool from cool white fluorescent tubes positioned at 28cm from the cultures.

Embryogenesis was visually measured after incubation by counting green centers of organization on the callus using a stereomicroscope at 10X magnification. After counting embryos, a population of embryos was aseptically transferred to a conversion medium solidified with 0.8% agar in 100 x 25mm petri plates and consisting of half strength SH medium (containing no hormones) and 1.5% (w/v) maltose. These embryos were incubated for 30 days under the conditions specified above. At the end of incubation, embryos were scored for the appearance of roots and shoots on the same embryo. This assay is the physiological equivalent to germination of true seed and is an objective measure of somatic embryo quality. Embryo quality can also be observed and quantified visually. Stuart, D.A. et al. , "Factors Affecting Developmental Processes in Alfalfa Cell Cultures," in Tissue Culture in Forestry and Agriculture. R.R. Henke et al- eds., Plenum Publishing Corp., pp. 59-73 (1985).

The growth regulator, racemic 2-(2,4-di¬ chlorophenoxy)propanoic acid (2,4-DP) [XXI], was tested over the range of 0.1 to 500μM for its effect on the number and quality of somatic embryos induced. These treatments were compared to an induction treatment containing 50μM 2,4-D [XX] which has been reported to be optimal for alfalfa somatic embryo culture. Walker, K.A. et al. , "Organogenesis in Callus Tissue of Medicago sativa. The Temporal Separation of Induction Processes from Differentiation Processes," Plant Sci. Lett. 16.:23-30 (1979). Cultures treated with 2,4-DP produced embryos that had higher visually-determined quality than the 2,4-D-treated embryos. In comparing embryos from the 50μM 2,4-D and 10 to 500μM 2,4-DP treatments, embryo yield was slightly lower with 2,4-DP but embryo quality, as measured by conversion to plants, was improved compared to 2,4-D 5 (See Table 1) . This result is an improvement over the obtained using 2,4-D which could not have been predicted from earlier plant culture studies comparing the effects of 2,4-D and 2,4-DP on plant cell growth.

Table 1

Effect of Racemic 2,4-DP [XXI] and 2,4-D [XX] in Induction Media on Alfalfa Somatic Embryo Quantity and Quality.

Auxin used Percent Conversion Embryo Yield in Induction to Plants per Plate

Experiment 1

50μM 2,4-D 22% ± 4 262 ± 19 lOμM 2,4-DP 45% ± 7 184 ± 10

50μM 2,4-DP 49% ± 8 173 ± 10

Experiment 2

50μM 2,4-D 8% ± 5 lOOμM 2,4-DP 25% ± 2

Example 6.

The synthetic auxin analog of Example 5, racemic 2-(2,4-dichlorophenoxy)propanoic acid (2,4-DP) [XXI], is actually composed of two distinct optical isomers: The (+)- or D-form [XXII] and the (-)- or L-form [XXIII]. This results from the asymmetric carbon at the 2 position of the propanoic acid side chain. Hence, the analog tested in Example 5 is actually a racemic mixture composed of approximately 50% (+)-2,4-DP and 50% (-)-2,4-DP.

The substantially pure (+)-form of 2-(2,4-dichlorophenoxy)propanoic acid was tested for its capacity to improve alfalfa somatic embryo quality over a range of 0.1 to 500μM by the methods described in Example 5.

The conversion frequency of alfalfa somatic embryos resulting from exposure to (+) 2,4-DP was compared to 2,4-D treated cultures. See Table 2. In each case (+)-2,4-DP gave higher embryo conversion compared to 2,4-D, also indicating that (+)-2,4-DP improves alfalfa embryo quality. Since (+)-2,4-DP has never been tested on plant cell culture heretofore, this was an unexpected finding.

Table 2

Effect of (+)-2,4-DP [XXII] and 2,4-D [XX] in Induction Media on Alfalfa Somatic Embryo Quality and Quantity.

Auxin used Percent Conversion Embryo Yield in Induction to Plants per Plate

Experiment 1

50μM 2,4-D 23% ± 6 248 ± 8 lOOμM (+)-2,4-DP 43% ± 5 234 ± 16

Experiment 2

50μM 2,4-D 48% ± 5 673 ± 36 lOOμM (+)-2,4-DP 88% ± 4 388 ± 16

Example 7.

The synthetic auxin analog racemic 2-(2,4-dichlorophenoxy)propanoic acid (2,4-DP) [XXI], was tested for its effect on induction of embryogenesis 5 in alfalfa single plant clones unrelated to Regen S. These clones were derived from field selections of alfalfa with superior agronomic characteristics but which displayed lower regeneration than RA-3. Clone 28- 6 is from a selfed cross of a single Dekalb 167 plant,

10 and clones 75-7 and 75-15 are from a cross between two Waterman/Loomis 318 plants.

In comparison to induction on 50μM 2,4-D, the somatic embryo yield and conversion was improved with 2,4-DP in elite alfalfa clones. See Table 3. These

15 improved results with 2,4-DP were unexpected based on the prior art in somatic embryogenesis. Table 3

Somatic Embryo Yield and Conversion from Elite Alfalfa Cultures Treated with 2,4-D [XX] and Racemic 2,4-DP [XXI]

Auxin used Percent Conversion in Induction Embryo Yield to Plants

Elite alfalfa clone 28-6

Experiment 1

50μM 2 , 4-D 25±6 0%±0 lOOμM 2 , 4-DP 36±4 21%±9 Experiment 2

50μM 2 , 4-D 13+1 0%±0 lOOμM 2 , 4-DP 57±22 5%±3

Elite alfalfa clone 75-7

50μM 2,4-D 12±2 17%±12 lOOμM 2,4-DP 57+12 37%±10

Elite alfalfa clone 75-15

50μM 2,4-D 3±1 lOOμM 2,4-DP 25±2 33%+7

Examole 8.

The synthetic auxin analog racemic 2,4-DP [XXI] was tested for its effect on the regeneration of elite alfalfa clone 87-01R, derived from a polycross of 85 5 non-dormant alfalfas selected for high regeneration from tissue culture.

Hypocotyl sections were placed onto standard maintenance medium supplemented with 50μM 2,4-D or with lOOμM 2,4-DP. In this example, tissue was transferred

10 directly to regeneration medium containing no hormones, without exposure to induction medium. Tissue treated with 2,4-DP produced more embryos than either maintenance medium or maintenance medium plus 2,4-D. Hypocotyl sections treated with 2,4-DP formed embryos of

15 higher visually-determined quality and would be expected to germinate at a higher frequency than either 2,4-D or no auxin. See Table 4. The results with 2,4-DP represent an unexpected improvement over 2,4-D treated cultures.

20

Table 4

Somatic Embryo Yield from Elite Alfalfa Cultures Treated with Maintenance Medium (Complete) , Maintenance Medium with 50μM 2,4-D [XX], or with 100μM racemic 2,4-DP [XXI].

Auxin used in Maintenance Medium Embryo Yield

Complete ^' 0.210.1

Complete plus 50μM 2,4-D 0.9±0.4

Complete plus lOOμM 2,4-DP 5.2±2.9 Example 9.

The synthetic auxin analog racemic 2-(2-methyl-4- chlorophenoxy)propanoic acid (Mecoprop) [XXIV] was tested using the methods described in Example 5, in the range of O.lμM to 500μM in alfalfa cell cultures. The effect of Mecoprop on somatic embryo quality was compared to 2-methyl-4-chlorophenoxyacetic acid (pCPA) [XXV] and 2,4-D [XX].

The growth and morphogenic activity of pCPA and 2,4-D have been previously investigated (Kamada, H. and H. Harada, "Studies on the Organogenesis in Carrot Tissue Cultures. I. Effects of Growth Regulators on Somatic Embryogenesis and Root Formation," Z. Pflanzenphysiol. 9JL:255-266 (1979)), with the result that pCPA was found slightly more potent than 2,4-D for embryo induction.

Alfalfa cultures treated with Mecoprop yielded somatic embryos with greater overall morphological quality than embryos from either pCPA- and 2,4-D-treated cultures. This demonstrates that Mecoprop facilitated development of embryos and that these embryos would germinate at a higher frequency than pCPA- or 2,4-D- derived somatic embryos.

The data shown in Table 5 indicates that Mecoprop treated cultures produced embryos with a greater germination frequency than 2,4-D-treated embryos. Mecoprop-treated cultures gave rise to embryos that were longer and larger than 2,4-D-treated cultures. Mecoprop also produced embryos that exhibited greater cotyledon development than those treated with 2,4-D. Since

Mecoprop [XXIV] had not been previously tested for its effect on embryogenesis, this result was unexpected. Table 5

Somatic Embryo Yield and Conversion from Alfalfa Cultures Treated with 2,4-D [XX] and Mecoprop [XXIV].

Auxin used Percent Conversion Embryo Yield in Induction to Plants per Plate

Experiment 1

50μM 2,4-D 8% ± 5 232 ± 15 lOOμM Mecoprop 38% ± 7 199 ± 20

Experiment 2

50μM 2,4-D 23% + 6 248 ± 6 lOOμM Mecoprop 44% ± 5 175 ± 15

Example 10.

The synthetic auxin analog racemic 2-(2,4,5-trichlorophenoxy)propanoic acid (2,4,5-TP) [XXVI] disclosed in Example 1 was tested, by the methods 5 described in Example 5, in the range of O.lμM to 500μM in alfalfa cell cultures. The effect of 2,4,5-TP on somatic embryo quality was assessed visually and compared to cultures treated with 2,4-D [XX].

Alfalfa cultures treated with 2,4,5-TP yielded

10 somatic embryos with greater overall morphological quality than did 2,4-D treated cultures. This indicated that 2,4,5-TP caused improved development of embryos and that these embryos would germinate at a higher frequency than 2,4-D derived embryos. 5 Data shown in Table 6 indicates that 2,4,5-TP treated cultures produced embryos with a greater germination frequency. The 2,4,5-TP-treated cultures produced embryos that were longer and larger than the 2,4-D-treated cultures. The 2,4,5-TP treatment also produced embryos that demonstrated greater cotyledon development of the cotyledon when compared to other treatments. Since 2,4,5-TP had never been previously tested for its effect on somatic embryogenesis, this result could not be predicted from the prior art.

Table 6

Effect of 2,4-D [XX] and 2,4,5-TP [XXVI] on Alfalfa Somatic Embryo Yield and Conversion to Plantlets.

Auxin used Percent Conversion Embryo Yield in Induction to Plants per Plate

Experiment 1

50μM 2,4-D 23% ± 6 129 ± 30 lOμM 2,4,5-TP 41% ± 4 119 ± 20

Experiment 2

50μM 2,4-D 23% ± 6 248 ± 6 lOOμM 2,4,5-TP 41% ± 4 210 ± 8

Example 11.

The (+)- [XXVII] and (-)- [XXVIII] stereoisomers of racemic 2,4,5-TP [XXIV], which can be produced as described in Example 2, were tested, by methods described in Example 5, for their individual effect on alfalfa somatic embryo formation. Table 7 summarizes the yield of somatic embryos obtained from media containing 2,4-D or racemic (+/-), (+) or (-) 2,4,5-TP ranging from 0.5μM to 300μM. The racemic (+/-) 2,4,5-TP mixture and (+)-2,4,5-TP stimulate somatic embryogenesis to the greatest degree while (-)-2,4,5-TP yielded few embryos. The embryos which form in the presence of a range of concentrations of (+)-2,4,5-TP are similar to the embryos formed on (+/-) 2,4,5-TP in Example 10. These embryos are enhanced in quality and germination compared to embryos induced to form in the presence of 2,4-D. Embryos raised from cultures treated with (-)-2,4,5-TP were inferior to those raised from (+/-) 2,4,5-TP- or (+) 2,4,5-TP-treated cultures.

Table 7

Effect Of (+)-2,4,5-TP [XXVII], (-)-2,4,5-TP [XXVIII], (+/-) 2,4,5-TP [XXVI] and 2,4-D [XX] on Somatic Embryo Yield.

Example 12.

The synthetic auxin analog racemic ₂-(4-chlorophenoxy)propanoic acid (pCPPA) [XXIX], was tested, as in Example 5, in the range of concentrations 5 from 0.1 to 500μM for its effect on induction of somatic embryos. This analog induced somatic embryos with improved quality over the range of concentrations tested. Embryos were larger and had bigger cotyledons than 2,4-D-induced embryos.

10 As shown in Table 8, conversion tests were performed on representative treatments. Cultures treated with (+/-) pCPPA produced embryos which converted to plants at a higher rate than embryos from 2,4-D-treated cultures. As (+/-) pCPPA had not

15 previously been tested for its effect on plant cell growth, embryogenesis or embryo quality, these results were unexpected.

Table 8

Effect of (+/-) pCPPA [XXIX] and 2,4-D [XX] on Alfalfa Somatic Embryo Quality.

Auxin used Percent Conversion in Induction to Plants

50μM 2,4-D 39% ± 5 lOOμM (+/-) PCPPA 51% ± 4

Example 13.

The (+)- [XXX] and (-)- [XXXI] stereoisomers of 2-(4-chlorophenoxy)propanoic acid [XXIX], which can be obtained as described in Examples 1 and 2, were tested, as described in Example 5, over the range of concentrations of 0.1 to 500μM for each stereoisomer. Cultures treated with the (+)-pCPPA stereoisomer produced numerous embryos which were of the same visually-determined quality as embryos raised on (+/-) racemic pCPPA. Cultures treated with (-)-pCPPA produced few embryos.

A representative sample of embryos obtained from (+)-pCPPA treatments was compared to a 2,4-D-treated culture. As shown in Table 9, the embryos raised on (+)-pCPPA displayed better conversion to plantlets, indicating that (+)-pCPPA induced higher quality embryos. This result was not expected or anticipated from the prior art, as (+)-pCPPA had never been tested for its effects upon tissue culture growth, induction of somatic embryogenesis or somatic embryo quality.

Table 9

Effect of 2,4-D [XX] and (+)-pCPPA [XXX] on Alfalfa Somatic Embryo Quality.

Auxin used Percent Conversion in Induction to Plants

50μM 2,4-D 39% ± 5 lOOμM (+) pCPPA 51% ± 4 Example 14.

The synthetic auxin analog racemic 2-(2-methyl -4-chlorophenoxy)butanoic acid (Mecobut) [XXXII], was tested at concentrations ranging from 0.1 to 500μM as in Example 5. Mecobut-induced somatic embryos displayed improved visually-determined quality over those raised from optimal exposure to 2,4-D.

Representative samples of 2,4-D- and Mecobut- treated embryos were tested for conversion to plantlets. In alfalfa cultures with 50μM 2,4-D in the induction medium, 23% of the somatic embryos produced converted to plants. Mecobut-treated cultures produced somatic embryos which germinated at a rate of 34%. Thus, Mecobut increased the quality of somatic embryos by two criteria. This result was not expected since Mecobut had not been tested previously for its effect on plant cell culture growth, induction of somatic embryos or its effect on somatic embryo quality.

Example 15.

The (+)- [XXXIII] and (-)- [XXXIV] stereoisomers of 2-(2-methyl-4-chlorophenoxy)butanoic acid (Mecobut) [XXXII] were obtained (R.L. Wain, as described previously) and were tested, by the methods described in Example 5, for their ability to induce high quality alfalfa somatic embryos.

Alfalfa cultures treated with (+) Mecobut in concentrations ranging from 0.1 to 500μM yielded somatic embryos of higher quality, compared to 2,4-D-induced somatic embryos. The (+) Mecobut-induced embryos were similar to those induced from (+/-) racemic Mecobut in Example 14. The (-) Mecobut stereoisomer was also tested, but yielded few embryos, and these embryos were of poorer quality than (+) Mecobut- or (+/-) Mecobut- treated cultures. Representative examples of (+) Mecobut-treated cultures were tested for embryo conversion. The 2,4-D-treated cultures produced embryos which converted to plants at a lower frequency than (+) Mecobut-treated cultures. Thus, by two criteria, (+) Mecobut-treated cultures yielded somatic embryos which were superior in quality to 2,4-D- treated cultures.

Example 16. The effect of 4-(2,4-dichlorophenoxy)butanoic acid

(4-(2,4-DB); Chem Service, Inc.) [XXXV] on somatic embryo quality was tested in concentrations ranging from 0.1 to 500μM on alfalfa cell cultures by the methods described in Example 5. Cultures treated with 4-(2,4-DB) yielded numerous embryos which were visibly superior in morphology to embryos treated with 2,4-D. Somatic embryos from 4-(2,4-DB)-treated cultures were larger and exhibited improved cotyledon development when compared to cultures induced with optimal 2,4-D.

Data regarding somatic embryo conversion for representative 4-(2,4-DB) treatments are shown in Table 10. The 4-(2,4-DB)-treated cultures formed embryos that had much higher quality than 2,4-D-treated cultures as shown by the results of conversion experiments.

This result was particularly surprising since 4-(2,4-DB) is not an active herbicide in alfalfa, although 4-(2,4-DB) will kill competing weed species in fields of alfalfa. The 4-(2,4-DB) is thought to be metabolized in most plants to 2,4-D via the _/9-oxidation pathway. The above results suggest that 4-(2,4-DB) is not oxidized to 2,4-D in alfalfa cell cultures because 4-(2,4-DB) has an improved effect on cell cultures compared to 2,4-D. If ^-oxidation is blocked in alfalfa cell cultures, 4-(2,4-DB) must be an active growth regulator of its own accord. These results could not be predicted from the prior art knowledge of tissue culture or herbicide research.

Table 10

Effect of 4-(2,4-DB) [XXXV] on Alfalfa Somatic Embryo Quality.

Auxin used Percent Conversion in Induction to Plants

Experiment 1

50μM 2,4-D 22% + 4 lOμM 4-(2,4-DB) 45% ± 7

50μM 4-(2,4-DB) 49% ± 8

Experiment 2

50μM 2,4-D 23% + 6 lOOμM 4-(2,4-DB) 52% ± 7

Example 17.

The effects of 3-(2,4-dichlorophenoxy)propanoic acid (3-(2,4-DP) [XXXVI] and 5-(2,4-dichlorophenoxy) pentanoic acid (5-(2,4-DP) [XXXVII] on somatic embryogenesis were also measured. These synthetic auxin analogs were tested in concentrations ranging from 0.1 to 500μM in alfalfa cell cultures.

Growth regulator literature has suggested that these odd side chain lengths should result in no auxin activity. Fawcett, CH. et al- , "The Degradation of

Certain Phenoxy Acids, Amides, and Nitrites Within Plant Tissues," in The Chemistry and Mode of Action of Plant Growth Substances. R.L. Wain and F. Wightman, eds., Butterworths, London, pp. 187-194 (1965). The 3-(2,4-DP) and 5-(2,4-DP) compounds did not induce any somatic embryos in cell cultures of alfalfa. Because 3-(2,4-DP) and 5-(2,4-DP) are inactive and because 4-(2,4-DB) [XXXV] is highly active as an inducer of embryogenesis, higher homologs of 4-(2,4-DB), such as 6-(2,4-dichlorophenoxy)hexanoic acid [XXXVIII] and

8-(2,4-dichlorophenoxy)octanoic acid [XXXIX] are likely to be highly active auxin analogs for the induction of somatic embryogenesis.

Example 18. Celery Embryogenesis

Seeds of celery Apium graveolens var Calmario were germinated for one to two weeks. The resulting seedlings were sterilized with a solution of 10% Clorox® for 20 minutes. Cotyledons or hypocotyls were removed and explants were placed on agar-solidified SH medium containing 2.25μM 2,4-D, 0.5μM kinetin, 3% sucrose and 0.8% agar (termed maintenance medium).

After initiation of callus for 3 to 4 weeks, callus was subcultured on maintenance medium. Heat labile additives were filter sterilized and added to warm medium. When required, specific amounts of tissue for inoculation were obtained using a modified spatula device and filling this to uniform volume. Subsequent subcultures of callus were grown on maintenance medium for 3 weeks.

To test the synthetic auxin analogs, 2,4-D was removed from the final subculture medium prior to regeneration and replaced with the selected auxin analog at the concentration noted in Table 11. For somatic embryo regeneration, lOOmg of induced callus was transferred to lOmL of 0.8% agar solidified medium containing SH salts, 3% sucrose, 2.4mM NH4⁺ and 30mM L-alanine. The cultures were incubated at 27°C for three weeks. Embryo numbers were determined as for alfalfa in Example 5. Embryo quality was determined using a five point rating system: + representing abnormal embryos possessing poor root and shoot development, and +++++ representing normal embryos which resembled a seedling. Celery embryos regenerated immediately after subculture on 2,4-D were abnormal in appearance and showed low yields (Table 11) . All of the synthetic auxin analogs tested for celery, including 2,4-DP [XXI] as a racemic mixture as well as pure (+)2,4-DP [XXII], improved somatic embryo quality compared to 2,4-D [XX]. The best auxin analog tested was (+)2,4-DP, which produced somatic embryos resembling true celery seedlings. Mecoprop [XXIV], racemic 2,4-DP [XXI] and 2,4-DB [XXXV] at concentrations of 2.25μM and lμM all improved the quality of celery somatic embryos, compared to 2,4-D [XX]. Table 11

Effect of 2,4-D, 2,4-DP, 4-(2,4-DB) and Mecoprop on Celery Somatic Embryo Quality and Quanity.

Auxin used Prior Quality of Embryos Yield of Embryos to Regeneration

2.25μM 2,4-D + 0.1 lμM ± 2,4-DP +++ 93

2.25μM ± 2,4-DP ++ 101 lμM + 2,4-DP ++++ 89

2.25μM + 2,4-DP ++ § 53 lμM ± Mecoprop +++ 134

2.25μM ± Mecoprop ++ 43 lμM 4-(2,4-DB) +++ 109

2.25μM 4-(2,4-DB) ++ 51

Example 19. Lettuce Embryogenesis

Seed of Lactuca sativa L. (var. Vanguard 75) were surface sterilized and germinated aseptically on moist filter paper. Cotyledons and hypocotyl explants were' 5 transferred to MS salts plus 0.8% agar, 3% sucrose, 3μM 4-chlorophenoxyacetic acid (pCPA) [XXV] and 10μM kinetin. Callus formation occurred after 2 to 3 weeks on this medium.

Callus was grown at 27°C under low light and 0 subcultured at 3 week intervals. Callus was then transferred to either the above medium or medium in which pCPA was removed and replaced with various levels of racemic 2-(2,4-dichlorophenoxy)propanoic acid [XXI]. Callus was subcultured on each respective medium for 10 5 days prior to plating onto SH salts plus 10mM NH ⁺, 3% maltose and 0.8% agar for lettuce plantlet regeneration. Embryoid morphology was determined by stereomicroscopic examination after 21 days culture on the latter medium. The yield of lettuce embryoids after subculture on pCPA- and racemic 2-2,4-dichlorophenoxy)propanoic acid-containing medium is shown in Table 12. The presence of varying levels of racemic 2-(2,4-dichlorophenoxy)propanoic acid in cultures produced more somatic embryoids than cultures pretreated with 4-chlorophenoxyacetic acid.

Table 12

Effect of pCPA [XXV] and 2,4-DP [XXI] on Lettuce Somatic Embryoid Formation.

Hormone Pretreatment Embryoid Number

3μM pCPA + lOμM kinetin 0

3μM 2,4-DP + lOμM kinetin 8

3μM 2,4-DP + 30μM kinetin 9

3μM 2,4-DP + lOOμM kinetin 11

Thus it can be seen that the present synthetic auxin analogs provide a novel and improved method for producing numerous high quality somatic embryos from plant tissue. Of the embryos produced, a high percentage ultimately germinate and develop into plantlets. Thus, a method of somatic embryogenesis capable of large scale plant production is provided. The lack of correlation between auxin-stimulated plant cell elongation activity and the ability to induce somatic embryo formation in cell cultures demonstrates that the ability of the present auxin analogs to induce somatic embryogenesis in cell culture was not predictable, based on their ability to stimulate cell elongation in whole plants, to stimulate cell elongation in culture or based on their herbicidal properties.

It should be understood that various alternatives to the methods and materials herein disclosed may be employed in practicing the present invention. It is intended that the following claims define the invention, and that the materials and methods within the scope of these claims and their equivalents be covered thereby.

Claims

Claims 1. A method for plant cell somatic embryogenesis comprising:

(a) inducing somatic embryo formation by contacting plant callus tissue capable of undergoing somatic embryogenesis with an induction medium comprising all mineral salts, vitamins and nutrients required to maintain tissue viability, together with at least one synthetic auxin analog selected from the group consisting of compounds having the general formula:

^R2

R₄

wherein

X is O, S, Se, NH or CH₂ ;

Rl is a carboxylic acid side chain; and

R₂, 3 and R4 are each independently H, Cl, Br, I, F, N0₂, Cι_ιo straight chain alkyl or O-Cι_ι₀ straight chain alkyl ; with the proviso that when X is 0, Ri is CH₂-COOH, and R3 is Cl, then:

R₂ cannot be Cl unless R4 is not H or Cl; or R₂ cannot be H or CH3 unless R4 is not H, in an amount sufficient to cause induction of somatic embryo formation;

(b) regenerating the somatic embryos by contacting said induced tissue with a regeneration medium comprising all mineral salts, vitamins and° nutrients required to maintain tissue viability, for such time and under conditions sufficient to cause formation of embryos from said induced tissue.

2. The method of Claim 1 wherein the regeneration medium further comprises at least one amino acid selected from the group consisting of neutral/nonpolar amino acids.

3. The method of claim 2 wherein the amino acid is at least one of L-proline or L-alanine.

4. The method of claim 1 wherein Ri is a carboxylic acid side chain selected from the group consisting of Rι_a and Rib wherein

^Rla is -(CH₂)_n-COOH and n is zero or any odd positive whole number; and

R_lb is -CH-(CH₂)_p-COOH I

(CH₂)_m-CH₃ and m is zero or any positive whole number and p is zero or any even positive whole number.

5. The method of Claim 4 wherein the synthetic auxin is selected from the group consisting of racemic 2-(2,4-dichlorophenoxy)propanoic acid, (+) 2-(2,4-dichorophenoxy)propanoic acid, racemic 2-(2-methyl-4-chlorophenoxy)propanoic acid, racemic 2-(2,4,5-trichlorophenoxy)propanoic acid, (+) 2-(2,4,5-trichlorophenoxy)propanoic acid, racemic 2-(4-chlorophenoxy)propanoic acid, (+) 2-(4-chlorophenoxy)propanoic acid, racemic 2-(2-methyl-4-chlorophenoxy)butanoic acid, (+) 2-(2-methyl-4-chlorophenoxy)butanoic acid, and 4-(2, -dichlorophenoxy)butanoic acid.

6. The method of Claim 1 wherein the induction medium further comprises at least one cytokinin in an amount sufficient to stimulate growth of the plant tissue.

7. The method of Claim 6 wherein the cytokinin is kinetin at a concentration in the medium of from approximately 0.2 to 20μM.

8. The method of Claim 1 wherein any one of the induction medium and the regeneration medium further comprises at least one carbohydrate source in an amount sufficient to stimulate growth of the plant tissue.

9. The method of Claim 8 wherein the carbohydrate source is at least one dissaccharide compound.

10. In a method for forming somatic embryos from plant tissue wherein callus tissue is induced to form somatic embryos and somatic embryos are regenerated from the induced callus, the improvement comprising inducing the callus tissue with an induction medium comprising all mineral salts, vitamins and nutrients required to maintain tissue viability, together with at least one synthetic auxin analog selected from the group consisting of compounds having the general formula:

^R2

R₄

wherein

X is O, S, Se, NH or CH₂; R_l is a carboxylic acid side chain; and R₂, R₃ and R4 are each independently H, Cl, Br, I, F, N0₂, Cι_ιo straight chain alkyl or θ-Cι_ι₀ straight chain alkyl; with the proviso that when X is O, i is CH₂-COOH, and R₃ is Cl, then:

R₂ cannot be Cl unless R4 is not H or Cl; or R₂ cannot be H or CH₃ unless R4 is not H, in an amount sufficient to cause induction of somatic embryo formation.

11. The method of claim 10 wherein Ri is a carboxylic acid side chain selected from the group consisting of Rι_a and Ri wherein

R_ia is -(CH₂)_n-COOH and n is zero or any odd positive whole number; and

R_lb is -CH-(CH₂)_p-COOH

I (CH₂)_m-CH₃ and m is zero or any positive whole number and p is zero or any even positive whole number.

12. The method of Claim 11 wherein the synthetic auxin is selected from the group consisting of racemic 2-(2,4-dichlorophenoxy)propanoic acid, (+) 2-(2,4-dichorophenoxy)propanoic acid, racemic 2-(2-methyl-4-chlorophenoxy)propanoic acid, racemic 2-(2,4,5-trichlorophenoxy)propanoic acid, (+) 2-(2,4,5-trichlorophenoxy)propanoic acid, racemic 2-(4-chlorophenoxy)propanoic acid, (+) 2-(4-chlorophenoxy)propanoic acid, racemic 2-(2-methyl-4-chlorophenoxy)butanoic acid, (+) 2-(2-methyl-4-chlorophenoxy)butanoic acid, and 4-(2,4-dichlorophenoxy)butanoic acid.

13. The method of Claim 10 wherein the induction medium further comprises at least one cytokinin in an amount sufficient to stimulate growth of the plant tissue.

14. The method of Claim 13 wherein the cytokinin is kinetin at a concentration in the medium of from approximately 0.2 to 20μM.

15. The method of Claim 10 wherein any one of the induction medium and the regeneration medium further comprises at least one carbohydrate source in an amount sufficient to stimulate growth of the plant tissue.

16. The method of Claim 15 wherein the carbohydrate source is at least one dissaccharide compound.

17. The method of claim 10 wherein the induction medium further comprises at least one carbohydrate selected from the group consisting of disaccharide compounds.