CA1297823C - Somatic embryogenesis employing organic acids - Google Patents

Abstract

Abstract Improved Somatic Embryogenesis Employing Organic Acids Disclosed is an improved somatic embryogenesis method and medium for subculturing cultured plant cells which contains selected organic acids in a balanced salt solution containing the nutritional and growth factors necessary for plant growth.

Description

8~3 Descri~tion Improved Somatic Embryogenesis Employing Or~anic Acids Technical Field The present discovery relates generally to somatic embryogenesis in plant cells and, more particularly, to methods and materials for increasing the quantity and quality of plant embryos produced from somatic cell cultures.
Background of the Invention The process of plant cell cloning can be accomplished by a variety of methods, one of which is somatic embryogenesis. This method offers advantages of scale and efficiency not found in other methods of cell culture since, in one step, somatic embryos develop both a root and shoot. This method of cloning also provides an opportunity for commercial scale production of naked or encapsulated somatic embryos. These embryos can be germinated and provide a substitute for true plant seed.
Evans, D.A. et al., "Growth and Behavior of cell cultures: Embryogenesis and organogenesis in Plant Tissue Culture," T . A . Thorpe, e.d., Academic Press, New York, pp. 45-113 (1981).
When practicing cloning by somatic embryogenesis, one must be mindful of the genotypic background of the explant source as well as the physiological conditions of culture. By understanding in detail the physiological conditions of regeneration, it is generally believed that the ~enetic background of the plant material will become a less important factor. For example, attempts to regenerate soybean through somatic embryogenesis for several decades were not successful.
Recently, a defined set of conditions have been described for soybean regeneration. Christianson, M.L.

~L2~ 3Z3 et al~., "A morphogenetically competent soybean suspension cul~uxe," Science 222:632 (1983). Thus, a genotype previously impossible to manipulate in vitro (the cultivated soybean) can now be regenerated as a result of a breakthrough in cell culture conditions.
There are two major drawbacks to using somatic embryos as a commercial scale cloning methodO First, in many crops the yield or number of somatic embryos produced in plant cell culture is low. Improvements in the efficiency of embryo yield is always a concern in production systems. Techniques for somatic embryogenesis in carrot have been sufficiently developed so that 90% of the cell culture produces somatic embryos. Fujimura, T. and A Komamine, "Synchronization of Somatic Embryogenesis in a Carrot Cell Suspension Culture," Plant Physiol. 64:162-164 (1979). It would be desirable to obtain similar rates of embryo formation in other species as well.
A second obstacle to commercial scale cloning of plant~ through somatis embryogenesis is the failure of most embryos to develop into plants upon germination.
Lutz, J.D. et al., "Somatic Embryogenesis for Mass Cloning of Crop Plants" in Tissue Culture in Forestry and Aqriculture, R.R. Henke, K.W. Hughes, M.J. Constantin, and A. Hollaender, eds., Plenum Press, New York, pp. 105-116 (1985). For example, studies using naked or encapsulated somatic embryos of carrot found that evsn when the best known germination conditions are employed, an extremely low percentage of somatic embryos ever germinate and develop into plantlets. 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 (lg79). Kitto, S.L. and J. Janick, "Production of Synthetic Seed by ~X~ 23 Encapsulating Asexual Embryos o~ Carrot, 11 J. American Hort. Society, 110:277-282 (1985). To be commercially useful, somatic embryos mus~ germina~e as rapidly as seed, and grow out with a vigorous shoot and root.
Typical subculture schemes for plant cell culture utilize media formulations composed of inorganic components, vitamins, phytohormones, and a carbohydrate source. In general, plant cell culture involves separate stages: (1) callus growth (maintenance~, ~2~
induction to alter the developmental pathway of callus cells, t3) regeneration o~ shoots on emhryos from callus, and ~4) production of complete plants. Sharp, W.R. et al., "The Physiology of In Vitro Asexual Embryogenesis," Hort. Rev., 2:268-310 (1980). Tisserat, B., "Somatic Embryogenesis in Angiosperms," Hort. Rev., 1:1_78 (1979). deFossard, R.A., "Tissue Culture for Plant Propagators," University of New England Printery, Armidale, New South Wales, Australia, p. 409 tl976).
The effect of organic acids upon plant cell cultures has been studied on a number of occasions. In general, organic acids have been used as a nitrogen source or as buffering agents. Cells of soybean, wheat, and flax can be grown for extended periods when ammonium is the sole source of nitrogen if citrate, malate, fumarate, or succinate are included in the medium.
Gamborg, O.L. and J.P. Shyluk, "The Culture of Plant Cells with Ammonium Salts as the Sole Nitrogen Source,"
Plant Physiology, 45:598 (1~70).
Tobacco c~lls will grow on ammonium as a sole source of nitrogen only i~ succinate, malate, fumarate, citrate, ~-ketoglutarate, glutamate, or pyruvate are included in the cell culture medium. This efect was attributed to the need for additional carbon skeletons for amino acid synthesis through the condensation of ammonium and ~-ketoglutarate. Behrend, J. and R.E.

~L2~8~3 Mateles, "Nitrogen Metabolism in Plant Cell Suspension Cultures," Plant Physiol., 58:510 (1976).
Cell growth can be stopped in soybean cultures if the cells are transferred onto urea as a sole nitroyen source in a medium containing 10mM citrate. Polacco, J.C., "Nitrogen Metabolism in Soybean Tissue Culture. I.
Assimilation of Urea," Plant Physiol., 5g:350 (1976).
The citrate effect can be reversed by adding ammonium or nickel sulfate. This effect has been attributed to the ability of citra~e to chelate trace amounts of nickel --a co-factor of urease in plants. Polacco, J.D.
"Nitrogen Metabolism in Soybean Tissue Culture. II.
~rea Utilization and Urease Synthesis Require Ni2~,"
Plant Physiol., 5g:827 (1977). In this instance, citrate is used to stop cell growth.
The effect of citrate and ~-ketoglutarate was studied in alfalfa, wheat, and tobacco cells grown on either ammonium, nitrate, or glutamine. The growth of cultures was more successful on ammonium medium, especially if ~-ketoglutarate was used. Growth rates were found to be poor if citrate was used in place of ~-ketoglutarate. Fukanaga, Y. et al., "The Differential Effects of TCA-cycle Acids on the Growth of Plant Cells Cultured in Liquid Media Containing Various Nitrogen Sources, Planta 139:199 (1978).
In carrot cell cultures grown on ammonium as the sole source of nitrogen, succinate, fumarate, malate, ~-ketoglutarate, glutamate, maleate, malonate, tartarate, and citrate were all found to support growth of the cell cultures. Dougall, D.K. and K.W. Weyrauch, "Abilities of Organic Acids to Support Growth and Anthocyanin Accumulation by Suspension Cultures of Wild Carrot Using Ammonium as the Sole Nitrogen Source, In Vitro 16:969 (1980). The observed growth in responsa to these organic acids was attributed to the pH buffering effect ~9~ 3 on the culture medium. Apart from cell growth, an effect of organic acids on the subsequent regeneration of the cultures was not observed.
In the above studies, organic acid additions to the cell culture medium were not shown to have an effect on somatic embryogenesis.
In carrot cell cultures, somatic embryogenesis is weak or absent if ammonium succinate medium is used for regeneration, but embryo yields are high if ammonium or glutamate is used. Wetherell, D.F~ and D.K. Dougall, "Sources of Nitrogen Supporting Growth and Embryogenesis in Cultured Wild Carrot Tissue," Physiologia Planturium 32:97 (1976). This result suggests that succinate is deleterious to embryogenesis of plant cell cultures.
In eggplant, various concentrations of ammonium ci~rate and potassium nitrate have been used in the regeneration medium (Gleddie, S., "Somatic Embryogenesis and Plant Regeneration from Leaf Explants and Cell Suspensions of Solanum meloqena (eggplant)," Can. J.
Bot~ 6:656 (1~83)). The optimal~ratio of ammonium to nitrate was found to be 2:1 (20mM NH4+:40mM N03-). If ammonium citrate concentration was increased above the determined optimum, embryogenesis was found to be inhibited. At 20mM citrate, embryogenesis was totally blocked. The optimization of embryogenesis in this instance was attributed to the ~avorable level of NH
and N03- in the medium. Citrate was not considered important in this response.
In rice cell cultures, succinate has been shown to inhibit callus growth in the presence of NH4+ and N03-but will stimulate growth if ammonium is the sole nitrogen source. Regeneration in th~ latter treatments, however, was poorer than with cells not treated with succinate, indicating that organic acid pretreatment is, by itself, deleterious to subsequent regeneration and 12~ 3 embryogenesis in cell cultures when compared to the other treatments tested. Chaleff, R.S. "Induction, Maintenance and Differentiation of Rice Callus Cultures on Ammonium as Sole Ni~rogen Source," Plant Cell Tissue organ Culture 2:29 (1983).
Finally, studies using soybean cell cultures grown on 40mM NH4+ (as ammonium citrate) in the absence of nitrate reported successful embryo development when cultures were transferred to Murashige and Skoog (MS) medium. Christianson, M.L., et al., "A
Morphogenetically Competent Soybean Suspension Culture,"
Science 222:632-634 (1983). Christianson, M.L. "An Embryogenic Culture o~ Soybean. Towards a General Theory o~ Somatic Embryogenesis" in Tissue Culture in Forestry and A~riculture, R.R. Henke, K.W. Hughes, M.L.
Constantin, and A. ~ollaender eds., Plenum Press, New York, pp. 83-103 (1985). Murashige, T. and F. Skoog, "A
Revised Medium for Rapid Growth and Bioassays with Tobacco Tissue Cultures," Physiologia Plantarum 15:473-497 ~1962). The author(s) point out that the hallmark of this procedure of regenerating soybean involves the "coordinate removal of 2,4-dichlorophenoxyacetic acid (an auxin) and a change from 40mM ammonium to 20mM
ammonium and 50mM nitrate." According to the authors, a change in nitrogen source is essential to the technique.
Christianson, M.L. et al., loc. cit., Christianson, M.L., loc. cit o In so concluding, the authors teach against using citrate as an additive to cell cultures to improve the yield and maturation of somatic embryos. In recent experimenks with soybean cultures it has been shown that ammonium citrate inhibits the development of soybean embryos when added to the regeneration medium whereas potassium citrate fails to inhibit embryo expression. ~.L. Christianson loc. cit. It was concluded that 40mM NH4~ has an adverse effect on embryo ~3~23 expression if included in the regeneration medium. It was also concluded that potassium citrate has no effect on embryo expression.
The above examples teach that organic acids, such as citrate or succinate, have a neutral or deleterious effect on somatic embryogenesis. In all of these examples, the authors concluded tha~ the main effector of somatic embryo production is the level of nitrogen, either as nitrate or ammonium. Thus, organic acid addition has been thought to have either a neutral role, as with eggplant or soybean somatic embryogenesis, or inhibit somatic embryogenesis, as was the case with rice somatic embryogenesisO

Disclosure of the Invention This invention provides materials and methods for improving the yield and quality of somatic embryos from plant cell culture by treating cell cultures with organic acids prior to somatic embryo development.
These materials and methods also improve the gexminability of somatic embryos produced in cell cultures so treated.
The present invention provides a medium for subculturing cultured plant cells which i5 comprised of at least one organic acid in a balanced salt solution containing the nutritional and growth factors necessary for plant growth. This medium contains an amount of the selected organic acid(s) sufficient to produce cells which possess an improved ability to undergo somatic embryogenesis when exposed to hormone and nutrient conditions avorable ~or regeneration.
Another aspect of this invention provides a medium for subculturing cultured plant cells which is comprised of at least one organic acid, together with reduced nitrogen additives, such as amino acids, in a balanced salt solution containing the nutritional and growth factors necessary for plant growth. This medium contains sufficient amounts of the selected organic acid(s) and reduced nitrogen to produce cells which possess an improved ability to undergo somatic embryogenesis when exposed to hormone and nutrient conditions favorable for regeneration.
A further aspect of ~he present invention provides a method for culturing cells utilizing the media of this invention. The somatic embryos resulting Prom such a pretreatment also show superior quality as measured by improved germinabili~y and conversion to plants.

Brief Descripkion of the Drawinas Figure 1 is a graphic representation of a treatment scheme used to sllbculture, induce and regenerate alfalfa somatic embryos, and to form plants from the embryos, including a pretreatment period in accordance with the present invention, which represents a specialized case of the maintenance culture of alfalfa cells; and Figure 2a-e are graphic representations of the yield o~ somatic embryos produced in response to a pretreatment of cell cultures of alfalfa with various organic acids.
Modes of Practicin~ the Invention This invention provides novel and improved methods and materials for producing numerous high quality somatic e~bryos from plant tissue by the addition of organic acids and reduced nitrogen to cultures prior to the processes of somatic embryogenesis and plant formation.
In the practice of the present invention, various organic acids are employed. These acids are generally ~7~3 dicarboxylic acids wherein ~he carboxyl groups are separated by a chain of five or less carbon ~toms, e.g.
~OH
c=o (CH2)n C=O
\OH

Alternatively, the carbon chain in an organic acid can include CHOH groups replacing one or more CH~ groups.
Representative organic acids include oxalic, malonic, succinic, glutaric, maleic, pimelic and tartaric acids, among others.
Additional benefits of the present invention can be obtained by the inclusion of a source of reduced nitrogen in the pretreatment medium. As an example of known sources of reduced nitrogen, amino acids can be employed with good effect. Among the preferred amino acids useful in the present invention are proline, alanine, glutamine, arginine and asparagine.
Numerous important crop and horticultural species have been shown to be capable of propagation through tissue culture and somatic embryogenesis. For a lengthy but by no ~eans exhaustive list of species capable of somatic embryogenesis, see Evans, D.A., et al., "Growth and Behavior of Cell Cultures: Embryogenesis and Organogenesis" in Plant Tissue Culture: Methods and APplications in Aariculture, T. ~horpe, ed., Academic Press, pg. 45 et ~. (1981).
In alfalfa tMedicago sativa L.)~ a representative species of the plant family Fabaceae, embryogenesis can be routinely induced in the Regen S line. Saunders, J.W. and E.T.
Bingham, "Production of Alfalfa Plants from Callus Tissue,"
Crop Sci. 12:804-808 (1972). A representative protocol for alfalfa somatic embryogenesis is provided therein and, b~- in the practice of the present invention, modifications to such procedures included a pretreatment period, generally as follows:

E~perimental Definition of Pretreatment Period According to published procedures, alfalfa cell cultures can be obtainQd from explants, such as leaf petioles placed on Schenk-Hildebrandt (SH) bas~d medium (Schenk, R.V. and A.C. Hildebrandt, Can. J. Bot., 50:199-204 (197~)) with 3% (w/v) sucrose also containing 25~M ~-Naphthaleneacetic acid (~-NAA) and 10~M kinetin (maintenance period). Cell cultures can be maintained by repeated subcultures onto resh medium of the same kind. Walker, K.~. and S.J. Sato, 'IMorphogenesis in Callus Tissue of Medicago sativa: The Role of Ammonium Ion in Somatic Embryogenesis," Plant Cell Tissue Organ Culture 1:109-121 (1981). As depicted in Figure 1, the period of cell subculture immediately preceding induction is herein termed the pretreakment period.
This period is generally 14 to 21 days long.
Cells are subsequently treated with SH containing 3% sucrose, 50~M 2,4~dichlorophenoxyacetic acid (2,4-D) and 5~M kinetin for 3 to 4 days (Induction). Transfer of cells to regeneration medium results in the development of embryos. This regeneration medium is characterized as having reduced or no hormones but containing ammonium ion and other supplements such as amino acids or carbohydrate sources other than sucrose, in the case of alfalfa. The final step in plant production is conversion, which occurs on a simple salt medium with sugar but not hormones (Figure 1). The effect of pretreatment conditions, therefore, i5 assessed by measuring embryogenesis and plant formation which occurs after pretreatment, induction and regeneration phases of culture. Measurements of embryo ~2~8~3 yield, shape, structure, and overall vigor in germination or conversion assays are used to determine the effectiveness of the pretreatment conditions on somatic embryo quantity and quality. Consequently, the pretreatment period occurs well before any embryos are initiated or formed.
In other emhryogenesis methods or protocols the maintenance medium and induction medium, as these terms are used here, are often one and the same. That is, other methods utilize nutrient and hormone conditions which cause plant cell growth and also predispose cultures to or initiate the process of embryogenesis.
In this sense, the pretreatment medium would be a medium on which plant cultures have been incubated on prior to transfer to regeneration or embryo develop~ent medium.
In a representative protocol of the present invention, the steps include:
Maintenance. Plants of edicago satlva L. Cultivar Regen S derived from the second cycle recurrent selection for regeneration from the cross of the varieties Vernal and Saranac were used. Callus was initiated by surface sterilizing petioles with 50%
Clorox~ for ~ive minutes, washing with H20 and plating on Schank-Hildebrandt medium (SH). The medium contained 25~M ~-Naphthyleneacetic acid (~-NAA) and lO~M kinetin (SH or other cell growth medium so modified is termed maintenance medium). The ionic and organic chemical constitution of SH medium is summarized in Table 1.

Table 1 The Composition of Schenk and ~ildebrand'c Medium Ma~or Salts mM
KN03 25.0 CaC12 1. a~
MgSO4 1.6 ~H4H2P04 2.6 Micronutrient Salts ~
KI 6.0 H3BO3 80.0 MnSO4 60.0 ZnS04 3.5 Na2MoO4 0.4 CUSO4 0.8 CoC12 0.4 Na2-Ethylene diaminetetraacetic acetic 55.0 FeSO4 55.0 2~
Or~anic ComPounds mq/l Inositol 1000.0 Nicotinic acid ~5.0 Thiamine ~Cl 5.0 25 Note: No hormones added unless specified.
Carbohydrate ~/1 Sucrose 30.0 Gamborg, O.L. et al., "Plant Tissue Culture Media,"
In Vitro 12:473-478 (1976).

Callus which is formed on the explant tissue was separated from the r~maining uncallused tissue and repeatedly subcultured on maintenance medium. Callus was subcultured at three week intervals and grown under indirect light at 27C.
Pretreatment. Culture pretreatment wa~ given by varying the components of the maintenance medium to include filter sterilized organic acids and/or amino acids. This medium in all instances included 25~M ~-NAA
and 10~M kinetin to promote callus growth. This medium is referred to herein as pretreatment medium.

Pretreatment was carri~d out for 21 days (one subculture cycle) unless otherwise noted.
Induction. Three to nine grams of callus were collected at 17 to 24 days post-subculture from plates of maintenance or pre~reatment medium and transferred to lOOmm x 15mm plates o~ agar solidified containing 50~M
2,4-dichlorophenoxyacetic acid (2,4-D) and 5~M kinetin for induc~ion. Walker, X.A., et al.,"Organogenesis in Callus ~issue of Medicago sativa: The Temporal Separation of Induction Processes from Differentiation Processes," Plant Sci. Lett. 16:23_30 (1979). This medium is termed the induction medium. Cells were cultured for thrae days at 27C under indirect light.
Re~enerat on. Induced callus was squashed with a spatula and transferred to regeneration medium. For replicate treatment, 75mg fresh weight of callus was maasured using a calibrated stainless steel scoop.
Alternatively, induced cells were aseptically sized on a series of column sieves (Fisher Scientific) under gentle vacuum. Cell clumps either fell or were forced through a 35 mesh (480~M) and collected on a 60 mesh (230~M) stainless steel screen. Cells retained on the 60 mesh screen were washed with 500ml of SH minus hormone medium for every three plates of induction culture volume. The washing medium was removed by vacuum. The fresh weight of the cell clumps was measured and cells were resuspended in SH medium without hormones at 150mg fresh weight per milliliter. Seventy-five mg (0.5ml) of resuspended cells were pipeted onto approximateiy lOml of agar solidified medium in 60mm x 15mm petri dishes.
Somatic embryogenesis will also occur in suspension culture if 300mg (2ml) of resuspended cells are delivered to 8ml of liquid SH medium contained in a 50ml erlenmeyer flask.

-14~
Regeneration medium used in many of the examples contained SH medium with 3% (w/v) sucrose and with no hormones. The total NH4+ level was lOmM and the L-proline level was 30mM. These components were added after autoclaving from a ~ilter sterilized stock solution. Medium, when solidi~ied, contained 0.8% (w/v) tissue culture quality agar.
Each treatment was generally replicated ten times.
Dishes were Parafilm~ wrapped and incubated for 21 days.
SuspPnsion flasks were foam plugged, sealed with Saran Wrap~ and incuba~ed for 14 days on a orbital shaker at lOOrpm. Incubation was at 27C under 12 hour illumination from cool white fluorescent tubes at 28cm away from solidified cultures or 2m from suspension cultures.
Embryogenesis was visually measured after incubation by counting green centers of organization on th~ callus using a stereomicroscope at a magnification of lOX.
Embryo size was measured using a calibrated ocular scale at lOX magnification. Embryo shape was determined by visual examination.
Conversion. Conversion of embryos to whole plants with root and shoot axis with the first primary leaf was done by aseptically transferring embryos from selected treatments at 21 days of initial culture to half-strength SH medium and solidified with 0.8% agar.
The above specification can be altered to contain less or more of any particular chemical component without broadly affecting the final embryo number or quality. Thus, pretreatments involving organic acids improved not only the yield of somatic embryos but also the quality o~ somatic embryos as measured in conversion assays. This result was unexpected as the organic acid pretreatment of callus tissue incapable of embryogenesis without further treatment was not expected to have an ~2~78~3 effect on ~mbryo development. Furthermore, th~ effect of the organic acid in the cell cultures was to reduce the growth rate siynificantly. This result was surprising since pretreatment of callus with organic acid ends 15 days before the first appearance of embryos in the culture on regeneration medium. (Figure 1).
Such dramatic effects of culture treatment prior to the addition of the active induction agent have not previously been described for plant cell culture.
Exam~le 1. Somatic Embryo Formation in Response to Organic Acid Pretreatment Citrate was added to the pretreatment medium for 21 days at concentration of 2.5, 10, 15, 20, 25 or 30mM
with 25mM L-glutamine. After pretreatment, the callus was removed from the medium and processed through the induction and regeneration media as previously described (Figure 1). At 20mM citrate, embryo yield (numbers~ at the end of the regeneration period was increased to 290 as compared to 150 for a pretreatment without citrate (Figure 2a~.
la. Malate was substituted for citrate at concentrations of 10, 20, 30, 40, 50, 60, 70 and 100mM.
Embryo yield was increased to between 200 and 300 (60mM
malate produced 300 embryos) when malate was used. The highest malate concentration of 100mM had a yield equal to the control (130 embryos) without organic acids (Figure 2b).
lb. Succinate was substituted for citrate at concentrations of 10, 20, 30, 40 and 50mM. Embryo yields were increased only at the highest concentration.
With 60mM succinate the yield was 190 embryos as compared to the control of 60 embryos tFigure 2c~.
lc. Tartarate was substituted for citrate at concentrations of 10, 20, 30, 40, 50, 60, 70 and 100mM.

..

All but the highest concentration improved embryo yield.
At 70mM tartrate, yield was 330 as compared to 180 for the control (Figure 2d).
ld. Oxalate was substituted for citrate at concentration of 2.5, 10 and 2OmM. Embryo yield was increased at 10mM (190 embryos) as compared to the control at 80 embryos.
As demonstrated by these examples, a number of organic acids were discovered to be effective over a broad concentration range in improving embryo quality and yield (Table 2).
Table 2 Organic Acid Activity on Improved Regeneration of Alfalfa Somatic Em~ryos -Organic Acid Concentration Range Approximate (mM~ Optimum (m~) - -Citric 10-25 20 L-Malic 10-100 60 Succinic 30-~0 50 25L-(+) Tartaric 10-100 70 Oxalic 5-20 10 Exampls 2. The Response to Citrate and Minus-Nitrate Pretreatment Conditions The effect of citrate pretreatment on somatic embryo yield was investigated as described in Example l with a variety of additives to the pretreatment medium (Table 3).

78;2:3 Table 3 Effect of Citrate Pretreatment in the Presence and Absence of Nitrate on Subsequent Embryogenesis of Al~al~a Cultures -Callus Pretreatmenk Embryo Yield Maintenance Medium (SH plus 3% sucrose 301 + 17 plus 25~M ~ NAA plus lO~M kinPtin) Maintenance Medium plus 20mM512 + 33 K2-citrate Naintenance Medium minus N03-22 + 5 Maintenance Medium plus 20mM249 + 13 K2-citrate minus N03-Maintenance Medium plus 2smM255 + 13 L-glutamine Maintenance Medium plus 20mM543 + 25 K-citrate plus N03 Maintenance Nedium plus 25mM533 + 23 L-glutamine plus 2OmM K2-citrate minus N03-Addition of 20mM potassium citrate (K-citrate) alone caused a 70~ increase in embryo yield with no other medium modifications. Deletion of nitrate from the pretreatment medium inhibited the embryogenesis response, and hence, minus nitrate medium alone did not stimulate embryogenesis. Citric acid, supplied here as K2-citrake, provided the best embryogenesis from callus pretreated with this organic acid. By providing only glutamine to the maintenance medium, which acts as an alternative nitrogen source for ammonium or nitrate, lower embryo yields resulted. If citrate was added in the presence of glutamine an increase in embryogenesis resulted even if nitrate was present or absent.

~9~78~

Example 3. _Use of OrgLanic Acid Pretreatments in Combination with Amino Acids Supplements The effect of citrate pretreatment on somatic embryo yield was investigated as described in Example 1 with the addition of amino acids (Table 4: A&B).
The addition of only amino acids as a pretreatment to cell cultures did not stimulate embryogenesis. If the amino acids L-glutamine or L-proline were used in the pretreatment medium with 20mM K2-citrate, however, embryogenesis was stimulated. In the case of proline plus citrate pretreatment, the highest embryo yields were achieved. The positive amino acid response was seen even when the pretreatment medium with citrate contained no nitrate (Table 4B).

~2~23 Table 4 The Effect of Pretreating Cell Cultures with organic Acids and Amino Acids . . .
Pretreatment Medium Somatic Embryo Yield A. ~
Main~enance Medium 486 + 19 Maintenance Medium plus 25mM 355 + 19 L-glutamine only Maintenance Medium plus 25mM 510 + 18 ~-glutamine plus 2OmM K2-citrate Maintenance medium plus 25mM 428 + 12 L-proline only Maintenance medium plus 25mM 620 + 33 L-proline plus 20mM K2-citrate B.
Maintenance Medium 288 + 16 Maintenance Medium plus 25mM L-proline 391 + 18 plus 20 mM K-citrate minus N03-Maintenance Medium plus 25mM L-alanine 328 + 14 plus 20mM K-citrate minus N03-As dPmonstrated in these examples, a number of amino acids, when used in conjunction with organic acids, were effective over a broad range of concentrations in improving embryo quality and yield (Table 5).
Table 5 Effective levels of amino acids used in embryogenesis pretreatment medium in combination with organic acids.
Amino AcidEffective Concentration Range .~
L-proline 10 to 300 mM
L-alanine 10 to 200 mM
L-glutamine 5 to 100 mM
L-asparagine 2.5 to 80 mM
L-arginine 2.5 to 80 mM

Example 4. Effect of Orqanic ACid Pretreatment on Somatic Embryo Conversion to a Plantlet The effect of organic acid pretreatment in somatic embryo quality, measured in terms of conversion-to-plant frequency, was determined as describe~ in Example 1 (Table 6A-C).
Table 6 The Effect of Organic Acid Pretreatment on Somatic Embryo Conversion to Plantlets Pretreatment Conversion Percentage Maintenance medium 22 + 3 Maintenance medium plU5 25mM 25 + 5 L-glutamine Maintenance medium plus 25mM 38 + 7 L-glutamine plus 20mM citrate .
B. -Maintenance medium 32 + 2 Maintenance medium plus 25mM
L-glutamine plus 20mM citrate 47 + 2 Maintenance medium plus 25mM
L-glutamine plus 50mM malate 50 + 4 Maintenance medium plus 25mM
L-glutamine plus 70mM tartrate 48 + 5 . .
C .
Maintenance medium 5 Naintenance medium plus 25mM 43 L-glutamine plus 20mM citrate Maintenance medium minus NO3- plus 38 25mM L-glutamine plus 20mM cikrate The performance of a naked embryo produced in vltro was measured by evaluating the development of entire plantlets from a population of somatic embryos. This ~7~23 value is called the conversion percen~age or frequency, which is analogous to the germination percentage or frequency of true seeds. Improvements in conversion frequency occurred as a result of pretreating callus with organic acids prior to induction and regeneration of somatic embryos.
With pretreatment of callus cultures with organic acids, citrate, malate, succinate and tartrate, embryo conversion to plantlets was significantly improved by 50 percent or more in each experiment.

Exam~le 5. Effect of Orqanic Acid Pretreatment on Callus Growth Rate an_d OveralL ~fficiency of Plant Production.
The effect of citrate pretreatment on somatic embryo yield and guality was investigated as described in Example 1 by comparing the overall effect of maintenance medium with and without organic acids.
One gram of callus when placed for 21 days on maintenance medium yielded 3.9 + 0.5g. callus. From one gram of the callus, 8,400 somatic embryos were produced based on the ~ubsample using the induction and regeneration conditions previously described. Of these embryos, 1,800 converted to whole plants, again using a subsample.
One gram of callus when placed for 21 days on pretreatment medium consisting of maintenance medium with 25mM glutamine, 25mM K-citrate, and no nitrate yielded significantly less callus, 2.2 + 0.lg. From one gram of this callus, 8,000 somatic embryos were produced, based on a subsample. Of these embryos 3,400 converted to whole plants, again using a subsample.
one gram of callus when placed for 21 days on pretreatment medium consisting of maintenance medium with 25mM glutamine, 25mM K-malate, and no nitrate z~

yielded significantly less callus, 1 6 + 0.lg, than the control. From one gram of this callus, 5,600 embryos were produced, based on a subsample. Of these e-mbryos, 2,200 converted to whole plants, again using a subsample.
Obviously, many modi~ications and varia~ions of the present invention are possible after consideration of the present disclosure. It is therefore to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as is specifically described.

Claims (20)

1. In a method of producing embryonic tissue from plant somatic tissue wherein the somatic tissue is regenerated from induced cells in a nutritive medium to form embryonic tissue, the improvement comprising pretreating the somatic cells with a nutritive medium containing at least one dicarboxylic acid in an amount sufficient to produce cells which possess an improved ability to undergo somatic embryogenesis when exposed to hormone and nutrient conditions favorable for regeneration.

2. A method as recited in claim 1, wherein the dicarboxylic acid is selected from the group consisting of citrate, malate, succinate, oxalate and tartarate.

3. A method as recited in claim 2, wherein the citrate is at a concentration of 10 to 25mM in the medium.

4. A method as recited in claim 2, wherein the malate is at a concentration of 10 to 100mM in the medium.

5. A method as recited in claim 2, wherein the succinate is at a concentration of 30 to 80mM in the medium.

6. A method as recited in claim 2, wherein the tartarate is at a concentration of 10 to 100mM in the medium.

7. A method as recited in claim 2, wherein the oxalate is at a concentration of 5 to 20mM in the medium.

8. A method as recited in Claim 1 further comprising adding a source of reduced nitrogen to the medium in an amount sufficient to stimulate embryogenesis or embryo conversion.

9. A method as recited in claim 8, wherein the reduced nitrogen source comprises at least one substance selected from the group consisting of proline, alanine, arginine, glutamine and asparagine.

10. A method as recited in claim 9, wherein each selected reduced nitrogen source is selected from the group consisting of:
proline at a concentration of 10 to 300mM in the medium;
alanine at a concentration of 10 to 200mM in the medium;
arginine at a concentration of 2.5 to 80mM in the medium;
glutamine at a concentration of 5 to 100mM in the medium; and asparagine at a concentration of 2.5 to 80mM
in the medium.

11. In a nutritive medium used for the culturing of plant somatic tissue, the improvement comprising the addition of at least one dicarboxylic acid in an amount sufficient to produce cells which possess an improved ability to undergo somatic embryogenesis when exposed to hormone and nutrient conditions favorable for regeneration.

12. A medium as recited in claim 11, wherein the dicarboxylic acid is selected from the group consisting of citrate, malate, succinate, oxalate and tartrate.

13. A medium as recited in claim 12, wherein the citrate is at a concentration of 10 to 25mM in the medium.

14. A medium as recited in claim 12, wherein the malate is at a concentration of 10 to 100mM in the medium.

15. A medium as recited in claim 12, wherein the succinate is at a concentration of 30 to 80mM in the medium.

16. A medium as recited in claim 12, wherein the tartrate is at a concentration of 10 to 100mM in the medium.

17. A medium as recited in claim 12, wherein the oxalate is at a concentration of 5 to 20mM in the medium.

18. A medium as recited in Claim 11 further comprising a source of reduced nitrogen in the medium in an amount sufficient to stimulate embryogenesis or embryo conversion.

19. A medium as recited in claim 18 wherein the reduced nitrogen source comprises at least one substance selected from the group consisting of proline, alanine, arginine, glutamine and asparagine.

20. A medium as recited in claim 19 wherein each selected reduced nitrogen source is selected from the group consisting of:
proline at a concentration of 10 to 300mM in the medium;
alanine at a concentration of 10 to 200mM in the medium;
arginine at a concentration of 2.5 to 80mM in the medium;
glutamine at a concentration of 5 to 100mM in the medium; and asparagine at a concentration of 2.5 to 80mM
in the medium.