JP2688621B2 - Improved somatic embryogenesis method and medium using organic acids - Google Patents

Description

TECHNICAL FIELD The present invention relates generally to adventitious embryogenesis of plant cells, and more specifically, methods and media for enhancing the quantity and quality of plant embryos produced by somatic cell culture. It is about.

BACKGROUND OF THE INVENTION The process of plant cloning is accomplished by a variety of methods, one of which is somatic embryogenesis. This method provides favorable scale and efficiency not found in other methods of cell culture, as roots and shoots develop from somatic embryos in one step. This cloning method can also be used to produce naked or encapsulated somatic embryos on a commercial scale. These somatic embryos can germinate and can be a substitute for plant seeds [Evans, Dew.
"Growth and Behavior of Cell Cultures (Evans, DA) etc.
r of Cell Cultures): Embryogenesis and Organogenesis in Plant Tissue
Culture (Embryogenesis and Organogenesis in Pla
nt Tissue Culture) ”TA Soap (TATho
rpe), Academic Press,
See New York, pp. 45-113 (1981)].

When cloning is performed by adventitious embryogenesis, the genotype of the source plant from which the explant is obtained and the physiological conditions of culture must be noted. It is the general understanding that the physiological conditions of regeneration will be well understood and the genetic nature of the plant material will become a less important factor. For example, attempts to regenerate soybean by somatic embryogenesis have been unsuccessful for decades. However, recently a clear set of conditions for soybean regeneration was reported [“A Morpho Genetically Competitive Soybean Suspension Culture (such as Christianson, ML). A Morphogeneticall
y Competent Soybean Suspension Culture) ”, Science, Vol. 222, p. 632 (1983)]. Thus, the genotype (cultivated soybeans) that could not be manipulated in vitro until now is now related to cell culture conditions. With breakthroughs, it can be regenerated.

There are two major obstacles to using somatic embryos as a commercial-scale cloning method. First, many crops produce low yields or numbers of somatic embryos produced in plant cell cultures. Improving embryo yield is always a concern in production systems. The technology for carrot somatic embryogenesis has been well developed, and 90% of the cultured cells have produced somatic embryos [Fujimura, T.] and A. Komamine "synchronization".・ Somatic Embryogenesis in a Carrot Cell Suspension Culture (Sy
nchronization of Somatic Embryogenesis in a Carrot
Cell Suspension Culture ", Plant Physiol., 64, 162-164 (1979). It is desirable to achieve similar embryogenesis rates in other plant species.

A second obstacle to commercial-scale plant cloning due to somatic embryogenesis is that most embryos are underdeveloped and unable to grow into plants [Lutz, JD et al. "Somatic Embryogenesis for Mass Cloning of Crop
Plants (Somatic Embryogenesis for Mass Cloning o
f Crop Plants) ”, Tissue Culture in
Forestry and Agriculture (Tissue C
ulture in Forestry and Agriculture), Earl Earl, RRHenke, KWughes, MJ Constantine (M.
J.Constantin), Hollaender, Plenum Press, New York, 105-1
See page 16 (1985)]. For example, studies with naked or encapsulated carrot somatic embryos show that the rate of somatic embryos that germinate and grow into seedlings is extremely high, even when using the best-known germination conditions. Very low
Kay El Drew (Drew, RKL) of "The Development Of Carrot (Daukasu Kyarota
Ell) Embryoise (Delivered From Cell)
Suspension Culture In Two Plantlets on a Sugar Free Basal Medium {The Development of Carrot ( Daucus carota L.) Emb
ryoids (derived from cell suspension culture into
plantlest on asugar-free basal medium)} ”, Horty Culture Research (Hort.Res.), Volume 19, 79-8
Page 4 (1979). SLC Kitto (SL)
And J. Janick's “Production of Synthetic Seed by Encapsul”
ating Asexual Embryos of Carrott) ”, Journal
Of the American Horty Culture Society, Vol. 110, pp. 277-282 (1985)]. To be commercially useful, somatic embryos grow as fast as seeds,
Vigorous shoots and roots must grow.

A typical subculture for plant cell culture uses a medium composition consisting of mineral components, vitamins, plant hormones and carbohydrate sources. In general, plant cell culture consists of the following steps: (1) callus growth (maintenance), (2) induction to alter the growth pathway of callus cells, (3) regeneration of shoots from adventitious embryos obtained from callus, (4) Complete plant production [Sharp WR (The Sharp WR) and other "The Physiology of In Vitro Axial Embryogenesis" (The Physiology of
In Vitro Asexual Embryogenesis) ", Horty Culture Review (Hort. Rev.), Vol. 2, pp. 268-310 (198).
0 years). Tisserat, B.'s "Somatic Embryogenesis in Angiosperms", Horticulture Review (Hort.Rev), Volume 1, 1-78
Page (1979). De Foussard, AA (deFo
ssard, RA) “Tissue Culture for Plant Propagators
Propagators) ”, University of New Englan
d Printery), New South Wales, Australia, Armidale, p. 409 (1976)
reference].

The effects of organic acids on plant cell culture have been well studied. Generally, organic acids have been used as a nitrogen source or buffer. Soybean, wheat, and flax cells can grow for long periods of time when ammonium is the only nitrogen source when citrate, malate, fumarate or succinate is included in the medium. [The Culture of Plant Cells with Ammonium Salts as the Sole Nitrogen by OH L Gamborg (OL) and JP Syhluk] Sauce (The Culture of Plant Cells with Ammonium Salts
as the Sole Nitrogen Source) ", Plant Physiology, 45, 598 (1970)
Year)].

Tobacco cells contain ammonium as the sole nitrogen source when succinate, malate, fumarate, citrate, α-ketoglutarate, glutamate, or pyruvate is included in the cell culture medium. Even grows. This was because an additional carbon skeleton was required for amino acid synthesis by condensation of ammonium and α-ketoglutarate [Behrend, J.
J) and RE Mateles' Nitrogen Metabolism in Plant Cell Suspension Cultures (Nitrogen Metabol
ismin Plant Cell Suspension Cultures) ”, Plant Physiol., 58, 510 (1
976)].

When there is only urea as the sole nitrogen source in the medium containing 10 mM citrate in soybean cell culture, when the cells are transferred to the medium, the growth of the cells is stopped [Polacco,
J. C. (Polacco, JC) "Nitrogen Metabolism in Soybean Tissue Culture, I, Asimilation of Urea (Nitrogen M
etabolism in Soybean Tissue Culture.I.Assimilatiio
n of Urea ”, Plant Phyology
siol.), 58, 350, (1976)].

The effect of citrate can be canceled by the addition of ammonium or nickel sulphate. This effect is due to the ability of citrate to form chelates with small amounts of nickel (plant cofactor for urease) [Polacco, JD's "Nitrogen Metabolism in. Soybean tissue culture,
II. Urea-utilization-and-urease-synthesis Rikuaia Ni ²⁺ (Nitrogen Metab
olism in Soybean Tissue Culture.II.Urea Utilizatio
n and Urease Synthesis Require Ni ²⁺ ) ”, Plant Physiology, Vol. 59, p. 827 (1977)]. In this example, citrate is used to stop cell growth.

The effects of citrate and α-ketoglutarate on cell growth of alfalfa, wheat and tobacco with either ammonium, nitrate or glutamine were investigated. The growth of cultured cells was more satisfactory on ammonium medium, especially with α-ketoglutarate. It was found that the use of citrate instead of α-ketoglutarate resulted in a slower growth rate [Fukanaga, Y. et al., “The Differential Effect of Tcie Acyc Acids On.・ The Grouse of Plant Cells ・
Cultured In Liquid Media Containing Berias Nitrogen Sources (The Diff
erential Effects of TCA-cycle Acids on the Growth
of Plant Cells Cultured in Liquind Media Cotainin
g Various Nitrogen Sources) ”, Planta
a), 139, p. 199 (1978)].

Carrot cell cultures grown on ammonium as the only nitrogen source have succinate, fumarate, malate,
Alpha-ketoglutarate, glutamate, maleate, malonate, tartrate and citrate were all found to promote cell growth in culture [Dougall, DK, and W Wayrauch's “Abilities ·
Of Organic Acids to Support Grouse and Anthocyanin Accumulation by Suspension Cultures of Wild Carrot Yousing Ammonium as the Sole Nitrogen Source (Abilities of Org
anic Acids to Support Growth and Anthocyanin Accum
ulation by Suspension Cultures of Wild Carrot Usin
g Ammonium as the Sole Nitrogen Source) ”, In Vitro, Vol. 16, p. 969 (1980)]. The promotion of growth by these organic acids was due to the pH buffer effect on the culture medium. Other than growth, no effect of organic acids on subsequent culture regeneration was observed.

The above studies did not show that the addition of organic acids to cell culture medium affected somatic embryogenesis.

In carrot cell cultures, somatic embryogenesis is weak or absent when ammonium succinate medium is used for regeneration. However, embryo yields are high with ammonium or glutamate [Wetherell, D. F.
therell, DF) and D.K.Dougall (DKDo)
ugall) “Source of Nitrogen Supporting Grouse and Embryogenesis”
In Cultured Wild Carrot Tissue (Sources of Nitrogen Supporting Growth and Embr
yogenesis in Cultured Wild Carrot Tissue ”, Physiologia Planturi
um), 32, page 97 (1976)]. This result suggests that succinate is detrimental to embryogenesis in plant cell culture.

In the case of eggplant, various concentrations of ammonium citrate and potassium nitrate were used in the regeneration medium [Grady,
Es (Gleddie, S.), "Somatic Embryo Genesis and Plant Regeneration From leaf Aix Plants-and-cell suspension's Of Solanum Merogena (egg plant) (Somatic Embryogenesis and Plant Regeneration
from Leaf Explants and Cell Suspensions of Solanu
m Melogena (eggplant) ”, Canadian Journal of Botany (Can.J.Bot.), Vol. 6, p. 656 (1
983)]]. The optimum ratio of ammonium to nitrate is 2: 1.
^{_{(20mM NH 4 +: 40mM NO}} 3 -) was found to be. It was found that when the ammonium citrate concentration was increased above the defined optimum concentration, embryogenesis was inhibited. Embryogenesis was completely blocked with 20 mM citrate. It was found that the optimization of embryogenesis in this example was determined by the concentration of NH ₄ ⁺ and NO ₃ ^{− in} the medium. Citrate was not considered important in this reaction.

The cell culture of rice, succinate NH ₄ ⁺ and NO ₃ ^- was found to inhibit the growth of callus in the presence of, if ammonium is the only nitrogen source for promoting the growth. However, plant regeneration with succinate treatment is worse than with cells not treated with succinate. This indicates that pretreatment with organic acids per se is more harmful to subsequent plant regeneration and embryogenesis in cell culture than other methods of treatment [Chalef, RS.
f, RS) “Induction, Maintenance and Difference of Rice Callus
Cultures on Ammonium as Sole Nitrogen Source (Induction, Maintenance and Di
fferentiation of Rice Callus Cultures on Ammonium
as Sole Nitrogen Source), Plant Cell Tissue Orga
n Culture), Vol. 2, p. 29 (1983)].

Finally, in a study of growing soybean culture cells in 40 mM NH ₄ ⁺ (as ammonium citrate) without nitrate,
Cultured cells are Murashige and Skoo
g) Successful development of embryos when transferred to (MS) medium [Christiansson, G. H.
ianson, ML) etc. "A Morphogenetically Competent Soybean Suspension
Culture) ”, Science, Volume 222, 632-6
34 pages (1983). Christianson, M.L.'s "Ann Embryogenic Culture of Soybean, Too's a General Shiori of
Somatic Embryogenic
Culture of Soybean.Towards a General Theory of So
matic Embryogenesis) ”, tissue culture
In Forestry and Agriculture (Ti
ssue Culture in Forestry and Agriculture), RRHenke, KW
KWHughes, MLConstantin and A. Hollae
Nder), Plenum Press, New York, pp. 83-103 (1985). Tea Murashige (TM
urashige) and F. Skoog's “A.
Revised Medium for Rapid Grouse and Bioassays with Tobacco Tissue Cultures
wth and Bioassays with Tobacco Tissue Culture
s) ”, Physiologia P.
Lantarum), Vol. 15, pp. 473-497 (1962)]. The author (etc.) of this paper said that the characteristics of this soybean regeneration method are "2.4-
It is a coordinated removal of dichlorophenoxyacetic acid (auxin) and a modification of using 20 mM ammonium and 50 mM nitrate instead of 40 mM ammonium. " It is said to be indispensable for this method [see Christianson, M. El, et al. (Cited above), Christianson M. El. (Cited above)]. In this way, the authors conclude that Opposing the addition of citrate as an additive to cell cultures to improve yield and maturation, a recent experiment on soybean cultures showed that ammonium citrate was added when added to the regeneration medium. It was shown that it blocks soybean embryo development but potassium citrate does not block embryo development (M. L. Christianson, supra). Reference) .40mM NH
_It was concluded that ₄ ⁺ had an adverse effect on embryonic expression when included in regeneration medium. It was also concluded that potassium citrate had no effect on embryo expression.

The above examples show that organic acids such as citrate or succinate have a neutral or detrimental effect on somatic embryogenesis. In all of these cases, the authors noted that nitrate was the main influencing factor of somatic embryogenesis,
It was concluded that it was the concentration of nitrogen, whether ammonium.
Thus, organic acid addition has either a neutral role as in somatic embryogenesis of eggplant or soybean, or it inhibits somatic embryogenesis as in somatic embryogenesis of rice.

DISCLOSURE OF THE INVENTION The present invention provides media and methods that improve the yield and quality of somatic embryos from plant cell cultures by treating the cell cultures with organic acids prior to somatic embryogenesis. These media and methods also improve the germinability of somatic embryos produced from the cell cultures thus treated.

The present invention provides a medium for subculturing cultured plant cells, which comprises at least one organic acid in a balanced salt solution containing nutrients required for plant growth and growth factors. . This medium comprises a sufficient amount of at least one of the at least one selected species to form cells with improved ability to undergo somatic embryogenesis when the cells are subjected to favorable hormonal and nutritional conditions for regeneration. Including organic acids.

The present invention subcultures cultured plant cells containing a reduced nitrogen additive such as an amino acid together with at least one organic acid in a balanced salt solution containing nutrients and growth factors necessary for plant growth. A medium for culturing is also provided. The medium is in sufficient quantity to produce cells having an improved ability to undergo somatic embryogenesis when the cells are exposed to favorable hormone and nutrient conditions for regeneration. It contains an organic acid and a reduced nitrogen source.

The present invention further provides a cell culture method utilizing the medium of the present invention. Adventitious embryos that develop upon such pretreatment show excellent properties in terms of germination and conversion into plants.

BEST MODE FOR CARRYING OUT THE INVENTION The present invention is
Provided is a new and improved method and medium for forming large quantities of high quality somatic embryos from plant tissue by adding organic acids and a reduced nitrogen source to the culture.

Various organic acids are used in the practice of the present invention. These acids are generally dicarboxylic acids in which the carboxyl groups are separated by chains of up to 5 carbon atoms, such as It is.

Instead of one or more of the carbon chains in the organic acid
The CH ₂ group may be replaced with a CHOH group. Typical organic acids are oxalic acid, malonic acid, succinic acid, glutaric acid, maleic acid, pimelic acid, tartaric acid and the like.

Another advantage of the present invention is obtained by adding a reduced nitrogen source to the pretreatment medium. Amino acids are preferred as examples of known reduced nitrogen sources. Preferred amino acids useful in the present invention include proline, alanine, glutamine, arginine and asparagine.

It has been found that many important crops and horticultural plants can be propagated by tissue culture and somatic embryogenesis. For a long, but by no means exhaustive, list of plant species capable of adventitious embryogenesis, see [Evans, D.
A.) et al., “Growth and Behavior of Cell Cult: Embryogenesis and Organogenesis”
ures: Embryogenesis and Organogenesis), plant
Tissue Culture: Methods and Applications in Agriculture (Plant Tissue C
ulture: Methods and Applications in Agriculture),
T. Thorpe, Academic Press, page 45 and below (1981)].

In alfalfa, embryogenesis is a routine
Induced by Regen S (strain name) [Sounders, Saunders, JW and ETBingham's “Production of Alfalfa Plant From Callus Tissues” Alfalfa Plants From Ca
llus tissue) ”, Crop Science (Crop Sci.)
12: 804-808 (1972)]. A typical procedure for alfalfa somatic embryogenesis is described there,
In the practice of the present invention, a variation of such a method includes a pretreatment period, which is generally as follows: Determining the experimental pretreatment period According to known methods, alfalfa cell cultures are
Schenk-Hildebrandt (SH) basal medium containing 3% (W / V) sucrose and also 25 μM α-naphthalene acetic acid (α-NAA) and 10 μM kinetin [Schenk, Schenk, Schenk, RV) and AC, Hildebrandt, The Canadian Journal of Botany, 50, 19
Pp. 9-204 (1972)] obtained from explants, for example petiole. Cultured cells are maintained by repeated subcultures on fresh media of the same type [Walker, KA and SJSato, “Morphogenesis in
Callus Tissue of Medicago Sativa: The Roll of Ammonium Ion in Somatic Embryogenesis
lus Tissue of Medicago Sativa : The Role of Ammonium
Ion in Somatic Embryogenesis) ", Plant Cell Tissue Organ Culture, Volume 1, 109-121
Page, (1981)]. As shown in FIG. 1, the cell subculture period immediately before induction is referred to as a pretreatment period here. This period is generally 14-21 days.

Cells were then treated with S containing 3% sucrose, 50 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 5 μM kinetin.
Treat with H medium for 3-4 days (induction). Transfer of cells to regeneration medium causes embryonic development. This regeneration medium, in the case of alfalfa, contains little or no hormone,
Ammonium ions and other auxiliary ingredients such as amino acids or carbohydrate sources other than sucrose. The final stage of plant formation is the conversion of somatic embryos into plant bodies (called conversion of somatic embryos). This occurs in a simple salt medium containing sugar but no hormone (Fig. 1). Therefore, in order to evaluate the effect of the pretreatment conditions, the embryo formation and the plant body formation that occur after the pretreatment, induction, and regeneration of the culture are measured. Embryonic yield, shape, structure measurements, and overall viability of germination or conversion assays are used to determine the effect of pretreatment conditions on somatic embryo quantity and quality. Therefore, the pretreatment period is set well before the embryo is induced or formed.

In other somatic embryogenesis methods or procedures, it is often identical to maintenance and induction media as used herein. That is, the other method is
Nutrient and hormonal conditions are used that cause plant cell growth and either predispose the cultured cells to the embryogenic process or initiate the embryogenic process. In this sense, pretreatment medium is a candy medium on which the plant culture is cultivated before it is transferred to the regeneration or embryogenesis medium.

 An exemplary procedure of the invention includes the following steps: Maintaining alfalfa (Medicago SativaL.) Vernal
(Vernal, breed name) and Saranac (Saranac, breed
Obtained from the second cycle of cyclic selection by crossing
Using Regen S (Cultivar Regen S, strain name)
Was. The surface of petiole is 50% Chlorox (trade name) (Crolox
) For 5 minutes, wash with water, and then Schenk Hildebra
Inducing callus by placing on callus (SH) medium
Was. The medium is 25 μM α-naphthalene acetic acid (α-NAA).
And 10 μM kinetin (SH medium or
The other cell growth medium prepared as above is called the maintenance medium.
Bu). Table 1 shows the composition of inorganic and organic substances in SH medium.
stop.

Table 1 Composition of Schenko-Hildebrandt medium Main salts mM KNO ₃ 25.0 CaCl ₂ 1.4 MgSO ₄ 1.6 NH ₄ H ₂ PO ₄ 2.6 Micronutrients μM KI 6.0 H ₃ BO ₃ 80.0 MnSO ₄ 60.6 ZnSO ₄ 3.5 Na ₂ MoO ₄ 0.4 CuSO ₄ 0.8 CoCl ₂ 0.4 Na ₂ -Ethylenediaminetetraacetic acid 55.0 FeSO ₄ 55.0 Organic compound mg / l Inositol 1000.0 Nicotinic acid 5.0 Thiamine-HCl 5.0 Note: No hormone added unless otherwise stated. Carbohydrate g / l Sucrose 30.0 Plant Tissue Culture Med, such as Gamborg (OL)
ia), In Vitro, Vol. 12, pp. 473-478 (1976)
Year).

Callus formed on the explant tissue was separated from the remaining non-callus tissue and subjected to repeated subculture in maintenance medium. Callus was subcultured at 3-week intervals and exposed under indirect light.
Grow at 0 ° C.

Pretreatment The culture was pretreated by changing the components of the maintenance medium so as to contain filter-sterilized organic acids and / or amino acids. This medium contained 25 μM α-NAA and 10 μM kinetin to promote callus growth in all experiments. This medium is referred to herein as the pretreatment medium.

The pretreatment was carried out for 21 days (one subculture cycle) unless otherwise specified.

Induction 3 to 9 g of callus was recovered from the plate of maintenance or pretreatment medium at 17 to 24 days after subculture, and 50 μM 2,4-
Transfer to a 100 mm x 15 mm plate of agar medium containing dichlorophenoxyacetic acid (2.4-D) and 5 μM kinetin,
Induced "Organogenesis in Callus Tissues such as Walker KA
Of Medicago Sativa: The Temporal Separation Of Induction Process From Difference Processes (Organogene)
sis in Callus Tissue of Medicago sativa : The Tempor
al Separation of Induction Processes from Differen
tiation Processes) ", Plant Science Letters (Plant Sci. Lett.) 16: 23-30 (1979)]. This medium is called induction medium.
Cultured under indirect light for 3 days.

Regeneration-induced callus was crushed with a spatula and transferred to regeneration medium. For repeated treatments, 75 mg fresh callus was measured using a graduated stainless steel spoon. Alternatively, the induced callus may be subjected to a series of column sieves [Fisher Scientific] under weak vacuum.
c)] and aseptically sized. Cell clumps were either dropped through 35 mesh (480 μM) or forced through and collected on a 60 mesh (230 μM) stainless steel screen. Cells retained on a 60 mesh screen are for each 3 plates of induced culture.
The culture medium was washed with 500 ml of medium excluding hormones from SH medium.
Wash medium was removed by aspiration. The fresh weight of the cell mass was measured, and the cells were placed in SH medium without hormones, 150 mg fresh weight /
Resuspended at a rate of ml. 75 mg (0.5 ml) of resuspended cells were pipetted and plated on approximately 10 ml of agar medium in a 60 mm x 15 mm petri dish.

Somatic embryogenesis also occurs in suspension culture when 300 mg (2 ml) of resuspended cells are added to 8 ml of liquid SH medium in a 50 ml Erlenmeyer flask.

As a regeneration medium, SH medium containing 3% (W / V) sucrose but no hormone was used in many examples. Total NH ₄ ⁺
The concentration was 10 mM and the L-proline concentration was 30 mM. These components were sterilized by filtration and added to an autoclaved medium. For solid media, add 0.8 a tissue culture agar.
% (W / V) was used.

 Each treatment was repeated approximately 10 times. Put the dish on parafilm (quote
(Name) (Palafilm ) And cultured for 21 days. Suspension
Rasco is capped with a vent plug, and Saran Wrap (trade name) (Sa
ran wrap ), Using an orbital shaker 100 times / min
And cultured for 14 days. Culture temperature is 27 ℃, and it is cold white for lighting.
Using a fluorescent tube, separate by 28 cm for static culture, and for suspension culture
The light period was 12 hours, separated by 2 m.

Embryogenesis was measured after culture by counting green organized centers on callus using a stereomicroscope at 10x magnification. Embryo size is calibrated using the ocular s
cale) was used and the measurement was performed at a magnification of 10 times. The shape of the embryo was determined by visual inspection.

From the transformed embryo to a complete plant with roots, stems and first true leaves, the embryo should be transformed at day 21 of the first culture with the treatment chosen.
It was done by aseptic transfer to half strength SH medium containing 0.8% agar.

The above procedure may be modified to include some specialty chemical constituents so as not to have a far-reaching effect on the final embryo number or its quality. For example, pretreatment with organic acids improved not only the yield of somatic embryos, but also the quality of somatic embryos as measured by the conversion assay. This result was unexpected. Because the pretreatment of callus tissue with organic acid does not cause embryogenesis unless further treatment is performed, we did not expect that the pretreatment of callus tissue with organic acid would affect the development of embryos. . Moreover, the action of organic acids in cell culture was to significantly reduce the growth rate. This result was amazing. This is because the pretreatment of callus with organic acid was completed 15 days before the first emergence of the embryo during the culture in the regeneration medium (No. 1).
Figure). It has never before been reported in plant cell cultures that culture treatments performed prior to the addition of activity inducers have such a dramatic effect.

Example 1 Adventitious embryogenesis in response to organic acid pretreatment. Callus was used with pretreatment medium to which citrate was added at a concentration of 2.5, 10, 15, 20, 25 or 30 mM with L-glutamine at a concentration of 25 mM. Cultured for a day. After pretreatment, the callus was removed from the pretreatment medium and cultured as described above with induction and regeneration medium (Fig. 1). In the case of 20 mM citrate, the yield (number of embryos) of embryos was 290 at the end of the regenerating period, but it was 150 when pretreated without adding citrate (2A). Figure).

1.a Malate in place of citrate 10, 20, 30, 4
Added at concentrations of 0, 50, 60, 70 and 100 mM. The number of embryos increased to 200-300 with malate (60 mM malate produced 300 embryos). At a maximum malate concentration of 100 mM, a control without organic acid (130
(Fig. 2B).

1.b Replace citrate with succinate at 10, 20, 30, 40
And 50 mM concentration. Only at the highest concentration did embryo yield increase. The number of embryos in the control was 60, compared to 60.
There were 190 succinates at mM concentration (Fig. 2C).

1.c Use tartrate instead of citrate for 10, 20, 30, 40, 5
Added at 0, 60, 70 and 100 mM concentrations. Embryonic yield increased at concentrations other than 100 mM. In contrast, the number of embryos is 180, but 70m
The number of M tartrates was 330 (Fig. 2D).

Oxalate was added at 2.5, 10 and 20 mM concentrations instead of 1.d citrate. Embryonic yield is 10 mM control (80)
Increased number of embryos compared to (190) As shown by these examples, many organic acids were found to be effective in improving embryo quality and yield over a wide range of concentrations. (Table 2).

Table 2 Activity of organic acids in improving regeneration of alfalfa somatic embryos Organic acid concentration range (mM) Approximately optimum concentration (mM) Citric acid 10-25 20 L-malic acid 10-100 60 Succinic acid 30-80 50 L- ( +) Tartaric acid 10-100 70 Oxalic acid 5-20 10 Example 2 Response to pretreatment conditions in which citrate was added and nitrates were removed The effect of citrate pretreatment on adventitious embryo yield was varied in various pretreatment media. The test was carried out in the same manner as in Example 1 with the addition of additives (Table 3).

Table 3 Effect of citrate pretreatment in the presence and absence of nitrate on the subsequent embryogenesis of alfalfa culture Callus pretreatment Embryonic yield maintenance medium (SH + 3% sucrose + 25 μM α-NAA + 10 μM kinetin 310 ± 17 maintenance medium + 20 mM potassium citrate 512 ± 33 maintenance media - (minus) NO ₃ ^- 22 ± 5 maintenance medium + 20 mM potassium citrate -NO ₃ ^- 249 ± 13 maintenance medium + 20 mM potassium citrate + (plus) NO ₃ ^- 543 ± 25 maintenance medium + 25 mM L-glutamine + 20 mM potassium citrate -NO ₃ ^-. 533 only the addition of ± 23 20 mM potassium citrate, without changing the other medium composition, embryo yield was increased 70% nitrate from the pretreatment medium Upon removal, the embryogenic reaction was inhibited.
Therefore, the medium without nitrate alone did not promote embryogenesis. The best embryogenesis was seen from callus pretreated with medium supplemented with citric acid (potassium citrate is used here). Embryonic yields were low when glutamine alone was added to the maintenance medium as a nitrogen source instead of ammonium or nitrate. Addition of citrate in the presence of glutamine increased embryogenesis in the presence or absence of nitrate.

Example 3 Combination of Organic Acid Pretreatment and Amino Acid Addition The effect of citrate pretreatment on adventitious embryo yield was tested in the same manner as in Example 1 in combination with amino acid addition (Table 4: A and B). ).

Embryogenesis was not promoted when amino acids alone were added to the cell culture pretreatment medium. However, when L-glutamine or L-proline was used in the pretreatment medium with 20 mM potassium citrate, embryogenesis was improved. The highest embryo yield was obtained when pretreated with proline and citrate. The pretreatment medium containing citrate was found to have an improved effect by the addition of amino acids even when it contained no nitrate (Table 4B).

Table 4 Effect of cell culture pretreatment with organic acids and amino acids Pretreatment medium Somatic embryo yield A. Maintenance medium 486 ± 19 Maintenance medium + 25mM L-glutamine only 355 ± 19 Maintenance medium + 25mM L-glutamine + 20mM Potassium citrate 510 ± 18 maintenance medium + 25 mM L-proline 428 ± 12 maintenance medium + 25 mM L-proline + potassium 20mM citrate 620 ± 33 B. maintenance medium 288 ± 16 maintenance medium + 25 mM L-proline + 20mM potassium citrate -NO ₃ ^- 391 ± 18 maintenance medium + 25 mM L- alanine + 20 mM potassium citrate -NO ₃ ^- as shown in 328 ± 14 these examples, a number of amino acids, when used in combination with organic acids, over a wide concentration range, the improvement in the quality and yield of embryos It was effective (5th
table).

Table 5 Effective concentration of amino acids used in combination with organic acids in pre-embryogenic pretreatment medium Effective concentration range of amino acids L-proline 10-300 mM L-alanine 10-200 mM L-glutamine 5-100 mM L-asparagine 2.5-80 mM L- Arginine 2.5-80 mM Example 4 Effect of organic acid pretreatment on the conversion of somatic embryos to plantlets The effect of organic acid pretreatment on the quality of somatic embryos is shown in Example 1 depending on the degree of conversion to plant bodies. Measured as described (Table 6A-C).

Table 6 Effects of organic acid pretreatment on the conversion of somatic embryos to plantlets Pretreatment conversion rate A. Maintenance medium 22 ± 3 Maintenance medium + 25mM L-glutamine 25 ± 5 Maintenance medium + 25mM L-glutamine + 20mM citrate 38 ± 7 B. Maintenance medium 32 ± 2 Maintenance medium + 25 mM L-glutamine + 20 mM citrate 47 ± 2 Maintenance medium + 25 mM L-glutamine + 50 mM Malate 50 ± 4 Maintenance medium + 25 mM L-glutamine + 70 mM Tartrate 48 ± 5 C. maintenance medium 5 maintenance medium + 25 mM L-glutamine + 20 mM citrate 43 maintenance medium -NO ₃ ^- + naked performance embryos created in 25 mM L-glutamine + 20 mM citrate 38 in vitro experiments (in vitro), a somatic embryo population It was measured by assessing the rate of development of whole plantlets. This rate is called the conversion rate or the conversion frequency, and has the same concept as the germination rate or germination frequency of seeds. The conversion frequency was improved by pretreatment of callus with organic acid prior to induction and regeneration of somatic embryos.

Pretreatment of callus cultures with organic acids, citrates, malates, succinates and tartrates significantly improved the conversion of embryos to plantlets by more than 50 percent in each experiment.

Example 5 Effect of Organic Acid Pretreatment on Callus Growth Rate and Overall Efficiency of Plant Production The effect of citrate pretreatment on yield and quality of somatic embryos, as described in Example 1, It was investigated by comparing the overall effect of maintenance medium with and without acid.

1 g of callus is 3.9 ± when placed on maintenance medium for 21 days.
It became 0.5g. From 1 g of callus, 8400 somatic embryos were produced based on subsamples using the induction and regeneration conditions described above. Of these embryos, 1,800 transformed into whole plants. Also in this case, a sample was used.

When 1 g of callus was placed in a pretreatment medium consisting of maintenance medium containing 25 mM glutamine, 25 mM potassium citrate and no nitrate for 21 days, 2.2 ± 0.1 g of callus was obtained, which was significantly higher than the control. There were few. From 1 g of this callus, 8,000 somatic embryos were produced based on the subsample. Of these embryos, 3,400 were transformed into whole plants. In this case as well, subsamples were used.

When 1 g of callus was placed in a pretreatment medium consisting of a maintenance medium containing 25 mM glutamine, 25 mM potassium malate and no nitrate for 21 days, 1.6 ± 0.1 g of callus was obtained, which was significantly less than the control. It was From 1 g of this callus, 5,600 embryos were produced based on the subsample. 2,200 of these embryos were transformed into whole plants. In this case as well, subsamples were used.

Obviously, many variations and modifications of the present invention are possible in light of the present disclosure. Therefore, it is to be understood that within the scope of the following claims, the present invention may be practiced otherwise than as specifically described.

[Brief description of the drawings]

FIG. 1 shows that alfalfa cells were subcultured, induced,
1 is a diagram showing an example of an outline of a process including a pretreatment institution according to the present invention, which is used for regenerating and forming a plant from an somatic embryo. Figures 2A-E are yields of somatic embryos (number of embryos) produced when alfalfa cultured cells were pretreated with various organic acids.
Is shown in the graph.

Continuation of the front page (56) References “Introduction to Illustrated Tissue Culture”, edited by Yoshiro Furukawa (Sho 60-5-10) Seibundo Shinkosha P. 132-135 Environmental and Experimental Botany, 21 [3/4] (1981) P. 277-280.

Claims (18)

(57) [Claims] 1. A method of forming embryonic tissue from plant tissue, wherein somatic tissue is regenerated from induced cells to form embryonic tissue in a nutrient medium, comprising: Selected from the group consisting of citrate, malate, succinate, oxalate and tartrate in sufficient quantity to produce cells with improved ability to undergo somatic embryogenesis when exposed to cells A method for increasing the yield of somatic embryos, comprising the addition of at least one organic acid. 2. The method of claim 1, wherein the citrate concentration is in the range of 10-25 mM. 3. The method according to claim 1, wherein the malate concentration is in the range of 10 to 100 mM. 4. The method of claim 1, wherein the succinate concentration is in the range of 30-80 mM. 5. The method according to claim 1, wherein the concentration of the tartrate salt is in the range of 10 to 100 mM. 6. The method according to claim 1, wherein the oxalate concentration is in the range of 5 to 20 mM. 7. The method according to claim 1, wherein the medium is further supplemented with a reduced nitrogen source in an amount sufficient to promote somatic embryogenesis or somatic embryo conversion. 8. The method according to claim 7, wherein the reduced nitrogen source comprises at least one substance selected from the group consisting of proline, alanine, arginine, glutamine and asparagine. 9. The selected reduced nitrogen sources are proline at a concentration of 10 to 300 M, alanine at a concentration of 10 to 200 mM, arginine at a concentration of 2.5 to 80 mM, glutamine at a concentration of 5 to 100 mM, and asparagine at a concentration of 2.5 to 80 mM. 9. The method of claim 8 selected from the group consisting of: 10. Forming cells in a nutrient medium used for plant cell tissue culture, which have an improved ability to undergo somatic embryogenesis when the cells are exposed to favorable hormones and nutrient conditions for regeneration. Medium for increasing the yield of somatic embryo, to which at least one organic acid selected from the group consisting of citrate, malate, succinate, oxalate and tartrate is added in an amount sufficient to . 11. The medium according to claim 10, wherein the citrate concentration is in the range of 10 to 25 mM. 12. The medium according to claim 10, wherein the malate concentration is in the range of 10 to 100 mM. 13. The medium according to claim 10, wherein the succinate concentration is in the range of 30 to 80 mM. 14. The medium according to claim 10, wherein the concentration of the tartrate salt is in the range of 10 to 100 mM. 15. The medium according to claim 10, wherein the oxalate concentration is in the range of 5 to 20 mM. 16. The method according to claim 10, further comprising a reduced nitrogen source in an amount sufficient to promote somatic embryogenesis or somatic embryo conversion.
The described medium. 17. The culture medium according to claim 16, wherein the reduced nitrogen source contains at least one substance selected from the group consisting of proline, alanine, arginine, glutamine and asparagine. 18. The selected reduced nitrogen source comprises proline at a concentration of 10 to 300 mM, alanine at a concentration of 10 to 200 mM, arginine at a concentration of 2.5 to 80 mM, glutamine at a concentration of 5 to 100 mM, and asparagine at a concentration of 2.5 to 80 mM. The culture medium according to claim 17, which is selected from the group consisting of: