JPH10179147A - Culture of plant cell and plant cell highly producing secondary metabolite - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for culturing a plant cell, capable of being also applied to breeding of the plant cell highly producing a secondary metabolite, etc., by alternately subjecting the plant cell to the culture of the plant cell in a proliferation medium and to the culture of the plant cell in a production culture medium for proliferating the plant cell and simultaneously producing the secondary metabolite. SOLUTION: This method for culturing a plant cell comprises alternately subjecting the plant cell to the culture of the plant cell in a proliferation culture medium for proliferating the plant cell and to the culture of the plant cell in a production culture medium for controlling the culture of the plant cell at such the optimal proliferation rate that the concentration of either of a nutritive ingredient and a plant growth-regulating substance is lowered and that the osmotic pressure of the culture medium is controlled to accumulate the secondary metabolite (e.g. anthocyanin) in the cells. The exchanges of the culture media between the proliferation culture medium and the production culture medium are carried out at the logarithmic growth phases of the cultures. The concentrations of inoculated plant cells after the exchanges are controlled to 10-50gFW/L. Thereby, the method enables to culture the plant cell and simultaneously produce the secondary metabolite for a long period, and can also be applied to breed the cells capable of highly producing the secondary metabolite.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for culturing plant cells, and more particularly to a method for culturing plant cells such as strawberries for producing secondary metabolites such as anthocyanins and breeding high-productivity cells. The present invention relates to a cell culturing method, and a high metabolite-producing plant cell bred by the method.

[0002]

2. Description of the Related Art Plant cell culture methods for culturing plant cells in a liquid medium, producing a large amount of cells in a short time, and obtaining useful metabolites in the cultured cells have been known.
Production of secondary metabolites by cultured cells causes the product to accumulate in the medium or intracellularly by culturing, but is usually produced in the stationary phase rather than in the logarithmic growth phase, and conditions suitable for production are suitable for growth. Condition. Therefore, generally, a two-stage culture is performed in which, first, the cells are cultured in a growth medium containing an essential nutrient sufficient to efficiently grow the cells, and then the cultured cells are inoculated into a production medium and produced.

For example, in strawberry cell culture for producing anthocyanin, a medium suitable for growing strawberry cells and a medium suitable for producing a secondary metabolite anthocyanin in the cultured cells are composed of components. However, since they are incompatible with each other, conventional production of anthocyanins is performed by placing cells suitable for culture in a medium suitable for growing strawberry cells (hereinafter referred to as a growth medium), and performing batch culture with priority on growth. Or a method in which cultured cells are transferred to a medium suitable for producing anthocyanins (hereinafter referred to as a production medium), batch-cultured with priority given to the amount of accumulation, and anthocyanins are produced in the cells. In such a conventional batch culture method, however, a medium suitable for growing the strawberry cells has a high strawberry cell growth rate, but has a small amount of anthocyanin production per cell weight, and produces a conventional anthocyanin. In a suitable medium, the growth of strawberry cells is suppressed and the increase in cell weight is low, so that both cannot increase the total anthocyanin production,
There was a problem that the production efficiency of anthocyanins was low.

[0004] In order to solve such a problem, the present inventors have already succeeded in developing a continuous culture method of plant cells as a culture method for improving the production efficiency of secondary metabolites. (Japanese Patent Application No. 8-12296)
issue). This continuous culture method of plant cells is a culture method in which the medium is stabilized by continuously (or intermittently) exchanging a part of the medium and culturing is performed.

[0005]

However, the continuous culture method may cause a decrease in cell activity in the latter half of the culture, and is not always suitable for long-term culture production or culture for breeding of highly productive cells. I didn't. The present invention provides a new plant cell culture method that enables long-term culture production in plant cell secondary metabolite production and can be used for breeding of high-productivity cells.

[0006]

Means for Solving the Problems The plant cell culture method of the present invention comprises culturing a plant cell in a growth medium for growing the plant cell, growing the plant cell and accumulating a secondary metabolite in the cell. The method is characterized by alternately culturing the cells in a production medium controlled to such an optimum growth rate. As the production medium, as compared with the growth medium, a medium in which the concentration of any one of a nutrient component and a plant growth regulator may be reduced, or as compared with the growth medium,
A medium with an increased osmotic pressure may be used. The medium exchange between the growth medium and the production medium may be performed in the logarithmic growth phase of each culture, and the cell inoculation concentration after the exchange is 10 to 50 gFW.
/ L. In addition, the plant cell with high productivity of secondary metabolites of the present invention is obtained by culturing using the above-described plant cell culturing method.

[0007]

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for culturing plant cells such as strawberry cells in a liquid medium to obtain metabolites such as anthocyanins produced in the cells.
In addition, the present invention is applied to breeding in which plant cells are cultured in a liquid medium to select cells having high production of a target metabolite. As plant cells, plant cells that can produce useful metabolites, not limited to strawberries, can be used, for example,
The present invention can be applied to carotene production by carrot cells, medicinal components produced by Korean carrot cells, lycopene production by tomato cells, pigment production by natural pigment-producing cells, and breeding of these producing cells.

The culture method of the present invention is a batch culture using a growth medium and a production medium alternately. That is, after culturing the cells in one medium, the cell suspension is replaced with the other fresh medium, the cells remaining slightly are cultured as seed cells in the other medium, and the culture is continued by repeating this medium exchange. Is the way. On the other hand, the continuous culture described above is a culture method in which a single medium is used, and the medium is stabilized by continuously (or intermittently) exchanging a part of the medium to perform the culture.
Different from the culture method of the present invention.

The growth medium is not particularly limited as long as it allows the cells to grow at a high growth rate, and includes, for example, commonly used basic mediums such as MS medium, LS medium, B5 medium, EM medium and the like. Modified medium that has been modified to the optimal concentration in the cells using the components, or based on these basic medium or modified medium,
A medium to which a plant growth regulator such as 4-dichlorophenoxyacetic acid (2,4-D) or 6-benzyladenine (BA), sucrose, or the like is appropriately added is used.

The production medium may be any medium that can control the growth of the cells to be used at a rate suitable for production, such as a culture medium containing a lower content of nutrient components or plant growth regulators than the growth medium, or a permeation medium. A medium with an increased pressure is used. In the case of (a) phosphoric acid,
The concentration of any of (b) the nitrogen source and (c) the plant growth regulator can be reduced below the concentration suitable for plant growth. By using such a medium, it is possible to grow cells while maintaining a specific growth rate suitable for production of a target secondary metabolite, and to efficiently culture cells having a high productivity of secondary metabolites at a high density. Become.

The medium exchange between the growth medium and the production medium is preferably performed in the logarithmic growth phase. That is, the cells are cultured in one medium, and before reaching the stationary phase, more preferably at the stage of reaching the logarithmic growth phase, a part of the culture solution is removed, and a fresh medium of the other medium composition is added. By exchanging the medium during the logarithmic growth phase, the state of high cell activity can be maintained, so that growth and production can be continued for a long time. In the medium exchange, the cell inoculation concentration is preferably about 10 to 50 gFW / L.
About 0 gFW / L is more preferable. Cell inoculation concentration of 10
If it is less than gFW / L, the cell growth may be delayed, and if it exceeds 50 gFW / L, the cell growth rate may be too high and the production may decrease.

Hereinafter, the present invention will be described in more detail with reference to anthocyanin production by strawberry cells. Substances considered as substrates for pigment production (eg, phenylalanine; P
he) is also essential for cell growth. Therefore, it is considered that Phe is actively accumulated in cells in the growth medium and is used for growth (partly used for pigment production). Therefore, when the medium is converted to a production medium, the growth is braked, so that Phe is in an excessive state, and the surplus is expected to be supplied to the dye synthesis.

In order to culture strawberry cells according to the present invention, cells suitable for callus formation are first aseptically removed from strawberries. The strawberry of the material is not limited to a specific variety, and can be used by selecting from various varieties. For example, Dutch strawberry and four seasons strawberry can be used.

Suitable cells for callus formation can be obtained from various parts such as strawberry leaves, stems, roots, runners, etc., and preferably, runners are used. After these parts are cut to an appropriate size, they are immersed in a bactericide usually used in tissue culture to sterilize them, transferred to an aseptic environment, washed with sterilized water, and then cut with a thin blade cutter or the like. ~
The growth point is excised while observing it as a fragment of about several mm or using a stereoscopic microscope.

Next, the cells obtained are placed on a solid medium to form a callus. As a solid medium used for this callus, a basic medium such as MS medium, LS medium, B5 medium, EM medium, auxin or auxin and cytokinin, a sugar such as saccharose as a carbon source, 0.8 to
A solid medium containing about 1% agar is used.

As the auxin, 2,4-dichlorophenoxyacetic acid (hereinafter, referred to as 2,4-D) is particularly preferably used, and as the cytokinin, benzyladenine (hereinafter, referred to as BA) is preferably used. 2,4
The addition amount of -D is 0.1 to 5 mg / L, and the addition amount of BA is 5
mg / L or less is preferred.

Culture for transforming strawberry cells into callus comprises:
At a temperature of about 20 to 30 ° C., the light condition is set to an illuminance of 3000 lx or less, preferably 800 lx or less.

A callus is formed from strawberry cells under such conditions, and subculture is performed as necessary to prepare a callus for mass culture. Among these, the white soft calli are subjected to the following preculture.

The above callus is subjected to shaking culture or stirring culture in a liquid medium for preculture. As this liquid medium, M
Auxin or auxin and cytokinin in a basic medium such as S medium, LS medium, B5 medium, and EM medium;
A liquid medium to which a sugar such as saccharose is added as a carbon source is used. As the auxin, 2,4-D is particularly preferably used, and as the cytokinin, BA is preferably used. The addition amount of 2,4-D is 0.1 to 5 m
g / L and BA are preferably added in an amount of 5 mg / L or less. Further, in the pre-culture of strawberry cells, it is preferable to set the light conditions at a temperature of about 20 to 30 ° C. and an illuminance of 800 lx or less.
By culturing under these conditions, callus cells proliferate in a liquid medium, and the cultured cells are scaled up and cultured as necessary to produce a desired amount of cultured cells.

The type of liquid medium and the nitrogen source concentration used in the preculture are not limited, and either a medium having the same composition as the liquid medium used in the next step of producing anthocyanin or a liquid medium having a different composition can be used. Usually, a medium having a high growth rate of cultured cells is used. For example, an LS medium or MS medium in which the growth of cultured cells is favorable, or a B5 medium in which the amount of a nitrogen source is increased can be used. This is used as a seed cell for culture.

Next, the seed cells grown in the above preculture are inoculated into a production medium and cultured. Here, the cell inoculation concentration is
It is preferably about 10 to 50 gFW / L.
gFW / L is more preferable. As described above, as the production medium, a medium in which the content of a nutrient component or a plant growth regulator is lower than that of a growth medium, or a medium in which the osmotic pressure is increased can be used. By using a medium having such a composition, cells can be grown while maintaining a specific growth rate suitable for anthocyanin production, and efficient culture can be achieved in which cells having high anthocyanin productivity are densified.

When a medium having a reduced content of nutrient components or plant growth regulators is used, (a) phosphoric acid,
The concentration of any of (b) the nitrogen source and (c) the plant growth regulator can be reduced compared to the growth medium.
In this case, in the production method, in order to improve the total yield, consideration is given to both the growth of the cells and the production amount per cell, and conditions are selected such that the product of the two is high. In the breeding method, conditions are selected so as to increase the production amount per cell.

For example, when the concentration of phosphoric acid in the production medium is limited, it is preferably about 1/4 of the growth medium in the culture for production, and about 1/4 of the growth medium in the culture for breeding.
It is preferred to be about 8 to 1/4. For example, when an MS medium or an MS modified medium (phosphate concentration: 1.25 mM) is used as a growth medium, the culture for production is 0.31 mM, and the culture for breeding is 0.16 to 0.16 mM.
Preferably, the concentration is about 0.31 mM.

[0024] or when limiting the nitrogen source concentration of production medium, NH ₄ ⁺ in the growth medium: NO ₃ ^- while maintaining the ratio, it is preferable to reduce the total nitrogen concentration. In a culture for production, it is preferably about 1/2 of the growth medium, and in a culture for breeding, it is preferably about 1/20 to 1/8 of the growth medium. For example, the growth medium contains ammonium nitrate5.
When an MS-modified medium containing 15 mM and 18.8 mM of potassium nitrate (total nitrogen concentration: 29.1 mM) was used, the total nitrogen concentration was about 14.5 mM in the culture for production, and the total nitrogen concentration in the culture for breeding. Is 3.7-14.5m
M (4 to 15 mM) is preferred.

When the 2,4-D concentration of the production medium is restricted, it is preferably about 1/2 of the growth medium for the culture for production, and one-half of the growth medium for the culture for breeding.
It is preferable to be about / 10 to １ ／. For example, when an MS medium or an MS-modified medium (phosphate concentration: 1.25 mM) is used as a growth medium, about 0.5 mg / L is used for culture for production, and about 0.1 mg / L for culture for breeding. It is preferred to be about 0.5 mg / L.

When a medium having an increased osmotic pressure is used, inexpensive substances that increase the osmotic pressure without adversely affecting the cultured cells, for example, saccharides such as mannitol, glycerin and dextrin, polyethylene glycol, and salts such as calcium chloride are used. Preferably, mannitol or sorbitol is used preferably.

Cultivation using the production medium having the above composition is carried out under conditions of illuminance of 2,000 to 8000 lx at 20 to 30 ° C. by stirring culturing with a jar fermenter, or rotating culturing with an Erlenmeyer flask. Then, preferably, before reaching the stationary phase, more preferably at the stage when the logarithmic growth phase is reached, a part of the culture solution is removed and a fresh growth medium is added. In the case of strawberry cells, the medium may be changed periodically on a 5- to 10-day cycle. Here, the cell concentration after adding the fresh growth medium is preferably about 10 to 50 gFW / L, more preferably about 20 gFW / L.

Here, as the growth medium, MS medium or LS
A basic medium such as a medium, a B5 medium, or an EM medium, or a modified medium, or a liquid medium to which auxin, auxin and cytokinin, and a sugar such as saccharose as a carbon source are added. The auxin is 2,4-D
Is particularly preferably used, and the cytokinin is B
A is preferably used. The amount of 2,4-D added is 0.1 to
5 mg / L and the amount of BA to be added are preferably 5 mg / L or less. One particularly preferred example is to reduce ammonium nitrate to 1/4 of 5.15 mM in MS medium, and use 6% sucrose, 1.0 mg / L 2,4-D, 0.1 m
A medium containing g / L of BA is exemplified.

Using the growth medium having the above composition, illuminance of 2000
Incubate at 20 to 30 ° C. under illuminance of 攪拌 8000 lx with stirring in a jar fermenter, or in a conical flask with swirling. Preferably, before reaching the stationary phase, more preferably at the stage when the logarithmic growth phase is reached, a part of the culture solution is removed and fresh production medium is added. In the case of strawberry cells, the medium may be changed periodically on a 5- to 10-day cycle.
Here, the cell concentration after adding the fresh production medium was adjusted to 10
About 50 gFW / L, preferably about 20 gF / L.
About W / L is more preferable. The above steps may be repeated to continue culturing, and culturing can be performed for a long time, for example, for 100 days or more. In the culturing method of the present invention, cells with high productivity are selected in the latter half of the culturing, so that cells with high productivity can be obtained from the cells after the cultivation according to the present invention.

[0030]

EXAMPLES The operation and effect of the present invention will be clarified by experimental examples. (Experimental example) A strawberry runner was soaked in a 5% synthetic detergent solution for 5 minutes, the surface was sterilized with a 10% NaClO solution for 10 minutes, and washed three times with sterile water. A growth point is aseptically extracted from the runner, and the growth point is determined by adding 1.0 mg / l of 2,4-D, 0.1 mg / l of BA and 0.09 M of sucrose,
It was placed on an LS solid medium containing 2.0 g / l agar and adjusted to pH 5.8 with 1 N KOH (before autoclaving), and cultured at 25 ° C and an illuminance of 800 lx. After 2 months of culture,
A white soft callus was induced. From here 1.0
The callus of 1.5 FW was taken out, and 50 ml of liquid MS medium (1.0 mg / L 2,4-D, 0.1 mg / L B
A, 3% sucrose) and transferred to a 300 ml Erlenmeyer flask, and cultivated by rotating and stirring at 25 ° C., 800 lx, and 80 rpm. Cultured cells obtained by subculture every 10 days were subjected to the following experiments. As the growth medium used below,
In the MS medium, a medium in which ammonium nitrate was reduced to 1/4 of the usual amount was used, and as a production medium, a medium in which ammonium nitrate and potassium dihydrogen phosphate were reduced to 1/4 was used. 2,4-D of L, 0.1
mg / L of BA was included. The culture in the growth medium and the production medium was performed in a 300 ml Erlenmeyer flask, each containing 50 ml of the medium, and under a 2.5 klx irradiation, a swirling culture.

Experiment 1: Cell Culture and Evaluation of Dye Productivity As described below, batch culture was repeatedly performed using the above cultured cells every 7 days at a cell inoculation concentration of 20 g FW / L. After inoculating the cultured cells into a production medium and culturing for 7 days,
The culture was replaced with fresh growth medium to 20 gFW / L. After a further 7 days of culture, the culture was replaced with fresh production medium to 20 gFW / L,
This exchange was repeated. As a further control, repeated batch culture was performed using only the growth medium or only the production medium. For these, the chromogenic productivity was evaluated based on the cell (wet weight) concentration, cell 1
The pigment productivity per gFW (wet weight; a cell group in which pigment-producing cells and non-producing cells coexist) and the pigment productivity per volume were performed. The cell concentration and the quantification of anthocyanins were in accordance with the usual methods.

FIG. 1 shows the results of repeated batch cultures of a medium conversion culture between a growth medium and a production medium (●), only a growth medium (△), and only a production medium (○). In the medium conversion culture (●) in FIG. 1, the value of the first batch is the production medium, the second is the growth medium, and the values are alternately repeated.
The cell concentration in the production medium was about 80 gFW / L, in the growth medium it was 200 gFW / L, and in the medium conversion section, both values were taken according to the medium. The productivity per cell in the medium conversion section was maintained at 0.3 mg / g FW.
In the growth medium, a decrease in pigment productivity per cell was observed as the number of batches increased. In particular, the tendency was remarkable in the production medium. Pigment productivity in medium conversion culture,
It can be seen that the productivity per cell does not decrease even in the latter half of the culture.

Experiment 2: Influence of culture cycle on medium change In the batch operation for medium change, the number of culture days was changed.
Cell inoculation concentration of 20 gF
Batch operations were performed as W / L and every 6 or 7 days. FIG. 2 shows the results of batch culture every 5 days (●), every 10 days (□), and every 15 days (▲). In FIG. 2, the value of the first batch is the production medium, the second is the growth medium, and the values are alternately repeated. In the test plots every 5 days, no change was observed in the cell growth even after the medium was changed. However, the pigment productivity of the cells was greatly affected, and oscillations were observed such that they were large in the production medium and small in the growth medium. This vibration increased with an increase in the number of batches. That is, it is considered that cells having higher pigment production were selected. On the other hand, in the test plots every 10 days, the cell growth was fluctuated due to the medium change, but the productivity per cell was not clearly different. In the test plots every 15 days, after the first batch operation, the cells did not proliferate at all, and further browning was remarkable, so further continuation was stopped.

FIG. 3 shows the results of the test performed every 6 days and 7 days based on the test every 5 days. In FIG. 3, the value of the first batch is the production medium, the second is the growth medium, and the values are alternately repeated. In the batch operation every 6 days and every 7 days, a clear oscillation was observed in the cell proliferation. Oscillation is partially observed in the productivity of the dye, but not every 5 days. These oscillations are thought to be tightly controlled by the culture cycle.

Experiment 3: Influence of Cell Inoculation Volume Batch culture was performed with a medium change in which the cell inoculation concentration (20, 60, 100 g FW / L; batch operation every 7 days) was changed. FIG. 4 shows the results. In FIG. 4, the value of the first batch is the production medium, the second is the growth medium, and the values are alternately repeated. The cell concentration on day 7 was dependent on the inoculum concentration. On the other hand, the pigment productivity per cell was 0.2 to 0.4 mg / gF at a cell inoculation concentration of 20 gFW / L.
While W is maintained, 60,100 gFW / L
It was 0.1 or less in the ward. Therefore, in repeated batch culture, the initial cell concentration at the time of batch is an important factor.

[0036]

As described above, the oscillation of growth and production is reproduced over a long period of time, and the amount of production per cell is rather increased without a decrease in cell activity. Therefore, it is suitable for long-term continuous culture production. In addition, since cells with high productivity are selected in the latter half of the culture, they are suitable for breeding high-producing strains. In addition, by exchanging the medium during the logarithmic growth phase, the state of high cell activity can be maintained, so that growth and production can be continued for a long time.

[Brief description of the drawings]

FIG. 1 is a graph showing the results of Experiment 1 of an example of the present invention.

FIG. 2 is a graph showing the results of Experiment 2 of the example of the present invention.

FIG. 3 is a graph showing the results of Experiment 2 of the example of the present invention.

FIG. 4 is a graph showing the results of Experiment 3 of the example of the present invention.

Claims (6)

[Claims] 1. A method for culturing a plant cell in a growth medium for growing a plant cell, and a method for growing the plant cell in a production medium in which the cell is grown at the optimum growth rate such that a secondary metabolite is accumulated in the cell. A method for culturing a plant cell, comprising alternately performing culturing. 2. The method according to claim 1, wherein the production medium is compared with the growth medium.
The plant cell culture method according to claim 1, wherein the concentration of any one of the nutrient component and the plant growth regulator is reduced. 3. The production medium according to claim 2, wherein the production medium is
2. The method according to claim 1, wherein the osmotic pressure is increased. 4. The plant cell culture according to claim 1, wherein the medium exchange between the growth medium and the production medium is performed in a logarithmic growth phase of each culture. Method. 5. The method according to claim 1, wherein the medium exchange between the growth medium and the production medium is performed so that the cell inoculation concentration after the exchange becomes 10 to 50 gFW / L. Plant cell culture method. 6. A plant cell which produces a high secondary metabolite, which is obtained by culturing using the method for culturing a plant cell according to any one of claims 1 to 5.