JPH074234B2 - Plant tissue culture method and culture device - Google Patents

Description

TECHNICAL FIELD The present invention relates to a plant tissue culture method and a culture apparatus. More specifically, the present invention relates to a method for efficiently culturing under a higher tissue culture concentration than that of the conventional method, and a culture device for carrying out the method.

[Conventional technology]

As conventional plant tissue culture methods, batch culture method and continuous culture method are known.

(1) Batch culture Since the yield of tissue culture (cultured cells, etc.) in batch culture depends on the nutrient substrate (medium components) concentration in the medium, the rinsemeyer and skoog medium often used in conventional batch culture. , White medium and Nichi &Nichi's medium consume nutrient substrates during culturing, and it is not possible to cultivate at a high tissue culture concentration. Further, as a method of increasing the tissue culture concentration, a method of culturing by increasing the nutrient substrate concentration of the medium as compared with the conventional method can be considered, but if the nutrient substrate concentration is too high, inhibition of the nutrient substrate concentration such as necrosis of the tissue culture will occur. Since this method has a limit in increasing the nutrient substrate concentration to increase the productivity of the tissue culture, the yield of the tissue culture per unit volume of the culture tank is low and the culture efficiency is low.

(2) Continuous culture As a conventional continuous culture method, there is known a method in which a tissue culture and a culture solution are discharged from the culture tank at any time, and the medium is renewed and cultured [eg, "Fermentation Engineering" Volume 61 1 1
28 (1983)]. However, since this method is a method in which an amount of culture solution equal to that of the fed medium is discharged out of the tank together with the tissue culture, the concentration of the tissue culture relative to the culture solution is usually expressed as a fresh weight. Since it is less than 100 g /, it is not possible to culture it at a tissue culture concentration higher than this.
Therefore, in this method, the yield of the tissue culture per unit volume of the culture tank is low, and the culture efficiency is poor.

[Problems to be solved by the invention]

Against this background, the present inventors have improved the conventional culture method using a liquid medium to efficiently produce a tissue culture, that is, the yield of the tissue culture per unit volume of the culture tank is high, We investigated a high-density culture method that enables more efficient culture than the conventional method.

[Means for solving problems]

As a result, they have found that the above object can be achieved by adopting the following method, and completed the present invention. That is, according to the first aspect of the present invention, when a plant is tissue-cultured using a liquid medium, the concentration of the tissue culture in the culture tank during the culture is expressed as 100% of the fresh weight of the tissue culture. ~ 500
As in the range of (g /), at least for a certain period, while continuously or discontinuously supplying a liquid medium in which the nutrient substrate concentration of the medium is adjusted to a predetermined concentration to the culture tank, from the other of the culture tanks, So that the average concentration of tissue culture in the total effluent is lower than the concentration of tissue culture in the tank: 1) at least for a period of time, withdrawing the culture solution continuously or discontinuously, and 2) at least The culture mixture (culture medium + tissue culture medium) is continuously or discontinuously withdrawn from the outlet of the culture tank for a certain period to continuously culture while updating the culture solution in the culture tank. A method for tissue culture of a plant is provided.

According to the second aspect of the present invention, the culture solution inlet and
An aerative culture apparatus having at least one culture solution discharge port, wherein at least one culture solution discharge port is provided with a filter for preventing discharge of tissue culture,
There is provided a culture device characterized in that a filter for preventing the discharge of tissue culture is not provided at each culture solution discharge port.

[Plant tissue culture method]

The plant to which the method for tissue culture of the plant of the present invention is applied is not particularly limited, and it is possible in principle to apply the method of the present invention to the plant as long as it is a plant to which conventional tissue culture can be applied. . As such plants, specifically, butterflies of the buttercup family, poppy of the family of the family of the family of theaceae, licorice of the plant of the family of the family of theaceae, carrots of the plant of the family of Ceriaceae, rhododendron japonicus, rhubarb of the plant of the Polygonaceae family, indica japonica, periwinkle, Tobacco of Solanaceae, purple of Musaceae, perilla of Lamiaceae, deer, deer, coffee of Rubiaceae, digitalis of Sesameaceae can be mentioned.

In the present invention, the tissue of the plant (including cells and organs) is cultivated using a liquid medium, and the liquid medium in this case is used in conventionally known plant tissue culture. A medium can be used. Specifically, as the medium, an inorganic component, a carbon source and a plant hormone are essential components,
This is a medium in which vitamins are added, and amino acids are further added as necessary. As the inorganic component of the medium,
Inorganic salts containing elements such as nitrogen, phosphorus, potassium, sodium, calcium, magnesium, sulfur, iron, manganese, zinc, boron, molybdenum, chlorine, iodine and cobalt can be mentioned, and specifically, potassium nitrate and sodium nitrate. , Ammonium nitrate, ammonium chloride, potassium chloride, calcium chloride, potassium monohydrogen phosphate, sodium dihydrogen phosphate, magnesium sulfate, magnesium chloride, sodium sulfate, ferrous sulfate, ferric sulfate, manganese sulfate, copper sulfate, molybdenum Examples thereof include compounds such as sodium acid salt, molybdenum oxide, potassium iodide, zinc sulfate, boric acid and cobalt chloride.

Examples of the carbon source of the medium include carbohydrates such as sucrose and derivatives thereof, organic acids such as fatty acids, and primary alcohols such as ethanol.

The plant hormone of the medium includes indoleacetic acid (IA
A), naphthalene acetic acid (NAA), P-chlorophenoxyisobutyric acid and 2,4-dichlorophenoxyacetic acid (2,4-D)
And auxins and kinetin, zeatin, and cytokinins such as penzyljiluadenine.

Examples of vitamins in the medium include biotin, thiamine (vitamin B ₁ ), pyridoxine (vitamin B ₆ ), pyridoxal, pyridoxamine, calcium pantothenate, ascorbic acid (vitamin C), inositol, nicotinic acid, nicotinic acid amide and riboflavin ( vitamin
B ₂ ) etc. can be illustrated.

Examples of amino acids in the medium include glycine, alanine, glutamic acid, cysteine, tyrosine, and lysine.

The liquid medium used in the present invention is usually about 0.1 μM to about 100 mM of the inorganic component, about 1 g / to about 100 g / of the carbon source, and 0.01 μM to 1000 μM of the plant hormone, preferably 1 μM. It is preferable that the vitamins are contained in the range of 0.1 to 100 μM, and the vitamins are contained in an amount of about 0.1 mg / to about 150 mg / and the amino acids of 0 to about 1000 mg /.

Specifically as such a medium containing each component, a medium conventionally used for tissue culture of plants,
For example, Murashige & Sko ('62)
og] medium, Rinsmeier Scoog (MR-1965)
[Linsmaier & Skoog] Medium, White ('63) [Whi
te] medium, Gamborg's B-5 medium, Mitsui's M-9 medium, etc., with the above-mentioned carbon source and plant hormone (A) added, and if necessary, the above-mentioned vitamins and amino acids. Although a medium prepared by adding the above can be exemplified, in the present invention, a medium prepared by using a Rinsmeier-Skoog or Murashige-Skoog medium is particularly preferable in the present invention. The composition of the conventionally known medium described above is described, for example, in “New Plant Tissue Culture” P386 to P391 by Takeuchi, Nakajima, and Otani, Asakura Shoten, 1979.

In the present invention, when tissue culture of the plant is performed using the liquid medium, the culture is performed under the following conditions that characterize the present invention. The conditions will be described in detail below.

(1) In the plant tissue culture method according to the present invention, the tissue culture concentration in the culture tank during the tissue culture is 100 to 500 [g /], preferably 200 to 400 [g /] with respect to the liquid medium.
The culture is carried out in the range of []. Since the tissue proliferates and increases its amount during the culturing period, in the present invention, an appropriate amount of the tissue culture is taken out of the culture tank during the culturing period by an appropriate method, so that the culture is performed. Any method may be used to maintain the tissue culture concentration in the bath within the above range. The weight of the tissue culture in this case is a fresh weight measured in a wet state. Here, the fresh weight is the weight of the tissue culture, which is usually obtained by the following method. That is, it is the weight of the tissue culture when the tissue culture is placed in a nuttier with a filter cloth and filtered for 5 minutes with a water washing pump or the like to remove the culture solution outside the tissue culture. The fresh weight of the tissue culture thus obtained varies depending on the plant, but the water content is usually 80 to 95.
wt%.

Further, the tissue culture is a culture of plant tissue pieces or cells, and the tissue pieces include shoot tips, stems, leaves, flowers, seeds,
Tissue pieces such as roots can be exemplified, and callus derived from the tissue pieces and cultured cells are also included. The tissue culture can then be used as a raw material for tissue culture. In the present invention, when the tissue culture concentration in the culture is less than 100 g /,
The yield of tissue culture per unit volume of the culture tank is low, and therefore the efficiency of culture is low as in the conventional method, which is not preferable. Further, when the tissue culture concentration exceeds 500 (g /) and is increased, the chiller concentration becomes high and the stirring and mixing of the culture solution become insufficient, making it difficult to efficiently supply oxygen. , Necrosis of the tissue culture occurs during the culture, which makes it difficult for the tissue culture to normally grow and grow. Further, if the agitation is vigorously performed in order to sufficiently supply the enzyme, the tissue culture may be destroyed or damaged, which makes the culture difficult. Thus, the tissue culture concentration was 500 [g /
If the value is higher than [], unfavorable conditions for culturing may occur. Therefore, the tissue culture concentration should be 100-500.
The culture is carried out so that it is in the range of [g /].

In the present invention, the tissue culture concentration during the culture is carried out while being maintained within the above range, in this case,
In order to satisfy the condition, it is possible to appropriately extract an appropriate amount of tissue culture so that the concentration of the tissue culture in the culture tank does not exceed the concentration range due to the growth of the tissue culture. As described above. Even if the tissue culture grows during the culture period and the concentration becomes high,
When the tissue culture concentration does not exceed the above concentration range, it is not always necessary to take out the tissue culture from the culture tank. In the present invention, the concentration of the tissue culture in the culture tank is not necessarily 100 to 500 (g /) from the beginning of the tissue culture.
It is not necessary to set the range to, for example, the concentration of plant tissue pieces or cultured cells in a culture tank in a liquid medium is 100
It may be less than [g /] and charged in a culture tank for tissue culture. In this case, since the cells grow during the culture and the concentration of the tissue culture is increased to 100 to 500 [g /], the tissue culture according to the method of the present invention can be performed. In the present invention, tissue culture can be performed by either a batch method or a continuous method.

(2) In the present invention, the tissue culture concentration should be 100 to 500 [g /] so as to satisfy the above condition (1).
The liquid medium in which the nutrient substrate concentration of the medium is adjusted to a predetermined concentration is continuously or discontinuously supplied to the culture tank for at least a certain period so that only the culture solution or the culture solution is supplied from the other of the culture tanks. Both the liquid and the culture mixture are continuously or discontinuously withdrawn so that the average concentration of the tissue culture in the total effluent is lower than the concentration of the tissue culture in the tank and placed in the culture tank. The tissue culture is carried out while the culture medium is renewed. The culture medium may be renewed continuously or discontinuously.

Here, the nutrient substrate is a medium component of the liquid medium used in the present invention described above, and is the aforementioned inorganic component, carbon source, plant hormone, vitamins and amino acids.

In addition, the nutrient solution concentration is adjusted to a predetermined concentration, as described above, an inorganic component of about 0.1 μM to about 100 mM, a carbon source of about 1 to about 100 g /, and a plant hormone of about 0.01 to 1000.
μM, preferably 1 to 100 μM, about 0.1 to about vitamins
It is a liquid medium in which the concentration range of 150 mg / amino acid is adjusted to 0 to about 1000 mg /. In the present invention, it is usually preferable to use a fresh liquid medium for the liquid medium in which the concentration of the medium components supplied to the culture tank is adjusted to fall within the above range, but the nutrient of the medium extracted from the culture tank is preferably used. It is possible to use a method in which the substrate concentration is adjusted and then reused again. In the case of circulating use, it is preferable to perform a treatment for appropriately removing waste products generated by metabolism of cells in the culture solution.

In performing the tissue culture while renewing the culture medium in the present invention, the amount of the culture medium in the culture tank is V [], the amount of the liquid medium supplied to the culture tank is F [/ day], and F / V 〔day
^-1 ]] It is preferable to carry out tissue culture of plants so that the relationship between the medium renewal rate and the specific growth rate of tissue culture μ [day ^-1 ] is in the range of μ ≦ F / V <20μ . Here, the specific growth rate μ [day ⁻¹ ] is an amount defined by the following method.

When the tissue culture is not discharged from the tank
When the amount of tissue culture of to is Xo and the amount of tissue culture after t hours (days) is xt, the specific growth rate μ is defined by the following equation.

When the tissue culture grown while maintaining the amount of tissue culture at Xo was discharged to the outside of the tank, if the amount of tissue culture discharged outside the tank after t hours (days) was Yt, the specific feeding rate μ was Is defined.

And the physical meaning of μ has a dimension of [time ^-1 ], and the larger the value of μ, the faster the growth of the tissue culture.

In the present invention, the value of the specific growth rate μ depends on the tissue culture of the plant, but is usually 0.02 to 0.4 (day ^-1 ), preferably 0.05 to 0.2 (day).
^-1 ] The tissue culture is performed in the range of [ ¹ ]. In the present invention, when the liquid medium in which the concentration of the nutrient substrate of the other culture medium is adjusted to a predetermined concentration is continuously or discontinuously supplied to the culture tank, the supply amount F [/ day] is μ ≦ F / V < As described above, it is preferable to determine so as to satisfy the equation of 20μ. In the present invention, the medium renewal rate F / V is usually 0.02 to 8 [day ^-1 ], preferably 0.05 to 4 [day ^-1 ].
Is in the range.

In the present invention, when the culture solution is continuously or discontinuously extracted from the other of the culture tanks, the concentration of the tissue culture in the culture tank during the culture is set within the range of 100 to 500 (g /) described above. Therefore, only the culture solution is discharged for at least a certain period. Here, at least for a certain period of time, only the culture solution or both the culture solution and the culture mixture is discharged so that the average concentration of the tissue culture in the whole discharge is lower than the concentration of the tissue culture in the tank. It means that normally only the culture solution containing no tissue culture may be extracted, and if necessary, an appropriate amount of the culture mixture may be extracted together with the culture solution. When tissue culture is carried out while extracting only the culture solution, it can be carried out using a culture tank having a culture solution discharge pipe mechanism equipped with a filter according to the present invention described later.

In the present invention, when the medium renewal rate (F / V) is F / V <μ which is smaller than the specific growth rate μ, the tissue culture concentration becomes too high, and the optimal concentration of tissue culture cannot be maintained. For that reason, it is preferable to take out an appropriate amount of cultured cells rather than taking out only the culture solution from the culture tank. For example, while taking out the culture solution containing the tissue culture from the discharge pipe provided in the culture tank, It is also possible to feed a liquid medium having a nutrient substrate concentration adjusted to the above-mentioned predetermined concentration into a culture tank to culture the tissue culture in the culture tank within the above range.

In the present invention, when the medium renewal rate (F / V) is 20 μ ≦ F / V, which is extremely higher than the specific growth rate μ, adverse effects such as blackening and necrosis of the tissue culture usually occur. Therefore, in the present invention, it is particularly preferable to perform tissue culture by controlling the medium renewal rate (F / V) within the range of μ ≦ F / V <20μ.

In the present invention, the amount of the culture solution continuously or discontinuously withdrawn from the other of the culture tanks is usually an amount corresponding to the culture amount F [/ day] supplied to the culture tank. However, it is not necessarily limited to this amount. For example, when culturing under the condition of F / V <μ mentioned above, the amount of medium to be withdrawn may be increased more appropriately than F [/ day] if necessary. It doesn't matter.

[Effects of the tissue culture method of the present invention]

A continuous culture method in which the culture solution is renewed and cultivated is also known in the tissue culture of plants that have been conventionally used, but in this case, as described above, not only the culture solution but also the tissue culture is removed from the culture tank at the same time. Since it is a method of extracting a tissue culture, it is not possible to culture a tissue culture at a high concentration as in the method of the present invention. On the other hand, in the present invention, during the culturing period, only the culture solution in the culture mixture is devised so that it can be withdrawn to the outside of the culture tank. A dense tissue culture method has become possible.

Generally, when trying to cultivate a high concentration of tissue culture in plant tissue culture, the consumption rate of the substrate (medium component) consumed by the tissue culture is high because of the high concentration of tissue culture. Therefore, when a culture medium having a normal substrate concentration is used, it is difficult to grow the tissue culture satisfactorily because the substrate will soon run out. Therefore, a method of culturing using a culture medium with a substrate concentration higher than usual can be considered, but if the concentration is too high, it causes problems such as necrosis and abnormal growth of the tissue culture, so the concentration is naturally limited. There is. The present invention recognizes such a point, and proposes a high-density tissue culture method which has never been attempted. According to the aforementioned tissue culture method of the present invention, the concentration of the tissue culture is Even when the culture is performed at a higher level than in the conventional method, the culture solution is renewed, so that the growth of the tissue culture is weakened or the growth of the cells is inhibited by the waste products discharged to the outside of the cell. Therefore, it is possible to grow the cells sufficiently and culture the tissue more efficiently than before.

[Cultivation device]

The above-described plant tissue culture method of the present invention is preferably carried out, for example, using the culture apparatus according to the present invention described in detail below. The culture apparatus has a culture solution inlet and two or more culture solutions. An aerative culture apparatus having a discharge port, wherein at least one culture solution discharge port is provided with a filter that prevents discharge of tissue culture, and at least one culture solution discharge port. The culture device is characterized in that it is not provided with a filter for preventing the discharge of

The culture apparatus of the present invention will be described using the culture apparatus illustrated in FIG. In the present invention, in the discharge pipe for taking out the culture solution, a filter is attached to the opening 3 located inside the culture tank and in contact with the culture solution. The culture solution containing no substance can be discharged to the outside of the culture tank through the discharge pipe. Specific examples of the filter include a net made of a metal material or a synthetic resin material, and a cloth-like material having pores obtained by processing a synthetic fiber or a natural fiber. Here, the size of the mesh or the size of the pores is arbitrary as long as it does not allow the tissue culture to pass but allows only the culture solution to pass. If a net or the like is used as the filter, two or more nets may be stacked and used as needed. Regarding the position of the opening 3 of the discharge tube equipped with the filter inside the culture tank, it does not have to be provided at any position as long as the opening is in contact with the culture solution, but it is effective to renew the culture solution. Since it needs to be carried out in a desired manner, it is desirable that the position is as far as possible from the supplied medium introduction port 2.

In the present invention, not only the culture solution can be extracted from this portion of the discharge pipe having the opening 2 to which the filter is attached, but also the pressurized fluid is fed from the other of the discharge pipes connected to the opening. Therefore, it is convenient that a mechanism is provided so as to prevent the filter from clogging by the cells by ejecting the fluid from the opening. This case will be further described with reference to FIG. The culture solution is discharged from the opening 3 through the culture solution outlet 4 to the culture solution outlet 4. On the other hand, in order to prevent the filter portion of the opening 3 from being clogged by the cultured cells, or as a method for removing the already clogged cells, the pressurized fluid inlet 8
A more appropriate amount of pressurized fluid is introduced and ejected from the opening 3 to remove cells clogged by this external force and prevent clogging. In this case, the ejection of the pressurized fluid may be performed at the same time as the withdrawal of the culture fluid through the opening 3, or the withdrawal of the culture fluid and the ejection of the pressurized fluid are alternately performed at appropriate intervals. be able to.

In the present invention, as the pressurized fluid ejected from the opening 3,
Any material may be used as long as it does not adversely affect the culture, but normally an oxygen-containing gas such as air, a pressurized gas such as titanium gas or a pressurized liquid medium can be used. The jetting amount and pressure of the pressurized fluid may be any amount as long as it can prevent the clogging of the filter in the opening 3, and the pressurized fluid may be jetted continuously or intermittently from the opening 3 as described above. You can

The culture apparatus used in the tissue culture of the plant of the present invention includes, in addition to the discharge tube having the opening 3 equipped with the filter described above, the medium introduction tube 1, the culture mixture during the culture or after the culture (culture medium + It has a discharge pipe 7 for extracting the tissue culture). In addition, the culture device used in the present invention has a vent pipe 5 or a stirring device for aerating an oxygen-containing gas (for example, air) used in the conventional culture device into the liquid medium, if necessary. May be. As a stirring device, for example, a culture tank for performing a new gyro-like running motion that requires almost no external force such as shearing force to damage cells to the culture proposed by the applicant in Japanese Patent Application No. 59-103938, or The rotating cylinder type culture device proposed in JP-A-58-134989 may be used.

〔The invention's effect〕

According to the plant tissue culture method of the present invention, since the yield of the tissue culture per unit volume of the culture tank can be increased as compared with the conventional method, when the tissue culture is performed on an industrial scale, Compared with this, it becomes possible to produce a tissue culture economically and efficiently. Moreover, high-density culture can be efficiently carried out by using the culture apparatus of the present invention.

Example 1 Cultured cells of Coptis japonica were cultured in Rinsmeier and Skoog's medium using the culture apparatus of the present invention. The culture equipment is 10 aeration-stirring type culture tanks as shown in Fig. 1 with a pore of about 0.4 mm at the outlet of the culture solution and a filtration area of about 1
The culture was carried out by using a 0 cm ² nylon mesh so that only the cell-free culture solution could be discharged to the outside of the culture tank. The culture was continued at 25 ° C. for 2 weeks, during which the cultured cells were allowed to accumulate in the tank without being discharged out of the tank.

The daily medium renewal rate F / V values in accordance with the growth of the cultured cells to 0.5% of the sugar concentration in the culture liquid discharged from the culture vessel 0.3 to 1.0 [day of the culture solution in this process ^{- It} was controlled within the range of ¹ ]. After 2 weeks of cultivation, the amount of the culture solution in the culture tank is 5
Double the volume of rinsemeyer and skoog medium was supplied. Table 1 shows the cell concentration and berberine yield after the culture.
The cells grew about 6.6 times in 2 weeks, and finally reached a cell concentration of 395 g /.

Comparative Example 1 Culture was started in a medium containing the total amount of nutrients supplied in Example 1, that is, a medium having a concentration 6 times that of Rinsmeier and Skoog's medium, and the medium for the culture period (2 weeks) was not updated at all. Culture was performed. Most of the cells were necrotic due to the inhibition of nutrient substrate concentration due to the high concentration of medium components. Table 1 shows the cell concentration and berberine yield after the culture.
It was shown to.

Comparative Example 2 Culture was started using a Rinsmeier-Skoog medium having the same concentration as that of Example 1 (one-fold concentration), and then a 20-fold concentrated medium of the Rinsmeier-Skoog medium was used in the same manner as in Example 1. An appropriate amount of a 20-fold concentrated medium was supplied to the culture tank so that the sugar concentration of the culture solution in the culture tank was 0.5%, and the culture was continued for 2 weeks without discharging the culture solution. Table 1 shows the cell concentration and berberine yield after the culture.

Example 2 Cultured cells of Coptis japonica were cultured in a Rinsmeier and Skoog medium using the culture apparatus according to the present invention. The same culture tank as in Example 1 was used as the culture device. Rinsmeyer and Scoog's medium was supplied so that 70% of the culture solution in the culture tank was renewed per day (medium renewal rate 0.7 day ^-1 ), so that the culture solution volume in the culture tank became constant. The culture medium containing no cells was discharged through the culture medium discharge pipe 4.
When the cell concentration in the culture solution exceeds 350 g / (fresh weight), the cells are discharged together with the culture solution from the tank through the discharge pipe 7 for the culture mixture, and the cell concentration is controlled so that the cell concentration becomes 350 g /. Meanwhile, continuous culture was performed. As a result, the specific growth rate μ of cells reached 0.12 [day ⁻¹ ] and the cell production amount per day was 42 g / (fresh weight).

Comparative Example 3 Rinsmeyer and Scoog's medium was added to the culture medium 70 per day.
%, The culture mixture was supplied in such an amount that the content was renewed, and the same culture mixture as that was discharged through the discharge pipe 7 to perform the culture, and the operation of separately extracting only the culture solution according to the present invention was not performed.

As a result, most of the cells in the culture tank flowed out after 2 weeks of culture, making it impossible to continue the culture.

Comparative Example 4 Conventionally known continuous culture was performed. That is, the rinse-meyer and Scoog's medium is supplied into the culture tank,
The culture was performed while discharging the same amount of the culture solution together with the cells out of the tank. As a result, a steady state is reached when the medium renewal rate is 0.1 [day ^-1 ] or less, and among them, the medium renewal rate 0.1 [da]
y ⁻¹ ], the highest cell yield was obtained. The specific growth rate μ at that time was 0.1 [day ^-1 ] and the cell concentration was 65 g / (fresh weight).
The daily cell yield was 6.5 g / (fresh weight) and 1
The cell yield per day was as low as about 15% of that in Example 2.

Examples 3 to 5 As plant cells other than the buttercup family, as a plant cell of the family Musaceae (Lithospemun), periwinkle plant (Lochnera) and a plant of the family Solanaceae (Nicoti).
Table 3 shows the results of culturing by the same method as in Example 2 using ana) and the respective cultured cells.

[Brief description of drawings]

FIG. 1 is a view showing an example of a culture device used for performing tissue culture with high density according to the present invention. 1 ... Medium introduction tube 2 ... Medium introduction port 3 ... Opening part equipped with a filter for discharging only culture solution 4 ... Culture solution discharge tube 5 ... Oxygen-containing gas aeration tube 6 ... Stirring blade 7 ... Culture mixture (tissue culture + culture medium) discharge pipe 8 ... Pressurized fluid inlet a, b, c, d, e ... Valve

Claims (4)

[Claims] 1. When performing tissue culture of a plant using a liquid medium, the concentration of the tissue culture in the culture tank during the culture is expressed as fresh weight of the tissue culture and is in the range of 100 to 500 [g /]. In the meanwhile, at least for a certain period of time, a liquid medium in which the nutrient substrate concentration of the medium has been adjusted to a predetermined concentration is continuously or discontinuously supplied to the culture tank, while from the other of the culture tanks, the tissue culture of the total discharge is 1) At least for a period of time, only the culture solution is continuously or discontinuously withdrawn so that the average concentration is lower than the concentration of the tissue culture in the tank, and 2) for at least a certain period of time, the culture mixture (culture solution + tissue). (2) Tissue culture method for plant characterized in that continuous culture is carried out while renewing the culture solution in the culture tank by continuously or discontinuously extracting the culture) from the outlet of the culture tank. 2. When renewing the culture medium, the amount of the culture medium in the culture vessel is V [] and the amount of the liquid medium supplied to the culture vessel is F [/ day], and F / V [day ^-1 ] The tissue culture of the plant is performed such that the relationship between the medium renewal rate and the specific growth rate of tissue culture μ [day ^-1 ] defined in [] is in the range of μ ≦ F / V <20μ. Claim (1)
Method described in section. 3. A ventilated culture apparatus comprising a culture solution inlet and two or more culture solution outlets, wherein at least one culture solution outlet is provided with a filter for preventing the discharge of tissue culture. In addition, at least one culture solution discharge port is not provided with a filter that prevents discharge of the tissue culture medium. 4. The culture device according to claim 3, wherein a pressurizing fluid feed-in pipe from the outside of the device is connected to the installed discharge port.