JP2724151B2 - Culture method of plant cultured cells - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to an apparatus for culturing and growing plant cultured cells, and more particularly to an apparatus for culturing and growing plant cultured cells for producing secondary metabolites. It is an effective invention.

(Conventional technology and its problems) With the development of biotechnology, cultivation of various plant cells has been widely performed, and accordingly, various culturing methods have been conventionally considered.

These conventional culture methods adopt a culture method of microorganisms,
Many improvements have been made, and the main focus of the improvement is on the fact that the cells are large and vulnerable to mechanical shock, and that the cells easily form cell clumps. Methods have been used. Liquid shaking culture method as a method for culturing plant cells, bubble column fermenter method, and a method for culturing such aeration stirred culture tank method, the former two has the drawback of a low K _La.

In addition, the above-described aeration-stirred culture tank method directly agitates a suspension of plant cultured cells with a rotary wing while feeding sterilized air. In addition, it is not only necessary to select cells that are resistant to mechanical shock, and to stir with a strength less than the limit stress, because the cultured cells are always subjected to mechanical shock, If cell clumps formed, they had to be removed to maintain the efficiency of the agitation.

Furthermore, the culture method in which the cultured cells are not in a suspension state has a drawback that a uniform cell population is not obtained because the surface state and the inner layer of the cell mass differ in the state of contact with the medium or air.

The culturing apparatus of the present invention allows plant culture cells to be attached to a cell holding net or a porous support frame and circulates the culture solution and dissolved oxygen without any corners, thereby preventing the plant culture cells from being mechanically impacted. , Which are grown homogeneously.

(Means for Solving the Problem) The apparatus for culturing plant cultured cells according to the present invention comprises a glass cylinder that has a lower end opening closed by a bottom plate, and a lid plate that can be opened and closed at the upper end opening to store a liquid medium. A rectifying plate fixed to the bottom plate by a stay at a position substantially concentric with the glass tube in the glass tube and forming a cylindrical portion rising upward from the inner peripheral edge of the circular plate; A support frame having a large number of small holes, a first magnet attached to a vertical axis of a motor provided below the bottom plate, and rotationally driven below the bottom plate; A second magnet at the lower portion facing the first magnet and the bottom plate and rotatably supported on the bottom plate and coupled to the centrifugal wing; and a rectifying plate inserted through the cover plate and inserted into the support frame. Nozzle with a plurality of small holes in the cylindrical part It consists of a trachea.

(Operation) This device uses a diffuser to introduce sterilized air into the culture tank at 0.1 to
1.0 vvm (volume of gas fed per minute to water 1) introduced, in accordance with the growth of cultured cells, K _La 100 to 300 start (gas efficiency to dissolve in water) ^-h, after starting growth The number of revolutions of the centrifugal wing is increased so as to be 300 to 2000- ^h, and culturing is performed such that plant cultured cells are attached to the outer peripheral surface of the support frame by a water flow.

(Example) As shown in FIG.
The lower end opening of the glass tube 1 is closed by fixing a disc-shaped bottom plate 2 having an outer diameter slightly larger than the outer diameter of the glass tube 1 to the lower end of the glass tube 1. The lower ends of the plurality of studs 3 are screwed and fixed to portions of the bottom plate 2 near the outer periphery and protruding outside the outer peripheral surface of the glass tube 1. The upper end of each stud 3, 3 projects slightly above the upper edge of the glass cylinder 1, respectively.
The upper end is inserted into circular holes 5, 5 formed in a portion near the outer periphery of a disk-shaped cover plate 4 having an outer diameter slightly larger than the outer diameter of the glass cylinder 1. In this embodiment, nuts (not shown) are screwed into upper ends of the studs 3, 3 projecting from the upper surface of the lid plate 4 through the circular holes 5, 5, and the upper lid plate 4 is attached to the glass cylinder. 1 is openably and closably mounted on the upper end opening, and the bottom plate 2 is pressed and fixed to the glass cylinder 1.

A rectifying plate 25 is provided at the inner lower portion of the glass tube 1 on the inner peripheral edge of a horizontal circular plate 23 provided substantially concentrically with the glass tube 1 and a cylinder 24 rising upward from the inner peripheral edge.
Are fixed on the bottom plate 2 by stays 7 and 7.

The lower end of a cylindrical support frame 6 made of a stainless steel filament braided (160 mesh) is externally fitted to the cylinder 24 of the current plate 25. Inside the glass tube 1, the glass tube 1 is fixed substantially concentrically.

On the other hand, a vertical shaft 8 is planted at the center of the upper surface of the bottom plate 2, and an inverted cylindrical cap 10 fixed to the center of the rotating block 9 is rotatably attached to the vertical shaft 8. I have. An annular first magnet 11 is provided around the rotating block 9.
The first magnet 11 and the bottom plate 2
An annular second magnet 16 fixed to an output shaft 15 of a motor 14 fixed via a bracket 13 below a substrate 12 to which is fixed is opposed to each other across the bottom plate 2.

A plurality of flat blades 17 are provided on the outer periphery of the rotating block 9 rotatably supported on the vertical shaft 8 via the cap 10.
The centrifugal impeller 18 is configured by fixing the radial direction 17.

Further, above the centrifugal impeller 18, a bubble nozzle 19 for blowing sterilized air into the liquid medium stored inside the glass cylinder 1 is provided at an inner portion of the cylinder 24 constituting the straightening plate 25. Is provided. The bubble nozzle 19 has a plurality of small holes 21 at the lower end of an air supply pipe 20 fixed to the center of the lid plate 4 that opens and closes the upper end opening of the glass cylinder 1 while penetrating the lid plate 4.
The nozzle 22 having the nozzle 21 is fixed, and the compressed gas sent through the air supply pipe 20 is ejected to a position immediately above the centrifugal impeller 18.

Although illustration and detailed description are omitted, the lid plate 4 includes, in addition to the air supply tube 20, a sensor for detecting the state of the culture solution in the glass cylinder 1, a tube for sending nutrients into the culture solution, and the like. Is provided.

The case where the cultured tobacco cells are grown by the culture apparatus configured as described above will be described. First, a Murashige-Skoog liquid medium (Sucrose 3%, no hormone added) is provided in the space between the glass tube 1 and the support frame 6. At the same time, a pre-cultured suspension of cultured tobacco cells (equivalent to 45 g of tobacco cultured cells) is added, and the total amount of the liquid is adjusted to 1.5.

When charging is completed, bubble nozzle through air line 20
At a rate of 0.5 VVM, sterilized air is fed into a nozzle 22 constituting the nozzle 19 at a rate of 0.5 VVM. The gas is blown into the culture solution stored in the glass cylinder 1 from the small holes 21, 21 of the nozzle 22 while the motor is removed. By energizing 14, the centrifugal wing 18 is rotated.

That is, when the motor 14 is energized, the output shaft of the motor 14
When the second magnet 16 fixed to 15 is rotated, the first magnet 11 opposed to the second magnet 16 via the bottom plate 2 is rotated by the magnet coupling action, and the first magnet 11 is rotated.
Centrifugal wings 18 constituted by a plurality of wing plates 17 fixed to the rotating block 9 together with 11 rotate around the vertical axis 8 below the circular plate 23 constituting the rectifying plate 25.

Due to the rotation of the centrifugal wing 18 accompanying the energization of the motor 14, the flow from the portion near the center of the glass cylinder 1 to the outside along the lower surface of the circular plate 23 at the bottom of the glass cylinder 1 storing the culture solution. Is caused, and accordingly, the cylinder constituting the current plate 25
The pressure inside the support frame 6 communicating with the inside of 24 becomes lower than the pressure outside.

When a pressure difference is generated between the inside and outside of the support frame 6 with the rotation of the centrifugal impeller 18 in this manner, a flow in which the culture solution existing outside the support frame 6 tries to flow into the inside of the support frame 6 is caused. The culture solution in the glass cylinder 1 flows down to the inside of the support frame 6,
This downward flow and a circulating flow due to the upward flow that rises in the cylindrical space between the outer peripheral surface of the support frame 6 and the inner peripheral surface of the glass tube 1 are generated in the glass tube 1. Many small holes (mesh) of the support frame 6 existing in the middle of this circulation flow together with the culture solution serve as the glass cylinder 1.
The small cell mass of each cell which is smaller than the small cell mass of the plant culture cells put into the inside and cannot flow through the small hole cannot flow through the small hole, and remains attached to the outer peripheral surface of the support frame 6. Thus, only the culture solution and a small cell mass smaller than the mesh flow while passing through the support frame 6. Also, this small cell mass adheres to the cell mass attached to the support frame 6 within 2 to 3 hours after the start of the culture.

In this manner, the culture solution flowing through the support frame 6 having the plant culture cells adhered to the outer peripheral surface thereof is removed from the bubble nozzle 19 provided at a position inside the cylinder 24 constituting the rectifying plate immediately above the centrifugal wing. The sterilized air is sent at a rate of 0.5VVM,
Since the oxygen concentration in this culture solution is always maintained at an optimum concentration for the growth of the cultured plant cells, the cultured plant cells attached to the cell holding network proliferate actively.

By using such a culture device, the culture solution always flows on the surface of the plant culture cells attached to the support frame (cell holding net), so that the plant culture cells are locally in an oligotrophic state and dissolved. Oxygen deficiency or a state in which secretions are accumulated are prevented, and the plant cultured cells in the culture apparatus can be grown as homogeneous.

Furthermore, since the plant cultured cells adhere to the outer peripheral surface of the support frame 6, they are not hit by the blades 17 during operation of the culture device, and the plant cultured cells are not destroyed with the operation of the culture device. .

Next, an experiment performed by the inventor using this apparatus will be described. First, as a preculture, 5 to 10 g of tobacco cultured cells (Nicotiana tabacun L. "Bright Yellow" T-13 conditioned callus) was removed from the maintenance subculture medium, and 100 ml of
Seed into Murashige-Skoog liquid medium (Sucrose 3%, hormone-free, 500 ml Erlenmeyer flask), 160 rpm, 30
The liquid shaking culture was performed at about 2 ° C. for about 2 weeks. By this culture, the cultured tobacco cells in the medium grew to 20 to 30 g. That is, the tobacco cultured cells used in the main culture were at 30 ° C.
After 15 days of culture, it grows to the fullest in the culture device, and its fresh weight is
400g (8-9 times). The tobacco cultured cells adhere to the outer peripheral surface of the support frame 6, and adhere to the inner peripheral surface of the glass cylinder 1 and the support frame 6.
To fill the space between the outer peripheral surface and a thick layer in which a dense structure and a spongy structure are mixed. The size and shape of each cell were uniform in the inner layer (the side of the support frame 6), the outer layer (the side of the glass cylinder 1), and the middle layer of the thick layer without any significant difference.

For the purpose of examining the mode of proliferation of the cultured tobacco cells, a growth curve was prepared and compared with the case of liquid shaking culture using an Erlenmeyer flask (the same conditions as the preculture). During the culture period, it is not possible to directly determine the amount of cultured tobacco cells in the culture device, so take out a small amount of the culture solution and examine the decrease in sugar added as a nutrient in the culture solution to reduce the amount of cell growth. Estimated and calculated. For the estimation and calculation, the correlation between the amount of cell growth and the decrease in sugar in the culture solution was examined by preliminary tests in liquid shaking culture using an Erlenmeyer flask, and this was used as a reference. For the sugar in this experiment, glucose was used alone for ease of measurement only in this experiment. As a result, when this culture apparatus is used, the growth curve is as shown in FIG. 2. Using a conical flask, a liquid shaking culture (a 100 ml culture medium, a 500 ml flask) is used to obtain a growth curve as shown in FIG. Use), the induction period in the initial stage of proliferation tends to be shorter, and the amount of subsequent proliferation is almost the same. Thus, it was confirmed that the tobacco cultured cells showed active proliferation.

One of the greatest industrial purposes of growing plant culture cells is to produce plant secondary metabolites. At that time, it is important to obtain a large amount of homogeneous cells having high secondary metabolic activity. The purpose of the development of the culture apparatus of the present invention is also present here. Next, the activity of the secondary metabolic pathway of the obtained tobacco cultured cells was examined.

In the introduction part of the aromatic secondary metabolic pathway, l-phenylalanine ammonia lyase (EC4, 3, 1, 5,
(Abbreviated as AL) is the rate-limiting enzyme. In the cultured tobacco cells used in the experiment, the phytohormone (kinetin) added to the culture solution increased the activity of PAL, and as a result, the amount of secondary metabolites of coumarins (scopoletin, scoporin) in the cells Is known to increase, and this was examined for cultured tobacco cells grown using this culture apparatus. As a result, it was found that the addition of kinetin increased the activity of PAL more than 2-fold, and showed that it had a high reactivity equivalent to or higher than that of liquid shaking culture in an Erlenmeyer flask. Furthermore, the PAL activity of the cells constituting the inner layer (the side of the support frame 6), the outer layer (the side of the glass cylinder 1), and the intermediate layer of the thick cell layer formed on the support frame (the cell holding network) is the same for each layer. When the coumarins showed high activity and were extracted and quantified, almost the same amounts of scopoletin and scoporin were extracted from each layer as shown in FIG.

Furthermore, in the same cultured tobacco cells, it is known that an enzyme that converts scopoletin to scoporin (scopoletin glycoside) is activated by a plant hormone (2,4-D). When the activity was examined, as shown in the attached table, similar results were obtained in the cells forming the inner layer, outer layer and intermediate layer of the thick cell layer, and the same conclusion as in the PAL activity was obtained.

From the experiments described above, it was found that the cultured plant cells grown using this culture device exhibited high reactivity and homogeneity in the activity of the secondary metabolic pathway.

(Effect of the Invention) The method for culturing plant cultured cells using the culture apparatus of the present invention configured as described above has a sufficient nutrient content without applying mechanical shock to the plant cultured cells as compared with the conventional culture method. It is capable of supplying oxygen and growing efficiently and homogeneously, and is particularly effective when producing secondary metabolites.

[Brief description of the drawings]

FIG. 1 is a longitudinal sectional view illustrating a culture apparatus of the present invention, FIG. 2 is a diagram showing a growth curve of cultured tobacco cells by this culture apparatus, and FIG. 3 is a growth curve of cultured tobacco cells by a conventional method. FIG. 4 is a bar graph showing the amounts of scopoletin and scoporin in cultured tobacco cells cultured by the culture apparatus of the present invention. 1: glass cylinder, 2: bottom plate, 3: stud, 4: lid plate, 5: circular hole, 6:
Support frame, 7: stay, 8: vertical axis, 9: rotating block, 10: cap, 11: first magnet, 12: board, 13: bracket, 14:
Motor, 15: output shaft, 16: second magnet, 17: blade, 18: centrifugal wing, 19: bubble nozzle, 20: air pipe, 21: small hole, 22: nozzle, 23: circular plate, 24: cylinder, 25: current plate.

Claims (1)

(57) [Claims] 1. A glass cylinder for storing a liquid culture medium having a lower end opening closed by a bottom plate and an openable and closable lid plate provided at an upper end opening, and a stay in a position substantially concentric with the glass inside the glass cylinder. A rectifying plate fixed on the bottom plate by forming a cylindrical portion rising upward from the inner peripheral edge of the circular plate, and a support frame having a large number of small holes with a lower end fitted to the cylindrical portion of the rectifying plate. A first magnet attached to a vertical axis of a motor provided below the bottom plate and rotatably driven below the bottom plate, facing the first magnet and the bottom plate at a lower portion in the glass cylinder, with the first magnet and the bottom plate interposed therebetween. A second magnet rotatably supported on the bottom plate and coupled to the centrifugal wing, and a nozzle portion which is inserted into the support frame through the cover plate and has a plurality of small holes in the cylindrical portion of the rectifying plate is attached. An apparatus for culturing plant culture cells comprising a diffuser tube.