JPH05284966A - Two-stage cultivation under aeration and agitation - Google Patents

Abstract

PURPOSE:To enable mass culture of a plant tissue in an extremely high efficiency without cutting the plant cell by carrying out liquid culture exclusively by aeration to effect the proliferation of the plant cell and carrying out the aeration and agitation culture under stirring at the lowest stirring speed necessary for keeping the cells in suspended state. CONSTITUTION:A sterilized culture liquid and a plant cell to be used as a seed are put into a sterilized culture tank 1 and the cell is cultured exclusively under aeration e.g. by introducing aseptic air through an aeration port 18 and dispersing the air with a sparger 19. When the proliferated plant cells begin to precipitate, the cells are subjected to aeration and agitation culture under stirring with a stirring blade 17 at the lowest speed necessary for suspending the cells.

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 efficiently mass-cultivating plant cells by liquid culture.

[0002]

2. Description of the Related Art Conventionally, in the technical field of plant cell culture, as large-scale culture by liquid culture, oxygen supply to the culture medium and suspension of cells are aeration only by aeration, aeration by aeration and mechanical stirring. Agitated culture is known. And, as a device used for aeration type culture, there is a bubble column type culture device, there is an air lift type culture device, as a device used for aeration stirring type culture, a turbine, a screw,
A culture device equipped with a stirring impeller such as a modified paddle is known. (Latest Botanical Engineering Manual (R & D Planning) P
552, plant tissue culture, 5 (1), 1988, P42)

[0003]

Among the above-mentioned conventionally known plant cell culture systems, in the aeration and agitation type culture, the cells may be sheared by the agitation blade, which may impair the cell growth. On the other hand, in the aeration-type culture, the cells are not sheared by the stirring blade, but when the cells grow and the cell concentration becomes high, it becomes difficult to stir the cells only by aeration. Thus, conventional systems are not satisfactory.

[0004]

The present invention has been made for the purpose of developing a system for efficiently mass-cultivating plant cells by solving the above-mentioned drawbacks of the conventional method.
As a result of diligent research from various directions, cells are grown by aeration-only culture without stirring at the initial stage of culture when cells are suspended only by aeration. Then, at the stage where the cells began to grow and settle, the lowest speed agitation necessary for suspending the cells was performed, and by performing aeration agitation culture, it was found that the intended purpose was achieved, and It has been completed.

That is, the culturing method of the present invention comprises a step of growing plant cells by culturing with only aeration using a pre-sterilized culture solution in a pre-sterilized culture tank, and a stage where the plant cells start to grow and settle. In addition, the step of performing aeration stirring culture at the lowest speed necessary for suspending the cells is included.

The culture apparatus used is a general culture apparatus as shown in FIG. 1, which comprises a liquid culture tank 1 and is equipped with a sterilization and temperature control apparatus 11. An inlet 12 and a vent 13 for inoculating plant tissue as seeds are provided on the upper surface of the culture tank 1, and a recovery port 14 for recovering the plant tissue after culturing is provided on the bottom surface or the side surface. The recovery port 14 is closed by a valve 15 during culture.
Inside the culture tank 1, a stirring blade 17 driven by a motor 16 and a sparger 19 for dispersing air from a vent 18 are arranged. The shape of the stirring blade 17 is not particularly limited, but a scree blade having a large blade area is suitable. Further, the shape of the culture tank 1 is better than that of a normal aeration and agitation type culture device, and the bottom surface is preferably spherical.

The operation of the culture apparatus used, that is, a specific mode of the culture method according to the present invention will be described below with reference to FIGS. 2 and 3.

(1) The pre-sterilized culture solution 1 and the plant cells to be seeds are put into the pre-sterilized culture tank 1 through the port 12 and dispersed in the sterile air from the vent 18 using the sparger 19. Liquid culture is performed only by aeration of plant cells (FIG. 2-Step A). And
(2) Propagate plant cells (FIG. 2-Step B). Then, (3) plant cells proliferate and cell precipitation begins (FIG. 2-Step C). Therefore, (4) the motor 16 causes the stirring blades 17 to carry out the stirring at the minimum speed necessary for suspending the cells, and liquid culture is performed by aeration stirring (Fig. 3-
Step D). In this way, (5) the plant cells are grown and collected from the collection port 14 after the culture is completed (FIG. 3-step E, FIG. 3-step F).

An example of the agitating two-step liquid culture according to the present invention will be described below.

[0010]

Example 1 Sesame seeds (Sesamun indicum)
L. Using the culture device (5 liters capacity) shown in FIG. 1, the proliferative cells (1) were subjected to aeration-agitation two-step culture according to the operations shown in FIGS. As the medium, sucrose (30 g / l), 2,4-dichloro phenoxy acetic acid ⁽⁸ × 10- 8 M), kinetin (1 × 10- ⁵ M) was added and the MS medium (Murashige Skoog medium)
3 liters were used. Aeration-only culture was performed for 7 days after the start of culture, and stirring was performed from the 8th day.

As a control, the same culture device as above was used,
Aeration culture and aeration-agitation culture were performed using the same medium.

The results of the culture are shown in FIG. In the figure, the cell growth amount is the dry weight of cells in 1 liter of the culture solution. As is clear from the figure, it was demonstrated that the aeration two-step culture method according to the present invention is far superior to the two methods, the aeration culture method and the aeration stirring culture method, which are the control methods.

[0013]

[Operation / Effect] The low-speed stirring of several tens of rpm hardly contributes to the increase in the rate of dissolution of oxygen into the medium. Therefore, in the early stage of the culture in which the cells are suspended only by aeration, the cells are grown by liquid culture without agitation, and the cells can be liquid-cultured without being sheared by the agitating blades.

As the cells grow and the cell concentration increases, the apparent viscosity of the culture medium increases, and even if stirring is performed, the effect of shearing of the cells by the stirring blade is less than that at the beginning of the culture. Therefore, the liquid culture of high density cells can be performed by performing the lowest speed stirring necessary for suspending the cells at the stage where the cells have started to grow and settle.

[0015]

EFFECTS OF THE INVENTION According to the present invention, cells can be grown healthy without shearing, and the growth rate of cells can be dramatically increased. Moreover, not only the conventional culture device can be used as it is, but also delicate operation is not required, and this method is extremely suitable for industrialization.

Since the system of the present invention can be widely applied to all plants, not only sesame,
Since it is possible to obtain a large amount of the target plant cells by an industrial method without cultivating the plant which is labor-intensive and easily affected by the weather, it becomes possible to efficiently produce various useful substances derived from these plants. Therefore, the present invention makes a great contribution to various technical fields such as food and drink, pharmaceuticals, cosmetics, and industrial chemicals.

[Brief description of drawings]

1 shows an example of a culture device used for carrying out the method according to the present invention.

FIG. 2 shows the steps (AC) of the method according to the invention.

FIG. 3 shows the steps (DF) of the method according to the invention.

FIG. 4 Method according to the present invention (aeration-stirring two-step culture method)
FIG. 4 is a comparison diagram of a conventional method (aeration culture method, aeration stirring culture method).

Claims (1)

[Claims] 1. (a) A step of growing plant cells by aeration-only culture using a pre-sterilized culture solution in a pre-sterilized culture tank, and (b) plant cells started to grow and precipitate. A step of performing aeration stirring culture at the lowest speed necessary for suspending the cells in the step, and carrying out aeration stirring culture of plant cells.