JPH02291220A - Apparatus for culturing plant hairy root and differentiated root and method for culturing using the same - Google Patents

Abstract

PURPOSE:To grow hairy roots over the whole culture vessel, enable an increase in production per culture medium, facilitate collection and improve production efficiency by placing hairy roots in a net cage and holding the hairy roots in a culture solution. CONSTITUTION:A lid part 2 of a culture vessel 1 is provided with a hermetically sealing plug 7 and a net cage 5 containing hairy roots or differentiated roots, such as potato, sweet potato or tomato, therein is held in a culture solution with a mooring tool 4, such as rope, mounted to the abovementioned plug to culture hairy roots, etc. A feeding port 3 and a vent hole 6 are provided in the upper part of the culture vessel 1 so as to enable feeding of air into the culture solution and discharging thereof from the culture solution.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a culture device for plant hairy roots and differentiated roots, which allows plant hairy roots and differentiated roots to be efficiently cultured in a culture tank. Plant hairy roots and differentiated roots (hereinafter referred to as
This invention relates to a method for cultivating hairy roots (hairy roots, etc.).

Since hairy roots of plants such as hairy roots have a high possibility of producing useful secondary metabolites of plants, methods for culturing them have been studied in recent years.

Initially, cultivation was carried out in Erlenmeyer flasks, but this did not allow for large-scale cultivation. Therefore, so-called stirred tank reactors, which are equipped with a culture tank and stirred inside the tank, and so-called cylindrical bubble column reactors, which blow bubbles into the tank, have emerged. However, in the former case, if the reactor is stirred at low speed, the hairy roots float to the top of the culture tank and cannot grow in the entire tank, and if the reactor is stirred at high speed, strong shearing force is applied to the hairy roots, inhibiting growth. This has the disadvantage that the production amount per medium decreases. In addition, in the latter case, the hairy roots etc. are pushed up to the top of the reactor in the late stage of culture due to the air bubbles generated, so the hairy roots etc. cannot grow in the entire culture tank.
Therefore, there was a drawback that the production amount per medium was reduced. As an improved version of the latter device, a method has been proposed in which hairy roots are immobilized in polyurethane foam and cultured in a cylindrical bubble column reactor [Kobayashi et al., Japanese Society of Agricultural Chemistry Abstracts (1981)] ). However, this method has problems such as the work of fixing the hairy roots being time-consuming, increasing the chances of contamination, and the need to separate the hairy roots and polyurethane after culture.

The present invention for treating p. The aim is to grow the hairy roots throughout the culture tank and increase the production amount per medium.Furthermore, after the cultivation is completed, all the hairy roots in the tank can be easily removed from the culture tank by simply pulling up the mesh basket inside the tank. Based on these advantages, it is possible to reduce the cost of extracting useful components from hairy roots.

That is, the present invention provides the following features: (11) A plant hairy root or differentiated root culture device comprising a rope cage containing plant hairy roots or differentiated roots held in a culture solution. (2) Plant hairy roots or differentiated roots A device for cultivating plant hairy roots or differentiated roots, which comprises a device for holding a net cage containing a plant in a culture solution and a device for supplying air into the culture solution. (3) Plant hairy roots or differentiation A method for culturing plant hairy roots or differentiated roots, which comprises placing the roots in a mesh cage, retaining them in a culture solution, and culturing them.

Regarding. The culture apparatus of the present invention is shown in FIG.

A sealing plug 7 is provided on the lid 2 of the culture tank 1, and a net cage 5 is held in the culture solution by a mooring device 4 such as a rope attached to the plug 7. Further, the mesh cage 5 is held in the culture medium using the mooring device 4. The mesh cage 5 is not particularly limited in its shape and capacity, and may be in any shape as long as it can accommodate hairy roots and maintain them in the culture tank, and the material may also have an effect on culture. It is fine as long as it is not given. In addition, the mesh size of the net should be large enough to prevent hairy roots from falling out of the net cage, and within a range that does not prevent the hairy roots from extending into the liquid from between the meshes of the rope cage. It can be used in any degree. Furthermore, in the present invention, an inlet port 3 and an exhaust port 6 are provided in the upper part of the culture tank 1, so that air can be introduced into and exhausted from the culture solution. The plant used in the present invention is not particularly limited, and any plant may be used as long as hairy roots can be obtained. Such plants include potatoes, sweet potatoes, and tomatoes. In addition, Agrobacterium, which is used to induce hairy roots,
Rhizogenes CAgrobactttrium rhiz
There are no particular limitations on the type of strain (A4 strain, LBA9402 strain, AT
Any strain such as C C 15834 strain can be used. Although there are no particular limitations on the induction and separation of hairy roots, the following methods are generally used.

Hairy root induction The roots, stems, and leaves of the plant were mixed with 70% ethanol and 1.2
% sodium hypochlorate aqueous solution and then washed with sterile water to obtain sterile plants. and Agrobacterium rhizogenes (Agrobacterium rhizog).
Apply enes) to disk-cut leaves or damaged stems. This is cultured in a well-known inorganic salt synthetic medium that does not contain plant hormones or sugars and organic substances (vitamins, amino acids, etc.) added thereto, and cultured in a light or dark place of 3000 Lux or less for 1 to 3 weeks. Isolation of Hairy Roots The hairy roots induced by the above method are grown in a well-known inorganic synthetic medium containing sugar without the addition of plant hormones and organic substances (vitamins, vitamins, etc.).
Add 50 to 1000 mg/1 of an antibiotic such as capenicillin or tetracycline to a medium containing amino acids, etc. to sterilize the roots, and isolate the sterile hairy roots. Hairy roots are cultured using the following method.

Culture of hairy roots The isolated hairy roots are grown in a well-known inorganic salt synthetic medium containing sugar and organic substances (vitamins, amino acids, etc.) without the addition of plant hormones.
The cells are cultured in the reactor shown in FIG. 1 using a medium to which 25% of the culture medium is added, for example, Murashige, Zkui' (MS medium), Gamborg's ns medium, Heller's medium, White's medium, etc. Furthermore, improved medium compositions and carbon sources such as sucrose, glucose, and fructose can also be used.

Next, examples of the present invention will be shown.

Example 1 Potato tubers were sterilized with 70% ethanol and 1.2% aqueous sodium hypochlorite solution and then washed with sterile water to obtain sterile plants. And Agrobatherium
Rhizogenes<Agrobacteriura rhiz
oegenes) was applied to disc-shaped tubers and inoculated. The inoculated tubers were cultured in MS medium with the composition shown in Table 1, which did not contain plant hormones or sugars, and
x? The cells were cultured for 1 to 3 weeks in the light or in the dark.

The induced hairy roots were treated with carpenicillin 500 g/l
The bacteria were sterilized using a plant hormone-free MS liquid medium (containing 3% sucrose) to obtain sterile hairy roots.

Table 1 MS medium composition The obtained hairy roots were cultured using Actor as shown in FIG. The reactor body was sterilized at 121"C for 20 minutes, and a plant hormone-free MS liquid medium (containing 3% sig sugar) was used as the medium. Cultivation was carried out at 25°C for 16 hours.
A photoperiod of 3 weeks was carried out. The results are shown in Table 2.

Example 2 Hairy roots were induced and cultured in the same manner as in Example 1 using white sweet potato. The results are shown in Table 2.

Example 3 Tomato seeds were treated with 70% ethanol and 1.2% sodium hypochlorite aqueous solution, then washed with sterile water and cultured for 1 to 2 weeks on MS medium containing no plant hormones (25°C
, 16-8 h photoperiod) to obtain tomato sterile plants. The induction of hairy roots, culture conditions, etc. were carried out in the same manner as in Example 1. The results are shown in Table 2.

From the results in Table 2, the production of hairy roots in the test plots cultured using a mesh cage was dramatically greater than in the control plot cultured without using a mesh cage. was found to increase. In addition, hairy roots could be easily collected from the mesh basket.

The present invention can be applied not only to hairy roots but also to differentiated roots. According to the present invention, by using a net cage in the culture tank, hairy roots, etc. can be grown throughout the culture tank, and the production amount per medium can be increased. Moreover, the hairy roots can be easily collected from the mesh basket, and production efficiency can be improved.

[Brief explanation of drawings]

FIG. 1 shows a longitudinal sectional view of the culture tank of the present invention. 1 is the culture tank, 2 is the lid, 3 is the inlet, 4 is the mooring device,
5 is a mesh basket, 6 is an exhaust port, 7 is a seal that holds the mooring device 4, and 8 is a drain line for the culture medium.

Claims (3)

[Claims] (1) A device for culturing plant hairy roots or differentiated roots, which comprises a mesh cage containing plant hairy roots or differentiated roots, held in a culture solution. (2) A device for culturing plant hairy roots or differentiated roots, which comprises holding a net cage containing plant hairy roots or differentiated roots in a culture solution, and providing a device in the culture solution for supplying air into the culture solution. . (3) A method for culturing plant hairy roots or differentiated roots, which comprises placing the plant hairy roots or differentiated roots in a mesh cage, retaining the same in a culture solution, and culturing it.