JP2691772B2 - Cultivation device for plant hairy roots and differentiated roots and culture method using the same - Google Patents

Description

TECHNICAL FIELD The present invention uses a plant hairy root and differentiated root culturing device and a device for cultivating plant hairy roots and differentiated roots efficiently in a culture tank. The present invention relates to a method for culturing plant hairy roots and differentiated roots (hereinafter referred to as hairy roots and the like).

Prior art and problems Since plant hairy roots and the like have a high possibility of producing useful secondary metabolites of plants, cultivation methods for them have been studied in recent years.

Initially, the culture was carried out in an Erlenmeyer flask, but this does not allow large-scale culture. Then, a so-called stirring tank type reactor for providing a culture tank and stirring the inside of the tank, a so-called cylindrical bubble column type reactor for blowing bubbles into the tank, and the like have appeared. However, in the former case, when the reactor is stirred at a low speed, hairy roots and the like float above the culture tank and cannot grow in the whole tank. There was a drawback that it was inhibited and the production amount per medium was reduced. Further, in the latter case, the hairy roots and the like are pushed up to the upper part of the reactor by the generated air bubbles in the latter stage of the culture, so that the hairy roots and the like cannot grow in the entire culture tank, resulting in a decrease in the production amount per medium. There was a drawback. Then, as a modification of the latter device, a method of immobilizing hairy roots on polyurethane foam in a cylindrical bubble column reactor and culturing is proposed. [Journal of the Japanese Society of Agricultural Chemistry, Vol. 62, No. 3, page 619, 3Yp11 (1988
Year)〕. However, this method has problems that the work of fixing the hairy roots is troublesome, the chances of contamination increase, and it is necessary to separate the hairy roots from polyurethane after culturing.

Means for Solving Problems The present invention has been made to solve these problems, and hairy roots, etc. are put in a net cage and held in a culture solution to obtain hairy roots, etc. Is grown in the entire culture tank to increase the production amount per medium. Furthermore, after the completion of the culture, all the hairy roots in the tank can be easily recovered from the culture tank simply by lifting the net basket in the tank,
Based on these advantages, it is possible to reduce the cost when extracting useful components from hairy roots.

That is, the present invention provides (1) an apparatus for cultivating plant hairy roots or differentiated roots, which comprises holding a net cage containing plant hairy roots or differentiated roots in a culture solution.

(2) Apparatus for culturing plant hairy roots or differentiated roots, which is provided with a device for holding a net cage containing plant hairy roots or differentiated roots in the culture solution and supplying air to 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 net cage, holding the same in a culture solution, and culturing.

About.

 The culture device of the present invention is shown in FIG.

The lid 2 of the culture tank 1 is provided with a tight stopper 7, and the net cage 5 is held in the culture medium by a mooring tool 4 such as a rope attached to the lid. In addition, the retention of the mesh basket 5 in the culture solution is
The mooring tool 4 is used. The mesh basket 5 is not particularly limited in its shape and capacity, and may have any shape as long as it can accommodate hairy roots and the like and can be maintained in the culture tank,
Any material may be used as long as it does not affect the culture. Also,
The size of the mesh of the mesh is such that hairy roots and the like do not fall out of the mesh basket and do not prevent the hairy roots from extending from the mesh of the mesh basket into the liquid. Any degree can be used. Further, in the present invention, the inlet 3 and the outlet 6 are provided in the upper part of the culture tank 1 so that air can be introduced and exhausted into the culture solution.

The plant used in the present invention is not particularly limited and may be any plant as long as hairy roots can be obtained. Such plants include potatoes, sweet potatoes,
There are tomatoes, etc.

In addition, Agrobacterium rhizogenes used for the induction of hairy roots is not particularly limited, and known ones such as A4 strain and LB are known.
Any of A 9402 strain, ATCC 15834 strain and the like can be used.

The induction and separation of hairy roots are not particularly limited, but generally the following method is used.

Induction of hairy roots 70% ethanol and 1.2% roots, stems and leaves of plants
Sterilize with an aqueous solution of sodium hypochlorite and then wash with sterile water to obtain a sterile plant. Then, Agrobacterium rhizogenes is applied to a disc-shaped leaf or a damaged stem. This is cultivated in a medium in which organic substances (vitamins, amine acids, etc.) are added to a known inorganic salt synthetic medium containing no plant hormone and containing no sugar, and cultivated for 1 to 3 weeks in a bright or dark place of 3000 Lux or less. .

Separation of hairy roots Hairy roots induced by the above method are antibiotics such as carbenicillin and tetracycline in a medium prepared by adding organic substances (vitamins, amino acids, etc.) to a known inorganic synthetic medium containing sugar without adding plant hormones. The substance is sterilized by adding 50 to 1000 mg / l, and sterile hairy roots are separated.

 Culture of hairy roots is performed by the following method.

Cultivation of hairy roots The isolated hairy roots are prepared by adding organic substances (vitamins, amino acids, etc.) to a known inorganic salt synthetic medium containing sugar without addition of plant hormones, for example, Murashige Skoog (MS medium), Gamborg's The cells are cultured in the reactor shown in FIG. 1 using B5 medium, Heller's medium, white medium and the like. In addition, those having an improved medium composition, sucrose, glucose, fructose and the like can be used as the carbon source.

 Next, examples of the present invention will be described.

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. Then, Agrobacterium rhizogenes was applied to a disk-shaped tuber and inoculated. The inoculated tubers were cultivated in MS medium having the composition shown in Table 1 containing no plant hormone and containing no sugar, and cultivated for 1 to 3 weeks in a bright or dark place of 3000 Lux or less. Induced hairy roots are carbenicillin 500m
Sterilized hairy roots were obtained by sterilization using an MS liquid medium (containing 3% sucrose) containing no plant hormone containing g / l.

The hairy roots thus obtained were cultured using the reactor shown in FIG. The reactor body was sterilized at 121 ° C. for 20 minutes, and the medium used was a plant hormone-free MS liquid medium (containing 3% sucrose). Culturing was carried out at 25 ° C. for 16 hours-8 hours photoperiod for 3 weeks. The results are shown in Table 2.

Example 2 In the same manner as in Example 1 using white sweet potato, hairy roots were induced,
The culture was performed. The results are shown in Table 2.

Example 3 Tomato seeds were treated with 70% ethanol and 1.2% aqueous sodium hypochlorite solution, and then washed with sterile water and cultured in MS medium without phytohormones for 1 to 2 weeks (25 ° C., 16-8
Photoperiod of time) to obtain sterile tomato plants. Induction of hairy roots, culture conditions and the like were the same as in Example 1. The results are shown in Table 2.

From the results shown in Table 2, the test plots cultivated in the net cages showed a dramatic increase in hairy root production in all cases compared to the control plots cultivated without the net cages. It turned out to increase. The hairy roots could be easily collected from the net cage.

The present invention can be applied not only to hairy roots but also to differentiated roots.

EFFECTS OF THE INVENTION According to the present invention, by using a net cage in the culture tank, hairy roots and the like can be grown in the entire culture tank and the production amount per medium can be increased. In addition, hairy roots can be easily collected from the net cage, and production efficiency can be improved.

[Brief description of the drawings]

FIG. 1 shows a longitudinal sectional view of the culture tank of the present invention. 1 is a culture tank, 2 is a lid, 3 is an inlet, 4 is a mooring tool,
5 is a net basket, 6 is an exhaust port, 7 is a stopper for holding the mooring tool 4, and 8 is a culture solution discharge line.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Shinichi Takafuji 3-9-10 Gakuen Higashi-cho, Kodaira-shi, Tokyo (56) References Journal of the Japanese Society of Agricultural Chemistry Vol. 62, No. 3, page 619, 3Yp11

Claims (3)

(57) [Claims] 1. An apparatus for cultivating plant hairy roots or differentiated roots, which comprises holding a net cage containing plant hairy roots or differentiated roots in a culture solution. 2. A plant hairy root or a differentiated root obtained by holding a net cage containing a plant hairy root or a differentiated root in the culture solution and providing a device for supplying air into the culture solution. Incubator. 3. A plant hairy root or a differentiated root is placed in a net basket,
A method for culturing plant hairy roots or differentiated roots, which comprises holding this in a culture medium and culturing.