JPH0797985B2 - Method for producing plant callus cell differentiation agent - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing a plant callus cell differentiation agent. More specifically, it relates to a method for culturing a microorganism to produce a plant callus cell differentiation agent.

[Conventional technology]

When part of the differentiated plant tissue (explant) is transferred to an appropriate medium and cultured, dedifferentiation occurs, and cell division is repeated to produce plant callus, which is an amorphous tissue mass. To induce and grow callus from explants, generally, plant hormones such as cytokinins (such as kinetin) or auxins (2,4-dichloroacetic acid, indoleacetic acid) are added to the basal medium.
Indole butyric acid, etc.) is added and used.

[Problems to be solved by the invention]

As described above, various plant hormones are currently used for redifferentiation of plant callus cells, but strict control of the concentration is required at that time, so many experimental examples are required and it is difficult to select the medium. Difficulty such as strange is seen.

Therefore, the present inventor has conducted various studies in search of a plant callus cell differentiating agent that does not have such drawbacks, and as a result, a microorganism belonging to the genus Enterobacter previously found by the present inventor (Japanese Patent Application No. 61-186,228). It was found that the culture solution of (see) has an effective plant callus cell division promoting effect.

[Means for solving problems]

Therefore, the present invention relates to a method for producing such a plant callus cell differentiating agent, and the production of the plant callus cell differentiating agent is carried out by a microorganism belonging to the genus Enterobacter sp.
MP-8884) is cultured, preferably in the presence of tryptophan to kill the bacteria in the culture solution, and then the plant callus cell differentiation agent is collected.

Microorganisms used in the present invention Enterobacter sp No. 1
1-5 (FERM P-8884) was separated by the present inventor from the soil of Takeno-cho, Kinosaki-gun, Hyogo Prefecture in January 1986 by the following method.

That is, L-broth (1% tryptone, 0.5 yeast extract)
%, NaCl 0.5%, pH 7.2 before sterilization) 5 ml was put into a test tube, 1 g of the above soil was added thereto, and the mixture was shake-cultured at 37 ° C. for 24 hours.

Enterobacter sp No. 11-5 has the following mycological properties.

A. Morphology (1) Cell shape and size: Gram-negative rod, 1.5-2μ
m × 1μm (2) Cell polymorphism: None (3) Motility: None (4) Spore: None (5) Gram stain: Negative (6) Antibacterial: Unknown B. Growth state in medium (1) Meat broth agar plate culture: brown, shiny, non-diffusible (2) Slope culture of broth agar: brown, shiny, non-diffusible (3) Meat broth liquid culture: brown, shiny, non-diffusible C. Physiological properties ( 1) Reduction of nitrate: Positive (2) Denitrification reaction: Unknown (3) MR test: Negative (4) VP test: Positive (5) Indole formation: Negative (6) Hydrogen sulfide formation: Negative (7) Starch Hydrolysis of: Negative (8) Use of citric acid: Positive (9) Use of inorganic nitrogen source: Positive (10) Dye formation: Negative (11) Urease: Positive (12) Oxidase: Negative (13) Catalase: Unknown (14) Range of growth: pH 4-10, temperature 15-45 ℃ (15) Oxygen condition : Facultative anaerobic (16) O-F test: production medium acid from the fermentation (17) sugars: peptone _{2g, NaCl 5g, K 2 HPO} 4 0.3g, carbohydrates
10 g, bromthymol blue 0.08 g, agar 15 g, distilled water 100
0 ml (pH 7.1) Added concentration: 1% L-arabinose-D-xylose (unknown) D-glucose + D-mannose (unknown) D-fructose (unknown) D-galactose (unknown) Maltose + sucrose + lactose + trehalose (Unknown) D-Sorbit + D-Mannitol-Inosit + Glycerin (Unknown) Starch-Based on the above-mentioned mycological properties, this bacterium was designated as Bergey's Mannual o
As a result of searching by f Determinative Bacteriology 8th edition and other documents, it was confirmed to be a bacterium belonging to the genus Enterobacter.

Cultivation of this bacterium Enterobacter sp No. 11-5 is carried out at 37 ° C. for about 72 to 90 hours under shaking conditions using an arbitrary medium. At that time, by adding about 1 to 4 mg of tryptophan per medium 1, the redifferentiation action of the plant callus cell differentiating agent obtained therefrom is further enhanced. After the culture, an inorganic acid typified by hydrochloric acid is added to the culture medium to kill the bacteria, and the supernatant is collected as a cell differentiation agent.

Regeneration of plant callus cells using such a plant callus cell differentiating agent, Murashige & Skoog medium (1962) and the like is used as a basic medium, to which about 4 to 16 ml of the above-mentioned cell differentiating agent is added to the basic medium, Transfer plant callus cells such as rice callus cells and red cabbage callus cells with tweezers, etc., and at
It is performed by culturing for 2 weeks.

〔The invention's effect〕

According to the present invention, a substance having a plant hormone-like activity which is effective as a plant callus cell differentiating agent is obtained from a culture of a microorganism. This cell differentiating agent does not require as strictness as the conventional plant hormones in terms of control of its concentration and selection of medium, and its activity is also large.

〔Example〕

 Next, the present invention will be described with reference to examples.

Example 1 Enterobacter sp. No. 11-5 (FERM P-8884) was cultivated at 37 ° C for 90 hours using a medium (Mineral Medium) having the following composition with shaking at 100 rpm.

In _{(NH 4) 2 SO 4 1.0} (g) KH 2 PO 4 10.0 KOH 1.94 MgSO 4 · 7H 2 O 0.1 Sodium citrate 0.5 Distilled water (pH7.0) 1000 (ml) of the culture solution, a 1N hydrochloric acid 500μ added Kill the fungus,
The cells were collected by a centrifuge (7000 rpm, 15 minutes, room temperature), and the supernatant was collected to obtain a cell differentiation agent I.

Example 2 In Example 1, tryptophan in an amount to give a final concentration of 3 mg / ml was further added and used as a medium component to obtain a cell differentiation agent II.

Comparative Example As the cell differentiation agent III, an equal mixture of 2,4-dichlorophenoxyacetic acid and kinetin (6-furfurylaminopurine), each having a concentration of 1 mg / ml, was used.

[Differentiation test]

Murashige & Skoog medium (medium 1
Inositol 100mg, sucrose 30g, thiamine
Hydrochloric acid salt (0.4 mg) and buffer solution (159 mg); product of GIBCO) were added in an amount as shown in Table 1 below, respectively, and rice callus cell redifferentiation test was carried out.
The redifferentiation test was performed using approximately 100 mg of rice callus cells, and
It was carried out by continuously irradiating with 200 lux light at 10 ° C. for 10 days, and by observing how many samples out of 4 samples were differentiated, the following results were obtained.

When red cabbage callus cells were used instead of rice callus cells, the following results were obtained.

In addition, when only Murashige & Skoog medium was used and no cell differentiation agent was used (No. 8 in Tables 1 and 2), the differentiated samples grew to some extent in both rice and red cabbage, but grew at a certain level. Has stopped.

Claims (2)

[Claims] 1. A microorganism belonging to the genus Enterobacter Entero
A method for producing a plant callus cell differentiation agent, comprising culturing bacter sp No. 11-5 (FERM P-8884) to kill the bacteria in the culture solution, and then collecting the plant callus cell differentiation agent. 2. A microorganism Entero belonging to the genus Enterobacter
Plant callus cell differentiation characterized by culturing bacter sp No. 11-5 (FERM P-8884) in the presence of tryptophan, killing the bacteria in the culture solution, and collecting a plant callus cell differentiation agent Agent manufacturing method.