JP3013444B2 - Plant callus cell differentiation agent and regeneration method using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a plant callus cell differentiation agent and a regeneration method using the same. More specifically, a plant callus containing a supernatant obtained by culturing a microorganism
The present invention relates to a cell differentiation agent and a regeneration method using the same.

[0002]

2. Description of the Related Art When a part of a differentiated plant tissue (explant) is transferred to an appropriate medium and cultured, dedifferentiation occurs, and cell division is repeated to produce a plant callus as an amorphous tissue mass. To induce and grow calli from explants, the basal medium is usually supplemented with the plant hormones cytokinins (such as kinetin) or auxins (such as 2,4-dichlorophenoxyacetic acid, indoleacetic acid, and indolebutyric acid). Used.

[0003] As described above, various plant hormones are currently used for regeneration of plant callus cells. At that time, strict control of the concentration is required, which requires many experimental examples, and also requires the use of medium. Difficulties such as difficulty in selection are seen.

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to provide a plant callus cell differentiation agent which does not have such difficulties and a method for regenerating plant callus cells using the same.

[0005]

SUMMARY OF THE INVENTION The first object of the present invention is to provide a microorganism belonging to the genus Pseudomonas P. mallei No.
206-1 (FERMP-11898) cultured in the presence of adenine
The supernatant obtained by killing the bacteria in the culture solution is
Achieved by a plant callus cell differentiation agent in combination with chin .

A second object of the present invention is to provide a
Microorganisms belonging to the genus Solanum P. mallei No. 206-1 (FERMP-1189
8) is cultured in the presence of adenine, and the bacteria in the culture are
The filter paper impregnated with supernatant obtained killed, kinase
This is achieved by a method for redifferentiating plant callus cells, which is placed on a base medium supplemented with tin , and plant callus cells are inoculated on the paper with the cell differentiating agent impregnated thereon, and cultured under light conditions.

The microorganism P. mall used in the present invention
ei No. 206-1 (FERM P-11898) was isolated by the present inventor from the hot spring water of Otari-mura, Kitaazumi-gun, Nagano by the following method in July 1999.

More specifically, 5 ml of L-broth (1% tryptone, 0.5% yeast extract, 0.5% NaCl, pH 7.2 before sterilization) was placed in a test tube, and 1 ml of the above hot spring water was added thereto. The cells were cultured with shaking for 24 hours.

P. mallei No. 206-1 has the following mycological properties. A. Morphology (1) Cell shape and size: Bacillus, 0.5 × 1 μm (2) Motility: None (3) Spores: None (4) Gram staining: negative or undefined Growth state in culture medium Gravy agar plate culture: shiny Physiological properties (1) Nitrate reduction: positive (2) VP test: positive (3) Indole formation: negative (4) Hydrogen sulfide formation: negative (5) Use of citric acid: negative (6) Pigment formation : Negative (7) Urease: Positive (8) Oxidase: Positive (9) Growth range: pH 4-9, temperature 30-45 ° C (10) Attitude to oxygen: aerobic (11) Production of acid from saccharide Medium: Peptone 2 g, NaCl 5 g, K2HPO4 0.3 g, carbohydrate 10 g, bromthymol blue 0.08 g, agar 15 g, distilled water 1000 ml (pH 7.1) Addition concentration: 1%

[0010] Based on the above mycological properties,
As a result of searching by y's Manual of Determinative Bacteriology ninth edition, it was confirmed that the bacterium belongs to the genus Pseudomonas.

The culture of the bacterium P. mallei No. 206-1 is carried out at 40 ° C. for about 72 to 90 hours under shaking conditions using an arbitrary medium. At this time, about 0.5 to 2 mg of adenine is added per liter of the medium . After the culture, the bacteria are killed by adding an inorganic acid represented by hydrochloric acid to the culture solution, and the supernatant is collected .

[0012] Such regeneration plant callus cells using supernatant using a basal medium Murashige & Skoog medium (1962), to that of about 0.5~2ppm added phytohormones thereto, the supernatant B. Place about 10 μl of paper onto a piece of paper, transfer carnation callus cells, wasabi callus cells and other plant callus cells with tweezers, and irradiate with light (about 10 to 16 hours under light conditions). ~ 30
C. for about 1-2 months. The plant hormone used is effective only for kinetin ,
No regeneration was observed with other plant hormones.

[0013]

According to the present invention, kinetin can be used in combination.
Condition the door, effective as a plant callus cell differentiation agents,
Substances with plant hormone-like activity are obtained from cultures of microorganisms. This cell differentiating agent is not as strict as conventional plant hormones in controlling its concentration and selecting a medium, and has a large activity.

And such plant callus cell differentiating agent
By using , the plant callus cells can be effectively regenerated.

[0015]

Next, the present invention will be described by way of examples. Comparative Example 1 P. mallei No. 206-1 (FERM P-11898) was cultured at 40 ° C. for 70 hours while shaking at 120 rpm using an L-bouillon medium (10 ml) having the following composition. did. Tryptone 10.0 (g) Yeast extract 5.0 NaCl 5.0 Distilled water (pH 7.0) 1000 (ml)

To this culture, 500 μl of 1N hydrochloric acid was added to kill the bacteria, and the cells were collected with a centrifuge (10,000 rpm, 10 minutes, 4 ° C.).
The supernatant was collected and used as cell differentiation agent I.

Example In Comparative Example 1 , adenine was added in a final concentration of 0.5 mg / ml as a medium component to obtain a cell differentiation agent II.

Comparative Example 2 The following five compounds were added at a concentration of 1 ppm to a Murashige & Skoog medium (MS medium), and a cell differentiation agent IIIa was added thereto.
~ IIIe. IIIa: 2,4-dichlorophenoxyacetic acid IIIb: indoleacetic acid IIIc: naphthylacetic acid IIId: 6-benzyladenine IIIe: kinetin

(Differentiation test) No. 1 Cell differentiation agent I 2 〃II 3 〃IIIa 4 〃IIIb 5 〃IIIc 6 〃IIId 7 〃IIIe 8 〃I + IIIa 9 ＩI + IIIb 10 ＩI + IIIc 11 ＩI + IIId 12 〃 I + IIIe 13 〃 II + IIIa 14 〃 II + IIIb 15 〃 II + IIIc 16 〃 II + IIId 17 〃 II + IIIe

In Nos. 1-2, round paper was placed on the MS medium, and 10 μl of the cell differentiation agent I or II was impregnated on the paper, and the callus cells of carnation were placed on such paper impregnated with the cell differentiation agent. 50-100 mg were planted with forceps.
This was cultured at 25 ° C. and 4000 lux for 12 hours under a light condition for one month, and it was observed how many samples out of three samples had differentiated.

In Nos. 3 to 7, cell differentiation agent IIIa was added to MS medium.
~ IIIe was added at 1 ppm, and a circular piece of paper was placed on the medium, and callus cells were inoculated on the piece of paper and cultured similarly.

In Nos. 8 to 17, 1 ppm of the cell differentiation agents IIIa to IIIe were added to the MS medium, a circular paper was placed on the medium, and 10 μl of the cell differentiation agent I or II was impregnated into the paper. Then, callus cells were inoculated on such a cell differentiating agent-impregnated paper and cultured similarly.

As a result, redifferentiation was not observed in any of Nos. 1 to 16, but in No. 17, differentiation was observed in one out of three samples, ie, differentiation was observed in roots, stems and leaves. Was seen.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI C12R 1: 385)

Claims (2)

(57) [Claims] 1. A microorganism P. ma belonging to the genus Pseudomonas.
A plant callus cell differentiation agent obtained by culturing llei No. 206-1 (FERMP-11898) in the presence of adenine and killing the bacteria in the culture solution and using a supernatant obtained in combination with kinetin. 2. A microorganism P. ma belonging to the genus Pseudomonas.
llei No. 206-1 (FERMP-11898) was cultured in the presence of adenine, and the paper impregnated with the supernatant obtained by killing the bacteria in the culture was placed on a basic medium supplemented with kinetin. And planting the plant callus cells on the paper differentiated with the cell differentiating agent, and culturing the cells under light conditions.