JPS6222517A - Culture of indefinite root of gourd family - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a tissue culture method for culturing adventitious roots of Cucurbitaceae plants.

[Prior Art and Problems] Many plants of the Cucurbitaceae family are edible, such as cucumbers, melons, Japanese cucumbers, Japanese cucumbers, and crow gourds. Also, winter melon, which is the seed of chili pepper, or
Some are used as raw materials for Chinese herbal medicine, such as Kokurojin, which is the seed of Kikarasu gourd. Cucurbitaceae plants, such as Trichosanthes kirilowii
The roots of the (kurokon) contain trichosanthin,
Trichosanthin is known as a protein that has abortifacient and anticancer effects.

Traditionally, trichosanthin was obtained by cultivating and extracting it from its roots, but the content of trichosanthin varied depending on the natural environment, weather, and other cultivation conditions, making it difficult to find an efficient method. There wasn't.

Regarding the tissue culture method for Cucurbitaceae plants, Patent Application No. 2183 filed in 1985 describes that in order to reliably mass produce and collect ascorbic acid oxidase, the copper ion concentration is 0.2.
A method for inducing callus from tissue pieces of Cucurbitaceae plants using a liquid medium with a concentration of μM or higher has been disclosed. Therefore, even if this method is used to induce callus from a tissue piece of Chikara gourd and a large number of undifferentiated cultured cells are obtained by further propagating the callus, these undifferentiated cultured cells will contain almost no trichosanthin. Not yet.

In order to efficiently produce trichosanthin, the present inventors focused their attention on efficiently culturing the roots of Cucumber gourd, which contain trichosanthin.

That is, an object of the present invention is to provide a culture method for efficiently obtaining adventitious roots of Cucurbitaceae plants in large quantities.

[Means for Solving the Problems] As a result of repeated research and consideration on a culture method for efficiently and large quantities of adventitious roots of Cucurbitaceae plants, the present inventors have succeeded in cultivating callus from tissue pieces of Cucurbitaceae plants. inducing and culturing the differentiated tissue formed on the surface of the callus to produce adventitious roots of the Cucurbitaceae plant, or inducing callus from tissue pieces of the Cucurbitaceae plant and culturing the differentiated tissue formed on the surface of the callus. By producing adventitious roots of Cucurbitaceae plants, and further culturing a part of the adventitious roots to create new adventitious roots of Cucurbitaceae plants, we succeeded in obtaining adventitious roots of Cucurbitaceae plants efficiently and in large quantities, and the present invention has been achieved. It was completed.

[Example 1] The method for culturing adventitious roots of Cucurbitaceae plants of the present invention will be described in detail below.

First, the differentiated tissue formed on the surface of the callus, which is the raw material in the method for culturing adventitious roots of Cucurbitaceae plants of the present invention, is derived from the roots, leaves, and stems of Cucurbitaceae plants, preferably actively growing leaves and stems. 1 part, especially preferably the petiole part that has just expanded.
Tissue pieces cut into ~3 mm squares are sterilized using the commonly used sterilization method (e.g., 30% in 70% ethanol aqueous solution).
After sterilizing by dipping in a 5% hypochlorous acid aqueous solution for 5 minutes and washing with sterilized water, place it on a culture medium and heat it at a temperature of 2.
It is obtained by inducing callus by adjusting the irradiation with light as bright as a fluorescent lamp for 12 to 24 hours a day at 0 to 30°C, preferably 25°C, for 3 to 5 weeks. As the medium, a solid medium can be used, which is made by adding agar or the like to a liquid containing inorganic components, a carbon source, plant hormones, and vitamins as essential components and adding amino acids to the solid medium and solidifying the same.

Specific examples of inorganic components include copper, nitrogen, phosphorus, potassium, calcium, magnesium, sulfur, iron, manganese,
Zinc, boron, molybdenum, chlorine, sodium, iodine, cobalt, etc., more specifically, potassium nitrate, sodium nitrate, calcium nitrate, ammonium nitrate, monopotassium phosphate, disodium phosphate, sodium ethylenediaminetetraacetate, potassium chloride, calcium chloride, Examples include magnesium sulfate, sodium sulfate, ferrous sulfate, ferric sulfate, manganese sulfate, zinc sulfate, boric acid, copper sulfate, sodium molybdate, molybdenum trioxide, potassium iodide, cobalt chloride, and the like.

Specific examples of carbon sources include carbohydrates such as sucrose, derivatives thereof, and the like.

A specific example of a plant hormone is indole acetic acid (IA
A), naphthaleneacetic acid (NAA), p-chlorophenoxyisobutyric acid, 2,4-dichlorophenoxyacetic acid (2,4
Examples include auxins such as -D), and cytokinins such as kinetin, zeatin, and dihydrozeatin.

Further, specific examples of vitamins include biotin, thiamine (vitamin B), pyridoxine (vitamin B6), pantothenic acid, ascorbic acid (vitamin C), inositol, nicotinic acid, etc., and specific examples of amino acids include: Examples include glycine, alanine, glutamine, and cysteine. In addition, Murashige-8koog medium (GT
(manufactured by BCO) etc., the above-mentioned components may be appropriately added to the Murashige-Skoog medium. In particular, sucrose as a carbon source, naphthalene acetic acid and 6-
Preferably, a solid medium containing benzylaminopurine and agar is used.

Below, a specific example of the production of a differentiated tissue formed on the surface of a callus derived from a tissue piece of a cucurbitaceous plant, which is a raw material for the method for culturing adventitious roots of a cucurbitaceous plant of the present invention, will be shown.

Specific example Murashige-Skoog medium (manufactured by GIBCO), sucrose (3% final concentration), agar (0.9% final 5 degrees)
and naphthalene acetic acid (manufactured by Sigma) at a final concentration of 1 mg/m2 to the medium.
A medium was prepared by adding benzylaminopurine (manufactured by Sigma) to the medium at a final concentration of 1 mg/ml. The fresh petiole of P. japonica was cut into approximately 2 mm square pieces, immersed in a 17.70% ethanol aqueous solution for 30 seconds, then immersed in a 5% hypochlorous acid aqueous solution for 5 minutes, and washed with sterilized water, which was then prepared as described above. It was placed on a medium. At 25℃, adjust the fluorescent lamp irradiation time to 16 hours a day, and
A callus was induced by interindividual culture, and a differentiated tissue formed on the surface of the callus was obtained.

The present invention will be described in detail below.

Examples of plants of the Cucurbitaceae family that can be used in the present invention include Currant gourd, Currant gourd, Currant melon, and the like.

In culturing the differentiated tissue formed on the surface of callus derived from a piece of tissue from a Cucurbitaceae plant, the differentiated tissue is first cut into pieces of 1 to 3 mm square tissue, placed in a culture medium, and incubated for 20 minutes.
~30°C, preferably 24-26°C in the dark, 2-4
Culture for a week.

As the medium, a liquid medium containing inorganic components, a carbon source, plant hormones, and vitamins as essential components and amino acids added thereto, or a solid medium obtained by adding agar, etc. to the liquid medium and solidifying it is used. be able to.

Specific examples of inorganic components include copper, nitrogen, phosphorus, potassium, calcium, magnesium, sulfur, iron, manganese,
Zinc, boron, molybdenum, chlorine, sodium, iodine, cobalt, etc., and more specifically, potassium nitrate, sodium nitrate, calcium nitrate, ammonium nitrate, phosphoric acid! Potassium, disodium phosphate, sodium ethylenediaminetetraacetate, potassium chloride, calcium chloride, matognenium sulfate, sodium sulfate-1 ferrous sulfate, ferric sulfate, manganese sulfate, zinc sulfate, boric acid, copper sulfate, molybdic acid Examples include sodium, molybdenum dioxide, potassium iodide, cobalt chloride, and the like.

Specific examples of carbon sources include carbohydrates such as sucrose, derivatives thereof, and the like.

Specific examples of plant hormones and 1. Then, indole acetic acid (I
AA), naphthaleneacetic acid (NAA), p-chlorophenoxyisobutyric acid, 2,4-dichlorophenoxyacetic acid (2,
Examples include auxins such as 4-D), and these auxins have a final concentration of 0.5 to 50 μm o
It is preferable to add it to a concentration of 1/',
In particular, the final concentration of naphthalene acetic acid in the medium is between 3 and 8.
It is preferable to add at a concentration of μmol/mole.

Specific examples of vitamins include biotin, thiamine (vitamin B), pyridoxine (vitamin B11),
Examples include pantothenic acid, ascorbic acid (vitamin C), inositol, nicotinic acid, and specific examples of amino acids include glycine, alanine, glutamine, cysteine, and the like. In addition, Murashige, which is already commercially available,
Murashige-3koog medium (G
IB, manufactured by CO), etc. may be used by adding the above-mentioned components as appropriate.

Next, to culture some of the adventitious roots of Cucurbitaceae plants □
As mentioned above, 1. From the adventitious root obtained by
Some tissue pieces, especially some tissue pieces having growth points, are collected, placed on a medium, and cultured in the dark at 20-30° C., preferably 24-26° C., for 2-4 weeks.

The medium may be a liquid medium containing inorganic components, a carbon source, plant hormones, and vitamins as essential components, to which amino acids are added, or a liquid medium prepared by adding agar or the like to the liquid medium and solidifying 1. A solid medium can be used.

Specific examples of the inorganic component and carbon source include the same specific examples as mentioned above.

A specific example of a plant hormone is indole acetic acid (IA
A), naphthaleneacetic acid (NAA), p-chlorophenoxyisobutyric acid, 2,4-dichlorophenoxyacetic acid (2,4
-D), etc., and it is preferable to add these auxins to the medium at a final concentration of 0.5 to 50 μmol/ml. 3-8μmol/
It is preferable to add it to the desired concentration.

In addition, specific examples of vitamins and amino acids and No. 17 include specific examples similar to those mentioned above. In addition, Murashige-Skoog medium (manufactured by GrBCO), which is already commercially available.
etc. as mentioned above in 1. Components may be appropriately added and used.

[Effects of the Invention] According to the present invention, adventitious roots of Cucurbitaceae plants can be stably obtained regardless of the season, climate, etc.

Since the adventitious roots obtained by the present invention have a low content of storage sugars such as starch, the target substance contained in the adventitious roots can be efficiently extracted.

[Production Examples] The present invention will be specifically described below with reference to Production Examples, but the present invention is not limited thereto.

Production Example 1 Trichosantrum obtained in the specific example
The differentiated tissue formed on the callus surface of Heskirilowii) was cut into 2 mm square pieces, and this tissue piece was
The cells were placed in Skoog medium [(manufactured by Gj I3 CO), but naphthalene acetic acid was added to the medium so that the final concentration was 6 μmol/1], and cultured at 25°C for 60 days in the dark. Adventitious roots of N.) were obtained. After freeze-drying and pulverizing this Adventitious root of Chikaras gourd, 100g was added to a phosphate buffer solution (pI7, 0) under water cooling.
) was extracted. Insoluble matter was removed by centrifugation, acetone was added to the extract, and after standing at -20°C for 1 hour, the precipitate was collected by centrifugation. This precipitate was dissolved in water, centrifuged again, and the supernatant was filtered by ultra-I filtration for 15 minutes.
Freeze-drying was performed to obtain a freeze-dried crude protein. When this freeze-dried product was developed by SDS electrophoresis and trichosanthin was quantified using a chromatography scanner (manufactured by Takagura Seisakusho), the content was found to be 5.12% based on the dry weight of adventitious roots.

Manufacturing example 2

Claims (2)

[Claims] (1) A method for culturing adventitious roots of Cucurbitaceae plants, which comprises inducing callus from a tissue piece of Cucurbitaceae plants, and culturing the differentiated tissue formed on the surface of the callus to produce adventitious roots of Cucurbitaceae plants. (2) Callus is induced from tissue pieces of Cucurbitaceae plants, the differentiated tissue formed on the surface of the callus is cultured to produce adventitious roots of Cucurbitaceae plants, and a part of the adventitious roots is further cultured to create new Cucurbitaceae plants. A method for culturing adventitious roots of Cucurbitaceae plants, characterized by producing adventitious roots.