JPH02163018A - Multiplication of large amount of seed and stock of natural yam by tissue culture - Google Patents

Abstract

PURPOSE:To efficiently carry out a large amount of multiplication of seeds and stocks of natural yam by tissue culture by forming a multiple shoot by specific culture of tissue piece of natural yam and culturing the shoot in a synthetic nutrient medium containing a carbon source. CONSTITUTION:First, a tissue piece of a plant material of natural yam in vitro (in test tube) is used as an explant, a terminal bud or an auxiliary bud, a meristem, is extracted, a base medium is blended with an auxin as and a cytokinin plant growth substances and growth of only an adventitious bud is promoted while controlling growth of subterranean part of plant material and growth and development of aerial part to form a multiple shoot. The shoot is successively cut with a sterilized surgical knife or tweezers, separated, cultured in a medium having a high concentration of carbon source, rooted, acclimatized and passed through a process of potting to supply a large amount of seeds and stocks.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention is directed to a plant of the family Dioscoreaceae (Dioscoreaj).
aponica THUMB) relates to a method for propagating large quantities of seedlings by tissue culture.

[Conventional technology]

Japanese yam is a climbing herb that belongs to the Yassimaceae family and is native to Japan.
It is a plant that has been used since ancient times as a bounty from the mountains and as a herbal medicine. In recent years, attention has been focused on the superiority of wild yam compared to other yams, such as its viscosity, taste, flavor, etc., and its cultivation and demand have rapidly increased. However, even if wild yam is cultivated, it will cross-breed with other varieties,
It is easy to hybridize and it is difficult to maintain pure lines. In addition, like other yam plants, it is a vegetatively propagated crop, and its multiplication rate is low, and viral diseases caused by continuous cropping are transmitted to seedlings through vegetative bodies, resulting in quality deterioration due to IB. has become a major problem (Jinenjo, written by Toshiyuki Masada, publisher, Rural Culture Association, 63, 2, 29.
issue).

As a method to solve these problems, Aichi Prefectural Agricultural Experiment Station, Koji Kageyama et al. (Plant Tissue Culture, 5(1) 11-
14, 1988) ``Plant regeneration and acclimatization from the shoot apex of Jinenjo'', all shoots (stem leaves) of Jinenjo differentiate directly from the shoot apex without passing through callus, and one elongates per shoot apex. Although seeds and seedlings have been obtained by rooting, it cannot be said that a method for mass propagation has been developed.

[Problem to be solved by the invention]

The purpose of the present invention is to efficiently propagate a large amount of seedlings of wild yam by tissue culture, which has not been previously known.

[Means to solve the problem]

The present invention involves the first step of culturing tissue pieces of apical or axillary buds, which are explants of wild yam, in a synthetic nutrient medium containing auxins and cytokinins as a growth regulator to form multiple shoots; The second step consists of separating the shoots and culturing them in a synthetic nutrient medium containing carbon sources and no growth regulators to form roots, and the rooted seedlings are made into seedlings by conventional methods. This makes it possible to produce wild yam seeds and seedlings hanging from the ceiling.

In the first step of the present invention, tissue pieces of in vitro (in vitro) wild yam plants are used as explants, and the apical or axillary buds, which are meristems, are removed, and specific growth regulators are added to the basic medium to explant the plants. The first step is to control the growth of the underground part and the above-ground part, promoting the growth of only adventitious buds to form multiple shoots, and then cutting the shoots using a sterilized scalpel or bottle set. and separate,
The second step is to culture and root in a rooting medium containing high-concentration charcoal source.
After going through several stages and acclimatizing, the seeds and seedlings of the human world are supplied through the potting process.

The present invention will be described in detail below, but it is important to use high-quality wild yam that is not hybridized, whether wild or cultivated, to be subjected to tissue culture in the present invention. It is possible to use meristems of apical buds, axillary buds, or adventitious buds derived from yam as a tissue piece of this wild yam, but methods such as aseptic sowing of yam, shoot apex culture, callus culture, etc., which are easy to aseptically treat, can be used. From a practical standpoint, it is preferable to use inhuman single seedlings that have been differentiated and regenerated.

Apical or axillary buds are removed from these explants and cultured in a synthetic nutrient medium containing a growth regulator to form shoots.

As the commonly used synthetic nutrient medium, for example, a representative example is Murashige Skoog (Hurashi
ge-3koog)'s medium, Linsmaier-Skoog's (Linsmaier-8koog) medium,
Nitsch medium, Gam
borg) Mr. B5 medium, etc.

In addition to the synthetic nutrient medium described above, the synthetic nutrient medium for shoot formation used in the present invention is one to which sucrose or glucose is added as a carbon source, usually at a concentration of 1 to 10%, preferably 3 to 5%, or a medium in which the medium is fixed. For this purpose, agar with a concentration of 0.6 to 1.2% or gellan gum with a concentration of about 0.2% is used, but it is also possible to use a liquid medium without adding a fixative.

A feature of the medium used in the first step of the present invention is that a growth regulator is added to the above medium.

These growth regulators include auxins such as naphthalene acetic acid, indole acetic acid, indole butyric acid, 2,4-dichlorophenoxy acetic acid, etc. (substances that physiologically affect the growth of underground parts of the plant and the formation and growth of adventitious roots) and benzyl. Cytokinins such as adenine, kinetin, zeatin, and isopentenyladenine (substances that act physiologically on the growth of above-ground parts, adventitious bud formation, and growth) or gibberellins and brassinolides are added individually or in combination. However, a balance between the two is important.

In the first step of the present invention, only a large number of shoots are formed, and roots are formed by adding a well-balanced combination of substances that physiologically affect the growth of above-ground and underground parts to a nutrient medium and culturing. It is possible to suppress this and form a large number of high-quality shoots for seedlings. For example, as a growth regulator, naphthalene acid 71 (NAA) is recommended as an auxin, and benzyladenine (BA) is recommended as a cytokinin.

Naphthalene acetic acid is 0.04-0.2111! ? / 1
And benzyladenine is 2.0rrrg/j! A combination of these is preferred.

That is, it is preferable that BA:NAA be in the range of 100:2 to 10.

The culture container can be of any shape, such as a test tube, an Erlenmeyer flask, or a wide-mouthed bottle. Regarding the culture conditions of the present invention, the temperature is 20 to 35°C, preferably 25°±1.
The optimal temperature is 50°C, and the irradiation temperature is 50°C using a fluorescent lamp.
0-10000 Lux, preferably 2000-300
0 Lux for about 16 to 24 hours (per day), and the pH range is 4 to 8, preferably 5 to 6.

When cultured for 8 to 12 weeks under these conditions, multiple shoots are formed from the apical or axillary buds placed on the medium as tissue pieces, but no rooting was observed under these conditions. The second stage following the first stage above will be described.

When producing seedlings, use the above nutrient medium (not containing growth regulators) and root using a rooting medium containing 3 to 9%, preferably 6 to 9%, of sucrose as a carbon source. .

In this way, the method of the present invention enables the efficient production of large quantities of seedlings.The method of the present invention requires approximately 3 months for the first stage of shoot formation, and approximately 1 month for rooting the separated shoots.
It takes several months to acclimate, and then the seeds are grown in pots.

[For production]

Since the present invention is configured as described above, in the first stage of propagating seedlings, high quality multiple shoots suitable for seedlings can be obtained without rooting, and in the second stage, multiple shoots of good quality suitable for seedlings can be obtained without rooting.
Since the shoots obtained at this stage are separated and cultured under conditions that remain suitable for root formation, rooting suitable for seedlings is promoted, and it is possible to easily propagate large oranges by tissue culture of wild yam seedlings.

〔Example〕

The present invention will be explained in more detail with reference to Examples below.

Example 1 [First stage] A wild type of wild yam, Mukago, was collected, and the surface was sterilized with a 0.2% aqueous pendelkonium chloride solution, 70% ethanol, and a 2% aqueous solution of sodium hypochlorite, and 09% agar was applied. Contains, pH
M3 adjusted to 5,8 (Hurashige-3k0
0 g) on a basal medium (Table 1) to obtain seedlings in test tubes.

Table 1 (Hurashige-3kooa) Ingredients contained in 1 liter of medium (m) Then, the apical bud and axillary bud were removed as tissue pieces from the sterile seedlings and cultured.

The medium was an agar medium prepared by adding a combination of naphthalic acid 1 (NAA) and benzyladenine (B^) as growth regulators at various concentrations to the above MS basic medium and adjusting the pt+ to 5.8 in a 100 m Erlenmeyer flask. A 30 d aliquot was used. Culture conditions are 25℃, 2000~3
000 Lux, and cultured under continuous illumination for 24 hours (all day long). The results after 3 months of culturing are shown in Table 2.

Table 2 Relationship between the number of shoots formed after 3 months of culture and the concentration of NAA and B△ (Note) S: Shoot formation (-) not possible, (+) slight, (ten) medium, (band) large, shot Sugars NAA O,04rtpi/l and B^2.0tn added to the basal medium as growth regulators as shown in Table 2
Remarkable multiple shoot formation was observed in the medium supplemented with the combination of y/1.

Shoots from cultured tissue pieces are small in number from apical buds, but many are from axillary buds, making it easy to obtain shoots with good growth. Table 2 shows the case where axillary buds are used.

[Second Stage] The shoots formed in the first stage were separated and cultured in a rooting medium to form roots.

The medium was MS basic medium supplemented with 3%, 9%, and 15% sucrose, and no growth regulator was added thereto. The culture conditions were the same as in the first stage. The results after one month of culture are shown in Figure 1. The rooting rate and number of shoots increase as the sucrose concentration in the medium increases from 3 to 9%, making it fully usable as seedlings. A cultured individual was obtained.

The useful range is 50% or more.

〔effect〕

The method of the present invention for suspending propagation of wild yam seeds and seedlings by tissue culture has the following remarkable advantages:
This is also clear from Table 2 and Figure 1.

(1) In vitro vegetative growth is possible, which makes it possible to maintain superior strains for many years.

(2) A large number of seeds and seedlings can be obtained from apical and axillary buds in the form of tissue pieces in a short period of time, saving materials and labor, improving efficiency, and supplying seeds and seedlings in large quantities at low cost. That is, according to the method of the present invention, it is calculated that it is possible to produce approximately 10,000 or more seedlings from one tissue piece in one year.

(3) Since it can be cultured in a controlled environment, infection and onset of plant diseases can be prevented during the process of seedling formation, making it easier to carefully select superior strains and making it easier to promote cultivation with superior seedlings. Become.

(4) It has characteristics such as being able to obtain disease-free strains.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the relationship between root formation after one month of shoot culture and the sucrose concentration of the medium. The horizontal axis is the sucrose concentration (%), and the left scale of the vertical axis is the root formation rate (
%), and the right vertical axis indicates the number of roots formed. Patent applicant: Murakashi Lime Industry Co., Ltd. (3rd place)

Claims (1)

[Claims] The first step is to culture tissue pieces of apical or axillary buds, which are explants of wild yam, in a synthetic nutrient medium containing auxins and cytokinins as growth regulators to form multiple shoots (many stems and leaves). 1. A method for mass propagating wild yam seedlings by tissue culture, comprising the second step of separating the shoots and culturing the shoots in a carbon source-containing synthetic nutrient medium containing no growth regulator to form roots.