JP2632031B2 - Mass propagation method of natural potato seedlings by tissue culture - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a potato plant, Dioscoreajaponica.
THUNB,) The present invention relates to a method of mass-producing seedlings by tissue culture.

[Prior Art] Japanese yam belongs to the yam family and is a vine plant native to Japan, and is a plant that has been used as a traditional Chinese medicine or as a herbal medicine. In recent years, cultivation and demand have rapidly increased, paying attention to the point that the potatoes are superior to other yams, such as the viscosity, taste, flavor, and the like of natural potatoes. However, even if natural potatoes are cultivated, it is easy to cross and hybridize with other varieties, and it is difficult to maintain a pure line.
In addition, it is a vegetatively propagated crop like other yam family plants, and its growth rate is low, and virus diseases caused by continuous cropping are transmitted through vegetative bodies to seeds and seedlings, leading to deterioration in cultivation. It ’s a big problem (Ginenjo,
Author: Toshio Masada, publishing house, Agriculture, Mountain and Fishing Village Cultural Association, 63, 2, 29, published)

To solve these problems, Aichi Prefectural Agricultural Research Institute, Koji Kageyama et al. (Plant tissue culture, 5 (1) 11-14
P., 1988) According to "Regeneration and acclimation of plants from the shoot apex of Ginenjo", shoots (foliage) of Ginenjo
Does not pass through the callus, but is directly differentiated from the shoot apex, elongates one per shoot apex, and roots to obtain seeds. However, it cannot be said that the method has reached the mass propagation method.

[Problems to be solved by the invention]

An object of the present invention is to efficiently mass-produce seeds and seedlings by tissue culture of hitherto unknown potatoes.

[Means for solving the problem]

The present invention relates to a first step of forming a multiple shoot by culturing a piece of apical or axillary bud, which is an explant of a natural potato, as a growth regulating substance in a synthetic nutrient medium containing auxins and cytokinins, and forming this shoot. And the second step of culturing the shoot in a carbon-containing synthetic nutrient medium containing no growth-regulating substance to form roots. And to enable the production of seeds and seedlings of natural potatoes in large quantities.

In the first step of the present invention, apical shoots or axillary buds, which are meristems, are excised using explants of in vitro (in vitro) plant pieces of natural potatoes, and a specific growth regulator is added to a basal medium to obtain plant The first stage in which the growth of the underground part and the growth and growth of the above-ground part are regulated to promote the growth of only adventitious buds to form multiple shoots, and then the shoots are cut using sterilized scalpels or tweezers. After a second stage of separation, cultivation and rooting in a rooting medium containing a high-concentration carbon source, the roots are acclimatized, and then a large amount of seedlings are supplied via a potting process.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail, but it is important to use excellent non-hybridized wild potatoes to be used for tissue culture in the present invention, regardless of whether they are wild or cultivated. It is possible to use meristems of apical buds, axillary buds, or adventitious buds derived from mukago as a piece of tissue of this natural potato. It is preferable from a practical standpoint to use an in vitro seedling that has been differentiated and regenerated.

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

Examples of the commonly used synthetic nutrient medium include, for example,
A typical example is Murashige-S
Koog's medium, Linsmaier
-Skoog's medium, Nitsch's medium, Gamborg's B5 medium and the like.

The synthetic nutrient medium for shoot formation used in the present invention is a medium to which sucrose or glucose is usually added at a concentration of 1 to 10%, preferably 3 to 5% as a carbon source, or the medium is fixed, in addition to the above-described synthetic nutrient medium. For this purpose, agar having a concentration of 0.6 to 1.2% or gellan gum having a concentration of about 0.2% is used, but it is possible to use a liquid medium to which no fixing agent is added.

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

Examples of the growth regulator include auxins such as naphthalene acetic acid, indole acetic acid, indole butyric acid, and 2.4 dichlorophenoxy acetic acid; Cytokinins such as adenine, kinetin, zeatin, and isopentenyl adenine (substances that have a physiological effect on the growth, formation of adventitious buds, and growth of above-ground parts) or gibberellins and brassinolides are added alone or in combination. Used, but the balance between them is important.

In the first stage of the present invention, only a large number of shoots are formed, and the above-mentioned substances that physiologically act on the growth of the above-ground and underground are combined in a well-balanced manner, added to a nutrient medium, and cultured to grow. And a large number of high quality shoots can be formed for seeds and seedlings. As an example, as a growth regulator, naphthalene acetic acid (NAA) is good for auxins, and benzyl adenine (BA) is recommended for cytokinins. The combination of naphthalene acetic acid of 0.04 to 0.2 mg / and benzyl adenine of 2.0 mg / is preferred.

 That is, BA: NAA is preferably in the range of 100: 2 to 10.

The culture vessel can be used in any shape such as a test tube, an Erlenmeyer flask, a wide-mouthed bottle, and the like, and if the culture conditions of the present invention are described, the temperature is optimally 20 to 35 ° C., preferably 25 ± 1 ° C. For light irradiation, 500 ~ 100 by fluorescent light
00Lux, preferably 2000-3000Lux for about 16-24 hours (1
And the pH is suitably in the range of 4-8, preferably 5-6.

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

When seedlings are used, rooting is carried out using the above nutrient medium (containing no growth regulator) and a rooting medium containing sucrose as a carbon source at a high concentration of 3 to 9%, preferably 6 to 9%. .

As described above, the method of the present invention enables efficient production of a large number of seeds and seedlings.
Approximately three months are required for the stage, and approximately one month is required for rooting the separated shoots, and then, after acclimatization, seedlings are raised.

(Operation)

Since the present invention is constituted as described above, in the first stage of growing the seedling, multiple shoots of good quality suitable for the seedling are obtained without rooting, and in the second stage, the multiple shoots are obtained in the first stage. Since shoots are separated and cultured under conditions suitable for root formation, rooting suitable for seeds and seedlings is promoted, and large-scale propagation of natural potato seeds and seedlings by tissue culture can be easily performed.

〔Example〕

 Hereinafter, the present invention will be described more specifically with reference to examples.

Example 1 [Step 1] A wild potato mukago is collected and subjected to surface sterilization with a 0.2% aqueous benzalkonium chloride solution, 70% ethanol and a 2% aqueous sodium zincate solution, containing 0.9% of agar, pH5. The seedlings were seeded on an MS (Murashige-Skoog) basic medium (Table 1) adjusted to 8 to obtain seedlings in test tubes.

Then, apical buds and axillary buds were removed from the aseptic seedlings as tissue pieces and cultured.

As the medium, 30 ml of an agar medium adjusted to pH 5.8 by adding naphthalene acetic acid (NAA) and benzyl adenine (BA) in combination at each concentration as growth regulators to the above MS basic medium was dispensed into a 100 ml Erlenmeyer flask. One used. Cultivation was performed at 25 ° C. under continuous illumination of 2000 to 3000 Lux for 24 hours (all day). Table 3 shows the results three months after the culturing.

As shown in Table 2, remarkable multiple shoot formation was observed in the medium in which 0.04 mg / A of NAA and 2.0 mg / BA of BA were added to the basal medium as growth regulators.

The shoots from the cultured tissue slices have a small number of shoots from the apical buds, while the shoots from the axillary buds have a large number of shoots, so that good growth can be easily obtained.

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

The medium was an MS basic medium to which 3%, 9% and 15% of sucrose was added without any growth regulator. Culture conditions were the same as in the first stage. One month after the cultivation, the rooting rate and the number of roots of shoots increased as the sucrose concentration in the medium increased to 3 to 9% as shown in FIG.
Cultured individuals that could be used as seeds and seedlings were obtained.

However, the root formation rate is 50% or more is an indication of the useful range.

(Effect)

The advantages described below are remarkable in the method for mass-producing natural potato seedlings by tissue culture according to the method of the present invention, and are evident from Table 2 and FIG.

(1) The vegetative body can be propagated in vitro, which makes it possible to maintain a superior strain for many years.

(2) A large number of seeds and seedlings can be obtained from the top buds and axillary buds as tissue pieces in a short period of time, saving resources, saving labor,
Efficiency is improved, and seeds and seedlings can be supplied in large quantities and at low cost. In other words, according to the method of the present invention, it is possible to calculate about 10,000 or more seedlings obtained from one piece of tissue in one year.

(3) Since cultivation can be carried out in a controlled environment, infection and onset of plant diseases can be prevented in the process of seed and seedling formation, selection of excellent strains is easy to select carefully, and dissemination by excellent seeds is facilitated. .

(4) It is characterized in that a disease-free strain can be obtained.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing the relationship between root formation one month after shoot culture and sucrose concentration in the medium. The abscissa indicates the sucrose concentration (%), the ordinate on the ordinate indicates the root formation rate (%), and the ordinate on the right indicates the number of roots formed.

 ──────────────────────────────────────────────────続 き Continued on the front page (56) References Yasuyuki Yamada “Plant Biotechnology, Modern Chemistry Special Issue 5” Published by Tokyo Chemical Dojinsha, April 25, 1986 17

Claims (1)

(57) [Claims] 1. An explant of a native potato, an explant of apical or axillary bud, is cultured in a synthetic nutrient medium containing auxins and cytokinins as growth regulators to form multiple shoots (multiple shoots). Tissue culture of native potato seedlings comprising a single step and a second step of separating the shoot and culturing the shoot in a synthetic nutrient medium containing a carbon source containing no growth regulator and forming roots. Mass propagation method.