JPH03244328A - Culture medium for tissue culture of cyclamen and method for tissue culture of cyclamen using the same culture medium - Google Patents

Abstract

PURPOSE:To enable efficient differentiation of adventitious buds and practice of tissue culture of a cyclamen capable of producing a large amount of young seedlings by using a culture medium containing magnesium ions (Mg<2+>) at 2 to 9 mM concentration. CONSTITUTION:A plant growth hormone such as auxins or cytokinins, as necessary, is added to a well-known basal culture medium such as Murashige-Skoog (MS) culture medium composed of a saccharide such as sucrose and other required components and a magnesium salt such as magnesium sulfate or magnesium chloride is used therein to regulate the concentration of the magnesium ions (Mg<2+>) within the range of 2 to 9 mM (the resultant culture medium is hereinafter referred to as an improved MS culture medium). Tissue pieces are then collected from leaf blades, shoot apexes, etc., of a cyclamen which is a cultivar such as Victoria and formed into slices of about 1 cm square. The aforementioned improved MS culture medium is subsequently fixed and the slices are bedded thereon and cultured in a dark place to form adventitious buds in the tissue slices. Thereby, since the adventitious buds are remarkably increased, the culture medium can be effectively utilized for mass production of young seedlings having homogeneous quality of the cyclamen.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a medium for tissue culture of cyclamen and a method for culturing cyclamen tissue using the same medium.

(Prior art) /・Kuramen has so far been cultivated by collecting seeds by seed propagation, sowing, and raising seedlings. According to this conventional method, because cyclamen individuals are crossed during the seed production process, important traits such as flower color and pore shape are not fixed, resulting in quality problems such as not being able to stably obtain the same seed as the parent plant. There is a problem.

In order to solve these problems, proposals have been made for tissue culture methods in which cyclamen tissues are cultured to proliferate adventitious buds and callus.

That is, for example, as a medium for tissue culture of cyclamen, Murashige & Skoo
g) Attempts to use known plant tissue culture media such as medium (hereinafter referred to as "rMS medium") in its original composition, or to use a medium with a composition in which the total inorganic salts are diluted to '/x or l/. has been done (Japanese Patent Application Laid-Open No. 1983-
226215, JP-A-63-137617, Illustrated Introduction to Tissue Culture, pages 72-75 (published by Seibundo Shinkosha in 1985), etc.).

However, the MS exemplified in these prior art documents
The tissue culture method using a medium has drawbacks such as a low regeneration rate of seedlings, and as a result, it has been difficult to put it to practical use as a method for mass production of cyclamen seedlings.

(Problems to be Solved by the Invention) As mentioned above, in order to mass produce cyclamen seedlings with the same characteristics, cyclamen tissue pieces are subjected to tissue culture,
The most effective method is thought to be to efficiently differentiate adventitious buds to form seedlings, but the regeneration rate of seedlings is low depending on tissue culture methods that use known culture media, so the efficiency of cyclamen production is limited. This is a new medium for tissue culture that can not be said to be a standard propagation method, and therefore has a high regeneration rate of cyclamen seedlings, and can efficiently differentiate adventitious buds and increase the number of adventitious buds, thereby producing a large number of seedlings. The problem that needed to be solved was the elucidation of the problem and the elucidation of a new growth method using the same medium.

(Means for Solving the Problems) As a result of intensive research to solve the above-mentioned problems, the present inventors found that the concentration of magnesium ions in the medium used for tissue culture leads to differentiation of adventitious buds and an increase in the number of adventitious buds. The present invention was completed based on the discovery that this had a very large effect. That is, the present inventors determined that the magnesium ion concentration of the medium used for conventional cyclamen tissue culture was 0,
It was revealed that a medium with a magnesium ion concentration higher than 5-1, 5mM efficiently differentiated adventitious buds when cyclamen tissue pieces were cultured, and significantly increased the number of adventitious buds.

Therefore, the present invention provides magnesium ion (Mg")
It relates to a tissue culture medium for cyclamen, characterized in that it contains a concentration of mM-9mM1, preferably 3mM to 5mM.

Furthermore, the present invention provides magnesium ions (Mg")
It also relates to a method of culturing cyclamen tissue using a medium containing mM-9111L, preferably at a concentration of 3mM to 5mM.

The medium components constituting the medium of the present invention are known basic media consisting of major inorganic components, minor inorganic components, sugars, and in some cases vitamins and amino acids, such as Kutubu's solution, MS
Medium, Ganpolg's 85 medium, White medium, Niche-
Niche media, synthetic media such as N6 media, boiled liquids or extracts of potatoes, cucumbers, apples, bananas, etc.
coconut milk, casein hydrolyzate, malt extract,
The basic medium composition is a medium derived from natural products such as yeast extract with sugar and other ingredients added, or a medium made by adding the ingredients derived from natural products to the above synthetic medium, and furthermore, magnesium in these medium. The magnesium ion concentration was adjusted to 2mM to 9mM by adjusting the ion content. Plant growth regulators such as plant growth hormones can also be added to these media if necessary.

As mentioned above, the characteristics of the culture medium of the present invention are that plant growth regulators are added to the known basic culture medium as necessary.
The concentration of magnesium ions (Mg'') is adjusted to a range of 2mM to 9mM. In this way, the concentration of magnesium ions is set to 2mM to 9mM, preferably 3mM, which is higher than that of known media. -5mM
When cyclamen tissue is cultured in this medium, differentiation of cyclamen adventitious buds is greatly promoted compared to culture in a medium other than the magnesium ion concentration range mentioned above, and the number of adventitious buds is reduced. A significant increase was observed and therefore it is highly preferred as a tissue culture medium for cyclamen.

The magnesium ion source used in preparing the culture medium of the present invention may be any magnesium salt, such as magnesium sulfate, magnesium chloride, magnesium phosphate, and the like.

Therefore, the medium of the present invention is characterized by containing a specific range of magnesium ions in a known basic medium, and any known medium components other than magnesium ions may be used. For this purpose, MS medium is preferably used as the basic medium, and the MS medium in which the magnesium ion concentration is adjusted within the above-mentioned range of the present invention (hereinafter referred to as improved MS medium) is preferably used.

The present invention will be explained below using this improved MS culture medium, and the effects of the culture medium having a content of magnonium ions in a specific range of the present invention will be explained.

In preparing the improved MS medium, one or more magnesium sulfate or other sources of magnesium ions used in preparing the MS medium, such as magnesium chloride, magnesium phosphate, are added to achieve the desired magnesium ion concentration.

In addition, the concentration of inorganic components other than magnesium sulfate in the improved MS medium is determined by either using the inorganic components of the MS medium as is or diluting it to 2.
It may be reduced to 1/3 or 1/3.

Furthermore, manganese ions, zinc ions, and
The concentration of metal ions such as copper ions and cobalt ions is within a specific range, that is, the concentration of manganese ions is 0.1 to 0.
．． 2mM, zinc ion concentration 0.03-0.06mM,
#] The ion concentration is 0.1-0.4JIM, :'The parto ion concentration is preferably 0.1-0.2 μM.

In addition, the plant growth hormones added to the improved MS medium include indole acetic acid (IAA), σ-naphthalene acetic acid (a
-NAA), indolebutyric acid (IBA), auxins such as 2,4-dichlorophenoxyacetic acid (2,4-D), and cytokinins such as benzylaminopurine (BA), kinetin, and zeatin.
-NAA 0.01-1mg/Q and BA 0.01-5
mg/Q, preferably 1 my/Q, may be used in combination.

Furthermore, carbon sources added to the improved MS medium include sugars such as sucrose, primary alcohols such as ethanol, etc.
, preferably at a concentration of 309/12(7).

In addition, the pH of the improved MS medium was adjusted to 5.6 to 5.8,
Used as a solid or liquid medium.

When a solid medium is used, agar, agarose, gelulite (gellan gum), carrageenan, etc. are used as a gelling agent. Solid culture is then performed on this solid medium.

In the case of culture using a liquid medium, the culture is suspended in the liquid,
Suspension culture is usually carried out, and in this case, a Jarf Armentaya tank can be used as a culture tank.

Next, a specific example of the preparation of this improved MS medium will be shown in Examples. In the culture medium of this example, the basic M
The components of S medium and the concentrations of each component are shown in Table 1 below. The modified MS medium is the basic MS medium with changes made to its components as described in each of the following examples.

Table 1 MS Medium 戊9 and Concentration Example 1 Improved MS medium using MgSO4.7H,O to adjust the magnesium ion concentration to 4.5mM Among the inorganic components of the MS medium shown in Table 1, Mg504-
Add 740my/Q (3.0mM) of 7H20 (total magnesium ion concentration to 4.5mM),
g5O* 4Hz The concentration of inorganic components other than O was diluted to one-third.

Add vitamins and amino acids to this as shown in Table 1,
Furthermore, a-NAA was added to Q, 1mg/Q, and BA was added to 1.0m.
y/Q, add 30 g/(2 of sucrose to make pn 5.8, and add I Og/(l of agar) to make a solid medium.

Example 2 Improved MS medium with magnesium ion concentration set to 4.5mM by combined use of MgSO=4HzO1MgCQ16HzO The concentration of all inorganic components of the MS medium shown in Table 1 was reduced to one-third
Dilute MgCl216H,O to 813+ig/1
2 (4.0mM) (total magnesium ion concentration 4.5mM).

Add vitamins and amino acids to this as shown in Table 1 at 1:1 and 2
+oL, Sarania a-NAAwoo, l+I+9/(lSB
Add 1.0 m9/Qs of A and 30/ff of sucrose to adjust the pH to 5.8, and add Gelrite 39/Q to prepare a solid medium.

Example 3 Mg504・7H20SMgs (7H2O5・8H20
The concentration of all inorganic components of the MS medium shown in Table 1 was diluted to one-third of the concentration of the total inorganic components of the improved MS medium shown in Table 1, and the magnesium ion concentration was set to 4.5mM using Mg3(PO2)!・8H20
1628+++9/4 (4-OmM) was added (total magnesium ion concentration 4.5mM).

Add vitamins and amino acids to this as shown in Table 1,
Furthermore, a-NAA was added at 0.1 mg/<1. BA 1.0
Add 30g of my/Q and sucrose, and adjust pu to 5.
8 and add 3 g/a of Gelrite to make a solid medium.

Cyclamen tissue culture according to the present invention is carried out as follows. The cyclamen used in the present invention is a plant of the genus Cyclamen, and varieties include Victoria, Barbak, Pure White, Pastel, Mini Cyclamen, etc., but are not limited to these varieties.

First, the cyclamen plant body, such as leaf blades, petioles, shoot tips,
Tissue pieces are collected from tubers, stone bases, flower stems, etc., and placed in an approximately 70-80% ethanol solution for 10 seconds, then approximately 1 hour.
% sodium hypochlorite solution for 20 minutes to sterilize. Next, this tissue piece was taken out and the sodium hypochlorite adhering to its surface was washed three times with sterile water.
The tissue pieces are cut into 1 cm square sections under sterile conditions.

Then, the improved medium prepared as shown in the above example was placed in a Petri dish with a diameter of 9c+n and solidified, and then the slices were placed on the medium. This is preferably heated at 15 to 25°C.
When cultured in a constant temperature room at 20° C. for 40 to 70 days, preferably 50 days in the dark, adventitious buds are formed on the tissue sections.

Test examples of the present invention are shown below.

Test example (method for propagating adventitious buds by tissue culture) 100 mQ of leaf blades of each variety of cyclamen (varieties: Victoria, Pure White, Barbak, Pastel, Mini Cyclamen)
in 70% ethanol solution for 10 seconds, then 500 mQ
After immersing the leaves in a 1% sodium hypochlorite solution for 20 minutes, the leaf blades were taken out and washed three times with sterile water. Thereafter, the leaf blade was cut into 1 cm square pieces using a scalpel.

A culture medium (pH 5,8) prepared according to the example so that the magnesium ion concentration was as shown in Table 2 was poured into a petri dish with a diameter of 9 cm and solidified.

The MS medium of the control group was the present invention group, except that the inorganic components listed in Table 1 were diluted to one-third (magnesium sulfate 0.5 mM) or were left as they were without dilution (magnesium sulfate 1.5 mM). The medium was prepared in the same way as the medium.

Four pieces of the above-mentioned leaf pieces (of the same variety) were placed in each petri dish on the culture medium obtained by straining the leaves in this manner, and five petri dishes were prepared in one concentration group. When this was cultured in a dark place at 20°C for 50 days, adventitious buds were formed from around the leaves.

The investigation was conducted on the 50th day of culture, and the total number of adventitious buds per 20 leaf pieces was determined to determine the average number of adventitious buds. The results are shown in Table 2.

(Effects of the Invention) According to the present invention, adventitious buds are efficiently differentiated from tissue pieces of cyclamen plants, and the number of adventitious buds is significantly increased, compared to the conventional method using a tissue culture medium. Therefore, the method of tissue culturing cyclamen using the medium of the present invention can be effectively used for mass production of cyclamen seedlings of uniform quality.

Claims (1)

[Claims] 1) Magnesium ion (Mg^2^+) at 2mM to 9
Cyclamen tissue culture medium, characterized in that it contains a concentration of mM. 2) Magnesium ion (Mg^2^+) from 2mM to 9
A method for culturing cyclamen tissue using a medium containing a concentration of mM.