JP3028613B2 - Growth culture method of adventitious buds of cyclamen - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a culture method for growing adventitious buds efficiently by subjecting adventitious buds of cyclamen to aeration culture in a liquid medium.

[0002]

2. Description of the Related Art Cyclamen has been conventionally cultivated by collecting seeds by a method of seed propagation, sowing and raising seedlings. In this conventional method, crossing is performed between cyclamen individuals during the seed production process, so important traits such as flower color and flower type are not fixed, and the same quality as the parent strain cannot be obtained stably. There's a problem.

[0003] In order to solve such a problem, a method of culturing cyclamen tissues to differentiate and grow adventitious buds and forming seedlings by using a solid medium has heretofore been used. Further, as a medium for differentiating and growing adventitious buds of tissue cyclamen, Murashige /
And Skoog (Murashige & Skoog) medium (hereinafter referred to as “MS medium”). These known plant tissue culture media may be used as they are, or may be used with the concentration of all inorganic salts being one-half or one-third of the composition of these media [JP-A-63-22].
No. 6215, JP-A-63-137617, Illustrated Tissue Culture Introduction, pp. 72-75 (issued by Seibundo Shinkosha in 1985). However, in the tissue culture method using these known solid media, the growth rate of the adventitious buds of cyclamen is slow, so that the culture period is prolonged or the rate of withering during the growth culture is increased. It is not always satisfactory as a method for producing adventitious buds used for mass production.

[0004] On the other hand, there is known a method of cultivating a tissue of a rose genus plant by aeration in a liquid medium to propagate seeds and seedlings (Japanese Patent Application Laid-Open No. 1-269430). However, there is no known cultivation method for growing adventitious buds of cyclamen by aeration in a liquid medium.

[0005]

In order to mass-produce cyclamen seedlings having the same trait, a process of tissue-culturing a piece of cyclamen tissue to differentiate adventitious buds is carried out. It is considered that a method of sequentially increasing the number of buds and sequentially growing the adventitious buds to form seedlings is optimal. An object of the present invention is to provide a method for efficiently growing and cultivating adventitious buds of cyclamen in a step of growing adventitious buds of cyclamen, which is an alternative to the method using a conventional solid medium having a low survival rate of adventitious buds and a low growth rate. It is in.

[0006]

Means for Solving the Problems The present inventors have intensively studied to solve the above-mentioned problems. As a result, it has been found that adventitious buds of cyclamen can be grown efficiently in a liquid medium to grow the adventitious buds efficiently and to form healthy seedlings. Therefore, the present invention is a method for growing and cultivating adventitious buds of cyclamen, wherein the adventitious buds are grown by aeration culture of the adventitious buds of cyclamen in a liquid medium containing no plant growth hormone.

The adventitious bud used in the method of the present invention is obtained as follows.

The cyclamen used in the present invention is a plant belonging to the genus Cyclamen, and varieties include Victoria, barberk, pure white, pastel, mini cyclamen, etc., but are not limited to these varieties.

First, a piece of tissue such as the leaf blade, petiole, stem tip, tuber, anther base, or flower stem of cyclamen is soaked in about 70-80% ethanol solution for 10 seconds, and then in about 1% sodium hypochlorite solution. After sterilization for 20 minutes, cut into 1 cm square sections.
This was added to a medium in which the inorganic component of the MS medium was diluted to one-third, and naphthalene acetic acid 0.1 mg / l and benzyl adenine 1.
0 mg / l and sucrose 30 g / l, respectively,
5.8, and then placed on a solid medium supplemented with 10 g / l of gellite. Then, the cells were cultured in the dark at 20 ° C. for 50 days to form calli from the periphery of the tissue, and adventitious shoots were differentiated on the calli.

[0010] A site having a growth point in the adventitious buds may be used.

An adventitious bud having a growth point site is, for example, an adventitious bud that is differentiated from a leaf slice of cyclamen and has an adventitious bud length of 0.5 cm to 3 cm, preferably 1 to 2 cm.
cm, which has a growth point site at the base of the adventitious bud that serves as a base for new leaf development. When the growth culture is performed using the adventitious bud having the growth point site, after removing the seedling formed by the growth culture from the liquid medium,
After rooting and acclimatization by a conventionally known method, it can be grown into a healthy plant having constant properties.

[0012] In addition, even if growth and culture using the adventitious buds having no growth point site in the liquid medium of the present invention do not result in healthy seedlings, they will die in the process of rooting and acclimation, I do not reach my body.

The adventitious bud obtained in this manner may be obtained from any of the above-mentioned pieces of tissue, but the adventitious bud obtained from a leaf blade or a petiole tissue piece is apt to form the most healthy seedling. In the present invention, adventitious buds from a leaf blade or a petiole tissue piece are used.

The method for cultivating adventitious buds of cyclamen according to the present invention is carried out in the following manner.

The medium used for growing adventitious shoots of the present invention includes, for example, Murashige-Skoog (hereinafter referred to as “MS medium”), Gumborg's B5 medium, white medium, and niche medium.
Examples include a medium for plant tissue culture such as a niche medium and an N6 medium. Regarding these known medium compositions, see, for example, “New plant tissue culture” No. 386, by Takeuchi, Nakajima, and Furuya.
Pages 391 to 391 (1979) published by Asakura Shoten. These media may be used as they are, or those obtained by diluting them to 1/3 or 1/2, but liquid media prepared using MS media are particularly preferable.

As the carbon source to be added to the liquid medium used in the present invention, saccharides such as sucrose, and primary carbon sources such as ethanol
Grade alcohol, etc., but sucrose is 20 g / l.
It may be used at a concentration of ９０90 g / l, preferably 30 g / l to 60 g / l.

The pH of the liquid medium is adjusted to 5.6 to 5.8 before use.

In the method of growing and cultivating adventitious buds according to the present invention, no plant growth hormone is usually added to the liquid medium.

A method of growing and culturing adventitious buds of cyclamen using the MS medium among the above-mentioned media will be described.

First, 30 g / l of sucrose was added to a medium obtained by diluting the inorganic component of the MS medium to one third to prepare a liquid medium having a pH of 5.8.

500 ml of this was placed in 11 glass containers, and adventitious buds 10 to 2
0 were transplanted. The incubator may be either shaking or standing. This is kept in a constant temperature room at 20 ° C for 20 to 40 days.
Preferably, after culturing for 30 days, adventitious buds grew and formed young seedlings along with elongation of the leaf stems.

During the cultivation of the adventitious buds, oxygen-containing gas is passed through a liquid medium. In this case, the oxygen-containing gas may be air alone, oxygen gas alone, or a mixture of two or more of oxygen, carbon dioxide, nitrogen, and air. The oxygen content in these gases is 5-100%, preferably 20-9%
0%. The gas flow rate of the gas into the liquid medium is 50 ml / l · min. More than 500ml / l · mi
n. Or less, preferably 100 ml / l · min. Above 3
00 ml / l · min. The following are suitable for the growth and culture of adventitious buds. Further, the growth and culture of adventitious buds of cyclamen in the present invention are preferably performed under light irradiation, and the illuminance in this case is 500 lux or more, 1000 lux or less,
Preferably, 1000 lux or more and 3000 lux or less are suitable. In addition, the culture under this light irradiation is
When the dark period is adjusted to a light-dark period of 2 to 18 hours, preferably 16 hours, and the dark period is 6 to 12 hours, preferably 8 hours, the rate of growth of adventitious shoots and foliage elongation is increased. Growth is promoted.

The following are test examples of the present invention.

[0024]

[Test example] Cyclamen (variety: Victoria, pastel,
A tissue piece of leaf blade (mini cyclamen) is immersed in an approximately 70-80% ethanol solution for 10 seconds, and then in an approximately 1% sodium hypochlorite solution for 20 minutes and sterilized. Next, the tissue piece is taken out, and the sodium hypochlorite attached to the surface is washed three times with sterilized water, and then the tissue piece is made into a 1 cm square section under aseptic conditions. This was added to a medium obtained by diluting the inorganic component of the MS medium to one-third with NAA of 0.1 m.
g / l, BA at 1.0 mg / l, and sucrose at 30 g / l to pH 5.8, and the mixture was placed on a solid medium containing gellite at 10 g / l. Cultured for days. Then, calli were formed around the leaves, and adventitious shoots were differentiated on the calli.

From the adventitious shoots, an adventitious shoot having a growth point and a length of 1 cm was cut off with a scalpel and used as a material.

Next, 500 ml of a liquid medium having a pH of 5.8 obtained by adding 30 g / l of sucrose to a medium obtained by diluting the inorganic component of the MS medium to one-third was put into a 1-liter glass container, and the above-mentioned liquid medium was added thereto. Transplant 20 adventitious shoots per container
The culture was performed at 20 ° C. with an illuminance of 2000 lux and a light period of 16 hours and a dark period of 8 hours, and sterile air was passed through at the flow rate shown in Table 1. The survey was carried out at the seedling stage when the adventitious buds were transplanted and then grown and the length of the foliage became 4 cm, and the survival rate (%) was calculated by the following formula 1. The results are as shown in Table 1.

[0027]

(Equation 1)

[0028]

[Comparative Example 1] Culture was performed in the same manner as in the test example except that sterile air was not passed through the liquid medium. The results are as shown in Table 1.

[0029]

Comparative Example 2 A solid medium containing 1% of agar was used as a medium and cultured in the same manner as in the above-mentioned test example except that sterile air was not aerated. The results are as shown in Table 1.

[0030]

[Table 1]

[0031]

According to the present invention, adventitious buds obtained by culturing a cyclamen tissue fragment can be efficiently grown and a desired seedling can be obtained, as compared with a conventional method using a solid medium for tissue culture. Can be. Therefore, mass production of cyclamen seedlings of uniform quality can be effectively performed using the method of the present invention.

 ────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Toshio Murayama 1-2-5, Chuo-Rinkan, Yamato-shi, Kanagawa (56) References JP-A-2-303432 (JP, A) Agriculture of this month October issue (1989) p38-43 Tissue culture 11 (9) 1985 p27-32 BIO INDUSTRY Vol3 No. 4 1986 p.332-399

Claims (1)

(57) [Claims] 1. A method for growing and cultivating adventitious buds of cyclamen, wherein the adventitious buds derived from the leaf blade or petiole of cyclamen are aerated and cultured in a liquid medium containing no plant growth hormone. .