JPH08112045A - Method for mass production of seed and seedling of plant of genus fritillaria - Google Patents

Abstract

PURPOSE: To provide a method for multiplying seeds and seedlings of a plant of the genus Fritillaria in a much larger amount than a conventional multiplying method by carrying out tissue culture dividedly in processes. CONSTITUTION: This method for producing a large amount of seeds and seedlings of a plant of the genus Fritillaria comprises culturing a tissue piece of a plant of the genus Fritillaria to form a cultured tissue piece, (1) a bulblet proliferating process for dividing the culture tissue piece, culturing the piece in a medium containing auxin and differentiating and forming a bulblet and (2) a bulblet cultivating process for culturing the bulblet in a medium substantially not containing auxin and growing and forming the bulblet.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing seeds and seedlings of the genus Fritillaria, and more particularly to an efficient method for mass producing bulbs by tissue culture.

[0002]

2. Description of the Related Art Fritillaria plants are
It is a lily plant and is a perennial plant with bulbs. Examples of the plants of the genus Fritillaria include, but not limited to, lily lily, black lily, white lily, leafhopper, and herb medicines called river shell mother, pine shellfish, flat shellfish, and shellfish. Lily lily is a perennial bulb plant belonging to the family Liliaceae, whose bulbs are white and thick,
Two bulbs are spherical with each other.

[0003] As for lily lily, river shell mother, pine shellfish, flat shellfish, hearth shell and the like, processed bulbs are used as crude drug raw materials for antitussive Chinese medicine, expectorant, etc. There is also a demand for gardening. Black lily is a perennial with black-purple flowers and is in demand for gardening. Yorakuyuri is a variety of flower colors,
Besides being cultivated for appreciation, bulbs are also used for food.

[0004] As a method for multiplying these plants of the genus Fritillaria by ordinary cultivation, there are methods such as the use of bulbs, scales and seeds, but the low growth rate and the long time required for harvesting There are practical issues.

[0005] For example, the seeds of Lilium edulis are scarcely used, and in general, the method of using bulbs is used for multiplication by shunting. Autumn planting and spring digging are the basic crop types, but the multiplication rate is It is as low as 2-3 times and the morbidity is relatively high. In addition, bulbs also have the problem of deterioration in quality due to decay and drying during storage (Tokue Morishita, Herb Cultivation Class (1986))
Page 101). As a means for solving these problems, a study on the multiplication of Lilium edulis by tissue culture has been conducted by Masahiro Masayama et al. (Biopharmaceutical Journal (1986) Vol.
page).

[0006] In the culturing process, scales were formed from the stem and base plate section of the sterilized budworm lily parent bulb,
A large number of scales are formed from the obtained scale slices, and the redifferentiated scale slices are root-cultured to form seedlings, which are then transplanted to soil and cultivated to form bulbs. Furthermore, after transplanting seedlings that had undergone rooting culture into soil and cultivating them, 2.5 bulbs were cultivated for 2.5 months and the average weight of one bulb was 266 mg. Bloating has not been achieved. Other than this, there is no knowledge that tissue culture was used for the growth of seedlings of the genus Fritillaria. As a method for proliferating Liliaceae plants other than the genus Fritillaria by tissue culture, for example, a method for the genus Lilium is known (Japanese Patent Publication No. 60-15268).

[0007]

In the above prior art, the vegetative growth method by cultivating bulbs has problems of low growth rate and difficulty of bulb storage.

According to Masayama et al.'S method for tissue culture of Lilium longifolia, seedlings were formed in tissue culture, but bulbs were not formed, and in order to form seedlings, scales were differentiated. After that, it is necessary to transfer to a rooting medium and culture, which complicates the culture process. Moreover, since small bulbs are formed only after transplanting and culturing the formed seedlings into soil, it is not easy to mass-produce them by this method. Furthermore, this method does not necessarily achieve sufficient enlargement of the follicle for soil transplantation, leaving a problem in terms of productivity in actual cultivation. Further, in the current cultivation, there are problems such as low proliferation rate and difficulty in storing bulbs.

Further, since the method described in the above publication also performs growth based on a lump of scale, a step of differentiating and forming a large number of primitive scales from the scale and a step of enlarging the primitive scale are required. However, there is a problem that the number of work processes increases. It is an object of the present invention to provide a method for efficiently mass-producing a seedling of a genus Fritillaria with good quality by tissue culture, which is characterized by combining a step of proliferating a ball and a step of forming a ball.

[0010]

[Means for Solving the Problems] As a result of intensive studies as means for solving the above problems, the following method was found.
That is, a follicle proliferating step of culturing a tissue piece of a plant of the genus Fritillaria to form a cultured tissue piece, (1) dividing the cultured tissue piece and culturing in a medium containing an auxin to differentiate and form a follicle (2) Mass production of seeds and seedlings of the genus Fritillaria is carried out by a step of culturing the follicle in a nutrient medium substantially free of auxin and growing the follicle.

The present invention will be described in detail below for each culture step. (1) Step of Forming Cultured Tissue Piece A tissue piece of a plant of the genus Fritillaria is aseptically removed according to a conventional method, and usually, one tissue piece is placed per about 10 ml of a solid medium and cultured. The type of plant used here is not particularly limited as long as it is a plant belonging to the genus Fritillaria, but it is particularly preferable to use lily lily.

The above-mentioned tissue pieces include leaves, stems,
Although shoots or other tissue pieces can be used, shoot apical meristem pieces are preferably used, and it is particularly preferable to use a growing point extracted from a shoot inside the scale of the parent bulb.
In the case of Aedes lily, it is normal (the culture period often differs depending on the type of plant, so it is better to specify it as "In the case of Aedes lily.") About 40 to 60 days of culture up to about 1.5 cm It is possible to obtain a cultured tissue piece in which scales are elongated.

As the medium used here, any medium can be used as long as it is a medium generally used for tissue culture of plants, and as an example, Murashige-Skoog medium and a medium modified based on this can be used. Alternatively, agar or gellan gum may be used as the solidifying agent.

As a plant growth promoting substance, auxins such as naphthalene acetic acid and indole acetic acid can be added to the medium in the range of 0.1 to 8.0 mg / l. At this time, cytokinins such as benzylaminopurine and kinetin can be appropriately combined and added to the medium. When cytokinin is used, the concentration should be low. For example, benzylaminopurine can be used in the range of 0 to 5 mg / l, preferably 0 to 1 mg / l. Further, when abscisic acid (ABA) is used in the range of 0.01 to 1 mg / l, it has the effect of promoting the formation and enlargement of bulbs. The sucrose concentration is preferably about 1-8%, more preferably 2-4%. No light is needed. In the culture under illumination, since the scale may be too elongated to obtain many tissue pieces suitable for use in the proliferation step, the culture is preferably performed in the dark. The culture temperature is preferably 15 to 35 ° C, more preferably 20 to 30 ° C. The pH of the medium is preferably 3 to 8, more preferably 4 to 7. In the present invention, the term “cultured tissue piece” refers to one obtained by culturing a tissue piece of a plant, and specifically refers to a bulb, a bulb bottom plate portion, a stem, a flower organ, or the like.

Hereinafter, the pH of the medium and the culture temperature are the same unless otherwise indicated (light conditions are important, so the description is made for each step without omission). (2) Step of Differentiating and Forming Follicles The cultured tissue pieces are divided to obtain divided cultured tissue pieces used in the proliferation step. The size of this divided culture tissue piece is 2 to 10 mm in length,
The basic shape is about 2 to 8 mm wide. The bottom plate of the cultured bulb can also be used in the same manner as the divided cultured tissue piece.
For example, in the case of lily lily, about 60 to 90 days of culture,
The formation of about 2 to 5 follicles is usually recognized per one piece of the divided culture tissue. A solid or liquid medium can be optionally used for culturing. When a solid medium is used, a divided culture tissue piece is usually placed at a ratio of 1 to 2 to 15 ml of the medium, and a liquid medium is used. Can be cultured by adding one divided culture tissue piece per 2 to 15 ml of the medium.

The plant growth promoting substance added to the medium may be any substance as long as it promotes the differentiation and formation of the follicle, but auxin is always added. The type of auxin may be any of naphthalene acetic acid, indole acetic acid, 2,4-D, etc., but naphthalene acetic acid is preferably used. The amount of the plant growth-promoting substance added varies depending on the type of plant or the plant growth-promoting substance, but for example, naphthalene acetic acid can be added in the range of 0.1 to 10 mg / l, particularly preferably 0.3 to 7 mg / l. The sucrose concentration is preferably about 1-8%, more preferably 2-4%. The culture is preferably performed under dark conditions as in the above step.

(3) Step of Enlarging Follicles The follicle differentiated and formed in the above step is cultivated in a medium for the step of culturing pups to enlarge the pups. In the case of Lilium edulis, the weight of the follicle grows 3 to 6 times as much as that at the start of the culturing with fresh weight after culturing for 60 to 90 days. The medium should be substantially free of auxin and the sucrose concentration should be 1-8%
Is preferable, and about 3 to 6% is more preferable. Substantially free of auxin as referred to in the present application means that, in the follicle-culturing step, it does not contain an amount of auxin that promotes differentiation formation of cultured tissue pieces, and does not particularly actively promote differentiation of cultured tissue pieces. As long as the amount of auxin is contained in the medium, there is no problem. The culture is preferably performed under dark conditions as in the above step.

Any culture vessel may be used in each step of the above-mentioned follicle growth and follicle cultivation, and a large vessel such as a glass tank or a jar fermenter may be used in addition to a flask. According to the present invention, a desired size of Fritillaria spp. Can be produced by arbitrarily repeating the follicular cell proliferating step and the follicular cell training step to proliferate in a large amount and pass the follicular cell training step.

In addition, in order to optimize the culture conditions of the above-mentioned tissue culture and increase the production efficiency of bulbs, the divided culture tissue pieces are treated with auxin, for example, naphthalene acetic acid in the range of 0.1 to 10 mg / l, in the step of proliferating bulbs. Differentiated and formed in a medium having a sucrose concentration of 1 to 8%, and in the follicle-culturing step, the follicle differentiated and formed in the previous step has a sucrose concentration of 1 to 8% in a medium that does not substantially contain auxin. It can be achieved by culturing using a range.

[0020]

【Example】

[Example 1] (1) A sprout occurring at the inner base of the scale of the Lilium longiflorum parental bulb is cut out, and 70% ethanol solution, sterilized water, 1% sodium hypochlorite solution (effective chlorine amount 1%) is cut out.
After sterilizing with sterilized water and thoroughly washing with sterile water, the shoot apical meristem pieces were aseptically excised, and put into a test tube of 22 mm in diameter and 220 mm in depth containing 10 ml of solid medium of the composition shown in Table 1-A and B. One test tube was placed on each tube, and static culture was carried out for 70 days in the dark at 25 ° C ± 1 ° C. In this culture, bulbs (cultured spheres) elongated to about 1 cm were obtained in the solid media having the compositions A and B shown in Table 1.

(2) The obtained bulb is about 0.4 cm in length and 0.4 in width.
Dividing scales were obtained by cutting and dividing with a basic shape of about cm. A small piece of cultured tissue produced incidentally in the above step was also used in this step. The weight of one divided scale was 0.05 to 0.10 g. Add 1 divided scale to 5 ml of liquid medium having the composition shown in Table 1-C, and rotate on a shaker at a rotation speed of about 100 r.
pm, temperature was 25 ± 1 ° C., and the cells were cultured in the dark for about 60 days. In this culture, an average of 3 follicles differentiated from one divided scale.

(3) The follicle obtained in (2) is placed in a flask having a capacity of 34 mm and a volume of 200 ml containing 50 ml of the liquid medium having the composition shown in Table 1-D, and one follicle per 5 ml of the medium is placed, and (2) The culture was carried out for about 60 days under the same culture conditions as described above. This culture yielded a follicle that was about 5 times enlarged. In this embodiment, (2),
The process of (3) is performed three times alternately, and as a result, from one parent bulb of about 15 g, a fresh weight of 0.1 to 0.4 g is obtained by culturing for a total of 430 days.
About 1,500 cultured cells were obtained.

[0023]

[Table 1]

Example 2 Based on 10 culture spheres of about 1 g obtained by the methods (1) to (3) of Example 1, Example 1 was used.
When the proliferating step of (2) was repeated once and the follicle training step of (3) was repeated twice, about 430 cultured spheres with an average fresh weight of about 1 g were obtained over the entire culture period of 250 days, and hypertrophy was achieved. The cultivated culture spheres were stably and efficiently obtained. Even in this case, many small cultured spheres (1 g or less) were obtained.

[0025]

INDUSTRIAL APPLICABILITY According to the present invention, by performing the tissue culture separately in the follicle proliferating step and the follicle proliferating step, it is possible to proliferate in a much larger amount than the conventional breeding methods. Furthermore, compared to conventional tissue culture methods, it is homogeneous and disease-free,
Mass production and production of good quality Fritillaria spp. Can be efficiently carried out. Further, when the bulb is used for normal cultivation and edible or medicinal use, the culture sphere obtained by the method of the present application may be used, or the bulb may be used after being grown to a desired size by culturing the culture sphere. it can.

Claims (4)

[Claims] 1. A tissue piece of a Fritillaria genus plant is cultured to form a cultured tissue piece, and (1) the cultured tissue piece is divided and cultured in a medium containing auxin to form follicle formation. Mass production method of seeds and seedlings of the genus Fritillaria, which comprises a step of proliferating a follicle and a step of (2) culturing the follicle in a medium substantially free of auxin to form a hypertrophy of the follicle. . 2. The method for mass-producing seedlings of the plant of the genus Fritillaria according to claim 1, wherein the step of proliferating the follicle and the step of training the follicle are arbitrarily repeated. 3. The plant of the genus Fritiliaria is a lily lily.
(Fritillaria thunbergii), The method for mass-producing a plant seedling of the genus Fritillaria according to claim 1. 4. The method for mass-producing seedlings of the plant of the genus Fritillaria according to claim 1, wherein the step of proliferating the ball and the step of training the ball are performed in the dark.