JPH04287623A - Method for proliferating bulbs by plant tissue culture - Google Patents

Abstract

PURPOSE:To proliferate bulbs in a small culture vessel with reduced culture amount by culturing cultured cells, etc., of a bulb plant in a differentiation medium and then culturing adventive buds or the small bulbs in a specific medium. CONSTITUTION:A bulb cut piece or other tissue piece of a bulb plant is cultured in a differentiation medium to differentiate the adventive buds and small bulbs. The adventive buds or small bulbs are cultured in a thickening medium by lowering a phosphoric acid concentration to 0.4mM to 0 for thickening adventive bud or small bulbs. Thereby, loss of the nutritive content by the formation of callus and the thickening and loss of the nutritive content by the frequent differentiation of bulbs are suppressed, and the adventive buds, etc., differentiated within a period can efficiently be thickened.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for mass propagation of bulbs by plant tissue culture.

0002

BACKGROUND OF THE INVENTION Conventionally, bulbous plants have been cultivated by dividing bulbs, rin-kazashi, and using mulch and wood. However, these propagation methods not only require large areas of land and a lot of manpower, but also the spread of viral diseases in recent years has led to a decrease in the propagation rate of seedlings of the plants, a decrease in the growth rate, and a decrease in the quality of flowers and bulbs. is a problem. In order to solve these problems, research has been reported in which growing points are cultured and virus-free bulbs are propagated by plant tissue culture. For example, Japanese Patent Application Laid-open No. 55-15734 discloses that after a solid medium is used to form and propagate bulblets from tissue pieces of plants belonging to the genus Lilium, they are transferred to a liquid medium, cultured, and enlarged. A method for mass propagation of plants of the genus Lilium is disclosed. However, in the above method, a solid medium using agar is used in the first stage of enlargement culture, which not only requires a lot of labor for the culture operation, but also makes it difficult to use the culture vessel in three dimensions, resulting in poor culture efficiency. There's a problem.

[0003] Against this background, the present applicant filed a patent application in 1983.
No. 0-128348 proposed a method for propagating lily seedlings. By using the above method, it is possible to efficiently propagate a large amount of seedlings.

0004

[Problems to be Solved by the Invention] Many of the conventional methods for propagating bulbs using tissue culture methods require improvement of the culture method to achieve proliferation efficiency and stability of the technique in order to be used as a practical technology. is necessary. For example, in the propagation of bulbous plants by tissue culture, a phenomenon has been observed in which a portion of the culture becomes a callus and the growth of the bulbs is suppressed, which is an important problem to be avoided. There is also the problem that adventitious buds or small bulbs continue to differentiate one after another, which prevents the enlargement of the previously differentiated adventitious buds or small bulbs.

As a method for inducing the enlargement of tulip bulbs, a technique for promoting the enlargement of tulip bulbs by transplanting adventitious buds differentiated on an agar medium to an agar medium containing abscisic acid (Y. , Nis
Written by hiuchi, Studies on Vegetables
activePropagation of Turti
ps. V. Effect of GrowthRe
gurators on The Bulb Form
ation of Adventitious Bud
Cultured in vitro. , Jour
nal of Hokkaido University
y of Education Vol. 34, No
．． 1:9-15, 1983) is known. Further, JP-A No. 61-56022 describes that abscisic acid added to the medium suppresses callus formation and promotes bulb enlargement.

However, bulbs that have been propagated and enlarged using conventional methods have the problem that germination after transplanting into soil is suppressed or significantly delayed, and that the germination timing is not uniform and varies.

[0007]

[Means for Solving the Problems] In order to solve the above problems, the present inventors conducted a detailed study on the method of propagating bulbs by tissue culture, and as a result, the phosphoric acid concentration in the enlargement medium was We have found that the above problems can be solved by culturing at a concentration of 0.40 mM or less or even zero, and based on this new knowledge, we have completed the present invention.

Therefore, the method for propagating bulbs of the present invention is a method for propagating bulbs by plant tissue culture, in which tissue pieces or cultured cells of bulbous plants are cultured in a differentiation medium, and adventitious buds or small bulbs are differentiated. , then the phosphoric acid concentration is 0
．． The method is characterized in that the differentiated adventitious buds or the small bulbs are cultured and enlarged in a medium containing no more than 40 mM phosphoric acid.

The details of the present invention will be explained below. The present invention is a method for propagating bulbs by tissue culture, which consists of the following two steps: AB. In the present invention, tissue culture of bulbous plants can be carried out using tissue pieces or cultured cells of the plants. Specifically, the tissue pieces include shoot tips, stems, leaves, flowers, seeds,
Examples include tissue pieces of bulbous plants, such as small bulbs, roots or other tissues cut into small pieces, which are usually used after being sterilized by sodium hypochlorite, ethyl alcohol or flame. However, when using bulbous plants cultured or cultivated aseptically, the above sterilization operation is not necessary. Furthermore, in the case of propagating virus-free bulbous plants, the tissue near the growing point, the above-mentioned tissue pieces of the bulbous plant obtained from the tissue near the growing point, etc. can be used as culture materials. (A) Formation of adventitious buds and small bulbs In the present invention, bulb sections or other tissue pieces of bulbous plants are cultured using a differentiation medium to differentiate adventitious buds and small bulbs. Differentiation medium is a conventionally known medium that facilitates the differentiation of adventitious buds and small bulbs from bulb sections or other tissue pieces, and a modified medium that improves the components of these mediums, often containing auxin, cytokinin, etc. This is a medium containing plant hormones. For example, a medium used for normal tissue culture such as Murashige-Skoog medium, White medium, Niche-Nich medium, Linsmeyer-Skoog medium, or a modified medium made by modifying these mediums as a basic medium is used. be able to. In the tissue culture method of the present invention, benzyladenine, kinetin, zeatin, isopenteladenine, indoleacetic acid, indolebutyric acid,
It is preferable to culture by adding a plant growth regulator such as naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid to the medium. The amount of these plant growth regulators added varies depending on the type of plant growth regulator, the type of bulbous plant, etc.
Generally, 10-4 mg/L to 50 mg/L is good. However, if the amount of these plant regulators is too large, when the bulbs obtained by the tissue culture of the bulbous plants of the present invention are further cut and cultured to differentiate into adventitious buds or small bulblets,
This may cause morphological abnormalities and genetic variations.
More preferably, it is about 10-3 mg/L to 1 mg/L. To cultivate bulbous plants, it is better to add carbon sources such as sucrose, glucose, fructose, and maltose to the medium. The amount of carbon source added is generally 5 g/L to 150 g/L, preferably 10 g/L to 50 g/L. The pH of the medium is preferably 4.0 to 8.0. Also, light is not necessarily necessary during culturing, but from 100 to
Good results may be obtained by culturing under illumination with a light intensity of 10,000 lux. Culture temperature is 15℃ to 35℃
is suitable.

[0010] The present invention can be carried out using either a solid medium or a liquid medium. (B) Culture of adventitious buds and small bulbs using enlargement medium (
After forming adventitious buds or small bulbs in A), the medium is aseptically removed, replaced with an enlargement medium, and cultured, whereby the adventitious buds or small bulbs can be efficiently enlarged. The enlargement medium is a medium that has been modified to increase the sugar concentration and promote the enlargement of adventitious buds and small bulbs, and the phosphoric acid concentration of the medium components is 0.5%. It is 40mM or less, more preferably 0.20mM or less. A growth regulator may be present in the medium as necessary. In the present invention, good results may sometimes be obtained even when phosphoric acid is completely removed. When reducing the phosphate concentration in the medium, other inorganic components may also be reduced. For example, when KH2PO4 is used as a phosphoric acid source, reducing KH2PO4 also reduces potassium (K), so it is necessary to supplement K with other substances. Other sources of potassium include, for example, potassium chloride KCl. If you want to increase the sugar concentration, it is best to increase it to about 5% to 15%. More preferably, it is up to 8%. Examples of growth regulators to be added include indoleacetic acid, indolebutyric acid, naphthaleneacetic acid, 2,4-dichlorophenoxyacetic acid, ancymidol, chlorocholine salt, and B-nine. The concentration of these growth regulators added is
About 10-3 mg/L to 20 mg/L is good. Further, the enlargement medium can be used to efficiently enlarge adventitious buds or bulbs by repeatedly renewing the medium after culturing for an arbitrary period of time.

[0011] After the medium components of the bulbs that have undergone the above steps A-B are washed, they are transplanted into soil. The soil includes not only field soil, but also sand, vermiculite, peat moss, Kanuma soil,
Sphagnum moss and the like can also be used alone or in an appropriate mixture. A above
As long as the bulbs obtained through step -B are cultivated under conventional cultivation conditions, there is no need for any special acclimatization operation to acclimatize them to the external environment at the time of transplantation.

[0012] Examples of bulbous plants to which the present invention can be applied include the following and varieties grown using them. Monocotyledonous class Liliflorae Liliaceae Onion (Allium cepa) Lilium auratum Tiger lily (
Lilium lancifolium)
Lilium speciosum) Longhorn lily (L
ilium longiflorum)
ilium elegans) Tulip
pa gesneriana) Hemerocallis (Heme
rokariosu sp. ) Hyacinth (Hiaci)
nthus orientaris) Amaryllidaceae (A
marylldiaceae sp. ) Alstroemeria (Alstroemeria sp.) Amaryllis (H
ippeastrum sp. ) Daffodil (Naruc)
issus sp. ) Nerine (Nerine sp.) Iridaceae (Iridaceae) Crocus (Crocus sp.) Freesia (Freesia sp.) Gladiolus (
Gladiolus sp. ) Dutch Iris (Iri)
s germanica) Arales (Arales) Family (Araceae) Taro (Colocasia succulenta)
) Caladium (Caladium bicolor) Dicotyledonous class (Convolvulaceae) Sweet potato (Ipomoea batatas Poir.) Solanaceae (Slanaceae) Potato (Solanum tuberosum L)
．． ) Next, examples will be described.

[0013]

[Example 1] Sucrose 4%, naphthaleneacetic acid 0.01mg/L, benzyladenine 0.02mg were used as sterile oriental hybrid type lily bulb bulb sections.
/L of sterile 1/2 Murashigeskoog liquid medium at pH 5.8 (differentiation medium; the composition is shown in Table 1)
0.5 g of the section was placed in an incubator containing 50 ml (capacity: 150 ml). For the culture container, 0.22μ
While blowing air that has passed through a sterilization filter into the liquid medium at a rate of 3 ml/min, incubate at 25°C in the dark for 1.
After culturing for 5 months, small bulbs were differentiated. Afterwards, the phosphate concentration was lowered to 0.40 mM and the sugar concentration was increased to 5%.
(enlargement medium) and cultured for 3 months. During this period, the medium was renewed every month.

By the main culture, 60 to 80 bulbs were differentiated from 0.5 g of slices within 1.5 months of culture, and in subsequent culture, 30 to 50 bulbs were newly differentiated, and at the end of culture, 80 to 130 bulbs were obtained. The average bulb diameter of the obtained bulbs was 4.5 mm. In addition, the total amount of castor-like culture other than bulbs (the total amount of culture including bulbs,
(same below) was 20% or less.

[0015] When the obtained bulbs were planted in vermicilite and a germination test was conducted under light conditions at 23°C, 97% of the bulbs germinated within one month after planting.
Table 1 Composition of MS medium Calcium chloride dihydrate
440mg ammonium nitrate
1650mg
potassium nitrate
1900mg
Magnesium sulfate heptahydrate
370mg
Potassium phosphate
170mg
Na2 EDTA/dihydrate salt

42.1mg manganese sulfate
Tetrahydrate salt
22.3mg zinc sulfate
7 hydrate salt
1.0mg boric acid
6.2mg
Sodium molybdate dihydrate
0.25mg
copper sulfate
0
．． 025mg cobalt chloride hexahydrate
0.025mg inositol

100mg nicotinic acid
0.50mg
Pyridoxine hydrochloride
0.50mg
glycine

2.0mg biotin

0.5mg

[0016]

[Example 2] In Example 1, the phosphoric acid concentration of the enlargement medium was changed to 0.0.
The same procedure as in Example 1 was performed except that the concentration was set to mM. By main culture, from 0.5 g of section to 1.5 months of culture, 6
0 to 80 bulbs are differentiated, and in subsequent culture, about 10 bulbs are newly differentiated, and after culturing, 65 to 9 bulbs are differentiated.
0 bulbs were obtained. The average diameter of the bulbs obtained was 3.
It was 9mm. In addition, callus-like culture other than bulbs accounted for less than 10% of the total weight.

[0017] When the obtained bulbs were planted in vermicilite and a germination test was conducted under light conditions at 23°C, 92% of the bulbs germinated within one month after planting.

[0018]

[Example 3] The same procedure as in Example 1 was carried out except that sterile cultured bulbs of gun lily were used. By main culture,
From 0.5 g of section to 1.5 months of culture, 60 to 80
30 to 40 bulbs are differentiated in subsequent culture, and at the end of culture, 80 to 120 bulbs are differentiated.
bulbs were obtained. The average diameter of the bulbs obtained was 4.4.
It was mm. In addition, callus-like culture other than bulbs accounted for less than 25% of the total weight.

[0019] When the obtained bulbs were planted in vermicilite and a germination test was conducted under light conditions at 23°C, 98% of the bulbs germinated within one month after planting.

[0020]

[Example 4] In the differentiation medium of Example 1, the liquid medium was changed to a solid medium (agar 8 g/L), and the concentration of naphthalene acetic acid was changed to 0.1 mg/L without using benzyladenine. Example 1 except that 4%
I did the same thing. By main culture, 20 to 27 bulbs were differentiated from 0.5 g of slices within 1.5 months of culture, and in subsequent culture, 8 to 15 bulbs were newly differentiated, and at the end of culture, 24 to 40 bulbs were differentiated. bulbs were obtained. The average bulb diameter of the obtained bulbs was 6.4 mm. In addition, callus-like culture other than bulbs accounted for less than 25% of the total weight.

[0021] When the obtained bulbs were planted in vermicilite and a germination test was conducted under light conditions at 23°C, 88% of the bulbs germinated within one month after planting. Comparative Example 1 The same procedure as in Example 1 was conducted except that the phosphoric acid concentration in the enlargement medium was set to 0.5 mM.

[0022] Through main culture, 60 to 80 bulbs were differentiated from 0.5 g of a section within 1.5 months of culturing.
In the subsequent culture, 100 to 120 bulbs were newly differentiated, and after the culture was completed, the number of differentiated bulbs was 160.
The number has increased from 200 to 200. Therefore, the average bulb diameter of the bulbs obtained was 3.6 mm, which was smaller than that of Example 1. Also, the callus-like culture other than bulbs increased from 60 to 75% of the total weight.

[0023] When the obtained bulbs were planted in vermicilite and a germination test was conducted under light conditions at 23°C, 96% of the bulbs germinated within one month after planting. Comparative Example 2 The same procedure as in Example 1 was carried out except that the phosphoric acid concentration in the enlargement medium was changed to 0.90 mM.

[0024] By main culture, culture 1 from 0.5 g of section
．． By 5 months, 60 to 80 bulbs had differentiated, and during the culture, 70 to 80 bulbs were newly differentiated, and after the cultivation was completed, the number of differentiated bulbs increased to 130 to 160. The average bulb diameter of the obtained bulbs was 4.0 mm. In addition, callus-like cultures other than bulbs increased to 70 to 80% of the total weight. In addition, when the obtained bulbs were planted in vermicilite and a germination test was conducted under light conditions at 23° C., 92% of the bulbs germinated within one month after planting.

Comparative Example 3 The same procedure as Comparative Example 2 was carried out except that 0.8 ppm of abscisic acid was added to the enlargement medium. By main culture, 60 to 80 bulbs were differentiated from 0.5 g of a section within 1.5 months of culture, and in subsequent culture, 30 to 40 bulbs were newly differentiated, and after the completion of culture, 100 to 80 bulbs were differentiated.
1120 bulbs were obtained. The average bulb diameter of the obtained bulbs was 4.2 mm. In addition, callus-like culture other than bulbs accounted for 40 to 50% of the total weight.

[0026] When these bulbs were planted in vermicilite and a germination test was conducted under light conditions at 23°C, the germination rate was 39% within one month after planting, and it took more than two months for more than 90% to germinate. It took. Table 2 shows a list of the above examples and comparative examples.

[0027]

[0028]

Effects of the Invention According to the present invention, it is possible to suppress the wasting of nutrients due to the formation and enlargement of callus and the wasting of nutrients due to the differentiation of bulbs one after another, and the adventitious buds or small bulbs differentiated at one time can be suppressed. can be enlarged efficiently. Therefore, bulbs can be propagated using a smaller culture container and a smaller amount of medium. Furthermore, when the obtained bulbs were transplanted into soil, they were able to germinate quickly.

Claims (1)

[Claims] Claim 1. A method for propagating bulbs by plant tissue culture, in which tissue pieces or cultured cells of bulbous plants are cultured in a differentiation medium, adventitious buds or small bulbs are differentiated, and then the phosphate concentration is 0.40 mM or less. A method for propagating bulbs, which comprises culturing and enlarging the differentiated adventitious buds or small bulbs in a phosphate-free medium.