JP2901021B2 - How to grow bulbs by plant tissue culture - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for mass-producing bulbs by plant tissue culture.

[0002]

2. Description of the Related Art Bulb plants have heretofore been obtained by utilizing spherules, phosphorus scissors, mukago and wood. However, these propagation methods not only require a large amount of land and a lot of manpower, but also in recent years, the spread of viral diseases has led to a decrease in the growth rate of seeds and 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, a study has been reported in which a growth point is cultured and virus-free bulbs are propagated by plant tissue culture. For example, Japanese Patent Application Laid-Open No. 55-15734 discloses that after forming and growing a sphere from a tissue piece of a lily plant using a solid medium,
There has been disclosed a method for mass-producing lily plants, which comprises transferring the mixture to a liquid medium, culturing the mixture, and expanding the mixture. However, in the method described above, since a solid medium using agar is used in the previous stage of the hypertrophic culture, not only a lot of manpower is required for the culturing operation, but also three-dimensional use of the incubator is difficult, and the efficiency of the culturing is poor. There's a problem.

[0003] Against this background, the present applicant has filed a Japanese Patent Application No.
-128348 proposed a method of growing lily seedlings. By using the above method, seeds and seedlings can be efficiently proliferated in large quantities.

[0004]

Many of the conventional methods for growing bulbs by the tissue culture method require improvement of the culture method to achieve growth efficiency and stabilization of the technology in order to be used as a practical technique. is necessary. For example, in the growth of a bulb plant by tissue culture, a phenomenon in which a part of the culture has turned into callus and the growth of the bulb is suppressed has been observed, which is an important problem to be avoided. There is also a problem that the adventitious buds or small bulbs continue to differentiate from one another to prevent the hypertrophy of previously differentiated adventitious buds or small bulbs.

[0005] As a method of inducing bulb enlargement, a technique of promoting bulb enlargement by transplanting adventitious shoots differentiated on an agar medium to an agar medium containing abscisic acid as a technique for enlarging and growing tulip bulbs (Y. , Nishiuchi
Author, Studies on VegitativePropagation of Turtips.
V. Effect of Growth Regurators on The Bulb Formatio
n of Adventitious Bud Cultured in vitro., Journal
of Hokkaido University of Education Vol.34, No.1: 9
-15, 1983). JP-A-61-56022 describes that abscisic acid added to a medium suppresses callus formation and promotes bulb enlargement.

However, bulbs grown and enlarged by a conventional method have problems that germination after soil transplantation is suppressed or significantly delayed, and that germination timing is not uniform but varies.

[0007]

Means for Solving the Problems In order to solve the above problems, the present inventors have conducted detailed studies on a method of growing bulbs by tissue culture, and as a result, have found that the phosphate concentration in the hypertrophic medium was reduced. It has been found that the above problems can be solved by culturing the cells at a concentration of 0.40 mM or less to zero, and the present invention has been completed based on this new finding.

Accordingly, the method for growing bulbs of the present invention is a method for growing bulbs by plant tissue culture, wherein a tissue fragment or a cultured cell of a bulb plant is cultured in a differentiation medium,
Differentiate adventitious shoots or bulbs, and then the phosphate concentration
The method is characterized in that the differentiated adventitious buds or the microbulbs are cultured in a medium containing no more than 0.40 mM or no phosphoric acid to enlarge them.

The details of the present invention will be described below. The present invention is a method for growing bulbs by tissue culture comprising the following two AB steps. In the present invention, tissue culture of a bulbous plant can be performed using a tissue piece or a cultured cell of the plant.
Specifically, the shoot tip, stem tip, stem, leaf, flower, seed,
Tissue pieces of a bulb plant obtained by cutting small bulbs, roots or other tissues into small pieces can be exemplified, and these tissue pieces are usually used after being sterilized by sodium hypochlorite, ethyl alcohol or flame. However, when using aseptically cultured or cultivated bulb plants, the above sterilization operation is not necessary. When a virus-free bulbous plant is propagated, a tissue near the growth point, the above-described tissue fragment of the bulbous plant obtained from the tissue near the growth point, or the like can be used as a culture material. (A) Formation of adventitious buds and small bulbs In the present invention, the adventitious buds and small bulbs are differentiated by culturing bulb sections or other tissue pieces of a bulbous plant using a differentiation medium. A differentiation medium is a medium known in the art so that adventitious buds and small bulbs are easily differentiated from bulb sections or other tissue pieces, and modified media obtained by improving the components of these media, and in many cases, such as auxin and cytokinin. This is a medium to which a plant hormone has been added. And, for example, a Murashige-Skoog medium, a white medium, a niche and niche medium, a medium used for normal tissue culture such as a Rinsmeyer-Skoog medium, or a modified medium obtained by modifying these mediums as a basic medium is used. be able to.
Further, in the tissue culture method of the present invention, benzyladenine, kinetin, zeatin, isopenteladenine, indoleacetic acid, indolebutyric acid, in order to promote the differentiation of tissue pieces or cultured cells into buds and roots during culture.
It is preferable to add a plant growth regulator such as naphthalene acetic acid or 2,4-dichlorophenoxy acetic acid to the above-mentioned medium and perform culturing. The amount of these plant growth regulators to be added depends on the type of plant growth regulator, the type of bulbous plant, and the like, but is generally preferably 10 ^-4 mg / L to 50 mg / L. However, if the amount of these plant regulators is too large, morphological abnormalities and genetic defects may occur when bulbs obtained by tissue culture of the bulbous plant of the present invention are further cut and cultured to differentiate adventitious buds or small bulbs. More preferably,
A good range is from 10 ^-3 mg / L to 1 mg / L. In order to culture a bulbous plant, it is better to add a carbon source such as sucrose, glucose, fructose, and maltose to the medium. The amount of carbon source added is generally 5g / L
To 150 g / L, preferably 10 g / L to 50 g / L. The pH of the medium is preferably from 4.0 to 8.0. Although light is not necessarily required during culture, good results may be obtained by culturing under illumination of a light amount of 100 to 10,000 lux. The culture temperature is preferably from 15 ° C to 35 ° C.

The present invention can be carried out using a solid medium or a liquid medium. (B) Cultivation of adventitious buds and small bulbs using a hypertrophic medium After adventitious buds or bulbs are formed in the above (A), the medium is aseptically extracted, replaced with a hypertrophy medium, and cultured to obtain adventitious buds or small bulbs. Small bulbs can be efficiently enlarged. The hypertrophic medium is a medium that has been modified to increase the sugar concentration so that adventitious buds and small bulbs grow easily and to promote the hypertrophy of adventitious buds or bulbs,
The concentration of phosphoric acid in the medium component is 0.40 mM or less, more preferably 0.20 mM or less. If necessary, a growth regulator may be present in the medium. In the present invention, good results may be obtained even if phosphoric acid is completely removed. When the concentration of phosphoric acid in the medium is reduced, other inorganic components may also be reduced. For example, when using a KH ₂ PO ₄ as a phosphate source, KH ₂ PO
_{If 4} is reduced, potassium (K) also decreases, so it is necessary to supplement K with other substances. Another potassium source is, for example, potassium chloride KCl. 5 to increase sugar concentration
It is good to increase from about 15% to 15%. More preferably 8%
Up to. The growth regulators to be added include indoleacetic acid, indolebutyric acid, naphthaleneacetic acid, 2,4
-Dichlorophenoxyacetic acid, ansimidol, chlorocholine salts, B-nine and the like. The concentration of these growth regulators to be added is preferably about 10 ⁻³ mg / L to about 20 mg / L.
The adventitious buds or bulbs can be efficiently enlarged by repeatedly renewing the medium after culturing for an arbitrary period.

The medium component of the bulb that has undergone the above steps AB is washed and then transplanted to soil. The soil of the field as well as the soil, sand, vermiculite, peat moss, Kanuma soil,
Water moss or the like can be used alone or in an appropriate mixture. A above
As long as the bulbs obtained through the step -B are cultivated under customary cultivation conditions, no special acclimatization operation for acclimating to the external environment at the time of transplantation is required.

Examples of bulb plants to which the present invention can be applied include the following and varieties grown using them as materials. Monocotyledonous plant Lily (Liliflorae) Lily (Liliacease) Onion (Allium cepa) Yamali (Lilium auratum) Oni lily (Lilium lancifolium) Lilium speciosum (Lilium speciosum) Lilium longiflorum Tulipa gulpe (Lilium longiflorum) Lilium longiflorum (Hemerokariosu sp.) Hyacinthus (Hiacinthus orientaris) Amaryllidaceae (Amarylldiaceae sp.) Alstroemeria (Alstromeria sp.) Amaryllis (Hippeastrum sp.) Narcissus (Narucissus sp.) Nerine sp. (Nerine sp.) Freesia (Freesia sp.) Gladiolus (Gladiolus sp.) Dutch iris (Iris germanica) Araceae (Arales) Araceae (Araceae) Taro (Colocasia esuculenta) Caladium (Caladium bicolor) Dicots Convolvulaceae Sweet potato (Ipomoea batatas Poir.) Solanaceae (Slanace ae) Potato (Solanum tuberosum L.) Next, examples will be described.

[0013]

Example 1 Using sterile oriental hybrid lily cultivar bulb sliced pieces, pH containing sucrose 4%, naphthalene acetic acid 0.01 mg / L and benzyladenine 0.02 mg / L
Incubator containing 50 ml of 5.8 sterile 1/2 Murashige Skoog liquid medium (differentiation medium; composition is shown in Table 1) (capacity: 150
0.5 g of a section was prepared. 0.22μm for the culture vessel
The cells were cultured in a dark place at 25 ° C. for 1.5 months while blowing air having passed through the sterilization filter at a rate of 3 ml / min to differentiate the bulbs. Thereafter, the medium was adjusted to a phosphate concentration of 0.40.
for 3 months culture was replaced with culture medium (hypertrophy medium) enhanced the lower Gaité sugar concentration in 5% mM. During this time, the medium was updated every other month.

[0014] By the main culture, the slices can be collected by 1.5 months after the culture.
From 5 g, 60 to 80 bulbs differentiated, and in subsequent culture, 30 to 50 bulbs newly differentiated, and at the end of culture,
80 to 130 bulbs were obtained. Average bulb diameter of obtained bulb
Was 4.5 mm . In addition, the amount of the castle-like culture other than the bulb was 20% or less of the total amount (the total amount of the culture including the bulb, the same applies hereinafter).

When the obtained bulb was planted in vermicilite and subjected to a germination test at 23 ° C. under light conditions, 97% germinated by one month after planting. Table 1 Composition of MS medium Calcium chloride dihydrate 440 mg ammonium nitrate 1650 mg potassium nitrate 1900 mg magnesium sulfate heptahydrate 370 mg potassium monophosphate 170 mg Na ₂ EDTA dihydrate 42.1 mg manganese sulfate tetrahydrate 22.3 mg zinc sulfate Heptahydrate 1.0 mg boric acid 6.2 mg sodium molybdate dihydrate 0.25 mg copper sulfate 0.025 mg cobalt chloride hexahydrate 0.025 mg inositol 100 mg nicotinic acid 0.50 mg pyridoxine hydrochloride 0.50 mg glycine 2.0 mg biotin 0.5 mg

[0016]

Example 2 In Example 1, the concentration of phosphoric acid in the hypertrophic medium was adjusted to 0.0 mM.
The procedure was performed in the same manner as in Example 1 except that By the main culture, 60 to 80 bulbs are differentiated from 0.5 g of the section by 1.5 months of culture, and about 10 bulbs are newly differentiated in the subsequent culture. After the culture, 65 to 90 bulbs are differentiated. Bulbs were obtained. The average bulb diameter of the obtained bulb was 3.9 mm . Also,
Callus cultures other than bulbs accounted for less than 10% of the total weight.

When the obtained bulbs were planted in vermicilite and subjected to a germination test at 23 ° C. under light conditions, 92% germinated by one month after planting.

[0018]

Example 3 The procedure of Example 1 was repeated, except that a sterile culture bulb of a gun lily was used. By main culture,
From 0.5 g of section, 60 to 80 bulbs differentiated by 1.5 months of culture, and 30 to 40 bulbs newly differentiated in the subsequent culture. At the end of culture, 80 to 120 bulbs were differentiated. Obtained. The average bulb diameter of the obtained bulb was 4.4 mm . Also,
Callus cultures other than bulbs accounted for less than 25% of the total weight.

When the resulting bulb was planted in vermicilite and subjected to a germination test at 23 ° C. under light conditions, 98% germinated by 1 month after planting.

[0020]

Example 4 A liquid medium was replaced with a solid medium in the differentiation medium of Example 1.
(Agar 8 g / L), the concentration of naphthalene acetic acid was changed to 0.1 mg / L without using benzyladenine, and the sugar concentration was increased to 4% in a hypertrophic medium. By main culture, from 0.5 g of section to 1.5 months of culture
From 20 to 27 bulbs differentiate, and in subsequent cultures, from 8 to 15 bulbs
At the end of the culture, 24 to 40 bulbs were obtained. The average bulb diameter of the obtained bulb was 6.4 mm . In addition, callus-like cultures other than bulbs
% Or less.

When the obtained bulb was planted in vermicilite and subjected to a germination test at 23 ° C. under light conditions, 88% germinated by 1 month after planting. Comparative Example 1 The procedure of Example 1 was repeated , except that the concentration of phosphoric acid in the hypertrophic medium was 0.5 mM .

By the main culture, bulbs were cultured from 0.5 g of the section.
By 1.5 months, 60 to 80 bulbs have differentiated, and in the subsequent culture, 100 to 120 bulbs have newly differentiated, and after the culture, the number of differentiated bulbs has increased from 160 to 200 .
Therefore, the average bulb diameter of the obtained bulb was 3.6 mm, which was smaller than that of Example 1. Callus cultures other than bulbs also increased from 60 to 75% of the total weight.

When the obtained bulb was planted in vermicilite and subjected to a germination test at 23 ° C. under light conditions, 96% germinated by one month after planting. Comparative Example 2 The procedure of Example 1 was repeated , except that the phosphate concentration of the hypertrophic medium was changed to 0.90 mM .

By the main culture, 60 to 80 bulbs are differentiated from 0.5 g of the section by 1.5 months of culture. In the culture, 70 to 80 bulbs are newly differentiated. The number of differentiated bulbs increased from 130 to 160. The average bulb diameter of the obtained bulb was 4.0 mm. In addition, callus-like cultures other than bulbs increased from 70 to 80% of the total weight. When the obtained bulb was planted in vermiculite and subjected to a germination test at 23 ° C. under light conditions, 92% germinated by one month after planting.

Comparative Example 3 The procedure of Comparative Example 2 was repeated, except that 0.8 ppm of abscisic acid was added to the hypertrophic medium. By this culture, 60 to 80 bulbs were differentiated from 0.5 g of the section by 1.5 months of culture, and 30 to 40 bulbs were newly differentiated in the subsequent culture.
After completion of the culture, 100 to 1120 bulbs were obtained. The average bulb diameter of the obtained bulb was 4.2 mm. Callus-like cultures other than bulbs accounted for 40 to 50% of the total weight.

When these bulbs were planted in vermicilite and subjected to a germination test under a light condition of 23 ° C., 1 bulb was obtained after planting.
The germination rate by 39 months was 39%, and it took more than 2 months for more than 90% to germinate. Table 2 shows a list of the above examples and comparative examples.

[0027]

[0028]

According to the present invention, it is possible to suppress the waste of nutrients due to the formation and enlargement of callus and the waste of nutrients due to the differentiation of bulbs from one to the next, and the adventitious buds or small bulbs differentiated at one time Can be efficiently enlarged. Therefore, bulbs can be grown in a small amount of medium using a smaller culture vessel. When the obtained bulb was transplanted to soil, it was able to germinate quickly.

Claims (1)

(57) [Claims] In a method for growing bulbs by plant tissue culture, a tissue fragment or a cultured cell of a bulbous plant is cultured in a differentiation medium to differentiate adventitious buds or small bulbs, and then a phosphate concentration of 0.40 mM or less to phosphate A method for growing bulbs, comprising culturing the differentiated adventitious buds or the small bulbs in a culture medium containing no and expanding the bulbs.