JPH0650965B2 - Method for growing seedlings of lily plants - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for growing a large amount of lily seedlings by tissue-culturing a lily plant by a specific method.

[Conventional technology]

There are many varieties of lily plants, such as gun lily, velvet lily, and lily squirrel are favored for appreciation as horticultural plants, and lily lilies and porcupines are also used as edible lilies. Conventionally, the growth of bulbous plants such as lilies has been carried out by dividing bulbs, shingles, using seedlings and sowing. However, these breeding methods not only require a lot of land and manpower,
In recent years, due to the spread of viral diseases, the growth rate of lily seedlings and the quality of flowers have become a problem. In recent years, a method utilizing a plant tissue culture technique has been reported for the purpose of improving these problems and improving the proliferation efficiency (for example, JP-A-55-15734). Proliferation by tissue culture technology is achieved through differentiation of cultured tissue pieces, adventitious buds from cultured cells, adventitious embryos, bulbs, etc., and these differentiations are controlled by the concentration ratio of cytokinin and auxin which are plant hormones. Have been thought to exist (eg Annals of Botany, vol
45.321-327, 1980). However, there are many plant species in which differentiation does not occur with only plant hormones and the frequency of occurrence of which is extremely low even if differentiation occurs, and it is expected to establish a more direct and effective method of inducing differentiation.

[Problems to be Solved by the Invention] The present inventors have recognized that the conventional tissue culture method for lily plants has the above-mentioned various problems, and therefore a novel method different from the conventional method has been proposed. Therefore, a method for tissue-culturing a Lily plant to efficiently grow seeds and seedlings of the plant was examined.

[Outline of Invention]

As a result, they have found the following method and completed the present invention. That is, according to the method of the present invention, there is provided a method for growing seedlings of a lily plant, which comprises anaerobically treating tissue pieces or cultured cells of a lily plant and then performing tissue culture.

[Specific Description of the Invention]

As the lily genus plant used in the tissue culture of the present invention, conventionally known plants belonging to the genus can be used in the method of the present invention. Specifically as the plant,
Illustrative examples include a gun lily, a squirrel lily, a lily of the valley, a lily of the valley, a lily of the valley, a lily of the bamboo grass and a lily of the new gun.

In the present invention, tissue culture of a Lily plant can be carried out using tissue pieces or cultured cells of the plant. Specific examples of the tissue piece include a tissue piece of a lily plant obtained by cutting shoots, stems, leaves, flowers, seeds, follicle (lumps of phosphorus pieces), pieces of phosphorus, roots or other tissues into small pieces. These tissue pieces are usually used after sterilization with sodium hypochlorite, ethyl alcohol or flame. However, when using a plant of the genus Lily that is cultivated aseptically, the above sterilization operation is not necessary. Further, in the case of growing seedlings of a disease-free and virus-free lily plant, a tissue near the growth point, the above-mentioned tissue piece of the lily plant obtained from the tissue near the growth point, or the like can be used as a culture material. Cultured cells that can be used in the tissue culture of the lily plant of the present invention,
It is an undifferentiated atypical cell including callus tissue obtained by culturing the above-mentioned tissue piece by a known method.

In the present invention, the following method is used for tissue-culturing a tissue piece or a cultured cell of a lily plant to form seedlings of a lily plant.

That is, in the present invention, a method of anaerobically treating a tissue piece or a cultured cell of a lily plant that has been collected for use in tissue culture and then performing tissue culture is used. In the present invention, it is particularly preferable to anaerobically treat the tissue piece. According to this method, the differentiation of tissue or cultured cells of the lily plant into bulbs is significantly promoted. The method is a novel finding related to the present inventors.

The anaerobic treatment according to the present invention can be performed as follows. After collecting a tissue piece of a lily plant to be subjected to tissue culture, this sample is placed in a gas atmosphere that does not contain oxygen such as titanium, argon, and CO ₂ or may contain oxygen in an amount of usually 5% or less. Anaerobic treatment is performed by placing the sample and contacting the gas with the gas at a temperature of 15 to 30 degrees for 30 to 90 minutes. In the present invention, the anaerobic treatment is particularly preferably performed immediately after the collection of the tissue piece, and when tissue culture is performed using such a tissue piece, when the anaerobic treatment is used after a while after the collection. Compared with, the seedlings of the lily plant can be efficiently propagated, which is preferable.

As the medium used in the tissue culture performed after the anaerobic treatment, the medium described in detail below is used.

The medium that can be used in the present invention is a medium in which an inorganic component and a carbon source are essential components, plant hormones and vitamins are added thereto, and amino acids are further added as necessary. The inorganic components of the medium include nitrogen, phosphorus, potassium, sodium, calcium, magnesium, sulfur, iron, manganese, zinc, boron, molybdenum, chlorine,
Examples thereof include inorganic salts containing elements such as iodine and cobalt. Specifically, potassium nitrate, sodium nitrate, ammonium nitrate, ammonium chloride, potassium chloride, calcium chloride, potassium monohydrogen phosphate, sodium dihydrogen phosphate, magnesium sulfate. Examples thereof include magnesium chloride, sodium sulfate, ferrous sulfate, ferric sulfate, manganese sulfate, copper sulfate, sodium molybdate, molybdenum trioxide, potassium iodide, zinc sulfate, boric acid, and cobalt chloride.

Examples of the carbon source of the medium include carbohydrates such as sucrose and derivatives thereof, organic acids such as fatty acids, and primary alcohols such as ethanol.

Examples of plant hormones in the medium include naphthalene acetic acid (NAA), indole acetic acid (IAA), p-chlorophenoxyacetic acid, and 2,4-dichlorophenoxyacetic acid (2,4-
D), auxins such as indolebutyric acid (IBA) and their derivatives, and cytokinins such as benzyladenine (BA), kinetin and zeatin can be exemplified.

Examples of vitamins in the medium include biotin, thiamine (vitamin B ₁ ), pyridoxine (vitamin B ₆ ), pyridoxal, pyridoxamine, calcium pantothenate, ascorbic acid (vitamin C), inositol, nicotinic acid, nicotinic acid amide and riboflavin ( Vitamin B
₂ ) etc. can be illustrated.

Examples of amino acids in the medium include glycine, alanine, glutamic acid, cysteine, phenylalanine and lysine.

The medium of the present invention usually contains about 0.1 μm of the inorganic component.
M to about 100 mM, the carbon source is about 1 g / to about 100
g /, about 0.1 mg / about 100 m of the plant hormones
g /, about 0.1 mg / to about 150 mg / of the above vitamins
And, it is preferable that the amino acids are used in an amount of 0 to about 1000 mg / ml.

Specific examples of the medium used for the tissue culture according to the present invention include media conventionally used for tissue culture of plants, for example, Murashige Skoog ('62).
[Murashige & Skoog] medium, Rinsmeier & Skoog (RM-1965) [Linsmaier & Skoog] medium, White ('63) [White] medium, Gambor (Gambor
g) B-5 medium, Mitsui M-9 medium, Nichi & Nitch medium [Nitch & Nitch], etc., to which the above-mentioned carbon source and plant hormones have been added, and, if necessary, the above-mentioned vitamins and amino acids. The medium prepared by adding the above can be exemplified, but in the present invention, a medium prepared by using a Nitchi-Nitchi, Rinsmeier-Skoog or Murashige-Skoog medium is particularly preferable in the present invention. Regarding the composition of the conventionally known medium described above, for example, Takeuchi, Nakajima, and Furuya “New Plant Tissue Culture” P386 to P391, Asakura Shoten, 1979.
Listed in the year.

The medium that can be used in the present invention is usually a liquid medium or agar.
It is a solid medium containing 0.5 to 1%.

In the present invention, the anaerobic treatment according to the above-mentioned method of the present invention is optionally carried out using a medium containing at least one compound selected from the group consisting of calcium ionophore, cyclic AMP and polyamine in the above-mentioned medium. It is also possible to carry out tissue culture of lily plants. The concentration of calcium ionophore added to the medium in this case is usually 10 ^-8 to 10 ^-4 M /, preferably 10 ^-7 to 10 ^-5.
It is in the range of M /, and in calcium ionophore
It is preferable to use A23187. Here, A23187 is 6S-
[6α (2S ^* , 3S ^* ), 8β (R ^* ), 9β, 11α] -5- (me
thylamino) -2- [〔3,9,11-trimethyl-8- 〔1-methyl-2-o
xo-2- (1H-pyrrol-2-yl) ethyl] -1,7-dioxaspiro 〔5,5〕
-un--dec-2-yl] methyl] -4-benzoxazolecarboxylicaci
d. Similarly, for cyclic AMP, usually 1
It is in the range of 0 ^-9 to 10 ^-5 M /, preferably 10 ^-8 to 10 ^-6 M /. For polyamines usually 10 ^-6 to 10 ^-3 M /
, Preferably in the range of 10 ⁻⁵ to 10 ⁻⁴ M /. Here, the polyamine added to the medium in the present invention is a compound having a structural unit having an amino group and / or an imino group at both ends of a polymethylene group [-(CH ₂ ) _n- , n is an integer] Spermine 〔Bis (amino--propyl) -tetramethylen
ediamine ； H ₂ N (CH ₂ ) ₃ NH (CH ₂ ) ₄ NH (CH ₂ ) ₃ NH ₂ ], spermidine [H ₂ N
_{(CH 2) 3 NH (CH} 2) 4 NH 2 ] and putrescine _{[H 2 N (CH 2) 4} N
H ₂ ] and other tetramethylenediamines can be exemplified.

In the present invention, the above-mentioned tissue pieces or cultured cells of the plant of the genus Lily can also be tissue-cultured by using a liquid medium aerated with an oxygen-containing gas as in Japanese Patent Application No. 60-128348 relating to the present applicant.

According to the method of the present invention, it is possible to efficiently obtain a large amount of scaly bulb-shaped follicles (including small bulbs) from tissue pieces or cultured cells of a lily plant. To further refer to this point, the leucocyte agglomerate mass of the progeny obtained by the method of the present invention is separated into a large number of aliquots, which are further added by the above-mentioned culture method according to the present invention. A large number of lily seedlings can be proliferated with a few balllets. In addition, the bulb of a lily plant obtained in the present invention can grow into a healthy plant body having a constant property and a beautiful flower can be produced by usual cultivation.

〔The invention's effect〕

By using the tissue culture method of the lily plant of the present invention, it is possible to efficiently cultivate a large amount of high quality follicle from the tissue or cultured cells of the lily plant as compared with the conventional method, and to multiply the lily seedlings in large quantities. be able to.

〔Example〕

Hereinafter, the configuration and effects of the present invention will be specifically described with reference to examples.

Examples 1 to 4 Using a material such as a gun lily piece, a gun lily leaf piece, a bamboo lily piece, and a new gun lily piece as materials,
% Sterilized with ethanol and sodium hypochlorite aqueous solution (effective chlorine amount 1%) and cut into a width of about 2 mm, sucrose 4%, naphthalene acetic acid 0.01 mg /, benzyladenine 0.
Aseptic Murashige Skoog (196 with pH 6.0 with 02 mg /
2 years) agar medium (agar concentration 0.8%) was prepared, and 10 1g sections of the above material were left to stand for 1 hour at room temperature in a sterile titanium gas atmosphere immediately after the start of culture (anaerobic processing)
After culturing at 25 ° C. in the light for 3 weeks, the results shown in Table 1 were obtained as the number of bulbs formed per 10 sections.

Comparative Examples 1 to 4 Table 1 shows the results obtained in the same manner as in Example 1 except that the anaerobic treatment was not performed in Examples 1 to 4.

Claims (1)

[Claims] 1. A method for growing seedlings of a lily plant, which comprises anaerobically treating tissue pieces or cultured cells of a lily plant and then performing tissue culture.