JPH01141524A - Tissue culture of asparagus - Google Patents

Abstract

PURPOSE:To multiply a large amount of seeds and stocks by using one or a combination of P995, CCC, AM01618 and uniconazole. CONSTITUTION:Seeds of asparagus are sterilely germinated to give shoots, which are cut into lateral bud-containing pieces having about 1-2cm length. A solid medium of Murashige and Skoog containing B995, sucrose, naphthaleneacetic acid and benzyladenine is prepared, the above-mentioned pieces of stem are set and cultivated in the medium.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a tissue culture method for asparagus, and particularly to a tissue culture method suitable for mass propagation of asparagus seedlings.

[Conventional technology]

Asparagus has traditionally been propagated by seeding or division. Asparagus is dioecious,
Male plants are said to be better in terms of quality and cultivation management. Furthermore, since it is a crop with large genetic variations, propagation by sowing leads to the contamination of female plants and causes differences in yield and quality among plants, which poses a significant problem in cultivation. In addition, propagation by division requires a lot of manpower and a long period of time, is extremely inefficient, and has a very high risk of transmitting viral diseases.

In order to improve the above-mentioned problems, mass propagation of superior strain clones by tissue culture has recently attracted attention. Normally, for mass propagation of asparagus, tissue pieces are cultured, a large number of shoots are propagated, and then each shoot is transplanted to a rooting medium, and adventitious roots are differentiated into ζ seedlings. This differentiation has been thought to be controlled by the concentration ratio of the plant hormones suitokinin and auxin (e.g.
J, Fac, Agrie, vol 61 (3L 29
5-306.1983). However, there are problems in that the rooting rate is generally low and the propagation efficiency is low.

Another problem is that many of the plants that germinate are not white vegetative roots, but transparent roots that wither and die at the 91st stage.

In view of the above, there is a need to develop a method for efficiently promoting the rooting of white roots.

[Problem that the invention seeks to solve]

The present inventors have recognized that the conventional asparagus tissue culture method has the above-mentioned problems, and have developed a method for efficiently propagating asparagus seedlings by culturing tissue using a novel method different from the conventional method. We considered this.

[Summary of the Invention] As a result, the present inventors discovered a substance that acts on asparagus shoots and promotes the differentiation of adventitious roots, and based on these findings, they developed a silk spray that can efficiently propagate asparagus seedlings. I found a way to cultivate it. That is, according to the present invention, in the asparagus tissue culture method, N-dimethylsuccinamic acid (N-climethylamine
o succinamic acid, hereafter B995
5, 2-(Chloroet, hy)trimethyl chloride
l) trimet, ylam-monium chl
oride, hereinafter referred to as CCC], 2'--isopropyl-4'-()limethylan 121 Kumukuro + JFi
5'-methylphenylpiperidine-I~carboxylene・
-t(2'-1sopropyl -4'-(tri
methylammonium chloride) -
5'-methylphenylpiperidine
1-carboxylate, hereinafter referred to as AM0161B] and 1-(4-chlorophenyl)-4,4-dimethyl-2-(LH-1,2,4-triazole-1-
yl)-1-penten-3-ol (1(4-ch
l.

rophenyl) -4+ 4-dirnethyl
-2(I H-1+ 2+4-triazol-1-
y+) 1-penten-3-ol, hereinafter referred to as uniconazole); A culture method is provided.

[Detailed description of the invention]

The method of the present invention can be carried out using asparagus tissue. Specific examples of the tissue culture piece include embryos, shoot tips, stems, aggregates, rhizomes, and other tissues. Furthermore, these tissue pieces are usually used after being sterilized with sodium hypozincate or ethyl alcohol.

However, when using aseptically cultivated plants, the above sterilization operation is unnecessary. Furthermore, in the case of propagating disease-free and virus-free asparagus, the tissue near the growing point, the above-mentioned tissue pieces of plants obtained from the tissue near the growing point, etc. can be used as culture materials.

In order to form asparagus seedlings by tissue culturing asparagus tissue pieces using the tissue culture method of the present invention, the method described in detail below is employed.

In the tissue culture method of the present invention, B2O2 is added to the culture medium.
Tissue culture is performed after adding at least one compound selected from the group consisting of , CCCSAM 0161B, and uniconazole. The tissue culture method will be explained below.

B2O2, CCClA added to the medium in the present invention
M 01618, and uniconazole are 10-8 to 1
It is preferable to add it at a concentration of 0-''M, preferably in the range of 10-'' to 10-''M, since the adventitious root differentiation is good.

The medium used in the present invention is a medium containing inorganic components and a carbon source as essential components, to which are added plant hormones and vitamins, and further added with amino acids as necessary.

Inorganic components of the medium include inorganic salts containing elements such as nitrogen, phosphorus, potassium, sodium, calcium, magnesium, sulfur, iron, manganese, zinc, boron, molybdenum, chlorine, iodine, cobalt, etc. Specifically, potassium nitrate, sodium nitrate, ammonium nitrate, potassium chloride, calcium chloride, potassium monohydrogen phosphate, sodium dihydrogen phosphate, magnesium sulfate,
Magnesium chloride, sodium sulfate, ferrous sulfate, ferric sulfate, manganese sulfate, copper sulfate, sodium molybdate, molybdenum dioxide, potassium iodide, zinc sulfate,
Examples include compounds such as boric acid and cobalt chloride.

Examples of carbon sources for the medium include carbohydrates such as sig sugar and their derivatives, organic acids such as fatty acids, and primary alcohols such as ethanol.

Examples of plant hormones in the medium include naphthaleneacetic acid (NAA), indoleacetic acid (IA8), l)-chlorophenoxyacetic acid, 2,4-dichlorophenoxyacetic acid (2,4-D), and indolebutyric acid (IBM). Examples include auxins such as , and derivatives thereof, and cytokinins such as benzylazo-one (HA), kinetin, and zeatin.

The vitamins in the medium include biotin, thiamine (vitamin B), pyridoxine (vitamin B), pyridoxal, pyridoxamine, calcium pantothenate, ascorbic acid (vitamin CL inositol, nicotinic acid, nicotinamide, and riboflavin (vitamin B).
6) etc. can be exemplified.

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 0.0% of the inorganic component.
IBM to about 100 mM, about 1 g of the carbon source
/l to about 10 h/l, and the plant hormones at about 0.0 h/l.
It is preferable that the amino acids be used in an amount of 0.01 mg/l to about 150 mg/l and 0 to about 1000 mg/l.

Specifically, the medium used in the tissue culture method of the present invention includes a conventionally known culture medium used for plant tissue culture, such as Murashige Skoog ('
62) [Murashige & Skoog] medium, Linsmeyer-Skoog (RM-1965) [
Linsmaier &Skoog's medium, White's medium, Gamborg's 8-5 medium, Mitsui's i, 9 medium, Niche Niche's medium [N1tch & N1t
An example of a culture medium prepared by adding the above-mentioned carbon sources and plant hormones to eh ] etc., and further adding the above-mentioned vitamins and amino acids as necessary. Preferably, the medium is prepared using a Meyer-Skoog or Murashige-Skoog medium. In the present invention, these two mediums are used in the patent application
A medium to which calcium ionophore, cylic AMP, and polyamine, as proposed in No. 308539, are added may also be used. Regarding the composition of the above-mentioned conventionally known culture medium, for example, see "New Plant Tissue Culture Journal" by Tenshi, Nakajima, and Furuya.
Described in p386-p391, Asakura Shoten, 1979.

The medium that can be used in the present invention is a liquid medium or a solid medium containing usually 0.1 to 2% of a gelling agent such as agar or Gelrite.

In the present invention, the above-mentioned asparagus tissue pieces can also be tissue cultured using a liquid medium aerated with an oxygen-containing gas, as in Japanese Patent Application No. 128348/1983 filed by the present applicant.

According to the present invention, adventitious roots (white roots) can be efficiently obtained from asparagus tissue pieces. In addition, the plants harvested according to the present invention can be grown into healthy plants with constant properties by normal cultivation.

[Effect of the invention] B2S3, CCC, 1M 01618 according to the present invention
If one or more of uniconazole and uniconazole are added to the culture medium to tissue culture asparagus, the differentiation of adventitious roots (white roots) of asparagus shoots will be significantly promoted. Seedlings can be propagated in large quantities through

Therefore, according to the tissue culture method of the present invention, high-quality plants can be cultured in large quantities from asparagus tissues more efficiently than in conventional methods, and seeds and seedlings can be propagated in large quantities.

〔Example〕

Example 1 Asparagus seeds were germinated aseptically, the resulting shoots were cut into sections with a length of about 1 to 2C11 including side buds, and B2S
3 to 10-'M, 3% sucrose, naphthaleneacetic acid 3X10
-'M and benzyladenine 10-'M,
Prepare a sterile Murashigeskoog solid medium (Gelrite concentration: 0.2%) at H5 and 6, place 200 asparagus stem sections on it, and culture at 25°C, 116-hour photoperiod, and 50,001 ux for 13 weeks. As a result, the results shown in Table 1 as the rate of white root formation were obtained. In the medium supplemented with B995, the white silver formation rate increased compared to Comparative Example 1.

Examples 2 to 3 In Example 1, CCC was 10-”M, 10-′M
Asparagus stem slices were cultured in the same manner as in Example 1 except that the concentration of 20% was added, and the results shown in Table 1 were obtained.

At this time, the formation rate of white silver increased compared to Comparative Example 1 in both cases.

Example 4 Asparagus stem sections were cultured in the same manner as in Example 1 except that AM 0161B was added at a concentration of 10-'M, and the results shown in Table 1 were obtained. At this time, the formation rate of white silver increased compared to Comparative Example 1 in both cases.

Example 5 Asparagus stem sections were cultured in the same manner as in Example 1 except that uniconazole was added at a concentration of 10 −6 M, and the results shown in Table 1 were obtained. At this time, the formation rate of white silver increased compared to Comparative Example 1 in both cases.

Comparative Example 1 Asparagus stem sections were cultured in the same manner as in Example 1 except that B995 was not added.

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Claims (1)

[Claims] 1. In the asparagus tissue culture method, N-dimethylaminosuccinamic acid, 2-(chloroethyl chloride)
Trimethylammonium, 2'-isopropyl-4'-
(trimethylammonium chloride)-5'-methylphenylpiperidine-1-carboxylate and 1-(4
-chlorophenyl)-4,4-dimethyl-2-(1H-
Asparagus is tissue cultured using a culture medium to which at least one compound selected from the group consisting of 1,2,4-triazol-1-yl)-1-penten-3-ol is added. Asparagus tissue culture method. 2. The asparagus tissue culture method according to claim 1, characterized in that the compound is added at a concentration in the range of 10^-^7 to 10^-^5M.