JPH02257815A - Method for forming and proliferating crown of plant of genus asparagus and production of seedling - Google Patents

Abstract

PURPOSE:To enable the production of the seedling of the subject plant in high efficiency by culturing a tissue or a callus of the plant in a medium containing >=3% of sucrose, carrying out the tissue culture of the formed crown to grow a plant body and acclimatizing the plant body. CONSTITUTION:Tissue (e.g. embryo or shoot apex) of a plant of genus Asparagus or a callus produced by the culture of the tissue is subjected to tissue culture in a medium produced by adding a carbon source (e.g. glucose), plant hormone (e.g. napthalene-acetic acid) and vitamins (e.g. biotin), especially >=3% of sucrose to a medium such as Nitsch-Nitsch medium. Linsmeier-Skoog medium or Murashige-Skoog medium to form a crown. If necessary, the crown is subjected to thickening growth and a proper amount of the grown product is cultured and rooted in a medium containing an anti-gibberellin agent such as ancymidole or uniconazole to effect the growth of the terminal bud to a plant body of a proper size. The grown plant body is acclimatized to obtain a seedling. Plant body and seedling can be produced in large quantities and high efficiency by this process.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for crown formation and propagation of plants of the genus Asparagus, and a method for propagating plants and seedlings.

[Prior Art] Plants of the genus Asparagus have conventionally been propagated by seeding or division. Plants of the genus Asparagus are dioecious, and male plants are considered 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 also leads to differences in yield and quality among plants, which poses serious problems 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 excellent strain clones by tissue culture has recently attracted attention. Normally, mass propagation of asparagus is done by culturing assembled stalks and producing large numbers of shoots. ) are propagated, each is transplanted into a rooting medium, and seedlings are formed through adventitious root differentiation.However, there is a problem that the propagation efficiency is low because the rooting rate is generally low. In addition, attempts have been made to culture tissue pieces to form callus, and then use somatic embryos produced from the callus to mass multiply (for example, 1985 Horticultural Society Autumn Conference Research Presentation Abstracts, page 210). ~211, November 23-25, 1988, Oryukyu University, 1988 Horticultural Society Research Presentation Abstracts P254-255, Showa 62.1
0.7-9, Kyushu University), although the problem of low rooting rate can be solved by growing somatic embryos, most isolated somatic embryos develop callus or malformation during culture. This has become a problem.

[Problem to be solved by the invention]

The present inventors recognized that the conventional tissue culture method for plants of the genus Asparagus has the above-mentioned problems, and cultivated the seeds and seedlings of plants of the genus Asparagus by culturing tissues using a new method different from the conventional method. We investigated methods for efficient proliferation.

[Means to solve the problem]

As a result, the present inventors discovered that when propagating Asparagus plants through tissue culture, the problems of the prior art described above can be satisfactorily solved by propagating the plant body via the crown. The present invention was completed through further research based on new findings.

Therefore, according to the method of the present invention, (1) a method for forming a crown of a plant of the genus Asparagus, which is characterized by forming a crown by culturing tissue or callus of a plant of the genus Asparagus; (2) tissue or callus of a plant of the genus Asparagus; A method for forming a crown of an Asparagus plant as described in (1) above, characterized in that culturing is carried out using a medium containing 3% or more of Shi=ItI; A method for propagating crowns of plants of the genus Asparagus, characterized by further culturing the obtained sections to enlarge the crown, and repeating the same propagation process of crown cutting → section culture → crown enlargement as necessary. ■ Asparagus Culture of tissue or callus of Gas genus plants at 3%
That's all! The method for propagating crowns of Asparagus plants as described in (1) above, which is carried out using a medium containing yIJi; (1) A method for propagating a plant of the Asparagus genus as described in (1) above, characterized by culturing the crown in the presence of an anti-gibberellin agent; (2) A method for propagating seeds and seedlings of plants of the genus Asparagus, which comprises acclimatizing the plants obtained by the method (1) or (2) above to produce seedlings.

In the present invention, any plant belonging to the genus Asparagus can be used. Specifically, the plant is edible asparagus ^sparagus officinalis
Examples include L, var, and altilis L, which are preferably used in the present invention.

In the present invention, tissues or callus (ca) of plants of the genus Asparagus are used.
llus) is tissue cultured to form a crown. Examples of tissues used for tissue culture in this case include tissues such as embryos, shoot tips, stems, aggregates, and rhizomes. Regarding callus, in the present invention, the tissue is cultured using, for example, a known culture method (for example, Horticultural Society Journal, Vol. 40, No. 4).
11-17, 1971) can be used.

The medium used in the crown formation of 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 lhydrogen phosphate, sodium dihydrogen phosphate, magnesium sulfate, magnesium chloride, sodium sulfate, ferrous sulfate, ferric sulfate, sulfuric acid. manganese, copper sulfate,
Examples include compounds such as sodium molybdate, molybdenum trioxide, potassium iodide, zinc sulfate, boric acid, and cobalt chloride.

Examples of carbon sources for the medium include carbohydrates such as sucrose and glucose, 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, 2,4-dichlorophenoxyacetic acid (2
, 4-b), indolebutyric acid (IBM), and auxins such as derivatives thereof, and benzyladenine (BA)
Examples include cytokinins such as , kinetin, and zeatin.

The vitamins in the medium include biotin, thiamine (vitamin B), pyridoxine (vitamin Ba), pyridoxal, pyridoxamine, calcium pantothenate,
Ascorbic acid (vitamin C), inositol, nicotinic acid, nicotinamide and riboflavin (vitamin B
&) etc.

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

The medium of the present invention usually contains about 0.1 of the inorganic components.
μH to about 100mM, the plant hormones to about 0
．． It is preferable that the amino acids be used in an amount of 0.01 to about 150 Ilg/l and 0 to about 1000 g/l.

Specifically, the medium used for tissue culture according to the present invention may be a conventionally known culture medium used for plant tissue culture, such as Murashige Skoog ('62
) [Murashige &Skoogl's medium,
Linsmeyer Skoog (RM-1965) [Li
n5saier & Skoogl medium, white ('
63) [Whitel's medium, Gamborg]
org]'s B-5 medium, Mitsui's To9 medium, Niche Niche's medium [N1tch & N1tch], etc., the carbon source and plant hormones described above were added, and if necessary, the vitamins and amino acids described above were added. An example of this is a culture medium that has been adjusted by adding additives, but in this invention, especially niche
Regarding the composition of the above-mentioned conventionally known culture medium, which is preferably prepared using a medium by Etchi, Linsmeyer-Skoog, or Murashige-Skoog, see, for example, "New Plant Tissue Culture" by Otsuchi, Chubu, and Furuya, p. 386. ~p391,
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 tissue or callus of an Asparagus plant can be tissue cultured using the above-mentioned medium to form a crown, and if necessary, a plant can be grown from the propagated crown, and the plant can be further grown. By acclimatization, seeds and seedlings of Asparagus plants can be obtained. In this case, the crown as used in the present invention is a storage fiber that corresponds to the underground rhizome in the whole plant and is morphologically a shortened stem. A shaped mass of cells that contains a bud or a bud primordium that becomes the basis of a bud.

In the present invention, the above-mentioned medium can be used as a medium for forming a crown, but the crown formation rate can be particularly increased when a medium containing 3% or more of sucrose as an initial sugar concentration is used.

In addition, in the present invention, even if the yield of crowns obtained by tissue culture is small, only the crowns are cut out from the tissue culture obtained by the above method, transferred to another medium, and cultured to enlarge the crowns. This allows the crown to grow.

Further, if necessary, the growth process of crown cutting → section culture → crown enlargement may be repeated. For the enlarged growth of the crown, the same medium as the above-mentioned crown formation medium can be used, especially Sheet IN 3%. (initial concentration) or more, the growth rate can be increased.

For example, the culture medium shown in Example 4 is used to grow the crown. Table 4 shows the large crown size and shoot size obtained by the crown multiplication factor (described later).
This shows that the number of buds with t) is increasing.

In the present invention, if necessary, the enlarged crown is cut to an appropriate size, preferably to include at least one bud, and the obtained crown sections are cultured again in the same manner. Crowns can be multiplied in large numbers by

Next, an appropriate amount of crowns are extracted from the crowns propagated as described above and cultured in a rooting medium to root them, and the resulting buds are used as a rooting medium to grow into plants of an appropriate size. For example, Murashige Skoog ('62)
[Murashige & Skoog] medium, Linsmeyer-Skoog (RM-1965) [Lins
Maier & Skoog] medium, white (
'63) [Whitel's medium, Gamborg [Gamb
org]'s 8-5 medium, Mitsui's To9 medium, Niche Niche's medium [N1tch & N1tch], etc., the carbon source and plant hormones described above were added, and if necessary, the vitamins and amino acids described above were added. A culture medium prepared by adding additives can be used, specifically, for example, in Example 4A5 or 6.
Rooting is particularly promoted when an anti-gibberellin agent is added to the medium. Specific examples of anti-gibberellin agents include ancymidol and uniconazole.

Furthermore, by acclimatizing the above-mentioned plants, they can be made into seeds and seedlings.In the present invention, by repeating the above method, it is possible to propagate a large amount of seeds and seedlings of plants belonging to the genus Asparagus.

Next, based on the attached drawings, each method of the present invention, and
Crowns, plants, etc. obtained by each method will be explained.

FIG. 1 is a diagram showing the process of crown formation, propagation, and multiplication of plants of Asparagus plants according to the method of the present invention. The upper part of FIG. The path of tissue culture to form a crown is shown, the lower left side of the figure shows the growth cycle for propagating a crown from the previous crown, and the lower right side of the figure shows the process of growing a crown in this way. The plant is shown rooted and regenerated from the crown.

FIG. 2 is a photograph showing a crown formed from an asparagus stem section.

FIGS. 3 and 4 are photographs showing the state in which the asparagus crown sprouts roots and the plant body is regenerated. and,
In Fig. 4, the mass in the center is the crown, and the finger-shaped parts to the left of it are phosphorus buds. Also, what extends above the crown is the shoot (stem), and what extends below the crown is the root (white storage root).

FIG. 5 is a photograph showing a tissue section of the crown;
In the figure, the black (visible part) is vascular tissue.

Hereinafter, the method of the present invention will be specifically illustrated by examples.

Example 1 Shoots of asparagus cultivar "Mary Washington 500" maintained in a sterile system were cut to length 7 including nodes.
The material was a section containing nodes adjusted to ~10 mm. The medium is based on MS medium for inorganic elements, etch-etch for organic elements, 3 times NIl, NO, 2 times 1 g CaC, and 10-'M glutamine added to the basic medium (hereinafter referred to as ASP medium). ).

Add to this 10.30.60 g/l of sucrose, p) 15.
8. IBM was added at 10-'M as auxin, BA was added at 10-'M as cytokinin, and ancymidol was added at 10-'M as anti-gibberellin, and 30 rd doses each were dispensed into commercially available glass bottles (220 m in volume). After that, 100 of the above asparagus stem sections were placed on a bed and heated at 25°C and 300°C.
When cultured for 9 weeks under 0.01 ux, the results shown in Table 1 were obtained.

Example 2 Shoots of asparagus cultivars maintained in a sterile system with a turbidity of 100°' were cut, and sections containing nodes adjusted to a length of 7 to 10 mm were used as materials.The medium contained inorganic components. M.S.
The culture medium and organic components are based on etching and etching, and NIIJ
3x Oz, 2x CaCl2, 10x Glutamine
The medium to which 4H was added was used as the basic medium.

See ItJ for this! 30g/1 or 60g/j!
, agar 7g#! , pH 5,8, indolebutyric acid (IBM) as auxin at 10-'M, benzyladenine (BA) as cytokinin at 3X10-'M or 10-'M, anti-gibberellin with or without angimidol at 10-'M. After adding M and dispensing 30 doses each into commercially available glass bottles (volume 220 ml), 100 of the above asparagus stem sections were placed on a bed and heated at 25°C at 130°C.
When cultured for 9 weeks under 001ux, the results shown in Table 2 were obtained.

Example 3 Shoots of asparagus cultivar "Nepheloma 100°" maintained in a sterile system were cut, and sections containing nodes adjusted to a length of 7 to 10 mm were used as material. The medium is based on ASP medium, sucrose Og, 10g, 20g, 30g,
40g, 50g, 60g, 70g, 80g,
90g #! , using a liquid medium with pH 5.8, IBA 10-'M as auxin and B as cytokinin.
3 x 10-hM of A and 10-'M of ancymidol as anti-gibberellin were added. This was placed in a commercially available glass bottle (volume 220 m) with about 0.9 m of polyester wool.
After dispensing 100 g of each asparagus stem along with g, 100 of the above-mentioned asparagus stem sections were placed on a bed and cultured for 4 weeks at 25° C. and 30,001 ux. The results shown in Table 3 were obtained.

Example 4 The crowns formed at the base of rooted seedlings of asparagus variety maintained in a sterile system with a turbidity of 100 were cut into pieces containing 2 to 3 buds each.

The medium is basically ASP medium, and SealtJ! 10.30
．． 60g/I! , agar 7g/2, pH 5.7,
To this were added 10-'M of IBM as auxin, 10-hM of BA as cytokinin, and 10-'M of ancymidol as anti-gibberellin. Add this to commercially available mayonnaise bottles (220m in volume) each with 301F! After dispensing 1 portion, 5 pieces of the above-mentioned asparagus crown slices were placed on a bed (10 mayonnaise bottles/experimental group), and incubated at 25°C and 300°C.
When cultured for 4 weeks under 0.01 ux, the results shown in Table 4 were obtained.

Example 5 The crown of the asparagus cultivar "Nedori 100" maintained by the method of Example 4 was cut into pieces containing 2 to 3 buds and used as a material. The medium is based on ASP medium, Sea I sugar 10.30.60g/l agar 1g/Lp
H5,7 and add NAA as auxin 3X
l0-'M, uncimidol as anti-gibberellin 3
Xl0-'M was added. After dispensing 30 pieces each into commercially available glass bottles (220 m2 in volume), 5 pieces of the above asparagus crown slices were each placed on the bed (10 glass bottles/experimental area), and the mixture was heated at 25°C under 30,001 ux. When cultured for 4 weeks, the results shown in Table 5 were obtained.

Example 6 Shi=II! The concentration is 30g/j! , no added uncimidol.

Table 6 shows the results obtained in the same manner as in Example 5 except that 3XIO''hM and 10-'M were used.

Tables 1 to 3 show that crowns are formed by tissue culture of stem sections, especially in the case of shi! It is shown that the crown formation rate is high when the F sugar concentration is 3% or more.

Table 4 also shows that the crowns obtained in Example 4 can be propagated by tissue culturing, especially in Sea I.
It is shown that the crown formation rate is high when the m concentration is 3% or more.

Furthermore, Tables 5 and 6 show that crowns obtained by the method of the present invention can be rooted and regenerated by tissue culture, that is, they can be used as seeds and seedlings. This shows that the rooting rate is particularly high when tissue culture is performed using a medium containing Cymidol.

Cytokinin A 3 % 26.7% 45.3% 57.3% Table 1 1% 5.0% (Margin below this page) (Margin below this page) Og/ ll 10g/ It 20g/ ll 30g/ j! 40g/ 1 50g/j! 60g/j! 70g/1 80g/1 Table 3 (4 weeks of culture) 0.0% 22.3% 45.2% 61.6% 88.9% 78.6% 81.8% 78.0% 85.4% ( Margin below this page) Sucrose concentration 10g/ 1 30g/ 1 60g/ 1 Table (%) Table Crown multiplication rate 1.4 3.2 4.8 (4 weeks of culture) Rooting rate 20.0 53.0 40. 0 (%) AA 3X10-'M Ansimidol 3X10-'M Table (4 weeks of culture) No additives 40.0 Photographs showing the crown formed from 3X10-'M 53.0, Figures 3 and 4 5 is a photograph showing a state in which roots have sprouted from an asparagus crown and a plant body has been regenerated, and FIG. 5 is a photograph showing a tissue section of the crown.

Note that FIGS. 2 to 5 are photographs showing the morphology of living things.

〔Effect of the invention〕

According to the method of the present invention, a crown can be formed from a plant of the genus Asparagus by tissue culture, and this can be efficiently mass-propagated. can be efficiently grown in large quantities.

[Brief explanation of drawings]

Figure 1 is a diagram showing the process of crown formation, propagation, plant growth, etc. of Asparagus plants according to the method of the present invention, and Figure 2 is an asparagus stem section representative of applicant Mitsui Petrochemical Industries, Ltd. Patent attorney Yu Hiraki Interpolation Patent attorney Sadashi Ishii Diagram 1 Rooted individual (plant body)

Claims (1)

[Scope of Claims] 1. A method for forming a crown of a plant of the genus Asparagus, which comprises culturing tissue or callus of a plant of the genus Asparagus to form a crown. 2. Culture of tissues or callus of Asparagus plants at 3%
2. The method for forming a crown of a plant of the genus Asparagus according to claim 1, characterized in that the method is carried out using a medium containing the above sucrose. 3. The section obtained by cutting the crown formed by the method described in claim 1 or claim 2 is further cultured to enlarge the crown, and if necessary, the procedure of similar crown cutting → section culture → crown enlargement is performed. A method for crown propagation of plants of the genus Asparagus, characterized by repeating the propagation process. 4. Culture of tissues or callus of Asparagus plants at 3%
4. The method for propagating crowns of plants of the genus Asparagus according to claim 3, characterized in that the method is carried out using a medium containing the above sucrose. 5. A method for propagating a plant of the genus Asparagus, which comprises growing a plant by culturing the crown obtained by the method according to any one of claims 1 to 4. 6. The method for propagating a plant of the genus Asparagus according to claim 5, characterized in that the crown culture is carried out in the presence of an anti-gibberellin agent. 7. The method for growing a plant of the genus Asparagus according to claim 6, wherein the anti-gibberellin agent is ancymidol or uniconazole. 8. The method for propagating a plant of the genus Asparagus according to any one of claims 5 to 7, characterized in that the growth process of the plant includes a rooting process. 9. A method for propagating seeds and seedlings of plants of the genus Asparagus, which comprises acclimating the plants obtained by the method according to any one of claims 5 to 8 to produce seeds.