JPH104811A - Proliferation of large amount of plant of genus hemerocallis - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for proliferating a large amount of a plant of the genus Hemerocallis in a short period of time while maintaining its genetic stability. SOLUTION: This method comprises a process for culturing the sterilely separated tissue of a plant of the genus Hemerocallis in an adventitious bud forming medium, forming an adventitious bud and collecting a small plant body, a process for culturing the collected small plant body in a multiple shoot inducing medium and a process for dividing the induced multiple shoot and culturing the divided multiple shoot in a plant regenerating medium.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

2. Description of the Related Art The genus Hemerocallis is a genus of the lily family and is distributed throughout East Asia including Japan. From the 1800's to today, breeding has been carried out mainly for ornamental use in the United States. Hemerocallis plants are excellent in cold resistance and drought resistance, can be planted on a thin land, and are widely used as horticultural plants and landscaping plants.

[0002] Conventionally, aseptic culture of a growing point of a plant or a section of a tissue on a medium has resulted in somatic embryos, adventitious buds,
Propagation methods by tissue culture of plants in which callus and the like are induced and grown by growing the plants are studied for various plants, and various techniques have been developed so far. However, the conditions for tissue culture must be examined in detail for each plant species and cannot be easily carried out for all plant species. In fact, the growth of Hemerocallis plants is carried out by seedlings or strains.

[0003]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a method for stably mass-producing Hemerocallis plants useful as flowering plants in a short period of time while maintaining genetic stability.

The constitution of the present invention comprises the following technical means (1) to (3). (1) A step of culturing aseptically isolated Hemerocallis plant tissue in an adventitious bud formation medium to form adventitious buds and obtaining plantlets, and culturing the obtained plantlets in a polyplast induction medium A method for mass-producing Hemerocallis plants, comprising: a step of inducing polyblasts by regenerating the plant; and a step of regenerating the plants by dividing the induced polyblasts and culturing the plants in a plant regeneration medium. (2) The method for large-scale propagation of Hemerocallis plants according to (1), wherein the tissue of Hemerocallis plants is a flower stem. (3) The method for mass-producing Hemerocallis plants according to (1) or (2), wherein the multibud induction medium and the plant regeneration medium are mediums containing no plant growth regulator.

[0004]

BEST MODE FOR CARRYING OUT THE INVENTION The plant of the genus Hemerocallis used in the present invention includes plants belonging to the lily family in terms of plant taxonomy and growing in Japan, as well as natural hybrids and artificial hybrids growing in the world. Examples of wild species that grow naturally in Japan include Norikanzo and Hamakanzo. In the United States, varieties are being actively improved, and many excellent horticultural varieties are produced. These can also be used in the present invention. What is generally called a daily lily in Europe and the United States also belongs to Hemerocallis plants.

[0005] Sections of these plants of the genus Hemerocallis, for example, shoot apices, flower stalks, stems, leaves, roots, and the like, may be treated with a fungicide such as ethanol, sodium hypochlorite, or benzalkonium chloride, if necessary. It is used after sterilization. Alternatively, aseptic seedlings and the like germinated by aseptically sowing disinfected seeds can also be used. In particular, inflorescences are easy to handle and suitable for sterilization.

[0006] The tissue culture medium used in the present invention includes an adventitious bud formation medium, a multiple shoots induction medium, and a plant regeneration medium.
As specific examples of these, Murashige-Skoog (Murasige-Skoog) generally used for tissue culture of ordinary plants is used.
koog) medium (hereinafter referred to as “MS medium”), Gamborg-B5 medium, and the like. An aqueous solution of Hyponex (trade name, manufactured by Hyponex Japan), which is usually used as a fertilizer, can also be used. If necessary, an appropriate amount of a carbon source, a plant growth regulator such as auxin, cytokinin, or a plant-derived organic substance such as coconut water can be added to these basic media. Here, it is preferable not to add a plant growth regulator to the multi-bud-induction medium and the plant regeneration medium in order to reduce the probability of occurrence of mutation in the plant.

Auxins include 2,4-dichlorophenoxyacetic acid (2,4-D) and α-naphthaleneacetic acid (N
AA), indoleacetic acid (IAA) and the like can be used. Examples of cytokinins include 6-furfurylaminopurine and 6-benzylaminopurine (BA
P), zeatin and the like can be used.

When culturing on a solid medium, agar, gellan gum or the like is used as a substrate for plant cultivation in an amount of 0.2 to 2.0.
It is used by adding about weight%.

[0009] The culture vessel used in the present invention is not particularly limited in its shape, and may be any vessel made of glass, polycarbonate or the like that transmits light. Further, it is desirable that the culture vessel is sealed with a lid, a stopper, or the like in order to maintain a sterile condition and humidity.

The cultivation temperature until a regenerated plant is obtained is 20
-30 ° C, preferably 23-25 ° C. As for the light condition, it is desirable to irradiate a fluorescent lamp or the like continuously or intermittently. When culturing in a liquid medium, shaking culturing may be performed using a rotary shaker or the like.

[0011]

【Example】

(Embodiment 1) An embodiment of the present invention will be described below with reference to the accompanying drawings. FIG. 1 is a schematic diagram of the processing steps of the present invention. In the figure, A is a step of collecting flower stem tissue, B is a step of forming adventitious buds to obtain small plantlets, C is a step of inducing polyblasts, D is a step of regenerating plants, and E is a habit of seedlings. FIG. The number of days required for the entire process was about 6 months.

In step A of collecting flower stem tissue, Hemerocallis thun , a wild species of Hemerocallis, is used.
bergii var.logituba Miq.), and licorice ( Hem )
erocallis aurantiaca var. littorea Makino) from the flower buds are harvested green immature flower stalks 1, after you cut the florets part,
The pieces were cut into lengths of about 5 cm, sterilized on the surface with sodium hypochlorite having an effective chlorine concentration of 1%, and cut into 2 to 3 mm pieces to prepare flower stem sections 2.

In the step B of forming adventitious buds and obtaining plantlets, the section 2 is treated with α-naphthaleneacetic acid (NAA).
0.1 mg / l, 6-benzylaminopurine (BAP)
Implanted in a 濃度 concentration MS solid medium (adventitious bud formation medium 3) containing 0.1 mg / l, and under continuous lighting (36 μm) at 25 ° C.
mol / m ² / s) to form adventitious buds 4. Table 1 shows the number of adventitious shoots formed.

[0014]

[Table 1]

The adventitious bud formation efficiencies (the number of sections that formed adventitious buds / the number of planted sections × 100) of Norikanzo and Hamakanzo were 19.0% and 9.1%, respectively. There was an interspecies difference in the success rate, which was thought to be due to the genotype difference of each species. FIG. 2 is a photograph showing adventitious buds of Norizo. The obtained adventitious shoots of Noctuids were continued to culture until rooting and complete plantlets were obtained.

In the subsequent step C for inducing polyblasts,
The obtained plantlets of Noctuids elegans were transplanted into each of the multiple shoots induction mediums 5 shown in Table 2, and were continuously illuminated at 25 ° C. (36 μm).
mol / m ² / s) with a rotary shaker (90 to 1).
(00 rpm) to induce polyblasts 6.

[0017]

[Table 2]

The optimal conditions for the multi-blast medium 5 are sucrose concentration of 10 g / l, no addition of plant growth regulator and no coconut water.
The multibud induction efficiency at the time of culturing for months (total number of shoots constituting the multibud / number of cultured plants × 100) is 275.0.
%Met. FIG. 3 is a photograph showing polyblasts of Norizo.

In the following step D for regenerating the plant,
The multi shoots 6 of the licorice were divided and transplanted from the multi shoot induction medium 5 to each plant regeneration medium 7 shown in Table 3. The buds elongated during 1-2 months, easily rooted, and the regenerated plant 8 was obtained.

[0020]

[Table 3]

The optimum conditions for the plant regeneration medium 7 were as follows: a 0.3% (w / v) Hyponex aqueous medium was used as a basic medium, and the sucrose concentration was 10 g / l and the agar concentration was 14 g / l. When the regenerated plant body 8 obtained here was subcultured in a medium under the optimum conditions and continued to be cultured, it grew to a height of 10 cm in one month. FIG. 4 is a photograph showing shoots of licorice elongated by transplanting the multiple shoots into a plant regeneration medium.

In the following step E for acclimating the seedlings, the regenerated plant 8 was transplanted to a vermiculite seedbed to acclimate to the external environment. FIG. 5 is a photograph showing the acclimation of the regenerated licorice plant.

[0023]

Industrial Applicability According to the present invention, the growth of Hemerocallis plants, which have been conventionally performed by seedlings or strains, can be performed in a short period, in a large amount, and genetically uniformly.

[Brief description of the drawings]

FIG. 1 is a schematic view of a processing step according to an embodiment of the present invention.

FIG. 2 is a photograph showing adventitious buds of Norizo.

FIG. 3 is a photograph showing multiple shoots of licorice.

FIG. 4 is a photograph showing shoots of licorice elongated by transplanting a multi-bud body into a plant regeneration medium.

FIG. 5 is a photograph showing habituation of a regenerated licorice plant.

[Explanation of symbols]

 REFERENCE SIGNS LIST 1 flower stem 2 flower stem section 3 adventitious bud formation medium 4 adventitious bud 5 multi-bud induction medium 6 multi-bud 7 plant regeneration medium 8 regenerated plant 9 seedling

Claims (3)

[Claims] 1. A step of culturing a tissue of a genus Hemerocallis isolated aseptically in an adventitious bud formation medium to form adventitious buds to obtain plantlets, and transforming the obtained plantlets into a polyblast induction medium. A method for mass-producing Hemerocallis plants, comprising the steps of: culturing in S. cerevisiae to induce polyblasts; and dividing the induced polyblasts and culturing in a plant regeneration medium to regenerate plants. 2. The method according to claim 1, wherein the tissue of the Hemerocallis plant is a flower stem. 3. The method according to claim 1, wherein the multibud induction medium and the plant regeneration medium are mediums containing no plant growth regulator.