JPH0445730A - Method for regenerating plant of genus platycodon and method for proliferating seed and seedling thereof - Google Patents

Abstract

PURPOSE:To efficiently obtain seeds and seedlings in a short time by culturing tissual pieces of a plant belonging to the genus Platycodon, providing calli, further culturing the resultant calli, differentiating adventitious buds, subsequently culturing the adventitious buds, affording young plant bodies and acclimatizing the prepared young plant bodies. CONSTITUTION:Tissual pieces of a plant belonging to the genus Platycodon (preferably Platycodon.grandiflorum) are cultured and the resultant calli are then cultured to provide adventitious buds. The obtained adventitious buds are subsequently differentiated and cultured to afford young plant bodies, which are then acclimatized to provide large amounts of seeds and seedlings.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for regenerating a plant of the genus Campanulum and a method for propagating its seeds and seedlings, and more particularly relates to mass propagation of a plant of the genus Campanulum and its seeds and seedlings via callus.

[Conventional technology]

Conventionally, there is no known method for mass-propagating plants of the genus Campanulum, especially their seedlings, other than propagation by seeds.

[Invention or problem to be solved]

However, the method of regenerating plants using seeds has a fatal drawback in that propagation and regeneration of the plants are inefficient and require a lot of time.

[Means to solve the problem]

In order to overcome the drawbacks of the above-mentioned conventional techniques, the present inventors have conducted intensive studies on regeneration methods using tissue culture for plants of the genus Campanulum. It was discovered that the method of differentiating adventitious buds allows for the regeneration of Campanulata plants in a much more efficient and short time than the above-mentioned conventional techniques, and as a result of further research based on this new knowledge, the present invention was developed. This is what we have come to complete.

That is, the method for regenerating a plant of the genus Platycodon of the present invention involves inducing callus from a tissue piece of a plant of the genus Platycodon, culturing the callus to differentiate into adventitious buds, and then culturing the adventitious buds. The method for propagating seeds and seedlings of plants of the genus Platycodon of the present invention is characterized by inducing callus from tissue pieces of plants of the genus Platycodon, and then culturing the callus. After differentiating a large number of adventitious buds, the adventitious buds are individually cultured and rooted to obtain seedlings, and further,
This is characterized by the fact that it must be habituated.

The present invention will be explained in detail below.

The plant of the genus Platycodong used in the present invention includes Platycodong grandiflorum (Platycodong grandiflorum).
randiflorum), and its varieties include Var, duplex Mak, ), rugosun Mak, and Var, placorollatum.
Mak, ), etc., and the basic Brachycodon grandiflorum is particularly preferred.

The plant tissue pieces may be obtained from various parts of the plant that are used as explants in normal tissue culture, such as leaves, stems, roots, etc., but foliage is particularly preferred. It is.

The tissue piece can be obtained, for example, as follows.

Seeds of the above-mentioned plants were sown in the soil normally used for cultivating plants of the genus Kikiyo, and tissues of the grown seedlings, such as stems and leaves, were mixed with 70% ethanol, sodium hypochlorite,
After sterilizing using a common sterilizing agent such as a surfactant, the tissue piece is washed with sterile water to obtain a sterile tissue piece. This tissue piece is 1~
Cut into pieces of about 5 mm to obtain tissue pieces for callus induction.

In addition, after sterilizing the seeds of the above-mentioned plants using a normal disinfectant such as 70% ethanol or sodium hypochlorite for about 10 to 40 minutes, the seeds after the sterilization treatment are thoroughly washed with sterilized water. , remove the bacterial agent.

The obtained sterilized seeds are placed in a medium normally used for plant tissue culture, such as a normal MS basic medium, with agar, agar, etc.
Indole acetic acid (IAA) and naphthalene acetic acid (NAA) as solidifying agents and plant hormones for gellan gum, etc.;2.
Auxins such as 4-D, benzyladenine (BA),
The seeds are sown on a solid medium supplemented with at least one plant hormone selected from cytokinins such as kinetin and zeatin.

At this time, the content of plant hormones in the medium is 0.05 to 2.0 /i, preferably 0.2 to 2.0 /i in the case of auxins.
Approximately 0.5■/I, 0.05~ for cytokinins
It is about 5.0 .mu./I, preferably about 0.1 to 2.0 .mu./I.

In addition, the content of solidifying agent is 0.7 to 1.5% in the case of agar (
W/V), in the case of gellan gum 0.2-0.3% (
W/V).

Next, the seeds are cultured under aseptic conditions at about 20 to 30° C. under a photoperiod of 8 to 20 hours for about 4 to 8 weeks to germinate to obtain sterile seedlings.

Next, tissue pieces for callus induction are obtained by chopping the young plants or their stems and leaves into about 1 to 5 pieces.

The callus is induced from the tissue piece in the following manner. That is, the tissue pieces obtained by shredding the stem and leaf parts into about 1 to 5 piths are mixed with a medium normally used for plant tissue culture, for example, at a concentration of sucrose alone at a concentration of 1.0 to 5 pith.
A solidifying agent such as gellan gum, and plant hormones such as auxins such as NAA and IAA, and cytokinins such as BA, kinetin, and zeatin are added to MS medium, B5 medium, etc. modified to 2.5% (W/V). Place the plate on the added solid medium and incubate at 20-30°C for 8-20 hours under photoperiod for 20-5 hours.
Cultivate for about 0 days to obtain callus.

In addition, the content of the solidifying agent in the above medium is 0.2 to 0.3%.
(W/V) or about 0.2 to 2.0 ■/I in the case of auxins and 0.1 to 1.0 ■/I in the case of cytokinins as plant hormones.

In addition, if necessary, the obtained callus was subcultured at one month intervals on MS solid medium containing plant hormones with the above-mentioned sucrose concentration modified to 1.0 to 2.5% (W/V). You can go.

The callus obtained as described above was added to the modified MS solid medium as a plant hormone containing 0 cytokinin (BA).
．． 1-2.0■/l alone or with IAA, NAA
A large number of adventitious buds of plants of the genus Campanulum were produced by placing auxins such as 0.01 to 0.5 μ/i7 on a medium and culturing at 20 to 30°C under a photoperiod of 8 to 24 hours. Adventitious buds that have grown to about 5 mm or more are transplanted to a solid medium in which only the inorganic salts of the MS medium have been reduced to about 4%, and incubated at 20 to 30°C for 16 to 24 hours under photoperiod. After culturing for more than a month, seedlings of plants of the genus Campanulum having shoots or adventitious roots are obtained.

Note that the number of regenerated shoot bodies may be increased by cultivating a part of the above-mentioned shoot body tissue, for example, by culturing the shoot apex or by culturing a callus similar to the above-mentioned operation.

When regenerating a complete plant from the shoot, the shoot is transplanted to the MS solid medium or a medium to which auxins are added at a low concentration of about 0.01 to 0.1 μ/l. Then, adventitious roots can be generated and grown, and a complete plant can be obtained. By further acclimatizing this plant under suitable conditions, seeds and seedlings of plants of the genus Campanulum can be obtained.

〔Effect of the invention〕

According to the present invention, plants of the genus Campanulum and their seeds and seedlings,
Because it proliferates through adventitious buds differentiated from callus, it is possible to produce the desired plants or seedlings in a much shorter period of time and more efficiently than with the conventional method of simply regenerating plants from seeds. The present invention is industrially significant because it can be reliably propagated.

The present invention will be specifically explained below using Examples.

〔Example〕

Platycodon grandiflorum (Platyco)
Don grandif lorum) seeds were surface sterilized with 70% ethanol for 3 minutes, then sterilized with a mixture of 1% sodium hypochlorite and several drops of Tween 20 for 20 minutes, and then thoroughly washed with sterile water. This sterilized seed
MS basal medium with 0.8% (w/v) agar, 0.5 μ/
IBA, 0.1mg/l IAA, O, 1mg/l
2.4-D, placed on a solid medium containing 0.5 mg/l Kinetin, and incubated at 27°C under a 16-hour photoperiod for about 3 hours.
After culturing for 5 days, seedlings were obtained.

After removing the roots of the seedlings and cutting the stems and leaves into several pieces, the sucrose concentration was reduced to 2% (W/V) i
: 1 m of modified MS medium, 2.2 g/I! Gerancum, 1.0■/l! NAA, 0.5■/lBA
Place the stalks and leaves on a solid medium containing 27°C 11
A yellow-green callus was obtained by culturing for about 30 days under a 6-hour photoperiod.

20 calluses, 0.5■/l BA and 0.1■/l I
The cells were transplanted onto the MS solid medium supplemented with AA. Adventitious buds were generated by culturing for about 5 to 6 weeks under the above conditions.

Approximately 30 adventitious buds were generated from 1 g of callus within the above culture period.

Next, adventitious buds that had grown to about 5 to 10 mm were taken out using a bottle set and added to MS medium 1 knee NAA (0.1 mg/
25°C, 30001ux.
When cultivated under these conditions, the plants were rooted and complete seedlings were obtained.

Furthermore, take out this young plant in a sterilized bottle set,
Transplanted to MS solid medium (does not contain plant hormones),
After culturing for several months, the plants, which have grown to a height of 5 to 10 ann, are transplanted to sterilized vermiculite.
Cover with glassware for 5 to 20 days at 25°C, 3
Seedlings of plants of the genus Campanulum were obtained by q conversion under conditions of 0001UX.

Claims (1)

[Scope of Claims] 1. Callus is induced from a tissue piece of a plant of the genus Platycodon, the callus is then cultured to differentiate into adventitious buds, and the adventitious buds are then cultured to form seedlings. A method for regenerating a plant of the genus Campanulum, characterized by obtaining. 2. Induce callus from a tissue piece of a plant of the genus Platycodon, then culture the callus to differentiate a large number of adventitious buds, and then culture the adventitious buds individually and root them to form seedlings. 1. A method for propagating seeds and seedlings of plants of the genus Campanulum, which comprises obtaining and further acclimatizing the same.