JPH0698653A - Production of seed and seedling of plant of genus exacum - Google Patents

Abstract

PURPOSE:To mass-produce seeds and seedlings of a plant of the genus Exacum having uniform quality. CONSTITUTION:The production of seeds and seedlings of a plant of the genus Exacum consists of (1) a step for culturing a tissue of the plant of the genus Exacum and inducing callus therefrom, (2) a step for proliferating the induced callus and (3) a step for forming an adventitious embryo from the callus and further regenerating a plant body from the adventitious embryo.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for mass-producing seeds and seedlings of Exacam plants.

[0002]

2. Description of the Related Art Breeding of a plant of the genus Exacam includes seed breeding and vegetative breeding, and some of them are known as vegetative breeding.

However, for example, in the cutting method, the survival rate of seedlings is low and the traits are difficult to align.

[0004] In addition, as a tissue culture method for Exacam plants, an adventitious bud culture method [Hort Science (HortSci
ence) Volume 19, No. 2, pp. 224 to 225 (1984)] are known.

However, the process of growth is not clearly described, and it cannot be said to be a mass production method of seeds and seedlings.

On the other hand, in the Gentianaceae plants such as Gentian, Ezo Lindau, and Murachixenburi, which belong to the genus Exacam, a method of inducing callus from a tissue piece and then inducing an adventitious embryo to regenerate the plant [12th plant tissue Symposium of the Symposium on Culture Society, page 46 (1991)
), Journal of Horticultural Science Vol. 60, Supplement 1, pages 450-45
1 (1991), 9th Annual Meeting of the Plant Culture Society Symposium, 157 (1985)].

[0007] However, in the Exacam plant, a method for inducing an adventitious embryo from a tissue piece to regenerate it into a plant is not known.

[0008]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a method for producing a large quantity of seeds of the genus Exacam of uniform quality.

[0009]

[Means for Solving the Problems] The inventors of the present invention have made extensive studies in order to achieve the above object. As a result, a method for mass-producing seedlings of an Exacam plant, in which callus obtained by culturing tissue of an Exacam plant in a medium containing a plant hormone is grown, and an adventitious embryo is formed on a solid medium to regenerate a seedling I found it.

That is, the gist of the present invention is that
The following steps (1) to (3), (1) a step of culturing a tissue of an Exacam plant to induce callus, (2) a step of growing the induced callus, (3) an adventitious embryo from the callus And a step of regenerating a plant from an adventitious embryo, the method for producing a seedling of an Exacam plant.

Next, the method of the present invention will be described.

The Exacam plant used in the present invention is
Exacum affine , E. macranthum , E. quinquenerui as seeds
um and E. tetragonum, but are not limited to these species and varieties.

Examples of the medium used in the method of the present invention include a medium for plant tissue culture such as Murashige-Skoog (hereinafter referred to as "MS medium"), Gamborg's B5 medium, White medium, Niche-Niche medium, N6 medium and the like. Can be mentioned. Regarding these known medium compositions, for example, "New Plant Tissue Culture" by Takeuchi, Nakajima, and Furuya, pages 386-3.
Page 91 (1979, published by Asakura Shoten).

In each step of the method of the present invention, the inorganic salt of these media may be used at a predetermined concentration or diluted to one third or one half of the predetermined concentration.
A medium prepared using MS medium is particularly preferable.

In the present invention, auxins and cytokinins are used as plant hormones. As auxins, indole acetic acid, α-naphthalene acetic acid (hereinafter referred to as “NAA”), indole butyric acid, 2,4-
Examples thereof include dichlorophenoxyacetic acid (hereinafter referred to as "2,4-PA"), 3,6-dichloromethoxybenzoic acid, 4-amino-3,5,6-trichloropicolinic acid and the like.

Examples of the cytokinins include benzyladenine (hereinafter referred to as "BA"), kinetin (hereinafter referred to as "KIN"), zeatin and the like.

Next, the steps (1) to (3) in the method for producing seeds and seedlings of the Exacam plant according to the present invention will be described in detail.

(1) Callus Inducing Step (First Step) As the plant of the genus Exacam used in the callus inducing step, for example, tissue pieces collected from leaves, stems, growth points, flower prunes, roots, etc. can be used. , Preferably leaf pieces.

Then, these tissues were immersed in a 70% ethanol solution for 10 seconds, and then the effective chlorine concentration was 0.2% to 5%.
%, Preferably 1% sodium hypochlorite solution for 2 minutes to 30 minutes, preferably 10 minutes and sterilized. Then, the tissue is taken out, the sodium hypochlorite adhering to the surface thereof is washed three times with sterile water, and then cut under aseptic conditions to obtain small pieces.

Next, a carbon source such as sucrose and plant hormones such as auxin and cytokinin are added to the inorganic salt of the MS medium to adjust the pH to 4.0 to 7.0, and further gelling such as gellite. After adding the agent, it is sterilized in an autoclave to prepare a callus induction medium.

The plant hormone used in the callus induction medium may be auxin and cytokinin in combination or auxin alone, but preferably 2,4-PA is 0.2 mg / l (mg / liter, the same applies hereinafter. ) -10.
0 mg / l, KIN 0.01 mg / l to 1.0 mg /
1, more preferably 2,4-PA 4.0 mg / l,
It is preferable to add KIN in an amount of 0.1 mg / l.

Examples of the carbon source of the callus induction medium include sugars such as sucrose and glucose.
g / l to 120 g / l, preferably 20 g / l to 50 g
It is preferable to use it at a concentration of 1 / l.

A solid medium or a liquid medium can be used as the callus-inducing medium, but a solid medium is preferably used. In this case, agar, agarose, gellite (gellan gum), carrageenan, etc. are used as the gelling agent, and preferably 0.1% to 2.0% gellite.
It is better to use it after adding%.

The piece of tissue obtained as described above is placed on the callus-inducing medium thus prepared.

The culture temperature in the callus induction step is 15 ° C to 3 ° C.
It is 0 ° C, preferably 25 ° C to 28 ° C.

In the callus induction step, it can be cultivated in a bright place or a dark place, but it is preferably cultivated in a dark place. In the case of culturing in a dark place, a callus having an adventitious embryogenesis ability is induced by culturing for 30 to 60 days, preferably 50 days after implantation of the tissue piece.

(2) Callus Growth Step (Second Step) The induced callus is transplanted to a callus growth medium in order to grow it up to a required amount, and cultured in a dark place. As the callus growth medium, a medium having the same tissue as the callus induction medium can be used.

In addition, since the growth efficiency of callus differs depending on the cultivar of the genus Exacam, in order to increase the growth efficiency, the auxin concentration and cytokinin concentration of the callus induction medium should be lowered, or a growth medium supplemented with auxin alone should be used. You can also The auxins and cytokinins used are not limited to 2,4-PA and KIN.

The culture temperature is 15 ° C to 30 ° C, preferably 25 ° C to 28 ° C, and the culture period is 5 days to 50 days, preferably 30 days.

A liquid medium or a solid medium is used as the callus growth medium.

In many plants other than the Exacam plant,
It is known that the transplantation of callus and the redifferentiation ability decrease as the cells are subcultured. Held at the level of.

(3) Adventitious embryo formation and plant regeneration step (third step) Propagated callus is selected using a stainless steel sieve having a pore size of 1 mm. In this case, the size of the callus is 0.
02 mm to 1 mm, preferably 0.125 mm to 0.5
mm.

Then, a carbon source and gellite are added to the inorganic salt of the MS medium to prepare a regeneration medium.

Examples of the carbon source of the medium for regeneration include sugars such as sucrose and glucose, but 10 g / l of sucrose is used.
It is preferable to use it at a concentration of 120 g / l, preferably 20 g / l to 50 g / l.

A solid medium is preferably used as the medium for regeneration, and agar, agarose, gellite (gellan gum), carrageenan, etc. are used as the gelling agent, and preferably 0.1% to 2.0% gellite. , Preferably 1.0% is added.

As for the plant hormone in the medium for regeneration, it is preferable to use no cytoplasm or to use cytokinin alone when adding it, in which case 0.01 mg BA /
It is preferable to add 1 to 1 mg / l for use.

The pH of the regenerating medium is pH 4.0 to 7.
The pH may be adjusted to 0, preferably pH 5.8.

In the regenerating medium thus obtained, the proliferating callus obtained above was suspended in a liquid MS medium containing sucrose in advance, and 1 ml (milliliter, the same hereinafter) (the number of callus was 20 to 1000, preferably Is 50-100
(Including individual) is preferably added to the regeneration medium.

Then, the medium containing this callus was added 5 times a day.
Irradiation (100 to 20 hours, preferably 16 hours)
(100,000 lux, preferably 4000 lux)
An adventitious embryo can be obtained by culturing under 5 to 50 days, preferably 10 days after implantation. Furthermore, when this somatic embryo is transplanted to a medium having the same composition as above and cultured for 10 days, a young plant body is regenerated.

The culture temperature in the regeneration step is 15 ° C to 3 ° C.
It is 0 ° C, preferably 25 ° C to 28 ° C.

[0041]

EXAMPLES Examples of the present invention will be described below.

[0042] The first step (callus induction process) Ekizakamu (Exacum affine, varieties: Rococo) leaves 7
It is immersed in a 0% ethanol solution for 10 seconds, then in a 1% sodium hypochlorite solution for 10 minutes, and then washed 3 times with sterile water. Cut the sterilized leaves into 1 cm leaf pieces with a scalpel,
This is the test material.

5 pieces of leaf pieces (per petri dish) thus obtained were prepared by using an inorganic salt of a predetermined concentration in MS medium, sucrose 5%,
Gellite 0.2% and 2,4-concentration shown in Table 1
Callus induction medium containing PA and KIN (pH 5.
Place at 8).

Then, the cells were subcultured at a temperature of 25 ° C. in the dark every 30 days after being placed in a medium having the same composition as described above.
After a day, white friable callus began to be induced, and culture 9
After 0 days, yellowish callus is induced. This yellow callus is a callus having somatic embryogenesis ability, and this callus is used in the next step.

In the investigation, the number of leaf pieces in which callus was induced after 90 days of culture was examined, and the callus induction rate (%) per leaf piece was calculated by the following formula. The results are shown in Table 1.

[0046]

[Equation 1]

[0047]

[Table 1]

Second step (callus multiplication step) The callus having a diameter of about 2 cm (about 4 g) obtained in the first step is divided into four equal parts. Then, a predetermined concentration of inorganic salt in the MS medium,
Sucrose 5%, 2,4-PA 4mg / l, KIN 0.1m
1 g of the callus obtained above is placed in a callus growth medium (pH 5.8) containing g / l and 0.2% of gellite and cultured at 25 ° C. for 30 days in the dark.

After 30 days of culture, about 4 g of callus is obtained from 1 g of callus.

Third Step (Adventitious Embryogenesis and Plant Regeneration Step) 1 g of callus obtained in the second step was selected using a stainless sieve having a pore size of 1 mm to select a callus smaller than 1 mm, and the inorganic salt of MS medium ( The callus is suspended by adding 200 ml of a liquid medium (pH 5.8) containing 30 g / l (g / l, the same applies hereinafter) of sucrose at a predetermined concentration diluted to one third.

Then, the inorganic salt of the MS medium (predetermined concentration of 3
1% dilution), sucrose 30 g / l, BA at the predetermined concentrations shown in Table 2, and a solid medium for plant regeneration (pH 5.8) containing gellite. Place 1 ml of the solution on each bed. Immediately after placement, callus on the medium is counted. Then, this is stored at 25 ° C. for 16 hours in a light period (4000 lux) and 8 hours in a dark period for 20 days.
After culturing for a day, somatic embryos are formed from about 10 days after culturing, and after 20 days of culturing, seedlings germinated and rooted from somatic embryos are obtained.

In the investigation, the number of solids regenerated in the seedlings was examined after 20 days of culture, and the plant regeneration rate (%) was calculated by the following formula. The results are shown in Table 2.

[0053]

[Equation 2]

[0054]

[Table 2]

[0055]

According to the method of the present invention, it is possible to produce a large amount of seeds and seedlings of a plant of the genus Exacam with uniform quality.

Claims (1)

[Claims] 1. The following steps (1) to (3), (1) a step of culturing tissue of a plant of the genus Exacam to induce callus,
(2) a step of proliferating the induced callus, (3) a step of forming an adventitious embryo from the callus, and further a step of regenerating a plant body from the adventitious embryo, a method for producing a seedling of an Exacam plant .