JPH0549484A - Method for deriving adventitious root of melissa officinalis l. and method for manifesting coloring matter of melissa officinalis l. - Google Patents

Abstract

PURPOSE:To derive an adventitious root of the title plant for fragrance component, etc. by subjecting calluses of Melissa officinalis L. to shaking culture containing cytokinin or not, in a medium for culturing a plant and artificially manifesting calluses having high mitoic activity and a coloring matter. CONSTITUTION:Full-ripe seeds of Melissa officinalis L. are sterilely inoculated into a plant material medium containing 2,4-dichlorophenoxyacetic acid and subjected to shaking culture at 25 deg.C at 3,000 luxes for 3-60 days to form calluses. The calluses of Melissa officinalis are subjected to subculture under a dark condition at 20 deg.C every 30 days and the prepared subculture calluses are transplanted in a plant material medium containing auxin and having kinetin as cytokinin or not and subjected to shaking culture at 20 deg.C under illumination with 3,000 luxes to derive an adventitious root of Melissa officinalis. The calluses of Melissa officinalis are cultured in a zeatin-containing medium as a coloring matter producing medium under illumination to give a purple coloring matter of Melissa officinalis L.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a technique for developing a new variety of herbal plant lemon balm.

[0002]

2. Description of the Related Art Most of the plants called herbs are Lamiaceae plants, and their aroma and medicinal properties have recently been drawing attention. However, breeding improvement of Lamiaceae has been delayed.

Lemon balm (scientific name:
Melissa officinalis L., Japanese name: Seiyoyama hakka) is a Lamiaceae plant, and is used for food because its leaves have a lemon flavor.

The present inventors have previously proposed that callus is induced by culturing lemon balm seeds in a medium containing auxin, and protoplasts are produced from this callus (Japanese Patent Application No. 2-222571).

Although the leaves of the lemon balm are limited to those with green leaves, the present inventors have previously applied for a patent for a method for obtaining an albino individual lemon balm, a method for growing the same, or a method for controlling elongation.

[0006]

However, the above-mentioned callus of lemon balm of the present inventors can obtain protoplasts which are the basis for large-scale growth of lemon balm plants and development of new varieties, but the protoplasts do not divide. There was a flaw. Therefore, it is necessary to develop a new callus induction method for lemon balm in order to establish a more diverse protoplast induction method.

The object of the present invention is to establish a technique for obtaining callus suitable for protoplast culture of lemon balm for the development of a new cultivar of lemon balm, and to establish an initial process of a technique for mass-proliferating plants from the obtained callus. That is.

[0008] Further, if lemon balm having various pigments can be obtained from albino lemon balm or lemon balm callus, it is obvious that the value of lemon balm as an ornamental plant is increased, but a specific method thereof is hardly known. It was Therefore, another object of the present invention is to establish a technique for obtaining a pigmented lemon balm from albino lemon balm or lemon balm callus.

[0009]

The above objects of the present invention can be achieved by the following constitutions.

A method for cultivating lemon balm callus, which comprises culturing lemon balm callus in a plant culture medium with or without addition of cytokinin under illumination with shaking.

Lemon balm callus is derived from a part of the lemon balm plant. Explants include seeds, stems,
Although petioles, hypocotyls, and cotyledons are used, mature seeds are preferable because they are easy to handle.

As the cytokinin, kinetin, 6-
Although benzylaminopurine can be used, 6-benzylaminopurine (hereinafter referred to as BA) is preferable because callus with high mitotic activity can be obtained.

BA is used at 0 to 20 mg / liter.
It is not necessary to add BA, but it is more effective to add BA up to 2 mg / liter. 20 mg BA
If added in an amount exceeding 1 / liter, the callus will die, which is harmful.

The object of the present invention can also be achieved in the following configuration.

A method for inducing adventitious roots of lemon balm obtained by subculturing lemon balm callus under dark conditions and culturing the obtained callus in a plant culture medium supplemented with auxin.

As an auxin, 3-indole acetic acid,
Although α-naphthalene acetic acid can be used, α-naphthalene acetic acid (hereinafter referred to as NAA) is preferable because it is easy to sterilize and hardly decomposes. NAA is used in an amount of 0.01 to 10 mg / liter, preferably 1 mg / liter. When the concentration is less than 0.01 mg / liter, NAA has no effect, and when the concentration exceeds 10 mg / liter, N
Rather, AA acts as a harmful substance.

As the lemon balm callus, one obtained by inducing aseptically seeded ripe seeds or the like in a auxin-added medium for plant culture is used.

The object of the present invention can also be achieved in the following configuration.

This is a method for expressing a pigment of lemon balm, in which albino lemon balm or lemon balm callus cultivated aseptically and grown in a plant culture medium is subcultured in a plant culture medium having a different inorganic salt composition from the medium.

Here, the lemon balm growth medium and pigment expression medium are generally used MS medium, B5 medium, and A medium.
A plant culture medium such as A medium is used.

For example, if the growth medium for albino lemon balm or lemon balm callus is 1/2 MS medium,
The purple lemon balm or lemon balm callus growth medium is MS medium.

[0022]

[Function] When callus of lemon balm is shake-cultured in a plant culture medium with or without addition of cytokinin,
The contact area between the callus and the medium becomes large, and the nutrients in the medium are easily absorbed by the callus, so that the callus can be rapidly grown. The result is a finely dispersed callus with high mitotic activity, suitable for protoplast culture.

Further, adventitious roots can be easily induced under illumination using a medium supplemented with auxin (NAA etc.) of callus subcultured under dark conditions.

Further, by subculturing albino lemon balm or lemon balm callus in a medium having a different inorganic salt composition, purple lemon balm or purple lemon balm callus can be obtained.

[0025]

EXAMPLES Examples of the present invention will be described.

Example 1 Ripe seeds were treated with 2,4-dichlorophenoxyacetic acid (hereinafter,
It is called 2,4-D. ) 1 mg / liter and sucrose 3
Aseptic seeding in MS medium supplemented with 50%, 25 ° C, 50
30-6 under illumination at rpm, 3000 looks for 16 hours
Callus is obtained by culturing for 0 days. The callus thus obtained was added with BA 0-2 mg / liter and sucrose 3%,
Shake culture for 20-30 days. At this time, examples of culturing callus with BA concentrations of 0, 0.02, 0.2, and 2 mg / liter were sequentially shown in FIGS.
Shown in (a) and (b).

Example 2 Ripe seeds were aseptically sown in an MS medium containing 2,4-D 1 mg / liter and sucrose 3%, and the seeds were cultivated at 25 ° C. for 300 days.
Callus is induced by culturing for 30 to 60 days under 0 lux and 16 hours of illumination. This callus was treated with MS medium for 2
Subculture is performed every 30 days at 0 ° C. in the dark. The subculture callus thus obtained was transplanted to NAA 1 mg / liter, MS medium supplemented with 3% sucrose, and
When cultivated under illumination at 3,000 ° C. for 16 hours for 20 to 60 days, adventitious roots of lemon balm could be induced with 100% reproducibility. An example of the induction of the lemon balm adventitious root thus obtained is shown in FIG. In addition, NAA is used as the auxin.
In addition, Table 1 shows an example of an adventitious root induction experiment when kinetin was further added as a cytokinin. (Below margin)

Table 1 NAA (mg / liter) 0.1 1.0 5.00 × ○ × Kinetin 0.1 × × ○ (mg / liter) 1.0 × ○ × 5.0 × × × Note Adventitious roots were induced in the treatment areas marked with ○.

From the results shown in Table 1 above, it can be seen that kinetin is not always necessary for the induction of adventitious roots of lemon balm.

Example 3 Next, the procedure for developing the purple pigment of albino lemon balm will be described. The method for obtaining albino lemon balm has been proposed in the patent application of the present inventors (Japanese Patent Application No. 2-73206). Albino Lemon Balm is indole acetic acid (IAA)
Grow in a 1/2 MS medium (medium in which the inorganic salt concentration of MS medium components is halved) to which 0.1 mg / liter, zeatin 0.01 mg / liter, and sucrose 3% were added. Then, IAA0.1 which is a subculture medium for pigment production
It is grown in an MS medium (twice the concentration of the albino lemon balm growth medium) to which mg / liter, zeatin 0.01 mg / liter, and sucrose 3% are added. At this time, 25
Place under illumination at 3000 lux for 16 hours / 1 day. The purple pigment appeared after about 5 days from the passage.

Further, the pigment expression of lemon balm callus can be carried out by the following procedure. First, a lemon balm callus obtained by the same procedure as described in Examples 1 and 2 was used to obtain a matured lemon balm seed.
Growing in B5 medium containing g / liter and 3% sucrose.
Then, zeatin 1 mg / liter and MS medium supplemented with 3% sucrose were used as a pigment production medium at 20 ° C. and 3000.
Looks 16 hours / day under light. The purple pigment was expressed for about 10 days from the 5th day after the passage.

[0032]

EFFECTS OF THE INVENTION By shaking culture in the liquid medium of the present invention, finely dispersed homogenous lemon balm callus with high mitotic activity suitable for protoplast culture can be obtained. Further, since the contact area between the callus and the medium is large and the nutrients in the medium are easily absorbed by the callus, the callus can be rapidly grown.

By using this callus as a material for culturing protoplasts, it became possible to divide lemon balm protoplasts which could not be divided so far.

In addition, adventitious roots could be induced with 100% reproducibility from callus subcultured under dark conditions.

Further, it is possible to artificially express a purple pigment in albino lemon balm and lemon balm callus having no chlorophyll, the number of types of lemon balm colors is increased, and the added value as an interior product is increased. Moreover, since the expression of the pigment is controlled in the artificial medium, the reproducibility of the purple pigment expression is high. Since the plant in culture is used as a material, it is possible to develop color in a short period of time.

[Brief description of drawings]

FIG. 1 is a photograph showing the morphology of organisms according to the method for culturing lemon balm callus of the present invention.

FIG. 2 is a photograph showing the morphology of organisms according to the method for culturing lemon balm callus of the present invention.

FIG. 3 is a photograph showing the morphology of an organism by the method for inducing adventitious roots of lemon balm of the present invention.

Claims (3)

[Claims] 1. A method for culturing lemon balm callus, which comprises culturing lemon balm callus in a plant culture medium with or without addition of cytokinin with shaking. 2. A method for inducing adventitious roots of lemon balm, which is obtained by subculturing lemon balm callus under dark conditions and culturing the obtained callus under illumination in a plant culture medium containing auxin. 3. An albino lemon balm characterized by subculturing albino lemon balm or lemon balm callus, aseptically seeded and grown in a plant culture medium, in a plant culture medium having an inorganic salt composition different from that of the medium. Alternatively, a method for expressing a pigment of lemon balm callus.