JP3001677B2 - Isolation of Protoplasts from Menta Plants and Method for Plant Regeneration by Their Culture - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to the isolation of protoplasts of a genus Menta plant and the regeneration of the plant body by cultivation thereof. The present invention relates to a method for regenerating a plant by isolating a protoplast of a spicy plant such as a plant and culturing the protoplast.

[0002]

2. Description of the Related Art Conventional breeding of mint (Menta genus plant) has been carried out only by the crossing method, but it cannot be performed at any time of the year. There was a problem that it was done. on the other hand,
With recent advances in biotechnology, new techniques such as cell fusion and gene transfer have been developed and attracted attention.

However, there have been few examples of the development of the technique of cultivating plant tissues of the Menta genus plant, and the tissue fragments described in Japanese Patent Application Laid-Open No. 3-15327 filed by the present applicant and plants derived from callus derived therefrom have been described. There are only reports on regeneration and its mass propagation method. Isolation of protoplasts from plant tissues, which can be positioned as a further development of this technology, and regeneration of plants by cultivation thereof have not been established at all. Therefore, cell fusion methods of this genus plant species, gene transfer methods, etc. It was not applicable to cell engineering breeding.

[0004]

The isolation and cultivation techniques of protoplasts that have already been established in many other plant species have not been able to be established with respect to the Menta plant, as in the present plant species. In plants containing a large amount of essential oil components in their tissues, the hydrophobicity of the plant tissue surface is high,
The main factor is that isolation of protoplasts was extremely difficult because contact between the protoplast isolation enzyme and plant cells was hindered. In the prior art, in such a case, the problem was solved by finely shearing the plant tissue.However, in the genus plant, such a technique is insufficient, and most of the sheared plant tissue is contained in an enzyme solution. Float without sinking. If this is further finely sheared, there is a concern that the plant tissue may be damaged. In addition, mere destruction of the epidermis due to shearing does not completely remove the essential oil components that are often present in tissues.

An object of the present invention is to provide a method for isolating protoplasts of spicy plants such as Menta genus plants containing a large amount of essential oil components in tissues, which has been difficult to handle conventionally, and a method for regenerating the plant bodies by culturing the same. And

[0006]

According to the present invention, an essential oil composition is provided.
In the isolation of spice plant protoplasts containing
Isolation of protoplasts by effectively destroying the leaf epidermis of the highly hydrophobic plant without shearing the leaves to increase the hydrophilicity of the leaves and removing essential oil components from the leaves from which the epidermis was disrupted Essential oil component characterized by effective action of enzymes
A method for isolating a protoplast of a spice plant is provided.

In order to effectively destroy the highly hydrophobic leaf epidermis without shearing the leaf to enhance the hydrophilicity of the leaf, for example, the stem apex of a plant is sterilized and then obtained by aseptic treatment. It is preferable that the leaves are collected from the plant body obtained and the epidermis is finely scratched one by one with a scalpel so that the leaves are not sheared in a CPW salt solution described later.

[0008] When removing essential oil components in leaves from the leaves whose epidermis has been destroyed in this way, the leaves treated as described above are transferred to a new CPW salt solution, which is then treated with 50 to 50%.
It is preferable to shake at 70 rpm for 2 to 4 hours. Thereafter, for example, about 10 ml of enzyme solution (cellulase YC 2%, macerozyme R-10: 0.8
%, Mannitol 0.5M, pH 6.0).
After standing for a period of time, the mixture is rotated and shaken at 70 rpm for 30 minutes, whereby protoplasts can be isolated from the mesophyll of the spice plant. In the enzyme treatment, of course, other conditions usually used can be used. Thereafter, the protoplast is purified by a conventional method.

In the method for isolating a protoplast of a spice plant, it is preferable that the spice plant is a plant of the genus Menta.

Further, according to the present invention, when culturing protoplasts of the genus Menta, (NH ₄ ) ₂ SO ₄
75 mg / l, NaH ₂ PO ₄ .H ₂ O 15 to 75 mg /
l, KNO ₃ 400~1500mg / l, sucrose 0.5
%, Auxin 5 mg / l or less, cytokinin
Culturing the protoplasts mental Genus at Ganborugu of B5 medium containing more than 0.05 mg / l, protoplast culture method of Mentha genus plants characterized in that to form colonies is provided. In this case, the illumination conditions are preferably dark conditions.

[0011] The method for culturing a protoplast of a Menta genus plant further comprises culturing the grown colony with shaking in a liquid medium while being embedded in the solid medium, and efficiently removing the colony from the solid medium. It is suitable.

Further, according to the present invention, when regenerating a plant of the genus Menta, a colony is cultured on a solid medium maintaining a high osmotic pressure, callus having a high plant regeneration ability is grown, and auxin 0 Provided is a method for regenerating a plant of the genus Menta, comprising regenerating callus derived from a protoplast of a genus Menta into a plant on a basal medium containing not less than 0.01 mg / l and not more than 20 mg / l cytokinin.
In this case, the culture is performed under illumination, and normal culture conditions can be used. The culture is maintained at a high osmotic pressure from the cultivation of the protoplasts until immediately before the regeneration of the plant body is induced, so that calli having a high differentiation ability are formed.

[0013]

As described above, the protoplast isolation and cultivation techniques already established in many other plant species are
What has not been established so far for Menta plants is
In plants such as this plant species, which contain a large amount of essential oil components in their tissues, the protoplasts are isolated because the surface of the plant tissues is highly hydrophobic and the contact between protoplast isolation enzymes and plant cells is hindered. The main factor was that it was very difficult. In the prior art, in such a case, the problem was solved by finely shearing the plant tissue.However, in the genus plant, such a technique is insufficient, and most of the sheared plant tissue is contained in an enzyme solution. Float without sinking. If this is further finely sheared, there is a concern that the plant tissue may be damaged. In addition, mere destruction of the epidermis by shearing does not completely remove the essential oil components that are often present in tissues.

Therefore, in the present invention, instead of shearing the plant tissue, only the surface is scratched as much as possible with a scalpel or the like to effectively remove the highly hydrophobic epidermis, and to improve the hydrophilicity of the plant tissue. In addition, by shaking for several hours in a solution with high osmotic pressure before enzymatic reaction, the essential oil component, which is considered to inhibit the enzymatic reaction, is eluted into the external solution, thereby making the enzyme effective. And made it possible to obtain large quantities of good-quality protoplasts. Also, regarding the cultivation of protoplasts, maintaining a high osmotic pressure until just before inducing regeneration, and forming calli with high differentiation potential, furthermore, there is almost no complicated operation in the isolation and culture of a series of protoplasts. Another feature of the present invention is that it is a very simple culture method.

The isolation of protoplasts from the tissue of the genus Menta and the regeneration of the plant by cultivation thereof described in the present application are a further development of the technique described in JP-A-3-15327. By applying cell engineering techniques to the breeding of Menta genus plants, it is possible to rapidly produce new mint species in the laboratory regardless of the season and which cannot be produced by natural crossing. Further, the isolation of protoplasts is widely applicable not only to the Menta plant but also to other spice plants.

[0016]

According to the present invention, there is provided a method for isolating a protoplast of a spicy plant such as a menta plant containing a large amount of an essential oil component in a tissue, which was conventionally difficult to handle, and a method for regenerating the plant body by culturing the same. Is done.

[0017]

EXAMPLES The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to the following examples.

The treatment solution, enzyme solution, culture medium and the like used in the experiment are as follows.

1) CPW salt solution (adjusted to pH 6.0,
_{Autoclaved) KH 2 PO 4 27.2 (mg} / l) KNO 3 101 (mg / l) CaCl 2 · 2H 2 O 1480 (mg / l) MgSO 4 · 7H 2 O 240 (mg / l) KI 0 .16 (mg / l) CuSO ₄ .5H ₂ O 0.025 (mg / l) mannitol 0.5M In addition, Z. Pflanzenphysiol., 104, 289-298, 1981 can be referred to.

2) Protoplast isolation enzyme solution Macellozyme R-10 (Yakult Biochemical) 0.5-0.8% Cellulase YC (Seishin Pharmaceutical) 0.5-2% Mannitol 0.5-0.7M pH 5.5-6.0 Sterile filter immediately before use.

3) Initial culture medium (adjusted to pH 5.5 to 6.0 and sterilized in an autoclave) Component 1 KNO ₃ 800 to 3000 mg / l (preferably around 1500 mg / l) (NH ₄ ) ₂ SO ₄ 30 to 150 mg / l (preferably around 75 mg / l) NaH ₂ PO ₄ .H ₂ O 30 to 150 mg / l (same as above) Component 2 Gamborg B5 medium component other than component 1 Normal value ^*) 2
Double concentration component 3 sucrose 0.5-3% (preferably 0.5-1%) Mannitol 0.5M component 4 Plant hormone Auxin 10mg / l or less (preferably NAA 2mg / l or 2,4-D 0.1mg) /
l) Cytokinin 0.1 mg / l or more (preferably BA
0.8-1 mg / l) *) See Gamborg et al., Exp. Cell Res., 50, 151 (1968).

4) Gelrite solution (autoclave sterilized) Sucrose Same value as in initial medium Mannitol Same as above Gellite 0.2-0.5% (preferably 0.2%)
Before and after).

5) Colony growing medium (pH 5.5-6.
Adjust to 0 and sterilize by autoclaving.) Component 1 of initial culture medium 1/2 concentration of initial culture medium Same component 2 1/2 concentration of initial culture medium Same component 3 Same as initial culture medium Same component 4 Initial culture medium 1/2 times the concentration.

6) Callus-forming medium A colony growing medium solidified with 0.2-0.3% gellite. It was made with a 90 mm sterile plastic petri dish.

7) Redifferentiation induction medium A 90 mm sterilized plastic petri dish was prepared in the same manner as the callus formation medium. However, the plant hormone is NAA 0.0
1 to 0.5 mg / l (preferably 0.05 to 0.2 mg
/ L), BA 0.5-20 mg / l (preferably 1-5
mg / l), and mannitol was reduced to 0.2M.

Using the above, the present invention was implemented as follows. Into a 300 ml conical beaker, inject Gamborg's B5 medium (2% sucrose, 0.2% gellite, pH 5.8) containing no plant hormones, cover with a heat-resistant transparent film (diafoil, etc.). In an autoclave-sterilized incubator, peppermint ( Mentha piperita )
After sterilization, the shoot apex was aseptically inserted and sprouts and grown for about 3 weeks to 1 month. The leaves are recovered from the obtained plant, and C
The epidermis was finely scratched one by one with a scalpel so as not to shear the leaves in the PW salt solution. This was transferred to a fresh CPW salt solution and shaken at 50-70 rpm for about 4 hours to remove the essential oil components of the leaves. Next, about 10 ml of enzyme solution (cellulase YC 2%, macerozyme R-10
(0.8%), mannitol 0.5M, pH 6.0), left still for 3 hours, and then rotationally shaken at 70 rpm for 30 minutes to isolate protoplasts from peppermint mesophyll.

For purification of the isolated protoplasts, first, undigested plant tissue was removed by filtration with Miracloth, and 150 ×
g, centrifugation for 2 minutes to precipitate protoplasts, remove the enzyme solution from the supernatant, and then wash with a 0.5 M mannitol solution (suspended in mannitol solution;
(After removing the supernatant after centrifugation at 50 xg for 2 minutes) for 3 times.

The purified protoplasts were added to 2 mg of NAA /
1.0 BA in the initial culture medium containing 0.8 mg / l BA
× 10 ⁵ cells / ml.
was transferred to a sterile plastic Petri dish having a diameter of 60 mm, and immediately mixed with 1.5 ml of a Gelrite solution.
By this operation, protoplasts were embedded at a density of 5.0 × 10 ⁴ cells / ml in the initial culture medium solidified with 0.1% gellite.

When protoplasts embedded in the initial culture medium were cultured at 25 ° C. under dark conditions, early division was observed on the sixth day after the start of the culture. Thereafter, the protoplasts were further divided, and grew to about 100 cells on about 30 days after the start of the culture.

The formed colony was excised together with the initial culture medium and placed in a 90 mm sterilized plastic petri dish for about 30 minutes.
The cells were transferred to a colony growth medium containing ml. After transplantation, 50
After shaking culture at about 70 rpm for about 20 days, colonies grew out of the solid medium while growing. The colonies thus removed from the initial culture medium were about 0.3 to
When the cells had grown to about 0.5 mm, they were suspended in a colony growing medium, spread on a callus forming medium with a pipette together with the colony growing medium, stirred well, and allowed to stand. When the colonies settled on the callus forming medium, the medium was gently tilted and the colony growing medium was removed with a pipette. By culturing on a callus forming medium at 25 ° C. for 16 hours under illumination (3000 lux), green calli having a diameter of 1 mm to 2 mm were formed after 2 weeks.

The formed green callus was placed on a regeneration-inducing medium and cultured under illumination at 25 ° C. and 3000 lux for 16 hours, so that adventitious buds and adventitious embryos were induced from about 8% of the placed callus. . When these were transplanted into Gambolg's B5 medium containing no plant hormones, they readily rooted and regenerated to complete plants. In this way, protoplast isolation of a Menta plant and regeneration of the plant body by culturing the protoplasts could be performed.

Continuation of front page (72) Inventor Hiroshi Sato 1-5-13 Numakage, Urawa-shi, Saitama (72) Inventor Kazuo Hosomi 7-6-1-401, Waseda, Misato-shi, Saitama (56) References JP-A-2-2 222626 (JP, A) JP-A-3-15327 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) A01H 4/00

Claims (5)

(57) [Claims] (1) In isolating a protoplast of a spice plant containing an essential oil component, the leaf epidermis of the highly hydrophobic plant is effectively destroyed without shearing the leaf to increase the hydrophilicity of the leaf; A method for isolating a protoplast of a spicy plant containing an essential oil component, wherein the protoplast isolation enzyme is effectively acted on by removing an essential oil component in the leaf from the leaf whose epidermis has been destroyed. 2. The spice plant is a Menta plant.
A method for isolating a protoplast of a spice plant according to the above. 3. When culturing protoplasts of the menta plant, 15-75 mg / l of (NH ₄ ) ₂ SO ₄ ,
H ₂ PO ₄ .H ₂ O 15 to 75 mg / l, KNO ₃ 400 to
1500 mg / l, sucrose 0.5% or more, auxin 5
The protoplasts of Menta genus plants are cultured in a Gambolg B5 medium containing not more than 0.05 mg / l and not more than 0.05 mg / l cytokinin to form colonies .
For culturing protoplasts of a Menta genus plant. 4. The protoplast culture of a Menta plant according to claim 3, further comprising shaking and culturing the grown colony in a liquid medium while being embedded in the solid medium, and efficiently removing the colony from the solid medium. Method. 5. When regenerating a plant of the Menta genus plant, the epidermis of a highly hydrophobic leaf of the plant is removed without shearing the leaf.
Leaf that effectively destroyed and increased the hydrophilicity of the leaf, destroyed the epidermis
Protoplus by removing essential oil components from leaves
Isolation of protoplasts by effective use of isolating enzyme
And the obtained protoplasts were subjected to NH ₄ ) ₂ SO _{4
15~75mg / l, NaH 2 PO 4} · H 2 O15~75
mg / l, KNO ₃ 400-1500 mg / l, sucrose
0.5% or more, auxin 5 mg / l or less, cytokai
Gamborg's B5 medium containing at least 0.05 mg / l of nin
Cultured in, to form colonies, culturing the colonies on solid medium kept a high osmotic pressure, to foster a high plant regeneration ability callus, auxin 0.01 mg / l or more,
Callus of the derived protoplasts on basal medium containing the following cytokinin 20 mg / l, characterized in that to regenerate into plants, plant regeneration method of mental Genus.