JPH11285324A - Culture of plant tissue - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for culturing a plant tissue by culturing the plant tissue in a culture medium containing vermiculite, etc., in a light- transmissible container closed with a specific porous membrane, capable of efficiently culturing the plant tissue in good workability and capable of producing healthy seedlings not requiring their acclimatization in good rooting and growth states. SOLUTION: This method for culturing a plant tissue comprises culturing the plant tissue in a culture medium 50 having a density of 0.01-2 g/cm<3> and comprising vermiculite having an average particle diameter of 0.1-10 mm and cellulose fibers having an average fiber length of 0.01-5 mm in a weight ratio of 50/50 to 95/5 in a light-transmissible container 10 whose opened end is closed with a porous membrane 40 having a gas permeability of 1-50 sec/100 cc. The culture medium 50 is preferably produced by mixing the cellulose fibers with the vermiculite in a dry state and subsequently subjecting the mixture to a compression molding treatment using a compression strength of 5-200 kg/cm<2> .

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for producing a plant tissue culture medium using a molded article of a mixture of vermiculite and cellulose fibers in a light-permeable container at least partially closed with a gas-permeable porous membrane, Acclimation from proliferation,
The present invention relates to a plant tissue culturing method capable of producing seedlings by continuous culturing until adult seedlings.

[0002]

2. Description of the Related Art In cell culture and growth point culture of higher plants, primary culture and subculture are carried out to form small plants and multiple shoots by a premature branching method, a protocomb-like body method, a shoot primordium method, and the like. Then, a seedling culture is performed. In this seedling culture stage, foliage is grown from the early branch, protocorm-like body, shoot primordia, etc., and then the adventitious root is differentiated. In this seedling culture, the medium is an important factor for plant growth and rooting.

In such plant culture, agar is most commonly used as a medium material. However, when agar is used, rooting is poor because air does not enter the culture medium,
Further, even if the rooting is improved by using a special rooting agar medium, the roots appear to be underwater roots and do not work effectively, and there is a problem that when transplanted directly to soil for cultivation, the aerial part dies. For this reason,
After acclimation in a medium such as perlite or vermiculite, transplantation to soil in an open or greenhouse is performed, but the acclimation process usually requires a long period of 1 to 3 months. During the cultivation period, the rooting of the cultured seedlings is poor and the cultured seedlings die or the seedling quality is not in a normal state with water immersion, and there are many problems in terms of labor and yield.

On the other hand, rock wool, perlite or vermiculite may be used as a medium instead of agar. However, in the case of rock wool, rock wool itself remains without decomposition when transplanted to cultivation soil,
Attempts to separate from the roots will damage the roots and cause a decrease in the survival rate, and furthermore, they will be too hard as a medium support and the roots will not elongate, leaving a problem.

[0005] Further, in the case of perlite or vermiculite, the fixation of the plant is poor due to the granular nature, the adhesion to the cultured plant is poor, and the growth of the perlite or vermiculite is uneven. Bad and practically difficult. On the other hand, as a container for culturing them, glass,
Containers made of polycarbonate or the like are generally used.However, in these containers, the plant is prolonged and the roots grow poorly. It was impossible to make a seedling. Therefore, two separate stages of culture, the growth rooting stage and the acclimatization stage, were required.

[0006]

As described above, in the prior art, when trying to obtain an adult seedling by plant tissue culture, the rooting from the culture is poor, the initial growth is poor, and the yield is low due to the low survival rate. In addition, there have been problems in this respect, and up to now, two stages of cultivation have been required, namely, the growth and rooting stage and the acclimatization stage, which are troublesome and labor-intensive. The present invention has been made in view of such a problem. That is, an object of the present invention is to provide a culture method for efficiently acclimating tissue culture plants from growth to obtaining adult seedlings.

[0007]

A feature of the present invention is that a molded body of a mixture of vermiculite and cellulose fibers is planted in a light-permeable container at least partially closed with a gas-permeable porous membrane. There is a culture method used as a culture medium. More preferably, there is a method of culturing in a container using a porous membrane having a gas permeability of the porous membrane of 1 to 50 sec / 100 cc.

As a result of intensive studies, the authors have found that the use of the above-mentioned medium and gas permeable container allows good operability and efficient cultivation, as well as good rooting and growth and no need for acclimatization in a single culture. They found that seedlings could be produced, and completed the invention. Hereinafter, the present invention will be described in detail.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The culture vessel of the present invention is a light-permeable vessel at least partially closed with a gas-permeable porous membrane. The shape of the container body is not particularly limited, and is generally a shape including a container body and a lid. The material is not particularly limited, and examples thereof include glass, polystyrene, polyester, polyvinyl chloride, and polycarbonate. Preferably, polycarbonate is excellent in durability and heat resistance. There is no particular limitation on the size, but a size that is easy to hold in a hand is preferable. A vent is provided in a part of the container main body or the lid, and a gas permeable porous membrane is used in close contact therewith. There is no particular limitation on the method of adhesion, and there are a method of attaching and attaching an adhesive to the porous film, a method of holding a cap from the top of the porous film, and the like. Is simple and preferable. Further, by changing the size of the vent portion of the cap, the gas permeation amount can be adjusted, which is more preferable. The area of the vent is not particularly limited, but is preferably 0.2 to ⁴ cm ² for use.

The porous membrane has a gas permeability (air permeability) of 1 to 50.
sec / 100 cc, preferably 1 to 10 sec / 10
0 cc. More preferably, 1 to 5 sec / 100c
c. If the air permeability is too low, gas permeation will be insufficient and the growth and rooting will be poor, and the effect will be poor. If the air permeability is high, water vapor will be greatly dissipated and drying will not be possible. Further, it is conceivable to increase the area with a porous membrane having a low air permeability in order to increase the amount of aeration. However, since the amount of water vapor dissipated increases in proportion to the area, drying becomes intense and cultivation becomes impossible, and 1 to 5 sec.
A c / 100 cc porous membrane is practically suitable.

The average pore diameter is preferably from 1 to 10 μm, more preferably from 0.1 to 5 μm. If the average pore diameter exceeds 10 μm, contamination of various bacteria and the like is feared, which is not preferred. The thickness is preferably in the range of 50 to 500 μm in terms of handling. Any material can be used as the material of this porous membrane as long as it can withstand sterilization conditions, for example, polystyrene, polyester, polyamide, polypropylene, nylon,
Cellulose, fluorine resin, poly 4-methylpentene 1, and the like. Further, a composite of these is also possible.

The following media are used in combination in these containers. The medium of the present invention to be used is obtained by molding a mixture of vermiculite and cellulose fibers. There is no particular limitation on the type of vermiculite to be used as the material.
10 to 10 mm, more preferably 0.5 to 5 mm is used.

The vermiculite which has been subjected to various treatments in advance may be used. Each treatment here means a heating treatment, a cooling treatment, a purification treatment, a swelling treatment,
It means chemical mechanical treatment such as pulverization, granulation, impregnation, and coating. For cellulose,
Those having an average fiber length of 0.01 to 5 mm are preferred. There is no particular limitation on the type of cellulose, cotton lint, cotton linter, softwood cellulose, hardwood cellulose,
Bast cellulose, hemp cellulose, regenerated cellulose, bacterial cellulose, and the like, and mixtures thereof are used.

In addition, these celluloses which have been treated in advance may be used. Each treatment here means a heating treatment, a cooling treatment, a purification treatment, an amorphization treatment, a swelling treatment, a polymerization degree reduction treatment, a derivatization treatment, a crosslinking treatment, a crystal type conversion treatment, a dissolution regeneration treatment, a pulverization treatment, It means chemical and mechanical treatment such as granulation treatment, impregnation treatment and coating treatment.

As a method of molding these mixtures,
Mix vermiculite and cellulose fiber in a liquid,
There are two types: a wet molding method in which molding and dewatering are followed by drying, and a dry compression molding method in which the mixture is compression molded in a dry state. The mixing ratio of vermiculite and cellulose fiber depends on the molding method, but is 50:50 to 95: 5 by weight, especially 6
5:35 to 90:10 is preferred.

In the case of wet molding, the average fiber length of cellulose is 0.1 mm to 4 mm, preferably 0.5 mm to 3 mm.
mm. The average particle size of vermiculite is preferably 0.5 mm to 5 mm.
If the cellulose fiber length is too short, when dried, the molded body becomes hard and the plant becomes difficult to insert, which is not preferable as a culture medium. On the other hand, if the cellulose fiber length is too long, the vermiculite and cellulose fibers tend to be unable to be uniformly mixed and molded first, which is not preferable as a culture medium.

If the particle size of vermiculite is too small,
Since the effect of holding the culture solution is reduced, the molded body becomes hard, and it becomes difficult to insert a plant, so that a favorable result cannot be obtained. If the particle size of vermiculite is too large, it is difficult to insert the plant body into an arbitrary position in the present medium, and the operability deteriorates, which is not preferable. If the cellulose fiber content is 5% by weight or less, the entanglement between the vermiculite and the cellulose fiber is poor, which is not preferable for molding.

In the case of a wet molded product, the vermiculite and cellulose are mixed in a liquid, preferably in water, the suspension is placed in a mold or the like, the liquid is removed, and the molded product is dried. can get. The liquid may be removed by any method, for example, a gravity drop method, a centrifugal dehydration method, or a suction dehydration method.

The drying method is not particularly limited. For example, a method using a dryer, solar drying, or the like is used. The liquid is not particularly limited, such as water, alcohol, and acetone, but water that does not affect plants is preferable. By adding a liquid such as a culture solution to the wet molded body, the liquid is retained in the cellulose fiber, vermiculite, and the structural part formed by these, the molded body becomes soft, and a medium suitable for inserting a plant. Become.

In the case of dry compression molding, the average fiber length of cellulose is 20 μm to 500 μm, preferably 100 μm.
μm to 400 μm are used. The average particle size of vermiculite is preferably 0.5 mm to 5 mm. If the cellulose fiber length is too short, the molded body becomes hard when a culture solution is added, and it becomes difficult to insert a plant, which is not preferable as a culture medium. On the other hand, if the cellulose fiber length is too long, it is difficult to obtain a uniform mixture of the vermiculite and the cellulose fibers, and it is difficult to mold the mixture.

If the particle size of vermiculite is too small,
Since the effect of holding the culture solution is reduced, the molded body becomes hard, and it becomes difficult to insert a plant, so that a favorable result cannot be obtained. If the particle size of vermiculite is too large, it is difficult to insert the plant body into an arbitrary position in the present medium, and the operability deteriorates, which is not preferable. 5kg / c for compressive strength
m ^{2 to} 200 kg / cm ² is usually used.

If the compressive strength is too low, the strength of the molded body will be low, and if the compressive strength is too high, the molded body will be hard and the plant will be difficult to insert, which is not preferable as a medium. The temperature at the time of compression is not particularly limited, but is usually 10 ° C to 200 ° C.
° C is used. Since the density of the molded body tends to increase as the temperature during compression increases, the temperature during compression may be set so as to obtain a desired density.

The compression time is also not particularly limited, but if the compression is usually performed for 10 seconds to 10 minutes, a molded body having strength suitable for use and easy insertion of plants can be obtained. By adding a liquid such as a culture solution to the compression molded body, the laminated structure portion of vermiculite is spread, the cellulose fiber, vermiculite, and the liquid is held in the structure portion formed by these, the molded body swells, becomes soft, This is a medium suitable for plant insertion.

The density of these molded products is 0.01 to
2g / cm^ThreeAnd in the case of wet molding, 0.
05g / cm^Three~ 1g / cm^Three, Preferably 0.07 g
/ C ^Three~ 0.3g / cm^ThreeIn the case of dry compression molding
0.5g / cm^Three~ 2g / cm ^Three, Preferably 0.5g
/ Cm^Three~ 1.5g / cm^ThreeMold so that it becomes. This
If the density is higher than this, the molded body becomes harder and the plant
It is difficult to insert and rooting is poor, making it unsuitable as a medium.
If the density is lower than this, shape retention cannot be maintained and operability
Not preferred in terms of surface.

Further, other components may be mixed to such an extent that the effect of the medium by mixing and molding the vermiculite and the cellulose fiber of the present invention is not impaired. The other components referred to here include organic substances, organic salts, organic acids, organic alkalis, organic polymers, inorganic substances, inorganic salts, inorganic acids, inorganic alkalis, inorganic polymers, and the like, among which rock wool, perlite, peat moss, wood Flour, sawdust, humus, chitin, chitosan and the like.

A culture solution is put in these media, sterilized by an autoclave or the like, and then a culture is implanted and cultured.
Here, a culture is a small plant formed by performing primary culture and subculture in cell culture or growth point culture of higher plants, and by the early branching method, protocomb-like body method, shoot primordium method, etc. And polyblasts. The amount of the culture solution to be used varies depending on the type of plant to be applied and is difficult to determine unambiguously.
5 ml is appropriate. If the amount of the culture solution is too large, the air phase becomes small and becomes excessively moist, the rooting is poor, and the plant becomes water-soaked, which is unsuitable for growth. When the amount of the culture solution is too small, nutrients are not sufficiently absorbed by the plant, resulting in poor growth.

The culture solution is not particularly limited and may be a normal M
S culture, White culture, Heller culture, Vicin &
A vent culture solution or the like is used. Further, the concentration of the culture solution may be reduced according to the type of plant, or certain components such as phosphoric acid and iron may be added, or those containing various plant hormones may be used. When the medium (support) of the present invention is used, the growth is generally better when the culture medium concentration is lower than that used in agar. This is because the liquid medium is used more efficiently than the agar, and the optimal concentration may be lower.

In molding, in order to save the trouble of adding the culture solution, the components of the culture solution may be added to the medium in advance and then molded. This medium (support) in a gas permeable container
By using, the gas conditions in the container are improved, the rooting from the cultured plant body is extremely improved, and the growth is also improved as compared with the case of using in a closed container.

Further, since the water vapor in the container permeates moderately, over-humidification is prevented, the acclimatization proceeds smoothly, and it is possible to produce seedlings that can be directly transplanted to cultivation soil. As a result, there is provided a revolutionary method capable of improving the yield and reducing the labor required for multiplication and acclimation once. Furthermore, there is no environmental pollution even after use, and it is superior to rock wool.

[0030]

The present invention will be described in detail with reference to the following examples, but the present invention is not limited to the following examples unless it exceeds the gist. The air permeability of the porous membrane was measured using a B-type Gurley-type densometer (Toyo Seiki Seisakusho, J
(According to IS standard P8117).

[Production Example 1] (Production of culture medium) 1 part by weight of wood pulp LBKP (average fiber length: 1.2 mm) is fibrillated with 50 parts by weight of water, and calcined vermiculite (average particle size: 2 mm) produced in China 5 Parts by weight were added and mixed. The mixed suspension was transferred onto a 355 μm sieve, and was molded so as to be uniform in height while being naturally dehydrated, to obtain a cake-like molded body. The molded body was sufficiently dehydrated, dried at 90 ° C., and cut into a size of 5 cm × 5 cm × 2 cm. The density of this molded product after drying is 0.
It was 13 g / cm ³ .

[Production Example 2] (Production of culture medium) 9 parts by weight of Chinese calcined vermiculite (average particle size 2 mm) and 1 part by weight of powdered cellulose fiber (average fiber length 300 μm) were mixed in a dry state. The mixture was placed in a mold so as to have a pressure of 0.7 g / cm ² , and press-molded under conditions of a compressive strength of 50 kg / cm ² and room temperature for 1 minute. This molded product was cut into a size of 5 cm × 5 cm. The density of this molded product was 1 g / cm ³ .

[Production Example 3] (Production of culture vessel) As shown in FIGS. 1 to 5, the vessel body was made of polycarbonate resin, and the bottom was 70 mm × 70 m.
m, an opening having a size of 90 mm × 90 mm, a height of 120 mm and a thickness of 1.5 mm was molded by an injection molding method.

The lid had a vent of 10φ (diameter: 10 mm), and was molded by an injection molding method so that it could be in close contact with the container body. The cap that holds the porous membrane is made of polypropylene resin, and is molded by an injection molding method so that the porous membrane can be held in the air hole of the lid without any gaps and the upper part of the cap is provided with an air hole so that it can adhere to the air hole of the lid. did. A porous membrane was set on this and used as a culture vessel.

Example 1 Porous membrane E01008E (manufactured by Pall Corporation, thickness: 200 μm, average pore diameter: 0.3 μm)
m, air permeability: 2 sec / 100 cc, material composition: glass fiber mesh / cellulose filter) with a diameter of 15 mm
Was set from the inside of the cap to be punched out into a circular shape to obtain a culture vessel.

Example 2 Porous membrane Hydrolon (manufactured by Nippon Pall Co., Ltd., thickness: 160 μm, average pore diameter: 1.2 μm)
m, air permeability: 7 sec / 100 cc, material composition: nylon) were punched out into a circle having a diameter of 15 mm and set from the inside of the cap to obtain a culture vessel.

Example 3 Porous membrane Biodyne A (manufactured by Pall Corporation, thickness: 150 μm, average pore diameter: 0.5 μm)
m, air permeability: 30 sec / 100c, material composition: nylon) were punched out into a circle having a diameter of 15 mm and set from the inside of the cap to obtain a culture vessel.

Comparative Example 1 Instead of a porous membrane, a nonporous polypropylene film (50 μm) was punched out into a circle having a diameter of 15 mm and set from the inside of a cap to obtain a culture vessel. Comparative Example 2 A porous membrane HDCIIj006 (manufactured by Pall Corporation, thickness: 200 μm, average pore diameter: 0.6 μm, air permeability: 70 sec / 100 cc, material composition: glass fiber mesh / cellulose filter) was punched out into a 15 mm diameter circular cap. Was set from the inside to obtain a culture vessel.

[Test Example 1] 8 g of the culture medium of Production Example 1 was placed in a culture vessel in which the porous membrane of Example 1 was set in the vessel of Production Example 3, and MS containing ammonium phosphate and iron was added thereto.
Culture solution (MS basic culture solution + NH ₄ H ₂ PO ₄ = 400 m
(g / l + FeEDTA = 33 mg / l, 9% saccharose), and 45 ml of the mixture was placed therein and sealed in an autoclave to obtain a culture medium.

The same procedure as in Examples 2 to 3 and Comparative Examples 1 and 2 were carried out except for the type of the porous membrane. In this culture medium,
One node (approximately 1 cm) of a sterile cultured sweet potato seedling was inserted and cultured at 25 ° C. for one month. Planting easiness of seedlings and growth investigation (plant height, number of leaves, rooting state) after one month of culture were performed. After that, the plants were transplanted into pots filled with vermiculite and the growth status was observed for 2 weeks.

As a comparative example, an example using agar (0.8%) instead of the product of the present invention is also shown. The results are shown in Table 1. In addition, rooting is shown in four stages, with +++ as large rooting (very good), ++ as normal rooting (good), + as small rooting (bad), ± as rooting minimum (worse). However, when the test plants are different, it is a relative evaluation between the same plants.

[0042]

[Table 1]

The medium of Production Example 1 was easy to insert and had good operability, and the medium and the seedlings cultured in the culture vessels using the porous membranes of Examples 1 to 3 showed a good rooting state and a firm seedling. And the growth after transplantation was good and excellent. [Test Example 2] 14 g of the culture medium of Production Example 2 was placed in a culture vessel in which the porous membrane of Example 1 was set in the container of Production Example 3, and an MS culture solution (MS basic culture method, 3% saccharose) 4 was added thereto.
5 ml of the mixture was charged and sealed in an autoclave to obtain a culture medium.

Examples 2 and 3 and Comparative Examples 1 and 2 were carried out in exactly the same manner except for the type of porous membrane. To this, the tip node (about 3 cm) of the carnation aseptic culture seedling was inserted and cultured at 20 ° C. for one month. One month after the cultivation, a growth survey (plant height, leaf number, living weight, seedling quality, rooting state) was performed. And then
Transfer to a pot filled with vermiculite and grow 2
Observed for a week. Agar (0.8%) and rock wool were used for comparison. The results are shown in Table 2.

[0045]

[Table 2]

The medium of Production Example 2 was easy to insert and had good operability. Furthermore, the medium and the seedlings cultured in the culture vessels using the porous membranes of Examples 1 to 3 were solid seedlings having a good rooting state. And the growth after transplantation was good and excellent. [Test Example 3] 8 g of the culture medium of Production Example 1 was placed in a culture vessel in which the porous membrane of Example 1 was set in the container of Production Example 3, and saccharose was removed from the MS culture solution (MS basic culture solution, 0%).
% Saccharose) and sealed and autoclaved to obtain a culture medium.

Examples 2 and 3 and Comparative Examples 1 and 2 were carried out in exactly the same manner except for the type of porous membrane. To this, the tip node (about 3 cm) of the carnation aseptic culture seedling was inserted and cultured at 20 ° C. for one month. One month after the cultivation, a growth survey (plant height, leaf number, living weight, seedling quality, rooting state) was performed. And then
Transfer to a pot filled with vermiculite and grow 2
Observed for a week. Agar (0.8%) and rock wool were used for comparison. The results are shown in Table 3.

[0048]

[Table 3]

The medium of Production Example 2 was easy to insert and had good operability, and the rooting state was good and firm even after sugar-free culture in a culture vessel using this medium and the porous membranes of Examples 1 to 3. Seedlings were formed and the growth after transplantation was good and excellent.

[0050]

As is clear from the comparison of the test examples, when the material of the present invention is used, the growth and rooting are better than when the conventional agar or rock wool is used, and good rooting is achieved during acclimation. This is extremely advantageous because the yield and the yield are improved. Furthermore,
What used to require two stages of culture, the growth rooting stage and the acclimatization stage, is now only required once, which is extremely advantageous in terms of labor. In addition, it can be grown without sugar, and is an extremely innovative technique. Further, there is no environmental pollution, which is extremely advantageous as compared with rock wool.

[Brief description of the drawings]

FIG. 1 is an assembled vertical sectional view of the present invention.

FIG. 2 is a perspective view of the container body of the present invention.

FIG. 3 is a perspective view of the lid of the present invention.

FIG. 4 is a perspective view of the cap of the present invention.

FIG. 5 is an enlarged cross-sectional view of the lid opening of the present invention.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 10 Container main body 11 Container main body upper end opening part 12 Container main body flange part 13 Container main body side taper part 14 Container main body bottom plate 20 Cover 21 Lid flange part 22 Vent port 23 Vent end face 24 Vent port flange part 30 Cap 31 Vent port 32 Vent 33 Flange 40 Porous membrane 50 Medium 60 Culture

Claims (5)

[Claims] 1. The open end has an air permeability of 1 to 50 sec / 10.
A plant tissue culture method using a medium in a light-permeable container closed with a porous membrane having gas permeability of 0 cc,
A plant tissue culture method comprising using a mixture containing vermiculite and cellulose fibers as a medium. 2. The medium has an average particle size of 0.1 mm to 10 mm.
Of vermiculite and average fiber length of 0.01 to 5 mm
The culture method according to claim 1, wherein the cellulose fiber is contained in a weight ratio of 50:50 to 95: 5. 3. The medium has a density of 0.01 g / cm ^{3 to} 2 g.
/ Cm ³ . 4. The culture method according to claim 1, wherein the culture medium is a mixture of cellulose fibers and vermiculite dispersed and mixed in a liquid, molded and dried. 5. A culture medium comprising a cellulose fiber and vermiculite mixed in a dry state, wherein the medium is 5 kg / cm ² to 200 kg / cm ^2.
The method of culturing according to claim 1, characterized in that is made by compressing and molding the compression strength of kg / cm ^2.