JPS62175170A - Medium for tissue culture of plant - Google Patents

Abstract

PURPOSE:To promote the taking of a plant in acclimatization and the growth of a young plant, by using ceramic fiber as a medium material to be used in combination with a liquid medium. CONSTITUTION:Ceramic fiber essentially free from dissolution of a component inhibiting tissue culture of a plant is used as a medium material for plant tissue culture to be used in combination with a liquid medium. The ceramic fiber is preferably essentially inert to the liquid medium and preferably hydrophilic. It is preferably silica alumina fiber. Preferably, ceramic fibers are collected at a bulk density of 0.005-0.3g/cm<3> and used as the medium material.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention relates to a medium material for plant tissue culture used in combination with a liquid medium, and in particular to a medium material made of ceramic fiber, The present invention relates to a medium material for plant tissue culture that can also be used as a conditioned medium for plants.

[Conventional technology] Agar has traditionally been the most commonly used medium for culturing plant tissues, and special mediums such as glass fiber and absorbent cotton have been used in combination with a liquid medium. It is also known that

However, agar contains unidentified substances that inhibit the proliferation, differentiation, and/or root elongation of plant tissue cells. In addition to delaying proliferation, differentiation, and/or rooting, since the entire medium is gelled, the diffusion rate of waste substances and harmful substances produced in the cultured tissue is slow, and depending on the type of plant, their toxic effects may occur. In some cases, culturing became completely difficult.

Moreover, the seedlings obtained by tissue culture in a gluing medium such as agar can be differentiated from the cultured tissue and live.

The root is a tap root with a small number of plates, so it is a so-called underwater root, and if it is transplanted directly to a conditioned medium and acclimatized, the survival rate is extremely poor. Furthermore, when the seedlings are acclimatized, if a medium is attached to their roots, the plants will rot and die due to contamination with mold and the like. To prevent this, removing the medium from the seedlings more completely will cause more damage to the plants and hinder their growth after acclimatization.

This results in a delay. For this purpose, with conventional culture methods that use agar, etc., there is a problem in that it is necessary to prepare and use conditioned media such as Vermiculai 1 to Vermiculai 1 that have been specially sterilized and disinfected during acclimation. .

In addition, when glass fiber is used as a medium material, alkaline content is eluted during use as a medium material, and the p +
There is a problem in that the value and chemical composition of - fluctuate. Sara 4
When using JIBJ cotton, worse results were obtained than with agar medium.

[Problems to be Solved by the Invention] The purpose of the present invention is to provide a medium + A for plant tissue culture that is used in combination with Δ1 and liquid 18 medium. Another purpose is to provide a culture medium that can promote the proliferation, differentiation, and rooting of plant tissues, and also improve the survival rate during tree formation and the growth of young plants after acclimatization. 1, and another object of the present invention is to
The purpose of the present invention is to provide a medium material that enables tissue culture of plants that requires rotation in gelling media such as agar.

In addition, another object of the present invention is to enable continuous use from tissue culture to transplantation after acclimatization, to save labor in acclimatization work, to improve survival rate during acclimatization, and to improve production rate after acclimatization. The purpose of the present invention is to provide a culture medium that can achieve the following objectives.

Lower part for solving and overcoming problems] That is, the present invention is used in combination with a liquid culture medium.
This is a plant tissue culture medium made of ceramic fiber that does not substantially elute components that have a harmful effect on plant tissue culture.

In terms of its chemical composition, the Reramic Noivar used in the present invention has components eluted during tissue culture operations, such as sterilization treatment performed by impregnating it with a liquid medium. It is acceptable as long as it does not have an effect on the roots, does not substantially react with the liquid medium used in combination with this medium material, and also has a
It is important not to affect the In particular, the elution of aluminum ions causes the problem of reacting with phosphate ions in the liquid medium to produce water-insoluble vA aluminum acetates that cannot be used by plants, resulting in a lack of phosphoric acid.
Elution of alkali ions changes the pH value of the liquid medium used, causing problems in the growth, differentiation, and rooting of plant tissues.

Examples of ceramic fibers used in the present invention include silica-alumina fibers (S!02
Au203>, silica alumina = 5- Zillonia fiber (S! 02-Af2203-
7rO2), silica/alumina/chromia fiber (
Silica/alumina fibers whose main components are silica and alumina such as Si 02-AlI303 Cr 203), silica fibers whose main component is silica, alumina fibers whose main component is alumina,
Examples include carbon fiber, etc.
Silica-alumina fibers are particularly preferable in consideration of components eluted from the fiber, reactivity with the liquid medium, stability, price, etc. This silica/alumina fiber is industrially mass-produced as a fireproofing material for furnaces, a heat insulating material, and a raw material for inorganic fiber paper, but in the manufacturing process, improvements in lJ1 performance and transportability are required. Although lubricants are often added for this purpose, it is preferable that the material be heat-treated to a temperature above the removal temperature of these additives.

The bulk density of the ceramic fiber used in the present invention varies depending on the purpose of use, but is usually 0.005 to 0.3 g/
cm3, preferably about 0.01 to 0°2'; J/cm3. If this bulk density is smaller than 0.005-6 = it will be difficult to handle;
'; If it is larger than l/cm3, the growth of resentment will be hindered.

The Reramic fiber culture medium of the present invention is itself a hydrophilic material, but in order to make it more hydrophilic, nonionic surfactants and higher alcohols are used that do not hinder the growth of plants. Substances may also be added.

The shape of the ceramic fiber culture medium vJ of the present invention (hereinafter referred to as the present culture medium material) may be an unprocessed product (so-called bulk), but the fibers may be arranged in a certain direction and needled if necessary. Preferably, the material is a molded plankera 1 or granulated cotton. In addition, when the main focus is on proliferation, differentiation, and rooting within the culture container, it can be used in a shape suitable for the culture container, such as paper, sheet, blanket, or granular shapes. There are no particular restrictions on the arrangement of the fibers, such as vertically, horizontally, or even randomly. In consideration of handling, root elongation, etc., it is preferable to arrange them vertically.

This medium 4・A was produced by Murashige-Skoog, White, Knudson, Linsmeyer-Skoog, Henhe, j-l~1
It is used in combination with liquid media such as No, Nirache/Nirache, Erickson, Gunborg/Miller/Ojima, Itamoto/Kano, and their modified media. These liquid media are
It is preferable to use it by impregnating it into the medium, but it can also be supplied by methods such as dripping or spraying.

As a method for culturing plant tissue using the main medium side, for example, place an appropriate amount of main medium material in a culture container, impregnate it with the most necessary liquid medium, and then This can be carried out by performing a sterilization operation using, for example, natural cooling, and then placing a pre-sterilized section of the plant tissue on the main culture medium in a culture container. In addition, the management method during cultivation -C is the same as when using agar, but
When using this medium 4, it is possible to add a liquid medium, add a plant hormone, and wash and replace the liquid medium without transplanting the cultured tissue when operating under the sterile tray 41. .

Moreover, this culture medium can be used as a conditioned medium for acclimatizing young plants in combination with a commercially available liquid fertilizer if necessary. When used in combination with a liquid fertilizer for this purpose, for example, the liquid fertilizer adjusted to the required concentration is supplied to the main medium material by dripping, spraying, or a method that also serves as irrigation. To acclimatize seedlings using the conditioned medium prepared in this way, for example, the medium used for tissue culture and rooting medium are removed from the seedlings obtained by tissue culture, and the seedlings are washed with water. After that, the seedlings are planted by wrapping the roots of the seedlings in an appropriate amount of conditioned medium placed in the cultivation container, and then placed in the greenhouse after being properly shaded and moisturized.
Acclimatize for about a week to a month. Regarding the management method during acclimatization,
This is the same as the conventional case of using ~, etc., but since this culture medium has high water retention properties, there is usually no need to saturate it with water, and
For young plants that have completed acclimatization, without removing the medium,
It can be used for normal soil cultivation or solution cultivation.

By using this culture medium for acclimation, it is possible to always provide adequate water retention and aeration to the roots of young plants, and because it has a very high porosity, It is thought that even if the roots are exposed to the soil, a large amount of gas phase still remains in the medium, which prevents oxygen deficiency in the roots.

As mentioned above, this medium material can be used as a medium for growth, differentiation, and rooting in tissue culture. Since it can also be used as a material, the liquid medium used in each process from plant propagation, differentiation, and rooting to the acclimatization of the resulting seedlings is washed with water, removed, and the next liquid medium is added before transplanting. You can do it as a member without any operations. In this way, by consistently performing the steps from plant propagation, differentiation, rooting to acclimatization without transplanting, it is possible to significantly reduce damage to seedlings, and improve the yield when producing seedlings using the tissue culture method. It is also possible to improve the growth rate of plants after acclimatization.

Furthermore, the plants that have been acclimatized can be used for normal soil cultivation, hydroponics, sand #1, gravel 1, rock wool cultivation, etc. without removing this medium material. Similar to the use of inorganic fibers, it is suitable for covering rock wool cultivation.

[Effect 1] In the present invention, since ceramic fibers are used as the medium material, the chemical stability and physical properties of the ceramic fibers make it possible to fully demonstrate the performance of the liquid medium used in combination V- as a whole. can. Furthermore, since this medium material has a very high porosity, a large amount of gas phase remains even after it is combined with a liquid medium, which promotes the proliferation, differentiation, and rooting of plant tissues or cells, and also promotes the growth of cultured tissues. If it promotes growth at the time of N11 conversion, it will be 1.

In addition, in this culture medium material, the combined liquid medium exists between the ceramic fiber fibers as capillary water, water that lands on the ceramic fibers, making it easy to absorb the liquid medium by the cultured tissue and remove the liquid medium during N11 conversion. It is estimated that

[Examples] Hereinafter, the present invention will be specifically explained based on Examples.
When Cf2150ml was added and the reaction was allowed to proceed at 30'C for 1 hour, the peak of the helium peak was 2290D, which was 8 times the amount of pl.
-I 7 Silica-aluminum horn iver with PL-1 of 6.5 after being immersed in sealed water at room temperature for 1 hour (Sin2: approx. 53,000%, Hep203: approx. 47%) ), and after adding an organic lubricant to layered cotton in which the fibers are arranged in a certain direction, needling is performed, and the resulting molded product is further heat-treated at approximately 700°C for approximately 20 minutes to remove the additives. After decomposition and removal, the blanket-like silica/alumina fiber medium material of Example 1 (bulk density: 0.1
39/cm3) was produced.

2 per side from this silica-alumino-7 fiber medium material
Cut out a square cube of cm and its 1. ○U2゜5cm
Place the fibers in one direction vertically at the bottom of a φX12 cm tube bottle, and add Murashige-Skoog medium (naphthalene acetic acid 0.1 mg/1) as a liquid medium in the tube bottle.
2. Dispense >8d containing 0.1 my/p of benzyl azonin and 309/u of 1-ton carose, cap with an aluminum container, sterilize with O-1 to crepe at 120'C for 15 minutes, and then Cooled.

Next, in the above-mentioned tube bottle in a clean bench, cut sections of the stem base of Cyperus pulchella (cultivar name: Nanasu), which had been weighed down in advance (using antiformin) and cut into approximately 2 mm squares, were placed at a temperature of 25°C. 12 hours of light period, 112 hours of light period and 3. The seeds were cultured for 3 weeks under the culture conditions of ○OO Lux, and the survival rate, shoe length from 1 to 1, maximum total shoot growth, shoot from 1 to length, and rooting rate were examined. The results are shown in Table 1.

Example 2 The amount of l eluted with respect to 1 g of sample diluted hydrochloric acid measured in the same manner as in Example 1 was 77.7 ppm, and 8 times the amount of D
Silica-alumina-zirconia fiber (Si02
: Approximately 50% by weight, △J2203: Approximately 35% by weight, Zr'
02: about 15% by weight) and the same procedure as in Example 1 was used to prepare a wrinkle strength, alumina, and zirconia substrate made of ivar medium (bulk density: 0.13 g/cm3).
was manufactured.

Using this silica-alumina-silica near fiber medium, the same plant tissues were cultured under the same conditions as in Example 1, and the same items as in Example 1 were investigated. The results are shown in Table 1.

Example 3 Silica-alumina-chromia fiber (S i 02: about 55% by weight, ρ203: A blanket-shaped silica/aluminum J/chromia fiber culture medium (bulk density: 0.13 g/cm3) was produced in the same manner as in Example 1 using 42% by weight, Cr2O3: about 3% by weight). did.

Using this silica-alumina-chromia fiber medium material, the same plant tissues were cultured under the same conditions as in Example 1,
The same items as in Example 1 were investigated. The results are shown in Table 1.

Comparative Example 1 An agar medium was prepared by adding 8 g/f2 of commercially available J8 purified agar to the same Murashige-Skoog medium as used in Examples 1 to 3-C, and 8 mρ of this agar medium was added to each of the above. The same plant tissue was cultured under the same conditions as Example 1 by dispensing it into the same tube bottle as used in the example (using 0.06 = '1 g of agar per tube bottle as a culture pond), The same items as in Example 1 were investigated.

The results are shown in Table 1.

Comparative Example 2 Rock wool culture medium for patterned cultivation commercially available as a culture medium 'r
jJ (S i 02: I'J 46 mm, %
, /1203: about 13% by weight, CaO: about 18% by weight,
Using granular cotton (hydrophilic type) containing MgO: about 12% by weight, Fe2O3: about 8% by weight, the same plant tissues were cultured under the same regulations as in Example 1, and the same items as in Example 1 were investigated. . The results are shown in Table 1.

Comparative Example 3 Commercially available glass fiber (SiO:l/1U59
Heaviness%, Aρ2o3: approx. 4%, Cab: approx. 16if
f%, MgO: about 5%, B2O2: about 3%, N
a2o: about 11% by weight), the same plant tissues were cultured under the same conditions as in Example 1, and the same items as in Example 1 were investigated.

The results are shown in Table 1.

a-3, each value showing the results in Table 1 was measured in Comparative Example 1, and the average value of the measured values was expressed as 100 (however,
Rooting rate only (j, shown as actual measured value).

As is clear from the results in Table 1, the results of Examples 1 to 3 using three types of ceramic fiber 8 substrates have a higher survival rate than those of Comparative Examples 1 to 3. ,
The growth and rooting rate of Shoe 1~ are significantly promoted.

Table 1 Example 4 Using the same silica/alumina fiber as Example 1,
Processed in the same manner as in Example 1 to obtain a bulk density of 0. A silica/alumina fiber medium material having a weight of 1 g/cm3 and having the shape of Planchera 1 was produced.

2cm per side from this silica/alumina fiber medium material
A 0.1 m cube was cut out, and the 0.1 cube was placed at the bottom of a 2°-17=5 cmφ x 12 cm tube bottle with the fiber direction perpendicular, and the same Murashige-Skoog medium as in Example 1 was used. , Cyperus in the same manner as in Example 1.
A section of the stem base of 1 Ruchera (cultivar name: Nanas) cut into approximately 2 mm squares was placed on the bed, and cultured in 3 cavities under the same conditions as in Example 1. The long and σ rooting rates were investigated. The results are shown in Table 2. In addition, each value of the result of this Example 4 was also expressed as 100 by culturing as a control according to Comparative Example 1, as in the case of Examples 1 to 3 above. .

As is clear from the results shown in Table 2, it was found that the use of the silica/alumina fiber culture medium significantly promoted shoot growth.

Next, in this Example 4, 1 q of seedlings of Cyperus were grown,
The silica/alumina fiber culture medium was taken out from the tube bottle and washed under running water.The seedlings were placed together with the culture medium in a plastic container and acclimatized for 10 days in a greenhouse with 75% light shielding. The survival rate during acclimatization was investigated.

In addition, in a plastic container of the same type as that used in Example 4, a medium for acclimatization was prepared by mixing 1 to 2 parts by weight of Pea, 1 part by weight of Barley 1, and 0.5 part by weight of vermiculite to a thickness of 2 cm. 1 in the control of Example 4.
The raised Cyperus seedlings (obtained by removing the agar attached to the roots from the tube bottle and washing with water) were transplanted and acclimatized under the same conditions. Check the survival rate at that time.

When the survival rate in the case of the above control was set as 100, the survival rate in the case of Example 4 was 143, and it was found that the survival rate during acclimatization was improved by using the silica/alumina fiber culture medium.

Example 5 Using the same silica/alumina fiber culture medium as in Example 4, a section of the runner tip of Nephrolepis marshallii cut into 5# in the same manner as in Example 4 was placed on the bed. J for 8 weeks under the same conditions? The survival rate, number of sheets, length of shoes, and rooting rate were investigated, and the results are shown in Table 3.

Example 6 The same silica-alumino-silica fiber as in Example 2 was used as the ceramic fiber, and the bulk density was 0.1.
';J/cm3 blanks 1~ made of silica, alumino, silconi) 7 fufu, Ibarano base material) blued, and the above except that this wrinkle strength, alumino, ziruni 1 nia fiber medium material was used. The same plant tissues were cultured in the same manner as in Example 5, and the same items were examined. 3rd result
Shown in the table.

For each of the results of Examples 5 and 6 above, cultivation was performed as a control according to Comparative Example 1 in the same manner as in Examples 1 to 3 above, and the $,! The average number of measurements for one fruit is "10"
Expressed as 0.

As is clear from the results in Table 3, in the cases of Examples 5 and 6 in which two types of ceramic fiber culture medium were used, the growth and rooting of the Shoe-1- were significantly greater than in the control case. It was found that it was promoted.

Table 3 Example 7 Using a silica/alumina fiber culture medium having the same bulk density of 0.1'j/cm3 as in Example 4 and having the shape of Planchera 1, -side 1 .8cm cubes are cut out and each tube bottle is approximately 0.6cm.
In addition, using the same Murashige-Skoog medium 6 Biao as in Example 1, cut the leaves of Centpaulia ionata (hybrid A) into approximately 2 mm squares in the same manner as in Example 1. Culture was carried out for 4'0 days under the same conditions as in Example 1, and the survival rate, callus diameter, number of shoots, shoot length, and rooting rate were examined. The average value of the measured values of the control cultured on agar 18 medium as in Comparative Example 1 above was expressed as 100, and the survival rate was 14./l.
The callus diameter was 269', the number of shoots was 1 to 260, the shoot length was 388, and the rooting rate was 135.

As is clear from the results, it was found that the use of the silica/aluminium-based Ivar culture medium of Example 7 significantly promoted callus growth, shoot differentiation, and growth.

Example 8 Using the same silica/alumina fiber as in Example 1,
Processed in the same manner as in Example 1 to give a bulk density of 0.07'j/cm3
A blanket-shaped silica/alumino fiber culture medium was manufactured.

2cm per side from this silica/alumina fiber medium material
Cut out a cube of m and its 0.56! IF 2゜5c
It was placed at the bottom of a tube bottle of mφ x 12 cm so that the fiber direction was perpendicular, and Centpaulia was grown in the same manner as in Example 1.
Ionata (hybrid B) leaves cut into approximately 2 mm squares were placed on a bed, and cultured for 13 weeks in the same row 1 as in Example 1. At that time, survival rate, callus formation rate, number of shoes,
Thickote length and rooting rate were examined. The average value of the measured values of the control cultured on the same agar medium as in Comparative Example 1 above was calculated as 1
The results expressed as 00 were: survival rate was 100, callus formation rate was 100, shoe number 1 to number was 105, shoot length was 276, and rooting rate was 100. As is clear from the results, it was found that the use of the silica-alumina fiber culture medium of Example 8 significantly promoted the growth of seacoat.

Example 9 A cube of 2 cm on each side was cut out from the plumket-shaped silica-alumino-7 fiber culture medium produced in Example 1, and a notch was made in the center of the cube to prepare a conditioned medium.

Tissue culture of seedlings of Scibellas pulchella (cultivar name: Ninanose) obtained through culture? 111 (Using Murashige-Skoog agar medium, cultured for 20 days) is taken out of the tube bottle, the agar layered on the roots is removed under running water, washed with water, and the roots are inserted into the notches of the conditioned medium. This young plant was then planted.

The seedlings thus planted in the conditioned medium were placed in a Plus Book cultivation container as they were, and acclimatized for 10 times in a greenhouse with 75% light shielding, and the survival rate at this time was examined. Irrigation should be performed once a day until the medium is completely saturated with water, and excess water should be drained from the cultivation container.

As a control for this Example 9, the conditioned medium was 2 parts by weight of Pea 1 Hemos, 1 to 1 part by weight of Burai, and Barge 1191.
Using a potting soil obtained by mixing 1 to 0.5 parts by weight, this potting soil was placed in the same plastic cultivation container as used in Example 4 above to a thickness of 2 cm. The same seedlings as used were planted on the conditioned medium in this cultivation container, and acclimatized under the same row M as in Example 9. When the survival rate of this control was taken as 100, the survival rate of Example 4 was 143.

Example 10 The same procedure was carried out except that seedlings of 70-Cassia macrorhiza germinated in tube bottles (Murashige-Skoog agar medium, cultured for 60 days) were used as seedlings, and the size of the conditioned medium was a cube of 3 cm on each side. Planting was carried out in the same manner as in Example 9, acclimation treatment was carried out under the same conditions, and the survival rate at this time was examined.

As a control for this Example 10, the thickness of the soil was 3 cm.
Except for the above, the same conditioned medium as in Example 9 was used, and the conditioning treatment was carried out under the same conditions as in Example 10. When the survival rate of this control is subtracted by 100, the survival rate of Example 10 described in -1- is 125.

Next, the plants acclimatized in Example 10 and its control were planted, and the amount of growth after acclimatization was examined.

That is, a hole of 3 cm in diameter and 3 cm in depth was formed in the center of a rock wool cube for plant cultivation measuring 7 cm in length, 7 cm in width, and 6 cm in height, and the plants acclimatized in Example 10 were placed in this hole. Push the conditioned medium together and plant it.
This plant was placed in a greenhouse with 45% light shielding, and used as a liquid fertilizer once a day with Hyponec-25 = Su stock solution (Murakami & Co., Ltd. (l Wooden: 5 (NL 10 (P)).
)-5(K), a 2,000-fold solution of
# It was given as a substitute for water and cultivated for 30 days.

As a control, we used a plant that had been acclimatized in accordance with the control of this Example 10, took it out of the cultivation container without dropping the soil around the roots as much as possible, and placed it in a lock for plant cultivation in the same manner as in this Example 10. They were planted in the hole in the center of the wool cube using the soil used for acclimatization and cultivated in a greenhouse.

When the growth amount of the plant of Example 10 and its control plant was compared in terms of the number of leaves, the growth amount of Example 10 was 150 when the growth amount of the control plant was 100.

[Effect of the invention] The present invention relates to a culture medium material made of ceramic fibers that does not substantially elute components that have a harmful effect on plant tissue culture, and which is used in combination with a liquid medium to improve the growth of cultured plant tissues. , promotion of differentiation and rooting, improvement of survival rate during N11ization and growth of seedlings after acclimatization, application to a wide variety of plants, continuous use from tissue culture to planting after acclimatization, and improvement of acclimation work. It is possible to save labor, improve the survival rate during acclimatization, and improve productivity after acclimatization.

Claims (6)

[Claims] (1) A medium material for plant tissue culture, which is made of ceramic fibers that do not substantially elute components that have an inhibitory effect on plant tissue culture, and is used in combination with a liquid medium. (2) The medium material for plant tissue culture according to claim 1, wherein the ceramic fiber does not substantially react with the liquid medium. (3) The medium material for plant tissue culture according to claim 1, wherein the ceramic fiber is hydrophilic. (4) The medium material for plant tissue culture according to claim 1, wherein the ceramic fiber is a silica-alumina fiber. (5) The medium material for plant tissue culture according to claim 1, wherein the ceramic fibers are arranged in a vertical direction. (6) The medium material for plant tissue culture according to claim 1, which has a bulk density of 0.005 to 0.3 g/cm^3.