JPH0373275B2 - - Google Patents

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 a medium material made of ceramic fiber, which can also be used as a conditioned medium for young plants. The present invention relates to a medium material for plant tissue culture that can be used for culturing plant tissues. [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 does not support growth of plant tissue cells.
Contains an unidentified substance that inhibits differentiation and/or root elongation, and even if highly purified agar is used, growth, differentiation, and/or rooting may be significantly delayed depending on the plant type. In addition, because the entire medium is gelatinized, the diffusion rate of waste substances and harmful substances produced in the cultured tissue is slow, and depending on the type of plant, its toxic effects make culturing completely difficult. There were cases where it happened. Moreover, seedlings obtained by tissue culture on gelatinized media such as agar have roots that are differentiated from the cultured tissue and have a tap root and a small number of roots. When transplanted and acclimatized, the survival rate is extremely low. 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 result in greater damage to the plants and slower growth after acclimatization. For this reason, conventional culture methods using agar or the like have had a problem in that it is necessary to prepare and use a conditioned medium of perlite, vermiculite, etc. that has been specially sterilized and disinfected during acclimatization. Furthermore, when glass fiber is used as a medium material, there is a problem that alkaline content is eluted during use as a medium material, causing fluctuations in the pH value and chemical structure of the medium. Furthermore, using cotton wool gives worse results than agar media. [Problems to be Solved by the Invention] An object of the present invention is to provide a novel medium material for plant tissue culture that is used in combination with a liquid medium. In addition, another object of the present invention is to be able to promote the expansion, differentiation, and rooting of plant tissues;
It is an object of the present invention to provide a culture medium that improves the survival rate during acclimatization and the growth of young plants after acclimatization. Furthermore, another object of the present invention is to provide a medium material that can enable tissue culture of plants that are difficult to culture in gelatinized media such as agar. Furthermore, another object of the present invention is that it can be used continuously from tissue culture to transplantation after acclimatization, saving labor in acclimatization work, improving survival rate during acclimatization, and increasing production rate after acclimatization. The objective is to provide a culture medium that can achieve the following. [Means for Solving the Problems] That is, the present invention provides a ceramic fiber plant tissue culture medium that is used in combination with a liquid medium and does not substantially elute components that have an inhibitory effect on plant tissue culture. It is a material. The chemical composition of the ceramic fiber used in the present invention is that components eluted during tissue culture operations, such as sterilization treatment performed by impregnating a liquid medium, have an inhibitory effect on the growth, differentiation, and rooting of plant tissues. However, it is important that it does not substantially react with the liquid medium used in combination with this medium material, and that it does not affect the pH of this liquid medium. In particular, the elution of aluminum ions causes the problem of reacting with phosphate ions in the liquid medium to produce water-insoluble aluminum phosphates that cannot be used by plants, resulting in a lack of phosphoric acid. Elution changes the pH value of the liquid medium used, causing problems in the growth, differentiation, and rooting of plant tissues. Ceramic fibers used in the present invention include, for example, silica-alumina fiber (SiO ₂ -Al ₂ O ₃ ), silica-alumina-zirconia fiber (SiO ₂ -Al ₂ O ₃ -ZrO 2 ), silica-alumina fiber (SiO 2 -Al 2 O 3 -ZrO ₂ ), Chromia fiber (SiO ₂ −Al ₂ O ₃ −
Examples include silica/alumina fibers whose main components are silica and alumina such as Cr ₂ O ₃ ), silica fibers whose main component is silica, alumina fibers whose main component is alumina, carbon fibers, etc. In consideration of components eluted from the fiber, reactivity with the liquid medium, stability, cost, etc., silica-alumina fibers are particularly preferred. 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. These additives are often added, but it is preferable to heat-treat the additives to a temperature higher than 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~
The amount is preferably about 0.3 g/cm ³ , preferably about 0.01 to 0.2 g/cm ³ . If the bulk density is less than 0.005, it will be difficult to handle, and if it is more than 0.3 g/cm ³ , root elongation will be hindered. The ceramic fiber culture medium material of the present invention is itself a hydrophilic material, but in order to further improve its hydrophilicity, substances that do not hinder plant growth, such as nonionic surfactants and higher alcohols, are added. Good too. The shape of the ceramic fiber medium material of the present invention (hereinafter referred to as the present 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, it is a blanket-like molded product or granulated cotton. In addition, when the main focus is solely 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 expansion, etc., it is preferable to arrange them in the vertical direction. This medium material is Murashige Skoog, White,
Knudson, Linsmeyer-Skoog, Herahe;
It is used in combination with liquid media such as Gautre, Nitsuchie Nitsuchie, Erickson, Gamborg Miller Ojima, Tsukamoto Kano, etc., and their modified media. It is desirable to use these liquid media by impregnating the main medium material, but they can also be supplied by dripping, spraying, or other methods. To culture plant tissue using this medium, for example, place an appropriate amount of this medium in a culture container, impregnate it with the required amount of liquid medium, and then sterilize it in an autoclave, etc. perform the operation,
After natural cooling, this can be carried out by placing a pre-sterilized section of the plant tissue on the main culture medium in a culture container. In addition, the management method during this culture is the same as when using agar, but when using this culture medium, the liquid medium can be grown under aseptic conditions without transplanting the cultured tissue. Additions, addition of plant hormones, washing and replacement of the liquid medium can be carried out. 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 culture medium by a method such as 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 the rooting medium are removed from the seedlings obtained by tissue culture. After washing with water, plant the seedlings by wrapping the roots with an appropriate amount of conditioned medium placed in a cultivation container, and acclimatize them in the greenhouse for about one week to one month with appropriate shade and humidity. . The management method during acclimatization is the same as in the conventional case of using perlite, vermiculite, etc., but since this culture medium has high water retention properties, there is usually no need for watering. Furthermore, the young plants that have been acclimatized can be used for normal soil cultivation or solution cultivation without removing the medium. By using this medium for acclimation, it is possible to always provide adequate water retention and air permeability to the roots of young plants, and because it has a very high porosity, even after irrigation. It is thought that 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 not only as a medium for propagation, differentiation, and rooting in tissue culture, but also as a medium material for conditioned medium used for acclimating seedlings cultured in this tissue culture. Because it can be used
The process from plant propagation, differentiation, and rooting to the acclimatization of the resulting seedlings can be done consistently without transplanting by simply washing off the liquid medium used in each process and adding the next liquid medium. It can be done by In this way, by consistently performing plant expansion, differentiation, rooting, and acclimatization without transplanting, it is possible to significantly reduce damage to seedlings, and to 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 regular soil cultivation, hydroponics, sand cultivation, gravel cultivation, rock wool cultivation, etc. without removing the medium material.
It is especially suitable for rock wool cultivation that uses inorganic fibers like this culture medium. [Function] In the present invention, since ceramic fiber is used as the medium material, overall, due to the chemical stability and physical properties of the ceramic fiber,
The performance of the liquid culture medium used in combination can be fully demonstrated. 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. It is presumed to promote growth during acclimatization. In addition, in this medium material, the combined liquid medium exists between the ceramic fiber fibers as capillary water and attached water, so it is assumed that the cultured tissue can easily absorb the liquid medium and remove the liquid medium during acclimatization. be done. [Examples] Hereinafter, the present invention will be specifically explained based on Examples. Example 1 As a ceramic fiber, 1 g of sample
When 150ml of 0.5N-HCl was added and reacted at 30℃ for 1 hour, the amount of Al released was 229ppm, which was 8 times the amount at pH 7.
PH after being immersed in test water for 6 days in a sealed state at room temperature
Using silica-alumina fibers (SiO ₂ : approx. 53% by weight, Al ₂ O ₃ : approx. 47% by weight) with a carbon fiber ratio of 6.5, an organic lubricant is added to layered cotton in which these fibers are arranged in a certain direction, and then the needles are put a ring on it,
The obtained molded product was further heat-treated at about 700°C for about 20 minutes to decompose and remove the additives, and the blanket-shaped silica/alumina fiber medium material of Example 1 (bulk density: 0.13 g/cm ³ ) was prepared. Manufactured. Cut out a cube of 2 cm on each side from this silica/alumina fiber medium material, and add 1.0 g of it to 2.5 cmφ.
Place the fibers in the bottom of a 12 cm x 12 cm tube bottle so that the fiber direction is perpendicular.
8 ml of the solution (containing 0.1 mg of benzyladenine, 0.1 mg of benzyladenine, and 30 g of sugar loin) was dispensed, capped with aluminum foil, sterilized in an autoclave at 120°C for 15 minutes, and then cooled naturally. Next, in a clean bench, a section of the stem base of Cyperus pulchella (cultivar name: Nanasu), which had been sterilized in advance using antiformin and cut into approximately 2 mm squares, was placed in the above-mentioned tube bottle, and placed at a temperature of 25°C in the light. period 12 hours,
3 under culture conditions of 12 hours dark period and 3000 lux
After culturing for a week, the survival rate, shoot length, total growth amount of shoots, number of shoots, and rooting rate were examined. The results are shown in Table 1. Example 2 A silica-alumina-zirconia fiber whose elution amount of Al was 77.7 ppm with respect to 1 g of sample diluted hydrochloric acid measured in the same manner as in Example 1, and whose pH after testing against 8 times the amount of PH7 test water was 6.5. −
(SiO ₂ : approx. 50% by weight, Al ₂ O ₃ : approx. 35% by weight,
Using the same procedure as in Example 1, using ZrO ₂ (approximately 15% by weight), a blanket-shaped silica-alumina-zirconia fiber culture medium (bulk density: 0.13 g/
cm ³ ) was produced. Using this silica-alumina-zirconia fiber medium material, the same plants 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 Eight times the amount of PH7 measured in the same manner as in Example 1
Silica with a pH of 6.5 after testing against test water.
Alumina/chromia fiber (SiO ₂ : approx. 55% by weight, Al ₂ O ₃ : approx. 42% by weight, Cr ₂ O ₃ : approx. 3% by weight)
A blanket-shaped silica-alumina-chromia fiber medium material (bulk density: 0.13 g/cm ³ ) was produced using the same procedure as in Example 1. Using this silica-alumina-chromia fiber medium material, 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. Comparative Example 1 8 g of commercially available purified agar for culture was added to the same Murashige-Skoog medium as used in Examples 1 to 3 above.
Prepare an agar medium by adding 8
ml in the same tube bottles used in each of the above examples (0.064 g of agar was used per tube bottle as the medium material)
The same plant tissues were cultured under the same conditions as in Example 1, and the same items as in Example 1 were examined.
The results are shown in Table 1. Comparative Example 2 Commercially available rock wool culture medium for plant cultivation (SiO ₂ : approx. 46% by weight, Al ₂ O ₃ : approx. 13% by weight, CaO: approx. 18% by weight, MgO: approx. 12% by weight,
Fe ₂ O ₃ (approximately 8% by weight) granular cotton (hydrophilic type)
The same plant tissue was cultured under the same conditions as in Example 1 above, 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 ₂ : approx. 59
Weight%, _Al2O3 : about 4% by weight, CaO: about 16% by weight, _MgO : about 5% _by weight, _B2O3 : about 3% by weight,
The same plant tissues were cultured under the same conditions as in Example 1 above using a bulk of Na ₂ O (approximately 11% by weight), and the same items as in Example 1 were investigated. The results are shown in Table 1. In addition, each value showing the results in Table 1 is expressed with the average value of the measured values measured in Comparative Example 1 as 100 (however, only the rooting rate is shown as an actual measured value). As is clear from the results in Table 1, the results of Examples 1 to 3 using the three types of ceramic fiber medium materials are higher than those of Comparative Examples 1 to 3 in terms of survival rate and shoot. Growth and rooting rate are significantly promoted.

[Table] Example 4 The same silica/alumina fiber as in Example 1 was used, processed in the same manner as in Example 1, and the bulk density was 0.1.
A blanket-shaped silica/alumina fiber medium material of g/cm ³ was produced. Cut out a cube of 2 cm on each side from this silica/alumina fiber medium material, and add 0.8 g of it to 2.5 cmφ.
Place the fibers in the bottom of a 12 cm x 12 cm tube bottle vertically, use 8 ml of the same Murashige-Skoog medium as in Example 1, and grow Cyperus pulchella (variety name: Nanasu) in the same manner as in Example 1. Approximately 2 stems from the base
Sections cut into mm squares were placed on a bed and cultured for 3 weeks under the same conditions as in Example 1, and the survival rate, number of shoots, shoot length, and rooting rate were examined. The results are shown in Table 2. In addition, for each value of the results of this Example 4, as in the case of Examples 1 to 3 above, culture was performed according to Comparative 1 as a control, and the average value of the measured values of the results was expressed as 100. did.

[Table] As is clear from the results in Table 2, it was found that the growth of shoots was significantly promoted by using the silica/alumina fiber culture medium. Next, the cyperus seedlings obtained in Example 4 were taken out of the tube together with the silica/alumina fiber medium material, and washed under running water.
The seedlings were placed in a plastic container together with the culture medium, and acclimatized for 10 days in a greenhouse with 75% light shielding, and the survival rate during acclimatization was examined. In addition, in a plastic container of the same type as that used in Example 4, a 2 cm thick acclimatization soil mixed with 2 parts by weight of beet moss, 1 part by weight of perlite, and 0.5 parts by weight of vermiculite was placed, and this was poured into a plastic container of the same type as that used in Example 4. The seedlings of Cyperus obtained in the control in step 4 (obtained by removing the agar attached to the roots from the tube bottle and washing with water) were moved and subjected to acclimation treatment under the same conditions. The survival rate during acclimatization was investigated. When the survival rate in the case of the control mentioned above is taken as 100, the survival rate in the case of Example 4 is 143, and the silica
It was found that the use of alumina fiber medium material improved the survival rate during acclimatization. Example 5 Using the same silica/alumina fiber culture medium as in Example 4, a 5 mm cut section of the runner tip of Nephrolepis merciarii was placed on the bed in the same manner as in Example 4, and under the same conditions as in Example 1. At 8
Culture was carried out for weeks, and the survival rate, number of shoots,
Shoot length and rooting rate were examined. The results are shown in Table 3. Example 6 Using the same silica-alumina-zirconia fiber as in Example 2 as the ceramic fiber, a blanket-shaped silica-alumina-zirconia fiber culture medium with a bulk density of 0.1 g/cm ³ was produced. - The same plant tissue was cultured in the same manner as in Example 5 above, except that a zirconia fiber culture medium was used, and the same items were investigated. The results are shown in Table 3. Regarding each value of the results of Examples 5 and 6 above,
As in Examples 1 to 3 above, culture was performed as a control according to Comparative Example 1, and the average value of the resulting measured values was expressed as 100. As is clear from the results in Table 3, in the cases of Examples 5 and 6, in which two types of ceramic fiber medium materials were used, the growth and rooting of the shoots were significantly promoted compared to the control case. It turned out that it was.

[Table] Example 7 Using a blanket-shaped silica/alumina fiber culture medium with the same bulk density of 0.1 g/cm ³ as in Example 4, a cube with a side of 1.8 cm was cut out from this silica/alumina fiber culture medium. Using approximately 0.6 g per tube bottle, and using 6 ml of the same Murashige-Skoog medium as in Example 1, leaves of Saintpaulia ionata (hybrid A) were cut into approximately 2 mm square leaves in the same manner as in Example 1. The cut sections were placed on a bed and cultured for 40 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 the same agar medium as in Comparative Example 1 above.
The results expressed as 100 are: survival rate is 144, callus diameter is 269, shoot number is 260, shoot length is
388, and the rooting rate was 135. As is clear from the results, it was found that the use of the silica/alumina fiber medium material of Example 7 significantly promoted callus growth, shoot differentiation, and growth. Example 8 The same silica-alumina fiber as in Example 1 was used, processed in the same manner as in Example 1, and the bulk density was 0.07.
A blanket-shaped silica/alumina fiber medium material of g/cm ³ was produced. Cut out a cube of 2 cm on each side from this silica/alumina fiber medium material, and add 0.56 g of it to 2.5 mm.
The fibers were placed at the bottom of a φ x 12 cm tube bottle so that the fiber direction was perpendicular, and the leaves of Centpaulia ionata (hybrid B) were cut into approximately 2 mm square pieces in the same manner as in Example 1. Under the same conditions as Example 1
Culture was carried out for 13 weeks, and the survival rate, callus formation rate, number of shoots, shoot 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 is expressed as 100, and the survival rate is 100, the callus formation rate is 100,
The number of shoots was 105, the shoot length was 276, and the rooting rate was 100. As is clear from this result,
It was found that the use of the silica-alumina fiber medium material of Example 8 significantly promoted the growth of shoots. Example 9 Blanket-shaped silica produced in Example 1
A conditioned medium was prepared by cutting out a cube of 2 cm on each side from the alumina fiber medium material and making a notch in the center. The tissue-cultured seedlings of Ciperus pulchella (cultivar name: Nanasu) obtained by culture (using Murashige-Skoog agar medium, cultured for 20 days) were taken out of the tube bottle, and the agar attached to the roots was washed under running water. The seedlings were removed and washed with water, and the seedlings were planted by inserting the roots into the notches of the conditioned medium. The seedlings planted in the conditioned medium in this way were placed in a plastic cultivation container for 75 minutes.
Acclimation treatment was carried out for 10 days in a greenhouse under light-shielding conditions of 1.5%, and the survival rate at this time was examined. In addition, irrigation is 1
The cultivation was carried out once a day until the medium was completely saturated with water, and excess water was drained from the cultivation container. As a control for this Example 9, a soil obtained by mixing 2 parts by weight of peat moss, 1 part by weight of perlite, and 0.5 parts by weight of vermiculite was used as a conditioned medium, and this soil was used in Example 4 above. The same seedlings used in Example 4 were placed in the same plastic cultivation container with a thickness of 2 cm, and the same seedlings were planted in the conditioned medium in this cultivation container, and the acclimatization treatment was performed under the same conditions as in Example 9. . The survival rate of Example 4 was 143 when the survival rate of this control was set as 100. Example 10 Same as Example 9, except that seedlings of Araucasia macrorhiza sprouted in a tube (Murasig-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. They were planted in the same manner as above, and acclimatized 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. The same conditioned medium as in Example 9 above was used, and the conditioning treatment was performed under the same conditions as in Example 10. The survival rate of Example 10 was 125 when the survival rate of this control was set as 100. 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 diameter x 3 cm 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 the hole in the conditioned medium. Push the entire wood into the plant, place the cube in a greenhouse with 45% light shielding, and apply Hyponex undiluted solution (manufactured by Murakami Bussan Co., Ltd.: 5(N)-10(P)-5(K) once a day as a liquid fertilizer. ), containing trace elements) was given as an alternative to irrigation, and cultivation was continued for 30 days. As a control, a plant that had been acclimatized in the control of this Example 10 was taken out of the cultivation container without dropping the soil around its roots as much as possible, and similarly to this Example 10, a plant that had been acclimated was treated with Rotsukwool for plant cultivation. The seeds were planted in a hole in the center of the cuvette using the soil used for acclimatization and cultivated in a greenhouse. When the growth amount of the plants of Example 10 and the 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 was 100. [Effects of the Invention] The present invention relates to a medium material made of ceramic fiber that does not substantially elute components that have an inhibitory effect on plant tissue culture, and is used in combination with a liquid medium to promote the growth, differentiation, and growth of cultured plant tissue. Promotion of rooting, improvement of survival rate during acclimatization and growth of seedlings after acclimatization, application to a wide variety of plants, continuous use from tissue culture to a fixed value after acclimatization, labor saving in acclimatization work, acclimatization It is possible to achieve an improvement in the survival rate during cultivation and an improvement in productivity after acclimatization.

Claims (1)

[Claims] 1. Comprised of a ceramic fiber that does not substantially elute components that have an inhibitory effect on plant tissue culture,
A medium material for plant tissue culture 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 .