JPH1070943A - Preparation of clone seedling - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a clone seedling having low withering rate without using acclimatization procedure and growing in a short time by transplanting a cultured plant body to a culture soil incorporated with a true fungi belonging to the genus Gliocladium. SOLUTION: A cultured plant body such as rice or lily cultured by tissue culture is transplanted to culture soil incorporated with a true fungi belonging to the genus Gliocladium such as Glicladium virens, Gliocladium roseum or Gliocladium aureum and grown without using acclimatization process to obtain a clone seedling. The concentration of the true fungi belonging to the genus Glicladium is preferably 1×10<4> to 1×10<10> CFU per 1g of the culture soil.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for producing cloned seedlings, and more particularly, to a method for producing cloned seedlings without performing a conditioning step.
The present invention relates to a method for producing a clone seedling having a low mortality after transplantation to a cultivation soil.

[0002]

2. Description of the Related Art A plant cultivation method using a so-called cloned seedling (tissue culture seedling) in which a cultured plant body obtained by separating and growing a part of a plant tissue is transplanted to a cultivation soil is used for cultivation of flowers and vegetables. Widely adopted. In this cultivation method, before transplanting a plant from a sterile incubator to a sterile greenhouse or a field, it is necessary to acclimate the seedling to a sterile environment so as not to die or stop growing. The acclimatization is performed, for example, by transplanting a cultured plant grown in an incubator to a sterilized acclimated soil, and then growing the cells under high-humidity conditions with shading.
Furthermore, carbon dioxide fertilization may be performed. At that time, the acclimatization period usually requires about 3 weeks to 2 months. For this reason, this acclimatization step is an inhibitory factor for improving the productivity of plant cultivation using cloned seedlings, and also causes an increase in cost.

Various methods have been attempted to shorten or eliminate the acclimatization period. For example, a method has been attempted in which a useful microorganism is inoculated to a cultured plant grown under aseptic conditions to increase disease resistance. Specifically, it has been reported that inoculation of tomato tissue cultures with attenuated virus resulted in the production of cloned seedlings having mosaic disease resistance ("Latest Green High Technology", Agriculture and Forestry Statistics Association 1991). Yearly publication, page 53). However, this method has drawbacks that the acclimatization step cannot be omitted and that it cannot be applied to crops other than tomato.

There is also a technique for inoculating a soil for acclimation with a symbiotic bacterium such as VA mycorrhizal fungi and inoculating a tissue culture there to shorten the acclimation period, promote the growth of cloned seedlings, and increase the survival rate. It has been disclosed (Japanese Patent Laid-Open No. 5-1113).
No. 29). However, this method also requires an acclimatization period of 20 days or more. Recently, a method has been proposed in which a symbiotic bacterium is inoculated into a cultured plant grown aseptically using the hypocotyl cutting method (Agricultural Technology System, Vol. 5, soil disease, 216-49, page 6). , Noyama Fishing Village Cultural Association 19
Published in 1995). However, this method requires cutting each seedling and inoculating a symbiotic bacterium, and the operation is complicated.

[0005]

SUMMARY OF THE INVENTION The present invention has been made in view of the above, and has as its object to provide a method for producing a cloned seedling having a low mortality after transplantation to a cultivation soil without performing a conditioning step. I do.

[0006]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, a fungus belonging to the genus Gliocladium (hereinafter abbreviated as a genus Gliocladium). Transplantation of a cultured plant grown in tissue culture into a cultivation soil to which cloning seedlings were added, and it was found that cloned seedlings that would suppress death and growth arrest without acclimation could be obtained. . The present invention has been completed based on such findings.

That is, the gist of the present invention is as follows. (1) A method for producing a cloned seedling, comprising transplanting a cultured plant cultured by tissue culture into a cultivation soil to which a fungus belonging to the genus Gliocladium has been added. (2) A method for producing a cloned seedling, which comprises transplanting a cultured plant cultured in tissue culture onto a cultivation soil to which a fungus belonging to the genus Gliocladium has been added, and growing the cloned seedling without a conditioning step. (3) The above-mentioned (1) or wherein the fungus belonging to the genus Gliocladium is one or more fungi selected from the group consisting of Gliocladium virens, Gliocladium roseum, and Gliocladium aureum. (2) A method for producing the cloned seedling according to (2). (4) The method for producing a cloned seedling according to any one of (1) to (3), wherein the concentration of fungi belonging to the genus Gliocladium is 1 × 10 ⁴ to 1 × 10 ¹⁰ CFU per 1 g of cultivation soil. .

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail. In the present invention, a plant tissue before acclimation is used as a cultured plant, and a plant tissue at a later stage is used as a cloned seedling. The type of clone seedling that can be produced by the method of the present invention is not particularly limited. Specifically, for example, grasses such as rice, lilies, hyacinths, tulips, asparagus, leek and the like, orchids such as cattleya and cymbidium, tobacco, potatoes, tomatoes, eggplants such as eggplants Plants, Apiaceous plants such as carrots, legumes such as sweet peas, soybeans, and kidney beans, roses such as strawberries and sakura, asteraceae such as chrysanthemums, and carabiners such as carnations can be exemplified.

The method for producing a cultured plant is not particularly limited, and any conventionally known method may be used. For example, a shoot apex, a leaf, a flower, a root, a tissue piece or the like cut out from a stem under sterile conditions are placed on an induction medium consisting of a solid medium such as agar, and the resulting callus and protocorn are obtained. , Seedling primordia,
The premature branches and the like are transplanted into a growth medium to proliferate cell tissues to obtain adventitious buds. A method of transplanting the obtained adventitious buds into a rooting medium, rooting and elongating shoots to obtain a cultured plant is exemplified. In addition, a method of producing a cultured plant body by cutting out a growth point from a parent plant, placing it on an agar medium, and elongating roots and growing shoots is also possible.

In the present invention, the species of the fungus is not particularly limited as long as it is a fungus belonging to the genus Gliocladium, and specifically, for example, Gliocladium aureum (Glio
cla-dium aureum), Gliocladium catenu-latum, Gliocladium deliquescens,
Gliocladium nigru
m), Gliocladium penicilloides
penicilloides), Gliocladium roseum (Gli
ocladium roseum), Gliocladium-sagariensis, Gliocladium vermoe-seni, Gliocladium virens, and the like. These may be used alone or in a combination of two or more. In addition, among the fungi belonging to the genus Gliocladium, in the present invention, gliocladium virens, gliocladium roseum, and gliocladium aureum, which are excellent in reducing the death of cloned seedlings, are preferable.

When the above fungi belonging to the genus Gliocladium are used in the present invention, a culture obtained by culturing the fungi in a medium in which the fungi of the genus Gliocladium can grow may be used. It is more preferable to use a culture containing a fungus belonging to the genus Gliocladium in which spores are sufficiently formed by culturing. The cultivation of a fungus belonging to the genus Gliocladium can be performed in the same manner as a usual method for culturing microorganisms. For example, in the laboratory, potato dextrose agar was used for 10 days at 25 ° C.
And a culturing method such as culturing. In the case of mass cultivation, ordinary liquid cultivation or solid cultivation using a plant-derived solid component such as bran, a porous body impregnated with a sugar or nitrogen source, or the like is also possible. The resulting culture of the fungus belonging to the genus Gliocladium, as it is, or, if necessary, after crushing or shredding the culture, or by separating the cells from the culture by centrifugation or the like Alternatively, it is also possible to use the present invention after drying the culture or the cells.

In the present invention, a fungus belonging to the genus Gliocladium can be used in the form of a carrier in which a culture thereof is carried on a carrier. As the carrier, any of an organic substance and an inorganic substance can be used, and a substance containing both an organic substance and an inorganic substance may be used. Specifically, for example,
Attapulgite, montmorillonite, zeolite, Akadama soil, Kanuma soil, Kuroboku soil, Akadama soil, calcined Akadama soil, vermiculite, perlite, minerals such as fossil shells, or
Organic substances such as peat moss, charcoal, pulp, straw, bagasse, oil cake, fish cake, bone meal, blood meal, crab chunks and the like, and mixtures thereof. Among them, porous carriers such as attapulgite, montmorillonite, zeolite, peat moss, and charcoal are preferable from the viewpoint of water retention ability, fertilization retention ability, or convenience in use.

In the present invention, a cultured plant is transplanted to a cultivation soil to which a fungus belonging to the genus Gliocladium has been added. The cultivation soil raw material may be any as long as the cultivation soil is formed by blending the culture of the genus Gliocladium and does not hinder the growth of cloned seedlings. Specifically, for example, Kanuma soil, Kuroboku soil, Akadama soil, fired Akadama soil,
Minerals such as vermiculite, perlite, zeolite, or humus, peat moss, charcoal, pulp, straw,
Organic substances such as bagasse, and mixtures thereof can be mentioned.

The cultivation soil used in the present invention is formed by blending the culture of the above-mentioned genus Gliocladium with the cultivation soil raw material.
It is sufficient that the concentration is equal to or higher than the concentration at which the effect of the combination is exhibited. In the present invention, 1 × 10 ⁴ ~ as colony forming units of a fungus belonging to the cultivated soil 1g per Gliocladium sp
It is preferably selected from the range of 1 × 10 ¹⁰ CFU,
In particular, it is preferably selected from the range of 1 × 10 ⁵ to 1 × 10 ⁹ CFU. It is less than 1 × 10 ⁴ CFU,
Due to the low concentration of the genus Gliocladium that settles on the roots of the plant, the effect of the genus Gliocladium on the growth of cloned seedlings may not be exhibited, and 1 × 10 ¹⁰ C
This is because even if a concentration exceeding FU is adopted, there is a high possibility that superior effects will not be exhibited as compared with a case where the concentration is lower than FU.

In the present invention, a cloned seedling having a low mortality can be produced by transplanting a cultured plant to a cultivation soil to which a fungus belonging to the genus Gliocladium has been added. In addition, a low mortality rate can be achieved without any acclimatization treatment after transplantation.

[0016]

The present invention will be further described with reference to the following examples. Examples 1 to 3 and Comparative Example 1 Runners of the strawberry cultivar "Onmine" were collected, cut at 2cm from the tip, the leaves were removed, and then surface-active in a 0.5% aqueous solution of sodium hypochlorite. The tip of the runner was immersed in an aqueous solution to which the agent Tween 20 was added to a concentration of 0.1% for 10 minutes, and then the runner was transferred to sterile water.

Next, using a dissecting microscope in a clean bench, the leaf buds are removed one by one from the tip of the runner to expose the growing point portion, and to have a size of about 0.3 mm from the apex of the growing point dome. It was excised and implanted in an agar medium in a test tube. As the medium, a medium obtained by adding sucrose to 2% and indoleacetic acid to 0.5 mg / liter to MS basic medium was used. The test tube after the implantation of the growing point has a light intensity of 2500 lux and 24 ° C.
And maintained in a positive incubator for 50 days to grow a cultured plant.

In Comparative Example 1, 3 parts of Kanuma soil, 2 parts of vermiculite, 2 parts of perlite, and peat moss 1 were mixed to form a cultivation soil. Without sterilization, a 30 x 40 x 10 cm bat with a fine hole at the bottom was prepared. The seedlings were placed so that the depth was 5 cm. Twenty of the above-mentioned cultured plants from which agar was removed by washing were planted thereon. Then, a 500-fold liquid fertilizer of Hyponex was added, and the cells were grown at room temperature for 60 days with only irrigation.

On the other hand, in Examples 1 to 3, a culture of Gliocladium oleum IFO 9055 prepared by the method described later (a spore and cells of Gliocladium oleum supported on an attapulgite carrier) was prepared in the above cultivation soil. Fixed, the concentration of bacterial cells in the carrier,
1 × 10 ⁸ CFU / g carrier) was added to the above cultivation soil for 2.5
It grew in the same manner as in Comparative Example 1 except that it was blended so as to be 10 to 10% by volume.

A culture of the genus Gliocladium was prepared by the following method. As a medium for producing a culture of a genus Gliocladium, glucose 0.2% by weight, soluble starch 2.0% by weight, peptone 0.4% by weight, meat extract 0.4% by weight, yeast extract 0.4% 150 ml of a medium material containing 0.4% by weight of calcium carbonate and 0.4% by weight of calcium carbonate was poured into a 1-liter Erlenmeyer flask, and glass beads having a diameter of 3 mm were put therein.
It was sterilized for 0 minutes to obtain a liquid medium for producing a culture of a genus Gliocladium. The medium was inoculated with a genus Gliocladium and cultured in a rotary shaker at 26 ° C. for 5 days.

Attapulgite 1 having an average particle size of 2 mm
The above-mentioned liquid medium (400 ml) was added to kg, placed in a stainless steel vat (40 cm × 60 cm × 15 cm) with a lid, and sterilized at 120 ° C. for 15 minutes to obtain a carrier for a culture of a genus Gliocladium. Next, the cells cultured in the rotary shaking incubator as described above were collected using a Buchner funnel in a clean bench, added to the carrier in the stainless steel vat, and mixed with a spatula. The stainless steel vat to which the cells were added was placed in a constant temperature room at 26 ° C.
The cells were cultured for 10 days by mixing the top and bottom once a day to obtain a culture.

In Examples 1 to 3, as described above, cloned seedlings were produced using cultivation soil containing the culture. The results are shown in Table 1.

[0023]

[Table 1]

In Table 1, those in which the seedlings were yellowed or wilted during the day were also judged to be dead strains and were excluded from the number of surviving strains. From Table 1, if the cultivation method of the present invention is adopted,
It was confirmed that the plants grew well without acclimation. On the other hand, in Comparative Example 1, the number of surviving strains was reduced to one third or less, and it was confirmed that the adaptation step was required.

Examples 4 to 6 and Comparative Example 2 From the growing shoots of Cattleya, the bud portion, which is the portion immediately before the tip leaves develop, is cut out and the base buds are exposed while washing the dirt. The rind and buds were stripped off. The buds were washed with a dilute solution of a neutral detergent and then sterilized with a 0.4% aqueous solution of sodium hypochlorite for 15 minutes.
After sterilization, the solution was transferred to sterile water, one or two outer leaves were peeled off, and then immersed again in a 0.3% aqueous solution of sodium hypochlorite.

The shoots were transferred to a clean bench, washed with sterile water, the outer skin was removed with a scalpel under a microscope, and the shoots were washed with sterile water and placed on a medium. As the medium, M
1 of LS formula except amino acids and vitamins from S medium
Sucrose 20 g / l, naphthalene acetic acid (NAA) 0.2 mg / l,
A solution prepared by adding 0.3 mg / liter of 6-benzylaminopurine (BA) was used.

Next, the PBLs formed in the tissues by PBL induction were placed on a solid medium prepared by adding 2% of sucrose to an inorganic medium having a 処方 concentration of LS and 2% of sucrose, and adding 0.8% of agar. The PBL was grown by placing this solid medium in a constant temperature bath at 24 ° C. under 1200 lux illumination.
The PBL grown in this manner was divided and transferred to a medium containing KS formulation for shoot formation and 10% banana liquefied with a juicer. Shoot about 1.5cm
When the shoot grew, one more shoot was separated, and the banana 1 was liquefied with a juicer in a KS-prescribed medium.
Transfer to a growth medium containing 5%, 2000 lux, 25 ° C
And grown until the seedlings of the cattleya tissue became 5-6 cm in size.

The cattleya cultured plant thus cultivated under aseptic conditions was transferred to field cultivation under sterile conditions. As a soil for cultivation in a field, 240 composted plants were transplanted using a mixed compost containing a genus Gliocladium prepared by the same method as in Example 1. Then, the survival rate of Cattleya after 60 days was measured, and the effect of the present invention was confirmed. The results are shown in Table 2.

[0029]

[Table 2]

In Table 2, those in which the seedlings were dead, yellowed, or withered during the day were also judged to be dead strains, and were excluded from the number of surviving strains. From Table 2, it was found that if the cultivation method of the present invention was employed, 80% or more of the clone strains could be used as seedlings for production without acclimation. On the other hand, in Comparative Example 2, the number of surviving strains was 80% or less, and it was confirmed that a habituation step was required to improve the survival rate.

Examples 7 to 14 and Comparative Examples 3 and 4 Potato tubers were washed with running water and sterilized in a 1% aqueous sodium hypochlorite solution for 30 minutes. Then, the top and bottom of the tubers were cut off and the center was punched out with a sterilized 16 mm cork borer. By this method, the central tissue was extracted from four tubers. This central tissue was immediately transferred to a 0.5% aqueous sodium hypochlorite solution and
Sterilized for a minute. After washing several times with sterile water, the top and bottom were cut off with a sterilized knife, and the central tissue was punched out with a 6 mm sterilized cork borer.

As a medium for culturing the above central tissue,
MS base medium supplemented with mannitol 45 g / l, indole acetic acid 0.1 mg / l, and zeatin 0.5 mg / l was used. This medium was dispensed in 40 ml portions into a 100 ml Erlenmeyer flask and sterilized at 120 ° C. for 10 minutes. The above central tissue punched with a 6 mm cork borer was further sliced to a thickness of 1 mm, and this was placed in the Erlenmeyer flask and 5 pieces were placed per flask. The Erlenmeyer flask was placed in a thermostat at 25 ° C. under a light quantity of 1500 lux and cultured for 16 weeks with a day length of 6 hours.

As a medium for transplanting the cultured central tissue, 19 g / liter of sucrose was added to MS basic medium, and 120 ml was dispensed into a 300 ml Erlenmeyer flask and sterilized at 120 ° C. for 10 minutes. Using. Then, stems of the adventitious buds differentiated from the cultured central tissue were cut out, transplanted to the medium, and cultured at 1500 lux, 24 ° C., and 24 hours in a light place for 3 weeks.

A basal part was cut out from the cultured tissue, transplanted into the MS basic medium supplemented with sucrose prepared as described above, and subjected to 1500 lux, 24 ° C., 24 ° C. as described above.
The operation of culturing for 3 weeks in the dark was repeated three times to obtain a cultured potato plant. Then, the cultured plant was taken out of the medium together with the agar, and the agar attached to the root was washed out and provided for growth under aseptic conditions.

As cultivation soil for growing cloned seedlings, vermiculite was put into a bat of 30 × 40 × 10 cm having a large number of holes on the bottom to a depth of 8 cm, and the bat was subjected to the same method as described in Example 1. A culture of the genus Gliocladium prepared by the method was blended so as to have the bacterial concentration shown in Table 3. The cultivated plant is planted in this bat, covered with a transparent plastic container, and
Cultivated for a week. Separately, a cultivation zone not covered was provided for comparison. The cultivation condition was 5000 lux, day length 1
The temperature was raised to 24 ° C. for 6 hours. The fertilizer is Hyponex 50
A 0-fold solution was applied every 6 days to the extent that moisture came out of the bottom of each vat. Cultured plants are planted 20 per vat and 1
Tested 2 times per condition, 3 for a total of 40 seedlings
The number of surviving strains after a week was evaluated. The results are shown in Table 3.

[0036]

[Table 3]

In Table 3, in parentheses in the column of the number of surviving potato lines, (presence) indicates the results of cultivation with a plastic cover, and (none) indicates the results of cultivation without a plastic cover.

In Table 3, seedlings that were dead, yellowed, or withered during the day were also judged as dead strains and were excluded from the number of surviving strains. From Table 3, it was confirmed that if the cultivation method of the present invention was adopted, 80% or more of the clone strains could grow without acclimatization. On the other hand, in Comparative Examples 3 and 4, the number of surviving strains was 80% or less, and it was confirmed that a habituation step was necessary to improve the survival rate.

Example 15 and Comparative Example 5 Bulbs of the yamali lily were collected, and among the pieces of the sapling bulb, the internal pieces without lesions were collected. After thoroughly washing with a diluted solution of a neutral detergent, 3% Was sterilized with an aqueous sodium hypochlorite solution for 19 minutes. After repeating the washing with a neutral detergent and the sterilization with an aqueous solution of sodium hypochlorite two more times, the pieces were transferred to sterile water and washed. After washing three times by replacing the sterile water used for washing, put the phosphorus pieces into a sterile petri dish,
The pieces were cut into 1 cm square pieces.

On the other hand, an agar medium was prepared by adding 90 g / liter of sucrose to the MS medium and placed on the scale with the inner curved surface of the scale piece facing upward. The test tube on which the phosphorus pieces were placed was placed at 20 ° C and 25 ° C.
The pieces were cultured for 2 months under 00 lux continuous light to form spheres. Separate the pieces of the child sphere and add N
After transplanting to an MS medium containing 0.2 mg / L of AA, 5 g / L of activated carbon, and 30 g / L of sucrose,
The cells were cultured for 4 months under the conditions of 2,500 lux and a photoperiod of 16 hours, and then the test tubes were kept at 5 ° C. for 80 days to break dormancy to obtain cultured plants.

In Example 15, the cells of Gliocladium aureum used in Example 1 were mixed with a vermiculite in a sterile state, and the concentration of the cells was 5 × 10 ⁵ CF.
Cultivation soil was used as U / g cultivation soil. The above cultured plant was transplanted to this cultivation soil, and a clone seedling was grown for one month at room temperature using a 2000-fold aqueous solution of Hyponex as a liquid fertilizer. On the other hand, in Comparative Example 5, cloned seedlings were grown using cultivation soil to which no Gliocladium sp. Was added.

Thereafter, 60 cloned seedlings per transplant were transplanted to the field and cultivated for 3 months, and the number of seedlings having no abnormal growth was measured. As a result, 46 seedlings were obtained in Example 15, and 46 seedlings in Comparative Example 5. There were 37. From these results, it was confirmed that the clone seedlings produced by the method of the present invention can be cultivated without acclimatization.

[0043]

According to the production method of the present invention, no acclimatization period is required when transplanting a cultured plant in a germ-free environment. Therefore, the plant can be grown in a short period of time from the cloned seedling to a state where it can be shipped.

Claims (4)

[Claims] 1. A method for producing a cloned seedling, wherein a cultured plant cultured by tissue culture is transplanted to a cultivation soil to which a fungus belonging to the genus Gliocladium has been added. 2. A method for producing cloned seedlings, wherein a cultured plant cultured by tissue culture is transplanted to a cultivation soil to which a fungus belonging to the genus Gliocladium has been added and grown without a conditioning step. 3. The fungus belonging to the genus Griocladium,
3. The fungus is one or more fungi selected from the group of Gliocladium virens, Gliocladium roseum, Gliocladium aureum.
A method for producing the cloned seedling according to the above. 4. The concentration of a fungus belonging to the genus Griocladium is 1 × 10 ⁴ to 1 × 10 ¹⁰ CF per gram of cultivation soil.
The method for producing a cloned seedling according to any one of claims 1 to 3, which is U.