JP7045007B2 - Artificial cultivation of fungal heterotrophic Orchidaceae plants - Google Patents

Description

Patent Law Article 30 Paragraph 2 Applicable Poster presentation at the 16th meeting of the Japanese Society of Plant Taxonomy

The present invention relates to artificial cultivation of fungal heterotrophic Orchidaceae plants.

Conventionally, artificial cultivation methods of fungal heterotrophic Orchidaceae plants are known (for example, Patent Documents 1 to 3).

Japanese Unexamined Patent Publication No. 2005-245332 Japanese Unexamined Patent Publication No. 2005-245333 Japanese Unexamined Patent Publication No. 2017-66118

However, although Patent Documents 1 and 2 describe an artificial culture method for Yoania japonica, the environment must be sterile in order to artificially culture Yoania japonica. Further, Patent Document 3 describes a method for promoting symbiosis with orchidaceous plants, but in order to promote germination of orchidaceous plants, a gibberellin inhibitor, salicylic acid, and a salicylic acid derivative are described. It is necessary to use an agent containing a substance selected from the group consisting of, and in the symbiotic germination system, a specific fungus (a fungus of the genus Orchidaceae) must be used. Further, in the invention shown in Patent Document 3, although it is possible to promote the germination of fungal heterotrophic orchidaceous plants, it is unclear whether it is possible to grow up to flowering and fruiting. Furthermore, to date, no artificial cultivation method has been established that enables flowering and fruiting of Gastrodia elata (for example, Gastrodia elata, Gastrodia elata, and Gastrodia elata), which is a heterotrophic orchidaceae plant. ..

An object of the present invention is to provide an artificial cultivation method capable of from germination to fruiting of a fungal heterotrophic Orchidaceae plant.

For the above purpose, as a medium for fungal heterotrophic Orchidaceae plants, logs in progress of rot and a plurality of bulbs in progress of rot are used, and the indoor environment in which the medium is located is set to a temperature of 15 ° C to 30 ° C and a humidity of 70% or more. It is achieved by the method of growing orchidaceous plants, which are heterotrophic to fungi.

According to the present invention, artificial cultivation of fungal heterotrophic Orchidaceae plants becomes possible.

It is a figure explaining the growth kit 100 used for the artificial cultivation method of the fungus heterotrophic orchidaceae plant by embodiment of this invention. It is a photograph which shows the cultivation result of the Yatsushiroran (Kuroyatsushiroran, Akizakiyatsushiroran, Haruzakiyatsushiroran) cultivated by using the method for artificially cultivating a fungal heterotrophic orchid plant according to the embodiment of the present invention. It is a photograph which shows the cultivation result for demonstrating the symbiosis of Kuroyatsushiroran and Akizakiyatsushiroran cultivated by the artificial cultivation method of the fungus heterotrophic Orchidaceae plant according to the embodiment of the present invention. It is a table of mycorrhizal fungi identified from the protocomb of the Yatsushirorans (Kuroyatsushiroran, Akizakiyatsushiroran) cultivated by the artificial cultivation method of the fungal heterotrophic Orchidaceae plant according to the embodiment of the present invention and the medium of the growth kit 100.

Hereinafter, a method for artificially cultivating a fungal heterotrophic Orchidaceae plant according to an embodiment of the present invention will be described.

Using FIG. 1, a breeding kit 100 for artificially culturing Gastrodia elata (Gastrodia elata, Gastrodia elata, Gastrodia elata), which is a fungal heterotrophic orchidaceae plant, will be described. FIG. 1A is a side view of the breeding kit 100, and FIG. 1B is a front view of the breeding kit 100.

In the breeding kit 100, a log 20 in progress of humus is placed in the center of the housing 11b in a translucent plastic case 11 (composed of a lid 11a and a housing 11b), and humus 40 and humus progress are placed around the log 20. The cones 30 formed on the leaves of the cedar inside are spread, covered with a moisture absorbent 13 (using gauze in this embodiment) and vinyl 12 so as to cover the opening of the housing 11b, and the lid 11a is closed and sealed. It is configured to be.

Seeds of Yatsushiroran (Kuroyatsushiroran, Akizakiyatsushiroran, Haruzakiyatsushiroran) are sown evenly on the logs 20, cones 30, and / or humus 40 placed in the breeding kit 100. For sowing, it is preferable to combine seeds with the medium of the production area of Yatsushiroran (2 logs, 30 cones, 40 humus), but there may be cases where the medium of the production area of Yatsushiroran and the seeds are not combined. In addition, as an experiment for confirming the effectiveness of the artificial cultivation method of the fungal dependent vegetative orchid plant according to the embodiment of the present invention, the seeds of Kuroyatsushiroran were used in Kanagawa prefecture and Shizuoka prefecture, and the seeds of Haruzakiyatsushiroran were used in Tokushima prefecture and Akizaki. The seeds of Yatsushiroran were collected from their own dough in Shizuoka prefecture, or self-pollinated in the laboratory. In addition, logs 20, cones 30, and humus 40 were collected from the native dough of Kanagawa prefecture in the artificial cultivation of black syrup, and collected from the native dough of Kochi and Kanagawa prefectures in the artificial cultivation of Akizakiyatsushiroran. It was collected from the native habitat of Tokushima prefecture in the artificial cultivation of Loran. For the log 20 in the artificial cultivation of kuroyatsushiroran, the cedar in the process of humus was adopted, in the cone 30, the cone of the cedar in the process of humus was adopted, and in the leaf mold 40, the leaf soil of the cedar was adopted. The log 20 in the artificial cultivation of Akizakiyatsushiroran adopted the humus in progress, the cone 30 adopted the cone of the cedar in the process of humus, and the humus 40 adopted the humus soil. In addition, the log 20 in the artificial cultivation of Haruzakiyatsushiroran adopted Castanopsis sieboldii in progress, the cone 30 adopted the cone of cedar in the process of humus, and the humus 40 adopted Castanopsis sieboldii forest humus. .. In addition, as the log 20, the cone 30, and the leaf mold 40, other than the above may be adopted. Further, the medium may be composed only of the log 20, the medium may be composed only of the bulb 30, the medium may be composed only of the leaf mold 40, or the medium may be composed of the log 20 and the bulb 30. , The medium may be composed of the log 20 and the humus 40, or the medium may be composed of the conifer 30 and the humus 40. The conifer 30 is preferably a conifer of Sugi, but may be a conifer of a coniferous tree other than Sugi (for example, Cupressaceae, Podocarpus macrophylla, Podocarpus macrophylla, or Pinaceae).

In the artificial cultivation of Yatsushiroran using the growing kit 100, the indoor temperature in the growing kit 100 is kept at 15 ° C to 30 ° C, the indoor humidity is kept at 70% or more, and the growing kit 100 is covered with corrugated cardboard until the flower stalk is extracted. The inside is set to a dark condition, and after the flower stalk is extracted, the inside of the growing kit 100 is changed to a light condition (bright in the daytime and dark in the nighttime). To keep the humidity at 70% or higher, sprinkle natural water or distilled water as appropriate while checking the humidity condition with a sprayer on a regular basis. The indoor temperature in the growth kit 100 is preferably maintained at 18 ° C. to 27 ° C., more preferably 20 ° C. to 25 ° C., and the indoor humidity is preferably maintained at 75% to 90%, still more preferably 80% to 85%. By setting the temperature to 20 ° C to 25 ° C and the humidity to 80% to 85%, the growth of Yatsushiroran and the activity of symbiotic bacteria are most active, and one individual can bloom and bear fruit three times a year for a short period of time. It became possible to germinate and grow. It is preferable to keep the inside of the growing kit 100 under dark conditions until the flower stem extraction of the growing kit 100, but it was confirmed that the growing kit 100 can be grown even under the light condition.

<Artificial cultivation of black shiroran>
Figure 2 shows the results of artificial cultivation of kuroyatsushiroran. The seeds of Kuroyatsushiroran germinated to form a protocomb. After the tuber primordium was formed, the tuber primordium formed bracts from the top of the ball-shaped protocomb, which were split and formed. The roots also developed from the left and right sides of the protocomb. Even after flowering, one of the roots grew preferentially and continuously. The tubers stop growing about 4 months after sowing, and shoot formation occurs. It blooms one month after moss extraction, and the fruit matures and dehiscences about 20-30 days after flowering under natural conditions. For details, it takes 17 days to about 2 months from sowing to germination of kuroyatsushiroran in cedar medium, and protocomb, tuber primordium, roots, flowering, and fruiting are 20-65 days, 25-80 days, and 30 days after sowing, respectively. It took -82 days, 154-299 days, and 194-321 days. Some of the flowers of Kuroyatsushiroran bloomed 154 days after sowing. The artificial culture method showed the effect of promoting growth and flowering, and some large tuber individuals flowered and set fruit three times a year. These growth speeds were considerably more effective than the natural conditions of blooming once a year. Furthermore, if the nutritional conditions and growing environment are poor, it may take several years to flower, or it may not bloom, the tubers may disappear, and the fungus may not be encountered and germination may not occur, so it can be said to be an epoch-making culture system.

<Artificial cultivation of Akizaki Yatsushiroran>
The result of artificial cultivation of Akizakiyatsushiroran is shown in FIG. Akizakiyatsushiroran is a cedar and moso bamboo medium from seeding to germination 8-36 days, after which protocomb, tuber primordium, roots, flowering and fruiting are 33-53 days, 44-79 days and 50-, respectively. It took 83 days, 154-340 days, and 245 days. Sugi medium was effective for flowering and fruiting. It was also confirmed that Akizaki and Kuroyatsushiroran can grow together in the same medium (see FIG. 3). It was confirmed that Akizakiyatsushiroran shares the same hyphae as Kuroyatsushiroran tubers at the time of protocomb (black arrow: Kuroyatsushiroran, white arrow: Akizakiyatsushiroran shown in FIG. 3). It was also confirmed that Kuroyatsushiroran and Akizakiyatsushiroran show similar growth speeds in the same medium. This seems to indicate that the species specificity of mycorrhizal fungi and Yatsushirorans is low.

<Artificial cultivation of Haruzaki Yatsushiroran>
Figure 2 shows the results of artificial cultivation of Haruzaki Yatsushiroran. Harusakiyatsushiroran takes 13-30 days from sowing to germination in Sugi medium and Castanopsis sieboldii medium, and protocomb, tuber primordium, and root formation take 15-32 days, 30-45 days, and 30-45 days, respectively, after sowing. It took. It was confirmed that germination was faster than under natural conditions.

<Medium and seed production area>
Furthermore, the following experimental results were obtained using the artificial cultivation method according to this embodiment.
(A) Kuroyatsushiroran and Shizuoka prefecture (different from the medium) collected in Kanagawa prefecture (same place as the medium) in the medium (20 cedar logs, 30 cedar bulbs, 40 cedar leaf soil) collected in Kanagawa prefecture. It was confirmed that it is possible to artificially cultivate the black shiroran collected in.
(B) Haruzaki Yatsushiroran and Shizuoka Prefecture (with the medium) collected in Tokushima Prefecture (the same place as the medium) in the medium (20 logs of Castanopsis sieboldii, 30 sugi bulbs, 40 leaves of Castanopsis sieboldii) collected in Tokushima Prefecture. It was confirmed that artificial cultivation of Akizakiyatsushiroran collected in different places) is possible.
(C) It is possible to artificially cultivate Akizakiyatsushiroran collected in Shizuoka prefecture (a place different from the medium) using the medium collected in Kochi prefecture (20 logs of moso bamboo, 30 cones of cedar, 40 leaf soil of moso chiku). I confirmed that there was.

<Identification of mycorrhizal fungi of Yatsushiroran>
The genome was extracted by the CTAB method using a protocomb. The ITS region in rDNA retained by the bacterium was amplified using the ITS1F-ITS4B primer and the TakaRa Ex Taq kit. The PCR reaction solution was adjusted to a yield of 2 μl of DNA extract, 0.05 μl of Ex Taq polymerase, 10 μM of each primer, 0.25 μM of dNTP mix, and 10 x buffer. The reaction solution was subjected to a cycle of heat denaturation at 94 ° C. for 5 minutes, annealing at 94 ° C. for 30 seconds, annealing at 55 ° C. for 30 seconds, and extension reaction at 72 ° C. for 1 minute 30 times with an iciller. The final elongation was performed at 72 ° C. for 7 minutes. The PCR product was purified using EconoSpin. Direct sequencing was performed on the purified PCR product. The obtained DNA sequence was subjected to BLAST search. A phylogenetic tree was created with ClustalX based on multiple sequences obtained from GeneBank.

<Identification of mycorrhizal fungi at the molecular level>
Using the artificial culture method in this study, we attempted to identify the mycorrhizal fungi of Gastrodia elata. Mycorrhizal fungi were identified based on the sequences obtained from the ITS region. FIG. 4 is a table of mycorrhizal fungi identified from the medium of the protocomb of Kuroyatsushiroran (Kanagawa Prefecture) and Haruzakiyatsushiroran (Tokushima Prefecture) and the growth kit 100. The ITS sequences obtained from the protocombs of Kuroyatsushiroran (Kanagawa Prefecture) and Haruzakiyatsushiroran (Tokushima Prefecture) were in agreement with the scientific name (Diplomitoporus rimosus) and the scientific name (Thelepurus membranaeus) with a homology rate of 99% or more, respectively. Similar results were detected for these mycorrhizal fungi in the respective Sugi and Castanopsis sieboldii media. This result was found to be efficient for identifying the fungus required for the growth of Gastrodia elata by using the artificial culture method. The mycorrhizal fungi of the scientific name (Diplomitoporus rimosus) and the scientific name (Theleporus membranaeus) are both a type of Polyporales Polyporales. Therefore, the names of these mycorrhizal fungi were searched from NCBI, the genomic sequences of the registered ITS regions of the same species were compared, analyzed by StandardW, and a phylogenetic tree was created by NJprot. The results show that these two species are not very closely related. In addition, in the identification of mycorrhizal fungi, other species of fungi were also detected. In the ITS sequences obtained from the protocombs of Akizakiyatsushiroran and Haruzakiyatsushiroran, mycena spp. However, due to the low homology with these detected strains, it was not possible to find a dependent relationship in the growth of Gastrodia elata. However, it was suggested that these Gastrodia elata genus Gastrodia elata maintain a symbiotic relationship with multiple mycorrhizal fungi in a non-specific manner in order to achieve good growth.

From the above, the following results were obtained in the artificial cultivation of Gastrodia elata using the Gastrodia elata 100.
・ Succeeded in artificial cultivation on Sugi medium, it was confirmed that the same individual can flower and bear fruit three times a year. In addition, it was confirmed that one of the mycorrhizal fungi has a scientific name (Diplomitoporus rimosus).
・ Succeeded in artificial cultivation in Akizakiyatsushiroran cedar medium and mouchiku medium. In addition, it was confirmed that one of the mycorrhizal fungi has a scientific name (Gerlonema strandodes).
・ Succeeded in artificial cultivation in Sugi medium and Castanopsis sieboldii medium. Moreover, it was confirmed that one of the mycorrhizal fungi has a scientific name (Theleporus membranaeus).

From the above experimental results, it was confirmed that the humus cones of Sugi had a positive effect on the growth of mycorrhizal fungi and the growth of Yatsushiroran. Many of the cedar cones have developed a wide variety of hyphae, and the complex 3-D structure of the cedar cones for symbiotic fungi has an appropriate humidity and vapor phase for the fungi. It was confirmed that the growing environment was good, and that the addition of cedar cones to the medium activated the growth activity of the symbiotic fungi and had a positive effect on this artificial cultivation. It was also confirmed that the installation of gauze on the ceiling surface prevented excessive dew condensation and dew condensation, maintained a moderate humidity, and had a positive effect on artificial cultivation. In addition, by keeping the room temperature in the growth kit 100 at around 20 to 25 ° C and the humidity in the growth kit 100 at around 80 to 85%, the growth of Yatsushirorans and the activity of symbiotic bacteria can be activated. confirmed. Results also suggest that Yatsushiroran coexists with multiple symbiotic bacteria and different mycorrhizal fungi during each growth process.

In addition, gastrodin (English name: Gastrodin) was discovered in the tubers of Yatsushiroran obtained from the above experimental results. The method of extracting gastrodin from the tubers of Yatsushiroran is shown below.
(A) Add an appropriate amount of 50% methanol to the tubers of Yatsushiroran and crush them in a mortar (10 ml of methanol is used per 1 g of tubers).
(B) Transfer the crushed bulbs and the extract to an eggplant flask, and perform extraction for about 10 minutes while refluxing in hot water at 80 ° C.
(C) Filter the extract using a predetermined filter paper to obtain a crude extract.
(D) Add an equal amount of petroleum ether to the crude extract, fractionate using a separating funnel, and separate the aqueous layer (repeat 3 times in this example).
(E) Add an equal amount of ethyl acetate to the aqueous layer, fractionate using a separating funnel, and separate the aqueous layer (repeat 3 times in this example).
(F) The obtained aqueous layer is concentrated by an evaporator.
(G) Resuspend in 50% methanol to prepare a crude purified solution.
By the above method, gastrodin was extracted from the tubers of Yatsushiroran. In addition, the highest concentration of gastrodin extract could be extracted from the tubers immediately before flowering. In addition, gastrodin may be extracted by a method other than the above.
It was also confirmed that the artificial cultivation method of the fungal heterotrophic Orchidaceae plant according to the present embodiment is also effective in the artificial cultivation of Gastrodia elata.

Until now, the fungi of the group have been regarded as symbiotic plants of mycotrophic plants, for example, in the genus Symbidium of the Orchidaceae, the diversity of fungi that coexist in the most closely related independent and mycotrophic plants. As a result of comparison, it has been considered that the diversity of symbiotic fungi of fungal-dependent vegetative plants is significantly low, and the species specificity between fungal-dependent orchidaceous plants and symbiotic fungi is high. However, it is said that the root fungus of Kuroyatsushiroran and the root fungus of Akizakiyatsushiroran and Haruzakiyatsushiroran established a symbiotic relationship with Akizakiyatsushiroran, and the root fungus of Galeola hydra established a symbiotic relationship with Galeola sp. What is done is that the orchids that coexist with the root fungus belong to the same genus as the orchids from which the root fungus is isolated, and that the symbiotic fungi of the same three species of Gastrodia genus Gastrodia have a proportion of Mycenaceae Marasmiaceae. Although there are many, a wide variety of fungi such as Ceratobaciaceae and Polyporaceae have been detected, and from the experimental results confirming the effect of the artificial cultivation method according to this embodiment, the partner who establishes a common symbiotic relationship among the three species of orchids of the same genus. The presence of the root fungus Myceneaceae or Polyporaceae has been confirmed, and the specificity of the species regarding the root fungus symbiosis for these three orchids is low, and while taking various risks during evolution (for example, being violated by phytopathogenic fungi). It was shown that it may be in the process of exploring the relationship building of partnerships with various symbiotic fungi for the future. Therefore, in the artificial cultivation method according to the present embodiment, various mycorrhizal fungi existing in the humus and humus around the own dough are collected without inoculating the seeds by extracting a specific fungus. It was confirmed that artificial cultivation of fungal heterotrophic orchidaceous plants is possible. In addition, the efficacy of Chinese herbal medicine is known for Gastrodia elata, which belongs to the same genus Gastrodia elata, and there are many reports on cultivation methods. There are no reports about. Further, it is an epoch-making breeding method that has never been seen before that a large amount of cultivation is possible in a short period of time by using the breeding kit 100 by the artificial cultivation method according to the present embodiment. Gastrodia elata is famous for coexisting with armillaria, but this is only after the growth has progressed, and it has been reported that the early stage of seedlings from germination coexists only with Mycena spp. It is said that the combination of types of bacteria that coexist with each other changes, and the optimal bacterial partner shifts for each growth stage in the armillaria elata. Therefore, by identifying symbiotic fungi, isolating and culturing them, and applying the optimum combination of fungi to the artificial cultivation method according to this embodiment, not only for the purpose of horticultural cultivation, but also for endangered plants. Biological education because it can be observed without destroying the morphology of the underground part, such as proliferation, conservation of biodiversity by local sowing and transplantation, utilization of fungal dependent vegetative plants with medicinal properties in the medical field, etc. It opens the way to various possibilities such as use as teaching materials in the field. In addition, since the seeds of Gastrodia elata can be obtained in 3 months by this method, it is expected to play a role as a model plant equivalent to arabidopsis known as a model plant. Modulation and mating between Gastrodia elata are also possible. Furthermore, inbreed mating by self-pollination is also possible.

The present invention can be widely used in various fields such as conservation of endangered biodiversity, medical field, horticultural field, and teaching material in biological education.

100 Growing Kid 11a Lid 11b Housing 12 Vinyl 13 Moisture Absorber 20 Log 30 Cone 40 Humus

Claims (1)

As a medium for fungal heterotrophic Orchidaceae plants, logs with humus and multiple cones with humus were used.
A method for growing a fungal heterotrophic Orchidaceae plant in which the indoor environment in which the medium is present is set to a temperature of 15 ° C to 30 ° C and a humidity of 70% or more.

Images