A method for ex-situ conservation of vandopsis gigantea

Abstract

The present application relates to the field of biodiversity protection, and particularly relates to a method for ex-situ conservation of Vrydaghiella. The present application uses symbiotic fungi of Vrydaghiella to symbiotically germinate mature Vrydaghiella seeds on a substrate composed of tree bark, water moss and the like, and cultivate the seeds into plants for ex-situ conservation. The use of symbiotic fungi enables the successful realization of Vrydaghiella seeds as ex-situ introduction materials, and effectively improves the survival rate of ex-situ conservation of Vrydaghiella.

Description

Technical Field

[0001] This invention relates to the field of biodiversity conservation, specifically to a method for the ex-situ conservation of dwarf Vanda orchids. Background Technology

[0002] Ex-situ conservation refers to the relocation of species whose survival and reproduction are severely threatened due to factors such as the loss of habitat or extremely low population numbers, from their original locations to botanical gardens or plant breeding centers for special protection and management. Ex-situ conservation is an important method primarily targeting rare and endangered plants, especially those threatened species, populations, or scattered individual plants distributed outside the effective protection area of ​​nature reserves. It complements in-situ conservation and is an important part of biodiversity conservation.

[0003] Orchidaceae, a major family of angiosperms, is frequently over-harvested for ornamental, medicinal, and scientific purposes, resulting in severe damage to wild orchid resources and making it a "flagship" group for plant conservation. Ex-situ conservation is a crucial method for protecting wild orchids, but because orchid seeds are tiny, consisting of only a spherical embryo without endosperm, they are difficult to germinate under natural conditions. Artificial germination of orchid seeds mostly relies on aseptic sowing. However, due to limitations in aseptic sowing conditions and seed germination characteristics, aseptic sowing techniques for orchids have only been successfully implemented in a few species.

[0004] In existing technologies, the introduction process for ex-situ conservation of orchids usually involves transplanting live plants. The wild digging of live plants sacrifices the population of already endangered species in need of conservation. In addition, the survival rate of live plants after being removed from their native environment is usually low. Therefore, the introduction and conservation methods for orchids are not efficient and have a high mortality rate. This not only damages the native plant population but also reduces the genetic diversity of the conservation species.

[0005] Vanda pumila Hook.f. is a rare orchid species listed in CITES Appendix II as Vulnerable (VU, IUCN standard). Current ex-situ conservation techniques not only damage native plant populations but also reduce the genetic diversity of Vanda pumila. This genetic diversity directly impacts the feasibility of returning artificially bred populations to the wild and restoring existing wild populations. Therefore, there is an urgent need to develop a new ex-situ conservation method for Vanda pumila that can preserve its genetic diversity. Summary of the Invention

[0006] In view of the shortcomings of existing ex-situ conservation technologies, one of the objectives of this invention is to provide a method for the ex-situ conservation of dwarf Vanda orchids.

[0007] Furthermore, the ex-situ conservation method for dwarf Vanda provided by this invention uses the seeds of the dwarf Vanda plant as conservation material.

[0008] Furthermore, the ex-situ conservation method for dwarf Vanda orchids according to the present invention includes:

[0009] S1: Cultivate symbiotic fungi of dwarf Vanda orchids to form mycelia;

[0010] S2: Sow mature seeds of dwarf Vanda orchids in the culture bed;

[0011] S3: The culture bed obtained from S2 is used to cultivate seedlings through symbiotic germination.

[0012] S4: Seedlings obtained from transplanting S3, and cultured using conventional methods.

[0013] Furthermore, the symbiotic fungus of the dwarf Vanda in step S1 is AWD.

[0014] Furthermore, step S1 includes:

[0015] S11: Inoculate the preserved AWD strain onto PDA medium and culture until colonies with a diameter of about 5 cm form on the surface of the medium, with a large number of aerial hyphae forming.

[0016] S12: Cut a piece of mycelium from S11 medium, inoculate it onto sterile solid medium, and culture it until the mycelium has fully grown on the solid medium.

[0017] Furthermore, the culture conditions for step S11 are: culture at 25-30℃ in the dark for 5-10 days.

[0018] Furthermore, in step S12, the size of the cut fungal block is (0.5-1.5)*(0.5-1.5) cm.

[0019] Furthermore, the sterilized solid culture medium is a 1:1 mixture of hardwood sawdust and chopped leaves, contained in a 500 mL glass bottle.

[0020] Furthermore, the culture conditions for step S12 are: cultured at 20-30℃ in the dark for 10-20 days.

[0021] Furthermore, the culture bed described in step S2 is formed by spreading the solid culture medium covered with mycelia in step S12, with a thickness of 1-5 cm.

[0022] Furthermore, the cultivation conditions for step S3 are as follows: cultivation at 20-30℃, humidity 70%-90%, and light intensity 800lx-2000lx until the seeds symbiotically germinate to form seedlings.

[0023] Furthermore, the ex-situ conservation conventional cultivation substrate described in step S4 is tree bark, coconut husk, sphagnum moss, or a mixture thereof.

[0024] Secondly, the present invention also provides a symbiotic fungus for dwarf Vanda orchids.

[0025] Furthermore, the symbiotic fungus of the dwarf Vanda orchid is named AWD, and it was deposited on January 9, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 41753.

[0026] Furthermore, the morphological characteristics of the symbiotic fungi of the dwarf Vanda are as follows: the colonies are white, with a large number of aerial hyphae, flocculent, and both the front and back of the colonies are white. The colony edges are relatively neat, but the thickness is uneven, with the edges being thinner.

[0027] Furthermore, the symbiotic fungi of the dwarf Vanda orchid were molecularly identified as belonging to the genus Ceratobasidium sp.

[0028] Furthermore, the ITS gene sequence of the symbiotic fungus of the dwarf Vanda is shown in SEQ ID No. 1.

[0029] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0030] (1) A culture technique for fungal symbiotic germination is provided. By using suitable symbiotic fungi, only a small number of pods can be harvested, and seedlings can be formed by establishing a symbiotic relationship between the seeds and the symbiotic fungi. This method has low requirements for equipment and environment; the seedlings formed have high adaptability to the environment.

[0031] (2) A method for ex-situ conservation based on seeds of dwarf Vanda orchids as ex-situ conservation material is provided. The method includes the steps of strain cultivation, sowing and transplanting, involving the use of symbiotic culture technology, which enables dwarf Vanda orchid seeds to be used as ex-situ conservation material, protects the genetic diversity of ex-situ conservation species, reduces the destruction of native wild populations, and effectively solves the problems of high contamination rate and high operation difficulty of aseptic sowing.

[0032] (3) A symbiotic fungus for dwarf Vanda is provided. This symbiotic fungus is isolated from the native environmental material of dwarf Vanda and has a highly efficient and benign interaction with the dwarf Vanda, enabling the plant to obtain nutrients and water more effectively, thereby promoting its growth and development. At the same time, the seedlings with the fungus are more adaptable to the environment, can better adapt to different growth environments and climatic conditions, and have stronger vitality and competitiveness. Detailed Implementation

[0033] To make the technical problem to be solved, the technical solution, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. The present invention will be specifically described below with reference to specific embodiments.

[0034] The introduction and relocation of orchids for ex-situ conservation typically involves transplanting live plants. However, the digging up of live plants from the wild sacrifices the already endangered populations of these species. Furthermore, the survival rate of live plants removed from their native environment is usually low. Therefore, the introduction and relocation method for orchids is inefficient and has a high mortality rate. The method of this invention utilizes symbiotic fungi, allowing only a small number of pods to be harvested. Seeds form seedlings through a symbiotic relationship with the fungi, minimizing disruption to the native endangered species' populations.

[0035] Existing technologies also employ a few methods for introducing and conserving orchid seeds, but these are limited by aseptic sowing techniques. Aseptic sowing requires a well-equipped plant tissue culture facility, and there are differences in sowing techniques between different species. Factors such as seed age and the presence of pathogens on the seed surface limit the success rate of aseptic sowing. Furthermore, seedlings must undergo hardening-off processes after being removed from the culture vessel, which can lead to varying degrees of seedling death and damage, further reducing the success rate of introduction and conservation. The symbiotic fungus ex-situ conservation technology of this invention has lower requirements for equipment and environment, requiring only a small amount of equipment. In addition, the symbiotic germination technology of this invention utilizes the self-renewal mechanism of dwarf Vanda orchids in their natural state. Seedlings can be easily obtained simply by selecting mature seeds and co-culturing them with suitable symbiotic fungi. This method can improve the plant's adaptability to the environment. During symbiotic germination, the seed and fungus form a mutually beneficial symbiotic relationship, which allows the plant to more effectively obtain nutrients and water, thereby promoting its growth and development. At the same time, due to the involvement of fungi, the seedlings carrying the fungus are more adaptable to the environment, better able to adapt to different growth environments and climatic conditions, and have stronger vitality and competitiveness.

[0036] This invention provides a method for the ex-situ conservation of dwarf Vanda orchids.

[0037] Specifically, the ex-situ conservation method for dwarf Vanda provided by this invention uses the seeds of the dwarf Vanda plant as conservation material.

[0038] Specifically, the ex-situ conservation method for dwarf Vanda orchids described in this invention includes:

[0039] S1: Cultivate symbiotic fungi of dwarf Vanda orchids to form mycelia;

[0040] S2: Sow mature seeds of dwarf Vanda orchids in the culture bed;

[0041] S3: The culture bed obtained from S2 is used to cultivate seedlings through symbiotic germination.

[0042] S4: Seedlings obtained from transplanting S3, and cultured using conventional methods.

[0043] Specifically, the symbiotic fungus of the dwarf Vanda in step S1 is AWD.

[0044] Specifically, step S1 includes:

[0045] S11: Inoculate the preserved AWD strain onto PDA medium and culture until colonies with a diameter of about 5 cm form on the surface of the medium, with a large number of aerial hyphae forming.

[0046] S12: Cut a piece of mycelium from S11 medium, inoculate it onto sterile solid medium, and culture it until the mycelium has fully grown on the solid medium.

[0047] Specifically, the culture conditions for step S11 are: culture at 25-30℃ in the dark for 5-10 days.

[0048] Specifically, in step S12, the size of the cut fungal block is (0.5-1.5)*(0.5-1.5) cm.

[0049] Specifically, the sterilized solid culture medium is a 1:1 mixture of hardwood sawdust and chopped leaves, which is contained in a 500mL glass bottle.

[0050] Specifically, the culture conditions for step S12 are: culture at 20-30℃ in the dark for 10-20 days.

[0051] Specifically, the culture bed mentioned in step S2 is made up of the solid culture medium covered with mycelia in step S12, and its thickness is 1-5 cm.

[0052] Specifically, the cultivation conditions for step S3 are as follows: cultivation at 20-30℃, humidity 70%-90%, and light intensity 800lx-2000lx until the seeds symbiotically germinate to form seedlings.

[0053] Specifically, the ex-situ conservation conventional cultivation substrate mentioned in step S4 is tree bark, coconut husk, sphagnum moss, or a mixture thereof.

[0054] Secondly, the present invention also provides a symbiotic fungus for dwarf Vanda orchids.

[0055] Specifically, the symbiotic fungus of the dwarf Vanda orchid is named AWD, and it was deposited on January 9, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 41753.

[0056] Specifically, the morphological characteristics of the symbiotic fungi of the dwarf Vanda are as follows: the colonies are white, with a large number of aerial hyphae, which are flocculent. Both the front and back of the colonies are white, the edges of the colonies are relatively neat, and the thickness is uneven, with the edges being thinner.

[0057] Specifically, the symbiotic fungi of the dwarf Vanda orchid were molecularly identified as belonging to the genus Ceratobasidium sp.

[0058] Specifically, the ITS gene sequence of the symbiotic fungus of the dwarf Vanda is shown in SEQ ID No. 1.

[0059] (SEQ ID No.1).

[0060] The following description is based on specific embodiments:

[0061] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available, and techniques not described in detail were performed according to standard methods well known to those skilled in the art.

[0062] Example 1: Ex-situ conservation of symbiotic fungi of Dwarf Vanda introduced from Yunnan

[0063] S11: Inoculate the preserved AWD strain onto PDA medium and incubate at 28°C in the dark for 7 days until colonies with a diameter of about 5 cm form on the surface of the medium and a large number of aerial mycelia are formed.

[0064] S12: Cut 1×1 cm mycelial blocks from S11 medium and inoculate them onto a sterile solid culture medium of 1:1 broadleaf sawdust and shredded leaves. The culture medium is placed in a 500 mL glass bottle. The inoculated culture is incubated at room temperature (25°C) in the dark for 15 days until the mycelium has fully grown onto the solid culture medium.

[0065] S2: A 2cm thick culture bed is made of solid culture medium covered with mycelium, and mature dwarf Vanda seeds introduced from Yunnan Province are sown on the surface of the culture bed.

[0066] S3: The culture bed is placed under the conditions of 25℃, 70% humidity and 1500lx light until the seeds germinate symbiotically to form seedlings. The seed germination rate is 46% and the seedling rate is 100%.

[0067] S4: The symbiotically germinated seedlings are transplanted into a conventional ex-situ conservation substrate for cultivation in an open nursery with 50% shade.

[0068] Example 2: Ex-situ conservation of symbiotic fungi of Dwarf Vanda introduced from Guangxi

[0069] S11: Inoculate the preserved AWD strain onto PDA medium and incubate at 28°C in the dark for 7 days until colonies with a diameter of about 5 cm form on the surface of the medium and a large number of aerial mycelia are formed.

[0070] S12: Cut 1×1 cm pieces of fungus and inoculate them on a sterile solid culture medium of 1:1 broadleaf sawdust and shredded leaves. The culture medium is placed in a 500 mL glass bottle. The inoculated culture is incubated at room temperature (25°C) in the dark for 15 days until the mycelium has fully grown on the solid culture medium.

[0071] S2: A 2cm thick culture bed is made of solid culture medium covered with mycelium, and mature dwarf Vanda seeds introduced from Guangxi Zhuang Autonomous Region are sown on the surface of the culture bed;

[0072] S3: The culture bed is placed under the conditions of 25℃, 90% humidity and 800lx light until the seeds germinate symbiotically to form seedlings. The seed germination rate is 51% and the seedling rate is 100%.

[0073] S4: The symbiotically germinated seedlings are transplanted into a conventional ex-situ conservation substrate for cultivation in an open nursery with 50% shade.

[0074] Comparative Example 1

[0075] A 2cm thick culture bed was prepared by spreading a sterilized solid culture medium of 1:1 broadleaf sawdust and shredded leaves, and mature dwarf Vanda seeds introduced from Yunnan Province were sown on the surface of the culture bed.

[0076] The seed germination rate was 0 when the culture bed was placed under conditions of 25℃, 70% humidity, and 1500 lx light for 6 months.

[0077] Comparative Example 2

[0078] MS culture medium was prepared, filled into glass culture flasks, and autoclaved to produce sterile culture medium.

[0079] Mature dwarf Vanda seeds introduced from Yunnan were disinfected by soaking in a 1% sodium hypochlorite solution for 7 minutes, rinsed 5 times with sterile water, and then evenly sown on the surface of a sterile culture medium.

[0080] After sowing, the culture bottles were placed in a clean culture room with a constant temperature of 25℃, humidity of 50%, and light intensity of 1800 lx. After 30 days, the contamination rate of the culture bottles was 30%. Culture continued for 90 days, and seedlings were then transferred to each bottle with 10 seedlings. After 200 days, the seedlings were removed from the bottles, hardened off, and then transplanted, with a survival rate of 70%.

[0081] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for ex-situ conservation of dwarf Vanda orchids, comprising: S1: Cultivate symbiotic fungi of dwarf Vanda orchids to form hyphae. The symbiotic fungus is AWD, which has been deposited at the China General Microbiological Culture Collection Center, Beijing, with accession number CGMCC No. 41753. The ITS gene sequence of the symbiotic fungus is shown in SEQ ID No.

1. S2: Sow mature seeds of dwarf Vanda orchids in the culture bed; S3: The culture bed obtained from S2 is used to cultivate seedlings through symbiotic germination. S4: Seedlings obtained from transplanting S3, and cultured using conventional methods.

2. The method of travel nursing according to claim 1, wherein, Step S1 includes: S11: Inoculate the AWD strain onto PDA medium and incubate at 25-30℃ in the dark for 5-10 days until colonies with a diameter of about 5 cm form on the surface of the medium and a large number of aerial hyphae are formed. S12: Cut off a piece of mycelium from S11 medium, inoculate it onto sterile solid medium, and culture it at 20-30℃ in the dark for 10-20 days until the mycelium has fully grown on the solid medium.

3. The method of travel nursing according to claim 2, wherein, The size of the cut piece of the mycelium is (0.5-1.5) (0.5-1.5) centimeters; and the sterilized solid medium is a medium of 1:1 of broadleaf wood chips: shredded leaves.

4. The method of travel nursery of claim 1, wherein, The culture bed mentioned in step S2 is made up of the solid culture medium covered with mycelium in step S12, and its thickness is 1-5 cm.

5. The method of transportable neonatology according to claim 1, wherein The cultivation conditions for step S3 are as follows: cultivation at 20-30℃, humidity 70%-90%, and light intensity 800lx-2000lx until the seeds symbiotically germinate to form seedlings.

6. A symbiotic fungus used in the ex-situ conservation method of Vanda dwarf as described in any one of claims 1-5, named AWD, has been deposited at the China General Microbiological Culture Collection Center (CGMCC) in Beijing, with accession number CGMCC No. 41753, and the ITS gene sequence of the symbiotic fungus is shown in SEQ ID No.

1.

7. The mutualistic fungus of claim 6, wherein, The morphological characteristics of the symbiotic fungi are as follows: the colonies are white, with a large number of aerial hyphae, which are flocculent. Both sides of the colonies are white, the edges of the colonies are relatively neat, and the thickness is uneven, with the edges being thinner.

8. The mutualistic fungus of claim 6, wherein, The symbiotic fungus was molecularly identified as belonging to the genus Daldinia (D. concentrica) Ceratobasidium sp. fungus.

Images