Adelea delavayi ex-situ conservation technology

Abstract

This invention relates to an ex-situ conservation technology for *Adiantum repens*, solving the problem of low survival rates after ex-situ conservation of this plant affected by engineering construction. It achieves new highs in both the quantity and quality of ex-situ conservation of *Adiantum repens*, significantly improving the protection level of rare and endangered plants. It also provides valuable reference for the ex-situ conservation of rare and endangered plants in other regions whose ecological environments have been severely damaged and whose distribution ranges are shrinking. Furthermore, it lays a solid foundation for future research and promotion of the breeding and application of endemic rare and endangered plants, providing ample domesticated breeding stock, greatly shortening the time required for breeding research and promotion, and saving human and material costs.

Description

Technical Field

[0001] This invention belongs to the field of agricultural and forestry technology, specifically relating to an ex-situ conservation technology for Adiantum repens. Background Technology

[0002] *Adiantum reniforme* L. var. *sinense* YXLin, belonging to the family Adiantaceae, is a perennial herbaceous fern, a Class I protected wild plant in China, and endemic to the country. Globally, *Adiantum reniforme* is only intermittently distributed in a narrow strip of land stretching nearly 100 kilometers along the Yangtze River, from Wanzhou District in the east to Xituo District in Shizhu County in the west, extending 3-5 kilometers into both banks. Its altitude is limited to between 80 and 430 meters, but is more concentrated between 170 and 250 meters. The concentrated distribution area is in Xinxiang, Xiaolunshan, and Shanshuping areas of Wanzhou, covering approximately 25 square kilometers. Most distribution points are located on southwest-facing cliffs or in thickets along riverbanks. *Adiantum reniforme* is endemic to the Three Gorges Reservoir area. The rhizome is short and erect, densely covered at the apex with brown lanceolate scales and multicellular, fine, long hairs. Leaves are clustered, simple, and round or reniform. The petiole has a notch of varying depth at its attachment point, and the auricles on both sides sometimes expand and overlap. The upper surface of the leaf blade forms 1-3 concentric circles around the petiole attachment point, and the leaf margin has rounded, blunt teeth. The indusia are round or nearly rectangular, with a straight upper margin, distributed along the leaf edge, close together or spaced apart, brown, membranous, and persistent. *Adiantum capillus-veneris* thrives in warm, humid, well-drained, slightly acidic soil. It is intolerant of drought, cold, and strong sunlight. The cultivation substrate requires loose, well-aerated soil. In the high temperatures of summer, direct sunlight should be avoided, and shading is necessary. Good air humidity should be maintained, and in winter, it should be moved to a warm location to prevent frost damage. *Adiantum capillus-veneris* has extremely high medicinal value. Due to severe damage to its habitat caused by engineering construction (water storage and inundation), mining for medicinal purposes, etc., its genetic diversity is low, and the genetic differentiation between different populations is low. In addition, due to changes in climate conditions, its suitable distribution area is extremely narrow and its natural regeneration capacity is poor, resulting in a sharp decline in its genetic diversity and population size. The number of wild seedlings is decreasing, and they only survive in a few rock crevices or thin soil layers on the rock surface.

[0003] There are many institutions in China engaged in the research and protection of the rare and endangered plant *Adiantum repens*, but there are very few institutions specializing in ex-situ transplantation and conservation techniques for this plant. Currently, Pan Liqin et al. (2005) conducted a study on the genetic diversity of natural populations of *Adiantum repens*, and the results showed that the genetic diversity level within *Adiantum repens* populations is relatively low compared to other ferns. Yu Shunhui et al. (2013) investigated *Adiantum repens* populations in Wanzhou District and Shizhu County, analyzed the possible reasons for its endangerment, and proposed conservation strategies, but did not provide any substantial methods. Wu Hua (2015) studied and explored the development of gametophytes and sporophytes in *Adiantum repens*, as well as the effects of different culture conditions and hormone-producing sporophytes. The effects on proliferation; Li Qiaojuan (2016) cloned the PIN gene of Adiantum repens and analyzed the expression of related genes; Yao Juan (2017) studied the gene function of ArsKN2 in Adiantum repens, and the results showed that the ArsKN2 gene can regulate the expression of key genes in the flowering integration pathway to delay flowering; Peng Ting (2017) studied DNA methylation during gametophyte development and sex organ differentiation in Adiantum repens, and MSAP analysis showed that the difference in DNA methylation level was associated with the different growth and development stages of Adiantum repens; Li Qiulin et al. (2018) conducted field visits and wild community surveys in the distribution area of ​​Adiantum repens to study its proliferation. The survival status was recorded in detail, and a comprehensive and systematic study of the *Adiantum repens* community was conducted using methods such as floristics, community ecology, and population ecology. Quantitative ecology methods were used to analyze the community characteristics of *Adiantum repens*, revealing its population characteristics and their relationship with environmental factors. However, no research was conducted on the ex-situ conservation and domestication techniques of *Adiantum repens*. Fu Qi et al. (2020) used high-throughput sequencing and bioinformatics to study the gametophytes of *Adiantum repens* at different reproductive modes and developmental stages, revealing the molecular regulatory mechanisms related to sex organ differentiation and efficient sporophyte production through agametosis. Chen Huiyuan et al. (2022) studied the *Adiantum repens*... Research on division propagation of Adiantum repens has been conducted, and a division propagation system for Adiantum repens has been established for the first time. The Adiantum repens parent plant used in this article was obtained from ex-situ conservation and domestication at our institution. This technique has improved the survival rate of division propagation and transplanting of Adiantum repens after ex-situ domestication to a certain extent. However, division propagation based on ex-situ conservation and domestication of Adiantum repens is much easier than ex-situ conservation of wild seedlings. Sun Weiyue et al. (2022) used simplified genome sequencing technology to study the endangered mechanism of Adiantum repens, revealing that low natural regeneration capacity and human interference may be the main reasons for the endangerment of Adiantum repens. However, they did not study the ex-situ conservation and domestication techniques of Adiantum repens. Given that the rare and endangered plant Adiantum repens has particularly high requirements for ex-situ adaptability and survival is a key factor, the above research and techniques have not substantially solved the core technology of ex-situ conservation of Adiantum repens. Summary of the Invention

[0004] This invention proposes an ex-situ conservation technology for Adiantum repens, which has important reference value for the protection of rare and endangered species in China.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A technique for ex-situ conservation of Adiantum repens, the method comprising the following steps:

[0007] Pre-ex-situ conservation survey of S1 Adiantum repens:

[0008] S2 relocation uses constant temperature and humidity, rapid recovery relocation technology to transplant from the original site to the relocation area for planting, so that digging and planting can be completed on the same day;

[0009] S3 after its relocation, management of Adiantum repens;

[0010] S4 Observation and Evaluation: Observe and evaluate the morphological changes, reproductive capacity, ecological suitability, and ex-situ survival rate of Adiantum repens in the new location.

[0011] S5 File Management: Establish a file management method for each ex-situ protected Adiantum repens, and regularly record growth according to the transplanting time to accurately grasp the growth and development pattern of ex-situ protected Adiantum repens;

[0012] Complete the ex-situ conservation of Adiantum repens.

[0013] Preferably, step S1, the pre-ex-situ conservation survey of *Adiantum repens*, includes:

[0014] S1.1 Conduct a native habitat survey of Adiantum repens, focusing on wild plants affected by engineering construction, medicinal herb collection, or other factors;

[0015] S1.2 Investigate the affected distribution range, number of plants, population structure, growth status, in-situ conservation status, or other conditions of Adiantum repens;

[0016] S1.3 Investigate the altitude, air humidity, annual frost-free period, average of the highest and lowest temperatures over three years, or other meteorological data of the native habitat of the ex-situ Adiantum repens, to identify the suitable slope aspect for the growth of Adiantum repens in its native habitat.

[0017] S1.4 Soil samples were taken from the native habitat of Adiantum repens for testing. The main tests included soil structure, organic matter content, pH, and other parameters. The physical properties, chemical characteristics, and microbial activity of the understory soil were investigated to understand the soil physicochemical and biological environmental conditions.

[0018] S1.5 Investigate the symbiotic vegetation communities around the Adiantum repens that require ex-situ conservation, including trees, shrubs, herbs and other ferns;

[0019] S1.6 Survey Results: Based on the above survey, wild plants affected by engineering construction, medicinal herb collection, or other factors were selected for rescue and protection. In accordance with the "habitat similarity principle," a comprehensive investigation and comparison were conducted on microclimate conditions such as light, water, air, and heat, as well as soil conditions, altitude, and surrounding environmental factors. Habitat differences were controlled within the range where Adiantum repens can thrive, and relocation was carried out according to the actual situation. Seedling selection should be carried out before digging, and the selected seedlings should be numbered sequentially, with one card for each seedling, and registered and managed in categories.

[0020] More preferably, step S2 specifically includes the following steps:

[0021] S2.1 Pretreatment of live plants;

[0022] S2.2 Excavation of living plants;

[0023] S2.3 Constant temperature and humidity treatment for living plants;

[0024] S2.4 Ex-situ planting of living plants.

[0025] More preferably, step S2.1, the pretreatment of live plants, includes:

[0026] S2.1.1 Timing: Early spring, March 15th - April 15th, when the soil temperature should be maintained above 10-15℃;

[0027] S2.1.2 Pruning: Prune some withered and yellow branches and leaves according to the growth condition; clean up the debris and weeds around the plant.

[0028] S2.1.3 Foliar treatment: To ensure survival, delay leaf wilting, and increase stomatal diffusion capacity, spray the leaves with a 30-50 ppm solution of GGR6 (GGR6 green plant growth regulator, Beijing Aibidi Biotechnology Co., Ltd.) until droplets fall.

[0029] S2.1.4 Bagging of above-ground parts: After the leaves are treated, cover the above-ground parts of the plant with a protective bag to keep them warm and moist.

[0030] More preferably, step S2.2, digging up the living plant, includes:

[0031] S2.2.1 The pretreated live plants are dug up, and the digging time is before 7:00-10:00 am in March;

[0032] S2.2.2 Dig a circular trench 5-7cm from the periphery of the plant, and then dig a soil ball in a pot-bottom shape after digging down the circular trench for 10-15cm.

[0033] After excavation is completed, spray the root area thoroughly with a GGR6 solution (50-80ppm, Beijing Aibiti Biotechnology Co., Ltd.). First, wrap the root ball with non-woven fabric, and then tie it with straps to reduce the root ball from falling apart during transportation.

[0034] More preferably, step S2.3, the constant temperature and humidity treatment of the living plant, includes:

[0035] S2.3.1 Plant treatment: Inject the self-made "root vitality preservation solution" into the soil ball, 15-25ml per plant; S2.3.2 Constant temperature: Adjust the temperature to 10-15℃;

[0036] S2.3.3 Humidification: Use a humidifier to maintain the air humidity in the portable constant temperature storage box within the range of 60-70%.

[0037] More preferably, the "root vitality preservation solution" in step S2.3.1 is a mixture of 0.25% potassium chloride (KCl) and 0.15% ascorbic acid (ASA).

[0038] More preferably, step S2.4, ex-situ planting of living plants, includes:

[0039] S2.4.1 Selection of relocation site: Relocate to greenhouse cultivation;

[0040] S2.4.2 Preparation of planting soil substrate in the relocation site: According to the soil requirements of the native habitat of Adiantum repens, the backfill soil of the planting hole should be prepared with soil substrate.

[0041] S2.4.3 Live Planting: The planting hole is set in layers, with a drainage layer, a native soil layer, and a nutrient soil layer; the drainage layer is made of gravel from the Three Gorges area, with a thickness of 5-10cm, for timely drainage during heavy rain; the thickness is 10-15cm, for the connection between the root ball of the maidenhair fern and the native soil; the nutrient soil layer is a composite nutrient substrate backfilled with soil matrix from S2.4.2, for filling and compacting around the root ball of the plant.

[0042] S2.4.4 Habitat Similarity Creation: In the planting area after the relocation of the Adiantum revolutum, plant the vegetation and companion tree species from its original habitat to create a "habitat similarity".

[0043] More preferably, the post-migration management of *Adiantum repens* in step S3 includes:

[0044] The soil matrix in step S2.4.2 includes the following components by weight: 3-5 parts of weathered limestone sand from the Yichang section of the Three Gorges Dam area, 1.5-2.5 parts of peat soil, 1.5-2.5 parts of vermiculite, and 0.5-1.5 parts of calcareous gray soil. The soil also includes physical and chemical properties such as pH value, EC value, and organic matter content. The particle size of the weathered limestone sand from the Yichang section of the Three Gorges Dam area is 5-10 mm. The pH value of the soil matrix is ​​adjusted to 6.0-7.0, the organic matter content is 3-5%, and the matrix moisture content is 60-70%.

[0045] More preferably, the post-migration management of *Adiantum repens* in step S3 includes:

[0046] S3.1 Simulates the wild growth environment of the maidenhair fern by constructing a shade net with a light transmittance of 65-75% to prevent direct sunlight in summer;

[0047] S3.2 Construct a misting system, i.e., fine mist nozzles, around the plant to maintain constant air humidity and create a microclimate similar to its native habitat, making the Adiantum repens suitable for its current growth environment;

[0048] S3.3 Rejuvenate the ex-situ Adiantum repens by dripping nutrient solution into the soil. The nutrient solution is a 0.5-1.5% CaCl2 solution. Since Adiantum repens has a low calcium absorption capacity and a low calcium bioavailability coefficient, the addition of CaCl2 solution can increase the translocation capacity of plant messengers and enhance the environmental adaptability and resistance of Adiantum repens.

[0049] S3.4 Spray the leaves with a homemade sunscreen and water-retaining solution;

[0050] S3.5 Irrigation water uses natural, pollution-free water sources, and irrigation time is before 8:00 AM or after 6:00 PM;

[0051] S3.6 Regularly carry out meticulous maintenance management measures such as loosening the soil, weeding, and pest and disease control.

[0052] The sunscreen and moisturizing solution in step S3.4 is: aloe vera extract 180-220g / L + Senhuo Bishui brand plant transpiration inhibitor 45-55g / L + salicylic acid 5-15mg / L;

[0053] Preparation method of aloe vera extract: Take 500g of fresh aloe vera, wash it clean, cut it into pieces, crush it with a juicer, filter out the juice and set it aside. Prepare the extract at a ratio of 180-220g / L.

[0054] A sunscreen and water-retaining liquid for the ex-situ protection of Adiantum repens, wherein the sunscreen and water-retaining liquid is composed of: aloe vera extract 180-220g / L + Senhuo Bishui brand plant transpiration inhibitor 45-55g / L + salicylic acid 5-15mg / L;

[0055] Preparation method of aloe vera extract: Take 500g of fresh aloe vera, wash it clean, cut it into pieces, crush it with a juicer, filter out the juice and set it aside. Prepare the extract at a ratio of 180-220g / L.

[0056] The sunscreen and water-retaining liquid is applied to the management of *Adiantum repens* after its introduction. The beneficial effects of this invention are:

[0057] 1. This invention overcomes the problems of rapid dehydration and high mortality rates in the ex-situ conservation techniques of the rare and endangered plant *Adiantum repens*, resulting in losses of human and material resources, especially plant resources. Compared with existing conventional ex-situ methods, it effectively solves the problems of water loss and wilting, root damage, nutrient imbalance, and easy death of wild *Adiantum repens* during ex-situ relocation, significantly improving the survival rate to 95%. The acclimatization period after relocation is shortened by nearly 90 days, with sporangia forming in mid-to-late July and spores maturing in mid-to-late August, allowing for spore collection and propagation within the same year. This lays a solid foundation for the breeding research and promotion of the unique rare and endangered plant *Adiantum repens*, providing sufficient acclimatized breeding stock, greatly shortening the time for breeding research and wild reintroduction, expanding the population size, and reducing the risk of extinction.

[0058] 2. A series of scientific management measures were adopted, which solved the problems of poor growth and weak plants of Adiantum repens after transplanting, improved environmental adaptability, accelerated the rejuvenation process, and the plants began to produce spores within 3-4 months after transplanting.

[0059] 3. The self-made "sunscreen and water-retaining liquid" and "root vitality preservation liquid" and the constructed "mist system" can alleviate the negative effects of high temperature stress on the photosynthesis, respiration, transpiration and antioxidant system of the Adiantum capillus-veneris. Attached Figure Description

[0060] Figure 1 Actual specimens showing the survival and growth of different sizes of *Adiantum repens* under ex-situ conservation. Figure 1 ;

[0061] Figure 2 Actual specimens showing the survival and growth of different sizes of *Adiantum repens* under ex-situ conservation. Figure 2 ;

[0062] Figure 3 Ex-situ conservation of Adiantum repens spores Figure 1 ;

[0063] Figure 4 Ex-situ conservation of Adiantum repens spores Figure 2 ;

[0064] Figure 5 Ex-situ conservation of Adiantum repens spores for seedling propagation;

[0065] Figure 6 : Promotion and application of ex-situ conservation of Adiantum repens seedlings;

[0066] Figure 7 Shade netting installation method (actual example) Figure 1 ;

[0067] Figure 8 Shade netting installation method (actual example) Figure 2 . Detailed Implementation

[0068] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0069] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0070] Example 1

[0071] A technique for ex-situ conservation of Adiantum repens, the method comprising the following steps:

[0072] 1. Pre-ex-situ conservation survey of *Adiantum repens*

[0073] 1.1 Conduct a survey of the native habitat of Adiantum repens, focusing on wild plants affected by engineering construction, medicinal herb collection, etc.

[0074] 1.2 Investigate the distribution range, number of plants, population structure, growth status, and in-situ conservation status of affected Adiantum repens.

[0075] 1.3 Investigate meteorological data such as altitude, air humidity, frost-free period, and average of the highest and lowest temperatures over three years in the native habitat of *Adiantum repens* to identify the suitable slope aspect for *Adiantum repens* in its native habitat, thus preparing for future ex-situ conservation.

[0076] 1.4 Soil samples were collected from the native habitat of *Adiantum repens* for testing, primarily focusing on soil structure, organic matter content, and pH. The main investigations covered the physical properties, chemical characteristics, and microbial activity of the understory soil to understand its physicochemical and biological environmental conditions.

[0077] 1.5 Investigate the symbiotic vegetation communities around the Adiantum repens that require ex-situ conservation, mainly consisting of trees, shrubs, herbs and other ferns.

[0078] 1.6 Survey Results

[0079] Based on the above investigation, wild plants affected by construction projects and medicinal herb collection were selected for rescue and protection. Following the principle of habitat similarity, a comprehensive comparison was conducted considering microclimate conditions such as light, water, air, and heat, as well as soil conditions, altitude, and surrounding environmental factors. Habitat differences were controlled within the suitable range for *Adiantum repens*, and relocation was carried out based on the actual situation. Seedling selection should be conducted before harvesting, and each selected seedling should be sequentially numbered, labeled, registered, and managed according to its classification.

[0080] 2. Employing relocation technology that combines constant temperature and humidity control with rapid recovery.

[0081] To enable the maidenhair fern to quickly adapt to the ex-situ growth environment, a constant temperature and humidity, rapid recovery ex-situ technology is adopted. The transplanting time from the original site to the ex-situ planting is minimized, and the digging and planting are completed on the same day. The planting and management are carried out in a way that simulates the excellent climatic conditions and soil conditions of the original site, such as air humidity, temperature, and light.

[0082] 2.1 Pretreatment of live plants

[0083] (1) Selection of relocation time: Early spring, from March 15 to April 15, is most suitable. The Adiantum capillus-veneris is not tolerant of low temperature, and the soil temperature should be maintained above 10-15℃.

[0084] (2) Pruning: Prune some of the withered and yellow branches and leaves according to the growth condition to reduce nutrient consumption. Remember not to prune too many branches and leaves; clean up the debris and weeds around the plant.

[0085] (3) Leaf treatment: In order to ensure survival, delay the wilting time of leaves and increase the stomatal diffusion capacity, use GGR6 to prepare a 60ppm solution and spray it on the leaf surface until droplets fall.

[0086] (4) Bagging the above-ground parts: After the leaves are treated, put the above-ground parts of the plant in a protective bag to keep them warm and moist in early spring.

[0087] 2.2 Excavation of living plants

[0088] (1) The pretreated live plants were dug up. According to the growth habits of the maidenhair fern in its native habitat, the digging time was before 7:00-10:00 am (March).

[0089] (2) Dig a circular trench 5-7cm away from the outer edge of the plant, and dig down 10-15cm along the circular trench to dig a soil ball in a pot-bottom shape to protect the soil ball so that it has a relatively complete root system.

[0090] (3) After excavation, spray the root area thoroughly with GGR6 solution with a content of 60ppm. First wrap the soil ball with non-woven fabric, and then tie the soil ball with binding straps to reduce the soil ball from falling off during transportation.

[0091] 2.3 Constant temperature and humidity treatment for living plants

[0092] (1) Plant treatment: Inject the soil ball with a self-made "root vitality preservation solution", 20 ml per plant. The "root vitality preservation solution" is a mixture of 0.2% potassium chloride (KCl) and 0.1% ascorbic acid (ASA);

[0093] (2) Constant temperature: Use a portable constant temperature storage box to adjust the temperature to 10-15℃;

[0094] (3) Humidification: Use a humidifier to keep the air humidity in the portable constant temperature storage box within the range of 60-70%.

[0095] 2.4 Ex-situ planting of living plants

[0096] (1) Selection of relocation site: Since light and air humidity are very important for the growth of maidenhair fern, it cannot grow well under strong light and requires a certain degree of shade. It is not tolerant of low temperature in winter and high temperature and drought in summer, and is extremely demanding in terms of environmental conditions. Considering the above factors, when relocating live wild plants to different regions, the environmental control of the relocation site should be the primary consideration. Relocation to greenhouse cultivation is the most ideal choice.

[0097] (2) Preparation of planting soil substrate in the relocation site: According to the soil requirements of the native habitat of Adiantum repens, the backfill soil of the planting hole should be prepared. The special substrate ratio is 4 parts weathered sand (particle size of about 5-10mm), 2 parts peat soil, 2 parts vermiculite, and 1 part gray calcium soil. The physical and chemical properties of the prepared soil such as pH value, EC value, and organic matter content are tested. The pH value is adjusted to the range of 6.0-7.0, the organic matter content is in the range of 3-5%, and the substrate moisture content is in the range of 60-70%.

[0098] (3) Live Planting: The planting pit is set up in layers, with a drainage layer, a native soil layer, and a nutrient soil layer. The drainage layer is made of gravel from the Three Gorges area, with a thickness of 5-10cm, for timely drainage during heavy rain; the thickness is 10-15cm, for the connection between the root ball of the Adiantum capillus-veneris and the native soil; the nutrient soil layer is made of the special backfill composite nutrient substrate prepared in the previous section, for filling and compacting around the planting root ball;

[0099] (4) Habitat similarity creation: In the planting area after the relocation of the maidenhair fern, plant its original vegetation and companion tree species to create a "habitat similarity" effect. The temperature is controlled within the range of 10-30℃, and the humidity is controlled above 80%.

[0100] 3. Post-immigration management techniques for Adiantum repens

[0101] 3.1 To simulate the natural growth environment of *Adiantum repens*, a shade net with 70% light transmittance was constructed, providing approximately 1500 LX of light to prevent direct sunlight in summer. See [link to shade net construction instructions] for details. Figure 7-8 .

[0102] 3.2 Construct a mist system around the plant to maintain constant air humidity and create a microclimate similar to its native habitat, making the Adiantum repens suitable for its current growth environment.

[0103] 3.3 Rejuvenation treatment was performed on ex-situ *Adiantum repens* by dripping nutrient solution into the soil. The nutrient solution was a 1% CaCl2 solution. Since *Adiantum repens* has a low calcium absorption capacity and a low calcium bioavailability coefficient, the addition of CaCl2 solution can increase the translocation capacity of plant messengers, enhancing the environmental adaptability and resistance of *Adiantum repens*. 3.4 A self-made sunscreen and water-retaining solution was sprayed on the leaves, consisting of 2 parts of 200g aloe vera extract + 1 part of 50g plant transpiration inhibitor + 10mg / L salicylic acid. This solution provides photoprotection to the epidermis and leaves of *Adiantum repens*, and also provides nutrients to the plant. It is readily available, economical, and practical.

[0104] 3.5 Irrigation water should be sourced from natural, unpolluted sources, and irrigation should be carried out before 8:00 AM or after 6:00 PM. 3.6 Regularly implement meticulous maintenance management measures such as loosening the soil, weeding, and pest and disease control.

[0105] 4. Observation and Evaluation

[0106] The morphological changes, reproductive capacity, ecological suitability, and ex-situ survival rate of Adiantum repens in the new habitat were observed and evaluated.

[0107] 5. Records Management

[0108] Establish a file management method for each ex-situ protected maidenhair fern, and regularly record its growth according to the transplanting time to accurately grasp the growth and development pattern of the ex-situ maidenhair fern.

[0109] Example 2

[0110] Based on Example 1, six different treatments were implemented while maintaining consistent environmental conditions, treatment time, and observation and statistical time. A blank control group received no treatment. Each treatment consisted of 20 plants. To facilitate observation and statistical analysis of the plants in each treatment, the damage levels of leaves and petioles were classified into five levels: A, B, C, D, and E. The specific levels and their corresponding information are as follows:

[0111] Table 1

[0112]

[0113] The table below compares the results of each treatment.

[0114] The differences in treatment methods include: step 3.4 applying "sunscreen and water-retaining liquid", step 2.3(1) "root vitality preservation liquid", step 3.2 constructing "fog system", number 1-3 are only single treatment methods, and other treatment methods are not used, number 4-5 use the above two treatment methods, number 6 is all three of the above methods are used, and number 7 is none of the above three methods are used.

[0115] Table 2

[0116]

[0117]

[0118]

[0119] As shown in Table 2, through six different treatments and a blank control, it was found that the application of "sunscreen and water-retaining solution" and "root vitality preservation solution," along with the simultaneous construction of a "mist system," significantly reduced the damage to the leaves and petioles of the Adiantum repens after ex-situ protection. The proportion of healthy leaves and petioles was relatively large, and the time period and maturity of spore production after migration were better. Furthermore, the plants exhibited zero mortality after ex-situ protection, with only about 10% of the plants showing poor growth, although this condition gradually improved over time.

[0120] Example 3

[0121] Based on Example 1, the conditions for (1) selection of relocation time in step 2.1 pretreatment of live plants were changed, while other conditions remained the same as in Example 1. The relocation of Adiantum repens is shown in Table 3.

[0122] Table 3

[0123]

[0124] As shown in Table 3, through two different treatment examples, it was found that when the ex-situ conservation was carried out in early spring from March 15 to April 15, the survival rate of the Adiantum repens plant reached 95%, the time period for the start of spore production after migration was stable within 3-4 months, and the spore maturity was very good. These indicators were all higher than those of other treatments.

[0125] Example 4

[0126] Based on Example 1, the conditions for foliar treatment (3) in step 2.1 of the pretreatment of live plants were changed, while the rest remained the same as in Example 1. The migration of Adiantum repens is shown in Table 4.

[0127] Table 4

[0128]

[0129]

[0130] As shown in Table 4, through three different treatment examples, it was found that when the leaves were sprayed with a 60 ppm solution prepared with GGR6 until droplets fell, the survival rate of the Adiantum capillus-veneris plant after ex-situ treatment reached 95%, the time period for spore production after migration was stable within 3-4 months, and the spore maturity was very good. These indicators were all higher than those of other treatments.

[0131] Example 5

[0132] Based on Example 1, the condition of spraying the root area thoroughly with a 60ppm GGR6 solution in step 2.2 (digging of living plants) was changed, while other conditions remained the same as in Example 1. The migration of Adiantum repens is shown in Table 5.

[0133] Table 5

[0134]

[0135] As shown in Table 5, through three different treatment examples, it was found that the treatment of spraying the root area thoroughly with a solution of GGR6 at a concentration of 60 ppm resulted in a 95% survival rate of Adiantum repens after ex-situ transplantation. The time period for spore production after transplantation was stable within 3-4 months, and the spore maturity was very good. These indicators were all higher than those of other treatments.

[0136] Example 6

[0137] Based on Example 1, the conditions for preparing the planting soil substrate in step 2.4 of the ex-situ planting of live plants (2) were changed, while the rest remained the same as in Example 1. The migration of Adiantum repens is shown in Table 6.

[0138] Table 6

[0139]

[0140] As shown in Table 6, through seven different treatment examples and a comparative example without any treatment, it was found that transplanting Adiantum repens using a special substrate ratio of 4 parts weathered sand (particle size of about 5-10 mm), 2 parts peat soil, 2 parts vermiculite, and 1 part calcareous soil resulted in a 95% survival rate of the transplanted plants. The time period for spore production after transplantation was stable within 3-4 months, and the spore maturity was very good. These indicators were all higher than those of other treatments.

[0141] In summary, through the above multiple embodiments, it was found that the technical measures adopted by this patent overcome the problems of rapid dehydration and high mortality rates in the ex-situ conservation technology of the rare and endangered plant *Adiantum repens*, resulting in losses of human and material resources, especially the loss and waste of plant resources. Compared with existing conventional ex-situ methods, this patent effectively solves the problems of water loss and wilting, root damage, nutrient imbalance, and easy death of wild *Adiantum repens* during ex-situ relocation, significantly improving the survival rate to 95%. The acclimatization period after relocation is shortened by nearly 90 days, with sporangia forming in mid-to-late July and spores maturing in mid-to-late August, allowing for spore collection and propagation within the same year. This lays a solid foundation for the breeding research and promotion of the unique rare and endangered plant *Adiantum repens*, providing sufficient acclimatized breeding stock, greatly shortening the time for breeding research and wild reintroduction, expanding the population size, and reducing the risk of extinction.

[0142] The above embodiments are merely preferred technical solutions of the present invention, but the scope of protection of the present invention is not limited thereto. The embodiments and features in the embodiments of this application can be arbitrarily combined with each other without conflict. The scope of protection of the present invention should be defined by the technical solutions described in the claims, including equivalent substitutions of the technical features described in the claims. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention are also covered within the scope of protection of the present invention.

Claims

1. A method for ex-situ conservation of *Adiantum repens*, characterized in that, The method includes the following steps: Pre-ex-situ conservation survey of S1 Adiantum repens: S2 relocation uses constant temperature and humidity, rapid recovery relocation technology to transplant from the original site to the relocation area for planting, so that digging and planting can be completed on the same day; S3 after its relocation, management of Adiantum repens; S4 Observation and Evaluation: Observe and evaluate the morphological changes, reproductive capacity, ecological suitability, and ex-situ survival rate of Adiantum repens in the new location. S5 File Management: Establish a file management method for each ex-situ protected Adiantum repens, and regularly record growth according to the transplanting time to accurately grasp the growth and development pattern of ex-situ protected Adiantum repens; Complete the ex-situ conservation of Adiantum repens; step S2 specifically includes the following steps: S2.1 Pretreatment of live plants; S2.2 Excavation of living plants; S2.3 Constant temperature and humidity treatment for living plants; S2.4 Ex-situ planting of living plants; Step S2.1, pretreatment of live plants, includes: S2.1.1 Timing: Early spring, March 15th - April 15th, soil temperature should be maintained at 10-15 degrees Celsius; S2.1.2 Pruning: Prune some withered and yellow branches and leaves according to the growth condition; clean up the debris and weeds around the plant. S2.1.3 Foliar treatment: To ensure survival, delay leaf wilting, and increase stomatal diffusion capacity, use GGR6 to prepare a 30-50 ppm solution and spray it on the foliage until droplets fall. S2.1.4 Bagging of above-ground parts: After the leaves are treated, cover the above-ground parts of the plant with plastic bags to keep them warm and moist. Step S2.2, the excavation of living plants, includes: S2.2.1 The pretreated live plants were dug up, and the digging time was from 7:00 to 10:00 am in March; S2.2.2 Dig a circular trench 5-7cm from the periphery of the plant, and then dig a soil ball in a pot-bottom shape after digging down the circular trench for 10-15cm. After excavation is completed, spray the root area thoroughly with a GGR6 solution containing 50-80 ppm GGR6. First, wrap the soil ball with non-woven fabric, and then tie it with straps to reduce soil ball scattering during transportation. The soil substrate for the ex-situ planting of live plants in step S2.4 includes the following components by weight: 3-5 parts of weathered limestone sand from the Yichang section of the Three Gorges Dam area, 1.5-2.5 parts of peat soil, 1.5-2.5 parts of vermiculite, and 0.5-1.5 parts of calcareous gray soil. The particle size of the weathered limestone sand from the Yichang section of the Three Gorges Dam area is 5-10 mm. The pH value of the soil substrate is adjusted to 6.0-7.0, the organic matter content is 3-5%, and the substrate moisture content is 60-70%.

2. The method for ex-situ conservation of *Adiantum repens* according to claim 1, characterized in that, The pre-ex-situ conservation survey of *Adiantum repens*, step S1, includes: S1.1 Conduct a native habitat survey of Adiantum repens, covering wild plants affected by engineering construction, medicinal herb collection, or other factors; S1.2 Investigate the distribution range, number of plants, population structure, growth status, in-situ conservation status, or other conditions of affected Adiantum repens; S1.3 Investigate the altitude, air humidity, frost-free period, average of the highest and lowest temperatures over three years, or other meteorological data of the native habitat of Adiantum repens, to identify the suitable slope aspect for Adiantum repens to grow in its native habitat. S1.4 Soil samples were taken from the native habitat of Adiantum repens for testing, including soil structure, organic matter content, pH, and other parameters. The physical properties, chemical characteristics, and microbial activity of the understory soil were investigated to understand the soil physicochemical and biological environmental conditions. S1.5 Investigate the symbiotic vegetation communities around the Adiantum repens that require ex-situ conservation, including trees, shrubs, herbs and other ferns; S1.6 Survey Results: Based on the above survey, wild plants affected by engineering construction, medicinal herb collection, or other factors were selected for rescue and protection. In accordance with the "habitat similarity principle," a comprehensive investigation and comparison were conducted on microclimate conditions such as light, water, air, and heat, soil conditions, altitude, and surrounding environmental factors. Habitat differences were controlled within the range where Adiantum repens can thrive, and relocation was carried out according to the actual situation. Seedling selection should be carried out before digging, and the selected seedlings should be numbered sequentially, with one card for each seedling, and registered and managed in categories.

3. The method for ex-situ conservation of *Adiantum repens* according to claim 1, characterized in that, Step S2.3, the constant temperature and humidity treatment of live plants, includes: S2.3.1 Plant treatment: Inject the soil ball with the self-made "root vitality preservation solution", 15-25ml per plant; S2.3.2 Constant temperature: Adjust the temperature to 10-15℃; S2.3.3 Humidification: Use a humidifier to maintain the air humidity in the portable constant temperature storage box within the range of 60-70%.

4. The method for ex-situ conservation of *Adiantum repens* according to claim 3, characterized in that, The "root vitality preservation solution" in step S2.3.1 consists of a mixture of potassium chloride (0.15-0.25% by mass) and ascorbic acid (0.05-0.15% by mass).

5. The method for ex-situ conservation of *Adiantum repens* according to claim 1, characterized in that, Step S2.4, ex-situ planting of living plants, includes: S2.4.1 Selection of relocation site: Relocate to greenhouse cultivation; S2.4.2 Preparation of planting soil substrate in the relocation site: According to the soil requirements of the native habitat of Adiantum repens, the backfill soil of the planting hole should be prepared with soil substrate. S2.4.3 Live Planting: The planting hole is set in layers, with a drainage layer, a native soil layer, and a nutrient soil layer; the drainage layer is made of gravel from the Three Gorges area, with a thickness of 5-10cm, for timely drainage during heavy rain; the thickness is 10-15cm, for the connection between the root ball of the maidenhair fern and the native soil; the nutrient soil layer is a composite nutrient substrate backfilled with soil matrix from S2.4.2, for filling and compacting around the root ball of the plant. S2.4.4 Habitat Similarity Creation: In the planting area after the relocation of the Adiantum capillus-veneris, plant the vegetation and companion tree species from its original habitat to create a "habitat similarity".

6. The method for ex-situ conservation of *Adiantum repens* according to claim 1, characterized in that, The post-migration management of *Adiantum repens* in step S3 includes: S3.1 Simulate the wild growth environment of the maidenhair fern by constructing a shade net with a light transmittance of 65-75%; S3.2 Construct a mist system around the plant to maintain constant air humidity and create a microclimate similar to its native habitat, making the Adiantum repens suitable for its current growth environment; S3.3 Rejuvenate the ex-situ Adiantum repens by dripping nutrient solution into the soil; the nutrient solution is a 0.5-1.5% CaCl2 solution. S3.4 Spray the leaves with a sun-protecting and water-retaining solution; S3.5 Irrigation water shall be sourced from natural, unpolluted water sources, and irrigation shall be carried out before 10:00 AM or after 5:00 PM. S3.6 Regularly carry out soil loosening, weeding, and pest and disease control measures for refined maintenance management.

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