First-aid conservation method for fan-shaped fern

Abstract

The invention discloses a first-aid conservation method for a field eradication species Neocheiropteris trilligera, and the method comprises the following steps: resuscitation and culture of nephila specimen spores, induction of green globular bodies (GGBs) by a gametophyte homogenization method, multiplication culture of the green globular bodies, differentiation culture of the green globular bodies, and culture and transplanting of tissue culture seedlings, and further comprises the following steps: resuscitation and culture of nephila specimen spores, induction of the green globular bodies (GGBs) by a gametophyte homogenization method, propagation culture of the green globular bodies (GGBs), differentiation culture of the green globular bodies (GGBs), propagation culture of the tissue culture seedlings (GGBs) and transplantation of the tissue culture seedlings (GGBs). By means of the method, a large number of cynanchum otophyllum plants can be obtained under the condition that no survival cynanchum otophyllum plants exist, rescue conservation of field eradication cynanchum otophyllum is achieved, reference is provided for rescue conservation of other field eradication pteridophyte, and the method has important significance on diversity protection of Chinese pteridophyte.

Description

Technical Field

[0001] This invention relates to the field of wild plant conservation and propagation technology, specifically to a rescue conservation method for *Ferns trifoliata*. Background Technology

[0002] The statements in this section provide only background information relevant to the disclosure of this application and may not constitute prior art.

[0003] Traditional ferns (pteridophytes) consist of two distinct evolutionary lineages: lycophytes and ferns. They are the second largest group of vascular plants after angiosperms, dating back 400 million years. While angiosperms dominate ecosystems, ferns have evolved morphological adaptations and species diversity, playing a vital role in various ecosystems and representing the oldest vascular plants on Earth. China, with its vast territory and diverse topography and climate, is one of the world's richest regions for lycophytes and ferns. China currently has 2215 species belonging to 177 genera and 38 families of ferns. Due to human activities disrupting their original habitats, some fern species are vulnerable or endangered, and a few are extinct in the wild, meaning no living specimens can be found.

[0004] Fan fern ( Neocheiropteris *Pteris vittata* is one of the four endemic genera of ferns in China, distributed only in Yunnan, Sichuan, and Guizhou provinces. Currently, only *Pteris vittata* is widely recognized as a species within this genus. Neocheiropteris palmatodata ) and three-lobed fan fern ( Neocheiropteris triglossa *Trifolium repens* is extremely rare, with collection records only found in Mile and Lufeng, Yunnan. According to relevant literature, researchers have not found its distribution in Mile, Lufeng, and other areas of central Yunnan during multiple surveys, suggesting that the species may be extinct in the wild. Due to its rarity, previous research on this species is very limited, consisting mainly of morphological descriptions and systematic taxonomic studies using specimens.

[0005] Since the Trifolium repens is already extinct in the wild, it is impossible to obtain surviving Trifolium repens and suitable breeding materials for artificial propagation. This has become a bottleneck problem for the rescue and conservation of Trifolium repens. Therefore, there are currently no reports on research on artificial propagation and rescue and conservation methods for Trifolium repens. Summary of the Invention

[0006] The purpose of this invention is to provide a rescue and conservation method for *Fernella trifoliata*, which is in dire need of conservation because there are no surviving plants in its wild habitat and it is close to extinction. This method can obtain a large number of *Fernella trifoliata* plants in the absence of living plants by reviving spores obtained from herbarium specimens and combining them with tissue culture propagation technology using the green spheroidal pathway for ferns, thereby achieving the rescue and conservation of *Fernella trifoliata*.

[0007] The technical solution of the present invention is as follows: A rescue and conservation method for *Ferns trifoliata* mainly includes the following steps: Step 1: Revival and culture of spores from herbarium specimens Step 1.1: Collect spores from the underside of the leaves of *Fernonia trifoliata* specimens. Inoculate the sterilized spores into spore resuscitation liquid medium, shake at 100 rpm, and incubate in the dark for 5 days to resuscitate the spores. The spore resuscitation liquid medium is: 1 / 4 MS + 5 g / L trehalose + 5 g / L sucrose, pH 5.80.

[0008] Step 1.2: Sow the aseptic spores of *Fernonia trifoliata* that have undergone the resuscitation treatment in Step 1.1 onto a spore culture medium, and after dark culture for 1 day, transfer to light culture.

[0009] Conventional fern tissue culture typically uses young, active meristematic tissues from living fern plants as explants, obtaining fern plants through adventitious bud or green globular pathways; or it involves collecting mature, fresh spores from the abaxial surface of leaves from living fern plants for culture, obtaining fern plants through the gametophyte pathway. However, since *Fernella trifoliata* is extinct in the wild, it is impossible to obtain young explants and fresh spores from living plants, which is a bottleneck problem for the artificial conservation of *Fernella trifoliata*. This application overcomes the problem of the lack of living *Fernella trifoliata* plants by creatively using spores from the abaxial surface of herbarium specimens as culture material.

[0010] Due to the age of the herbarium specimens, the spores from these specimens exhibit significantly reduced viability and poor activity compared to fresh spores. Extensive preliminary research by the applicant revealed that directly culturing herbarium spores resulted in difficulty in germination and gametophyte formation. Therefore, this application employs a method of first resuscitating sterilized herbarium spores before culturing them, which significantly improves the germination rate. Herbarium spore culture, as the first step in the conservation process, significantly influences the results of subsequent culture; therefore, enhancing the viability of herbarium spores through resuscitation treatment is crucial for obtaining better cultivation results.

[0011] Step 2: Induction of green spheroids using gametophyte homogenization method Step 2.1: Observe the development of sexual organs in the gametophytes developed from the spores in Step 1. When the proportion of mature sexual organs in the gametophytes reaches more than 70%, homogenize the gametophytes under sterile conditions. The mature sexual organs in the gametophytes are defined as gametophytes that simultaneously possess mature antheridium and archegonia.

[0012] Step 2.2: The gametophyte homogenate from Step 2.1 was inoculated onto a green spheroid pre-induction medium. After culturing in the dark for 2 weeks, it was transferred to light culture for 2 weeks to perform green spheroid pre-induction culture. The green spheroid pre-induction medium was: 1 / 2 MS + 0.5~1.0 mg / L TDZ + 0.5~1.0 mg / L adenine + 0.1~0.2 mg / L NAA + 20 g / L sucrose + 7 g / L agar, pH 5.80.

[0013] Step 2.3: The gametophyte homogenate from Step 2.2, after pre-induced culture, is homogenized again under aseptic conditions and inoculated onto green spheroid induction medium. After dark incubation for 2 weeks, it is transferred to light incubation. The green spheroid induction medium is: 1 / 2 MS + 2.0~3.0 mg / L 6-BA + 0.5~1.0 mg / L adenine + 20 g / L sucrose + 7 g / L agar, pH 5.80.

[0014] Ferns exhibit a distinct generation cycle. After spore germination, they form gametophytes. Upon maturation, the gametophytes develop archegonia (producing egg cells) and archegonia (producing sperm cells). When a sperm enters the archegonia and fertilizes an egg cell, forming a zygote, the zygote gradually develops into an embryo and eventually forms a sporophyte, i.e., a fern plant. Extensive preliminary research and observation have revealed that the probability of a *Fern ternatus* gametophyte forming a sporophyte is extremely low. However, a certain proportion of fertilized eggs or embryos are present in the archegonia of mature gametophytes. Since fertilized eggs or embryos have a strong ability to divide, this application involves homogenizing mature gametophytes of *Fern ternatus*. This process facilitates the release of fertilized eggs or embryos from the archegonia, thereby inducing them to form green spheroids. This effectively improves the efficiency of green spheroid induction, shortens the induction period, and increases the success rate of artificial conservation.

[0015] Step 3: Proliferation culture of green spherical bodies The green spherical lobes of *Fernella trifoliata* induced in step 2 were divided into uniformly sized pieces and inoculated onto a green spherical lobule proliferation medium for cultivation under light. The medium was changed every 4 weeks. The green spherical lobule proliferation medium was: 1 / 2 MS + 1.0~2.0 mg / L 6-BA + 30~40 mL / L coconut milk + 20 g / L sucrose + 7 g / L agar, pH 5.80.

[0016] Step 4: Differentiation culture of green spheroids The green spherical prawns obtained in step 3 were divided into uniformly sized pieces and inoculated onto a green spherical differentiation medium for cultivation under light. The medium was changed every 4 weeks. The green spherical differentiation medium was: 1 / 4 MS + 1.0~1.5 g / L activated carbon + 30~40 mL / L coconut milk + 20 g / L sucrose + 7 g / L agar, pH 5.80.

[0017] Step 5: Cultivation and Transplantation of Tissue Culture Seedlings Step 5.1 Cluster the *Trichoderma* seedlings differentiated from the green spherical bodies in Step 4 onto the tissue culture seedling growth medium and culture them under light. Change the medium every 4 weeks. The tissue culture seedling growth medium is: MS + 1.0~1.5 g / L activated carbon + 40~50 mL / L coconut milk + 10~20 g / L potato + 20 g / L sucrose + 7 g / L agar, pH 5.80.

[0018] Step 5.2 After the *Fernella trifoliata* tissue culture seedlings from Step 5.1 have developed horizontal rhizomes and formed robust seedlings, transplant them into seedling trays containing a mixed cultivation substrate. After 8 weeks of cultivation, transplant the surviving seedlings into flowerpots containing a mixed cultivation substrate. The mixed cultivation substrate is: peat moss: vermiculite: perlite = 6:1:1.

[0019] Through the above steps, using herbarium specimen spores as culture material in step 1 overcomes the bottleneck problem of the extinct *Trichoderma trifoliata* species lacking living plants, fresh materials, and fresh spores. Furthermore, the resuscitation treatment enhances the activity of herbarium specimen spores, significantly impacting the achievement of better cultivation results. In step 2, homogenizing the mature gametophytes of the sexual organs of *Trichoderma trifoliata* facilitates the release of fertilized eggs or embryos from the archegonia, thereby inducing the formation of green spheroids. This effectively improves the induction efficiency of green spheroids, shortens the induction cycle, and enhances the efficiency of artificial conservation. After obtaining the green spheroids in step 2, the subsequent steps of green spheroid proliferation culture in step 3, green spheroid differentiation culture in step 4, and tissue culture seedling culture and transplantation in step 5 can yield a large number of *Trichoderma trifoliata* plants, thus achieving the rescue conservation of *Trichoderma trifoliata* and improving its extinction status in the wild.

[0020] According to a preferred embodiment, the light-induced culture environment is: temperature 20–25°C, light intensity 40–50 μmol•m. -2 •s -1 The area receives 16 hours of sunlight daily and has a relative humidity of 40-50%.

[0021] According to a preferred embodiment, the method further includes step 6: conservation efficiency statistics: statistics on the tissue culture propagation efficiency of *Fernonia trifoliata* obtained by the method of this application and the survival rate of tissue culture seedlings after transplanting.

[0022] According to a preferred embodiment, the statistical method for the tissue culture propagation coefficient in step 6 is as follows: the green spherical body of *Fernella trifoliata* obtained in step 2 of this application is divided into spherical bodies with a diameter of 3 mm. The number of tissue culture seedlings that can be obtained from each divided green spherical body after one green spherical body proliferation culture in step 3 (culture cycle 4 weeks), one green spherical body differentiation culture in step 4 (culture cycle 4 weeks), and tissue culture seedling culture in step 5 (culture cycle 8 weeks) is the tissue culture propagation coefficient.

[0023] According to a preferred embodiment, the statistical method for the survival rate of tissue culture seedlings in step 6 is as follows: 30 tissue culture seedlings of *Fernonia trifoliata* that have grown horizontal rhizomes in step 5.1 of this application are selected and transplanted according to step 5.2 of this application. Four weeks after transplanting into flowerpots, the number of surviving plants is counted. The survival rate of tissue culture seedlings = number of surviving plants / number of transplanted seedlings × 100%.

[0024] Compared with existing technologies, the advantages of this invention are: 1. A rescue conservation method for *Fernella trifoliata*, which overcomes the bottleneck of lacking living plants, fresh materials, and fresh spores when *Fernella trifoliata* is extinct in the wild. The method creatively uses herbarium spores as culture material and enhances their activity through resuscitation treatment. The resuscitated herbarium spores, following the method described in this application, can be used to obtain a large number of *Fernella trifoliata* plants through green spheroid tissue culture, thereby changing the extinct status of *Fernella trifoliata* in the wild and achieving rescue conservation. Simultaneously, this application also provides important reference for the artificial conservation of endangered ferns or extinct wild fern species.

[0025] 2. A rescue conservation method for *Fernella trifoliata* can overcome the problem of the extremely low probability of sporophyte formation from gametophytes during sporoculture. This method creatively prepares mature gametophytes into gametophyte homogenates, freeing fertilized eggs or embryos from the archegonia, and then inducing them to form green spheroids. This effectively improves the efficiency of green spheroid induction, shortens the induction cycle, and increases the efficiency of artificial conservation of *Fernella trifoliata*. It also provides a new approach for optimizing tissue culture propagation technology using the green spheroid pathway in ferns. Attached Figure Description

[0026] Figure 1 A flowchart illustrating a rescue conservation method for *Ferns trifoliata* provided in this application embodiment; Figure 2The green spherical bodies of *Trichoderma* obtained in the embodiments of this application are shown. Detailed Implementation

[0027] The following embodiments are provided to facilitate a better understanding of the present invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. All experimental materials used in the following embodiments are commercially available. In the quantitative experiments in the following embodiments, three replicate experiments were conducted, and the results were averaged.

[0028] The preparation methods of the culture media in the following examples are conventional methods, namely: after mixing each component according to its content according to the culture media formula, the pH is adjusted to 5.80, and the culture media is prepared by conventional disinfection and sterilization operations.

[0029] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0030] Example 1: Refer to Figure 1 This is a flowchart illustrating a rescue conservation method for *Ferns trifoliata* provided in an embodiment of the present invention. The process may include at least the specific implementation methods of steps 1 to 5, and the conservation efficiency statistical method of step 6. Step 1: Revival and culture of spores from herbarium specimens Step 1.1: Collect spores from the underside of the leaves of *Fernonia trifoliata* specimens. Inoculate the sterilized spores into spore resuscitation liquid medium, shake at 100 rpm, and incubate in the dark for 5 days to resuscitate the spores. The spore resuscitation liquid medium is: 1 / 4 MS + 5 g / L trehalose + 5 g / L sucrose, pH 5.80.

[0031] Step 1.2: Sow the aseptic spores of *Fernonia trifoliata* that have undergone resuscitation treatment in Step 1.1 onto a spore culture medium. After dark incubation for 1 day, transfer to light incubation. The light incubation environment is: temperature 20–25℃, light intensity 40–50 μmol•m -2 •s -1 The area receives 16 hours of sunlight daily and has a relative humidity of 40-50%.

[0032] Step 2: Induction of green spheroids using gametophyte homogenization method Step 2.1: Observe the development of sexual organs in the gametophytes developed from the spores in Step 1. When the proportion of mature sexual organs in the gametophytes reaches more than 70%, homogenize the gametophytes under sterile conditions. The mature sexual organs in the gametophytes are defined as gametophytes that simultaneously possess mature antheridium and archegonia.

[0033] Step 2.2: The gametophyte homogenate from Step 2.1 was inoculated onto a green spheroid pre-induction medium. After culturing in the dark for 2 weeks, it was transferred to light for 2 weeks to perform green spheroid pre-induction culture. The green spheroid pre-induction medium was: 1 / 2 MS + 0.5 mg / L TDZ + 1.0 mg / L adenine + 0.1 mg / L NAA + 20 g / L sucrose + 7 g / L agar, pH 5.80. The light culture environment was the same as in Step 1.2.

[0034] Step 2.3: The gametophyte homogenate from Step 2.2, after pre-culture, is homogenized again under aseptic conditions and inoculated onto green spheroid induction medium. After dark incubation for 2 weeks, it is transferred to light incubation. The green spheroid induction medium is: 1 / 2 MS + 2.0 mg / L 6-BA + 1.0 mg / L adenine + 20 g / L sucrose + 7 g / L agar, pH 5.80. The light incubation environment is the same as in Step 1.2.

[0035] Step 3: Proliferation culture of green spherical bodies The green spherical lobes of *Fernella trifoliata* induced in step 2 were divided into uniformly sized pieces and inoculated onto a green spherical lobule proliferation medium for light cultivation. The medium was changed every 4 weeks. The green spherical lobule proliferation medium was: 1 / 2 MS + 2.0 mg / L 6-BA + 30 mL / L coconut milk + 20 g / L sucrose + 7 g / L agar, pH 5.80. The light cultivation environment was the same as in step 1.2.

[0036] Step 4: Differentiation culture of green spheroids The green spherical lobes of *Fernella trifoliata* obtained in step 3 were divided into uniformly sized pieces and inoculated onto a green spherical differentiation medium for light cultivation. The medium was changed every 4 weeks. The green spherical differentiation medium was: 1 / 4 MS + 1.0 g / L activated carbon + 40 mL / L coconut milk + 20 g / L sucrose + 7 g / L agar, pH 5.80. The light cultivation environment was the same as in step 1.2.

[0037] Step 5: Cultivation and Transplantation of Tissue Culture Seedlings Step 5.1 Clusters of *Trichoderma* seedlings differentiated from the green spherical bodies in Step 4 were inoculated onto the tissue culture seedling growth medium and cultured under light, with the medium changed every 4 weeks. The tissue culture seedling growth medium was: MS + 1.0 g / L activated carbon + 50 mL / L coconut milk + 10 g / L potato + 20 g / L sucrose + 7 g / L agar, pH 5.80. The light culture environment was the same as in Step 1.2.

[0038] Step 5.2 After the *Fernella trifoliata* tissue culture seedlings from Step 5.1 have developed horizontal rhizomes, transplant them into seedling trays containing a mixed culture substrate. After 8 weeks of cultivation, transplant the surviving seedlings into flowerpots containing the mixed culture substrate. The mixed culture substrate is: peat moss: vermiculite: perlite = 6:1:1.

[0039] Step 6: Conservation Efficiency Statistics Step 6.1 Statistical analysis of tissue culture propagation coefficient: The green spherical bodies of *Fernella trifoliata* obtained in Step 2 of this application are divided into spherical bodies with a diameter of 3 mm. The number of tissue culture seedlings that can be obtained from each divided green spherical body after one green spherical body proliferation culture in Step 3 (culture cycle 4 weeks), one green spherical body differentiation culture in Step 4 (culture cycle 4 weeks), and tissue culture seedling culture in Step 5 (culture cycle 8 weeks) is the tissue culture propagation coefficient.

[0040] Step 6.2 Statistics on the survival rate of tissue culture seedlings after transplanting: 30 tissue culture seedlings of *Fernonia trifoliata* that had grown horizontal rhizomes in step 5.1 of this application were selected and transplanted according to step 5.2 of this application. Four weeks after transplanting into flower pots, the number of surviving plants was counted. The survival rate of tissue culture seedlings after transplanting = number of surviving plants / number of transplanted seedlings × 100%.

[0041] Examples 2 and 3 are further improvements to Example 1; the same content will not be repeated.

[0042] The differences between Examples 2 and 3 and Example 1 are shown in Table 1: Table 1. Differences between Examples 2 and 3 and Example 1

[0043] The conservation efficiency of *Trichoderma* in Examples 1, 2, and 3 is shown in Table 2: Table 2 Conservation efficiency of Trifolium repens in Examples 1-3

[0044] As shown in Table 2, the method described in this application for the rescue and conservation of *Fernella trifoliata* achieves a tissue culture propagation coefficient of 30.33–32.00. This means that a single 3mm diameter green spherical *Fernella trifoliata* specimen, after one green spherical proliferation culture in step 3, one green spherical differentiation culture in step 4, and one tissue culture seedling culture in step 5, can yield more than 30 *Fernella trifoliata* tissue culture seedlings, demonstrating a high propagation coefficient. Furthermore, the survival rate of the transplanted *Fernella trifoliata* tissue culture seedlings can reach 96.67–100%. Therefore, the method described in this application for the rescue and conservation of *Fernella trifoliata* has high conservation efficiency and can significantly improve the current situation of *Fernella trifoliata*'s extinction in the wild.

Claims

1. A method for the rescue and conservation of *Trifolium repens*, characterized in that, Includes the following steps: Step 1: Revival and culture of spores from herbarium specimens Step 1.1: Collect spores from the underside of the leaves of the herbarium of *Ferns trifoliata*. Inoculate the sterilized spores into spore recovery liquid medium, shake at 100 rpm, and incubate in the dark for 5 days to recover the spores. Step 1.2: Sow the aseptic spores of *Fernonia trifoliata* that have undergone the resuscitation treatment in Step 1.1 onto a spore culture medium, incubate in the dark for 1 day, and then transfer to light culture. Step 2: Induction of green spheroids using gametophyte homogenization method Step 2.1: Observe the development of sexual organs in the gametophytes developed from the spores in Step 1. When the proportion of mature sexual organs in the gametophytes reaches more than 70%, the gametophytes are homogenized under sterile conditions. Step 2.2: Inoculate the gametophyte homogenate from Step 2.1 onto the green spheroid pre-induction medium, culture in the dark for 2 weeks, and then transfer to light culture for 2 weeks to carry out green spheroid pre-induction culture; Step 2.3: The gametophyte homogenate that underwent pre-induced culture in Step 2.2 was homogenized again under aseptic conditions, inoculated onto green spheroid induction medium, and cultured in the dark for 2 weeks before being transferred to light culture. Step 3: Proliferation culture of green spherical bodies The green spherical bodies of *Fernella trifoliata* induced in step 2 were divided into small pieces of uniform size, inoculated onto the green spherical body proliferation medium, and cultured under light. The medium was changed every 4 weeks. Step 4: Differentiation culture of green spheroids The green spherical bodies of *Fernella trifoliata* obtained in step 3 were divided into small pieces of uniform size and inoculated onto the green spherical body differentiation medium for light culture. The medium was changed every 4 weeks. Step 5: Cultivation and Transplantation of Tissue Culture Seedlings Step 5.1 Inoculate clusters of the *Trichoderma* seedlings differentiated from the green spherical bodies in Step 4 onto the tissue culture seedling growth medium and culture them under light. Change the medium every 4 weeks. Step 5.2 After the tissue culture seedlings of the three-leaved fan fern from Step 5.1 have grown horizontal stolons and formed strong seedlings, transplant the tissue culture seedlings into seedling boxes containing mixed cultivation substrate. After 8 weeks of cultivation, transplant the surviving seedlings into flower pots containing mixed cultivation substrate.

2. The method for the rescue and conservation of *Trifolium repens* according to claim 1, characterized in that, In step 1, the spore resuscitation liquid culture medium is: 1 / 4 MS + 5 g / L trehalose + 5 g / L sucrose, pH 5.

80.

3. The method for the rescue and conservation of *Trifolium repens* according to claim 1, characterized in that, In step 2, the mature gametophyte of the sexual organ is one that simultaneously possesses mature antheridium and archegonium.

4. The method for the rescue and conservation of *Trifolium repens* according to claim 1, characterized in that, In step 2, the green spheroid pre-induction medium is: 1 / 2 MS + 0.5~1.0 mg / L TDZ + 0.5~1.0 mg / L adenine + 0.1~0.2 mg / L NAA + 20 g / L sucrose + 7 g / L agar, pH 5.

80.

5. A method for the rescue and conservation of *Trifolium repens* according to claim 1, characterized in that, In step 2, the green spheroid induction medium is: 1 / 2 MS + 2.0~3.0 mg / L 6-BA + 0.5~1.0 mg / L adenine + 20 g / L sucrose + 7 g / L agar, pH 5.

80.

6. A method for the rescue and conservation of *Trifolium repens* according to claim 1, characterized in that, In step 3, the green spheroid proliferation medium is: 1 / 2 MS + 1.0~2.0 mg / L 6-BA + 30~40 mL / L coconut juice + 20 g / L sucrose + 7 g / L agar, pH 5.

80.

7. A method for the rescue and conservation of *Trifolium repens* according to claim 1, characterized in that, In step 4, the green spheroid differentiation medium is: 1 / 4 MS + 1.0~1.5 g / L activated carbon + 30~40 mL / L coconut juice + 20 g / L sucrose + 7 g / L agar, pH 5.

80.

8. A method for the rescue and conservation of *Trifolium repens* according to claim 1, characterized in that, In step 5, the culture medium for tissue culture seedlings is: MS + 1.0~1.5g / L activated carbon + 40~50 mL / L coconut juice + 10~20g / L potato + 20g / L sucrose + 7g / L agar, pH 5.

80.

9. A method for the rescue and conservation of *Trifolium repens* according to claim 1, characterized in that, In step 5, the mixed culture medium is: peat: vermiculite: perlite = 6:1:

1.

10. A method for the rescue and conservation of *Trifolium repens* according to claim 1, characterized in that, In steps 1-5, the light-induced culture environment is: temperature 20-25℃, light intensity 40-50 μmol•m. -2 •s -1 The area receives 16 hours of sunlight daily and has a relative humidity of 40-50%.

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