A method for improving the seed utilization rate of the extremely endangered plant, narrow-leaved hillside-rice (zizania tenuis)

Abstract

This invention discloses a method for improving the seed utilization rate of the critically endangered plant *Hopea stenoptera*, comprising seed collection, seed pretreatment, seed sterilization, germination induction, seedling cultivation, hardening-off transplanting, and germplasm preservation. It maximizes the utilization of immature, semi-mature, and mature seeds that cannot germinate under natural conditions, including those that have not yet lost water. Given a fixed number of seeds, it promotes the conversion of more seeds into seedlings. It also enables the in vitro preservation of *Hopea stenoptera* seeds for a certain period, breaking the limitation that seed germination and utilization must occur at the seed maturity harvesting season. This significantly improves seed utilization and provides sterile materials for long-term preservation of germplasm resources and large-scale propagation. It lays a technical foundation for the conservation of this species, ecological restoration, and resource development and utilization, and has significant practical implications for saving this endangered tree species.

Description

Technical Field

[0001] This invention relates to the field of plant propagation technology, specifically a method for improving the utilization rate of the stubborn seeds of the critically endangered plant *Hopea stenoptera*. Background Technology

[0002] *Hopea chinensis* Hand.-Mazz., also known as South China Hopea or Narrow-leaved Hopea, belongs to the genus *Hopea* in the family Dipterocarpaceae. It is a critically endangered tree species endemic to Guangxi. An evergreen tree, it can reach a height of 30m and a diameter at breast height of 50-100cm. It grows in foothills, valleys, and hillside forests below 600m in altitude. It is one of the representative tree species of tropical evergreen monsoon forests in northern and southern Guangxi and is of great value for studying the composition, geographical distribution, and ecological habits of tropical rainforests. Meanwhile, its wood has an interlocking grain, fine structure, hard texture, and extremely strong resistance to decay. It can remain intact for hundreds of years when buried in the soil, making it an excellent special timber species known as "ten-thousand-year wood." It can be used for military, machinery, and high-end furniture. The resin it produces is similar to sesame resin and can be used in paint manufacturing. The bark has a dragon robe-like pattern, hence it is also known as the "dragon robe tree." The by-products are aromatic oils, which can be used as high-end cosmetics and precious Chinese medicine materials, making them extremely valuable.

[0003] *Hopea stenoptera* is a remnant species from the Tertiary period, distributed in localized topographical "refuges." Native to the Shiwan Mountains and Daqing Mountains in Guangxi, its population has drastically declined due to human activities and climate change. Surveys indicate that the *Hopea stenoptera* population in Daqing Mountains is now extinct, with only a small number remaining in the Shiwan Mountains Nature Reserve and surrounding areas. The number of wild *Hopea stenoptera* individuals has decreased dramatically in the past 20 years, posing a serious threat of extinction. It has been classified as a Class I protected plant in China and listed as Critically Endangered (CR) by the International Union for Conservation of Nature (IUCN). Research on its endangered status reveals sparse fruit production, biennial bearing, and in some years, no fruit at all. The wild population's distribution range has shrunk dramatically, posing a serious risk of extinction and urgently requiring protection.

[0004] Expanding population size through conservation measures is the most fundamental condition for saving endangered plants. Research has found that the seed-to-seed conversion rate of wild *Hopea stenoptera* populations is relatively low because its seeds are typically recalcitrant. Recalcitrant seeds, once exposed to drought or unable to fall into suitable soil, rapidly lose water, significantly reducing their lifespan and viability, and may even become completely inactive and unable to germinate. Wild *Hopea stenoptera* plants mostly grow along stream banks, and seeds are primarily dispersed by gravity. Many mature seeds fall into the stream near the mother plant, and most of these seeds are waterlogged and fail to germinate. Furthermore, due to the recalcitrant nature of its seeds, there is currently no mature preservation technology. Freshly collected seeds must be sown immediately; once placed in a refrigerator or dehydrated, they lose viability. If suitable seedling conditions are not provided before the seeds become inactive, they will completely fail to germinate and grow into seedlings. Strictly limiting seed germination and utilization to near the time of seed maturity significantly restricts seed utilization. To date, there are no reports on seed propagation for *Hopea stenoptera*. To better restore its population, save this critically endangered plant, improve seed utilization, and promote the conversion of more of its seeds into seedlings is of great practical significance. Summary of the Invention

[0005] The purpose of this invention is to solve the technical problems of low seed-to-seed conversion and low utilization rate caused by the inability to preserve stubborn seeds of *Hopea stenoptera* and their easy inactivation. The invention aims to promote the conversion of more seeds into seedlings within a given number of seeds, preserve germplasm resources for a certain period, save seedling space, control seedling conditions, and improve seed utilization.

[0006] This invention is achieved through the following technical solution:

[0007] A method for improving the seed utilization rate of the critically endangered plant *Hopea stenoptera* var. *narrow-leaved* includes seed collection, seed pretreatment, seed sterilization, germination induction, seedling cultivation, hardening-off transplanting, and germplasm preservation. The specific operational steps are as follows:

[0008] (1) Seed collection:

[0009] Collect fresh, disease-free seeds from the natural distribution area of ​​*Hopea stenoptera*.

[0010] (2) Seed pretreatment:

[0011] Remove the seed wings from both sides of the narrow-leaved holly seeds, wash the surface of the seeds with clean water to remove impurities, stir and soak in cleaning solution for 5 minutes, wash off the cleaning solution with pure water, wrap the seeds and sterilized fine sand in gauze, soak in 2% Australian tea tree essential oil aqueous solution for 20 minutes, stir and soak in sterilized water for 3-5 minutes, and then transfer to a clean bench.

[0012] (3) Seed sterilization:

[0013] Open the laminar flow hood, immerse the seeds in 75% alcohol for 1 minute, sterilize them by burning with an alcohol lamp flame for 5 seconds, open the gauze, wash away the fine sand from the seeds, place them in an inoculation tray containing sterilized filter paper, turn on the ultraviolet lamp of the laminar flow hood and irradiate for 30 minutes, then put the seeds into the sterilization solution and stir and soak for 10-15 minutes, repeat once, then immerse them in sterile water at 35-40℃ 3-5 times, peel off the seed coat with a scalpel to obtain sterilized embryos;

[0014] (4) Inducing germination:

[0015] After sterilization, the embryos were classified according to maturity and inoculated into germination induction media. Immature and semi-mature embryos were inoculated into germination induction medium ①, and mature embryos were inoculated into germination induction medium ②, with one embryo per bottle. The culture conditions were 25±3℃ in the dark.

[0016] (5) Seedling culture: After the embryos of *Hopea stenoptera* are cultured in the germination induction medium for 10-15 days, they are transferred to the culture bottle of seedling culture medium. The culture conditions are a temperature of 25±3℃, and the culture bottle is placed in a light source with a light duration of 12-15h / d and a light intensity of 1500-3000lx.

[0017] (6) Hardening off seedlings and transplanting

[0018] After the tissue culture seedlings have matured, move them outdoors and harden them under natural light for 5-7 days. Then, open the bottle cap and continue hardening them for 1-3 days. Take out the seedlings, wash off the culture medium, and transplant them into the seedling substrate. Within 7 days after transplanting, cover them with a shade net with a shading degree of 75-85%, and then carry out routine seedling management.

[0019] (7) Germplasm preservation

[0020] After inducing germination, the tissue culture seedlings were transferred to preservation medium. The medium was placed in 270mL PC plastic bottles with a 70mm diameter × 110mm height sealed cap. Five seedlings were inoculated into each bottle. The bottle mouth was wrapped with plastic wrap twice. The preservation medium was replaced every 30-40 days. When it was ready to be used, the seedlings were transferred to culture bottles containing seedling culture medium. After 25 days of culture, the seedlings could be directly hardened off and transplanted.

[0021] The seed collection described in step (1) above includes collecting seeds from trees and picking up fallen seeds; the fresh seeds include immature, semi-mature and mature seeds that have not yet lost water, wherein the seed coat and seed wings of immature seeds are all green; the seed coat of semi-mature seeds is yellowish-green and the seed wings are dark yellow; the seed coat of mature seeds is all yellowish-green and the seed wings are dark yellow or brown.

[0022] The cleaning solution mentioned in step (2) above is 5g of flour added to 1000mL of clean water.

[0023] In step (3) above, 1 cm of sterile water needs to be poured into the sterile filter paper in the inoculation tray; the sterile solution is a mixture of 0.1% HgCl2 solution and 1 drop of Tween 20.

[0024] The germination induction medium ① described in step (4) above has the following formula: MS + BA 0.25 mg / L + NAA 0.25~0.5 mg / L + GA3 0.5~0.8 mg / L + 30 g / L sucrose + 3.8 g / L agar powder, pH 5.8~6.0; the germination induction medium ② has the following formula: MS + BA 0.5 mg / L + NAA 0.4~0.8 mg / L + 30 g / L sucrose + 3.8 g / L agar powder, pH 5.8~6.0.

[0025] The seedling culture medium formula described in step (5) above is 1 / 2MS + IAA 1.5~2.0mg / L + IBA 1.5~2.0mg / L + 15g / L sucrose 3.8g / L agar powder, pH 5.8~6.0.

[0026] The seedling substrate mentioned in step (6) above is a mixture of peat soil, red soil and mushroom residue in a volume ratio of 4:4:2.

[0027] The preservation culture medium formula described in step (7) above is 2 / 3 MS + BA 0.1~0.2 mg / L + IAA 0.5 mg / L + 30 g / L sucrose + 3.8 g / L agar powder, pH 5.8~6.0.

[0028] This invention has the following advantages and positive effects over the prior art:

[0029] 1. The technical solution proposed in this invention involves collecting and processing different types of seeds for in vitro culture, which reduces the harm caused by pests and diseases. By optimizing the culture medium formula at each stage, the germination rate of seed embryos can reach 85.88-92.21%, the seedling rate can reach 81.20-84.59%, the cultivated seedlings are robust, and the survival rate of seedlings after hardening and transplanting can reach 82.57-89.33%. After seedlings are stored in vitro for 90.24-93.31 days, the survival rate of seedlings after recovery culture and hardening and transplanting can reach 75.41-80.18%, achieving good technical results.

[0030] 2. This invention designs different culture medium formulations for seeds at different maturity levels, significantly improving the germination rate and seedling rate. Collecting immature seeds allows them to be utilized before they encounter extreme weather or other accidents during the seed maturation process. Collecting fallen seeds, including those that have fallen into or near streams, avoids the risk of some seeds falling off and becoming inactive or being eaten by insects or birds after maturation. This maximizes the utilization of seeds that cannot germinate into seedlings under natural conditions, significantly improving seed utilization. Given a certain number of seeds, this invention promotes the conversion of more seeds into seedlings, laying a technical foundation for research on the conservation biology, restoration ecology, and resource development and utilization of this species.

[0031] 3. This invention establishes a sterile system for *Hopea stenoptera* using embryos as explants, which can provide sterile materials for tissue culture propagation of *Hopea stenoptera*, thus laying the foundation for long-term preservation of germplasm resources and large-scale propagation through tissue culture technology.

[0032] 4. This invention employs a series of innovative combined measures for seed pretreatment and sterilization, significantly improving the sterilization success rate and initiation rate. Before sterilization, seeds are washed with a cleaning solution prepared by adding 5g of flour to 1000mL of clean water. The fine flour particles have a certain adsorption capacity, and the flour-water solution has a certain viscosity, which can wash away dirt adhering to the seed coat surface. Fine sand is wrapped in gauze for further cleaning, which increases friction on the seed coat, promotes germination, facilitates sterilization, and avoids direct contact of alcohol with the seeds, thus preventing damage to seed viability. During seed sterilization, seeds are soaked in 75% alcohol followed by a brief flame burn, which avoids alcohol residue on the surface, and the brief heat treatment inactivates some bacteria. After soaking in the sterilization solution, seeds are soaked in sterilization water at 35-40℃. The increased temperature of the sterilization water washes away the sterilization solution while activating seed viability, further promoting germination.

[0033] 5. This invention, through optimization of the germination induction medium, seedling culture medium, and preservation medium, can effectively improve the germination success rate of seed embryos. After being transferred to preservation culture, seedling formation can be delayed, and seedling formation can be achieved rapidly after recovery culture. It can achieve short-term preservation and extend the seed utilization time by 60-90 days. It breaks the limitation that seed germination utilization must be carried out during the seed maturity harvesting season, effectively alleviating the situation where seeds cannot be sown immediately due to objective factors, resulting in seed inactivation and failure to form seedlings. Moreover, it is not limited by land and seedling space, which is of great scientific significance for saving this endangered tree species.

[0034] 6. The transplanting substrate used in this invention is peat moss: yellow soil: mushroom residue in a volume ratio of 4:4:2. The use of peat moss and red soil can ensure sufficient water and fertilizer retention capacity. Mushroom residue is the waste residue after planting edible fungi. It can change the content of soil organic matter and humus, and improve soil fertility. This seedling substrate has a simple and reasonable ratio and is easy to prepare. It can provide sufficient nutrition for the seedlings of *Hopea stenoptera* and can significantly improve the survival rate of transplanted *Hopea stenoptera* seedlings to 82.57%–89.33%. Attached Figure Description

[0035] Figure 1 This is a picture of immature seeds of *Hopea stenoptera*.

[0036] Figure 2 This is a picture of a semi-mature seed of *Hopea stenoptera*.

[0037] Figure 3 This is a picture of mature seeds of *Hopea stenoptera*.

[0038] Figure 4 This is a diagram of seed germination of *Hopea stenoptera*.

[0039] Figure 5 This is a picture of tissue culture seedlings of *Hopea stenoptera*.

[0040] Figure 6 This is a picture of transplanting tissue culture seedlings of *Hopea stenoptera* into cup seedlings. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0042] Explanation of abbreviations and calculation formulas:

[0043] MS: MS medium;

[0044] 2 / 3MS: The amount of macroelements in MS medium is reduced to 2 / 3;

[0045] 1 / 2MS: The amount of macroelements in MS medium is halved;

[0046] BA: 6-Benzylaminopurine;

[0047] NAA: Naphthaleneacetic acid;

[0048] GA3: Gibberellin;

[0049] IAA: Indoleacetic acid;

[0050] IBA: Indolebutyric acid;

[0051] Contamination rate (%) = Number of contaminated bud embryos / Total number of inoculated embryos × 100;

[0052] Initiation rate (%) = Number of initiating embryos / Total number of inoculated embryos × 100. An embryo is considered to be initiating when its bud elongation breaks through the embryo.

[0053] Germination rate (%) = Number of germinated embryos / Total number of inoculated embryos × 100. Embryos with a bud length greater than the diameter of the embryo are considered to be germinated embryos.

[0054] Seedling survival rate (%) = Number of rooted seedlings / Total number of inoculated seedlings × 100;

[0055] Transplant survival rate (%) = Number of surviving seedlings / Total number of transplants × 100;

[0056] Brown mortality rate at 60 days (%) = Number of brown deaths / Total number of vaccinations × 100;

[0057] The longest storage time refers to the time when the browning mortality rate is ≤20%.

[0058] Example 1:

[0059] A method for improving the seed utilization rate of the critically endangered plant *Hopea stenoptera* var. *narrow-leaved*, comprising seed collection, seed pretreatment, seed sterilization, germination induction, seedling cultivation, hardening-off transplanting, and germplasm preservation. The specific operational steps are as follows:

[0060] (1) Seed collection:

[0061] Fresh immature seeds with green seed coat and seed wings, or fresh semi-mature seeds with yellow-green seed coat and dark yellow seed wings, or fresh mature seeds with yellow-green seed coat and dark yellow or brown seed wings, were collected from the natural population distribution area of ​​Hopinus acutissima.

[0062] (2) Seed pretreatment:

[0063] Remove the seed wings on both sides of the narrow-leaved holly seeds, wash the surface of the seeds with clean water to remove impurities, then soak them in a cleaning solution of 1000mL clean water with 5g of flour for 5 minutes, rinse off the cleaning solution with pure water, wrap the seeds and sterilized fine sand in gauze, then soak them in a 2% Australian tea tree essential oil aqueous solution for 20 minutes, stir and soak them in sterilized water for 5 minutes, and then transfer them to an ultra-clean workbench.

[0064] (3) Seed sterilization:

[0065] Open the laminar flow hood. On the laminar flow hood, soak the seeds in 75% alcohol for 1 minute, then sterilize them by flame burning with an alcohol lamp for 5 seconds. Open the gauze, wash away the fine sand from the seeds of *Holothuria angustifolia*, and place them in an inoculation tray containing sterile filter paper. Pour 1 cm of sterile water containing a mixture of 0.1% HgCl2 solution and 1 drop of Tween 20 onto the sterile filter paper in the inoculation tray. Turn on the ultraviolet lamp in the laminar flow hood and irradiate for 30 minutes. Stir and soak the seeds for 15 minutes, repeat once, and then soak them in sterile water at 35-40℃ 5 times. Use a scalpel to peel off the seed coat to obtain sterile embryos.

[0066] (4) Inducing germination:

[0067] Sterilized *Holothuria angustifolia* embryos were classified according to maturity. Immature or semi-mature embryos were inoculated into MS medium ① containing 0.25 mg / L BA, 0.25 mg / L NAA, 0.8 mg / L GA3, 30 g / L sucrose, and 3.8 g / L agar powder at pH 5.8–6.0. ​​Mature embryos were inoculated into MS medium ② containing 0.5 mg / L BA, 0.8 mg / L NAA, 30 g / L sucrose, and 3.8 g / L agar powder at pH 5.8–6.0. ​​One embryo was inoculated per bottle, and the culture conditions were 25 ± 3℃ in the dark.

[0068] (5) Seedling cultivation:

[0069] After culturing the embryos of *Hopea stenoptera* in germination induction medium for 15 days, they were transferred to a seedling culture medium consisting of 1 / 2 MS + IAA 1.5 mg / L + IBA 1.5 mg / L + 15 g / L sucrose + 3.8 g / L agar powder, pH 5.8–6.0. ​​The culture conditions were 25 ± 3℃, and the culture bottles were placed in a light source with a photoperiod of 12–15 h / d and a light intensity of 1500–3000 lx.

[0070] (6) Hardening off seedlings and transplanting

[0071] After the tissue culture seedlings have matured, move them outdoors and harden them under natural light for 5 days. Then, open the bottle cap and continue hardening them for another day. Take out the seedlings, wash off the culture medium, and transplant them into a seedling substrate made of peat moss, red soil, and mushroom residue in a volume ratio of 4:4:2. Cover the seedlings with a shade net with an 85% shading degree within 7 days after transplanting, and then carry out routine seedling management.

[0072] (7) Germplasm preservation

[0073] After induced germination culture, the tissue culture seedlings were transferred to a preservation medium containing 2 / 3 MS + BA 0.2 mg / L + IAA 0.5 mg / L + 30 g / L sucrose + 3.8 g / L agar powder, with a pH of 5.8–6.0. ​​The medium was then placed in 270 mL PC plastic bottles with a 70 mm diameter × 110 mm height sealed cap, with 5 seedlings inoculated per bottle. The bottle opening was wrapped twice with plastic wrap. The preservation medium was replaced with fresh medium every 30 days. When ready for use, the seedlings were transferred to culture bottles containing seedling culture medium and cultured for 25 days before being hardened off and transplanted.

[0074] Example 2:

[0075] A method for improving the seed utilization rate of the critically endangered plant *Hopea stenoptera* var. *narrow-leaved*, comprising seed collection, seed pretreatment, seed sterilization, germination induction, seedling cultivation, hardening-off transplanting, and germplasm preservation. The specific operational steps are as follows:

[0076] (1) Seed collection:

[0077] Fresh immature seeds with green seed coat and seed wings, or fresh semi-mature seeds with yellow-green seed coat and dark yellow seed wings, or fresh mature seeds with yellow-green seed coat and dark yellow or brown seed wings, were collected from the natural population distribution area of ​​Hopinus acutissima.

[0078] (2) Seed pretreatment:

[0079] Remove the seed wings on both sides of the narrow-leaved holly seeds, wash the surface of the seeds with clean water to remove impurities, soak them in a cleaning solution of 1000mL clean water with 5g of flour for 5 minutes, rinse off the cleaning solution with pure water, wrap the seeds and sterilized fine sand in gauze, soak them in a 2% Australian tea tree essential oil aqueous solution for 20 minutes, soak them in sterilized water for 3 minutes, and then transfer them to an ultra-clean workbench.

[0080] (3) Seed sterilization:

[0081] Open the laminar flow hood. On the laminar flow hood, soak the seeds in 75% alcohol for 1 minute, then sterilize them by flame burning with an alcohol lamp for 5 seconds. Open the gauze, wash away the fine sand from the seeds of *Holothuria angustifolia*, and place them in an inoculation tray containing sterile filter paper. Pour 1 cm of sterile water containing a mixture of 0.1% HgCl2 solution and 1 drop of Tween 20 onto the sterile filter paper in the inoculation tray. Turn on the ultraviolet lamp in the laminar flow hood and irradiate for 30 minutes. Stir and soak the seeds for 10 minutes, repeat once, and then soak them in sterile water at 35-40℃ three times. Use a scalpel to peel off the seed coat to obtain sterile embryos.

[0082] (4) Inducing germination:

[0083] Sterilized *Holothuria angustifolia* embryos were classified according to maturity. Immature or semi-mature embryos were inoculated into MS medium ① containing 0.25 mg / L BA, 0.5 mg / L NAA, 0.5 mg / L GA3, 30 g / L sucrose, and 3.8 g / L agar powder at pH 5.8–6.0. ​​Mature embryos were inoculated into MS medium ② containing 0.5 mg / L BA, 0.4 mg / L NAA, 30 g / L sucrose, and 3.8 g / L agar powder at pH 5.8–6.0. ​​One embryo was inoculated per bottle, and the culture conditions were 25 ± 3℃ in the dark.

[0084] (5) Seedling cultivation:

[0085] After culturing the embryos of *Hopea stenoptera* in the germination induction medium for 10 days, they were transferred to a seedling culture medium consisting of 1 / 2 MS + IAA 2.0 mg / L + IBA 2.0 mg / L + 15 g / L sucrose + 3.8 g / L agar powder, pH 5.8–6.0. ​​The culture conditions were 25 ± 3℃, and the culture bottles were placed in a light source with a photoperiod of 12–15 h / d and a light intensity of 1500–3000 lx.

[0086] (6) Hardening off seedlings and transplanting

[0087] After the tissue culture seedlings have matured, move them outdoors and harden them under natural light for 7 days. Then, open the bottle cap and continue hardening them for another 3 days. Take out the seedlings, wash off the culture medium, and transplant them into a seedling substrate made of peat moss, red soil, and mushroom residue in a volume ratio of 4:4:2. Cover the seedlings with a shade net with an 85% shading degree within 7 days after transplanting, and then carry out routine seedling management.

[0088] (7) Germplasm preservation

[0089] After induced germination culture, the tissue culture seedlings were transferred to a preservation medium containing 2 / 3 MS + BA 0.1 mg / L + IAA 0.5 mg / L + 30 g / L sucrose + 3.8 g / L agar powder, with a pH of 5.8–6.0. ​​The medium was then placed in 270 mL PC plastic bottles with a 70 mm diameter × 110 mm height sealed cap, with 5 seedlings inoculated per bottle. The bottle opening was wrapped twice with plastic wrap. The preservation medium was replaced with fresh medium every 40 days. When ready for use, the seedlings were transferred to culture bottles containing seedling culture medium and cultured for 25 days before being hardened off and transplanted.

[0090] Example 3:

[0091] A method for improving the seed utilization rate of the critically endangered plant *Hopea stenoptera* var. *narrow-leaved*, comprising seed collection, seed pretreatment, seed sterilization, germination induction, seedling cultivation, hardening-off transplanting, and germplasm preservation. The specific operational steps are as follows:

[0092] (1) Seed collection:

[0093] Fresh immature seeds with green seed coat and seed wings, or fresh semi-mature seeds with yellow-green seed coat and dark yellow seed wings, or fresh mature seeds with yellow-green seed coat and dark yellow or brown seed wings, were collected from the natural population distribution area of ​​Hopinus acutissima.

[0094] (2) Seed pretreatment:

[0095] Remove the seed wings on both sides of the narrow-leaved holly seeds, wash the surface of the seeds with clean water to remove impurities, then soak them in a cleaning solution of 1000mL clean water with 5g of flour for 5 minutes, rinse off the cleaning solution with pure water, wrap the seeds and sterilized fine sand in gauze, then soak them in a 2% Australian tea tree essential oil aqueous solution for 20 minutes, stir and soak them in sterilized water for 5 minutes, and then transfer them to an ultra-clean workbench.

[0096] (3) Seed sterilization:

[0097] Open the laminar flow hood. On the laminar flow hood, soak the seeds in 75% alcohol for 1 minute, then sterilize them by flame burning with an alcohol lamp for 5 seconds. Open the gauze, wash away the fine sand from the seeds of *Holothuria angustifolia*, and place them in an inoculation tray containing sterile filter paper. Pour 1 cm of sterile water containing a mixture of 0.1% HgCl2 solution and 1 drop of Tween 20 onto the sterile filter paper in the inoculation tray. Turn on the ultraviolet lamp in the laminar flow hood and irradiate for 30 minutes. Stir and soak the seeds for 15 minutes, repeat once, and then soak them in sterile water at 35-40℃ three times. Use a scalpel to peel off the seed coat to obtain sterile embryos.

[0098] (4) Inducing germination:

[0099] Sterilized *Holothuria angustifolia* embryos were classified according to maturity. Immature or semi-mature embryos were inoculated into MS medium ① containing 0.25 mg / L BA, 0.5 mg / L NAA, 0.6 mg / L GA3, 30 g / L sucrose, and 3.8 g / L agar powder at pH 5.8–6.0. ​​Mature embryos were inoculated into MS medium ② containing 0.5 mg / L BA, 0.5 mg / L NAA, 30 g / L sucrose, and 3.8 g / L agar powder at pH 5.8–6.0. ​​One embryo was inoculated per bottle, and the culture conditions were 25 ± 3℃ in the dark.

[0100] (5) Seedling cultivation:

[0101] After culturing the embryos of *Hopea stenoptera* in germination induction medium for 15 days, they were transferred to a seedling culture medium consisting of 1 / 2 MS + IAA 2.0 mg / L + IBA 1.5 mg / L + 15 g / L sucrose + 3.8 g / L agar powder, pH 5.8–6.0. ​​The culture conditions were 25 ± 3℃, and the culture bottles were placed in a light source with a photoperiod of 12–15 h / d and a light intensity of 1500–3000 lx.

[0102] (6) Hardening off seedlings and transplanting

[0103] After the tissue culture seedlings have matured, move them outdoors and harden them under natural light for 6 days. Then, open the bottle cap and continue hardening them for another 2 days. Take out the seedlings, wash off the culture medium, and transplant them into a seedling substrate made of peat moss, red soil, and mushroom residue in a volume ratio of 4:4:2. Cover the seedlings with a shade net with an 85% shading degree within 7 days after transplanting, and then carry out routine seedling management.

[0104] (7) Germplasm preservation

[0105] After induced germination culture, the tissue culture seedlings were transferred to a preservation medium containing 2 / 3 MS + BA 0.1 mg / L + IAA 0.5 mg / L + 30 g / L sucrose + 3.8 g / L agar powder, with a pH of 5.8–6.0. ​​The medium was then placed in 270 mL PC plastic bottles with a 70 mm diameter × 110 mm height sealed cap, with 5 seedlings inoculated per bottle. The bottle opening was wrapped twice with plastic wrap. The preservation medium was replaced with fresh medium every 35 days. When ready for use, the seedlings were transferred to culture bottles containing seedling culture medium and cultured for 25 days before being hardened off and transplanted.

[0106] Comparative Example 1:

[0107] A method for utilizing the resilient seeds of the critically endangered plant *Hopea stenoptera* includes seed collection, seed pretreatment, seed sterilization, germination induction, seedling cultivation, hardening-off transplanting, and germplasm preservation. The specific operational steps are as follows:

[0108] The steps in this example are basically the same as those in Example 1, except that some steps in steps (2) and (3) have been adjusted. In step (2), during seed pretreatment, only the seed wings on both sides of the seeds of *Hopea stenoptera* are removed, and after washing the surface of the seeds with clean water to remove impurities, they are transferred to a clean bench. In step (3), during seed sterilization, the clean bench is opened, and the seeds are soaked in 75% alcohol for 1 minute. The seeds are then placed in a sterilization solution made of 0.1% HgCl2 solution and 1 drop of Tween 20 and stirred and soaked for 15 minutes. This process is repeated once, and the seeds are then soaked in sterile water 3 to 5 times. The seed coat is then removed with a scalpel to obtain sterilized embryos.

[0109] Comparative Example 2

[0110] A method for utilizing the resilient seeds of the critically endangered plant *Hopea stenoptera* includes seed collection, seed pretreatment, seed sterilization, germination induction, seedling cultivation, hardening-off transplanting, and germplasm preservation. The specific operational steps are as follows:

[0111] The steps in this example are basically the same as those in Example 1. The only difference is that the germination induction medium used in step (4) is different. That is, immature and semi-mature embryos and mature embryos are all inoculated into MS+BA0.5mg / L+NAA0.8mg / L+30g / L sucrose+3.8g / L agar powder, pH 5.8~6.0 germination induction medium ②.

[0112] Comparative Example 3

[0113] A method for utilizing the resilient seeds of the critically endangered plant *Hopea stenoptera* includes seed collection, seed pretreatment, seed sterilization, germination induction, seedling cultivation, hardening-off transplanting, and germplasm preservation. The specific operational steps are as follows:

[0114] The steps in this example are basically the same as those in Example 1. The only difference is that the germination induction medium used in step (4) is different. That is, immature and semi-mature embryos and mature embryos are all inoculated into MS+BA0.25mg / L+NAA0.25mg / L+GA30.8mg / L+30g / L sucrose+3.8g / L agar powder, pH 5.8~6.0 germination induction medium ①.

[0115] Comparative Example 4

[0116] A method for utilizing the resilient seeds of the critically endangered plant *Hopea stenoptera* includes seed collection, seed pretreatment, seed sterilization, germination induction, seedling cultivation, hardening-off transplanting, and germplasm preservation. The specific operational steps are as follows:

[0117] The steps in this example are basically the same as those in Example 1, except that the only difference is the seedling culture medium formula used in step (5) when cultivating seedlings. In Comparative Example 4, MS was used as the basic culture medium, and the hormones used were a combination of NAA and IBA, namely MS + NAA 1.5 mg / L + IBA 1.5 mg / L + 30 g / L sucrose + 3.8 g / L agar powder, pH 5.8-6.0.

[0118] Comparative Example 5

[0119] A method for utilizing the resilient seeds of the critically endangered plant *Hopea stenoptera* includes seed collection, seed pretreatment, seed sterilization, germination induction, seedling cultivation, hardening-off transplanting, and germplasm preservation. The specific operational steps are as follows:

[0120] The steps in this example are basically the same as those in Example 1, except that the only difference is the preservation medium formula used in step (7) for germplasm preservation. The preservation medium formula of Comparative Example 5 directly uses the germination induction medium, that is, immature or semi-mature embryos are prepared with MS + BA 0.25mg / L + NAA 0.25mg / L + GA3 0.8mg / L + 30g / L sucrose + 3g / L agar powder, and mature embryos are prepared with MS + BA 0.5mg / L + NAA 0.8mg / L + 30g / L sucrose + 3g / L agar powder, with pH 5.8~6.0.

[0121] Implementation results:

[0122] Seeds of *Hopea stenoptera* were utilized using the treatment methods of Examples 1-3 and Comparative Examples 1-5 within the scope of protection of this invention, and the seed utilization effects of different treatments were statistically compared.

[0123] 1. The seed utilization effects of the three examples are shown in Table 1.

[0124] Table 1: Seed utilization effect of the examples

[0125]

[0126] As shown in Table 1, Example 1 is the optimal example. The contamination rate after seed treatment and sterilization is the lowest. The germination rate of seeds of different maturity is the highest among the same category. The seedling rate, seedling survival rate, and transplant survival rate are all the highest. The browning mortality rate is the lowest during storage and culture, and the longest storage time is the longest.

[0127] 2. Comparison of Seed Sterilization Effects

[0128] Seed sterilization is a crucial step in the utilization of seeds through culture media. Bacterial contamination can prevent seeds from germinating and growing properly, hindering further utilization and severely impacting seed efficiency. The feasibility of a seed sterilization scheme depends primarily on the contamination rate and the start-up rate. Comparative Example 1 primarily examines whether the steps added to the seed sterilization scheme of this invention—washing with cleaning solution, wrapping fine sand in gauze, washing with tea tree oil solution, burning with an alcohol lamp, irradiation with ultraviolet light, and increasing the sterilization water temperature—help reduce the sterilization rate while increasing the start-up rate. During the seed sterilization scheme screening experiment, each step was compared individually. To more intuitively demonstrate the effectiveness of the sterilization scheme of this invention, Comparative Example 1 used relatively conventional sterilization steps reported in the literature, comparing its sterilization effect with that of Example 1. The sterilization effects are compared in Table 2.

[0129] Table 2: Comparison of sterilization effects between Comparative Example 1 and Example 1

[0130] deal with Pollution rate (%) Start-up rate (%) Example 1 9.94 89.25 Comparative Example 1 36.66 62.08

[0131] As shown in Table 2, compared with Example 1, Comparative Example 1 showed a 26.72% increase in contamination rate and a 27.17% decrease in initiation rate. The comparison between Comparative Example 1 and Example 1 demonstrates that this series of innovative combined measures significantly reduced the contamination rate and increased the initiation rate. The function of each step is analyzed as follows: Before seed sterilization, the seeds were washed with a cleaning solution prepared by adding 5g of flour to 1000mL of clean water. The fine flour particles have a certain adsorption capacity, and the flour-water solution has a certain viscosity, which can wash away dirt adhering to the seed coat surface. The method of wrapping sterilized fine sand in gauze increases the friction on the seed coat, promoting germination, and also facilitates sterilization while avoiding direct contact of alcohol with the seeds, thus preventing damage to their viability. During seed sterilization, the seeds were soaked in 75% alcohol followed by a brief flame burning, which avoids alcohol residue on the surface, and the brief heat treatment can inactivate some bacteria. After soaking in the sterilization solution, the seeds were soaked in sterilization water at 35-40℃. The higher temperature of the sterilization water not only washes away the sterilization solution but also activates the seed viability, further promoting germination. Through extensive experimental screening, this invention has yielded a preferred sterilization scheme for the sterilization of narrow-leaved Gloydius seeds.

[0132] 3. Comparison of induced germination effects

[0133] Comparative Examples 2 and 3 primarily examined whether seeds of different maturity levels could be induced to germinate using the same culture medium formulation. The comparison of germination induction effects for each example is shown in Table 3.

[0134] Table 3: Comparison of germination induction effects between Comparative Examples 2-3 and Example 1

[0135] deal with Germination rate of immature and semi-mature seeds (%) Germination rate of mature seeds (%) Example 1 88.23 92.21 Comparative Example 2 52.16 90.48 Comparative Example 3 86.19 68.30

[0136] Table 3 shows that the suitable germination media for seeds of different maturity levels differ. When immature and semi-mature seeds were cultured in a suitable germination induction medium for mature seeds, the germination rate was only 52.16%, a decrease of 36.07%, significantly lower than the 88.23% in Example 1. Similarly, when mature seeds were cultured in a suitable germination induction medium for immature and semi-mature seeds, the germination rate was only 68.30%, a decrease of 23.91%, significantly lower than the 92.21% in Example 1. The reason for this may be that different seed maturity levels result in significant differences in the levels of endogenous hormones, leading to variations in the types and concentrations of hormones that promote germination. The difference between germination induction media ① and ② lies in the different types and concentrations of cytokinins. Adding an appropriate amount of GA3 to the germination induction medium for immature and semi-mature seeds promotes germination, while mature seeds may have higher levels of GA3 in their endogenous hormones, which may inhibit germination in the germination induction medium with added GA3. Therefore, different culture medium formulations should be used for seeds of different maturity levels of *Hopea stenoptera*. This invention designs different germination induction media for seeds of different maturity levels, and screens and optimizes them to obtain two germination induction media formulations suitable for seeds of different maturity levels.

[0137] 4. Comparison of the effects of seedling culture media

[0138] Table 4 shows the comparison of the effects of basic culture medium and hormone types on seedling growth.

[0139] Table 4 Comparison of seedling growth effect between Comparative Example 4 and Example 1

[0140] deal with Seedling survival rate (%) Example 1 84.59 Comparative Example 4 36.66

[0141] Comparative Example 4 primarily investigated the effects of the basal medium and hormone type in the seedling culture medium on seedling formation. MS was used as the basal medium in Comparative Example 4, while 1 / 2 MS was used in Example 1. The difference lies in the content of macronutrients. Since root formation does not require large amounts of nutrients, reducing nutrient content encourages the plant to actively seek and absorb nutrients, rather than passively absorbing them. Higher inorganic salt concentrations are detrimental to the formation of initial root cells, easily leading to cell dehydration. Furthermore, another key factor affecting rooting is the auxin content. Adding appropriate types and concentrations of auxin to the culture medium can further promote the formation of root meristems. This invention, through extensive experimental screening, has determined that the main factor affecting seedling formation is the basal medium, with 1 / 2 MS being the optimal basal medium. The rooting nutrient requirements of *Hopea stenoptera* align with the patterns of most plants. When screening auxin types, commonly used NAA, IBA, and IAA were selected. Generally, NAA is more stable than IAA. In the screening experiments of this invention, it was found that IBA was the main factor affecting seedling emergence, followed by IAA. The effect of NAA+IBA was significantly lower than that of IAA+IBA. The reason may be that different plants have different sensitivities and response mechanisms to plant hormones, and IAA is more suitable for *Hopea stenoptera*. To more intuitively demonstrate the influence of basic culture medium and auxin NAA and IAA on seedling emergence, a comparative experiment was conducted between Comparative Example 4 and Example 1. The statistical results are shown in Table 4. As shown in Table 4, the seedling emergence rate of Comparative Example 4 was only 36.66%, significantly lower than the seedling emergence rate of 84.59% in Example 1. Therefore, the seedling emergence culture medium formula for *Hopea stenoptera* was selected as a combination of 1 / 2 MS + IAA + IBA.

[0142] 5. Comparison of the effects of preserving culture media

[0143] Table 5 shows a comparison of the effects of preservation culture media on germplasm resources.

[0144] Table 5 Comparison of seedling growth effects between Comparative Example 5 and Example 1

[0145] deal with Brown mortality rate at 60 days (%) Maximum storage time (d) Survival rate (%) after transplanting after seedling recovery Example 1 7.91 93.31 80.18 Comparative Example 5 58.94 45.28 62.16

[0146] Comparative Example 5 primarily investigated whether an induction germination medium could be directly used during preservation culture. Example 1 used a preservation medium of 2 / 3 MS + BA 0.2 mg / L + IAA 0.5 mg / L + 30 g / L sucrose + 3.8 g / L agar powder. Comparative Example 5 used preservation media of MS + BA 0.25 mg / L + NAA 0.25 mg / L + GA3 0.8 mg / L + 30 g / L sucrose + 3.8 g / L agar powder and MS + BA 0.5 mg / L + NAA 0.8 mg / L + 30 g / L sucrose + 3.8 g / L agar powder, representing the optimal induction germination media for immature, semi-mature, and mature seeds, respectively. The purpose of the preservation medium is to maintain the seedlings in a state of minimum viability, that is, to delay their growth to a certain extent without losing their vitality. Lowering the concentration of inorganic salts and hormone levels is beneficial for delaying growth, while an appropriate combination of cytokinins and auxins is beneficial for maintaining normal viability. Table 5 shows a comparison of the seedling emergence rates between Comparative Example 5 and Example 1. As can be seen from Table 5, when calculating the 60-day browning mortality rate, Comparative Example 5 had a rate as high as 58.94%, significantly higher than Example 1's 7.91%. The longest preservation time for Comparative Example 5 was only 45.28 days, significantly lower than Example 1's 93.31 days. Furthermore, the survival rate of the recovered seedlings after transplanting in Comparative Example 5 was only 62.12%, a decrease of 18.06% compared to Example 1's 80.18%. This indicates that the germination-inducing medium for *Hopea stenoptera* cannot be used as a preservation medium for germplasm resources. The reason may be due to its excessively favorable nutrient conditions and high hormone levels, which stimulate rapid plant growth and consume nutrients, leading to nutrient deficiency in the later stages. Therefore, screening and optimizing a preservation medium suitable for maintaining minimum growth of *Hopea stenoptera* is beneficial for reducing the browning mortality rate and extending its preservation time. The preservation medium screened in this invention can delay growth for a certain period, extend the preservation time, and thus improve seed utilization.

[0147] The analysis of the above results proves that Example 1 has the best utilization effect. This also shows that the utilization method disclosed in this invention can maximize the utilization of immature, semi-mature, and mature seeds that cannot germinate into seedlings under natural conditions, including those that have not yet lost water. Under the condition of a certain number of seeds, it promotes the conversion of more seeds into seedlings. It realizes the in vitro preservation of *Hopea stenoptera* seeds for a certain period of time, breaks the limitation that seed germination utilization must be carried out during the seed maturity harvest season, significantly improves the seed utilization rate, provides sterile materials for long-term preservation of germplasm resources and large-scale breeding, and lays a technical foundation for the protection of this species, ecological restoration, and resource development and utilization. It has great practical significance for saving this endangered tree species.

Claims

1. A method for improving the seed utilization rate of the critically endangered plant *Hopea stenoptera*, characterized in that: It includes seed collection, seed pretreatment, seed sterilization, germination induction, seedling cultivation, hardening-off transplanting, and germplasm preservation; the specific operational steps are as follows: (1) Seed collection: Collect fresh, disease-free seeds from the natural distribution area of ​​*Hopea stenoptera*. (2) Seed pretreatment: Remove the seed wings from both sides of the narrow-leaved holly seeds, wash the surface of the seeds with clean water to remove impurities, stir and soak in cleaning solution for 5 minutes, wash off the cleaning solution with pure water, wrap the seeds and sterilized fine sand in gauze, soak in a 2% Australian tea tree essential oil aqueous solution for 20 minutes, stir and soak in sterilized water for 3-5 minutes, and then transfer to an ultra-clean workbench. (3) Seed sterilization: Open the laminar flow hood, immerse the seeds in 75% alcohol for 1 minute, sterilize them by flame burning with an alcohol lamp for 5 seconds, open the gauze, wash away the fine sand from the seeds, place them in an inoculation tray containing sterilized filter paper, turn on the ultraviolet lamp of the laminar flow hood and irradiate for 30 minutes, then put the seeds into the sterilization solution and stir and soak for 10-15 minutes, repeat once, then immerse them in sterile water at 35-40℃ 3-5 times, peel off the seed coat with a scalpel to obtain sterilized embryos; (4) Inducing germination: After sterilization, the embryos were classified according to maturity and inoculated into germination induction medium. Immature and semi-mature embryos were inoculated into germination induction medium ①, and mature embryos were inoculated into germination induction medium ②. One embryo was inoculated into each bottle. The culture conditions were 25±3 ℃ and cultured in the dark. (5) Seedling cultivation: After culturing the embryos of *Hopea stenoptera* in the germination induction medium for 10–15 days, they were transferred to culture bottles of seedling culture medium. The culture conditions were: temperature 25±3 ℃, and the culture bottles were placed under light for 12–15 h / d and light intensity of 1500–3000 lx. (6) Hardening off seedlings and transplanting: After the tissue culture seedlings have matured, move them outdoors and harden them under natural light for 5-7 days. Then open the bottle cap and continue hardening for 1-3 days. Take out the seedlings, wash off the culture medium, and transplant them into the seedling substrate. Cover them with a shade net with a shading degree of 75-85% within 7 days after transplanting, and then carry out routine seedling management. (7) Germplasm preservation: After inducing germination, the tissue culture seedlings were transferred to preservation medium. The medium was placed in 270 mL PC plastic bottles with a 70 mm diameter × 110 mm height sealed cap. Five seedlings were inoculated into each bottle. The bottle mouth was wrapped with plastic wrap twice. The preservation medium was replaced with fresh medium every 30–40 days. When ready for use, the seedlings were transferred to culture bottles containing seedling culture medium. After 25 days of culture, the seedlings could be directly hardened off and transplanted. The germination induction medium ① described in step (4) above has the following formula: MS + BA 0.25 mg / L + NAA 0.25~0.5 mg / L + GA3 0.5~0.8 mg / L + 30 g / L sucrose + 3.8 g / L agar powder, pH 5.8~6.0; the germination induction medium ② has the following formula: MS + BA 0.5 mg / L + NAA 0.4~0.8 mg / L + 30 g / L sucrose + 3.8 g / L agar powder, pH 5.8~6.

0. The seedling culture medium formula described in step (5) above is 1 / 2MS + IAA 1.5~2.0 mg / L + IBA 1.5~2.0 mg / L + 15 g / L sucrose + 3.8 g / L agar powder, pH 5.8~6.0; The preservation culture medium formula described in step (7) above is 2 / 3MS + BA 0.1~0.2 mg / L + IAA 0.5 mg / L + 30g / L sucrose + 3.8 g / L agar powder, pH 5.8~6.

0.

2. The method for improving the seed utilization rate of the critically endangered plant *Hopea stenoptera* according to claim 1, characterized in that: The seed collection in step (1) includes collecting seeds from trees and picking up fallen seeds.

3. The method for improving the utilization rate of stubborn seeds of the critically endangered plant *Hopea stenoptera* according to claim 1, characterized in that: The fresh seeds mentioned in step (1) include immature, semi-mature and mature seeds that have not yet lost water. The seed coat and seed wings of immature seeds are all green; the seed coat of semi-mature seeds is partially yellowish-green and the seed wings are dark yellow; the seed coat of mature seeds is entirely yellowish-green and the seed wings are dark yellow or brown.

4. The method for improving the seed utilization rate of the critically endangered plant *Hopea stenoptera* according to claim 1, characterized in that: The cleaning solution in step (2) is 5 g of flour added to 1000 mL of clean water.

5. The method for improving the seed utilization rate of the critically endangered plant *Hopea stenoptera* according to claim 1, characterized in that: In step (3), sterile water to a depth of 1 cm needs to be poured onto the sterile filter paper in the inoculation tray.

6. The method for improving the utilization rate of stubborn seeds of the critically endangered plant *Hopea stenoptera* according to claim 1, characterized in that: The sterilization solution in step (3) is a mixture of 0.1% HgCl2 solution and 1 drop of Tween 20.

7. The method for improving the seed utilization rate of the critically endangered plant *Hopea stenoptera* according to claim 1, characterized in that: The seedling substrate in step (6) is a mixture of peat soil, red soil and mushroom residue in a volume ratio of 4:4:2.

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