A method for improving seedling emergence rate and growth state of a plant of a small population of a species of Leucopogon parvifolius

Abstract

The application is a method for improving seedling rate of tiny population plant narrow-leaved slope and improving growth state of seedling, which comprises seed collection and screening, seed cleaning and sterilization, seed germination promotion, planting, transplanting and seedling management. The application significantly improves seedling rate of narrow-leaved slope and growth performance of seedling, so that normal seedling rate reaches 96.72%, seedling rate of small seed reaches 90.25%, height of seedling can reach 45.52 cm in one year, ground diameter can reach 5.04 mm, seedling is healthy and has consistent growth state, which provides seedling basis, scientific basis and technical support for returning to nature and realizing population reconstruction of the application, lays technical foundation for protection organism, ecological restoration and resource development and utilization of the species, and has great practical significance for saving the endangered tree species.

Description

Technical Field

[0001] This invention relates to the field of seedling cultivation technology, specifically a method for improving the seedling germination rate of *Hopea stenoptera*, a plant with very small populations, and improving the growth status of seedlings. Background Technology

[0002] Narrow-leaved Hopea (Hopea chinensis Hand.-Mazz.), also known as South China Hopea or narrow-leaved Hopea, belongs to the genus Hopea in the family Dipterocarpaceae. It grows in foothills, valleys, and hillside forests below 600m in altitude and is one of the representative tree species of tropical evergreen monsoon forests in northern and southern Guangxi. It is of great value for studying the composition, geographical distribution, and ecological habits of tropical rainforests. Furthermore, its wood is hard and extremely resistant to decay, earning it the nickname "ten-thousand-year wood," making it an excellent special-purpose timber species. Its resin, similar to tamarisk resin, can be used in paint manufacturing. The bark has a dragon robe-like pattern, hence the name "dragon robe tree." Byproducts include aromatic oils, which can be used in high-end cosmetics and as valuable traditional Chinese medicine materials, making it extremely valuable. However, the narrow-leaved holly is a remnant species from the Tertiary period, distributed in "refuges" formed by local topography. Due to the destruction caused by human production activities and the impact of climate change, the number of wild narrow-leaved holly individuals has decreased sharply in the past 20 years, and it is threatened with extinction. It has been classified as a national second-class key protected plant, a species with extremely small population in China, and listed as a critically endangered (CR) species by the International Union for Conservation of Nature (IUCN).

[0003] Studies have found that *Hopea stenoptera* produces few seeds and exhibits alternate bearing, with some years even failing to produce any seeds at all. The conversion rate of seeds to seedlings in wild populations is relatively low. While seedlings exist in the understory, most are in poor condition, with few maturing into saplings or mature trees. Even when seedlings reach about 20cm in height, large numbers often die off. Expanding the population through conservation measures is the most fundamental condition for saving endangered plants. Research on improving seed-to-seedling rates and seedling growth performance in *Hopea stenoptera* is of great value for the protection and expansion of wild populations, as well as for ex-situ and near-situ conservation efforts. To date, no research reports on seed-seedling cultivation techniques for *Hopea stenoptera* have been found, making systematic research on these techniques urgently needed. Summary of the Invention

[0004] The purpose of this invention is to solve the technical problem of low seed conversion rate and poor seedling growth under natural conditions of Hopunia angustifolia seeds. Under a certain number of seeds, the invention aims to promote the conversion of more seeds into seedlings and improve the growth status of seedlings, thereby providing robust seedlings for the natural colonization and reintroduction of seedlings.

[0005] This invention is achieved through the following technical solution:

[0006] A method for improving the seed germination rate and seedling growth status of the extremely small population plant *Hopea stenoptera*, the main steps of which are as follows:

[0007] (1) Seed collection and screening:

[0008] Fresh, mature seeds free from pests and diseases were collected from the natural distribution area of ​​*Hopea stenoptera*. The seeds were then sorted and classified according to size after the two seed wings were removed.

[0009] (2) Seed washing and sterilization:

[0010] After cleaning the seeds with clean water to remove impurities, wrap the seeds and fine sand in gauze and gently rub them repeatedly. Wash away the fine sand with clean water, soak them in a 10 mg / mL solution of crude extract of *Hopea stenoptera* branches and leaves for 20 minutes, then soak them in a 0.1% benzalkonium chloride solution for 5 minutes with stirring, and rinse them with running water for 3-5 minutes.

[0011] (3) Seed germination promotion:

[0012] Place the smaller seeds in a sealed bag containing a germination promoter, and then soak them together with normal seeds in warm water at 30-35℃ for 20-30 minutes. After soaking, sow the seeds on the prepared sand bed, spray to keep the sand bed moist, and build an arched frame on the sand bed with wire and cover it with plastic film.

[0013] (4) Planting: After the cotyledons and hypocotyl of the seed have fully germinated, gently pull the seed out of the sand bed, rinse off the fine sand with clean water, and plant it in the seedling container cup. When planting, be careful to gently bury the radicle in the soil and do not damage or break the radicle.

[0014] (5) Transplanting: When the seedlings grow to 20-30cm in height, transplant them into new seedling cups. When transplanting, trim the longer roots, then soak the seedling cups in the root protection nutrient solution for 3-5 minutes, then cut open the non-woven bag and plant them into a larger seedling bag.

[0015] (6) Seedling management: After planting and transplanting, the seedlings should be placed in an outdoor seedling greenhouse with shade netting, and water should be sprayed once a day. The light transmittance should be maintained at 40-50% for one week. After one week, the light transmittance should be gradually increased. One month after planting and transplanting, the light transmittance should be increased to 80-90%. Nutrient solution should be sprayed every 15-20 days.

[0016] Preferably, the collection of fresh, mature seeds free from pests and diseases in step (1) above includes collecting seeds from trees and picking up fallen seeds; the mark of mature seeds is that the seed coat is entirely yellow-green and the seed wings are dark yellow or brown; the criteria for judging fresh seeds free from pests and diseases are that there are no insect mouths on the seed surface, the seed coat has not yet withered and wrinkled, and the seed wings have not yet dehydrated and dried; the method of screening and classifying according to size is to use a sieve with a diameter of 43cm and a pore size of 10mm to filter the seeds, and those with a diameter ≥10mm are classified as normal seeds, and those <10mm are classified as small seeds.

[0017] Preferably, the method for preparing the crude extract of *Hopea stenoptera* branches and leaves in step (2) above is to collect the branches and leaves of *Hopea stenoptera*, dry them in a drying oven at 50°C, pulverize them, and soak them in a methanol solution with a volume of 5 times their mass for 48 hours.

[0018] Preferably, the sprouting agent in step (3) above is composed of 1.0-1.5 mg / L IAA + 0.8-1.0 mg / L GA3 + 50-100 mg / L KH2PO3 + 200-300 mg / L NH4NO3 + 100-200 mg / L CaCl2.

[0019] Preferably, the sowing method described in step (3) above is to lightly press the seeds into shallow pits, with a spacing of 3-5 cm between the seeds, and then sprinkle a thin layer of sand. The sand covering thickness is ≤0.5 mm, but it is necessary to ensure that the seeds are completely covered without any bare areas. The spraying refers to spraying water with a sprayable container. It is important to avoid using too much water so that the seeds are exposed on the sand.

[0020] Preferably, in step (4) above, the seedling container cup is a non-woven fabric cup with a diameter of 8cm and a height of 12cm. The substrate is coconut coir: red soil: vermiculite in a volume ratio of 5:3:1. After the substrate is filled into the seedling container, it is thoroughly soaked with 3% FeSO4 aqueous solution.

[0021] Preferably, the root-protecting nutrient agent mentioned in step (5) above is 0.5~1.0mg / L GGR+50~100mg / LKH2PO3+800~1000mg / L KNO3.

[0022] Preferably, the larger seedling bag mentioned in step (5) above refers to a non-woven fabric cup with a diameter of 20cm and a height of 24cm. The substrate is placed in layers inside the seedling bag. The upper substrate is made of red soil: peat soil: mushroom residue with a volume ratio of 5:2:1, and the lower substrate is made of coconut coir: river sand: vermiculite with a volume ratio of 4:4:1. The volume ratio of the upper substrate to the lower substrate is 2:1.

[0023] Preferably, the nutrient solution mentioned in step (6) above is 30-50 mg / L KH2PO3 + 400-500 mg / L KNO3, and the spraying method is to spray the leaves with the nutrient solution and irrigate the substrate thoroughly.

[0024] This invention has the following advantages and positive effects over the prior art:

[0025] 1. This invention systematically proposes a series of detailed schemes from seed collection and germination promotion to seedling growth. In particular, it innovatively proposes to use different germination promotion schemes for seeds of different sizes, and different seedling containers and substrates for seedlings at different developmental stages. When transplanting seedlings, the roots are soaked in a root-protecting nutrient agent, and a specific nutrient solution is sprayed on the seedlings. This significantly improves the seedling survival rate and seedling growth performance, resulting in a seedling survival rate of 96.72% for normal seeds and 90.25% for smaller seeds. Seedlings can grow up to 45.52cm in height and 5.04mm in diameter in one year. The seedlings are robust and grow in a uniform manner, providing a seedling foundation, scientific basis and technical support for their return to nature and the reconstruction of the population.

[0026] 2. In the seed treatment of this invention, the seed wings are first removed. This avoids wasting more seedling space due to long seed wings and improves germination efficiency. Long seed wings prevent the seeds from making close contact with the substrate and from absorbing water and other substances to promote germination. Moreover, seed wings have a significant inhibitory effect on seed germination. Removing the seed wings plays an important role in promoting seed germination. Afterward, the seeds are wrapped in gauze and rubbed together to increase friction, process the seed coat in batches, and reduce the inhibitory effect of the seed coat on seed germination.

[0027] 3. The seed sterilization scheme proposed in this invention innovatively incorporates the step of soaking seeds in an aqueous solution of crude extract of *Polygonum angustifolium*. This allows the crude extract to have antibacterial activity without harming the seeds, thereby improving the sterilization success rate without damaging seed vigor, achieving the desired effect.

[0028] 4. The germination-promoting scheme proposed in this invention uses warm water soaking to promote germination, and seeds of different sizes are classified. Smaller seeds are soaked in warm water while a germination-promoting agent is added, which significantly improves the germination rate of seeds with low germination rates under natural conditions. This makes full use of the relatively scarce germplasm resources and promotes the conversion of more seeds into seedlings under the condition of a certain number of seeds, laying a technical foundation for research on the conservation biology, restoration ecology, and resource development and utilization of this species.

[0029] 5. In the germination promotion scheme proposed in this invention, after disinfecting the sand bed with potassium permanganate, rinsing the sand bed with warm water at 35-40℃ can appropriately increase the temperature of the sand bed. The seeds of Hopinia angustifolia mostly mature between December and January when the temperature is relatively low. Since the seeds are typical stubborn seeds, they cannot be stored and can only be sown immediately after harvesting. The low temperature is not conducive to seed germination. Soaking the seeds in warm water before sowing them in a sand bed with a more suitable temperature significantly improves the seed germination effect.

[0030] 6. In the germination promoter designed for small seeds in this invention, IAA and GA3 act as growth regulators to promote the secretion of endogenous plant hormones in the seed and increase amylase activity, thereby accelerating seed metabolism and improving seed germination ability. CaCl2, NH4NO3, and KH2PO3 are essential nutrients for plant growth and can maintain metabolic stability inside the seed. On the other hand, they can also act as osmotic substances to regulate the rate of water entering the seed and can enter the embryonic cells of the seed to affect the pre-germination metabolism of the seed. The two work synergistically to improve the germination rate of small seeds.

[0031] 7. This invention improves the survival rate of seedlings by soaking them in a root-protecting nutrient solution during repotting. The GGR in the root-protecting nutrient solution can promote root regeneration, while KH2PO3 and KNO3 provide rich nitrogen, phosphorus, potassium and other nutrients for plant growth, resulting in a survival rate of 97.37% for narrow-leaved holly seedlings after repotting.

[0032] 8. This invention uses different seedling substrates at different developmental stages, which can significantly improve the seed germination rate and seedling growth performance of *Hopea stenoptera*, with a one-year seedling mortality rate as low as 2.71%. During the germination and seedling stage, a coconut coir:red soil:vermiculite volume ratio of 5:3:1 is used, with coconut coir as the main component. Combined with vermiculite's excellent water and fertilizer adsorption properties, it has good water retention and aeration, fully meeting the critical water requirements during the germination stage. During the seedling growth stage, a layered substrate is used. The upper layer uses red soil:peat moss:mushroom residue in a volume ratio of 5:2:1 as the nutrient layer substrate, ensuring the nutrients needed for seedling growth. The bottom layer uses coconut coir:river sand:vermiculite in a 4:4:1 ratio, increasing bottom aeration and preventing root rot due to poor aeration, which could negatively impact seedling growth or even cause death.

[0033] 9. This invention uses nutrient solution spraying to manage seedlings. Compared with the ordinary method of spreading fertilizer on the substrate surface, liquid nutrients are more easily absorbed. The nitrogen, phosphorus and potassium ratio of the selected and prepared nutrient solution is precisely suitable for the growth and development of narrow-leaved holly seedlings, which significantly improves the growth status of seedlings. Attached Figure Description

[0034] Figure 1 This is a picture of fresh, mature seeds of *Hopea stenoptera*.

[0035] Figure 2 This is a picture of the seeds of *Hopea stenoptera* after sterilization.

[0036] Figure 3 This is a picture of narrow-leaved holly seeds sown on a sand bed to promote germination.

[0037] Figure 4 This is a picture of narrow-leaved holly seeds sown and covered with a thin layer of sand.

[0038] Figure 5 This is a diagram of seeds germinating from *Hopea stenoptera*.

[0039] Figure 6 This is a picture of a seedling growing in a cup after germination of seeds from *Hopea stenoptera*.

[0040] Figure 7 This is a picture of seedlings of *Hopea stenoptera* that have survived transplanting.

[0041] Figure 8 This is a picture of an annual seedling of *Hopea stenoptera*. Detailed Implementation

[0042] 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.

[0043] Seedling survival rate (%) = Number of germinated and plantable seeds / Total number of seeds sown × 100;

[0044] Transplant survival rate (%) = Number of surviving seedlings / Total number of transplants × 100;

[0045] Mortality rate (%) = Number of dead seedlings / Total number of transplanted seedlings surviving × 100;

[0046] Germination lag: the number of days required for the first seed in the tested seeds to germinate;

[0047] Germination period: The time difference between the germination of the first and last seeds in the test.

[0048] Example 1:

[0049] A method for improving the seed germination rate and seedling growth status of the extremely small population plant *Hopea stenoptera*, comprising the following steps:

[0050] (1) Seed collection and screening:

[0051] Seeds were collected from trees and fallen, disease-free, fresh, mature seeds within the natural distribution area of ​​*Hopea stenoptera*. Mature seeds were characterized by a completely yellowish-green seed coat and dark yellow or brown seed wings. Disease-free, fresh seeds were identified by the absence of insect bites on the seed surface, an intact seed coat without wilting or wrinkling, and fresh, undehydrated seed wings. After removing the seed wings, the seeds were sorted by size. The sorting method involved filtering the seeds through a 43cm diameter sieve with 10mm pores. Seeds ≥10mm in diameter were classified as normal seeds, while those <10mm were classified as small seeds.

[0052] (2) Seed washing and sterilization:

[0053] After washing the seeds with clean water to remove impurities, wrap the seeds and fine sand in gauze and gently rub them repeatedly. Wash away the fine sand with clean water and soak them in a 10 mg / mL aqueous solution of crude extract of *Holothuria angustifolia* branches and leaves for 20 minutes. The crude extract of *Holothuria angustifolia* branches and leaves is prepared by collecting branches and leaves of *Holothuria angustifolia*, drying them in a drying oven at 50℃, pulverizing them, soaking them in a methanol solution with a volume of 5 times their mass for 48 hours, and then stirring and soaking them in a 0.1% benzalkonium chloride aqueous solution for 5 minutes, followed by rinsing with running water for 3-5 minutes.

[0054] (3) Seed germination promotion:

[0055] Smaller seeds were placed in a sealed bag containing a germination promoter composed of 1.5 mg / L IAA + 0.8 mg / L GA3 + 100 mg / L KH2PO3 + 200 mg / L NH4NO3 + 200 mg / L CaCl2. These seeds were then soaked in 35°C warm water for 30 minutes along with normal seeds. The sand bed was pre-treated with a 0.5% potassium permanganate solution, and then soaked in 40°C warm water every hour, repeating this process 3-5 times. The soaked seeds were then sown on the treated sand bed, gently pressing them into shallow pits with a 5 cm spacing between seeds. A thin layer of sand (≤0.5 mm) was then sprinkled on top, ensuring complete seed coverage. The sand bed was kept moist using a spray bottle, avoiding excessive water that could expose the seeds. An arched frame was then constructed over the sand bed using wire mesh and covered with a plastic film.

[0056] (4) Planting: After the cotyledons and hypocotyl of the seed have fully germinated, gently pull the seed out of the sand bed, rinse off the fine sand with clean water, and plant it in the seedling container cup. The seedling container cup is a non-woven cup with a diameter of 8cm and a height of 12cm. The substrate is coconut coir: red soil: vermiculite in a volume ratio of 5:3:1. After the substrate is filled into the seedling container, it is thoroughly watered with 3% FeSO4 aqueous solution. When planting, be careful to gently bury the radicle in the soil and do not damage or break the radicle.

[0057] (5) Transplanting: When the seedlings grow to 20cm in height, transplant them into new cups. When transplanting, trim the longer roots. Then soak the seedling cups in the root protection nutrient solution for 5 minutes. The root protection nutrient solution consists of 0.5mg / L GGR + 50mg / L KH2PO3 + 1000mg / L KNO3. After that, cut open the non-woven bag and plant it into a larger seedling bag. The larger seedling bag refers to a non-woven cup with a diameter of 20cm and a height of 24cm. Place the substrate in layers inside the seedling bag. The upper substrate is red soil: peat soil: mushroom residue with a volume ratio of 5:2:1. The lower substrate is coconut coir: river sand: vermiculite with a volume ratio of 4:4:1. The volume ratio of the upper substrate to the lower substrate is 2:1.

[0058] (6) Seedling management: After planting and transplanting, the seedlings should be placed in an outdoor seedling greenhouse with shade netting. Spray water once a day. Keep the light transmittance at 40-50% for one week. Gradually increase the light after one week. One month after planting and transplanting, the light transmittance should be increased to 80-90%. Spray nutrient solution every 15 days. The nutrient solution is 50mg / L KH2PO3 + 400mg / L KNO3. Spray the leaves with a spray and thoroughly wet the substrate with the nutrient solution.

[0059] Example 2:

[0060] A method for improving the seed germination rate and seedling growth status of the extremely small population plant *Hopea stenoptera*, comprising the following steps:

[0061] (1) Seed collection and screening:

[0062] Seeds were collected from trees and fallen, disease-free, fresh, mature seeds within the natural distribution area of ​​*Hopea stenoptera*. Mature seeds were characterized by a completely yellowish-green seed coat and dark yellow or brown seed wings. Disease-free, fresh seeds were identified by the absence of insect bites on the seed surface, an intact seed coat without wilting or wrinkling, and fresh, undehydrated seed wings. After removing the seed wings, the seeds were sorted by size. The sorting method involved filtering the seeds through a 43cm diameter sieve with 10mm pores. Seeds ≥10mm in diameter were classified as normal seeds, while those <10mm were classified as small seeds.

[0063] (2) Seed washing and sterilization:

[0064] After washing the seeds with clean water to remove impurities, wrap the seeds and fine sand in gauze and gently rub them repeatedly. Wash away the fine sand with clean water and soak them in a 10 mg / mL aqueous solution of crude extract of *Holothuria angustifolia* branches and leaves for 20 minutes. The crude extract of *Holothuria angustifolia* branches and leaves is prepared by collecting branches and leaves of *Holothuria angustifolia*, drying them in a drying oven at 50℃, pulverizing them, soaking them in a methanol solution with a volume of 5 times their mass for 48 hours, and then stirring and soaking them in a 0.1% benzalkonium chloride aqueous solution for 5 minutes, followed by rinsing with running water for 3-5 minutes.

[0065] (3) Seed germination promotion:

[0066] Smaller seeds were placed in a sealed bag containing a germination promoter composed of 1.0 mg / L IAA + 1.0 mg / L GA3 + 50 mg / L KH2PO3 + 200 mg / L NH4NO3 + 100 mg / L CaCl2. These seeds were then soaked in 30°C warm water for 20 minutes along with normal seeds. The sand bed was pre-treated with a 0.5% potassium permanganate solution, and then soaked in 35°C warm water every hour, repeating this process 3-5 times. The soaked seeds were then sown on the treated sand bed, gently pressing them into shallow pits with a 3 cm spacing between seeds. A thin layer of sand (≤0.5 mm) was then sprinkled on top, ensuring complete seed coverage. The sand bed was kept moist using a spray bottle, avoiding excessive water that could expose the seeds. An arched frame was then constructed over the sand bed using wire mesh and covered with a plastic film.

[0067] (4) Planting: After the cotyledons and hypocotyl of the seed have fully germinated, gently pull the seed out of the sand bed, rinse off the fine sand with clean water, and plant it in the seedling container cup. The seedling container cup is a non-woven cup with a diameter of 8cm and a height of 12cm. The substrate is coconut coir: red soil: vermiculite in a volume ratio of 5:3:1. After the substrate is filled into the seedling container, it is thoroughly watered with 3% FeSO4 aqueous solution. When planting, be careful to gently bury the radicle in the soil and do not damage or break the radicle.

[0068] (5) Transplanting: When the seedlings grow to 30cm in height, transplant them into new cups. When transplanting, trim the longer roots. Then soak the seedling cups in the root protection nutrient solution for 3 minutes. The root protection nutrient solution consists of 1.0mg / L GGR + 100mg / L KH2PO3 + 800mg / L KNO3. After that, cut open the non-woven bag and plant it into a larger seedling bag. The larger seedling bag refers to a non-woven cup with a diameter of 20cm and a height of 24cm. Place the substrate in layers inside the seedling bag. The upper substrate is red soil: peat moss: mushroom residue with a volume ratio of 5:2:1. The lower substrate is coconut coir: river sand: vermiculite with a volume ratio of 4:4:1. The volume ratio of the upper substrate to the lower substrate is 2:1.

[0069] (6) Seedling management: After planting and transplanting, the seedlings should be placed in an outdoor seedling greenhouse with shade netting. Spray water once a day. Keep the light transmittance at 40-50% for one week. Gradually increase the light after one week. One month after planting and transplanting, the light transmittance should be increased to 80-90%. Spray nutrient solution every 20 days. The nutrient solution is 30mg / L KH2PO3 + 500mg / L KNO3. Spray the leaves with a spray and thoroughly wet the substrate with the nutrient solution.

[0070] Example 3:

[0071] A method for improving the seed germination rate and seedling growth status of the extremely small population plant *Hopea stenoptera*, comprising the following steps:

[0072] (1) Seed collection and screening:

[0073] Seeds were collected from trees and fallen, disease-free, fresh, mature seeds within the natural distribution area of ​​*Hopea stenoptera*. Mature seeds were characterized by a completely yellowish-green seed coat and dark yellow or brown seed wings. Disease-free, fresh seeds were identified by the absence of insect bites on the seed surface, an intact seed coat without wilting or wrinkling, and fresh, undehydrated seed wings. After removing the seed wings, the seeds were sorted by size. The sorting method involved filtering the seeds through a 43cm diameter sieve with 10mm pores. Seeds ≥10mm in diameter were classified as normal seeds, while those <10mm were classified as small seeds.

[0074] (2) Seed washing and sterilization:

[0075] After washing the seeds with clean water to remove impurities, wrap the seeds and fine sand in gauze and gently rub them repeatedly. Wash away the fine sand with clean water and soak them in a 10 mg / mL aqueous solution of crude extract of *Holothuria angustifolia* branches and leaves for 20 minutes. The crude extract of *Holothuria angustifolia* branches and leaves is prepared by collecting branches and leaves of *Holothuria angustifolia*, drying them in a drying oven at 50℃, pulverizing them, soaking them in a methanol solution with a volume of 5 times their mass for 48 hours, and then stirring and soaking them in a 0.1% benzalkonium chloride aqueous solution for 5 minutes, followed by rinsing with running water for 3-5 minutes.

[0076] (3) Seed germination promotion:

[0077] Smaller seeds were placed in a sealed bag containing a germination promoter composed of 1.0 mg / L IAA + 1.0 mg / L GA3 + 100 mg / L KH2PO3 + 300 mg / L NH4NO3 + 200 mg / L CaCl2. These seeds were then soaked in 30°C warm water for 25 minutes along with normal seeds. The sand bed was pre-treated with a 0.5% potassium permanganate solution, and then soaked in 35°C warm water every hour, repeating this process 3-5 times. The soaked seeds were then sown on the treated sand bed, gently pressing them into shallow pits with a 5 cm spacing between seeds. A thin layer of sand (≤0.5 mm) was then sprinkled on top, ensuring complete seed coverage. The sand bed was kept moist using a spray bottle, avoiding excessive water that could expose the seeds. An arched frame was then constructed over the sand bed using wire mesh and covered with a plastic film.

[0078] (4) Planting: After the cotyledons and hypocotyl of the seed have fully germinated, gently pull the seed out of the sand bed, rinse off the fine sand with clean water, and plant it in the seedling container cup. The seedling container cup is a non-woven cup with a diameter of 8cm and a height of 12cm. The substrate is coconut coir: red soil: vermiculite in a volume ratio of 5:3:1. After the substrate is filled into the seedling container, it is thoroughly watered with 3% FeSO4 aqueous solution. When planting, be careful to gently bury the radicle in the soil and do not damage or break the radicle.

[0079] (5) Transplanting: When the seedlings grow to 20cm in height, transplant them into new cups. When transplanting, trim the longer roots. Then soak the seedling cups in the root protection nutrient solution for 3 minutes. The root protection nutrient solution consists of 0.5mg / L GGR + 50mg / L KH2PO3 + 900mg / L KNO3. After that, cut open the non-woven bag and plant it into a larger seedling bag. The larger seedling bag refers to a non-woven cup with a diameter of 20cm and a height of 24cm. Place the substrate in layers inside the seedling bag. The upper substrate is red soil: peat moss: mushroom residue with a volume ratio of 5:2:1. The lower substrate is coconut coir: river sand: vermiculite with a volume ratio of 4:4:1. The volume ratio of the upper substrate to the lower substrate is 2:1.

[0080] (6) Seedling management: After planting and transplanting, the seedlings should be placed in an outdoor seedling greenhouse with shade netting. Spray water once a day. Keep the light transmittance at 40-50% for one week. Gradually increase the light after one week. One month after planting and transplanting, the light transmittance should be increased to 80-90%. Spray nutrient solution every 15 days. The nutrient solution is 50mg / L KH2PO3 + 500mg / L KNO3. Spray the leaves with a spray and thoroughly wet the substrate with the nutrient solution.

[0081] Comparative Example 1:

[0082] A method for cultivating *Hopea stenoptera* seeds is basically the same as the steps in Example 1, except that in step (3), smaller seeds are not treated with a germination promoter and are soaked directly in warm water at 30-35°C for 20 minutes, just like normal seeds. Comparative Example 2:

[0083] A method for seedling cultivation of *Hopea stenoptera* is basically the same as the steps in Example 1, except that in step (3), the water used for seed germination promotion is adjusted from 30-35℃ to room temperature water, while the water used for sand bed treatment is adjusted from 35-40℃ to room temperature water. Comparative Example 3:

[0084] A method for seedling cultivation of *Hopea stenoptera* is basically the same as the steps in Example 1, except that in step (5), the roots are not soaked in a root-protecting nutrient solution during transplanting, and the roots are directly transplanted after being trimmed.

[0085] Comparative Example 4:

[0086] A method for seedling from narrow-leaved holly seeds is basically the same as the steps in Example 1. The difference is that the seedling substrate used in step (5) is different. The seedling substrate used in Comparative Example 5-1 is still the seedling substrate used in step (4), that is, the volume ratio of coconut coir: red soil: vermiculite is 5:3:1. The seedling substrate used in Comparative Example 5-2 is red soil.

[0087] Comparative Example 5:

[0088] A method for seedling of *Hopea stenoptera* is basically the same as the steps in Example 1, except that in step (6) seedling management, the nutrient solution sprayed is adjusted to a commercially available compound fertilizer, that is, a compound fertilizer with N:P:K = 15:15:15 is evenly sprinkled on the substrate surface.

[0089] Implementation results:

[0090] Seedlings of *Hopea stenoptera* were cultivated using the treatment methods of Examples 1-3 and Comparative Examples 1-5 within the scope of protection of this invention, and the seedling emergence effect and seedling growth status of different treatments were statistically compared.

[0091] Table 1: Seedling germination rate and seedling growth status of *Hopea stenoptera* seeds in Examples 1-3

[0092]

[0093] Table 1 shows the seedling germination rate and seedling growth status of the three embodiments of *Hopea stenoptera* seeds. As can be seen from Table 1, Embodiment 1 is the optimal embodiment, exhibiting the highest seedling germination rate, transplant survival rate, one-year-old seedling height, and ground diameter for different seed sizes, with the lowest one-year-old seedling mortality rate. The seedlings performed best, exhibiting uniform and robust growth. Embodiments 2 and 3 were slightly inferior, but both still achieved seedling germination rates above 90% and one-year-old seedling mortality rates below 4%, significantly exceeding the seedling germination rate and seedling growth status under natural, undisturbed conditions. Therefore, they are also within the scope of protection of this invention.

[0094] Table 2: Comparison of germination and seedling formation effects between Comparative Examples 1-2 and Example 1

[0095]

[0096] Comparative Example 1 primarily investigated whether germination promoters had a promoting effect on the germination of smaller seeds. Successful seed germination is a crucial factor affecting plant growth and reproduction. Under natural conditions, the seeds of endangered plants often fail to germinate due to their inherent biological characteristics, and the germination time varies considerably. Given the already scarce seed resources, maximizing the germination of various seed types through artificial intervention is of great significance for the regeneration of endangered plant seedlings and the recovery of populations. Therefore, this invention screened and studied germination promoters for smaller seeds with low germination rates under natural conditions. The comparative effects of these germination promoters can be obtained from the experimental data analysis of Comparative Example 1 and Example 1. Table 2 shows that, compared with Example 1, Comparative Example 1 showed that the germination lag and germination duration of smaller seeds were prolonged by 0.82 days and 9.97 days, respectively, and the seedling rate decreased by 11.81%. This indicates that the germination promoter can accelerate the germination of smaller seeds, improve the uniformity of germination within a certain range, and increase the seedling rate. The germination promoter has a significant promoting effect on the germination of smaller seeds. The germination promoter screened in this invention consists of plant growth regulators (IAA and GA3) and inorganic salts (KH2PO3, NH4NO3, and CaCl2). Plant growth regulators can affect the physiological and biochemical processes related to seed germination. Inorganic salts are not only indispensable nutrients for plant growth, maintaining metabolic stability within the seed, but also act as osmotic substances to regulate the rate of water entering the seed, and can also enter the embryonic cells of the seed to affect the pre-germination metabolism. However, they all require appropriate concentrations to exert their promoting effect synergistically; excessively high or low concentrations may have the opposite effect. The germination promoter obtained by this invention through extensive experimental screening is suitable for small seeds of *Hopea stenoptera* and has achieved relatively ideal results. It is of great significance for making full use of small seeds and improving seedling rate when seed resources are scarce.

[0097] Comparative Example 2 primarily investigated whether warm water promotes seed germination. Temperature is another crucial factor affecting seed germination. The physiological changes during seed germination are catalyzed by relevant enzymes, and these enzymatic reactions are closely related to temperature. Low temperatures reduce enzyme activity, hindering seed germination. Only suitable temperatures enhance enzyme activity, promote material and energy conversion, and improve germination efficiency. The effect of temperature on the germination of *Hopea stenoptera* seeds can be determined by comparing the experimental data from Comparative Example 2 and Example 1. Compared to Example 1, Comparative Example 2 showed that smaller seeds had a longer germination lag and germination period of 0.67 days and 3.07 days, respectively, resulting in an 8.83% decrease in seedling survival rate. Normal seeds had a longer germination lag and germination period of 1.42 days and 3.03 days, respectively, resulting in a 6.54% decrease in seedling survival rate. Therefore, the warm water temperature selected in this invention is beneficial for *Hopea stenoptera* seed germination, and the added warm water soaking and sand bed treatment significantly improved the seedling survival rate of *Hopea stenoptera*.

[0098] Table 3: Comparison of seedling transplant survival rates between Comparative Example 3 and Example 1

[0099] deal with Transplant survival rate (%) Example 1 97.37 Comparative Example 3 92.43

[0100] Comparative Example 3 primarily examined whether the selected root-protecting nutrient agent had an effect on improving transplant survival rate. Changing to different seedling containers during transplanting, according to the seedling's growth stage, is more conducive to seedling growth. Seedling death often occurs due to root damage during transplanting; therefore, protecting the roots during transplanting to improve survival rate is crucial. Compared to Example 1, Comparative Example 3 showed a 4.94% decrease in transplant survival rate, indicating that the root-protecting nutrient agent of this invention promotes the transplant survival of *Hopea stenoptera*. The root-protecting nutrient agent selected in this invention consists of the plant growth regulator GGR and inorganic salts (KH2PO3 and KNO3). GGR is an innovative green product following chemical fertilizers, micronutrients, hormones, and yield-enhancing bacteria, which can significantly improve seedling survival rate. Its effect on promoting root growth is superior to that of single auxins, while the added inorganic salts (KH2PO3 and KNO3) provide sufficient nutrition for root metabolism.

[0101] Table 4: Comparison of growth status of one-year-old seedlings from Comparative Examples 4-5 and Example 1

[0102] deal with mortality rate(%) Seedling height (cm) Ground diameter (mm) Seedling performance Example 1 2.71 45.52 5.04 Healthy, green leaves and uniform seedling growth Comparative Example 4-1 2.87 21.14 2.22 The leaves are slightly yellow, and the seedlings are of varying heights and thin. Comparative Example 4-2 5.94 18.29 1.48 The leaves are slightly curled and yellow, the seedlings are thin and weak, and yellow tips are appearing. Comparative Example 5 6.79 20.57 3.47 The leaves are yellowish-green and relatively small.

[0103] Comparative Example 4 primarily examined whether the transplanted seedling substrate used in the selection process promoted the improvement of seedling growth. The seedling substrate provides a basic source of nutrition for seedling growth, and its quality is crucial for seedling growth and development. High-quality nutrient soil should possess the following characteristics: rich in organic matter and with good aeration and drainage. Comparative Example 4-1 investigated whether the same seedling substrate could be used throughout the entire developmental stage of *Hopea stenoptera* seeds. Table 4 shows that using the same seedling substrate after transplanting and changing cups had little impact on mortality, increasing it by only 0.16%, but significantly altered seedling growth. Compared to Example 1, Comparative Example 4-1 showed a 24.38 cm decrease in seedling height, a 2.82 mm decrease in ground diameter, and slightly yellowing leaves, indicating inconsistent seedling growth and poor overall growth. Comparative Example 4-2 used red soil, a widely recognized and effective seedling substrate. Compared to Example 1, Comparative Example 4-2 showed a 3.23% higher mortality rate, a 27.23 cm lower seedling height, a 3.56 mm lower ground diameter, slightly curled and yellowed leaves, weak seedlings, and yellowing tips, exhibiting the worst growth condition. This indicates that the nutritional requirements of *Hopea stenoptera* vary significantly at different growth stages, and only by selecting suitable seedling substrates at different stages can the growth status of seedlings be improved. The seedling substrate selected in this invention is suitable for the growth needs of *Hopea stenoptera* seedlings and significantly improves their growth status.

[0104] Comparative Example 5 primarily investigated whether the nutrient solution used for spraying was more suitable for the growth of *Hopea stenoptera* seedlings. Fertilization is one of the important factors affecting plant growth and development; the type of fertilizer and the method of application are crucial. Comparative Example 5 used a general-purpose compound fertilizer, applied by sprinkling it onto the surface. Compared to Example 1, Comparative Example 5 showed a 4.08% increase in mortality, a 24.95 cm decrease in seedling height, a 1.57 mm decrease in ground diameter, and smaller, yellowish-green leaves, indicating poorer growth than Example 1. Therefore, the nutrient solution formulation obtained in this invention is suitable for the growth and survival of *Hopea stenoptera* seedlings, and the spraying method facilitates absorption by the seedlings, which is of great significance for improving seedling growth.

Claims

1. A method for improving the seed germination rate and seedling growth status of the extremely small population plant *Hopea stenoptera*, characterized in that: The operation steps are as follows: (1) Seed collection and screening: Fresh, mature seeds free from pests and diseases were collected from the natural distribution area of ​​*Hopea stenoptera*. The seeds were then sorted and classified according to size after the two seed wings were removed. (2) Seed washing and sterilization: After cleaning the seeds with clean water to remove impurities, wrap the seeds and fine sand in gauze and gently rub them repeatedly. Wash away the fine sand with clean water, soak them in a 10 mg / mL solution of crude extract of *Hopea stenoptera* branches and leaves for 20 min, then soak them in a 0.1% benzalkonium chloride solution for 5 min with stirring, and rinse them with running water for 3-5 min. (3) Seed germination promotion: Place the smaller seeds in a sealed bag containing a germination promoter, and then soak them together with normal seeds in warm water at 30-35°C for 20-30 minutes. Meanwhile, the sand bed is thoroughly watered with a 0.5% potassium permanganate solution beforehand, and then watered with warm water at 35-40°C every hour, repeating 3-5 times. After soaking, the seeds are sown on the treated sand bed, and the sand bed is kept moist by spraying. An arched frame is built on the sand bed with wire and covered with plastic film. (4) Planting: After the cotyledons and hypocotyl of the seed have fully germinated, gently pull the seed out of the sand bed, rinse off the fine sand with clean water, and plant it in the seedling container cup. When planting, be careful to gently bury the radicle into the substrate. The substrate should be coconut coir: red soil: vermiculite in a volume ratio of 5:3:

1. After filling the seedling container cup with the substrate, water it thoroughly with a 3% FeSO4 aqueous solution. Do not damage or break the radicle. (5) Transplanting: When the seedlings grow to 20-30 cm tall, transplant them into new seedling cups. When transplanting, trim the longer roots and then soak the seedling container cups in the root protection nutrient solution for 3-5 minutes. After that, cut open the seedling container cups and plant them into larger seedling bags. (6) Seedling management: After planting and transplanting, the seedlings should be placed in an outdoor seedling greenhouse with shade netting, and watered once a day. The light transmittance should be maintained at 40-50% for one week. After one week, the light transmittance should be gradually increased. One month after planting and transplanting, the light transmittance should be increased to 80-90%. Nutrient solution should be sprayed every 15-20 days. The above step (1) describes a method for sorting seeds by size, which involves using a sieve with a diameter of 43 cm and a pore size of 10 mm to filter the seeds. Seeds with a diameter ≥ 10 mm are classified as normal seeds, and those < 10 mm are classified as small seeds. The sprouting agent mentioned in step (3) above is composed of 1.0-1.5 mg / L IAA + 0.8-1.0 mg / L GA3 + 50-100 mg / L KH2PO3 + 200-300 mg / L NH4NO3 + 100-200 mg / L CaCl2; In step (5) above, the substrate is placed in layers inside the seedling bag. The upper substrate is red soil: peat soil: mushroom residue with a volume ratio of 5:2:1, and the lower substrate is coconut coir: river sand: vermiculite with a volume ratio of 4:4:

1. The volume ratio of the upper substrate to the lower substrate is 2:

1.

2. The method for improving the seed germination rate and seedling growth status of the extremely small population plant *Hopea stenoptera* according to claim 1, characterized in that: The collection of fresh, mature seeds free from pests and diseases in step (1) includes collecting seeds from trees and picking up fallen seeds. Mature seeds are characterized by a seed coat that is entirely yellow-green and a seed wing that is dark yellow or brown. Fresh seeds free from pests and diseases are judged by the absence of insect bites on the seed surface, the seed coat that has not yet withered or wrinkled, and the seed wing that has not yet dehydrated and dried.

3. The method for improving the seed germination rate and seedling growth status of the extremely small population plant *Hopea stenoptera* according to claim 1, characterized in that: The method for preparing the crude extract of *Hopea stenoptera* branches and leaves in step (2) is to collect the branches and leaves of *Hopea stenoptera*, dry them in a drying oven at 50 ℃, pulverize them, and soak them in a methanol solution with a volume of 5 times their mass for 48 h.

4. The method for improving the seed germination rate and seedling growth status of the extremely small population plant *Hopea stenoptera* according to claim 1, characterized in that: The sowing method described in step (3) is to lightly press the seeds into shallow pits, with a spacing of 3 to 5 cm between the seeds. Then, sprinkle a thin layer of sand, with a sand covering thickness of ≤0.5 mm, but ensure that the seeds are completely covered without any bare spots. Spraying refers to spraying water with a spray container. Avoid using too much water, which would leave the seeds exposed on the sand.

5. The method for improving the seed germination rate and seedling growth status of the extremely small population plant *Hopea stenoptera* according to claim 1, characterized in that: In step (4), the seedling container cups used are non-woven fabric cups with a diameter of 8 cm and a height of 12 cm.

6. The method for improving the seed germination rate and seedling growth status of the extremely small population plant *Hopea stenoptera* according to claim 1, characterized in that: The root-protecting nutrient agent mentioned in step (5) is 0.5-1.0 mg / L GGR + 50-100 mg / L KH2PO3 + 800-1000 mg / L KNO3.

7. The method for improving the seed germination rate and seedling growth status of the extremely small population plant *Hopea stenoptera* according to claim 1, characterized in that: The larger seedling bag mentioned in step (5) refers to a non-woven cup with a diameter of 20 cm and a height of 24 cm.

8. The method for improving the seed germination rate and seedling growth status of the extremely small population plant *Hopea stenoptera* according to claim 1, characterized in that: The nutrient solution in step (6) is 30-50 mg / L KH2PO3 + 400-500 mg / L KNO3. The spraying method is to spray the leaves with the nutrient solution and irrigate the substrate thoroughly.

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