Method for reestablishing wild plant population of small population of wild plant michelia hainanensis

Abstract

The application discloses a method for reestablishing a wild plant Hainan wind-blowing nanmu field population, and belongs to the technical field of wild plant conservation. The method comprises the following steps: returning seedling cultivation, returning land selection, field domestication, returning land habitat repair, land preparation and hole digging, planting, and field returning population monitoring and management. The method significantly improves the growth of Hainan wind-blowing nanmu seedlings, and the height of the cultivated half-year seedlings can reach 65.3 cm, and the ground diameter can reach 1.23 cm. After returning for 3 years, the survival rate of the seedlings reaches 94.6%, the plant height is 1.76 m, and the ground diameter is 2.93 cm. The application not only can cultivate high-quality Hainan wind-blowing nanmu seedlings in large quantities, solves the problem of low propagation efficiency of Hainan wind-blowing nanmu, but also can successfully reestablish the field population of Hainan wind-blowing nanmu, expand the population quantity and scale, and lay a technical foundation for the protection and recovery of the species.

Description

Technical Field

[0001] This invention belongs to the field of wild plant conservation technology, and relates to plant seedling cultivation and wild reintroduction technology. Specifically, it is a method for reconstructing the wild population of the extremely small population wild plant, *Machilus hainanensis*. Background Technology

[0002] *Horsfieldia hainanensis* Merr., belonging to the genus *Horsfieldia* in the family Myristicaceae, is a tall, evergreen tree endemic to China. It is distributed intermittently in "island-like" or "spot-like" patterns in Yunnan, Hainan, and Guangxi provinces. It is a Class II protected plant in my country and a species with an extremely small population nationwide. *Horsfieldia hainanensis* is an indicative species of humid tropical rainforests and is of great value for studying the floristic composition, geographical distribution, and ecological habits of tropical rainforests. Due to excessive human disturbance, the habitat fragmentation of *Horsfieldia hainanensis* populations is severe, leading to a gradual decrease in population size and number of individuals. Coupled with its low seed setting rate, seed stubbornness, weak seedling competitiveness, and high mortality rate, the population regeneration is slow or even stagnant, ultimately leading to the endangerment or extinction of *Horsfieldia hainanensis*.

[0003] For endangered species due to habitat loss or destruction, population migration and reconstruction are highly effective methods for saving the species. There are two approaches to population migration and reconstruction: ex-situ conservation and reintroduction. Because the reconstructed populations are small and limited in number, ex-situ conservation often leads to the loss of genetic diversity. Furthermore, differences between the transplant site and the original habitat can cause the population to lose its self-sustaining and expansion capabilities, increasing the chances of inbreeding and the probability of extinction. In contrast, reintroduction allows for the reconstruction of large-scale populations, effectively protecting the species' genetic diversity and potentially leading to sustainable populations through the ecosystem's self-sustaining capacity. Therefore, to effectively save and protect *Machilus hainanensis*, population reconstruction and natural reintroduction are urgently needed. Summary of the Invention

[0004] The purpose of this invention is to provide a method for reconstructing the wild population of the extremely small wild plant *Machilus hainanensis*. Through the cultivation of reintroduced seedlings, selection of reintroduced sites, wild acclimatization, restoration of reintroduced habitats, land preparation and planting, and monitoring and management of the reintroduced wild population, a large number of high-quality *Machilus hainanensis* seedlings can be cultivated, solving the problem of low propagation efficiency of *Machilus hainanensis* and improving the wild survival rate of *Machilus hainanensis*, thereby expanding the population size and scale of *Machilus hainanensis*.

[0005] The technical solution of the present invention is as follows:

[0006] A method for reconstructing the wild population of the extremely small wild plant *Machilus hainanensis* includes the following steps: seedling cultivation, relocation site selection, wild acclimatization, habitat restoration, land preparation and planting, planting, and monitoring and management of the relocated wild population. The specific operational steps are as follows:

[0007] (1) Cultivation of seedlings: In May, collect mature seeds of Hainan windblown nanmu and germinate them; when the seedlings are 6-8 cm tall and have 2 leaves, take them out, cut off the excessively long taproot, soak them in rooting solution for 15 minutes, and then transplant them into non-woven bags containing seedling substrate; after planting, move them into a shaded area for cultivation, and water them every day during this period; 30 days after transplanting, apply water-fertilizer solution regularly; after half a year of seedling cultivation, when the seedlings are 62-68 cm tall and have a ground diameter of 1.0-1.3 cm, they can be transplanted.

[0008] (2) Selection of return site: Based on the growth habits of Hainan nanmu, in the South Asian subtropical climate zone, select forest land with an altitude of less than 1000m, an average annual temperature of ≥21℃, an annual rainfall of ≥1300 mm, a relative humidity of 60%~85%, a forest canopy closure of 50%~60%, a slope of less than 30°, a soil of red soil or yellow soil, a surface humus layer thickness of ≥2 cm, a soil layer thickness of ≥60 cm, and a forest that is not prone to landslides, waterlogging and human disturbance as the return site;

[0009] (3) Wild acclimatization: The seedlings that were taken out of the nursery in step (1) were placed in the return site for 20 days of wild acclimatization to obtain seedlings after wild acclimatization;

[0010] (4) Restoration of the habitat in the return site: Harvest all the above-ground parts of shrubs, herbs and vines in the return site forest land, cut the roots to a height of less than 10cm, and shallowly turn over the areas where the vine roots are concentrated (5~10cm deep) to remove the main roots; at the same time, sanitary felling of dead trees and dead branches, clearing the forest land, and filling in the low-lying waterlogged areas and insect holes in the forest land.

[0011] (5) Land preparation and pit digging: Select areas with thicker soil layers randomly according to the terrain and clear them into 1m×1m plots, and design according to the planting density; after clearing the plots, dig planting pits in the center of each plot, and mix organic fertilizer with the soil before filling the pits.

[0012] (6) Planting: From January to March, choose a time when there is sufficient rainfall and the forest soil is moist. Plant the seedlings that have been domesticated in the wild in the planting hole, cover them with soil and tamp them down, and water them thoroughly after planting. After planting, spray the forest with 50% carbendazim disinfectant.

[0013] (7) Monitoring and management of reintroduced populations: Regular monitoring and management of reintroduced Hainan nanmu plants. Monitoring mainly includes observation of survival rate and growth of Hainan nanmu, and management mainly includes watering, weeding and fertilization.

[0014] Further optimization involves shortening the taproot of the seedlings in step (1) to a length of 4 cm, and formulating the rooting solution as follows: ABT6 20 mg / L + chitosan 0.2 g / L + sodium alginate 0.1 g / L. In this invention, ABT6 directly promotes root differentiation and elongation through hormone signal regulation. Chitosan enhances the seedlings' disease resistance and stress tolerance by activating the plant defense system. Sodium alginate forms a protective film on the root surface, reducing transplanting damage and maintaining a moist rhizosphere environment, all contributing to root development and improving transplant survival rate.

[0015] Further optimization involves mixing sieved yellow soil, seedling-specific peat moss, and fully decomposed, odorless mushroom residue in a weight ratio of 4-6:3-4:1-2 to obtain a basic substrate. Then, 5% AMF (arbuscular mycorrhizal fungi) inoculant is added to the basic substrate, and after thorough mixing, the seedling substrate is obtained and filled into a non-woven bag with a diameter of 15 cm and a height of 25 cm for later use. In this invention, the combination of yellow soil, peat moss, mushroom residue, and AMF forms a multi-layered composite substrate system with the functions of a heavy clay soil base for water and fertilizer retention, organic matter improving structure and providing slow-release nutrients, cellulose waste increasing aeration pores and supplementing readily available nutrients, and mycorrhizal fungi constructing a symbiotic absorption mechanism. This system is suitable for the growth needs of Hainan nanmu seedlings. Seedlings cultivated using this seedling substrate exhibit robust root development and a significantly increased density of fibrous roots; a higher mycorrhizal infection rate; and improved root absorption area and nutrient utilization efficiency. The seedlings also demonstrate significantly enhanced drought and disease resistance, providing high-quality seedling materials for subsequent field planting and population reconstruction.

[0016] Further optimization is achieved by using the water-fertilizer solution described in step (1) above, which consists of amino acid foliar fertilizer, 0.01% brassinolide, 50% carbendazim wettable powder, and water. Specifically, 30 mL of amino acid foliar fertilizer is first added to 10 kg of water and stirred evenly. Then, 10 mL of 0.01% brassinolide soluble powder is added and stirred evenly. Finally, 30 g of 50% carbendazim wettable powder is added while stirring until completely dissolved and dispersed. The water-fertilizer solution is prepared and used immediately. The amino acid foliar fertilizer in the water-fertilizer solution provides a fast-acting organic nitrogen and carbon source, directly supplementing leaf nutrition, improving photosynthetic efficiency, and promoting dry matter accumulation. Brassinolide enhances the systemic resistance of seedlings through hormone regulation, promotes root development and above-ground growth, and also enhances the control effect of carbendazim. Carbendazim provides a disease protection barrier for seedlings and prevents fungal diseases during the seedling stage. Regularly spraying the water-fertilizer solution of this invention enhances the photosynthesis of seedlings, resulting in dark green and thick leaves, and accelerates the growth of plant height and diameter. It also significantly reduces the incidence of diseases and significantly improves the environmental adaptability of seedlings, laying a solid physiological foundation for subsequent wild domestication and population reconstruction.

[0017] To further optimize, the spraying method of the water-fertilizer solution mentioned in step (1) above is as follows: spray once every 20 days after the seedlings are transplanted, spraying both sides of the leaves each time, ensuring that the leaves are moist but not dripping, and stop spraying in December.

[0018] Further optimization, the field acclimatization method described in step (3) above is as follows: water once every 3 days for the first 6 days, water once every 5 days for the next 10 days, and water once every 7 days for the last 14 days.

[0019] Further optimization: the planting density in step (5) above is 60 plots / acre, and the planting hole specifications are 60cm long × 50cm wide × 40cm deep; the organic fertilizer is Stanley brand bio-organic fertilizer with organic matter ≥65.0% and effective live bacteria count ≥300 million / g, and 0.25 kg is applied per hole.

[0020] Further optimization, the specific method of seedling planting in step (6) above is as follows: dig a planting pit in the center of the planting hole with a depth of more than 5 cm above the nutrient cup, place the seedling in the pit, straighten it, and cover it with soil. The soil covering should be more than 5 cm higher than the original soil surface of the nutrient cup. Then, tamp the soil around the seedling with your feet and cover it with a layer of loose soil so that the surface of the hole is saucer-shaped. Water each seedling with 2-2.5L of water.

[0021] Further optimization, the regular monitoring of the Hainan windblown nanmu plants after the return mentioned in step (7) above mainly includes the statistics and measurement of survival rate and growth (diameter and height). The survival rate is investigated for the first time 1 month after the return planting, and then the survival rate is investigated and the growth is measured every December.

[0022] Further optimization, the regular maintenance method for the Hainan windblown nanmu plants after the return of the above step (7) is as follows: water once a month within 6 months after the return; cut down weeds and shrubs in the forest land from June to August every year, with the root height within 20cm, and remove the grass and shrub stumps within 60cm on all sides of the Hainan windblown nanmu plant as the center, dig a fertilizer trench at a suitable position away from the tree root, and evenly apply 0.15-0.25kg Stanley organic bio-fertilizer in the fertilizer trench.

[0023] This invention has the following advantages and positive effects over the prior art:

[0024] 1. This invention significantly improves the growth of Hainan Phoebe zhennan seedlings through methods such as bud root cutting, rooting solution soaking, transplanting, transplanting substrate ratio, and water and fertilizer management. The average height of the cultivated six-month-old seedlings can reach 65.3 cm, and the average ground diameter can reach 1.23 cm. It can be used to propagate high-quality and healthy reintroduced seedlings in large quantities. After three years of reintroduction, the survival rate of these seedlings can reach 94.6%, with an average plant height of 1.76 m and an average ground diameter of 2.93 cm.

[0025] 2. The field acclimatization technology proposed in this invention involves transplanting Hainan windblown nanmu seedlings to their return location and gradually adapting them to the natural environment through gradient watering, thereby improving their stress resistance and preventing them from dying due to maladaptation to the natural environment.

[0026] 3. This invention creates a suitable microhabitat and nutrients for the growth of Hainan windblown nanmu seedlings by selecting a reclaimed site, restoring the reclaimed site habitat, and using land preparation and pit digging techniques.

[0027] 4. The present invention establishes a reasonable planting method, which reduces damage to the reintroduced seedlings, shortens the seedling recovery period, and effectively improves the seedling survival rate.

[0028] 5. This invention, by selecting the optimal planting density, avoids seedlings from shading each other and competing for nutrients due to overcrowding, which leads to weak seedlings and reduced quality, thus effectively improving seedling growth rate and quality.

[0029] 6. This invention, through watering, weeding, irrigating, and fertilizing the reclaimed seedlings, can regulate the seedling temperature, enhance disease resistance, reduce competition for water, nutrients, and light from weeds and irrigated plants, improve soil structure, promote nutrient cycling, and enhance the survival rate, growth rate, and environmental adaptability of the seedlings, thus successfully rebuilding the wild population of Hainan nanmu. Attached Figure Description

[0030] Figure 1 This is a diagram illustrating the germination process of Hainan windblown nanmu seeds according to the present invention.

[0031] Figure 2 This is a picture of Hainan windblown nanmu seedlings before transplanting according to the present invention.

[0032] Figure 3 This is a picture of Hainan Phoebe zhennan seedlings after transplanting according to the present invention.

[0033] Figure 4 This is a picture of Hainan windblown nanmu seedlings before they are sold.

[0034] Figure 5 This invention relates to the field domestication of Hainan windblown nanmu seedlings.

[0035] Figure 6 This is a diagram of habitat restoration and land preparation for the present invention.

[0036] Figure 7 This invention is a field regression map of *Machilus hainanensis* in Hainan.

[0037] Figure 8 This is a growth chart showing the growth of the invention three years after regression. Detailed Implementation

[0038] 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 for illustrative purposes only and are not intended to limit the invention.

[0039] To facilitate understanding, the relevant technical terms involved in the embodiments of this application will first be explained and described.

[0040] (1) In this invention, the ground diameter refers to the diameter of the base of the main stem of the plant (5cm from the ground or substrate surface), expressed in centimeters (cm).

[0041] (2) In this invention, seedling height refers to the vertical length from the ground diameter to the base of the terminal bud when the seedling leaves the nursery, expressed in centimeters (cm).

[0042] (3) In this invention, the survival rate refers to the proportion of Hainan nanmu trees that survived 3 years after being reintroduced to the wild to the total number of trees planted at the time of reintroduction, expressed as a percentage (%).

[0043] (4) In this invention, plant height refers to the vertical length from the root neck to the base of the terminal bud of the Hainan windblown nanmu plant after its return, expressed in meters (m).

[0044] (5) In this invention, relative growth refers to the value obtained by subtracting the growth (sapling height, ground diameter) of Hainan nanmu seedlings before return to the wild from the growth (sapling height, ground diameter) of Hainan nanmu seedlings after 3 years of return to the wild.

[0045] Example 1:

[0046] A method for reconstructing wild populations of the extremely small wild plant *Machilus hainanensis* mainly includes the following steps:

[0047] (1) Seedling cultivation: In May 2022, mature seeds of *Machilus hainanensis* were collected and germinated. When the seedlings reached a height of 6-8 cm and had two leaves, they were removed, and the excessively long taproots were cut off, leaving a length of 4 cm. The roots were soaked in a rooting solution prepared by ABT6 (20 mg / L), chitosan (0.2 g / L), and sodium alginate (0.1 g / L) for 15 minutes. Then, they were transplanted into non-woven bags with a diameter of 15 cm and a height of 25 cm containing seedling substrate. The seedling substrate was prepared by mixing sieved yellow soil, seedling-specific peat moss, and fully decomposed, odorless mushroom residue in a weight ratio of 6:3:1. AMF (arbuscular mycorrhizal fungi) inoculant was added to the basic substrate, and the mixture was thoroughly stirred to obtain the seedling substrate. The seedlings were then transplanted into a shaded area for cultivation and watered daily. Thirty days after transplanting, spray a water-fertilizer solution every 20 days, spraying both sides of the leaves each time, ensuring the leaves are moist but not dripping wet. Stop spraying in December. The water-fertilizer solution consists of amino acid foliar fertilizer, 0.01% brassinolide, 50% carbendazim wettable powder, and water. Specifically, first add 30 mL of amino acid foliar fertilizer to 10 kg of water and stir well; then add 10 mL of 0.01% brassinolide soluble powder and stir well; finally, add 30 g of 50% carbendazim wettable powder while stirring until completely dissolved and dispersed. The water-fertilizer solution should be prepared and used immediately. After six months of cultivation, when the seedlings reach a height of 62-68 cm and a ground diameter of 1.0-1.3 cm, they are ready for transplanting.

[0048] (2) Selection of return site: Based on the growth habits of Hainan nanmu, in the subtropical climate zone of Guangxi Zhuang Autonomous Region, Qinzhou City, select forest land with an altitude of less than 1000 m, an average annual temperature of ≥21℃, an annual rainfall of ≥1300 mm, a relative humidity of 60%~85%, a forest canopy closure of 50%~60%, a slope of less than 30°, a red soil or yellow soil, a surface humus layer thickness of ≥2 cm, a soil layer thickness of ≥60 cm, and a forest that is not prone to landslides, waterlogging and human interference as the return site.

[0049] (3) Wild acclimatization: The seedlings that were taken out of the nursery in step (1) were placed in the return site for 20 days of wild acclimatization. During the acclimatization period, the seedlings were watered once every 3 days for the first 6 days, once every 5 days for the next 10 days, and once every 7 days for the last 14 days.

[0050] (4) Restoration of habitat in the return site: Harvest all the above-ground parts of shrubs, herbs and vines in the forest land, cut the roots within 10cm in height, and shallowly turn the vine roots to a depth of 5-10cm in the concentrated distribution area to remove the main roots; at the same time, sanitary felling of dead trees and dead branches, clearing the forest land, and filling in the low-lying waterlogged areas and insect holes in the forest land.

[0051] (5) Land preparation and planting: Randomly select areas with thick soil layers and clear them into 1m×1m plots. Design according to a density of 60 plots / acre. Clearing includes harvesting all branches and leaves of shrubs, herbs and vines in the plots and digging out the roots of shrubs, herbs and vines. After clearing the plots, dig planting holes in the center of the plots with a length of 60cm×width×depth of 50cm×40cm. Take 0.25kg of organic fertilizer as base fertilizer for each planting hole and mix it with the soil before returning it to the hole. The organic fertilizer is Stanley brand bio-organic fertilizer with organic matter ≥65.0% and effective live bacteria ≥300 million / g.

[0052] (6) Planting: From January to March 2023, select a period with sufficient rainfall and moist forest soil, plant the seedlings that have been domesticated in the wild in tree pits. When planting, dig a planting pit in the center of the pit that is more than 5cm deeper than the nutrient cup, place the seedling in the pit, straighten it, and cover it with soil. The soil covering should be more than 5cm higher than the original soil surface of the nutrient cup. Then tamp the soil around the seedling with your feet, and then cover it with a layer of loose soil so that the surface of the pit is saucer-shaped. Water each seedling with 2-2.5L of water.

[0053] (7) Monitoring and management of reintroduced populations in the wild: Regular management and monitoring of reintroduced *Machilus hainanensis* plants were carried out. Management mainly included watering, weeding, and fertilization. Watering was carried out once a month for the first 6 months after reintroduction. From June to August each year, weeds and shrubs in the forest land were cut down to a height of no more than 20 cm. The weeds and shrubs within a 60 cm radius around the *Machilus hainanensis* plant were removed. Fertilization trenches were dug at a suitable location from the tree roots, and 0.15–0.25 kg of Stanley organic bio-fertilizer was evenly applied in the trenches. Monitoring mainly included the survival rate and growth (diameter and height) of *Machilus hainanensis* plants. The survival rate of the reintroduced *Machilus hainanensis* population after 3 years was 94.6%, with an average diameter at breast height of 2.93 cm and an average height of 1.76 m.

[0054] Example 2:

[0055] The difference between this embodiment and embodiment 1 is only that: after transplanting in step (1), the seedling substrate is made by mixing sieved yellow soil, seedling-specific peat soil, and fully decomposed mushroom residue in a weight ratio of 5:3:2 to obtain the basic substrate. Then, 5% of the total weight of AMF bacterial agent is added to the basic substrate and stirred thoroughly to obtain the seedling substrate.

[0056] Following steps (2) to (7) in Example 1, the survival rate of the reintroduced Hainan nanmu population after 3 years was 92.3%, with an average ground diameter of 2.75 cm and an average plant height of 1.66 m.

[0057] Example 3:

[0058] The difference between this embodiment and embodiment 1 is only that: after transplanting in step (1), the seedling substrate is made by mixing sieved yellow soil, seedling-specific peat soil, and fully decomposed mushroom residue in a weight ratio of 4:4:2 to obtain the basic substrate. Then, 5% of the total weight of AMF bacterial agent is added to the basic substrate and stirred thoroughly to obtain the seedling substrate.

[0059] Following steps (2) to (7) in Example 1, the survival rate of the reintroduced Hainan nanmu population after 3 years was 91.4%, with an average ground diameter of 2.79 cm and an average plant height of 1.71 m.

[0060] Comparative Example 1-1:

[0061] The only difference from Example 1 is that in step (1), the roots of the seedlings are not cut off, and the roots are soaked in clean water for 15 minutes before transplanting. The rest of the steps remain the same.

[0062] Comparative Examples 1-2:

[0063] The difference from Example 1 is that only in step (1) the excessively long taproot is cut off, leaving a length of 2 cm. After soaking the roots in a rooting solution of ABT6 (20 mg / L) + chitosan (0.2 g / L) + sodium alginate (0.1 g / L) for 15 min, the roots are transplanted. The remaining steps are unchanged.

[0064] Comparative Examples 1-3:

[0065] The only difference from Example 1 is that in step (1), the excessively long taproot is cut off, leaving a length of 4 cm, and the roots are soaked in a 20 mg / LABT6 aqueous solution for 15 min before transplanting. The remaining steps are unchanged.

[0066] Comparative Example 2:

[0067] The only difference from Example 1 is that in step (1), the seedling cultivation substrate is obtained by mixing sieved yellow soil and seedling-specific peat soil in a weight ratio of 6:4. The other steps remain unchanged.

[0068] Comparative Example 3-1:

[0069] The steps are basically the same as those in Example 1, except that in step (1), amino acid foliar fertilizer and mixed solution of multi-live bacteria are not applied, while the other seedling cultivation steps remain unchanged.

[0070] Comparative Example 3-2:

[0071] The steps are basically the same as those in Example 1. The difference is that in step (1), the water and fertilizer solution is prepared regularly by adding 30 mL of amino acid foliar fertilizer and 30 g of 50% carbendazim wettable powder to every 10 kg of water. The other seedling cultivation steps remain unchanged.

[0072] Comparative Example 3-3:

[0073] The steps are basically the same as those in Example 1. The difference is that in step (1), the water and fertilizer solution is prepared regularly by adding 40 mL of amino acid foliar fertilizer, 10 mL of 0.01% brassinolide soluble concentrate, and 40 g of 50% carbendazim wettable powder to every 10 kg of water. The other seedling cultivation steps remain unchanged.

[0074] Comparative Example 4:

[0075] The steps and procedures are basically the same as in Example 1, except that the return site in step (2) is selected in a forest with full sunlight and no shade. The other geographical conditions remain unchanged.

[0076] Comparative Example 5-1:

[0077] The steps are basically the same as those in Example 1, except that step (3) is not performed, that is, the seedlings are not acclimatized in the wild.

[0078] Comparative Example 5-2:

[0079] The steps are basically the same as those in Example 1, except that the seedling acclimatization time in step (3) is adjusted to 7 days, and the seedlings are watered after the acclimatization is completed.

[0080] Comparative Example 6:

[0081] The steps are basically the same as those in Example 1, except that step (4) is not performed, that is, the restoration of the return habitat is not performed.

[0082] Comparative Example 7:

[0083] The steps are basically the same as those in Example 1, except that no fertilizer is applied to each planting hole in step (5).

[0084] Comparative Example 8:

[0085] The steps and procedures are basically the same as in Example 1. The difference is that in step (7), after the plants are planted, they are allowed to grow naturally without artificial watering, weeding, pruning, fertilizing, or other artificial management.

[0086] Hainan Phoebe zhennan seedlings were cultivated and reintroduced using the treatment methods of Examples 1-3 and Comparative Examples 1-8 within the scope of protection of this invention, and the growth of seedlings under different treatments and the survival rate and growth after 3 years of reintroduction were statistically compared.

[0087] Table 1. Seedling growth of Comparative Example 1 and Example 1

[0088]

[0089] Comparative Example 1 investigated the effects of root pruning and root soaking in rooting solution on seedling growth. Comparing the experimental results of Comparative Example 1 and Example 1 (Table 1), the height and diameter at ground level of six-month-old seedlings treated without root pruning and without rooting solution soaking (Comparative Example 1-1) were 40.7 cm and 0.68 cm, respectively. However, the seedlings treated with root pruning and root soaking in rooting solution showed significantly increased height and diameter at ground level after transplanting. Specifically, the seedlings with a 4 cm main root, soaked in a rooting solution composed of 20 mg / L ABT6 + 0.2 g / L chitosan + 0.1 g / L sodium alginate (Example 1), showed greater height and diameter at ground level than those with a 2 cm main root. The seedlings soaked in the same rooting solution (Comparative Example 1-2) and those with a 4 cm main root, soaked in a solution containing only 20 mg / L ABT6 (Comparative Example 1-3) also showed significant differences in height and diameter at ground level. The results indicate that root cutting and root soaking in rooting solution can significantly improve seedling growth, with the main root retained at 4cm. The root soaking solution composed of ABT6 20mg / L + chitosan 0.2g / L + sodium alginate 0.1g / L has the best effect.

[0090] Table 2. Growth of Hainan Phoebe zhennan seedlings and survival rate and growth after 3 years of reintroduction in Examples 1-3

[0091]

[0092] Table 2 shows the growth of Hainan Phoebe zhennan seedlings and their survival rate and growth after 3 years of reintroduction in the three examples. As can be seen from Table 2, Example 1 is the optimal example, with the highest seedling height and diameter at ground level, the highest survival rate after 3 years of reintroduction, and the highest relative growth in the cultivated half-year-old seedlings. Examples 2 and 3 are second best, but both have seedling heights above 62 cm and survival rates above 90% after 3 years of reintroduction, far exceeding the survival rate and growth of Hainan Phoebe zhennan seedlings under natural, undisturbed conditions. Therefore, they are also within the scope of protection of this invention.

[0093] Table 3. Growth of seedlings in Comparative Examples 2-3 and Example 1, and survival rate and growth after 3 years of regression.

[0094]

[0095] Comparative Example 2 investigated the effects of conventional seedling substrates and their regression results. Comparing the experimental results of Comparative Example 2 and Example 1 (Table 3), the seedlings cultivated using a substrate made from a uniform mixture of yellow soil and peat moss at a weight ratio of 6:4 for six months had a height and diameter at birth of 43.8 cm and 0.92 cm, respectively. After three years of regression using this substrate, the survival rate was 86.1%, and the relative growth in plant height and diameter at birth were 1.05 m and 1.58 cm, respectively. In contrast, the six-month-old seedlings cultivated using the conventional seedling substrate showed increases in plant height and diameter at birth of 49.1% and 33.7%, respectively, compared to the substrate in Comparative Example 2. After three years of regression, the survival rate reached 94.6%, and the relative growth in plant height and diameter at birth were 1.11 m and 1.70 cm, respectively, both exceeding the survival rate and growth of seedlings cultivated using only yellow soil and peat moss after regression. This indicates that the seedling cultivation effect and regression results of using conventional seedling substrates such as yellow soil and peat moss are poor.

[0096] Comparative Example 3 investigated the seedling cultivation effect of applying sap-fertilizer solution and its regression results. Comparing the experimental results of Comparative Examples 3-1, 3-2, 3-3 and Example 1 (Table 3), it can be seen that the seedling height, ground diameter, survival rate and relative growth of the 6-month-old seedlings with sap-fertilizer solution applied during the seedling cultivation period were significantly greater than those of the seedlings without sap-fertilizer solution applied during the regression period, as were the growth, survival rate and relative growth of the seedlings after 3 years of regression. Comparing the experimental results of Comparative Examples 3-2, 3-3, and Example 1, it can be seen that there are significant differences in seedling growth, survival rate, and relative growth after 3 years of reintroduction with different aqueous solutions. In Example 1 (with 30 mL of amino acid foliar fertilizer, 10 mL of 0.01% brassinolide soluble concentrate, and 30 g of 50% carbendazim wettable powder added per 10 kg of water), the seedling height, ground diameter, survival rate, relative growth of plant height, and relative growth of ground diameter after 3 years of reintroduction were 65.3 cm, 1.23 cm, 94.6%, 1.11 m, and 1.70 cm, respectively, significantly higher than those of Comparative Examples 3-2 (with 30 mL of amino acid foliar fertilizer and 30 g of 50% carbendazim wettable powder added per 10 kg of water) and 3-3 (with 40 mL of amino acid foliar fertilizer, 10 mL of 0.01% brassinolide soluble concentrate, and 40 g of carbendazim wettable powder added per 10 kg of water). The study compared the seedling height, ground diameter, and survival rate, relative growth in plant height, and relative growth in ground diameter of seedlings after 3 years of reintroduction using 50% carbendazim wettable powder. This indicates that applying the water-fertilizer solution of this invention significantly improves seedling growth, survival rate, and relative growth after reintroduction. The best results were achieved when 30 mL of amino acid foliar fertilizer, 10 mL of 0.01% brassinolide soluble concentrate, and 30 g of 50% carbendazim wettable powder were added to every 10 kg of water.

[0097] Table 4. Survival rate and growth of Comparative Examples 4-8 and Example 1 after 3 years of regression.

[0098]

[0099] Comparative Example 4 investigated the impact of light conditions at the reintroduction site on the growth of reintroduced plants. Light is the energy source for photosynthesis, providing the material and energy basis for plant growth. Insufficient light leads to a decrease in photosynthetic rate, reduced organic matter accumulation, and slow plant growth, while excessive light causes photoinhibition, resulting in leaf scorch, damage to chloroplast structure, and a decrease in net photosynthetic rate. Comparing the experimental results of Comparative Example 4 and Example 1 (Table 4), using the same reintroduced seedlings, after 3 years in a forest under full sunlight, the survival rate was only 52.3%, and the relative growth of plant height and diameter at breast height was 0.66 m and 1.58 cm, respectively, both far lower than the survival rate and growth after 3 years in Example 1 (stand canopy closure 50%~60%). This indicates that Hainan nanmu seedlings are not suitable for growth under full sunlight, and strong light will cause photoinhibition.

[0100] Comparative Examples 5-1 and 5-2 investigated the effects of seedling acclimatization in the wild on the growth of reintroduced plants. The core function of seedling acclimatization in the wild is to enhance plant adaptability. Through gradual environmental regulation, the plant's ability to adapt to external conditions is enhanced, thereby increasing the transplant survival rate. Comparing the experimental results of Comparative Examples 5-1 and 5-2 with Example 1 (Table 4), for the same seedlings, the survival rate of seedlings without wild acclimatization (Comparative Example 5-1) after 3 years of reintroduction was only 43.6%, with relative growth of plant height and diameter at birth rate of 0.77 m and 1.18 cm, respectively. Seedlings that underwent wild acclimatization for 7 days (Comparative Example 5-2) after 3 years of reintroduction had a survival rate of 76.5%, with relative growth of plant height and diameter at birth rate of 0.90 m and 1.31 cm, respectively. Seedlings that underwent wild acclimatization for 20 days and received watering management (Example 1) after 3 years of reintroduction had a survival rate of 94.6%, with relative growth of plant height and diameter at birth rate of 1.11 m and 1.70 cm, respectively. This indicates that as the time of acclimatization in the wild increases, the survival rate and growth of the plants after re-entry significantly improve.

[0101] Comparative Example 6 investigated the impact of habitat restoration on the growth of reintroduced plants. Habitat restoration, by improving site conditions such as soil and moisture and removing factors unfavorable to reintroduced plants, provides a basic growth environment, enhancing the stress resistance of reintroduced plants and increasing their survival rate. Comparing the experimental results of Comparative Example 6 and Example 1 (Table 4), the same seedlings, after 3 years in a reintroduced site without habitat restoration, had a survival rate of 73.2%, with relative growth in plant height and diameter at breast height of 0.73 m and 1.08 cm, respectively. However, after 3 years in a reintroduced site with habitat restoration, the survival rate was 94.6%, an increase of 21.4%, with relative growth in plant height and diameter at breast height increasing by 52.1% and 57.4%, respectively. This indicates that habitat restoration can significantly improve the survival rate and growth of reintroduced *Machilus hainanensis*.

[0102] Comparative Example 7 investigated the effects of artificially added basal fertilizer and natural nutrients in forest soil on the growth of reintroduced plants. Basal fertilizer improves soil structure and provides continuous nutrients, laying the nutrient foundation for plant growth. Organic fertilizer contains major elements such as nitrogen, phosphorus, and potassium, as well as various trace elements. Its nutrient release is slow and balanced, avoiding nutrient imbalance caused by single fertilization. Comparing the experimental results of Comparative Example 7 and Example 1 (Table 4), for the same seedlings, without basal fertilizer at planting, the survival rate of plants 3 years after reintroduction was 83.4%, and the relative growth of plant height and ground diameter was 0.57m and 0.83cm, respectively. The survival rate and relative growth were significantly lower than those of plants 3 years after reintroduction in Example 1 (planted with organic fertilizer as basal fertilizer). This indicates that the natural nutrients in forest soil cannot fully meet the growth needs of reintroduced *Machilus hainanensis*, and additional artificial nutrients are needed to improve the survival rate and growth of reintroduced *Machilus hainanensis*.

[0103] Comparative Example 8 investigated the impact of artificial management on the growth of reintroduced plants. Artificial management, including regular watering, weeding, pruning, fertilization, and habitat maintenance, provided continuous growth support for the reintroduced plants. Comparing the results of Comparative Example 8 and Example 1 (Table 4), the same seedlings, without artificial management and allowed to grow naturally after reintroduction, had a survival rate of only 30.4% after 3 years, with relative growth in plant height and diameter at ground level of 0.52 m and 0.62 cm, respectively. However, after receiving meticulous artificial management measures such as watering, weeding, pruning, and fertilization, the reintroduced *Machilus hainanensis* plants were provided with sufficient water, nutrients, and light, effectively ensuring a survival rate of 94.6% and promoting rapid growth (relative growth in plant height and diameter at ground level of 1.11 m and 1.70 cm, respectively).

[0104] In summary, the seedling transplanting method, seedling substrate ratio, and seedling management measures selected in this invention can promote the healthy and high-yield growth of *Machilus hainanensis* seedlings, enabling the large-scale propagation of high-quality, healthy seedlings. Furthermore, by optimizing the selection of reintroduction sites, wild acclimatization, habitat restoration, land preparation and planting, as well as monitoring and management of reintroduced wild populations, the survival rate and growth of reintroduced *Machilus hainanensis* populations are significantly improved, successfully reconstructing wild populations of *Machilus hainanensis*.

Claims

1. A method for reconstructing wild populations of the extremely small population plant *Machilus hainanensis*, characterized by: It includes the cultivation of reintroduced seedlings, selection of reintroduction sites, wild acclimatization, habitat restoration, land preparation and planting, and monitoring and management of reintroduced populations in the wild; the specific operational steps are as follows: (1) Cultivation of seedlings: In May, collect mature seeds of Hainan windblown nanmu for germination; when the seedlings are 6-8 cm tall and have 2 leaves, take them out, cut off the excessively long taproot, soak them in rooting solution for 15 minutes, and then transplant them into non-woven bags containing seedling substrate; after planting, move them into a shade house for cultivation, and water them every day during this period; 30 days after transplanting, spray them with water-fertilizer solution regularly; after half a year of seedling cultivation, when the seedlings are 62-68 cm tall and have a ground diameter of 1.0-1.3 cm, they can be transplanted. (2) Selection of return site: Based on the growth habits of Hainan nanmu, in the South Asian subtropical climate zone, select forest land with an altitude of less than 1000 m, an average annual temperature of ≥21℃, an annual rainfall of ≥1300 mm, a relative humidity of 60%~85%, a forest canopy density of 50%~60%, a slope of less than 30°, a soil of red soil or yellow soil, a surface humus layer thickness of ≥2 cm, a soil layer thickness of ≥60 cm, and a forest that is not prone to landslides, waterlogging and human disturbance as the return site; (3) Wild acclimatization: The seedlings that were taken out of the nursery in step (1) were placed in the return site for 20 days of wild acclimatization to obtain seedlings after wild acclimatization; (4) Restoration of the habitat in the return site: Harvest all the above-ground parts of shrubs, herbs and vines in the return site forest land, cut the roots to a height of less than 10cm, and shallowly turn over the areas where the vine roots are concentrated to remove the main roots; at the same time, sanitary felling of dead trees and dead branches, clearing the forest land, and filling in the low-lying waterlogged areas and insect holes in the forest land. (5) Land preparation and pit digging: Select areas with thicker soil layers randomly according to the terrain and clear them into 1m×1m plots, and design according to the planting density; after clearing the plots, dig planting pits in the center of each plot, and mix organic fertilizer with the soil before filling the pits. (6) Planting: From January to March, choose a time when there is sufficient rainfall and the forest soil is moist. Plant the seedlings that have been domesticated in the wild in the planting hole, cover them with soil and tamp them down, and water them thoroughly after planting. After planting, spray the forest with 50% carbendazim disinfectant. (7) Monitoring and management of reintroduced populations: Regular monitoring and management of reintroduced Hainan nanmu plants. Monitoring mainly includes observation of survival rate and growth of Hainan nanmu, and management mainly includes watering, weeding and fertilization.

2. The method for reconstructing the wild population of the extremely small population wild plant *Machilus hainanensis* according to claim 1, characterized in that: In step (1), the main root is shortened and retained to a length of 4 cm; the formula of the rooting solution is: ABT6 20mg / L + chitosan 0.2g / L + sodium alginate 0.1g / L.

3. The method for reconstructing the wild population of the extremely small population wild plant *Machilus hainanensis* according to claim 1, characterized in that, The seedling substrate mentioned in step (1) is obtained by mixing sieved yellow soil, seedling-specific peat soil, and fully decomposed, odorless mushroom residue in a weight ratio of 4~6:3~4:1~2. Then, 5% of the total weight of AMF inoculant is added to the basic substrate and thoroughly mixed to obtain the seedling substrate. The mixture is then filled into a non-woven bag with a diameter of 15 cm and a height of 25 cm for later use.

4. The method for reconstructing the wild population of the extremely small population wild plant *Machilus hainanensis* according to claim 1, characterized in that, The water-fertilizer solution mentioned in step (1) consists of amino acid foliar fertilizer, 0.01% brassinolide, 50% carbendazim wettable powder and water. Specifically, first add 30 mL of amino acid foliar fertilizer to 10 kg of water and stir evenly; then add 10 mL of 0.01% brassinolide soluble powder and stir evenly; finally add 30 g of 50% carbendazim wettable powder while stirring until completely dissolved and dispersed; the water-fertilizer solution is prepared and used immediately.

5. The method for reconstructing the wild population of the extremely small population wild plant *Machilus hainanensis* according to any one of claims 1 or 4, characterized in that, The method of spraying the water-fertilizer solution mentioned in step (1) is as follows: spray once every 20 days after the seedlings are transplanted, spraying both sides of the leaves each time, until they are moist but not dripping water, and stop spraying in December.

6. The method for reconstructing the wild population of the extremely small population wild plant *Machilus hainanensis* according to claim 1, characterized in that: The field acclimatization method described in step (3) is as follows: water once every 3 days for the first 6 days, water once every 5 days for the next 10 days, and water once every 7 days for the last 14 days.

7. The method for reconstructing the wild population of the extremely small population wild plant *Machilus hainanensis* according to claim 1, characterized in that, The planting density mentioned in step (5) is 60 plots / acre, and the planting hole specifications are 60 cm long × 50 cm wide × 40 cm deep; the organic fertilizer is Stanley brand bio-organic fertilizer with organic matter ≥ 65.0% and effective live bacteria count ≥ 300 million / g, and 0.25 kg is applied per hole.

8. The method for reconstructing the wild population of the extremely small population wild plant *Machilus hainanensis* according to claim 1, characterized in that: The specific method for planting seedlings in step (6) is to dig a planting pit with a depth of more than 5 cm above the nutrient cup in the center of the planting hole, place the seedling in the pit, straighten it, and cover it with soil. The soil covering should be more than 5 cm higher than the original soil surface of the nutrient cup. Then, tamp the soil around the seedling with your feet and cover it with a layer of loose soil so that the surface of the hole is saucer-shaped. Water each planted seedling with 2-2.5L of water.

9. The method for reconstructing the wild population of the extremely small population wild plant *Machilus hainanensis* according to claim 1, characterized in that: The regular monitoring of the Hainan windblown nanmu plants after the return to the plant, as described in step (7), mainly includes the statistics and measurement of survival rate and growth. The survival rate is investigated for the first time one month after the return to the plant, and then the survival rate is investigated and the growth is measured every December.

10. The method for reconstructing the wild population of the extremely small population wild plant *Machilus hainanensis* according to claim 1, characterized in that: The post-return management method described in step (7) is to water once a month for 6 months after the return; cut down weeds and shrubs in the forest from June to August each year, with the root height within 20cm, and remove the grass and shrub stumps within 60cm above, below and to the left and right of the Hainan windblown nanmu tree as the center, and dig a fertilizer trench at a suitable position away from the tree root, and evenly apply 0.15-0.25kg Stanley organic bio-fertilizer in the fertilizer trench.

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