Method for returning paphiopedilum helenae tissue culture seedling to wild field

Abstract

The application discloses a method for returning Phalaenopsis x hybrida tissue culture seedlings to the wild, which comprises the following steps: selecting Phalaenopsis x hybrida tissue culture seedlings as materials, selecting a region similar to the original habitat as a returning point, domesticating the Phalaenopsis x hybrida tissue culture seedlings before returning to enhance the adaptability and drought resistance of the Phalaenopsis x hybrida tissue culture seedlings, and selecting a place with large slope and not easy to deposit dry branches and leaves as a planting point of the returning plants for planting. Through the operations of returning point screening, seedling culture, domestication before returning, plant planting point selection and treatment, and returning tube protection, the preservation rate of the Phalaenopsis x hybrida tissue culture seedlings can reach 45.4-54.9% in one year, which is 4-5 times of that of a comparative example; the average number of buds and the average number of leaves are 1.5-1.8 and 4.5-5.6 respectively, which are higher than those of the comparative example, and the effect is obvious. Therefore, the application can promote the survival and growth of the Phalaenopsis x hybrida tissue culture seedlings returned to the wild, increase the population quantity, and protect endangered species resources.

Description

Technical Field

[0001] This invention belongs to the field of field reintroduction technology for rare and endangered plants, and in particular relates to a method for field reintroduction of tissue culture seedlings of Paphiopedilum helenense. Background Technology

[0002] *Paphiopedilum helenae* Aver is a perennial evergreen plant belonging to the genus *Paphiopedilum* in the family Orchidaceae, and is a Class I protected plant in China. *Paphiopedilum helenae* is characterized by its compact and small size, unique flower shape, and high ornamental value, making it extremely valuable for both economic and scientific research. Surveys have revealed only five distribution points in Cao Bang, Vietnam (Averyanov et al., 2003), and only five populations (totaling 162 plants or clumps) in Longzhou County, Jingxi County, and Napo County, Guangxi Province, China. This indicates that *Paphiopedilum helenae* is extremely rare and on the verge of extinction, urgently requiring protection. Ex-situ conservation is currently the most common and effective strategy for critically endangered species. However, for orchids, due to their close, even highly specific, synergistic relationships with fungi and insects during evolution, their survival and reproduction are highly dependent on their environment. Ex-situ conservation environments are often insufficient to meet their reproductive needs (such as pollinating insects), making reintroduction into the wild a potentially more effective conservation strategy. However, the reintroduction of orchids faces more difficulties than that of other plants. First, it is necessary to solve the problems of propagation and domestication of reintroduced materials. At the same time, it is also necessary to consider the reintroduction habitat, the planting and management of reintroduced seedlings, etc. Therefore, the reintroduction of orchids is still in its initial stage.

[0003] The seeds of *Paphiopedilum helenense* are embryoless or have incomplete embryo development, making germination extremely difficult under natural conditions. Furthermore, the plant grows slowly, making seedling acquisition through division very challenging. Through the tireless efforts of researchers, aseptic germination and tissue culture of *Paphiopedilum helenense* seeds have achieved some success, providing seedlings for the reintroduction into the wild and expanding the population. However, artificially propagated *Paphiopedilum helenense* seedlings, after tissue culture and year-round greenhouse conditions, exhibit poor adaptability. Additionally, *Paphiopedilum helenense* grows in karst topography, on mountain ridges or steep slopes in rock crevices, where soil is scarce, creating a very specific habitat and narrow distribution range, requiring advanced cultivation techniques. Moreover, the reintroduction habitat of *Paphiopedilum helenense* is a karst region with extremely poor water retention and a lack of surrounding water sources, making it susceptible to drought. The drought resistance of the seedlings also significantly impacts the reintroduction success rate. Therefore, improving the survival rate of *Paphiopedilum helenense* during its reintroduction into the wild has become an urgent problem to be solved. Summary of the Invention

[0004] The technical problem to be solved by this invention is to provide a method for the field reintroduction of tissue culture seedlings of Paphiopedilum helenii with high survival rate and good growth.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0006] The method of reintroducing tissue culture seedlings of Paphiopedilum helenii into the wild uses tissue culture seedlings of Paphiopedilum helenii as material. The reintroduction point is selected in an area similar to the original habitat. Before reintroduction, the seedlings are acclimatized to enhance their adaptability and drought resistance. The planting point is selected in a place with a large slope and where it is not easy for dead branches and fallen leaves to accumulate.

[0007] The above-mentioned method for the field reintroduction of Paphiopedilum tissue culture seedlings includes the following steps:

[0008] (1) Regression point selection

[0009] (2) Seedling cultivation

[0010] (3) Pre-retrogression seedling domestication

[0011] (4) Planting

[0012] (5) Maintenance and monitoring.

[0013] In step (1), the regression point is selected at the original wild population of Paphiopedilum helenii.

[0014] The point of return is located in the subtropical humid monsoon region south of the Tropic of Cancer in southwestern Guangxi, at an altitude of 450-850m, with an average annual temperature of 19.0-22.2℃ and a karst mountain range with a shade coverage of 50-80%.

[0015] *Paphiopedilum helenense* is distributed in southwestern Guangxi, located between 105°59′ and 106°56′ east longitude and 22°28′ and 22°58′ north latitude, at an altitude of 430-878 meters, a very narrow distribution range. *Paphiopedilum helenense* prefers a warm and humid climate and cannot tolerate direct sunlight. Wild plants grow in crevices on karst mountain ridges or steep slopes, a very specific habitat. Therefore, selecting an environment similar to its native habitat during reintroduction can effectively increase the success rate of *Paphiopedilum helenense*.

[0016] In step (2), the tissue culture bottle seedlings with well-developed root systems obtained from tissue culture are transplanted in a greenhouse for 10-12 months. Select healthy, disease-free, and uniform Paphiopedilum nutrient cup seedlings with 4-6 leaves.

[0017] In step (2), the tissue culture seedlings are obtained by collecting wild population seeds and then sterilizing and culturing them.

[0018] Helen Paphiopedilum is very difficult to propagate under natural conditions. Aseptic germination and tissue culture techniques can effectively solve the problem of seedling propagation, providing seedlings for the reintroduction of Helen Paphiopedilum into the wild and expanding the population.

[0019] In step (3), the seedlings obtained in step (2) are moved to a simple rain shelter for acclimatization to obtain acclimatized seedlings.

[0020] Step (3) is to be performed as follows:

[0021] S1: In the first month of acclimatization, reduce the amount of fertilizer applied to half that used in greenhouse cultivation, while keeping the water supply unchanged;

[0022] S2: In the second month of acclimatization, reduce the application of fertilizer and water supply to half that of greenhouse cultivation;

[0023] S3: In the third month of acclimatization, stop applying fertilizer and reduce the water supply to half that of greenhouse cultivation.

[0024] S4: Water every 10 days or so during the 4th-5th month of acclimatization;

[0025] S5: During the 6th-8th month of acclimatization, water sparingly after the plant leaves show signs of water loss.

[0026] The aforementioned domestication enhances the adaptability and drought resistance of Paphiopedilum helenii seedlings, providing a physiological basis for the plants to adapt to the environment as quickly as possible after being reintroduced into the wild.

[0027] Step (4) is to be performed as follows:

[0028] S1: Planting time is from March to May or from September to November;

[0029] S2: Select a location with a slope greater than 75 degrees and where it is not easy for dead branches and fallen leaves to accumulate as the planting site for the reintroduced plants.

[0030] S3: Planting hole treatment. When planting in rock crevices or rock pits, fill the pits with native humus soil before planting. When planting in soil, dig a hole about 8-10cm in diameter and 5cm deep before planting.

[0031] S4: Plant the plant, remove the nutrient cup, plant it together with the original cultivation substrate, and cover the planting site with native humus and moss.

[0032] Helen's Paphiopedilum plants are small and slow-growing, easily covered by weeds and fallen leaves. This is especially true for tissue-cultured seedlings; if covered by weeds and fallen leaves, waterlogging at the growing point and insufficient light can easily lead to disease and death due to lack of sunlight, severely reducing the survival rate of reintroduced plants. Therefore, choosing a slope greater than 75 degrees as the planting site for reintroduced plants overcomes the problem of accumulated fallen leaves and reduces the workload of field management. After reintroduction planting, covering the planting hole with moss or humus enhances the soil's water retention capacity, providing a suitable microhabitat and nutrients for the reintroduced plants.

[0033] Step (5) Follow these steps: Observe once a week for the first 3 months after transplanting, water as needed, remove dead branches, fallen leaves and weeds that affect growth, and gradually reduce maintenance after 3 months.

[0034] To address the challenges of low natural populations and difficulties in reintroducing *Paphiopedilum helenense* into the wild, the inventors have developed a method for the reintroduction of tissue-cultured *Paphiopedilum helenense* seedlings into the wild. Using tissue-cultured seedlings as material, the method selects areas similar to their native habitat as reintroduction points. Before reintroduction, the seedlings undergo acclimatization to enhance their adaptability and drought resistance. Planting sites are chosen on steep slopes that are less prone to accumulating dead leaves and branches. This invention, through methods such as reintroduction point selection, seedling cultivation, pre-introduction acclimatization, selection and treatment of planting sites, and reintroduction management, achieves a one-year survival rate of 45.4-54.9% for reintroduced *Paphiopedilum helenense* seedlings, which is 4-5 times higher than the control group. The average number of buds and leaves is 1.5-1.8 and 4.5-5.6 respectively, significantly higher than the control group's 1 and 1.5-2.3 leaves, demonstrating a substantial effect. Therefore, applying this invention can promote the survival and growth of *Paphiopedilum helenense* tissue-cultured seedlings in the wild, increase population numbers, and protect endangered species resources. Attached Figure Description

[0035] Figure 1 The image shows actual Helena petioles plants that have been grown for 1 year as an example. In the image, A to D represent Helena petioles plants from Examples 1 to 4, respectively. Detailed Implementation

[0036] To clearly illustrate how this invention is implemented, the following examples further illustrate the concept. Among them,

[0037] Preparation of Helen's Paphiopedilum tissue culture seedlings: After the well-rooted Helen's Paphiopedilum tissue culture seedlings have been hardened off and the culture medium cleaned, they are transplanted onto a mixed substrate (pine bark: nutrient soil: volcanic rock = 2:1:1) and managed as usual in a greenhouse. After 10-12 months of cultivation, select healthy seedlings with 4-6 leaves that are growing well to prepare for acclimatization and reintroduction.

[0038] Example 1

[0039] (1) Regression point selection

[0040] The return point was selected from the wild population of Paphiopedilum helenii in the lower tundra of Longzhou County at an altitude of 455m.

[0041] (2) Seedling cultivation

[0042] Tissue culture seedlings with well-developed root systems obtained from tissue culture are transplanted in a greenhouse for 10-12 months. Select healthy, disease-free, and uniform Paphiopedilum nutrient cup seedlings with 4-6 leaves.

[0043] Tissue culture seedlings are obtained by collecting seeds from wild populations and then performing aseptic germination and tissue culture.

[0044] (3) Pre-retrogression seedling domestication

[0045] Tissue culture seedlings were moved to a simple rain shelter for acclimatization to obtain acclimatized seedlings, as detailed below:

[0046] S1: In the first month of acclimatization, reduce the amount of fertilizer applied to half that used in greenhouse cultivation, while keeping the water supply unchanged;

[0047] S2: In the second month of acclimatization, reduce the application of fertilizer and water supply to half that of greenhouse cultivation;

[0048] S3: In the third month of acclimatization, stop applying fertilizer and reduce the water supply to half that of greenhouse cultivation.

[0049] S4: Water every 10 days or so during the 4th-5th month of acclimatization;

[0050] S5: During the 6th-8th month of acclimatization, water sparingly after the plant leaves show signs of water loss.

[0051] (4) Planting

[0052] S1: Planting time is from March to May;

[0053] S2: Select a location with a slope greater than 75 degrees and where it is not easy for dead branches and fallen leaves to accumulate as the planting site for the reintroduced plants.

[0054] S3: Planting hole treatment: Choose rock crevices or rock holes for planting. First fill the holes with native humus soil before planting.

[0055] S4: Plant the plant, remove the nutrient cup, plant it together with the original cultivation substrate, and cover the planting site with native humus and moss.

[0056] (5) Maintenance and Monitoring

[0057] After the transplanted plants are established, observe them once a week for the first 3 months, water them as needed, and remove dead branches, fallen leaves and weeds that affect growth. After 3 months, gradually reduce maintenance.

[0058] Example 2

[0059] The method is basically the same as Example 1, except that the regression point in step (1) is Jingxi Bangliang, with an altitude of 500m, and the regression plant planting point in step (4) is a rock wall with a slope greater than 85 degrees. The plant planting location is covered with white sandy soil formed after the rock dissolves. First, dig a hole with a diameter of 8-10cm and a depth of about 5cm before planting. The planting time is from March to May.

[0060] Example 3

[0061] The method is basically the same as Example 1, except that the regression point in step (1) is Longming Town, Tiandeng County, at an altitude of 780m, the slope of the regression plant planting point in step (4) is 35 degrees, the plant planting location is a rock covered with native humus soil, and the planting time is from March to May.

[0062] Example 4

[0063] The method is basically the same as Example 1, except that the regression point in step (1) is at the same altitude, about 300m away from the wild population of Paphiopedilum helenii in the lower tundra of Longzhou County, and the slope of the regression plant planting point in step (4) is greater than 75 degrees. The plant planting location is a rock crevice or rock pit, and the planting time is from September to November.

[0064] Comparative Example 1

[0065] Similar to Example 1, the seedlings in the nutrient cups in step (2) are cultivated in the greenhouse for 18-20 months and are directly reintroduced without undergoing the acclimatization in step (3). The slope of the planting point for the reintroduced plants in step (4) is greater than 75 degrees, and the planting location is a rock crevice or rock pit. The planting time is from March to May.

[0066] Comparative Example 2

[0067] Similar to Example 1, the domestication in step (3) only completes steps S1-S3 before reverting to the original state. In step (4), the slope of the reverted plant planting point is greater than 75 degrees, the plant planting location is a rock crevice or rock pit, and the planting time is from March to May.

[0068] The results of one-year field regression experiments in Examples 1 to 4 and Comparative Examples 1 to 2 of this invention are as follows: Figure 1 See Table 1. The results show that the preservation rate and growth of Examples 1, 2, and 4 are significantly better than those of Example 3 and Comparative Examples 1 and 2.

[0069] Table 1. Survival and growth of *Paphiopedilum helenense* after 1 year in the examples and comparative regression.

[0070] Retention rate (%) Average number of buds per plant Average number of leaves per plant (sheets) Example 1 47.5 1.8 5.6 Example 2 54.9 1.5 4.5 Example 3 28.7 1.2 3.0 Example 4 45.4 1.7 5.0 Comparative Example 1 10.2 1 1.5 Comparative Example 2 14.5 1 2.3

Claims

1. A method for the field reintroduction of tissue-cultured Paphiopedilum helenii seedlings, characterized in that: Using tissue-cultured seedlings of *Paphiopedilum helenense* as material, a region similar to its native habitat was selected as the reintroduction point. Before reintroduction, the seedlings underwent acclimatization to enhance their adaptability and drought resistance. Planting sites were selected on steep slopes where fallen leaves and branches were less likely to accumulate. This method includes the following steps: (1) Regression point selection The regression point is located in the subtropical humid monsoon region south of the Tropic of Cancer in southwestern Guangxi, at an altitude of 450-850m, with an average annual temperature of 19.0-22.2℃ and a karst mountain with a shade rate of 50-80%. (2) Seedling cultivation (3) Pre-retrogression seedling domestication S1: In the first month of acclimatization, reduce the amount of fertilizer applied to half that used in greenhouse cultivation, while keeping the water supply unchanged; S2: In the second month of acclimatization, reduce the application of fertilizer and water supply to half that of greenhouse cultivation; S3: In the third month of acclimatization, stop applying fertilizer and reduce the water supply to half that of greenhouse cultivation. S4: Water every 10 days or so during the 4th-5th month of acclimatization; S5: Water a small amount of water after the plant leaves show signs of water loss during the 6th-8th month of acclimatization. (4) Planting S1: Planting time is from March to May or from September to November; S2: Select a location with a slope greater than 75 degrees and where it is not easy for dead branches and fallen leaves to accumulate as the planting site for the reintroduced plants. S3: Planting hole treatment. When planting in rock crevices or rock pits, fill the pits with native humus soil before planting. When planting in soil, dig a hole about 8-10cm in diameter and 5cm deep before planting. S4: Planting: Remove the nutrient cup and plant the plant together with the original cultivation substrate. Cover the planting site with native humus and moss. (5) Maintenance and monitoring.

2. The method for the field reintroduction of Paphiopedilum tissue culture seedlings according to claim 1, characterized in that... In step (1), the regression point is selected at the original wild population of Paphiopedilum helenii.

3. The method for field reintroduction of Paphiopedilum tissue culture seedlings according to claim 1, characterized in that... In step (2), the tissue culture bottle seedlings with well-developed root systems obtained from tissue culture are transplanted in a greenhouse for 10-12 months. Select healthy, disease-free, and uniform Paphiopedilum nutrient cup seedlings with 4-6 leaves.

4. The method for field reintroduction of Paphiopedilum tissue culture seedlings according to claim 3, characterized in that... In step (2), the tissue culture seedlings are obtained by collecting wild population seeds and then sterilizing and culturing them.

5. The method for field reintroduction of Paphiopedilum tissue culture seedlings according to claim 3, characterized in that... In step (3), the seedlings obtained in step (2) are moved to a simple rain shelter for acclimatization to obtain acclimatized seedlings.

6. The method for field reintroduction of Paphiopedilum tissue culture seedlings according to claim 1, characterized in that... Step (5) Follow these steps: Observe once a week for the first 3 months after transplanting, water as needed, remove dead branches, fallen leaves and weeds that affect growth, and gradually reduce maintenance after 3 months.

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