Field regression method for tissue culture seedlings of paphiopedilum helenae

Abstract

The invention discloses a field regression method for tissue culture seedlings of paphiopedilum helenae. The tissue culture seedlings of paphiopedilum helenae are adopted as materials, areas similar to original habitat are selected as regression points, the adaptability and drought resistance of the seedlings are enhanced through domestication before regression, and places where the gradient is large and dry branches and fallen leaves are not prone to deposition are selected as regression plant planting points for planting. Through operations such as regression point screening, seedling culture, domestication before regression, plant field planting point selection and treatment, regression management and protection and the like, the preservation rate of the paphiopedilum helenae tissue culture seedlings in one year can be 45.4-54.9%, which is 4-5 times that of a contrast; 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 1 and 1.5-2.3 of the contrast, and the method has an obvious effect. Therefore, by applying the method, field return survival and growth of the paphiopedilum helenae tissue culture seedlings can be promoted, the population number is increased, and endangered species resources are protected.

Description

Technical Field

[0001] The invention belongs to the technical field of wild reintroduction of rare and endangered plants, and in particular relates to a method for wild reintroduction of Paphiopedilum helengensis tissue culture seedlings. Background Art

[0002] Paphiopedilum helenae Aver is a perennial evergreen plant of the genus Paphiopedilum in the Orchidaceae family and is a national first-class protected plant. Paphiopedilum helenae Aver has a compact and small plant shape, unique flower shape, good ornamental value, and extremely high economic and scientific research value. According to investigations, only five distribution points in Cao Bang, Vietnam have been found abroad (Averyanov et al., 2003), and only five populations have been found in Longzhou County, Jingxi County, and Napo County in Guangxi in China, with a total of 162 plants (or clusters). It can be seen that the existing resources of Paphiopedilum helenae Aver are very few and are on the verge of extinction at any time, and are in urgent need of protection. Ex situ conservation is currently the most commonly used and effective conservation strategy for critically endangered species, but for orchids, because they have established a close and even highly specific cooperative relationship with fungi and insects during the evolution process, their survival and reproduction are highly dependent on the living environment. The environment of ex situ conservation is difficult to meet their reproduction needs (such as pollinating insects), and wild return may be a more effective conservation strategy. However, the reintroduction of orchids faces more difficulties than other plants. First of all, it is necessary to solve the expansion and domestication of the reintroduction materials. At the same time, it is also necessary to consider the reintroduction habitat, planting and management of reintroduction seedlings, etc. Therefore, the reintroduction of orchids is still in its infancy.

[0003] The seeds of Paphiopedilum helengensis have no embryo or the embryo is underdeveloped. It is extremely difficult to germinate under natural conditions. In addition, the plant grows slowly, and it is also very difficult to obtain seedlings through division. After the unremitting efforts of scientific researchers, the aseptic germination and tissue culture of Paphiopedilum helengensis seeds have achieved certain results, providing seedlings for the return of Paphiopedilum helengensis to the wild and expanding the population. However, the artificially bred Paphiopedilum helengensis seedlings have been cultured through tissue culture and kept in greenhouses all year round, and their adaptability is poor; at the same time, Paphiopedilum helengensis grows in the ridges of karst karst landforms or in the cracks of rock walls on the upper part of steep slopes, with scarce soil, very special habitats and a narrow distribution range, resulting in the plant itself requiring higher planting technology. In addition, the habitat of Paphiopedilum helengensis is a karst karst area with extremely poor water retention, and there is a lack of water sources in the surrounding area, which is easily threatened by drought. The drought resistance of the seedlings also greatly affects the return effect. Therefore, how to improve the survival rate of Paphiopedilum helengensis when it returns to the wild has become an urgent problem to be solved. Summary of the invention

[0004] The technical problem to be solved by the invention is to provide a method for returning Paphiopedilum helengensis tissue culture seedlings to the wild with high survival rate and good growth.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0006] The method for field reintroduction of tissue-cultured seedlings of Paphiopedilum helengensis uses tissue-cultured seedlings of Paphiopedilum helengensis as materials, selects areas similar to their native habitats as reintroduction points, and domesticates them before reintroduction to enhance their adaptability and drought resistance. Places with large slopes and where dead branches and fallen leaves are not easily deposited are selected as planting points for reintroduction plants.

[0007] The above-mentioned method for returning Paphiopedilum helengensis tissue culture seedlings to the wild comprises the following steps:

[0008] (1) Regression point selection

[0009] (2) Seedling cultivation

[0010] (3) Seedling domestication before return

[0011] (4) Planting

[0012] (5) Management, maintenance and monitoring.

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

[0014] The regression point is in the subtropical humid monsoon zone near the Tropic of Cancer in the southwest of Guangxi, with an altitude of 450-850m, an average annual temperature of 19.0-22.2℃, and a karst karst stone mountain with a shade degree of 50-80%.

[0015] Paphiopedilum Helenii is distributed in the southwest of Guangxi, located in the area of ​​105°59′-106°56′ east longitude, 22°28′-22°58′ north latitude, and 430-878m above sea level. The distribution range is very narrow. Paphiopedilum Helenii prefers a warm and humid climate and cannot tolerate direct sunlight. Wild plants grow in the ridges of karst karst landforms or in the cracks of rock walls on the upper part of steep slopes. The habitat is very special. Therefore, choosing an environment similar to the original habitat when returning can effectively increase the chances of success of Paphiopedilum Helenii.

[0016] In step (2), the tissue culture bottle seedlings with well-developed root systems obtained by tissue culture are transplanted in a greenhouse for 10-12 months, and the Helenium Paphiopedilum nutrient cup seedlings that are healthy, free of pests and diseases, have 4-6 leaves and are in basically the same state are selected.

[0017] In step (2), the tissue culture bottle seedlings are obtained by collecting seeds from wild populations and performing aseptic germination and tissue culture.

[0018] It is very difficult to reproduce Paphiopedilum Helenii under natural conditions. The use of sterile germination and tissue culture technology can effectively solve the problem of seedling propagation and provide seedlings for Paphiopedilum Helenii to return to the wild and expand the population.

[0019] In step (3), the nutrient cup seedlings obtained in step (2) are moved into a simple rain shelter for domestication to obtain domesticated seedlings.

[0020] Step (3) Follow the steps below:

[0021] S1: In the first month of acclimatization, the fertilizer application was reduced to 1 / 2 of that in greenhouse cultivation, and the water supply remained unchanged;

[0022] S2: In the second month of acclimatization, the fertilizer and water supply were reduced to 1 / 2 of that in greenhouse cultivation;

[0023] S3: In the third month of acclimatization, stop applying fertilizer and reduce the water supply to 1 / 2 of that in greenhouse cultivation;

[0024] S4: During the 4th to 5th month of acclimatization, water once every 10 days or so;

[0025] S5: In the 6th to 8th month of acclimatization, water the plants slightly after the leaves start to lose water.

[0026] The above-mentioned domestication enhances the adaptability and drought resistance of the returned seedlings of Paphiopedilum helenense, and provides a physiological basis for the plants to adapt to the environment as quickly as possible after returning to the wild.

[0027] Step (4) Follow the steps below:

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

[0029] S2: The planting site of the plant is selected in a place with a slope greater than 75 degrees and where dead branches and fallen leaves are not easily deposited as the planting site of the returning plant;

[0030] S3: Plant planting hole treatment: when the planting site is a rock gap or rock hole, first fill it with native humus soil before planting; when the planting site is soil planting, first dig a hole with a diameter of 8-10cm and a depth of about 5cm before planting;

[0031] S4: Plant the plants, remove the nutrient cups, plant them together with the original cultivation medium, and cover the planting area with humus soil and moss from the original habitat.

[0032] Paphiopedilum helengensis is small and grows slowly, and is easily covered by weeds and dead branches and leaves. Especially for Paphiopedilum helengensis tissue culture seedlings, if they are covered by weeds and dead branches and leaves, it is very easy to form water accumulation at the plant growth point and cause insufficient light, leading to disease and death due to lack of sunlight, thus seriously reducing the preservation rate of returning plants. Therefore, choosing a place with a slope greater than 75 degrees as the planting site for returning plants overcomes the problem of deposited dead branches and leaves and reduces the workload of field management. After returning to planting, cover the planting hole with moss or humus to enhance the soil's water retention and moisture retention capacity, and provide a suitable microhabitat and nutrients for returning plants.

[0033] Step (5) is carried out according to the following steps: observe once a week within 3 months after returning to planting, water at appropriate times, remove dead branches and leaves and weeds that affect growth, and gradually reduce management and maintenance after 3 months.

[0034] In view of the problem that the natural population of Helen's Paphiopedilum is small and it is difficult to return to the wild, the inventor has established a method for the wild return of Helen's Paphiopedilum tissue culture seedlings, using Helen's Paphiopedilum tissue culture seedlings as materials, selecting areas similar to the native habitat as return points, and enhancing their adaptability and drought resistance through domestication before return, and selecting places with large slopes and not easy to deposit dead branches and fallen leaves as return plant planting points for planting. The present invention can make the preservation rate of Helen's Paphiopedilum tissue culture seedlings return for 1 year to 45.4-54.9%, which is 4-5 times that of the comparative example; the average number of buds and leaves is 1.5-1.8 and 4.5-5.6, respectively, which is higher than 1 and 1.5-2.3 of the comparative example, and has obvious effects. Therefore, the application of the present invention can promote the survival and growth of Helen's Paphiopedilum tissue culture seedlings in the wild, increase the population, and protect endangered species resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a physical picture of the Paphiopedilum Helenii plants that have been planted for one year in the embodiment. In the picture: A to D are the Paphiopedilum Helenii plants of Examples 1 to 4, respectively. DETAILED DESCRIPTION

[0036] In order to clearly illustrate how to implement the present invention, the following examples are further explained.

[0037] Preparation of tissue culture seedlings of Paphiopedilum Helenii: After the well-rooted Paphiopedilum Helenii tissue culture bottle seedlings are hardened and the culture medium is cleaned, they are transplanted to the mixed matrix (pine bark: nutrient soil: volcanic stone = 2:1:1) and routinely managed in the greenhouse. After 10-12 months of cultivation, healthy seedlings with good growth and 4-6 leaves are selected for preparation for acclimatization and return.

[0038] Example 1

[0039] (1) Regression point selection

[0040] The regression point was selected as the wild population of Paphiopedilum Helenii in the lower tundra of Longzhou County at an altitude of 455m.

[0041] (2) Seedling cultivation

[0042] The tissue culture bottle seedlings with well-developed root systems obtained by tissue culture are transplanted in a greenhouse for 10-12 months, and the Helenium Paphiopedilum nutrient cup seedlings that are healthy, free of pests and diseases, have 4-6 leaves and are in basically the same condition are selected.

[0043] Tissue culture bottle seedlings are collected from wild population seeds and obtained through sterile germination and tissue culture.

[0044] (3) Seedling domestication before return

[0045] The tissue culture seedlings were moved to a simple rain shelter for domestication to obtain domesticated seedlings, as follows:

[0046] S1: In the first month of acclimatization, the fertilizer application was reduced to 1 / 2 of that in greenhouse cultivation, and the water supply remained unchanged;

[0047] S2: In the second month of acclimatization, the fertilizer and water supply were reduced to 1 / 2 of that in greenhouse cultivation;

[0048] S3: In the third month of acclimatization, stop applying fertilizer and reduce the water supply to 1 / 2 of that in greenhouse cultivation;

[0049] S4: During the 4th to 5th month of acclimatization, water once every 10 days or so;

[0050] S5: In the 6th to 8th month of acclimatization, water the plants slightly after the leaves start to lose water.

[0051] (4) Planting

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

[0053] S2: The planting site of the plant is selected in a place with a slope greater than 75 degrees and where dead branches and fallen leaves are not easily deposited as the planting site of the returning plant;

[0054] S3: Planting hole treatment: choose rock gaps or rock holes for planting, fill them with native humus soil before planting;

[0055] S4: Plant the plants, remove the nutrient cups, plant them together with the original cultivation medium, and cover the planting area with humus soil and moss from the original habitat.

[0056] (5) Maintenance and monitoring

[0057] Observe once a week within 3 months after returning to planting, water in time, remove dead branches, leaves and weeds that affect growth, and gradually reduce maintenance after 3 months.

[0058] Example 2

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

[0060] Example 3

[0061] It is basically the same as Example 1, except that the regression point in step (1) is Longming Town, Tiandeng County, 780m above sea level, the regression plant planting point in step (4) has a slope of 35 degrees, the plant planting position is rock covered with native habitat humus soil, and the planting time is March-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 helengensis in the lower tundra of Longzhou County, the slope of the regression plant planting point in step (4) is greater than 75 degrees, the plant planting position is a rock crevice or rock hole, and the planting time is September-November.

[0064] Comparative Example 1

[0065] Basically the same as Example 1, the nutrient cup seedlings in step (2) are cultured in a greenhouse for 18-20 months, and are directly returned without the acclimation in step (3). The slope of the planting point of the returned plants in step (4) is greater than 75 degrees, and the plant planting position is a rock crevice or rock hole, and the planting time is March to May.

[0066] Comparative Example 2

[0067] Basically the same as Example 1, the domestication in step (3) is completed only after steps S1-S3, and the slope of the planting point of the returning plant in step (4) is greater than 75 degrees, the plant planting position is a rock crevice or rock hole, and the planting time is March-May.

[0068] The results of the field return of Examples 1 to 4 and Comparative Examples 1 to 2 for one year are as follows: Figure 1 and 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 helengensis in the embodiment and comparative example after regression for 1 year

[0070] Preservation rate (%) Average number of buds per plant Average number of leaves per plant 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 field return of Paphiopedilum helengensis tissue culture seedlings, characterized in that: The tissue culture seedlings of Paphiopedilum helengensis were used as materials, and areas similar to their native habitats were selected as the return points. Before the return, they were domesticated to enhance their adaptability and drought resistance. Places with large slopes and where dead branches and fallen leaves were not easily deposited were selected as the planting points for the return plants.

2. The method for field return of Paphiopedilum helengensis tissue culture seedlings according to claim 1, characterized in that The following steps are involved: (1) Regression point selection (2) Seedling cultivation (3) Seedling domestication before return (4) Planting (5) Management, maintenance and monitoring.

3. The method for field return of Paphiopedilum helengensis tissue culture seedlings according to claim 1, characterized in that In step (1), the regression point is selected as the original wild population of Paphiopedilum helengensis.

4. The method for field return of Paphiopedilum helengensis tissue culture seedlings according to claim 3, characterized in that: The regression point is located in the subtropical humid monsoon zone south of the Tropic of Cancer in the southwest of Guangxi, with an altitude of 450-850m, an annual average temperature of 19.0-22.2°C, and a karst karst stone mountain with a shade degree of 50-80%.

5. The method for field return of Paphiopedilum helengensis tissue culture seedlings according to claim 2, characterized in that In step (2), the tissue culture bottle seedlings with well-developed root systems obtained by tissue culture are transplanted in a greenhouse for 10-12 months, and the Helenium Paphiopedilum nutrient cup seedlings that are healthy, free of pests and diseases, have 4-6 leaves and are in basically the same state are selected.

6. The method for field return of Paphiopedilum helengensis tissue culture seedlings according to claim 5, characterized in that In step (2), the tissue culture bottle seedlings are obtained by collecting seeds from wild populations and performing aseptic germination and tissue culture.

7. The method for field return of Paphiopedilum helengensis tissue culture seedlings according to claim 2, characterized in that In step (3), the nutrient cup seedlings obtained in step (2) are moved into a simple rain shelter for domestication to obtain domesticated seedlings.

8. The method for field return of Paphiopedilum helengensis tissue culture seedlings according to claim 7, characterized in that Step (3) Follow the steps below: S1: In the first month of acclimatization, the fertilizer application was reduced to 1 / 2 of that in greenhouse cultivation, and the water supply remained unchanged; S2: In the second month of acclimatization, the fertilizer and water supply were reduced to 1 / 2 of that in greenhouse cultivation; S3: In the third month of acclimatization, fertilizer application was stopped and water supply was reduced to 1 / 2 of that in greenhouse cultivation; S4: During the 4th to 5th month of acclimatization, water once every 10 days or so; S5: In the 6th to 8th month of acclimatization, water the plants slightly after the leaves start to lose water.

9. The method for field return of Paphiopedilum helengensis tissue culture seedlings according to claim 2, characterized in that Step (4) Follow the steps below: S1: Planting time is March to May or September to November; S2: The planting site of the plant is selected in a place with a slope greater than 75 degrees and where dead branches and fallen leaves are not easily deposited as the planting site of the returning plant; S3: Plant planting hole treatment: when the planting site is a rock gap or rock hole, first fill it with native humus soil before planting; when the planting site is soil planting, first dig a hole with a diameter of 8-10cm and a depth of about 5cm before planting; S4: Plant the plants, remove the nutrient cups, plant them together with the original cultivation medium, and cover the planting area with humus soil and moss from the original habitat.

10. The method for field return of Paphiopedilum helengensis tissue culture seedlings according to claim 2, characterized in that Step (5) is carried out according to the following steps: observe once a week within 3 months after returning to planting, water at appropriate times, remove dead branches and leaves and weeds that affect growth, and gradually reduce management and maintenance after 3 months.

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