Dendrobium huoshuangshanense conservation method

Abstract

The application provides a Hainan dendrobium conservation method and relates to the technical field of rare and endangered plant protection. The Hainan dendrobium conservation method comprises the following steps: collecting Hainan dendrobium fruit hulls, disinfecting and obtaining standby seeds; sowing the standby seeds in a germination culture medium, illuminating, culturing and obtaining seedlings; and the formula of the germination culture medium is 1 / 2MS culture medium+NAA+sucrose+agar+regulator solution. The application optimizes the formula of the special germination culture medium and rooting and seedling strengthening culture medium according to the needs of Hainan dendrobium seed germination and seedling growth, and especially by adding a mixed solution in the rooting and seedling strengthening culture medium, the health and survival rate of the seedlings are significantly improved.

Description

Technical Field

[0001] This invention relates to the field of rare and endangered plant protection technology, specifically to a method for the conservation of Dendrobium hainanense. Background Technology

[0002] Dendrobium hainanense is a species endemic to my country. Due to its small wild population, strong habitat specificity, and suffering from over-harvesting and habitat destruction, it has been listed in Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) and assessed as Vulnerable (VU) on the IUCN Red List, indicating an extremely high urgency for conservation.

[0003] Currently, conservation research on Dendrobium hainanense is still in its early stages. Traditional conservation methods mostly involve directly transplanting mature plants from the wild or propagating them by division under simple conditions, which has the following obvious drawbacks:

[0004] 1. Significant damage to wild resources: Transplanting mature plants will directly reduce the already scarce wild populations, violating conservation ethics.

[0005] 2. Low reproductive efficiency: The division propagation coefficient is low, the reproduction speed is slow, and it is difficult to rapidly expand the number of conservation populations.

[0006] 3. Poor adaptability and low survival rate: The artificial cultivation environment differs greatly from the native environment, and the mature plants lack the domestication process, resulting in poor adaptability to the artificial environment. In particular, the epiphytic characteristics cannot be effectively simulated, leading to poor plant growth and difficulty in completing the complete life cycle (flowering and fruiting), thus limiting the conservation effect.

[0007] Therefore, there is an urgent need in this field for a systematic technical method that can efficiently and cost-effectively simulate the natural habitat to the greatest extent, thereby significantly improving the conservation success rate of Dendrobium officinale in Hainan. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides a method for the conservation of Dendrobium officinale in Hainan, which effectively overcomes the limitations of directly transplanting wild plants or simple division, and achieves the goal of fully artificial and controllable conservation from seed to mature plant.

[0009] To achieve the above objectives, the present invention provides the following technical solution:

[0010] A method for the conservation of Dendrobium officinale in Hainan includes the following steps:

[0011] S1. Collect the pods of Dendrobium hainanense, disinfect them, and obtain seeds for later use;

[0012] S2. Sow the prepared seeds in the germination medium, expose to light, and cultivate for 50-70 days to obtain seedlings;

[0013] The germination medium formula is: 2.3-2.5 g / L 1 / 2 MS medium + 0.3-0.4 mg / L NAA + 30 g / L sucrose + 7 g / L agar + 6-16 mg / L preparation solution;

[0014] The preparation solution was obtained by mixing calcium alginate with aloe vera dilution;

[0015] 20 ≤ mass ratio of preparation solution to NAA ≤ 40;

[0016] S3. Transfer the seedlings to the rooting and seedling strengthening culture medium to obtain cultivated seedlings;

[0017] The formula for the rooting and seedling strengthening medium is: 2.3-2.5 g / L 1 / 2 MS medium + 0.5-0.8 mg / L NAA + 20 g / L sucrose + 20-30 mg / L mixed solution + 100-110 g / L banana homogenate + 7 g / L agar + 2 g / L activated carbon;

[0018] The mixture was prepared by adding a 5% (v / v) aloe vera dilution to orange peel after fermentation with Bacillus subtilis.

[0019] S4. Place the seedlings in culture bottles, move them to a greenhouse for 5-7 days, and open the lid for ventilation for 2-3 days to obtain tissue culture seedlings;

[0020] S5. Take out the tissue culture seedlings, disinfect them, put them into the acclimatization substrate, and cultivate them for 3-6 months. Then, attach the seedlings to the dead wood for planting, spray fertilizer, and realize biomimetic cultivation.

[0021] Preferably, the Dendrobium pods collected in step S1 are mature but unopened. The disinfection process involves disinfecting the surface of the Dendrobium pods with 75% ethanol solution for 20-40 seconds, then soaking the pods in 0.1% mercuric chloride solution for 10-20 minutes, and finally rinsing them with sterile water 3-5 times.

[0022] Preferably, in step S2, calcium alginate is mixed with aloe vera dilution with a volume concentration of 10% at a mass ratio of 1:2-3.

[0023] Preferably, the aloe vera diluents in steps S2 and S3 are obtained by diluting the original aloe vera extract;

[0024] The aloe vera extract is prepared according to the following steps: after washing and drying the aloe vera, it is crushed, passed through an 80-mesh sieve, 3-7 times the amount of water is added, stirred for 30-60 minutes, soaked at 32-38℃ for 20-28 hours, and the residue is removed to obtain the aloe vera extract.

[0025] Preferably, in step S3, the light conditions are an illuminance of 1000-2000 lux and a photoperiod of 12-14 h / day, and a culture temperature of 23-27℃.

[0026] Preferably, the specific preparation steps of the mixed solution in step S3 are as follows:

[0027] S3-1. Wash the orange peel, rehydrate it, dry it, pulp it, and pass it through a 60-mesh sieve to obtain the pulp.

[0028] S3-2. Inoculate Bacillus subtilis into the slurry, with a mass ratio of slurry to Bacillus subtilis of 100:3-5. Ferment for 30-40 hours, then sterilize by passing through a 0.22μm filter membrane to obtain the fermentation mixture.

[0029] S3-3. Add 5% (v / v) aloe vera dilution to the fermentation mixture. The volume ratio of the fermentation mixture to the aloe vera dilution is 1.5-2:1 to obtain the mixed solution.

[0030] Preferably, the acclimatization substrate in step S5 is obtained by mixing sphagnum moss, coconut shell pellets, and pine bark in a mass ratio of 1-3:1:1, with a humidity of 70-80%.

[0031] Preferably, the specific operation of attaching the seedling to the dead wood in step S5 is as follows: fix the dead wood to the bonsai pot, fill it with sand, plant moss and small ferns on the surface to form a moist micro-ecosystem, prune the rotten roots of the seedling, disinfect and spray with a rooting promoter, wrap the roots with wet sphagnum moss, and fix the plant to the dead wood with fishing line and nail gun.

[0032] Preferably, the dripping rate in step S3-3 is 3 drops / s, and the content of Bacillus subtilis is 2×10⁻⁶. 10 CFU / g.

[0033] This invention provides a method for the conservation of Dendrobium officinale in Hainan, which has the following advantages compared with the prior art:

[0034] This invention proposes a Hainan Dendrobium conservation system that integrates "sterile sowing → substrate domestication → biomimetic epiphytosis," overcoming the limitations of traditional methods and achieving fully artificial and controllable conservation from seed to mature plant.

[0035] To meet the needs of seed germination and seedling growth of Dendrobium hainanense, we optimized the formulations of specialized germination and rooting / seedling strengthening media. In particular, by adding a mixed solution to the rooting / seedling strengthening media, we significantly improved the robustness and survival rate of seedlings.

[0036] A "step-by-step acclimatization" strategy was adopted, which involves first acclimatizing and cultivating the seedlings in a substrate until they are robust, and then simulating their native microenvironment by fixing them to dead wood for epiphytic cultivation. This method effectively solves the problems of poor adaptability and low survival rate of tissue-cultured seedlings when directly epiphyted, allowing the plants to better complete their life cycle. Attached Figure Description

[0037] Figure 1The germination results of Dendrobium pods in the germination medium prepared in Example 1;

[0038] Figure 2 The germination results of Dendrobium pods in the germination medium prepared in Example 2;

[0039] Figure 3 The germination status of Dendrobium pods in the germination culture media prepared for each experimental group is shown. Specifically: A represents the germination result of pods in experimental group 1; B represents the germination result of pods in experimental group 2; C represents the germination result of pods in experimental group 3; and D represents the germination result of pods in experimental group 4.

[0040] Figure 4 The growth status of Dendrobium seedlings using the rooting and seedling strengthening culture medium of Example 3;

[0041] Figure 5 The growth status of Dendrobium seedlings using the rooting and seedling strengthening culture medium of Example 4;

[0042] Figure 6 The growth status of Dendrobium seedlings in the rooting and seedling strengthening culture media prepared for each experimental group is shown. Where: A represents the growth results of seedlings in experimental group 1; B represents the growth results of seedlings in experimental group 2; C represents the growth results of seedlings in experimental group 3; D represents the growth results of seedlings in experimental group 4; and E represents the growth results of seedlings in experimental group 5.

[0043] Figure 7 This is a diagram showing the planting state of seedlings attached to dead wood. Detailed Implementation

[0044] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0045] Calcium alginate was purchased from Hebei Runbu Biotechnology Co., Ltd.; 1 / 2 MS medium was purchased from Qingdao Haibo Biotechnology Co., Ltd. (HB8469-12); Bacillus subtilis was purchased from Weifang Ruichen Biotechnology Co., Ltd., with a bacterial count of 2×10⁻⁶. 10 CFU / g; activated carbon purchased from the Tanerno brand.

[0046] Aloe vera extract: Take 1 kg of aloe vera, wash and dry it, then crush it and pass it through an 80-mesh sieve. Add 5 times the amount of deionized water, stir for 45 minutes, soak at 75℃ for 24 hours, remove the residue at room temperature, and obtain aloe vera extract.

[0047] Different volume concentrations of aloe vera dilutions were obtained by diluting aloe vera stock solution with deionized water.

[0048] 1. Preparation of the conditioning solution:

[0049] Preparation solution A: prepared by mixing calcium alginate with 10% aloe vera dilution at a mass ratio of 1:2.5;

[0050] Preparation solution B: Prepared by mixing calcium alginate with 10% aloe vera dilution at a mass ratio of 1:1;

[0051] Preparation solution C: prepared by mixing calcium alginate with 5% aloe vera dilution at a mass ratio of 1:2.5;

[0052] 2. Preparation of the mixed solution:

[0053] Mixed solution A:

[0054] S1. Wash the orange peel, rehydrate it, dry it, pulp it, and pass it through a 60-mesh sieve to obtain the pulp.

[0055] S2. Inoculate the slurry with Bacillus subtilis at a mass ratio of 100:4 and ferment for 35 hours. Sterilize the fermentation mixture by passing it through a 0.22 μm filter membrane to obtain the fermentation mixture.

[0056] S3. Add 5% aloe vera dilution at a rate of 3 drops / s to the fermentation mixture. The volume ratio of the fermentation mixture to the aloe vera dilution is 1.8:1 to obtain the mixed solution.

[0057] Mixed solution B:

[0058] Mixture B is prepared using the same method as mixture A, except for step S2. The remaining steps are the same:

[0059] S2. Filter the slurry to obtain the fermentation mixture;

[0060] Mixed solution C:

[0061] Mixture C is prepared using the same method as mixture A, except that step S3 is omitted and step S2 is adjusted. The remaining steps are the same, specifically:

[0062] S2. Inoculate the slurry with Bacillus subtilis at a mass ratio of 100:4. Ferment for 35 hours and sterilize by passing through a 0.22 μm filter membrane to obtain the mixed solution.

[0063] Mixed solution D:

[0064] Mixture D is prepared using the same method as mixture A, except for step S3, while the other steps remain the same. Specifically:

[0065] S3. Add 10% aloe vera dilution at a rate of 3 drops / s to the fermentation mixture. The volume ratio of the fermentation mixture to the aloe vera dilution is 1.8:1 to obtain the mixed solution.

[0066] Mixed solution E:

[0067] Mixture E is prepared using the same method as mixture A, except for step S3, while the other steps remain the same. Specifically:

[0068] S3. Add 5% aloe vera dilution at a rate of 3 drops / s to the fermentation mixture. The volume ratio of the fermentation mixture to the aloe vera dilution is 1:1 to obtain the mixed solution.

[0069] Example 1:

[0070] The sprouting of Dendrobium hainanense:

[0071] S1. Collect mature but unopened pods of Dendrobium hainanense, disinfect the surface of the pods with 75% ethanol solution for 30 seconds, then soak the pods in 0.1% mercuric chloride solution for 15 minutes, and finally rinse with sterile water 4 times to obtain seeds for later use.

[0072] S2. Sow the reserved seeds in germination medium and culture them at 25℃ for 75 days under the conditions of light intensity of 1500 lux and photoperiod of 13h / day to obtain seedlings. 1L of germination medium consists of 2.3g 1 / 2MS medium, 0.3mg NAA, 30g sucrose, 7g agar, and 6mg preparation solution A, with pH 5.8±0.2, where 20≤ the mass ratio of preparation solution to NAA≤40.

[0073] Example 2:

[0074] The sprouting of Dendrobium hainanense:

[0075] S1. Collect mature but unopened pods of Dendrobium hainanense, disinfect the surface of the pods with 75% ethanol solution for 30 seconds, then soak the pods in 0.1% mercuric chloride solution for 15 minutes, and finally rinse with sterile water 4 times to obtain seeds for later use.

[0076] S2. Sow the reserved seeds in germination medium and culture them at 25℃ for 75 days under the conditions of light intensity of 1500 lux and photoperiod of 13h / day to obtain seedlings. 1L of germination medium consists of 2.5g 1 / 2MS medium, 0.4mg NAA, 30g sucrose, 7g agar, and 16mg preparation solution A, with pH 5.8±0.2, where 20≤ the mass ratio of preparation solution to NAA≤40.

[0077] Comparative example:

[0078] This comparative example included four experimental groups, with only the composition ratio of the germination medium adjusted. The other steps were the same as in Example 1, as detailed in Table 1.

[0079] Table 1

[0080]

[0081] Detection:

[0082] Following the method in Example 1, 20 spare seeds were sown in various germination media. After 60 days of cultivation, the growth status and germination rate were observed, as detailed in Table 2. Figure 1-3 As shown:

[0083] Table 2

[0084]

[0085] As shown in the table above and Figure 1-3 As shown, Example 1 ( Figure 1 ) and Example 2 ( Figure 2 The germination rate and growth status of the ) are both good, such as Figure 3 As shown in Figure C, although the growth status of experimental group 3 was also relatively good, its germination rate decreased by 47.1% compared with Example 1. Although the germination rate of experimental group 1 was better, its growth status was poor.

[0086] Comparing the data from Example 1 and Experimental Group 3, it can be seen that adding a conditioning solution prepared by mixing calcium alginate and aloe vera diluted solution at a volume ratio of 1:2.5 to the germination medium has a positive effect on seed germination. Comparing the data from Example 1 and Experimental Groups 1 and 2, adding the conditioning solution at a mass ratio of 20 ≤ conditioning solution to NAA ≤ 40 has a positive effect on the germination and growth of the prepared seeds.

[0087] Example 3:

[0088] Rooting and strengthening seedlings of Dendrobium hainanense:

[0089] S1. The seedlings cultivated in Example 1 were transferred to the rooting and seedling strengthening medium to obtain the cultured seedlings. 1L of rooting and seedling strengthening medium consisted of 2.3g 1 / 2MS medium, 0.5mg NAA, 20g sucrose, 20mg mixed solution A, 100g banana homogenate, 7g agar and 2g activated carbon, with a pH of 5.8±0.2.

[0090] S2. Place the seedlings into culture bottles containing rooting and seedling strengthening culture medium, maintain an ambient humidity of 75%, and move them to a greenhouse at 18 degrees Celsius for 6 days. Then, open the lid and ventilate for 2.5 days at 25 degrees Celsius to obtain tissue culture seedlings.

[0091] S3. Take out the tissue culture seedlings, wash the culture medium and disinfect them. Place them in an acclimatization substrate made of sphagnum moss, coconut shell pellets and pine bark in a mass ratio of 2:1:1 and cultivate for 4.5 months. Fix the deadwood to the bonsai pot, fill it with sand, and plant moss and small ferns on the surface to form a moist micro-ecosystem. After pruning the rotten roots of the seedlings, disinfect them and spray them with a rooting promoter. Wrap the roots with wet sphagnum moss and fix the plant to the deadwood with fishing line and nail gun.

[0092] Example 4:

[0093] This embodiment refers to Embodiment 3, except that the composition ratio of the rooting and seedling strengthening culture medium is different; the other steps are the same. Specifically:

[0094] S1. The seedlings cultivated in Example 1 were transferred to the rooting and seedling strengthening medium to obtain the cultured seedlings. 1L of rooting and seedling strengthening medium consisted of 2.5g 1 / 2MS medium, 0.8mg NAA, 20g sucrose, 30mg mixed solution A, 110g banana homogenate, 7g agar and 2g activated carbon, with a pH of 5.8±0.2.

[0095] Comparative example:

[0096] This comparative example has 5 experimental groups, and the only adjustment is the composition ratio of the rooting and seedling strengthening culture medium. The other steps are the same as in Example 3, as shown in Table 3:

[0097] Table 3

[0098]

[0099] Detection:

[0100] Following the methods in Examples 3-4 and Experimental Groups 1-5, 20 spare seeds were sown in various rooting and seedling strengthening culture media. After 90 days of cultivation, the growth status and survival rate after transplanting to the acclimatization substrate were observed, and the average values ​​were taken, as shown in Table 4. Figure 4-6 As shown:

[0101] Table 4

[0102]

[0103] From the table above and Figure 4-6 As shown, Example 3 ( Figure 4 ) and Example 4 ( Figure 5 The seedlings cultivated in the above manner showed the best growth and had the highest survival rate after being transferred to the acclimatization substrate. (Observe the table above and...) Figure 6 Regarding the "seedling height" data, the seedlings in experimental group 5 grew better than those in example 3 (e.g., Figure 6As shown in Figure E), the root system was not growing well, affecting the survival rate after transplanting. Therefore, the mixed solution was directly replaced with aloe vera extract. Although the seedlings grew well, the root system was poor. In the "average root length" item, the data of experimental group 4 and the growth status were relatively better (e.g., Figure 6 As shown in D), the number of roots was not high, resulting in a 21.56% decrease in survival rate compared to Example 3. Therefore, although the root length increased after reducing the amount of fermentation mixture in the mixed solution, the number of roots decreased. Regarding the "number of roots," Experimental Group 3 performed relatively better (e.g., ...). Figure 6 As shown in C), its root length decreased by 9.23% compared to Example 3, and the survival rate decreased. Therefore, although increasing the concentration of aloe vera dilution in the mixture will increase the number of roots, the root length will decrease.

[0104] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for the conservation of Dendrobium officinale from Hainan, characterized in that, The conservation method includes the following steps: S1. Collect the pods of Dendrobium hainanense, disinfect them, and obtain seeds for later use; S2. Sow the prepared seeds in the germination medium, expose to light, and cultivate for 50-70 days to obtain seedlings; The germination medium formula is: 2.3-2.5 g / L 1 / 2 MS medium + 0.3-0.4 mg / L NAA + 30 g / L sucrose + 7 g / L agar + 6-16 mg / L preparation solution; The preparation solution is obtained by mixing calcium alginate and aloe vera dilution with a volume concentration of 10% at a mass ratio of 1:2-3. 20 ≤ mass ratio of preparation solution to NAA ≤ 40; S3. Transfer the seedlings to the rooting and seedling strengthening culture medium to obtain cultivated seedlings; The formula for the rooting and seedling strengthening medium is: 2.3-2.5 g / L 1 / 2 MS medium + 0.5-0.8 mg / L NAA + 20 g / L sucrose + 20-30 mg / L mixed solution + 100-110 g / L banana homogenate + 7 g / L agar + 2 g / L activated carbon; The specific preparation steps for the mixture are as follows: S3-1. Wash the orange peel, rehydrate it, dry it, pulp it, and pass it through a 60-mesh sieve to obtain the pulp. S3-2. Inoculate Bacillus subtilis into the slurry, with a mass ratio of slurry to Bacillus subtilis of 100:3-5. Ferment for 30-40 hours, then sterilize by passing through a 0.22μm filter membrane to obtain the fermentation mixture. S3-3. Add 5% (v / v) aloe vera dilution to the fermentation mixture. The volume ratio of the fermentation mixture to the aloe vera dilution is 1.5-2:1 to obtain the mixed solution. S4. Place the seedlings in culture bottles, move them to a greenhouse for 5-7 days, and open the lid for ventilation for 2-3 days to obtain tissue culture seedlings; S5. Take out the tissue culture seedlings, disinfect them, put them into the acclimatization substrate, and cultivate them for 3-6 months. Then, attach the seedlings to the dead wood for planting, spray fertilizer, and realize biomimetic cultivation.

2. The conservation method according to claim 1, characterized in that, The Dendrobium pods collected in step S1 are specifically mature but unopened pods. The disinfection process involves disinfecting the surface of the Dendrobium pods with a 75% ethanol solution for 20-40 seconds, then soaking the pods in a 0.1% mercuric chloride solution for 10-20 minutes, and finally rinsing them with sterile water 3-5 times.

3. The conservation method according to claim 1, characterized in that, The aloe vera dilution in steps S2 and S3-3 is obtained by diluting the original aloe vera solution. The aloe vera extract is prepared according to the following steps: after washing and drying the aloe vera, it is crushed, passed through an 80-mesh sieve, 3-7 times the amount of water is added, stirred for 30-60 minutes, soaked at 32-38℃ for 20-28 hours, and the residue is removed to obtain the aloe vera extract.

4. The conservation method according to claim 1, characterized in that, In step S2, the light conditions are an illuminance of 1000-2000 lux and a photoperiod of 12-14 h / day, and a culture temperature of 23-27℃.

5. The conservation method according to claim 1, characterized in that, In step S5, the acclimatization substrate is prepared by mixing sphagnum moss, coconut husk granules, and pine bark in a mass ratio of 1-3:1:1, with a humidity of 70-80%.

6. The conservation method according to claim 1, characterized in that, The specific operation of attaching the seedling to the dead wood in step S5 is as follows: fix the dead wood to the bonsai pot, fill it with sand, plant moss and small ferns on the surface to form a moist micro-ecosystem, prune the rotten roots of the seedling, disinfect and spray rooting promoter, wrap the roots with wet sphagnum moss, and fix the plant to the dead wood with fishing line and nail gun.

7. The conservation method according to claim 1, characterized in that, The dropping rate in step S3-3 is 3 drops / s; The content of Bacillus subtilis in step S3-2 is 2×10⁻⁶. 10 CFU / g.

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