Orchid seed germination method based on enzyme treatment in capsule

Abstract

The present invention provides an orchid seed germination method based on enzyme treatment in capsules, including cleaning and disinfecting orchid capsules, preparing enzyme solution; then injecting the enzyme solution into the capsules for enzymolysis by using a syringe, replacing the culture solution to clean the capsules, and finally taking out the seeds in the capsules and inoculating and coating them on a culture medium for germination culture, so that the orchid seeds germinate. The germination method provided by the present invention has the characteristics of easy availability of raw materials, convenient operation and processing, high germination rate of orchid seeds, short germination time, etc., and can achieve rapid germination of orchid seeds.

Description

Technical Field

[0001] The invention belongs to the technical field of orchid propagation, relates to orchid seed germination, and specifically relates to an orchid seed germination method based on enzyme treatment in capsules. Background Art

[0002] Orchids are traditional Chinese famous flowers, and my country has a history of cultivating orchids for more than 2,000 years. Orchids are often propagated by division, sowing and tissue culture. Although there are tens of thousands to millions of seeds in an orchid capsule, orchid seeds are very small, with only incompletely developed embryonic cells and no endosperm, and their germination ability is extremely low. In addition, the seed coat is not easy to absorb water, and it is not easy to germinate by conventional sowing methods. In nature, the germination of orchid seeds generally relies on mycorrhizal fungi to provide nutrients for them, and the germination time is very long. At present, the traditional method of orchid reproduction is still traditional division reproduction, which is small in number, long in time, and has a low germination rate, and cannot achieve large-scale production. With the continuous growth of orchid market demand, the traditional method of orchid reproduction can no longer meet the needs of large-scale industrial development. Summary of the invention

[0003] The invention provides an orchid seed germination method based on enzyme treatment in capsules, which can conveniently and efficiently promote the germination of orchid seeds.

[0004] The technical solution of the present invention is a method for germinating orchid seeds based on enzyme treatment in capsules, characterized in that it comprises the following steps:

[0005] S1. Clean and disinfect the surface of the orchid capsule, dry it in the air and set it aside; prepare enzyme solution for later use;

[0006] S2, connecting a first needle, a first needle filter and a first syringe at one end of the capsule stem node in sequence, and drawing the enzyme solution prepared in S1 into the first syringe; connecting a second needle and a second needle filter at the other end of the capsule column in sequence, pushing the enzyme solution into the capsule through the first syringe, and stopping when liquid flows out of the second needle filter; placing the capsule and the connected structure in an environment of 45°C-55°C for enzymolysis reaction for 4-6 hours;

[0007] S3, draw 20-30mL of liquid MS culture medium with a second syringe, replace the first syringe at the first needle, and inject the MS culture medium in the second syringe into the orchid capsule until 10-12ml of MS culture medium flows out of the second needle filter; place the capsule and its connected structure in a dark culture at 25°C-28°C for 5-7 days;

[0008] S4, removing the connecting structures at both ends of the capsule, taking out the seeds in the capsule, inoculating and spreading them on a culture medium for germination culture, so that the orchid seeds germinate.

[0009] Furthermore, when cleaning the surface of the orchid capsule in S1, sterile water was used to shake and then fished out, and when disinfecting, 75% alcohol was used to scrub the capsule skin multiple times.

[0010] Furthermore, the enzyme solution in S1 is one or more of lignin peroxidase, cellulase, neutral protease, and saccharifying enzyme; water is added to form a solution with a mass concentration of 0.05%-0.08%.

[0011] Furthermore, the mass ratio of lignin peroxidase, cellulase, protease and saccharifying enzyme in the enzyme solution is 1-2:1-2:1:1.

[0012] Furthermore, the pore size of the first syringe filter and the second syringe filter in S2 is 0.25 μm.

[0013] Furthermore, during the enzymatic hydrolysis reaction in S2, the capsule and the connected structure are placed in a culture dish, and the culture dish is placed in an incubator for enzymatic hydrolysis; 0.5-1 mL of the enzyme solution in the first syringe is pushed out every 30 minutes.

[0014] Furthermore, during dark culture in S3, 1 to 2 mL of the MS culture medium in the second syringe was pushed out every other day.

[0015] Furthermore, the culture medium in S4 is MS culture medium containing 8-10 wt % coconut milk.

[0016] Furthermore, the culture conditions in S4 are 25°C to 28°C, and the fluorescent lighting intensity is 1500lx for 10 to 12 hours per day.

[0017] Furthermore, the method is performed in an aseptic manner, and the instruments, water and culture medium used are all aseptically treated.

[0018] The present invention has the following beneficial effects:

[0019] 1. According to the composition of orchid seed coat, the composite enzyme treatment has strong specificity and short action time. It can quickly and effectively remove the seed coat around the embryo without damaging the orchid embryo. It is safe and efficient.

[0020] 2. Enzyme treatment in orchid capsules can effectively avoid contamination by bacteria in the environment and is more beneficial to the germination of orchid seeds;

[0021] 3. Enzyme treatment in orchid capsules combined with short-term dark culture of seeds in capsules can better simulate the effects of symbiotic bacteria in orchid seed germination in the natural environment, which is beneficial to orchid seed germination;

[0022] 4. The structure of using orchid capsule to connect needle, needle filter and syringe for enzyme liquid injection and cleaning is convenient for processing flow and operation, and the connecting needle, double needle filter and syringe are all aseptically treated. The complex enzyme liquid is also filtered and sterilized after passing through the needle filter, avoiding contamination by miscellaneous bacteria.

[0023] 5. By carrying out enzyme treatment in orchid capsules, the wall breaking rate of orchid seeds can reach more than 90%, the germination rate of orchid seeds can reach more than 60%, the germination time can be shortened by 1 / 5-1 / 4, and the germination effect is good. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 These are photos of enzyme treatment inside orchid capsules.

[0025] Figure 2 This is a photo of orchid seeds that have not been treated with enzymes (magnification 30X).

[0026] Figure 3 Example 3 Photograph of orchid seeds after enzyme treatment (magnification 30X).

[0027] Figure 4 This is a photo of orchid seeds on MRS medium.

[0028] Figure 5 This is a photo of orchid seeds on MRS+ coconut milk medium.

[0029] Figure 6 This is a photo of the seeds after germination in Example 2. DETAILED DESCRIPTION

[0030] The embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention.

[0031] Embodiment 1:

[0032] Take several 80% mature and complete orchid capsules and put them into 3 culture bottles filled with sterile water in turn. Shake them thoroughly and then take them out. Then wipe the capsule skin with 75% alcohol 3 times and dry them for later use.

[0033] According to the mass ratio of lignin peroxidase, cellulase, neutral protease and saccharifying enzyme of 2:2:1:1, a composite enzyme solution with a mass percentage concentration of 0.05% is prepared with purified water, and then 10 ml of the composite enzyme solution is sucked into the first syringe, and then a 0.25 μm first syringe filter is connected to the nipple of the first syringe in sequence, and the other end of the first syringe filter is connected to the first needle; the first needle connected to the first syringe is inserted into the orchid capsule from one end of the stem node, and at the same time, another second needle connected to a 0.25 μm second syringe filter at the needle plug is inserted into the pistil at the other end of the orchid capsule. The connection structure can be seen in Figure 1 Slowly inject the compound enzyme solution from the first syringe into the orchid capsule, and stop injecting the compound enzyme solution when liquid flows out of the second syringe filter at the column end; put the orchid capsule connected with the first syringe and the second needle into a culture dish, cover the dish, and put the culture dish into a 45°C incubator for 4 hours of enzymatic hydrolysis of the orchid seed pod, wherein about 0.5 ml of the enzyme solution in syringe 1 is pushed out every 30 minutes.

[0034] Orchid capsules were taken before and after the above treatments, the capsule skin was cut open with a blade, orchid seeds were picked up with tweezers and placed on a glass slide, and toothpicks were used to evenly spread on the glass slide, and then observed under a stereo microscope and photographed to obtain Figure 2 and Figure 3 In the micrographs, it can be seen Figure 3 The enzyme-treated seeds have had their seed coats detached.

[0035] Use a second syringe to draw 30 ml of liquid MS culture medium, replace the first syringe at the first needle, and connect the second syringe, then slowly inject the MS culture medium in the second syringe into the orchid capsule until 10 ml of MS culture medium flows out of the second syringe filter.

[0036] Put the cleaned orchid capsules together with the second syringe and the second needle into a culture dish, cover the dish, and place the culture dish in a 26°C incubator for dark culture for 5 days, wherein about 2 ml of MS culture medium in the second syringe is pushed out every other day.

[0037] The second syringe and the second needle connected to the orchid capsule after dark culture were removed, and the seeds in the orchid capsule were taken out by conventional methods and inoculated and coated on MS medium containing 10% coconut milk for culture (such as Figure 5 ), and a blank control (MS medium direct culture, such as Figure 4 ), the culture conditions were 28°C, and the fluorescent lighting intensity was about 1500lx for 10 hours per day.

[0038] All instruments, purified water and culture medium used must be sterile, and all operations must be performed aseptically.

[0039] In this embodiment, the orchid seeds cultured in the MS medium containing 10% coconut milk had a wall-breaking rate of 87.2%, a germination rate of 65.5%, and germinated after 158 days of culture. The orchid seeds cultured directly in the MS medium had a wall-breaking rate of 86.5%, a germination rate of 35.7%, and germinated after 168 days of culture.

[0040] Embodiment 2:

[0041] Take 80% mature and complete orchid capsules and put them into 3 culture bottles filled with sterile water in turn, shake them thoroughly, then take them out, wipe the capsule skin with 75% alcohol 3 times, dry them and set aside.

[0042] According to the mass ratio of lignin peroxidase, cellulase, neutral protease and saccharifying enzyme of 2:2:1:1, a composite enzyme solution with a mass percentage concentration of 0.08% is prepared with purified water, and then 10 ml of the composite enzyme solution is drawn into a syringe 1, and then a 0.25 μm first syringe filter is connected to the nipple of the first syringe in sequence, and the other end of the first syringe filter is connected to the first needle;

[0043] The first needle connected to the first syringe is inserted into the orchid capsule from one end of the stem node, and at the same time, a second needle connected to a 0.25 μm second syringe filter is inserted into the column at the other end of the orchid capsule; then the complex enzyme solution is slowly injected into the orchid capsule from the first syringe, and the injection of the complex enzyme solution is stopped when liquid flows out of the second syringe filter at the column end; the orchid capsule connected to the first syringe and the second needle is placed in a culture dish, the dish cover is closed, and the culture dish is placed in an incubator at 50°C for 5 hours of enzymatic hydrolysis of the orchid seed pod, wherein about 1 ml of the enzyme solution in the syringe 1 is pushed out every 30 minutes.

[0044] Use a second syringe to draw 20 ml of liquid MS culture medium, replace the first syringe at the first needle, and connect the second syringe, then slowly inject the MS culture medium in the second syringe into the orchid capsule until all 12 ml of MS culture medium flows out of the second syringe filter.

[0045] Put the cleaned orchid capsules together with the second syringe and the second needle into a culture dish, cover the dish, and place the culture dish in a 25°C incubator for dark culture for 7 days, wherein about 1 ml of the MS culture medium in the second syringe is pushed out every day.

[0046] The second syringe and the second needle connected to the orchid capsule after dark culture are removed, and the seeds in the orchid capsule are taken out by conventional methods and inoculated and spread on MS medium containing 10% coconut milk for culture. The culture conditions are 25°C and the fluorescent lighting intensity is about 1500lx for 12 hours per day.

[0047] All instruments, purified water and culture medium used must be sterile, and all operations must be performed aseptically.

[0048] In this embodiment, the orchid seed wall breaking rate can reach 93.3%, and the orchid seed germination rate can reach 66.2%. The germination takes 161 days to complete. Figure 6 shown.

[0049] Embodiment 3:

[0050] Take 70% mature and complete orchid capsules and place them in 3 culture bottles filled with sterile water in turn. Shake them thoroughly and then take them out. Then wipe the capsule skin with 75% alcohol 3 times and dry them for later use.

[0051] According to the mass ratio of lignin peroxidase, cellulase, neutral protease and saccharifying enzyme of 2:2:1:1, a composite enzyme solution with a mass percentage concentration of 0.06% is prepared with purified water, and then 10 ml of the composite enzyme solution is drawn into a syringe 1, and then a 0.25 μm first syringe filter is connected to the nipple of the first syringe in sequence, and the other end of the first syringe filter is connected to the first needle;

[0052] The first needle connected to the first syringe is inserted into the orchid capsule from one end of the stem node, and at the same time, a second needle connected to a 0.25 μm second syringe filter is inserted into the column at the other end of the orchid capsule; then the complex enzyme solution is slowly injected into the orchid capsule from the first syringe, and the injection of the complex enzyme solution is stopped when liquid flows out of the second syringe filter at the column end; the orchid capsule connected to the first syringe and the second needle is placed in a culture dish, the dish cover is closed, and the culture dish is placed in an incubator at 55°C for 4 hours of enzymatic hydrolysis of the orchid seed pod, wherein about 0.5 ml of the enzyme solution in syringe 1 is pushed out every 30 minutes.

[0053] Use a second syringe to draw 20 ml of liquid MS culture medium, replace the first syringe at the first needle, and connect the second syringe, then slowly inject the MS culture medium in the second syringe into the orchid capsule until all the MS culture medium flows out of the second syringe filter by 10.

[0054] Put the cleaned orchid capsules together with the second syringe and the second needle into a culture dish, cover the dish, and place the culture dish in a 27°C incubator for dark culture for 6 days, wherein about 1.5 ml of MS culture medium in the second syringe is pushed out every day.

[0055] The second syringe and the second needle connected to the orchid capsule after dark culture are removed, and the seeds in the orchid capsule are taken out by conventional methods and inoculated and spread on MS medium containing 10% coconut milk for culture. The culture conditions are 27°C and the fluorescent lighting intensity is about 1500lx for 10 hours per day.

[0056] All instruments, purified water and culture medium used must be sterile, and all operations must be performed aseptically.

[0057] In this embodiment, the wall breaking rate of orchid seeds can reach 95.3%, the germination rate of orchid seeds can reach 64.2%, and they germinate after 171 days of cultivation.

[0058] Embodiment 4:

[0059] Take 70% mature and complete orchid capsules and place them in 3 culture bottles filled with sterile water in turn. Shake them thoroughly and then take them out. Then wipe the capsule skin with 75% alcohol 3 times and dry them for later use.

[0060] According to the mass ratio of lignin peroxidase, cellulase, neutral protease and saccharifying enzyme of 1:1:1:1, pure water is used to prepare a composite enzyme solution with a mass percentage concentration of 0.06%, and then 10 ml of the composite enzyme solution is drawn into syringe 1, and then a 0.25 μm first syringe filter is connected to the nipple of the first syringe in sequence, and the other end of the first syringe filter is connected to the first needle;

[0061] The first needle connected to the first syringe is inserted into the orchid capsule from one end of the stem node, and at the same time, a second needle connected to a 0.25 μm second syringe filter is inserted into the column at the other end of the orchid capsule; then the complex enzyme solution is slowly injected into the orchid capsule from the first syringe, and the injection of the complex enzyme solution is stopped when liquid flows out of the second syringe filter at the column end; the orchid capsule connected to the first syringe and the second needle is placed in a culture dish, the dish cover is closed, and the culture dish is placed in an incubator at 55°C for 6 hours of enzymatic hydrolysis of the orchid seed pod, wherein about 0.8 ml of the enzyme solution in the syringe 1 is pushed out every 30 minutes.

[0062] Use a second syringe to draw 25 ml of liquid MS culture medium, replace the first syringe at the first needle, and connect the second syringe, then slowly inject the MS culture medium in the second syringe into the orchid capsule until all 11 ml of MS culture medium flows out of the second syringe filter.

[0063] Put the cleaned orchid capsules together with the second syringe and the second needle into a culture dish, cover the dish, and place the culture dish in a 28°C incubator for dark culture for 6 days, wherein about 2 ml of MS culture medium in the second syringe is pushed out every other day.

[0064] The second syringe and the second needle connected to the orchid capsule after dark culture were removed, and the seeds in the orchid capsule were taken out by conventional methods and inoculated and spread on MS medium containing 10% coconut milk for culture. The culture conditions were 26°C and the fluorescent lamp illumination intensity was about 1500lx for 11 hours per day.

[0065] All instruments, purified water and culture medium used must be sterile, and all operations must be performed aseptically.

[0066] In this embodiment, the wall breaking rate of orchid seeds can reach 83.1%, the germination rate of orchid seeds can reach 51.4%, and they germinate after 164 days of cultivation.

[0067] Embodiment 5:

[0068] Take 70% mature and complete orchid capsules and place them in 3 culture bottles filled with sterile water in turn. Shake them thoroughly and then take them out. Then wipe the capsule skin with 75% alcohol 3 times and dry them for later use.

[0069] Cellulase, neutral protease and saccharifying enzyme are mixed in a mass ratio of 1:1:1 with purified water to prepare a composite enzyme solution with a mass percentage concentration of 0.06%, and then 10 ml of the composite enzyme solution is drawn into syringe 1, and then a 0.25 μm first syringe filter is connected to the nipple of the first syringe in sequence, and the other end of the first syringe filter is connected to the first needle;

[0070] The first needle connected to the first syringe is inserted into the orchid capsule from one end of the stem node, and at the same time, a second needle connected to a 0.25 μm second syringe filter is inserted into the column at the other end of the orchid capsule; then the complex enzyme solution is slowly injected into the orchid capsule from the first syringe, and the injection of the complex enzyme solution is stopped when liquid flows out of the second syringe filter at the column end; the orchid capsule connected to the first syringe and the second needle is placed in a culture dish, the dish cover is closed, and the culture dish is placed in an incubator at 55°C for 6 hours of enzymatic hydrolysis of the orchid seed pod, wherein about 0.5 ml of the enzyme solution in the syringe 1 is pushed out every 30 minutes.

[0071] Use a second syringe to draw 20 ml of liquid MS culture medium, replace the first syringe at the first needle, and connect the second syringe, then slowly inject the MS culture medium in the second syringe into the orchid capsule until all the MS culture medium flows out of the second syringe filter by 10.

[0072] Put the cleaned orchid capsules together with the second syringe and the second needle into a culture dish, cover the dish, and place the culture dish in a 27°C incubator for dark culture for 6 days, wherein about 1.5 ml of MS culture medium in the second syringe is pushed out every day.

[0073] The second syringe and the second needle connected to the orchid capsule after dark culture are removed, and the seeds in the orchid capsule are taken out by conventional methods and inoculated and spread on MS medium containing 10% coconut milk for culture. The culture conditions are 27°C and the fluorescent lighting intensity is about 1500lx for 10 hours per day.

[0074] All instruments, purified water and culture medium used must be sterile, and all operations must be performed aseptically.

[0075] In this embodiment, the wall breaking rate of orchid seeds can reach 47.6%, the germination rate of orchid seeds can reach 23.1%, and they germinate after 171 days of cultivation.

[0076] Comparative Example 1:

[0077] Take an orchid capsule that is 80% mature and complete, put it into 3 culture bottles filled with sterile water in a clean workbench, shake it thoroughly, then take it out, wipe the capsule skin with 75% alcohol 3 times, dry it and set it aside.

[0078] According to the mass ratio of lignin peroxidase, cellulase, neutral protease and saccharifying enzyme of 2:2:1:1, pure water was used to prepare a 0.08% compound enzyme solution. In the ultra-clean workbench, 50 ml of the compound enzyme solution was drawn with a sterile syringe, and a 0.25 μm sterile syringe filter was connected to the needle plug to filter and sterilize the compound enzyme solution. Then, the compound enzyme solution was placed in a 100 ml sterile culture dish, and then the orchid capsule was taken, the capsule skin was cut with a blade, and several orchid seeds were taken with tweezers. Place the orchid seeds in the above-mentioned culture dish, cover the dish with a lid, and place the culture dish in a 50°C incubator for 5 hours of enzymatic hydrolysis of the orchid seeds. After the hydrolysis is completed, take them out of the incubator, filter out the enzyme solution with sterile filter paper in an ultra-clean workbench, and repeatedly wash the seeds on the filter paper with sterile liquid MS culture medium. Then transplant the washed seeds to MS culture medium containing 10% coconut milk for culture. The culture conditions are 25°C and the light intensity of fluorescent lighting is about 1500lx for 12 hours a day.

[0079] All instruments, purified water, and culture media used must be sterile, and all operations must be aseptic and performed with the aid of an ultra-clean workbench.

[0080] Due to the large number of operation steps, in this case, some seeds adhered to the filter paper and were lost during the washing and filtering process.

[0081] In this case, the orchid seed wall breaking rate can reach 93.1%, and the orchid seed germination rate can reach 67.2%, and it will germinate after 160 days of cultivation.

[0082] The method provided by the present invention can simplify the requirements for the treatment process and the treatment environment, shorten the seed germination time, and achieve a good germination effect.

[0083] The above embodiments are only for illustrating the technical ideas and features of the present invention, and the contents are only preferred embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Within the technical scope disclosed by the present invention, equivalent changes or improvements made according to the technical solution and inventive concept of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for germinating orchid seeds based on enzyme treatment in capsules, characterized in that: The following steps are involved: S1. Clean and disinfect the surface of the orchid capsule, dry it and set it aside; prepare enzyme solution for later use, wherein the mass ratio of lignin peroxidase, cellulase, protease and saccharifying enzyme in the enzyme solution is 1-2:1-2:1:1, and add water to make a solution with a mass concentration of 0.05%-0.08%; S2, connecting a first needle, a first needle filter and a first syringe at one end of the capsule stem node in sequence, and drawing the enzyme solution prepared in S1 into the first syringe; connecting a second needle and a second needle filter at the other end of the capsule column in sequence, pushing the enzyme solution into the capsule through the first syringe, and stopping when liquid flows out of the second needle filter; placing the capsule and the connected structure in an environment of 45°C-55°C for enzymolysis reaction for 4-6 hours; S3, with the second syringe draw liquid MS medium 20-30mL, MS medium contains 8-10wt% coconut milk, replace the first syringe at the first needle, the MS medium in the second syringe is injected into the orchid capsule, until the MS medium flows out 10-12ml from the second syringe filter; The capsule and the connected structure are placed in 25 ℃-28 ℃ environment and dark cultured for 5-7 days; S4, removing the connecting structures at both ends of the capsule, taking out the seeds in the capsule, inoculating and spreading them on a culture medium for germination culture, so that the orchid seeds germinate.

2. The method according to claim 1, characterized in that: When cleaning the surface of orchid capsules in S1, sterile water was used to shake and then fished out. When disinfecting, 75% alcohol was used to wipe the capsule surface several times.

3. The method according to claim 1, characterized in that: The pore size of the first syringe filter and the second syringe filter in S2 is 0.25 μm.

4. The method according to claim 1, characterized in that: During the enzymatic hydrolysis reaction in S2, the capsule and its connected structure are placed in a culture dish, and the culture dish is placed in an incubator for enzymatic hydrolysis; 0.5-1 mL of the enzyme solution in the first syringe is pushed out every 30 minutes.

5. The method according to claim 1, characterized in that: During dark culture in S3, 1-2 mL of MS medium was pushed out of the second syringe every other day.

6. The method according to claim 1, characterized in that: The culture conditions in S4 are 25°C-28°C, fluorescent lighting intensity 1500lx, 10-12h light per day.

7. The method according to any one of claims 1 to 6, characterized in that: The operation process of this method is all aseptic operation, and the instruments, water and culture medium used are all sterile.

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