Method for improving sterile sowing propagation efficiency of cymbidium faberi

Abstract

The invention provides a method for sterile sowing and efficient propagation of cymbidium faberi. The method comprises the following steps: pretreatment of cymbidium faberi seeds, sterile germination of the cymbidium faberi seeds, protocorm proliferation, differentiation, seedling strengthening and acclimatization and transplantation. The pretreatment comprises the steps of refrigerating hybrid seeds of cymbidium orchid at 4 DEG C, performing secondary disinfection with sodium hypochlorite, performing dark culture for 10-30 days after sowing, and then performing light culture, and the germination culture medium is prepared by taking 1 / 4 MS as a basic culture medium and adding 100-200ml / L of coconut water, 0.5-1.0 mg / L of 6-benayl aminopurine, 0.1-0.5 mg / L of naphthylacetic acid, 0.5-2.0 g / L of peptone and 0.5-1.0 g / L of activated carbon. According to the method, the seed germination rate can be effectively increased, the 60d seed germination rate reaches up to 85%, the pollution rate is reduced to 5% or below, 50.0 g / L inositol, 2.0 mg / L glycine and 2.0 mg / L nicotinic acid are added into a differentiation culture formula, the differentiation rate can be effectively increased, the leaf bud differentiation rate reaches up to 90%, meanwhile, rooting can be induced, the growth time of cymbidium faberi from seeds to seedlings is greatly shortened, a large number of high-quality seedlings can be obtained within a short time, and the method is suitable for large-scale popularization and application. Technical data are accumulated for cymbidium resource protection and new variety cultivation.

Description

Technical Field

[0001] The invention belongs to the field of plant biotechnology, and in particular relates to a method for improving the efficiency of aseptic sowing and propagation of cymbidium orchids. Background Art

[0002] Cymbidiums faberi is a precious potted ornamental flower of the orchid family. It has a slender and graceful plant, soft and firm leaves, a faint and distant fragrance, and a beautiful and unique flower shape. It is known as the "gentleman among flowers" and is one of the orchids with the longest cultivation history and the most popular in my country. There are many famous Cymbidium orchids handed down from generation to generation. The appreciation aesthetics of petal shape, exotic flowers, colorful flowers and leaf art has been continuously developed, becoming an important spiritual symbol in Chinese culture. The main method of propagation of Cymbidium orchids is currently division, but the propagation cycle is long and can only be carried out once every 3-4 years, and the propagation efficiency is low. Cymbidium orchid seeds are extremely small, have no endosperm, and the seeds mature later. They can only germinate in symbiosis with fungi under natural suitable conditions, and the germination rate is extremely low.

[0003] So far, there have been successful examples of applying sterile sowing tissue culture technology to Cymbidium, such as the application (patent) number "200910101739.0" and the name "A culture medium composition suitable for aseptic germination of Cymbidium seeds and method thereof", the application (patent) number "201610648641.7" and the name "A method for rapid germination of Cymbidium seeds", the application (patent) number "201711051075.7" and the name "A method for aseptic germination of Cymbidium seeds", etc. In comparison, the present invention greatly shortens the germination time and significantly improves the germination rate of Cymbidium seeds. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a method for improving the efficiency of aseptic sowing and propagation of Cymbidium orchids.

[0005] The present invention provides a method for efficient propagation of cymbidium orchid by aseptic sowing by means of hybrid breeding, cold treatment, secondary disinfection with sodium hypochlorite and dark treatment of cymbidium orchid seeds, which can effectively shorten the germination period and improve the germination rate of cymbidium orchid seeds, and provides a certain theoretical basis for the selection and breeding of new varieties of cymbidium orchids in the later stage.

[0006] First, the present invention provides a method for improving the efficiency of aseptic sowing and propagation of Cymbidium orchids, comprising the following steps: sterilizing the surface of mature and unopened Cymbidium orchid capsules, taking out seeds by aseptic operation, aseptically refrigerating the seeds for 10-30 days, sterilizing the seeds with sodium hypochlorite after refrigeration, aseptically sowing the sterilized seeds on a germination medium, culturing them in the dark for 10-30 days, then switching to light culture, and germinating to obtain protocorms; the germination medium is: using 1 / 4MS as a basic medium, adding Coconut water 100-200 ml / L, naphthaleneacetic acid 0.1-0.5 mg / L, 6-benzylaminopurine 0.5-1.0 mg / L, peptone 0.5-2.0 g / L, activated carbon 0.5-1.0 g / L; wherein, the specific steps of treating the seeds with sodium hypochlorite are: disinfecting with 70%-75% alcohol for 30-60 seconds, rinsing with sterile water for 1-3 times, rinsing with 0.1%-1% sodium hypochlorite solution for 10-30 minutes, and then rinsing with sterile water for 5-6 times.

[0007] The capsule is a capsule that grows fully 120-150 days after pollination.

[0008] In a specific embodiment of the present invention, the specific steps of surface sterilization of capsules include: brushing dust off the surface of the capsules with a toothbrush dipped in soapy water, and rinsing under running water for 1-3 hours; rinsing with 70%-75% alcohol for 3-5 minutes, then rinsing with sterile water 1-3 times, transferring to 0.1% mercuric chloride solution for disinfection for 10-15 minutes, shaking continuously during the process to ensure sufficient disinfection, and finally rinsing with sterile water 7-8 times, placing on sterile filter paper to absorb excess moisture on the surface.

[0009] Among them, the working concentration of sodium hypochlorite is 1% and the processing time is 30 minutes.

[0010] Among them, the light culture conditions are: culture temperature 25℃-28℃, light intensity 1200-1500Lux, 12-14h light / 8-12h dark.

[0011] The seeds were refrigerated at 4°C for 30 days.

[0012] The present invention also provides a method for efficient propagation of Cymbidium orchid by aseptic sowing, characterized in that it comprises the following steps:

[0013] 1) Aseptic sowing: Obtaining protocorms according to the method for improving the efficiency of aseptic sowing and propagation of Cymbidium orchids;

[0014] 2) Protocorm proliferation culture: inoculating the protocorm obtained in step 1) into a proliferation medium for proliferation culture, wherein the proliferation medium is: 1 / 4MS as a basic medium, supplemented with 100-200 ml / L of coconut water, 0.1-0.5 mg / L of naphthaleneacetic acid, 0.5-2.0 g / L of peptone, and 0.5-1.0 g / L of activated carbon;

[0015] 3) Protocorm differentiation culture: The protocorms proliferated in step 2) are inoculated into a differentiation medium for differentiation culture to obtain seedlings, wherein the differentiation medium is: 1 / 4MS as a basic medium, supplemented with 0.2-2 mg / L of 6-benzylaminopurine, 0.4-4 mg / L of naphthaleneacetic acid, 100-200 mg / L of coconut water, 0.5-2.0 g / L of peptone, 50 mg / L of inositol, 2 mg / L of glycine, and 2 mg / L of nicotinic acid.

[0016] Wherein, the method for efficient propagation of Cymbidium orchid by aseptic sowing may optionally further include:

[0017] 4) seedling cultivation: inoculating the seedlings obtained in step 3) onto a seedling cultivation medium for seedling cultivation to obtain complete plants; the seedling cultivation medium is: 1 / 4MS as a basic medium, supplemented with 0.2-2 mg / L of 6-benzylaminopurine, 0.2-4 mg / L of naphthaleneacetic acid, 0.6-1.0 mg / L of indolebutyric acid, 100-200 ml / L of coconut water, 0.5-2.0 g / L of peptone, 0.5-1.0 g / L of activated carbon, 50 mg / L of inositol, 2 mg / L of glycine, and 2 mg / L of nicotinic acid;

[0018] 5) Transplantation of test tube seedlings: The bottle seedlings obtained in step 4) are transferred to natural light for hardening for 7-14 days, then the seedlings are taken out, the culture medium on the surface is washed off, and the seedlings are transplanted to a cultivation medium containing coconut bricks, volcanic rocks, peat soil and pine bark for cultivation.

[0019] Among them, the mixing ratio of coconut bricks, volcanic stones, peat soil and pine bark is 1:1:3:1 by volume.

[0020] The present invention has the following beneficial effects:

[0021] The present invention obtains seeds with high vitality through interspecific hybridization of cymbidium orchids, uses cymbidium orchid seeds of suitable maturity as explants, performs cold treatment on seeds, dark treatment, and secondary disinfection with sodium hypochlorite can effectively improve the germination rate of cymbidium orchid seeds and shorten the germination time. The seed coat of cymbidium orchid seeds has an inhibitory effect on the germination of cymbidium orchid seeds to a certain extent. By using sodium hypochlorite to treat it, the seed coat of cymbidium orchid seeds can be effectively corroded, and its inhibitory effect can be reduced, thereby improving the germination rate, shortening the germination time of cymbidium orchid seeds breaking through the seed coat, and reducing the difficulty of seed germination. Cold treatment and dark cultivation of seeds simulate the natural conditions. After the cymbidium orchid capsule matures in autumn and cracks, the seeds fall into the soil and are underground for a long time, and the growth needs to experience a certain vernalization effect, which provides cymbidium orchid seeds with suitable living conditions similar to the natural environment, and can effectively promote its germination. After 60d of aseptic sowing, the seed germination rate is as high as 85%. After the seeds germinate, a complete plant is obtained through protocorm proliferation, differentiation and seedling culture. The differentiation culture formula adds 50.0 mg / L of inositol, 2.0 mg / L of glycine and 2.0 mg / L of nicotinic acid to effectively improve the differentiation rate. After 30 days of differentiation culture, the differentiation rate of protocorm leaf buds is as high as 90%, and a large number of high-quality Cymbidium seedlings can be obtained in a short time. The invention has the advantages of high seed germination rate, low contamination rate after sowing, short germination time, fast emergence and good seedling quality. The Cymbidium seedlings can meet market needs and are conducive to the protection, innovation, new variety cultivation and sustainable utilization of Cymbidium germplasm resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The results are shown for 60-day germination of Cymbidium orchid seeds. (A) Germination effect of Z-6; (B) Germination effect of Z-10 with 200 ml coconut water added; (C) Germination effect of Z-10-1 with 0.5 g / L peptone added; (D) Germination effect of Z-19; (E) Germination effect of Z-23 (control); (F) Germination effect of Z-20 with 1% sodium hypochlorite rinse for 30 min + dark culture for 30 days pretreatment.

[0023] Figure 2 Shown is the proliferation culture of Cymbidium protocorms. (A and B) Protocorm proliferation.

[0024] Figure 3 The following are the differentiation of Cymbidium protocorms. (A) Leaf buds grew out after 30 days of differentiation culture; (B) 60 days of differentiation culture.

[0025] Figure 4 Shown is the cultivation of strong seedlings of Cymbidium.

[0026] Figure 5 Shown are Cymbidium orchid seedlings. DETAILED DESCRIPTION

[0027] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the examples are based on conventional experimental conditions or the conditions recommended by the manufacturer's instructions.

[0028] A method for efficient propagation of Cymbidium orchid by aseptic sowing:

[0029] Step 1, artificial pollination and seed acquisition: Select the robust Cymbidium varieties 'Zheng Xiaohe' (♀) and 'Xin Jipin' (♂) (or ♀ 'Wenzhousu' × 'Jin Aosu' ♂) for artificial cross pollination, and select the plump and mature capsules that have grown 120-150 days after successful pollination as materials for sterile sowing;

[0030] Step 2: Explant disinfection and aseptic sowing: All the following operations must be performed in a clean bench. Disinfect the fruit pods that have been successfully pollinated for 120-150 days, cut the fruit pods, take out the powdered seeds and refrigerate them at 4℃ for 30 days. After 30 days, the seeds are rinsed with 1% sodium hypochlorite for 30 minutes, and then the seeds are inoculated on a seed germination medium for cultivation, and dark cultivation is carried out in a culture room at a temperature of 25° C.-28° C. for 30 days, and then transferred to a light culture environment with an illumination of 1200-1500 Lux (12-14 h light / 8-12 h dark) for cultivation for 30 days, and the seeds germinate to form protocorms. The seed germination medium is: 1 / 4MS basic culture medium, 100-200 ml / L of coconut water, 0.5-1.0 mg / L of 6-benzylaminopurine, 0.1-0.5 mg / L of naphthaleneacetic acid, 0.5 g / L of peptone, 1.0 g / L of activated carbon, 30.0 g / L of sucrose, 7.0 g / L of agar, and pH 5.8.

[0031] Step 3, protocorm proliferation medium: inoculate the protocorm onto a proliferation medium for cultivation at a temperature of 25°C-28°C and a light intensity of 1200-1500 Lux (12-14 h light / 8-12 h dark). The protocorm significantly increases in size and weight. The proliferation medium is 1 / 4MS basic medium supplemented with 200 ml / L coconut water, 0.1-0.5 mg / L naphthaleneacetic acid, 0.5 g / L peptone, 1.0 g / L activated carbon, 30.0 g / L sucrose, 6.0 g / L agar, and a pH of 5.8.

[0032] Step 4, protocorm differentiation culture: the protocorm in step 3 is inoculated into a differentiation medium for culture at a temperature of 25°C-28°C and a light intensity of 1500 Lux (12-14 h light / 8-12 h dark), and the protocorm is differentiated into leaf buds. The differentiation medium is: 1 / 4MS as a basic medium, with the addition of 200 ml / L coconut water, 0.2-2.0 mg / L 6-benzylaminopurine, 0.4-4.0 mg / L naphthaleneacetic acid, 50.0 mg / L inositol, 2.0 mg / L glycine, 2.0 mg / L nicotinic acid, 1.0 g / L peptone, 30.0 g / L sucrose, 6.0 g / L agar, pH 5.8;

[0033] Step 5, seedling cultivation: the seedlings obtained in step 4 are inoculated onto a seedling culture medium for cultivation at a temperature of 25°C-28°C and a light intensity of 3000 Lux (12-14 h light / 8-12 h dark), and complete plants are obtained after the cultivation is completed; the seedling culture medium is: 1 / 4MS basic culture medium, supplemented with 100-200 mg / L coconut water, 0.2-2.0 mg / L 6-benzylaminopurine, 0.2-4.0 mg / L naphthaleneacetic acid, 0.6-1.0 mg / L indolebutyric acid, 50.0 mg / L inositol, 2.0 mg / L glycine, 2.0 mg / L nicotinic acid, 1.0 g / L peptone, 1.0 g / L activated carbon, 40.0 g / L sucrose, 6.0 g / L agar, pH 5.8;

[0034] Step 6. Transplantation of test tube seedlings: Transfer the intact plants to natural light for 14 days of hardening, then take out the tissue culture seedlings, wash the culture medium on the surface, and transplant them into the cultivation medium containing coconut bricks, volcanic rocks, peat soil and pine bark.

[0035] Example 1

[0036] 1. Artificial pollination and seed acquisition: Select the robust Cymbidium varieties 'Zheng Xiaohe' (♀) and 'Xin Jipin' (♂) (or ♀ 'Wenzhousu' × 'Jinaosu' ♂) for artificial hybridization pollination. The pollination time is selected in the early morning or evening. Remove the lip of the flower to facilitate pollination. Use sterilized tweezers to remove the pollen block in the female parent's stamen column and insert it into the pistil cavity of the male parent's flower. Record the pollination time and parent information. The artificial pollination success rate of 'Zheng Xiaohe' and 'Wenzhousu' is 90%. Select the capsules that grow well 150 days after successful pollination as explants for sterile sowing;

[0037] 2. Explant disinfection and sterile sowing: After removing the pods, use a toothbrush dipped in soapy water to brush off the dust on the surface, rinse under running water for two hours, and rinse with sterile water once. Perform subsequent operations in a clean bench. First, rinse the pods with 75% alcohol for 5 minutes, rinse with clean water once, and transfer to 0.1% mercuric chloride solution for disinfection for 10 minutes. Shake continuously during the process to ensure sufficient sterilization. Then rinse with sterile water 7-8 times, place on sterile filter paper to absorb excess moisture on the surface. Use a sterile scalpel to cut along the edges and corners of the pods, use a sterile medicine spoon to scrape the powdered seeds into a sterile 5ml centrifuge tube, insert a sterile cotton ball to seal, record the time and variety information, put into a sterile tissue culture bottle with a breathable membrane, and put it in a 4℃ refrigerator for 30 days.

[0038] After 30 days, take out the Cymbidium seeds from the refrigerator and sow them aseptically in the clean bench. First take out the cotton ball, add 3-4 ml of 75% alcohol into the centrifuge tube, cover it tightly and shake it vigorously for sterilization for 30 seconds, then pour it into a sterile 5.5 cm × 7 cm non-woven bag, and seal the bag with a thin rope. Rinse it with sterile water once, transfer it to 1% sodium hypochlorite solution and rinse it for 30 minutes, then rinse it with sterile water 5-6 times, place it on sterile filter paper, absorb excess water on the bag, and use a sterile medicine spoon to evenly sow the seeds on the sterile germination medium.

[0039] Treatment 1: Culture formula screening, the basic culture medium is 1 / 2MS, 1 / 4MS, 1 / 6MS culture medium, 6-benzylaminopurine concentration gradient is set to 2.0mg / L, 1.0mg / L, 0.5mg / L, naphthaleneacetic acid concentration gradient is set to 0.1mg / L, 0.2mg / L, 0.5mg / L, orthogonal experiment, three factors and three levels, a total of 9 groups of experiments. General additives: sucrose 30.0g / L, agar powder 7.0g / L, activated carbon 1.0g / L, pH5.8. After sowing, first place it in a tissue culture room with a room temperature of 25℃ for dark culture for 30 days, and then transfer it to a tissue culture room with a room temperature of 25℃ and a light intensity of 1200Lux (14h light / 10h dark) for culture for 30 days. The seed germination rate was counted. The specific results are shown in Table 1. The best culture formula is Z-6, the germination rate can reach 20%, and the germination effect is as follows. Figure 1 As shown in A.

[0040] Table 1 Effects of different concentrations of inorganic salts and growth hormones on seed development

[0041]

[0042] Treatment 2: Screening of organic additives. Using Z-6 medium as the basal medium, the organic additives were treated as follows: Z-10: 200 ml / L coconut water, Z-11: 200 ml / L potato extract, Z-12: 200 g / L banana puree, Z-13: 100 ml / L potato extract + 100 ml / L coconut water, Z-14: 100 ml / L potato extract + 100 g / L banana puree, Z-15: 100 ml / L coconut water + 100 g / L banana puree, Z-16: no organic matter was added, general additives: 30.0 g / L sucrose, 7.0 g / L agar powder, 1.0 g / L activated carbon, pH 5.8. After sowing, the seeds were first placed in a tissue culture room at 25°C for dark culture for 30 days, and then transferred to a tissue culture room at 25°C with a light intensity of 1200 Lux (14 h light / 10 h dark) for culture for 30 days. After the culture was completed, the seed germination rate was counted. The specific results are shown in Table 2. Adding 200 ml / L coconut water had the best culture effect, with a germination rate of 75%. Figure 1 As shown in B.

[0043] Table 2 Effects of different organic additives on seed germination

[0044]

[0045]

[0046] Treatment 3: Screening of peptone additives, based on Z-10 medium, peptone concentration gradients of 0.5g / L, 1.0g / L, 2.0g / L, no peptone was added to the control group, general additives: 200ml / L coconut water, 30.0g / L sucrose, 7.0g / L agar powder, 1.0g / L activated carbon, pH 5.8. After sowing, the seeds were first placed in a tissue culture room at room temperature of 25°C for dark culture for 30 days, and then transferred to a tissue culture room at room temperature of 25°C with a light intensity of 1200Lux (14h light / 10h dark) for culture for 30 days. After the culture was completed, the seed germination rate was counted. The specific results are shown in Table 3. The optimum amount of peptone added was 0.5g / L, and the germination rate could reach 83%. The germination effect was as follows: Figure 1 As shown in C.

[0047] Table 3 Effects of peptone supplements on seed germination

[0048]

[0049] Treatment 4: Screening of different concentrations of inorganic salts, based on Z-10-1 medium, MS, 1 / 2MS, 1 / 4MS, 1 / 6MS medium as the basic medium, general additives: coconut water 200ml / L, sucrose 30.0g / L, agar powder 7.0g / L, activated carbon 1.0g / L, pH 5.8. After sowing, first put it into the tissue culture room at room temperature 25℃ for dark culture for 30 days, then transfer it to the tissue culture room at room temperature 25℃, light intensity of 1200Lux (14h light / 10h dark) for 30 days, and count the germination rate of Cymbidium orchid seeds. The specific results are shown in Table 4. 1 / 4MS is the best inorganic salt concentration, the germination rate can reach 85%, and the germination effect is as follows Figure 1 As shown in D.

[0050] Table 4 Effects of different concentrations of inorganic salts on seed germination

[0051]

[0052] Treatment 5: Different pretreatment screening, based on Z-19 medium, all seeds were not cold treated, and the following four treatments were performed: Z-20: 1% sodium hypochlorite rinse for 30 minutes + dark culture for 30 days, Z-21: 1% sodium hypochlorite rinse for 30 minutes + dark culture for 0 days, Z-22: 1% sodium hypochlorite rinse for 0 minutes + dark culture for 30 days, Z-23: 1% sodium hypochlorite rinse for 0 minutes + dark culture for 0 days. After sowing, the seeds were first placed in a tissue culture room at room temperature of 25°C for dark culture for 30 days, and then transferred to a tissue culture room at room temperature of 25°C with a light intensity of 1200Lux (14h light / 10h dark) for culture for 30 days. The germination rate of Cymbidium orchid seeds was counted, and the specific results are shown in Table 5. The germination effect of the untreated seeds is shown in Table 5. Figure 1 As shown in E, the germination rate can reach 75% after rinsing with 1% sodium hypochlorite for 30 minutes and dark culture for 30 days. Figure 1 F. The germination effect of Z-20 without cold treatment was lower than that of Z-19 after cold treatment for 30 days. Therefore, the most suitable pretreatment conditions for seed germination were: cold treatment for 30 days, rinsing with 1% sodium hypochlorite for 30 minutes and dark culture for 30 days. The most suitable germination medium was: 1 / 4MS + 6-BA 0.5mg / L + NAA 0.1mg / L + coconut water 200ml / L + peptone 0.5g / L + sucrose 30g / L + agar 7g / L + activated carbon 1g / L.

[0053] Table 5 Effects of different pretreatment conditions on seed germination

[0054] Serial number Processing Fastest germination time (d) 60d germination rate (%) Z-20 1% sodium hypochlorite rinse for 30 minutes + dark culture for 30 days 21 75 Z-21 1% sodium hypochlorite rinse for 30 min + dark culture for 0 d 32 20 Z-22 1% sodium hypochlorite rinse 0min + dark culture 30d 40 25 Z-23 1% sodium hypochlorite rinse 0min + dark culture 0d 50 10

[0055] 3. Proliferation culture medium: The germinated protocorms are transplanted into the protocorm proliferation culture medium for proliferation culture. All operations are completed in the clean bench. The proliferation culture medium is: 1 / 4MS basic culture medium, naphthaleneacetic acid concentration gradient is set at 0.1mg / L, 0.2mg / L, 0.3mg / L, 0.4mg / L, 0.5mg / L, general additives: coconut water 200ml / L, peptone 0.5g / L, sucrose 30.0g / L, agar powder 6.0g / L, activated carbon 1.0g / L, pH5.8; cultured at room temperature 25℃, light intensity 1200Lux (14h light / 10h dark) for 45 days. After the culture is completed, the protocorms are significantly enlarged and weighted. Figure 2 The specific proliferation coefficient is shown in Table 6. The most suitable proliferation medium formula is: 1 / 4MS + NAA 0.1mg / L + coconut water 200ml / L + peptone 0.5g / L + sucrose 30.0g / L + agar 6.0g / L + activated carbon 1.0g / L.

[0056] Table 6 Effects of different concentrations of growth hormone on the proliferation of protocorms

[0057] Serial number Culture medium NAA(mg / L) Proliferation coefficient Y-1 1 / 4MS 0.1 10 Y-2 1 / 4MS 0.2 6 Y-3 1 / 4MS 0.3 5 Y-4 1 / 4MS 0.4 5 Y-5 1 / 4MS 0.5 3

[0058] 4. Protocorm differentiation culture: The protocorms that have completed proliferation are transferred into the differentiation culture medium. All operations are completed in the clean bench. The differentiation medium uses 1 / 4MS as the basic medium, and 6 groups of growth hormone combinations are set up, F1: 6-benzylaminopurine 0.1mg / L + naphthaleneacetic acid 0.4mg / L, F2: 6-benzylaminopurine 0.5mg / L + naphthaleneacetic acid 2.0mg / L, F3: 6-benzylaminopurine 1.0mg / L + naphthaleneacetic acid 4.0mg / L, F4: 6-benzylaminopurine 2.0mg / L + naphthaleneacetic acid 8.0mg / L, F5: 6-benzylaminopurine 2.0mg / L + naphthaleneacetic acid 0.5mg / L, F6: 6-benzylaminopurine 2.0mg / L + naphthaleneacetic acid 4.0mg / L, general additives: coconut water 200ml / L, inositol 50.0mg / L, glycine 2.0mg / L, niacin 2.0mg / L, peptone 1.0g / L, sucrose 30.0g / L, agar 6.0g / L, pH 5.8. The cells were cultured at room temperature of 25°C and light intensity of 1500 Lux (14 h light / 10 h dark) for 60 days. Figure 3The results showed that the best differentiation effect was achieved when the growth hormone combination was 6-benzylaminopurine 0.5 mg / L + naphthaleneacetic acid 2.0 mg / L (Table 7). The most suitable differentiation medium formula was: 1 / 4MS + 6-BA 0.5 mg / L + NAA 2.0 mg / L + coconut water 200 ml / L + inositol 50.0 mg / L + glycine 2.0 mg / L + nicotinic acid 2.0 mg / L + peptone 1.0 g / L + sucrose 30.0 g / L + agar 6.0 g / L.

[0059] Table 7 Effects of different growth hormone ratios on protocorm differentiation

[0060] Serial number 6-ba(mg / L) NAA(mg / L) Differentiation rate (%) F1 0.1 0.4 60 F2 0.5 2 90 F3 1 4 50 F4 2 8 0 F5 2 0.5 50 F6 2 4 30

[0061] 5. Seedling culture: Transplant the seedlings that have completed differentiation culture into seedling culture medium for culture. All operations are completed in the clean bench. The seedling culture medium uses 1 / 4MS as the basic culture medium, and the growth hormone combination is set as follows: ZM1: 6-benzylaminopurine 2.0mg / L + naphthaleneacetic acid 0.4mg / L, ZM2: 6-benzylaminopurine 1.0mg / L + naphthaleneacetic acid 0.5mg / L, ZM3: 6-benzylaminopurine 2.0mg / L + naphthaleneacetic acid 1.2mg / L, ZM4: 6-benzylaminopurine 2.0mg / L + naphthaleneacetic acid 0.2mg / L, Z M5: 6-benzylaminopurine 0.5mg / L + naphthaleneacetic acid 4.0mg / L, ZM6: 6-benzylaminopurine 0.5mg / L + naphthaleneacetic acid 2.0mg / L, ZM7: 6-benzylaminopurine 0.2mg / L + naphthaleneacetic acid 2.0mg / L, ZM8: 6-benzylaminopurine 0.5mg / L + naphthaleneacetic acid 4.0mg / L, ZM9: 6-benzylaminopurine 0.2mg / L + naphthaleneacetic acid 4.0mg / L. General additives: coconut water 200ml / L, indolebutyric acid 0.8mg / L, inositol 50.0mg / L, glycine 2.0mg / L, niacin 2.0mg / L, peptone 1.0g / L, activated carbon 1.0g / L, sucrose 40.0g / L, agar 6.0g / L, pH 5.8. The culture was carried out at room temperature of 25°C and light intensity of 3000 Lux (14 h light / 10 h dark) for 60 days. Figure 4 ), and the rooting rate was counted (Table 8). The most suitable medium formula for seedling growth was: 1 / 4MS + 6-BA 2mg / L + NAA 0.2mg / L + IBA 0.8mg / L + coconut water 200ml / L + inositol 50.0mg / L + glycine 2.0mg / L + nicotinic acid 2.0mg / L + peptone 1.0g / L + activated carbon 1.0g / L + sucrose 40.0g / L + agar 6.0g / L.

[0062] Table 8 Effects of different hormone concentration ratios on seedling vigor

[0063]

[0064] 6. Transplantation of test tube seedlings: Place the obtained seedlings in a glass greenhouse covered with a single-layer shade net for seedling training. Take out the seedlings in the bottle after 14 days ( Figure 5 ), wash the culture medium attached to the material with clean water, and dry the surface moisture in a cool place. Prepare the transplanting culture medium for seedlings, mix coconut bricks, volcanic rocks, peat soil and pine bark in a ratio of 1:1:3:1, and use a permeable clay pot as a potting container for planting to obtain a large number of seedlings.

[0065] In summary, the present invention provides a method for efficient propagation of Cymbidium orchid by aseptic sowing, obtaining Cymbidium orchid seeds with high vitality through artificial pollination and interspecific hybridization, using sterile powdered Cymbidium orchid seeds as explants, and pre-treating the Cymbidium orchid seeds can effectively shorten the germination time and improve the germination rate. Through seed germination, proliferation, differentiation, and seedling cultivation, new plants can be grown, and a large number of high-quality seedlings can be obtained in a short time.

[0066] (1) This method is a technology for rapidly breeding Cymbidium orchid seedlings, which is specifically designed to address the low germination rate of Cymbidium orchid seeds under natural conditions and the long traditional division propagation cycle;

[0067] (2) The mature seeds are easy to collect. Each capsule contains tens of thousands of powdered seeds, which provides sufficient material and is easy to store for a long time.

[0068] (3) After pre-treating the Cymbidium orchid seeds with sodium hypochlorite, the seed coat of the Cymbidium orchid seeds can be effectively corroded, helping the Cymbidium orchid seeds to better break through the seed coat and germinate. At the same time, the sterilized non-woven bag is used as a container for seed sterilization, making seed sterilization more convenient. Sodium hypochlorite can also sterilize seeds very well, reducing the risk of contamination caused by explants during aseptic sowing. The seed germination rate of this method can reach 85%; it can also effectively reduce the contamination rate of aseptic sowing, and the leaf bud induction rate of differentiation culture is as high as 90%. High-quality Cymbidium orchid seedlings with a high survival rate can be obtained in a short time, effectively solving the problem of large-scale factory-based seedling cultivation of Cymbidium orchids, and accumulating technical information for the protection of wild Cymbidium orchid germplasm resources.

[0069] (4) The seeds obtained by hybridization in this method have high vitality, and there is a certain variability in the seed propagation method, which provides certain technical support for the selection and development of new hybrid varieties of Cymbidium orchids. The present invention has laid a good foundation for the genetic transformation, biotechnology research and application of this species, and has broad application prospects.

[0070] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A method for improving the efficiency of aseptic seeding and propagation of Cymbidium orchids, characterized in that: The method comprises the following steps: sterilizing the surface of mature unopened Cymbidium capsules, taking out seeds by aseptic operation, aseptically refrigerating the seeds for 10-30 days, sterilizing the seeds with sodium hypochlorite for a second time after refrigeration, aseptically sowing the sterilized seeds on a germination medium, culturing them in the dark for 10-30 days and then switching to light culture to germinate and obtain protocorms; the germination medium comprises: taking 1 / 4MS as a basic medium, adding 100-200 ml / L of coconut water, 0.5-1.0 mg / L of 6-benzylaminopurine, 0.1-0.5 mg / L of naphthaleneacetic acid, 0.5-2.0 g / L of peptone and 0.5-1.0 g / L of activated carbon; and the specific steps of sodium hypochlorite treatment are: sterilizing with 70%-75% alcohol for 30-60 seconds, aseptically rinsing for 1-3 times, rinsing with 0.1%-1% sodium hypochlorite solution for 10-30 minutes, and then rinsing with sterile water for 5-6 times.

2. The method for improving the efficiency of aseptic seeding and propagation of Cymbidium orchids as claimed in claim 1, characterized in that: The capsule is a full capsule grown 120-150 days after pollination.

3. The method for improving the efficiency of aseptic seed propagation of Cymbidium orchids as claimed in claim 1, characterized in that: The specific steps for surface sterilization of capsules include: using a toothbrush dipped in soapy water to brush away dust on the surface of the capsules, and rinsing under running water for 1-3 hours; rinsing with 70%-75% alcohol for 3-5 minutes, rinsing with sterile water 1-3 times, transferring to 0.1% mercuric chloride solution for disinfection for 10-15 minutes, shaking continuously during the process to ensure sufficient disinfection, and finally rinsing with sterile water 7-8 times, placing on sterile filter paper to absorb excess moisture on the surface of the capsule.

4. The method for improving the efficiency of aseptic seed propagation of Cymbidium orchids as claimed in claim 1, characterized in that: The working concentration of sodium hypochlorite is 1% and the treatment time is 30 minutes.

5. The method for improving the efficiency of aseptic seed propagation of Cymbidium orchids as claimed in claim 1, characterized in that: The light culture conditions are: culture temperature 25℃-28℃, light intensity 1200-1500Lux, 12-14h light / 8-12h dark.

6. The method for improving the efficiency of aseptic seed propagation of Cymbidium orchids as claimed in claim 1, characterized in that: The seeds were refrigerated at 4°C for 30 days.

7. A method for efficient propagation of Cymbidium orchid by aseptic sowing, characterized in that: The steps include: 1) Aseptic sowing: obtaining protocorms according to the method described in any one of claims 1 to 6; 2) Protocorm proliferation culture: inoculating the protocorm obtained in step 1) into a proliferation medium for proliferation culture, wherein the proliferation medium is: 1 / 4MS as a basic medium, supplemented with 100-200 ml / L of coconut water, 0.1-0.5 mg / L of naphthaleneacetic acid, 0.5-2.0 g / L of peptone, and 0.5-1.0 g / L of activated carbon; 3) Protocorm differentiation culture: The protocorm obtained through proliferation culture in step 2) is inoculated into a differentiation medium for differentiation culture to obtain seedlings, wherein the differentiation medium is 1 / 4MS as a basic medium, supplemented with 0.2-2 mg / L 6-benzylaminopurine, 0.4-4 mg / L naphthaleneacetic acid, 100-200 ml / L coconut water, 0.5-2.0 g / L peptone, 50 mg / L inositol, 2 mg / L glycine, and 2 mg / L nicotinic acid.

8. The method for efficient propagation of Cymbidium orchid by aseptic sowing as claimed in claim 7, characterized in that: Also includes: 4) seedling cultivation: inoculating the seedlings obtained in step 3) onto a seedling cultivation medium for seedling cultivation to obtain complete plants; the seedling cultivation medium is: 1 / 4MS as a basic medium, supplemented with 0.2-2 mg / L of 6-benzylaminopurine, 0.2-4 mg / L of naphthaleneacetic acid, 0.6-1.0 mg / L of indolebutyric acid, 100-200 ml / L of coconut water, 0.5-2.0 g / L of peptone, 0.5-1.0 g / L of activated carbon, 50 mg / L of inositol, 2 mg / L of glycine, and 2 mg / L of nicotinic acid; 5) Transplantation of test tube seedlings: The bottle seedlings obtained in step 4) are transferred to natural light for 7-14 days of hardening, and then the seedlings are taken out, the culture medium on the surface is washed off, and the seedlings are transplanted to a cultivation medium containing coconut bricks, volcanic rocks, peat soil, and pine bark for cultivation.

9. The method for efficient propagation of Cymbidium orchid by aseptic sowing as claimed in claim 8, characterized in that: The mixing ratio of coconut bricks, volcanic stones, peat soil and pine bark is 1:1:3:1 by volume.

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