Shallow-layer liquid standing culture method for tissue culture seedlings of bamboo reed

Abstract

The invention discloses a superficial layer liquid static culture method for tissue culture seedlings of bamboo reeds, and belongs to the technical field of plant biologication.The method comprises the following steps that S1, explant collection and sterilization treatment are conducted, specifically, new tender stem segments with axillary buds of the bamboo reeds are selected as the explants, and cleaning and disinfection are conducted; s2, cluster bud induction: inoculating the disinfected explant into a superficial layer liquid cluster bud induction culture medium for culture, and inducing cluster buds; s3, multiplication culture: transferring the cluster buds induced in the step S2 into a superficial layer liquid multiplication culture medium for culture, and carrying out multiplication; s4, rooting culture: transferring the multiple shoots proliferated in the step S3 into a superficial layer liquid rooting culture medium for culture, and inducing rooting; and S5, transplanting: transplanting the rooted seedlings in the step S4 into a hole tray, and carrying out seedling hardening and growth. The superficial layer liquid culture method is remarkably superior to a traditional solid culture method in the aspects of proliferation efficiency, rooting quality, transplanting survival rate and the like, the working efficiency can be improved, and the production cost can be reduced.

Description

Technical Field

[0001] This invention belongs to the field of plant biotechnology, specifically relating to a method for shallow liquid static culture of Reed tuber seedlings. Background Technology

[0002] Arundo donax L., a clump-forming evergreen herbaceous plant belonging to the genus Arundo in the family Poaceae, is also known as reed or river reed. Arundo donax has high economic, ecological, medicinal, and ornamental value, and has broad application prospects in biomass energy, papermaking, and ecological restoration. With the deepening understanding and research of Arundo donax, its utilization is becoming more comprehensive, and demand is increasing daily. However, due to its low seed setting rate, slow traditional division propagation, and difficulty in rooting cuttings, it is difficult to meet the demand for Arundo donax seedlings for large-scale industrialization. To enable the large-scale promotion of Arundo donax, the development of an in vitro rapid propagation technology system using tissue culture is one of the technologies for achieving rapid propagation in the short term.

[0003] Currently, most rapid in vitro propagation techniques for *Arundinaria repens* utilize solid culture media. Solid culture typically employs agar, plant gels, and other supporting materials, which usually account for over 50% of the culture medium cost. Furthermore, preparation requires boiling, and the roots must be thoroughly cleaned of any adhering culture medium before transplanting, resulting in high electricity, labor, and time costs, thus increasing the production cost of tissue culture seedlings. Therefore, continuously optimizing culture conditions and improving culture methods to enhance the rapid propagation efficiency of tissue culture seedlings remains a crucial task in establishing a comprehensive *Arundinaria repens* tissue culture rapid propagation technology system.

[0004] Chinese patent application CN118000092A discloses a method for efficient rapid propagation of Reed Piper asiatica via tissue culture, including: Reed Piper asiatica tissue culture seedling propagation culture, rooting culture and transplanting. In this method, the rooting culture of Reed Piper asiatica tissue culture seedlings requires 30-40 days, and the rooting culture time needs to be shortened.

[0005] Chinese patent application CN102919127B discloses a method for establishing a tissue culture system for Phragmites australis, including: explant disinfection, primary culture, subculture, bud strengthening and rooting culture, and domestication and transplanting. In this method, the culture medium needs to be supplemented with 8g / L agar, and the production cost needs to be reduced.

[0006] Chinese patent application CN109315292A discloses a method for rapidly propagating sweet potato explants using a shallow liquid culture method, including: obtaining sweet potato explants, obtaining sterile seedlings, hardening off seedlings, transplanting seedlings, and field propagation. This method requires placing defatted cotton in the culture bottle as a support. Placing defatted cotton requires a lot of manual labor, is complicated to operate, and has a high cost. Therefore, the production cost needs to be reduced.

[0007] In conclusion, to shorten the rooting culture time and reduce the cost of industrialized seedling production, it is necessary to propose a liquid culture method for tissue culture seedlings that is suitable for rapid industrial propagation of Phragmites australis, has low growth costs, and is easy to operate.

[0008] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0009] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a shallow liquid culture method for *Arundinaria mirifica* tissue culture seedlings that is simple to operate, low in cost, has high propagation efficiency, short rooting culture time, and produces robust seedlings.

[0010] To achieve the above objectives, a specific embodiment of the present invention provides the following technical solution:

[0011] A method for shallow liquid static culture of Reed rhizome tissue culture seedlings includes the following steps:

[0012] S1. Collection and sterilization of explants: Select tender young stem segments with axillary buds from Phragmites australis as explants, and clean and disinfect them;

[0013] S2. Shoot induction: Sterilized explants were inoculated into a shallow liquid shoot induction medium and cultured to induce shoot clusters;

[0014] S3. Proliferation culture: The clustered shoots induced in step S2 are transferred to a shallow liquid proliferation culture medium for proliferation.

[0015] S4. Rooting culture: The clustered shoots proliferated in step S3 are transferred to a shallow liquid rooting medium for culture to induce rooting;

[0016] S5. Transplanting: Transplant the seedlings that have rooted in step S4 into seedling trays for hardening and growth.

[0017] In one or more embodiments of the present invention, in step S2, the volume of the shallow liquid shoot induction medium is 20-25 mL / culture flask;

[0018] The volume of the shallow liquid proliferation medium in step S3 is 20-25 mL / culture flask;

[0019] The volume of the shallow liquid rooting medium in step S4 is 10-15 mL / culture flask.

[0020] In one or more embodiments of the present invention, the shallow liquid shoot induction medium in step S2 is MS medium, with 3-5 mg / L 6-BA, 0.5-1 mg / L IBA and 20-30 g / L sucrose added.

[0021] In one or more embodiments of the present invention, the shallow liquid proliferation medium in step S3 is MS medium, which contains 3-5 mg / L 6-BA, 1-2 mg / L IAA and 20-30 g / L sucrose.

[0022] In one or more embodiments of the present invention, the shallow liquid rooting medium in step S4 is 1 / 2 MS medium, with 0.2-0.5 mg / L NAA and 15-25 g / L sucrose added.

[0023] In one or more embodiments of the present invention, 4-6 explants are inoculated into each culture flask in step S2;

[0024] In step S3, each culture bottle is inoculated with 5-7 clustered shoots;

[0025] In step S4, each culture bottle is inoculated with 8-10 clustered shoots.

[0026] In one or more embodiments of the present invention, the cultivation conditions for step S2 are: light intensity 1500-2500 Lux, light duration 12-16 h / d, temperature 24-26 °C, and cultivation for 20-30 days.

[0027] The cultivation conditions for step S3 are: light intensity 1500-2500 Lux, light duration 12-16 h / d, temperature 24-26℃, and cultivation for 25-30 days;

[0028] The cultivation conditions for step S4 are: light intensity 2000-3000 Lux, light duration 12-16 h / d, temperature 24-26℃, and cultivation for 15-20 days.

[0029] In one or more embodiments of the present invention, the explant in step S1 is a tender young stem segment of Phragmites australis with axillary buds, cut into 1.5-2.5cm lengths, first cleaned, and then disinfected with alcohol and HgCl2 solution.

[0030] In one or more embodiments of the present invention, the seedling trays used for transplanting in step S5 are 50-cell seedling trays, and the substrate is composed of coconut coir, vermiculite and perlite in a weight ratio of 1:1:2. After transplanting, the temperature is maintained at 20-30°C and the air humidity is above 85%. The seedlings are shaded for the first 3 days, and the light intensity is 1500-2000 Lux. After that, the light intensity is gradually increased to 25000-40000 Lux.

[0031] In one or more embodiments of the present invention, the shallow liquid shoot induction medium, the shallow liquid proliferation medium, and the shallow liquid rooting medium do not contain agar, plant gel, absorbent cotton, or other similar materials as supports.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] 1. The shallow liquid culture medium provides a multi-angle nutrient absorption interface for the buds, and the nutrient diffusion rate in the liquid is fast, which significantly improves the proliferation coefficient of Reed.

[0034] 2. In shallow liquid culture medium, there is almost no mechanical resistance. Once the root primordia are formed, the cells can extend freely and rapidly without hindrance, and the roots grow faster and longer.

[0035] 3. It eliminates the need for supporting materials such as agar and plant gel, reducing the cost of the culture medium; the operation process is simplified, as it does not require insertion into the culture medium, improving work efficiency; and there is no need to clean the culture medium from the roots during transplanting, saving a lot of labor and costs.

[0036] 4. It reduces the use of agar and lowers the pressure of subsequent waste disposal. Attached Figure Description

[0037] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0038] Figure 1 This is a flowchart of a shallow liquid static culture method for Reed tissue culture seedlings according to an embodiment of the present invention;

[0039] Figure 2 This is a growth status diagram of *Arundinaria salina* after 15 days of liquid culture for inducing shoot growth in one embodiment of the present invention.

[0040] Figure 3 This is a diagram showing the induction of shoot clusters in Reed sphagnum moss after 25 days of cultivation in liquid culture medium, according to one embodiment of the present invention.

[0041] Figure 4 This is a diagram showing the rooting status of *Arundinaria salsa* tissue culture seedlings after 15 days of culture in liquid rooting medium, according to an embodiment of the present invention.

[0042] Figure 5 This is a comparison diagram of liquid culture and solid culture during the shoot induction stage of *Arundinaria salsa* tissue culture seedlings in one embodiment of the present invention.

[0043] Figure 6 This is a comparison diagram of liquid culture and solid culture during the propagation stage of Reed tissue culture seedlings in one embodiment of the present invention;

[0044] Figure 7 This is a comparison of the growth status of Reed tissue culture seedlings in liquid culture and solid culture during the rooting stage in one embodiment of the present invention.

[0045] Figure 8 This is a comparison of root growth status in liquid culture and solid culture during the rooting stage of *Arundo donax* tissue culture seedlings in one embodiment of the present invention. Detailed Implementation

[0046] To enable those skilled in the art to better understand the technical solutions in this disclosure, the technical solutions in the embodiments of this disclosure will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this disclosure, and not all embodiments. Based on the embodiments in this disclosure, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this disclosure.

[0047] Example 1:

[0048] like Figures 1-8 As shown, a method for shallow liquid static culture of Reed tuber seedlings includes the following steps:

[0049] S1. Collection and Sterilization of Explants: Healthy, disease-free reeds were selected and collected after three consecutive sunny days. Young, tender stem segments with axillary buds were cut as explant material. The stem segments were cut into approximately 2cm pieces with axillary buds, and the leaves were removed. The treated stem segments were placed in a glass and rinsed with running water for 15 minutes, then washed with detergent for 1 minute, and rinsed thoroughly with running water to remove foam, surface dust, and other impurities. Finally, they were rinsed three times with distilled water. The rinsed material was placed in a clean bench and washed three times with sterile water, then with alcohol for 30 seconds, followed by three more washes with sterile water. The material was then treated with 0.1% HgCl2 as a disinfectant for 8 minutes, and finally rinsed four times with sterile water.

[0050] S2. Shallow Liquid Sprout Induction: The explants sterilized in S1 were inoculated into culture flasks containing liquid sprout induction medium. 20 ml of liquid sprout induction medium was added to each flask, with 5 explants per flask being recommended. The preferred induction medium was MS medium supplemented with 3 mg / L 6-BA, 0.5 mg / L IBA, and 25 g / L sucrose. Culture conditions were: light intensity 1500-2500 Lux, photoperiod 12 h / d, temperature 24-26℃, for 30 days, which induced a large number of sprouts.

[0051] S3. Proliferation Culture: The robust shoots induced in S2, growing to 3-5 cm in height, are transferred individually or in small clusters to culture flasks containing liquid proliferation medium. Each culture flask should contain 20 ml of liquid proliferation medium, with 6 shoots inoculated per flask. The preferred induction medium is MS medium supplemented with 5 mg / L 6-BA, 2 mg / L IIAA, and 25 g / L sucrose. Culture conditions are: light intensity 1500-2500 Lux, photoperiod 12 h / d, temperature 24-26℃, for 25 days, which will induce a large number of shoot clusters.

[0052] S4. Rooting Culture: Healthy shoots induced in S3, reaching 3-5 cm in height, are individually or in small clusters transferred to culture bottles containing liquid rooting medium. Each culture bottle should contain 10 ml of liquid rooting medium, with a maximum of 8 shoots per bottle. Alternatively, healthy shoots induced in S3, reaching 2-3 cm in height, are individually or in small clusters transferred to a shallow layer of liquid rooting medium, with a liquid depth of 1.2 cm, with a maximum of 10 shoots per culture bottle. The preferred rooting medium is 1 / 2 MS medium supplemented with 0.3 mg / L NAA and 25 g / L sucrose. Culture conditions are: light intensity 2000-3000 Lux, photoperiod 12 h / d, temperature 24-26℃. After 15 days of culture, well-developed adventitious roots will appear, forming complete regenerated plants.

[0053] S5. Transplanting: Transplant the rooted seedlings from S4 into plug trays, using 9cm high, 50-cell trays. The substrate should have a weight ratio of coconut coir:vermiculite:perlite = 1:1:2. After transplanting, maintain a greenhouse temperature of 20-30℃ and an air humidity of over 85% (achieved by covering with a plastic film arch). Gradually increase ventilation to reduce humidity to 60-70% to harden the seedlings. Initially, keep the substrate moist but not waterlogged. Water using a misting method to avoid washing away the seedlings. Provide appropriate shade for the first 3 days after transplanting (light intensity 1500-2000 Lux), then gradually increase the light intensity to normal growth levels (25000-40000 Lux). One week after transplanting, when the seedlings resume growth, begin removing the shade netting and plastic film.

[0054] Example 2:

[0055] like Figures 1-8 As shown, a method for shallow liquid static culture of Reed tuber seedlings includes the following steps:

[0056] S1. Collection and Sterilization of Explants: Healthy, disease-free reeds were selected and collected after three consecutive sunny days. Young, tender stem segments with axillary buds were cut as explant material. The stem segments were cut into approximately 2cm pieces with axillary buds, and the leaves were removed. The treated stem segments were placed in a glass and rinsed with running water for 15 minutes, then washed with detergent for 1 minute, and rinsed thoroughly with running water to remove foam, surface dust, and other impurities. Finally, they were rinsed three times with distilled water. The rinsed material was placed in a clean bench and washed three times with sterile water, then with alcohol for 30 seconds, followed by three more washes with sterile water. The material was then treated with 0.1% HgCl2 as a disinfectant for 8 minutes, and finally rinsed four times with sterile water.

[0057] S2. Shallow Liquid Sprout Induction: Explants sterilized in S1 were inoculated into culture flasks containing liquid sprout induction medium. 25 ml of liquid sprout induction medium was added to each flask, with 6 explants per flask being optimal. The preferred induction medium was MS medium supplemented with 3 mg / L 6-BA, 1 mg / L IBA, and 25 g / L sucrose. Culture conditions were: light intensity 1500-2500 Lux, photoperiod 12 h / d, temperature 24-26℃, for 30 days, which induced a large number of sprouts.

[0058] S3. Proliferation Culture: Healthy shoot clusters induced in S2, reaching a height of 3-5 cm, are transferred individually or in small clusters to culture flasks containing liquid proliferation medium. Each flask should contain 25 ml of liquid proliferation medium, with 6 shoots inoculated per flask. Alternatively, healthy shoot clusters induced in S2, reaching a height of 3-5 cm, are transferred individually or in small clusters to a shallow layer of liquid proliferation medium. The liquid depth of the rooting medium should be 1.6 cm, with 6 shoots inoculated per flask. The preferred induction medium is MS medium supplemented with 4 mg / L 6-BA, 1.5 mg / L IIAA, and 20 g / L sucrose. Culture conditions are: light intensity 1500-2500 Lux, photoperiod 12 h / d, temperature 24-26℃, for 25 days, which will induce a large number of shoot clusters.

[0059] S4. Rooting Culture: Healthy shoots induced in S3, reaching 3-5 cm in height, are transferred individually or in small clusters to culture bottles containing liquid rooting medium. Each culture bottle should contain 10 ml of liquid rooting medium, with a recommended inoculation of 9 shoots per bottle. The preferred rooting medium is 1 / 2 MS medium supplemented with 0.5 mg / L NAA and 25 g / L sucrose. Culture conditions are: light intensity 2000-3000 Lux, photoperiod 12 h / d, temperature 24-26℃. After 15 days of culture, well-developed adventitious roots will appear, forming complete regenerated plants.

[0060] S5. Transplanting: Transplant the rooted seedlings from S4 into plug trays, using 9cm high, 50-cell trays. The substrate should have a weight ratio of coconut coir:vermiculite:perlite = 1:1:2. After transplanting, maintain a greenhouse temperature of 20-30℃ and an air humidity of over 85% (achieved by covering with a plastic film arch). Gradually increase ventilation to reduce humidity to 60-70% to harden the seedlings. Initially, keep the substrate moist but not waterlogged. Water using a misting method to avoid washing away the seedlings. Provide appropriate shade for the first 3 days after transplanting (light intensity 1500-2000 Lux), then gradually increase the light intensity to normal growth levels (25000-40000 Lux). One week after transplanting, when the seedlings resume growth, begin removing the shade netting and plastic film.

[0061] Example 3:

[0062] like Figures 1-8 As shown, a method for shallow liquid static culture of Reed tuber seedlings includes the following steps:

[0063] S1. Collection and Sterilization of Explants: Healthy, disease-free reeds were selected and collected after three consecutive sunny days. Young, tender stem segments with axillary buds were cut as explant material. The stem segments were cut into approximately 2cm pieces with axillary buds, and the leaves were removed. The treated stem segments were placed in a glass and rinsed with running water for 15 minutes, then washed with detergent for 1 minute, and rinsed thoroughly with running water to remove foam, surface dust, and other impurities. Finally, they were rinsed three times with distilled water. The rinsed material was placed in a clean bench and washed three times with sterile water, then with alcohol for 30 seconds, followed by three more washes with sterile water. The material was then treated with 0.1% HgCl2 as a disinfectant for 8 minutes, and finally rinsed four times with sterile water.

[0064] S2. Shallow Liquid Sprout Induction: Explants sterilized in S1 were inoculated into culture flasks containing liquid sprout induction medium. 25 ml of liquid sprout induction medium was added to each flask, with 6 explants per flask being recommended. The preferred induction medium was MS medium supplemented with 4 mg / L 6-BA, 0.5 mg / L IBA, and 30 g / L sucrose. Culture conditions were: light intensity 1500-2500 Lux, photoperiod 12 h / d, temperature 24-26℃, for 30 days, which induced a large number of sprouts.

[0065] S3. Proliferation Culture: Healthy shoots induced in S2, growing to 3-5 cm in height, are transferred individually or in small clusters to culture flasks containing liquid proliferation medium. Each flask should contain 25 ml of liquid proliferation medium, with a maximum of 7 shoots per flask. The preferred induction medium is MS medium supplemented with 5 mg / L 6-BA, 1.5 mg / L IIAA, and 30 g / L sucrose. Culture conditions are: light intensity 1500-2500 Lux, photoperiod 12 h / d, temperature 24-26℃, for 25 days, which will induce a large number of shoot clusters.

[0066] S4. Rooting Culture: Healthy shoots induced in S3, reaching 3-5 cm in height, are transferred individually or in small clusters to culture bottles containing liquid rooting medium. Each culture bottle should contain 15 ml of liquid rooting medium, with 10 shoots per bottle being ideal. The preferred rooting medium is 1 / 2 MS medium supplemented with 0.4 mg / L NAA and 25 g / L sucrose. Culture conditions are: light intensity 2000-3000 Lux, photoperiod 12 h / d, temperature 24-26℃. After 15 days of culture, well-developed adventitious roots will appear, forming complete regenerated plants.

[0067] S5. Transplanting: Transplant the rooted seedlings from S4 into plug trays, using 9cm high, 50-cell trays. The substrate should have a weight ratio of coconut coir:vermiculite:perlite = 1:1:2. After transplanting, maintain a greenhouse temperature of 20-30℃ and an air humidity of over 85% (achieved by covering with a plastic film arch). Gradually increase ventilation to reduce humidity to 60-70% to harden the seedlings. Initially, keep the substrate moist but not waterlogged. Water using a misting method to avoid washing away the seedlings. Provide appropriate shade for the first 3 days after transplanting (light intensity 1500-2000 Lux), then gradually increase the light intensity to normal growth levels (25000-40000 Lux). One week after transplanting, when the seedlings resume growth, begin removing the shade netting and plastic film.

[0068] Comparative Example 1: (Traditional solid-state shoot induction culture)

[0069] Except for adding 6 g / L agar to the S2 shoot induction medium in Example 1 and using a medium injection volume of 35 ml, the other steps were exactly the same as in Example 1. As shown in Table 1, after 30 days of culture, the number of induced shoots and the shoot length in Comparative Example 1 were significantly lower than those in Example 1.

[0070] Table 1

[0071]

[0072] Comparative Example 2: (Traditional solid-state proliferation culture)

[0073] Except for adding 6 g / L agar to the S3 proliferation medium in Example 1 and using a medium injection volume of 35 ml, the other steps were exactly the same as in Example 1. As shown in Table 2, after 25 days of culture, the proliferation coefficient of Comparative Example 2 was significantly lower than that of Example 1.

[0074] Table 2

[0075]

[0076] Comparative Example 3: (Traditional solid rooting culture)

[0077] Except for adding 6 g / L agar to the S4 rooting medium in Example 1 and using a medium injection volume of 35 ml, the other steps were exactly the same as in Example 1. As shown in Table 3, after 15 days of culture, the rooting rate, number of roots, plant height, and transplant survival rate of Comparative Example 3 were significantly lower than those of Example 1.

[0078] Table 3

[0079]

[0080] The above data fully demonstrate that the shallow liquid culture method of the present invention is significantly superior to the traditional solid culture method in terms of shoot induction, proliferation efficiency, rooting quality and transplant survival rate.

[0081] The shallow liquid culture medium of the present invention provides a multi-angle nutrient absorption interface for the buds, and the nutrient diffusion rate in the liquid is fast, which significantly improves the proliferation coefficient of Arundo donax, by more than 30% compared with solid culture.

[0082] The shallow liquid culture medium of the present invention has almost no mechanical resistance. Once the root primordia are formed, the cells can extend freely and rapidly without hindrance, and the roots grow faster and longer.

[0083] This invention eliminates the need for supports such as agar and plant gel, reducing the cost of the culture medium; the operation process is simplified, eliminating the need to insert the plant into the culture medium, thus improving work efficiency; and there is no need to clean the culture medium from the roots during transplanting, saving a lot of labor, with a saving of approximately 0.16 yuan per seedling, as shown in Table 4.

[0084] Table 4

[0085]

[0086] This invention reduces the use of agar and lowers the burden of subsequent waste disposal.

[0087] It will be apparent to those skilled in the art that this disclosure is not limited to the details of the exemplary embodiments described above, and that this disclosure can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of this disclosure is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within this disclosure. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0088] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for culturing Arundo donax plantlets in a shallow liquid static culture, characterized by, The method comprises the following steps: S1. Collection and sterilization of explants: select new and tender stem segments with axillary buds of Arundo donax as explants, and perform cleaning and sterilization; S2. Induction of multiple shoots: inoculate the sterilized explants into a shallow liquid medium for induction of multiple shoots; S3. Proliferation culture: transfer the multiple shoots induced in step S2 to a shallow liquid medium for proliferation culture; S4. Rooting culture: transfer the multiple shoots proliferated in step S3 to a shallow liquid rooting medium for culture to induce rooting; S5. Transplanting: transplant the seedlings with roots in step S4 to a plug tray for seedling raising and growth.

2. The method according to claim 1, wherein the method is characterized by, In step S2, the volume of the shallow liquid medium for induction of multiple shoots is 20-25 mL per flask; In step S3, the volume of the shallow liquid medium for proliferation culture is 20-25 mL per flask; In step S4, the volume of the shallow liquid rooting medium is 10-15 mL per flask.

3. The method according to claim 1 or 2, wherein the method is a shallow liquid static culture method of the Arundo donax tissue culture seedlings. In step S2, the shallow liquid medium for induction of multiple shoots is MS medium added with 3-5 mg / L 6-BA, 0.5-1 mg / L IBA and 20-30 g / L sucrose.

4. The method according to claim 1 or 2, wherein the method is a shallow liquid static culture method of the Arundo arientaria tissue culture seedling. In step S3, the shallow liquid medium for proliferation culture is MS medium added with 3-5 mg / L 6-BA, 1-2 mg / L IAA and 20-30 g / L sucrose.

5. The method according to claim 1 or 2, wherein the method is a shallow liquid static culture method of the Arundo donax tissue culture seedlings. In step S4, the shallow liquid rooting medium is 1 / 2MS medium added with 0.2-0.5 mg / L NAA and 15-25 g / L sucrose.

6. The method according to claim 1, wherein the method is a method for culturing Arundo donax phytomers in a shallow liquid static culture, characterized by, In step S2, 4-6 explants are inoculated per flask; In step S3, 5-7 multiple shoots are inoculated per flask; In step S4, 8-10 multiple shoots are inoculated per flask.

7. The method according to claim 1, wherein the method is a method for culturing Arundo donax phytomers in a shallow liquid static culture, characterized by, In step S2, the culture conditions are: light intensity of 1500-2500 Lux, light time of 12-16 h / d, temperature of 24-26℃, and culture time of 20-30 days; In step S3, the culture conditions are: light intensity of 1500-2500 Lux, light time of 12-16 h / d, temperature of 24-26℃, and culture time of 25-30 days; In step S4, the culture conditions are: light intensity of 2000-3000 Lux, light time of 12-16 h / d, temperature of 24-26℃, and culture time of 15-20 days.

8. The method according to claim 1, wherein the method is a method for culturing Arundo donax phytomers in a shallow liquid static culture, characterized by, In step S1, the explants are new and tender stem segments with axillary buds of Arundo donax, which are cut into 1.5-2.5 cm in length, cleaned first, and then sterilized with alcohol and HgCl2 solution.

9. The method according to claim 1, wherein the method is a method for culturing Arundo donax phytomers in a shallow liquid static culture, characterized by, In step S5, the plug tray used for transplanting is a 50-hole plug tray, and the substrate is composed of coconut husk, vermiculite and perlite at a weight ratio of 1:1:

2. After transplanting, the temperature is maintained at 20-30℃, the air humidity is above 85%, the light is shaded for the first 3 days, and the light intensity is 1500-2000 Lux, and then the light is gradually increased to 25000-40000 Lux.

10. The method according to claim 1, wherein the method is a shallow liquid static culture method of the Arundo arientaria tissue culture seedling. The shallow liquid medium for induction of multiple shoots, the shallow liquid medium for proliferation culture and the shallow liquid rooting medium do not add agar, plant gel or defatted cotton as a support.

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