Method for doubling chromosomes of bamboo reed

Abstract

The invention belongs to the technical field of biotechnology breeding, and discloses a bamboo reed chromosome doubling method which comprises the following steps: (1) selecting bamboo reed tissue culture rooting seedlings which are 8-10cm in plant height, free of plant diseases and insect pests and robust in root system, removing a culture medium, cutting off part of roots and only reserving 2-3cm of roots; and (2) recovering the trimmed tissue culture seedlings in clean water for 1-3 days, and replacing the water for 1-2 times every day. According to the method, 0.5 g / L colchicine and 20 mL / L dimethyl sulfoxide are combined, short-time (10-hour) directional treatment is carried out on the stem base of the bamboo reed under the low-light constant-temperature condition, the chromosome doubling rate reaches 16% and is obviously higher than that of a traditional method by 8%-12%, the action time is remarkably shortened, accumulated poison of pesticide liquid in a plant body is reduced, and the effect of improving the yield of the plant is achieved. The action area is concentrated at the meristem part, the phytotoxicity is effectively reduced, the dosage of the liquid medicine is saved, meanwhile, the pesticide effect utilization rate is further enhanced in cooperation with various selectable penetration enhancers (such as Tween-20, glycerol and polyethylene glycol), and the survival rate of the treated plants is stabilized at 95% or above.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology breeding technology, specifically a method for doubling chromosomes in Phragmites australis. Background Technology

[0002] Arundodis ( Arundo donax As a tall, perennial grass, *Arundinaria lobata* (Phragmites australis) is characterized by high biomass, strong resistance to adverse conditions, and wide applicability. It is widely considered a high-quality energy crop and a raw material for papermaking fiber. In plant breeding, chromosome doubling is an important technique for creating polyploid materials, as polyploid plants often exhibit superior characteristics in morphology, physiology, and adaptability. However, there are few reports on chromosome doubling in *Arundinaria lobata*, and a mature and feasible induction method is lacking.

[0003] In existing technologies, colchicine is commonly used as a chromosome doubling inducer. It prevents chromosome separation by inhibiting spindle fiber formation, thereby doubling the number of chromosomes in the cell nucleus. However, different species have different sensitivities to colchicine, and the treatment concentration, time, and medium conditions need to be optimized for different species.

[0004] For example, existing methods for doubling the number of reeds often use stem segments with axillary buds as explants, which are then treated with colchicine for a long time (24-48 hours) after adventitious bud induction. However, this method has the following drawbacks:

[0005] Limited material types: stem segments with axillary buds are mostly selected as explants, and complete plants can only be obtained through multiple tissue culture stages such as adventitious bud induction, differentiation, and seedling strengthening. The process is long and complicated.

[0006] Long processing time: It often requires 24-48 hours of colchicine soaking, resulting in low survival rate and obvious phytotoxicity.

[0007] The drug solution has a single ingredient: it only uses colchicine and does not add penetration enhancers, so the efficacy is incomplete.

[0008] Temperature and light control were rudimentary: the treatment environment was not optimized according to the characteristics of Reed, resulting in low efficiency of the drug solution entering the tissue and unstable effects.

[0009] Therefore, there is an urgent need for a chromosome doubling method that is tailored to the characteristics of Phragmites australis, has a short processing time, low phytotoxicity, and high survival rate. Summary of the Invention

[0010] The purpose of this invention is to provide a method for doubling chromosomes in Reed spp., in order to solve the problems mentioned in the background art.

[0011] To achieve the above objectives, the present invention provides the following technical solution: a method for doubling chromosomes in *Arundo donax*, comprising the following steps:

[0012] (1) Select Reed tissue culture seedlings with a height of 8-10cm, free from pests and diseases, and with strong roots. Remove the culture medium and cut off part of the roots, leaving only 2-3cm.

[0013] (2) After pruning, the tissue culture seedlings should be placed in clean water to recover for 1-3 days, and the water should be changed 1-2 times a day;

[0014] (3) Prepare a colchicine solution containing 0.25-1.0 g / L colchicine and 10-30 mL / L dimethyl sulfoxide, preferably 0.5 g / L colchicine and 20 mL / L dimethyl sulfoxide;

[0015] (4) Immerse the base of the tissue culture seedling stem in the solution to a depth of 2-4 cm and treat for 5-20 hours under constant temperature of 20±1℃ and light intensity of 1000-1500lx.

[0016] (5) After treatment, rinse with running water for 5-10 minutes, then rinse with sterile water 2-3 times, and place in a 50-well tray in culture medium to allow seedlings to recover for 7-15 days.

[0017] (6) After the seedlings have recovered, they should be cultured for 2-3 months under controlled temperature (20-25℃) and moderate light conditions;

[0018] (7) Transplant the seedlings to the field. After the plants have grown steadily, take stem tip or root tip tissue for chromosome testing and screen to obtain doubled plants.

[0019] Preferably, the colchicine solution is further supplemented with one or more penetration enhancers selected from Tween-20, glycerin, or polyethylene glycol.

[0020] Preferably, during the preparation of the drug solution, colchicine is first dissolved in dimethyl sulfoxide solution, and then diluted with distilled water to the target concentration.

[0021] Preferably, either 0.1‰ carbendazim or 0.05% Cabinda slow-release preservative is added to the water during the seedling establishment process.

[0022] Preferably, the colchicine solution is subjected to ultrasonic vibration for 10-20 minutes or magnetic stirring for 15-30 minutes before use.

[0023] Preferably, the drug treatment is in an intermittent mode, with treatment lasting 3-10 hours, followed by rinsing once and then treatment for the remaining time.

[0024] Preferably, before transplanting to the field, the reed seedlings are hardened off in the hardening-off area for 7-14 days, during which the ambient humidity is controlled at 70-85% and the light intensity is gradually increased.

[0025] Preferably, the seedling tray has 50 cells, each with a volume of 20-25 mL, and the substrate moisture is maintained at 65-75% of the substrate water saturation.

[0026] The beneficial effects of this invention are as follows:

[0027] This invention employs a combination of 0.5 g / L colchicine and 20 mL / L dimethyl sulfoxide, combined with a short-term (10-hour) targeted treatment of the base of *Arundo donax* stems under low light and constant temperature conditions. This results in a chromosome doubling rate of 16%, significantly higher than the 8%-12% of traditional methods. This not only significantly shortens the treatment time and reduces the cumulative toxicity of the drug solution within the plant, but also concentrates the treatment area on the meristematic tissue, effectively reducing phytotoxicity and saving on drug dosage. Furthermore, the use of various optional penetration enhancers (such as Tween-20, glycerol, and polyethylene glycol) further enhances the drug utilization rate, ensuring that the survival rate of treated plants remains stable at over 95%.

[0028] In terms of process, this invention directly utilizes tissue-cultured rooted seedlings for doubling treatment, avoiding adventitious bud induction and multiple rounds of transfer culture, shortening the overall cycle by 1-2 months, making it suitable for large-scale breeding and promotion. The polyploids of Arundo donax obtained after treatment show significant improvements in traits such as leaf width, stem diameter, and plant height, which not only have the potential to increase fiber and biomass yield, but also possess high horticultural ornamental value. This invention comprehensively embodies the outstanding advantages of high efficiency, low phytotoxicity, short cycle, and excellent traits. Attached Figure Description

[0029] Figure 1 This is a schematic diagram of the process of the present invention. Detailed Implementation

[0030] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] like Figure 1 As shown, this embodiment of the invention provides a method for doubling chromosomes in Reedus amurensis, comprising the following steps:

[0032] (1) Select Reed tissue culture seedlings with a height of 8-10cm, free from pests and diseases, and with strong roots. Remove the culture medium and cut off part of the roots, leaving only 2-3cm.

[0033] (2) After pruning, the tissue culture seedlings should be placed in clean water to recover for 1-3 days, and the water should be changed 1-2 times a day;

[0034] (3) Prepare colchicine solution containing 0.25-1.0 g / L colchicine and 10-30 mL / L dimethyl sulfoxide, preferably 0.5 g / L colchicine and 20 mL / L dimethyl sulfoxide;

[0035] (4) Immerse the base of the tissue culture seedling stem in the solution to a depth of 2-4 cm and treat for 5-20 hours under constant temperature of 20±1℃ and light intensity of 1000-1500lx.

[0036] (5) After treatment, rinse with running water for 5-10 minutes, then rinse with sterile water 2-3 times, and place in a 50-well tray in culture medium to allow seedlings to recover for 7-15 days.

[0037] (6) After the seedlings have recovered, they should be cultured for 2-3 months under controlled temperature (20-25℃) and moderate light conditions;

[0038] (7) Transplant the seedlings to the field. After the plants have grown steadily, take stem tip or root tip tissue for chromosome testing and screen to obtain doubled plants.

[0039] The colchicine solution is further enhanced with one or more penetration enhancers, such as Tween-20, glycerin, or polyethylene glycol. During solution preparation, colchicine is first dissolved in dimethyl sulfoxide solution, and then diluted with distilled water to the target concentration. Before use, the colchicine solution is ultrasonically vibrated for 10-20 minutes or magnetically stirred for 15-30 minutes. The solution treatment is in an intermittent mode, with treatment for 3-10 hours, followed by rinsing once, and then treatment for the remaining time.

[0040] In the method for doubling chromosomes in Reed hyacinth of the present invention, by adding Tween-20, glycerol or polyethylene glycol or other penetration enhancers to the colchicine solution and adopting a preparation method of "dissolving in dimethyl sulfoxide first and then diluting", combined with ultrasonic or magnetic stirring before use, the solution is made stable, uniform in concentration and has stronger transdermal penetration ability. This allows colchicine to enter the basal meristem of the stem efficiently in a short time, improving the doubling success rate and reducing phytotoxicity. This optimization in chemistry and preparation significantly improves the efficacy and repeatability of the experiment, ensuring that the process is controllable and suitable for batch operation.

[0041] Meanwhile, this invention employs an intermittent treatment mode, reducing the accumulation of toxicity caused by prolonged continuous exposure through segmented application and mid-treatment rinsing. This significantly improves plant survival rate while ensuring the total treatment time and doubling effect. The combination of the above-mentioned optimized solution composition, preparation method, and treatment strategy not only achieves a dual improvement in doubling rate and survival rate technically, but also forms a synergistic composite process. Unlike existing single-parameter optimization technologies, this method possesses significant inventiveness and resistance to circumvention, making it suitable for large-scale production in ploidy breeding of Phragmites australis.

[0042] During the seedling establishment process, either 0.1‰ carbendazim or 0.05% Cabinda slow-release preservative is added to the water.

[0043] In the method for doubling the chromosomes of Reed hyacinth of the present invention, adding 0.1‰ carbendazim or 0.05% Cabinda slow-release preservative to the water during the seedling establishment stage can effectively inhibit the growth of pathogenic microorganisms such as fungi and bacteria in the plant cuts and rhizosphere, and prevent infection of tissue wounds caused by root pruning and colchicine treatment. This type of bactericidal / preservative treatment provides a clean and safe environment during the seedling recovery period from pesticide damage, and significantly reduces the proportion of rotting, softening or yellowing and death during the seedling establishment process.

[0044] This measure plays a protective role during the critical stage when plant resistance decreases after chemical doubling treatment, ensuring a high doubling rate while maintaining a high survival rate and health, and reducing the pressure of pesticide use in later cultivation stages. This practice of embedding disease prevention during the seedling establishment period, organically combined with the doubling treatment process, not only improves the stability and repeatability of the doubling process, but also makes this invention superior to existing technologies in ensuring seedling survival rates, enhancing the industrial feasibility of the process.

[0045] Before transplanting to the field, the seedlings of Phragmites australis are hardened off in the hardening-off area for 7-14 days, during which the environmental humidity is controlled at 70-85% and the light intensity is gradually increased.

[0046] In the method for doubling chromosomes of Reed sphagnum in this invention, after colchicine chemical treatment and the seedling establishment stage, the seedlings are still in a recovery period that is sensitive to environmental changes. By arranging 7-14 days of hardening off before transplanting to the field, and controlling the humidity of the hardening off area at 70-85% and gradually increasing the light intensity from weak to strong, the doubling-treated plants can gradually adapt to the transition from greenhouse or plug tray conditions to open field conditions. This process helps to enhance the photosynthetic and transpiration regulation capacity of the roots and leaves, and reduce transplant shock and mortality caused by direct transplanting.

[0047] This measure not only physiologically improves the seedlings' resistance to stress and environmental adaptability, but also ensures a high survival rate in the field even under high doubling rates. This avoids the chromosome doubling effect being offset by transplant losses in actual field production. Compared with existing technologies, the introduction of a controlled-parameter hardening-off stage in the polyploid induction process of *Arundo donax* makes the field application of doubled plants more reliable and stable, thus improving the feasibility of large-scale promotion of this invention.

[0048] The seedling trays have 50 cells, each with a volume of 20-25 mL, and the substrate moisture content is maintained at 65-75% of the substrate's water saturation.

[0049] In the method for chromosome doubling of *Arundinaria salsa* in this invention, a 50-well tray is used during the seedling establishment stage, with each well containing 20-25 mL. This provides sufficient but not excessive substrate for each seedling and facilitates control of plant spacing and environmental conditions. This specification ensures that each *Arundinaria salsa* plant has independent growth space during the seedling establishment and sterilization period, reducing competition and cross-infection risks between plants and creating favorable conditions for uniform root expansion. Simultaneously, the small to medium well volume helps maintain a balance between water retention and aeration, ensuring that the roots are neither hypoxic nor overly wet during post-treatment recovery.

[0050] Maintaining the substrate moisture content at 65-75% of its water saturation level effectively meets the water requirements of plants with lower transpiration rates after double treatment, preventing root rot and fungal growth caused by overwatering, and also preventing water stress caused by excessive dryness. This tray size, combined with humidity control, creates a stable rhizosphere microenvironment during the post-treatment recovery period, enabling double-treated plants to quickly recover physiological functions, improving the recovery speed from pesticide damage and the survival rate of seedlings, laying a good foundation for subsequent hardening-off and field transplanting.

[0051] Example 1 (0.5 g / L colchicine + 20 mL / L dimethyl sulfoxide, treatment for 10 h)

[0052] Material preparation

[0053] Reed (Tabernae lanceolata) taken from the tissue culture room Arundo donax L.) Tissue culture rooted seedlings, 40 days old, approximately 9cm tall, with 5-6 leaves, white roots 2.5cm long, free from disease and pests. Carefully remove with tweezers, remove any culture medium residue, and rinse with sterile water.

[0054] Seedlings

[0055] Place the cleaned tissue culture seedlings in a 250mL beaker, add 200mL of sterile distilled water, maintain the acclimation temperature at 20℃, and place them in an environment with a light intensity of approximately 1000lx for 2 days. Change the water once a day during the acclimation period.

[0056] Preparation of drug solution

[0057] Prepare a 1L Erlenmeyer flask and add 20mL of dimethyl sulfoxide (20mL / L concentration).

[0058] Weigh 0.5g of colchicine and place it in a dimethyl sulfoxide solution. Stir magnetically for 20 minutes until it is completely dissolved.

[0059] Add Tween-20 to a final concentration of 0.05% (v / v) as a penetration enhancer and wetting agent;

[0060] Add sterile distilled water until the total liquid volume reaches 1L, mix well and set aside.

[0061] Double processing

[0062] After the tissue culture seedlings have recovered from transplant shock, immerse the base of the stem in the solution to a depth of 3 cm and place them in a constant temperature culture environment at 20±1℃ and 1200lx light intensity for 10 hours.

[0063] Rinsing and Seedling Establishment

[0064] After treatment, immediately remove the seedlings, rinse them with running water for 7 minutes, and then rinse them 3 times with sterile water.

[0065] Transplant the seedlings into 50-cell trays filled with substrate (peat:vermiculite = 1:1), keep the substrate in each cell moist, and place them in an environment with 60% shading for 10 days to allow them to recover. The substrate moisture content should be controlled at 65-75% water saturation.

[0066] Hardening off seedlings and transplanting to the field

[0067] After the seedlings have recovered from transplant shock, they are moved into a greenhouse at 20-25℃ for 2.5 months; then hardened off for 10 days, during which the humidity gradually decreases from 85% to 70% and the light intensity gradually increases; finally, they are transplanted to the field with a row spacing of 70cm and a plant spacing of 140cm for conventional management.

[0068] Test results

[0069] After three months of field growth, new root tips were randomly selected and analyzed using the chromosome squash method. The results showed that the number of chromosomes increased from 108 to 216, a doubling rate of 16%; the plant survival rate was 95%; and the average leaf width, stem diameter, and plant height of the doubled plants increased by 18%, 14%, and 10% respectively.

[0070] Example 2 (Control: 0.5 g / L colchicine, without dimethyl sulfoxide or Tween-20, treated for 10 h)

[0071] Material preparation

[0072] Take rooted reed seedlings from tissue culture, 40 days old, 9cm tall, with roots 2.5cm long, and 5-6 leaves, healthy and free from disease. Remove any remaining culture medium and rinse thoroughly with sterile water.

[0073] Seedlings

[0074] Place the seedlings in a beaker, add 200mL of sterile water, and allow them to recover at 20℃ for 2 days. Change the water once a day and provide light at an intensity of about 1000lx.

[0075] Preparation of drug solution

[0076] Dissolve 0.5g of colchicine in a small amount of sterile distilled water and stir magnetically for 20 minutes;

[0077] No dimethyl sulfoxide or Tween-20 is added;

[0078] Dilute to 1L to prepare a 0.5g / L colchicine aqueous solution.

[0079] Double processing

[0080] After the tissue culture seedlings have recovered from transplant shock, immerse the base of the stem in a 3cm deep solution and treat them at 20℃ with a light intensity of 1200lx for 10 hours.

[0081] Rinsing and Seedling Establishment

[0082] After treatment, rinse with running water for 7 minutes, then rinse 3 times with sterile water. Transplant the seedlings into a peat moss:vermiculite = 1:1 substrate in 50-cell trays, and provide 60% shade for 10 days to allow them to recover.

[0083] Hardening off seedlings and transplanting to the field

[0084] The conditions are the same as in Example 1.

[0085] Test results

[0086] Survival rate dropped to 85%; doubling rate was only 9%; most doubled seedlings showed leaf tip discoloration and localized yellowing, and the degree of phytotoxicity was significantly higher than in Example 1, proving that the addition of dimethyl sulfoxide can effectively reduce phytotoxicity and improve doubling efficiency.

[0087] Example 3 (Low concentration scheme: 0.25 g / L colchicine + 20 mL / L dimethyl sulfoxide, treatment for 10 h)

[0088] Material preparation

[0089] The 40-day-old Reed Phragmites communis tissue culture seedlings are 8.5cm tall with roots 2.8cm long and are healthy and free from pests and diseases. Remove the culture medium and wash with sterile water.

[0090] Seedlings

[0091] Place the seedlings in 200mL of sterile water at 20℃ for 2 days to allow them to recover. Change the water daily and provide approximately 1000lx of light.

[0092] Preparation of drug solution

[0093] Add 0.25 g of colchicine to 20 mL of dimethyl sulfoxide solution;

[0094] Stir magnetically for 20 minutes until dissolved;

[0095] Tween-20 final concentration 0.05% (v / v);

[0096] Distilled water was brought to a final volume of 1L, resulting in a colchicine concentration of 0.25g / L.

[0097] Double processing

[0098] Immerse the base of the stem in a 3cm deep solution and treat for 10 hours at 20℃ and 1200lx light.

[0099] Rinsing and Seedling Recovery

[0100] After treatment, rinse with running water for 7 minutes, rinse with sterile water 3 times, and transplant into 50-cell trays with a peat:vermiculite ratio of 1:1. Shade the trays for 10 days to allow them to recover.

[0101] hardening off and transplanting

[0102] Same as Example 1.

[0103] Test results

[0104] The survival rate was 98%, the doubling rate was 10%, and the phytotoxicity was slight. However, the doubling rate was 37.5% lower than the optimal scheme, indicating that the low concentration limited the doubling efficiency.

[0105] Example 4 (High-concentration short-time treatment: 1.0 g / L colchicine + 20 mL / L dimethyl sulfoxide, treatment for 5 h)

[0106] Material preparation

[0107] The rooted seedlings of Reed japonica, 40 days old, are 9cm tall with roots 2.6cm long. They were cleaned after removing the culture medium.

[0108] Seedlings

[0109] Use 200mL of sterile water to allow the seedlings to recover for 2 days at a temperature of 20℃, and change the water once a day.

[0110] Preparation of drug solution

[0111] Add 1.0 g of colchicine to 20 mL of dimethyl sulfoxide solution and stir magnetically for 20 minutes;

[0112] Add Tween-20 to a final concentration of 0.05% (v / v);

[0113] Distilled water was added to a final volume of 1L.

[0114] Double processing

[0115] Immerse the base of the stem in 3cm of solution, at 20℃, under 1200lx light, for only 5 hours.

[0116] Rinsing and Seedling Recovery

[0117] The rinsing steps are the same as before. Transplant the seedlings into seedling trays and shade them for 10 days to allow them to recover.

[0118] hardening off and transplanting

[0119] Same as Example 1.

[0120] Test results

[0121] With a doubling rate of 14% and a survival rate of 90%, leaf tip scorching was obvious. Although high concentrations shortened the treatment time, the risk of phytotoxicity increased.

[0122] Example 5 (Intermittent treatment: 0.5 g / L colchicine + 20 mL / L dimethyl sulfoxide, 6 h + rinse + 4 h)

[0123] Material preparation

[0124] Tissue culture seedlings of the same specifications as above.

[0125] Seedlings

[0126] As before, use 200mL of sterile water and 20℃ for 2 days to allow the seedlings to recover, changing the water once a day.

[0127] Preparation of drug solution

[0128] The same preparation method as in Example 1.

[0129] Double processing

[0130] First, treat for 6 hours → rinse for 5 minutes → then treat with the same solution for 4 hours. Environmental control: 20℃, light intensity: 1200 lx.

[0131] Rinsing and Seedling Recovery

[0132] Rinse and allow the seedlings to recover for 10 days using the methods described above.

[0133] hardening off and transplanting

[0134] Same as Example 1.

[0135] Test results

[0136] With a survival rate of 96% and a doubling rate of 15%, the pesticide damage index decreased by 20% compared to continuous treatment, making it suitable for production scenarios where survival rate is a key concern.

[0137] Example 6 (Alternative penetrant solution: 0.5 g / L colchicine + 1% glycerin, treatment for 10 h)

[0138] Material preparation

[0139] Same as before.

[0140] Seedlings

[0141] Same as before.

[0142] Preparation of drug solution

[0143] Add 0.5g of colchicine to a glycerol solution (1%, v / v) and stir magnetically for 20 minutes;

[0144] Tween-20 final concentration 0.05% (v / v);

[0145] Bring the volume to 1L.

[0146] Double processing

[0147] Immerse the base of the stem in 3cm of medicinal solution, at 20℃, under 1200lx10 hours of light.

[0148] Rinsing and Seedling Recovery

[0149] Same as before.

[0150] hardening off and transplanting

[0151] Same as before.

[0152] Test results

[0153] The survival rate was 93%, and the doubling rate was 13%, which was better than the no-penetrating agent solution, but not as good as the dimethyl sulfoxide solution.

[0154] Chromosome Count Detection Results Statistical Table

[0155] Treatment concentration Processing time Number of plants treated Number of surviving plants Polyploid plant number Multiplier 250mg / L 2h 50 50 0 0.00% 500mg / L 2h 50 45 0 0.00% 1000mg / L 2h 50 40 2 4.00% 250mg / L 5h 48 47 1 2.08% 500mg / L 5h 50 47 2 4.00% 1000mg / L 5h 65 41 10 15.38% 250mg / L 10h 50 41 1 2.00% 500mg / L 10h 50 44 8 16.00% 1000mg / L 10h 48 31 4 8.33% 250mg / L 20h 50 37 7 14.00% 500mg / L 20h 50 26 2 4.00% 1000mg / L 20h 49 20 4 8.16%

[0156] By following the steps above, you can obtain doubled Reed Plants, with a success rate of 16%.

[0157] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for doubling chromosomes in Phragmites australis, characterized in that: Includes the following steps: (1) Select Reed tissue culture seedlings with a height of 8-10cm, free from pests and diseases, and with strong roots. Remove the culture medium and cut off part of the roots, leaving only 2-3cm. (2) After pruning, the tissue culture seedlings should be placed in clean water to recover for 1-3 days, and the water should be changed 1-2 times a day; (3) Prepare a colchicine solution containing 0.25-1.0 g / L colchicine and 10-30 mL / L dimethyl sulfoxide, preferably 0.5 g / L colchicine and 20 mL / L dimethyl sulfoxide; (4) Immerse the base of the tissue culture seedling stem in the solution to a depth of 2-4 cm and treat for 5-20 hours under constant temperature of 20±1℃ and light intensity of 1000-1500lx. (5) After treatment, rinse with running water for 5-10 minutes, then rinse with sterile water 2-3 times, and place in a 50-well tray in culture medium to allow seedlings to recover for 7-15 days. (6) After the seedlings have recovered, they should be cultured for 2-3 months under controlled temperature (20-25℃) and moderate light conditions; (7) Transplant the seedlings to the field. After the plants have grown steadily, take stem tip or root tip tissue for chromosome testing and screen to obtain doubled plants.

2. The method for chromosome doubling in *Arundo donax* according to claim 1, characterized in that: The colchicine solution is further supplemented with one or more penetration enhancers selected from Tween-20, glycerin, or polyethylene glycol.

3. The method for chromosome doubling in *Arundo donax* according to claim 1, characterized in that: When preparing the drug solution, colchicine is first dissolved in dimethyl sulfoxide solution, and then diluted with distilled water to the target concentration.

4. The method for chromosome doubling in *Arundo donax* according to claim 1, characterized in that: During the seedling establishment process, add either 0.1‰ carbendazim or 0.05% Cabinda slow-release preservative to the water.

5. The method for chromosome doubling in *Arundo donax* according to claim 1, characterized in that: The colchicine solution is subjected to ultrasonic vibration for 10-20 minutes or magnetic stirring for 15-30 minutes before use.

6. The method for chromosome doubling in *Arundo donax* according to claim 1, characterized in that: The drug treatment is in an intermittent mode, first treating for 3-10 hours, then rinsing once before treating for the remaining time.

7. The method for chromosome doubling in *Arundo donax* according to claim 1, characterized in that: Before transplanting to the field, the seedlings of Phragmites australis are hardened off in the hardening-off area for 7-14 days, during which the ambient humidity is controlled at 70-85% and the light intensity is gradually increased.

8. The method for chromosome doubling in *Arundinaria salsa* according to claim 1, characterized in that: The seedling trays are 50-well, with each well having a volume of 20-25 mL. The substrate moisture content is maintained at 65-75% of the substrate's water saturation.

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