Method for disinfecting and aseptically sowing rhododendron micranthum seeds

Abstract

The invention belongs to the technical field of plant seedling raising, and discloses a rhododendron micranthum seed disinfection and sterile sowing method, which comprises the following steps: screening seeds to obtain rhododendron micranthum seeds with the purity of more than or equal to 90%; washing with normal-temperature water until the water absorption rate of the seeds reaches 15-18% Sterile seeds are obtained through alcohol disinfection and bromogeramine disinfection; inoculating the disinfected seeds into a germination culture medium, and culturing in an illumination incubator to obtain aseptic seedlings. According to the method, double disinfection is combined with sterile operation, so that the seed pollution rate is remarkably reduced, and the disinfection safety is improved; an optimized culture medium formula and precise light and temperature conditions are adopted, so that the seed germination rate is increased to be greater than or equal to 90%, the germination time is shortened to 7 days, and the germination is neater and quicker than that of traditional soil sowing; robust transplantable seedlings can be obtained after sterile culture is performed for 3 months, the seedling raising period is greatly shortened, meanwhile, the seedling death risk caused by environment fluctuation and plant diseases and insect pests in traditional seedling raising is reduced, and reliable technical support is provided for efficient breeding of high-quality rhododendron micranthum seedlings.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant seedling raising, and relates to a method for disinfecting and aseptically sowing Rhododendron micranthum Turcz. seeds. Background Art

[0002] Rhododendron micranthum Turcz. Rhododendron micranthum ), also known as small-flowered rhododendron, is an evergreen shrub of the genus Rhododendron in the family Ericaceae. It is 1-2 meters tall, with leathery leaves, small and dense white flowers, flowering from May to June, and fruiting from September to October. As a key species in the mountain ecosystem of Beijing, Rhododendron micranthum Turcz. plays an important role in soil and water conservation and biodiversity protection. Its evergreen characteristics add a touch of vitality to the mountains in winter and become an important symbol of Beijing's natural heritage. As a native evergreen broad-leaved flowering shrub in Beijing, it is distributed in Qupotou in Miyun District, Jinshan in Huairou District and Haidian District, Shangfang Mountain in Fangshan District, Baihua Mountain and Dongling Mountain in Mentougou District, and Pinggu and Yanqing. However, they are all wild species and have not been artificially bred and promoted yet. Therefore, it is urgent to carry out large-scale breeding, development and promotion of it.

[0003] Research by Zhang Jingli et al. shows that there are significant differences in the germination rates of different rhododendron varieties under the same substrate conditions. For example, the germination rate of Rhododendron decorum Franch. in leaf mold soil reaches 60%, while that of Rhododendron agastum Balf. f. et W. W. Smith, Rhododendron irroratum Franch., and Rhododendron delavayi Franch. is only 9%, 8%, and 5% respectively; the germination rate of all varieties in the mountain soil substrate of the mixed soil approaches zero. This difference stems from the diversity of seed biological characteristics (such as seed coat structure, dormancy mechanism) and nutritional requirements. Rhododendron decorum Franch. is more adaptable to the humus and microbial environment in leaf mold soil, while other varieties may rely on specific symbiotic bacteria or more demanding temperature and humidity conditions. Their research results suggest that rhododendron seedling raising needs to select a suitable substrate according to the variety characteristics. A single general-purpose substrate is difficult to meet the needs of multiple varieties, and precise regulation needs to be carried out in combination with the characteristics of the seed source.

[0004] The seeds of Rhododendron micranthum Turcz. are extremely small (the thousand-grain weight is only 0.25049 grams), with a low germination rate and difficult seedling establishment, which is the primary problem in the seedling raising process. Zhang Haiqin et al. summarized a set of sowing and seedling raising techniques for Rhododendron micranthum Turcz., pointing out that: (1) For seed treatment, the soaking time (soaking in 0.1% potassium permanganate solution for 30 min and then in warm water for 24 h), temperature, and germination environment (23-27°C) need to be strictly controlled. A slight mistake will affect the germination rate.

[0005] (2) The management of the seedling stage is complex. The water (spray watering, soil water content 30%), light (the light transmittance of the sunshade net is 50%), and temperature (15-28°C in the greenhouse) need to be precisely controlled. Otherwise, it is easy to cause seedling burns or waterlogging and death.

[0006] (3) In addition, the seedlings have poor stress resistance and are sensitive to pests, diseases (such as damping-off and black spot) and environmental changes. It is necessary to frequently spray fungicides and adjust the soil pH (improvement with pine needles and treatment with ferrous sulfate), which is cumbersome and requires high technical skills. The alkaline soil is prone to causing iron-deficiency chlorosis, and additional intervention is needed, further increasing the management difficulty.

[0007] In the process of implementing the present invention, the inventors repeatedly verified the method proposed by Zhang Haiqin et al. and found that the germination rate of Rhododendron micranthum Turcz. seeds sown and raised in nursery is still relatively low (<69%), and the emergence time is late (≥15 days, consistent with their experimental results), the germination duration is 10 days, the cycle is long, and the germination rate and emergence rate are uneven. The inventors found that factors such as the type of substrate, sowing depth, humidity, and temperature will all affect the germination rate and emergence uniformity of Rhododendron micranthum Turcz. seeds. Even for the same batch of seeds, under different environmental conditions, there are differences in germination time and growth rate, resulting in uneven growth of seedlings and further increasing the difficulty of later management. Therefore, the sowing and raising seedling technology of Rhododendron micranthum Turcz. still needs further research. Summary of the Invention

[0008] In view of this, the present invention aims to provide a method for disinfecting and aseptically sowing Rhododendron micranthum Turcz. seeds. By optimizing the seed disinfection treatment and aseptic sowing conditions, the problems of low germination rate, late emergence time and uneven emergence in the prior art are solved, so as to achieve efficient, rapid and uniform seedling raising effect and improve the breeding efficiency and quality of Rhododendron micranthum Turcz.

[0009] Through long-term exploration and attempts, as well as multiple experiments and efforts, and continuous reform and innovation, the inventors provide the following technical solution to solve the above technical problems: a method for disinfecting and aseptically sowing Rhododendron micranthum Turcz. seeds, including the following steps: 1) Seed disinfection treatment: S1. Seed screening: Screen the seeds to obtain Rhododendron micranthum Turcz. seeds with a purity ≥90%; S2. Seed washing: Wash the screened seeds with normal temperature water to make the water absorption rate of the seeds reach 15% - 18%; S3. Alcohol disinfection: Place the washed seeds in a sterile centrifuge tube, disinfect with disinfected alcohol for 50 - 70 seconds, and then rinse with sterile water; S4. Bromogeramine disinfection: Disinfect the seeds after alcohol disinfection with 0.1% bromogeramine disinfectant solution for 8 - 12 minutes, and then rinse with sterile water; 2) Aseptic sowing of seeds: Inoculate the disinfected seeds on the germination medium, and the germination medium is 1 / 2WPM basic medium, adding 8 g / L of agar and 30 g / L of sucrose, and adjusting the pH value to 5.7; Cultivate under the conditions of 25±1°C, light intensity of 3000 - 4000 LX, and photoperiod of 16 hours / 8 hours. The seeds start to germinate on the 7th day, with a germination rate ≥ 90%. Sterile seedlings suitable for transplantation can be obtained after 3 months of cultivation.

[0010] Compared with the prior art, the beneficial effects of the present invention are as follows: By combining double disinfection with alcohol and bromogeramine and aseptic operation, the present invention significantly reduces the seed contamination rate and improves the disinfection safety. The optimized 1 / 2WPM medium formula and precise light and temperature conditions increase the seed germination rate to ≥ 90%, and the germination time is shortened to 7 days. The germination is more uniform and faster than that of traditional soil sowing. Robust seedlings suitable for transplantation can be obtained after 3 months of aseptic culture, greatly shortening the seedling raising cycle. At the same time, it reduces the risk of seedling death caused by environmental fluctuations and pests and diseases in traditional seedling raising, providing reliable technical support for the efficient breeding of high-quality Rhododendron micranthum Turcz. seedlings.

[0011] Based on the above technical solutions, the present invention can be further improved as follows: Furthermore: The seed screening uses test sieves with gradient sieve hole sizes, and separation is achieved by utilizing the particle size differences among fruits, seeds, and impurities.

[0012] Compared with the prior art, the beneficial effects of adopting the above further technical solution are as follows: By screening and separating seeds with test sieves of gradient sieve hole sizes, seeds can be separated from fruits and impurities more efficiently, significantly improving the seed purity to ≥ 95%. At the same time, it reduces manual intervention and the number of screening times, and lowers the production cost.

[0013] Based on the above technical solutions, the present invention can be further improved as follows: Furthermore: In the step S2, the water temperature of the normal temperature water is 25±2°C.

[0014] Compared with the prior art, the beneficial effects of adopting the above further technical solution are as follows: By precisely controlling the water temperature of the normal temperature water at 25±2°C, the water absorption rate of the seeds and the stability of the cell membrane can be effectively maintained, avoiding uneven water absorption or physiological damage of the seeds caused by water temperature fluctuations. Thus, the seed disinfection efficiency and the subsequent germination uniformity are significantly improved.

[0015] Based on the above technical solutions, the present invention can be further improved as follows: Furthermore: In the step S2, the seeds are continuously rinsed in a constant temperature water flow with a flow rate of 2 - 3 L / min and a temperature of 25±2°C for 60±5 min.

[0016] Compared with the prior art, the beneficial effects of adopting the above further technical solution are as follows: By continuously flushing with a constant temperature (25 ± 2 °C) and controlled flow rate (2 - 3 L / min) for 60 ± 5 min, it is possible to uniformly remove impurities on the seed surface and precisely control the water absorption rate (15% - 18%), avoiding seed damage or uneven water absorption caused by unstable water flow or temperature fluctuations in traditional flushing, thereby improving the disinfection effect and germination consistency and further reducing the pollution risk.

[0017] Preferably: The alcohol concentration of the disinfection alcohol is 75%.

[0018] Based on the above technical solutions, the present invention can also be improved as follows: Further: In the above steps S2 and S3, vortex oscillation disinfection is adopted.

[0019] Compared with the prior art, the beneficial effects of adopting the above further technical solutions are: Vortex oscillation disinfection can enhance the contact uniformity between the disinfectant and the seed surface, effectively remove microorganisms attached covertly, reduce the disinfection blind area, thereby significantly reducing the risk of secondary pollution and shortening the disinfection time.

[0020] Based on the above technical solutions, the present invention can also be improved as follows: Further: No plant growth regulator is added to the germination medium.

[0021] Compared with the prior art, the beneficial effects of adopting the above further technical solutions are: No plant growth regulator is added to the germination medium, which not only avoids problems such as seedling spindling, malformation or abnormal root development that may be caused by exogenous hormones, but also can, through precise regulation of basic nutrition and light and temperature conditions, prompt the seeds to achieve natural germination and robust growth relying on their own metabolism, thereby simplifying the culture process, reducing production costs, and at the same time improving the ecological adaptability and transplanting survival rate of the seedlings.

[0022] Based on the above technical solutions, the present invention can also be improved as follows: Further: In the step of aseptic sowing of seeds, a pipette gun is used to adsorb the seeds and then evenly spread them on the surface of the germination medium.

[0023] Compared with the prior art, the beneficial effects of adopting the above further technical solutions are: By precisely adsorbing and evenly spreading the seeds with a pipette gun, problems such as seed accumulation or omission in traditional manual sowing can be avoided, ensuring that each single seed is in full contact with the medium, significantly improving the germination uniformity and seedling quality, and at the same time reducing the risks of mechanical damage and operation pollution.

[0024] Based on the above technical solutions, the present invention can also be improved as follows: Furthermore: During the cultivation process, cotyledons, hypocotyls, and radicles of the aseptic seedlings emerged on the 30th day, and after 2 months of cultivation, the seedling height was ≥ 2 cm and the root system was well-developed.

[0025] Compared with the prior art, the beneficial effects of adopting the above further technical solution are as follows: By clarifying that the differentiation of cotyledons, hypocotyls, and radicles of the aseptic seedlings is completed on the 30th day and the growth standard of seedling height ≥ 2 cm and well-developed root system is achieved within 2 months, the stability and efficiency of the cultivation system can be quantitatively verified. The seedling formation period is significantly shortened by about 50% compared with the traditional seedling cultivation method. At the same time, it ensures the complete morphological development of the seedlings and strong roots, providing a higher survival guarantee for subsequent transplantation.

[0026] On the basis of the above technical solution, the present invention can be further improved as follows: Furthermore: In the method, the contamination rate ≤ 2%, and the seed survival rate ≥ 90%.

[0027] Compared with the prior art, the beneficial effects of adopting the above further technical solution are as follows: By strictly controlling the contamination rate ≤ 2% and increasing the seed survival rate to ≥ 90%, the reliability of the aseptic seedling cultivation system is significantly optimized, and the pollution loss is reduced by about 80% compared with the traditional technology. At the same time, it ensures the robustness of the seedlings and the stability of the physiological state, providing a standardized technical solution with high survival rate and low risk for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention, and thus should not be regarded as limiting the scope. For those of ordinary skill in the art, other relevant drawings can be obtained based on these drawings without creative efforts.

[0029] Figure 1 It shows the Rhododendron micranthum Turcz. seeds after screening treatment in a preferred embodiment of the present invention.

[0030] Figure 2 It shows the process of disinfecting the seeds using a centrifuge tube in a preferred embodiment of the present invention.

[0031] Figure 3 It presents the operation of evenly spreading the disinfected seeds on the germination medium in a preferred embodiment of the present invention.

[0032] Figure 4 It records the situation where the seeds start to germinate on the 7th day after sowing in a preferred embodiment of the present invention.

[0033] Figure 5It shows the growth state of the aseptic seedlings on the 30th day after sowing in a preferred embodiment of the present invention.

[0034] Figure 6 It shows the robust aseptic seedlings after growing for 2 months in a preferred embodiment of the present invention. Detailed implementation manners

[0035] The following is an illustration with specific embodiments.

[0036] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed present invention, but merely represents selected embodiments of the present invention.

[0037] In the present invention, unless otherwise specified, all equipment and raw materials can be purchased from the market or are commonly used in this industry. The methods in the following embodiments, unless otherwise specified, are conventional methods in this field.

[0038] In this embodiment, the germination medium is 1 / 2WPM basal medium, added with 8 g / L of agar, 30 g / L of sucrose, and the pH value is adjusted to 5.7; no plant growth regulator is added.

[0039] In this embodiment, the seeds of Rhododendron micranthum Turcz. are taken from the Daan Mountain Forest Farm in Mentougou District, Beijing, with the geographical coordinates of 39°90’N, 115°74’E and an altitude of 1,245 meters. Select healthy plants of Rhododendron micranthum Turcz. with plump fruits and no diseases and pests, and collect their capsules. After collection, place the capsules in a ventilated, dry and cool place to dry. After the capsules naturally crack, collect the seeds.

[0040] The steps of disinfection and aseptic sowing of the seeds of Rhododendron micranthum Turcz. are as follows: (1)Seed screening: Use a test sieve to screen the collected seeds.

[0041] Select a test sieve with an appropriate pore size to separate the seeds from fruit residues, impurities, etc., and obtain seeds of Rhododendron micranthum Turcz. with a purity ≥ 90%. See Figure 1 . The screened seeds should be stored in a clean and dry container for standby.

[0042] During the treatment process of the seeds of Rhododendron micranthum Turcz., the inventor has tried a variety of different treatments.

[0043] For example, without undergoing screening treatment, the fruits are directly disinfected, and then the seeds are separated from the disinfected fruits for sowing. However, this method has many drawbacks.

[0044] First of all, it is very difficult to separate the seeds after the fruits are disinfected. The structure of the fruits is relatively complex. After disinfection, the fruits become softened and sticky, etc., resulting in difficulty in separating the seeds from parts such as the pericarp and pulp, increasing the difficulty and workload of seed extraction and reducing the operation efficiency. Secondly, the storage property of the fruits is poor. During long-term storage, the fruits are prone to cracking, which not only causes the seeds to scatter naturally, resulting in loss and difficulty in collecting the seeds, but also exposes the seeds to the external environment, increasing the risk of contamination and being unfavorable to the smooth progress of long-term experiments.

[0045] Therefore, during the processing of Rhododendron micranthum Turcz. seeds, removing impurities is one of the key steps to ensure the success of disinfection and aseptic sowing of Rhododendron micranthum Turcz. seeds, and it is of great significance for improving the efficiency and quality of the entire seedling-raising process.

[0046] By using a test sieve for screening treatment, the seeds can be effectively separated from fruit residues, impurities, etc., and Rhododendron micranthum Turcz. seeds with a purity ≥ 90% can be obtained. This process not only ensures the high efficiency and uniformity of the subsequent disinfection process, but also lays a solid foundation for the aseptic sowing and healthy growth of the seeds.

[0047] The seeds after removing impurities are purer, which helps to improve the disinfection effect, reduce the influence of potential pathogens and pollutants on seed germination, and thus improve the seed vigor and germination rate.

[0048] In addition, the pure seeds are conducive to achieving uniform distribution after sowing, which is beneficial to the neat growth of seedlings and convenient for subsequent management operations.

[0049] (2) Seed washing: The screened seeds are placed in a constant-temperature water flow with a flow rate of 2 - 3 L / min and a temperature of 25 ± 2 °C and continuously rinsed for 60 ± 5 minutes to make the seed water absorption rate reach 15% - 18%.

[0050] In this step, through the action of hydraulic shearing, the tiny impurities remaining on the seed surface can be completely removed, ensuring a clean seed surface and creating good conditions for subsequent disinfection treatment. A clean seed surface helps the disinfectant to come into close contact with the seeds, improving the disinfection effect and reducing the potential threat of pathogenic bacteria to seed germination. Secondly, by controlling the flow rate and temperature of the flushing water, the water absorption rate of the seeds can reach 15% - 18% while cleaning, achieving the coordinated treatment of cleaning and seed soaking. An appropriate water absorption rate can not only provide the necessary moisture for the subsequent germination of the seeds, but also avoid difficulties in seed germination caused by insufficient water absorption or seed damage caused by excessive water absorption, thereby improving the vitality and germination rate of the seeds and ensuring the smooth progress of the seedling raising process.

[0051] (3) Alcohol disinfection: Place the washed seeds in a laminar flow hood. Use sterile forceps to put the seeds into a 5 - 10 mL sterile centrifuge tube. Add 75% alcohol solution to completely immerse the seeds. Use a vortex oscillator to shake and disinfect for 60 ± 5 seconds. After disinfection, rinse with sterile deionized water 3 times. After each rinse, pour out the liquid in the centrifuge tube to ensure that there is no residual alcohol on the seed surface. The rinsed seeds need to be placed in a sterile environment for subsequent disinfection treatment.

[0052] Disinfecting alcohol has strong permeability and volatility, and can quickly penetrate into the tiny gaps on the seed surface, destroying the cell membrane structure of pathogenic bacteria, thereby effectively killing microorganisms such as bacteria, fungi, and viruses attached to the seed surface. Its rapid bactericidal property can immediately reduce the microbial load on the seed surface, laying the foundation for subsequent disinfection steps.

[0053] (4) Bromogeramine disinfection: Put the seeds that have been disinfected with alcohol back into a sterile centrifuge tube, add 0.1% bromogeramine disinfectant solution to completely immerse the seeds, see Figure 2 . Use a vortex oscillator to shake and disinfect for 10 ± 1 minute. After disinfection, rinse with sterile deionized water 8 times until there is no foam residue. The rinsed seeds need to be placed in a sterile environment again and prepared for sowing.

[0054] As a comparison, this example also used different disinfection treatments to disinfect Rhododendron mucronatum seeds: ① Use 0.1% sodium hypochlorite, and the disinfection time is 8, 10, 12 min; ② Treat with 1% bromogeramine disinfectant solution, and the disinfection time is 8, 10, 12 min; Sow the disinfected seeds into the germination medium. For each treatment, inoculate 10 bottles, with 10 seeds in each bottle, and repeat 3 times.

[0055] After inoculation, observe and record the contamination rate and germination rate of the explants. The results are shown in Table 1.

[0056] Table 1 Effects of Different Disinfection Methods and Times on the Disinfection Effect of Seeds Note: In Table 1, the disinfection time 1 + 8 means 75% alcohol disinfection for 1 minute and 1% benzalkonium bromide or 0.1% sodium hypochlorite disinfection for 8 minutes.

[0057] According to Table 1, different disinfection times and methods of Rhododendron micranthum Turcz. seeds have different effects on them.

[0058] Benzalkonium bromide (0.1% concentration) is a cationic surfactant with broad-spectrum antibacterial activity. Its bactericidal effect is mild and persistent. On the basis of preliminary alcohol disinfection, benzalkonium bromide can further penetrate into the fine parts of the seed surface and continuously kill the pathogenic bacteria that may not be completely removed by alcohol. At the same time, the protective film formed by benzalkonium bromide on the seed surface can inhibit the growth of microorganisms for a long time and reduce the risk of seed contamination during subsequent operations.

[0059] The combined disinfection method of 75% alcohol + 0.1% benzalkonium bromide for 10 minutes can not only significantly improve the disinfection effect, but also minimize the damage to seed viability. The rapid volatility of alcohol avoids the seeds being soaked in high-concentration disinfectants for a long time, and the mild bactericidal characteristics of benzalkonium bromide will not damage the embryo of the seeds. Through the synergistic effect of the two, it not only ensures the thorough cleaning of the seed surface, but also protects the integrity and germination ability of the seeds, thus providing a strong guarantee for the aseptic sowing and healthy germination of Rhododendron micranthum Turcz. seeds.

[0060] 0.1% sodium hypochlorite is also one of the commonly used disinfectants for seed disinfection. It has a certain disinfection effect on seeds, but its strong oxidizing property may cause damage to the seeds, resulting in problems such as reduced seed germination rate and poor seedling growth. The seeds of Rhododendron micranthum Turcz. are extremely small, and sodium hypochlorite may damage the embryo of the seeds, affecting the viability and normal germination of the seeds, resulting in the loss of seed activity or a decrease in germination ability. Its disinfection effect is not stable enough. Too long treatment time or too high concentration may have a toxic effect on the seeds, and even kill the beneficial microorganisms inside the seeds, affecting the subsequent growth of the seeds.

[0061] The inventor also tried to disinfect the seeds of Rhododendron micranthum Turcz. with other commonly used disinfectants. For example, HgCl2 will make more seeds lose their activity, and the germination rate is lower than that of sodium hypochlorite.

[0062] Therefore, based on a comprehensive consideration of the test results, it was finally determined that the best disinfection plan for Rhododendron micranthum seeds was to select the disinfection time of (1 + 10) min in the disinfection method of 75% alcohol + 1% bromogeramine. Using this plan for disinfection not only had a relatively low pollution rate of only 2%, but also ensured a seed survival rate of 90%.

[0063] (5)Sterile sowing of seeds: Screening of basic media: The disinfected Rhododendron micranthum seeds were inoculated onto four different germination media, namely MS, 1 / 2MS, WPM, and 1 / 2 WPM. For each treatment, 10 bottles were inoculated, with 10 seeds in each bottle, and the experiment was repeated 3 times. Observe and record the germination time and germination rate of Rhododendron micranthum seeds, and the results are shown in Table 2.

[0064] Table 2 Effects of different basic media treatments on seed germination According to Table 2, when using the 1 / 2WPM medium: The germination time was the earliest: The seeds started to germinate on the 7th day after sowing, which was 2 days, 5 days, and 5 days earlier than the WPM, 1 / 2MS, and MS media respectively.

[0065] The germination rate was the highest: The germination rate reached 90%, which was 5 percentage points higher than that of the WPM medium, 28 percentage points higher than that of the 1 / 2MS medium, and 26 percentage points higher than that of the MS medium, respectively, compared with other media.

[0066] The continuous germination time was the shortest: The continuous germination time was 5 days, which was the shortest among all media, indicating that the seeds germinated most neatly in this medium, and most seeds germinated concentratedly within a short period of time.

[0067] During the aseptic sowing and seedling raising process of Rhododendron micranthum Turcz. seeds, an early germination time means that the seeds can initiate growth in a shorter time. This not only helps to enter the seedling growth stage earlier, but also effectively shortens the entire seedling raising cycle, improves the seedling raising efficiency, and enables the seedlings to reach the transplanting standard faster. A high germination rate indicates that more seeds among a certain number of seeds can successfully germinate and grow into seedlings, thus significantly increasing the success rate of seedling raising and the number of seedlings, and reducing the waste of resources and cost increase caused by non-germinating seeds. A short continuous germination time indicates that the seeds germinate concentratedly in a shorter time. This is conducive to cultivating a uniform seedling population, facilitating subsequent unified management, such as fertilization, watering, pest and disease control, etc. operations. At the same time, it also provides a guarantee for the synchronous growth and development of the seedlings, helping to improve the overall quality and consistency of the seedlings. The advantages of this embodiment in these three aspects jointly lay a solid foundation for the large-scale breeding and promotion of Rhododendron micranthum Turcz., and improve the overall efficiency and competitiveness of the seedling raising work.

[0068] Screening test of germination medium: The disinfected seeds were inoculated into the medium for germination culture. The test used 1 / 2WPM as the basic medium, and TDZ and NAA were added respectively. The concentration of TDZ was set at 0.5, 1.0, 1.5, and the concentration of NAA was set at 0.5, 1.0, 1.5. There were 9 treatments in total for the test. 20 bottles were inoculated for each treatment, and 30 seeds were inoculated in each bottle. The experiment was repeated 3 times. The germination time and germination rate were observed and recorded. The results are shown in Table 3.

[0069] Table 3 Effects of different plant growth regulator treatments on seed germination It can be concluded from Table 3 that after adding different concentrations and types of plant growth regulators, the germination time of the seeds is even later and the germination rate is lower. However, for the treatment using only 1 / 2WPM as the germination medium without adding other plant growth regulators, the germination time is only 7 days, and the germination rate is as high as 90%. And during the subsequent growth process of the aseptic seedlings, we observed that the aseptic seedlings added with plant growth additives grew slowly, their cotyledons grew in a curled shape, the hypocotyls were relatively short, and no radicles appeared. While the aseptic seedlings grown with only 1 / 2WPM as the germination medium grew well. At the 30th day of germination, obvious cotyledons, hypocotyls and radicles appeared in the aseptic seedlings, and there was no need to replace other types of media or plant growth additives during the subsequent growth process.

[0070] In the cultivation of Rhododendron micranthum Turcz., it is a better choice not to add plant growth regulators. Experimental data shows that when using 1 / 2 WPM medium without adding any plant growth regulators, the seeds start to germinate on the 7th day, and the germination rate is as high as 90%. However, in the treatment groups with different concentrations of NAA and TDZ added, the germination time is postponed, and the germination rate also drops significantly. For example, when adding 0.5 mg / L of NAA and 0.5 mg / L of TDZ in treatment 1, the germination time is extended to the 10th day, and the germination rate is only 63%. This indicates that the externally added plant growth regulators fail to effectively promote the germination of Rhododendron micranthum Turcz. seeds and may instead interfere with the internal germination mechanism of the seeds themselves.

[0071] From the perspective of the subsequent growth of the seedlings, the seedlings without adding plant growth regulators grow more robustly, with well-developed roots, dark green leaves, and good growth states. While the seedlings treated with plant growth regulators show abnormal phenomena such as slow growth, curled leaves, and short hypocotyls, and are more sensitive to environmental changes and have poor stress resistance during the subsequent growth process. This further confirms that in the cultivation of Rhododendron micranthum Turcz., not adding plant growth regulators is more conducive to seed germination and the healthy growth of seedlings. Without adding extra plant growth regulators, potential adverse effects on seeds and seedlings can be avoided, simplifying the cultivation process and improving the success rate of cultivation and the quality of seedlings.

[0072] Sterile sowing: In a laminar flow hood, use a sterile pipette gun to evenly spread the disinfected seeds on the surface of the sterilized 1 / 2 WPM medium. See Figure 3 . An appropriate amount of seeds can be inoculated in each petri dish, with a uniform spacing to ensure that the seeds have enough growth space. In this embodiment, sterile sowing is carried out in a laminar flow hood. Use a pipette gun to adsorb the disinfected Rhododendron micranthum Turcz. seeds, adsorbing 1 - 2 seeds each time, and evenly spreading the seeds in the germination medium.

[0073] Cultivation conditions: Place the sown petri dishes or culture bottles in a constant temperature incubator for cultivation. The cultivation temperature is set at 25 ± 1 °C, the light intensity is controlled at 3000 - 4000 LX, and the photoperiod is 16 hours of light / 8 hours of darkness. During the cultivation process, it is necessary to regularly observe the germination of the seeds and the growth state of the sterile seedlings. Generally, around the 7th day after sowing, the seeds start to germinate. See Figure 4 , at this time, it can be observed that the radicle breaks through the seed coat. As the cultivation time extends, the radicle gradually elongates, and the hypocotyl and cotyledons also start to differentiate. When cultivated to about the 30th day, the radicle, hypocotyl, and cotyledons of the sterile seedlings are already relatively obvious, and the plant height can reach more than 2 cm, and the roots are initially formed. See Figure 5 . After 2 months of cultivation, the sterile seedlings grow robustly, with the leaves unfolded and the roots well-developed. SeeFigure 6 After growing in the germination medium for 3 months, transplantation can be carried out.

[0074] In the method of the present invention, the entire process of seed disinfection and aseptic sowing should be carried out in a laminar flow hood to ensure the aseptic environment of the operation. Operators need to wear protective equipment such as sterile gloves and masks to avoid human contamination of the seeds and the medium. At the same time, the tools and containers used need to be strictly sterilized.

[0075] The 1 / 2WPM medium selected in the present invention is the most suitable medium for the germination of Rhododendron micranthum Turcz. seeds screened through repeated experiments. In actual operation, the medium should be prepared strictly according to the formula to ensure the accurate concentration of each component, so as to ensure the normal germination and growth of the seeds.

[0076] During the cultivation process, temperature, light intensity and photoperiod are crucial for the germination of seeds and the growth of aseptic seedlings. It should be ensured that the temperature of the constant temperature incubator is stable at 25±1°C, and the light intensity and photoperiod meet the requirements. If the light intensity is too strong or too weak, or the temperature is too high or too low, it may affect the germination rate of seeds and the growth quality of aseptic seedlings.

[0077] During the seed disinfection process, the disinfection time and disinfectant concentration should be strictly controlled. Too long disinfection time or too high concentration may lead to a decrease in seed viability and affect its germination ability; while incomplete disinfection is likely to cause contamination by miscellaneous bacteria. Therefore, during the operation, it is necessary to strictly operate according to the disinfection steps and parameters described in the present invention to ensure the disinfection effect and viability of the seeds.

[0078] When the aseptic seedlings are cultured to a certain extent (generally after 3 months of cultivation), they can be transplanted into the disinfected nutrient soil or substrate for subsequent acclimatization and transplantation cultivation. During transplantation, it is necessary to handle them gently to avoid damaging the roots of the seedlings, and ensure that the humidity and light conditions of the transplantation environment are appropriate to improve the transplantation survival rate.

[0079] The method for disinfection and aseptic sowing of Rhododendron micranthum Turcz. seeds of the present invention is simple to operate, has good disinfection effect, high germination rate, and the aseptic seedlings grow robustly, providing an effective technical means for the large-scale breeding and promotion of Rhododendron micranthum Turcz. By strictly controlling the various parameters and conditions in the seed disinfection treatment and aseptic sowing process, the germination rate of Rhododendron micranthum Turcz. seeds and the quality of aseptic seedlings can be significantly improved, the seedling raising cycle can be shortened, and the seedling raising cost can be reduced, having broad application prospects.

[0080] To further verify the specific adaptation advantage of the 1 / 2WPM medium for the germination of Rhododendron micranthum Turcz. seeds, the inventor excluded the accidental applicability of the medium through comparative experiments with other varieties and revealed the significant differences in the germination mechanisms among varieties.

[0081] Based on the existing Rhododendron germplasm materials in the laboratory, the inventor selected Rhododendron mucronulatum Turcz. and conducted a seed germination test using 1 / 2WPM medium under the same disinfection measures and environmental conditions as in this example, recording the germination time and germination rate: Variety Germination time / d Germination rate / % Rhododendron micranthum Turcz 7 90 Rhododendron mucronulatum Turcz 11 86 Through comparative experiments, we can see that there are obvious differences in the germination performance of Rhododendron micranthum Turcz. and Rhododendron mucronulatum Turcz. in 1 / 2WPM medium. In 1 / 2WPM medium, the germination time of Rhododendron micranthum Turcz. seeds is 7 days, and the germination rate reaches 90%. This indicates that this medium can well meet the germination requirements of Rhododendron micranthum Turcz. seeds, providing suitable nutrients and environmental conditions for them and effectively promoting the rapid and uniform germination of seeds. However, for Rhododendron mucronulatum Turcz. seeds under the same medium and environmental conditions, the germination time requires 11 days, and the germination rate is 86%. This shows that the adaptability of Rhododendron mucronulatum Turcz. seeds to 1 / 2WPM medium is relatively weak, probably because the seed coat structure, dormancy mechanism or nutrient requirements of its seeds are different from those of Rhododendron micranthum Turcz., resulting in less ideal germination effect in the same medium.

[0082] The above differences reflect significant biological specificities in the seed germination mechanisms of different Rhododendron varieties. Therefore, 1 / 2WPM medium has a specific adaptation advantage for Rhododendron micranthum Turcz. seeds. For other Rhododendron varieties, it is necessary to further optimize the medium components or adjust the environmental conditions to meet their specific germination requirements. This also emphasizes that when conducting Rhododendron seedling cultivation, the differences between varieties should be fully considered, and appropriate media and seedling cultivation strategies should be selected to achieve the best seedling cultivation effect.

[0083] In the description of the present invention, it should be understood that "-" and "~" represent the range between two numerical values, and this range includes the endpoints. For example: "A - B" represents a range greater than or equal to A and less than or equal to B. "A ~ B" represents a range greater than or equal to A and less than or equal to B.

[0084] In the description of the present invention, the term "and / or" herein is merely a description of the association relationship of associated objects, indicating that three relationships can exist. For example, A and / or B can represent: A exists alone, A and B exist simultaneously, and B exists alone.

[0085] In the description of the invention, numerical values such as time, temperature, ratio, and mass involved can be based on actual measurements, equipment standard parameters, simplified rounding results, or within an acceptable error range, ensuring the practicability and repeatability of the invention.

[0086] In the description of the present invention, the term "about" or "approximately" is used to express an approximation of a numerical value or range, allowing for a certain degree of error to ensure flexibility and practicality in the description while remaining within an acceptable error range, with the maximum error range not exceeding 10% of the corresponding numerical value or numerical range.

[0087] The above are only the preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be construed as limiting the present invention, and the protection scope of the present invention should be defined by the scope of the claims. For those of ordinary skill in the art, without departing from the spirit and scope of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as within the protection scope of the present invention.

Claims

1. A method for disinfecting and aseptically sowing Rhododendron micranthum Turcz. seeds, characterized in that, It includes the following steps: 1) Seed disinfection treatment: S1. Seed screening: Screen the seeds to obtain Rhododendron micranthum Turcz. seeds with a purity of ≥90%; S2. Seed washing: Wash the screened seeds with normal temperature water to make the water absorption rate of the seeds reach 15% - 18%; S3. Alcohol disinfection: Place the washed seeds in a sterile centrifuge tube, disinfect them with disinfected alcohol for 50 - 70 seconds, and then rinse them thoroughly with sterile water; S4. Benzalkonium bromide disinfection: Disinfect the seeds after alcohol disinfection with 0.1% benzalkonium bromide disinfectant solution for 8 - 12 minutes, and then rinse them thoroughly with sterile water; 2) Sterile sowing of seeds: Inoculate the disinfected seeds onto a germination medium, and the germination medium is 1 / 2WPM basal medium, supplemented with 8 g / L of agar and 30 g / L of sucrose, and the pH value is adjusted to 5.7; Cultivate under the conditions of 25±1°C, light intensity of 3000 - 4000 LX, and photoperiod of 16 hours / 8 hours. The seeds start to germinate on the 7th day, and the germination rate is ≥90%. After 3 months of cultivation, sterile seedlings that can be transplanted are obtained.

2. The disinfection and aseptic sowing method of Rhododendron micranthum Turcz. seeds according to claim 1, characterized in that, The seed screening uses test sieves with gradient sieve hole sizes, and separation is achieved by using the particle size differences of fruits, seeds, and impurities.

3. The disinfection and aseptic sowing method of Rhododendron micranthum Turcz. seeds according to claim 1, characterized in that, In step S2, the water temperature of the normal temperature water is 25±2°C.

4. The disinfection and aseptic sowing method of Rhododendron micranthum Turcz. seeds according to claim 1 or 3, characterized in that In step S2, the seeds are continuously washed in a constant temperature water flow with a flow rate of 2 - 3 L / min and a temperature of 25±2°C for 60±5 min.

5. The disinfection and aseptic sowing method of Rhododendron micranthum Turcz. seeds according to claim 1, characterized in that, The alcohol concentration of the disinfected alcohol is 75%.

6. The disinfection and aseptic sowing method of Rhododendron micranthum Turcz. seeds according to claim 1, characterized in that, In steps S2 and S3, vortex oscillation disinfection is used.

7. The disinfection and aseptic sowing method of Rhododendron micranthum Turcz. seeds according to claim 1, characterized in that, No plant growth regulator is added to the germination medium.

8. The disinfection and aseptic sowing method of Rhododendron micranthum Turcz. seeds according to claim 1, characterized in that, In the step of sterile sowing of seeds, use a pipette to adsorb the seeds and evenly spread them on the surface of the germination medium.

9. The disinfection and aseptic sowing method of Rhododendron micranthum Turcz. seeds according to claim 1, characterized in that, During the cultivation process, cotyledons, hypocotyls, and radicles appear in the sterile seedlings on the 30th day. After 2 months of cultivation, the seedling height is ≥2 cm and the roots are well-developed.

10. The disinfection and aseptic sowing method of Rhododendron micranthum Turcz. seeds according to claim 1, characterized in that, In the method, the contamination rate is ≤2%, and the seed survival rate is ≥90%.

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