Method and system for one-step seedling formation of cremastra appendiculata tissue culture seedlings

Abstract

The invention belongs to the technical field of seedling breeding of traditional Chinese medicinal materials, and discloses a one-step seedling forming method for tissue culture seedlings of cremastra appendiculata. According to the method, related substances such as a plant nutrient element 1 / 2 MS culture medium, hormones NAA, 6-BA, ZT, cane sugar, fresh milk, agar and activated carbon are mainly used for carrying out tissue culture and seedling induction of the cremastra appendiculata plants. The method can directly germinate seeds, induce proliferation differentiation, root and grow seedlings. According to the one-step seedling method, the processes of tissue culture transfer, continuous culture and the like such as proliferation differentiation and rooting induction of tissue culture seedlings are reduced, the time cost and the midway transfer cost are saved, seeds are used as explants for one-step seedling, and after culture is conducted for 8-9 months, the root system of one-step seedling is 4-5 cm long, and the leaf bud is 10-15 cm long. The invention solves the problems of long time consumption, high cost and tedious multi-stage transfer process in the existing plant tissue culture. The method specifically aims at the one-step seedling forming method of the cremastra appendiculata tissue culture seedlings, a simplified culture process is adopted, the culture period is effectively shortened, the cost is reduced, and the consistency and health degree of plant growth are enhanced.

Description

Technical Field

[0001] The invention belongs to the technical field of Chinese medicinal material seedling breeding, and in particular relates to a method and system for one-step seedling formation of azalea tissue culture seedlings. Background Art

[0002] At present, plant tissue culture technology usually needs to go through multiple stages in practice, including the proliferation stage and the rooting stage. Each stage requires the re-preparation of targeted culture media and the transfer of plant tissues. Specifically, these steps include transferring plant tissues from one culture medium to another culture medium suitable for the next stage for cultivation.

[0003] Defects and shortcomings of existing technology:

[0004] 1. Multiple transfers increase complexity and risk

[0005] In the prior art, since each culture stage requires a transfer operation, this significantly increases the complexity of the production process. At the same time, multiple transfers also cause plant tissues to be affected by the external environment during the operation process, increasing the risk of bacterial contamination and vitrification of seedlings.

[0006] 2. The time period is too long

[0007] The complete process of plant tissue culture technology, from seed germination to seedling formation, usually takes a long time. In particular, multiple transfers will increase the time between each stage, directly extending the entire culture cycle and affecting production efficiency.

[0008] 3. High cost

[0009] The existing technology requires the preparation of new culture medium for each culture stage and the purchase of corresponding culture containers. This not only increases material costs, but also increases labor costs. In addition, the process of preparing culture medium multiple times also increases the infection rate of foreign bacteria, further pushing up production costs.

[0010] 4. Low efficiency and waste of resources

[0011] Due to the high repetitiveness of the culture medium preparation and multiple transfer steps, resource utilization efficiency is low. On the one hand, a large amount of culture medium materials are consumed; on the other hand, this inefficient culture model is not suitable for large-scale, low-cost commercial production needs.

[0012] In the prior art, there is no effective solution for optimizing the multiple transfers during the tissue culture of azalea orchid plants. Current research and technical practices mainly focus on improving conventional culture media and adjusting growth conditions, rather than systematically optimizing the culture process.

[0013] In summary, the existing plant tissue culture technology has many problems in the cultivation of azalea orchid tissue culture seedlings, such as multiple transfers, long time and high cost, which need to be solved urgently. How to simplify the cultivation process, shorten the cycle, reduce costs and improve the seedling rate has become the key direction of current technology development. Summary of the invention

[0014] In view of the problems existing in the prior art, the present invention provides a method for one-step seedling formation of azalea tissue culture seedlings. In the proliferation and rooting culture stages of the azalea tissue culture seedlings of the present invention, there is no need to prepare a culture medium and transfer, and the seeds can be directly induced into seedlings in one step.

[0015] The present invention is achieved in that a method for one-step seedling formation of azalea tissue culture seedlings comprises:

[0016] Step 1, explant selection: select capsules of azalea orchid that mature from November to December each year as explants, fresh seeds with dark yellow or light yellow-green peel and not cracked, dry them and store them at 5°C for later use, and then carry out tissue culture;

[0017] Step 2, disinfection and sterilization: rinse with running water for 5 to 10 minutes, soak in 10% sodium hypochlorite solution for 10 to 15 minutes, soak in 75% ethanol solution for 5 to 10 minutes, rinse with sterile water for 3 to 5 times and dry for later use;

[0018] Step 3, culture medium and hormone formula: using 1 / 2MS nutrient medium, adding 3% to 5% sucrose, 0.5% to 0.7% agar, 0.3g / L to 0.8g / L activated carbon and 1% to 3% pure milk, hormones are NAA, 6-BA and ZT, and the pH value of the culture medium is adjusted to 5.8;

[0019] Step 4, inoculation and cultivation: The seeds are inoculated into a sterile culture medium and cultured for germination and rooting in an environment with a certain temperature, humidity and light. The light cycle is 9 hours of light / 15 hours of darkness, and the temperature is controlled at 25±1°C.

[0020] Furthermore, the hormones are 0.1mg / L to 0.5mg / L NAA (naphthaleneacetic acid), 0.3mg / L to 0.8mg / L 6-BA (6-benzylaminopurine) and 0.5mg / L to 1.2mg / L ZT (zeatin).

[0021] Furthermore, in step 4, the sterilized and disinfected mature capsules are split open with a sterile knife, and the seeds are evenly spread into the sterilized culture medium for aseptic culture. The culture temperature is controlled at 24° C. to 26° C., the humidity is 60% to 80%, the light intensity is 2200lx to 2500lx, and the light-dark environment is cycled for 9h to 15h every day.

[0022] Another object of the present invention is to provide a system for one-step seedling formation of azalea tissue culture seedlings, comprising:

[0023] The explant selection module is used to select capsules of azalea orchid that mature from November to December each year as explants, and fresh seeds with dark yellow or light yellow-green peel and not cracked, and store them at 5°C for later use after drying, and then conduct tissue culture;

[0024] The disinfection and sterilization module is used to rinse with running water for 5 to 10 minutes, soak with 10% sodium hypochlorite solution for 10 to 15 minutes, and then soak with 75% ethanol solution for 5 to 10 minutes, then rinse with sterile water for 3 to 5 times and dry for use;

[0025] The culture medium and hormone module is used to adopt 1 / 2MS nutrient culture medium, add 3% to 5% sucrose, 0.5% to 0.7% agar, 0.3g / L to 0.8g / L activated carbon and 1% to 3% pure milk, the hormones are 0.1mg / L to 0.5mg / L NAA, 0.3mg / L to 0.8mg / L 6-BA and 0.5mg / L to 1.2mg / L ZT, and the pH value of the culture medium is adjusted to 5.8;

[0026] The inoculation and cultivation module is used to inoculate seeds into sterile culture medium and carry out germination and rooting cultivation in an environment with certain temperature, humidity and light.

[0027] The present invention also provides an application of a method for one-step seedling formation of azalea orchid tissue culture in the rapid propagation of azalea orchid seedlings, and the efficient cultivation of azalea orchid seedlings is achieved through the following steps:

[0028] Mature capsules were used as explants. After screening, disinfection and sterilization, 1 / 2MS nutrient medium was used with sucrose, agar, activated carbon, pure milk and appropriate amounts of plant hormones for seed inoculation and germination and rooting culture.

[0029] Under a constant temperature of 25±1℃, combined with a photoperiod of 9 hours of light and 15 hours of darkness, rapid seedling establishment and high survival rate of azalea seedlings were achieved through precise control of nutrient composition and environmental conditions.

[0030] Furthermore, the method can be applied to large-scale seedling breeding of azalea orchids. By utilizing low-cost culture medium and automated control technology, the breeding efficiency and quality of azalea orchid seedlings can be improved by selecting the best explants and optimizing culture conditions. The method is suitable for seedling breeding and commercial production of Chinese medicinal materials.

[0031] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0032] At present, there is no patent or literature report on the one-step seedling technology of azalea tissue culture seedlings. The present invention, through independent research and development, innovatively proposes a one-step seedling technology of azalea tissue culture seedlings from seeds to seedlings, innovatively optimizes the culture process, and provides a new solution for plant tissue culture technology.

[0033] First, the present invention does not go through the successive transfer culture process of proliferation and differentiation culture → induction rooting culture → rapid cultivation of regenerated plants, but directly induces proliferation and rooting culture into seedlings from seed germination.

[0034] The invention can directly germinate seeds, induce proliferation, differentiate cotyledons, bulbs and root systems, and directly form seedlings.

[0035] Advantages: The one-step seedling method reduces the transfer and cultivation process such as proliferation and differentiation of tissue culture seedlings and induction of rooting, saving time and mid-transfer costs. The one-step seedling method uses seeds as explants. After culturing for 8 to 9 months, the induction rate of the one-step seedlings is above 90%, the root system is up to 4 cm to 5 cm long, and the leaf buds are up to 10 cm to 15 cm long.

[0036] Simplified operation process: By eliminating multiple transfer and culture steps, the operation process is simplified, avoiding bacterial infection of the tissue due to multiple transfers and reducing labor requirements.

[0037] Reduce costs: Reduce the amount of culture medium used, reduce the labor cost of tissue transfer compared to conventional tissue culture seedlings, reduce electricity consumption during culture, etc., significantly reducing production costs.

[0038] Improve seedling health: reduce the chances of repeated and multiple contacts of tissue materials with different growth hormones, effectively reduce the number of subcultures, and reduce the risks of browning of tissue materials and vitrified seedlings.

[0039] Speed ​​up the production cycle: Since multiple transfer steps are eliminated, the entire tissue culture process is shortened, speeding up the production cycle of seedlings.

[0040] This new technology has significant economic and practical significance for improving the efficiency of tissue culture of azalea orchids. It can not only improve the production efficiency of azalea orchid tissue culture seedlings, but also reduce production costs and improve seedling quality. This one-step seedling technology is innovative and practical, meets the innovation requirements of the Patent Law, and is expected to obtain patent authorization.

[0041] The present invention aims to solve the problem of time-consuming, costly and cumbersome multi-stage transfer culture process in existing plant tissue culture. Specifically, the method for one-step seedling formation of azalea orchid tissue culture adopts a simplified culture process, effectively shortens the culture cycle, reduces costs, and enhances the consistency and health of plant growth.

[0042] In the tissue culture process of the prior art, multiple stages are required, including seed germination culture, induced proliferation and differentiation culture, induced rooting culture, and rapid cultivation of regenerated plants. These stages are not only time-consuming, but also introduce pollution during each transfer process, while increasing labor and material costs. The present invention omits multiple intermediate links through a one-step method directly from seeds to seedlings, aiming to simplify the entire process.

[0043] Improved time efficiency: The method of directly going from seeds to seedlings greatly shortens the overall period of tissue culture. The traditional method takes October to December, while the method of the present invention only takes August to September.

[0044] Save costs and reduce risks: Reduce multiple transfers and related culture medium, container usage and labor costs, reduce electricity consumption during cultivation, etc., significantly reduce production costs. At the same time, saving multiple transfer steps reduces the risk of plant contamination.

[0045] Rooting and growth optimization: Through the one-step seedling method, the plant has a well-developed root system (up to 4cm to 5cm long) and lush leaf buds (up to 10cm to 15cm long), showing better growth potential and adaptability.

[0046] Improve seedling health: reduce the chances of repeated and multiple contacts of tissue materials with different growth hormones, effectively reduce the number of subcultures, reduce the risks of browning of tissue materials and vitrified seedlings, and enhance the growth health of plants.

[0047] Second, the expected benefits and commercial value after the technical solution of the present invention is transformed as follows: after the technical solution of the present invention is transformed, the production cost of azalea tissue culture seedlings can be significantly reduced in terms of saving time, labor and the use of related tissue culture materials, which has greater economic benefits compared with tissue culture seedlings that are not one-step seedlings.

[0048] The technical solution of the present invention fills the technical gap in the industry at home and abroad: The technical solution of the present invention fills the technical gap in the one-step seedling method of azalea tissue culture seedlings in China.

[0049] The technical solution of the present invention solves the technical problems that people have been eager to solve but have never been able to solve successfully: the technical solution of the present invention solves the technical problems of long tissue culture seedling cycle and high cost.

[0050] Thirdly, in the technology of tissue culture seedling of azalea orchid, the present invention solves the technical problems of low seedling efficiency, complex manual operation and high production cost in the prior art through scientific parameter control, optimized culture medium formula and integration of automation modules, and has achieved remarkable technical progress, which is specifically reflected in the following aspects:

[0051] 1. Improved seedling efficiency

[0052] The existing technology usually requires multiple manual transfers and multiple culture medium changes, resulting in a long seedling cycle and a low seedling rate. The present invention uses a one-time configuration of optimized culture medium and precise growth environment control, so that the azalea orchid tissue culture seedlings only need one transfer to complete the entire growth process within one cycle. Compared with the existing technology, the seedling cycle is shortened by 20% to 30%, from 10 to 12 months to 8 to 9 months. At the same time, the automated equipment ensures a stable light, temperature and humidity environment, significantly improving the survival rate of the seedlings.

[0053] 2. Significantly reduce production costs

[0054] In traditional technology, 3 to 4 manual transfers and culture medium changes are required during the culture process, which consumes a lot of human resources and culture materials. The implementation scheme of the present invention only requires one transfer, and the culture medium and labor costs are reduced by more than 60%. In addition, through the automated control system, the errors caused by manual operation are reduced, ensuring the uniformity of seedling quality. This greatly reduces the production cost, making the commercialization and large-scale production of azalea seedling cultivation more economical.

[0055] 3. Improved energy efficiency

[0056] Traditional tissue culture technology requires long-term illumination and environmental maintenance to ensure the needs of plants at different growth stages. The present invention adopts a scientifically optimized light cycle (9 hours of light / 15 hours of darkness) and a suitable constant temperature environment (25±1°C) to achieve energy consumption control. At the same time, the cultivation cycle is shortened and the overall energy consumption is reduced by more than 20%, which is particularly important in large-scale production and further promotes environmental protection and sustainable production.

[0057] 4. High-efficiency culture medium formula and seedling quality

[0058] The culture medium formula of the present invention comprises sucrose, agar, activated carbon and pure milk in an optimized ratio, and the hormone ratio is controlled within the optimal range (such as 0.1mg / L-0.5mg / LNAA, 0.3mg / L-0.8mg / L 6-BA, 0.5mg / L-1.2mg / LZT). This formula not only greatly promotes the seedling rate of azalea orchid, but also makes the seedlings superior to the prior art in terms of root length, leaf bud length, etc. Data comparison shows that the seedling rate of the present invention can reach more than 90%, the root length increases to 4cm-5cm, and the leaf bud length reaches 10cm-15cm, which is significantly better than the traditional technology (seedling rate 55%, root length 2cm-3cm, leaf bud length 5cm-10cm).

[0059] 5. Technology automation and standardization

[0060] The system of the present invention integrates functional modules such as explant screening, disinfection and sterilization, culture medium preparation and inoculation culture, and has the advantages of automation and standardization. In actual operation, the parameter setting of the automated control equipment reduces the dependence on professional skills, simplifies the operation, and improves the stability of production. At the same time, the automated system can ensure the consistency of the operating procedures of each batch, avoid fluctuations caused by manual labor, and further ensure the quality consistency of the azalea seedlings.

[0061] 6.Quality control and standardized production

[0062] The present invention ensures the stability of each link through high-precision control, thereby significantly improving the quality of azalea orchid seedlings. The cultivation process of this technical solution is transparent and standardized, so that enterprises can accurately manage various parameters and monitor the growth situation in real time, so as to achieve high output of high-quality azalea orchid seedlings, and provide a solid foundation for the large-scale production and market competitiveness of azalea orchids.

[0063] In summary, the innovative design of the present invention in the tissue culture seedling technology of azalea orchid has achieved remarkable progress in seedling efficiency, production cost, energy consumption, seedling quality and production process automation, and has obvious superiority and application value compared with the existing technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 The present invention provides a flow chart of a method for one-step seedling formation of azalea tissue culture seedlings provided in an embodiment of the present invention.

[0065] Figure 2 The present invention provides a system structure diagram for one-step seedling formation of azalea tissue culture seedlings provided in an embodiment of the present invention.

[0066] Figure 3 This is a graph showing the growth of azalea orchid seeds after 90 days of cultivation provided by an embodiment of the present invention. DETAILED DESCRIPTION

[0067] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0068] like Figure 1 As shown, a method for one-step seedling formation of azalea tissue culture seedlings provided by an embodiment of the present invention comprises the following steps:

[0069] S101, explant selection: select capsules of azalea orchid that mature from November to December each year as explants, and fresh seeds with dark yellow or light yellow-green peel and not cracked, dry them and store them at 5℃ for later use, and then carry out tissue culture;

[0070] S102, disinfection and sterilization: rinse with running tap water for 5 min to 10 min, soak in 10% sodium hypochlorite solution for 10 min to 15 min, soak in 75% ethanol solution for 5 min to 10 min, rinse with sterile water for 3 to 5 times and dry for later use;

[0071] S103, culture medium and hormone formula: 1 / 2MS nutrient medium, 3% to 5% sucrose, 0.5% to 0.7% agar, 0.3g / L to 0.8g / L activated carbon and 1% to 3% pure milk were added, hormones were 0.1mg / L to 0.5mg / L NAA, 0.3mg / L to 0.8mg / L 6-BA and 0.5mg / L to 1.2mg / L ZT, and the pH value of the culture medium was adjusted to 5.8;

[0072] S104, inoculation and cultivation: The seeds are inoculated into a sterile culture medium and cultured for germination and rooting in an environment with a certain temperature, humidity and light intensity. The light cycle is 9 hours of light / 15 hours of darkness, and the temperature is controlled at 25±1°C.

[0073] Example 1 for components

[0074] The culture medium uses 1 / 2MS nutrient medium, with 3% sucrose, 0.5% agar, 0.3g / L activated carbon and 1% pure milk added. The hormone configuration is 0.2mg / L NAA, 0.4mg / L 6-BA and 0.5mg / L ZT. The pH value of the culture medium is adjusted to 5.8 to ensure a suitable growth environment.

[0075] Example 2 for components

[0076] The culture medium uses 1 / 2MS nutrient medium, with 4% sucrose, 0.6% agar, 0.5g / L activated carbon and 2% pure milk added. The hormone configuration is 0.3mg / L NAA, 0.5mg / L 6-BA and 0.8mg / L ZT. The pH value of the culture medium is also adjusted to 5.8, which is suitable for medium hormone concentration experiments.

[0077] Example 3 for components

[0078] The culture medium uses 1 / 2MS nutrient medium, with 5% sucrose, 0.7% agar, 0.8g / L activated carbon and 3% pure milk added. The hormone configuration is 0.4mg / L NAA, 0.7mg / L 6-BA and 1.0mg / L ZT. The pH value of the culture medium is controlled at 5.8, which is suitable for high-concentration nutrient and hormone ratio experiments.

[0079] Example 4 for components

[0080] The culture medium uses 1 / 2MS nutrient medium, with 3.5% sucrose, 0.55% agar, 0.6g / L activated carbon and 1.5% pure milk added. The hormone configuration is 0.2mg / L NAA, 0.6mg / L 6-BA and 0.9mg / L ZT. The pH value of the culture medium is adjusted to 5.8, which is suitable for comprehensive experiments of multiple hormone ratios.

[0081] The one-step seedling method for tissue culture seedlings of azalea orchid provided in the embodiment of the present invention effectively improves the survival rate and growth quality of azalea orchid seedlings through scientific process flow and precise nutrition and environmental control.

[0082] 1. Selection of Explants

[0083] In step S101, mature, unopened Rhododendron capsules are selected as explants because the seeds in capsules picked from November to December have higher activity and better growth status. The seeds are best mature when the peel is dark yellow or light yellow-green, which is conducive to improving the survival rate of tissue culture. By screening the maturity and vitality of the seeds, it can be ensured that the seeds have good germination and seedling capabilities.

[0084] 2. Disinfection and sterilization

[0085] In step S102, the explants are strictly disinfected and sterilized to remove pathogens and microorganisms on the surface of the seeds and ensure growth in a sterile environment. First, use running water to rinse for 5 to 10 minutes to preliminarily remove surface dirt and microorganisms; then use 10% sodium hypochlorite solution and 75% ethanol solution to disinfect the seeds, soaking for 10 to 15 minutes and 5 to 10 minutes respectively. Finally, rinse with sterile water 3 to 5 times to thoroughly remove disinfectant residues. This series of operations ensures the sterility of the seed surface and reduces the impact of pathogens on seedlings.

[0086] 3. Preparation of culture medium and hormones

[0087] In step S103, 1 / 2MS nutrient medium is used and precise proportions of growth nutrients and hormones are added. The medium contains 3% to 5% sucrose, 0.5% to 0.7% agar, 0.3g / L to 0.8g / L activated carbon and 1% to 3% pure milk, providing sufficient carbon sources, gelling agents and auxiliary nutrients, especially activated carbon can effectively promote root growth. The addition of hormones is 0.1mg / L to 0.5mg / L NAA, 0.3mg / L to 0.8mg / L 6-BA and 0.5mg / L to 1.2mg / L ZT, which can promote the germination of rhododendron seeds and the growth of seedlings. The pH value of the culture medium is adjusted to 5.8 to ensure an acid-base environment suitable for seed germination.

[0088] 4. Inoculation and cultivation

[0089] In step S104, the treated azalea seeds are inoculated in a sterile culture medium to create suitable environmental conditions to promote germination and rooting. The culture environment needs to maintain a cyclical illumination of 9 hours of light / 15 hours of darkness to simulate the day and night cycle in the natural environment. The setting of the light intensity and cycle is conducive to the growth and development of the seeds. The temperature is controlled at 25±1°C, providing the most suitable temperature conditions to make the development of the seedlings more uniform and stable.

[0090] 5. Control conditions to ensure stable growth

[0091] During the entire cultivation process, the lighting, temperature, humidity and other environmental conditions are precisely controlled to ensure that the seeds can grow in the optimal environment. The light cycle and temperature settings are based on the growth needs of the azalea orchid, which helps the seeds fully absorb nutrients in the culture medium and successfully complete the germination and rooting process. At the same time, the composition and hormone ratio of the culture medium have been optimized through repeated experiments, so that it has a good promoting effect on the germination of azalea orchid seeds.

[0092] 6. One-step seedling cultivation improves efficiency

[0093] The method achieves rapid seedling formation of azalea orchids by optimizing the culture medium and controlling the environment. Compared with the traditional tissue culture method with multiple transfers, the present invention only requires one inoculation and culture, which not only significantly shortens the seedling formation cycle, but also greatly reduces manual operations, reduces the consumption of culture medium, improves the survival rate of seedlings and production efficiency, and makes the cultivation of azalea orchids simpler and more efficient.

[0094] Furthermore, the hormones are 0.1mg / L to 0.5mg / L NAA (naphthaleneacetic acid), 0.3mg / L to 0.8mg / L 6-BA (6-benzylaminopurine) and 0.5mg / L to 1.2mg / L ZT (zeatin).

[0095] S104, inoculation and cultivation: split the skin of the disinfected and sterilized mature capsule with a sterile knife, and evenly spread the seeds into the sterilized culture medium for aseptic cultivation. Control the culture temperature at 24℃~26℃, the humidity at 60%~80%, the light intensity at 2200lx~2500lx, and the light-dark environment at 9h~15h cycle every day.

[0096] The mature seeds of azalea orchid from November to December every year are selected as explants because the capsules have not yet cracked and the inside is not polluted by the environment. The seeds inside the uncracking capsules with dark yellow or light yellow-green peels have strong seed vitality and are suitable for tissue culture. Selecting seeds at this stage can improve the vitality of tissue culture seedlings, promote rapid germination and the smooth progress of subsequent culture processes.

[0097] The use of tap water rinsing and sodium hypochlorite solution and ethanol solution in the disinfection step is intended to remove impurities and other microorganisms on the surface of the capsule. Sodium hypochlorite is an effective disinfectant that can quickly kill pathogens, while ethanol can further kill bacteria and fungi to ensure that the surface of the seeds is sterile. After multiple rinses with sterile water, the surface of the seeds is basically free of bacterial contamination, providing a pure environment for subsequent cultivation. This process is a key step in the success of tissue culture, avoiding the presence of bacteria or fungi in the culture medium and infecting the culture medium.

[0098] Using 1 / 2MS medium is a common plant tissue culture method, which provides the basic nutrients required for seed development. Compared with the full amount of MS medium, 1 / 2MS reduces the concentration of inorganic salts, helps reduce the stress response of plants, and promotes the balanced development of roots and shoots. At the same time, 3% sucrose is added as a carbon source for seeds to use and promote metabolic processes.

[0099] The key role of hormone combinations in plant tissue culture is to regulate cell division and growth. NAA (naphthaleneacetic acid), as an auxin, can promote cell elongation, root formation and initial seed germination, while 6-BA (6-benzylaminopurine) and ZT (zeatin) are cytokinins that can accelerate cell division, promote tissue differentiation and bud formation. This hormone ratio ensures that seeds can germinate quickly and form complete seedlings by precisely regulating the cell division and differentiation process.

[0100] Activated carbon added to the culture medium helps to absorb excess harmful substances or metabolites in the culture medium, such as phenolic compounds secreted by plants during their development. If these substances accumulate, they will cause browning and tissue necrosis in the plant. Activated carbon can also adjust the transparency of the culture medium, making it easier for the roots to contact the culture medium, thereby promoting root growth and absorption.

[0101] Adding 1% pure milk to the culture medium provides an additional source of nutrition. The protein, fat, vitamins and trace elements in the milk provide richer nutrition for the seedlings and enhance the growth momentum of the seeds. This natural nutritional supplement can not only improve the stress resistance of the seedlings, but also promote cell division and organ formation, further improve the tissue culture efficiency and the survival rate of the seedlings, and also plays a key role in the present invention.

[0102] like Figure 2 As shown, a system for one-step seedling formation of azalea tissue culture seedlings provided by an embodiment of the present invention comprises:

[0103] The explant selection module is used to select capsules of azalea orchid that mature from November to December each year as explants, and fresh seeds with dark yellow or light yellow-green peel and not cracked, and store them at 5°C for later use after drying, and then conduct tissue culture;

[0104] The disinfection and sterilization module is used to rinse with running water for 5 to 10 minutes, soak with 10% sodium hypochlorite solution for 10 to 15 minutes, and then soak with 75% ethanol solution for 5 to 10 minutes, then rinse with sterile water for 3 to 5 times and dry for use;

[0105] The culture medium and hormone module is used to adopt 1 / 2MS nutrient culture medium, add 3% to 5% sucrose, 0.5% to 0.7% agar, 0.3g / L to 0.8g / L activated carbon and 1% to 3% pure milk, the hormones are 0.1mg / L to 0.5mg / L NAA, 0.3mg / L to 0.8mg / L 6-BA and 0.5mg / L to 1.2mg / L ZT, and the pH value of the culture medium is adjusted to 5.8;

[0106] The inoculation and cultivation module is used to inoculate seeds into sterile culture medium and carry out germination and rooting cultivation in an environment with certain temperature, humidity and light.

[0107] The specific technical content and implementation principle of the present invention are specifically implemented.

[0108] Explant selection: Select mature capsules harvested from mid-November of Rhododendron orchid, with fresh seeds whose peel is dark yellow or light yellow-green and not cracked, dry them and store them at 5°C for later use and subsequent tissue culture.

[0109] Disinfection and sterilization: Rinse the unopened mature capsules with running tap water for 5 minutes, dry them and then soak them in 10% sodium hypochlorite solution for 10 minutes, dry them with sterile paper and then soak them in 75% ethanol solution for 5 minutes, then rinse them with sterile water for 3 times, dry them with sterile paper and set aside.

[0110] Culture medium and hormones: Use 1 / 2MS nutrient medium, add hormones of 0.1mg / L~0.5mg / LNAA (naphthaleneacetic acid), 0.3mg / L~0.8mg / L 6-BA (6-benzylaminopurine) and 0.5mg / L~1.2mg / L ZT (zeatin), 3% sucrose, 0.5% agar, 0.4g / L activated carbon and 1% pure milk, and adjust the pH value of the culture medium to 5.8.

[0111] Cell totipotency: Utilizing the totipotency of cells, by adding appropriate proportions of nutrients, hormones, etc. to the culture medium, the direction of cell differentiation can be regulated to achieve direct transformation from seeds to seedlings.

[0112] Hormone regulation: The ratio of NAA, 6-BA and ZT affects the balance between cell differentiation and organogenesis, and optimizes the process of inducing seedlings.

[0113] The present invention provides a more economical and efficient one-step method for cultivating azalea orchid tissue culture seedlings, which greatly simplifies the complexity of traditional plant tissue culture and significantly improves the culture efficiency and plant quality, providing a new feature for plant tissue culture technology.

[0114] The explant selection module of the present invention is equipped with a high-precision optical detection device for identifying the maturity of azalea capsules. The device detects the color and structural changes of the fruit skin through spectral analysis technology, and screens dark yellow-brown or brown and unopened capsule seeds. The screening process automatically calculates the color value and thickness of the fruit skin through an algorithm to ensure that the selected seeds are in the best biological activity state, thereby improving tissue culture efficiency and seedling rate.

[0115] The disinfection and sterilization module has an embedded automated disinfection device and sterile water rinsing equipment, which can accurately execute the disinfection procedure. First, the seeds are soaked in sodium hypochlorite solution for a certain period of time to remove surface germs and impurities. Subsequently, they are further sterilized with ethanol solution to enhance the sterilization effect. After the sterile water rinsing equipment is started, multiple rinsing cycles are used to remove residual chemicals to ensure that the seed surface is completely free of contamination. The entire process is controlled by a program with a high degree of automation, ensuring the uniformity and stability of the disinfection effect.

[0116] The culture medium preparation module is equipped with an automatic pH adjustment device and a sensor monitoring system. When preparing the culture medium, the sensor is used to monitor the composition of the culture medium in real time, including the concentration ratio of sucrose, agar, hormones, activated carbon and pure milk. Subsequently, the automatic control device accurately adjusts the pH value of the culture medium to 5.8 to meet the optimal conditions for the development of azalea orchid seeds. Throughout the process, closed-loop control is used to ensure the stability of the culture medium composition and maximize the germination and seedling formation of seeds.

[0117] The internal environment of the tissue culture room is regulated by a constant temperature and humidity system. The system monitors and adjusts the temperature, humidity and light intensity in real time to maintain the optimal conditions for seed germination. The high-efficiency air filtration system (HEPA) further ensures a sterile environment and avoids the influence of external pollution sources on the culture medium. Through precise control of environmental conditions, an ideal growth environment is provided for azalea orchid seeds from germination to seedling formation.

[0118] Each module collects data in real time through sensors, including the disinfection of the seed surface, the pH value of the culture medium, the concentration of ingredients, and environmental conditions. The collected data is analyzed by the central control system to determine whether each parameter meets the standard. If an abnormality is found, the system will automatically adjust the operation of the relevant module or send an alarm notification to ensure the stable operation of the entire process. This intelligent feedback mechanism significantly improves the success rate of tissue culture.

[0119] After the seeds germinate in the culture medium, the system monitors the growth status of the seedlings in real time, including the development of the root system and the degree of leaf expansion. The monitoring data is used to dynamically adjust the hormone concentration and environmental conditions to meet the needs of different growth stages. When the seedlings reach the predetermined standards, the system will automatically mark the mature seedlings and prompt the technicians to carry out subsequent transplanting operations, forming a full-process efficient closed-loop tissue culture seedling system.

[0120] Through the collaborative work of the above multiple modules, the azalea orchid tissue culture one-step seedling system has achieved fully automated operations from seed screening to seedling formation, greatly improving production efficiency and seedling quality, while reducing the risks and errors of human participation.

[0121] The following are two specific embodiments of the one-step seedling method of azalea tissue culture:

[0122] Example 1: Tissue culture of azalea seeds suitable for rapid seedling formation

[0123] 1. Explant selection: Select fresh, ripe, light yellow-green, unopened capsules of Rhododendron orchid collected in mid-November, and ensure that the peel is intact and the seeds have not cracked.

[0124] 2. Disinfection and sterilization: Rinse the capsule with running tap water for 5 minutes, dry it, soak it in 10% sodium hypochlorite solution for 10 minutes, and then dry it with sterile paper. Then, soak it in 75% ethanol solution for 5 minutes, then rinse it with sterile water 3 times, and dry it with sterile paper for later use.

[0125] 3. Culture medium and hormones: Use 1 / 2MS nutrient medium, add 0.2mg / L NAA, 0.5mg / L6-BA and 0.7mg / L ZT as hormone combination, and add 3% sucrose, 0.5% agar, 0.4g / L activated carbon and 1% pure milk to the culture medium, and adjust the pH to 5.8.

[0126] 4. Cultivation process: Inoculate the sterilized azalea orchid seeds onto the culture medium and cultivate them in a constant temperature incubator at 25°C, with a humidity of 60%, a light intensity of 2200lx, and a photoperiod of 9 hours light / 15 hours dark. After 55 days of cultivation, the seeds begin to germinate and form seedlings within 9 months.

[0127] 5. Results: This method can quickly induce seed germination and form complete azalea seedlings in a short time, with a germination rate of 95%, strong seedlings and high survival rate.

[0128] Example 2: Tissue culture seedling method applicable to various azalea orchid varieties

[0129] 1. Explant selection: Select ripe capsules of different varieties of azalea orchids collected in December, with fresh, ripe pods with dark yellow peel and not cracked.

[0130] 2. Disinfection and sterilization: Rinse the capsule with tap water for 5 minutes, then soak it in 10% sodium hypochlorite solution for 8 minutes, then soak it in 75% ethanol solution for 5 minutes, rinse it with sterile water 3 times, and dry it for later use.

[0131] 3. Culture medium and hormones: Use 1 / 2MS culture medium and add hormone combination 0.2mg / L NAA, 0.5mg / L 6-BA and 0.8mg / L ZT. Add 0.3g / L activated carbon, 3% sucrose, 0.4% agar and 1.5% pure milk to the culture medium, and adjust the pH value to 5.8.

[0132] 4. Cultivation process: Inoculate the sterilized azalea seeds into the above culture medium and place them in a 27°C constant temperature incubator for cultivation, with a humidity of 70%, a light intensity of 2400lx, and a light cycle of 9 hours of light / 15 hours of darkness. After about 60 days, the seeds begin to germinate and seedlings form within 8 months.

[0133] 5. Results: This method is applicable to different varieties of rhododendron seeds, with a germination rate of more than 90%, a short culture cycle, and uniform seedling growth, which can meet the growth needs of a variety of rhododendron varieties.

[0134] The embodiment of the present invention significantly reduces the production cost in the process of tissue culture seedling cultivation through innovative processes and technical routes. Compared with the existing tissue culture technology, the present invention only requires one manual transfer and culture medium input, while the traditional technology usually requires 3 to 4 transfers and culture medium changes. This optimized design enables the present invention to relatively save more than 60% of labor and material input in one tissue culture seedling cycle, greatly reducing production costs.

[0135] In addition, the present invention also shows significant advantages in terms of culture time and energy consumption. The existing technology usually requires 10 to 12 months of culture time, while the present invention only requires 8 to 9 months, shortening the culture cycle by 2 to 3 months. By reducing the illumination time and power consumption, the present invention saves more than 20% of time and power resources in the entire culture process, making the entire production process more efficient and energy-saving.

[0136] In terms of tissue culture effect, the present invention is significantly superior to the existing technology in terms of key technical indicators. Experimental data show that the tissue culture induction rate of the present invention exceeds 90%, which is significantly higher than the 55% of the traditional technology; in terms of the root length and leaf bud length of seedling development, the present invention is superior, with the root length reaching 4 to 5 cm and the leaf bud length reaching 10 to 15 cm, while the root length of the existing technology is about 2 to 3 cm and the leaf bud length is 5 to 10 cm, which significantly improves the quality of seedlings.

[0137] Through the above technical improvements, the present invention can reduce the labor cost of multiple transfers and the consumption of culture medium while maintaining a high induction rate and seedling quality, effectively reducing the overall cost of seedling production, while shortening the cultivation time and improving energy efficiency.

[0138] To further demonstrate the technical advantages and improvement effects of the present invention, comparative data show that in the process of cultivating azalea orchid seeds, the present invention shows stronger growth performance in the 90-day growth observation results. The significant improvement of root length, leaf bud length and induction rate in the process of tissue culture seedlings ensures that the cultivated plants are superior to traditional methods in development and growth quality, and are suitable for application in large-scale commercial production.

[0139] In summary, the present invention has achieved significant progress in production cost, energy efficiency and seedling quality, and provides an excellent technical solution for saving cost, improving efficiency and ensuring quality. Specific comparisons are shown in Tables 1 and 2, which are suitable for the promotion and application of plant tissue culture.

[0140] Table 1 Input cost comparison table

[0141] Comparison of different technology input costs Time and electricity Transfer to manual Culture medium input Other existing technical indicators October to December 3-4 times 3-4 times Technical indicators of the embodiments of the present invention August ~ September 1 time 1 time The present invention saves >20％ >60％ >60％

[0142] Table 2 Technical index comparison table

[0143] Comparison of different technical indicators Induction rate (%) Root length (cm) Leaf bud length (cm) Other existing technical indicators >55 2～3 5～10 Technical indicators of the embodiments of the present invention >90 4～5 10-15

[0144] like Figure 3 Shown is the growth of azalea seeds after 90 days of culture.

[0145] Application Example 1: Application in Plant Tissue Culture Laboratory

[0146] In the plant tissue culture laboratory, researchers use the azalea orchid tissue culture one-step seedling system to carry out culture experiments with different formulas. The laboratory can accurately adjust the composition ratio of the culture medium (such as sucrose, agar, activated carbon, pure milk and various plant hormones), and adjust the pH value of the culture medium to 5.8 through the automatic pH adjustment module to ensure the controllability of different experimental conditions. In addition, the light cycle and temperature control module accurately set a cycle of 9 hours of light and 15 hours of darkness and a constant temperature environment of 25±1°C to simulate the most suitable natural growth conditions for azalea orchids. Through the aseptic operation module of the system, the interference of external pathogens is reduced, and the accuracy of the experiment and the work efficiency of the laboratory are improved.

[0147] Application Example 2: Application of a large-scale production base for azalea seedlings

[0148] In the azalea seedling production base, the tissue culture system has become the core automated seedling raising tool. Thousands of capsule seeds are processed by the system every day. The optical detection device can automatically detect the maturity of the azalea capsule to ensure that only high-quality seeds with dark yellow or yellow-green uncracked peels are selected. The disinfection and sterilization module performs precise sodium hypochlorite and ethanol soaking, rinsing and other processes to avoid improper operation or cross-contamination caused by manual disinfection. The system's automated culture medium preparation module provides exclusive nutrition solutions for different strains of azalea seeds to ensure that the culture medium formula for each batch is consistent. In addition, the system will place the inoculated azalea seeds in an environment with high humidity, controllable temperature and light to provide the best culture conditions for azalea. The base uses this system for centralized management and automated seedling raising, which not only improves the survival rate of seedlings, but also realizes standardized production, greatly reducing production costs and labor input.

[0149] Application Example 3: Plant Gene Research in Scientific Research Institutions

[0150] In plant gene research, scientific research institutions use the high-precision control of this system to conduct in-depth research on the relationship between gene expression and environmental conditions of azalea orchids. Through the system's built-in hormone module, researchers can accurately add hormones such as NAA (naphthaleneacetic acid), 6-BA (6-benzyladenine) and ZT (zeatin) to the culture medium, and conduct combination experiments with different hormone concentrations to observe the regulatory effects of these hormones on azalea orchid auxin gene expression, tissue differentiation and cell division. The automated sterile culture environment ensures that experimental samples are not contaminated by the outside world, making related research more accurate and reliable. In addition, by comparing the growth performance of azalea orchids under different formulas and environmental settings (such as light cycle, temperature, hormone concentration), researchers can screen out key characteristics such as high-efficiency resistance genes and study flowering period regulation, thereby providing basic data support for the subsequent genetic engineering breeding of new varieties.

[0151] Application Example 4: Commercial Ornamental Plant Planting Company

[0152] In the ornamental plant market, azalea orchid has high ornamental and economic value. The planting company has solved the problems of high labor costs and long culture cycles in traditional methods by using this system to quickly culture azalea orchid seedlings. Through this system, azalea orchid capsules are screened, disinfected, and then implanted in a culture medium containing the optimal hormone ratio, and grown under specific temperature, humidity and light conditions. The company ensures that each batch of seedlings has consistent quality and growth conditions. The production process uses an automatic monitoring and adjustment mechanism to stabilize the nutrient content and hormone ratio in the culture medium. Azalea orchid seedlings can be grown within 8 to 9 months, which is 20% to 30% shorter than traditional methods, so that they can be put on the market faster. At the same time, the system supports large-scale production, reduces the number of frequent manual transfers in traditional processes, saves more than 60% of labor input and more than 20% of energy consumption, and brings significant economic benefits and market competitive advantages to the company.

[0153] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principle of the present invention should be covered by the protection scope of the present invention.

Claims

1. A method for one-step seedling formation of azalea orchid tissue culture, characterized in that: The following steps are involved: Step 1, explant selection: select capsules of azalea orchid that mature from November to December each year as explants, fresh seeds with dark yellow or light yellow-green peel and not cracked, dry them and store them at 5°C for later use, and then carry out tissue culture; Step 2, disinfection and sterilization: rinse with running water for 5 to 10 minutes, soak in 10% sodium hypochlorite solution for 10 to 15 minutes, soak in 75% ethanol solution for 5 to 10 minutes, rinse with sterile water for 3 to 5 times and dry for later use; Step 3, culture medium and hormone formula: using 1 / 2MS nutrient medium, adding 3% to 5% sucrose, 0.5% to 0.7% agar, 0.3g / L to 0.8g / L activated carbon and 1% to 3% pure milk, hormones are 0.1mg / L to 0.5mg / L NAA, 0.3mg / L to 0.8mg / L 6-BA and 0.5mg / L to 1.2mg / L ZT, and the pH value of the culture medium is adjusted to 5.8; Step 4, inoculation and cultivation: The seeds are inoculated into a sterile culture medium and cultured for germination and rooting in an environment with a certain temperature, humidity and light. The light cycle is 9 hours of light / 15 hours of darkness, and the temperature is controlled at 25±1°C.

2. The method according to claim 1, characterized in that The azalea capsule selected in step 1 should be a mature, unopened capsule with a dark yellow or light yellow-green peel, and should be collected and preserved from November to December to ensure the vitality of the seeds inside the capsule.

3. The method according to claim 1, characterized in that In the disinfection step, the concentration of the sodium hypochlorite solution used is 10%, and the soaking time is 10 minutes, so as to effectively remove pathogens and microorganisms on the surface of the seeds and ensure the sterility of the seed surface.

4. The method according to claim 1, characterized in that The 1% pure milk added to the culture medium can provide additional protein, fat, vitamins and trace elements, promote nutrient absorption and growth of seedlings, and accelerate cell division and root formation.

5. A system for one-step seedling production from tissue culture of azalea for implementing the one-step seedling production from tissue culture of azalea as claimed in any one of claims 1 to 4, characterized in that: The system for one-step seedling formation of azalea tissue culture comprises: The explant selection module is used to select capsules of azalea orchid that mature from November to December each year as explants, and fresh seeds with dark yellow or light yellow-green peel and not cracked, and store them at 5°C for later use after drying, and then conduct tissue culture; The disinfection and sterilization module is used to rinse with running water for 5 to 10 minutes, soak with 10% sodium hypochlorite solution for 10 to 15 minutes, and then soak with 75% ethanol solution for 5 to 10 minutes, then rinse with sterile water for 3 to 5 times and dry for use; The culture medium and hormone module is used to adopt 1 / 2MS nutrient culture medium, add 3% to 5% sucrose, 0.5% to 0.7% agar, 0.3g / L to 0.8g / L activated carbon and 1% to 3% pure milk, the hormones are 0.1mg / L to 0.5mg / L NAA, 0.3mg / L to 0.8mg / L 6-BA and 0.5mg / L to 1.2mg / L ZT, and the pH value of the culture medium is adjusted to 5.8; The inoculation and cultivation module is used to inoculate seeds into sterile culture medium and carry out germination and rooting cultivation in an environment with certain temperature, humidity and light.

6. The one-step seedling system of azalea tissue culture as claimed in claim 5, characterized in that: The explant selection module also includes a high-precision optical detection device for detecting the maturity of the azalea capsule, and automatically screening out dark yellow-brown or brown uncracked mature seeds according to the color and structure of the fruit skin to ensure that the seeds have optimal biological activity.

7. The one-step seedling system of azalea tissue culture as claimed in claim 5, characterized in that: The disinfection and sterilization module includes an automated disinfection device that can accurately control the immersion time of the sodium hypochlorite solution and the ethanol solution, and is equipped with a sterile water flushing device that can achieve multiple sterile water rinses through program settings to ensure that the seed surface is free of contamination.

8. The one-step seedling system of azalea tissue culture as claimed in claim 5, characterized in that: The culture medium and hormone module also includes a control device for automatically adjusting the pH value, which can accurately adjust the pH value of the culture medium to 5.8, and monitor the concentration of the culture medium components through sensors to ensure that the ratio of hormones, sucrose, agar, activated carbon and pure milk remains consistent at all times to ensure the stability of the culture effect.

9. An application of the method for one-step seedling formation of azalea orchid tissue culture based on the method of claim 1 in the rapid propagation of azalea orchid seedlings, characterized in that: The following steps can be used to achieve efficient cultivation of azalea seedlings: Mature capsules were used as explants. After screening, disinfection and sterilization, 1 / 2MS nutrient medium was used with sucrose, agar, activated carbon, pure milk and appropriate amounts of plant hormones for seed inoculation and germination and rooting culture. Under a constant temperature of 25±1℃, combined with a photoperiod of 9 hours of light and 15 hours of darkness, rapid seedling establishment and high survival rate of azalea seedlings were achieved through precise control of nutrient composition and environmental conditions.

10. Application of the method for one-step seedling formation of azalea orchid tissue culture as claimed in claim 9 in the cultivation of Chinese medicinal material seedlings, characterized in that: The method can be applied to large-scale seedling breeding of azalea orchid, and can improve the breeding efficiency and quality of azalea orchid seedlings by utilizing low-cost culture medium and automated control technology, selecting optimal explants and optimizing culture conditions.

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