A method and system for one-step seedling of tissue culture seedlings of a cymbidium

Abstract

The application belongs to the technical field of Chinese medicinal material seedling breeding, and discloses a method for one-step seedling of Cymbidium goeringii tissue culture seedlings, which uses seeds as explants, carries out Cymbidium goeringii plant tissue culture induction seedling through 1 / 2MS culture medium, hormones NAA, 6-BA, ZT, sucrose, fresh milk, agar and active carbon and other related substances after disinfection and sterilization. The application can directly germinate from seeds, induce value-added differentiation and root seedling. The one-step seedling method reduces the processes of value-added differentiation and induction rooting of tissue culture seedlings, saves time cost and transfer cost, uses seeds as explants, and the roots of the one-step seedling are 4-5 cm long and the leaf buds are 10-15 cm long after 8-9 months of culture. The application solves the problems of long time consumption, high cost and complicated multi-stage transfer process in the existing plant tissue culture. The one-step seedling method for Cymbidium goeringii tissue culture seedlings adopts a simplified culture process, effectively shortens the culture period, reduces the cost, and enhances the consistency and health of plant growth.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of Chinese medicinal material seedling cultivation, and particularly relates to a method and system for one-step seedling formation of Rhynchostylis retusa tissue culture seedlings. BACKGROUND

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

[0003] Defects and deficiencies of the prior art:

[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 tissue to be affected by the external environment during the operation process, increasing the risk of bacterial contamination and vitrification seedlings.

[0006] 2. 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 increase the time between the connection of each stage, directly extending the entire culture period and affecting production efficiency.

[0008] 3. High cost

[0009] The prior art requires the preparation of new culture media 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 media multiple times also increases the rate of bacterial infection, further increasing production costs.

[0010] 4. Low efficiency and resource waste

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

[0012] In the prior art, there is no effective solution to the problem of how to optimize the multiple transfers in the process of Rhynchostylis retusa plant tissue culture. Current research and technical practice mainly focuses on conventional medium improvement and growth condition adjustment, rather than systematic optimization of the culture process.

[0013] In summary, the existing plant tissue culture technology in the problems of multiple transfer, long time and high cost in the culture of Cymbidium group culture seedlings need to be solved. How to simplify the culture process, shorten the cycle, reduce the cost and improve the seedling rate becomes the key direction of current technology development. SUMMARY

[0014] In view of the problems existing in the prior art, the application provides a method for one-step seedling of Cymbidium group culture seedlings. The proliferation and rooting culture stages of the Cymbidium group culture seedlings do not need to prepare the culture medium and transfer again, and the seedlings are directly induced from the seeds in one step.

[0015] The application is implemented in the following manner: a method for one-step seedling of Cymbidium group culture seedlings comprises the following steps:

[0016] Step 1: selecting explants: selecting mature capsules of Cymbidium from November to December every year as explants, fresh seeds with deep yellow or light yellow-green fruit peels and uncracked, and the seeds are dried and stored at 5 DEG C for later tissue culture;

[0017] Step 2: disinfection and sterilization: using flowing tap water to wash for 5 min to 10 min, immersing in 10% sodium hypochlorite solution for 10 min to 15 min, immersing in 75% ethanol solution for 5 min to 10 min, then washing with sterile water for 3 to 5 times and drying 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, 0.3 g / L to 0.8 g / L activated carbon and 1% to 3% pure milk, and the 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 culture: inoculating the seeds into the sterile culture medium, and performing germination and rooting culture in an environment with 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 DEG C.

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

[0021] Further, in step 4, the mature capsules after disinfection and sterilization are cut open with a sterile knife, the seeds are evenly scattered into the sterilized culture medium for sterile culture, the culture temperature is controlled at 24 DEG C to 26 DEG C, the humidity is controlled at 60% to 80%, the light intensity is controlled at 2200 lx to 2500 lx, and the light and dark environment is cycled for 9 h to 15 h per day.

[0022] Another object of the present application is to provide a Cymbidium step-by-step seedling system comprising:

[0023] An explant selection module is used to select mature capsules of Cymbidium from November to December each year as explants, and fresh seeds with deep yellow or light yellow-green pericarp and uncracked are dried and stored at 5℃ for later use in tissue culture;

[0024] A disinfection and sterilization module is used to flow wash for 5-10 min with tap water, soak for 10-15 min with 10% sodium hypochlorite solution, soak for 5-10 min with 75% ethanol solution, then rinse 3-5 times with sterile water and dry for later use;

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

[0026] An inoculation and culture module is used to seed in sterile culture medium and carry out germination and root culture in an environment with certain temperature, humidity and light.

[0027] The present application also provides a method for Cymbidium step-by-step seedling, which is applied in the rapid propagation of Cymbidium seedlings, and the efficient cultivation of Cymbidium seedlings is realized through the following steps:

[0028] Mature capsules are used as explants, and after screening, disinfection and sterilization, 1 / 2MS nutrient medium is used, combined with sucrose, agar, activated carbon, pure milk and appropriate plant hormones, seed inoculation and germination and root culture are carried out;

[0029] Under the constant temperature condition of 25±1℃, combined with 9 hours of light and 15 hours of darkness in the light cycle, through the precise control of nutritional ingredients and environmental conditions, the rapid seedling and high survival rate of Cymbidium seedlings are realized.

[0030] Further, the method can be applied to large-scale seedling breeding of Cymbidium, and through the selection of the best explants and the optimization of the culture conditions, the propagation efficiency and quality of Cymbidium seedlings are improved by using low-cost culture medium and automatic control technology, which is suitable for seedling breeding and commercial production of Chinese herbal medicines.

[0031] In combination with the above technical solutions and the technical problems solved, the technical solution to be protected by the present application has the following advantages and positive effects:

[0032] Currently, there is no patent or literature report on the one-step seedling technique of Cymbidium goeringii tissue culture seedlings. Through independent research and development, the present application innovatively proposes a one-step seedling technique from seed to seedling of Cymbidium goeringii tissue culture seedlings, and optimizes the culture process, thereby providing a new solution for plant tissue culture technology.

[0033] Firstly, the present application directly induces proliferation, rooting culture and seedling from seed germination without the processes of propagation differentiation culture, induction rooting culture, and regeneration plant rapid cultivation.

[0034] The present application can directly induce proliferation, differentiation of cotyledon, bulbil and root system from seed germination, and directly form seedlings.

[0035] Advantages: The one-step seedling method reduces the processes of propagation differentiation and induction rooting of tissue culture seedlings, saves time cost and transfer cost, uses seeds as explants, and has an induction rate of more than 90% after 8-9 months of culture, a root system of 4-5 cm, and a leaf bud of 10-15 cm.

[0036] Simplified operation process: The operation process is simplified by eliminating multiple transfer culture steps, avoiding bacterial infection of tissue bodies caused by multiple transfers, and reducing labor demand.

[0037] Cost reduction: The use amount of culture medium is reduced, the labor cost of tissue body transfer is reduced compared with conventional tissue culture seedlings, the power consumption during culture is reduced, and the production cost is significantly reduced.

[0038] Improved seedling health: The opportunities of repeated contact of tissue body materials with different growth hormones are reduced, the number of subculture is effectively reduced, and the risks of tissue body material browning and vitrification seedlings are reduced.

[0039] Accelerated production cycle: The production cycle of seedlings is accelerated due to the omission of multiple transfer steps, and the entire tissue culture process time is shortened.

[0040] This new technology has significant economic and practical significance for improving the tissue culture efficiency of Cymbidium goeringii. It not only improves the production efficiency of Cymbidium goeringii tissue culture seedlings, but also reduces the production cost and improves the quality of seedlings. The one-step seedling technique is innovative and practical, meets the innovation requirements of the Patent Law, and is expected to be granted a patent.

[0041] The present application aims to solve the problems of long time consumption, high cost and complicated multi-stage transfer culture process in existing plant tissue culture. Specifically, the one-step seedling method of Cymbidium goeringii tissue culture is adopted, a simplified culture process is used, the culture period is effectively shortened, the cost is reduced, and the consistency and health of plant growth are enhanced.

[0042] The prior art needs to go through seed germination culture, induction proliferation and differentiation culture, induction rooting culture and rapid cultivation of regenerated plants in the process of tissue culture. These stages not only take a long time, but also introduce pollution during each transfer process, and increase labor and material costs. The present application directly goes from seed to seedling in one step, eliminating multiple intermediate links, aiming to simplify the entire process.

[0043] Time efficiency improvement: The method of directly from seed to seedling greatly shortens the overall cycle of tissue culture. The traditional method takes 10-12 months, while the method of the present application only takes 8-9 months.

[0044] Cost saving and risk reduction: The reduction of multiple transfers and related culture medium, container use and labor cost, as well as the reduction of power consumption during culture, significantly reduces the production cost. At the same time, the saving of multiple transfer steps reduces the risk of plant contamination.

[0045] Rooting and growth optimization: Through the one-step seedling method, the plant root system is developed (up to 4-5 cm), the leaf bud is lush (up to 10-15 cm), and better growth potential and adaptability are shown.

[0046] Improved seedling health: The reduction of tissue material repetition, multiple contact with different growth hormones, the effective reduction of subculture times, the reduction of tissue material browning, vitrification seedling risk, and the enhancement of plant growth health.

[0047] Second, the expected income and commercial value of the technical solution of the present application after transformation are: after the transformation of the technical solution of the present application, the production cost of the Cymbidium goeringii tissue culture seedlings can be significantly reduced in terms of time, labor and related tissue culture material saving, which has greater economic benefits compared with non-one-step seedling tissue culture seedlings.

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

[0049] The technical solution of the present application solves the technical problems that people have been eager to solve but have always failed to succeed: the technical solution of the present application solves the technical problems of long cycle and high cost of tissue culture seedlings.

[0050] Third, in the technology of Cymbidium goeringii tissue culture seedling, the present application solves the technical problems of low seedling efficiency, complex manual operation and high production cost existing in the prior art through scientific parameter control, optimized culture medium formula and integration of automatic module, and achieves significant technical progress. It is embodied in the following aspects:

[0051] 1. Improved seedling efficiency

[0052] Existing technologies usually require multiple manual transfers and multiple culture medium changes, resulting in a long seedling cycle and a low seedling success rate. The present invention uses a one-time configuration of an optimized culture medium and precise control of the growth environment, allowing azalea orchid tissue culture seedlings to complete the entire growth process in one cycle with only one transfer. Compared with existing technologies, 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] Traditional cultivation techniques require three to four manual transfers and culture medium changes, consuming significant human resources and culture materials. The present invention requires only a single transfer, reducing culture medium and labor costs by over 60%. Furthermore, the automated control system reduces errors caused by manual operation and ensures consistent seedling quality. This significantly reduces production costs, making commercial and large-scale production of azalea orchid seedling cultivation more economical.

[0055] 3. Improved energy efficiency

[0056] Traditional tissue culture techniques require long periods of light and environmental maintenance to ensure the plants meet their needs at different growth stages. This invention utilizes a scientifically optimized light cycle (9 hours light / 15 hours dark) and a suitable constant temperature (25±1°C) to achieve energy consumption control. This also shortens the culture cycle and reduces overall energy consumption by over 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 contains sucrose, agar, activated carbon and pure milk in optimized proportions, 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 existing technology 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 and cultivation, offering the advantages of automation and standardization. In actual operation, the parameter setting of the automated control equipment reduces reliance on specialized skills, simplifies operation, and improves production stability. Furthermore, the automated system ensures consistent operational procedures for each batch, avoiding fluctuations caused by manual labor and further ensuring the consistent quality of mature azalea orchid seedlings.

[0061] 6.Quality control and standardized production

[0062] This invention significantly improves the quality of azalea seedlings through high-precision control, ensuring stability at every stage. This technical solution makes the cultivation process transparent and standardized, enabling companies to accurately manage various parameters and monitor growth in real time, achieving high yields of high-quality azalea seedlings and providing a solid foundation for large-scale production and market competitiveness.

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

[0064] Figure 1 The present invention provides a flowchart of a method for one-step seedling formation of azalea orchid tissue culture seedlings.

[0065] Figure 2 This is a system structure diagram for one-step seedling formation of azalea orchid tissue culture seedlings provided by an embodiment of the present invention.

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

[0067] In order to make the purpose, technical solutions 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 intended to limit the present invention.

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

[0069] S101, Explant Selection: Select capsules of Rhododendron orchid that mature between November and December each year as explants. Fresh seeds with dark yellow or light yellow-green peel and not cracked are selected. After drying, store at 5°C for subsequent tissue culture.

[0070] S102, disinfection and sterilization: use tap water flow rinse for 5-10 minutes, soak in 10% sodium hypochlorite solution for 10-15 minutes, then soak in 75% ethanol solution for 5-10 minutes, then rinse with sterile water for 3-5 times and dry for standby;

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

[0072] S104, inoculation and culture: seed inoculation into sterile medium, germination and root culture in a certain temperature, humidity and light environment, light cycle is 9 hours light / 15 hours darkness, temperature control at 25±1℃.

[0073] Example 1 for components

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

[0075] Example 2 for components

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

[0077] Example 3 for components

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

[0079] Example 4 for components

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

[0081] The one-step seedling method for Cymbidium seedlings provided by the embodiment of the application effectively improves the survival rate and growth quality of Cymbidium seedlings through a scientific process flow and precise nutrition and environment control.

[0082] 1. Selection of explants

[0083] In step S101, a mature unsplit Cymbidium capsule is selected as the explant, because the seeds in the capsule picked up from November to December have higher activity and better growth state, and the pericarp is dark yellow or light yellow-green, which is the best maturity of the seeds, and is conducive to improving the survival rate of tissue culture. By screening the maturity and activity of the seeds, it can be ensured that the seeds have good germination and seedling ability.

[0084] 2. Disinfection and sterilization treatment

[0085] In step S102, the explant is subjected to strict disinfection and sterilization treatment to remove pathogenic bacteria and microorganisms on the surface of the seed and ensure growth in a sterile environment. First, the seed is washed with running tap water for 5 to 10 minutes to preliminarily remove surface dirt and microorganisms; then the seed is disinfected with 10% sodium hypochlorite solution and 75% ethanol solution for 10-15 minutes and 5-10 minutes respectively. Finally, the seed is rinsed with sterile water for 3-5 times to completely remove the disinfectant residues. This series of operations ensures that the surface of the seed is sterile and reduces the influence of pathogenic bacteria on the seedling.

[0086] 3. Preparation of 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, which provides sufficient carbon source, gelling agent and auxiliary nutrients, especially activated carbon which can effectively promote root growth. The addition of hormones is 0.1mg / L-0.5mg / L NAA, 0.3mg / L-0.8mg / L 6-BA and 0.5mg / L-1.2mg / L ZT, which can promote the germination of Cymbidium seeds and the growth of seedlings. The pH value of the medium is adjusted to 5.8 to ensure an acid-alkaline environment suitable for seed germination.

[0088] 4. Inoculation and culture

[0089] In step S104, the treated Cymbidium seed is inoculated in a sterile culture medium, and suitable environmental conditions are created to promote germination and rooting. The culture environment needs to maintain a periodic light of 9 hours light / 15 hours darkness to simulate the day and night cycle in the natural environment, and the setting of light intensity and cycle helps the growth and development of the seed. The temperature is controlled at 25±1℃, which provides the most suitable temperature conditions to make the seedling development more uniform and stable.

[0090] 5. Control conditions ensure stable growth

[0091] During the entire culture process, the light, temperature and humidity conditions of the environment are precisely controlled to ensure that the seeds can grow in the optimal environment. The setting of light cycle and temperature is based on the growth needs of Cymbidium, which helps the seeds to 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 are optimized through repeated tests, which has a good promoting effect on the germination of Cymbidium seeds.

[0092] 6. One-step seedling improves efficiency

[0093] In this method, the optimized culture medium and environmental control realize the rapid seedling of Cymbidium. Compared with the traditional multi-transferring tissue culture method, this invention only needs one inoculation and culture, which not only significantly shortens the seedling period, but also greatly reduces the manual operation, reduces the consumption of culture medium, improves the survival rate of seedlings and production efficiency, and makes the culture of Cymbidium more simple and efficient.

[0094] Further, the hormone is 0.1mg / L-0.5mg / L NAA (naphthalene acetic acid), 0.3mg / L-0.8mg / L 6-BA (6-benzylaminopurine) and 0.5mg / L-1.2mg / L ZT (zeatin).

[0095] S104, inoculation and culture: the mature anther after sterilization and disinfection is cut open with a sterile knife, and the seeds are evenly scattered into the sterilized culture medium for sterile culture. The culture temperature is controlled at 24-26℃, the humidity is 60%-80%, the light intensity is 2200lx-2500lx, and the light and dark environment is cycled for 9-15 hours per day.

[0096] The mature seeds of Cymbidium from November to December each year are selected as explants, because the anther has not yet cracked at this time, and the inside has not been contaminated by the environment. The uncracked pericarp is dark yellow or light yellow-green, and the seeds inside the anther have strong seed vigor, which is suitable for tissue culture. Selecting seeds at this stage can improve the vigor of tissue culture seedlings, promote rapid germination and smooth subsequent culture process.

[0097] The tap water flushing and the use of sodium hypochlorite solution and ethanol solution in the disinfection step are aimed at removing 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 the sterility of the seed surface. After several rinses with sterile water, the seed surface is basically free of bacterial contamination, providing a pure environment for subsequent culture. This process is a key step for successful tissue culture, avoiding the presence of bacteria or fungi in the culture medium to infect the medium.

[0098] Using 1 / 2MS medium is a common means of plant tissue culture, which provides the basic nutrients required for seed development. Compared with full-strength MS medium, 1 / 2MS reduces the concentration of inorganic salts, which helps to reduce the stress response of plants and promote the balanced development of root and shoot. At the same time, 3% sucrose is added as a carbon source for seed utilization to promote the metabolic process.

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

[0100] The addition of activated carbon in the medium helps to adsorb excess harmful substances or metabolic products in the medium, such as phenolic compounds secreted by plants during development. These substances, if accumulated, can cause browning, tissue necrosis and other conditions. Activated carbon can also adjust the transparency of the medium, making it easier for the root system to contact the medium and promoting root growth and absorption.

[0101] The addition of 1% pure milk in the medium provides an additional source of nutrients. The proteins, fats, vitamins and trace elements in milk provide more nutrients for seedlings, enhancing the growth momentum of seeds. This natural nutrient supplement not only improves the stress resistance of seedlings, but also promotes cell division and organ formation, further improving the efficiency of tissue culture and the survival rate of seedlings, which also plays a key role in this invention.

[0102] As shown in Figure 2 The system for one-step seedling of Cymbidium kanran tissue culture seedlings provided by the embodiment of the present application comprises:

[0103] The explant selection module is used to select the mature capsule of Cymbidium kanran from November to December each year as the explant, the fresh seed with deep yellow or light yellow-green pericarp and uncracked is dried and stored at 5℃ for standby, and subsequent tissue culture is carried out.

[0104] Disinfection and sterilization module, for using tap water flow washing 5min~10min, using 10% sodium hypochlorite solution soaking 10min~15min, then using 75% ethanol solution soaking 5min~10min, then using sterile water rinsing 3~5 times and drying for standby;

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

[0106] Inoculation and culture module, for seed inoculation into sterile culture medium, and germination and root culture in an environment with certain temperature, humidity and light.

[0107] The specific technical content and implementation principle of the embodiment of the application.

[0108] Explant selection: mature capsules of Cymbidium goeringii collected in the middle of November are selected, fresh seeds with pericarp of dark yellow or light yellow green and uncracked are dried and stored at 5 DEG C for standby, and subsequent tissue culture is carried out.

[0109] Disinfection and sterilization: the uncracked mature capsule is first washed with tap water flow for 5min, then soaked with 10% sodium hypochlorite solution for 10min after drying the water, then soaked with 75% ethanol solution for 5min after drying the water with sterile paper, then rinsed with sterile water for 3 times, and then dried with sterile paper for standby.

[0110] Culture medium and hormone: 1 / 2MS nutrient medium is used, and hormones are 0.1mg / L~0.5mg / L NAA (naphthalene acetic 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 the pH value of the culture medium is adjusted to 5.8.

[0111] Cell totipotency: the totipotency of cells is utilized, nutrients and hormones are added in the culture medium in proper proportion, the differentiation direction of cells is regulated, and direct conversion from seeds to seedlings is realized.

[0112] Hormone regulation: the ratio of NAA, 6-BA and ZT regulates the balance of cell differentiation and organogenesis, and the process of inducing seedlings is optimized.

[0113] The application provides a more economical and efficient method for one-step seedling of Cymbidium seedlings, greatly simplifies the complexity of traditional plant tissue culture, and significantly improves the culture efficiency and plant quality, thereby providing a new nature for plant tissue culture technology.

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

[0115] The disinfection and sterilization module is embedded with automatic disinfection devices and sterile water rinsing equipment, which can accurately perform disinfection procedures. First, the seeds are soaked in sodium hypochlorite solution for a certain period of time to remove surface bacteria and impurities. Then, further sterilization is carried out through ethanol solution to enhance the sterilization effect. After the sterile water rinsing equipment is started, multiple rinsing cycles are used to remove residual chemicals, ensuring that the surface of the seeds is completely free of pollution. The entire process is controlled by a program with high automation, ensuring the uniformity and stability of the disinfection effect.

[0116] The culture medium preparation module is equipped with automatic pH value adjustment devices and sensor monitoring systems. During the preparation of the culture medium, the sensor first monitors the composition of the culture medium in real time, including the concentration ratio of sucrose, agar, hormones, activated carbon and pure milk. Then, 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 Cymbidium seeds. Throughout the process, closed-loop control is used to ensure the stability of the culture medium composition and maximize the germination and seedling effect of the seeds.

[0117] The internal environment of the tissue culture room is adjusted 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 required for seed germination. The high-efficiency air filtration system (HEPA) further ensures a sterile environment, avoiding the influence of external pollution sources on the culture medium. Through precise control of environmental conditions, an ideal growth environment is provided for Cymbidium seeds from germination to seedling.

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

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

[0120] Through the above multi-module collaborative work, the Cymbidium seedling system realizes the full automation operation from seed selection to seedling, greatly improves the production efficiency and seedling quality, and reduces the risk and error of human participation.

[0121] The following are two specific examples of Cymbidium seedling system:

[0122] Example 1: Cymbidium seed tissue culture suitable for rapid seedling

[0123] 1. Selection of explants: Select fresh mature capsules of Cymbidium in mid-November, which are light yellow-green and have not yet cracked, ensuring that the pericarp is intact and the seeds have not yet cracked.

[0124] 2. Disinfection and sterilization: Wash the capsules with flowing tap water for 5 minutes, dry them, then soak them in 10% sodium hypochlorite solution for 10 minutes, and then dry them with sterile paper. Then, soak them in 75% ethanol solution for 5 minutes, then rinse them with sterile water 3 times, and dry them with sterile paper for use.

[0125] 3. Culture medium and hormones: Use 1 / 2MS nutrient medium, add 0.2mg / L NAA, 0.5mg / L 6-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 medium, adjust the pH to 5.8.

[0126] 4. Culture process: Inoculate the disinfected Cymbidium seeds into the culture medium, and cultivate in a 25℃ constant temperature incubator, with humidity of 60% and light intensity of 2200lx, light cycle of 9 hours light / 15 hours darkness. After 55 days of culture, the seeds begin to germinate and form seedlings within 9 months.

[0127] 5. Results: This method rapidly induces seed germination and forms complete Cymbidium seedlings in a short time, with a germination rate of 95%, strong seedlings and high survival rate.

[0128] Example 2: Tissue culture and seedling method suitable for various Cymbidium varieties

[0129] 1. Selection of explants: Select mature capsules of different varieties of Cymbidium collected in December, which are fresh and mature capsules with deep yellow pericarp that have not yet cracked.

[0130] 2. Disinfection and sterilization: The capsule was first rinsed with tap water for 5 minutes, then soaked in a 10% sodium hypochlorite solution for 8 minutes, followed by a 75% ethanol solution for 5 minutes, rinsed with sterile water for 3 times, and then dried for use.

[0131] 3. Culture medium and hormones: 1 / 2MS medium was used, and hormone combination 0.2mg / L NAA, 0.5mg / L 6-BA and 0.8mg / L ZT was added. The medium was additionally supplemented with 0.3g / L activated carbon, 3% sucrose, 0.4% agar and 1.5% pure milk, and the pH value was adjusted to 5.8.

[0132] 4. Culture process: The sterilized Cymbidium seed was inoculated into the above-mentioned medium and placed in a 27℃ incubator for culture, with humidity of 70% and light intensity of 2400lx, and light cycle of 9 hours light / 15 hours dark. After about 60 days, the seeds began to germinate and formed seedlings within 8 months.

[0133] 5. Results: This method is suitable for different varieties of Cymbidium seeds, with germination rate above 90%, shorter culture period, and uniform growth of seedlings, which meets the growth needs of various Cymbidium varieties.

[0134] In the culture process of tissue culture and seedling formation, the embodiment of the present application significantly reduces the production cost through innovative process and technical route. Compared with the existing tissue culture technology, the present application only needs one-time manual transfer and medium input, while the traditional technology usually needs 3 to 4 times of transfer and medium replacement. This optimized design can save more than 60% of labor and material input in one tissue culture and seedling formation cycle, greatly reducing the production cost.

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

[0136] In terms of tissue culture effect, the present application is significantly superior to the existing technology in key technical indicators. Experimental data shows that the tissue culture induction rate of the present application is more than 90%, which is significantly higher than the traditional technology of more than 55%; in terms of root length and leaf bud length of seedling development, the present application is superior, with root length of 4 to 5 centimeters and leaf bud length of 10 to 15 centimeters, while the existing technology has root length of about 2 to 3 centimeters and leaf bud length of 5 to 10 centimeters, significantly improving the quality of seedling formation.

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

[0138] To further demonstrate the technical advantages and improvement effects of the present application, the comparison data show that in the cultivation process of Cymbidium seed, the present application shows stronger growth performance in the 90-day growth observation result. The significant improvement of root length, leaf bud length and induction rate in the process of tissue culture and seedling ensures that the developed plants are superior to the traditional method in development and growth quality, and is suitable for application in large-scale commercial production.

[0139] In summary, the present application has made significant progress in production cost, energy efficiency and seedling quality, providing an excellent technical solution that saves cost, improves efficiency and ensures quality. For specific comparison, see Table 1 and Table 2, which is suitable for the promotion and application of plant tissue culture.

[0140] Table 1 Comparison of input costs

[0141] Different technical input costs Time and electricity Switching artificial Culture medium input Other existing technical indicators October to December 3-4 times 3-4 times Technical indicators of the embodiment of the application August to September 1 time 1 time The application saves >20％ >60％ >60％

[0142] Table 2 Comparison of technical indicators

[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 embodiment of the application >90 4～5 10-15

[0144] As shown in Table 1 and Table 2, the growth of Cymbidium seed culture for 90 days. Figure 3

[0145] Application Example 1: Application of plant tissue culture laboratory

[0146] In the plant tissue culture laboratory, researchers use Cymbidium tissue culture one-step seedling system to carry out culture experiments of different formulations. The laboratory can accurately adjust the component ratio of the culture medium (such as sucrose, agar, activated carbon, pure milk and various plant hormones), and through the automatic pH adjustment module, the pH value of the culture medium is adjusted to 5.8, ensuring the controllability of different experimental conditions. In addition, the light cycle and temperature control module accurately sets the cycle of 9 hours of light and 15 hours of darkness and the constant temperature environment of 25±1℃ to simulate the most suitable natural growth conditions for Cymbidium. Through the sterile 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 Cymbidium seedling large-scale production base

[0148] ​In the Cymbidium seedling production base, the tissue culture system becomes the core automated seedling tool. Each day, thousands of capsules and seeds are processed by the system, and the optical detection device can automatically detect the maturity of Cymbidium capsules, ensuring that only high-quality seeds with deep yellow or yellow-green pericarps 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 medium preparation module provides exclusive nutrition solutions for different Cymbidium seed lines, ensuring that each batch of medium formula is consistent. In addition, the system places inoculated Cymbidium seeds in an environment with high humidity, controllable temperature, and light, providing optimal cultivation conditions for Cymbidium. The base uses the system for centralized management and automated seedling, 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 system's high-precision control to deeply study the relationship between Cymbidium gene expression and environmental conditions. Through the system's built-in hormone module, researchers can accurately add hormones such as NAA (naphthalene acetic acid), 6-BA (6-benzyladenine), and ZT (zeatin) to the medium and conduct combination experiments with different hormone concentrations to observe the effects of these hormones on Cymbidium 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 Cymbidium under different formulations and environmental settings (such as light cycle, temperature, and hormone concentration), researchers can screen high-efficiency resistance genes and study key characteristics such as flowering regulation, thereby providing basic data support for subsequent genetic engineering breeding of new varieties.

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

[0152] In the ornamental plant market, the Cymbidium has a high ornamental and economic value. The planting company solves the problems of high labor cost and long culture period in traditional methods through the system in the rapid tissue culture of Cymbidium. Through the system, the capsule of Cymbidium is screened and disinfected and then planted in the culture medium containing the optimal hormone ratio, and grows under the specific temperature, humidity and light conditions, and the company ensures that each batch of seedlings has consistent quality and growth conditions. The production process is adjusted by the automatic monitoring mechanism, so that the nutrient content and hormone ratio in the culture medium are stable, and the Cymbidium seedlings can complete the seedling in 8-9 months, which is 20%-30% shorter than the traditional method, so as to be put into the market faster. At the same time, the system supports large-scale production, reduces the number of frequent manual transfer in the traditional process, saves more than 60% of the labor input and more than 20% of the energy consumption, and brings significant economic benefits and market competitive advantage to the company.

[0153] The above merely illustrates the specific embodiments of the present application, but the protection scope of the present application is not limited thereto, and any modification, equivalent replacement and improvement within the technical range disclosed by the present application and within the spirit and principle of the present application shall be covered within the protection scope of the present application.

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 between November and 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°C for subsequent tissue culture; Step 2, disinfection and sterilization: rinse with running tap water for 5-10 minutes, soak in 10% sodium hypochlorite solution for 10-15 minutes, soak in 75% ethanol solution for 5-10 minutes, rinse with sterile water 3-5 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 culture medium pH 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 intensity. The photoperiod 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, wherein 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, wherein 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, wherein 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 producing seedlings from azalea orchid tissue culture in one step for implementing the method for producing seedlings from azalea orchid tissue culture in one step according to any one of claims 1 to 4, characterized in that: The system for one-step seedling formation from tissue culture of azalea orchid comprises: The explant selection module is used to select capsules of azalea orchid that mature from November to December each year as explants. The fresh seeds with dark yellow or light yellow-green peel and not cracked are dried and stored at 5°C for subsequent tissue culture. The disinfection and sterilization module is used to flush with running water for 5 minutes to 10 minutes, soak with 10% sodium hypochlorite solution for 10 minutes to 15 minutes, and then soak with 75% ethanol solution for 5 minutes 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 use 1 / 2MS nutrient 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 for tissue culture of azalea 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 rhododendron capsule and automatically screening out dark yellow-brown or brown uncracked mature seeds based on the color and structure of the peel to ensure that the seeds have optimal biological activity.

7. The one-step seedling system for tissue culture of azalea 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 sodium hypochlorite solution and ethanol solution, and is equipped with sterile water flushing equipment. Through program settings, multiple sterile water rinses can be achieved to ensure that the seed surface is free of contamination.

8. The one-step seedling system for tissue culture of azalea 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 rapid propagation of azalea seedlings by tissue culture in one step based on the method of claim 1, characterized in that: The following steps are used to achieve efficient cultivation of azalea seedlings: Mature capsules are used as explants. After screening, disinfection and sterilization, 1 / 2MS nutrient medium is 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, the rapid establishment and high survival rate of azalea seedlings are achieved through precise control of nutrient composition and environmental conditions.

10. Use of the method for one-step tissue culture seedling formation of azalea 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 propagation of azalea orchids, and can improve the propagation 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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