A sterilization method of tissue culture explant seeds of stonecrop genus plants based on burning method and a culture method of tissue culture explant seeds of stonecrop genus plants

Abstract

This invention provides a sterilization method for Lycoris radiata tissue culture explant seeds based on flaming, and a method for culturing Lycoris radiata tissue culture explant seeds, specifically belonging to the field of plant tissue culture technology. The sterilization method of this invention involves removing surface soil and dust from Lycoris radiata capsules, mixing them with 70%–75% alcohol, and sterilizing for 2–3 minutes; then flaming the sterilized Lycoris radiata capsules for 2–5 seconds; the flaming is repeated 3–4 times to obtain sterilized Lycoris radiata tissue culture explant seeds. This invention uses flaming to sterilize the capsules, which is time-efficient, highly effective, and causes minimal damage to the explant seeds, resulting in a high germination rate and shortening the seedling cycle. The flaming method of this invention allows for intermittent and repeated flaming, significantly increasing the proportion of completely sterile products compared to conventional sterilization agents, and resulting in a low contamination rate.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture technology, specifically relating to a sterilization method for explant seeds of Lycoris genus tissue culture based on flaming and a method for culturing explant seeds of Lycoris genus tissue culture. Background Technology

[0002] *Lycoris* belongs to the Amaryllidaceae family and is a perennial herb. It has an underground bulb that is nearly spherical or ovoid; leaves are strap-shaped and clustered. The flowering stem is solitary, erect, and solid; bearing a terminal umbel with 4-8 flowers; flowers are white, creamy white, milky yellow, golden yellow, pink to bright red; tepals are 6, fused at the base into a tubular shape, oblanceolate or oblong, lily-shaped or arranged in a dragon-like pattern; stamens are 6, inserted at the throat, with filiform filaments and T-shaped anthers; pistil is 1, with a slender style, a very small, capitate stigma, an inferior ovary, 3-locular, with a few ovules in each locule. The capsule is usually triangular; seeds are nearly spherical and black.

[0003] The bulbs contain alkaloids such as lycorine, galantamine, and lycorine, which can be used as raw materials for pharmaceuticals. Galantamine and lycorine are present in higher concentrations, possessing detoxifying and nodule-dispersing properties, and are used to treat sore throat, boils, carbuncles, and scrofula. Bulbs 2-3 years old are typically harvested in spring or autumn, washed, and dried, or sliced ​​and dried. For external use, they are crushed and applied to the affected area. In landscaping, all species in this genus have beautiful flowers and vibrant colors, making them suitable for flowering ground cover, planting in flower borders, or natural planting among rocks. About half of the species have robust flower stalks and tall, upright flower stems, making them ideal cut flower materials.

[0004] Lycoris seeds are one of the explants used in rapid tissue culture propagation systems for Lycoris species, but there are currently few reports on using Lycoris seeds as explants for rapid tissue culture propagation. Due to their high starch content, conventional sterilizing agents have difficulty penetrating them, making sterilization challenging. Therefore, there is an urgent need to develop an efficient sterilization method suitable for Lycoris explant seeds used in tissue culture. Summary of the Invention

[0005] The purpose of this invention is to provide a sterilization method for explant seeds of Lycoris radiata tissue culture based on flaming, and a method for culturing explant seeds of Lycoris radiata tissue culture. The sterilization method described in this invention achieves highly efficient seed sterilization, is time-saving, highly efficient, causes minimal damage to the seeds, and results in a high germination rate and low contamination rate after sterilization.

[0006] This invention provides a sterilization method for seeds of tissue-cultured explants of the genus Lycoris based on pyrolysis, comprising the following steps: After removing surface soil and dust from the capsules of Lycoris radiata plants, mix them with 70%~75% alcohol and disinfect for 2~3 minutes. The sterilized capsules of Lycoris radiata were ignited for 2-5 seconds; the ignition was repeated 2-4 times to obtain sterilized Lycoris radiata tissue culture explant seeds.

[0007] Preferably, the seeds include mature seeds and immature seeds; the immature seeds are seeds that have been pollinated for 28 to 35 days.

[0008] Preferably, the volume ratio of the Lycoris radiata capsule to 70%~75% alcohol is 1:(2~5).

[0009] Preferably, the pretreatment method for the capsules includes: harvesting the capsules when the fruit pods of the Lycoris genus turn yellow or 28-35 days after pollination, retaining a 0.5-1 cm long pedicel.

[0010] Preferably, after calcination, the pericarp of the capsule is removed to obtain the seeds.

[0011] Preferably, the Lycoris species include Lycoris chinensis, Lycoris radiata, Lycoris dwarfusa, Lycoris floribunda, Lycoris radiata, Lycoris floribunda ...huinanensis, or Lycoris rubrum.

[0012] This invention also provides a method for tissue culture of Lycoris radiata explant seeds obtained by the sterilization method described above, comprising the following steps: The seeds of Lycoris radiata tissue culture explants obtained by the sterilization method described above are inoculated into a culture medium for cultivation.

[0013] Preferably, the culture medium uses B5 as the basic medium and is supplemented with GA3 1~2 mg / L, KT 1~2 mg / L, 2,4-D 0~1 mg / L, sucrose 20~30 g / L and agar 6.5~7.5 g / L.

[0014] Preferably, the cultivation includes germination culture and sprouting culture.

[0015] Preferably, the temperature for the germination culture is 4°C and the germination culture time is 3-4 weeks; the temperature for the germination culture is 24-26°C and the germination culture time is 2-4 weeks.

[0016] This invention provides a sterilization method for seeds of tissue-cultured explants of the genus *Lycoris* based on a burning method. This invention employs a burning method, targeting the capsules, to sterilize the seeds of tissue-cultured explants of the genus *Lycoris*. This method is time-efficient, highly effective, and causes minimal damage to the seeds, resulting in a high germination rate and low contamination rate after sterilization. The burning method of this invention allows for intermittent and repeated burning, significantly increasing the proportion of completely sterilized seeds compared to conventional sterilization agents. This method is of great significance for seedling cultivation using sowing methods, especially for the germination of artificially hybridized seeds and the cultivation of hybrid seedlings. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a diagram illustrating the effect of aseptic germination using Lycoris radiata seeds as explants, as provided by the present invention. Figure 2 This is an image illustrating the effect of aseptic germination using immature seeds of Lycoris radiata as explants, as provided by the present invention. Detailed Implementation

[0019] This invention provides a sterilization method for seeds of tissue-cultured explants of the genus Lycoris based on pyrolysis, comprising the following steps: After removing surface soil and dust from the capsules of Lycoris radiata plants, mix them with 70%~75% alcohol and disinfect for 2~3 minutes. The sterilized capsules of Lycoris radiata were ignited for 2-5 seconds; the ignition was repeated 3-4 times to obtain sterilized Lycoris radiata tissue culture explant seeds.

[0020] This invention draws upon the principles of Pasteurization (i.e., short-time high-temperature sterilization) and employs an incineration method to sterilize tissues and organs (capsules) where sterilizing agents have difficulty penetrating quickly. This method is time-efficient, highly effective, and causes minimal damage to the explants (seeds), resulting in a low contamination rate and high germination rate after sterilization. Conventional sterilizing agents are insufficient for sterilizing tissues and organs that are difficult to penetrate. For example, the seeds of Lycoris genus plants are coated with a thick layer of starch; alcohol, hydrogen peroxide, sodium hypochlorite, and mercuric chloride can only achieve surface sterilization. The incineration method allows for intermittent and repeated incineration, significantly increasing the proportion of completely sterilized plants. In this invention, the Lycoris genus plants include Lycoris chinensis, Lycoris radiata, Lycoris dwarf, Lycoris floribunda, Lycoris radiata, Lycoris floribunda, Lycoris floribunda, Lycoris huinanensis, or Lycoris rubrum.

[0021] This invention involves removing surface soil and dust from the capsules of *Lycoris* plants, then mixing them with 70%–75% alcohol for 2–3 minutes for disinfection. In a specific embodiment, the seeds include mature and immature seeds; the immature seeds are those harvested 28–35 days after pollination (i.e., after embryo maturation). In a specific embodiment, the pretreatment method for the capsules includes harvesting the capsules when the *Lycoris* fruit pods turn yellow or 28–35 days after pollination, retaining a 0.5–1 cm long stalk. Yellowing of the *Lycoris* fruit pods and blackening of the seed coat indicate seed maturity; 28–35 days after pollination, the immature seeds in the capsules can be cultured, achieving a low contamination rate and a high germination rate. In a specific embodiment, the volume ratio of the *Lycoris* plant capsules to 70%–75% alcohol is 1:(2–5), achieving surface disinfection through soaking and ensuring alcohol adheres to the explant surface for subsequent flaming.

[0022] This invention involves flaking sterilized Lycoris radiata capsules for 2-5 seconds; the flaking is repeated 2-4 times to obtain sterilized Lycoris radiata tissue culture explant seeds. The flaking can be performed in a sterile environment. In a specific embodiment, the sterile environment can be a laminar flow hood. In a specific embodiment, an alcohol lamp can be used for flaking. In a specific embodiment, a mesh metal filter with a handle can be used for flaking. Specifically, the explant can be placed on the mesh metal filter with a handle, and the filter and explant are placed 1-3 cm above the alcohol lamp to ensure the alcohol on the surface of the explant burns off completely. Lycoris radiata seeds have a smooth surface and can absorb very little alcohol; the 70%-75% alcohol used for flaking has a very short burning time, and this application does not require extinguishing the flame. In a specific embodiment, after flaking, the pericarp of the capsule is removed to obtain the seeds. In a specific embodiment, mature seeds can be flaked 2, 3, or 4 times, while immature seeds can be flaked 2 times.

[0023] This invention also provides a method for tissue culture of Lycoris radiata explant seeds obtained by the sterilization method described above, comprising the following steps: The seeds of Lycoris radiata tissue culture explants obtained by the sterilization method described above are inoculated into a culture medium for cultivation.

[0024] This invention involves inoculating Lycoris radiata explant seeds obtained by the sterilization method described in the above technical solution into a culture medium for cultivation. In a specific embodiment, the culture medium uses B5 as the basic medium, and also adds GA3 1~2 mg / L, KT 1~2 mg / L, 2,4-D 0~1 mg / L, sucrose 20~30 g / L, and agar 6.5~7.5 g / L. In a specific embodiment, the cultivation includes germination culture and germination culture. In a specific embodiment, the temperature for germination culture is 4℃, and the germination culture time is 3~4 weeks; the temperature for germination culture is 24~26℃, specifically 25℃, and the germination culture time is 2~4 weeks, specifically 2 weeks, 3 weeks, or 4 weeks. Lycoris radiata seeds are recalcitrant seeds, difficult to store, and their germination rate drops sharply due to water loss, which limits the time for obtaining them as tissue culture explants (they can only be obtained during the seed maturity stage). However, the sterilization method of this application can achieve efficient sterilization of seeds from different harvesting times. Combined with the tissue culture method, it can achieve efficient germination of seeds from different harvesting times (including mature and immature seeds). Specifically, after culturing seeds using the method of this invention, the seed germination rate can reach 76%~90%. Currently, Lycoris radiata seeds are mostly germinated naturally, and aseptic germination is rarely performed. This invention simulates low temperature in a refrigerator for germination, and also adds gibberellin GA3 to the culture medium to break dormancy and shorten germination time. This is of great significance for seedling cultivation using sowing methods, especially for the germination of artificially hybridized seeds and the cultivation of hybrid seedlings.

[0025] Specifically, the germination cycle of Lycoris genus seeds under natural conditions is long. Naturally, seeds mature from September to November and can be sown immediately after harvesting. Germination typically occurs in March or April of the following year, relying on nutrients stored in the endosperm to form small bulbs. Autumn-leaved varieties emerge leaves (one leaf) in September or October, while spring-leaved varieties don't emerge until the spring of the third year. From sowing to bulb flowering generally takes 5-6 years, making the propagation cycle of Lycoris genus by sowing very long. This further lengthens the cycle for breeding new varieties through hybridization. Mature Lycoris seeds have a high starch content, making them difficult to sterilize with common sterilizing agents. This invention uses a burning method for sterilization of seeds, supplemented by low temperature and plant growth regulators for germination promotion, which can significantly shorten the germination and leaf emergence cycle, thereby shortening the hybridization breeding process. Specifically, this invention employs a sterile germination tissue culture method, specifically using low temperature + GA3 to break dormancy, shortening the germination time of Lycoris seeds to 1-2 months. After being transferred to 24-26℃ for cultivation, leaves will emerge immediately, thus significantly reducing the leaf emergence time. If seeds obtained from hybridization breeding (generally in small quantities) are germinated using this method through tissue culture, the breeding cycle can be effectively shortened, which is of great significance. At the same time, Lycoris seeds have a long development time (generally 60-90 days). This invention enables the sterilization and tissue culture of immature seeds, promoting their germination, which further shortens the germination cycle and the seedling emergence time of Lycoris, also having significant implications for shortening the hybridization breeding cycle.

[0026] Secondly, in the *Lycoris* genus, the endosperm cells are not fully differentiated 28-35 days after pollination, and at this stage, they can still dedifferentiate to form callus or adventitious buds. Since the endosperm cells are triploid, endosperm culture can induce the production of a large number of triploid plants, which can be a new breeding approach. This eliminates the traditional steps of tetraploidization followed by hybridization with diploid plants, shortening the triploid formation time. Furthermore, allopolyploid or homopolyploid plants can be obtained through pollination control. Simultaneously, endosperm development varies among different species, allowing for morphological observation through endosperm culture. In addition, the endosperm is a reservoir of seed nutrients, and endosperm culture can be used to study the biosynthesis and metabolism of stored substances. This application collected *Lycoris* seeds 28-35 days after pollination, when the endosperm is not yet fully differentiated. This invention achieved successful tissue culture of seeds at this stage, laying a technical foundation for future endosperm culture to obtain triploid plants.

[0027] To further illustrate the present invention, the sterilization method for explant seeds of Lycoris radiata based on the burning method and the cultivation method for explant seeds of Lycoris radiata based on the burning method provided by the present invention will be described in detail below with reference to the embodiments. However, these should not be construed as limiting the scope of protection of the present invention.

[0028] Example 1 Sterilization of Lycoris radiata capsules by scorching and aseptic germination of seeds (1) Harvesting of Lycoris dwarfus seeds: In mid-October 2025, when the pericarp of Lycoris dwarfus fruit turns yellow, the capsules are harvested, taking care to retain a 0.5cm long fruit stalk, and brought back to the laboratory for later use.

[0029] (2) Pretreatment of explants: Wash the surface of the capsules with tap water to remove the soil and dust, and be careful to keep the pericarp and pedicel intact.

[0030] (3) Preliminary disinfection of explants: Place the explant capsules into a sterilized empty container, wash away the dirt and dust on the surface of the capsules with sterile deionized water, add 70% alcohol to the container, the volume ratio of explants to alcohol is 1:2, disinfect for 3 minutes, and then transfer to a clean bench.

[0031] (4) Sterilization of explants by burning: After sterilization, remove the capsules from the laminar flow hood and place them on a mesh metal filter with a handle. Then, place the filter and the explants 1 cm above an alcohol lamp to burn off the alcohol on the surface of the explants. The burning time above the alcohol lamp is 5 seconds, and the burning is repeated 3 times to complete the sterilization by burning.

[0032] (5) Aseptic germination of seeds: The sterilized capsules were placed on an inoculation tray, the pericarp was removed, and mature black seeds were obtained. The seeds were inoculated onto a culture medium. The culture medium was B5 as the basic medium, with the addition of GA3 1.5 mg / L, KT 1.5 mg / L, 2,4-D 0.5 mg / L, sucrose 25 g / L and agar 7.0 g / L. After inoculation, the seeds were placed in a refrigerator at 4℃ for 3 weeks to germinate. After that, they were taken out and germinated at 25℃. After 2 weeks, the seeds began to germinate successively, with a germination rate of 76%.

[0033] Seeds were sterilized using sodium hypochlorite, mercuric chloride, and hydrogen peroxide, respectively. The sterilization targets were also the capsules. The sterilization methods, parameters, and results are shown in Table 1. After sterilization, the seeds were rinsed four times with sterile deionized water to remove residual sterilizing agents.

[0034] Table 1. Comparison of sterilization effects of flaming method and other sterilization methods on Lycoris radiata seeds (50 seeds).

[0035] Note: Germination rate (%) = number of germinated seeds 100 / 50.

[0036] When performing aseptic germination of seeds, Lycoris seeds store a large amount of starch, making it difficult for sterilizing agents such as sodium hypochlorite or hydrogen peroxide to penetrate, resulting in a high contamination rate. Lycoris seeds also have a long development period (60-90 days from flowering to seed maturity), a large bacterial load, and are extremely difficult to sterilize. However, results show that compared to other sterilization methods, the scorching method of this invention significantly reduces the contamination rate, achieves a high germination rate, and has a short operation time (2-3 minutes for three sterilization cycles). This method can shorten the germination time, thereby shortening the leaf emergence cycle. Lycoris genus seeds require approximately six months to germinate in the field: seeds mature around October, can be sown immediately after harvesting, and generally germinate in March or April of the following year. Species that leaf in autumn will leaf in the following autumn; species that leaf in spring will leaf in the following spring. Because the propagation cycle is very long, this invention uses tissue culture for aseptic seed germination, which can shorten the seedling cycle. For hybridization breeding, where the number of seeds is limited, the method of this invention can shorten the breeding cycle while aseptically propagating hybrid offspring, which is of great practical significance in terms of technology.

[0037] Example 2 Sterilization of immature capsules of Lycoris radiata by scorching and aseptic germination of seeds (1) Cross-pollination of Lycoris radiata: In late July, during the peak flowering period of Lycoris radiata, select the small flowers that have opened for two days from the inflorescence and collect pollen between 9:00 and 10:00 am on a sunny day (at this time, the pollen viability is the highest, and the success rate of pollination is also the greatest). Repeatedly apply the pollen to the stigma of another small flower bud that has already shown color but has not yet opened, so that as much pollen as possible adheres to the stigma. After pollination, attach a tag to the plant and record the pollination time.

[0038] (2) Harvesting of immature capsules of Lycoris radiata: 35 days after pollination, the capsules were harvested, taking care to retain a 0.8 cm long pedicel, and brought back to the laboratory for later use.

[0039] (3) Pretreatment and initial disinfection of explants: Wash the surface of the capsules with tap water to remove the soil and dust, and be careful to keep the pericarp and pedicel intact. Then put the capsules into a sterilized empty container, add 70% alcohol to the container, the volume ratio of explants to alcohol is 1:3, disinfect for 2.5 min, and then transfer to a clean bench.

[0040] (4) Sterilization of explants by burning: After preliminary sterilization, the capsules are removed from the laminar flow hood and placed on a mesh metal filter with a handle. The filter and the explants are then held 1 cm above an alcohol lamp to burn off the alcohol on the surface of the explants. The burning time above the alcohol lamp is 4 seconds, and the process is repeated twice to complete the sterilization by burning.

[0041] (5) Aseptic germination of seeds: Place the sterilized capsules on an inoculation tray and gently peel off the pericarp (at this time, the capsules are not yet mature, and the pericarp and seed coat are tightly bound), taking care to keep the seed coat intact to obtain immature seeds. At this time, the seed coat is only a light brown color. Inoculate the seeds onto the culture medium. The culture medium is B5 as the basic medium, with the addition of GA3 1.5 mg / L, KT 1.5 mg / L, 2,4-D 0.5 mg / L, sucrose 25 g / L and agar 7.0 g / L. After inoculation, place the seeds in a refrigerator at 4℃ for 3 weeks to germinate. Then take them out and germinate at 25℃. After 2 weeks, the seeds begin to germinate successively, with a germination rate of 90%.

[0042] Sodium hypochlorite and mercuric chloride were used to sterilize the seeds, respectively. The sterilization methods, conditions, and results are shown in Table 2. The seeds were sterilized as capsules. After sterilization, the seeds were rinsed four times with sterile deionized water to remove any residual sterilizing agent.

[0043] Table 2 Comparison of sterilization effects of flaming method and other sterilization methods on immature Lycoris radiata seeds in China (50 seeds)

[0044] Note: Germination rate (%) = number of germinated seeds 100 / 50.

[0045] Table 2 shows that, compared to other sterilization methods, the incineration method of this invention significantly reduces the contamination rate, achieves a high germination rate, and has a short operation time. This invention enables aseptic germination of immature seeds, resulting in shorter development time, lower bacterial load, a higher sterilization rate, and a higher germination rate. Mature Lycoris seeds, after field sowing, rely solely on nutrients provided by the endosperm to form offsets without leaf emergence. This invention not only enables normal germination of immature seeds but also further shortens the germination time compared to the mature seeds used in Example 1, thereby shortening the leaf emergence cycle. For seeds obtained through hybridization breeding, which are often few in number, the method of using immature seeds for germination, as described in this invention, is particularly practical in shortening the aseptic breeding cycle for hybrid offspring.

[0046] Example 3 Sterilization of Lycoris radiata capsules by scorching and aseptic germination of seeds (1) Harvesting of Lycoris radiata seeds: In mid-October 2025, when the pericarp of Lycoris radiata fruit turns yellow, the capsules are harvested, taking care to retain a 1.0cm long fruit stalk, and brought back to the laboratory for later use.

[0047] (2) Pretreatment of explants: Wash the surface of the capsules with tap water to remove the soil and dust, and be careful to keep the pericarp and pedicel intact.

[0048] (3) Preliminary disinfection of explants: Place the explant capsules into a sterilized empty container, wash away the soil and dust on the surface of the capsules with sterile deionized water, add 75% alcohol to the container, the volume ratio of explants to alcohol is 1:5, disinfect for 2.5 minutes, and then transfer to a clean bench.

[0049] (4) Sterilization of explants by burning: After sterilization, remove the capsules from the laminar flow hood and place them on a mesh metal filter with a handle. Then, place the filter and explants 1 cm above an alcohol lamp to burn off the alcohol on the surface of the explants. The burning time above the alcohol lamp is 5 seconds. Then, put the capsules into the alcohol and burn them again for 5 seconds to complete the sterilization by burning.

[0050] (5) Aseptic germination of seeds: The sterilized capsules were placed on an inoculation tray, the pericarp was removed, and mature black seeds were obtained. The seeds were inoculated onto a culture medium. The culture medium was B5 as the basic medium, with the addition of GA3 1.8 mg / L, KT 1.2 mg / L, 2,4-D 0.3 mg / L, sucrose 20 g / L and agar 6.8 g / L. After completing the four treatments, the seeds were placed in a refrigerator at 4°C for 3 weeks to germinate. After that, they were taken out and germinated at 25°C in the dark. After 2 weeks, the seeds began to germinate successively, and after 4 weeks, the germination rate reached 80%.

[0051] Example 4 Sterilization of the capsule by burning and aseptic germination of the seeds of *Lysimachia christinae* (1) Harvesting of seeds of *Houttuynia cordata*: In early November 2025, when the pericarp of *Houttuynia cordata* fruit turns yellow, the capsules are harvested, taking care to retain a 1.0 cm long fruit stalk, and brought back to the laboratory for later use.

[0052] (2) Pretreatment of explants: Wash the surface of the capsules with tap water to remove the soil and dust, and be careful to keep the pericarp and pedicel intact.

[0053] (3) Preliminary disinfection of explants: Place the explant capsules into a sterilized empty container, wash away the soil and dust on the surface of the capsules with sterile deionized water, add 75% alcohol to the container, the volume ratio of explants to alcohol is 1:3, disinfect for 3 minutes, and then transfer to a clean bench.

[0054] (4) Sterilization of explants by burning: After sterilization, remove the capsules in a clean bench and place them on a mesh metal filter with a handle. Then, place the filter and explants 1 cm above an alcohol lamp to burn off the alcohol on the surface of the explants. The burning time above the alcohol lamp is 5 seconds. After that, put the capsules back into the alcohol and then remove them. Repeat the burning process to complete the sterilization by burning.

[0055] (5) Aseptic germination of seeds: The sterilized capsules were placed on an inoculation tray, the pericarp was removed, and mature black seeds were obtained. The seeds were inoculated onto a culture medium. The culture medium was B5 as the basic medium, with the addition of GA3 1.0 mg / L, KT 2.0 mg / L, sucrose 30 g / L and agar 6.5 g / L. After inoculation, the seeds were placed in a refrigerator at 4℃ for 3 weeks to germinate. After that, they were taken out and germinated at 25℃. After 2 weeks, the seeds began to germinate successively, and after 4 weeks, the germination rate was 84%.

[0056] Example 5 Sterilization of the capsule of *Lysimachia christinae* by scorching and aseptic germination of immature seeds (1) Cross-pollination of *Lysimachia christinae*: In early September, during the peak flowering period of *Lysimachia christinae*, select the small flowers that have opened for two days from the inflorescence and collect pollen between 9:00 and 10:00 AM on a sunny day (at this time, the pollen viability is the highest, and the success rate of pollination is also the greatest). Repeatedly apply the pollen to the stigma of another small flower whose bud has already shown color but has not yet opened, so that as much pollen as possible adheres to the stigma for pollination. After pollination, hang a tag on the plant to record the time of pollination.

[0057] (2) Harvesting of immature capsules of *Lysimachia christinae*: 30 days after pollination, the capsules are harvested, taking care to retain a fruit stalk of about 0.9 cm in length, and brought back to the laboratory for later use.

[0058] (3) Pretreatment and initial disinfection of explants: Wash the surface of the capsules with tap water to remove the soil and dust, and be careful to keep the pericarp and pedicel intact. Then put the capsules into a sterilized empty container, add 72% alcohol to the container, and the volume ratio of explants to alcohol is 1:2.5. After disinfection for 2.5 min, transfer them to a clean bench.

[0059] (4) Sterilization of explants by burning: After preliminary sterilization, the capsules are removed from the laminar flow hood and placed on a mesh metal filter with a handle. The filter and explants are then held 1 cm above an alcohol lamp to burn off the alcohol on the surface of the explants. The burning time above the alcohol lamp is 4 seconds. After that, the capsules are placed back into the alcohol and then removed. The same method is used to burn them again to complete the sterilization by burning.

[0060] (5) Aseptic germination of immature seeds: Place the sterilized capsules on an inoculation tray and gently peel off the pericarp (at this time, the capsules are not yet mature, and the pericarp and seed coat are tightly bound), taking care to keep the seed coat intact to obtain immature seeds. At this time, the seed coat is only a light brown color. Inoculate the seeds onto the culture medium. The culture medium is B5 as the basic medium, with the addition of GA3 1.0 mg / L, KT 2.0 mg / L, 2,4-D 0.25 mg / L, sucrose 30 g / L and agar 7.0 g / L. After inoculation, place the seeds in a refrigerator at 4℃ for 3 weeks to germinate. Then take them out and germinate at 25℃. After 2 weeks, the seeds begin to germinate successively, with a germination rate of 88%.

[0061] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A sterilization method for seeds of tissue-cultured explants of the genus *Lycoris* based on pyrolysis, characterized in that, Includes the following steps: After removing surface soil and dust from the capsules of Lycoris radiata plants, mix them with 70%~75% alcohol and disinfect for 2~3 minutes. The sterilized capsules of Lycoris radiata were ignited for 2-5 seconds; the ignition was repeated 2-4 times to obtain sterilized Lycoris radiata tissue culture explant seeds.

2. The sterilization method according to claim 1, characterized in that, The seeds include mature seeds and immature seeds; the immature seeds are those 28-35 days after pollination.

3. The sterilization method according to claim 1, characterized in that, The volume ratio of the capsule of the Lycoris genus plant to 70%~75% alcohol is 1:(2~5).

4. The sterilization method according to claim 1, characterized in that, The pretreatment method for the capsules includes: harvesting the capsules when the fruit pods of the Lycoris genus turn yellow or 28-35 days after pollination, retaining a 0.5-1 cm long pedicel.

5. The sterilization method according to claim 1, characterized in that, After scorching, the pericarp of the capsule is removed to obtain the seeds.

6. The sterilization method according to claim 1, characterized in that, The Lycoris species include Lycoris chinensis, Lycoris radiata, Lycoris dwarfusa, Lycoris floribunda, Lycoris radiata, Lycoris floribunda ...huinanensis, or Lycoris rubrum.

7. A method for tissue culture of seeds from Lycoris radiata explants obtained by the sterilization method according to any one of claims 1 to 6, comprising the following steps: Seeds of Lycoris radiata tissue culture explants obtained by the sterilization method according to any one of claims 1 to 6 are inoculated into a culture medium for cultivation.

8. The method according to claim 7, characterized in that, The culture medium uses B5 as the basic medium and is supplemented with GA3 1~2 mg / L, KT 1~2 mg / L, 2,4-D 0~1 mg / L, sucrose 20~30 g / L and agar 6.5~7.5 g / L.

9. The method according to claim 7, characterized in that, The cultivation includes germination culture and sprouting culture.

10. The method according to claim 9, characterized in that, The temperature for the germination culture is 4℃, and the germination culture time is 3-4 weeks; the temperature for the germination culture is 24-26℃, and the germination culture time is 2-4 weeks.

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