A sterilization method for stamens of tissue-cultured explants of Lycoris radiata based on flaming and a tissue culture method for stamens of tissue-cultured explants of Lycoris radiata.

Abstract

This invention provides a sterilization method for stamens of Lycoris radiata explants based on flaming, and a tissue culture method for stamens of Lycoris radiata explants, specifically belonging to the field of plant tissue culture technology. The sterilization method of this invention involves removing surface soil and dust from unopened inflorescences or florets, mixing them with 70%–75% alcohol, and sterilizing for 2–3 minutes. The sterilized Lycoris radiata tissue or organ is then flaming to obtain sterilized Lycoris radiata explant stamens. This sterilization method is time-efficient, highly effective, and causes minimal damage to the explant stamens, resulting in a high induction rate of callus or hairy roots after sterilization, which is beneficial for achieving haploid induction of anthers. This invention is of great significance for establishing a stamen regeneration system for Lycoris radiata, inducing the development of different plant organs, and haploid breeding.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture technology, specifically relating to a sterilization method for stamens of tissue-cultured explants of Lycoris genus based on flaming method and a tissue culture method for stamens of tissue-cultured explants of Lycoris genus. 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] The establishment of rapid tissue culture propagation systems for Lycoris genus plants typically uses bulb discs as explants. The advantage is the ease of inducing adventitious buds. However, because bulbs are underground organs, they have a high microbial load, making sterilization difficult and resulting in a high contamination rate in the initial culture. Stamens (including anthers and filaments) are part of the flower structure, have a short lifespan, low bacterial load, and are very delicate. They are one of the explants for tissue culture propagation projects, possessing advantages such as easy dedifferentiation and the potential for multi-directional differentiation, and can lay the foundation for inducing haploid plants. However, currently, for relatively delicate stamens, alcohol, HClO (produced by NaClO), and reactive oxygen species (produced by H2O2) can penetrate young cells and rapidly spread, leading to their death. Therefore, there is an urgent need to develop an efficient sterilization method suitable for young tissue organs such as stamens. Summary of the Invention

[0005] The purpose of this invention is to provide a sterilization method for stamens of tissue-cultured explants of the genus *Lycoris* based on cauterization, and a tissue culture method for stamens of tissue-cultured explants of the genus *Lycoris*. The sterilization method described in this invention achieves highly efficient sterilization of stamens of tissue-cultured explants of the genus *Lycoris*, with short sterilization time, high sterilization efficiency, minimal damage to the stamens, and a high induction rate of callus tissue or plant organs (hairy roots) after sterilization.

[0006] This invention provides a sterilization method for stamens of tissue-cultured explants of the genus *Lycoris* based on flaming, comprising the following steps: After removing surface soil and dust from Lycoris radiata plant tissues or organs, mix them with 70%~75% alcohol and disinfect for 2~3 minutes; the Lycoris radiata plant tissues or organs are the inflorescences whose involucres have not yet opened or the florets on the inflorescences. Sterilized stamens of Lycoris radiata tissues or organs were obtained by burning sterilized Lycoris radiata tissue culture explants.

[0007] Preferably, the volume ratio of the Lycoris plant tissue or organ to 70%~75% alcohol is 1:(2~3).

[0008] Preferably, the burning is performed 1 to 2 times, and each burning session lasts 2 to 5 seconds.

[0009] Preferably, the pretreatment method for inflorescences with unopened involucres includes: when the height of the flower stalk is 9-11 cm, the height of the florets is 2-3 cm, and the involucre is unopened, the entire inflorescence is cut off 1-1.5 cm below the inflorescence.

[0010] Preferably, when the Lycoris plant tissue or organ is an inflorescence with an unopened involucre, the burning process further includes cutting off the inflorescence axis and involucral bracts to obtain a complete floret, removing the perianth segments from the base along the ovary of the floret, and removing the stamens; when the Lycoris plant tissue or organ is a floret on an inflorescence, the burning process further includes removing the perianth segments from the base along the ovary of the floret and removing the stamens.

[0011] This invention also provides a method for tissue culture of stamens from explants of the genus *Lycoris* based on the sterilization method described above, comprising the following steps: The stamens of the Lycoris genus tissue culture explants prepared by the sterilization method described above were inoculated into a culture medium for cultivation.

[0012] Preferably, the culture medium is a callus culture medium or a hairy root induction culture medium.

[0013] Preferably, the callus culture medium uses B5 as the basic culture medium and further adds nicotinic acid 3.0~5.0 mg / L, 6-BA 5.0~6.0 mg / L, NAA 0.5~1.0 mg / L, 2,4-D 0.2~0.8 mg / L, activated charcoal 0.5~1.0 g / L, acid-hydrolyzed casein 0.5~1.0 mg / L, sucrose 20~30 g / L and agar 6.5~7.5 g / L.

[0014] Preferably, the following are also added: nicotinic acid 3.0~5.0 mg / L, 6-BA 10.0~15.0 mg / L, NAA 5.0~10.0 mg / L, activated charcoal 0.5~1.0 g / L, acid-hydrolyzed casein 0.5~1.0 mg / L, sucrose 20~30 g / L and agar 6.5~7.5 g / L.

[0015] Preferably, the culture time is 6 to 8 weeks; the culture temperature is 24 to 26°C; the culture light intensity is 800 to 1200 Lx; and the culture light duration is 12 to 16 h / d.

[0016] This invention provides a sterilization method for stamens of tissue-cultured explants of the genus *Lycoris* based on cauterization. This invention uses cauterization to sterilize the stamens of tissue-cultured explants of *Lycoris*, which is time-efficient, highly effective, and causes minimal damage to the stamens, resulting in a high induction rate of callus or plant organs (hairy roots) after sterilization. The cauterization method of this invention allows for intermittent and repeated cauterization, significantly increasing the proportion of complete sterilization compared to conventional sterilization agents. This method is of great significance for establishing a stamen regeneration system for *Lycoris*, inducing the development of different plant organs, and haploid breeding. 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 showing the callus induction results using the stamens of Lycoris radiata as explants, as provided by this invention. Figure 2 This is a diagram showing the induction results of hairy roots using the stamens of *Echeveria elegans* as explants, as provided by this invention. Detailed Implementation

[0019] This invention provides a sterilization method for stamens of tissue-cultured explants of the genus *Lycoris* based on flaming, comprising the following steps: After removing surface soil and dust from Lycoris radiata plant tissues or organs, mix them with 70%~75% alcohol and disinfect for 2~3 minutes; the Lycoris radiata plant tissues or organs are the inflorescences whose involucres have not yet opened or the florets on the inflorescences. Sterilized stamens of Lycoris radiata tissues or organs were obtained by burning sterilized Lycoris radiata tissue culture explants.

[0020] This invention draws upon the principles of Pasteurization (i.e., short-time high-temperature sterilization), employing a burning method to sterilize young, unopened inflorescences or florets on inflorescences. This method is time-efficient, highly effective, and causes minimal damage to the stamens of the explants, resulting in a high induction rate of callus and hairy roots after sterilization. Conventional sterilizing agents are prone to killing young Lycoris genus tissues and organs, while the burning method allows for intermittent and repeated burning, significantly increasing the proportion of complete sterilization while minimizing damage to the explants. In this invention, inflorescences or florets on inflorescences of all species in this genus can be sterilized using this method. The Lycoris genus species include Lycoris chinensis, Lycoris radiata, Lycoris dwarf, Lycoris floribunda, Lycoris radiata, Lycoris floribunda, Lycoris floribunda, Lycoris floribunda, Lycoris huinanensis, or Lycoris rubrum.

[0021] This invention involves removing surface soil and dust from Lycoris radiata plant tissues or organs, then mixing them with 70%–75% alcohol for 2–3 minutes for disinfection. The Lycoris radiata plant tissues or organs refer to inflorescences with unopened bracts or florets on the inflorescence. In a specific embodiment, the volume ratio of the Lycoris radiata plant tissues or organs to 70%–75% alcohol is 1:(2–3), achieving surface disinfection through immersion and ensuring alcohol adherence to the explant surface for subsequent flaming. In a specific embodiment, the pretreatment method for inflorescences with unopened bracts includes: cutting the entire inflorescence 1–1.5 cm below the inflorescence when the flower stalk height is 9–11 cm, the floret height is 2–3 cm, and the bract is unopened. Since the stamens are very delicate inside the inflorescence, sodium hypochlorite, mercuric chloride, etc., can easily kill them. Therefore, this invention uses flaming of the inflorescence to achieve indirect sterilization of the stamens. This invention utilizes the penetrating power of a brief period of high temperature during burning to sterilize the florets. The involucre and perianth segments protect the stamens from being burned, thus achieving a sterile effect. The burning process described in this invention can be performed in a sterile environment. In a specific embodiment, the sterile environment can be a laminar flow hood. In a specific embodiment, the burning can be performed using an alcohol lamp. In a specific embodiment, the burning can be performed using a mesh metal filter with a handle. Specifically, the unopened inflorescence or florets can be placed on the mesh metal filter with a handle, and the filter, along with the unopened inflorescence or florets, can be placed 1-3 cm above the alcohol lamp to ensure that the alcohol on the surface of the unopened inflorescence or florets burns off completely. The 70%-75% alcohol used in this invention has a very short burning time, and no extinguishing operation is required.

[0022] This invention involves scorching sterilized Lycoris radiata plant tissues or organs to obtain sterilized Lycoris radiata plant tissue culture explant stamens. In a specific embodiment, the scorching is performed 1-2 times, with each scorching lasting 2-5 seconds. In a specific embodiment, when the Lycoris radiata plant tissue or organ is an inflorescence with an unopened involucre, the scorching process further includes cutting off the inflorescence axis and involucral bracts to obtain complete florets, removing the perianth segments from the base along the ovary of the floret, and harvesting the stamens; when the Lycoris radiata plant tissue or organ is a floret on an inflorescence, the scorching process further includes removing the perianth segments from the base along the ovary of the floret and harvesting the stamens.

[0023] This invention also provides a method for tissue culture of stamens from explants of the genus *Lycoris* based on the sterilization method described above, comprising the following steps: The stamens of the Lycoris genus tissue culture explants prepared by the sterilization method described above were inoculated into a culture medium for cultivation.

[0024] This invention involves inoculating the stamens of Lycoris radiata explants prepared by the sterilization method described in the above technical solution into a culture medium for cultivation. In a specific embodiment, the culture medium is a callus culture medium or a hairy root induction medium. In a specific embodiment, the callus culture medium uses B5 as the basic medium and also adds nicotinic acid 3.0~5.0 mg / L, 6-BA 5.0~6.0 mg / L, NAA 0.5~1.0 mg / L, 2,4-D 0.2~0.8 mg / L, activated charcoal 0.5~1.0 g / L, acid-hydrolyzed casein 0.5~1.0 mg / L, sucrose 20~30 g / L, and agar 6.5~7.5 g / L. In a specific embodiment, the hairy root induction medium uses B5 as the basic medium, and further adds nicotinic acid 3.0~5.0 mg / L, 6-BA 10.0~15.0 mg / L, NAA 5.0~10.0 mg / L, activated carbon 0.5~1.0 g / L, acid-hydrolyzed casein 0.5~1.0 mg / L, sucrose 20~30 g / L, and agar 6.5~7.5 g / L. In a specific embodiment, the culture time is 6~8 weeks, specifically 8 weeks; the culture temperature is 24~26℃, specifically 25℃; the light intensity is 800~1200 Lx, specifically 1000 Lx; and the photoperiod is 12~16 h / d, specifically 14 h. The method of this invention induces frost-like, granular callus tissue by culturing stamens for 6~8 weeks, specifically 8 weeks. The cauterization method causes relatively little damage to explants, and the method of this invention can induce the development of multiple organs based on stamens. Hairy roots have advantages such as hormone autonomy, genetic stability, and high yield, and their growth rate is usually much higher than that of callus tissue and cells in cell suspension culture. Moreover, most of them can be detected with secondary metabolites at levels comparable to or even higher than those of the original plant. The method of this invention can successfully induce white hairy roots by culturing stamens for 6-8 weeks, specifically 8 weeks.

[0025] Literature reports (Chen Jingjue. Optimization of In Vitro Regeneration System of Lycoris and Preliminary Study on Callus Induction of Floral Organs. Master's Thesis, Zhejiang University. 2012) indicate that sodium hypochlorite was used to sterilize the anthers and filaments of stamens separately, resulting in a 100% anther mortality rate. This demonstrates that sodium hypochlorite is not suitable for separately sterilizing the anthers and filaments of young stamens. This invention uses an incineration method to sterilize the inflorescence or florets, then separates the stamens from the florets, ensuring the stamens (including anthers and filaments) are sterile with minimal damage, ultimately yielding a frosty, granular callus. Lycoris plants have a slow growth rate and a long seedling cycle. This invention sterilizes young stamens with minimal damage and a high sterility rate. Later, the anthers from the stamens are used as explants to obtain callus or clustered shoots, providing a rapid, convenient, and highly operable method for cultivating haploid Lycoris plants. This method has great application potential in the breeding of new Lycoris varieties.

[0026] The method of this invention is of great significance for the establishment of callus-based regeneration systems for Lycoris radiata, as well as for haploid breeding and hybridization breeding.

[0027] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, provides a method for sterilizing stamens of tissue-cultured explants of the genus Lycoris based on the burning method and a method for tissue culture of stamens of tissue-cultured explants of the genus Lycoris, but these should not be construed as limiting the scope of protection of the present invention.

[0028] Example 1 Callus induction using Lycoris radiata stamens as explants (1) Obtaining explants: During the flowering period of Lycoris radiata in mid-July 2025, when young inflorescences are used as explants, the entire inflorescence is cut off 1.0-1.5cm below the inflorescence when the height of the flower stalk is about 10.0cm, the height of the florets is 2-3cm, and the involucre has not opened. The involucre is then brought back to the laboratory for later use.

[0029] (2) Pretreatment of explants: Gently rinse away surface dust with tap water. Be careful not to open the involucre and keep the involucre intact.

[0030] (3) Preliminary disinfection of explants: Place the explant inflorescences into a sterilized empty container, wash away the soil and dust on the surface of the inflorescences with sterile deionized water, add 75% alcohol to the container, the volume ratio of explant inflorescences to alcohol is 1:2.5, disinfect for 3 minutes, and then transfer to a clean bench.

[0031] (4) Sterilization of explants by burning: After sterilization with 75% alcohol, remove the inflorescence and place it on a mesh metal filter with a handle. Then, place the filter and the explant inflorescence 1 cm above the alcohol lamp to burn off the alcohol on the surface of the explant. The burning time above the alcohol lamp is 3 seconds to complete the sterilization by burning.

[0032] (5) Inoculation: Place the sterilized inflorescences by flaming on an inoculation tray, remove the inflorescence axis and involucre to obtain complete florets; then remove the perianth segments from the base along the top of the ovary to obtain complete stamens and pistils. Inoculate the stamens onto callus induction medium. The callus induction medium is B5 as the basic medium, supplemented with nicotinic acid 5.0 mg / L, 6-BA 6.0 mg / L, NAA 0.8 mg / L, 2,4-D 0.5 mg / L, activated carbon 0.5 g / L, acid-hydrolyzed casein 0.5 mg / L, and sucrose 20 g / L and agar 7.0 g / L. After 8 weeks of culture, frosty, granular callus tissue was induced at the base of the stamens, such as... Figure 1 As shown.

[0033] Sterilization by flaming does not require removing the involucre. It utilizes short-duration high-temperature sterilization, leveraging the penetrating power of the heat to kill exogenous bacteria, thereby increasing the sterility rate and reducing contamination. This invention uses flaming of unopened inflorescences with involucres for sterilization, requiring only one flaming session (2-3 seconds). For larger inflorescences such as *Lycoris radiata* and *Lycoris radiata*, two flaming sessions can be performed. Because of the protection provided by the involucre and perianth segments, damage to the explant stamens is minimal, resulting in a high callus induction rate.

[0034] Example 2 Induction of hairy roots using stamens of *Euphorbia milii* as explants (1) Obtaining explants: During the peak flowering period of *Echeveria elegans*, in mid-July 2025, when young inflorescences are used as explants, the entire inflorescence is cut off 1.0-1.5 cm below the inflorescence when the flower stalk is about 10.0 cm high, the florets are 2-3 cm high, and the involucre is not open (note that the involucre should be kept intact). The inflorescence is then brought back to the laboratory for later use.

[0035] (2) Pretreatment of explants: Gently rinse away surface dust with tap water. Be careful not to open the involucre and keep the involucre intact.

[0036] (3) Preliminary disinfection of explants: Place the explant inflorescences into a sterilized empty container, wash away the soil and dust on the surface of the inflorescences with sterile deionized water, add 75% alcohol to the container, the volume ratio of explant inflorescences to alcohol is 1:2.5, disinfect for 3 minutes, and then transfer to a clean bench.

[0037] (4) Sterilization of explants by burning: After preliminary sterilization, remove the inflorescence and place it on a mesh metal filter with a handle. Place the filter and inflorescence 1 cm above an alcohol lamp to burn off the alcohol on the surface of the explant. The burning time above the alcohol lamp is 3 seconds to complete the sterilization by burning.

[0038] (5) Inoculation: Place the sterilized inflorescences by flaming on an inoculation tray, remove the inflorescence axis and involucre to obtain complete florets; then remove the perianth segments from the base along the top of the ovary to obtain complete stamens and pistils. Inoculate the stamens onto hairy root induction medium. The hairy root induction medium is B5 as the basic medium, supplemented with nicotinic acid 5.0 mg / L, 6-BA 12.0 mg / L, NAA 10.0 mg / L, activated carbon 0.5 g / L, acid-hydrolyzed casein 0.5 mg / L, and sucrose 20 g / L and agar 7.0 g / L. After 8 weeks of culture, white hairy roots are induced from the filaments at the base of the stamens, such as... Figure 2 As shown.

[0039] Sterilization by flaming does not require removing the involucre. It utilizes short-duration high-temperature sterilization, leveraging the penetrating power of the heat to kill exogenous bacteria, thereby increasing the sterility rate and reducing contamination. This invention uses flaming of unopened inflorescences with involucres for sterilization, requiring only one flaming session (2-3 seconds). For larger inflorescences such as *Lycoris radiata* and *Lycoris radiata*, two flaming sessions may be necessary. Because of the protection provided by the involucre and perianth segments, damage to the stamens is minimal, and the formation of hairy roots can be induced.

[0040] Comparative Example 1 Using unopened inflorescences as material, the sterilization method was changed. First, the inflorescences were disinfected with 75% alcohol for 45 seconds, washed twice with sterile deionized water, and then disinfected with sodium hypochlorite aqueous solution with an effective chlorine content of 2.5% for 15 minutes, during which they were constantly shaken and agitated. The remaining steps were the same as in Example 1.

[0041] Comparative Example 2 Using unopened inflorescences as material, the sterilization method was changed. First, the inflorescences were disinfected with 75% alcohol for 45 seconds, washed twice with sterile deionized water, and then disinfected with a sodium hypochlorite aqueous solution with an effective chlorine content of 2.5% for 15 minutes, during which the inflorescences were shaken and agitated continuously. The remaining steps were the same as in Example 2.

[0042] The comparison results of the effects of incineration sterilization and sodium hypochlorite sterilization on inflorescence sterilization are shown in Table 1: Table 1 Comparison of the effects of inflorescence sterilization by flaming and sodium hypochlorite sterilization

[0043] Note: Flower integrity rate refers to the absence of broken perianth segments (i.e., petals) or broken stamens after sterilization. Induction rate = number of stamens inducing callus or hairy roots × 100 / number of inoculated stamens. Contamination rate = number of contaminated stamens × 100 / number of inoculated stamens. The number of stamens inoculated after sterilization using both methods was 50.

[0044] 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 stamens of tissue-cultured explants of the genus *Lycoris* based on flaming, characterized in that, Includes the following steps: After removing surface soil and dust from Lycoris radiata plant tissues or organs, mix them with 70%~75% alcohol and disinfect for 2~3 minutes; the Lycoris radiata plant tissues or organs are the inflorescences whose involucres have not yet opened or the florets on the inflorescences. Sterilized stamens of Lycoris radiata tissues or organs were obtained by burning sterilized Lycoris radiata tissue culture explants.

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

3. The sterilization method according to claim 1, characterized in that, The burning is performed 1 to 2 times, with each burning session lasting 2 to 5 seconds.

4. The sterilization method according to claim 1, characterized in that, The pretreatment method for inflorescences with unopened involucres includes: when the height of the flower stalk is 9-11cm, the height of the florets is 2-3cm, and the involucre is unopened, cut the entire inflorescence 1-1.5cm below the inflorescence.

5. The sterilization method according to claim 1, characterized in that, When the tissue or organ of the Lycoris genus is an inflorescence with an unopened involucre, the burning process further includes cutting off the inflorescence axis and involucral bracts to obtain a complete floret, removing the perianth segments from the base along the ovary of the floret, and removing the stamens; when the tissue or organ of the Lycoris genus is a floret on an inflorescence, the burning process further includes removing the perianth segments from the base along the ovary of the floret and removing the stamens.

6. A method for tissue culture of stamens from explants of the genus *Lycoris* based on the sterilization method described in any one of claims 1 to 5, characterized in that, Includes the following steps: The stamens of the Lycoris genus tissue culture explants prepared by the sterilization method according to any one of claims 1 to 5 are inoculated into a culture medium for culture.

7. The tissue culture method according to claim 6, characterized in that, The culture medium is a callus culture medium or a hairy root induction culture medium.

8. The tissue culture method according to claim 7, characterized in that, The callus culture medium uses B5 as the basic medium and is supplemented with nicotinic acid 3.0~5.0 mg / L, 6-BA 5.0~6.0 mg / L, NAA 0.5~1.0 mg / L, 2,4-D 0.2~0.8 mg / L, activated charcoal 0.5~1.0 g / L, acid-hydrolyzed casein 0.5~1.0 mg / L, sucrose 20~30 g / L and agar 6.5~7.5 g / L.

9. The tissue culture method according to claim 7, characterized in that, The hairy root induction medium uses B5 as the basic medium and is supplemented with nicotinic acid 3.0~5.0 mg / L, 6-BA 10.0~15.0 mg / L, NAA 5.0~10.0 mg / L, activated carbon 0.5~1.0 g / L, acid-hydrolyzed casein 0.5~1.0 mg / L, sucrose 20~30 g / L and agar 6.5~7.5 g / L.

10. The tissue culture method according to claim 6, characterized in that, The culture time is 6 to 8 weeks; the culture temperature is 24 to 26°C; the light intensity is 800 to 1200 Lx; and the light duration is 12 to 16 h / d.

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