Tissue culture method of Guizhou golden flower tea

Abstract

The application discloses a Guizhou golden flower tea tissue culture method and belongs to the Guizhou golden flower tea tissue culture technical field.The method comprises the following steps: (1) collecting fully mature Guizhou golden flower tea fruits, taking out seeds, sand storing, removing seed surface sand and seed coat after the seeds grow 1-2 cm radicles, cleaning with clean water, then sequentially immersing in 75% alcohol and 0.1% mercury solution, cleaning with clean water, and obtaining external sterilization explants; (2) cutting off the excess endosperm of the explants, inoculating the remaining part into a primary culture medium for culture, obtaining Guizhou golden flower tea aseptic seedlings after the seedlings grow; and (3) cutting the obtained aseptic seedlings into stem segments with 1-2 leaf buds, inoculating the stem segments into a subculture medium for culture, then transferring the stem segments into a rooting culture medium for culture, and obtaining Guizhou golden flower tea seedlings, so that the propagation efficiency can be improved, and the problems of high Guizhou golden flower tea tissue culture pollution rate and low survival rate can be reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of Guizhou Golden Camellia tissue culture, in particular to a Guizhou Golden Camellia tissue culture method. BACKGROUND

[0002] The natural distribution range of Guizhou Golden Camellia is narrow, the number is limited, the seed yield is small, the abortion rate is high, the setting rate is low, it is mainly distributed in the north of the tropic, belongs to the tropical monsoon climate zone, the abortion rate of flower bud period is 16.33%, the abortion rate of flowering period is 63.05%, the abortion rate of fruit period is 44.09%, and it is listed as a second-class protected plant in China. There are two kinds of Guizhou Golden Camellia in Guizhou, one is Tian'e Guizhou Golden Camellia (formerly named: Guizhou Golden Camellia (Camellia huana T.L.Ming et W.J.Zhang)) distributed in Luodian County of Guizhou Province and partial areas in adjacent Guangxi, and the other is Guizhou Golden Camellia (formerly named: Camellia liberofilamenta Chang et C.H.Yang) distributed in Cheheng County of Guizhou Province. In 2024, the article of 'Classification Revision of Guizhou Golden Camellia (Theaceae)' in Plant Research Journal revised the Chinese name and Latin name of the two kinds of Guizhou Golden Camellia. The number of Guizhou Golden Camellia in the wild is only more than 20, and it has great significance in germplasm resource preservation and genetic breeding.

[0003] At present, the main propagation methods of Guizhou Golden Camellia are sowing, cutting and grafting seedling. The propagation speed of cutting and grafting is slow, the propagation coefficient is low, and the mother plant is damaged, which is not conducive to the large-scale production of Guizhou Golden Camellia propagation. Guizhou Golden Camellia is a perennial woody plant, and the conventional breeding and seedling period is long, the propagation coefficient is small, and the germplasm advantage is easy to degenerate. Therefore, it is urgent to develop tissue culture technology of Guizhou Golden Camellia. According to the query of relevant literature, there is no report on the tissue culture method of Guizhou Golden Camellia, and it is found that there is still a high infection rate after the traditional sterilization method is used to treat the explant. This may be related to the endophytic contamination of Guizhou Golden Camellia. Therefore, a new Guizhou Golden Camellia tissue culture method is sought to solve the problems of high contamination rate and low survival rate of tissue culture. SUMMARY

[0004] In view of this, the purpose of the present application is not only to provide a new Guizhou Golden Camellia tissue culture method to improve the propagation efficiency, but also to solve the problems of high contamination rate and low survival rate of Guizhou Golden Camellia tissue culture.

[0005] The present application solves the above technical problems through the following technical solutions:

[0006] A Guizhou Golden Camellia tissue culture method, the tissue culture method is as follows:

[0007] (1) After collecting fully mature Guizhou golden camellia fruits, the seeds are taken out and stored in sand. After the seeds grow 1-2cm radicles, the mud and seed coat on the seed surface are removed, and the seeds are washed with clean water. Then, they are soaked in 75% alcohol and 0.1% mercuric chloride solution in turn, and then washed with clean water to obtain externally disinfected explants.

[0008] (2) The excess endosperm of the explant was cut off, and the remaining part was inoculated into the primary culture medium and cultured to grow seedlings to obtain sterile seedlings of Camellia chrysantha var. guili.

[0009] (3) Cut the obtained sterile seedlings into stem segments with 1-2 leaf buds, inoculate them into subculture medium for subculture, transfer the subcultured seedlings to rooting medium for rooting culture, and obtain Guizhou golden camellia seedlings after the rooting culture is completed.

[0010] Because *Camellia laurentii* is rare, this invention uses its seeds for tissue culture to avoid the problem of genotypic homogeneity caused by asexual reproduction. Furthermore, seed propagation can expand the *Camellia laurentii* population. However, due to the numerous procedures involved in seed tissue culture and the abundance of endophytic bacteria in the seeds, the contamination rate is high, leading to a low survival rate of seedlings. Therefore, strict disinfection and maintaining a sterile environment are crucial during the tissue culture process.

[0011] Furthermore, the culture time for the primary culture is 25-30 days; the culture time for the secondary culture is 50-60 days; and the culture time for the rooting culture is 35-40 days.

[0012] Furthermore, the primary and secondary culture media are composed of: MS medium + 2-8 mg / L 6-BA + 2-8 mg / L brassinolide + 0.1-0.5 mg / L NAA.

[0013] Furthermore, the rooting medium consists of 1 / 2 MS medium + 4-6 mg / L NAA.

[0014] Besides the contamination rate, the rooting rate also determines the success of tissue culture seedlings, and a reasonable hormone ratio is key to rooting. Therefore, optimizing the hormone ratio during primary culture, subculture, and rooting culture is also very important. We found that using the same formula for both primary and subculture media resulted in better embryo growth and subculture growth.

[0015] Furthermore, the specific procedures for external disinfection of explants are as follows: On a clean bench, soak the seeds, which have been cleaned with water, in 75% alcohol for 30 seconds. After removing them, rinse them 3-4 times with sterile water, then soak them in 0.1% mercuric chloride solution for 8-10 minutes, shaking them once every 10-30 seconds during this period. Rinse them 3-4 times with sterile water, and then blot dry with filter paper to obtain externally disinfected explants.

[0016] Because the experiment revealed severe endophytic bacteria in *Camellia laurentii*, this invention employs a combined internal and external sterilization approach. Externally, the explants are disinfected with substances such as ethanol and mercuric chloride solution to reduce the impact of external bacteria. Simultaneously, excess endosperm is removed to minimize the influence of endophytic bacteria within the endosperm. However, even after endosperm removal, endophytic bacteria can still diffuse into the plumule through the vascular tissue or intercellular spaces within the seed during germination. Therefore, in addition to removing the endosperm, endophytic bacteria disinfectant is used simultaneously to further reduce their impact.

[0017] Specifically, the explant also undergoes internal disinfection, and the specific internal disinfection process is as follows:

[0018] Grind the surface of the Guizhou golden camellia seeds with sterilized 0.3-0.5mm sand for 5-6 minutes, then soak them in an endophytic fungicide for 10-20 minutes. After soaking, let them stand for 10-20 minutes, then soak them in 1-2wt% calcium chloride solution for 5-10 minutes. After soaking, let them stand at room temperature for 1-2 hours, then bury them in sand for sand stratification. Starting from the 20th day of sand stratification, spray with a citric acid solution with a pH of 5.5-6.5, spraying once every 5-7 days.

[0019] Furthermore, the temperature during the sand storage process is 10-15℃, the humidity is maintained at 60-70% for the first 20 days of sand storage, and the humidity is maintained at 50-60% after the 20th day of sand storage.

[0020] Furthermore, the preparation method of the endophytic bactericide is as follows:

[0021] (1) Mix 2 wt% chitosan solution and 1 wt% sodium alginate solution at a volume ratio of 2:1 to obtain a viscous liquid. Then add a mixture of streptomycin, ε-polylysine and 3 wt% enzymatic hydrolysis lignin solution. After dispersing evenly, slowly add 3-5 wt% calcium chloride solution to obtain a gel block. Then freeze at -20℃ and crush to obtain a gel powder with a particle size of 0.2-0.5 mm. Then add water to swell for 30 min and set aside for use.

[0022] (2) After dissolving shellac in 70wt% ethanol solution, centrifuge to remove the precipitate, then add jojoba oil and glycerin and stir at 800-1000 rpm for 5 min, then add 5wt% Span solution, swollen gel powder and water and stir at 800-1000 rpm for 1-2 min, then shear at 20000-24000 rpm for 1-2 min to obtain endophytic bactericide.

[0023] This invention uses ε-polylysine and streptomycin as bactericides to disinfect the seeds of *Camellia chrysantha* during sand aging. ε-polylysine is a natural antibacterial substance that disrupts bacterial cell membranes and has a broad-spectrum antibacterial effect. Streptomycin is also commonly used for sterilization in tissue culture. Therefore, the two are chosen to work synergistically to kill endophytic bacteria in the seeds. Furthermore, *Camellia chrysantha* seeds have hard shells and a dormancy period, requiring sand aging for germination. This invention uses a mechanical micro-damage method to accelerate the germination process, but damaging the seeds makes them susceptible to bacterial infection. Therefore, this invention utilizes an endometrium-like bactericide to uniformly coat the seeds, allowing the bactericidal components to be released into the seed interior through the micro-damage wounds, directly acting on the bacteria inside the seed. The bactericidal components in the endophytic bactericide are encapsulated with chitosan and sodium alginate for slow release. Chitosan itself also has broad-spectrum antibacterial properties, preventing external bacteria from invading the seed interior and causing infection during sand aging.

[0024] The endophytic fungicide of this invention differs from traditional gel-based slow-release fungicides. Firstly, during the early stages of seed stratification in sand, the seeds are dormant, and endophytic fungal activity is low. After the fungicide is encapsulated and secondary gelled with calcium chloride, a uniform gel coating forms on the surface. The fungicide continuously targets the seed interior to kill endophytic fungi. However, in the later stages of stratification, the gel loses water, forming a gel film that hinders the invasion of external bacteria. At this time, seed vigor increases, and the embryo begins to germinate. If the film continues to encapsulate the seed, it will hinder embryo growth. Therefore, an acidic citric acid solution is sprayed. Chitosan dissolves under acidic conditions, and calcium alginate shrinks, causing the film to rupture. Shellac also gradually loses water and curls, and internal stress leads to film rupture, which is beneficial for embryo emergence and growth. In addition to promoting plant tissue development, lignin can also adsorb the fungicide. Due to its good permeability, it can penetrate deep into the intercellular spaces, promoting embryo growth while simultaneously allowing the fungicide to penetrate the seed interior and eliminate endophytic fungi.

[0025] Furthermore, in the endophytic bactericide, the mass ratio of the viscous liquid to ε-polylysine, streptomycin, and enzymatically hydrolyzed lignin solution is 1:(0.02-0.03):(0.03-0.05):(0.5-0.6), and the mass ratio of shellac, Span, glycerin, jojoba oil, swollen gel powder, and water is 1:(0.7-0.8):(0.2-0.4).

[0026] (0.8-1): (0.5-0.8): (3.5-4).

[0027] Furthermore, the culture temperature was maintained at 25±3℃, the light intensity at 2000-2500 Lux, and the light duration at 16h / d throughout the primary culture, subculture, and rooting culture processes.

[0028] Beneficial effects:

[0029] The endophytic fungicide prepared in this invention gradually eliminates endophytic bacteria and prevents external bacteria from entering the seed interior and causing explant contamination during sand stratification, thus reducing the probability of infection. While reducing the obstruction caused by endophytic bacteria, strict aseptic treatment during subsequent subculturing and rooting cultures can improve propagation efficiency and rooting rate. This establishes an effective rapid propagation technology for *Camellia chrysantha* tissue culture, laying a theoretical foundation for the mass production of *Camellia chrysantha* tissue culture seedlings. Attached Figure Description

[0030] Figure 1 : Explants after disinfection;

[0031] Figure 2 Seeds stored in sand;

[0032] Figure 3 : Sterile seedlings during the propagation and culture process. Detailed Implementation

[0033] The present invention will be described in detail below with reference to specific embodiments:

[0034] Unless otherwise specified, the experimental methods used in the embodiments of this invention are conventional methods. Unless otherwise specified, the materials and reagents used in the embodiments of this invention are commercially available.

[0035] Example 1: Tissue culture of Camellia chrysantha from Guizhou

[0036] (1) After collecting fully mature Camellia laurentii fruits from Guizhou, remove the seeds, disinfect them with 800 times 50% carbendazim wettable powder, rinse them with clean water, and then store them in sand. During the storage process, maintain the temperature at 10-15℃ and the humidity at 60-70%. After about 60 days of storage, when the seeds have grown 1-2cm radicles, remove the mud and seed coat from the seed surface, rinse them with running clean water, and then soak the seeds in 75% alcohol for 30 seconds on a clean bench. After removing them, rinse them with sterile water 3-4 times, then soak them in 0.1% mercuric chloride solution for 8-10 minutes, shaking them once every 10-30 seconds. Rinse them with sterile water 3-4 times, and then dry them with filter paper to obtain externally disinfected explants.

[0037] (2) Cut off the excess endosperm of the explant and inoculate the remaining part into the primary culture medium and culture for 25-30 days. After the seedlings grow, sterile seedlings of Camellia chrysantha var. guili are obtained.

[0038] (3) Cut the obtained sterile seedlings into stem segments with 1-2 leaf buds, inoculate them into subculture medium and culture for 50-60 days. If the medium is insufficient during this period, replace it with a new subculture medium. After the subculture is completed, transfer it to rooting medium and culture for 35-40 days to obtain Guizhou golden camellia seedlings.

[0039] Throughout the primary culture, subculture, and rooting culture processes, the culture temperature was maintained at 25±3℃, the light intensity at 2000-2500 Lux, and the light duration at 16h / d.

[0040] The primary and secondary culture media consist of MS medium + 4 mg / L 6-BA + 6 mg / L brassinolide + 0.3 mg / L NAA. The rooting medium consists of 1 / 2 MS medium + 6 mg / L NAA.

[0041] Example 2: Tissue culture of Camellia chrysantha var. guizhouense

[0042] Preparation process of endophytic bactericide:

[0043] (1) A viscous liquid was obtained by mixing 2 wt% chitosan solution and 1 wt% sodium alginate solution at a volume ratio of 2:1. Then, streptomycin, ε-polylysine and 3 wt% enzymatic hydrolysate solution were added to the viscous liquid at a mass ratio of 1:0.02:0.03:0.5. After mixing and dispersing evenly, a mixture was obtained. Then, 3 wt% calcium chloride solution was slowly added and stirred to obtain a gel block. The volume ratio of the mixture to the calcium chloride solution was 1:1. After freezing at -20℃, the mixture was pulverized to obtain a gel powder with a particle size of 0.2-0.3 mm. After swelling with water for 30 min, it was ready for use.

[0044] (2) Shellac was dissolved in a 70 wt% ethanol solution, and the precipitate was removed by centrifugation. After evaporation of the ethanol by water bath heating, jojoba oil and glycerin were added, and the mixture was stirred at 800 rpm for 5 min. Then, 5 wt% Span-80 solution, swollen gel powder, and water were added, and the mixture was stirred at 1000 rpm for 2 min. Finally, the mixture was sheared at 24000 rpm for 2 min to obtain an endophytic bactericide. The mass ratio of shellac, Span-80, glycerin, jojoba oil, swollen gel powder, and water was 1:0.7:0.2:0.8:0.5:3.5.

[0045] Tissue culture process:

[0046] (1) After collecting fully mature Camellia laurentii fruits from Guizhou, remove the seeds and disinfect them by soaking the surface in 75% alcohol for 30 seconds. Then, grind them in high-temperature sterilized sand with a particle size of 0.3-0.5 mm for 5 minutes. After that, soak them in an endophytic fungicide for 10 minutes, remove them and let them stand for 10 minutes. Then, soak them in a 1 wt% calcium chloride solution for 5 minutes. Remove them and let them stand at room temperature for 1 hour before burying them in sand for sand storage. The temperature during the sand storage process should be 10-15℃. Maintain the humidity at 60-70% for the first 20 days of sand storage, and maintain the humidity at 50-60% after the 20th day of sand storage. Starting from the 20th day of sand storage, spray a citric acid solution with a pH of 5.5 every 7 days. Citric acid solution can be used instead of water to maintain humidity.

[0047] After about 45 days of sand storage, when the seeds have grown 1-2 cm of radicle, remove the mud and seed coat from the seed surface, wash them with running water, and then soak them in 75% alcohol for 30 seconds on a clean bench. After taking them out, rinse them 3-4 times with sterile water, then soak them in 0.1% mercuric chloride solution for 8-10 minutes, shaking them once every 10-30 seconds during this time. Rinse them 3-4 times with sterile water, and then blot dry with filter paper to obtain externally sterilized explants.

[0048] (2) Cut off the excess endosperm of the explant and inoculate the remaining part into the primary culture medium and culture for 25-30 days. After the seedlings grow, sterile seedlings of Camellia chrysantha var. guili are obtained.

[0049] (3) Cut the obtained sterile seedlings into stem segments with 1-2 leaf buds, inoculate them into subculture medium and culture for 50-60 days. If the medium is insufficient during this period, replace it with a new subculture medium. After the subculture is completed, transfer it to rooting medium and culture for 35-40 days to obtain Guizhou golden camellia seedlings.

[0050] Throughout the primary culture, subculture, and rooting culture processes, the culture temperature was maintained at 25±3℃, the light intensity at 2000-2500 Lux, and the light duration at 16h / d.

[0051] The primary and secondary culture media consist of MS medium + 4 mg / L 6-BA + 6 mg / L brassinolide + 0.3 mg / L NAA. The rooting medium consists of 1 / 2 MS medium + 6 mg / L NAA.

[0052] Example 3: Tissue culture of Camellia chrysantha var. guizhouense

[0053] Preparation process of endophytic bactericide:

[0054] (1) A viscous liquid was obtained by mixing 2 wt% chitosan solution and 1 wt% sodium alginate solution at a volume ratio of 2:1. Then, streptomycin, ε-polylysine and 3 wt% enzymatic hydrolysate solution were added to the viscous liquid at a mass ratio of 1:0.03:0.05:0.6. After mixing and dispersing evenly, a mixture was obtained. Then, 5 wt% calcium chloride solution was slowly added and stirred to obtain a gel block. The volume ratio of the mixture to the calcium chloride solution was 1:1. After freezing at -20℃, the mixture was pulverized to obtain a gel powder with a particle size of 0.2-0.3 mm. After swelling with water for 30 min, it was ready for use.

[0055] (2) Shellac was dissolved in a 70 wt% ethanol solution, centrifuged to remove the precipitate, heated in a water bath to evaporate the ethanol, and then jojoba oil and glycerin were added. The mixture was stirred at 1000 rpm for 5 min, and then 5 wt% Span-80 solution, the swollen gel powder, and water were added. The mixture was stirred at 800 rpm for 2 min and then sheared at 20000 rpm for 2 min to obtain an endophytic bactericide. The mass ratio of shellac, Span-80, glycerin, jojoba oil, swollen gel powder, and water was 1:0.8:0.4:1:0.8:4.

[0056] Tissue culture process:

[0057] (1) After collecting fully mature Camellia laurentii fruits from Guizhou, remove the seeds and disinfect them by soaking them in 75% alcohol for 30 seconds. Then, grind them in high-temperature sterilized sand with a particle size of 0.3-0.5 mm for 6 minutes. After that, soak them in an endophytic fungicide for 20 minutes, remove them and let them stand for 20 minutes. Then, soak them in a 2wt% calcium chloride solution for 10 minutes. Remove them and let them stand at room temperature for 2 hours before burying them in sand for sand storage. The temperature during the sand storage process should be 10-15℃. Maintain the humidity at 60-70% for the first 20 days of sand storage, and maintain the humidity at 50-60% after the 20th day of sand storage. Starting from the 20th day of sand storage, spray a citric acid solution with a pH of 6.5 every 5 days. Citric acid solution can be used instead of water to maintain humidity.

[0058] After about 45 days of sand storage, when the seeds have grown 1-2 cm of radicle, remove the mud and seed coat from the seed surface, wash them with running water, and then soak them in 75% alcohol for 30 seconds on a clean bench. After taking them out, rinse them 3-4 times with sterile water, then soak them in 0.1% mercuric chloride solution for 8-10 minutes, shaking them once every 10-30 seconds during this time. Rinse them 3-4 times with sterile water, and then blot dry with filter paper to obtain externally sterilized explants.

[0059] (2) Cut off the excess endosperm of the explant and inoculate the remaining part into the primary culture medium and culture for 25-30 days. After the seedlings grow, sterile seedlings of Camellia chrysantha var. guili are obtained.

[0060] (3) Cut the obtained sterile seedlings into stem segments with 1-2 leaf buds, inoculate them into subculture medium and culture for 50-60 days. If the medium is insufficient during this period, replace it with a new subculture medium. After the subculture is completed, transfer it to rooting medium and culture for 35-40 days to obtain Guizhou golden camellia seedlings.

[0061] Throughout the primary culture, subculture, and rooting culture processes, the culture temperature was maintained at 25±3℃, the light intensity at 2000-2500 Lux, and the light duration at 16h / d.

[0062] The primary and secondary culture media consist of MS medium + 4 mg / L 6-BA + 6 mg / L brassinolide + 0.3 mg / L NAA. The rooting medium consists of 1 / 2 MS medium + 6 mg / L NAA.

[0063] Comparative Example 1:

[0064] This invention is compared with Example 2, the only difference being that this invention does not involve mechanical damage, but directly uses an endophytic bacteria bactericide for soaking treatment. The specific tissue culture process is as follows:

[0065] (1) After collecting fully mature Guizhou golden camellia fruits, remove the seeds and disinfect the surface by soaking them in 75% alcohol for 30 seconds. Then soak them in an endophytic fungicide for 10 minutes, remove them and let them stand for 10 minutes. Then soak them in a 1wt% calcium chloride solution for 5 minutes. Remove them and let them stand at room temperature for 1 hour before burying them in sand for sand storage. The temperature during sand storage should be 10-15℃. Maintain the humidity at 60-70% for the first 20 days of sand storage, and maintain the humidity at 50-60% after the 20th day of sand storage. When the 20th day of sand storage begins, spray a citric acid solution with a pH of 5.5 every 7 days. Citric acid solution can be used instead of water to maintain humidity.

[0066] After the seeds stored in sand have grown 1-2 cm of radicle, remove the mud and seed coat from the seed surface, wash them thoroughly with running water, and then soak them in 75% alcohol for 30 seconds on a clean bench. After removing them, rinse them 3-4 times with sterile water, then soak them in 0.1% mercuric chloride solution for 8-10 minutes, shaking them once every 10-30 seconds during this time. Rinse them 3-4 times with sterile water again, and then blot dry with filter paper to obtain externally sterilized explants.

[0067] The subsequent tissue culture process is the same.

[0068] Comparative Example 2:

[0069] This invention is compared with Example 2, the only difference being that this invention directly uses an endophytic bactericide made from sodium alginate and chitosan gel. The specific preparation and tissue culture process of the endophytic bactericide is as follows:

[0070] Preparation of endophytic bacteria bactericide:

[0071] A viscous liquid was obtained by mixing 2 wt% chitosan solution and 1 wt% sodium alginate solution at a volume ratio of 2:1. Then, streptomycin and ε-polylysine were added to the viscous liquid at a mass ratio of 1:0.02:0.03. After mixing and dispersing evenly, a mixture was obtained. Then, 3 wt% calcium chloride solution was slowly added and stirred to obtain a gel solution. The volume ratio of the mixture to the calcium chloride solution was 1:0.8.

[0072] Tissue culture process:

[0073] (1) After collecting fully mature Camellia laurentii fruits from Guizhou, remove the seeds and disinfect them by soaking them in 75% alcohol for 30 seconds. Then, grind them in high-temperature sterilized sand with a particle size of 0.3-0.5 mm for 5 minutes. After that, soak them in a gel solution for 10 minutes, remove them and let them stand for 10 minutes. Then, soak them in a 1 wt% calcium chloride solution for 5 minutes. Remove them and let them stand at room temperature for 1 hour before burying them in sand for sand storage. The temperature during the sand storage process should be 10-15℃. Maintain the humidity at 60-70% for the first 20 days of sand storage, and maintain the humidity at 50-60% after the 20th day of sand storage. Starting from the 20th day of sand storage, spray a citric acid solution with a pH of 5.5 every 7 days. Citric acid solution can be used instead of water to maintain humidity.

[0074] The subsequent tissue culture process is the same.

[0075] Comparative Example 3:

[0076] This invention is compared with Example 2, the only difference being that this invention directly uses an endophytic bactericide prepared by mixing shellac, sodium alginate, and chitosan. The specific preparation and tissue culture process of the endophytic bactericide is as follows:

[0077] (1) A viscous liquid was obtained by mixing 2 wt% chitosan solution and 1 wt% sodium alginate solution at a volume ratio of 2:1. Then, streptomycin, ε-polylysine and 3 wt% enzymatic hydrolysate solution were added to the viscous liquid at a mass ratio of 1:0.02:0.03:0.5. The mixture was then dispersed evenly to obtain a mixture.

[0078] (2) After dissolving shellac in a 70wt% ethanol solution, centrifuge to remove the precipitate, heat in a water bath to evaporate the ethanol and add it to the mixture, stir at 1000rpm for 8min and then add 3wt% calcium chloride solution. After stirring evenly, the endophytic bacteria bactericide is obtained. The mass ratio of shellac to the mixture is 1:0.5, and the amount of calcium chloride solution added is 1.5 times the volume of the mixture.

[0079] The subsequent tissue culture process is the same.

[0080] Comparative Example 4:

[0081] This invention is compared with Example 2, the only difference being that no enzymatic hydrolysis of lignin is added during the preparation of the endophytic fungicide in this invention. The specific preparation process is as follows:

[0082] (1) A viscous liquid was obtained by mixing 2 wt% chitosan solution and 1 wt% sodium alginate solution at a volume ratio of 2:1. Then, streptomycin and ε-polylysine were added to the viscous liquid at a mass ratio of 1:0.02:0.03. After mixing and dispersing evenly, a mixture was obtained. Then, 3 wt% calcium chloride solution was slowly added and stirred to obtain a gel block. The volume ratio of the mixture to the calcium chloride solution was 1:1. After freezing at -20℃, the mixture was pulverized to obtain a gel powder with a particle size of 0.2-0.5 mm. The powder was then swollen with water for 30 min and set aside for use.

[0083] (2) Shellac was dissolved in a 70 wt% ethanol solution, and the precipitate was removed by centrifugation. After evaporation of the ethanol by water bath heating, jojoba oil and glycerin were added, and the mixture was stirred at 800 rpm for 5 min. Then, 5 wt% Span-80 solution, swollen gel powder, and water were added, and the mixture was stirred at 1000 rpm for 2 min. Finally, the mixture was sheared at 24000 rpm for 2 min to obtain an endophytic bactericide. The mass ratio of shellac, Span-80, glycerin, jojoba oil, swollen gel powder, and water was 1:0.7:0.2:0.8:0.5:3.5.

[0084] The subsequent tissue culture process is the same.

[0085] Comparative Example 5:

[0086] This invention is compared with Example 2, the only difference being that the endophytic bactericide of this invention is not prepared as an emulsion during the preparation process. The specific preparation process is as follows:

[0087] (1) A viscous liquid was obtained by mixing 2 wt% chitosan solution and 1 wt% sodium alginate solution at a volume ratio of 2:1. Then, streptomycin, ε-polylysine and 3 wt% enzymatic hydrolysate solution were added to the viscous liquid at a mass ratio of 1:0.02:0.03:0.5. After mixing and dispersing evenly, a mixture was obtained. Then, 3 wt% calcium chloride solution was slowly added and stirred to obtain a gel block. The volume ratio of the mixture to the calcium chloride solution was 1:1. After freezing at -20℃, the mixture was pulverized to obtain a gel powder with a particle size of 0.2-0.3 mm. After swelling with water for 30 min, it was ready for use.

[0088] (2) Shellac was dissolved in a 70wt% ethanol solution, centrifuged to remove the precipitate, heated in a water bath to evaporate the ethanol, and then glycerol was added. The mixture was stirred at 800 rpm for 5 min, and then the swollen gel powder and water were added. The mixture was stirred at 1000 rpm for 10 min to obtain an endophytic bactericide. The mass ratio of shellac, glycerol, swollen gel powder and water was 1:0.2:0.5:3.5.

[0089] Comparative Example 6:

[0090] This invention is compared with Example 2, the only difference being that in this invention, after using the endophytic bacteria bactericide in the tissue culture process, citric acid solution is not sprayed during the sand storage process, and water is used to adjust the humidity; the rest of the operations remain the same.

[0091] Comparative Example 7:

[0092] This invention is compared with Example 2, the only difference being that calcium chloride is not used for a secondary gelation process in the tissue culture process of this invention. The specific operation is as follows:

[0093] (1) After collecting fully mature Camellia laurentii fruits from Guizhou, remove the seeds and disinfect the surface by soaking them in 75% alcohol for 30 seconds. Then, grind them in high-temperature sterilized sand with a particle size of 0.3-0.5 mm for 5 minutes. After that, soak them in an endophytic fungicide for 10 minutes. After removing them, let them stand for 1 hour and then bury them in sand for sand storage. The temperature during the sand storage process should be 10-15℃. Maintain the humidity at 60-70% for the first 20 days of sand storage, and maintain the humidity at 50-60% after the 20th day of sand storage. Starting from the 20th day of sand storage, spray a citric acid solution with a pH of 5.5 every 7 days. Citric acid solution can be used instead of water to maintain humidity.

[0094] After the seeds stored in sand have grown 1-2 cm of radicle, remove the mud and seed coat from the seed surface, wash them with running water, and then soak them in 75% alcohol for 30 seconds on a clean bench. After removing them, rinse them 3-4 times with sterile water, then soak them in 0.1% mercuric chloride solution for 8-10 minutes, shaking them once every 10-30 seconds during this time. Rinse them 3-4 times with sterile water, and then blot dry with filter paper to obtain externally sterilized explants.

[0095] The subsequent tissue culture process is the same.

[0096] Comparative Example 8:

[0097] This invention is compared with Example 2, the only difference being the change in the mass ratio of shellac to swollen gel during the preparation of the endophytic bactericide in this invention. The specific step (2) is as follows:

[0098] (2) Shellac was dissolved in a 70 wt% ethanol solution, centrifuged to remove the precipitate, heated in a water bath to evaporate the ethanol, and then jojoba oil and glycerin were added. The mixture was stirred at 800 rpm for 5 min, and then 5 wt% Span-80 solution, swollen gel powder, and water were added. The mixture was stirred at 1000 rpm for 2 min and then sheared at 24000 rpm for 2 min to obtain an endophytic bactericide. The mass ratio of shellac, Span-80, glycerin, jojoba oil, swollen gel powder, and water was 1:0.7:0.2:0.8:1:3.5.

[0099] Comparative Example 9:

[0100] This invention is compared with Example 2, the only difference being the change in the mass ratio of shellac to swollen gel during the preparation of the endophytic bactericide in this invention. The specific step (2) is as follows:

[0101] (2) Shellac was dissolved in a 70 wt% ethanol solution, centrifuged to remove the precipitate, heated in a water bath to evaporate the ethanol, and then jojoba oil and glycerin were added. The mixture was stirred at 800 rpm for 5 min, and then 5 wt% Span-80 solution, swollen gel powder, and water were added. The mixture was stirred at 1000 rpm for 2 min and then sheared at 24000 rpm for 2 min to obtain an endophytic bactericide. The mass ratio of shellac, Span-80, glycerin, jojoba oil, swollen gel powder, and water was 1:0.7:0.2:0.8:0.3:3.5.

[0102] experiment:

[0103] 1. Seeds of *Camellia chrysantha* harvested in mid-October by the Guizhou Academy of Forestry Sciences were selected for tissue culture experiments. The tissue culture methods of Examples 1, 2, and Comparative Examples 1-9 were used. Each group contained 11 seeds, with 3 replicates per group. The time it took for the radicle to first reach 1 cm in length after different sand stratification treatments (seeds were covered with approximately 5 cm of sand) was recorded. Once the radicle reached 1 cm in height, the externally sterilized explants were cultured. The specific sterilization process is as follows:

[0104] After the seed germinates to 1cm, remove the mud and seed coat from the seed surface, wash it with running water, and then soak the seed in 75% alcohol for 30 seconds on a clean bench. After that, rinse it three times with sterile water, then soak it in 0.1% mercuric chloride solution for 8 minutes, shaking it once every 30 seconds during the soaking. Rinse it three times with sterile water, and then blot the water with filter paper to obtain externally sterilized explants.

[0105] 2. After sand stratification, when the radicle of the seed germinated to 1 cm, one seed from each group was randomly selected for bacterial culture, with three replicates per group. The seeds were disinfected externally with 75% alcohol for 30 seconds, then rinsed with sterile water. The embryo was cut off and ground into a homogenate using a mortar. 5 mL of sterile water was added and shaken for 10 minutes. The stock solution was serially diluted 1 mL to 1000 times, and 100 μl of the homogenate was added to each culture medium. The number of colonies was observed. Fungal culture medium: 4 g potato starch, 20 g glucose, 15 g agar, 1 L water, natural pH, cultured at 25-28℃ for 5 days; Bacterial culture medium: 5 g peptone, 3 g beef extract, 5 g NaCl, 15 g agar, 1 L water, pH 7.0, cultured at 28-30℃ for 3 days.

[0106] 3. The obtained explants were subjected to primary culture, with 10 explants in each group, 3 replicates, and a culture time of 30 days. The contamination rate was recorded. Contaminated embryos were discarded, and sterile seedlings obtained from uncontaminated embryos were selected for subculture. The sterile seedlings were cut into stem segments with one leaf bud and then subcultured for 50 days. To ensure sufficient nutrients in the culture medium, the subculture medium was replaced on day 25. The proliferation coefficient was recorded after the culture was completed. For rooting culture, 20 explants were inoculated in each group, 3 replicates, and the rooting culture time was 35 days. The rooting rate was recorded. Throughout the primary, subculture, and rooting cultures, the culture temperature was maintained at 25±3℃, the light intensity at 2000-2500 Lux, and the photoperiod was 16h / d. The primary and subculture media consisted of MS medium + 4mg / L 6-BA + 6mg / L brassinolide + 0.3mg / L NAA. The rooting medium consisted of 1 / 2 MS medium + 6mg / L NAA.

[0107] The obtained data is shown in Table 1.

[0108] Table 1

[0109]

[0110] The analysis results show that:

[0111] 1. Compared with Example 1, Example 2 has a shorter germination time, lower endophytic bacteria content and contamination rate, and a higher proliferation coefficient than Example 1. This indicates that the scheme of Example 1 can also be used for tissue culture of Camellia chrysantha var. guillica. The endophytic bacteria bactericide in Example 2 effectively removes endophytic bacteria and reduces the contamination rate. For Camellia chrysantha var. guillica, which has a small population and precious seeds, the scheme of Example 2 is more suitable for the actual situation.

[0112] 2. In contrast, Comparative Example 1 did not use mechanical micro-damage to treat the seeds, resulting in a longer germination time and a higher embryo contamination rate. This may be due to the excessive sand storage time leading to high activity of endophytic bacteria or the entry of external bacteria that infected the seeds, thus increasing the contamination rate. During the propagation process, endophytic bacteria continued to contaminate the seedlings, reducing propagation efficiency and rooting rate.

[0113] 3. Comparative Example 2 directly used sodium alginate and chitosan gel to prepare an endophyte fungicide. Comparative Example 3 used a mixture of sodium alginate, chitosan gel, and shellac to prepare an endophyte fungicide. Although the sand storage days were reduced, the removal effect on endophytes was poor. This is because simply using the above-mentioned gel encapsulation resulted in a faster release rate of the fungicide components, and the endophytes were not completely killed, so infection still occurred, leading to a high contamination rate. The presence of endophytes affected the growth efficiency and rooting rate. Adding shellac directly slowed down this release rate, but the endophyte fungicide was still unevenly distributed.

[0114] 4. Comparative Example 4 was lignin-free, and Comparative Example 5 was not formulated into an emulsion. Both affected the killing of endophytic bacteria. Lignin can promote plant tissue development and also adsorb fungicides. Due to its good permeability, it can penetrate deep into the intercellular spaces, promoting embryo growth while allowing the fungicide to seep into the seed and eliminate endophytic bacteria. The emulsion, on the other hand, can adhere the fungicide to the seed surface, evenly coating it. During the later stages of sand stratification, it can break down evenly and degrade rapidly, preventing uneven lysis that could damage germination.

[0115] 5. Comparative Example 6 did not use citric acid, and Comparative Example 7 did not use calcium chloride secondary gelation. Instead, chitosan dissolved under acidic conditions, and calcium alginate contracted under acidic conditions, causing the membrane to rupture. Meanwhile, the shellac curled up as it gradually lost water, and the internal stress led to membrane rupture, which is beneficial for embryo emergence and growth. The secondary gelation using calcium chloride, calcium alginate, and chitosan resulted in a more uniform gel on the seed surface and a better gelation effect, preventing the endophytic fungicide from falling off during sand storage.

[0116] 6. Comparative Examples 8 and 9 show the changes in the mass ratio of shellac to gel powder. Shellac is more robust, while the mixed gel of calcium alginate and chitosan is more absorbent. Seeds in the early stage of sand stratification are in a dormant period, at which time the activity of endophytic bacteria is not high. The gel continuously releases bactericidal substances to continuously target and kill endophytic bacteria inside the seeds. However, in the middle and late stages of sand stratification, the gel has lost water and formed a gel film on the surface to prevent the invasion of external bacteria. If the ratio is not appropriate, it will cause water loss to be too fast or too slow, affecting germination and sterilization effects.

[0117] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention. Technical aspects, shapes, and structures not described in detail in this invention are all well-known technologies.

Claims

1. A Guizhou golden flower tea tissue culture method, characterized in that, The tissue culture method is as follows: (1) After collecting fully matured Guizhou golden tea fruits, the seeds are taken out for sand storage. When the seeds grow 1-2 cm radicles, the seed surface sand and seed coat are removed, and the seeds are cleaned with clean water, then immersed in 75% alcohol and 0.1% mercury chloride solution, cleaned with clean water to obtain external sterilized explants; (2) The excess endosperm of the explants is cut off, and the remaining part is inoculated into the primary culture medium to grow seedlings to obtain Guizhou golden tea aseptic seedlings; (3) The obtained aseptic seedlings are cut into stem segments with 1-2 leaf buds, inoculated into the subculture medium for subculture, and the subcultured seedlings are transferred to the rooting medium for rooting culture, and Guizhou golden tea seedlings are obtained after rooting culture; The compositions of the primary culture medium and the subculture medium are both MS medium+2-8 mg / L 6-BA+2-8 mg / L brassinolide+0.1-0.5 mg / L NAA; the composition of the rooting medium is 1 / 2MS medium+4-6 mg / L NAA; The explants are also internally sterilized, and the specific internal sterilization process is as follows: The surface of Guizhou golden tea seeds is ground with 0.3-0.5 mm sand after sterilization for 5-6 min, then immersed in an endophyte bactericide for 10-20 min, taken out and placed for 10-20 min, then immersed in 1-2 wt% calcium chloride solution for 5-10 min, taken out and placed at room temperature for 1-2 h, then buried in sand for sand storage. On the 20th day of sand storage, a citric acid solution with pH 5.5-6.5 is sprayed, and the spraying frequency is 5-7 days / time; The preparation method of the endophyte bactericide is as follows: (1) A viscous liquid is obtained by uniformly mixing 2 wt% chitosan solution and 1 wt% sodium alginate solution according to a volume ratio of 2:1, then a mixture of streptomycin, ε-polylysine and 3 wt% enzymatic lignin solution is added, uniformly dispersed, then 3-5 wt% calcium chloride solution is slowly added to obtain gel blocks, then frozen at-20℃, crushed to obtain gel powder with a particle size of 0.2-0.5 mm, and then swelled in water for 30 min for use; (2) The shellac is dissolved in 70 wt% ethanol solution, centrifuged to remove the precipitate, then 800-1000 rpm stirring is conducted for 5 min after adding jojoba oil and glycerol, then 5 wt% Span solution, swelled gel powder and water are added, stirred at 800-1000 rpm for 1-2 min, then sheared at 20000-24000 rpm for 1-2 min to obtain the endophyte bactericide; In the endophyte bactericide, the mass ratio of the viscous liquid to ε-polylysine, streptomycin, enzymatic lignin solution is 1:(0.02-0.03):(0.03-0.05):(0.5-0.6), and the mass ratio of the shellac, Span, glycerol, jojoba oil, swelled gel powder and water is 1:(0.7-0.8):(0.2-0.4):(0.8-1):(0.5-0.8):(3.5-4).

2. The tissue culture method of Guizhou golden tea according to claim 1, characterized in that, The culture time of the primary culture is 25-30 days; the culture time of the subculture is 50-60 days; and the culture time of the rooting culture is 35-40 days.

3. The tissue culture method of Guizhou golden tea according to claim 2, characterized in that, The specific operation of external disinfection of the explant is as follows: on the clean bench, the seed cleaned with clean water is soaked with 75% alcohol for 30s, then washed with sterile water for 3-4 times, then soaked with 0.1% mercury solution for 8-10 min, shaken once every 10-30s during the interval, then washed with sterile water for 3-4 times, then the water is absorbed with filter paper, and the external disinfection explant is obtained.

4. The tissue culture method of Guizhou golden tea according to claim 3, characterized in that, The sand storage process temperature is 10-15℃, the humidity is kept at 60-70% for 20 days before sand storage, and the humidity is kept at 50-60% after the 20th day of sand storage.

5. The tissue culture method of Guizhou golden tea according to claim 4, characterized in that, In the tissue culture process, the culture temperature is kept at 25±3℃, the light intensity is 2000-2500 Lux, and the light time is 16h / d.

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