Tissue culture method of Guizhou golden camellia

Abstract

The invention discloses a tissue culture method of Guizhou camellia nitidissima, and belongs to the technical field of tissue culture of Guizhou camellia nitidissima. The method comprises the following steps: (1) collecting fully mature Guizhou camellia nitidissima fruits, taking out seeds, storing in sand, removing silt and seed coats on the surfaces of the seeds after radicles of 1-2cm grow from the seeds, and taking out the seeds; cleaning with clear water, soaking with 75% alcohol and 0.1% mercury bichloride solution in sequence, and cleaning with clear water to obtain an explant of which the outside is disinfected; (2) cutting off redundant endosperm of the explant, inoculating the remaining part into a primary culture medium for culturing, and obtaining a Guizhou golden camellia aseptic seedling after a seedling grows; 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, and then transferring the stem segments into a rooting medium for culture to obtain Guizhou golden camellia seedlings, so that the propagation efficiency can be improved, and the problems of high pollution rate and low survival rate of golden camellia tissue culture are solved.

Description

Technical Field

[0001] The invention relates to the technical field of Guizhou golden camellia tissue culture, in particular to a Guizhou golden camellia tissue culture method. Background Art

[0002] The Camellia genus of the Theaceae family has a narrow natural distribution range, limited number, low seed yield, high abortion rate, and low fruiting rate. It is mainly distributed south of the Tropic of Cancer, on the northern edge of the tropics, and belongs to the tropical monsoon climate zone. The abortion rate at the bud stage is 16.33%, the abortion rate at the flowering stage is 63.05%, and the abortion rate at the fruit stage is 44.09%. It is listed as a second-level protected plant in my country. There are two types of Camellia chrysantha naturally distributed in Guizhou. One is Tian'e Camellia huana TLMing etW.J.Zhang, which is distributed in Luodian County, Guizhou Province and some parts of the adjacent Guangxi Province. The other is Guizhou Camellia huana TLMing etW.J.Zhang, which is only distributed in Ceheng County, Guizhou Province. The article "Correction of the Classification of Guizhou Camellia (Theaceae)" in the 2024 Plant Research Journal corrected the Chinese and Latin names of these two Camellias. There are only more than 20 plants of Guizhou Golden Camellia naturally distributed in the wild, which is of great significance in germplasm resource preservation and genetic breeding.

[0003] At present, the main propagation methods of Guizhou Camellia chrysantha are sowing, cuttings and grafting seedlings. Cuttings, grafting and other methods have slow propagation speed, low propagation coefficient, and damage to the mother plant, which is not conducive to the large-scale production of Guizhou Camellia chrysantha. Guizhou Camellia chrysantha is a perennial woody plant with a long growth cycle. The conventional breeding and seedling raising cycle is long, the propagation coefficient is small, and the germplasm advantage is easy to degenerate. Therefore, the development of tissue culture technology of Guizhou Camellia chrysantha is an urgently needed breeding method in the future. According to the relevant literature, there is no report on the tissue culture method of Guizhou Camellia chrysantha. At the same time, after the traditional sterilization method is used to treat the explants, it is found that there is still a high infection rate, which may be related to the endophytic contamination of Guizhou Camellia chrysantha. Therefore, a new tissue culture method of Guizhou Camellia chrysantha is found to solve the problems of high infection rate and low survival rate of tissue culture. Summary of the invention

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

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

[0006] A tissue culture method for Guizhou golden camellia, the tissue culture method is as follows:

[0007] (1) After collecting fully mature Guizhou Camellia chrysantha fruits, the seeds were taken out and stored in sand. After the seeds grew 1-2 cm radicles, the sand and seed coat on the surface of the seeds were removed, and the seeds were washed with clean water and then soaked in 75% alcohol and 0.1% mercuric chloride solution in sequence. After washing with clean water, the explants were disinfected externally;

[0008] (2) cutting off the excess endosperm of the explant, and inoculating the remaining part into the primary culture medium to grow seedlings to obtain the aseptic seedlings of Guizhou Camellia chrysantha;

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

[0010] Since Guizhou Camellia chrysantha is rare, the present invention uses Guizhou Camellia chrysantha seeds for tissue culture, which can avoid the problem of genotype homogeneity caused by asexual reproduction, and at the same time, the population of Guizhou Camellia chrysantha can be expanded after seed propagation. However, due to the numerous operations in the seed tissue culture process and the rich endophytes of Guizhou Camellia chrysantha seeds themselves, the contamination rate in the tissue culture process is high, resulting in a low survival rate of Camellia chrysantha seedlings. Therefore, strict disinfection is performed during the tissue culture process and a sterile environment is maintained as much as possible.

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

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

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

[0014] In addition to the contamination rate, the rooting rate also determines the success of tissue culture seedlings, and a reasonable hormone ratio is the key to rooting. Therefore, it is also very important to optimize the hormone ratio during primary culture, subculture, and rooting culture. We found that when the primary culture medium and subculture culture medium use the same formula, the embryo growth and subculture growth effects are better.

[0015] Furthermore, the specific operation of external disinfection of explants is as follows: on an ultra-clean workbench, the seeds washed with clean water are soaked in 75% alcohol for 30 seconds, taken out and rinsed with sterile water for 3-4 times, and then soaked in 0.1% mercuric chloride solution for 8-10 minutes, shaking once every 10-30 seconds, and then rinsed with sterile water for 3-4 times, and the moisture was absorbed with filter paper to obtain externally disinfected explants.

[0016] Since the endophytes of Guizhou Camellia chrysantha were found to be serious during the experiment, the present invention adopts a combined internal and external sterilization operation. The explants are disinfected externally with ethanol, mercuric chloride solution and other substances to reduce the impact of external bacteria on the explants, and the excess endosperm is cut off to reduce the impact of endophytes in the endosperm. However, even if the endosperm is cut off, the endophytes still diffuse to the embryo part through the vascular tissue or cell gap inside the seed during the germination process. Therefore, on the basis of removing the endosperm, an endophyte fungicide is used for disinfection to reduce the impact of endophytes.

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

[0018] The surface of Guizhou Camellia chrysantha seeds was ground with sterilized 0.3-0.5 mm sand for 5-6 minutes, then taken out and immersed in an endophytic fungicide for 10-20 minutes, taken out and allowed to stand for 10-20 minutes, then immersed in 1-2 wt % calcium chloride solution for 5-10 minutes, taken out and allowed to stand at room temperature for 1-2 hours, then buried in sand for sand storage, and sprayed with citric acid solution with a pH of 5.5-6.5 on the 20th day of sand storage, with a spraying frequency of 5-7 days / time.

[0019] Furthermore, the temperature during the sand storage process is 10-15°C, 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] Further, the preparation method of the endophytic fungicide is as follows:

[0021] (1) 2 wt% chitosan solution and 1 wt% sodium alginate solution are mixed in a volume ratio of 2:1 to obtain a viscous liquid, and then a mixture consisting of streptomycin, ε-polylysine and 3 wt% enzymatic lignin solution is added, and after being evenly dispersed, 3-5 wt% calcium chloride solution is slowly added to obtain a gel block, which is then frozen at -20°C and crushed to obtain a gel powder with a particle size of 0.2-0.5 mm, and then water is added to swell for 30 minutes for standby use;

[0022] (2) Dissolving shellac in a 70 wt% ethanol solution and centrifuging to remove the precipitate, then adding jojoba oil and glycerin, stirring at 800-1000 rpm for 5 min, then adding 5 wt% Span solution, swollen gel powder, and water, stirring at 800-1000 rpm for 1-2 min, and then shearing at 20000-24000 rpm for 1-2 min to obtain an endophyte bactericide.

[0023] The present invention uses ε-polylysine and streptomycin as bactericides to disinfect the seeds of Guizhou Jinhua Tea during the sand storage process. ε-polylysine is a natural antibacterial substance that destroys bacterial cell membranes and has a broad-spectrum antibacterial effect, and streptomycin is also commonly used for tissue culture sterilization, so the two are selected to act synergistically on seeds to kill endophytes. In addition, the seed shell of Guizhou Jinhua Tea is relatively hard and has a dormant period, so it needs to be stored in sand for germination. The present invention uses a mechanical micro-damage method to accelerate germination and accelerate the tissue culture process, but after the seeds are damaged, the seeds are also susceptible to bacteria. Based on this, the present invention uses a bactericide in an inner emulsion state to uniformly wrap the seeds, so that the bactericidal components are released inwardly through the micro-damaged wounds into the seeds, and directly act on the bacteria inside the seeds. The bactericidal components in the endophytic bactericide are wrapped with chitosan and sodium alginate to achieve slow release, and the chitosan itself has a broad-spectrum antibacterial property, which can also prevent external bacteria from invading the seeds and causing infection during the sand storage process.

[0024] The endophyte fungicide of the present invention is different from the traditional gel slow-release fungicide. First, in the early stage of seed sand storage, the seeds are in a dormant period, and the activity of endophytes is not high at this time. After wrapping the endophyte fungicide, a uniform gel package is formed on the surface after calcium chloride secondary gelation. The bactericidal substance continuously targets the inside of the seed to kill endophytes, and in the middle and late stages of sand storage, the gel has lost water and the surface forms a gel film to hinder the invasion of external bacteria, but at this time, the seed vitality increases and the embryo begins to sprout. If the film continues to wrap the seed, it will cause the embryo to be difficult to grow, so it starts to spray acidic citric acid solution, and chitosan is used to dissolve under acidic conditions. Calcium alginate shrinks and causes the membrane to rupture, and shellac gradually curls during the process of losing water, and internal stress causes the membrane to rupture, which is conducive to the extension and growth of the embryo. Lignin can not only promote the development of plant tissues, but also absorb bactericides, because it has good permeability and can penetrate into the intercellular space, and the bactericide is infiltrated into the seed while promoting the growth of the embryo, eliminating endophytes.

[0025] Furthermore, in the endophyte fungicide, the mass ratio of the viscous liquid to ε-polylysine, streptomycin, and 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, 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, during the primary culture, subculture and rooting culture, the culture temperature was maintained at 25±3°C, the light intensity was 2000-2500 Lux, and the lighting time was 16 h / d.

[0028] Beneficial effects:

[0029] The endophyte fungicide prepared by the present invention gradually eliminates endophytes during the sand storage process and prevents external bacteria from entering the seeds to cause explant contamination, thereby reducing the probability of infection. While reducing the obstruction of endophytes, strictly implementing aseptic treatment during the later subculture and rooting culture process can improve the reproduction efficiency and rooting rate. The established effective Guizhou golden camellia tissue culture rapid propagation technology lays a theoretical foundation for realizing the batch production of Guizhou golden camellia tissue culture seedlings. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 : Explants after sterilization;

[0031] Figure 2 : Seeds after sand storage;

[0032] Figure 3 : Sterile seedlings in the proliferation culture process. DETAILED DESCRIPTION

[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 examples of the present invention are all conventional methods. The materials, reagents, etc. used in the examples of the present invention, unless otherwise specified, can be obtained from commercial channels.

[0035] Example 1: Guizhou Camellia chrysantha tissue culture

[0036] (1) After collecting fully mature Guizhou Camellia chrysantha fruits, the seeds are taken out, disinfected with 800 times 50% carbendazim wettable powder, washed with clean water, and then stored in sand. During the sand storage process, the temperature is maintained at 10-15°C and the humidity is maintained at 60-70%. After the seeds grow 1-2 cm radicles after about 60 days of sand storage, the mud and seed coat on the surface of the seeds are removed, and the seeds are washed with running clean water. After soaking in 75% alcohol for 30 seconds on a clean bench, the seeds are taken out and rinsed with sterile water for 3-4 times, and then soaked in 0.1% mercuric chloride solution for 8-10 minutes, shaken once every 10-30 seconds, and then rinsed with sterile water for 3-4 times, and the water is absorbed with filter paper to obtain externally sterilized explants;

[0037] (2) cutting off the excess endosperm of the explant, and inoculating the remaining part into the primary culture medium and culturing for 25-30 days, and obtaining the aseptic seedlings of Guizhou Camellia chrysantha after the seedlings grow out;

[0038] (3) The obtained sterile seedlings were cut into stem segments with 1-2 leaf buds, inoculated into subculture medium and cultured for 50-60 days. If the medium was insufficient in nutrients during this period, a new subculture medium could be replaced. After the subculture was completed, the seedlings were transferred to a rooting medium and cultured for 35-40 days to obtain Guizhou Camellia chrysantha seedlings.

[0039] During the primary culture, subculture and rooting culture, the culture temperature was maintained at 25±3°C, the light intensity was 2000-2500Lux, and the light duration was 16h / d;

[0040] The composition of primary culture medium and secondary culture medium: MS culture medium + 4 mg / L 6-BA + 6 mg / L brassinolide + 0.3 mg / L NAA; the composition of rooting culture medium: 1 / 2 MS culture medium + 6 mg / L NAA.

[0041] Example 2: Tissue culture of Guizhou golden camellia

[0042] Preparation process of endophytic fungicide:

[0043] (1) 2 wt% chitosan solution and 1 wt% sodium alginate solution are mixed uniformly in a volume ratio of 2:1 to obtain a viscous liquid, and then streptomycin, ε-polylysine and 3 wt% enzymatic lignin solution are added in a mass ratio of 1:0.02:0.03:0.5 to the viscous liquid, ε-polylysine, streptomycin and enzymatic lignin solution, and the mixture is dispersed uniformly after mixing to obtain a mixture, and then 3 wt% calcium chloride solution is slowly added and stirred to obtain a gel block, the volume ratio of the mixture to the calcium chloride solution is 1:1, and then the mixture is frozen at -20°C and crushed to obtain a gel powder with a particle size of 0.2-0.3 mm, and then water is added to swell for 30 minutes and then standby;

[0044] (2) Dissolve shellac in 70 wt% ethanol solution and centrifuge to remove precipitate, heat in a water bath to evaporate ethanol, add jojoba oil and glycerol, stir at 800 rpm for 5 min, then add 5 wt% Span-80 solution, swollen gel powder, and water, stir at 1000 rpm for 2 min, and shear at 24000 rpm for 2 min to obtain an endophyte fungicide. The mass ratio of shellac, Span-80, glycerol, jojoba oil, swollen gel powder, and water is 1:0.7:0.2:0.8:0.5:3.5.

[0045] Tissue culture process:

[0046] (1) After collecting fully mature Guizhou Camellia chrysantha fruits, the seeds are taken out and the surface is disinfected by soaking in 75% alcohol for 30 seconds, then ground with high temperature sterilized sand with a particle size of 0.3-0.5 mm for 5 minutes, then taken out and soaked in an endogenous fungicide for 10 minutes, taken out and left to stand for 10 minutes, then immersed in a 1wt% calcium chloride solution for 5 minutes, taken out and left to stand at room temperature for 1 hour, and then buried in sand for sand storage. The temperature of the sand storage process is 10-15°C, and 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. On the 20th day of sand storage, a citric acid solution with a pH of 5.5 is sprayed, and the spraying frequency is 7 days / time. Citric acid solution can be used instead of clean water to maintain humidity.

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

[0048] (2) cutting off the excess endosperm of the explant, inoculating the remaining part into the primary culture medium and culturing for 25-30 days, and obtaining the aseptic seedlings of Guizhou Camellia chrysantha after the seedlings grow out;

[0049] (3) The obtained sterile seedlings were cut into stem segments with 1-2 leaf buds, inoculated into subculture medium and cultured for 50-60 days. If the medium was insufficient in nutrients during this period, a new subculture medium could be replaced. After the subculture was completed, the seedlings were transferred to a rooting medium and cultured for 35-40 days to obtain Guizhou Camellia chrysantha seedlings.

[0050] During the primary culture, subculture and rooting culture, the culture temperature was maintained at 25±3°C, the light intensity was 2000-2500Lux, and the light duration was 16h / d;

[0051] The composition of primary culture medium and secondary culture medium: MS culture medium + 4 mg / L 6-BA + 6 mg / L brassinolide + 0.3 mg / L NAA; the composition of rooting culture medium: 1 / 2 MS culture medium + 6 mg / L NAA.

[0052] Example 3: Tissue culture of Guizhou Camellia chrysantha

[0053] Preparation process of endophytic fungicide:

[0054] (1) 2 wt% chitosan solution and 1 wt% sodium alginate solution are mixed uniformly in a volume ratio of 2:1 to obtain a viscous liquid, and then streptomycin, ε-polylysine and 3 wt% enzymatic lignin solution are added in a mass ratio of 1:0.03:0.05:0.6 to the viscous liquid, ε-polylysine, streptomycin and enzymatic lignin solution, and the mixture is dispersed uniformly after mixing to obtain a mixture, and then 5 wt% calcium chloride solution is slowly added and stirred to obtain a gel block, wherein the volume ratio of the mixture to the calcium chloride solution is 1:1, and then the mixture is frozen at -20°C and crushed to obtain a gel powder with a particle size of 0.2-0.3 mm, and then water is added to swell for 30 minutes and then used;

[0055] (2) Dissolve shellac in 70 wt% ethanol solution and centrifuge to remove precipitate, heat in a water bath to evaporate ethanol, add jojoba oil and glycerol, stir at 1000 rpm for 5 min, then add 5 wt% Span-80 solution, swollen gel powder and water, stir at 800 rpm for 2 min, and shear at 20000 rpm for 2 min to obtain an endophyte fungicide. The mass ratio of shellac, Span-80, glycerol, jojoba oil, swollen gel powder and water is 1:0.8:0.4:1:0.8:4.

[0056] Tissue culture process:

[0057] (1) After collecting fully mature Guizhou Camellia chrysantha fruits, the seeds are taken out and the surface is disinfected by soaking in 75% alcohol for 30 seconds, then ground with high temperature sterilized sand with a particle size of 0.3-0.5 mm for 6 minutes, then taken out and soaked in an endogenous fungicide for 20 minutes, taken out and left to stand for 20 minutes, then immersed in a 2wt% calcium chloride solution for 10 minutes, taken out and left to stand at room temperature for 2 hours, and then buried in sand for sand storage. The temperature of the sand storage process is 10-15°C, and 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. On the 20th day of sand storage, a citric acid solution with a pH of 6.5 is sprayed, and the spraying frequency is 5 days / time. Citric acid solution can be used instead of clean water to maintain humidity.

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

[0059] (2) cutting off the excess endosperm of the explant, inoculating the remaining part into the primary culture medium and culturing for 25-30 days, and obtaining the aseptic seedlings of Guizhou Camellia chrysantha after the seedlings grow out;

[0060] (3) The obtained sterile seedlings were cut into stem segments with 1-2 leaf buds, inoculated into subculture medium and cultured for 50-60 days. If the medium was insufficient in nutrients during this period, a new subculture medium could be replaced. After the subculture was completed, the seedlings were transferred to a rooting medium and cultured for 35-40 days to obtain Guizhou Camellia chrysantha seedlings.

[0061] During the primary culture, subculture and rooting culture, the culture temperature was maintained at 25±3°C, the light intensity was 2000-2500Lux, and the light duration was 16h / d;

[0062] The composition of primary culture medium and secondary culture medium: MS culture medium + 4 mg / L 6-BA + 6 mg / L brassinolide + 0.3 mg / L NAA; the composition of rooting culture medium: 1 / 2 MS culture medium + 6 mg / L NAA.

[0063] Comparative Example 1:

[0064] The present invention is in contrast to Example 2, the only difference being that the present invention does not perform mechanical damage, but directly uses an endophytic fungicide for soaking treatment. The specific tissue culture process is as follows:

[0065] (1) After collecting fully mature Guizhou Camellia chrysantha fruits, the seeds were taken out and the surface was disinfected by soaking in 75% alcohol for 30 seconds, then immersed in an endophytic fungicide for 10 minutes, taken out and allowed to stand for 10 minutes, then immersed in a 1wt% calcium chloride solution for 5 minutes, taken out and allowed to stand at room temperature for 1 hour, and then buried in sand for sand storage. The temperature of the sand storage process was 10-15°C, and the humidity was maintained at 60-70% for the first 20 days of sand storage, and the humidity was maintained at 50-60% after the 20th day of sand storage. On the 20th day of sand storage, a citric acid solution with a pH of 5.5 was sprayed, and the spraying frequency was 7 days / time. Citric acid solution can be used instead of clean water to maintain humidity.

[0066] After the sand-stored seeds grow 1-2 cm radicles, remove the mud and seed coat on the surface of the seeds, wash them with running water, and soak the seeds in 75% alcohol for 30 seconds on a clean workbench. After taking them out, rinse them with sterile water for 3-4 times, and then soak them in 0.1% mercuric chloride solution for 8-10 minutes, shaking them once every 10-30 seconds, and then rinse them with sterile water for 3-4 times. Use filter paper to absorb the moisture to obtain externally sterilized explants.

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

[0068] Comparative Example 2:

[0069] The present invention is in contrast to Example 2, the only difference being that the present invention directly uses an endophytic fungicide made of sodium alginate and chitosan gel. The specific preparation and tissue culture process of the endophytic fungicide is as follows:

[0070] Preparation of Endophytic Fungicide:

[0071] 2wt% chitosan solution and 1wt% sodium alginate solution were mixed evenly in a volume ratio of 2:1 to obtain a viscous liquid, and then streptomycin and ε-polylysine were added in a mass ratio of 1:0.02:0.03 to the viscous liquid, ε-polylysine, and streptomycin, and the mixture was dispersed evenly after mixing to obtain a mixture, and then 3wt% calcium chloride solution was slowly added and stirred to obtain a gel solution, wherein 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 Guizhou Camellia chrysantha fruits, the seeds are taken out and the surface is soaked in 75% alcohol for 30 seconds for disinfection. After that, the seeds are ground with high temperature sterilized sand with a particle size of 0.3-0.5 mm for 5 minutes, taken out and soaked in gel solution for 10 minutes, taken out and left to stand for 10 minutes, then immersed in 1wt% calcium chloride solution for 5 minutes, taken out and left to stand at room temperature for 1 hour, and then buried in sand for sand storage. The temperature of the sand storage process is 10-15°C, and 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. On the 20th day of sand storage, a citric acid solution with a pH of 5.5 is sprayed, and the spraying frequency is 7 days / time. Citric acid solution can be used instead of clean water to maintain humidity.

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

[0075] Comparative Example 3:

[0076] The present invention is in contrast to Example 2, the only difference being that the present invention directly uses an endophytic fungicide prepared by mixing shellac, sodium alginate and chitosan. The specific preparation and tissue culture process of the endophytic fungicide is as follows:

[0077] (1) 2 wt% chitosan solution and 1 wt% sodium alginate solution are mixed uniformly in a volume ratio of 2:1 to obtain a viscous liquid, and then streptomycin, ε-polylysine and 3 wt% enzymatic lignin solution are added in a mass ratio of 1:0.02:0.03:0.5 between the viscous liquid and ε-polylysine, streptomycin and enzymatic lignin solution, and the mixture is dispersed uniformly after mixing to obtain a mixture;

[0078] (2) Dissolving shellac in a 70 wt% ethanol solution and centrifuging to remove the precipitate, heating in a water bath to evaporate the ethanol and then adding it to the mixture, stirring at 1000 rpm for 8 min, adding a 3 wt% calcium chloride solution, and stirring evenly to obtain an endophyte fungicide, wherein 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] The present invention is in contrast to Example 2, the only difference being that no enzymatic lignin is added during the preparation of the endophytic fungicide of the present invention. The specific preparation process is as follows:

[0082] (1) 2 wt% chitosan solution and 1 wt% sodium alginate solution are mixed in a volume ratio of 2:1 to obtain a viscous liquid, and then streptomycin and ε-polylysine are added in a mass ratio of 1:0.02:0.03 to the viscous liquid, and then dispersed evenly to obtain a mixture, and then 3 wt% calcium chloride solution is slowly added and stirred to obtain a gel block, and the volume ratio of the mixture to the calcium chloride solution is 1:1, and then frozen at -20°C and crushed to obtain a gel powder with a particle size of 0.2-0.5 mm, and then swollen with water for 30 minutes for standby use;

[0083] (2) Dissolve shellac in 70 wt% ethanol solution and centrifuge to remove precipitate, heat in a water bath to evaporate ethanol, add jojoba oil and glycerol, stir at 800 rpm for 5 min, then add 5 wt% Span-80 solution, swollen gel powder, and water, stir at 1000 rpm for 2 min, and shear at 24000 rpm for 2 min to obtain an endophyte fungicide. The mass ratio of shellac, Span-80, glycerol, jojoba oil, swollen gel powder, and water is 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] The present invention is in contrast to Example 2, the only difference being that the endophytic fungicide of the present invention is not prepared into an emulsion state during the preparation process. The specific preparation process is as follows:

[0087] (1) 2 wt% chitosan solution and 1 wt% sodium alginate solution are mixed uniformly in a volume ratio of 2:1 to obtain a viscous liquid, and then streptomycin, ε-polylysine and 3 wt% enzymatic lignin solution are added in a mass ratio of 1:0.02:0.03:0.5 to the viscous liquid, ε-polylysine, streptomycin and enzymatic lignin solution, and the mixture is dispersed uniformly after mixing to obtain a mixture, and then 3 wt% calcium chloride solution is slowly added and stirred to obtain a gel block, the volume ratio of the mixture to the calcium chloride solution is 1:1, and then the mixture is frozen at -20°C and crushed to obtain a gel powder with a particle size of 0.2-0.3 mm, and then water is added to swell for 30 minutes and then standby;

[0088] (2) Dissolve shellac in 70 wt% ethanol solution and centrifuge to remove precipitate, heat in a water bath to evaporate ethanol, add glycerol, stir at 800 rpm for 5 min, then add swollen gel powder and water, stir at 1000 rpm for 10 min to obtain an endophyte fungicide, wherein the mass ratio of shellac, glycerol, swollen gel powder and water is 1:0.2:0.5:3.5.

[0089] Comparative Example 6:

[0090] The present invention is in contrast to Example 2, the only difference being that after the endophytic fungicide is used in the tissue culture process of the present invention, the citric acid solution is not sprayed during the sand storage process, and the humidity is adjusted with clean water, and the rest of the operations remain unchanged.

[0091] Comparative Example 7:

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

[0093] (1) After collecting fully mature Guizhou Camellia chrysantha fruits, remove the seeds, soak them in 75% alcohol for 30 seconds to disinfect them, grind them with high-temperature sterilized sand with a particle size of 0.3-0.5 mm for 5 minutes, take them out and soak them in an endophytic fungicide for 10 minutes, let them stand for 1 hour, and then bury them in sand for sand storage. The temperature during the sand storage process is 10-15°C, and 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. On the 20th day of sand storage, start spraying a citric acid solution with a pH of 5.5, and the spraying frequency is 7 days / time. Citric acid solution can be used instead of clean water to maintain humidity.

[0094] After the sand-stored seeds grow 1-2 cm radicles, remove the mud and seed coat on the surface of the seeds, wash them with running water, and then soak the seeds in 75% alcohol for 30 seconds on a clean bench. After taking them out, rinse them with sterile water for 3-4 times, and then soak them in 0.1% mercuric chloride solution for 8-10 minutes, shaking them once every 10-30 seconds, and then rinse them with sterile water for 3-4 times. Use filter paper to absorb the water to obtain externally sterilized explants;

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

[0096] Comparative Example 8:

[0097] The present invention is in contrast to Example 2, the only difference being that the mass ratio of shellac to the swollen gel during the preparation of the endophytic fungicide of the present invention is changed. Specifically, step (2):

[0098] (2) Dissolve shellac in 70 wt% ethanol solution and centrifuge to remove precipitate, heat in a water bath to evaporate ethanol, add jojoba oil and glycerol, stir at 800 rpm for 5 min, then add 5 wt% Span-80 solution, swollen gel powder and water, stir at 1000 rpm for 2 min, and shear at 24000 rpm for 2 min to obtain an endophyte fungicide. The mass ratio of shellac, Span-80, glycerol, jojoba oil, swollen gel powder and water is 1:0.7:0.2:0.8:1:3.5.

[0099] Comparative Example 9:

[0100] The present invention is in contrast to Example 2, the only difference being that the mass ratio of shellac to the swollen gel during the preparation of the endophytic fungicide of the present invention is changed. Specifically, step (2):

[0101] (2) Dissolve shellac in 70 wt% ethanol solution and centrifuge to remove precipitate, heat in a water bath to evaporate ethanol, add jojoba oil and glycerol, stir at 800 rpm for 5 min, then add 5 wt% Span-80 solution, swollen gel powder, and water, stir at 1000 rpm for 2 min, and shear at 24000 rpm for 2 min to obtain an endophyte fungicide. The mass ratio of shellac, Span-80, glycerol, jojoba oil, swollen gel powder, and water is 1:0.7:0.2:0.8:0.3:3.5.

[0102] experiment:

[0103] 1. Select Guizhou Camellia chrysantha seeds picked by Guizhou Academy of Forestry in mid-October for tissue culture experiment. The tissue culture methods of Example 1, Example 2, and Comparative Examples 1-9 were used for the experiment. Each group of experiments had 11 seeds, and each group was repeated 3 times. At the same time, the time when the radicle first grew to 1 cm after different sand storage (the surface of the seeds was covered with about 5 cm thick sand for sand storage) treatment in each group was recorded, and the explants disinfected externally were cultivated when the radicle was 1 cm high. The specific disinfection process is as follows:

[0104] After the seeds grow a 1 cm radicle, remove the mud and seed coat on the surface of the seeds, wash them with running water, and soak the seeds in 75% alcohol for 30 seconds on a clean bench. After taking them out, rinse them with sterile water three times, and then soak them in 0.1% mercuric chloride solution for 8 minutes, shaking them once every 30 seconds, and then rinse them with sterile water three times. Use filter paper to absorb the moisture to obtain externally sterilized explants.

[0105] 2. After the seed radicle germinated to 1cm after sand storage, one seed was randomly selected from each group for bacterial culture experiment, and three replicates were performed in each group. The seed was disinfected with 75% alcohol for 30s and then cleaned with sterile water. The embryo was cut off and ground into a homogenate with a mortar. 5mL of sterile water was added and oscillated for 10min. 1ml of the stock solution was gradiently diluted to 1000 times and 100μl of the homogenate was added to the culture medium to observe the number of colonies. Fungal culture medium: 4g potato powder, 20g glucose, 15g agar, 1L water, natural pH, cultured at 25-28℃ for 5 days; bacterial culture medium: 5g peptone, 3g beef extract, 5g NaCl, 15g agar, 1L water, pH 7.0, cultured at 28-30℃ for 3 days.

[0106] 3. The explants were cultured in primary culture, 10 were inoculated in each group, 3 replicates were made, the culture time was 30 days, and the contamination rate was calculated; the contaminated embryos were discarded, and sterile seedlings obtained by non-contaminated embryos were selected for subculture, and the sterile seedlings were cut into stem segments with 1 leaf bud for subculture, and the culture time was 50 days; to ensure that the culture medium had sufficient nutrients, the subculture medium was replaced on the 25th day, and the proliferation coefficient was calculated after the culture was completed; 20 were inoculated in each group for rooting culture, 3 replicates were made, the rooting culture time was 35 days, and the rooting rate was calculated. During the primary culture, subculture culture, and rooting culture, the culture temperature was kept at 25±3℃, the light intensity was 2000-2500Lux, and the light time was 16h / d. The composition of the primary culture medium and subculture culture medium: MS culture medium + 4mg / L 6-BA + 6mg / L brassinolide + 0.3mg / LNAA, and the composition of the rooting culture medium: 1 / 2MS culture medium + 6mg / LNAA.

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

[0108] Table 1

[0109]

[0110] The analysis results show that:

[0111] 1. Compared with Example 1 and Example 2, the method of Example 2 has a shorter germination time, a lower endophyte content and a lower contamination rate, and a higher proliferation coefficient than Example 1, indicating that the scheme of Example 1 can also be used for tissue culture of Guizhou Camellia chrysantha, and the endophyte fungicide of Example 2 effectively removes endophytes and reduces the contamination rate. For Guizhou Camellia chrysantha with a small population and precious seeds, the scheme of Example 2 is more suitable for the actual situation.

[0112] 2. Comparative Example 1 did not use mechanical micro-damage to treat seeds, resulting in a long seed germination time and a high embryo contamination rate. This may be due to the long sand storage time, high activity of endophytes or the entry of external bacteria, which infected the seeds and increased the contamination rate. During the proliferation process, endophytes continued to contaminate the seedlings, reducing the proliferation efficiency and rooting rate.

[0113] 3. Comparative Example 2 is an endophyte fungicide made directly from sodium alginate and chitosan gel. Comparative Example 3 is an endophyte fungicide made from a mixture of sodium alginate, chitosan gel and shellac. Although the number of days of sand storage is reduced, the removal effect of endophytes is poor. This is because the simple use of the above-mentioned gel wrapping leads to a faster release rate of the bactericidal components, and the endophytes are not completely killed. Infection still occurs, resulting in a high contamination rate. The carrying of endophytes affects the efficiency and rooting rate. After adding direct mixed shellac, the release rate will be slowed down, but the endophyte fungicide will still be unevenly distributed.

[0114] 4. Comparative Example 4 is free of lignin, and Comparative Example 5 is not made into an emulsion, both of which will affect the killing of endophytes. Lignin can promote the development of plant tissues and can also absorb fungicides. Because of its good permeability, it can penetrate into the intercellular space, promote the growth of embryos, and penetrate the fungicide into the seeds to eliminate endophytes. The emulsion can adhere the endophytic fungicide to the surface of the seeds, evenly wrap it, and it can be evenly broken and quickly degraded in the late stage of sand storage, preventing germination damage caused by uneven cracking.

[0115] 5. Comparative Example 6 does not spray citric acid, and Comparative Example 7 does not use calcium chloride secondary gel. Chitosan is dissolved under acidic conditions. Calcium alginate shrinks under acidic conditions to cause membrane rupture, and shellac gradually curls during water loss. Internal stress causes membrane rupture, which is beneficial to the extension and growth of the embryo. Calcium chloride, calcium alginate and chitosan secondary gel can make the seed surface gel more uniform, and the gel effect is better, preventing the endogenous fungicide from falling off during sand storage.

[0116] 6. Comparative Examples 8 and 9 are the changes in the mass ratio of shellac to gel powder. Shellac is stronger and the mixed gel of calcium alginate and chitosan is more absorbent. The seeds in the early stage of sand storage are in a dormant period, and the activity of endophytes is not high at this time. The gel continuously releases bactericidal substances to continuously target the inside of the seeds to kill endophytes. In the middle and late stages of sand storage, the gel has lost water and a gel film has formed on the surface to hinder the invasion of external bacteria. If the ratio is inappropriate, it will cause water loss too quickly or too slowly, affecting the 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 the present invention. Although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should be included in the scope of the claims of the present invention. The techniques, shapes, and structural parts not described in detail in the present invention are all known technologies.

Claims

1. A tissue culture method for Guizhou Camellia chrysantha, characterized in that: The tissue culture method is as follows: (1) After collecting fully mature Guizhou Camellia chrysantha fruits, the seeds were taken out and stored in sand. After the seeds grew 1-2 cm radicles, the sand and seed coat on the surface of the seeds were removed, and the seeds were washed with clean water and then soaked in 75% alcohol and 0.1% mercuric chloride solution in sequence. After washing with clean water, the explants were disinfected externally; (2) cutting off the excess endosperm of the explant, and inoculating the remaining part into the primary culture medium to grow seedlings to obtain the aseptic seedlings of Guizhou Camellia chrysantha; (3) Cut the obtained sterile seedlings into stem segments with 1-2 leaf buds, inoculate them into a subculture medium for subculture, transfer the subcultured seedlings into a rooting medium for rooting culture, and obtain Guizhou Camellia chrysantha seedlings after the rooting culture is completed.

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

3. The tissue culture method of Guizhou golden camellia according to claim 2, characterized in that: The compositions of the primary culture medium and the secondary culture medium are: MS culture medium + 2-8 mg / L 6-BA + 2-8 mg / L brassinolide + 0.1-0.5 mg / L NAA.

4. The tissue culture method of Guizhou golden camellia according to claim 3, characterized in that: The rooting culture medium consists of: 1 / 2MS culture medium + 4-6 mg / L NAA.

5. The tissue culture method of Guizhou golden camellia according to claim 4, characterized in that: The specific operation of external disinfection of explants is as follows: on an ultra-clean workbench, soak the seeds cleaned with clean water in 75% alcohol for 30 seconds, take them out and rinse them with sterile water for 3-4 times, then soak them in 0.1% mercuric chloride solution for 8-10 minutes, shake them once every 10-30 seconds, rinse them with sterile water for 3-4 times, and absorb the moisture with filter paper to obtain externally disinfected explants.

6. A tissue culture method for Guizhou golden camellia according to any one of claims 1 to 5, characterized in that: The explant is also internally sterilized, and the specific internal sterilization process is as follows: The surface of Guizhou Camellia chrysantha seeds was ground with sterilized 0.3-0.5 mm sand for 5-6 minutes, then taken out and immersed in an endophytic fungicide for 10-20 minutes, taken out and allowed to stand for 10-20 minutes, then immersed in 1-2 wt % calcium chloride solution for 5-10 minutes, taken out and allowed to stand at room temperature for 1-2 hours, then buried in sand for sand storage, and sprayed with citric acid solution with a pH of 5.5-6.5 on the 20th day of sand storage, with a spraying frequency of 5-7 days / time.

7. The tissue culture method of Guizhou golden camellia according to claim 6, characterized in that: The temperature during the sand storage process is 10-15° C., 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.

8. The tissue culture method of Guizhou golden camellia according to claim 7, characterized in that: The preparation method of the endophyte fungicide is as follows: (1) 2 wt% chitosan solution and 1 wt% sodium alginate solution are mixed in a volume ratio of 2:1 to obtain a viscous liquid, and then a mixture consisting of streptomycin, ε-polylysine and 3 wt% enzymatic lignin solution is added, and after being evenly dispersed, 3-5 wt% calcium chloride solution is slowly added to obtain a gel block, which is then frozen at -20°C and crushed to obtain a gel powder with a particle size of 0.2-0.5 mm, and then swollen with water for 30 minutes for standby use; (2) Dissolving shellac in a 70 wt% ethanol solution and centrifuging to remove the precipitate, then adding jojoba oil and glycerin, stirring at 800-1000 rpm for 5 min, then adding 5 wt% Span solution, swollen gel powder, and water, stirring at 800-1000 rpm for 1-2 min, and then shearing at 20000-24000 rpm for 1-2 min to obtain an endophyte bactericide.

9. The tissue culture method of Guizhou golden camellia according to claim 8, characterized in that: In the endophyte fungicide, the mass ratio of the viscous liquid to ε-polylysine, streptomycin, and 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, glycerin, jojoba oil, swollen gel powder, and water is 1: (0.7-0.8): (0.2-0.4): (0.8-1): (0.5-0.8): (3.5-4).

10. The tissue culture method of Guizhou golden camellia according to claim 9, characterized in that: During the tissue culture process, the culture temperature was maintained at 25±3° C., the light intensity was maintained at 2000-2500 Lux, and the light duration was maintained at 16 h / d.

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