Anthurium andraeanum tissue culture propagation expanding seedling method

Abstract

The invention provides a propagation expanding and seedling raising method for anthurium andraeanum tissue culture. The propagation expanding and seedling raising method comprises the following steps of explant selection and disinfection, explant cutting, callus induction, proliferator culture, adventitious bud differentiation, rooting and root strengthening, seedling hardening and transplanting. The compound disinfectant prepared from chlorobromoisocyanuric acid, permanent flower essential oil, Tween and distilled water in a specific proportion is used, chlorobromoisocyanuric acid and permanent flower essential oil generate a complementary effect through different antibacterial mechanisms, and the broad-spectrum property and the sterilizing effect of the compound disinfectant are improved. The sambucus williamsii extract is added into the rooting culture medium, coumarin and ferulic acid contained in the sambucus williamsii extract can be converted into lignin in anthurium andraeanum tissue culture seedlings, so that mechanical connection between cells is enhanced, and adventitious roots and calluses are connected more tightly. Besides, polysaccharides and derivatives thereof contained in the culture medium can promote synthesis and deposition of pectin in tissue culture seedlings, so that the adhesion between cells is increased, and adventitious roots and calluses are connected more tightly.

Description

Technical Field

[0001] The invention relates to the technical field of plant seedling cultivation, and in particular to a method for propagating and raising seedlings through tissue culture of anthurium andraeanum. Background Art

[0002] Anthurium, scientifically known as Anthurium, is a perennial herbaceous plant of the genus Anthurium in the family Araceae. It is native to the tropical rainforests of South America and is now widely cultivated around the world. Anthurium is favored for its unique spathe inflorescence, bright and gorgeous color and long flowering period. It is a popular cut flower and potted flower. Anthurium fruits take a long time to mature, the number is small and they are easy to lose vitality, so it is difficult to grow seedlings through seeds. And it has fewer tillers and a low reproduction rate, so it is difficult to expand production through division. Therefore, in terms of seedling cultivation, Anthurium mainly uses tissue culture to expand seedling cultivation. When raising seedlings, select suitable explants from healthy mother plants, inoculate them on the culture medium after disinfection, and culture them under suitable temperature, light and humidity conditions to induce the explants to produce callus tissue, and then differentiate adventitious buds and adventitious roots to form a complete plant. However, the adventitious roots formed by the callus of the existing anthurium tissue culture seedlings are prone to breakage at the connection between the adventitious roots and the callus, thereby limiting the nutrient absorption capacity of the tissue culture seedlings, thereby affecting their normal growth and development. Summary of the invention

[0003] In view of this, the present invention proposes a method for propagating and raising seedlings of anthurium andraeanum tissue culture to solve the above problems.

[0004] The technical solution of the present invention is achieved as follows: a method for expanding and breeding seedlings of anthurium andraeanum tissue culture, comprising the following steps:

[0005] S1. Selection and disinfection of explants: Fresh, healthy, and pest-free young leaves of anthurium were selected as explants, and the explants were rinsed with tap water for 20-30 minutes, then disinfected with ethanol, and then disinfected with a composite disinfectant. The composite disinfectant is composed of chlorobromoisocyanuric acid, helichrysum essential oil, Tween, and distilled water in a mass volume ratio of g / mL of (0.40-0.60):(0.15-0.25):(0.05-0.15):(90.0-100.0).

[0006] S2. Explant cutting: Cut the sterilized explant into 0.8-1.2cm 2 size.

[0007] S3. Callus induction: The explants cut above were inoculated into an induction medium under sterile conditions to induce callus. The induction medium composition was: MS + 1.0 mg / L 6-BA + 0.3 mg / L NAA + 30 g / L sucrose + 50 g / L banana puree + 80 ml / L potato juice.

[0008] S4. Proliferator cultivation: The callus tissue is inoculated into a proliferation medium under sterile conditions to proliferate proliferators. The proliferation medium is composed of: MS + 0.8 mg / L 6-BA + 0.3 mg / L NAA + 30 g / L sucrose + 50 g / L banana puree + 60 ml / L coconut juice.

[0009] S5. Differentiation of adventitious buds: The above-mentioned proliferators are inoculated into differentiation medium under sterile conditions to differentiate adventitious buds. The differentiation medium composition is: 1 / 2MS+0.4mg / L6-BA+0.2mg / LNAA+20g / L sucrose+30g / L agar+50ml / L carrot juice.

[0010] S6. Rooting and root strengthening: The above adventitious buds are inoculated into a rooting medium under sterile conditions to obtain tissue culture seedlings. The rooting medium composition is: 1 / 2MS+0.2mg / LNAA+0.1mg / LIAA+20g / L sucrose+40ml / L elderberry extract.

[0011] S7. Hardening and transplanting: Open the bottle cap of tissue culture seedlings, harden the seedlings at 23-27℃ for 4-6 days, then take out the tissue culture seedlings, wash off the culture medium residue attached to the roots of the tissue culture seedlings, and implant the tissue culture seedlings into the culture matrix and culture them in a seedling greenhouse with a sunshade net. The culture matrix is ​​composed of plant ash, vermiculite, coconut bran and moss in a mass ratio of (2-4):(4-6):(6-10):1-3).

[0012] Furthermore, in S1, 70-75wt% ethanol is first used for disinfection for 20-30s, and then a composite disinfectant is used for disinfection for 10-20min.

[0013] Furthermore, the helichrysum essential oil in the composite is prepared by the following method: fresh helichrysum petals are washed, dried and crushed, the crushed helichrysum petals are placed in a distiller, water is added at a solid-liquid ratio of 1:(3-5), distilled and extracted at 70-90° C. for 3-5 hours, the collected water vapor is condensed, centrifuged at 3000-5000 rpm for 10-20 minutes, and the upper layer of liquid is taken, which is the helichrysum essential oil.

[0014] Furthermore, the culture conditions of S3 in the induction medium are: temperature 23-27°C, humidity 70-80%, light intensity 1500-2000 lux, light time 10-12h / d, and culture for 35-45d.

[0015] Furthermore, the culture conditions of S4 inoculated into the proliferation medium are: temperature 24-28°C, humidity 75-85%, light intensity 1800-2200 lux, light time 12-14 h / d, and culture for 28-35 days.

[0016] Furthermore, the culture conditions of S5 in the differentiation medium are: temperature 22-25°C, humidity 65-75%, light intensity 2000-2500 lux, light time 12-14 h / d, and culture for 40-50 days.

[0017] Furthermore, the culture conditions of S6 inoculated into the rooting medium are: temperature 23-25°C, humidity 70-80%, light intensity 1500-2000 lux, light time 10-12h / d, and culture for 25-35d.

[0018] Furthermore, the elderberry extract in the rooting medium is prepared by the following method: fresh and ripe elderberry fruits are washed and crushed, 75-95 wt% is added thereto at a solid-liquid ratio of 1:(5-10), heated to 55-65°C and extracted for 2-4 hours. After the extraction is completed, the filtrate is filtered and the filtrate is vacuum concentrated to 10-20% of the original solution volume under a vacuum degree of 0.06-0.08 MPa.

[0019] Furthermore, the culture conditions of S7 implanted in the culture matrix and cultured in a seedling greenhouse with a sunshade net are: temperature 20-25°C, humidity 60-70%, shading 50-70%, and culture for 40-60 days.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. The present invention uses a composite disinfectant composed of chlorobromoisocyanuric acid, helichrysum essential oil, Tween, and distilled water in a specific ratio, wherein the chlorobromoisocyanuric acid releases hypochlorous acid and hypobromous acid in water, which can destroy the cell walls and cell membranes of bacteria, fungi, and viruses, causing the cell contents to leak out, thereby making the microorganisms inactive. Chlorobromoisocyanuric acid can also enter the interior of the explant through systemic conduction. After being absorbed by the explant, chlorobromoisocyanuric acid can be conducted to various parts of the explant through the conducting tissue. In the explant, the mother body after releasing hypobromous acid will form active ingredients such as triazinedione and s-triazine, which can interfere with the replication process of the virus, thereby inhibiting the spread and proliferation of the virus in the explant. Compounds such as α-curcumene, β-curcumene, and γ-curcumene contained in the helichrysum essential oil can penetrate the cell membrane of the microorganism, destroy its integrity, and cause the leakage of the internal substances of the cell, thereby killing the microorganism. Italone in essential oils can interfere with the metabolic process of microorganisms, such as inhibiting enzyme activity and blocking the absorption of nutrients, thus preventing microorganisms from growing and reproducing normally. Chlorobromoisocyanuric acid and helichrysum essential oil produce complementary effects through different antibacterial mechanisms, improving the broad-spectrum and bactericidal effect of the composite disinfectant. Tween can reduce the surface tension of the liquid, enhance the wetting and penetration ability of other disinfectant ingredients, make it easier for disinfectants to enter the microbial cells, and thus improve the disinfection effect.

[0022] 2. The present invention adds elderberry extract to the rooting medium. The coumarin and ferulic acid contained in the elderberry extract can be converted into lignin in the anthurium tissue culture seedling, thereby enhancing the mechanical connection between cells and making the connection between adventitious roots and callus more closely. And the polysaccharides and derivatives contained in the elderberry extract can be used as raw materials for pectin synthesis, promote the synthesis and deposition of pectin in the tissue culture seedling, increase the adhesion between cells, thereby enhancing the connection between cells, and making the connection between adventitious roots and callus more closely. In addition, the elderberry extract contains rich antioxidant substances, such as vitamin C, vitamin E, polyphenols, flavonoids, etc., which can remove free radicals, reduce the damage of oxidative stress to cells, thereby improving the microenvironment of the joint, improving its stress resistance, enhancing the tolerance of cells to adversity, and reducing the risk of fracture. In addition, the elderberry extract also contains some minerals and trace elements that are beneficial to plant growth, such as potassium, calcium, magnesium, iron, zinc, etc. These elements are necessary for the normal physiological functions of plant cells, and they may indirectly improve the strength and stress resistance of joints by improving the nutritional status of cells.

[0023] 3. The present invention adds banana puree and potato juice to the induction medium. Banana puree contains rich nutrients such as vitamins, minerals and carbohydrates, which provide the necessary nutritional source for the induction of anthurium callus. Banana puree contains a variety of plant hormones, such as auxin, gibberellin, etc. These hormones can promote the division and proliferation of anthurium cells, thereby accelerating the formation of callus. Potato juice contains promoting factors, which can stimulate the dedifferentiation of anthurium cells to form callus. By adding banana puree and coconut juice to the proliferation medium, the banana puree continues to provide nutrition and promote cell division and proliferation. The coconut juice contains nutrients such as a variety of amino acids, vitamins and minerals, which provide the necessary nutritional source for the proliferation of anthurium cells. In addition, the kinetin contained in coconut juice can promote the division of anthurium cells and has an important influence on the development process of anthurium proliferators. By adding carrot juice to the differentiation medium, the β-carotene and other carotenoids contained therein can be converted into vitamin A in the anthurium body. Vitamin A has a variety of physiological functions in plants, including promoting cell differentiation, maintaining epithelial cell integrity, etc. During the process of anthurium tissue culture, vitamin A can stimulate anthurium cells to differentiate into adventitious buds. Carrot juice is also rich in vitamin C and vitamin E, which can remove free radicals in the body and reduce cell damage. At the same time, they can also promote cell metabolism and growth, and provide necessary nutritional support for cell differentiation. DETAILED DESCRIPTION

[0024] In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

[0025] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

[0026] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources.

[0027] Example 1

[0028] A method for propagating and raising seedlings of anthurium andraeanum tissue culture, comprising the following steps:

[0029] S1. Selection and disinfection of explants: Select fresh, healthy, disease-free and insect-free Anthurium tender leaves as explants, rinse the explants with tap water for 20 minutes, then disinfect with 70wt% ethanol for 30 seconds, and then disinfect with a composite disinfectant for 10 minutes. The composite disinfectant consists of chlorobromoisocyanuric acid, helichrysum essential oil, Tween, and distilled water with a mass volume ratio of g / mL of 0.40:0.15:0.05:90.0. The helichrysum essential oil in the composite is prepared by the following method: take fresh helichrysum petals, wash, dry and crush them, put the crushed helichrysum petals into a distiller, add water at a solid-liquid ratio of 1:3, distill and extract at 70°C for 5 hours, condense the collected water vapor, centrifuge at 3000rpm for 20 minutes, and take the upper liquid, which is the helichrysum essential oil.

[0030] S2. Explant cutting: Cut the sterilized explant into 0.8 cm 2 size.

[0031] S3. Callus induction: The explants cut above were inoculated into the induction medium under sterile conditions, and cultured for 45 days at a temperature of 23°C, a humidity of 70%, a light intensity of 1500 lux, and a light duration of 10 h / d to induce callus. The induction medium composition was: MS + 1.0 mg / L 6-BA + 0.3 mg / L NAA + 30 g / L sucrose + 50 g / L banana puree + 80 ml / L potato juice.

[0032] S4. Proliferation body cultivation: The above callus tissue was inoculated into a proliferation medium under sterile conditions, and cultured for 35 days at a temperature of 24°C, a humidity of 75%, a light intensity of 1800 lux, and a light duration of 12 h / d to proliferate the proliferators. The proliferation medium composition is: MS + 0.8 mg / L 6-BA + 0.3 mg / L NAA + 30 g / L sucrose + 50 g / L banana puree + 60 ml / L coconut juice.

[0033] S5. Differentiation of adventitious buds: The above-mentioned proliferators were inoculated into differentiation medium under sterile conditions and cultured for 50 days at a temperature of 22°C, a humidity of 6%, a light intensity of 2000 lux, and a light duration of 12 h / d to differentiate adventitious buds. The differentiation medium composition is: 1 / 2MS + 0.4mg / L6-BA + 0.2mg / LNAA + 20g / L sucrose + 30g / L agar + 50ml / L carrot juice.

[0034] S6, rooting and root strengthening: Inoculate the above adventitious buds into the rooting medium under sterile conditions, culture for 35 days at a temperature of 23°C, a humidity of 70%, a light intensity of 1500 lux, and a light time of 102h / d to obtain tissue culture seedlings. The rooting medium composition is: 1 / 2MS + 0.2mg / LNAA + 0.1mg / LIAA + 20g / L sucrose + 40ml / L elderberry extract. The elderberry extract is prepared by the following method: wash and crush fresh and mature elderberry fruits, add 75wt% to it at a solid-liquid ratio of 1:5, heat to 55°C and extract for 4h. After the extraction is completed, filter and take the filtrate, and vacuum concentrate the filtrate to 10% of the original solution volume under a vacuum degree of 0.06MPa.

[0035] S7, seedling training and transplantation: open the bottle cap of tissue culture seedlings, and harden the seedlings at 23°C for 6 days, then take out the tissue culture seedlings, wash off the culture medium residue attached to the roots of the tissue culture seedlings, and implant the tissue culture seedlings into the culture matrix and culture them in a seedling greenhouse with a sunshade net for 60 days at a temperature of 20°C, a humidity of 60%, and a light shielding of 50%. The culture matrix is ​​composed of plant ash, vermiculite, coconut bran, and moss in a mass ratio of 2:4:6:1.

[0036] Example 2

[0037] A method for propagating and raising seedlings of anthurium andraeanum tissue culture, comprising the following steps:

[0038] S1. Selection and disinfection of explants: Select fresh, healthy, disease-free and insect-free Anthurium tender leaves as explants, rinse the explants with tap water for 25 minutes, then disinfect with 75wt% ethanol for 25 seconds, and then disinfect with a composite disinfectant for 15 minutes. The composite disinfectant consists of chlorobromoisocyanuric acid, helichrysum essential oil, Tween, and distilled water with a mass volume ratio of g / mL of 0.50:0.20:0.10:95.0. The helichrysum essential oil in the composite is prepared by the following method: take fresh helichrysum petals, wash, dry and crush them, put the crushed helichrysum petals into a distiller, add water at a solid-liquid ratio of 1:4, distill and extract at 80°C for 4 hours, condense the collected water vapor, centrifuge at 4000rpm for 15 minutes, and take the upper liquid, which is the helichrysum essential oil.

[0039] S2. Explant cutting: Cut the sterilized explant into 1.0 cm2 size.

[0040] S3. Callus induction: The explants cut above were inoculated into the induction medium under sterile conditions, and cultured for 40 days at a temperature of 25°C, a humidity of 75%, a light intensity of 1750 lux, and a light duration of 11 h / d to induce callus. The induction medium composition was: MS + 1.0 mg / L 6-BA + 0.3 mg / L NAA + 30 g / L sucrose + 50 g / L banana puree + 80 ml / L potato juice.

[0041] S4. Proliferator cultivation: The callus tissue was inoculated into a proliferation medium under sterile conditions, and cultured for 32 days at a temperature of 26°C, a humidity of 80%, a light intensity of 2000 lux, and a light duration of 13 h / d to proliferate proliferators. The proliferation medium composition was: MS + 0.8 mg / L 6-BA + 0.3 mg / L NAA + 30 g / L sucrose + 50 g / L banana puree + 60 ml / L coconut juice.

[0042] S5. Differentiation of adventitious buds: The above-mentioned proliferators were inoculated into differentiation medium under sterile conditions and cultured for 45 days at a temperature of 23°C, a humidity of 70%, a light intensity of 2250 lux, and a light duration of 13 h / d to differentiate adventitious buds. The differentiation medium composition is: 1 / 2MS + 0.4 mg / L 6-BA + 0.2 mg / L NAA + 20 g / L sucrose + 30 g / L agar + 50 ml / L carrot juice.

[0043] S6, rooting and root strengthening: Inoculate the above adventitious buds into the rooting medium under sterile conditions, culture for 30 days at a temperature of 24°C, a humidity of 75%, a light intensity of 1750lux, and a light time of 11h / d to obtain tissue culture seedlings. The rooting medium composition is: 1 / 2MS + 0.2mg / LNAA + 0.1mg / LIAA + 20g / L sucrose + 40ml / L elderberry extract. The elderberry extract is prepared by the following method: wash and crush fresh and mature elderberry fruits, add 85wt% to it at a solid-liquid ratio of 1:7.5, heat to 60°C and extract for 3h. After the extraction is completed, filter the filtrate and vacuum concentrate the filtrate to 15% of the original solution volume under a vacuum degree of 0.07MPa.

[0044] S7, seedling training and transplantation: open the bottle cap of tissue culture seedlings, harden the seedlings at 25℃ for 5 days, then take out the tissue culture seedlings, wash off the culture medium residue attached to the roots of the tissue culture seedlings, and implant the tissue culture seedlings into the culture matrix and culture them in a seedling greenhouse with a sunshade net for 50 days at a temperature of 23℃, a humidity of 65%, and a light shielding of 60%. The culture matrix is ​​composed of plant ash, vermiculite, coconut bran, and moss in a mass ratio of 3:5:8:2.

[0045] Example 3

[0046] A method for propagating and raising seedlings of anthurium andraeanum tissue culture, comprising the following steps:

[0047] S1. Selection and disinfection of explants: Select fresh, healthy, disease-free and insect-free Anthurium tender leaves as explants, rinse the explants with tap water for 30 minutes, then disinfect with 75wt% ethanol for 20 seconds, and then disinfect with a composite disinfectant for 20 minutes. The composite disinfectant consists of chlorobromoisocyanuric acid, helichrysum essential oil, Tween, and distilled water with a mass volume ratio of g / mL of 0.60:0.25:0.15:100.0. The helichrysum essential oil in the composite is prepared by the following method: take fresh helichrysum petals, wash, dry and crush them, put the crushed helichrysum petals into a distiller, add water at a solid-liquid ratio of 1:5, distill and extract at 90°C for 3 hours, condense the collected water vapor, centrifuge at 5000rpm for 10 minutes, and take the upper liquid, which is the helichrysum essential oil.

[0048] S2. Explant cutting: Cut the sterilized explant into 1.2 cm 2 size.

[0049] S3. Callus induction: The explants cut above were inoculated into induction medium under sterile conditions, and cultured for 35 days at a temperature of 27°C, a humidity of 80%, a light intensity of 12000 lux, and a light duration of 12 h / d to induce callus. The induction medium composition was: MS + 1.0 mg / L 6-BA + 0.3 mg / L NAA + 30 g / L sucrose + 50 g / L banana puree + 80 ml / L potato juice.

[0050] S4. Proliferation body cultivation: The above callus tissue was inoculated into a proliferation medium under sterile conditions, and cultured for 28 days at a temperature of 28°C, a humidity of 85%, a light intensity of 2200 lux, and a light duration of 14 h / d to proliferate the proliferators. The proliferation medium composition was: MS + 0.8 mg / L 6-BA + 0.3 mg / L NAA + 30 g / L sucrose + 50 g / L banana puree + 60 ml / L coconut juice.

[0051] S5. Differentiation of adventitious buds: The above-mentioned proliferators were inoculated into differentiation medium under sterile conditions and cultured for 40 days at a temperature of 25°C, a humidity of 75%, a light intensity of 2500 lux, and a light duration of 14 h / d to differentiate adventitious buds. The differentiation medium composition is: 1 / 2MS + 0.4mg / L6-BA + 0.2mg / LNAA + 20g / L sucrose + 30g / L agar + 50ml / L carrot juice.

[0052] S6, rooting and root strengthening: Inoculate the above adventitious buds into the rooting medium under sterile conditions, culture for 25 days at a temperature of 25°C, a humidity of 80%, a light intensity of 2000 lux, and a light time of 12h / d to obtain tissue culture seedlings. The rooting medium composition is: 1 / 2MS + 0.2mg / LNAA + 0.1mg / LIAA + 20g / L sucrose + 40ml / L elderberry extract. The elderberry extract is prepared by the following method: wash and crush fresh and mature elderberry fruits, add 95wt% to it at a solid-liquid ratio of 1:10, heat to 65°C and extract for 2h. After the extraction is completed, filter and take the filtrate, and vacuum concentrate the filtrate to 20% of the original solution volume under a vacuum degree of 0.08MPa.

[0053] S7, seedling training and transplantation: open the bottle cap of tissue culture seedlings, harden the seedlings at 27℃ for 4 days, then take out the tissue culture seedlings, wash off the culture medium residue attached to the roots of the tissue culture seedlings, and implant the tissue culture seedlings into the culture matrix and culture them in a seedling greenhouse with a sunshade net for 40 days at a temperature of 25℃, a humidity of 70%, and a light shielding of 70%. The culture matrix is ​​composed of plant ash, vermiculite, coconut bran, and moss in a mass ratio of 4:6:10:3.

[0054] Comparative Example 1

[0055] The difference between this comparative example and Example 2 is that, in the step of selecting and sterilizing the explants in S1, ethanol is used for sterilization and then 0.1 wt% mercuric chloride solution is used for sterilization for 12 minutes. After the sterilization, sterile water is used for washing 3 times.

[0056] Comparative Example 2

[0057] Compared with Example 2, the difference between this comparative example and Example 2 is that the composite disinfectant in the explant selection and disinfection step S1 is composed of helichrysum essential oil, Tween and distilled water in a mass volume ratio of g / mL of (0.15-0.25):(0.50-0.15):(90.0-100.0).

[0058] Comparative Example 3

[0059] Compared with Example 2, the difference between this comparative example and Example 2 is that the composite disinfectant in the explant selection and disinfection step S1 is composed of chlorobromoisocyanuric acid, Tween and distilled water in a mass volume ratio of g / mL of (0.40-0.60):(0.50-0.15):(90.0-100.0).

[0060] Comparative Example 4

[0061] The difference between this comparative example and Example 2 is that the composition of the rooting medium in the S6 rooting and root strengthening step is: 1 / 2MS+0.2mg / LNAA+0.1mg / LIAA+20g / L sucrose.

[0062] 1. Disinfection effect test

[0063] 100 explants were taken respectively and seedlings were grown according to the seedling growing methods of Examples 1-3 and Comparative Examples 1-3. When the seedlings were grown to the end of the S3 induction medium culture, the contamination rate and survival rate were calculated. The results are shown in Table 1.

[0064] Table 1

[0065] Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 Comparative Example 3 Pollution rate % 0 0 0 11 3 4 Survival rate% 98 100 99 78 89 84

[0066] As can be seen from Table 1, Examples 1-3 were not contaminated, and the survival rates were also higher than those of Comparative Examples 1-3. This shows that the composite disinfectant of the present invention can effectively kill viruses and is beneficial to the growth and development of explants.

[0067] Compared with the mercuric chloride solution used in Comparative Example 1, although the mercuric chloride solution can effectively disinfect, its residual toxicity has a long-term adverse effect on explant cells. Even after multiple rinses, the mercuric chloride solution is difficult to completely remove, and its residues have a toxic effect on red anthurium cells, which leads to contamination of the culture medium and affects the normal physiological functions of the cells, thereby causing a decrease in survival rate. In the composite disinfectant of the present invention, chlorobromoisocyanuric acid releases hypochlorous acid and hypobromous acid in water, which can destroy the cell walls and cell membranes of bacteria, fungi and viruses, causing the cell contents to leak out, thereby making the microorganisms inactive. Chlorobromoisocyanuric acid can also enter the inside of the explant through systemic conduction. After chlorobromoisocyanuric acid is absorbed by the explant, it can be conducted to various parts of the explant through the conducting tissue. In the explant body, the mother body after releasing hypobromous acid will form active ingredients such as triazinedione and s-triazine, which can interfere with the replication process of the virus, thereby inhibiting the spread and proliferation of the virus in the explant body. In addition, chlorobromoisocyanuric acid may also stimulate the explants to produce more growth-promoting hormones such as auxin and cytokinin by regulating the hormone balance in the explants, while inhibiting the production of growth-inhibiting hormones such as ethylene, thereby promoting the induction of callus tissue in the explants. Compounds such as α-curcumene, β-curcumene, and γ-curcumene in Helichrysum essential oil can penetrate the cell membrane of microorganisms, destroy their integrity, and cause the leakage of internal cell substances, thereby killing the microorganisms. Italone in essential oils can interfere with the metabolic process of microorganisms, such as inhibiting enzyme activity and blocking the absorption of nutrients, so that microorganisms cannot grow and reproduce normally. In addition, neryl acetate in Helichrysum essential oil has the effect of promoting cell regeneration and division, which can stimulate the activity of explant cells, promote their division and proliferation, and thus accelerate the growth and development of explants. At the same time, Helichrysum essential oil can also reduce the oxidative damage suffered by explants during the culture process, thereby maintaining their vitality and health. Chlorobromoisocyanuric acid and Helichrysum essential oil produce complementary effects through different antibacterial mechanisms, improving the broad-spectrum and bactericidal effects of composite disinfectants. Tween can reduce the surface tension of the liquid, enhance the wetting and penetration ability of other disinfectant ingredients, make it easier for disinfectants to enter the interior of microbial cells, and thus improve the disinfection effect.

[0068] It can also be seen from the comparison between Examples 1-3 and Comparative Examples 2-3 that chlorobromoisocyanuric acid and helichrysum essential oil can produce a better antibacterial and disinfecting effect when used in combination.

[0069] 2. Root connection strength test

[0070] Twenty explants were taken respectively for seedling cultivation according to the methods of Examples 1-3 and Comparative Example 4. When the seedling cultivation reached the end of S6 rooting and root strengthening culture, 10 tissue culture seedlings were randomly selected from each group for tensile testing. The tensile force required for breaking the connection between the adventitious roots and the callus tissue of the tissue culture seedlings was recorded, and the average tensile force value of the connection of each group was calculated. The results are shown in Table 2.

[0071] Table 2

[0072] Example 1 Example 2 Example 3 Comparative Example 4 Average tension / N 25.0 25.3 24.8 13.7

[0073] It can be seen from Table 2 that the strength of the connection between the adventitious root and the callus in Examples 1-3 of the present invention is higher than that in Comparative Example 4. This is because, by adding elderberry extract to the rooting medium, the coumarin and ferulic acid contained in the elderberry extract can be converted into lignin in the anthurium tissue culture seedling, thereby enhancing the mechanical connection between cells and making the connection between the adventitious root and the callus more tightly. And the polysaccharides and derivatives contained in the elderberry extract can be used as raw materials for pectin synthesis, promote the synthesis and deposition of pectin in the tissue culture seedling, increase the adhesion between cells, thereby enhancing the connection between cells, and making the connection between the adventitious root and the callus more tightly. In addition, the elderberry extract contains rich antioxidant substances, such as vitamin C, vitamin E, polyphenols, flavonoids, etc., which can remove free radicals and reduce the damage of oxidative stress to cells, thereby improving the microenvironment of the connection, improving its stress resistance, enhancing the tolerance of cells to adversity, and reducing the risk of fracture. In addition, the elderberry extract also contains some minerals and trace elements that are beneficial to plant growth, such as potassium, calcium, magnesium, iron, zinc, etc. These elements are necessary for the normal physiological functions of plant cells, and they may indirectly improve the strength and stress resistance of joints by improving the nutritional status of cells.

[0074] In summary, the various bioactive ingredients in elderberry extract work together at the junction of adventitious roots and callus tissue, significantly enhancing the strength of the junction and making it less likely to break through multiple mechanisms such as strengthening intercellular connections, improving the microenvironment, and enhancing stress resistance.

[0075] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for propagating and raising seedlings of anthurium andraeanum tissue culture, characterized in that: The following steps are involved: S1. Selection and disinfection of explants: fresh, healthy, disease-free and insect-free young leaves of anthurium were selected as explants, and the explants were rinsed with tap water for 20-30 min, then disinfected with ethanol, and then disinfected with a composite disinfectant; the composite disinfectant was composed of chlorobromoisocyanuric acid, helichrysum essential oil, Tween, and distilled water in a mass volume ratio of g / mL of (0.40-0.60):(0.15-0.25):(0.05-0.15):(90.0-100.0); S2. Explant cutting: Cut the sterilized explant into 0.8-1.2cm 2 size; S3. Callus induction: The explants cut above are inoculated into an induction medium under sterile conditions to induce callus; the induction medium is composed of: MS + 1.0 mg / L 6-BA + 0.3 mg / L NAA + 30 g / L sucrose + 50 g / L banana puree + 80 ml / L potato juice; S4, proliferation body cultivation: inoculate the above callus tissue into a proliferation medium under sterile conditions to culture and proliferate the proliferation body; the proliferation medium composition is: MS + 0.8 mg / L 6-BA + 0.3 mg / L NAA + 30 g / L sucrose + 50 g / L banana puree + 60 ml / L coconut juice; S5. Differentiation of adventitious buds: Inoculate the above-mentioned proliferators into a differentiation medium under sterile conditions to differentiate adventitious buds; the differentiation medium is composed of: 1 / 2MS + 0.4mg / L6-BA + 0.2mg / LNAA + 20g / L sucrose + 30g / L agar + 50ml / L carrot juice; S6. Rooting and root strengthening: inoculate the above adventitious buds into a rooting medium under sterile conditions to obtain tissue culture seedlings; the rooting medium is composed of: 1 / 2MS + 0.2mg / L NAA + 0.1mg / LIAA + 20g / L sucrose + 40ml / L elderberry extract; S7, hardening and transplanting seedlings: open the bottle cap of tissue culture seedlings, harden the seedlings at 23-27°C for 4-6 days, then take out the tissue culture seedlings, wash away the culture medium residue attached to the roots of the tissue culture seedlings, and implant the tissue culture seedlings into the culture matrix, and culture them in a seedling greenhouse with a sunshade net; the culture matrix is ​​composed of wood ash, vermiculite, coconut bran and moss in a mass ratio of (2-4):(4-6):(6-10):1-3).

2. The method for expanding and raising seedlings of anthurium andraeanum tissue culture according to claim 1, characterized in that: In the S1, 70-75wt% ethanol is first used for disinfection for 20-30s, and then a composite disinfectant is used for disinfection for 10-20min.

3. A method for propagating and raising seedlings by tissue culture of anthurium andraeanum as claimed in claim 2, characterized in that: The helichrysum essential oil in the composite is prepared by the following method: fresh helichrysum petals are washed, dried and crushed, the crushed helichrysum petals are put into a distiller, water is added at a solid-liquid ratio of 1:(3-5), distilled and extracted at 70-90° C. for 3-5 hours, the collected water vapor is condensed, centrifuged at 3000-5000 rpm for 10-20 minutes, and the upper layer of liquid is taken, which is the helichrysum essential oil.

4. The method for expanding and raising seedlings of anthurium andraeanum tissue culture according to claim 1, characterized in that: The culture conditions of the S3 induction culture medium are: temperature 23-27° C., humidity 70-80%, light intensity 1500-2000 lux, light time 10-12 h / d, and culture for 35-45 days.

5. The method for expanding and raising seedlings of anthurium andraeanum tissue culture according to claim 1, characterized in that: The culture conditions of the S4 inoculated into the proliferation culture medium are: temperature 24-28° C., humidity 75-85%, light intensity 1800-2200 lux, light time 12-14 h / d, and culture for 28-35 days.

6. The method for expanding and raising seedlings of anthurium andraeanum tissue culture according to claim 1, characterized in that: The culture conditions of the S5 inoculated differentiation medium are: temperature 22-25° C., humidity 65-75%, light intensity 2000-2500 lux, light duration 12-14 h / d, and culture for 40-50 days.

7. The method for expanding and raising seedlings of anthurium andraeanum tissue culture according to claim 1, characterized in that: The culture conditions of the S6 inoculated into the rooting medium are: temperature 23-25° C., humidity 70%-80%, light intensity 1500-2000 lux, light duration 10-12 h / d, and culture for 25-35 days.

8. The method for expanding and raising seedlings of anthurium andraeanum tissue culture according to claim 7, characterized in that: The elderberry extract in the rooting medium is prepared by the following method: fresh and ripe elderberry fruits are washed and crushed, 75-95wt% of elderberry is added thereto at a solid-liquid ratio of 1:(5-10), heated to 55-65°C and extracted for 2-4h, after the extraction is completed, the filtrate is filtered and the filtrate is vacuum concentrated to 10-20% of the volume of the original solution under a vacuum degree of 0.06-0.08MPa.

9. The method for expanding and raising seedlings of anthurium andraeanum tissue culture according to claim 1, characterized in that: The culture conditions of the S7 implanted in the culture matrix and cultured in a seedling greenhouse with a sunshade net are: temperature 20-25° C., humidity 60-70%, shading 50-70%, and culture for 40-60 days.