A method for propagating and cultivating seedlings of ananas comosus

Abstract

The application provides a propagation method for tissue culture of anthurium andraeanum, which comprises the following steps: external explant selection and disinfection, external explant cutting, callus induction, proliferation body cultivation, differentiation of adventitious buds, rooting and strong root training, seedling training and transplantation. The composite disinfectant is composed of chlorobromoisocyanuric acid, everlasting flower oil, Tween and distilled water in a specific ratio. Chlorobromoisocyanuric acid and everlasting flower oil produce complementary effects through different antibacterial mechanisms, thereby improving the broad-spectrum and bactericidal effect of the composite disinfectant. Sambucus nigra extract is added to the rooting culture medium, and the coumarin and ferulic acid contained in the sambucus nigra extract can be converted into lignin in the anthurium andraeanum tissue culture seedlings, thereby enhancing the mechanical connection between cells and making the connection between the adventitious roots and the callus more compact. In addition, the polysaccharides and their derivatives contained in the sambucus nigra extract can promote the synthesis and deposition of pectin in the tissue culture seedlings, thereby increasing the adhesion between cells and making the connection between the adventitious roots and the callus more compact.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant seedling culture, in particular to a method for propagating and cultivating seedlings of anthurium andraeanum. BACKGROUND

[0002] Anthurium andraeanum, also known as flower candle, is a perennial herb of araceae flower candle genus, originally from the tropical rainforest area of South America, and has been widely cultivated all over the world. Anthurium andraeanum is favored for its unique spadix, bright and beautiful flower color and long flowering period, and is a popular cut flower and potted flower. Anthurium andraeanum is difficult to be cultivated by seeds due to long maturation time, small quantity and easy loss of vitality. In addition, it has less tillering and low propagation rate, so it is also difficult to expand production by division propagation. Therefore, in the aspect of seedling culture, anthurium andraeanum is mainly propagated and cultivated by tissue culture. In the process of seedling culture, suitable explants are selected from healthy mother plants, and then inoculated on culture medium after disinfection, and cultured under suitable temperature, light and humidity conditions to induce callus and then differentiate into adventitious buds and roots, so as to form complete plants. However, the adventitious roots formed by callus of the existing anthurium andraeanum tissue culture seedlings are prone to breakage at the connection between the adventitious roots and the callus, which limits the nutrient absorption capacity of the tissue culture seedlings and affects their normal growth and development. SUMMARY

[0003] In view of this, the present application provides a method for propagating and cultivating seedlings of anthurium andraeanum to solve the above problems.

[0004] The technical scheme of the present application is realized as follows: a method for propagating and cultivating seedlings of anthurium andraeanum, comprising the following steps:

[0005] S1, explant selection and disinfection: selecting fresh and healthy anthurium andraeanum leaves without pests as explants, washing the explants with tap water for 20-30 min, then disinfecting them with ethanol, and then disinfecting them with a composite disinfectant. The composite disinfectant is composed of chlorobromoisocyanuric acid, permanent flower oil, tween and distilled water with 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: cutting the disinfected explants into 0.8-1.2 cm 2 in size.

[0007] S3, callus induction: culturing the cut explants in the induction medium under sterile conditions to induce callus. The induction medium is composed of MS+1.0mg / L 6-BA+0.3mg / L NAA+30g / L sucrose+50g / L banana paste+80ml / L potato juice.

[0008] S4, proliferation culture: the above-mentioned callus is inoculated into the proliferation culture medium under sterile conditions to proliferate the proliferation body. The proliferation culture medium is composed of MS+0.8 mg / L 6-BA+0.3 mg / L NAA+30 g / L sucrose+50 g / L banana paste+60 ml / L coconut juice.

[0009] S5, differentiation of adventitious buds: the above-mentioned proliferation body is inoculated into the differentiation culture medium under sterile conditions to differentiate the adventitious buds. The differentiation culture medium is composed of 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.

[0010] S6, rooting and strong root culture: the above-mentioned adventitious buds are inoculated into the rooting culture medium under sterile conditions to obtain the tissue culture seedlings. The rooting culture medium is composed of 1 / 2MS+0.2 mg / L NAA+0.1 mg / L IAA+20 g / L sucrose+40 ml / L elderberry extract.

[0011] S7, seedling training and transplantation: the bottle cap of the tissue culture seedlings is opened, and the seedlings are trained for 4-6 days at 23-27°C, then the tissue culture seedlings are taken out, the culture medium residues attached to the roots of the seedlings are washed away, and the seedlings are planted into the culture medium in the seedling raising greenhouse with a sunshade net. The culture medium is composed of grass ash, vermiculite, coconut husk and moss with a mass ratio of (2-4):(4-6):(6-10):1-3.

[0012] Further, in S1, 70-75 wt% ethanol is used for sterilization for 20-30 s, and then the composite disinfectant is used for sterilization for 10-20 min.

[0013] Further, the permanent flower essential oil in the composite agent is prepared by the following method: fresh permanent flower petals are washed, dried, and crushed, the crushed permanent flower petals are put into a distiller, water is added at a solid-liquid ratio of 1:(3-5), and distilled at 70-90°C for 3-5 h, the collected water vapor is centrifuged at 3000-5000 rpm for 10-20 min, and the upper liquid is taken as the permanent flower essential oil.

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

[0015] Further, the inoculation and culture conditions of S4 in the proliferation culture medium are as follows: temperature 24-28°C, humidity 75-85%, light intensity 1800-2200 lux, light time 12-14 h / d, and culture time 28-35 d.

[0016] Further, the S5 access differentiation culture medium culture conditions are: temperature 22-25 DEG C, humidity 65-75%, light intensity 2000-2500 lux, light time 12-14h / d, culture 40-50d.

[0017] Further, the S6 access rooting culture medium culture conditions are: temperature 23-25 DEG C, humidity 70-80%, light intensity 1500-2000 lux, light time 10-12h / d, culture 25-35d.

[0018] Further, the bone-set wood extract in the rooting culture medium is prepared by the following method: fresh mature bone-set wood fruits are washed and crushed, 75-95wt% is added to the crushed fruits at a ratio of 1:(5-10), heated to 55-65 DEG C and extracted for 2-4h. After extraction, the filtrate is obtained by filtration, and the filtrate is vacuum concentrated to 10-20% of the original liquid volume under a vacuum degree of 0.06-0.08MPa.

[0019] Further, the S7 implantation culture medium culture conditions are: temperature 20-25 DEG C, humidity 60-70%, light shading 50-70%, culture 40-60d.

[0020] Compared with the prior art, the beneficial effects of the present application are:

[0021] 1、The present application uses a composite disinfectant composed of chlorobromoisocyanuric acid, permanent flower essential oil, Tween, and distilled water in a specific ratio. Chlorobromoisocyanuric acid releases hypochlorous acid and hypobromous acid in water, which can destroy the cell walls and membranes of bacteria, fungi, and viruses, causing the contents of the cells to leak out, thereby rendering the microorganisms inactive. Chlorobromoisocyanuric acid can also enter the explant through internal conduction. After being absorbed by the explant, chlorobromoisocyanuric acid can be conducted to various parts of the explant through the conducting tissues. In the explant, the mother substance that releases hypobromous acid forms active ingredients such as triazinedione and s-triazine, which can interfere with the replication process of viruses, thereby inhibiting the spread and proliferation of viruses in the explant. The compounds such as alpha-curcumene, beta-curcumene, and gamma-curcumene contained in the permanent flower essential oil can penetrate the cell membranes of microorganisms, destroy their integrity, and cause the contents of the cells to leak out, thereby killing the microorganisms. The italones in the essential oil can interfere with the metabolic processes of microorganisms, such as inhibiting the activity of enzymes and blocking the absorption of nutrients, thereby preventing the normal growth and reproduction of microorganisms. Chlorobromoisocyanuric acid and permanent flower essential oil produce complementary effects through different antibacterial mechanisms, improving the broad-spectrum and bactericidal effects of the composite disinfectant. Tween can reduce the surface tension of the liquid, enhance the wetting and penetration ability of other disinfecting components, and make the disinfectant more easily enter the interior of the microbial cells, thereby improving the disinfection effect.

[0022] 2、The application can enhance the mechanical connection between cells by adding Sambucus williamsii Hance extract to the rooting medium. The coumarin and ferulic acid contained in the Sambucus williamsii Hance extract can be converted into lignin in the body of the anthurium tissue culture seedlings, thereby enhancing the mechanical connection between cells and making the connection between the adventitious roots and the callus more compact. In addition, the polysaccharides and their derivatives contained in the Sambucus williamsii Hance extract can be used as raw materials for pectin synthesis, promote the synthesis and deposition of pectin in the body of the tissue culture seedlings, increase the adhesion between cells, thereby enhancing the connection between cells and making the connection between the adventitious roots and the callus more compact. Furthermore, the Sambucus williamsii Hance extract contains rich antioxidant substances such as vitamin C, vitamin E, polyphenols, flavonoids and the like, which can scavenge free radicals and reduce the damage of oxidative stress to cells, thereby improving the microenvironment of the connection, improving the stress resistance, enhancing the tolerance of cells to adversity and reducing the risk of breakage. In addition, the Sambucus williamsii Hance extract also contains some minerals and trace elements beneficial to plant growth, such as potassium, calcium, magnesium, iron, zinc and the like. These elements are necessary for the normal physiological functions of plant cells, and they can indirectly improve the strength and stress resistance of the connection by improving the nutritional status of cells.

[0023] 3、The application can provide necessary nutrient sources for the induction of anthurium callus by adding banana puree and potato juice to the induction medium. The banana puree contains rich nutrients such as vitamins, minerals and carbohydrates, etc. and provides necessary nutrient sources for the induction of anthurium callus. In addition, the banana puree contains various plant hormones such as auxin, gibberellin and the like, which can promote the division and proliferation of anthurium cells, thereby accelerating the formation of callus. The potato juice contains promoting factors which can stimulate anthurium cells to dedifferentiate and form callus. The banana puree in the proliferation medium continues to provide nutrients and promote cell division and proliferation. The coconut juice contains various nutrients such as amino acids, vitamins and minerals, etc. and provides necessary nutrient sources for the proliferation of anthurium cells. In addition, the kinetin contained in the coconut juice can promote the division of anthurium cells and has an important influence on the development process of anthurium proliferation bodies. The β-carotene and other carotenoids contained in the carrot juice can be converted into vitamin A in the body of anthurium. Vitamin A has various physiological functions in plants, including promoting cell differentiation, maintaining epithelial cell integrity and the like. In the process of anthurium tissue culture, vitamin A can stimulate anthurium cells to differentiate into adventitious buds. Carrot juice also contains rich vitamin C and vitamin E which can scavenge free radicals 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 application, the following specific examples are provided to further illustrate the present application.

[0025] The experimental methods used in the embodiments of the present application are all conventional methods unless otherwise specified.

[0026] The materials, reagents, etc. used in the embodiments of the present application can be obtained from commercial channels unless otherwise specified.

[0027] Embodiment 1

[0028] A tissue culture propagation method of an anthurium comprises the following steps:

[0029] S1, selection and disinfection of explants: fresh and healthy anthurium leaves without pests and diseases are selected as explants, the explants are washed with tap water for 20 min, then 70wt% ethanol is used for disinfection for 30 s, and then a composite disinfectant is used for disinfection for 10 min. The composite disinfectant is composed of chlorobromoisocyanuric acid, permanent flower essential oil, Tween, and distilled water with a mass / volume ratio g / mL of 0.40:0.15:0.05:90.0. The permanent flower essential oil in the composite disinfectant is prepared by the following method: fresh permanent flower petals are washed, dried, and crushed, the crushed permanent flower petals are placed in a distillation apparatus, water is added at a solid-liquid ratio of 1:3, distillation extraction is performed at 70℃ for 5 h, the water vapor collected by condensation is centrifuged at 3000 rpm for 20 min, and the upper liquid is taken as the permanent flower essential oil.

[0030] S2, cutting of explants: the explants treated by disinfection are cut into 0.8cm 2 in size.

[0031] S3, callus induction: the cut explants are inoculated into an induction medium under sterile conditions, and are cultured at a temperature of 23℃, a humidity of 70%, a light intensity of 1500 lux, and a light time of 10 h / d for 45 d to induce callus. The induction medium is composed of MS+1.0mg / L 6-BA+0.3mg / L NAA+30g / L sucrose+50g / L banana paste+80ml / L potato juice.

[0032] S4, proliferation culture: the callus is inoculated into a proliferation medium under sterile conditions, and is cultured at a temperature of 24℃, a humidity of 75%, a light intensity of 1800 lux, and a light time of 12 h / d for 35 d to proliferate the proliferation body. The proliferation medium is composed of MS+0.8mg / L 6-BA+0.3mg / L NAA+30g / L sucrose+50g / L banana paste+60ml / L coconut juice.

[0033] S5, differentiating adventitious buds: under sterile conditions, the above proliferated plants are put into differentiation medium, and cultured for 50 days under the conditions of temperature 22℃, humidity 6%, light intensity 2000 lux, and light time 12h / d, to differentiate adventitious buds. The differentiation medium consists of: 1 / 2MS+0.4mg / L 6-BA+0.2mg / L NAA+20g / L sucrose+30g / L agar+50ml / L carrot juice.

[0034] S6, rooting and strengthening roots: under sterile conditions, the above adventitious buds are put into rooting medium, and cultured for 35 days under the conditions of temperature 23℃, humidity 70%, light intensity 1500 lux, and light time 12h / d, to obtain tissue culture seedlings. The rooting medium consists of: 1 / 2MS+0.2mg / L NAA+0.1mg / L IAA+20g / L sucrose+40ml / L elderberry extract. The elderberry extract is prepared by the following method: fresh mature elderberry fruits are washed and crushed, and 75wt% heated to 55℃ is added to the crushed fruits at a solid-liquid ratio of 1:5 for extraction for 4h. After extraction, the filtrate is obtained by filtration, and the filtrate is vacuum concentrated to 10% of the original liquid volume under a vacuum degree of 0.06MPa.

[0035] S7, seedling training and transplanting: the bottle cap of the tissue culture seedlings is opened, and the seedlings are trained for 6 days at 23℃. Then the tissue culture seedlings are taken out, the medium residues attached to the roots of the seedlings are washed away, and the seedlings are planted into a culture medium in a seedling raising greenhouse with a sunshade net, and cultured for 60 days under the conditions of temperature 20℃, humidity 60%, and light shading degree 50%. The culture medium consists of grass ash, vermiculite, coconut husk, and moss at a mass ratio of 2:4:6:1.

[0036] Example 2

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

[0038] S1, selection and sterilization of explants: fresh and healthy anthurium tender leaves without pests and diseases are selected as explants, and the explants are washed with tap water for 25min. Then the explants are sterilized with 75wt% ethanol for 25s, and then sterilized with a composite sterilizing agent for 15min. The composite sterilizing agent consists of chlorobromoisocyanuric acid, permanent flower essential oil, Tween, and distilled water at a mass / volume ratio of 0.50:0.20:0.10:95.0 g / mL. The permanent flower essential oil in the composite agent is prepared by the following method: fresh permanent flower petals are washed, dried, and crushed, and the crushed petals are put into a distiller. Water is added at a solid-liquid ratio of 1:4, and distilled at 80℃ for 4h. The water vapor collected by condensation is centrifuged at 4000rpm for 15min, and the upper liquid is taken as the permanent flower essential oil.

[0039] S2, cutting of explants: the sterilized explants are cut into 1.0cm2 size.

[0040] S3. Callus Induction: Under aseptic conditions, the cut explants were inoculated into the induction medium and cultured for 40 days at 25℃, 75% humidity, 1750 lux light intensity, and 11 h / d light duration to induce callus formation. The induction medium consisted 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.

[0041] S4. Proliferator Culture: Under aseptic conditions, the above-mentioned callus tissue was inoculated into proliferation medium and cultured for 32 days at a temperature of 26℃, humidity of 80%, light intensity of 2000 lux, and light duration of 13 h / d to proliferate proliferate. 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: Under aseptic conditions, the above-mentioned proliferators were inoculated into differentiation medium and cultured for 45 days at a temperature of 23℃, humidity of 70%, light intensity of 2250 lux, and light duration of 13 h / d to differentiate adventitious buds. The differentiation medium consisted of: 1 / 2 MS + 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 Strengthening: Under aseptic conditions, the above-mentioned adventitious buds were inoculated into rooting medium and cultured for 30 days at 24℃, 75% humidity, 1750 lux light intensity, and 11 h / d light duration to obtain tissue culture seedlings. The rooting medium consisted of 1 / 2 MS + 0.2 mg / L NAA + 0.1 mg / L IAA + 20 g / L sucrose + 40 ml / L elderberry extract. The elderberry extract was prepared by the following method: fresh, mature elderberry fruits were washed, crushed, and 85 wt% of the extract was added at a material-to-liquid ratio of 1:7.5 and extracted at 60℃ for 3 h. After extraction, the filtrate was collected and concentrated under vacuum at 0.07 MPa to 15% of the original volume.

[0044] S7. Hardening-off and Transplanting: Open the caps of the tissue culture seedling bottles and harden off the seedlings at 25℃ for 5 days. Then, remove the seedlings, wash away any remaining culture medium residue from the roots, and transplant them into the culture medium. Cultivate the seedlings in a seedling greenhouse with shade netting at 23℃, 65% humidity, and 60% shading for 50 days. The culture medium consists of wood ash, vermiculite, coconut coir, and moss in a mass ratio of 3:5:8:2.

[0045] Example 3

[0046] A method for propagating and cultivating seedlings of an anthurium, comprising the following steps:

[0047] S1, selection and disinfection of explants: fresh and healthy anthurium leaves without pests and diseases are selected as explants, the explants are washed with tap water for 30 minutes, then 75% ethanol is used for disinfection for 20 seconds, and then a composite disinfectant is used for disinfection for 20 minutes. The composite disinfectant is composed of chlorobromoisocyanuric acid, permanent flower essential oil, Tween, and distilled water with a mass / volume ratio of 0.60:0.25:0.15:100.0. The permanent flower essential oil in the composite disinfectant is prepared by the following method: fresh permanent flower petals are washed, dried, and crushed, the crushed permanent flower petals are placed in a distiller, water is added at a solid-liquid ratio of 1:5, distillation extraction is performed at 90°C for 3 hours, the collected water vapor is centrifuged at 5000 rpm for 10 minutes, and the upper liquid is taken as the permanent flower essential oil.

[0048] S2, cutting of explants: the disinfected explants are cut into 1.2cm 2 in size.

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

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

[0051] S5, differentiation of adventitious buds: the proliferation body is inoculated into a differentiation medium under sterile conditions, and is cultured at a temperature of 25°C, a humidity of 75%, a light intensity of 2500 lux, and a light time of 14h / d for 40 days to differentiate adventitious buds. The differentiation medium is composed of 1 / 2MS+0.4mg / L 6-BA+0.2mg / L NAA+20g / L sucrose+30g / L agar+50ml / L carrot juice.

[0052] S6, rooting and strengthening roots: the adventitious buds are inoculated into the rooting medium under sterile conditions, and cultured at 25°C, 80% humidity, 2000 lux light intensity, and 12h / d light time for 25d to obtain the tissue culture seedlings. The rooting medium comprises 1 / 2MS+0.2mg / L NAA+0.1mg / L IAA+20g / L sucrose+40ml / L Cornus officinalis extract. The Cornus officinalis extract is prepared by the following method: fresh mature Cornus officinalis fruits are washed and crushed, and 95wt% is added to the crushed fruits at a solid-liquid ratio of 1:10 and heated to 65°C for 2h. After the extraction is completed, the filtrate is obtained by filtration, and the filtrate is vacuum concentrated to 20% of the original liquid volume under a vacuum degree of 0.08MPa.

[0053] S7, seedling training and transplanting: the tissue culture seedling bottle cap is opened, the seedlings are trained at 27°C for 4d, then the tissue culture seedlings are taken out, the medium residues attached to the roots of the tissue culture seedlings are washed away, and the tissue culture seedlings are planted into the culture medium in the seedling raising greenhouse with sunshade nets, and cultured at 25°C, 70% humidity, and 70% shading for 40d. The culture medium is composed of grass ash, vermiculite, coconut coir, and moss at a mass ratio of 4:6:10:3.

[0054] Comparative Example 1

[0055] This comparative example is compared with Example 2, the difference being that the external explants are selected and sterilized in the S1 step using ethanol sterilization followed by 0.1wt% mercuric chloride solution sterilization for 12min. The sterilized explants are washed with sterile water for 3 times.

[0056] Comparative Example 2

[0057] This comparative example is compared with Example 2, the difference being that the composite sterilizing agent in the S1 step is composed of permanent flower essential oil, Tween, and distilled water at a mass-volume ratio g / mL of (0.15-0.25):(0.50-0.15):(90.0-100.0).

[0058] Comparative Example 3

[0059] This comparative example is compared with Example 2, the difference being that the composite sterilizing agent in the S1 step is composed of chlorobromoisocyanuric acid, Tween, and distilled water at a mass-volume ratio g / mL of (0.40-0.60):(0.50-0.15):(90.0-100.0).

[0060] Comparative Example 4

[0061] This comparative example is compared with Example 2, the difference being that the rooting medium in the S6 step is composed of 1 / 2MS+0.2mg / L NAA+0.1mg / L IAA+20g / L sucrose.

[0062] I. Disinfection effect test

[0063] Each 100 explants, respectively, according to the seedling method of Example 1-3 and Comparative Example 1-3 were carried out, and the seedling was carried out to S3 induction base culture, and the pollution rate and survival rate were counted at the end of the culture, and 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 Contamination 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 are not contaminated, and the survival rate is also higher than that of Comparative Examples 1-3, so the composite disinfectant of the present application can effectively kill the virus and be beneficial to the growth and development of the 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 rinsing, the mercuric chloride solution is difficult to completely remove, and its residues have a toxic effect on the anthurium cells, further causing the culture medium to be contaminated and affecting the normal physiological functions of the cells, thereby causing the survival rate to decrease. In the composite disinfectant of the present application, chlorobromoisocyanuric acid releases hypochlorous acid and hypobromous acid in water, which can destroy the cell walls and membranes of bacteria, fungi and viruses, leading to the leakage of cell contents, thereby causing the microorganisms to lose activity. Chlorobromoisocyanuric acid can also enter the interior of the explant through the action of 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 viruses, thereby inhibiting the spread and proliferation of viruses in the explant. In addition, chlorobromoisocyanuric acid can also stimulate the explant to produce more growth hormones such as auxin and cytokinin by regulating the hormone balance in the explant, while inhibiting the production of growth-inhibiting hormones such as ethylene, thereby promoting the explant to induce callus. The compounds such as α-curcumene, β-curcumene and γ-curcumene in the essential oil of Persea americana Mill. can penetrate the cell membrane of microorganisms, destroy its integrity, and cause the leakage of intracellular substances, thereby causing the death of microorganisms. The italidione in the essential oil can interfere with the metabolic process of microorganisms, such as inhibiting the activity of enzymes and blocking the absorption of nutrients, thereby causing microorganisms to be unable to grow and reproduce normally. In addition, the neral in the essential oil of Persea americana Mill. has the effect of promoting cell regeneration and division, can stimulate the activity of explant cells, promote their division and proliferation, and thereby accelerate the growth and development of the explant. At the same time, the essential oil of Persea americana Mill. can also reduce the oxidative damage to the explant during the culture process, thereby maintaining its vitality and healthy state. Chlorobromoisocyanuric acid and the essential oil of Persea americana Mill. produce complementary effects through different antibacterial mechanisms, thereby 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 disinfecting components, and make the disinfectant more easily enter the interior of the microbial cells, thereby improving the disinfecting effect.

[0068] As can be seen from the comparison of Examples 1-3 and Comparative Examples 2-3, chlorobromoisocyanuric acid and the essential oil of Persea americana Mill. can only produce better antibacterial disinfecting effect when used together.

[0069] II. Root connection strength test

[0070] Take 20 explants according to the method of examples 1-3 and comparative example 4 to grow seedlings, grow seedlings to S6 rooting and strong root culture end, randomly select 10 strains of tissue culture seedlings from each group for tensile test, record the tensile value required when the callus connection of tissue culture seedlings is broken, and calculate the average tensile value of each group, see table 2.

[0071] Table 2

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

[0073] As can be seen from table 2, the strength of the callus connection of tissue culture seedlings of examples 1-3 is higher than that of comparative example 4. This is because, by adding elderberry extract in the rooting medium, the coumarin and ferulic acid contained in the elderberry extract can be converted into lignin in the body of anthurium tissue culture seedlings, thereby enhancing the mechanical connection between cells, making the connection between adventitious roots and callus more closely. And the polysaccharides and their derivatives contained in the elderberry extract can be used as raw materials for pectin synthesis, promote the synthesis and deposition of pectin in the body of tissue culture seedlings, increase the adhesion between cells, thereby enhancing the connection between cells, 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 and other compounds, which can scavenge free radicals, 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 beneficial to plant growth, such as potassium, calcium, magnesium, iron, zinc, etc. These elements are essential for the normal physiological function of plant cells, which may indirectly improve the strength and stress resistance of the connection by improving the nutritional status of cells.

[0074] In summary, the various bioactive components in elderberry extract act on the connection between adventitious roots and callus, significantly enhance the strength of the connection by enhancing cell connection and improving microenvironment, improving stress resistance and other mechanisms, so that it is not easy to break.

[0075] The above only describes the preferred embodiments of the present application and should not be used to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for propagating and raising seedlings of Anthurium through tissue culture, characterized in that, Includes the following steps: S1. Selection and disinfection of explants: Select fresh, healthy, and disease-free young leaves of Anthurium as explants. Rinse the explants with tap water for 20-30 minutes, then disinfect them with ethanol, and then disinfect them with a compound disinfectant. The compound disinfectant is composed of chlorobromoisocyanuric acid, helichrysum essential oil, Tween, and distilled water in a mass-to-volume ratio of (0.40-0.60):(0.15-0.25):(0.05-0.15):(90.0-100.0) g / mL. S2. Explant cutting: Cut the sterilized explants into pieces of 0.8-1.2cm. 2 size; S3. Callus induction: Under aseptic conditions, the above-cut explants were inoculated into the induction medium and cultured to induce callus. The induction medium consisted 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. Proliferator culture: Under aseptic conditions, the above-mentioned callus tissue is inoculated into proliferation medium and cultured to proliferate proliferate proliferate; 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: Under aseptic conditions, the above-mentioned proliferators were inoculated into differentiation medium and cultured to differentiate adventitious buds; the differentiation medium consisted of: 1 / 2 MS + 0.4 mg / L 6-BA + 0.2 mg / L NAA + 20 g / L sucrose + 30 g / L agar + 50 ml / L carrot juice; S6. Rooting and strengthening of roots: Under aseptic conditions, the above adventitious buds are inoculated into rooting medium to obtain tissue culture seedlings; the rooting medium consists of: 1 / 2 MS + 0.2 mg / L NAA + 0.1 mg / L IAA + 20 g / L sucrose + 40 ml / L elderberry extract. S7. Hardening off and transplanting: Open the cap of the tissue culture seedling bottle and harden off the seedlings at 23-27℃ 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 transplant the tissue culture seedlings into the culture medium. Cultivate them in a seedling greenhouse with shade netting. The culture medium is composed of wood ash, vermiculite, coconut coir and moss in a mass ratio of (2-4):(4-6):(6-10):(1-3). The helichrysum essential oil in the compound is prepared by the following method: take fresh helichrysum petals, wash and dry them and crush them. Put the crushed helichrysum petals into a distiller and add water at a material-to-liquid ratio of 1:(3-5). Distill and extract at 70-90℃ for 3-5 hours. Condense the collected water vapor and centrifuge at 3000-5000rpm for 10-20 minutes. Take the upper liquid, which is the helichrysum essential oil. The elderberry extract in the rooting medium is prepared by the following method: fresh and mature elderberry fruits are washed and crushed, and 75-95wt% are added to the medium at a material-to-liquid ratio of 1:(5-10). The medium is heated to 55-65℃ for 2-4 hours. After extraction, the filtrate is filtered and concentrated under vacuum at a vacuum degree of 0.06-0.08MPa to 10-20% of the original liquid volume.

2. The method for propagating and raising seedlings of Anthurium tissue culture as described in claim 1, characterized in that, In step S1, 70-75wt% ethanol is first used for disinfection for 20-30 seconds, followed by disinfection with a compound disinfectant for 10-20 minutes.

3. The method for propagating and raising seedlings of Anthurium tissue culture as described in claim 1, characterized in that, The culture conditions in the S3 induction medium are: temperature 23-27℃, humidity 70-80%, light intensity 1500-2000 lux, light duration 10-12 h / d, and culture for 35-45 days.

4. The method for propagating and raising seedlings of Anthurium tissue culture as described in claim 1, characterized in that, The culture conditions for S4 inoculation in the proliferation medium are: temperature 24-28℃, humidity 75-85%, light intensity 1800-2200 lux, light duration 12-14 h / d, and culture for 28-35 days.

5. The method for propagating and raising seedlings of Anthurium tissue culture as described in claim 1, characterized in that, The culture conditions for S5 in the differentiation medium are: temperature 22-25℃, humidity 65-75%, light intensity 2000-2500 lux, light duration 12-14 h / d, and culture for 40-50 days.

6. The method for propagating and raising seedlings of Anthurium tissue culture as described in claim 1, characterized in that, The S6 culture medium was inoculated under the following conditions: temperature 23-25℃, humidity 70%-80%, light intensity 1500-2000 lux, light duration 10-12 h / d, and cultured for 25-35 days.

7. The method for propagating and raising seedlings of Anthurium tissue culture as described in claim 1, characterized in that, The cultivation conditions for the S7 implantation culture medium in a seedling greenhouse with shade netting are: temperature 20-25℃, humidity 60-70%, shading degree 50-70%, and cultivation for 40-60 days.