Tissue culture and virus-free propagation method of chrysanthemum morifolium

Abstract

The application discloses a tissue culture and virus elimination method for table chrysanthemum, and belongs to the technical field of plant tissue culture, which comprises the following steps: stem tip stripping: selecting new lateral buds of the table chrysanthemum free from diseases and pests, stripping stem tips with a size of 2-5 mm after cleaning and disinfection; sterilization treatment: sterilizing by using a sterilization solution and then washing with sterile water; gradient low-hormone subculture and proliferation: segmented bottle-out rooting: dipping the base of the strong seedling buds with a growth regulator, and cutting into the disinfected substrate, and controlling the environment according to stages, and the table chrysanthemum is obtained.The application realizes the production goal of high virus elimination rate of the table chrysanthemum seedling by the combined application of differential stem tip stripping, precise sterilization, a gradient low-hormone subculture and proliferation system and a segmented bottle-out rooting technology.Compared with traditional cutting propagation, the virus elimination rate of the technology is increased by 15.9% to 22.7%.The application effectively solves the problems in the prior art, improves the quality and production efficiency of the table chrysanthemum seedling, and has remarkable economic and social benefits.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, specifically to a method for the detoxification and propagation of Ping Pong Chrysanthemum through tissue culture. Based on a gradient low-hormone proliferation system and segmented environmental regulation, this method achieves efficient detoxification and propagation of Ping Pong Chrysanthemum through tissue culture, and is suitable for large-scale breeding of high-quality seedlings of cut flower Ping Pong Chrysanthemum, protection of varietal resources, and prevention and control of viral diseases. Background Technology

[0002] Currently, the production of ping-pong chrysanthemum seedlings mainly relies on asexual propagation through cuttings. While this method is simple to operate and has low difficulty in rooting cuttings, it has several key drawbacks. High risk of genetic degradation: Repeated cuttings can lead to virus accumulation in seedlings, degradation of superior varietal traits, and reduced ornamental value of the flowers (decreased flower shape regularity and color purity); poor adaptability for large-scale production: Some ping-pong chrysanthemum varieties severely infected with viruses can no longer be propagated through cuttings, making it difficult to meet the rapidly growing market demand for cut-flower ping-pong chrysanthemum seedlings in China; and a lack of technological research: There is a lack of systematic research on virus removal technology for cut-flower chrysanthemums (including ping-pong chrysanthemums) in China, and a standardized tissue culture virus removal program that can be applied industrially has not yet been developed.

[0003] In summary, a new method for tissue culture detoxification and propagation of *Chrysanthemum indicum* is urgently needed to solve the above problems. Summary of the Invention

[0004] The purpose of this invention is to provide a method for tissue culture detoxification and propagation of *Chrysanthemum indicum*, in order to solve the problems in the prior art.

[0005] This invention is achieved through the following technical solution: a method for virus elimination and propagation of ping-pong chrysanthemum through tissue culture, characterized by comprising the following steps: Stem tip stripping: Select disease-free and pest-free new lateral buds of ping-pong chrysanthemum, clean and disinfect them, and then peel off the stem tips of 2-5mm in size. Sterilization treatment: Sterilize with a sterilizing solution, then rinse with sterile water; Gradient-low hormone proliferative proliferation: Primary culture: Inoculate into 1 / 2 MS medium containing 0.3 mg / L 6-BA, 0.05 mg / L NAA, and (20-30) g / L sucrose, and culture for 7 days; Mid-term proliferation: Transfer to MS medium supplemented with 0.6 mg / L 6-BA, 0.1 mg / L NAA, and (20-30) g / L sucrose; proliferation coefficient 4.7-6.2. Later stage seedling growth: Transfer to MS medium + 0.08 mg / L NAA + (20-30) g / L sucrose + 50 mg / L Scutellaria baicalensis extract, and culture for 5 days; Segmented external rooting: After the seedlings have grown strong, dip the base of the cuttings in a growth regulator and insert them into sterilized substrate. The environment is then controlled according to stages. Induction period: 90% humidity, 25℃ temperature, 70% shading, culture for 5-7 days; Elongation period: 75% humidity, 25℃ temperature, 40% shading rate, culture for 7-8 days; Hardening-off period: 65% humidity, 25℃ temperature, no shading, culture for 3 days.

[0006] Further: In the sterilization process, the sterilization solution is a mercuric chloride solution, and the device is rinsed 5-6 times with sterile water.

[0007] Furthermore, in the segmented in-bottle rooting step, the rooting powder is 100 mg / L IBA slow-release rooting powder, and the substrate is perlite: peat moss: humus = 1:2:1.

[0008] The advantages of this invention are: High virus elimination rate: By combining differentiated stem tip stripping, precise sterilization, a gradient low-hormone subculture propagation system, and segmented in-bottle rooting technology, the production target of high virus elimination rate for Ping Pong Chrysanthemum seedlings is achieved. Compared with traditional cutting propagation, the virus elimination rate of this technology is increased by 15.9% to 22.7%.

[0009] High propagation coefficient: The method of this invention significantly improves the propagation coefficient of ping-pong chrysanthemum, enabling the number of propagation generations per year to reach 9-11, and the annual seedling production of a single mother plant to reach 6,000-9,000 plants, increasing production efficiency by about 120 times.

[0010] High transplant survival rate: Through segmented high-efficiency in-bottle rooting technology and variety-differentiated growth regulator treatment, the transplant survival rate of Ping Pong Chrysanthemum seedlings has been improved. Compared with traditional cutting propagation, the transplant survival rate has increased by 1% to 4%.

[0011] Adaptable to large-scale production: The method of this invention is suitable for the large-scale propagation of high-quality ping-pong chrysanthemum seedlings for cut flowers, and can meet the rapidly growing market demand for ping-pong chrysanthemum seedlings in China.

[0012] Variety resource protection: Through tissue culture and virus elimination technology, rare varieties can be rapidly propagated, enriching the diversity of Ping Pong Chrysanthemum varieties and providing support for the protection of endangered and rare Ping Pong Chrysanthemum varieties.

[0013] Viral disease prevention and control services: Providing virus-free seedling replacement services to planting bases affected by viral diseases to help restore production, and conducting seedling virus testing services to prevent the spread of viral diseases from the source.

[0014] In conclusion, this solution effectively solves the problems in the existing technology, improves the quality and production efficiency of ping-pong chrysanthemum seedlings, and has significant economic and social benefits. Detailed Implementation

[0015] This invention discloses a method for virus elimination and propagation of ping-pong chrysanthemum through tissue culture, comprising the following steps: Stem tip peeling: Select disease-free and pest-free new lateral buds of Ping Pong Chrysanthemum, clean and disinfect them, and peel off stem tips of 2-5mm size. After rinsing with tap water, soaking in 75% alcohol, and rinsing with sterile water, the stem tip peeling is performed under a dissecting microscope. Specifically, select healthy and disease-free new lateral buds from the Ping Pong Chrysanthemum mother plant, rinse with tap water for 10 minutes, transfer to a clean bench, soak in 75% alcohol for 30 seconds, rinse with sterile water 3 times, and peel off the stem tip with a dissecting microscope: place the bud on a glass slide, peel off the outer leaves layer by layer with a dissecting needle to expose the growing point, and cut the stem tip to the corresponding size according to the characteristics of the variety, ensuring the integrity of the growing point cells. For white and dark pink varieties, a stem tip size of 4-5mm is ideal, as the cells at the growing point proliferate rapidly. A slightly larger stem tip can increase the survival rate while ensuring the virus removal effect. For burgundy and yellow varieties, a stem tip size of 2-3mm is ideal, as the virus easily infects cells around the growing point. A smaller stem tip can maximize the removal of virus-infected tissue and ensure the virus removal rate. For purple and green varieties, a stem tip size of 3-4mm is ideal, as the activity of the growing point and the distribution of the virus are at an intermediate level. This range can balance the survival rate and the virus removal effect.

[0016] Sterilization treatment: Use 0.1% mercuric chloride solution as the sterilization solution for sterilization, followed by rinsing with sterile water 5-6 times to thoroughly remove residual sterilizing reagents; the sterilization time is divided into conventional time (suitable for green, white, and yellow varieties, with a time range of 8-10 minutes) and appropriately extended time (suitable for purple, burgundy, and deep pink varieties, with a time range of 12-15 minutes) according to the characteristics of the variety. Conventional time is suitable for tender buds with thin wax layers. Excessive sterilization will damage the cells at the growth point. Conventional time can balance sterilization effect and cell activity. Appropriately extended time is for varieties with thicker wax layers on the surface of the buds. Extending the sterilization time can thoroughly kill the microorganisms attached to the surface; Gradient-based low-hormone subculture proliferation was performed using MS medium supplemented with 7 g / L agar and pH adjusted to 5.8-6.0. The hormone formulation was dynamically adjusted according to the proliferation stage, and natural plant extracts were introduced to replace some synthetic hormones, reducing the risk of hormone residues. Specifically, the process was divided into three stages: initial culture, mid-stage proliferation, and late-stage seedling strengthening. Primary culture: Inoculate into 1 / 2 MS medium containing 0.3 mg / L 6-BA, 0.05 mg / L NAA, and (20-30) g / L sucrose, and culture for 7 days; slowly activate dormant shoot tip cells to avoid the risk of mutations caused by high hormone levels; Mid-term proliferation: Transfer to MS medium containing 0.6 mg / L 6-BA, 0.1 mg / L NAA, and (20-30) g / L sucrose, with a proliferation coefficient of 4.7-6.2; maintain a stable proliferation rate, reduce hormone residues, and shorten the seedling recovery time; Later stage seedling growth: Transfer to MS medium + 0.08 mg / L NAA + (20-30) g / L sucrose + 50 mg / L Scutellaria baicalensis extract, and culture for 5 days; promote thickening of seedling stems and leaves, and enhance the basic vigor of ex-rooting.

[0017] Segmented external rooting: The dynamic parameters of the rooting substrate and environment are divided into induction, elongation, and hardening-off periods, with humidity, temperature, and shading rate controlled respectively. Variety-specific growth regulator treatments include dipping in a 1:500 mixture of 100 mg / L ABT and talc, a 1:500 mixture of 100 mg / L NAA and talc, and a 1:500 slow-release rooting powder of 100 mg / L IBA. After the seedlings have grown strong, dip the base of the rooting powder into the rooting powder and insert them into the sterilized substrate, controlling the environment according to the stages: Induction period: 90% humidity, 25℃ temperature, 70% shading rate, culture for 5-7 days; disinfect by spraying with an agent containing Bacillus subtilis + gelatinous Bacillus before inoculation; Elongation period: 75% humidity, 25℃ temperature, 40% shading rate, culture for 7-8 days; gradually increase light intensity to promote root lignification; Hardening-off period: 65% humidity, 25℃ temperature, no shading, cultivate for 3 days to adapt to the external environment and improve transplant resistance.

[0018] Furthermore, in the segmented in-bottle rooting step, the rooting powder is 100 mg / L IBA slow-release rooting powder, and the substrate is a substrate with a volume ratio of perlite: peat moss: humus = 1:2:1.

[0019] Through the above steps, tissue culture detoxification and propagation of ping-pong chrysanthemum can be achieved, improving the detoxification rate, propagation coefficient and transplant survival rate of seedlings, adapting to the needs of large-scale production, protecting varietal resources and preventing the spread of viral diseases.

[0020] Preferably, in the segmented in-bottle rooting step, the growth regulator is one of the following three: a 1:500 mixture of 100 mg / L LABT and talc, a 1:500 mixture of 100 mg / L NAA and talc, or a 1:500 slow-release rooting powder of 100 mg / L IBA.

[0021] Preferably, the matrix is ​​a perlite: peat: humus matrix with a ratio of 1:2:1.

[0022] Preferred: Scutellaria baicalensis extract, a standardized extract made from the dried roots of Scutellaria baicalensis through water or alcohol extraction. Its main active ingredient is baicalin (content ≥85%), and it is a brownish-yellow powder. It is suitable for the carbon source requirements of conventional varieties and enhances cell activity.

[0023] Ginkgo leaf extract is made from dried ginkgo leaves through extraction and purification processes. Its main active ingredients are ginkgo flavonoids (≥24%) and terpene lactones (≥6%), and it appears as a light brownish-yellow to brownish-brown powder. It can reduce osmotic pressure and increase the photosynthetic rate by 15%.

[0024] Seaweed oligosaccharides are a mixture of oligosaccharides obtained by degrading seaweed polysaccharides through enzymatic or acid hydrolysis of brown algae (such as kelp and Sargassum). The main component is fucoidan, which is a pale yellow powder and easily soluble in water. It is adapted to photosynthetic characteristics and enhances hormone sensitivity.

[0025] The following examples further illustrate this solution.

[0026] Example 1: Virus-free propagation of white ping-pong chrysanthemum through tissue culture.

[0027] The steps in this embodiment refer to the above process, wherein: Stem tip stripping: Strip the stem tip to a size of 4-5mm. White ping-pong chrysanthemum has a rapid cell proliferation rate at the growth point, and a slightly larger stem tip can increase the survival rate while ensuring the detoxification effect.

[0028] Sterilization treatment: Sterilize with 0.1% mercuric chloride solution for the standard duration (8-10 minutes), followed by rinsing 5 times with sterile water. The white ping-pong chrysanthemum buds are delicate with a thin waxy layer; the standard duration ensures both sterilization effectiveness and cell viability.

[0029] Gradient-low hormone proliferative proliferation: Primary culture: 1 / 2 MS medium + 0.3 mg / L 6-BA + 0.05 mg / L NAA + 30 g / L sucrose medium, cultured for 7 days; Mid-term proliferation: MS medium containing 0.6 mg / L 6-BA, 0.1 mg / L NAA, and 30 g / L sucrose achieved a proliferation coefficient of 5.0. Later stage seedling growth: MS medium containing 0.08 mg / L NAA, 30 g / L sucrose, and 50 mg / L Scutellaria baicalensis extract was cultured for 5 days.

[0030] The principle of differentiation of natural extracts: White Ping Pong Chrysanthemum meets the needs of conventional carbon sources, and the baicalin contained in Scutellaria baicalensis extract can remove intracellular reactive oxygen species, enhance the antioxidant capacity of cells, and improve the proliferation activity after the shoot tip survives.

[0031] Segmented external rooting: Dip the plant in 100mg / L IBA slow-release rooting powder. This treatment enhances the root system's resistance to stress and improves the transplant survival rate of white ping-pong chrysanthemums.

[0032] Operating procedure: Select disease-free and pest-free new lateral buds of white ping-pong chrysanthemum, rinse with tap water for 10 minutes, then transfer to a clean bench, soak in 75% alcohol for 30 seconds, rinse 3 times with sterile water, and peel off 4-5mm stem tips; sterilize with 0.1% mercuric chloride solution for 8-10 minutes, and rinse 5 times with sterile water; culture according to a gradient low-hormone subculture propagation process; dip the base of healthy 3-4cm seedlings in 100mg / LIBA slow-release rooting powder, and insert them into a sterilized substrate of perlite: peat moss: humus = 1:2:1. Induction period (90% humidity, 25℃ temperature, 70% shading rate, 7 days of cultivation) → elongation period (75% humidity, 25℃ temperature, 40% shading rate, 8 days of cultivation) → hardening period (65% humidity, 25℃ temperature, no shading, 3 days of cultivation); after 18 days, the rooting rate reaches 97%, and the transplant survival rate is 94%.

[0033] Example 2: Virus-free propagation of yellow ping-pong chrysanthemum through tissue culture.

[0034] Core differentiating parameters and principles: Stem tip stripping: Strip the stem tip to a size of 2-3mm. Yellow ping-pong chrysanthemum virus easily infects cells around the growing point; smaller stem tips allow for maximum stripping of virus-infected tissue, ensuring a high virus elimination rate.

[0035] Sterilization treatment: Sterilize with 0.1% mercuric chloride solution for the standard duration (8-10 minutes), and rinse 6 times with sterile water. The buds of the yellow ping-pong chrysanthemum are delicate and have a thin waxy layer, so the standard duration can avoid damaging the growth point cells.

[0036] Gradient-low hormone proliferative proliferation: Primary culture: 1 / 2 MS medium + 0.3 mg / L 6-BA + 0.05 mg / L NAA + 25 g / L sucrose medium, cultured for 7 days; Mid-term proliferation: MS medium containing 0.6 mg / L 6-BA, 0.1 mg / L NAA, and 25 g / L sucrose resulted in a proliferation coefficient of 6.2. Later stage seedling growth: MS medium containing 0.08 mg / L NAA, 25 g / L sucrose, 50 mg / L Scutellaria baicalensis extract, and 30 mg / L seaweed oligosaccharide was cultured for 5 days.

[0037] The principle of differentiation of natural extracts: Yellow ping-pong chrysanthemum has a low photosynthetic rate. Algae oligosaccharides can regulate the expression of hormone receptor proteins on the cell membrane and enhance the plant's sensitivity to hormones such as 6-BA and NAA. At the same time, Scutellaria baicalensis extract can stabilize the intracellular metabolic environment. The two work together to improve proliferation efficiency.

[0038] Segmented external rooting: Dip the sample in a 1:500 mixture of 100 mg / L NAA and talc. This treatment enhances root primordia induction and shortens the rooting cycle in yellow ping-pong chrysanthemums.

[0039] Operating procedure: Select disease-free and pest-free new lateral buds of yellow ping-pong chrysanthemum, rinse with tap water for 10 minutes, then transfer to a clean bench, soak in 75% alcohol for 30 seconds, rinse 3 times with sterile water, and peel off 2-3mm stem tips; sterilize with 0.1% mercuric chloride solution for 8-10 minutes, and rinse 6 times with sterile water; culture according to a gradient low-hormone subculture propagation process; dip the base of healthy 3-4cm seedlings in a mixture of 100mg / L NAA and talc powder (1:500), and insert them into a sterilized substrate of perlite: peat moss: humus = 1:2:1. Induction period (90% humidity, 25℃ temperature, 70% shading, 5 days) → elongation period (75% humidity, 25℃ temperature, 40% shading, 7 days) → hardening period (65% humidity, 25℃ temperature, no shading, 3 days); after 15 days, the rooting rate reaches 100%, and the transplant survival rate is 97%.

[0040] Example 3: Virus-free propagation of purple ping-pong chrysanthemums through tissue culture.

[0041] Core differentiating parameters and principles: Stem tip stripping: Strip 3-4mm diameter stem tips. The activity and virus distribution at the growth point of *Chrysanthemum 'Ping Pong'* are at an intermediate level, balancing survival probability and virus elimination effect.

[0042] Sterilization treatment: Sterilize with 0.1% mercuric chloride solution for an extended time (12-15 minutes), then rinse 6 times with sterile water. The purple ping-pong chrysanthemum buds have a thick waxy layer on their surface; extending the sterilization time can thoroughly kill any microorganisms attached to the surface.

[0043] Gradient-low hormone proliferative proliferation: Primary culture: 1 / 2 MS medium + 0.3 mg / L 6-BA + 0.05 mg / L NAA + 30 g / L sucrose medium, cultured for 7 days; Mid-term proliferation: MS medium containing 0.6 mg / L 6-BA, 0.1 mg / L NAA, and 30 g / L sucrose resulted in a proliferation coefficient of 5.4. Later stage seedling growth: MS medium containing 0.08 mg / L NAA, 30 g / L sucrose, and 50 mg / L Scutellaria baicalensis extract was cultured for 5 days.

[0044] The principle of differentiation of natural extracts: Purple ping-pong chrysanthemum has moderate growth point activity, while the flavonoids in Scutellaria baicalensis extract can promote cytokinin synthesis, enhance cell proliferation activity, and inhibit viral nucleic acid replication, thus consolidating the detoxification effect while ensuring proliferation efficiency.

[0045] Segmented endorooting in bottles: Dip the plant in a 1:500 mixture of 100 mg / L ABT and talc. This treatment broadly promotes the growth of both taproots and lateral roots in purple ping-pong chrysanthemums, enhancing overall root vitality.

[0046] Operating procedure: Select disease-free and pest-free new lateral buds of purple ping-pong chrysanthemum, rinse with tap water for 10 minutes, then transfer to a clean bench, soak in 75% alcohol for 30 seconds, rinse 3 times with sterile water, and peel off 3-4mm stem tips; sterilize with 0.1% mercuric chloride solution for 12-15 minutes, and rinse 6 times with sterile water; culture according to a gradient low-hormone subculture propagation process; dip the base of healthy 3-4cm seedlings in a mixture of 100mg / LABT and talc powder at a ratio of 1:500, and insert them into a sterilized substrate of perlite: peat moss: humus = 1:2:1. Induction period (90% humidity, 25℃ temperature, 70% shading rate, 7 days of cultivation) → elongation period (75% humidity, 25℃ temperature, 40% shading rate, 8 days of cultivation) → hardening period (65% humidity, 25℃ temperature, no shading, 3 days of cultivation); after 17 days, the rooting rate reaches 99%, and the transplant survival rate is 96%.

[0047] Example 4: Virus-free propagation of green ping-pong chrysanthemum through tissue culture.

[0048] Core differentiating parameters and principles: Stem tip stripping: Strip 3-4mm diameter stem tips. The activity and virus distribution at the growth point of *Chrysanthemum 'Green Ping Pong'* are at an intermediate level, a range that balances survival probability and virus elimination effectiveness.

[0049] Sterilization treatment: Sterilize with 0.1% mercuric chloride solution for the standard duration (8-10 minutes), followed by rinsing 5 times with sterile water. The green ping-pong chrysanthemum buds are delicate and have a thin waxy layer; the standard duration ensures both sterilization effectiveness and cell viability.

[0050] Gradient-low hormone proliferative proliferation: Primary culture: 1 / 2 MS medium + 0.3 mg / L 6-BA + 0.05 mg / L NAA + 22 g / L sucrose medium, cultured for 7 days; Mid-term proliferation: MS medium containing 0.6 mg / L 6-BA, 0.1 mg / L NAA, and 22 g / L sucrose resulted in a proliferation coefficient of 4.7. Later stage seedling growth: MS medium containing 0.08 mg / L NAA, 22 g / L sucrose, 50 mg / L Scutellaria baicalensis extract, and 20 mg / L Ginkgo biloba extract, cultured for 5 days.

[0051] The principle of differentiation of natural extracts: Green Ping Pong Chrysanthemum cells have high osmotic pressure. Ginkgo biloba extract contains ginkgolide, which can regulate cell membrane permeability, reduce intracellular osmotic pressure, and increase photosynthetic rate by 15%. Scutellaria baicalensis extract can synergistically protect chloroplast structure and avoid the decline in photosynthetic efficiency caused by hormone stress during proliferation.

[0052] Segmented in-bottle rooting: Dip the plant in a 1:500 mixture of 100mg / L ABT and talc. This treatment broadly promotes the growth of both taproots and lateral roots, enhancing the overall vitality of the root system.

[0053] The procedure involves selecting disease-free, newly sprouted lateral buds of green ping-pong chrysanthemum, rinsing them with tap water for 10 minutes, then transferring them to a clean bench for soaking in 75% alcohol for 30 seconds, rinsing them three times with sterile water, and peeling off 3-4mm stem tips. The buds are then sterilized with 0.1% mercuric chloride solution for 8-10 minutes and rinsed five times with sterile water. A gradient low-hormone subculture propagation process is followed. Healthy 3-4cm seedlings are dipped at the base in a 1:500 mixture of 100mg / LABT and talc powder, and inserted into a sterilized substrate of perlite: peat moss: humus = 1:2:1. The cultivation process is as follows: induction period (90% humidity, 25℃, 70% shading, 7 days) → elongation period (75% humidity, 25℃, 40% shading, 8 days) → hardening-off period (65% humidity, 25℃, no shading, 3 days). After 18 days, the rooting rate reaches 98%, and the transplant survival rate is 95%.

[0054] Example 5: Virus-free propagation of burgundy ping-pong chrysanthemums through tissue culture.

[0055] Core differentiating parameters and principles: Stem tip stripping: Strip 2-3mm of stem tip. The burgundy ping-pong chrysanthemum virus easily infects cells around the growing point; smaller stem tips allow for maximum removal of virus-infected tissue, ensuring a high virus elimination rate.

[0056] Sterilization treatment: Sterilize with 0.1% mercuric chloride solution for an extended time (12-15 minutes), then rinse 6 times with sterile water. The burgundy ping-pong chrysanthemum buds have a thick waxy layer on their surface; extending the sterilization time can thoroughly kill any microorganisms attached to the surface.

[0057] Gradient-low hormone proliferative proliferation: Primary culture: 1 / 2 MS medium + 0.3 mg / L 6-BA + 0.05 mg / L NAA + 30 g / L sucrose medium, cultured for 7 days; Mid-term proliferation: MS medium containing 0.6 mg / L 6-BA, 0.1 mg / L NAA, and 30 g / L sucrose resulted in a proliferation coefficient of 5.7. Later stage seedling growth: MS medium containing 0.08 mg / L NAA, 30 g / L sucrose, and 50 mg / L Scutellaria baicalensis extract was cultured for 5 days.

[0058] The principle of differentiation of natural extracts: The synthesis of pigments in burgundy ping-pong chrysanthemum consumes a lot of energy. Scutellaria baicalensis extract can improve the efficiency of intracellular ATP synthesis, providing sufficient energy for seedling proliferation and pigment synthesis, while inhibiting viral protein translation and ensuring the purity of virus-free seedlings.

[0059] Segmented external rooting: Dip the plant in a 1:500 mixture of 100 mg / L NAA and talc. This treatment enhances root primordia induction and shortens the rooting cycle in burgundy ping-pong chrysanthemum.

[0060] The procedure involves selecting disease-free, newly sprouted lateral buds of burgundy ping-pong chrysanthemum, rinsing them with tap water for 10 minutes, then transferring them to a clean bench. The buds are soaked in 75% alcohol for 30 seconds, rinsed three times with sterile water, and the 2-3mm stem tips are peeled off. The buds are then sterilized with 0.1% mercuric chloride solution for 12-15 minutes, followed by rinsing with sterile water six times. A gradient low-hormone subculture propagation process is then followed. Healthy 3-4cm seedlings are dipped at the base in a 1:500 mixture of 100mg / L NAA and talc, and inserted into a sterilized substrate of perlite: peat moss: humus = 1:2:1. The cultivation process is as follows: induction period (90% humidity, 25℃, 70% shading, 6 days) → elongation period (75% humidity, 25℃, 40% shading, 7 days) → hardening-off period (65% humidity, 25℃, no shading, 3 days). After 16 days, the rooting rate reaches 98%, and the transplant survival rate is 95%.

[0061] Example 6: Virus-free propagation of deep pink ping-pong chrysanthemum through tissue culture.

[0062] Core differentiating parameters and principles: Stem tip stripping: Strip the stem tip to a size of 4-5mm. The cells at the growing point of the deep pink ping-pong chrysanthemum proliferate rapidly, and a slightly larger stem tip can increase the survival rate while ensuring the detoxification effect.

[0063] Sterilization treatment: Sterilize with 0.1% mercuric chloride solution for an extended time (12-15 minutes), then rinse 6 times with sterile water. The deep pink ping-pong chrysanthemum buds have a thick waxy layer on their surface; extending the sterilization time can thoroughly kill the microorganisms attached to the surface.

[0064] Gradient-low hormone proliferative proliferation: Primary culture: 1 / 2 MS medium + 0.3 mg / L 6-BA + 0.05 mg / L NAA + 22 g / L sucrose medium, cultured for 7 days; Mid-term proliferation: MS medium containing 0.6 mg / L 6-BA, 0.1 mg / L NAA, and 22 g / L sucrose resulted in a proliferation coefficient of 4.9. Later stage seedling growth: MS medium containing 0.08 mg / L NAA, 22 g / L sucrose, 50 mg / L Scutellaria baicalensis extract, and 20 mg / L Ginkgo biloba extract, cultured for 5 days.

[0065] The principle of differentiation of natural extracts: Deep pink ping-pong chrysanthemum accumulates photosynthetic products slowly, while ginkgo leaf extract can promote the opening of leaf stomata and improve carbon dioxide absorption efficiency. Combined with the enhanced cell metabolic activity of scutellaria baicalensis extract, the photosynthetic rate can be increased by 15%, providing sufficient nutrition for seedling proliferation.

[0066] Segmented in-bottle rooting: Dip the plant in a 1:500 mixture of 100 mg / L NAA and talc. This treatment enhances root primordia induction and shortens the rooting cycle in deep pink ping-pong chrysanthemums.

[0067] Operating procedure: Select new, disease-free lateral buds of deep pink ping-pong chrysanthemum, rinse with tap water for 10 minutes, then transfer to a clean bench. Soak in 75% alcohol for 30 seconds, rinse three times with sterile water, and peel off 4-5mm stem tips. Sterilize with 0.1% mercuric chloride solution for 12-15 minutes, and rinse six times with sterile water. Cultivate according to a gradient low-hormone subculture propagation process. Dip the base of healthy 3-4cm seedlings in a 1:500 mixture of 100mg / L NAA and talc powder, and insert them into a sterilized substrate of perlite: peat moss: humus = 1:2:1. Induction period (90% humidity, 25℃, 70% shading, 7 days) → elongation period (75% humidity, 25℃, 40% shading, 8 days) → hardening period (65% humidity, 25℃, no shading, 3 days). After 18 days, the rooting rate reaches 99%, and the transplant survival rate is 96%.

[0068] The detoxification rate of the above embodiments was tested using the following method.

[0069] (a) Sample collection: Tissue culture stage sampling: In the late stage of gradient low hormone subculture proliferation, select healthy seedlings with a height of 3-4cm, thick stems and spread leaves. Collect 30 seedlings for each variety, and take 2-3 functional leaves from the top of each seedling.

[0070] Sampling after transplanting: After the hardening-off period of the segmented bottle-rooted seedlings, select the transplanted plants that have survived, collect 30 plants of each variety, and take the middle mature leaves of each plant.

[0071] (II) Virus detection technology: For common viruses in chrysanthemum (chrysanthemum dwarfing virus, chrysanthemum chlorotic mottle virus, etc.), the following mainstream detection method is adopted: enzyme-linked immunosorbent assay (ELISA); Reagent preparation: A commercially available Ping Pong Chrysanthemum virus ELISA detection kit was used, which includes coating antibody, enzyme-labeled secondary antibody, positive control, negative control, substrate solution, etc.

[0072] Operating Procedure: ① Sample Processing: Cut the collected leaves into small pieces, add extraction buffer at a ratio of 1:10 (w / v), grind into a homogenate, let stand at 4℃ for 30 minutes, then centrifuge to collect the supernatant. ② Coating Plate: Dilute the coating antibody according to the kit requirements and add it to the wells of the ELISA plate, incubate overnight at 4℃. ③ Sample Addition: Discard the coating solution, wash the ELISA plate 3 times, add sample supernatant, positive control, and negative control, and incubate at 37℃ for 1.5 hours. ④ Add Enzyme-Labeled Secondary Antibody: After washing, add the diluted enzyme-labeled secondary antibody and incubate at 37℃ for 1 hour. ⑤ Color Development: After washing, add substrate solution and incubate at 37℃ in the dark for 15-30 minutes, then add stop solution to terminate the reaction. ⑥ Result Interpretation: Measure the absorbance at 450nm using an ELISA reader. When the sample absorbance value / negative control absorbance value ≥ 2.1, it is considered to be toxic; otherwise, it is non-toxic.

[0073] (III) Calculation of detoxification rate: Detoxification rate = (number of virus-free plants / total number of plants tested) × 100%, and the average value of the test results in the tissue culture stage and the post-transplanting stage is taken as the final detoxification rate.

[0074] The detoxification effects of each embodiment are compared in the table below: Purple 99 95 82 15.9% green 98 93 78 19.2% Claret 97 97 85 14.1% yellow 98 96 83 15.7% White 99 92 75 22.7% Deep pink 100 94 80 17.5% The tissue culture detoxification and propagation method for Ping Pong Chrysanthemum of this invention significantly improves production efficiency. Through the application of a gradient low-hormone subculture propagation system and segmented in-vitro rooting technology, a high propagation coefficient for Ping Pong Chrysanthemum seedlings is achieved. Compared with traditional cutting propagation, the tissue culture propagation scheme of this invention increases the number of propagation generations per year from 3-5 to 9-11, and the annual seedling production per mother plant from 50-80 to 6000-9000, representing an overall efficiency increase of approximately 120 times.

[0075] This solution achieves high virus-free rate, high propagation coefficient, and high transplant survival rate for Ping Pong Chrysanthemum seedlings through a combination of differentiated stem tip stripping, precise sterilization, a gradient low-hormone subculture propagation system, and segmented in-bottle rooting technology. It is particularly suitable for the following application scenarios: Large-scale seedling production: Establish tissue culture virus-free seedling production lines for domestic cut flower ping-pong chrysanthemum planting bases, with an annual production capacity of over 12 million seedlings; at the same time, provide customized variety services to meet the personalized needs of growers; Variety resource protection: Conduct tissue culture and virus-free preservation of endangered and rare Ping Pong Chrysanthemum varieties to establish a variety resource bank; rapidly propagate rare varieties through tissue culture technology to enrich the diversity of Ping Pong Chrysanthemum varieties; Viral disease prevention and control services: providing virus-free seedling replacement services for planting bases affected by viral diseases to help restore production; conducting seedling virus testing services to prevent the spread of viral diseases from the source; Green seedling supply: Relying on low-hormone and natural extract technology, we produce Ping Pong Chrysanthemum seedlings that meet organic standards and expand into the high-end flower market.

Claims

1. A method for virus elimination and propagation of *Chrysanthemum indicum* via tissue culture, characterized in that: Includes the following steps: Stem tip stripping: Select disease-free and pest-free new lateral buds of ping-pong chrysanthemum, clean and disinfect them, and then peel off the stem tips of 2-5mm in size. Sterilization treatment: Sterilize with a sterilizing solution, then rinse with sterile water; Gradient-low hormone proliferative proliferation: Primary culture: Inoculate into 1 / 2 MS medium containing 0.3 mg / L 6-BA, 0.05 mg / L NAA, and (20-30) g / L sucrose, and culture for 7 days; Mid-term proliferation: Transfer to MS medium supplemented with 0.6 mg / L 6-BA, 0.1 mg / L NAA, and (20-30) g / L sucrose; proliferation coefficient 4.7-6.

2. Later stage seedling growth: Transfer to MS medium + 0.08 mg / L NAA + (20-30) g / L sucrose + 50 mg / L Scutellaria baicalensis extract, and culture for 5 days; Segmented external rooting: Dip the base of vigorous seedlings in a growth regulator, insert them into sterilized substrate, and control the environment according to stages: in, Induction period: 90% humidity, 25℃ temperature, 70% shading, culture for 5-7 days; Elongation period: 75% humidity, 25℃ temperature, 40% shading rate, culture for 7-8 days; Hardening-off period: 65% humidity, 25℃ temperature, no shading, culture for 3 days.

2. The method for tissue culture detoxification and propagation of *Chrysanthemum indicum* according to claim 1, characterized in that: In the sterilization process, the sterilization solution is a mercuric chloride solution, and the patient is rinsed 5-6 times with sterile water.

3. The method for tissue culture detoxification and propagation of *Chrysanthemum indicum* according to claim 1, characterized in that: In the segmented in-bottle rooting step, the growth regulator is one of the following three: a 100 mg / L ABT and talc mixture at a ratio of 1:500, a 100 mg / L NAA and talc mixture at a ratio of 1:500, or a 100 mg / L IBA slow-release rooting powder; the substrate is a perlite: peat moss: humus = 1:2:1 substrate.