A method for stem tip virus-free tissue culture rapid propagation of phalaenopsis

Abstract

The application belongs to the field of plant tissue culture, and discloses a method for meristem detoxification tissue culture and rapid propagation of butterfly orchid, which comprises the following steps: ①virus detection of detoxification butterfly orchid bottle seedlings, ②cold and hot treatment of detoxification butterfly orchid bottle seedlings, ③liquid oscillation culture of stem tip stripping, ④stem tip differentiation induction, ⑤virus detection of meristematic seedlings, ⑥subculture and proliferation, and ⑦rooting of adventitious buds. The method for meristem detoxification tissue culture and rapid propagation of butterfly orchid provided by the application firstly performs virus detection on the detoxification materials, and determines the virus species to be removed and detected from the stem tip, thereby solving the common problem of possible missed detection of other viruses due to subjective determination of the target virus to be detected; the detoxification materials are pretreated by cold and hot combination, the hot treatment accelerates the growth of stem tip meristematic cells to expand the virus-free area of the stem tip, the cold and hot treatment passivates the virus, and then the stem tip with a diameter of 0.2-0.5 mm is combined with liquid culture, the effect of stem tip detoxification is ensured through skilled stem tip stripping operation, the survival rate of the stem tip is ensured through liquid oscillation culture and addition of CPPU, and thus the common problems of low survival rate and low detoxification rate of the current butterfly orchid stem tip are solved.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology and relates to a method for rapid propagation of Phalaenopsis orchids via stem tip detoxification tissue culture. Background Technology

[0002] Phalaenopsis is a plant belonging to the genus Phalaenopsis in the family Orchidaceae. It is native to tropical and subtropical regions and thrives in hot and humid environments. Because its flowers resemble butterflies, have vibrant colors, and are beautiful, it is known as the "Queen of Orchids." In addition, its long flowering period, numerous varieties, and rich colors have made it a favorite among flower consumers at home and abroad, making it one of the most popular flowers in the world.

[0003] According to domestic flower market research, China's Phalaenopsis orchid production reached 130 million plants in 2022, with 80 million plants supplied to the 2023 Lunar New Year market, generating a value exceeding 1.4 billion yuan. Furthermore, consumption is growing at a rate of at least 10% annually. Meanwhile, international flower market research shows that in 2018, Europe alone produced 150 million plants, generating a value exceeding 300 million US dollars, with consumption still growing at a rate of 15-20% annually. Global market demand for Phalaenopsis orchids is strong, and the export market outlook is promising. However, viral diseases have become a major obstacle to my country's Phalaenopsis orchid exports.

[0004] According to global statistics, as many as 30 viruses have been identified as capable of infecting Phalaenopsis orchids. Currently, the main viruses causing widespread damage to Phalaenopsis orchids are Cymbidium mosaic virus (CymMV), Odontoglossum ringspot virus (ORSV), Cucumber mosaiccucumovirus (CMV), and Orchid fleck virus, with CymMV and ORSV being the most prevalent. Extensive research has been conducted on Phalaenopsis orchid viruses both domestically and internationally, and identification techniques are relatively mature. For the detection of CymMV and ORSV, serological techniques and molecular biotechnology are currently the most commonly used methods.

[0005] The main plant virus elimination methods reported domestically and internationally include: heat treatment, cryogenic treatment, shoot tip meristem culture, a combination of heat treatment and shoot tip meristem culture, shoot tip micrografting, callus culture, protoplast culture, anther culture, and antiviral chemical agents. Heat treatment is relatively effective against spherical viruses, striae-like viruses, and mycoplasma-like viruses, but its effect on rod-shaped and filamentous viruses is not significant. Cryogenic treatment is limited by the characteristics of different plant tissues, water content, and tolerance to cryogenic temperatures; shoot tip survival rate is low, shoot tip recovery period is long, and the vitrification solution of dried shoot tips contains polyethylene glycol and dimethyl sulfoxide (DMSO), both of which are toxic to plant cells. Protoplast culture and callus culture methods carry a relatively high risk of virus latency. Virus elimination through shoot tip meristem culture is theoretically possible because the growth cone within 0.5 mm is essentially virus-free. However, when harvesting shoot tips, the developmental stage of the shoot tip often makes it difficult to obtain tips within 0.5 mm. Furthermore, the harvesting technique can easily lead to the shoot tips being contaminated with sap from older tissues. Small shoot tips are also prone to browning, resulting in low survival rates. Micrografting of shoot tips for virus elimination is limited by the rootstock and cannot achieve ideal results. The mechanism of virus elimination through anther culture is still unclear. Research on virus elimination using antiviral chemical agents is limited; different antiviral chemicals inhibit different types of viruses, and using this method alone results in a low success rate. Patent CN201711358071 employs a method combining heat treatment with shoot tip and antiviral agent detoxification. The treated seedlings are tissue culture bottle seedlings, and the treatment temperature is 30℃ to 38℃, with daily increases in temperature. Shoot tips of 0.5-1.0 mm are cut. Surviving shoot tip clusters are tested for CymMV and ORSV viruses; negative clusters are then used for subsequent culture. The key primary culture medium is 1 / 2 MS + 6-BA 4.0 mg / L + NAA. 0.5 mg / L + 8 g / L agar + 30 g / L sucrose + 25 g / L banana puree + 4 g / L activated charcoal + 40 mg / L ribavirin + 5.0 g / L sodium bicarbonate; CymMV is a linear virus and ORSV is a baculovirus, and heat treatment was ineffective against these two viruses; the shoot tip tissue (0.5–1.0 g / L) was relatively large, and the detoxification effect was also poor; ribavirin mainly inhibits the replication of potato virus X (PVX), belladonna mottle virus, tobacco mosaic virus, potato virus Y (PVY), and tobacco necrosis virus, but its inhibitory effect on CymMV and ORSV has not been reported; in addition, the initial culture medium was solid, and although activated charcoal was added, it could not transfer browning in time, and could not effectively reduce the continuous damage of browning to shoot tip tissue.Patent CN201811063504 employs ultra-low temperature treatment for virus removal, targeting 1.0-1.5cm stem tips of Phalaenopsis tissue culture seedlings carrying CymMV and ORSV. Through ultra-low temperature treatment with liquid nitrogen, the survival rate of stem tips was 67-80%, the regeneration rate was 57%, the virus removal rate of Cymbidium faberi leaf virus was 62.5%, and the virus removal rate of Cymbidium goeringii ringspot virus was 83.3%. The redifferentiation rate and virus removal rate of surviving stem tips need to be further improved. Furthermore, the vitrification solution used to dry the stem tips contains polyethylene glycol and dimethyl sulfoxide (DMSO), which are toxic to plants. DMSO also has mutagenic effects. Patent CN201610915724 employs a two-stage shoot tip stripping method combined with antiviral agent detoxification. The patent uses shoot tips from newly formed clusters of shoots induced by flower stalks as the culture medium. Shoot tips of 1-2 mm are stripped twice and cultured on a detoxification medium of 1 / 3 MS + 6-BA 5.0 mg / L + NAA 0.5 mg / L + 5% amino oligosaccharide 0.2-0.3 mL / L + coconut milk 100 mL / L + sucrose 20 g / L + agar 6.0-6.5 g / L. Virus-free seedlings were obtained through cultivation, testing for non-toxicity (CymMV, ORSV), propagation, seedling strengthening, and rooting. The survival rate of shoot tips reached 64.74%, and the virus elimination rate reached 94%. Based on the principle of shoot tip virus elimination, the virus-carrying rate of shoot tips of 1-2 mm is relatively high, and even secondary shoot tip stripping will not improve the situation much. The antiviral agent amino oligosaccharide is effective against watermelon wilt, cotton verticillium wilt, tomato late blight, and tobacco viral diseases, but its inhibitory effect on CymMV and ORSV has not been reported.

[0006] The aforementioned methods for detoxifying Phalaenopsis orchids, besides having some shortcomings in the methods themselves, also have two common problems. On the one hand, they do not conduct a virus screening of the material to be detoxified, which may lead to missed detections and cause minor viruses to become major viruses. For example, in the 1990s, the detoxification of banana mosaic virus and banana stripe in the southern market led to banana bunchy top disease becoming the main virus causing damage. On the other hand, they all use solid culture, which makes it difficult to guarantee the survival rate of the stem tips.

[0007] To address the aforementioned issues, this invention first conducts a virus assessment of the material to be detoxified, identifying the target virus species to be removed and detected. This resolves the common problem of missed detection of other viruses due to subjective identification of the target virus. The invention employs a combination of hot and cold pretreatment of the material to be detoxified. Heat treatment accelerates the growth of shoot tip meristems to expand the virus-free area at the shoot tip, while simultaneous hot and cold treatment inactivates the virus. Then, the shoot tips (0.2–0.5 mm) are cultured in liquid. Skilled shoot tip peeling ensures effective detoxification, and liquid shaking culture and the addition of CPPU ensure cell division and shoot tip survival. This addresses the common problems of low shoot tip survival and low detoxification rates in Phalaenopsis orchids. Summary of the Invention

[0008] This invention aims to provide a method for rapid propagation of Phalaenopsis orchids through stem tip detoxification tissue culture. The method involves identifying the viruses carried by the Phalaenopsis orchid seedlings to be detoxified, determining the target virus species to be removed and detected, and pretreating the material to be detoxified using a combination of hot and cold treatments. The method then incorporates 0.2–0.5 mm stem tips into liquid culture, ensuring effective detoxification through skillful stem tip peeling, and guaranteeing the survival rate of the stem tips by liquid shaking culture and the addition of CPPU to promote cell division. To achieve this objective, the invention discloses the following technical details.

[0009] A method for rapid propagation of Phalaenopsis orchids via stem tip detoxification tissue culture includes the following steps.

[0010] ① Identify the viruses carried by the virus-free Phalaenopsis orchid seedlings; ② Perform cold and heat treatment on the virus-free Phalaenopsis orchid seedlings; ③ Perform shoot tip separation culture; ④ Perform shoot tip differentiation induction; ⑤ Perform virus detection on the seedlings; ⑥ Perform subculture and propagation; ⑦ Perform adventitious bud rooting.

[0011] ① To investigate the virus carried by the Phalaenopsis orchid seedlings to be detoxified, randomly select Phalaenopsis orchid seedlings to be detoxified, and inoculate their sap onto virus bioindicator plants such as Nicotiana scabra, Amaranthus chinensis, and Datura stramonium. After 30 days, take the leaves of Nicotiana scabra, Amaranthus chinensis, and Datura stramonium that show lesions to purify the virus, and perform electron microscopy to observe the virus morphology and determine the virus species. Based on the preliminary determination of the virus, perform corresponding PCR tests to further confirm the virus species carried by the Phalaenopsis orchids to be detoxified.

[0012] The second step involves the cold and heat treatment of the virus-free Phalaenopsis orchid seedlings. The seedlings to be virus-free are placed in an artificial climate chamber and subjected to 30℃ for 6 hours, 12℃ for 6 hours, and 1000 lux for 20 consecutive days.

[0013] For the third step, stem tip peeling culture, after heat and cold treatment, 10mm stem segments with stem tips were cut from Phalaenopsis orchids on a clean bench. Under a continuous zoom microscope, the young leaves were peeled away layer by layer to expose the smooth growing point. The growing point (approximately 0.1-0.5mm) with 1-2 leaf primordia was then harvested using a 'V' shaped cut to avoid contaminating the growing point with sap from other parts of the plant. The growing point was then quickly inoculated into a liquid stem tip culture medium and cultured at 26±1℃, 300-500 lux, for 12h / d with continuous shaking at 120r. During the stem tip peeling process, a new set of sterile scalpels and forceps was used after each layer of young leaves was peeled away.

[0014] The aforementioned shoot tip liquid culture uses MS-based liquid culture medium containing 0.1-0.5 mg / L CPPU, 0.01-0.1 mg / L NAA, 50-100 mg / L VC, 50-100 mg / L CA, 5-15% coconut water, 20 g / L sucrose, and pH 5.8.

[0015] The fourth step, shoot tip differentiation induction, involves liquid shaking culture of shoot tips for 15–20 days. Once the shoot tips turn green, they are promptly transferred to shoot tip differentiation induction medium and cultured at 26±1℃, 300–1500 lux, for 12 h / d. The shoot tip differentiation induction medium is MS-based and contains 2–5 mg / L 6-BA, 0.01–0.1 mg / L NAA, 50–100 mg / L VC, 50–150 mg / L CA, 5–15% coconut water, 20 g / L sucrose, and 6–7 g / L agar, with a pH of 5.8. Differentiated shoot clusters can contain 10–40 shoots, randomly distributed.

[0016] For the fifth step, the virus detection of the seedlings involves inoculating 10-15% of the stem and leaf sap from the seedling clusters onto the virus biomarker plants, including Tobacco denudata, Amaranthus chinensis, and Datura stramonium. After 30 days, the leaves of Tobacco denudata, Amaranthus chinensis, and Datura stramonium, as well as the stems and leaves of the seedling clusters, are collected for PCR detection and electron microscopy of the target virus. Seedling clusters free of virus are then selected.

[0017] The ⑥ subculture propagation refers to the process of removing browned old tissue from the bud clusters that have been tested for the target virus, cutting them directionally, and inoculating them into a propagation medium with bud height of 10-25 mm, stem diameter ≥5 mm, more than 2 unfolded leaves, leaf width ≥5 mm, and 2-3 buds / cluster. The medium is 10 clusters / cup, cultured at 26±1℃, 300-1500 lux, with a light intensity of 12 h / d, for 40-45 days, until the bud cluster propagation rate reaches 3-4%.

[0018] The subculture proliferation medium contains 2-4 g / L of Flower Treasure 1#, 2-3 mg / L of 6-BA, 0.1-4 mg / L of IBA, 0.1-0.4 mg / L of NAA, 5-10% coconut juice, 1-2 g / L of peptone, 10-50 g / L of organic matter, 50-100 mg / L of LVC, 50-150 mg / L of LCA, 20-30 g / L of sugar, 5-7 g / L of agar, and pH 5.8.

[0019] The ⑦ adventitious bud rooting refers to the process of cutting a single bud with two unfolded leaves, each leaf width ≥5mm and ￠ ≥5mm, from the proliferating bud cluster, inoculating it in a rooting medium at 15 buds / cup, at 26±1℃, 1500~2500 lux, and with a light intensity of 12h / d.

[0020] The adventitious bud rooting medium is based on MS modified medium, containing 70-100 g / L banana and 1-2 g / L LAC. After culturing for 50-60 days, when the leaf width is ≥30 mm, the ￠ is ≥5 mm, and there are 2-3 fleshy roots, the seedlings can be hardened off and transplanted.

[0021] MS was changed to: all elements except iron salts and organic matter were reduced by half.

[0022] The innovation of this invention lies in disclosing a method for rapid propagation of Phalaenopsis orchids through stem tip virus removal tissue culture. One innovation is the pre-treatment of the material to be virus-free, clearly identifying the target virus species to be removed and detected, thus solving the common problem of missed detection of other viruses due to subjective identification of the target virus. The second innovation is the use of a combination of hot and cold pretreatment of the material to be virus-free. Heat treatment accelerates the growth of stem tip meristems to expand the virus-free area of ​​the stem tip, while simultaneous hot and cold treatment inactivates the virus. Then, the stem tips (0.2–0.5 mm) are combined with liquid culture. Skilled stem tip peeling ensures the effectiveness of stem tip virus removal, and liquid shaking culture and the addition of CPPU ensure stem tip cell division and survival rate, thereby solving the common problems of low survival rate and low virus removal rate of Phalaenopsis orchid stem tips.

[0023] Virus-free stem tips divide rapidly, forming bud clusters with 30-50 buds per cluster, growing randomly; virus-free seedlings grow quickly and have good traits. Attached Figure Description

[0024] Figure 1 Phalaenopsis orchid seedlings awaiting virus removal.

[0025] Figure 2 Datura is an indicator plant whose leaves develop necrotic spots 30 days after inoculation with virus-free Phalaenopsis orchid sap.

[0026] Figure 3 Amaranthus chinensis is an indicator plant whose leaves show mosaic patterns 30 days after inoculation with virus-free Phalaenopsis orchid sap.

[0027] Figure 4 : Figure 2 , Figure 3 Virus particle A, purified from the sample, was initially identified as CyMV and magnified 50,000 times under an electron microscope.

[0028] Figure 5 : Figure 2 , Figure 3 Virus particles B purified from the sample were initially identified as ORSV, and were examined under an electron microscope at 50,000x magnification.

[0029] Figure 6 : The exposed stem tip of a Phalaenopsis orchid after the leaf primordia have been removed, magnified 400 times.

[0030] Figure 7 A detached Phalaenopsis orchid stem tip, magnified 400 times.

[0031] Figure 8 Adventitious bud clusters of Phalaenopsis orchids induced by stem tips without viruses.

[0032] Figure 9 Virus-free Phalaenopsis orchid rooted seedlings. Detailed Implementation

[0033] A method for rapid propagation of Phalaenopsis orchids via stem tip detoxification tissue culture, comprising the following steps: (The following examples are used to further illustrate the present invention, but are not intended to limit the scope of the invention).

[0034] Example 1.

[0035] ① To investigate the viruses carried by the Phalaenopsis orchid seedlings to be detoxified, 10 bottles of Phalaenopsis orchid seedlings to be detoxified were randomly selected, and 5 bud clusters were randomly selected from each bottle. The mixed sap of the stems and leaves of the buds was inoculated onto virus bioindicator plants such as Nicotiana scabra, Amaranthus chinensis, and Datura stramonium. After 30 days, the leaves of Nicotiana scabra, Amaranthus chinensis, and Datura stramonium with lesions were taken to purify the virus. Then, electron microscopy was performed to observe the virus morphology and determine that the virus was CymMV and ORSV. The corresponding PCR test was then performed to further confirm that the virus species carried by the Phalaenopsis orchids to be detoxified were CymMV and ORSV.

[0036] ② Cold and heat treatment of virus-free Phalaenopsis orchid seedlings: Take 6 bottles of Phalaenopsis orchid seedlings to be virus-free and place them in an artificial climate chamber. The temperature is 30℃ for 6 hours, 12℃ for 6 hours, and 1000 lux for 20 consecutive days.

[0037] ③ Stem tip peeling culture: After heat and cold treatment, take 10mm stem segments with stem tips from Phalaenopsis orchids. Under a clean bench, peel away the young leaves layer by layer under a continuous zoom microscope to expose the smooth growing point. Cut the growing point (approximately 0.1-0.3mm) with 1-2 leaf primordia. Use a 'V' shaped cut to avoid contaminating the growing point with sap from other parts of the plant. Immediately inoculate the growing point into the stem tip liquid culture medium (60 cups total). Incubate at 26±1℃, 300-500 lux, 12h / d, with continuous shaking at 120 rpm. Replace the sterile scalpel and forceps after each layer of young leaves is peeled. The stem tip liquid culture medium is: MS + CPPU 0.1mg / L + NAA 0.01mg / L + VC 50mg / L + CA 50mg / L + 10% coconut milk + 20g / L sucrose, pH 5.8.

[0038] ④ Shoot tip differentiation induction: Shoot tips were cultured in liquid with shaking for 18 days, resulting in 56 green shoot tips, with a survival rate of 93.3%. The greened shoot tips were promptly transferred to shoot tip differentiation induction medium and cultured at 26±1℃, 300–1500 lux, for 12 h / d. The shoot tip differentiation induction medium consisted of MS medium containing 3 mg / L 6-BA, 0.03 mg / L NAA, 100 mg / L VC, 100 mg / L CA, 10% coconut milk, 20 g / L sucrose, and 7 g / L agar, pH 5.8. The shoot tips formed bud clusters with 20–40 shoots, randomly distributed.

[0039] ⑤ Virus detection in seedlings: Ten bottles of seedlings were randomly selected, and the sap from their stems and leaves was inoculated onto plants with viral biomarkers, namely, Nicotiana scabra, Amaranthus chinensis, and Datura stramonium. After 30 days, the sap from Nicotiana scabra, Amaranthus chinensis, and Datura stramonium inoculated with the sap from the seedlings, as well as the sap from the 60 bottles of seedlings, were subjected to CymMV and ORSV PCR detection and electron microscopy detection. No CymMV, ORSV, or other viruses were detected, and the virus elimination rate was 100%.

[0040] ⑥ Subculture propagation: Take the above-mentioned virus-free meristem clusters, remove the browned old tissue, and cut them directionally. The buds should be 10-25 mm high, with a stem diameter ≥5 mm, at least 2 unfolded leaves, and a leaf width ≥5 mm. Inoculate 2-3 buds / cluster into the proliferation medium, 10 clusters / cup, at 26±1℃, 300-1500 lux, with 12 h / d light, for 40-45 days. The callus proliferation rate should reach 3-4%. Subculture propagation medium: Flower Treasure 1# 3g / L + 6-BA 3mg / L + IBA 0.2mg / L + NAA 0.1mg / L + coconut juice 5% + peptone 2g / L + banana 30g / L + VC 100mg / L + CA 100mg / L + sugar 30g / L + agar 7g / L, pH 5.8.

[0041] ⑦ Adventitious bud rooting refers to the process of cutting single buds with two unfolded leaves (each ≥5mm wide and ≥5mm in diameter) from a proliferating bud cluster, inoculating them into rooting medium at a rate of 15 buds / cup, and culturing at 26±1℃ and 1500~2500 lux with 12h / d light. The rooting medium is MS modified (all elements except iron salts and organic matter are halved) + banana 100g / L + AC 1g / L + sugar 30g / L + agar 7g / L, pH 5.8. After 60 days of culture, when the leaves are ≥30mm wide and ≥5mm in diameter, and there are 2-3 fleshy roots, the seedlings are ready for hardening off and transplanting.

[0042] Example 2.

[0043] ① To investigate the viruses carried by the Phalaenopsis orchid seedlings to be detoxified, 10 bottles of Phalaenopsis orchid seedlings to be detoxified were randomly selected, and 5 bud clusters were randomly selected from each bottle. The mixed sap of the stems and leaves of the buds was inoculated onto virus bioindicator plants such as Nicotiana scabra, Amaranthus chinensis, and Datura stramonium. After 30 days, the leaves of Nicotiana scabra, Amaranthus chinensis, and Datura stramonium with lesions were taken to purify the virus. Then, electron microscopy was performed to observe the virus morphology and determine that the virus was CymMV and ORSV. The corresponding PCR test was then performed to further confirm that the virus species carried by the Phalaenopsis orchids to be detoxified were CymMV and ORSV.

[0044] ② Cold and heat treatment of virus-free Phalaenopsis orchid seedlings: Take 6 bottles of Phalaenopsis orchid seedlings to be virus-free and place them in an artificial climate chamber. The temperature is 30℃ for 6 hours, 12℃ for 6 hours, and 1000 lux for 20 consecutive days.

[0045] ③ Stem tip peeling culture: After heat and cold treatment, take 10mm stem segments with stem tips from Phalaenopsis orchids. Under a clean bench, peel away the young leaves layer by layer under a continuous zoom microscope to expose the smooth growing point. Cut the growing point (approximately 0.3-0.6mm) with 1-2 leaf primordia, using a 'V' cut to avoid contaminating the growing point with sap from other parts of the plant. Immediately inoculate the growing point into the stem tip liquid culture medium (60 cups total). Incubate at 26±1℃, 300-500 lux, 12h / d, with continuous shaking at 120 rpm. During the stem tip peeling process, change the sterile scalpel and forceps after each layer of young leaves is peeled. The stem tip liquid culture medium is: MS + CPPU 0.2mg / L + NAA 0.01mg / L + VC 50mg / L + CA 50mg / L + 15% coconut milk + 20g / L sucrose, pH 5.8.

[0046] ④ Shoot tip differentiation induction: Shoot tips were cultured in liquid with shaking for 20 days, resulting in 60 green shoot tips with a 100% survival rate. The greened shoot tips were promptly transferred to shoot tip differentiation induction medium and cultured at 26±1℃, 300–1500 lux, for 12 h / d. The shoot tip differentiation induction medium consisted of MS medium containing 3 mg / L 6-BA, 0.01 mg / L NAA, 100 mg / L VC, 100 mg / L CA, 15% coconut milk, 20 g / L sucrose, and 7 g / L agar, pH 5.8. The shoot tips formed bud clusters with 10–20 shoots, randomly distributed.

[0047] ⑤ Virus detection in seedlings: Ten bottles of seedlings were randomly selected, and the sap from their stems and leaves was inoculated onto plants with viral biomarkers, namely, Nicotiana scabra, Amaranthus chinensis, and Datura stramonium. After 30 days, the sap from Nicotiana scabra, Amaranthus chinensis, and Datura stramonium inoculated with the sap from the seedlings, as well as the sap from the 60 bottles of seedlings, were subjected to CymMV and ORSV PCR detection and electron microscopy detection. Among them, 5 bottles showed both CymMV and ORSV, 3 bottles showed only CymMV, and 2 bottles showed only ORSV, with a virus elimination rate of 83.3%.

[0048] ⑥ Subculture propagation: Selected virus-free shoot clusters were harvested, browned old tissue was removed, and the shoots were directionally cut. The shoots were 10-25 mm high, with a stem diameter ≥5 mm, at least two unfolded leaves, and a leaf width ≥5 mm. Each cluster had 2-3 shoots and was inoculated into the propagation medium at 10 clusters per cup. The culture was carried out at 26±1℃, 300-1500 lux, with a light intensity of 12 h / d, for 40-45 days. The shoot cluster propagation rate reached 3-4%. The subculture propagation medium consisted of: Flower Power 1# 4 g / L + 6-BA 2 mg / L + IBA 0.1 mg / L + NAA 0.1 mg / L + coconut juice 10% + peptone 1 g / L + banana 50 g / L + VC 100 mg / L + CA 100 mg / L + sugar 30 g / L + agar 7 g / L, pH 5.8.

[0049] ⑦ Adventitious bud rooting refers to the process of cutting single buds with two unfolded leaves (each ≥5mm wide and ≥5mm in diameter) from a proliferating bud cluster, inoculating them into rooting medium at a rate of 15 buds / cup, and incubating at 26±1℃ and 1500~2500 lux with 12h / d light. The rooting medium is MS modified (all elements except iron salts and organic matter are halved) + banana 80g / L + AC 1g / L + sugar 30g / L + agar 7g / L, pH 5.8. After 60 days of culture, when the leaves are ≥30mm wide and ≥5mm in diameter, and there are 2-3 fleshy roots, the seedlings are ready for hardening off and transplanting.

Claims

1. A method for rapid propagation of Phalaenopsis orchids via stem tip detoxification tissue culture, comprising the following steps: 1) To investigate the virus carried by the Phalaenopsis orchid seedlings to be detoxified, randomly select Phalaenopsis orchid seedlings to be detoxified, and inoculate their sap onto virus bioindicator plants such as Nicotiana scabra, Amaranthus chinensis, and Datura stramonium. After 30 days, take the leaves of Nicotiana scabra, Amaranthus chinensis, and Datura stramonium that show lesions to purify the virus, and perform electron microscopy to observe the virus morphology and determine the virus species. Based on the preliminary determination of the virus species, perform the corresponding PCR test to further confirm the virus species carried by the Phalaenopsis orchids to be detoxified, and use it as the basis for detecting whether the shoot tip division seedlings are detoxified. 2) Cold and heat treatment of virus-free Phalaenopsis orchid seedlings: Place the Phalaenopsis orchid seedlings to be virus-free in an artificial climate chamber, at 30℃ for 6 hours and 12℃ for 6 hours per day, at 1000 lux, for 20 days. 3) Stem tip peeling culture: Take Phalaenopsis orchids after heat and cold treatment, cut 10mm stem segments with stem tips on a clean bench, peel off the young leaves layer by layer under a continuous zoom microscope to expose the smooth growing point, and cut the growing point with 1-2 leaf primordia and a length of 0.1-0.5mm. The cutting method is "V" shaped to avoid the growing point from the non-cutting parts. Quickly inoculate into the stem tip liquid culture medium and culture at 26±1℃, 300-500 lux, 12h / d, 120r with continuous shaking. When peeling the stem tip, change a set of sterile scalpels and forceps after peeling off each layer of young leaves. The stem tip liquid culture medium is MS based and contains 0.1-0.5mg / L CPPU, 0.01-0.1mg / L NAA, 50-100mg / L VC, 50-100mg / L CA, 5-15% coconut milk, 20g / L sucrose, pH 5.8; 4) Shoot tip differentiation induction: Shoot tips were cultured in liquid with shaking for 15–20 days. After turning green, they were promptly transferred to shoot tip differentiation induction medium and cultured at 26±1℃, 300–1500 lux, 12 h / d for 40–45 days. The shoot tip differentiation induction medium was MS-based and contained 2–5 mg / L 6-BA, 0.01–0.1 mg / L NAA, 50–100 mg / L VC, 50–150 mg / L CA, 5–15% coconut water, 20 g / L sucrose, 6–7 g / L agar, and pH 5.

8. The shoot tip formed a cluster of 10–40 shoots, which were randomly distributed. 5) Detection of virus in shoot clusters: Take 10-15% of the stem and leaf sap from shoot clusters and inoculate them onto virus biomarker plants such as Nicotiana scabra, Amaranthus chinensis, and Datura stramonium. After 30 days, take the sap from the leaves of Nicotiana scabra, Amaranthus chinensis, and Datura stramonium, as well as the stem and leaf sap from shoot clusters, and perform PCR and electron microscopy detection of the target virus to screen for virus-free shoot clusters. 6) Subculture propagation: Take virus-free meristems, remove browned old tissue, and cut directionally, selecting buds 10-25mm in height and stems... Bud clusters with 2 or more unfolded leaves, leaf width ≥5mm, and 2-3 buds / cluster are inoculated into subculture proliferation medium, 10 clusters / cup, and cultured at 26±1℃, 300-1500 lux, 12h / d for 40-45 days, achieving a bud cluster proliferation rate of 3-4. The subculture proliferation medium contains 2-4 g / L of Flower Treasure 1#, 2-3 mg / L of 6-BA, 0.1-4 mg / L of IBA, 0.1-0.4 mg / L of NAA, 5-10% coconut juice, 1-2 g / L of peptone, 10-50 g / L of organic matter, 50-100 mg / L of VC, 50-150 mg / L of CA, 20-30 g / L of sugar, 5-7 g / L of agar, and pH 5.

8. 7) Adventitious bud rooting: Cut off two fully expanded leaves with a width ≥ 5 mm from the proliferating bud cluster. Single buds were inoculated into adventitious bud rooting medium at a density of 15 buds / cup, and cultured at 26±1℃, 1500–2500 lux, for 12 h / d. The adventitious bud rooting medium was MS modified, containing 70–100 g / L banana, 1–2 g / L AC, 20–30 g / L sugar, and 5–7 g / L agar, pH 5.

8. After 50–60 days of culture, when the leaf width was ≥30 mm,… Harden off the seedlings and transplant them when they have 2-3 fleshy roots.

2. The method for rapid propagation of Phalaenopsis orchids via stem tip detoxification tissue culture according to claim 1, characterized in that: The composition of the shoot tip liquid culture medium in step 3) is MS + CPPU 0.1-0.2 mg / L + NAA 0.01 mg / L + VC 50 mg / L + CA 50 mg / L + coconut juice 10-15% + sucrose 20 g / L, pH 5.

8.

3. The method for rapid propagation of Phalaenopsis orchids via stem tip detoxification tissue culture according to claim 1, characterized in that: The subculture proliferation medium described in step 6) consists of Flower Treasure 1# 4g / L + 6-BA 2mg / L + IBA 0.1mg / L + NAA 0.1mg / L + coconut juice 10% + peptone 1g / L + banana 50g / L + VC 100mg / L + CA 100mg / L + sugar 30g / L + agar 7g / L, pH 5.

8.

4. The method for rapid propagation of Phalaenopsis orchids via stem tip detoxification tissue culture according to claim 1, characterized in that: The adventitious shoot rooting medium mentioned in step 7) consists of MS 改 +80-100g / L banana +1g / L AC +30g / L sugar +7g / L agar, pH 5.

8.

5. The method for rapid propagation of Phalaenopsis orchids via stem tip detoxification tissue culture according to claim 4, characterized in that: The MS 改 All elements except iron salts and organic matter are reduced by half.

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