Tissue culture and rapid propagation method of Cyperus perfoliatus

Abstract

The invention provides a method for tissue culture and rapid propagation of Cyperus perfoliatus, which comprises the following steps: taking base axillary buds of Cyperus perfoliatus as a starting material, collecting explants, cleaning and disinfecting, and carrying out primary axillary bud elongation culture, adventitious bud induction and multiplication culture, rooting culture and tissue culture seedling domestication and transplanting to obtain Cyperus perfoliatus seedlings. According to the method, the adventitious buds are directly induced by adopting the galingale perfoliata axillary bud explant to achieve the purpose of plant regeneration, the high-quality character of a maternal plant can be kept, the possibility of variation in the culture process is reduced, the propagation coefficient of the adventitious buds is 2.8-3, the rooting rate of tissue culture seedlings is 95% or above, the requirements of factory seedling production of plant seedlings are completely met, and the method is suitable for large-scale production. High-quality seedlings can be rapidly propagated in batches, the propagation efficiency is high, the seedlings grow robustly, and the supply requirement of the cyperus esculentus is met.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of plant tissue culture, and particularly relates to a method for tissue culture and rapid propagation of Ficinia truncata (Thunb.) Schrad. BACKGROUND

[0002] Ficinia truncata (Thunb.) Schrad., also known as ice crystal grass, is a perennial plant of Ficinia in Cyperaceae, and is originally from Cape Province of South Africa. It mainly grows in subtropical regions, most of which have strong environmental adaptability, long growth period, vigorous growth, developed root system, and high ornamental value, and can be used for lawn development and landscaping.

[0003] Cyperaceae plants can be propagated by seeding, division, cutting and rhizome, etc. But seed germination is greatly affected by dormancy, and the current research on seed germination technology is very little, so it limits the seed propagation; the propagation by division, cutting and rhizome is restricted by material and season, and the speed of seedling propagation is slow, which cannot meet the demand of seedling. The establishment of efficient tissue culture system by using plant tissue culture technology is the main way to produce a large number of high-quality seedlings quickly, and there have been reports on the use of tissue culture for in vitro rapid propagation of Cyperaceae. Qupingmei et al. (Qupingmei, Long Chunlin, Cheng Zhiying, et al. Tissue culture and rapid propagation of Cyperus esculentus L. Plant Physiology Communications, 2007, 43(2):1.) used Cyperus esculentus L. tubers with intact bud eyes as starting material, and obtained aseptic seedlings through bud induction, proliferation and rooting culture, with a proliferation rate of 5-10 times and a rooting rate of more than 95%. Zhou Bo et al. (Zhou Bo, Xu Weiqian, Li Na, et al. Study on tissue culture and rapid propagation of Carex duriuscula. North Horticulture, 2010, 000(017):150-152.) used total bracts of Carex duriuscula as starting material for tissue culture, and successfully established a rapid propagation system with a propagation coefficient of 8.4. M. Rewers et al. (M Rewers, AKisiala, J Drouin, E Sliwinska, E Cholewa. In vitro-regenerated wetland sedge Eriophorum vaginatum L. is genetically stable. Acta Physiologiae Plantarum, 2012 (34):2197-2206) used seeds of Eriophorum vaginatum L. as explants, and obtained 40 regenerated plants from one seed after callus induction, bud and root regeneration, and 6 months. Zhang Weiping (Zhang Weiping. Establishment of Cyperus clones. Anhui Agricultural Sciences, 2012, 40(11):2.) used Cyperus tubers as starting material, and obtained 5.8 buds from callus through callus induction and differentiation, rooting and transplanting, successfully established Cyperus clones, and obtained a large number of high-quality seedlings.Simin Lai et al (S Lai, A Menon, S Turner, A Kodym, E Bunn. Development of an in vitro protocol for a difficult-to-propagate endemic Australian dryland sedge species Mesomelaena pseudostygia (Cyperaceae). In Vitro Cellular & Developmental Biology-Plant, 2014(50):99-109) used Cyperus iria cyphellus as starting material, after 6 weeks, the bud proliferation rate was about 3.4.

[0004] At present, there is no tissue culture research on C. iria, therefore, establishing a rapid propagation system, rapidly producing high-quality seedlings in batches can further expand and popularize C. iria, and lay a solid foundation for the next step of related research work. SUMMARY

[0005] The technical problems to be solved by the present application are to provide a C. iria tissue culture and rapid propagation method, establish a C. iria tissue culture and rapid propagation technical system, maintain the excellent traits of the female parent, improve the propagation efficiency, and meet the application requirements of industrial production of seedlings.

[0006] To solve the above technical problems, the technical scheme adopted by the present application is as follows: a C. iria tissue culture and rapid propagation method, comprising the following steps: Taking C. iria axillary buds as starting material, collecting explants, washing and disinfecting, then performing primary axillary bud elongation culture, adventitious bud induction and proliferation culture, and rooting culture to obtain tissue culture seedlings; The primary culture medium formula is: MS+6-BA 0.1-0.5mg / L+NAA 0.05-0.1mg / L+activated carbon 0.1-0.3g / L+sucrose 30g / L+agar 7.0-8.0g / L, pH 5.78-5.82; The adventitious bud induction medium formula is: MS+6-BA 0.5-1.0mg / L+NAA 0.05-0.2mg / L+PVP 0.2-1.0g / L+sucrose 30g / L+agar 7.0-8.0g / L, pH 5.78-5.82 The formula of the adventitious bud proliferation medium is: MS+6-BA 0.2-0.8 mg / L+NAA 0.05-0.1 mg / L+PVP 0.5-1.0 g / L+sucrose 30 g / L+agar 7.0-8.0 g / L, pH 5.78-5.82; The formula of the rooting medium is: MS+NAA 0.1-0.5 mg / L+activated carbon 0.1-0.5 g / L+sucrose 30 g / L+agar 7.0-8.0 g / L, pH 5.78-5.82.

[0007] Preferably, the collection method of the explants is: the axillary buds of the Cynodon spinosus are stripped of the outer leaves, 1-2 layers of leaves with hard texture are reserved, the upper and lower excess parts are cut off, the surface of the base is ensured to be flat, the material with a length of 2.5 cm and a growth point at the base is reserved as the explants.

[0008] Preferably, the cleaning method of the explants is: first, the explants are soaked in a water solution of dishwashing liquid with a mass fraction of 0.2-0.5%, ultrasonic oscillation is performed for 8-10 min, and then the explants are washed clean with running water until no dishwashing liquid is left on the surface of the explants.

[0009] Preferably, the disinfection method of the explants is: first, the explants are soaked in an alcohol solution with a volume fraction of 70-75% for 30-40 s, then the explants are soaked in a sodium hypochlorite solution with an effective chlorine concentration of 0.5-1.0% for 6-8 min, and then the explants are washed with sterile water for 3-4 times.

[0010] Preferably, the elongation culture of the primary axillary buds is: the upper and lower excess parts of the disinfected explants are cut off, 1.5-2 cm is reserved, and then the explants are vertically inoculated in the primary culture medium for culture.

[0011] Preferably, the induction and proliferation culture of the adventitious buds is: the primary culture axillary buds are separated from the mother material at the base after growing to 2 cm, are vertically inoculated in the adventitious bud induction medium for culture, and the axillary buds are divided into 4-6 clusters longitudinally at the base, the upper excess leaves are cut off, the height of the adventitious buds is reserved to be 2-3 cm, and then the adventitious buds are inoculated in the adventitious bud proliferation medium for culture.

[0012] Preferably, the rooting culture is: the adventitious buds with a height not less than 3 cm after the proliferation culture are divided into single plants longitudinally at the base, the surface yellow and dry leaves are cut off, and then the adventitious buds are inoculated in the rooting medium for culture.

[0013] Preferably, the culture environment is: the temperature is 18-21 ℃, the light cycle is 12 h / d, the light intensity is 30-35 μmol·m -2 ·s -1 .

[0014] Preferably, the silver-edge cyperus tissue culture and rapid propagation method further comprises acclimatization and transplanting of the tissue culture seedlings: selecting tissue culture seedlings with consistent growth, 2-3 1-2cm adventitious roots at the base, washing the roots, cutting off part of the yellow leaves and excessively long roots, and then transplanting into the substrate, maintaining a relative humidity of 80% or more, a culture temperature of 15-25℃, and a light intensity of 2000-4000Lx.

[0015] Compared with the prior art, the present application has the following remarkable technical effects: 1. The present application uses ex vivo rapid propagation culture technology to factory-grow silver-edge cyperus seedlings, which is not affected by natural environmental factors, can produce high-quality seedlings in batches throughout the year, and meets the supply demand of silver-edge cyperus. Currently, there is no research report on the tissue culture of silver-edge cyperus.

[0016] 2. In the silver-edge cyperus tissue culture and rapid propagation method of the present application, the adventitious bud propagation coefficient is 2.8-3, and the rooting rate of the tissue culture seedlings is 95% or more, fully meeting the requirements of plant seedling factory-growing production.

[0017] 3. The present application reduces the induction of callus and the differentiation stage of callus, directly induces adventitious buds from silver-edge cyperus explants to achieve the purpose of regenerating plants, maintains the original high-quality plant traits, and reduces the possibility of variation during the culture process.

[0018] 4. The present application establishes a silver-edge cyperus tissue culture and rapid propagation system, which lays a foundation and provides a reference for future research on silver-edge cyperus and other varieties of plants in this genus.

[0019] The present application will be further described in detail below in combination with the drawings and examples. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 is a silver-edge cyperus plant of the present application; Figure 2 is a primary axillary bud elongation culture diagram; Figure 3 is an adventitious bud induction culture diagram; Figure 4 is an adventitious bud proliferation culture diagram; Figure 5 is a rooting culture diagram; Figure 6 is a plug planting diagram. DETAILED DESCRIPTION

[0021] The present application provides a silver-edge cyperus tissue culture and rapid propagation method, which uses silver-edge cyperus basal axillary buds as starting materials to collect explants, and obtains rooted tissue culture seedlings through primary axillary bud elongation culture, adventitious bud induction culture, adventitious bud proliferation culture, and rooting culture. The steps are as follows: S1, explant collection and treatment The plant of silver-edged Cyperus alternifolius was used as the starting material Figure 1 ). The roots were washed, the outer leaves were peeled off, 1-2 layers of hard leaves were reserved, the upper and lower parts were cut off, the surface of the base was ensured to be flat, 2.5 cm long material was reserved, and after being washed clean, the material was used as the explant.

[0022] The explant was treated by washing: the explant was soaked in a water solution of dishwashing liquid with a mass fraction of 0.2-0.5%, ultrasonic oscillation was performed for 8-10 min, and then the explant was washed clean with running water until no dishwashing liquid was left on the surface of the explant.

[0023] The explant was treated by disinfection: the pre-treated explant was transferred into a sterile conical flask on a prepared clean bench, 70-75% alcohol solution was added for immersion disinfection, the time was 30-40 s, the conical flask was slightly shaken horizontally during the period, then the alcohol solution was poured out, 0.5-1% sodium hypochlorite solution with an effective chlorine concentration was added for immersion disinfection, the time was 6-8 min, the conical flask was slightly shaken horizontally, the mouth of the conical flask was burned on an alcohol lamp from time to time during the period, the sodium hypochlorite solution was poured out, and finally the explant was washed with sterile water for 3-4 times.

[0024] S2, primary axillary bud elongation culture The explant after disinfection was taken out of the conical flask, 3-6 explants were taken out at a time, the upper and lower parts were cut off, 1.5-2 cm was reserved, and then the explant was vertically inoculated in the primary culture medium for primary axillary bud elongation culture, as shown in Figure 2 The formula of the primary culture medium of the application is: MS+6-BA 0.1-0.5 mg / L+NAA 0.05-0.1 mg / L+activated carbon 0.1-0.3 g / L+sucrose 30 g / L+agar 7.0-8.0 g / L, pH 5.78-5.82; further preferably MS+6-BA 0.5 mg / L+NAA 0.1 mg / L+activated carbon 0.3 g / L+sucrose 30 g / L+agar 7.0 g / L, pH 5.78-5.82.

[0025] S3, adventitious bud induction culture After the axillary bud grew to about 2 cm, the axillary bud was separated from the mother material at the base, and then the axillary bud was vertically inoculated in the adventitious bud induction culture medium for adventitious bud induction, as shown in Figure 3The adventitious bud induction medium formula of the application is: MS+6-BA 0.5-1.0 mg / L+NAA 0.05-0.2 mg / L+PVP 0.2-1.0 g / L+sucrose 30 g / L+agar 7.0-8.0 g / L, pH 5.78-5.82; further preferably MS+6-BA 1.0 mg / L+NAA 0.2 mg / L+PVP 0.8 g / L+sucrose 30 g / L+agar 7.0 g / L, pH 5.78-5.82.

[0026] S4, adventitious bud proliferation culture The induced cluster buds are longitudinally divided from the base, the upper excess leaves are cut off, the height of the remaining adventitious buds is 2-3 cm, and then inoculated into the proliferation culture medium for proliferation culture, as shown in the following table. Figure 4 The adventitious bud proliferation medium formula of the application is: MS+6-BA 0.2-0.8 mg / L+NAA 0.05-0.1 mg / L+PVP 0.5-1.0 g / L+sucrose 30 g / L+agar 7.0-8.0 g / L, pH 5.78-5.82; further preferably MS+6-BA 0.5 mg / L+NAA 0.1 mg / L+PVP 0.8 g / L+sucrose 30 g / L+agar 7.0 g / L, pH 5.78-5.82.

[0027] S5, rooting culture The adventitious buds with a height of not less than 3 cm after proliferation culture are longitudinally divided into single plants from the base, the surface yellow leaves are cut off, and then inoculated into the rooting culture medium for culture, as shown in the following table. Figure 5 The rooting culture medium formula of the application is: MS+NAA 0.1-0.5 mg / L+activated carbon 0.1-0.5 g / L+sucrose 30 g / L+agar 7.0-8.0 g / L, pH 5.78-5.82; further preferably MS+NAA 0.2 mg / L+activated carbon 0.1 g / L+sucrose 30 g / L+agar 7.0 g / L, pH 5.78-5.82.

[0028] The tissue culture environment of the application is: the culture room temperature is 18-21℃, the illumination time is 12h / d, the illumination intensity is 30-35 μmol·m -2 ·s -1 , and preferably the illumination light source is a 5000K color temperature LED tubular light source.

[0029] The application also includes: S6, acclimatization and transplanting of tissue culture seedlings The tissue culture seedlings with consistent growth vigor, 2-3 1-2 cm adventitious roots at the base were selected and moved to a greenhouse. After cleaning the roots, cutting off part of the yellow leaves and excessive roots, the seedlings were transplanted into the substrate. The relative humidity was above 80%, the culture temperature was 15-25℃, and the light intensity was 2000-4000Lx. Figure 6 is a diagram of the plug planting of Cynodon xiaozhuangii.

[0030] Example 1 The present embodiment is a tissue culture and rapid propagation method of Cynodon xiaozhuangii, and the steps are as follows: Culture medium formula The basic culture medium is MS medium, the agar powder content is 7.5g / L, and the pH of the culture medium is 5.78-5.82; The primary culture medium is MS+6-BA 0.5mg / L+NAA 0.1mg / L+activated carbon 0.3g / L+sucrose 30g / L; The adventitious bud induction medium is MS+6-BA 1.0mg / L+NAA 0.2mg / L+PVP 0.8g / L+sucrose 30g / L; The adventitious bud proliferation medium is MS+6-BA 0.5mg / L+NAA 0.1mg / L+PVP 0.8g / L+sucrose 30g / L; The rooting medium is MS+NAA 0.2mg / L+activated carbon 0.1g / L+sucrose 30g / L.

[0031] Culture environment The culture room temperature is 18-21℃, the light time is 12h / d, the light intensity is 30-35 μmol·m -2 ·s -1 , and the illumination light source is a 5000K color temperature LED tubular light source.

[0032] Selection of explants The Cynodon xiaozhuangii plants were selected as the starting material. The plants were divided into single plants, the roots were cleaned, the outer leaves were peeled off, 1-2 layers of hard leaves were reserved, the upper and lower parts were cut off, the base surface was ensured to be flat, 2.5cm length material was reserved, and after cleaning, the starting explants were obtained.

[0033] Treatment of explants The starting explants were soaked in a 0.2-0.5% aqueous solution of dishwashing liquid, ultrasonically vibrated for 8-10min, and finally the residual dishwashing liquid solution on the surface of the explants was washed off with running water.

[0034] Surface disinfection of explants On the prepared clean bench, the cleaned explants are transferred into a sterile conical flask or other container, 70-75% alcohol solution is added, the liquid surface is about 0.5 cm higher than the surface of the plant material, the conical flask is shaken slightly horizontally for 30-40 s, then the alcohol solution is poured out, 0.5-1% sodium hypochlorite solution is added, the liquid surface is about 0.5 cm higher than the surface of the plant material, the conical flask is shaken slightly horizontally for 6-8 min, the sodium hypochlorite solution is poured out, and finally the plant material sterilized by the sodium hypochlorite solution is washed with sterile water for 3-4 times.

[0035] Primary axillary bud elongation culture The explants sterilized on the conical flask are taken out, 3-6 explants are taken out each time, the upper and lower excess parts are cut off, 1.5-2 cm are reserved, and then vertical inoculation is performed in the primary culture medium for culture.

[0036] Adventitious bud induction and proliferation After the primary culture axillary bud grows to about 2 cm, it is separated from the parent material at the base, vertical inoculation is performed in the adventitious bud induction medium for culture; 4-6 adventitious buds are taken as a unit, longitudinally separated from the base, the upper excess leaves are cut off, the height of the adventitious bud is reserved to 2-3 cm, and then inoculation is performed in the adventitious bud proliferation medium for culture.

[0037] Rooting culture The adventitious bud with a height not less than 3 cm after proliferation culture is longitudinally separated into a single plant at the base, the surface yellow leaves are cut off, and then inoculation is performed in the rooting culture medium for culture.

[0038] Tissue culture seedling domestication and transplanting The tissue culture seedlings with consistent growth, 2-3 adventitious roots with a length of 1-2 cm at the base are selected for greenhouse domestication; after the roots are washed and part of the yellow leaves and excessively long roots are cut off, the seedlings are transplanted into the substrate (peat: coconut husk = 1:1), the relative humidity is kept above 80%, the culture temperature is 15-25°C, and the light intensity is 2000-4000 Lx.

[0039] Example 2 The difference from example 1 is that: The basic culture medium is MS medium, the agar powder content is 7.0 g / L, and the culture medium pH is 5.78-5.82; The primary culture medium is MS+6-BA 0.1 mg / L+NAA 0.05 mg / L+active carbon 0.1 g / L+sucrose 30 g / L; The adventitious bud induction medium is MS+6-BA 0.5 mg / L+NAA 0.05 mg / L+PVP 0.8 g / L+sucrose 30 g / L; The adventitious bud proliferation medium is MS+6-BA 0.2mg / L+NAA 0.05mg / L+PVP 0.8g / L+sucrose 30g / L; The rooting medium is MS+NAA 0.1mg / L+activated carbon 0.3g / L+sucrose 30g / L.

[0040] Example 3 The difference from Example 1 is that: The basic medium is MS medium, the agar powder content is 8.0g / L, and the medium pH is 5.78-5.82; The primary culture medium is MS+6-BA 0.25mg / L+NAA 0.1mg / L+activated carbon 0.1g / L+sucrose 30g / L; The adventitious bud induction medium is MS+6-BA 1.0mg / L+NAA 0.05mg / L+sucrose 30g / L; The adventitious bud proliferation medium is MS+6-BA 0.8mg / L+NAA 0.1mg / L+sucrose 30g / L; The rooting medium is MS+NAA 0.3mg / L+activated carbon 0.1g / L+sucrose 30g / L.

[0041] Example 4 Effect of different media on the elongation of axillary buds of Cynodon laceratus in primary culture.

[0042] Test design: The primary culture medium is MS medium, the agar powder content is 7.0-8.0g / L, the sucrose content is 30g / L, the medium pH is 5.78-5.82, and different contents of 6-BA, NAA and activated carbon are added, as shown in Table 1. The Cynodon laceratus explants are inoculated and cultured, the growth of axillary buds is observed, and the culture room environment is the same as that of Example 1.

[0043] Table 1 Effect of different media on the elongation of axillary buds of Cynodon laceratus According to the analysis in Table 1, for the primary culture of axillary buds of Cynodon laceratus explants, the medium formula is MS+6-BA 0.5mg / L+NAA 0.1mg / L+activated carbon 0.3g / L+sucrose 30g / L+agar 7.0-8.0g / L, the axillary buds grow best, which is beneficial to subsequent tissue culture.

[0044] Example 5 Effect of different media on the induction of adventitious buds of Cynodon laceratus.

[0045] Test design: Adventitious bud induction medium: the basic medium is MS medium, the agar powder content is 7.0-8.0 g / L, the sucrose content is 30 g / L, the medium pH is 5.78-5.82, and different contents of 6-BA, NAA and PVP are added, as shown in Table 2. After the axillary buds of the primary culture grow to about 2 cm, they are separated from the mother material at the base and vertically inoculated into the adventitious bud induction medium for culture. The induction rate is counted, the growth of the adventitious buds is observed, and the culture room environment is the same as in Example 1.

[0046] Table 2 Influence of different media on induction of adventitious buds of Cynodon xiaozhuangii According to the analysis in Table 2, during the induction culture stage of the adventitious buds, the medium formula is MS+6-BA 1.0 mg / L+NAA 0.2 mg / L+PVP 0.8 g / L+sucrose 30 g / L+agar 7.0-8.0 g / L, a higher induction rate is obtained, and the growth of the adventitious buds is best.

[0047] Example 6 Influence of different media on the proliferation of adventitious buds of Cynodon xiaozhuangii.

[0048] Test design: Adventitious bud proliferation medium: the basic medium is MS medium, the agar powder content is 7.0-8.0 g / L, the sucrose content is 30 g / L, the medium pH is 5.78-5.82, and different contents of 6-BA, NAA and PVP are added, as shown in Table 3. The induced multiple shoots are vertically separated from the base in units of 4-6 multiple shoots, the upper excess leaves are removed, the height of the adventitious buds is preserved at 2-3 cm, and then inoculated into the proliferation medium for proliferation culture. The proliferation rate is counted, the growth of the adventitious buds is observed, and the culture room environment is the same as in Example 1.

[0049] Table 3 Influence of different media on the proliferation of adventitious buds of Cynodon xiaozhuangii According to the analysis in Table 3, during the induction culture stage of the adventitious buds, the medium formula is MS+6-BA 1.0 mg / L+NAA 0.2 mg / L+PVP 0.8 g / L+sucrose 30 g / L+agar 7.0-8.0 g / L, a higher induction rate is obtained, and the growth of the adventitious buds is best.

[0050] Example 7 Influence of different media on the rooting culture of Cynodon xiaozhuangii.

[0051] Test design: The rooting medium is MS medium, the agar powder content is 7.0-8.0 g / L, the medium pH is 5.78-5.82, and different contents of NAA and activated carbon are added, as shown in Table 4. After proliferation culture, the adventitious buds with a height of not less than 3 cm are longitudinally divided into single plants from the base, the surface yellow leaves are cut off, and then inoculated into the rooting medium for culture, the rooting rate and the rooting number are counted, and the culture room environment is the same as that in Example 1.

[0052] Table 4 Influence of different culture media on rooting of Cynodon xiaozhuangii According to the analysis in Table 4, in the rooting culture stage, the medium formula is MS+NAA 0.2 mg / L+activated carbon 0.1 g / L+sucrose 30 g / L+agar 7.0-8.0 g / L, the rooting effect is the best, the rooting rate is the highest, the rooting state is suitable, and it is conducive to later cleaning and transplanting, and ensures the survival rate.

[0053] In conclusion, the application provides a tissue culture and rapid propagation method of Cynodon xiaozhuangii, establishes an efficient, simple and practical tissue culture and rapid propagation technical system of Cynodon xiaozhuangii, can maintain the excellent traits of the female parent, improve the propagation efficiency, and meet the application demand of seedling factory production.

[0054] The above is only a preferred embodiment of the application, and does not limit the application. Any simple modification, change and equivalent change according to the technical essence of the application to the above embodiment are still within the protection scope of the technical solution of the application.

Claims

1. A method for tissue culture and rapid propagation of Cyperus rotundus, characterized in that, Includes the following steps: Using axillary buds of Cyperus rotundus as starting material, explants were collected, cleaned and disinfected, and then tissue culture seedlings were obtained through primary axillary bud elongation culture, adventitious bud induction and proliferation culture, and rooting culture. The initial culture medium formula was: MS + 6-BA 0.1-0.5 mg / L + NAA 0.05-0.1 mg / L + activated carbon 0.1-0.3 g / L + sucrose 30 g / L + agar 7.0-8.0 g / L, pH 5.78-5.82; The adventitious bud induction medium formula is: MS + 6-BA 0.5-1.0 mg / L + NAA 0.05-0.2 mg / L + PVP 0.2-1.0 g / L + sucrose 30 g / L + agar 7.0-8.0 g / L, pH 5.78-5.

82. The formulation of the adventitious bud proliferation medium is: MS + 6-BA 0.2-0.8 mg / L + NAA 0.05-0.1 mg / L + PVP 0.5-1.0 g / L + sucrose 30 g / L + agar 7.0-8.0 g / L, pH 5.78-5.82; The rooting medium formula is: MS + NAA 0.1-0.5 mg / L + activated carbon 0.1-0.5 g / L + sucrose 30 g / L + agar 7.0-8.0 g / L, pH 5.78-5.

82.

2. The method for tissue culture and rapid propagation of Cyperus rotundus according to claim 1, characterized in that, The method for collecting the explants is as follows: peel off the outer leaves of the axillary buds of Cyperus sibirica, retain 1-2 layers of hard leaves, remove the excess parts at the top and base, ensure that the base surface is flat, and retain 2.5 cm of material with a growth point at the base as the explant.

3. The method for tissue culture and rapid propagation of Cyperus rotundus according to claim 2, characterized in that, The method for cleaning the explants is as follows: first soak them in a detergent solution with a mass fraction of 0.2-0.5%, then ultrasonically vibrate them for 8-10 minutes, and finally rinse them with running water until there is no detergent residue on the surface of the explants.

4. The method for tissue culture and rapid propagation of Cyperus rotundus according to claim 3, characterized in that, The disinfection method for the explants is as follows: first, soak them in an alcohol solution with a volume fraction of 70-75% for 30-40 seconds, then soak them in a sodium hypochlorite solution with an effective chlorine concentration of 0.5-1.0% for 6-8 minutes, and then rinse them with sterile water 3-4 times.

5. The method for tissue culture and rapid propagation of Cyperus rotundus according to claim 1, characterized in that, The primary axillary bud elongation culture is performed as follows: after disinfection, the excess upper and basal parts of the explants are cut off, leaving 1.5-2 cm, and then vertically inoculated into the primary culture medium for culture.

6. The method for tissue culture and rapid propagation of Cyperus rotundus according to claim 1, characterized in that, The adventitious bud induction and proliferation culture is as follows: after the axillary buds of the primary culture reach 2cm in length, they are separated from the parent material at the base and vertically inoculated into the adventitious bud induction medium for culture; to induce the formation of clustered buds, 4-6 clustered buds are separated longitudinally from the base, excess leaves at the top are removed, and the height of the adventitious buds is retained at 2-3cm, and then inoculated into the adventitious bud proliferation medium for culture.

7. The method for tissue culture and rapid propagation of Cyperus rotundus according to claim 1, characterized in that, The rooting culture is as follows: adventitious buds with a height of not less than 3cm after proliferation culture are selected, longitudinally divided into individual plants from the base, the withered yellow leaves on the surface are removed, and they are inoculated into rooting culture medium for culture.

8. The method for tissue culture and rapid propagation of Cyperus rotundus according to claim 1, characterized in that, The cultivation environment was as follows: temperature 18-21℃, photoperiod 12h / d, light intensity 30-35μmol·m⁻². -2 ·s -1 .

9. The method for tissue culture and rapid propagation of Cyperus rotundus according to claim 1, characterized in that, It also includes the acclimatization and transplanting of tissue culture seedlings: select tissue culture seedlings with uniform growth and 2-3 adventitious roots of 1-2cm at the base for greenhouse acclimatization; clean the roots, cut off some withered yellow leaves and excessively long roots, and plant them in the substrate, maintaining a relative humidity of over 80%, a culture temperature of 15-25℃, and a light intensity of 2000-4000Lx.

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