A method for rapid in vitro propagation of Meconopsis quinqueneri tissue culture

Abstract

The invention discloses a method for rapid in vitro propagation of Meconopsis quinqueneri tissue culture. The method of the invention uses the stem tip of Meconopsis quinqueneri as an explant, and sterilizes and rinses it by soaking in a mercuric chloride solution to obtain a sterilized explant stem tip; the sterilized explant stem tip is first inoculated on a hormone-free culture medium, and then inoculated on an induction bud differentiation culture medium to form 2 to 3 clustered buds; the clustered buds are inoculated on a subculture proliferation culture medium to form Meconopsis quinqueneri clustered seedlings; the Meconopsis quinqueneri clustered seedlings are then divided and transferred to a rooting culture medium for culture to form complete regenerated seedlings; finally, the complete regenerated seedlings are transplanted into a seedling culture medium, and after hardening, they grow into normal Meconopsis quinqueneri plants. The method of the invention overcomes the problems of long growth cycle and low reproduction coefficient of Meconopsis quinqueneri seedlings, is easy to operate, and can be used for rapid factory seedling cultivation.

Description

Technical Field

[0001] The invention relates to the technical field of tissue culture and in vitro rapid propagation, and in particular to a method for in vitro rapid propagation of Meconopsis quinqueneri tissue culture. Background Art

[0002] Meconopsis quintupinenia Rege1. is a plant of the genus Meconopsis Vig. of the family Papaveraceae. Its Tibetan name is "Ou Shi Wan Bao" and it is also known as Wild Golden Lotus (Handbook of Common Chinese Herbal Medicines in Qinghai). It is a traditional Tibetan medicine and is widely used among the people. It is distributed in Tibet, Shaanxi, Gansu, Qinghai, Sichuan and other places. It mainly grows in alpine meadows and shady slope shrubs at an altitude of 2300-4600m. The whole plant is used as medicine, which has the effects of clearing heat, treating hepatitis, cholecystitis, pneumonia, tuberculosis, gastric ulcer, etc. As an important variety of alpine medicinal plant Meconopsis, Meconopsis quintupinenia Rege1 is used in more than 30 Tibetan medicine formulas, mainly used to treat hepatitis, pneumonia, headache, edema and other symptoms, and has a particularly good analgesic effect. The research on Meconopsis quinquenervii in the literature mainly focuses on resources, morphology and histology, chemical composition, pharmacological activity, clinical application, etc. Meconopsis quinquenervii contains a variety of chemical components and has a variety of significant pharmacological effects, with broad development and application prospects. Qinghai Province is one of the main production areas of Meconopsis quinquenervii, which has the characteristics of wide distribution, high ornamental value and treatment of diseases. At present, with the further development of science and technology, the application of Meconopsis quinquenervii is becoming more and more extensive, but the development of its natural resources has been in a disorderly state, resulting in its wild resources tending to the edge of exhaustion. In order to rationally develop Meconopsis resources and promote the sustainable utilization and healthy development of wild resources, the artificial development of Meconopsis quinquenervii is also gradually carried out.

[0003] So far, Meconopsis quinqueneri is wild. Although it is widely distributed, Meconopsis quinqueneri medicinal materials have long relied on wild excavation to meet domestic and foreign market demand. Germplasm resources have been severely damaged and reserves have been sharply reduced. The population of Meconopsis quinqueneri has been devastated, causing its resources to be unable to meet market needs. Especially in recent years, with the increase in demand for Meconopsis quinqueneri at home and abroad, the contradiction between supply and demand has become increasingly prominent. In addition, driven by profit, a large amount of predatory excavation has put the resources of Meconopsis quinqueneri in danger of depletion. It is imperative to protect germplasm resources and expand the breeding of Meconopsis quinqueneri.

[0004] Plant tissue culture technology utilizes the totipotency of cells (i.e., the genetic potential of differentiated cells of an individual organ or tissue to regenerate into a complete individual), takes cell clusters or tissues from individual plants, and uses artificially prepared culture media to make these cell clusters or tissues form thousands of plants. The use of tissue culture for in vitro rapid propagation has the following important significances: (1) The propagation speed is fast and is not affected by external climatic factors. Rapid and large-scale propagation can be carried out indoors all year round; (2) It is easy to maintain excellent biological characteristics; (3) It is of great significance in preserving and breeding excellent mutants or excellent hybrids. A mutant of Meconopsis quinqueneri with obvious advantages or a progeny with obvious advantages can be widely promoted after tissue culture, etc.

[0005] In existing studies, tissue culture of wild alpine medicinal materials, such as Saussurea involucrata and Rhodiola rosea, has been widely reported, but tissue culture of wild Meconopsis quinqueneri stem tips as explants has not been reported. Explants collected from the wild breed a large number of microorganisms such as bacteria and fungi on their surfaces, some of which even grow into the tissues; in addition, Meconopsis quinqueneri contains a lot of quinones and phenolic substances, which are easily oxidized and browned. Direct cultivation of Meconopsis quinqueneri seedlings will result in a long cycle and low reproduction coefficient; therefore, the selection of explant materials and disinfection methods are very critical.

[0006] To this end, the present invention adopts the stem apex explant tissue culture method for the first time to carry out in vitro propagation research on wild Meconopsis quinqueneri germplasm, which provides a new way to protect Meconopsis quinqueneri germplasm resources, carry out genetic improvement, promote factory-based seedling cultivation of Meconopsis quinqueneri, and obtain bioactive substances of Meconopsis quinqueneri from tissue culture products. Summary of the invention

[0007] Based on the above technical problems, the object of the present invention is to provide a method for rapid in vitro propagation of Meconopsis quinquenervais tissue culture that is easy to operate and can be used for large-scale factory production.

[0008] The present invention protects a method for rapid in vitro propagation of Meconopsis quinqueneri tissue culture, which specifically comprises the following steps:

[0009] Step 1, selection and treatment of explants: using the stem tip of Meconopsis quinqueneri as the explant, washing it with running water for 15 to 30 minutes, washing it with sterile deionized water for 4 to 5 times, then placing the washed explant in a 0.2% mercuric chloride solution, immersing and sterilizing it for 4 to 5 minutes, and washing it with sterile deionized water for 4 to 6 times, and finally drying it with sterile filter paper to obtain the sterilized stem tip of the explant;

[0010] Step 2, explant inoculation: the explant stem tips after disinfection are first inoculated on a hormone-free medium, and then inoculated on a bud differentiation induction medium after 7 to 15 days, wherein 2 to 3 explant stem tips after disinfection are inoculated in every 25 ml of the bud differentiation induction medium; the temperature is 23°C ± 2°C, the light source is a fluorescent lamp, and the light intensity is 30 to 60umol.m -2 .s -1 , light exposure time is 12h.d -1 Cultivate under the conditions of 25-30 days to form 2-3 clustered buds at the base of the stem tip;

[0011] Step 3, subculture proliferation culture: inoculate the 2 to 3 clustered buds into the subculture proliferation medium, wherein 1 to 2 clustered buds are inoculated into every 25 ml of the subculture proliferation medium; at a temperature of 23°C ± 2°C, a light source of fluorescent lamp, and a light intensity of 30 to 60 umol.m -2 .s -1 , light exposure time is 12h.d -1 Cultivate under the conditions of 25 to 35 days to form clustered seedlings of Meconopsis quinqueneri;

[0012] Step 4, rooting induction: when the five-veined Meconopsis clump seedlings grow to 3-4 cm, the plantlets are divided, the vigorously growing seedlings are separated from the clump seedlings, and then transferred to the rooting medium, wherein 2-3 five-veined Meconopsis clump seedlings are inoculated into each 25 ml of the rooting medium; the temperature is 23°C ± 2°C, the light source is a fluorescent lamp, and the light intensity is 30-60umol.m -2 .s -1 , light exposure time is 12h.d -1 After culturing for 20 to 30 days under the conditions of , white, strong and short roots appeared at the base of the clump seedlings and a complete root system grew out, indicating that the complete regenerated seedlings were obtained;

[0013] Step 5, sterilizing the seedling medium: mixing humus and perlite in a weight ratio of 2:1 to form a mixed medium, sterilizing the mixed medium at a temperature of 115-121° C., and obtaining a sterilized seedling medium after 15-20 minutes; the total porosity of the seedling medium is 70-75%, and the organic matter content is 20%;

[0014] Step 6, plant transplanting: directly transplant the complete regenerated seedlings into the sterilized seedling medium, and after hardening, grow into normal Meconopsis quinqueneri plants; the complete regenerated seedlings are planted 10 cm above the sterilized seedling medium. 2 within the range.

[0015] Furthermore, in step 1, the source plant of Meconopsis quintupinenia Rege1. is a herbaceous plant of the Papaveraceae family.

[0016] Furthermore, in step 2, the hormone-free culture medium refers to a 1000ml beaker, in which 100ml of macroelement mother solution, 10ml of trace element mother solution, 10ml of organic component mother solution, 5ml of iron salt mother solution, 4ml of citric acid mother solution, 2ml of Vc mother solution, and 30g of sucrose are added in sequence, and then double distilled water is added to the 1000ml mark, and after stirring, 1mM NaOH solution is added to adjust the pH value to 5.8, and finally 5.0g of agar powder is added, and each 25ml is packaged into a bottle in a 100ml conical flask, and sterilized at 121°C.

[0017] Furthermore, in the step 2, the bud differentiation induction medium refers to a 1000ml beaker, in which 100ml of a large amount of element mother solution, 10ml of a trace element mother solution, 10ml of an organic component mother solution, 5ml of an iron salt mother solution, 4ml of a citric acid mother solution, 2ml of a Vc mother solution, 30g of sucrose, 20ml of a 0.1mg / ml 6-benzylaminoadenine mother solution, and 10ml of a 0.1mg / ml naphthaleneacetic acid mother solution are added in sequence, and then double distilled water is added to the 1000ml mark, and after stirring, 1mM NaOH solution is added to adjust the pH value to 5.8, and finally 5.0g of agar powder is added, and each 25ml is packaged into a bottle in a 100ml conical flask, and sterilized at 121°C.

[0018] Furthermore, in the step 3, the subculture proliferation medium refers to a 1000ml beaker, in which 100ml of a large amount of element mother solution, 10ml of a trace element mother solution, 10ml of an organic component mother solution, 5ml of an iron salt mother solution, 4ml of a citric acid mother solution, 2ml of a Vc mother solution, 30g of sucrose, 10ml of a 0.1mg / ml 6-benzylaminoadenine mother solution, and 5ml of a 0.1mg / ml zeatin mother solution are added in sequence, and then double distilled water is added to the 1000ml mark, and after stirring, 1mM NaOH solution is added to adjust the pH value to 5.8, and finally 5.0g of agar powder is added, and the mixture is packaged into 100ml Erlenmeyer flasks in an amount of 25ml each, and sterilized at 121°C.

[0019] Furthermore, in step 4, the rooting medium refers to a 1000ml beaker, in which 50-100ml of a large amount of element mother solution, 5-10ml of a trace element mother solution, 5-10ml of an organic component mother solution, 2.5-5ml of an iron salt mother solution, 4ml of a citric acid mother solution, 2ml of a Vc mother solution, 15-30g of sucrose, and 5-10ml of a 0.1mg / ml naphthaleneacetic acid mother solution are added in sequence, then double distilled water is added to the 1000ml mark, and after stirring, 1mM NaOH solution is added to adjust the pH value to 5.8, and finally 6.0g of agar powder is added, and the mixture is packaged into a bottle of 25ml each in a 100ml conical flask, and sterilized at 121°C.

[0020] Further, the macroelement mother liquor refers to a mixed solution containing 16.5 g / L ammonium nitrate, 19.0 g / L potassium nitrate, 3.7 g / L magnesium sulfate, 1.7 g / L potassium dihydrogen phosphate, and 4.4 g / L calcium chloride; the trace element mother liquor refers to a mixed solution containing 1.68 g / L manganese sulfate, 0.86 g / L zinc sulfate, 0.62 g / L boric acid, 0.083 g / L potassium iodide, 0.025 g / L sodium molybdate, 0.0025 g / L copper sulfate, and 0.0025 g / L cobalt chloride. mixed solution; the organic component mother liquor refers to a mixed solution containing 0.2g / L of glycine, 0.04g / L of thiamine hydrochloride, 0.05g / L of pyridoxine hydrochloride, 0.05g / L of nicotinic acid, and 0.1g / L of inositol; the iron salt mother liquor refers to a mixed solution containing 5.58g / L of ferrous sulfate and 7.46mg / L of Na2.EDTA.2H2O; the citric acid mother liquor refers to a solution containing 10mg / mL of citric acid; the Vc mother liquor refers to a solution containing 1mg / mL of ascorbic acid.

[0021] Compared with the existing technology, the present invention has the following beneficial effects:

[0022] (1) The present invention is the first to use stem tip explants for in vitro rapid propagation through bioengineering technology - tissue culture technology, thereby overcoming the problems of long seedling raising period and low propagation coefficient of Meconopsis quinqueneri; at the same time, research on in vitro propagation of wild Meconopsis quinqueneri germplasm resources is carried out, providing a new way to protect rare and endangered Meconopsis quinqueneri germplasm resources, carry out genetic improvement, and promote factory-based seedling raising of Meconopsis quinqueneri, and providing a tissue culture in vitro propagation method for artificial propagation of Meconopsis quinqueneri, in vitro preservation and utilization of germplasm resources, and breeding of high medicinal ingredient content.

[0023] (2) Since the culture medium used in the present invention contains the plant hormone 6-BA, which belongs to the cytokinin class and has a dominant role in inducing bud differentiation, the induction time is short, the seedlings emerge quickly, the proliferation frequency is high, and in particular, the seedlings emerge uniformly, and the seedlings can survive directly after hardening. It is easy to operate and can be produced on a large scale in a factory.

[0024] (3) The present invention utilizes tissue culture for in vitro rapid propagation, which has the advantages of fast propagation speed, being unaffected by external climatic factors, and being able to rapidly and massively propagate indoors all year round; it is easy to maintain excellent biological characteristics, i.e., good synchronization; it is of great significance in preserving and breeding excellent mutants or excellent hybrids; a mutant of Meconopsis quinqueneri with obvious advantages or an offspring with obvious advantages can be widely promoted after tissue culture, etc. DETAILED DESCRIPTION

[0025] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] Example 1

[0027] A method for rapid in vitro propagation of Meconopsis quinqueneri tissue culture, comprising the following steps:

[0028] Step 1, using the stem tip of Meconopsis quinquenervii, a papaveraceae herbaceous plant, as an explant, washing it with running water for 15 minutes, washing it with sterile deionized water for 3 times, placing the washed explant in a 0.2% mercuric chloride solution, immersing it for 4 minutes for sterilization, washing it with sterile deionized water for 3 times, and finally drying it with sterile filter paper to obtain a sterilized explant stem tip;

[0029] Step 2, explant inoculation: the explant stem tips after disinfection are first inoculated on a hormone-free medium, and then inoculated on a bud differentiation induction medium after 7 days, wherein 2 to 3 explant stem tips after disinfection are inoculated in every 25 ml of the bud differentiation induction medium; the temperature is 23°C ± 2°C, the light source is a fluorescent lamp, and the light intensity is 30 to 60umol.m -2 .s -1 , light exposure time is 12h.d -1 Cultivate under the conditions of 25 days to form 2 to 3 clustered buds at the base of the stem tip;

[0030] The hormone-free culture medium refers to a 1000ml beaker, in which 100ml of a large amount of element mother solution, 10ml of a trace element mother solution, 10ml of an organic component mother solution, 5ml of an iron salt mother solution, 4ml of a citric acid mother solution, 2ml of a Vc mother solution, and 30g of sucrose are added in sequence, and then double distilled water is added to the 1000ml mark, and after stirring, 1mM NaOH solution is added to adjust the pH value to 5.8, and finally 5.0g of agar powder is added, and each 25ml is divided into a bottle in a 100ml triangular flask, and sterilized at 121°C.

[0031] The bud differentiation induction medium is prepared by sequentially adding 100 ml of a macroelement mother solution, 10 ml of a trace element mother solution, 10 ml of an organic component mother solution, 5 ml of an iron salt mother solution, 4 ml of a citric acid mother solution, 2 ml of a Vc mother solution, 30 g of sucrose, 20 ml of a 6-benzylaminoadenine (6-BA) mother solution with a concentration of 0.1 mg / ml, and 10 ml of a naphthaleneacetic acid (NAA) mother solution with a concentration of 0.1 mg / ml into a 1000 ml beaker, then adding double distilled water to the 1000 ml mark, stirring evenly, adding 1 mM NaOH solution to adjust the pH value to 5.8, and finally adding 5.0 g of agar powder, packaging the mixture into 25 ml bottles in 100 ml triangular flasks, and sterilizing the mixture at 121° C.

[0032] Step 3, subculture proliferation culture: inoculate the 2 to 3 clustered buds into the subculture proliferation medium, wherein 1 to 2 clustered buds are inoculated into every 25 ml of the subculture proliferation medium; at a temperature of 23°C ± 2°C, a light source of fluorescent lamp, and a light intensity of 30 to 60 umol.m -2 .s -1 , light exposure time is 12h.d -1 Under the conditions of 25 days, the clustered seedlings of Meconopsis quinqueneri were formed;

[0033] The subculture proliferation medium is prepared by sequentially adding 100 ml of a macroelement mother solution, 10 ml of a trace element mother solution, 10 ml of an organic component mother solution, 5 ml of an iron salt mother solution, 4 ml of a citric acid mother solution, 2 ml of a Vc mother solution, 30 g of sucrose, 10 ml of a 6-benzylaminoadenine (6-BA) mother solution with a concentration of 0.1 mg / ml, and 5 ml of a zeatin (ZT) mother solution with a concentration of 0.1 mg / ml into a 1000 ml beaker, then adding double distilled water to the 1000 ml mark, stirring evenly, adding 1 mM NaOH solution to adjust the pH value to 5.8, and finally adding 5.0 g of agar powder, packaging the mixture into 100 ml Erlenmeyer flasks of 25 ml each, and sterilizing the mixture at 121° C.

[0034] Step 4, rooting induction: when the five-veined Meconopsis clump seedlings grow to 3-4 cm, the plantlets are divided, the vigorously growing seedlings are separated from the clump seedlings, and then transferred to the rooting medium, wherein 2-3 five-veined Meconopsis clump seedlings are inoculated into each 25 ml of the rooting medium; the temperature is 23°C ± 2°C, the light source is a fluorescent lamp, and the light intensity is 30-60umol.m -2 .s -1 , light exposure time is 12h.d -1 After culturing for 20 to 25 days under the conditions of , white, strong short roots appear at the base of the clump seedlings and a complete root system grows out, i.e., a complete regenerated seedling is obtained; wherein the complete regenerated seedling refers to a young plant that has completed the differentiation of buds and roots, has grown small leaves, and can grow independently and autotrophically;

[0035] The rooting medium is prepared by sequentially adding 100 ml of a macroelement mother solution, 10 ml of a trace element mother solution, 10 ml of an organic component mother solution, 5 ml of an iron salt mother solution, 4 ml of a citric acid mother solution, 2 ml of a Vc mother solution, 30 g of sucrose, and 10 ml of a 0.1 mg / ml naphthaleneacetic acid mother solution into a 1000 ml beaker, then adding double distilled water to the 1000 ml mark, stirring evenly, adding 1 mM NaOH solution to adjust the pH value to 5.8, and finally adding 6.0 g of agar powder, packaging the mixture into 25 ml bottles in 100 ml triangular flasks, and sterilizing the mixture at 121° C.

[0036] Step 5, sterilizing the seedling medium: mixing humus and perlite in a weight ratio of 2:1 to form a mixed medium, sterilizing the mixed medium at a temperature of 115-121° C., and obtaining a sterilized seedling medium after 15-20 minutes; the total porosity of the seedling medium is 70-75%, and the organic matter content is 20%;

[0037] Step 6, plant transplanting: directly transplant the complete regenerated seedlings into the sterilized seedling medium, and after hardening, grow into normal Meconopsis quinqueneri plants; the complete regenerated seedlings are planted 10 cm above the sterilized seedling medium. 2 Within the range, the survival rate is generally above 70%.

[0038] The mother liquor of macroelements refers to a mixed solution containing 16.5 g / L ammonium nitrate, 19.0 g / L potassium nitrate, 3.7 g / L magnesium sulfate, 1.7 g / L potassium dihydrogen phosphate, and 4.4 g / L calcium chloride; the mother liquor of trace elements refers to a mixed solution containing 1.68 g / L manganese sulfate, 0.86 g / L zinc sulfate, 0.62 g / L boric acid, 0.083 g / L potassium iodide, 0.025 g / L sodium molybdate, 0.0025 g / L copper sulfate, and 0.0025 g / L cobalt chloride; the mother liquor of organic components refers to a mixed solution containing 0.2 g / L glycine, 0.04 g / L thiamine hydrochloride, 0.05 g / L pyridoxine hydrochloride, 0.05 g / L nicotinic acid, and 0.1 g / L inositol; the mother liquor of iron salts refers to a mixed solution containing 5.58 g / L ferrous sulfate, 5.58 g / L Na2.EDTA.2H2O 7.46 mg / L of mixed solution; citric acid mother solution refers to a solution containing 10 mg / mL of citric acid; Vc mother solution refers to a solution containing 1 mg / mL of ascorbic acid.

[0039] Example 2

[0040] A method for rapid in vitro propagation of Meconopsis quinqueneri tissue culture, comprising the following steps:

[0041] Step 1, using the stem tip of Meconopsis quinquenervii, a papaveraceae herbaceous plant, as an explant, washing it with running water for 20 minutes, washing it with sterile deionized water for 4 times, placing the washed explant in a 0.2% mercuric chloride solution, immersing it for sterilization for 4.5 minutes, washing it with sterile deionized water for 4 times, and finally drying it with sterile filter paper to obtain a sterilized explant stem tip;

[0042] Step 2, explant inoculation: the explant stem tips after disinfection are first inoculated on a hormone-free medium, and then inoculated on a bud differentiation induction medium after 10 days, wherein 2 to 3 explant stem tips after disinfection are inoculated in every 25 ml of the bud differentiation induction medium; the temperature is 23°C ± 2°C, the light source is a fluorescent lamp, and the light intensity is 30 to 60umol.m -2 .s -1 , light exposure time is 12h.d -1 Cultivate under the conditions of 28 days to form 2 to 3 clustered buds at the base of the stem tip;

[0043] The hormone-free culture medium refers to a 1000ml beaker, in which 100ml of a large amount of element mother solution, 10ml of a trace element mother solution, 10ml of an organic component mother solution, 5ml of an iron salt mother solution, 4ml of a citric acid mother solution, 2ml of a Vc mother solution, and 30g of sucrose are added in sequence, and then double distilled water is added to the 1000ml mark, and after stirring, 1mM NaOH solution is added to adjust the pH value to 5.8, and finally 5.0g of agar powder is added, and each 25ml is divided into a bottle in a 100ml triangular flask, and sterilized at 121°C.

[0044] The bud differentiation induction medium is prepared by sequentially adding 100 ml of a macroelement mother solution, 10 ml of a trace element mother solution, 10 ml of an organic component mother solution, 5 ml of an iron salt mother solution, 4 ml of a citric acid mother solution, 2 ml of a Vc mother solution, 30 g of sucrose, 20 ml of a 0.1 mg / ml 6-benzylaminoadenine mother solution, and 10 ml of a 0.1 mg / ml naphthaleneacetic acid mother solution into a 1000 ml beaker, then adding double distilled water to the 1000 ml mark, stirring evenly, adding 1 mM NaOH solution to adjust the pH value to 5.8, and finally adding 5.0 g of agar powder, packaging the mixture into 25 ml bottles in 100 ml triangular flasks, and sterilizing the mixture at 121° C.

[0045] Step 3, subculture proliferation culture: inoculate the 2 to 3 clustered buds into the subculture proliferation medium, wherein 1 to 2 clustered buds are inoculated into every 25 ml of the subculture proliferation medium; at a temperature of 23°C ± 2°C, a light source of fluorescent lamp, and a light intensity of 30 to 60 umol.m -2 .s -1 , light exposure time is 12h.d -1Cultivate under the conditions of 25 to 35 days to form clustered seedlings of Meconopsis quinqueneri;

[0046] The subculture proliferation medium is prepared by sequentially adding 100 ml of a macroelement mother solution, 10 ml of a trace element mother solution, 10 ml of an organic component mother solution, 5 ml of an iron salt mother solution, 4 ml of a citric acid mother solution, 2 ml of a Vc mother solution, 30 g of sucrose, 10 ml of a 0.1 mg / ml 6-benzylaminoadenine mother solution, and 5 ml of a 0.1 mg / ml zeatin mother solution into a 1000 ml beaker, then adding double distilled water to the 1000 ml mark, stirring evenly, adding 1 mM NaOH solution to adjust the pH value to 5.8, and finally adding 5.0 g of agar powder, packaging the mixture into 100 ml Erlenmeyer flasks of 25 ml each, and sterilizing the mixture at 121° C.

[0047] Step 4, rooting induction: when the five-veined Meconopsis clump seedlings grow to 3-4 cm, the plantlets are divided, the vigorously growing seedlings are separated from the clump seedlings, and then transferred to the rooting medium, wherein 2-3 five-veined Meconopsis clump seedlings are inoculated into each 25 ml of the rooting medium; the temperature is 23°C ± 2°C, the light source is a fluorescent lamp, and the light intensity is 30-60umol.m -2 .s -1 , light exposure time is 12h.d -1 After culturing for 20 to 30 days under the conditions of , white, strong short roots appear at the base of the clump seedlings and a complete root system grows out, i.e., a complete regenerated seedling is obtained; wherein the complete regenerated seedling refers to a young plant that has completed the differentiation of buds and roots, has grown small leaves, and can grow independently and autotrophically;

[0048] The rooting medium is prepared by sequentially adding 50 ml of macroelement mother solution, 5 ml of trace element mother solution, 5 ml of organic component mother solution, 2.5 ml of iron salt mother solution, 4 ml of citric acid mother solution, 2 ml of Vc mother solution, 30 g of sucrose, and 10 ml of 0.1 mg / ml naphthaleneacetic acid mother solution into a 1000 ml beaker, then adding double distilled water to the 1000 ml mark, stirring evenly, adding 1 mM NaOH solution to adjust the pH value to 5.8, and finally adding 6.0 g of agar powder, packaging the mixture into 25 ml bottles in 100 ml conical flasks, and sterilizing the mixture at 121° C.

[0049] Step 5, sterilizing the seedling medium: mixing humus and perlite in a weight ratio of 2:1 to form a mixed medium, sterilizing the mixed medium at a temperature of 115-121° C., and obtaining a sterilized seedling medium after 15-20 minutes; the total porosity of the seedling medium is 70-75%, and the organic matter content is 20%;

[0050] Step 6, plant transplanting: directly transplant the complete regenerated seedlings into the sterilized seedling medium, and after hardening, grow into normal Meconopsis quinqueneri plants; the complete regenerated seedlings are planted 10 cm above the sterilized seedling medium. 2 Within the range, the survival rate is generally above 70%.

[0051] The mother liquor of macroelements refers to a mixed solution containing 16.5 g / L ammonium nitrate, 19.0 g / L potassium nitrate, 3.7 g / L magnesium sulfate, 1.7 g / L potassium dihydrogen phosphate, and 4.4 g / L calcium chloride; the mother liquor of trace elements refers to a mixed solution containing 1.68 g / L manganese sulfate, 0.86 g / L zinc sulfate, 0.62 g / L boric acid, 0.083 g / L potassium iodide, 0.025 g / L sodium molybdate, 0.0025 g / L copper sulfate, and 0.0025 g / L cobalt chloride; the mother liquor of organic components refers to a mixed solution containing 0.2 g / L glycine, 0.04 g / L thiamine hydrochloride, 0.05 g / L pyridoxine hydrochloride, 0.05 g / L nicotinic acid, and 0.1 g / L inositol; the mother liquor of iron salts refers to a mixed solution containing 5.58 g / L ferrous sulfate, 5.58 g / L Na2.EDTA.2H2O 7.46 mg / L of mixed solution; citric acid mother solution refers to a solution containing 10 mg / mL of citric acid; Vc mother solution refers to a solution containing 1 mg / mL of ascorbic acid.

[0052] Example 3

[0053] A method for rapid in vitro propagation of Meconopsis quinqueneri tissue culture, comprising the following steps:

[0054] Step 1, using the stem tip of Meconopsis quinquenervii, a papaveraceae herbaceous plant, as an explant, washing it with running water for 30 minutes, washing it with sterile deionized water for 5 times, placing the washed explant in a 0.2% mercuric chloride solution, immersing it for 5 minutes for sterilization, washing it with sterile deionized water for 5 times, and finally drying it with sterile filter paper to obtain a sterilized explant stem tip;

[0055] Step 2, explant inoculation: the explant stem tips after disinfection are first inoculated on a hormone-free medium, and then inoculated on a bud differentiation induction medium after 15 days, wherein 2 to 3 explant stem tips after disinfection are inoculated in every 25 ml of the bud differentiation induction medium; the temperature is 23°C ± 2°C, the light source is a fluorescent lamp, and the light intensity is 30 to 60umol.m -2 .s -1 , light exposure time is 12h.d -1 Cultivate under the conditions of 30 days to form 2 to 3 clustered buds at the base of the stem tip;

[0056] The hormone-free culture medium refers to a 1000ml beaker, in which 100ml of a large amount of element mother solution, 10ml of a trace element mother solution, 10ml of an organic component mother solution, 5ml of an iron salt mother solution, 4ml of a citric acid mother solution, 2ml of a Vc mother solution, and 30g of sucrose are added in sequence, and then double distilled water is added to the 1000ml mark, and after stirring, 1mM NaOH solution is added to adjust the pH value to 5.8, and finally 5.0g of agar powder is added, and each 25ml is divided into a bottle in a 100ml triangular flask, and sterilized at 121°C.

[0057] The bud differentiation induction medium is prepared by sequentially adding 100 ml of a macroelement mother solution, 10 ml of a trace element mother solution, 10 ml of an organic component mother solution, 5 ml of an iron salt mother solution, 4 ml of a citric acid mother solution, 2 ml of a Vc mother solution, 30 g of sucrose, 20 ml of a 0.1 mg / ml 6-benzylaminoadenine mother solution, and 10 ml of a 0.1 mg / ml naphthaleneacetic acid mother solution into a 1000 ml beaker, then adding double distilled water to the 1000 ml mark, stirring evenly, adding 1 mM NaOH solution to adjust the pH value to 5.8, and finally adding 5.0 g of agar powder, packaging the mixture into 25 ml bottles in 100 ml triangular flasks, and sterilizing the mixture at 121° C.

[0058] Step 3, subculture proliferation culture: inoculate the 2 to 3 clustered buds into the subculture proliferation medium, wherein 1 to 2 clustered buds are inoculated into every 25 ml of the subculture proliferation medium; at a temperature of 23°C ± 2°C, a light source of fluorescent lamp, and a light intensity of 30 to 60 umol.m -2 .s -1 , light exposure time is 12h.d -1 Under the conditions of 35 days, the five-veined Meconopsis quinquefolia cluster seedlings were formed;

[0059] The subculture proliferation medium is prepared by sequentially adding 100 ml of a macroelement mother solution, 10 ml of a trace element mother solution, 10 ml of an organic component mother solution, 5 ml of an iron salt mother solution, 4 ml of a citric acid mother solution, 2 ml of a Vc mother solution, 30 g of sucrose, 10 ml of a 0.1 mg / ml 6-benzylaminoadenine mother solution, and 5 ml of a 0.1 mg / ml zeatin mother solution into a 1000 ml beaker, then adding double distilled water to the 1000 ml mark, stirring evenly, adding 1 mM NaOH solution to adjust the pH value to 5.8, and finally adding 5.0 g of agar powder, packaging the mixture into 100 ml Erlenmeyer flasks of 25 ml each, and sterilizing the mixture at 121° C.

[0060] Step 4, rooting induction: when the five-veined Meconopsis clump seedlings grow to 3-4 cm, the plantlets are divided, the vigorously growing seedlings are separated from the clump seedlings, and then transferred to the rooting medium, wherein 2-3 five-veined Meconopsis clump seedlings are inoculated into each 25 ml of the rooting medium; the temperature is 23°C ± 2°C, the light source is a fluorescent lamp, and the light intensity is 30-60umol.m -2 .s -1 , light exposure time is 12h.d -1 After culturing for 20 to 30 days under the conditions of , white, strong and short roots appeared at the base of the clump seedlings and a complete root system grew out, indicating that the complete regenerated seedlings were obtained;

[0061] The rooting medium is prepared by sequentially adding 50 ml of macroelement mother solution, 5 ml of trace element mother solution, 5 ml of organic component mother solution, 2.5 ml of iron salt mother solution, 4 ml of citric acid mother solution, 2 ml of Vc mother solution, 15 g of sucrose, and 5 ml of 0.1 mg / ml naphthaleneacetic acid mother solution into a 1000 ml beaker, then adding double distilled water to the 1000 ml mark, stirring evenly, adding 1 mM NaOH solution to adjust the pH value to 5.8, and finally adding 6.0 g of agar powder, packaging the mixture into 25 ml bottles in 100 ml conical flasks, and sterilizing the mixture at 121° C.

[0062] Step 5, sterilizing the seedling medium: mixing humus and perlite in a weight ratio of 2:1 to form a mixed medium, sterilizing the mixed medium at a temperature of 115-121° C., and obtaining a sterilized seedling medium after 15-20 minutes; the total porosity of the seedling medium is 70-75%, and the organic matter content is 20%;

[0063] Step 6, plant transplanting: directly transplant the complete regenerated seedlings into the sterilized seedling medium, and after hardening, grow into normal Meconopsis quinqueneri plants; the complete regenerated seedlings are planted 10 cm above the sterilized seedling medium. 2 Within the range, the survival rate is generally above 70%.

[0064] The mother liquor of macroelements refers to a mixed solution containing 16.5 g / L ammonium nitrate, 19.0 g / L potassium nitrate, 3.7 g / L magnesium sulfate, 1.7 g / L potassium dihydrogen phosphate, and 4.4 g / L calcium chloride; the mother liquor of trace elements refers to a mixed solution containing 1.68 g / L manganese sulfate, 0.86 g / L zinc sulfate, 0.62 g / L boric acid, 0.083 g / L potassium iodide, 0.025 g / L sodium molybdate, 0.0025 g / L copper sulfate, and 0.0025 g / L cobalt chloride; the mother liquor of organic components refers to a mixed solution containing 0.2 g / L glycine, 0.04 g / L thiamine hydrochloride, 0.05 g / L pyridoxine hydrochloride, 0.05 g / L nicotinic acid, and 0.1 g / L inositol; the mother liquor of iron salts refers to a mixed solution containing 5.58 g / L ferrous sulfate, 5.58 g / L Na2.EDTA.2H2O 7.46 mg / L of mixed solution; citric acid mother solution refers to a solution containing 10 mg / mL of citric acid; Vc mother solution refers to a solution containing 1 mg / mL of ascorbic acid.

[0065] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for rapid in vitro propagation of Meconopsis quinqueneri tissue culture, characterized in that: The specific steps include: Step 1, selection and treatment of explants: The stem tip of Meconopsis quinqueneri is used as an explant, rinsed with running water for 15 to 30 minutes, rinsed with sterile deionized water for 4 to 5 times, and then placed in a 0.2% mercuric chloride solution for immersion sterilization for 4 to 5 minutes, and rinsed with sterile deionized water for 4 to 6 times, and finally dried with sterile filter paper to obtain the sterilized stem tip of the explant: Step 2, explant inoculation: the explant stem tips after disinfection are first inoculated on a hormone-free medium, and then inoculated on a bud differentiation induction medium after 7 to 15 days, wherein 2 to 3 explant stem tips after disinfection are inoculated in every 25 ml of the bud differentiation induction medium; the temperature is 23°C ± 2°C, the light source is a fluorescent lamp, and the light intensity is 30 to 60umol.m -2 .s -1 , light exposure time is 12h.d -1 Cultivate under the conditions of 25-30 days to form 2-3 clustered buds at the base of the stem tip; The hormone-free culture medium refers to a 1000ml beaker, in which 100ml of a large amount of element mother solution, 10ml of a trace element mother solution, 10ml of an organic component mother solution, 5ml of an iron salt mother solution, 4ml of a citric acid mother solution, 2ml of a Vc mother solution, and 30g of sucrose are added in sequence, and then double distilled water is added to the 1000ml mark, and after stirring, 1mM NaOH solution is added to adjust the pH value to 5.8, and finally 5.0g of agar powder is added, and each 25ml is divided into a bottle in a 100ml triangular flask, and sterilized at 121°C. The bud differentiation induction medium is prepared by sequentially adding 100 ml of a macroelement mother solution, 10 ml of a trace element mother solution, 10 ml of an organic component mother solution, 5 ml of an iron salt mother solution, 4 ml of a citric acid mother solution, 2 ml of a Vc mother solution, 30 g of sucrose, 20 ml of a 0.1 mg / ml 6-benzylaminoadenine mother solution, and 10 ml of a 0.1 mg / ml naphthaleneacetic acid mother solution into a 1000 ml beaker, then adding double distilled water to the 1000 ml mark, stirring evenly, adding 1 mM NaOH solution to adjust the pH value to 5.8, and finally adding 5.0 g of agar powder, packaging the mixture into 25 ml bottles in 100 ml triangular flasks, and sterilizing the mixture at 121° C. Step 3, subculture proliferation culture: inoculate the 2 to 3 clustered buds into the subculture proliferation medium, wherein 1 to 2 clustered buds are inoculated into every 25 ml of the subculture proliferation medium; at a temperature of 23°C ± 2°C, a light source of fluorescent lamp, and a light intensity of 30 to 60 umol.m -2 .s -1 , light exposure time is 12h.d -1 Cultivate under the conditions of 25 to 35 days to form clustered seedlings of Meconopsis quinqueneri; The subculture proliferation medium is prepared by sequentially adding 100 ml of a macroelement mother solution, 10 ml of a trace element mother solution, 10 ml of an organic component mother solution, 5 ml of an iron salt mother solution, 4 ml of a citric acid mother solution, 2 ml of a Vc mother solution, 30 g of sucrose, 10 ml of a 0.1 mg / ml 6-benzylaminoadenine mother solution, and 5 ml of a 0.1 mg / ml zeatin mother solution into a 1000 ml beaker, then adding double distilled water to the 1000 ml mark, stirring evenly, adding 1 mM NaOH solution to adjust the pH value to 5.8, and finally adding 5.0 g of agar powder, packaging the mixture into 100 ml Erlenmeyer flasks of 25 ml each, and sterilizing the mixture at 121° C. Step 4, rooting induction: when the clump seedlings of Meconopsis quinqueneri grow to 3 to 4 cm, the clump seedlings are divided, the vigorously growing seedlings are separated from the clump seedlings, and then transferred to the rooting medium, wherein 2 to 3 clump seedlings of Meconopsis quinqueneri are inoculated into each 25 ml of the rooting medium; the temperature is 23°C ± 2°C, the light source is a fluorescent lamp, and the light intensity is 30 to 60umol.m -2 .s -1 , light exposure time is 12h.d -1 After culturing for 20 to 30 days under the conditions of , white, strong and short roots appeared at the base of the clump seedlings and a complete root system grew out, indicating that the complete regenerated seedlings were obtained; The rooting medium is prepared by sequentially adding 50-100 ml of a macroelement mother solution, 5-10 ml of a trace element mother solution, 5-10 ml of an organic component mother solution, 2.5-5 ml of an iron salt mother solution, 4 ml of a citric acid mother solution, 2 ml of a Vc mother solution, 15-30 g of sucrose, and 5-10 ml of a 0.1 mg / ml naphthaleneacetic acid mother solution into a 1000 ml beaker, then adding double distilled water to the 1000 ml mark, stirring evenly, adding 1 mM NaOH solution to adjust the pH value to 5.8, and finally adding 6.0 g of agar powder, packaging the mixture into 100 ml triangular flasks of 25 ml each, and sterilizing the mixture at 121° C. Step 5, sterilizing the seedling medium: mixing humus and perlite in a weight ratio of 2:1 to form a mixed medium, sterilizing the mixed medium at a temperature of 115-121° C., and obtaining a sterilized seedling medium after 15-20 minutes; the total porosity of the seedling medium is 70-75%, and the organic matter content is 20%; Step 6, plant transplanting: directly transplant the complete regenerated seedlings into the sterilized seedling medium, and after hardening, grow into normal Meconopsis quinqueneri plants; the complete regenerated seedlings are planted 10 cm above the sterilized seedling medium. 2 within the scope of The macroelement mother liquor refers to a mixed solution containing 16.5 g / L ammonium nitrate, 19.0 g / L potassium nitrate, 3.7 g / L magnesium sulfate, 1.7 g / L potassium dihydrogen phosphate, and 4.4 g / L calcium chloride; the trace element mother liquor refers to a mixed solution containing 1.68 g / L manganese sulfate, 0.86 g / L zinc sulfate, 0.62 g / L boric acid, 0.083 g / L potassium iodide, 0.025 g / L sodium molybdate, 0.0025 g / L copper sulfate, and 0.0025 g / L cobalt chloride; the organic component mother liquor refers to a mixed solution containing 0.2 g / L glycine, 0.04 g / L thiamine hydrochloride, 0.05 g / L pyridoxine hydrochloride, 0.05 g / L nicotinic acid, and 0.1 g / L inositol; the iron salt mother liquor refers to a mixed solution containing 5.58 g / L ferrous sulfate, Na2·EDTA·2H2O The citric acid mother solution refers to a solution containing 10 mg / mL of citric acid; the Vc mother solution refers to a solution containing 1 mg / mL of ascorbic acid.

2. The method for rapid in vitro propagation of Meconopsis quinqueneri tissue culture according to claim 1, characterized in that: In the step 1, the source plant of Meconopsis quintupinenia Rege1 is the herbaceous plant Meconopsis quintupinenia Rege1 of the Papaveraceae family.