High-quality seedling propagation method for tissue culture of rubus coreanus

Abstract

The invention discloses a tissue culture high-quality seedling propagation method for rosaceae rubus L plant rosaceae rubus L, which is suitable for tissue culture and rapid propagation of rosaceae rubus L plant rosaceae rubus L, and belongs to the technical field of plant biology. The teasel-free rubus coreanus is a medicinal and edible small berry which is widely planted in China at present, the teasel-free rubus coreanus is obtained through the steps of callus induction and differentiation, adventitious bud rooting and seedling strengthening, test-tube plantlet transplanting and the like by utilizing an optimized culture medium formula and an aseptic seedling propagation method by taking a sterilized teasel-free rubus coreanus branch stem section as an explant, and the teasel-free rubus coreanus is obtained through the steps of rooting and seedling strengthening of adventitious buds, test-tube plantlet transplanting and the like. And a tissue culture and rapid propagation system of the high-quality seedlings of the rubus coreanus is established for the first time. The method has the characteristics of simple plant tissue culture equipment, high success rate, high propagation efficiency, good seedling quality and the like. The method provides technical support for large-scale production of the rubus coreanus, and has a good market prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a method for propagating high-quality seedlings of Rubus niveus Thunb. var. kuntzeanus (Hance) Bailey without thorns by tissue culture. Background Art

[0002] Rubus niveus Thunb. var. kuntzeanus (Hance) Bailey Rubus niveus belongs to the genus Rubus of the Rosaceae family. Its fruits are dark purple (commonly known as thornless blackberries), sweet and sour, rich in anthocyanins, ellagic acid, flavonoids, amino acids and various vitamins. Especially, the content of superoxide dismutase is the highest among fruits, with antioxidant, health care and beauty effects. It is an important functional small berry with both medicinal and edible uses. However, its purple-red or green branches are sparsely covered with hooked prickles, which affects the popularization of this plant. In recent years, with the discovery of wild thornless mutants and subsequent artificial improvement, picking and processing have become more convenient, and it is widely planted in the southwestern region of China, and the industrial scale is constantly expanding. However, at present, the artificial cultivation of Rubus niveus Thunb. var. kuntzeanus (Hance) Bailey mainly uses propagation methods such as cutting, which has problems such as low breeding efficiency, difficult rooting of cuttings, pathogen transmission, and decline in yield and quality, and cannot meet the market demand for Rubus niveus Thunb. var. kuntzeanus (Hance) Bailey seedlings.

[0003] The innovation of the present invention lies in the first use of tissue culture technology to achieve the rapid propagation of Rubus niveus Thunb. var. kuntzeanus (Hance) Bailey, improving the breeding efficiency and survival rate, reducing the production cost, and also laying a foundation for the construction of the genetic transformation system of Rubus niveus Thunb. var. kuntzeanus (Hance) Bailey. At present, there is no report on the related research of tissue culture of Rubus niveus Thunb. var. kuntzeanus (Hance) Bailey. Regarding the establishment of the tissue culture system of the genus Rubus, mainly the stem segments with axillary buds are used as explants, and the sterile buds are germinated and proliferated to expand the propagation. There are few reports on the induction of callus in the stem segment part for breeding seedlings. Through the induction and differentiation of callus from the stem segments of Rubus niveus Thunb. var. kuntzeanus (Hance) Bailey, a large number of adventitious buds can be obtained, with a high multiplication coefficient and a fast growth rate. At the same time, it can lay a foundation for the construction of genetic transformation systems such as genetic improvement and gene editing of Rubus niveus Thunb. var. kuntzeanus (Hance) Bailey. It is expected that the present invention will promote the upgrading of genetic improvement technology of Rubus niveus Thunb. var. kuntzeanus (Hance) Bailey and the sustainable development of the industry. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for propagating high-quality seedlings of Rubus niveus Thunb. var. kuntzeanus (Hance) Bailey by tissue culture to solve the problems raised in the above background art. The present invention is achieved through the following technical solutions, including the following steps: (1) Selection and sterilization of explants: Select the young stem segments of Rubus niveus Thunb. var. kuntzeanus (Hance) Bailey plants as explants, wash them clean, and perform sterilization treatment; (2) Induction of callus: Use a sterile scalpel to cut the disinfected stem segments of Erythrina suberosa into lengths of about 0.5 cm, place them on the callus induction medium, and culture them in the dark at a temperature of 23 - 25°C. After 5 - 7 days of culture, transfer them to a light culture environment with a light intensity of 2500 - 3500 lux and a culture temperature of 23 - 25°C, maintaining a light-dark cycle of 16 h / 8 h.

[0005] (3) Differentiation of adventitious buds: The induced callus continues to be cultured under light conditions on the induction medium until small buds differentiate.

[0006] (4) Proliferation of adventitious buds: Use a sterile scalpel to cut the clustered buds obtained by differentiation, transfer them to the proliferation medium for adventitious bud proliferation culture, and the light and temperature conditions during the culture process are the same as those for callus induction.

[0007] (5) Rooting culture: Transfer the proliferated Erythrina suberosa plants to the rooting medium, culture them under light for 12 h with a light intensity of 2500 - 3500 lux and a culture temperature of 23 - 25°C, maintaining a light-dark cycle of 12 h / 12 h.

[0008] (6) Transplanting of tissue-cultured seedlings: When the height of the Erythrina suberosa tissue-cultured seedlings reaches about 5 cm, take out the tissue-cultured seedlings and carry out acclimatization and transplantation, paying attention to shading, watering, and maintaining humidity and temperature.

[0009] Preferably, in step (1), the young stem segments of the newly growing and disease-free branches on the Erythrina suberosa plants are used as explants. Remove the redundant leaves, cut them into small pieces, soak them in a solution containing 0.1% detergent for 10 min, then rinse them with tap water for 0.5 h, immediately transfer them to the ultra-clean workbench, disinfect them with 75% ethanol solution for 30 - 40 s, wash them with sterile water 3 - 5 times, disinfect them with 0.1% mercuric chloride solution for 3 - 7 min, and then wash them with sterile water 3 - 5 times, and dry them with a sterilized filter paper.

[0010] Preferably, in step (2), the cut segment length is 0.5 cm, the induction medium contains MS medium, and the rest are added with 30 g / L sucrose, 7.5 g / L agarose, 0.5 - 1.5 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D), 1.0 - 1.5 mg / L thidiazuron (TDZ), 0.05 - 0.1 mg / L naphthaleneacetic acid (NAA), adjust the pH value to 5.7 - 5.9, the dark culture condition is dark culture at 23 - 25°C for 5 - 7 days, the light culture condition is a light intensity of 2500 - 3500 lux, a culture temperature of 23 - 25°C, with light and darkness alternating, and the light-dark cycle is 16 h / 8 h.

[0011] Preferably, in the step (4), the proliferation medium comprises MS medium, and additionally contains 30 g / L sucrose, 7.5 g / L agarose, 0.3 - 0.7 ml / L 3-indolebutyric acid (IBA), 0.05 - 0.3 mg / L 6-benzyladenine (6-BA), and the pH value is adjusted to 5.7 - 5.9.

[0012] Preferably, in the step (5), the rooting medium comprises 1 / 2 MS medium, and additionally contains 30 g / L sucrose, 7.5 g / L agarose, 0.3 - 0.5 ml / L 3-indolebutyric acid (IBA), 0.3 - 0.5 mg / L 6-benzyladenine (6-BA), and the pH value is adjusted to 5.7 - 6.0.

[0013] Preferably, in the step (6), the obtained rooted tissue culture seedlings are transferred to a greenhouse, acclimatized for 5 - 8 days under natural conditions, then the bottle mouths are opened and left for another 3 - 5 days, and then the tissue culture seedlings are taken out, the medium on the roots is washed, and transplanted into a mixed substrate of peat soil : perlite : vermiculite = 2:1:1.

[0014] On the other hand, the present invention also provides the application of any of the above methods in the rapid propagation of Rubus niveus Thunb. tissue culture.

[0015] The present invention relates to an efficient and simple method for breeding sterile seedlings of Rubus niveus Thunb. The invention selects the young stem segments of the newly growing, young and disease-free branches on the thornless Rubus niveus Thunb. plants as explants, and through a series of plant tissue culture techniques, including the acquisition and disinfection of sterile explants, the induction of callus, the differentiation and proliferation of adventitious buds, rooting culture, and the transplantation of tissue culture seedlings, etc., finally breeds high-quality sterile seedlings of thornless Rubus niveus Thunb. At each culture stage, the present invention selects appropriate culture formulas and culture conditions according to the growth characteristics of Rubus niveus Thunb. to ensure the efficient dedifferentiation and redifferentiation of explants, so as to successfully obtain high-quality seedlings. These seedlings can meet the market demand for high-quality Rubus niveus Thunb. seedlings, and are also suitable for scientific research purposes such as the construction of genetic transformation systems.

[0016] The invention is not only simple and fast in technology, but also high in application value, and can significantly improve the efficiency and accuracy of seedling raising. The equipment required for implementing the invention is simple, only basic plant tissue culture facilities are needed. This makes the method easy to promote and apply in production practice, providing a new technical approach for the industrialized breeding of Rubus niveus Thunb.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The present invention realizes for the first time the callus tissue culture induction of the stem segments of thornless Rubus niveus Thunb., and the propagation method of callus differentiation and seedling raising. It effectively solves the problems of difficult differentiation and germination and difficult rooting in the process of tissue culture rapid propagation of thornless Rubus niveus Thunb.

[0018] 2. Compared with the traditional cutting propagation method, the present invention provides a more convenient, faster and more efficient propagation technology for Rubus niveus Thunb. var. tsaii (Focke) Yu et Lu without thorns. This method significantly improves the efficiency of seedling breeding, and at the same time avoids common problems such as root rot and leaf yellowing during cutting. Since there is no need for cumbersome management, the present invention not only reduces the workload of the operator, but also can efficiently obtain excellent sterile seedlings. This method is very suitable for large-scale production, and can provide a stable and high-quality supply of seedlings for related industries to meet the market demand for seedlings of Rubus niveus Thunb. var. tsaii (Focke) Yu et Lu without thorns.

[0019] 3. Compared with the traditional method of inducing buds from axillary buds of explants, the present invention has a higher proliferation coefficient of directly inducing buds from callus, and at the same time the cultivation period is shortened. These advantages make the present invention very suitable for large-scale propagation of sterile seedlings of Rubus niveus Thunb., and can efficiently meet the market demand for seedlings of Rubus niveus Thunb. var. tsaii (Focke) Yu et Lu without thorns.

[0020] 4. The present invention is not only applicable to the propagation of Rubus niveus Thunb. var. tsaii (Focke) Yu et Lu without thorns, but its principle and technology can be widely applied to other species of Rubus plants by adjusting the culture conditions and medium components. Especially for those woody plants with similar growth characteristics, the present invention provides an efficient and reliable propagation method. This breakthrough will open up new ways for the commercial cultivation and promotion of Rubus plants, and at the same time provide important technical support for the breeding work of other woody plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is the explant of Rubus niveus Thunb. used.

[0022] Figure 2 is the callus induced from Rubus niveus Thunb.

[0023] Figure 3 is the mixture of callus and differentiated buds induced from Rubus niveus Thunb.

[0024] Figure 4 is the high-quality seedlings of Rubus niveus Thunb. obtained by tissue culture proliferation.

[0025] Figure 5 is the rooting culture of high-quality seedlings in tissue culture.

[0026] Figure 6 is the high-quality seedlings of Rubus niveus Thunb. after transplantation. DETAILED DESCRIPTION OF THE INVENTION

[0027] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0028] The experimental methods in the following embodiments are all conventional methods or carried out according to the conditions recommended by the manufacturers, unless otherwise specified.

[0029] The test materials and reagents used in the following embodiments can be obtained from commercial channels, unless otherwise specified.

[0030] Preparation of the culture medium: Callus induction culture: MS medium supplemented with 30 g / L sucrose, 7.5 g / L agarose, 0.5 - 1.5 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D), 1.0 - 1.5 mg / L thidiazuron (TDZ), 0.05 - 0.1 mg / L naphthaleneacetic acid (NAA), pH 5.7 - 5.9, sterilized at high temperature; Adventitious bud differentiation culture: MS medium supplemented with 30 g / L sucrose, 7.5 g / L agarose, 0.5 - 1.5 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D), 1.0 - 1.5 mg / L thidiazuron (TDZ), 0.05 - 0.1 mg / L naphthaleneacetic acid (NAA), pH 5.7 - 5.9, sterilized at high temperature; Proliferation culture: MS medium supplemented with 30 g / L sucrose, 7.5 g / L agarose, 0.3 - 0.7 ml / L 3-indolebutyric acid (IBA), 0.05 - 0.3 mg / L 6-benzyladenine (6-BA), pH 5.7 - 5.9, sterilized at high temperature; Rooting culture: 1 / 2MS medium supplemented with 30 g / L sucrose, 7.5 g / L agarose, 0.3 - 0.5 ml / L 3-indolebutyric acid (IBA), 0.3 - 0.5 mg / L 6-benzyladenine (6-BA), pH 5.7 - 6.0, sterilized at high temperature. Example 1

[0031] (1)Disinfection measures for explants: Select the young stem segments of the newly growing, tender and disease-free branches on the Rubus niveus Thunb. plant as explants. Cut off the leaves of the explants and cut them into small pieces. Immerse the materials in a solution containing 0.1% dishwashing liquid for 10 min, then rinse with tap water for 0.5 h. Immediately transfer them to the ultra-clean workbench, disinfect with 75% ethanol solution for 30 - 40 s, wash with sterile water 4 times, then disinfect with 0.1% mercuric chloride solution for 5 min, and wash with sterile water 4 times. Use sterile filter paper to absorb the moisture ( Figure 1 ).

[0032] (2)Callus induction: Cut a stem segment about 0.5 cm long and inoculate it into the induction medium. The disinfection success rate is 62%. Cultivate at 23℃ in the dark. After 7 days of cultivation, transfer to 23℃ with light, light intensity 2500 lux, and light time 16 h. After 7 days, 93% of the callus is induced ( Figure 2 ).

[0033] The medium used is MS medium, supplemented with 30 g / L sucrose, 7.5 g / L agarose, 1.5 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D), 1.0 mg / L thidiazuron (TDZ), 0.1 mg / L naphthaleneacetic acid (NAA), pH 5.8. Mix all components evenly and sterilize at high temperature.

[0034] (3)Differentiation of adventitious buds: The callus is continuously cultured in the induction medium. After 20 days, small buds differentiate from the callus ( Figure 3 ).

[0035] (4)Proliferation of adventitious buds: Cut the clustered buds with a length of 1 - 2 cm induced and transfer them to the proliferation medium for proliferation culture of adventitious buds. Keep at 23℃ with light, light intensity 2500 lux, and light time 16 h. Wait until the plants are strong ( Figure 4 ), and the plants can be subcultured every 18 days.

[0036] The medium used is MS medium, supplemented with 30 g / L sucrose, 7.5 g / L agarose, 0.5 mg / L 3-indolebutyric acid (IBA), 0.1 mg / L 6-benzyladenine (6-BA), pH 5.8. Mix all components evenly and sterilize at high temperature.

[0037] (5)Rooting culture: The sterile seedlings of Rubus niveus Thunb. grow to about 3 - 4 cm in height. Cut off a single small seedling from the base of the plant and inoculate it into the rooting medium. Keep at 23℃ with light, light intensity 2500 lux, and light time 12 h. After 20 days, the rooting rate can reach 81% ( Figure 5 ).

[0038] The culture medium used was 1 / 2 MS medium supplemented with 30 g / L sucrose, 7.5 g / L agarose, 0.3 mg / L 3-indolebutyric acid (IBA), 0.5 mg / L 6-benzyladenine (6-BA), pH 5.8. The components were mixed evenly and sterilized at high temperature.

[0039] (6)Transplantation of tissue-cultured seedlings: When the rooted tissue-cultured seedlings of Rubus niveus Thunb. were about 5 cm tall, the sterile seedlings were transferred to the greenhouse and acclimatized for 7 days under natural conditions. Then, the bottle mouths were opened and left for another 3 days. The tissue-cultured seedlings were taken out, the culture medium on the roots was washed off, and they were transplanted into a mixed substrate of peat soil: perlite: vermiculite = 2:1:1, paying attention to shading, watering, and maintaining moisture and temperature ( Figure 6 ). Example 2

[0040] (1)Disinfection measures for explants: Select the young stem segments of the newly growing, tender, and disease-free branches on the Rubus niveus Thunb. plant as explants. Cut off the leaves of the explants and cut them into small pieces. Immerse the materials in a solution containing 0.1% detergent for 10 min, then rinse with tap water for 0.5 h. Immediately transfer them to the ultra-clean workbench, disinfect with 75% ethanol solution for 30 - 40 s, wash with sterile water 3 times, then disinfect with 0.1% mercuric chloride solution for 3 min, and wash with sterile water 3 times. Use sterile filter paper to blot dry the moisture.

[0041] (2)Callus induction: Cut stem segments about 0.5 cm long and inoculate them onto the induction medium. The disinfection success rate was 40%. Cultivate at 23℃ in the dark. After 7 days of cultivation, transfer to 23℃ with light, light intensity 2500 lux, and light time 16 h. After 7 days, 83% of the callus was induced.

[0042] The culture medium used was MS medium supplemented with 30 g / L sucrose, 7.5 g / L agarose, 0.5 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D), 1.5 mg / L thidiazuron (TDZ), 0.1 mg / L naphthaleneacetic acid (NAA), pH 5.8. The components were mixed evenly and sterilized at high temperature.

[0043] (3)Differentiation of adventitious buds: The callus was continuously cultured in the induction medium, and small buds differentiated from the callus after 25 days.

[0044] (4)Proliferation of adventitious buds: Cut the clustered buds of 1 - 2 cm induced and transfer them to the proliferation medium for proliferation culture of adventitious buds. At 23℃ with light, light intensity 2500 lux, and light time 16 h, the plants can be subcultured every 26 days.

[0045] The culture medium used was MS medium supplemented with 30 g / L sucrose, 7.5 g / L agarose, 0.3 mg / L 3-indolebutyric acid (IBA), 0.05 mg / L 6-benzyladenine (6-BA), pH 5.8. All components were mixed evenly and sterilized at high temperature.

[0046] (5)Rooting culture: When the aseptic seedlings of Rubus niveus Thunb. grew to about 3 - 4 cm in height, individual small seedlings were cut from the base of the plant and inoculated into the rooting medium. The light was set at 23 °C, the light intensity was 2500 lux, and the light duration was 12 h. After 20 days, the rooting rate could reach 67%.

[0047] The culture medium used was 1 / 2MS medium supplemented with 30 g / L sucrose, 7.5 g / L agarose, 0.3 mg / L 3-indolebutyric acid (IBA), 0.3 mg / L 6-benzyladenine (6-BA), pH 5.8. All components were mixed evenly and sterilized at high temperature.

[0048] (6)Transplanting of tissue-cultured seedlings: When the rooted tissue-cultured seedlings of Rubus niveus Thunb. were about 5 cm in height, the aseptic seedlings were transferred to the greenhouse and acclimatized for 7 days under natural conditions. Then, the bottle mouths were opened and left for another 3 days. The tissue-cultured seedlings were taken out, the medium on the roots was washed clean, and they were transplanted into a mixed substrate of peat soil: perlite: vermiculite = 2:1:1. Attention should be paid to shading, watering, and maintaining moisture and temperature. Example 3

[0049] (1)Disinfection measures for explants: Select the young stem segments of the newly growing, tender and disease-free branches on the Rubus niveus Thunb. plant as explants. Cut off the leaves of the explants and cut them into small pieces. Put the materials into a solution containing 0.1% detergent and soak for 10 min, then rinse with tap water for 0.5 h. Immediately transfer to the ultra-clean workbench, disinfect with 75% ethanol solution for 30 - 40 s, wash with sterile water 5 times, then disinfect with 0.1% mercuric chloride solution for 7 min, and wash with sterile water 5 times. Use sterile filter paper to absorb the moisture.

[0050] (2)Callus induction: Cut stem segments about 0.5 cm and inoculate them into the induction medium. The disinfection success rate was 80%. Cultivate at 23 °C in the dark. After 7 days, set the light at 23 °C, the light intensity at 2500 lux, and the light duration at 16 h. After 7 days, 78% of the callus was induced.

[0051] The culture medium used was MS medium supplemented with 30 g / L sucrose, 7.5 g / L agarose, 1.5 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D), 1.5 mg / L thidiazuron (TDZ), 0.05 mg / L naphthaleneacetic acid (NAA), pH 5.8. All components were mixed evenly and sterilized at high temperature.

[0052] (3) Differentiation of adventitious buds: The callus was continuously cultured in the induction medium, and small buds differentiated from the callus after 23 days.

[0053] (4) Proliferation of adventitious buds: The clustered buds with a length of 1 - 2 cm induced were cut and transferred to the proliferation medium for the proliferation culture of adventitious buds. The light was at 23 °C, the light intensity was 2500 lux, and the light time was 16 h. The plants could be subcultured every 22 days.

[0054] The medium used was MS medium, supplemented with 30 g / L sucrose, 7.5 g / L agarose, 0.7 mg / L 3 - indolebutyric acid (IBA), 0.3 mg / L 6 - benzyladenine (6 - BA), pH 5.8. Each component was mixed evenly and sterilized at high temperature.

[0055] (5) Rooting culture: When the aseptic seedlings of Rubus niveus Thunb. grew to about 3 - 4 cm in height, single small seedlings were cut from the base of the plants and inoculated into the rooting medium. The light was at 23 °C, the light intensity was 2500 lux, and the light time was 12 h. The rooting rate could reach 59% after 20 days.

[0056] The medium used was 1 / 2MS medium, supplemented with 30 g / L sucrose, 7.5 g / L agarose, 0.5 mg / L 3 - indolebutyric acid (IBA), 0.3 mg / L 6 - benzyladenine (6 - BA), pH 5.8. Each component was mixed evenly and sterilized at high temperature.

[0057] (6) Transplanting of tissue - cultured seedlings: When the rooted tissue - cultured seedlings of Rubus niveus Thunb. were about 5 cm in height, the aseptic seedlings were transferred to the greenhouse and acclimatized for 7 days under natural conditions, then the bottle mouths were opened and left for another 3 days. The tissue - cultured seedlings were taken out, the medium on the roots was washed clean, and transplanted into the mixed substrate of peat soil: perlite: vermiculite = 2:1:1, paying attention to shading, watering, and maintaining moisture and temperature.

[0058] Although specific embodiments and examples of the present invention have been disclosed, it should not be construed as a limitation of the present invention. The protection scope of the present invention is defined by the scope defined in the appended claims. For those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for propagating high-quality seedlings of thornless red thorn vine tissue culture, characterized in that: The following steps are involved: (1) Explant selection and sterilization: Select young stem segments of the plant of Rhizoma Cyperi as explants, clean them, and sterilize them; (2) Callus induction: Use a sterile scalpel to cut the disinfected red thorn vine stem segments, place them on the callus induction medium, and culture them in the dark at a temperature of 23-25°C. After 5-7 days of culture, transfer them to a light culture environment with a light intensity of 2500-3500 lux and a culture temperature of 23-25°C, maintaining a light-dark cycle of 16h / 8h; (3) Differentiation of adventitious buds: The callus tissue is cultured in the induction medium in step (2) under light conditions until the callus tissue differentiates into small buds; (4) Proliferation of adventitious buds: Use a sterile scalpel to cut the differentiated cluster buds and transfer them to a proliferation medium for adventitious bud proliferation culture. The light and temperature conditions during the culture process are consistent with the callus induction conditions. (5) Rooting culture: The proliferated Echinops erythrorhizon plants were transferred to the rooting medium and cultured under illumination for 12 h, with a light intensity of 2500-3500 lux, a culture temperature of 23-25 ​​°C, and a light / dark cycle of 12 h / 12 ​​h. (6) Transplantation of tissue culture seedlings: When the height of the tissue culture seedlings of Rhizoma Cibotii reaches 5 cm, take out the tissue culture seedlings and carry out seedling hardening and transplanting.

2. The method for producing high-quality seedlings of Rhizoma Cibotii according to claim 1, characterized in that: In the step (1), the young stem segments of new branches of the red thorn vine that grow normally and are free of diseases are used as explants, and the excess leaves are removed, cut into small pieces, and placed in a solution containing 0.1% detergent for 10 minutes, and then rinsed with tap water for 0.5 hours, and then transferred to an ultra-clean workbench, disinfected with 75% ethanol solution for 30-40 seconds, washed with sterile water for 3-5 times, disinfected with 0.1% mercuric chloride solution for 3-7 minutes, and then washed with sterile water for 3-5 times, and the moisture is absorbed by sterilized filter paper.

3. The method for producing high-quality seedlings of Rhizoma Cibotii according to claim 1, characterized in that: In the step (2), the length of the cut segment is 0.5 cm, the induction culture medium comprises MS culture medium, and the rest is added with 30 g / L sucrose, 7.5 g / L agarose, 0.5-1.5 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D), 1.0-1.5 mg / L thidiazuron (TDZ), and 0.05-0.1 mg / L naphthaleneacetic acid (NAA), the pH value is adjusted to 5.7-5.9, the dark culture condition is dark culture at 23-25° C. for 5-7 days, the light culture condition is light intensity of 2500-3500 lux, culture temperature of 23-25° C., light and dark are alternated, and the light-dark cycle is 16h / 8h.

4. The method for producing high-quality seedlings of Rhizoma Cibotii according to claim 1, characterized in that: In the step (4), the proliferation medium comprises MS medium, and the rest is added with 30 g / L sucrose, 7.5 g / L agarose, 0.3-0.7 ml / L 3-indolebutyric acid (IBA), and 0.05-0.3 mg / L 6-benzyladenine (6-BA), and the pH value is adjusted to 5.7-5.

9.

5. The method for producing high-quality seedlings of Rhizoma Cibotii according to claim 1, characterized in that: In the step (5), the rooting medium comprises 1 / 2 MS medium, and the rest is added with 30 g / L sucrose, 7.5 g / L agarose, 0.3-0.5 ml / L 3-indolebutyric acid (IBA), and 0.3-0.5 mg / L 6-benzyladenine (6-BA), and the pH value is adjusted to 5.7-6.

0.

6. The method for producing high-quality seedlings of Rhizoma Cibotii according to claim 1, characterized in that: In the step (6), the rooted tissue culture seedlings obtained are transferred to a greenhouse and hardened under natural conditions for 5-8 days. The bottle mouth is then opened and the seedlings are left for another 3-5 days. The tissue culture seedlings are taken out, the culture medium at the roots is washed, and the seedlings are transplanted into a mixed matrix of peat soil: perlite: vermiculite = 2:1:

1.

7. Use of the method according to any one of claims 1 to 6 in rapid propagation of Phyllostachys erythrorhizon tissue culture.

Images