A method for establishing a tissue culture and rapid propagation system of eucommia ulmoides oliver stem segments

Abstract

The application belongs to the technical field of woody plant asexual propagation, and particularly relates to a method for establishing a tissue culture and rapid propagation system of Eucommia ulmoides stem segments. The method comprises the following steps: disinfecting Eucommia ulmoides seeds, obtaining sterile Eucommia ulmoides tissue culture seedlings through seed germination induction, cutting stems of the sterile Eucommia ulmoides tissue culture seedlings into stem segments with a length of 0.5 cm to 1 cm, inoculating the stem segments into an adventitious bud induction medium to perform adventitious bud induction culture, forming clustered adventitious buds after 20 days of the adventitious bud induction culture, separating the clustered adventitious buds into multiple individual adventitious buds, and transferring the individual adventitious buds into a rooting medium to perform rooting and seedling strengthening culture, forming a large number of adventitious roots after 10 days of the rooting and seedling strengthening culture, and obtaining complete regenerated Eucommia ulmoides plants. The method for establishing the tissue culture and rapid propagation system of Eucommia ulmoides stem segments has the advantages of short rapid propagation cycle, high rapid propagation efficiency, no seasonality limitation, good excellent traits maintenance, small space occupation, and virus removal.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of asexual propagation of woody plants, and particularly relates to a method for establishing a tissue culture and rapid propagation system of Eucommia ulmoides stem segments. BACKGROUND

[0002] Eucommia ulmoides Oliver belongs to Eucommiaceae and Eucommia, is a single-species tree, and is a relic plant left after the Quaternary glaciation. Eucommia ulmoides Eucommiaceae Eucommia Eucommia ulmoides Oliver is a unique precious medicinal material and woody oil tree species in China, and is also a high-quality natural rubber resource with great development potential. The bark of Eucommia ulmoides Oliver is a traditional medicinal material and has the effects of tonifying liver and kidney and strengthening bones and muscles. As a high-quality natural rubber resource, the fruit, bark and leaves of Eucommia ulmoides Oliver can be used to extract Eucommia rubber, which has the unique characteristics of “rubber-plastic duality” and has broad application prospects in the fields of national defense, military industry, medical treatment, aerospace and the like. The leaves and male flowers of Eucommia ulmoides Oliver are a kind of food and medicine, are rich in active ingredients such as total flavonoids, chlorogenic acid and geniposidic acid, and have important medicinal and health values. In addition, the seed oil of Eucommia ulmoides Oliver is high in unsaturated fatty acid content, and the content of alpha-linolenic acid is up to 68.1%.

[0003] Due to the high economic value and good comprehensive benefits of Eucommia ulmoides Oliver, the industrial development of Eucommia ulmoides Oliver has attracted much attention, the market demand is continuously increasing, and the demand for seedlings is prosperous. At present, in production, Eucommia ulmoides Oliver is mainly propagated by sowing, cutting and grafting, but the seeds of Eucommia ulmoides Oliver contain a large amount of Eucommia rubber, and therefore need to be treated in a specific way to break dormancy in the propagation process. Therefore, the seed propagation has the problems of complicated process and extremely low germination rate. Although grafting and cutting can maintain the excellent traits of the parent plants, they are limited by seasons, and have the problems of complicated operation, high cost and low efficiency. At present, the key technology in the propagation of Eucommia ulmoides Oliver has not been broken through, which affects the large-scale and efficient propagation of seedlings, leads to insufficient supply of seedlings, and greatly limits the sustainable and healthy development of the Eucommia ulmoides Oliver industry.

[0004] In recent years, plant tissue culture technology has become increasingly mature and has been widely applied in plant rapid propagation. Through the establishment of an efficient and stable Eucommia ulmoides rapid propagation system, Eucommia ulmoides tissue culture seedlings with consistent seedling age and uniform growth vigor can be obtained, which can effectively solve the problems existing in the current seedling propagation process, promote the development of seedling production towards intensification and standardization, meet the market demand for high-quality seedlings, and provide stable technical support and basic materials for the preservation of Eucommia ulmoides germplasm resources, genetic improvement and directional breeding.

[0005] At present, the existing method for establishing a Eucommia ulmoides rapid propagation system can be used for the propagation of Eucommia ulmoides seedlings, but still has the problems of complicated operation, long cycle and low rooting rate. SUMMARY

[0006] ​​In order to solve the problems of complicated operation, long period and low rooting rate in the establishment method of the existing technology of Eucommia ulmoides rapid propagation system, the present application takes Eucommia ulmoides seed as explant, after seedling germination, takes the stem segment of the seedling as material to induce adventitious bud differentiation and adventitious bud rooting, and provides a kind of establishment method of Eucommia ulmoides stem segment tissue culture rapid propagation system.To achieve the above purpose, the present application adopts the following technical scheme.

[0007] The present application provides a kind of establishment method of Eucommia ulmoides stem segment tissue culture rapid propagation system, comprising the following steps:

[0008] Take Eucommia ulmoides seed as explant, after removing seed coat of the Eucommia ulmoides seed, disinfect, after seed germination induction, obtain aseptic Eucommia ulmoides tissue culture seedling.

[0009] The stem of the aseptic Eucommia ulmoides tissue culture seedling is cut into 0.5 cm~1 cm stem segment, inoculated in adventitious bud induction medium, and adventitious bud induction culture is carried out; adventitious bud induction culture is carried out for 20 d~25 d, and cluster adventitious bud is formed.

[0010] The cluster adventitious bud is divided into single adventitious bud, and is transferred to rooting medium, and rooting and seedling culture are carried out; rooting and seedling culture is carried out for 10 d~15 d, and adventitious root is formed, and complete regenerated plant of Eucommia ulmoides with strong root system is obtained.

[0011] The adventitious bud induction medium is composed of the following materials with final concentration: 4.4 g / L MS, 0.05 mg / L~0.1 mg / L naphthalene acetic acid, 0.9 mg / L~1.125 mg / L 6-benzylaminopurine, 30.0 g / L sucrose and 4.0 g / L plant gel, and the solvent is water; the pH of the adventitious bud induction medium is 5.8~6.0.

[0012] The rooting medium is composed of the following materials with final concentration: 2.2 g / L~4.4 g / L MS, 0.1 mg / L~0.3 mg / L naphthalene acetic acid, 15.0 g / L sucrose, 4.0 g / L plant gel, and the solvent is water; the pH of the rooting medium is 5.8~6.0.

[0013] The application provides a method for establishing a tissue culture and rapid propagation system of Eucommia ulmoides stem segments, which comprises the following steps: taking Eucommia ulmoides seeds as explants, disinfecting the explants to obtain sterile Eucommia ulmoides tissue culture seedlings; culturing the sterile Eucommia ulmoides tissue culture seedlings to induce adventitious bud culture; after 20 days of the adventitious bud induction culture, forming cluster-like adventitious buds; dividing the cluster-like adventitious buds into multiple single adventitious buds and transferring them to a rooting culture medium to perform rooting and seedling culture; after 10 days of the rooting and seedling culture, forming a large number of adventitious roots and obtaining complete regenerated Eucommia ulmoides plants with healthy root systems. Through the mutual cooperation of the above steps, the explants are treated and cultured, and the adventitious bud induction medium and the rooting culture medium are cultured, so that the regeneration of the Eucommia ulmoides stem segments is fast, the cycle is shortened, and the rooting rate is improved. The method for establishing the tissue culture and rapid propagation system of the Eucommia ulmoides stem segments has the advantages of simple operation, short rapid propagation cycle, high rooting rate (the highest rooting rate in the prior art is 72%, while the highest rooting rate in the application is 100%), high rapid propagation efficiency, no seasonality, maintenance of excellent properties, small space occupation, and virus removal, and can solve the problems of complicated operation, long cycle and low rooting rate in the prior art method for establishing the Eucommia ulmoides rapid propagation system.

[0014] Preferably, the method for disinfecting the Eucommia ulmoides seeds after removing the seed coat is as follows:

[0015] The Eucommia ulmoides seeds are sequentially immersed in a 10% sodium hypochlorite solution and a 75% alcohol solution for double disinfection, so as to guarantee a sterile culture environment.

[0016] Preferably, the Eucommia ulmoides seeds are immersed in the 10% sodium hypochlorite solution for 5-6 minutes.

[0017] Preferably, the Eucommia ulmoides seeds are immersed in the 75% alcohol solution for 5-6 minutes.

[0018] Preferably, the adventitious bud induction culture medium is composed of the following materials with final concentrations: 4.4 g / L MS, 0.1 mg / L naphthalene acetic acid, 0.9 mg / L 6-benzylaminopurine, 30.0 g / L sucrose and 4.0 g / L plant gel, and the solvent is water.

[0019] Preferably, the rooting culture medium is composed of the following materials with final concentrations: 2.2 g / L MS, 0.1 mg / L naphthalene acetic acid, 15.0 g / L sucrose and 4.0 g / L plant gel, and the solvent is water.

[0020] Preferably, the adventitious bud induction culture is performed under the following conditions:

[0021] The temperature is 24-26°C, the light cycle is 15-17 hours, and the light intensity is 1790-1810 Lx.

[0022] Preferably, the rooting and seedling culture conditions are as follows:

[0023] The temperature is 24-26 DEG C, the light cycle is 15-17 h, and the light intensity is 1790-1810 Lx.

[0024] Preferably, the Eucommia ulmoides seed is the seed of the national approved variety 'Huazhong 8' Eucommia ulmoides, which is a one-year-old seed with an intact seed coat and is mature and full

[0025] The present application establishes a high-efficiency and stable regeneration and propagation method for Eucommia ulmoides, which has the advantages of short propagation cycle, high efficiency and low cost.

[0026] Compared with the prior art, the present application has the following beneficial effects:

[0027] 1. The present application provides a method for establishing a Eucommia ulmoides stem segment tissue culture and rapid propagation system, which first uses Eucommia ulmoides seeds as explants, disinfects the explants to obtain sterile Eucommia ulmoides tissue culture seedlings, cultures the sterile Eucommia ulmoides tissue culture seedlings, and induces adventitious bud culture; after 20 days of adventitious bud induction culture, cluster-shaped adventitious buds are formed; the cluster-shaped adventitious buds are divided into multiple individual adventitious buds, which are then transferred to a rooting culture medium for rooting and seedling culture; after 10 days of rooting and seedling culture, a large number of adventitious roots are formed, and complete regenerated plants of Eucommia ulmoides with healthy root systems are obtained. Through the mutual cooperation of the above steps, the treatment of the explants for tissue culture, and the culture of the adventitious bud induction medium and the rooting medium, the regeneration of Eucommia ulmoides stem segments is promoted, the cycle is shortened, and the rooting rate is improved. The method for establishing the Eucommia ulmoides stem segment tissue culture and rapid propagation system provided by the present application has the advantages of simple operation, short rapid propagation cycle, high rooting rate, high rapid propagation efficiency, no seasonality, maintenance of excellent traits, small space occupation, and virus removal, and can solve the problems of complicated operation, long cycle and low rooting rate in the prior art method for establishing a Eucommia ulmoides rapid propagation system.

[0028] 2. The method for establishing the Eucommia ulmoides stem segment tissue culture and rapid propagation system provided by the present application includes the steps of explant disinfection, sterile seedling culture, adventitious bud induction, and adventitious root induction, and the treatment of the explants for tissue culture, the continuous optimization of the culture medium formula to promote regeneration, and the high rooting rate can solve the problems of complicated operation, long cycle and low rooting rate in the prior art method for establishing a Eucommia ulmoides rapid propagation system.

[0029] 3. The present application uses Eucommia ulmoides seeds as explants, induces adventitious buds by germination of the seedlings, and then induces the formation of adventitious roots by transferring the adventitious buds to a rooting culture medium, so that a large number of Eucommia ulmoides seedlings with consistent age and uniform growth vigor are obtained, which provides strong technical support for the rapid propagation, popularization and industrialization development of Eucommia ulmoides elite varieties, and provides basic materials for the preservation of Eucommia ulmoides germplasm resources, genetic improvement and directional breeding. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 For the stem section in the present application to induce rooting directly; wherein, Figure 2 A figure in the above is: the aseptic Eucommia ulmoides seedling obtained after the germination of Eucommia ulmoides seeds is transferred into different rooting culture mediums; Figure 2 B figure in the above is: the formation of adventitious roots of the aseptic Eucommia ulmoides seedling in the RIM4 culture medium after 10 days; Figure 2 C figure in the above is: the root system develops most vigorously and the state of the stem and leaf is the best after 30 days; wherein RIM1~RIM6 respectively represent 6 different types of rooting culture mediums; wherein two samples in each picture are parallel samples.

[0031] Figure 2 For the stem section in the present application to induce adventitious buds; wherein, Figure 2 A figure in the above is: the stem of the aseptic Eucommia ulmoides seedling is taken as the material of Eucommia ulmoides stem section, the Eucommia ulmoides stem section is cut into small sections with a length of about 0.5 cm~1 cm by using a sterile knife, and the small sections are placed into different adventitious bud induction culture mediums by using tweezers; Figure 2 B figure in the above is: after 10 days, the two ends of the Eucommia ulmoides stem section in each culture medium swell to produce callus; Figure 2 C figure in the above is: the induction of adventitious buds after 20 days of culture in the adventitious bud induction culture medium; wherein SIM1~SIM6 respectively represent 6 different types of adventitious bud induction culture mediums; wherein six samples in each picture are parallel samples.

[0032] Figure 3 For the mode chart of the Eucommia ulmoides regeneration system in the present application; wherein, Figure 3 A figure in the above is: after obtaining the aseptic Eucommia ulmoides seedling, the stem is taken and cut into sections for the induction of adventitious buds; wherein six samples are parallel test samples; Figure 3 B figure in the above is: the adventitious buds grow out on the cut surface of the stem section after 20 days of culture in the adventitious bud induction culture medium, wherein the red arrow indicates the adventitious roots; wherein six samples are parallel samples; Figure 3 C figure in the above is: the bud is cut off and placed in the culture medium to induce rooting to form new seedlings, wherein the red arrow indicates the adventitious roots; Figure 3 D figure in the above is: a flow chart of establishing the regeneration system by using the Eucommia ulmoides stem section as the material; the whole flow steps are: the induction of adventitious buds of the stem section, the continuous swelling of callus, the induction of callus to develop adventitious buds, and the induction of adventitious buds to root. DETAILED DESCRIPTION

[0033] The present application will be described in detail below with reference to the accompanying drawings and specific examples, but should not be understood as limiting the present application. If not specifically stated, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, if not specifically stated, can be obtained from commercial channels.

[0034] Example 1: Screening of rooting medium

[0035] (1) Disinfection of Eucommia ulmoides seed and germination of aseptic seedling

[0036] Mature and full Eucommia ulmoides seeds were selected as explants, and the explants were removed of the Eucommia ulmoides seed coat using sterile tweezers in a clean bench, and the peeled Eucommia ulmoides seeds were placed in a sterile culture dish for standby.

[0037] The peeled Eucommia ulmoides seeds were transferred to a sterilized container containing 10% sodium hypochlorite solution, shaken for 5 minutes, then transferred to a 75% ethanol solution, shaken for 5 minutes, then washed with sterile water for 1 minute each time, and the fresh sterile water was replaced after each washing to obtain the disinfected seeds. Finally, the disinfected Eucommia ulmoides seeds were laid flat on sterile filter paper, naturally air-dried in a clean bench, and the surface of the Eucommia ulmoides seeds was observed to ensure that there were no visible water stains, and then the Eucommia ulmoides seeds were placed in the seed germination medium using sterile tweezers for culture. After 10 days of culture, the aseptic Eucommia ulmoides tissue culture seedlings were obtained.

[0038] The Eucommia ulmoides seeds were intact and had no damage. The variety of the Eucommia ulmoides seeds was the national approved variety 'Huazhong No. 8', and the source was the Yuanyang experimental base of the Institute of Economic Forests, Chinese Academy of Forestry.

[0039] The culture conditions of the Eucommia ulmoides seeds in the seed germination medium were a temperature of 25°C, and the light cycle was preferably 16 hours, and the light intensity was preferably 1800 Lx.

[0040] The formula of the seed germination medium was 4.4 g / L MS, 30.0 g / L sucrose and 7.0 g / L agar, and the solvent was water. The pH of the seed germination medium was 5.9.

[0041] (2) Direct induction of adventitious root formation from stem segments

[0042] In a clean bench, the aseptic Eucommia ulmoides tissue culture seedlings obtained in step (1) were cut at the stem segment (3 cm from the terminal bud) using sterile scissors, and then the cut aseptic Eucommia ulmoides seedlings were placed in different types of rooting medium for rooting and seedling culture.

[0043] The formula of the different types of rooting medium is shown in Table 1.

[0044] The average number of roots and the induction frequency of different rooting media were counted after 30 days of culture, and the results are shown in Table 1 and Figure 1

[0045] Table 1 Induction of adventitious root formation in different types of rooting media

[0046]

[0047] Note: RIM1-RIM6 represent six different types of rooting media, respectively; MS is MS Base Salts with vitamins, purchased from Shengong Biotechnology (Shanghai) Co., Ltd.; NAA is naphthalene acetic acid; and plant gel is purchased from Shengong Biotechnology (Shanghai) Co., Ltd.

[0048] It can be seen from the above results that the optimal adventitious root induction medium is RIM4 rooting medium, and the formula thereof is as follows: Figure 1 After 10 days, the bottom of the aseptic Eucommia ulmoides seedling in the RIM4 rooting medium formed adventitious roots; after 30 days, the root system in the RIM4 rooting medium developed vigorously; as shown in Table 1, the average number of roots in the RIM2 rooting medium to the RIM4 rooting medium was 3, 3 and 6, respectively, and the induction frequency was 5%, 50% and 100%, respectively; and the RIM1 rooting medium, the RIM5 rooting medium to the RIM6 rooting medium did not form roots, so the rooting medium is preferably RIM4.

[0049] It can be seen from the above results that the optimal adventitious root induction medium is RIM4 rooting medium, and the formula thereof is as follows:

[0050] 2.2 g / L MS, 0.1 mg / L naphthalene acetic acid, 15.0 g / L sucrose, 4.0 g / L plant gel, and the solvent is water. The pH of the RIM4 rooting medium is 5.9.

[0051] Example 2: Screening of adventitious bud induction medium

[0052] (1) Disinfection of Eucommia ulmoides seeds and germination of aseptic seedlings

[0053] Mature and full Eucommia ulmoides seeds were selected as explants, and the explants were removed from the outer skin of the Eucommia ulmoides seeds using sterile tweezers in a clean bench, and the peeled Eucommia ulmoides seeds were placed in a sterile culture dish for standby.

[0054] ​The peeled eucommia seed is transferred to a sterilized container containing 10% sodium hypochlorite solution by volume, shaken for 5 minutes, then transferred to a 75% ethanol solution by volume, shaken for 5 minutes, then rinsed with sterile water, three times for 1 minute each time, and fresh sterile water is changed after each rinse to obtain the disinfected eucommia seed. Finally, the disinfected eucommia seed is placed on sterile filter paper and naturally dried in a clean bench, and the seed surface is observed to ensure that there is no visible water stain, and then the eucommia seed is placed in the seed germination medium using sterile tweezers. After 10 days of culture, the sterile eucommia tissue culture seedling is obtained.

[0055] The eucommia seed is a seed with an intact seed coat and no damage. The variety of the eucommia seed is the national approved variety 'Huazhong No. 8', and the source is the Yuanyang test base of the Institute of Economic Forests of the Chinese Academy of Forestry.

[0056] The culture conditions of the eucommia seed in the seed germination medium are that the temperature is preferably 25°C, the light cycle is preferably 16h, and the light intensity is preferably 1800 Lx.

[0057] The formula of the seed germination medium is as follows:

[0058] 4.4 g / L MS, 30.0 g / L sucrose and 7.0 g / L agar, with water as the solvent. The pH of the seed germination medium is 5.9.

[0059] (2) Adventitious bud induction

[0060] The stem segments of the sterile eucommia tissue culture seedling are used as materials, and the sterile eucommia tissue culture seedling is cut into small segments with a length of about 0.5 cm using a sterile knife in a clean bench. The small segments are placed in different types of adventitious bud induction medium using sterile tweezers to induce adventitious buds (Table 2).

[0061] The formula of the different types of adventitious bud induction medium is shown in Table 1.

[0062] After 20 days of culture, the average number of buds and the induction frequency of different adventitious bud induction media are counted, and the results are shown in Table 2 and Figure 2

[0063] Table 2 Formation of adventitious buds in different types of adventitious bud induction medium

[0064]

[0065] ​Note: SIM1~SIM6 represent 6 different types of adventitious shoot induction media; MS is MS BaseSalts with vitamins, purchased from Sangon Biotech (Shanghai) Co., Ltd.; NAA is naphthaleneacetic acid; 6-BA is 6-benzylaminopurine; plant gel was purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0066] Depend on Figure 2 It can be seen that adventitious buds are directly induced from the cut of the stem segment after 20 days. As shown in Table 2, the average number of buds germinating in each adventitious bud induction medium in SIM2 to SIM5 is 15, 6, 10 and 6, respectively, with induction frequencies of 83.3%, 33.3%, 55.5% and 33.3%, respectively. The average number of buds germinating in both SIM1 and SIM6 adventitious bud induction media is 3. Therefore, SIM2 is the preferred adventitious bud induction medium.

[0067] The results above indicate that the optimal adventitious shoot induction medium is SIM2 adventitious shoot induction medium, and its formula is as follows:

[0068] The SIM2 adventitious shoot induction medium contained 4.4 g / L MS, 0.1 mg / L NAA, 0.9 mg / L 6-BA, 30.0 g / L sucrose, and 4.0 g / L plant gel, in water. The pH of the medium was 5.9.

[0069] Example 3: Establishment of a rapid propagation system for Eucommia ulmoides stem segment tissue culture

[0070] A method for establishing a rapid propagation system of Eucommia ulmoides stem segments via tissue culture includes the following steps:

[0071] (1) Disinfection of Eucommia ulmoides seeds and germination of sterile seedlings

[0072] Mature and plump Eucommia ulmoides seeds were selected as explants. The outer skin of the Eucommia ulmoides seeds was removed using sterile forceps in a clean bench. The peeled Eucommia ulmoides seeds were then placed in sterile petri dishes for later use.

[0073] After peeling, the Eucommia ulmoides seeds were transferred to a sterile container containing a 10% (v / v) sodium hypochlorite solution and shaken for 5 minutes for sterilization. Then, the seeds were transferred to a 75% (v / v) ethanol solution and shaken for another 5 minutes. Next, the seeds were rinsed with sterile water three times, for one minute each time, with fresh sterile water after each rinse, to obtain sterilized seeds. Finally, the sterilized Eucommia ulmoides seeds were spread evenly on sterile filter paper and allowed to air dry naturally in a laminar flow hood. After observing the seed surface to ensure there were no visible water stains, the seeds were placed in seed germination medium using sterile forceps. Sterile Eucommia ulmoides tissue culture seedlings were obtained after 10 days of culture.

[0074] The Eucommia ulmoides seed is a seed with an intact seed coat and no damage. The Eucommia ulmoides seed is a national approved variety 'Huazhong No. 8', and is from the Yuanyang experimental base of the Institute of Economic Forests, Chinese Academy of Forestry.

[0075] The Eucommia ulmoides seed is cultured in the seed germination medium under the condition of a temperature of 25 DEG C, and a light cycle of 16 h, and a light intensity of 1800 Lx.

[0076] The seed germination medium is prepared by using 4.4 g / L MS, 30.0 g / L sucrose and 7.0 g / L agar as solvents.

[0077] (2) Adventitious bud induction

[0078] The stem segment of the aseptic Eucommia ulmoides tissue culture seedling is cut into small segments with a length of about 0.5 cm (as shown in the A figure of FIG. 2) by using a sterile knife in a clean bench. Figure 3 The small segments are placed into the SIM2 adventitious bud induction medium by using a sterile tweezers to induce the adventitious buds. After 20 days, the cluster-like adventitious buds are formed (as shown in the B figure of FIG. 2). Figure 3

[0079] (3) Adventitious root induction and establishment of the Eucommia ulmoides regeneration system mode

[0080] The cluster-like adventitious buds obtained in the step (2) are divided into single adventitious buds, and are transferred into the RIM4 adventitious root induction medium to perform rooting and seedling culture. After 10 days, the adventitious roots are formed (as shown in the C figure of FIG. 3). Figure 3 Finally, the efficient, stable and short propagation cycle Eucommia ulmoides regeneration system is established, that is, a Eucommia ulmoides stem segment tissue culture rapid propagation system is established (as shown in the D figure of FIG. 3). Figure 3

[0081] In conclusion, the Eucommia ulmoides seed is used as an explant, the germination of the Eucommia ulmoides seedling is performed, the stem segment of the seedling is used as a material to induce the adventitious buds, the adventitious buds are transferred into the rooting medium to induce the formation of the adventitious roots, and finally a large number of Eucommia ulmoides seedlings with consistent seedling age and uniform growth are obtained, which provides a strong technical support for the Eucommia ulmoides elite rapid propagation, popularization and industrial development, and provides a basic material for the Eucommia ulmoides germplasm resource preservation, genetic improvement and directional breeding.

[0082] It should be noted that when the numerical range is involved in the present application, it should be understood that each numerical range and any number between the two endpoints can be selected. In order to prevent redundancy, the preferred embodiments are described in the present application.

[0083] ​​While the preferred embodiments of the application have been described, additional variations and modifications can be made to these embodiments by those skilled in the art once they have the benefit of the present disclosure without departing from the spirit and scope of the application.

Claims

1. A method for establishing a tissue culture rapid propagation system of Eucommia ulmoides Oliver stem segments, characterized by, It comprises the following steps: The seed coat of Eucommia ulmoides Oliv is removed, and the Eucommia ulmoides Oliv seed is sterilized, and then aseptic Eucommia ulmoides Oliv seedling is obtained by seed germination induction; The stem of the aseptic Eucommia ulmoides Oliv seedling is cut into 0.5 cm-1 cm stem segments, which are inoculated into adventitious bud induction medium for adventitious bud induction culture; the adventitious bud induction culture is performed for 20-25 days to form cluster adventitious buds; The cluster adventitious buds are separated into single adventitious buds, which are transferred into rooting medium for rooting and seedling culture; the rooting and seedling culture is performed for 10-15 days to form adventitious roots, and complete regenerated Eucommia ulmoides Oliv plants are obtained; The adventitious bud induction medium is composed of the following materials with final concentrations: 4.4 g / L MS, 0.1 mg / L naphthalene acetic acid, 0.9 mg / L 6-benzylaminopurine, 30.0 g / L sucrose and 4.0 g / L plant gel, and the solvent is water; the pH of the adventitious bud induction medium is 5.8-6.0; The rooting medium is composed of the following materials with final concentrations: 2.2 g / L MS, 0.1 mg / L naphthalene acetic acid, 15.0 g / L sucrose and 4.0 g / L plant gel, and the solvent is water; the pH of the rooting medium is 5.8-6.

0.

2. The establishment method according to claim 1, characterized by, The method for sterilizing the Eucommia ulmoides Oliv seed after removing the seed coat is as follows: The Eucommia ulmoides Oliv seed is sequentially immersed in 10% sodium hypochlorite solution and 75% alcohol solution for sterilization.

3. The establishment method according to claim 2, characterized in that, The Eucommia ulmoides Oliv seed is immersed in the 10% sodium hypochlorite solution for 5-6 minutes for sterilization.

4. The establishment method according to claim 2, wherein, The Eucommia ulmoides Oliv seed is immersed in the 75% alcohol solution for 5-6 minutes for sterilization.

5. The method of establishing of claim 1, wherein, The conditions for the adventitious bud induction culture are as follows: The temperature is 24-26℃, the light cycle is 15-17 hours, and the light intensity is 1790-1810 Lx.

6. The establishment method of claim 1, wherein, The conditions for the rooting and seedling culture are as follows: The temperature is 24-26℃, the light cycle is 15-17 hours, and the light intensity is 1790-1810 Lx.

7. The establishment method of claim 1, wherein, The variety of the Eucommia ulmoides Oliv seed is 'Huazhong No.8'.

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