A tissue culture rapid propagation method of atractylodes chinensis

Abstract

The application belongs to the technical field of cultivation of Atractylodes chinensis, and particularly relates to a tissue culture rapid propagation method of Atractylodes chinensis. Compared with the previous tissue culture rapid propagation technology of Atractylodes chinensis, the method has the advantages of simple culture process, no need of callus dedifferentiation and redifferentiation culture, accelerated rooting speed, greatly shortened tissue culture time, simple and easy-to-prepare culture medium composition and type, reduced tissue culture production cost, easy production and popularization, and the like. The method comprises the steps of rooting culture, strong seedling culture, subsequent seedling hardening and transplanting, and the like. From the inoculation culture of explants to the survival of the seedling hardening and transplanting greenhouse, about 50-60 days are needed. The obtained Atractylodes chinensis has developed roots, and is a rhizome part medicine Atractylodes chinensis. The method provides a more effective way for the protection of the rare medicinal plant resource and the popularization and application of Atractylodes chinensis planting.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of Atractylodes chinensis breeding, and particularly relates to a tissue culture and rapid propagation method of Atractylodes chinensis. BACKGROUND

[0002] Atractylodes chinensis Wall. is a perennial herb of the family Asteraceae. Atractylodes is also known as Guan Atractylodes, and its rhizome is used as a medicine. Atractylodes is one of the sources of Atractylodes rhizome recorded in Chinese Pharmacopoeia, and has a large market demand. Atractylodes is divided into A.chinensis (DC.) Koidz (Huacangzhu) and Atractylodes lancea (Thunb.) DC (Maocangzhu). A.chinensis is distributed in Hebei, Jilin, Liaoning, Inner Mongolia and other places, and is one of the native northern medicines in China. A.chinensis has the functions of drying dampness, invigorating the spleen, dispelling wind and cold, and improving eyesight, and can also relieve depression. A.chinensis can be used to treat related symptoms caused by internal dampness.

[0003] At present, most of the cultivated Atractylodes are propagated by seeds, but are greatly affected by environmental temperature, light, soil and other factors. Atractylodes needs to establish a rapid breeding technology system as soon as possible due to the lack of wild resources, in order to meet the needs of seedlings for artificial cultivation. Improving the germination rate of Atractylodes seeds and finding the suitable living environment of Atractylodes will provide a theoretical basis for large-area planting of Atractylodes. In addition, actively developing the internal and external factors affecting the yield and quality of Atractylodes, and improving the yield and quality of cultivated Atractylodes can replace wild Atractylodes with artificial Atractylodes, which can effectively alleviate the resource pressure of wild Atractylodes (Yang Jing, Peng Jingjing. Research progress of Atractylodes [J]. Shaanxi Agricultural Science, 5 (2018) 12-0136-04).

[0004] In addition to the traditional seedling method, scholars have successively carried out research on the plant tissue rapid propagation technology of Atractylodes. The basic culture media used in the tissue culture of Atractylodes include: N 68, B5, MS, 1 / 2MS, 1 / 4MS, ER, VW, White and LS, etc. Among them, the MS medium is mainly used for induction and subculture, and the 1 / 2MS and 1 / 4MS media are used for rooting induction (Application of Orthogonal Design in Tissue Culture of Trifolium repens L. [J]. Journal of Northeastern University: Natural Science Edition, 1998(1): 41-45.). Different rhizoma et radix seu rhizoma atrati varieties and different explants are selected in rhizoma et radix seu rhizoma atrati tissue culture, and the types, concentrations and culture conditions of hormones required in the morphological formation process are different. In the selection of exogenous hormones, scholars have used 6-BA, NAA, IBA, IAA, 2, 4-D and KT, etc. There are few studies on tissue culture of rhizoma et radix seu rhizoma atrati. For example: Zhang Weikun and Zhao Hui et al. used rhizoma et radix seu rhizoma atrati stem segments with buds as explants to explore the factors affecting the regeneration of tissue culture seedlings (Zhang Weikun, Zhao Hui. Research on tissue culture and rapid propagation technology of rhizoma et radix seu rhizoma atrati [J]. Seed, 5 (2018) 12-0136-04); Wu Zhiquang and Wang Sixuan et al. selected rhizoma et radix seu rhizoma atrati stem segments with buds as explants, set different primary culture medium formula, subculture medium formula and rooting medium formula in the culture process, and obtained the best tissue culture scheme by comparing the best culture mode at each stage (Wu Zhiquang, Wang Sixuan. Research progress of rhizoma et radix seu rhizoma atrati tissue culture and rapid propagation technology [J]. Modern Agricultural Research, (2020) 10-0030-31.); Zuo Jingjing and Yan Guiyun et al. studied the rooting culture of rhizoma et radix seu rhizoma atrati tissue culture seedlings, and the culture time was 45d, and considered that 1 / 2MS+NAA 0.5mg / L was the most suitable medium for rhizoma et radix seu rhizoma atrati tissue culture seedling rooting culture (Orthogonal design optimization of rhizoma et radix seu rhizoma atrati tissue culture [J]. Chinese Agricultural Bulletin, 2017, 33(11): 21-24.);

[0005] Rhizoma et radix seu rhizoma atrati is used as a medicine with rhizome. The traditional method of sowing seeds to harvesting needs 4 years. Plant tissue culture rapid propagation has the advantages of less material, short culture period, high propagation rate, and can be factory produced. In recent years, the study of rhizoma et radix seu rhizoma atrati tissue culture shows that scholars have used and successfully obtained 11 kinds of explants including embryo, embryo axis, radicle, leaf blade, petiole, axillary bud, cotyledon, true leaf, rhizome, rhizome lateral bud and stem tip. Different explants are selected, and the tissue culture approaches are different. The rhizoma et radix seu rhizoma atrati tissue culture process generally needs three culture stages of primary culture, subculture and rooting culture. SUMMARY

[0006] In order to overcome the shortcomings and defects of the prior art, the purpose of the present application is to provide a rhizoma et radix seu rhizoma atrati tissue culture and rapid propagation method, which is more rapid and simple in operation, has the advantages of short period, low cost, fast rooting speed, high rooting rate and the like.

[0007] The purpose of the present application is realized by the following technical scheme:

[0008] A rhizoma et radix seu rhizoma atrati tissue culture and rapid propagation method comprises the following steps:

[0009] (1) cutting two-year-old Atractylodes lancea branches, selecting stem segments with 2 tender buds and carrying out pretreatment and disinfection treatment;

[0010] (2) inoculating the stem segments after disinfection treatment in step (1) into rooting medium, and carrying out rooting culture under the conditions of a culture temperature of 23-24℃, a light intensity of 1800-2400lx, and a daily light time of 12-14h;

[0011] (3) when the rooting culture in step (2) reaches a certain length, transferring the stem segments into a new rooting medium for 5-7d;

[0012] (4) inoculating the stem segments with buds in the medium in step (3) into a new rooting medium, and carrying out secondary rooting culture under the conditions of a culture temperature of 23-24℃, a light intensity of 1800-2400lx, and a daily light time of 12-14h, so as to expand the number of propagation;

[0013] (5) transplanting the stem segments after the secondary rooting culture in step (4) or the stem segments after the secondary rooting culture in step (4) into a new rooting medium for 5-7d;

[0014] The length of the stem segments with 2 tender buds in step (1) is preferably 3-4cm;

[0015] The pretreatment in step (1) is preferably washing the stem segments with 2 tender buds with flowing water for 30-60min;

[0016] The disinfection treatment in step (1) is preferably as follows:

[0017] immersing the stem segments with 2 tender buds into alcohol with a volume fraction of 75% for 30s, and then disinfecting the stem segments with a solution containing 0.1wt.% of mercuric chloride and 1wt.% of Tween 20 for 10min, and washing the stem segments with sterile water for 3-4 times; after washing with sterile water, absorbing the surface water and cutting the contact part of the incision with the disinfection solution;

[0018] The rooting medium in step (2) or (4) is WPM+NAA 1.0mg / L+sucrose 30g / L+agar 4.9g / L, pH=5.8;

[0019] The stem segments with buds in step (4) are preferably 2-3cm in length;

[0020] The stem segments with buds in step (4) are preferably 2-3cm in length;

[0021] The number of roots of the tissue culture seedling in step (5) is preferably not less than 10, and the root length is preferably not less than 3-4 cm;

[0022] The specific operation of the seedling transplanting in step (5) is preferably as follows:

[0023] When the number of roots of the tissue culture seedling is not less than 10, and the average root length is not less than 3-4 cm, the following treatments are sequentially performed:

[0024] ① In-vessel seedling: loosen the sealing film of the culture bottle to gradually reduce the humidity in the bottle and enhance the light; after 3-4 days, completely remove the sealing film, and open the bottle to perform seedling for 2-3 days;

[0025] ② Out-of-vessel seedling: after the opening of the bottle to perform seedling is completed, the tissue culture seedling is taken out, and the agar on the roots is cleaned; the tissue culture seedling is transplanted into the mixed substrate, and is covered with a transparent film, and then is placed in a culture room with a temperature of 23-24 ℃ and a humidity of 70-80% to perform out-of-vessel seedling for 7-10 days, water is sprayed every day, and ventilation is performed for 2-3 hours every day;

[0026] ③ Greenhouse management: after the out-of-vessel seedling is completed, the transparent film is removed, and the seedling and the mixed substrate are transferred into a greenhouse to perform shading culture, wherein the environmental temperature in the greenhouse is 20-24 ℃, the environmental humidity is 70-80%, and shading is performed for 3-4 hours every day; after shading culture for 4-5 days, normal culture can be performed;

[0027] The mixed substrate in step ② is a mixture of vermiculite and grass charcoal soil, and the mass ratio of the vermiculite to the grass charcoal soil is 1:1;

[0028] The mixed substrate in step ② is pretreated as follows:

[0029] After the mixed substrate is sterilized at high temperature, 0.5 mg / L of NAA solution is sprayed to wet the substrate; the pretreated mixed substrate is more beneficial to promote the growth and robustness of the roots;

[0030] The shading time in step ③ is preferably from 11 o'clock in the morning to 15 o'clock in the afternoon;

[0031] When the seedling and the mixed substrate are transferred into the greenhouse in step ③, the mixed substrate is preferably sprayed with 0.2 wt.% potassium permanganate solution until the mixed substrate is wet, so as to prevent diseases;

[0032] Principle of the application:

[0033] In order to rapidly propagate the tissue culture seedling of Atractylodes lancea, the application provides a tissue culture rapid propagation method of Atractylodes lancea, which has the advantages of short cycle, low cost, rapid rooting, 100% rooting rate, large expansion quantity, robust plant, and developed root system:

[0034] (1) The present application takes the two-year-old, newly germinated and 2-bud stem segments of Atractylodes lancea as explant material, uses 1 / 2MS, MS, WPM, etc. as the basic culture medium, and adds 6-BA, NAA, etc. as exogenous hormones to carry out tissue culture test, and finally determines the types of rooting culture medium, the types and concentrations of hormones, the disinfection conditions of explants, the process of seedling raising (the sterilization method and composition of the transplanting medium, etc.), etc. The results show that the explants of 3-4 cm long, newly germinated and 2-bud stem segments are directly used for rooting culture, the inoculation contamination rate is low, the differentiation culture is not needed in the culture process, the roots can be directly rooted in the rooting culture, the rooting is fast, only about 20 days are needed, the root length is more than 2 cm, and the rooting rate reaches 100%.

[0035] (2) Based on the low survival rate of tissue culture seedlings of Atractylodes lancea and the problem of yellowing, the present application adds a process of seedling culture after rooting culture, which can improve the health of seedlings and effectively improve the problem of yellowing.

[0036] (3) In order to realize the rapid obtaining of Atractylodes lancea sterile seedlings in a short time, the present application selects the tissue cultured stem segments as explants for secondary rooting culture after rooting culture and seedling culture, so as to achieve the purpose of secondary rapid propagation.

[0037] (4) Since Atractylodes lancea belongs to root medicine, the developed roots can well improve its economic value. In order to quickly recover the roots in the transplanting process, the present application further optimizes the process of seedling raising and transplanting, and gradually adapts the roots of Atractylodes lancea to the transition from agar medium to seedling raising medium by adding NAA hormone in the seedling raising medium, etc., so as to further improve the number, length and fresh weight of roots, etc.

[0038] In summary, on the one hand, the explants can be directly rooted in the rooting culture medium, and the tissue culture seedlings with root length more than 2 cm can be obtained in 20 days, so that the Atractylodes lancea sterile seedlings can be obtained in a short time; on the other hand, the present application can realize the rapid culture of Atractylodes lancea sterile seedlings in a short time, and the time from explant inoculation to the survival of the finally seedling-raised and transplanted tissue culture seedlings is about 50-60 days, which greatly shortens the tissue culture time, expands the propagation quantity, and greatly reduces the production cost of tissue culture. The present application provides more research space and greater research value for the tissue culture research of Atractylodes lancea, and provides an important way for solving the market demand problem of Atractylodes lancea and protecting and sustainably utilizing the rare medicinal plant resources.

[0039] The present application has the following advantages and effects compared with the prior art:

[0040] (1) Compared with the previous tissue culture method of Atractylodes chinensis, the tissue culture and rapid propagation method of Atractylodes chinensis provided by the application has a simple rooting culture process, does not need callus dedifferentiation and redifferentiation culture, speeds up the rooting speed, greatly shortens the tissue culture time, has simple and easy-to-prepare medium composition and types, reduces the tissue culture production cost, and is easy to produce and popularize.

[0041] (2) The tissue culture and rapid propagation method of Atractylodes chinensis provided by the application has a large number of seedling propagation: in the rooting culture process, the stem segments can be cut and rooted again after the first rooting, the purpose of secondary rapid propagation is achieved, and a large number of Atractylodes chinensis sterile seedlings can be cultured rapidly in a shorter time.

[0042] (3) The tissue culture and rapid propagation method of Atractylodes chinensis provided by the application has a remarkable rooting effect: in the rooting culture process, the stem segments rapidly elongate, new leaves are lush, the growth is good, the root system is developed, and the rooting rate reaches 100%. Moreover, the explants can root in a short time (about 20 days), the root length is greater than 2 cm, the efficiency is more than doubled compared with the previous tissue culture, and the sterile seedlings can be cut and transferred to the rooting culture medium after the rooting culture and seedling culture (about 35-40 days), and the stem segments with sprouts are rooted again after 20 days.

[0043] (4) The tissue culture and rapid propagation method of Atractylodes chinensis provided by the application adds a seedling culture process on the basis of the rooting culture, and the effect is remarkable: the seedling culture for about 5-7 days can significantly improve the health and effectively improve the yellowing problem of some tissue culture seedlings.

[0044] (5) The tissue culture and rapid propagation process of Atractylodes chinensis is completed in 3 years, and a rapid propagation system is successfully established. From explant inoculation culture to seedling transplanting and survival in a greenhouse, it takes about 50-60 days, and the obtained Atractylodes chinensis has developed roots, which provides a more effective way for the protection of this rare medicinal plant resource and the popularization and application of Atractylodes chinensis planting. BRIEF DESCRIPTION OF DRAWINGS

[0045] Figure 1 Figure 1 is a display diagram of the rooting culture of treatment 1 and treatment 2 in Example 1 on the 45th day using 1 / 2MS medium as the basic medium, wherein A: treatment 1 (1 / 2MS+NAA 0.5 mg / L+ sucrose 30 g / L+ agar 4.9 g / L, pH=5.8), B: treatment 2 (1 / 2MS+NAA 1.0 mg / L+ sucrose 30 g / L+ agar 4.9 g / L, pH=5.8).

[0046] Figure 2 Figure 2 is a display diagram of some treatments of the rooting culture of Atractylodes chinensis in Example 1 by adding different types and concentrations of hormones to the 1 / 2MS medium as the basic medium.

[0047] Figure 3is a rooting culture display figure of Example 1 in which the MS medium was used as a basic medium and the medium of treatment 1 (MS+NAA 0.5 mg / L+sucrose 30 g / L+agar 4.9 g / L, pH=5.8) was used for rooting culture for 45 days.

[0048] Figure 4 is a rooting culture display figure of Example 1 in which the MS medium was used as a basic medium and the medium of partial treatment in which different kinds and concentrations of hormones were added to the medium was used for rooting culture.

[0049] Figure 5 is a rooting culture display figure of Example 1 in which the WPM medium was used as a basic medium and the medium of treatment 2 (WPM+NAA 1.0 mg / L+sucrose 30 g / L+agar 4.9 g / L, pH=5.8) was used for rooting culture, in which A: 20 days after the rooting culture, B: 38 days after the rooting culture, C: 50 days after the rooting culture, D1: 70 days after the rooting culture (root of a seedling with normal growth), D2: 70 days after the rooting culture (root of a seedling with excellent growth), E: 70 days after the rooting culture for measuring the root length, F: secondary rooting culture of a stem segment with a sprout cut after 45 days of the rooting culture, and G: seedling culture.

[0050] Figure 6 is a rooting culture display figure of Example 1 in which the WPM medium was used as a basic medium and the medium of treatment 1 (WPM+NAA 0.5 mg / L+sucrose 30 g / L+agar 4.9 g / L, pH=5.8) was used for rooting culture for 30 days, 50 days, and 70 days.

[0051] Figure 7 is a rooting culture display figure of Example 1 in which the WPM medium was used as a basic medium and the medium of treatment 3 (WPM+NAA 1.5 mg / L+sucrose 30 g / L+agar 4.9 g / L, pH=5.8) was used for rooting culture for 30 days, 50 days, and 70 days.

[0052] Figure 8 is a rooting culture display figure of Example 1 in which the WPM medium was used as a basic medium and the medium of treatment 5 (WPM+NAA 1.0 mg / L+6-BA 0.5 mg / L+sucrose 30 g / L+agar 4.9 g / L, pH=5.8) was used for rooting culture for 30 days, 50 days, and 70 days.

[0053] Figure 9 is a rooting culture display figure of Example 1 in which the WPM medium was used as a basic medium and the medium of treatment 6 (WPM+NAA 1.5 mg / L+6-BA 0.5 mg / L+sucrose 30 g / L+agar 4.9 g / L, pH=5.8) was used for rooting culture for 30 days, 50 days, and 70 days. DETAILED DESCRIPTION

[0054] The present application will be described in further detail by working examples and accompanying drawings, but the embodiments of the present application are not limited to them.

[0055] Selection of basal medium and exogenous hormones of Example 1

[0056] NAA belongs to auxin, 6-BA belongs to cytokinin, wherein, NAA can promote adventitious root growth, 6-BA can promote bud differentiation and lateral bud germination. In this embodiment, 1 / 2MS, MS, WPM are used as basal medium, and different types and different concentrations of exogenous hormones are added to study the possibility of tissue culture of two-year-old, newly germinated and two-tender-bud-bearing stem segments of A. chinensis as explants. The specific method and results are as follows:

[0057] I. Test process

[0058] 1. Sampling and disinfection of explants

[0059] (1) Collection and pretreatment of explants: cut two-year-old A. chinensis branches and remove leaves to obtain stem segments with tender buds, wherein each stem segment is 3-4 cm long and has two newly germinated tender buds; wash the two-year-old, newly germinated and two-tender-bud-bearing stem segments with running water for 40 min;

[0060] (2) Disinfection of explants: immerse in 75% alcohol for 30 s, then disinfect in a solution containing 0.1 wt.% mercuric chloride and 1 wt.% Tween 20 for 10 min, wash with sterile water 4 times, finally dry with sterile filter paper and cut off the contact part of the incision and disinfectant;

[0061] 2. Inoculation and rooting culture

[0062] Inoculate the explants after step 1 disinfection into different rooting culture media (see Tables 1-3 for specific compositions, also containing sucrose 30 g / L, agar 4.9 g / L), and carry out rooting culture under the conditions of indoor culture temperature 24℃, light intensity 1800-2400 lx, daily light 12 h.

[0063] II. Test results and analysis

[0064] 1. Using 1 / 2MS as basal medium

[0065] The two-year-old newly germinated stems with two buds of A. veitchii were used as explants, and 1 / 2MS was used as the basic medium with different concentrations of NAA (0.5, 1.0, 1.5 mg / L) and 6-BA (0, 0.5, 1.0 mg / L) for rooting culture. The completely random design was used, and there were 9 treatments, each repeated 3 times (Table 1). The possibility of using the two-year-old newly germinated stems with two buds of A. veitchii as explants for tissue culture was observed by adding different types and concentrations of growth hormones. The results are shown in Table 1. Among them, 1 / 2MS was used as the basic medium, and 0.5 mg / L and 1.0 mg / L of NAA were added to induce root primordium formation, and the rooting rates were 67.4% and 51.2%, respectively. Among them, Figure 1 A and Figure 1 B are the rooting conditions after 45 days of treatment 1 and treatment 2, respectively. The stems of treatment 2 are relatively weak, but the root length is longer than that of treatment 1. In the remaining treatments, NAA and 6-BA were added, and no rooting phenomenon was observed, but callus formation was observed. Some results are shown in Figure 2 , which shows that 6-BA effectively promotes cell division and induces bud differentiation.

[0066] Table 1 Rooting culture results of 1 / 2MS medium with different hormone types and concentrations

[0067]

[0068] 2, MS was used as the basic medium

[0069] The two-year-old newly germinated stems with two buds of A. veitchii were used as explants, and MS was used as the basic medium, with different concentrations of NAA (0.5, 1.0, 1.5 mg / L) and 6-BA (0, 0.5, 1.0 mg / L) for rooting culture. The completely random design was used, and there were 9 treatments, each repeated 3 times (Table 2). The possibility of using the two-year-old newly germinated stems with two buds of A. veitchii as explants for tissue culture was observed by adding different types and concentrations of growth hormones. The results are shown in Table 2. From Table 2, only treatment 1 (MS+NAA 0.5 mg / L+ sucrose 30 g / L+ agar 4.9 g / L) had rooting (rooting rate 71.2%), Figure 3 is the rooting condition after 45 days of treatment 1. The remaining treatments all have callus formation, and no root differentiation is observed. Some results are shown in Figure 4 . It is shown that 6-BA effectively promotes cell division and induces bud differentiation, which is similar to the results in Table 1.

[0070] Table 2 Rooting culture results of MS medium with different hormone types and concentrations

[0071]

[0072] 3. WPM as a basal medium

[0073] The two-year-old newly germinated stems with two buds of A. veitchii were used as explants, and WPM was used as a basal medium, and different concentrations of NAA (0.5, 1.0, 1.5 mg / L) and BA (0, 0.5, 1.0 mg / L) were added for rooting culture. A total of 9 treatments were designed at random, and each treatment was repeated 3 times (Table 3). The possibility of using the two-year-old newly germinated stems with two buds of A. veitchii as explants for tissue culture was observed, and the results are shown in Table 3. As shown in Table 3, rooting occurred in treatments 1, 2, and 3 with NAA concentrations of 0.5 mg / L (treatment 1), 1.0 mg / L (treatment 2), and 1.5 mg / L (treatment 3), respectively. However, root growth was very sensitive to NAA concentration, and there were significant differences in root growth. When the NAA concentration increased to 1.5 mg / L, the rooting time was later than that of 1.0 mg / L (treatment 2), and the effect on the formation of root primordia was small. When the NAA concentration was 1.0 mg / L, the root primordia of the explants could be induced more quickly. No callus was formed during rooting, and the rooting was early, with a rooting rate of 100% after 20 days of culture, the root length was more than 2 cm, the number of roots was large, the roots were thick and long, and the rooting rate was 100%. Figure 5 is the rooting condition of treatment 2 on the 38th day of culture, and compared with 1 / 2MS treatment 1, 1 / 2MS treatment 2, and MS treatment 1 on the 45th day of culture, the root length and the number of roots increased significantly. In addition, the absence of callus formation is also beneficial to the subsequent transplantation after rooting, because the formation of callus will affect the transport of nutrients.

[0074] In treatments 4, 5, 6, 7, 8, and 9, in addition to the addition of NAA, 6-BA hormone was also added. When the 6-BA concentration was 0.5 mg / L (treatment 5, treatment 6), rooting occurred, but similar to treatments 1 and 3, the rooting was not ideal, and the roots were few and short after about 5 weeks. Figure 6 Figure 7 Figure 8 Figure 9 When the 6-BA concentration was 1.0 mg / L, no rooting occurred, indicating that the higher the 6-BA concentration, the greater the inhibition of root growth. Therefore, the mixture of 6-BA and NAA cannot significantly induce the formation of root primordia and promote root growth.

[0075] Table 3. WPM medium with different hormone types and concentrations and rooting culture results

[0076]

[0077] ​​​The above test results show that: taking the two-year-old newly germinated stem segments with two tender buds of Atractylodes lancea as explants, using WPM as a basic culture medium, and adding growth hormone NAA for rooting culture, the rooting effect is ideal, the best culture medium is WPM+NAA 1.0 mg / L+sucrose 30 g / L+agar 4.9 g / L, pH=5.8. The WPM is selected as the basic culture medium in the application, which is different from the basic culture medium of each treatment in Table 1 (1 / 2MS) and Table 2 (MS). According to the published research papers and related reports, no researchers have used WPM as the basic culture medium for Atractylodes lancea tissue culture.

[0078] The WPM culture medium is very significant for the rooting speed and root growth effect (root length, root thickness, and root number) of Atractylodes lancea, the culture period is short, and it is obviously better than 1 / 2MS culture medium and MS culture medium.

[0079] Example 2: Selection of disinfectant type, concentration and disinfection time

[0080] This example studies the influence of disinfectant type, concentration and disinfection time on Atractylodes lancea tissue culture, and the specific method and results are as follows:

[0081] I. Test process

[0082] 1. Sampling and disinfection of explants

[0083] (1) Collection and pretreatment of explants: same as example 1;

[0084] (2) Disinfection of explants: 20 explants were inoculated for each disinfection treatment, each treatment was repeated 3 times, and the specific treatment was as follows: 75% alcohol was used for immersion and washing for 30 s, and then 5wt.% sodium hypochlorite solution was used for disinfection for 12 min, 10wt.% 84 disinfection for 30 min, 0.1wt.% mercury chloride solution for 10 min or 12 min, or 0.1wt.% mercury chloride solution and 1wt.% Tween 20 solution for 10 min, and then washed with sterile water for 4 times; finally, the surface moisture was absorbed by sterile filter paper and the contact part of the incision and disinfectant was cut off;

[0085] 2. Inoculation and rooting culture

[0086] The specific culture is the same as example 1; wherein, after 3 days of inoculation, the pollution number and death number of each treatment were observed and counted, respectively, and after 30 days of inoculation, the normal germination number of each treatment was counted.

[0087] II. Test results and analysis

[0088] The present example studies the different effects of various disinfectants on explant disinfection. As can be seen from Table 4, the explant contamination rates of treatment 1 and treatment 2 are both very high. Because the surface of A. chinensis is covered with hair and the branches are easy to carry bacteria, sodium hypochlorite and 84 are not easy to completely sterilize the explants, and thus are not suitable for disinfecting A. chinensis explants.

[0089] The disinfection effects of treatment 3 and treatment 4 are obviously superior to sodium hypochlorite and 84, but the disinfection time of 0.1wt.% mercury chloride solution is very critical. If the time is short, the contamination rate is high, and if the time is long, the explants are severely browned and the mortality rate is high. After the explants are disinfected with 0.1wt.% mercury chloride solution for 12 min in treatment 4, the contamination rate of the explants is 33.4%, but most of the explant stems are black-brown and severely browned, with a mortality rate as high as 58.3%. If the disinfection time is too long, the damage to the explants is very large, which seriously affects the growth and differentiation. After the explants are disinfected with 0.1wt.% mercury chloride solution for 10 min in treatment 3, the browning of the explants is light, but the contamination rate is as high as 41.3%. This also shows that the disinfection of A. chinensis explants is difficult. Treatment 5 is to add 1wt.% Tween 20 to 0.1wt.% mercury chloride solution, which can make the mercury chloride more easily penetrate into the surface of the explants, improve the sterilization effect, and the contamination rate is 11.6% and the mortality rate is only 21.5%.

[0090] Therefore, the disinfection effect of the treatment of washing with 75% alcohol for 30 s and then disinfecting with 0.1wt.% mercury chloride + 1wt.% Tween 20 for 10 min is the best, the contamination rate is low, and it is suitable for disinfecting the stem segments of A. chinensis explants.

[0091] Table 4 Sterilization test of A. chinensis stem segments

[0092]

[0093] Example 3 Secondary rooting culture

[0094] I. Test process

[0095] 1. Taking and disinfecting explants

[0096] (1) Collecting and pretreating explants: same as example 1;

[0097] (2) Disinfecting explants: washing with 75% alcohol for 30 s, then disinfecting with a solution containing 0.1wt.% mercury chloride and 1wt.% Tween 20 for 10 min, washing with sterile water for 4 times, finally absorbing the surface moisture with sterile filter paper and cutting off the contact part of the incision and disinfecting solution;

[0098] 2. Inoculation and rooting culture

[0099] The sterilized explants of step 1 are inoculated into rooting medium (WPM+NAA 1.0 mg / L+sucrose 30 g / L+agar 4.9 g / L, pH=5.8) and cultured in a room with a temperature of 24°C, a light intensity of 1800-2400 lx, and a light duration of 12 hours per day;

[0100] 3. Secondary rooting culture

[0101] The stem segments (about 2-3 cm long) are cut again after the primary rooting culture and transferred to fresh rooting medium (WPM+NAA 1.0 mg / L+sucrose 30 g / L+agar 4.9 g / L, pH=5.8) for secondary rooting culture to expand the number of propagations. The secondary rooting culture conditions are the same as step 2.

[0102] II. Test results and analysis

[0103] The rooting effect is obvious at 20 days of rooting culture in step 2 Figure 5 A), the average root length is about 4 cm, and the number of roots can reach more than 10 Figure 5 B) after 38 days of rooting culture; the number of roots increases, and the roots are thick and strong Figure 5 B) after 50 days of rooting culture; in general, the roots are very developed, the number of roots is large, and the average root length is >10 cm after 70 days of rooting culture Figure 5 D1, E), and some seedlings have particularly developed roots Figure 5 D2); Figure 5 F is a display of seedlings at 20 days of secondary rooting culture of the stem segments cut from the aseptic seedlings after about 38 days of rooting culture. The seedlings grow well.

[0104] Example 4. Seedling culture

[0105] This example studies the effect of increasing seedling culture. The specific method is as follows:

[0106] 1. Explant sampling and sterilization

[0107] (1) Explant collection and pretreatment: same as example 3;

[0108] (2) Explant sterilization: same as example 3;

[0109] 2. Inoculation and rooting culture: same as example 3;

[0110] 3. Seedling culture

[0111] After the adventitious roots grow out of the rooting culture, the seedlings are transferred to seedling culture medium (WPM+coconut juice 10 wt.%+agar 4.9 g / L, pH=5.8) for 5 days of culture. The culture conditions are the same as step 2.

[0112] 4. Secondary rooting culture

[0113] After the seedling culture, secondary rooting culture was carried out, and the specific method was the same as that in Example 3.

[0114] II. Test results and analysis

[0115] In this example, a seedling culture process was added after the primary rooting culture (Example 3), and the specific method was as follows: Figure 5 As can be seen from Table G, after the seedling culture, the number and thickness of the roots of the seedlings were significantly increased, and the yellowing problem was effectively improved, and the overall robustness of the seedlings was improved. The seedling culture can shorten the time from rooting to seedling transplantation. The growth cycle of Atractylodes chinensis from seed sowing to harvesting generally needs 4 years, and the rhizome part is mainly used for medicine. The developed roots are not only beneficial to the later transplanting and adaptation process, but also can effectively shorten the field planting cycle of Atractylodes chinensis, save the time cost and economic cost of medicine planting farmers, and improve the economic income of farmers, which provides a better way for the popularization and application of Atractylodes chinensis medicine.

[0116] Example 5: Effect of different sterilization methods of transplanting substrate on seedling transplantation

[0117] In this example, the effect of different substrates on seedling transplantation was studied, and the specific method was as follows:

[0118] I. Test process

[0119] 1. Explant sampling and sterilization

[0120] (1) Explant collection and pretreatment: the same as Example 4;

[0121] (2) Explant sterilization: the same as Example 4;

[0122] 2. Inoculation and rooting culture: the same as Example 4;

[0123] 3. Seedling culture: the same as Example 4;

[0124] 4. Secondary rooting culture: the same as Example 4;

[0125] 5. Seedling transplantation

[0126] When the tissue culture seedlings after step 3 seedling culture or the tissue culture seedlings after step 4 secondary rooting culture grow developed root system (the number of roots is not less than 10, and the average root length is not less than 4 cm), the following treatments were carried out in turn:

[0127] (1) In-bottle seedling training: loosen the culture bottle sealing film to gradually reduce the humidity in the bottle and increase the light; after 3 days, completely remove the sealing film and open the bottle for 2 days of seedling training;

[0128] (2) Bottle outside acclimation: after the completion of bottle acclimation, the tissue culture seedlings are taken out, and the agar on the roots is cleaned; vermiculite and peat soil are mixed according to a mass ratio of 1:1 to obtain a mixed substrate, the tissue culture seedlings are transplanted into soft plastic pots (10x10 cm) containing the mixed substrate and covered with a transparent film, and then placed in a culture room at a temperature of 24°C and a humidity of 80% for bottle outside acclimation for 7 days, and observed, watered and ventilated for 2 hours every day; wherein, the mixed substrate is sterilized before use as follows, 20 bottles of tissue culture seedlings are transplanted for each treatment method, and each treatment is repeated 3 times:

[0129] Treatment 1: a 0.4wt.% potassium permanganate solution is uniformly sprayed on the mixed substrate, then the substrate is stirred uniformly, and then covered with a plastic film, and fumigated for about 7 days;

[0130] Treatment 2: a 0.3wt.% copper sulfate solution is uniformly sprayed on the mixed substrate, then the substrate is stirred uniformly, and then covered with a plastic film, and fumigated for about 7 days;

[0131] Treatment 3: the substrate is subjected to high-temperature sterilization treatment, and then the temperature of the substrate is cooled to below 30°C;

[0132] Treatment 4: the substrate is subjected to high-temperature sterilization treatment, and then the temperature of the substrate is cooled to below 30°C;

[0133] (3) Greenhouse management: after the end of bottle outside acclimation, the transparent film is removed, and the seedlings and the mixed substrate are transferred together to a greenhouse for shading culture, wherein the environmental humidity in the greenhouse is maintained at 70-80%, and the environmental temperature is 20-24°C, and shading is performed for 4 hours every day (from 11 am to 15 pm); after shading culture for 5 days, routine culture can be performed. When transferred to the greenhouse, each mixed substrate is treated as follows to prevent diseases:

[0134] Treatment 1: a 0.2wt.% potassium permanganate solution is sprayed to wet the mixed substrate, and the plant leaf surface is rinsed with clean water after spraying to prevent the occurrence of phytotoxicity;

[0135] Treatment 2: a 0.2wt.% copper sulfate solution is sprayed to wet the mixed substrate, and the plant leaf surface is rinsed with clean water after spraying to prevent the occurrence of phytotoxicity;

[0136] Treatment 3: no treatment is performed;

[0137] Treatment 4: a 0.2wt.% potassium permanganate solution is sprayed to wet the mixed substrate, and the plant leaf surface is rinsed with clean water after spraying to prevent the occurrence of phytotoxicity.

[0138] II. Test results and analysis

[0139] The transplanting substrate requires to be loose, and has good drainage and ventilation. Commonly used substrates include vermiculite, peat soil, perlite and humus soil, etc. The substrate of pure vermiculite has good water and air permeability, but the substrate has large flowability, and the tissue culture seedlings are not easy to be fixed. The mixture of vermiculite and peat soil with a mass ratio of 1:1 is selected as the substrate in the present application, so that the problem can be solved. Meanwhile, the different effects of different disinfection treatment methods on the mixed substrate after disinfection and the seedling raising and transplanting are studied.

[0140] As can be seen from Table 5, the high-temperature sterilization method of treatment 4 has obvious effect, and the survival rate is high. If 0.2wt.% potassium permanganate solution is sprayed on the surface of the mixed substrate during the transplanting management, the substrate contamination can be further reduced to the minimum, the tissue culture seedlings grow normally, and the survival rate is the highest. The survival rate of treatment 1 is 94.6%, the survival rate of treatment 2 is 91.3%, and the survival rate of treatment 3 is 93.8%. The disinfection effects of treatments 1, 2 and 3 are all good, but considering the influence of disinfectant on the environment and the economic cost of substrate disinfection, it is considered that treatment 4 is the best disinfection method after comprehensive consideration.

[0141] Table 5 Influence of different disinfection methods of substrate on survival rate of transplanting

[0142]

[0143] Example 6 Comparison of seedling raising effects of hormone (NAA) contained in transplanting substrate and control

[0144] In this embodiment, the growth difference of tissue culture seedlings with and without adding hormone (NAA) in the substrate is studied, and the specific implementation is as follows:

[0145] I. Test process

[0146] 1. Sampling and disinfection of explants

[0147] (1) Collection and pretreatment of explants: same as example 4;

[0148] (2) Disinfection of explants: same as example 4;

[0149] 2. Inoculation and rooting culture: same as example 4;

[0150] 3. Strong seedling culture: same as example 4;

[0151] 4. Secondary rooting culture: same as example 4;

[0152] 5. Seedling raising and transplanting

[0153] When the tissue culture seedlings after step 3 strong seedling culture or the tissue culture seedlings after step 4 secondary rooting culture grow developed root system (the number of roots is not less than 10, and the average root length is not less than 4cm), the following treatments are sequentially performed:

[0154] (1) In-vitro seedling: same as Example 5;

[0155] (2) Out-vitro seedling: after the in-vitro seedling is completed, the tissue culture seedlings are taken out, and the agar on the roots is cleaned; the vermiculite and the peat soil are mixed according to a mass ratio of 1:1 to obtain a mixed substrate, the tissue culture seedlings are transplanted into soft plastic pots (10x10 cm) containing the mixed substrate, and are covered with a transparent film, and then are placed in a culture room at a temperature of 24°C and a humidity of 80% for out-vitro seedling for 7 days, and are observed, watered and ventilated for 2 hours every day; wherein, before use, the mixed substrate is treated as follows, 20 bottles of tissue culture seedlings are transplanted for each treatment method, and each treatment is repeated for 3 times:

[0156] Treatment 1: the substrate is subjected to high-temperature sterilization treatment, and then the temperature of the substrate is cooled to below 30°C, and the mixed substrate is sprayed with clean water to be moistened;

[0157] Treatment 2: the substrate is subjected to high-temperature sterilization treatment, and then the temperature of the substrate is cooled to below 30°C, and the mixed substrate is sprayed with a 0.5 mg / L NAA solution to be moistened;

[0158] (3) Greenhouse management: after the out-vitro seedling is completed, the transparent film is removed, and the seedlings and the mixed substrate are transferred together into a greenhouse for shading culture, wherein, when being transferred into the greenhouse, the mixed substrate is treated as follows to prevent diseases: a 0.2 wt.% potassium permanganate solution is sprayed to moisten the mixed substrate, and after spraying, the plant leaf surface is washed with clean water to prevent the occurrence of drug damage; the environmental humidity in the greenhouse is maintained at 70-80%, and the environmental temperature is 20-24°C, and shading is performed for 4 hours every day (from 11 am to 15 pm); after shading culture for 5 days, routine culture can be performed.

[0159] II. Test results and analysis

[0160] Based on the principle that NAA can promote rooting, a comparative test of the survival rate of tissue culture seedlings in the substrate with added hormone NAA and the control is performed. As can be seen from Table 6, after adding NAA to the transplanting substrate, the growth of the tissue culture seedlings shows significant differences. After adding NAA, the tissue culture seedlings show better growth, and the survival rate is higher than that of the control.

[0161] Table 6 Growth comparison of tissue culture seedlings in the substrate with added hormone (NAA) and the control

[0162]

[0163] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are all included in the protection scope of the present application.

Claims

1. A rapid propagation method for Atractylodes lancea tissue culture, characterized in that... The method comprises the following steps: (1) cutting two-year-old Atractylodes lancea branches, selecting stem segments with two sprouts and carrying out pretreatment and disinfection treatment; (2) inoculating the stem segments with two sprouts and carrying out rooting culture at a culture temperature of 23-24 DEG C, a light intensity of 1800-2400lx and a daily light time of 12-14h; (3) when the rooting culture in step (2) is completed, transferring the stem segments with two sprouts to a new rooting culture medium for 5-7d; (4) cutting the stem segments with two sprouts in the culture medium in step (3) and inoculating them in a new rooting culture medium for secondary rooting culture to expand the propagation quantity; (5) transplanting the tissue culture seedlings in step (3) or the tissue culture seedlings in step (4) after secondary rooting culture. The disinfection treatment in step (1) is specifically as follows: immersing the stem segments with two sprouts in 75% alcohol for 30s, then disinfecting them in a solution containing 0.1wt.% mercuric chloride and 1wt.% Tween 20 for 10min, and washing them with sterile water for 3-4 times; after washing with sterile water, absorbing the surface water and cutting the contact part of the incision with the disinfection solution; the rooting culture medium in step (2) or (4) is WPM+NAA 1.0mg / L+cane sugar 30g / L+agar 4.9g / L, pH=5.8; the stem segments with two sprouts in step (1) are 3-4cm long.

3. The method according to claim 1, wherein the pretreatment in step (1) is washing the stem segments with two sprouts with running water for 30-60min.

4. The method according to claim 1, wherein the stem segments with two sprouts in step (4) are 2-3cm long.

5. The method according to claim 1, wherein the tissue culture seedlings in step (5) have not less than 10 roots and the root length is not less than 3-4cm.

6. The method according to claim 1, wherein the transplanting in step (5) is specifically as follows: when the tissue culture seedlings have not less than 10 roots and the average root length is not less than 3-4cm, the following treatments are carried out in sequence: ① in-bottle hardening: loosening the culture bottle sealing film to gradually reduce the humidity in the bottle and increase the light; after 3-4d, completely removing the sealing film and opening the bottle for 2-3d. ​ ​ ​ ​ ​ ​ ②Bottle outside seedling: after the completion of the bottle seedling, the tissue culture seedlings are taken out, and the agar on the roots is cleaned; the tissue culture seedlings are transplanted into the mixed substrate and covered with a transparent film, and then placed in a culture room with a temperature of 23-24°C and a humidity of 70-80% for 7-10 days of bottle outside seedling, with water spraying every day and ventilation for 2-3 hours every day; ③Greenhouse management: after the end of the bottle outside seedling, the transparent film is removed, and the seedlings and the mixed substrate are transferred together to the greenhouse for shading culture, wherein the environmental temperature in the greenhouse is 20-24°C, the environmental humidity is 70-80%, and the shading is performed for 3-4 hours every day; after 4-5 days of shading culture, the conventional culture can be performed.

7. The tissue culture rapid propagation method of Rhizoma et Radix seu Caulis Notoginseng according to claim 6, characterized in that: The mixed substrate in step ② is a mixture of vermiculite and grass charcoal soil, and the mass ratio of vermiculite to grass charcoal soil is 1:

1.

8. The tissue culture rapid propagation method of Rhizoma et Radix seu Caulis Notoginseng according to claim 6, characterized in that: The mixed substrate in step ② is pretreated as follows: After high-temperature sterilization of the mixed substrate, 0.5 mg / L of NAA solution is sprayed to the substrate until it is wet.

9. The tissue culture rapid propagation method of Rhizoma et Radix seu Caulis Notoginseng according to claim 6, characterized in that: When the seedlings and the mixed substrate are transferred together to the greenhouse in step ③, 0.2 wt.% of potassium permanganate solution is sprayed to the mixed substrate until it is wet.

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