A tissue culture rapid propagation medium and method for cultivating Parrotia subaequalis

Abstract

The present invention belongs to the technical field of plant tissue culture, and particularly relates to a tissue culture rapid propagation medium and a tissue culture rapid propagation method for cultivating Parrotia subaequalis. The present invention provides a tissue culture rapid propagation medium and a tissue culture rapid propagation method for cultivating Parrotia subaequalis. In the tissue culture rapid propagation medium provided by the present invention, kinetin (KT) can promote the formation of buds and the growth of adventitious buds. 6-BA is 6-benzylaminopurine, which promotes the growth of buds; TDZ is thidiazuron, which promotes the growth and development of buds. Under the combined action of KT, 6-BA and TDZ, the bud induction rate of the stem segments with buds and the bud proliferation coefficient are improved. The present invention also provides a tissue culture rapid propagation method for Parrotia subaequalis, in which the contamination rate of the induced stem segments is low, the survival rate of the stem segments is high, and the browning rate of the stem segments is low; the highest lateral bud induction rate of the stem segments reaches 91%, and the proliferation coefficient, rooting rate and transplanting survival rate are high.

Description

Technical Field

[0001] The invention belongs to the technical field of plant tissue culture, and particularly relates to a tissue culture rapid propagation medium and a tissue culture rapid propagation method for culturing Hamamelis argyi. Background Art

[0002] Hamamelis chinensis Parrotia subaequalis (H.T.Cheng)R.M.Hao et H_T.wei) is a small deciduous tree of the genus Hamamelis of the family Hamamelidaceae. It is a key protected wild plant in the first batch of "National Key Protected Wild Plant List", listed as a Class I endangered species in the "China Red List of Plants", and listed as a critically endangered (CR) species by the International Union for Conservation of Nature (IUCN). It is known as a living fossil among angiosperms. As a relict plant of the Tertiary period, Hamamelis has important scientific research value for species evolution and global environmental changes. Hamamelis has a narrow habitat distribution, distributed in parts of Yixing, Jiangsu, Anji and Lin'an, Zhejiang, Shucheng, Jinzhai and Jixi, Anhui, and the largest population has only 91 trees. The flowers, leaf shape, color, and tree shape of Hamamelis argyrae are all of great ornamental value, and are a valuable resource for use in garden landscaping and bonsai. Its wood is fine, hard, and has a high specific gravity, making it an excellent timber species. Therefore, research on the protection of Hamamelis argyrae resources and breeding technology is of great significance.

[0003] Under natural conditions, the competitiveness of Hamamelis argentea in the population is relatively weak. Wild Hamamelis argentea mostly grows beside rivers and streams in low-altitude areas and is distributed among rocks in high-altitude areas. Conservation work is very difficult. In places with insufficient light, the survival rate of Hamamelis argentea is very low. Secondly, it usually blooms once every 1 to 3 years. The male and female flowers of Hamamelis argentea are sterile during the flowering period, and the natural pollination rate is very low. Wild seeds are extremely difficult to obtain. At present, there are only a few reports on the research technology of Hamamelis argentea, most of which focus on the research of seed germination conditions and cutting environmental factors. Zheng Xuefeng et al. used cutting propagation methods for old Hamamelis argentea trees. The survival rate of hard branch cuttings in spring was only 30% to 35%, and the survival rate of cuttings after the leaves of the current year branches fell in autumn and winter was 60% to 70%, but the subsequent growth of seedlings has not been reported. There are currently no reports on the successful rapid propagation of Hamamelis argyrae through tissue culture. The only study by Lai Fuxin (see "Lai Fuxin. Preliminary study on the restoration of endangered plant Hamamelis argyrae populations [D]. Anqing Normal University, 2022.") used sterile sowing and in vitro culture of stem segments. The seed germination rate was only 11.7%, and the induction rate using stem segments as explants was 21.67%. In addition, browning seriously affected the growth of seedlings, and there was no systematic report on successful rooting culture and transplanting. Summary of the invention

[0004] The invention provides a tissue culture rapid propagation culture medium and a tissue culture rapid propagation method for culturing Hamamelis salviae var.

[0005] To solve the above technical problems, the present invention proposes the following technical solutions:

[0006] The present invention provides a tissue culture and rapid propagation medium for cultivating Parrotia subaequalis, and the tissue culture and rapid propagation medium includes an induction medium, a proliferation medium, and a rooting medium;

[0007] The induction medium uses WPM as the basic medium and further includes 1.0 - 1.5 mg / L KT, 28 - 30 g / L sucrose, and 6 - 8 g / L agar;

[0008] The proliferation medium uses WPM as the basic medium and further includes 1.0 - 1.5 mg / L 6 - BA, 0.003 - 0.005 mg / L TDZ, 28 - 30 g / L sucrose, and 6 - 8 g / L agar;

[0009] The rooting medium uses 1 / 2MS as the basic medium and further includes 28 - 30 g / L sucrose and 6 - 8 g / L agar.

[0010] Preferably, the rooting medium further includes 0.11 - 0.4 mg / L NAA.

[0011] The present invention provides a tissue culture and rapid propagation method for Parrotia subaequalis, which uses the tissue culture and rapid propagation medium described in the above technical solution, and includes the following steps:

[0012] Inoculate the stem segments with latent buds of Parrotia subaequalis into the induction medium for induction culture to obtain stem segments with lateral buds;

[0013] Transfer the stem segments with lateral buds to the proliferation medium for proliferation culture to obtain cluster buds;

[0014] Cut the cluster buds into single buds and transfer them to the rooting medium for rooting culture to obtain rooted seedlings.

[0015] Preferably, before inoculation, the stem segments are also disinfected, and the disinfection includes first soaking them in 75% ethanol by volume for 30 - 40 s, rinsing them with sterile water 3 - 4 times, then soaking them in a sodium hypochlorite solution with an available chlorine content of 0.26% - 0.52% for 5 - 10 min, and rinsing them with sterile water 5 - 6 times.

[0016] Preferably, the temperatures of the induction culture, proliferation culture, and rooting culture are 23 - 27 °C respectively.

[0017] Preferably, the induction culture is first dark - cultured and then light - cultured; the proliferation culture and rooting culture are respectively light - cultured.

[0018] Preferably, the light exposure time during light cultivation is 12 - 16 h / d; the light intensity is 2200 - 2500 lx; the dark time during light cultivation is 8 - 12 h / d.

[0019] Preferably, the induction cultivation time is 28 - 32 d, the dark cultivation time during induction cultivation is 2 - 4 d, and the light cultivation time is 26 - 28 d.

[0020] Preferably, the proliferation cultivation time is 28 - 35 d; the rooting cultivation time is 28 - 33 d.

[0021] Preferably, before the proliferation cultivation of the stem segments with lateral buds, it further includes: performing short - term subculture on the stem segments with lateral buds.

[0022] Preferably, the length of the stem segment with latent buds is 1.5 - 2.0 cm.

[0023] The beneficial effects of the present invention: The present invention provides a tissue culture and rapid propagation medium for cultivating Parrotia subaequalis. In this tissue culture and rapid propagation medium, KT is Kinetin, that is, kinetin, which can promote the formation of buds and the growth of adventitious buds. 6 - BA is 6 - benzylaminopurine, which promotes the growth of buds; TDZ is thidiazuron, which promotes the growth and development of buds. Under the combined action of KT, 6 - BA and TDZ, the bud induction rate of the stem segments with buds and the bud proliferation coefficient are improved.

[0024] The present invention also provides a tissue culture and rapid propagation method for Parrotia subaequalis, using the stem segment with latent buds as the explant for tissue culture. The present invention has established a complete in vitro regeneration and breeding technology system for the rare and endangered plant Parrotia subaequalis for the first time, that is, using the stem segment with latent buds on the new branches of the seedlings grown from seeds sown in the wild for 1 - 3 years as the explant. Through the screening of the culture techniques and medium formulations in four stages: inducing lateral bud germination, lateral bud proliferation, and rooting cultivation, the present invention has established a set of stem segment regeneration technology system for Parrotia subaequalis. The contamination rate of the induced stem segments is low, the survival rate of the stem segments is as high as 78% - 83%, and the browning rate of the stem segments is only 7% - 16%; the lateral bud induction rate (i.e., the germination rate) of the stem segments is up to 91% at most, the lateral bud proliferation coefficient is as high as 4.17, and the rooting rate reaches 60%; transplanting the test - tube seedlings with good rooting, the transplanting survival rate is over 95%. Description of the Drawings

[0025] Figure 1 It is a diagram showing the influence of different basic media on the germination of lateral buds of Parrotia subaequalis, where A represents MS, B represents 1 / 2MS, and C represents WPM;

[0026] Figure 2The figure shows the effects of different concentrations of KT on the induction of lateral buds from in vitro stem segments. F1 - F5 are 0.5 mg / L, 1.0 mg / L, 1.5 mg / L, 2.0 mg / L, and 4.0 mg / L respectively;

[0027] Figure 3 The figure shows the effects of different concentrations of 6 - BA on the induction of lateral buds from in vitro stem segments. D1 - D5 are 0.5 mg / L, 1.0 mg / L, 1.5 mg / L, 2.0 mg / L, and 4.0 mg / L respectively;

[0028] Figure 4 The figure shows the effects of different concentrations of TDZ on the induction of lateral buds from in vitro stem segments. E1 - E5 are 0.001 mg / L, 0.01 mg / L, 0.1 mg / L, 1.0 mg / L, and 2.0 mg / L

[0029] Figure 5 The figure shows the effects of different concentrations of 6 - BA and TDZ on the proliferation of lateral buds of Parrotia subaequalis;

[0030] Figure 6 The figure shows the effects of different concentrations of TDZ on the proliferation of lateral buds of Parrotia subaequalis;

[0031] Figure 7 The figure shows the effects of different concentrations of IBA and NAA on the rooting of axenic seedlings of Parrotia subaequalis;

[0032] Figures 1 - 7 The scale bar for all is 1 cm. Detailed implementation method

[0033] The present invention provides a tissue culture and rapid propagation medium for cultivating Parrotia subaequalis, and the tissue culture and rapid propagation medium includes an induction medium, a proliferation medium, and a rooting medium.

[0034] The induction medium of the present invention uses WPM as the basic medium and also includes 1.0 - 1.5 mg / L KT, 28 - 30 g / L sucrose, and 6 - 8 g / L agar.

[0035] As an alternative embodiment, the concentration of KT in the induction medium provided by the present invention is 1.0 - 1.5 mg / L; in the specific embodiments of the present invention, the concentration of KT in the induction medium can be 1.0, 1.3 or 1.5 mg / L. KT helps to induce the formation of buds from explants and improve the bud induction rate. The concentration of sucrose in the induction medium provided by the present invention is 28 - 30 g / L. In the specific embodiments of the present invention, the concentration of sucrose in the induction medium can be 28, 29 or 30 g / L. The concentration of agar in the induction medium provided by the present invention is 6 - 8 g / L. In the specific embodiments of the present invention, the concentration of agar in the induction medium can be 6, 7 or 8 g / L. In the present invention, the pH value of the induction medium can be 5.7 - 5.9, or can also be 5.8.

[0036] The proliferation medium of the present invention uses WPM as the basic medium and further includes 1.0 - 1.5 mg / L 6 - BA, 0.003 - 0.005 mg / L TDZ, 28 - 30 g / L sucrose and 6 - 8 g / L agar.

[0037] As an alternative embodiment, the concentration of 6 - BA in the proliferation medium provided by the present invention is 1.0 - 1.5 mg / L, or can also be 1.2 - 1.4 mg / L; in the specific embodiments of the present invention, the concentration of 6 - BA in the proliferation medium is 1.0, 1.2, 1.4 or 1.5 mg / L. The concentration of TDZ in the proliferation medium provided by the present invention is 0.003 - 0.005 mg / L; in the specific embodiments of the present invention, the concentration of TDZ in the proliferation medium is 0.003, 0.004 or 0.005 mg / L. 6 - BA and TDZ jointly promote the proliferation of buds under a suitable concentration ratio, and the growth state of lateral buds is good. The concentration of sucrose in the proliferation medium provided by the present invention is 28 - 30 g / L; in the specific embodiments of the present invention, the concentration of sucrose in the proliferation medium is 28, 29 or 30 g / L. The concentration of agar in the proliferation medium provided by the present invention is 6 - 8 g / L. In the specific embodiments of the present invention, the concentration of agar in the proliferation medium can be 6, 7 or 8 g / L. In the present invention, the pH value of the proliferation medium can be 5.7 - 5.9, or can also be 5.8.

[0038] The rooting medium of the present invention uses 1 / 2MS as the basic medium and further includes 28 - 30 g / L sucrose and 6 - 8 g / L agar; as another alternative embodiment, the rooting medium of the present invention uses 1 / 2MS as the basic medium and further includes 0.11 - 0.4 mg / L NAA, 28 - 30 g / L sucrose and 6 - 8 g / L agar. NAA can promote plant growth and the formation of roots.

[0039] As an alternative embodiment, the concentration of NAA in the rooting medium provided by the present invention is 0.11 - 0.4 mg / L, and it can also be 0.11 - 0.2 mg / L. NAA can promote the adventitious bud rooting of Parrotia subaequalis. The concentration of sucrose in the rooting medium provided by the present invention is 28 - 30 g / L. In the specific embodiments of the present invention, the concentration of sucrose in the rooting medium can be 28, 29 or 30 g / L. The concentration of agar in the rooting medium provided by the present invention is 6 - 8 g / L. In the specific embodiments of the present invention, the concentration of agar in the rooting medium can be 6, 7 or 8 g / L. In the present invention, the pH value of the rooting medium can be 5.7 - 5.9, or it can also be 5.80.

[0040] The induction medium, proliferation medium and rooting medium provided by the present invention all contain sucrose, and sucrose can provide the carbon source required for plant growth, and promote the generation and proliferation of buds, the growth and rooting of buds. The tissue culture rapid propagation medium provided by the present invention has good application effects, and can improve the lateral bud induction rate and the proliferation coefficient of lateral buds of Parrotia subaequalis with buds stem segments. The present invention has no special limitation on the sources of each component in the induction medium, proliferation medium and rooting medium, and conventional commercially available products can be used.

[0041] The present invention provides a tissue culture rapid propagation method for Parrotia subaequalis, using the tissue culture rapid propagation medium described in the above technical solution, and comprising the following steps:

[0042] Inoculate the stem segments with latent buds of Parrotia subaequalis into the induction medium for induction culture to obtain stem segments with lateral buds;

[0043] Transfer the stem segments with lateral buds to the proliferation medium for proliferation culture to obtain cluster buds;

[0044] Cut the cluster buds into single buds and transfer them to the rooting medium for rooting culture to obtain rooted seedlings.

[0045] In the present invention, the bud-bearing stem segments of Parrotia subaequalis are inoculated into an induction medium for induction culture to obtain stem segments with lateral buds. As an alternative embodiment, the method for preparing the bud-bearing stem segments of Parrotia subaequalis in the present invention includes: cutting the leaves off the branches of Parrotia subaequalis and then cutting them into bud-bearing stem segments; the length of the stem segments is 1.5 - 2.0 cm. The branches of Parrotia subaequalis in the present invention are current-year branches, and the bud-bearing stem segments in the present invention include at least one latent bud; before inoculation, the present invention also includes first cleaning the bud-bearing stem segments and then disinfecting them; the present invention has no special limitation on the cleaning method, and conventional methods can be used. For the disinfection in the present invention, first soak in 75% ethanol by volume for 30 - 40 s, which can also be 30 s; then rinse with sterile water 3 - 4 times, and then soak in a sodium hypochlorite solution with an available chlorine content of 0.26% - 0.52% for 5 - 10 min, which can also be soaked for 6 - 7 min, and rinse with sterile water 5 - 6 times. The disinfection method of the present invention is simple, improving the survival rate during the induction culture of Parrotia subaequalis stem segments, reducing the contamination rate and browning rate during the induction culture, making the lateral buds of the bud-bearing stem segments grow well, and the newly grown leaves being emerald green.

[0046] As an alternative embodiment, after disinfecting the bud-bearing stem segments in the present invention, it further includes sucking dry the moisture with sterile filter paper and then inoculating the bud-bearing stem segments of Parrotia subaequalis into an induction medium for induction culture to obtain stem segments with lateral buds. The present invention has no special limitation on the inoculation method, and conventional methods can be used. There are latent buds in the stem segments of the present invention. As the induction culture progresses, the latent buds germinate to obtain lateral buds, and stem segments with lateral buds are obtained.

[0047] As an alternative embodiment, the temperature for the induction culture is 23 - 27 °C, which can also be 25 °C; the time for the induction culture is 28 - 32 d, which can also be 29 - 31 d, preferably 30 d. As an alternative embodiment, the induction culture is first dark-cultured and then light-cultured. The time for the induction culture in the present invention is calculated as the sum of the time for light culture and the time for dark culture. The time for light culture is 26 - 28 d, which can also be 27 d; the time for dark culture is 2 - 4 d, which can also be 3 d. As an alternative embodiment, the light time for light culture is 12 - 16 h / d, which can also be 14 - 16 h / d; the dark time during light culture can be 8 - 12 h / d, which can also be 8 - 10 h / d; the intensity of light culture can be 2200 - 2500 lx, which can also be 2300 - 2400 lx. The time for the induction culture in the present invention is calculated from the time when the stem segments are inoculated on the induction medium until the latent buds germinate to grow lateral buds, the lateral buds elongate and grow into new leaves. Dark culture helps to promote the generation of lateral buds in bud-bearing stem segments and reduce the degree of browning.

[0048] As an alternative embodiment, after obtaining the stem segments with lateral buds, the present invention transfers the stem segments with lateral buds to a proliferation medium for proliferative culture of the lateral buds to obtain cluster buds.

[0049] As an alternative embodiment, before transferring the stem segments with lateral buds of the present invention to a proliferation medium for proliferative culture, it further includes: performing short-term subculture on the stem segments with lateral buds. The time for the short-term subculture of the present invention is 6 - 8 days, preferably 7 days. The medium used for the short-term subculture of the present invention includes a subculture medium. The subculture medium of the present invention is a hormone-free WPM medium, and the composition of the subculture medium is: using WPM as the basic medium, and further adding 28 - 30 g / L of sucrose and 6 - 8 g / L of agar. In the present invention, the pH value of the subculture medium can be 5.7 - 5.9, or can also be 5.8. No hormones are added to the medium used for the short-term subculture of the present invention. The temperature for the subculture of the present invention is 25 ± 2 °C. The role of the subculture is to promote the proliferation of lateral buds on the stem segments.

[0050] As an alternative embodiment, the stem segments with lateral buds obtained by the subculture of the present invention are transferred to a proliferation medium for proliferative culture. The temperature for the proliferative culture of the present invention is 25 ± 2 °C, or can also be 25 °C; the time for the proliferative culture is 28 - 35 days, or can also be 29 - 32 days, and more preferably can be 30 days. The proliferative culture is a light culture, the light time for the light culture is 12 - 16 h / d, or can also be 14 - 16 h / d; the dark time for the light culture is 8 - 12 h / d, or can also be 8 - 10 h / d; the intensity of the light culture is 2200 - 2500 lx, or can also be 2400 - 2500 lx. In the proliferative culture of the present invention, the lateral buds proliferate to obtain cluster buds, and finally cluster bud seedlings are obtained. The proliferative culture is stopped until the height of the cluster buds reaches 2 - 5 cm.

[0051] After obtaining the cluster buds, the present invention cuts the cluster buds into single buds and transfers them to a rooting medium for rooting culture to obtain rooted seedlings.

[0052] As an alternative embodiment, the present invention cuts the single buds from the cluster buds and inoculates the single buds into a rooting medium for rooting culture. The length of the single bud is 1.5 - 2 cm. As an alternative embodiment, the temperature for the rooting culture in the present invention is 25 ± 2 °C or can also be 25 °C; the time for the rooting culture is 28 - 33 days, can also be 29 - 31 days, and more preferably can be 30 days. The rooting culture is a light culture, the light duration of the light culture is 12 - 16 h / d, can also be 14 - 16 h / d; the dark duration of the light culture can be 8 - 12 h / d, can also be 8 - 10 h / d; the intensity of the light culture can be 2200 - 2500 lx, can also be 2400 - 2500 lx. The rooting culture is stopped until the root length of the rooted seedlings reaches 1 - 3 cm. The rooting culture of the present invention enables the sterile seedlings to grow strong roots.

[0053] As an alternative embodiment, the present invention further includes acclimatizing and transplanting the rooted seedlings to obtain complete regenerated plants. The time for acclimatizing in the present invention can be 7 - 14 days. The substrate used for transplantation includes perlite and peat; the volume ratio of perlite to peat is 1:3; carbendazim is added to the substrate; the concentration of carbendazim is 0.1%; the function of carbendazim is to disinfect the substrate and purify the substrate. As an alternative embodiment, the temperature for acclimatization in the present invention is 23 - 27 °C, and the light intensity for acclimatization is 15000 - 20000 lx. The survival rate of transplanting the rooted seedlings in the present invention reaches more than 95%, and the transplanting survival rate is high.

[0054] Currently, there is only one research report on the propagation technology using the stem segments of Parrotia subaequalis as explants (see "Lai Fuxin. Preliminary Study on the Population Restoration of Endangered Plant Parrotia subaequalis [D]. Anqing Normal University, 2022."). Its contamination rate is 25.00%, the survival rate is 46.47%, and the browning rate is 100%; however, the induction rate of the stem segments is only 21.6%, which is only 1 / 4 times the induction rate of the present invention. Through the culture techniques and screening of the medium formulations in four stages, namely explant disinfection, induction of lateral bud germination, lateral bud proliferation, and rooting culture, the present invention has established a set of regeneration technology systems for the stem segments of Parrotia subaequalis. The contamination rate of the induced stem segments in the technical solution of the present invention is as low as 7% - 10%, the survival rate of the stem segments is as high as 78% - 83%, and the browning rate of the stem segments is as low as only 7% - 16%; the lateral bud induction rate (i.e., the germination rate) of the stem segments is as high as 91%, the lateral bud proliferation coefficient is as high as 4.17, the rooting rate reaches 60%, and the transplanting survival rate is more than 95%.

[0055] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0056] The seedlings of Parrotia subaequalis used in the following examples are from the same batch.

[0057] 1. All the prepared culture media in the experiment were autoclaved at 121 °C for 15 min, cooled and dispensed for later use.

[0058] 2. The index calculation methods are as follows:

[0059] Contamination rate = (number of contaminated explants / total number of inoculated explants) × 100%;

[0060] Browning rate = (number of browned explants / total number of inoculated explants) × 100%;

[0061] Survival rate = (number of survived explants / total number of inoculated explants) × 100%;

[0062] Germination rate = [number of germinated stem segments / (total number of inoculated stem segments - number of contaminated ones)] × 100%;

[0063] Bud multiplication coefficient = (number of buds after growth - number of buds at the time of inoculation) / number of buds at the time of inoculation;

[0064] Average bud length = total length of new buds / number of new buds;

[0065] Average germination number = total germination number of each treatment / number of buds at the time of inoculation;

[0066] Rooting rate (%) = (number of rooted plants / total number of inoculated plants) × 100%.

[0067] 3. Excel and SPSS statistical analysis software were used for data statistics and analysis, and Duncan's method was used for significance test of differences.

[0068] Example 1 Selection and disinfection method of Parrotia subaequalis explants

[0069] 1. Selection of explants

[0070] The seedling raising cycle is shorter than that of the technical solutions of Hu Guowei et al. which use seeds as explants and propagate seedlings through lateral bud multiplication, greatly saving the seedling raising cost. The comparison results between the present invention and the prior art are shown in Table 1.

[0071] The seedling raising cycle is shorter and the propagation efficiency is higher than that of the technical solutions such as Hu Guowei et al. which use seeds, etc. as explants and adopt the seedling raising method of lateral bud proliferation. If annual seedlings are used to prepare stem segments, about 70 stem segments can be taken at one time from the annual seedlings of the present invention in spring. The lateral bud proliferation coefficient of each stem segment is 4.17, which means that the young trees generated from one seed after one year can be used to produce an average of 70 explants of stem segments with latent buds. The induction rate of 70 explants is 90%, and the proliferation coefficient of each lateral bud is 4.17. Therefore, an average of 70×90%×4.17 = 262.71 buds can be produced. Calculated according to the rooting rate of 60%, an average of 157.63 rooted seedlings can be obtained, greatly saving the seedling raising cost.

[0072] Table 1 Comparison of different seedling raising methods for Parrotia subaequalis

[0073]

[0074] Note: 14d, 28d and 21d are the germination time, proliferation time and rooting time respectively during the culture process. The reason why they are different from the time in the examples is that in the examples, for the convenience of investigation, the longest time for sufficient growth is set; during the actual growth process, the time in Table 1 has been stably germinated, proliferated or rooted.

[0075] 2. Disinfection method

[0076] The material is the seedlings of Parrotia subaequalis from Longwang Mountain in Anji, and the age of the seedlings is 1 - 3 years. Select the seedlings without diseases and pests and with strong growth, first culture them in the greenhouse for 1 - 3 months, and then select the stem segments with buds of the current year's young and tender branches as explants.

[0077] The specific method for preparing explants is as follows: Take the current year's branches of Parrotia subaequalis young trees, cut off the leaves, cut the stem segments into lengths of 1.5 - 2.0 cm, rinse with running water for 2 h, and then transfer them to the ultra-clean workbench for standby.

[0078] Using the stem segments with latent buds of Parrotia subaequalis as explants, the specific disinfection of explants is as follows: First, treat the stem segments with buds with 75% ethanol by volume concentration for 30 s, and rinse with sterile water for 3 - 4 times; then disinfect with 0.52% NaClO (sodium hypochlorite) solution with available chlorine for 5 min (A1), 10 min (A2), 15 min (A3), 20 min (A4), 25 min (A5) respectively. After the disinfection is completed, rinse with sterile water for 5 - 6 times, and dry the water with sterile filter paper.

[0079] Inoculate the disinfected stem segments with buds into the culture medium. The composition of the culture medium is: Based on WPM as the basic medium, 30 g / L sucrose and 8 g / L agar are also added, and the pH is 5.7.

[0080] Twenty stem segments were processed for each treatment, with 3 replicates. After 20 days, the contamination rate was counted. After 30 days, the browning rate and the survival rate of axenic seedlings were counted to determine the optimal disinfection time. The results are shown in Table 2.

[0081] It can be seen that when the explants were placed in the NaClO solution with an available chlorine of 0.52%, as the soaking time prolonged, the contamination rate of the explants cultured for 20 days first increased and then decreased, and the contamination rate was the lowest at 5% when treated for 25 minutes. However, the browning rate gradually increased with the prolongation of the soaking time, and the browning rate was the lowest at 7% when treated for 5 minutes. After about 30 days of growth and development, it was found that the survival rate of the treatment for 5 minutes was the highest, reaching 83%, the browning rate was the lowest, only 7%, and the lateral buds grew well with the new leaves being emerald green.

[0082] Table 2 Effects of different NaClO treatment times on the disinfection effect of stem segment explants of Parrotia subaequalis

[0083]

[0084] Note: The contamination rate, survival rate, and browning rate in the table all represent the mean ± standard deviation; the Duncan multiple comparison method was used; different letters in the same column represent significant differences ( P <0.05), the same below.

[0085] Establishing an axenic system is one of the most basic and crucial steps in tissue culture. Therefore, the sterilization effect of explants is directly related to the success of the experiment. This invention studied the effects of different NaClO treatment times on the surface sterilization effect of Parrotia subaequalis stem segments with buds. It can be seen that the effect of disinfecting with the NaClO solution with an available chlorine of 0.52% for 5 - 10 minutes is good, with a low contamination rate, a high survival rate, and a low browning rate during the growth process of the explants.

[0086] Example 2 Screening the most suitable basic medium for the germination of Parrotia subaequalis stem segments

[0087] After disinfecting the Parrotia subaequalis stem segments with buds, the specific explant disinfection was as follows: First, treat the stem segments with buds with 75% ethanol by volume for 30 seconds and rinse with sterile water 3 - 4 times; then disinfect with the NaClO solution with an available chlorine of 0.52% for 5 minutes. After disinfection, rinse with sterile water 5 - 6 times, blot dry with sterile filter paper, and then inoculate on the lateral bud induction media 1 - 3. The pH values of the lateral bud induction media 1 - 3 were 5.7 respectively.

[0088] The composition of lateral bud induction medium 1 was based on MS as the basic medium, and also added 30 g / L sucrose and 8 g / L agar.

[0089] The composition of lateral bud induction medium 2 was based on 1 / 2MS as the basic medium, and also added 30 g / L sucrose and 8 g / L agar.

[0090] The composition of the axillary bud induction medium 3 is based on WPM as the basic medium, and additionally contains 30 g / L sucrose and 8 g / L agar.

[0091] After inoculation, each medium was first subjected to dark culture for 3 days throughout the day, and then light culture for 27 days. The dark culture was carried out in the dark cabinet of the sterile room, and the temperature of the sterile culture room was 23 - 25°C; the light culture was carried out in the sterile culture room, with a photoperiod of 16 h / d for light culture and 8 h / d for dark culture; the light intensity was 2400 lx, and the temperature for light culture was 25 ± 2°C. Twenty stem segments were inoculated for each treatment, and one stem segment was inoculated in each petri dish. Three replicate experiments were set up. The total culture time was 30 days. After 30 days, the axillary bud germination rate and growth status were statistically analyzed. The latent buds in the stem segments germinated and elongated into axillary buds, obtaining stem segments with axillary buds. The results are shown in Table 3. Under the condition of using WPM as the basic medium, the germination rate was relatively high at 72%, and the growth state was good (see Figure 1 ), which was the appropriate basic medium for the germination of Parrotia stem segments.

[0092] Table 3 Effects of different basic media on the axillary bud germination of Parrotia

[0093]

[0094] Note: Different letters in the same column represent significant differences ( P <0.05), the same below.

[0095] Example 3

[0096] After disinfecting the Parrotia stem segments with buds, they were respectively inoculated into the following media 1 - 15 and CK for induction culture. The disinfection method was the same as in Example 2. The pH values of media 1 - 15 and CK were 5.7. The induction culture was first subjected to dark culture for 3 days, and then light culture for 27 days. The dark culture was carried out in the dark cabinet of the sterile room, and the temperature of the sterile culture room was 25 ± 2°C; the light culture was carried out in the sterile culture room, with a photoperiod of 16 h / d for light culture and 8 h / d for dark culture, and the light intensity was 2400 lx.

[0097] F1: The composition of medium 1 is: based on WPM as the basic medium, and additionally contains 0.5 mg / L KT, 30 g / L sucrose and 8 g / L agar;

[0098] F2: The composition of medium 2 is: based on WPM as the basic medium, and additionally contains 1.0 mg / L KT, 30 g / L sucrose and 8 g / L agar;

[0099] F3: The composition of medium 3 is: based on WPM as the basic medium, and additionally contains 1.5 mg / L KT, 30 g / L sucrose and 8 g / L agar;

[0100] F4: The composition of Medium 4 is: based on WPM as the basic medium, and also added with 2.0 mg / L KT, 30 g / L sucrose and 8 g / L agar;

[0101] F5: The composition of Medium 5 is: based on WPM as the basic medium, and also added with 4.0 mg / L KT, 30 g / L sucrose and 8 g / L agar;

[0102] CK: The composition of the medium is: based on WPM as the basic medium, and also added with 30 g / L sucrose and 8 g / L agar;

[0103] D1: The composition of Medium 6 is: based on WPM as the basic medium, and also added with 0.5 mg / L 6-BA, 30 g / L sucrose and 8 g / L agar;

[0104] D2: The composition of Medium 7 is: based on WPM as the basic medium, and also added with 1.0 mg / L 6-BA, 30 g / L sucrose and 8 g / L agar;

[0105] D3: The composition of Medium 8 is: based on WPM as the basic medium, and also added with 1.5 mg / L 6-BA, 30 g / L sucrose and 8 g / L agar;

[0106] D4: The composition of Medium 9 is: based on WPM as the basic medium, and also added with 2.0 mg / L 6-BA, 30 g / L sucrose and 8 g / L agar;

[0107] D5: The composition of Medium 10 is: based on WPM as the basic medium, and also added with 4.0 mg / L 6-BA, 30 g / L sucrose and 8 g / L agar;

[0108] E1: The composition of Medium 11 is: based on WPM as the basic medium, and also added with 0.001 mg / L TDZ, 30 g / L sucrose and 8 g / L agar;

[0109] E2: The composition of Medium 12 is: based on WPM as the basic medium, and also added with 0.01 mg / L TDZ, 30 g / L sucrose and 8 g / L agar;

[0110] E3: The composition of Medium 13 is: based on WPM as the basic medium, and also added with 0.1 mg / L TDZ, 30 g / L sucrose and 8 g / L agar;

[0111] E4: The composition of Medium 14 is: based on WPM as the basic medium, and also added with 1.0 mg / L TDZ, 30 g / L sucrose and 8 g / L agar;

[0112] E5: The composition of medium 15 is as follows: based on WPM as the basic medium, 2.0 mg / L TDZ, 30 g / L sucrose and 8 g / L agar are also added;

[0113] One shoot-bearing stem segment was inoculated in each petri dish, and a total of 20 shoot-bearing stem segments were inoculated in each parallel experiment. Three parallel experiments were set up. After 30 days of culture, the germination rate, average number of germinations and growth status were counted.

[0114] The results of the effect of KT on the induction of lateral buds of Parrotia subaequalis are shown in Table 4 and Figure 2 , it can be seen that KT has no significant effect on the induction of cluster buds from lateral buds of Parrotia subaequalis. The germination rate first increases and then decreases with the increase of concentration. The highest germination rate is induced at 1.0 mg / L, reaching 91%. Compared with WPM without any hormone addition, the germination rate is increased by 19%, but the average number of germinations is stable at 1 (Table 4). According to observations, the lateral buds induced by KT have larger leaves and darker colors (see Figure 2 ).

[0115] Table 4 Effects of different concentrations of KT on the germination of lateral buds of excised stem segments

[0116]

[0117] The results of the effect of different concentrations of 6-BA (6-benzylaminopurine, N-(Phenylmethyl)-9H-purin-6-amine) on the induction of lateral buds of excised stem segments are shown in Table 5 and Figure 3 , it can be seen that 6-BA plays a certain role in the induction of lateral buds of Parrotia subaequalis. The germination rate first increases and then decreases with the increase of concentration. The highest germination rate is reached at 1.5 mg / L of 6-BA, reaching 86%. With the increase of concentration, the change range of the average number of germinations is not large, and the highest value is 1.17 at 1.0 mg / L (Table 5). According to observations, the cluster buds induced by 6-BA are greener in color (see Figure 3 ).

[0118] Table 5 Effects of different concentrations of 6-BA on the growth of lateral buds induced from excised stem segments

[0119]

[0120] The results of the effect of different concentrations of TDZ (Tiabendazole) on the induction of lateral buds of excised stem segments are shown in Table 6 and Figure 4, TDZ had a significant effect on the induction of lateral buds in Parrotia subaequalis, and the number of clustered buds generated from lateral buds was relatively large and the color was darker. As the concentration of TDZ increased, the germination rate of lateral buds increased and reached the highest at 1.0 mg / L and 2.0 mg / L, reaching 87%. However, the average number of germinated buds showed a trend of increasing first and then decreasing with the increase of concentration, and reached the highest at 1.0 mg / L, reaching 1.72 (Table 6). It can be seen that compared with the treatments of 6-BA and KT, TDZ was prone to deformity and vitrification phenomena (see Figure 4 ).

[0121] Table 6 Effects of TDZ at different concentrations on the growth of induced lateral buds of in vitro stem segments

[0122]

[0123] Example 4

[0124] 1. Effects of combinations of 6-BA and TDZ at different concentrations on the proliferation of lateral buds in Parrotia subaequalis

[0125] After single-factor experiments, it was found that 6-BA and TDZ played a major role in the germination of lateral buds in Parrotia subaequalis. To explore the effects of different ratios of the two on the proliferation of lateral buds in Parrotia subaequalis, the following experiments were carried out. The sterile stem segments with 1 lateral bud growing in the F2 medium were inoculated into the proliferation media J1 - J9 respectively to induce the proliferation culture of lateral buds. The pH values of the proliferation media J1 - J9 were 5.7. The specific compositions of the proliferation media J1 - J9 were as follows:

[0126] J1: Proliferation medium 1: Using WPM as the basic medium, and also adding 1.0 mg / L 6-BA, 0.01 mg / L TDZ, 30 g / L sucrose and 8 g / L agar;

[0127] J2: Proliferation medium 2: Using WPM as the basic medium, and also adding 1.0 mg / L 6-BA, 0.1 mg / L TDZ, 30 g / L sucrose and 8 g / L agar;

[0128] J3: Proliferation medium 3: Using WPM as the basic medium, and also adding 1.0 mg / L 6-BA, 1.0 mg / L TDZ, 30 g / L sucrose and 8 g / L agar;

[0129] J4: Proliferation medium 4: Using WPM as the basic medium, and also adding 1.5 mg / L 6-BA, 0.01 mg / L TDZ, 30 g / L sucrose and 8 g / L agar;

[0130] J5: Proliferation medium 5: Using WPM as the basic medium, and also adding 1.5 mg / L 6-BA, 0.1 mg / L TDZ, 30 g / L sucrose and 8 g / L agar;

[0131] J6: Proliferation Medium 6: Based on WPM as the basic medium, supplemented with 1.5 mg / L 6-BA, 1.0 mg / L TDZ, 30 g / L sucrose and 8 g / L agar;

[0132] J7: Proliferation Medium 7: Based on WPM as the basic medium, supplemented with 2.0 mg / L 6-BA, 0.01 mg / L TDZ, 30 g / L sucrose and 8 g / L agar;

[0133] J8: Proliferation Medium 8: Based on WPM as the basic medium, supplemented with 2.0 mg / L 6-BA, 0.1 mg / L TDZ, 30 g / L sucrose and 8 g / L agar;

[0134] J9: Proliferation Medium 9: Based on WPM as the basic medium, supplemented with 2.0 mg / L 6-BA, 1.0 mg / L TDZ, 30 g / L sucrose and 8 g / L agar;

[0135] Proliferation culture is carried out in a sterile culture room at a temperature of 25 ± 2°C; light culture is carried out in a sterile culture room with a photoperiod of 16 h / d light culture and 8 h / d dark culture, and the light intensity is 2400 lx.

[0136] For each medium treatment, 20 bottles are inoculated, with 1 explant in each bottle; 3 replicate experiments are set, and a total of 60 bottles are inoculated for each medium treatment. After 30 d of culture in each medium, the proliferation coefficient, new shoot length and seedling growth are counted. The results are shown in Table 7 and Figure 5 , after 30 d of culture, with the increase of 6-BA concentration, the average number of bud lengths gradually decreases, and the bud proliferation coefficient first increases and then decreases; with the increase of TDZ concentration, the bud proliferation coefficient gradually increases, but the vitrification degree is serious. When growing in the medium supplemented with 1.0 mg / L 6-BA + 1.0 mg / L TDZ, the highest average bud length is 1.82. When growing in the medium supplemented with 1.5 mg / L 6-BA + 0.1 mg / L TDZ, the highest bud proliferation coefficient is 1.58 (Table 7, Figure 5 ). When 6-BA in the proliferation medium is 1.5 mg / L, the bud length of the lateral buds elongates quickly and more cluster buds are produced.

[0137] Table 7 Effects of Different Concentrations of 6-BA and TDZ on the Proliferation of Lateral Buds of Parrotia subaequalis

[0138]

[0139] 2. Effects of Different Concentrations of TDZ and 1.5 mg / L 6-BA on the Proliferation of Lateral Buds of Parrotia subaequalis

[0140] The aseptic stem segments with one lateral bud growing in F2 medium were subcultured in the subculture proliferation medium for 7 days and then transferred to new media S1 - S5 for proliferation culture. The subculture medium was hormone - free WPM medium, and the composition of the subculture medium was: using WPM as the basic medium, supplemented with 30 g / L sucrose and 8 g / L agar, and the pH value was 5.7.

[0141] Media S1 - S5 were used to explore the effect of TDZ on the proliferation of lateral buds of Parrotia subaequalis. The compositions of media S1 - S5 are as follows.

[0142] S1: Proliferation medium 1: Using WPM as the basic medium, supplemented with 1.5 mg / L 6 - BA, 0.003 mg / L TDZ, 30 g / L sucrose and 8 g / L agar;

[0143] S2: Proliferation medium 2: Using WPM as the basic medium, supplemented with 1.5 mg / L 6 - BA, 0.004 mg / L TDZ, 30 g / L sucrose and 8 g / L agar;

[0144] S3: Proliferation medium 3: Using WPM as the basic medium, supplemented with 1.5 mg / L 6 - BA, 0.005 mg / L TDZ, 30 g / L sucrose and 8 g / L agar;

[0145] S4: Proliferation medium 4: Using WPM as the basic medium, supplemented with 1.5 mg / L 6 - BA, 0.006 mg / L TDZ, 30 g / L sucrose and 8 g / L agar;

[0146] S5: Proliferation medium 5: Using WPM as the basic medium, supplemented with 1.5 mg / L 6 - BA, 0.007 mg / L TDZ, 30 g / L sucrose and 8 g / L agar;

[0147] The pH of each medium was 5.7. For each medium treatment, 20 bottles were inoculated, with 1 explant in each bottle, and 3 parallel experiments were set up. A total of 60 bottles were inoculated for each medium treatment. After 30 days of culture, the proliferation multiple, average bud length and lateral bud growth status were counted. The results are shown in Table 8 and Figure 6 . The proliferation culture was carried out in a sterile culture room at a temperature of 25 ± 2 °C; the light culture was carried out in a sterile culture room with a photoperiod of 16 h / d light culture and 8 h / d dark culture, and the light intensity was 2400 lx.

[0148] Table 8 Effect of different concentrations of TDZ on the proliferation of lateral buds of Parrotia subaequalis

[0149]

[0150] Example 5 Effect of different concentrations of IBA and NAA on the rooting of Parrotia subaequalis

[0151] Inoculation: Select single buds with a length of 1.5 - 2 cm from the clustered buds grown in S1 of Example 4 and cut them, then inoculate them into the rooting medium.

[0152] The composition of the rooting medium is as follows: Using 1 / 2MS as the basic medium, different concentrations of IBA (0, 0.2, 0.4 mg / L) and NAA (0, 0.1, 0.2 mg / L) are added. The specific compositions of the rooting media L0 - L8 are as follows:

[0153] L0: The rooting medium uses 1 / 2MS as the basic medium and also adds 30 g / L sucrose and 8 g / L agar;

[0154] L1: The rooting medium uses 1 / 2MS as the basic medium and also adds 0.2 mg / L IBA, 30 g / L sucrose and 8 g / L agar;

[0155] L2: The rooting medium uses 1 / 2MS as the basic medium and also adds 0.4 mg / L IBA, 30 g / L sucrose and 8 g / L agar;

[0156] L3: The rooting medium uses 1 / 2MS as the basic medium and also adds 0.1 mg / L NAA, 30 g / L sucrose and 8 g / L agar;

[0157] L4: The rooting medium uses 1 / 2MS as the basic medium and also adds 0.2 mg / L NAA, 30 g / L sucrose and 8 g / L agar;

[0158] L5: The rooting medium uses 1 / 2MS as the basic medium and also adds 0.2 mg / L IBA, 0.1 mg / L NAA, 30 g / L sucrose and 8 g / L agar;

[0159] L6: The rooting medium uses 1 / 2MS as the basic medium and also adds 0.2 mg / L IBA, 0.2 mg / L NAA, 30 g / L sucrose and 8 g / L agar;

[0160] L7: The rooting medium uses 1 / 2MS as the basic medium and also adds 0.4 mg / L IBA, 0.1 mg / L NAA, 30 g / L sucrose and 8 g / L agar;

[0161] L8: The rooting medium uses 1 / 2MS as the basic medium and also adds 0.4 mg / L IBA, 0.2 mg / L NAA, 30 g / L sucrose and 8 g / L agar;

[0162] The pH of each culture medium was 5.7. For each treatment, 20 bottles were inoculated, with 1 explant in each bottle, and the experiment was repeated three times. After 30 days, the rooting situation was counted. The results are shown in Table 9. The rooting culture was carried out in a sterile culture room at a temperature of 23 - 27°C; the light culture was carried out in a sterile culture room with a photoperiod of 16 h / d light culture and 8 h / d dark culture, and the light intensity was 2400 lx.

[0163] It can be seen that after 30 days of culture, with the increase of IBA concentration, the rooting rate gradually decreased. In the rooting medium supplemented with only 0.2 mg / L NAA, the rooting rate was the highest, reaching 60%, and green callus grew at the base, the adventitious roots were white, and the root length (see Table 9, Figure 7 ).

[0164] Table 9 Effects of Different Concentrations of IBA and NAA on the Rooting of Parrotia subaequalis

[0165]

[0166] Example 6 Transplanting

[0167] Select test-tube seedlings with good rooting. When the height of the test-tube seedlings is 2 - 4 cm or more and the root length is 1 - 2 cm, they are first placed in an acclimation room for 7 - 14 days of acclimation. The temperature in the acclimation room is 23 - 27°C, and the light intensity is 15000 - 20000 lx. After new leaves or new roots grow during the acclimation process of the test-tube seedlings, they can be transplanted into a nutrient bowl with a diameter of 6 - 10 cm. The transplanting substrate is perlite and peat soil, and the volume ratio of perlite to peat soil is 1:3. Carbendazim solution is added to the substrate, and the mass concentration of the added carbendazim solution is 0.1%. Stir evenly, and after 30 days of culture, the transplanting survival rate is counted. The transplanting survival rate reaches more than 95%.

[0168] In summary, as a rare and endangered plant, for Parrotia subaequalis, the present invention uses stem segments of 1.5 - 2 cm collected from the tender branches of 1 - 3-year-old seedlings as explants. Through the culture techniques and optimization of the medium formula in four stages: disinfection of explants, induction and germination of lateral buds, proliferation of lateral buds, rooting culture, and acclimatization and transplantation, a tissue culture and propagation technology system for Parrotia subaequalis is established, providing technical support for the protection and propagation of endangered plants. The present invention uses stem segments with latent buds as explants. After disinfecting with a single, harmless and non-toxic disinfectant NaClO for 5 minutes, a purification rate of over 90% (the purification rate is 100% minus the contamination rate), a relatively high lateral bud germination rate (91%), and a relatively low browning rate (7%) can be achieved. The disinfection process of the explants is simple and ensures the good growth of the explants, initiating the aseptic culture of Parrotia subaequalis. The present invention also screened the medium formula for the germination of lateral buds on stem segments, with a germination rate as high as 91% (F2 medium). The present invention screened the medium formula for the proliferation of lateral buds, with a proliferation coefficient of 4.17, which can greatly improve the propagation efficiency. The rooting medium obtained in the present invention has a high rooting rate. After acclimatizing the seedlings in an acclimatization room at a temperature of 23 - 27°C and a light intensity of 15000 - 20000 lx for 7 - 14 days and transplanting them into the substrate, the substrate is mixed with carbendazim, and the volume ratio of perlite to peat soil in the substrate is 1:3, a tissue culture pot of Parrotia subaequalis is obtained, and the transplantation survival rate of the tissue culture pot of Parrotia subaequalis reaches over 95%. The transplantation survival rate is high, and the tissue culture potted seedlings of Parrotia subaequalis can be used for afforestation.

[0169] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all embodiments. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A tissue culture and rapid propagation method of Hamamelis truncatula, characterized in that: The method adopts tissue culture rapid propagation medium for culturing, wherein the tissue culture rapid propagation medium is composed of an induction medium, a proliferation medium and a rooting medium, and comprises the following steps: Inoculating the stem segments with hidden buds of Hamamelis argyi into an induction culture medium for induction culture to obtain stem segments with lateral buds; the induction culture medium consists of WPM basic culture medium, 1.0-1.5 mg / L KT, 28-30 g / L sucrose and 6-8 g / L agar; the induction culture is first dark culture and then light culture; The stem segment with the lateral buds is transferred to a proliferation medium for proliferation culture to obtain clustered buds; the proliferation medium is composed of WPM basic medium, 1.0-1.5 mg / L 6-BA, 0.003-0.005 mg / L TDZ, 28-30 g / L sucrose and 6-8 g / L agar; The clustered buds are cut into single buds and transferred to a rooting medium for rooting culture to obtain rooted seedlings; the rooting medium consists of 1 / 2MS basic medium, 0.2mg / L NAA, 28-30g / L sucrose and 6-8g / L agar; the proliferation culture and the rooting culture are respectively light culture.

2. The tissue culture rapid propagation method according to claim 1, characterized in that: The stem segments are also disinfected before inoculation, which includes soaking in 75% ethanol by volume for 30-40 seconds, rinsing with sterile water for 3-4 times, and then soaking in a sodium hypochlorite solution with an effective chlorine content of 0.26%-0.52% for 5-10 minutes, and rinsing with sterile water for 5-6 times.

3. The tissue culture rapid propagation method according to claim 2, characterized in that: The temperatures of the induction culture, proliferation culture and rooting culture are 23-27° C. respectively.

4. The tissue culture rapid propagation method according to claim 2, characterized in that: The illumination time in the illumination culture is 12-16 h / d; the illumination intensity is 2200-2500 lx; ​​and the dark time in the illumination culture is 8-12 h / d.

5. The tissue culture rapid propagation method according to claim 2, characterized in that: The induction culture time is 28 to 32 days, the dark culture time in the induction culture is 2 to 4 days, the light culture time is 26 to 28 days; the proliferation culture time is 28 to 35 days; and the rooting culture time is 28 to 33 days.

6. The tissue culture and rapid propagation method according to claim 2, characterized in that: Before the stem segment with lateral buds is subjected to proliferation culture, the method further comprises: subjecting the stem segment with lateral buds to short-term subculture culture.

7. The tissue culture and rapid propagation method according to claim 2, characterized in that: The length of the stem segment with hidden buds is 1.5-2.0 cm.

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