Tissue culture and rapid propagation culture medium and tissue culture and rapid propagation method for culturing Shaniodendron subaequalis

Abstract

The invention belongs to the technical field of plant tissue culture, and particularly relates to a tissue culture and rapid propagation culture medium and a tissue culture and rapid propagation method for culturing Shaniodendron subaequalis. The invention provides a tissue culture and rapid propagation culture medium and a tissue culture and rapid propagation method for culturing Shaniodendron subaequalis. Kinetin KT (Kinetin) in the tissue culture and rapid propagation culture medium provided by the invention can promote the generation of buds and the growth of adventitious buds. 6-BA is 6-benayl aminopurine and is used for promoting the growth of buds; tDZ is thiabendazole, and growth and development of buds are promoted. Under the combined action of KT, 6-BA and TDZ, the bud induction rate and the bud multiplication coefficient of the stem with the bud are improved. The invention further provides a tissue culture and rapid propagation method of the Shaniodendron subaequalis, stem segments subjected to induction culture are low in pollution rate, high in survival rate and low in browning rate; the lateral bud induction rate of the stem section is as high as 91%, and the multiplication coefficient, the rooting rate and the 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] In order to solve the above technical problems, the present invention proposes the following technical solutions: The invention provides a tissue culture rapid propagation medium for culturing Hamamelis argyi, wherein the tissue culture rapid propagation medium comprises an induction medium, a proliferation medium and a rooting medium; The induction medium 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; The proliferation medium uses WPM as a basic medium and also 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; The rooting medium uses 1 / 2MS as a basic medium and also includes 28-30 g / L sucrose and 6-8 g / L agar.

[0006] Preferably, the rooting medium further comprises 0.11-0.4 mg / L NAA.

[0007] The present invention provides a method for tissue culture and rapid propagation of Hamamelis salsa, which adopts the tissue culture and rapid propagation medium described in the above technical solution and comprises the following steps: Inoculating the stem segments with hidden buds of Hamamelis argyi into an induction medium for induction culture to obtain stem segments with lateral buds; Transferring the stem segment with lateral buds to a proliferation culture medium for proliferation culture to obtain clustered buds; The clustered buds are cut into single buds and transferred to a rooting medium for rooting culture to obtain rooted seedlings.

[0008] Preferably, the stem segments are disinfected before inoculation, and the disinfection includes soaking in 75% ethanol by volume for 30 to 40 seconds, rinsing with sterile water for 3 to 4 times, and then soaking in a sodium hypochlorite solution with an effective chlorine content of 0.26% to 0.52% for 5 to 10 minutes, and rinsing with sterile water for 5 to 6 times.

[0009] Preferably, the temperatures of the induction culture, proliferation culture and rooting culture are 23-27° C., respectively.

[0010] Preferably, the induction culture is first dark culture and then light culture; the proliferation culture and rooting culture are light cultures respectively.

[0011] Preferably, 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.

[0012] Preferably, the induction culture time is 28 to 32 days, the dark culture time in the induction culture is 2 to 4 days, and the light culture time is 26 to 28 days.

[0013] Preferably, the proliferation culture time is 28 to 35 days; the rooting culture time is 28 to 33 days.

[0014] Preferably, 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.

[0015] Preferably, the length of the stem segment with hidden buds is 1.5~2.0 cm.

[0016] Beneficial effects of the present invention: The present invention provides a tissue culture rapid propagation medium for culturing Hamamelis argyi. In the tissue culture rapid propagation medium, KT is Kinetin, i.e., kinetin, and 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 thiabendazole, which promotes the growth and development of buds. Under the joint action of KT, 6-BA and TDZ, the bud induction rate of stem segments with buds and the bud proliferation coefficient are improved.

[0017] The invention also provides a tissue culture rapid propagation method of Hamamelis argentea, wherein tissue culture is carried out using stem segments with hidden buds as explants. The invention establishes a complete in vitro regeneration and breeding technology system of Hamamelis argentea, a rare and endangered plant, for the first time, that is, using stem segments with hidden buds on new branches of seedlings sown in the wild for 1 to 3 years as explants. The invention establishes a set of Hamamelis argentea stem segment regeneration technology system through culture technology of four stages including lateral bud germination induction, lateral bud proliferation and rooting culture and screening of culture medium formulas. The induced cultured stem segments have a low contamination rate, a stem segment survival rate as high as 78% to 83%, and a stem segment browning rate as low as 7% to 16%. The lateral bud induction rate of the stem segments, i.e., the germination rate, reaches a maximum of 91%, the lateral bud proliferation coefficient is as high as 4.17, and the rooting rate reaches 60%. The test tube seedlings with good rooting are transplanted, and the transplant survival rate reaches more than 95%. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a graph showing the effects of different basic culture media on the germination of lateral buds of Hamamelis argyi, where A represents MS, B represents 1 / 2MS, and C represents WPM; Figure 2 The figure shows the effect of different concentrations of KT on the induction of lateral buds 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; Figure 3This is the effect of different concentrations of 6-BA on the induction of lateral buds in vitro stem segments, D1~D5 are 0.5 mg / L, 1.0 mg / L, 1.5 mg / L, 2.0 mg / L, 4.0 mg / L respectively; Figure 4 The effect of different concentrations of TDZ on the induction of lateral buds in vitro stem segments, E1~E5 are 0.001mg / L, 0.01mg / L, 0.1mg / L, 1.0mg / L, 2.0mg / L Figure 5 This is a graph showing the effects of different concentrations of 6-BA and TDZ on the proliferation of lateral buds of Hamamelis argyi; Figure 6 This is the effect of different concentrations of TDZ on the proliferation of lateral buds of Hamamelis argyi; Figure 7 This is a graph showing the effects of different concentrations of IBA and NAA on the rooting of Hamamelis sterile seedlings; Figures 1 to 7 The scale of the bars is 1 cm. DETAILED DESCRIPTION

[0019] The invention provides a tissue culture rapid propagation medium for culturing Hamamelis argyi. The tissue culture rapid propagation medium comprises an induction medium, a proliferation medium and a rooting medium.

[0020] The induction culture medium of the present invention uses WPM as a basic culture medium and further comprises 1.0-1.5 mg / L KT, 28-30 g / L sucrose and 6-8 g / L agar.

[0021] As an optional embodiment, the concentration of KT in the induction medium provided by the present invention is 1.0~1.5mg / L; in a specific embodiment of the present invention, the concentration of KT in the induction medium may be 1.0, 1.3 or 1.5mg / L. KT helps to induce explants to form buds and improve the induction rate of buds. The concentration of sucrose in the induction medium provided by the present invention is 28~30g / L. In a specific embodiment of the present invention, the concentration of sucrose in the induction medium may be 28, 29 or 30g / L. The concentration of agar in the induction medium provided by the present invention is 6~8g / L. In a specific embodiment of the present invention, the concentration of agar in the induction medium may be 6, 7 or 8g / L. In the present invention, the pH value of the induction medium may be 5.7~5.9, or 5.8.

[0022] The proliferation culture medium of the present invention uses WPM as a basic culture medium and further comprises 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.

[0023] As an optional embodiment, the concentration of 6-BA in the proliferation medium provided by the present invention is 1.0~1.5mg / L, or 1.2~1.4mg / L; in a specific embodiment of the present invention, the concentration of 6-BA in the proliferation medium is 1.0, 1.2, 1.4 or 1.5mg / L. The concentration of TDZ in the proliferation medium provided by the present invention is 0.003~0.005mg / L; in a specific embodiment of the present invention, the concentration of TDZ in the proliferation medium is 0.003, 0.004 or 0.005mg / L. 6-BA and TDZ jointly promote the proliferation of buds at an appropriate concentration ratio, and the lateral buds grow well. The concentration of sucrose in the proliferation medium provided by the present invention is 28~30g / L; in a specific embodiment of the present invention, the concentration of sucrose in the proliferation medium is 28, 29 or 30g / L. The concentration of agar in the proliferation medium provided by the present invention is 6~8g / L. In a specific embodiment 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 5.8.

[0024] The rooting medium of the present invention is 1 / 2MS as a basic medium, and also includes 28-30g / L sucrose and 6-8g / L agar; as another optional embodiment, the rooting medium of the present invention is 1 / 2MS as a basic medium, and also includes 0.11-0.4mg / L NAA, 28-30g / L sucrose and 6-8g / L agar. NAA can promote plant growth and promote root formation.

[0025] As an optional embodiment, the concentration of NAA in the rooting medium provided by the present invention is 0.11~0.4mg / L, and can also be 0.11~0.2mg / L. NAA can promote the rooting of the adventitious buds of Hamamelis salsa. The concentration of sucrose in the rooting medium provided by the present invention is 28~30g / L. In a specific embodiment of the present invention, the concentration of sucrose in the rooting medium can be 28, 29 or 30g / L. The concentration of agar in the rooting medium provided by the present invention is 6~8g / L. In a specific embodiment of the present invention, the concentration of agar in the rooting medium can be 6, 7 or 8g / L. In the present invention, the pH value of the rooting medium can be 5.7~5.9, or it can be 5.80.

[0026] The induction medium, proliferation medium and rooting medium provided by the present invention all contain sucrose, which can provide a carbon source required for plant growth, promote the generation and proliferation of buds, and promote the growth and rooting of buds. The tissue culture rapid propagation medium provided by the present invention has good application effect, and can improve the lateral bud induction rate and the proliferation coefficient of the lateral buds of the bud-bearing stem segment of Hamamelis argyi. The present invention does not specifically limit the sources of the components in the induction medium, proliferation medium and rooting medium, and conventional commercially available products can be used.

[0027] The present invention provides a method for tissue culture and rapid propagation of Hamamelis salsa, which adopts the tissue culture and rapid propagation medium described in the above technical solution and comprises the following steps: Inoculating the stem segments with hidden buds of Hamamelis argyi into an induction medium for induction culture to obtain stem segments with lateral buds; Transferring the stem segment with lateral buds to a proliferation culture medium for proliferation culture to obtain clustered buds; The clustered buds are cut into single buds and transferred to a rooting medium for rooting culture to obtain rooted seedlings.

[0028] The present invention inoculates the stem segments with hidden buds of Hamamelis argentea into an induction culture medium for induction culture to obtain stem segments with lateral buds. As an optional embodiment, the preparation method of the stem segments with hidden buds of Hamamelis argentea according to the present invention comprises: cutting off the leaves of the Hamamelis argentea branches, and then cutting them into stem segments with hidden buds; the length of the stem segments is 1.5-2.0 cm. The Hamamelis argentea branches of the present invention are branches of the current year, and the stem segments with hidden buds of the present invention include at least one hidden bud; the present invention also includes cleaning the stem segments with buds before inoculation, and then disinfecting; the present invention does not specifically limit the cleaning method, and conventional methods can be used. The disinfection of the present invention is first soaked in ethanol with a volume concentration of 75% for 30-40 seconds, or 30 seconds; then rinsed with sterile water for 3-4 times, and then soaked in a sodium hypochlorite solution with an effective chlorine content of 0.26%-0.52% for 5-10 minutes, or 6-7 minutes, and rinsed with sterile water for 5-6 times. The disinfection method of the invention is simple, improves the survival rate of the hamamelis stem segments during induction culture, reduces the contamination rate and browning rate during induction culture, ensures that the lateral buds of the stem segments with buds grow well, and the new leaves are emerald green.

[0029] As an optional embodiment, after the stem segment with hidden buds is disinfected, the method further includes using sterile filter paper to absorb moisture and then inoculating the stem segment with hidden buds of Hamamelis into an induction culture medium for induction culture to obtain a stem segment with lateral buds. The present invention has no special limitation on the inoculation method, and a conventional method can be used. The stem segment of the present invention has hidden buds, and as the induction culture proceeds, the hidden buds germinate to obtain lateral buds, thereby obtaining a stem segment with lateral buds.

[0030] As an optional embodiment, the temperature of the induction culture is 23~27℃, or 25℃; the time of the induction culture is 28~32d, or 29~31d, preferably 30d. As an optional embodiment, the induction culture is first dark culture and then light culture. The time of the induction culture of the present invention is calculated as the sum of the time of light culture and the time of dark culture. The time of light culture is 26~28d, or 27d; the time of dark culture is 2~4d, or 3d. As an optional embodiment, the illumination time of the light culture is 12~16h / d, or 14~16h / d; the dark time in the light culture can be 8~12h / d, or 8~10h / d; the intensity of the light culture can be 2200~2500lx, or 2300~2400lx. The induction culture time of the present invention is calculated from the time when the stem segment is inoculated on the induction culture medium until the latent buds sprout and grow lateral buds, and the lateral buds elongate and grow into new leaves. Dark culture helps to promote the formation of lateral buds in the stem segment with buds and reduce the degree of browning.

[0031] As an optional embodiment, after obtaining the stem segment with lateral buds, the present invention transfers the stem segment with lateral buds to a proliferation culture medium for lateral bud proliferation culture to obtain clustered buds.

[0032] As an optional embodiment, before the stem segment with lateral buds of the present invention is transferred to the proliferation culture medium for proliferation culture, it also includes: short-term subculture of the stem segment with lateral buds. The time of the short-term subculture of the present invention is 6 to 8 days, preferably 7 days. The culture 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 culture medium, and the composition of the subculture medium is: WPM as the basic culture medium, and 28 to 30 g / L sucrose and 6 to 8 g / L agar are added. In the present invention, the pH value of the subculture medium can be 5.7 to 5.9, or 5.8. No hormones are added to the culture medium used for the short-term subculture of the present invention. The temperature of the subculture of the present invention is 25±2°C. The role of subculture is to promote the proliferation of lateral buds on the stem segment.

[0033] As an optional embodiment, the stem segments with lateral buds obtained by subculture of the present invention are transferred to a proliferation culture medium for proliferation culture. The temperature of the proliferation culture of the present invention is 25±2°C, or 25°C; the time of the proliferation culture is 28~35d, or 29~32d, or 30d. The proliferation culture is light culture, and the illumination time of the light culture is 12~16h / d, or 14~16h / d; the dark time of the light culture is 8~12h / d, or 8~10h / d; the intensity of the light culture is 2200~2500lx, or 2400~2500lx. In the proliferation culture of the present invention, the lateral buds proliferate to obtain clustered buds, and finally obtain clustered bud seedlings, and the proliferation culture is stopped until the height of the clustered buds is 2~5cm.

[0034] After obtaining the clustered buds, the present invention cuts the clustered buds into single buds and transfers them to a rooting medium for rooting culture to obtain rooted seedlings.

[0035] As an optional embodiment, the present invention cuts off the single bud in the clustered buds, and inoculates the single bud into a rooting medium for rooting culture. The length of the single bud is 1.5~2cm. As an optional embodiment, the temperature of the rooting culture of the present invention is 25±2℃, or 25℃; the time of the rooting culture is 28~33d, or 29~31d, or 30d. The rooting culture is light culture, and the light time of the light culture is 12~16h / d, or 14~16h / d; the dark time of the light culture can be 8~12h / d, or 8~10h / d; the intensity of the light culture can be 2200~2500lx, or 2400~2500lx. Rooting culture is performed until the root length of the rooted seedling is 1~3cm. The rooting culture of the present invention enables the sterile seedlings to produce strong roots.

[0036] As an optional embodiment, the present invention also includes hardening and transplanting the rooted seedlings to obtain complete regenerated plants. The hardening time of the present invention can be 7 to 14 days, and the matrix used for transplanting includes perlite and peat; the volume ratio of perlite to peat is 1:3; carbendazim is added to the matrix; the concentration of carbendazim is 0.1%; the role of carbendazim is to disinfect and purify the matrix. As an optional embodiment, the temperature of the acclimation of the present invention is 23 to 27°C, the light intensity of the acclimation is 15000 to 20000 lx, and the transplant survival rate of the rooted seedlings of the present invention reaches more than 95%, and the transplant survival rate is high.

[0037] At present, there is only one research report on the breeding technology of Hamamelis argentea stem segments as explants (see "Lai Fuxin. Preliminary study on the restoration of endangered plant Hamamelis argentea population [D]. Anqing Normal University, 2022."), with a contamination rate of 25.00%, a survival rate of 46.47%, and a browning rate of 100%; but the stem segment induction rate is only 21.6%, which is only 1 / 4 times the induction rate of the present invention. The present invention establishes a set of Hamamelis argentea stem segment regeneration technology system through the screening of culture technology and culture medium formula in four stages of explant disinfection, induction of lateral bud germination, lateral bud proliferation, and rooting culture. The contamination rate of the stem segments induced by 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 7%~16%; the lateral bud induction rate of the stem segments, i.e., the germination rate, is as high as 91%, the lateral bud proliferation coefficient is as high as 4.17, the rooting rate is 60%, and the transplant survival rate is more than 95%.

[0038] In order to further illustrate the present invention, the technical solution provided by the present invention is 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.

[0039] The Hamamelis argyi seedlings used in the following examples are from the same batch.

[0040] 1. All prepared culture media in the experiment were sterilized at 121℃ in an autoclave for 15 min, cooled and packaged for later use.

[0041] 2. The indicator calculation method is as follows: Contamination rate = (number of contaminated explants / total number of inoculated explants) × 100%; Browning rate = (number of browned explants / total number of inoculated explants) × 100%; Survival rate = (number of surviving explants / total number of inoculated explants) × 100%; Germination rate = [number of germinated stem segments / (total number of inoculated stem segments-number of contaminated stem segments)] × 100%; Bud proliferation coefficient = (number of buds after growth - number of buds just after inoculation) / number of buds just after inoculation; Average shoot length = total shoot length / number of shoots; Average number of sprouts = total number of sprouts in each treatment / number of sprouts at the time of inoculation; Rooting rate (%) = (rooted plants / total number of grafted plants) × 100%.

[0042] 3. Excel and SPSS statistical analysis software were used for data statistics and analysis, and Duncan's method was used for significant difference test.

[0043] Example 1 Selection and disinfection method of Hamamelis explants 1. Selection of explants The seedling raising cycle is shorter than the seedling raising method of Hu Guowei et al., which uses seeds as explants and multiplies lateral buds, thus greatly saving the seedling raising cost. The comparison results between the present invention and the prior art are shown in Table 1.

[0044] Compared with the technical scheme of Hu Guowei and others, which uses seeds as explants and proliferates lateral buds, the seedling raising cycle is shorter and the breeding efficiency is higher. If one-year-old seedlings are used to prepare stem segments, the one-year-old seedlings of the present invention can take about 70 stem segments at a time in spring, and the lateral bud proliferation coefficient of each stem segment is 4.17, which means that a seedling generated one year later can make an average of 70 stem segment explants with hidden buds, and 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, which is 262.71 buds, can be produced. According to the rooting rate of 60%, an average of 157.63 rooted seedlings are obtained, which greatly saves the cost of seedling raising.

[0045] Table 1 Comparison of different seedling raising methods of Hamamelis chinensis

[0046] Note: 14d, 28d and 21d are the germination time, proliferation time and rooting time in the culture process, respectively. The reason why they are different from the time in the embodiments is that the embodiments set the longest growth time for the convenience of investigation; in the actual growth process, the time in Table 1 has stabilized germination, proliferation or rooting.

[0047] 2. Disinfection method The material is the seedlings of Anji Longwangshan Silver Hamamelis, which are 1 to 3 years old. The seedlings without diseases and insect pests and strong growth are selected to be cultured in a greenhouse for 1 to 3 months, and then the stem segments with buds of young branches of the year are selected as explants.

[0048] The specific method for preparing the explant is as follows: take the current year branches of the young Hamamelis argenteae tree, cut off the leaves, and cut the stem segments into 1.5-2.0 cm in length, rinse them with running water for 2 hours, and then transfer them to the clean bench for use.

[0049] The stem segments with hidden buds of Hamamelis argyi were used as explants. The explant disinfection was as follows: first, the stem segments with buds were treated with 75% ethanol by volume for 30 seconds, and then rinsed with sterile water for 3 to 4 times; then, the stem segments were disinfected with 0.52% NaClO (sodium hypochlorite) solution of effective chlorine for 5 minutes (A1), 10 minutes (A2), 15 minutes (A3), 20 minutes (A4), and 25 minutes (A5), respectively. After disinfection, they were rinsed with sterile water for 5 to 6 times, and the moisture was absorbed with sterile filter paper.

[0050] The sterilized stem segments with buds were inoculated into a culture medium, the composition of which was as follows: WPM was used as a basic culture medium, 30 g / L sucrose and 8 g / L agar were added, and the pH was 5.7.

[0051] Each treatment was performed with 20 stem segments, repeated 3 times. The contamination rate was calculated after 20 days, and the browning rate and survival rate of sterile seedlings were calculated after 30 days. The results of determining the optimal disinfection time are shown in Table 2.

[0052] It can be seen that when the explants were placed in a NaClO solution with an effective chlorine content of 0.52%, the contamination rate of the explants cultured for 20 days first increased and then decreased with the extension of the immersion time. The contamination rate was the lowest at 5% when the treatment was 25 minutes. However, the browning rate gradually increased with the extension of the immersion time. The browning rate was the lowest at 7% when the treatment was 5 minutes. After about 30 days of growth and development, it was found that the survival rate was the highest at 83% when the treatment was 5 minutes, and the browning rate was the lowest at only 7%. The lateral buds grew well, and the new leaves were emerald green.

[0053] Table 2 Effects of different NaClO treatment times on the disinfection of Hamamelis argyi stem explants

[0054] Note: The contamination rate, survival rate and browning rate in the table are all mean ± standard deviation; Duncan multiple comparison method was used; different letters in the same column represent significant differences ( P <0.05), the same below.

[0055] Establishing a sterile system is one of the most basic and critical links in tissue culture, so the sterilization effect of explants is directly related to the success of the experiment. The present invention studies the effect of different NaClO treatment times on the surface sterilization effect of Hamamelis argyi with buds. It can be seen that the effect of 0.52% NaClO solution disinfection for 5 to 10 minutes is good, and the contamination rate during the growth of explants is low, the survival rate is high, and the browning rate is low.

[0056] Example 2 Screening of the most suitable basic culture medium for germination of Hamamelis argyi stem segments After the bud-bearing stem segments of Hamamelis argyi were disinfected, the explants were disinfected as follows: first, the bud-bearing stem segments were treated with 75% ethanol by volume for 30 seconds, and then rinsed with sterile water for 3 to 4 times; then, the stem segments were disinfected with 0.52% NaClO solution for 5 minutes, and then rinsed with sterile water for 5 to 6 times after the disinfection, and the water was dried with sterile filter paper, and then inoculated into lateral bud induction medium 1 to 3. The pH of lateral bud induction medium 1 to 3 was 5.7 respectively.

[0057] The composition of the lateral bud induction medium 1 is MS as the basic medium, and 30 g / L sucrose and 8 g / L agar are added.

[0058] The composition of the lateral bud induction medium 2 is 1 / 2MS as the basic medium, and 30 g / L sucrose and 8 g / L agar are added.

[0059] The composition of the lateral bud induction medium 3 is WPM as the basic medium, and 30 g / L sucrose and 8 g / L agar are added.

[0060] After inoculation, each culture medium was first cultured in the dark for 3 days all day, and then cultured in light for 27 days. Dark culture was carried out in a dark cabinet in a sterile room, and the temperature of the sterile culture room was 23~25℃; light culture was carried out in a sterile culture room, and the photoperiod was 16h / d light culture and 8h / d dark culture; the light intensity was 2400lx, and the temperature of light culture was 25±2℃. 20 stem segments were inoculated in each treatment, and one stem segment was inoculated in each culture dish. Three repeated experiments were set up, and the total culture time was 30d. After 30d, the lateral bud germination rate and growth status were counted. The hidden buds in the stem segments germinated and elongated into lateral buds to obtain stem segments with lateral buds. The results are shown in Table 3. Under the condition of WPM as the basic culture medium, the germination rate was as high as 72%, and the growth status was good (see Figure 1 ), which is the basic culture medium suitable for the germination of Hamamelis chinensis stem segments.

[0061] Table 3 Effects of different basic culture media on the germination of lateral buds of Hamamelis chinensis

[0062] Note: Different letters in the same column represent significant differences ( P <0.05), the same below.

[0063] Example 3 After disinfection, the stem segments of Hamamelis argyi with buds were inoculated in the following culture media 1-15 and CK for induction culture. The disinfection method was the same as in Example 2. The pH of culture media 1-15 and CK was 5.7, respectively. The induction culture was first dark cultured for 3 days, and then light cultured for 27 days. The dark culture was carried out in a dark cabinet in a sterile room, and the temperature of the sterile culture room was 25±2°C; the light culture was carried out in a sterile culture room, and the photoperiod of the light culture was 16h / d light culture, 8h / d dark culture, and the light intensity was 2400lx.

[0064] F1: The composition of medium 1 is: WPM as the basic medium, supplemented with 0.5 mg / L KT, 30 g / L sucrose and 8 g / L agar; F2: The composition of medium 2 is: WPM as the basic medium, supplemented with 1.0 mg / L KT, 30 g / L sucrose and 8 g / L agar; F3: The composition of medium 3 is: WPM as the basic medium, supplemented with 1.5 mg / L KT, 30 g / L sucrose and 8 g / L agar; F4: The composition of medium 4 is: WPM as the basic medium, supplemented with 2.0 mg / L KT, 30 g / L sucrose and 8 g / L agar; F5: The composition of medium 5 is: WPM as the basic medium, supplemented with 4.0 mg / L KT, 30 g / L sucrose and 8 g / L agar; CK: The composition of the culture medium is: WPM as the basic medium, supplemented with 30 g / L sucrose and 8 g / L agar; D1: The composition of medium 6 is: WPM as the basic medium, supplemented with 0.5 mg / L 6-BA, 30 g / L sucrose and 8 g / L agar; D2: The composition of medium 7 is: WPM as the basic medium, supplemented with 1.0 mg / L 6-BA, 30 g / L sucrose and 8 g / L agar; D3: The composition of medium 8 is: WPM as the basic medium, supplemented with 1.5 mg / L 6-BA, 30 g / L sucrose and 8 g / L agar; D4: The composition of medium 9 is: WPM as the basic medium, supplemented with 2.0 mg / L 6-BA, 30 g / L sucrose and 8 g / L agar; D5: The composition of medium 10 is: WPM as the basic medium, supplemented with 4.0 mg / L 6-BA, 30 g / L sucrose and 8 g / L agar; E1: The composition of medium 11 is: WPM as the basic medium, supplemented with 0.001 mg / L TDZ, 30 g / L sucrose and 8 g / L agar; E2: The composition of medium 12 is: WPM as the basic medium, supplemented with 0.01 mg / L TDZ, 30 g / L sucrose and 8 g / L agar; E3: The composition of medium 13 is: WPM as the basic medium, supplemented with 0.1 mg / L TDZ, 30 g / L sucrose and 8 g / L agar; E4: The composition of medium 14 is: WPM as the basic medium, supplemented with 1.0 mg / L TDZ, 30 g / L sucrose and 8 g / L agar; E5: The composition of medium 15 is: WPM as the basic medium, supplemented with 2.0 mg / L TDZ, 30 g / L sucrose and 8 g / L agar; One stem segment with buds was inoculated in each culture dish, and a total of 20 stem segments with buds were inoculated in each parallel experiment. Three parallel experiments were set up, and the germination rate, average number of buds and growth status were counted after 30 days of culture.

[0065] The results of the effect of KT on the induction of lateral buds of Hamamelis argyi are shown in Tables 4 and Figure 2It can be seen that KT has no significant effect on the induction of clustered buds from the lateral buds of Hamamelis argyi. The germination rate increases first and then decreases with the increase of concentration. The highest germination rate is 91% at 1.0 mg / L. Compared with WPM without any hormone addition, the germination rate increases by 19%, but the average number of buds is stable at 1 (Table 4). According to observations, the lateral buds induced by KT have larger leaves and darker colors (see Figure 2 ).

[0066] Table 4 Effects of different concentrations of KT on lateral bud germination of isolated stem segments

[0067] The results of the effects of different concentrations of 6-BA (6-benzylaminopurine, N-(Phenylmethyl)-9H-purin-6-amine) on the induction of lateral buds from isolated stem segments are shown in Tables 5 and Figure 3 It can be seen that 6-BA plays a certain role in the induction of lateral buds of Hamamelis argentea. With the increase of concentration, the germination rate shows a trend of increasing first and then decreasing. When the concentration of 6-BA is 1.5 mg / L, the germination rate is the highest, reaching 86%. With the increase of concentration, the average germination number does not change much, reaching the highest at 1.0 mg / L, reaching 1.17 (Table 5). According to observations, the color of the clustered buds induced by 6-BA is relatively emerald green (see Figure 3 ).

[0068] Table 5 Effects of different concentrations of 6-BA on the induction of lateral bud growth in vitro stem segments

[0069] The results of the effects of different concentrations of TDZ (Tiabendazole) on the induction of lateral buds in vitro are shown in Tables 6 and Figure 4 TDZ has a significant effect on the induction of lateral buds of Hamamelis argentea. The number of lateral buds that grow into clustered buds is larger and the color is darker. As the concentration of TDZ increases, the germination rate of lateral buds increases, reaching the highest level of 87% at 1.0 mg / L and 2.0 mg / L. However, the average number of buds increases first and then decreases with the increase in concentration, reaching the highest level at 1.0 mg / L, reaching 1.72 (Table 6). It can be seen that TDZ is more prone to deformity and vitrification than 6-BA and KT treatments (see Figure 4 ).

[0070] Table 6 Effects of different concentrations of TDZ on the induction of lateral bud growth in vitro stem segments

[0071] Example 4 1. Effects of different concentrations of 6-BA and TDZ on the proliferation of lateral buds of Hamamelis chinensis Through single factor experiments, it was found that 6-BA and TDZ played a major role in the initiation of lateral buds of Hamamelis argentea. In order to explore the effects of different ratios of the two on the proliferation of lateral buds of Hamamelis argentea, the following experiments were conducted. The sterile stem segments with one lateral bud grown in F2 medium were inoculated into proliferation medium J1~J9 to induce lateral buds for proliferation culture. The pH values ​​of proliferation medium J1~J9 were 5.7 respectively. The specific composition of proliferation medium J1~J9 is as follows: J1: proliferation medium 1: WPM as the basic medium, supplemented with 1.0 mg / L 6-BA, 0.01 mg / L TDZ, 30 g / L sucrose and 8 g / L agar; J2: proliferation medium 2: WPM as the basic medium, supplemented with 1.0 mg / L 6-BA, 0.1 mg / L TDZ, 30 g / L sucrose and 8 g / L agar; J3: proliferation medium 3: WPM as the basic medium, supplemented with 1.0 mg / L 6-BA, 1.0 mg / L TDZ, 30 g / L sucrose and 8 g / L agar; J4: proliferation medium 4: WPM as the basic medium, supplemented with 1.5 mg / L 6-BA, 0.01 mg / L TDZ, 30 g / L sucrose and 8 g / L agar; J5: proliferation medium 5: WPM as the basic medium, supplemented with 1.5 mg / L 6-BA, 0.1 mg / L TDZ, 30 g / L sucrose and 8 g / L agar; J6: proliferation medium 6: 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; J7: proliferation medium 7: 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; J8: proliferation medium 8: 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; J9: proliferation medium 9: 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; 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 16h / d light culture and 8h / d dark culture, and the light intensity was 2400lx.

[0072] Each culture medium was inoculated with 20 bottles, with one explant in each bottle; three replicates were set up, with a total of 60 bottles inoculated in each culture medium. After 30 days of culture in each culture medium, the proliferation coefficient, new bud length, and seedling growth were statistically analyzed. The results are shown in Tables 7 and Figure 5 After 30 days of cultivation, with the increase of 6-BA concentration, the average bud length gradually decreased, and the bud proliferation coefficient first increased and then decreased; with the increase of TDZ concentration, the bud proliferation coefficient gradually increased, but the degree of vitrification was serious. When grown in the medium supplemented with 1.0 mg / L 6-BA+1.0 mg / L TDZ, the average bud length was the highest at 1.82, and when grown in the medium supplemented with 1.5 mg / L 6-BA+0.1 mg / L TDZ, the bud proliferation coefficient was the highest at 1.58 (Table 7, Figure 5 When the 6-BA content in the proliferation medium was 1.5 mg / L, the lateral buds grew faster and produced more clusters of buds.

[0073] Table 7 Effects of different concentrations of 6-BA and TDZ on the proliferation of Hamamelis argyi lateral buds

[0074] 2. Effects of different concentrations of TDZ and 1.5 mg / L 6-BA on the proliferation of lateral buds of Hamamelis argyi The sterile stem segments with one lateral bud grown in F2 medium were transferred to new medium S1~S5 for proliferation culture after subculture in subculture proliferation medium for 7 days. The subculture medium was hormone-free WPM medium. The composition of the subculture medium was WPM as the basic medium, supplemented with 30g / L sucrose and 8g / L agar, and the pH value was 5.7.

[0075] Culture media S1~S5 investigated the effect of TDZ on the proliferation of lateral buds of Hamamelis argyi. The composition of S1~S5 culture media is as follows.

[0076] S1: proliferation medium 1: WPM as basic medium, supplemented with 1.5 mg / L 6-BA, 0.003 mg / L TDZ, 30 g / L sucrose and 8 g / L agar; S2: proliferation medium 2: 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; S3: proliferation medium 3: 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; S4: proliferation medium 4: 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; S5: proliferation medium 5: 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; The pH of each culture medium was 5.7. Each culture medium was inoculated with 20 bottles, with one explant in each bottle. Three parallel experiments were set up, and a total of 60 bottles were inoculated with each culture medium. After 30 days of culture, the proliferation times, average bud length and lateral bud growth 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 16h / d light culture and 8h / d dark culture, and the light intensity was 2400lx.

[0077] Table 8 Effects of different concentrations of TDZ on the proliferation of Hamamelis argyi lateral buds

[0078] Example 5 Effects of different concentrations of IBA and NAA on the rooting of Hamamelis chinensis Inoculation: Single buds with a length of 1.5 to 2 cm were selected from the clustered buds grown in S1 of Example 4, cut off, and inoculated into a rooting medium.

[0079] The composition of the rooting medium is: 1 / 2MS is used as the basic medium, and different concentrations of IBA (0, 0.2, 0.4 mg / L) and NAA (0, 0.1, 0.2 mg / L) are added. The specific composition of the rooting medium L0~L8 is as follows: L0: Rooting medium is 1 / 2MS as basic medium, supplemented with 30 g / L sucrose and 8 g / L agar; L1: Rooting medium is 1 / 2MS as basic medium, supplemented with 0.2 mg / L IBA, 30 g / L sucrose and 8 g / L agar; L2: Rooting medium is 1 / 2MS as basic medium, supplemented with 0.4 mg / L IBA, 30 g / L sucrose and 8 g / L agar; L3: Rooting medium is 1 / 2MS as basic medium, supplemented with 0.1 mg / L NAA, 30 g / L sucrose and 8 g / L agar; L4: Rooting medium is 1 / 2MS as basic medium, supplemented with 0.2 mg / L NAA, 30 g / L sucrose and 8 g / L agar; L5: Rooting medium is 1 / 2MS as basic medium, supplemented with 0.2 mg / L IBA, 0.1 mg / L NAA, 30 g / L sucrose and 8 g / L agar; L6: Rooting medium is 1 / 2MS as basic medium, supplemented with 0.2 mg / L IBA, 0.2 mg / L NAA, 30 g / L sucrose and 8 g / L agar; L7: Rooting medium is 1 / 2MS as basic medium, supplemented with 0.4 mg / L IBA, 0.1 mg / L NAA, 30 g / L sucrose and 8 g / L agar; L8: Rooting medium is 1 / 2MS as basic medium, supplemented with 0.4 mg / L IBA, 0.2 mg / L NAA, 30 g / L sucrose and 8 g / L agar; The pH of each culture medium was 5.7. Each treatment was inoculated with 20 bottles, 1 explant per bottle, and repeated three times. The rooting situation was counted after 30 days. The results are shown in Table 9. Rooting culture was carried out in a sterile culture room at a temperature of 23-27°C; light culture was carried out in a sterile culture room with a photoperiod of 16h / d light culture and 8h / d dark culture, and the light intensity was 2400lx.

[0080] It can be seen that after 30 days of cultivation, with the increase of IBA concentration, the rooting rate gradually decreased. The rooting medium with only 0.2 mg / L NAA had the highest rooting rate, reaching 60%, and the base grew green callus, the adventitious roots were white, and the root length was (see Table 9, Figure 7 ).

[0081] Table 9 Effects of different concentrations of IBA and NAA on rooting of Hamamelis argyi

[0082] Example 6 Transplantation Select the test tube seedlings with good rooting. When the test tube seedlings are more than 2-4 cm tall and the roots are 1-2 cm long, place them in the acclimation room for 7-14 days. The temperature in the acclimation room is 23-27℃ and the light intensity is 15000-20000lx. After the test tube seedlings grow new leaves or new roots during the acclimation process, they can be transplanted into a nutrient pot with a diameter of 6-10 cm. The transplanting matrix is ​​perlite and peat soil. The volume ratio of perlite and peat soil is 1:3. Add carbendazim solution to the matrix. The mass concentration of the added carbendazim solution is 0.1%. Stir evenly. After 30 days of cultivation, the transplant survival rate is calculated. The transplant survival rate is more than 95%.

[0083] In summary, as a rare and endangered plant, the present invention uses 1.5-2 cm stem segments collected from young branches of 1-3 year old seedlings as explants, and optimizes the culture technology and culture medium formula of four stages: explant disinfection, lateral bud induction germination, lateral bud proliferation, rooting culture, and transplanting and domestication. A set of Hamamelis tissue culture and breeding technology system has been established to provide technical support for the protection and propagation of endangered plants. The present invention uses stem segments with hidden buds as explants, and uses a single, harmless and non-toxic disinfectant, NaClO, to disinfect for 5 minutes, which can achieve a purification rate of more than 90% (the purification rate is 100% minus the contamination rate), a higher lateral bud germination rate (91%), and a lower browning rate (7%). The explant disinfection process is simple and ensures good growth of the explant, which starts the aseptic culture of Hamamelis. The present invention also screens the culture medium formula for stem segment lateral bud germination, and the germination rate is as high as 91% (F2 culture medium). The present invention screens the culture medium formula for lateral bud proliferation. The formula of the propagation culture medium makes the proliferation coefficient 4.17, which can greatly improve the breeding efficiency; the rooting culture medium obtained by the present invention has a high rooting rate, and the seedlings are hardened in an acclimatization room with a temperature of 23-27°C and a light intensity of 15000-20000lx for 7-14 days, and then transplanted into a matrix, the matrix is ​​mixed with carbendazim, and the volume ratio of perlite and peat soil in the matrix is ​​1:3, and a witch hazel tissue culture pot is obtained. The transplant survival rate of the witch hazel tissue culture pot reaches more than 95%, and the transplant survival rate is high. The witch hazel tissue culture pot seedlings can be used for afforestation.

[0084] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A tissue culture rapid propagation medium for culturing Hamamelis salsa, characterized in that: The tissue culture rapid propagation medium includes an induction medium, a proliferation medium and a rooting medium; The induction medium 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; The proliferation medium uses WPM as a basic medium and also includes 1.0-1.5 mg / L 6-BA, 0.003-0.005 mg / LTDZ, 28-30 g / L sucrose and 6-8 g / L agar; The rooting medium uses 1 / 2MS as a basic medium and also includes 28-30 g / L sucrose and 6-8 g / L agar.

2. The tissue culture rapid propagation medium according to claim 1, characterized in that The rooting medium also includes 0.11-0.4 mg / L NAA.

3. A tissue culture and rapid propagation method of Hamamelis truncatula, characterized in that: The method of using the tissue culture rapid propagation medium according to claim 1 or 2 comprises the following steps: Inoculating the stem segments with hidden buds of Hamamelis argyi into an induction medium for induction culture to obtain stem segments with lateral buds; Transferring the stem segment with lateral buds to a proliferation culture medium for proliferation culture to obtain clustered buds; The clustered buds are cut into single buds and transferred to a rooting medium for rooting culture to obtain rooted seedlings.

4. The tissue culture rapid propagation method according to claim 3, 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.

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

6. The tissue culture and rapid propagation method according to claim 3, characterized in that: The induction culture is first dark culture and then light culture; the proliferation culture and rooting culture are light culture respectively.

7. The tissue culture and rapid propagation method according to claim 6, 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.

8. The tissue culture rapid propagation method according to claim 3 or 6, 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.

9. The tissue culture rapid propagation method according to claim 3 or 6, 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.

10. The tissue culture and rapid propagation method according to claim 3, characterized in that: The length of the stem segment with hidden buds is 1.5-2.0 cm.

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