A common culture medium for differentiation, proliferation and rooting culture of cephalotaxus fortunei and a tissue culture method

Abstract

The application discloses a common culture medium for differentiation, proliferation and rooting culture of Cibotium barometz and a tissue culture method, and belongs to the technical field of tissue culture of Cibotium barometz. The common culture medium comprises: modified 1 / 2MS+6-BA 0.1 mg / L+GA3 0.1 mg / L+IBA 0.1 mg / L; wherein the modified 1 / 2MS contains KNO3 3640 mg / L and NH4NO3 9500 mg / L. The tissue culture method comprises the following steps: after Cibotium barometz is germinated, Cibotium barometz seedlings are obtained through differentiation, proliferation and rooting culture. The application can improve the differentiation rate of Cibotium barometz, and makes the tissue culture of Cibotium barometz simpler, more reliable and more economical.

Description

Technical Field

[0001] The present invention relates to the technical field of plant tissue culture, specifically to the tissue culture technology of Cibotium barometz, and more specifically, the present invention relates to a common culture medium for the differentiation, proliferation and rooting culture of Cibotium barometz and a tissue culture method. Background Art

[0002] Cibotium barometz (L.) J. Sm. is a perennial large tree-like terrestrial fern of the genus Cibotium Kaulf. in the family Dicksoniaceae, and is an important medicinal plant.

[0003] Currently, the national market demand for Cibotium barometz is about 4,000 tons per year. There are about 100 prescriptions using the medicinal materials of Cibotium barometz, such as Yutong Pills, Shengli Powder, Tongling Huangjin Ointment, Jingou Decoction, and Gouji Decoction; at the same time, the medicinal materials are also used in more than 70 kinds of proprietary Chinese medicines, such as Cibotium barometz Pills, Zhuangyao Jianshen Pills, Zhuangyao Jianshen Tablets, Kanggu Zengsheng Capsules, Guli Capsules, Gentongping Granules, Compound Psoralea冲剂, Zhuangyao Jianshen Oral Liquid, Dongtian Changchun Ointment, Qufeng Chushi Medicinal Liquor, etc. In addition, the Cibotium barometz plant also has extremely high ornamental value and can also be used as wild vegetables for consumption.

[0004] Due to the huge demand for Cibotium barometz and the resulting economic benefits, the development of its wild resources has attracted much attention in recent years. However, the growth, reproduction and distribution rules of Cibotium barometz have been ignored in the utilization, showing a disorderly and random excavation situation; at the same time, due to the internal endangered factors of Cibotium barometz itself, including underdeveloped plant roots, low natural regeneration efficiency and poor environmental adaptability, etc., the wild resources of Cibotium barometz are continuously decreasing.

[0005] Actively carrying out the tissue culture of Cibotium barometz is an effective method to protect the germplasm resources of Cibotium barometz and the current shortage of medicinal materials resources, and to more effectively protect wild resources.

[0006] Tissue culture technology is an effective way to achieve rapid plant propagation and is a prerequisite for large-scale artificial cultivation. Obtaining healthy plant seedlings quickly and conveniently is the ultimate goal of rapid propagation. In recent years, researchers have reported on tissue culture methods for *Cibotium barometz*. Su Yuqin et al. simultaneously used rhizomes and mature spores from different parts of *Cibotium barometz* for seedling propagation research. Their study found that the seedling survival rate induced by rhizomes from different parts was low, only around 70%, and the subsequent survival and growth of the plants were significantly affected. Furthermore, harvesting rhizomes as explants caused some damage to the plants (see existing technical literature 1). When using mature spores in experiments, the need to change the differentiation and rooting culture medium during the experiment not only easily led to contamination during operation, but also affected seedling differentiation, resulting in a final differentiation rate of only 56% at most (see existing technical literature 2).

[0007] Based on the above research results, the low induction differentiation rate of Cibotium barometz tissue culture and the lack of a simple and reliable method for generating Cibotium barometz seedlings are key factors limiting the large-scale and rapid propagation of Cibotium barometz seedlings through tissue culture.

[0008] Existing technical documents:

[0009] 1. Su Yuqin, Mo Yanlan. Research on the breeding of Cibotium barometz seedlings [J]. Modern Agricultural Science and Technology, 2019, 742(08):47+49.

[0010] 2. Su Yuqin. Study on tissue culture of Cibotium barometz [J]. Modern Agricultural Science and Technology, 2018, 715(05):54-55. Summary of the Invention

[0011] One objective of this invention is to provide a common culture medium for the differentiation, proliferation, and rooting of Cibotium barometz, which not only improves the differentiation rate of Cibotium barometz but also makes Cibotium barometz tissue culture simpler, more reliable, and more economical.

[0012] Another objective of this invention is to provide a method for tissue culture of Cibotium barometz, which aims to solve the technical problem of low differentiation rate of Cibotium barometz and to make Cibotium barometz tissue culture simple, reliable and economical.

[0013] To achieve these objectives and other advantages of the present invention, the present invention provides a common culture medium for the differentiation, proliferation and rooting of Cibotium barometz, comprising: modified 1 / 2 MS + 6-BA 0.1 mg / L + GA3 0.1 mg / L + IBA 0.1 mg / L; wherein the modified 1 / 2 MS contains KNO3 3640 mg / L and NH4NO3 9500 mg / L, and other components remain unchanged.

[0014] Preferably, the culture medium for the differentiation, proliferation and rooting of Cibotium barometz is further supplemented with 30 g / L sucrose, 5 g / L agar, and the pH is adjusted to 5.8.

[0015] The present invention provides a method for tissue culture of Cibotium barometz, comprising: obtaining Cibotium barometz seedlings through differentiation culture, proliferation culture and rooting culture after germination; wherein the components of the differentiation culture medium, proliferation culture medium and rooting culture medium are the same as those of the common culture medium, specifically: modified 1 / 2MS + 6-BA 0.1 mg / L + GA3 0.1 mg / L + IBA 0.1 mg / L; the modified 1 / 2MS contains KNO3 3640 mg / L, NH4NO3 9500 mg / L, and other components remain unchanged.

[0016] Preferably, in the described method for tissue culture of Cibotium barometz, the differentiation medium, proliferation medium, and rooting medium are further supplemented with 30 g / L sucrose, 5 g / L agar, and the pH is adjusted to 5.8.

[0017] Preferably, in the described Cibotium barometz tissue culture method, the differentiation culture process includes three stages:

[0018] Early differentiation stage: The culture temperature for days 1-10 is 20℃;

[0019] Mid-differentiation stage: The culture temperature was 28℃ from day 11 to day 20;

[0020] Late differentiation stage: The culture temperature from day 21 to day 60 is 25℃;

[0021] The light intensity during the differentiation culture process is 1500-2000 lx, and the light duration is 12 hours / day.

[0022] Preferably, in the tissue culture method of Cibotium barometz, the differentiation medium also contains 1-2 g / L of Cibotium barometz extract; the preparation method of Cibotium barometz extract is as follows: fresh Cibotium barometz leaves are cleaned and then added to a blender at a mass ratio of Cibotium barometz leaves to sterile water of 1:10 and blended into a slurry. The slurry is then centrifuged to obtain the supernatant. The supernatant is then sterilized by pasteurization to obtain Cibotium barometz extract.

[0023] Preferably, the method for tissue culture of Cibotium barometz includes:

[0024] S1: Sterilize mature spores of Cibotium barometz with 8% NaClO solution for 15-25 minutes, and wash with sterile water 4-5 times to obtain sterile explants;

[0025] S2: Sterile explants were cultured in a lower germination medium for 85-95 days to obtain gametophytes of Cibotium barometz. The germination medium was 1 / 2 MS, with 30 g / L sucrose and 5 g / L agar added, and the pH was adjusted to 5.8.

[0026] S3: The obtained gametophytes of Cibotium barometz were placed in a differentiation medium and cultured for 60 days to obtain sporophytes;

[0027] S4: The differentiated sporophytes were placed in a proliferation medium and cultured for 30 days at a temperature of 25℃, a light intensity of 1700 lx, and a light duration of 12 hours / day.

[0028] S5: Place the sporophytes obtained from the proliferation culture in a rooting medium and culture for 25-35 days to obtain Cibotium barometz seedlings. The culture temperature is 25℃, the light intensity is 1500-2000 lx, and the light duration is 12 hours / day.

[0029] The present invention has at least the following beneficial effects:

[0030] 1. The difficulty in differentiating Cibotium barometz lies in the process of prothallium differentiation into sporophyte after spore germination. During this process, the prothallium produces antheridium and archegonia. Sperm fertilizes the egg to form a zygote, which then differentiates into a sporophyte. Commonly used hormone ratios are difficult to induce differentiation or result in low differentiation rates. In one embodiment of this invention, a modified 1 / 2MS medium containing 0.1 mg / L 6-BA, 0.1 mg / L GA3, and 0.1 mg / L IBA was screened. This modified 1 / 2MS medium contained 3640 mg / L KNO3 and 9500 mg / L NH4NO3, with other components remaining unchanged. Differentiation culture showed a high differentiation rate, thus solving the technical problem of low differentiation rate in Cibotium barometz.

[0031] 2. The archegonium of the golden retriever dog develops later than the antheridium. To address this characteristic, this invention precisely divides the differentiation culture of the golden retriever dog into three stages: pre-differentiation, mid-differentiation, and post-differentiation. In the pre-differentiation stage, a lower temperature of 20°C is used for culture, which is conducive to the survival of the produced sperm. In the mid-differentiation stage, the culture temperature is increased to 28°C to improve the activity of sperm and eggs, promoting the fertilization of sperm and eggs to form zygotes. In the post-differentiation stage, the normal temperature of 25°C is used for culture to maintain normal differentiation, effectively improving the differentiation rate.

[0032] 3. In another embodiment of the present invention, Cibotium barometz extract was added to the modified 1 / 2MS + 6-BA 0.1 mg / L + GA3 0.1 mg / L + IBA 0.1 mg / L differentiation medium to induce differentiation and improve the differentiation rate.

[0033] 4. This invention has screened out a common culture medium formula for differentiation, proliferation and rooting, which is suitable for production use and is more economical.

[0034] 5. This invention uses the sequence of germination, differentiation, proliferation, and rooting for tissue culture. Compared with proliferation followed by differentiation, this effectively reduces the loss of raw materials caused by differentiation failure and lowers costs.

[0035] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0036] Figure 1 The golden-haired dog spores of Example 2;

[0037] Figure 2 This is the process of obtaining gametophytes from Cibotium barometz spore germination culture in Example 2;

[0038] Figure 3 The gametophyte differentiation process of Cibotium barometz in Example 2;

[0039] Figure 4 The sporophyte of Example 2 is a cultured organism.

[0040] Figure 5 The results of sporophyte rooting culture in Example 2 are shown. Detailed Implementation

[0041] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0042] Example 1

[0043] A common culture medium for the differentiation, proliferation and rooting of Cibotium barometz includes: modified 1 / 2 MS + 6-BA 0.1 mg / L + GA3 0.1 mg / L + IBA 0.1 mg / L; wherein the modified 1 / 2 MS contains KNO3 3640 mg / L, NH4NO3 9500 mg / L, and also contains 30 g / L sucrose, 5 g / L agar, and the pH is adjusted to 5.8.

[0044] Example 2

[0045] A method for culturing Cibotium barometz tissue includes:

[0046] 1. Disinfection of explants

[0047] Mature spores of Cibotium barometz were selected, and an appropriate amount was placed in a non-woven fabric bag. The bag was sterilized with 8% NaClO solution for 20 minutes, and then washed 4-5 times with sterile water to obtain sterile explants. Cibotium barometz spores... Figure 1 As shown.

[0048] II. Germination Culture

[0049] Sterile explants were cultured in a low germination medium for 90 days to obtain gametophytes from *Cibotium barometz*. The culture temperature was 25℃, the light intensity was 1700 lx, and the photoperiod was 12 hours / day. The germination medium was 1 / 2 MS, with 30 g / L sucrose, 5 g / L agar, and the pH adjusted to 5.8. The process of obtaining gametophytes from *Cibotium barometz* spore germination culture is as follows: Figure 2 As shown.

[0050] III. Differentiation and Cultivation

[0051] The obtained gametophytes of Cibotium barometz were placed in a differentiation medium for differentiation culture to obtain sporophytes. The differentiation process is as follows: Figure 3 As shown.

[0052] The differentiation medium was: modified 1 / 2 MS + 6-BA 0.1 mg / L + GA3 0.1 mg / L + IBA 0.1 mg / L; with 30 g / L sucrose and 5 g / L agar added, and the pH was adjusted to 5.8.

[0053] The modified 1 / 2MS medium contained 3640 mg / L KNO3 and 9500 mg / L NH4NO3, with other components remaining unchanged.

[0054] The cultivation conditions were as follows: 60 days; 25℃; 1700 lx light intensity; and 12 hours / day of light exposure.

[0055] IV. Proliferation Culture

[0056] The differentiated sporophytes were cultured in proliferation medium for 30 days at 25℃, light intensity of 1700 lx, and a photoperiod of 12 hours / day. The proliferation medium consisted of modified 1 / 2 MS medium + 0.1 mg / L 6-BA + 0.1 mg / L GA3 + 0.1 mg / L IBA; with the addition of 30 g / L sucrose and 5 g / L agar, and the pH adjusted to 5.8. The proliferated sporophytes were as follows: Figure 4 As shown.

[0057] The modified 1 / 2MS medium contained 3640 mg / L KNO3 and 9500 mg / L NH4NO3, with other components remaining unchanged.

[0058] V. Rooting Culture

[0059] The sporophytes obtained from the proliferation culture were placed in rooting medium and cultured for 30 days at a temperature of 25℃, a light intensity of 1700 lx, and a light duration of 12 hours / day. The results of the sporophyte rooting culture are as follows: Figure 5 As shown.

[0060] The rooting medium was prepared as follows: modified 1 / 2 MS medium + 6-BA 0.1 mg / L + GA3 0.1 mg / L + IBA 0.1 mg / L; with the addition of 30 g / L sucrose and 5 g / L agar, and the pH adjusted to 5.8. This group involved a modified 1 / 2 MS medium containing 3640 mg / L KNO3 and 9500 mg / L NH4NO3, with other components remaining unchanged.

[0061] Example 3

[0062] Unlike Example 2, the differentiation culture was divided into three stages: early differentiation (days 1-10, temperature 20°C); mid-differentiation (days 11-20, temperature 28°C); and late differentiation (days 21-60, temperature 25°C). All other steps were the same.

[0063] Example 4

[0064] Unlike Example 3, in this case, the differentiation culture medium also contained Cibotium barometz extract at a concentration of 1 g / L. All other steps remained the same.

[0065] The extract of Cibotium barometz is prepared by: washing fresh Cibotium barometz leaves, adding them to a blender at a mass ratio of Cibotium barometz leaves to sterile water of 1:10 and blending them into a pulp, then centrifuging the pulp to obtain the supernatant, and then sterilizing the supernatant using pasteurization (80℃, 30 minutes) to obtain the Cibotium barometz extract.

[0066] Example 5

[0067] Unlike Example 4, the differentiation culture medium also contained Cibotium barometz extract at a concentration of 2 g / L. Everything else remained the same.

[0068] Example 6

[0069] Unlike Example 5, the differentiation culture medium also contained Cibotium barometz extract at a concentration of 3 g / L. Everything else remained the same.

[0070] Experiment on the effect of condition optimization

[0071] I. Germination Culture

[0072] A. Explant disinfection: Select mature spores of Cibotium barometz, take an appropriate amount and put them into a non-woven bag, disinfect with 8% NaClO solution for 20 minutes, and wash with sterile water 4-5 times to obtain sterile explants.

[0073] B. Selection of germination medium: Sterile explants were cultured in the germination medium in Table 1 below for 90 days to obtain gametophytes of Cibotium barometz. The culture temperature was 25℃, the light intensity was 1700lx, and the light duration was 12 hours / day.

[0074] Table 1 Germination medium

[0075] 1 MS + NAA 0.5 mg / L Unsprouted A small number of germinations Germination 2 MS+NAA 1mg / L Unsprouted A small number of germinations Germination 3 MS + NAA 1.5 mg / L Unsprouted A small number of germinations A small number of germinations 4 MS + 6-BA 0.5 mg / L Unsprouted A small number of germinations A small number of germinations 5 MS + 6-BA 1 mg / L Unsprouted Unsprouted A small number of germinations 6 MS + 6-BA 1.5 mg / L Unsprouted Unsprouted A small number of germinations 7 MS + GA3 0.5 mg / L Unsprouted A small number of germinations A small number of germinations 8 MS + GA3 1mg / L Unsprouted A small number of germinations Germination 9 MS + GA3 1.5 mg / L Unsprouted A small number of germinations Germination 10 1 / 8MS+6-BA0.04mg / L+IAA0.02mg / L Unsprouted A small number of germinations Germination 11 1 / 2MS Unsprouted Germination Numerous germination 12 MS Unsprouted A small number of germinations Germination

[0076] Note: In the above culture medium preparation, 30 g / L sucrose and 5 g / L agar should also be added, and the pH should be adjusted to 5.8.

[0077] The results in Table 1 show that aseptic sowing of spores in all treatments resulted in germination at 90 days, with groups 1, 2, 8, 9, 10, and 12 exhibiting better germination rates, and group 11 showing the best germination. At 60 days, groups 5 and 6 failed to germinate, while the remaining groups showed some germination, but group 11 had the highest germination rate at 1 / 2 MS. Considering germination efficiency and production efficiency, group 11, with its lower component ratio and higher germination rate, is the optimal choice for production and is suitable for this purpose.

[0078] II. Preliminary Selection of Differentiation Culture Conditions

[0079] The gametophytes of Cibotium barometz were differentiated into sporophytes using a differentiation medium, as follows: The gametophytes of Cibotium barometz were placed in the differentiation medium shown in Table 2 below and cultured for 60 days at a temperature of 25℃, a light intensity of 1700 lx, and a light duration of 12 hours / day.

[0080] Table 2 Differentiation culture medium

[0081]

[0082]

[0083] Note: In the above culture medium preparation, 30 g / L sucrose and 5 g / L agar should also be added, and the pH should be adjusted to 5.8.

[0084] The results in Table 2 show that differentiation occurred in groups 10, 11, 12, 13, 14, and 15, with group 12 showing the best differentiation. This group involved a modified 1 / 2 MS medium. The modified medium consisted of KNO3 3640 mg / L and NH4NO3 9500 mg / L, while other components remained unchanged. The optimal differentiation medium composition should be: modified 1 / 2 MS + 6-BA 0.1 mg / L + GA3 0.1 mg / L + IBA 0.1 mg / L.

[0085] III. Further Improvements to Differentiation Culture Conditions

[0086] Considering that the gametophyte of Cibotium barometz first forms a prothallus, the prothallus is fertilized to form a zygote, and the zygote differentiates to form a sporophyte, the differentiation culture of Cibotium barometz gametophyte was further divided into three stages, and then culture experiments were conducted for comparison: early differentiation stage: days 1-10; mid-differentiation stage: days 11-20; late differentiation stage: days 21-60.

[0087] The specific culture conditions and culture medium settings are shown in Table 3 below. Each group of experiments consisted of 100 plants, and the differentiation rate was calculated after 60 days.

[0088] Table 3 Improvement Plan for Cultivation Conditions in the Pre-Cultivation, Intermediate, and Post-Cultivation Stages

[0089]

[0090]

[0091] Note: The above differentiation medium is: modified 1 / 2 MS + 6-BA 0.1 mg / L + GA3 0.1 mg / L + IBA 0.1 mg / L, and also needs to add 30 g / L sucrose, 5 g / L agar, and adjust the pH to 5.8; the light intensity for culture is 1700 lx, and the light time is 12 hours / day.

[0092] The Cibotium barometz extract is prepared by: washing fresh Cibotium barometz leaves, adding them to a blender at a mass ratio of Cibotium barometz leaves to sterile water of 1:10 and blending them into a slurry, transferring the slurry to a centrifuge to obtain the supernatant, and then sterilizing the supernatant using pasteurization (80°C, 30 minutes) to obtain the Cibotium barometz extract.

[0093] The results showed that the differentiation of group 7 was the best. Therefore, the optimal differentiation medium was: modified 1 / 2MS + 6-BA 0.1 mg / L + GA3 0.1 mg / L + IBA 0.1 mg / L + Cibotium barometz extract 3 g / L; the optimal temperature conditions were: early differentiation: 20℃; mid-differentiation: 28℃; early differentiation: 25℃.

[0094] IV. Proliferation Culture

[0095] The young sporophytes obtained from subculture were placed in the proliferation medium shown in Table 4 below for 30 days of proliferation culture at a temperature of 25℃, a light intensity of 1700 lx, and a light duration of 12 hours / day.

[0096] Table 4. Various proliferation culture media

[0097]

[0098] Note: In the above culture medium preparation, 30 g / L sucrose and 5 g / L agar should also be added, and the pH should be adjusted to 5.8.

[0099] The results showed that group 7 had the highest proliferation rate, reaching 5.3. Group 1 had yellowish leaves, while group 7 was normal. The modified 1 / 2 MS medium used in group 7 had a modified KNO3 concentration of 3640 mg / L and an NH4NO3 concentration of 9500 mg / L, with other components remaining unchanged. Therefore, the optimal proliferation medium composition should be: modified 1 / 2 MS + 6-BA 0.1 mg / L + GA3 0.1 mg / L + IBA 0.1 mg / L.

[0100] V. Rooting Culture

[0101] The sporophytes obtained from the proliferation culture were placed in the rooting medium in Table 5 below for rooting culture for 30 days. The culture temperature was 25℃, the light intensity was 1700lx, and the light duration was 12 hours / day.

[0102] Table 5 Rooting Culture Medium

[0103] 1 1 / 2MS + NAA 0.1mg / well-developed root system 2 1 / 2MS + NAA 1 mg / L well-developed root system 3 1 / 2MS + NAA 3mg / L Long root system 4 1 / 2MS + IBA 0.1 mg / L well-developed root system 5 1 / 2MS + IBA 1 mg / L well-developed root system 6 1 / 2MS + IBA 3mg / L Long root system 7 Improved 1 / 2MS+6-BA0.1mg / L+GA3 0.1mg / L+IBA0.1mg / L well-developed root system

[0104] Note: The above culture medium ratio also requires the addition of 30 g / L sucrose, 5 g / L agar, and pH adjustment to 5.8.

[0105] The results showed that all groups could root, with groups 3 and 6 having longer root systems, which was not conducive to transplanting. Group 7 showed better differentiation and proliferation, with a well-developed root system during the rooting process. Considering all factors, the optimal culture medium ratio was modified 1 / 2 MS + 6-BA 0.1 mg / L + GA3 0.1 mg / L + IBA 0.1 mg / L. This group involved a modified 1 / 2 MS medium, with the modified formulation containing KNO3 3640 mg / L and NH4NO3 9500 mg / L, while other components remained unchanged.

[0106] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. It can be applied to various fields suitable for the present invention. Further modifications can be readily implemented by those skilled in the art.

Claims

1. A method for tissue culture of Cibotium barometz, characterized in that, include: After germination, *Cibotium barometz* seedlings were obtained through differentiation culture, proliferation culture, and rooting culture; the differentiation culture, proliferation culture, and rooting culture all used the same culture medium for differentiation, proliferation, and rooting culture of *Cibotium barometz*. The common culture medium composition was: modified 1 / 2 MS + 6-BA 0.1 mg / L + GA3 0.1 mg / L + IBA 0.1 mg / L, with the addition of 30 g / L sucrose and 5 g / L agar; the modified 1 / 2 MS contained 3640 mg / L KNO3 and 9500 mg / L NH4NO3, while other components remained unchanged. The differentiation culture process was set into three stages: early differentiation: the culture temperature was 20℃ from day 1 to day 10; mid-differentiation: the culture temperature was 28℃ from day 11 to day 20; late differentiation: the culture temperature was 25℃ from day 21 to day 60; the light intensity during the differentiation culture process was 1700 lx and the light duration was 12 hours / day.

2. The method for tissue culture of Cibotium barometz as described in claim 1, characterized in that, The pH of the culture medium used for the differentiation, proliferation, and rooting of Cibotium barometz was adjusted to 5.

8.

3. The method for tissue culture of Cibotium barometz as described in claim 1, characterized in that, The differentiation medium was replaced with: 1-3 g / L of Cibotium barometz extract was added to the common medium; the preparation method of Cibotium barometz extract is as follows: fresh Cibotium barometz leaves were cleaned and added to a blender at a mass ratio of Cibotium barometz leaves: sterile water = 1:10 and crushed into a pulp. The pulp was then transferred to a centrifuge to obtain the supernatant. The supernatant was then sterilized by pasteurization to obtain Cibotium barometz extract.

4. The method for tissue culture of Cibotium barometz as described in claim 3, characterized in that, include: S1: Sterilize mature spores of Cibotium barometz with 8% NaClO solution for 15-25 minutes, and wash with sterile water 4-5 times to obtain sterile explants; S2: Sterile explants were cultured in a lower germination medium for 85-95 days to obtain gametophytes of Cibotium barometz. The germination medium was 1 / 2 MS, with 30 g / L sucrose and 5 g / L agar added, and the pH was adjusted to 5.

8. S3: The obtained gametophytes of Cibotium barometz were placed in a differentiation medium and cultured for 60 days to obtain sporophytes; S4: The differentiated sporophytes were placed in a proliferation medium and cultured for 30 days at a temperature of 25℃, a light intensity of 1700 lx, and a light duration of 12 hours / day. S5: The sporophytes obtained from the proliferation culture were placed in the rooting medium and cultured for 25-35 days to obtain Cibotium barometz seedlings. The culture temperature was 25℃, the light intensity was 1700lx, and the light duration was 12 hours / day. The components of the differentiation medium, proliferation medium, and rooting medium are the same as those of the common medium for the differentiation, proliferation, and rooting culture of Cibotium barometz.

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