Tissue culture method of bistort rhizome

Abstract

The invention relates to the technical field of plant tissue culture, in particular to a tissue culture method of bistort rhizome. According to the method, a stem segment with stem leaves of a bistort mother plant is selected as an explant for tissue culture of the bistort, and a suitable culture medium and culture method for inducing germination of stem segment bud points, proliferation of adventitious buds and rooting of the adventitious buds are developed for the explant. By utilizing the bistort rhizome tissue culture method, the induction success rate of adventitious buds of the bistort rhizome can be effectively increased, the induction period is shortened, and the multiplication coefficient and rooting rate of the bistort rhizome are increased, so that rapid propagation of the bistort rhizome is realized, and the application of the bistort rhizome is promoted.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, in particular to a tissue culture method of Polygonum multiflorum. Background Art

[0002] Polygonum bistorta L. is a perennial herb belonging to the Polygonaceae family. The dried rhizome of Polygonum bistorta is used as a traditional Chinese medicine, which has the effects of clearing away heat and detoxifying, reducing swelling and stopping bleeding. The flowering period of Polygonum bistorta is from June to July. The racemes are spike-shaped, and the flowers are pink or white. They can be used for viewing, and can also be planted in the ground or dried for appreciation.

[0003] Due to the multiple uses of Polygonum multiflorum, in order to speed up the large-scale production of this variety, it is necessary to establish the tissue culture rapid propagation technology of Polygonum multiflorum to provide an effective method for the further development and utilization of this variety. Summary of the invention

[0004] The invention provides a tissue culture method of Polygonum multiflorum.

[0005] Specifically, the present invention provides the technical solution described below.

[0006] The present invention provides a tissue culture method of Polygonum multiflorum, which comprises: using a stem segment with cauline leaves of Polygonum multiflorum as an explant, sequentially inducing bud germination of the stem segment, adventitious bud proliferation and adventitious bud rooting; The culture medium used for inducing stem segment bud germination includes the following plant hormone components: 6-BA (6-benzylaminoadenine) 1.0-2.0 mg / L, NAA (naphthaleneacetic acid) 0.2-0.4 mg / L; The culture medium used for adventitious bud proliferation includes the following plant hormone components: 6-BA 2.0-3.0 mg / L, TDZ (thidiazuron) 0.5-1.0 mg / L.

[0007] The selection of appropriate explants has an important impact on the final results and efficiency of tissue culture. The present invention finds that for Bistoria oxyphylla, using stem segments with cauline leaves as explants for tissue culture can significantly improve the induction success rate, which has obvious advantages over using leaves and other parts as explants.

[0008] The stem segments with cauline leaves mentioned above are preferably derived from a mother plant of Polygonum multiflorum that is healthy and free of diseases and insect pests.

[0009] In view of the selection of the above explants, the present invention optimizes the hormone components added to the culture medium used in each step of tissue culture. For the culture medium used to induce stem segment bud germination, the present invention finds that by adding the above specific concentrations of 6-BA and NAA at the same time, the two hormones can work together to significantly improve the induction rate of bud germination, while shortening the time required for induction, and facilitating the improvement of the proliferation coefficient in the adventitious bud proliferation stage. For the culture medium used for adventitious bud proliferation, the present invention finds that the above specific concentrations of 6-BA and TDZ can work together well to jointly improve the proliferation coefficient of adventitious buds.

[0010] For the culture medium used to induce adventitious bud rooting, it is preferred not to add plant hormones. The present invention finds that the clustered bud plants induced under the above-mentioned appropriate culture medium in the induction of stem segment bud germination and adventitious bud proliferation stage are strong, and in the induction stage of rooting, 100% rooting rate can be obtained without adding plant hormones to the rooting culture medium, which can reduce the effect of hormones on the survival rate of subsequent tissue culture seedling transplanting. Of course, if the cost of plant hormones and the adverse effects of adding hormones in the rooting stage on the survival rate of subsequent tissue culture seedling transplanting are not considered, plant hormones can also be added to the culture medium used to induce adventitious bud rooting, such as adding IBA, and the added concentration is greater than 0 and less than or equal to 0.2 mg / L.

[0011] For the above culture medium, the culture medium used for inducing bud germination of stem segments, the culture medium used for adventitious bud proliferation and the culture medium used for inducing adventitious bud rooting all further include sugar and agar.

[0012] The culture medium used to induce rooting of adventitious buds also includes activated carbon, which can absorb the metabolic products of adventitious buds and enhance the permeability of the culture medium.

[0013] Preferably, the culture medium used for inducing stem segment bud germination uses MS culture medium as the basic culture medium and includes the following components: 6-BA 1.0-2.0 mg / L, NAA 0.2-0.4 mg / L, sucrose 25-30 g / L, and agar 4-5 g / L.

[0014] Preferably, the culture medium used for the adventitious bud proliferation uses MS medium as the basic culture medium and includes the following components: 6-BA 2.0-3.0 mg / L, TDZ 0.5-1.0 mg / L, sucrose 25-30 g / L, and agar 4-5 g / L.

[0015] Adopting the above-mentioned specific culture medium as the culture medium for the induction stem segment bud germination culture stage and the adventitious bud proliferation culture stage can make the two culture stages well connected and coordinated, effectively improve the differentiation of adventitious buds, and improve the proliferation efficiency of adventitious buds at the same time. Among them, in the culture medium for inducing stem segment bud germination, 6-BA and NAA act in coordination as inducing hormones, which can better promote the proliferation and expansion of cells, induce explant dedifferentiation and adventitious bud germination. Compared with the induction culture medium using NAA alone, the culture medium for inducing stem segment bud germination can effectively improve the differentiation of adventitious buds, thereby improving the breeding efficiency of Polygonum multiflorum. In the culture medium for inducing adventitious bud proliferation, adding two mitogens, 6-BA and TDZ, for use in combination, significantly improves the proliferation rate, and has little effect on the growth of adventitious buds.

[0016] In some embodiments of the present invention, the culture medium used for inducing stem segment bud germination is obtained by adding the following components to MS culture medium: 6-BA 1.0-2.0 mg / L, NAA 0.2-0.4 mg / L, sucrose 25-30 g / L, and agar 4-5 g / L.

[0017] In some embodiments of the present invention, the culture medium used for adventitious bud proliferation is obtained by adding the following components to MS culture medium: 6-BA 2.0-3.0 mg / L, TDZ 0.5-1.0 mg / L, sucrose 25-30 g / L, and agar 4-5 g / L.

[0018] Preferably, the culture medium used for inducing adventitious bud rooting uses 1 / 2 MS culture medium as the basic culture medium and includes the following components: 0.3-0.5 g / L activated carbon, 25-30 g / L sucrose, and 4-5 g / L agar.

[0019] In combination with the above-mentioned induction of stem segment bud germination culture and adventitious bud proliferation culture, the obtained adventitious buds can promote rooting and form seedlings under the joint action of the above-mentioned adventitious bud rooting induction medium and various hormones synthesized by themselves.

[0020] In some embodiments of the present invention, the culture medium used for inducing adventitious bud rooting is obtained by adding 0.3-0.5 g / L activated carbon, 25-30 g / L sucrose and 4-4.5 g / L agar to 1 / 2 MS culture medium.

[0021] The pH of the culture medium used for inducing stem segment bud germination, the culture medium used for adventitious bud proliferation and the culture medium used for inducing adventitious bud rooting described above is preferably 5.8-6.0.

[0022] In the above tissue culture method, the culture temperature for inducing stem segment bud germination, adventitious bud proliferation and inducing adventitious bud rooting is 23-27°C, and the light duration is 8-10h / d.

[0023] Among them, the light intensity for inducing stem bud germination and adventitious bud proliferation is 1500-2300 Lux, and the light intensity for inducing adventitious bud rooting is 2000-2500 Lux.

[0024] In the tissue culture method described above, the explant is disinfected before inducing stem segment buds to germinate; the disinfection treatment includes: first disinfection with 70%-80% alcohol, and then disinfection with 0.08%-0.12% mercuric chloride solution.

[0025] Preferably, the disinfection treatment includes: first disinfection treatment with 75% alcohol, and then disinfection treatment with 0.1% mercuric chloride solution.

[0026] The above disinfection treatment is preferably an immersion and shaking treatment.

[0027] Preferably, the alcohol disinfection treatment time is 30-60 seconds; the mercuric chloride solution disinfection treatment time is 6-10 minutes.

[0028] Preferably, the explants are rinsed with water before the alcohol disinfection treatment.

[0029] Preferably, after the alcohol disinfection and the mercuric chloride solution treatment, the explants are washed with sterile water.

[0030] After disinfection, the stem segments with cauline leaves were cut into small segments with buds of 1-2 cm in length to obtain explants to be induced.

[0031] In some embodiments of the present invention, a tissue culture method of Polygonum multiflorum is provided, the method comprising: obtaining and sterilizing explants, inducing bud germination of stem segments, multiplying adventitious buds, and inducing rooting of adventitious buds.

[0032] The overall process of the tissue culture method of the bistort provided by the present invention is as follows: after the stem segment with cauline leaves taken from a bistort mother plant that is healthy and free of pests and diseases is disinfected, the stem segment with cauline leaves is induced using a culture medium for inducing bud germination of the stem segment, and adventitious buds of the stem segment are induced to germinate under a suitable culture environment. The induced adventitious buds are peeled off from the stem segment, and then the adventitious buds are induced using a culture medium for adventitious bud proliferation, and under a suitable culture environment, the adventitious buds proliferate to form clustered buds. The clustered buds are separated into individual plants, and rooting induction is performed on the individual plants on a culture medium for inducing rooting of the adventitious buds to form rooted seedlings.

[0033] On the other hand, the present invention also provides a culture medium for tissue culture of Polygonum multiflorum, the culture medium comprising a stem segment bud germination induction culture medium, an adventitious bud proliferation culture medium and an adventitious bud rooting induction culture medium; The stem segment bud germination induction medium uses MS medium as the basic medium and includes the following components: 6-BA 1.0-2.0 mg / L, NAA 0.2-0.4 mg / L, sucrose 25-30 g / L, agar 4-5 g / L; The adventitious bud proliferation medium uses MS medium as the basic medium and includes the following components: 6-BA 2.0-3.0 mg / L, TDZ 0.5-1.0 mg / L, sucrose 25-30 g / L, and agar 4-5 g / L; The adventitious bud rooting induction medium uses 1 / 2 MS medium as the basic medium and includes the following components: 0.3-0.5 g / L activated carbon, 25-30 g / L sucrose, and 4-5 g / L agar.

[0034] The present invention also provides application of the above-mentioned culture medium for Polygonum multiflorum tissue culture in Polygonum multiflorum tissue culture.

[0035] Preferably, the application is to carry out tissue culture using the stem segments of Polygonum multiflorum with cauline leaves as explants.

[0036] The beneficial effect of the present invention is that the present invention selects the stem segment with cauline leaves of the mother plant of the Polygonum multiflorum as an explant for tissue culture of the Polygonum multiflorum, and develops a suitable culture medium and culture method for inducing bud germination, adventitious bud proliferation and adventitious bud rooting of the explant. The Polygonum multiflorum tissue culture method of the present invention can effectively improve the induction success rate of the Polygonum multiflorum adventitious buds, shorten the induction period, improve the proliferation coefficient and rooting rate of the Polygonum multiflorum, thereby realizing the rapid reproduction of the Polygonum multiflorum and promoting the application of the Polygonum multiflorum. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0038] Figure 1 It is a schematic diagram of inducing bud germination in stem segments during tissue culture of Polygonum multiflorum using the tissue culture method of Example 2.

[0039] Figure 2 It is a schematic diagram of the proliferation and differentiation of adventitious buds to form clustered adventitious buds during the tissue culture process of Polygonum multiflorum using the tissue culture method of Example 2.

[0040] Figure 3 It is a schematic diagram of adventitious bud rooting during the tissue culture process of Polygonum multiflorum using the tissue culture method of Example 2. DETAILED DESCRIPTION

[0041] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0042] The alcohol used in the following examples is Ojie 75% disinfectant alcohol purchased from Hangzhou Outuopu Biotechnology Co., Ltd.; mercuric chloride is a standard solution of mercuric chloride purchased from Tianjin Guangfu Fine Chemical Research Institute.

[0043] The mother plants of Polygonum multiflorum used in the following examples were sourced from Beijing Huaxiang Flower Science and Technology Research Institute Co., Ltd.

[0044] The present invention provides a tissue culture method of Polygonum multiflorum, which specifically comprises the following steps: 1. Obtaining and sterilizing explants (1) Obtaining explants: Select mother plants that are healthy and free of pests and diseases, and select stem segments with cauline leaves; (2) Disinfection of explants: Rinse: rinse the stem segment with cauline leaves obtained in step (1) under running tap water for 10 minutes and dry; Alcohol disinfection: After drying, soak and shake in 75% alcohol for 30-60 seconds, then wash with sterile water 1-2 times, each time for 1-2 minutes; 0.1% mercuric chloride solution treatment: Place the stem segments with cauline leaves washed in the previous step in 0.1% mercuric chloride solution and soak and shake for 6-10 minutes, then wash with sterile water for 3-5 times, each time for 1-2 minutes; Cutting: Cut off the damaged part at the base of the disinfected stem segment, and then divide it into small segments with buds of 1-2 cm to obtain the explants to be induced.

[0045] 2. Induce stem segment bud germination The explants to be induced obtained in step 1 are vertically inserted into a culture medium for inducing stem segment bud germination, so as to induce the stem segment bud germination and obtain adventitious buds.

[0046] Among them, the culture medium formula used in this step is: MS medium, 6-BA 1.0-2.0 mg / L, NAA 0.2-0.4 mg / L, sucrose 25-30 g / L, agar 4-5 g / L, pH 5.8-6.0; the culture temperature is 23-27°C, the light intensity is 1500-2300 Lux, and the lighting time is 8-10h / d.

[0047] 3. Adventitious bud proliferation The adventitious buds obtained in step 2 are inoculated into a culture medium for inducing adventitious bud proliferation, and clustered buds are obtained after proliferation in 4-5 weeks.

[0048] Among them, the culture medium formula used in this step is: MS medium, 6-BA 2.0-3.0 mg / L, TDZ 0.5-1.0 mg / L, sucrose 25-30 g / L, agar 4-5 g / L, pH 5.8-6.0; the culture temperature is 23-27°C, the light intensity is 1500-2300 Lux, and the light duration is 8-10h / d.

[0049] 4. Induce rooting of adventitious buds The clustered shoots obtained in step 3 are separated into individual shoots and inoculated into a culture medium for inducing rooting of adventitious shoots.

[0050] Among them, the culture medium formula used in this step is: 1 / 2 MS culture medium, activated carbon (AC) 0.3-0.5 g / L, sucrose 25-30 g / L, agar 4-5 g / L, pH 5.8-6.0; the culture temperature is 23-27°C, the light intensity is 2000-2500 Lux, and the lighting time is 8-10 h / d.

[0051] The following is combined with Examples 1-3, Comparative Examples 1-4, Figure 1-3 And detection experiments are further described in detail the technical solution of the present invention.

[0052] Example 1 This embodiment provides a tissue culture method of Polygonum multiflorum, which specifically comprises the following steps: 1. Obtaining and sterilizing explants (1) Obtaining explants: Select a mother plant that is healthy and free of pests and diseases, and select the stem segment with cauline leaves from the mother plant; (2) Disinfection of explants: Rinse: rinse the stem segment with cauline leaves obtained in step (1) under running tap water for 10 minutes and dry; Alcohol disinfection: After drying, place the stem segment with stem leaves in 75% alcohol and shake for 30 seconds, then shake and wash it with sterile water once, each time for 1 minute; 0.1% mercuric chloride treatment: Place the stem segments with cauline leaves washed in the previous step in a 0.1% mercuric chloride solution and immerse and shake for 6 minutes, then wash with sterile water for 3 times, 1 minute each time; Cutting: Cut off the damaged part at the base of the disinfected stem segment, and then divide it into small segments with buds of 1 cm to obtain the explants to be induced.

[0053] 2. Induce stem segment bud germination The explants to be induced obtained in step 1 are inoculated into a culture medium for inducing stem segment bud germination, and the stem segment bud germination is induced, and adventitious buds are obtained after 3 weeks.

[0054] 3. Adventitious bud proliferation The adventitious buds obtained in step 2 are inoculated into a culture medium for inducing adventitious bud proliferation, and proliferating clustered buds are obtained after 3 weeks.

[0055] 4. Induce rooting of adventitious buds The clustered buds obtained in step 3 are separated from each other to obtain individual adventitious buds, and the adventitious buds are inoculated into a culture medium for inducing rooting of the adventitious buds.

[0056] The condition parameters involved in the above steps and the culture medium formula used are shown in Table 1.

[0057] Table 1 Parameters and culture medium formula used in each step of the method of Examples 1-3 and Comparative Examples 1-4

[0058] Example 2 This embodiment provides a tissue culture method of Polygonum multiflorum, which has the same steps as those of Embodiment 1, except for the condition parameters of some steps and the medium formula used, as shown in Table 1. Except for the condition parameters and medium listed in Table 1, the method steps of Embodiment 2 are the same as those of Embodiment 1.

[0059] Example 3 This embodiment provides a tissue culture method of Polygonum multiflorum, which is the same as the step process of Example 1, except that the condition parameters of some steps and the medium formula used are shown in Table 1. Except for the condition parameters and medium listed in Table 1, the method steps of Example 3 are the same as those of Example 1.

[0060] Comparative Examples 1-3 Comparative Examples 1-3 respectively provide a method for tissue culture of Polygonum multiflorum, which differs from Example 1 in the condition parameters of some steps and the medium formula used, as shown in Table 1. Except for the condition parameters and medium listed in Table 1, the method steps of Comparative Examples 1-3 are the same as those of Example 1.

[0061] Comparative Example 4 Comparative Example 4 provides a tissue culture method for Polygonum multiflorum, which differs from Example 1 in the selection of explants, as shown in Table 1. Except for the condition parameters listed in Table 1, the method steps of Comparative Example 4 are the same as those of Example 1.

[0062] Experimental example The Bistorta was cultured using the tissue culture methods provided in Examples 1-3 and Comparative Examples 1-4. During the culture, the contamination rate, browning mortality rate, and investigation parameters of each culture stage in the explant disinfection stage were recorded, and the adventitious bud induction rate, adventitious bud proliferation coefficient, and adventitious bud rooting rate were calculated. The specific test item results are shown in Table 2. At the same time, the adventitious bud induction and the growth of differentiated seedlings during the Bistorta culture process were recorded. The specific recorded results are shown in Table 3. Among them, the schematic diagram of inducing stem segment bud germination during the Bistorta tissue culture process using the tissue culture method of Example 2 is shown in Figure 1 The schematic diagram of adventitious bud proliferation and differentiation to form clustered adventitious buds is shown in Figure 2 The schematic diagram of adventitious bud rooting is shown in Figure 3 shown.

[0063] Table 2 Results of tissue culture of Polygonum multiflorum (1)

[0064] Table 3 Results of tissue culture of Polygonum multiflorum (2)

[0065] In combination with the tissue culture methods of Examples 1-3 and Comparative Examples 1-4 and the results of Tables 2 and 3, the present invention selects stem segments with cauline leaves on the mother plant as explants, and selects appropriate culture medium formulas and culture conditions in each tissue culture stage to carry out tissue culture of Polygonum multiflorum, thereby shortening the induction period and increasing the proliferation rate of adventitious buds. At the same time, Polygonum multiflorum has a good rooting effect, which is beneficial to the survival of transplantation in the later stage, thereby accelerating the market application of Polygonum multiflorum.

[0066] First, selecting a suitable part of the mother plant as an explant for the tissue culture of the ginseng directly affects the final result of the tissue culture of the ginseng. Comparing Example 1 and Comparative Example 4, under the same tissue culture conditions and the requirement of no pests and diseases, the explant selected in Example 1 is a stem segment with a cauline leaf, while the explant selected in Comparative Example 4 is a leaf. It can be seen from Tables 2 and 3 that the adventitious buds are induced by the tissue culture method of Example 1, and the adventitious buds sprout normally, grow rapidly, and there is no vitrification phenomenon. The differentiated seedlings grow well, the plants thrive, and the proliferation rate is high; while the results of the tissue culture method of Comparative Example 4 are: using the leaf as an explant, although callus tissue is formed, the callus tissue is not finally differentiated into adventitious buds. In summary, the selection of explants has a great influence on the effect of the tissue culture of the ginseng, and the selection of a suitable part of the mother plant can determine the success of the early induction. The present invention selects a stem segment with a cauline leaf as an explant for the tissue culture of the ginseng, which can effectively improve the induction rate and proliferation coefficient of the ginseng and reduce the tissue culture production cost of the ginseng.

[0067] Furthermore, in the step of inducing the bud germination of stem segments, the addition of mitogen 6-BA also has a great effect on the induction of buds. In Comparative Example 1, only auxin NAA is added, and mitogen 6-BA is not added. It can be seen from Tables 2 and 3 that although the adventitious buds of Comparative Example 1 can sprout normally in the induction stage, the proliferation rate in the proliferation stage is significantly reduced. It can be seen that in the step of inducing the bud germination of stem segments, the addition of mitogen 6-BA in the first step of inducing the bud germination helps to increase the proliferation rate of the adventitious buds in the second step.

[0068] Furthermore, the selection of plant hormones in the adventitious bud proliferation stage will also affect the results of the ginseng tissue culture. It can be seen from Tables 1, 2 and 3 that compared with Comparative Example 2, Example 2 selects two mitogens 6-BA and TDZ for use in combination, which significantly improves the proliferation rate and has little effect on the growth of adventitious buds. It can be seen that under the premise that the leaves of the clustered buds formed by adventitious buds are normal and the plants are strong, in the step of inducing adventitious bud proliferation, the proliferation rate can be improved by the combination of two mitogens 6-BA and TDZ.

[0069] In addition, the tissue culture method of the present invention induces a robust bud plant under a suitable medium formula for adventitious bud germination and proliferation. In the rooting stage of inducing the plant, a 100% rooting rate can be obtained without adding plant hormones in the rooting medium, thereby reducing the adverse effects of hormones on the survival rate of transplanted tissue culture seedlings. As shown in Tables 1 and 3, comparative example 4 adds auxin IBA to the rooting medium, and compared with Example 1 without adding hormones, there is no significant difference, and the rooting rate can reach 100%. In the rooting stage of tissue culture seedlings, the design of a rooting medium without adding activated carbon and hormones can not only save production costs, but also reduce the hormone content in the tissue culture seedlings, and enhance the adaptability of the tissue culture seedlings to the external environment after transplanting.

[0070] In summary, in the tissue culture method of Polygonum multiflorum provided by the present invention, the explant is selected from the stem segment with cauline leaves, and a suitable culture medium for cultivation at each stage is used to induce adventitious buds to germinate and form clustered buds. The cycle is short and the proliferation coefficient is high. At the same time, the rooting effect of Polygonum multiflorum is good, and the rooting rate reaches 100%, which improves the transplant survival rate of Polygonum multiflorum, thereby facilitating the market application of Polygonum multiflorum.

[0071] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for tissue culture of Polygonum multiflorum, characterized in that: The method comprises: using the stem segment with cauline leaves of the Polygonum multiflorum as an explant, sequentially inducing bud germination of the stem segment, adventitious bud proliferation and adventitious bud rooting; The culture medium used for inducing stem segment bud germination includes the following plant hormone components: 6-BA 1.0-2.0 mg / L, NAA 0.2-0.4 mg / L; The culture medium used for adventitious bud proliferation includes the following plant hormone components: 6-BA 2.0-3.0 mg / L, TDZ 0.5-1.0 mg / L.

2. The tissue culture method of Polygonum multiflorum according to claim 1, characterized in that: The culture medium used for inducing bud germination of stem segments, the culture medium used for adventitious bud proliferation and the culture medium used for inducing adventitious bud rooting all further include sugar and agar.

3. The tissue culture method of Polygonum multiflorum according to claim 2, characterized in that: The culture medium used to induce rooting of adventitious buds also includes activated carbon.

4. The method for tissue culture of Polygonum multiflorum according to any one of claims 1 to 3, characterized in that: The culture medium used for inducing stem segment bud germination uses MS culture medium as the basic culture medium and includes the following components: 6-BA 1.0-2.0 mg / L, NAA 0.2-0.4 mg / L, sucrose 25-30 g / L, and agar 4-5 g / L; And / or, the culture medium used for the adventitious bud proliferation uses MS culture medium as the basic culture medium and comprises the following components: 6-BA 2.0-3.0 mg / L, TDZ 0.5-1.0 mg / L, sucrose 25-30 g / L, agar 4-5 g / L; And / or, the culture medium used for inducing adventitious bud rooting uses 1 / 2 MS culture medium as the basic culture medium and includes the following components: 0.3-0.5 g / L activated carbon, 25-30 g / L sucrose, and 4-5 g / L agar.

5. The method for tissue culture of Polygonum multiflorum according to any one of claims 1 to 3, characterized in that: The pH of the culture medium is 5.8-6.

0.

6. The method for tissue culture of Polygonum multiflorum according to any one of claims 1 to 3, characterized in that: The culture temperature for inducing stem segment bud germination, adventitious bud proliferation and adventitious bud rooting is 23-27°C, and the light duration is 8-10h / d; And / or, the light intensity for inducing stem segment bud germination and adventitious bud proliferation is 1500-2300 Lux, and the light intensity for inducing adventitious bud rooting is 2000-2500 Lux.

7. The method for tissue culture of Polygonum multiflorum according to any one of claims 1 to 3, characterized in that: Before inducing the buds of the stem segments to germinate, the explants were sterilized; The disinfection treatment includes: first disinfection treatment with 70%-80% alcohol, and then disinfection treatment with 0.08%-0.12% mercuric chloride solution.

8. The method for tissue culture of Polygonum multiflorum according to claim 7, characterized in that: The alcohol disinfection treatment time is 30-60 seconds; the mercuric chloride solution disinfection treatment time is 6-10 minutes.

9. A culture medium for tissue culture of Polygonum multiflorum, characterized in that: The culture medium includes a stem segment bud germination induction culture medium, an adventitious bud proliferation culture medium and an adventitious bud rooting induction culture medium; The inducing stem segment bud germination medium uses MS medium as the basic medium and includes the following components: 6-BA 1.0-2.0 mg / L, NAA 0.2-0.4 mg / L, sucrose 25-30 g / L, agar 4-5 g / L; The adventitious bud proliferation medium uses MS medium as the basic medium and includes the following components: 6-BA 2.0-3.0 mg / L, TDZ 0.5-1.0 mg / L, sucrose 25-30 g / L, and agar 4-5 g / L; The adventitious bud rooting induction medium uses 1 / 2 MS medium as the basic medium and includes the following components: 0.3-0.5 g / L activated carbon, 25-30 g / L sucrose, and 4-5 g / L agar.

10. Use of the culture medium according to claim 9 in the tissue culture of Polygonum multiflorum.

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