A tissue culture method for Bistorta vivipara

Abstract

The present invention relates to the technical field of plant tissue culture, and particularly to a tissue culture method for Bistorta officinalis. The present invention selects the stem segment with cauline leaves of the Bistorta officinalis mother plant as the explant for the tissue culture of Bistorta officinalis, and develops suitable culture media and culture methods for inducing the germination of stem segment bud points, the proliferation of adventitious buds, and the rooting of adventitious buds for this explant. By using the tissue culture method for Bistorta officinalis of the present invention, the induction success rate of adventitious buds of Bistorta officinalis can be effectively improved, the induction period can be shortened, the proliferation coefficient and rooting rate of Bistorta officinalis can be increased, so as to realize the rapid propagation of Bistorta officinalis and promote the application of Bistorta officinalis.

Description

Technical Field

[0001] The present invention relates to the technical field of plant tissue culture, and in particular to a tissue culture method for Bistorta officinalis Background Art

[0002] Bistorta officinalis, belonging to the genus Bistorta of the family Polygonaceae, is a perennial herb. The dried rhizome of Bistorta officinalis is used as traditional Chinese medicine, which has the effects of clearing heat and detoxifying, detumescence and hemostasis. The flowering period of Bistorta officinalis is from June to July, the raceme is spike-shaped, and the flower color is pink or white, which can be used for ornamental purposes, and can also be used for ground planting and dried flower appreciation

[0003] Due to the various uses of Bistorta officinalis, in order to accelerate the large-scale production of this variety, it is necessary to establish a tissue culture and rapid propagation technology for Bistorta officinalis to provide an effective method for the further development and utilization of this variety Summary of the Invention

[0004] The present invention provides a tissue culture method for Bistorta officinalis

[0005] Specifically, the present invention provides the following technical solutions

[0006] The present invention provides a tissue culture method for Bistorta officinalis, the method comprising: using the stem segment with cauline leaves of Bistorta officinalis as the explant, and successively inducing the germination of stem segment bud points, the proliferation of adventitious buds and the rooting of adventitious buds

[0007] Among them, the culture medium used for inducing the germination of stem segment bud points includes the following phytohormone components: 6-BA (6-benzylaminopurine) 1.0-2.0 mg / L, NAA (naphthaleneacetic acid) 0.2-0.4 mg / L

[0008] The culture medium used for adventitious bud proliferation includes the following phytohormone components: 6-BA 2.0-3.0 mg / L, TDZ (thidiazuron) 0.5-1.0 mg / L

[0009] Selecting a suitable explant has an important impact on the final result and efficiency of tissue culture. The present invention finds that for Bistorta officinalis, using the stem segment with cauline leaves as the explant for tissue culture can significantly improve the induction success rate, and has obvious advantages compared with using parts such as leaves as the explant

[0010] The above-mentioned stem segment with cauline leaves is preferably derived from a healthy Bistorta officinalis mother plant without diseases and pests

[0011] Regarding the selection of the above explants, the present invention optimizes the hormone components added to the culture media used in each step of tissue culture. For the culture medium used to induce the germination of bud points on stem segments, the present invention finds that by simultaneously adding the above specific concentrations of 6-BA and NAA, the two hormones can cooperate to significantly increase the induction rate of bud point germination, shorten the induction time required, and facilitate the increase in the multiplication coefficient during 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 cooperate well to jointly increase the multiplication coefficient of adventitious buds.

[0012] For the culture medium used to induce the rooting of adventitious buds, it is preferably without adding plant hormones. The present invention finds that the cluster buds induced under the appropriate culture media in the above-mentioned stages of inducing the germination of bud points on stem segments and adventitious bud proliferation are robust. During the stage of inducing their rooting, a 100% rooting rate can be obtained without adding plant hormones to the rooting medium, which can reduce the impact of hormones on the survival rate of subsequent tissue-cultured seedlings during transplantation. Of course, if the cost of plant hormones is not considered and the possible adverse effects of adding hormones during the rooting stage on the survival rate of subsequent tissue-cultured seedlings during transplantation are not considered, plant hormones can also be added to the culture medium used to induce the rooting of adventitious buds, for example, adding IBA, and the added concentration is greater than 0 and less than or equal to 0.2 mg / L.

[0013] For the above culture media, the culture medium used to induce the germination of bud points on stem segments, the culture medium used for adventitious bud proliferation, and the culture medium used to induce the rooting of adventitious buds all further include sugar and agar.

[0014] Among them, the culture medium used to induce the rooting of adventitious buds further includes activated carbon. Activated carbon can adsorb the metabolites of adventitious buds and enhance the permeability of the culture medium.

[0015] Preferably, the culture medium used to induce the germination of bud points on stem segments uses the 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, agar 4 - 5 g / L.

[0016] Preferably, the culture medium used for adventitious bud proliferation uses the MS culture 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, agar 4 - 5 g / L.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] Among them, the light intensity for inducing the germination of stem segment bud points and the proliferation of adventitious buds is 1500 - 2300 Lux, and the light intensity for inducing the rooting of adventitious buds is 2000 - 2500 Lux.

[0026] For the tissue culture method described above, before inducing the germination of stem segment bud points, the explant is disinfected; the disinfection treatment includes: first disinfecting with 70% - 80% alcohol, and then disinfecting with 0.08% - 0.12% mercuric chloride solution.

[0027] Preferably, the disinfection treatment includes: first disinfecting with 75% alcohol, and then disinfecting with 0.1% mercuric chloride solution.

[0028] The above disinfection treatment is preferably soaking and shaking treatment.

[0029] Preferably, the time for alcohol disinfection treatment is 30 - 60 s; the time for mercuric chloride solution disinfection treatment is 6 - 10 min.

[0030] Preferably, before alcohol disinfection treatment, the explant is first rinsed with water.

[0031] Preferably, after alcohol disinfection and after mercuric chloride solution treatment, the explant is rinsed with sterile water.

[0032] After the disinfection is completed, the stem segment with cauline leaves is segmented into small segments with bud points and 1 - 2 cm in length to obtain the explant to be induced.

[0033] In some embodiments of the present invention, a tissue culture method for Bistorta amplexicaulis is provided, and the method includes: obtaining and disinfecting the explant, inducing the germination of stem segment bud points, proliferating adventitious buds, and inducing the rooting of adventitious buds.

[0034] The overall process of the tissue culture method for Bistorta amplexicaulis provided by the present invention is as follows: After disinfecting the stem segment with cauline leaves taken from a healthy and pest - free Bistorta amplexicaulis mother plant, the above - mentioned stem segment with cauline leaves is induced using the medium for inducing the germination of stem segment bud points. Under a suitable culture environment, the adventitious buds of the stem segment are induced to germinate. The induced adventitious buds are separated from the stem segment, and then the adventitious buds are induced using the medium for adventitious bud proliferation. Under a suitable culture environment, the adventitious buds proliferate to form cluster buds. The cluster buds are separated into single plants, and the single plants are induced to root on the medium for inducing the rooting of adventitious buds to form rooted seedlings.

[0035] On the other hand, the present invention also provides a medium for Bistorta amplexicaulis tissue culture, and the medium includes a medium for inducing the germination of stem segment bud points, a medium for adventitious bud proliferation, and a medium for inducing the rooting of adventitious buds;

[0036] Among them, the medium for inducing the germination of shoot buds on the stem segment uses the 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;

[0037] The medium for adventitious bud proliferation uses the 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, agar 4-5 g / L;

[0038] The medium for inducing adventitious bud rooting uses the 1 / 2 MS medium as the basic medium and includes the following components: activated carbon 0.3-0.5 g / L, sucrose 25-30 g / L, agar 4-5 g / L.

[0039] The present invention also provides the application of the above-mentioned medium for polygonum bistorta tissue culture in polygonum bistorta tissue culture.

[0040] Preferably, the application is to use the stem segment with cauline leaves of polygonum bistorta as the explant for tissue culture.

[0041] The beneficial effects of the present invention are as follows: The present invention selects the stem segment with cauline leaves of the polygonum bistorta mother plant as the explant for polygonum bistorta tissue culture, and develops suitable media and culture methods for inducing the germination of shoot buds on the stem segment, adventitious bud proliferation, and inducing adventitious bud rooting for this explant. Using the polygonum bistorta tissue culture method of the present invention, the induction success rate of polygonum bistorta adventitious buds can be effectively improved, the induction period can be shortened, the proliferation coefficient and rooting rate of polygonum bistorta can be increased, so as to realize the rapid propagation of polygonum bistorta and promote the application of polygonum bistorta. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0043] Figure 1 It is a schematic diagram of inducing the germination of shoot buds on the stem segment during the polygonum bistorta tissue culture process using the tissue culture method of Example 2.

[0044] Figure 2 It is a schematic diagram of adventitious bud proliferation and differentiation to form cluster adventitious buds during the polygonum bistorta tissue culture process using the tissue culture method of Example 2.

[0045] Figure 3It is a schematic diagram of adventitious bud rooting during the tissue culture process of Bistorta officinalis using the tissue culture method of Example 2. Detailed implementation mode

[0046] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below with reference to the accompanying drawings in the present invention. Apparently, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without making creative efforts shall fall within the protection scope of the present invention.

[0047] The alcohol used in the following examples is Oujie 75% disinfection alcohol, purchased from Hangzhou Outop Biotechnology Co., Ltd.; mercury chloride is the mercury chloride standard solution purchased from Tianjin Guangfu Fine Chemical Research Institute.

[0048] The Bistorta officinalis mother plants used in the following examples are from Beijing Huaxiang Flower Technology Research Institute Co., Ltd.

[0049] The present invention provides a tissue culture method for Bistorta officinalis, which specifically includes the following steps:

[0050] 1. Acquisition and disinfection of explants

[0051] (1) Acquisition of explants: Select healthy mother plants without diseases and pests, and select stem segments with cauline leaves.

[0052] (2) Disinfection of explants:

[0053] Rinsing: Rinse the stem segments with cauline leaves obtained in step (1) under running tap water for 10 min, and air dry.

[0054] Alcohol disinfection: After air drying, soak and shake in 75% alcohol for 30 - 60 s, and then shake and wash with sterile water for 1 - 2 times, 1 - 2 min each time.

[0055] Treatment with 0.1% mercury chloride solution: Then soak and shake the stem segments with cauline leaves after the above washing in 0.1% mercury chloride solution for 6 - 10 min, and then shake and wash with sterile water for 3 - 5 times, 1 - 2 min each time.

[0056] Trimming: Cut off the damaged parts at the base of the disinfected stem segments, and then divide them into small segments with bud points, 1 - 2 cm in length, to obtain the explants to be induced.

[0057] 2. Inducing the germination of bud points on the stem segments

[0058] Vertically insert the explants to be induced obtained in step 1 into the medium for inducing the germination of bud points on the stem segments to induce the germination of bud points on the stem segments and obtain adventitious buds.

[0059] 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 light duration is 8 - 10 h / d.

[0060] 3. Adventitious bud proliferation

[0061] Inoculate the adventitious buds obtained in step 2 into the culture medium for inducing adventitious bud proliferation, and obtain proliferated clustered buds after 4 - 5 weeks.

[0062] 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 - 10 h / d.

[0063] 4. Inducing adventitious bud rooting

[0064] Separate the clustered buds obtained in step 3 into single buds and inoculate them into the culture for inducing adventitious bud rooting.

[0065] Among them, the culture medium formula used in this step is: 1 / 2 MS 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 light duration is 8 - 10 h / d.

[0066] The following further elaborates on the technical solution of the present invention in combination with Examples 1 - 3, Comparative Examples 1 - 4, Figures 1-3 as well as the detection test.

[0067] Example 1

[0068] This example provides a tissue culture method for Bistorta officinalis, which specifically includes the following steps:

[0069] 1. Acquisition and disinfection of explants

[0070] (1) Acquisition of explants: Select healthy mother plants without diseases and pests, and select the stem segments with cauline leaves on the mother plants;

[0071] (2) Disinfection of explants:

[0072] Rinsing: Rinse the stem segments with cauline leaves obtained in step (1) under running tap water for 10 min, and then air dry them.

[0073] Alcohol disinfection: After air drying, immerse the stem segments with cauline leaves in 75% alcohol and soak and shake for disinfection for 30 s, then wash them by shaking with sterile water once, 1 min each time.

[0074] Treatment with 0.1% mercuric chloride: Immerse the stem segments with cauline leaves after the above washing step in 0.1% mercuric chloride solution and soak and shake for 6 min, then wash them by shaking with sterile water three times, 1 min each time.

[0075] Trimming: Cut off the damaged parts at the base of the disinfected stem segments, and then divide them into small segments with bud points, each 1 cm long, to obtain the explants to be induced.

[0076] 2. Inducing the germination of bud points on the stem segments

[0077] Inoculate the explants to be induced obtained in step 1 into the medium for inducing the germination of bud points on the stem segments to induce the germination of bud points on the stem segments, and adventitious buds can be obtained after 3 weeks.

[0078] 3. Proliferation of adventitious buds

[0079] Inoculate the adventitious buds obtained in step 2 into the medium for inducing the proliferation of adventitious buds, and proliferated cluster buds can be obtained after 3 weeks.

[0080] 4. Inducing the rooting of adventitious buds

[0081] Separate the cluster buds obtained in step 3 from each other to obtain individual adventitious buds, and inoculate the adventitious buds into the medium for inducing the rooting of adventitious buds.

[0082] The condition parameters involved in the above steps and the medium formula used are shown in Table 1.

[0083] Table 1 Parameters and medium formula used in each step of the methods of Examples 1-3 and Comparative Examples 1-4

[0084]

[0085] Example 2

[0086] This example provides a tissue culture method for Bistorta vivipara, which has the same step process as that of Example 1, and the difference lies in the condition parameters of some steps and the medium formula used, as shown in Table 1 specifically. Except for the condition parameters and medium listed in Table 1, the method steps of Example 2 are the same as those of Example 1.

[0087] Example 3

[0088] This example provides a tissue culture method for bistorta officinalis, which has the same step process as that in Example 1. The differences lie in the conditional parameters of some steps and the culture medium formula used, as shown in Table 1 specifically. Except for the conditional parameters and culture medium listed in Table 1, the method steps of Example 3 are the same as those of Example 1.

[0089] Comparative Examples 1 - 3

[0090] Comparative Examples 1 - 3 respectively provide a tissue culture method for bistorta officinalis. The differences from Example 1 lie in the conditional parameters of some steps and the culture medium formula used, as shown in Table 1 specifically. Except for the conditional parameters and culture medium listed in Table 1, the method steps of Comparative Examples 1 - 3 are the same as those of Example 1.

[0091] Comparative Example 4

[0092] Comparative Example 4 provides a tissue culture method for bistorta officinalis. The difference from Example 1 lies in the selection of explants, as shown in Table 1 specifically. Except for the conditional parameters listed in Table 1, the method steps of Comparative Example 4 are the same as those of Example 1.

[0093] Experimental Example

[0094] The tissue culture methods provided by Examples 1 - 3 and Comparative Examples 1 - 4 are used to culture bistorta officinalis. During the process, the contamination rate, browning mortality rate in the explant disinfection stage, and the inspection parameters in each culture stage are recorded, and the adventitious bud induction rate, adventitious bud proliferation coefficient, and adventitious bud rooting rate are calculated. The results of specific detection items are shown in Table 2. At the same time, the adventitious bud induction situation and the growth trend of differentiated seedlings during the bistorta officinalis culture process are recorded, and the specific recording results are shown in Table 3. Among them, during the tissue culture of bistorta officinalis using the tissue culture method of Example 2, the schematic diagram of inducing the germination of stem segment bud points is as Figure 1 shown, the schematic diagram of adventitious bud proliferation and differentiation to form cluster adventitious buds is as Figure 2 shown, and the schematic diagram of adventitious bud rooting is as Figure 3 shown.

[0095] Table 2 Results of the Tissue Culture of Bistorta Officinalis (1)

[0096]

[0097] Table 3 Results of the Tissue Culture of Bistorta Officinalis (2)

[0098]

[0099] Combined with the tissue culture methods of Examples 1 - 3 and Comparative Examples 1 - 4 and the results in Tables 2 and 3, the present invention selects the stem segment with cauline leaves on the mother plant as the explant, and at the same time selects appropriate culture medium formulations and culture conditions at each tissue culture stage to conduct the tissue culture of Bistorta officinalis, shortening the induction period, increasing the multiplication rate of adventitious buds. Meanwhile, the rooting effect of Bistorta officinalis is good, which is beneficial to the survival of later transplantation, thus accelerating the market application of Bistorta officinalis.

[0100] First of all, selecting an appropriate part of the mother plant as the explant for the tissue culture of Bistorta officinalis directly affects the final result of the tissue culture of Bistorta officinalis. 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 the stem segment with cauline leaves, while the explant selected in Comparative Example 4 is the leaf. It can be seen from Tables 2 and 3 that by using the tissue culture method of Example 1 for induction, adventitious buds germinate normally, and the adventitious buds grow rapidly, without vitrification phenomenon, and the differentiated seedlings grow well, the plants are robust, and the proliferation rate is high; while the result of using the tissue culture method of Comparative Example 4 is that with the leaf as the explant, although callus is formed, the callus finally does not differentiate into adventitious buds. To sum up, the selection of the explant has a great influence on the effect of the tissue culture of Bistorta officinalis, and selecting an appropriate part of the mother plant can determine the success or failure of the early induction. The present invention selects the stem segment with cauline leaves as the explant for the tissue culture of Bistorta officinalis, which can effectively improve the induction rate and proliferation coefficient of Bistorta officinalis and reduce the tissue culture production cost of Bistorta officinalis.

[0101] Furthermore, in the step of inducing the germination of stem segment bud points, the addition of the cytokinin 6 - BA also has a great influence on the induction of bud points. In Comparative Example 1, only the auxin NAA was added, and the cytokinin 6 - BA was not added. It can be seen from Tables 2 and 3 that although adventitious buds germinated normally in the induction stage in Comparative Example 1, the proliferation rate in the proliferation stage decreased significantly. Thus, it can be seen that in the step of inducing the germination of stem segment bud points, the addition of the cytokinin 6 - BA in the first step of inducing the germination of bud points helps to improve the proliferation rate of adventitious buds in the second step.

[0102] Further, the selection of plant hormones in the adventitious bud proliferation stage will also affect the result of the tissue culture of Bistorta officinalis. It can be seen from Tables 1, 2 and 3 that compared with Comparative Example 2, in Example 2, two cytokinins 6 - BA and TDZ are used in combination, which significantly increases the proliferation rate and has less influence on the growth of adventitious buds. Thus, it can be seen that on the premise that the leaves of the clustered buds formed during the formation of adventitious buds are normal and the plants are robust, in the step of inducing the proliferation of adventitious buds, the combination of two cytokinins 6 - BA and TDZ can be used to increase the proliferation rate.

[0103] In addition, the cluster buds induced by the tissue culture method of the present invention under the appropriate culture medium formula for adventitious bud germination and proliferation are robust. During the rooting stage, a rooting rate of 100% can be obtained without adding plant hormones to the rooting medium, thereby reducing the adverse effects of hormones on the transplanting survival rate of tissue-cultured seedlings. As can be seen from Table 1 and Table 3, in Comparative Example 4, auxin IBA was added to the rooting medium. Compared with Example 1 without adding hormones, there was no significant difference, and the rooting rate could reach 100%. The design of the rooting medium with activated carbon added and no hormones during the rooting stage of tissue-cultured seedlings not only saves production costs but also reduces the hormone content in tissue-cultured seedlings and enhances the adaptability of tissue-cultured seedlings to the external environment after transplantation.

[0104] In summary, in the polygonum bistorta tissue culture method provided by the present invention, the explant is selected as the stem segment with cauline leaves, and with the culture medium suitable for each stage of culture, by inducing adventitious buds to germinate and form cluster buds, the cycle is short and the proliferation coefficient is high. At the same time, the rooting effect of polygonum bistorta is good, the rooting rate reaches 100%, the transplanting survival rate of polygonum bistorta is improved, which is conducive to accelerating the market application of polygonum bistorta.

[0105] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate 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 a stem segment with cauline leaves of a 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 is: MS basic culture 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; The culture medium used for the adventitious bud proliferation is: MS basic culture 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; The culture medium used for inducing adventitious bud rooting is: 1 / 2 MS basic culture medium + activated carbon 0.3-0.5 g / L + sucrose 25-30 g / L + agar 4-5 g / L.

2. The tissue culture method of Polygonum multiflorum according to claim 1, characterized in that: The pH of the culture medium is 5.8-6.

0.

3. The tissue culture method of Polygonum multiflorum according to claim 1 or 2, characterized in that: 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.

4. The tissue culture method of Polygonum multiflorum according to claim 1 or 2, characterized in that: 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.

5. The method for tissue culture of Polygonum multiflorum according to claim 1 or 2, 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.

6. The method for tissue culture of Polygonum multiflorum according to claim 5, characterized in that: The alcohol disinfection treatment time is 30-60s; the mercuric chloride solution disinfection treatment time is 6-10min.

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