CN115968790A - Tissue culture and rapid propagation method and application of aconitum sinomontanum nakai - Google Patents
Tissue culture and rapid propagation method and application of aconitum sinomontanum nakai Download PDFInfo
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Abstract
The invention relates to the field of medicinal plant tissue culture and traditional Chinese medicinal materials, in particular to a tissue culture and rapid propagation method and application of Aconitum carmichaeli, which comprises the following steps: explant selection and cleaning; sterilizing explants; primary induction culture; subculture multiplication culture; and (5) rooting culture. The invention relates to a method for breeding seedlings by utilizing a tissue culture rapid propagation technology, which belongs to the category of asexual propagation, can maintain the excellent characters of a female parent to the maximum extent, and meanwhile, although the asexual propagation has the degeneration possibility of a certain period, the primary culture of the original female parent can be continuously carried out in the tissue culture propagation process, thereby reducing the generation number of the asexual propagation and finally obtaining the aconitum sinomontanum seedlings with extremely stable quality.
Description
Technical Field
The invention relates to the field of medicinal plant tissue culture and traditional Chinese medicinal materials, in particular to a rapid propagation method and application of Aconitum carmichaeli tissue culture.
Background
Aconitum carmichaeli (Aconitum bulleyanum) belongs to the Chinese medicinal materials of Aconitum in Ranunculaceae, 350 Aconitum plants exist in the world, 162 varieties exist in China, and according to records of Chinese materia medica, 40 or more Chinese aconitum kusnezoffii (A. Pendanum), aconitum kusnezoffii (A. Kusnezoffii), aconitum carmichaeli (A. Austryunnanense) and Aconitum vilmorinianum kusnezoffii (A. Vilmorinianum) belong to medicinal plants in the whole Aconitum genus, wherein the medicinal materials are the most famous aconitum kusnezoffii, radix Aconiti lateralis, radix aconiti feri, radix aconiti kusnezoffii and the like. The aconitum medicinal plant contains diterpenoid alkaloids which mainly comprise aconitine, hypaconitine, neoaconitine and the like, has the effects of dispelling wind and eliminating dampness and warming channels and relieving pain, is mainly used for treating traumatic injury, arthritis, neuropathic pain, stroke paralysis, gastroenteritis, carbuncle, ulcer and other symptoms, and is a necessary traditional Chinese medicine material for Chinese traumatological Chinese medicinal preparations such as Yunnan white drug powder, invincible ointment, short-acting pain relieving liniment of short-term rupestris herb and the like.
Aconitum carmichaeli is a perennial herb, mainly grows on mountain forest edge or brook edge with the altitude of 3200-3500 m, is one of aconitum plants specific to Yunnan, and is mainly distributed in high altitude areas of Yunnan, such as Dali, lijiang, weixi and the like. Similar to other medicinal aconitum plants, aconitum carmichaeli Debx contains large amount of diterpene alkaloids and has strong toxicity. However, aconitum carmichaeli is not a medicinal Aconitum Chinese medicinal material recorded in Chinese materia medica, but is a common traumatology herb in Dianxi folk, and is often used for cooking in cold mountainous areas such as Dali and Lijiang to achieve the purposes of strengthening body, relaxing muscles and tendons and promoting blood circulation.
Research of research units of Yunnan departments discovers that aconitum yunnanense contains less special alkaloid-bulleyaconitine A which is a main active raw material for preparing bulleyaconitine A injection, bulleyaconitine A tablets, bulleyaconitine A oral liquid and other related preparations. By the beginning of this century, the bulleyaconitine A content in Aconitum carmichaeli is still high, and can reach 1% at most, while the purchasing standard of related pharmaceutical enterprises is 0.3%. However, with the unordered mining of wild resources, the wild aconitum yunnanense is gradually exhausted, and then the artificial introduction and domestication planting of the aconitum yunnanense is gradually carried out in Dali, lijiang, diqing and the like, but in recent years, the content of the aconitum yunnanense Chinese herbal medicine aconitine A artificially planted is reduced year by year, and the purchasing standard of medicinal materials of various pharmaceutical enterprises is reduced from the original 0.3% to 0.1%, and some are even reduced to 0.06%, due to the raw material problems. The low-content aconitum yunnanense medicinal material greatly reduces the extraction yield of the bulleyaconitine A on one hand, and on the other hand, the extraction cost is multiplied because of the low content, and finally the price of the bulleyaconitine A is greatly increased.
The reason for the annual reduction of the bulleyaconitine A content in the aconitum yunnanense is probably that the aconitum yunnanense adopts a plant division breeding mode throughout the year to degrade the variety and cause the reduction of the bulleyaconitine A content, and the seed breeding is generally more or less doped with other aconitum varieties because of planting plots, so that the aconitum yunnanense is hybridized with the other aconitum varieties to obtain the hybridized aconitum yunnanense, and finally the bulleyaconitine A content in the aconitum yunnanense is reduced year by year.
The existing tissue culture technology of aconitum plants of Ranunculaceae has certain research, but no related report exists on Aconitum yunnanense, while the existing tissue culture technology of other aconitum plants has low application rate or even can not be applied to Aconitum yunnanense, and a new method is urgently needed to meet the requirement of rapid propagation of Aconitum yunnanense.
Disclosure of Invention
Aiming at the defects of the prior art, the invention specifically invents a tissue culture rapid propagation technology of aconitum yunnanense by utilizing the modern biotechnology, and can provide seedlings with sufficient quantity and stable quality for the planting of high-quality aconitum yunnanense.
In order to achieve the purpose, the invention adopts the technical scheme that: a tissue culture and rapid propagation method of Aconitum carmichaeli comprises the following steps:
(1) Explant selection and cleaning: selecting a stem section of 15-25 cm from the top end of the Aconitum carmichaeli as an explant, removing leaves, cutting into small sections with buds of 3-4 cm, washing with a saturated soap water solution for 10-15 min, washing the surface of the small sections with soap water by using tap water, dropwise adding 2-3 drops of Tween-80, shaking for 10-15 min, and finally flushing with the tap water for 60-70 min;
(2) Explant disinfection: transferring the cleaned explant to a super clean workbench, sterilizing the explant by using 75% alcohol for 15-20 s, rinsing the explant by using sterile water for 2-3 times, sterilizing the explant by using a saturated bleaching powder solution for 15-20 min, rinsing the explant by using sterile water for 3-4 times, sterilizing the explant by using a 25% hydrogen peroxide solution for 2-3 min, rinsing the explant by using sterile water for 2-3 times, sterilizing the explant by using a 5% hydrogen peroxide solution for 20-25 min, and rinsing the explant by using sterile water for 4-5 times;
(3) Primary induction culture: absorbing surface moisture of a sterilized explant by using sterile paper, cutting off wounds at two ends, cutting the explant into 1-2 cm small sections with buds, inoculating the small sections with buds into an MS + NAA 0.1-0.2 mg/L + ZT 0.5-0.8 mg/L + KT 2.0-3.0 mg/L + silver nitrate 0.2-0.3 mu g/L + activated carbon 1.0g/L primary induction culture medium, performing full-black dark culture at the temperature of 5 +/-2 ℃ for 7 days, then transferring the culture medium to an LX alternating environment with the temperature of 25 +/-2 ℃, the illumination intensity of 2000-3000 LX and the sunshine of 12h for 40-50 days;
(4) Subculture multiplication culture: cutting the induced cluster buds into single buds, inoculating the single buds in a secondary multiplication culture medium of MS + CPPU 0.05-0.08 mg/L + TDZ 0.5-0.6 mg/L + BR 0.08-0.1mg/L + coconut juice 60-80 ml/L + silver nitrate 0.1-0.15 mu g/L, and culturing for 30-40 days in a light-dark alternating environment with the temperature of 25 +/-2 ℃, the illumination intensity of 2000-3000 LX and the sunshine of 12 hours;
(5) Rooting culture: cutting the cluster buds obtained by multiplication culture into single buds, inoculating the single buds into a 1/2 improved MS + NAA 0.4-0.6 mg/L + IBA 0.2-0.3 mg/L + coconut juice 100-120 mL/L + silver nitrate 0.05-0.1 microgram/L rooting culture medium, and culturing for 30-40 days in a light-dark alternating environment with the temperature of 25 +/-2 ℃, the illumination intensity of 2000-3000 LX and the sunshine of 12 h;
(6) Domestication: removing the rooted seedlings from the bottles, cleaning a basal culture medium, transplanting the seedlings into a matrix of peat soil and perlite according to a mass ratio of 5.
Furthermore, in the step (5), the modified MS culture medium is characterized in that the rest elements are unchanged, and the concentration of sucrose is adjusted to be 12-15 g/L.
The beneficial technical effects of the invention are as follows:
1. the invention relates to a method for breeding seedlings by utilizing a tissue culture rapid propagation technology, which belongs to the category of asexual propagation, can maintain the excellent characters of a female parent to the maximum extent, and meanwhile, although the asexual propagation has the degeneration possibility of a certain period, the primary culture of the original female parent can be continuously carried out in the tissue culture propagation process, thereby reducing the generation number of the asexual propagation and finally obtaining the aconitum sinomontanum seedlings with extremely stable quality.
2. The conventional tissue culture disinfection technology usually adopts 0.1% mercuric chloride solution for disinfection, but no matter how high concentration and long time of mercuric chloride disinfection is adopted for the aconitum yunnanense and other varieties of aconitum, the browning rate of 100% can be caused, the browning of the aconitum yunnanense directly causes that clustered buds cannot be induced, and the browning can also directly influence the subculture proliferation and the rooting of rootless seedlings. The invention does not adopt mercuric chloride solution for disinfection, but adopts a disinfection mode of combining 75% alcohol, saturated bleaching powder solution and high-concentration hydrogen peroxide short-time disinfection with low-concentration hydrogen peroxide long-time disinfection, can reduce the disinfection pollution rate and the disinfection death rate to the greatest extent, also reduces the damage of the disinfectant to explants, and finally reduces the browning rate of the initial generation. And during primary culture, low-temperature culture is carried out, which is favorable for inhibiting browning during primary culture of aconitum yunnanense, and simultaneously, silver nitrate and activated carbon with certain concentration are combined during primary induction, secondary proliferation and rooting culture, so that the browning rate during primary induction, secondary proliferation and rooting culture is reduced to the extent that the induction, proliferation and rooting of cluster buds are not influenced.
3. In the invention, CPPU, TDZ and BR with certain concentration are organically matched during subculture, and coconut juice with certain concentration is added, so that the multiplication coefficient can reach 6.
4. In the rooting culture process, the low-concentration NAA and IBA are matched and used, and the high-concentration coconut juice is added, so that the rooting rate can reach 100 percent, and meanwhile, because of the low-concentration auxin matching and the proper addition of the coconut juice, the sugar concentration in a conventional MS culture medium is reduced, and the rooting culture process is favorable for root tuber formation of bottle seedlings.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the description of the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a graph showing the results of the culture in step (4) of example 1 of the present invention;
FIG. 2 is a graph showing the results of the culture in step (5) of example 1 of the present invention.
Description of the preferred embodiment
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
A tissue culture and rapid propagation method of Aconitum carmichaeli comprises the following steps:
(1) Explant selection and cleaning: transplanting Aconitum carmichaeli into a room before emergence of seedlings, spraying and irrigating roots with 500 times of 75% carbendazim solution every 7 days after emergence of the seedlings, continuously spraying for 3 times, cutting off stem segments with 20cm at the top end, cutting into 3cm bud segments after leaf removal, rinsing with saturated soap water solution for 10min, cleaning the surface of the segments with soap water by using tap water, dripping 3 drops of Tween-80, shaking for 10min, and finally rinsing with tap water for 60min;
(2) Explant disinfection: transferring the cleaned explant to a super clean workbench, disinfecting for 15s by using 75% alcohol, rinsing for 2 times by using sterile water, disinfecting for 15min by using a saturated bleaching powder solution, rinsing for 3 times by using sterile water, disinfecting for 2min by using a 25% hydrogen peroxide solution, rinsing for 3 times by using sterile water, disinfecting for 25min by using a 5% hydrogen peroxide solution, and rinsing for 5 times by using sterile water;
(3) Primary induction culture: absorbing surface moisture of the disinfected explant by using sterile paper, cutting wounds at two ends, cutting the explant into small sections with 1cm buds, inoculating the small sections into an MS + NAA 0.1mg/L + ZT 0.5mg/L + KT 2.0mg/L + silver nitrate 0.2 mu g/L + activated carbon 1.0g/L primary induction culture medium, culturing the small sections in a full-black and dark environment at the temperature of 5 ℃ for 7 days, and then transferring the culture medium to a light-dark alternating environment with the temperature of 25 ℃, the illumination intensity of 2000LX and the light of 12 hours for culturing for 50 days;
(4) Subculture multiplication culture: shearing the induced cluster buds into single buds, inoculating the single buds into a subculture multiplication medium of MS + CPPU 0.05mg/L + TDZ 0.5mg/L + BR 0.08mg/L + coconut juice 60ml/L + silver nitrate 0.1 mu g/L, and culturing for 40 days in a light-dark alternating environment with the temperature of 25 ℃, the illumination intensity of 2000LX and the sunshine of 12h (the seedlings are shown in figure 1 after culture);
(5) Rooting culture: cutting the cluster buds obtained by multiplication culture into single buds, inoculating the single buds into a 1/2 improved MS + NAA 0.4mg/L + IBA 0.2mg/L + coconut juice 100mL/L + silver nitrate 0.05 mu g/L rooting culture medium, and culturing for 40 days in a light-dark alternating environment with the temperature of 25 ℃, the illumination intensity of 2000LX and the sunshine duration of 12 h; modifying MS culture medium until the rest elements are unchanged, and adjusting sucrose concentration to 12g/L (the cultured plantlet is shown in figure 1);
(6) Domestication: the rooted seedlings were removed from the bottles, washed with a basal medium, transplanted into a matrix of peat soil + perlite (5). Keeping the air humidity at 100% ten days before transplanting, then gradually reducing the air humidity, and keeping the substrate humidity at 55% during the whole acclimation period.
Example 2
A tissue culture and rapid propagation method of Aconitum carmichaeli comprises the following steps:
(1) Explant selection and cleaning: transplanting Aconitum carmichaeli into a room before emergence of seedlings, spraying and irrigating roots with 500 times of 75% carbendazim solution every 7 days after emergence of the seedlings, continuously spraying for 4 times, cutting off stem segments with the top end of about 20cm, cutting into 4cm bud segments after leaf removal, rinsing with saturated soap water solution for 15min, cleaning surface soap water with tap water, dripping 2 drops of Tween-80, shaking for 15min, and finally rinsing with tap water for 70min;
(2) Explant disinfection: transferring the cleaned explant to a super clean workbench, disinfecting with 75% alcohol for 20s, rinsing with sterile water for 3 times, disinfecting with a saturated bleaching powder solution for 20min, rinsing with sterile water for 4 times, disinfecting with a 25% hydrogen peroxide solution for 3min, rinsing with sterile water for 3 times, disinfecting with a 5% hydrogen peroxide solution for 20min, and rinsing with sterile water for 5 times;
(3) Primary induction culture: after disinfection, the surface water of the explants is absorbed by sterile paper, wounds at two ends are cut off, the explants are cut into 2cm small sections with buds, the small sections are inoculated into primary induction culture media of MS + NAA 0.2mg/L + ZT 0.8mg/L + KT 3.0mg/L + silver nitrate 0.3 mug/L + activated carbon 1.0g/L, the primary induction culture media are cultured for 7 days in the dark at the temperature of 4 ℃, and then the primary induction culture media are transferred to the light-dark alternating environment with the temperature of 23 ℃, the illumination intensity of 3000LX and the sunshine of 12h for 50 days.
(4) Subculture multiplication culture: shearing the induced cluster buds into single buds, inoculating the single buds into a subculture multiplication medium consisting of MS, CPPU 0.08mg/L, TDZ 0.6mg/L, BR 0.1mg/L, coconut juice 80ml/L, silver nitrate 0.15 mu g/L and active carbon 0.5g/L, and culturing for 40 days in a light-dark alternating environment with the temperature of 24 ℃, the illumination intensity of 3000LX and the sunshine of 12 h;
(5) Rooting culture: cutting the cluster buds obtained by proliferation into single buds, inoculating the single buds into a 1/2 improved MS + NAA 0.6mg/L + IBA 0.3mg/L + coconut juice 120mL/L + silver nitrate 0.1 mu g/L + active carbon 0.8g/L rooting culture medium, and culturing for 40 days in a light-dark alternating environment with the temperature of 24 ℃, the illumination intensity of 3000LX and the sunshine of 12 h; modifying the MS culture medium to keep the other elements unchanged, and adjusting the sucrose concentration to 15g/L;
(6) Domestication: the rooted seedlings were removed from the bottles, washed with a basal medium, transplanted into a matrix of peat soil + perlite (5). Keeping the air humidity at 100% ten days before transplanting, then gradually reducing the air humidity, and keeping the substrate humidity at 60% during the whole acclimation period.
Comparative example 1
Tissue culture rapid propagation of Aconitum carmichaeli is carried out by the optimal method of "establishment of tissue culture rapid propagation system of Aconitum carmichaeli stem (molecular plant breeding [ J ],2020, 18 (15): 5081-5089"), and domestication is carried out by the step (6) of example 1 in the invention.
Comparative example 2
The tissue culture and rapid propagation of the aconitum yunnanense are carried out according to the optimal method of Du search, chenhong gang and the like, and the research on the aconitum szechenyianum tissue culture technology (university of Gansu agriculture' J, 2011, 2.
Comparative example 3
Tissue culture rapid propagation of Aconitum carmichaeli is carried out by an optimal method of tissue culture rapid propagation of Aconitum chuanxiong seedlings (Chinese wild plant resource [ J ],2004,23 (4): 62-63), and domestication is carried out by the step (6) of example 1 in the invention.
Comparative example 4
Tissue culture and rapid propagation of Aconitum sungpanense hand-mazz are carried out according to example 1 in a patent of a rapid propagation method of Aconitum vilmorinianum (CN202110896553. X), and domestication is carried out by adopting step (6) in example 1 in the invention.
Comparative example 5
Tissue culture and rapid propagation of Aconitum carmichaeli are carried out according to example 1 in patent "a rapid propagation method for Aconitum carmichaeli tissue culture" (CN 201410463223.1), and domestication is carried out by adopting step (6) in example 1 in the invention.
Comparative example 6
Tissue culture and rapid propagation of Aconitum sungpanense hand-Mazz are carried out according to example 1 in patent 'a culture medium for culturing Aconitum kusnezoffii tissues' (CN 202011585035.8), and domestication is carried out by adopting step (6) of example 1 in the invention.
The results of comparing examples 1 and 2 with comparative examples 1 to 6 with respect to the disinfection contamination rate, disinfection mortality rate, primary browning rate, cluster bud induction rate, callus differentiation rate, proliferation coefficient, rooting rate, root tuber formation rate and acclimation survival rate are shown in FIG. 1.
TABLE 1 comparative results
| Examples | Rate of disinfection and contamination | Mortality rate by disinfection | Initial browning rate | Induction rate of cluster buds | Callus induction rate | Callus differentiation rate | Coefficient of proliferation | Rooting rate | Root tuber formation rate | Survival rate of domestication |
| Example 1 | 31.2% | 0 | 4.9% | 82.4% | —— | —— | 5.8 | 100% | 34.3% | 100% |
| Example 2 | 29.8% | 0 | 5.3% | 85.5% | —— | —— | 6.2 | 100% | 32.9% | 100% |
| Comparative example 1 | 24.9% | 47.6% | 98.8% | 4.9% | —— | —— | 3.2 | 100% | 0 | 86.4% |
| Comparative example 2 | 62.8% | 11.7% | 36.8% | 36.7% | —— | —— | 1.7 | 97.9% | 0 | 80.7% |
| Comparative example 3 | 19.2% | 54.7% | 100% | 2.7% | —— | —— | 2.3 | 100% | 0 | 81.0% |
| Comparative example 4 | 60.6% | 10.6% | 21.9% | 0 | —— | —— | —— | —— | —— | —— |
| Comparative example 5 | 10.6% | 84.9% | 100% | 1.7% | —— | —— | 2.1 | 100% | 0 | 84.4% |
| Comparative example 6 | 49.3% | 17.8% | 92.4% | —— | 16.3% | 10.7% | —— | 83.2% | 0 | 71.9% |
In the tissue culture and rapid propagation process of aconitum yunnanense, the browning degree directly influences the inductivity of aconitum yunnanense, and the higher the browning rate is in the same culture medium, the lower the inductivity is. As can be seen from the above table 1, in both the comparative examples 2 and 4, the PVP and the activated carbon with a certain concentration are added into the induction culture medium, so that the browning can be inhibited to a great extent, but the inhibition is incomplete, the primary induction is still greatly influenced, the PVP has a certain influence on the growth of Aconitum carmichaeli Debx, and the PVP shows a certain performance in the secondary propagation culture. When the subculture is carried out, the proliferation culture of aconitum yunnanense is carried out by applying the proportional subculture medium, the proliferation coefficient is generally low, and the extremely low proliferation coefficient can greatly increase the production period and improve the production cost. Although the rooting rate of each proportion is higher except the comparative example 6 during the rooting culture, the survival rate cannot reach 100 percent during the domestication of the rooted seedlings, on one hand, the seedlings are weaker because each proportion has no root tuber to a certain degree and simultaneously no growth substance which is beneficial to the strong seedlings of the aconitum yunnanense is added in each proportion.
In the case of comparative example 4, which failed to induce multiple shoots, it was possible that the selection of the type and concentration of each plant growth regulator and the ratio of auxin to cytokinin were not appropriate.
The comparative example 6 is not consistent with the technical route of the invention, and is a way of inducing callus by explants, differentiating into seedlings by callus and then rooting seedlings, but when the technical route is used for carrying out tissue culture and rapid propagation of aconitum wissmannianum, the initial generation induction rate is low, the callus differentiation rate is extremely low, so that the technical route is not suitable for the tissue culture and rapid propagation of aconitum wissmannianum under the current technical conditions. Meanwhile, the general callus is a non-organic cell mass, and due to the loose property, the cell character of the callus is easy to be interfered by external conditions to generate variation, and the genetic instability of aconitum yunnanense can also be caused.
Finally, it should be noted that: although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that: modifications and equivalents may be made thereto without departing from the spirit and scope of the invention and it is intended to cover in the claims the invention as defined in the appended claims.
Claims (6)
1. The tissue culture and rapid propagation method of aconitum sinomontanum nakai is characterized by comprising the following steps:
(1) Explant selection and cleaning;
(2) Explant disinfection: transferring the cleaned explant to a super clean workbench, and sequentially disinfecting with 75% alcohol, saturated bleaching powder solution, 25% hydrogen peroxide solution and 5% hydrogen peroxide solution;
(3) Primary induction culture: absorbing surface moisture of a sterilized explant by using sterile paper, cutting off wounds at two ends, cutting the explant into 1-2 cm small sections with buds, inoculating the small sections with buds into an MS + NAA 0.1-0.2 mg/L + ZT 0.5-0.8 mg/L + KT 2.0-3.0 mg/L + silver nitrate 0.2-0.3 mu g/L + activated carbon 1.0g/L primary induction culture medium, performing full-black dark culture at the temperature of 5 +/-2 ℃ for 7 days, then transferring the culture medium to an LX alternating environment with the temperature of 25 +/-2 ℃, the illumination intensity of 2000-3000 LX and the sunshine of 12h for 40-50 days;
(4) Subculture multiplication culture: shearing the induced cluster buds into single buds, inoculating the single buds into a subculture multiplication medium consisting of MS, CPPU 0.05-0.08 mg/L, TDZ 0.5-0.6 mg/L, BR 0.08-0.1mg/L, coconut juice 60-80 ml/L, silver nitrate 0.1-0.15 mu g/L, and culturing for 30-40 days in an alternate environment of 25 +/-2 ℃, illumination intensity of 2000-3000 LX and 12h sunshine;
(5) Rooting culture: cutting the cluster buds obtained by multiplication culture into single buds, inoculating the single buds into a 1/2 improved MS + NAA 0.4-0.6 mg/L + IBA 0.2-0.3 mg/L + coconut juice 100-120 mL/L + silver nitrate 0.05-0.1 mu g/L rooting culture medium, and culturing for 30-40 days in a light-dark alternating environment with the temperature of 25 +/-2 ℃, the illumination intensity of 2000-3000 LX and the sunshine of 12 h.
2. The method according to claim 1, wherein step (1) is specifically: selecting a stem section of 15-25 cm from the top end of the Aconitum carmichaeli as an explant, removing leaves, cutting into small sections with buds of 3-4 cm, washing with saturated soap water solution for 10-15 min, washing the surface of the small sections with tap water, dripping 2-3 drops of Tween-80, shaking for 10-15 min, and finally flushing with tap water for 60-70 min.
3. The method according to claim 1, wherein the step (2) of sterilizing is embodied as: disinfecting with 75% alcohol for 15-20 s, washing with sterile water for 2-3 times, disinfecting with saturated bleaching powder solution for 15-20 min, washing with sterile water for 3-4 times, disinfecting with 25% hydrogen peroxide solution for 2-3 min, washing with sterile water for 2-3 times, disinfecting with 5% hydrogen peroxide solution for 20-25 min, and washing with sterile water for 4-5 times.
4. The method according to claim 1, further comprising a step of acclimatization, in particular: removing the rooted seedlings from the bottles, cleaning a basal culture medium, transplanting the seedlings into a matrix of peat soil and perlite according to a mass ratio of 5.
5. The method according to claim 1, wherein in the step (5), the modified MS medium is prepared such that the remaining elements are unchanged and the sucrose concentration is adjusted to 12 to 15g/L.
6. The method according to any one of claims 1-5, for use in tissue culture and rapid propagation of Aconitum yunnanense.
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