CN117717003B - Tissue culture rapid propagation method and application of anoectochilus formosanus combined with bottle external rooting technology - Google Patents
Tissue culture rapid propagation method and application of anoectochilus formosanus combined with bottle external rooting technology Download PDFInfo
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Abstract
The invention relates to the technical field of plant tissue culture, in particular to a tissue culture and rapid propagation method and application of anoectochilus formosanus in combination with an extrabottle rooting technology, comprising the following steps: sterile material preparation, inoculation in a primary induction culture medium, single bud shearing of primary induction cluster buds, inoculation in a secondary proliferation culture medium, continuous inoculation in a pre-rooting culture medium, culture, taking out the anoectochilus formosanus seedlings subjected to pre-rooting culture, washing the residual culture medium at the base, soaking for 24 hours with a nutrient solution A, planting in humus soil, building a small arch shed, and watering with a nutrient solution B every 10 days for 3-4 times continuously. According to the invention, when rooting is performed outside the bottle, the combination of IBA, salicylic acid and VB 12 with a certain concentration can better promote the expansion of leaves in humus soil, and the rooting rate of the tissue culture seedlings can reach more than 95%.
Description
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a tissue culture and rapid propagation method for anoectochilus formosanus combined with an extrabottle rooting technology and application thereof.
Background
The Anoectochilus formosanus (STEPHANIA CEPHARANTHA HAYATA) is a perennial grass vine of Stephania of Menispermaceae. The anoectochilus formosanus can be used as a medicine by using root tuber, can extract alkaloid, stephanine, rotundine and the like, is also She Yuanrun emerald, is suitable for potting in balcony, courtyard and the like, and has good ornamental value.
In recent years, with development of medicinal value and popularization of ornamental value, requirements of the anoectochilus formosanus are suddenly increased, but limited wild resources cannot meet market requirements, and prices of the anoectochilus formosanus are also suddenly increased, so that various scientific research institutions and enterprises develop planting technology researches of the anoectochilus formosanus in a dispute mode, but due to the fact that the anoectochilus formosanus is hermaphrodite, and female plants are distributed in a small quantity in a population, seed quantity is small, and meanwhile, due to the fact that the germination rate of seeds is low and the germination period is long. Meanwhile, as the hermaphrodite strain has strong seed propagation hybridization and unstable female parent genetic character, the mutation probability of the female parent is larger when the female parent is used as a medicine and ornamental. In recent years, research on tissue culture techniques such as Guangxi di-deficiency and Lepidium palustris has been carried out in part of units, and the effects are not ideal in terms of root growth, and the tissue culture techniques are not suitable for the first-generation induction and proliferation, but hardly root the Anoectochilus formosanus in terms of root growth.
Disclosure of Invention
The invention aims to provide a tissue culture and rapid propagation method for anoectochilus formosanus combined with an extrabottle rooting technology and application thereof.
In order to achieve the above purpose, the invention adopts the following technical scheme: a tissue culture and rapid propagation method of a tortoise plastron with gold thread combined with an extrabottle rooting technology comprises the following steps:
(1) Preparing a sterile material;
(2) Primary induction culture: inoculating the sterile material into a primary induction culture medium of MS+NAA 0.02-0.04 mg/L+CPPU 0.1-0.2 mg/L+KT2.0-3.0 mg/L for culturing for 25-35 days;
(3) And (3) subculturing and proliferation: cutting the primary induced cluster buds into single buds, and inoculating the single buds into a secondary proliferation culture medium of MS+IBA 0.01-0.02 mg/L+IAA 0.05-0.10 mg/L+CPPU 0.3-0.5 mg/L for culturing for 30-40 days;
(4) Pre-rooting culture in a bottle: cutting the cluster buds subjected to the secondary multiplication into single buds, and inoculating the single buds into a 1/2 modified MS+IBA 0.3-0.5 mg/L+IAA 0.5-0.6 mg/L+paclobutrazol 0.2-0.3 mg/L+coconut milk 20-30 g/L pre-rooting culture medium for culturing for 30-40 days;
the modified MS is characterized in that other elements are unchanged, and the sugar consumption is reduced to 15g/L;
(5) Rooting outside the bottle: taking out the anoectochilus formosanus seedlings subjected to pre-rooting culture from the bottle, cleaning a residual culture medium at the base, soaking the seedlings in the nutrient solution A for 24 hours, planting the seedlings in humus soil, building a small arch shed after planting, and watering the seedlings with the nutrient solution B every 10 days, and continuously watering the seedlings for 3-4 times;
The formula of the nutrient solution A is as follows: 40-50 mg/L of salicylic acid and 8-10 mug/L of VB 12;
The formula of the nutrient solution B is as follows: IBA 60-80 mg/L+salicylic acid 80-100 mg/L+VB 12 20-25 mug/L.
Further, in step (1), the preparation of the sterile material is specifically: selecting tender shoot segments with buds and stems with 10-20 cm of top ends, shearing the tender shoot segments into bud segments, rinsing the bud segments with saturated soap water solution, flushing the bud segments, sterilizing the bud segments with 75% alcohol for 10-25 s, flushing the bud segments with sterile water for 3-4 times, sterilizing the bud segments with saturated bleaching powder solution for 20-30 min and flushing the bud segments with sterile water for 2-3 times, and sterilizing the bud segments with 0.05% mercuric solution for 10-15 min and flushing the bud segments with sterile water for 6-8 times.
Further, in the steps (2), (3) and (4), the culture conditions are: the temperature is 23+/-2 ℃, the illumination intensity is 2000-3000 Lx, and the light and dark alternate culture is carried out after illumination for 10-14 hours.
Further, the formula of the primary induction culture medium is as follows: MS+NAA 0.03mg/L+CPPU 0.15 mg/L+KT2.5 mg/L.
Further, the formula of the secondary proliferation culture medium is as follows: MS+IBA 0.01mg/L+IAA 0.75mg/L+CPPU 0.4mg/L.
Further, the formula of the pre-rooting culture medium is as follows: 1/2 modified MS+IBA 0.4mg/L+IAA 0.55 mg/L+paclobutrazol 0.25 mg/L+coconut milk 25g/L.
Further, the formula of the nutrient solution A is as follows: 45mg/L of salicylic acid and VB 12 mu g/L of water solution; the formula of the nutrient solution B is as follows: IBA 70 mg/L+salicylic acid 90mg/L+VB 12. Mu.g/L.
The beneficial technical effects of the invention are as follows:
1. The team of the invention found in the test: NAA and active carbon have remarkable inhibition effect on rooting of anoectochilus formosanus, but when non-NAA auxin is adopted as a primary induction culture medium, callus is induced to be free from budding or a small amount of budding or is directly incapable of being induced, so that NAA is adopted in the primary induction, CPPU and KT with certain concentration are matched, cluster buds can be furthest induced instead of callus, and IBA is adopted to replace NAA in the secondary induction, so that on one hand, the cluster buds can be continuously proliferated, and on the other hand, the inhibition effect of NAA on root growth can be furthest reduced.
2. When the method is used for pre-rooting culture in the bottle, IBA, IAA and paclobutrazol with certain concentration are combined, meanwhile, through the blood glucose reduction treatment of the basic culture medium, obvious root primordia can be formed on the base of the stem by grouping the culture seedlings in 30 days, less part of the culture seedlings can form roots, and then the culture seedlings are matched with coconut milk with certain concentration, so that the stem is thicker, the leaves are thickened to a certain extent, and the survival of the culture seedlings in the bottle is facilitated.
3. The tissue culture seedling of the anoectochilus formosanus is difficult to root in the bottle, the rooting rate in the bottle is within 50% through long-term and repeated experiments, and the production cost and the seedling rate are determined according to the rooting rate of the tissue culture seedling. According to the invention, when rooting is performed outside the bottle, the combination of IBA, salicylic acid and VB 12 with a certain concentration can better promote the expansion of leaves in humus soil, and the rooting rate of the tissue culture seedlings of the anoectochilus formosanus can reach more than 95%.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions of the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, it being obvious that the drawings in the description below are only some embodiments of the invention, and that other drawings can be obtained according to these drawings without inventive faculty for a person skilled in the art.
FIG. 1 is a graph showing the results of rooting culture in bottles of example 1;
FIG. 2 is a graph showing the results of the out-vitro rooting culture in example 1;
FIG. 3 is a graph showing the results of rooting culture in bottles of example 2;
FIG. 4 is a graph showing the results of rooting culture in comparative example 1;
FIG. 5 is a graph showing the results of rooting culture in the flask of comparative example 2;
FIG. 6 is a graph showing the result of seedling hardening and transplanting in comparative example 2.
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
A tissue culture and rapid propagation method of a tortoise plastron with gold thread combined with an extrabottle rooting technology comprises the following steps:
(1) Preparation of sterile materials: selecting tender shoot segments with buds at the top end of 10cm, shearing the tender shoot segments into bud segments, rinsing the bud segments with saturated soap water solution, flushing the bud segments with saturated soap water solution, sterilizing the bud segments with 75% alcohol for 10s and flushing the bud segments with sterile water for 3 times, sterilizing the bud segments with saturated bleaching powder solution for 20min and flushing the bud segments with sterile water for 2 times, and finally sterilizing the bud segments with 0.05% mercuric chloride solution for 10min and flushing the bud segments with sterile water for 6 times.
(2) Primary induction culture: the sterile material is inoculated into a primary induction culture medium of MS+NAA 0.02mg/L+CPPU 0.1 mg/L+KT2.0 mg/L, and the culture is carried out for 25 days by light and dark alternation according to the temperature of 23+/-2 ℃ and the illumination intensity of 2000Lx and the illumination time of 10 hours.
(3) And (3) subculturing and proliferation: cutting the primary induced cluster buds into single buds, inoculating the single buds into a secondary proliferation culture medium of MS+IBA 0.01mg/L+IAA 0.05mg/L+CPPU 0.3mg/L, and carrying out light-dark alternate culture for 30 days according to the temperature of 23+/-2 ℃ and the illumination intensity of 2000Lx and the illumination time of 10 hours.
(4) Pre-rooting culture in a bottle: cutting the cluster buds subjected to secondary multiplication into single buds, inoculating the single buds into a pre-rooting culture medium of 1/2 modified MS+IBA 0.3mg/L+IAA 0.5 mg/L+paclobutrazol 0.2 mg/L+coconut milk 20g/L, and carrying out light-dark alternate culture for 10 hours according to the temperature of 23+/-2 ℃ and the illumination intensity of 2000 Lx;
Wherein the modified MS is the same as other elements, the sugar consumption is reduced to 15g/L, and the culture result is shown in figure 1.
(5) Rooting outside the bottle: taking out the anoectochilus formosanus seedlings subjected to pre-rooting culture from the bottle, cleaning a residual culture medium at the base, soaking for 24 hours by using a nutrient solution A, planting in humus soil, building a small arch shed after planting, and watering for 3 times by using a nutrient solution B every 10 days, wherein the result is shown in figure 2, and the vigorous seedlings can be seen from the figure;
Wherein, the formula of the nutrient solution A is as follows: 40mg/L of salicylic acid and VB 12 mug/L of water solution; the formula of the nutrient solution B is as follows: IBA 60 mg/L+salicylic acid 80mg/L+VB 12. Mu.g/L.
And other steps or water and fertilizer management modes are managed in a conventional mode.
Example 2
A tissue culture and rapid propagation method of a tortoise plastron with gold thread combined with an extrabottle rooting technology comprises the following steps:
(1) Preparation of sterile materials: selecting tender shoot segments with buds and 15cm at the top, shearing the tender shoot segments into bud segments, rinsing the bud segments with saturated soap water solution, flushing the bud segments with saturated soap water solution, sterilizing the bud segments with 75% alcohol for 20s, flushing the bud segments with sterile water for 4 times, sterilizing the bud segments with saturated bleaching powder solution for 30min, flushing the bud segments with sterile water for 3 times, and finally sterilizing the bud segments with 0.05% mercuric chloride solution for 15min and flushing the bud segments with sterile water for 8 times.
(2) Primary induction culture: the sterile material is inoculated into a primary induction culture medium of MS+NAA 0.04mg/L+CPPU 0.2 mg/L+KT3.0 mg/L, and the culture is carried out for 35 days by light and dark alternate culture according to the temperature of 23+/-2 ℃ and the illumination intensity of 3000Lx and the illumination time of 14 h.
(3) And (3) subculturing and proliferation: cutting the primary induced cluster buds into single buds, inoculating the single buds into a secondary proliferation culture medium of MS+IBA 0.02mg/L+IAA 0.10mg/L+CPPU 0.5mg/L, and carrying out light-dark alternate culture for 40 days according to the temperature of 23+/-2 ℃ and the illumination intensity of 3000Lx and the illumination time of 14 hours.
(4) Pre-rooting culture in a bottle: cutting the cluster buds subjected to secondary multiplication into single buds, inoculating the single buds into a pre-rooting culture medium of 1/2 modified MS+IBA 0.5mg/L+IAA 0.6 mg/L+paclobutrazol 0.3 mg/L+coconut milk 30g/L, and carrying out light-dark alternate culture for 14h according to the temperature of 23+/-2 ℃ and the illumination intensity of 3000 Lx;
Wherein, the modified MS is unchanged from other elements, and the sugar consumption is reduced to 15g/L. The culture results are shown in FIG. 3.
(5) Rooting outside the bottle: taking out the anoectochilus formosanus seedlings subjected to pre-rooting culture from the bottle, cleaning a residual culture medium at the base, soaking the seedlings in the nutrient solution A for 24 hours, planting the seedlings in humus soil, building a small arch shed after planting, and watering the seedlings with the nutrient solution B every 10 days, and continuously watering the seedlings for 3-4 times;
Wherein, the formula of the nutrient solution A is as follows: salicylic acid 50mg/L+VB 12 mu g/L aqueous solution; the formula of the nutrient solution B is as follows: IBA 80 mg/L+salicylic acid 100mg/L+VB 12. Mu.g/L.
And other steps or water and fertilizer management modes are managed in a conventional mode.
Example 3
A tissue culture and rapid propagation method of a tortoise plastron with gold thread combined with an extrabottle rooting technology comprises the following steps:
(1) Preparation of sterile materials: selecting tender shoot segments with buds and 15cm at the top, shearing the tender shoot segments into bud segments, rinsing the bud segments with saturated soap water solution, flushing the bud segments with saturated soap water solution, sterilizing the bud segments with 75% alcohol for 25 seconds and flushing the bud segments with sterile water for 4 times, sterilizing the bud segments with saturated bleaching powder solution for 25 minutes and flushing the bud segments with sterile water for 3 times, and finally sterilizing the bud segments with 0.05% mercuric chloride solution for 15 minutes and flushing the bud segments with sterile water for 7 times.
(2) Primary induction culture: the sterile material is inoculated into a primary induction culture medium of MS+NAA 0.03mg/L+CPPU 0.15 mg/L+KT2.5 mg/L, and the culture is carried out for 30 days under alternate light and dark illumination according to the temperature of 23+/-2 ℃ and the illumination intensity of 2500Lx and the illumination time of 12 hours.
(3) And (3) subculturing and proliferation: cutting the primary induced cluster buds into single buds, inoculating the single buds into a secondary proliferation culture medium of MS+IBA 0.01mg/L+IAA 0.75mg/L+CPPU 0.4mg/L, and carrying out light-dark alternate culture for 35 days according to the temperature of 23+/-2 ℃ and the illumination intensity of 2500Lx and the illumination time of 12 hours.
(4) Pre-rooting culture in a bottle: cutting the cluster buds subjected to secondary multiplication into single buds, inoculating the single buds into a pre-rooting culture medium of 1/2 modified MS, IBA 0.4mg/L, IAA 0.55mg/L, paclobutrazol 0.25mg/L and coconut milk 25g/L, and carrying out light-dark alternate culture for 12 hours according to the temperature of 23+/-2 ℃ and the illumination intensity of 2500 Lx;
Wherein, the modified MS is unchanged from other elements, and the sugar consumption is reduced to 15g/L.
(5) Rooting outside the bottle: taking out the anoectochilus formosanus seedlings subjected to pre-rooting culture from the bottle, cleaning a residual culture medium at the base, soaking the seedlings in the nutrient solution A for 24 hours, planting the seedlings in humus soil, building a small arch shed after planting, and irrigating the seedlings with the nutrient solution B once every 10 days, and continuously irrigating the seedlings for 4 times;
Wherein, the formula of the nutrient solution A is as follows: 45mg/L of salicylic acid and VB 12 mu g/L of water solution; the formula of the nutrient solution B is as follows: IBA 70 mg/L+salicylic acid 90mg/L+VB 12. Mu.g/L.
And other steps or water and fertilizer management modes are managed in a conventional mode.
Comparative example 1
The tip tender bud stem section of the anoectochilus formosanus is used as a material, and the patent application name of the reference Chinese invention is: the procedure of example 2 of the tissue culture propagation method (publication No. CN 109122318A) was carried out, and the results of the in-bottle rooting culture are shown in FIG. 4.
And (5) performing domestication and transplanting according to a conventional method after rooting culture for 30 days.
Comparative example 2
The tip tender bud stem section of the anoectochilus formosanus is used as a material, and the patent application name of the reference Chinese invention is: an in vitro rapid propagation method of Tortoise (publication No. CN 102017894A) was performed in the method of example 1, and the results of rooting in the bottle for 30 days are shown in FIG. 5.
And (3) carrying out domestication and transplanting according to a conventional method after rooting culture for 30 days, wherein the result is shown in figure 6.
Comparative example 3
The young stem section with bud at the top of the golden thread tortoise is taken as a material, and the young stem section with bud at the top of 15cm is selected according to the embodiment 1, and the patent application name of the reference Chinese invention is: a tissue culture propagation method of Stephania sinica Diels with high content of cycleanine and stephanine (publication No. CN 109964813A) was cultured by the method of example 1.
And (5) performing domestication and transplanting according to a conventional method after rooting culture for 30 days.
Comparative example 4
The young stem section with bud at the top of the golden thread tortoise is taken as a material, and the young stem section with bud at the top of 15cm is selected according to the embodiment 1, and the patent application name of the reference Chinese invention is: a tissue culture technique (publication No. CN 109496852A) of Tortoise was cultured by the method of example 2.
And (5) performing domestication and transplanting according to a conventional method after rooting culture for 30 days.
Comparative example 5
The young stem section with bud at the top of the golden thread tortoise is taken as a material, and the young stem section with bud at the top of 15cm is selected according to the embodiment 1, and the patent application name of the reference Chinese invention is: a method for tissue culture and rapid propagation of Tortoise (publication No. CN 110250004A) was carried out in the same manner as in example 2.
And (5) performing domestication and transplanting according to a conventional method after rooting culture for 30 days.
Comparative example 6
The method disclosed in 594-601.DOI:10.13271/j.mpb.021.000594 of molecular plant breeding, 2023,21 (02) of the patent publication [ J ] of KT and TDZ to tissue culture of medicinal plants of Tortoise ] is used for disinfection, cluster bud induction, culture of secondary proliferation, rooting culture and seedling hardening transplantation, wherein the young stem with buds at the top of Tortoise is selected as a material according to the method disclosed in example 1, and the patent publication of Wei Liping and the like.
And (5) performing domestication and transplanting according to a conventional method after rooting culture for 30 days.
Comparative example 7
The method comprises the steps of selecting a young stem section with buds at the top of a tortoise with gold thread as a material according to the method of example 1, selecting a young stem section with buds at the top of a tortoise with gold thread 15cm, and performing operations according to Chinese herbal medicines of Guangxi Di Shuangju tissue culture and rapid propagation study [ J ], [ 2007, (03): 445-449 ] issued by Huang Ningzhen and the like.
And (5) performing domestication and transplanting according to a conventional method after rooting culture for 30 days.
Comparative example 8
The young stem with buds at the top of the anoectochilus formosanus is selected according to the example 1, and the young stem with buds at the top of the anoectochilus formosanus is selected according to the tissue culture research of medicinal plant anoectochilus formosanus of pond source [ D ]. Southwest university, 2009.
And (5) performing domestication and transplanting according to a conventional method after rooting culture for 30 days.
The results of comparing the cluster bud induction rate, proliferation coefficient, in-vitro rooting rate of 30d bottle and survival rate of 90d in examples 1-3 and comparative examples 1-8 are shown in the following table.
TABLE 1 statistical results
| Examples | Induction rate of cluster buds | Proliferation coefficient | Rooting rate in 30d bottle | Rooting rate outside 30d bottle | Survival rate of 90d | Remarks |
| Example 1 | 74.6% | 4.7 | 48.8% | 97.2% | 97.9% | |
| Example 2 | 70.9% | 4.2 | 47.9% | 96.8% | 99.4% | |
| Example 3 | 71.4% | 4.5 | 49.6% | 98.1% | 97.1% | |
| Comparative example 1 | 62.2% | 3.6 | 3.4% | 31.4% | 30.6% | |
| Comparative example 2 | 32.7% | 2.9 | 1.4% | 20.3% | 27.9% | |
| Comparative example 3 | 6.7% | 1.7 | 37.2% | 56.9% | 70.1% | The first generation is greatly healed |
| Comparative example 4 | 44.1% | 4.0 | 5.8% | 21.7% | 42.6% | |
| Comparative example 5 | 40.6% | 3.2 | 39.7% | 61.0% | 62.9% | |
| Comparative example 6 | 29.4% | 3.6 | 43.4% | 79.4% | 86.3% | Less root recovery during rooting |
| Comparative example 7 | 21.4% | 3.1 | 40.8% | 71.2% | 72.3% | |
| Comparative example 8 | 26.4% | 3.2 | 39.2% | 72.9% | 64.6% |
As can be seen from the statistics in Table 1, the cluster bud induction rate, proliferation coefficient, in-bottle rooting rate of 30d, out-bottle rooting rate of 30d and survival rate of 90d of examples 1-3 are all higher than those of comparative examples 1-8.
Finally, what should be said is: the above embodiments are only for illustrating the technical aspects of the present invention, and 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, which is intended to be encompassed by the claims.
Claims (7)
1. A tissue culture and rapid propagation method of a tortoise with gold thread combined with an extrabottle rooting technology is characterized by comprising the following steps:
(1) Preparation of sterile materials: selecting tender shoot segments with buds and stems with 10-20 cm of top ends, shearing the tender shoot segments into bud segments, rinsing the bud segments with saturated soap water solution, flushing the bud segments, sterilizing the bud segments with 75% alcohol for 10-25 s, flushing the bud segments with sterile water for 3-4 times, sterilizing the bud segments with saturated bleaching powder solution for 20-30 min and flushing the bud segments with sterile water for 2-3 times, and sterilizing the bud segments with 0.05% mercuric solution for 10-15 min and flushing the bud segments with sterile water for 6-8 times;
(2) Primary induction culture: inoculating the sterile material into a primary induction culture medium of MS+NAA 0.02-0.04 mg/L+CPPU 0.1-0.2 mg/L+KT2.0-3.0 mg/L for culturing for 25-35 days;
(3) And (3) subculturing and proliferation: cutting the primary induced cluster buds into single buds, and inoculating the single buds into a secondary proliferation culture medium of MS+IBA 0.01-0.02 mg/L+IAA 0.05-0.10 mg/L+CPPU 0.3-0.5 mg/L for culturing for 30-40 days;
(4) Pre-rooting culture in a bottle: cutting the cluster buds subjected to the secondary multiplication into single buds, and inoculating the single buds into a 1/2 modified MS+IBA 0.3-0.5 mg/L+IAA 0.5-0.6 mg/L+paclobutrazol 0.2-0.3 mg/L+coconut milk 20-30 g/L pre-rooting culture medium for culturing for 30-40 days;
the modified MS is characterized in that other elements are unchanged, and the sugar consumption is reduced to 15g/L;
(5) Rooting outside the bottle: taking out the anoectochilus formosanus seedlings subjected to pre-rooting culture from the bottle, cleaning a residual culture medium at the base, soaking the seedlings in the nutrient solution A for 24 hours, planting the seedlings in humus soil, building a small arch shed after planting, and watering the seedlings with the nutrient solution B every 10 days, and continuously watering the seedlings for 3-4 times;
The formula of the nutrient solution A is as follows: 40-50 mg/L of salicylic acid and 8-10 mug/L of VB 12;
The formula of the nutrient solution B is as follows: IBA 60-80 mg/L+salicylic acid 80-100 mg/L+VB 12 20-25 mug/L.
2. The method of claim 1, wherein in steps (2), (3) and (4), the culture conditions are: the temperature is 23+/-2 ℃, the illumination intensity is 2000-3000 Lx, and the light and dark alternate culture is carried out after illumination for 10-14 hours.
3. The method of claim 1, wherein the primary induction medium is formulated as follows: MS+NAA 0.03mg/L+CPPU 0.15 mg/L+KT2.5 mg/L.
4. The method of claim 1, wherein the formulation of the secondary proliferation medium is: MS+IBA 0.01mg/L+IAA 0.75mg/L+CPPU 0.4mg/L.
5. The method of claim 1, wherein the pre-rooting medium is formulated as follows: 1/2 modified MS+IBA 0.4mg/L+IAA 0.55 mg/L+paclobutrazol 0.25 mg/L+coconut milk 25g/L.
6. The method according to claim 1, wherein the nutrient solution a is formulated as follows: 45mg/L of salicylic acid and VB 12 mu g/L of water solution; the formula of the nutrient solution B is as follows: IBA 70 mg/L+salicylic acid 90mg/L+VB 12. Mu.g/L.
7. Use of the method according to any one of claims 1-6 in tissue culture and rapid propagation of anoectochilus formosanus.
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