A tissue culture and rapid propagation system of Verbena officinalis

Abstract

The invention provides a tissue culture and rapid propagation system of Verbena officinalis, and relates to the field of plant tissue culture. The Verbena officinalis tissue culture and rapid propagation system specifically comprises the following steps: S1. preparation of test materials, S2. disinfection of stem segments with axillary buds, S3. determination of axillary bud proliferation culture medium, S4. determination of plant rooting culture medium, S5. determination of seedling hardening time and transplanting matrix, S6. culture conditions, and S7. data processing. Studies have shown that different sterilization times have significant effects on the contamination rate, survival rate and mortality rate of stem segments with axillary buds. Considering the comprehensive contamination rate, survival rate and mortality rate, it can be concluded that the longer the disinfection time is, the better. Too long a time will lead to an increase in the number of explant deaths, and too short a time will lead to incomplete disinfection of the explants. Different plant growth regulator ratios have a significant effect on the rooting of tissue culture seedlings. Under the same concentration, IBA has a more obvious effect on promoting rooting of Verbena officinalis, and the IBA combination has a better proliferation effect than the NAA combination.

Description

Technical Field

[0001] The invention relates to the field of plant tissue culture, in particular to a Verbena tissue culture rapid propagation system. Background Art

[0002] Willow verbena, also known as South American verbena, long-stem verbena, one-foot verbena, and Brazilian verbena, is native to Brazil, Argentina and other places in South America. Willow verbena is a long-day flower that likes warmth. The suitable temperature for growth is 20-30℃. It grows slowly below 10℃ and above 35℃. The leaves are dark green and cross-opposed. The initial leaves are oval. After the flower stalks grow taller, the leaves turn into slender willow leaves with slightly notched edges and grow in clusters at the base. The stems are square, upright, slender and tough. The whole plant has cilia. After flowering, the plant height can reach 100 to 150 cm. Willow verbena is slightly cold and bitter. The whole plant on the clean ground is used as medicine. It has the effects of clearing away heat and detoxifying, promoting blood circulation and removing blood stasis, and diuresis and swelling. The roots can cure diseases such as red and white diarrhea. It is a variety included in the "Pharmacopoeia of the People's Republic of China".

[0003] From the development history of plant tissue culture technology, the most commonly used tissue culture technology at present is plant in vitro rapid propagation technology, which is also the most effective plant tissue culture technology. Plant viruses are extremely harmful, not only seriously threatening the growth and survival rate of crops, but also adversely affecting the use value and economic value of crops. Plant tissue culture technology can better solve this problem, because plant tissue culture can cultivate and propagate virus-free plants. Among them, in vitro rapid propagation technology can complete plant reproduction in a shorter time, and can effectively ensure the efficiency of plant tissue culture. If in vitro rapid propagation technology is applied to those precious, scarce, new varieties, and species with low reproduction rates, it will have greater practical significance and value. Summary of the invention

[0004] 1. Technical issues to be resolved

[0005] In view of the deficiencies in the prior art, the present invention provides a tissue culture and rapid propagation system for Verbena officinalis, which solves the problem that there is no clear and appropriate numerical range standard for sterilization time, plant growth regulator ratio and seedling hardening time in the existing tissue culture and rapid propagation system for Verbena officinalis.

[0006] (II) Technical solution

[0007] To achieve the above objectives, the present invention is implemented through the following technical scheme: a tissue culture and rapid propagation system of Verbena officinalis, which specifically comprises the following steps:

[0008] S1. Preparation of test materials

[0009] The stem segments of Verbena officinalis with axillary buds planted in the Agricultural Bioengineering Research Institute of Huaxi District, Guizhou Province were selected, and the newly grown explants of the year were picked to establish a tissue culture in vitro rapid propagation system;

[0010] S2. Disinfection of stem segments with axillary buds

[0011] Cut the tender green branches of Verbena 5-6cm long, try to choose healthy branches that are free of pests and diseases and newly grown in the current year, cut off the leaves, keep some petioles near the axillary buds, and repeatedly rinse the axillary bud stem segments of Verbena 5-6cm long with a trickle of tap water for more than 1h, place them on a clean bench for standby, and design a total of 9 treatment groups with two disinfection reagents: 75% disinfectant alcohol (15, 30, 45s) and 20% sodium hypochlorite (6, 8, 10min). After disinfection with 75% disinfectant alcohol, wash the axillary bud stem segments 3 times with sterile water, and then disinfect with 20% sodium hypochlorite. After disinfection, use sterile absorbent paper to dry the stem segments. After the water is dried, use sterile tweezers on the clean bench to inoculate the axillary bud stem segment explants into the culture medium. Each treatment group is repeated 3 times, and 120 are inoculated each time. After 7d, the contamination rate, survival rate and mortality rate of the axillary bud stem segments are counted to determine the optimal disinfection time;

[0012] S3. Determination of axillary bud proliferation medium

[0013] When the axillary buds in the germination medium germinate to about 3-4 cm, the axillary buds are cut off, the base is cut obliquely, and inoculated into the axillary bud proliferation medium with different ratios of plant growth regulators added. The axillary bud proliferation medium is based on MS as the basic medium, and 8 treatment groups are added with different concentrations of 6-BA (1.00 mg / L, 1.50 mg / L), IBA (0.00, 0.10, 0.15 mg / L), and NAA (0.00, 0.05, 0.10 mg / L). Each treatment group is repeated 3 times, and 50 buds are inoculated each time. The growth of the proliferation buds is observed and recorded, and the number of proliferation buds of each axillary bud is counted after 30 days, and the proliferation coefficient is finally calculated;

[0014] S4. Determination of plant rooting medium

[0015] The rootless Verbena seedlings with good growth and a height of about 5 cm were cut obliquely at the bottom with a blade and inoculated into different culture media. The rooting culture medium was MS based on vermiculite or agar, and no IBA (0.00, 0.05, 0.10 mg / L) and NAA (0.00, 0.05, 0.10 mg / L) were added. There were 9 treatment groups. Each treatment group was repeated 3 times, and 40 plants were inoculated each time. The growth of the roots was observed and recorded, and the number of plants with roots, the number of roots and the length of roots were counted after 15 days. Finally, the rooting rate, the average number of roots and the average root length were calculated.

[0016] S5. Determination of seedling hardening time and transplanting medium

[0017] When the root length and root number of the Verbena officinalis plants in the rooting medium are appropriate, sterile seedlings with basically the same growth are selected for subsequent hardening and transplanting. During hardening, the sterile seedlings in the culture bottle are moved to the greenhouse, and different hardening times (2d, 4d, 6d) and transplanting substrates (Vvermiculite:Vnutrient soil=1:1, Vvermiculite:Vnutrient soil=1:2) are designed for experiments. A total of 6 treatment groups are designed, each treatment group is repeated 3 times, and 30 plants are transplanted in each treatment group. During the hardening process, the bottle cap is opened and the hardening is carried out under natural light. After the hardening is completed, the plants with the culture medium are rinsed and transplanted to the previously configured transplanting substrate. After transplanting, the plants are watered for the first time and covered with plastic film. The growth of the plants is observed and recorded, and the number of survivors is counted after 30 days, and the survival rate of the plants is finally calculated.

[0018] S6. Culture conditions

[0019] During the whole process of in vitro culture of Verbena officinalis explants, MS was used as the basic medium, the pH value was 6.01-6.02, the light intensity was 3500 lx, the temperature was (25±2℃), and the light time was 16h / d;

[0020] S7. Data processing

[0021] Data analysis and processing software was used to perform statistics, variance analysis, and LSD multiple comparison analysis on the experimental data.

[0022] Preferably, in S2, the calculation formula is as follows:

[0023] Contamination rate (%) = (number of contaminated explants / total number of explants inoculated) × 100%;

[0024] Survival rate (%) = (number of explants with growth vitality / total number of explants inoculated) × 100%;

[0025] Mortality rate (%) = (number of explants without growth vitality / total number of explants inoculated) × 100%.

[0026] Preferably, in S3, the calculation formula is as follows:

[0027] Axillary bud proliferation coefficient = number of axillary bud proliferation / number of axillary bud inoculations.

[0028] Preferably, in S4, the calculation formula is as follows:

[0029] Rooting rate (%) = (number of rooted explants / number of inoculated explants) × 100%;

[0030] Average rooting number = total number of roots of rooted plants / number of rooted explants;

[0031] Average root length = total root length of rooted plants / total number of roots.

[0032] Preferably, in S5, the calculation formula is as follows:

[0033] Plant survival rate (%) = (number of plants surviving 30 days after transplanting / total number of transplanted plants) × 100%.

[0034] Preferably, in S6, the components of the MS-based culture medium are 4.43 g / L MS powder, 30.00 g / L sucrose, and 8.00 g / L agar.

[0035] Preferably, in S7, the data analysis and processing software is Microsoft Excel and SPSS22.0 software.

[0036] (III) Beneficial effects

[0037] The present invention provides a tissue culture and rapid propagation system for Verbena officinalis. It has the following beneficial effects:

[0038] 1. The present invention provides a tissue culture and rapid propagation system of verbena. The system shows through research that different sterilization times have significant effects on the contamination rate, survival rate and mortality rate of stem segments with axillary buds. Considering the comprehensive contamination rate, survival rate and mortality rate, when the disinfection treatment conditions are 75% alcohol for 30 seconds and 20% sodium hypochlorite for 8 minutes, the contamination rate is 12.48%, the mortality rate is 4.44%, and the survival rate reaches 82.78%, which is the highest among the 9 treatment groups. The optimal disinfection time for the stem segments with axillary buds of verbena is 75% alcohol disinfection for 30 seconds and 20% sodium hypochlorite soaking for 8 minutes. When the 75% alcohol disinfection time remains unchanged, with 2 With the increase of 0% sodium hypochlorite disinfection time, the contamination rate of stem segments with axillary buds gradually decreased and the survival rate gradually increased. When the disinfection conditions were 75% alcohol for 15 seconds and 20% sodium hypochlorite for 6 minutes, the contamination rate was as high as 39.72%, and the survival rate was as low as 55.00%. When the disinfection conditions were 75% alcohol for 45 seconds and 20% sodium hypochlorite for 10 minutes, the contamination rate was as low as 4.17%, the mortality rate was as high as 15.28%, and the survival rate was 80.56%. It can be concluded that the longer the disinfection time is, the better. Too long time will lead to an increase in the number of explant deaths, and too short time will lead to incomplete disinfection of the explants.

[0039] 2. The present invention provides a tissue culture and rapid propagation system for Verbena willowii. Studies have shown that different ratios of plant growth regulators have a significant effect on the proliferation of axillary buds. Based on the comprehensive proliferation coefficient and the growth of seedlings, when the culture medium is MS+1.50 mg / L6-BA+0.10 mg / L IBA, the proliferation coefficient reaches 4.77 after 30 days, and under this condition, the germination rate is the highest. Studies have found that as the concentration of 6-BA increases, the proliferation coefficient also increases. When the concentration of 6-BA reaches 1.50 mg / L, the proliferation coefficient reaches above 3.61. When the concentration of 6-BA is constant, the IBA combination has a better proliferation effect than the NAA combination.

[0040] 3. The present invention provides a tissue culture and rapid propagation system of verbena. Studies have shown that different plant growth regulator ratios have a significant effect on the rooting of tissue culture seedlings. Under the condition of MS+0.10mg / LIBA+0.05mg / LNAA, the rooting rate is 100% after 15 days, the average number of roots is 40.43, and the average root length is 3.27cm, which is the best in the entire treatment. Therefore, the culture medium suitable for the rooting of verbena tissue culture seedlings is 0.1mg / LIBA+0.05mg / LNAA, the average number of roots and the average root length of MS+0.05mg / LIBA are greater than the average number of roots and the average root length of MS+0.05mg / LNAA, and the average number of roots and the average root length of MS+0.10mg / LIBA are greater than those of MS+0.10mg / LNAA average root number and average root length, which shows that at the same concentration, IBA has a more obvious effect on promoting rooting of Verbena officinalis. On this basis, using the above-mentioned determined hormone concentration, the culture medium is compared with vermiculite as the matrix and agar as the matrix. The results show that after 15 days of using vermiculite matrix as the rooting medium compared with agar matrix, the average root length in agar matrix is ​​3.27±0.21cm, and the average root length in vermiculite matrix can reach 4.50±0.50cm, which is significantly greater than the average root length in agar matrix. After 45 days, the average root length in agar matrix is ​​14.33±2.08cm, and the average root length in vermiculite matrix can reach 42.33±2.51cm, which is significantly greater than the average root length in agar matrix. Therefore, this experiment uses vermiculite instead of agar as the matrix in the rooting stage. At the same time, vermiculite can reduce the cost of the experiment.

[0041] 4. The present invention provides a tissue culture and rapid propagation system of verbena. Studies have shown that as the hardening time gradually increases, the survival rate decreases from high to low. When the hardening time is 4 days, the survival rate is above 77.67%, which is the most suitable number of days for hardening. The results show that when the ratio of V vermiculite: V nutrient soil is 1:2 and the hardening time is 4 days, the survival rate is the highest among the 6 treatment groups, which is 93.33%. Moreover, under this condition, the plants grow vigorously after transplanting, the leaves stretch, and the roots continue to extend downward. Therefore, the hardening time of verbena is 4 days, and the transplanting medium selects V vermiculite: V nutrient soil = 1:2. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 It is a schematic diagram showing the effect of different disinfection times on stem segments with axillary buds according to the present invention;

[0043] Figure 2 Schematic diagram of the effect of different plant growth regulator ratios on axillary bud proliferation of the present invention;

[0044] Figure 3 The growth diagram of the axillary buds of the present invention after proliferation and culture for 30 days in different culture media;

[0045] Figure 4 It is a schematic diagram of the effects of different plant growth regulators of the present invention on plant rooting;

[0046] Figure 5 This is a diagram showing the rooting of the plants of the present invention after being cultured on different rooting medium for 15 days;

[0047] Figure 6 This is a schematic diagram of the effects of different seedling hardening times and substrates on plant transplanting according to the present invention;

[0048] Figure 7 This is a surviving plant diagram of Verbena officinalis of the present invention;

[0049] Figure 8 The figure shows the rooting conditions of the plants of the present invention cultured in different substrates. DETAILED DESCRIPTION

[0050] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. Example

[0051] like Figure 1-8 As shown, the embodiment of the present invention provides a tissue culture and rapid propagation system of Verbena officinalis, which specifically includes the following steps:

[0052] S1. Preparation of test materials

[0053] We selected stem segments of Verbena officinalis with axillary buds planted in the Agricultural Bioengineering Research Institute of Huaxi District, Guizhou Province, and picked the newly grown explants of the year to establish a tissue culture in vitro rapid propagation system to improve the experimental effect;

[0054] S2. Disinfection of stem segments with axillary buds

[0055] Cut 5-6 cm long tender green branches of Verbena willowii, try to choose healthy branches that are free of diseases and insect pests and newly grown in the current year, cut off the leaves, and retain part of the petiole near the axillary buds. Rinse the axillary bud stems of Verbena willowii repeatedly with a stream of tap water for more than 1 hour, place them on a clean bench for use, and design the experiment with two disinfecting agents: 75% disinfectant alcohol (15, 30, 45s) and 20% sodium hypochlorite (6, 8, 10min), for a total of 9 treatment groups. After disinfection with 75% disinfectant alcohol, the axillary bud stem segments were washed 3 times with sterile water, and then disinfected with 20% sodium hypochlorite. After disinfection, the stem segments were dried with sterile absorbent paper. After the water was dried, the stem segments with axillary buds were inoculated into the culture medium with sterile tweezers on the clean bench. Each treatment group was repeated 3 times, and 120 were inoculated each time. After 7 days, the contamination rate, survival rate and mortality rate of the stem segments with axillary buds were calculated to determine the optimal disinfection time. The establishment of the sterile system of the stem segments with axillary buds was designed with a combination of 75% alcohol and 20% sodium hypochlorite. The results showed that the best effect was achieved under the conditions of 75% alcohol disinfection for 30 seconds and 20% sodium hypochlorite disinfection for 8 minutes. After 7 days, the contamination rate was 12.48%, the mortality rate was 4.44%, and the survival rate reached 82.78%.

[0056] S3. Determination of axillary bud proliferation medium

[0057] When the axillary buds in the germination medium germinate to about 3-4 cm, the axillary buds are cut off, the base is cut obliquely, and inoculated into the axillary bud proliferation medium with different ratios of plant growth regulators added. The axillary bud proliferation medium is based on MS and supplemented with different concentrations of 6-BA (1.00 mg / L, 1.50 mg / L), IBA (0.00, 0.10, 0.15 mg / L), NAA (0.00, 0.05, 0.10 mg / L) were used for 8 treatment groups. Each treatment group was repeated 3 times, and 50 buds were inoculated each time. The growth of the proliferating buds was observed and recorded, and the number of proliferating buds of each axillary bud was counted after 30 days. Finally, the proliferation coefficient was calculated, and the axillary bud proliferation medium was determined. Different concentrations of 6-BA, NAA, and IBA were added to the MS medium to induce the proliferation of axillary buds. The results showed that when the medium was MS+1.5mg / L6-BA+0.10mg / L IBA, the proliferation coefficient reached 4.77 after 30 days, and the number of clustered buds produced was large.

[0058] S4. Determination of plant rooting medium

[0059] The rootless seedlings of Verbena officinalis with good growth and a height of about 5 cm were cut obliquely at the lower end with a blade and inoculated into different culture media. The rooting culture medium was MS with vermiculite or agar as the matrix, and no IBA (0.00, 0.05, 0.10 mg / L) and NAA (0.00, 0.05, 0.10 mg / L) were added. There were 9 treatment groups, each treatment group was repeated 3 times, 40 plants were inoculated each time, the growth of roots was observed and recorded, and the number of roots, root number and root length of plants were counted after 15 days. Finally, the rooting rate, average number of roots and average root length were calculated. The rooting culture medium for plants was determined. Different concentrations of IBA and NAA were added to the MS medium with vermiculite and agar as the matrix for rooting induction culture. The results showed that MS+0.10 mg / L IBA+0.05 mg / L NAA, a medium with vermiculite as the matrix, had a rooting rate of 100% after 15 days, an average root length of 3.27 cm, and good overall plant growth;

[0060] S5. Determination of seedling hardening time and transplanting medium

[0061] When the root length and root number of the Verbena plants in the rooting medium are appropriate, sterile seedlings with basically the same growth are selected for subsequent hardening and transplanting. During hardening, the sterile seedlings in the culture bottles are moved to the greenhouse, and different hardening times (2d, 4d, 6d) and transplanting substrates (Vvermiculite: Vnutrient soil = 1:1, Vvermiculite: Vnutrient soil = 1:2) are designed for experiments. There are a total of 6 treatment groups, each of which is repeated 3 times. 30 plants are transplanted in each treatment group. The bottle caps are opened during the hardening process, and the hardening is carried out under natural light conditions. After the hardening was completed, the plants with the culture medium were rinsed and transplanted to the previously configured transplanting medium. After the first watering after transplanting, they were covered with plastic film, and the growth of the plants was observed and recorded. The number of survivors was counted after 30 days, and the survival rate of the plants was finally calculated. The hardening time and the transplanting medium were determined. The experiment was designed based on the hardening time and the transplanting medium. The results showed that after 4 days of hardening under natural light conditions, the survival rate was 93.33% after 30 days when they were transplanted to the medium of V vermiculite: V nutrient soil = 1:2.

[0062] S6. Culture conditions

[0063] During the whole process of in vitro culture of Verbena officinalis explants, MS was used as the basic medium, the pH value was 6.01-6.02, the light intensity was 3500 lx, the temperature was (25±2℃), and the light time was 16h / d;

[0064] S7. Data processing

[0065] Data analysis and processing software was used to perform statistics, variance analysis, and LSD multiple comparison analysis on the experimental data to obtain the experimental results.

[0066] In S2, the calculation formula is as follows:

[0067] Contamination rate (%) = (number of contaminated explants / total number of explants inoculated) × 100%;

[0068] Survival rate (%) = (number of explants with growth vitality / total number of explants inoculated) × 100%;

[0069] Mortality (%) = (number of explants without growth vitality / total number of explants inoculated) × 100%;

[0070] In S3, the calculation formula is as follows:

[0071] Axillary bud proliferation coefficient = number of buds proliferated by axillary buds / number of axillary buds inoculated;

[0072] In S4, the calculation formula is as follows:

[0073] Rooting rate (%) = (number of rooted explants / number of inoculated explants) × 100%;

[0074] Average rooting number = total number of roots of rooted plants / number of rooted explants;

[0075] Average root length = total root length of rooted plants / total number of roots;

[0076] In S5, the calculation formula is as follows:

[0077] Plant survival rate (%) = (number of surviving plants 30 days after transplanting / total number of transplanted plants) × 100%, which is helpful to provide a basis for calculation of various test results. In S6, the components of MS as the basic culture medium are MS powder 4.43g / L, sucrose 30.00g / L, and agar 8.00g / L, ensuring the basic use and cultivation function of the culture medium. In S7, the data analysis and processing software is Microsoft Excel and SPSS22.0 software, which is convenient for processing test data.

[0078] like Figure 8 As shown, A. the growth of the plant on MS + 0.1 mg / L IBA + 0.05 mg / L NAA agar matrix medium for 15 days; B. the growth of the plant on MS + 0.1 mg / L IBA + 0.05 mg / L NAA vermiculite matrix medium for 15 days; C. the growth of the plant on MS + 0.1 mg / L IBA + 0.05 mg / L NAA agar and vermiculite matrix medium for 45 days. The scale bars in the figures are 1 cm.

[0079] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for rapid propagation of Verbena officinalis tissue culture, characterized in that: The specific steps include: S1. Preparation of test materials The stem segments of Verbena officinalis with axillary buds planted in the Agricultural Bioengineering Research Institute of Huaxi District, Guizhou Province were selected, and the newly grown explants of the year were picked to establish a tissue culture in vitro rapid propagation system; S2. Disinfection of stem segments with axillary buds Cut the tender green branches of Verbena 5-6cm long, try to choose healthy branches that are free of diseases and insect pests and newly grown in the current year, cut off the leaves, keep some petioles near the axillary buds, rinse the axillary bud stem segments of Verbena 5-6cm long with a trickle of tap water for more than 1h, place them on a clean bench for standby, and design the experiment with two disinfection reagents of 75% disinfectant alcohol for 15, 30, 45s and 20% sodium hypochlorite for 6, 8, 10min, with a total of 9 treatment groups; after disinfection with 75% disinfectant alcohol, use sterile water to wash the axillary bud stem segments 3 times, and then disinfect with 20% sodium hypochlorite. After disinfection, use sterile absorbent paper to dry the stem segments. After the water is dried, use sterile tweezers on the clean bench to inoculate the axillary bud stem segment explants into the culture medium. Each treatment group is repeated 3 times, and 120 are inoculated each time. After 7d, the contamination rate, survival rate and mortality rate of the axillary bud stem segments are counted to determine the optimal disinfection time; S3. Determination of axillary bud proliferation medium When the axillary buds in the germination medium germinate to about 3-4 cm, cut off the axillary buds, cut the base obliquely, and inoculate them into the axillary bud proliferation medium, which is composed of MS, 6-BA 1.50 mg / L, and IBA 0.10 mg / L. Repeat 3 times, inoculate 50 buds each time, observe and record the growth of the proliferation buds, and count the number of proliferation buds of each axillary bud after 30 days, and finally calculate the proliferation coefficient; S4. Determination of plant rooting medium The rootless Verbena seedlings with good growth and a height of about 5 cm were cut obliquely at the bottom with a blade and inoculated into the rooting medium, which was composed of MS, IBA 0.10 mg / L, and NAA 0.05 mg / L with vermiculite or agar as the matrix. The process was repeated 3 times, with 40 seedlings inoculated each time. The root growth was observed and recorded, and the number of roots, root number and root length of the plants were counted after 15 days. Finally, the rooting rate, average number of roots and average root length were calculated. S5. Determination of seedling hardening time and transplanting medium When the root length and root number of the Verbena officinalis plants in the rooting medium are suitable, sterile seedlings with basically the same growth are selected for subsequent hardening and transplanting. During hardening, the sterile seedlings in the culture bottle are moved to the greenhouse, and the hardening time is designed to be 4 days and the transplanting matrix V vermiculite: V nutrient soil = 1:2 for testing, which is repeated 3 times, and 30 plants are transplanted each time. During the hardening process, the bottle cap is opened and the hardening is carried out under natural light conditions. After the hardening is completed, the plants with the culture medium are rinsed and transplanted to the previously configured transplanting matrix. After transplanting, water is poured for the first time and covered with plastic film, the growth of the plants is observed and recorded, and the number of survivors is counted after 30 days, and the survival rate of the plants is finally calculated; S6. Culture conditions During the whole process of in vitro culture of Verbena officinalis explants, MS was used as the basic culture medium, the pH value was 6.01-6.02, the light intensity was 3500lx, the temperature was 25±2℃, and the light time was 16h / d; the components of MS as the basic culture medium were 4.43g / L MS powder, 30.00g / L sucrose, and 8.00g / L agar; S7. Data processing Data analysis and processing software was used to perform statistics, variance analysis, and LSD multiple comparison analysis on the experimental data.

2. A method for rapid propagation of Verbena officinalis tissue culture according to claim 1, characterized in that: In S2, the calculation formula is as follows: Contamination rate (%) = (number of contaminated explants / total number of explants inoculated) × 100%; Survival rate (%) = (number of explants with growth vitality / total number of explants inoculated) × 100%; Mortality rate (%) = (number of explants without growth vitality / total number of explants inoculated) × 100%.

3. A method for rapid propagation of Verbena officinalis tissue culture according to claim 1, characterized in that: In S3, the calculation formula is as follows: Axillary bud proliferation coefficient = number of axillary bud proliferation / number of axillary bud inoculations.

4. A method for rapid propagation of Verbena officinalis tissue culture according to claim 1, characterized in that: In S4, the calculation formula is as follows: Rooting rate (%) = (number of rooted explants / number of inoculated explants) × 100%; Average rooting number = total number of roots of rooted plants / number of rooted explants; Average root length = total root length of rooted plants / total number of roots.

5. A method for rapid propagation of Verbena officinalis tissue culture according to claim 1, characterized in that: In S5, the calculation formula is as follows: Plant survival rate (%) = (number of plants surviving 30 days after transplanting / total number of transplanted plants) × 100%.

6. A method for tissue culture and rapid propagation of Verbena officinalis according to claim 1, characterized in that: In S7, the data analysis and processing software is Microsoft Excel and SPSS22.0 software.

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