Tissue culture method taking centella asiatica inflorescence as explant

Abstract

The invention relates to the technical field of tissue culture, in particular to a tissue culture method taking centella asiatica inflorescence as an explant. Firstly, centella creeping stems are cultured to obtain aseptic seedlings, non-flowering inflorescences of the aseptic seedlings serve as explants, tissue culture of centella is conducted, tissue cells of the non-flowering inflorescences are vigorous in division activity and good in development, and the induction success rate of centella can be increased; meanwhile, non-flowering inflorescences of aseptic seedlings are used as explants for tissue culture, the disinfection success rate of the centella asiatica explants can be effectively increased, the induction period in the tissue culture process is shortened, and therefore the propagation efficiency of centella asiatica is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of tissue culture technology, in particular to a tissue culture method using flower spikes of Centella asiatica as explants. BACKGROUND

[0002] The information disclosed in this Background section is only for the purpose of increasing an understanding of the general background of the application and does not necessarily constitute an admission or a recognition that the information forms part of the prior art already known to a person of ordinary skill in the art.

[0003] Centella asiatica is a perennial herb widely distributed in tropical and subtropical regions around the world. It has important medicinal, edible and cosmetic values. Its extract is rich in active ingredients such as triterpenoid saponins and flavonoids, and has anti-inflammatory, antibacterial, antioxidant, and wound healing effects. In traditional medicine, it is used to treat various diseases such as skin ulcers, burns, eczema, and varicose veins. Modern research shows that Centella asiatica extract also has anti-aging, anti-tumor, and neuroprotective effects, and has a broad application prospect.

[0004] However, the global supply of Centella asiatica raw materials is highly dependent on (about 90%) wild resources; due to overexploitation, habitat destruction (urbanization, herbicide use), and its own characteristics such as small size, slow growth, and difficult harvesting, the amount of wild Centella asiatica resources has decreased dramatically, making it difficult to meet the needs of industrialization. Therefore, introduction and cultivation, artificial breeding, and other methods have become new ways to effectively utilize Centella asiatica resources.

[0005] Currently, artificial breeding mainly uses wild seedlings for root division propagation or uses stolon for asexual reproduction. However, these methods have obvious defects: low survival rate of division propagation, small propagation coefficient; dependent on the number of mother plants, difficult to obtain a large number of seedlings in the short term; and unable to guarantee genetic consistency and seedling quality.

[0006] Therefore, developing a high-efficiency, stable, low-cost, and fast-providing method for mass production of high-quality, genetically consistent seedlings is of great importance for protecting wild resources, achieving sustainable utilization, meeting the growing market demand, and promoting the development of related industries. SUMMARY

[0007] To overcome the above problems, the present application provides a tissue culture method using flower spikes of Centella asiatica as explants.

[0008] To achieve the above technical purposes, the present application adopts the following technical solutions: In a first aspect of the present application, a tissue culture method using flower spikes of Centella asiatica as explants is provided, comprising the following steps: (1) Collecting creeping stems of Centella asiatica, washing and disinfecting, inoculating in bud induction medium, and culturing to obtain aseptic seedlings; (2) Collecting inflorescences of aseptic seedlings which have not yet flowered, using the same as explants, inoculating in callus induction medium, and inducing to obtain callus; (3) Inoculating the callus in adventitious bud induction medium, and inducing to form adventitious buds; (4) Inoculating the induced adventitious buds in rooting medium, and culturing Centella asiatica tissue culture seedlings; (5) Transplanting the Centella asiatica tissue culture seedlings after hardening.

[0009] In one or more embodiments, the length of the creeping stem of Centella asiatica is 1.2-1.8 cm, preferably 1.5 cm. The length of 1.2-1.8 cm of the creeping stem is in the vigorous growth period, the cell division is active, the differentiation ability is strong, and it is easier to induce dedifferentiation and redifferentiation; meanwhile, the limited length will not increase the difficulty of surface sterilization.

[0010] In one or more embodiments, the method for washing and disinfecting is: washing with running water for 25-40 min, then disinfecting with 0.1% HgCl2 solution on the clean bench for 3-15 min, and then rinsing with sterile water for 4-6 times.

[0011] Preferably, the method for washing and disinfecting is: washing with running water for 30 min, then disinfecting with 0.1% HgCl2 solution on the clean bench for 7 min, and then rinsing with sterile water for 5 times. The disinfection time will affect the survival rate of the final explants, and research shows that the survival rate of the explants is the highest when disinfecting with 0.1% HgCl2 solution for 7 min.

[0012] In one or more embodiments, in step (1), the formula of the bud induction medium is: MS medium+6-benzylaminopurine (6-BA) 0.5-1.0 mg / L+naphthalene acetic acid (NAA) 0.1 mg / L+sucrose 30 g / L+agar 8 g / L, and the pH value is 5.8.

[0013] Preferably, in step (1), the formula of the bud induction medium is: MS medium+6-benzylaminopurine (6-BA) 0.8 mg / L+naphthalene acetic acid (NAA) 0.1 mg / L+sucrose 30 g / L+agar 8 g / L, and the pH value is 5.8. Research shows that the concentration of 6-benzylaminopurine (6-BA) will affect the bud induction rate (the number of buds / the total number), and when the concentration is 0.8 mg / L, the bud induction rate is the highest.

[0014] In one or more embodiments, in step (1), the culture conditions are as follows: temperature 22-24°C, light intensity 1500-2000 Lux, and light duration 12-14 hours.

[0015] In one or more embodiments, in step (1), the culture is performed for 15-20 days to obtain sterile seedlings.

[0016] In one or more embodiments, in step (2), the length of the inflorescence is 0.4-0.6 cm, preferably 0.5 cm. The inflorescence with a length of 0.4-0.6 cm is in the early stage of floral primordium formation, and the cells have not yet fully differentiated, thus having stronger dedifferentiation ability and being easy to induce callus or directly regenerate.

[0017] In one or more embodiments, in step (2), the callus induction medium has the following formulation: MS medium + thidiazuron (TDZ) 0.5-1.5 mg / L + naphthalene acetic acid (NAA) 0.3 mg / L + sucrose 30 g / L + agar 8 g / L, and the pH value is 5.8.

[0018] Preferably, the callus induction medium has the following formulation: MS medium + thidiazuron (TDZ) 1.2 mg / L + naphthalene acetic acid (NAA) 0.3 mg / L + sucrose 30 g / L + agar 8 g / L, and the pH value is 5.8. Studies have shown that the concentration of thidiazuron (TDZ) affects the callus induction rate, and when the concentration of thidiazuron (TDZ) is 1.2 mg / L and the concentration of naphthalene acetic acid (NAA) is 0.3 mg / L, the callus induction rate is the highest, and the callus formation speed is fast, which is suitable for subsequent adventitious bud induction.

[0019] In one or more embodiments, in step (2), the culture conditions are as follows: temperature 22-24°C, light intensity 1500-2000 Lux, and light duration 12-14 hours; and the culture time is 28-32 days.

[0020] In one or more embodiments, in step (3), the adventitious bud induction medium has the following formulation: MS medium + thidiazuron (TDZ) 0.5-2.0 mg / L + naphthalene acetic acid (NAA) 0.1 mg / L + sucrose 30 g / L + agar 8 g / L, and the pH value is 5.8.

[0021] Preferably, the formula of the adventitious bud differentiation medium is: MS medium+thidiazuron (TDZ) 1.5 mg / L+naphthalene acetic acid (NAA) 0.1 mg / L+sucrose 30 g / L+agar 8 g / L, and the pH value is 5.8. Studies have shown that the concentration of thidiazuron (TDZ) affects the differentiation rate (the number of differentiated buds / the number of inoculated calluses), and when the concentration of thidiazuron (TDZ) is 1.5 mg / L and the concentration of naphthalene acetic acid (NAA) is 0.1 mg / L, the differentiation rate of the adventitious bud is the highest, and the bud body is robust and the leaf color is emerald green, which is suitable for subsequent rooting culture.

[0022] In one or more embodiments, in step (3), the culture conditions are: temperature 22-24 ℃, light 1500-2000 Lux, light for 12-14 h; and the culture time is 38-42 days.

[0023] In one or more embodiments, in step (4), the length of the adventitious bud is 1.8-2.2 cm, preferably 2.0 cm. The adventitious bud with the length usually has a certain tissue differentiation (such as preliminary formation of vascular bundles), which can provide sufficient nutrients and energy to support the rooting process; at the same time, the adventitious bud with the length usually accumulates an appropriate amount of auxin, which is conducive to inducing the differentiation of root primordia.

[0024] In one or more embodiments, in step (4), the formula of the rooting medium is: MS medium+indole-3-butyric acid (IBA) 0.5 mg / L+sucrose 30 g / L+agar 8 g / L, and the pH value is 5.8.

[0025] In one or more embodiments, in step (4), the culture conditions are: temperature 22-24 ℃, light 1500-2000 Lux, light for 12-14 h; and the rooting time is 19-21 days.

[0026] In one or more embodiments, in step (5), the seedling raising method is: the spergularia rubra tissue culture seedling is gently taken out from the bottle, the agar on the root is washed away with water, and is transplanted into a mixed substrate of perlite and peat soil 1:3 (volume ratio), covered with plastic film to maintain 60% humidity, temperature 21-25 ℃, light 1500 Lux, and light cycle 14 h / 10 h (light / dark).

[0027] The present application has the following beneficial effects: (1) The present application firstly cultures the creeping stem of the Centella asiatica to obtain aseptic seedlings, takes the inflorescence of the aseptic seedlings which has not yet flowered as an explant, and carries out tissue culture of the Centella asiatica, so that the cell division of the inflorescence tissue which has not yet flowered is vigorous and develops well, the success rate of induction of the Centella asiatica can be improved, meanwhile, taking the inflorescence of the aseptic seedlings which has not yet flowered as an explant to carry out tissue culture can effectively improve the disinfection success rate of the explant of the Centella asiatica, shorten the induction period in the process of tissue culture, and thus improve the propagation efficiency of the Centella asiatica.

[0028] (2) The tissue culture method provided by the present application realizes efficient induction of callus, high differentiation rate of adventitious buds and rapid generation of root systems by optimizing the disinfection process, the culture medium formula and the culture system, significantly improves the propagation efficiency and the survival rate of transplanting, and provides reliable technical support for large-scale production. BRIEF DESCRIPTION OF DRAWINGS

[0029] The accompanying drawings, which form a part of this specification, are included to provide a further understanding of the application and are incorporated herein for illustrative purposes. The embodiments illustrated in the drawings are not intended to be unduly limited to the illustrated embodiments but rather are meant to be illustrative of the present application.

[0030] Figure 1 It is the bud diagram of the aseptic seedling cultured in Example 1; Figure 2 It is the inflorescence callus diagram cultured in Example 1; Figure 3 It is the adventitious bud diagram cultured in Example 1; Figure 4 It is the Centella asiatica tissue culture seedling diagram cultured in Example 1; Figure 5 It is the survival seedling diagram transplanted in Example 1. DETAILED DESCRIPTION

[0031] It should be noted that the following detailed description is exemplary in nature and is intended to provide further description of the present application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0032] It should be noted that the terms used herein are for the purpose of describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, the singular form is intended to include the plural form unless the context clearly indicates otherwise, and it should also be understood that when the terms "comprise" and / or "include" are used in the specification, there is a reference to the presence of a feature, step, operation, device, component and / or combinations thereof.

[0033] In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below with specific embodiments.

[0034] Embodiment 1 A tissue culture method using the inflorescence of Centella asiatica as an explant, comprising the following steps: (1) Collecting the creeping stem segments of Centella asiatica that are healthy and free of pests and diseases, cutting them into small segments with a length of about 1.5 cm, washing them with running water for 30 min, then sterilizing them with a 0.1% HgCl2 solution on a clean bench for 7 min, then washing them with sterile water for 5 times, and then inoculating them into a bud induction medium; the formula of the bud induction medium is: MS medium + 6-benzylaminopurine (6-BA) 0.8 mg / L + naphthalene acetic acid (NAA) 0.1 mg / L + sucrose 30 g / L + agar 8 g / L, with a pH value of 5.8; the specific induction culture conditions are: temperature 23 ℃, light intensity 1800 Lux, light for 13 h; and aseptic seedlings are obtained after 18 days of culture, and the aseptic seedling buds are as shown in Figure 1 . If the inflorescence of Centella asiatica is directly collected from the field, the contamination rate of the explant is high, which is not conducive to subsequent work such as callus induction; therefore, the creeping stems of Centella asiatica are collected, washed and sterilized, and then inoculated into the bud induction medium to obtain aseptic seedlings.

[0035] (2) Collecting the inflorescences of aseptic seedlings with a length of 0.5 cm that have not yet flowered, using them as explants, inoculating them into a callus induction medium, and inducing callus (as shown in Figure 2 ); the formula of the callus induction medium is: MS medium + thidiazuron (TDZ) 1.2 mg / L + naphthalene acetic acid (NAA) 0.3 mg / L + sucrose 30 g / L + agar 8 g / L, with a pH value of 5.8; the specific induction culture conditions are: temperature 23 ℃, light intensity 1800 Lux, light for 13 h; and the culture time is 30 days.

[0036] (3) Inoculating the callus into an adventitious bud induction medium to induce the formation of adventitious buds (as shown in Figure 3 ); the formula of the adventitious bud induction medium is: MS medium + thidiazuron (TDZ) 1.5 mg / L + naphthalene acetic acid (NAA) 0.1 mg / L + sucrose 30 g / L + agar 8 g / L, with a pH value of 5.8; the specific differentiation culture conditions are: temperature 23 ℃, light intensity 1800 Lux, light for 13 h; and the culture time is 40 days.

[0037] (4) Inoculating the induced adventitious buds with a length of 2.0 cm into a rooting medium to culture Centella asiatica tissue culture seedlings (as shown in Figure 4The rooting medium formula is: MS medium + indole butyric acid (IBA) 0.5 mg / L + sucrose 30 g / L + agar 8 g / L, pH value is 5.8; the specific differentiation culture conditions are: temperature 23 ℃, illumination is 1800 Lux, illumination 13 h; the rooting time is 20 days; the rooting rate reaches about 80.00%, and the average number of roots is 4-6 per plant.

[0038] (5) The tissue culture seedlings of Centella asiatica are transplanted after acclimatization; the acclimatization method is: the tissue culture seedlings of Centella asiatica are taken out from the bottle, the agar on the roots is washed off with water, and the seedlings are transplanted into the mixed substrate of perlite and peat soil 1:3 (volume ratio), covered with plastic film to maintain 60% humidity, temperature 23 ℃, illumination 1500 Lux, light cycle 14 h / 10 h (light / dark), acclimatization time 7-10 days, and then transplanted into the field, and the transplanted survival seedlings are as shown in Figure 5 .

[0039] Example 2 Optimization of disinfection time: The stolon of Centella asiatica which is healthy and free of pests and diseases is cut into small pieces with a length of about 1.5 cm, washed with running water for 30 min, and then disinfected with 0.1% HgCl2 solution on the clean bench for 3-15 min, and the contamination rate and survival rate are counted after 14 days, and the results are shown in Table 1.

[0040] Table 1 Optimization of disinfection time

[0041] The results show that the disinfection time will affect the contamination rate and the survival rate of explants, and the research shows that disinfection with 0.1% HgCl2 solution for 7 min can control the contamination rate (26.67%) while maintaining a high explant survival rate (63.33%), so the optimal disinfection time is 7 min as the standard disinfection time for subsequent experiments.

[0042] Example 3 Optimization of hormone ratio in bud induction medium: The disinfected stolon is inoculated into MS basic medium containing different concentrations of 6-benzylaminopurine (6-BA) and naphthalene acetic acid (NAA), 30 replicates per treatment, the culture conditions are: temperature 25±1 ℃, illumination intensity 2000 Lux, illumination time 14 h / d, culture period 15-20 days, and the bud induction rate (number of buds / total number) is counted, and the results are shown in Table 2.

[0043] Table 2 Optimization of hormone ratio in bud induction medium

[0044] The results show that the concentration of 6-benzylaminopurine (6-BA) will affect the bud induction rate (the number of buds / the total number), when the concentration of 6-BA is 0.8 mg / L, NAA is 0.1 mg / L (treatment 2), the bud induction rate is the highest (83.33%), and the aseptic seedling grows rapidly and healthily, which is suitable as the explant for subsequent callus induction.

[0045] Example 4 Optimization of hormone ratio in callus induction medium: The aseptic seedlings with flower spikes of 0.5 cm in length were collected and used as explants, which were inoculated in callus induction medium with different concentrations of TDZ, 30 replicates per treatment, the culture conditions were: temperature 25±1 ℃, light intensity 2000 Lux, light time 14 h / d, culture period 30 days, the callus induction rate (the number of calli / the total number) was counted, and the results are shown in Table 3.

[0046] Table 3 Optimization of hormone ratio in callus induction medium

[0047] The results show that the concentration of thidiazuron (TDZ) will affect the callus induction rate (the number of calli / the number of explants), when the concentration of TDZ is 1.2 mg / L, naphthalene acetic acid (NAA) is 0.3 mg / L, the callus induction rate is the highest, and the callus tissue is light green and relatively firm, which is suitable for subsequent adventitious bud induction.

[0048] Example 5 Optimization of hormone ratio in adventitious bud differentiation medium: The callus tissues induced in Examples 2 and 3 were transferred to differentiation medium to evaluate the effect of TDZ and NAA ratio on adventitious bud differentiation. 30 callus tissues were inoculated per treatment, the temperature was 25±1 ℃, the light intensity was 2000 Lux, the light time was 14 h / d, the culture period was 40 days, the adventitious bud differentiation rate (the number of adventitious buds / the total number of calli) was counted, and the results are shown in Table 4.

[0049] Table 4 Optimization of hormone ratio in adventitious bud differentiation medium

[0050] The results show that the concentration of thidiazuron (TDZ) will affect the differentiation rate (the number of differentiated buds / the number of inoculated calli), when the concentration of thidiazuron (TDZ) is 1.5 mg / L, naphthalene acetic acid (NAA) is 0.1 mg / L, the adventitious bud differentiation rate is the highest, and the bud body is healthy and the leaf color is emerald green, which is suitable for subsequent rooting culture.

[0051] The above merely provides the preferred embodiments of the present application, and is not used to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the principles and technical scope of the present application shall fall into the scope of the present application.

Claims

1. A method of tissue culture using inflorescence of Centella asiatica as explant, characterized by, The method comprises the following steps: (1) collecting creeping stems of Centella asiatica, cleaning and disinfecting, inoculating in bud induction medium, and culturing to obtain sterile seedlings; (2) collecting inflorescences of the sterile seedlings which have not yet flowered, taking the inflorescences as explants, inoculating in callus induction medium, and inducing to obtain callus; (3) inoculating the callus in adventitious bud induction medium, and inducing to form adventitious buds; (4) inoculating the induced adventitious buds in rooting medium, and culturing Centella asiatica tissue culture seedlings; (5) transplanting the Centella asiatica tissue culture seedlings after seedling raising.

2. The method of claim 1, wherein the inflorescence of Centella asiatica is used as an explant. The length of the creeping stem of Centella asiatica is 1.2-1.8 cm, preferably 1.5 cm; Or, the method for cleaning and disinfecting is as follows: washing with flowing water for 25-40 min, then disinfecting with a 0.1% HgCl2 solution for 3-15 min on the clean bench, and then rinsing with sterile water for 4-6 times.

3. The method of claim 1, wherein the flower spikes of Centella asiatica are used as explants. In step (1), the formula of the bud induction medium is: MS medium+6-benzylaminopurine (6-BA) 0.5-1.0 mg / L+naphthalene acetic acid (NAA) 0.1 mg / L+sucrose 30 g / L+agar 8 g / L, and the pH value is 5.8; Or, the culture condition is: temperature 22-24 ℃, illumination 1500-2000 Lux, and illumination time 12-14 h; Or, in step (1), the sterile seedlings are obtained after culturing for 7-15 days.

4. The method of claim 1, wherein the flower spikes of Centella asiatica are used as explants. In step (2), the length of the inflorescence is 0.4-0.6 cm, preferably 0.5 cm.

5. The method of claim 1, wherein the flower spikes of Centella asiatica are used as explants. In step (2), the formula of the callus induction medium is: MS medium+thidiazuron (TDZ) 0.5-1.5 mg / L+naphthalene acetic acid (NAA) 0.3 mg / L+sucrose 30 g / L+agar 8 g / L, and the pH value is 5.8; Or, in step (2), the culture condition is: temperature 22-24 ℃, illumination 1500-2000 Lux, illumination time 12-14 h, and culture time 28-32 days.

6. The method of claim 1, wherein the flower spikes of Centella asiatica are used as explants. In step (3), the formula of the adventitious bud induction medium is: MS medium+thidiazuron (TDZ) 0.5-2.0 mg / L+naphthalene acetic acid (NAA) 0.1 mg / L+sucrose 30 g / L+agar 8 g / L, and the pH value is 5.

8.

7. The method of claim 1, wherein the flower spikes of Centella asiatica are used as explants. In step (3), the culture condition is: temperature 22-24 ℃, illumination 1500-2000 Lux, illumination time 12-14 h, and culture time 38-42 days.

8. The method of claim 1, wherein the inflorescence of Centella asiatica is used as an explant. In step (4), the length of the adventitious bud is 1.8-2.2 cm, preferably 2.0 cm.

9. The method of claim 1, wherein the inflorescence of Centella asiatica is used as an explant. In step (4), the formula of the rooting medium is: MS medium+indole-3-butyric acid (IBA) 0.5 mg / L+sucrose 30 g / L+agar 8 g / L, and the pH value is 5.8; Or, in step (4), the culture condition is: temperature 22-24 ℃, illumination 1500-2000 Lux, illumination time 12-14 h, and rooting time 19-21 days.

10. The method of claim 1, wherein the inflorescence of Centella asiatica is used as an explant. In step (5), the method for seedling raising is as follows: the Yunnan Rhizoma Bupleuri tissue culture seedlings are gently taken out from the bottle, the agar on the roots is washed away with water, and then the seedlings are transplanted into a mixed substrate of perlite and peat soil at a volume ratio of 1:3, covered with plastic film to maintain a humidity of 60%, a temperature of 21-25 ℃, illumination of 1500 Lux, and a light cycle of 14 h / 10 h (light / dark).

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