Method for rapidly propagating calluses of ardisia rubescens and increasing saponin content of calluses of ardisia rubescens

Abstract

The invention relates to the technical field of plant breeding, in particular to a method for rapidly breeding calluses of ardisia rubescens and increasing the saponin content of the calluses of ardisia rubescens. Wherein the synergistic effect of the 2, 4-D and the NAA is very important for the calluses of the ardisia rubescens, the induction of the calluses is facilitated, the induction rate is greatly improved, and the calluses are promoted to be more compact in structure, high in growth speed and not prone to browning and death. SA and MeJA can jointly regulate and control biosynthesis of saponin, have a remarkable promotion effect on growth of saponin, and can effectively improve the content of saponin in ardisia rubescens especially at a specific concentration, so that the quality of medicinal materials is improved. According to the rapid propagation technology, protection of germplasm resources of the ardisia rubescens is enhanced, hereditary characteristics of good varieties are kept, and the rapid propagation technology is suitable for large-scale application.

Description

Technical Field

[0001] The present invention relates to the technical field of plant breeding, and more specifically to a method for rapid propagation of Ardisia crenata Sims var. bicolor (Walk) C. Y. Wu et C. Chen callus and increasing the saponin content. Background Art

[0002] Ardisia crenata Sims var. bicolor (Walk) C. Y. Wu et C. Chen is the dried root and rhizome of Ardisia crenata Sims var. bicolor (Walk) C. Y. Wu et C. Chen, a plant of the genus Ardisia in the family Myrsinaceae. It is a variety of Ardisia crenata Sims, also known as Hongbazhua. It is used as medicine with its root, and is a traditional folk herb commonly used in ethnic minority areas. The plant is an erect shrub, often with an umbrella-shaped crown. The leaves are mostly elliptic leathery or firm papery, with a pointed tip at the top, serrated at the base, wavy at the edge, and purple-red on the back. There are rich resources of the genus Ardisia in China, including 68 species and 12 varieties, mainly distributed in the provinces and regions south of the Yangtze River Basin. Ardisia crenata Sims var. bicolor has the effects of dispelling wind and dampness, clearing heat and detoxifying, relieving cough and asthma, etc., and is commonly used to treat traumatic injuries, joint pain, tonsillitis, sore throat, low back pain, etc. The main chemical components in Ardisia crenata Sims var. bicolor include triterpenoid saponins, flavonoids, coumarins, phenolic acids and other components. Modern pharmacological studies have shown that it has the effects of antiviral, anti-tumor, preventing and treating cardiovascular and cerebrovascular diseases, and inhibiting breast cancer metastasis.

[0003] However, at present, the related industries have led to the risk of exhaustion of wild resources, resulting in a sharp decline in the quantity of germplasm resources. At the same time, the saponin content in Ardisia crenata Sims var. bicolor needs to be increased. It can be seen that it is necessary to strengthen the protection of the germplasm resources of Ardisia crenata Sims var. bicolor in this field, deeply study the technology of rapid propagation of seedlings, and maintain the genetic characteristics of excellent varieties.

[0004] Therefore, how to obtain a method for rapid propagation of Ardisia crenata Sims var. bicolor callus and increasing the saponin content is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a method for rapid propagation of Ardisia crenata Sims var. bicolor callus and increasing the saponin content.

[0006] In order to achieve the above object, the present invention adopts the following technical scheme:

[0007] A method for rapid propagation of Ardisia crenata Sims var. bicolor callus and increasing the saponin content, comprising the following steps:

[0008] (1) After disinfecting the fruits of Ardisia crenata Sims var. bicolor, rinsing with sterile water, and inoculating them into a basic medium, sterile seedlings are obtained through cultivation;

[0009] (2) Cut the stem segments of the axenic seedlings and inoculate them into the induction medium, which includes MS + NAA + 6-BA + 2,4-D + SA + MeJA + sucrose + agar, and induce callus for 20 - 30 days; among them, the concentration of SA is 50 - 150 μmol / L, and the concentration of MeJA is 50 - 150 μmol / L.

[0010] Preferably, the specific method of disinfection in step (1) is: first treat in 75% (v / v) alcohol for 2 - 3 minutes, and then treat in 0.1% mercuric chloride solution for 10 - 12 minutes.

[0011] Preferably, the composition of the basal medium in step (1) is MS + 0.2 - 0.3 mg / L NAA + 0.05 - 0.1 mg / L 6-BA + 30 mg / L sucrose + 6 mg / L agar.

[0012] Preferably, the pH value of the induction medium in step (2) is 5.8 - 6.0.

[0013] Preferably, the culture temperature in step (2) is 25 ± 1 °C, the light time is 12 - 16 h / d, and the light intensity is 2000 - 2200 lx.

[0014] Preferably, the concentration of NAA in step (2) is 0.5 - 1 mg / L, the concentration of 6-BA is 0.1 - 0.2 mg / L, and the concentration of 2,4-D is 0.05 - 0.2 mg / L.

[0015] Preferably, the concentration of sucrose in step (2) is 30 - 40 mg / L, and the concentration of agar is 5 - 7 mg / L.

[0016] Preferably, the concentration of SA in step (2) is 100 μmol / L.

[0017] Preferably, the concentration of MeJA in step (2) is 100 μmol / L.

[0018] As can be seen from the above technical solutions, compared with the prior art, the present invention provides a method for rapid propagation of Ardisia crenata Sims callus and increasing the saponin content, and has the following beneficial effects:

[0019] The various raw materials of the culture medium of the present invention are rationally formulated. The synergistic effect of 2,4-D and NAA is very important for the callus of Ardisia crenata Sims var. bicolor (Walker) C. Chen, which is beneficial to the induction of callus, greatly improves the induction rate, promotes the callus structure to be more compact, has a fast growth rate, and is not easily browned and dead. When one of them is lacking, the induction effect is significantly reduced. SA and MeJA can jointly regulate the biosynthesis of saponins and have a significant promoting effect on the growth of saponins. Especially at a specific concentration, they can effectively increase the saponin content in Ardisia crenata Sims var. bicolor (Walker) C. Chen and improve the quality of the medicinal material. The rapid propagation technology of the present invention strengthens the protection of the germplasm resources of Ardisia crenata Sims var. bicolor (Walker) C. Chen, maintains the genetic characteristics of excellent varieties, and is suitable for large-scale application. Specific embodiments

[0020] The following will describe clearly and completely the technical solutions in the embodiments of the present invention in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0021] Example 1

[0022] A method for the rapid propagation of Ardisia crenata Sims var. bicolor (Walker) C. Chen callus and the improvement of saponin content includes the following steps:

[0023] (1) After disinfecting the fruits of Ardisia crenata Sims var. bicolor (Walker) C. Chen, rinsing them with sterile water, and inoculating them into the basal medium, sterile seedlings are obtained through cultivation;

[0024] (2) Cutting the stem segments of the sterile seedlings and inoculating them into the induction medium. The induction medium includes MS + NAA + 6-BA + 2,4-D + SA + MeJA + sucrose + agar, and callus induction is carried out for 28 days; among them, the concentration of SA is 50 μmol / L, and the concentration of MeJA is 50 μmol / L;

[0025] The specific method of disinfection in step (1) is: first treating in 75% (v / v) alcohol for 2 minutes, and then treating in 0.1% mercuric chloride solution for 10 minutes;

[0026] The composition of the basal medium in step (1) is MS + 0.2 mg / L NAA + 0.05 mg / L 6-BA + 30 mg / L sucrose + 6 mg / L agar;

[0027] The pH value of the induction medium in step (2) is 5.8;

[0028] The culture temperature in step (2) is 25 °C, the light time is 12 h / d, and the light intensity is 2000 lx;

[0029] In step (2), the concentration of NAA is 0.5 mg / L, the concentration of 6-BA is 0.1 mg / L, and the concentration of 2,4-D is 0.05 mg / L;

[0030] In step (2), the concentration of sucrose is 30 mg / L, and the concentration of agar is 5 mg / L.

[0031] Example 2

[0032] A method for rapid propagation of Ardisia crenata Sims. callus and increasing the saponin content, comprising the following steps:

[0033] (1) After disinfecting the fruits of Ardisia crenata Sims., rinsing with sterile water, and inoculating them into a basic medium, sterile seedlings are obtained through cultivation;

[0034] (2) Cutting the stem segments of the sterile seedlings and inoculating them into an induction medium, where the induction medium includes MS + NAA + 6-BA + 2,4-D + SA + MeJA + sucrose + agar, and performing callus induction for 28 days; among them, the concentration of SA is 150 μmol / L, and the concentration of MeJA is 150 μmol / L.

[0035] The specific method of disinfection in step (1) is: first treating in 75% (v / v) alcohol for 3 minutes, and then treating in 0.1% mercuric chloride solution for 12 minutes;

[0036] The composition of the basic medium in step (1) is MS + 0.3 mg / L NAA + 0.1 mg / L 6-BA + 30 mg / L sucrose + 6 mg / L agar;

[0037] The pH value of the induction medium in step (2) is 6.0;

[0038] The culture temperature in step (2) is 25 °C, the light time is 16 h / d, and the light intensity is 2200 lx;

[0039] In step (2), the concentration of NAA is 1 mg / L, the concentration of 6-BA is 0.2 mg / L, and the concentration of 2,4-D is 0.2 mg / L;

[0040] In step (2), the concentration of sucrose is 40 mg / L, and the concentration of agar is 7 mg / L.

[0041] Example 3

[0042] A method for rapid propagation of Ardisia crenata Sims. callus and increasing the saponin content, comprising the following steps:

[0043] (1) After disinfecting the fruits of Ardisia crenata Sims., rinsing with sterile water, and inoculating them into a basic medium, sterile seedlings are obtained through cultivation;

[0044] (2) Cut the stem segments of the axenic seedlings and inoculate them into the induction medium, which includes MS + NAA + 6-BA + 2,4-D + SA + MeJA + sucrose + agar, for callus induction for 28 days; among them, the concentration of SA is 100 μmol / L, and the concentration of MeJA is 100 μmol / L.

[0045] The specific method of disinfection in step (1) is: first treat in 75% (v / v) alcohol for 3 minutes, and then treat in 0.1% mercuric chloride solution for 12 minutes;

[0046] The composition of the basic medium in step (1) is MS + 0.25 mg / L NAA + 0.08 mg / L 6-BA + 30 mg / L sucrose + 6 mg / L agar;

[0047] The pH value of the induction medium in step (2) is 6.0;

[0048] The culture temperature in step (2) is 25 °C, the light time is 14 h / d, and the light intensity is 2100 lx;

[0049] The concentration of NAA in step (2) is 0.8 mg / L, the concentration of 6-BA is 0.15 mg / L, and the concentration of 2,4-D is 0.1 mg / L;

[0050] The concentration of sucrose in step (2) is 30 mg / L, and the concentration of agar is 6 mg / L.

[0051] Comparative Example 1

[0052] A method for rapid propagation of Ardisia crenata Sims var. bicolor (Walker) C. Y. Wu & C. Chen callus and increasing the saponin content, which is different from Example 3 in that SA is removed from the induction medium, and the rest are the same.

[0053] Comparative Example 2

[0054] A method for rapid propagation of Ardisia crenata Sims var. bicolor (Walker) C. Y. Wu & C. Chen callus and increasing the saponin content, which is different from Example 3 in that MeJA is removed from the induction medium, and the rest are the same.

[0055] Comparative Example 3

[0056] A method for rapid propagation of Ardisia crenata Sims var. bicolor (Walker) C. Y. Wu & C. Chen callus and increasing the saponin content, which is different from Example 3 in that SA and MeJA are removed from the induction medium, and the rest are the same.

[0057] Comparative Example 4

[0058] A method for rapid propagation of Ardisia crenata Sims var. bicolor (Walker) C. Y. Wu & C. Chen callus and increasing the saponin content, which is different from Example 3 in that 2,4-D is removed from the induction medium, and the rest are the same.

[0059] Comparative Document 5

[0060] A method for rapid propagation of Ardisia crenata Sims var. bicolor (Walker) C. Y. Wu & C. Chen callus and increasing the saponin content, which is different from Example 3 in that NAA is removed from the induction medium and the rest is the same.

[0061] For each experiment of each example and comparative example, 15 explants were treated simultaneously each time, with 3 replicates.

[0062] I. Calculate the induction rate and browning rate, as shown in Table 1.

[0063] Induction rate / % = (Number of explants with induced callus / Total number of explants) × 100%;

[0064] Browning rate / % = (Number of browning explants / Total number of explants) × 100%.

[0065] Table 1

[0066] Group Induction rate / % Browning rate / % Example 1 88.26 18.52 Example 2 90.75 12.15 Example 3 94.38 9.67 Comparative Example 1 76.41 33.45 Comparative Example 2 79.33 31.22 Comparative Example 3 70.78 38.91 Comparative Example 4 43.46 58.24 Comparative Example 5 52.85 62.17

[0067] As can be seen from Table 1, the synergistic effect of 2,4-D and NAA is very important for Ardisia crenata Sims var. bicolor (Walker) C. Y. Wu & C. Chen callus. Under the medium ratio of the present invention, especially in Example 3, it is most conducive to the induction of callus, greatly improving the induction rate, promoting the callus structure to be more compact, with a fast growth rate, not easily browning and dying. When one of them is missing, the induction effect drops significantly.

[0068] II. Determine the content of triterpenoid saponins

[0069] Precisely weigh 1.011 mg of triterpenoid saponin A standard product, place it in a 10 mL brown volumetric flask, add 80% methanol and dissolve it by ultrasonic wave and make up the volume to obtain a standard product solution (0.1 mg / mL). Dilute the standard product solution, select 6 mass concentrations of 0.1, 0.05, 0.025, 0.01, 0.005, 0.001 mg / mL for injection, and perform linear regression with the peak area (mAU×min) as the ordinate and the injection mass concentration (mg / mL) as the abscissa.

[0070] After drying each callus, it was ground into powder and passed through a 40-mesh sieve. 0.2 g was accurately weighed, 5 mL of 80% methanol was added, and ultrasonic extraction was carried out at 80 °C for 50 min. After centrifugation, the upper clear liquid was collected; 2 mL of 80% methanol was added to the precipitate, ultrasonic extraction was carried out at 80 °C for 30 min, centrifuged, and the upper clear liquid was collected. This was repeated once, and the upper clear liquids from the three extractions were combined. After extraction with n-butanol, the volume was fixed to 10 mL. It was filtered through a 0.45 μm microporous membrane and determined by high performance liquid chromatography (HPLC). Chromatographic conditions: LC-20AB high performance liquid chromatography system (Shimadzu, Japan), chromatographic column: SHIM-PACK VP-ODS, 250 mm × 4.6 mm, 5 μm; flow rate: 1.0 mL / min-1; column temperature: 40 °C; injection volume: 10 μL; mobile phase: V(acetonitrile):V(water) = 37:63; isocratic elution, detection wavelength 205 nm. The results are shown in Table 2.

[0071] Table 2

[0072] Group Triterpenoid saponin content mg / g Example 1 4.92 Example 2 5.16 Example 3 5.68 Comparative Example 1 3.61 Comparative Example 2 3.32 Comparative Example 3 2.55 Comparative Example 4 3.77 Comparative Example 5 3.96

[0073] As can be seen from Table 2, SA and MeJA can jointly regulate the biosynthesis of saponins, have a significant promoting effect on the growth of saponins, especially at specific concentrations, can effectively increase the saponin content in Ardisia crenata Sims var. bicolor (Walker) C. Y. Wu et C. Chen, and improve the quality of the medicinal materials.

[0074] In this specification, each embodiment is described in a progressive manner. The key point of each embodiment is to illustrate the differences from other embodiments. For the same or similar parts among the embodiments, reference can be made to each other.

[0075] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for rapid propagation of Ardisia crenata Sims var. bicolor (Walker) C. Y. Wu & C. Chen callus and increasing the content of saponins, characterized in that, It includes the following steps: (1) After disinfecting the fruits of Ardisia crenata Sims, rinse them with sterile water and inoculate them into the basal medium to obtain sterile seedlings through cultivation; (2) Cut the stem segments of the sterile seedlings and inoculate them into the induction medium. The induction medium includes MS + NAA + 6-BA + 2,4-D + SA + MeJA + sucrose + agar, and conduct callus induction for 20 - 30 d; among them, the concentration of SA is 50 - 150 μmol / L, and the concentration of MeJA is 50 - 150 μmol / L.

2. The method for rapid propagation of Ardisia crenata Sims callus and increasing the content of saponins according to claim 1, characterized in that, The specific method of disinfection in step (1) is: first treat in 75% (v / v) alcohol for 2 - 3 min, and then treat in 0.1% mercuric chloride solution for 10 - 12 min.

3. A method for rapid propagation of Ardisia crenata Sims callus and increasing the saponin content according to claim 1, characterized in that, The composition of the basal medium in step (1) is MS + 0.2 - 0.3 mg / L NAA + 0.05 - 0.1 mg / L 6-BA + 30 mg / L sucrose + 6 mg / L agar.

4. A method for rapid propagation of Ardisia crenata Sims callus and increasing the saponin content according to claim 1, characterized in that The pH value of the induction medium in step (2) is 5.8 - 6.

0.

5. A method for rapid propagation of Ardisia crenata Sims callus and increasing the saponin content according to claim 1, characterized in that, The cultivation temperature in step (2) is 25 ± 1 °C, the light time is 12 - 16 h / d, and the light intensity is 2000 - 2200 lx.

6. A method for rapid propagation of Ardisia crenata Sims callus and increasing the saponin content according to claim 1, characterized in that The concentration of NAA in step (2) is 0.5 - 1 mg / L, the concentration of 6-BA is 0.1 - 0.2 mg / L, and the concentration of 2,4-D is 0.05 - 0.2 mg / L.

7. A method for rapid propagation of Ardisia crenata Sims callus and increasing the saponin content according to claim 1, characterized in that, The concentration of sucrose in step (2) is 30 - 40 mg / L, and the concentration of agar is 5 - 7 mg / L.

8. A method for rapid propagation of Ardisia crenata Sims. callus and increasing the content of saponins according to claim 1, characterized in that, The concentration of SA in step (2) is 100 μmol / L.

9. A method for rapid propagation of Ardisia crenata Sims callus and increasing the saponin content according to claim 1, characterized in that The concentration of MeJA in step (2) is 100 μmol / L.