Method for producing salidroside through rhodiola crenulata adventitious root suspension culture and culture medium

Abstract

The invention provides a method for producing salidroside through rhodiola crenulata adventitious root suspension culture and a culture medium, a sectional liquid suspension culture technology is adopted, and the method comprises the following steps: establishing a rhodiola crenulata aseptic seedling system, and obtaining aseptic seedlings through specific disinfection and culture conditions; liquid culture and domestication of the adventitious roots are carried out, the growth cycle of the adventitious roots is shortened to 35-40 days by utilizing an optimized culture medium and a domestication strategy, and the growth vitality is improved; according to culture optimization for efficiently accumulating the salidroside, adventitious roots are transferred into a specific induction culture medium and cultured for 8-10 days under the condition of a shaking table, and finally the content of the salidroside is larger than or equal to 0.6%. The method does not depend on natural root collection, avoids damage to wild rhodiola crenulata resources, is short in production period, high in efficiency, stable in quality and free of heavy metal and pesticide residues, can realize large-scale industrialized production, meets the market requirements of medicines and health care products, reduces dependence on wild resources, and is suitable for industrial production. Sustainable development and utilization of endangered Chinese herbal medicines are assisted.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method and culture medium for producing salidroside by suspension culture of adventitious roots of Rhodiola rosea. Background Art

[0002] Rhodiola crenulata (Hook.f.&Thomson) H.Ohba is a perennial herbaceous plant of the genus Rhodiola in the Crassulaceae family. It often grows in hillside grasslands, shrubs, and rock crevices at an altitude of 2,800 to 5,600 meters. It prefers high altitude, cool alpine environments and is suitable for rocky soil. Rhodiola crenulata is listed as an endangered (EN) species in the IUCN Red List of Threatened Species. Because Rhodiola crenulata has been in a harsh environment of hypoxia, low temperature, strong ultraviolet rays, and large temperature differences between day and night for a long time, it has developed super vitality and adaptability.

[0003] The roots of Rhodiola rosea are rich in a variety of active ingredients, including salidroside, starch, protein, flavonoids and a small amount of volatile oil. They have the effects of treating physical weakness, delaying aging, anti-fatigue, and anti-hypoxia, and have a wide range of application value in the fields of medicine and health care. Since Rhodiola rosea mainly grows in high-altitude mountainous areas with an altitude of 4000m to 5100m, this special growth environment makes its collection and planting face huge challenges. In addition, due to the huge application value of Rhodiola rosea, excessive human excavation has caused a sharp decline in the population of Rhodiola rosea. Rhodiola rosea has been listed as an endangered (EN) species in the "Red List of Endangered Species of the World Conservation Union", which seriously restricts people's development and utilization.

[0004] At present, the development of Rhodiola rosea mainly relies on the extraction of salidroside from its natural roots, but due to its long growth cycle and limited distribution area, it is difficult for resources to meet market demand. The harsh environment in high-altitude mountainous areas increases the difficulty of collection, and over-exploitation has led to its endangerment, affecting its sustainable use. At the same time, wild growth conditions make it difficult to control the purity and stability of the active ingredients.

[0005] In order to solve the above problems, in recent years, the suspension culture technology of plant adventitious roots has gradually become a research hotspot. The growth rate of adventitious roots is much faster than that of natural plant roots, which can significantly increase the accumulation of biomass. Adventitious roots have a high production capacity of secondary metabolites and can effectively accumulate target components such as salidroside. Through liquid suspension culture technology, adventitious roots are not restricted by environmental conditions, and the accumulation of secondary metabolites can be artificially regulated; in addition, the adventitious root culture process is not subject to environmental pollution, and the products have no heavy metals, pesticide residues and toxic side effects. It is an important way to solve the sustainable utilization of rare and endangered medicinal plants whose roots are used as medicine, and it is in line with the development direction of modern green medicine.

[0006] Therefore, it is necessary to provide a liquid suspension culture method based on the adventitious roots of Rhodiola rosea to achieve efficient production of salidroside, thereby solving problems such as limited resources, difficult collection, endangered species caused by over-exploitation, and uncontrollable ingredient quality, and ensuring the sustainable development and utilization of Rhodiola rosea. Summary of the invention

[0007] According to the technical problems raised above, a method and culture medium for producing salidroside by suspension culture of adventitious roots of Rhodiola rosea is provided. The present invention mainly uses MS as the basic culture medium, and regulates the growth of adventitious roots and the accumulation of salidroside of Rhodiola rosea by changing the carbon source, hormones, pH, etc., and obtains the culture method that is most conducive to the growth of adventitious roots and the accumulation of salidroside by adjusting the culture medium components and culture conditions of the adventitious roots of Rhodiola rosea. The present invention not only overcomes the problems of limited natural resources, difficult collection and unstable quality of ingredients, but also ensures the green safety of the production process, without heavy metals and pesticide residues, meets market demand, and promotes the sustainable development and utilization of Rhodiola rosea.

[0008] The technical means adopted by the present invention are as follows:

[0009] A method for producing salidroside by suspension culture of adventitious roots of Rhodiola rosea, comprising the following steps:

[0010] Establishment of sterile seedling system: sterilize the top stem of Rhodiola rosea and inoculate it into the regeneration medium, and culture it under the preset light and temperature conditions to obtain sterile seedlings;

[0011] Adventitious root liquid culture and domestication: Cut the vigorously growing roots from the sterile seedlings, transfer them to the adventitious root liquid culture medium, culture them under the preset temperature, rotation speed and ventilation conditions, induce the formation of adventitious roots, and shorten the growth cycle through multiple domestication;

[0012] Cultivation optimization for efficient accumulation of salidroside: the cultured adventitious roots are transferred to the Rhodiola rosea induction medium and cultured under the conditions of preset temperature and rotation speed to increase the content of salidroside;

[0013] Adventitious root collection and testing: The adventitious roots after cultivation were harvested, freeze-dried, and the salidroside content was determined.

[0014] Furthermore, the method for establishing the sterile seedling system includes: taking water from the top stem of Rhodiola rosea and rinsing it for 30 minutes; in a sterile environment, rinsing it once with sterile water, absorbing the water with absorbent paper, soaking it in 75% alcohol for 1 minute, rinsing it with sterile water 6 times, soaking the roots, leaves and stems in 0.1% HgCl2 solution for 5 minutes, rinsing it with sterile water 6 times, absorbing the water and inoculating it into a regeneration culture medium, and culturing it under light at 20°C for 40 days to obtain sterile seedlings.

[0015] Furthermore, the formula of the regeneration medium is: MS (without sucrose and agar) as the basic medium; 30g / L sucrose, 2.0mg / L 6-BA (6-benzylaminopurine), 0.25mg / L IAA (indole-3-acetic acid), and 6g / L agar are added; and the pH value is adjusted to 5.5-6.0.

[0016] Furthermore, the method for liquid culture and domestication of adventitious roots includes: cutting the vigorously growing roots of sterile seedlings, cutting them into 1 cm long segments, and transferring them to adventitious root liquid culture medium; culturing in the dark for 55-60 days at 12-15°C, with a shaking speed of 90 rpm or a ventilation volume of 0.5 V / V·min; cutting the newly grown adventitious roots with vigorous growth, transferring them to liquid culture medium for culture again, and performing multiple domestication to shorten the adventitious root growth period to 35-40 days.

[0017] Furthermore, the formula of the adventitious root liquid culture medium is: MS (without sucrose and agar) as the basic culture medium; adding 40g / L sucrose, 1.0mg / L NAA (naphthaleneacetic acid), 2.5mg / L IBA (3-indolebutyric acid), 0.25mg / L 6-BA, 1.5mg / L potassium humate, 4.0mg / L Vc (ascorbic acid), 0.1-1.0mg / L β-nicotinamide; adjusting the pH value to 5.5-6.0.

[0018] Furthermore, the method for optimizing the culture for efficiently accumulating salidroside includes: transferring the adventitious roots cultured in liquid to a rhodiola rosea induction culture medium; and harvesting after culturing in the dark at 25° C. and a shaking speed of 150 rpm for 8-10 days.

[0019] Furthermore, the formula of the Rhodiola rosea induction culture medium is: 1 / 2MS (without sucrose and agar) as the basic culture medium; 20g / L sucrose, 1.0mg / L NAA, 2.5mg / L IBA, 0.25mg / L6-BA, 4mg / L 2,4-D, 1.5mg / L potassium humate, 4.0mg / L Vc, 30mg / L phenylalanine are added; and the pH value is adjusted to 4.5.

[0020] Furthermore, after the adventitious roots are harvested and freeze-dried, the detected salidroside content is ≥0.6%.

[0021] The present invention also provides a culture medium for the method for producing salidroside by suspension culture of the adventitious roots of Rhodiola rosea, comprising a regeneration culture medium, the formula of which is: taking MS (without sucrose and agar) as the basic culture medium, adding 30g / L sucrose, 2.0mg / L 6-BA, 0.25mg / L IAA, and 6g / L agar; and adjusting the pH value to 5.5-6.0.

[0022] Furthermore, it also includes an adventitious root liquid culture medium, the formula of which is: taking MS (without sucrose and agar) as the basic culture medium, adding 40g / L sucrose, 1.0mg / L NAA, 2.5mg / L IBA, 0.25mg / L 6-BA, 1.5mg / L potassium fulvic acid, 4.0mg / L Vc, 0.1-1.0mg / L β-nicotinamide, and adjusting the pH value to 5.5-6.0;

[0023] Furthermore, it also includes Rhodiola rosea induction culture medium, whose formula is: using 1 / 2MS (without sucrose and agar) as the basic culture medium, adding 20g / L sucrose, 1.0mg / L NAA, 2.5mg / L IBA, 0.25mg / L6-BA, 4mg / L 2,4-D, 1.5mg / L potassium humate, 4.0mg / L Vc, 30mg / L phenylalanine, and adjusting the pH value to 4.5.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] 1. The present invention adopts segmented liquid suspension culture to achieve efficient proliferation of adventitious roots and enrichment of secondary metabolites. The growth environment of adventitious roots is optimized through multiple domestication, so that the growth cycle is shortened from 55-60 days in traditional methods to 35-40 days, thereby increasing the yield of salidroside.

[0026] 2. The present invention promotes the rapid growth of adventitious roots and the efficient accumulation of salidroside by accurately optimizing the culture medium components, using MS or 1 / 2MS culture medium, and adding specific hormones and induction factors such as NAA, IBA, 6-BA, 2,4-D, potassium humate, Vc, β-nicotinamide and phenylalanine, so as to enable the adventitious roots to efficiently synthesize salidroside in a short time, and finally obtain adventitious roots with a salidroside content of ≥0.6%.

[0027] 3. The present invention adopts liquid suspension culture technology, and under the conditions of temperature, rotation speed and ventilation volume controlled by a shaker, it can produce adventitious roots rich in salidroside on a large scale and stably. It has the characteristics of high efficiency, standardization and strong controllability. Compared with the traditional wild picking method, it greatly improves the feasibility of industrial production.

[0028] In summary, the present invention has significant advantages in increasing the yield of salidroside, shortening the production cycle, improving production stability, and realizing sustainable development. The obtained adventitious roots rich in salidroside have no heavy metal and pesticide residues, are suitable for large-scale factory production, and can not only be used as a medicinal active ingredient for medical research such as anti-fatigue, anti-oxidation and immunity enhancement, but also can be used as a raw material for functional food or health care products to meet the market demand for salidroside, and provide a new path for the sustainable development and utilization of Rhodiola rosea. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0030] Figure 1 This is a physical picture of the sterile seedlings cultivated using the sterile seedling system in the method of the present invention in Example 1.

[0031] Figure 2 This is a physical picture of the adventitious roots of Rhodiola rosea cultivated using the method of the present invention in Example 1.

[0032] Figure 3 This is a chromatogram of salidroside detection in the adventitious roots of Rhodiola rosea cultivated using the method of the present invention in Example 1.

[0033] Figure 4 This is a physical picture of the adventitious roots of Rhodiola rosea that were cultured for 60 days without domestication in Comparative Example 1.

[0034] Figure 5 This is the chromatogram of salidroside detection of adventitious roots that have not been domesticated and cultured in Comparative Example 1.

[0035] Figure 6 This is a real picture of the adventitious roots of the domesticated Rhodiola rosea in Comparative Example 2 after 40 days of culture.

[0036] Figure 7 This is the chromatogram of salidroside detection after adventitious root domestication in Comparative Example 2. DETAILED DESCRIPTION

[0037] 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.

[0038] Example 1

[0039] The present invention provides a method for producing salidroside by suspension culture of adventitious roots of Rhodiola rosea, comprising the following specific steps:

[0040] (1) Establishment of the sterile seedling system of Rhodiola rosea:

[0041] Prepare the regeneration medium of Rhodiola rosea according to the formula of 4.74g / L MS (without sucrose and agar), 30g / L sucrose, 2.0mg / L 6-BA, 0.25mg / LIAA, and 6g / L agar, and sterilize it for use. Take the top stem of Rhodiola rosea and rinse it with water for 30 minutes, move it into a sterile environment and rinse it with sterile water once, dry it with absorbent paper, soak it in 75% alcohol for 1 minute, rinse it with sterile water 6 times, soak the roots, leaves and stems in 0.1% HgCl2 for 5 minutes, rinse it with sterile water 6 times, dry it and inoculate it into the regeneration medium, and culture it under light at 20℃ for 40 days to obtain regenerated sterile seedlings (such as Figure 1 shown).

[0042] (2) Liquid culture and domestication of adventitious roots:

[0043] The adventitious root liquid culture medium of Rhodiola rosea was prepared according to the formula of 4.74g / L MS (without sucrose and agar), 40g / L sucrose, 1.0mg / L NAA, 2.5mg / LIBA, 0.25mg / L 6-BA, 1.5mg / L potassium humate, 4.0mg / L Vc, and 0.5mg / Lβ-nicotinamide. The pH value was adjusted to 5.8 and the culture medium was sterilized for use.

[0044] The roots of Rhodiola rosea that grow vigorously on the regeneration medium, have no browning, and have strong division ability are cut from the sterile seedlings and inoculated into the adventitious root liquid culture medium, and cultured in the dark under the condition of a 15°C shaker with a rotation speed of 90 rpm until new adventitious roots grow for 60 days. The new adventitious roots with vigorous growth are cut and transferred to the liquid culture medium for culture again. After repeated acclimation and repeated culture for 3 times, the growth period of the adventitious roots is shortened to 35-40 days.

[0045] (3) Cultivation optimization of adventitious roots of Rhodiola rosea for efficient accumulation of salidroside:

[0046] Take 1 / 2MS (without sucrose and agar) as the basic culture medium, add 20g / L sucrose, 1.0mg / L NAA, 2.5mg / LIBA, 0.25mg / L 6-BA, 4mg / L 2,4-D, 1.5mg / L potassium humate, 4.0mg / L Vc, 30mg / L phenylalanine, adjust the pH value to 4.5, prepare the Rhodiola rosea induction culture medium, sterilize and set aside. Transfer the adventitious roots of Rhodiola rosea cultured in liquid in step (2) to the above Rhodiola rosea induction culture medium, culture in a shaker at 25°C with a speed of 150rpm in the dark for 10 days, and then harvest to obtain adventitious roots (such as Figure 2 shown).

[0047] (4) After freeze-drying the adventitious roots of Rhodiola rosea obtained in (3), the salidroside content was determined by Waters UPLC H-Class ultra-high performance liquid chromatography according to the method in the Pharmacopoeia of the People's Republic of China (2020 Edition). The chromatogram is as follows: Figure 3 shown.

[0048] Comparative Example 1

[0049] According to the method of implementation case 1, sterile seedlings were established, and roots of Rhodiola rosea that grew vigorously on the regeneration medium, had no browning, and had strong division ability were cut from the sterile seedlings and inoculated into the adventitious root liquid medium. The roots were cultured in the dark under the conditions of 15°C shaker with a speed of 90 rpm for 60 days. After new adventitious roots grew out, they were recovered and freeze-dried, and the salidroside content was detected to be 0.12%. The adventitious roots were as follows: Figure 4 As shown in the chromatogram Figure 5 shown.

[0050] Comparative Example 2

[0051] According to the method of implementation case 1, sterile seedlings were established, and roots of Rhodiola rosea that grew vigorously on the regeneration medium, had no browning, and had strong division ability were cut from the sterile seedlings and inoculated into the adventitious root liquid culture medium. The roots were cultured in the dark under the conditions of 15°C shaker with a speed of 90 rpm for 60 days. After acclimation and culture for 40 days, the roots were recovered and freeze-dried. The content of salidroside was detected to be 0.28%. The adventitious roots were as follows Figure 6 As shown in the chromatogram Figure 7 shown.

[0052] It can be seen that in Comparative Example 1, Rhodiola rosea was directly cut from sterile seedlings and cultured in an adventitious root liquid culture medium for 60 days. The obtained adventitious roots of salidroside were short and had the lowest salidroside content; the adventitious roots in Comparative Example 2 were domesticated from the adventitious roots in Comparative Example 1 to make them more adaptable to the liquid culture system, and the obtained adventitious roots were longer, and the salidroside content was increased compared with Comparative Example 1. Example 1 further induced the domesticated adventitious roots to accumulate more salidroside, and the liquid chromatography test results also proved that its rhodiola content was higher. The results show that the culture method provided by the present invention is most conducive to adventitious root growth and salidroside accumulation.

[0053] The above embodiments are only preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the above embodiments, a person of ordinary skill in the art should understand that any modification, equivalent substitution, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for producing salidroside by suspension culture of adventitious roots of Rhodiola rosea, characterized in that: The following steps are involved: Establishment of sterile seedling system: sterilize the top stem of Rhodiola rosea and inoculate it into the regeneration medium, and culture it under the preset light and temperature conditions to obtain sterile seedlings; Adventitious root liquid culture and domestication: Cut the vigorously growing roots from the sterile seedlings, transfer them to the adventitious root liquid culture medium, culture them under the preset temperature, rotation speed and ventilation conditions, induce the formation of adventitious roots, and shorten the growth cycle through multiple domestication; Cultivation optimization for efficient accumulation of salidroside: the cultured adventitious roots are transferred to the Rhodiola rosea induction medium and cultured under the conditions of preset temperature and rotation speed to increase the content of salidroside; Adventitious root collection and testing: The adventitious roots after cultivation were harvested, freeze-dried, and the salidroside content was determined.

2. The method for producing salidroside by suspension culture of adventitious roots of Rhodiola rosea according to claim 1, characterized in that: The method for establishing the sterile seedling system comprises: taking water from the top stem of Rhodiola rosea and rinsing it for 30 minutes; in a sterile environment, rinsing it once with sterile water, absorbing water with absorbent paper, soaking it in 75% alcohol for 1 minute, rinsing it with sterile water 6 times, soaking the roots, leaves and stems in 0.1% HgCl2 solution for 5 minutes, rinsing it with sterile water 6 times, absorbing water and inoculating it into a regeneration culture medium, and culturing it under light at 20°C for 40 days to obtain sterile seedlings.

3. The method for producing salidroside by suspension culture of adventitious roots of Rhodiola rosea according to claim 2, characterized in that: The formula of the regeneration culture medium is: using MS (without sucrose and agar) as the basic culture medium; adding 30 g / L sucrose, 2.0 mg / L 6-BA, 0.25 mg / L IAA, and 6 g / L agar; and adjusting the pH value to 5.5-6.

0.

4. The method for producing salidroside by suspension culture of adventitious roots of Rhodiola rosea according to claim 1, characterized in that: The method for liquid culture and domestication of adventitious roots comprises: cutting vigorously growing roots of sterile seedlings, cutting into 1 cm long segments, and transferring to adventitious root liquid culture medium; culturing in the dark for 55-60 days at 12-15°C, with a shaking speed of 90 rpm or a ventilation volume of 0.5 V / V·min; cutting newly grown adventitious roots with vigorous growth, transferring to liquid culture medium for culture again, and performing multiple domestication to shorten the adventitious root growth period to 35-40 days.

5. The method for producing salidroside by suspension culture of adventitious roots of Rhodiola rosea according to claim 4, characterized in that: The formula of the adventitious root liquid culture medium is: MS (without sucrose and agar) as the basic culture medium; Add 40g / L sucrose, 1.0mg / L NAA, 2.5mg / L IBA, 0.25mg / L 6-BA, 1.5mg / L potassium humate, 4.0mg / L Vc, 0.1-1.0mg / L β-nicotinamide; adjust the pH value to 5.5-6.

0.

6. The method for producing salidroside by suspension culture of adventitious roots of Rhodiola rosea according to claim 1, characterized in that: The method for optimizing the cultivation for efficiently accumulating salidroside comprises: transferring the adventitious roots cultured in liquid to a rhodiola rosea induction culture medium; and culturing in the dark at 25° C. and a shaking speed of 150 rpm for 8-10 days before harvesting.

7. The method for producing salidroside by suspension culture of adventitious roots of Rhodiola rosea according to claim 6, characterized in that: The formula of the Rhodiola rosea induction culture medium is: 1 / 2MS (without sucrose and agar) is used as the basic culture medium; 20g / L sucrose, 1.0mg / L NAA, 2.5mg / L IBA, 0.25mg / L 6-BA, 4mg / L 2,4-D, 1.5mg / L potassium humate, 4.0mg / LVc, and 30mg / L phenylalanine are added; and the pH value is adjusted to 4.

5.

8. The method for producing salidroside by suspension culture of adventitious roots of Rhodiola rosea according to claim 7, characterized in that: After the adventitious roots were harvested and freeze-dried, the detected salidroside content was ≥0.6%.

9. A culture medium for the method for producing salidroside by suspension culture of adventitious roots of Rhodiola rosea according to any one of claims 1 to 8, characterized in that: The invention comprises a regeneration culture medium, the formula of which is: taking MS (without sucrose and agar) as the basic culture medium, adding 30g / L sucrose, 2.0mg / L 6-BA, 0.25mg / L IAA and 6g / L agar; and adjusting the pH value to 5.5-6.

0.

10. The culture medium according to claim 9, characterized in that Also included is an adventitious root liquid culture medium, the formula of which is: taking MS (without sucrose and agar) as the basic culture medium, adding 40 g / L sucrose, 1.0 mg / L NAA, 2.5 mg / L IBA, 0.25 mg / L 6-BA, 1.5 mg / L potassium humate, 4.0 mg / L Vc, 0.1-1.0 mg / L β-nicotinamide, and adjusting the pH value to 5.5-6.0; It also includes Rhodiola rosea induction culture medium, whose formula is: 1 / 2MS (without sucrose and agar) as the basic culture medium, added with 20g / L sucrose, 1.0mg / L NAA, 2.5mg / L IBA, 0.25mg / L 6-BA, 4mg / L 2,4-D, 1.5mg / L potassium humate, 4.0mg / L Vc, 30mg / L phenylalanine, and the pH value is adjusted to 4.5.

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