Culture method and culture medium for producing adventitious roots through direct induction of rhodiola crenulata leaves

Abstract

The invention provides a culture method and a culture medium for producing adventitious roots through direct induction of rhodiola crenulata leaves, and aims to solve the problems of low efficiency, long period, resource limitation and the like of a traditional rhodiola crenulata propagation mode. Comprising the following steps: selecting and treating leaves, inducing the leaves to produce adventitious roots, and carrying out in-vitro culture and domestication on the adventitious roots to improve the survival rate and growth quality of the adventitious roots. According to the method, an adventitious root culture system of the rhodiola plants is established for the first time, efficient induction and large-scale propagation of the adventitious roots are achieved by optimizing the culture medium and environmental conditions, the method can be directly used for industrialized production, accumulation of salidroside, flavone and various vitamins is improved, and component uniformity and pesticide effect stability are ensured. The culture method has the advantages of being short in period, high in reproduction rate, high in controllability and the like, dependence on wild resources is effectively reduced, and technical support is provided for sustainable development and biological medicine application of rhodiola crenulata.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, and in particular to a culture method and culture medium for directly inducing the production of adventitious roots from leaves of Rhodiola rosea. Background Art

[0002] Rhodiola rosea is a perennial herb or subshrub plant of the genus Rhodiola L. of the Crassulaceae family. It is the whole herb of Rhodiola rosea. Rhodiola rosea is mainly distributed in high-altitude areas such as Tibet, northwest Yunnan, and western Sichuan in China. It is also distributed in neighboring countries such as Nepal and Bhutan. Its growth environment is extreme. It usually grows between the cracks of hillside grasslands, shrubs, snowfields, and steep and barren gravel beaches at an altitude of 2,800 to 5,600 meters, and is affected by harsh environments such as long-term hypoxia, dryness, and strong ultraviolet rays. Therefore, the plant has formed a unique physiological metabolic mechanism under long-term natural selection, accumulated rich salidrosides, flavonoids, multiple vitamins and trace elements, and also contains 17 kinds of amino acids necessary for the body, showing extremely high medicinal value. Research in recent decades has shown that Rhodiola rosea has multiple biological functions such as anti-hypoxia, anti-cold, anti-fatigue, anti-radiation, anti-virus, delaying body aging, and preventing senile diseases. It is considered to be a natural adaptogen drug with great development value, which is used to enhance the body's adaptability, especially suitable for enhancing the body's adaptability in special environments such as plateaus, polar regions, and aerospace.

[0003] Most of the plants of the genus Rhodiola are natural plant resources with strong medicinal functions. However, since Rhodiola rosea mostly grows in high-altitude and low-temperature environments in nature, the quantity of Rhodiola rosea is scarce and the quality is uneven. Due to the growth of market demand in recent years, over-exploitation has caused the resources of Rhodiola rosea to face the risk of depletion, and it has been included in the "National Secondary Protected Plant List" and the "IUCN Red List of Endangered Species". However, due to its long growth cycle and low reproduction rate, its recovery speed in the natural environment is far slower than the growth of market demand, resulting in a situation of supply exceeding demand.

[0004] In response to this problem, plant tissue culture technology provides many advantages in the cultivation of adventitious roots of Rhodiola rosea. Through adventitious root culture, adventitious roots of Rhodiola rosea rich in salidroside, flavonoids and multiple vitamins can be reproduced in large quantities in a short period of time, thus breaking through the limitations of natural resources and achieving stable and efficient production of medicinal materials. Plant tissue culture technology has the advantages of high efficiency, controllability, short cycle, resource conservation, gene improvement, and repeatability. These advantages make the adventitious roots of Rhodiola rosea produced by this technology have uniform quality and controllability as well as high content of salidroside, which provides favorable conditions for the large-scale cultivation of adventitious roots of Rhodiola rosea to increase the content of salidroside, flavonoids, multiple vitamins and trace elements.

[0005] Therefore, it is necessary to provide an efficient cultivation method for adventitious roots of Rhodiola rosea to solve the above problems. Summary of the invention

[0006] According to the technical problems raised above, a culture method and culture medium for directly inducing the production of adventitious roots from Rhodiola rosea leaves are provided. The present invention adopts plant tissue culture technology to overcome the difficulties in quantity and quality of Rhodiola rosea by mass-propagating adventitious roots of Rhodiola rosea rich in salidroside, flavonoids and multiple vitamins in a short period of time. The present invention mainly utilizes Rhodiola rosea leaves to directly induce the production of adventitious roots, from the selection and treatment of leaves; the induction of adventitious roots from leaves; to the in vitro culture and domestication of adventitious roots. The obtained Rhodiola rosea adventitious roots have the advantages of rapid growth, good dispersibility, stable traits in subculture, and no degeneration.

[0007] The technical means adopted by the present invention are as follows:

[0008] A method for directly inducing adventitious roots from Rhodiola rosea leaves comprises the following steps:

[0009] Selection and treatment of leaves: Select new leaves of Rhodiola rosea with vigor and vitality, wash them with sterile water, disinfect them with ethanol, treat them with mercuric chloride, and rinse them with sterile water several times to obtain sterile leaves;

[0010] Inducing adventitious roots from leaves: inoculate the treated leaves onto rooting medium under sterile conditions to induce adventitious roots;

[0011] In vitro culture and acclimation of adventitious roots: Select vigorously growing and strong adventitious roots, cut the root tip tissue and inoculate it onto the adventitious root propagation medium for culture, so as to achieve adventitious root propagation and acclimation.

[0012] Furthermore, the selection and treatment of the leaves include: rinsing the leaves with sterile water for 3 times and absorbing the water with sterile filter paper; soaking in 75% ethanol for 30 seconds, rinsing with sterile water for 5 times, and absorbing the water with sterile filter paper; soaking in 0.1% mercuric chloride solution for 5-10 minutes, rinsing with sterile water for 6 times, and finally absorbing the water with sterile filter paper.

[0013] Furthermore, the rooting medium formula is: MS as the basic medium, added with 30g / L sucrose, 6g / L agar, 1.0mg / L NAA (naphthaleneacetic acid), 2.5mg / L IBA (3-indolebutyric acid), 0.25mg / L 6-BA (6-benzylaminopurine), 1.5mg / L potassium humate, and 4.0mg / L Vc (ascorbic acid).

[0014] Furthermore, the culture conditions for inducing the production of adventitious roots by the rooting medium are 18-22° C., 16 h / d light cycle, 2200 Lux light intensity, and culture for 15 days.

[0015] Furthermore, the formula of the propagation medium is: MS as the basic medium, supplemented with 30g / L sucrose, 6g / L agar, 1.0mg / L NAA, 2.5mg / L IBA, 0.25mg / L 6-BA, 1.5mg / L potassium humate, 4.0mg / L Vc, and 0.1-1.0mg / L β-nicotinamide.

[0016] Furthermore, the culture conditions for propagation of the propagation medium and acclimatization of adventitious roots are 18-22° C. and dark culture for 40 days.

[0017] The present invention also discloses a culture medium for the culture method of directly inducing adventitious roots produced by the leaves of Rhodiola rosea, including a rooting culture medium, the formula of which is: taking MS as a basic culture medium, adding 30g / L sucrose, 6g / L agar, 1.0mg / L NAA, 2.5mg / L IBA, 0.25mg / L 6-BA, 1.5mg / L potassium humate and 4.0mg / L Vc.

[0018] Furthermore, it also includes a propagation culture medium, whose formula is: MS as the basic culture medium, supplemented with 30g / L sucrose, 6g / L agar, 1.0mg / L NAA, 2.5mg / L IBA, 0.25mg / L 6-BA, 1.5mg / L potassium humate, 4.0mg / L Vc, and 0.1-1.0mg / L β-nicotinamide.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] 1. Directly induce adventitious roots from leaves, improve induction efficiency and shorten the growth cycle

[0021] The present invention adopts the method of directly inducing adventitious roots from leaves, avoiding the uncertainty and inefficiency of traditional induction relying on roots or stem segments. By optimizing the composition of the rooting medium and combining culture conditions such as light control and temperature regulation, the induction period of adventitious roots is significantly shortened, allowing them to be successfully induced to grow within 15 days.

[0022] 2. Established the adventitious root culture system of Rhodiola rosea for the first time, realizing standardized reproduction

[0023] The present invention establishes a method for cultivating adventitious roots of Rhodiola plants for the first time. According to the physiological characteristics of Rhodiola leaves, precise disinfection steps and optimized rooting medium are developed, and the efficient induction and propagation of adventitious roots of Rhodiola are successfully achieved. Different from traditional methods, the domestication and propagation culture process provided by the present invention can ensure the growth stability of adventitious roots and the consistency of medicinal ingredients, laying a foundation for subsequent research and industrial application.

[0024] 3. Improve the survival rate of adventitious roots through domestication and cultivation to ensure large-scale production

[0025] The present invention adopts the in vitro domestication technology of adventitious roots, cuts the root tip tissue of the vigorously growing adventitious roots, transfers it to the optimized propagation medium, and cultures it in the dark at 18-22°C for 40 days, effectively improving the survival rate and growth quality of the adventitious roots. The domesticated adventitious roots have strong adaptability and can be directly used for further factory-scale production, avoiding the instability of traditional plant propagation, improving the continuity and controllability of production, and providing a sustainable development path for the resource protection and industrial development of Rhodiola rosea.

[0026] In summary, the present invention directly induces adventitious roots through the leaves of Rhodiola rosea, combines the optimization of culture conditions and induction strategies, improves the generation efficiency of adventitious roots, and ensures the stability and high activity of its growth. A large amount of time required for its natural growth is saved, providing a favorable method for alleviating the shortage of Rhodiola rosea in a natural state. The adventitious roots of Rhodiola rosea obtained by the cultivation of the present invention grow rapidly, have good dispersibility, and have uniform and stable quality. The content of effective ingredients such as salidroside, flavonoids, multiple vitamins and trace elements is high, and no degradation phenomenon will occur in the subsequent adventitious root subculture. The system is stable, which provides technical support for the efficient production of active ingredients such as salidroside and its application in the fields of biomedicine, functional health products, etc.

[0027] Based on the above reasons, the present invention can be widely promoted in the field of plant tissue culture technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] 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.

[0029] Figure 1 In order to adopt the cultivation method of the present invention, the leaves of Rhodiola rosea are used to induce the growth of adventitious roots.

[0030] Figure 2 The adventitious roots are those of Rhodiola rosea that have been domesticated and cultivated by the present invention.

[0031] Figure 3 It is the adventitious root of Rhodiola rosea that has not been domesticated and cultivated.

[0032] Figure 4 These are the adventitious roots of Rhodiola rosea cultured in the propagation medium without adding β-nicotinamide. DETAILED DESCRIPTION

[0033] 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.

[0034] Example 1

[0035] The present invention provides a method for directly inducing adventitious roots from leaves of Rhodiola rosea, comprising the following steps:

[0036] Selection and treatment of leaves: Select new leaves of Rhodiola rosea that are growing vigorously, rinse them three times with sterile water in an ultra-clean workbench, dry them with sterile filter paper, soak them in 75% ethanol for 30 seconds, rinse them five times with sterile water, dry them with sterile filter paper, soak them in 0.1% mercuric chloride for 6 minutes, rinse them six times with sterile water, and dry them with sterile filter paper again.

[0037] Inducing adventitious roots from leaves: Cultivate the treated leaves on rooting medium under sterile conditions at 20°C, with a light intensity of 2000 Lux, a photoperiod of 16 h / d, and a cultivation time of 15 days. Figure 1 The rooting medium formula is: MS as the basic medium, with 30 g / L sucrose, 6 g / L agar, 1.0 mg / L NAA, 2.5 mg / L IBA, 0.25 mg / L 6-BA, 1.5 mg / L potassium humate, and 4.0 mg / L Vc added.

[0038] In vitro and acclimatization culture of adventitious roots: Select the newly grown and strong adventitious roots of Rhodiola rosea and inoculate them on the propagation medium for culture. Culture them at 20℃ in the dark for 40 days and then subculture them. After 5 generations of continuous culture, the adventitious roots grow fast and have stable properties. The adventitious roots obtained have no browning phenomenon. Figure 2The formula of the enrichment medium is: MS as the basic medium, supplemented with 30 g / L sucrose, 6 g / L agar, 1.0 mg / L NAA, 2.5 mg / L IBA, 0.25 mg / L 6-BA, 1.5 mg / L potassium fulvic acid, 4.0 mg / L Vc, and 0.1-1.0 mg / L β-nicotinamide.

[0039] Comparative Example 1

[0040] According to the method of Example 1, adventitious roots were induced from the leaves of Rhodiola rosea. The vigorous and strong new adventitious roots of Rhodiola rosea were selected and inoculated into the propagation medium in Example 1. The adventitious roots were cultured at 20°C in the dark for 40 days. Figure 3 As shown, the adventitious roots were short and more callus tissue was formed while browning occurred.

[0041] Comparative Example 2

[0042] According to the method of Example 1, adventitious roots were induced from the leaves of Rhodiola rosea, and vigorous and strong new adventitious roots of Rhodiola rosea were selected and inoculated into the propagation medium without β-nicotinamide in Example 1. After culturing at 20° C. in the dark for 40 days, subculture was performed, and the cultured adventitious roots were transferred and cultured for 5 generations. There was almost no browning phenomenon in the cultured adventitious roots, but the growth of the adventitious roots was obviously slower than that in Example 1. Figure 4 shown.

[0043] In summary, the present invention not only solves the problems of short adventitious roots, severe browning or slow growth, but also ensures the vigorous growth, complete morphology and stable proliferation of adventitious roots by optimizing the culture medium formula and accurately controlling the culture conditions. The method of the present invention is more suitable for large-scale factory production, provides a stable technical guarantee for the efficient production of salidroside, flavonoids and multiple vitamins, and meets the demand for high-quality Rhodiola rosea raw materials in the fields of biomedicine, functional health products, etc., and has broad application prospects.

[0044] 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 directly inducing adventitious roots from leaves of Rhodiola rosea, characterized in that: The following steps are involved: Selection and treatment of leaves: Select new leaves of Rhodiola rosea with vigor and vitality, wash them with sterile water, disinfect them with ethanol, treat them with mercuric chloride, and rinse them with sterile water several times to obtain sterile leaves; Inducing adventitious roots from leaves: inoculate the treated leaves onto rooting medium under sterile conditions to induce adventitious roots; In vitro culture and acclimation of adventitious roots: Select vigorously growing and strong adventitious roots, cut the root tip tissue and inoculate it onto the adventitious root propagation medium for cultivation, so as to achieve adventitious root propagation and acclimation.

2. The culture method according to claim 1, characterized in that The leaf selection and treatment comprises: washing the leaf with sterile water for 3 times, and drying the water with sterile filter paper; soaking the leaf in 75% ethanol for 30 seconds, rinsing with sterile water for 5 times, and drying the water with sterile filter paper; soaking the leaf in 0.1% mercuric chloride solution for 5-10 minutes, rinsing with sterile water for 6 times, and finally drying the water with sterile filter paper.

3. The culture method according to claim 2, characterized in that The rooting medium formula is: MS as the basic medium, added with 30g / L sucrose, 6g / L agar, 1.0mg / L NAA, 2.5mg / L IBA, 0.25mg / L 6-BA, 1.5mg / L potassium humate, and 4.0mg / L Vc.

4. The culture method according to claim 3, characterized in that The culture conditions for inducing the production of adventitious roots in the rooting medium are 18-22° C., 16 h / d light cycle, 2200 Lux light intensity, and 15 days of culture.

5. The culture method according to claim 4, characterized in that The formula of the propagation culture medium is: MS as the basic culture medium, supplemented with 30 g / L sucrose, 6 g / L agar, 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, and 0.1-1.0 mg / L β-nicotinamide.

6. The culture method according to claim 5, characterized in that: The culture conditions for propagation and domestication of adventitious roots in the propagation medium are 18-22° C. and culture in the dark for 40 days.

7. A culture medium for the method for directly inducing adventitious roots from leaves of Rhodiola rosea according to any one of claims 1 to 6, characterized in that: The invention comprises a rooting medium, the formula of which is: taking MS as a basic medium, adding 30 g / L sucrose, 6 g / L agar, 1.0 mg / L NAA, 2.5 mg / L IBA, 0.25 mg / L 6-BA, 1.5 mg / L potassium humate and 4.0 mg / L Vc.

8. The culture medium according to claim 7, characterized in that It also includes a propagation culture medium, whose formula is: MS as the basic culture medium, supplemented with 30g / L sucrose, 6g / L agar, 1.0mg / L NAA, 2.5mg / L IBA, 0.25mg / L 6-BA, 1.5mg / L potassium humate, 4.0mg / L Vc, and 0.1-1.0mg / L β-nicotinamide.

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