A culture method and medium for directly inducing production of adventitious roots from leaves of rhodiola crenulata

Abstract

This invention provides a method and culture medium for directly inducing adventitious root production from Rhodiola rosea leaves, aiming to overcome the problems of low efficiency, long cycle, and limited resources in traditional Rhodiola rosea propagation methods. The method includes leaf selection and treatment, leaf-induced adventitious root production, and in vitro culture and acclimatization of the adventitious roots to improve their survival rate and growth quality. This invention establishes for the first time an adventitious root culture system for Rhodiola rosea plants. By optimizing the culture medium and environmental conditions, it achieves efficient induction and large-scale propagation of adventitious roots, which can be directly used for industrial production, increasing the accumulation of rhodiolosides, flavonoids, and various vitamins, ensuring component uniformity and efficacy stability. The culture method of this invention has the advantages of short cycle, high propagation rate, and strong controllability, effectively reducing dependence on wild resources and providing technical support for the sustainable development and biomedical application of Rhodiola rosea.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, and in particular to a culture method and culture medium for directly inducing adventitious root production from Rhodiola rosea leaves. Background Technology

[0002] Rhodiola grandiflora, a perennial herb or subshrub belonging to the genus Rhodiola in the family Crassulaceae, is the whole plant. It thrives in extreme environments, typically growing on mountain slopes, in grasslands, thickets, snowfields, and in the crevices of rugged, barren scree slopes at altitudes of 2800–5600 meters, enduring long-term exposure to oxygen deficiency, dryness, and strong ultraviolet radiation. Therefore, through long-term natural selection, this plant has developed a unique physiological and metabolic mechanism, accumulating abundant rhodiolosides, flavonoids, various vitamins and trace elements, as well as 17 essential amino acids, exhibiting extremely high medicinal value. Research in recent decades has shown that Rhodiola grandiflora possesses various biological functions, including resistance to hypoxia, cold, fatigue, radiation, and viruses, as well as the ability to delay aging and prevent age-related diseases. It is considered a natural adaptogen with significant development potential, used to enhance human adaptability, particularly suitable for enhancing adaptability in special environments such as high altitudes, polar regions, and space travel.

[0003] Most plants in the genus *Rhodiola* are natural plant resources with strong medicinal properties. However, because *Rhodiola grandiflora* grows primarily in high-altitude, low-temperature environments, its quantity is scarce and its quality varies greatly. Due to increased market demand in recent years, over-harvesting has led to the depletion of *Rhodiola grandiflora* resources, and it has been listed in the *National Class II Protected Plant List* and the *IUCN Red List of Threatened Species*. However, due to its long growth cycle and low reproduction rate, the recovery rate in the natural environment is far slower than the growth in market demand, resulting in a supply shortage.

[0004] To address this issue, plant tissue culture technology offers numerous advantages in the cultivation of adventitious roots of *Rhodiola crenulata*. Through adventitious root culture, large quantities of *Rhodiola crenulata* adventitious roots rich in rhodiolosides, flavonoids, and various vitamins can be propagated in a short period, thus overcoming the limitations of natural resources and achieving stable and efficient medicinal material production. Plant tissue culture technology boasts advantages such as high efficiency, controllability, short cycle, resource conservation, gene modification, and reproducibility. These advantages ensure that the *Rhodiola crenulata* adventitious roots produced using this technology exhibit uniform and controllable quality, as well as high rhodioloside content. This provides favorable conditions for the large-scale cultivation of *Rhodiola crenulata* adventitious roots to increase the content of rhodiolosides, flavonoids, various vitamins, and trace elements.

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

[0006] To address the aforementioned technical problems, this invention provides a method and culture medium for directly inducing adventitious root production from Rhodiola rosea leaves. This invention utilizes plant tissue culture technology to rapidly propagate large quantities of Rhodiola rosea adventitious roots rich in rhodiolosides, flavonoids, and various vitamins, overcoming the difficulties in both quantity and quality of Rhodiola rosea. This invention primarily utilizes the direct induction of adventitious root production from Rhodiola rosea leaves, encompassing leaf selection and treatment; leaf-induced adventitious root production; and in vitro culture and acclimatization of the adventitious roots. The resulting Rhodiola rosea adventitious roots exhibit advantages such as rapid growth, good dispersibility, stable traits during subculture, and no degeneration.

[0007] The technical means employed in this invention are as follows:

[0008] A method for directly inducing adventitious root production from Rhodiola rosea leaves includes the following steps:

[0009] Leaf selection and treatment: Select vigorous new leaves of Rhodiola rosea, rinse with sterile water, disinfect with ethanol, treat with mercuric chloride, and then rinse with sterile water multiple times to obtain sterile leaves.

[0010] Leaf-induced adventitious root production: Treated leaves are inoculated onto rooting medium under aseptic conditions to induce the production of adventitious roots;

[0011] Adventitious root in vitro and acclimatization culture: Select vigorous and robust adventitious roots, cut root tip tissue and inoculate it onto adventitious root propagation medium for culture to achieve adventitious root propagation and acclimatization.

[0012] Furthermore, the selection and treatment of the leaves includes: rinsing the leaves three times with sterile water and blotting them dry with sterile filter paper; soaking them in 75% ethanol for 30 seconds, rinsing them five times with sterile water, and blotting them dry with sterile filter paper; soaking them in 0.1% mercuric chloride solution for 5-10 minutes, rinsing them six times with sterile water, and finally blotting them dry with sterile filter paper.

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

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

[0015] Furthermore, the propagation medium is formulated as follows: MS medium is used as the base medium, with the addition of 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 vitamin C, and 0.1-1.0 mg / L β-nicotinamide.

[0016] Furthermore, the culture conditions for propagating and acclimatizing adventitious roots in the propagation medium are 18-22℃ and cultured in the dark for 40 days.

[0017] The present invention also discloses the culture medium for the above-mentioned method of directly inducing adventitious root production from Rhodiola rosea leaves, including a rooting medium with the following formula: MS as the base medium, with the addition of 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 vitamin C.

[0018] Furthermore, it also includes a propagation medium, the formulation of which is: MS as the basal medium, with the addition of 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 vitamin C, and 0.1-1.0 mg / L β-nicotinamide.

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

[0020] 1. Direct induction of adventitious roots through leaves improves induction efficiency and shortens the growth cycle.

[0021] This invention employs a method of directly inducing adventitious roots from leaves, avoiding the uncertainty and inefficiency of traditional root or stem segment induction. By optimizing the composition of the rooting medium and combining it with culture conditions such as light control and temperature regulation, the induction period of adventitious roots is significantly shortened, enabling successful induction of growth within 15 days.

[0022] 2. For the first time, an adventitious root culture system for Rhodiola rosea plants was established, achieving standardized propagation.

[0023] This invention establishes for the first time a method for cultivating adventitious roots of Rhodiola rosea plants. Targeting the physiological characteristics of Rhodiola rosea leaves, precise sterilization procedures and optimized rooting culture media were developed, successfully achieving efficient induction and propagation of Rhodiola rosea adventitious roots. Unlike traditional methods, the domestication and propagation process provided by this invention ensures the growth stability of adventitious roots and the consistency of medicinal components, laying the foundation for subsequent research and industrial applications.

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

[0025] This invention employs an in vitro adventitious root acclimatization technique, where root tip tissue is extracted from vigorously growing adventitious roots and transferred to an optimized propagation medium. After 40 days of cultivation at 18-22℃ in the dark, the survival rate and growth quality of the adventitious roots are effectively improved. The acclimatized adventitious roots exhibit strong adaptability and can be directly used for further large-scale industrial production, avoiding the instability of traditional plant propagation and improving the continuity and controllability of production. This provides a sustainable development path for the resource conservation and industrialization of Rhodiola rosea.

[0026] In summary, this invention directly induces adventitious roots from the leaves of *Rhodiola rosea*, and by optimizing culture conditions and induction strategies, improves the efficiency of adventitious root generation, ensuring its growth stability and high activity. This significantly reduces the time required for natural growth, providing a beneficial method to alleviate the shortage of *Rhodiola rosea* in its natural state. The *Rhodiola rosea* adventitious roots obtained through this invention grow rapidly, disperse well, and exhibit stable and uniform quality. They are characterized by high levels of effective components such as rhodioloside, flavonoids, various vitamins, and trace elements. Furthermore, no degeneration occurs in subsequent subcultures of adventitious roots, and the system remains stable. This provides technical support for the efficient production of active ingredients such as rhodioloside and their application in fields such as biomedicine and functional health products.

[0027] Based on the above reasons, this invention can be widely promoted in the field of plant tissue culture technology. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 To cultivate the adventitious roots induced by Rhodiola rosea leaves using the cultivation method of this invention.

[0030] Figure 2 These are adventitious roots of Rhodiola rosea that have been domesticated and cultivated according to this invention.

[0031] Figure 3 These are adventitious roots of Rhodiola rosea that have not been domesticated or propagated.

[0032] Figure 4 Adventitious roots of Rhodiola rosea cultured in a medium without the addition of β-nicotinamide were propagated. Detailed Implementation

[0033] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] Example 1

[0035] This invention provides a method for directly inducing adventitious root production from Rhodiola rosea leaves, comprising the following steps:

[0036] Leaf selection and treatment: Select vigorous new leaves of Rhodiola rosea. Rinse them three times with sterile water in a clean bench, blot dry with sterile filter paper, soak them in 75% ethanol for 30 seconds, rinse them five times with sterile water, blot dry with sterile filter paper, soak them in 0.1% mercuric chloride for 6 minutes, rinse them six times with sterile water, and blot dry with sterile filter paper again.

[0037] Leaf-induced adventitious root induction: Treated leaves were cultured on rooting medium under aseptic conditions at 20℃, with a light intensity of 2000 Lux, a photoperiod of 16 h / d, and a culture time of 15 days. Figure 1 As shown. The rooting medium formula is: MS as the basal medium, with the addition of 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 vitamin C.

[0038] Adventitious root in vitro and acclimatization culture: Vigorous and robust newly grown adventitious roots of *Rhodiola crenulata* were inoculated onto a propagation medium and cultured at 20℃ in the dark for 40 days. Subculture was then performed for 5 generations. The adventitious roots showed rapid growth and stable characteristics, and no browning was observed. Figure 2As shown. The propagation medium formula is as follows: MS as the basal medium, with the addition of 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 vitamin C, and 0.1-1.0 mg / L β-nicotinamide.

[0039] Comparative Example 1

[0040] Adventitious roots were induced from Rhodiola rosea leaves according to the method in Example 1. Vigorous and robust newly formed Rhodiola rosea adventitious roots were selected and inoculated onto the propagation medium in Example 1. The roots were cultured at 20°C in the dark for 40 days. The results are as follows: Figure 3 As shown, the adventitious roots are short and form a large amount of callus tissue while undergoing browning.

[0041] Comparative Example 2

[0042] Adventitious roots were induced from Rhodiola rosea leaves according to the method in Example 1. Vigorous and robust newly formed Rhodiola rosea adventitious roots were selected and inoculated into the propagation medium without β-nicotinamide as described in Example 1. After 40 days of culture at 20°C in the dark, the roots were subcultured and continuously transferred for 5 generations. The cultured adventitious roots showed almost no browning, but their growth was significantly slower than in Example 1. Figure 4 As shown.

[0043] In summary, this invention, through optimizing the culture medium formulation and precisely controlling the culture conditions, not only solves problems such as short adventitious roots, severe browning, or slow growth, but also ensures vigorous growth, intact morphology, and stable proliferation of adventitious roots. The method of this invention is more suitable for large-scale industrial production, providing a stable technical guarantee for the efficient production of rhodiolosides, flavonoids, and various vitamins, and meeting the demand for high-quality Rhodiola rosea raw materials in the fields of biomedicine and functional health products, thus possessing broad application prospects.

[0044] The above embodiments are merely 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 foregoing embodiments, those skilled in the art should understand that any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for directly inducing adventitious root production from Rhodiola rosea leaves, characterized in that, Includes the following steps: Leaf selection and treatment: Select vigorous new leaves of Rhodiola rosea, rinse with sterile water, disinfect with ethanol, treat with mercuric chloride, and then rinse with sterile water multiple times to obtain sterile leaves. Leaf induction of adventitious root production: The treated leaves were inoculated onto a rooting medium under aseptic conditions to induce the production of adventitious roots; the rooting medium consisted of MS basal medium, 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 vitamin C; Adventitious root in vitro and acclimatization culture: vigorous and robust adventitious roots were selected, root tip tissues were cut and inoculated onto adventitious root propagation medium for culture to achieve adventitious root propagation and acclimatization; the propagation medium consisted of MS as the basal medium, 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 vitamin C, and 0.1-1.0 mg / L β-nicotinamide.

2. The cultivation method according to claim 1, characterized in that, The selection and treatment of the leaves include: rinsing the leaves three times with sterile water and blotting them dry with sterile filter paper; soaking them in 75% ethanol for 30 seconds, rinsing them five times with sterile water, and blotting them dry with sterile filter paper; soaking them in 0.1% mercuric chloride solution for 5-10 minutes, rinsing them six times with sterile water, and finally blotting them dry with sterile filter paper.

3. The cultivation method according to claim 2, characterized in that, The rooting medium was used to induce the production of adventitious roots under the following conditions: 18-22℃, 16h / d light cycle, 2200Lux light intensity, and 15 days of culture.

4. The cultivation method according to claim 3, characterized in that, The culture conditions for propagating and acclimatizing adventitious roots in the propagation medium are 18-22℃ and cultured in the dark for 40 days.

5. A culture medium for the method of directly inducing adventitious root production from Rhodiola rosea leaves according to any one of claims 1-4, characterized in that, The medium includes a rooting medium and a propagation medium. The rooting medium consists of MS basal medium, 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 vitamin C. The treated leaves are inoculated onto the rooting medium for culture. The propagation medium consists of MS basal medium, 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 vitamin C, and 0.1-1.0 mg / L β-nicotinamide.

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