A medium for inhibiting browning of exogenous explants of xuelingshi and improving callus induction rate and application thereof
By optimizing the culture medium combination and induction conditions, the problems of easy browning of Ganoderma lucidum explants and low callus induction rate were solved, realizing efficient artificial propagation of Ganoderma lucidum and improving the quality of callus tissue, thus providing a basis for industrial production.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHANGHAI SHENGYU MEIKE BIOTECHNOLOGY CO LTD
- Filing Date
- 2026-04-24
- Publication Date
- 2026-06-26
AI Technical Summary
In existing technologies, Ganoderma lucidum explants are prone to browning and have low callus induction rates, making it difficult to achieve efficient artificial propagation and accumulation of effective components.
MS basal medium supplemented with 2,4-D 0.5 mg/L, NAA 0.3 mg/L, KT 1.0 mg/L, PVP 2 mg/L and sucrose 30 g/L, combined with specific pretreatment and induction culture conditions, was used to inhibit explant browning and improve callus induction rate.
It significantly inhibited browning of Ganoderma lucidum explants and increased the callus induction rate to 91.7%, obtaining stable and loosely textured callus tissue, providing a reliable platform for the cell culture and industrial production of Ganoderma lucidum.
Smart Images
Figure CN122278743A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of plant tissue culture technology, and in particular to a culture medium that inhibits browning of Ganoderma lucidum explants and improves callus induction rate, and its application. Background Technology
[0002] Snow Ganoderma (Tibetan Snow Ganoderma), known as "Azhong Gabu" in Tibetan, is a perennial cushion-like herbaceous plant belonging to the genus *Agrostis* in the family Caryophyllaceae. The entire plant is an important traditional Tibetan medicine, possessing various pharmacological activities such as clearing heat and relieving cough, nourishing and anti-inflammatory properties, and anti-hypoxia effects. Due to its extremely slow growth, low natural reproduction rate, and over-harvesting, wild snow ganoderma resources are on the verge of depletion, and it is listed as a vulnerable (VU) species. Therefore, achieving rapid artificial propagation and accumulation of its active ingredients through plant tissue culture technology is crucial for solving the resource crisis and protecting biodiversity.
[0003] Currently, research on *Ganoderma lucidum* (snow lingzhi) focuses primarily on chemical composition analysis and pharmacological activity exploration. Its artificial cultivation techniques, especially tissue culture and callus induction, remain largely unexplored. As a typical plant from the extreme environment of high-altitude plateaus, *Ganoderma lucidum* possesses unique physiological and metabolic mechanisms. Directly applying conventional plant culture medium formulations to *Ganoderma lucidum* generally encounters core technical bottlenecks such as severe explant browning and death, difficulty in induction initiation, and extremely low induction rates. This is mainly attributed to the significant differences between high-altitude plants and ordinary plants in terms of endogenous hormone levels, secondary metabolite composition, and antioxidant systems.
[0004] Therefore, developing a dedicated induction culture medium and method that can overcome the strong stress response of Ganoderma lucidum explants and effectively initiate their dedifferentiation process has important practical application value. Summary of the Invention
[0005] The purpose of this invention is to provide a culture medium that inhibits browning of Ganoderma lucidum explants and improves callus induction rate, and its application, to solve the problems existing in the prior art. This invention provides a specialized culture medium that can significantly inhibit browning of Ganoderma lucidum explants, greatly improve callus induction rate and quality, and is suitable for callus culture of Ganoderma lucidum, an endangered Tibetan medicinal plant from the plateau. It also provides a well-defined and reproducible method for inducing Ganoderma lucidum callus, laying the foundation for establishing its aseptic culture system, conducting cell suspension culture, and producing secondary metabolites.
[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides a culture medium that inhibits browning of Ganoderma lucidum explants and improves callus induction rate. The culture medium comprises MS basal medium + 2,4-D 0.5 mg / L + NAA 0.3 mg / L + KT 1.0 mg / L + PVP 2 mg / L + sucrose 30 g / L + agar 7 g / L.
[0007] The present invention provides the use of the above-described culture medium in any of the following: (1) Enhance the browning resistance of Ganoderma lucidum explants; (2) To prepare products that enhance the anti-browning ability of Ganoderma lucidum explants; (3) Improve the callus induction efficiency of Ganoderma lucidum explants; (4) Prepare products that improve the callus induction efficiency of Ganoderma lucidum explants; (5) Ganoderma lucidum tissue culture.
[0008] This invention provides a product that enhances the browning resistance of Ganoderma lucidum explants, the product comprising the aforementioned culture medium.
[0009] This invention provides a product for improving the callus induction efficiency of Ganoderma lucidum explants, the product comprising the aforementioned culture medium.
[0010] Optionally, the product may also include auxiliary materials.
[0011] This invention provides a method for improving the browning resistance of Ganoderma lucidum explants, comprising the steps of using young leaves of Ganoderma lucidum as explants and inoculating the explants into a culture medium for induction culture; The culture medium consists of MS basal medium + 2,4-D 0.5 mg / L + NAA 0.3 mg / L + KT 1.0 mg / L + PVP 2 mg / L + sucrose 30 g / L + agar 7 g / L.
[0012] Optionally, the induction culture temperature is 18±1℃.
[0013] This invention provides a method for improving the browning resistance of Ganoderma lucidum explant callus, comprising the steps of using young leaves of Ganoderma lucidum as explants and inoculating the explants into a culture medium for induction culture; The culture medium consists of MS basal medium + 2,4-D 0.5 mg / L + NAA 0.3 mg / L + KT 1.0 mg / L + PVP 2 mg / L + sucrose 30 g / L + agar 7 g / L.
[0014] Optionally, the induction culture temperature is 18±1℃.
[0015] This invention provides a method for tissue culture of Ganoderma lucidum, comprising the steps of inducing culture and subculturing the explants using tender leaves of Ganoderma lucidum as explants; The culture medium used for the induction culture and the subculture culture includes MS basal medium + 2,4-D 0.5 mg / L + NAA 0.3 mg / L + KT 1.0 mg / L + PVP 2 mg / L + sucrose 30 g / L + agar 7 g / L.
[0016] More preferably, the temperature for both the induction culture and the subculture is 18±1℃.
[0017] The present invention discloses the following technical effects: This invention discloses for the first time a culture medium and method for effectively inducing callus tissue from *Ganoderma lucidum* (Tibetan medicinal plant), filling a gap in the field of biotechnology research on rare Tibetan medicinal plants. The culture medium provided by this invention effectively inhibits browning of *Ganoderma lucidum* explants and increases the callus induction rate (91.7%). Subculturing the callus tissue obtained from the callus induction medium yields a large quantity of loosely textured and stable callus tissue, providing a high-quality "seed source" for the industrial production of *Ganoderma lucidum* suspension cells. Thus, this invention provides a solution to the problem of "easy browning of explants"; and the combination of growth regulators designed for plateau plants improves the callus induction rate of *Ganoderma lucidum*; resulting in a *Ganoderma lucidum* callus line with excellent characteristics, loose texture, and bright color. The method provided by this invention clarifies key parameters such as explant type, pretreatment conditions, and culture temperature, establishes a standardized operating procedure, and has good reproducibility, providing a reliable technical platform for subsequent cell culture, germplasm preservation, and industrial development of *Ganoderma lucidum*. Attached Figure Description
[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0019] Figure 1 This is a diagram showing the state of the Ganoderma lucidum callus induced under treatment 8 conditions in Example 3 of the present invention; Figure 2 This is a diagram showing the state of the Ganoderma lucidum callus induced under treatment 3 in Example 3 of the present invention; Figure 3 This is a diagram showing the state of the Ganoderma lucidum callus induced under treatment 5 in Example 3 of the present invention; Figure 4This is a diagram of the callus tissue that has grown vigorously and has a loose texture after 2-3 subcultures in Example 4 of the present invention. Detailed Implementation
[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.
[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.
[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.
[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.
[0025] The tender leaves of Ganoderma lucidum used in the experiments of this invention are sourced from Tibet.
[0026] The MS basal medium used was formulated with 2084 mg / L potassium nitrate, 440 mg / L calcium chloride, 2724 mg / L ammonium nitrate, 370 mg / L magnesium sulfate, 170 mg / L potassium dihydrogen phosphate, 0.025 mg / L copper sulfate, 6.2 mg / L boric acid, 22.3 mg / L manganese sulfate, 0.25 mg / L sodium molybdate, 8.6 mg / L zinc sulfate, 0.025 mg / L cobalt chloride, 0.83 mg / L potassium iodide, 27.8 mg / L FeSO4·7H2O, 37.26 mg / L disodium ethylenediaminetetraacetate, 2.0 mg / L glycine, 100 mg / L inositol, 0.50 mg / L nicotinic acid, 0.1 mg / L vitamin B1, and 0.5 mg / L vitamin B6.
[0027] Example 1: Preparation of Culture Medium To the MS basal medium, add the following plant growth regulators: 2,4-dichlorophenoxyacetic acid (2,4-D) 0.5 mg / L, naphthaleneacetic acid (NAA) 0.3 mg / L, 6-furfurylaminopurine (KT) 1.0 mg / L, polyvinylpyrrolidone (PVP) 2 mg / L, sucrose 30 g / L, and agar 7 g / L, i.e., MS basal medium + 2,4-D 0.5 mg / L + NAA 0.3 mg / L + KT 1.0 mg / L + PVP 2 mg / L + sucrose 30 g / L + agar 7 g / L. Adjust the pH to 5.83–5.85, dispense, and autoclave at 121°C for 25 min.
[0028] Example 2: Preparation of Tibetan Ganoderma lucidum explants Select tender leaves of Tibetan Ganoderma lucidum, remove withered parts, and rinse with running water for 20-60 minutes (30 minutes in this embodiment), then wipe dry. On a sterile workbench, immerse the tender leaves in 75% (v / v) alcohol for 15-60 seconds (45 seconds in this embodiment). After alcohol disinfection, immediately rinse them in sterile water 2-6 times (4 times in this embodiment), then immerse them in 3.5wt% sodium hypochlorite aqueous solution for 4-10 minutes (8 minutes in this embodiment) for sterilization. Immediately remove them and rinse them in sterile water 5-6 times (6 times in this embodiment). Then, soak them in a sterile aqueous solution containing 100 mg / L ascorbic acid for 10 minutes to obtain explants for later use.
[0029] Example 3: Effects of different hormone ratios on callus induction in Ganoderma lucidum Based on the culture medium provided in Example 1, the concentrations of 2,4-D, NAA, and KT were varied to establish nine treatment groups (Table 1). Each treatment was inoculated with 30 young leaf explants (explants prepared in Example 2), and the treatment was repeated three times. The culture conditions were: temperature 18±1℃, dark culture, and relative humidity 40-60%. After 30 days of culture, the callus induction rate (number of explants induced to produce callus / total number of inoculated explants × 100%) was calculated, and the callus growth status was observed. The callus status of treatment 8 is shown in the figure below. Figure 1 As shown, the callus state diagram of treatment 3 is as follows. Figure 2 As shown in Table 1, the results indicate that under fixed NAA and KT conditions, the induction rate was only 61.7% when the 2,4-D concentration was 0.8 mg / L, and the callus was yellowish-brown and water-soaked. The best overall effect was achieved when the NAA concentration was 0.3 mg / L, while higher concentrations (0.5 mg / L) easily led to browning. Therefore, high concentrations of auxin stimulate the synthesis of phenolic substances and promote polyphenol oxidase activity, leading to browning and death of the callus. The best effect was achieved when the KT concentration was 1.0 mg / L, which is related to the coordination of the cytokinin / auxin ratio. By comprehensively comparing the induction rate and callus state, the optimal hormone ratio was determined to be: 2,4-D 0.5 mg / L + NAA 0.3 mg / L + KT 1.0 mg / L. This combination (treatment 5) had the highest induction rate, reaching 91.7%, and the best callus quality, with the callus being yellowish-white, granular, loosely structured, and without browning (see Table 1). Figure 3 Moreover, it is in good growth condition and is suitable as a material for subsequent differentiation culture.
[0030] In summary, the optimal induction medium for explants of young leaves of Ganoderma lucidum from Tibet is MS basal medium + 2,4-D 0.5 mg / L + NAA 0.3 mg / L + KT 1.0 mg / L + PVP 2 mg / L + sucrose 30 g / L + agar 7 g / L.
[0031] Table 1. Effects of different hormone ratios on callus induction in Ganoderma lucidum. Example 4: Subculture and Propagation of Callus Take the high-quality callus induced by treatment 5 in Example 3 (i.e., the high-quality callus obtained after 30 days of culture), remove the aged parts, cut into small pieces of about 0.5 cm, and transfer to subculture medium (the same formula as the induction medium screened in Example 3). Inoculate 5-6 pieces per bottle and incubate in the dark at 18±1℃ with a relative humidity of 40-60%. The subculture cycle is about 18 days. After three consecutive subcultures, the callus proliferates rapidly, still maintaining a yellowish-white, loose granular shape, without browning (see...). Figure 4 ).
[0032] Example 5: Control Experiment Using standard MS basal medium (without any plant growth regulators or PVP) as a control, young leaves of *Ganoderma lucidum* were inoculated as explants, and the culture conditions were the same as in Example 3. After 30 days of culture, all explants browned and died, with no callus formation. Treatment with a standard hormone formulation (MS basal medium + 2,4-D 2.0 mg / L + 6-BA 0.5 mg / L) resulted in an induction rate of only 12.3%, and the callus was water-soaked and easily browned. These results suggest that the culture medium and method provided in this invention represent a significant advancement.
[0033] In summary, this invention utilizes plant tissue culture technology to induce and screen stable, loosely textured *Ganoderma lucidum* callus tissue, achieving rapid artificial propagation and accumulation of effective components. The unique active substances contained in *Ganoderma lucidum* exhibit great potential in anti-oxidation, anti-photoaging, anti-inflammatory and soothing effects, as well as cell repair, and are expected to provide innovative solutions for common skin problems faced by modern populations, such as sensitive skin repair, photoaging, and damaged skin barriers.
[0034] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.
Claims
1. A culture medium that inhibits browning of Ganoderma lucidum explants and improves callus induction rate, characterized in that, The culture medium consists of MS basal medium + 2,4-D 0.5 mg / L + NAA 0.3 mg / L + KT 1.0 mg / L + PVP 2 mg / L + sucrose 30 g / L + agar 7 g / L.
2. The use of the culture medium according to claim 1 in any of the following: (1) Enhance the browning resistance of Ganoderma lucidum explants; (2) To prepare products that enhance the anti-browning ability of Ganoderma lucidum explants; (3) Improve the callus induction efficiency of Ganoderma lucidum explants; (4) Prepare products that improve the callus induction efficiency of Ganoderma lucidum explants; (5) Ganoderma lucidum tissue culture.
3. A product for improving the anti-browning ability of Ganoderma lucidum explants, characterized in that, The product includes the culture medium as described in claim 1.
4. A product for improving the callus induction efficiency of Ganoderma lucidum explants, characterized in that, The product includes the culture medium as described in claim 1.
5. The product according to claim 3 or 4, characterized in that, The product also includes auxiliary materials.
6. A method for improving the browning resistance of Ganoderma lucidum explants, characterized in that, The process includes the step of using tender leaves of Ganoderma lucidum as explants and inoculating the explants into a culture medium for induction culture. The culture medium consists of MS basal medium + 2,4-D 0.5 mg / L + NAA 0.3 mg / L + KT 1.0 mg / L + PVP 2 mg / L + sucrose 30 g / L + agar 7 g / L.
7. The method according to claim 6, characterized in that, The induction culture temperature was 18±1℃.
8. A method for improving the browning resistance of Ganoderma lucidum explant callus, characterized in that, The process includes the step of using tender leaves of Ganoderma lucidum as explants and inoculating the explants into a culture medium for induction culture. The culture medium consists of MS basal medium + 2,4-D 0.5 mg / L + NAA 0.3 mg / L + KT 1.0 mg / L + PVP 2 mg / L + sucrose 30 g / L + agar 7 g / L.
9. The method according to claim 8, characterized in that, The induction culture temperature was 18±1℃.
10. A method for tissue culture of Ganoderma lucidum, characterized in that, The process includes the steps of inducing and subculturing the explants using tender leaves of Ganoderma lucidum as explants. The culture medium used for the induction culture and the subculture culture includes MS basal medium + 2,4-D 0.5 mg / L + NAA 0.3 mg / L + KT 1.0 mg / L + PVP 2 mg / L + sucrose 30 g / L + agar 7 g / L.