A method for inducing and proliferating flower bud callus of rhododendron anthopogonoides maxim

Abstract

The application belongs to the technical field of plant tissue culture, and discloses a method for inducing and proliferating flower bud callus of Rhododendron anthopogonoides flower bud, wherein full flower bud inner layer green small buds of wild and robust Rhododendron anthopogonoides at an altitude of 2800-3500 m are used as explants, the explants are subjected to gradient mild sterile disinfection, inoculated into modified MS induction medium suitable for plateau germplasm, and dark cultured to be dedifferentiated, and then subjected to multi-stage special proliferation medium segmented light and temperature regulation culture, so that efficient induction and stable subculture proliferation of callus are realized. The application can make the flower bud callus induction rate stable to reach more than 86%, the culture period is 28-31 days, the obtained callus has dense texture, uniform color, strong cell activity and stable genetic traits, can be subcultured for a long time, is suitable for in vitro conservation of Rhododendron anthopogonoides germplasm, enrichment of medicinal active ingredients and factory-like rapid propagation of seedlings, and provides technical support for Tibetan medicine resource protection and industrialization development.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture technology, and in particular relates to a method for inducing and proliferating callus tissue from flower buds of the Tibetan medicine Rhododendron simsii. Background Technology

[0002] Fragrant Rhododendron ( Rhododendron anthopogonoides Maxim. Rhododendron (Rhododendron) is an evergreen alpine shrub belonging to the genus Rhododendron in the family Ericaceae. It is mainly distributed wild in cold, damp alpine shrublands. Rich in volatile oils, flavonoids, triterpenoids, and phenolic acids, it contains specific active ingredients characteristic of Tibetan medicine. Its leaves, flowers, and branches can all be used medicinally. Traditional Tibetan medicine uses it for clearing heat and reducing swelling, relieving cough and asthma, dispelling dampness and detoxifying, and treating respiratory diseases caused by high-altitude hypoxia. Modern pharmacology has confirmed its anti-inflammatory, antioxidant, anti-altitude stress, lung-protective, and antibacterial pharmacological activities. It is a core raw material plant for the development of Tibetan medicine raw materials and intermediates for high-altitude pharmaceuticals, while also possessing extremely high ecological protection and horticultural ornamental value.

[0003] Currently, the raw materials for Rhododendron simsii rely entirely on wild harvesting, and the industry's development faces three major bottlenecks: First, this species is a typical alpine endemic plant, growing extremely slowly, with a very low natural fruit-bearing rate, a long seed dormancy period, and difficulty in rooting through cuttings in the wild, resulting in extremely poor regeneration capacity of wild populations. Long-term unregulated wild harvesting has led to the degradation of wild Rhododendron simsii shrublands on the Qinghai-Tibet Plateau, habitat fragmentation, and the near depletion of wild resources, which does not meet the requirements for the sustainable development of Tibetan medicinal resources and the protection of the plateau's ecology. Second, alpine cultivation is severely limited by the harsh climatic conditions of high altitude, low temperatures, strong ultraviolet radiation, permafrost, and a short frost-free period, resulting in extremely low survival rates in artificial field cultivation, making it difficult to supply raw materials on a large scale. Third, the medicinal components of individual wild plants vary greatly, significantly affected by altitude, sunlight, and soil conditions, making it difficult to standardize and control the quality of raw materials.

[0004] In vitro callus culture is a fundamental technology for the aseptic propagation of rare medicinal plants, suspension fermentation of medicinal cells, targeted enrichment of secondary metabolites, artificial germplasm resource propagation, and selection of superior varieties. Currently, tissue culture techniques for Rhododendron species are insufficient for directly propagating Rhododendron 'Liexiang' seedlings because it is a high-altitude, cold-resistant, acid-loving Tibetan medicinal plant with extremely high phenolic content in its explants, making it highly susceptible to browning and necrosis during in vitro culture. Furthermore, this plant is difficult to dedifferentiate, and conventional MS basal medium and general Rhododendron hormone formulations suffer from high explant browning rates, low callus induction rates, loose and waterlogged callus tissue, poor proliferation stability, and rapid browning and apoptosis. Currently, there is no standardized callus generation technology specifically designed for Rhododendron 'Liexiang' with low browning and high induction rates, severely hindering research and industrialization related to its medicinal cell engineering, artificial propagation, industrial production of secondary metabolites, and in vitro preservation of germplasm resources. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes a method for inducing and proliferating callus tissue from flower buds of the Tibetan medicine Rhododendron 'Liexiang'. Based on the alpine physiological characteristics of Rhododendron 'Liexiang', this method utilizes optimized anti-browning systems, specialized growth regulators, and stepwise culture techniques to achieve efficient induction and stable subculture proliferation of Rhododendron 'Liexiang' callus tissue, obtaining dense, granular embryogenic callus tissue. This provides standardized embryogenic callus material for subsequent plant regeneration, suspension culture of medicinal secondary metabolites, construction of in vitro germplasm banks, and production of Tibetan medicine cell factories.

[0006] To achieve the above objectives, this invention provides a method for inducing and proliferating callus tissue from the flower buds of the Tibetan medicinal herb *Rhododendron simsii*, comprising the following steps: (1) Screening and pretreatment of explants: Take unopened flower buds from Rhododendron simsii branches, remove the outer dry bracts and rinse with running water, and take out the flower buds in the inner layer of the flower buds as explants; (2) Gradient sterilization of explants: The explants are subjected to gradient sterilization treatment, and then the surface moisture is dried to obtain sterile explants; (3) Callus induction culture: The sterile explants were inoculated into the primary induction culture medium and cultured in complete darkness to obtain the initial callus; (4) Subculture of callus: The initial callus is cut into pieces and transferred to primary proliferation medium, secondary proliferation medium and expansion medium in sequence for culture to complete the proliferation and expansion of callus.

[0007] Preferably, in step (1), the rinsing time is 30~40 minutes.

[0008] Preferably, in step (2), the gradient disinfection process specifically involves: soaking the explant in 75% ethanol for 10 seconds, then disinfecting it in 2% sodium hypochlorite solution for 10-15 minutes, and rinsing it with sterile water 7-9 times; subsequently, soaking it in 70%-75% ethanol for 15-25 seconds, rinsing it with sterile water 5-6 times, then soaking it in 0.1% mercuric chloride solution for 4-6 minutes, and rinsing it with sterile water 5-6 times.

[0009] Preferably, in step (4), the cutting is done by cutting into small pieces of 0.3cm × 0.4cm.

[0010] Preferably, in step (3), the primary induction culture medium consists of: 50 mL / L 10× macro-element reagent, 10 mL / L 100× micro-element reagent, 10 mL / L 100× iron salt, 10 mL / L 100× organic vitamins, 2.80 mg / L thiamethoxam, 1.85 mg / L gibberellin, 20~25 g sucrose and 7.5 g agar powder, with a pH of 5.2~5.4; the culture temperature in the completely dark environment is 20℃~22℃, and the culture time is 4~6 days. In step (4), the primary proliferation medium consists of: 50 mL / L 10× macro-element reagent, 10 mL / L 100× micro-element reagent, 10 mL / L 100× iron salt, 10 mL / L 100× organic vitamins, 0.08 mg / L naphthaleneacetic acid, 0.8 mg / L 6-benzylaminopurine, 20~30 g / L sucrose and 7.5 g / L agar powder, pH 5.2~5.4, light intensity 2800~3600 lx, photoperiod 12h light / 12h dark, temperature 20~24℃, and culture time 4~7 days; The second-generation proliferation medium consisted of: 50 mL / L 10× macro-element reagent, 10 mL / L 100× micro-element reagent, 10 mL / L 100× iron salt, 10 mL / L 100× organic vitamins, 0.06 mg / L naphthaleneacetic acid, 0.6 mg / L 6-benzylaminopurine, 20-30 g / L sucrose, and 7.5 g / L agar powder. The pH was 5.2-5.4. The light intensity was 2500-3000 lx, the photoperiod was 12 h light / 12 h dark, the temperature was 20-24℃, and the culture time was 4-7 days. The propagation medium consisted of: 25 mL / L 10× macro-element reagent, 10 mL / L 100× micro-element reagent, 10 mL / L 100× iron salt, 10 mL / L 100× organic vitamins, 0.15 mg / L naphthaleneacetic acid, 20 g / L sucrose, and 7.2 g / L agar powder. The pH was 5.2–5.4. The light intensity was 2800–3150 lx, the photoperiod was 12 h light / 12 h dark, the temperature was 22–25 °C, and the culture time was 4–6 days.

[0011] Preferably, the composition of the 10× macro-element reagent is 16.5 g / L NH4NO3, 19 g / L KNO3, 4.4 g / L CaCl2·2H2O, 3.7 g / L MgSO4·7H2O, and 1.7 g / L KH2PO4; the composition of the 100× micro-element reagent is 0.83 g / L KI, 6.2 g / L H3BO3, 22.3 g / L MnSO4·4H2O, 8.6 g / L ZnSO4·7H2O, 0.25 g / L Na2MoO4·2H2O, 0.025 g / L CuSO4·5H2O, and 0.025 g / L CoCl2·6H2O; and the composition of the 100× iron salt is 3.73 g / L Na2-EDTA and 7 H2O. 2.78 g / L; The composition of 100× organic vitamins is inositol 10 g / L, niacin 0.1 g / L, VB6 0.1 g / L, and VB1 0.02 g / L.

[0012] The method for inducing and proliferating callus tissue from flower buds of the Tibetan medicinal herb Rhododendron 'Liexiang' is also provided for its application in the preservation of germplasm resources of Rhododendron 'Liexiang'.

[0013] The method for inducing and proliferating callus tissue from Rhododendron simsii flower buds, as described in the Tibetan medicine, is also provided for its application in the in vitro cultivation of medicinal components.

[0014] The method for inducing and proliferating callus tissue in Rhododendron simsii flower buds is also provided for application in the large-scale rapid propagation of seedlings.

[0015] Compared with the prior art, the present invention has the following advantages and technical effects: (1) This invention is the first to select plump, unopened flower buds of suitable age in high-altitude areas as the only exclusive explant. The tissue has dense meristematic cells, strong division activity, and few oxidative substrates, which can significantly accelerate the callus initiation speed and greatly reduce the browning rate. The induction rate of Rhododendron simsii flower bud callus tissue is ≥86%, which is 35%~68% higher than the control group, far exceeding the existing conventional tissue culture technology. The culture cycle is shortened to 28~31 days, which greatly improves the culture efficiency.

[0016] (2) Based on the metabolic differences between the two core physiological stages of callus induction and redifferentiation proliferation, this invention constructs a segmented culture mode of full-dark dedifferentiation induction and moderate light proliferation, which precisely matches the temperature, light intensity, photoperiod and environmental humidity parameters of each stage, replacing the traditional single constant culture conditions. This makes the callus grow uniformly, with dense texture, no browning, no water staining, strong cell activity, and stable genetic traits, and can be subcultured for a long time to meet the needs of subsequent differentiation, extraction of medicinal components and germplasm preservation.

[0017] (3) This invention improves the culture medium and precisely controls the environment to adapt to the characteristics of Rhododendron simsii on the plateau, effectively solving the problem of browning and necrosis. It has high technical stability and strong reproducibility and can be promoted and applied on a large scale.

[0018] (4) This invention uses wild flower buds of Rhododendron simsii as explants, which is convenient to obtain and causes little damage to the native plants. While protecting wild germplasm resources, it achieves efficient propagation of in vitro resources, taking into account both ecological protection and industrial value.

[0019] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0020] Figure 1 Before flower bud tissue induction and proliferation culture; Figure 2 After flower bud tissue induction and proliferation culture; Figure 3 This describes the initial culture of flower bud callus tissue. Figure 4 This describes the subculture and proliferation of flower bud callus tissue. Figure 5 This diagram illustrates the formation of callus tissue from flower bud and stem / leaf differentiation. In the diagram, A represents callus tissue formed from flower bud differentiation, and B represents callus tissue formed from stem / leaf differentiation. Detailed Implementation

[0021] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0022] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0023] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available materials.

[0024] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein can be applied to the methods of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0025] The MS media involved in the examples are all modified MS media. The modified MS media are: glycine component removed from the traditional MS media, the amount of ammonium nitrate reduced to 50%~60% of the traditional MS media, and the total inorganic nitrogen concentration reduced. The specific composition includes: 10× macro-element reagent (composition: NH4NO3 16.5g / L, KNO3 19g / L, CaCl2·2H2O 4.4g / L, MgSO4·7H2O 3.7g / L, KH2PO4 1.7g / L), 100× micro-element reagent (composition: KI 0.83g / L, H3BO3 6.2g / L, MnSO4·4H2O 22.3g / L, ZnSO4·7H2O 8.6g / L, Na2MoO42H2O 0.25g / L, CuSO4·5H2O 0.025g / L, CoCl2·6H2O 0.025g / L), and 100× iron salt (composition: Na2-EDTA 3.73g / L, FeSO4·7H2O). 2.78 g / L) and 100× organic vitamins (composed of inositol 10 g / L, niacin 0.1 g / L, VB6 0.1 g / L, VB1 0.02 g / L).

[0026] Example 1 A method for inducing and subculturing callus tissue from flower buds of the Tibetan medicinal herb Rhododendron 'Liexiang', with schematic diagrams before and after flower bud induction and subculturing as shown below. Figure 1 , Figure 2 As shown, the specific steps include: S1. Explant screening and pretreatment.

[0027] Fresh, healthy, and disease-free wild Rhododendron simsii branches, collected on May 12, 2025, from Baoku Forest Farm, Datong Hui and Tu Autonomous County, Qinghai Province, at an altitude of 2800-3500m, were selected. Plump flower buds were chosen as explants. The outer layer of dried bracts was removed, and the explants were rinsed with running water for 30-40 minutes and placed in a clean bench for later use. The flower buds were carefully peeled open layer by layer with small tweezers, and the small green buds (flower buds) inside the buds were removed and sterilized. Simultaneously, stems and leaves were used as controls for subsequent experiments.

[0028] S2, Gradient aseptic disinfection of explants.

[0029] First, sterilize the flower buds, stems and leaves with 75% ethanol for 10-16 seconds, then disinfect them with 2%-2.7% sodium hypochlorite solution for 13 minutes. Rinse the pretreated flower buds, stems and leaves with sterile water 7 times.

[0030] Then, the treated flower buds are subjected to gradient disinfection treatment, which involves soaking in 70%~75% ethanol for 15~25 seconds, rinsing with sterile water 5~6 times, soaking in 0.1% mercuric chloride solution for 4~6 minutes, and rinsing with sterile water 5~6 times.

[0031] Finally, blot dry the surface moisture on sterile filter paper before inoculation.

[0032] S3, callus induction culture.

[0033] Sterilized callus explants (flower buds, stem and leaf callus) were inoculated into primary culture medium, 4-5 explants per bottle, and inducing culture in complete darkness at a temperature of 20-22℃. After 4-7 days of culture, pale yellow-green initial callus tissue was obtained (e.g., ...). Figure 3 (As shown).

[0034] The initial culture medium consists of: 50 mL / L 10× macro-element reagent, 10 mL / L 100× micro-element reagent, 10 mL / L 100× iron salt, 10 mL / L 100× organic vitamins, and 2.80 mg / L TDZ (thiabendazole). Then add 1.85 mg / L GA3 (gibberellin), followed by 20-25 g of sucrose, and finally 7.5 g of agar powder. Adjust the pH to 5.2. After the solution boils and becomes clear, it can be dispensed into inoculation bottles, sterilized, and used.

[0035] S4. Subculture and proliferation of callus tissue.

[0036] Select dense, pale yellow-green, and non-browning initial callus tissues (buds, stems, and leaves), cut them into 0.3cm × 0.4cm pieces. First, transfer them to primary proliferation medium and culture them for 2 days at a light intensity of 2800 lx, a photoperiod of 12h light / 12h dark, and a temperature of 21-22℃. Second, transfer them to secondary proliferation medium and culture them for 2 days at a light intensity of 2900 lx, a photoperiod of 12h light / 12h dark, and a temperature of 21-22℃. Third, finally culture them for 4-5 days at a propagation medium (light intensity of 2850 lx, a photoperiod of 12h light / 12h dark, and a temperature of 22-23℃).

[0037] The primary proliferation medium consisted of: 50 mL / L 10× macro-element reagent, 10 mL / L 100× micro-element reagent, 10 mL / L 100× iron salt, 10 mL / L 100× organic vitamins, 0.08 mg / L NAA (naphthaleneacetic acid), 0.8 mg / L 6-BA (6-benzylaminopurine), 28 g / L sucrose, and 7.5 g / L agar powder, with the pH adjusted to 5.2–5.4. The composition of the second-generation proliferation medium is as follows: 50 mL / L 10× macro-element reagent, 10 mL / L 100× micro-element reagent, 10 mL / L 100× iron salt, 10 mL / L 100× organic vitamins, 0.06 mg / L NAA (naphthaleneacetic acid), 0.6 mg / L 6-BA (6-benzylaminopurine), 28 g / L sucrose, and 7.5 g / L agar powder, with the pH adjusted to 5.2~5.4.

[0038] The propagation medium consisted of: 25 mL / L 10× macro-element reagent, 10 mL / L 100× micro-element reagent, 10 mL / L 100× iron salt, 10 mL / L 100× organic vitamins, 0.15 mg / L NAA (naphthaleneacetic acid), 20 g / L sucrose, 7.2 g / L agar powder, and pH 5.2~5.4.

[0039] The results showed that the average callus induction rate of flower buds was 87.2%, and the average browning rate was 8.3% (e.g., ...). Figure 4 (As shown). A schematic diagram of flower bud and stem / leaf culture is shown below. Figure 5 As shown, a total of 29 subculture groups were induced. Statistical analysis revealed that 22 groups were flower bud induction subcultures, with an induction rate of 82%–96% and a browning rate of 0%–9%. In the 7 groups of stem and leaf induction subcultures, the induction rate was 72%–89% and the browning rate was 2%–11%. The results confirm that bud induction is more efficient and faster.

[0040] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for inducing and proliferating callus tissue from the flower buds of the Tibetan medicinal herb *Rhododendron simsii*, characterized in that... Includes the following steps: (1) Screening and pretreatment of explants: Take unopened flower buds from Rhododendron simsii branches, remove the outer dry bracts and rinse with running water, then take out the flower buds in the bud layer as explants; (2) Gradient sterilization of explants: The explants are subjected to gradient sterilization treatment, and then the surface moisture is dried to obtain sterile explants; (3) Callus induction culture: The sterile explants were inoculated into the primary induction culture medium and cultured in complete darkness to obtain the initial callus tissue; (4) Subculture of callus: The initial callus is cut into pieces and transferred to primary proliferation medium, secondary proliferation medium and expansion medium in sequence for culture to complete the proliferation and expansion of callus.

2. The method according to claim 1, characterized in that, In step (1), the water rinsing time is 30~40 minutes.

3. The method according to claim 1, characterized in that, In step (2), the gradient disinfection process is as follows: the explant is soaked in 75% ethanol for 10 seconds, then disinfected in 2% sodium hypochlorite solution for 10-15 minutes, and then rinsed with sterile water 7-9 times; subsequently, it is soaked in 70%-75% ethanol for 15-25 seconds, rinsed with sterile water 5-6 times, then soaked in 0.1% mercuric chloride solution for 4-6 minutes, and then rinsed with sterile water 5-6 times.

4. The method according to claim 1, characterized in that, In step (4), the cutting and segmentation refers to cutting into small pieces of 0.3cm × 0.4cm.

5. The method according to claim 1, characterized in that, In step (3), the primary induction culture medium consists of: 50 mL / L 10× macro-element reagent, 10 mL / L 100× micro-element reagent, 10 mL / L 100× iron salt, 10 mL / L 100× organic vitamins, 2.80 mg / L thiamethoxam, 1.85 mg / L gibberellin, 20-25 g sucrose and 7.5 g agar powder, with a pH of 5.2-5.4; the culture temperature in the completely dark environment is 20℃-22℃, and the culture time is 4-6 days. In step (4), the primary proliferation culture medium consists of: 50 mL / L 10× macro-element reagent, 10 mL / L 100× micro-element reagent, 10 mL / L 100× iron salt, 10 mL / L 100× organic vitamins, 0.08 mg / L naphthaleneacetic acid, 0.8 mg / L 6-benzylaminopurine, 20~30 g / L sucrose and 7.5 g / L agar powder, pH 5.2~5.4, light intensity 2800~3600 lx, photoperiod 12h light / 12h dark, temperature 20~24℃, and culture time 4~7 days; The second-generation proliferation medium consisted of: 50 mL / L 10× macro-element reagent, 10 mL / L 100× micro-element reagent, 10 mL / L 100× iron salt, 10 mL / L 100× organic vitamins, 0.06 mg / L naphthaleneacetic acid, 0.6 mg / L 6-benzylaminopurine, 20-30 g / L sucrose, and 7.5 g / L agar powder. The pH was 5.2-5.

4. The light intensity was 2500-3000 lx, the photoperiod was 12 h light / 12 h dark, the temperature was 20-24℃, and the culture time was 4-7 days. The propagation culture medium consisted of: 25 mL / L 10× macro-element reagent, 10 mL / L 100× micro-element reagent, 10 mL / L 100× iron salt, 10 mL / L 100× organic vitamins, 0.15 mg / L naphthaleneacetic acid, 20 g / L sucrose, and 7.2 g / L agar powder. The pH was 5.2–5.

4. The light intensity was 2800–3150 lx, the photoperiod was 12 h light / 12 h dark, the temperature was 22–25 °C, and the culture time was 4–6 days.

6. The method according to claim 5, characterized in that, The 10× macro-element reagent consists of NH4NO3 16.5 g / L, KNO3 19 g / L, CaCl2·2H2O 4.4 g / L, MgSO4·7H2O 3.7 g / L, and KH2PO4 1.7 g / L; the 100× micro-element reagent consists of KI 0.83 g / L, H3BO3 6.2 g / L, MnSO4·4H2O 22.3 g / L, ZnSO4·7H2O 8.6 g / L, Na2MoO4·2H2O 0.25 g / L, CuSO4·5H2O 0.025 g / L, and CoCl2·6H2O 0.025 g / L; the 100× iron salt consists of Na2-EDTA 3.73 g / L and FeSO4·7H2O. 2.78 g / L; the composition of the 100× organic vitamins is inositol 10 g / L, niacin 0.1 g / L, VB6 0.1 g / L, and VB1 0.02 g / L.

7. The application of the method as described in any one of claims 1 to 6 in the preservation of germplasm resources of Tibetan medicinal herb Rhododendron simsii.

8. The application of the method according to any one of claims 1 to 6 in the cultivation of in vitro pharmaceutical components.

9. The application of the method as described in any one of claims 1 to 6 in the large-scale rapid propagation of seedlings.

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