In-vitro propagation method of Chinese herbal medicine rhododendron deciduous-rhododendron molle

Abstract

The invention belongs to the field of plant tissue culture, and particularly relates to an in-vitro propagation method of Chinese herbal medicine rhododendron decidulum-rhododendron molle. The in-vitro propagation method comprises the steps of explant collection and treatment, adventitious bud differentiation induction, elongation and stem extraction, rooting induction and acclimatization and transplantation, wherein wild rhododendron molle branches are collected to cultivate tender leaves, and the tender leaves are washed and disinfected to obtain explants; and sequentially inoculating into WPM1, WPM2 and WPM3 culture mediums, respectively completing adventitious bud induction, stem drawing and rooting culture, and finally, carrying out acclimatization and transplanting to obtain adult plants. According to the method, tender leaves are used as explants, the culture medium is simple in formula, few in hormone variety, safe and environment-friendly to operate, high in propagation efficiency and short in period, the adventitious bud induction rate and the transplanting survival rate both reach 100%, the method is suitable for large-scale production, and technical support is provided for rhododendron molle species protection and Chinese herbal medicine industrialization.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture, specifically relating to an in vitro propagation method for the traditional Chinese medicine Rhododendron deciduousense (Rhododendron molle). Background Technology

[0002] Rhododendron molle ( Rhododendron molle *Rhododendron simsii* (Blume) G. Don is a deciduous shrub belonging to the Ericaceae family, also known as Yellow Rhododendron, Jade Branch, Sheep-Not-Eat Grass, and Rhododendron Flower, and is endemic to China. The entire plant resembles a herbaceous vine, with young branches covered in soft and stiff hairs. The leaves are oblong, about 8 cm long, and young leaves are also pubescent. The funnel-shaped corolla is 4.5 cm long and golden yellow. The cylindrical capsule is about 3 cm long. *Rhododendron simsii* generally grows in high-altitude, humid climates, such as some southern regions like Guizhou, Fujian, and Yunnan. It cannot grow in the cold conditions of the north, thus its wild population faces conservation challenges. *Rhododendron simsii* is tolerant of acidic, drought, and poor soil, but it does not grow well in clay or poorly permeable soil, and it is intolerant of strong sunlight, only surviving in shrublands or under forests on hillsides.

[0003] The entire plant of *Rhododendron molle* is toxic, with the flowers and fruits being particularly toxic. It was listed in the *Chinese Pharmacopoeia* in 1990. As an important medicinal plant, it contains various compounds such as terpenes, flavonoids, and lignans, possessing multiple effects including immunomodulatory, analgesic, hypotensive, antiviral, and anti-inflammatory properties. More than 170 compounds can be isolated from various organs, with the main medicinal tissues being the roots, flowers, fruits, and leaves. Diterpenes and lignans can be extracted from the roots, while diterpenes and flavonoids have been identified in the flowers. Diterpenes, triterpenes, and flavonoids can be isolated from the fruits, and sesquiterpenes, diterpenes, triterpenes, flavonoids, and lignans can also be obtained from the leaves. Furthermore, the leaves have strong regeneration capabilities, making them suitable for large-scale production.

[0004] Rhododendron molle exhibits self-incompatibility; even when hybridized with other genera, the offspring are weak and fail to flower, resulting in a high sterility rate. Coupled with human-induced digging and destruction, Rhododendron molle resources are dwindling. Propagation is generally achieved through seed germination, cuttings, and layering. Studies have shown that storing dried Rhododendron molle seeds in a low-temperature environment improves germination and emergence rates; seedlings grown in petri dishes are also more robust. Furthermore, two cutting propagation techniques—full-light misting and closed-loop humidity control—have been applied in small-scale trials, with different hormone concentrations showing significant differences in rooting rates. Layering propagation combined with ABT rooting powder can also achieve rooting and survival rates exceeding 80%. As a species that is difficult to root, the propagation of Rhododendron molle is limited by many environmental factors. Although layering and cutting propagation can ensure the survival of the plants, both asexual propagation methods must be completed between mid-July and mid-August, which is a strict time requirement. Furthermore, the age of the branches, the circumference of the girdling, and the substrate all affect the survival rate of callus tissue, rooting rate, and overall survival rate. Therefore, the establishment of an asexual propagation system is particularly important if large-scale propagation is to be achieved.

[0005] Gu Dizhou et al. published "High-Efficiency Plant Regeneration Technology of Rhododendron molle" in 2013. Using newly sprouted stems as explants, they utilized a four-hormone mixture (1 / 4 DR + ZT 0.06 mg / L + NAA 0.02 mg / L + GA3 1 mg / L) to induce germination, elongation, and rooting. Similarly, Shao Diankun et al., in their 2013 paper "Induction of Axillary Bud Clustering Sprouts and High-Efficiency Plant Regeneration of Rhododendron molle," also used newly sprouted stem segments as explants and employed a combination of five hormones. The induction medium for clustering buds was 1 / 2 DR + 2ip 4.40 mg / L + NAA 0.01 mg / L + GA3 2.50 mg / L, and the rooting medium was 1 / 4 DR + ZT 0.06 mg / L + NAA 0.02 mg / L. However, this type of technology relies on a large number of hormone combinations, resulting in complex formulations and high seedling costs.

[0006] In their 2018 paper, "Establishment of an In Vitro Leaf Regeneration System for Rhododendron molle," Sun Xiaobo et al. published a protocol for the in vitro regeneration of Rhododendron molle leaves. Using WPM medium, they collected the 5th and 6th leaves for adventitious bud induction. After 10-15 days of dark culture, adventitious buds were differentiated using a formulation of WPM + TDZ 1.0 mg / L + IAA 1.0 mg / L. However, this technique only achieved adventitious bud induction and did not regenerate complete plants, resulting in a low transplant survival rate.

[0007] In the invention published in 2020 by Luo Xiangdong et al., under publication number CN 105532467 A, a method for the in vitro tissue culture, propagation and preservation of Rhododendron molle was disclosed, which described a multiple sterilization method for axillary buds, involving 70% ethanol, 10% hydrogen peroxide containing 0.5% Tween 20 and 0.1% mercuric chloride containing 0.5% Tween 20. The differentiation and propagation culture medium consists of 6,7-V + ZT 1.5 mg / L + NAA 0.05 mg / L + 2,4-D 0.05 mg / L, with a proliferation culture period of 20-40 days. The stem-elongation culture medium is half 6,7-V + ZT 1 mg / L, with the medium changed every 3-4 months. Rooting is induced by adding 0.05 mg / L NAA to half 6,7-V medium. The tissue culture seedlings are then transplanted into a mixed substrate of perlite and nutrient soil and cultured in a small greenhouse for 10-15 days, with the film gradually removed. After 15 days, the greenhouse film is completely removed to complete the acclimatization process. However, this technology contains a combination of Tween 20, hydrogen peroxide, and mercuric chloride (toxic), posing significant operational risks and being environmentally unfriendly. Furthermore, the axillary bud differentiation, stem-elongation, and rooting culture medium described in this patent uses four different plant hormones, and the entire process takes at least four months to obtain seedlings, resulting in high time and economic costs.

[0008] Therefore, based on this, the technical solution of the present invention is proposed. Summary of the Invention

[0009] This invention aims to solve the technical problems of existing Chinese herbal medicines such as the picky living environment, difficulty in rooting, and low propagation efficiency of Rhododendron deciduousii-Rhododendron simsii. The solution of this invention is to provide an in vitro propagation method for the Chinese herbal medicine Rhododendron deciduousii-Rhododendron simsii, which includes the following steps:

[0010] (1) Collection and treatment of explants: branches of wild Rhododendron deciduousii-Rhododendron simsii plants were collected, placed in water for cultivation, and the tender leaves obtained from the cultivation were cut off and rinsed and disinfected in sequence to obtain disinfected explants; (2) Induction of adventitious bud differentiation: The sterilized explants were placed in adventitious bud induction medium WPM1 for culture to induce callus tissue with adventitious buds; (3) Elongation of stems: The callus tissue with adventitious buds was transferred to the stem elongation culture medium WPM2 and cultured for a period of time to increase the stem length; (4) Inducing rooting: After the adventitious buds have increased in length, cut them off and place them in WPM3 rooting medium for cultivation. Then harden the seedlings and transplant them into the soil for cultivation.

[0011] It should be emphasized that the wild-type deciduous rhododendron collected in this invention is from the distribution area of ​​Yunnan. This plant grows in hilly areas at an altitude of 1000 meters, with a height of about 1.5 meters. The whole plant can be used as medicine, and it is characterized by difficulty in rooting.

[0012] Preferably, in step (1), branches of wild deciduous rhododendron-Rhododendron plants with a length of 8-12 cm and free from pests and diseases and in good growth condition are collected.

[0013] Preferably, in step (1), the disinfection method is as follows: the tender leaves that have been rinsed are soaked in sterile water 4-6 times, then quickly soaked in 70-80% alcohol for 10-20 seconds, and then rinsed with sterile water 4-6 times until the alcohol is completely removed; then soaked in 2-4% sodium hypochlorite for 5-15 minutes, and then rinsed with sterile water 4-6 times until the sodium hypochlorite is completely removed; finally, the water is absorbed with sterile paper and the blackened parts of the material are removed during the operation.

[0014] Preferably, in step (2), the adventitious bud induction culture medium WPM1 includes macroelements, microelements, iron salts, organic matter, naphthaleneacetic acid, and 6-benzylaminopurine; wherein: The macroelements include: 500-800 mg / L ammonium nitrate, 2.0 g / L potassium sulfate, 400-500 mg / L magnesium sulfate, 300-400 mg / L dipotassium hydrogen phosphate, and 150-200 mg / L calcium chloride; The trace elements include: 45-50 mg / L manganese sulfate, 15-20 mg / L zinc sulfate, 15-20 mg / L boric acid, 0.5-0.8 mg / L copper sulfate, and 0.5-0.8 mg / L sodium molybdate; The iron salts include: 800-1000 mg / L calcium nitrate and 75-78 mg / L disodium ethylenediaminetetraacetate; The organic compounds include: 150-200 mg / L inositol, 1.5-2.0 mg / L thiamine hydrochloride, 1.0-1.5 mg / L nicotinic acid, 1.0-1.5 mg / L pyridoxine hydrochloride, 2.0-5.0 mg / L glycine, 20-30 g / L sucrose, and 5.0-6.0 g / L agar; The concentration of naphthaleneacetic acid is 0.02-0.04 mg / L; The concentration of 6-benzylaminopurine is 0.03-0.05 mg / L.

[0015] Preferably, in step (2), the cultivation method is as follows: under conditions of 24-26 ℃, light cultivation is carried out for 12-16 h / d, and the radiation flux density is 30-40 μmol·m. -2 ·s -1 The cultivation period is 28-32 days.

[0016] Preferably, in step (3), the stem-cutting culture medium WPM2 comprises macroelements, microelements, iron salts, organic matter, naphthaleneacetic acid, and 6-benzylaminopurine; wherein: The macroelements include: 500-800 mg / L ammonium nitrate, 2 g / L potassium sulfate, 400-500 mg / L magnesium sulfate, 300-400 mg / L dipotassium hydrogen phosphate, and 150-200 mg / L calcium chloride; The trace elements include: 45-50 mg / L manganese sulfate, 15-20 mg / L zinc sulfate, 15-20 mg / L boric acid, 0.5-0.8 mg / L copper sulfate, and 0.5-0.8 mg / L sodium molybdate; The iron salts include: 800-1000 mg / L calcium nitrate and 75-78 mg / L disodium ethylenediaminetetraacetate; The organic compounds include: 150-200 mg / L inositol, 1.5-2.0 mg / L thiamine hydrochloride, 1.0-1.5 mg / L nicotinic acid, 1.0-1.5 mg / L pyridoxine hydrochloride, 2.0-5.0 mg / L glycine, 20-30 g / L sucrose, and 5.0-6.0 g / L agar; The concentration of naphthaleneacetic acid is 0.02-0.04 mg / L; The concentration of 6-benzylaminopurine is 0.001-0.1 mg / L.

[0017] Preferably, in step (4), the rooting medium WPM3 includes macroelements, microelements, iron salts, organic matter, and naphthaleneacetic acid; wherein: The macroelements include: 500-800 mg / L ammonium nitrate, 2.0 g / L potassium sulfate, 400-500 mg / L magnesium sulfate, 300-400 mg / L dipotassium hydrogen phosphate, and 150-200 mg / L calcium chloride; The trace elements include: 45-50 mg / L manganese sulfate, 15-20 mg / L zinc sulfate, 15-20 mg / L boric acid, 0.5-0.8 mg / L copper sulfate, and 0.5-0.8 mg / L sodium molybdate; The iron salts include: 800-1000 mg / L calcium nitrate and 75-78 mg / L disodium ethylenediaminetetraacetate; The organic compounds include: 150-200 mg / L inositol, 1.5-2.0 mg / L thiamine hydrochloride, 1.0-1.5 mg / L nicotinic acid, 1.0-1.5 mg / L pyridoxine hydrochloride, 2.0-5.0 mg / L glycine, 20-30 g / L sucrose, and 5.0-6.0 g / L agar; The concentration of naphthaleneacetic acid is 0.5-2.0 mg / L.

[0018] Preferably, in step (4), the seedling hardening method is as follows: open the cap of the culture bottle, add distilled water to the tissue culture bottle containing the plant, and harden the seedlings at a temperature of 24-26 ℃, humidity above 60%, light intensity of 12-16 h / d, and radiation flux density of 30-40 μmol·m -2 ·s -1 Culture for 2-3 days under suitable conditions.

[0019] Preferably, in step (4), the transplanting method is as follows: the culture medium in the tissue culture bottle containing the plant is broken up, the plant is removed from the bottle and rinsed under running water to remove the culture medium on the surface of the roots, then placed in a seedling pot and covered with soil until the roots are not exposed to air; then the soil is compacted and watered until the soil is saturated, and the transplanting is carried out at a temperature of 24-26 ℃, humidity above 80%, light intensity of 12-16 h / d, and radiation flux density of 30-40 μmol·m -2 ·s -1 Cultivate under the following conditions for 6-8 days; water daily during this period to maintain soil moisture above 80%; after 6-8 days, reduce the amount of water in the soil and the air humidity to above 60%, and control the temperature at 22-24℃ for continued cultivation.

[0020] The beneficial effects of this invention are as follows: 1. This invention uses the tender leaves of wild Rhododendron molle as explants to induce and proliferate adventitious buds. Through asexual reproduction, it rapidly expands the plant using aseptic culture. It features high propagation efficiency, low cost, short cycle, and is not limited by season. Its source is singular, the plants grow uniformly, and the content of effective medicinal components is stable, making it suitable for large-scale production.

[0021] 2. Existing sterilization methods involve highly toxic chemicals, pollute the environment, require multiple hormones, involve cumbersome procedures, have high seedling costs, and result in slow plant growth and low efficiency. The difference between this invention and existing technologies lies in its use of newly sprouted leaves as material, which is readily available and promotes rapid growth. It employs a WPM-based culture medium combined with naphthaleneacetic acid and 6-benzylaminopurine hormone, reducing seedling costs and increasing the number of buds. After 30 days, the number of buds reaches over 40. Bud elongation can be observed after 20 days in the stem-elongating medium. After another 10 days of cultivation, when the stem length reaches 1.5-2.0 cm, the plant can enter the rooting culture stage. After 30 days of cultivation in the rooting medium, the plant root system reaches 1.5-2.0 cm, ready for hardening off and transplanting.

[0022] 3. This invention provides optimal culture conditions and medium for the growth of Rhododendron molle. By applying different combinations and ratios of hormones, a stem-elongation step is added after inducing adventitious buds, increasing the number of plants propagated and the rooting survival rate. Although an additional step is added, the overall propagation efficiency is greatly improved, and the seedling time is reduced. In this invention, the adventitious bud induction rate is 100%, the number of adventitious buds on each explant is approximately 40, the stem length is 1.5-2.0 cm, the tissue culture rooting rate is 98%, and the transplant survival rate is 100%.

[0023] 4. Compared with this invention, the two literature reports, "High-Efficiency Plant Regeneration Technology of Rhododendron molle" and "Induction of Axillary Bud Clustering in Tender Stems of Rhododendron molle and High-Efficiency Plant Regeneration," use more plant hormones, which are expensive. This invention, however, only requires two hormones, reducing costs. Furthermore, this invention uses tender leaves for adventitious bud induction, making it easier to collect explant material without damaging the plant itself, thus better protecting the plant.

[0024] 5. Compared with the literature "Establishment of the In Vitro Leaf Regeneration System of Rhododendron molle" in the background art, the present invention not only completes the induction of adventitious buds, but also induces adventitious buds to take root and grow into seedlings, completing the entire process from in vitro regeneration of plants to container seedling cultivation, which is a significant breakthrough.

[0025] 6. Compared with the prior art patent CN 105532467 A, this invention treats young leaves, making explant materials easier to obtain and without damaging the plant itself. The explant disinfection method only requires 75% alcohol and 3% sodium hypochlorite. Compared with the combination of Tween 20, hydrogen peroxide, and mercuric chloride (toxic), this invention is safer to operate, facilitates wastewater treatment, is environmentally friendly, and suitable for large-scale application. Furthermore, existing patents use four different plant hormones in the axillary bud differentiation, stem elongation, and rooting culture media, and the entire process takes at least four months to obtain seedlings. This invention, however, involves only two hormones, resulting in lower costs and a seedling formation time of only three months, leading to higher tissue culture regeneration efficiency. Attached Figure Description

[0026] 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 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.

[0027] Figure 1 This is a photograph of leaf differentiation in step (2) of Example 1.

[0028] Figure 2This is a photograph of the adventitious buds sprouting stems in step (3) of Example 1.

[0029] Figure 3 This is a photograph of the plant rooting in step (4) of Example 1.

[0030] Figure 4 This is a photograph of the plant transplantation in step (4) of Example 1.

[0031] Figure 5 This is a photograph of the plant's growth status two months after transplanting in step (4) of Example 1. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0033] Example 1 This embodiment provides a method for in vitro propagation of the traditional Chinese medicinal herb Rhododendron deciduousense (also known as Rhododendron molle orientalis), which includes the following steps: (1) Collection and treatment of explants: Branches of wild deciduous rhododendron-Rhododendron plants, approximately 10 cm long and free from pests and diseases and in good growth condition, were collected and cultured in water until new leaves emerged. Tender leaves were then cut and rinsed with running water for about 1 hour before being sterilized in a clean bench. Among them: The specific disinfection method is as follows: After rinsing the explants in running water for 1 hour, soak and wash them 5 times with sterile water, then replace with 75% alcohol, shake quickly for 15 seconds, and then wash 5 more times with sterile water until the alcohol is completely removed. Then soak them in 3% sodium hypochlorite for 10 minutes, and wash them 5 more times with sterile water until the sodium hypochlorite is completely removed. Finally, blot dry with sterile paper, remove any blackened parts from the incision during the procedure, and set aside for later use.

[0034] (2) Adventitious shoot differentiation induction: The treated explants were placed in shoot induction medium WPM1 and cultured under light at 25℃ for 14 h / d with a radiation flux density of 35 μmol·m -2 ·s -1 The adventitious buds were cultured for approximately 30 days, and the number of adventitious buds was then counted. The adventitious bud induction rate was 100%, and each explant had no fewer than 40 adventitious buds. Photos of successful adventitious bud induction are shown below. Figure 1 As shown. Wherein: The adventitious shoot induction medium WPM1 includes macroelements, microelements, iron salts, organic matter, naphthaleneacetic acid, and 6-benzylaminopurine; more specifically: The macroelements include: 650 mg / L ammonium nitrate, 2.0 g / L potassium sulfate, 450 mg / L magnesium sulfate, 350 mg / L dipotassium hydrogen phosphate, and 175 mg / L calcium chloride; The trace elements include: 45 mg / L manganese sulfate, 15 mg / L zinc sulfate, 15 mg / L boric acid, 0.5 mg / L copper sulfate, and 0.5 mg / L sodium molybdate; The iron salts include: 900 mg / L calcium nitrate and 75 mg / L disodium ethylenediaminetetraacetate; The organic compounds include: 175 mg / L inositol, 1.5 mg / L thiamine hydrochloride, 1.0 mg / L nicotinic acid, 1.0 mg / L pyridoxine hydrochloride, 3.5 mg / L glycine, 25 g / L sucrose, and 5.4 g / L agar; The concentration of naphthaleneacetic acid is 0.03 mg / L; The concentration of 6-benzylaminopurine is 0.04 mg / L.

[0035] In preparing the bud induction medium WPM1, macroelements, microelements, iron salts, and organic matter were first diluted with water to a final volume of 1L and the pH was adjusted to 5.6 before sterilization. After sterilization, the medium was cooled to 60 °C, and 0.03 mg / L naphthaleneacetic acid and 0.04 mg / L 6-benzylaminopurine were added. The medium was then poured into plates (25 mL / plate) and allowed to solidify.

[0036] (3) Stem elongation: Replace the callus tissue from which adventitious buds were induced in the previous step with the stem elongation culture medium WPM2, maintain the same temperature and light intensity, and culture for about 20 days. Observe the stem elongation phenomenon. The stem elongates by about 1 cm. The photos of the stem elongation are as follows. Figure 2 As shown. Wherein: The stem-cutting culture medium WPM2 includes macroelements, microelements, iron salts, organic matter, naphthaleneacetic acid, and 6-benzylaminopurine; more specifically: The macroelements include: 650 mg / L ammonium nitrate, 2 g / L potassium sulfate, 450 mg / L magnesium sulfate, 350 mg / L dipotassium hydrogen phosphate, and 175 mg / L calcium chloride; The trace elements include: 45 mg / L manganese sulfate, 15 mg / L zinc sulfate, 15 mg / L boric acid, 0.5 mg / L copper sulfate, and 0.5 mg / L sodium molybdate; The iron salts include: 900 mg / L calcium nitrate and 75 mg / L disodium ethylenediaminetetraacetate; The organic compounds include: 175 mg / L inositol, 1.5 mg / L thiamine hydrochloride, 1.0 mg / L nicotinic acid, 1.0 mg / L pyridoxine hydrochloride, 3.5 mg / L glycine, 25 g / L sucrose, and 5.4 g / L agar; The concentration of naphthaleneacetic acid is 0.03 mg / L; The concentration of 6-benzylaminopurine is 0.001 mg / L.

[0037] When preparing the WPM2 culture medium for stem cutting, the macro-elements, micro-elements, iron salts, and organic matter were first diluted with water to a final volume of 1L and the pH was adjusted to 5.6 before sterilization. After sterilization, the culture medium was cooled to 60 °C, and 0.03 mg / L naphthaleneacetic acid and 0.001 mg / L 6-benzylaminopurine were added, and the mixture was poured into culture flasks (100 mL / flask) to solidify.

[0038] (4) Rooting Induction: Select adventitious buds with a stem length exceeding 1 cm. Aseptically cut the buds with scissors and place them in WPM3 rooting medium. Culture for approximately 30 days, observing root growth. When the root length exceeds 3 cm, place the plants in a greenhouse for hardening off. After 3 days, transplant them into soil for further cultivation. Images of root growth in the culture medium are shown below. Figure 3 As shown in the photo, the plant was transplanted. Figure 4 As shown, the plant's condition two months after transplanting is as follows. Figure 5 As shown. Wherein: The rooting medium WPM3 includes macro-elements, micro-elements, iron salts, organic matter, and naphthaleneacetic acid; more specifically: The macroelements include: 650 mg / L ammonium nitrate, 2.0 g / L potassium sulfate, 450 mg / L magnesium sulfate, 350 mg / L dipotassium hydrogen phosphate, and 175 mg / L calcium chloride; The trace elements include: 45 mg / L manganese sulfate, 15 mg / L zinc sulfate, 15 mg / L boric acid, 0.5 mg / L copper sulfate, and 0.5 mg / L sodium molybdate; The iron salts include: 900 mg / L calcium nitrate and 75 mg / L disodium ethylenediaminetetraacetate; The organic compounds include: 175 mg / L inositol, 1.5 mg / L thiamine hydrochloride, 1.0 mg / L nicotinic acid, 1.0 mg / L pyridoxine hydrochloride, 3.5 mg / L glycine, 25 g / L sucrose, and 5.4 g / L agar; The concentration of naphthaleneacetic acid is 0.5 mg / L.

[0039] When preparing the WPM3 rooting medium, first add water to the macro-elements, micro-elements, iron salts, and organic matter to a final volume of 1L and adjust the pH to 5.6 before sterilization. After sterilization, when the medium is cooled to 60 ℃, add 0.5 mg / L naphthaleneacetic acid and pour it into culture flasks (100 mL / flask) to solidify.

[0040] The seedling hardening method is as follows: Open the cap of the culture bottle, add distilled water to the tissue culture bottle containing the plantlets, and maintain the conditions at 25℃, humidity above 60%, light intensity of 14 h / d, and radiation flux density of 35 μmol·m. -2 ·s -1 Cultured for 2 days under the specified conditions.

[0041] The transplanting method is as follows: Break up the culture medium in the tissue culture bottle containing the plant, remove the plant from the bottle, rinse it under running water to remove the culture medium from the root surface, then place it in a seedling pot and cover it with the cultivation substrate until the roots are not exposed to air. The cultivation substrate is peat moss:vermiculite = 10:1; then compact the cultivation substrate and water until the soil is saturated. The transplanting is carried out under the following conditions: temperature 25 ℃, humidity above 80%, light intensity 14 h / d, and radiation flux density 35 μmol·m³. -2 ·s -1 Cultivate under the following conditions for 7 days; water daily during this period to maintain soil moisture above 80%; after 7 days, reduce soil moisture and air humidity to above 60%, and maintain the temperature at 23℃ for continued cultivation.

[0042] Example 2 This embodiment provides a method for in vitro propagation of the traditional Chinese medicinal herb Rhododendron deciduousense (also known as Rhododendron molle) and Rhododendron simsii, which is basically the same as in Embodiment 1, with only some parameters changing. The main differences are as follows: In step (2), the adventitious bud induction medium WPM1 includes macroelements, microelements, iron salts, organic matter, naphthaleneacetic acid, and 6-benzylaminopurine; wherein: The macroelements include: 500 mg / L ammonium nitrate, 2.0 g / L potassium sulfate, 400 mg / L magnesium sulfate, 300 mg / L dipotassium hydrogen phosphate, and 150 mg / L calcium chloride; The trace elements include: 45 mg / L manganese sulfate, 15 mg / L zinc sulfate, 15 mg / L boric acid, 0.5 mg / L copper sulfate, and 0.5 mg / L sodium molybdate; The iron salts include: 800 mg / L calcium nitrate and 75 mg / L ethylenediaminetetraacetic acid; The organic compounds include: 150 mg / L inositol, 1.5 mg / L thiamine hydrochloride, 1.0 mg / L nicotinic acid, 1.0 mg / L pyridoxine hydrochloride, 2.0 mg / L glycine, 20 g / L sucrose, and 5.4 g / L agar; The concentration of naphthaleneacetic acid is 0.02 mg / L; The concentration of 6-benzylaminopurine is 0.03 mg / L.

[0043] In step (3), the stem-cutting culture medium WPM2 includes macro-elements, micro-elements, iron salts, organic matter, naphthaleneacetic acid, and 6-benzylaminopurine; wherein: The macroelements include: 500 mg / L ammonium nitrate, 2.0 g / L potassium sulfate, 400 mg / L magnesium sulfate, 300 mg / L dipotassium hydrogen phosphate, and 150 mg / L calcium chloride; The trace elements include: 45 mg / L manganese sulfate, 15 mg / L zinc sulfate, 15 mg / L boric acid, 0.5 mg / L copper sulfate, and 0.5 mg / L sodium molybdate; The iron salts include: 800 mg / L calcium nitrate and 75 mg / L disodium ethylenediaminetetraacetate; The organic compounds include: 150 mg / L inositol, 1.5 mg / L thiamine hydrochloride, 1.0 mg / L nicotinic acid, 1.0 mg / L pyridoxine hydrochloride, 2.0 mg / L glycine, 20 g / L sucrose, and 5.4 g / L agar; The concentration of naphthaleneacetic acid is 0.02 mg / L; The concentration of 6-benzylaminopurine is 0.001 mg / L.

[0044] In step (4), the rooting medium WPM3 includes macroelements, microelements, iron salts, organic matter, and naphthaleneacetic acid; more specifically: The macroelements include: 500 mg / L ammonium nitrate, 2.0 g / L potassium sulfate, 400 mg / L magnesium sulfate, 300 mg / L dipotassium hydrogen phosphate, and 150 mg / L calcium chloride; The trace elements include: 45 mg / L manganese sulfate, 15 mg / L zinc sulfate, 15 mg / L boric acid, 0.5 mg / L copper sulfate, and 0.5 mg / L sodium molybdate; The iron salts include: 800 mg / L calcium nitrate and 75 mg / L disodium ethylenediaminetetraacetate; The organic compounds include: 150 mg / L inositol, 1.5 mg / L thiamine hydrochloride, 1.0 mg / L nicotinic acid, 1.0 mg / L pyridoxine hydrochloride, 2.0 mg / L glycine, 20 g / L sucrose, and 5.4 g / L agar; The concentration of naphthaleneacetic acid is 0.5 mg / L.

[0045] Example 3 This embodiment provides a method for in vitro propagation of the traditional Chinese medicinal herb Rhododendron deciduousense (also known as Rhododendron molle) and Rhododendron simsii, which is basically the same as in Embodiment 1, with only some parameters changing. The main differences are as follows: The adventitious shoot induction medium WPM1 comprises macroelements, microelements, iron salts, organic matter, naphthaleneacetic acid, and 6-benzylaminopurine; wherein: The macroelements include: 800 mg / L ammonium nitrate, 2.0 g / L potassium sulfate, 500 mg / L magnesium sulfate, 400 mg / L dipotassium hydrogen phosphate, and 200 mg / L calcium chloride; The trace elements include: 50 mg / L manganese sulfate, 20 mg / L zinc sulfate, 20 mg / L boric acid, 0.8 mg / L copper sulfate, and 0.8 mg / L sodium molybdate; The iron salts include: 1000 mg / L calcium nitrate and 78 mg / L disodium ethylenediaminetetraacetate; The organic compounds include: 200 mg / L inositol, 2.0 mg / L thiamine hydrochloride, 1.5 mg / L nicotinic acid, 1.5 mg / L pyridoxine hydrochloride, 5.0 mg / L glycine, 30 g / L sucrose, and 6.0 g / L agar; The concentration of naphthaleneacetic acid is 0.04 mg / L; The concentration of 6-benzylaminopurine is 0.05 mg / L.

[0046] In step (3), the stem-cutting culture medium WPM2 includes macro-elements, micro-elements, iron salts, organic matter, naphthaleneacetic acid, and 6-benzylaminopurine; wherein: The macroelements include: 800 mg / L ammonium nitrate, 2 g / L potassium sulfate, 500 mg / L magnesium sulfate, 400 mg / L dipotassium hydrogen phosphate, and 200 mg / L calcium chloride; The trace elements include: 50 mg / L manganese sulfate, 20 mg / L zinc sulfate, 20 mg / L boric acid, 0.8 mg / L copper sulfate, and 0.8 mg / L sodium molybdate; The iron salts include: 1000 mg / L calcium nitrate and 78 mg / L disodium ethylenediaminetetraacetate; The organic compounds include: 200 mg / L inositol, 2 mg / L thiamine hydrochloride, 1.5 mg / L nicotinic acid, 1.5 mg / L pyridoxine hydrochloride, 5.0 mg / L glycine, 30 g / L sucrose, and 6.0 g / L agar; The concentration of naphthaleneacetic acid is 0.04 mg / L; The concentration of 6-benzylaminopurine is 0.1 mg / L.

[0047] In step (4), the rooting medium WPM3 includes macroelements, microelements, iron salts, organic matter, and naphthaleneacetic acid; wherein: The macroelements include: 800 mg / L ammonium nitrate, 2.0 g / L potassium sulfate, 500 mg / L magnesium sulfate, 400 mg / L dipotassium hydrogen phosphate, and 200 mg / L calcium chloride; The trace elements include: 50 mg / L manganese sulfate, 20 mg / L zinc sulfate, 20 mg / L boric acid, 0.8 mg / L copper sulfate, and 0.8 mg / L sodium molybdate; The iron salts include: 1000 mg / L calcium nitrate and 78 mg / L disodium ethylenediaminetetraacetate; The organic compounds include: 200 mg / L inositol, 2.0 mg / L thiamine hydrochloride, 1.5 mg / L nicotinic acid, 1.5 mg / L pyridoxine hydrochloride, 5.0 mg / L glycine, 30 g / L sucrose, and 6.0 g / L agar; The concentration of naphthaleneacetic acid is 2.0 mg / L.

[0048] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for in vitro propagation of Rhododendron anthopogon D. Don, characterized in that, The in-vitro propagation method comprises the following steps: (1) explant collection and treatment: collecting branches of wild Rhododendron anthopogon-berberidifolium plants, placing them in water for culture, cutting the obtained tender leaves and sequentially washing and disinfecting them to obtain disinfected explants; (2) adventitious bud differentiation induction: placing the disinfected explants in an adventitious bud induction medium WPM1 for culture to induce adventitious bud-bearing callus; (3) elongation of stems: transferring the adventitious bud-bearing callus to a stem elongation medium WPM2 for continued culture to increase the length of the stems; (4) rooting induction: cutting the adventitious buds after their length is increased, placing them in a rooting medium WPM3 for culture, then transplanting them to soil for culture.

2. The method for in vitro propagation of Rhododendron anthopogon D. Don according to claim 1, wherein, In step (1), branches of wild Rhododendron anthopogon-berberidifolium plants with a length of 8-12 cm, free from disease and pest interference and in a good growth state are collected.

3. The method for in vitro propagation of Rhododendron anthopogon D. Don according to claim 1, wherein, In step (1), the disinfection method is as follows: the washed tender leaves are soaked and washed with sterilized water for 4-6 times, then rapidly soaked and washed with 70-80% alcohol for 15-25 s, and then washed with sterilized water for 4-6 times until the alcohol is completely removed; then the tender leaves are soaked with 2-4% sodium hypochlorite for 5-15 min, and then washed with sterilized water for 4-6 times until the sodium hypochlorite is completely removed, and finally the tender leaves are dried with sterilized paper and the blackened parts in the operation process are removed.

4. The method for in vitro propagation of Rhododendron anthopogon D. Don according to claim 1, wherein, In step (2), the adventitious bud induction medium WPM1 comprises macroelements, microelements, iron salts, organic matter, naphthalene acetic acid and 6-benzylaminopurine; wherein: the macroelements comprise 500-800 mg / L ammonium nitrate, 2.0 g / L potassium sulfate, 400-500 mg / L magnesium sulfate, 300-400 mg / L dipotassium hydrogen phosphate and 150-200 mg / L calcium chloride; the microelements comprise 45-50 mg / L manganese sulfate, 15-20 mg / L zinc sulfate, 15-20 mg / L boric acid, 0.5-0.8 mg / L copper sulfate and 0.5-0.8 mg / L sodium molybdate; the iron salts comprise 800-1000 mg / L calcium nitrate and 75-78 mg / L disodium ethylenediaminetetraacetate; the organic matter comprises 150-200 mg / L myo-inositol, 1.5-2.0 mg / L thiamine hydrochloride, 1.0-1.5 mg / L nicotinic acid, 1.0-1.5 mg / L pyridoxine hydrochloride, 2.0-5.0 mg / L glycine, 20-30 g / L sucrose and 5.0-6.0 g / L agar; the naphthalene acetic acid has a concentration of 0.02-0.04 mg / L; and the 6-benzylaminopurine has a concentration of 0.03-0.05 mg / L.

5. The method for in vitro propagation of Rhododendron anthopogon D. Don according to claim 1, wherein, In step (2), the culture is carried out at 24-26 ℃, with light culture for 12-16 h / d, at a radiation flux density of 30-40 μmol·m -2 ·s -1 ; and the culture time is 28-32 days.

6. The method for in vitro propagation of Rhododendron anthopogon D. Don according to claim 1, wherein, In step (3), the stem elongation medium WPM2 comprises macroelements, microelements, iron salts, organic matter, naphthalene acetic acid and 6-benzylaminopurine; wherein: The macroelements include: 500-800 mg / L ammonium nitrate, 2.0 g / L potassium sulfate, 400-500 mg / L magnesium sulfate, 300-400 mg / L dipotassium hydrogen phosphate, 150-200 mg / L calcium chloride; The microelements include: 45-50 mg / L manganese sulfate, 15-20 mg / L zinc sulfate, 15-20 mg / L boric acid, 0.5-0.8 mg / L copper sulfate, 0.5-0.8 mg / L sodium molybdate; The iron salt includes: 800-1000 mg / L calcium nitrate, 75-78 mg / L disodium ethylenediaminetetraacetate; The organic matter includes: 150-200 mg / L inositol, 1.5-2.0 mg / L thiamine hydrochloride, 1.0-1.5 mg / L nicotinic acid, 1.0-1.5 mg / L pyridoxal hydrochloride, 2.0-5.0 mg / L glycine, 20-30 g / L sucrose, 5.0-6.0 g / L agar; The naphthalene acetic acid concentration is 0.02-0.04 mg / L; The 6-benzylaminopurine concentration is 0.001-0.1 mg / L.

7. The method for in vitro propagation of Rhododendron anthopogon D. Don according to claim 1, wherein, In step (4), the rooting medium WPM3 includes macroelements, microelements, iron salt, organic matter and naphthalene acetic acid; wherein: The macroelements include: 500-800 mg / L ammonium nitrate, 2.0 g / L potassium sulfate, 400-500 mg / L magnesium sulfate, 300-400 mg / L dipotassium hydrogen phosphate, 150-200 mg / L calcium chloride; The microelements include: 45-50 mg / L manganese sulfate, 15-20 mg / L zinc sulfate, 15-20 mg / L boric acid, 0.5-0.8 mg / L copper sulfate, 0.5-0.8 mg / L sodium molybdate; The iron salt includes: 800-1000 mg / L calcium nitrate, 75-78 mg / L disodium ethylenediaminetetraacetate; The organic matter includes: 150-200 mg / L inositol, 1.5-2.0 mg / L thiamine hydrochloride, 1.0-1.5 mg / L nicotinic acid, 1.0-1.5 mg / L pyridoxal hydrochloride, 2.0-5.0 mg / L glycine, 20-30 g / L sucrose, 5.0-6.0 g / L agar; The naphthalene acetic acid concentration is 0.5-2.0 mg / L.

8. The method for in vitro propagation of Rhododendron anthopogon D. Don according to claim 1, wherein, In step (4), the seedling raising is performed by opening the cap of the culture bottle, adding the culture bottle with the plant into distilled water, and culturing for 3-5 days under the conditions of temperature 24-26℃, humidity 60% or more, light 12-16 h / d, and radiation flux density 30-40 μmol·m -2 ·s -1 -2.

9. The method of claim 1, wherein the Rhododendron anthopogon D. Don is Rhododendron mucronatum. In step (4), the transplanting method is as follows: the culture medium in the tissue culture bottle containing the plant is broken, the plant is clamped out of the bottle and washed under running water, the culture medium on the surface of the root system is removed, and then the plant is placed in a seedling pot and covered with soil until the root system is not exposed to air; then the soil is compacted and watered until the soil is saturated, and the plant is cultivated under the conditions of temperature 24-26 ℃, humidity above 80%, illumination 12-16 h / d, and radiation flux density 30-40 μmol·m -2 ·s -1 -2 / d for 6-8 days; during this period, the soil is watered every day to keep the soil humidity above 80%; after 6-8 days, the water content in the soil and the air humidity are reduced to above 60%, and the temperature is controlled at 22-24 ℃ for continuous cultivation.

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