Dictamnus dasycarpus propagation method

Abstract

The invention belongs to the technical field of plant planting, and particularly relates to a method for expanding propagation of dictamnus dasycarpus. The method comprises the following steps: sterilizing dictamnus dasycarpus seeds which are swelled after absorbing water to obtain pretreated seeds; refrigerating the pretreated seeds at 8-10 DEG C for 5-7 days, and then transferring the pretreated seeds at 23-25 DEG C for culturing until the seeds germinate to obtain seedlings; sterilizing the seedlings to obtain explants, and transferring the explants into an induction culture medium containing plant hormones for induction culture to obtain calluses; transferring the calluses to a differential medium containing plant hormones for differentiation culture to obtain adventitious buds; transferring the adventitious buds into a rooting culture medium containing plant hormones for rooting culture to obtain dictamnus dasycarpus seedlings. The invention provides a novel culture method for artificial propagation of dictamnus dasycarpus from seed germination to propagation culture.

Description

Technical Field

[0001] The invention belongs to the technical field of plant planting, and particularly relates to a method for propagating Dictamnus chinensis. Background Art

[0002] White fresh ( Dictamnus dasycarpus Turcz. Dictamnus (Dictamnus L.) is a perennial herbaceous plant in the Rutaceae family. Dictamnus bark is a well-known, major Chinese medicinal herb with a bitter taste and cold properties. Its main active ingredients are cinnamone, chalazine, and dacranine. It has the effects of dispelling wind, drying dampness, clearing heat, and detoxifying. It is mainly used to treat damp-heat sores, jaundice, eczema, scabies, rheumatic fever, jaundice, and dark urine (Chinese Pharmacopoeia, 2020). Li Shizhen's "Compendium of Materia Medica" notes on Dictamnus: "Fresh, it represents the smell of sheep. The roots of this herb are white and have a sheep-like smell, and its seeds are dense and resemble peppercorns." The market for Dictamnus bark is high, primarily relying on wild resources and rarely cultivated. Currently, the market for Dictamnus bark relies solely on wild resources, which is difficult to meet demand. Furthermore, years of indiscriminate and excessive mining have led to a significant decline in wild Dictamnus bark reserves.

[0003] Artificial plant propagation methods mainly include sexual reproduction and asexual reproduction. Sexual reproduction mainly refers to seed propagation, while asexual reproduction includes grafting, cuttings, division and tissue culture. The advantage of seed propagation is that a large number of plants can be obtained from one sowing, and seed collection, storage and transportation are convenient. The seedlings have the characteristics of vigorous growth, strong stress resistance and easy domestication. In contrast, asexual reproduction can obtain offspring with basically the same genetics as the mother plant, maintain the excellent traits of the mother plant, and the seedlings grow uniformly with little variation. In addition, asexual reproduction is also conducive to the preservation of high-quality germplasm resources. In recent years, in the large-scale production of medicinal plants, in addition to seed germination research technology, asexual reproduction is often used to expand the propagation of some plants that are difficult to bear fruit or whose seeds are difficult to obtain and germinate. The most representative method is plant tissue culture technology. Plant tissue culture technology is a technique for asexual in vitro propagation developed based on the theory of plant cell totipotency. It involves inoculating explants such as root tips, stem tips, young leaves, young embryos, and anthers onto artificially prepared culture media through aseptic manipulation and then culturing them in vitro under artificially controlled environmental conditions to grow them into complete plants. Plant tissue culture includes different methods such as organ culture, stem apex meristem culture, callus culture, cell culture, and protoplast culture. Plant tissue culture has the advantages of a short growth cycle, high reproduction rate, ease of controlling culture conditions, and convenient automation. Currently, the propagation and cultivation technology for Dictamnus odoratus mainly relies on sexual reproduction. However, this method is time-consuming, and seed germination is easily affected by human labor, resulting in a low seed germination rate, which in turn affects the subsequent growth of Dictamnus odoratus. Summary of the Invention

[0004] To address the above-mentioned issues, the present invention provides a method for propagating Dictamnus chinensis. The method effectively improves the germination rate of Dictamnus chinensis seeds through precise temperature-variation technology, and after the seedlings are cultivated, asexual propagation of the tissue culture seedlings is performed, thereby achieving rapid propagation of Dictamnus chinensis.

[0005] To achieve the above object, the present invention provides a method for propagating Dictamnus chinensis.

[0006] A method for propagating Dictamnus chinensis, characterized by comprising the following steps: After sterilizing the water-swelled white sage seeds, pre-treated seeds are obtained; The pretreated seeds were refrigerated at 8-10°C for 5-7 days and then transferred to 23-25°C for cultivation. This temperature setting can simulate the process of wild Dictamnus seeds overwintering at low temperatures and germinating in spring after breaking dormancy, so as to determine the most suitable treatment temperature; until the seeds germinated and seedlings were obtained; After sterilizing the seedlings, explants are obtained, and the explants are transferred to an induction medium containing plant hormones for induction culture to obtain callus tissue; The callus tissue is transferred to a differentiation medium containing plant hormones for differentiation culture to obtain adventitious buds; The adventitious buds are transferred to a rooting medium containing plant hormones for rooting culture to obtain white radish seedlings; The plant hormones in the induction medium are 1.5 mg / L to 1.6 mg / L of naphthaleneacetic acid and 1 mg / L to 1.1 mg / L of furanaminopurine; The plant hormones in the differentiation medium are 1.8 mg / L to 2 mg / L of naphthaleneacetic acid and 1.8 mg / L to 2 mg / L of 6-benzylaminopurine; The plant hormones in the rooting medium are 0.8 mg / L~1 mg / L indoleacetic acid and 0.4 mg / L~0.5 mg / L 1 of indole-3-butyric acid.

[0007] The present invention, through precise temperature change, refrigerates 5d~7d at 8~10 ℃ and treats low-temperature cold storage to effectively break the dormancy of seeds and promote seed germination. This is mainly due to the physiological dormancy of white radish seeds. Low-temperature cold storage can simulate the low temperature conditions in winter in the natural environment, promote physiological changes inside the seeds, break dormancy, and create conditions for subsequent germination. If the cold storage time is shorter than 5 days, the low-temperature treatment time may be insufficient, and the dormancy of seeds may not be completely broken. The physiological changes inside the seeds have not yet been completed, resulting in a low germination rate. Even if the seeds that have not fully broken dormancy germinate, they may also be weak in seedling growth and poor in stress resistance due to incomplete conversion of internal nutrients. If the cold storage time is longer than 7 days, the seed vitality may decrease, delaying the development of the seeds. Even if they are subsequently transferred to suitable temperature culture, the germination rate may also decrease. Therefore, the low-temperature cold storage time of white radish seeds needs to be strictly controlled at 5~7 days to ensure that the dormancy of the seeds is fully broken and to avoid a decrease in seed vitality. If the cold storage time is too short or too long, it will have an adverse effect on the germination rate of seeds and the growth of seedlings, and reduce the utilization efficiency and propagation effect of seeds. By transferring the white fresh seeds to 23℃~25℃ after cold storage, the seeds, after low temperature treatment, are transferred to this temperature range, which can quickly start the germination process and significantly improve the germination rate. The temperature of 23℃~25℃ is higher than the suitable temperature of 16℃~20℃ for the germination of white fresh seeds. This is mainly to match the low temperature storage temperature in the early stage. 23℃~25℃ helps to accelerate germination, but too high a temperature may also cause heat stress to seeds, affecting the germination rate and the health of seedlings. Therefore, the present invention is achieved by transferring the seeds to 23℃~25℃ after cold storage for 5d~7d at 8~10℃. The two temperatures cooperate with each other to ensure that rapid germination is promoted without causing damage to the seeds through precise temperature control. Moreover, 23℃~25℃ can make the Dictamnus seeds germinate in a concentrated manner in a shorter period of time, with high germination uniformity, which is beneficial to subsequent seedling management; and by regulating the types and concentrations of plant hormones in the induction culture medium, differentiation culture medium and rooting culture medium, the differentiation of Dictamnus can be further accelerated, thereby achieving the expansion and propagation of Dictamnus.

[0008] In another preferred embodiment, the water absorption and swelling refers to soaking the Dictamnus seeds in water for 12 hours to 24 hours.

[0009] In another preferred embodiment, the sterilization of the water-swollen Dictamnus seeds specifically refers to soaking the water-swollen Dictamnus seeds in 75% by mass ethanol for 1 minute to 3 minutes, then treating them with sodium hypochlorite solution for 8 minutes to 10 minutes, and finally rinsing them with sterile water.

[0010] In another preferred embodiment, the induction medium, the differentiation medium and the rooting medium are all MS medium containing plant hormones.

[0011] In another preferred embodiment, the rooting medium further comprises activated carbon in an amount of 10% to 5% by weight of the rooting medium.

[0012] In another preferred embodiment, the seedling sterilization specifically refers to treating the seedlings with a sodium hypochlorite solution for 8 minutes to 10 minutes.

[0013] In another preferred embodiment, the conditions for the induction culture, the differentiation culture and the rooting culture are all at a temperature of 24° C. to 26° C., a light intensity of 20,000 lx, and a light duration of 16 h / d.

[0014] In another preferred embodiment, the pH of the MS medium is 5.8-6.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The present invention refrigerates Dictamnus seeds at 8°C-10°C for 5-7 days to break their dormancy and promote seed germination. Precisely controlling the refrigeration time and temperature not only promotes physiological changes within the seeds, breaking dormancy and creating conditions for subsequent germination, but also avoids the problem of delayed seed development caused by prolonged refrigeration. The refrigerated seeds are then cultured at 23°C-25°C, providing an optimal germination temperature for Dictamnus seeds, thereby increasing the germination rate to as high as 97.8%, effectively improving the seed germination rate. The addition of different ratios of plant hormones to MS basal medium successfully induced callus differentiation. Furthermore, by adjusting the types and ratios of plant hormones in the culture medium, the differentiation potential of the callus was stimulated, resulting in the formation of a large number of adventitious buds. Furthermore, root induction is a crucial step in the plant regeneration process. By selecting healthy seedlings that successfully induced adventitious buds and optimizing the types and ratios of plant hormones in the rooting medium, root growth and development are further promoted, providing a complete asexual propagation technology solution for Dictamnus.

[0016] By adding 10% activated carbon to the rooting medium, the present invention simulates a dark soil environment, providing more suitable conditions for root growth. This optimization measure successfully induced regenerated seedlings with healthy root systems, providing a foundation for the propagation, transplantation, and growth of Dictamnus chinensis. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a graph showing the effects of different temperature treatments on the germination rate of Dictamnus seeds.

[0018] Figure 2 These are pictures of seed germination of Dictamnus seeds after disinfection and without disinfection; A is the picture after disinfection, and B is the picture without disinfection.

[0019] Figure 3 This is a graph showing the effects of different disinfection methods on the germination rate of Dictamnus seeds.

[0020] Figure 4 The results of inducing Dictamnus callus under different hormone ratios are shown in Figure A. The Dictamnus callus induced by different hormones are numbered 1 to 4 in Table 1 from left to right; B. The Dictamnus callus induced by different hormone ratios are numbered 5 to 7 in Table 1 from left to right.

[0021] Figure 5 This is a picture of Dictamnus tissue proliferation.

[0022] Figure 6 Figures 1 to 2 show the adventitious bud and proliferation results under different hormone ratios. A to F correspond to numbers 1 to 6 in Table 2.

[0023] Figure 7 Figure 1 shows the results of rooting induction medium proliferation. A and B are regenerated plants of Dictamnus chinensis, and C is the root system of the regenerated Dictamnus chinensis. A to C correspond to numbers 1 to 3 in Table 3, respectively.

[0024] Figure 8 Figures A and B show the rooting results of Dictyophora after adding activated carbon. A shows the rooting of Dictyophora after adding activated carbon, B shows the regenerated seedlings of Dictyophora, and C shows the root system of the regenerated seedlings of Dictyophora. A to C correspond to numbers 4 to 6 in Table 3, respectively. DETAILED DESCRIPTION

[0025] The present invention will be described in detail below with reference to the accompanying drawings and specific examples, but they should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0026] In the following examples, 6-benzylaminopurine is denoted as 6-BA, naphthaleneacetic acid is denoted as NAA, indoleacetic acid is denoted as IAA, furanaminopurine is denoted as KT, and indole-3-butyric acid is denoted as IBA, all of which were purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0027] 1. Experimental Materials 1.1 Selection of Dictamnus seeds Select the same size, plump and white ( Dictamnus dasycarpus Mature seeds were collected from Nanwutai, Xi'an, Shaanxi Province, for the experiment. 200 Dictamnus seeds were randomly selected and weighed using an electronic analytical balance. Ten replicates were set up, and the 1,000-grain weight was calculated. Seeds approximately 3–4 mm long, brown to black in color, and weighing 19.928 g ± 0.318 g were selected for the experiment.

[0028] 1.2 Preparation of culture medium The formula of MS medium selected as basic culture medium is: NH4NO3 1650 mg·L -1、KNO31900mg·L -1 、CaCl2·2H2O 440mg·L -1 、MgSO4·7H2O 370mg·L -1 、KH2PO4170mg·L -1 、FeSO4·7H2O27.8mg·L -1 、Na2EDTA·2H2O 37.3mg·L -1 、H3BO36.2mg·L -1 、MnSO4·H2O 22.3mg·L -1 、ZnSO4·7H2O 8.6mg·L -1 、Na2MoO4·2H2O 0.25mg·L -1 、CuSO4·5H2O 0.025mg·L -1 、CoCl2·6H2O 0.025mg·L -1 , agar 7500mg·L -1 , sucrose 30000mg·L -1 , add water to 1 L, adjust the pH to 5.8-6.0, and sterilize by high-pressure steam at 121°C for 30 min.

[0029] 2. Experimental process 2.1 Seed germination Seed pretreatment: Soak fresh white radish seeds in clean water for 12-24 hours to allow the seeds to fully absorb water and swell.

[0030] Preliminary screening is carried out to remove impurities and damaged seeds, peel off the seed coat of the white safflower seeds, and select mature, plump, disease-free, and uniformly sized white safflower seeds for germination.

[0031] Disinfection: Take the imbibed white fresh seeds and perform aseptic disinfection on a clean bench. First, soak the seeds in 75% alcohol for 3 minutes, then treat them with sodium hypochlorite solution for 10 minutes, and finally rinse them with sterile water to ensure that the seed surface is sterile and there is no obvious residue of the disinfectant; unsterilized seeds are only rinsed with sterile water.

[0032] 2.2.1 Effect of temperature on seed germination The experiment set up three treatment groups in total: a 10℃ constant temperature treatment group, a 25℃ constant temperature treatment group, and a 10℃ refrigerated for 7 days followed by a 25℃ treatment group.

[0033] The effect of temperature on seed germination of Dictyophora chinensis was investigated. Three replicates were set for each temperature treatment level. Seed germination was observed and recorded daily. Germination was considered complete when the radicle and seed were equal in length. Germination was considered complete when no seeds germinated for five consecutive days across all replicates during the incubation period. The germination rate was calculated using the following formula:

[0034] Germination rate (Gr) = (total number of test seeds germinated / total number of test seeds) × 100%.

[0035] 2.2.2 Effect of sterilization on seed germination To investigate the effects of endophytes on the germination of Dictamnus seeds, this study conducted a comparative experiment on seed germination rate. The experimental setup included two treatment groups: unsterilized seeds with endophytes and seeds that had been sterilized normally.

[0036] 2.3 Cultivation of Dictamnus explants into seedlings 2.3.1 Callus induction The seedlings germinated from the seeds in 2.2 were sterilized with sodium hypochlorite solution for 10 minutes under sterile conditions. The sterilized explants were dried and inoculated into MS minimal culture medium to obtain sterile explants.

[0037] 2.3.2 Callus induction Different combinations and levels of plant hormones were used to prepare Dictyophora callus induction media, as shown in Table 1. Three biological replicates were set for each treatment, and five explants were seeded into each bottle of medium. Explants were cut into 0.5 cm × 0.5 cm pieces and inoculated onto the induction medium containing plant hormones. Callus induction was observed, and the induction success rate was calculated to screen for suitable medium for callus induction.

[0038] Table 1 Different plant hormone combinations in callus induction medium 2.3.3 Adventitious bud induction Adventitious bud induction medium was prepared with different phytohormone levels, as shown in Table 2. Three biological replicates were set for each treatment, and five cut calli were inoculated into each bottle of medium. Calli that had been proliferating for 20 days were cut into 0.5 cm × 0.5 cm pieces and inoculated onto the adventitious bud induction medium. Cluster bud induction was observed, and the induction success rate was calculated to screen for the appropriate phytohormone level.

[0039] Table 2 Different plant hormone levels in adventitious bud induction medium 2.3.4 Rooting of adventitious buds Table 3 lists the rooting induction medium settings with different phytohormone levels. Three biological replicates were set for each treatment, and five sterile seedlings were seeded into each bottle of medium. Differentiated sterile seedlings were selected, retaining two to three smaller leaves at the top. Rooting was observed in the stem segments, and the induction success rate was calculated to screen for the optimal phytohormone level for rooting.

[0040] Table 3 Different plant hormone levels in root induction medium 2.3.5 Culture conditions During the test, the materials were kept at a temperature of (25±1°C), a light intensity of 20000 lx, and a light exposure time of 16 h·d. -1 cultured in a culture room.

[0041] 3. Experimental results 3.1 Effect of temperature on seed germination rate Among the three groups of seeds treated at different temperatures, the variable temperature group began to germinate on the 8th day, and the constant temperature 10℃ treatment group, which germinated the latest, began to germinate on the 14th day. After 30 days of statistics, it was found that the germination rate of seeds that were refrigerated at 10℃ for 7 days and then transferred to a 25℃ environment was higher than that of the constant temperature group, reaching 97.8%, and the germination speed was faster. This shows that the low-temperature refrigeration treatment may have effectively broken the dormancy of the seeds and promoted the germination of the seeds; while the seeds treated at a constant temperature of 10℃ showed a lower germination rate, only 44.4% after 30 days. Figure 1 The results showed that temperature not only had an important influence on the germination rate of seeds, but also that appropriate low-temperature pretreatment could promote better germination of Dictamnus seeds.

[0042] 3.2 Effect of sterilization on seed germination The results showed that due to the presence of endophytes, the germination time of seeds that had not been disinfected was relatively later than that of the disinfected group, and the germination rate was significantly lower than that of the seeds that had been disinfected normally. The seeds began to germinate on the 13th day, and at the end of the germination on the 30th day, the germination rate was only 33.3%. Figure 3 As shown, most of the seeds have obvious colonies around them, such as Figure 2 The presence of endophytes may inhibit seed germination by competing for nutrients and altering the seed surface microenvironment, thereby inhibiting normal germination. Conventional disinfection of seeds, however, removes potential endophytes and exhibits a higher germination rate.

[0043] The above experimental results show that temperature has a significant impact on seed germination. After 7 days of cold storage at 10°C and then incubation at 25°C, the germination rate was higher than that of the two constant temperature groups. These results indicate that appropriate low-temperature cold storage can break the dormancy of Dictyophora seeds and promote germination. Low temperature treatment may activate enzyme systems within the seeds, breaking the dormancy mechanism and thus improving the germination success rate. However, the germination rate of Dictyophora seeds kept in a constant temperature of 10°C was lower, possibly because prolonged low temperature conditions suppressed the germination potential of the seeds, preventing them from germinating normally. Temperature regulation is a key factor influencing Dictyophora seed germination, and appropriate temperature treatment can significantly improve seed germination.

[0044] Endophytes can affect normal seed growth and development by competing for nutrients and altering the microenvironment. Unsterilized Dictamnus seeds harboring endophytes have a significantly lower germination rate than properly sterilized seeds. Endophytes can not only delay seed germination but also lead to disease or poor growth during germination. Therefore, seed disinfection is crucial for ensuring a high germination rate for Dictamnus seeds. In practical planting, effective disinfection methods to remove microorganisms on and within seeds can help improve seed germination and reduce disease risks. Analysis of the effects of temperature and endophytes on Dictamnus seed germination reveals that temperature plays a crucial role in Dictamnus seed germination. Low temperature treatment can effectively break seed dormancy and promote germination, providing a reliable temperature control strategy for Dictamnus seed propagation. Endophytes can inhibit normal seed germination, making disinfection essential for improving seed germination and ensuring healthy seed development. Removing endophytes from seeds provides a more favorable germination environment, ensuring successful germination.

[0045] 3.3 Callus induction results To ensure the growth and differentiation of explants during the culture process, MS medium is used as the basic medium. Then, different ratios of plant hormones, especially auxins and cytokinins, are added to the culture medium to stimulate the explants to produce callus tissue. IAA and NAA can promote cell division and elongation, while cytokinin KT helps cell division and differentiation. By adjusting the concentration and ratio of hormones, the formation of callus tissue can be effectively induced, such as Figure 4 The results showed that 1.5 mg·L -1 NAA+1 mg·L - 1 KT can effectively induce dedifferentiation of Dictamnus callus, with an induction success rate of over 86%.

[0046] When a large number of callus cells are generated on the surface of the explant, the callus cells are carefully divided in a sterile environment and the proliferation culture is continued to expand the reproduction. Figure 5 This process can increase the amount of callus tissue and provide sufficient callus material for subsequent plant regeneration.

[0047] 3.4 Adventitious bud induction and proliferation results The callus was inoculated into a medium with MS medium as the basal medium, the ratio of auxin and cytokinin in the medium was directionally changed, and the differentiation of callus into buds was promoted, thereby inducing the differentiation of adventitious buds of Dictyophora alba.

[0048] During the adventitious bud induction process, the concentration and ratio of plant hormones are crucial for the formation of adventitious buds. High concentrations of cytokinins can promote the production of bud primordia in callus tissue, while appropriate amounts of auxins promote bud differentiation and growth. After screening, the combination of NAA and 6BA was ultimately selected. By continuously optimizing the hormone ratio and culture conditions, the success rate of adventitious bud induction can be improved. Studies have found that 2mg·L -1 NAA+2 mg·L -1 6BA has a good effect in inducing the differentiation of Dictamnus callus into adventitious buds, with a success rate of over 80%. When the callus successfully differentiates into adventitious buds, the buds can be further cultured after they grow up. Figure 6 As shown, it is further expanded and reproduced to grow into sterile seedlings.

[0049] 3.5 Root induction and proliferation results Select sterile white fresh seedlings that are pollution-free, have successfully induced the differentiation of adventitious buds, and are growing healthily as materials for rooting induction to prepare for transplanting into the soil environment. Plant hormones are mainly auxins IAA or IBA, which stimulate root formation. Auxins can effectively promote cell differentiation and expansion, induce the formation of root primordia, and further promote root growth. By adjusting the concentration and ratio of plant hormones, the root differentiation process can be controlled to obtain better rooting effects ( Figure 7 ), the study found that 1mg·L -1 IAA+0.5 mg·L -1 IBA can successfully induce rooting of sterile seedlings of Dictamnus dichotoma, with an induction success rate of over 73%.

[0050] During the rooting induction process, the optimization of the rooting medium is crucial. In addition to basic plant hormone regulation, other factors that affect the rooting effect must also be considered. Adding 10% activated carbon to the culture medium can effectively simulate the dark environment of the soil and help accelerate the differentiation of induced roots ( Figure 8 ).

[0051] Different plant hormone ratios were used to explore the optimal conditions for callus induction, adventitious bud differentiation, and rooting induction in Dictamnus chinensis. Selecting young and tender parts as explants for cultivation effectively promoted callus formation. By adding different ratios of plant hormones to MS basal medium, callus differentiation was successfully induced. Furthermore, by adjusting the types and ratios of plant hormones in the culture medium, the differentiation potential of the callus was stimulated, resulting in the formation of a large number of adventitious buds. Furthermore, rooting induction is a crucial step in the plant regeneration process. By selecting healthy seedlings that successfully induced adventitious buds and optimizing the rooting medium, root growth and development were further promoted. In the present invention, by adding 10% activated carbon to the culture medium, a dark soil environment was simulated, providing more suitable conditions for root growth. This optimization measure successfully induced regenerated seedlings with healthy root systems, providing a foundation for Dictamnus propagation, transplanting, and growth. Overall, the optimized conditions for callus induction, adventitious bud differentiation, and rooting induction provide a highly efficient technical means for the asexual propagation of Dictamnus chinensis plants. By adjusting the ratio of plant hormones, optimizing the culture medium composition and adding activated carbon to simulate the dark soil environment, the differentiation efficiency and root development ability of the regenerated seedlings were significantly improved.

[0052] Although preferred embodiments of the present invention have been described, additional changes and modifications to these embodiments may occur to those skilled in the art once the basic inventive concepts become known.

[0053] Obviously, those skilled in the art may make various modifications and variations to the present invention without departing from the spirit and scope of the present invention. Thus, if such modifications and variations fall within the scope of the claims and their equivalents, the present invention is intended to include such modifications and variations.

Claims

1. A method for propagating Dictamnus chinensis, characterized in that: The following steps are involved: After sterilizing the water-swelled white sage seeds, pre-treated seeds are obtained; The pretreated seeds were refrigerated at 8°C to 10°C for 5 to 7 days and then cultured at 23°C to 25°C until the seeds germinated and seedlings were obtained; After sterilizing the seedlings, explants are obtained, and the explants are transferred to an induction medium containing plant hormones for induction culture to obtain callus tissue; The callus tissue is transferred to a differentiation medium containing plant hormones for differentiation culture to obtain adventitious buds; The adventitious buds are transferred to a rooting medium containing plant hormones for rooting culture to obtain white radish seedlings; The plant hormones in the induction medium are 1.5 mg / L to 1.6 mg / L of naphthaleneacetic acid and 1 mg / L to 1.1 mg / L of furanaminopurine; The plant hormones in the differentiation medium are 1.8 mg / L to 2 mg / L of naphthaleneacetic acid and 1.8 mg / L to 2 mg / L of 6-benzylaminopurine; The plant hormones in the rooting medium are 0.8 mg / L~1 mg / L indoleacetic acid and 0.4 mg / L~0.5 mg / L 1 of indole-3-butyric acid.

2. The method for propagating Dictamnus chinensis according to claim 1, wherein The water absorption and swelling refers to soaking the Dictamnus seeds in water for 12 hours to 24 hours.

3. The method for propagating Dictamnus chinensis according to claim 1, wherein: The specific process of sterilizing the water-swollen Dictamnus seeds is to soak the water-swollen Dictamnus seeds in 75% by mass ethanol for 1 minute to 3 minutes, then treat them with 10% by mass sodium hypochlorite solution for 8 minutes to 10 minutes, and finally rinse them with sterile water.

4. The method for propagating Dictamnus chinensis according to claim 1, wherein: The induction medium, the differentiation medium and the rooting medium are all MS medium containing plant hormones.

5. The method for propagating Dictamnus chinensis according to claim 1, wherein: The rooting medium also includes activated carbon in an amount of 8% to 10% of the mass of the rooting medium.

6. The method for propagating Dictamnus chinensis according to claim 1, wherein: The seedling sterilization specifically refers to treating the seedlings with a 10% by mass sodium hypochlorite solution for 8 minutes to 10 minutes.

7. The method for propagating Dictamnus chinensis according to claim 1, wherein: The conditions of the induction culture, the differentiation culture and the rooting culture are all 24° C. to 26° C., the light intensity is 20,000 lx, and the light duration is 16 h / d.

8. The method for propagating Dictamnus chinensis according to claim 4, wherein: The pH of the MS medium is 5.8-6.

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