Rhododendron pulchrum tissue culture method based on callus regeneration

Abstract

The invention relates to the technical field of plant tissue culture, in particular to a Rhododendron pulchrum tissue culture method based on callus regeneration. According to the method, the tender cotyledons of aseptic seedlings are taken as explants, and an efficient Rhododendron pulchrum tissue culture and rapid propagation technical system is established through induction of calluses, subculture multiplication of the calluses, differentiation of adventitious buds, bud strengthening and rooting culture, so that complete tissue culture progeny plants are formed. The induction rate of the callus tissue of the system is 100.0%, the proliferation multiple of the callus tissue in a subculture cycle reaches 5.2 times, 28.5 adventitious buds can be differentiated from a callus tissue block with the size of about 0.5 mm * 0.5 mm * 0.5 mm, the rooting rate reaches 95.8%, the system is not limited by factors such as seasons, climate and regions, and efficient and rapid propagation can be achieved all the year round. Meanwhile, the establishment of the system also lays a technical foundation for genetic transformation improvement, mutant screening breeding and the like of the Rhododendron pulchrum based on the callus.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, in particular to a Rhododendron splendidum tissue culture method based on callus regeneration. Background Art

[0002] Rhododendron splendidum Rhododendron pulchrum ) belongs to the Ericaceae family, Rhododendron subgenus ( Rhododendron Subgenus Tsutsusi ) plant, widely cultivated in Asia, Europe and North America, and is also a common cultivated azalea group in China. Rhododendron splendidum is a semi-evergreen shrub with lush leaves and flowers, rich and colorful flowers. It can be potted, planted in clusters or groups in parks or courtyards, or planted as flower hedges on the roadside. As a representative of the hairy azalea in the horticultural classification of azaleas, Rhododendron splendidum is also resistant to pruning and has strong adaptability. It is a common garden greening plant in the Yangtze River Basin and the areas south of it. In addition to its extremely high ornamental value, Rhododendron splendidum also has high medicinal value. Its roots, stems and leaves can be used as medicine to treat rheumatism, sores, gynecology, cough and other diseases. Studies have shown that the leaves of Rhododendron splendidum contain broad-spectrum antibacterial ingredients, which have inhibitory effects on some common Gram-like bacteria and drug-resistant bacteria. In addition, Rhododendron splendidum contains relatively rich flavonoid compounds, such as hyperoside, quercetin and azalea, which have analgesic, expectorant, antitussive, antibacterial and anti-inflammatory, and cardiovascular protection effects.

[0003] Plant tissue culture is a technology that utilizes the omnipotence of plant cells to cultivate plant organs, tissues or cells in artificial culture media under sterile conditions to regenerate them into complete plants. Plant tissue culture has the characteristics of fast reproduction, high production efficiency, saving of reproduction materials, and no seasonal and geographical restrictions. It is a rapid reproduction method widely used in agriculture and forestry, and is also the basis for plant genetic transformation and improvement.

[0004] The regeneration methods of plant tissue culture can be divided into two main pathways: direct and indirect. Indirect refers to the dedifferentiation of the explant to form callus tissue, and then inducing differentiation from the callus tissue to produce buds, roots or embryoids, etc., and finally forming regenerated plants. Callus tissue is a new tissue formed on the wound surface after the local part of the plant body is stimulated by trauma. It is composed of living thin-walled cells and can originate from living cells of various tissues in any organ of the plant body. Therefore, its material source is wide. Secondly, callus tissue has great value-added potential. It can be subcultured for a long time under suitable conditions. At the same time, it has strong differentiation ability. A single callus tissue can differentiate into multiple adventitious buds or embryoids at the same time, so it can significantly improve the reproduction efficiency. In addition, callus tissue is an ideal receptor for genetic transformation such as Agrobacterium-mediated or gene gun method due to its active cell division, genetic uniformity, easy screening, and strong regeneration ability. It is also an ideal material for mutation breeding such as screening polyploidy, disease resistance, and stress resistance.

[0005] Regarding the tissue culture of Rhododendron pulchrum, predecessors have established a direct organogenesis tissue culture system for shoot multiplication from shoot, such as Dong Chunzhi et al. (1989) established a tissue culture system for direct regeneration of shoots using newly developed young stems as explants, and Liu Yuxuan et al. (2022) established an open tissue culture system using young stem segments as explants. For the indirect occurrence pathway of callus, Li Zheng et al. (2019) cut the leaves of sterile seedlings to form wounds and used them as explants to establish a method for inducing callus of Rhododendron pulchrum (CN110419446 A). However, in the existing technology, there is no report on the establishment of a complete tissue culture system of Rhododendron pulchrum based on indirect regeneration of callus. The present invention establishes a tissue culture method of Rhododendron pulchrum based on callus regeneration, providing an effective way for the efficient rapid propagation of Rhododendron pulchrum, and also providing technical support for its genetic transformation improvement and mutant screening breeding, etc. Summary of the Invention

[0006] The object of the present invention is to provide a tissue culture method of Rhododendron pulchrum based on callus regeneration in view of the problem in the existing technology that there is a lack of a complete tissue culture system of Rhododendron pulchrum based on indirect regeneration of callus.

[0007] To achieve the above object, the technical solution adopted by the present invention is: A tissue culture method of Rhododendron pulchrum based on callus regeneration, comprising the following steps: (1) Obtaining sterile seedlings: Take the mature but unopened capsules of Rhododendron pulchrum, rinse them with washing powder water and then rinse with running water for 20 - 30 min, transfer them to a laminar flow bench, soak and shake them with 75% alcohol solution for sterilization for 55 - 65 s, rinse them with sterile water 2 - 3 times, soak and shake them with 0.1% mercuric chloride solution for sterilization for 15 - 20 min, rinse them with sterile water 4 - 5 times, and blot the surface moisture with sterile filter paper; Open the capsules, take out the seeds and inoculate them on the seed germination medium for culturing for 25 - 35 days; The seed germination medium is: WPM medium + sucrose 25.0 - 35.0 g / L + agar 6.5 - 7.5 g / L; (2) Inducing callus: Take the sterile seedlings formed after 25 - 35 days of inoculation, pick up the seedlings with forceps, cut off the cotyledons, and cut wounds along the edges of the cotyledons, inoculate them with the abaxial side facing down on the callus induction medium for culturing for 25 - 35 days; The formula of the callus induction medium is: WPM medium + TDZ 1.8 - 2.2 mg / L + NAA 0.4 - 0.6 mg / L + PAA 14.0 - 16.0 mg / L + sucrose 25.0 - 35.0 g / L + agar 6.5 - 7.5 g / L; (3) Subculture and proliferation of callus: Take callus pieces and divide them into small pieces of about 0.5 mm × 0.5 mm × 0.5 mm, and inoculate them on the callus proliferation medium for culturing for 30 - 40 days to make them proliferate; the formula of the callus subculture proliferation medium is: WPM medium + TDZ 0.8 - 1.2 mg / L + NAA 0.2 - 0.3 mg / L + PAA 7.0 - 8.0 mg / L + sucrose 25.0 - 35.0 g / L + agar 6.5 - 7.5 g / L; (4) Differentiation of adventitious buds: Inoculate callus of about 0.5 mm × 0.5 mm × 0.5 mm on the adventitious bud differentiation medium for culturing for 30 - 40 days to induce the differentiation of adventitious buds; the formula of the adventitious bud differentiation medium is: WPM medium + BA 1.8 - 2.2 mg / L + TDZ 0.4 - 0.6 mg / L + NAA 0.04 - 0.06 mg / L + sucrose 25 - 35 g / L + agar 6.5 - 7.5 g / L; (5) Strengthening buds and rooting: Divide the adventitious buds induced from callus into single buds, and inoculate them in the bud - strengthening and rooting medium according to polarity for culturing for 40 - 50 days; the bud - strengthening and rooting medium is: 1 / 2 WPM + TDZ 0.4 - 0.6 mg / L + IBA 0.4 - 0.6 mg / L + NAA 0.4 - 0.6 mg / L + coconut milk 45 - 55 mL / L + sucrose 25 - 35 g / L + agar 6.5 - 7.5 g / L.

[0008] The culture conditions for steps (1), (2), (3), (4) and (5) are: temperature 23 - 27 °C, light intensity 2000 - 2500 Lx, and light - dark alternation culture with 12 - 14 h of daily illumination.

[0009] The pH value of the media used in steps (1), (2), (3), (4) and (5) is 5.4 - 5.6.

[0010] The formula of the seed germination medium is: WPM medium + sucrose 30.0 g / L + agar 7.0 g / L.

[0011] The formula of the callus induction medium is: WPM medium + TDZ 1.8 - 2.2 mg / L + NAA 0.4 - 0.6 mg / L + PAA 14.0 - 16.0 mg / L + sucrose 25.0 - 35.0 g / L + agar 6.5 - 7.5 g / L.

[0012] The formula of the callus subculture and proliferation medium is: WPM medium + TDZ 0.8 - 1.2 mg / L + NAA 0.2 - 0.3 mg / L + PAA 7.0 - 8.0 mg / L + sucrose 25.0 - 35.0 g / L + agar 6.5 - 7.5 g / L.

[0013] The formula of the adventitious bud differentiation medium is: WPM medium + BA 1.8 - 2.2 mg / L + TDZ 0.4 - 0.6 mg / L + NAA 0.04 - 0.06 mg / L + sucrose 25 - 35 g / L + agar 6.5 - 7.5 g / L.

[0014] The formula of the strong shoot and rooting medium is: 1 / 2 WPM + TDZ 0.4 - 0.6 mg / L + IBA 0.4 - 0.6 mg / L + NAA 0.4 - 0.6 mg / L + coconut milk 45 - 55 mL / L + sucrose 25 - 35 g / L + agar 6.5 - 7.5 g / L.

[0015] The beneficial technical effects of the present invention are: Compared with the prior art, the present invention provides a tissue culture method of Rhododendron pulchrum based on callus regeneration. Using the young cotyledons of sterile seedlings as explants, through callus induction, callus subculture and proliferation, adventitious bud differentiation, strong shoot and rooting culture, an efficient tissue culture and rapid propagation technology system is established, and complete tissue culture progeny plants are formed. The callus induction rate of this system is 100.0%, the proliferation multiple of callus reaches 5.2 times within one subculture cycle, about 28.5 adventitious buds can be differentiated from a callus block of about 0.5 mm × 0.5 mm × 0.5 mm, the rooting rate reaches 95.8%, and it is not restricted by factors such as season, climate and region, and can achieve efficient and rapid propagation throughout the year. At the same time, the establishment of this system also lays a technical foundation for genetic transformation and improvement and mutant screening breeding of Rhododendron pulchrum based on callus. Description of the Drawings

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0017] Figure 1 It is the mature but unopened capsule in Example 1.

[0018] Figure 2It is the seedling formed by the aseptic germination of the seeds in Example 1.

[0019] Figure 3 It is the callus induced from cotyledons in Example 2.

[0020] Figure 4 It is the subculture and proliferation of the callus in Example 3.

[0021] Figure 5 It is the adventitious buds induced from the callus in Example 4.

[0022] Figure 6 It is the elongation of adventitious buds in Example 4.

[0023] Figure 7 It is the strong shoot and rooting culture in Example 5. Specific implementation mode

[0024] The following further illustrates the present invention in conjunction with specific implementation schemes. These examples should be understood as only the preferred examples of the present invention and do not limit the present invention in any way.

[0025] Example 1: Influence of the method of aseptic germination of seeds on the present invention 1) Experimental materials: In November, the mature but unopened capsules of Rhododendron pulchrum on healthy plants were collected ( Figure 1 ) for aseptic germination culture of seeds. The culture medium was prepared using commercially available basal medium, hormones, sucrose, and agar.

[0026] 2) Experimental method: The capsules were rinsed with detergent water and then rinsed with running water for 20 - 30 min, transferred to a laminar flow hood, soaked and shaken in 75% alcohol solution for sterilization for 55 - 65 s, rinsed with sterile water 2 - 3 times, soaked and shaken in 0.1% mercuric chloride solution for sterilization for 15 - 20 min, rinsed with sterile water 4 - 5 times, and blotted dry with sterile filter paper for later use.

[0027] The seed germination medium used MS and WPM as the basal medium, with or without added hormones, added 30 g / L sucrose and 7 g / L agar, adjusted the pH to 5.5, sterilized at 121 °C for 20 min, cooled and solidified for later use. The sterilized capsules were dissected, the seeds were taken out and inoculated on the seed germination medium, and germinated and cultured on the culture rack in the culture room. The temperature in the culture room was 23 °C - 27 °C, the light intensity at the bottom of the culture layer was 2000 - 2500 Lx, and the light time was 12 - 14 h per day.

[0028] Observe and record the seed germination and growth conditions, and evaluate the effects of the culture media on seed germination and growth. Calculate the seed germination rate and the seedling browning rate. The seed germination rate (%) = (number of germinated seeds / total number of inoculated seeds) × 100, and the seedling browning rate (%) = (number of browned seedlings / total number of seedlings) × 100.

[0029] 3) Experimental results and conclusions: Table 1. Effects of different culture media on the aseptic germination of Rhododendron pulchrum seeds

[0030] After the seeds were inoculated on the germination medium, they began to absorb water and swell. After about one week, the embryo was observed to break through the seed coat and initiate germination. By 30 days, the cotyledons had fully expanded, and the seedling height reached 5 - 8 mm ( Figure 2 ). As shown in Table 1, the seed germination rates on each culture medium ranged from 87.5% to 100.0%, the seedling browning rates were 0 - 28.6%, and the leaf colors showed yellowish, light green to dark green. The highest seedling browning rate was 28.6% on culture medium ③ WPM + ZT 0.5 mg / L + NAA 0.1 mg / L, followed by 25.3% on culture medium ④ WPM + NAA 0.1 mg / L. The seedling browning rate on culture medium ⑤ WPM + GA3 100 mg / L was relatively low, at 2.5%, and no seedling browning was observed on culture media ① MS and ② WPM without added hormones. At the same time, the leaf colors of the seedlings on culture media ③ WPM + ZT 0.5 mg / L + NAA 0.1 mg / L and ④ WPM + NAA 0.1 mg / L were yellowish, those on culture media ① MS and ⑤ WPM + GA3 100 mg / L were light green, and those on culture medium ② WPM were dark green.

[0031] In summary, on the WPM basal medium ② without any added hormones, not only was the seed germination rate the highest, with all seeds germinated, but also no seedling browning occurred and the leaf color was dark green. Therefore, the WPM basal medium ② without any added hormones is suitable for the aseptic germination of Rhododendron pulchrum seeds.

[0032] Example 2: Effects of callus induction methods on the present invention 1) Experimental materials: The plant materials used in the experiment were the cotyledons of aseptic seedlings formed after 25 - 35 days of aseptic germination of Rhododendron pulchrum seeds. The culture media were prepared using commercially available basal media, hormones, sucrose, and agar.

[0033] 2) Experimental methods: The callus induction medium uses WPM as the basic medium, adds different types and concentrations of hormones, 30 g / L of sucrose and 7 g / L of agar, adjusts the pH to 5.5, sterilizes at 121 °C for 20 min, cools and solidifies for standby. Use forceps to pick up the seedlings, cut off the cotyledons, cut wounds along the edges of the cotyledons, inoculate with the abaxial side down on the callus induction medium, place it on the culture rack in the culture room and culture for 25 - 35 days to induce the generation of callus. The temperature of the culture room is 23 °C - 27 °C, the light intensity at the bottom of the culture layer is 2000 - 2500 Lx, and the light time is 12 - 14 h per day.

[0034] Observe and record the callus induction situation, statistically calculate the callus induction rate. The callus induction rate (%) = the number of cotyledons with induced callus / the total number of inoculated cotyledons × 100.

[0035] 3) Experimental results and conclusions: Table 2. Effects of different media on callus induction from cotyledons of aseptic seedlings of Rhododendron pulchrum

[0036] After inoculating and culturing the wounded cotyledons for one week, the edges of the wounds began to swell, the cotyledons gradually thickened, and callus formation was observed at the wounds after about two weeks. On the appropriate medium, the callus gradually increased with the prolongation of the culture time ( Figure 3), callus was induced from all cotyledons about one month after inoculation. However, on some other media, callus was only induced from partial cotyledons, and the callus structure was loose, and partial browning or severe browning occurred as the culture time extended. As can be seen from Table 2, the callus induction rate on each medium was between 45.2% and 100.0%. On medium ① WPM + TDZ 2.0 mg / L + NAA 0.5 mg / L + PAA 15.0 mg / L, the callus induction rate was the highest, reaching 100.0%, and the callus showed a green and healthy state and was large in volume. On medium ⑥ WPM + ZT 10.0 mg / L + IAA 10.0 mg / L, the callus induction rate was the lowest, being 45.2%, and severe browning of the callus occurred as the culture time extended. On medium ② WPM + TDZ 0.5 mg / L + 2,4-D 1.0 mg / L and medium ③ WPM + TDZ 0.3 mg / L + 2,4-D 0.3 mg / L, the callus induction rates were 89.7% and 93.5% respectively, and the callus was green but had a loose structure. On medium ④ WPM + ZT 2.0 mg / L + NAA 1.0 mg / L and medium ⑤ WPM + ZT 1.0 mg / L + NAA 2.0 mg / L, the callus induction rates were 88.9% and 73.5% respectively, and the callus was yellowish and partial browning occurred. On medium ⑦ WPM + 2-iP 1.5 mg / L + 2,4-D 0.5 mg / L + IAA 1.0 mg / L, the callus induction rate was relatively low, being 65.3%, and the callus was light green and had a loose structure.

[0037] Therefore, the optimal medium for inducing callus from the cotyledons of aseptic seedlings of Rhododendron pulchrum Sweet is ① WPM + TDZ 2.0 mg / L + NAA 0.5 mg / L + PAA 15.0 mg / L, the callus induction rate reaches 100.0%, and the callus shows a green and healthy state and is large in volume.

[0038] Example 3: Influence of callus subculture and proliferation method on the present invention 1) Experimental materials: The plant materials used in the experiment were callus induced from the cotyledons of aseptic seedlings of Rhododendron pulchrum Sweet. The culture medium was prepared using commercially available basic medium, hormones, sucrose and agar.

[0039] 2) Experimental method: For the subculture proliferation culture of callus, the same medium as that for callus induction is used, namely ① WPM + TDZ 2.0 mg / L + NAA 0.5 mg / L + PAA 15.0 mg / L, or a medium with hormones halved on the basis of ①, namely ② WPM + TDZ 1.0 mg / L + NAA 0.25 mg / L + PAA 7.5 mg / L. 30 g / L of sucrose and 7 g / L of agar are added to the medium. After adjusting the pH to 5.5, it is sterilized at 121 °C for 20 min and cooled and solidified for standby. The callus is divided into small pieces of about 0.5 mm × 0.5 mm × 0.5 mm and inoculated on the callus proliferation medium. The culture bottle is placed on the culture rack in the culture room and cultured for 30 - 40 days to induce proliferation. The temperature of the culture room is 23 °C - 27 °C, the light intensity at the bottom of the culture layer is 2000 - 2500 Lx, and the light time is 12 - 14 h per day.

[0040] After culturing for 30 - 40 days, weigh the mass of the proliferated callus blocks and compare it with the mass before proliferation, calculate the proliferation multiple of the callus, and evaluate the influence of the medium on the subculture proliferation culture of the callus. Proliferation multiple of callus = (mass after proliferation - mass before proliferation) / mass before proliferation.

[0041] 3) Experimental results and conclusions: Table 3. Effects of different media on the subculture proliferation of Rhododendron pulchrum Sweet callus

[0042] The callus is cultured on the proliferation medium, and its volume increases, showing obvious proliferation ( Figure 4 ). As can be seen from Table 3, when subcultured on the same medium ① WPM + TDZ 2.0 mg / L + NAA 0.5 mg / L + PAA 15.0 mg / L as that for callus induction, the callus proliferated 4.5 times within one subculture cycle. However, with the increase of subculture times, the color of the callus changed from dark green to light green, and some showed a water-soaked state. On the medium ② WPM + TDZ 1.0 mg / L + NAA 0.25 mg / L + PAA 7.5 mg / L with halved hormones, the proliferation multiple of the callus reached 5.2 within one subculture cycle, and the callus showed a green and healthy state.

[0043] Therefore, the optimal medium for the subculture proliferation of Rhododendron pulchrum Sweet callus is ② WPM + TDZ 1.0 mg / L + NAA 0.25 mg / L + PAA 7.5 mg / L, with a proliferation multiple of 5.2, and the callus shows a green and healthy state.

[0044] Example 4: Influence of adventitious bud differentiation method on the present invention 1) Experimental materials: The plant materials used in the experiment were callus of Rhododendron pulchrum. The culture medium was prepared using commercially available basal medium, hormones, sucrose and agar.

[0045] 2) Experimental method: The adventitious bud differentiation medium was based on WPM as the basal medium, supplemented with different types and concentrations of hormones, 30 g / L sucrose and 7 g / L agar. After adjusting the pH to 5.5, it was sterilized at 121 °C for 20 min and cooled and solidified for standby. Callus blocks of about 0.5 mm × 0.5 mm × 0.5 mm were inoculated on the adventitious bud differentiation medium, and the culture bottles were placed on the culture rack in the culture room for 30 - 40 days to induce the differentiation of adventitious buds. The temperature in the culture room was 23 °C - 27 °C, the light intensity at the bottom of the culture layer was 2000 - 2500 Lx, and the light time was 12 - 14 h per day.

[0046] The adventitious bud differentiation situation was observed and recorded, and the number of adventitious buds induced and differentiated from the callus was counted.

[0047] 3) Experimental results and conclusions: Table 4. Influence of different media on the induction and differentiation of adventitious buds from callus of Rhododendron pulchrum

[0048] When the callus was inoculated on the differentiation medium, adventitious buds could be observed to start forming on its surface after about two weeks ( Figure 5 ). When cultured on a suitable medium, the adventitious buds gradually elongated to form complete and healthy buds ( Figure 6 ). As can be seen from Table 4, after culturing for 30 - 40 days on medium ① WPM + BA 2.0 mg / L + TDZ 0.5 mg / L + NAA 0.05 mg / L, callus blocks of about 0.5 mm × 0.5 mm × 0.5 mm could average differentiate into 28.5 adventitious buds, and the buds were green and healthy. On other media, the number of adventitious buds differentiated was between 5.1 and 17.8, far lower than the number of adventitious buds on medium ①. The buds induced on medium ④ WPM + ZT 10.0 mg / L + IAA 10.0 mg / L had short internodes and vitrified leaves, and were of no use.

[0049] Therefore, the optimal medium for inducing and differentiating adventitious buds from the callus of Rhododendron pulchrum Sweet is ① WPM + BA 2.0 mg / L + TDZ 0.5 mg / L + NAA 0.05 mg / L. Callus pieces of about 0.5 mm × 0.5 mm × 0.5 mm can differentiate into an average of 28.5 adventitious buds, and the buds are green and healthy.

[0050] Example 5: Influence of the methods of strengthening buds and rooting on the present invention 1) Experimental materials: The plant materials used in the experiment were adventitious buds induced and differentiated from the callus of Rhododendron pulchrum Sweet. The medium was prepared using commercially available 1 / 2 WPM medium, hormones, sucrose, and agar.

[0051] 2) Experimental method: Using 1 / 2 WPM medium as the basic medium, different types and concentrations of hormones were added, coconut milk was added or not added, 30 g / L sucrose and 7 g / L agar were added, and after adjusting the pH to 5.5, it was sterilized at 121 °C for 20 min and cooled and solidified for standby. The adventitious buds induced from the callus and elongated were cut off, and the longer ones could be segmented. The lower morphological ends were inserted into the medium for strengthening buds and rooting, and the culture bottles were placed on the culture rack in the culture room for 40 - 50 days. The temperature in the culture room was 23 °C - 27 °C, the light intensity at the bottom of the culture layer was 2000 - 2500 Lx, and the light time was 12 - 14 h per day.

[0052] The rooting situation and the condition of the seedlings were observed and recorded, and the rooting rate was calculated. Rooting rate (%) = number of rooted buds / total number of buds × 100.

[0053] 3) Experimental results and conclusions: Table 5. Influence of different media on the strengthening of adventitious buds and rooting of Rhododendron pulchrum Sweet

[0054] The adventitious buds induced from the callus and elongated were inoculated on the medium for strengthening buds and rooting. After about 20 days of culture, roots began to form at the base of the buds, and as the culture time extended, the roots continued to elongate and lateral roots grew out ( Figure 7 ). As can be seen from Table 5, on medium ① 1 / 2 WPM + TDZ 0.5 mg / L + IBA 0.5 mg / L + NAA 0.5 mg / L + coconut milk 50 mL / L, the rooting rate was the highest, reaching 95.8%, and the buds were in good shape, with many roots and dark green leaves. On other media, the rooting rates of adventitious buds were between 64.3% and 90.5%, all lower than that on medium ①, and there were fewer roots on media ② and ④.

[0055] Therefore, the optimal medium for the strong shoot and rooting culture of Rhododendron pulchrum Sweet adventitious buds is ① 1 / 2 WPM + TDZ 0.5 mg / L + IBA 0.5 mg / L + NAA 0.5 mg / L + coconut milk 50 mL / L, on which the shoots are in good shape, there are many roots, and the leaf color is dark green.

[0056] The implementation schemes described above can be further combined or replaced. Moreover, the implementation schemes only describe the preferred embodiments of the present invention, rather than limiting the concept and scope of the present invention. Without departing from the design idea of the present invention, various changes and improvements made by those of ordinary skill in the art to the technical solutions of the present invention all fall within the protection scope of the present invention. The protection scope of the present invention is given by the appended claims and any equivalent technical solutions thereof.

Claims

1. A tissue culture method of Rhododendron pulchrum Sweet based on callus regeneration, characterized in that, It includes the following steps: (1) Obtaining aseptic seedlings: Take mature unopened capsules, rinse them, and then rinse with tap water for 20 - 30 min. Soak and shake them in an alcohol solution for sterilization in a laminar flow hood, rinse with sterile water, soak and shake them in a 0.1% mercuric chloride solution for 15 - 20 min, rinse with sterile water, and blot dry the surface moisture. Culture them on a seed germination medium for 25 - 35 days; the seed germination medium is: WPM medium + sucrose 25.0 - 35.0 g / L + agar 6.5 - 7.5 g / L; (2) Inducing callus: Cut off the cotyledons of the aseptic seedlings, make incisions, and inoculate them on a callus induction medium for 25 - 35 days; the formula of the callus induction medium is: WPM medium + TDZ 1.8 - 2.2 mg / L + NAA 0.4 - 0.6 mg / L + PAA 14.0 - 16.0 mg / L + sucrose 25.0 - 35.0 g / L + agar 6.5 - 7.5 g / L; (3) Subculture and proliferation of callus: Divide the callus blocks into small pieces and inoculate them on a callus proliferation medium for 30 - 40 days; the formula of the callus subculture and proliferation medium is: WPM medium + TDZ 0.8 - 1.2 mg / L + NAA 0.2 - 0.3 mg / L + PAA 7.0 - 8.0 mg / L + sucrose 25.0 - 35.0 g / L + agar 6.5 - 7.5 g / L; (4) Differentiation of adventitious buds: Inoculate the callus on an adventitious bud differentiation medium for 30 - 40 days; the formula of the adventitious bud differentiation medium is: WPM medium + BA 1.8 - 2.2 mg / L + TDZ 0.4 - 0.6 mg / L + NAA 0.04 - 0.06 mg / L + sucrose 25 - 35 g / L + agar 6.5 - 7.5 g / L; (5) Strengthening buds and rooting: Divide the adventitious buds into single buds and inoculate them in a bud strengthening and rooting medium according to polarity for 40 - 50 days; the bud strengthening and rooting medium is: 1 / 2 WPM + TDZ 0.4 - 0.6 mg / L + IBA 0.4 - 0.6 mg / L + NAA 0.4 - 0.6 mg / L + coconut milk 45 - 55 mL / L + sucrose 25 - 35 g / L + agar 6.5 - 7.5 g / L.

2. The method according to claim 1, characterized in that, The culture conditions for steps (1), (2), (3), (4), and (5) are: temperature 23 - 27 °C, light intensity 2000 - 2500 Lx, and alternate light and dark culture with 12 - 14 h of daily light.

3. The method according to claim 1, characterized in that, The pH value of the media used in steps (1), (2), (3), (4), and (5) is 5.4 - 5.

6.

4. The method according to claim 1, characterized in that, The formula of the seed germination medium is: WPM medium + sucrose 30.0 g / L + agar 7.0 g / L.

5. The method according to claim 1, wherein The formula of the callus induction medium is: WPM medium + TDZ 1.8 - 2.2 mg / L + NAA 0.4 - 0.6 mg / L + PAA 14.0 - 16.0 mg / L + sucrose 25.0 - 35.0 g / L + agar 6.5 - 7.5 g / L.

6. The method according to claim 1, characterized in that, The formula of the callus subculture and proliferation medium is: WPM medium + TDZ 0.8 - 1.2 mg / L + NAA 0.2 - 0.3 mg / L + PAA 7.0 - 8.0 mg / L + sucrose 25.0 - 35.0 g / L + agar 6.5 - 7.5 g / L.

7. The method according to claim 1, characterized in that, The formula of the adventitious bud differentiation medium is: WPM medium + BA 1.8 - 2.2 mg / L + TDZ 0.4 - 0.6 mg / L + NAA 0.04 - 0.06 mg / L + sucrose 25 - 35 g / L + agar 6.5 - 7.5 g / L.

8. The method according to claim 1, characterized in that, The formula of the strong shoot and rooting medium is: 1 / 2WPM + TDZ 0.4 - 0.6 mg / L + IBA 0.4 - 0.6 mg / L + NAA 0.4 - 0.6 mg / L + coconut milk 45 - 55 mL / L + sucrose 25 - 35 g / L + agar 6.5 - 7.5 g / L.

9. The method according to claim 1, wherein In step (3), the callus blocks are divided into small pieces of 0.5 mm × 0.5 mm × 0.5 mm; in step (4), the 0.5 mm × 0.5 mm × 0.5 mm callus is inoculated on the adventitious bud differentiation medium and cultured for 30 - 40 days.

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