A tissue culture method of Rhododendron splendidum based on callus regeneration

Abstract

The present invention relates to the field of plant tissue culture technology, and specifically to a tissue culture method for Rhododendron splendidum based on callus regeneration. Using young cotyledons of sterile seedlings as explants, an efficient tissue culture and rapid propagation technology system for Rhododendron splendidum was established through callus induction, subculture proliferation of callus, differentiation of adventitious buds, bud strengthening and rooting culture, forming complete tissue culture offspring plants. The callus induction rate of this system is 100.0%, and the callus proliferation multiples reach 5.2 times within one subculture cycle. A callus block of approximately 0.5 mm×0.5 mm×0.5 mm can differentiate into 28.5 adventitious buds, with a rooting rate of 95.8%. 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 of Rhododendron splendidum based on callus tissue and mutant screening and breeding.

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] Splendid Rhododendron Rhododendron pulchrum ) belongs to the Ericaceae family, Rhododendron subgenus ( Rhododendron Subgenus Tsutsusi Azaleas are a semi-evergreen shrub with lush leaves and abundant flowers in rich and vibrant colors. They can be grown in pots, in clusters or groups in parks and gardens, or as hedges along roadsides. As a representative of the hairy rhododendrons in the horticultural classification, Rhododendrons are resistant to pruning and highly adaptable, making them a common garden plant in the Yangtze River Basin and south. Besides its high ornamental value, Rhododendrons also possess significant medicinal value. Its roots, stems, and leaves are used to treat rheumatism, sores, gynecological conditions, and coughs. Research has shown that Rhododendrons leaves contain broad-spectrum antimicrobial compounds with inhibitory effects against some common Gram-fungi and drug-resistant bacteria. Furthermore, Rhododendrons are rich in flavonoids, such as hyperoside, quercetin, and azalea, which have analgesic, expectorant, antitussive, antibacterial, anti-inflammatory, and cardiovascular protective properties.

[0003] Plant tissue culture utilizes the totipotency of plant cells to cultivate plant organs, tissues, or cells in an artificial culture medium under sterile conditions, regenerating them into complete plants. Plant tissue culture boasts rapid reproduction, high production efficiency, conservation of propagation materials, and freedom from seasonal or regional restrictions. It is a rapid propagation method widely used in agriculture and forestry, and serves as the foundation for plant genetic transformation and improvement.

[0004] Plant tissue culture regeneration can be divided into two main pathways: direct and indirect. Indirect regeneration involves the explant first dedifferentiating into callus tissue, which is then induced to differentiate into shoots, roots, or embryoids, ultimately forming a regenerated plant. Callus tissue is newly formed on the surface of a wound following localized trauma. Composed of living parenchyma cells, it can originate from living cells in any tissue within the plant's organs, making it readily available. Furthermore, callus has a high growth potential and can be subcultured for long periods under suitable conditions. It also possesses a strong differentiation capacity, allowing a single callus to simultaneously differentiate into multiple adventitious shoots or embryoids, significantly improving propagation efficiency. Furthermore, due to its active cell division, genetic uniformity, ease of selection, and strong regeneration capacity, callus tissue is an ideal recipient for genetic transformation methods such as Agrobacterium-mediated or biolistic bombardment. It is also an ideal material for mutation breeding, such as screening for new traits such as polyploidy, disease resistance, and stress tolerance.

[0005] Previous researchers have established direct organogenesis tissue culture systems for Rhododendron splendidum (Azalea) through bud propagation. For example, Dong Chunzhi et al. (1989) established a tissue culture system for direct bud regeneration using newly emerged young stems as explants, and Liu Yuxuan et al. (2022) established an open tissue culture system using young stem segments as explants. Regarding the indirect callus generation pathway, Li Zheng et al. (2019) established a callus induction method for Rhododendron splendidum (Azalea) using wounds formed from sterile seedling leaves as explants (CN110419446 A). However, there are no reports of a complete Rhododendron splendidum tissue culture system based on indirect callus regeneration. The present invention establishes a Rhododendron splendidum tissue culture method based on callus regeneration, providing an effective approach for the efficient and rapid propagation of Rhododendron splendidum and also providing technical support for its genetic transformation, improvement, and mutant screening and breeding. Summary of the Invention

[0006] The purpose of the present invention is to provide a Rhododendron splendidum tissue culture method based on callus regeneration in order to solve the problem that the prior art lacks a complete Rhododendron splendidum tissue culture system based on indirect regeneration of callus.

[0007] To achieve the above object, the technical solution adopted by the present invention is:

[0008] A tissue culture method of Rhododendron splendidum based on callus regeneration comprises the following steps:

[0009] (1) Obtaining sterile seedlings: Take mature but unopened capsules of Rhododendron splendidum, rinse them with detergent solution and then with running water for 20-30 min, transfer them to a clean bench, soak them in 75% alcohol solution and shake sterilize them for 55-65 s, rinse them with sterile water 2-3 times, soak them in 0.1% mercuric chloride solution and shake sterilize them for 15-20 min, rinse them with sterile water 4-5 times, and dry the surface moisture with sterile filter paper; cut open the capsules, take out the seeds and inoculate them on 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;

[0010] (2) Callus induction: Take the sterile seedlings formed 25 to 35 days after inoculation, pick up the seedlings with tweezers, cut off the cotyledons, and cut wounds along the edges of the cotyledons. Inoculate the seedlings with the back of the leaves facing down on callus induction medium and culture for 25 to 35 days; the formula of the callus induction medium is: WPM medium + TDZ 1.8 to 2.2 mg / L + NAA 0.4 to 0.6 mg / L + PAA 14.0 to 16.0 mg / L + sucrose 25.0 to 35.0 g / L + agar 6.5 to 7.5 g / L;

[0011] (3) Subculture proliferation of callus tissue: Take the callus tissue block and divide it into small pieces of about 0.5 mm × 0.5 mm × 0.5 mm, inoculate it on callus tissue proliferation medium and culture it for 30 to 40 days to allow it to proliferate; the formula of the callus tissue subculture proliferation medium is: WPM medium + TDZ 0.8 to 1.2 mg / L + NAA 0.2 to 0.3 mg / L + PAA 7.0 to 8.0 mg / L + sucrose 25.0 to 35.0 g / L + agar 6.5 to 7.5 g / L;

[0012] (4) Adventitious bud differentiation: Callus tissue of approximately 0.5 mm × 0.5 mm × 0.5 mm in size was inoculated on an adventitious bud differentiation medium and cultured for 30–40 days to induce adventitious bud differentiation; the adventitious bud differentiation medium had the following formula: 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;

[0013] (5) Bud growth and rooting: The adventitious buds induced from the callus tissue were separated into single buds and inoculated into a bud growth and rooting medium according to polarity and cultured for 40 to 50 days; the bud growth and rooting medium was: 1 / 2 WPM + TDZ 0.4 to 0.6 mg / L + IBA 0.4 to 0.6 mg / L + NAA 0.4 to 0.6 mg / L + coconut milk 45 to 55 mL / L + sucrose 25 to 35 g / L + agar 6.5 to 7.5 g / L.

[0014] The culture conditions of steps (1), (2), (3), (4) and (5) are: temperature 23-27°C, light intensity 2000-2500 Lx, and light exposure for 12-14 hours per day for light-dark alternation culture.

[0015] The pH value of the culture medium used in steps (1), (2), (3), (4) and (5) is 5.4 to 5.6.

[0016] The formula of the seed germination medium is: WPM medium + sucrose 30.0 g / L + agar 7.0 g / L.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] The formula of 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.

[0021] The beneficial technical effects of the present invention are:

[0022] Compared with the existing technology, the present invention provides a tissue culture method for Rhododendron splendidum based on callus regeneration. Using the young cotyledons of sterile seedlings as explants, the method establishes an efficient tissue culture and rapid propagation technology system through callus induction, callus subculture, adventitious bud differentiation, bud strengthening, and rooting culture, resulting in complete tissue culture offspring plants. The callus induction rate of this system is 100.0%, and the callus proliferation rate reaches 5.2 times within one subculture cycle. A callus block of approximately 0.5 mm × 0.5 mm × 0.5 mm can differentiate into 28.5 adventitious buds, with a rooting rate of 95.8%. This method is not restricted by seasonal, climatic, or regional factors, enabling efficient and rapid propagation throughout the year. Furthermore, the establishment of this system also lays a technical foundation for callus-based genetic transformation and improvement of Rhododendron splendidum and mutant screening and breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0024] Figure 1 It is the capsule of embodiment 1 that is ripe but not yet cracked.

[0025] Figure 2 These are seedlings formed by aseptic germination of the seeds in Example 1.

[0026] Figure 3 This is Example 2, inducing callus from cotyledons.

[0027] Figure 4 This is the subculture proliferation of the callus tissue in Example 3.

[0028] Figure 5 This is Example 4, inducing adventitious buds from callus tissue.

[0029] Figure 6 It is the elongation of adventitious buds in Example 4.

[0030] Figure 7 It is embodiment 5 strong bud and rooting culture. DETAILED DESCRIPTION

[0031] The present invention will be further described below with reference to specific embodiments. These embodiments should be understood as merely preferred examples of the present invention, and are not intended to limit the present invention in any way.

[0032] Example 1: Effect of the Aseptic Seed Germination Method on the Present Invention

[0033] 1) Experimental Materials:

[0034] In November, mature but unopened Rhododendron splendidum capsules were collected from healthy plants. Figure 1 ) for sterile seed germination culture. The culture medium is prepared using commercially available minimal culture medium, hormones, sucrose, and agar.

[0035] 2) Experimental methods:

[0036] Rinse the capsule with detergent water and then with running water for 20 to 30 minutes, transfer it to a clean bench, soak it in 75% alcohol solution and shake it for 55 to 65 seconds, rinse it with sterile water 2 to 3 times, soak it in 0.1% mercuric chloride solution and shake it for 15 to 20 minutes, rinse it with sterile water 4 to 5 times, and dry the surface moisture with sterile filter paper for later use.

[0037] Seed germination medium consists of MS and WPM as the basic medium, with or without hormones, supplemented with 30 g / L sucrose and 7 g / L agar. The pH is adjusted to 5.5, sterilized at 121°C for 20 minutes, and cooled to solidify. Sterilized capsules are opened, and seeds are inoculated onto the seed germination medium. Germination is then cultured on a culture rack in the incubator. The incubator temperature is 23°C–27°C, and the light intensity at the bottom of the culture layer is 2000–2500 Lx, with a photoperiod of 12–14 hours per day.

[0038] Observe and record seed germination and growth, and evaluate the effects of the culture medium on seed germination and growth. Statistically calculate the seed germination rate and seedling browning rate: Seed germination rate (%) = number of germinated seeds / total number of inoculated seeds × 100; seedling browning rate (%) = number of browning seedlings / total number of seedlings × 100.

[0039] 3) Experimental results and conclusions:

[0040] Table 1. Effects of different culture media on aseptic germination of Rhododendron splendidum seeds

[0041]

[0042] After the seeds are inoculated on the germination medium, they begin to absorb water and swell. After about a week, the embryo begins to break through the seed coat and start germination. By 30 days, the cotyledons have fully expanded and the seedlings have reached a height of 5 to 8 mm ( Figure 2 As shown in Table 1, the seed germination rates on various media ranged from 87.5% to 100.0%, the seedling browning rate ranged from 0 to 28.6%, and the leaf color ranged from yellowish to light green to dark green. The highest seedling browning rate, 28.6%, was observed on medium ③ (WPM + 0.5 mg / L ZT + 0.1 mg / L NAA). Medium ④ (WPM + 0.1 mg / L NAA) was second, at 25.3%. Medium ⑤ (WPM + 100 mg / L GA3) had a lower browning rate of 2.5%. No seedling browning was observed on the hormone-free media ① MS and ② (WPM). At the same time, the leaves of seedlings on culture medium ③ WPM + ZT 0.5 mg / L + NAA 0.1 mg / L and ④ WPM + NAA 0.1 mg / L were yellowish, the leaves of seedlings on culture medium ① MS and ⑤ WPM + GA3100 mg / L were light green, and the leaves of seedlings on culture medium ② WPM were dark green.

[0043] In summary, on ② WPM basic medium without adding any hormones, not only the seed germination rate was the highest and all the seeds germinated, but also the seedlings did not show browning and the leaves were dark green. Therefore, ② WPM basic medium without adding any hormones was suitable for the aseptic germination of Rhododendron splendidum seeds.

[0044] Example 2: Effect of callus induction method on the present invention

[0045] 1) Experimental Materials:

[0046] The plant material used in the experiment was the cotyledons of sterile seedlings formed 25-35 days after aseptic germination of Rhododendron splendidum seeds. The culture medium was prepared using commercially available minimal medium, hormones, sucrose, and agar.

[0047] 2) Experimental methods:

[0048] Callus induction medium uses WPM as the basic medium, supplemented with various hormone types and concentrations, 30 g / L sucrose, and 7 g / L agar. The pH is adjusted to 5.5, sterilized at 121°C for 20 minutes, and cooled to solidify. Seedlings are picked with tweezers, and the cotyledons are cut and wound along the edges. The seedlings are inoculated with the dorsal surface facing down onto the callus induction medium and incubated on a culture rack in the culture room for 25–35 days to induce callus formation. The room temperature is 23–27°C, and the light intensity at the bottom of the culture layer is 2000–2500 Lx, with a photoperiod of 12–14 hours per day.

[0049] The callus induction situation was observed and recorded, and the callus induction rate was calculated statistically. Callus induction rate (%) = number of cotyledons induced into callus / total number of inoculated cotyledons × 100.

[0050] 3) Experimental results and conclusions:

[0051] Table 2. Effects of different culture media on callus induction from cotyledons of sterile seedlings of Rhododendron splendidum

[0052]

[0053] After one week of inoculation of wounded cotyledons, the wound edges began to swell and the cotyledons gradually thickened. About two weeks later, callus formation was observed at the wound site. On suitable culture media, the callus gradually grew larger with the extension of culture time ( Figure 3), callus was induced from all cotyledons approximately one month after inoculation. However, on other cultures, callus was induced only from some cotyledons, and the callus structure was loose, with partial or severe browning with prolonged culture time. Table 2 shows that the callus induction rate on each culture medium ranged from 45.2% to 100.0%. The highest callus induction rate, 100.0%, was achieved on culture medium ① (WPM + 2.0 mg / L TDZ + 0.5 mg / L NAA + 15.0 mg / L PAA). The calli appeared green, healthy, and large. The lowest callus induction rate, 45.2%, was achieved on culture medium ⑥ (WPM + 10.0 mg / L ZT + 10.0 mg / L IAA). The calli also showed severe browning with prolonged culture time. On media ② (WPM + 0.5 mg / L TDZ + 1.0 mg / L 2,4-D) and ③ (WPM + 0.3 mg / L TDZ + 0.3 mg / L 2,4-D), callus induction rates were 89.7% and 93.5%, respectively. The calli were green but loose in structure. On media ④ (WPM + 2.0 mg / L ZT + 1.0 mg / L NAA) and ⑤ (WPM + 1.0 mg / L ZT + 2.0 mg / L NAA), callus induction rates were 88.9% and 73.5%, respectively. The calli were yellowish with some browning. On medium ⑦ (WPM + 1.5 mg / L 2-iP + 0.5 mg / L 2,4-D + 1.0 mg / L IAA), the callus induction rate was relatively low, at 65.3%. The calli were light green with a loose structure.

[0054] Therefore, the optimal culture medium for inducing callus from the cotyledons of sterile seedlings of Rhododendron splendidum was ① WPM + TDZ 2.0 mg / L + NAA 0.5 mg / L + PAA 15.0 mg / L, with a callus induction rate of 100.0%. The calli were green and healthy, with a large volume.

[0055] Example 3: Effect of callus subculture proliferation method on the present invention

[0056] 1) Experimental Materials:

[0057] The plant material used in the experiment was callus tissue induced from the cotyledons of sterile seedlings of Rhododendron splendidum. The culture medium was prepared using commercially available minimal culture medium, hormones, sucrose, and agar.

[0058] 2) Experimental methods:

[0059] Subculture of callus tissue was performed using the same medium used for callus induction: (i.e., WPM + 2.0 mg / L TDZ + 0.5 mg / L NAA + 15.0 mg / L PAA) or (ii.e., WPM + 1.0 mg / L TDZ + 0.25 mg / L NAA + 7.5 mg / L PAA) with half the hormone content of (i.e., WPM + 1.0 mg / L TDZ + 0.25 mg / L NAA + 7.5 mg / L PAA). The medium was supplemented with 30 g / L sucrose and 7 g / L agar, adjusted to pH 5.5, and sterilized at 121°C for 20 minutes before solidification. Callus tissue was divided into small pieces measuring approximately 0.5 mm × 0.5 mm × 0.5 mm and inoculated onto callus proliferation medium. Culture flasks were placed on a culture rack in the culture room for 30–40 days to induce proliferation. The culture room temperature was 23–27°C, and the light intensity at the bottom of the culture layer was 2000–2500 Lx for 12–14 hours per day.

[0060] After 30-40 days of culture, weigh the mass of the callus after proliferation and compare it with the mass before proliferation to calculate the proliferation fold of the callus and evaluate the effect of the culture medium on callus subculture proliferation. The proliferation fold of the callus = (mass after proliferation - mass before proliferation) / mass before proliferation.

[0061] 3) Experimental results and conclusions:

[0062] Table 3. Effects of different culture media on subculture and proliferation of Rhododendron splendidum callus

[0063]

[0064] The callus tissue was cultured on the proliferation medium, and the volume increased and the proliferation was obvious ( Figure 4 As shown in Table 3, calli cultured on the same medium used for callus induction (① WPM + 2.0 mg / L TDZ + 0.5 mg / L NAA + 15.0 mg / L PAA) increased in number by 4.5 times within one subculture cycle. However, with increasing subcultures, the callus color changed from dark green to light green, and some areas appeared water-soaked. On the medium with half the hormone content (② WPM + 1.0 mg / L TDZ + 0.25 mg / L NAA + 7.5 mg / L PAA), the callus increased in number by 5.2 times within one subculture cycle, and the calli appeared green and healthy.

[0065] Therefore, the optimal culture medium for subculture proliferation of Rhododendron splendidum 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 callus appearing green and healthy.

[0066] Example 4: Effect of the adventitious bud differentiation method on the present invention

[0067] 1) Experimental Materials:

[0068] The plant material used in the experiment was callus tissue of Rhododendron splendidum. The culture medium was prepared using commercially available basic culture medium, hormones, sucrose, and agar.

[0069] 2) Experimental methods:

[0070] Adventitious bud differentiation medium uses WPM as the basic medium, supplemented with various hormone types and concentrations, 30 g / L sucrose, and 7 g / L agar. The pH is adjusted to 5.5, sterilized at 121°C for 20 minutes, and cooled to solidify. Callus pieces measuring approximately 0.5 mm × 0.5 mm × 0.5 mm are inoculated onto the adventitious bud differentiation medium. The culture flasks are placed on a culture rack in the culture room and incubated for 30–40 days to induce adventitious bud differentiation. The room temperature is 23–27°C, and the light intensity at the bottom of the culture layer is 2000–2500 Lx, with a photoperiod of 12–14 hours per day.

[0071] Observe and record the differentiation of adventitious buds, and count the number of adventitious buds induced and differentiated from callus tissue.

[0072] 3) Experimental results and conclusions:

[0073] Table 4. Effects of different culture media on adventitious bud induction from Rhododendron splendidum callus

[0074]

[0075] The callus tissue was inoculated on the differentiation medium, and adventitious buds began to form on its surface after about two weeks ( Figure 5 ). Continue to cultivate on a suitable culture medium, and the adventitious buds will gradually elongate to form complete and healthy buds ( Figure 6 As shown in Table 4, after 30–40 days of culture on medium ① (WPM + 2.0 mg / L BA + 0.5 mg / L TDZ + 0.05 mg / L NAA), callus pieces measuring approximately 0.5 mm × 0.5 mm × 0.5 mm produced an average of 28.5 healthy, green adventitious buds. On other media, the number of adventitious buds produced ranged from 5.1 to 17.8, significantly lower than that on medium ①. Buds induced on medium ④ (WPM + 10.0 mg / L ZT + 10.0 mg / L IAA) had short internodes and vitrified leaves, making them unusable.

[0076] Therefore, the optimal culture medium for inducing adventitious buds from Rhododendron splendidum callus was ① WPM + BA 2.0 mg / L + TDZ 0.5 mg / L + NAA 0.05 mg / L. A callus block of approximately 0.5 mm × 0.5 mm × 0.5 mm could produce an average of 28.5 green and healthy adventitious buds.

[0077] Example 5: Effect of bud-strengthening and rooting culture methods on the present invention

[0078] 1) Experimental Materials:

[0079] The plant material used in the experiment was adventitious buds induced from callus tissue of Rhododendron splendidum. The culture medium was prepared using commercially available 1 / 2 WPM medium, hormones, sucrose, and agar.

[0080] 2) Experimental methods:

[0081] Use 1 / 2 WPM medium as the basic medium, supplemented with various hormone types and concentrations, with or without coconut milk, 30 g / L sucrose, and 7 g / L agar. Adjust the pH to 5.5, sterilize at 121°C for 20 minutes, and cool to solidify. Excise elongated adventitious buds induced from the callus. Longer ones can be segmented, and the lower end of the morphologically defined segment is inserted into a bud-strengthening and rooting medium. Culture bottles are placed on a culture rack in the incubator for 40–50 days. The incubator temperature is 23–27°C, and the light intensity at the bottom of the culture layer is 2000–2500 Lx, with a photoperiod of 12–14 hours per day.

[0082] Observe and record the rooting situation and the condition of the seedlings, and calculate the rooting rate. Rooting rate (%) = number of rooted buds / total number of buds × 100.

[0083] 3) Experimental results and conclusions:

[0084] Table 5. Effects of different culture media on the growth and rooting of adventitious buds of Rhododendron splendidum

[0085]

[0086] The adventitious buds induced and elongated from the callus tissue were inoculated on the bud-strengthening and rooting medium. After about 20 days of culture, roots began to form at the base of the buds. As the culture time prolonged, the roots continued to elongate and lateral roots grew ( Figure 7Table 5 shows that the highest rooting rate, 95.8%, was achieved on medium ① (1 / 2 WPM + 0.5 mg / L TDZ + 0.5 mg / L IBA + 0.5 mg / L NAA + 50 mL / L coconut water). The rooting was bud-like, with numerous roots and dark green leaves. On the other media, the rooting rates of adventitious buds ranged from 64.3% to 90.5%, all lower than those on medium ①. Furthermore, fewer roots were observed on media ② and ④.

[0087] Therefore, the optimal culture medium for bud strengthening and rooting culture of adventitious buds of Rhododendron splendidum is ① 1 / 2 WPM + TDZ 0.5 mg / L + IBA 0.5 mg / L + NAA 0.5 mg / L + coconut water 50 mL / L, which has bud-shaped, abundant roots and dark green leaves.

[0088] The embodiments described above may be further combined or replaced, and the embodiments are merely descriptions of preferred embodiments of the present invention and do not limit the concept and scope of the present invention. Various changes and improvements made to the technical solutions of the present invention by persons of ordinary skill in the art without departing from the design concept of the present invention are within the scope of protection of the present invention. The scope of protection of the present invention is defined by the appended claims and any equivalent technical solutions.

Claims

1. A tissue culture method of Rhododendron splendidum based on callus regeneration, characterized in that: The following steps are involved: (1) Obtaining sterile seedlings: Take mature, unopened capsules, rinse them with tap water for 20-30 minutes, soak and sterilize them in an alcohol solution in a clean bench, rinse them with sterile water, soak and sterilize them with 0.1% mercuric chloride solution by shaking for 15-20 minutes, rinse them with sterile water, and 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) Callus induction: Cut the cotyledons of sterile seedlings, cut the wound, inoculate on callus induction medium and culture for 25 to 35 days; the formula of the callus induction medium is: WPM medium + TDZ 1.8 to 2.2 mg / L + NAA 0.4 to 0.6 mg / L + PAA 14.0 to 16.0 mg / L + sucrose 25.0 to 35.0 g / L + agar 6.5 to 7.5 g / L; (3) Subculture proliferation of callus: Take the callus tissue block and divide it into small pieces, inoculate it on callus proliferation medium and culture it for 30-40 days; the formula of the callus 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) Adventitious bud differentiation: The callus tissue was inoculated on an adventitious bud differentiation medium and cultured for 30 to 40 days; the formula of the adventitious bud differentiation medium was: WPM medium + BA 1.8 to 2.2 mg / L + TDZ 0.4 to 0.6 mg / L + NAA 0.04 to 0.06 mg / L + sucrose 25 to 35 g / L + agar 6.5 to 7.5 g / L; (5) Bud growth and rooting: The adventitious buds were separated into single buds and inoculated into a bud growth and rooting medium according to polarity and cultured for 40 to 50 days; the bud growth and rooting medium was: 1 / 2 WPM + TDZ 0.4 to 0.6 mg / L + IBA 0.4 to 0.6 mg / L + NAA 0.4 to 0.6 mg / L + coconut milk 45 to 55 mL / L + sucrose 25 to 35 g / L + agar 6.5 to 7.5 g / L.

2. The method according to claim 1, wherein 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 for 12-14 h per day.

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

6.

4. The method according to claim 1, wherein 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 Step (3) taking the callus tissue block and dividing it into small pieces of 0.5 mm × 0.5 mm × 0.5 mm; Step (4) inoculating the 0.5 mm × 0.5 mm × 0.5 mm callus tissue on adventitious bud differentiation medium and culturing it for 30 to 40 days.

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