A method for tissue culture and rapid propagation and adventitious bud regeneration of prunus serrulata

Abstract

This invention discloses a method for rapid propagation of *Prunus campanulata* via tissue culture and regeneration of adventitious buds. This invention pioneers a system for rapid propagation and organ regeneration of *Prunus campanulata* via tissue culture, utilizing its vegetative organs, stem segments, leaves, or seeds as explants. This results in a primary induction rate as high as 74.4%, a proliferation coefficient of approximately 3.9, and a rooting rate of 83.5%, achieving highly efficient and rapid propagation. Unrooted, robust buds can also be used as scions for grafting propagation. In the seed disinfection process, the plant tissue culture antibacterial agent PPM is applied for the first time to disinfect seeds of the *Prunus* subgenus, achieving good control of the contamination rate and significantly reducing it. The invention also explores the number of times mercuric chloride disinfectant can be reused for the first time, finding that its disinfection effect decreases with increasing reuse frequency, with a significant decrease in disinfection effect after three or more uses. Furthermore, the same culture medium is used for obtaining sterile explants, inducing sterile seedlings, and propagation culture, simplifying the operation process and reducing production costs.

Description

Technical Field

[0001] This invention belongs to the field of tissue culture technology, specifically relating to a method for rapid propagation of Cherry Blossom through tissue culture and regeneration of adventitious buds. Background Technology

[0002] Cherry blossom (Prunus spp.) is a general term for plants in the subgenus Cerasus of the genus Prunus. The subgenus Cerasus has been a famous woody ornamental plant since ancient times, and includes more than 150 species.

[0003] Cherry blossoms are prized for their beautiful shape, abundant varieties, and diverse flower colors. When in bloom, they create a breathtaking spectacle, like clouds and rosy dawn. Even after the flowers fade, the trees offer attractive fruit and foliage. In landscaping, they can be planted in large numbers to create "seas of flowers," or combined with other garden plants for landscaping. They can also be used as hedges, street trees, or small bonsai, making them widely applicable. In recent years, cherry blossoms have become increasingly popular in modern landscaping as early spring flowering plants, with a huge market potential and a continuously increasing demand for seedlings. Furthermore, as people's spiritual lives become richer, they place higher demands on the ornamental value and landscaping applications of cherry blossoms, all of which have spurred continuous innovation in production and propagation methods.

[0004] Currently, propagation by cuttings or grafting is widely used in production. However, according to Peng Yufu et al. (2019), grafting often results in incompatibility between different varieties and is easily affected by factors such as time, climate, and environment, hindering large-scale promotion. Cherry blossom cuttings are difficult to root, with lower survival rates, slower growth, and weaker upright growth compared to grafting. They also have high requirements regarding the season, environment, and selection of cuttings (Zou Na et al., 2007). According to Duan Xiaomei et al. (2002), most seeds of this genus are deeply dormant, resulting in a very low seedling survival rate from direct sowing. Low-temperature vernalization or gibberellin treatment is required to break dormancy. Zhan Linxing (2018) found that seed propagation requires cumbersome pre-sowing preparation, including variable-temperature refrigeration and sand stratification. Sowing time is seasonally limited, and the development from seed to mature plant takes a long time. Plant tissue culture allows isolated plant organs (such as roots, stems, and leaves), tissues (cambium, anther tissue, endosperm, etc.), cells (somatic cells or reproductive cells), and protoplasts to be cultured under sterile conditions and rapidly grown into complete plants. After the proliferation stage, the propagation coefficient is high, enabling the production of a large number of seedlings in a short period of time. It is less affected by seasons and climate, allowing for year-round production, while maintaining the superior traits of the parent plant, thus better meeting market demands.

[0005] *Prunus campanulata* is a deciduous small tree belonging to the genus *Prunus* in the family Rosaceae, and it blooms early. *Prunus campanulata* 'Feihan' is an excellent cultivated variety, producing abundant, bright pink flowers with open petals and a graceful, butterfly-like shape. It exhibits good cold and heat tolerance, vigorous growth, strong resistance to pests and diseases, wide adaptability, and a beautiful tree shape. Its flowering period falls between early-blooming cherry varieties (opening in early February) and mid-blooming cherry varieties (peak blooming in March-April), making it highly promising for landscaping applications and with broad prospects for growth.

[0006] Current reports on *Prunus cerasifera* 'Feihan' focus on genetic diversity analysis (Zhou Chengcheng et al., 2020), horticultural applications (Zhang Yanfang, 2016), and cultivation management (Gao Zhaoliang et al., 2021). Research on propagation methods has only covered seedling and grafting (Kuang Hongling et al., 2020; Zhou Ling et al., 2022). Research on tissue culture-based rapid propagation of this variety is still in its infancy.

[0007] References:

[0008] Peng Yufu, Peng Huohui, Chen Hualing, et al. Preliminary study on bud grafting propagation of five cherry blossom varieties [J]. Modern Horticulture, 2019, 42(23): 45-46.

[0009] Zou Na, Cao Guangqiu, Lin Sizu. Research progress on propagation techniques of ornamental cherry blossoms [J]. Journal of Southwest Forestry University, 2007, (06): 42-46.

[0010] Zhan Linxing. Planting experiment of sowing and raising seedlings of Prunus serrulata in Fujian Province [J]. Modern Agricultural Science and Technology, 2018, (19): 164-165. Zhou Chengcheng, Yang Deming, Li Shikun, et al. ISSR analysis of phylogenetic relationships of 18 Prunus serrulata materials [J]. Journal of Forestry and Environment, 2020, 40(1): 46-53.

[0011] Zhang Yanfang. Common cherry blossom varieties and their landscaping applications [J]. Landscape Architecture, 2016(4): 72-75.

[0012] Gao Zhaoliang, Zhu Jianjun, Jun Jiaxiang, et al. Study on the effect of compound fertilizer combined with microbial inoculant on the growth of 'Feihan Cherry' seedlings [J]. Shanghai Journal of Agricultural Sciences, 2021, 37(05): 116-120.

[0013] Zhou Ling. Effects of rootstock type and grafting method on grafting effect of Prunus campanulata [J]. Green Technology, 2022, 24(21): 142-145.

[0014] Kuang Hongling, Nie Chaoren, Xu Haiqin, et al. Research on sowing and seedling raising techniques of eight cherry blossom varieties [J]. Hubei Forestry Science and Technology, 2020, 49(01): 25-26. Summary of the Invention

[0015] The purpose of this invention is to provide a method for rapid propagation of Cherry Blossom through tissue culture and regeneration of adventitious buds. This tissue culture method has a high germination rate, good rooting rate, short culture cycle, and high survival rate.

[0016] To achieve the above objectives, the technical solution of the present invention is as follows:

[0017] A method for rapid propagation of Cherry Blossom through tissue culture and regeneration of adventitious buds, characterized in that the method includes the following steps:

[0018] (1) Selection and disinfection of explants: Select non-lignified stem segments, leaves or seeds from the current year with buds and disinfect them respectively;

[0019] D. Disinfection treatment of stem segments: First, disinfect with 75% alcohol for 30 seconds, rinse with sterile water 2-3 times, then disinfect with 1% sodium hypochlorite solution for 3-10 minutes or 0.1% mercuric chloride solution for 3-7 minutes, and rinse with sterile water 5-6 times.

[0020] E. Disinfection treatment of stem segments: First, disinfect with 75% alcohol for 30 seconds, rinse with sterile water 2-3 times, then disinfect with 1% sodium hypochlorite solution for 1-5 minutes or 0.1% mercuric chloride solution for 3-7 minutes, and rinse with sterile water 5-6 times.

[0021] F. Seed disinfection treatment: Cut open the shell to remove the kernel, soak the embryo in detergent water for 20 minutes, then rinse under tap water for 1 hour, then soak in 0.1% carbendazim solution for 20 minutes, then disinfect with 75% alcohol for 30 seconds, rinse with sterile water 3-4 times, then disinfect with 0.1% mercuric chloride solution for 10-15 minutes, then rinse with sterile water more than 5 times, and then soak in 1-4% PPM for 1-4 hours;

[0022] (2) Stem segment bud induction and proliferation culture: The sterilized stem segments were inoculated into the induction medium. After 10-15 days, the axillary buds began to sprout and form small buds. The composition of the induction medium was: MS or 1 / 2 MS or WPM + 6-BA 0.5-2.0 mg / L + NAA 0.1-1-0.3 mg / L + sucrose 30 g / L + agar 7.0 g / L, pH = 5.8-6.0. When the aseptic buds obtained from the primary culture reached 2.5 cm, they were cut off and transferred to the axillary bud proliferation medium. After 30 days, clustered buds were formed. The composition of the proliferation medium was: MS + ZT 0.3-1.0 mg / L + NAA 0.1-0.5 mg / L + GA3 0.5-1.5 mg / L + sucrose 30 g / L + agar 7.0 g / L, pH = 5.8-6.0.

[0023] (3) Seed initiation and proliferation culture: After disinfection, the seed coat is removed, and the seeds are inserted into the seed germination medium with the tip pointing downwards. The seed germination medium consists of: MS + 6-BA 1.0-3.0 mg / L + NAA 0.1-0.3 mg / L + GA3 1.0-2.0 mg / L + 0.3% PPM + sucrose 30 g / L + agar 7.0 g / L, pH = 5.8-6.0; the PPM is a plant tissue culture antibacterial agent.

[0024] (4) Leaf adventitious bud regeneration culture

[0025] After sterilization, leaves were inoculated onto callus induction medium. Approximately one week later, the cut edges curled and arched towards the center. After 10-15 days, small callus protrusions appeared at the leaflet veins and cut edges. The callus induction medium consisted of MS or 1 / 2 MS or WPM + 6-BA 0.5-2.0 mg / L + 2,4-D 1.0-2.0 mg / L + sucrose 30 g / L + agar 6.0-7.5 g / L, pH 5.8-6.0. When the aseptic buds obtained from primary culture reached 2.5 cm, they were cut into cubes and transferred to differentiation medium. Adventitious buds formed after 6-7 weeks. The differentiation medium consisted of MS + 6-BA 0.5-2.0 mg / L + 2,4-D 0.1-0.5 mg / L + TDZ. 0.5-2.0 mg / L + 200 mg / L hydrolyzed casein + 30 g / L sucrose + 7.0 g / L agar, pH = 5.8-6.0.

[0026] (5) Rooting culture

[0027] Select large seedlings with a height of 3cm or more, remove the callus tissue and ineffective proliferating buds from the base of the stem, and inoculate them onto rooting medium. First, culture them in the dark for 3-5 days; then transfer them to light for culture. New roots will begin to form after 15 days. The rooting medium consists of: 1 / 2 MS + NAA 0.2-0.5 mg / L + IBA 0.4-0.8 mg / L + sucrose 20 g / L + agar 6.0 g / L, pH = 5.8-6.0.

[0028] (6) Hardening off seedlings and transplanting

[0029] After 2-3 days of hardening off, the rooted seedlings of *Cherry Blossom indicum* are transplanted into a mixed substrate and cultured for 3-4 weeks. The indoor humidity is controlled at 70-90% and the temperature at 22-30℃ to obtain rooted cherry blossom seedlings.

[0030] Preferably, the specific method for selecting explants in step (1) is as follows: Select robust, disease-free, and superior plants. In February to April, take current-year shoots or semi-lignified branches, cut them into branches with 2-3 buds, and insert them into a substrate with a peat moss:perlite volume ratio of 1:1. Before cutting, the substrate and cuttings are disinfected by soaking in a 1000-fold dilution of carbendazim. Place them in a growth chamber and spray with a 1% gibberellin solution and a 2% cyanamide solution. When the buds grow to 5-7cm, cut off non-lignified single-bud stem segments or terminal buds as explants. Cut the already sprouted branches into 2-3cm long stem segments with buds for pretreatment to ensure that each segment has at least one bud. Collect pest-free, tender leaves from branches in February to April for later use. Collect more mature fruits in March to April, wash away excess pulp, dry the seeds, and store them in a 4℃ refrigerator for later use.

[0031] In this invention, an optimal method for disinfecting stem segments is to first disinfect with 75% alcohol for 30 seconds, rinse three times with sterile water, and then disinfect with 0.1% mercuric chloride for 7 minutes, rinsing six times with sterile water. 11. The method according to claim 1, characterized in that, in the leaf disinfection process, first disinfect with 75% alcohol for 30 seconds, rinse three times with sterile water, and then disinfect with 0.1% mercuric chloride for 3 minutes, rinsing six times with sterile water.

[0032] In seed disinfection, an optimal method is to first disinfect with 75% alcohol for 30 seconds, rinse with sterile water 4 times, then disinfect with 0.1% mercuric chloride solution for 15 minutes, rinse with sterile water more than 5 times, and then soak in 4% PPM solution for 2 hours. The mercuric chloride solution can be reused 3 times or more.

[0033] During the experiment, the conditions of various culture media were optimized, and the optimal implementation method was determined to be:

[0034] The induction medium in step (2) is: 1 / 2 MS medium + 6-BA 2mg / L + NAA 0.2mg / L + sucrose 30g / L + agar 7.0g / L; the axillary bud proliferation medium is: MS + ZT 0.5mg / L + NAA 0.1mg / L + GA3 1mg / L + sucrose 30g / L + agar 7.0g / L.

[0035] The seed germination medium in step (3) consists of: MS + 6-BA 2mg / L + NAA 0.2mg / L + GA 32.0mg / L + 0.3% PPM + sucrose 30g / L + agar 7.0g / L.

[0036] The leaf callus induction medium in step (4) consists of: WPM + 6-BA 0.5 mg / L + 2,4-D 2.0 mg / L + sucrose 30 g / L + agar 7.0 g / L.

[0037] The rooting medium in step (5) consists of 1 / 2 MS + 0.2 mg / L NAA + 0.8 mg / L IBA + 20 g / L sucrose + 6.0 g / L agar.

[0038] The mixed matrix in step (6) is peat, perlite and vermiculite, and the volume ratio of peat, perlite and vermiculite is 2:1:1.

[0039] Advantages of this invention:

[0040] (1) A tissue culture-based rapid propagation and organ regeneration system for *Prunus campanulata* was first established. Using its vegetative organs, stem segments, leaves, or seeds as explants, the initial induction rate reached 74.4%, the proliferation coefficient was around 3.9, and the rooting rate reached 83.5%. This system enables efficient and rapid propagation without being limited by season, climate, or region. Unrooted strong buds can also be used as scions for grafting propagation. Its leaf regeneration system also provides infection material for genetic transformation and gene editing.

[0041] (2) In the process of seed disinfection, the plant tissue culture antibacterial agent PPM was applied to the disinfection of seeds of the subgenus Cherry for the first time. The contamination rate was well controlled and the contamination rate was significantly reduced to a minimum of about 6.38%, which can provide a scientific reference for the tissue culture disinfection of the subgenus Cherry.

[0042] (3) The number of times mercuric chloride disinfectant can be reused was investigated for the first time. It was found that the disinfection effect of the disinfectant decreased with the increase of the number of reuses when disinfecting seeds. After the experiment, it was found that the disinfection effect of mercuric chloride used 3 times or more decreased significantly.

[0043] (4) The same culture medium is used for obtaining sterile explants from seeds, inducing and culturing sterile seedlings, and promoting proliferation, which simplifies the operation process and reduces production costs.

[0044] (5) During the propagation culture, it was found that plants of the subgenus *Prunus* rapidly lignify after budding, which is consistent with the findings of Xu Chenjie et al. (2020). In the early stage, stem segments were retained for propagation culture, and it was found that the propagation results were poor after lignification, with almost no change in the culture. Later, the lignified stem segments gradually turned brown and died, exhibiting a stubborn phenomenon. The stubborn phenomenon in in vitro plant culture refers to the lack or loss of response of cells, tissues, and organs to culture operations under in vitro conditions. Subsequently, 6-BA was replaced with the more effective ZT, and subculture was carried out before the stem segments lignified. During this period, the culture bottles were rotated multiple times. After the mother plant stem segments were completely lignified, rooted seedlings obtained from tissue culture and other propagated seedlings were used as explants for continued propagation, and the propagation effect was improved. In addition, the concentration of regulators used in some treatments was high, and the inoculated buds did not show any propagation phenomenon and gradually turned yellow and died. This may be due to the accumulation of regulators in the plant, which produced toxicity. Later, the concentration of regulators was reduced, and the weaker buds were placed in blank culture medium for a period of time before propagation culture was carried out. Attached Figure Description

[0045] Figure 1 These are the explants collected.

[0046] Figure 2 Photo of seeds contaminated with bacteria.

[0047] Figure 3 The germination of *Prunus cerasifera* stem segments under different treatments is shown in the figure. In the figure, a: treatment 1; b: treatment 2; c: treatment 3; d: treatment 4; e: treatment 5; f: treatment 6; g: treatment 7; h: treatment 8; i: treatment 9.

[0048] Figure 4 The image shows the germination status of *Prunus campanulata* seeds. In the image, a: *Prunus campanulata* seeds; bf: growth status of experimental treatment 5 (b: immediately after inoculation; c: 4-5 days after inoculation; d: 10 days after inoculation; ef: 25 days after inoculation).

[0049] Figure 5 The axillary bud proliferation of *Prunus cerasifera* under different treatments is shown in the figure. In the figure, a: treatment 1; b: treatment 2; c: treatment 3; d: treatment 4; e: treatment 5; f: treatment 6; g: treatment 7; h: treatment 8; i: treatment 9.

[0050] Figure 6 The callus induction of *Prunus cerasifera* leaves under different treatments is shown in the figure. In the figure, a: treatment 1; b: treatment 2; c: treatment 3; d: treatment 4; e: treatment 5; f: treatment 6; g: treatment 7; h: treatment 8; i: treatment 9.

[0051] Figure 7This shows the callus induction and differentiation of *Prunus cerasifera* leaflets; ab: callus tissue transferred to differentiation medium; cg: the process of callus differentiation into adventitious buds; gh: adventitious buds transferred to proliferation medium.

[0052] Figure 8 The rooting of *Prunus campanulata* under different treatments is shown in the figure; a: treatment 1; b: treatment 2; c: treatment 3.

[0053] Figure 9 The transplanting of seedlings for the domestication of Cherry Blossom. Detailed Implementation

[0054] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, unless otherwise specified, the specific experimental methods involved in the following embodiments are conventional methods or implemented according to the conditions recommended in the manufacturer's instructions.

[0055] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, the experimental methods in the following embodiments are all conventional methods. Unless otherwise specified, the reagents and materials used can be purchased commercially.

[0056] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be used in this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0057] 1. Test materials

[0058] The explants of *Prunus campanulata* include current-year, non-lignified stem segments with buds and seeds. Figure 1 The collection period was from late February to mid-May, and the collection location was the cherry blossom boardwalk in Guishan Park, Wuhan (30.55545°N, 114.28009°E).

[0059] Select robust, disease- and pest-free superior plants, taking current-year shoots or semi-lignified branches, and quickly bring them back to the laboratory for water immersion. Bring back lignified branches with plump dormant buds that have not yet fully sprouted, cut them into sections with 2-3 buds each, and insert them into a substrate with a peat moss:perlite volume ratio of 1:1. Before insertion, disinfect both the substrate and the cuttings by soaking them in a 1000-fold dilution of carbendazim. Place them in a growth chamber with an external environmental condition of 22-25℃, 12 hours of light / 8 hours of darkness. Simultaneously spray with a 1% gibberellin solution and a 2% cyanamide solution. When the buds grow to about 5-7cm, cut off non-lignified single-bud stem segments or terminal buds as explants. Collect semi-lignified branches that have already sprouted, cut them into 2-3cm long stem segments with buds for pretreatment, ensuring each segment has at least one bud. Collect young, pest-free leaves from the branches for later use. Harvest the more mature fruits, wash away the excess pulp, dry them, and store them in a refrigerator at 4℃ for later use.

[0060] 2. Test Methods

[0061] 2.1 Establishment of a disinfection system for explants

[0062] 2.1.1 Stem segment disinfection

[0063] Remove the leaves from the harvested stem segments with buds, leaving some petioles. Soak in a solution of dish soap and laundry detergent, shake on a shaker for 20 minutes, then scrub to remove surface dust and oil. Rinse with running water for 2-3 hours before transferring to a clean bench. Disinfect with 75% alcohol (30s treatment time), rinse 2-3 times with sterile water, then disinfect with 1% sodium hypochlorite solution or 0.1% mercuric chloride solution for different durations, followed by 5 rinses with sterile water, each for at least 30s. Blot dry with filter paper, then inoculate the sterilized explants into MS blank medium. Each treatment is inoculated in 20 bottles, replicated 3 times. The contamination rate of the stem segments is calculated after 30 days.

[0064] The experimental results are shown in Table 1. Different disinfection treatments showed significant differences in their disinfection effects on *Prunus armeniaca* stem segments. When the disinfection time with 75% alcohol remained constant, the contamination rate of the stem segments generally decreased as the disinfection time with another disinfectant increased. Treatment 6 showed the lowest contamination rate.

[0065] Table 1 Comparison of disinfection status of Cherry Blossom stem segments

[0066]

[0067]

[0068] 2.1.2 Effects of different growth stages on stem segment contamination rate

[0069] Over time, the contamination rate of explants gradually increased, and the survival rate decreased. This is mainly because contaminants gradually accumulate on the plants, making sterilization difficult. This is consistent with the findings of Xu Chenjie (2020).

[0070] Table 2. Effects of collection at different growth stages on the contamination rate of stem segments from *P. campanulata* 'Feihan'.

[0071]

[0072] 2.1.3 Seed disinfection

[0073] Before the experiment, the outer shell was mechanically broken, and the embryos were soaked in detergent water for about 20 minutes, then rinsed under tap water for 1-2 hours. Afterward, they were soaked in a 0.1% carbendazim solution for 30 minutes and then placed in a clean bench. They were then disinfected with 75% alcohol for 30 seconds, rinsed 3-4 times with sterile water, and then disinfected with 0.1% mercuric chloride solution for 10, 12, and 15 minutes, followed by rinsing with sterile water at least 5 times. After treatment, they were placed in a sterile bottle containing sterile water overnight and then inoculated into a 3% (v / v) PPM medium. A large number of endophytic bacteria were found in the seeds, so after routine disinfection, further disinfection was performed using the plant preservative mixture (PPM). PPM is a broad-spectrum antimicrobial agent that effectively inhibits microbial contamination in plant tissue culture and does not affect seed germination, callus proliferation, and regeneration at certain concentrations and within a certain time frame. The study used mercuric chloride disinfection time, PPM immersion concentration, and PPM immersion time as factors, with 3 levels for each factor, for a total of 9 treatments. Each treatment contained 20 bottles and was repeated 3 times.

[0074] Table 3 shows that different combinations of bactericides and sterilization times significantly affected the contamination rate of explants. Among the three different treatments with mercuric chloride disinfection time, the 0.1% mercuric chloride disinfection time of 15 min was the most effective. Furthermore, after immersion disinfection with PPM, the contamination rate was significantly lower than that of treatments using only conventional disinfection steps, and the 4% (v / v) concentration showed the best disinfection effect. Among all treatments, treatment 9 had the lowest contamination rate.

[0075] Table 3. Disinfection of *Prunus cerasifera* embryos

[0076]

[0077]

[0078] Note: Immersion time of 0 hours means that only the conventional disinfection method of alcohol + mercuric chloride is used.

[0079] As shown in Table 4 of the analysis of variance, the effects of mercuric chloride disinfection time, PPM soaking concentration, and time on the contamination rate of Cherry Blossom seeds were all extremely significant, with P values ​​all less than 0.01.

[0080] Table 4. Analysis of variance regarding the disinfection of Cherry Blossom seeds.

[0081]

[0082] In practice, it was found that mercuric chloride residue remained after rinsing and reusing the seeds, suggesting that this residue might affect the disinfection process of *Prunus campanulata* seeds. An experiment was designed to investigate this. Disinfection was carried out using a regimen of 75% alcohol for 30 seconds + 0.1% mercuric chloride for 15 minutes + 1% (v / v) PPM for 4 hours. The results showed that using mercuric chloride that was reused three or more times significantly affected the final disinfection effect of *Prunus campanulata* seeds.

[0083] Secondly, during the specific experimental procedures, it was found that the embryos were significantly affected by bacterial contamination. Figure 2 The results showed the condition of seeds contaminated with bacteria. After preliminary experiments, it was speculated that repeated use of mercuric chloride had a significant impact on the disinfection effect of seed embryos. An experiment was designed to investigate this, using a disinfection regimen of 75% alcohol for 30 seconds + 0.1% mercuric chloride for 15 minutes + 1% (v / v) PPM for 4 hours. As shown in Table 5, the use of mercuric chloride that was reused three or more times had a significant impact on the final disinfection effect of Cherry Blossom seeds.

[0084] Table 5. Effect of the number of mercuric chloride re-disinfection cycles on seed disinfection efficacy.

[0085]

[0086]

[0087] 2.1.4 Leaf disinfection

[0088] Leaves from harvested leafy branches were used as explants. Surface dust was brushed off with a brush, and the leaves were soaked in detergent water and shaken for approximately 20 minutes. They were then rinsed under tap water for 1 hour and placed in a clean bench. Disinfection was performed using 75% alcohol and 1% sodium hypochlorite solution (1, 3, 5 min) or 0.1% mercuric chloride (3, 5, 7 min) as disinfectants. Leaves were inoculated onto MS blank medium, with 20 plates per experiment. Each petri dish contained 4-5 leaf fragments, with three replicates. After 30 days, the contamination rate, browning rate, and survival rate were statistically analyzed. The optimal sterilization method was then selected. The experimental results are shown in Table 6. Different disinfection treatments showed significant differences in their effectiveness on *Prunus cerasifera* leaves. When the 75% alcohol disinfection time remained constant, the contamination rate of stem segments generally decreased with increasing disinfection time using another disinfectant. The disinfection effect of 0.1% mercuric chloride solution is better than that of 1% NaClO solution. Overall, treatment 5 has the best effect, with lower contamination and browning rates.

[0089] Table 6 Disinfection of Cherry Blossom Leaves

[0090]

[0091] 2.2 Stem Segment Initiation Culture

[0092] Uncontaminated stem segments were transferred to induction media. Three different plant basal media were prepared: WPM, MS, and 1 / 2 MS. Two plant growth regulators were added to each medium: the first was a cytokinin 6-BA at concentrations of 0.5 mg / L, 1.0 mg / L, and 2.0 mg / L; the second was an auxin NAA at concentrations of 0.1 mg / L, 0.2 mg / L, and 0.3 mg / L. Shoot induction and growth were recorded and observed 30 days after inoculation.

[0093] Stem segments of *Prunus cerasifera* var. *flavovirens* inoculated onto different combinations of growth regulators and different culture media could induce budding, but the quality and development speed of the buds varied depending on the combination of growth regulators. The results are shown in Table 7. Figure 3 Different combinations of exogenous hormones significantly affected the induction rate of axillary buds. Among them, the bud induction rates of experimental treatments 9, 6, and 3 were relatively high, at 74.44%, 62.22%, and 60.83%, respectively. When the NAA concentration increased, axillary bud germination may be inhibited. Different basal media also showed certain differences in bud induction survival rate and number of buds induced. 1 / 2 MS + 2.0 mg / L 6-BA + 0.2 mg / L NAA was the optimal initiation culture scheme for *Prunus cerasifera* stem segments.

[0094] Table 7. Effects of different treatment combinations on stem germination of *Prunus cerasifera*.

[0095]

[0096] Note: + indicates normal growth, ++ indicates good growth, and +++ indicates robust growth. The same applies below. Different lowercase letters in the same column indicate significant differences (P < 0.05), the same applies below.

[0097] As shown in Table 8 of the analysis of variance, the effects of basic culture medium and NAA concentration on the germination rate of Prunus cerasifera stem segments were not significant, with P values ​​of 0.945 and 0.87, respectively, both greater than 0.05. However, the effect of 6-BA on the germination rate was extremely significant, with P value less than 0.001.

[0098] Table 8. Analysis of variance on stem segment germination of Cherry Blossom.

[0099]

[0100] 2.3 Embryo initiation culture

[0101] The sterilized *Prunus campestris* embryos were transferred to induction medium, using MS as the basal medium. Three plant growth regulators were added to each medium: 6-BA at two concentration gradients (2.0 mg / L and 3.0 mg / L); NAA at three concentration gradients (0.1 mg / L, 0.2 mg / L, and 0.3 mg / L); and gibberellin GA3 at three concentration gradients (1.0 mg / L, 2.0 mg / L, and 3.0 mg / L). Thirty embryos were inoculated for each treatment, with three replicates. Induction rate was calculated after 30 days.

[0102] The results are shown in Table 9 and Figure 4 Different treatment combinations significantly affected the embryo induction rate. Some embryos showed greening of cotyledons and gradual germination as early as 4-5 days, and generally 2-3 true leaves emerged after 10-20 days. Adding different concentrations of NAA did not show significant differences, indicating that the embryos are not sensitive to NAA concentration. Adding different concentrations of 6-BA showed significant differences; the induction rate increased with increasing 6-BA concentration, and the seedlings grew better. This suggests that the optimal 6-BA concentration for embryo induction in *Prunus campanulata* should be 2.0 mg / L or even higher. Too low a concentration of 6-BA will not effectively stimulate embryo germination. Based on the existing data analysis, the optimal culture medium for seed induction is: MS + 2.0 mg / L 6-BA + 0.2 mg / L NAA + 2.0 mg / L GA3. Furthermore, the experiment found that *Prunus campanulata* embryos can be directly induced to form clustered buds on germination medium.

[0103] Table 9. Effects of different treatment combinations on seed germination of *Prunus cerasifera*.

[0104]

[0105] As shown in Table 10 of the analysis of variance, the effects of NAA concentration and GA3 concentration on the germination rate of Prunus campanulata seeds were not significant, with P values ​​of 0.07 and 0.25, respectively, both greater than 0.05. However, the effect of 6-BA on the germination rate was significantly different, with P value less than 0.05.

[0106] Table 10. Analysis of variance on seed germination of *Prunus campanulata*.

[0107]

[0108]

[0109] 2.4 Proliferation Culture

[0110] Healthy buds were excised from stem segments on the induction medium and inoculated into MS subculture medium supplemented with three regulators for proliferation culture. Fifteen days after inoculation, a small amount of callus tissue grew from the base of some buds, and leaflets began to grow. (See Table 11 and...) Figure 5 It can be seen that treatment 4 had the highest bud proliferation coefficient. In the proliferation culture, the proliferation coefficient first increased and then decreased with the increase of ZT concentration. The proliferation coefficient of the treatment with 0.5 mg / L ZT was generally greater than that of the treatments with 0.3 mg / L and 1.0 mg / L ZT.

[0111] Table 11 Effects of different treatment combinations on axillary bud proliferation of Cherry Blossom.

[0112]

[0113] 2.5 Leaf callus induction culture

[0114] After disinfecting and inoculating uncontaminated *Prunus cerasifera* leaves onto induction medium, the cut edges curled and arched towards the center after about one week. Around 10-15 days later, small callus protrusions appeared at the leaflet veins and cut edges. The callus tissue was mostly yellowish-brown, loose in texture, and had a rough, granular surface; a small portion was milky white and firmer, presumably embryogenic callus. (Based on Table 12 and...) Figure 6 Only treatment 7 showed a significant difference in callus induction rate, but the callus quality varied considerably among the different treatments. Treatments using WPM as the basal medium consistently induced embryogenic callus of good quality. Treatments using MS and 1 / 2 MS as the basal medium mostly induced non-embryogenic callus, which could not regenerate into subsequent plants. Based on the current experimental results, WPM + 0.5 mg / L 6-BA + 2 mg / L 2,4-D is the suitable callus induction scheme for *Pterocarya stenoptera* leaves.

[0115] Table 12 Callus Induction Culture Scheme for Cherry Blossom Leaves

[0116]

[0117] As shown in Table 13 of the analysis of variance, the type of culture medium and the concentration of 6-BA had no significant effect on the induction rate of *Prunus campanulata* leaves, with P values ​​of 0.496 and 0.175, respectively, both greater than 0.05. However, the concentration of 2,4-D had a significant effect on the induction rate, with a P value less than 0.05.

[0118] Table 13. Analysis of variance of callus induction by Cherry Blossom.

[0119]

[0120] 2.6 Callus Differentiation Culture

[0121] In tissue culture of *Prunus* subgenus plants, the differentiation of adventitious buds from callus tissue is relatively difficult, and its differentiation rate is constrained by various factors, among which hormones are one of the more important factors. The obtained callus tissue is cut into small pieces and inoculated onto differentiation medium. As the culture time progresses, the callus tissue gradually becomes firmer, its growth slows down, and granular green callus tissue appears on some surfaces. A small portion of the green callus tissue differentiates into buds. Figure 7 Some treatments failed to differentiate adventitious buds, and after about 6-7 weeks, the surface color gradually turned black or brown, eventually drying out and dying with prolonged culture time. Table 14 shows that adventitious bud differentiation occurred in treatments 1, 4, 5, and 8, with treatment 5 showing the highest differentiation rate at 24.44%. The callus tissues in the other control groups did not effectively form adventitious buds. Analysis of these results suggests that using higher concentrations of auxin regulators may reduce the activity of enzymes related to the synthesis of endogenous cytokinins within cells, directly affecting bud formation and decreasing the adventitious bud differentiation rate. Appropriate concentrations of 6-BA and TDZ can help cells synthesize endogenous cytokinins, thereby inducing adventitious bud differentiation.

[0122] Table 14. Induction scheme for callus differentiation of Cherry Blossom leaves

[0123]

[0124] As shown in Table 15 of the ANOVA, the TDZ concentration had no significant effect on the callus differentiation rate of *Prunus cerasifera* leaves (P=0.781, greater than 0.05). However, the concentrations of 6-BA and 2,4-D had significant effects on the differentiation rate (P=less than 0.05 for both). Furthermore, 2,4-D and TDZ together significantly affected the callus differentiation rate of *Prunus cerasifera* leaves (P=less than 0.05 for both).

[0125] Table 15. Analysis of variance on callus differentiation of Cherry Blossom.

[0126]

[0127] 2.7 Rooting Culture

[0128] Select healthy subcultured seedlings and inoculate them into rooting medium. Generally, white rootlets appear on the seedlings 20 days after inoculation. As shown in Table 16, the seedlings can all root in the existing rooting medium. Treatment 3 showed the best results, with a rooting rate as high as 83.33%.

[0129] Table 16 Effects of different plant growth regulators on rooting culture of Prunus cerasifera 'Feihan'.

[0130]

[0131] 2.8 Acclimatization and Transplantation

[0132] After 2-3 days of hardening off, the rooted seedlings of *Prunus cerasifera* were transplanted. They were considered successfully transplanted after approximately 30 days when two true leaves emerged. The results are as follows: Figure 9 During transplanting, pay attention to shading and moisturizing. After 3-5 days of hardening-off treatment, transplant the seedlings into a mixed substrate of peat moss:perlite:vermiculite = 2:1:1 (treatment 1), peat moss:vermiculite = 3:1 (treatment 2), and peat moss:perlite = 3:1 (treatment 3) (Table 17). Before transplanting, the agar on the roots should be washed off, and the substrate should be disinfected with a 1000-fold dilution of carbendazim solution. For one month after transplanting, cover the seedlings with a seedling box to allow them to gradually adapt to the external environment. After 30 days, the survival rates of the three substrates were 91.11%, 60.15%, and 67.78%, respectively. The leaves of the transplanted seedlings gradually turned green, and their height gradually increased, showing good growth. From the comparison of survival rates, treatment 1 showed the best results.

[0133] Table 17 Comparison of different cultivation substrates

[0134]

[0135] The embodiments described above are merely preferred embodiments of the present invention and are only used to explain the present invention. They are not intended to limit the scope of the present invention. For those skilled in the art, other implementation methods can be easily made by substitution or modification based on the technical content disclosed in this specification. Therefore, all changes and improvements made on the principle of the present invention should be included within the scope of the patent application of the present invention.

Claims

1. A rapid propagation method for *Prunus campanulata* via tissue culture, characterized in that... The method includes the following steps: (1) Selection and disinfection of explants: Select seeds as explants, disinfect them with 75% alcohol for 30 seconds, rinse them with sterile water 4 times, then disinfect them with 0.1% mercuric chloride solution for 15 minutes, rinse them with sterile water more than 5 times, and then soak them in 4% PPM for 2 hours. (2) Seed initiation and proliferation culture: After disinfection, the seed coat is removed and the seeds are inserted into the seed germination medium with the tip pointing downwards. The seed germination medium consists of: MS + 6-BA 2 mg / L + NAA 0.2 mg / L + GA3 2.0 mg / L + 0.3% PPM + sucrose 30 g / L + agar 7.0 g / L, pH=5.8-6.0; PPM is a plant tissue culture antibacterial agent. (3) Rooting culture Select large seedlings with a height of 3cm or more, remove the callus tissue and ineffective proliferating buds from the base of the stem, and inoculate them onto rooting medium. First, culture them in the dark for 3-5 days; then transfer them to light for culture. New roots will begin to form after 15 days. The rooting medium consists of: 1 / 2 MS + 0.2 mg / L NAA + 0.8 mg / L IBA + 20 g / L sucrose + 6.0 g / L agar, pH=5.8-6.

0. (4) Hardening off seedlings and transplanting After 2-3 days of hardening off, the rooted seedlings of *Cherry Blossom indicum* are transplanted into a mixed substrate and cultured for 3-4 weeks. The indoor humidity is controlled at 70-90% and the temperature at 22-30℃ to obtain rooted cherry blossom seedlings.

2. The method according to claim 1, characterized in that, The specific method for selecting explants in step (1) is as follows: collect mature fruits in March-April, wash away excess pulp, dry the seeds and store them in a refrigerator at 4℃ for later use.

3. The method according to claim 1, characterized in that, The mixed matrix in step (4) is peat, perlite and vermiculite, and the volume ratio of peat, perlite and vermiculite is 2:1:1.

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