A rapid proliferation method of peachy yew callus

Abstract

The application discloses a kind of rapid proliferation methods of arisaema consanguineum callus, belong to plant tissue culture technical field, break arisaema consanguineum seed dormancy by sand storage method, seed is soaked after distilled water and gibberellin solution and river sand is fully mixed, after storage germination is sowed in the soil added with equal amount of matrix, arisaema consanguineum seedling is obtained after cultivation;Take arisaema consanguineum seedling fresh leaf as explant, cut into small pieces after disinfection on the MS medium added with hormone and cultivate 30d to induce callus, the callus induced is cultivated to proliferate at the optimum temperature of 15 DEG C, inoculate to the same fresh medium after cultivation 20d to prevent browning generation, to obtain high-quality callus, after every 20d, fresh medium can be replaced to realize arisaema consanguineum callus rapid mass proliferation.By the above method, a large amount of high-quality arisaema consanguineum callus can be quickly obtained, which provides sufficient materials for podophyllotoxin extraction and medicinal value development.

Description

Technical Field

[0001] This invention relates to plant tissue culture technology, specifically a method for rapid proliferation of callus tissue from *Prunus persica*. Background Technology

[0002] *Sinopodophyllum hexandrum (Royle) TSYing* is a perennial herb belonging to the genus *Sinopodophyllum* in the family Berberidaceae. Its roots and rhizomes are used medicinally. *Sinopodophyllum hexandrum* seeds have a long dormancy period and poor germination ability under natural conditions, and its growth requirements are very demanding, resulting in a narrow natural distribution area and weak natural reproductive capacity. Coupled with ecological destruction and human harvesting, the maintenance of its population and biodiversity are severely constrained, and it is currently endangered. Furthermore, traditional cultivation methods can no longer meet market demand, directly impacting its medicinal and economic applications. Podophyllotoxin is the main medicinal component of *Sinopodophyllum hexandrum*. With the depletion of wild resources, the industrial production of podophyllotoxin through rhizome harvesting has been limited. Inducing callus tissue of *Pyrrosia lingua* and achieving rapid proliferation through plant tissue culture technology can be an effective means of producing podophyllotoxin from *Pyrrosia lingua*. However, due to the high technical difficulty, there are still many problems in the tissue culture research of *Pyrrosia lingua*, including long induction and culture time of callus tissue, easy browning and growth stagnation, and slow proliferation rate of callus tissue, resulting in poor quality of callus tissue and difficulty in meeting the standards of commercial production.

[0003] Therefore, researching and solving problems such as poor callus quality, slow growth, unsatisfactory growth status, and unstable proliferation culture of *Taoerqi* can lay an experimental foundation for the large-scale production of *Taoerqi* callus. Summary of the Invention

[0004] In view of this, the present invention provides a method for rapid proliferation of Podophyllum heliotropium callus, which solves the problems of difficult seed germination, scarce explant material for tissue culture, callus aging and browning, slow growth, unstable subculture, low callus quality, and delayed tissue culture process. It can quickly obtain a large amount of high-quality Podophyllum heliotropium callus, providing sufficient material for the extraction of Podophyllotoxin and the development of its medicinal value.

[0005] A method for rapid proliferation of *Prunus persica* callus tissue, characterized by the following steps:

[0006] S1. Breaking the dormancy of Peach Glory seeds and seedling cultivation: The sand storage method is used to break the dormancy of seeds. The seeds are soaked in distilled water and gibberellin solution respectively, and then thoroughly mixed with cleaned, sieved and sterilized moist river sand. They are then stored at 4℃ to promote germination. After the seeds germinate, an equal amount of substrate is added to the soil for sowing, and Hoagland nutrient solution is added regularly to ensure the normal growth of the plants.

[0007] S2. Explant disinfection: Select fresh leaves of *Prunus cerasifera* as explants. Disinfect the leaves with 75% alcohol for 30 seconds and then with 10% NaClO for 10 minutes each.

[0008] S3. Induction of callus tissue of *Prunus persica*: Disinfected leaves of *Prunus persica* were cut into uniform small pieces and inoculated onto MS medium supplemented with different hormones, including 6-BA 1.0 mg / L + TDZ 0.5 mg / L + 2,4-D 1.5 mg / L. The medium was cultured at 23℃ for 30 days.

[0009] S4. Callus proliferation culture of *Prunus persica*: Callus induced from leaves for 30 days was cut into uniformly sized pieces and inoculated onto MS medium supplemented with different hormones for proliferation. The hormones included 6-BA 1.0 mg / L + NAA 0.5 mg / L + GA3 0.1 mg / L. The callus was pre-cultured at 23℃ for 2 days to allow it to fully adapt to the new medium. Then it was cultured at 15℃ for 20 days to obtain healthy callus.

[0010] S5. Rapid proliferation of Peach 7 callus: The callus obtained from S4 culture for 20 days was cut into uniformly sized pieces and inoculated into the same fresh culture medium. The callus was then cultured at 15℃ under the same conditions as S4. After 20 days of culture, a large number of callus with good growth and high quality were obtained. Thereafter, the fresh culture medium was replaced every 20 days to achieve rapid and large-scale proliferation of Peach 7 callus.

[0011] Preferably, the MS medium formulation is MS 4.74 g / L, sucrose 30 g / L, agar 7 g / L, and pH adjusted to 5.8.

[0012] Preferably, the culture conditions in steps S3, S4 and S5 also include a light intensity of 3000 lx and a light duration of 14 h / d.

[0013] Preferably, in step S3, the leaves of *Prunus persica* are cut into 0.3cm pieces. 2 size.

[0014] Preferably, in steps S4 and S5, the callus tissue is cut into 0.5cm pieces. 2 size.

[0015] Preferably, in step S1, the seeds are first soaked in distilled water for 24 hours, with the water changed 2-3 times to ensure they fully absorb water, and then soaked in 100ppm gibberellin solution for 24 hours, with the solution changed 2-3 times. The river sand is washed and passed through a 10-mesh sieve, then sterilized in an autoclave (121℃, 30min). The river sand is then moistened with sterile water. The seeds of *Prunus persica* are then mixed with the river sand, and the surface is covered with river sand to a humidity of 30%. The mixture is then sealed with plastic wrap and perforated for ventilation. The substrate is a mixture of vermiculite, perlite, and wood ash in a 1:1:1 ratio.

[0016] Preferably, in step S2, before disinfecting the leaves, the surface impurities are cleaned with detergent, then rinsed with tap water, placed in a clean bench, and the surface moisture is absorbed with sterile filter paper. The leaves are then rinsed with sterile water 3 to 5 times.

[0017] The beneficial effects are as follows:

[0018] This invention improves and optimizes the problems of low callus induction rate, slow growth, severe browning, and unstable proliferation culture of *Prunus armeniaca* by adjusting the culture medium ratio, changing the culture temperature, and updating the culture medium with fresh medium. It proposes a highly efficient method for rapid proliferation of *Prunus armeniaca* callus, with a short culture cycle, high culture efficiency, high callus yield, and good quality. Specifically:

[0019] (1) Since the seeds of *Prunus serrulata* have strong dormancy characteristics and are rare and endangered plants, the traditional method of inducing callus tissue by seed embryo and hypocotyl is limited by the amount of available materials. This invention uses leaves as explants, which are readily available and abundant. Furthermore, by breaking the dormancy of *Prunus serrulata* seeds through sand storage, a stable supply of explant materials can be maintained after sowing. Moreover, there is no need to cultivate sterile seedlings, which not only simplifies the tissue culture process and enables the rapid acquisition of a large amount of high-quality callus tissue, but also reduces the complexity and cost of the operation.

[0020] (2) This method screened out the best disinfection method for leaves as explants, screened out the hormone ratio of the culture medium with a callus induction rate of about 80%, and the callus induced for 30 days can be used for proliferation culture, avoiding browning of the callus and obtaining high-quality callus in a short time. The determined optimal culture condition of 15℃ makes the proliferation coefficient of the callus higher than other temperature conditions, which is consistent with the characteristics of the low temperature growth of Prunus persica. Temperature induction helps the accumulation of energy substances and the enhancement of the antioxidant system.

[0021] (3) This method is simple to operate, requires low equipment, has a short cycle, and has high induction efficiency. Using this method to cultivate callus tissue can be used to extract podophyllotoxin, alleviate the shortage of wild resources of *Prunus persica*, improve the current situation where cultivated resources cannot meet market demand, and may achieve a sustainable drug supply, thereby reducing dependence on wild natural resources of *Prunus persica*. Attached Figure Description

[0022] Figure 1 Flowchart of efficient and rapid proliferation of callus tissue in Peach Seven;

[0023] Figure 2 : Peach 7 seedlings (a is a seedling grown after sand storage, b and c are seedlings cultivated in substrate soil);

[0024] Figure 3 : Callus induction of Peach No. 7 (a is callus induced from leaves for 10 days, b is callus induced from leaves for 30 days);

[0025] Figure 4 Callus tissue cultured at different temperatures (ad: callus tissue grown at 10℃, eh: callus tissue grown at 15℃, il: callus tissue grown at 20℃, mp: callus tissue grown at 23℃, qt: callus tissue grown at 25℃).

[0026] Figure 5 Growth curves of callus tissue proliferation under different temperature conditions. Detailed Implementation

[0027] To make the objectives, solutions, and advantages of the technical solutions of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings of specific embodiments of the present invention.

[0028] like Figure 1 As shown, this invention provides a method for rapid proliferation of callus tissue from *Prunus persica*. First, the dormancy of *Prunus persica* seeds is broken by sand storage. The seeds are soaked in distilled water and gibberellin solution, then thoroughly mixed with river sand. After storage and germination, they are sown in soil with an equal amount of substrate added, and *Prunus persica* seedlings are obtained through cultivation. Fresh leaves from the *Prunus persica* seedlings are used as explants, disinfected, cut into small pieces, and induced to form callus tissue on MS medium supplemented with hormones. The induced callus tissue is cultured and proliferated at an optimal temperature of 15°C. After 20 days of culture, it is inoculated onto the same fresh medium to prevent browning, thus obtaining high-quality callus tissue. Thereafter, the medium is replaced with fresh medium every 20 days to achieve rapid and large-scale proliferation of *Prunus persica* callus tissue.

[0029] The optimal implementation scheme of the present invention was finally obtained through the following specific experiments:

[0030] (1) Breaking the dormancy of Peach Seven Seeds and Seedling Cultivation

[0031] Seed dormancy was broken using sand stratification. First, seeds were soaked in distilled water for 24 hours, changing the water 2-3 times to ensure full water absorption. Then, they were soaked in a 100ppm gibberellin solution for 24 hours, changing the solution 2-3 times. River sand was washed, sieved (10 mesh), and sterilized in an autoclave (121℃, 30 min). The river sand was moistened with sterile water, and then the *Prunus armeniaca* seeds were mixed with the river sand. The surface was covered with river sand, maintaining a humidity of 30%. The container was sealed with plastic wrap, with holes for ventilation, and stored at 4℃. Germinated *Prunus armeniaca* seeds were selected and sown in soil with an equal amount of substrate (vermiculite, perlite, and wood ash mixed in a 1:1:1 ratio). Hoagland nutrient solution was added regularly to maintain normal plant growth. After 120 days of sand stratification, *Prunus armeniaca* seeds gradually germinated, and after 160 days, 90% of the seeds germinated, with the two cotyledons separating. Figure 2 a) The seedlings of *Prunus persica* transplanted into the substrate soil grew well, but at a slow rate, gradually developing from 2 cotyledons to 3 functional true leaves. Figure 2 b, c).

[0032] By adopting the above measures, the dormancy period of Peach No. 7 seeds can be shortened from the normal 10 months to 4 months to produce seedlings, which greatly reduces the seedling time.

[0033] (2) Determination of explant disinfection time

[0034] The disinfection time of explants can determine whether contamination occurs and affect the viability of *Prunus persica* leaves; therefore, determining the optimal disinfection time is crucial. Fresh *Prunus persica* leaves are selected as explants. First, surface impurities are cleaned with detergent, then rinsed thoroughly with tap water. The leaves are placed in a laminar flow hood and blotted dry with sterile filter paper. They are then rinsed 3-5 times with sterile water. The leaves are then disinfected with 75% alcohol for 30 seconds, followed by disinfection with 10% NaClO for 5 minutes, 10 minutes, and 15 minutes respectively. Finally, they are cut into 0.3cm pieces. 2 Explants were inoculated onto hormone-free MS basal medium (MS 4.74 g / L, sucrose 30 g / L, agar 7 g / L, pH 5.8), and explant contamination was observed and the contamination rate was calculated (Formula 1). Disinfection for 5 minutes was insufficient to kill microorganisms on the explant surface, resulting in significant contamination (67%). After 10 minutes of disinfection, the contamination rate decreased significantly to only 5%. After 15 minutes of disinfection, contamination was very rare, but some leaves turned white and explant viability was significantly damaged. This indicates that disinfection with NaClO for 5 minutes was ineffective, and disinfection for 15 minutes may cause cell death. The optimal disinfection conditions for *Prunus persica* leaves were 10% NaClO disinfection for 10 minutes (Table 1).

[0035]

[0036] Table 1. Statistics on disinfection of *Taoer 7* explants

[0037]

[0038] Note: Contamination rate is mean ± standard deviation; different lowercase letters after the data in the same column indicate significant differences (p<0.05).

[0039] (3) Determination of the culture medium for inducing callus formation in *Prunus serrulata*

[0040] Based on the optimal disinfection time determined in (2), using *Prunus persica* leaves as explants, disinfection was performed with 75% alcohol for 30 seconds, followed by disinfection with 10% NaClO for 10 minutes, and then cut into 0.3cm pieces. 2 Callus tissue was inoculated onto MS medium supplemented with different hormones (6-BA, TDZ, 2,4-D) and cultured at 23℃ under a light intensity of 3000 lx and a photoperiod of 14 h / d. The induction rate was calculated after 30 days of culture (Formula 2). The highest callus induction rate and optimal growth were observed on MS medium supplemented with 1.0 mg / L 6-BA, 0.5 mg / L TDZ, and 1.5 mg / L 2,4-D. Therefore, this medium was determined to be the optimal medium for inducing callus tissue from *Prunus armeniaca* leaves. Figure 3 (Table 2).

[0041]

[0042] Table 2. Callus induction by Tao'erqi

[0043]

[0044] Note: Induction rate is mean ± standard deviation; different lowercase letters after the data in the same column indicate significant differences (p<0.05).

[0045] (4) Determination of the optimal culture medium for callus proliferation culture of *Prunus serrulata*

[0046] Callus tissue induced from leaves for 30 days was cut into 0.5cm sections. 2 Uniformly sized callus fragments were inoculated onto MS medium supplemented with different hormones (6-BA, NAA, GA3, CH). The callus was pre-cultured at 23°C for 2 days under a light intensity of 3000 lx and a photoperiod of 14 h / d to allow it to fully adapt to the new medium. Subsequently, the callus was cultured at 15°C and 23°C, respectively, and the proliferation coefficient was calculated after 30 days of culture (Formula 3). The highest proliferation coefficient and optimal growth status were observed in callus inoculated on MS medium supplemented with 1.0 mg / L 6-BA, 0.5 mg / L NAA, and 0.1 mg / L GA3 at 15°C (Table 3). This experiment demonstrates that temperature affects callus culture; therefore, the optimal culture temperature needs to be determined.

[0047]

[0048] Table 3. Callus proliferation of Tao'erqi

[0049]

[0050]

[0051] Note: The proliferation coefficient is expressed as mean ± standard deviation; different lowercase letters after the data in the same column indicate significant differences (p<0.05).

[0052] (5) Temperature regulation promotes rapid proliferation of callus tissue in *Prunus persica* (determination of optimal temperature).

[0053] Select the optimal culture medium (4), and cut the callus tissue induced from the leaves for 30 days into 0.5cm pieces. 2 Uniformly sized callus fragments were inoculated into MS medium supplemented with 1.0 mg / L 6-BA, 0.5 mg / L NAA, and 0.1 mg / L GA3. The fragments were pre-cultured at 23°C for 2 days to allow the callus tissue to fully adapt to the new medium. Five culture temperatures (10°C, 15°C, 20°C, 23°C, and 25°C) were set (light intensity 3000 lx, light duration 14 h / d). The changes in callus tissue proliferation culture under different temperature conditions were recorded after 5, 10, 20, and 30 days.

[0054] ① Morphological characteristics of callus tissue

[0055] As the proliferation culture time increased, the volume of callus tissue cultured at various temperatures gradually increased, as detailed in the table below. Figure 4 :

[0056] At 10℃, the callus tissue began to brown after 10 days of culture, with local greening and a relatively firm texture. Browning became obvious after 20 days of culture, and severe browning and growth cessation occurred after 30 days.

[0057] The callus cultured at 15℃ increased significantly in volume after inoculation. After 10 days, the color changed from yellowish-white to yellowish-green. After 20 days, a distinct light green color appeared, and the callus gradually became firmer. After 30 days, the light green color intensified, and some callus tissues showed slight browning.

[0058] Callus cultured at 20℃ showed localized green color and relatively soft texture after 10 days of growth, and gradually turned yellowish-green with slight browning after 20 days of culture.

[0059] Callus cultured at 23℃ rapidly increases in volume, becomes soft in texture, and is yellowish-white in color. After 20 days, it gradually turns brown and grows slowly. After 30 days, growth stops.

[0060] Callus cultured at 25℃ rapidly turned brown, became soft in texture, and turned yellowish-white in color. After 20 days of culture, obvious growth cessation was observed.

[0061] ②Proliferation coefficient

[0062] The proliferation coefficient was calculated by recording the initial fresh weight and the fresh weight of the callus after 20 days of culture. After 20 days of proliferation culture, the proliferation coefficient of callus cultured at 10℃ was 3.98, at 15℃ it was 5.24, while at 20℃ it reached 4.89, at 23℃ it was 4.34, and at 25℃ it was 4.32. This indicates that the growth and proliferation capacity of callus at 15℃ was significantly better than at other temperatures. 15℃ may be the optimal temperature for the growth and proliferation of *Taoerqi* callus on MS medium containing 1.0 mg / L 6-BA, 0.5 mg / L NAA, and 0.1 mg / L GA3. At this temperature, the metabolic activity and biochemical reaction rate of cells are most suitable, thus promoting rapid cell proliferation.

[0063] ③ Growth curve

[0064] The fresh weight of callus was measured, and growth curves were plotted to compare the growth dynamics of callus at different temperatures. The growth curves showed that days 1-20 were the period of rapid callus proliferation, and the growth rate of callus cultured at 15℃ was significantly higher than at other temperatures. This indicates that the biological metabolism and cell division activities of the callus were most active, thus promoting its rapid growth. This also demonstrates the importance of temperature control in optimizing growth conditions and improving growth efficiency. Figure 5 ).

[0065] (6) Rapid proliferation of callus tissue in Tao'erqi

[0066] Take the callus tissue from (5) that has been cultured at 15℃ for 20 days, cut the callus tissue into uniformly sized pieces and inoculate them onto the same fresh culture medium (MS + 6-BA 1.0 mg / L + NAA 0.5 mg / L + GA3 0.1 mg / L) to prevent browning. Continue to culture the callus tissue at 15℃ (other culture conditions are as above, i.e., pre-culture at 23℃ for 2 days to allow the callus tissue to fully adapt to the new culture medium, and then culture it again at 15℃, light intensity of 3000 lx, and light time of 14 h / d for 20 days to obtain a large number of callus tissues with good growth and high quality. After that, replace the fresh culture medium every 20 days to achieve rapid and large-scale proliferation of the callus tissue of *Taoerqi*.

[0067] Summary of optimal techniques:

[0068] First, soak the seeds in distilled water for 24 hours, changing the water 2-3 times to ensure they are fully hydrated. Then soak them in a 100ppm gibberellin solution for 24 hours, changing the solution 2-3 times. Wash the river sand and pass it through a 10-mesh sieve. Sterilize the sand in an autoclave (121℃, 30min). Moisten the sand with sterile water. Then mix the seeds with the sand, cover the surface with sand, maintain a humidity of 30%, seal with plastic wrap, and poke holes for ventilation. Store at 4℃ to promote germination. After germination, add an equal amount of substrate (vermiculite, perlite, and wood ash mixed in a 1:1:1 ratio) to the soil for sowing. Regularly add Hoagland nutrient solution to ensure normal plant growth and obtain the leaves of the *Prunus mume*.

[0069] Select fresh leaves of *Prunus persica* as explants. Clean the surface with detergent to remove impurities, then rinse thoroughly with tap water. Place them in a clean bench and blot dry with sterile filter paper. Rinse 3-5 times with sterile water, then disinfect with 75% alcohol for 30 seconds, followed by 10% NaClO for 10 minutes each time. Cut into 0.3cm pieces. 2 The cells were inoculated into MS medium containing 1.0 mg / L 6-BA, 0.5 mg / L TDZ, and 1.5 mg / L 2,4-D to induce callus formation (culture conditions: light intensity 3000 lx, light duration 14 h / d, cultured at 23℃) for 30 days.

[0070] The callus tissue that had grown for 30 days was cut into 0.5cm pieces. 2 Callus tissue of various sizes was inoculated into MS medium containing 1.0 mg / L 6-BA, 0.5 mg / L NAA, and 0.1 mg / L GA3 for proliferation culture. It was pre-cultured at 23°C for 2 days (culture conditions as above) to allow the callus tissue to adapt to the new medium. Subsequently, it was transferred to 15°C for 20 days, after which the callus tissue was again cut into 0.5 cm pieces. 2 Large-sized callus pieces were inoculated onto the same fresh culture medium to prevent browning. The callus tissue was then cultured at 15°C (other culture conditions were as above). The callus tissue grew rapidly, and after 20 days of culture, a large number of callus tissues with good growth and high quality were obtained. Thereafter, the culture medium was replaced with fresh medium every 20 days to achieve rapid and large-scale proliferation of *Taoerqi* callus tissue.

Claims

1. A method for rapid proliferation of *Prunus persica* callus tissue, characterized in that... Includes the following steps: S1. Breaking the dormancy of Peach Glory seeds and seedling cultivation: The sand storage method is used to break the dormancy of seeds. The seeds are soaked in distilled water and gibberellin solution respectively, and then thoroughly mixed with cleaned, sieved and sterilized moist river sand. They are then stored at 4℃ to promote germination. After the seeds germinate, an equal amount of substrate is added to the soil for sowing, and Hoagland nutrient solution is added regularly to ensure the normal growth of the plants. S2. Explant disinfection: Select fresh leaves of *Prunus persica* as explants. Disinfect the leaves with 75% alcohol for 30 seconds and then with 10% NaClO for 10 minutes. S3. Induction of callus from *Prunus persica*: Disinfected leaves of *Prunus persica* were cut into uniform small pieces and inoculated onto a culture medium consisting of MS + 6-BA 1.0 mg / L + TDZ 0.5 mg / L + 2,4-D 1.5 mg / L. The medium was cultured at 23℃ for 30 days. S4. Callus proliferation culture of *Prunus persica*: Callus induced from leaves for 30 days was cut into uniformly sized pieces and inoculated onto a culture medium for proliferation. The culture medium was MS + 6-BA 1.0 mg / L + NAA 0.5 mg / L + GA3 0.1 mg / L. The callus was pre-cultured at 23℃ for 2 days to allow it to fully adapt to the new culture medium. Then it was cultured at 15℃ for 20 days to obtain healthy callus. S5. Rapid proliferation of *Taoerqi* callus: The callus obtained after 20 days of culture in S4 was cut into uniformly sized pieces and inoculated into the same fresh culture medium. The callus was then cultured at 15°C under the same conditions as in S4. After 20 days of culture, a large number of callus with good growth and high quality were obtained. Thereafter, the fresh culture medium was replaced every 20 days to achieve rapid and large-scale proliferation of *Taoerqi* callus.

2. The method for rapid proliferation of *Prunus persica* callus tissue according to claim 1, characterized in that: The MS medium formulation consisted of MS 4.74 g / L, sucrose 30 g / L, agar 7 g / L, and pH adjusted to 5.

8.

3. The method for rapid proliferation of *Prunus persica* callus tissue according to claim 2, characterized in that: The cultivation conditions for steps S3, S4 and S5 also include a light intensity of 3000 lx and a light duration of 14 h / d.

4. The method for rapid proliferation of *Prunus persica* callus tissue according to claim 3, characterized in that: In step S3, cut the leaves of the peach tree into 0.3 cm pieces. 2 size.

5. The method for rapid proliferation of *Prunus persica* callus tissue according to claim 3, characterized in that: In steps S4 and S5, the callus tissue is cut into 0.5 cm pieces. 2 size.

6. The method for rapid proliferation of *Prunus persica* callus tissue according to claim 1, characterized in that: In step S1, the seeds are first soaked in distilled water for 24 hours, with the water changed 2-3 times to ensure they are fully hydrated. Then, they are soaked in a 100 ppm gibberellin solution for 24 hours, with the solution changed 2-3 times. The river sand is washed, passed through a 10-mesh sieve, and sterilized in an autoclave at 121°C for 30 minutes. The river sand is then moistened with sterile water. The seeds are then mixed with the river sand, covered with river sand, and the humidity is 30%. The mixture is sealed with plastic wrap, with holes punched for ventilation. The substrate is a mixture of vermiculite, perlite, and wood ash in a 1:1:1 ratio.

7. The method for rapid proliferation of *Prunus persica* callus tissue according to claim 1, characterized in that: In step S2, before disinfecting the leaves, clean the surface impurities with detergent, rinse them with tap water, place them in a clean bench, absorb the surface moisture with sterile filter paper, and rinse them with sterile water 3-5 times.

Images