Rapid proliferation method of podophyllum hexandrum callus

Abstract

The invention discloses a podophyllum hexandrum callus rapid proliferation method, and belongs to the technical field of plant tissue culture, the podophyllum hexandrum seed dormancy is broken through a sand storage method, the seeds are soaked in distilled water and a gibberellin solution and then are fully and uniformly mixed with river sand, after storage and germination acceleration, the seeds are sown in soil added with an equal amount of matrix, and podophyllum hexandrum seedlings are obtained through culture; fresh leaves of podophyllum hexandrum seedlings are taken as explants, the explants are disinfected and cut into small pieces, the small pieces are cultured on an MS culture medium added with hormone for 30 d to induce callus, the induced callus is cultured and proliferated at the optimal temperature of 15 DEG C, and the callus is inoculated to the same fresh culture medium after being cultured for 20 d to prevent browning, so that the high-quality callus is obtained. And then replacing a fresh culture medium every 20 days to realize rapid and mass proliferation of the podophyllum hexandrum callus. Through the method, a large amount of high-quality podophyllum hexandrum callus can be rapidly obtained, and sufficient materials are provided for podophyllotoxin extraction and medicinal value development.

Description

Technical Field

[0001] The invention relates to a plant tissue culture technology, in particular to a method for rapid proliferation of Psoralea corylifolia callus. Background Art

[0002] Sinopodophyllum hexandrum (Royle) T Ying is a perennial herbaceous plant of the genus Sinopodophyllum in the Berberidaceae family, and its roots and rhizomes are used as medicine. Sinopodophyllum seeds have a long dormancy period and poor germination ability under natural conditions, and are very demanding on the growth environment, resulting in a relatively narrow natural distribution area and weak natural reproduction ability. In addition, due to the destruction of the ecological environment and artificial excavation, the maintenance of its population and the development of biodiversity are severely restricted. It is currently in an endangered state, and traditional cultivation methods can no longer meet the market demand for it, which directly affects the medicinal and economic applications of Sinopodophyllum. Podophyllotoxin is the main medicinal ingredient of Sinopodophyllum. With the reduction of wild resources of Sinopodophyllum, the industrial production of podophyllotoxin extracted by excavating rhizomes has been restricted. Inducing callus tissue and achieving rapid proliferation through plant tissue culture technology can be used as an effective means for Psoralea corylifolia to produce podophyllotoxin. However, due to the high technical difficulty, there are still many problems in the tissue culture research of Psoralea corylifolia, including the long induction and culture time of callus tissue, which is prone to browning and growth stagnation, and the slow proliferation rate of callus tissue, resulting in poor quality of callus tissue and difficulty in meeting the standards for commercial production.

[0003] Therefore, studying and solving the problems of poor quality of Psoralea corylifolia callus, slow growth, poor growth state, unstable proliferation culture, etc. can lay an experimental foundation for realizing large-scale production of Psoralea corylifolia callus. Summary of the invention

[0004] In view of this, the present invention provides a method for rapid proliferation of Psoralea corylifolia callus, which solves the problems of difficult germination of Psoralea corylifolia seeds, scarce tissue culture explant materials, aging and browning of callus, slow growth, unstable subculture, low callus quality, and delayed tissue culture process. A large amount of high-quality Psoralea corylifolia callus can be quickly obtained, providing sufficient materials for podophyllotoxin extraction and medicinal value development.

[0005] A method for rapid proliferation of Psoralea corylifolia callus, characterized by comprising the following steps:

[0006] S1. Breaking the dormancy of Psoralea corylifolia seeds and cultivating seedlings: breaking the dormancy of seeds by sand storage method, soaking the seeds in distilled water and gibberellin solution respectively, and fully mixing with moist river sand that has been washed, sieved and sterilized, and then storing at 4°C for germination; after the seeds germinate, adding an equal amount of substrate to the soil for sowing, and regularly adding Hoagland nutrient solution to ensure normal growth of the plants;

[0007] S2. Explant disinfection: Select fresh Psoralea corylifolia leaves as explants, disinfect the leaves with 75% alcohol for 30 seconds and then with 10% NaClO for 10 minutes;

[0008] S3. Inducing callus tissue of Psoralea corylifolia: Cut the disinfected Psoralea corylifolia leaves into small uniform pieces and inoculate them on 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, and culture at 23°C for 30 days;

[0009] S4. Callus proliferation culture of Psoralea corylifolia: The callus tissue induced by leaves for 30 days was cut into blocks of uniform size and inoculated on MS medium supplemented with different hormones for proliferation, where the hormones included 6-BA 1.0mg / L+NAA 0.5mg / L+GA3 0.1mg / L. The callus tissue was pre-cultured at 23℃ for 2 days to allow the callus tissue to fully adapt to the new medium, and then cultured at 15℃ for 20 days to obtain healthy callus tissue.

[0010] S5. Rapid proliferation of Psoralea corylifolia callus: The callus obtained by culturing for 20 days in S4 was cut into even-sized blocks and inoculated into the same fresh culture medium. The proliferation culture was continued at 15°C. The culture conditions were the same as those in S4. A large amount of callus with good growth status and high quality could be obtained after culturing for 20 days. Thereafter, the rapid and massive proliferation of Psoralea corylifolia callus could be achieved by replacing the fresh culture medium every 20 days.

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

[0012] Preferably, the culture conditions of 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 Psoralea corylifolia are cut into 0.3 cm 2 size.

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

[0015] Preferably, in step S1, the seeds are first soaked in distilled water for 24 hours, the water is changed 2 to 3 times in the middle, so that the seeds can fully absorb water, and then the seeds are soaked in 100 ppm gibberellin solution for 24 hours, the solution is changed 2 to 3 times in the middle, the river sand is washed and passed through a 10-mesh sieve, sterilized in an autoclave (121° C., 30 min), the river sand is wetted with sterile water, and then the peach seed is mixed with the river sand, the surface is covered with river sand, the humidity is 30%, and it is sealed with plastic wrap and perforated for ventilation; the matrix is ​​a mixture of vermiculite, perlite and wood ash in a ratio of 1:1:1.

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

[0017] The beneficial effects are as follows:

[0018] The present invention improves and optimizes the problems of low induction rate, slow growth, severe browning, unstable proliferation culture, etc. of Psoralea corylifolia callus by adjusting the culture medium ratio, changing the culture temperature, and renewing the fresh culture medium, and invents an efficient method for rapid proliferation of Psoralea corylifolia callus, which has a short culture cycle, high culture efficiency, high callus yield, and good quality. The specific performance is:

[0019] (1) Since Psoralea corylifolia seeds have strong dormancy characteristics and are a rare and endangered plant, the traditional method of inducing callus tissue from embryos and hypocotyls is limited by the amount of available materials. The present invention uses leaves as explants, which make the materials easy to obtain and relatively abundant, and breaks the dormancy of Psoralea corylifolia seeds through sand storage. After sowing, a stable supply of explant materials can be maintained, and there is no need to cultivate sterile seedlings. This not only simplifies the tissue culture process, but also enables rapid acquisition of a large amount of high-quality callus tissue, and reduces the complexity and cost of the operation.

[0020] (2) This method screens out the best disinfection method for leaves as explants, and screens out the culture medium hormone ratio with a callus induction rate of up to about 80%. Callus tissue induced for 30 days can be used for proliferation culture, avoiding callus browning and obtaining high-quality callus tissue in a short time. The determined optimal culture condition of 15°C makes the proliferation coefficient of callus tissue higher than other temperature conditions, which is in line with the low-temperature growth characteristics of Psoralea comoides, and temperature induction is conducive to the accumulation of energy substances and the enhancement of the antioxidant system.

[0021] (3) This method is simple to operate, has low equipment requirements, a short cycle, and a high induction efficiency. The method can be used to culture callus tissue to extract podophyllotoxin, alleviate the shortage of wild resources of Psoralea corylifolia, improve the current situation where cultivated resources are difficult to meet market demand, and possibly achieve sustainable drug supply, thereby reducing dependence on wild natural resources of Psoralea corylifolia. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 :Flow chart of efficient and rapid proliferation of Psoralea corylifolia callus;

[0023] Figure 2 : Peach Seven seedlings (a is the seedling grown after sand storage, b and c are the seedlings cultured in matrix soil);

[0024] Figure 3 : Callus induction of Psoralea corylifolia (a is the callus induced by leaves for 10 days, b is the callus induced by leaves for 30 days);

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

[0026] Figure 5 : Growth curves of callus proliferation of Psoralea corylifolia under different temperature conditions. DETAILED DESCRIPTION

[0027] In order to make the purpose, scheme and advantages of the technical solution of the present invention clearer, the technical solution of the embodiment of the present invention will be clearly and completely described below in conjunction with the drawings of specific embodiments of the present invention.

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

[0029] The best embodiment of the present invention is finally obtained through the following specific experiments:

[0030] (1) Breaking the dormancy of Psoralea corylifolia seeds and cultivating seedlings

[0031] The sand storage method is used to break the dormancy of seeds. First, soak the seeds in distilled water for 24 hours, change the water 2 to 3 times in the middle, so that it can fully absorb water, and then soak the seeds in 100ppm gibberellin solution for 24 hours, and change the solution 2 to 3 times in the middle. Wash the river sand and sieve it (10 mesh), sterilize it in an autoclave (121℃, 30min), wet the river sand with sterile water, then mix the peach seven seeds with the river sand, cover the surface with river sand, the humidity is 30%, seal it with plastic wrap, punch holes for ventilation, and store it at 4℃. Select the germinated peach seven seeds, add an equal amount of substrate (vermiculite, perlite and wood ash mixed in a ratio of 1:1:1) to the soil for sowing, and add Hoagland nutrient solution regularly during the period to maintain normal growth of the plants. The peach seven seeds gradually germinate after 120 days of sand storage. After 160 days, 90% of the seeds germinate, and the two cotyledons are separated ( Figure 2 a), the seedlings of Prunus mume transplanted into the matrix soil grew well, but at a slow rate, gradually growing from 2 cotyledons to 3 functional true leaves ( Figure 2 b, c).

[0032] The above measures can shorten the dormancy period of Prunus mume seeds from the normal 10 months to 4 months to produce seedlings, greatly reducing the seedling raising time.

[0033] (2) Determination of explant disinfection time

[0034] The disinfection time of explants may determine whether they are contaminated and affect the activity of Psoralea corylifolia leaves. It is crucial to determine the optimal disinfection time. Select fresh Psoralea corylifolia leaves as explants, first clean the surface impurities with detergent and then rinse with tap water, put them in a clean bench and use sterile filter paper to absorb the surface moisture, rinse with sterile water 3 to 5 times, disinfect the leaves with 75% alcohol for 30 seconds, and then disinfect with 10% NaClO for 5 minutes, 10 minutes, and 15 minutes respectively, and cut into 0.3 cm 2 The size of the inoculated in MS basal medium without hormones (MS 4.74g / L, sucrose 30g / L, agar 7g / L, pH 5.8), observed the contamination of the explants, and the contamination rate was calculated (Formula 1). Among them, disinfection for 5 minutes could not kill the microorganisms on the surface of the explants very well, and there was a large contamination, and the contamination rate reached 67%. After disinfection for 10 minutes, the contamination rate was significantly reduced to only 5%. After disinfection for 15 minutes, there was very little contamination, but some leaves turned white and the activity of the explants was greatly damaged, indicating that the effect of disinfection with NaClO for 5 minutes was not good, and disinfection for 15 minutes may cause cell death. The best disinfection condition for the leaves of Taoerqi was 10% NaClO disinfection for 10 minutes (Table 1).

[0035]

[0036] Table 1 Statistics on the disinfection of Psoralea corylifolia 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 culture medium for inducing callus tissue of Psoralea corylifolia

[0040] Based on the optimal disinfection time determined in (2), the leaves of Psoralea corylifolia were used as explants, disinfected with 75% alcohol for 30 seconds, then disinfected with 10% NaClO for 10 minutes, and cut into 0.3 cm 2 The callus induction rate was the highest in the medium inoculated with MS+6-BA 1.0mg / L+TDZ 0.5mg / L+2,4-D 1.5mg / L, and the growth state was the best. Therefore, this medium was determined to be the best medium for inducing callus in Psoralea corylifolia leaves ( Figure 3 , Table 2).

[0041]

[0042] Table 2 Callus induction of Psoralea corylifolia

[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 Psoralea corylifolia

[0046] The callus tissue induced by leaves for 30 days was cut into 0.5 cm 2 Blocks of uniform size were inoculated on MS medium supplemented with different hormones (6-BA, NAA, GA3, CH), pre-cultured at 23°C for 2 days under a light intensity of 3000lx and a light duration of 14h / d, so that the callus tissue could fully adapt to the new medium, and then cultured at 15°C and 23°C respectively. The proliferation coefficient was calculated after 30 days of culture (Formula 3). Among them, the callus tissue proliferation coefficient inoculated on MS+6-BA 1.0mg / L+NAA 0.5mg / L+GA3 0.1mg / L medium cultured at 15°C was the highest, and the growth state was the best (Table 3). Through this experiment, it was found that temperature would affect callus tissue culture, so it is also necessary to determine the optimal culture temperature.

[0047]

[0048] Table 3 Callus proliferation of Psoralea corylifolia

[0049]

[0050]

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

[0052] (5) Temperature regulation for rapid proliferation of Psoralea corylifolia callus (determination of the most suitable temperature)

[0053] Select the optimal culture medium (4) and cut the callus tissue induced by leaves for 30 days into 0.5 cm 2 Blocks of uniform size were inoculated into MS+6-BA 1.0mg / L+NAA 0.5mg / L+GA3 0.1mg / L proliferation medium and pre-cultured at 23℃ for 2d to allow the callus to fully adapt to the new medium. Five culture temperatures of 10℃, 15℃, 20℃, 23℃ and 25℃ were set (light intensity was 3000lx, light time was 14h / d), and the changing characteristics of callus proliferation culture under different temperature conditions were recorded after 5d, 10d, 20d and 30d.

[0054] ① Callus morphological characteristics

[0055] With the extension of proliferation culture time, the volume of callus cultured at various temperatures gradually increased. Figure 4 :

[0056] Under 10℃, callus tissue began to brown after 10 days of culture, with local green color and a tight texture. Browning was obvious after 20 days of culture, and serious browning and growth stagnation occurred after 30 days of culture.

[0057] The callus cultured at 15℃ increased significantly in volume after inoculation, and its color changed from yellow-white to yellow-green after 10 days. After 20 days, it showed obvious light green and gradually became firm from loose. After 30 days, the light green color became darker, and some callus showed slight browning.

[0058] The callus cultured at 20℃ showed partial green color and soft texture after 10 days of growth, and gradually turned yellow-green and slightly browned after 20 days of culture.

[0059] The callus tissue cultured at 23℃ rapidly increased in size, became soft in texture, and yellowish-white in color. After 20 days, it gradually turned brown and grew slowly, and after 30 days, the growth stopped.

[0060] The callus cultured at 25℃ quickly turned brown, had a soft texture, and was yellow-white in color. After 20 days of culture, obvious growth stagnation was observed.

[0061] ② Growth coefficient

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

[0063] ③ Growth curve

[0064] The fresh weight of callus tissue was measured, and growth curves were drawn to compare the growth dynamics of callus tissue at different temperatures. The growth curves showed that 1-20 days was the period of rapid proliferation of callus tissue, and the growth rate of callus tissue cultured at 15°C was significantly higher than that at other temperatures, indicating that the biological metabolism and cell division activities of callus tissue were the most active, thus promoting its rapid growth. At the same time, it also reflects the importance of temperature control for optimizing growth conditions and improving growth efficiency ( Figure 5 ).

[0065] (6) Rapid proliferation of Psoralea corylifolia callus

[0066] Take the callus tissue cultured at 15°C for 20 days in (5), cut the callus tissue into blocks of uniform size again and inoculate them into the same fresh culture medium (MS+6-BA 1.0mg / L+NAA 0.5mg / L+GA3 0.1mg / L) to prevent browning, and continue to proliferate the callus tissue at 15°C (other culture conditions are the same as above, i.e., pre-culture at 23°C for 2 days to allow the callus tissue to fully adapt to the new culture medium, and then culture again at 15°C, with a light intensity of 3000lx and a light duration of 14h / d for 20 days to obtain a large amount of callus tissue with good growth status and high quality. Thereafter, the fresh culture medium is replaced every 20 days to achieve rapid and large-scale proliferation of the callus tissue of Taoerqi.

[0067] Summary of the best techniques:

[0068] First, soak the seeds in distilled water for 24 hours, change the water 2 to 3 times in the middle to make them fully absorb water, then soak the seeds in 100ppm gibberellin solution for 24 hours, change the solution 2 to 3 times in the middle, wash the river sand through a 10-mesh sieve, sterilize it in an autoclave (121°C, 30min), wet the river sand with sterile water, then mix the peach seven seeds with the river sand, cover the surface with river sand, the humidity is 30%, seal it with plastic wrap, punch holes for ventilation, and then store it at 4°C for germination; after the seeds germinate, add an equal amount of substrate (vermiculite, perlite and wood ash mixed in a ratio of 1:1:1) to the soil for sowing, and regularly add Hoagland nutrient solution to ensure the normal growth of the plant, so as to obtain peach seven leaves;

[0069] Select fresh Prunus persica leaves as explants, clean the surface impurities with detergent and rinse with tap water, put them in a clean bench and use sterile filter paper to absorb the surface moisture, rinse with sterile water 3 to 5 times, then disinfect with 75% alcohol for 30 seconds and then with 10% NaClO for 10 minutes; cut into 0.3 cm 2 The size was inoculated in MS+6-BA 1.0mg / L+TDZ 0.5mg / L+2,4-D 1.5mg / L medium to induce callus tissue (culture conditions: light intensity of 3000lx, light time of 14h / d, culture at 23℃) and cultured for 30 days;

[0070] The callus tissue grown for 30 days was cut into 0.5 cm 2 The callus was inoculated into MS+6-BA 1.0mg / L+NAA 0.5mg / L+GA3 0.1mg / L medium for proliferation culture. It was pre-cultured at 23℃ for 2 days (culture conditions are as above) to allow the callus to adapt to the new culture medium. It was then transferred to 15℃ for 20 days and the callus was cut into 0.5cm pieces again. 2 Blocks of different sizes were inoculated onto the same fresh culture medium to prevent browning. The callus tissue was continued to proliferate and cultured at 15°C (other culture conditions were the same as above). It grew rapidly, and a large amount of callus tissue with good growth state and high quality could be obtained after 20 days of culture. Thereafter, fresh culture medium was replaced every 20 days to achieve rapid and large-scale proliferation of Taoerqi callus tissue.

Claims

1. A method for rapid proliferation of Psoralea corylifolia callus, characterized in that The steps include: S1. Breaking the dormancy of Psoralea corylifolia seeds and cultivating seedlings: breaking the dormancy of seeds by sand storage method, soaking the seeds in distilled water and gibberellin solution respectively, and fully mixing with moist river sand that has been washed, sieved and sterilized, and then storing at 4°C for germination; after the seeds germinate, adding an equal amount of substrate to the soil for sowing, and regularly adding Hoagland nutrient solution to ensure normal growth of the plants; S2. Explant disinfection: Select fresh Psoralea corylifolia leaves as explants, disinfect the leaves with 75% alcohol for 30 seconds and then with 10% NaClO for 10 minutes; S3. Inducing callus tissue of Psoralea corylifolia: Cut the disinfected Psoralea corylifolia leaves into small uniform pieces and inoculate them on 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, and culture at 23°C for 30 days; S4. Callus proliferation culture of Psoralea corylifolia: The callus tissue induced by leaves for 30 days was cut into blocks of uniform size and inoculated on MS medium supplemented with different hormones for proliferation, where the hormones included 6-BA 1.0 mg / L + NAA 0.5 mg / L + GA30.1 mg / L. The callus tissue was pre-cultured at 23℃ for 2 days to allow the callus tissue to fully adapt to the new medium, and then cultured at 15℃ for 20 days to obtain healthy callus tissue. S5. Rapid proliferation of Psoralea corylifolia callus: The callus obtained by culturing for 20 days in S4 was cut into even-sized blocks and inoculated into the same fresh culture medium. The proliferation culture was continued at 15°C. The culture conditions were the same as those in S4. A large amount of callus with good growth status and high quality could be obtained after culturing for 20 days. Thereafter, the rapid and massive proliferation of Psoralea corylifolia callus could be achieved by replacing the fresh culture medium every 20 days.

2. The method for rapid proliferation of Psoralea corylifolia callus according to claim 1, characterized in that: The MS culture medium formula is 4.74 g / L MS, 30 g / L sucrose, 7 g / L agar, and the pH is adjusted to 5.

8.

3. The method for rapid proliferation of Psoralea corylifolia callus according to claim 2, characterized in that: The culture conditions of 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 Psoralea corylifolia callus according to claim 3, characterized in that: In step S3, the leaves of the peach tree are cut into 0.3 cm 2 size.

5. The method for rapid proliferation of Psoralea corylifolia callus according to claim 3, characterized in that: In steps S4 and S5, the callus tissue was cut into 0.5 cm 2 size.

6. The method for rapid proliferation of Psoralea corylifolia callus according to claim 1, characterized in that: In step S1, the seeds are first soaked in distilled water for 24 hours, the water is changed 2 to 3 times in the middle, so that the seeds can fully absorb water, and then the seeds are soaked in 100ppm gibberellin solution for 24 hours, the solution is changed 2 to 3 times in the middle, the river sand is washed and passed through a 10-mesh sieve, sterilized in an autoclave (121°C, 30min), the river sand is wetted with sterile water, and then the peach seven seeds are mixed with the river sand, the surface is covered with river sand, the humidity is 30%, and it is sealed with plastic wrap, and holes are punched for ventilation; the matrix is ​​a mixture of vermiculite, perlite and wood ash in a ratio of 1:1:

1.

7. The method for rapid proliferation of Psoralea corylifolia callus according to claim 1, characterized in that: In step S2, before disinfecting the leaves, the surface impurities are first cleaned with detergent and then rinsed with tap water, placed in a clean bench, and the surface moisture is absorbed with sterile filter paper, and then rinsed with sterile water for 3 to 5 times.

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