A method for in vitro culture of ormosia japonica seed embryo and aseptic rapid propagation of tissue culture seedlings

Abstract

The present application relates to the technical field of leaflet red bean rapid propagation, and particularly relates to a method for in-vitro culture of seed embryo of leaflet red bean and aseptic rapid propagation of tissue culture seedlings, which comprises the following steps: S1: soaking leaflet red bean seeds in concentrated hydrochloric acid, ethanol solution and hypochlorous acid in sequence for disinfection and softening of seed coat, then soaking in water for swelling, peeling off the softened seed coat to obtain peeled seeds; S2: soaking the peeled seeds in a mixed solution of GA3 and mannitol to obtain explant embryos; S3: inoculating the explant embryos into an induction medium for seed germination culture to obtain seedlings with roots; and S4: inoculating the seedlings with roots into a strong seedling culture medium for strong seedling culture to obtain tissue culture seedlings. The present application can achieve higher germination rate of seed embryo, better root development of tissue culture seedlings, better seedling growth and higher survival rate of transplanted seedlings by deep disinfection, removal of seed coat and breaking of dormancy, and applying corresponding growth hormones according to the growth stage of leaflet red bean.

Description

Technical Field

[0001] This invention relates to the field of rapid propagation technology of small-leaf red bean, specifically a method for in vitro culture of small-leaf red bean embryos and aseptic rapid propagation of tissue culture seedlings. Background Technology

[0002] Small-leaf red bean ( Ormosia microphylla It has a narrow suitable habitat, requiring warm, humid, well-drained, humus-rich acidic soil. Its natural regeneration capacity is poor, its seeds exhibit dormancy, and its natural germination rate is extremely low. Under the dual pressures of long-term over-logging and severe fragmentation of its native habitat, actively promoting its resource conservation and artificial cultivation technology research is urgently needed.

[0003] The seeds of the small-leaved red bean are flat and oval-shaped, with a hard seed coat and a waxy layer on the surface. They have poor water permeability and are not easy to absorb water and germinate. Without special germination treatment, it is difficult to start the germination process. Under natural conditions, the seeds germinate unevenly, and some seeds need to go through a dormancy period of up to 2 years in the soil before they can germinate. In addition, the seeds are easily eaten by rodents and birds, which further exacerbates the difficulty of natural population regeneration.

[0004] Currently, the propagation of small-leaved red beans mainly relies on two methods: sowing and cutting. However, neither of these methods can meet the needs of large-scale seedling propagation and population restoration. Tissue culture technology has the characteristics of extremely fast propagation speed, high efficiency, and preservation of the excellent shape of the parent plant, making it key to rapid plant propagation. However, tissue culture technology requires a sterile environment, and tissue-cultured seedlings are difficult to root and transplant. Therefore, current tissue culture methods for small-leaved red beans suffer from a series of problems, including high contamination rates, low seed germination rates, and long rooting times.

[0005] Chinese patent CN116548312A discloses a rapid propagation culture medium and rapid propagation method for small-leaved red beans. However, the seed coat removal method used is relatively crude, resulting in a high contamination rate and a low seed germination rate. Furthermore, the lack of proper seed awakening leads to a long overall cultivation cycle.

[0006] In summary, there is an urgent need for a tissue culture rapid propagation technology for small-leaved red bean varieties with high seed germination rate, low pollution rate, and short cultivation cycle. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for in vitro culture of small-leaved red bean embryos and aseptic rapid propagation of tissue culture seedlings to achieve at least the effects of high seed germination rate, low contamination rate and short cultivation cycle.

[0008] The objective of this invention is achieved through the following technical solution: A method for in vitro culture of small-leaved red bean embryos includes the following steps: S1: Soak the small-leaved red bean seeds in concentrated hydrochloric acid, ethanol solution and hypochlorous acid in turn to disinfect and soften the seed coat. Then soak them in water to absorb the swelling, peel off the softened seed coat to obtain peeled seeds. S2: The peeled seeds are soaked in a mixed solution of GA3 and mannitol to obtain explant embryos; S3: The explant embryos are inoculated into an induction medium for seed germination culture to obtain rooted seedlings; S4: The rooted seedlings are inoculated into a seedling strengthening culture medium for seedling strengthening culture to obtain tissue culture seedlings; The induction culture medium includes the hormone IBA; The seedling culture medium includes hormones 6-BA, IAA, and KT.

[0009] In some embodiments, the induction medium is based on MS medium, comprising 0.9-1.1 mg / L IBA, 28-32 g / L sucrose and 7-8 g / L agar, with a pH of 5.7-5.9; The seedling culture medium is based on MS medium, which includes 0.9~1.1 mg / L 6-BA, 0.07~0.09 mg / L IAA, 0.08~0.10 mg / L KT, 28~32 g / L sucrose and 7~8 g / L agar, with a pH of 5.7~5.9.

[0010] In some embodiments, in step S1, the concentration of the concentrated hydrochloric acid is 36-38 wt%; the concentration of the ethanol solution is 3-5 wt%; and the concentration of the hypochlorous acid is 5-15 wt%. The soaking time in concentrated hydrochloric acid is 1-2 hours; the soaking time in ethanol solution is 30 seconds; the soaking time in hypochlorous acid is 5-15 minutes; and the soaking and swelling time in water is 24 hours at a water temperature of 20-25°C.

[0011] In some examples, the seed coat surface needs to be rinsed with water after each soaking to avoid pesticide residue.

[0012] It is worth noting that this invention first uses concentrated hydrochloric acid for soaking, utilizing the strong corrosiveness and dehydrating properties of concentrated hydrochloric acid to kill most of the microorganisms attached to the material surface. After washing, it is then rapidly soaked in alcohol to further kill bacteria, fungi, and other microorganisms on the material surface, quickly completing preliminary surface sterilization. After washing, it is finally soaked in sodium hypochlorite solution. Sodium hypochlorite can efficiently destroy the nucleic acid and enzyme systems of microorganisms, achieving deep sterilization. Through these three steps, various microorganisms attached to the seeds are effectively eliminated, reducing the possibility of contamination during tissue culture. Furthermore, the fibrous structure of the seed coat is destroyed, softening the seed coat, allowing for rapid imbibition to peel off the seed coat without damaging the embryo.

[0013] In some embodiments, in step S3, the length of the rooted seedling is 5-10 cm; In step S4, the tissue culture seedlings are 10-15cm long, have 5-15 leaves, each leaf is 3-4cm long, and have 5-10 normal roots.

[0014] In some embodiments, in step S3, the seed germination culture is as follows: cultured at a temperature of 23±2℃ for 9 to 19 days, with the first 4 days in the dark, and the 5th to 15th days under light for 11 to 12 hours per day, with a light intensity of 1000 to 1500 lx. In step S4, the seedling cultivation is carried out by culturing at a temperature of 23±2℃ for 15 days, with 11~12 hours of light per day and a light intensity of 1500~2000lx.

[0015] In some embodiments, in step S2, the concentration of mannitol is 100-150 mmol / L; the concentration of GA3 is 100-300 mmol / L; and the soaking time is 4-5 h.

[0016] It is worth noting that even after the seed coat is removed, small-leaved red bean seeds remain in a state of physiological dormancy, and the degree of dormancy varies among seeds. Direct tissue culture at this stage not only results in a long cultivation time but also leads to uneven seedling development, making uniform management difficult. Therefore, this invention uses a mixed solution of gibberellin GA3 and mannitol as a dormancy-breaking solution to awaken the embryos of small-leaved red beans. GA3 inhibits the expression of the endogenous hormone ABA in the seed, breaking the physiological inhibition of germination within the small-leaved red bean seed. Since there is no seed coat barrier, the absorption efficiency is higher, and the embryo can rapidly adjust its internal osmotic pressure through the mannitol solution, achieving an osmotic initiation effect, activating the seed's physiological activity, breaking dormancy in a short time, and improving overall cultivation efficiency. Normally, dormancy breaking involves directly immersing the intact seeds in the dormancy-breaking solution. This method preserves the natural protective function of the seed coat to the greatest extent, preventing direct contact and corrosion of the delicate embryo by the reagents, and reducing the risk of contamination. Generally, dormancy breaking and osmotic initiation regulation take several days, while hormone regulation takes more than ten hours. The present invention discovers that after sterile peeling of small-leaf red beans, simultaneous awakening with GA3 and mannitol can break dormancy and start germination culture within 4-5 hours. This not only does not affect the germination of small-leaf red bean embryos, but also greatly shortens the seedling time and reduces the contamination rate.

[0017] A method for aseptic rapid propagation of small-leaved red bean seedlings via tissue culture includes the following steps: S4: Transplant the tissue culture seedlings to a hardening culture medium for hardening, and then transplant the tissue culture seedlings to a soil substrate for cultivation.

[0018] In some embodiments, the seedling hardening culture medium includes peat, vermiculite, and tree chips; the volume ratio of the peat, vermiculite, and tree chips is 4~5:2~3:4~5.

[0019] In some embodiments, the hardening-off process involves: hardening off the seedlings for 5 days in an airtight, sterile environment under natural light conditions, and then hardening off the seedlings for 2 days in a sterile environment.

[0020] In some embodiments, the culture conditions are: temperature 23±2℃, light duration 11~12h / d, and light intensity 1500~2000lx.

[0021] The beneficial effects of this invention are: 1. This invention uses a triple treatment of concentrated hydrochloric acid, ethanol solution and hypochlorous acid to deeply kill bacteria, fungi and other microorganisms carried by small-leaf red bean seeds, reduce the contamination rate of tissue culture, and soften the seed coat so that the seed coat can be peeled off after a simple soaking treatment.

[0022] 2. This invention uses a mixed solution of GA3 and mannitol to soak the seed embryos after removing the seed coat. Through a dual mechanism of inhibiting the expression of dormant hormones from the inside and regulating osmotic pressure and water absorption rate from the outside, the dormancy state is broken, and the embryos that were originally in different dormant states are uniformly activated into the germination state. This not only shortens the cultivation time, but also avoids the problem of inconsistent development state of subsequent tissue culture seedlings, which makes it difficult to unify the time of seedling release, thus facilitating large-scale production.

[0023] 3. This invention first uses hormones containing a regulating and inducing culture medium and a seedling strengthening culture medium to make the tissue culture process conform to the natural growth law of small-leaf red bean, which is "roots first and seedlings later". Compared with the simultaneous induction of adventitious buds and differentiation of cluster buds, the germination rate of seed embryos is higher, the root development of tissue culture seedlings is better, the seedling vigor is better, and the transplant survival rate is higher.

[0024] 4. This invention obtains embryos after disinfection, seed coat removal, and dormancy breaking. These embryos undergo primary induction culture, germinating into robust tissue-cultured seedlings with roots and apical buds within 10-14 days. After another 15 days of intensive seedling cultivation, 1-3 buds proliferate, forming lateral branches; the number of leaves increases, averaging 10-13 per plant; the number of fibrous roots increases to 10-20, and the root system is relatively long; the seedlings reach a height of 10-15 cm and exhibit good growth. After hardening off and transplanting, the overall survival rate exceeds 98%. This method produces tissue-cultured seedlings with short germination time, rapid proliferation, high survival rate after transplanting, and robust, upright, and uniformly growing seedlings. This invention provides technical support for research on efficient propagation technology and artificial cultivation techniques for small-leaved red bean seedlings, as well as for the effective protection and management of precious tree species. It has significant theoretical importance and high economic value and development potential. Attached Figure Description

[0025] Figure 1 These are images of the soaking and swelling process of small-leaf red bean seeds in the experimental examples of this invention; Figure 2 The image shows a mixed solution of GA3 and mannitol in an embodiment of the present invention. The seeds in the image are seeds that have been absorbing water but have not yet been peeled. In actual operation, the seeds are peeled before being placed in the mixed solution. Figure 3 This is a picture taken on day 4 of the explant embryogenic rooting induction culture in this embodiment of the invention; Figure 4 This is a picture taken on day 7 of the explant embryogenic rooting induction culture in this embodiment of the invention; Figure 5 This is a picture taken on day 10 of the explant embryogenic rooting induction culture in this embodiment of the invention; Figure 6 This is an image of a tissue culture seedling in an embodiment of the present invention. Detailed Implementation

[0026] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings, but the scope of protection of the present invention is not limited to the following description.

[0027] The test materials used in the following examples and comparative examples are naturally matured seeds. The seeds are selected from the soil, which are plump, free from pests and diseases and rot. They are brought back to the laboratory, rinsed with running water to remove surface impurities, and then air-dried naturally.

[0028] Example This embodiment provides a method for in vitro embryo culture and aseptic rapid propagation of tissue culture seedlings from small-leaved red beans. The specific method is as follows: 1) Prepare culture media: Prepare induction medium, seedling strengthening medium and seedling hardening substrate separately. The pH value of these media is 5.7~5.9 and the thickness of the media is 1.5~2.0cm.

[0029] The induction medium consisted of MS medium containing 1.0 mg / L IBA, 30 g / L sucrose, and 7.5 g / L agar.

[0030] Seedling growth medium: MS + 1.0 mg / L 6-BA + 0.08 mg / L IAA + 0.09 mg / L KT + 30 g / L sucrose + 7.5 g / L agar.

[0031] Hardening culture medium: peat, vermiculite, and tree chips in a volume ratio of 4:2:4.

[0032] 2) Material disinfection and seed coat removal: Completely immerse 200 seeds in 12 mol / L concentrated hydrochloric acid for 1 hour in a well-ventilated environment. After removal, rinse continuously under running water for 30 minutes to thoroughly remove residual sulfuric acid. Then, soak the seeds in 75 wt% alcohol for 30 seconds, followed by rinsing three times with sterile water, agitating thoroughly each time to remove residual alcohol. Next, soak the seeds in a 10% sodium hypochlorite solution for 5 minutes, then rinse three more times with sterile water to thoroughly remove any sodium hypochlorite residue. Gently blot the seeds dry with pre-sterilized filter paper, place them on sterilized clean paper, and soak in sterile water at 25°C for 24 hours. Figure 1 As shown, allow it to fully swell. Finally, in a sterile operating table, carefully peel off the outer seed coat using sterile forceps.

[0033] 3) Breaking dormancy: Prepare a mixed solution of GA3 and mannitol, where the concentration of GA3 is 200 mmol / L and the concentration of mannitol is 120 mmol / L. Figure 2 As shown. Peeled seeds were soaked in the above mixed solution for 4 hours, then rinsed three times with sterile water and dried with filter paper to obtain explant embryos.

[0034] 3) Rooting induction culture: The explant embryos are evenly inoculated onto the pre-sterilized induction medium at a density of one explant embryo per 200g of induction medium. The culture room temperature was 23±2℃, and dark incubation was used for the first 4 days. Figure 3 As shown, for the next 10 days, the daily light duration was 12 hours, and the light intensity was 1500 lx, resulting in rooted seedlings, as shown. Figures 4-5 As shown, the rooted seedlings have similar growth rates, with seedling lengths ranging from 5 to 10 cm.

[0035] 4) Seedling Strengthening Culture: Rooted seedlings are inoculated into a seedling strengthening culture medium, one seedling per culture bottle. The culture room temperature is 23±2℃, the light duration is 12h / d, the light intensity is 1800lx, and the seedling strengthening culture time is 15 days, yielding tissue culture seedlings. Figure 6 As shown, the tissue culture seedlings have similar growth, with a length of 10-15cm, 5-15 leaves, leaves 3-4cm long, and 5-10 normal roots.

[0036] 5) Hardening off and transplanting: Place the tissue culture seedlings in natural light, seal the culture bottle for 5 days to harden off, and then open the bottle cap for 2 days to harden off.

[0037] Next, the tissue culture seedlings were gradually removed from the culture bottle using gun-shaped forceps, and the culture medium attached to the roots was washed away. They were then inoculated into sterilized soil substrate and covered with a glass cover with two pores for continued cultivation. The cultivation conditions were: the temperature in the culture room was 23±2℃, the light duration was 12h / d, the light intensity was 2000lx, and the observation period was 7 days. After 7 days, the survival rate was 98.5%.

[0038] Comparative Example 1 This comparative example is used to select the optimal conditions for material disinfection and seed coat removal, as follows: The seeds to be treated were divided into 12 groups (A1-A12), with 50 seeds in each group. They were treated according to the method shown in Table 1, except for the soaking solution and soaking time. All other operational details were the same as in the previous example. The optimal treatment method was selected based on the germination status of the explant embryos and the contamination rate (visual inspection) during subsequent culture. The results are shown in Table 2. Table 1 Different seed treatment methods

[0039] Table 2 Germination rate and contamination rate of explant embryos

[0040] The results showed that 1-2 hours of concentrated hydrochloric acid corrosion effectively softened the seed coat of small-leaved red beans, with 1 hour being more effective than 2 hours. The 2-hour treatment resulted in an excessively thin seed coat after corrosion, and the prolonged sodium hypochlorite soaking could damage the seeds, preventing germination. Disinfection method A1 resulted in the fastest seed germination, achieving a 100% germination rate, and the germinated aseptic seedlings were in good condition with rapid rooting and sprouting. Treatment method A3 damaged the seeds, causing them to lose activity, with a germination rate of only 50%. Treatment methods A7 and A12 failed to achieve effective disinfection and soften the seed coat, resulting in high contamination rates and difficulty in removing the seed coat, easily damaging the embryo during the process.

[0041] Comparative Example 2 This comparative example is used to select the optimal culture medium conditions, as follows: The seeds to be treated were divided into 6 groups (B1-B6), with 200 seeds in each group. The culture medium was prepared according to the table in Table 3. Except for the hormones added to the culture medium, the other operational details were the same as in the example. Based on the survival rate of the tissue culture seedlings and the cultivation time (the cultivation time is the time when more than 90% of the tissue culture seedlings reach the standard of step 4), if the standard is not met, the seedlings were continued to be cultivated for 5 days until the standard was met (not included in the seedling hardening time), the optimal culture medium combination was screened. The results are shown in Table 4. Table 3 Different culture medium treatments

[0042] Table 4. Survival rate and cultivation time of tissue culture seedlings

[0043] Comparative Example 3 This comparative example is used to select the optimal reagent for breaking dormancy, as follows: The seeds to be treated were divided into three groups (C1-C6), with 200 seeds in each group. The dormancy-breaking solution was prepared as shown in Table 5. Except for the dormancy-breaking solution used, the other operational details were the same as in the previous example. Based on the survival rate of the tissue culture seedlings and the cultivation time (the cultivation time is the time when more than 90% of the tissue culture seedlings reach the standard of step 4), if the standard is not met, the seedlings were continued to be cultivated for 5 days until the standard was met (not included in the seedling hardening time). The optimal culture medium combination was screened, and the results are shown in Table 6. Table 5 Different dormancy-breaking solutions

[0044] Table 6 Survival rate and cultivation time of tissue culture seedlings

[0045] Comparative Example 4 This comparative example is used to select the optimal time to break dormancy, as follows: The seeds to be treated were divided into 12 groups (D1-D12), with 50 seeds in each group. The dormancy-breaking solution was prepared as shown in Table 7, and different soaking times were used. In addition, the seeds in groups D1-D3 were soaked after the seed coat was removed, while the seeds in groups D4-D12 were soaked without the seed coat removal treatment. The remaining operational details are the same as in the example below: 1) Wash the seeds (200 seeds) with water and soak them in a mixed solution of GA3 and mannitol, where the concentration of GA3 is 200 mmol / L and the concentration of mannitol is 120 mmol / L. Soak the seeds in the above mixed solution for a period of time. After soaking, remove the seeds and rinse them three times with sterile water. Then, blot the water dry with filter paper.

[0046] 2) Completely immerse the seeds in 12 mol / L concentrated hydrochloric acid for 1 hour in a well-ventilated environment. Afterward, rinse continuously under running water for 30 minutes to thoroughly remove residual sulfuric acid. Then, soak the seeds in 75 wt% alcohol for 30 seconds, followed by rinsing three times with sterile water, agitating thoroughly each time to remove residual alcohol. Next, soak the seeds in a 10% sodium hypochlorite solution for 5 minutes, then rinse three times again with sterile water to thoroughly remove any sodium hypochlorite residue. Gently blot the seeds dry with pre-sterilized filter paper, place them on sterilized clean paper, and soak in sterile water at 25°C for 24 hours. Figure 1As shown, allow it to fully absorb and swell. Finally, in a sterile operating table, carefully peel off the outer seed coat using sterile forceps to obtain the explant embryo.

[0047] The explant embryos obtained from each group were subjected to rooting induction culture, and the germination rate and contamination rate were recorded, as shown in Table 8. Table 7 Different dormancy breakout time points

[0048] Table 8 Germination rate and contamination rate

[0049] As shown in Table 8, breaking dormancy after seed coat removal is significantly more efficient than breaking dormancy directly on seeds with the coat intact. This is clearly because direct contact between the embryo and the dormancy-breaking solution greatly accelerates hormone and osmotic pressure regulation, shortening the time required to break dormancy. Furthermore, breaking dormancy before disinfection and peeling is not only inefficient and ineffective, but prolonged soaking can also lead to increased contamination and reduced germination rates. This is likely because prolonged soaking before disinfection softens and damages the seed coat, allowing bacteria carried by the seed to enter the embryo and contaminate the seed.

[0050] The above description is merely a preferred embodiment of the present invention. It should be understood that the present invention is not limited to the forms disclosed herein and should not be construed as excluding other embodiments. It can be used in various other combinations, modifications, and environments, and can be altered within the scope of the concept described herein through the above teachings or related technologies or knowledge. Modifications and variations made by those skilled in the art that do not depart from the spirit and scope of the present invention should be within the protection scope of the appended claims.

Claims

1. A method for in vitro culture of small-leaved red bean embryos, characterized in that, Includes the following steps: S1: Soak the small-leaved red bean seeds in concentrated hydrochloric acid, ethanol solution and hypochlorous acid in turn to disinfect and soften the seed coat. Then soak them in water to absorb the swelling, peel off the softened seed coat to obtain peeled seeds. S2: The peeled seeds are soaked in a mixed solution of GA3 and mannitol to obtain explant embryos; S3: The explant embryos are inoculated into an induction medium for seed germination culture to obtain rooted seedlings; S4: The rooted seedlings are inoculated into a seedling strengthening culture medium for seedling strengthening culture to obtain tissue culture seedlings; The induction culture medium includes the hormone IBA; The seedling culture medium includes hormones 6-BA, IAA, and KT.

2. The method for in vitro culture of small-leaved red bean embryos according to claim 1, characterized in that: The induction medium is based on MS medium, comprising 0.9-1.1 mg / L IBA, 28-32 g / L sucrose, and 7-8 g / L agar, with a pH of 5.7-5.9; The seedling culture medium is based on MS medium, which includes 0.9~1.1 mg / L 6-BA, 0.07~0.09 mg / L IIAAA, 0.08~0.10 mg / L KT, 28~32 g / L sucrose and 7~8 g / L agar, with a pH of 5.7~5.

9.

3. The method for in vitro culture of small-leaved red bean embryos according to claim 1, characterized in that: In step S1, the concentration of the concentrated hydrochloric acid is 36-38 wt%; the concentration of the ethanol solution is 3-5 wt%; and the concentration of the hypochlorous acid is 5-15 wt%.

4. The method for in vitro culture of small-leaved red bean embryos according to claim 3, characterized in that: The soaking time in concentrated hydrochloric acid is 1-2 hours; the soaking time in ethanol solution is 30 seconds; the soaking time in hypochlorous acid is 5-15 minutes; and the soaking and swelling time in water is 24 hours at a water temperature of 20-25°C.

5. The method for in vitro culture of small-leaved red bean embryos according to claim 1, characterized in that: In step S3, the length of the rooted seedling is 5-10 cm; In step S4, the tissue culture seedlings are 10-15cm long, have 5-15 leaves, each leaf is 3-4cm long, and have 5-10 normal roots.

6. The method for in vitro culture of small-leaved red bean embryos according to claim 5, characterized in that: In step S3, the seed germination culture is carried out at a temperature of 23±2℃ for 9 to 19 days, with the first 4 days in the dark and the 5th to 15th days under light for 11 to 12 hours per day, with a light intensity of 1000 to 1500 lx. In step S4, the seedling cultivation is carried out by culturing at a temperature of 23±2℃ for 15 days, with 11~12 hours of light per day and a light intensity of 1500~2000lx.

7. The method for in vitro culture of small-leaved red bean embryos according to claim 1, characterized in that: In step S2, the concentration of mannitol is 100-150 mmol / L; the concentration of GA3 is 100-300 mmol / L; and the soaking time is 4-5 h.

8. A method for aseptic rapid propagation of small-leaved red bean seedlings via tissue culture, wherein the tissue culture seedlings are obtained by the in vitro embryo culture method of small-leaved red bean seed as described in any one of claims 1-5, characterized in that... Includes the following steps: S4: Transplant the tissue culture seedlings to a hardening culture medium for hardening, and then transplant the tissue culture seedlings to a soil substrate for cultivation.

9. The method for aseptic rapid propagation of small-leaved red bean seedlings by tissue culture according to claim 8, characterized in that: The seedling hardening culture medium includes peat, vermiculite, and tree chips; the volume ratio of the peat, vermiculite, and tree chips is 4~5:2~3:4~5.

10. The method for aseptic rapid propagation of small-leaved red bean seedlings by tissue culture according to claim 8, characterized in that, The seedling hardening process involves: first hardening the seedlings in an airtight, sterile environment for 5 days under natural light conditions, and then hardening them in a sterile environment for 2 days. The cultivation conditions were: temperature 23±2℃, light duration 11~12h / d, and light intensity 1500~2000lx.

Images