Anti-browning tissue culture rapid propagation method of lotus

Abstract

The invention belongs to the technical field of plant tissue culture and rapid propagation, and particularly relates to an anti-browning tissue culture and rapid propagation method for lotus. The method comprises the following steps: culturing a lotus explant by adopting a solid-liquid double-layer culture medium, covering the lotus explant with an aseptic substance (hydrophilic non-woven fabric or sterile degreasing cotton), carrying out dark culture, removing the aseptic substance, and carrying out light culture to form a stem seedling. After the aseptic absorbs the liquid culture solution, a barrier similar to liquid seal can be formed on the periphery of the lotus explant, and a small amount of phenolic substances generated on the surface of the lotus explant can be adsorbed by the aseptic and gradually permeate to the outer surface of the aseptic or diluted into the liquid culture solution, so that the accumulation of phenolic and quinones substances on the explant is reduced, and the growth of the lotus explant is promoted. According to the method disclosed by the invention, the browning lethal in the tissue culture process is successfully reduced, the seedling rate is improved, robust stem-walking sterile seedlings are obtained after explant culture and proliferation, the propagation coefficient of each subculture can reach 5.0 or above, the period is short, the propagation efficiency is high, the survival rate after transplanting is high, the growth vigor is vigorous, and a new way is provided for large-scale production of lotus seedlings.

Description

Technical Field

[0001] The invention belongs to the technical field of plant tissue culture and rapid propagation, and particularly relates to a browning-proof tissue culture and rapid propagation method for lotus. Background Art

[0002] Lotus (Nelumbo nucifera Gaertn.) is an aquatic herbaceous plant of the genus Nelumbo of the family Nelumbo. It has two propagation methods: seed propagation and lotus root propagation. Lotus root propagation is usually adopted in production, but this method has a low propagation coefficient, a long cycle, a large amount of seeds, and it is time-consuming and labor-intensive to dig lotus root seeds, and the cost is high. In addition, long-term lotus root seed propagation is prone to germplasm degradation due to the accumulation of diseases, resulting in a decrease in yield and quality. Lotus has hermaphroditic flowers, which are characterized by the pistil maturing first and the stamen maturing later. The pistil has matured 2 to 3 days before flowering and has the ability to pollinate. On the first day of flowering, the stamen anthers cannot be released. It is a plant that mainly cross-pollinates. The offspring often show the phenomenon of trait separation through seed propagation. Therefore, conventional seedling (lotus root) breeding is a bottleneck problem restricting the development of lotus planting industry.

[0003] Tissue culture, also known as in vitro culture, refers to the technology of separating tissues, organs or cells, protoplasts, etc. that meet the needs from plants, and cultivating them under artificial controlled conditions through aseptic operation to obtain regenerated complete plants or produce other products with economic value. The rapid propagation technology of lotus using tissue culture has a high propagation coefficient, low cost, and can cultivate virus-free seedlings. It is the key research direction of today's scientific workers. However, lotus roots are rich in phenolic substances. When lotus roots or lotus buds are mechanically or chemically damaged, enzymatic browning will occur. The quinone oxides produced by browning will inhibit the activity of other enzymes, leading to metabolic dysfunction, thereby affecting the success rate of tissue culture.

[0004] At present, the main measures for preventing and controlling browning in tissue culture are: (1) Selecting suitable explants and optimal culture conditions. (2) Continuous transfer. For materials that are prone to browning, the bottles can be transferred after 2 to 24 hours of culture, and the treatment can be continued several times. (3) Use antioxidants. Add antioxidants such as ascorbic acid and L-aspartic acid to the culture medium. (4) Add adsorbents. Add 0.1% to 0.5% of activated carbon and other adsorbents. In the production operation process, after adopting one or more of the above measures, although there is a certain effect, it is not ideal, the anti-browning effect is not good, the success rate is low, and the experimental repeatability is poor. Therefore, it is necessary to provide a method for rapid propagation of tissue culture to solve the current problems of long lotus seedling propagation cycle and low efficiency. Summary of the invention

[0005] The purpose of the invention is to provide a method for rapid propagation of lotus by tissue culture to prevent browning, effectively solve the problems of high browning mortality of explants, weak growth potential, low proliferation efficiency, etc. in the existing lotus tissue culture technology, significantly reduce the browning mortality in the lotus tissue culture process, improve the survival rate of lotus tissue culture seedlings, make the tissue culture seedlings grow vigorously, have a high reproduction coefficient, and reduce the reproduction cycle.

[0006] The present invention provides a method for rapid propagation of lotus by tissue culture to prevent browning, comprising the following steps:

[0007] Inoculating the lotus explants into a solid culture medium so that 1 / 3 to 1 / 2 of the height of the lotus explants are inserted into the solid culture medium; adding a liquid culture medium onto the solid culture medium so that the liquid culture medium submerges the lotus explants, covering the lotus explants with sterile material, removing the sterile material after dark culture, and then subjecting the lotus explants to light culture to obtain stem-running sterile seedlings;

[0008] The sterile material includes sterile hydrophilic non-woven fabric or sterile absorbent cotton;

[0009] The solid culture medium is based on MS and includes 0.5-2.0 mg / L 6-BA, 0.5-2.0 mg / L NAA, 30-80 g / L sucrose, 6-8 g / L agar, and 0.3-0.8 g / L activated carbon;

[0010] The liquid culture medium uses MS as the basic culture medium and includes 0.5-2.0 mg / L of 6-BA and 0.5-2.0 mg / L of NAA.

[0011] Preferably, the lotus explants include lotus buds and / or lotus cores.

[0012] Preferably, the specification of the sterile hydrophilic nonwoven fabric is 80-160 g / m 2 .

[0013] Preferably, the dark culture time is 15 to 20 days; the dark culture temperature is 24 to 26°C.

[0014] Preferably, the light intensity of the light culture is 4000-6000 lx; ​​the temperature of the light culture is 25-28°C.

[0015] Preferably, when the runner sterile seedlings grow to ≥10 nodes, the runner sterile seedlings are cut into 1-2 nodes to obtain newly cut lotus explants; the new lotus explants are subcultured to obtain a large number of runner sterile seedlings;

[0016] The subculture method comprises: inoculating the newly cut lotus explants into a solid culture medium, pouring a liquid culture medium into the solid culture medium, making the liquid culture medium submerge 1 / 2 to 2 / 3 of the lotus explants, and performing subculture;

[0017] The solid culture medium is based on MS and includes 0.5-2.0 mg / L 6-BA, 0.5-2.0 mg / L NAA, 30-80 g / L sucrose, 6-8 g / L agar, and 0.3-0.8 g / L activated carbon;

[0018] The liquid culture medium uses MS as the basic culture medium and includes 0.5-2.0 mg / L 6-BA and 0.5-2.0 mg / L NAA.

[0019] Preferably, the illumination intensity of the subculture is 4000-6000 lx and the temperature is 25-28°C.

[0020] Preferably, 1 / 3 to 2 / 3 of the newly cut lotus explants are embedded in the solid culture medium.

[0021] Preferably, after obtaining the sterile runner seedlings, the method further comprises: hardening and acclimatizing the sterile runner seedlings and then transplanting them.

[0022] Preferably, the time for hardening and acclimatization of the seedlings is 30 to 45 days.

[0023] Beneficial effects:

[0024] The invention provides a lotus anti-browning tissue culture rapid propagation method, which comprises the following steps: inoculating a lotus explant into a solid culture medium, so that the lotus explant with a height of 1 / 3 to 1 / 2 is inserted into the solid culture medium; adding a liquid culture medium onto the solid culture medium, so that the liquid culture medium submerges the lotus explant, covering the lotus explant with sterile material, removing the sterile material after dark culture, and then obtaining a running stem sterile seedling through light culture; the sterile material comprises a sterile hydrophilic non-woven fabric or a sterile absorbent cotton; the solid culture medium uses MS as a basic culture medium, and comprises 0.5 to 2.0 mg / L of 6-BA, 0.5 to 2.0 mg / L of NAA, 30 to 80 g / L of sucrose, 6 to 8 g / L of agar, and 0.3 to 0.8 g / L of activated carbon; and the liquid culture medium uses MS as a basic culture medium, and comprises 0.5 to 2.0 mg / L of 6-BA and 0.5 to 2.0 mg / L of NAA. After the lotus explant is inoculated, the covered sterile material absorbs the liquid culture solution to form a "liquid-sealed" barrier. Meanwhile, a small amount of phenolic substances generated on the surface of the lotus explant can be adsorbed by the sterile material, gradually seep into the outer surface of the lotus explant or be diluted into the liquid culture solution, thereby reducing the aggregation of phenolic and quinone substances on the explant, successfully reducing the browning in the tissue culture process, and improving the seedling rate. The cultured stem-running sterile seedlings grow healthily, the propagation coefficient of each subculture can reach more than 5.0, the cycle is short, the propagation efficiency is high, the survival rate after transplanting is high, and the growth is vigorous, thereby providing a new way for the large-scale production of lotus seedlings. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0026] Figure 1 This is a physical picture of the three groups of inoculated explants treated in Example 1;

[0027] Figure 2 This is a physical picture of 4 groups of inoculated explants after treatment in Example 1;

[0028] Figure 3 This is a physical picture of the three groups of inoculated explants treated in Example 1 after 50 days of culture;

[0029] Figure 4 This is a photo of the inoculated explants of treatment group 3 and treatment group 4 of Example 1 after 70 days of culture;

[0030] Figure 5 This is a photo of the four groups of inoculated explants after 85 days of culture in Example 1;

[0031] Figure 6 It is a physical picture of the seedling hardening of Example 1. DETAILED DESCRIPTION

[0032] The present invention provides a method for rapid propagation of lotus by tissue culture to prevent browning, comprising the following steps:

[0033] Inoculating the lotus explants into a solid culture medium so that 1 / 3 to 1 / 2 of the height of the lotus explants are inserted into the solid culture medium; adding a liquid culture medium onto the solid culture medium so that the liquid culture medium submerges the lotus explants, covering the lotus explants with sterile material, removing the sterile material after dark culture, and then subjecting the lotus explants to light culture to obtain stem-running sterile seedlings;

[0034] The sterile material includes sterile hydrophilic non-woven fabric or sterile absorbent cotton;

[0035] The solid culture medium is based on MS and includes 0.5-2.0 mg / L 6-BA, 0.5-2.0 mg / L NAA, 30-80 g / L sucrose, 6-8 g / L agar, and 0.3-0.8 g / L activated carbon;

[0036] The liquid culture medium uses MS as the basic culture medium and includes 0.5-2.0 mg / L 6-BA and 0.5-2.0 mg / L NAA.

[0037] In the present invention, the solid culture medium is based on MS, including 6-BA 0.5-2.0 mg / L, NAA 0.5-2.0 mg / L, sucrose 30-80 g / L, agar 6-8 g / L and activated carbon 0.3-0.8 g / L, preferably only including 6-BA 0.5-2.0 mg / L, NAA 0.5-2.0 mg / L, sucrose 30-80 g / L, agar 6-8 g / L and activated carbon 0.3-0.8 g / L. The concentration of 6-BA in the solid culture medium of the present invention is preferably 0.5-1.5 mg / L, and more preferably 1.0 mg / L. The concentration of NAA in the solid culture medium of the present invention is preferably 0.5-1.5 mg / L, and more preferably 1.0 mg / L. The concentration of sucrose in the solid culture medium of the present invention is preferably sucrose 60 g / L. The concentration of agar in the solid culture medium of the present invention is preferably 7 g / L. The concentration of activated carbon in the solid culture medium of the present invention is preferably 0.5 g / L.

[0038] In the present invention, the liquid culture medium is based on MS, including 6-BA 0.5-2.0 mg / L and NAA 0.5-2.0 mg / L, preferably only including 6-BA 0.5-2.0 mg / L and NAA 0.5-2.0 mg / L. The concentration of 6-BA in the liquid culture medium of the present invention is preferably 0.5-1.5 mg / L, and more preferably 0.8-1.0 mg / L. The concentration of NAA in the liquid culture medium of the present invention is preferably 0.5-1.5 mg / L, and more preferably 1.0 mg / L.

[0039] The present invention preferably disinfects the lotus explant to obtain the disinfected lotus explant. In the present invention, the lotus explant preferably includes lotus buds and / or lotus cores, and more preferably lotus buds. The present invention preferably removes the lotus buds, peels off the outer leaf sheaths, leaves 1 to 2 layers of inner leaf sheaths, and disinfects them. After the disinfection is completed, all leaf sheaths are peeled off to obtain the disinfected lotus explant. The disinfection method of the present invention is preferably: after rinsing with running water, soak in 75% v / v ethanol for 2 minutes, wash with sterile water 3 times, soak in a mixture of 2.5 to 10.0 wt.% sodium hypochlorite solution and Tween 20 for 10 to 15 minutes, and rinse with sterile water 3 to 5 times; the volume ratio of the sodium hypochlorite solution is 100 mL: 1 drop. The present invention does not completely peel off the outer leaf sheath during disinfection, which can reduce the damage to the explant during the disinfection process. After obtaining the sterilized lotus explant, the present invention preferably inoculates the sterilized lotus explant into a solid culture medium under aseptic conditions, then adds a liquid culture medium onto the solid culture medium, so that the liquid culture medium submerges 1 / 2 to 2 / 3 of the sterilized lotus explant, and culture is performed after covering the lotus explant with sterile absorbent cotton. The present invention preferably embeds 1 / 3 to 2 / 3 of the lotus explant into the solid culture medium, and more preferably 1 / 3. The sterile absorbent cotton covered in the present invention can absorb the liquid culture solution to form a liquid sealing layer, so that the lotus explant is isolated from the air. At the same time, a small amount of phenolic substances generated by the lotus explant itself can be absorbed by the sterile absorbent cotton, gradually infiltrated into the outer surface of the lotus explant or diluted into the liquid culture solution, reducing the accumulation of phenolic and quinone substances in the explant and the surrounding area, and successfully reducing the browning of the tissue culture process.

[0040] In the present invention, the dark culture time is preferably 15 to 20 days, more preferably 16 to 18 days; the dark culture temperature is preferably 24 to 26° C., more preferably 25° C.; the light intensity of the light culture is preferably 4000 to 6000 lx, more preferably 5000 lx; ​​the light culture temperature is preferably 25 to 28° C., more preferably 27° C. After 15 to 20 days of light culture in the present invention, the lotus explant begins to proliferate, and leaves and underground stems are differentiated as they grow.

[0041] When the runner sterile seedlings grow to ≥10 nodes, the present invention preferably cuts the runner sterile seedlings according to 1-2 nodes to obtain newly cut lotus explants; the new lotus explants are subcultured to obtain a large number of runner sterile seedlings; the subculture method preferably includes: inoculating the newly cut lotus explants into a solid culture medium, pouring a liquid culture medium into it, making the liquid culture medium submerge 1 / 2-2 / 3 of the lotus explants, and subculture. The solid culture medium is based on MS, including 6-BA 0.5-2.0 mg / L, NAA 0.5-2.0 mg / L, sucrose 30-80 g / L, agar 6-8 g / L and activated carbon 0.3-0.8 g / L, preferably only including 6-BA 0.5-2.0 mg / L, NAA 0.5-2.0 mg / L, sucrose 30-80 g / L, agar 6-8 g / L and activated carbon 0.3-0.8 g / L. The concentration of 6-BA in the solid culture medium of the present invention is preferably 0.5-1.5 mg / L, and more preferably 1.0 mg / L. The concentration of NAA in the solid culture medium of the present invention is preferably 0.5-1.5 mg / L, and more preferably 1.0 mg / L. The concentration of sucrose in the solid culture medium of the present invention is preferably sucrose 60 g / L. The concentration of agar in the solid culture medium of the present invention is preferably 7 g / L. The concentration of activated carbon in the solid culture medium of the present invention is preferably 0.5 g / L.

[0042] In the present invention, the illumination intensity of the subculture is 4000-6000 lx, more preferably 5000 lx; ​​the illumination time of the subculture is preferably 10-14 hours / day, more preferably 12 hours / day; the temperature of the subculture is preferably 25-28°C, more preferably 27°C. The present invention preferably embeds 1 / 3-2 / 3 of the new lotus explant into the solid culture medium, more preferably 1 / 3. After the subculture of the present invention for 2 months, a large number of rooted runner sterile seedlings can be obtained, and this can be repeated, preferably the subculture proliferation culture can be performed several times to increase the number of runner sterile seedlings.

[0043] After obtaining the runner sterile seedlings, the present invention preferably performs seedling hardening and acclimatization on the runner sterile seedlings before transplanting. In the present invention, the seedling hardening and acclimatization time is preferably 30 to 45 days, and more preferably 40 days. The seedling hardening and acclimatization of the present invention can enable the lotus leaves to adapt to the strong light, high temperature and dry conditions of the field.

[0044] In order to further illustrate the present invention, a method for rapid propagation of lotus with anti-browning tissue culture provided by the present invention is described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0045] Example 1

[0046] 1. Culture medium preparation

[0047] (1) Solid culture medium: MS is used as the basic culture medium, and only 0.5 mg / L 6-BA, 1.0 mg / L NAA, 60 g / L sucrose, 7 g / L agar, and 0.5 g / L activated carbon are added, with a pH of 5.8; 6BA and NAA are first dissolved in MS culture medium in sequence, and then sucrose, agar, and activated carbon are added. After heating and dissolving, the pH is adjusted, and then the culture is divided into culture bottles, sterilized at high temperature and high pressure, and then used;

[0048] (2) Liquid culture medium: MS was used as the basic culture medium, with only 0.5 mg / L 6-BA and 0.5 mg / L NAA added, pH 5.8; each component was prepared according to the set concentration, sterilized at high temperature and high pressure, and then used.

[0049] 2. Explant preparation.

[0050] The underground stem tip (lotus root bud) of lotus was used as an explant. The lotus root bud was first cut off, the outer leaf sheath was peeled off, and 1 to 2 layers of inner leaf sheath were left for disinfection. The disinfection method was as follows: after rinsing with running water for 30 minutes, soaking in 75% v / v ethanol for 2 minutes, washing with sterile water for 3 times, and then soaking in 5.0wt.% sodium hypochlorite plus a small amount of Tween 20 (1 drop of Tween 20 per 100mL sodium hypochlorite solution) for disinfection for 15 minutes, taking out and washing with sterile water for 5 times; after completing the disinfection, all leaf sheaths were peeled off.

[0051] 3. Explant inoculation

[0052] The explants were randomly divided into 4 treatment groups and treated as follows, with 20 explants in each treatment group and repeated 3 times:

[0053] Treatment 1 (solid culture medium group): 1 / 3 of the explants in step 2 were embedded in solid culture medium under sterile conditions;

[0054] Treatment 2 (solid culture medium + cotton wool covering group): 1 / 3 of the explants in step 2 were embedded in the solid culture medium under sterile conditions, and a piece of sterile cotton wool was covered on the surface of the remaining explants;

[0055] Treatment 3 (solid-liquid double-layer culture medium group): Under sterile conditions, 1 / 3 of the explants in step 2 were embedded in the solid culture medium, and the liquid culture medium was poured in such a way that half of the liquid culture medium submerged the explants ( Figure 1 );

[0056] Treatment 4 (solid-liquid double-layer culture medium + cotton wool covering group): Under sterile conditions, 1 / 3 of the explants in step 2 were embedded in the solid culture medium, and the liquid culture medium was poured in such a way that half of the liquid culture medium was immersed in the explants, and a piece of sterile cotton wool was covered on top of the explants ( Figure 2 ).

[0057] 4. Cultivation of stem seedlings.

[0058] Step 3 After inoculation of each treatment group, dark culture was carried out at room temperature of 25°C for 16 days. Treatments 2 and 4 were cultured in light with a light intensity of 5000 lx and room temperature of 27°C by shaking the bottles to remove the covering cotton. After entering the light culture, the explants proliferated faster and gradually differentiated into leaves and underground stems, and finally obtained runner seedlings. The growth of treatment 3 group after 50 days of explant inoculation culture is shown in Figure 2. Figure 3 The growth of treatment groups 3 and 4 after 60 days of inoculation of explants is shown in Figure 4 As shown, the left picture is treatment group 3, and the right picture is treatment group 4. The growth of treatment group 4 after 80 days of culture from explant inoculation is as follows Figure 5 The browning status of each treatment group 30 days after inoculation and the seedling status 60 days after inoculation are shown in Table 1.

[0059] Table 1 Browning and seedling formation of stem-bearing seedlings in each treatment group

[0060]

[0061] According to Table 1, the browning rate and degree of treatment 4 were much lower than those of other treatments. The stem seedlings grew healthily, with a browning rate of 30.0% and a seedling rate of 70.0%, which were significantly higher than those of other treatments. The experiment also found that slight browning could also result in seedlings.

[0062] 5. Propagation of stem seedlings

[0063] Step 4: When the four groups of sterile runner seedlings grow to more than 10 nodes, the sterile runner seedlings are cut into two nodes to form newly cut lotus explants; one-third of the newly cut lotus explants are embedded in a solid culture medium under aseptic conditions; a liquid culture medium is poured in such a way that half of the liquid culture medium is immersed outside the newly cut lotus explants, the ambient temperature is set to 27° C., the light intensity is 5000 lx, the light time is 12 hours / day, and subculture is performed. After 2 months, the sterile runner seedlings grow to more than 10 nodes again. The subculture proliferation culture is repeated several times to obtain a large number of rooted sterile runner seedlings.

[0064] 6. Hardening and transplanting of tissue culture seedlings

[0065] One week before the sterile runner seedlings are taken out of the tissue culture room and placed indoors, the light intensity is increased to 7000-9000lx to stimulate the plantlets to photosynthesize. Loosen the bottle caps of the light-treated bottle seedlings and place them for 3 days, then open the bottle caps and place them for 3 days. Then take out the seedlings, wash off the culture medium, soak the roots of the runner seedlings with 800-1000 times diluted carbendazim for 5 minutes, take out and drain the water, and transplant them into a small pot filled with mud for acclimatization and cultivation. In the first 2 weeks after transplanting, keep the air temperature above 80%-90% and the light intensity below 15000lx to prevent leaf dehydration and burns. After 25 days, gradually increase ventilation and light ( Figure 6 ), after 35 days, completely remove the protection, and after 45 days, it can be transplanted to the field.

[0066] It can be seen from the above content that the tissue culture rapid propagation method provided by the present invention successfully reduces the browning of the lotus tissue culture process, improves the survival rate of the tissue culture seedlings, and the tissue culture seedlings grow vigorously and have a high propagation coefficient, which solves the current problems of long lotus seedling propagation cycle and low efficiency, and is beneficial to the large-scale production of lotus seedlings.

[0067] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for rapid propagation of lotus to prevent browning by tissue culture, characterized in that: The steps include: Inoculating the lotus explants into a solid culture medium so that 1 / 3 to 1 / 2 of the height of the lotus explants are inserted into the solid culture medium; adding a liquid culture medium onto the solid culture medium so that the liquid culture medium submerges the lotus explants, covering the lotus explants with sterile material, removing the sterile material after dark culture, and then subjecting the lotus explants to light culture to obtain stem-running sterile seedlings; The sterile material includes sterile hydrophilic non-woven fabric or sterile absorbent cotton; The solid culture medium is based on MS and includes 0.5-2.0 mg / L 6-BA, 0.5-2.0 mg / L NAA, 30-80 g / L sucrose, 6-8 g / L agar, and 0.3-0.8 g / L activated carbon; The liquid culture medium uses MS as the basic culture medium and includes 0.5-2.0 mg / L 6-BA and 0.5-2.0 mg / L NAA.

2. The tissue culture rapid propagation method according to claim 1, characterized in that: The lotus explants include lotus buds and / or lotus cores.

3. The tissue culture rapid propagation method according to claim 1, characterized in that The specification of the sterile hydrophilic nonwoven fabric is 80-160 g / m 2 .

4. The tissue culture rapid propagation method according to claim 1, characterized in that: The dark culture time is 15 to 20 days; the dark culture temperature is 24 to 26°C.

5. The tissue culture rapid propagation method according to claim 1, characterized in that: The light intensity of the light culture is 4000-6000 lx; ​​the temperature of the light culture is 25-28°C.

6. The tissue culture and rapid propagation method according to claim 1, characterized in that: When the runner sterile seedlings grow to ≥10 nodes, the runner sterile seedlings are cut into 1-2 nodes to obtain newly cut lotus explants; Subculturing the new lotus explants to obtain a large number of sterile stem seedlings; The subculture method comprises: inoculating the newly cut lotus explants into a solid culture medium, pouring a liquid culture medium into the solid culture medium, making the liquid culture medium submerge 1 / 2 to 2 / 3 of the lotus explants, and performing subculture; The solid culture medium is based on MS and includes 0.5-2.0 mg / L 6-BA, 0.5-2.0 mg / L NAA, 30-80 g / L sucrose, 6-8 g / L agar, and 0.3-0.8 g / L activated carbon; The liquid culture medium uses MS as the basic culture medium and includes 0.5-2.0 mg / L 6-BA and 0.5-2.0 mg / L NAA.

7. The tissue culture and rapid propagation method according to claim 6, characterized in that: The illumination intensity of the subculture is 4000-6000 lx, and the temperature is 25-28°C.

8. The tissue culture and rapid propagation method according to claim 6, characterized in that: 1 / 3 to 2 / 3 of the newly cut lotus explants are embedded in the solid culture medium.

9. The tissue culture and rapid propagation method according to any one of claims 1 to 8, characterized in that: After obtaining the runner sterile seedlings, the method further comprises: hardening and acclimatizing the runner sterile seedlings and then transplanting them.

10. The tissue culture and rapid propagation method according to claim 9, characterized in that: The time for hardening and acclimatization of the seedlings is 30 to 45 days.

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