A tissue culture method for inducing clustered buds from the seeds embryos of Paeonia delavayi

Abstract

The present invention provides a tissue culture method for inducing cluster buds of Paeonia delavayi Franch. embryos, which relates to the technical field of tissue culture of Paeonia delavayi Franch. The tissue culture method mainly includes steps such as obtaining explants, germination culture of embryos, induction culture of cluster buds of embryos, proliferation culture of cluster buds, rooting culture of cluster buds, and acclimatization and transplantation. The present invention overcomes the deficiencies of the prior art, uses the embryos in fresh and mature Paeonia delavayi Franch. seeds of the current year as explants, and through the research on the induction, proliferation, and rooting media of cluster buds of embryos, preliminarily establishes a regeneration system for the induction pathway of cluster buds of embryos, laying a foundation for the establishment of the genetic transformation system of Paeonia delavayi Franch.

Description

Technical Field

[0001] The invention relates to the technical field of Yunnan peony tissue culture, and in particular to a tissue culture method for inducing clustered buds of Yunnan peony seed embryos. Background Art

[0002] Yunnan peony (Paeonia delavayi Franch) is a national second-class protected plant distributed in the high-altitude and cold regions of southwest my country. It has rich and diverse flower colors and leaf shapes, and is of great ornamental value. It can be used in landscaping as specialized gardens, flower beds, flower mirrors and flower belts to create beautiful urban landscapes, which requires a large number of seedlings. Seedlings propagated by seeds have the disadvantages of long existence time, low efficiency, low germination rate, and large phenotypic differentiation, which cannot meet the requirements of large-scale production and breeding, and have become one of the constraints that have always plagued the promotion and use of Yunnan peony; and the double dormancy characteristics of the upper and lower hypocotyls of peony plants lead to their weak natural regeneration ability, which seriously restricts the increase of their population and the inheritance of excellent traits.

[0003] Plant tissue culture technology is the most important basic work for the genetic engineering breeding of new varieties. It is less restricted by time and space, can achieve the purpose of rapid expansion in a short time, and the excellent traits of the mother parent are also maintained with a small coefficient of variation, which can lay the foundation for peony breeding and industrial development. There are reports on the successful establishment of a complete regeneration system using embryos through callus induction or cluster bud induction for 'Fengdan' peony, peony, and large-flowered yellow peony. However, the research on the embryos of Yunnan peony only stays at the callus proliferation stage, and no differentiation of adventitious buds is observed. The tissue culture of petioles and stamens also only achieves the proliferation of callus. In addition, no related research on cluster bud induction has been found. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention provides a tissue culture method for inducing clustered buds from seed embryos of Yunnan peony. The seed embryos in fresh and mature Yunnan peony seeds of the current year are used as explants. Through the research on seed embryo clustered bud induction, proliferation and rooting culture medium, a regeneration system for the seed embryo clustered bud induction pathway is preliminarily established, laying the foundation for the establishment of a Yunnan peony genetic transformation system.

[0005] To achieve the above objectives, the technical solution of the present invention is implemented through the following technical solutions:

[0006] A tissue culture method for inducing clustered buds from Yunnan tree peony embryos, the culture method comprising the following steps:

[0007] S1. Obtaining explants: soaking the seeds of Paeonia dahliae in a gibberellin solution and then washing them, dissecting out the complete embryos and performing disinfection to obtain explants for later use;

[0008] S2. Germination culture of the seed embryo: Inoculate the above explants into the germination medium and culture them under dark conditions to obtain embryo seedlings; and the formula of the germination medium is: MS + 0.2 mg / L GA 3 + 0.5 g / L PVP + 7 g / L agar + 30 g / L sucrose;

[0009] S3. Induction culture of adventitious buds of the seed embryo: Inoculate the above embryo seedlings into the induction medium for adventitious buds and culture them to induce multiple adventitious buds; and the formula of the induction medium for adventitious buds is: MS + 0.5 mg / L KT + 0.5 mg / L 6 - BA + 30 g / L sucrose + 7 g / L agar + 0.5 g / L PVP + 20 mg / L ethyl isothiocyanate + 30 mg / L tea saponin;

[0010] S4. Proliferation culture of adventitious buds: Transfer the embryo seedlings with multiple adventitious buds induced above into the proliferation medium and culture them until the adventitious buds proliferate more than 2 times; and the formula of the proliferation medium is: MS + 0.2 mg / L GA 3 + 0.5 mg / L KT + 0.5 mg / L 6 - BA + 0.3 g / L CH + 30 g / L sucrose + 7 g / L agar + 1 g / L PVP + 15 mg / L tocopheryl acetate;

[0011] S5. Rooting culture of adventitious buds: Inoculate the adventitious buds obtained in step S4 above into the rooting medium for adventitious buds and culture them to obtain Paeonia delavayi Franch. seedlings; and the formula of the rooting medium for adventitious buds is modified 1 / 2MS + 1 mg / L IBA + 0.2 mg / L NAA + 0.5 mg / L IAA + 7 g / L agar + 1 g / L PVP + 2 g / L AC + 15 mg / L ethyl isothiocyanate + 30 mg / L tocopheryl acetate; and the formula of modified 1 / 2MS is: 720 mg / L KNO 3 、580 mg / L NH 4 NO 3 、106 mg / L KH 2 PO 4 、146 mg / L MgSO 4 ·7H 2 O、255 mg / L CaCl 2 ·2H 2 O、0.83 mg / L KI、6.2 mg / L H 3 BO 3 、22.3 mg / L MnSO 4 ·H 2 O、8.6 mg / L ZnSO 4 ·7H 2 O、0.25 mg / L Na 2 MoO 4·2H 2 O, 0.025 mg / L CuSO 4 ·5H 2 O, 0.025 mg / L CoCl 2 ·6H 2 O, 27.6 mg / L EDTA-Na 2 、30.5 mg / L FeSO 4 ·7H 2 O, 100 mg / L inositol, 3.5 mg / L glycine, 0.1 mg / L thiamine hydrochloride, 0.5 mg / L pyridoxine hydrochloride;

[0012] S6. Acclimatization and transplanting: Select the robust Paeonia delavayi seedlings in the above S4. After removing them from the incubator and acclimatizing for 2 days, remove the rooting medium of the cluster buds at the roots and transplant them into the growth substrate for cultivation.

[0013] Preferably, the method of soaking the Paeonia delavayi seeds in the step S1 with gibberellin solution is to soak the Paeonia delavayi seeds in a 500 mg / L gibberellin solution for 48 - 50 h.

[0014] Preferably, the method of disinfecting the Paeonia delavayi seeds in the step S1 is to soak them in a sodium hypochlorite solution with a mass concentration of 0.4% - 0.5% for 5 - 6 min, and then rinse them 2 - 3 times with sterile water.

[0015] Preferably, the conditions for the germination culture of the embryos in the step S2 are dark culture at 25 ± 2 °C.

[0016] Preferably, the temperature condition for the induction culture of the cluster buds in the step S3 is 15 ± 1 °C. During the culture process, first conduct dark culture, then control the daily light time to be 16 h for supplementary light culture. The time of the dark culture is 10 d, and white LED light is used as the light source during the supplementary light culture.

[0017] Preferably, the temperature for the proliferation culture of the cluster buds in the step S4 is 25 ± 2 °C, and the light time is controlled to be 16 h / d with a white LED lamp.

[0018] Preferably, the temperature for the rooting culture of the cluster buds in the step S5 is 20 ± 1 °C, and the light time is controlled to be 16 h / d with a white LED lamp.

[0019] Preferably, the criteria for the robust Paeonia delavayi seedlings in the step S6 are that the seedling height > 8 cm, the root length > 5 cm, and the number of fibrous roots > 3.

[0020] Preferably, in step S6, the growth substrate is any one of the substrates with a mass ratio of humus: laterite: perlite = 1:1:1 or humus: perlite = 1:0.5. The growth substrate is autoclaved at 121 °C for 20 min one day before use and then naturally cooled before use.

[0021] Preferably, the conditions for transplanting into the growth substrate for cultivation in step S6 are light cultivation at 28 ± 2 °C, controlling the light for 16 h per day, and covering with a transparent plastic cover 7 days before transplantation.

[0022] The present invention provides a tissue culture method for inducing clustered buds from the seeds of Paeonia delavayi Franch. Compared with the prior art, the advantages are as follows:

[0023] By sequentially culturing the seeds of Paeonia delavayi Franch. with an induction medium, a proliferation medium, and a rooting medium for clustered buds, the present invention can establish a regeneration system for inducing clustered buds from seeds, obtain complete Paeonia delavayi Franch. plants, facilitate the breeding of Paeonia delavayi Franch., ensure the healthy growth of the induced roots of Paeonia delavayi Franch., and improve the survival rate of subsequent transplantation. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of seedling plants cultured with different induction media for clustered buds in the examples of the present invention;

[0025] Figure 2 Schematic diagram of the rooting of clustered buds cultured with rooting medium 1 in the examples of the present invention;

[0026] Figure 3 Schematic diagram of the rooting of clustered buds cultured with rooting medium 2 in the examples of the present invention;

[0027] Figure 4 Schematic diagram of the rooting of clustered buds cultured with rooting medium 3 in the examples of the present invention;

[0028] Figure 5 Schematic diagram of the rooting of clustered buds cultured with rooting medium 4 in the examples of the present invention;

[0029] Figure 6 Schematic diagram of the rooting of clustered buds cultured with rooting medium 5 in the examples of the present invention;

[0030] Figure 7 Schematic diagram of the rooting of clustered buds cultured with rooting medium 6 in the examples of the present invention;

[0031] Figure 8 Schematic diagram of the growth of seedlings 15 days after transplantation in the examples of the present invention, where a is the growth situation after transplantation into substrate 1; b is the growth situation after transplantation into substrate 2. DETAILED DESCRIPTION OF THE INVENTION

[0032] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0033] Example:

[0034] Tissue culture experiment of Paeonia delavayi Franch. seeds:

[0035] 1. Selection of test materials:

[0036] Fresh Paeonia delavayi Franch. seeds were collected from the Paeonia delavayi Franch. germplasm resource nursery (102°53′55″ E, 24°45′38″ N, altitude 2,733 m) set up by Yunnan Dianxiang Guose Agricultural Technology Co., Ltd. in Chengjiang City, Yunnan Province. After collection, they were placed in a well-ventilated place indoors to dry in the shade and stored in a 4°C refrigerator in the dark for later use;

[0037] 2. Selection of disinfection treatment methods for seeds:

[0038] The Paeonia delavayi Franch. seeds soaked in 500 mg / L GA 3 for 48 h were transferred to tap water containing dishwashing liquid and gently rubbed to wash the surface dirt and impurities. Then, they were rinsed with running water for 30 min. On the experimental bench, the complete seeds were picked out with a scalpel and dissecting needle and placed in a 2 ml centrifuge tube. Subsequently, in the ultra-clean workbench, they were disinfected with sodium hypochlorite (NaClO) at three different concentrations (0.3%, 0.4%, 0.5%) for 4 min, 5 min, and 6 min respectively, washed 3 times with sterile water, and inoculated into a 1 / 2MS medium containing 0.2 mg / L GA 3 . There were a total of 9 treatments, with 20 seeds inoculated in each treatment, 3 replicates, and a total of 60 seeds inoculated in each treatment. The contamination rate of different treatments was counted 7 days after inoculation, and the browning rate and mortality rate of the seeds were counted 30 days after inoculation; the specific results are shown in Table 1 below:

[0039] Table 1

[0040]

[0041] Note: The data in the table are mean ± standard deviation.

[0042] That is, the best disinfection method was selected as a NaClO concentration of 0.4% and a disinfection time of 5 min.

[0043] 3. Germination culture of seeds:

[0044] The embryos treated with the above-mentioned optimal disinfection method were respectively inoculated into different germination media in Table 2 below. 30 embryos were inoculated in each basic medium, with 3 replicates. A total of 90 embryos were inoculated in each medium. They were cultured in the dark at 25±2°C, and the germination status was counted every 7 days. After 30 days of culture, the seedling formation rate, seedling height, root length, etc. of the embryos were counted to screen the optimal basic germination medium;

[0045] Table 2

[0046] Germination medium Formulation Germination medium 1 <![CDATA[MS + 0.2 mg / L GA 3 + 0.5 g / L PVP + 7 g / L agar + 30 g / L sucrose]]> Germination medium 2 <![CDATA[1 / 2MS + 0.2 mg / L GA 3 + 0.5 g / L PVP + 7 g / L agar + 30 g / L sucrose]]> Germination medium 3 <![CDATA[WPM + 0.2 mg / L GA 3 + 0.5 g / L PVP + 7 g / L agar + 30 g / L sucrose]]>

[0047] The results of the 30-day culture are shown in Table 3 below (where the germination rate (%) = (the number of embryos with changes / the total number of inoculated embryos) × 100%; the seedling formation rate (%) = (the number of embryos with both the epicotyl and hypocotyl elongated / the total number of inoculated embryos) × 100%):

[0048] Table 3

[0049]

[0050] That is, germination medium 1 is the optimal germination medium.

[0051] 4. Induction of adventitious buds of embryos:

[0052] The embryo seedlings with consistent growth obtained by culturing with the above-mentioned germination medium 1 for 20 days were respectively inoculated into different adventitious bud induction media in Table 4 below. 10 bottles were inoculated in each medium, and 3 embryo seedlings were inoculated in each bottle. A total of 90 embryo seedlings were inoculated in each medium. They were cultured in the dark at 15±1°C for 10 days and then transferred to be cultured under white LED lights with a 16:8 light-dark cycle;

[0053] Table 4

[0054]

[0055] After 30 days of culture, the germination, epicotyl elongation and adventitious bud induction of the embryos in each medium were observed and counted. The specific results are shown in Table 5 below (the epicotyl elongation rate (%) = (the length of the epicotyl after culture - the length of the epicotyl before culture) / the length of the epicotyl before culture × 100%; the adventitious bud induction rate (%) = (the number of embryos with adventitious buds induced / the total number of inoculated embryos) × 100%):

[0056] Table 5

[0057] Medium Germination rate (%) Epicotyl elongation rate (%) Cluster bud induction rate (%) Cluster bud induction medium 1 78.89％ 63.72％ 46.67％ Cluster bud induction medium 2 74.44％ 43.24％ 34.44％ Cluster bud induction medium 3 77.78％ 47.21％ 42.22％ Cluster bud induction medium 4 66.67％ 28.57％ 26.67％

[0058] That is, adventitious bud induction medium 1 is the optimal adventitious bud induction medium, and the specific induction of adventitious buds is as Figure 1 shown, among which the adventitious buds induced by culturing with adventitious bud induction medium 1 are as Figure 1As shown in d, the number of cluster buds > 5; the cluster buds induced by the induction medium 4 of cluster buds are as Figure 1 shown in a, the number of cluster buds is 1 - 2; the cluster buds induced by the induction medium 3 of cluster buds are as Figure 1 shown in b, the number of cluster buds is 1 - 2; the cluster buds induced by the induction medium 2 of cluster buds are as Figure 1 shown in c, the number of cluster buds is 3 - 4.

[0059] 5. Proliferation culture of embryo cluster buds:

[0060] Select the embryo seedlings with single-bud growth of the hypocotyls that have been cultured on the above induction medium 1 for 30 days and have consistent growth vigor, and inoculate the cluster buds into the proliferation medium in Table 6 below for the proliferation culture of cluster buds. 20 embryo seedlings with single-bud growth of the hypocotyls are inoculated in each medium, and it is repeated 3 times. It is cultured under white LED light with a 16:8 light-dark cycle at 25 ± 2 °C. After 30 days, calculate the proliferation rate and average proliferation coefficient of the cluster buds (proliferation rate (%) = (number of embryo seeds with increased cluster buds / total number of inoculated embryo seeds) × 100, proliferation coefficient = number of increased cluster buds / number of cluster buds before proliferation culture);

[0061] Table 6

[0062]

[0063]

[0064] The culture results after 30 days are as shown in Table 7 below:

[0065] Table 7

[0066] Medium Proliferation rate (%) Proliferation coefficient Proliferation medium 1 75.79 3.14 Proliferation medium 2 51.67 1.47 Proliferation medium 3 55.00 1.50 Proliferation medium 4 63.33 2.81

[0067] That is, the proliferation medium 1 is the optimal proliferation medium.

[0068] 6. Rooting culture of cluster buds:

[0069] Select the cluster buds that have been cultured on the above proliferation medium 1 for 30 days and have consistent growth vigor, and inoculate them into the rooting media with different formulations in Table 8 below. 30 seedlings are inoculated in each medium, and it is cultured under light in an environment of 20 ± 1 °C. After 30 days, count the rooting rate and the growth status of the seedlings;

[0070] Table 8

[0071]

[0072] And the formulation of the above improved 1 / 2MS is as follows:

[0073] 720mg / L KNO 3, 580 mg / L NH 4 NO 3 , 106 mg / L KH 2 PO 4 , 146 mg / L MgSO 4 ·7H 2 O, 255 mg / L CaCl 2 ·2H 2 O, 0.83 mg / L KI, 6.2 mg / L H 3 BO 3 , 22.3 mg / L MnSO 4 ·H 2 O, 8.6 mg / L ZnSO 4 ·7H 2 O, 0.25 mg / L Na 2 MoO 4 ·2H 2 O, 0.025 mg / L CuSO 4 ·5H 2 O, 0.025 mg / L CoCl 2 ·6H 2 O, 27.6 mg / L EDTA-Na 2 , 30.5 mg / L FeSO 4 ·7H 2 O, 100 mg / L inositol, 3.5 mg / L glycine, 0.1 mg / L thiamine hydrochloride, 0.5 mg / L pyridoxine hydrochloride.

[0074] The rooting rate and seedling growth status at 30 d are as shown in Table 9 (rooting rate (%) = (number of tissue culture seedlings with adventitious roots / total number of inoculated tissue culture seedlings) × 100%):

[0075] Table 9

[0076] Rooting medium Rooting rate (%) Root growth status Rooting medium 1 60.00％ Taproot length 0.5 cm, 4 - 8 lateral roots, average lateral root length 2 cm Rooting medium 2 53.33％ Taproot length 0.3 cm, 1 - 3 lateral roots, length 0.3 - 1.2 cm Rooting medium 3 46.67％ Taproot length 0.2 cm, 1 - 2 lateral roots, length 0.45 - 0.55 cm Rooting medium 4 40.00％ Taproot length 0.2 cm, 2 - 3 lateral roots, length 0.2 - 0.5 cm Rooting medium 5 33.33％ Taproot length 0.2 cm, one lateral root, length 0.1 - 0.2 cm Rooting medium 6 23.33％ Taproot length 0.2 cm, no lateral roots

[0077] That is, rooting medium 1 is the optimal rooting medium.

[0078] 7. Hardening-off and transplanting:

[0079] Hardening-off and transplanting the seedlings cultured in rooting medium 1 with a seedling height > 8 cm, root length > 5 cm, and number of fibrous roots > 3. The substrate is configured as shown in Table 10:

[0080] Table 10

[0081] Substrate Formulation (mass ratio) Substrate 1 Humus: red soil: perlite = 1:1:1 Substrate 2 Humus: perlite = 1:0.5

[0082] One day before use, the substrate was autoclaved at 121 °C for 20 min and then naturally cooled. After that, the seedlings were removed from the incubator and naturally acclimated for 2 days, and then transplanted into each group of substrates. 100 plants were transplanted into each group of substrates, and after watering thoroughly with clear water, they were covered with a transparent plastic cover for the first 7 days to prevent water loss. Then, the survival rate of each group was counted after 15 days of planting, as shown in Table 11 below:

[0083] Table 11

[0084] Substrate Survival rate Substrate 1 97％ Substrate 2 98％

[0085] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A tissue culture method for inducing clustered buds from seed embryos of Paeonia dahliae, characterized in that: The culture method comprises the following steps: S1. Obtaining explants: soaking the seeds of Paeonia dahliae in a gibberellin solution and then washing them, dissecting out the complete embryos and disinfecting them to obtain explants for later use, and the disinfection method is to soak them in a sodium hypochlorite solution with a mass concentration of 0.4%-0.5% for 5-6 minutes, and then rinse them with sterile water 2-3 times; S2. Germination culture of seed embryos: inoculate the above explants into germination medium, and culture in dark conditions to obtain embryo seedlings; and the formula of the germination medium is: MS+0.2mg / L GA3+0.5g / L PVP+7g / L agar+30g / L sucrose; S3. Induction culture of clustered buds of seed embryos: inoculate the above embryo seedlings into the induction medium for clustered buds for culture, and induce multiple clustered buds; and the formula of the induction medium for clustered buds is: MS+0.5mg / L KT+0.5mg / L 6-BA+30g / L sucrose+7g / L agar+0.5g / L PVP+20mg / L ethyl isothiocyanate+30mg / L tea saponin; S4. Proliferation culture of clustered buds: the embryo seedlings induced with multiple clustered buds are transferred to a proliferation medium for culture until the clustered buds proliferate more than 2 times; and the formula of the proliferation medium is: MS+0.2mg / L GA3+0.5mg / L KT+0.5mg / L6-BA+0.3g / L CH+30 g / L sucrose+7 g / L agar+1 g / L PVP+15mg / L tocopherol acetate; S5. Cluster bud rooting culture: The cluster buds obtained in the above step S4 are inoculated into the cluster bud rooting medium to culture and obtain Yunnan peony seedlings; and the formula of the cluster bud rooting medium is improved 1 / 2MS+1mg / L IBA+0.2mg / L NAA+0.5mg / L IAA+7g / L agar+1g / L PVP+2g / L AC+15mg / L ethyl isothiocyanate+30mg / L tocopherol acetate; and the formula of the improved 1 / 2MS is: 720mg / L KNO3, 580mg / L NH4NO3, 106mg / L KH2PO4, 146mg / L MgSO4·7H2O, 255mg / L CaCl2·2H2O, 0.83mg / L KI, 6.2mg / L H3BO3, 22.3mg / L MnSO4·H2O, 8.6mg / LZnSO4·7H2O, 0.25mg / L Na2MoO4·2H2O, 0.025mg / L CuSO4·5H2O, 0.025mg / L CoCl2·6H2O, 27.6mg / L EDTA-Na2, 30.5mg / L FeSO4·7H2O, 100mg / L inositol, 3.5mg / L glycine, 0.1mg / L thiamine hydrochloride, 0.5mg / L pyridoxine hydrochloride; S6. Seedling hardening and transplanting: Select the robust Yunnan tree peony seedlings grown in S4 above, move them out of the incubator for acclimatization for 2 days, remove the rooting medium of the root clusters, and transplant them into the growth medium for cultivation.

2. The tissue culture method according to claim 1, characterized in that: In the step S1, the Yunnan peony seeds are soaked in a gibberellin solution by soaking the Yunnan peony seeds in a 500 mg / L gibberellin solution for 48-50 hours.

3. The tissue culture method according to claim 1, wherein: The embryo germination culture condition in step S2 is 25±2° C. in the dark.

4. The tissue culture method according to claim 1, characterized in that: The temperature condition for the induction culture of clustered buds in step S3 is 15±1°C, and during the culture process, dark culture is first performed, and then the daily lighting time is controlled to 16h for supplementary light culture, and the dark culture time is 10d. White LED light is used as the light source during the supplementary light culture.

5. The tissue culture method according to claim 1, characterized in that: The temperature of the proliferation culture of the clustered buds in step S4 is 25±2° C., and the white LED lamp is used to control the lighting time to 16 h / d.

6. The tissue culture method according to claim 1, characterized in that: The temperature of the clustered bud rooting culture in step S5 is 20±1° C., and the white LED lamp is used to control the lighting time to 16 h / d.

7. The tissue culture method according to claim 1, characterized in that: The standards for healthy growth of Paeonia dahliae seedlings in step S6 are seedling height>8 cm, root length>5 cm, and number of fibrous roots>3.

8. The tissue culture method according to claim 1, characterized in that: In step S6, the growth substrate is any one of a substrate with a mass ratio of humus: laterite: perlite = 1:1:1 or a substrate with a mass ratio of humus: perlite = 1:0.5, and the growth substrate is sterilized at 121° C. and high temperature and high pressure for 20 min the day before use and then naturally cooled before use.

9. The tissue culture method according to claim 1, characterized in that: The conditions for transplanting to the growth medium for cultivation in step S6 are: light cultivation at 28±2°C, with light exposure controlled for 16 h per day, and covering with a transparent plastic cover 7 days before transplanting.

Images