A method for propagating polygonatum cyrtonema
By constructing a somatic embryogenesis system using Polygonatum multiflorum embryos, the problems of low seed propagation efficiency and easy rot of rhizomes in Polygonatum multiflorum were solved, realizing an efficient and stable seedling method, and providing technical support for the commercial seedling production and genetic improvement of Polygonatum multiflorum.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- CENTRAL SOUTH UNIVERSITY OF FORESTRY AND TECHNOLOGY
- Filing Date
- 2024-02-22
- Publication Date
- 2026-07-07
AI Technical Summary
The seed propagation of Polygonatum multiflorum has a low germination rate and a long seedling cycle. The rhizomes are used in large quantities and are easily infected and rotted by microorganisms during the germination process, resulting in economic losses. Traditional propagation methods are difficult to meet market demand.
Using the immature embryos of Polygonatum multiflorum as explants, a highly efficient regeneration system was constructed by inducing callus tissue and forming somatic embryos. This system included culture media with specific concentrations of plant growth regulators and specific culture conditions to achieve the germination of somatic embryos and the cultivation of robust seedlings.
A somatic embryogenesis system for Polygonatum multiflorum was established, which improved reproductive efficiency, reduced the risk of microbial infection, and provided an efficient and stable seedling method, laying a technical foundation for commercial seedling production and genetic improvement.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of medicinal plant tissue culture, and more particularly to a method for propagating Polygonatum multiflorum. Background Technology
[0002] Currently, the propagation methods for Polygonatum cyrtonema mainly include asexual and sexual reproduction, namely rhizome propagation and seed propagation. However, seed propagation suffers from low germination rates and long seedling cycles. Therefore, in current artificial cultivation, asexual propagation mainly relies on rhizomes, but this method also has some drawbacks. For example, the large quantity of rhizomes used and the frequent microbial infection of wounds during germination can lead to rotting before new shoots emerge, resulting in significant economic losses. However, with in-depth research into the modern pharmacology of Polygonatum cyrtonema, it is widely used in the food, pharmaceutical, and cosmetic industries, further increasing market demand. Given this increasing market demand and the numerous seedling defects, the traditional Chinese medicinal herb cultivation industry faces significant challenges.
[0003] Plant somatic cells can undergo genetic reprogramming and differentiation into somatic embryos through somatic embryogenesis under the induction of exogenous plant growth regulators (PGRs). Regenerated plants obtained through this biological process offer numerous benefits, including a large number of plants, relatively stable genetic characteristics, and a low coefficient of variation. Currently, in terms of crop genetic improvement, somatic embryos are relatively more readily accepting of exogenous genes than other materials, a fact confirmed in numerous studies. To date, somatic embryos have been reported in in vitro regeneration studies of various plants; however, there are currently no reported cases of establishing a somatic embryogenesis system for *Polygonatum cyrtonema*. Summary of the Invention
[0004] This invention addresses the problems in current Polygonatum multiflorum tissue culture, such as the large amount of rhizomes used and the frequent rotting of new shoots due to wound infection during the germination process. By using immature embryos as explants, a high-quality and efficient regeneration system is constructed, which uses somatic embryos for the propagation of Polygonatum multiflorum tissue culture seedlings.
[0005] To solve the above-mentioned technical problems, the present invention provides a method for propagating Polygonatum multiflorum, the method comprising the steps of using the immature embryo of Polygonatum multiflorum as an explant, inducing somatic embryos by inducing callus tissue and somatic embryos, and germinating the somatic embryos into seedlings with roots.
[0006] In the above method, the embryo is an embryo that is 50 to 60 days after the end of the flowering period.
[0007] In the above method, the somatic embryo is a somatic embryo in the spherical embryo stage.
[0008] In the above method, the plant growth regulators in the culture medium used to induce callus are: 1.0 mg / L of 6-BA and 0.5 mg / L of 2,4-D.
[0009] In the above method, the induced callus is cultured in the dark at a temperature of 25±1℃ for 30 days.
[0010] In the above method, the plant growth regulators in the culture medium used for somatic embryo induction are: 1.0 mg / L of 6-BA and 1.0 mg / L of 2,4-D.
[0011] In the above method, the somatic embryo induction is carried out under dark conditions at a temperature of 25±1℃ for 30 days.
[0012] In the above method, the plant growth regulators in the culture medium used for somatic embryo germination are: 2.0 mg / L of 6-BA and 1.0 mg / L of NAA.
[0013] In the above method, the somatic embryos are cultured at a temperature of 25±1℃, a light intensity of 2000Lux, and a light duration of 14h / d, specifically for 40 days.
[0014] In the above method, the culture medium used for inducing callus, the culture medium used for inducing somatic embryos, and the culture medium used for germinating somatic embryos are all solid culture media obtained by adding sucrose, coagulant, and plant growth regulator to MS basic culture medium.
[0015] The above method further includes the following steps: after the seedlings with roots have been cultivated to strengthen their seedlings, they are hardened off and then transplanted.
[0016] In the above method, the plant growth regulators in the culture medium used for seedling cultivation are: 0.5 mg / L of IAA and 1.0 mg / L of IBA.
[0017] In the above method, the seedlings are cultured at a temperature of 25±1℃, a light intensity of 2000Lux, and a light duration of 14h / d, specifically for 20 days.
[0018] In the above method, the culture medium used for the seedling cultivation is a solid culture medium obtained by adding sucrose, a coagulant and a plant growth regulator to MS basic culture medium.
[0019] In the above methods, agar is used as the solidifying agent in all culture media.
[0020] In the above methods, the sucrose content in all culture media was 30 g / L, the agar content was 7 g / L, and the pH value of all culture media was 6.1 ± 0.1.
[0021] In the above method, the explants need to be disinfected.
[0022] The present invention also provides a composition for propagating Polygonatum multiflorum, the composition comprising the culture medium for inducing callus, the culture medium for inducing somatic embryos, the culture medium for somatic embryo germination, and the culture medium for cultivating seedlings.
[0023] This invention utilizes immature embryos of Polygonatum multiflorum as explants to construct a regeneration system for somatic embryos of Polygonatum multiflorum. The establishment of this system represents the first report of somatic embryogenesis, filling a technological gap and providing technical support for the commercial seedling cultivation and genetic improvement of Polygonatum multiflorum. Attached Figure Description
[0024] Figure 1 This is a photograph taken on day 0 of the embryonic embryos inoculated onto callus induction medium with PGRs combination A-4 in Example 1 of the present invention.
[0025] Figure 2 This is a photograph of the embryonic embryos in Example 1 of the present invention, 20 days after being inoculated onto callus induction medium with PGRs combination A-4.
[0026] Figure 3 This is a photograph of the embryonic cells in Example 1 of the present invention after 30 days of inoculation onto callus induction medium with PGRs combination A-4.
[0027] Figure 4 This is a microscopic photograph of callus tissue in Example 1 of the present invention.
[0028] Figure 5 This is a photograph of a spherical embryo in callus tissue under a microscope in Example 1 of the present invention.
[0029] Figure 6 This is a photograph of the centromorphic embryo of callus tissue under a microscope in Example 1 of the present invention.
[0030] Figure 7 This is a photograph of a torpedo-shaped embryo in callus tissue under a microscope in Example 1 of the present invention.
[0031] Figure 8 This is a photograph of a cotyledon-shaped embryo in callus tissue under a microscope in Example 1 of the present invention.
[0032] Figure 9 This is a photograph of the abnormal phenomenon of only leaves occurring after the germination of the somatic embryo on the somatic embryo-callus chimera in Example 1 of the present invention.
[0033] Figure 10 This is a photograph of the normal phenomenon of synchronous root and leaf germination after somatic embryo germination on the somatic embryo-callus chimera in Example 1 of the present invention.
[0034] Figure 11 This is a photograph of a somatic embryo (developing at the stage of a spherical embryo at the time of inoculation) in Example 1 of the present invention, inoculated onto a C-3 somatic embryo germination medium and cultured for 1 week.
[0035] Figure 12 This is a photograph of a somatic embryo (developing at the stage of a spherical embryo at the time of inoculation) in Example 1 of the present invention, inoculated onto a C-3 somatic embryo germination medium and cultured for 20 days.
[0036] Figure 13 This is a photograph of a somatic embryo (developing at the stage of a spherical embryo at the time of inoculation) in Example 1 of the present invention, inoculated onto a C-3 somatic embryo germination medium and cultured for 40 days.
[0037] Figure 14 The germination rate of somatic embryos (developing at the spherical stage at inoculation) in Example 1 of this invention, inoculated onto C-3 somatic embryo germination medium and control MS basal medium, was statistically analyzed after 30 days of culture. The ** indicates that the significance analysis result is P<0.01.
[0038] Figure 15 This is a photograph of seedlings cultured for 20 days in Example 1 of the present invention. Figure 15 A is a side view of the entire culture flask. Figure 15 Photo B is a side view of the lower half of the culture flask. Figure 15 C is a photograph of the bottom of the culture flask. Detailed Implementation
[0039] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.
[0040] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.
[0041] All culture media used in the following examples were prepared according to standard methods and autoclaved at 116°C for 30 minutes. The pH was adjusted to 6.1 ± 0.1 using 1 mol / L NaOH or 1 mol / L HCl. The substrates were sterilized at 121°C for 20 minutes. All culture dishes used were disposable sterile dishes.
[0042] The culture media, plant hormones, and agar used in the following examples were all purchased from Solarbio Biotechnology Co., Ltd. A table showing the abbreviations and full names of the plant hormones used in the culture media is provided in Table 1.
[0043] Table 1. Comparison of Abbreviations and Full Names of Plant Hormones Used
[0044] abbreviation Full name 6-BA 6-Benzylaminopurine NAA Naphthaleneacetic acid 2,4-D 2,4-Dichlorophenoxyacetic acid IBA Indolebutyric acid
[0045] The Polygonatum multiflorum samples in the following examples were collected from Tongxi Village, Xuefeng Town, Hongjiang City, Huaihua City, Hunan Province, on July 15, 2023.
[0046] Example 1
[0047] The specific method for tissue culture of Polygonatum multiflorum is as follows:
[0048] S1. Collect immature fruits within 50-60 days after flowering, disinfect, and remove the embryos.
[0049] S1-1. Collect immature fruits within 50-60 days after the end of the flowering period, rub them with 5% alkaline detergent (Libai dishwashing liquid) for 5 minutes, rinse with tap water to remove detergent residue, and drain the tap water remaining on the surface of the fruit under natural conditions.
[0050] S1-2 Disinfection: Transfer the fruit to a clean bench, disinfect with 75% alcohol for 30 seconds, rinse with sterile water 2-3 times, then disinfect with 0.1% HgCl2 for 30 minutes, and rinse with sterile water 4-5 times.
[0051] S1-3. After disinfection, separate the immature embryos from the fruit in a clean bench. Keep the separated immature embryos moist and inoculate them into callus induction medium within 30 minutes.
[0052] S2, callus induction
[0053] MS medium was used as the basal medium. Different concentrations of 6-BA (0.2 mg / L, 1.0 mg / L, 1.5 mg / L) and 2,4-D (0.5 mg / L, 1.0 mg / L, 1.5 mg / L) were added to this basal medium, and a two-factor, three-level orthogonal experiment was conducted. The concentrations of 6-BA and 2,4-D after addition are shown in Table 1. The callus induction medium with PGRs combination A-1 was used as an example (the callus induction medium for other PGRs combinations can be deduced similarly):
[0054] The callus induction medium for PGRs combination A-1 is based on MS basal medium, with 6-BA content of 0.2 mg / L, 2,4-D content of 0.5 mg / L, sucrose content of 30 g / L, and agar content of 7 g / L. The preparation method (taking 1L as an example) is as follows: Dissolve 4.74g of MS medium base salt (Solarbio product, Lot No. 104P031, Cat#M8526), 30g of sucrose and 7g of agar powder in water, and add 6-BA and 2,4-D. Add water to make up to 1L, so that the content of 6-BA in the callus induction medium with PGRs combination A-1 is 0.2mg / L and the content of 2,4-D is 0.5mg / L. Adjust the pH value to 6.1±0.1, autoclave at 116℃ for 30 minutes, and while still hot, dispense into disposable sterile culture dishes in a laminar flow hood. After cooling, the callus induction medium with PGRs combination A-1 is obtained.
[0055] The embryos obtained in step S1 were inoculated under aseptic conditions onto callus induction culture media containing the above 9 different combinations of PGRs.
[0056] Three replicates were set up, with five culture dishes for each PGR combination in each replicate, and nine immature embryos inoculated into each culture dish. The cultures were incubated in the dark at 25±1℃ for 30 days.
[0057] The total mass of callus and the callus induction rate were calculated 30 days after inoculation (total mass of callus and callus induction rate induced per replicate of 45 immature embryos for each culture medium).
[0058] Induction rate = Number of callus tissues / Number of explants
[0059] Table 1. Concentration of plant growth regulators (PGRs) in callus induction medium and their effects on total callus mass and callus induction rate.
[0060] PGRs combination 6-BA (mg / L) 2,4-D (mg / L) Total mass (g) Induction rate (%) A-1 0.2 0.5 4.63±0.67df 100.00±0.00a A-2 0.2 1 4.60±0.95df 100.00±0.00a A-3 0.2 1.5 7.27±0.80c 100.00±0.00a A-4 1 0.5 15.80±1.32a 100.00±0.00a A-5 1 1 12.53±1.21b 100.00±0.00a A-6 1 1.5 6.10±0.87cd 100.00±0.00a A-7 1.5 0.5 5.73±1.40cdf 100.00±0.00a A-8 1.5 1 3.90±0.30f 100.00±0.00a A-9 1.5 1.5 4.60±0.26df 100.00±0.00a
[0061] Note: Data analysis was performed using SPSS software. The Waller-Duncan (W) a, b, c test was used to determine significance. Different letters after the data in the same column indicate that the difference reached a significant level (P<0.05).
[0062] The results are shown in Table 1. The induction rate of callus induction media with nine different PGRs combinations all reached 100%. Among them, the total weight of callus inoculated on the callus induction medium with PGR combination A-4 (i.e., MS basal medium as the base medium, with 6-BA content of 1.0 mg / L, 2,4-D content of 0.5 mg / L, sucrose content of 30 g / L, and agar content of 7 g / L) was the heaviest, significantly higher than that of callus induction media with other callus induction media, and the induction effect was the best.
[0063] See the photograph taken on day 0 when the immature embryos were inoculated onto callus induction medium with PGRs combination A-4. Figure 1 See the photograph taken 20 days after inoculation onto callus induction medium with PGRs combination A-4. Figure 2 The photograph shows the callus induction medium containing PGRs combination A-4 after 30 days. Figure 3 .
[0064] Callus tissue from callus induction medium with PGRs combination A-4 was selected for further experiments.
[0065] S3, somatic embryo induction
[0066] MS medium was used as the basal medium. Different concentrations of 6-BA (0.2 mg / L, 1.0 mg / L, 1.5 mg / L) and 2,4-D (1.0 mg / L, 1.5 mg / L, 2.0 mg / L) were added to this basal medium, and a two-factor, three-level orthogonal experiment was conducted. The concentrations of 6-BA and 2,4-D after addition are shown in Table 2. The somatic embryo induction medium with PGRs combination B-1 was used as an example (the somatic embryo induction medium for other PGRs combinations follows the same principle):
[0067] The somatic embryo induction medium for PGRs combination B-1 is based on MS basal medium, with 6-BA content of 0.2 mg / L, 2,4-D content of 1.0 mg / L, sucrose content of 30 g / L, and agar content of 7 g / L. The preparation method (taking 1L as an example) is as follows: Dissolve 4.74g of MS medium base salt (Solarbio product, Lot No. 104P031, Cat#M8526), 30g of sucrose and 7g of agar powder in water, and add 6-BA and 2,4-D. Add water to make up to 1L, so that the content of 6-BA in the somatic embryo induction medium with PGRs combination B-1 is 0.2mg / L and the content of 2,4-D is 1.0mg / L. Adjust the pH value to 6.1±0.1, autoclave at 116℃ for 30 minutes, and while still hot, dispense into disposable sterile culture dishes in a laminar flow hood. After cooling, the somatic embryo induction medium with PGRs combination B-1 is obtained.
[0068] The callus tissue obtained in step S2, which is a combination of PGRs A-4, was inoculated under aseptic conditions onto somatic embryo induction media of the above 9 different combinations of PGRs.
[0069] Three replicates were set up, with 45 culture dishes of somatic embryo induction medium for each PGR combination in each replicate, and one callus tissue was inoculated in each culture dish. The cultures were incubated in the dark at 25±1℃ for 30 days.
[0070] Under a microscope, callus tissue (see) Figure 4 Somatic embryos on ) grow over time, according to the spherical embryo (see Figure 5 — Heart-shaped embryo (see) Figure 6 — Torpedo-shaped embryo (see) Figure 7 — Cotyledonous embryo (see) Figure 8 Following the regular developmental pattern, somatic embryos were obtained at different developmental stages, resulting in somatic embryo-callus chimeras. On day 30 after inoculation, the somatic embryo induction rate was calculated. Since the globular embryo is the earliest somatic embryo, the somatic embryo induction rate was calculated based on the globular embryo induction rate.
[0071] Somatic embryo induction rate = Material containing spherical embryos / Number of callus tissues.
[0072] Table 2. Concentration of plant growth regulators (PGRs) in somatic embryo induction medium and their effects on somatic embryo induction rate.
[0073]
[0074] Note: Data analysis was performed using SPSS software. The Waller-Duncan (W) a, b, c test was used to determine significance. Different letters after the data in the same column indicate that the difference reached a significant level (P<0.05).
[0075] The results are shown in Table 2. Among the nine different PGRs combinations in the somatic embryo induction medium, the somatic embryo induction rate inoculated on the PGRs combination B-4 (i.e., the medium with MS basal medium as the base medium, 6-BA content of 1.0 mg / L, 2,4-D content of 1.0 mg / L, sucrose content of 30 g / L, and agar content of 7 g / L) was 97.78% ± 3.85%, which was significantly better than other somatic embryo induction media.
[0076] Callus tissue containing somatic embryos was selected from somatic embryo induction medium with PGRs combination B-4 for further experiments.
[0077] S4, somatic embryo germination
[0078] S4.1 Preliminary Experiment of Somatic Embryo Germination
[0079] MS medium was used as the basal medium, and different concentrations of 6-BA (2.0 mg / L, 3.0 mg / L, 4.0 mg / L) and NAA (0.2 mg / L, 0.5 mg / L, 1.0 mg / L) were added to this basal medium for a two-factor, three-level orthogonal experiment. The concentrations of 6-BA and NAA after addition are shown in Table 3. The somatic embryo germination medium with PGRs combination C-1 was used as an example (the somatic embryo germination medium for other PGRs combinations follows the same principle):
[0080] The somatic embryo germination medium for PGRs combination C-1 is based on MS basal medium, with 6-BA content of 2.0 mg / L, NAA content of 0.2 mg / L, sucrose content of 30 g / L, and agar content of 7 g / L. The preparation method (taking 1L as an example) is as follows: Dissolve 4.74g of MS medium base salt (Solarbio product, Lot No. 104P031, Cat#M8526), 30g of sucrose and 7g of agar powder in water, and add 6-BA and NAA. Add water to make up to 1L, so that the content of 6-BA in the somatic embryo germination medium with PGRs combination C-1 is 2.0mg / L and the content of NAA is 0.2mg / L. Adjust the pH value to 6.1±0.1, autoclave at 116℃ for 30 minutes, and while still hot, dispense into disposable sterile culture dishes in a laminar flow hood. After cooling, the somatic embryo germination medium with PGRs combination C-1 is obtained.
[0081] The somatic embryo-callus chimera obtained in step S3, which is a combination of PGRs B-4, was inoculated under aseptic conditions onto somatic embryo germination media of the above 9 different combinations of PGRs.
[0082] Three replicates were set up, with 30 culture dishes per replicate for each PGR combination, and one somatic embryo-callus chimera inoculated into each dish. The chimeras were cultured for 30 days at 25±1℃, light intensity of 2000 Lux, and a photoperiod of 14 h / d. At this time, the abnormal phenomenon of leaf-only germination was observed in the somatic embryos of the somatic embryo-callus chimeras (see...). Figure 9 ) and the normal phenomenon of simultaneous occurrence of root and leaf (see Figure 10 ), abnormal germination rate (normal germination rate = number of leaves only / 30) and normal germination rate (abnormal germination rate = number of roots and leaves occurring simultaneously / 30).
[0083] Table 3. Concentration of plant growth regulators (PGRs) in somatic embryo germination medium and their effects on leaf emergence rate and root-leaf synchronous emergence rate.
[0084]
[0085] Note: Data analysis was performed using SPSS software. The Waller-Duncan (W) a, b, c test was used to determine significance. Different letters after the data in the same column indicate that the difference reached a significant level (P<0.05).
[0086] The results are shown in Table 3. Among the nine different PGRs combinations of somatic embryo germination media, the normal germination rate (normal phenomenon) of somatic embryo-callus chimeras inoculated on the somatic embryo germination medium with PGR combination C-4 (i.e., a medium with MS basal medium as the base medium, 6-BA content of 2.0 mg / L, NAA content of 1.0 mg / L, sucrose content of 30 g / L, and agar content of 7 g / L) was significantly higher than that of other somatic embryo germination media, reaching 73.33% ± 6.67%, while the abnormal germination rate (abnormal phenomenon) on it was only 26.67% ± 6.67%.
[0087] S4.2 Somatic Embryo Germination
[0088] The somatic embryo germination medium selected in this invention is the somatic embryo germination medium with PGR combination C-3 (i.e., the medium based on MS basal medium, with 6-BA content of 2.0 mg / L, NAA content of 1.0 mg / L, sucrose content of 30 g / L, and agar content of 7 g / L). The preparation method (taking 1L as an example) is as follows: Dissolve 4.74g of MS medium base salt (Solarbio product, Lot No. 104P031, Cat#M8526), 30g of sucrose and 7g of agar powder in water, and add 6-BA and NAA. Add water to make up to 1L, so that the content of 6-BA in the somatic embryo germination medium with PGRs combination C-3 is 2.0mg / L and the content of NAA is 1.0mg / L. Adjust the pH value to 6.1±0.1, autoclave at 116℃ for 30 minutes, and while still hot, dispense into disposable sterile culture dishes in a laminar flow hood. After cooling, the somatic embryo germination medium with PGRs combination C-3 is obtained.
[0089] A blank control was prepared by adding sugar source (sucrose) and solidifying agent (agar powder) to MS basal medium without plant growth regulators (PGRs). The preparation method (taking 1L as an example) is as follows: Dissolve 4.74g of MS medium basal salt (Solarbio product, Lot No. 104P031, Cat#M8526), 30g of sucrose, and 7g of agar powder in water, adjust the pH to 6.1±0.1, autoclave at 116℃ for 30 minutes, and while still hot, dispense into disposable sterile petri dishes in a laminar flow hood. After cooling, the blank control medium is obtained.
[0090] Set up 3 replicates, with 3 culture dishes of somatic embryo germination medium or blank control medium with PGR combination C-3 for each replicate. 10 spherical embryos (spherical embryos excised from somatic embryo-callus chimeras on somatic embryo induction medium with PGR combination B-4 obtained in step S3) were inoculated in each culture dish.
[0091] The cells were cultured at a temperature of 25±1℃, a light intensity of 2000 Lux, and a photoperiod of 14 h / d. Photos after one week of culture are shown below. Figure 11 On day 20 post-inoculation, incomplete root and leaf structures began to appear, along with miniature rhizomes (see photos). Figure 12 On day 40 of cultivation, the seedlings developed; see photo. Figure 13 Germination rate was calculated on day 30 after inoculation, and the results are shown below. Figure 14The normal germination rate of globular embryos on somatic embryo germination medium with PGRs combination C-3 was 82.22% (germination rate = number of simultaneous root and leaf occurrences / 30), which was significantly higher than the germination rate on blank control medium (31.11%).
[0092] S5. Strong seedlings and transplanting
[0093] MS basal medium was used as the basal medium, with IAA content of 0.5 mg / L, IBA content of 1.0 mg / L, sucrose content of 30 g / L, and agar content of 7 g / L as the seedling strengthening medium of the present invention. The preparation method (taking 1L as an example) is as follows: Dissolve 4.74g of MS medium base salt (Solarbio product, Lot No. 104P031, Cat#M8526), 30g of sucrose and 7g of agar powder in water, add IBA, adjust the pH to 6.1±0.1, add water to make up to 1L, so that the IBA content in the seedling culture medium is 1.0mg / L, put it into a 1L screw-top reagent bottle, autoclave at 116℃ for 30 minutes. After sterilization, let it cool for 40 minutes, add sterile IAA in a clean bench, mix well, so that the IAA content in the seedling culture medium is 0.5mg / L, dispense into sterile culture bottles to obtain the seedling culture medium.
[0094] Each culture flask was inoculated with one seedling with roots obtained from the PGRs combination obtained in S4 after 40 days of somatic embryo culture on C-3 somatic embryo germination medium. A total of 30 seedlings were inoculated.
[0095] Seedlings were cultured for 20 days under conditions of 25±1℃, 2000 Lux light intensity, and 14 h / d photoperiod. Photos of the seedlings after 20 days are shown below. Figure 15 After cultivation, the culture bottles were opened in the culture room for a 3-day acclimatization period (i.e., hardening off). Then, the seedlings were removed and the culture medium was washed off with ddH2O. To prevent dehydration, each seedling was left with only one leaf before being transplanted into a seedling substrate with a nutrient soil:perl salt ratio of 3:1 (v / v). One month after transplanting, 28 out of 30 seedlings survived, a survival rate of 93.33%.
[0096] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, contents, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some basic features can be applied within the scope of the following appended claims.
Claims
1. A method for propagating Polygonatum multiflorum, characterized in that, The method includes the steps of using the immature embryo of Polygonatum multiflorum as an explant, inducing somatic embryos by inducing callus tissue and somatic embryos, and germinating the somatic embryos into seedlings with roots. The immature embryo is an immature embryo that is 50 to 60 days after the end of the flowering period; the somatic embryo is a somatic embryo at the globular embryo stage; The plant growth regulators used in the culture medium for inducing callus consist of: 1.0 mg / L of 6-BA and 0.5 mg / L of 2,4-D. The plant growth regulators used in the culture medium for somatic embryo induction consisted of 1.0 mg / L of 6-BA and 1.0 mg / L of 2,4-D. The plant growth regulators used in the culture medium for somatic embryo germination consist of 2.0 mg / L 6-BA and 1.0 mg / L NAA. The culture medium used for inducing callus, the culture medium used for inducing somatic embryos, and the culture medium used for germinating somatic embryos are all solid culture media obtained by adding sucrose, coagulant, and plant growth regulator to MS basic culture medium.
2. The method according to claim 1, characterized in that, The induced callus was cultured in the dark at a temperature of 25±1℃; the somatic embryo induction was cultured in the dark at a temperature of 25±1℃; and the somatic embryo germination was cultured at a temperature of 25±1℃ and a light intensity of 2000 Lux.
3. The method according to claim 2, characterized in that, The method further includes the following steps: after the seedlings with roots have been cultivated to be strong, they are hardened off and then transplanted.
4. The method according to claim 3, characterized in that, The plant growth regulators used in the seedling cultivation medium consist of 0.5 mg / L IAA and 1.0 mg / L IBA.
5. The method according to claim 4, characterized in that, The culture medium used for the seedling cultivation is a solid culture medium obtained by adding sucrose, coagulant and plant growth regulator to MS basic culture medium.
6. A composition for propagating Polygonatum multiflorum, characterized in that, The composition comprises the culture medium for inducing callus as described in claim 1, the culture medium for inducing somatic embryos, the culture medium for germinating somatic embryos, and the culture medium for cultivating seedlings as described in claim 4.