Quasi-frog egg induction method based on chrysanthemum indicum

Abstract

The invention relates to the technical field of frog-like egg induction, in particular to a chrysanthemum indicum-based frog-like egg induction method, which comprises the following steps: S1, obtaining sterile seedlings; s2, performing frog-like egg induction on the explants of the aseptic seedlings to obtain embryos, and performing culture by adopting a frog-like egg induction culture medium containing 0.125-0.375 mg / L of 2, 4-D in the frog-like egg induction process; s3, accelerating germination of the embryos; s4, proliferation induction of cluster buds; s5, rooting induction and seedling hardening. The method comprises the following steps: carrying out frog-like egg body induction culture on an aseptic seedling explant by utilizing a frog-like egg body induction culture medium containing 0.125-0.375 mg / L of 2, 4-D, and starting the generation of frog-like egg bodies in the explant; meanwhile, by establishing a stable and efficient indirect regeneration system mediated by the frog-like spawns, efficient proliferation is realized, and regenerated seedlings derived from a frog-like spawn structure are obtained.

Description

Technical Field

[0001] The invention relates to the technical field of frog-like egg induction, and in particular to a frog-like egg induction method based on wild chrysanthemum. Background Art

[0002] Plant somatic embryogenesis is the asexual reproduction process in which plant somatic cells, through induction or spontaneous reprogramming, undergo development, transform into embryonic cells, and further differentiate into somatic embryos. Conventional structures of plant somatic embryogenesis include the classic SE structure, the protocorm-like structure of the orchid family, and the PLB structure of the Rosaceae family. In addition, novel plant somatic embryo structures such as frog-like ovules, tuberous rhizomes, and bulbils have been disclosed. Among them, the induction and construction of frog-like ovules is more conducive to the large-scale production, enrichment, purification, and development of genetically engineered products such as vaccines, antimicrobial peptides, and plant-derived functional proteins. Existing technologies typically induce tobacco leaf explants using high concentrations of plant growth analogs, but high concentrations of plant growth analogs are highly susceptible to stress, leading to undirected mutations in the regenerated plants. However, there are no reports on the construction of a frog-like ovule system in wild chrysanthemum.

[0003] Wild chrysanthemum is a common plant in the Asteraceae family with important ornamental and medicinal value. Its rich chemical components such as flavonoids, volatile oils, terpenoids, polysaccharides and phenolic acids give it biological activities such as anti-inflammatory, antibacterial, anti-tumor, antioxidant and immunomodulatory. It is used to develop anti-inflammatory and anti-tumor drugs or health foods. Therefore, wild chrysanthemum is required to have the properties of efficient regeneration and detoxification and rapid reproduction.

[0004] The prior art discloses a method for establishing an efficient regeneration system for wild chrysanthemum, which induces wild chrysanthemum rooting by selecting a specific culture medium. However, this method cannot induce the regeneration of wild chrysanthemum leaves, has low efficiency, and exhibits unsatisfactory regeneration effects. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the defects of the prior art in that it cannot induce the regeneration of wild chrysanthemum leaves, and has low efficiency and unsatisfactory regeneration effect, thereby providing a method for inducing frog-like eggs based on wild chrysanthemum.

[0006] To this end, the present invention provides a frog-like egg induction method based on wild chrysanthemum, comprising the following steps: S1, obtaining sterile seedlings; S2, performing frog-like egg induction on the explants of the sterile seedlings to obtain embryos, wherein the frog-like egg induction is performed using a frog-like egg induction medium containing 0.125-0.375 mg / L 2,4-D for culturing; S3, accelerating germination of the embryos; S4, inducing cluster bud proliferation; and S5, inducing rooting and hardening the seedlings.

[0007] In some embodiments, in step S1, sterile seedlings are obtained by germinating the pretreated seeds and culturing the seedlings, and the seed germination step includes culturing the pretreated seeds in a seed germination medium to obtain seedlings.

[0008] In some embodiments, the seed germination medium includes MS inorganic salts, MS organic components, sucrose, plant growth regulator gibberellin, activated carbon and a solidifying agent.

[0009] In some embodiments, the pretreatment step includes seed disinfection and vernalization. Preferably, the seed disinfection step sequentially disinfects the seeds using an ethanol-water solution and a mercuric chloride solution. Preferably, the vernalization temperature is 3-4°C, and the vernalization treatment duration is 2-3 days.

[0010] In some embodiments, the seedling cultivation step includes culturing the sterile seedlings in a sterile seedling culture medium.

[0011] In some embodiments, the sterile seedling culture medium comprises MS inorganic salts, MS organic components, sucrose, additives and a curing agent. Preferably, the additive comprises at least one of activated carbon, silver nitrate, hydrolyzed casein, and polyvinyl pyrrolidone.

[0012] In some embodiments, the concentration of sucrose in the sterile seedling culture medium is 28-30 g / L, the concentration of the additive is 0.1-0.15 wt %, and the concentration of the solidifying agent is 8-8.5 g / L.

[0013] In some embodiments, in step S2, the frog-like egg body induction conditions of the sterile seedling explants are: an induction temperature of 24-26° C., dark conditions, and culturing for 45 days to 60 days.

[0014] In some embodiments, the frog-like egg induction medium further comprises MS inorganic salts, MS organic components, sucrose and a solidifying agent.

[0015] In some embodiments, the explant comprises at least one of a leaf or a root.

[0016] In some embodiments, the solidifying agent in the frog-like egg induction medium and / or the sterile seedling medium includes at least one of agar powder or gellan gum.

[0017] In some embodiments, the concentration of sucrose in the frog-like egg induction medium is 28-30 g / L, and the concentration of the solidifying agent is 3.5-3.6 g / L.

[0018] In some embodiments, in step S3, during embryo germination acceleration, the sterile seedlings are cultured in an embryo germination medium comprising MS inorganic salts, MS organic components, sucrose, a plant growth regulator, thiabendazole, silver nitrate, and a curing agent. Preferably, the embryo germination medium comprises 28-30 g / L of sucrose, 1.0-1.05 mg / L of thiabendazole, 0.1-0.15 wt% of silver nitrate, and 3.5-3.6 g / L of the curing agent.

[0019] In some embodiments, in step S4, when cluster bud proliferation induction is performed, a cluster bud proliferation medium is used for proliferation induction, and the cluster bud proliferation medium comprises MS inorganic salts, MS organic components, sucrose, 6-benzylaminopurine cytokinin, a plant growth regulator indole-3-butyric acid, and a solidifying agent. Preferably, the concentration of sucrose in the cluster bud proliferation medium is 28-30 g / L, the concentration of 6-benzylaminopurine cytokinin is 0.95-1 mg / L, the concentration of indole-3-butyric acid, a plant growth regulator, is 0.15-0.2 mg / L, and the concentration of the solidifying agent is 3.5-3.6 g / L.

[0020] In some embodiments, in step S5, during root induction, rooting is induced using a rooting and proliferation medium comprising MS inorganic salts, B5 organic components, sucrose, a plant growth regulator, indole-3-butyric acid, and a curing agent. Preferably, the rooting and proliferation medium comprises a sucrose concentration of 10-15 g / L, a plant growth regulator, indole-3-butyric acid, at a concentration of 0.1-0.15 mg / L, and a curing agent at a concentration of 8-8.5 g / L.

[0021] In some embodiments, the culture conditions in step S1 and / or step S3 and / or step S4 and / or step S5 are culturing at 24-26°C, sequentially under light and dark conditions, the light culture time is 15-16h, and the light intensity is 150-180μmol.m -2 s -1 , the dark cultivation time is 8-9h.

[0022] In some embodiments, the hardening method includes at least one of a hardening method before transplanting or a hardening method after transplanting.

[0023] The technical solution of the present invention has the following advantages:

[0024] 1. The present invention provides a method for inducing frog-like eggs based on wild chrysanthemum, comprising the following steps: S1, obtaining sterile seedlings; S2, inducing frog-like eggs on the explants of the sterile seedlings to obtain embryos, wherein the frog-like eggs are cultured in a frog-like egg induction medium containing 0.125-0.375 mg / L 2,4-D; S3, accelerating the germination of the embryos; S4, inducing cluster bud proliferation; S5, inducing rooting and hardening the seedlings. The present invention utilizes a frog-like egg induction medium containing 0.125-0.375 mg / L 2,4-D to induce frog-like eggs on sterile seedlings, thereby initiating the occurrence of frog-like eggs in the explants; at the same time, the present invention utilizes proliferation induction culture to establish a stable and efficient indirect regeneration system mediated by frog-like eggs, thereby achieving efficient proliferation and obtaining regenerated seedlings derived from frog-like egg structures.

[0025] 2. The method for inducing frog-like eggs from wild chrysanthemum provided herein comprises obtaining sterile seedlings by germinating pretreated seeds and culturing seedlings in step S1, wherein the seed germination step comprises culturing the pretreated seeds in a seed germination medium to obtain seedlings. By germinating the seeds, the present invention maximizes seed dormancy, improves seed germination efficiency and germination consistency, accelerates root development, and accelerates the completion of seedling morphology.

[0026] 3. The present invention provides a method for inducing frog-like eggs based on wild chrysanthemum, wherein the seed germination medium includes MS inorganic salts, MS organic components, sucrose, plant growth regulator gibberellin, additives and curing agents, wherein gibberellin can promote seed dormancy.

[0027] The embryo germination culture medium includes MS inorganic salts, MS organic components, sucrose, a plant growth regulator thiabendazole, silver nitrate and a curing agent. Among them, the plant growth regulator thiabendazole has a bidirectional regulatory effect, that is, it has the dual effects of an auxin analogue and a cytokinin analogue, which can initiate the redifferentiation of the embryo and complete embryo germination.

[0028] The cluster bud proliferation culture medium comprises MS inorganic salts, MS organic components, sucrose, cytokinin 6-benzylaminopurine, plant growth regulator indole-3-butyric acid and a solidifying agent. 6-benzylaminopurine cells can promote and accelerate plant cell division, indole-3-butyric acid can stimulate plant cell growth, and the synergistic use of 6-benzylaminopurine cells and indole-3-butyric acid can complete plant organogenesis.

[0029] The rooting and proliferation culture medium comprises MS inorganic salt, B5 organic component, sucrose, plant growth regulator indole-3-butyric acid and a solidifying agent, wherein indole-3-butyric acid can promote the morphological construction of roots.

[0030] 4. The frog-like egg induction method based on wild chrysanthemum provided by the present invention is simple, fast, and has a high embryo production rate. It can complete efficient regeneration and detoxification and rapid propagation mediated by frog-like eggs in a short period of time; frog-like eggs are mostly multi-embryo aggregates, and the induction method provided by the present invention can regenerate a large number of clustered buds, which can be used for large-scale propagation of recipient plants and rescue of endangered species; at the same time, the induction method provided by the present invention can achieve a natural detoxification effect because the embryo formation process is physiologically isolated from the mother material. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0032] Figure 1 1 is a physical diagram of each stage of the induction process of the frog-like egg induction method provided in Example 1 of the present invention, wherein: Figure 1 A in the figure is a sterile seedling leaf; Figure 1 A1 in the figure is the FELB-induced state of leaf explants in a culture dish; Figure 1 A2 and Figure 1 A3 in the figure is the initial stage of FELB induction in leaves; Figure 1 A4, A5, and A6 in the middle are the middle stages of leaf FELB development; Figure 1 A7 and A8 in the figure are the mature stages of leaf FELB development; Figure 1 B in the figure is the original explant of the root segment; Figure 1 B1 in the figure is the FELB-induced state of the root segment explant in the culture dish; Figure 1 B2 and B3 in the figure are the initial stages of FELB induction in the root segment; Figure 1 B4 in the figure is the middle stage of root segment FELB development; Figure 1 B5, B6, B7 and B8 are the mature stages of root segment FELB; Figure 1 C and C1 in the figure are the early stages of germination under FELB light; Figure 1 C2, C3 and C4 in the FELB mediate the differentiation of regenerated buds; Figure 1 C5 in the figure is the regenerated solid seedling of FELB; Figure 1 C6 in the figure is the flowering stage of FELB regenerated solid seedlings; Figure 1 C7 in the figure is the initial flowering stage of a single flower of FELB regenerated wild chrysanthemum; Figure 1 C8 is the peak flowering period of a single flower of FELB regenerated wild chrysanthemum. DETAILED DESCRIPTION

[0033] The following examples are provided for a better understanding of the present invention and are not intended to limit the best mode of implementation. They do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the scope of protection of the present invention.

[0034] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in this field can be used. If the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional reagents.

[0035] The seed germination medium (1 / 2MS) in this embodiment and the comparative example has the following formula: 1 / 2MS medium base salt, 1 / 2MS organic, 20g / L sucrose, 0.25mg / L gibberellin (GA3), 0.1wt% activated carbon and 8g / L agar powder, pH 5.8;

[0036] Sterile seedling medium (MS) formula: MS medium base salt, MS organic, 30g / L sucrose, 0.1wt% activated carbon and 8g / L agar powder, pH 5.8;

[0037] FELB induction medium (FELBIM) formulation: MS medium basal salts, MS organic, 30 g / L sucrose, (0-4 mg / L) 2,4-D, and 3.6 g / L gellan gum, pH 5.8;

[0038] SEGE medium (SEGE) formulation: MS medium basal salts, MS organic, 30 g / L sucrose, 1.0 mg / L thiabendazole (TDZ), 0.1 wt% AgNO3, and 3.6 g / L gellan gum, pH 5.8;

[0039] Cluster shoot proliferation medium (CSPM) formulation: MS medium basal salts, MS organic, 30 g / L sucrose, 1.0 mg / L 6-benzylaminopurine (BAP), 0.2 mg / L indole-3-butyric acid (IBA), and 3.6 g / L gellan gum, pH 5.8;

[0040] The rooting and proliferation medium (RM) formula is as follows: 1 / 2 MS culture medium basal salt, 1 / 2 B5 organic, 0.1 mg / L indole-3-butyric acid (IBA), 10 g / L sucrose and 8 g / L agar powder, pH 5.8, wherein the B5 organic components are 1000 mg / L thiamine hydrochloride, 100 mg / L pyridoxine hydrochloride, 100 mg / L niacin, and 10,000 mg / L inositol, and the dilution multiple is 100 times.

[0041] It should be noted that the embodiments of the present invention only use the above culture medium formula as an example, and all culture medium formulas within the protection scope of the present invention can achieve the technical effects of the present invention.

[0042] Example 1

[0043] This embodiment provides a method for inducing frog-like eggs based on wild chrysanthemum. The specific steps and parameters are as follows:

[0044] (1) Seed disinfection and vernalization:

[0045] Select healthy, plump, and well-germinated wild chrysanthemum seeds, first disinfect them with 75% (v / v) ethanol for 1 min and rinse them with sterile water 5 times; then disinfect them with 0.1% (v / v) mercuric chloride for 9 min and rinse them with sterile water 5 times; place the disinfected wild chrysanthemum seeds at the bottom of a 1.5 mL sterile centrifuge tube, seal it with a cap, keep a small amount of sterile distilled water at the bottom of the tube to keep it moist, seal it with parafilm, and place it in a refrigerator at 4°C for vernalization for 72 h.

[0046] (2) Seed germination and sterile seedling culture:

[0047] Sow the vernalized seeds in a tissue culture bottle filled with 1 / 2MS seed germination medium (all ingredients are reduced by half). Place the tissue culture bottle at 25℃, 16h light / 8h dark, and the light intensity is 150μmol.m -2 s -1 After the two cotyledons of wild chrysanthemum are fully expanded, they are transferred to sterile seedling culture medium MS, and two wild chrysanthemum seedlings are planted in each bottle. The culture medium is placed at 25℃ and 16h light (150μmol.m -2 s -1 ) / 8h dark tissue culture room for alternating dark culture.

[0048] (3) Explant selection and FELB induction:

[0049] After the wild chrysanthemum tissue culture seedlings have grown for 35 days, they have 4-5 true leaves and are about 5-10 cm in height. The roots and leaves of the wild chrysanthemum sterile seedlings are selected and cut into 1-2 cm root segments and 1 cm 2 The leaf discs were placed on FELBIM induction medium containing 0.125 mg / L of the plant growth regulator 2,4-D. The root segments were placed horizontally with the leaf discs facing upwards. FELB embryoids were induced at 25°C in the dark.

[0050] In this embodiment, 30 culture dishes were used for each type of explant, with a total of 300 explants as induction objects.

[0051] (4) FELB embryo germination:

[0052] After 16 h of light exposure (150 μmol.m -2 s -1 ) / 8h dark condition, the FELB produced by the above explants were transferred to the embryo germination medium SEGE for FELB embryo germination treatment.

[0053] (5) Induction of sprout proliferation:

[0054] After 16 h of light exposure (150 μmol.m -2 s -1 ) / 8h dark conditions, the cluster buds were cut off from the mother plant and transferred to the cluster bud proliferation medium CSPM to proliferate the number of cluster buds.

[0055] (6) Root induction:

[0056] After 16 h of light exposure (150 μmol.m -2 s -1 ) / 8h dark conditions, the clustered buds were cut off from the mother plant and transferred to the rooting and proliferation medium RM for sterile seedling root induction.

[0057] (7) Acclimation and transplanting of seedlings:

[0058] When the roots of FELB regenerated seedlings grow to more than 5 cm, any of the following methods can be used for seedling hardening.

[0059] Before transplanting, open the bottle cap and harden the seedlings for 7 days, wash off the root agar powder, and transplant the seedlings into the culture medium (vermiculite: flower soil = 1:1) for maintenance. First, place them under weak light for 16 hours (light intensity of 80μmol.m -2 s -1 ) / 8h in darkness indoors and can be transferred to field culture after 7d.

[0060] After transplanting, the seedlings were taken out of the tissue culture bottles, the root agar powder was washed off, and the seedlings were transplanted into the culture medium (vermiculite: flower soil = 1:1). -2 s -1 ) / 8h in darkness and covered with plastic wrap for moisture retention. After 9 days, remove the plastic wrap and transplant to the field for maintenance.

[0061] The materials formed at each cultivation stage of this embodiment are recorded, see Figure 1, after measurement, it can be known that the length of sterile seedling leaves (A), leaf FELB mid-stage development (A6-A8), root segment original explant (B), root segment FELB mid-stage development (B4-B7), early germination under FELB light (C, C1), FELB-mediated regeneration bud differentiation (C2-C4) and FELB regeneration wild chrysanthemum single flower in full bloom (C8) = 1 cm;

[0062] Length of leaf explants in the culture dish in the FELB-induced state (A1) and root segment explants in the culture dish in the FELB-induced state (B1) = 3 cm;

[0063] The length of the leaf at the early stage of FELB induction (A2, A3), the middle stage of leaf FELB development (A4, A5), and the root segment at the early stage of FELB induction (B2, B3) = 0.25 cm;

[0064] Length of root segment FELB at mature stage (B8) = 0.5 cm;

[0065] Length of FELB regenerated plantlets (C5) = 2 cm;

[0066] The length of FELB regenerated solid seedlings at the flowering stage (C6) = 6 cm;

[0067] The initial flowering stage (C7) of a single flower of FELB regenerated wild chrysanthemum is 0.65 cm.

[0068] Example 2

[0069] This embodiment provides a method for inducing frog-like eggs based on wild chrysanthemum. The specific steps and parameters are the same as those in Example 1, except that the concentration of the plant growth regulator 2,4-D in the induction medium FELBIM in step (3) is 0.250 mg / L.

[0070] Example 3

[0071] This embodiment provides a method for inducing frog-like eggs based on wild chrysanthemum. The specific steps and parameters are the same as those in Example 1, except that the concentration of the plant growth regulator 2,4-D in the induction medium FELBIM in step (3) is 0.375 mg / L.

[0072] Example 4

[0073] This embodiment provides a method for inducing frog-like eggs based on wild chrysanthemum. The specific steps and parameters are as follows:

[0074] (1) Seed disinfection and vernalization:

[0075] Healthy, plump, and well-germinated wild chrysanthemum seeds were selected and surface-disinfected with 75% (v / v) ethanol for 30 seconds and rinsed four times with sterile water. The seeds were then disinfected with 0.1% (v / v) mercuric chloride for 10 minutes and rinsed four times with sterile water. The disinfected wild chrysanthemum seeds were placed at the bottom of a 1.5 mL sterile centrifuge tube, sealed with a cap, leaving a small amount of sterile distilled water at the bottom of the tube to keep it moist. The tube was sealed with parafilm and placed in a refrigerator at 3°C for vernalization for 2 days.

[0076] (2) Seed germination and sterile seedling culture:

[0077] The vernalized seeds were sown in a tissue culture bottle filled with 1 / 2MS seed germination medium (all components were reduced by half). The tissue culture bottle was placed at 24℃, 15h light / 9h dark, and the light intensity was 150μmol.m -2 s -1 After the two cotyledons of wild chrysanthemum are fully expanded, they are transferred to sterile seedling culture medium MS, and two wild chrysanthemum seedlings are planted in each bottle. The culture medium is placed at 24℃ and 15h light (180μmol.m -2 s -1 ) / 9h dark tissue culture room for alternating dark culture.

[0078] (3) Explant selection and FELB induction:

[0079] When the wild chrysanthemum seedlings grow for about 30 days, they have 4-5 true leaves and are about 5-10 cm in height. The roots and leaves of the wild chrysanthemum sterile seedlings are selected and cut into 1-2 cm root segments and 1 cm 2 The leaf discs were placed on FELBIM induction medium containing 0.125 mg / L of the plant growth regulator 2,4-D. The root segments were placed horizontally with the leaf discs facing upwards. FELB embryoids were induced at 25°C in the dark.

[0080] In this embodiment, 30 culture dishes were used for each type of explant, with a total of 300 explants as induction objects.

[0081] (4) FELB embryo germination:

[0082] After 16 h of light exposure (180 μmol.m -2 s -1 ) / 8h dark condition, the FELB produced by the above explants were transferred to the embryo germination medium SEGE for FELB embryo germination treatment.

[0083] (5) Induction of sprout proliferation:

[0084] After 15 h of light exposure (180 μmol.m -2 s-1 ) / 9h dark condition, the cluster buds were cut off from the mother plant and transferred to the cluster bud proliferation medium CSPM to proliferate the number of cluster buds.

[0085] (6) Root induction:

[0086] After 15 h of light exposure (180 μmol.m -2 s -1 ) / 9h dark conditions, the clustered buds were cut off from the mother plant and transferred to the rooting and proliferation medium RM for sterile seedling root induction.

[0087] (7) Acclimation and transplanting of seedlings:

[0088] When the roots of FELB regenerated seedlings grow to more than 5 cm, any of the following methods can be used for seedling hardening.

[0089] Before transplanting, open the bottle cap and harden the seedlings for 5 days, wash off the root agar powder, and transplant the seedlings into the culture medium (vermiculite: flower soil = 1:1) for maintenance. First, place them under weak light for 16 hours (light intensity of 80μmol.m -2 s -1 ) / 8h in darkness and can be transferred to field culture after 7d.

[0090] After transplanting, the seedlings were taken out of the tissue culture bottles, the root agar powder was washed off, and the seedlings were transplanted into the culture medium (vermiculite: flower soil = 1:1). -2 s -1 ) / 8h in darkness and covered with plastic wrap to keep it moist. After 10 days, remove the plastic wrap and transplant to the field for maintenance.

[0091] Comparative Example 1

[0092] This comparative example provides a method for inducing frog-like eggs based on wild chrysanthemum. The specific steps and parameters are the same as those in Example 1, except that the induction medium FELBIM in step (3) does not contain 2,4-D.

[0093] Comparative Example 2

[0094] This comparative example provides a method for inducing frog-like eggs based on wild chrysanthemum. The specific steps and parameters are the same as those in Example 1, except that the concentration of the plant growth regulator 2,4-D in the induction medium FELBIM in step (3) is 0.4 mg / L.

[0095] Experimental example

[0096] The number of FELBs produced by the induction method of Example 1-3 and Comparative Example 1-2 was detected, and the results are shown in Table 1. The method for detecting the number of FELBs is to use a double staining method of acetic acid magenta and Evans blue to distinguish embryos from ordinary callus tissue, and the number of FELBs is counted under a stereomicroscope.

[0097] The data in Table 1 were analyzed using SPSS 20.0 for Duncan test, where capital letters indicate a significant difference at 1%, and lowercase letters indicate a significant difference at 5%.

[0098] Table 1 Number of FELB induced in root and leaf explants of Chrysanthemum indicum

[0099]

[0100] According to the data in Table 1, it can be seen that compared with the induction method provided in Comparative Examples 1-2, the present invention can significantly increase the number of induced FELBs by adjusting the concentration of 2,4-D to 0.125-0.375 mg / L. Moreover, when the concentration of 2,4-D is 0.250 mg / L, the number of FELBs produced with roots as explants is 53.23±0.96, and the number of FELBs produced with leaves as explants is 33.90±0.56, indicating that a 2,4-D concentration of 0.250 mg / L is a better choice. At the same time, for the same induction method, the induction effect is better when the explant is a root than when the explant is a leaf.

[0101] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A method for inducing frog-like eggs based on wild chrysanthemum, characterized in that: The following steps are included: S1, obtain sterile seedlings; S2, performing frog-like egg induction on the sterile seedling explants to obtain embryos, wherein the frog-like egg induction medium containing 0.125-0.375 mg / L 2,4-D is used for culturing; S3, embryo germination; S4, induction of cluster shoot proliferation; S5, rooting induction and seedling hardening.

2. The method for inducing frog-like eggs based on wild chrysanthemum according to claim 1, characterized in that: In step S1, sterile seedlings are obtained by germinating the pretreated seeds and culturing the seedlings. The seed germination step includes culturing the pretreated seeds in a seed germination medium to obtain seedlings.

3. The method for inducing frog-like eggs based on wild chrysanthemum according to claim 2, characterized in that: The seed germination accelerating medium comprises MS inorganic salt, MS organic component, sucrose, plant growth regulator gibberellin, activated carbon and a curing agent; and / or, The pre-treatment steps include sterilizing and vernalizing the seeds; and / or, The step of culturing the seedlings comprises culturing the sterile seedlings in a sterile seedling culture medium.

4. The method for inducing frog-like eggs based on wild chrysanthemum according to claim 3, characterized in that: The seed disinfection step comprises disinfecting the seeds with an ethanol aqueous solution and a mercuric chloride solution in sequence; and / or, The vernalization temperature is 3-4°C and the vernalization treatment time is 2-3 days; and / or, The sterile seedling culture medium comprises MS inorganic salts, MS organic components, sucrose, additives and a solidifying agent. The additive includes at least one of activated carbon, silver nitrate, hydrolyzed casein, and polyvinyl pyrrolidone. The sucrose concentration of the sterile seedling culture medium is 28-30 g / L, the additive concentration is 0.1-0.15 wt%, and the solidifying agent concentration is 8-8.5 g / L.

5. The method for inducing frog-like eggs based on wild chrysanthemum according to claim 4, characterized in that: In step S2, the conditions for inducing frog-like eggs from the sterile seedling explants are: inducing at a temperature of 24-26° C., in darkness, and culturing for 45 days to 60 days; and / or, The frog-like egg induction culture medium further comprises MS inorganic salts, MS organic components, sucrose, and a solidifying agent; and / or, The explant includes at least one of a leaf or a root.

6. The method for inducing frog-like eggs based on wild chrysanthemum according to claim 5, characterized in that: The solidifying agent in the frog-like egg induction medium and / or the sterile seedling medium comprises at least one of agar powder or gellan gum; and / or, The concentration of sucrose in the frog-like egg induction medium is 28-30 g / L, and the concentration of the solidifying agent is 3.5-3.6 g / L; and / or, In step S3, when accelerating embryo germination, the sterile seedlings are cultured using an embryo germination medium, wherein the embryo germination medium comprises MS inorganic salts, MS organic components, sucrose, a plant growth regulator thiabendazole, silver nitrate, and a curing agent.

7. The method for inducing frog-like eggs based on wild chrysanthemum according to claim 1, characterized in that: In step S4, when cluster bud proliferation induction is performed, cluster bud proliferation medium is used for proliferation induction, and the cluster bud proliferation medium includes MS inorganic salt, MS organic component, sucrose, 6-benzylaminopurine cytokinin, plant growth regulator indole-3-butyric acid and curing agent.

8. The method for inducing frog-like eggs based on wild chrysanthemum according to claim 1, characterized in that: In step S5, during root induction, a rooting and proliferation medium is used for rooting induction, wherein the rooting and proliferation medium comprises MS inorganic salt, B5 organic component, sucrose, plant growth regulator indole-3-butyric acid and a curing agent.

9. The method for inducing frog-like eggs based on wild chrysanthemum according to claim 1, characterized in that: The culture conditions in step S1 and / or step S3 and / or step S4 and / or step S5 are as follows: culture is carried out at 24-26°C under light and dark conditions, the light culture time is 15-16 hours, and the light intensity is 150-180 μmol.m -2 s -1 , the dark incubation time is 8-9h; and / or, The method of hardening the seedlings includes at least one of a hardening-in method before transplanting or a hardening-in method after transplanting.

10. The method for inducing frog-like eggs based on wild chrysanthemum according to any one of claims 6 to 8, characterized in that: The embryo germination medium comprises a sucrose concentration of 28-30 g / L, a plant growth regulator thiabendazole concentration of 1.0-1.05 mg / L, a silver nitrate concentration of 0.1-0.15 wt % and a curing agent concentration of 3.5-3.6 g / L; and / or, The sucrose concentration in the cluster bud proliferation culture medium is 28-30 g / L, the 6-benzylaminopurine cytokinin concentration is 0.95-1 mg / L, the plant growth regulator indole-3-butyric acid concentration is 0.15-0.2 mg / L, and the solidifying agent concentration is 3.5-3.6 g / L; and / or, The rooting and proliferation culture medium contains sucrose at a concentration of 10-15 g / L, a plant growth regulator indole-3-butyric acid at a concentration of 0.1-0.15 mg / L, and a solidifying agent at a concentration of 8-8.5 g / L.

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