Method for obtaining cyclamen regenerated plant by taking leaf as explant

Abstract

The invention relates to the technical field of plant tissue culture, in particular to a method for obtaining cyclamen regenerated plants by taking leaves as explants, which comprises the following steps: S1, taking healthy cyclamen leaves as explants, inoculating into an induction culture medium, and inducing to form calluses; s2, transferring the callus obtained in the step S1 into a proliferation culture medium for subculture proliferation culture; s3, inoculating the callus obtained in S2 into a differential medium, and performing induced differentiation to form adventitious buds; s4, transferring the adventitious buds obtained in S3 into a rooting culture medium, and inducing rooting to obtain cyclamen regenerated plants. According to the method, cyclamen leaves are used as explants, callus is induced to generate and differentiated to generate adventitious buds, cyclamen plants with stable inheritance can be obtained, good characters are kept, meanwhile, orderliness of seedlings is guaranteed, the breeding period is shortened, and the method is suitable for commercialized large-scale production.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, specifically a method for obtaining regenerated cyclamen plants using leaves as explants. Background Technology

[0002] Cyclamen Cyclamen persicum Cyclamen (Cyclamen) is a perennial bulbous flowering plant belonging to the genus Cyclamen in the family Primulaceae. Cyclamen has many varieties, unique flower shapes, vibrant colors, and a long flowering period of up to five months. Its flowering period coincides with traditional festivals such as New Year's Day and the Spring Festival, making it an important potted plant and a popular choice for New Year's celebrations. As a highly ornamental bulbous flower, cyclamen is deeply loved by people.

[0003] Currently, cyclamen seedling production mainly relies on seed propagation. However, this method has significant limitations: on the one hand, long-term self-pollination or inbreeding easily leads to varietal degeneration; on the other hand, newly bred or introduced superior varieties often exhibit severe phenotypic segregation during sexual reproduction, making it difficult to maintain genetic stability and phenotypic consistency. Furthermore, some double-flowered or sterile varieties cannot be propagated through seeds, further limiting their promotion and application. Currently, my country still heavily relies on imports for high-end cyclamen F1 generation seeds, resulting in high costs and significantly hindering the development of my country's cyclamen industry and the improvement of its economic benefits.

[0004] Plant tissue culture technology offers an effective solution to the aforementioned problems. By establishing an efficient in vitro regeneration system, the totipotency of plant cells can be utilized to induce explants to form complete plants through callus formation or direct organogenesis under sterile conditions. This technology not only enables rapid, asexual propagation of superior germplasm while ensuring genetic consistency, but also significantly shortens the breeding cycle. More importantly, the obtained callus or regeneration system can serve as an ideal recipient system, laying a crucial technological foundation for the genetic transformation, gene function research, and molecular design breeding of cyclamen.

[0005] However, existing research indicates that the in vitro regeneration process of cyclamen is influenced by various factors such as genotype, explant type, and culture conditions, resulting in significant differences in regeneration capacity among different varieties. A universally applicable and consistently efficient regeneration method has yet to be established. Although attempts have been made to regenerate cyclamen using cotyledons, hypocotyls, receptacles, or leaves as explants, these methods generally suffer from low callus induction rates, difficulty in differentiation, long regeneration cycles, or poor genetic stability. In particular, regeneration systems using leaves as explants have significant application potential due to their ease of operation and sustainable material availability; however, a systematic optimization and validation of highly efficient and stable techniques is still lacking.

[0006] Therefore, there is an urgent need to establish a tissue culture regeneration method that uses cyclamen leaves as explants, is easy to operate, has high regeneration efficiency, and is genetically stable, in order to meet the practical needs of rapid propagation of superior varieties and modern bio-breeding. Summary of the Invention

[0007] The purpose of this invention is to provide a method for obtaining regenerated cyclamen plants using leaves as explants, in order to solve the problems mentioned in the background art.

[0008] To achieve the above objectives, the present invention provides the following technical solution: a method for obtaining regenerated cyclamen plants using leaves as explants, comprising the following steps: S1: Healthy cyclamen leaves were taken as explants and inoculated into induction medium to induce callus formation; S2: The callus obtained in S1 was transferred to a proliferation medium containing 2,4-dichlorophenoxyacetic acid at a concentration ranging from 0.1 to 1.5 mg / L for subculture. S3: The callus obtained in S2 was inoculated into the differentiation medium to induce differentiation and form adventitious shoots; S4: The adventitious buds obtained in S3 were transferred to the rooting medium to induce rooting and obtain cyclamen regenerated plants.

[0009] In a preferred embodiment: In S1, the induction medium consists of the following components: 1 / 2 MS medium, naphthaleneacetic acid 0.15 mg / L, kinetin 0.2 mg / L, 6-benzylaminopurine 2.0 mg / L, mannitol 20 g / L, glucose 20 g / L, and agar 7.5 g / L.

[0010] In a preferred embodiment: In S2, the proliferation medium is composed of the following: MS medium, 2,4-dichlorophenoxyacetic acid 0.5 mg / L, Kinetin 0.2 mg / L, 6-benzylaminopurine 1.0 mg / L, Mannitol 20 g / L, glucose 20 g / L, and agar 7.5 g / L.

[0011] In a preferred embodiment: In S3, the differentiation medium is composed of the following: MS medium, naphthaleneacetic acid 0.5 mg / L, kinetin 0.2 mg / L, 6-benzylaminopurine 3.0 mg / L, mannitol 20 g / L, glucose 20 g / L, and agar 7.5 g / L.

[0012] In a preferred embodiment: In S4, the rooting medium consists of the following: 1 / 2 MS medium, indole-3-butyric acid 0.5 mg / L, naphthaleneacetic acid 0.01 mg / L, sucrose 30 g / L, and agar 7.5 g / L.

[0013] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention uses cyclamen leaves as explants to induce callus formation and differentiation into adventitious buds. This not only yields stably inherited cyclamen plants, maintaining excellent traits while ensuring uniform seedlings, but also shortens the breeding cycle, making it suitable for large-scale commercial production. 2. This invention uses healthy leaves, which have a wide range of explant sources and can be continuously sampled, as starting material, avoiding dependence on bulbs or seeds. It is especially suitable for the rapid asexual propagation of double-flowered or sterile varieties. The resulting callus tissue is a good recipient for Agrobacterium-mediated genetic transformation of cyclamen and can be used for research on the genetic transformation of cyclamen. 3. This invention significantly improves the callus induction rate to a maximum of 62.97% by optimizing the hormone ratio of the culture medium at each stage and adding osmotic regulators such as mannitol. At the same time, it increases the callus proliferation multiple to a maximum of 2.64 and improves the rooting efficiency of adventitious shoots to 80%. Attached Figure Description

[0014] Figure 1 This is a photograph of callus tissue obtained after culturing cyclamen leaf explants on callus induction medium for 2 weeks in this invention. Figure 2 To be Figure 1 Photographs showing the state of callus tissue after it has been transferred to proliferation medium and subcultured. Figure 3 Photographs of adventitious shoots induced by inoculating proliferating callus tissue into adventitious shoot differentiation medium; Figure 4 To be Figure 3 A photograph showing the successful induction of roots in adventitious buds after they were transferred to a rooting medium. Figure 5 To be Figure 4 The photo shows the adventitious buds that were hardened off and then transplanted into the substrate to grow into complete cyclamen plants. Detailed Implementation

[0015] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Example 1

[0016] This embodiment provides a method for obtaining regenerated cyclamen plants using leaves as explants, including the following steps: S1: On a clean bench, healthy leaves from robust, disease-free cyclamen plants cultivated in a greenhouse were used as explants. The leaves were cut into 1.5 cm × 1.5 cm pieces, and punctured on the underside. Nine explants were inoculated into the induction medium, with the underside facing down, in each culture dish. After two weeks of culture in the dark, callus formation was observed at the leaf margins. Figure 1 As shown.

[0017] The induction medium consisted of the following: 1 / 2 MS medium, naphthaleneacetic acid 0.15 mg / L, kinetin 0.2 mg / L, 6-benzylaminopurine 2.0 mg / L, mannitol 20 g / L, glucose 20 g / L, and agar 7.5 g / L.

[0018] S2: The callus tissue obtained in S1 is transferred to a proliferation medium for subculture, such as... Figure 2 As shown.

[0019] The proliferation medium consisted of the following: MS medium, 2,4-dichlorophenoxyacetic acid 0.1 mg / L, Kinetin 0.2 mg / L, 6-benzylaminopurine 1.0 mg / L, Mannitol 20 g / L, glucose 20 g / L, and agar 7.5 g / L.

[0020] S3: The callus tissue obtained in S2 was inoculated into differentiation medium to induce differentiation and form adventitious shoots, such as Figure 3 As shown.

[0021] The differentiation medium consisted of the following: MS medium, naphthaleneacetic acid 0.5 mg / L, kinetin 0.2 mg / L, 6-benzylaminopurine 3.0 mg / L, mannitol 20 g / L, glucose 20 g / L, and agar 7.5 g / L.

[0022] S4: The adventitious shoots obtained in S3 were transferred to rooting medium and cultured under light conditions. The culture temperature was 25±2℃, the light intensity was 1500~2000 lx, and the photoperiod was 12 hours. The adventitious shoots developed roots on the rooting medium, such as... Figure 4 As shown, this yields regenerated cyclamen plants.

[0023] The rooting medium consisted of the following: 1 / 2 MS medium, indole-3-butyric acid 0.5 mg / L, naphthaleneacetic acid 0.01 mg / L, sucrose 30 g / L, and agar 7.5 g / L.

[0024] The preparation methods for the induction medium, proliferation medium, differentiation medium, and rooting medium are as follows: Dissolve each component in about 800 mL of distilled water, heat and stir until completely dissolved, cool to 50-60℃, and then make up to about 950 mL with distilled water. Adjust the pH to 6.0 with 1 mol / L NaOH solution, and finally make up to 1 L with distilled water.

[0025] For the preparation method of MS medium, please refer to: Murashige T, Skoog F. Physiologia Plantarum, 1962, 15: 473-497.; 1 / 2 MS medium is MS medium with macroelements halved, microelements halved, Fe salts halved, and organic elements unchanged.

[0026] Cyclamen regenerated plants obtained in S4 were hardened off and then transplanted into a cultivation substrate to grow into complete cyclamen plants, such as... Figure 5 As shown. Example 2

[0027] It is basically the same as Example 1, except that the amount of 2,4-dichlorophenoxyacetic acid used in the proliferation medium is 0.1 mg / L. Example 3

[0028] It is basically the same as Example 1, except that the amount of 2,4-dichlorophenoxyacetic acid used in the proliferation medium is 1.0 mg / L. Example 4

[0029] It is basically the same as Example 1, except that the amount of 2,4-dichlorophenoxyacetic acid used in the proliferation medium is 1.5 mg / L.

[0030] Comparative Example 1 It is basically the same as Example 1, except that 2,4-dichlorophenoxyacetic acid is not added to the proliferation medium, while the other components and contents remain unchanged.

[0031] Experimental Example 1: Effect of different concentrations of 2,4-dichlorophenoxyacetic acid on callus proliferation rate Experiment content: Cyclamen callus tissue with consistent growth and no browning, obtained in stage S1 of Example 1, was used as the starting material. Five treatment groups were set up, namely Comparative Examples 1 to 4, and the concentration of 2,4-dichlorophenoxyacetic acid in the proliferation medium corresponding to Comparative Example 1. Each group was set up with 3 replicate culture dishes, and 10 pieces of callus tissue were inoculated in each culture dish.

[0032] Before inoculation, the surface moisture of the callus was blotted dry with sterile filter paper, and the fresh weight of the callus was measured. The culture conditions in S2 of Example 1 were followed. After culture, the callus was removed, the surface moisture was blotted dry with sterile filter paper, the fresh weight was measured, and the proliferation factor was calculated.

[0033] Multiplication factor = Average fresh weight of callus at the end of culture / Average fresh weight of callus before inoculation.

[0034] The statistical results are shown in Table 1.

[0035] Table 1 2,4-Dichlorophenoxyacetic acid concentration (g / L) Multiplication rate Example 1 0.1 1.92 Example 2 0.5 2.64 Example 3 1.0 1.81 Example 4 1.5 1.37 Comparative Example 1 0 1.05 As shown in Table 1, without the addition of 2,4-dichlorophenoxyacetic acid, the callus tissue showed almost no proliferation, indicating that 2,4-dichlorophenoxyacetic acid is an essential plant growth regulator for the proliferation of cyclamen callus tissue. The concentration of 2,4-D in the range of 0.1 to 1.5 mg / L can promote the proliferation of cyclamen callus tissue, with 0.5 mg / L being the optimal concentration.

[0036] Experiment 2: Effects of different culture medium combinations on callus induction rate of cyclamen leaves Experiment content: The explants obtained in S1 of Example 1 were inoculated into the following three groups of culture media: Culture medium A: MS medium, 6-benzylaminopurine 2.0 mg / L, kinetin 0.25 mg / L, naphthaleneacetic acid 0.5 mg / L; Culture medium B: 1 / 2 MS medium, 6-benzylaminopurine 2.0 mg / L, kinetin 0.2 mg / L, naphthaleneacetic acid 0.15 mg / L; Culture medium C: MS medium, 1.0 mg / L thiabendazole, 0.2 mg / L naphthaleneacetic acid.

[0037] Each group was inoculated with 90 explants (divided into 10 dishes, 9 explants per dish), and all were cultured under the same environmental conditions: cultured in the dark at 25±2℃ for 14 days, and the callus induction rate was counted.

[0038] Induction rate (%) = Number of explants that form callus / Total number of inoculated explants × 100%.

[0039] The statistical results are shown in Table 2.

[0040] Table 2 Culture medium categories Induction rate / % Culture medium A 0±0B Culture medium B 62.97±3.70A Culture medium C 0±0B Note: Data are the mean ± standard error of three replicates; different letters (A, B) indicate that the difference is significant at the P<0.01 level.

[0041] As shown in Table 2, only culture medium B can effectively induce callus formation in cyclamen leaves, with the highest induction rate of 62.97%. Neither MS full-volume culture medium nor the combination of thiabendazole replacing traditional cytokinin could induce callus formation. This indicates that the synergistic effect of 1 / 2 MS culture medium and the specific hormone ratio has a key impact on achieving efficient callus induction in cyclamen, thus verifying the effectiveness of the induction culture medium formula disclosed in this invention.

[0042] Experiment 3: Effects of different culture media on the rooting rate of adventitious shoots of cyclamen Experiment content: Cyclamen adventitious shoots with consistent growth status obtained in stage S3 of Example 1 were inoculated into the following two groups of rooting media: Culture medium D: 1 / 2 MS medium, indole-3-butyric acid 0.5 mg / L, naphthaleneacetic acid 0.01 mg / L, sucrose 30 g / L, agar 7.5 g / L; Culture medium E: MS medium, with all other components being exactly the same as culture medium D.

[0043] Twenty-five adventitious buds were inoculated into each group and randomly assigned to five culture dishes (five buds per dish). All buds were cultured under the same environmental conditions: temperature 25±2℃, light intensity 1500–2000 lx, photoperiod 12 h, and culture period 28 days. Root length ≥0.5 cm was used as the standard for rooting, and the rooting rate and average number of roots per seedling were calculated.

[0044] Rooting rate (%) = Number of rooted explants / Number of inoculated explants × 100%.

[0045] The statistical results are shown in Table 3.

[0046] Table 3 Culture medium categories Average number of roots per seedling Rooting rate / % Culture medium D 4 80 Culture medium E 3 60 As shown in Table 3, under the same hormone ratio, the rooting rate of 1 / 2MS medium was significantly higher than that of MS medium, indicating that reducing the inorganic salt concentration is beneficial to the rooting of cyclamen adventitious buds, and verifying the rationality and superiority of using 1 / 2MS medium in the rooting stage in this invention.

[0047] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for obtaining regenerated cyclamen plants using leaves as explants, characterized in that, Includes the following steps: S1: Healthy cyclamen leaves were taken as explants and inoculated into induction medium to induce callus formation; S2: The callus obtained in S1 was transferred to a proliferation medium containing 2,4-dichlorophenoxyacetic acid at a concentration ranging from 0.1 to 1.5 mg / L for subculture. S3: The callus obtained in S2 was inoculated into the differentiation medium to induce differentiation and form adventitious shoots; S4: The adventitious buds obtained in S3 were transferred to the rooting medium to induce rooting and obtain cyclamen regenerated plants.

2. The method for obtaining regenerated cyclamen plants using leaves as explants according to claim 1, characterized in that, In S1, the induction medium composition is as follows: 1 / 2 MS medium, naphthaleneacetic acid 0.15 mg / L, kinetin 0.2 mg / L, 6-benzylaminopurine 2.0 mg / L, mannitol 20 g / L, glucose 20 g / L, and agar 7.5 g / L.

3. The method for obtaining regenerated cyclamen plants using leaves as explants according to claim 1, characterized in that, In S2, the proliferation medium composition is as follows: MS medium, 2,4-dichlorophenoxyacetic acid 0.5 mg / L, Kinetin 0.2 mg / L, 6-benzylaminopurine 1.0 mg / L, Mannitol 20 g / L, glucose 20 g / L, agar 7.5 g / L.

4. The method for obtaining regenerated cyclamen plants using leaves as explants according to claim 1, characterized in that, In S3, the differentiation medium composition is as follows: MS medium, naphthaleneacetic acid 0.5 mg / L, kinetin 0.2 mg / L, 6-benzylaminopurine 3.0 mg / L, mannitol 20 g / L, glucose 20 g / L, agar 7.5 g / L.

5. The method for obtaining regenerated cyclamen plants using leaves as explants according to claim 1, characterized in that, In S4, the rooting medium composition is as follows: 1 / 2 MS medium, indole-3-butyric acid 0.5 mg / L, naphthaleneacetic acid 0.01 mg / L, sucrose 30 g / L, and agar 7.5 g / L.

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