Tissue culture and rapid propagation method of scabiosa cochinchinensis

Abstract

The invention relates to a tissue culture and rapid propagation method of scabiosa cochinchinensis, which belongs to the technical field of plant tissue culture and rapid propagation, and is characterized by comprising the following steps: S1, preparing a culture medium; the culture medium comprises an MS culture medium, a callus induction culture medium, a callus differentiation proliferation culture medium and a rooting induction culture medium; s2, performing induced callus culture; inoculating the explant sample into a callus culture medium until a callus tissue is formed; s3, callus differentiation and proliferation; inoculating explants with calluses into the callus differentiation and proliferation culture medium, and culturing until bud clusters are formed; s4, induced rooting culture; the bud clusters are inoculated into induced rooting culture for rooting culture; s5, transplanting tissue seedlings; after the plant height of the bud clusters is larger than 4 cm, the bud clusters are transplanted into a matrix, the environment humidity is guaranteed, and normal cultivation is conducted; the substrate is formed by mixing peat soil, perlite and sand according to the volume ratio of 4: 3: 2.

Description

Technical Field

[0001] This application belongs to the technical field of rapid plant tissue culture propagation, and specifically relates to a method for rapid tissue culture propagation of Scabiosa columbaria ‘Butterfly Blue’. Background Art

[0002] Scabiosa columbaria ‘Butterfly Blue’ is a perennial herbaceous plant and belongs to an important flower variety. Its flowers are blue-violet, with bright colors, beautiful flower shapes, and relatively long flowering periods, having high ornamental value and being suitable for horticultural scenes such as potted plants and flower beds. In addition, this plant also has certain medicinal value. In Europe and Africa, its leaves, roots, and the whole plant can be used to treat diseases such as respiratory diseases and skin infections.

[0003] In horticultural production, Scabiosa columbaria ‘Butterfly Blue’ cannot maintain its excellent characteristics through seed propagation. At present, it mainly relies on cuttings to maintain excellent germplasm. However, in practice, Scabiosa columbaria ‘Butterfly Blue’ is not easy to root by cuttings, and the propagation efficiency is relatively low, which severely restricts the germplasm preservation, market supply, and industrial development of Scabiosa columbaria ‘Butterfly Blue’. The tissue culture rapid propagation system may be a potential direction to solve this situation.

[0004] In terms of the existing tissue culture technology, there are only a small number of technical documents conducting preliminary research on this.

[0005] Xu Dongting et al. (《Tissue culture and plant regeneration of Scabiosa tschiliensis》, Acta Botanica Sinica, 2011, 46(4): 476 - 478; DOI: 10.3724 / SP.J.1259.2011.00476) and patent CN114208673A (a method for tissue culture of Scabiosa tschiliensis) provided some basic data, but did not propose specific treatment methods and lacked technical detail support. Romeijn and Andr'e A.M. van Lammeren (《Plant regeneration through callus initiation from anthers and ovules of Scabiosa columbaria》, Plant cell, tissue and organ culture, 1999, 56: 169 - 177.) only analyzed the proliferation of its gamete cells through in vitro culture of anthers and ovules of Scabiosa columbaria.

[0006] Takashi Hosoki and Sachie Nojima ("Micropropagation of Scabiosa caucasicabieb. cv. Caucasica blue", In Vitro Cellular & Developmental Biology - Plant; 2004, 40: 482 - 484.) studied explants such as the shoot tips, leaves, and petioles of Scabiosa caucasica and found that 6-benzyladenine (6-BA) and indolebutyric acid (IBA) played important roles in the tissue culture process. To sum up, the current tissue culture and rapid propagation technology for Scabiosa caucasica 'Feige' is still in a blank state, and there is no specific treatment method and culture system for this plant. It is urgent to carry out in-depth research to achieve rapid propagation and industrial application. Summary of the Invention

[0007] To solve the blank existing in the prior art, the present invention explored and tried the culture conditions for each link in the tissue culture process of Scabiosa caucasica 'Feige', such as callus induction, callus differentiation and proliferation, and rooting induction, and obtained a set of efficient and systematic tissue culture and rapid propagation system for Scabiosa caucasica 'Feige'.

[0008] Specifically, the solution of the present invention is as follows: A tissue culture and rapid propagation method for Scabiosa caucasica 'Feige', characterized by comprising the following steps: S1. Prepare the culture medium: MS basal medium: Dissolve MS powder without agar and sucrose at 4.74 g / L, sucrose at 30 g / L, and carrageenan at 7 g / L in water, adjust the pH to 5.8 - 6, and sterilize at 121 °C for 20 minutes to obtain; Callus induction medium: Add thidiazuron 2.0 mg / L and α-naphthaleneacetic acid 0.5 mg / L to the MS basal medium to obtain; The callus differentiation and proliferation medium: same as the MS basal medium; The rooting induction medium is: Add indolebutyric acid 2.0 mg / L to the MS medium to obtain; S2. Induce callus: Inoculate the explant sample of Scabiosa caucasica 'Feige' into the callus induction medium and culture to form callus; S3. Callus differentiation and proliferation: Inoculate the callus into the callus differentiation and proliferation medium and cultivate until adventitious buds are differentiated; S4. Induce rooting: Inoculate the adventitious buds into the rooting induction medium and carry out rooting cultivation to grow into axenic seedlings; S5. Transplant tissue seedlings: After the axenic seedlings are taller than 4 cm, transplant them into the substrate and carry out conventional cultivation.

[0009] Preferably, in the tissue culture rapid propagation method, step S1 further includes: S21. Disinfection of explant samples: Select disease-free leaves or petiole samples of *Ceratostigma plumbaginoides* var. *alba*, rinse for 30 minutes, and then successively disinfect with 75% alcohol for 30 seconds twice, rinse with sterile water, disinfect with 2% sodium hypochlorite for 9 minutes, and rinse with sterile water again. After draining, cut and reserve for use.

[0010] Preferably, in the tissue culture rapid propagation method, the callus differentiation and proliferation medium does not contain 6-benzyladenine.

[0011] Preferably, in the tissue culture rapid propagation method, the explant sample of *Ceratostigma plumbaginoides* var. *alba* is a leaf tissue of *Ceratostigma plumbaginoides* var. *alba* cut into 1 cm×1 cm size; or a petiole of *Ceratostigma plumbaginoides* var. *alba* cut into 1 cm in length.

[0012] Preferably, in the tissue culture rapid propagation method, the culture conditions for inducing callus in S2 are: culture at 25±2°C for 30 days until callus is formed.

[0013] Preferably, in the tissue culture rapid propagation method, the cultivation conditions for callus differentiation and proliferation in S3 are: culture at 25±2°C until adventitious buds are formed.

[0014] Preferably, in the tissue culture rapid propagation method, the culture conditions for inducing rooting in S4 are: culture at 25±2°C for 15 - 20 days until roots grow out from the bud clusters.

[0015] The present invention provides a tissue culture rapid propagation method for *Ceratostigma plumbaginoides* var. *alba*. A specific and efficient technical solution is proposed for the problems of low propagation efficiency, non-specific treatment methods, and lack of specific technical guidance in the prior art.

[0016] By optimizing the ratio of plant growth regulators (such as TDZ 2.0 mg / L + NAA 0.5 mg / L), the present invention achieves a callus induction rate as high as 92.38% (petiole) and 86.67% (leaf). Moreover, the present invention also significantly improves the callus differentiation rate and the number of adventitious bud proliferation by creatively omitting 6-BA in the differentiation and proliferation medium (see Table 3 in the examples and Figure 3 );In the rooting culture stage, by adding IBA (2.0 mg / L) to promote root growth, the rooting rate and plant survival rate are ensured, complex environmental control is avoided, and the production difficulty is further reduced; In the seedling transplanting stage, a mixed substrate of peat soil, perlite, and sand is used, significantly improving the seedling survival rate.

[0017] The present invention provides a complete, systematic and standard method for rapid tissue culture propagation of *Scabiosa comosa* Fisch. ex Roem. et Schult. var. *ciliata* Wulf., which significantly improves the production efficiency. It is not only applicable to large-scale seedling cultivation in horticultural production, but also can be used for subculture preservation of excellent germplasm of *Scabiosa comosa* Fisch. ex Roem. et Schult. var. *ciliata* Wulf., providing effective technical support for commercial supply and medicinal industry development of *Scabiosa comosa* Fisch. ex Roem. et Schult. var. *ciliata* Wulf., and having significant economic and social benefits. Description of the Drawings

[0018] Figure 1 is the result diagram of different concentrations of plant hormones on inducing petiole callus in this application; Figure 2 is the result diagram of different concentrations of plant hormones on inducing leaf callus in this application; Figure 3 is the effect diagram of different concentrations of 6-BA on callus differentiation and adventitious bud proliferation in this application; Figure 4 is the result diagram of the effect of different concentrations of IBA on rooting in this application; Figure 5 is the diagram of the growth of transplanted seedlings in this application. Detailed Description of the Invention

[0019] To make the purpose, technical solutions and advantages of the implementation of this application clearer, the technical solutions of this application will be described exemplarily below in combination with the embodiments and the drawings. Those skilled in the art understand that these exemplary descriptions are used to support the technical solutions claimed by the present invention, rather than to limit its protection scope.

[0020] The following combines the attached Figures 1-5 to further elaborate in detail on the method for rapid tissue culture propagation of *Scabiosa comosa* Fisch. ex Roem. et Schult. var. *ciliata* Wulf. of this application.

[0021] This application discloses a method for rapid tissue culture propagation of *Scabiosa comosa* Fisch. ex Roem. et Schult. var. *ciliata* Wulf., and the materials are selected from the leaves and petioles of *Scabiosa comosa* Fisch. ex Roem. et Schult. var. *ciliata* Wulf. of the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences. The specific steps are as follows: S1. Prepare the culture medium. The culture medium includes: MS medium, callus induction medium, callus differentiation and proliferation medium, and rooting induction medium. MS basal medium: It is prepared by mixing 4.74 g / L of MS powder without agar and sucrose, 30 g / L of sucrose, and 7 g / L of carrageenan, adjusting the pH to 5.8 - 6, and sterilizing at 121 °C for 20 minutes with a horizontal autoclave; Callus induction medium: Prepared by adding plant growth regulators to the MS basal medium; Callus differentiation and proliferation medium: The same as the MS basal medium, that is, prepared by mixing 4.74 g / L of MS powder without agar and sucrose, 30 g / L of sucrose, and 7 g / L of carrageenan, adjusting the pH to 5.8 - 6, and sterilizing at 121 °C for 20 minutes with a horizontal autoclave; Inducing rooting medium: Prepared by adding plant growth regulators to the MS basal medium.

[0022] The plant growth regulators used in this study include: indole butyric acid (IBA), α-naphthaleneacetic acid (NAA), thidiazuron (TDZ), or 6-benzyladenine (6-BA). S21. Sample disinfection. Select disease-free plant leaf and / or petiole samples, wash the dust on the leaf surface with tap water and dish soap, and rinse under running tap water for 30 minutes. After rinsing, pour the samples into a beaker, transfer them into a laminar flow hood, and perform the following disinfection steps in sequence: Disinfect with 75% alcohol for 30 seconds, repeat twice, and then thoroughly wash with sterile water to remove alcohol residue; Subsequently, soak the samples in a 2% sodium hypochlorite solution for 9 minutes for deep disinfection, and rinse again with sterile water to remove disinfectant residue; Finally, gently blot the moisture on the sample surface with sterile filter paper, cut the leaves into explants of 1 cm × 1 cm size; cut the petioles into explants of 1 cm in length for standby.

[0023] S22. Inducing callus culture. Inoculate the disinfected explants into callus induction media with different formulations, and add different concentrations of plant growth regulators to each medium (for specific components and formulations, see Tables 1 and 2). Set 7 culture bottles for each medium formulation, inoculate 5 explants in each bottle, and culture at a constant temperature of 25 ± 2°C for 30 days until callus is formed. Take pictures regularly during the culture process, and count the callus induction rate at the end of the culture.

[0024] S3. Callus differentiation and proliferation. Cut the explants with callus into small pieces of uniform size, and inoculate them into callus differentiation and proliferation media with different components (for specific components and formulations, see Table 3). Set 8 culture bottles for each medium formulation, inoculate 3 callus explants in each bottle, and culture at 25 ± 2°C for about 60 days until a bud cluster is formed. Take pictures regularly during the culture process, and count the differentiation rate and the number of adventitious buds differentiated from each piece of callus at the end of the experiment.

[0025] S4. Inducing rooting culture. Select well-grown and uniform-sized adventitious bud clusters, and inoculate them into rooting media with different formulations (for specific components and formulations, see Table 4). Set 10 culture bottles for each medium formulation, inoculate 3 bud clusters in each bottle, and culture at 25 ± 2°C for about 30 days until the bud clusters form complete roots and the plant height reaches more than 4 cm. Take pictures regularly during the culture process, and count the rooting rate, root length, and number of roots.

[0026] S5. Transplant the tissue seedlings. After the bud cluster height exceeds 4 cm, select the well-rooted seedlings and transplant them into the substrate. The substrate is made by mixing peat soil, perlite, and sand in a volume ratio of 4:3:2. After transplantation, water thoroughly, place the seedlings in the greenhouse, keep the environmental temperature at 20 - 25 °C, and the humidity between 80% - 90%, and conduct normal cultivation. Observe the growth status of the seedlings 15 days after cultivation and calculate the survival rate statistically.

[0027] Experiment and Result Analysis 1. Effects of Plant Growth Regulators at Different Concentrations on Callus Induction from Petioles As shown in Table 1, the disinfected petioles were inoculated into MS-based media supplemented with different concentrations and combinations of plant growth regulators: TDZ (0.00, 1.00, 2.00 mg / L), TDZ (1.00, 2.00 mg / L) + NAA (0.5 mg / L), 6-BA (0.8, 1.00, 2.00 mg / L) + NAA (0.5 mg / L). About 7 days later, swelling occurred around the wound, and light green callus was induced at the wound edge. The callus grew rapidly within 30 days, and a large amount of callus appeared. The states of the callus were different, and the colors were yellowish-green, green, and dark green.

[0028] Table 1: Effects of Plant Growth Regulators at Different Concentrations on Callus Induction from Petioles

[0029] As Figure 1 shown, the results observed 3 days after cultivation showed that no callus could be induced in the MS medium without any hormones; when only TDZ was added, although there was a certain callus induction effect, the formed callus was small; when TDZ and NAA were used in combination, the callus induction effect was the best, and the formed callus was dark green in color and had a compact structure. Among them, the A6 medium (i.e., MS medium + TDZ 2.00 mg / L + NAA 0.05 mg / L) had the best effect on callus induction from petioles; in contrast, when 6-BA and NAA were used in combination, the callus induction effect was the worst, and the produced callus was small and prone to browning.

[0030] In addition, it can also be seen from Table 1 that when TDZ was used alone, within the range of TDZ concentration from 0.00 mg / L to 1.50 mg / L, the callus induction rate gradually increased with the increase in concentration. When TDZ and NAA were used in combination, the induction effect was significantly enhanced. Among them, the induction rate of the A6 medium reached 92.38%, showing the best performance. In contrast, in the treatments with the combination of 6-BA and NAA, the callus induction effects were all very poor, and the induction rates were all lower than 40%. Among them, the induction rate of the control group A1 (without any plant growth regulator) was 0, and no callus was formed.

[0031] In summary, the optimal medium for petiole-induced callus is as follows: 4.74 g / L of MS powder (without agar and sucrose), 30 g / L of sucrose, 7 g / L of carrageenan, 2.0 mg / L of TDZ, and 0.5 mg / L of NAA.

[0032] 2. Effects of Different Concentrations of Plant Growth Regulators on Leaf-induced Callus As shown in Table 2, the disinfected leaves were inoculated into MS-based media supplemented with different concentrations of TDZ (0.00, 1.00, 2.00 mg / L), TDZ (2.00 mg / L) + NAA (0.5 mg / L), 6-BA (0.8, 1.00, 2.00 mg / L) + NAA (0.5 mg / L). About 7 days later, the wound edges of the leaves swelled, and light green callus was induced at the wound margins.

[0033] Table 2: Effects of Different Concentrations of Plant Growth Regulators on Leaf Callus Induction

[0034] As Figure 2 shown and the statistical data in Table 2 show, after 30 days of culture, the results indicate that without adding any plant growth regulators, callus could not be induced. When TDZ was added alone, although there was a certain callus induction effect, the formed callus was small, and adventitious bud differentiation occurred in some callus. When TDZ and NAA were used in combination, the callus induction effect was significantly improved. Among them, the medium of B5: 4.74 g / L of MS powder (without agar and sucrose), 30 g / L of sucrose, 7 g / L of carrageenan, 2.0 mg / L of TDZ, and 0.5 mg / L of NAA had the best callus induction effect on petioles, and the maximum induction rate was 86.67%. In contrast, in the media with the combination of 6-BA and NAA, the callus induction effects were all poor, the formed callus was small, and browning was prone to occur, and the induction rates were all below 10%. As the concentration of 6-BA increased, the degree of callus browning increased, resulting in a further reduction in the induction effect.

[0035] In summary, the optimal medium for leaf-induced callus is as follows: 4.74 g / L of MS powder (without agar and sucrose), 30 g / L of sucrose, 7 g / L of carrageenan, 2.0 mg / L of TDZ, and 0.5 mg / L of NAA.

[0036] 3. Effects of Different Concentrations of 6-BA on Callus Differentiation and Proliferation The callus was inoculated on different callus differentiation and proliferation media, and it was observed that adventitious buds proliferated in all groups, but the differentiation and proliferation situations were very different. Around the 10th day, the color of the callus became darker. Adventitious buds grew out after 20 days. On the 30th day, it was transferred to the same medium. On the 60th day, the adventitious buds proliferated rapidly, and a large number of clustered buds appeared.

[0037] As Figure 3 shown, the callus differentiation and proliferation situation of Group C1 was the best. The adventitious buds grew well, with a dark green color and a relatively high height. As the concentration of 6-BA increased, the color of the adventitious buds after callus differentiation and proliferation turned yellow to a certain extent, and most adventitious buds were relatively short.

[0038] As shown in Table 3, the highest induction rate of callus differentiation and proliferation was in Group C1. This group used a basal medium without 6-BA, and the induction rate reached 82.22%. Under this condition, each callus differentiated into more than 10 adventitious buds, showing strong differentiation ability. In contrast, the worst differentiation effect was in Group C5, where the concentration of 6-BA was 4.00 mg / L, and the induction rate was only 11.76%. The number of adventitious buds differentiated from each callus was less than 3. The overall trend showed that as the concentration of 6-BA increased, the induction rate of callus differentiation and proliferation decreased significantly.

[0039] Table 3: Effects of Different Concentrations of 6-BA on Inducing Callus Differentiation and Adventitious Bud Proliferation

[0040] To sum up, the optimal medium for inducing callus differentiation and proliferation is: the medium without adding 6-BA, and the specific formula is: 4.74 g / L of MS powder (without agar and sucrose), 30 g / L of sucrose, and 7 g / L of carrageenan.

[0041] 4. Effects of Different Concentrations of IBA on Rooting Culture As Figure 4 shown, adventitious buds with good growth status and the same size were inoculated on IBA-induced rooting media with different concentrations, and adventitious buds in all treatment groups successfully rooted. Among them, the rooting time of adventitious buds in Group D3 was about 3 days earlier than that of the other two groups. On the 30th day of culture, the adventitious buds all showed different degrees of growth, the number of roots increased, and the roots further elongated. The observation results showed that the root length of Group D3 was longer, the root and stem were relatively thick, and the rooting effect and growth vigor were better than those of other groups.

[0042] As shown in Table 4, the rooting rates of axenic seedlings in the three different rooting induction media were relatively high. When the concentration of IBA in the medium was 1.00 mg / L and 2.00 mg / L, the rooting rate of axenic seedlings was the highest, reaching 93.33%. Among them, when the IBA concentration was 2.00 mg / L, the average number of roots of axenic seedlings was 9.10, and the proportion of plants with root length exceeding 4 cm reached 86.67%, which was significantly higher than the other two groups. Analyzing the comprehensive rooting rate and root growth, adding an appropriate amount of IBA can effectively promote the rooting induction of adventitious buds and significantly increase the number of roots and root length.

[0043] Table 4: Effects of Different Concentrations of IBA on Rooting

[0044] In summary, the optimal medium for rooting induction is: MS powder (without agar and sucrose) 4.74 g / L, sucrose 30 g / L, carrageenan 7 g / L, and IBA 2.0 mg / L.

[0045] 5. Tissue Seedling Transplanting Test As Figure 5 shown, select axenic seedlings with well-developed roots and plant height greater than 4 cm, take them out of the conical flask, wash the residual medium on the roots, and then transplant the seedlings into the substrate, watering thoroughly during transplantation. After 16 days of cultivation, the plants grew well, with green leaves and strong seedlings. A total of 25 seedlings were transplanted this time, and the survival rate reached 100%.

[0046] 6. Final Experimental Conclusion The optimal medium for inducing callus from petioles and leaves is MS powder (without agar and sucrose) 4.74 g / L, sucrose 30 g / L, carrageenan 7 g / L, TDZ 2.0 mg / L, and NAA 0.5 mg / L. In this medium, the callus induction rate of petioles was 92.38%, and that of leaves was 86.67%. During the culture process, the callus of petioles and leaves began to appear around the 10th day, entered the rapid growth stage after the 20th day, the color gradually changed from light green to dark green, and the tissue structure changed from loose to compact, showing good differentiation potential. In addition, the callus grew evenly without browning, which was suitable for subsequent adventitious bud differentiation culture.

[0047] The optimal medium for inducing callus differentiation and proliferation is MS powder (without agar and sucrose) at 4.74 g / L, sucrose at 30 g / L, and carrageenan at 7 g / L, without adding any plant growth regulators. In this medium, the differentiation induction rate of callus reaches 82.22%, and the average number of adventitious buds differentiated from each callus is 11.8. Adventitious buds start to appear after 30 days. The differentiated adventitious buds are bright green in color and uniform in size. More than 90% of the adventitious buds are in good growth condition, without browning or necrosis, showing good growth vitality.

[0048] The best medium for inducing rooting is MS powder (without agar and sucrose) at 4.74 g / L, sucrose at 30 g / L, carrageenan at 7 g / L, and IBA at 2.0 mg / L. Under these conditions, the rooting rate of adventitious buds is 93.33%, the average number of roots is 9.10, and the proportion of plants with root length exceeding 4 cm is 86.67%. During the cultivation process, the adventitious buds in Group D3 start to root on the 12th day, about 3 days earlier than other groups. The roots after rooting are thick, rich in root hairs, and have many branches. The root color is healthy milky white, showing high vitality and being able to provide a good root basis for subsequent transplantation.

[0049] The transplanting substrate is composed of peat soil, perlite, and sand mixed in a volume ratio of 4:3:2. During transplantation, healthy seedlings with well-developed roots and plant height greater than 4 cm are washed with the root medium and then transplanted into the substrate. After sufficient watering, they are placed in a greenhouse environment (20 - 25 °C, humidity 80% - 90%) for 16 days. A total of 25 seedlings are transplanted in the experiment, and the survival rate reaches 100%. During the 16-day observation after transplantation, all seedlings show a good state of new leaf germination and continued root growth. The leaves are green and healthy, without wilting or withering, showing strong environmental adaptability and growth vitality, verifying the effectiveness and reliability of the rapid propagation method of the present invention.

[0050] As described above, it is only the specific implementation manner of this application, but the protection scope of this application is not limited thereto. Any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed in this application should be covered by the protection scope of this application. Therefore, the protection scope of this application should be subject to the protection scope of the claimed rights.

Claims

1. A method for rapid tissue culture propagation of Scabiosa columbaria subsp. olivieri, characterized in that, It includes the following steps: S1. Prepare the culture medium: MS basal medium: Dissolve MS powder without agar and sucrose at 4.74 g / L, sucrose at 30 g / L, and carrageenan at 7 g / L in water, adjust the pH to 5.8 - 6, and sterilize at 121 °C for 20 minutes to obtain it; Callus induction medium: Add thidiazuron 2.0 mg / L and α-naphthaleneacetic acid 0.5 mg / L to the MS basal medium to obtain it; Callus differentiation and proliferation medium: The same as the MS basal medium; Root induction medium: Add indolebutyric acid 2.0 mg / L to the MS medium to obtain it; S2. Induce callus: Inoculate the explant sample of Ceratostigma plumbaginoides into the callus induction medium and culture to form callus; S3. Callus differentiation and proliferation: Inoculate the callus into the callus differentiation and proliferation medium and cultivate until adventitious buds are differentiated; S4. Induce rooting: Inoculate the adventitious buds into the root induction medium and carry out rooting cultivation to grow into aseptic seedlings; S5. Transplant tissue seedlings: After the height of the aseptic seedlings is greater than 4 cm, transplant them into the substrate and carry out conventional cultivation.

2. According to the tissue culture rapid propagation method described in claim 1, step S2 further includes: S21. Disinfection of explant sample: Select disease-free Ceratostigma plumbaginoides leaf samples or petiole samples, rinse for 30 minutes, and then successively carry out 75% alcohol disinfection for 30 seconds twice, sterile water rinse, 2% sodium hypochlorite disinfection for 9 minutes, and again sterile water rinse. Drain and cut for standby.

3. According to the tissue culture rapid propagation method described in claim 1, the callus differentiation and proliferation medium does not contain 6-benzyladenine.

4. According to the tissue culture rapid propagation method described in claim 1, the Ceratostigma plumbaginoides explant sample is a Ceratostigma plumbaginoides leaf tissue cut into 1 cm × 1 cm size; or a Ceratostigma plumbaginoides petiole cut into 1 cm in length.

5. According to the tissue culture rapid propagation method described in claim 1, the culture conditions for inducing callus in S2 are: culture at 25 ± 2 °C for 30 days until callus is formed.

6. According to the tissue culture rapid propagation method described in claim 1, the cultivation conditions for callus differentiation and proliferation in S3 are: culture at 25 ± 2 °C until adventitious buds are formed.

7. According to the tissue culture rapid propagation method described in claim 1, the culture conditions for inducing rooting in S4 are: culture under the condition of 25 ± 2 °C for 15 - 20 days until roots grow out from the bud cluster.

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