A tissue culture method of nerine bowdenii
By using the flower stalk of Astilbe as an explant, combined with the optimization of specific culture media and hormones, the problem of low propagation efficiency of Astilbe was solved, a highly efficient tissue culture method was achieved, and the rooting rate and propagation efficiency of Astilbe were improved.
Patent Information
- Application Number
- CN202510501642.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-21
- Publication Date
- 2025-11-04
- Estimated Expiration
- 2045-04-21
AI Technical Summary
In existing technologies, conventional propagation methods for Astilbe are inefficient, and leaves used as explants are difficult to disinfect and have a low rooting rate. Therefore, there is a need to develop efficient tissue culture methods.
Astilbe flower stalks were used as explants, and adventitious shoot induction culture, adventitious shoot proliferation culture, and rooting culture were carried out using a culture medium with a specific composition. Hormones and nutrients such as 6-BA, NAA, VB1, IBA, and activated carbon were added to the culture medium, and the culture conditions for each step were optimized.
It improved the survival rate, adventitious bud induction rate and rooting rate of Astilbe, simplified the operation steps, significantly improved the propagation efficiency, and reduced the seasonal restrictions on production.
Smart Images
Figure FT_1 
Figure FT_2 
Figure FT_3
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of plant tissue culture, in particular to a tissue culture method of Astilbe chinensis. BACKGROUND
[0002] Astilbe chinensis is a perennial herb of Astilbe chinensis, which can be used as an ornamental plant and has good medicinal value. The conventional propagation methods of Astilbe chinensis mainly include seeding and stolonization, but both have the problem of low propagation efficiency. The reported tissue culture method uses Astilbe chinensis leaves as explants, however, the Astilbe chinensis leaves are thin and small, and are not resistant to disinfectants in actual operation, and the disinfection is difficult, and the rooting rate of the leaves as explants needs to be improved. Therefore, it is still necessary to develop an efficient tissue culture method of Astilbe chinensis. SUMMARY
[0003] The present application provides a tissue culture method of Astilbe chinensis.
[0004] Specifically, the present application provides the technical solutions described below.
[0005] The present application provides a tissue culture method of Astilbe chinensis, which comprises: using a peduncle of Astilbe chinensis as an explant, and sequentially performing induction of adventitious bud culture, adventitious bud proliferation culture and rooting culture.
[0006] The culture medium used in the induction of adventitious bud culture uses MS medium as a basic culture medium and contains the following components: 6-BA 1.5-2.5 mg / L, NAA 0-0.4 mg / L, VB1 0.5-1.5 mg / L.
[0007] The culture medium used in the adventitious bud proliferation culture uses MS medium as a basic culture medium and contains the following components: 6-BA 0.3-0.6 mg / L.
[0008] The culture medium used in the rooting culture uses 1 / 2MS medium as a basic culture medium and contains the following components: IBA 0.2-0.4 mg / L, activated carbon 0.8-1.5 g / L.
[0009] The present application develops a tissue culture method for Astilbe chinensis. In the research and development process, it is found that using the peduncle of Astilbe chinensis as an explant for tissue culture not only can better tolerate the disinfection treatment of the explant, but also can effectively improve the success rate of inducing adventitious buds, and can ensure a high proliferation rate and rooting rate. Compared with using leaves and other tissues as explants, the tissue culture method has obvious advantages in terms of propagation efficiency and operability.
[0010] The present application further develops suitable culture media for each step in view of the petiole as explants. The selection and dosage of hormones and vitamins in the above-mentioned culture media are very important for the success rate and propagation efficiency of each step of tissue culture. In the induction of adventitious bud culture stage, the addition of 6-BA or 6-BA and NAA in the above-mentioned specific concentration range, and the further addition of vitamin B1 (VB1) on this basis, can reduce the browning rate of explants, significantly improve the induction rate of induced adventitious buds, and VB1 as a nutrient can better cooperate with the above-mentioned hormones to promote the improvement of the induction rate. In the adventitious bud proliferation stage, only the addition of low-concentration 6-BA as a hormone can significantly improve the proliferation efficiency of Rosa roxburghii adventitious buds. In the rooting culture stage, the addition of IBA can effectively promote the rooting of Rosa roxburghii adventitious buds and improve the rooting rate. In combination with the action of activated carbon, the rooting rate can reach 100%, and the rooting culture time is relatively short, which is conducive to improving the tissue culture efficiency.
[0011] Preferably, the culture medium used in the induction of adventitious bud culture uses MS medium as the basic culture medium and contains the following components: 6-BA 1.8-2.2 mg / L (preferably 1.9-2.1 mg / L), NAA 0-0.4 mg / L, VB1 0.8-1.2 mg / L (preferably 0.9-1.1 mg / L).
[0012] Preferably, the culture medium used in the adventitious bud proliferation culture uses MS medium as the basic culture medium and contains the following components: 6-BA 0.4-0.5 mg / L.
[0013] Preferably, the culture medium used in the rooting culture uses 1 / 2MS medium as the basic culture medium and contains the following components: IBA 0.2-0.3 mg / L, activated carbon 0.9-1.1 g / L.
[0014] In addition to containing the above-mentioned hormones and nutrients, the culture medium further contains sugar 25-35 g / L and agar 4-6 g / L.
[0015] Preferably, the sugar is sucrose.
[0016] In some embodiments of the present application, the culture medium used in the induction of adventitious bud culture uses MS medium as the basic culture medium and contains the following components: 6-BA 1.9-2.1 mg / L, NAA 0-0.4 mg / L, VB1 0.9-1.1 mg / L, sucrose 25-30 g / L, and agar 4-6 g / L.
[0017] In some embodiments of the present application, the culture medium used in the adventitious bud proliferation culture uses MS medium as the basic culture medium and contains the following components: 6-BA 0.4-0.5 mg / L, sucrose 25-30 g / L, and agar 4-6 g / L.
[0018] In some embodiments of the present application, the rooting culture uses a 1 / 2MS medium as a basic medium and comprises the following components: IBA 0.2-0.25 mg / L, activated carbon 1-1.1 g / L, sucrose 25-30 g / L, and agar 4-6 g / L.
[0019] Preferably, the pH of the medium used in each step is 5.8-6.0.
[0020] Preferably, the flower stalk used as the explant is a young flower stalk that has not yet flowered.
[0021] Further preferably, the young flower stalk that has not yet flowered is selected from a healthy mother plant free of pests and diseases as the explant.
[0022] In the above tissue culture method, the culture conditions for the induction of adventitious bud culture, adventitious bud proliferation culture, and rooting culture are as follows: culture temperature 21-25°C, light intensity 1500-1800 lux, and light time 8-10 hours / day.
[0023] Preferably, the method comprises the following steps: cutting the flower stalk into small pieces of 1-2 cm in length, inoculating the small pieces on the medium for induction of adventitious bud culture, and then inducing adventitious bud culture; after 25-35 days, inoculating the grown adventitious buds on the medium for adventitious bud proliferation culture after cutting and dividing the adventitious buds; and after another 25-35 days, inoculating the adventitious buds on the medium for rooting culture after cutting the adventitious buds into single plants.
[0024] Preferably, the flower stalk is cut into small pieces of 1-2 cm in length, and then inoculated on the medium for induction of adventitious bud culture after lightly scratching the small pieces.
[0025] Preferably, rooting can be achieved after 13-17 days of rooting culture. The above tissue culture method can achieve a 100% rooting rate for tissue culture of Rudbeckia hirta.
[0026] The above tissue culture method further comprises a step of disinfecting the explant; the disinfection comprises the following steps: first washing the explant with water, and then sequentially disinfecting the explant with 0.5-1% new jieer disinfectant, 70-80% alcohol, and 0.5-1% sodium hypochlorite.
[0027] Preferably, the disinfection time with new jieer disinfectant is 10-20 min; the disinfection time with alcohol is 1-2 min; and the disinfection time with sodium hypochlorite is 5-10 min.
[0028] Further preferably, the disinfection time with new jieer disinfectant is 13-17 min; the disinfection time with alcohol is 1-1.5 min; and the disinfection time with sodium hypochlorite is 8-10 min.
[0029] Preferably, each disinfectant is rinsed with sterile pure water for 4-5 times after disinfection.
[0030] The present application has at least the following advantages: the present application provides a tissue culture method for Rudbeckia, uses peduncle as explant, and optimizes the medium at each step, significantly improves the survival rate, the induction rate of explant-induced adventitious buds, the proliferation rate of adventitious buds, and the rooting rate, effectively improves the propagation efficiency of tissue culture, and has the advantages of simple operation steps and high efficiency, etc., providing an effective method for rapid propagation of Rudbeckia. BRIEF DESCRIPTION OF DRAWINGS
[0031] In order to more clearly illustrate the technical solutions in the present application or prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.
[0032] Figure 1 The figure shows the culture results of inducing adventitious buds in the method of embodiment 1 of the present application.
[0033] Figure 2 The figure shows the culture results of adventitious bud proliferation in the method of embodiment 1 of the present application.
[0034] Figure 3 The figure shows the culture results of inducing adventitious bud rooting in the method of embodiment 1 of the present application. DETAILED DESCRIPTION
[0035] In order to make the purpose, technical solutions and advantages of the present application clearer, the following will combine the drawings in the present application to clearly and completely describe the technical solutions in the present application. Obviously, the described embodiments are some embodiments of the present application, not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the protection scope of the present application.
[0036] The bronopol used in the following examples and comparative examples is a 500 mL solution with 27 g / L-33 g / L of effective ingredient benzalkonium bromide, purchased from Shandong Lierkang Medical Technology Co., Ltd.; the alcohol is 500 mL of 75% disinfectant alcohol purchased from Hangzhou Outech Biotechnology Co., Ltd.; the sodium hypochlorite is a 500 mL solution with 10.0% of effective chlorine content, purchased from Tianjin Zhiyuan Chemical Reagent Co., Ltd.
[0037] The Rudbeckia lilies and the mother plants of Pumi La used in the following examples and comparative examples are selected from Beijing Huaxiang Flower Technology Research Co., Ltd.
[0038] The application is further described in detail below in connection with the accompanying drawings and the comparison results of the relevant data of Examples 1-2 and Comparative Examples 1-2.
[0039] Example 1
[0040] The present embodiment provides a tissue culture method of Rudbeckia hirta, which specifically comprises the following steps:
[0041] 1. Maintenance of mother plant and selection of explants
[0042] (1) Maintenance of mother plant: isolate the mother plant from other varieties and place it in a separate area in a greenhouse for 30-60 days. Remove the withered leaves in time during the growth process and ensure sufficient sunlight to make the plant grow healthy;
[0043] (2) Selection of explants: select the mother plant with healthy growth, good performance and no pests and diseases, and take the young flower stalks without flowers on the mother plant as the explants;
[0044] 2. Cleaning and disinfection of explants
[0045] (1) Rinse: rinse the collected explants with running water for 30 minutes to remove the surface dust, and then dry them in the clean bench.
[0046] (2) Disinfection with new jieer: after drying, cut them into small pieces of about 5 cm for better soaking, and then shock disinfect them with 1% new jieer for 15 minutes, and then rinse them with sterile water for 4-5 times, each time for 1-2 minutes;
[0047] (3) Alcohol disinfection: then shock disinfect them with 75% alcohol for 1 minute, and rinse them with sterile water for 4-5 times, each time for 1-2 minutes;
[0048] (4) Sodium hypochlorite disinfection: finally, shock disinfect them with 1% sodium hypochlorite for 10 minutes, and rinse them with sterile water for 4-5 times, each time for 1-2 minutes, and slightly dry the water. Get the disinfected explants.
[0049] 3. Inducing adventitious buds
[0050] Cut off the parts of the above disinfected small pieces that are infiltrated with disinfectant at both ends, and then cut them into small pieces of about 1.5 cm, lightly scratch the surface, and finally flatly inoculate them into the culture medium for inducing adventitious buds; the culture medium for inducing adventitious buds is as follows: MS medium, 6-BA (6-benzylaminopurine) 2 mg / L, NAA (naphthalene acetic acid) 0.4 mg / L, VB1 (vitamin B1) 1 mg / L, sucrose 30 g / L, agar 5 g / L, pH 6.0; the culture temperature is 23°C, the light intensity is 1800 Lux, and the light duration is 10 h / d.
[0051] 4. Adventitious bud proliferation culture
[0052] The adventitious buds obtained above are cut from the explants, divided into 2-3 adventitious buds for a cluster, and vertically inoculated on the adventitious bud proliferation culture medium; the adventitious bud proliferation culture medium formula is: MS medium, 6-BA (6-benzylaminopurine) 0.5 mg / L, sucrose 30 g / L, agar 5 g / L, pH 6.0; the culture temperature is 23°C, the light intensity is 1800 Lux, and the light duration is 10 h / d.
[0053] 5. Inducing adventitious bud rooting
[0054] The adventitious buds obtained above are cut into single adventitious buds, and the single adventitious buds are inoculated on the adventitious bud rooting culture medium; the adventitious bud rooting culture medium formula is: 1 / 2MS medium, IBA (indole-3-butyric acid) 0.2 mg / L, AC (activated carbon) 1 g / L, sucrose 30 g / L, agar 5 g / L, pH 6.0; the culture temperature is 23°C, the light intensity is 1800 Lux, and the light duration is 10 h / d.
[0055] The parameters involved in the above part of the steps and the culture medium formula used are shown in Table 1.
[0056] Table 1. Parameters involved in each step and culture medium formula used
[0057]
[0058] Example 2
[0059] The example provides a tissue culture method of Rosa roxburghii, which is different from the step process of Example 1 in that the condition parameters of part of the steps and the culture medium formula used are different, and the specific differences are shown in Table 1. Except for the condition parameters and the culture medium listed in Table 1, the method steps of Example 2 are the same as those of Example 1.
[0060] Comparative Example 1
[0061] The comparative example provides a tissue culture method of Rosa roxburghii, which is different from the step process of Example 1 in that the condition parameters of part of the steps and the culture medium formula used are different, and the specific differences are shown in Table 1. Except for the condition parameters and the culture medium listed in Table 1, the method steps of Comparative Example 1 are the same as those of Example 1.
[0062] Comparative Example 2
[0063] The comparative example provides a tissue culture method of Rosa roxburghii, which is different from Example 1 only in that the material for explants is different, and the specific differences are shown in Table 2. Except for the condition parameters listed in Table 2, the method steps of Comparative Example 2 are the same as those of Example 1.
[0064] Table 2 Selection and inoculation of explants of Example 1 and Comparative Example 2
[0065]
[0066] Experimental example
[0067] The Rudbeckia hirta 'Pumila' was subjected to tissue culture using the Rudbeckia tissue culture method provided in Examples 1-2 and Comparative Examples 1-2, the observation parameters of the culture at each stage in the corresponding culture process were recorded, and the contamination rate and mortality rate of the Rudbeckia were calculated, and the specific observation parameters and recorded results are shown in Table 3.
[0068] At the same time, the disinfection effect of the explants and the quality of the adventitious buds during the culture of the Rudbeckia were recorded, and the specific recorded results are shown in Table 4. In addition, the Rudbeckia lilies were subjected to tissue culture using the Rudbeckia tissue culture method provided in Examples 1-2 and Comparative Examples 1-2, and the obtained tissue culture effects were similar to those of the Rudbeckia 'Pumila'.
[0069] Table 3 Results of tissue culture of Rudbeckia (1)
[0070]
[0071] Table 4 Results of tissue culture of Rudbeckia (2)
[0072]
[0073] In combination with the tissue culture methods of Examples 1-2 and Comparative Examples 1-2 and the results of Tables 3 and 4, the present application selects the young flower stalks that have not yet flowered on the mother plants as the explants, and at the same time, selects suitable medium formulations and culture conditions at each tissue culture stage to culture the Rudbeckia, which not only improves the disinfection success rate of the Rudbeckia explants, but also shortens the induction period, improves the induction rate, and improves the adventitious bud proliferation rate. At the same time, the Rudbeckia has good rooting effect, thereby improving the propagation efficiency of the Rudbeckia, reducing the limitation of the season on the production of the variety, and thereby promoting the application of the Rudbeckia.
[0074] Firstly, the selection of suitable mother plant parts as explants for tissue culture of Rudbeckia hirta can directly affect the final result of tissue culture of Rudbeckia hirta. By comparing the culture results of Example 1 and Comparative Example 2, under the same tissue culture conditions and the requirement of no pests and diseases, the explants selected in Example 1 are unflowering young peduncles, while the explants selected in Comparative Example 2 are young leaves. As can be seen from Tables 3 and 4, the contamination rate obtained by using the tissue culture method of Example 1 is 6%, the mortality rate is 0%, a large number of adventitious buds are induced, the growth rate is fast, the plants are relatively strong, and the proliferation rate is high; while the contamination rate obtained by using the tissue culture method of Comparative Example 2 is 8%, the mortality rate is 22%, a large number of calli can be induced, a small number of adventitious buds can be formed, and the time is relatively long and easy to brown. Therefore, the selection of explants has a great influence on the effect of tissue culture of Rudbeckia hirta, and the selection of suitable mother plant parts as explants for tissue culture of Rudbeckia hirta can improve the effect of tissue culture of Rudbeckia hirta. In the present application, the unflowering young peduncles of the mother plant are selected as the explants for tissue culture of Rudbeckia hirta, and the inflorescences are wrapped, which can effectively reduce the damage of various disinfectants to the explants during tissue culture, and the young peduncles have stronger physiological activity and are easier to sprout. Thus, the contamination rate and mortality rate of Rudbeckia hirta are reduced, the germination rate, proliferation rate and rooting rate of Rudbeckia hirta are improved, and the time for inducing adventitious buds of Rudbeckia hirta is shortened.
[0075] In addition, the medium formula and culture conditions selected at each stage of Rudbeckia hirta tissue culture also affect the results of tissue culture. In the steps of inducing adventitious buds and proliferating adventitious buds of explants, the selection of plant hormones is particularly important. By comparing the culture results of Example 1-2 and Comparative Example 1, the time for inducing adventitious buds of Example 1-2 is shorter, the proliferation coefficient is higher, and the adventitious buds are strong; while the induction rate of Comparative Example 1 is lower, there is occasional browning, the proliferation coefficient is not as high as that of Example 1-2, and the adventitious buds appear vitrification and deformity. Therefore, the addition of VB1 in the induction of explants can effectively inhibit the generation of browning, and the selection of appropriate hormone concentration in the proliferation stage is also particularly important. The plant hormones used in the induction of adventitious buds in Example 1-2 are relatively low concentration of 6-BA, while the plant hormones used in the induction of sterile seedlings to form cluster buds in Comparative Example 1 are relatively high concentration of 6-BA. The results show that too high concentration of 6-BA inhibits growth.
[0076] Finally, the addition amount of IBA and activated carbon in the medium for inducing adventitious buds to root has a great influence on rooting. By comparing Example 1-2 and Comparative Example 1, it can be found that Example 1-2 with high concentration of IBA and activated carbon has a rooting rate of 100%, fast rooting, and good rooting effect. While Comparative Example 1 with low concentration of IBA and activated carbon has only a rooting rate of 80%, slow rooting speed, and relatively thin and weak roots.
[0077] In summary, the tissue culture method of Rudbeckia hirta provided by the present application selects the unflowering young peduncle of the mother plant, has fast budding, shortens the induction period, and the adventitious bud growth is robust; the proliferation coefficient is high, and the growth speed is fast. Meanwhile, the rooting effect of Rudbeckia hirta is good, the rooting rate reaches 100%, so that the propagation efficiency and the rooting seedling quality of Rudbeckia hirta are improved, the season limitation on the production of the variety is reduced, and the application of Rudbeckia hirta is promoted.
[0078] As an example, in the process of tissue culture of Rudbeckia hirta by the method of example 1, the culture result of inducing adventitious buds is as shown in Figure 1 , the culture result of adventitious bud proliferation is as shown in Figure 2 , and the culture result of inducing adventitious bud rooting is as shown in Figure 3 .
[0079] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.
Claims
1. A tissue culture method of Rosa roxburghii, characterized by, The method comprises: taking the peduncle of Rosa roxburghii as an explant, and sequentially performing induction of adventitious buds, proliferation of adventitious buds, and rooting culture; The medium used in the induction of adventitious buds is a basic medium of MS and consists of 6-BA 1.5-2.5 mg / L, NAA 0-0.4 mg / L, VB1 0.5-1.5 mg / L, sucrose 25-35 g / L, and agar 4-6 g / L; The medium used in the proliferation of adventitious buds is a basic medium of MS and consists of 6-BA 0.3-0.6 mg / L, sucrose 25-35 g / L, and agar 4-6 g / L; The medium used in the rooting culture is a basic medium of 1 / 2MS and consists of IBA 0.2-0.4 mg / L, activated carbon 0.8-1.5 g / L, sucrose 25-35 g / L, and agar 4-6 g / L.
2. The tissue culture method of Rosa multiflora according to claim 1, characterized by, The medium used in the induction of adventitious buds is a basic medium of MS and consists of 6-BA 1.5-2.5 mg / L, NAA 0-0.4 mg / L, VB1 0.5-1.5 mg / L, sucrose 25-35 g / L, and agar 4-6 g / L; The medium used in the proliferation of adventitious buds is a basic medium of MS and consists of 6-BA 0.3-0.6 mg / L, sucrose 25-35 g / L, and agar 4-6 g / L; The medium used in the rooting culture is a basic medium of 1 / 2MS and consists of IBA 0.2-0.4 mg / L, activated carbon 0.8-1.5 g / L, sucrose 25-35 g / L, and agar 4-6 g / L.
3. The tissue culture method of Rosa roxburghii according to claim 1 or 2, characterized in that, The pH of the medium is 5.8-6.
0.
4. The tissue culture method of Rosa roxburghii according to claim 1 or 2, characterized in that, The peduncle is a young peduncle that has not yet flowered.
5. The tissue culture method of Rosa multiflora according to claim 1 or 2, characterized by, The culture conditions for the induction of adventitious buds, the proliferation of adventitious buds, and the rooting culture are as follows: a culture temperature of 21-25 °C, a light intensity of 1500-1800 lux, and a light time of 8-10 hours per day.
6. The tissue culture method of Rosa multiflora according to claim 1 or 2, characterized by, The method comprises: cutting the peduncle into small pieces of 1-2 cm in length, and flatly inoculating the small pieces on the medium for the induction of adventitious buds to induce adventitious buds; after 25-35 days, cutting the grown adventitious buds and inoculating the adventitious buds on the medium for the proliferation of adventitious buds to proliferate the adventitious buds; and after another 25-35 days, cutting the adventitious buds into single plants and inoculating the single plants on the medium for the rooting culture to perform the rooting culture.
7. The tissue culture method of Rosa roxburghii according to claim 6, characterized in that, The method further comprises a step of disinfecting the explant; The disinfection comprises: first washing the explant with water, and then sequentially disinfecting the explant with 0.5-1% new jieer disinfectant, 70-80% alcohol, and 0.5-1% sodium hypochlorite.
8. The tissue culture method of Rosa roxburghii according to claim 7, characterized in that, The disinfection time of the new jieer disinfectant is 10-20 min; the disinfection time of the alcohol is 1-2 min; and the disinfection time of the sodium hypochlorite is 5-10 min.
Citation Information
Patent Citations
Tissue culture method of allium fistulosum
CN117441617A
Tissue culture method of hemerocallis fulva scape
CN119837044A