Tissue culture method of astilbe chinensis

Abstract

The invention relates to the technical field of plant tissue culture, in particular to a tissue culture method of astilbe chinensis. The astilbe chinensis tissue culture method provided by the invention comprises the following steps: taking astilbe chinensis scapes as explants, and sequentially performing adventitious bud induction culture, adventitious bud multiplication culture and rooting culture. The tissue culture method is provided for the astilbe chinensis, the scapes are adopted as the explants, culture medium optimization of all the steps is matched, the survival rate, the induction rate of adventitious buds induced by the explants, the proliferation rate of the adventitious buds and the rooting rate are remarkably increased, the propagation efficiency of tissue culture is effectively improved, and the tissue culture method has the advantages of being simple in operation step, efficient and the like; and an effective method is provided for rapid propagation of the astilbe chinensis.

Description

Technical Field

[0001] The present invention relates to the technical field of plant tissue culture, and particularly relates to a tissue culture method for Astilbe chinensis. Background Art

[0002] Astilbe chinensis is a perennial herbaceous plant of the genus Astilbe in the family Saxifragaceae. It can be used as an ornamental plant and also has good medicinal value. The conventional propagation methods of Astilbe chinensis mainly include sowing and dividing, but both have problems such as low propagation efficiency. The reported tissue culture methods use the leaves of Astilbe chinensis as explants. However, the leaves of Astilbe chinensis are thin and small in texture, are not resistant to disinfectants during actual operation, are difficult to disinfect, 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 for Astilbe chinensis. Summary of the Invention

[0003] The present invention provides a tissue culture method for Astilbe chinensis.

[0004] Specifically, the present invention provides the following technical solutions.

[0005] The present invention provides a tissue culture method for Astilbe chinensis, the method comprising: using the scape of Astilbe chinensis as an explant, and successively performing adventitious bud induction culture, adventitious bud proliferation culture, and rooting culture; wherein, the culture medium used for the adventitious bud induction culture is based on MS 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; The culture medium used for the adventitious bud proliferation culture is based on MS medium and contains the following components: 6-BA 0.3 - 0.6 mg / L; The culture medium used for the rooting culture is based on 1 / 2MS medium and contains the following components: IBA 0.2 - 0.4 mg / L, activated carbon 0.8 - 1.5 g / L.

[0006] The present invention develops a tissue culture method for Astilbe chinensis. During the R & D process, it is found that using the scape of Astilbe chinensis as an explant for tissue culture can not only better tolerate the disinfection treatment of the explant, but also effectively improve the success rate of adventitious bud induction, and can ensure a high proliferation rate and rooting rate. Compared with using tissues such as leaves as explants, it has obvious advantages in terms of tissue culture propagation efficiency, operability, etc.

[0007] Regarding the scape as the explant, the present invention further develops suitable culture media for each step. 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 tissue culture in each step. Among them, in the stage of inducing adventitious buds, adding 6-BA within the above-mentioned specific concentration range or adding 6-BA and NAA simultaneously, and further adding vitamin B1 (VB1) on this basis, can reduce the browning rate of the explant and significantly improve the induction rate of adventitious buds. As a nutrient, VB1 can cooperate well with the above-mentioned hormones to promote the improvement of the induction rate. In the stage of adventitious bud proliferation, only adding a low concentration of 6-BA as a hormone can significantly improve the proliferation efficiency of Astilbe chinensis adventitious buds. In the stage of rooting culture, adding IBA can effectively promote the rooting of Astilbe chinensis adventitious buds and improve the rooting rate. With the action of activated carbon, a rooting rate of 100% can be achieved, and the rooting culture time is short, which is beneficial to improving the tissue culture efficiency.

[0008] Preferably, the culture medium used for inducing adventitious buds uses the MS culture 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).

[0009] Preferably, the culture medium used for adventitious bud proliferation uses the MS culture medium as the basic culture medium and contains the following components: 6-BA 0.4 - 0.5 mg / L.

[0010] Preferably, the culture medium used for rooting culture uses the 1 / 2MS culture 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.

[0011] In addition to containing the above-mentioned hormones and nutrients, the culture medium also contains 25 - 35 g / L of sugar and 4 - 6 g / L of agar.

[0012] Preferably, the sugar is sucrose.

[0013] In some embodiments of the present invention, the culture medium used for inducing adventitious buds uses the MS culture 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.

[0014] In some embodiments of the present invention, the culture medium used for adventitious bud proliferation uses the MS culture 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.

[0015] In some embodiments of the present invention, the culture medium used for rooting culture is based on 1 / 2 MS culture medium and contains 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.

[0016] Preferably, the pH of the culture medium used in each of the above steps is 5.8 - 6.0.

[0017] Preferably, the flower stalk used as the explant is a young and tender flower stalk that has not bloomed.

[0018] More preferably, a young and tender flower stalk of a disease - and pest - free, vigorously growing mother plant that has not bloomed is selected as the explant.

[0019] In the above tissue culture method, the culture conditions for adventitious bud induction culture, adventitious bud proliferation culture, and rooting culture are: culture temperature 21 - 25 °C, light intensity 1500 - 1800 lux, and light duration 8 - 10 hours per day.

[0020] Preferably, the method includes: cutting the flower stalk into segments with a length of 1 - 2 cm, spreading and inoculating them on the culture medium for adventitious bud induction culture to conduct adventitious bud induction culture; after 25 - 35 days, dividing the adventitious buds that have grown and inoculating them on the culture medium for adventitious bud proliferation culture to conduct adventitious bud proliferation culture; after another 25 - 35 days, cutting the adventitious buds into single plants and inoculating them on the culture medium for rooting culture to conduct rooting culture.

[0021] Preferably, the flower stalk is cut into segments with a length of 1 - 2 cm, lightly scratched on it, and then spread and inoculated on the culture medium for adventitious bud induction.

[0022] Preferably, rooting can occur after 13 - 17 days of rooting culture. Using the above tissue culture method for the tissue culture of Astilbe can achieve a rooting rate of 100%.

[0023] The above tissue culture method further includes a step of disinfecting the explant; the disinfection includes: first rinsing the explant with water, and then successively conducting disinfection with 0.5 - 1% benzalkonium bromide, 70 - 80% alcohol, and 0.5 - 1% sodium hypochlorite.

[0024] Preferably, the time for benzalkonium bromide disinfection is 10 - 20 min; the time for alcohol disinfection is 1 - 2 min; the time for sodium hypochlorite disinfection is 5 - 10 min.

[0025] More preferably, the time for benzalkonium bromide disinfection is 13 - 17 min; the time for alcohol disinfection is 1 - 1.5 min; the time for sodium hypochlorite disinfection is 8 - 10 min.

[0026] Preferably, after each disinfection, the explants are rinsed 4 - 5 times with sterile pure water.

[0027] The beneficial effects of the present invention at least include: The present invention provides a tissue culture method for Astilbe chinensis, using the scape as the explant, and optimizing the culture medium in each step, which significantly improves the survival rate, the induction rate of adventitious buds from the explants, the proliferation rate of adventitious buds, and the rooting rate, effectively improving the propagation efficiency of tissue culture, and having the advantages of simple and efficient operation steps, providing an effective method for the rapid propagation of Astilbe chinensis. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0029] Figure 1 It is a schematic diagram of the culture result of inducing adventitious buds in the method of Example 1 of the present invention.

[0030] Figure 2 It is a schematic diagram of the culture result of adventitious bud proliferation in the method of Example 1 of the present invention.

[0031] Figure 3 It is a schematic diagram of the culture result of inducing adventitious bud rooting in the method of Example 1 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0032] To make the objectives, technical solutions, and advantages of the present invention clearer, the following will clearly and completely describe the technical solutions in the present invention in conjunction with the drawings in the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

[0033] In the following examples and comparative examples, the benzalkonium bromide used as the active ingredient is a 500 mL solution with a content of 27 g / L - 33 g / L of benzalkonium bromide, purchased from Shandong Lilkon Medical Technology Co., Ltd.; the alcohol is 500 mL of 75% disinfected alcohol of Ou Jie, purchased from Hangzhou Otop Biotechnology Co., Ltd.; the sodium hypochlorite is a 500 mL solution with an available chlorine content of 10.0%, purchased from Tianjin Zhiyuan Chemical Reagent Co., Ltd.

[0034] In the following examples and comparative examples, the mother plants of Astilbe chinensis Lace and Primula are selected from Beijing Huaxiang Flower Science and Technology Research Institute Co., Ltd.

[0035] The present invention will be further described in detail below in conjunction with the accompanying drawings and the comparison results of the relevant data of Examples 1-2 and Comparative Examples 1-2.

[0036] Example 1 This example provides a tissue culture method for Astilbe chinensis, which specifically includes the following steps: 1. Maintenance of the mother plant and selection of explants (1) Maintenance of the mother plant: Isolate the mother plant from other varieties and place it in a separate area in the greenhouse for 30-60 days. During the growth process, timely remove the withered and yellow leaves to ensure sufficient sunlight and the healthy growth of the plant; (2) Selection of explants: Select a healthy mother plant with good trait performance and no pests and diseases from the above-mentioned mother plants, and take the young and tender flower scapes that have not blossomed on the mother plant as explants; 2. Cleaning and disinfection of explants (1) Rinsing: First, rinse the collected explants with running water for 30 minutes to wash off the surface dust, and then place them in the ultra-clean workbench to dry.

[0037] (2) Disinfection with benzalkonium bromide: After drying, cut it into small sections about 5 cm long for better soaking, disinfect it with 1% benzalkonium bromide by shaking for 15 minutes, and then rinse it with sterile water 4-5 times, 1-2 minutes each time; (3) Disinfection with alcohol: Then disinfect it with 75% alcohol by shaking for 1 minute, and rinse it with sterile water 4-5 times, 1-2 minutes each time; (4) Disinfection with sodium hypochlorite: Finally, disinfect it with 1% sodium hypochlorite by shaking for 10 minutes, and rinse it with sterile water 4-5 times, 1-2 minutes each time, and slightly dry the moisture. The disinfected explants are obtained.

[0038] 3. Induction of adventitious buds Cut off the parts of the above-mentioned disinfected small sections that are infiltrated by the disinfectant at both ends, then cut them into small sections about 1.5 cm long, gently scratch the surface, and finally spread and inoculate them on the medium for inducing adventitious buds to induce the formation of adventitious buds on the flower scapes; The medium formula for inducing adventitious buds is: MS medium, 6-BA (6-benzylaminopurine) 2 mg / L, NAA (naphthaleneacetic 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.

[0039] 4. Proliferation culture of adventitious buds The adventitious buds obtained above are cut from the explants and divided into clusters of 2 - 3 adventitious buds each, which are then vertically inoculated onto the medium for adventitious bud proliferation. The formula of the medium for adventitious bud proliferation 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.

[0040] 5. Inducing adventitious bud rooting The adventitious buds obtained from the above proliferation are separated into individual adventitious buds, and each individual adventitious bud is inoculated onto the medium for inducing adventitious bud rooting. The formula of the medium for inducing adventitious bud rooting is: 1 / 2 MS 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.

[0041] The parameters involved in some of the above steps and the formula of the medium used are shown in Table 1.

[0042] Table 1 Parameters involved in each step and the formula of the medium used

[0043] Example 2 This example provides a tissue culture method for Astilbe chinensis, and the difference from the step - by - step process of Example 1 lies in that: the conditional parameters of some steps and the formula of the medium used are different, and the specific differences are shown in Table 1. Except for the conditional parameters and the medium listed in Table 1, the method steps of Example 2 are the same as those of Example 1.

[0044] Comparative Example 1 This comparative example provides a tissue culture method for Astilbe chinensis, and the difference from the step - by - step process of Example 1 lies in that: the conditional parameters of some steps and the formula of the medium used are different, and the specific differences are shown in Table 1. Except for the conditional parameters and the medium listed in Table 1, the method steps of Comparative Example 1 are the same as those of Example 1.

[0045] Comparative Example 2 This comparative example provides a tissue culture method for Astilbe chinensis, and the only difference from Example 1 is that: the material selection of the explant is different, as shown in Table 2 specifically. Except for the conditional parameters listed in Table 2, the method steps of Comparative Example 2 are the same as those of Example 1.

[0046] Table 2 Selection and inoculation of explants in Example 1 and Comparative Example 2

[0047] Experimental Example Using the tissue culture methods of Astilbe chinensis provided in Examples 1-2 and Comparative Examples 1-2 to conduct tissue culture on Astilbe chinensis pumila, recording the inspection parameters of the cultures at each stage during the corresponding culture process, and calculating the contamination rate and mortality rate of Astilbe chinensis. The specific inspection parameters and recording results are shown in Table 3.

[0048] Meanwhile, record the disinfection effect of the explants and the quality of the adventitious buds formed during the culture process of Astilbe chinensis. The specific recording results are shown in Table 4. In addition, using the tissue culture methods of Astilbe chinensis provided in Examples 1-2 and Comparative Examples 1-2 to conduct tissue culture on Astilbe chinensis lace, the obtained tissue culture effects are similar to those of Astilbe chinensis pumila.

[0049] Table 3 Tissue culture results of Astilbe chinensis (1)

[0050] Table 4 Tissue culture results of Astilbe chinensis (2)

[0051] Combining the tissue culture methods of Examples 1-2 and Comparative Examples 1-2 and the results in Table 3 and Table 4, the present invention selects the young and tender flower scapes that have not blossomed on the mother plant as explants, and at the same time selects appropriate culture medium formulations and culture conditions at each tissue culture stage to conduct tissue culture on Astilbe chinensis. This not only improves the disinfection success rate of the Astilbe chinensis explants, but also shortens the induction period, increases the induction rate, and increases the multiplication rate of adventitious buds. At the same time, the rooting effect of Astilbe chinensis is good, thereby improving the propagation efficiency of Astilbe chinensis, reducing the limitation of seasons on the production of this variety, and thus promoting the application of Astilbe chinensis.

[0052] First of all, the selection of appropriate parts of the mother plant as explants for the tissue culture of Astilbe chinensis can directly affect the final result of the tissue culture of Astilbe chinensis. 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 explant selected in Example 1 was the young and tender flower scape that had not blossomed, while the explant selected in Comparative Example 2 was the young and tender leaf. It can be seen from Table 3 and Table 4 that the contamination rate obtained by using the tissue culture method of Example 1 was 6%, the mortality rate was 0%, a large number of adventitious buds were induced, the growth rate was fast, the plants were relatively robust, and the proliferation rate was high; while the contamination rate obtained by using the tissue culture method of Comparative Example 2 was 8%, and the mortality rates were 22% respectively. A large amount of callus could be induced to form, and a small number could form adventitious buds, and the time was long and it was easy to turn brown. Thus, it can be seen that the selection of explants has a great influence on the effect of the tissue culture of Astilbe chinensis. Selecting appropriate parts of the mother plant as explants for the tissue culture of Astilbe chinensis can improve the effect of the tissue culture of Astilbe chinensis. The present invention selects the young and tender flower scape that has not blossomed on the mother plant as the explant for the tissue culture of Astilbe chinensis. Since the inflorescence is wrapped, it can effectively reduce the damage of various disinfectants to the explant during the tissue culture process, and the young and tender flower scape has stronger physiological activity and is more likely to germinate. Thereby reducing the contamination rate and mortality rate of Astilbe chinensis, increasing the germination rate, proliferation rate and rooting rate of Astilbe chinensis, and shortening the time for inducing adventitious buds of Astilbe chinensis.

[0053] In addition, the culture medium formula and culture conditions selected at each stage of the tissue culture of Astilbe chinensis will also affect the result of the tissue culture. In the steps of inducing adventitious buds and adventitious bud proliferation of the explant, the selection of plant hormones is particularly important. Comparing the culture results of Examples 1-2 and Comparative Example 1, the time used for inducing adventitious buds in Examples 1-2 was shorter, and the proliferation coefficient was higher, and the adventitious buds were robust; while the induction rate in Comparative Example 1 was lower, occasional browning occurred, and the proliferation coefficient was not as high as that in Examples 1-2, and the adventitious buds showed vitrification and malformation. Therefore, adding VB1 in the stage of inducing explants can effectively inhibit the occurrence of browning, and it is also particularly important to select appropriate hormone concentrations in the proliferation stage. The plant hormones used in Examples 1-2 to induce adventitious bud proliferation were relatively low concentrations of 6-BA, while the plant hormones used in Comparative Example 1 to induce the proliferation of sterile seedlings to form cluster buds were relatively high concentrations of 6-BA. The results show that too high a concentration of 6-BA will instead inhibit growth.

[0054] Finally, the addition amounts of IBA and activated carbon in the culture medium for inducing adventitious bud rooting have a great influence on rooting. By comparing Examples 1-2 and Comparative Example 1, it can be found that in Examples 1-2 with high concentrations of IBA and activated carbon added, the rooting rate was 100%, the rooting was fast, and the rooting effect was good. While in Comparative Example 1 with low concentrations of IBA and activated carbon added, the rooting rate was only 80%, and the rooting speed was slow and the roots were relatively thin and weak.

[0055] In summary, for the tissue culture method of Astilbe chinensis provided by the present invention, the explant is selected as the young and tender flower scape on the mother plant that has not yet flowered. It has a fast budding rate, shortens the induction period, and the adventitious buds grow strong and healthy; it has a high proliferation coefficient and a fast growth rate. At the same time, the rooting effect of Astilbe chinensis is good, and the rooting rate reaches 100%, thereby improving the propagation efficiency and the quality of rooted seedlings of Astilbe chinensis, reducing the limitation of seasons on the production of this variety, and promoting the application of Astilbe chinensis.

[0056] Among them, as an example, during the tissue culture process of Astilbe chinensis using the method of Example 1, the culture results of inducing adventitious buds are as Figure 1 shown, and the culture results of adventitious bud proliferation are as Figure 2 shown, and the culture results of inducing adventitious bud rooting are as Figure 3 shown.

[0057] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some 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 invention.

Claims

1. A tissue culture method of Astilbe chinensis, characterized in that: The method comprises: using the scape of Astilbe chinensis as an explant, sequentially performing adventitious bud induction culture, adventitious bud proliferation culture and rooting culture; The culture medium used for inducing adventitious bud culture is MS culture medium as the 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; The culture medium used for the adventitious bud proliferation culture uses MS culture medium as the basic culture medium and contains the following components: 6-BA 0.3-0.6 mg / L; The culture medium used for the rooting culture uses 1 / 2MS culture medium as the basic culture medium and contains the following components: IBA 0.2-0.4 mg / L and activated carbon 0.8-1.5 g / L.

2. The tissue culture method of Astilbe chinensis according to claim 1, characterized in that: The culture medium used for inducing adventitious bud culture uses MS culture medium as the basic culture medium and contains the following components: 6-BA 1.8-2.2 mg / L, NAA 0-0.4 mg / L, VB1 0.8-1.2 mg / L; The culture medium used for the adventitious bud proliferation culture uses MS culture medium as the basic culture medium and contains the following components: 6-BA 0.4-0.5 mg / L; The culture medium used for the rooting culture uses 1 / 2MS culture medium as the basic culture medium and contains the following components: IBA 0.2-0.3 mg / L and activated carbon 0.9-1.1 g / L.

3. The tissue culture method of Astilbe chinensis according to claim 1 or 2, characterized in that: The culture medium also contains 25-35 g / L of sugar and 4-6 g / L of agar.

4. The tissue culture method of Astilbe chinensis according to claim 3, characterized in that: The sugar is sucrose.

5. The tissue culture method of Astilbe chinensis according to any one of claims 1 to 4, characterized in that: The pH of the culture medium is 5.8-6.

0.

6. The tissue culture method of Astilbe chinensis according to any one of claims 1 to 4, characterized in that: The scape is a young scape that has not bloomed.

7. The tissue culture method of Astilbe chinensis according to any one of claims 1 to 4, characterized in that: The culture conditions for inducing adventitious bud culture, adventitious bud proliferation culture and rooting culture are: culture temperature 21-25°C, light intensity 1500-1800 lux, and lighting time 8-10 hours / day.

8. The tissue culture method of Astilbe chinensis according to any one of claims 1 to 7, characterized in that: The method comprises: cutting the scape into small segments of 1-2 cm in length, spreading and inoculating the segments on a culture medium for inducing adventitious buds to carry out inducing adventitious buds; after 25-35 days, dividing the grown adventitious buds and inoculating the segments on a culture medium for adventitious bud proliferation to carry out adventitious bud proliferation; after another 25-35 days, cutting the adventitious buds into individual plants and inoculating the segments on a culture medium for rooting to carry out rooting.

9. The tissue culture method of Astilbe chinensis according to claim 8, characterized in that: The method further comprises the steps of sterilizing the explant; The disinfection comprises: firstly washing the explant with water, and then disinfecting with 0.5-1% chlorhexidine, 70-80% alcohol and 0.5-1% sodium hypochlorite in sequence.

10. The tissue culture method of Astilbe chinensis according to claim 9, characterized in that: The disinfection time of the chlorhexidine is 10-20 minutes; the disinfection time of the alcohol is 1-2 minutes; and the disinfection time of the sodium hypochlorite is 5-10 minutes.

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