Vernonia patula propagation medium and its tissue culture rapid propagation method

Abstract

The application discloses a guayule propagation culture medium and a tissue culture rapid propagation method thereof, and belongs to the technical field of tissue culture, and the culture medium comprises MS+0.07 mg / L 6-BA+1 g / L activated carbon+2 g / L agar. The method comprises the following steps: primary induction culture: stem segment explants are induced into seedlings; propagation culture: tissue culture seedlings obtained through the primary culture are selected, stems of axillary buds are taken, and subculture propagation culture is carried out to obtain multiple shoots; rooting culture: the multiple shoots are cultured to obtain rooted tissue culture seedlings; and transplanting: the rooted tissue culture seedlings are transplanted to obtain seedlings. The application can solve the technical problems that the subculture propagation process is extremely easy to vitrify and the propagation coefficient is low, can solve the problems that the excellent seed source of guayule is stable in property and rapid propagation, and realizes large-scale production of high-quality and non-toxic guayule seedlings.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of tissue culture technology, more particularly, the present application relates to a kind of Vernonia saligna Propagation Medium and its tissue culture rapid propagation method. BACKGROUND

[0002] Vernonia saligna (Wall.) DC of Compositae plant grows in the mountain slope and humid grassland and marsh of Guangdong, Guangxi, Guizhou, southern Yunnan, etc., the plant of the genus is widely distributed in the tropics and subtropics of the world, at present, about 1000 kinds of Compositae Vernonia plant are known, more than 30 kinds of plants of the genus have been found in China, mainly distributed in the southwest to the southeast, Taiwan, Xinjiang and other regions, there are 22 species in China, among them, there are 14 species in Xishuangbanna. Vernonia saligna is a perennial herb, the leaf can treat high fever, the root can induce labor, the whole plant is used to treat malaria in folk, the Yi people use it to treat sore throat, pulmonary tuberculosis, cough and hemoptysis, uterine prolapse, etc., in addition, the plant of the genus contains herbicide and anti-infection active substances. In recent years, with the increasing market demand, the collection amount of wild resources gradually increases, and the wild Vernonia saligna has a long time of self-reproduction in the wild, which leads to the wild Vernonia saligna resources being close to exhaustion, so introduction and cultivation, artificial reproduction, etc. will become a new way to effectively utilize Vernonia saligna resources. At present, there are few research reports on the propagation of Vernonia saligna, such as the Chinese patent with the publication number CN111418364A discloses a cutting propagation method of Vernonia saligna, but it belongs to cutting propagation, and no research on tissue culture technology has been reported.

[0003] The present application establishes a large-scale production system of Vernonia saligna tissue culture seedlings by using plant tissue culture technology, which can effectively solve the problems of stable and rapid propagation of excellent germplasm traits, and is an effective way to produce high-quality and non-toxic Vernonia saligna seedlings on a large scale, and provides a necessary technical basis for realizing the standardized production of Vernonia saligna cultivation. SUMMARY

[0004] The first object of the present application is to provide a kind of Vernonia saligna Propagation Medium, which improves the sensitive and easy vitrification of Vernonia saligna, and solves the technical problems of low propagation coefficient and easy vitrification in the process of subculture propagation.

[0005] The second object of the present application is to provide a kind of Vernonia saligna tissue culture rapid propagation method, which solves the problems of stable and rapid propagation of excellent germplasm traits of Vernonia saligna, and realizes the large-scale production of high-quality and non-toxic Vernonia saligna seedlings.

[0006] In order to achieve the objects of the present application, the present application provides a kind of Vernonia saligna Propagation Medium for Vernonia saligna subculture propagation culture, which comprises:

[0007] MS+0.07mg / L 6-BA+1g / L activated carbon+2g / L agar.

[0008] During the experiment, it is found that the guayule is particularly sensitive to hormones, and is prone to vitrification after adding hormones, and the bud is prone to curling and other pathological phenomena, and vitrification is prone to occur after adding activated carbon. The subculture propagation medium does not vitrify when the hormones 6-BA and activated carbon are properly matched, and the tissue culture seedlings grow well, the leaf color is dark green, and the germination is more. In an embodiment, the following media are compared:

[0009] ①MS+6-BA 0.5mg / L+NAA 0.2 mg / L;

[0010] ②MS+6-KT 0.5mg / L+NAA 0.5 mg / L;

[0011] ③MS+6-BA 0.1mg / L+NAA 0.01 mg / L;

[0012] ④MS+6-BA 0.2mg / L+IBA 0.05 mg / L;

[0013] ⑤MS+6-BA 0.1mg / L+NAA 0.02 mg / L;

[0014] ⑥MS+6-BA 0.01mg / L+agar 2g / L;

[0015] ⑦MS+6-BA 0.01mg / L+activated carbon 1g / L+agar 2g / L;

[0016] ⑧MS+6-BA 0.05mg / L+activated carbon 1g / L+agar 2g / L;

[0017] ⑨MS+6-BA 0.005mg / L+activated carbon 1g / L+agar 2g / L;

[0018] ⑩MS+6-BA 0.07mg / L+activated carbon 1g / L+agar 2g / L.

[0019] It is found that the guayule tissue culture seedlings of the propagation medium of the application do not vitrify during subculture propagation, and the tissue culture seedlings grow well, the leaf color is dark green, and the germination is more.

[0020] The guayule tissue culture rapid propagation method provided by the application utilizes the above-mentioned guayule propagation medium for subculture propagation culture.

[0021] The guayule tissue culture rapid propagation method provided by the application specifically includes two stages of primary culture and subculture, wherein:

[0022] The primary culture stage includes:

[0023] S11, primary induction culture: inducing the stem explants into seedlings;

[0024] The subculture stage includes:

[0025] S21, proliferation culture: selecting the seedlings obtained in the primary culture, and taking the stems of the axillary buds for subculture and proliferation culture to obtain the multiple shoots;

[0026] S22, rooting culture: culturing the multiple shoots to obtain the rooted seedlings;

[0027] S23, transplanting: transplanting the rooted seedlings to obtain the seedling plants of Flourensia thymifolia.

[0028] Preferably, before the primary induction culture or the proliferation culture, the stems are subjected to disinfection and sterilization and explant browning inhibition culture treatment.

[0029] Preferably, the disinfection and sterilization treatment of the explants is performed by using 75.0% alcohol for 20 s and then 0.1% mercuric chloride for 8 min. In an embodiment, the contamination rate is 0 and the survival rate reaches 66.7%.

[0030] Preferably, the browning inhibition culture is performed by using the medium of MS+0.3 mg / L 6-BA+0.05 mg / L NAA and culturing at 4°C for 12 hours or more. In an embodiment, no obvious browning occurs at 4°C, and the bud induction rate reaches 80.0%.

[0031] Preferably, in the primary induction culture, the medium used is MS+0.02 mg / L 6-BA+0.01 mg / L NAA+25 g / L sucrose+5.0 g / L agar, the induction rate is 77.1%, the adventitious buds grow well, and the seedlings are fast.

[0032] Preferably, in the proliferation culture, the suitable medium for the subculture and proliferation culture of Flourensia thymifolia is MS+0.07 mg / L 6-BA+1.0 g / L activated carbon+2 g / L agar. In an embodiment, the Flourensia thymifolia seedlings do not undergo vitrification during the subculture and proliferation process, and the seedlings grow well with dark green leaves and many buds.

[0033] Preferably, in the rooting culture, the suitable medium for the rooting of Flourensia thymifolia is 1 / 2MS+0.2 mg / L IBA+0.1 mg / L NAA, the rooting rate reaches more than 95%, the number of roots is large, and the average number of roots is 4.

[0034] Preferably, in the transplanting, the most suitable medium is sterilized peat soil: light medium of vermiculite = 3:1, and the survival rate reaches 100%;

[0035] Preferably, when the roots of the guayule are thick and about 1cm long, or the roots are thin and about 3cm long, and the number of roots is 1-2, the survival rate of the transplanting is high, and the highest survival rate reaches 100%.

[0036] The present application at least includes the following beneficial effects:

[0037] 1. The proliferation culture medium for the guayule subculture propagation of the present application, in the subculture propagation process of the guayule tissue culture seedlings, there is no vitrification, and the growth of the tissue culture seedlings is good, the leaf color is dark green, and the germination is more.

[0038] 2. The guayule tissue culture rapid propagation method of the present application optimizes from the aspects of disinfection and sterilization, explant browning inhibition, induction culture, proliferation culture, rooting culture and transplanting, establishes a guayule rapid propagation system, solves the problems of stable and rapid propagation of the guayule excellent seed source, has high survival rate, good seedling growth, fast seedling, no vitrification in the proliferation process, good growth of the tissue culture seedlings, dark green leaf color, more germination, high rooting rate, high survival rate of the transplanting, and can realize the large-scale production of high-quality and non-toxic guayule seedlings.

[0039] Other advantages, objects and features of the present application will be partly embodied by the following description, and partly understood by those skilled in the art through the research and practice of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 Guayule tissue culture seedlings obtained by using the guayule proliferation culture medium of the present application for culture;

[0041] Figure 2 Guayule plant of the present application;

[0042] Figure 3 Guayule adventitious buds of the present application for the primary induction culture;

[0043] Figure 4 Guayule multiple shoots of the present application for the rooting culture;

[0044] Figure 5 Guayule tissue culture seedlings of the present application for the transplanting;

[0045] Figure 6 Guayule tissue culture seedlings of the present application under different medium treatments;

[0046] Figure 7 Guayule tissue culture seedlings of the present application with thick and short root system. DETAILED DESCRIPTION

[0047] The application will be further described in conjunction with examples so that those skilled in the art can implement the application according to the description.

[0048] It should be understood that the terms such as "have", "contain" and "include" used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0049] Example 1

[0050] A propagation medium for guayule, the components of which are: MS+0.07 mg / L 6-BA+1 g / L activated carbon+2 g / L agar.

[0051] Effect experiment

[0052] Select the tissue culture seedlings with consistent growth after primary induction culture, cut the stems with axillary buds of about 1 cm for subculture and propagation culture, and the propagation culture medium is treated as follows:

[0053] ①MS+6-BA 0.5 mg / L+NAA 0.2 mg / L;

[0054] ②MS+6-KT 0.5 mg / L+NAA 0.5 mg / L;

[0055] ③MS+6-BA 0.1 mg / L+NAA 0.01 mg / L;

[0056] ④MS+6-BA 0.2 mg / L+IBA 0.05 mg / L;

[0057] ⑤MS+6-BA 0.1 mg / L+NAA 0.02 mg / L;

[0058] ⑥MS+6-BA 0.01 mg / L+agar 2 g / L;

[0059] ⑦MS+6-BA 0.01 mg / L+activated carbon 1 g / L+agar 2 g / L;

[0060] ⑧MS+6-BA 0.05 mg / L+activated carbon 1 g / L+agar 2 g / L;

[0061] ⑨MS+6-BA 0.005 mg / L+activated carbon 1 g / L+agar 2 g / L;

[0062] ⑩MS+6-BA 0.07 mg / L+activated carbon 1 g / L+agar 2 g / L.(This example)

[0063] The additional agar treatment mainly investigated the effect of agar dosage on vitrification. Five bottles were used for each treatment, with four buds inoculated in each bottle. The treatment was repeated three times, and the proliferation coefficient and growth were observed and recorded at any time. The results are shown in Table 1 below.

[0064] Table 1 Effects of different treatments on tissue culture seedling proliferation culture

[0065]

[0066]

[0067] Tissue culture seedlings of *Chrysanthemum indicum* are highly sensitive to hormones during subculture and are prone to vitrification, resulting in a low proliferation coefficient. Table 1 shows that, comparing treatments ⑦-⑩, vitrification also occurred in the culture medium containing activated carbon. However, in treatment ⑩ of this example, when the ratio of hormone 6-BA to activated carbon was appropriate, vitrification did not occur, and the tissue culture seedlings exhibited better growth, darker green leaves, and more buds. Figure 1 As shown in the figure, treatment with 6-BA concentrations above 0.1 mg / L during subculture resulted in flocculent callus tissue and vitrification at the bud base. In the medium containing KT, there was almost no proliferation effect, indicating that KT cannot promote bud proliferation. Considering both bud growth and proliferation coefficient, the most suitable subculture medium for *Illicium verum* is MS medium + 0.07 mg / L 6-BA + 1.0 g / L activated charcoal + 2 g / L agar.

[0068] Example 2

[0069] A rapid propagation method for *Asteris lanceolata* via tissue culture includes:

[0070] Step 1: Disinfection and sterilization:

[0071] Select healthy, virus-free young stem segments of *Aster spp.* (also known as *Aster spp.*) and cut them into 1.0–2.0 cm long sections with axillary buds. Soak the segments in a detergent solution containing 1% dish soap for 5–8 minutes, then rinse them under running tap water for about 30 minutes. Next, disinfect the segments with 75% alcohol on a clean bench for 30 seconds, then rinse 3–5 times with sterile water. Finally, sterilize the segments with 0.1% mercuric chloride for 8 minutes, then rinse 5 times with sterile water. Place the segments in a shallow, autoclaved stainless steel dish, trim the terminal portion of the stem, and make a cross-shaped incision on the leaves for inoculation. This completes the initial treatment. (The text then repeats the description of *Aster spp.* plants.) Figure 2 As shown.

[0072] Step 2: Browning Inhibition Culture of Explants

[0073] The non-browning stem segments after sterilization in step one were placed in browning inhibition medium, and the browning inhibition medium was treated with MS+0.3 mg / L 6-BA+0.05 mg / L NAA+4 ℃ low temperature treatment, wherein the low temperature treatment was that the explants were placed in a 4 ℃ refrigerator for 12 h or more.

[0074] Step three, primary induction culture:

[0075] The stem segments after browning inhibition treatment were placed in the primary induction medium, and the culture obtained the tissue culture seedlings. The primary induction medium was treated with MS+0.02 mg / L 6-BA+0.01 mg / L NAA+25 g / L sucrose+5.0 g / L agar. The average light intensity of the culture room was 2 000 lx, the light time was 12 h / d, and the temperature was (24±2) ℃. More than 75% of the primary induction of Flourensia thurifera had good growth of adventitious buds and fast seedling, as shown in FIG. 2. Figure 3

[0076] Step four, subculture proliferation culture:

[0077] The tissue culture seedlings with consistent growth in the primary induction culture were selected, and the stem segments with axillary buds of about 1 cm were cut for proliferation culture, and the Flourensia thurifera multiple shoots were obtained. The proliferation medium was MS+0.07 mg / L 6-BA+1 g / L activated carbon+2 g / L agar. The multiple shoots had good growth, dark green leaf color, and 3-5 new shoots.

[0078] Step five, rooting culture:

[0079] The multiple shoots of Flourensia thurifera obtained by proliferation culture were transferred into the rooting medium, and the rooting medium was treated with 1 / 2MS+0.2 mg / L IBA+0.1 mg / L NAA. The rooting time was 5-7 d, and the rooted tissue culture seedlings were obtained, as shown in FIG. 4. After culture, the root system was strong, the number of roots was large, and the rooting rate was more than 95.0%. Figure 4

[0080] Step six, transplanting:

[0081] After rooting culture, the rooted Flourensia thurifera tissue culture seedlings with a height of about 6 cm, vigorous growth, and developed root system (thick root system, 3-4 roots, and a length of about 1 cm) were selected and placed in a normal temperature room. After 2 d, the cover was opened to allow the Flourensia thurifera tissue culture seedlings to fully contact with the air, and water was sprayed on the tissue culture seedlings in the bottle to keep the water in the bottle sufficient. After 3 d, the tissue culture seedlings were taken out of the bottle, washed to remove the culture medium on the roots, and transplanted into the light medium of sterilized peat soil:vermiculite=3:1, moderately shaded, and kept a certain humidity. After 30 d of culture, the survival rate of the Flourensia thurifera tissue culture seedlings was more than 95%. The transplanted Flourensia thurifera tissue culture seedlings are shown in FIG. 5. Figure 5

[0082] ​​​Effect experiment

[0083] 1. The effect of different sterilization time on the induction of stem segments

[0084] Select healthy V. glabra stems and cut them into 1-2 cm stem segments with axillary buds. After the preliminary treatment, the stem segments are sterilized with 75% alcohol for 0s, 10s, 20s, and 30s, respectively. The corresponding sterilization time with 0.1% HgCl2 is 8 min, 8 min, 8 min, 8 min, and 0 min, respectively. The explants are inoculated on the medium containing only MS for culture. Each treatment has 20 bottles, each bottle inoculates one explant, and the experiment is repeated three times. The contamination rate and survival rate are observed in time to determine the best sterilization method.

[0085] Table 2. The effect of different sterilization time on the induction of stem segments

[0086]

[0087] As shown in Table 2, the young stem segments of V. glabra are sterilized with different methods, and both 75% alcohol and 0.1% HgCl2 sterilization have no contamination. The contamination rate of 30s with only 75% alcohol sterilization is 75.0%, and the contamination rate of 0.1% HgCl2 sterilization is 10%. Although the survival rate of both is 100%, the contamination rate is high. The contamination rate of 75.0% alcohol sterilization for 20s and 0.1% HgCl2 sterilization for 8 min is 0, and the survival rate is 66.7%. Therefore, the best sterilization method for V. glabra explants is 75.0% alcohol sterilization for 20s and 0.1% HgCl2 sterilization for 8 min.

[0088] 2. The effect of different browning inhibition treatments on explant browning

[0089] The non-browning stem segments after sterilization are inoculated into different treatment media, and the medium treatments are as follows:

[0090] ① MS + 6-BA 0.3 mg / L + NAA 0.05 mg / L + PVPP 0.5 g / L;

[0091] ② MS + 6-BA 0.3 mg / L + NAA 0.05 mg / L + PVPP 1 g / L;

[0092] ③ MS + 6-BA 0.3 mg / L + NAA 0.05 mg / L + activated carbon 0.5 g / L;

[0093] ④ MS + 6-BA 0.3 mg / L + NAA 0.05 mg / L + activated carbon 1 g / L;

[0094] ⑤MS+6-BA 0.3 mg / L+NAA 0.05 mg / L+4℃ low temperature treatment;

[0095] Wherein the low temperature treatment is that the explants are placed in 4℃ refrigerator for more than 12 hours, and the unit of the hormone is as follows: 6-BA 0.3 means 0.3 mg / L 6-BA, and the same below. 20 bottles are used for each treatment, and 1 explant is inoculated in each bottle, and the browning is observed and recorded in time, and the browning rate is counted, and the explants with the least browning are selected for the next step.

[0096] Table 3 Influence of different browning inhibition treatments on explant browning

[0097]

[0098] It can be seen from Table 3 that a certain concentration of PVPP, activated carbon and low temperature treatment can inhibit the browning of Flourensia floridana. In the PVPP treatment, the browning effects of different concentrations of PVPP are different, and the browning rate of 0.5 g / L PVPP is lower than that of 1.0 g / L PVPP, but the induction rate is also lower than that of the latter; different activated carbon treatments have the same effect on inhibiting browning, and the browning rate is 25.0%, but the bud induction rate of 0.5 g / L activated carbon is higher, and the browning rate is 75.0%. Low temperature treatment has no obvious browning, and the bud induction rate reaches 80.0%. Therefore, the best method of browning treatment is 4℃ low temperature treatment.

[0099] 3, Influence of different hormone ratios on stem induction

[0100] The stems treated by browning inhibition treatment are inoculated into different hormone ratio culture media, and the culture media are treated as follows:

[0101] ①MS;

[0102] ②MS+6-BA 0.5 mg / L;

[0103] ③MS+6-BA 0.5 mg / L+NAA 0.2 mg / L;

[0104] ④MS+6-BA 0.5 mg / L+NAA 0.05 mg / L;

[0105] ⑤MS+6-BA 0.5 mg / L+NAA 0.1 mg / L;

[0106] ⑥MS+KT 0.5 mg / L+NAA 0.1 mg / L;

[0107] ⑦MS+6-BA 0.5 mg / L+KT 0.2 mg / L+NAA 0.2 mg / L;

[0108] MS+6-BA 0.5 mg / L + NAA 0.05 mg / L;

[0109] MS+6-BA 0.8 mg / L + NAA 0.1 mg / L;

[0110] MS+6-BA 1.0 mg / L + NAA 0.1 mg / L;

[0111] MS+6-BA 0.02 mg / L + NAA 0.01 mg / L.

[0112] Each treatment medium also includes 25 g / L sucrose + 5.0 g / L agar, each treatment is inoculated 35 bottles, each bottle is inoculated with 1 explant, repeated 3 times, and the induction effect is observed and recorded at any time.

[0113] Table 4 Effect of different hormone ratios on stem induction

[0114]

[0115]

[0116] The primary induction of stem segments of Flourensia thurifera was carried out with different hormone ratios. It was found that Flourensia thurifera was sensitive to hormones during the induction process; higher hormone concentrations were easy to cause flocculent callus and vitrification at the base, which was not conducive to subsequent seedling formation. As can be seen from Table 4, the MS medium without any hormone had better bud growth than other media with added hormones, but the induction rate was the lowest, only 28.6%. When the concentration of cytokinin 6-BA was 0.5 mg / L, different concentrations of auxin NAA were added, with the increase of NAA concentration, callus appeared at the base, and the induction rate also decreased, which had a certain influence on stem induction, therefore, the concentration of NAA should be below 0.05 mg / L; when the concentration of 6-BA was greater than 0.5 mg / L, the growth of buds was also poor, and tender buds were easy to curl and vitrify; when 6-BA was 0.02 mg / L and NAA was 0.01 mg / L, the induction rate reached 77.1%, the adventitious buds grew well, the seedlings grew fast, and the leaves were green. In general, from the growth condition, although the medium containing hormones had a high induction rate, too high hormone concentration was easy to cause flocculent callus and vitrification, which was not conducive to subsequent seedling formation, therefore, the best medium for primary induction of tender stem segments of Flourensia thurifera was MS+0.02 mg / L 6-BA+0.01 mg / L NAA, which had a high induction rate, strong seedlings, good growth, fast seedling formation, and green leaves.

[0117] 4、Different rooting treatments on the growth of Flourensia thurifera tissue culture seedlings

[0118] The healthy and 2cm high tissue culture seedlings were transferred into rooting medium, and the rooting medium treatments were as follows:

[0119] ①MS+6-BA 0.25+NAA 0.2;

[0120] ②1 / 2MS;

[0121] ③1 / 2MS+NAA 0.15;

[0122] ④1 / 2MS+NAA 0.05;

[0123] ⑤1 / 2MS+IBA 0.1+activated carbon 1g / L;

[0124] ⑥1 / 2MS+NAA 0.05+IBA 0.1;

[0125] ⑦1 / 2MS+NAA 0.15+IBA 0.1;

[0126] ⑧1 / 2MS+IBA 0.2;

[0127] ⑨1 / 2MS+NAA 0.05+IBA 0.1+activated carbon 1g / L;

[0128] ⑩1 / 2MS+IBA 0.1;

[0129] 1 / 2MS+0.2mg / L IBA+0.1mg / L NAA+25g / L sucrose+5g / L agar.

[0130] Each treatment was repeated 3 times, 4 seedlings were inoculated in each bottle, and the rooting and growth were observed and counted at any time.

[0131] Table 5 Effects of different hormone combinations on rooting of tissue culture seedlings

[0132]

[0133] From Table 5, it can be seen that the rooting time of the Eupatorium odoratum tissue culture seedlings is 5-7 days, and the 1 / 2MS medium is more suitable for rooting culture. Different proportions and concentrations of auxin can induce the rooting of the tissue culture seedlings, but different auxins, different concentrations and different proportions have different effects on rooting. In the rooting medium with only IBA, within a certain range, the roots become thicker and stronger with the increase of the concentration, and the rooting rate also increases obviously. In the rooting medium with only NAA, the root growth coefficient is low at low concentration, and the bud growth is weak; in the rooting medium with both NAA and IBA, the root growth is the same, but the seedling growth is stronger with higher concentration of NAA. In the rooting medium without any auxin, the roots are thin and long, which is not conducive to transplanting, indicating that rooting requires growth hormone. In addition, the addition of activated carbon has no significant effect on root growth. According to the rooting rate, average number of roots and root growth of the roots, the most suitable rooting medium for Eupatorium odoratum is treatment 1 / 2MS+0.2mg / L IBA+0.1mg / L NAA+25g / L sucrose+5g / L agar, and the rooting rate of the tissue culture seedlings cultured in the medium is more than 95%, the root system is thick and strong, the leaves are green, and the plant is strong.

[0134] 5、Different substrate treatments on the transplantation of tissue culture seedlings

[0135] Select the healthy, about 6cm high root culture obtained after rooting tissue culture seedlings for transplanting. First, acclimate the seedlings at room temperature for 1 day, then open the lid for 2 days, then wash and remove the excess medium, and then move to different substrates for culture. After one month, the survival rate and growth of the transplanted seedlings are counted.

[0136] Table 6 Effect of different substrate treatments on the transplantation of tissue culture seedlings

[0137]

[0138] The above light substrate is in a mass ratio of 3:1 of peat soil:vermiculite.

[0139] From the above table and Figure 6 It can be seen that the survival rate of the light substrate is the highest, reaching 100%, the transplanted seedlings are strong and grow fast; the transplanted seedlings of the mixed substrate grow the fastest, but the survival rate is low, only 60.0%; the survival rate of the nursery soil is only 40.0%, and the transplanted seedlings are weak and grow normally. According to the survival rate and the growth of the transplanted seedlings, the best transplanting substrate for Eupatorium odoratum tissue culture seedlings is the light substrate.

[0140] 6、Different root system types affect the transplantation of tissue culture seedlings

[0141] Healthy, rooted tissue culture seedlings approximately 6cm tall were selected for transplanting. First, the seedlings were hardened off at room temperature for one day, then hardened off for another two days with the covers off. Afterward, excess culture medium was removed from the roots, and the seedlings were transferred to a lightweight substrate for further cultivation. The survival rate and growth were assessed one month later.

[0142] Table 7. Effects of different root types on transplantation of tissue culture seedlings

[0143]

[0144] As can be seen from the table above, different root types have a significant impact on the survival rate of transplanted tissue culture seedlings. Tissue culture seedlings with thick, short roots, approximately 1 cm long (such as...) Figure 7 Tissue culture seedlings are best suited for transplanting, with a 100% survival rate. After transplanting, the seedlings are robust and grow rapidly. Excessively long roots, on the other hand, negatively impact transplant survival and growth. Therefore, the optimal root system for transplanting *Chrysanthemum indicum* tissue culture seedlings is short and thick, with 3-4 roots approximately 1 cm long.

[0145] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. It can be applied to various fields suitable for the present invention. Further modifications can be readily implemented by those skilled in the art.

Claims

1. A method for rapid propagation of *Chrysanthemum indicum* through tissue culture, characterized in that, Specifically, it includes two stages: initial generation training and subsequent generation training, in which: The initial training phase includes: S11, Primary induction culture: Inducing stem segment explants into seedlings; The succession training phase includes: S21. Proliferation Culture: Select tissue culture seedlings obtained from the primary culture, and take stem segments from axillary buds for subculture to obtain clustered buds; the subculture culture medium for *Ilex chinensis* is: MS, 0.07 mg / L 6-BA, 1 g / L activated carbon and 2 g / L agar; S22. Rooting culture: Rooted tissue culture seedlings are obtained by culturing clustered buds; S23. Transplanting: Transplanting rooted tissue culture seedlings to obtain *Chrysanthemum indicum* plants; Before the initial induction culture or proliferation culture, the stem segments were first disinfected and sterilized. The disinfection and sterilization process was as follows: disinfection with 75.0% alcohol for 20 seconds, followed by sterilization with 0.1% mercuric chloride for 8 minutes. After disinfection and sterilization, browning inhibition culture was performed. The browning inhibition culture was carried out using MS medium: MS + 0.3 mg / L 6-BA + 0.05 mg / L NAA, and cultured at 4℃ for at least 12 hours. The culture medium used in the initial induction culture was: MS + 0.02 mg / L 6-BA + 0.01 mg / L NAA + 25 g / L sucrose + 5.0 g / L agar; The culture medium used for proliferation culture was: MS + 0.07 mg / L 6-BA + 1.0 g / L activated carbon + 2 g / L agar; The culture medium used in the rooting culture was: 1 / 2 MS + 0.2 mg / L IBA + 0.1 mg / L NAA.

2. The rapid propagation method of *Aster spp.* tissue culture as described in claim 1, characterized in that, For transplanting, use tissue culture seedlings with thick roots, 1 cm long, and 3-4 roots; or tissue culture seedlings with thin roots, 3 cm long, and 1-2 roots; the substrate used is a light substrate of peat moss:vermiculite = 3:1.

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