Vernonia esculenta proliferation culture medium and tissue culture rapid propagation method thereof

Abstract

The invention discloses a vernonia esculenta proliferation 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, 1g / L activated carbon and 2g / L agar. The method comprises the following steps: primary induction culture: inducing stem explants into seedlings; multiplication culture: selecting tissue culture seedlings obtained by primary culture, and taking stems of axillary buds for subculture multiplication culture to obtain cluster buds; rooting culture: culturing the cluster buds to obtain tissue culture seedlings with roots; transplanting: transplanting the tissue culture seedlings with roots to obtain seedlings. According to the method disclosed by the invention, the technical problems of extremely easy vitrification and low multiplication coefficient in the subculture multiplication process can be solved, the problems of stable character and rapid propagation of the excellent provenance of the vernonia esculenta can be solved, and large-scale production of high-quality and non-toxic vernonia esculenta seedlings is realized.

Description

Technical Field

[0001] The invention relates to the technical field of tissue culture, and more specifically to a Vernonia proliferating culture medium and a tissue culture rapid propagation method thereof. Background Art

[0002] Vernonia saligna (Wall.) DC, a plant of the Asteraceae family, grows on hillsides, wet grasslands and swamps in Guangdong, Guangxi, Guizhou, and southern Yunnan in my country. This genus is widely distributed in tropical and subtropical regions around the world. At present, there are about 1,000 known species of Vernonia in the Asteraceae family. More than 30 species of this genus have been discovered in my country, mainly distributed in the southwest to southeast, Taiwan, Xinjiang and other regions. There are 22 species in my country, including 14 species in Xishuangbanna. Vernonia saligna is a perennial herb. Its leaves can treat high fever, and its roots can induce labor. The whole plant is used by the people to treat malaria. The Yi people use it to treat sore throats, tuberculosis, coughing and hemoptysis, uterine prolapse, etc. In addition, this genus contains active substances that can kill weeds and fight infections. In recent years, with the continuous increase in market demand, the collection of wild resources has gradually increased, and the wild Vernonia has a long time of self-reproduction in the wild, resulting in the depletion of wild Vernonia resources. Therefore, introduction and cultivation, artificial propagation, etc. will become new ways to effectively utilize Vernonia resources. At present, there are few research reports on the reproduction of Vernonia. For example, the Chinese patent with announcement number CN111418364A discloses a cutting propagation method for Vernonia, but it belongs to cutting propagation, and research related to tissue culture technology has not been reported.

[0003] The present invention utilizes plant tissue culture technology to establish a large-scale production system for Vernonia tissue culture seedlings, which can effectively solve the problem of stable and rapid reproduction of excellent provenance traits, is an effective way to mass-produce high-quality, non-toxic Vernonia seedlings, and provides a necessary technical basis for realizing standardized production of Vernonia cultivation. Summary of the invention

[0004] The first purpose of the present invention is to provide a proliferation culture medium for Vernonia serrata, which improves the phenomenon that Vernonia serrata is sensitive to vitrification and has bud curling and other pathological phenomena, especially solves the technical problems of easy vitrification and low proliferation coefficient during subculture proliferation.

[0005] The second purpose of the present invention is to provide a method for rapid propagation of Vernonia spp. through tissue culture, so as to solve the problem of stable and rapid propagation of excellent provenance traits of Vernonia spp. and realize large-scale production of high-quality and non-toxic Vernonia spp. seedlings.

[0006] In order to achieve the objects of the present invention, the present invention provides a Vernonia proliferating culture medium for subculture and proliferation of Vernonia, comprising:

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

[0008] During the experiment, the present invention found that Vernonia scabra was particularly sensitive to hormones. After adding hormones, it was very easy to vitrify, and there were pathological phenomena such as bud curling. It was also easy to vitrify after adding activated carbon. When the ratio of hormone 6-BA and activated carbon was appropriate, the subculture proliferation medium did not vitrify, and the tissue culture seedlings grew well, the leaves were dark green, and there were many germinations. In one embodiment, the following culture media were compared:

[0009] ①MS+6-BA0.5mg / L+NAA0.2 mg / L;

[0010] ②MS+6-KT 0.5mg / L+NAA0.5 mg / L;

[0011] ③MS+6-BA0.1mg / L+NAA0.01 mg / L;

[0012] ④MS+6-BA0.2mg / L+IBA0.05 mg / L;

[0013] ⑤MS+6-BA0.1mg / L+NAA0.02 mg / L;

[0014] ⑥MS+6-BA0.01mg / L+agar 2g / L;

[0015] ⑦MS+6-BA0.01mg / L+activated carbon 1g / L+agar 2g / L;

[0016] ⑧MS+6-BA0.05mg / L+activated carbon 1g / L+agar 2g / L;

[0017] ⑨MS+6-BA0.005mg / L+activated carbon 1g / L+agar 2g / L;

[0018] ⑩MS+6-BA0.07mg / L+activated carbon 1g / L+agar 2g / L.

[0019] The results show that the Vernonia tissue culture seedlings in the proliferation medium of the present invention have no vitrification during the subculture proliferation process, and the tissue culture seedlings have good growth, dark green leaves, and many buds.

[0020] The invention provides a method for rapid propagation of Vernonia scabra tissue culture, which utilizes the Vernonia scabra proliferation culture medium to perform subculture proliferation culture.

[0021] The present invention provides a method for rapid propagation of Vernonia scabra by tissue culture, which specifically includes two stages: primary culture and subculture, wherein:

[0022] The primary culture stage includes:

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

[0024] The subculture stage includes:

[0025] S21, proliferation culture: selecting tissue culture seedlings obtained from primary culture, taking stem segments of axillary buds for secondary proliferation culture to obtain clustered buds;

[0026] S22, rooting culture: culturing the clustered buds to obtain rooted tissue culture seedlings;

[0027] S23. Transplanting: Transplanting the tissue culture seedlings with roots to obtain Vernonia scabra seedlings.

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

[0029] Preferably, the explants are sterilized by using 75.0% alcohol by volume for 20 seconds and then sterilized by 0.1% mercuric chloride for 8 minutes. In one embodiment, the contamination rate is 0 and the survival rate is 66.7%.

[0030] Preferably, the browning inhibition culture is cultured in a medium of: MS+0.3 mg / L 6-BA+0.05 mg / L NAA+4°C for 12 hours or more. In one embodiment, no obvious browning occurs during the low temperature treatment of 4°C, and the bud induction rate reaches 80.0%.

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

[0032] Preferably, in the proliferation culture, the more suitable culture medium for the subculture proliferation culture of Vernonia is MS+0.07mg / L6-BA+1.0g / L activated carbon+2g / L agar. In one embodiment, the Vernonia tissue culture seedlings do not undergo vitrification during the subculture proliferation process, and the tissue culture seedlings grow well, have dark green leaves, and have many buds.

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

[0034] Preferably, during transplanting, the most suitable substrate is a sterilized peat soil: vermiculite = 3:1 light substrate, with a survival rate of 100%;

[0035] Preferably, the survival rate of transplantation is high when the roots of Vernonia are thick, about 1 cm long, and 3 to 4 in number, or thin, about 3 cm long, and 1 to 2 in number, and the maximum survival rate is 100%.

[0036] The present invention has at least the following beneficial effects:

[0037] 1. The proliferation medium for subculture proliferation of Vernonia scabra of the present invention has no vitrification in the subculture proliferation process of Vernonia scabra tissue culture seedlings, and the tissue culture seedlings have good growth, dark green leaves, and many buds.

[0038] 2. The tissue culture rapid propagation method of Vernonia scabra of the present invention is optimized from the aspects of disinfection and sterilization, explant browning inhibition, induction culture, proliferation culture, rooting culture and transplanting, and a rapid propagation system of Vernonia scabra is established to solve the difficult problems of stable and rapid propagation of excellent provenance traits of Vernonia scabra, and the method has a high survival rate, good seedling growth, fast seedling formation, no vitrification during the proliferation process, and good growth of tissue culture seedlings, dark green leaves, many germinations, high rooting rate, and high transplant survival rate, and can realize large-scale production of high-quality, non-toxic Vernonia scabra seedlings.

[0039] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Vernonia tissue culture seedlings obtained by culturing the Vernonia proliferation medium of the present invention;

[0041] Figure 2 The Vernonia scabra plant of the present invention;

[0042] Figure 3 The adventitious buds of Vernonia scabra obtained by primary induction culture of the present invention;

[0043] Figure 4 The Vernonia oleifera clustered buds cultured for rooting of the present invention;

[0044] Figure 5 The transplanted Vernonia tissue culture seedlings of the present invention;

[0045] Figure 6 Vernonia tissue culture seedlings treated with different substrates according to the present invention;

[0046] Figure 7 The invention discloses a Vernonia tissue culture seedling with a thick and short root system. DETAILED DESCRIPTION

[0047] The present invention is further described in detail below with reference to examples so that those skilled in the art can implement the invention with reference to the description.

[0048] It should be understood that terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.

[0049] Example 1

[0050] A Vernonia scabra proliferation culture medium comprises the following components: MS+0.07 mg / L 6-BA+1 g / L activated carbon+2 g / L agar.

[0051] Effect Experiment

[0052] The tissue culture seedlings with relatively consistent growth after the primary induction culture were selected, and the stem segments with axillary buds of about 1 cm were cut for subculture proliferation culture. The proliferation culture medium treatments were as follows:

[0053] ①MS+6-BA0.5mg / L+NAA0.2mg / L;

[0054] ②MS+6-KT 0.5mg / L+NAA0.5mg / L;

[0055] ③MS+6-BA0.1mg / L+NAA0.01mg / L;

[0056] ④MS+6-BA0.2mg / L+IBA0.05 mg / L;

[0057] ⑤MS+6-BA0.1mg / L+NAA0.02 mg / L;

[0058] ⑥MS+6-BA0.01mg / L+agar 2g / L;

[0059] ⑦MS+6-BA0.01mg / L+activated carbon 1g / L+agar 2g / L;

[0060] ⑧MS+6-BA0.05mg / L+activated carbon 1g / L+agar 2g / L;

[0061] ⑨MS+6-BA0.005mg / L+activated carbon 1g / L+agar 2g / L;

[0062] ⑩MS+6-BA0.07mg / L+activated carbon 1g / L+agar 2g / L. (Present Example)

[0063] The treatment with additional agar was mainly used to investigate the effect of agar dosage on vitrification. Five bottles were used for each treatment, and four buds were inoculated in each bottle. The treatment was repeated three times. 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 proliferation of tissue culture seedlings

[0065]

[0066]

[0067] Vernonia tissue culture seedlings are relatively sensitive to hormones during the process of subculture and proliferation, and are very easy to vitrify, with a low proliferation coefficient. From the results in Table 1, it can be seen that in comparison of treatments ⑦-⑩, it is found that the buds in the culture medium with activated carbon added also vitrify, but in the treatment of this embodiment ⑩, when the hormone 6-BA and activated carbon are properly matched, there is no vitrification, and the tissue culture seedlings grow well, with dark green leaves and many buds. Figure 1 As shown. In the subculture, treatments with mitogen 6-BA concentrations above 0.1 mg / L will cause flocculent callus at the base of the buds and vitrification. In the culture medium with KT added, there was almost no proliferation effect, indicating that KT could not promote bud proliferation. Considering the growth status and proliferation coefficient of the buds, the most suitable subculture medium for Vernonia scabra is MS+0.07 mg / L 6-BA+1.0 g / L activated carbon+2 g / L agar.

[0068] Example 2

[0069] A method for rapid propagation of Vernonia scabra by tissue culture, comprising:

[0070] Step 1: Disinfection and sterilization:

[0071] Select strong and virus-free young stem segments of Vernonia, cut the stem segments into 1.0-2.0 cm long small stem segments with axillary buds, soak them in detergent with 1% detergent for 5-8 minutes, and then rinse them with running water for about 30 minutes. Then disinfect the stem segments with 75% alcohol on an ultra-clean workbench for 30 seconds, and then rinse them with sterile water for 3-5 times; finally, sterilize them with 0.1% mercuric chloride for 8 minutes, and then rinse them with sterile water 5 times, take them out and place them in a high-pressure sterilized stainless steel shallow dish, cut off the end of the stem tissue, and draw a cross in the middle of the leaf for inoculation, and the preliminary treatment is completed. Vernonia plants such as Figure 2 shown.

[0072] Step 2: Explant browning inhibition culture:

[0073] The stem segments that have not turned brown after the sterilization treatment in step 1 are inoculated into the browning inhibition medium. The browning inhibition medium treatment is MS+0.3mg / L 6-BA+0.05mg / L NAA+4°C low temperature treatment, wherein the low temperature treatment is to place the explants in a 4°C refrigerator for 12 hours or more.

[0074] Step 3: Primary induction culture:

[0075] The stem segments after browning inhibition treatment were inoculated into the primary induction medium and cultured to obtain 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 in the culture room was 2 000 lx, the light duration was 12 h / d, and the temperature was (24 ± 2) °C. More than 75% of the primary induction of Vernonia scabra had good growth of adventitious buds and fast seedling formation. Figure 3 shown.

[0076] Step 4: Subculture and proliferation:

[0077] The tissue culture seedlings with relatively 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 to obtain clustered buds of Vernonia scabra. The proliferation culture medium was: MS+0.07mg / L 6-BA+1g / L activated carbon+2g / L agar. The clustered buds grew well, with dark green leaves and 3 to 5 new buds.

[0078] Step 5: Rooting culture:

[0079] The clustered buds of Vernonia scabra obtained by proliferation culture were transferred to the rooting medium for culture. The rooting medium was treated with: 1 / 2MS+0.2mg / L IBA+0.1mg / L NAA. The rooting time was 5-7d. Rooted tissue culture seedlings were obtained. Figure 4 After cultivation, the root system was strong, the number of roots was large, and the rooting rate was over 95.0%.

[0080] Step 6: Transplanting:

[0081] After rooting culture, select the Vernonia tissue culture seedlings with roots of about 6 cm in height, vigorous growth and well-developed root system (thick root system, 3 to 4 roots, about 1 cm long) and move them into a constant temperature room for placement. After 2 days, open the lid to allow the Vernonia tissue culture seedlings to be fully exposed to the air. During this period, water the tissue culture seedlings in the bottle to keep the bottle sufficiently hydrated. After 3 days, take out the tissue culture seedlings from the bottle, wash the culture medium at the roots, and move them to a sterilized light substrate with a ratio of peat soil and vermiculite of 3:1. Provide appropriate shade and maintain a certain humidity. After 30 days of culture, the survival rate of the Vernonia tissue culture seedlings is over 95%. Transplanted Vernonia tissue culture seedlings are Figure 5 shown.

[0082] Effect Experiment

[0083] 1. Effects of different sterilization times on stem segment induction

[0084] Select robust Vernonia scabra branches and cut them into 1-2 cm stem segments with axillary buds. After preliminary treatment, use 75% alcohol for disinfection for 0s, 10s, 20s, and 30s, respectively. The corresponding mass concentration of 0.1% mercuric chloride sterilization time is 8min, 8min, 8min, 8min, and 0min. Inoculate the explants on the medium containing only MS for culture. Each treatment has 20 bottles, and each bottle is inoculated with 1 explant. Repeat 3 times, observe and count the contamination rate and survival rate at any time, and determine the best disinfection and sterilization method.

[0085] Table 2 Effects of different sterilization times on stem segment induction

[0086]

[0087] As shown in Table 2, after different disinfection and sterilization treatments, the young stem segments of Vernonia scabra were free of contamination when disinfected with 75% alcohol and 0.1% mercuric chloride. The contamination rate of 75.0% for 30s of sterilization with only 75% alcohol was 10%; the contamination rate of 0.1% mercuric chloride was 10%. Although the survival rates of both reached 100%, the contamination rate was higher. The contamination rate of 75.0% alcohol disinfection for 20s and 0.1% mercuric chloride disinfection for 8min was 0, and the survival rate reached 66.7%. Therefore, the best disinfection treatment for Vernonia scabra explants is 75.0% alcohol sterilization for 20s, followed by 0.1% mercuric chloride sterilization for 8min.

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

[0089] The unbrown stem segments after sterilization were inoculated into different culture media. The culture media treatments were:

[0090] ①MS+6-BA0.3mg / L+NAA0.05mg / L+PVPP 0.5g / L;

[0091] ②MS+6-BA0.3mg / L+NAA0.05mg / L+PVPP 1g / L;

[0092] ③MS+6-BA0.3mg / L+NAA0.05mg / L+activated carbon 0.5g / L;

[0093] ④MS+6-BA0.3mg / L+NAA0.05mg / L+activated carbon 1g / L;

[0094] ⑤MS+6-BA0.3mg / L+NAA0.05mg / L+4℃ low temperature treatment;

[0095] The low temperature treatment means that the explants are placed in a refrigerator at 4℃ for more than 12 hours. Regarding the dosage unit of hormones, 6-BA0.3 means 0.3mg / L 6-BA, the same below. Each treatment has 20 bottles, and each bottle is inoculated with 1 explant, which is repeated 3 times. The browning situation is observed and recorded at any time, and the browning rate is calculated. The one with the least browning is selected for the next step of research.

[0096] Table 3 Effects 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 all have a certain inhibitory effect on the browning of Vernonia. In the PVPP treatment, different concentrations of PVPP have different browning effects, among which 0.5g / L PVPP has a lower browning rate than 1.0g / LPVPP, but the induction rate is also lower than the latter; different activated carbon treatments have the same effect of inhibiting browning, with a browning rate of 25.0%, but the bud induction rate of 0.5g / L activated carbon is higher, with a browning rate of 75.0%. No obvious browning occurred in the low temperature treatment, and the bud induction rate reached 80.0%. Therefore, the best method for browning treatment is 4℃ low temperature treatment.

[0099] 3. Effects of different hormone ratios on stem segment induction

[0100] The stem segments after the browning inhibition treatment were inoculated into different hormone ratio culture media, and the culture media treatments were:

[0101] ①MS;

[0102] ②MS+6-BA0.5mg / L;

[0103] ③MS+6-BA0.5mg / L+NAA0.2mg / L;

[0104] ④MS+6-BA0.5mg / L+NAA0.05mg / L;

[0105] ⑤MS+6-BA0.5mg / L+NAA0.1mg / L;

[0106] ⑥MS+KT 0.5mg / L+NAA0.1mg / L;

[0107] ⑦MS+6-BA0.5mg / L+KT 0.2mg / L+NAA 0.2mg / L;

[0108] ⑧MS+6-BA0.3mg / L+NAA0.05mg / L;

[0109] ⑨MS+6-BA0.8mg / L+NAA0.1mg / L;

[0110] ⑩MS+6-BA1.0mg / L+NAA0.1mg / L;

[0111] MS+6-BA0.02mg / L+NAA 0.01mg / L.

[0112] Each treatment medium also included 25 g / L sucrose + 5.0 g / L agar. 35 bottles were inoculated for each treatment, with one explant in each bottle. The process was repeated three times, and the induction effect was observed and recorded at any time.

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

[0114]

[0115]

[0116] Different hormone ratios were used to induce the stem segments of Vernonia serrata. During the induction process, it was found that Vernonia serrata was more sensitive to hormones. Higher hormone concentrations easily caused 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 hormones had better bud growth than other mediums with hormones, but the induction rate was the lowest, only 28.6%. When the concentration of mitogen 6-BA was 0.5 mg / L, different concentrations of auxin NAA were added. As the concentration of NAA increased, callus appeared at the base, and the induction rate also decreased, which had a certain effect on the induction of stem segments. 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 it was easy for the buds 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, and the seedlings formed quickly. In general, from the perspective of growth conditions, although the culture medium containing hormones has a high induction rate, too high a hormone concentration can easily lead to the formation of flocculent callus and vitrification, which is not conducive to subsequent seedling formation. Therefore, the optimal culture medium for the initial induction of young stem segments of Vernonia is MS+0.02mg / L 6-BA+0.01mg / L NAA, which has a high induction rate, produces sturdy seedlings, good growth, fast seedling formation, and green leaves.

[0117] 4. Effects of different rooting treatments on the growth of Vernonia tissue culture seedlings

[0118] The tissue culture seedlings obtained by proliferation culture with healthy growth and a height of about 2 cm were selected and transferred to the rooting medium for culture. The rooting medium treatments were as follows:

[0119] ①MS+6-BA0.25+NAA0.2;

[0120] ②1 / 2MS;

[0121] ③1 / 2MS+NAA0.15;

[0122] ④1 / 2MS+NAA0.05;

[0123] ⑤1 / 2MS+IBA0.1+activated carbon 1g / L;

[0124] ⑥1 / 2MS+NAA0.05+IBA0.1;

[0125] ⑦1 / 2MS+NAA0.15+IBA0.1;

[0126] ⑧1 / 2MS+IBA0.2;

[0127] ⑨1 / 2MS+NAA0.05+IBA0.1+activated carbon 1g / L;

[0128] ⑩1 / 2MS+IBA0.1;

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

[0130] Each treatment was performed with 5 bottles, 4 seedlings were inoculated in each bottle, and the process was repeated 3 times. The rooting and growth conditions were observed and recorded at any time.

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

[0132]

[0133] As can be seen from Table 5, the rooting time of tissue culture seedlings of Vernonia is 5 to 7 days, and 1 / 2MS medium is more suitable for rooting culture. Auxins of different ratios and concentrations can induce rooting of tissue culture seedlings, but different auxins, different concentrations and different ratios have different effects on rooting. For the rooting medium with only IBA added, within a certain range, as the concentration increases, the roots become thicker and the rooting rate also increases significantly. In the medium with only NAA added, the root growth coefficient with a lower concentration is also lower, and the bud growth is thinner and weaker; in the rooting medium with both NAA and IBA added, the root growth conditions are the same, but with a higher concentration of NAA, the seedlings grow stronger. In the rooting medium without any auxin added, the roots are slender, which is not conducive to transplanting, indicating that growth hormones are needed for rooting. 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 present invention concludes that the most suitable rooting medium for Vernonia is the one treated with 1 / 2MS+0.2mg / L IBA+0.1mg / L NAA+25g / L sucrose+5g / L agar, the rooting rate of tissue culture seedlings cultivated is over 95%, the root system is thick, the leaves are green, and the plants are strong.

[0134] 5. Effects of different substrate treatments on transplanting tissue culture seedlings

[0135] Select robust, 6 cm tall rooted tissue culture seedlings for transplanting. First, harden the seedlings at room temperature for 1 day, then open the lid for 2 days, then clean and remove excess culture medium from the roots, and move to different substrates for cultivation. After one month, the survival rate and growth of the transplanted seedlings were counted.

[0136] Table 6 Effects of different substrate treatments on transplanting tissue culture seedlings

[0137]

[0138] The mass ratio of peat soil to vermiculite in the above light matrix is ​​3:1.

[0139] From the above table and Figure 6 It can be seen that the light substrate has the highest survival rate, which is 100%, and the transplanted seedlings are strong and grow fast; the transplanted seedlings in the mixed substrate grow 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 generally. According to the survival rate and the growth of the transplanted seedlings, it is concluded that the best transplanting substrate for Vernonia scabra tissue culture seedlings is the light substrate.

[0140] 6. Effects of different root types on transplanting tissue culture seedlings

[0141] Select robust, 6 cm tall rooted seedlings for transplanting. First, harden the seedlings at room temperature for 1 day, then open the lid for 2 days, then clean and remove excess culture medium from the roots, and move to a light substrate for cultivation. After one month, the survival rate and growth of the transplanted seedlings were counted.

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

[0143]

[0144] From the table above, we can see that different root types have a significant impact on the survival rate of tissue culture seedlings after transplantation. Figure 7 ) is most suitable for transplanting, with a survival rate of 100%, and the transplanted seedlings are strong and grow fast. Too long roots will affect the survival rate and growth of transplanting. Therefore, the best root system for transplanting Vernonia tissue culture seedlings is a short and thick root system with 3 to 4 roots and a length of about 1 cm.

[0145] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and the embodiments. They can be applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily realized.

Claims

1. A Vernonia proliferating culture medium, characterized in that: include: MS, 0.07 mg / L 6-BA, 1 g / L activated carbon and 2 g / L agar.

2. The Vernonia scabra proliferating medium according to claim 1, characterized in that Used for the subculture and proliferation of Vernonia scabra.

3. A method for rapid propagation of Vernonia scabra by tissue culture, characterized in that: The Vernonia scabra proliferation culture medium according to claim 1 is used for subculture proliferation culture.

4. The method for rapid propagation of Vernonia scabra by tissue culture as claimed in claim 3, characterized in that: Specifically, it includes two stages: primary culture and secondary culture, among which: The primary culture stage includes: S11, primary induction culture: inducing stem segment explants into seedlings; The subculture stage includes: S21, proliferation culture: selecting tissue culture seedlings obtained from primary culture, taking stem segments of axillary buds for subculture proliferation culture to obtain clustered buds; S22, rooting culture: culturing the clustered buds to obtain rooted tissue culture seedlings; S23. Transplanting: Transplanting the rooted tissue culture seedlings to obtain Vernonia plants.

5. The method for rapid propagation of Vernonia scabra by tissue culture as claimed in claim 4, characterized in that: Before primary induction culture or proliferation culture, the stem segments should be disinfected and sterilized; The disinfection and sterilization treatment method is: disinfection with 75.0% alcohol by volume for 20 seconds, and then sterilization with 0.1% mercuric chloride by mass concentration for 8 minutes.

6. The method for rapid propagation of Vernonia scabra by tissue culture as claimed in claim 5, characterized in that: After disinfection and sterilization, browning inhibition culture treatment is performed; Browning inhibition culture was performed using the culture medium: MS + 0.3 mg / L 6-BA + 0.05 mg / L NAA at 4°C for at least 12 hours.

7. The method for rapid propagation of Vernonia scabra by tissue culture as claimed in claim 6, characterized in that: In the primary induction culture, the culture medium used was: MS+0.02 mg / L 6-BA+0.01 mg / L NAA+25 g / L sucrose+5.0 g / L agar.

8. The method for rapid propagation of Vernonia scabra by tissue culture as claimed in claim 7, characterized in that: In the proliferation culture, the culture medium used is: MS+0.07mg / L 6-BA+1.0g / L activated carbon+2g / L agar.

9. The method for rapid propagation of Vernonia scabra by tissue culture as claimed in claim 8, characterized in that: In the rooting culture, the culture medium used was: 1 / 2MS+0.2mg / L IBA+0.1mg / L NAA.

10. The method for rapid propagation of Vernonia scabra by tissue culture as claimed in claim 9, characterized in that: During transplanting, use tissue culture seedlings with thick roots, 1 cm long, and 3 to 4 roots; or tissue culture seedlings with thin roots, 3 cm long, and 1 to 2 roots; the substrate used is a light substrate with a ratio of peat soil to vermiculite of 3:1.

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