A multiplication medium for cultivating arnebia euchroma test tube seedlings, arnebia euchroma test tube seedlings and a cultivating method thereof

Abstract

The present application relates to the technical field of plant culture, and especially relates to a multiplication medium for cultivating Xinjiang Bistorta test-tube seedlings, Xinjiang Bistorta test-tube seedlings and a cultivating method thereof. The multiplication medium is based on 1 / 2MS medium without ammonium and further comprises the following components with the following concentrations: 6-benzylaminopurine 2.0-3.0 mg / L, naphthaleneacetic acid 0.05-0.15 mg / L, lemon juice 300-500 mg / L, sucrose 25-35 g / L and agar 7.0-8.0 g / L. The pH value of the multiplication medium is 5.8-6.0. The multiplication coefficient of the Xinjiang Bistorta multiplication seedlings obtained by using the multiplication medium is higher than 3.7. Healthy Xinjiang Bistorta test-tube seedlings can be obtained in a short time. The present application provides a basis for cultivating Xinjiang Bistorta tissue culture seedlings.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant culture, in particular to a proliferation medium for cultivating new Xinjiang Bistort test tube seedlings, Xinjiang Bistort test tube seedlings and a cultivation method thereof. BACKGROUND

[0002] Bistort is a plant of the genus Bistortae in the family Bistortaceae, which was first recorded in Shennong's Herbal Classic. As a commonly used traditional Chinese medicine, it has the effects of clearing heat and cooling blood, promoting blood circulation to remove toxins, and promoting rash and eliminating spots. Its alias is hard Bistort and soft Bistort.

[0003] The main component of Bistort, Bistortin and its derivatives, is an anthraquinone compound, which has significant anti-tumor, anti-inflammatory and antibacterial activities. In addition to being used as a medicine, Bistort is also an important raw material for daily chemical products. Researchers have successfully developed a natural lipstick pigment from the pigment, which is very popular among consumers because it does not contain artificial pigments. Hairy roots are induced by Agrobacterium rhizogenes Ri plasmid, which has the characteristics of hormone autonomy, rapid growth, high and stable content of effective components, and is a new effective way to grow plant secondary metabolites. Due to its unique medicinal value, Bistort is also a source of natural and precious dyes, but its plant resources are severely lacking. Plant tissue culture has the advantages of small space occupation, short culture period, fast propagation speed, and the ability to maintain the inherent properties and characteristics of the original variety, and is the development direction of future ecological agricultural factory cultivation of Chinese medicine. In order to solve the shortage of Xinjiang Bistort resources and the imbalance between supply and demand, plant tissue culture has become an effective way to solve this problem. At present, the research on tissue culture of Xinjiang Bistort mainly focuses on two aspects: one is to screen excellent cell lines from Bistort callus for the production of useful secondary metabolites. The other is to induce and screen excellent lines using plant tissue culture technology, and further rapidly propagate them into complete plants. The use of Bistort in vitro rapid propagation technology not only meets the demand for Bistort in production, but also is beneficial to the protection of wild Bistort resources. The Bistort test tube seedlings cultivated are important research materials and the best explants for inducing Bistort hairy roots. The main problems in Bistort tissue culture at present are: 1. The propagation coefficient of Bistort test tube seedlings is low, the vitrification is obvious during the culture process, the leaf yellowing phenomenon is serious, and the existing medium is brown during the Bistort tissue culture process; 2. The existing research on in vitro propagation of Xinjiang Bistort is mainly completed through indirect organ (embryo axis, cotyledon and stem segment) regeneration, but the propagation coefficient of the regenerated seedlings obtained by this method is low, and the variation probability is high. The other way is to induce callus first and then differentiate into multiple buds, but this method also has the problems of: (1) low differentiation rate of callus, (2) long cultivation period, and (3) low survival rate of test tube seedlings after transplanting.

[0004] Based on this, the present application is proposed. SUMMARY

[0005] The application aims to provide a proliferation medium for cultivating Arnebia euchroma tube seedlings, and the Arnebia euchroma tube seedlings and a cultivation method thereof.

[0006] To achieve the above-mentioned application purposes, the application provides the following technical solutions.

[0007] The application provides a proliferation medium suitable for cultivating Arnebia euchroma tube seedlings, which is based on 1 / 2MS medium without ammonium and further comprises the following components at the following concentrations:

[0008] 6-benzylaminopurine 2.0-3.0 mg / L, naphthaleneacetic acid 0.05-0.15 mg / L, lemon juice 300-500 mg / L, sucrose 25-35 g / L, and agar 7.0-8.0 g / L.

[0009] The pH value of the proliferation medium is 5.8-6.0.

[0010] The application further provides application of the proliferation medium in cultivating excellent Arnebia euchroma tube seedlings.

[0011] The application further provides a cultivation method of Arnebia euchroma tube seedlings, which comprises the following steps:

[0012] (1) disinfecting Arnebia euchroma explants to obtain sterile Arnebia euchroma explants;

[0013] (2) inoculating the sterile Arnebia euchroma explants into a start-up medium and culturing for 25-35 days to obtain sterile seedlings;

[0014] (3) inoculating the sterile seedlings into a proliferation medium and culturing for 21-25 days to obtain proliferation seedlings;

[0015] (4) inoculating the proliferation seedlings into a rooting medium and culturing for 18-25 days to obtain Arnebia euchroma tube seedlings;

[0016] The proliferation medium is the proliferation medium.

[0017] Preferably, the Arnebia euchroma explants are peeled Arnebia euchroma seeds or Arnebia euchroma stem segments with roots removed.

[0018] The disinfection method is as follows: placing the Arnebia euchroma external explants in ethanol, soaking for 50-70s, to obtain the primary disinfection Arnebia euchroma external explants; placing the primary disinfection Arnebia euchroma external explants in hydrogen peroxide aqueous solution, soaking for 10-20min, to obtain the secondary disinfection Arnebia euchroma external explants; placing the secondary disinfection Arnebia euchroma external explants in ampicillin solution, soaking for 50-70min, to obtain the sterile Arnebia euchroma external explants.

[0019] The initial concentration of the ethanol is 70-80vt%.

[0020] The initial concentration of the hydrogen peroxide aqueous solution is 10-20vt%.

[0021] The initial concentration of the ampicillin solution is 450-550mg / L.

[0022] Preferably, the starting culture medium is based on SH culture medium, and further comprises the following components at the following concentrations:

[0023] Sucrose 15-25g / L, agar 7.0-8.0g / L.

[0024] The pH value of the starting culture medium is 5.8-6.0.

[0025] Preferably, the rooting culture medium is based on 1 / 2MS culture medium, and further comprises the following components at the following concentrations:

[0026] Naphthaleneacetic acid 0.3-0.5mg / L, indolebutyric acid 0.5-1.0mg / L, activated carbon 0.2-0.5g / L, sucrose 25-35g / L, agar 7.0-8.0g / L.

[0027] The pH value of the rooting culture medium is 5.8-6.0.

[0028] Preferably, the temperature of the culture in steps (2)-(4) is independently 23-27℃, the light intensity of the culture is independently 5000-10000lx, and the light time of the culture is independently 13-15h / d.

[0029] The application further provides the Arnebia euchroma test-tube seedlings obtained by the cultivation method.

[0030] The application further provides the use of the Arnebia euchroma test-tube seedlings in cultivating Arnebia euchroma seedlings to be transplanted with high survival rate.

[0031] The application further provides a cultivation method of Arnebia euchroma seedlings to be transplanted, comprising the following steps:

[0032] The Xinjiang Arnebia euchroma test tube seedlings are acclimatized for 3-5 days to obtain seedlings to be cultivated; the seedlings to be cultivated are soaked in a carbendazim solution for 30-40 min, and then transplanted into a seedling culture medium to culture for 5-7 days to obtain the Xinjiang Arnebia euchroma seedlings to be transplanted;

[0033] The acclimatization method is open-cap acclimatization;

[0034] The seedling culture medium comprises the following components in a mass ratio: vermiculite: nutrient soil 1-2:1-2;

[0035] The culture temperature is 22-28 DEG C, and the illumination time is 7-9 h / d.

[0036] The application provides a proliferation culture medium for cultivating Xinjiang Arnebia euchroma test tube seedlings, Xinjiang Arnebia euchroma test tube seedlings and a cultivation method thereof. The proliferation culture medium can solve the problems of serious yellowing, vitrification and browning of leaves in the in-vitro culture of Xinjiang Arnebia euchroma and low proliferation coefficient of organogenesis, and can obtain proliferation seedlings with a proliferation coefficient of more than 3.7.

[0037] The method can obtain Xinjiang Arnebia euchroma test tube seedlings in a short time, and can obtain Xinjiang Arnebia euchroma test tube seedlings with high survival rate after transplantation in 64-85 days. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 Xinjiang Arnebia euchroma;

[0039] Figure 2 The seed with the peeled seed coat is cultured in a culture dish containing the initiation culture medium for 12 days;

[0040] Figure 3 The aseptic seedling obtained by culturing the stem segment with the removed root as the explant;

[0041] Figure 4 The proliferation seedling obtained by using the seed with the peeled seed coat as the explant;

[0042] Figure 5 The proliferation seedling obtained by using the stem segment with the removed root as the explant;

[0043] Figure 6 The proliferation seedling obtained by using the method of Example 3;

[0044] Figure 7 The growth condition of the Xinjiang Arnebia euchroma test tube seedling cultured for 7 days;

[0045] Figure 8 The growth condition of the Xinjiang Arnebia euchroma test tube seedling cultured for 15 days;

[0046] Figure 9The growth state of the Xinjiang Arnebia euchroma tube seedling is cultured for 20 days. DETAILED DESCRIPTION

[0047] The application provides a proliferation culture medium suitable for cultivating Xinjiang Arnebia euchroma tube seedlings, which is based on 1 / 2MS culture medium without ammonium and further comprises the following components in the following concentrations:

[0048] 6-benzylaminopurine 2.0-3.0 mg / L, preferably 2.5 mg / L;

[0049] naphthaleneacetic acid 0.05-0.15 mg / L, preferably 0.1 mg / L;

[0050] lemon juice 300-500 mg / L, preferably 400 mg / L;

[0051] sucrose 25-35 g / L, preferably 30 g / L;

[0052] agar 7.0-8.0 g / L, preferably 7.5 g / L;

[0053] The pH value of the proliferation culture medium is 5.8-6.0, preferably 5.9.

[0054] In the application, the 1 / 2MS culture medium without ammonium is based on water and comprises the following components in the following concentrations: potassium nitrate 1900 g / L, potassium dihydrogen phosphate 170 g / L, magnesium sulfate 180.7 g / L, calcium chloride 332.2 g / L, potassium iodide 0.83 g / L, boric acid 6.2 g / L, manganese sulfate 16.9 g / L, zinc sulfate 8.6 g / L, sodium molybdate 0.25 g / L, copper sulfate 0.025 g / L, cobalt chloride hexahydrate 0.025 g / L, disodium ethylenediaminetetraacetate 37.7 g / L, ferrous sulfate 27.8 g / L, myo-inositol 100 g / L, thiamine hydrochloride 0.1 g / L, pyridoxal hydrochloride 0.5 g / L, and nicotinic acid 0.5 g / L.

[0055] The application further provides application of the proliferation culture medium in cultivating excellent Xinjiang Arnebia euchroma tube seedlings.

[0056] The application further provides a cultivation method of Xinjiang Arnebia euchroma tube seedlings, which comprises the following steps:

[0057] (1) disinfecting an Xinjiang Arnebia euchroma explant to obtain a sterile Xinjiang Arnebia euchroma explant;

[0058] (2) inoculating the sterile Xinjiang Arnebia euchroma explant into a start culture medium and culturing for 25-35 days to obtain sterile seedlings;

[0059] (3) inoculating the sterile seedlings into a proliferation culture medium and culturing for 21-25 days to obtain proliferation seedlings;

[0060] (4) transferring the proliferated seedlings to a rooting medium and culturing them for 18 to 25 days to obtain Xinjiang Lithospermum officinale test tube seedlings;

[0061] The proliferation culture medium is the proliferation culture medium.

[0062] In the present invention, the Xinjiang Lithospermum explant is Xinjiang Lithospermum seeds with the seed coat removed or a stem segment with the Xinjiang Lithospermum root removed;

[0063] The disinfection method comprises the following steps: placing the Xinjiang lithospermum explant in ethanol and soaking it for 50 to 70 seconds to obtain a preliminarily disinfected Xinjiang lithospermum explant; placing the preliminarily disinfected Xinjiang lithospermum explant in a hydrogen peroxide aqueous solution and soaking it for 10 to 20 minutes to obtain a second-disinfected Xinjiang lithospermum explant; placing the second-disinfected Xinjiang lithospermum explant in an ampicillin solution and soaking it for 50 to 70 minutes to obtain a sterile Xinjiang lithospermum explant;

[0064] The initial concentration of the ethanol is 70-80 vt%, preferably 75 vt%; the immersion time in the ethanol is preferably 60 s; the initial concentration of the hydrogen peroxide aqueous solution is 10-20 vt%, preferably 15 vt%; the immersion time in the hydrogen peroxide aqueous solution is preferably 15 min; the initial concentration of the ampicillin solution is 450-550 mg / L, preferably 500 mg / L; and the immersion time in the ampicillin solution is preferably 60 min.

[0065] In the present invention, the priming medium is based on SH medium and further comprises the following components at the following concentrations:

[0066] Sucrose 15-25 g / L, preferably 20 g / L;

[0067] Agar 7.0-8.0 g / L, preferably 7.5 g / L;

[0068] The pH value of the start-up culture medium is 5.8-6.0, preferably 5.9.

[0069] In the present invention, the SH medium uses water as a solvent and includes the following components:

[0070] Potassium nitrate 2500g / L, ammonium phosphate 300g / L, magnesium sulfate 195.4g / L, calcium chloride 151g / L, potassium iodide 1g / L, boric acid 5g / L, manganese sulfate 10g / L, zinc sulfate 1g / L, sodium molybdate 0.1g / L, copper sulfate 0.2g / L, cobalt chloride hexahydrate 0.1g / L, disodium edetate 20g / L, ferrous sulfate 15g / L, inositol 1000g / L, thiamine hydrochloride 5g / L, pyridoxine hydrochloride 0.5g / L, niacin 5g / L.

[0071] The culture time in step (2) is preferably 30 days.

[0072] In the present application, the rooting culture medium is based on 1 / 2MS culture medium, and further comprises the following components at the following concentrations:

[0073] Naphthaleneacetic acid 0.3-0.5 mg / L, preferably 0.4 mg / L;

[0074] Indolebutyric acid 0.5-1.0 mg / L, preferably 0.8 mg / L;

[0075] Activated carbon 0.2-0.5 g / L, preferably 0.3 g / L;

[0076] Sucrose 25-35 g / L, preferably 30 g / L;

[0077] Agar 7.0-8.0 g / L, preferably 7.5 g / L;

[0078] The pH of the rooting culture medium is 5.8-6.0, preferably 5.9.

[0079] In the present application, the 1 / 2MS culture medium is based on water as solvent, and further comprises the following components at the following concentrations: potassium nitrate 950 g / L, ammonium nitrate 825 g / L, potassium dihydrogen phosphate 85 g / L, magnesium sulfate 185 g / L, calcium chloride 220 g / L, potassium iodide 0.83 g / L, boric acid 6.2 g / L, manganese sulfate 22.3 g / L, zinc sulfate 8.6 g / L, sodium molybdate 0.25 g / L, copper sulfate 0.025 g / L, cobalt chloride hexahydrate 0.025 g / L, disodium ethylenediaminetetraacetate 37.3 g / L, ferrous sulfate 27.8 g / L, myo-inositol 100 g / L, glycine 2 g / L, thiamine hydrochloride 0.1 g / L, pyridoxal hydrochloride 0.5 g / L, nicotinic acid 0.5 g / L.

[0080] The culture time in step (3) is preferably 23 days.

[0081] The culture time in step (4) is preferably 22 days.

[0082] In the present application, the culture temperature in steps (2)-(4) is independently 23-27℃, preferably 25℃, the culture light intensity is independently 5000-10000 lx, preferably 8000 lx, and the culture light time is independently 13-15 h / d, preferably 14 h / d.

[0083] The present application also provides the Xinjiang Baphlania delavayi test tube seedlings obtained by the cultivation method.

[0084] The present application also provides the use of the Xinjiang Baphlania delavayi test tube seedlings in cultivating high-survival-rate Xinjiang Baphlania delavayi seedlings to be transplanted.

[0085] The application further provides a cultivating method of the transplanted seedling of Arnebia euchroma (R. Br.) Johnst.

[0086] The test-tube seedling of Arnebia euchroma (R. Br.) Johnst. is acclimatized for 3-5 days to obtain the seedling to be cultivated; the seedling to be cultivated is soaked in a carbendazim solution for 30-40 min and then transplanted into a seedling raising substrate to be cultured for 5-7 days to obtain the transplanted seedling of Arnebia euchroma (R. Br.) Johnst.

[0087] The acclimatization method is open-cover acclimatization, and the acclimatization time is preferably 4 days.

[0088] The seedling raising substrate comprises the following components in a mass ratio: vermiculite: nutrient soil = 1-2:1-2, preferably 1:1.

[0089] The culture temperature is 22-28°C, preferably 25°C, the culture illumination time is 7-9 h / d, preferably 8 h / d, and the culture time is preferably 6 days.

[0090] The technical solutions provided by the application will be described in detail below in combination with the embodiments, but they should not be understood as limitations to the protection scope of the application.

[0091] The Arnebia euchroma (R. Br.) Johnst. seeds in the embodiments of the application are from Zhaosu County, Yili Kazak Autonomous Prefecture, Xinjiang Uygur Autonomous Region.

[0092] The 1 / 2MS medium without ammonium in the embodiments of the application uses water as a solvent and comprises the following components in the following concentrations: potassium nitrate 1900 g / L, potassium dihydrogen phosphate 170 g / L, magnesium sulfate 180.7 g / L, calcium chloride 332.2 g / L, potassium iodide 0.83 g / L, boric acid 6.2 g / L, manganese sulfate 16.9 g / L, zinc sulfate 8.6 g / L, sodium molybdate 0.25 g / L, copper sulfate 0.025 g / L, cobalt chloride hexahydrate 0.025 g / L, disodium ethylenediaminetetraacetate 37.7 g / L, ferrous sulfate 27.8 g / L, myo-inositol 100 g / L, thiamine hydrochloride 0.1 g / L, pyridoxal hydrochloride 0.5 g / L, and nicotinic acid 0.5 g / L.

[0093] The SH medium in the embodiments of the application uses water as a solvent and comprises the following components in the following concentrations: potassium nitrate 2500 g / L, ammonium phosphate 300 g / L, magnesium sulfate 195.4 g / L, calcium chloride 151 g / L, potassium iodide 1 g / L, boric acid 5 g / L, manganese sulfate 10 g / L, zinc sulfate 1 g / L, sodium molybdate 0.1 g / L, copper sulfate 0.2 g / L, cobalt chloride hexahydrate 0.1 g / L, disodium ethylenediaminetetraacetate 20 g / L, ferrous sulfate 15 g / L, myo-inositol 1000 g / L, thiamine hydrochloride 5 g / L, pyridoxal hydrochloride 0.5 g / L, and nicotinic acid 5 g / L.

[0094] The 1 / 2MS medium described in the embodiments of the present invention uses water as a solvent and further includes the following components at the following concentrations: 950 g / L potassium nitrate, 825 g / L ammonium nitrate, 85 g / L potassium dihydrogen phosphate, 185 g / L magnesium sulfate, 220 g / L calcium chloride, 0.83 g / L potassium iodide, 6.2 g / L boric acid, 22.3 g / L manganese sulfate, 8.6 g / L zinc sulfate, 0.25 g / L sodium molybdate, 0.025 g / L copper sulfate, 0.025 g / L cobalt chloride hexahydrate, 37.3 g / L disodium edetate, 27.8 g / L ferrous sulfate, 100 g / L inositol, 2 g / L glycine, 0.1 g / L thiamine hydrochloride, 0.5 g / L pyridoxine hydrochloride, and 0.5 g / L niacin.

[0095] The lemon juice prepared in the embodiment of the present invention is prepared by mixing 100 g of lemons with 1 L of distilled water, boiling the mixture, and collecting the liquid to obtain 100 g / L of lemon juice.

[0096] Example 1

[0097] Effects of different explants on the proliferation coefficient of Lithospermum arborvitae seedlings

[0098] Take Xinjiang Lithospermum officinale ( Figure 1 ) fruits were peeled and the seeds were taken out, which were placed in a -20°C refrigerator for vernalization for 2 days. The Xinjiang Lithospermum seeds were divided into two groups, one group of seeds with peeled seed coats and the other group of seeds with unpeeled seed coats. The stem segments of the Xinjiang Lithospermum mother plant were used as a control to obtain three explants: one group of seeds with peeled seed coats, one group of seeds with unpeeled seed coats, and one group of stem segments with unpeeled roots. The three explants were sterilized separately in a clean bench. They were first soaked in 75vt% ethanol for 60s, rinsed three times with sterile water, then soaked in a 15vt% hydrogen peroxide solution for 15min, rinsed three times with sterile water, and then soaked in a 500mg / L ampicillin solution for 60min. The moisture on the surface of the explants was absorbed with filter paper to obtain sterile Xinjiang Lithospermum explants.

[0099] Sterile Xinjiang Lithospermum explants were placed in culture dishes containing the start-up culture medium and cultured for 12 days at a temperature of 25°C, a light intensity of 8000 lx, and a light duration of 14 hours per day. (After 12 days of culture, the growth of the seeds with the seed coat peeled off was as follows: Figure 2 After that, the cells were transferred to a tissue culture bottle containing a start-up culture medium and cultured for 18 days at a temperature of 25°C, a light intensity of 8000 lx, and a light duration of 14 h per day to obtain sterile seedlings (sterile seedlings obtained by using stem segments with roots removed as explants as shown in FIG. Figure 3The germination rate of the seeds with and without seed coat peeling was observed during the process of obtaining the aseptic seedlings. The results are shown in Table 1. Germination rate = the number of germinated seeds / the total number of experimental seeds x 100%. The initiation medium is based on SH medium and further includes 20 g / L sucrose, 7.5 g / L agar, and pH 5.9.

[0100] The roots of the aseptic seedlings obtained by excising the peeled seed coat and the seed coat of the seeds without peeling were cut off, and the withered leaves were cut off to obtain stem segments, which were transferred to the proliferation medium and cultured for 25 days in an environment with a temperature of 25°C, a light intensity of 8000 lx, and a light time of 14 h per day, and the proliferation medium was replaced every 10 days. The withered leaves of the aseptic seedlings obtained by using the stem segments as explants were cut off, and the stem segments were transferred to the proliferation medium and cultured for 25 days in an environment with a temperature of 25°C, a light intensity of 8000 lx, and a light time of 14 h per day, and the proliferation medium was replaced every 10 days. The proliferation coefficient of the proliferation seedlings obtained by different explants was calculated, and the results are shown in Table 1. The proliferation medium is based on 1 / 2MS medium without ammonium and further includes 6-benzylaminopurine 2.0 mg / L, naphthalene acetic acid 0.2 mg / L, sucrose 30 g / L, agar 7.5 g / L, and pH 5.9. The proliferation seedlings obtained by using the peeled seed coat of Xinjiang Baphlania Radix as explants are shown in Figure 4 The stem segments without roots obtained by using the peeled seed coat of Xinjiang Baphlania Radix as explants are shown in Figure 5 The proliferation coefficient = the number of stem segments after proliferation / the number of inoculated stem segments.

[0101] Table 1 Influence of different explants on the proliferation culture of Xinjiang Baphlania Radix

[0102] Explant Germination rate (%) Proliferation coefficient Seeds with peeled testa 94.7 2.18 Seeds without peeled testa 89.5 0 Stem segments with removed roots / 3.67

[0103] As shown in Table 1, except that the proliferation coefficient of the seed without peeling seed coat as the explant for the proliferation culture of Xinjiang Baphlania Radix is 0, the other two kinds of explants can obtain proliferation seedlings with a certain proliferation effect.

[0104] Example 2

[0105] Influence of the proliferation medium on the proliferation seedlings of Xinjiang Baphlania Radix

[0106] The stems of the roots of E. przewalskii were removed as explants, and the aseptic seedlings were cultured according to the method of Example 1. The obtained aseptic seedlings were transferred to a proliferation medium based on 1 / 2MS medium without ammonium and containing different concentrations of 6-benzylaminopurine, naphthalene acetic acid, and lemon juice, pH 5.9, and cultured for 25 days at a temperature of 25°C, an illumination intensity of 8000 lx, and a light time of 14 h per day. The proliferation medium was replaced every 10 days, and the effect of different hormone concentrations on the proliferation of E. przewalskii aseptic seedlings was calculated according to the method of Example 1. The hormone concentration combinations and results are shown in Table 2.

[0107] Table 2 Effect of different hormone concentration combinations on the proliferation of E. przewalskii aseptic seedlings

[0108]

[0109] Table 2 shows that when the concentration of 6-benzylaminopurine added to the proliferation medium is 2.0-3.0 mg / L, the concentration of naphthalene acetic acid is 0.05-0.1 mg / L, and the concentration of lemon juice is 300-500 mg / L, the proliferation coefficient of the obtained proliferation seedlings is 3.7-4.3, greatly improving the proliferation effect.

[0110] Example 3

[0111] The stems of the roots of E. przewalskii were removed as explants and sterilized in a clean bench. First, they were soaked in 70% ethanol for 70 s, washed with sterile water 3 times, then soaked in 20% hydrogen peroxide solution for 10 min, washed with sterile water 3 times, and then soaked in an ampicillin solution with a concentration of 450 mg / L for 50 min. The water on the surface of the explants was absorbed with filter paper to obtain sterile E. przewalskii explants.

[0112] The sterile E. przewalskii explants were placed in a culture dish containing the initiation medium and cultured at a temperature of 23°C, an illumination intensity of 10000 lx, and a light time of 13 h per day for 12 days. Then they were transferred to a tissue culture bottle containing the initiation medium and cultured at a temperature of 23°C, an illumination intensity of 10000 lx, and a light time of 13 h per day for 20 days to obtain aseptic seedlings. The initiation medium was based on SH medium and also contained 15 g / L sucrose and 7.0 g / L agar, with a pH of 5.9.

[0113] The wilted leaves of the aseptic seedlings were cut off and transferred to the multiplication medium, and cultured for 25 days in an environment with a temperature of 23°C, a light intensity of 10000 lx, and a light time of 13 hours per day, and the multiplication medium was replaced every 10 days. The multiplication medium was based on ½MS medium without ammonium, and further included 6-benzylaminopurine 3.0 mg / L, naphthalene acetic acid 0.05 mg / L, lemon juice 400 mg / L, sucrose 30 g / L, agar 7.5 g / L, and pH 5.9. The multiplication coefficient of the multiplication seedlings obtained by using the method was 4.5. The multiplication seedlings obtained by using the method are shown in Figure 6 .

[0114] The multiplication seedlings were transferred to the rooting medium (based on ½MS medium, further including naphthalene acetic acid 0.4 mg / L, indole-3-butyric acid 0.5 mg / L, activated carbon 0.5 g / L, sucrose 30 g / L, agar 8.0 g / L; pH 6.0), and cultured for 20 days in an environment with a temperature of 23°C, a light intensity of 10000 lx, and a light time of 13 hours per day, to obtain the test tube seedlings of Radix Baphlai. The growth conditions of the test tube seedlings of Radix Baphlai obtained at 7 days, 15 days and 20 days during the culture process are shown in Figure 7 , Figure 8 and Figure 9 .

[0115] Figure 7 , Figure 8 and Figure 9 It can be seen that the test tube seedlings did not appear wilted and browned during the growth process, and the growth condition was good.

[0116] The test tube seedlings were moved to the open cover acclimation for 3 days, and the medium remaining in the root system of the test tube seedlings after acclimation was washed away by flowing water, and the water was absorbed by the absorbent paper, to obtain the seedlings to be cultivated. The wilted leaves of the seedlings to be cultivated were cut off by scissors, then soaked in the carbendazim solution for 30 minutes, and finally transplanted into the seedling tray containing the seedling substrate, and cultured for 6 days in a constant temperature incubator with a temperature of 25°C and a light time of 8 hours per day, to obtain the seedlings to be transplanted. The breathable bag was sleeved on the top of the plug tray 6 days before the plug tray seedling. The seedling substrate was vermiculite and nutrient soil, and the mass ratio of vermiculite to nutrient soil was 1:1.

[0117] The Radix Baphlai seedlings to be transplanted were transplanted into the field, and the survival rate was 88%.

[0118] Example 4

[0119] The seed of Arnebia euchroma (Royle) Johnst. with seed coat peeled off was used as explants and was sterilized in a clean bench. The explants were first immersed in 80% ethanol for 50 s, washed with sterile water for 3 times, then immersed in 10% hydrogen peroxide solution for 20 min, washed with sterile water for 3 times, and then immersed in 500 mg / L ampicillin solution for 70 min. The water on the surface of the explants was absorbed by filter paper to obtain sterile Arnebia euchroma explants.

[0120] The sterile Arnebia euchroma explants were placed in a culture dish containing initiation medium and were cultured in an environment with a temperature of 25℃, a light intensity of 8000 lx, and a light time of 14 h per day for 12 d, and then were transferred to a tissue culture bottle containing initiation medium and were further cultured in an environment with a temperature of 25℃, a light intensity of 8000 lx, and a light time of 14 h per day for 17 d to obtain sterile seedlings with roots. The initiation medium was based on SH medium and further included 25 g / L sucrose and 8.0 g / L agar, and the pH was 5.9.

[0121] The sterile seedlings were cut to remove the roots and the withered leaves, and stem segments were obtained. The stem segments were transferred to a proliferation medium and were cultured in an environment with a temperature of 25℃, a light intensity of 8000 lx, and a light time of 14 h per day for 21 d, and the proliferation medium was replaced every 10 d. The proliferation medium was based on 1 / 2MS medium without ammonium and further included 6-benzylaminopurine 2.5 mg / L, naphthalene acetic acid 0.15 mg / L, lemon juice 500 mg / L, sucrose 30 g / L, and agar 7.5 g / L, and the pH was 5.9. The proliferation coefficient of the proliferation seedlings obtained by the method was 4.2.

[0122] The proliferation seedlings were transferred to a rooting medium (based on 1 / 2MS medium and further including naphthalene acetic acid 0.3 mg / L, indole-3-butyric acid 0.8 mg / L, activated carbon 0.3 g / L, sucrose 30 g / L, and agar 8.0 g / L; the pH was 6.0) and were cultured in an environment with a temperature of 25℃, a light intensity of 8000 lx, and a light time of 14 h per day for 22 d to obtain Arnebia euchroma test tube seedlings.

[0123] The test tube seedlings were moved outdoors to harden off for 5 d, and the medium remaining in the root system of the test tube seedlings after hardening off was washed away by flowing water, and the water was absorbed by a water-absorbing paper to obtain seedlings to be cultivated. The withered leaves of the seedlings to be cultivated were cut off by scissors, and then were immersed in a carbendazim solution for 40 min, and finally were transplanted into a seedling raising tray containing seedling raising substrate and were cultured in a constant temperature incubator with a temperature of 25℃ and a light time of 8 h per day for 6 d to obtain seedlings to be transplanted. A breathable bag was sleeved on the top of the plug tray 6 d before plug tray seedling raising. The seedling raising substrate was vermiculite and nutrient soil, and the mass ratio of vermiculite to nutrient soil was 1:1.

[0124] The survival rate of the transplanted seedlings of the Xinjiang Arnebia euchroma is 90% in the field.

[0125] From the above examples, the application provides a proliferation medium for cultivating Xinjiang Arnebia euchroma test-tube seedlings, Xinjiang Arnebia euchroma test-tube seedlings and a cultivating method thereof. The proliferation medium of the application can be used to cultivate Xinjiang Arnebia euchroma test-tube seedlings, and can solve the problems of serious yellowing, vitrification and browning of leaves in the in-vitro culture of Xinjiang Arnebia euchroma and low proliferation coefficient of organogenesis pathways. The method of the application can obtain proliferation seedlings with a proliferation coefficient of 3.7 or more.

[0126] The above only describes the preferred embodiments of the application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the application, and these improvements and refinements should also be considered as the protection scope of the application.

Claims

1. A proliferation culture medium suitable for cultivating Arnebia euchroma tube seedlings, characterized in that, Based on 1 / 2MS medium without ammonium, further comprising the following components at the following concentrations: 6-benzylaminopurine 2.0-3.0 mg / L, naphthalene acetic acid 0.05-0.15 mg / L, lemon juice 300-500 mg / L, sucrose 25-35 g / L, and agar 7.0-8.0 g / L; The pH value of the proliferation medium is 5.8-6.

0.

2. Use of the proliferation medium of claim 1 in cultivating fine new Xinjiang Bistorta test-tube seedlings.

3. A method for cultivating Arnebia euchroma test-tube seedlings, characterized in that, Comprising the following steps: (1) disinfecting Xinjiang Bistorta explants to obtain sterile Xinjiang Bistorta explants; (2) inoculating the sterile Xinjiang Bistorta explants into a start-up medium and culturing for 25-35 days to obtain sterile seedlings; (3) transferring the sterile seedlings into a proliferation medium and culturing for 21-25 days to obtain proliferation seedlings; (4) transferring the proliferation seedlings into a rooting medium and culturing for 18-25 days to obtain Xinjiang Bistorta test-tube seedlings; The proliferation medium is the proliferation medium of claim 1.

4. The breeding method according to claim 3, characterized by, The Xinjiang Bistorta explants are peeled seeds or stems with roots removed; The disinfection method is: soaking the Xinjiang Bistorta explants in ethanol for 50-70 seconds to obtain preliminarily disinfected Xinjiang Bistorta explants; soaking the preliminarily disinfected Xinjiang Bistorta explants in a hydrogen peroxide solution for 10-20 minutes to obtain re-disinfected Xinjiang Bistorta explants; and soaking the re-disinfected Xinjiang Bistorta explants in an ampicillin solution for 50-70 minutes to obtain sterile Xinjiang Bistorta explants; The initial concentration of the ethanol is 70-80 vol%; The initial concentration of the hydrogen peroxide solution is 10-20 vol%; The initial concentration of the ampicillin solution is 450-550 mg / L.

5. The cultivation method according to claim 4, characterized in that The start-up medium is based on SH medium and further comprises the following components at the following concentrations: Sucrose 15-25 g / L and agar 7.0-8.0 g / L; The pH value of the start-up medium is 5.8-6.

0.

6. The breeding method according to claim 5, characterized by, The rooting medium is based on 1 / 2MS medium and further comprises the following components at the following concentrations: Naphthalene acetic acid 0.3-0.5 mg / L, indole butyric acid 0.5-1.0 mg / L, activated carbon 0.2-0.5 g / L, sucrose 25-35 g / L, and agar 7.0-8.0 g / L; The pH value of the rooting medium is 5.8-6.

0.

7. The breeding method according to any one of claims 3 to 6, characterized by, The temperature of the culture in steps (2)-(4) is independently 23-27°C, the light intensity of the culture is independently 5000-10000 lx, and the light time of the culture is independently 13-15 hours per day.

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