Method for cultivating Xinjiang lithospermum erythrorhizon test-tube plantlets and method for hardening test-tube plantlets

Abstract

The invention relates to a method for cultivating Xinjiang lithospermum erythrorhizon test-tube plantlets and a method for hardening the test-tube plantlets, and belongs to the technical field of plant tissue culture. The invention provides a proliferation culture medium suitable for tissue culture of Sinkiang lithospermum, the proliferation culture medium is based on an SH culture medium and comprises 6-benayl aminopurine, indoleacetic acid, coconut water, sucrose and agar, and the pH value of the proliferation culture medium is 5.6-6.0. When the method for culturing the Sinkiang lithospermum test-tube plantlets is adopted for tissue culture, the germination rate of Sinkiang lithospermum seeds can reach 96.7%, the multiplication coefficient can reach 13.5, the rooting rate can reach 96.3%, and when the Sinkiang lithospermum test-tube plantlets are subjected to seedling hardening by adopting the method for hardening the Sinkiang lithospermum test-tube plantlets, the obtained to-be-transplanted plantlets are good in root system development and good in transplanting effect. And the seedling hardening survival rate of the test-tube seedlings can reach more than 91.3%. The method provides technical support for rapid propagation and large-scale production of the Sinkiang lithospermum erythrorhizon, and resource protection and sustainable utilization of the Sinkiang lithospermum erythrorhizon are realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant tissue culture, in particular to a cultivation method of Arnebia euchroma(Royle) Johnst test-tube seedlings and a method for seedling hardening of the test-tube seedlings. BACKGROUND

[0002] Arnebia euchroma(Royle) Johnst is a perennial herb of Arnebia Forssk in Boraginaceae, with thick and strong roots, erect stems, and leaf blades without petioles and sparsely pubescent on both surfaces. It is distributed in the south and north slopes of the Tianshan Mountains in Xinjiang and the western part of Tibet, China, and grows in gravel slopes, alluvial fans, grasslands and meadows at an altitude of 2500-4200m. In the wild population, the plants of Arnebia euchroma often distribute in a cluster, and multiple single plants gather to form a cluster of buds. It is suitable for cool, humid, good light, and prefers cool and humid climate, and weakly acidic to weakly alkaline soil. It is afraid of high temperature, but it is drought-tolerant and not water-tolerant, and the suitable temperature is between 9-22℃. When growing at a temperature of 25℃ for a long time, the leaf blades of Arnebia euchroma are thin and narrow, and when growing in an environment with a temperature greater than 30℃, the plants show burn symptoms, and even die. The propagation of Arnebia euchroma includes root propagation and seed propagation. Since Arnebia euchroma is used as a medicinal root, and there is little artificial planting of Arnebia euchroma at present, the source of medicine is mainly wild resources, so the root propagation is less used, and the seed propagation is mainly used.

[0003] Arnebia euchroma has important value in medicine and economy, and has been excessively excavated in recent years. Arnebia euchroma is difficult to cultivate, and has strict requirements for the growth environment, and has a long growth cycle of five years for flowering and seed setting. Due to the reasons of heteromorphic style self-incompatibility, the seed setting rate of Arnebia euchroma is low, and the seed germination rate is low, the seedling period is withering, and the seedling transplanting is difficult. The wild resources are rapidly decreasing, becoming an endangered species, and resource protection is imminent.

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

[0005] The purpose of the present application is to provide a cultivation method of Arnebia euchroma test-tube seedlings and a method for seedling hardening of the test-tube seedlings, so as to solve the problems of long growth cycle, low seed setting rate, low seed germination rate, seedling period withering, and seedling transplanting difficulty of Arnebia euchroma in the prior art.

[0006] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical scheme:

[0007] The present application provides a proliferation culture medium suitable for tissue culture of Arnebia euchroma, which is based on SH culture medium and comprises the following components in the following final concentrations:

[0008] 0.5-2.5 mg / L of 6-benzylaminopurine, 0.1-0.5 mg / L of indoleacetic acid, 50-150 g / L of coconut water, 9-11 g / L of sucrose and 6.6-8.6 g / L of agar; the pH value of the proliferation culture medium is 5.6-6.0.

[0009] The application provides application of the proliferation culture medium in cultivating high-survival-rate new Xinjiang Bistorta test tube seedlings.

[0010] The application provides a cultivating method of new Xinjiang Bistorta test tube seedlings, which comprises the following steps:

[0011] (1) seed disinfection and germination culture: Xinjiang Bistorta seeds are stored at -18- -22 ℃ for 1-3 days, and then inoculated into a seed germination culture medium after disinfection, and cultured at 24-26 ℃ for 2-6 weeks to obtain Xinjiang Bistorta aseptic seedlings;

[0012] (2) proliferation culture: the aseptic seedlings are inoculated into a proliferation culture medium and cultured for 25-35 days to obtain Xinjiang Bistorta proliferation seedlings;

[0013] (3) rooting culture: the adventitious buds of the Xinjiang Bistorta proliferation seedlings are cut into single plants and inoculated into a rooting culture medium and cultured for 20-30 days to obtain Xinjiang Bistorta test tube seedlings;

[0014] The proliferation culture medium in step (2) is the proliferation culture medium.

[0015] Preferably, the disinfection method in step (1) is soaking Xinjiang Bistorta seeds in an aqueous HgCl2 solution for 5-6 min;

[0016] The initial concentration of the aqueous HgCl2 solution is 0.05%-0.15%.

[0017] Preferably, the seed germination culture medium in step (1) is based on SH culture medium and comprises the following components with terminal concentrations:

[0018] 9-11 g / L of sucrose and 6.6-8.6 g / L of agar;

[0019] The pH value of the seed germination culture medium is 5.6-6.0.

[0020] Preferably, the rooting culture medium in step (3) is based on SH culture medium and comprises the following components with terminal concentrations:

[0021] 0.1-1.0 mg / L of 1-naphthaleneacetic acid, 0.1-0.5 mg / L of indolebutyric acid, 9-11 g / L of sucrose and 6.6-8.6 g / L of agar;

[0022] The pH value of the rooting culture medium is 5.6-6.0.

[0023] Preferably, the temperature of the culture is independently 23-27℃, the humidity of the culture is independently 70-80%, the light time of the culture is independently 13-15h / d, and the light intensity of the culture is independently 2000-3000Lx.

[0024] The application provides the new Xinjiang Arnebia euchroma test tube seedling obtained by the cultivation method.

[0025] The application provides a method for hardening the Xinjiang Arnebia euchroma test tube seedling.

[0026] The Xinjiang Arnebia euchroma test tube seedling is the Xinjiang Arnebia euchroma test tube seedling.

[0027] Preferably, the substrate comprises peat soil and vermiculite, and the mass ratio of the peat soil and the vermiculite is 1-3:1.

[0028] The temperature of the culture is 23-27℃, the humidity of the culture is 70-80%, the light time of the culture is 13-15h / d, and the light intensity of the culture is 3000-4000Lx.

[0029] The application has the following technical effects and advantages:

[0030] The application optimizes and screens the proliferation culture medium and the rooting culture medium for the tissue culture of Xinjiang Arnebia euchroma, and establishes a cultivation method for the Xinjiang Arnebia euchroma test tube seedling according to the screened proliferation culture medium and rooting culture medium. The germination rate of the Xinjiang Arnebia euchroma seeds can reach 96.7% by using the cultivation method for the Xinjiang Arnebia euchroma test tube seedling, the proliferation coefficient can reach 13.5, and the rooting rate can reach 96.3%. The application also establishes a hardening method for the Xinjiang Arnebia euchroma test tube seedling. The Xinjiang Arnebia euchroma test tube seedling is placed in a covered seedling raising pot for hardening, which can reduce the water loss and death of the leaves of the test tube seedling, greatly improve the survival rate of the Xinjiang Arnebia euchroma test tube seedling, and finally obtain the seedling to be transplanted with well-developed root systems. The survival rate of the test tube seedling for hardening can reach more than 91.3%. The technical scheme of the application provides technical support for the rapid propagation and large-scale production of Xinjiang Arnebia euchroma, and realizes the resource protection and sustainable utilization of Xinjiang Arnebia euchroma. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 Fig. 1 is a growth state diagram of Xinjiang Arnebia euchroma seeds in the germination culture for 1 week in Embodiment 4;

[0032] Figure 2 Fig. 2 is a growth state diagram of Xinjiang Arnebia euchroma seeds in the germination culture for 2 weeks in Embodiment 4;

[0033] Figure 3 Figure 4 is a growth state diagram of the aseptic seedlings of Arnebia euchroma (R. Br.) Johnst. in the proliferation culture for 4 weeks in Example 4;

[0034] Figure 4 Figure 5 is a growth state diagram of the aseptic seedlings of Arnebia euchroma (R. Br.) Johnst. in the proliferation culture for 10 days in Example 4;

[0035] Figure 5 Figure 6 is a growth state diagram of the aseptic seedlings of Arnebia euchroma (R. Br.) Johnst. in the proliferation culture for 20 days in Example 4;

[0036] Figure 6 Figure 7 is a growth state diagram of the aseptic seedlings of Arnebia euchroma (R. Br.) Johnst. in the proliferation culture for 30 days in Example 4;

[0037] Figure 7 Figure 8 is a rooting culture diagram of the adventitious buds of Arnebia euchroma (R. Br.) Johnst. in Example 4;

[0038] Figure 8 Figure 9 is a test tube seedling diagram of Arnebia euchroma (R. Br.) Johnst. in Example 4;

[0039] Figure 9 Figure 10 is a growth state diagram of the test tube seedlings of Arnebia euchroma (R. Br.) Johnst. transplanted into the covered seedling raising pots in Example 6;

[0040] Figure 10 Figure 11 is a diagram of the seedlings to be transplanted of Arnebia euchroma (R. Br.) Johnst. in Example 6.DETAILED DESCRIPTION

[0041] The application provides a proliferation culture medium suitable for tissue culture of Arnebia euchroma (R. Br.) Johnst., which is based on SH culture medium and comprises the following components in the following final concentrations:

[0042] 0.5-2.5 mg / L 6-benzylaminopurine, 0.1-0.5 mg / L indole-3-acetic acid, 50-150 g / L coconut water, 9-11 g / L sucrose and 6.6-8.6 g / L agar;

[0043] The final concentration of the 6-benzylaminopurine is preferably 1.5 mg / L; the final concentration of the indole-3-acetic acid is preferably 0.3 mg / L; the final concentration of the coconut water is 150 g / L; the final concentration of the sucrose is preferably 10 g / L; and the final concentration of the agar is preferably 7.6 g / L;

[0044] The pH value of the proliferation culture medium is 5.6-6.0, preferably 5.8;

[0045] The SH culture medium comprises the following components in the following final concentrations with water as the solvent:

[0046] Potassium nitrate 2500 mg / L, ammonium phosphate 300 mg / L, magnesium sulfate 195.4 mg / L, calcium chloride 151 mg / L, potassium iodide 1 mg / L, boric acid 5 mg / L, manganese sulfate 10 mg / L, zinc sulfate 1 mg / L, sodium molybdate 0.1 mg / L, copper sulfate 0.2 mg / L, cobalt chloride 0.1 mg / L, disodium ethylenediaminetetraacetate 20 mg / L, ferrous sulfate 15 mg / L, inositol 1000 mg / L, thiamine hydrochloride 5 mg / L, pyridoxine hydrochloride 0.5 mg / L, nicotinic acid 5 mg / L, and sucrose 10000 mg / L.

[0047] This invention provides the application of the aforementioned proliferation medium in cultivating test-tube seedlings of Lithospermum erythrorhizon with a high survival rate.

[0048] This invention provides a method for cultivating test-tube seedlings of Lithospermum erythrorhizon from Xinjiang, comprising the following steps:

[0049] (1) Seed disinfection and germination culture: Xinjiang Lithospermum seeds were refrigerated at -18 to -22℃ for 1 to 3 days, disinfected, and then inoculated into seed germination culture medium. They were cultured at 24 to 26℃ for 2 to 6 weeks to obtain sterile Xinjiang Lithospermum seedlings.

[0050] The preferred refrigeration temperature is -20℃; the preferred refrigeration time is 2 days.

[0051] The preferred culture temperature is 25°C;

[0052] The preferred culture time is 4 weeks, more preferably 2 weeks of dark culture followed by 2 weeks of light culture;

[0053] (2) Proliferation culture: Sterile seedlings were inoculated into a proliferation culture medium and cultured for 25 to 35 days to obtain Xinjiang Lithospermum propagation seedlings;

[0054] The preferred culture time is 30 days;

[0055] (3) Rooting culture: The adventitious buds of Xinjiang Lithosperm propagation seedlings were cut into single plants and inoculated into rooting culture medium for 20-30 days to obtain Xinjiang Lithosperm test-tube seedlings.

[0056] The preferred culture time is 25 days;

[0057] The proliferation medium mentioned in step (2) is the proliferation medium described above.

[0058] In this invention, the disinfection method in step (1) is to soak Xinjiang Lithospermum seeds in HgCl2 aqueous solution for 5-6 minutes, preferably 6 minutes;

[0059] The initial concentration of the HgCl2 aqueous solution is 0.05% to 0.15%, preferably 0.1%.

[0060] In the present application, the seed germination medium in step (1) is based on SH medium and comprises the following components at the following final concentrations:

[0061] 9-11 g / L sucrose and 6.6-8.6 g / L agar;

[0062] The final concentration of the sucrose is preferably 10 g / L; the final concentration of the agar is preferably 7.6 g / L;

[0063] The pH value of the seed germination medium is 5.6-6.0, preferably 5.8.

[0064] In the present application, the rooting medium in step (3) is based on SH medium and comprises the following components at the following final concentrations:

[0065] 0.1-1.0 mg / L 1-naphthaleneacetic acid, 0.1-0.5 mg / L indolebutyric acid, 9-11 g / L sucrose and 6.6-8.6 g / L agar;

[0066] The final concentration of the 1-naphthaleneacetic acid is preferably 0.5 mg / L; the final concentration of the indolebutyric acid is preferably 0.1 mg / L; the final concentration of the sucrose is preferably 10 g / L; the final concentration of the agar is preferably 7.6 g / L;

[0067] The pH value of the rooting medium is 5.6-6.0, preferably 5.8.

[0068] In the present application, the temperature of the culture in steps (2)-(3) is independently 23-27°C, preferably 25°C, the humidity of the culture is independently 70-80%, preferably 75%, the light time of the culture is independently 13-15 h / d, preferably 14 h / d, and the light intensity of the culture is independently 2000-3000 Lx, preferably 2500 Lx.

[0069] The present application provides the new Xinjiang Baphalan test tube seedling obtained by the cultivation method.

[0070] The present application provides a method for hardening the Xinjiang Baphalan test tube seedling, wherein when the root length of the Xinjiang Baphalan test tube seedling reaches 3-4 cm, the Xinjiang Baphalan test tube seedling is transplanted into a substrate and placed in a covered seedling raising pot for 10-15 days to obtain the Xinjiang Baphalan seedling to be transplanted;

[0071] Preferably, when the root length of the Xinjiang Baphalan test tube seedling reaches 3.5 cm, the Xinjiang Baphalan test tube seedling is taken out of the tissue culture bottle, washed to remove the agar on the root, transplanted into a substrate, and placed in a covered seedling raising pot for 14 days to obtain the Xinjiang Baphalan seedling to be transplanted.

[0072] The new Xinjiang Arnebia euchroma test tube seedling is the Xinjiang Arnebia euchroma test tube seedling.

[0073] In the present application, the substrate comprises peat soil and vermiculite; the mass ratio of the peat soil and the vermiculite is 1-3:1, preferably 2:1;

[0074] The temperature of the culture is 23-27℃, preferably 25℃, the humidity of the culture is 70-80%, preferably 75%, the light time of the culture is 13-15h / d, preferably 14h / d, and the light intensity of the culture is 3000-4000Lx, preferably 3500Lx.

[0075] The technical solutions provided by the present application are described in detail below in combination with examples, but they should not be understood as limiting the scope of protection of the present application.

[0076] The preparation method of the SH culture medium in the present application is as follows: the components in Table 1 are added into water as a solvent, and then the pH value is adjusted to 5.8 to obtain the SH culture medium.

[0077] Table 1: Composition of SH culture medium

[0078] No. Ingredient Content (mg / L) No. Ingredient Content (mg / L) 1 Potassium nitrate 2500 10 Copper sulfate 0.2 2 Ammonium phosphate 300 11 Cobalt chloride 0.1 3 Magnesium sulfate 195.4 12 EDTA disodium salt 20 4 Calcium chloride 151 13 Ferrous sulfate 15 5 Potassium iodide 1 14 Myo-inositol 1000 6 Boric acid 5 15 Thiamine hydrochloride 5 7 Manganese sulfate 10 16 Pyridoxine hydrochloride 0.5 8 Zinc sulfate 1 17 Nicotinic acid 5 9 Sodium molybdate 0.1 18 Sucrose 10000

[0079] The preparation method of the seed germination culture medium used in the present application is as follows: the SH culture medium is used as a base, sucrose with a final concentration of 10g / L and agar with a final concentration of 7.6g / L are added, and the pH value is adjusted to 5.8 to obtain the seed germination culture medium.

[0080] Example 1: Effect of different disinfection methods on seed germination of Xinjiang Arnebia euchroma

[0081] Peel the pericarp from the fruit of *Lithospermum erythrorhizon* and extract the seeds. Store the seeds in a -20℃ low-temperature storage box for 2 days. After low-temperature storage, clean the seeds and divide them into 4 groups of 30 seeds each. Treat the seeds with different disinfection methods: Group 1: Soak the seeds in a 0.1% HgCl2 aqueous solution for 5 minutes; Group 2: Soak the seeds in a 0.1% HgCl2 aqueous solution for 5 minutes, then soak them in a 500 mg / L thiazolyl fibrozil (CEF) aqueous solution for 30 minutes; Group 3: Soak the seeds in a 0.1% HgCl2 aqueous solution for 6 minutes; Group 4: Soak the seeds in a 0.1% HgCl2 aqueous solution for 6 minutes, then soak them in a 500 mg / L CEF aqueous solution for 30 minutes. After disinfection, each group of seeds was rinsed three times with sterile water, and the surface moisture was absorbed with filter paper. Then, the seeds were inoculated into seed germination medium and cultured in the dark at 25°C for 2 weeks, followed by culture under light for 2 weeks to obtain sterile seedlings of Lithospermum erythrorhizon from Xinjiang. The number of germinated seeds, the number of dead seeds, and the number of contaminated seeds were counted. The germination rate (%), mortality rate (%), and contamination rate (%) were calculated. The effect of different disinfection methods on the germination of Lithospermum erythrorhizon seeds was determined. The results are shown in Table 2.

[0082] Table 2. Effects of different disinfection methods on the germination of Lithospermum erythrorhizon seeds in Xinjiang.

[0083]

[0084] According to Table 2, the number of germinated seeds in the third group after disinfection treatment was 29, with a germination rate of 96.7% and a mortality rate of 3.3% with 1 dead seed. No contamination was observed, which was significantly higher than other treatment groups. Therefore, HgCl2 aqueous solution was selected to disinfect Xinjiang Lithospermum seeds by soaking for 6 minutes.

[0085] Example 2: Effects of different concentrations of hormones in the proliferation medium on the proliferation culture of Lithospermum erythrorhizon in Xinjiang

[0086] The proliferation medium was prepared by adding sucrose to a final concentration of 10 g / L and agar to a final concentration of 7.6 g / L, based on SH medium. Then, different concentrations of 6-benzylaminopurine (6-BA), indoleacetic acid (IAA), and coconut water were added, and the pH was adjusted to 5.8 to obtain 9 groups of proliferation media containing different final concentrations of 6-BA, IAA, and coconut water.

[0087] The sterile seedlings of Group 3 in Example 1 were inoculated into 9 groups of proliferation culture media containing different final concentrations of 6-BA, IAA and coconut water. They were cultured for 30 days at a temperature of 25°C, a humidity of 75%, a light duration of 14 h / d and a light intensity of 2500 Lx to obtain Xinjiang Lithospermum proliferation seedlings.

[0088] The proliferation coefficient of *Lithospermum erythrorhizon* seedlings in Xinjiang was determined, and the effects of different concentrations of hormones in the proliferation culture medium on the proliferation culture of *Lithospermum erythrorhizon* seedlings in Xinjiang were compared. The calculation method of the proliferation coefficient is as follows:

[0089] Proliferation coefficient = (Number of plants after propagation / Number of inoculated plants) × 100%;

[0090] The effects of different concentrations of hormones in the proliferation medium on the proliferation culture of Lithospermum erythrorhizon seedlings in Xinjiang are shown in Table 3.

[0091] Table 3. Effects of different concentrations of hormones on the proliferation culture of Lithospermum erythrorhizon in Xinjiang

[0092]

[0093] As shown in Table 3, the proliferation coefficient of the fifth group of proliferation media is 13.5, which is higher than that of the other groups. Therefore, the optimal proliferation medium for the Xinjiang Lithospermum tissue culture method is based on SH medium, with the addition of 6-BA at a final concentration of 1.5 mg / L, IAA at a final concentration of 0.3 mg / L, coconut water at a final concentration of 150 g / L, sucrose at a final concentration of 10 g / L, and agar at a final concentration of 7.6 g / L, with a pH of 5.8.

[0094] Example 3: Effects of different concentrations of hormones in rooting medium on the rooting culture of Lithospermum erythrorhizon in Xinjiang

[0095] The rooting medium was prepared by adding sucrose to a final concentration of 10 g / L and agar to a final concentration of 7.6 g / L, based on SH medium. Then, different concentrations of 1-naphthaleneacetic acid (NAA) and indolebutyric acid (IBA) were added, and the pH was adjusted to 5.8 to obtain 9 groups of rooting media containing different final concentrations of NAA and IBA.

[0096] The *Lithospermum erythrorhizon* seedlings cultured in the fifth group of proliferation medium in Example 2 were inoculated into nine groups of rooting mediums containing different final concentrations of NAA and IBA. The seedlings were cultured for 25 days under conditions of 25℃, 75% humidity, 14 h / d light duration, and 2500 Lx light intensity to obtain test-tube seedlings. The rooting rate was measured, and the effects of different concentrations of hormones in the rooting medium on the rooting culture of *Lithospermum erythrorhizon* were compared. The results are shown in Table 4.

[0097] Table 4. Effects of different concentrations of hormones on rooting culture of Lithospermum erythrorhizon in Xinjiang

[0098] Group Medium type NAA (mg / L) IBA (mg / L) Rooting rate (%) 1 SH 0.1 0.1 85.2 2 SH 0.1 0.3 88.9 3 SH 0.1 0.5 90.5 4 SH 0.5 0.3 94.6 5 SH 0.5 0.5 92.7 6 SH 0.5 0.1 96.3 7 SH 1.0 0.5 89.2 8 SH 1.0 0.1 86.4 9 SH 1.0 0.3 88.3

[0099] As shown in Table 4, the rooting medium in group 6 had the highest rooting rate, at 96.3%. Therefore, the optimal rooting medium for the Xinjiang Lithospermum tissue culture method is based on SH medium, with the addition of NAA at a final concentration of 0.5 mg / L, IBA at 0.1 mg / L, sucrose at 10 g / L, and agar at 7.6 g / L, with a pH of 5.8.

[0100] Example 4: A method for cultivating test-tube seedlings of Lithospermum erythrorhizon from Xinjiang

[0101] A method for cultivating test-tube seedlings of Lithospermum erythrorhizon from Xinjiang includes the following steps:

[0102] (1) Seed disinfection and germination culture: The pericarp of the fruit of *Lithospermum erythrorhizon* was removed and the seeds were extracted. The seeds were stored in a -20℃ low-temperature storage box for 2 days. After low-temperature storage, the seeds were cleaned and disinfected by soaking them in a 0.1% HgCl2 aqueous solution for 6 minutes. The seeds were then rinsed three times with sterile water, and the surface moisture was absorbed with filter paper. They were then inoculated into seed germination medium and cultured in the dark at 25℃ for 2 weeks, followed by 2 weeks of light culture to obtain sterile *Lithospermum erythrorhizon* seedlings. The growth status of seed germination was recorded during the germination culture period. The results are as follows: Figure 1-3 As shown, Figure 1 This image shows the growth status of Xinjiang Lithospermum seeds after one week of germination culture. Figure 2 This image shows the growth status of Xinjiang Lithospermum seeds after 2 weeks of germination culture. Figure 3 Image showing the growth status of sterile seedlings after 4 weeks of germination culture of Lithospermum erythrorhizon seeds from Xinjiang.

[0103] (2) Proliferation Culture: Aseptic seedlings were inoculated into the proliferation culture medium obtained in Example 2 for proliferation culture. The seedlings were cultured for 30 days at a temperature of 25℃, humidity of 75%, light duration of 14 h / d, and light intensity of 2500 Lx to obtain Xinjiang Lithospermum propagation seedlings. The growth status of the aseptic seedlings was recorded during the proliferation culture period, and the results are as follows: Figure 4-6 As shown, Figure 4 This image shows the growth status of aseptic seedlings of Lithospermum erythrorhizon from Xinjiang after 10 days of propagation culture. Figure 5 This image shows the growth status of aseptic seedlings of Lithospermum erythrorhizon from Xinjiang after 20 days of propagation culture. Figure 6 A graph showing the growth status of aseptic seedlings of Lithospermum erythrorhizon from Xinjiang after 30 days of propagation culture.

[0104] (3) Rooting Culture: Adventitious buds from the propagated seedlings were cut into individual plants and inoculated into the rooting medium obtained in Example 3. The plants were cultured for 25 days at 25℃, 75% humidity, 14h / d light duration, and 2500Lx light intensity to obtain Xinjiang Lithospermum in vitro seedlings. The growth status of the adventitious buds was recorded during the rooting culture period. The results are as follows: Figure 7-8 As shown, Figure 7 This diagram illustrates the adventitious bud rooting culture of Lithospermum erythrorhizon from Xinjiang.Figure 8 This image shows a test-tube seedling of Lithospermum erythrorhizon from Xinjiang.

[0105] Example 5: Effect of different substrate ratios on the survival rate of Xinjiang Lithospermum erythrorhizon test-tube seedlings

[0106] Following the method in Example 4, test-tube seedlings of *Lithospermum erythrorhizon* were cultivated. The seedlings were removed from the bottles, the agar on the roots was washed off, and they were transplanted into 10 different seedling substrates (Group 1: nutrient soil; Group 2: nutrient soil:vermiculite = 1:1; Group 3: nutrient soil:vermiculite = 2:1; Group 4: nutrient soil:vermiculite:river sand = 1:1:1; Group 5: nutrient soil:vermiculite:river sand = 2:1:1; Group 6: peat moss; Group 7: peat moss:vermiculite = 1:1; Group 8: peat moss:vermiculite = 2:1:1). Group 1, Group 9 (peat soil:vermiculite:river sand = 1:1:1); Group 10 (peat soil:vermiculite:river sand = 2:1:1), 30 seedlings per group. The 10 groups of transplanted test-tube seedlings were cultured in covered seedling trays for 14 days under the conditions of 25℃, 75% humidity, 14h / d light duration, and 3500Lx light intensity to obtain seedlings ready for transplanting. The survival rate of the seedlings was measured, and the effects of different substrate ratios on the hardening and survival rate of Xinjiang Lithospermum test-tube seedlings were compared. The results are shown in Table 5.

[0107] Table 5. Effects of different substrate ratios on the survival rate of *Lithospermum erythrorhizon* test-tube seedlings from Xinjiang.

[0108]

[0109]

[0110] Table 5 shows that using a mixture of peat moss and vermiculite in a 2:1 mass ratio as the substrate for transplanting and hardening off Xinjiang Lithospermum in vitro seedlings resulted in the highest survival rate of 91.3%. Therefore, the optimal substrate for hardening off Xinjiang Lithospermum in vitro seedlings to prepare for transplanting is a mixture of peat moss and vermiculite in a 2:1 mass ratio.

[0111] Example 6: A method for hardening off in vitro seedlings of Lithospermum erythrorhizon from Xinjiang

[0112] Following the method in Example 4, in vitro seedlings of *Lithospermum erythrorhizon* were cultivated. When the roots of the seedlings reached a length of 3.5 cm, they were removed from the tissue culture bottles, the agar on the roots was washed off, and they were transplanted into a substrate of peat moss and vermiculite in a 2:1 ratio. They were then placed in covered seedling trays and cultured for 14 days to obtain *Lithospermum erythrorhizon* seedlings ready for transplanting. During the hardening-off period, the growth status of the *Lithospermum erythrorhizon* seedlings was recorded, and the results are as follows: Figure 9-10 As shown, Figure 9 This diagram shows the growth status of Xinjiang Lithospermum erythrorhizon test-tube seedlings transplanted into covered seedling trays. Figure 10 This image shows seedlings of Lithospermum erythrorhizon from Xinjiang awaiting transplantation.

[0113] As shown in the above embodiments, this invention provides a method for cultivating test-tube seedlings of *Lithospermum erythrorhizon* and a method for hardening off test-tube seedlings. This invention optimizes and screens the proliferation and rooting media for *Lithospermum erythrorhizon* tissue culture, and establishes a method for cultivating test-tube seedlings of *Lithospermum erythrorhizon* based on the screened proliferation and rooting media. Using this method, the germination rate of *Lithospermum erythrorhizon* seeds can reach 96.7%, the proliferation coefficient can reach 13.5, and the rooting rate can reach 96.3%. This invention also establishes a method for hardening off test-tube seedlings of *Lithospermum erythrorhizon*. Placing the test-tube seedlings in covered seedling trays for hardening off reduces leaf water loss and mortality, significantly improving the survival rate of hardened-off seedlings, ultimately yielding seedlings with well-developed root systems ready for transplanting. The survival rate of hardened-off test-tube seedlings can reach over 91.3%. The technical solution of this invention provides technical support for the rapid propagation and large-scale production of *Lithospermum erythrorhizon*, realizing the resource protection and sustainable utilization of *Lithospermum erythrorhizon*.

[0114] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A proliferation culture medium suitable for tissue culture of Lithospermum erythrorhizon from Xinjiang, characterized in that, The proliferation medium is based on SH medium and includes the following components at final concentrations: The culture medium contains 0.5–2.5 mg / L of 6-benzylaminopurine, 0.1–0.5 mg / L of indoleacetic acid, 50–150 g / L of coconut water, 9–11 g / L of sucrose, and 6.6–8.6 g / L of agar; the pH of the culture medium is 5.6–6.

0.

2. The application of the proliferation medium described in claim 1 in the cultivation of high-survival-rate Xinjiang Lithospermum in vitro seedlings.

3. A method for cultivating test-tube seedlings of Lithospermum erythrorhizon from Xinjiang, characterized in that, Includes the following steps: (1) Seed disinfection and germination culture: Xinjiang Lithospermum seeds were refrigerated at -18 to -22℃ for 1 to 3 days, disinfected, and then inoculated into seed germination culture medium. They were cultured at 24 to 26℃ for 2 to 6 weeks to obtain sterile Xinjiang Lithospermum seedlings. (2) Proliferation culture: Sterile seedlings were inoculated into a proliferation culture medium and cultured for 25 to 35 days to obtain Xinjiang Lithospermum propagation seedlings; (3) Rooting culture: The adventitious buds of Xinjiang Lithosperm propagation seedlings were cut into single plants and inoculated into rooting culture medium for 20-30 days to obtain Xinjiang Lithosperm test-tube seedlings. The proliferation medium mentioned in step (2) is the proliferation medium described in claim 1.

4. The cultivation method according to claim 3, characterized in that, The disinfection method described in step (1) is to soak Xinjiang Lithospermum seeds in HgCl2 aqueous solution for 5-6 minutes; The initial concentration of the HgCl2 aqueous solution is 0.05% to 0.15%.

5. The cultivation method according to claim 3, characterized in that, The seed germination medium described in step (1) is based on SH medium and includes the following components at final concentrations: 9–11 g / L of sucrose and 6.6–8.6 g / L of agar; The pH value of the seed germination medium is 5.6 to 6.

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6. The cultivation method according to claim 3, characterized in that, The rooting medium described in step (3) is based on SH medium and includes the following components at the following final concentrations: 0.1–1.0 mg / L of 1-naphthaleneacetic acid, 0.1–0.5 mg / L of indolebutyric acid, 9–11 g / L of sucrose, and 6.6–8.6 g / L of agar; The pH value of the rooting medium is 5.6 to 6.

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7. The cultivation method according to claim 3, characterized in that, The temperature for cultivation in steps (2) to (3) is 23 to 27°C, the humidity for cultivation is 70 to 80%, the light exposure time for cultivation is 13 to 15 h / d, and the light intensity for cultivation is 2000 to 3000 Lx.

8. The Xinjiang Lithospermum test-tube seedlings obtained by the cultivation method according to any one of claims 3 to 7.

9. A method for hardening off in vitro seedlings of Lithospermum erythrorhizon from Xinjiang, characterized in that, When the roots of the Xinjiang Lithospermum test-tube seedlings reach 3-4 cm in length, transplant the Xinjiang Lithospermum test-tube seedlings into the substrate and place them in a covered seedling pot for 10-15 days to obtain Xinjiang Lithospermum seedlings ready for transplanting. The Xinjiang Lithospermum in vitro seedlings are the Xinjiang Lithospermum in vitro seedlings as described in claim 8.

10. The method according to claim 9, characterized in that, The matrix comprises peat moss and vermiculite; the mass ratio of peat moss to vermiculite is 1 to 3:1; The culture temperature is 23–27°C, the culture humidity is 70–80%, the culture light duration is 13–15 h / d, and the culture light intensity is 3000–4000 Lx.

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