A method for tissue culture of Radix Sagittifolia seedlings

Abstract

The present invention provides a method for tissue culture of Radix Sagittiflorae seedlings, and relates to the technical field of Radix Sagittiflorae cultivation. The method for tissue culture of Radix Sagittiflorae seedlings adopts adventitious buds on the pseudobulbs of Radix Sagittiflorae as explants, and sequentially adopts rhizome induction medium, rhizome induction medium, differentiation medium, and rooting medium to induce test tube seedlings with a high transplant survival rate, and by improving the White medium, honeysuckle, quercetin, tea saponin, and bisabolol in each culture medium, the efficiency of each link of cultivation can be effectively guaranteed, and the loss of Radix Sagittiflorae seedling tissue culture can be comprehensively reduced. The present invention overcomes the shortcomings of the prior art, obtains robust Radix Sagittiflorae seedlings through tissue culture methods, reduces the cost of artificial cultivation of Radix Sagittiflorae, improves the survival rate of seedling cultivation, and facilitates large-scale artificial cultivation.

Description

Technical Field

[0001] The invention relates to the technical field of cultivating sagittaria sagittifolia, and in particular to a method for culturing sagittaria sagittifolia seedlings. Background Art

[0002] Mao Ci Mushroom is a perennial herbaceous plant of the Orchidaceae family, also known as Mountain Ci Mushroom and Ice Ball. It is a commonly used Chinese medicinal material with strong cold resistance. It grows mostly in high mountain areas with an altitude of 600-1200m, suitable for growing under forests, on the hillsides of deep mountains, or in places with shade and only scattered light. It is distributed in Northeast and Northwest my country. The pseudobulb is used as medicine, which tastes sweet and slightly spicy, is cold in nature, has a slight toxicity, and enters the liver and spleen meridians. It has the effects of clearing heat and detoxifying, resolving phlegm and dispersing nodules. It is used to treat carbuncle, furuncle, scrofula, phlegm nodules, lymph node tuberculosis, throat swelling and pain, snake and insect bites, and rabies. According to modern medical research, Mao Ci Mushroom has a certain preventive and therapeutic effect on cancer, and can enhance human immunity to a certain extent.

[0003] Due to its good medicinal effects and simple preparation, it has a broad market in the fields of traditional Chinese medicine and health care products. However, the overall wild resources of Trichosanthes are limited. In recent years, wild resources have been over-exploited, resulting in resource scarcity and high market prices. Therefore, artificial cultivation of Trichosanthes is an important direction at this stage.

[0004] Since the seeds of Trichosanthes are very small and the embryos are not fully developed, it is difficult to plant them artificially by sowing. The existing planting methods mainly include bulb propagation and tissue culture propagation. However, bulb propagation is greatly restricted by season and temperature and is not conducive to industrial planting. Tissue culture propagation can well realize large-scale industrial planting. Generally, capsules or bulbs can be used for tissue culture to obtain test tube seedlings. However, the survival rate of rooted seedlings obtained by existing tissue culture is low, which increases the cost of artificial planting to a certain extent. Summary of the invention

[0005] In view of the shortcomings of the existing technology, the present invention provides a method for tissue culture of Trichosanthes kirilowii seedlings. Robust Trichosanthes kirilowii seedlings are obtained through the tissue culture method, the cost of artificial cultivation of Trichosanthes kirilowii is reduced, the survival rate of seedling cultivation is improved, and large-scale artificial cultivation is facilitated.

[0006] To achieve the above objectives, the technical solution of the present invention is implemented through the following technical solutions:

[0007] A method for tissue culture of Pleurotus eryngii seedlings, comprising the following steps:

[0008] S1. Obtaining explants: Cut off the adventitious buds on the pseudobulbs of Sagittaria sagittifolia and use them as explants after disinfection;

[0009] S2. Rhizome induction culture: the explants are inoculated in a rhizome induction medium to induce bud differentiation and form rhizomes; wherein the formula of the rhizome induction medium is composed of the following substances: modified White medium + NAA 1.0-1.5 mg / L + Huabao No. 1 5-8 mL / L + honeysuckle 20-40 mg / L + agar powder 3-5 g / L; wherein the composition of the components in the modified White medium is: KNO3 40 mg / L + citric acid 2.5 mg / L + Ca(NO3)2·4H2O 165 mg / L + MgSO4 1120 mg / L + KCl 50 mg / L + NaH2PO4 20 mg / L + thiamine hydrochloride 0.1 mg / L + pyridoxine hydrochloride 0.1 mg / L + nicotinic acid 0.5 mg / L + glycine 5 mg / L + MnSO4·4H2O 11 mg / L + ZnSO4·7H2O 4mg / L+KI 0.75mg / L+H3BO3 1.5mg / L+sucrose 20g / L;

[0010] S3, rhizome proliferation culture: take the rhizome formed in the above rhizome induction medium, cut off the rhizome and inoculate it into the rhizome proliferation medium for proliferation culture, and culture to obtain the raw rhizome, and the rhizome proliferation medium is composed of the following contents of substances: MS medium + 6-BA 4.5-5.5 mg / L + NAA 0.12-0.15 mg / L + quercetin 6-8 mg / L + tea saponin 20-30 mg / L + sucrose 15-20 g / L + agar powder 3-5 g / L;

[0011] S4, differentiation culture: take the above-mentioned rhizome, cut it off and inoculate it into differentiation medium for differentiation culture to obtain Mao Sagittaria seedlings; the differentiation medium is composed of the following substances: MS medium + NAA 0.12-0.15mg / L + 6-BA 2.0-2.5mg / L + honeysuckle 60-80mg / L + sucrose 15-20g / L + agar powder 3-5g / L;

[0012] S5, rooting culture: inoculate the above-mentioned Mao Sagittaria seedlings into a rooting medium, and perform rooting culture until adventitious roots are produced to obtain test tube seedlings; the formula of the rooting medium is composed of the following substances: 1 / 2MS medium + 6-BA 2.0-3.0 mg / L + aspirin 10-20 mg / L + honeysuckle 10-20 mg / L + bisabolol 10-15 mg / L + sucrose 15-20 g / L + agar powder 3-5 g / L;

[0013] S6. Transplantation: After hardening the test tube seedlings, transplant them into the substrate for cultivation.

[0014] Preferably, the disinfection method in step S1 is to rinse the adventitious buds with sterile water, then absorb the moisture, then rinse with 75% ethanol solution 2-3 times, then soak in 20g / L sodium hypochlorite solution for 15min, then rinse with sterile water 3-4 times, and complete the disinfection after absorbing the surface moisture.

[0015] Preferably, the culturing method in step S2 is to culture the inoculated rhizome induction culture medium in the dark at 25±5° C. for 48 hours, and then control the lighting time to 10 hours per day, and the light intensity is 1000 lx.

[0016] Preferably, in step S3, the length of the cut rhizome is controlled to be 0.6-0.8 cm.

[0017] Preferably, the proliferation culture condition in step S3 is dark culture at a temperature of 25±5°C.

[0018] Preferably, the differentiation culture conditions in step S4 are culture at a temperature of 25±5° C. with illumination of 10 h / d and a light intensity of 1000 lx.

[0019] Preferably, the rooting culture condition in step S5 is to culture at a temperature of 25±5° C. with a light intensity of 10 h / d and a light intensity of 1000 lx.

[0020] Preferably, the method of hardening the seedlings in step S6 is to place the test tube seedlings with rooting culture base outdoors for 2-3 days.

[0021] Preferably, the transplanting method in step S6 is to take out the test tube seedlings and remove the culture medium, and then plant them in a sterilized substrate.

[0022] The present invention provides a method for tissue culture of Pleurotus eryngii seedlings, which has the following advantages compared with the prior art:

[0023] The invention adopts adventitious buds on the pseudobulbs of Agaricus as explants, and configures a rhizome induction medium with improved White medium, honeysuckle, Huabao No. 1 and other components to induce rhizomes from the explants in a relatively short time; subsequently, MS medium, 6-BA, NAA, quercetin, tea saponin and other components are used as rhizome proliferation medium, which can effectively promote the proliferation multiples of the rhizomes; subsequently, MS medium, NAA, 6-BA, honeysuckle and the like are used to prepare a differentiation medium, which can effectively and quickly differentiate the rhizomes, sprout and grow into seedlings; finally, a rooting medium configured with 1 / 2MS medium, 6-BA, aspirin, honeysuckle and bisabolol is used for culturing, so that relatively robust test tube seedlings can be obtained, and the survival rate of transplanting in the later stage can be effectively guaranteed. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1This is a schematic diagram of the growth of rhizomes of explants cultured in rhizomes inducing medium 1 for 40 days in Example 1 of the present invention;

[0025] Figure 2 This is a schematic diagram of the bud differentiation of the explants cultured in rhizome induction medium 1 for 20 days in Example 1 of the present invention;

[0026] Figure 3 This is a schematic diagram of the bud differentiation of the explants in Example 1 of the present invention after being cultured in rhizome induction medium 4 for 20 days;

[0027] Figure 4 This is a growth diagram of culturing the rhizome proliferation medium 1 for 40 days in Example 2 of the present invention;

[0028] Figure 5 This is a growth diagram of culturing for 35 days using differentiation medium 1 in Example 3 of the present invention;

[0029] Figure 6 This is a schematic diagram of the growth of seedlings cultured on rooting medium 1 for 20 days in Example 4 of the present invention. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention is clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] The formula of White medium in the following examples is: KNO3 80mg / L+citric acid 2mg / L+Ca(NO3)2·4H2O287mg / L+MgSO4 720mg / L+KCl 65mg / L+NaH2PO4 16.6mg / L+thiamine hydrochloride 0.1mg / L+pyridoxine hydrochloride 0.1mg / L+niacin 0.5mg / L+glycine 3mg / L+MnSO4·4H2O 6.6mg / L+ZnSO4·7H2O 2.7mg / L+KI 0.75mg / L+H3BO3 1.5mg / L+sucrose 20g / L; the formula of improved White medium is: KNO3 40mg / L+citric acid 2.5mg / L+Ca(NO3)2·4H2O 165mg / L+MgSO4 1120mg / L+KCl 50mg / L+NaH2PO4 20mg / L+Thiamine hydrochloride 0.1mg / L+Pyridoxine hydrochloride 0.1mg / L+Niacin 0.5mg / L+Glycine 5mg / L+MnSO4·4H2O 11mg / L+ZnSO4·7H2O 4mg / L+KI 0.75mg / L+H3BO3 1.5mg / L+Sucrose 20g / L.

[0032] Example 1

[0033] Rhizome induction culture of Radix Sagittifolia:

[0034] 1. Cut the adventitious buds from the same batch of pseudobulbs of Radix Sagittifolia, rinse them with sterile water, dry them, rinse them with 75% ethanol solution for 3 times, soak them in 20g / L sodium hypochlorite solution for 15min, rinse them with sterile water for 4 times, dry the surface water and complete disinfection, and obtain the explants for use;

[0035] 2. Setting of rhizome induction medium: different rhizome induction mediums were set according to the formula in Table 1 below:

[0036] Table 1

[0037] 3. The above explants were inoculated into rhizome induction medium with different formulations in Table 1 above, 20 explants were inoculated into each group of medium, and cultured in the dark at 25±5°C for 48h, and then the light time was controlled to be 10h per day, and the light intensity was 1000lx. The induction rate of rhizomes in each group at 30d, 40d, 50d, and 60d was observed and recorded. The specific results are shown in Table 2 below:

[0038] Table 2

[0039] From Table 2 above, we can see that rhizome induction medium 1 and rhizome induction medium 2 can achieve a high rhizome induction rate after 40 days of cultivation. The growth of the rhizome induction medium 1 after 40 days of cultivation is as follows: Figure 1 shown and by Figure 2-3 It can be seen that the explant buds cultured in rhizome induction medium 1 have begun to differentiate at 20 days ( Figure 2 ), while the bud differentiation of explants cultured in rhizome induction medium 4 was poor at 20 days ( Figure 3 ).

[0040] That is, among the above culture media, rhizome induction medium 1 and rhizome induction medium 2 have the best rhizome induction culture effects.

[0041] Example 2

[0042] Rhizome proliferation culture:

[0043] 1. Prepare different rhizome proliferation medium according to the formula in Table 3 below:

[0044] Table 3

[0045] 2. Take the rhizomes that have been cultured in the rhizome induction medium 1 in the above Example 1 for 50 days and have the same growth potential, cut them into 0.6-0.8 cm in length, and inoculate them into the above rhizome proliferation mediums, 20 inoculated into each culture medium, and culture them in the dark at 25±5°C for 40 days. The number of clustered rhizomes and the proliferation times of rhizomes in various culture media are recorded. The specific results are shown in Table 4 below:

[0046] Table 4

[0047] It can be seen from the above table that the rhizomes cultured in rhizome proliferation medium 1 and rhizome proliferation medium 2 can increase the number of induced clustered rhizomes differentiation and increase the proliferation multiples.

[0048] Example 3

[0049] Differentiation culture of clustered rhizomes:

[0050] 1. Prepare different differentiation medium according to the formula in Table 5 below:

[0051] Table 5

[0052] 2. Take the rhizomes that have been cultured in the rhizome proliferation medium 1 in the above Example 2 for 40 days and grow uniformly, cut them and inoculate them into various differentiation mediums (20 inoculated in each medium) for differentiation culture for 35 days (cultured at 25±5°C according to the light intensity of 10h / d, and the light intensity is 1000lx), and obtain the Mao Sagittaria seedlings. The differentiation bud rate (the proportion of differentiated bud rhizomes to total rhizomes) and the leaf expansion rate (the proportion of expanded leaves to total leaves) after 25 days of culture in various culture media and 35 days of culture are observed. The specific results are shown in Table 6 below:

[0053] Table 6

[0054] It can be seen from the above table that the differentiation and seedling growth conditions of the cultures cultured in differentiation medium 1 and differentiation medium 2 are better.

[0055] Example 4

[0056] Rooting culture and transplanting of Mao Ci mushroom seedlings:

[0057] 1. Design the rooting medium with reference to Table 7 below:

[0058] Table 7

[0059] 2. The same growth of the Trichosanthes kirilowii seedlings cultured in the differentiation medium 1 in the above Example 3 for 40 days was inoculated into the above various rooting mediums (20 Trichosanthes kirilowii seedlings were inoculated in each), and cultured at a temperature of 25±5°C with a light intensity of 10h / d and a light intensity of 1000lx for 20 days. The rooting rate of the Trichosanthes kirilowii seedlings in each group of culture medium was observed and recorded. The specific results are shown in Table 8 below:

[0060] Table 8

[0061] 3. The seedlings obtained by culturing for 20 days in the manner of the above groups 13-16 were placed outdoors for 2 days of seedling hardening. The seedlings were then taken out and the culture medium at the roots was cleaned. The rooted seedlings were then transplanted into the sterilized substrate (mass ratio of coconut bran: perlite: garden soil: organic fertilizer = 2:1:3:2). 100 seedlings were transplanted in each group. The transplanting temperature was controlled at 20±2℃. After transplanting, the roots were irrigated thoroughly with water. The substrate humidity was then controlled at 40%-60%. The survival rates at 7d, 15d, and 25d after transplanting were recorded. The specific results are shown in Table 9 below:

[0062] Table 9

[0063] As shown in Table 9 above, the seedlings cultured with rooting medium 1 and rooting medium 2 had a higher survival rate after transplanting.

[0064] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for tissue culture of Pleurotus eryngii seedlings, characterized in that: The tissue culture method comprises the following steps: S1. Obtaining explants: Cut off the adventitious buds on the pseudobulbs of Sagittaria sagittifolia and use them as explants after disinfection; S2. Rhizome induction culture: the explants are inoculated in a rhizome induction medium to induce bud differentiation and form rhizomes; wherein the formula of the rhizome induction medium is composed of the following substances: modified White medium + NAA 1.0-1.5 mg / L + Huabao No. 1 5-8 mL / L + honeysuckle 20-40 mg / L + agar powder 3-5 g / L; wherein the composition of the components in the modified White medium is: KNO3 40 mg / L + citric acid 2.5 mg / L + Ca(NO3)2·4H2O 165 mg / L + MgSO4 1120 mg / L + KCl 50 mg / L + NaH2PO4 20 mg / L + thiamine hydrochloride 0.1 mg / L + pyridoxine hydrochloride 0.1 mg / L + nicotinic acid 0.5 mg / L + glycine 5 mg / L + MnSO4·4H2O 11 mg / L + ZnSO4·7H2O 4mg / L+KI 0.75mg / L+H3BO3 1.5mg / L+sucrose 20g / L; S3, rhizome proliferation culture: take the rhizome formed in the above rhizome induction medium, cut off the rhizome and inoculate it into the rhizome proliferation medium for proliferation culture, and culture to obtain the raw rhizome, and the rhizome proliferation medium is composed of the following contents of substances: MS medium + 6-BA 4.5-5.5 mg / L + NAA 0.12-0.15 mg / L + quercetin 6-8 mg / L + tea saponin 20-30 mg / L + sucrose 15-20 g / L + agar powder 3-5 g / L; S4, differentiation culture: take the above-mentioned rhizome, cut it off and inoculate it into differentiation medium for differentiation culture to obtain Mao Sagittaria seedlings; the differentiation medium is composed of the following substances: MS medium + NAA 0.12-0.15mg / L + 6-BA 2.0-2.5mg / L + honeysuckle 60-80mg / L + sucrose 15-20g / L + agar powder 3-5g / L; S5, rooting culture: inoculate the above-mentioned Mao Sagittaria seedlings into a rooting medium, and perform rooting culture until adventitious roots are produced to obtain test tube seedlings; the formula of the rooting medium is composed of the following substances: 1 / 2MS medium + 6-BA 2.0-3.0 mg / L + aspirin 10-20 mg / L + honeysuckle 10-20 mg / L + bisabolol 10-15 mg / L + sucrose 15-20 g / L + agar powder 3-5 g / L; S6. Transplantation: After hardening the test tube seedlings, transplant them into the substrate for cultivation.

2. The method for tissue culture of Pleurotus eryngii seedlings according to claim 1, characterized in that: The disinfection method in step S1 is to rinse the adventitious buds with sterile water, then absorb the moisture, then rinse with 75% ethanol solution 2-3 times, then soak them in 20g / L sodium hypochlorite solution for 15 minutes, then rinse with sterile water 3-4 times, and complete the disinfection after absorbing the surface moisture.

3. The method for tissue culture of Psoralea corylifolia seedlings according to claim 1, characterized in that: The culture method in step S2 is to culture the inoculated rhizome induction culture medium in the dark at 25±5° C. for 48 hours, and then control the light time to 10 hours per day, and the light intensity is 1000 lx.

4. The method for tissue culture of Pleurotus eryngii seedlings according to claim 1, characterized in that: In step S3, the length of the cut rhizome is controlled to be 0.6-0.8 cm.

5. The method for tissue culture of Pleurotus eryngii seedlings according to claim 1, characterized in that: The proliferation culture condition in step S3 is dark culture at a temperature of 25±5°C.

6. The method for tissue culture of Pleurotus eryngii seedlings according to claim 1, characterized in that: The differentiation culture conditions in step S4 are culture at a temperature of 25±5° C. with 10 h / d of light illumination and a light intensity of 1000 lx.

7. The method for tissue culture of Pleurotus eryngii seedlings according to claim 1, characterized in that: The rooting culture conditions in step S5 are to culture at a temperature of 25±5° C. with a light intensity of 10 h / d and a light intensity of 1000 lx.

8. The method for tissue culture of Sagittaria sagittifolia seedlings according to claim 1, characterized in that: The method of hardening the seedlings in step S6 is to place the test tube seedlings with rooting culture base outdoors for 2-3 days.

9. The method for tissue culture of Sagittaria sagittifolia seedlings according to claim 1, characterized in that: The transplanting method in step S6 is to take out the test tube seedlings and remove the culture medium, and then plant them in the sterilized substrate.

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