CN112369324B - Tissue culture method for sedum aizoon - Google Patents
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention discloses a tissue culture method of sedum aizoon. The method comprises the following steps: a. soaking the seeds of the sedum aizoon in water of 80 ℃ for 5-10min, sowing the seeds on 1/2MS culture medium after disinfection, and culturing aseptic seedlings; b. taking sterile seedling leaves as explants, inoculating the explants on callus and bud induction culture media, and inducing callus and buds; the callus and bud induction culture medium comprises the following components: each liter contains 2.0mg of 6-BA, 0.5mg of NAA, 30g of cane sugar and 8g of agar, the balance is 1/2MS, and the pH value is 5.8; c. and (4) taking the regenerated buds, inoculating the regenerated buds on 1/2MS culture medium, and inducing the regenerated buds to root to obtain the regenerated seedlings of the sedum lineare. The method can provide raw material support for basic researches such as the pharmacological activity analysis and liver protection application of the sedum aizoon, and has high application value.
Description
Technical Field
The invention belongs to the technical field of plant tissue culture, and particularly relates to a method for culturing a sedum aizoon tissue.
Background
Armored plant (Cassia mimosoides Linn.) also called Cassia mimosa Cassia obtusifolia is a plant of Cassia of Leguminosae, an annual or perennial subshrubular herb with a height of 30-60 cm, 10-16 seeds, and a flower and fruit period of 8-10 months. Originally recorded in Ming dynasty 'Jiu ban Cao', the medicine is homologous in medicine and food, and the whole herb can be used for medicine, and has the effects of clearing heat and removing toxicity, tonifying spleen and promoting diuresis, relaxing bowels and the like. The method is mainly distributed in places such as Guangdong, Guangxi, Sichuan, Hunan, Fujian and the like in China. It can be used for treating jaundice, summer heat, emesis, diarrhea, infantile malnutrition, edema, dysuresia, habitual constipation, furuncle, carbuncle, swelling, and snake bite. The chemical components of the sedum lineare comprise anthraquinones, flavonoids, sterols and the like, and the research on the pharmacological activity of the sedum lineare at home and abroad mainly focuses on the liver protection effect of the sedum lineare. Early Japanese researchers found that anthraquinone compounds in Tephrosia ferruginea have strong lipase inhibiting effect, and can prevent and improve body obesity, liver hypertrophy, hypertriglyceridemia caused by high fat diet, etc. Shenchuangpeng and the like similarly find that the water extract of the sedum aizoon has the effects of reducing the lipid and protecting the liver tissue. Through deep analysis of chemical components of the zornia ferruginea, the zornia ferruginea alcohol extract can inhibit rat hepatic fibrosis induced by dimethyl nitrosamine, has certain liver protection activity, and the mechanism of the zornia ferruginea alcohol extract is possibly related to chemical components of emodin and oleanolic acid in the zornia ferruginea alcohol extract. The research also indicates that the ethyl acetate extract of the sedum aizoon has high content of flavone and phenols, has good DPPH removing capability and stronger antioxidant activity, thereby playing a role in protecting the liver.
In Hakka area of Guangdong, Mei for hundreds of years, the medicine is taken by decocting or preparing herbal tea with folk conventional herbal medicine Tejiacao, and is used for preventing and treating liver diseases such as chronic hepatitis B, hepatic fibrosis and liver cirrhosis. However, in a natural environment, the natural propagation period of the sedum aizoon is long, and the seed obtaining time is long, so that the development of a sedum aizoon tissue culture technology is urgently needed, the growth period can be shortened, the market demand is met, and a raw material support is provided for further understanding basic researches such as the pharmacological activity analysis and liver protection application of the sedum aizoon.
Disclosure of Invention
The invention aims to provide a tissue culture method for sedum lineare, aiming at the defect of long breeding period of the sedum lineare in the prior art.
The invention relates to a method for culturing the tissue of the sedum aizoon, which comprises the following steps:
a. soaking the seeds of the sedum aizoon in water of 80 ℃ for 5-10min, sowing the seeds on 1/2MS culture medium after disinfection, and culturing aseptic seedlings;
b. taking sterile seedling leaves as explants, inoculating the explants on callus and bud induction culture media, and inducing callus and buds; the callus and bud induction culture medium comprises the following components: each liter contains 2.0mg of 6-BA, 0.5mg of NAA, 30g of cane sugar and 8g of agar, the balance is 1/2MS, and the pH value is 5.8;
c. and (4) taking the regenerated buds, inoculating the regenerated buds on 1/2MS culture medium, and inducing the regenerated buds to root to obtain the regenerated seedlings of the sedum lineare.
Preferably, the disinfection in the step a is carried out by washing with 70% ethanol water solution by volume fraction for 2-3 times, 5-10s each time, washing with 5% NaClO water solution by mass fraction for 5-10min, and washing with sterile distilled water for 3 times in an ultra-clean bench.
Preferably, the 1/2MS medium of steps a and c contains 30g of sucrose and 8g of agar per liter, the balance is 1/2MS, and the pH is 5.8.
Preferably, the tissue culture method of the sedum aizoon comprises the following culture conditions: the light is 12 h/dark 12h, the light intensity is 2000-2500 lx, the temperature is 22 ℃, and the relative humidity is 80%.
Preferably, the new buds of the step c grow 1.5-2 cm.
The MS culture medium is an international universal culture medium, and the components and the configuration method are shown in Murashige T, Skoog F (1962) A recycled medium for rapid growth and bioassay with a microbacterious tissue cultures. The 1/2MS refers to halving macroelements in the MS culture medium, and keeping the rest components unchanged.
Experiments show that after the armored seeds are treated in a water bath at 80 ℃ for 5-10 minutes, the seeds can be promoted to germinate, the germination rate can be obviously improved, and the germination time can be shortened. The germless seedling young leaf of the armored grass is taken as the explant on 1/2MS +2.0 mg/L6-BA +0.5mg/L NAA culture medium, the callus induction rate is high, the growth state is good, and the germination number is the largest. Adding IBA with different concentrations into 1/2MS culture medium, finding that IBA can not effectively promote rooting; however, the NAA can promote the generation of roots, when the culture medium is 1/2MS +0.5mg/L NAA, the rooting rate is reduced, and the roots are shortened; the best rooting medium is 1/2 MS.
The invention has the following advantages:
in the method, the gerbera ferruginea seeds are treated by using water bath at 80 ℃ for 5-10 minutes, so that the seed germination is promoted, and aseptic seedlings which grow well and strongly can be cultured quickly; the tissue culture method of the carex incarnata breaks through the traditional mode of the propagation of the carex incarnata, shortens the culture period and provides guarantee for the preservation of the germplasm resources of the carex incarnata; meanwhile, the raw material is provided for the pharmacological and pharmacodynamic study of the Chinese herbal medicine of the sedum aizoon.
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FIG. 1 is the effect of NAA on callus-induced sprouting wherein: a: 1/2MS +2 mg/L6-BA; b: 1/2MS +2 mg/L6-BA +0.2mg/L NAA; c: 1/2MS +2 mg/L6-BA +0.5mg/L NAA; d: 1/2MS +2 mg/L6-BA +1.0mg/L NAA; the scale is 1 cm.
FIG. 2 is the effect of different concentrations of IBA on rooting, wherein: a: 1/2 MS; b: 1/2MS +0.5mg/L IBA; c: 1/2MS for 30 days; d: 1/2MS +0.5mg/L IBA for 30 days; in cases A-B corresponding to 20 days, the scale is 1 cm.
FIG. 3 is the effect of different concentrations of NAA on rooting, wherein: a: 1/2 MS; b: 1/2MS +0.1mg/L NAA; c: 1/2MS +0.2mg/L NAA; d: 1/2MS +0.5mg/L NAA; the scale is 1 cm.
FIG. 4 is the effect of different media on seed germination.
FIG. 5 shows the effect of different temperature treatments on the germination and growth of armored seeds on 1/2MS medium, which is inoculated in 1/2MS medium after 10min of soaking treatment with water at the temperature of (A)25 ℃, (B)40 ℃, (C)60 ℃, (D)80 ℃ and (E)100 ℃ and grown for 20 days.
FIG. 6 shows the growth of the rooted shoots after 20 days of culture in transplanting soil.
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1
1 materials and methods
1.1 Experimental materials
Tiejiacao seeds, collected from Yin-nan mountain of Meizhou; sterile seedlings of the sedum aizoon.
1.2 culture conditions
Selecting 1/2MS culture medium, adding sucrose 30g/L and agar 8g/L, adjusting pH to 5.8, sterilizing at 121 deg.C and 101MPa for 20min, and pouring plate for aseptic seedling propagation. The conditions of the constant-temperature incubator are that the incubator is illuminated for 12 h/dark for 12h, the illumination intensity is 2000-2500 lx, the temperature is 22 ℃, and the relative humidity is 80%.
1.3 Experimental methods
1.3.1 Germination of seeds
Soaking the seeds in (1) normal-temperature distilled water for 20min, (2)80 ℃ water bath for 5min or (3) 98% concentrated sulfuric acid for 50min, then cleaning the seeds with 70% ethanol water solution by volume fraction for 2-3 times, 5-10s each time, washing the seeds with 5% NaClO water solution by mass fraction for 5-10min, taking the seeds to an ultra-clean bench, washing the seeds with sterile distilled water for 3 times, and finally sowing the seeds on 1/2MS culture medium.
1.3.2 Induction of callus
1/2MS culture medium and 2.0 mg/L6-BA are used as callus induction basal culture medium, and NAA with different concentrations such as 0.2mg/L NAA, 0.5mg/L NAA and 1.0mg/L NAA are added exogenously. Taking sterile young and tender seedling leaves by using tweezers in a superclean workbench, horizontally placing the sterile young and tender seedling leaves on a culture medium, making 6-8 bottles for each treatment, and repeating the steps for three times.
1.3.3 rooting Medium screening
1/2MS culture medium is used as basal culture medium, IBA (0.5mg/L and 1.0mg/L IBA) and NAA (0.1mg/L, 0.2mg/L and 0.5mg/L NAA) with different concentrations are added exogenously. Taking the new buds with the length of 1.5-2cm in a superclean bench by using forceps, vertically placing the buds on a culture medium with 1-4 buds per bottle, making 6-8 bottles per treatment, and repeating the steps for three times.
1.4 statistical methods
And observing callus induction and rooting conditions, and counting the callus induction rate and the callus growth condition. Callus induction rate is the number of explants forming callus/total number of inoculated explants multiplied by 100%; the sprouting rate is the number of explants sprouting in cluster buds/the number of uncontaminated explants multiplied by 100 percent; the rooting coefficient is the rooting number/rooting explant number multiplied by 100%; the rooting rate is the number of rooted buds/inoculated buds multiplied by 100%.
2 results
2.1 Heat treatment to promote seed Germination
1/2MS is used as a basic culture medium, and a seed germination experiment shows that seeds are subjected to 80 ℃ water bath heat treatment for 5 minutes in advance and are soaked in 98% concentrated sulfuric acid for 50 minutes, so that the seed dormancy can be effectively broken (the seeds can germinate in about 3-4 days and the untreated seeds cannot germinate) compared with the seeds treated at normal temperature, the seed germination time is obviously shortened, but the seeds are poor in germination state after being treated by the concentrated sulfuric acid and are not beneficial to subsequent growth. And (3) combining the seed germination rate condition, and performing water bath heat treatment at 80 ℃ to obtain an optimal scheme.
TABLE 1 Geranium sibiricum seed Germination
Treatment 1: soaking in distilled water at room temperature for 20 min; 2: water bath at 80 deg.C for 5 min; 3: soaking in 98% concentrated sulfuric acid for 50 min.
b represents P <0.05, ab represents P > 0.05.
2.2 Effect of different media on seed Germination
1/2MS and MS are used as seed germination culture media, seeds are treated by water bath at 80 ℃ for 5 minutes, and are respectively sowed in the two culture media, and the seed germination rate is counted after sowing for 48 hours and 96 hours, and the results are shown in Table 2 and figure 4. The gerbera ferruginea seeds begin to germinate at 1/2MS 48 hours after being sown, and the germination rate is 70.9 +/-13.5%; after sowing for 96h, the germination rate in 1/2MS reaches 96.6 +/-5.9%, and the germination rate in MS reaches 55.0 +/-14.8%.
TABLE 2 Germination of Alstonia herbeck seeds in different media
2.3 selection of callus Induction Medium
Taking the tender leaves of the gerbera ferruginea sterile seedlings as explants, adding NAA with different concentrations on the basis of 1/2MS and 2.0 mg/L6-BA, and finding that the induction rate of the callus is over 90 percent, which indicates that the NAA promotes the formation of the callus, but the budding rate is obviously different, and the budding rate is the highest when 0.5mg/L NAA is added (Table 3, figure 1). The statistics of the culture on the medium for 60 days are shown in Table 3.
TABLE 3 callus induction and sprouting statistics
b represents P <0.05, ab represents P > 0.05.
2.4 screening of rooting Medium
On the basis of 1/2MS culture medium, after adding IBA and NAA with different concentrations, it is found that IBA can not effectively promote the rooting of the armored grass bud, compared with the control, the rooting can be realized in 0.5mg/L IBA, but the rooting coefficient is reduced, the generation of roots can be inhibited by high concentration (Table 4, figure 2); while NAA was effective in promoting root formation, the number of rooted shoots increased but the number of rooted shoots decreased for a single shoot (Table 5, FIG. 3). Tables 4 and 5 show the results of culturing on 1/2MS medium for 20 days.
TABLE 4 Effect of different concentrations of IBA on rooting (20 days)
TABLE 5 Effect of NAA at different concentrations on rooting (20 days)
b represents P <0.05, ab represents P > 0.05.
3 transplanting
The above-mentioned seedling of P.ferruginea induced to root on 1/2MS medium was taken out from the culture flask, washed of the medium on the root, transplanted in soil, and cultured for 20 days, and the growth was good as shown in FIG. 6.
Example 2 Effect of different temperature treatments on Geomethyl iron-weed seed Germination growth on 1/2MS Medium
Soaking the seeds in water at the temperature of (1)25 ℃, 2), (40), (3), (60), (4)80 and (5)100 ℃ for 10min, then washing the seeds for 2-3 times by volume fraction 70% ethanol aqueous solution for 5-10s each time, washing the seeds for 5-10min by mass fraction 5% NaClO aqueous solution, taking the seeds to an ultra-clean bench, washing the seeds for 3 times by sterile distilled water, and finally sowing the seeds on 1/2MS culture medium. The growth was recorded after 20 days. The results are shown in table 6 and fig. 5.
TABLE 6 Effect of different temperature treatments on Geranium sibiricum seed germination growth on 1/2MS Medium
The result shows that the seed soaking treatment by 80 ℃ water has 100 percent of seed expansion rate and seed germination rate, which is obviously superior to other temperature treatments; and the average main stem length and the average main root length are obviously superior to those of other temperature treatments.
Claims (3)
1. A method for culturing the tissue of the sedum aizoon comprises the following steps:
a. soaking the seeds of the sedum aizoon in water of 80 ℃ for 5-10min, sowing the seeds on 1/2MS culture medium after disinfection, and culturing aseptic seedlings;
b. taking sterile seedling leaves as explants, inoculating the explants on callus and bud induction culture media, and inducing callus and buds; the callus and bud induction culture medium comprises the following components: each liter contains 2.0mg of 6-BA, 0.5mg of NAA, 30g of cane sugar and 8g of agar, the balance is 1/2MS, and the pH value is 5.8;
c. inoculating the regenerated bud on 1/2MS culture medium, inducing to root, and obtaining regenerated seedling of Trifolium Pratense L; the 1/2MS culture medium of the steps a and c contains 30g of cane sugar and 8g of agar per liter, the balance is 1/2MS, and the pH is 5.8; the culture conditions were: the light is 12 h/dark 12h, the light intensity is 2000-2500 lx, the temperature is 22 ℃, and the relative humidity is 80%.
2. The method as claimed in claim 1, wherein the sterilization in step a is performed by washing with 70% ethanol aqueous solution by volume fraction for 2-3 times, 5-10s each time, washing with 5% NaClO aqueous solution by mass fraction for 5-10min, and washing with sterile distilled water 3 times in a super clean bench.
3. The method of claim 1, wherein said young shoots of step c are 1.5-2cm in length.
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| CN113317204B (en) * | 2021-07-08 | 2022-07-19 | 嘉应学院 | Method for inducing adventitious buds of seedlings of sedum aizoon and efficiently regenerating plants |
| CN115191350B (en) * | 2021-12-29 | 2023-02-28 | 广东海洋大学 | Method for inducing regeneration of adventitious buds of stem explant of sedum aizoon |
| CN115475185A (en) * | 2022-08-30 | 2022-12-16 | 广州中医药大学第一附属医院 | Application of sedum aizoon in treating metabolic syndrome |
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