CN113711911B

CN113711911B - Method for establishing and rapidly proliferating Epimedium pubescens sterile system

Info

Publication number: CN113711911B
Application number: CN202110881712.9A
Authority: CN
Inventors: 林良科; 王旭东
Original assignee: Sichuan Xieli Pharmaceutical Co Ltd
Current assignee: Sichuan Xieli Pharmaceutical Co Ltd
Priority date: 2021-08-02
Filing date: 2021-08-02
Publication date: 2022-11-25
Anticipated expiration: 2041-08-02
Also published as: CN113711911A

Abstract

The invention belongs to the technical field of medicinal plant tissue culture, and particularly relates to a method for establishing and rapidly proliferating epimedium herb sterile system, which comprises the following steps: 1) Collecting young and tender rhizome of herba Epimedii, removing leaves, soaking in washing powder saturated solution for 30min, brushing the surface with brush, washing with flowing water for 2 hr, placing on a clean bench, sterilizing, and shearing off 2mm of each end of the rhizome with sterile surgical scissors to obtain sterile stem segment with complete bud point; 2) Vertically inoculating the sterile stem section obtained in the step 1) to an induction culture medium for culture, and inducing the stem section to bud; 3) Cutting the shoot buds obtained in the step 2) into single shoot points, vertically inoculating the shoot points to a multiplication culture medium for culture, and obtaining epimedium herb cluster buds; the method can obtain the method for quickly proliferating the epimedium, can provide high-quality seedlings for artificial breeding of the epimedium, and solves the problem of difficult seedling culture of the epimedium due to difficult seed collection and low germination rate.

Description

Method for establishing and rapidly proliferating Epimedium pubescens sterile system

Technical Field

The invention belongs to the technical field of medicinal plant tissue culture, and particularly relates to a method for establishing and rapidly proliferating a epimedium herb sterile system.

Background

Epimedium pubescens is one of the original plants of Epimedium pubescens collected in the pharmacopoeia of the people's republic of China (2020 edition), is a traditional Chinese medicinal material in China, mainly has the effects of tonifying kidney yang, strengthening tendons and bones and dispelling wind-damp, and has more than 2000 years of application history in China. Modern researchers find various physiologically active chemical components such as general flavone, polysaccharide, alkaloid and the like in bodies of the modern researchers, and the modern researchers are widely applied to traditional medicine and modern medicine at present. The current market demand of epimedium is about 5000 tons/year, and the main acquisition mode is still for excavating wild resources, and along with the increase of the excavation amount and the unordered excavation of people, the wild epimedium resources are continuously reduced, so that the regeneration and the sustainable utilization of the resources are not very facilitated. Therefore, the artificial introduction and cultivation of epimedium is urgent.

Under natural conditions, due to the natural dormancy and the after-ripening of seed embryos, the seed germination rate of epimedium dauricum seeds is extremely low, the germination period is long, and the method is very not beneficial to the large-scale planting of epimedium. At present, the artificial planting of epimedium basically adopts a way of plant division propagation, long-term asexual propagation easily causes variety degradation, and the yield and the quality of medicinal materials are reduced.

Disclosure of Invention

The method can obtain an epimedium sterile system and a rapid propagation method, can provide high-quality seedlings for artificial breeding of the epimedium, and solves the problem of difficult seedling culture of the epimedium due to difficult seed collection and low germination rate.

The invention provides a method for establishing and rapidly proliferating an epimedium dauricum sterile system, which comprises the following steps:

1) Collecting young and tender rhizome of herba Epimedii, removing leaves, soaking in washing powder saturated solution for 30min, brushing the surface with brush, washing with flowing water for 2h, placing on a clean bench, sterilizing with 75% alcohol for 30s, washing with sterile water for 3 times, sterilizing, and shearing off 2mm each of two ends of the rhizome with sterile surgical scissors to obtain sterile stem segment with complete bud point;

2) Vertically inoculating the sterile stem section obtained in the step 1) to an induction culture medium for culture, and inducing germination; the induction culture medium is MS +0.5-2.0 mg/L6-BA +0.3-1.1mg/L IBA +40-70mg/L PVP +80-140mg/L streptomycin +5.8g/L agar +30g/L sucrose;

3) Cutting the shoot buds obtained in the step 2) into single shoot points, vertically inoculating the shoot points to a proliferation culture medium for culture, and culturing for 28 days under the conditions that the temperature is 25 ℃, the illumination intensity is 2300-2500lx and the illumination duration is 12h/d to obtain epimedium clustered buds; the proliferation culture medium is MS +1.5-2.5mg/L6-BA +0.3-0.9mg/L NAA +60mg/L PVP +5.8g/L agar +30g/L sucrose.

Further, in step 1), the method for re-sterilization is: sterilizing with 0.1% mercuric chloride for 4min, washing with sterile water for 5 times, sterilizing with 0.1% mercuric chloride for 2.5min, and washing with sterile water for 5 times.

Further, in step 2), the culture conditions are dark culture at 25 ℃ for 7d, and then the culture medium is transferred to 25 ℃ for 18d under the condition of the illumination intensity of 2300-2500lx and the illumination duration of 12 h/d.

Further, in step 2), the induction medium is: MS +1.5mg/L6-BA +0.8mg/L IBA +60mg/L PVP +120mg/L streptomycin +5.8g/L agar +30g/L sucrose, pH was adjusted to 5.8.

Further, in step 3), the proliferation medium is: MS +2.0 mg/L6-BA +0.5mg/L NAA +60mg/L PVP +5.8g/L agar +30g/L sucrose, pH was adjusted to 5.8.

The invention has the beneficial effects that: the invention adopts the buds of epimedium as explants, and establishes a rapid proliferation sterile system of the epimedium dauricum through a cluster bud approach; the rapid propagation sterile system of the epimedium dauricum is established by combining disinfection, induction culture and propagation culture, so that the pollution rate is reduced, and the yield and the quality of the epimedium are improved; the method has the advantages of simple flow and short propagation period, provides technical guidance for herba epimedii seedling breeding and large-scale propagation, solves the problem of difficult seedling culture caused by low germination rate and long germination period of herba epimedii seeds, reduces the herba epimedii seedling breeding cost, and provides high-quality seedlings for artificial propagation of herba epimedii.

Drawings

FIG. 1 is a line graph showing the effect of a mercuric chloride disinfectant composition of the present invention on explant disinfection.

Detailed Description

The principles and features of this invention are described below in conjunction with examples which are set forth to illustrate, but are not to be construed to limit the scope of the invention.

Unless otherwise specifically indicated, various raw materials, reagents, instruments, equipment and the like used in the present invention may be commercially available or may be prepared by existing methods.

2) Vertically inoculating the sterile stem section obtained in the step 1) to an induction culture medium for culture, and inducing the stem section to bud; the induction culture medium is MS +0.5-2.0 mg/L6-BA +0.3-1.1mg/L IBA +40-70mg/L PVP +80-140mg/L streptomycin +5.8g/L agar +30g/L cane sugar;

The effect of cutting off 2mm of each of the two ends of the rhizome by using the sterile surgical scissors is to cut off the part seriously stimulated by the disinfectant, so as to ensure the subsequent normal culture of the sterile stem section;

wherein, MS culture medium is a common basic culture medium formula in plant tissue culture technology, the content composition is fixed, and the formula is shown in table 1:

TABLE 1MS media composition Table

In addition, 6-BA (6-benzylamino adenine) is one of plant cytokinins, can promote plant cell division and elongation in stem tip or bud culture, can break apical dominance and promote the generation and proliferation of lateral buds and multiple buds;

IBA (indolebutyric acid) is one of auxin and can promote the regeneration and proliferation of plant seedling buds;

PVP (polyvinylpyrrolidone K30) has an inhibiting effect on the browning of an implant, the action mechanism is that the PVP is a specific adsorbent of phenolic substances, a CO-N = group in the PVP has strong capability of complexing polyphenol compounds, and the polyphenol compounds can not become substrates of polyphenol oxidase, so that the browning is inhibited;

streptomycin and 72 percent streptomycin sulfate have an inhibiting effect on bacteria, and streptomycin with a proper concentration is added into an induction culture medium to inhibit the endophyte pollution of explants, reduce the pollution rate and improve the induction rate;

NAA (naphthalene acetic acid) is one of auxin, can promote plant cell differentiation, and can promote seedling bud regeneration and proliferation by combining with cytokinin under proper concentration;

the 6-BA, IBA, PVP and streptomycin in the induction culture medium are cooperatively matched to improve the bud induction rate of the Epimedium pubescens explant, and simultaneously reduce the pollution rate and browning rate of the explant;

6-BA, NAA and PVP in the multiplication culture medium cooperate to improve multiplication times of the clustered shoots of Epimedium pubescens, and reduce browning rate of the clustered shoots at the same time.

The method for establishing and rapidly propagating epimedium dauricum sterile system of the present application will be described in detail below with reference to examples and experimental data.

Example 1

1) Collecting young and tender rhizome of epimedium, removing leaves, soaking for 30min by using a washing powder saturated solution, slightly brushing the surface by using a brush, washing for 2h by using running water, placing on a super-clean workbench, disinfecting for 30s by using 75% alcohol by volume fraction, washing for 3 times by using sterile water, disinfecting by using 0.1% mercury bichloride by mass fraction according to a disinfection combination shown in table 2, washing for 5 times by using the sterile water, washing for more than 90s each time, and shearing two ends of the rhizome by using a sterile surgical scissors to obtain sterile stem sections with complete bud points for later use, wherein the two ends of the rhizome are 2mm respectively;

TABLE 2 Mercury-mercurial-sterilizing combined experimental design table

2) Vertically inoculating the sterile stem section obtained in the step 1) to an induction culture medium (MS +1.5mg/L6-BA +0.8mg/L IBA +60mg/L PVP +120mg/L streptomycin +5.8g/L agar +30g/L sucrose, pH is 5.8) for culture, inoculating 10 bottles in each group, and inoculating 1 explant in each bottle; culturing the inoculated induction culture medium at 25 ℃ for 7d in the dark, and then culturing the culture medium at 25 ℃ under the condition that the illumination intensity is 2300-2500lx and the illumination time is 12 h/d;

3) The number of the polluted explants and the number of the explants which are disinfected by mercury bichloride for too long to damage and die are observed and counted in 1-10d of culture, and the pollution after 10d of culture is not considered as the pollution caused by incomplete disinfection of the explants.

The results are shown in table 3:

TABLE 3 Mercury mercurial disinfection combination test results table

The explant contamination rate and explant sterilization mortality rate were plotted and the results are shown in FIG. 1.

FIG. 1 is a line chart showing the effect of mercuric chloride disinfection combination on explant disinfection, and as can be seen from FIG. 1 and Table 3, only one disinfection (combinations 1-4) is adopted, so that the explant pollution rate is continuously reduced and the explant disinfection death rate is continuously increased with the increase of disinfection time; the step of secondary disinfection (combination 5-10) is adopted, the contamination rate of the explants is changed from high to low along with the increase of the time of the secondary disinfection, and the disinfection death rate of the explants is gradually increased; the explant loss rate of the disinfection combination 7 and the disinfection combination 9 is the lowest and is 30%, the disinfection time of the disinfection combination 7 is shorter, so the combination 7 is adopted as the most suitable disinfection time, namely the explant is disinfected for 4min by using mercury bichloride with the mass fraction of 0.1%, the explant is washed by sterile water for 5 times, the explant is disinfected for 2.5min by using mercury bichloride with the mass fraction of 0.1% again, the sterile water is washed by the sterile water for 5 times, the explant pollution rate is low, the disinfection death rate is low, the disinfection time is shorter, and the economic benefit is favorably realized.

Example 2

1) Collecting young and tender rhizome of epimedium, removing leaves, soaking for 30min by using a washing powder saturated solution, slightly brushing the surface by using a brush, washing for 2h by using running water, then placing on a super-clean workbench, disinfecting for 30s by using 75% alcohol by volume fraction, washing for 3 times by using sterile water, disinfecting for 4min by using 0.1% mercury bichloride, washing for 5 times by using sterile water, disinfecting for 2.5min by using 0.1% mercury bichloride again, washing for 5 times by using sterile water, and shearing two ends of the rhizome by using sterile surgical water by 2mm respectively to obtain sterile stem sections with complete bud points for later use;

2) Vertically inoculating the sterile stem sections obtained in the step 1) to a single-hormone culture medium shown in the table 4, inoculating 10 bottles of each group, inoculating 1 explant in each bottle, and setting 2 groups of repeated experiments; culturing at 25 deg.C in dark for 7d, and culturing at 25 deg.C under illumination intensity of 2300-2500lx for 12 h/d;

TABLE 4 Single hormone Medium design Table

3) And (4) counting the budding rate of the explant and the growth vigor of buds. The growth vigor of the buds is respectively shown as poor to good by +, + + +, and + + +.

The results are shown in Table 5:

TABLE 5 test results on single hormone medium

As can be seen from Table 5, the effect of 6-BA when cytokinin was used alone was better than that of 6-KT, and the growth of shoots was better when the concentration of 6-BA was 1.5 mg/L; when the auxin is used alone, the IBA effect is better than that of NAA, and the growth vigor of buds is better when the IBA concentration is 0.9mg/L, so that the Epimedium dauricum explant is sensitive to hormones 6-BA and IBA, and the bud induction effect is better.

Example 3

2) Vertically inoculating the sterile stem sections obtained in the step 1) to induction culture media with different concentrations of hormone combinations as shown in table 6 for culture, inoculating 10 bottles in each group, inoculating 1 explant in each bottle, and setting 2 groups for repeated experiments; culturing at 25 deg.C in dark for 7d, and culturing at 25 deg.C under illumination intensity of 2300-2500lx for 18d with illumination duration of 12 h/d;

TABLE 6 Induction Medium design Table for different concentrations of hormone combinations

Serial number	6-BA(mg/L)	IBA(mg/L)
			1	1.3	0.7
2	1.5	0.8
			3	1.7	0.9
4	--	1.0
			5	--	1.1

The results are shown in Table 7:

TABLE 7 Induction Medium test results for different concentrations of hormone combinations

Combination (I)	6-BA(mg/L)	IBA(mg/L)	Rate of induction	Growth vigor of buds
					1	13	07	45％	+
2	13	08	50％	++
					3	13	09	55％	++
4	13	10	50％	++
					5	13	11	4％	+
6	15	07	50％	++
					7	15	08	70％	+++
8	15	09	65％	+++
					9	15	10	50％	+++
10	1.5	1.1	45％	++
					11	1.7	0.7	35％	+
12	1.7	0.8	50％	++
					13	17	09	50％	++
14	17	10	45％	+
					15	17	11	40％	+

As is clear from Table 7, when the combination 7, i.e., 6-BA was 1.5mg/L and IBA was 0.8mg/L, the highest explant induction rate was 70% and the best shoot growth was observed. The hormone combination 7 is shown to have the best effect on the induction of Epimedium pubescens explants.

Example 4

2) Vertically inoculating the sterile stem segments obtained in the step 1) to induction culture media (MS +1.5mg/L6-BA +0.8mg/L IBA +5.8g/L agar +30g/L sucrose, pH 5.8) which are respectively added with PVP with different concentrations and streptomycin with different concentrations and shown in Table 8 for culture, inoculating 10 bottles in each group, inoculating 1 explant in each bottle, and setting 2 groups for repeated experiments; culturing at 25 deg.C in dark for 7d, and culturing at 25 deg.C under illumination intensity of 2300-2500lx for 18d with illumination duration of 12 h/d;

TABLE 8 concentration design Table for PVP and streptomycin

Serial number	PVP(mg/L)	Streptomycin (mg/L)
			1	40	80
2	50	100
			3	60	120
4	70	140

3) And observing and counting the browning quantity and the browning rate of the explant and the pollution quantity and the pollution rate of endophytes of the explant.

The results are shown in Table 9:

TABLE 9 test results of different concentrations of PVP and streptomycin

As can be seen from Table 9, the PVP concentration is 60mg/L, the browning rate of the explant is the lowest, although the browning rate is 10% when the PVP concentration is 70mg/L, the growth vigor of the buds is obviously inferior to that when the PVP concentration is 60mg/L, and the growth of the buds can be inhibited by the excessively high PVP concentration; the contamination rate is lowest when the streptomycin concentration is 140 and 160mg/L, but the bud growth is inhibited, so that the streptomycin concentration of 120mg/L is most beneficial to the growth of the Epimedium dauricum explants.

Example 5

2) Vertically inoculating the sterile stem section obtained in the step 1) to an induction culture medium (MS +1.5mg/L6-BA +0.8mg/L IBA +60mg/L PVP +120mg/L streptomycin +5.8g/L agar +30g/L sucrose, pH is 5.8) for culture, carrying out dark culture at the temperature of 25 ℃ for 7d, then transferring the culture medium to the temperature of 25 ℃, culturing for 18d under the illumination intensity of 2300-2500lx and the illumination duration of 12h/d, and inducing to bud;

(3) Cutting the bud of the sprout tiller obtained in the step 2) into single bud points, vertically inoculating the bud points to enrichment culture media with different hormone combinations shown in a table 10 for culture, inoculating 10 bottles in each group, inoculating 1 explant in each bottle, setting 2 groups for repeated experiments, culturing for 28d under the conditions of 25 ℃, illumination intensity of 2300-2500lx and illumination duration of 12h/d, observing and counting multiplication times and growth vigor of the epimedium clustered buds, and respectively using plant height and bud coarseness as indexes to indicate that the growth vigor of the buds is poor to good by +, + + and+ +.

TABLE 10 proliferation medium design table for different hormone combinations

The results are shown in Table 11:

TABLE 11 proliferation medium test results for different hormone combinations

As can be seen from Table 11, when the NAA concentration is 0.5mg/L, the multiplication times of the clustered shoots of Epimedium hirsutum are generally higher and the growth vigor of the clustered shoots is better; when the concentration of 6-BA is increased from 1.5mg/L to 2.0mg/L, the multiplication times are gradually increased, the growth vigor of the cluster buds is gradually improved, the multiplication times are reduced along with the continuous increase of the concentration of 6-BA, the concentration of 6-BA is probably higher, and the differentiation of the cluster buds is inhibited, so the hormone combination with the optimal multiplication is that the concentration of 6-BA is 2.0mg/L, the concentration of NAA is 0.5mg/L, the maximum multiplication times is 3.9, and the growth vigor of the cluster buds is also best.

In conclusion, the optimal re-disinfection method is to disinfect the mercury bichloride with the mass fraction of 0.1% for 4min and wash the mercury bichloride for 5 times, and then disinfect the mercury bichloride with the mass fraction of 0.1% for 2.5min and wash the mercury bichloride for 5 times; the most suitable induction culture medium is MS +1.5mg/L6-BA +0.8mg/L IBA +60mg/L PVP +120mg/L streptomycin +5.8g/L agar +30g/L sucrose, and the pH is 5.8; the most suitable proliferation culture medium is MS +2.0 mg/L6-BA +0.5mg/L NAA +60mg/L PVP +5.8g/L agar +30g/L sucrose, and the pH is 5.8; the invention has simple flow and short propagation period, the multiplication rate reaches 3.9, provides technical guidance for the herba epimedii seedling propagation and large-scale propagation, solves the problems of low germination rate of herba epimedii seeds and difficult seedling propagation caused by long germination period, reduces the herba epimedii seedling propagation cost and provides high-quality seedlings for the artificial propagation of herba epimedii.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and should not be taken as limiting the scope of the present invention, which is intended to cover any modifications, equivalents, improvements, etc. within the spirit and scope of the present invention.

Claims

1. A method for establishing and rapidly proliferating an Epimedium pubescens sterile system is characterized by comprising the following steps:

2) Vertically inoculating the sterile stem section obtained in the step 1) to an induction culture medium for culture, and inducing germination; the induction culture medium is MS +1.5mg/L6-BA +0.8mg/L IBA +60mg/L PVP +120mg/L streptomycin +5.8g/L agar +30g/L sucrose, and the pH is adjusted to 5.8;

3) Cutting the shoot buds obtained in the step 2) into single shoot points, vertically inoculating the shoot points to a multiplication culture medium for culture, and culturing for 28d under the conditions that the temperature is 25 ℃, the illumination intensity is 2300-2500lx and the illumination time is 12h/d to obtain epimedium clustered buds; the multiplication culture medium is MS +2.0 mg/L6-BA +0.5mg/L NAA +60mg/L PVP +5.8g/L agar +30g/L cane sugar, and the pH is adjusted to be 5.8;

in step 1), the method for re-sterilization is: sterilizing with 0.1% by mass of mercuric chloride for 4min, washing with sterile water for 5 times, sterilizing with 0.1% by mass of mercuric chloride for 2.5min, and washing with sterile water for 5 times;

in the step 2), the culture condition is that the culture is carried out for 7d in the dark at the temperature of 25 ℃, and then the culture medium is transferred to the condition of 25 ℃, the illumination intensity is 2300-2500lx and the illumination time is 12h/d for 18d.