CN109022344B - Culture method of golden gentian in-vitro cells - Google Patents

Culture method of golden gentian in-vitro cells Download PDF

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CN109022344B
CN109022344B CN201810907969.5A CN201810907969A CN109022344B CN 109022344 B CN109022344 B CN 109022344B CN 201810907969 A CN201810907969 A CN 201810907969A CN 109022344 B CN109022344 B CN 109022344B
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conyza blinii
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陈惠�
郑天润
孙文君
詹俊义
刘燕
汪茂佳
刘默洋
马招堂
王涛
唐自钟
布同良
吴琦
李成磊
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Sichuan Agricultural University
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Abstract

The invention discloses a culture method of gold gentian in-vitro cells, which comprises the following steps: selecting and sterilizing explants; separating single cells of the leaves of the conyza blinii; establishing a suspension cell line of the conyza blinii; and (5) stable subculture. The invention has shorter period than tissue culture, does not need to cultivate the conyza blinii and is not limited by geographical factors.

Description

Culture method of golden gentian in-vitro cells
Technical Field
The invention belongs to the technical field of plant tissue culture, and particularly relates to a culture method of gentiana rigescens in vitro cells.
Background
Conyza blinii (Conyzablinii. Lev.) belongs to two-year-old herbaceous plant of genus Conyza of Compositae, also known as felwort, etc., and grows in sparse forest zone with elevation of about 2000 m, and is mainly distributed in Panxi area of Sichuan, southwest part of Yunnan and western part of Guizhou. The product as a medicinal plant has cold property and extremely bitter taste, has the effects of clearing heat, diminishing inflammation, detoxifying, removing blood stasis, reducing swelling and the like, and has obvious curative effects on eliminating phlegm, relieving cough and asthma, acute and chronic bronchitis, sphagitis, bronchopneumonia and the like. Most of the traditional medicinal materials of the conyza blinii are collected in the field and cultivated artificially, are influenced by natural factors and cultivation period, and the resources of the conyza blinii can not meet the requirements of the medical market and scientific research.
Therefore, there is a need to provide a technique and a method for rapidly propagating conyza blinii to ensure basic scientific research and market demand.
Disclosure of Invention
In view of the above, the present invention provides a method for culturing isolated cells of gentiana rigescens.
In order to solve the technical problem, the invention discloses a culture method of gold gentian isolated cells, which comprises the following steps:
step 1, selection and sterilization of explants: collecting young and tender new leaves with relatively large leaf surface area as a standby explant; washing the explant in sterile water in a super-clean workbench, and sucking residual water by using sterile filter paper; soaking the treated leaves in 70% ethanol for 10-30 s, taking out, immediately washing with a large amount of sterile water for 3-5 times, and adding 0.1% HgCl 2 Soaking for 6-10min, immediately washing with a large amount of sterile water for 3-5 times, placing the explant on sterile filter paper to absorb water, and cutting off vein with sterile scalpel;
step 2, carrying out single cell separation on the leaves of the conyza blinii: spraying 70% alcohol to the beef tripe homogenizer for sterilization, and volatilizing with alcohol for later use; separating single cells by a mechanical method, transferring the processed leaves into a sterile beef tripe homogenizer containing STN buffer solution, and quickly grinding and homogenizing; passing through a sterile cell sieve, and collecting the in vitro single cells of the conyza blinii;
step 3, establishing a suspension cell line of the conyza blinii: sucking cell fluid obtained by mechanical separation, inoculating the cell fluid into suspension cell culture fluid, culturing the inoculated culture fluid in a shaking table, and identifying whether the suspension cells are the suspension cells of the conyza blinii or not by adopting SCAR;
and 4, stable subculture: sucking suspension cell line identified as the parent of conyza blinii, transferring into new culture solution, and culturing for 3 days.
Optionally, the sterilization conditions of the explant in step 1) are: soaking in 70% ethanol for 10s, and adding 0.1% HgCl 2 Soaking for 8 min.
Optionally, the suspension cell culture solution is MS + sucrose 30 mg.L -1 +2,4-D 1.0mg·L -1 +6-BA 0.1mg·L -1 +KT 0.5mg·L -1 + mannitol 0.3M, pH 5.5.
Alternatively, the STN buffer in step 2 has a pH of 7.5.
Optionally, the homogenization time in the step 3 is 0.5-1.5min, and the mesh number of the sterile cell sieve is 180-220 meshes.
Alternatively, the incubation temperature in step 3 is 25 ℃ and the rotation speed is 100-120 rpm.
Compared with the prior art, the invention can obtain the following technical effects:
1) the invention cultures the in vitro cells of the conyza blinii through the cell engineering technology, quickly proliferates in a short time, and obtains the effective components from the conyza blinii suspension cell line.
2) The invention has shorter period than tissue culture, does not need to cultivate the conyza blinii and is not limited by geographical factors.
Of course, it is not necessary for any one product in which the invention is practiced to achieve all of the above-described technical effects simultaneously.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and not to limit the invention. In the drawings:
FIG. 1 is a process flow chart of the method for in vitro rapid propagation and suspension cell culture of conyza blinii of the present invention;
FIG. 2 is a schematic representation of a parental suspension cell line of Gentiana scabra Bunge of the present invention;
FIG. 3 shows SCAR, a specific molecular marker identification of the Gentiana scabra Bunge of the present invention; wherein, 1 represents DNA marker (D2000), 2 and 3 represent the suspension cell line of the conyza blinii;
FIG. 4 is a state diagram of F1 generation suspension cells cultured for 24h, 48h, 72h and 96 h; wherein a represents 24h, b represents 48h, c represents 72h, and d represents 96 h;
FIG. 5 is the cell density statistics of 0h-78h suspension cell line of the invention;
FIG. 6 is a graph showing the results of the coloration of suspension cells in stationary phase and the coloration of suspension cells in latent phase according to the present invention; wherein, a represents the result of oleanolic acid coloration of suspension cells in the stationary phase, and b represents the result of oleanolic acid coloration of suspension cells in the latent phase.
Detailed Description
The following embodiments are described in detail with reference to the accompanying drawings, so that how to implement the technical features of the present invention to solve the technical problems and achieve the technical effects can be fully understood and implemented.
The invention discloses a method for in vitro rapid propagation and suspension cell culture of gentiana rigescens, which comprises the following steps as shown in figure 1:
step 1, selection and sterilization of explants: collecting new leaves which are strong in growth and free from diseases and insect pests as standby explants. In a clean bench, explants were rinsed under sterile water and residual water was blotted dry with sterile filter paper. Soaking the treated leaves in 70% ethanol for 10-30 s, taking out, immediately washing with a large amount of sterile water for 3-5 times, and adding 0.1% HgCl 2 Soaking for 6-10min, immediately washing with a large amount of sterile water for 3-5 times, placing the explant on sterile filter paper to absorb water, and cutting off vein with sterile scalpel;
preferably, the sterilization combination is: soaking in 70% ethanol for 10s + 0.1% HgCl 2 Soaking for 8 min;
step 2, carrying out single cell separation on the leaves of the conyza blinii:
separating single cell by mechanical method, transferring the processed leaf into 20ml cattle tripe homogenizer (aseptic) containing STN buffer solution (pH7.5), and rapidly grinding and homogenizing for 0.5-1.5 min; sieving with a cell sieve of 180-220 meshes (sterile);
step 3, establishing a suspension cell line of the conyza blinii:
respectively sucking 1ml of cell fluid obtained by mechanical separation, and inoculating the cell fluid to suspension cell culture fluid; culturing the inoculated culture solution in a shaking table with the temperature of 25 ℃ and the speed of 100-120rpm, after a large number of cells are attached to the wall of the bottle, rapidly identifying a molecular marker-SCAR by utilizing the specificity of the conyza blinii, referring to an SCAR primer and a method (application number: 201310373522.1, application date: 2013-08-23; publication number: CN103589783A, publication date: 2014-02-19) for identifying the conyza blinii and a confounding product thereof by utilizing an SCAR technology, and identifying whether the suspension cells are conyza blinii suspension cells or not; as shown in FIG. 3, the PCR amplification showed the target band, which confirmed that the suspension cells were suspension cells of Scarabaeus scaber.
Preferably, the suspension cell culture is 20mL MS culture: MS + sucrose 30 mg.L -1 +2,4-D 1.0mg·L -1 +6-BA 0.1mg·L -1 +KT 0.5mg·L -1 + mannitol 0.3M;
wherein, after the suspension cells of the conyza blinii are cultured for 18h, the cell line enters a logarithmic growth phase, and is subcultured by adopting an absorption method after 3 days of culture, wherein 1ml of solution is absorbed each time;
and 4, stable subculture: sucking 1ml of suspension cell line identified as the parent of the conyza blinii, transferring into a new MS culture solution (the same formula as the MS culture solution) and subculturing for 3 days.
Wherein, the MS culture solution (suspension cell culture solution) used by the invention is prepared by MS culture medium without agar and sucrose, 6-BA, 2,4-D, KT, mannitol and sucrose, and the pH value is 5.5.
The culture temperature of the suspension cells and the culture temperature of the subsequent subculture are both 25 ℃, and the rotating speed is 110 rpm.
Example 1
A method for in vitro rapid propagation and suspension cell culture of Gentiana rigescens Bunge comprises the following steps:
step 1, selection and sterilization of explants: collecting new leaves which are strong in growth and free from diseases and insect pests as standby explants. Washing the explant in sterile water in a clean benchThe residual water was blotted dry with bacteria filter paper. Soaking the treated leaves in 70% ethanol for 10s, taking out, immediately washing with a large amount of sterile water for 3-5 times, and adding 0.1% HgCl 2 Soaking for 8min, immediately washing with a large amount of sterile water for 3-5 times, placing the explant on sterile filter paper, drying, and cutting off leaf veins with a sterile scalpel;
step 2, carrying out single cell separation on the leaves of the conyza blinii:
separating single cell by mechanical method, transferring the processed leaf into 20ml cattle tripe homogenizer (aseptic) containing STN buffer solution, and rapidly grinding and homogenizing for 1 min; after sieving with a 200-mesh cell sieve (sterile), a proper amount of cell fluid was taken under an optical microscope to observe the integrity of the cells.
Step 3, establishing a suspension cell line of the conyza blinii:
1ml of cell sap obtained by mechanical separation is respectively sucked and inoculated in a culture solution; culturing the inoculated culture solution in a shaking table at 25 ℃ and 110rpm, and identifying whether the suspension cells are the conyza blinii suspension cells or not by using the SCAR after a large number of cells are attached to the bottle wall; wherein, the MS culture solution is as follows: MS + sucrose 30 mg.L -1 +2,4-D 1.0mg·L -1 +6-BA 0.1mg·L -1 +KT 0.5mg·L -1 + mannitol 0.3M; as shown in FIG. 2, the culture solution was turbid and had a distinct cell mass, which confirmed the successful culture of the suspension cell line of the parent plant of conyza blinii.
And 4, stable subculture:
sucking 1ml of suspension cell line identified as the parent of the conyza blinii, transferring into a new MS culture solution (the formula of the MS culture solution is the same as that of the MS culture solution), counting cells every 6h, and taking pictures for recording (as shown in FIG. 4 and FIG. 5); as the culture time increases, the cell concentration in the culture solution also increases; the growth of the conyza blinii suspension cell line conforms to an S-shaped growth curve, 0h-18h is the incubation period of the conyza blinii suspension cell line, 18h-48h is the logarithmic growth period of the conyza blinii suspension cell line, 48h-66h is the stabilization period of the conyza blinii suspension cell line, and 66h later is the plateau period; the optimal subculture time was finally determined to be 3 days.
Example 2
A method for in vitro rapid propagation and suspension cell culture of Gentiana rigescens Bunge comprises the following steps:
step 1, selection and sterilization of explants: collecting new leaves which are strong in growth and free from diseases and insect pests as standby explants. The explants were rinsed under sterile water in a clean bench and the residual water was blotted with sterile filter paper. Soaking the treated leaves in 70% ethanol for 30s, taking out, washing with a large amount of sterile water for 3-5 times, and adding 0.1% HgCl 2 Soaking for 6min, immediately washing with a large amount of sterile water for 3-5 times, placing the explant on sterile filter paper to absorb water, and cutting off vein with sterile scalpel;
step 2, carrying out single cell separation on the leaves of the conyza blinii:
separating single cell by mechanical method, transferring the processed leaf into 20ml cattle tripe homogenizer (aseptic) containing STN buffer solution, and rapidly grinding and homogenizing for 0.5 min; sieving with 200 mesh cell sieve (sterile);
step 3, establishing a suspension cell line of the conyza blinii:
respectively sucking 1ml of cell fluid obtained by mechanical separation, inoculating the cell fluid into a suspension cell culture solution, culturing the inoculated culture solution in a shaking table at 25 ℃ and 100rpm, and identifying whether the suspension cell is a conyza blinii suspension cell by using an SCAR (sequence characterized amplified region) after a large number of cells are attached to the bottle wall; the suspension cell culture solution is as follows: MS + sucrose 30 mg. L -1 +2,4-D 1.0mg·L -1 +6-BA 0.1mg·L -1 +KT 0.5mg·L -1 + mannitol 0.3M;
wherein, after the suspension cells of the conyza blinii are cultured for 18h, the cell line enters a logarithmic growth phase, and is subcultured by adopting an absorption method after 3 days of culture, wherein 1ml of solution is absorbed each time;
and 4, stable subculture: sucking 1ml of suspension cell line identified as the parent of the conyza blinii, transferring into a new culture solution (synchronous step 3), and subculturing for 3 days.
Example 3
A method for in vitro rapid propagation and suspension cell culture of Gentiana rigescens Bunge comprises the following steps:
step 1, selection and sterilization of explants: collecting new leaves which are strong in growth and free from diseases and insect pests as standby explants. In thatIn an ultraclean bench, explants are rinsed under sterile water and residual water is blotted dry with sterile filter paper. Soaking the treated leaves in 70% ethanol for 20s, taking out, immediately washing with a large amount of sterile water for 3-5 times, and adding 0.1% HgCl 2 Soaking for 10min, immediately washing with a large amount of sterile water for 3-5 times, placing the explant on sterile filter paper to absorb water, and cutting off vein with sterile scalpel;
step 2, carrying out single cell separation on the leaves of the conyza blinii:
separating single cell by mechanical method, transferring the processed leaf into 20ml cattle tripe homogenizer (aseptic) containing STN buffer solution, and rapidly grinding and homogenizing for 1.5 min; sieving with 200 mesh cell sieve (sterile);
step 3, establishing a conyza blinii suspension cell line:
respectively sucking 1ml of cell fluid obtained by mechanical separation, inoculating the cell fluid into a suspension cell culture solution, culturing the inoculated culture solution in a shaking table at 25 ℃ and 120rpm, and identifying whether the suspension cell is a conyza blinii suspension cell by using an SCAR (sequence characterized amplified region) after a large number of cells are attached to the bottle wall; the culture solution is as follows: MS + sucrose 30 mg. L -1 +2,4-D 1.0mg·L -1 +6-BA 0.1mg·L -1 +KT 0.1mg·L -1 + mannitol 0.3M;
and 4, stable subculture: 1ml of suspension cell line identified as the parent of conyza blinii was pipetted and transferred into a new culture medium (same as in example 3) for 3 days.
The suspension cells of the parent plant of conyza blinii obtained in example 1 can be stably subcultured, and examples 2 and 3 are the same as example 1.
Comparative example 1
A method for in vitro rapid propagation and suspension cell culture of Gentiana rigescens Bunge comprises the following steps:
step 1, selection and sterilization of explants: collecting new leaves which are strong in growth and free from diseases and insect pests as standby explants. In a clean bench, explants were rinsed under sterile water and residual water was blotted dry with sterile filter paper. Soaking the treated leaves in 70% ethanol for 10s, taking out, immediately washing with a large amount of sterile water for 3-5 times, and adding 0.1% HgCl 2 Soaking for 8min, immediately washing the explant with a large amount of sterile water for 3-5 times after the completion, then putting the explant on sterile filter paper to absorb water, and cutting off veins with a sterile scalpel;
step 2, carrying out single cell separation on the leaves of the conyza blinii:
and (3) separating single cells by an enzymolysis method, and performing enzymolysis on the treated leaves in a centrifugal tube (sterile) containing mixed enzyme liquid (2% of cellulase and 1.5% of pectinase) at 37 ℃ for 1h by a shaking table at 110 rpm. After enzymolysis, the mixture is sieved by a 200-mesh cell sieve (sterile).
Step 3, establishing a suspension cell line of the conyza blinii:
respectively sucking 1ml of cell fluid obtained by separation, inoculating the cell fluid into a culture solution containing suspension cells, culturing the inoculated culture solution in a shaking table at 25 ℃ and 110rpm, and identifying whether the suspension cells are the conyza blinii suspension cells or not by using an SCAR (sequence characterized amplified region) after a large number of cells are attached to the bottle wall; wherein, after the suspension cells of the conyza blinii are cultured for 18h, the cell line enters a logarithmic growth phase, and is subcultured by adopting an absorption method after 3 days of culture, wherein 1ml of solution is absorbed each time;
and 4, stable subculture: sucking 1ml of suspension cell line identified as the parent of the conyza blinii, transferring into a new culture solution, and subculturing for 3 days.
Comparative example 1 and example 1 compare the effects: the unicells of the leaves of the conyza blinii obtained by the mechanical method (example 1) have higher concentration than that of the unicells obtained by the enzymatic hydrolysis method (comparative example 1), are easy to culture parent suspension cells, and have lower cost.
The content of oleanolic acid, which is a metabolic intermediate product of Chinese herbal medicines, is detected by an indirect determination method, and the reference is made to the spectrophotometry for determining the oleanolic acid saponin content in panax japonicus. The result of the coloration of the suspension cell oleanolic acid in the stationary phase is shown in fig. 6a, and the result of the coloration of the suspension cell oleanolic acid in the latent phase is shown in fig. 6b, therefore, the invention cultures the isolated cells of the conyza blinii Lindl by the cell engineering technology, rapidly proliferates in a short time, and obtains the effective components from the conyza blinii suspension cell line.
While the foregoing description shows and describes several preferred embodiments of the invention, it is to be understood, as noted above, that the invention is not limited to the forms disclosed herein, but is not to be construed as excluding other embodiments and is capable of use in various other combinations, modifications, and environments and is capable of changes within the scope of the inventive concept as expressed herein, commensurate with the above teachings, or the skill or knowledge of the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.

Claims (3)

1. A culture method of golden gentian in vitro cells is characterized by comprising the following steps:
step 1, selection and sterilization of explants: collecting young and tender new leaves with relatively large leaf surface area as a standby explant; washing the explant in sterile water in a super-clean workbench, and sucking residual water by using sterile filter paper; soaking the treated leaves in 70% ethanol for 10-30 s, taking out, immediately washing with a large amount of sterile water for 3-5 times, and adding 0.1% HgCl 2 Soaking for 6-10min, immediately washing with a large amount of sterile water for 3-5 times, placing the explant on sterile filter paper to absorb water, and cutting off vein with sterile scalpel;
step 2, carrying out single cell separation on the leaves of the conyza blinii: spraying 70% alcohol to the beef tripe homogenizer for sterilization, and allowing the alcohol to evaporate to dryness; separating single cells by mechanical method, transferring the processed leaves into a sterile beef tripe homogenizer containing STN buffer solution, and rapidly grinding and homogenizing for 0.5-1.5 min; passing through a sterile cell sieve, wherein the mesh number of the sterile cell sieve is 180-220 meshes, and collecting the in-vitro single cells of the conyza blinii;
step 3, establishing a suspension cell line of the conyza blinii: absorbing the cell fluid obtained by mechanical separation, inoculating the cell fluid into a suspension cell culture solution, culturing the inoculated culture solution in a shaking table at the culture temperature of 25 ℃ and the rotation speed of 100-120rpm, and identifying whether the suspension cell is the conyza blinii suspension cell by adopting an SCAR (sequence characterized amplified region);
and 4, stable subculture: sucking suspension cell line identified as the parent of conyza blinii, and culturing in new suspension culture solution for 3 days;
the suspension cell culture solution is MS + sucrose 30 mg.L -1 +2,4-D 1.0mg·L -1 +6-BA 0.1mg·L -1 +KT 0.5mg·L -1 + mannitol 0.3M, pH 5.5.
2. The method for culturing the isolated cells of conyza blinii as claimed in claim 1, wherein the sterilization conditions of the explant in step 1 are as follows: soaking in 70% ethanol for 10s, and adding 0.1% HgCl 2 Soaking for 8 min.
3. The method for culturing the isolated cells of conyza blinii as claimed in claim 1, wherein the pH of the STN buffer solution in step 2 is 7.5.
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