CN103320380A - Method for establishing salicornia suspension cell line - Google Patents
Method for establishing salicornia suspension cell line Download PDFInfo
- Publication number
- CN103320380A CN103320380A CN2013102968417A CN201310296841A CN103320380A CN 103320380 A CN103320380 A CN 103320380A CN 2013102968417 A CN2013102968417 A CN 2013102968417A CN 201310296841 A CN201310296841 A CN 201310296841A CN 103320380 A CN103320380 A CN 103320380A
- Authority
- CN
- China
- Prior art keywords
- medium
- suspension
- final concentration
- tdz
- naa
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a method for establishing a salicornia suspension cell line. The method for establishing the salicornia suspension cell line comprises the following steps: (1) placing a salicornia explants onto an induction medium for induction culture so as to obtain callus 1; (2) placing the callus 1 onto a subculture medium to carry out subculture for more than five times so as to obtain callus 2; and (3) placing the callus 2 onto a suspension medium to carry out suspension and shake culture so as to obtain the salicornia suspension cell line. By the utilization of the method provided by the invention, the salicornia suspension cell line is established for the first time, thus being beneficial to deep analysis of salt tolerance mechanism of salicornia from the angle of cytology, laying a technical foundation for gene transformation and genetic modification of salicornia and providing a new possible way to gene function identification of salicornia.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of method of setting up the salicornia europaeal suspension cell line.
Background technology
Existing a lot of for the cultivation of plant suspension cell in the world, the cultivation of suspension cell has vital role for the improvement of science, medical science, agricultural and daily life; They are excellent cytology research materials, and can regard bio-reactor, synthetic relevant secondary metabolite (seasonings, essence etc.).At present, about the existing a lot of piece of writing bibliographical informations of the culture technique of suspension cell, wherein not only there are various model plant suspension cells to set up scheme, also have the suspension cell of various food crop and cash crop to set up scheme, wherein also related to the cultural method of some stubborn medicinal plants and xylophyta.Cao Fuliang etc. set up ginkgo suspension cell line (2008) with gingkgo callus; Li Xiangyun etc. disclose a kind of E. wushanense suspended cell culture system and establishment method and application (2011); Qi Xiangjun etc. have invented the culture process of bark of ash suspension cell, the fast and good dispersity (2011) of gained suspension cell growth speed; Yan has waited delivering a kind of technique (2006) that improves American ginseng suspending cell yield quietly.But up to now, also there is not halophytes to set up the relevant report of suspension cell line.
Salicornia europaeal (Salicornia europaea L.) is the dicotyledonous euhalophyte of a kind of carnification of Chenopodiaceae, extensively is distributed near coastal and the inland brine lake, is considered in the world a kind of higher plant of salt tolerant.Set up the suspension cell line of salicornia europaeal, not only be conducive to deeply resolve the salicornia europaeal Mechanism of Salt-tolerant from the cytology angle, for gene transformation and the genetic modification of salicornia europaeal have been established technical foundation, also the gene function checking for salicornia europaeal provides a new possible approaches.
Summary of the invention
The purpose of this invention is to provide a kind of method and special culture media thereof of setting up the salicornia europaeal suspension cell line.
The method of setting up the salicornia europaeal suspension cell line provided by the present invention can comprise the steps:
(1) the salicornia europaeal explant is placed carries out inducing culture on the inducing culture, obtain callus 1(khaki);
(2) described callus 1 is placed carry out succeeding transfer culture on the subculture medium more than 5 times (such as 5 times), obtain callus 2(light yellow loose watery);
(3) described callus 2 is placed suspension medium suspend and shake cultivation, thereby obtain the salicornia europaeal suspension cell line;
Described suspension medium be following a) or b):
A) be in the MS liquid nutrient medium, to add the substratum that obtains behind TDZ and the NAA; The final concentration of TDZ is 1.5-2.5mg/L(such as 2.0mg/L in the described suspension medium), the final concentration of NAA is 0.1-0.3mg/L(such as 0.1mg/L);
B) be in the MS liquid nutrient medium, to add the substratum that obtains behind TDZ, NAA and the NaCl; The final concentration of TDZ is 1.5-2.5mg/L(such as 2.0mg/L in the described suspension medium), the final concentration of NAA is 0.1-0.3mg/L(such as 0.1mg/L), the final concentration of NaCL is 0.1M.
In aforesaid method, in the step (1), described with the salicornia europaeal explant place carry out inducing culture on the inducing culture before, also comprise the step that described salicornia europaeal explant is carried out disinfection.
In the present invention, described salicornia europaeal explant is carried out disinfection, specific as follows: as to place first solution A to soak 1 minute described salicornia europaeal explant, go to again in the solution B and soaked 15 minutes, then use aseptic water washing 7-8 time.Described solution A is that volume fraction is 75% aqueous ethanolic solution; The solvent of solution B is water, and solute and concentration thereof are that volume fraction is that 2% NaClO and volume fraction are 1.5% Triton X-100.
In aforesaid method, in the step (3), described " described callus 2 is placed suspension medium ", specifically can be N(N is natural number, such as 15) piece diameter 0.8-1.2cm as described in callus 2 place 10 * N ml(such as 150mL) as described in suspension medium.
In the present invention, in the step (3), the method for described " concussion that suspends is cultivated, thereby obtains the salicornia europaeal suspension cell line " specifically comprises the steps:
(I) described callus 2 is placed described suspension medium, with 100-140 turn/min(as 120 turn/min) rotating speed concussion cultivated 12 ± 2 days, obtained cell suspending liquid;
(II) add isopyknic described suspension medium in step (I) the gained cell suspending liquid, again with 100-140 turn/min(as 120 turn/min) rotating speed concussion cultivates 7-12 days (such as 10 days), obtains cell suspending liquid to be filtered;
(III) described cell suspending liquid to be filtered is filtered with 40 mesh sieves, filtrate and described suspension medium are mixed, making the cell quantity in the mixed solution is 9-53/μ L(such as 12-53/μ L, such as 14-40/μ L, such as 14-27, for another example 23-27/μ L), again with 100-140 turn/min(as 120 turn/min) rotating speed concussion cultivates 7-12 days (such as 10 days), obtains filtering again culturing cell suspension;
(IV) repeating step (III) 3-5 time (such as 4 times), for each repetitive operation, all with the filtration of gained in the front operation steps (III) again culturing cell suspension operate as cell suspending liquid to be filtered, last gained filters again that culturing cell suspension is the salicornia europaeal suspension cell line.
In aforesaid method, in the step (III), described " filtrate and described suspension medium are mixed, and making the cell concn in the mixed solution is 9-53/μ L(such as 12-53/μ L, such as 14-40/μ L; such as 14-27; for another example 23-27/μ L) ", being specially described filtrate (cell concn is 70-80/μ L) and described suspension medium is that 1:7 to 2:1(is such as 1:5 to 2:1, such as 1:4 to 1:1 according to volume ratio, such as 1:4 to 1:2, for another example 1:2) ratio is mixed.
The calculation formula of the above cell concn is: cell concn=total cell number/mixeding liquid volume; Wherein, total cell number is the cell number sum less than the cell number in the cell mass of 10 cells and free single cell, namely only to cell mass or unicellular counting less than 10 cells, calculate strictly according to the facts less than the cell number in the cell mass of 10 cells, count total number of cells.
In the step (I)-(IV) of aforesaid method, the rotation radius that described rotation concussion is cultivated is 13mm.
In aforesaid method, described inducing culture is to add the substratum that obtains behind TDZ and the NAA in the MS solid medium; The final concentration of TDZ is 1.5-2.5mg/L(such as 2.0mg/L in the described inducing culture), the final concentration of NAA is 0.1-0.3mg/L(such as 0.1mg/L);
Described subculture medium adds the substratum that obtains behind TDZ and the NAA in the MS solid medium; The final concentration of TDZ is 1.5-2.5mg/L(such as 2.0mg/L in the described subculture medium), the final concentration of NAA is 0.1-0.3mg/L(such as 0.1mg/L).
In each related substratum of aforesaid method, the carbon source in described MS liquid nutrient medium and the described MS solid medium is specially sucrose; Gelifying agent in the described MS solid medium is specially plant gel;
In the present invention, the final concentration of described sucrose in described MS liquid nutrient medium and described MS solid medium all is specially 30g/L; The final concentration of described plant gel in described MS solid medium all is specially 3-4g/L.
In the present invention, the seed that related described salicornia europaeal explant is salicornia europaeal in the aforesaid method is specially the salicornia europaeal seed of peeling shell off.
Carry out the culture condition of succeeding transfer culture described in the culture condition that carries out inducing culture described in the step (1) of aforesaid method, step (2), and the condition of carrying out suspension culture described in the step (3) is dark cultivation, temperature is 25-28 ℃.
In the step (1) of aforesaid method, described culture cycle of carrying out inducing culture can be 20-30 days.
In the step (2) of aforesaid method, the subculture frequency of described succeeding transfer culture specifically can be: first three time subculture, and per 20 ± 2 days subcultures are once; From the 4th subculture, per 15 ± 2 days subcultures once.
Substratum be used to setting up the salicornia europaeal suspension cell line provided by the present invention is specially following (A) or (B):
(A) suspension medium, for following a) or b):
A) be in the MS liquid nutrient medium, to add the substratum that obtains behind TDZ and the NAA; The final concentration of TDZ is 1.5-2.5mg/L(such as 2.0mg/L in the described suspension medium), the final concentration of NAA is 0.1-0.3mg/L(such as 0.1mg/L);
B) be in the MS liquid nutrient medium, to add the substratum that obtains behind TDZ, NAA and the NaCl; The final concentration of TDZ is 1.5-2.5mg/L(such as 2.0mg/L in the described suspension medium), the final concentration of NAA is 0.1-0.3mg/L(such as 0.1mg/L), the final concentration of NaCL is 0.1M;
(B) substratum group, by inducing culture, subculture medium and suspension medium (A) form;
Described inducing culture is to add the substratum that obtains behind TDZ and the NAA in the MS solid medium; The final concentration of TDZ is 1.5-2.5mg/L(such as 2.0mg/L in the described inducing culture), the final concentration of NAA is 0.1-0.3mg/L(such as 0.1mg/L);
Described subculture medium adds the substratum that obtains behind TDZ and the NAA in the MS solid medium; The final concentration of TDZ is 1.5-2.5mg/L(such as 2.0mg/L in the described subculture medium), the final concentration of NAA is 0.1-0.3mg/L(such as 0.1mg/L).
In above-mentioned each substratum, the carbon source in described MS liquid nutrient medium and the described MS solid medium specifically can be sucrose; Gelifying agent in the described MS solid medium specifically can be plant gel;
Further, the final concentration of described sucrose in described MS liquid nutrient medium and described MS solid medium all is specially 30g/L; The final concentration of described plant gel in described MS solid medium all is specially 3-4g/L.
The application of the above substratum in setting up the salicornia europaeal suspension cell line also belongs to protection scope of the present invention.
The pH of above all described MS liquid nutrient medium is 5.8-5.9, and solvent is water, and solute and concentration thereof are as follows:
A. a large amount of: NH
4NO
31.65g/L, KNO
31.9g/L, MgSO
47H
2O0.37g/L, KH
2PO
40.17g/L, CaCl
20.33g/L;
B. micro-: KI0.0083mg/L, H
3BO
38mg/L, MnSO
44H
2O 22.3mg/L, ZnSO
47H
2O10mg/L, Na
2MoO
42H
2O 0.25mg/L, CuSO
45H
2O 0.025mg/L, CoCl
26H
2O 0.025mg/L;
C. VITAMIN: nicotinic acid 0.5mg/L, pyridoxol 0.5mg/L, VitB1 0.1mg/L, glycine 2mg/L;
D. molysite: FeSO
47H
2O 27.85mg/L, Na
2EDTA 37.25mg/L;
E. inositol 0.1g/L, MES 0.488g/L;
F. sucrose 30g/L.
Above each concentration is the final concentration of respective components in described MS liquid nutrient medium.
Above all described MS solid mediums are and add the solid medium that obtains after final concentration is the plant gel of 3-4g/L in described MS liquid nutrient mediums.
The present invention has set up the salicornia europaeal suspension cell line first, not only be conducive to deeply resolve the salicornia europaeal Mechanism of Salt-tolerant from the cytology angle, for gene transformation and the genetic modification of salicornia europaeal have been established technical foundation, also the gene function checking for salicornia europaeal provides a new possible approaches.
Description of drawings
Fig. 1 is the comparison that the different states callus carries out suspension culture.Wherein, A is the callus of twice of subculture; B is the callus of subculture three times; C is the callus of subculture five times; D is the suspension cell line that the callus of subculture three times forms; E is the suspension cell line that the callus of subculture five times forms.
Fig. 2 is that the suspension medium of hormon component proportion is on the value-added impact of salicornia europaeal suspension cell.Wherein, A is MS liquid nutrient medium (suspension medium 1); B is MS+2,4-D 1.5mg/L(suspension medium 2); C is MS+TDZ 2.0mg/L+NAA 0.1mg/L(suspension medium 3); D is MS+TDZ 2.0mg/L+NAA0.1mg/L+NaCl0.1M(suspension medium 4); E is suspension cell concentration (n=3, identical lowercase are illustrated under p＜0.05 condition, do not have significant difference) in 4 kinds of suspension mediums of statistics.Among the E, 1 expression suspension medium 1; 2 expression suspension mediums 2; 3 expression suspension mediums 3; 4 expression suspension mediums 4.
Fig. 3 is that different subinoculation amounts are on the value-added impact of salicornia europaeal suspension cell.Wherein, A is for being the ratio subinoculation of 2:1 according to filtrate and suspension medium volume ratio; B is for being the ratio subinoculation of 1:1 according to filtrate and suspension medium volume ratio; C is for being the ratio subinoculation of 1:2 according to filtrate and suspension medium volume ratio; D is for being the ratio subinoculation of 1:4 according to filtrate and suspension medium volume ratio; E is for being the ratio subinoculation of 1:5 according to filtrate and suspension medium volume ratio; F is for being the ratio subinoculation of 1:7 according to filtrate and suspension medium volume ratio; G is cell concn (n=3, identical lowercase are illustrated under p＜0.05 condition, do not have significant difference) in 6 kinds of different subinoculation amount gained suspension cell lines of statistics.
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
All medicines among the following embodiment and consumptive material are purchased the company in Sigmaaldrich, and its network address is referring to as follows:
http://www.sigmaaldrich.com/china-mainland.html。
Salicornia europaeal (S.europaea L.): seed originates from China Jiangsu Province Dafeng City's beach, is provided by Jiangsu Jinglong Ocean Industry Development Co., Ltd.
The pH of related MS liquid nutrient medium is 5.8-5.9 among the following embodiment, fills a prescription as follows:
A.20 doubly a large amount of: NH
4NO
333g/L, KNO
338g/L, MgSO
47H
2O 7.4g/L, KH
2PO
43.4g/L, CaCl
26.6g/L;
B.200 micro-mother liquor: KI1.66mg/L doubly, H
3BO
31600mg/L, MnSO
44H
2O 4460mg/L, ZnSO
47H
2O 2000mg/L, Na
2MoO
42H
2O 50mg/L, CuSO
45H
2O 5mg/L, CoCl
26H
2O5mg/L;
C.1000 times VITAMIN: nicotinic acid 500mg/L, pyridoxol 500mg/L, VitB1 100mg/L, glycine 2000mg/L;
D.200 times molysite: FeSO
47H
2O 5570mg/L, Na
2EDTA 7450mg/L;
E. inositol 0.1g/L, MES 0.488g/L pH is 5.8-5.9;
F. sucrose 30g/L.
Above each concentration is the final concentration of respective components in described MS liquid nutrient medium.
Related MS solid medium is and adds the solid medium that obtains after final concentration is the plant gel of 3-4g/L in described MS liquid nutrient medium among the following embodiment.
The acquisition of embodiment 1, salicornia europaeal suspension cell line and evaluation
The present embodiment through induction of callus, succeeding transfer culture and suspension culture, is finally set up the salicornia europaeal suspension cell line take the salicornia europaeal seed as explant.Wherein, the present inventor is to the composition and ratio of hormone in the state of callus, the suspension medium, and the subinoculation amount in the suspension culture process is optimized.Specific as follows:
One, callus status is on the impact of salicornia europaeal suspension cell line
(1) experimental technique
1, the sterilization of explant
Get the salicornia europaeal seed, peel shell off, place first solution A to soak 1 minute, go to again in the solution B and soaked 15 minutes, then use aseptic water washing 7-8 time, finish sterilization, for subsequent use.
Solution A: volume fraction is 75% aqueous ethanolic solution;
Solution B: solvent is water, and solute and concentration thereof are that volume fraction is that 2% NaClO and volume fraction are 1.5% Triton X-100.
2, callus induces
Inducing culture (MS+TDZ 2.0mg/L+NAA 0.1mg/L): in the MS solid medium, add the substratum that obtains behind TDZ and the NAA; The final concentration of TDZ is that the final concentration of 2.0mg/L, NAA is 0.1mg/L in the described inducing culture.
The salicornia europaeal seed of step 1 sterilization is placed on the inducing culture, under 25-28 ℃, secretly cultivate 20-30 days (according to seed vitality, needing asynchronism(-nization)), until khaki callus occurs in hypocotylar bottom.
3, the subculture of callus and expand numerous
Subculture medium (MS+TDZ 2.0mg/L+NAA 0.1mg/L): in the MS solid medium, add the substratum that obtains behind TDZ and the NAA; The final concentration of TDZ is that the final concentration of 2.0mg/L, NAA is 0.1mg/L in the described subculture medium.
The callus that step 2 is induced downcuts from the hypocotyl low side, is placed on the subculture medium, allows callus propagation expand numerous, and culture condition is: the dark cultivation, temperature is 25-28 ℃.After callus is grown up (diameter is about 0.5cm), continuous breeding callus on subculture medium, during first three time subculture, per 20 days left and right sides subcultures once, from the 4th subculture, per 15 days left and right sides subcultures are once.Select respectively subculture for the second time, for the third time with the 5th time after callus (about diameter 0.8-1.2cm), be used for the research callus status to the impact of salicornia europaeal suspension cell line.
4, the acquisition of suspension cell line
Suspension medium (MS+TDZ 2.0mg/L+NAA 0.1mg/L): in the MS liquid nutrient medium, add the substratum that obtains behind TDZ and the NAA; The final concentration of TDZ is that the final concentration of 2.0mg/L, NAA is 0.1mg/L in the described suspension medium.
1) gets the callus (about diameter 0.8-1.2cm) of three kinds of different states that step 3 chooses, 15 every kind, every kind of callus is corresponding respectively puts into three culturing bottles that contain the 150ml suspension medium, in 25-28 ℃, under the dark condition, concussion was cultivated about 12 days, and shaking speed 120r/min(rotation radius is 13mm).
2) will shake the cell suspending liquid of cultivating after 12 days and add isopyknic suspension medium, in 25-28 ℃, under the dark condition, again take the 120r/min(rotation radius as 13mm) the rotating speed concussion cultivated 10 days.
3) after 10 days, filter callus lines with 40 mesh sieve, filtrate and suspension medium mixed according to the volume ratio of 1:2, in 25-28 ℃, under the dark condition, again take the 120r/min(rotation radius as 13mm) the rotating speed concussion cultivated 10 days.
4) repeating step 3) 4 times, obtain three parts of candidate's suspension cell lines.
5) by the OLYMPUS microscope three parts of candidate's suspension cell lines of step 4) gained are observed.
(2) experimental result
Select respectively subculture for the second time, for the third time with the 5th time after callus, with the callus lines of equivalent inoculate and suspension culture relatively.As shown in Figure 1, also harder during subculture twice when callus is cut from hypocotyl, color deep yellow (A among Fig. 1); When callus subculture for the third time the time, integral body is still hard, and color becomes faint yellow (B among Fig. 1) simultaneously; When callus subculture the 5th time, it is soft that integral body becomes, and color has become light yellow (C among Fig. 1) simultaneously.These three kinds of callus are carried out suspension culture, and the first callus (subculture twice, A among Fig. 1) can't form suspension cell line fully; For the second callus (subculture three times, B among Fig. 1), although can suspend amplification, because too hard, can't form simple cell mass, after repeatedly cultivating, only have macrobead in the substratum, be difficult to form suspension culture system (D among Fig. 1); For the third callus (subculture five times, C among Fig. 1), by behind the succeeding transfer culture several times, can form the suspension culture system (F among Fig. 1) of homogeneous.
As seen, different callus status is set up salicornia europaeal suspension cell line success, and tool has a great impact.According to above result, in actually operating, suggestion selects the callus (light yellow loose watery) of subculture more than five times to be used for setting up the salicornia europaeal suspension cell line.
Two, the hormone composition and ratio in the suspension medium is on the impact of salicornia europaeal suspension cell line
(1) experimental technique
1, the sterilization of explant
With step 11.
2, callus induces
With step 12.
3, the subculture of callus and expand numerous
Concrete operations are referring to step 13, and difference only is to select the callus (about diameter 0.8-1.2cm) behind the subculture the 5th time, is used for step 4 and sets up the salicornia europaeal suspension cell line.
4, the acquisition of suspension cell line
Following four kinds of suspension mediums are set, are used for the hormone composition and ratio of research suspension medium to the impact of salicornia europaeal suspension cell line:
Suspension medium 1(MS): the MS liquid nutrient medium.
Suspension medium 2(MS+2,4-D 1.5mg/L): the substratum that obtains after in the MS liquid nutrient medium, adding 2,4-D; In the described suspension medium 22, the final concentration of 4-D is 1.5mg/L.
Suspension medium 3(MS+TDZ 2.0mg/L+NAA 0.1mg/L): in the MS liquid nutrient medium, add the substratum that obtains behind TDZ and the NAA; The final concentration of TDZ is that the final concentration of 2.0mg/L, NAA is 0.1mg/L in the described suspension medium 3.
Suspension medium 4(MS+TDZ 2.0mg/L+NAA 0.1mg/L+NaCl0.1M): in the MS liquid nutrient medium, add the substratum that obtains behind TDZ, NAA and the NaCl; The final concentration of TDZ is that the final concentration of 2.0mg/L, NAA is that the final concentration of 0.1mg/L, NaCl is 0.1M in the described suspension medium 4.
Concrete operations are as follows:
1) above 4 parts of suspension mediums is put into respectively 4 culturing bottles, 150ml/ bottle.Get the callus (about diameter 0.8-1.2cm) behind the subculture that step 3 chooses the 5th time, 4 parts, 15 every part.Put into a callus for every bottle in 4 culturing bottles, in 25-28 ℃, under the dark condition, concussion was cultivated about 12 days, and shaking speed 120r/min(rotation radius is 13mm).
2) will shake the cell suspending liquid of cultivating after 12 days and add isopyknic corresponding suspension medium, in 25-28 ℃, under the dark condition, again take the 120r/min(rotation radius as 13mm) the rotating speed concussion cultivated 10 days.
3) after 10 days, filter callus lines with 40 mesh sieve, filtrate and corresponding suspension medium mixed according to the volume ratio of 1:2,, in 25-28 ℃, under the dark condition, again take the 120r/min(rotation radius as 13mm) the rotating speed concussion cultivated 10 days.
4) repeating step 3) 4 times, obtain four parts of candidate's suspension cell lines of corresponding four kinds of suspension mediums.
5) by the OLYMPUS microscope four parts of candidate's suspension cell lines of step 4) gained are observed.Cell concn in the calculated candidate suspension cell line.Cell concn=total cell number/candidate's suspension cell line volume; Wherein, total cell number is the cell number sum less than the cell number in the cell mass of 10 cells and free single cell, namely only to cell mass or unicellular counting less than 10 cells, calculate strictly according to the facts less than the cell number in the cell mass of 10 cells, count total number of cells.The experiment triplicate, results averaged.
(2) experimental result
The result cell can be shaken up dispersion with MS substratum (suspension medium 1), but cell amplification is slow as shown in Figure 2, and through succeeding transfer culture, the number of cell in suspension system still maintains A and the E among 6/μ l(Fig. 2); Compare with the MS substratum, the suspension medium 2 that has added growth hormone (1.5mg/L2,4D) can improve fissional speed, but still value-added slow, large B and the E in 10/μ l(Fig. 2 of cell concn behind the succeeding transfer culture).Yet, when adding phytokinin (TDZ) and keeping mitogen concentration far above growth hormone after (suspension medium 3), cell number has the increment of highly significant, behind the succeeding transfer culture, cell concn can reach C and the E among 74/μ l(Fig. 2).For the suspension system of setting up halophytes, the effect that applies of NaCl also is Consideration, and therefore, the present inventor has added 0.1M NaCl on the basis of suspension medium 3, investigates NaCl for the value-added impact of suspension cell.D among result such as Fig. 2 and E, after finding to have applied the NaCl of 0.1M, the cell increment does not increase significantly, and behind succeeding transfer culture, cell number is 76/μ l.
In sum, by groping of hormone combination, the suspension medium of finally determining to be fit to set up the salicornia europaeal suspension cell line is suspension medium 3, i.e. MS+TDZ 2.0mg/L+NAA 0.1mg/L.
Three, the subinoculation amount in the suspension culture process is on the impact of salicornia europaeal suspension cell line
(1) experimental technique
1, the sterilization of explant
With step 11.
2, callus induces
With step 12.
3, the subculture of callus and expand numerous
With step 23.
4, the acquisition of suspension cell line
Suspension medium (MS+TDZ 2.0mg/L+NAA 0.1mg/L): in the MS liquid nutrient medium, add the substratum that obtains behind TDZ and the NAA; The final concentration of TDZ is that the final concentration of 2.0mg/L, NAA is 0.1mg/L in the described suspension medium.
Concrete operations are as follows:
1) gets callus (about diameter 0.8-1.2cm) behind the subculture that 15 steps 3 choose the 5th time, put into the culturing bottle that the 150mL suspension medium is housed, in 25-28 ℃, under the dark condition, concussion was cultivated about 12 days, and shaking speed 120r/min(rotation radius is 13mm).
2) will shake the cell suspending liquid of cultivating after 12 days and add isopyknic suspension medium, in 25-28 ℃, under the dark condition, again take the 120r/min(rotation radius as 13mm) the rotating speed concussion cultivated 10 days.
3) after 10 days, filter callus lines with 40 mesh sieve, get filtrate, cell concn is that 70-80/μ L(measuring method is seen step 5) in the filtrate after measured).6 parallel laboratory tests are set, filtrate and suspension medium are mixed according to following 6 volume ratios (2:1,1:1,1:2,1:4,1:5,1:7) respectively, (cumulative volume of 6 portions of mixed solutions equates to obtain 6 portions of mixed solutions, be 200mL), in 25-28 ℃, under the dark condition, again take the 120r/min(rotation radius as 13mm) rotating speed concussion cultivated 10 days.
Wherein, according to filtrate and the descending order of suspension medium volume ratio, its cell concn is followed successively by 47-53/μ L, 35-40/μ L, 23-27/μ L, 14-16/μ L, 12-13/μ L, 9-10/μ L in 6 portions of mixed solutions.
4) repeating step 3) 4 times, obtain six parts of candidate's suspension cell lines of corresponding 6 kinds of filtrates and suspension medium ratio.
5) by the OLYMPUS microscope six parts of candidate's suspension cell lines of step 4) gained are observed.Cell concn in the calculated candidate suspension cell line.Cell concn=total cell number/candidate's suspension cell line volume; Wherein, total cell number is the cell number sum less than the cell number in the cell mass of 10 cells and free single cell, namely only to cell mass or unicellular counting less than 10 cells, calculate strictly according to the facts less than the cell number in the cell mass of 10 cells, count total number of cells.The experiment triplicate, results averaged.
(2) experimental result
The result is the ratio subinoculation of 1:2 according to filtrate and suspension medium volume ratio as shown in Figure 3, can obtain the maximum suspension cell of unit volume quantity (C among Fig. 3 and G).Although be the ratio inoculation of 2:1 and 1:1 according to filtrate and suspension medium volume ratio, can see more cell mass (A among Fig. 3 and B), but the final quantity of suspension cell (less than cell mass or the free single cell of 10 cells) that forms is but not as according to filtrate and suspension medium volume ratio being many (G among Fig. 3) of the ratio inoculation of 1:2.And be the ratio inoculation of 1:4,1:5 and 1:7 according to filtrate and suspension medium volume ratio, the suspension cell concentration of acquisition is too low, is unfavorable for further amplification and correlative study (D among Fig. 3, E, F and G).
In sum, the subinoculation amount is added take new and old substratum ratio as 2:1 (namely according to filtrate and the suspension medium volume ratio ratio subinoculation as 1:2, cell concn is 23-27/μ L in the rear mixed solution of i.e. inoculation), the growth of the most suitable salicornia europaeal suspension cell and increment.
Claims (10)
1. a method of setting up the salicornia europaeal suspension cell line comprises the steps:
(1) the salicornia europaeal explant is placed carries out inducing culture on the inducing culture, obtain callus 1;
(2) described callus 1 is placed carry out succeeding transfer culture on the subculture medium more than 5 times, obtain callus 2;
(3) described callus 2 is placed suspension medium suspend and shake cultivation, thereby obtain the salicornia europaeal suspension cell line;
Described suspension medium be following a) or b):
A) be in the MS liquid nutrient medium, to add the substratum that obtains behind TDZ and the NAA; The final concentration of TDZ is that the final concentration of 1.5-2.5mg/L, NAA is 0.1-0.3mg/L in the described suspension medium;
B) be in the MS liquid nutrient medium, to add the substratum that obtains behind TDZ, NAA and the NaCl; The final concentration of TDZ is that the final concentration of 1.5-2.5mg/L, NAA is that the final concentration of 0.1-0.3mg/L, NaCL is 0.1M in the described suspension medium.
2. method according to claim 1 is characterized in that: in the step (3), the described concussion that suspends is cultivated, thereby obtains the method for salicornia europaeal suspension cell line, comprises the steps:
(I) described callus 2 is placed described suspension medium, with 100-140 turn/concussion of the rotating speed of min cultivated 12 ± 2 days, obtained cell suspending liquid;
(II) add isopyknic described suspension medium in step (I) the gained cell suspending liquid, again with 100-140 turn/the rotating speed concussion of min cultivated 7-12 days, obtained cell suspending liquid to be filtered;
(III) described cell suspending liquid to be filtered is filtered with 40 mesh sieves, gained filtrate and described suspension medium are mixed, making cell quantity in the mixed solution is 9-53/μ L, again with 100-140 turn/the rotating speed concussion of min cultivated 7-12 days, obtained filtering again culturing cell suspension;
(IV) repeating step (III) is 3-5 time, for each repetitive operation, all with the filtration of gained in the front operation steps (III) again culturing cell suspension operate as cell suspending liquid to be filtered, last gained filters again that culturing cell suspension is the salicornia europaeal suspension cell line.
3. method according to claim 1 and 2 is characterized in that: described inducing culture is to add the substratum that obtains behind TDZ and the NAA in the MS solid medium; The final concentration of TDZ is that the final concentration of 1.5-2.5mg/L, NAA is 0.1-0.3mg/L in the described inducing culture;
Described subculture medium adds the substratum that obtains behind TDZ and the NAA in the MS solid medium; The final concentration of TDZ is that the final concentration of 1.5-2.5mg/L, NAA is 0.1-0.3mg/L in the described subculture medium.
4. arbitrary described method according to claim 1-3 is characterized in that: the carbon source in described MS liquid nutrient medium and the described MS solid medium is sucrose; Gelifying agent in the described MS solid medium is plant gel;
The final concentration of described sucrose in described MS liquid nutrient medium and described MS solid medium all is specially 30g/L; The final concentration of described plant gel in described MS solid medium all is specially 3-4g/L.
5. arbitrary described method according to claim 1-4 is characterized in that: described salicornia europaeal explant is the seed of salicornia europaeal.
6. arbitrary described method according to claim 1-5, it is characterized in that: the culture condition that carries out carrying out described in the culture condition, step (2) of inducing culture succeeding transfer culture described in the step (1), and the culture condition that carries out suspension culture described in the step (3), be dark cultivation, temperature is 25-28 ℃.
7. arbitrary described method according to claim 1-6 is characterized in that: in the step (2), the subculture frequency of described succeeding transfer culture is: first three time subculture, and per 20 ± 2 days subcultures are once; From the 4th subculture, per 15 ± 2 days subcultures once.
8. be used for setting up the substratum of salicornia europaeal suspension cell line, for following (A) or (B):
(A) suspension medium, for following a) or b):
A) be in the MS liquid nutrient medium, to add the substratum that obtains behind TDZ and the NAA; The final concentration of TDZ is that the final concentration of 1.5-2.5mg/L, NAA is 0.1-0.3mg/L in the described suspension medium;
B) be in the MS liquid nutrient medium, to add the substratum that obtains behind TDZ, NAA and the NaCl; The final concentration of TDZ is that the final concentration of 1.5-2.5mg/L, NAA is that the final concentration of 0.1-0.3mg/L, NaCL is 0.1M in the described suspension medium;
(B) substratum group, by inducing culture, subculture medium and suspension medium (A) form;
Described inducing culture is to add the substratum that obtains behind TDZ and the NAA in the MS solid medium; The final concentration of TDZ is that the final concentration of 1.5-2.5mg/L, NAA is 0.1-0.3mg/L in the described inducing culture;
Described subculture medium adds the substratum that obtains behind TDZ and the NAA in the MS solid medium; The final concentration of TDZ is that the final concentration of 1.5-2.5mg/L, NAA is 0.1-0.3mg/L in the described subculture medium.
9. substratum according to claim 8, it is characterized in that: the carbon source in described MS liquid nutrient medium and the described MS solid medium is sucrose; Gelifying agent in the described MS solid medium is plant gel;
The final concentration of described sucrose in described MS liquid nutrient medium and described MS solid medium all is specially 30g/L; The final concentration of described plant gel in described MS solid medium all is specially 3-4g/L.
10. claim 8 or 9 application of described substratum in setting up the salicornia europaeal suspension cell line.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310296841.7A CN103320380B (en) | 2013-07-16 | 2013-07-16 | A kind of method setting up salicornia suspension cell line |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310296841.7A CN103320380B (en) | 2013-07-16 | 2013-07-16 | A kind of method setting up salicornia suspension cell line |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103320380A true CN103320380A (en) | 2013-09-25 |
| CN103320380B CN103320380B (en) | 2015-09-30 |
Family
ID=49189426
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310296841.7A Expired - Fee Related CN103320380B (en) | 2013-07-16 | 2013-07-16 | A kind of method setting up salicornia suspension cell line |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103320380B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107494261A (en) * | 2017-07-17 | 2017-12-22 | 暨南大学 | A kind of cultural method for establishing cabbage heart suspension cell line |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101531991B (en) * | 2009-04-17 | 2012-05-23 | 北京林业大学 | Rhodida plant callus and suspension cell granule culture method |
-
2013
- 2013-07-16 CN CN201310296841.7A patent/CN103320380B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101531991B (en) * | 2009-04-17 | 2012-05-23 | 北京林业大学 | Rhodida plant callus and suspension cell granule culture method |
Non-Patent Citations (3)
| Title |
|---|
| 余桂红等: "海蓬子愈伤组织的诱导剂植株再生", 《核农学报》, 31 December 2010 (2010-12-31), pages 269 - 275 * |
| 史秀玲等: "盐角草体外再生体系的建立", 《2005年全国作物生物技术与诱变技术学术研讨会论文摘要集》, 31 December 2006 (2006-12-31), pages 122 * |
| 王彦文: "桑树叶片愈伤组织的诱导及不定芽分化影响因素的研究", 《蚕业科学》, vol. 32, no. 2, 31 December 2006 (2006-12-31), pages 157 - 160 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107494261A (en) * | 2017-07-17 | 2017-12-22 | 暨南大学 | A kind of cultural method for establishing cabbage heart suspension cell line |
| CN107494261B (en) * | 2017-07-17 | 2019-10-15 | 暨南大学 | A kind of cultural method for establishing cabbage heart suspension cell line |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103320380B (en) | 2015-09-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104472359B (en) | A kind of ginseng adventitious root proliferative induction method | |
| KR20160045149A (en) | System and methods for continuous propagation and mass production of arbuscular mycorrhizal fungi in liquid culture | |
| CN105494098B (en) | A kind of method of quick breeding tuniclike psammosilene root seedling | |
| CN108401902B (en) | Rapid propagation method for tissue culture of hemp stem tip | |
| CN103168693B (en) | Proliferation culture medium for aristolochia longgonensis tissue culture and application of proliferation culture medium | |
| CN102134561B (en) | Culture medium used for fast breeding tissue-culture root-shaped stems of nervilia fordii | |
| CN105941149A (en) | Castanea mollissima somatic embryo induction method | |
| CN103270946A (en) | Dendrobium officinale seedling production and hardening synchronization method | |
| CN104996304B (en) | Culture medium and culture method for inducing callus differentiation through peony leaves | |
| CN103583357B (en) | Method for sterile seeding of lithops and establishing regeneration system | |
| CN100333634C (en) | Method for cultivating oriental lily test tube bulb | |
| CN107372122B (en) | Gum tree embryonic callus induced medium and gum tree embryonic callus quick proliferation method | |
| CN103320380B (en) | A kind of method setting up salicornia suspension cell line | |
| CN101824408A (en) | Cultivating method of new species of anti-late blight and anti-beetle potato | |
| CN105028203A (en) | Method for promoting Chinese cabbage microspore embryogeny and direct seedling development | |
| CN112868527A (en) | Method for rapidly propagating flamingo pepper grass | |
| CN105794647A (en) | Method for culturing Brassica rapa microspores with sodium butyrate-NaB as histone deacetylase inhibitor | |
| CN102165920A (en) | Tissue culturing method for regenerating somatic cells of aquatic iris | |
| CN104351058A (en) | Method for inducing sugarcane embryoids by spermine | |
| CN100444720C (en) | Process for expelling Danshen virus by using finely cellular agglomerate regeneration method | |
| CN109880788A (en) | The cabbage type rape protoplast electrofusion and genetic transforming method and regenerating system used not limited by genotype | |
| CN104630159A (en) | Method for producing hog cholera C-strain virus by culturing ST Cells in low serum | |
| CN105830922A (en) | A method of promoting napa cabbage microspore embryogeny and direct production of plantlets | |
| CN106119276A (en) | A kind of method setting up agriculture bacillus mediated Herba Erigerontis high-efficiency transfection system | |
| CN103238518B (en) | Method for induction and subculture maintenance on jasmine flower calluses |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20150930 Termination date: 20190716 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |