CN103320380B - A kind of method setting up salicornia suspension cell line - Google Patents
A kind of method setting up salicornia suspension cell line Download PDFInfo
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Abstract
The invention discloses a kind of method setting up salicornia suspension cell line.The method setting up salicornia suspension cell line provided by the present invention comprises the steps: that salicornia europaeal explant is placed on inducing culture by (1) and carries out inducing culture, obtains callus 1; (2) described callus 1 is placed on subculture medium carries out succeeding transfer culture more than 5 times, obtain callus 2; (3) described callus 2 is placed in suspension medium and carries out suspension concussion cultivation, thus obtain salicornia suspension cell line.Method provided by the present invention is utilized to establish salicornia suspension cell line first, this is not only conducive to deeply resolving salicornia europaeal Mechanism of Salt-tolerant from cytology angle, for the gene transformation of salicornia europaeal and genetic modification have established technical foundation, also for the gene function checking of salicornia europaeal provides a new possible approaches.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of method setting up salicornia suspension cell line.
Background technology
Cultivation in the world for plant suspension cell is existing a lot, and 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, the secondary metabolite (seasonings, essence etc.) that synthesis is relevant.At present, culture technique about suspension cell has a lot of section bibliographical information, various model plant suspension cell is wherein not only had 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.The gingkgo callus such as Cao Fuliang sets up ginkgo suspension cell line (2008); Li Xiangyun etc. disclose a kind of E. wushanense suspended cell culture system and establishment method thereof 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 waits quietly having delivered a kind of technique (2006) improving American ginseng suspending cell yield.But up to now, also do not have 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, is extensively distributed near coastal and inland brine lake, is considered to a kind of higher plant of most salt tolerant in the world.Set up the suspension cell line of salicornia europaeal, be not only conducive to deeply resolving salicornia europaeal Mechanism of Salt-tolerant from cytology angle, for the gene transformation of salicornia europaeal and genetic modification have established technical foundation, also for the gene function checking of salicornia europaeal provides a new possible approaches.
Summary of the invention
The object of this invention is to provide a kind of method and the special culture media thereof of setting up salicornia suspension cell line.
The method setting up salicornia suspension cell line provided by the present invention, can comprise the steps:
(1) salicornia europaeal explant is placed on inducing culture carries out inducing culture, obtain callus 1(khaki);
(2) described callus 1 is placed in subculture medium carries out succeeding transfer culture more than 5 times (as 5 times), obtain callus 2(light yellow loose watery);
(3) described callus 2 is placed in suspension medium and carries out suspension concussion cultivation, thus obtain salicornia suspension cell line;
Described suspension medium be following a) or b):
A) be the substratum obtained add TDZ and NAA in MS liquid nutrient medium after; In described suspension medium, the final concentration of TDZ is that 1.5-2.5mg/L(is as 2.0mg/L), the final concentration of NAA is that 0.1-0.3mg/L(is as 0.1mg/L);
B) be the substratum obtained add TDZ, NAA and NaCl in MS liquid nutrient medium after; In described suspension medium, the final concentration of TDZ is that 1.5-2.5mg/L(is as 2.0mg/L), the final concentration of NAA is that 0.1-0.3mg/L(is as 0.1mg/L), the final concentration of NaCL is 0.1M.
In the above-mentioned methods, in step (1), described salicornia europaeal explant is placed in inducing culture carries out 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: described salicornia europaeal explant is first placed in solution A soak 1 minute, then go in solution B soak 15 minutes, then use aseptic water washing 7-8 time.Described solution A to be volume fraction be 75% aqueous ethanolic solution; The solvent of solution B is water, solute and concentration thereof to be volume fraction be 2% NaClO and volume fraction be 1.5% Triton X-100.
In the above-mentioned methods, in step (3), described " described callus 2 is placed in suspension medium ", specifically can be N(N is natural number, as 15) block diameter 0.8-1.2cm as described in callus 2 be placed in 10 × N ml(as 150mL) as described in suspension medium.
In the present invention, in step (3), the method for described " carry out suspension concussion to cultivate, thus obtain salicornia suspension cell line ", specifically comprises the steps:
(I) described callus 2 is placed in described suspension medium, with 100-140 turn/min(is as 120 turns/min) rotating speed concussion cultivation 12 ± 2 days, obtain cell suspending liquid;
(II) in step (I) gained cell suspending liquid, add isopyknic described suspension medium, then with 100-140 turn/min(is as 120 turns/min) rotating speed concussion cultivate 7-12 days (as 10 days), obtain cell suspending liquid to be filtered;
(III) described cell suspending liquid to be filtered 40 mesh sieve are filtered, by filtrate and the mixing of described suspension medium, the cell quantity in mixed solution is made to be that 9-53/μ L(is as 12-53/μ L, as 14-40/μ L, as 14-27, for another example 23-27/μ L), then with 100-140 turn/min(is as 120 turns/min) rotating speed concussion cultivate 7-12 days (as 10 days), obtain filtering culturing cell suspension again;
(IV) repeating step (III) 3-5 time (as 4 times), for each repetitive operation, all using the filtration of gained in a front operation steps (III) again culturing cell suspension operate as cell suspending liquid to be filtered, last gained filters culturing cell suspension again and is salicornia suspension cell line.
In the above-mentioned methods, in step (III), described " by filtrate and the mixing of described suspension medium, make the cell concn in mixed solution be 9-53/μ L(as 12-53/μ L, as 14-40/μ L; as 14-27; 23-27/μ L for another example) ", be specially by described filtrate (cell concn is 70-80/μ L) and described suspension medium according to volume ratio be 1:7 to 2:1(as 1:5 to 2:1, as 1:4 to 1:1, as 1:4 to 1:2,1:2 for another example) ratio mixing.
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 being less than cell number in the cell mass of 10 cells and free single cell, namely only to being less than the cell mass of 10 cells or unicellular counting, the cell number be less than in the cell mass of 10 cells calculates strictly according to the facts, counts total number of cells.
In step (I)-(IV) of aforesaid method, the described rotation radius rotating concussion cultivation is 13mm.
In the above-mentioned methods, described inducing culture is the substratum obtained add TDZ and NAA in MS solid medium after; In described inducing culture, the final concentration of TDZ is that 1.5-2.5mg/L(is as 2.0mg/L), the final concentration of NAA is that 0.1-0.3mg/L(is as 0.1mg/L);
The substratum that described subculture medium obtains add TDZ and NAA in MS solid medium after; In described subculture medium, the final concentration of TDZ is that 1.5-2.5mg/L(is as 2.0mg/L), the final concentration of NAA is that 0.1-0.3mg/L(is as 0.1mg/L).
In each substratum involved by aforesaid method, the carbon source in described MS liquid nutrient medium and described MS solid medium is specially sucrose; Gelifying agent in 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 is all specially 30g/L; The final concentration of described plant gel in described MS solid medium is all specially 3-4g/L.
In the present invention, described salicornia europaeal explant involved in aforesaid method is the seed of salicornia europaeal, is specially the salicornia europaeal seed peeling shell off.
Described in the step (1) of aforesaid method, carry out the culture condition carrying out succeeding transfer culture described in the culture condition of inducing culture, step (2), and the condition of carrying out suspension culture described in step (3) is light culture, temperature is 25-28 DEG C.
In the step (1) of aforesaid method, described in carry out inducing culture culture cycle 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 subculture, and every 20 ± 2 days subcultures once; From the 4th subculture, every 15 ± 2 days subcultures once.
Substratum for setting up salicornia suspension cell line provided by the present invention, is specially following (A) or (B):
(A) suspension medium, for following a) or b):
A) be the substratum obtained add TDZ and NAA in MS liquid nutrient medium after; In described suspension medium, the final concentration of TDZ is that 1.5-2.5mg/L(is as 2.0mg/L), the final concentration of NAA is that 0.1-0.3mg/L(is as 0.1mg/L);
B) be the substratum obtained add TDZ, NAA and NaCl in MS liquid nutrient medium after; In described suspension medium, the final concentration of TDZ is that 1.5-2.5mg/L(is as 2.0mg/L), the final concentration of NAA is that 0.1-0.3mg/L(is as 0.1mg/L), the final concentration of NaCL is 0.1M;
(B) substratum group, is made up of suspension medium described in inducing culture, subculture medium and (A);
Described inducing culture is the substratum obtained add TDZ and NAA in MS solid medium after; In described inducing culture, the final concentration of TDZ is that 1.5-2.5mg/L(is as 2.0mg/L), the final concentration of NAA is that 0.1-0.3mg/L(is as 0.1mg/L);
The substratum that described subculture medium obtains add TDZ and NAA in MS solid medium after; In described subculture medium, the final concentration of TDZ is that 1.5-2.5mg/L(is as 2.0mg/L), the final concentration of NAA is that 0.1-0.3mg/L(is as 0.1mg/L).
In above-mentioned each substratum, the carbon source in described MS liquid nutrient medium and described MS solid medium specifically can be sucrose; Gelifying agent in 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 is all specially 30g/L; The final concentration of described plant gel in described MS solid medium is all specially 3-4g/L.
The above substratum also belongs to protection scope of the present invention setting up the application in salicornia suspension cell line.
The pH of described MS liquid nutrient mediums all is above 5.8-5.9, and solvent is water, solute and concentration 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. trace: 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.
Each concentration is the final concentration of respective components in described MS liquid nutrient medium above.
Described MS solid mediums all is above that in described MS liquid nutrient medium, add final concentration be the solid medium obtained after the plant gel of 3-4g/L.
The present invention establishes salicornia suspension cell line first, not only be conducive to deeply resolving salicornia europaeal Mechanism of Salt-tolerant from cytology angle, for the gene transformation of salicornia europaeal and genetic modification have established technical foundation, also for the gene function checking of salicornia europaeal provides a new possible approaches.
Accompanying drawing explanation
Fig. 1 is the comparison that different states callus carries out suspension culture.Wherein, A is the callus of subculture twice; B is the callus of subculture three times; C is the callus of subculture five times; D is the suspension cell line of the Callus formation of subculture three times; E is the suspension cell line of the Callus formation of subculture five times.
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 in statistics 4 kinds of suspension mediums (n=3, under identical lowercase alphabet is shown in p < 0.05 condition, does not exist significant difference).In E, 1 represents suspension medium 1; 2 represent suspension medium 2; 3 represent suspension medium 3; 4 represent suspension medium 4.
Fig. 3 is that different subinoculation amount is on the value-added impact of salicornia europaeal suspension cell.Wherein, A is be the ratio subinoculation of 2:1 according to filtrate and suspension medium volume ratio; B is be the ratio subinoculation of 1:1 according to filtrate and suspension medium volume ratio; C is be the ratio subinoculation of 1:2 according to filtrate and suspension medium volume ratio; D is be the ratio subinoculation of 1:4 according to filtrate and suspension medium volume ratio; E is be the ratio subinoculation of 1:5 according to filtrate and suspension medium volume ratio; F is be the ratio subinoculation of 1:7 according to filtrate and suspension medium volume ratio; G is cell concn (n=3, under identical lowercase alphabet is shown in p < 0.05 condition, does not exist significant difference) in statistics 6 kinds different subinoculation amount gained suspension cell line.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
All medicines in following embodiment and consumptive material are purchased in Sigmaaldrich company, and its network address is see as follows:
http://www.sigmaaldrich.com/china-mainland.html。
Salicornia europaeal (S.europaea L.): seed originates from Jiangsu Province of China Dafeng City's beach, is provided by Jiangsu Jinglong Ocean Industry Development Co., Ltd.
The pH of MS liquid nutrient medium involved in following embodiment is 5.8-5.9, 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 doubly micro-mother liquor: KI1.66mg/L, 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.
Each concentration is the final concentration of respective components in described MS liquid nutrient medium above.
MS solid medium involved in following embodiment is that in described MS liquid nutrient medium, add final concentration be the solid medium obtained after the plant gel of 3-4g/L.
The acquisition of embodiment 1, salicornia suspension cell line and qualification
The present embodiment for explant, through induction of callus, succeeding transfer culture and suspension culture, finally sets up salicornia suspension cell line with salicornia europaeal seed.Wherein, the present inventor is to the composition and ratio of hormone in the state of callus, suspension medium, and the subinoculation amount in suspension culture process is optimized.Specific as follows:
One, callus status is on the impact of salicornia suspension cell line
(1) experimental technique
1, the sterilization of explant
Get salicornia europaeal seed, peel shell off, be first placed in solution A soak 1 minute, then go in solution B soak 15 minutes, then use aseptic water washing 7-8 time, complete sterilization, for subsequent use.
Solution A: volume fraction is the aqueous ethanolic solution of 75%;
Solution B: solvent is water, solute and concentration thereof to be volume fraction be 2% NaClO and volume fraction be 1.5% Triton X-100.
2, the induction of callus
Inducing culture (MS+TDZ 2.0mg/L+NAA 0.1mg/L): the substratum obtained add TDZ and NAA in MS solid medium after; In described inducing culture, the final concentration of TDZ is the final concentration of 2.0mg/L, NAA is 0.1mg/L.
The salicornia europaeal seed of step 1 being sterilized is placed on inducing culture, and at 25-28 DEG C, light culture 20-30 days (according to seed vitality, needing time difference), until occur khaki callus in hypocotylar bottom.
3, callus subculture and expand numerous
Subculture medium (MS+TDZ 2.0mg/L+NAA 0.1mg/L): the substratum obtained add TDZ and NAA in MS solid medium after; In described subculture medium, the final concentration of TDZ is the final concentration of 2.0mg/L, NAA is 0.1mg/L.
Callus step 2 induced cuts from hypocotyl low side, is placed on subculture medium, and allow callus proliferation expand numerous, culture condition is: light culture, and temperature is 25-28 DEG C.After callus grows up (diameter is about 0.5cm), subculture medium constantly breeds callus, during first three subculture, once, from the 4th subculture, every about 15 days subcultures once for every about 20 days subcultures.Select subculture second time respectively, the callus (diameter about 0.8-1.2cm) for the third time and after the 5th time, for studying the impact of callus status on salicornia suspension cell line.
4, the acquisition of suspension cell line
Suspension medium (MS+TDZ 2.0mg/L+NAA 0.1mg/L): the substratum obtained add TDZ and NAA in MS liquid nutrient medium after; In described suspension medium, the final concentration of TDZ is the final concentration of 2.0mg/L, NAA is 0.1mg/L.
1) callus (diameter about 0.8-1.2cm) of three kinds of different states that step 3 is chosen is got, 15 pieces often kind, often kind of callus is corresponding respectively puts into three culturing bottles containing 150ml suspension medium, in 25-28 DEG C, under dark condition, concussion cultivation about 12 days, shaking speed 120r/min(rotation radius is 13mm).
2) the concussion cell suspending liquid of cultivation after 12 days is added isopyknic suspension medium, in 25-28 DEG C, under dark condition, then with 120r/min(rotation radius for 13mm) rotating speed concussion cultivation 10 days.
3) after 10 days, filter callus lines by 40 mesh sieve, filtrate and suspension medium mixed according to the volume ratio of 1:2, in 25-28 DEG C, under dark condition, then with 120r/min(rotation radius for 13mm) rotating speed concussion cultivation 10 days.
4) repeating step 3) 4 times, obtain three parts of candidate's suspension cell lines.
5) by OLYMPUS microscope, step 4) gained three parts of candidate's suspension cell lines are observed.
(2) experimental result
Select subculture second time respectively, the callus for the third time and after the 5th time, carries out inoculation with the callus lines of equivalent and suspension culture compares.As shown in Figure 1, when callus is cut from hypocotyl, during subculture twice, also harder, color deep yellow (in Fig. 1 A); When callus subculture third time, entirety is still hard, and color becomes faint yellow (in Fig. 1 B) simultaneously; When callus subculture the 5th time, entirety becomes soft, and color becomes light yellow (in Fig. 1 C) simultaneously.Carry out suspension culture to these three kinds of callus, the first callus (in subculture twice, Fig. 1 A) cannot form suspension cell line completely; For the second callus (subculture three times, B in Fig. 1), although suspension amplification can be carried out, because too hard, simple cell mass cannot be formed, only have macrobead repeatedly cultivating in wild Oryza species, be difficult to form suspension culture system (in Fig. 1 D); For the third callus (subculture five times, C in Fig. 1), by after succeeding transfer culture several times, homogeneous suspension culture system (in Fig. 1 F) can be formed.
Visible, different callus status is set up salicornia 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 for setting up salicornia suspension cell line.
Two, the hormone composition and ratio in suspension medium is on the impact of salicornia suspension cell line
(1) experimental technique
1, the sterilization of explant
With step one 1.
2, the induction of callus
With step one 2.
3, callus subculture and expand numerous
Concrete operations are see step one 3, and difference is only to select the callus (diameter about 0.8-1.2cm) after subculture the 5th time, sets up salicornia suspension cell line for step 4.
4, the acquisition of suspension cell line
Following four kinds of suspension mediums are set, for studying hormone composition and ratio in suspension medium to the impact of salicornia suspension cell line:
Suspension medium 1(MS): MS liquid nutrient medium.
Suspension medium 2(MS+2,4-D 1.5mg/L): the substratum obtained add 2,4-D in MS liquid nutrient medium after; In described suspension medium 2, the final concentration of 2,4-D is 1.5mg/L.
Suspension medium 3(MS+TDZ 2.0mg/L+NAA 0.1mg/L): the substratum obtained add TDZ and NAA in MS liquid nutrient medium after; In described suspension medium 3, the final concentration of TDZ is the final concentration of 2.0mg/L, NAA is 0.1mg/L.
Suspension medium 4(MS+TDZ 2.0mg/L+NAA 0.1mg/L+NaCl0.1M): the substratum obtained add TDZ, NAA and NaCl in MS liquid nutrient medium after; In described suspension medium 4, the final concentration of the final concentration of TDZ to be the final concentration of 2.0mg/L, NAA be 0.1mg/L, NaCl is 0.1M.
Concrete operations are as follows:
1) above 4 parts of suspension mediums are put into 4 culturing bottles, 150ml/ bottle respectively.Get the callus (diameter about 0.8-1.2cm) after subculture that step 3 chooses the 5th time, 4 parts, 15 pieces every part.A callus is put into for every bottle in 4 culturing bottles, in 25-28 DEG C, under dark condition, concussion cultivation about 12 days, shaking speed 120r/min(rotation radius is 13mm).
2) the concussion cell suspending liquid of cultivation after 12 days is added isopyknic corresponding suspension medium, in 25-28 DEG C, under dark condition, then with 120r/min(rotation radius for 13mm) rotating speed concussion cultivation 10 days.
3) after 10 days, filter callus lines by 40 mesh sieve, filtrate and corresponding suspension medium mixed according to the volume ratio of 1:2, in 25-28 DEG C, under dark condition, then with 120r/min(rotation radius for 13mm) rotating speed concussion cultivation 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 OLYMPUS microscope, step 4) gained four parts of candidate's suspension cell lines are observed.Cell concn in 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 being less than cell number in the cell mass of 10 cells and free single cell, namely only to being less than the cell mass of 10 cells or unicellular counting, the cell number be less than in the cell mass of 10 cells calculates strictly according to the facts, counts total number of cells.Test in triplicate, results averaged.
(2) experimental result
Cell as shown in Figure 2, can be shaken up dispersion with MS substratum (suspension medium 1) by result, but cell amplification is slow, and through succeeding transfer culture, the number of cell in suspension system still maintains A and E in 6/μ l(Fig. 2); Compared with MS substratum, the suspension medium 2 that with the addition of growth hormone (1.5mg/L2,4D) can improve fissional speed, but still value-added slow, B and E of cell concn greatly in 10/μ l(Fig. 2 after succeeding transfer culture).But maintain mitogen concentration far above (suspension medium 3) after when growth hormone when interpolation phytokinin (TDZ), cell number has the increment of highly significant, and after succeeding transfer culture, cell concn can reach C and E in 74/μ l(Fig. 2).For the suspension system setting up halophytes, the applying effect of NaCl is also Consideration, and therefore, the present inventor, on the basis at suspension medium 3, with the addition of 0.1M NaCl, investigates NaCl for the value-added impact of suspension cell.Result is as D and E in Fig. 2, and after finding to be applied with the NaCl of 0.1M, cell proliferation does not increase significantly, and after succeeding transfer culture, cell number is 76/μ l.
In sum, groping by hormone combination, the suspension medium finally determining to be applicable to setting up salicornia suspension cell line is suspension medium 3, i.e. MS+TDZ 2.0mg/L+NAA 0.1mg/L.
Three, the subinoculation amount in suspension culture process is on the impact of salicornia suspension cell line
(1) experimental technique
1, the sterilization of explant
With step one 1.
2, the induction of callus
With step one 2.
3, callus subculture and expand numerous
With step 23.
4, the acquisition of suspension cell line
Suspension medium (MS+TDZ 2.0mg/L+NAA 0.1mg/L): the substratum obtained add TDZ and NAA in MS liquid nutrient medium after; In described suspension medium, the final concentration of TDZ is the final concentration of 2.0mg/L, NAA is 0.1mg/L.
Concrete operations are as follows:
1) callus (diameter about 0.8-1.2cm) after subculture that 15 pieces of steps 3 choose the 5th time is got, put into the culturing bottle that 150mL suspension medium is housed, in 25-28 DEG C, under dark condition, concussion cultivation about 12 days, shaking speed 120r/min(rotation radius is 13mm).
2) the concussion cell suspending liquid of cultivation after 12 days is added isopyknic suspension medium, in 25-28 DEG C, under dark condition, then with 120r/min(rotation radius for 13mm) rotating speed concussion cultivation 10 days.
3) after 10 days, filter callus lines, obtain filtrate by 40 mesh sieve, in filtrate, cell concn is that 70-80/μ L(measuring method is shown in step 5) 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 is equal to obtain 6 portions of mixed solutions, be 200mL), in 25-28 DEG C, under dark condition, then with 120r/min(rotation radius for 13mm) rotating speed concussion cultivation 10 days.
Wherein, according to filtrate and the descending order of suspension medium volume ratio in 6 portions of mixed solutions, 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.
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 OLYMPUS microscope, step 4) gained six parts of candidate's suspension cell lines are observed.Cell concn in 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 being less than cell number in the cell mass of 10 cells and free single cell, namely only to being less than the cell mass of 10 cells or unicellular counting, the cell number be less than in the cell mass of 10 cells calculates strictly according to the facts, counts total number of cells.Test in triplicate, results averaged.
(2) experimental result
Result as shown in Figure 3, is the ratio subinoculation of 1:2 according to filtrate and suspension medium volume ratio, can obtain the maximum suspension cell of unit volume quantity (C and G in Fig. 3).Although according to the ratio inoculation that filtrate and suspension medium volume ratio are 2:1 and 1:1, can see more cell mass (A and B in Fig. 3), but the quantity finally forming suspension cell (being less than cell mass or the free single cell of 10 cells) is but not so good as many (in Fig. 3 the G) of the ratio inoculation according to filtrate and suspension medium volume ratio being 1:2.And inoculate according to the ratio that filtrate and suspension medium volume ratio are 1:4,1:5 and 1:7, the suspension cell concentration of acquisition is too low, is unfavorable for further amplification and correlative study (D, E, F and G in Fig. 3).
In sum, subinoculation amount with new and old substratum ratio for 2:1 add (be namely the ratio subinoculation of 1:2 according to filtrate and suspension medium volume ratio, namely after inoculation, in mixed solution, cell concn is 23-27/μ L), the growth of the most applicable salicornia europaeal suspension cell and increment.
Claims (8)
1. set up a method for salicornia suspension cell line, comprise the steps:
(1) salicornia europaeal explant is placed on inducing culture carries out inducing culture, obtain callus 1;
Described inducing culture is the substratum obtained add TDZ and NAA in MS solid medium after; In described inducing culture, the final concentration of TDZ is the final concentration of 1.5-2.5mg/L, NAA is 0.1-0.3mg/L;
(2) described callus 1 is placed on subculture medium carries out succeeding transfer culture more than 5 times, obtain callus 2;
The substratum that described subculture medium obtains add TDZ and NAA in MS solid medium after; In described subculture medium, the final concentration of TDZ is the final concentration of 1.5-2.5mg/L, NAA is 0.1-0.3mg/L;
(3) described callus 2 is placed in suspension medium and carries out suspension concussion cultivation, thus obtain salicornia suspension cell line;
Described suspension medium be following a) or b):
A) be the substratum obtained add TDZ and NAA in MS liquid nutrient medium after; In described suspension medium, the final concentration of TDZ is the final concentration of 1.5-2.5mg/L, NAA is 0.1-0.3mg/L;
B) be the substratum obtained add TDZ, NAA and NaCl in MS liquid nutrient medium after; In described suspension medium, the final concentration of the final concentration of TDZ to be the final concentration of 1.5-2.5mg/L, NAA be 0.1-0.3mg/L, NaCL is 0.1M;
Described salicornia europaeal explant is the seed of salicornia europaeal.
2. method according to claim 1, is characterized in that: in step (3), described in carry out suspension concussion and cultivate, thus obtain the method for salicornia suspension cell line, comprise the steps:
(I) described callus 2 is placed in described suspension medium, turns/the rotating speed of min concussion cultivation 12 ± 2 days with 100-140, obtain cell suspending liquid;
(II) in step (I) gained cell suspending liquid, add isopyknic described suspension medium, then with 100-140 turn/concussion of the rotating speed of min cultivates 7-12 days, obtains cell suspending liquid to be filtered;
(III) described cell suspending liquid to be filtered 40 mesh sieve are filtered, by gained filtrate and the mixing of described suspension medium, make the cell quantity in mixed solution be 9-53/μ L, then with 100-140 turn/the rotating speed concussion of min cultivates 7-12 days, obtains filtering culturing cell suspension again;
(IV) repeating step (III) 3-5 time, for each repetitive operation, all using the filtration of gained in a front operation steps (III) again culturing cell suspension operate as cell suspending liquid to be filtered, last gained filters culturing cell suspension again and is salicornia suspension cell line.
3. method according to claim 1, is characterized in that: the carbon source in described MS liquid nutrient medium and described MS solid medium is sucrose; Gelifying agent in described MS solid medium is plant gel;
The final concentration of described sucrose in described MS liquid nutrient medium and described MS solid medium is 30g/L; The final concentration of described plant gel in described MS solid medium is 3-4g/L.
4. according to described method arbitrary in claim 1-3, it is characterized in that: the culture condition carrying out carrying out described in the culture condition of inducing culture, step (2) succeeding transfer culture described in step (1), and described in step (3), carry out the culture condition of suspension culture, be light culture, temperature is 25-28 DEG C.
5. according to described method arbitrary in claim 1-3, it is characterized in that: in step (2), the subculture frequency of described succeeding transfer culture is: first three subculture, and every 20 ± 2 days subcultures once; From the 4th subculture, every 15 ± 2 days subcultures once.
6., for setting up the substratum of salicornia suspension cell line, be following (A) or (B):
(A) suspension medium is the substratum obtained add TDZ, NAA and NaCl in MS liquid nutrient medium after; In described suspension medium, the final concentration of the final concentration of TDZ to be the final concentration of 1.5-2.5mg/L, NAA be 0.1-0.3mg/L, NaCL is 0.1M;
(B) substratum group, is made up of suspension medium described in inducing culture, subculture medium and (A);
Described inducing culture is the substratum obtained add TDZ and NAA in MS solid medium after; In described inducing culture, the final concentration of TDZ is the final concentration of 1.5-2.5mg/L, NAA is 0.1-0.3mg/L;
The substratum that described subculture medium obtains add TDZ and NAA in MS solid medium after; In described subculture medium, the final concentration of TDZ is the final concentration of 1.5-2.5mg/L, NAA is 0.1-0.3mg/L.
7. substratum according to claim 6, is characterized in that: the carbon source in described MS liquid nutrient medium and described MS solid medium is sucrose; Gelifying agent in described MS solid medium is plant gel;
The final concentration of described sucrose in described MS liquid nutrient medium and described MS solid medium is 30g/L; The final concentration of described plant gel in described MS solid medium is 3-4g/L.
8. the substratum described in claim 6 or 7 is setting up the application in salicornia suspension cell line.
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