CN108064687A - The method and its application of suspension cell line are obtained using cabbage type rape hypocotyl as explant - Google Patents
The method and its application of suspension cell line are obtained using cabbage type rape hypocotyl as explant Download PDFInfo
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- CN108064687A CN108064687A CN201610986021.4A CN201610986021A CN108064687A CN 108064687 A CN108064687 A CN 108064687A CN 201610986021 A CN201610986021 A CN 201610986021A CN 108064687 A CN108064687 A CN 108064687A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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Abstract
The invention belongs to plant cell culture technology fields.More particularly to the method and its application that suspension cell line is obtained using cabbage type rape hypocotyl as explant.The present invention is using the sterile hypocotyl of cabbage type rape as explant, pass through the induction of embryo callus, the culture of suspension cell and etc. establish cabbage type rape suspension cell line, and the form and activity difference of required micronutrient element suspension cell normally and under the conditions of lacking are compared using the architectural study.Cabbage type rape suspension cell activity prepared by the present invention is high, form stable is homogeneous, cultivation cycle is short, from the limitation of the factor of natural environment such as season, weather, the research and application of cell growth, form pattern, nutrient transmembrane transport, Physiological and Biochemical Metabolism and gene expression etc. available for cell development on cabbage type rape individual cell level and under abiotic stress.
Description
Technical field
The invention belongs to plant cell culture technology fields, and in particular to be obtained by explant of cabbage type rape hypocotyl
The method and its application of suspension cell line.
Background technology
Cabbage type rape (Brassica napus L.), Cruciferae are one of most important oil crops in the world,
The fifth-largest crop and the first big oil crops after rice, corn, wheat, soybean are become in China, have planted throughout the year
About 6,700,000 hm of area2, it is distributed widely in China Yangtze river basin, total yield is more than 1 100 ten thousand t, produces about 4,500,000 t of rapeseed oil per year, accounts for
More than the 40% of self-produced vegetable oil total amount accounts for more than the 57% of herbaceous plant oil total amount.Traditionally to the research of cabbage type rape
Plant is all based on, however the complexity of plant in itself causes progress slow, suspension cell avoids plant as unicellular
The whole complexity of strain.In recent years, unicellular research is used widely on other species, however cabbage type rape still lacks
A kind of efficient, easy unicellular culture technique.
Suspension cell is used as one kind is unicellular to get the attention, traditional cabbage type rape plant cell suspension cultures
Orr W., Keller W.A.and Singh J.1986, the method for acquisition is the (J.Plant obtained using microspores culture
Physiol.126:23-32), the suspension cell line obtained followed by the method for microspores culture promotes to a certain extent
Cabbage type rape research (Carswell, M.C., Grant, B.R.and Plaxton, W.C., 1997.Planta, 203 (1),
pp.67-74；Moraes,T.F.and Plaxton,W.C.,2000.European Journal of Biochemistry,
267(14),pp.4465-4476；Palma,D.A.,Blumwald,E.and Plaxton,W.C.,2000.FEBS
letters,486(2),pp.155-158；Smith,C.R.,Knowles,V.L.and Plaxton,W.C.,
2000.European Journal of Biochemistry,267(14),pp.4477-4485；Plaxton,W.C.,
Smith,C.R.and Knowles,V.L.,2002.Archives of biochemistry and biophysics,400
(1),pp.54-62；Singh,V.K.,Wood,S.M.,Knowles,V.L.and Plaxton,W.C.,2003.Planta,
218 (2), pp.233-239.), but had the disadvantage that using the suspension cell line that the method for microspores culture obtains：(1)
It is easily restricted by materials season, cabbage type rape is bloomed once every year, it is impossible to sampling culture at any time；(2) pollen will be into before culture
Row microscopy is inconvenient for operation to determine suitable Pollen stage, and easily misses the preferably period of pollen development；(3) parent
The physiological status of plant has a direct impact calli induction frequencies, in climatic environment unstable season, rape microspore
Callus induction rate has notable difference.These shortcomings seriously constrain the research of cabbage type rape individual cell level, therefore, invention one
The method of kind acquisition cabbage type rape suspension cell line easy to operate, from season and envirment factor limitation is necessary.
The method that emerging cabbage type rape plant cell suspension cultures obtain gets down to the method using indoor tissue-cultured seedling,
Attempt using cabbage type rape hypocotyl for explant obtain suspension cell line (Wang Huoyan etc., 2002, plant nutrient with
Fertilizer journal.8(1)：100~104；Yang Yuhua etc., 2002, Agricultural College of Hubei Prov. journal .22 (4)：289-292；Yang Lu, 2012
Year, Hua Zhong Agriculture University's master's thesis；Hua, Y etc., 2016, Plant, cell＆environment, 39 (7),
P.1601.), it is inconvenient for operation and the shortcomings that easily limited by season and envirment factor to overcome above-mentioned microspores culture, but from
From the point of view of disclosed article, the cell line cell degree of scatter that this method is established is poor, severe deformities, and vigor is extremely low, it is difficult to be wild cabbage
The individual cell level research of type rape provides excellent biomaterial.
It there is no at present and successfully obtain suspension cell line using cabbage type rape hypocotyl for explant culture callus
Report, it is therefore necessary to a kind of suspension cell line for overcoming the above problem is established in cabbage type rape, is that cabbage type rape is slender
The research of born of the same parents' level provides new way.
The content of the invention
Cabbage type rape hypocotyl is utilized as explant it is an object of the invention to overcome the deficiencies of the prior art and provide a kind of
The method that body obtains suspension cell line.The cabbage type rape hypocotyl that the present invention utilizes can be directly obtained by tissue cultures, be led to
The approach for crossing callus induction, subculture and suspension cell culture obtains cabbage type rape suspension cell line.The method of the present invention
Easy to operate from the influence of the environmental factors such as season, weather, cell degree of scatter is good, and form is normal, and activity is higher, is one
The efficient cabbage type rape plant cell suspension cultures of kind.
It is described that technical scheme is as follows：
One kind obtains the side of suspension cell line using cabbage type rape (Brassica napus L.) hypocotyl as explant
Method comprises the following steps：
(1) disinfection of explant：The cabbage type rape seed of clear water soaked overnight is poured into triangular flask, adds 75% wine
Essence sterilizing 30s, then sterilized 10min with 0.1% mercury chloride, with aseptic water washing 4~5 times, by the cabbage type rape after sterilizing
Seed is seeded on sowing culture medium, and the sowing culture medium is basic culture medium using B5 medium, add 30g/L sucrose with
10g/L agar, supplement distilled water adjust the pH to 5.8-5.9 of culture medium, light culture 5-7 days at 24 DEG C, acquisition Wild cabbage type to 1L
Rape aseptic seedling；
(2) induction of callus：The hypocotyl of cabbage type rape aseptic seedling obtained by step (1) is cut into 0.5~1cm stems
Section is inoculated on callus inducing medium, and secretly induction obtains the callus of cabbage type rape at 24 DEG C；
(3) subculture of callus：The cabbage type rape callus of 30 ages in days is rejected to the callus group of browning and aging
It knits, is transferred on callus subculture medium, the light culture at 24 DEG C, squamous subculture 1~2 time, each subculture 14-16 days；
(4) foundation of suspension cell line：By color is yellowish, open-textured embryo callus is transferred to suspension cell training
It supports on base, the bottled 30mL suspension cell cultures base of triangle per 150mL, every bottle of inoculation yellowish and open-textured embryo of 1g colors
Callus, at 25 DEG C, rotating speed obtains suspension cell line to be cultivated under conditions of 120r/min and light culture；
Wherein：
The preparation of callus inducing medium described in step (2)：Using B5 medium as basic culture medium, add
The sucrose of the 6-BA of 2,4-D, the 0.5mg/L of 2.0mg/L, 30g/L, 0.1g/L citric acids, the caseinhydrolysate of 300mg/L,
The L-PROLINE of 500mg/L and 8g/L agar, supplement ultra-pure water adjust the pH to 5.8-5.9 of culture medium to 1L；
The preparation of callus subculture medium described in step (3)：Using B5 medium as basic culture medium, add
The sucrose of the 6-BA of 2,4-D, the 0.5mg/L of 2.0mg/L, 30g/L, 0.1g/L citric acids, the caseinhydrolysate of 300mg/L,
The L-PROLINE of 500mg/L and 8g/L agar, supplement ultra-pure water adjust the pH to 5.8-5.9 of culture medium to 1L；
The preparation of suspension cell culture base described in step (4)：Using the B5 medium of improvement as basic culture medium, ingredient
For：Calcium chloride dihydrate 440mg/L, potassium dihydrogen phosphate 170mg/L, magnesium sulfate 370mg/, potassium chloride 2940mg/L, potassium iodide
0.83mg/L, CoCL2 6H2O 0.025mg/L, boric acid 6.2mg/L, Sodium Molybdate Dihydrate 0.25mg/L, manganese sulfate monohydrate 16.9mg/
L, cupric sulfate pentahydrate 0.025mg/L, white vitriol 8.6mg/L, inositol 100mg/L, thiamine hydrochloride 0.5mg/L, ethylenediamine
Tetraacethyl disodium 37.25mg/L, ferrous sulfate heptahydrate 27.85mg/L add the L- asparagus fern ammonia of 2,4-D, the 266mg/L of 3mg/L
Acid, the L-Glutamine of 877mg/L, the L-arginine of 228mg/L, the glycine of 75mg/L, the sucrose of 25g/L, 0.1g/L's
Citric acid, supplement ultra-pure water adjust the pH to 5.8-5.9 of culture medium to 1L.
It applicant provides a kind of suitable for being obtained using cabbage type rape (Brassica napus L.) hypocotyl as explant
The culture medium of suspension cell line is obtained, the formula of the culture medium is as described below：
(1) culture medium is sowed：Using B5 medium as basic culture medium, 30g/L sucrose and 10g/L agar are added, supplement is steamed
Distilled water adjusts the pH to 5.8-5.9 of culture medium to 1L；
(2) callus inducing medium：Using B5 medium as basic culture medium, the 2,4-D of 2.0mg/L is added,
The sucrose of the 6-BA of 0.5mg/L, 30g/L, 0.1g/L citric acids, the caseinhydrolysate of 300mg/L, the L-PROLINE of 500mg/L
With 8g/L agar, supplement ultra-pure water adjusts the pH to 5.8-5.9 of culture medium to 1L；
(3) callus subculture medium：Using B5 medium as basic culture medium, the 2,4-D of 2.0mg/L is added,
The sucrose of the 6-BA of 0.5mg/L, 30g/L, 0.1g/L citric acids, the caseinhydrolysate of 300mg/L, the L-PROLINE of 500mg/L
With 8g/L agar, supplement ultra-pure water adjusts the pH to 5.8-5.9 of culture medium to 1L；
(4) suspension cell culture base：Using the B5 medium of improvement as basic culture medium, ingredient is：Calcium chloride dihydrate
440mg/L, potassium dihydrogen phosphate 170mg/L, magnesium sulfate 370mg/, potassium chloride 2940mg/L, potassium iodide 0.83mg/L, six water chlorinations
Cobalt 0.025mg/L, boric acid 6.2mg/L, Sodium Molybdate Dihydrate 0.25mg/L, manganese sulfate monohydrate 16.9mg/L, cupric sulfate pentahydrate
0.025mg/L, white vitriol 8.6mg/L, inositol 100mg/L, thiamine hydrochloride 0.5mg/L, disodium ethylene diamine tetraacetate
37.25mg/L ferrous sulfate heptahydrate 27.85mg/L；The L-Aspartic acid of 2,4-D, the 266mg/L of additional 3mg/L, 877mg/L
L-Glutamine, the L-arginine of 228mg/L, the glycine of 75mg/L, the sucrose of 25g/L, the citric acid of 0.1g/L, supplement
Ultra-pure water adjusts the pH to 5.8-5.9 of culture medium to 1L.
Compared with prior art, beneficial effects of the present invention are as described below：
1st, using cabbage type rape hypocotyl, for explant, to successfully obtain stable suspension thin for the first time in the world by the present invention
Born of the same parents are to provide excellent biologic material resource for the research of cabbage type rape individual cell level.
2nd, explant material of the invention is easily obtained.Suspension cell line is obtained with Microspore of Brassica napus cultivation
Method ratio, advantage are：Cabbage type rape is bloomed once every year, and it is shorter to carry out the time of Anther Culture, and utilizes Wild cabbage type
Rape hypocotyls are explant, only need in triangular flask light culture cabbage type rape seed 5-7 days, hypocotyl can grow to bottle
Mouthful, as explant, from the influence of the natural causes such as season, weather.
4th, operating procedure simplicity is time saving.The method ratio of suspension cell line is obtained with Microspore of Brassica napus cultivation, it is excellent
Gesture is：Pollen will carry out microscopy to determine suitable Pollen stage before culture, inconvenient for operation, and easily miss pollen
Development preferably period.And the cabbage type rape hypocotyl of the present invention is utilized as explant evoked callus, it only need to be in culture dish
It is middle that callus is cut into segment, it is sowed on calli induction media.
5th, callus induced efficiency of the present invention is high.The Callus induction rate of this method is up to more than 90%.And microspore callus lures
Conductance there is no statistics to report in cabbage type rape, but the inductivity counted in rice (Wang Lingxian between 1~60%
Deng, 2009, biotechnology, 19 (6)：92-95).
6th, the suspension cell activity that the present invention obtains is high, and dispersion effect is good, and form is normal.With utilizing cabbage type rape before
Hypocotyl is the suspension cell line ratio that explant obtains, and advantage is：The suspension cell cell dispersion effect that forefathers obtain is poor, tight
Weight deformity, activity are extremely low.And the present invention is then good for further acquisition cell dispersion effect, form is normal, and the high suspension of activity is thin
Born of the same parents system proposes specific technical solution.
Description of the drawings
Fig. 1：Using cabbage type rape hypocotyl as explant, obtained cabbage type rape callus after induction 30 days.It is attached
Figure description of symbols：The callus of induction is mostly that color is yellowish, open-textured callus.
Fig. 2：The cabbage type rape that method using cabbage type rape hypocotyl as explant acquisition suspension cell line obtains hangs
The cabbage type rape that floating cell is obtained with method of the forefathers using cabbage type rape hypocotyl as explant acquisition suspension cell line hangs
The comparison of floating cell.Reference sign：A figures in Fig. 2 are to intercept the cabbage type rape that forefathers (Yang Lu, 2012) obtain to suspend
The image of cell；B figures in Fig. 2 are the images for the cabbage type rape suspension cell that the present invention obtains.
Fig. 3：The suspension cell that method using cabbage type rape hypocotyl as explant acquisition suspension cell line obtains is not
With cell quantity statistics of the boron under horizontal.Reference sign：50,25,10,1,0.25,0.1,0.05 and 0 μM of boron is horizontal
Under cell quantity, wherein the cell number of 50 μM of boron under horizontal is set to 100.
Fig. 4：Suspension cell (the culture of the method acquisition of suspension cell line is obtained using cabbage type rape hypocotyl as explant
12 days) microscopy result.Reference sign：Cell exists mostly in the form of unicellular in system, and a figures in Fig. 4 are sufficient in boron
Cellular morphology under the conditions of (50 μM of boron)；B figures in Fig. 4 are the cellular morphologies under the conditions of boron deficiency (0.25 μM of boron).Mark
Ruler is 100 μm." B " in " the B concentration " of Fig. 4 abscissas is the abbreviation of boron.
Fig. 5：Suspension cell (the culture of the method acquisition of suspension cell line is obtained using cabbage type rape hypocotyl as explant
12 days) result of Activity determination.Reference sign：A figures in Fig. 5 are the cytoactives under the conditions of boron sufficient (50 μM of boron)
Detection；B figures in Fig. 5 are the Activity determinations under the conditions of boron deficiency (50 μM of boron).Scale is 100 μm.
Specific embodiment
Influence of the 1 hormon concentration combination of embodiment to cabbage type rape Callus induction rate and Callus morphology
The mode of appearance and physiological status of callus directly influence the quality of the suspension cell line of later stage acquisition.Induction
The key of callus be explant in itself, additional hormone kind and concentration, basal medium and additional organic matter.Wherein hormone
Play a crucial role, 2,4-D for auxin promote cell division, 6-BA is the basic element of cell division, be promote plant into
Enter the factor of conservative state, show as inhibiting cell division more.Cabbage type rape callus is lured in order to study hormone combination
The influence of conductance, the present embodiment have carried out following experiment：
By cabbage type rape variety " blue or green oil 10 " (Inst. of Oil Crops, Chinese Academy of Agriculture of clear water soaked overnight
Qingyuan Guo and teacher Liao Xing give) seed poured into triangular flask, and it is sterilized 30s with 75% alcohol, then is gone out with 0.1% mercury chloride
Bacterium 10min with aseptic water washing 4~5 times, carries out disinfection to explant, the cabbage type rape seed after disinfection is seeded in and is broadcast
Kind culture medium, the culture medium are using B5 medium as on basic culture medium (being shown in Table 1), add 30g/L sucrose and 10g/L agar,
Distilled water is supplemented to 1L, adjusts the pH to 5.8-5.9 of culture medium, the light culture at 24 DEG C obtains cabbage type rape aseptic seedling.
The hypocotyl length of cabbage type rape is treated to bottleneck (5-7 days), in superclean bench, by cabbage type rape aseptic seedling
Hypocotyl be cut into 0.5~1cm stem sections and be inoculated on callus tissue culture base, the calli induction media is using B5 medium as base
Basal culture medium (compared with other culture mediums, it is mainly characterized by containing relatively low ammonium, suitable for the growth of cabbage type rape).It removes
Hormone, and add 30g/L sucrose, 0.1g/L citric acids, 300mg/L caseinhydrolysates, 500mg/L L-PROLINEs, 8g/L fine jades
Fat, pH 5.8-5.9.2,4-D and 6-BA concentration is set to 18 combinations (being shown in Table 2).Light culture 30 days in 24 DEG C of sterile petri dish
Afterwards, the inductivity (being shown in Table 2) of callus is counted.
1 B5 medium formula of table (abbreviation B5 or B5 basal medium)
Influence of the 2 hormon concentration combination of table to cabbage type rape callus induction rate
Influence of the 3 hormon concentration combination of table to cabbage type rape Callus morphology
It is found by the applicant that hormone combination for 2.0mg/L 2,4-D, 0.5mg/L 6-BA and 2.5mg/L 2,4-D,
When 0.5mg/L 6-BA are combined, cabbage type rape Callus induction rate is respectively 97.2,96.8%, is significantly higher than other combinations.Than
Compared with callus status (being shown in Table 3), discovery hormone combination is 2.0mg/L 2, when 4-D, 0.5mg/L 6-BA are combined, Wild cabbage type oil
Dish callus quality is loose, and block is big, and color is yellowish (see Fig. 1), finally definite 2.0mg/L 2,4-D, 0.5mg/L 6-BA
It is combined as optimal combination.
The horizontal influence to cabbage type rape " blue or green oil 10 " suspension cell multiplication of 2 boron of embodiment element
Boron element is the necessary micronutrient element of plant, and cabbage type rape is sensitive to boron deficiency, shows as the colored and unreal diseases that Deng
Shape causes seed Severe Reduction.In order to study under individual cell level, influence of the boron element to cabbage type rape has carried out different boron
Suspension cell culture experiment under concentration.It using the B5 medium (table 4) that improves is basic culture medium that suspending nutrient solution, which is, and
Be with the addition of 3mg/L 2,4-D, 266mg/L L-Aspartic acids, 877mg/L L-Glutamines, 228mg/L L-arginines,
75mg/L glycine, 25g/L sucrose and 0.1g/L citric acids (pH 5.8).1g colors is yellowish, open-textured Wild cabbage type oil
Dish callus is transferred to boron concentration as 50 respectively；25；10；1；0.25；0.1；It is trained on 0.05 and 0 μM of suspension cell culture base
After supporting 12 days, 10 μ L drops of suspension is taken, by calculating the number of cells under same camera lens, to determine cell quantity in glass slide.
The number of cells under 50 μM of B concentration is wherein set to 100, the number of cells under remaining concentration is obtained through conversion such as Fig. 3 result figures 3
Show that cabbage type rape suspension cell growth is significantly influenced by boron deficiency, cell Proliferation is remarkably decreased with the reduction of boron concentration.
3 boron of embodiment element is horizontal to cabbage type rape " blue or green oil 10 " suspension cell form and activity influence
Cabbage type rape suspension cell is in boron normal level (50 μM of boron), the condition of boron deficiency level (0.25 μM of boron)
Under, after cultivating 12 days, cellular morphology is observed with light microscope, with the activity of fluorescence microscope cell.Suspension cell
Active diacetic acid fluorescein-propidium iodide (FDA-PI) Determination Staining.FDA can be by living cells film, and passes through intracellular
Esterase be converted into Green fluorescent dye, esterase is that living cells is proprietary, and therefore, it is living thin that cell membrane, which is contaminated for the cell of green,
Born of the same parents.Opposite, PI can enter dead cell core, generate red fluorescence by forming PI- nucleic conjugates, therefore, core, which is contaminated, is
Red cell is dead cell.FDA-PI solution is formed by the way that 1 μ L PI and 1 μ L FDA are added in 98 μ L ultra-pure waters.10μL
FDA-PI solution is added in 90 μ L cell suspending liquids, is incubated at room temperature 2min, and the cell of FDA-PI dyeing passes through fluorescence microscope) it sees
It examines.Result of the test is shown in Fig. 4 and Fig. 5.
The B5 medium formula that table 4 is improved
Due to Fig. 4, suspension cell growth is significantly influenced by boron deficiency, and teratocyte ratio increases, under total number of cells amount is notable
Drop, so cytoactive significantly reduces (see Fig. 5) simultaneously.
Claims (2)
1. a kind of method that suspension cell line is obtained using cabbage type rape (Brassica napus L.) hypocotyl as explant,
It is characterised in that it includes following steps：
(1) disinfection of explant：The cabbage type rape seed of clear water soaked overnight is poured into triangular flask, 75% alcohol is added to go out
Bacterium 30s, then sterilized 10min with 0.1% mercury chloride, with aseptic water washing 4~5 times, by the cabbage type rape seed after sterilizing
It is seeded on sowing culture medium, the sowing culture medium adds 30g/L sucrose and 10g/ using B5 medium as basic culture medium
L agar, supplement distilled water adjust the pH to 5.8-5.9 of culture medium, light culture 5-7 days at 24 DEG C, acquisition cabbage type rape to 1L
Aseptic seedling；
(2) induction of callus：The hypocotyl of cabbage type rape aseptic seedling is cut into 0.5~1cm stem sections, is inoculated into callus group
It knits on inducing culture, secretly induction obtains the callus of cabbage type rape at 24 DEG C；
(3) subculture of callus：The cabbage type rape callus of 30 ages in days is rejected to the callus of browning and aging, is turned
It moves on callus subculture medium, the light culture at 24 DEG C, squamous subculture 1~2 time, each subculture 14-16 days；
(4) foundation of suspension cell line：By color is yellowish, open-textured embryo callus is transferred to suspension cell culture base
On, the bottled 30mL suspension cell cultures base of triangle per 150mL, every bottle of inoculation yellowish and open-textured embryo callus subculture of 1g colors
Tissue, at 25 DEG C, rotating speed obtains suspension cell line to be cultivated under conditions of 120r/min and light culture；
Wherein：
The preparation of callus inducing medium described in step (2)：Using B5 medium as basic culture medium, 2.0mg/L is added
2,4-D, 0.5mg/L 6-BA, the sucrose of 30g/L, 0.1g/L citric acids, the caseinhydrolysate of 300mg/L, 500mg/L's
L-PROLINE and 8g/L agar, supplement ultra-pure water adjust the pH to 5.8-5.9 of culture medium to 1L；
The preparation of callus subculture medium described in step (3)：Using B5 medium as basic culture medium, 2.0mg/L is added
2,4-D, 0.5mg/L 6-BA, the sucrose of 30g/L, 0.1g/L citric acids, the caseinhydrolysate of 300mg/L, 500mg/L's
L-PROLINE and 8g/L agar, supplement ultra-pure water adjust the pH to 5.8-5.9 of culture medium to 1L；
The preparation of suspension cell culture base described in step (4)：Using the B5 medium of improvement as basic culture medium：Calcium chloride dihydrate
440mg/L, potassium dihydrogen phosphate 170mg/L, magnesium sulfate 370mg/, potassium chloride 2940mg/L, potassium iodide 0.83mg/L, six water chlorinations
Cobalt 0.025mg/L, boric acid 6.2mg/L, Sodium Molybdate Dihydrate 0.25mg/L, manganese sulfate monohydrate 16.9mg/L, cupric sulfate pentahydrate
0.025mg/L, white vitriol 8.6mg/L, inositol 100mg/L, thiamine hydrochloride 0.5mg/L, disodium ethylene diamine tetraacetate
37.25mg/L, ferrous sulfate heptahydrate 27.85mg/L add the L-Aspartic acid of 2,4-D, the 266mg/L of 3mg/L, 877mg/L
L-Glutamine, the L-arginine of 228mg/L, the glycine of 75mg/L, the sucrose of 25g/L, the citric acid of 0.1g/L, supplement
Ultra-pure water adjusts the pH to 5.8-5.9 of culture medium to 1L.
2. one kind is suitable for obtaining suspension cell line by explant of cabbage type rape (Brassica napus L.) hypocotyl
Culture medium, which is characterized in that the formula of the culture medium is as described below：
(1) culture medium is sowed：Using B5 medium as basic culture medium, 30g/L sucrose and 10g/L agar are added, supplements distilled water
To 1L, the pH to 5.8-5.9 of culture medium is adjusted；
(2) callus inducing medium：Using B5 medium as basic culture medium, add 2,4-D, the 0.5mg/L's of 2.0mg/L
The sucrose of 6-BA, 30g/L, 0.1g/L citric acids, the caseinhydrolysate of 300mg/L, the L-PROLINE of 500mg/L and 8g/L fine jades
Fat, supplement ultra-pure water adjust the pH to 5.8-5.9 of culture medium to 1L；
(3) callus subculture medium：Using B5 medium as basic culture medium, add 2,4-D, the 0.5mg/L's of 2.0mg/L
The sucrose of 6-BA, 30g/L, 0.1g/L citric acids, the caseinhydrolysate of 300mg/L, the L-PROLINE of 500mg/L and 8g/L fine jades
Fat, supplement ultra-pure water adjust the pH to 5.8-5.9 of culture medium to 1L；
(4) suspension cell culture base：Using the B5 medium of improvement as basic culture medium, ingredient is：Calcium chloride dihydrate 440mg/
L, potassium dihydrogen phosphate 170mg/L, magnesium sulfate 370mg/, potassium chloride 2940mg/L, potassium iodide 0.83mg/L, CoCL2 6H2O
0.025mg/L, boric acid 6.2mg/L, Sodium Molybdate Dihydrate 0.25mg/L, manganese sulfate monohydrate 16.9mg/L, cupric sulfate pentahydrate
0.025mg/L, white vitriol 8.6mg/L, inositol 100mg/L, thiamine hydrochloride 0.5mg/L, disodium ethylene diamine tetraacetate
37.25mg/L, ferrous sulfate heptahydrate 27.85mg/L add the L-Aspartic acid of 2,4-D, the 266mg/L of 3mg/L, 877mg/L
L-Glutamine, the L-arginine of 228mg/L, the glycine of 75mg/L, the sucrose of 25g/L, the citric acid of 0.1g/L, supplement
Ultra-pure water adjusts the pH to 5.8-5.9 of culture medium to 1L.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108739388A (en) * | 2018-06-08 | 2018-11-06 | 中国科学院昆明植物研究所 | It is a kind of to promote rape callus proliferation, the method broken up and taken root and culture medium |
| CN109880788A (en) * | 2019-04-08 | 2019-06-14 | 天津吉诺沃生物科技有限公司 | The cabbage type rape protoplast electrofusion and genetic transforming method and regenerating system used not limited by genotype |
| CN113325201A (en) * | 2021-05-26 | 2021-08-31 | 西南大学 | Method suitable for atomic force microscope observation of cell skeletons of perennial fruit trees |
| CN116574668A (en) * | 2023-06-13 | 2023-08-11 | 浙江觅得优生物科技有限公司 | Method for promoting liquorice cells to release secondary metabolite liquorice total flavonoids into suspension culture medium |
| CN117402808A (en) * | 2023-10-18 | 2024-01-16 | 青岛农业大学 | Separation, purification and regeneration method of brassica napus protoplast |
-
2016
- 2016-11-09 CN CN201610986021.4A patent/CN108064687A/en active Pending
Non-Patent Citations (1)
| Title |
|---|
| 王火焰等: "不同硼效率甘蓝型油菜品种悬浮细胞的硼钙营养效应", 《植物营养与肥料学报》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108739388A (en) * | 2018-06-08 | 2018-11-06 | 中国科学院昆明植物研究所 | It is a kind of to promote rape callus proliferation, the method broken up and taken root and culture medium |
| CN109880788A (en) * | 2019-04-08 | 2019-06-14 | 天津吉诺沃生物科技有限公司 | The cabbage type rape protoplast electrofusion and genetic transforming method and regenerating system used not limited by genotype |
| CN113325201A (en) * | 2021-05-26 | 2021-08-31 | 西南大学 | Method suitable for atomic force microscope observation of cell skeletons of perennial fruit trees |
| CN116574668A (en) * | 2023-06-13 | 2023-08-11 | 浙江觅得优生物科技有限公司 | Method for promoting liquorice cells to release secondary metabolite liquorice total flavonoids into suspension culture medium |
| CN116574668B (en) * | 2023-06-13 | 2023-12-05 | 浙江觅得优生物科技有限公司 | Method for promoting liquorice cells to release secondary metabolite liquorice total flavonoids into suspension culture medium |
| CN117402808A (en) * | 2023-10-18 | 2024-01-16 | 青岛农业大学 | Separation, purification and regeneration method of brassica napus protoplast |
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