CN102899282A - Sugarcane callus protoplast separation and purification method - Google Patents
Sugarcane callus protoplast separation and purification method Download PDFInfo
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Abstract
The invention relates to a sugarcane callus protoplast separation and purification method, which comprises sugarcane callus culture, sugarcane suspended cell culture, sugarcane protoplast separation, and sugarcane protoplast purification. By the method, suspended cell lines can be obtained by sugarcane embryogenic callus culture and an adequate amount of complete protoplasts with high quality and uniformity can be obtained by enzyme digestion, separation and purification; therefore, ideal experimental materials can be provided for study transgenetic transient expression such as sugarcane exogenous gene expression, promoter analysis and gene silencing.
Description
Technical field
The present invention relates to a kind of isolation and purification method of plant protoplast, be specifically related to a kind of isolation and purification method of sugarcane callus protoplastis, belong to biological technical field.
Background technology
Plant protoplast has overcome this barrier of cell walls, is the ideal material of plant physiology, cytobiology, the research of somatic cell genetics scheduling theory, and aspect genetically engineered and the crop improvement rearing new variety important application prospect is being arranged.In recent years, along with the molecular biology of plants development, protoplastis is widely used in the research of the aspects such as the assignment of genes gene mapping, gene expression regulation, the reticent mechanism of RNA, cell calcium signal transduction and regulation mechanism thereof as a unicellular system.
The research of sugarcane callus protoplastis starts from the seventies in last century, and protoplastis separation, cultivation and plant regeneration also succeed.But, to compare with model plant tobacco or the research of Arabidopis thaliana mesophyll protoplast, sugarcane callus protoplast separation purification effect is desirable not to the utmost, and plant regeneration frequency is very low, until there is not tremendous development today.Take the sugarcane callus protoplastis as acceptor material, carry out sugarcane genetically modified transient expression and have no report.The present invention sets up efficiently, the Isolation and purification method of sugarcane callus protoplastis fast, and the protoplastis that obtains high quality, high yield can be used as the acceptor material of the transgenosis transient expressions researchs such as exogenous gene expression, promoter Analysis, gene silencing.
Summary of the invention
The isolation and purification method that the purpose of this invention is to provide a kind of quick, efficient, easy sugarcane callus protoplastis, overcome the problem that existing sugarcane callus isolation and purification of protoplast method exists quality and quantitative deficiency, the sugarcane callus protoplastis of high quality, high yield is provided for the research of the transgenosis transient expressions such as sugarcane exogenous gene expression, promoter Analysis, gene silencing.
The objective of the invention is to realize by the following method.
The isolation and purification method of sugarcane callus protoplastis of the present invention comprises sugarcane callus cultivation, sugarcane suspension cell culture, the separation of Protoplasts of Sugarcane and the purifying of Protoplasts of Sugarcane; It is characterized in that operation steps is as follows:
1, sugarcane callus is cultivated
The sugarcane lobus cardiacus tissue of cancellation behind the poison is cut into the thick thin slice of 1-2 cm under the aseptic condition, on the MS3 solid medium, 28 ℃ dark cultivated 1-2 individual month, every 2 all subcultures 1 time; Described MS3 solid medium is: MS+0.5 g/L caseinhydrolysate+3 mg/L 2,4-D+30 g/L sucrose+6.0 g/L agar powders; The MS substratum is 1962, the disclosed MS substratum of Murashige and Skoog (lower same);
2, sugarcane suspension cell culture
After Sugarcane Embryogenic Callus smashed to pieces, in the MS3 liquid nutrient medium, 28 ℃ of dark cultivations for 2 weeks, filtered through gauze, filtrate is left standstill 10-15 min, draw bottom embryo callus nutrient solution, cultivate 120 revolutions per seconds of rotating speeds, 28 ℃ of dark cultivations the 1-2 month in the constant-temperature table concussion, every 1 all subcultures 1 time, until suspension cell line occurs; Described MS3 liquid nutrient medium: MS+0.5 g/L caseinhydrolysate+3 mg/L 2,4-D+30 g/L sucrose, deionized water are supplied 1 liter;
3, the separation of Protoplasts of Sugarcane
The fresh suspension cell of getting step 2 divides and installs to 50 mL centrifuge tubes, centrifugal 2 min of 100 g; Abandon supernatant liquor, behind enzymolysis buffer solution for cleaning suspension cell, centrifugal 2 min of 100 g; Abandon supernatant liquor, get suspension cell 2-3 gram and add in the Tissue Culture Plate in 6 holes, spend the night with 3 mL enzyme digestion reaction liquid enzymolysis, isolate the sugarcane callus protoplastis; Described enzymolysis damping fluid: 0.02 mol/L MES+0.4 mol/L N.F,USP MANNITOL+0.02 mol/L Repone K, pH 5.7; Described enzyme digestion reaction liquid: 2 % cellulases+0.1 % polygalacturonase+0.01 mol/L calcium chloride+0.1 % bovine serum albumin are dissolved in the enzymolysis damping fluid; In enzyme digestion reaction liquid, the shared per-cent of each raw material is the weight percent of each raw material and enzymolysis damping fluid;
4, the purifying of Protoplasts of Sugarcane
With the nylon wire in sterile water wash 70 μ m apertures, then use the rinse of W5 solution, the protoplastis of getting step 3 acquisition is transferred in the 10 mL centrifuge tubes centrifugal 2 min of 100 g; Abandon supernatant liquor, the W5 solution suspension protoplastis of ice bath, centrifugal 1 min of 100 g; Abandon the sugarcane callus protoplastis that is purifying after the supernatant liquor, at observed under electron microscope, the normal protoplasma scale of construction of form reaches 10
6Individual/mL; Described W5 solution: 2 mmol/L MES+154 mmol/L sodium chloride+125 mmol/L calcium chloride+5 mmol/L Repone K, pH 5.7.
The present invention also protects the application of sugarcane callus protoplastis in sugarcane genetically modified transient expression that utilizes the aforesaid method isolation and purification.
All chemical reagent that the present invention uses are analytical pure.
Advantage of the present invention is, cultivate from Sugarcane Embryogenic Callus fast and obtain suspension cell line, and by enzyme clear up that rear separation and purification obtains that quality is high, quantity foot, complete protoplastis that homogeneity is good, for the research of the transgenosis transient expressions such as sugarcane exogenous gene expression, promoter Analysis, gene silencing provides desirable experiment material.
Description of drawings
Fig. 1 is the Protoplasts of Sugarcane after the separation and purification, and scale is 0.2 μ m.
Fig. 2 is that pUbi-EYFP-Nos and pUbi-P19-Nos(P19 are the RNA silencing suppressors of tomato bushy stunt virus coding) behind the Plasmid Transformation, the transient expression of EYFP gene on protoplastis, scale are 0.2 mm.
Fig. 3 is that pUbi-EYFP-Nos and pUbi-γ b-Nos(γ b are the RNA silencing suppressors of barly strip mosaic virus coding) behind the Plasmid Transformation, the transient expression of EYFP gene on protoplastis, scale are 0.2 mm.
Fig. 4 is the empty carrier that pUbi-EYFP-Nos and pUbi-Nos(do not contain any silencing suppressors) behind the Plasmid Transformation, the transient expression of EYFP gene on protoplastis, scale are 0.2 mm.
Fig. 5 is the Transient Expression of GUS behind the different promoters Plasmid Transformation protoplastis, and wherein, A is pPr4-GUS-Nos, and B is pUbi-GUS-Nos, and C is p35S-GUS-Nos, and D is the pUbi-Nos(empty carrier) plasmid; The scale in left side represents the GUS activity value, and unit is pmol 4-MU/ μ g proteinmin, and error line is standard error among the figure.
Embodiment
In order further to illustrate the present invention rather than restriction the present invention, be illustrated below in conjunction with embodiment.
Embodiment 1: a kind of isolation and purification method of sugarcane callus protoplastis may further comprise the steps:
1, sugarcane callus is cultivated: choose growth normal and without the sugarcane taper of disease and pest, remove peripheral Lao Ye until the highest visible plumpness with the unnecessary blade that leaf and top grow, keeps the long lobus cardiacus tissue of 25 cm; Be that the spirituous solution of 75 % carries out in aseptic Bechtop, sugarcane lobus cardiacus tissue being cut into the thick thin slice of 1.5 cm after the surface cleans sterilization with volume ratio, on the MS3 solid medium, 28 ℃ of dark cultivations 1.5 months are every 2 all subcultures 1 time;
2, sugarcane suspension cell culture: select densification, golden yellow, granular embryo callus 5 grams, after smashing to pieces, move into 100 mL MS3 liquid nutrient mediums, after 28 ℃ of dark 2 weeks of cultivation, with embryo callus nutrient solution filtered through gauze, filtrate is left standstill 12 min, with the tiny embryo callus nutrient solution of pipette, extract bottom 10 mL, in constant-temperature table, shake cultivation with 100 mL MS3 liquid nutrient mediums, 120 revolutions per seconds of rotating speeds, 28 ℃ of dark cultivations the 1-2 months are every 1 all subcultures 1 time, until suspension cell line tiny, homogeneous occurs.
3, the fresh suspension cell of getting step 2 divides and installs to 50 mL centrifuge tubes, perhaps with the suspension cell succeeding transfer culture of step 2 after 3 days, minute installs to 50 mL centrifuge tubes; Centrifugal 2 min of 100 g; Abandon supernatant liquor, add 40 mL enzymolysis buffer solution for cleaning suspension cells, centrifugal 2 min of 100 g; Abandon supernatant liquor, get suspension cell 2-3 gram and add in the Tissue Culture Plate in 6 holes, spend the night with 3 mL enzyme digestion reaction liquid enzymolysis, isolate the sugarcane callus protoplastis;
4, with the nylon wire in sterile water wash 70 μ m apertures, then use the rinse of W5 solution, the sugarcane suspension cell that step 3 enzymolysis is spent the night passes through nylon net filter, gets 5 mL filtrates and is transferred in the 10 mL centrifuge tubes, centrifugal 2 min of 100 g; Abandon supernatant liquor, the W5 solution suspension protoplastis of 1 mL ice bath, centrifugal 1 min of 100 g; Abandon the sugarcane callus protoplastis that is purifying after the supernatant liquor.
The W5 solution of the getting 1 mL ice bath protoplastis that again suspends; Under 100 power microscopes, calculate protoplastis quantity with blood counting chamber, complete protoplastis as shown in Figure 1, low temperature storage is for subsequent use.
Embodiment 2: utilize from the protoplastis of the separation of sugar cane breed CP72-1210 lobus cardiacus callus, purifying, study the activity of the silencing suppressors of different plant viruses codings.
1, PEG genetic transformation
1.1 with sugarcane CP72-1210 callus protoplastis ice bath 30 min that prepare, remove W5 solution, with extremely every milliliter 2 * 10 of 1-2 mL MMG solution suspension protoplastis concentration
5Individual.
1.2 get respectively pUbi-EYFP-Nos and the pUbi-P19-Nos plasmid DNA (1 μ g/ μ l) of 5 μ L, the pUbi-EYFP-Nos of 5 μ L and pUbi-γ b-Nos plasmid DNA (1 μ g/ μ l), the pUbi-EYFP – Nos of 5 μ L and pUbi-Nos plasmid DNA (1 μ g/ μ l) are to 2 mL centrifuge tubes.
1.3 add 100 μ L sugarcane CP72-1210 protoplastiss, after the mixing, add 110 μ L, 40% polyglycol solutions (PEG-4000), mixing, the room temperature temperature is bathed 5-15 min.
1.4 add 440 uL W5 solution, mixing stops genetic transformation, centrifugal 2 min of 100 g.Abandon supernatant liquor, then 1 mL WI solution suspension protoplastis moves into the Tissue Culture Plate in 6 holes, and room temperature is placed 24 h.
MMG solution: 4 mmol/L MES+0.4 mol/L N.F,USP MANNITOL+15 mmol/L magnesium chlorides, pH 5.7.
WI solution: 4 mmol/L MES+0.5 mol/L N.F,USP MANNITOL+20 mmol/L Repone K, pH 5.7.
Polyoxyethylene glycol conversion fluid: 40% (W/V) polyoxyethylene glycol (PEG4000)+0.2 mol/L N.F,USP MANNITOL+100 mmol/L calcium chloride.
2, (YFP fluorescence filter disc) observed and expressed EYFP protoplastis number under fluorescent microscope, such as Fig. 2, Fig. 3 and shown in Figure 4, the activity of the silencing suppressors of different plant virus codings, show as P19 and γ b silencing suppressors can improve foreign gene EYFP expression level, average amplification reaches respectively 63% and 56%.
Example 3: utilize from the protoplastis of the separation of sugar cane breed CP72-1210 lobus cardiacus callus, purifying the activity of research different promoters.
1, transgenosis transient expression
1.1 with sugarcane CP72-1210 callus protoplastis ice bath 30 min that prepare, remove W5 solution, with extremely every milliliter 2 * 10 of 1-2 mL MMG solution suspension protoplastis concentration
5Individual.
10 μ L plasmid DNA (pPr4-GUS-NOS, pUbi-GUS-NOS, p35S-GUS-NOS, 10 μ g) in 2 mL centrifuge tubes, are established empty carrier (pUbi-NOS), water is blank 1.2 get respectively.
1.3 add 100 μ L protoplastiss, after the mixing, add 110 μ L, 40 % polyoxyethylene glycol conversion fluids, mixing, the room temperature temperature is bathed 5-15 min.
1.4 add gently mixing of 440 μ L W5 solution, stop genetic transformation, centrifugal 2 min of 100 g.Abandon supernatant liquor, then 1 mL WI solution suspension protoplastis moves into the Tissue Culture Plate in 6 holes, and room temperature is placed 24 h.
2, GUS determination of activity
2.1 with the sugarcane CP72-1210 callus protoplastis 1.5 mL centrifuge tubes of packing into, centrifugal 2 min of 100 g abandon supernatant liquor, get 25 μ L equivalent protoplastiss and divide and install to 1.5 mL centrifuge tubes.
2.2 add 25 μ L MUG substrate reactions liquid, 37 ℃ of reaction 60 min.
2.3 add 950 μ L sodium carbonate solution (0.2 mol/L) termination reactions.
2.4 use the fluorescent spectrophotometer assay fluorescence intensity.
2.5 protoplasma plastid total protein content adopts Xylene Brilliant Cyanine G method (Bradford method).
2.6 the drafting of 4-MU typical curve
In the stop buffer of 0.2 mol/L sodium carbonate solution, add 4-MU, be made into the solution of different 4-MU concentration, then utilize spectrophotofluorometer to survey the fluorescence of its generation (exciting light 365 nm, utilizing emitted light 455 nm), drawing standard curve.
Calculate 2.7 GUS is active
The pmol of the active 4-MU with every microgram albumen per minute formation of GUS represents, calculation formula is
The 4-MU(pmol that GUS activity=MUG is decomposed to form) ÷ [resolvability albumen (μ g) * reaction times (min)].Experiment repeats 3 times.
GUS damping fluid: 50 mmol/L phosphoric acid buffers (pH 7.0), 10 mmol/L beta-mercaptoethanols, 10 mmol/L ethylenediamine tetraacetic acid (EDTA)s (pH 8.0), 0.1% (v/v) Triton X-100.
MUG substrate reactions liquid: 2 mmol/L MUG are dissolved in the GUS damping fluid.
2.8 different promoters activity difference: as shown in Figure 5, the different promoters activity is difference to some extent, and promoter activity is followed successively by Pr4, Ubi, 35S, and it orders about the GUS activity and is respectively 40.86,15.71,4.99 pmol 4-MU/ μ g proteinmin.
Claims (2)
1. the isolation and purification method of a sugarcane callus protoplastis comprises sugarcane callus cultivation, sugarcane suspension cell culture, the separation of Protoplasts of Sugarcane and the purifying of Protoplasts of Sugarcane; It is characterized in that operation steps is as follows:
(1) sugarcane callus is cultivated
The sugarcane lobus cardiacus tissue of cancellation behind the poison is cut into the thick thin slice of 1-2 cm under the aseptic condition, on the MS3 solid medium, 28 ℃ dark cultivated 1-2 individual month, every 2 all subcultures 1 time; Described MS3 solid medium is: MS+0.5 g/L caseinhydrolysate+3 mg/L 2,4-D+30 g/L sucrose+6.0 g/L agar powders; The MS substratum is 1962, the disclosed MS substratum of Murashige and Skoog;
(2) sugarcane suspension cell culture
After Sugarcane Embryogenic Callus smashed to pieces, in the MS3 liquid nutrient medium, 28 ℃ of dark cultivations for 2 weeks, filtered through gauze, filtrate is left standstill 10-15 min, draw bottom embryo callus nutrient solution, cultivate 120 revolutions per seconds of rotating speeds, 28 ℃ of dark cultivations the 1-2 month in the constant-temperature table concussion, every 1 all subcultures 1 time, until suspension cell line occurs; Described MS3 liquid nutrient medium: MS+0.5 g/L caseinhydrolysate+3 mg/L 2,4-D+30 g/L sucrose, deionized water are supplied 1 liter;
(3) separation of Protoplasts of Sugarcane
The fresh suspension cell of getting step (2) divides and installs to 50 mL centrifuge tubes, centrifugal 2 min of 100 g; Abandon supernatant liquor, behind enzymolysis buffer solution for cleaning suspension cell, centrifugal 2 min of 100 g; Abandon supernatant liquor, get suspension cell 2-3 g and add in the Tissue Culture Plate in 6 holes, spend the night with 3 mL enzyme digestion reaction liquid enzymolysis, isolate the sugarcane callus protoplastis; Described enzymolysis damping fluid: 0.02 mol/L MES+0.4 mol/L N.F,USP MANNITOL+0.02 mol/L Repone K, pH 5.7; Described enzyme digestion reaction liquid: 2 % cellulases+0.1 % polygalacturonase+0.01 mol/L calcium chloride+0.1 % bovine serum albumin are dissolved in the enzymolysis damping fluid; In enzyme digestion reaction liquid, the shared per-cent of each raw material is the weight percent of each raw material and enzymolysis damping fluid;
(4) purifying of Protoplasts of Sugarcane
With the nylon wire in sterile water wash 70 μ m apertures, then use the rinse of W5 solution, the protoplastis of getting step (3) acquisition is transferred in the 10 mL centrifuge tubes centrifugal 2 min of 100 g; Abandon supernatant liquor, the W5 solution suspension protoplastis of ice bath, centrifugal 1 min of 100 g; Abandon the sugarcane callus protoplastis that is purifying after the supernatant liquor; Described W5 solution: 2 mmol/L MES+154 mmol/L sodium chloride+125 mmol/L calcium chloride+5 mmol/L Repone K, pH 5.7.
2. by the application of sugarcane callus protoplastis in sugarcane genetically modified transient expression of the method isolation and purification of claim 1.
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| CN104745523A (en) * | 2015-04-03 | 2015-07-01 | 天津吉诺沃生物科技有限公司 | Separation, transformation and regeneration system for oilseed rape protoplast |
| CN105331574A (en) * | 2015-11-16 | 2016-02-17 | 华南农业大学 | Method for preparation and transient gene expression of jatropha curcas tissue culture seedling protoplast |
| CN106520666A (en) * | 2016-12-05 | 2017-03-22 | 天津吉诺沃生物科技有限公司 | Method and special culture medium for efficiently separating, converting and regenerating potato protoplast |
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| CN109207419A (en) * | 2018-08-22 | 2019-01-15 | 广西大学 | Protoplasts of Sugarcane cryopreservation method |
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| CN111202005A (en) * | 2020-03-24 | 2020-05-29 | 广西壮族自治区农业科学院 | Sugarcane variety embryonic callus induction method |
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| CN104745523B (en) * | 2015-04-03 | 2020-04-24 | 天津吉诺沃生物科技有限公司 | System for separating, transforming and regenerating rape protoplast |
| CN104745523A (en) * | 2015-04-03 | 2015-07-01 | 天津吉诺沃生物科技有限公司 | Separation, transformation and regeneration system for oilseed rape protoplast |
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| CN109207470A (en) * | 2018-10-30 | 2019-01-15 | 广西大学 | A kind of method of Protoplasts of Sugarcane electro' asion |
| CN109182276B (en) * | 2018-10-30 | 2021-11-26 | 广西大学 | Sugarcane somatic cell hybridization method |
| CN110669718A (en) * | 2019-09-30 | 2020-01-10 | 江苏省中国科学院植物研究所 | Method for separating and purifying root, stem and leaf protoplasm body of larch and performing instantaneous high-efficiency conversion |
| CN111202005A (en) * | 2020-03-24 | 2020-05-29 | 广西壮族自治区农业科学院 | Sugarcane variety embryonic callus induction method |
| CN114058640A (en) * | 2021-12-01 | 2022-02-18 | 云南省农业科学院甘蔗研究所 | Efficient agrobacterium-mediated sugarcane genetic transformation method |
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| CN115287248A (en) * | 2022-07-06 | 2022-11-04 | 贵州大学 | Preparation method of eucommia ulmoides protoplast |
| CN115287248B (en) * | 2022-07-06 | 2023-08-29 | 贵州大学 | Preparation method of eucommia ulmoides protoplast |
| CN116463276A (en) * | 2023-04-28 | 2023-07-21 | 广西大学 | Method for separating and culturing sugarcane suspension single cells and method for regenerating plants |
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| CN116536242A (en) * | 2023-05-19 | 2023-08-04 | 云南省农业科学院甘蔗研究所 | Rapid and efficient sugarcane protoplast preparation method |
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Application publication date: 20130130 |