CN115340972B - Preparation method and application of banana protoplast - Google Patents
Preparation method and application of banana protoplast Download PDFInfo
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- CN115340972B CN115340972B CN202211050174.XA CN202211050174A CN115340972B CN 115340972 B CN115340972 B CN 115340972B CN 202211050174 A CN202211050174 A CN 202211050174A CN 115340972 B CN115340972 B CN 115340972B
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- 210000001938 protoplast Anatomy 0.000 title claims abstract description 50
- 235000018290 Musa x paradisiaca Nutrition 0.000 title claims abstract description 42
- 238000002360 preparation method Methods 0.000 title abstract description 12
- 240000005561 Musa balbisiana Species 0.000 title 1
- 241000234295 Musa Species 0.000 claims abstract description 46
- 238000000034 method Methods 0.000 claims abstract description 4
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 26
- 235000010355 mannitol Nutrition 0.000 claims description 16
- 239000007788 liquid Substances 0.000 claims description 11
- 239000006228 supernatant Substances 0.000 claims description 10
- 210000004027 cell Anatomy 0.000 claims description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- 229930195725 Mannitol Natural products 0.000 claims description 6
- ATJFFYVFTNAWJD-UHFFFAOYSA-N Tin Chemical compound [Sn] ATJFFYVFTNAWJD-UHFFFAOYSA-N 0.000 claims description 6
- 238000001914 filtration Methods 0.000 claims description 6
- 239000000594 mannitol Substances 0.000 claims description 6
- 238000007789 sealing Methods 0.000 claims description 6
- 108010059892 Cellulase Proteins 0.000 claims description 5
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 claims description 5
- GZCGUPFRVQAUEE-SLPGGIOYSA-N aldehydo-D-glucose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-SLPGGIOYSA-N 0.000 claims description 4
- 229940106157 cellulase Drugs 0.000 claims description 4
- 239000011780 sodium chloride Substances 0.000 claims description 4
- 108090000790 Enzymes Proteins 0.000 claims description 3
- 102000004190 Enzymes Human genes 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 3
- 229940088598 enzyme Drugs 0.000 claims description 3
- 238000011010 flushing procedure Methods 0.000 claims description 3
- 239000013049 sediment Substances 0.000 claims description 3
- 238000002791 soaking Methods 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims 1
- 238000011160 research Methods 0.000 abstract description 10
- 230000009466 transformation Effects 0.000 abstract description 10
- 230000004960 subcellular localization Effects 0.000 abstract description 5
- 241000196324 Embryophyta Species 0.000 abstract description 4
- 108090000623 proteins and genes Proteins 0.000 abstract description 4
- 102000004169 proteins and genes Human genes 0.000 abstract description 4
- 230000000694 effects Effects 0.000 abstract description 3
- 238000012795 verification Methods 0.000 abstract description 3
- 238000005516 engineering process Methods 0.000 abstract description 2
- 238000010521 absorption reaction Methods 0.000 abstract 1
- 238000000464 low-speed centrifugation Methods 0.000 abstract 1
- 239000000725 suspension Substances 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 27
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 11
- 239000012153 distilled water Substances 0.000 description 7
- 230000002068 genetic effect Effects 0.000 description 7
- 229920001030 Polyethylene Glycol 4000 Polymers 0.000 description 5
- 235000021015 bananas Nutrition 0.000 description 5
- 238000009395 breeding Methods 0.000 description 5
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 description 5
- 230000001488 breeding effect Effects 0.000 description 4
- 239000010413 mother solution Substances 0.000 description 4
- 240000000905 Nymphoides indica Species 0.000 description 3
- 235000017590 Nymphoides indica Nutrition 0.000 description 3
- 230000002255 enzymatic effect Effects 0.000 description 3
- 230000014509 gene expression Effects 0.000 description 3
- 239000012452 mother liquor Substances 0.000 description 3
- 230000010474 transient expression Effects 0.000 description 3
- 235000020138 yakult Nutrition 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000007071 enzymatic hydrolysis Effects 0.000 description 2
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000000644 propagated effect Effects 0.000 description 2
- 241000894007 species Species 0.000 description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 241000207199 Citrus Species 0.000 description 1
- 208000035240 Disease Resistance Diseases 0.000 description 1
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
- 108010059820 Polygalacturonase Proteins 0.000 description 1
- 208000020584 Polyploidy Diseases 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
- 230000002745 absorbent Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 235000011148 calcium chloride Nutrition 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 235000020971 citrus fruits Nutrition 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000000408 embryogenic effect Effects 0.000 description 1
- 108010093305 exopolygalacturonase Proteins 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 239000005090 green fluorescent protein Substances 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000010979 pH adjustment Methods 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000001902 propagating effect Effects 0.000 description 1
- 230000006916 protein interaction Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
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- 230000001052 transient effect Effects 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/04—Plant cells or tissues
Abstract
The invention discloses a preparation method and application of banana protoplast, and belongs to the technical field of plant protoplast. The invention adopts the inner layer tissue of banana bud absorption, improves the enzymolysis solution formula, and obtains the high-purity banana protoplast by a plurality of low-speed centrifugation and re-suspension methods, thereby reaching 1.22 multiplied by 10 per gram of tissue 7 And (3) protoplasts. The invention has higher reproducibility, can stably obtain high-quality banana protoplast, and realizes the effect that the transformation efficiency reaches more than 75 percent. The invention provides an effective research technology for molecular biology researches such as banana protein subcellular localization, promoter function verification and the like, and has wide application value.
Description
Technical Field
The invention belongs to the technical field of plant protoplasts, and particularly relates to a preparation method and application of banana protoplasts.
Background
The plant protoplast is used as an ideal genetic transformation receptor, and the transient expression by utilizing the plant protoplast is an important technical means for carrying out analysis such as gene expression, subcellular localization, protein interaction, promoter activity and the like in analysis molecular genetic research. Currently, successful protoplast transformation systems are reported in a wide variety of species, including model species. For bananas, embryogenic cell suspension lines and calli are considered to be good protoplast isolation materials, but it is indeed reported that viable protoplasts are isolated directly from banana plant tissue.
Bananas are the second largest fruit in the world, and the area and yield of bananas are inferior to citrus, and are one of important economic and food crops in tropical and subtropical areas. The current common cultivars of bananas are poorly resistant and often suffer serious attack from soil-borne diseases, especially banana vascular wilt. Most banana cultivars belong to asexual propagation and have lost genetic diversity due to their own polyploidy and high sterility, which causes problems such as difficulty in implementation of conventional breeding methods. Therefore, a new variety of bananas with high quality is bred by utilizing a new technical system, so that the healthy and continuous development of the banana industry is a current priority. The banana protoplast separation and transformation system and the protoplast fusion technology are utilized to solve the problem that the traditional banana breeding is difficult, and the new variety is obtained in an accelerating way. The transient expression system of banana protoplast is perfected, a good research technical means can be provided for the subsequent research on the functional genome of the banana, and a foundation is further laid for the research on disease resistance breeding of the banana. Therefore, the construction of the efficient and stable protoplast preparation and transient transformation method has important significance for banana genetic research and breeding utilization.
Disclosure of Invention
Aiming at the technical problems, the invention provides a preparation method and application of banana protoplast, solves the problems that the active protoplast is directly separated from banana plant tissues and genetic transformation work is difficult to develop, and provides a stable and efficient technical method for molecular genetic research such as banana protein subcellular localization, promoter function verification and the like.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
the preparation method of the banana protoplast comprises the following steps:
(1) Collecting banana bud, peeling layer by layer to obtain tender inner layer, and cutting into 0.5-1mm bud-sucking segments;
(2) Soaking the bud sucking small section into a culture dish containing 0.6M mannitol, sealing the culture dish with tinfoil paper, and standing for 5 min;
(3) After the mannitol is sucked by a pipetting gun, the bud sucking small section is moved to a 50 mL beaker, 5mL enzymolysis liquid is added, and the vacuum is pumped in a vacuum pump for 2-3 times for 5 min each time;
(4) Sealing the beaker with tinfoil paper, horizontally oscillating at 50 rpm, and performing enzymolysis at room temperature (26 ℃) until the protoplast is dissociated from the bud-sucking small section;
(5) Filtering the enzymolysis liquid into a 50 mL centrifuge tube by using a 200-mesh cell sieve after enzymolysis is finished, flushing a small beaker with a W5 solution precooled on ice for many times, and filtering into a 50 mL centrifuge tube;
(6) Centrifuging the solution filtered in the step (5) at a rotation speed of 400 g (slow speed of rise and fall) for 3 min, and discarding the supernatant; slowly adding 15mL precooled W5 solution, gently resuspending cells, centrifuging again at 400 g for 3 min, and then sucking off redundant supernatant;
(7) Repeating the step (6) for 2 times, re-suspending the protoplast obtained by the last centrifugation in a W5 solution precooled by 15mL, placing in a refrigerator at 4 ℃ for precooling for 30 min, centrifuging for 3 min at a rotation speed of 400 g, and discarding the supernatant;
(8) Then adding a proper amount of MMG solution, and fully re-suspending the cell sediment for later use.
Preferably, the enzymolysis time in the step (4) is 6 h.
Preferably, the components and the contents of the enzymolysis liquid are as follows: 10 mM MES pH7.5, 0.6M D-Mannitol, 2.5% (w/v) Cellulase (Cellulase R-10), 1% (w/v) educt enzyme (Macerozyme R-10）、10 mM CaCl 2 0.1% (w/v) BSA and 0.35% (v/v) 2-mercaptoethanolβ-Mercaptoethanol）。
Preferably, the components and the contents of the W5 solution are as follows: 1.5 mM MES pH7.5, 155 mM NaCl, 125 mM CaCl 2 、5 mM KCl、5 mM D-(+)-Glucose。
Preferably, the MMG solution comprises the following components in percentage by weight: 4mM MES pH7.5, 0.6M D-Mannitol, 10 mM MgCl 2 ·6H 2 O。
The banana protoplast prepared by the invention can be used for genetic transformation, banana protein subcellular localization and banana breeding.
The beneficial effects are that:
(1) The invention can obtain 1.22×10 per gram of tissue 7 The protoplast effectively solves the problem of directly separating the active protoplast from banana plant tissues. Meanwhile, the matched transformation system is optimized, so that the transformation efficiency reaches more than 75%.
(2) The invention provides an effective research technical means for molecular genetics research such as banana protein subcellular localization, promoter function verification and the like. From the technical point of view, the invention is realized through a plurality of technical process improvements which are simple and easy to implement and have innovativeness, and the operation difficulty is reduced, so that the popularization and application value and the commercial development potential are more highlighted.
Drawings
FIG. 1 is a schematic diagram of banana propagating seedlings (A), rooting seedlings (C) and absorbing buds (E), wherein the left graph of the graph E is a complete banana absorbing bud, and the right graph is a young part of the banana absorbing bud after the outer layer is peeled; bar=1 cm; FIGS. B, D and F are graphs of protoplast yield obtained by hydrolyzing banana propagated seedlings, rooted seedlings and shoots under different enzymatic hydrolysis conditions for 8 hours, respectively;
FIG. 2 is a graph showing the yield of protoplasts isolated from banana absorbent buds at different enzymolysis times;
FIG. 3 is a graph showing the effect of YFP on expression after transformation in banana protoplasts, wherein the left panel shows GFP fields, the middle panel shows bright fields, and the right panel shows pooled fields; bar=50 μm.
Detailed Description
The reagent consumable used in the invention is as follows:
MES (Sigma，V900336-500G)
KCl (Xilan science Co., ltd.)
MgCl 2 •6H 2 O (Sigma，M2393-500G)
CaCl 2 (Sigma，C4901-500G)
PEG4000 (Sigma，81240-1KG)
NaCl (Xilan science stock Co., ltd.)
D-Mannitol (Sigma，M1902-1kg)
Cellulase R-10 (Yakult，L0012)
Macerozyme R-10 (Yakult，JM6033)
Pectinase Y-23 (Yakult, P8220-100)
D-(+)-Glucose（Sigma，G8270-1KG）
BSA (Lanbolide biotechnology Co., ltd.)
β-ME (Sigma，M3148)
The invention is further described below with reference to the drawings and specific embodiments.
The configuration of each solution in the following examples is as follows:
(1) The preparation method of the enzymolysis solution comprises the following steps:
39.048 g of MES is dissolved in 1L of distilled water, and the pH value is adjusted to 7.5 by KOH, so that 0.2M MES pH7.5 mother solution is obtained, and the mother solution is stored in the dark at 4 ℃; 146.536 g of D-Mannitol is dissolved in 1L of distilled water to obtain 0.8M D-Mannitol mother liquor, and the mother liquor is stored at room temperature; dissolving 149.1 g of KCl into 1L distilled water to obtain 2M KCl mother liquor, and preserving at room temperature; 110.98 g CaCl 2 Dissolving in distilled water of 1L to obtain 1M CaCl 2 Storing mother solution at room temperature; 203.3 g of MgCl 2 ·6H 2 O is dissolved in distilled water of 1L to obtain 1M MgCl 2 ·6H 2 And (5) storing the O mother solution at room temperature.
1 mL of 0.2M MES (final concentration 10 mM), 15mL of 0.8M D-Mannitol (final concentration 0.6M) and 200. Mu.L of 0.2M KCl are added into each 20 mL enzymolysis solutionFinal concentration 20 mM), 0.5 g Cellulase Cellulase R-10 (final concentration 2.5%), 0.2 g isolated enzyme Macerozyme R-10 (final concentration 1%),. The enzymatic buffer was placed in a 55℃water bath for 10 min, mixed upside down every 2 to 3 min, left on ice to cool to room temperature, and 200. Mu.L of 1M CaCl2 (final concentration 10 mM), 0.02 g BSA (final concentration 10%) and 7. Mu.L were added, respectivelyβME (final concentration 0.35%) to finally obtain the prepared enzymolysis liquid which can be used at present and stored at 4 ℃ for a short period.
(2) The preparation method of the PEG4000 solution comprises the following steps:
1.125 g PEG4000 (final concentration 45%), 625. Mu.L 0.8M D-Mannitol (final concentration 0.2M), 250. Mu.L 1M CaCl were added to each 2.5 mL PEG4000 solution 2 (final concentration 0.1. 0.1M), the prepared 45% PEG solution was finally obtained, and ready-to-use.
(3) The preparation method of the W5 solution comprises the following steps:
10 mL of 0.2M MES (final concentration 2 mM), 90 g of NaCl (final concentration 154 mM) and 125 mL of 1M CaCl were added to each 1L of W5 solution 2 (final concentration 125 mM), 2.5 mL 2M KCl (final concentration 5 mM), 0.9 g D- (+) -Glucose (final concentration 5 mM), and finally obtaining a formulated W5 solution, constant volume to 1L with distilled water, pH adjustment to 5.7 with KOH of 1M, high temperature and high pressure sterilization, and preservation at 4 ℃.
(4) The preparation method of the MMG solution comprises the following steps:
0.2 mL of 0.2M MES (final concentration 4. 4 mM), 7.5 mL of 0.8M D-Mannitol (final concentration 0.6. 0.6M) and 150. Mu.L of 1M MgCl are added to each 10 mL of MMG solution 2 ·6H 2 O (final concentration 15 mM), constant volume with distilled water, and preserving at 4deg.C.
Example 1
The preparation method of the banana protoplast comprises the following steps:
(1) Material culture: as shown in fig. 1E, banana buds are collected, peeled off layer by layer, only soft white tender parts of the inner layer remain, and the obtained buds are cut into small sections of 0.5-1mm by a surgical knife;
(2) Enzymolysis: soaking the bud sucking small section into a culture dish containing 0.6M mannitol, sealing the culture dish with tinfoil paper, and standing for 5 min; after the mannitol is sucked by a pipetting gun, the bud sucking small section is moved to a 50 mL beaker, 5mL enzymolysis liquid is added, and the vacuum is pumped in a vacuum pump for 2-3 times for 5 min each time; sealing the beaker with tinfoil paper, horizontally oscillating at 50 rpm, and performing enzymolysis at room temperature (26 ℃) until the protoplast is dissociated from the bud-sucking small section; microscopic examination of enzymolysis results;
(3) And (3) stopping enzymolysis: filtering the enzymolysis liquid into a 50 mL centrifuge tube by using a 200-mesh cell sieve, flushing a small beaker with a pre-cooled W5 solution on ice for many times, and filtering into a 50 mL centrifuge tube;
(4) Collecting and rinsing protoplast: centrifuging the solution filtered in the step (3) at a rotation speed of 400 g (slow speed of rise and fall) for 3 min, and discarding the supernatant; slowly adding 15mL precooled W5 solution, gently resuspending cells, centrifuging again at 400 g for 3 min, and then sucking off redundant supernatant;
(5) Ice bath protoplasts: repeating the step (4) for 2 times, re-suspending the protoplast obtained by the last centrifugation in a W5 solution precooled by 15mL, placing in a refrigerator at 4 ℃ for precooling for 30 min, centrifuging for 3 min at a rotation speed of 400 g, and discarding the supernatant;
(6) Protoplasts were resuspended in MMG solution: and adding a proper amount of MMG solution, and fully re-suspending the cell sediment for later use.
Example 2
Banana protoplasts were prepared as in example 1 and control groups of different tissues (propagated seedlings, rooted seedlings and shoots) and different enzymatic digests were set.
As a result, as shown in FIG. 1, the number of banana protoplasts obtained by the propagation and rooting of seedlings under the combination of all enzymatic liquids (FIG. 1, B, D) was significantly smaller than that obtained by young shoots (FIG. 1F) at 8h enzymatic hydrolysis time. For young shoots, the number of protoplasts prepared in enzyme-hydrolyzed solution groups 1-8 and 10 was significantly less than that prepared in example 1 (enzyme-hydrolyzed solution group 9).
Example 3
Banana protoplasts were prepared as in example 1 and control groups of different enzymolysis times were set.
As a result, as shown in FIG. 2, banana protoplasts were obtained at the time of enzymolysis (1 h, 2h, 3h, 4h, 5h, 6h, 7h, 8 h), and the highest number of protoplasts was reached at the time of enzymolysis for 6h (FIG. 2).
Example 4
Use of transient expression of banana protoplasts, said use comprising the steps of:
(1) 10 mu L of 35S (YFP plasmid (concentration is more than 1 mu g/. Mu.L) and 200 mu L of protoplast are added into a 2 mL centrifuge tube at one time, after being fully and evenly mixed, 220 mu L of PEG4000 solution is added, and the mixture is immediately and gently mixed, and is stood for 15 min at the dark and room temperature;
(2) The conversion reaction was terminated: adding 1 mL of W5 solution, mixing, horizontally centrifuging (4 ℃ C., 400 and g) for 3 min, discarding the supernatant and collecting protoplast, and repeating the steps for 2 times;
(3) Culturing 12 h-16 h under weak light;
(4) The dark cultured protoplast was taken and examined with a microscope for YFP expression (see FIG. 3).
As can be seen from the signal diagram of transformation of banana bud aspiration prepared protoplast with green fluorescent protein gene in FIG. 3, the protoplast prepared by the invention can be successfully transformed.
It is to be understood that the invention is not limited in its application to the examples described above, but is capable of modification and variation in light of the above teachings by those skilled in the art, and that all such modifications and variations are intended to be included within the scope of the appended claims.
Claims (1)
1. A method for preparing banana protoplasts, which is characterized by comprising the following steps:
(1) Collecting banana buds, peeling the banana buds layer by layer to obtain an inner tender part, and cutting the inner tender part into 0.5-1 and mm small segments;
(2) Soaking the bud sucking small section into a culture dish containing 0.6M mannitol, sealing the culture dish with tinfoil paper, and standing for 5 min;
(3) After the mannitol is sucked by a pipetting gun, the bud sucking small section is moved to a beaker, 5mL enzymolysis liquid is added, and the vacuum is pumped in a vacuum pump for 2 to 3 times for 5 minutes each time;
(4) Sealing the beaker with tinfoil paper, horizontally oscillating at 50 rpm, and carrying out enzymolysis for 6 hours at room temperature until protoplast is dissociated from the bud-sucking small section;
(5) Filtering the enzymolysis liquid into a centrifuge tube by using a 200-mesh cell sieve after enzymolysis is finished, flushing a beaker for a plurality of times by using a W5 solution precooled on ice, and filtering the enzymolysis liquid into the centrifuge tube;
(6) Centrifuging the filtered solution in the step (5) for 3 min, and discarding the supernatant; slowly adding 15mL precooled W5 solution, gently resuspending cells, centrifuging for 3 min again, and then sucking off redundant supernatant;
(7) Repeating the step (6) for 2 times, re-suspending the protoplast obtained by the last centrifugation in 15mL of pre-cooled W5 solution, placing in a refrigerator at 4 ℃ for pre-cooling for 30 min, centrifuging for 3 min, and discarding the supernatant;
(8) Adding MMG solution, and fully re-suspending the cell sediment for later use;
the enzymolysis liquid comprises the following components in percentage by weight: 10 mM MES pH7.5, 0.6M D-Mannitol, 2.5% (w/v) cellulase, 1% (w/v) educt enzyme, 10 mM CaCl 2 0.1% (w/v) BSA, 0.35% (v/v) 2-mercaptoethanol;
the W5 solution comprises the following components in percentage by weight: 1.5 mM MES pH7.5, 155 mM NaCl, 125 mM CaCl 2 、5 mM KCl、5 mM D-(+)-Glucose；
The MMG solution comprises the following components in percentage by weight: 4mM MES pH7.5, 0.6M D-Mannitol, 10 mM MgCl 2 ·6H 2 O。
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Citations (2)
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| CN113088559A (en) * | 2021-04-27 | 2021-07-09 | 广东省农业科学院果树研究所 | Method for rapidly screening CRISPR-Cas9 genome editing gRNA target site |
| CN114480248A (en) * | 2022-01-18 | 2022-05-13 | 南通大学 | Preparation method and application of sugarcane stem tissue protoplast |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113088559A (en) * | 2021-04-27 | 2021-07-09 | 广东省农业科学院果树研究所 | Method for rapidly screening CRISPR-Cas9 genome editing gRNA target site |
| CN114480248A (en) * | 2022-01-18 | 2022-05-13 | 南通大学 | Preparation method and application of sugarcane stem tissue protoplast |
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| Title |
|---|
| Establishment of a Protoplasts-Based Transient Expression System in Banana (Musa spp.);Chunhui Zhao等;Agronomy;第1-12页 * |
| Influence of donor material and genotype on protoplast regeneration in banana and plantain cultivars (Musa spp.);Akym Assani等;Plant Science;第355-362页 * |
| 香蕉原生质体培养研究进展;刘代;魏岳荣;胡家金;易干军;;中国农学通报(第10期);第1节 * |
| 香蕉遗传育种研究进展;王钱洁;陈厚彬;徐春香;胡桂兵;;福建果树(第03期);第15-22页 * |
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