CN115820532B - Dissociation method of banana young root protoplast - Google Patents
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Abstract
The invention relates to a method for decomposing protoplast of banana young rootAn isolation method belongs to the technical field of plant cell protoplast separation. The invention selects the young root tissue which sprouts for 14 days as a material, cuts the young root tissue into slices, and then adds enzymolysis liquid to shake and carry out enzymolysis for 1 to 3 hours under the dark condition; and (3) after enzymolysis, filtering by using a cell filter with the particle size of 70 mu m and a cell filter with the particle size of 40 mu m, cleaning by using a protoplast buffer solution, and obtaining protoplasts by using a low-temperature low-speed centrifugal precipitation method. The invention realizes dissociation of banana young root protoplast, has simple operation, can prepare protoplast rapidly and efficiently, and detects the protoplast separated by the optimal enzyme concentration by adopting FDA dyeing and adopting a fluorescence inversion microscope and a blood cell counting plate to find that the yield of the protoplast is up to 1.2 multiplied by 10 6 The activity rate of each mL reaches 92%, and the subsequent 10X Genomics single cell sequencing on-machine requirement is met.
Description
Technical Field
The invention belongs to the technical field of plant cell protoplast separation, and particularly relates to a method for separating banana young root protoplasts.
Background
Bananas are one of the most important fruits in the world, are widely planted in tropical and subtropical areas, and are second only to citrus in planting area and yield, and are the second largest fruits in the world; fresh fruit consumption and trade volume both reside on the crown of global fruits, one of the few fruits that cause world trade war. Meanwhile, the bananas are also one of very important economic fruit trees in the south China, and even are the main economic prop industry in partial areas. Currently, banana industry is facing serious threat of important diseases and insect pests such as fusarium wilt, and genetic improvement of banana is always concerned by researchers. By exploring the development track of banana root cells and the molecular mechanism of stress response, gene resources can be provided for genetic improvement researches such as banana disease resistance.
With the development of sequencing technology of life science, single-cell sequencing technology capable of analyzing information such as gene expression, DNA methylation and the like from single-cell level is silently emerging. The technology can deeply analyze the characteristics and the interrelation of different types of cells, and can solve the problem of heterogeneity among cells in tissues or organs covered by the traditional sequencing technology. Single cell sequencing has been widely used in the research of human and animal cells; in plant cells, the single-cell sequencing is limited by factors such as difficulty in dissociation of cell walls, and the single-cell sequencing can be carried out only by dissociation into protoplasts, so that the application is relatively slow.
Protoplasts refer to naked plant cells after removal of the cell wall by a method. At present, few reports are reported on the preparation method of banana protoplasts, and the existing method for preparing the protoplasts is mainly focused on utilizing callus or suspension cells as materials (incomplete plants, which is unfavorable for the performance of stress experiments), and the prepared protoplasts are also mainly used for researches such as genetic transformation, plant regeneration and the like, and the quality and the concentration of the protoplasts cannot meet the on-machine requirement of 10X Genomics single cell sequencing. Therefore, developing a protoplast preparation method to prepare high quality protoplasts for subsequent single cell sequencing is a major problem for those skilled in the art.
Disclosure of Invention
The invention aims to provide a dissociation method of banana young root protoplast, which is simple and efficient, so as to solve the problem that the quality and the concentration of the protoplast prepared by the prior art can not meet the on-machine requirement of 10X Genomics single cell sequencing.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a method for dissociation of banana young root protoplasts, comprising the steps of:
firstly, obtaining banana tissue culture seedlings by adopting a tissue culture technology, and cutting banana young root tissues after disinfection and cleaning to obtain an experimental material for preparing protoplasts;
step two, adding a small amount of protoplast buffer solution to keep the experimental material moist, then rapidly cutting banana young root tissues into slices by a blade, sucking the protoplast buffer solution, adding an enzymolysis solution, and shaking away from light at a constant speed for enzymolysis;
step three, filtering the enzymolysis liquid and protoplast mixed liquid obtained in the step two by adopting a cell filter, adding protoplast buffer solution into the filtrate for cleaning, centrifuging at low temperature and low speed, and removing supernatant;
step four, adding a protoplast buffer solution for re-suspending and precipitating, filtering again by using a cell filter, centrifuging at a low temperature and at a low speed, and removing the supernatant;
and fifthly, adding a protoplast buffer again to resuspend the protoplasts.
Further, in the first step, the banana tissue culture seedlings are tissue culture seedlings after 14 days of germination, and the sterilization is carried out for 15min by using 2% sodium hypochlorite;
the root tissue cut after the disinfection and cleaning is the root tissue cut from the root tip at a position of 1 cm.
Further, the protoplast buffer in the step two comprises KCl100mM and MgCl 2 ·6H 2 O20mM、CaCl 2 20mM, mannitol 0.6M and 2-N-morpholinoethanesulfonic acid 80mM, the solvent is sterile water, and the pH value is 5.5-5.8.
Further, the preparation process of the protoplast buffer in the second step comprises the following steps: weigh 0.75g KCl and 0.19g MgCl 2 ·6H 2 O、0.22gCaCl 2 0.22g mannitol and 1.76g 2-N-morpholinoethanesulfonic acid are dissolved in sterile water, the pH value of 0.1MTris-HCl is adjusted to 5.5-5.8, the sterile water is used for constant volume to 100mL, and finally a microporous filter membrane with the size of 0.22 mu m is used for filtration sterilization.
Further, the enzymolysis liquid in the second step is prepared by dissolving cellulase, educing enzyme, crashing enzyme and BSA in a protoplast buffer solution, and after the enzyme is completely dissolved, adjusting the pH value to 5.5-5.8 by adopting 0.1MTris-HCl, and fixing the volume of the protoplast buffer solution to the required volume; the weight of the components is as follows: the weight of the cellulase, the eductase, the crashing enzyme and the BSA respectively account for 1 to 3 percent, 0.3 to 1 percent, 0.01 to 0.1 percent and 0.1 percent of the total volume of the enzymolysis liquid according to the volume mL of the solution; the enzymolysis liquid is filtered and sterilized by a microporous filter membrane with the diameter of 0.22 mu m.
Further, the cellulase is cellulase R-10, and the eductase is eductase R-10.
Further, in the second step, the banana young root tissue is rapidly cut into 0.9-1.1mm slices; the enzymolysis reaction condition comprises that the reaction temperature is 28-30 ℃, and the sample is placed on a horizontal shaking table at 60-100 rpm/min for enzymolysis for 1-3 hours under the dark condition.
And in the third step, the enzymolysis liquid and the protoplast mixed liquid obtained in the second step are filtered by a 70 mu m cell filter, the protoplast buffer liquid is added into the filtrate for cleaning, and the supernatant is removed after centrifugation for 5-10 min by a centrifugal force of 300-500 Xg at the temperature of 4-8 ℃.
In the fourth step, the protoplast obtained in the third step is added with a protoplast buffer solution for re-suspending and precipitating, and is filtered again by a 40 mu m cell filter, and centrifuged for 5-10 min at 300-500 Xg centrifugal force at 4-8 ℃ and the supernatant is removed to obtain the protoplast precipitate.
Further, after the protoplasts are resuspended, the protoplast concentration and viability are detected by FDA staining and then by fluorescence inversion microscopy and a blood cell counting plate; when in dyeing, the final concentration of FDA in the mixed solution is 100 mug/mL, and the mixed solution is dyed for 5-30min at room temperature under dark condition.
Based on the technical scheme, the invention has the following advantages:
the dissociation method of banana young root protoplast disclosed by the invention realizes dissociation of banana young root protoplast, has simple operation, can rapidly and efficiently prepare protoplast, and the concentration of the protoplast separated by the optimal enzyme concentration can be up to 1.2 multiplied by 10 6 The single cell sequencing of 10 xGenomics can be directly carried out on the machine, the basis is laid for researching gene expression, development track and the like of banana root cells, and reference is provided for exploring protoplast preparation conditions of other types of plants and other parts.
Drawings
FIG. 1 is an experimental material used in example 1 of the present invention;
FIG. 2 is a morphology of banana young root protoplasts prepared in example 4 under a 20X optical microscope;
FIG. 3 is a morphology of banana young root protoplasts prepared in example 4 under a 40X optical microscope.
Detailed Description
In order to more clearly understand the technical scheme of the present invention, the technical scheme of the present invention will be described in further detail below with reference to the accompanying drawings and examples. It should be noted that, the embodiments described herein are provided with the technical solution of the present invention, and detailed implementation manners and specific operation procedures are given, but the protection scope of the present invention is not limited to the embodiments. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
An embodiment of the invention provides a dissociation method of banana young root protoplast, comprising the following steps: firstly, obtaining banana tissue culture seedlings by adopting a tissue culture technology, and cutting banana young root tissues after disinfection and cleaning to obtain an experimental material for preparing protoplasts; step two, adding a small amount of protoplast buffer solution to keep the experimental material moist, then rapidly cutting banana young root tissues into slices of about 1mm by a blade, sucking the protoplast buffer solution, adding an enzymolysis solution, and carrying out enzymolysis for 1-3 hours by shaking at a constant speed in a dark place; step three, filtering the enzymolysis liquid and protoplast mixed liquid obtained in the step two by adopting a 70 mu m cell filter, adding protoplast buffer solution into the filtrate for cleaning, centrifuging at low temperature and low speed, and removing supernatant; step four, adding a protoplast buffer solution for re-suspending and precipitating, filtering again by a 40 mu m cell filter, centrifuging at a low temperature and at a low speed, and removing the supernatant; and fifthly, adding a protoplast buffer solution again to resuspend the protoplasts, and detecting the concentration and the activity of the protoplasts by using a fluorescence inversion microscope and a blood cell counting plate after FDA staining.
The experimental reagent used in the embodiment of the invention is prepared as follows:
preparation of protoplast buffer: weigh 0.75g KCl and 0.19g MgCl 2 ·6H 2 O、0.22gCaCl 2 0.22g mannitol and 1.76g 2-N-morpholinoethanesulfonic acid are dissolved in sterile water, the pH value of 0.1MTris-HCl is adjusted to 5.5-5.8, the sterile water is used for constant volume to 100mL, and finally a microporous filter membrane with the size of 0.22 mu m is used for filtration sterilization.
Specifically, the protoplast buffer provided by the invention comprises KCl100mM and MgCl 2 ·6H 2 O20mM and CaCl 2 20mM, KCl, mgCl 2 ·6H 2 O and CaCl 2 Has the effect of protecting cytoplasmic membrane, and can prevent protoplast from being limited by physiological state of experimental materialThereby maintaining a better physiological state of the protoplasts.
Specifically, the protoplast buffer provided by the invention contains 0.6M mannitol, wherein mannitol is an osmotic stabilizer which is inert in metabolism, so that the osmotic pressure of the buffer can be maintained, and the dissociated protoplast can maintain a better cell morphology and a higher cell viability.
Specifically, the protoplast buffer provided by the invention comprises 80mM of 2-N-morpholinoethanesulfonic acid, and the 2-N-morpholinoethanesulfonic acid plays a role in stabilizing the pH value of a solution.
Preparing enzymolysis liquid: weighing cellulase R-101 g-3 g, educing enzyme R-100.3 g-1 g, crashing enzyme 10 mg-100 mg and BSA0.1g, dissolving in protoplast buffer, adjusting pH value to 5.5-5.8 by 0.1MTris-HCl after enzyme is completely dissolved, fixing volume to 100mL by protoplast buffer, and finally filtering and sterilizing by using microporous filter membrane of 0.22 mu m.
The weight of the components is as follows: solution volume mL meter, specifically:
the enzymatic hydrolysate provided by the invention comprises 1% -3% of cellulase R-10, 0.3% -1% of eduction enzyme R-10 and 0.01% -0.1% of crashing enzyme, wherein the cellulase R-10, the eduction enzyme R-10 and the crashing enzyme can be matched to effectively and rapidly decompose the cell wall of banana larva roots and free protoplasts;
the enzymatic hydrolysate provided by the invention contains 0.1% of BSA, and the BSA can improve the concentration of protein in the enzymatic hydrolysate, protect enzymes, prevent the decomposition and denaturation of the enzymes and the like.
The enzymolysis solutions with different enzyme contents have different degrees of influence on the time of dissociation of the protoplast from the banana young root, and the yield and the activity of the protoplast. The following is a detailed description of specific embodiments.
Example 1
The enzyme solution is prepared according to the step of preparing enzyme solution according to the proportion of containing cellulase R-101g, eductase R-100.3g, crashing enzyme 10mg and BSA0.1g in each 100mL enzyme solution.
The protoplast preparation method of example 1 comprises the following specific steps:
1. selection of experimental materials: selecting banana tissue culture seedlings which sprout for 14 days as shown in fig. 1, sterilizing for 15min by using 2% sodium hypochlorite, cleaning for 6-8 times by using sterile water, placing the banana tissue culture seedlings in a culture dish, and cutting young root tissues from a position of 1cm from the root tip by using a blade.
2. Dissociation of protoplasts: transferring the cut banana young root tissue into a new sterile culture dish, adding a small amount of protoplast buffer solution to keep the experimental material moist, then rapidly cutting the banana young root tissue into slices of about 1mm by a blade, sucking the protoplast buffer solution, adding an enzymolysis solution, placing the sample on a horizontal shaking table at 60-100 rpm/min for enzymolysis under dark condition, and setting the temperature of the shaking table to be 28-30 ℃.
3. Filtration and washing of protoplasts: filtering the obtained enzymolysis liquid and protoplast mixed liquid by a 70 mu m cell filter, adding protoplast buffer solution into the filtrate for cleaning, centrifuging for 5-10 min at the temperature of 4-8 ℃ by using the centrifugal force of 300-500 Xg, and removing the supernatant.
4. Adding the protoplast buffer solution again to re-suspend the sediment, filtering again by a 40 mu m cell filter, centrifuging for 5-10 min at the temperature of 4-8 ℃ by using a centrifugal force of 300-500 Xg, and removing the supernatant to obtain the protoplast sediment.
5. Microscopic examination and viability detection of protoplasts: the protoplast pellet is resuspended in 1mL protoplast buffer, 10. Mu.L protoplast suspension is aspirated and mixed with 90. Mu.L sterile water, i.e., diluted 10-fold and added drop wise onto a glass slide to observe the morphology of the protoplasts under a 20X, 40X microscope field of view; and (3) sucking 49 mu L of protoplast suspension and 1 mu L of FDA mother liquor with the concentration of 5mg/mL, uniformly mixing, dyeing for 5min at room temperature under a dark condition, then adding PBS for cleaning, centrifuging for 5-10 min by using 300-500 Xg centrifugal force, removing supernatant, re-suspending protoplasts by using 1mLPBS, dripping on a blood cell counting plate, and detecting the concentration and the activity of the protoplasts by using a fluorescence inversion microscope.
Specifically, the FDA is a commonly used plant cell protoplast vitality identification dye, the specific configuration of the FDA mother liquor is that 5mgFDA powder is weighed and dissolved in 1mL of acetone to prepare a solution with the concentration of 5mg/mL, the solution can be placed in a refrigerator with the temperature of 4 ℃ to be preserved in a dark place for standby, and the FDA mother liquor and protoplast suspension are mixed during dyeing to ensure that the final concentration of the FDA mother liquor is 100 mug/mL.
Specifically, the concentration of protoplast (number/ml) = (10×total number of protoplasts in 5 large cells in field of view at bright field/5) ×10 4 X dilution;
protoplast viability (%) = number of green-emitting protoplasts in 5 large cells in fluorescence field/total number of protoplasts in 5 large cells in bright field.
The protoplast enzymolysis time, the protoplast yield and the protoplast activity rate obtained in example 1 are shown in Table 1.
Example 2
The enzyme solution is prepared according to the step of preparing enzyme solution according to the proportion of containing cellulase R-101g, eductase R-100.5g, crashing enzyme 10mg and BSA0.1g in each 100mL enzyme solution.
Protoplast preparation method of example 2 referring to example 1, the protoplast enzymolysis time, the protoplast yield, and the protoplast activity ratio using example 2 are shown in table 1.
Example 3
The enzyme solution is prepared according to the proportion of cellulase R-102g, eductase R-100.5g, crashing enzyme 10mg and BSA0.1g which are contained in each 100mL enzyme solution by referring to the step of preparing the enzyme solution.
Protoplast preparation method of example 3 referring to example 1, the enzymolysis time, the protoplast yield and the protoplast activity ratio using example 3 are shown in table 1.
Example 4
The enzyme solution is prepared according to the proportion of cellulase R-102g, eductase R-100.5g, crashing enzyme 50mg and BSA0.1g which are contained in each 100mL enzyme solution by referring to the step of preparing the enzyme solution.
Protoplast preparation method of example 4 referring to example 1, the protoplast enzymolysis time, the protoplast yield, and the protoplast activity ratio using example 4 are shown in table 1.
Example 5
The enzyme solution is prepared according to the step of preparing enzyme solution according to the proportion of containing cellulase R-102g, eductase R-101g, crashing enzyme 10mg and BSA0.1g in each 100mL enzyme solution.
Protoplast preparation method of example 5 referring to example 1, the protoplast enzymolysis time, the protoplast yield, and the protoplast activity ratio using example 5 are shown in table 1.
Example 6
The enzyme solution is prepared according to the step of preparing enzyme solution according to the proportion of containing cellulase R-102g, eductase R-101g, crashing enzyme 50mg and BSA0.1g in each 100mL enzyme solution.
Protoplast preparation method of example 6 referring to example 1, the enzymolysis time, the protoplast yield and the protoplast activity ratio using example 6 are shown in table 1.
Example 7
The enzyme solution is prepared according to the step of preparing enzyme solution according to the proportion of 100mL enzyme solution containing cellulase R-102g, eductase R-101g, crashing enzyme 100mg and BSA0.1 g.
Protoplast preparation method of example 7 referring to example 1, the enzymolysis time, the protoplast yield and the protoplast activity ratio using example 7 are shown in table 1.
Example 8
The enzyme solution is prepared according to the proportion of cellulase R-103g, eductase R-100.5g, crashing enzyme 10mg and BSA0.1g which are contained in each 100mL enzyme solution by referring to the step of preparing the enzyme solution.
Protoplast preparation method of example 8 referring to example 1, the enzymolysis time, the protoplast yield and the protoplast activity ratio using example 8 are shown in table 1.
Example 9
The enzyme solution is prepared according to the proportion of cellulase R-103g, eductase R-100.5g, crashing enzyme 50mg and BSA0.1g which are contained in each 100mL enzyme solution by referring to the step of preparing the enzyme solution.
Protoplast preparation method of example 9 referring to example 1, the enzymolysis time, the protoplast yield and the protoplast activity ratio using example 9 are shown in table 1.
Example 10
The enzyme solution is prepared according to the step of preparing enzyme solution according to the proportion of containing cellulase R-103g, eductase R-101g, crashing enzyme 10mg and BSA0.1g in each 100mL enzyme solution.
Protoplast preparation method of example 10 referring to example 1, the protoplast enzymolysis time, the protoplast yield, and the protoplast activity ratio using example 10 are shown in table 1.
Example 11
The enzyme solution is prepared according to the step of preparing enzyme solution according to the proportion of containing cellulase R-103g, eductase R-101g, crashing enzyme 50mg and BSA0.1g in each 100mL enzyme solution.
Protoplast preparation method of example 11 referring to example 1, the protoplast enzymolysis time, the protoplast yield, and the protoplast activity ratio using example 11 are shown in table 1.
Example 12
The enzyme solution is prepared according to the step of preparing enzyme solution according to the proportion of 100mL enzyme solution containing cellulase R-103g, eductase R-101g, crashing enzyme 100mg and BSA0.1 g.
Protoplast preparation method of example 12 referring to example 1, the protoplast enzymolysis time, the protoplast yield, and the protoplast activity ratio using example 12 are shown in table 1.
Example 13
The enzyme solution is prepared according to the step of preparing enzyme solution according to the proportion of containing cellulase R-103g, eductase R-102g, crashing enzyme 10mg and BSA0.1g in each 100mL enzyme solution.
Protoplast preparation method of example 13 referring to example 1, the enzymolysis time, the protoplast yield and the protoplast activity ratio using example 13 are shown in table 1.
Example 14
The enzyme solution is prepared according to the step of preparing enzyme solution according to the proportion of containing cellulase R-103g, eductase R-102g, crashing enzyme 50mg and BSA0.1g in each 100mL enzyme solution.
Protoplast preparation method of example 14 referring to example 1, the protoplast enzymolysis time, the protoplast yield, and the protoplast activity ratio using example 14 are shown in table 1.
Example 15
The enzyme solution is prepared according to the step of preparing enzyme solution according to the proportion of 100mL enzyme solution containing cellulase R-103g, eductase R-102g, crashing enzyme 100mg and BSA0.1 g.
Protoplast preparation method of example 15 referring to example 1, the enzymolysis time, the protoplast yield and the protoplast activity ratio using example 15 are shown in table 1.
Table 1 shows the extraction results of banana young root protoplasts under the conditions of different enzyme concentrations in examples 1-15.
TABLE 1
Based on the above results, the following components are expressed in weight g: the method comprises the steps that (1) the volume mL of a solution is calculated, and enzymolysis liquid containing 2% of cellulase R-10, 0.5% of educing enzyme R-10 and 0.05% of crashing enzyme is adopted, so that the number of protoplasts obtained is the largest, the shape is optimal, the background is clean, and the enzymolysis time is proper; meanwhile, the concentration of each enzyme in the enzyme combination for obtaining protoplast with the same level is the lowest, so the invention adopts the enzymolysis liquid containing 2 percent of cellulase R-10, 0.5 percent of isolated enzyme R-10 and 0.05 percent of crashing enzyme to prepare banana young root single cell suspension for the subsequent 10X Genomics single cell sequencing and loading experiment.
The protoplast suspension extracted by using the enzymatic hydrolysate was observed under a microscope, and the results are shown in fig. 2 and 3. The obtained protoplast has clear structure and complete shape.
The dissociation method of banana young root protoplast disclosed by the invention realizes dissociation of banana young root protoplast, is simple to operate, can rapidly and efficiently prepare protoplast, has the advantages of multiple number and good morphology of protoplast separated by the combination of optimal enzyme concentration and enzymolysis liquid, has proper enzymolysis time, can directly carry out subsequent 10X Genomics single-cell sequencing on-machine experiments, lays a foundation for researching banana root cell gene expression, development track and the like, and provides a reference basis for exploring protoplast preparation conditions of other types of plants and other parts.
Finally, it should be pointed out that, for a person skilled in the art, various corresponding changes and modifications can be given to the solutions and concepts of the above embodiments, all of which are intended to be covered by the scope of the invention as claimed.
Claims (9)
1. A method for dissociation of banana young root protoplasts, the method comprising the steps of:
firstly, obtaining banana tissue culture seedlings by adopting a tissue culture technology, and cutting banana young root tissues after disinfection and cleaning to obtain an experimental material for preparing protoplasts;
step two, adding a small amount of protoplast buffer solution to keep the experimental material moist, wherein the protoplast buffer solution comprises KCl100mM and MgCl 2 ·6H 2 O 20mM、CaCl 2 20mM, mannitol 0.6M and 2-N-morpholinoethanesulfonic acid 80mM, wherein the solvent is sterile water, the pH value is 5.5-5.8, then the banana young root tissue is rapidly sliced by a blade, the protoplast buffer solution is sucked off, and then the enzymolysis solution is added, wherein the enzymolysis solution is prepared by dissolving cellulase, eductase, crashing enzyme and BSA in the protoplast buffer solution according to the weight of the components g: the weight of the cellulase, the eductase, the crashing enzyme and the BSA respectively account for 1 to 3 percent, 0.3 to 1 percent, 0.01 to 0.1 percent and 0.1 percent of the total volume of the enzymolysis liquid according to the volume mL of the solution; the pH value is 5.5-5.8, and the enzyme is hydrolyzed for 1-3 hours by shaking at a constant speed in a dark place;
step three, filtering the enzymolysis liquid and protoplast mixed liquid obtained in the step two by adopting a cell filter, adding protoplast buffer solution into the filtrate for cleaning, centrifuging at low temperature and low speed, and removing supernatant;
step four, adding a protoplast buffer solution for re-suspending and precipitating, filtering again by using a cell filter, centrifuging at a low temperature and at a low speed, and removing the supernatant;
and fifthly, adding a protoplast buffer again to resuspend the protoplasts.
2. The dissociation method of claim 1, wherein in step one, the banana tissue culture seedlings are tissue culture seedlings after 14 days of germination;
sterilizing with 2% sodium hypochlorite for 15min;
the root tissue cut after the disinfection and cleaning is the root tissue cut from the root tip at a position of 1 cm.
3. The dissociation method of claim 1, wherein in step two, the preparation of the protoplast buffer is performed by: weighing 0.75g KCl and 0.19g MgCl 2 ·6H 2 O、0.22g CaCl 2 0.22g mannitol and 1.76g 2-N-morpholinoethanesulfonic acid are dissolved in sterile water, the pH value of 0.1M Tris-HCl is adjusted to 5.5-5.8, the sterile water is used for constant volume to 100mL, and finally a microporous filter membrane with the size of 0.22 mu M is used for filtration sterilization.
4. The dissociation method as claimed in claim 1, wherein the enzymolysis solution in the second step is prepared by dissolving cellulase, educing enzyme, crashing enzyme and BSA in a protoplast buffer, and after the enzyme is completely dissolved, adjusting the pH value by using 0.1M Tris-HCl, and fixing the volume of the protoplast buffer to a required volume; the enzymolysis liquid is filtered and sterilized by a microporous filter membrane with the diameter of 0.22 mu m.
5. The method according to claim 1 or 4, wherein the cellulase is cellulase R-10 and the eductase is eductase R-10.
6. The dissociation method of claim 1, wherein in step two, banana young root tissue is rapidly cut into 0.9-1.1mm sheets; the enzymolysis reaction condition includes that the reaction temperature is 28-30 ℃, and the sample is placed on a horizontal shaking table at 60-100 rpm/min for enzymolysis under the dark condition.
7. The dissociation method according to claim 1, wherein in the third step, the mixture of the enzymatic hydrolysate and the protoplast obtained in the second step is filtered through a 70 μm cell filter, and the filtrate is washed with a protoplast buffer, and centrifuged at 300 to 500 Xg for 5 to 10 minutes at 4 to 8 ℃.
8. The dissociation method according to claim 1, wherein in the fourth step, the protoplast obtained in the third step is added with a protoplast buffer to re-suspend and precipitate, and the solution is filtered again by a 40 μm cell filter, and centrifuged at 300 to 500 Xg centrifugal force at 4 to 8℃for 5 to 10 minutes, and the supernatant is removed to obtain a protoplast precipitate.
9. The dissociation method of claim 1, wherein said step five is performed by detecting the concentration and viability of protoplasts after re-suspending the protoplasts by FDA staining and by fluorescence inversion microscopy and a blood cell counting plate; when dyeing, the final concentration of FDA in the mixed solution is 100 mug/mL, and the dyeing is carried out for 5-30min at room temperature under the dark condition.
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| WO2000001221A2 (en) * | 1998-07-03 | 2000-01-13 | Heide Schnabl | Methods for interspecific hybridization |
| CN101139582A (en) * | 2007-08-10 | 2008-03-12 | 中山大学 | Method for acquiring banana somatic cell hybrid by using protoplast asymmetric fusion technology |
| CN115340972A (en) * | 2022-08-31 | 2022-11-15 | 福建农林大学 | Preparation method and application of banana protoplast |
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| WO2000001221A2 (en) * | 1998-07-03 | 2000-01-13 | Heide Schnabl | Methods for interspecific hybridization |
| CN101139582A (en) * | 2007-08-10 | 2008-03-12 | 中山大学 | Method for acquiring banana somatic cell hybrid by using protoplast asymmetric fusion technology |
| CN115340972A (en) * | 2022-08-31 | 2022-11-15 | 福建农林大学 | Preparation method and application of banana protoplast |
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