CN109136167B - Preparation method of lily mesophyll protoplast - Google Patents
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Abstract
The invention discloses a preparation method of lily mesophyll protoplasts, which comprises the following steps: (1) taking lily plants with unopened buds, and carrying out dark treatment; (2) taking plant leaves, cleaning and disinfecting; (3) adding an enzymolysis solution into the mesophyll material, carrying out vacuum extraction in a dark place, and then standing in the dark place for enzymolysis for a plurality of hours to obtain an enzymolysis solution containing mesophyll protoplasts; (4) diluting the obtained enzymolysis liquid with W5 solution, filtering with mesh screen, centrifuging the obtained filtrate at low speed, discarding supernatant, resuspending the protoplast precipitate with W5 solution, centrifuging at low speed, discarding supernatant, and repeatedly washing once; resuspend the protoplast pellet with MMG solution. The method overcomes the problem of low separation efficiency of the lily mesophyll protoplast, and effectively establishes the preparation method of the lily mesophyll protoplast.
Description
Technical Field
The invention relates to a preparation technology of plant protoplast, in particular to a preparation method of lily mesophyll protoplast.
Background
Protoplasts refer to naked cells coated with a plasma membrane with the cell wall removed. The protoplast has cell totipotency and can be regenerated into a complete plant. The protoplast can take in exogenous gene, virus, plasmid and other matters, and is one excellent material for plant cell engineering to perform a series of operation and gene conversion. In addition, genetic material can be changed by protoplast fusion technology to obtain new species. Therefore, the protoplast separation and regeneration have important significance in the fields of plant genetic breeding, character improvement, genetic transformation and the like.
Lily (Lilium spp.) is a perennial bulbous flower of the genus Lilium of the family liliaceae, and is a famous ornamental flower in the world. With the expansion of the market, people's demand for new varieties of lily is increasing day by day, and the improvement of the genetic traits of lily becomes an important content for the development of the industry. In addition, the development of molecular biology of lily is deepened day by day, but because the genetic transformation system of lily is difficult to establish and the gene function research is obviously delayed, the transient expression system has important significance for the research of lily gene function. Protoplast isolation is a basic technology for genetic trait improvement and transient expression.
The lily is used as a high-heterozygotic monocotyledon, the difficulty of protoplast isolation culture is high, and the research reports about the preparation of lily protoplasts at home and abroad are few at present. Horita (Horita M, Morohashi H, Komai F.Regeneration of Flowering Plants From differential simple Plants by Means of a culture of a Nurse culture of Plants [ J ], 2002, 5 (215): 880-884) by isolating Protoplasts of lilium oriental and lilium arborescens, a first successful cultivation of a hybrid plant of somatic cells of the initial lilium arborescens was carried out in 2002 in a Nurse culture manner. In 2006, preliminary experiments were performed on the preparation and culture of lily protoplasts from oriental lily 'Bernini', asian lily 'Elite' and red lead (l.lancifolium) as materials, but no regenerated plants could be obtained. On the basis, in recent years, certain researches are carried out on protoplast separation and purification technologies of oriental lilies, wild lilies, edible lilies and the like, but the used materials are mostly concentrated on calluses or suspension cells, and the researches on directly taking mature plants are less.
Disclosure of Invention
The invention aims to provide a simple and efficient preparation method of lily mesophyll protoplasts.
In order to achieve the purpose of the invention, the invention provides a preparation method of lily mesophyll protoplasts, which comprises the following steps:
(1) carrying out dark treatment on lily plants;
(2) cleaning and disinfecting the plant leaves subjected to dark treatment in the step (1);
(3) adding an enzymolysis solution into the disinfected leaf material, performing vacuum extraction in a dark place, and then standing in the dark place for enzymolysis for a plurality of hours to obtain an enzymolysis solution containing mesophyll protoplasts;
(4) diluting the enzymatic hydrolysate obtained in the step (3) by using a W5 solution, filtering by using a mesh screen, centrifuging the obtained filtrate at a low speed, discarding the supernatant, re-suspending the protoplast precipitate by using a W5 solution, centrifuging at a low speed, discarding the supernatant, re-suspending the protoplast precipitate by using a W5 solution, centrifuging at a low speed, and re-suspending the protoplast precipitate by using an MMG solution;
wherein, the active ingredients of the enzymolysis solution in the step (3) are cellulase and eductase, and the concentration ratio of the cellulase to the eductase is 1-2: 0.2-1, preferably 2: 1.
Preferably, the dark treatment in the step (1) is carried out for 24-72 h, preferably 48 h.
In the method, the leaves taken in the step (2) are the top, middle and lower leaves of the plant, and preferably the top leaves.
Preferably, the specific method for disinfecting the leaves in the step (2) is as follows: soaking in soap water, washing with running water, soaking in 70-75% alcohol for 5min, washing with sterile water, and sucking to remove water.
Preferably, the formula of the enzymolysis solution in the step (3) is as follows: 1-2% of cellulase R-10, 0.2-1% of macerozyme R-10, 10-20mmol/L of potassium chloride, 10-20mmol/L of 2- (N-morpholino) ethanesulfonic acid, 10-15mmol/L of calcium chloride, 0.4-0.6mol/L of mannitol (used as an osmotic pressure regulator) and 0.1% of bovine serum albumin, and the pH value is 5.7-5.8. The enzymolysis solution is prepared by water, and is used after being filtered and sterilized by a 0.45 mu m micropore filter membrane.
More preferably, the formulation of the enzymatic solution is: 2% cellulase R-10, 0.5% macerase R-10, 20mmol/L potassium chloride, 20 mmol/L2- (N-morpholino) ethanesulfonic acid, 10mmol/L calcium chloride, 0.4mol/L mannitol (as an osmo-regulator) and 0.1% bovine serum albumin, pH 5.7-5.8.
Preferably, the enzymolysis time of the mesophyll in the step (3) is 4-10 hours, preferably 7 hours.
The formula of the W5 solution in the invention is as follows: 2 mmol/L2- (N-morpholino) ethanesulfonic acid, 154mmol/L sodium chloride, 125mmol/L calcium chloride, 5mmol/L potassium chloride, and pH 5.7-5.8.
The formula of the MMG solution is as follows: 4 mmol/L2- (N-morpholino) ethanesulfonic acid, 0.4mol/L mannitol, 15mmol/L magnesium chloride, and pH 5.7-5.8.
Preferably, the mesh in step (4) is a nylon mesh with 200 and 300 meshes.
Preferably, the low-speed centrifugation conditions in step (4) are: centrifuging at the temperature of 4 ℃ and the temperature of 500 ℃ at the speed of 700r/min for 5-6 min.
The lily variety of the invention includes but is not limited to oriental lily hybrid 'Sobang' (Lilium 'Sorbone').
In one embodiment of the invention, the method comprises the steps of:
1) selecting lily plants which are good in growth state and free of diseases and insect pests and grow for about 30-40 days, and performing dark treatment for 24-72 hours at room temperature;
2) taking the leaves of the plants subjected to the dark treatment in the step 1), washing with clear water, and disinfecting;
3) cutting the disinfected blade material into strips with the width of about 2-3mm, weighing 1g of the strips, immersing the strips into 10mL of enzymolysis solution, extracting the strips in the dark for 30min by using a vacuum pump, and then standing the strips at room temperature under the dark condition for enzymolysis for 4-10 h;
4) diluting the enzymolysis liquid obtained in the step 3) with an isovolumetric W5 solution to obtain a diluent; wetting a 250-mesh nylon mesh screen with a W5 solution, filtering the diluent, centrifuging the obtained filtrate at 4 ℃ for 5-6min at 500-700r/min, discarding the supernatant, re-suspending the protoplast precipitate with 10mL of a pre-cooled W5 solution, centrifuging at 500-700r/min for 5-6min, discarding the supernatant, re-suspending the protoplast precipitate with 10mL of a pre-cooled W5 solution, centrifuging at 500-700r/min for 5-6min, and finally re-suspending the protoplast precipitate with an MMG solution.
By the technical scheme, the invention at least has the following advantages and beneficial effects:
according to the method, lily leaves are subjected to dark pretreatment, and the protoplast of the lily is separated and purified by using the combination of cellulase, isolation enzyme, different enzymolysis time, material-taking parts and the like, so that the problems of low separation efficiency and long period of the lily protoplast are effectively solved, and a large amount of complete protoplast can be obtained only in about 48 hours. A lily protoplast preparation system is established, so that the extraction efficiency of the mesophyll protoplast is improved by about 72 percent, and the activity of the protoplast is ensured to be more than 70 percent. On the basis, operations such as protoplast culture, transient transformation, cell fusion and the like can be carried out, and the method has important significance for the research of lily gene function and the improvement of genetic traits.
The invention takes the leaves of the oriental lily Sophora as materials, separates and purifies protoplasts, establishes an efficient protoplast preparation system, and aims to provide technical support for the research fields of establishment and improvement of genetic characters, molecular breeding and the like of lily instant transformation systems.
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FIG. 1 is a diagram of the separation and purification of the leaf protoplast from the material-taken part in the preferred embodiment of the present invention.
FIG. 2 shows mesophyll protoplasts after isolation in a preferred embodiment of the invention.
FIG. 3 is an FDA-stained Lily 'Sopont' mesophyll protoplast under a fluorescence microscope in accordance with a preferred embodiment of the invention.
FIG. 4 shows the change in the yield of protoplasts of lily mesophyll after dark treatment in the preferred embodiment of the invention.
FIG. 5 is a graph showing the relationship between the yield of protoplasts of lily leaves and the factors in a preferred embodiment of the invention.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.
The enzymatic solutions used in the following examples were prepared as follows: cellulase R-10, isolation enzyme R-10, 20mmol/L potassium chloride, 20 mmol/L2- (N-morpholino) ethanesulfonic acid, 10mmol/L calcium chloride and 0.1% bovine serum albumin, and 0.4mol/L mannitol as osmotic pressure regulator, pH adjusted to 5.7, and 0.45 μm microfiltration membrane filtration sterilized.
The formula of the protoplast rinsing liquid W5 is as follows: 2 mmol/L2- (N-morpholino) ethanesulfonic acid, 154mmol/L sodium chloride, 125mmol/L calcium chloride, 5mmol/L potassium chloride, pH 5.7.
The formula of the MMG heavy suspension liquid is as follows: 4 mmol/L2- (N-morpholino) ethanesulfonic acid, 0.4mol/L mannitol, 15mmol/L magnesium chloride, pH 5.7.
Example 1 preparation of Lily mesophyll protoplasts
This example provides a method for the isolation and purification of lily mesophyll protoplasts, the method comprising the steps of:
1. dark pretreatment: selecting good-growth-state and pest-free oriental lily 'Sophora' plants which grow for about 30d, and performing dark treatment for 48h at room temperature.
2. Material treatment: washing the new top leaves (figure 1) of the plants obtained in the step 1 with clear water, soaking the plants in soap water for disinfection, washing the plants with running water, soaking the plants in 75% alcohol for 5min, washing the plants with sterile water, and sucking water for later use.
3. And (3) enzymolysis treatment: and (3) cutting the material obtained in the step (2) into strips with the width of 2mm by using a sterilized blade, weighing 1g of the strips, and putting the strips into 10ml of enzymolysis solution by using tweezers. Extracting with vacuum pump in dark for 30 min. Standing at room temperature in dark for several hours. When the enzymolysis liquid changes color, the culture dish is gently shaken to promote the release of the protoplast. The concentration of two enzymes in the enzymolysis solution of the embodiment is: 2 percent of cellulase R-10 and 1 percent of macerozyme R-10, and the enzymolysis time is 7 hours.
4. And (3) purification treatment: diluting the enzymolysis liquid obtained in the step 4 with an equal volume of W5 solution. The nylon mesh was wetted with the W5 solution, and the enzymatic hydrolysate containing leaf protoplasts was filtered through a 250-mesh screen. The filtrate was centrifuged at low speed at 700r/min at 4 ℃ for 6 min. The supernatant was discarded and the protoplasts were gently resuspended in 10ml of W5 solution pre-cooled on ice. The resuspension was centrifuged at 700r/min for 6min at low speed. The W5 solution was removed as much as possible without touching the protoplast pellet and the washing was repeated once. The precipitated protoplasts were resuspended in MMG solution. The counts were observed under the microscope (FIG. 2).
5. And (3) identifying the yield of the protoplast: the protoplast yield is measured by adopting a simple counting method to measure the field area of an objective lens; then, the proper dropping volume of the suspension is determined (the standard is cell monolayer arrangement, the cover glass is uniformly distributed, the edge has no overflowing cells and liquid, and no vacuole), the suspension with the determined large volume is sucked by a pipette, the process is repeated for 6 times, and the number of protoplasts under the visual field is counted. And finally, calculating the density of the protoplast according to a formula.
6. Protoplast density (per g) — (1000/appropriate volume of suspension added) × (24 × 24/area of field) × number average value of protoplasts per field/mass of enzymatic material
Wherein 24X 24 is the size of the cover glass used, i.e., (24X 24) mm2。
7. And (3) determining the activity of the protoplast: the Fluorescein Diacetate (FDA) staining method was used. Dissolving FDA in acetone to obtain 5mg/mL solution, storing at 4 deg.C, collecting 0.5mL purified protoplast, adding 12 μ L FDA solution, standing for 5-10min, and observing under fluorescence microscope. At this time, viable protoplasts showed yellow-green fluorescence (FIG. 3), 5 representative fields were selected for statistics and averaged.
8. Protoplast viability ═ 100% (number of fluorescent protoplasts in dark field/number of protoplasts in bright field) ×
Example 2 Effect of dark treatment on preparation of Lily mesophyll protoplasts
For the dark treatment time in the method of example 1, the invention sets 5 time gradients: 0. 24, 36, 48 and 72 h. The dark-treated material was subjected to enzymatic hydrolysis and purification as in example 1. The results show that the leaves are suitable for 48h treatment (Table 1).
TABLE 1 Lily protoplast yield and viability after dark treatment
Note: the absence of the same lower case letters indicates significant differences at the P < 0.05 level.
As shown in Table 1, the dark treatment has a very significant effect on the yield of protoplasts from the lily leaves, the yield of protoplasts increases significantly with the increase of the treatment time (FIG. 4), and the yield of protoplasts from the lily leaves reaches a maximum of 7.30X 10 after 48h treatment (FIG. 4)5The protoplast viability also reached a maximum of 77.83% per g. The leaf protoplast yield can be improved by about 72% at most by dark treatment, the protoplast yield and the activity are comprehensively considered, and the optimal dark pretreatment time for the separation of the lily leaf protoplasts is 48 h.
Example 3 influence of enzyme concentration ratio, enzymolysis time and material-drawing period on preparation of Lily mesophyll protoplast
Aiming at the material taking period, the enzyme concentration ratio and the enzymolysis time in the steps 2 and 4, the invention sets the L with 4 factors and 3 levels9(34) Orthogonal test (table 2). The concentration levels of cellulase are 1%, 1.5% and 2%; the concentration of the eductase is set to be 0.2 percent, 0.5 percent and 1 percent; the enzymolysis time gradient of the leaves is 4h, 7h and 10 h; the parts of the leaves from which the plant grows are the upper, middle and bottom leaves of the plant growing about 30d respectively (figure 1). And (4) performing enzymolysis according to each group of test conditions, and counting the yield and the activity of the protoplast.
The results are shown in table 2, and it can be seen from the extreme differences (R values) that the four factors affect the yield of leaf protoplasts in the following order: the concentration of the cellulase is more than the concentration of the material-taking part is more than the concentration of the eductase is more than the enzymolysis time. They all had a very significant effect on leaf protoplast yield, with 2% cellulase very significantly different from both the other two levels, with very significant differences between the levels of both the concentration of the eductase and the time of the enzymatic hydrolysis, and with the upper leaf protoplast yield very significantly higher than both the other two levels. Therefore, the theoretically best combination of the blade orthogonal test is A3B3C2D1I.e., 2% cellulase + 1% macerase + 7h + time of enzymolysis, and upper new leaves were taken (fig. 5).
TABLE 2L9(34) Orthogonal experimental result of lily leaf protoplast
Note: the absence of the same lower case letters indicates a significant difference at the P < 0.05 level and the absence of the same upper case letters indicates a significant difference at the P < 0.01 level.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (1)
1. The preparation method of the lily mesophyll protoplast is characterized by comprising the following steps:
(1) dark pretreatment: selecting a good-growing-state and pest-free oriental lily 'Sophora' plant which is in a good state for 30 days to carry out dark treatment for 48 hours at room temperature;
(2) material treatment: washing new leaves at the top of the plant obtained in the step (1) with clear water, soaking the plant in soap water for disinfection, washing with running water, soaking in 75% alcohol for 5min, washing with sterile water, and sucking water for later use;
(3) and (3) enzymolysis treatment: cutting the material obtained in the step (2) into strips with the width of 2mm by using a sterilized blade, weighing 1g of strips, and putting the strips into 10ml of enzymolysis solution by using tweezers; extracting in the dark for 30min by using a vacuum pump; standing at room temperature in dark for enzymolysis for 7 h; when the enzymolysis liquid changes color, the culture dish is slightly shaken to promote the release of the protoplast;
wherein, the preparation of the enzymolysis solution is as follows: 2% cellulase R-10, 1% macerozyme R-10, 20mmol/L potassium chloride, 20 mmol/L2- (N-morpholino) ethanesulfonic acid, 10mmol/L calcium chloride and 0.1% bovine serum albumin, and 0.4mol/L mannitol is used as osmotic pressure regulator, pH is adjusted to 5.7, and the mixture is used after filtration and sterilization by a 0.45 mu m microporous filter membrane;
(4) and (3) purification treatment: diluting the enzymolysis liquid obtained in the step (3) with an equal volume of W5 solution; wetting a nylon mesh screen by using a W5 solution, and filtering enzymatic hydrolysate containing the leaf protoplast by using a 250-mesh screen; centrifuging the filtrate at 4 deg.C at 700r/min for 6 min; discarding the supernatant, and gently resuspending the protoplasts with 10ml of W5 solution pre-cooled on ice; centrifuging the heavy suspension at low speed of 700r/min for 6 min; the W5 solution is removed as much as possible under the condition of not touching the protoplast sediment, and the washing is repeated once; resuspending the precipitated protoplasts in MMG solution; observing and counting under a microscope;
wherein, the formula of the W5 solution is as follows: 2 mmol/L2- (N-morpholino) ethanesulfonic acid, 154mmol/L sodium chloride, 125mmol/L calcium chloride, 5mmol/L potassium chloride, pH 5.7;
the formula of the MMG solution is as follows: 4 mmol/L2- (N-morpholino) ethanesulfonic acid, 0.4mol/L mannitol, 15mmol/L magnesium chloride, pH 5.7.
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| CN110878281A (en) * | 2019-12-12 | 2020-03-13 | 武汉德翊环境科技有限公司 | Method for extracting submerged plant agave protoplast |
| CN110982775B (en) * | 2019-12-26 | 2023-10-20 | 河北农业大学 | Preparation method of bentgrass protoplast creeping |
| CN112126616B (en) * | 2020-06-30 | 2022-06-07 | 电子科技大学 | Larix gmelinii protoplast separation purification and transient expression method |
| CN112251395A (en) * | 2020-10-29 | 2021-01-22 | 西南大学 | Separation method of loquat protoplast |
| CN115505596A (en) * | 2022-08-04 | 2022-12-23 | 沈阳农业大学 | Lily protoplast-based transient transformation method |
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