Preparation method and application of phyllostachys edulis root tip protoplast
Highly active and pure moso bamboo root tip protoplasts were prepared by washing with mannitol and gradient centrifugation with sucrose solution, which solved the problem of many cell fragments and impurities in the moso bamboo root single-cell isolation system and enabled the application of moso bamboo root single-cell sequencing.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- INT CENT FOR BAMBOO & RATTAN
- Filing Date
- 2023-05-15
- Publication Date
- 2026-06-05
AI Technical Summary
Existing technologies make it difficult to prepare high-quality moso bamboo root protoplasts, resulting in a large amount of cell debris and release of intracellular contents in the moso bamboo root single-cell isolation system, which is difficult to meet the requirements of single-cell sequencing.
The raw material of moso bamboo root tips was washed with 0.6-0.8M mannitol, and enzymatic hydrolysis was performed for 2.5-3.5 hours. Subsequently, it was purified by gradient centrifugation with 18-22% sucrose solution to remove impurities and prepare highly active protoplasts.
We obtained moso bamboo root tip protoplasts with cell viability of over 85% and few impurities, which meet the requirements for constructing single-cell transcriptome sequencing libraries.
Smart Images
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Abstract
Description
Technical Field
[0001] This invention relates to the field of plant tissue protoplast isolation technology, and in particular to a method for preparing and applying protoplasts from the root tips of moso bamboo. Background Technology
[0002] Single-cell transcriptome sequencing technology can reveal the expression of all genes across the entire genome at the single-cell level, making it suitable for studying expression heterogeneity between cells and helping to elucidate how individual cell function regulates organismal development. Single-cell transcriptome sequencing of various plants, including Arabidopsis thaliana, rice, tomato, maize, peanut, poplar, and tea, has been reported, confirming the high heterogeneity of plant tissues and the presence of specific marker genes for different cell types, cell division, and cell differentiation. Compared to animal cells, plant cells have cell walls, requiring enzymatic digestion to break them down. Therefore, preparing plant protoplast suspensions is more challenging.
[0003] Moso bamboo (Phyllostachys edulis) is the bamboo species with the largest area of cultivation, the highest ecological and economic value, and the best development and utilization in my country. For a long time, research on moso bamboo has focused primarily on its above-ground parts, while theoretical research on its underground system, especially its root system, has been severely lacking. The root system is crucial for water absorption and energy flow in bamboo. Therefore, using single-cell transsequencing technology to elucidate the cell types and developmental mechanisms of moso bamboo roots is of great significance.
[0004] Currently, there are no reports on the isolation of single cells from bamboo roots for use in single-cell sequencing applications. This is because current single-cell isolation systems for bamboo root tissue cannot guarantee the preparation of high-quality protoplasts. Specifically, during protoplast preparation, the removal of the cell wall weakens cell stability, making them prone to breakage. This results in the release of large amounts of cell debris and intracellular contents, such as starch granules, into the enzymatic hydrolysate, making it difficult to obtain high-quality cells. This is especially true when cells are sorted using the 10×Genomics platform, often leading to low cell capture rates.
[0005] Therefore, there is an urgent need to establish a method for isolating single-cell protoplasts from moso bamboo roots that can balance cell survival rate and high cell capture rate. This is of great importance to the development of single-cell sequencing technology in moso bamboo, thereby promoting the application of single-cell sequencing technology in moso bamboo. Summary of the Invention
[0006] To address the shortcomings of existing technologies, the purpose of this invention is to provide a method for preparing protoplasts that achieves cell viability of over 85% while minimizing impurities in the protoplasts, thus ensuring cell quality meets the requirements for constructing single-cell transcriptome sequencing libraries from moso bamboo roots.
[0007] First, this invention provides a method for preparing protoplasts, comprising:
[0008] (1) Material selection: Collect the root tips of the bamboo shoots at the base of the bamboo shoots as raw materials;
[0009] (2) Isolation of protoplasts: The raw material was washed with 0.6-0.8M mannitol, and then the washed raw material was placed in an enzymatic hydrolysis solution for 2.5-3.5 hours to obtain a protoplast single-cell suspension;
[0010] The enzymatic hydrolysate comprises: 0.6–0.8 M mannitol, 2–4% cellulase R10, 0.5–1% dissociative enzyme R10, 0.1–0.3% pectinase, 15–25 mM KCl, 15–25 mM MES, 0.05–0.15% bovine serum albumin, and 5–15 mM CaCl2;
[0011] (3) Purification of protoplasts: Centrifuge the protoplast single-cell suspension at 40g-60g, remove the supernatant, and resuspend the cells in mannitol to obtain a cell resuspension; mix the cell resuspension with a sucrose solution with a mass concentration of 18-22%, and centrifuge at 40g-60g to obtain the protoplasts.
[0012] This invention has found that when the root tip of the bamboo shoot at the base of the bamboo shoot is selected as raw material, compared with other parts of bamboo (such as the leaf sheath of bamboo seedlings), the cells are more easily broken, releasing a large amount of starch granules, resulting in a relatively low purity of protoplasts. Moreover, the cells are more sensitive to separation and purification conditions, making it difficult to obtain high cell activity.
[0013] Through extensive experiments on separation and purification conditions, this invention has found that by using a combination of the above-mentioned enzyme hydrolysate components, osmotic pressure, enzyme hydrolysis time, and purification methods, it is possible to obtain moso bamboo root tip protoplasts with high cell activity and fewer impurities.
[0014] The use of mannitol for repeated washing before enzymatic hydrolysis effectively removes starch granules, which facilitates a more efficient enzymatic hydrolysis reaction in the subsequent process.
[0015] Furthermore, through extensive experimentation, this invention optimized the culture medium for the root tip of bamboo shoots, specifically targeting the base of the rootstock. This optimization enabled efficient enzymatic hydrolysis of the bamboo root tip. When using the aforementioned hydrolysate, controlling the mannitol concentration to 0.6–0.8 M ensured suitable osmotic pressure for the bamboo root tip cells during hydrolysis. Excessive osmotic pressure prevented protoplast shrinkage and division, while insufficient osmotic pressure prevented protoplast rupture and reduced survival rates. Protoplasts prepared using this hydrolysate formulation exhibited excellent condition. Additionally, this invention found that hydrolysis for 2.5–3.5 hours in the aforementioned hydrolysate system resulted in a high yield of active protoplasts with excellent integrity.
[0016] Furthermore, the present invention uses a gradient centrifugation with an 18-22% sucrose solution during the purification process, which can effectively remove broken cells, starch granules and tiny impurities. While ensuring the purity of protoplasts, it does not affect the activity of protoplasts, resulting in protoplasts with an activity of over 85% and a clean background, which can be used in the construction of single-cell transcriptome sequencing libraries from moso bamboo roots.
[0017] Preferably, the enzymatic hydrolysate is prepared using ultrapure water.
[0018] Preferably, the pH of the MES is 5.5 to 6.0.
[0019] Preferably, the enzymatic hydrolysate is sterilized by filtration through a 0.45 μm sterile filter membrane before use.
[0020] Preferably, the mannitol concentration during washing is the same as the mannitol concentration in the enzymatic hydrolysate.
[0021] Preferably, the mannitol concentration during resuspension in the purification process is the same as the mannitol concentration in the enzymatic hydrolysate.
[0022] Preferably, in step (2), the raw material is washed with mannitol at least three times.
[0023] In some implementation schemes, when the bamboo shoots grow to a height of 50cm to 100cm, the root tips of the bamboo shoot base are collected as raw materials.
[0024] In some implementations, in step (1), after collecting the root tips of the bamboo shoots at the base of the bamboo shoots, the root tips are cut into thin strips to obtain the raw material.
[0025] In some embodiments, in step (2), before the enzymatic hydrolysis, the washed raw material is mixed with the enzymatic hydrolysate and then reacted in a vacuum environment below -0.05 MPa for more than 30 minutes.
[0026] Reacting in a vacuum environment facilitates the penetration of the enzymatic hydrolysate into the raw materials, thereby improving the efficiency of subsequent enzymatic hydrolysis reactions and shortening the hydrolysis time.
[0027] In some implementations, in step (2), the enzymatic hydrolysis is carried out at 22–25°C and under light-protected conditions.
[0028] In some embodiments, step (2) after the enzymatic hydrolysis further includes: filtering out undissociated filaments using a filter, then rinsing the filaments with mannitol, and passing the rinsing solution through a filter to filter the residue again to obtain the protoplast single-cell suspension.
[0029] In some implementations, in step (3), the mass concentration of the sucrose solution is 20%.
[0030] Controlling the concentration of the sucrose solution to the above concentration can further improve the removal effect of impurities and make the protoplast activity higher.
[0031] Preferably, in step (3), the cell resuspension is mixed with an equal volume of sucrose solution and then centrifuged.
[0032] In some implementations, in step (3), the temperature of the mannitol used to resuspend the cells is 2°C to 4°C.
[0033] In some implementations, in step (3), the centrifugation temperature is 2 to 6°C; and / or the centrifugation time is 2 to 5 minutes.
[0034] In some implementations, trypan blue is used to stain and observe protoplast single-cell suspensions after step (2).
[0035] Furthermore, the present invention provides a root tip protoplast of moso bamboo prepared by any of the above embodiments or a combination thereof.
[0036] Furthermore, the present invention also provides any of the above embodiments or combinations thereof, or the application of the above-described moso bamboo root tip protoplasts in constructing single-cell transcriptome sequencing libraries.
[0037] In some implementation schemes, the prepared bamboo root tip protoplasts are placed on ice, and single-cell transcriptome sequencing and library construction are completed within 30 minutes.
[0038] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0039] The preparation method of this invention can produce protoplasts with high cell viability and pure background, which can meet the requirements for constructing single-cell transcriptome sequencing libraries of moso bamboo roots, and is of great significance for single-cell sequencing of moso bamboo roots. Attached Figure Description
[0040] Figure 1 This is a high-power microscopic examination result of a single-cell suspension.
[0041] Figure 2 This is a low-power microscopic examination result of a single-cell suspension.
[0042] Figure 3 This is a graph showing the results of the protoplast activity test.
[0043] Figure 4 This is a microscopic examination result of the protoplast suspension from Example 1.
[0044] Figure 5 This is a microscopic examination result of the protoplast suspension in Comparative Example 5. Detailed Implementation
[0045] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.
[0046] Unless otherwise specified, all methods used in the examples were conventional or performed according to techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents and instruments used without specified manufacturers were all conventional products that could be purchased from legitimate channels.
[0047] Example 1
[0048] This embodiment provides a method for preparing protoplasts, the specific steps of which are as follows:
[0049] (1) Material sourcing
[0050] When the bamboo shoots grow to a height of 50cm-100cm, the root tips of the bamboo shoot base are collected for the separation of the root protoplasts.
[0051] (2) Separation of protoplasts
[0052] 1) Select well-developed roots, take a suitable root tip area, divide it into 4 parts, weigh 2g, use a double-edged blade to cut the root tip into thin strips, wash it 3 times with 0.6M mannitol solution, remove the supernatant, and then put it into the enzymatic hydrolysis solution;
[0053] The formulation of the enzymatic hydrolysate is as follows: 0.6M mannitol, 3% cellulase R10, 0.7% dissociative enzyme R10, 0.2% pectinase, 20mM KCl, 20mM MES (pH=5.8), 0.1% bovine serum albumin, and 10mM CaCl2;
[0054] 2) Under room temperature conditions, the enzymatic hydrolysate containing root strips was evacuated to -0.05 MPa and maintained for 30 min;
[0055] 3) Place the mixture of enzymatic hydrolysate and root strips in a shaker and incubate at room temperature in the dark for 3 hours;
[0056] 4) After the enzymatic hydrolysis is completed, use a wide pipette tip to draw up the hydrolysate and filter it through a 40μm cell filter into a new 50mL centrifuge tube to remove the incompletely dissociated filaments. Use 0.6M mannitol solution to repeatedly rinse the remaining bamboo root filaments. Filter the rinsing solution through a cell filter again to remove the residue and obtain a preliminary single-cell suspension of bamboo root protoplasts.
[0057] (3) Purification of protoplasts
[0058] 1) Place the cell suspension obtained in (2) into a centrifuge tube, centrifuge at 4℃, 50g for 3min, remove the supernatant, add 4mL of 0.6M mannitol solution at 4℃, and resuspend the cells;
[0059] 2) Aliquot 2 mL of cell resuspension into 15 mL centrifuge tubes and place them on ice. Then, slowly inject 2 mL of 20% sucrose solution into the bottom of the cell resuspension. Centrifuge at 50 g for 3 min at 4 °C. The supernatant is the protoplast suspension. Finally, use a wide pipette tip to aspirate the protoplasts from the supernatant and transfer them into a new centrifuge tube. Place the tubes on ice for later use.
[0060] 3) Place the above protoplast suspension on ice and complete single-cell transcriptome sequencing library construction within 30 minutes.
[0061] Example 2
[0062] This embodiment provides a method for preparing protoplasts, the only difference from Example 1 being that the concentration of mannitol in the enzymatic hydrolysate is 0.8M.
[0063] Comparative Example 1
[0064] This comparative example provides a method for preparing protoplasts, the only difference from Example 1 being that the concentration of mannitol in the enzymatic hydrolysate is 0.4M.
[0065] Comparative Example 2
[0066] This comparative example provides a method for preparing protoplasts, the only difference from Example 1 being that the concentration of mannitol in the enzymatic hydrolysate is 1M.
[0067] Comparative Example 3
[0068] This comparative example provides a method for preparing protoplasts, the only difference from Example 1 being that the enzymatic hydrolysis time is 2 hours.
[0069] Comparative Example 4
[0070] This comparative example provides a method for preparing protoplasts, the only difference from Example 1 being that the enzymatic hydrolysis time is 4 hours.
[0071] Comparative Example 5
[0072] This comparative example provides a method for preparing protoplasts, the only difference from Example 1 being that the sucrose solution is replaced with a polysucrose solution of the same concentration.
[0073] Comparative Example 6
[0074] This comparative example provides a method for preparing protoplasts, the only difference from Example 1 being that 0.2% pectinase is replaced with 0.2% hemicellulase in the formulation of the enzymatic hydrolysate.
[0075] Test case
[0076] 1. The single-cell suspensions of *Phyllostachys edulis* root protoplasts prepared in step (2) of Examples 1 and 2 and Comparative Examples 1, 2, 3, 4, and 6 above were examined under a microscope. During the examination, 0.4% trypan blue solution was mixed with the single-cell suspension at a volume ratio of 1:1, and the cells were counted using a hemocytometer. The results are as follows: Figure 1 and Figure 2 As shown.
[0077] The protoplast activity in Example 1 and Comparative Example 4 was tested using the following method:
[0078] Add 6 μL of protoplast suspension to a PCR tube, then add 6 μL of 0.4% trypan blue solution, mix well, and stain for 3-4 min. Next, transfer 10 μL of the suspension onto a glass slide, cover with a coverslip, and count the total number of cells and the number of viable cells in several random fields under a light microscope. Calculate cell viability. Cell viability = (Number of viable cells / Total number of cells) × 100%.
[0079] Protoplast activity results as follows Figure 3 As shown.
[0080] The results show that, in the enzymatic hydrolysate system of the present invention, using mannitol as an osmotic pressure stabilizer, the protoplasts in Examples 1 and 2 exhibited the best state. The osmotic pressure of Comparative Examples 1 and 2 was unsuitable for the *Phyllostachys edulis* root cells under this enzymatic hydrolysate system. Osmotic pressure higher than the intracellular osmotic pressure would cause protoplast shrinkage, which is detrimental to protoplast division, while too low an osmotic pressure would lead to protoplast rupture.
[0081] Furthermore, the results showed that the optimal enzymatic hydrolysis time for *Cymbidium* root was 3 hours, resulting in the highest yield of active protoplasts, exceeding 85%, with both yield and activity meeting the requirements for single-cell library construction. In Comparative Example 3, with an enzymatic hydrolysis time of 2 hours, the protoplast yield was low, and most tissues were not yet dissociated. In Comparative Example 4, although the total protoplast yield was the highest with an enzymatic hydrolysis time of 4 hours, the protoplast activity rate was below 85%, failing to meet the requirements for single-cell library construction. In Comparative Example 6, the enzymatic hydrolysis solution formulation was unsuitable, resulting in poor tissue dissociation and a low protoplast yield.
[0082] 2. The protoplast suspensions prepared in step (3) of Example 1 and Comparative Example 5 were examined under a microscope. During the examination, 0.4% trypan blue solution was mixed with the protoplast suspension at a volume ratio of 1:1, and the protoplasts were counted using a hemocytometer. The results of Example 1 are as follows. Figure 4 As shown, Figure 4A is the protoplast before purification, and the interstitial spaces of the protoplast are filled with a large number of starch granules. Figure 4 B represents the purified protoplast, with only a small amount of starch granules remaining; the results of Comparative Example 5 are as follows. Figure 5 As shown, the activity of the purified protoplasts was significantly reduced, and the amount of starch granules remaining was relatively large.
[0083] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims
1. A method for preparing protoplasts, characterized in that, include: (1) Material selection: Collect the root tips of the bamboo shoots at the base of the bamboo shoots as raw materials; (2) Isolation of protoplasts: The raw material was washed with 0.6-0.8M mannitol, and then the washed raw material was placed in an enzymatic hydrolysis solution for 2.5-3.5 hours to obtain a protoplast single-cell suspension; The enzymatic hydrolysate comprises: 0.6–0.8 M mannitol, 2–4% cellulase R10, 0.5–1% dissociative enzyme R10, 0.1–0.3% pectinase, 15–25 mM KCl, 15–25 mM MES, 0.05–0.15% bovine serum albumin, and 5–15 mM CaCl2; (3) Purification of protoplasts: The protoplast single-cell suspension was centrifuged at 40g-60g, the supernatant was removed, and the cells were resuspended in mannitol at 2℃-4℃ to obtain a cell resuspension; the cell resuspension was mixed with a sucrose solution with a mass concentration of 18%-22%, and centrifuged at 40g-60g to obtain the protoplasts; In step (1), after collecting the root tips of the bamboo shoots at the base of the bamboo shoots, the root tips are cut into thin strips to obtain the raw material. In step (2), before the enzymatic hydrolysis, the washed raw material is mixed with the enzymatic hydrolysate and placed in a vacuum environment below -0.05 MPa for more than 30 minutes; the enzymatic hydrolysis is carried out at 22℃~25℃ and under light-protected conditions; after the enzymatic hydrolysis, the following steps are also included: filtering out the undissociated filaments with a filter, then rinsing the filaments with mannitol, and filtering the residue again through a filter to obtain the protoplast single-cell suspension.
2. The preparation method according to claim 1, characterized in that, In step (3), the mass concentration of the sucrose solution is 20%.
3. The preparation method according to claim 1, characterized in that, In step (3), the centrifugation temperature is 2℃~6℃; and / or the centrifugation time is 2~5min.
4. The application of the bamboo root tip protoplasts prepared by any one of claims 1 to 3 in the construction of single-cell transcriptome sequencing libraries.