Preparation method and kit of water plant nai cae leaf protoplast

Abstract

The application provides a preparation method and a kit of Nymphoides peltatum leaf protoplast. The application takes Nymphoides peltatum leaf as material, adjusts an enzymolysis system, a washing system and a purification method, and establishes a Nymphoides peltatum leaf protoplast preparation system. The enzymolysis effect is good when 1% cellulase, 0.5% pectinase and 0.5% macerase are in the enzymolysis liquid system; the compounding of MES, sorbitol and KCl can ensure that the external medium and the osmotic potential of the cell itself are the same when the protoplast is separated, maintain the activity of the protoplast, and the 26% sucrose solution can better maintain the osmotic pressure stability of the protoplast; the method can quickly and efficiently dissociate the protoplast cells of Nymphoides peltatum leaf tissue, the protoplasts obtained by separation are numerous, have high activity and a clean background. The result can be directly used for single cell sequencing, and provides an important basis for exploring the molecular cell biology research and the molecular mechanism of development of Nymphoides peltatum heterophyllous leaves.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular cell biology technology, specifically relating to a method and kit for preparing protoplasts from the leaves of the aquatic plant *Cymbidium goeringii*. Background Technology

[0002] *Ottelia cordata*, an annual or perennial aquatic herbaceous plant belonging to the genus *Ottelia* in the family Hydrocharitaceae, grows in flowing water and ditches and has a gregarious habit. *Ottelia cordata* has two types of heteromorphic leaves: submerged leaves and floating leaves. Submerged leaves are usually strap-shaped or lanceolate, while floating leaves are usually oblong-ovate. Besides morphological differences, the two leaf types also differ significantly in cell structure and photosynthetic capacity. Studying how the same tissues of plants with the same genetic background develop into different phenotypes to adapt to different environments is of significant ecological importance.

[0003] Traditional plant biology research has typically employed conventional sequencing techniques, using whole tissues as samples. This approach fails to reveal the heterogeneity of expression among different cell types. Even when using microdissection techniques to obtain specific tissue sites, contamination from other tissue sites is possible, and the number of cells obtained is highly limited. To determine the functional characteristics of specific genes and uncover potential key factors regulating plant life activities at the cellular level, single-cell sequencing technology has gradually become a new generation of research tools. By dissecting plant tissues into individual cells and sequencing them, omics information can be obtained at the single-cell level.

[0004] However, obtaining a single cell is the first step in single-cell sequencing. Currently, protoplast single-cell suspensions are typically obtained using mechanical or enzymatic methods. Mechanical methods are complex and inefficient, while enzymatic methods suffer from limitations due to the significant differences in cell wall composition among different plants, requiring specific enzymatic digest ratios for each plant. Existing methods lack universality and are largely unused in aquatic plants like *Hydrocharis arvensis*. Furthermore, single-cell sequencing instruments have high requirements for the quality and purity of the single-cell suspension. Existing protoplast preparation methods primarily target somatic cell fusion or transgenic technologies, with less emphasis on protoplast purity. Using existing techniques to extract protoplasts from *Hydrocharis arvensis* leaves yields a low number of protoplasts with low activity, which can negatively impact subsequent experiments. While a double-enzymatic digestion method was used to extract leaf protoplasts, followed by sedimentation purification, the protoplast suspension contained numerous impurities from plant tissue or cell fragments. Low-purity protoplasts can hinder sequencing.

[0005] Therefore, there is an urgent need for a rapid and efficient method for preparing protoplasts from watercress, ensuring that the obtained protoplasts can be directly used for single-cell sequencing research. Summary of the Invention

[0006] The purpose of this invention is to address the aforementioned shortcomings of the prior art by providing a method and kit for preparing protoplasts from watercress leaves, thereby filling the gap in the preparation of protoplasts from aquatic plant leaves and providing a powerful tool for molecular and cell biology research on aquatic plants.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] The first objective of this invention is to provide a method for preparing protoplasts from watercress leaves, comprising the following specific steps:

[0009] Step S1: Take a submerged or floating leaf of the water hyacinth to obtain a water hyacinth leaf; the water hyacinth leaf is the first leaf newly grown by the water hyacinth plant.

[0010] Step S2: Using a double-edged blade, cut the water lily leaves into strips of 0.5mm to 1mm, completely immerse them in the enzymatic hydrolysate, and carry out the reaction in the dark to obtain the primary enzymatic hydrolysate.

[0011] The enzymatic hydrolysate consists of deionized water and substances of the following concentrations:

[0012]

[0013]

[0014] Step S3: Add W5 solution to the primary enzymatic hydrolysis product obtained in step S2 to terminate the reaction, centrifuge at low speed, discard the supernatant, and obtain the precipitate.

[0015] The W5 solution is composed of deionized water and substances of the following concentrations:

[0016]

[0017] Step S4: Add 26% sucrose solution to the precipitate obtained in step S3 for purification treatment, centrifuge at low speed, and collect the protoplast band floating at the solution interface.

[0018] Step S5: Add the protoplast bands obtained in step S4 to the washing solution for resuspending to obtain water hyacinth leaf protoplasts for single-cell sequencing.

[0019] Furthermore, in step S2, the conditions for the enzymatic hydrolysis reaction are as follows: the temperature of the enzymatic hydrolysis reaction is 28-29℃, and the mixture is cultured in the dark on a horizontal shaker at 45-50 r / min for 4.5-5.5 h.

[0020] Furthermore, in step S3, the pH value of the W5 solution is 5.7 to 5.8.

[0021] Furthermore, the specific process of step S3 is as follows: the mixture of the enzymatic hydrolysis primary product obtained in step S2 and the W5 solution is filtered through a sterile 40m cell sieve to filter plant tissue fragments, centrifuged at 100g speed for 3min, and the filtrate is collected.

[0022] Furthermore, in step S4, the centrifugation conditions are 100g speed for 2 minutes.

[0023] Furthermore, in step S5, the cleaning solution is a solution containing 20 mmol MES and 0.4 mol sorbitol per liter of sterile deionized water.

[0024] Furthermore, in step S1, the step of obtaining water hyacinth leaves includes: selecting submerged leaves from water hyacinth plants that only grow submerged leaves or floating leaves from water hyacinth plants that only grow floating leaves, removing the petioles, and washing with sterile water.

[0025] A second objective of this invention is to provide a kit for preparing protoplasts from watercress leaves, the kit comprising the above-mentioned enzymatic hydrolysate and W5 solution.

[0026] Compared with the prior art, the beneficial effects of the technical solution provided by the present invention are:

[0027] (1) The method for preparing protoplasts from watercress leaves provided by this invention uses the first newly grown leaf of a watercress plant as material. A protoplast preparation system for watercress leaves is established by adjusting the enzymatic hydrolysis system, washing system, and purification method. Observation results show that the enzymatic hydrolysis effect is better when the hydrolysate contains 1% cellulase, 0.5% pectinase, and 0.5% dissociation enzyme. The combination of MES, sorbitol, and KCl ensures that the osmotic potential of the external medium and the cell itself is the same when the protoplasts are released, maintaining the viability of the protoplasts. The combination of CaCl2 and BSA maintains the integrity of the protoplast membrane, preventing damage to the protoplasts by proteolytic enzymes in the hydrolysate. The use of a 26% sucrose solution effectively maintains the osmotic pressure stability of the protoplasts, resulting in a large number of protoplasts and fewer fragments and impurities. The method of this invention can rapidly and efficiently dissociate protoplast cells from watercress leaf tissue, obtaining a large number of highly active protoplasts with a pure background. These results can be directly used for single-cell sequencing, providing an important foundation for exploring the molecular cell biology of heteromorphic leaves and the molecular mechanisms of their development in watercress. Attached Figure Description

[0028] Figure 1 Protoplasts of submerged leaves of *Watercress* observed under 10x magnification.

[0029] Figure 2 Protoplasts of floating leaves of water hyacinth observed under 10x magnification;

[0030] Figure 3 Protoplasts of submerged leaves of *Watercress* observed under 40x magnification;

[0031] Figure 4 Protoplasts of floating leaves of water hyacinth observed under 40x magnification;

[0032] Figure 5 Leaf protoplasts purified by the flotation method;

[0033] Figure 6 Leaf protoplasts purified by the interface method;

[0034] Figure 7 Leaf protoplasts purified by sedimentation method;

[0035] Figure 8 This is a graph showing the protoplast activity detection results for Example 1. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of this invention clearer, the specific embodiments and accompanying drawings are described in further detail below. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0037] The compounds used in the specific implementation method of this invention were all purchased from Sangon Biotech (Shanghai) Co., Ltd., and the enzymes used were all from the Japanese brand Yault.

[0038] The cellulase used in this invention has an enzyme activity greater than 10,000 U / g, the cleavage enzyme has an enzyme activity greater than 3,000 U / g, and the pectinase has an enzyme activity greater than 1,000 U / g. The cellulase is cellulase R-10, the cleavage enzyme is cleavage enzyme R-10, and the pectinase is pectinase Y-23.

[0039] MES: 2-(N-morpholino)ethanesulfonic acid; BSA: serum protein.

[0040] Protoplast counting was performed as follows: 10 μL of protoplast resuspension was placed on a hemocytometer, and the number of protoplasts was counted using the hemocytometer. The total number of protoplasts (units / g) = (total number of protoplasts in 5 squares / 80 × 400 × 10⁻⁶) 4 × dilution factor) / total leaf mass (g).

[0041] Protoplast viability assay: Cell viability was detected using fluorescein diacetic acid (FDA). If the cell membrane was intact, the protoplasts would emit green fluorescence; if the protoplasts were broken, they would not emit green fluorescence. The purified protoplast resuspension was mixed with 0.01% FDA working solution, allowed to stand at room temperature for 5 minutes, and then dropped onto a hemocytometer. The staining of the cells was observed using a fluorescence confocal microscope. The viability counting formula was: (fluorescing protoplasts / total number of protoplasts) × 100%.

[0042] The method for preparing protoplasts from watercress leaf cells according to the present invention includes the following steps:

[0043] Step S1: Obtain the submerged and floating leaves of the water hyacinth.

[0044] Step S2: Using a double-edged blade, cut the water lettuce leaves into strips about 1 mm thick, completely immerse them in the enzymatic hydrolysate, and carry out the reaction in the dark.

[0045] Step S3: Add W5 solution to terminate the reaction. Use a 40μm sterile cell filter to sieve the mixture of the primary enzymatic hydrolysis product obtained in step S2 and W5 solution, collect it into a new test tube, centrifuge at low speed, and discard the supernatant.

[0046] Step S4: The precipitate obtained in step S3 is added to a 26% sucrose solution for purification, centrifuged at low speed, and the protoplast bands floating at the solution interface are collected.

[0047] Step S5: The protoplasts obtained in step S4 are added to the washing buffer for resuspending, and the cell concentration and activity are observed under a microscope.

[0048] Example 1

[0049] Method for preparing cell protoplasts from submerged leaves of submerged-leaf water hyacinth plants:

[0050] (1) Preparation of materials:

[0051] Select submerged leaves from plants that only grow submerged leaves, ensuring the leaves are the first newly grown leaves of the plant. Remove the petioles and wash with sterile water.

[0052] (2) Preparation of consumables:

[0053] Prepare sterile double-edged blades, culture dishes, centrifuge tubes (1.5mL), Pasteur pipettes, 40m sterile cell strainers, pipettes of different sizes, ddH2O, etc.

[0054] (3) Preparation of enzyme hydrolysate:

[0055] The enzymatic hydrolysate system consisted of: 1% (w / v) cellulase, 0.5% (w / v) anabolase, 0.5% (w / v) pectinase, 0.4 mol / L sorbitol, 20 mmol / L MES, 20 mmol / L KCl, 10 mmol / L CaCl2, and 0.1% (w / v) BSA, with sterile ddH2O as the solvent.

[0056] Cellulase, cleavage enzyme, pectinase, MES, sorbitol, and KCl were mixed and incubated in a water bath at 55°C for 10 minutes to obtain a mixed solution. After cooling the mixed solution to room temperature, it was mixed with CaCl2 and BSA to obtain the enzymatic hydrolysate. The enzymatic hydrolysate should be clear and brown, and the enzyme solution should be prepared and used immediately.

[0057] (4) Dissociation of protoplasts:

[0058] Wash the prepared leaves thoroughly with sterile water, then quickly cut them into strips approximately 1 mm wide using a double-edged blade. Immerse the strips completely in a culture dish containing 10 mL of enzymatic hydrolysate. Incubate at 28°C in the dark with a horizontal shaker at 45 rpm for 5 hours. During this time, observe the release of protoplasts under a microscope.

[0059] (5) Filtration, purification and collection of protoplasts:

[0060] First, add 5 mL of pre-chilled W5 solution to terminate the reaction. Gently shake the culture dish to promote protoplast release. Filter the mixture of the primary enzymatic hydrolysis product obtained in the previous step with the W5 solution through a sterile 40 μm cell sieve to remove plant tissue fragments. Centrifuge at 100 g for 3 min and collect the filtrate into a 1.5 mL centrifuge tube. Gently remove the supernatant using a Pasteur pipette and pipette. Resuspend the protoplasts in an appropriate amount of pre-chilled 26% sucrose solution. Centrifuge at 100 g for 2 min and collect the protoplast bands floating at the solution interface. Finally, resuspend in an appropriate amount of pre-chilled washing solution to prepare a protoplast suspension.

[0061] The W5 solution was formulated as follows: 2 mmol / L MES, 154 mmol / L NaCl, 125 mmol / L CaCl2 and 5 mmol / L KCl, with sterile ddH2O added and mixed thoroughly. The pH of the W5 solution was 5.8.

[0062] The cleaning solution is formulated as follows: 20 mmol / L MES, 0.4 mol / L sorbitol, and finally sterilized ddH2O is added and mixed evenly.

[0063] (6) Microscopic examination of protoplasts:

[0064] Take a droplet of protoplast suspension and place it on a glass slide for microscopic examination under a regular optical microscope.

[0065] The results are as follows Figure 1 As shown, under a 10x microscope, a considerable number of well-formed, structurally intact, and undamaged protoplasts of *Potamogeton crispus* can be observed in the field of view. The protoplast yield of the first submerged leaf, as determined by a hemocytometer, is approximately 5.53 × 10⁻⁶. 6 The activity of the sample was 88.6% based on activity testing.

[0066] like Figure 5 As shown, the protoplasts purified by the flotation method have the purest background, with a large number of protoplasts and few fragments and impurities.

[0067] like Figure 8 As shown, the number of fluorescent protoplasts is considerable, and their activity is high.

[0068] Example 2

[0069] Method for preparing cell protoplasts from submerged leaves of floating water hyacinth plants:

[0070] The method is basically the same as in Example 1, except that: the material is the floating leaf of a water hyacinth plant that grows only floating leaves, and the leaf is the first leaf that grows newly on the plant; the temperature of the enzymatic hydrolysis reaction is 29°C, and the plant is cultured in the dark on a horizontal shaker at 50 r / min for 5 h; the pH value of the W5 solution is 5.7.

[0071] The results are as follows Figure 1 As shown in the image, under a 10x microscope, a considerable number of well-formed, structurally intact, and undamaged protoplasts of *Potamogeton crispus* can be observed in the field of view. The protoplast yield of the first submerged leaf, as determined by a hemocytometer, is approximately 5.92 × 10⁻⁶. 6 The activity of the sample was 89.1% based on activity testing.

[0072] Example 3

[0073] Method for preparing cell protoplasts from submerged leaves of submerged-leaf water hyacinth plants:

[0074] The process was essentially the same as in Example 1, except that the enzymatic hydrolysis time was 4.5 hours. The protoplast yield of the first submerged leaf, as determined by a hemocytometer, was approximately 5.46 × 10⁻⁶. 6 The activity of the sample was 86.7% based on activity testing.

[0075] Example 4

[0076] Method for preparing cell protoplasts from submerged leaves of submerged-leaf water hyacinth plants:

[0077] The process was essentially the same as in Example 1, except that the enzymatic hydrolysis time was 5.5 hours. The protoplast yield of the first submerged leaf, as determined by a hemocytometer, was approximately 5.46 × 10⁻⁶.6 The activity of the sample was 88.2% based on the activity test results.

[0078] Comparative Example 1

[0079] Method for preparing cell protoplasts from submerged leaves of submerged-leaf water hyacinth plants:

[0080] The results were essentially the same as in Example 1, except that the submerged leaf of the *Aquatic Plant* plant was the newly grown second leaf. Figure 3 As shown in the image, under 40x magnification, the number of protoplasts is sparse. Using a hemocytometer, the protoplast yield of the second leaf of the submerged leaf was estimated to be approximately 2.10 × 10⁻⁶. 6 The number of protoplasts was significantly reduced, and some protoplasts still had cell walls, indicating a high fragmentation rate.

[0081] Comparative Example 2

[0082] Method for preparing cell protoplasts from submerged leaves of floating water hyacinth plants:

[0083] This is essentially the same as Example 2, except that the submerged leaf of the *Aquatic Plant* plant is the newly grown second leaf. The results are as follows... Figure 4 As shown in the image, under 40x magnification, the number of protoplasts is sparse. Using a hemocytometer, the protoplast yield of the second leaf of the submerged leaf was approximately 2.56 × 10⁻⁶. 6 The number of protoplasts was significantly reduced, and some protoplasts still had cell walls, indicating a high fragmentation rate.

[0084] Comparative Example 3

[0085] Method for preparing cell protoplasts from submerged leaves of submerged-leaf water hyacinth plants:

[0086] The method was essentially the same as in Example 1, except that the enzymatic hydrolysate contained 1.5% cellulase and 0.4% dissociative enzyme. The protoplast yield, as determined by a hemocytometer, was 3.56 × 10⁻⁶. 6 The number of protoplasts was 72.5% by activity test, and some protoplasts were found to still have cell walls.

[0087] Comparative Example 4

[0088] Method for preparing cell protoplasts from submerged leaves of submerged-leaf water hyacinth plants:

[0089] The procedure was essentially the same as in Example 1, except that: first, enzymatic hydrolysis was performed for 3 hours using a hydrolysate containing 1% cellulase, 0.5% cleavage enzyme, and 0.5% pectinase; then, enzymatic hydrolysis was performed for 2 hours using a hydrolysate containing 1.2% cellulase and 0.4% cleavage enzyme. The protoplast yield was calculated to be 4.83 × 10⁻⁶ using a hemocytometer. 6 The activity of the sample was 60.9% based on activity testing.

[0090] Comparative Example 5

[0091] Method for preparing cell protoplasts from submerged leaves of submerged-leaf water hyacinth plants:

[0092] The results were essentially the same as in Example 1, except that the enzymatic hydrolysis time was 6 hours. The protoplast yield, as determined by a hemocytometer, was 4.78 × 10⁻⁶. 6 The activity of the sample was 74.5% based on activity testing.

[0093] Comparative Example 6

[0094] Method for preparing cell protoplasts from submerged leaves of submerged-leaf water hyacinth plants:

[0095] The results were essentially the same as in Example 1, except that the enzymatic hydrolysis time was 4 hours. The protoplast yield, as determined by a hemocytometer, was 3.56 × 10⁻⁶. 6 The activity of the sample was 62.7% based on activity testing.

[0096] Comparative Example 7

[0097] Method for preparing cell protoplasts from submerged leaves of submerged-leaf water hyacinth plants:

[0098] The process was basically the same as in Example 1, except that the preliminary enzymatic hydrolysate obtained by enzymatic hydrolysis for 5 hours using a hydrolysate containing 1% cellulase, 0.5% pectinase, and 0.5% dissociation enzyme was purified by interfacial method using 13% mannitol solution and 26% sucrose solution, and the purification was observed using an optical microscope.

[0099] The results are as follows Figure 6 As shown, the protoplasts purified by this interface method have a relatively pure background, but the number of protoplasts is small.

[0100] Comparative Example 8

[0101] Method for preparing cell protoplasts from submerged leaves of submerged-leaf water hyacinth plants:

[0102] The process was basically the same as in Example 1, except that the preliminary enzymatic hydrolysate obtained by enzymatic hydrolysis for 5 hours using an enzymatic hydrolysate containing 1% cellulase, 0.5% pectinase, and 0.5% dissociation enzyme was purified by sedimentation using a 0.4 mol / L sorbitol solution, and the purification was observed using an optical microscope.

[0103] The results are as follows Figure 7 As shown, this sedimentation method yields a large number of protoplasts, but also produces a significant amount of background impurities.

[0104] Comparative Example 9

[0105] Method for preparing cell protoplasts from submerged leaves of submerged-leaf water hyacinth plants:

[0106] The results were essentially the same as in Example 1, except for the use of a 25% sucrose solution. Protoplast production was determined to be 5.06 × 10⁻⁶ using a hemocytometer. 6 The activity of the sample was 69.4% based on activity testing.

[0107] Comparative Example 10

[0108] Method for preparing cell protoplasts from submerged leaves of submerged-leaf water hyacinth plants:

[0109] The results were essentially the same as in Example 1, except for the use of a 27% sucrose solution. Protoplast production was determined to be 4.97 × 10⁻⁶ using a hemocytometer. 6 The activity of the sample was 73.8% based on the concentration of 1 / g and the activity test results.

[0110] The method for preparing protoplasts from watercress leaves of this invention can rapidly and efficiently dissociate protoplast cells from watercress leaf tissue. The isolated protoplasts are numerous, highly active, and have a clean background. These results can be directly used for single-cell sequencing, providing an important foundation for exploring the molecular cell biology and developmental mechanisms of heteromorphic leaves in watercress.

[0111] Where there is no conflict, the above embodiments and features described herein can be combined with each other.

[0112] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing protoplasts from watercress leaves, characterized in that, The specific steps include the following: S1. Take submerged or floating leaves of water hyacinth to obtain water hyacinth leaves; the water hyacinth leaves are the first leaves newly grown on the water hyacinth plant; S2. Using a double-edged blade, cut the watercress leaves into strips of 0.5mm~1mm, completely immerse them in the enzymatic hydrolysate, and react under dark conditions to obtain the primary enzymatic hydrolysate. Specifically, the enzymatic hydrolysate is prepared by mixing cellulase, dissociative enzyme, pectinase, MES, sorbitol, and KCl at 55°C. The mixture was heated in a water bath at 28-29°C for 10 minutes to obtain a mixed solution. After the mixture was cooled to room temperature, it was mixed with CaCl2 and BSA to obtain the final product. The enzymatic hydrolysis reaction was carried out at 28-29°C and cultured in the dark on a horizontal shaker at 45-50 rpm for 4.5-5.5 hours. The enzymatic hydrolysate consists of deionized water and the following concentrations of substances. composition: S3. Add W5 solution to the primary enzymatic hydrolysis product obtained in step S2 to terminate the reaction, centrifuge at low speed, discard the supernatant, and obtain the precipitate. The W5 solution is composed of deionized water and substances of the following concentrations: S4. Add 26% sucrose solution to the precipitate obtained in step S3 for purification treatment, centrifuge at low speed, and collect the protoplast band floating at the solution interface. S5. Add the protoplasts obtained in step S4 to the washing solution and resuspend them to obtain the *Caulis Paeoniae Alba* protoplasts for single-cell sequencing.

2. The preparation method according to claim 1, characterized in that, In step S3, the pH value of the W5 solution is 5.7 to 5.

8.

3. The preparation method according to claim 1, characterized in that, The specific process of step S3 is to filter the mixture of the primary enzymatic hydrolysis product obtained in step S2 and the W5 solution through a sterile 40μm cell sieve to filter plant tissue fragments, and centrifuge at 100g speed for 3min.

4. The preparation method according to claim 3, characterized in that, In step S4, the centrifugation conditions are 100g speed for 2 minutes.

5. The preparation method according to claim 3, characterized in that, In step S5, the cleaning solution is a solution containing 20 mmol MES and 0.4 mol sorbitol per liter of sterile deionized water.

6. The preparation method according to claim 5, characterized in that, In step S1, the steps of obtaining water hyacinth leaves include: selecting submerged leaves from water hyacinth plants that only grow submerged leaves or floating leaves from water hyacinth plants that only grow floating leaves, removing the petioles, and washing with sterile water.

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