Method for preparing protoplast from rhododendron simsii and application of protoplast
The preparation process of rhododendron mottled protoplasts was optimized by steps such as enzymatic hydrolysis, microscopic examination, and centrifugation, which solved the problem of dissociation difficulties in the existing technology and achieved rapid and efficient separation of rhododendron mottled protoplasts, supporting subsequent experiments.
Patent Information
- Application Number
- CN202410938273.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-07-13
- Publication Date
- 2026-01-13
AI Technical Summary
Existing technologies are insufficient for the rapid and efficient dissociation of rhododendron mottled protoplasts, which limits subsequent experiments such as plant single-cell sequencing, plasmid DNA transfection, and protoplast fusion.
The protoplast preparation process for rhododendron spots was optimized by using enzymatic hydrolysis, microscopic observation, filtration, and protoplast precipitation, combined with different centrifugal forces and resuspensions. This included the use of enzymatic hydrolysates such as Cellulase R10, Macerozyme R-10, and D-Mannitol, and purity was ensured by microscopic examination and counting.
This method enables rapid and efficient isolation of rhododendron mottled protoplasts, providing high-quality experimental materials for subsequent experiments and supporting downstream experiments such as plant single-cell sequencing, plasmid DNA transfection, and protoplast fusion.
Smart Images
Figure CN121320221A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of protoplast preparation technology for rhododendron spots, specifically a method and application for preparing protoplasts from rhododendron spots. Background Technology
[0002] Azaleas, deciduous shrubs belonging to the genus *Rhododendron* in the family Ericaceae, are prized for their vibrant and diverse flower colors and high ornamental value, making them one of my country's traditional famous flowers. Beyond their ornamental value, azaleas are also widely used in traditional Chinese medicine. Research has found that azaleas are rich in anthocyanins, which possess antioxidant and anti-platelet aggregation properties, potentially helping to prevent cardiovascular diseases. Azaleas also contain certain flavonoids, which can lower blood lipids and inhibit platelet aggregation, offering some benefit in preventing hypertension, coronary heart disease, and other related conditions, thus possessing significant research value.
[0003] This invention relates to a rapid and efficient method for dissociating rhododendron mottle protoplasts, which can effectively solve the problem of difficult dissociation of rhododendron mottle protoplasts and can be used for subsequent downstream experiments such as plant single-cell sequencing (scRNA-seq), plasmid DNA transfection, and protoplast fusion. Summary of the Invention
[0004] To address the shortcomings of existing technologies, this invention provides a method and application for preparing protoplasts from rhododendron spots, thus solving the problems mentioned in the background section.
[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing protoplasts from rhododendron spots and its application, comprising the following specific steps:
[0006] S1: Material preparation; Under aseptic conditions, take the spotted and non-spotted parts of the azalea and cut them into 0.5mm fine filaments with a sharp scalpel. Place the cut leaves in a culture dish, seal the culture dish, and then place it in a 20℃ constant temperature incubator for enzymatic pretreatment.
[0007] S2: Enzymatic hydrolysis; The pretreated raw material is placed in a shaker of the enzymatic hydrolysis system at a speed of 60-70 r / min and a temperature of 25℃-27℃ in the dark for 2-4 hours.
[0008] S3: Microscopic observation; every 0.5-1 hour, take 10 μl of the enzyme digest and add it to a clean glass slide for observation;
[0009] S4: Filtration; After the enzymatic hydrolysis is completed, the enzymatic hydrolysis product is filtered through a filter screen to remove large fragments of 70um and 40um, and the enzymatic hydrolysis is terminated with twice the volume of W5.
[0010] S5: Protoplast precipitation; select the lowest speed setting, centrifuge at 100-130g for 5 minutes, carefully remove the supernatant, resuspend in 300-500μl of resuspension, and observe under a microscope again.
[0011] A further improvement of the technical solution of the present invention is as follows: after the protoplast precipitate in S5 is observed under a microscope, the protoplast is resuspended in 5-10 ml of pre-cooled and well-mixed W5 solution, centrifuged at 100-130 g for 5 min for purification, and then the supernatant is carefully removed. If the supernatant is resuspended in 300-500 μl of resuspension, it can be examined under a microscope.
[0012] A further improvement of the technical solution of the present invention is as follows: after microscopic observation of the protoplast precipitate in S5, the protoplast is resuspended in 5-10 ml of pre-cooled and well-mixed W5 solution, purified by centrifugation at 80-100 g for 10 min, and the supernatant is carefully removed. If 300-500 μl is resuspended, count stars are performed. If impurities are still present, purification can continue.
[0013] A further improvement of the technical solution of the present invention is that: in S1-S5, the centrifuge speed is adjusted to 0, and the material transfer is carried out by tilting.
[0014] A further improvement to the technical solution of the present invention is that the 10 μl of enzyme hydrolysate taken in the S3 microscopic observation is extracted using a pipette tip with a cut end, and the solution is carefully blown away during the extraction process.
[0015] A further improvement of the technical solution of the present invention is that: after centrifugation in S5, the supernatant is not discarded, and the precipitate and supernatant are observed under a microscope respectively, and the presence of floating impurities in the supernatant is checked.
[0016] A further improvement of the technical solution of the present invention is that: in the S3 microscopic observation, the enzyme hydrolysate is dropped onto a clean glass slide and then photographed using 4x and 10x microscopy.
[0017] Beneficial effects
[0018] This invention provides a method and application for preparing protoplasts from rhododendron spots. Compared with existing technologies, it has the following advantages:
[0019] This method and application for preparing protoplasts from rhododendron spots involves enzymatic digestion, microscopic observation, filtration, and protoplast precipitation of rhododendron spots. After protoplast precipitation, the method enables the first rapid and effective separation of protoplasts from rhododendron spots, providing experimental materials for subsequent downstream experiments such as plant single-cell sequencing, plasmid DNA transfection, and protoplast fusion. Attached Figure Description
[0020] Figure 1 This is a sample image of the actual rhododendron used in the experiment of this invention;
[0021] Figure 2 This is a sample image of the actual rhododendron used in the experiment of this invention;
[0022] Figure 3 This is a diagram of the dissociation of rhododendron spots in this invention;
[0023] Figure 4 This is a diagram of the non-patterned dissociation of rhododendrons according to the present invention. Detailed Implementation
[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0025] Reference Figures 1-4 This invention provides five technical solutions:
[0026] Example 1:
[0027] A method for preparing protoplasts from rhododendron spots and its application, comprising the following specific steps:
[0028] S1: Material preparation; Under aseptic conditions, take the spotted and non-spotted parts of the azalea and cut them into 0.5mm fine filaments with a sharp scalpel. Place the cut leaves in a culture dish, seal the culture dish, and then place it in a 20℃ constant temperature incubator for enzymatic pretreatment.
[0029] S2: Enzymatic hydrolysis; The pretreated raw material is placed in a shaker of the enzymatic hydrolysis system at a speed of 60-70 r / min and a temperature of 25℃-27℃ in the dark for 2-4 hours.
[0030] S3: Microscopic observation; every 0.5-1 hour, take 10g of the enzyme digest and add it to a clean glass slide for observation;
[0031] S4: Filtration; After the enzymatic hydrolysis is completed, the enzymatic hydrolysis product is filtered through a filter screen to remove large fragments of 70um and 40um, and the enzymatic hydrolysis is terminated with twice the volume of W5.
[0032] S5: Protoplast precipitation; select the lowest speed setting, centrifuge at 100-130g for 5 minutes, carefully remove the supernatant, resuspend in 300-500 ml resuspension and observe under a microscope again.
[0033] After observing the protoplast precipitate in S5 under a microscope, resuspend the protoplasts in 5-10 ml of pre-cooled and well-mixed W5 solution. Purify by centrifuging at 100-130 g for 5 min and carefully remove the supernatant. If resuspend in 300-500 μl of resuspension, the supernatant can be examined under a microscope.
[0034] After observing the protoplast precipitate under a microscope in S5, resuspend the protoplasts in 5-10 ml of pre-cooled and well-mixed W5 solution. Purify by centrifugation at 80-100 g for 10 min, then carefully remove the supernatant. Resuspend in 300-500 μl and count the protoplasts. If impurities are still present, continue purification.
[0035] In S1-S5, the centrifuge speed is set to 0, and all material transfers are done by tilting.
[0036] The specific method for taking 10 μl of enzyme hydrolysate for S3 microscopic examination is to use a pipette tip with a cut end to extract it, and to carefully blow it during the extraction process.
[0037] After centrifugation with S5, do not discard the supernatant. Observe the precipitate and supernatant under a microscope and check for any floating impurities in the supernatant.
[0038] In S3 microscopic observation, the enzyme digest was dropped onto a clean glass slide and then photographed using 4x and 10x microscopy.
[0039] Example 2:
[0040] Based on Example 1: The protoplast precipitate obtained in Example 1 was subjected to relevant dissociation experiments. The enzymatic hydrolysate included Cellulase R10, Macerozyme R-10, D-Mannitol, 0.5M MES buffer (pH 5.8, sterile), 2M KCl, 2.5M CaCl2, 10% BSA, and nuclease-free water, with final concentrations of 1.0-1.5%, 0.05-1.0%, 0.5-0.7M, 20mM, 20mM, 10mM, 0.1%, and 10ml, respectively. The trace elements in W5 of Example 1 were NaCl, CaCl2, KCl, MES, and nuclease-free water, with final concentrations of 154mmol, 125mmol, 5mmol, 2mmol / L, and 1L, respectively.
[0041] Example 3:
[0042] Based on Examples 1 and 2: A comparative experiment was conducted on the dissociation results of Countstar detection. The specific steps were as follows:
[0043] S1: Material preparation; Under aseptic conditions, take the spotted and non-spotted parts of Rhododendron simsii and cut them into 0.5mm fine filaments with a sharp scalpel. Place the cut leaves in a culture dish, seal the culture dish, and then place it in a 20℃ constant temperature incubator for enzymatic pretreatment.
[0044] S2: Enzymatic hydrolysis; The pretreated raw material is placed in a shaker of the enzymatic hydrolysis system at a speed of 60-70 r / min and a temperature of 25℃-27℃ in the dark for 2-4 hours.
[0045] S3: Microscopic observation; every 0.5-1 hour, take 10g of the enzyme digest and add it to a clean glass slide for observation;
[0046] S4: Filtration; After the enzymatic hydrolysis is completed, the enzymatic hydrolysis product is filtered through a filter screen to remove large fragments of 70um and 40um, and the enzymatic hydrolysis is terminated with twice the volume of W5.
[0047] S5: Protoplast precipitation; select the lowest speed setting, centrifuge at 100-130g for 5 minutes, carefully remove the supernatant, resuspend in 300-500 ml resuspension and observe under a microscope again.
[0048] After observing the protoplast precipitate in S5 under a microscope, resuspend the protoplasts in 5-10 ml of pre-cooled and well-mixed W5 solution. Purify by centrifuging at 100-130 g for 5 min and carefully remove the supernatant. If resuspend in 300-500 μl of resuspension, the supernatant can be examined under a microscope.
[0049] After observing the protoplast precipitate under a microscope in S5, resuspend the protoplasts in 5-10 ml of pre-cooled and well-mixed W5 solution. Purify by centrifugation at 80-100 g for 10 min, then carefully remove the supernatant. Resuspend in 300-500 μl and count the protoplasts. If impurities are still present, continue purification.
[0050] The final Countstar detection dissociation result is:
[0051]
[0052]
[0053] Example 4:
[0054] Based on Example 3: A comparative experiment was conducted on the dissociation results of the Countstar detection. The specific steps were as follows:
[0055] S1: Material preparation; Under aseptic conditions, take the spotted and non-spotted parts of Rhododendron yunnanense and cut them into 0.5mm fine filaments with a sharp scalpel. Place the cut leaves in a culture dish, seal the culture dish, and then place it in a 20℃ constant temperature incubator for enzymatic pretreatment.
[0056] S2: Enzymatic hydrolysis; The pretreated raw material is placed in a shaker of the enzymatic hydrolysis system at a speed of 60-70 r / min and a temperature of 25℃-27℃ in the dark for 2-4 hours.
[0057] S3: Microscopic observation; every 0.5-1 hour, take 10g of the enzyme digest and add it to a clean glass slide for observation;
[0058] S4: Filtration; After the enzymatic hydrolysis is completed, the enzymatic hydrolysis product is filtered through a filter screen to remove large fragments of 70um and 40um, and the enzymatic hydrolysis is terminated with twice the volume of W5.
[0059] S5: Protoplast precipitation; select the lowest speed setting, centrifuge at 100-130g for 5 minutes, carefully remove the supernatant, resuspend in 300-500 ml resuspension and observe under a microscope again.
[0060] After observing the protoplast precipitate in S5 under a microscope, resuspend the protoplasts in 5-10 ml of pre-cooled and well-mixed W5 solution. Purify by centrifuging at 100-130 g for 5 min and carefully remove the supernatant. If resuspend in 300-500 μl of resuspension, the supernatant can be examined under a microscope.
[0061] After observing the protoplast precipitate under a microscope in S5, resuspend the protoplasts in 5-10 ml of pre-cooled and well-mixed W5 solution. Purify by centrifugation at 80-100 g for 10 min, then carefully remove the supernatant. Resuspend in 300-500 μl and count the protoplasts. If impurities are still present, continue purification.
[0062] The final Countstar detection dissociation result is:
[0063]
[0064] Example 5:
[0065] Based on Examples 3 and 4: A control experiment was conducted on the dissociation results of the countstar detection. The specific steps were as follows: S1: Material preparation; Under sterile conditions, the spotted and non-spotted parts of the azalea were cut into 0.5mm filaments with a sharp scalpel. The cut leaves were placed in a culture dish, the culture dish was sealed, and then placed in a 20℃ constant temperature incubator for enzymatic pretreatment.
[0066] S2: Enzymatic hydrolysis; The pretreated raw material is placed in a shaker of the enzymatic hydrolysis system at a speed of 60-70 r / min and a temperature of 25℃-27℃ in the dark for 2-4 hours.
[0067] S3: Microscopic observation; every 0.5-1 hour, take 10g of the enzyme digest and add it to a clean glass slide for observation;
[0068] S4: Filtration; After the enzymatic hydrolysis is completed, the enzymatic hydrolysis product is filtered through a filter screen to remove large fragments of 70um and 40um, and the enzymatic hydrolysis is terminated with twice the volume of W5.
[0069] S5: Protoplast precipitation; select the lowest speed setting, centrifuge at 100-130g for 5 minutes, carefully remove the supernatant, resuspend in 300-500 μL of resuspension, and observe under a microscope again. After observing the protoplast precipitation in S5, resuspend the protoplasts in 5-10 ml of pre-cooled and well-mixed W5 solution, centrifuge at 100-130g for 5 minutes for purification, and carefully remove the supernatant. If resuspending in 300-500 μL, the supernatant can be examined under a microscope. After observing the protoplast precipitation in S5, resuspend the protoplasts in 5-10 ml of pre-cooled and well-mixed W5 solution, centrifuge at 80-100g for 10 minutes for purification, and carefully remove the supernatant. If resuspending in 300-500 μL, count the protoplasts. If impurities are still present, continue purification.
[0070] The final Countstar detection dissociation result is:
[0071]
[0072] Based on the findings of Examples 3, 4, and 5, the concentration of Cellulase R-10 in the spotted rhododendron variety is greater than that in the non-spotted rhododendron variety. Existing research on rhododendron protoplast preparation shows that cellulase concentration has a significant impact on rhododendron protoplast yield, protoplast viability, and the yield of live protoplasts, making it a key factor affecting protoplast yield. Therefore, the protoplast separation from spotted rhododendrons is rapid and efficient. Furthermore, comparative experiments with various seedlings of *Azalea variegated*, *Azalea sylvestris*, and *Azalea yunnanensis* demonstrate that this method for preparing protoplasts from spotted rhododendrons is representative and universal.
[0073] Furthermore, all content not described in detail in this specification is existing technology known to those skilled in the art, and the model parameters of each electrical appliance are not specifically limited; conventional equipment can be used.
[0074] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.
[0075] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.
Claims
1. A method for preparing Rhododendron anthopogis protoplasts and its application, characterized in that, The specific steps include the following: S1: Material preparation; Under aseptic conditions, take the spotted and non-spotted parts of the azalea and cut them into 0.5mm fine filaments with a sharp scalpel. Place the cut leaves in a culture dish, seal the culture dish, and then place it in a 20℃ constant temperature incubator for enzymatic pretreatment. S2: Enzymatic hydrolysis; The pretreated raw material is placed in a shaker of the enzymatic hydrolysis system at a speed of 60-70 r / min and a temperature of 25℃-27℃ in the dark for 2-4 hours. S3: Microscopic observation; every 0.5-1 hour, take 10g of the enzyme digest and add it to a clean glass slide for observation; S4: Filtration; After the enzymatic hydrolysis is completed, the enzymatic hydrolysis product is filtered through a filter screen to remove large fragments of 70um and 40um, and the enzymatic hydrolysis is terminated with twice the volume of W5. S5: Protoplast precipitation; select the lowest speed setting, centrifuge at 100-130g for 5 minutes, carefully remove the supernatant, resuspend in 300-500 ml resuspension and observe under a microscope again.
2. The method for preparing the Rhododendron punctatum protoplast and application according to claim 1, characterized in that: After observing the protoplast precipitate in S5 under a microscope, resuspend the protoplast in 5-10 ml of pre-cooled and well-mixed W5 solution, centrifuge at 100-130 g for 5 min for purification, and carefully remove the supernatant. If resuspend in 300-500 μl of resuspension, the supernatant can be examined under a microscope.
3. The method and application for preparing protoplasts from rhododendron spots according to claim 2, characterized in that: After microscopic observation of the protoplast precipitate in S5, resuspend the protoplasts in 5-10 ml of pre-cooled and well-mixed W5 solution. Purify by centrifugation at 80-100 g for 10 min, then carefully remove the supernatant. Resuspend in 300-500 μl and count the protoplasts. If impurities are still present, continue purification.
4. The method and application for preparing protoplasts from rhododendron spots according to claim 1, characterized in that: In S1-S5, the centrifuge speed is set to 0, and all material transfers are performed by tilting.
5. The method and application for preparing protoplasts from rhododendron spots according to claim 1, characterized in that: The specific method for extracting the 10 enzyme hydrolysate during the S3 microscopic examination was to use a pipette tip with a cut end to extract it, and to carefully blow it during the extraction process.
6. The method and application for preparing protoplasts from rhododendron spots according to claim 1, characterized in that: After centrifugation in step S5, the supernatant is not discarded. The precipitate and supernatant are observed under a microscope, and any floating impurities are checked in the supernatant.
7. The method and application for preparing protoplasts from rhododendron spots according to claim 1, characterized in that: In the S3 microscopic observation, the enzymatic hydrolysate was dropped onto a clean glass slide and then photographed using 4x and 10x microscopy.