Separation method of sphagnum moss protoplast
By treating peat moss stems and leaves with tissue culture and enzymatic hydrolysate, the problem of separating high-quality peat moss protoplasts in existing technologies has been solved, achieving efficient and low-cost protoplast extraction and providing a good foundation for molecular biology research on peat moss.
Patent Information
- Application Number
- CN202410503034.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-04-25
- Publication Date
- 2025-10-28
AI Technical Summary
Existing technologies struggle to efficiently isolate high-quality peat moss protoplasts, and the resulting protoplasts exhibit low genetic transformation efficiency.
The stems and leaves of sphagnum moss were tissue cultured, pretreated with mild mechanical damage and mechanical crushing, and then enzymatically hydrolyzed with an enzyme solution containing cellulase, hemicellulase and pectinase, and the protoplasts were separated by washing with a mannitol solution.
The method achieves efficient acquisition of large quantities of high-quality peat moss protoplasts, with material usage less than 1/10 of that of existing methods. The protoplasts are pure and maintain good vitality, making them suitable for genetic transformation.
Smart Images
Figure HDA0004809291710000011
Abstract
Description
Technical Field
[0001] This invention pertains to biological research and relates to a method for isolating protoplasts of peat moss. Background Technology
[0002] Sphagnum moss is one of the most ecologically and economically valuable bryophytes. Research on sphagnum moss requires transgenic molecular biology studies. Protoplasts are important materials for plant molecular biology research. The isolation of most plant protoplasts ultimately relies on enzymatic hydrolysis of the cell wall. However, the cell walls of not all plant materials can be enzymatically hydrolyzed to isolate protoplasts. The key to protoplast isolation lies in cultivating suitable materials for protoplast isolation, as well as the pretreatment methods and hydrolysis time, hydrolysis osmotic conditions, and the determination of the protoplast isolation process. The specific details of protoplast isolation methods vary among different plants. Tissue culture of sphagnum moss has been achieved. However, an effective method for the isolation of sphagnum moss protoplasts has not yet been reported. Foreign literature reports obtaining 1.5 x 10^ ... 5 The current method for obtaining protoplasts is cumbersome and yields protoplasts with low genetic transformation efficiency. While a modified method based on the protoplast isolation method for *Moss sclerotium* in a graduate thesis in our lab can also extract protoplasts, this method is inefficient, and the obtained protoplasts are unclean and of poor quality, unsuitable for subsequent experimental procedures. Therefore, a method for obtaining clean, high-quality protoplasts is urgently needed. Summary of the Invention
[0003] To address the shortcomings of existing technologies, the present invention aims to provide a method for extracting protoplasts from peat moss. The method uses peat moss obtained through tissue culture as material, and after culturing under certain conditions for a certain period of time, it undergoes further pretreatment. After enzymatic hydrolysis for a certain period of time, the protoplasts are washed and separated using a specific method to obtain a large quantity of high-quality protoplasts.
[0004] This invention provides a method for isolating peat moss protoplasts, the method comprising the following steps:
[0005] 1) Tissue culture of stem and leaf tissue materials of peat moss;
[0006] 2) The material was subjected to mild mechanical damage followed by subculture.
[0007] 3) The culture material described in step 2) is subjected to mechanical pulverization pretreatment;
[0008] 4) After filtering the pulverized material described in step 3), immerse it in a certain amount of mannitol solution containing enzymatic hydrolysate for enzymatic hydrolysis;
[0009] 5) Filter the enzymatic hydrolysate obtained in step 4) and collect the filtrate;
[0010] 6) After multiple mannitol extractions, combine all the filtrates, centrifuge, and discard the supernatant to obtain crude peat moss protoplasts;
[0011] 7) After washing the crude protoplast obtained in step 6) with mannitol solution, high-quality protoplasts can be obtained.
[0012] In step 1), the stem and leaf material of the peat moss is fresh, healthy stem and leaf subcultured in liquid; wherein the subculture time does not exceed 60 days.
[0013] In step 2), the subculture time is 10-18 days; preferably, it is 14 days.
[0014] In step 3), the key point of the mechanical pulverization pretreatment is a short pulverization time and low intensity, which disperses the material but minimizes structural damage. The pulverization time is 2-5 seconds.
[0015] In step 4), the concentration of the mannitol solution is 6%-7.5%; preferably, it is 7%.
[0016] In step 4), the certain amount is more than 5 times the volume of the material.
[0017] In step 4), the enzymatic hydrolysis time is 5-8 hours; preferably, it is 6 hours.
[0018] In step 4), the enzyme is a breakdown enzyme, or a mixture of cellulase, hemicellulase and pectinase as its main components, or other mixtures containing the above three enzymes.
[0019] In this invention, the separation of protoplasts includes filtration and filtration after multiple washings, as well as centrifugation after combining the filtrates.
[0020] In step 6), the mannitol extraction refers to adding 5-10 times the volume of mannitol solution to the filtered material, mixing thoroughly, filtering, and collecting the filtrate.
[0021] In step 6), the concentration of the mannitol solution is 6%-7.5%; the multiple times refers to 1-3 times.
[0022] In step 7), the mannitol solution washing refers to adding 5-10 times the volume of mannitol solution to the centrifuged crude protoplast, mixing thoroughly, centrifuging, and discarding the supernatant.
[0023] In step 7), the concentration of the mannitol solution is 6%-7.5%; the number of washes is 1-2.
[0024] The beneficial effects of this invention are as follows: This invention provides a method for extracting protoplasts from peat moss, creating a sound technical foundation for further molecular biology research on peat moss. The method described in this invention uses far less material than existing methods (less than 1 / 10 of published methods abroad), and the yield of protoplasts obtained can exceed 10 times that of existing methods. Furthermore, the protoplasts are pure, maintain good viability, and can be used for genetic transformation. Attached Figure Description
[0025] Figure 1 These are a large number of peat moss protoplasts obtained in Example 1 of the present invention. Detailed Implementation
[0026] The present invention will be further described in detail below with reference to the specific embodiments and accompanying drawings. Except for the contents specifically mentioned below, the processes, conditions, and experimental methods for implementing the present invention are all common knowledge and general knowledge in the art, and the present invention does not have any particular limitations.
[0027] Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the invention or its application or use.
[0028] In all examples shown and discussed herein, any specific values should be interpreted as merely exemplary and not as limitations. Therefore, other examples of exemplary embodiments may have different values.
[0029] Unless otherwise specified, the experimental materials used in the examples are all conventional biochemical reagents and routine procedures.
[0030] Example 1:
[0031] (1) Take fresh peat moss stems and leaves that have been cultured in liquid tissue for 30 days and mechanically pulverize them for 20 seconds using an IKA tissue disperser.
[0032] (2) The crushed peat moss was cultured for 14 days under conventional liquid culture conditions.
[0033] (3) The cultured peat moss was mechanically pulverized for 5 seconds using an IKA tissue disperser.
[0034] (4) After filtering the crushed peat moss material, add it to 20 ml of 7% mannitol solution containing 2% pyrolysis enzyme and enzymatically hydrolyze for 6 hours.
[0035] (5) Filter the enzymatically hydrolyzed material and collect the filtrate.
[0036] (6) Add 20 ml of 7% mannitol solution to the filtered residue, mix well, filter, and collect the filtrate.
[0037] (7) Repeat step (6) twice, combine all the protoplasts obtained after centrifugation, and wash twice with 15 ml of 7% mannitol to obtain high-quality protoplasts.
[0038] Results: Using the method of Example 1 of this invention, 1.71 × 10¹² sphagnum moss was isolated from 0.31 g of fresh peat moss culture material (equivalent to 0.006 g dry weight). 6 The protoplasts were clean and their viability was measured to be over 90%, with an instantaneous conversion efficiency of 54%.
[0039] Example 2:
[0040] (1) Take fresh peat moss stems and leaves that have been cultured in liquid tissue for 21 days and mechanically pulverize them for 20 seconds using an IKA tissue disperser.
[0041] (2) The crushed peat moss was cultured for 16 days under conventional liquid culture conditions.
[0042] (3) The cultured peat moss was mechanically pulverized for 5 seconds using an IKA tissue disperser.
[0043] (4) After filtering the crushed peat moss material, add it to 10 ml of 7% mannitol solution containing 2% pyrolysis enzyme and enzymatically hydrolyze for 5 hours.
[0044] (5) Filter the enzymatically hydrolyzed material and collect the filtrate.
[0045] (6) Add 10 ml of 7% mannitol solution to the filtered residue, mix well, filter, and collect the filtrate.
[0046] (7) Repeat step (6) 3 times, combine the protoplasts obtained after centrifuging all the filtrates, wash twice with 15 ml of 7% mannitol, discard the supernatant, and you can obtain high-quality protoplasts.
[0047] Results: Using the method of Example 2 of this invention, 1.38 x 10⁻⁶ sphagnum moss was isolated from 1 g of fresh peat moss culture material (equivalent to 0.02 g dry weight). 6 One protoplast, the protoplast is clean.
[0048] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
[0049] As used in this invention, the terms "comprising" and "including" are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.
[0050] As used in this invention, the term "and / or" includes any one or more of the related listed items and all combinations thereof.
[0051] The scope of protection of this invention is not limited to the above embodiments. Any variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of this invention are included in this invention and are protected by the appended claims.
Claims
1. A method for extracting protoplasts from peat moss, characterized in that, The method includes the following steps: 1) Tissue culture of stem and leaf tissue materials of peat moss; 2) The material was subjected to mild mechanical damage followed by subculture. 3) The culture material described in step 2) is subjected to mechanical pulverization pretreatment; 4) After filtering the pulverized material described in step 3), immerse it in a certain amount of mannitol solution containing enzymatic hydrolysate for enzymatic hydrolysis; 5) Filter the enzymatic hydrolysate obtained in step 4) and collect the filtrate; 6) After multiple mannitol extractions, combine all the filtrates, centrifuge, and discard the supernatant to obtain crude peat moss protoplasts; 7) After washing the crude protoplast obtained in step 6) with mannitol solution, high-quality protoplasts can be obtained.
2. The protoplast separation method as described in claim 1, characterized in that, In step 1), the stem-leaf material is a fresh, healthy stem-leaf subcultured in liquid, and the subculture time does not exceed 60 days.
3. The protoplast separation method as described in claim 1, characterized in that, In step 2), the subculture time is 10-18 days.
4. The protoplast separation method as described in claim 1, characterized in that, In step 3), the pulverization time of the mechanical pulverization pretreatment is 2-5 seconds.
5. The protoplast separation method as described in claim 1, characterized in that, In step 4), the concentration of the mannitol solution is 6%-7.5%; and / or, the amount is more than 5 times the volume of the material.
6. The protoplast separation method as described in claim 1, characterized in that, In step 4), the enzymatic hydrolysis time is 5-8 hours; the enzyme is a breakdown enzyme or a mixture of cellulase, hemicellulase and pectinase.
7. The protoplast separation method as described in claim 1, characterized in that, In step 6), the mannitol extraction refers to adding 5-10 times the volume of mannitol solution to the filtered material, mixing thoroughly, filtering, and collecting the filtrate.
8. The protoplast separation method as described in claim 1, characterized in that, In step 6), the concentration of the mannitol solution is 6%-7.5%; and / or, the multiple times are 1-3 times.
9. The protoplast separation method as described in claim 1, characterized in that, In step 7), the mannitol solution washing refers to adding 5-10 times the volume of mannitol solution to the centrifuged crude protoplast, mixing thoroughly, centrifuging, and discarding the supernatant.
10. The protoplast separation method as described in claim 1, characterized in that, In step 7), the concentration of the mannitol solution is 6%-7.5%; and / or, the number of washes is 1-2.