CN-121975791-A - Tricholoma giganteum and stropharia rugoso-annulata protoplast fusion-based stropharia rugoso-annulata breeding method

CN121975791ACN 121975791 ACN121975791 ACN 121975791ACN-121975791-A

Abstract

The invention belongs to the technical field of edible fungus biological breeding, and particularly discloses a stropharia rugoso-annulata breeding method based on the fusion of stropharia rugoso-annulata and stropharia rugoso-annulata protoplasts, which comprises the steps of respectively preparing stropharia rugoso-annulata and stropharia rugoso-annulata protoplasts; carrying out heat inactivation on stropharia rugoso-annulata protoplast, carrying out ultraviolet inactivation on tricholoma giganteum protoplast, mixing the two protoplasts after the inactivation, adding an induction reagent to carry out protoplast fusion, coating a fusion product on a regeneration culture medium for culture to obtain a fusion strain, screening a target fusion strain through an antagonism test and a molecular marking technology, carrying out fruiting management, and screening out a new strain of the high-temperature resistant stropharia rugoso-annulata. According to the invention, by optimizing protoplast preparation and PEG thawing promotion conditions, distant fusion efficiency is remarkably improved, and a high-efficiency screening and identification system is combined, so that a novel stropharia rugoso-annulata strain which can normally fruiting at a high temperature of 30 ℃ and has excellent agronomic characters and good flavor is successfully obtained.

Inventors

MO MEIHUA
ZHAO YUKE
XU XUEFENG

Assignees

华南农业大学

Dates

Publication Date: 20260505
Application Date: 20260331

Claims (6)

1. The stropharia rugoso-annulata breeding method based on the fusion of stropharia rugoso-annulata and stropharia rugoso-annulata protoplast is characterized by comprising the following steps: s1, respectively preparing protoplasts of stropharia rugoso-annulata and tricholoma giganteum; s2, carrying out heat inactivation and ultraviolet inactivation treatment on stropharia rugoso-annulata protoplast and tricholoma giganteum protoplast respectively; s3, mixing the two inactivated protoplasts, and adding a fusion reagent to fuse the protoplasts; S4, after fusion is finished, centrifugally washing the mixed reagent through a permeation stabilizing agent, coating the washed mixed reagent on a regeneration culture medium, culturing and recording regenerated bacterial colonies, and separating to obtain a fusion strain; S5, carrying out an antagonism test and ISSR molecular marker screening on the fusion strain to obtain a target fusion strain; s6, fruiting management is carried out on the target fusion strain, and a novel stropharia rugoso-annulata strain which can normally grow and fruiting at 30 ℃ is screened out.
2. The method for breeding stropharia rugoso-annulata based on the fusion of stropharia rugoso-annulata protoplast according to claim 1, wherein in the step S1, the preparation method of the protoplast comprises the steps of respectively placing the stropharia rugoso-annulata and the mycelium of stropharia rugoso-annulata with the bacterial age of 4-10 days into enzyme solutions for enzymolysis, wherein the enzyme solutions are prepared by adding 2.5% of cellulase in a mass-volume ratio, 1.5% of lywallenzyme in a mass-volume ratio and 2% of snailase in a mass-volume ratio into a 0.6M sterile mannitol solution, the enzymolysis temperature is 26-32 ℃, the enzymolysis time is 80-160 min, filtering the obtained stropharia rugoso-annulata enzymolysis solution and the obtained stropharia rugoso-annulata enzymolysis solution to remove mycelium fragments, washing, centrifuging, removing supernatant, collecting protoplasts, washing, and resuspensioning to obtain purified stropharia rugoso-annulata protoplast and stropharia rugoso suspensions respectively.
3. The method for breeding stropharia rugoso-annulata based on the fusion of stropharia rugoso-annulata protoplast according to claim 1, wherein in the step S2, the condition of heat inactivation is that the temperature is constant water bath at 40 ℃ and is 25 min, and the condition of ultraviolet inactivation is that the temperature is constant water bath at 15W and cm under 18W ultraviolet lamp and 18 min is irradiated.
4. The method for breeding stropharia rugoso-annulata based on the fusion of stropharia rugoso-annulata protoplast according to claim 1, wherein in the step S3, the fusion reagent comprises an induction reagent polyethylene glycol with a molecular weight of 6000 and a concentration of 24% -36%, tris of 0.004-0.016M and CaCl 2 of 0.014-0.026M, and the condition of protoplast fusion is that the water bath fusion time is 18-30 min, the water bath fusion temperature is 24-36 ℃, and the pH value at the time of fusion is 7.4-8.6.
5. The method for breeding stropharia rugoso-annulata based on the fusion of stropharia rugoso-annulata protoplast according to claim 1, wherein in step S4, the penetration stabilizing agent is a mannitol solution of 0.6 mol/L.
6. The method for breeding stropharia rugoso-annulata based on the fusion of stropharia rugoso-annulata and stropharia rugoso-annulata protoplast according to claim 2, wherein the preparation process of the stropharia rugoso-annulata and stropharia rugoso-annulata mycelium is as follows: Inoculating Tricholoma giganteum and Stropharia rugoso-annulata strain into PDA flat-plate culture medium for activating 4 d, inoculating 3 colony blocks with the front end of 0.5 cm size into liquid potato culture medium, placing the liquid potato culture medium inoculated with Tricholoma giganteum into a 30 ℃ incubator for culturing 7 d, placing the liquid potato culture medium inoculated with Stropharia rugoso-annulata into a 25 ℃ incubator for culturing 7 d to obtain Tricholoma giganteum mycelium pellet and Stropharia rugoso-annulata mycelium pellet, inoculating the Tricholoma giganteum mycelium pellet into a 250 mL conical flask containing 120 mL liquid potato culture medium for culturing 4-10 d to obtain Tricholoma giganteum mycelium, and inoculating the Stropharia rugoso-annulata mycelium pellet into a 250 mL conical flask containing 120 mL liquid potato culture medium for culturing 4-10 d to obtain Stropharia rugoso-annulata mycelium.

Description

Tricholoma giganteum and stropharia rugoso-annulata protoplast fusion-based stropharia rugoso-annulata breeding method Technical Field The invention relates to the technical field of edible fungus biological breeding, in particular to a stropharia rugoso-annulata breeding method based on the fusion of stropharia rugoso-annulata and stropharia rugoso-annulata protoplast. Background The tricholoma giganteum is a high-temperature rare edible fungus, mycelium and fruiting body of the tricholoma giganteum are both favored by high temperature in growth and development, the mycelium can still grow vigorously at 30-35 ℃, and the proper temperature for fruiting body formation is as high as 25-32 ℃, so that the tricholoma giganteum shows remarkable high-temperature tolerance. The stropharia rugoso-annulata is a cultivated mushroom seed recommended by the grain and agriculture organizations of the united nations, and has strong adaptability and high yield. However, it is a typical medium and low temperature type mushroom, and the optimum temperature range for fruiting is narrow, usually 12-25 ℃. When the ambient temperature is continuously higher than 28 ℃, the fruiting body is easy to grow, the fungus cover is opened in advance, the fungus handle is excessively grown and is thin and weak, and even primordia cannot be formed. Traditionally, edible fungi breeding mainly relies on systematic breeding, cross breeding and mutation breeding. For stropharia rugoso-annulata, the methods have been developed to some extent in terms of improving color, yield and the like, but the development of obtaining breakthrough properties such as high temperature resistance and the like by intraspecific hybridization is slow due to the relatively narrow genetic background. While tricholoma giganteum and stropharia rugoso-annulata belong to distant species, serious reproductive isolation exists, and gene communication is difficult to realize by the traditional hybridization means. Mutation breeding can create mutation, but mutation direction is random and uncontrollable, efficiency of obtaining target characters is extremely low, and screening workload is huge. Protoplast fusion technology is a key means to break reproductive isolation and achieve recombination of distant genetic material. However, when the technology is applied to fusion breeding of tricholoma giganteum and stropharia rugoso-annulata, the prior art has the prominent defects that (1) the protoplast preparation technology is not mature, parameters such as enzyme liquid type, concentration, action temperature and time are not optimized, so that the prepared protoplast has low activity and poor regeneration capability, (2) a fusion promoting system is low in efficiency, and the optimal research of polyethylene glycol (PEG) with optimal molecular weight, optimal concentration, action pH value and treatment time aiming at the specific fusion combination is lacking, so that the formation rate of heterokaryons is extremely low, and (3) a high-efficiency fusion screening and identification system is lacking, so that the method is crucial for establishing a set of efficient early screening and molecular identification procedures for the tricholoma giganteum and stropharia rugoso-annulata strains which lack known genetic markers. Therefore, developing a complete and efficient breeding method aiming at the fusion of tricholoma giganteum and stropharia rugoso-annulata from protoplast optimization preparation to PEG parameter optimization fusion promotion until efficient identification of the fusion becomes a necessary technical demand for breaking through the industrial bottleneck. Disclosure of Invention In order to solve the technical problems, the invention provides a stropharia rugoso-annulata breeding method based on the fusion of tricholoma giganteum and stropharia rugoso-annulata protoplasts. In order to achieve the above purpose, the invention is implemented according to the following technical scheme: A stropharia rugoso-annulata breeding method based on the fusion of stropharia rugoso-annulata and stropharia rugoso-annulata protoplast comprises the following steps: s1, respectively preparing protoplasts of stropharia rugoso-annulata and tricholoma giganteum; s2, carrying out heat inactivation and ultraviolet inactivation treatment on stropharia rugoso-annulata protoplast and tricholoma giganteum protoplast respectively; s3, mixing the two inactivated protoplasts, and adding a fusion reagent to fuse the protoplasts; S4, after fusion is finished, centrifugally washing the mixed reagent through a permeation stabilizing agent, coating the washed mixed reagent on a regeneration culture medium, culturing and recording regenerated bacterial colonies, and separating to obtain a fusion strain; S5, carrying out an antagonism test and ISSR molecular marker screening on the fusion strain to obtain a target fusion strain; s6, fruiting management is carried out on the target fusion strain