Method for preparing germination-promoting fungal protoplasts directly from orchid protocorms

Abstract

This application belongs to the fields of microbiology and cell biology, and provides a method for directly preparing germination-promoting fungal protoplasts from orchid protocorms, comprising: (1) physical homogenization; (2) filtration; (3) removal of plant contents and residual cell walls; (4) purification of mycelial clusters; and (5) release and purification of fungal protoplasts. Through the rational design of homogenization and purification steps, this application achieves high-yield, high-purity, and high-vitality preparation of germination-promoting fungal protoplasts, providing excellent materials for orchid gene variety breeding and genetic engineering operations.

Description

Technical Field

[0001] This application belongs to the fields of microbiology and cell biology. Specifically, this application provides a method for preparing germination-promoting fungal protoplasts directly from the protocorms of orchid plants. Background Technology

[0002] Orchidaceae ( Orchidaceae Orchids belong to the class Magnoliopsida, subclass Liliaceae, and order Asparagales, making them the second largest family of angiosperms. They possess extremely high economic, ornamental, and medicinal value. However, the seeds of most orchids are minuscule and lack functional endosperm, making germination impossible through their own nutrition. They must establish a symbiotic relationship with specific soil fungi under natural conditions to initiate germination, which is a major bottleneck for their resource conservation and artificial propagation. The protocorm formed after successful seed germination is a unique structure representing the symbiotic relationship between orchids and fungi, and is a prerequisite for seedling formation. Therefore, elucidating the molecular interaction mechanism between the host and fungi at the protocorm stage is the theoretical foundation for solving the germination problem of orchids.

[0003] In this symbiotic system, fungi form specialized pelotons within the cortical cells of the protocorm. Figure 1 These hyphae increase the contact area with plant cells to exchange nutrients and communicate signals. These hyphal masses have a short lifespan and are rapidly digested and renewed within plant cells, exhibiting high dynamism. Further research has revealed functional differentiation among fungi in plant cells. For example, Li et al. discovered, through ultrastructural analysis, that papillary thickened cell walls (such as...) appeared in plant cells invaded by hyphae. Figure 1 (As indicated by the middle arrow), it may perform nutrient transport functions. Zhao et al. proposed a model through multi-omics research showing that there are differences in the division of labor for nutrient transport between senescent and active hyphae (…). Figure 1 The interaction between fungi and plants mainly occurs at the symbiotic interface formed by the plant's plasma membrane enveloping the hyphae (e.g., Figure 1 (As shown in the blue section), this interface is a key location for the exchange of matter and signals.

[0004] Cai et al. (2018). "Plants send small RNAs inextracellular vesicles to fungal pathogens to silence virulence genes." Science 360(6393): 1126–1129) from Arabidopsis thaliana ( Arabidopsis Botrytis cinerea, a pathogenic fungus, was isolated from leaf tissue. Botrytis cinereaResearchers developed a sequential protoplast preparation method based on the different components of plant and fungal cell walls. The method includes: (1) physical homogenization: homogenizing in a separation buffer at the highest rotation speed for 1 min to release fungi from plant cells; (2) removal of plant components: removing plant components using detergents and plant cell wall degrading enzymes, and collecting fungi by centrifugation; (3) release and purification of fungal protoplasts: treating fungi with fungal cell wall degrading enzymes, and then purifying the protoplasts by adding 30% sucrose solution. These purified fungal protoplasts were then subjected to sRNA sequencing to identify sRNAs transported from plants. This method successfully demonstrated the cross-border transport of plant sRNAs to fungi in a pathogen-pathogen interaction system.

[0005] While existing technologies have achieved success in plant-pathogen interaction research, their application to mutualistic symbiotic systems such as orchid-symbiotic fungi has the following inherent limitations:

[0006] First, this technology relies on the initial step of "releasing intact fungal mycelia from plant tissue." This is feasible in pathogen-pathogen interactions because pathogenic mycelia grow between plant tissues and are relatively easy to release intact. However, in orchid protocorms, the symbiotic fungi form highly coiled intracellular mycelial masses tightly encased in plant cytoplasm. Ensuring both the release of the mycelial masses from the plant cells and maintaining the integrity of the mycelial mass structure presents certain difficulties. Figure 2 ).

[0007] Second, it is time-consuming, taking about 10 hours from sample preparation to protoplast preservation.

[0008] Third, the buffer solution and purification conditions are not suitable for protoplasts isolated from mycelial clusters, resulting in the inability to obtain complete, high-yield and pure protoplasts.

[0009] Fourth, the rotation speed used in the Chinese description of "purifying protoplasts with 30% sucrose solution" is 5000 rpm. However, when purifying protoplasts from mycelial clusters, this rotation speed will cause the protoplasts to break down, resulting in the fragmented protoplasts and impurities settling at the bottom layer, making it impossible to observe the stratification phenomenon, thus leading to the failure of protoplast separation and purification.

[0010] Silvestri et al. (Silvestri, A., et al. (2025). "A fungal sRNA silences ahost plant transcription factor to promote arbuscular mycorrhizal symbiosis." New Phytologist 246(3): 924–935) studied the cross-kingdom transport of fungal sRNAs in arbuscular mycorrhizal symbiosis. This study directly sequenced the sRNAs of roots (a mixture of plant cells and root hyphae) and the extra-root hyphae after removing extra-root hyphae. By comparing the expression profiles of the sRNAs in both, they screened for fungal sRNAs highly expressed in the roots and verified their function of silencing plant target genes across kingdoms through a series of molecular biology experiments.

[0011] While this study is the first to demonstrate fungal-plant sRNA transport in an arbuscular mycorrhizal symbiotic system and provides an example for elucidating the symbiotic mechanism of orchids, its technical approach has inherent "precision limitations": First, the research precision remains at the tissue level, unable to pinpoint individual cells. The "roots with extracellular hyphae removed" used in this method comprise a mixed system of various plant cell types (colonized cells, non-colonized cells, epidermal cells, cortical cells, etc.) and fungal structures (hyphae, arbuscular inflorescences). The resulting sRNA expression data is an average of all cell signals, making it impossible to determine which specific cell type the sRNA transport and function occur in.

[0012] Second, it cannot resolve the cellular heterogeneity of the symbiotic fungi themselves: sequencing mixed tissues masks the possible cellular heterogeneity and functional division of labor within the symbiotic fungi (such as active hyphae versus senescent hyphae). Figure 3 This makes it impossible to reveal the dynamic regulatory network of symbiotic development. Summary of the Invention

[0013] In summary, a dedicated method is needed to efficiently prepare high-purity, high-vitality fungal protoplasts from the unique symbiotic complex of orchid protocorms. Existing technologies either cannot be applied to the orchid protocorm system or lack the precision to reveal the exact regulatory mechanisms at the cellular level. This invention explores steps 1 and 3 through three key steps: 1. physical homogenization to release mycelial clusters; 2. enzymatic washing to remove plant material; and 3. preparation of fungal protoplasts. A dedicated method has been invented that can directly and efficiently prepare high-yield, high-purity, and high-vitality fungal protoplasts from the unique symbiotic complex of orchid protocorms, overcoming the shortcomings of existing technologies.

[0014] Based on the structural differences between "pathogen hyphae" and "symbiotic hyphal clusters": linear, free growth (easily released intact) vs. highly coiled, compact clusters (tightly wrapped by plant cytoplasm); the applicant designed a suitable physical homogenization method for separation; the protoplasts released from the symbiotic hyphal clusters have membrane structures that are exceptionally sensitive to mechanical shear forces, and the applicant optimized the purification method for the hyphal clusters, saving processing time while maintaining the stability of the protoplasts; considering the osmotic pressure requirements of specific fungal protoplasts, various buffer formulations were adjusted.

[0015] On the one hand, this application provides a method for preparing germination-promoting fungal protoplasts directly from orchid protocorms, the method comprising: (1) Physical homogenization; (2) Filtration; (3) Remove plant contents and residual cell walls; (4) Purification of mycelial clumps; (5) Release and purify fungal protoplasts.

[0016] Further, step (1) physical homogenization includes: placing the orchid protocorm and steel ball into a centrifuge tube, adding separation buffer to submerge the orchid protocorm; vortexing at 40 Hz for 30 s, with 10 s intervals, for 2 cycles to obtain a homogenate.

[0017] Preferably, in step (1) physical homogenization, 0.1g of orchid protocorm and 2 5 mm steel balls are placed in a 2 mL round-bottom centrifuge tube; 1 mL of separation buffer is added to submerge the orchid protocorm; the vortex mixer is used at 40 Hz for 30 s, with an interval of 10 s, for 2 cycles to obtain a homogenate.

[0018] Further, step (2) filtration includes: first filtration: the homogenate obtained in step (1) is filtered through a 70 μm filter and the filtrate is collected; the bottom of the container used to collect the filtrate is rinsed with separation buffer and then filtered through a 70 μm filter; second filtration: the large tissue above the filter is added back into the separation buffer to immerse the protocorm, vortexed at 40 Hz for 30 s with a 10 s interval, for 2 cycles, the resulting homogenate is filtered through a 70 μm filter and the filtrate is collected, the bottom of the container used to collect the filtrate is rinsed with separation buffer and then filtered through a 70 μm filter; the filtrates are combined.

[0019] Preferably, step (2) filtration includes: first filtration: the homogenate obtained in step (1) is filtered through a 70 μm filter, the filtrate is collected in a 50 mL round-bottom centrifuge tube, and the round-bottom centrifuge tube is rinsed with 1 mL of separation buffer and then filtered through a 70 μm filter; second filtration: the large tissue above the filter is placed back into a 2 mL round-bottom centrifuge tube, 1 mL of separation buffer is added to submerge the protocorm, the vortex is run at 40 Hz for 30 s with a 10 s interval, and the cycle is repeated twice. The homogenate is then filtered through a 70 μm filter, the filtrate is collected, and the 2 mL round-bottom centrifuge tube is rinsed with 1 mL of separation buffer and then filtered through a 70 μm filter; the filtrates are combined.

[0020] The preferred containers for collecting the filtrate are various centrifuge tubes, especially round-bottom centrifuge tubes. Further, step (3) to remove plant contents and residual cell walls includes: centrifuging the filtrate obtained in step (2); discarding the supernatant, resuspending the precipitate in plant cell wall digestion solution; incubating for 1 h; centrifuging the mixture after incubation; discarding the supernatant, resuspending the precipitate in Triton X-100 solution, and incubating in an ice bath for 5 min.

[0021] Further, step (3) to remove plant contents and residual cell walls includes: centrifuging the filtrate obtained in step (2) at 4°C and 1000 g for 10 min; discarding the supernatant, resuspending the precipitate in 1 mL of plant cell wall digestion solution; incubating at 28°C and 40 rpm for 1 h; centrifuging the mixture after incubation at 4°C and 1000 g for 10 min; discarding the supernatant, resuspending the precipitate in 1 mL of 1% Triton X-100, and incubating on ice for 5 min; centrifuging again at 4°C and 1000 g for 10 min; discarding the supernatant, resuspending the precipitate in 1 mL of 1% Triton X-100, and incubating on ice for 5 min.

[0022] Furthermore, the plant cell wall digestion solution is an aqueous solution containing 0.01% BSA, 1.5% cellulase R10, 0.4% macerozyme R10, 0.4 M mannitol, 20 mM KCl, 20 mM MES, and 10 mM CaCl2, with a pH of 5.7.

[0023] Further, the purification of the mycelial clumps in step (4) includes density gradient centrifugation, filtration purification, and Triton X-100 washing.

[0024] Further, step (4) purification of the mycelial clumps includes: (4-1) First purification: 4℃, 1000 g, 10 min, discard supernatant, resuspend the precipitate with 1 mL pre-cooled separation buffer, repeat twice; add 1 mL 30% sucrose solution from the bottom, 4℃, 300 rpm, 15 min, aspirate about 400-600 μL of the intermediate layer and place it on a 20 / 40 μm filter screen; (4-2) Add 1 mL 1% Triton X-100, mix by pipetting, incubate for 5 min, 4℃, 500 rpm, 8 min, discard filtrate, repeat twice. Add 1 mL separation buffer, 4℃, 500 rpm, 8 min, discard filtrate, repeat twice; invert the filter screen into a clean 6 cm culture dish or small beaker, add 1 mL separation buffer several times to rinse the mycelial clumps on the filter screen into the culture dish or small beaker, transfer all the liquid in the culture dish or small beaker to a 50 mL centrifuge tube, 4℃, 1000 g, 8 min, discard supernatant. Furthermore, the separation buffer is an aqueous solution containing 0.6 M mannitol and 20 mM MOPS.

[0025] Further, step (5) for releasing and purifying fungal protoplasts includes: centrifuging the product from step (4); discarding the supernatant and resuspending the precipitate in fungal cell wall digestion solution; incubating for 60 min; filtering the incubated mixture and collecting the filtrate; rinsing the incubation container with STC, passing the filter again, and combining the filtrates; centrifuging the filtrate; discarding the supernatant and resuspending the precipitate in pre-cooled STC buffer; adding 30% sucrose solution to the bottom of the cell suspension, a clear interface is visible; centrifuging and aspirating about 200 μL of the milky white layer, which is the fungal protoplast; centrifuging for 15 min; discarding the supernatant and resuspending the precipitate in pre-cooled STC buffer.

[0026] Further, step (5) for releasing and purifying fungal protoplasts includes: centrifuging the product from step (4) at 4°C and 1000g for 8 min; discarding the supernatant and resuspending the precipitate in 1.7 mL of fungal cell wall digestion solution; incubating at 34°C and 40 rpm for 60 min; passing the incubated mixture through a 20 μm filter and collecting the filtrate; rinsing the incubation container with 1 mL of STC, passing the filter again, and combining the filtrates; centrifuging the filtrate at 4°C and 1200 rpm for 8 min; discarding the supernatant and resuspending the precipitate in 1 mL of pre-cooled STC; adding 1 mL of 30% sucrose solution to the bottom of the cell suspension, where a clear interface is visible; centrifuging at 4°C and 300 rpm for 15 min and then aspirating approximately 200 μL of the milky white layer, which is the fungal protoplast; centrifuging at 4°C and 1200 rpm for 15 min; discarding the supernatant and resuspending the precipitate in 1 mL of pre-cooled STC buffer.

[0027] Furthermore, the STC buffer is an aqueous solution containing 1 M sorbitol, 0.1 M Tris-HCl, and 5 mM CaCl2, with a pH of 7.5.

[0028] Furthermore, the method for preparing the fungal cell wall digestion solution is as follows: dissolve 0.06 g of cell wall lysin in 1.7 mL of 0.6 M mannitol solution.

[0029] The methods specifically include: (1) Physical homogenization: Place 0.1g of orchid protocorm and two 5mm steel balls into a 2mL round-bottom centrifuge tube; add 1mL of separation buffer to submerge the orchid protocorm; vortex at 40 Hz for 30 s, with 10 s intervals, for 2 cycles to obtain a homogenate.

[0030] (2) Filtering: First filtration: The homogenate obtained in step (1) was filtered through a 70 μm filter, the filtrate was collected in a 50 mL round-bottom centrifuge tube, and the round-bottom centrifuge tube was rinsed with 1 mL of separation buffer and then filtered through a 70 μm filter. Second filtration: The large tissue above the filter was placed back into a 2 mL round-bottom centrifuge tube, 1 mL of separation buffer was added to submerge the protocorm, and the vortex was run at 40 Hz for 30 s with a 10 s interval for 2 cycles. The homogenate was then filtered through a 70 μm filter, the filtrate was collected, and the 2 mL round-bottom centrifuge tube was rinsed with 1 mL of separation buffer and then filtered through a 70 μm filter. The filtrates were combined.

[0031] (3) Remove plant contents and residual cell walls: Centrifuge the filtrate obtained in step (2) at 4℃ and 1000 g for 10 min; discard the supernatant and resuspend the precipitate in 1 mL of plant cell wall digestion solution; incubate at 28℃ and 40 rpm for 1 h; centrifuge the mixture after incubation at 4℃ and 1000 g for 10 min; discard the supernatant and resuspend the precipitate in 1 mL of 1% Triton X-100 and incubate on ice for 5 min; centrifuge again at 4℃ and 1000 g for 10 min; discard the supernatant and resuspend the precipitate in 1 mL of 1% Triton X-100 and incubate on ice for 5 min.

[0032] (4) Purification of mycelial clumps: (4-1) First purification: 4℃, 1000 g, 10 min, discard supernatant, resuspend the precipitate with 1 mL of pre-cooled separation buffer, repeat twice; add 1 mL of 30% sucrose solution from the bottom, 4℃, 300 rpm, 15 min, aspirate about 400-600 μL of the intermediate layer and place it on a 20 / 40 μm filter screen; (4-2) add 1 mL of 1% Triton X-100, mix by pipetting, incubate for 5 min, 4℃, 500 rpm, 8 min, discard the filtrate, repeat twice. Add 1 mL of separation buffer, incubate at 4℃, 500 rpm, for 8 min, discard the filtrate, and repeat twice. Place the filter upside down in a clean 6 cm petri dish or small beaker, and add 1 mL of separation buffer several times to rinse the mycelial clumps on the filter into the petri dish or small beaker. Transfer all the liquid in the petri dish or small beaker to a 50 mL centrifuge tube, incubate at 4℃, 1000 g, for 8 min, and discard the supernatant.

[0033] (5) Release and purification of fungal protoplasts: Centrifuge the product from step (4) at 4°C and 1000 g for 8 min; discard the supernatant and resuspend the precipitate in 1.7 mL of fungal cell wall digestion solution; incubate at 34°C and 40 rpm for 30-60 min; pass the incubated mixture through a 20 μm filter and collect the filtrate; rinse the incubation container with 1 mL of STC, pass through the filter again, and combine the filtrates; centrifuge the filtrate at 4°C and 1200 rpm for 8 min; discard the supernatant and resuspend the precipitate in 1 mL of pre-cooled STC; add 1 mL of 30% sucrose solution to the bottom of the cell suspension, and a clear interface will be visible; centrifuge at 4°C and 300 rpm for 15 min, and then aspirate about 200 μL of the milky white layer, which is the fungal protoplast; centrifuge at 4°C and 1200 rpm for 15 min; discard the supernatant and resuspend the precipitate in 1 mL of pre-cooled STC buffer.

[0034] The methods described in this application can be used for, for example, breeding: using fungal protoplasts and plant protoplasts from orchids or other economic crops (such as medicinal herbs and forest trees) to perform somatic cell fusion or introduce genetic material, artificially creating or optimizing new symbiotic relationships, and cultivating new varieties that are resistant to stress (drought resistance, salt tolerance), promote growth, or have high quality; metabolic engineering platforms: utilizing fungal protoplasts to act on effective active ingredients or metabolic engineering platforms; preservation and propagation of rare orchid fungal germplasm resources: through ultra-low temperature freezing of protoplasts, high-purity strains can be efficiently preserved and used for subsequent liquid fermentation amplification, providing stable and controllable fungal inoculants for industrialized orchid seedling production; and establishing an efficient genetic manipulation and gene function research platform using protoplasts. Attached Figure Description

[0035] Figure 1 This is a schematic diagram of orchid mycorrhizae.

[0036] Figure 2 This shows the entanglement of mycelial clumps and plant tissue (red arrows indicate entangled mycelial clumps and plant tissue).

[0037] Figure 3 This displays the condition of active and senescent mycelial clusters.

[0038] Figure 4 This application demonstrates the technical approach used.

[0039] Figure 5 This demonstrates the effect of different physical homogenization conditions on the morphology of mycelial clusters.

[0040] Figure 6 This displays the number of mycelial clumps obtained from the first and second filtrations (AC section: first filtration; DF section: second filtration. Red arrows indicate mycelial clumps). : P <0.05).

[0041] Figure 7 This shows the effect of different sorbitol concentrations in STC buffer on protoplast separation.

[0042] Figure 8 The results of centrifugal washing purification and gradient centrifugation purification are displayed.

[0043] Figure 9 An observational image showing the protoplasts of the hyphal mass. Detailed Implementation

[0044] Example 1 Materials and Reagents Material: GS2 strain of the genus *Cyclocarya* Ceratobasidium sp. GS2 (GenBank accession number OK655751.1) was isolated from the root system of *Gynostemma pentaphyllum* growing in an alpine meadow at an altitude of 1920 m in Songshan, Beijing. It significantly promoted seed germination and seedling biomass accumulation in *Gynostemma pentaphyllum*. Frozen-preserved GS2 was inoculated onto freshly prepared oat agar (OMA) medium and cultured in the dark at 28°C. The strain was preserved at the Institute of Microbiology's Culture Collection Center.

[0045] Hand ginseng ( Gymnadenia conopsea Seeds were collected in mid-August 2024 from the Baihuashan Nature Reserve in Beijing. Ten plants were randomly selected, and ten unopened capsules were collected from each plant. After collection, the capsules were surface-sterilized by immersing them in a 3% sodium hypochlorite solution for 5 minutes, followed by rinsing three times with sterile water. After sterilization, the capsules were air-dried and stored in 1.5 mL centrifuge tubes containing discolored silica gel to prevent moisture. Seeds were stored in a refrigerator at 4°C protected from light until further use.

[0046] Protocorm culture system: Using a 3 mm punch, samples were taken from the periphery of OMA that was covered with GS2 mycelium (cultured for about one week), and inoculated into the center of a 9 cm layer of OMA. Sterilized cotton was used to dab the seeds onto the OMA. After about 30 days, the seeds germinated. Protocorms that had germinated for about 60 days after the fungus-seed co-culture were collected.

[0047] Experimental consumables, instruments, and reagents: Consumables: 2 mL and 50 mL round-bottom centrifuge tubes, pipettes and tips (20 / 1000 μL), 0.22 μm sterile filter, 70 μm and 40 μm cell filters.

[0048] Instruments: Vortex shaker, optical / fluorescence microscope, centrifuge, constant temperature shaker, clean bench, autoclave, molecular balance, pH meter.

[0049] Reagents: Mannitol, potassium chloride (KCl), calcium chloride (CaCl2), sodium chloride (NaCl), MES [2-(N-Morpholino)ethanesulfonic acid, 2-(N-morpholino)ethanesulfonic acid], potassium hydroxide (KOH), cellulase (CELLULASE R-10), cleavage enzyme (MACEROZYME R-10), bovine serum albumin (BSA), nuclease-free water, lysozyme (Guangdong Institute of Microbiology), Triton X-100, MOPS, sorbitol, sucrose, trypan blue, FDA, acetone.

[0050] Solution preparation: Mother liquor preparation: Table 1 Mother liquor formulation

[0051] Note: Triton X-100 solution is viscous and should be slowly aspirated / pumped into the separation buffer, and blown up and down (to avoid liquid residue in the pipette tip as much as possible), and stirred or vortexed to make it completely dissolved and transparent.

[0052] Buffer solution preparation: Table 2 Separation Buffer

[0053] Note: Sterilize by filtration, store at 4℃ for 1 year.

[0054] Table 30.6 M Mannitol Buffer

[0055] Note: Sterilize by filtration, store at 4℃ for 1 year.

[0056] Table 4 STC Buffer

[0057] Note: Sterilize by filtration, store at 4℃ for 1 month.

[0058] Preparation of washing and purification solutions: Table 5 1% Triton X-100 Washing Solution

[0059] Note: Store at room temperature away from light. Shelf life: 1 year. Table 6 30% Sucrose Solution

[0060] Note: Store at 4 degrees Celsius for 3-6 months. Preparation of enzymatic hydrolysate: Table 7. Formulation of plant cell wall enzymatic hydrolysate

[0061] Note: Prepare and use immediately; store on ice. Table 8 Formulation of fungal cell wall enzymatic hydrolysate

[0062] Note: Prepare and use immediately; store on ice. Dye solution preparation: Table 9 0.4% trypan blue dye solution

[0063] Note: Store at 4℃ away from light.

[0064] Table 10 FDA Stock Solution (5 mg / mL)

[0065] Note: Store at 4℃ away from light for 1 year.

[0066] Table 11 0.01% FDA Working Solution

[0067] Note: Prepare and use immediately, store at 4℃ away from light.

[0068] Example 2 Preparation of protoplasts The protoplast preparation process is as follows: Figure 4 As shown: Physical homogenization releases mycelial clusters (~20 min): Sample preparation: Place the protocorm (weighed, 0.1g) and two 5 mm steel balls into a 2 mL round-bottom centrifuge tube, and add 1 mL of separation buffer to submerge the protocorm.

[0069] Physical homogenization: vortex mixer 40 Hz, 30 s, 10 s interval, 2 cycles.

[0070] First filtration: The homogenate was filtered through a 70 μm filter, the filtrate was collected in a 50 mL round-bottom centrifuge tube, and the round-bottom centrifuge tube was rinsed with 1 mL of separation buffer and then filtered through a 70 μm filter.

[0071] Second filtration: The large tissue fragment above the filter was placed back into a 2 mL round-bottom centrifuge tube, and 1 mL of separation buffer was added to submerge the protocorm. A vortex mixer was used at 40 Hz for 30 s, with 10 s intervals, for two cycles. The homogenate was filtered through a 70 μm filter, and the filtrate was collected. The filtrate was then rinsed in a 2 mL round-bottom centrifuge tube with 1 mL of separation buffer and filtered through a 70 μm filter. The filtrates were combined.

[0072] To investigate the effect of physical homogenization conditions on mycelial clump morphology, we compared two different programs using a vortex mixer: 40 Hz, 30 s, 10 s interval, 2 cycles; and 70 Hz (maximum speed), 60 s. The results showed that the gentle, intermittent oscillation program resulted in mycelial clumps that were essentially clumped together (e.g., ...). Figure 5 The AC section; a high-speed, non-intermittent oscillation program, resulting in loose and broken mycelial clumps (such as...). Figure 5 (DF portion). The results showed that only a gentle, intermittent oscillation rate could ensure the complete release of the mycelial clumps.

[0073] To investigate the effect of filtration conditions on the number of mycelial clusters, we compared the number of clusters after one filtration and two filtrations. First filtration: the homogenate was filtered through a 70 μm filter; Second filtration: based on the first filtration, the residue on the filter screen was homogenized again at 40 Hz for 30 s, with two cycles, and the homogenate was then filtered again through a 70 μm filter. The results showed that the average total yield of the second filtration (5,450 clusters) was 68% higher than that of the first filtration (3,238 clusters), and this was statistically significant (p = 0.020). Figure 6 The coefficients of variation (CV) for the two filtrations were 3.0% and 4.5%, respectively, indicating that the operation was highly stable and reproducible. Through the optimized two-stage filtration process, the total yield of mycelial clumps was significantly increased by approximately 68% compared to single filtration (p<0.05), and the process was stable and reliable (CV<5%). Under these conditions, 6.1 × 10⁻⁶ mycelial clumps could be isolated from each gram of protocorm. 4 The mycelial clusters provided ample starting material for subsequent protoplast preparation.

[0074] Removal of plant contents and residual cell walls (~2 h): Plant cell wall digestion: 4℃, 1000 g, 10 min. Discard the supernatant, resuspend the precipitate in 1 mL of plant cell wall digestion solution. Incubate at 28℃, 40 rpm (shaking speed depends on enzyme volume and container volume; observe slow shaking) for 1 h.

[0075] Membrane digestion: 4°C, 1000 g, 10 min. Discard the supernatant, resuspend the precipitate in 1 mL of 1% Triton X-100, and incubate on ice for 5 min. Repeat twice.

[0076] Mycelial cluster purification: (1) First purification: 4℃, 1000 g, 10 min, discard supernatant, resuspend the precipitate with 1 mL of pre-cooled separation buffer, repeat twice. Add 1 mL of 30% sucrose solution from the bottom, 4℃, 300 rpm, 15 min, and aspirate about 400-600 μL of the intermediate layer and place it on a 20 / 40 μm filter (select the filter according to the diameter of the mycelial cluster). (2) Add 1 mL of 1% Triton X-100, mix by pipetting (use a cut pipette tip as much as possible to reduce damage to the mycelial cluster), incubate for 5 min, 4℃, 500 rpm, 8 min, discard the filtrate, repeat twice. Add 1 mL of separation buffer, 4℃, 500 rpm, 8 min, discard the filtrate, repeat twice. Place the filter upside down in a clean 6 cm petri dish / beaker. Add 1 mL of separation buffer several times to rinse the mycelial clumps on the filter into the petri dish / beaker. Transfer all the liquid in the petri dish / beaker to a 50 mL centrifuge tube. Incubate at 4°C, 1000 g, for 8 min. Discard the supernatant.

[0077] Mycelial cluster counting: 4℃, 1000 g, 10 min, discard supernatant, precipitate (a white precipitate should be observed at this point, with no plant residue; otherwise, repeat the previous step), resuspend in 1 mL of pre-cooled separation buffer. Microscopic examination: Place 20 μL of homogenate on a glass slide, cover with a coverslip, and observe and count the mycelial clusters under a 10× microscope.

[0078] The inventors experimented with different sorbitol concentrations in the STC buffer, and the results were as follows: Figure 7 As shown, only 1M sorbitol-containing STC buffer can achieve effective separation of protoplasts.

[0079] To obtain high-purity mycelial clumps as starting material for protoplast preparation, the inventors compared centrifugal washing purification and gradient centrifugation purification. Centrifugal washing purification: After removing 1% Triton X-100, resuspend in 1 mL of separation buffer, incubate at 4°C, 1500 g, for 8 min, repeated at least 3 times.

[12] Gradient centrifugation purification: After removing 1% Triton X-100, resuspend in 1 mL of separation buffer, add 1 mL of 30% sucrose solution from the bottom, incubate at 4℃, 300 rpm for 15 min, aspirate approximately 400-600 μL of the intermediate layer, incubate at 4℃, 1000 g for 8 min, and resuspend in 1 mL of separation buffer. Results showed that a significant amount of plant debris (such as...) remained after centrifugation and washing purification. Figure 8 (Left side) After gradient centrifugation purification, mycelial clumps free of plant debris can be obtained (e.g., ... Figure 8 (Right side section).

[0080] Release and purification of fungal protoplasts (~2h) Fungal cell wall digestion: 4°C, 1000 g, 8 min, discard supernatant, resuspend the precipitate in 1.7 mL of fungal cell wall digestion solution. Incubate at 34°C, 40 rpm (shaking speed as before) for 60 min. After this step, the pipette tip needs to be cut and rounded with a lighter to avoid damaging the protoplasm.

[0081] Enzyme hydrolysate filtration: Pass the enzyme hydrolysate through a 20 μm filter (depending on the size of the mycelial protoplasts; in this experiment, the protoplast diameter was approximately 10 μm), collect the filtrate, and rinse a 2 ml round-bottom centrifuge tube with 1 mL STC twice. Pass the filtrate through the filter again and combine the filtrates.

[0082] Protoplast purification: 4°C, 1200 rpm, 8 min, discard supernatant, resuspend the precipitate in 1 mL pre-cooled STC. Add 1 mL of 30% sucrose solution to the bottom of the cell suspension; a clear interface is visible. 4°C, 300 rpm, 15 min, a milky white layer appears around the 1 mL mark (more clearly visible under light). Carefully aspirate approximately 200 μL of the milky white layer (fungal protoplasts) using a cut yellow pipette tip.

[0083] Protoplasmic resuspension: 4°C, 1200 rpm, 15 min, discard supernatant, resuspend the precipitate in 1 mL of pre-cooled STC.

[0084] Observation of protoplasts and determination of their viability and yield: Observation of protoplasts: Take 10 μL of protoplast suspension from the enzymatic digestion process into a hemocytometer. First, under low magnification (4×), adjust the coarse adjustment knob to a clear field of view, locate the area where the protoplasts are located, and place them in the center of the field of view. Then, switch to high magnification (10× or 40×), adjust the fine adjustment knob to a clear field of view, take a picture and record the real-time state of the protoplasts, and adjust the dilution factor of the protoplast suspension.

[0085] Protoplast viability assay: Trypan blue staining method: Take 10 μL of protoplast suspension and 10 μL of 0.4% trypan blue staining solution, mix thoroughly on the EP tube cap or slide, and use a pipette to draw a small drop of the mixture (about 10~15 μL) onto a hemocytometer. Carefully cover with a coverslip to avoid air bubbles. Under a fluorescence microscope, count the number of stained (blue) protoplasts (dead) and the total number of protoplasts. Count three replicates for each sample. The incubation time in this step should not exceed 1-3 minutes. If the incubation time is too long, even living cells will begin to die and be stained, leading to inaccurate results.

[0086] FDA staining method: Take 10 μL of protoplast suspension and 10 μL of 0.01% FDA staining solution, mix thoroughly on an EP tube cap or glass slide, and stain in the dark for 15-20 min. Use a pipette to take a small drop of the mixture (about 10-15 μL) and place it on a hemocytometer, carefully covering it with a coverslip to avoid air bubbles. Count the number of stained (green) protoplasts and the total number of protoplasts under a fluorescence microscope (bright field and DAPI channels), with three replicates for each sample.

[0087] Protoplast viability = (Number of live cells n) / (Total number of cells N) × 100% Note: Viability >80% indicates that protoplast preparation is very successful and suitable for subsequent experiments (such as RNA extraction, protoplast regeneration, etc.). If used for DNA extraction, viability is not required.

[0088] Protoplast yield determination: 10 μL of purified protoplast suspension was placed in a 25×16 hemocytometer. The number of protoplasts in each of the five large squares was counted under an optical microscope. Three replicates were performed for each sample. The yield was calculated using the following formula: Protoplast yield (units / g) = [Total number of protoplasts / (0.5 × m)] × V × 1000 Note: 0.5: Volume of 5 squares, in mm. 3 m: weight of the original bulb V: Protoplast suspension volume, 1000:1000 μL = 1 mL Protoplast purity determination: A certain volume of protoplast suspension (approximately 10) was taken. 6Centrifuge at 4°C, 2000 g, for 5 min, discard the supernatant, resuspend in 1 mL DNA lysis buffer, extract protoplast DNA according to the DNA extraction procedure, and amplify with plant-specific and fungal-specific sequences respectively, followed by gel electrophoresis.

[0089] Table 12 Primer Sequences

[0091] To shorten the experimental time and efficiently prepare protoplasts from mycelial masses in protocorms, the applicant optimized the experimental procedures according to the experimental objectives. Experimental methods: In removing plant contents and residual cell walls, we adopted the following methods: ① omitting the step of incubation with 1% Triton X-100 before removing plant cell wall degrading enzymes; ② replacing multiple centrifugation and washing with a single gradient centrifugation using 30% sucrose solution instead of the separation buffer. In releasing and purifying fungal protoplasts, we used a cell wall lysin (Guangdong Provincial Institute of Microbiology) to shorten the enzymatic digestion time. Experimental results: By optimizing the experimental process, we shortened the experimental time by approximately 3 hours (as shown in Table 13).

[0092] Table 13 Comparison of test times

[0093] Final yield: Approximately 1×10 7 Count / g protocorm; Viability: 99.9%; Purity: 100%.

[0094] The observation results of mycelial protoplasts at each stage are as follows: Figure 9 As shown, Part A: Enzymatic digestion for 0.5 h, the mycelial clumps are not completely digested, and protoplasts are being released; Part B: Enzymatic digestion for 1 h, the mycelial clumps are basically completely digested, and a large number of protoplasts are released; Part C: Protoplasts after purification and staining with trypan blue; Part D: Protoplasts after purification and staining with FDA. The left image is bright field, and the right image is taken using the DAPI channel.

[0095] Method: Dilute the stock solution with 1 mL of 1M STC, take 20 μL, add 180 μL of STC with different sorbitol concentrations, take 20 μL, mix with 20 μL of trypan blue, and use for counting.

Claims

1. A method for preparing germination-promoting fungal protoplasts directly from protocorms of orchid plants, characterized in that, The method includes: (1) Physical homogenization; (2) Filtration; (3) Remove plant contents and residual cell walls; (4) Purification of mycelial clumps; (5) Release and purify fungal protoplasts.

2. The method according to claim 1, wherein step (1) physical homogenization comprises: Place the orchid protocorms and steel balls into centrifuge tubes, and add separation buffer to submerge the orchid protocorms. The vortex generator was used at 40 Hz for 30 s, with a 10 s interval, and the cycle was repeated twice to obtain a homogenate.

3. According to the method of claim 2, in step (1) physical homogenization, 0.1g of orchid protocorm and 2 5 mm steel balls are placed in a 2 mL round-bottom centrifuge tube; 1 mL of separation buffer is added to submerge the orchid protocorm; the vortex mixer is used at 40 Hz for 30 s with 10 s intervals, and the cycle is repeated twice to obtain a homogenate.

4. The method according to claim 1, wherein step (2) filtering comprises: first Filtration: The homogenate obtained in step (1) was filtered through a 70 μm filter and the filtrate was collected; The bottom of the container used to collect the filtrate was rinsed with separation buffer, and then filtered through a 70 μm filter. For the second filtration, the large tissue pieces above the filter were added back into the separation buffer to submerge the protocorms. The mixture was vortexed at 40 Hz for 30 s with a 10 s interval, for a total of 2 cycles. The resulting homogenate was then filtered through a 70 μm filter, and the filtrate was collected. The bottom of the container used to collect the filtrate was rinsed with separation buffer, and then filtered through a 70 μm filter. The filtrates were then combined.

5. The method according to claim 4, wherein step (2) filtering comprises: a first... Filtration: The homogenate obtained in step (1) was filtered through a 70 μm filter, the filtrate was collected in a 50 mL round-bottom centrifuge tube, and the round-bottom centrifuge tube was rinsed with 1 mL of separation buffer and then filtered through a 70 μm filter; Second filtration: The large tissue above the filter was placed back into a 2 mL round-bottom centrifuge tube, 1 mL of separation buffer was added to submerge the protocorm, and the vortex was run at 40 Hz for 30 s with a 10 s interval for 2 cycles. The homogenate was then filtered through a 70 μm filter, the filtrate was collected, and the 2 mL round-bottom centrifuge tube was rinsed with 1 mL of separation buffer and then filtered through a 70 μm filter; The filtrates were combined.

6. The method according to claim 1, wherein step (3) of removing plant contents and residual cell walls comprises: Centrifuge the filtrate obtained in step (2); discard the supernatant, resuspend the precipitate in plant cell wall digestion solution; incubate for 1 hour; Centrifuge the incubated mixture; discard the supernatant, resuspend the precipitate in Triton X-100 solution, and incubate on ice for 5 min.

7. The method according to claim 6, wherein step (3) of removing plant contents and residual cell walls comprises: Centrifuge the filtrate obtained in step (2) at 4℃ and 1000 g for 10 min; discard the supernatant and resuspend the precipitate in 1 mL of plant cell wall digestion solution; incubate at 28℃ and 40 rpm for 1 h; centrifuge the mixture after incubation at 4℃ and 1000 g for 10 min; discard the supernatant and resuspend the precipitate in 1 mL of 1% Triton X-100 and incubate on ice for 5 min; centrifuge again at 4℃ and 1000 g for 10 min; discard the supernatant and resuspend the precipitate in 1 mL of 1% Triton X-100 and incubate on ice for 5 min.

8. The method according to claim 6 or 7, wherein the plant cell wall digestion solution is an aqueous solution containing 0.01% BSA, 1.5% cellulase R10, 0.4% macerozyme R10, 0.4 M mannitol, 20 mM KCl, 20 mM MES, and 10 mM CaCl2, with a pH of 5.

7.

9. The method according to claim 1, wherein step (4) purification of the mycelial clump comprises: Density gradient centrifugation, filtration purification, and Triton X-100 washing were performed.

10. The method according to claim 9, wherein step (4) purification of the mycelial clump comprises: (4-1) First purification: 4℃, 1000 g, 10 min, discard supernatant, resuspend the precipitate with 1 mL of pre-cooled separation buffer, repeat twice; add 1 mL of 30% sucrose solution from the bottom, 4℃, 300 rpm, 15 min, and aspirate about 400-600 μL of the intermediate layer and place it on a 20 / 40 μm filter screen; (4-2) Add 1 mL of 1% Triton X-100, mix by pipetting, incubate for 5 min, 4℃, 500 rpm, 8 min, discard the filtrate, repeat twice; add 1 mL of separation buffer, 4℃, 500 rpm, 8 min, discard the filtrate, repeat twice; place the filter screen upside down in a clean 6 cm culture dish or small beaker, add 1 mL of separation buffer several times to rinse the mycelial clumps on the filter screen into the culture dish or small beaker, transfer all the liquid in the culture dish or small beaker to a 50 mL centrifuge tube, 4℃, 1000 g, 8 min, discard the supernatant.

11. The method according to any one of claims 2-10, wherein the separation buffer is an aqueous solution containing 0.6 M mannitol and 20 mM MOPS.

12. The method according to claim 1, wherein step (5) releasing and purifying the fungal protoplast comprises: Centrifuge the product from step (4); discard the supernatant and resuspend the precipitate in fungal cell wall digestion solution; Incubate for 30-60 min; filter the incubated mixture and collect the filtrate; rinse the incubation container with STC, pass through the filter again, and combine the filtrates; centrifuge the filtrate; discard the supernatant, and resuspend the precipitate in pre-cooled STC buffer; add 30% sucrose solution to the bottom of the cell suspension, and a clear interface will be visible; after centrifugation, aspirate about 200 μL of the milky white layer, which is the fungal protoplast; centrifuge for 15 min; discard the supernatant, and resuspend the precipitate in pre-cooled STC buffer.

13. The method according to claim 12, wherein step (5) releasing and purifying the fungal protoplast comprises: Centrifuge the product from step (4) at 4°C and 1000 g for 8 min; discard the supernatant and resuspend the precipitate in 1.7 mL of fungal cell wall digestion solution; incubate at 34°C and 40 rpm for 60 min; pass the incubated mixture through a 20 μm filter and collect the filtrate; rinse the incubation container with 1 mL of STC, pass through the filter again, and combine the filtrates; centrifuge the filtrate at 4°C and 1200 rpm for 8 min; discard the supernatant and resuspend the precipitate in 1 mL of pre-cooled STC; add 1 mL of 30% sucrose solution to the bottom of the cell suspension, and a clear interface will be visible; centrifuge at 4°C and 300 rpm for 15 min, and then aspirate about 200 μL of the milky white layer, which is the fungal protoplast; centrifuge at 4°C and 1200 rpm for 15 min; discard the supernatant and resuspend the precipitate in 1 mL of pre-cooled STC buffer.

14. The method according to claim 12 or 13, wherein the STC buffer is an aqueous solution containing 1 M sorbitol, 0.1 M Tris-HCl, and 5 mM CaCl2, with a pH of 7.

5.

15. The method according to claim 12 or 13, wherein the fungal cell wall digestion solution is prepared by dissolving 0.06 g of cell wall lysin in 1.7 mL of 0.6 M mannitol solution.

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