A buffer formulation and application for efficient protoplast extraction and transformation of rice

Abstract
A novel buffer formulation for rice protoplasts has been proposed, which streamlines the extraction process by eliminating the need for resuspension with W5 and MMG solutions. This innovative buffer is designed to be compatible with the resuspension functions traditionally served by W5 and MMG solutions. Furthermore, the polyethylene glycol (PEG)-mediated transformation system has been refined, thereby facilitating the genetic transformation of rice protoplasts with enhanced efficiency and convenience.

Background
Plant cells have cell walls, which are rich in pectin and connect adjacent cells. The living cytoplasm is surrounded by a plasma membrane, forming a protoplast. The cell membrane is closely connected to the cell wall and participates in wall synthesis. However, in hypertonic solutions, the plasma membrane will contract away from the cell wall. After removing the cell wall, the fragile protoplast is released, at which point the plasma membrane becomes the only barrier between the cytoplasm and the external environment (Davey et al.,2005).
Plant protoplasts serve as a versatile transient expression system, extensively utilized in the realms of plant biochemistry and the elucidation of molecular mechanisms. They are instrumental in pivotal biological processes, including but not limited to cell signal transduction, ion transport mechanisms, cell wall biosynthesis, and protein secretion pathways. The experimental methodologies predicated on protoplasts encompass a spectrum of techniques such as subcellular localization studies, promoter activity analyses, ion uptake assays, the establishment of plant regeneration systems, distant hybridization techniques, and the validation of protein-protein interactions. Collectively, these approaches furnish a robust technical foundation that underpins advancements in plant science research.(An et al., 2005).
Nonetheless, the preparation of rice protoplasts is often encumbered by intricate formulae and laborious procedures, which impedes the progression of research in this area. Furthermore, the susceptibility of protoplasts to rupture during transformation stages frequently compromises gene expression and introduces other complications. In response to these challenges, we introduce a high-efficiency buffer formulation and application methodology designed to facilitate the extraction and transformation of rice protoplasts, thereby mitigating the aforementioned issues and enhancing the overall efficacy of related research endeavors.

Materials and reagents 
1. 2 ml round-bottom Eppendorf tubes, 50 ml round-bottom Eppendorf tubes
2.A diverse array of pipette tips is required
   Note:it is imperative that all pipette tips which come into contact with protoplasts are modified by truncating their tips to        prevent damage to the fragile protoplasts.
3. Razor blades 
4. Aluminum foil 
5. Sieves with mesh sizes of 200 and 100 microns
6. Albino seedlings of the rice cultivar indica 93-11 (referred to as 9311)
    Note: It is crucial to sow 9311 seeds in Yoshida medium for hydroponic cultivation and to incubate them at a temperature       of 28°C in the dark for a period of 10 to 15 days. Seedlings younger than 10 days exhibit very short leaf sheaths, and the         cells are too diminutive for effective observation. In contrast, seedlings older than 15 days have leaf sheaths that change         from white to a slightly brownish color, indicating cellular senescence. These aged cells have a reduced viability following         protoplast transformation, resulting in a diminished number of viable cells for experimental analysis.
7. KOH (Sangon) 
8. Celluase R10 (Yakult Japan) 
9. Macerozyme R10 (Yakult Japan)  
10. Bovine Serum Albumin(BSA) (Sigma) 
11. D-Mannitol (Sigma) 
12. KCl (Sangon) 
13. CaCl₂·2H₂O(BBI) 
14. NaCl (BBI) 
15. MES hydrate (Sigma) 
16. 0.6 M D-Mannitol (Sigma, FW: 182.17) (See solution formula Table 2) 
17. 0.2 M MES (Sigma, FW: 195.24) (See solution formula Table 3) 
18. L enzymatic hydrolysate (Enzyme solution) (See solution formula Table 1&4) 
19. S solution (See solution formula Table 5) 
20.W solution (See solution formula Table 6)

 Instruments and equipment
1. Shaking incubator
3. Pipette
4. Centrifuge
4. Confocal microscope

Experimental steps
1. Procure 80 etiolated seedlings of the rice cultivar 9311 that have been cultured in darkness for a period of 12 days. Select the rice stem segment, approximately 5-7 centimeters above the collar of the youngest fully expanded leaf, and prepare it into thin sections with a thickness of 0.4-0.6 millimeters using a sterile, sharp blade—ideally a sterilized razor blade. It is essential that the cutting be executed in a single, swift motion, with a calendered paper placed beneath to facilitate this process. Transfer these thin sections into a 250 milliliter Erlenmeyer flask, ensuring they cover the bottom of the flask.
2.Immerse the thin sections in 20 milliliters of L enzyme solution within the flask. To prevent exposure to light, which can adversely affect the enzymatic digestion process, wrap the Erlenmeyer flask with aluminum foil. Incubate the flask on a rotary shaker at a controlled temperature of 28°C Celsius and a shaking speed of 80 revolutions per minute for a duration of four hours. This controlled environment facilitates the enzymatic digestion of the cell walls, thereby releasing the protoplasts from the plant tissue.
3.After the cultivation period, the enzymatically treated tissue should be subjected to mechanical filtration through a sieve with a 200-micron mesh size. This step should be performed by gently applying manual pressure to the thin slice fragments to express the protoplasts through the sieve, with meticulous attention to avoid exerting excessive force that could compromise the integrity of the protoplasts.
4.The L enzyme solution must be completely drained from the sieve. To facilitate this, a pipette tip, can be utilized to scrape any residual small tissue pieces back into the flask. The initial filtrate, which contains the L enzyme solution, should be discarded, as it no longer serves a purpose in the protocol. The remaining tissue slices, now enriched with isolated protoplasts, should be carefully collected and preserved for subsequent experimental procedures.
5. The residual tissue slices obtained after enzymatic digestion should be resuspended in 30 milliliters of S solution. This suspension should then be cultured on a rotary shaker at a speed of 80 revolutions per minute (rpm) in a dark environment maintained at a temperature of 28°C Celsius for a duration of 2 hours. Following this incubation period, the suspension should be passed through a sieve with a mesh size ranging from 100 to 200 microns. The slices will be retained on the sieve, while the liquid fraction is allowed to pass through.
6.After the filtration, the slices on the sieve should be washed four times with 5 milliliters of S solution per wash, with each filtrate being collected into a sterile 50 milliliter round-bottom Eppendorf tube. The collected filtrate should then be centrifuged at a speed of 1200 rpm, which corresponds to a centrifugal force of less than 250g, for a period of 6 minutes. The centrifugation process should be conducted with an acceleration and deceleration rate set to 3. Following centrifugation, the supernatant should be carefully removed, and 500 microliters of the precipitate should be retained.
7.Subsequently, 500 microliters of S solution should be slowly added along the wall of the 50 ml Eppendorf tube to the retained precipitate. The mixture should then be gently resuspended using a yellow pipette tip, which has been modified by cutting off approximately 0.5 centimeters from the tip and sterilized by flaming for 3 seconds. This results in the formation of the initial suspension solution of 9311 protoplasts, which will appear yellow and turbid, indicating the presence of the isolated protoplasts.
8. Prepare a mixture by combining the plasmid DNA, which has been extracted using the Endofree Maxi Plasmid Kit (TIANGEN; China) and has a concentration exceeding 1 ng/μl, with 100 μl of the 9311 protoplast suspension. Subsequently, introduce 110 μl of W solution to the mixture and allow it to incubate on ice for a period of 20 minutes. This step facilitates the interaction between the plasmid DNA and the protoplasts, enhancing the efficiency of subsequent transformation processes.
9. Upon completion of the ice incubation, gently add 1 ml of S solution to the reaction mixture along the wall of the container and repetitive invert to ensure thorough mixing. It should be noted that the appearance of small particles under fluorescent light does not significantly impact the experimental outcomes. The mixture is then subjected to centrifugation at 800 rpm for 6 minutes, with the centrifuge acceleration and deceleration settings adjusted to 3. After centrifugation, carefully remove the supernatant. Re-suspend the pellet in 1 ml of S solution, ensuring thorough mixing, and then transfer the suspension to a 28°C incubator for a period of 16 hours in the dark. This dark incubation period is crucial for allowing the protoplasts to recover and for the expression of any introduced genes to occur.
10. Following the dark cultivation period, the suspension is centrifuged again at 800 rpm for 6 minutes, with the centrifuge acceleration and deceleration settings maintained at 3. After centrifugation, remove 800 μl of the supernatant to isolate the transformed protoplasts of the experimental group. Gently mix the remaining protoplasts and observe them under a confocal microscope to assess the fluorescence, which serves as an indicator of successful transformation and gene expression. This step is essential for validating the efficiency of the transformation process and for analyzing the outcomes of the introduced genetic material.

Solution formulation
Table 1  L enzymatic hydrolysate I

Note：The formulation of the 0.6 M D-Mannitol solution is shown in Table 2, and the formulation of the 0.2 M MES solution is shown in Table 3.
Table 2  0.6 M D-Mannitol solution
 

Note: Sterilize at 121°C for 15 minutes and store in a 4°C refrigerator for use.
Table 3 0.2 M MES solution

Table 4  L enzymatic hydrolysateⅡ

Note: The L enzyme solution I must be freshly prepared to ensure its efficacy. Immediately following preparation, the solution should be subjected to incubation in a water bath maintained at a temperature of 55°C  Celsius for a duration of 10 minutes. This step is essential for activating the enzymatic components. After incubation, the solution should be allowed to equilibrate to room temperature.Subsequently, an equal volume of L enzyme solution II should be added to the cooled L enzyme solution I. This mixture yields the final L enzyme solution, which is intended for use in the enzymatic digestion of plant tissues.Prior to its application, the L enzyme solution must be filtered through a 0.22-micrometer filter membrane to remove any potential particulate matter or contaminants. This filtration step is crucial for preventing the introduction of foreign materials into the experimental system, thereby ensuring the purity and reliability of the enzymatic digestion process.

Table 5 S solution

Note: Check if the pH is between 5.56-5.7, if it is lower, adjust the pH to 5.7 using a 1 M KOH solution, sterilize at 121°C for 15 minutes, and then store in a 4°C refrigerator for use.
Table 6  W solution
 

References
1.Davey, M. R., Anthony, P., Power, J. B., & Lowe, K. C. (2005). Plant protoplasts: status and biotechnological perspectives. Biotechnology Advances, 23(2), 131-171. https://doi.org/10.1016/j.biotechadv.2005.01.002
2.An, C. I., Sawada, A., Kawaguchi, Y., Fukusaki, E., & Kobayashi, A. (2005). Transient RNAi induction against endogenous genes in Arabidopsis protoplasts using in-vitro prepared double-stranded RNA. Biosensors and Bioelectronics, 69(2), 415-418. https://doi.org/10.1016/j.bios.2004.08.005