“Fast-lyzer”: A low-cost 3D printable device for plant tissue disruption and homogenization

“Fast-lyzer”: A low-cost 3D printable device for plant tissue disruption and homogenization | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Method Article “Fast-lyzer”: A low-cost 3D printable device for plant tissue disruption and homogenization Rodrigo Matías González, Carola Agranatti, Federico E. Aballay, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9440300/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Background: Sample disruption and homogenization are critical steps in numerous biological extraction procedures (i.e., DNA, RNA, or protein isolation). These steps are often performed manually which can yield highly variable results depending on operator expertise. Several companies offer dedicated tissue-processing machines that enable high reproducibility and high-throughput sample preparation using various bead types and shaking mechanisms. However, the high cost of this equipment makes it inaccessible to many laboratories worldwide. To address this limitation, we developed an open‑source, 3D‑printable adaptor that enables the use of a low‑cost wood jigsaw as the source of mechanical agitation. Results: We validated our device, Fast-lyzer, by extracting DNA, RNA, and proteins from different plant tissues, obtaining results comparable — if not superior — to traditional, time‑consuming manual grinding. We also demonstrated the effectiveness of the Fast-lyzer and bead‑based disruption for extracting live bacteria from plant tissues for leaf‑colonization assays. Conclusion: Fast-lyzer provides an open‑source, low‑cost solution that allows resource‑limited laboratories to access grinding and homogenization methods typically reliant on equipment costing thousands of dollars. The estimated cost of our system is approximately 200 USD, representing at least a 100‑fold reduction in price. More importantly, the Fast-lyzer speeds up sample grinding process by 8-fold if used for batch processing in comparison to manual procedure. Additionally, we optimized extraction conditions for several Arabidopsis thaliana tissues and for bacterial cell counts to facilitate adoption by the research community. Tissue disrupter 3D printing Jigsaw Nucleic Acid Extraction Protein Extraction Seedlings Leaves Stems Seeds Bacterial Extraction Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Sample disruption and homogenization are two critical steps in numerous biological extraction procedures (i.e., DNA, RNA or protein extraction). It is often performed manually and can show a very broad range of results depending on the expertise of the person performing the protocol. Several companies offer tissue processing machines that enable high reproducibility and high number of sample processing relying on different types and sizes of beads and shaking movements [1][2][3][4][5][6]. The prices for this equipment are over 10000 USD which makes them difficult to purchase in counties with low scientific investment [7]. For this reason, we decided to contribute with this open scientific hardware development [8][9][10]. Their working principle relies on shaking beads from different sizes and materials according to the type of tissue / organism to process. For instance, Qiagen recommends: 0.1–0.6 mm (mean diameter) glass beads for bacteria, 0.5 mm glass beads for yeast and unicellular animal cells, and 3–7 mm stainless steel or tungsten carbide beads for plant and animal/human tissues. (Manual available at https://www.qiagen.com/us/resources/download.aspx?id=65e7826c-4d50-4faf-8154-2fbc782c41a6&lang=en). Some works determined the best conditions for extracting nucleic acids in non-conventional models, using Tissuelyzer™ [11],but to our best knowledge there are no systematic studies on Arabidopsis thaliana .Stainless steel beads can be purchased at specialized scientific stores for 50 USD for 100 beads (https://www.usascientific.com/stainless-steel-beads-/p/Steel_Beads) or from other non-scientific manufacturers at 3 to 5 USD for 1000 beads. Non-lab-grade materials must be properly prepared and washed prior to their use. Stainless steel beads should be washed and rinsed several times in distilled water. They can be autoclaved to avoid contamination on future extracts and reused after washing. As a replacement for the shaking force, we propose to use a non-orbital 600W jigsaw. Jigsaws typically oscillate in a back-and-forth movement at 300–3000 RPM (≈ 5–50 Hz), a range comparable to devices such as the Qiagen Tissuelyser™, which operates between 3 and 30 Hz. Combined with a simple custom-made blade and 3D printed plastic pieces to attach the tubes into the jigsaw, we were able to assemble an inexpensive device for shaking based bead sample grinding [12]. Methods Jigsaw metal adapter We cut the jigsaw adapter from a 1.5mm sheet of carbon steel 15N20 (with high carbon and nickel content) that provides excellent tenacity, resistance to traction and fatigue. This material is commonly used to build knives. The adapter emulates the shape of the blades to fit perfectly on the machine (Figure 1A). It has also an additional part that fits an optical barrier for RPM counting and two holes for fixation to the plastic sample holder. We make cuts by laser cutting on a local company with a precision of 0.1 mm. Holder The holder consists of two 3D printed parts (Figure 1B). A rack with holes for Eppendorf tubes and a lid. The rack has additional hexagonal holes to fit 2 hexagonal bolts. Once we added the tubes to the rack, the lid is tightly be adjusted with nuts. We previously assembled the lid with the metal adapter with bolts and Nylon insert-nuts tightly adjusted to the final assembly (Figure 1C). RPM counter The RPM counter consists of an Arduino Nano V3 connected to optocoupled IR barrier (FC-03 IR) and LCD display (LCD I2C 16x2) modules. They are mounted in a way that the metal adapter can obturate the IR barrier during the jigsaw operation. All the necessary programs and schematics are described in supplementary material (device building files). To determine if changes in adapter weight—resulting from loading sample tubes, beads, and eventually buffer—substantially modify the device's RPM, we measured the speed five times for each selector position used in this study (1, 2, 3, or 4). We performed these measurements under four conditions: without added weight and with masses of 20, 30, and 50 grams. In each iteration, we calculated the slope of the line and evaluated whether these slopes differed significantly from zero, which would indicate that the applied mass does not affect the speed at all (Figure 3E). 3D printing We designed models using FreeCad 1.0 software (https://github.com/Reqrefusion/FreeCAD-Documentation-Project). After meshing, they were sliced using Orca slicer. We printed the adapters using a standard printer (Creality Ender 3 V3 se) in polylactic acid (PLA) or Polyethylene Terephthalate Glycol (PETG) with similar results. Printing density should be high on the light blue highlighted regions (see Figure 1B). We optimized the design to prevent its breaking by increasing the amount of material on its weaker points. Plants We used Arabidopsis thaliana (ecotype Columbia-0) at various developmental stages to collect the different tissue samples used throughout all experiments. We used 14 days old seedlings germinated on Agar- 0,5X MS plates. For stems and leaves we used 6-week-old plants grown in soil under standard conditions (16/8 light-dark photoperiod, 22ºC and commercial plant growth substrate). We used three-day imbibed seeds in darkness at 4°C. For tissue collection we snap froze in liquid nitrogen, (1) 100 mg of whole seedlings, (2) four 8 mm diameter leaf discs (weighting approximately 50mg), (3) 100 mg floral stems cut into 1 cm long pieces and (4) ~20 mg of dry seeds and stored at –80 °C until used. We later used the samples for DNA, RNA or protein extraction. DNA extraction We harvested approximately 100 mg of tissues into 1.5 ml microcentrifuge tubes for manual extraction or into 2 ml round-bottom tubes for mechanical disruption. For manual extraction, we used a plastic pestle (Kimble Kontes) and ground the tissue with liquid nitrogen. For mechanical disruption, we used the Fast-lyzer with different settings indicated throughout the publication. Once we ground the tissues, we extracted DNA using 400 µl of CTAB buffer [13]. Briefly, we incubated samples for 30 minutes at 65°C, followed by centrifugation at 14,000 × g for 5 minutes. We transferred the supernatant to a new tube and performed an organic extraction using 400 µl of chloroform:isoamyl alcohol (24:1). We recovered the aqueous phase, and precipitated the DNA with 280 µl of isopropanol, incubated at -20°C for 20 minutes, followed by pelleting for 20 minutes at 21,500 × g at 4°C in a refrigerated microcentrifuge. Finally, we washed the pellet with 70% ethanol, dried, and resuspended in Milli-Q water. Assessment of DNA concentration and integrity We run DNA extracted from different tissues using various approaches on a 1% agarose gel at 120 V for 40 minutes. To determine the mass of each sample, we ran a 1 kb DNA ladder (New England Biolabs) along with several dilutions (0.5, 0.25, and 0.125) to generate a calibration curve. To this end, we used 500 ng of DNA ladder, specifically targeting the 1.5 kb (36 ng), 2 kb (48 ng), and 3 kb (125 ng) bands, along with their respective two-fold, four-fold, and eight-fold dilutions. We loaded 10 μL of each ladder dilution and 5 μL of each sample. Subsequently, we visualized the gel using an UVP transilluminator with the following settings: f = 1.2, zoom 65%, focus 87.9, and a 15-second exposure. We captured the image and performed an analysis using ImageJ software [14]. We inverted the gel photograph colours (black bands on a white background). Then, we used a rectangular selection tool to define each lane. We obtained profile plots for each lane to determine the pixel intensity (area) of each band and using the data from the ladder, we constructed a calibration curve to calculate the DNA mass in the samples. To account for background noise, we estimated the pixel values under the curve, excluding the peaks. We determined electrophoresis background by measuring the area under the curve in an empty lane and subtracting this value from the sample lanes. We then determined the integrity percentage by calculating the ratio between the main band mass and the total mass in the lane (band plus smeared DNA). We analyzed the data using GraphPad Prism software (v. 9.0.0). We first tested for normality using the Shapiro-Wilk test. Subsequently, we performed a one-way ANOVA followed by Tukey’s multiple comparisons post-hoc test to identify statistically significant differences between assays. For comparisons between manual and Fast-lyzer optimally grinding conditions, we employed a Student’s T test. RNA extraction of seedlings, leaves and stems We extracted RNA from ground samples using TriPure (Sigma) [15][16]. Briefly, we added 1 mL of TriPure to each nitrogen-frozen sample and mixed by inversion. After centrifugation at 12,000 x g for 10 min at 4ºC we transferred the supernatant into a new tube with 200 µl of chloroform and shook it for 1 min. We recovered the aqueous phase after centrifugation at 12,000 x g for 10 min at 4ºC, transferred into a new tube and precipitated by adding 500 µl of isopropanol, letting the mixed sample rest at room temperature for 10 minutes and later centrifugation at 12.000 x g for 10 minutes. We washed the pellets twice with Ethanol 75% and centrifuged the tubes at the same speed for 5 min and dried in a laminar flow cabinet. We finally resuspended the pellets in 50 µl RNAse-free Milli-Q water and stored at -80ºC for 1 hour. RNA integrity and quantification We assessed RNA integrity by agarose gel electrophoresis. Briefly, 3 μL of each sample + 3 μL of Orange-G loading buffer was loaded in each well. Samples were separated by gel electrophoresis at 150 V for 20 min in 1% w/v agarose gel supplemented with 1 % v/v bleach [17]. We photographed the gels using an UVP transilluminator with the following settings: f/1.2, 65% zoom, 87.9 focus, and a 15-second exposure. Next, we performed relative RNA quantification using ImageJ software. After selecting the lanes with the rectangular selection tool, we plotted them to obtain the area under the curves. We summed the areas of all ribosomal RNA bands present and calculated the mean of the manually extracted samples, which we arbitrarily set to one. Finally, we normalized all values to this mean for statistical analysis. We employed GraphPad Prism software (v. 9.0.0) for statistical analysis, performing a Shapiro-Wilk test to check for normality distribution and a Student’s T test to find statistically differences between both grinding methods. Protein extraction We extracted ground samples by addition of 250 µl of urea buffer (200 mM Tris–HCl pH 6.8, 10% SDS, 6 M Urea, 20% Glycerol, 50 mM DTT, 0.05% Bromophenol Blue). We heated the samples for 10 min at 80°C, run them in standard 12% SDS-PAGE gels and stained them with Coomassie brilliant blue according to standard protocols [18] Following staining, we scanned the gel to obtain a TIFF image for analysis using ImageJ software. We quantified the most intense band, corresponding to the RUBISCO protein by calculating the area under the peak. We averaged the values from manually extracted samples, and we arbitrarily set to 1 this mean. We then normalized all remaining samples to this mean for statistical analysis. We employed GraphPad Prism software (v. 9.0.0) for statistical analysis, performing a Shapiro-Wilk test to check for normality distribution and a Student’s T test to find statistically differences between both grinding methods. RNA extraction from seeds We sowed A rabidopsis thaliana Col-0 seeds (~20 mg seed dry weight) onto filter papers in clear plastic boxes (40 × 33 × 15 mm), each containing 10 mL of 0.8% ( w / v ) agar in demineralized water and incubated for three days in darkness at 4 °C. After sampling, seeds were briefly blotted on filter paper to remove excess surface moisture and then immediately frozen in liquid nitrogen and stored at − 80 °C until grounding. We extracted RNA from ground samples using the Spectrum TM Plant Total RNA Kit (Sigma-Aldrich, Steinheim, Germany) according to the manufacturer’s protocol. We assessed RNA integrity by agarose gel electrophoresis as previously described for the other tissues. Bacterial plate counting Pseudomonas syringae pv. tomato DC3000 (kindly provided by Dr. Nicolás Cecchini, CIQUIBIC – UNC/CONICET) was grown in Luria-Bertani (LB) agar medium supplemented with rifampicin 100 μg/ml and kanamycin 75 μg/ml for 48h at 28ºC. Then, we resuspended the bacteria in MgCl 2 10 mM to O.D 600 = 0.01. Afterwards, we treated bacteria with our Fast-lyzer (2000 RPM) with one 2.8 mm bead, two 2.8 mm beads or one 4 mm bead. Finally, we evaluated bacterial growth by serial dilution followed by droplet plating and counting on the same culture medium than before (CFU/mL). The experiments consisted of three biological replicates (different days) with at least three replicates each day. Bacterial counting from pathogen infection assay Plants were grown in long-day conditions (16h/8h light/darkness) at 21 ºC with a light intensity of 100-120 μmol s −1 m −2 and 50% relative humidity for 25-28 days. On the other hand, P. syringae pv. tomato DC3000 was grown as previously described for 48h at 28 ºC. Then, we re-suspended the bacteria and diluted to OD 600 =0.0005 in MgCl 2 10 mM for infection. We infected the abaxial surface of leaves by pressure-mediated infiltration with a needleless syringe. To assess possible differences with classical methods, we cut 8 mm leaf discs to grind them either with a piston during 60 seconds per sample, or with our Fast-lyzer, both in MgCl 2 10 mM at 72 hours post-infection. For the case of Fast-lyzer, we ground the samples with two 2.8 mm beads, 2000 RPM and for 15 seconds three times. Then, we assessed in planta bacterial growth (CFU/mL) by droplet plating as previously described. Results Device mounting: The adapter consists of a tube holder and a steel flat bar (Figure 1C). The tube holder (consisting of a tube rack and a lid) is firmly attached to the wood jigsaw by the custom-cut blade-shaped steel flat bar with an integrated jigsaw shank (model available at Supplementary files, Device Building Files). This piece fits precisely into the jigsaw’s standard blade-mounting slot, allowing the entire adapter module to be attached or removed using the machine’s normal locking mechanism. Because 3D printing is inexpensive, multiple adapters can be produced to enable rapid sample processing. Loaded samples can be stored at the desired temperature (−20 °C or even −80 °C) until use. The holder can be printed in polyethylene terephthalate glycol (PETG) or polylactic acid (PLA) which are the most commonly used 3D printing materials [19]. Our current design accommodates 25 tubes of 1.6 ml or 2 ml volume, but this can be easily modified depending on experimental needs. Before assembly, each tube must be filled with the sample, beads, and buffer if required. It is very important to balance the weight by placing pairs of tubes with equal weight symmetrically around the central position in order to avoid vibrations perpendicular to the one generated by the jigsaw. The type of beads used depends on the characteristics of the tissue being processed. To prevent tubes from opening during grinding, the tube holder and lid are secured together using bolts and locking nuts. Depending on the protocol, the assembled adapter can be placed on ice, or the tubes can be flash frozen in liquid nitrogen (ensuring that only the tubes — not the 3D printed adapter — are submerged). Once everything is prepared, the holder is mounted onto the jigsaw in the same way as a standard blade (Figure 1D). For safety, we placed the jigsaw inside a PET made protective container box before powering it on. It is also necessary that the operator wear safety glasses and hearing protection (see Supplementary Video 1 for full procedural details). The jigsaw must be used with the blade along the gravity axis. Because oscillation speed is critical for reproducibility across different jigsaw models, we implemented an optocoupled sensor system connected to an Arduino to directly measure and calibrate the jigsaw’s RPM. This allowed us to establish a precise relationship between the speed dial position and the actual oscillation frequency, ensuring consistent performance across experiments. In our setup, we routinely used speed setting 4 out of 6, corresponding to approximately 2000 RPM. Given that the weight of the tube holder can change substantially when loaded with tubes, buffers and stainless-steel beads, we first evaluated whether the total mass (samples + holder) affected the final RPM of the machine at a fixed speed setting. Our results showed that the weight variation introduced by the number of samples (Considering that the sum of the mass of microtubes, stainless steel beads, samples and up to 1 ml of buffer per tube, will not exceed 50 grams of total load in the holder) did not significantly alter the machine’s actual RPM and therefore does not represent an additional parameter that needs to be controlled (Figure 1E and Supplementary Table 1). Grinding procedure: Due to their structure, different plant tissues have distinct mechanical properties. We optimized the grinding conditions separately for whole seedlings [20], leaf discs [21], stem segments [22] and imbibed seeds [23]. We adjusted size and number of metal beads, total grinding time and RPMs. We initially optimized all conditions using DNA extraction efficiency as the main indicator. We chose DNA because, due to its large molecular size, it is easily sheared and therefore serves as a sensitive proxy for assessing grinding harshness [24]. To avoid sample heating and subsequent DNA degradation (Supplementary Figure S1), every 30 seconds, we chilled the samples by submerging the tube bottoms in liquid nitrogen for 10 seconds. Once the optimal parameters were established, we also validated them for RNA and protein extraction. We determined the time needed to process samples manually and with Fast-lyzer (considering that 25 tubes are loaded into the device). For manual grinding, we employed a Kimble Kontes plastic piston for DNA, protein and bacteria extractions, which required a grinding time of 181,0 +/- 7,1 sec per sample. For RNA extraction, we used mortar and pestle, requiring 409.2 +/- 33.7 sec per sample. In all cases, batch processing with Fast-lyzer significantly reduced the grinding to 25,5 +/- 0,9 sec per sample (Supplementary Figure S2) (One-way ANOVA P< 0,0001, Tukey’s post-test: mortar and pestle vs Fast-lyzer P<0.0001, piston vs Fast-lyzer P=0,001). Whole seedling: Initially, we set the shaking speed to 2000 RPM and the grinding time to 1 minute and evaluated different bead numbers and sizes to identify the condition that maximized the recovery of intact genomic DNA. In all cases, bead-assisted extraction clearly outperformed manual grinding, yielding significantly higher concentrations of a high–molecular weight (HMW) DNA band on agarose gels, while maintaining comparable DNA integrity percentages (Figure 2A). Using two 4 mm beads and a fixed frequency of 2000 RPM, we next evaluated different grinding durations, reasoning that longer processing times might improve tissue disruption but could also increase DNA fragmentation. Extended grinding times indeed resulted in higher yields of the HMW band but contrary to our initial thoughts it also improved DNA integrity indices (Figure 2B). Having established that 120 seconds and two 4 mm beads were optimal, we then optimized frequency, again considering that higher frequencies could potentially fragment DNA. Contrary to this expectation, both DNA concentration and integrity peaked at 2000 RPM of frequency (Figure 2C and Supplementary Table 2). Frequencies exceeding 2000 RPM are not recommended because centrifuge tubes tend to fracture when operated above this threshold. Having determined these optimized conditions -two 4 mm beads, 2000 RPM, and 120 seconds of grinding- we proceeded to perform RNA and protein extractions. When we tested RNA extractions performed with TRI-reagent according to manufacturer’s instructions, we did not observe differences neither on integrity (at least by running standard Agarose gels) nor on yield (Figure 2E and Supplementary Table 3). Protein extractions were performed with urea extraction buffer (see methods) and also showed that the method can speed up the grinding process improving the extraction outcome (Figure 2D and Supplementary Table 4). Fully developed leaf discs: Next, we performed the same set of experiments using 8 mm discs cut from fully developed true leaves. As before, we began with 2000 RPM for 1 minute while testing different bead combinations. The optimal setup was one 4 mm bead and one 2.8 mm bead, which produced slightly higher -yet not statistically significant- yield in comparison to the manual extraction (Figure 3A). Adjusting the grinding duration did not affect either the yield or the integrity of the recovered DNA. We observed a slightly higher –not significant- yield at 60 seconds (Figure 3B). Finally, when we lowered the RPM while using one 4 mm beads and one 2.8 mm bead for a 60 second grind, we observed clear reductions in both DNA yield and integrity indices (Figure 3C). Our optimal settings for leaves were set at one 4 mm and one 2.8 mm beads, 2000 RPM, and 60 seconds of grinding (Supplementary Table 2). Using the optimal grinding conditions for leaf discs established for DNA extraction, we proceeded to extract RNA with Tri-Reagent (Figure 3E and Supplementary Table 3) and proteins (Figure 3D and Supplementary Table 4) with an 8 M urea buffer, as described for whole seedlings. For proteins, we observed non-significant differences between manual extraction and Fast-lyzer mediated extraction under the same conditions employed for DNA extraction. Stems: For stems we started with 2000 RPM and 1 minute grinding and adjusted bead size and amounts. Manual grinding outperformed bead grinding giving statistically higher and less fragmented DNA in comparison to beads (Figure 4A). We continued using two 4 mm beads since they gave better -yet not significant- parameters compared to the other bead combinations. With two 4 mm beads and 2000 RPM we tested grinding time and found that more grinding time resulted in more yield and better DNA integrity and fixed the time at 120 seconds (Figure 4B). Similarly, grinding at higher frequencies gave progressively higher and less fragmented DNA. Therefore, we determined that our optimal settings for stems were 2000 RPM, 2 4 mm beads for 120 seconds (Figure 4C). These later settings gave slightly higher but not significant yields compared to the manual extraction. See Supplementary Table 2 for full data. Under the conditions used for DNA extraction, we carried out total protein extraction with urea buffer. We observed that, in this case, manual grinding yields more proteins than Fast-lyzer grinding. 36% less protein is obtained when grinding using Fast lyzer compared to manual grinding; however, the amount of protein obtained is sufficient for further applications while time saving is considerable (Figure 4D and Supplementary Table 4). Finally, we extracted RNA using Tri-Reagent and the same conditions for grinding used for DNA extraction (Figure 4E and Supplementary Table 3). We did not observed statistically differences between manual and mechanical disruption. Therefore, the use of the device is advantageous due to the time saved in sample processing. Other samples: To further extend our trials we also explored extraction on seeds, and bacteria inside of plant leaves. Seeds. Grinding conditions in the other tissues were initially evaluated using genomic DNA extraction, as high-molecular-weight DNA is highly susceptible to mechanical shearing and therefore provides a sensitive indicator of grinding harshness. While this approach proved effective for several vegetative tissues as shown before, assays for imbibed seeds of Arabidopsis thaliana was performed using total RNA extraction. Seed tissues are particularly challenging for DNA purification because they are rich in storage compounds, including proteins, lipids, polysaccharides, and secondary metabolites that can interfere with nucleic acid extraction by promoting co-precipitation or increasing extract viscosity [25,26]. In contrast, imbibed seeds contain relatively abundant RNA derived both from transcripts stored during seed maturation and from early transcriptional activity following imbibition [27,28]. Many of these stored mRNAs are rapidly recruited for translation immediately after imbibition, supporting early metabolic reactivation before large-scale de novo transcription occurs [29,30]. The small size of Arabidopsis thaliana seeds further increases the sensitivity of nucleic acid extraction protocols to incomplete tissue disruption, making efficient homogenization a critical step for obtaining reproducible molecular analyses. Consequently, RNA yield and integrity provided a more robust proxy for evaluating grinding efficiency than genomic DNA recovery during optimization of the custom-built Fast-lyzer used to disrupt small seed samples. For efficient tissue disruption and optimal RNA recovery, seeds should be briefly blotted on filter paper prior to freezing to remove excess surface moisture. Seed disruption by means of Fast-lyzer was obtained using three 2.8 mm beads and a sequential grinding program consisting of three 20 seconds cycles at 1600 RPM followed by four 30 seconds cycles at 2000 RPM. We imbibed seeds for three days at 4°C prior to sampling, and then, we performed the grinding as mentioned above. Even though we did not observed statistically differences between manual and mechanical disruption in terms of RNA yield, the use of the device is advantageous due to the time saved in sample processing (Figure 5A and Supplementary Table 3) Bacterial extraction from plants. Plant immunity studies frequently rely on phenotyping approaches based on the quantification of bacterial growth, assays that are inherently variable and therefore require large numbers of samples and biological replicates [31]. We evaluated whether the Fast‑lyzer could be used to accelerate these procedures while preserving experimental outcomes. First, we cultured Pseudomonas syringae pv. tomato DC3000 (Pst) to assess whether mechanical grinding negatively affects bacterial viability. Under all tested conditions, we consistently recovered equivalent total bacterial counts (Figure 5B), indicating that the grinding procedure does not compromise cell viability. Having established this, we next infected adult leaves with Pst to determine whether bead‑based leaf disc grinding enables recovery of bacterial populations comparable to those obtained with the classical manual piston method. No differences in bacterial counts were observed between the two approaches (Figure 5C), demonstrating that bead‑based grinding is as efficient as manual grinding for the extraction of viable bacterial cells from plant tissues. Discussion Our device outperformed manual extraction across most tested tissues with respect to processing time, nucleic acid yield, and integrity of the recovered material. In general, higher grinding frequencies and extended grinding durations increased both DNA yield and integrity. In the case of leaves and seedlings, Fast-lyzer optimal grinding conditions yielded better results than manual processing (Student´s T test, p=0.0047 and p=0.0022, respectively). Even in the case of floral stems where our initial trials gave poor results, after increasing the grinding time, we obtained better yields (Student´s T test, p=0.0188). The inability of the Fast-lyzer to outperform manual grinding in stems in short times, may be due to the specific action of the pestle, which crushes the tissue against the tube walls by applying direct pressure to the stem tips, thereby enhancing disruption. Conversely, the beads strike the stems randomly, which reduces the efficiency of tissue rupture in these tougher samples. By increasing the grinding time, this effect gradually disappears, eventually reaching levels higher than manual grinding. For protein extraction, whose result was worse than in manual grinding and considering that the conditions are the same as for DNA extraction, we hypothesize that further extraction with urea buffer may not be as efficient with that level of tissue grinding. Our main scope is to validate the developed device, but we also provide optimized conditions for tissue grinding along three different tissues in Arabidopsis thaliana . Combination with smaller beads or different kinds of beads -zirconium, or glass- might further enhance the yield or quality of the extractions and may need further exploration for non-standard extraction needs like Oxford Nanopore Technology sequencing. Electrophoretic profiles obtained through all the assays showed that the low‑molecular‑weight smear remained relatively constant across conditions, while harsher grinding predominantly increased the abundance of high molecular weight (HMW) DNA, resulting in superior integrity indices. We hypothesize that once genomic DNA is fragmented into relatively large pieces, it becomes less susceptible to additional mechanical shearing. Under this scenario, DNA retains its integrity while the grinding beads continue to disrupt remaining intact cells, thereby releasing additional nucleic acids [32]. RNA proved less sensitive to mechanical disruption, consistent with its shorter average fragment length; RNA integrity is perhaps more dependent on the prevention of RNase activity rather than on the intensity of grinding. Proteins also demonstrated substantial resistance to bead-induced mechanical forces. When performing bacterial extractions, we did not observe any reduction on bacterial counts. This is likely because bacterial cells, due to their small size relative to the steel beads, are not efficiently crushed between the beads or against the tube walls. Temperature control was crucial. Continuous grinding for 120 seconds without cooling intervals markedly reduced DNA integrity and led to substantial heating of the tubes and samples. These observations emphasize the importance of incorporating cooling pauses during high intensity grinding to preserve nucleic acid quality. Regarding the quality of nucleic acids or proteins required for downstream applications such as PCR, qPCR, RT-PCR, sequencing, or Western blotting, many studies focus on extraction buffers or procedures following tissue disruption regardless of the grinding methodology used. Often, tissue grinding is performed using either manual methods (mortar and pestle) or commercial grinders [33]. Other studies employ a mortar and pestle followed by various strategies to ensure high-quality yields, such as specialized extraction buffers, commercial kits, or a combination of both [34][35]. Notably, these works lack specific protocols for the grinding process itself. Therefore, a methodology that mimics manual grinding while reducing processing time would be highly desirable for any laboratory requirement involving nucleic acid or protein extraction. Tube quality is a critical variable: stainless-steel beads exert a considerable mechanical stress, and lower grade tubes are prone to rupture. Tubes rated certified for centrifugal forces up to 21500 × g can usually withstand our experimental conditions, whereas tubes with a maximum rate of 15000 × g frequently failed during grinding, particularly when samples were first flash frozen in liquid nitrogen and subsequently processed at 2000 RPM. The parameters used for 3D printing as well as the structural design of the tube holder were also critical determinants of overall device robustness. Additively manufactured plastic components are particularly vulnerable at the interfaces between printed layers. After testing multiple iterations, we identified the joint between the holder lid and the metal adapter as the principal mechanical weak point. Redesign of this interface, including the incorporation of a logarithmic conical geometry and reinforcing with 80% infill in critical areas, prevented holder fractures. The final configuration resulted in a robust plastic component capable of withstanding shear forces generated at 2000 RPM while holding 25 tubes, each containing two 4 mm metal beads and 1 ml of extraction buffer. Conclusions We present Fast-lyzer, a low-cost method for tissue grinding suitable for various types of plant tissues. The device performs tissue disruption with significant time savings and yields results that are superior, or at least similar, to manual grinding for most tissues tested, specifically for DNA, RNA, or protein extraction. We also propose optimal grinding configurations (bead number and quantity, time, and oscillation frequency) for different tissues that may be applied to available commercial devices. These details are often omitted in studies focused on nucleic acid or protein extraction, typically centered on buffers or centrifugation parameters. The developed device is an excellent alternative for laboratories with limited resources, as it provides an efficient way to homogenize tissues quickly and at a minimal cost. Abbreviations DNA: Deoxyribonucleic acid RNA: Ribonucleic acid 3D: Three-dimensional USD: United States Dollar mm: millimeter MS: Murashige and Skoog mg: milligram cm: centimeter °C: degrees Celsius ml: milliliter CTAB: Cetyltrimethylammonium bromide x g: times gravity µl: microliter V: Volt kb: kilobase ng: nanogram min: minute h: hour v/v: volume per volume w/v: weight per volume mM: millimolar SDS: Sodium Dodecyl Sulfate DTT: Dithiothreitol HCl: hydrochloric acid PAGE: Polyacrylamide Gel Electrophoresis RUBISCO: Ribulose-1,5-bisphosphate carboxylase/oxygenase Col-0: Columbia-0 MgCl 2 :Magnesium chloride OD 600 :Optical Density at 600 nanometers RPM: Revolutions Per Minute Hz: hertz µmol: micromole s: second m: meter CFU: Colony Forming Unit M: molar PCR: Polymerase Chain Reaction q-PCR: quantitative Polymerase Chain Reaction RT-PCR: Reverse Transcription Polymerase Chain Reaction Declarations Consent for publication All authors give consent to the publication of the manuscript. Availability of data and materials All data supporting the findings of this study are available within the paper and its Supplementary Information. Competing Interests The authors declare no competing interest. Funding Agencia Nacional de Promoción de la Investigación, el Desarrollo Tecnológico y la Innovación. PICT grant to Martiniano María Ricardi. Author Contributions R.M.G. performed DNA extractions, optimized grinding conditions, and contributed to the figures and the writing and proofreading of the manuscript. C.A. performed RNA extractions from seedlings, leaves, and stems. F.E.A. performed RNA extractions from seedlings, leaves, and stems, conducted all experiments involving Pseudomonas syringae and contributed with the writing of the manuscript. C.C. performed protein extraction and SDS-PAGE electrophoresis. F.S.R. and R.S.T performed RNA extractions from imbibed seeds and contributed with the writing of the manuscript. F.C. and F.R. conducted prototype testing for the Fast-Lyzer, evaluating early versions through to the final iteration. S.L. developed the RPM meter and evaluated device performance under sample-load conditions. E.P. contributed to and supervised all RNA experiments. M.M.R. conceived the project, designed the device, produced the 3D prints and the metal holder, and contributed to the figures and the writing of the manuscript. All authors read and approved the final version of the manuscript. Acknowledgements Authors wish to thank Dr. Nicolás Cecchini for providing Pseudomonas syringae pvar. tomato DC3000 for the assays with bacteria. We also wish to thank the interns Tyara Alburquenque and Ignacio del Valle for contributing with plant growth, agarose gel electrophoresis and SDS-PAGE running. References Goldberg S. Mechanical/physical methods of cell disruption and tissue homogenization. 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Plant Methods [Internet]. 2023;19:84. https://doi.org/10.1186/s13007-023-01063-5 Oliveira RR, Viana AJ, Reategui AC, Vincentz MG. Short Communication An efficient method for simultaneous extraction of high-quality RNA and DNA from various plant tissues. Genet Mol Res [Internet]. 2015;14:18828–38. https://doi.org/10.4238/2015.December.28.32 Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial.zip Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 08 May, 2026 Reviewers agreed at journal 08 May, 2026 Reviewers agreed at journal 04 May, 2026 Reviewers invited by journal 29 Apr, 2026 Editor assigned by journal 20 Apr, 2026 Submission checks completed at journal 20 Apr, 2026 First submitted to journal 16 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9440300","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Method Article","associatedPublications":[],"authors":[{"id":635852186,"identity":"c66567fc-4f0c-4974-820d-74b5c0541060","order_by":0,"name":"Rodrigo Matías González","email":"","orcid":"","institution":"Instituto de Fisiología, Biología Molecular y Neurociencias","correspondingAuthor":false,"prefix":"","firstName":"Rodrigo","middleName":"Matías","lastName":"González","suffix":""},{"id":635852187,"identity":"11c76d1e-a3aa-411d-931d-c79e156704af","order_by":1,"name":"Carola Agranatti","email":"","orcid":"","institution":"Universidad de Buenos Aires","correspondingAuthor":false,"prefix":"","firstName":"Carola","middleName":"","lastName":"Agranatti","suffix":""},{"id":635852189,"identity":"a2a9546d-e7eb-46a9-ae7d-d43942fcba6a","order_by":2,"name":"Federico E. 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B) Isometric views of the tube holder consisting of a tube rack with 25 tube capacity and a lid. Holes for Lid-rack fastening (Empty arrows) and Lid-steel bar joints (Full arrows). C) Picture of the final assembly ready to mount on the Jigsaw. D) Final assembly E) RPM-weight dependence.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-9440300/v1/3b981a9b58dc3d36374e717d.png"},{"id":108807953,"identity":"85c9772f-e47f-43ec-8305-50976fd72f3e","added_by":"auto","created_at":"2026-05-08 15:38:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":305467,"visible":true,"origin":"","legend":"\u003cp\u003eNucleic acid and protein extraction from whole \u003cem\u003eArabidopsis thaliana\u003c/em\u003e seedlings.\u003c/p\u003e\n\u003cp\u003eA-C) Quantification of High MW band and % of DNA integrity with respective representative gel pictures. A) Selection of stainless-steel bead size and quantity. We set frequency to 2000 RPM for 60 seconds and compared to manual piston-based grinding. B) Selection of grinding time: using two 4-mm balls at 2000 RPM based on results from (A). C) Selection of grinding frequency using two 4-mm balls for 120 seconds based on results from (B). D) Protein extraction using the optimal conditions identified for DNA -two 4-mm balls, 120 seconds and 2000 RPM- in comparison to piston-based manual grinding. Rubisco band quantification relativized to manual grinding average yield. E) RNA extraction using the same setting as in D in comparison to mortar-based manual grinding. A), B), and C) 1% agarose gel. D) 12% polyacrylamide gel stained with Coomassie Brilliant Blue. E) 1% agarose gel with 1% bleach. In all cases, identical letters or the letters NS indicate no statistical significance. A to C) One-Way ANOVA. D and E) Student´s T test.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-9440300/v1/9f57e7792d799663513e3db3.png"},{"id":108807985,"identity":"606e1871-c689-40f0-b9b9-5f2278044a48","added_by":"auto","created_at":"2026-05-08 15:38:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":315795,"visible":true,"origin":"","legend":"\u003cp\u003eNucleic acid and protein extraction from fully developed \u003cem\u003eArabidopsis thaliana\u003c/em\u003e leaf discs.\u003c/p\u003e\n\u003cp\u003eA-C) Quantification of High MW band and % of DNA integrity with respective representative gel pictures. A) Selection of stainless-steel bead size and quantity. We set frequency to 2000 RPM for 60 seconds and compared to manual piston-based grinding. B) Selection of grinding time: using one 4 mm and one 2.8 mm balls at 2000 RPM based on results from (A). C) Selection of grinding frequency using one 4 mm and one 2.8 mm balls for 60 seconds based on results from (B). D) Protein extraction using the optimal conditions identified for DNA -two 4-mm balls, 120 sec and 2000 RPM- in comparison to piston-based manual grinding. Rubisco band quantification relativized to manual grinding average yield. E) RNA extraction using the same setting as in D in comparison to mortar-based manual grinding. A), B), and C) 1% agarose gel. D) 12% polyacrylamide gel stained with Coomassie Brilliant Blue. E) 1% agarose gel with 1% bleach. In all cases, identical letters or the letters NS indicate no statistical significance. A to C) One-Way ANOVA. D and E) Student´s T test.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-9440300/v1/9e7c53ecbee99ca9491fbd8e.png"},{"id":108808121,"identity":"049d8181-9a7c-48c7-a6b5-7628e6d7cd77","added_by":"auto","created_at":"2026-05-08 15:39:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":292325,"visible":true,"origin":"","legend":"\u003cp\u003eNucleic acid and protein extraction from \u003cem\u003eArabidopsis thaliana\u003c/em\u003e flower stems.\u003c/p\u003e\n\u003cp\u003eA-C) Quantification of High MW band and % of DNA integrity with respective representative gel pictures. A) Selection of stainless-steel bead size and quantity. We set frequency to 2000 RPM for 60 seconds and compared to manual piston-based grinding. B) Selection of grinding time: using two 4-mm balls at 2000 RPM based on results from (A). C) Selection of grinding frequency using two 4-mm balls for 120 seconds based on results from (B). D) Protein extraction using the optimal conditions identified for DNA -two 4-mm balls, 120 sec and 2000 RPM- in comparison to piston-based manual grinding. Rubisco band quantification relativized to manual grinding average yield. E) RNA extraction using the same setting as in D in comparison to mortar-based manual grinding. A), B), and C) 1% agarose gel. D) 12% polyacrylamide gel stained with Coomassie Brilliant Blue. E) 1% agarose gel with 1% bleach. In all cases, identical letters or the letters NS indicate no statistical significance. A to C) One-Way ANOVA. D and E) Student´s T test.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-9440300/v1/042023622bfbbef7ccb92d56.png"},{"id":108807984,"identity":"fa6e3383-6c3b-437c-ba44-75e26fc0c660","added_by":"auto","created_at":"2026-05-08 15:38:20","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":192258,"visible":true,"origin":"","legend":"\u003cp\u003eSeeds and plant – bacterial interaction studies.\u003c/p\u003e\n\u003cp\u003eA) RNA extraction from \u003cem\u003eArabidopsis thaliana\u003c/em\u003eimbibed seeds. 1% agarose gel with 1% bleach. B) Colony count of \u003cem\u003ePseudomonas syringae\u003c/em\u003e pvar. tomato DC3000 from culture treated with the Fast-lyzer with different size and amounts of beads or not treated. One-Way ANOVA. P=0.5777 C) Colony count of \u003cem\u003ePseudomonas syringae\u003c/em\u003e pvar. tomato DC3000 extracted from leaves previously infected, employing a plastic piston or with the Fast-lyzer. Student’s T test P=0.4706. NS indicate no statistical significance.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-9440300/v1/3037d4332aac51fdc621b52a.png"},{"id":108979594,"identity":"7808d4fa-07a6-493e-84e5-949bb0886a49","added_by":"auto","created_at":"2026-05-11 12:00:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2457857,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9440300/v1/16611b67-3793-44b4-a74a-380ad40691a0.pdf"},{"id":108807986,"identity":"04bae276-9ccb-46a1-bd1c-4fa54a18f725","added_by":"auto","created_at":"2026-05-08 15:38:21","extension":"zip","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":759830656,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.zip","url":"https://assets-eu.researchsquare.com/files/rs-9440300/v1/39be2813afc728b0c4995449.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"“Fast-lyzer”: A low-cost 3D printable device for plant tissue disruption and homogenization","fulltext":[{"header":"Background","content":"\u003cp\u003eSample disruption and homogenization are two critical steps in numerous biological extraction procedures (i.e., DNA, RNA or protein extraction). It is often performed manually and can show a very broad range of results depending on the expertise of the person performing the protocol. Several companies offer tissue processing machines that enable high reproducibility and high number of sample processing relying on different types and sizes of beads and shaking movements\u0026nbsp;[1][2][3][4][5][6]. The prices for this equipment are over 10000 USD which makes them difficult to purchase in counties with low scientific investment\u0026nbsp;[7]. For this reason, we decided to contribute with this open scientific hardware development\u0026nbsp;[8][9][10].\u003c/p\u003e\n\u003cp\u003eTheir working principle relies on shaking beads from different sizes and materials according to the type of tissue / organism to process. For instance, Qiagen recommends: 0.1–0.6 mm (mean diameter) glass beads for bacteria, 0.5 mm glass beads for yeast and unicellular animal cells, and 3–7 mm stainless steel or tungsten carbide beads for plant and animal/human tissues. (Manual available at https://www.qiagen.com/us/resources/download.aspx?id=65e7826c-4d50-4faf-8154-2fbc782c41a6\u0026amp;lang=en). Some works determined the best conditions for extracting nucleic acids in non-conventional models, using Tissuelyzer™\u0026nbsp;[11],but to our best knowledge there are no systematic studies on \u003cem\u003eArabidopsis thaliana\u003c/em\u003e.Stainless steel beads can be purchased at specialized scientific stores for 50 USD for 100 beads (https://www.usascientific.com/stainless-steel-beads-/p/Steel_Beads) or from other non-scientific manufacturers at 3 to 5 USD for 1000 beads. Non-lab-grade materials must be properly prepared and washed prior to their use. Stainless steel beads should be washed and rinsed several times in distilled water. They can be autoclaved to avoid contamination on future extracts and reused after washing.\u003c/p\u003e\n\u003cp\u003eAs a replacement for the shaking force, we propose to use a non-orbital 600W jigsaw. Jigsaws typically oscillate in a back-and-forth movement at 300–3000 RPM (≈ 5–50 Hz), a range comparable to devices such as the Qiagen Tissuelyser™, which operates between 3 and 30 Hz. Combined with a simple custom-made blade and 3D printed plastic pieces to attach the tubes into the jigsaw, we were able to assemble an inexpensive device for shaking based bead sample grinding [12].\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eJigsaw metal adapter\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe cut the jigsaw adapter from a 1.5mm sheet of carbon steel 15N20 (with high carbon and nickel content) that provides excellent tenacity, resistance to traction and fatigue. This material is commonly used to build knives. The adapter emulates the shape of the blades to fit perfectly on the machine (Figure 1A). It has also an additional part that fits an optical barrier for RPM counting and two holes for fixation to the plastic sample holder. We make cuts by laser cutting on a local company with a precision of 0.1 mm.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHolder\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe holder consists of two 3D printed parts (Figure 1B). A rack with holes for Eppendorf tubes and a lid. The rack has additional hexagonal holes to fit 2 hexagonal bolts. Once we added the tubes to the rack, the lid is tightly be adjusted with nuts. We previously assembled the lid with the metal adapter with bolts and Nylon insert-nuts tightly adjusted to the final assembly (Figure 1C).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRPM counter\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe RPM counter consists of an Arduino Nano V3 connected to optocoupled IR barrier (FC-03 IR) and LCD display (LCD I2C 16x2) modules. They are mounted in a way that the metal adapter can obturate the IR barrier during the jigsaw operation. All the necessary programs and schematics are described in supplementary material (device building files). To determine if changes in adapter weight—resulting from loading sample tubes, beads, and eventually buffer—substantially modify the device's RPM, we measured the speed five times for each selector position used in this study (1, 2, 3, or 4). We performed these measurements under four conditions: without added weight and with masses of 20, 30, and 50 grams. In each iteration, we calculated the slope of the line and evaluated whether these slopes differed significantly from zero, which would indicate that the applied mass does not affect the speed at all (Figure 3E).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3D printing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe designed models using FreeCad 1.0 software (https://github.com/Reqrefusion/FreeCAD-Documentation-Project). After meshing, they were sliced using Orca slicer. We printed the adapters using a standard printer (Creality Ender 3 V3 se) in polylactic acid (PLA) or Polyethylene Terephthalate Glycol (PETG) with similar results. Printing density should be high on the light blue highlighted regions (see Figure 1B). We optimized the design to prevent its breaking by increasing the amount of material on its weaker points.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe used \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (ecotype Columbia-0) at various developmental stages to collect the different tissue samples used throughout all experiments. We used 14 days old seedlings germinated on Agar- 0,5X MS plates. For stems and leaves we used 6-week-old plants grown in soil under standard conditions (16/8 light-dark photoperiod, 22ºC and commercial plant growth substrate). We used three-day imbibed seeds in darkness at 4°C.\u003c/p\u003e\n\u003cp\u003eFor tissue collection we snap froze in liquid nitrogen, (1) 100 mg of whole seedlings, (2) four 8 mm diameter leaf discs (weighting approximately 50mg), (3) 100 mg floral stems cut into 1 cm long pieces and (4)\u0026nbsp;~20 mg of dry seeds and stored at –80 °C until used. We later used the samples for DNA, RNA or protein extraction.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDNA extraction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe harvested approximately 100 mg of tissues into 1.5 ml microcentrifuge tubes for manual extraction or into 2 ml round-bottom tubes for mechanical disruption. For manual extraction, we used a plastic pestle (Kimble Kontes) and ground the tissue with liquid nitrogen. For mechanical disruption, we used the Fast-lyzer with different settings indicated throughout the publication. Once we ground the tissues, we extracted DNA using 400 µl of CTAB buffer\u0026nbsp;[13]. Briefly, we incubated samples for 30 minutes at 65°C, followed by centrifugation at 14,000 × g for 5 minutes. We transferred the supernatant to a new tube and performed an organic extraction using 400 µl of chloroform:isoamyl alcohol (24:1). We recovered the aqueous phase, and precipitated the DNA with 280 µl of isopropanol, incubated at -20°C for 20 minutes, followed by pelleting for 20 minutes at 21,500 × g at 4°C in a refrigerated microcentrifuge. Finally, we washed the pellet with 70% ethanol, dried, and resuspended in Milli-Q water.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssessment of DNA concentration and integrity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe run DNA extracted from different tissues using various approaches on a 1% agarose gel at 120 V for 40 minutes. To determine the mass of each sample, we ran a 1 kb DNA ladder (New England Biolabs) along with several dilutions (0.5, 0.25, and 0.125) to generate a calibration curve. To this end, we used 500 ng of DNA ladder, specifically targeting the 1.5 kb (36 ng), 2 kb (48 ng), and 3 kb (125 ng) bands, along with their respective two-fold, four-fold, and eight-fold dilutions. We loaded 10 μL of each ladder dilution and 5 μL of each sample. Subsequently, we visualized the gel using an UVP transilluminator with the following settings: f = 1.2, zoom 65%, focus 87.9, and a 15-second exposure.\u003c/p\u003e\n\u003cp\u003eWe captured the image and performed an analysis using ImageJ software [14]. We inverted the gel photograph colours (black bands on a white background). Then, we used a rectangular selection tool to define each lane. We obtained profile plots for each lane to determine the pixel intensity (area) of each band and using the data from the ladder, we constructed a calibration curve to calculate the DNA mass in the samples.\u003c/p\u003e\n\u003cp\u003eTo account for background noise, we estimated the pixel values under the curve, excluding the peaks. We determined electrophoresis background by measuring the area under the curve in an empty lane and subtracting this value from the sample lanes. We then determined the integrity percentage by calculating the ratio between the main band mass and the total mass in the lane (band plus smeared DNA).\u003c/p\u003e\n\u003cp\u003eWe analyzed the data using GraphPad Prism software (v. 9.0.0). We first tested for normality using the Shapiro-Wilk test. Subsequently, we performed a one-way ANOVA followed by Tukey’s multiple comparisons post-hoc test to identify statistically significant differences between assays. For comparisons between manual and Fast-lyzer optimally grinding conditions, we employed a Student’s T test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA extraction of seedlings, leaves and stems\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe extracted RNA from ground samples using TriPure (Sigma)\u0026nbsp;[15][16].\u0026nbsp;Briefly, we added 1 mL of TriPure to each nitrogen-frozen sample and mixed by inversion. After centrifugation at 12,000 x g for 10 min at 4ºC we transferred the supernatant into a new tube with 200 µl of chloroform and shook it for 1 min. We recovered the aqueous phase after centrifugation at 12,000 x g for 10 min at 4ºC, transferred into a new tube and precipitated by adding 500 µl of isopropanol, letting the mixed sample rest at room temperature for 10 minutes and later centrifugation at 12.000 x g for 10 minutes. We washed the pellets twice with Ethanol 75% and centrifuged the tubes at the same speed for 5 min and dried in a laminar flow cabinet. We finally resuspended the pellets in 50 µl RNAse-free Milli-Q water and stored at -80ºC for 1 hour.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA integrity and quantification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe assessed RNA integrity by agarose gel electrophoresis. Briefly, 3 μL of each sample + 3 μL of Orange-G loading buffer was loaded in each well. Samples were separated by gel electrophoresis at 150 V for 20 min in 1% w/v agarose gel supplemented with 1 % v/v bleach [17]. We photographed the gels using an UVP transilluminator with the following settings: f/1.2, 65% zoom, 87.9 focus, and a 15-second exposure. Next, we performed relative RNA quantification using ImageJ software. After selecting the lanes with the rectangular selection tool, we plotted them to obtain the area under the curves. We summed the areas of all ribosomal RNA bands present and calculated the mean of the manually extracted samples, which we arbitrarily set to one. Finally, we normalized all values to this mean for statistical analysis. We employed GraphPad Prism software (v. 9.0.0) for statistical analysis, performing a Shapiro-Wilk test to check for normality distribution and a Student’s T test to find statistically differences between both grinding methods.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein extraction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe extracted ground samples by addition of 250 µl of urea buffer (200 mM Tris–HCl pH 6.8, 10% SDS, 6 M Urea, 20% Glycerol, 50 mM DTT, 0.05% Bromophenol Blue). We heated the samples for 10 min at 80°C, run them in standard 12% SDS-PAGE gels and stained them with Coomassie brilliant blue according to standard protocols [18]\u003c/p\u003e\n\u003cp\u003eFollowing staining, we scanned the gel to obtain a TIFF image for analysis using ImageJ software. We quantified the most intense band, corresponding to the RUBISCO protein by calculating the area under the peak. We averaged the values from manually extracted samples, and we arbitrarily set to 1 this mean. We then normalized all remaining samples to this mean for statistical analysis. We employed GraphPad Prism software (v. 9.0.0) for statistical analysis, performing a Shapiro-Wilk test to check for normality distribution and a Student’s T test to find statistically differences between both grinding methods.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA extraction from seeds\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe sowed A\u003cem\u003erabidopsis thaliana\u0026nbsp;\u003c/em\u003eCol-0 seeds (~20 mg seed dry weight) onto filter papers in clear plastic boxes (40 × 33 × 15 mm), each containing 10 mL of 0.8% (\u003cem\u003ew\u003c/em\u003e/\u003cem\u003ev\u003c/em\u003e) agar in demineralized water and incubated for three days in darkness at 4 °C. After sampling, seeds were\u0026nbsp;briefly blotted on filter paper to remove excess surface moisture\u0026nbsp;and then immediately frozen in liquid nitrogen and stored at − 80 °C until grounding.\u0026nbsp;We extracted RNA from ground samples using the Spectrum\u003csup\u003eTM\u003c/sup\u003e Plant Total RNA Kit\u0026nbsp;(Sigma-Aldrich, Steinheim, Germany) according to the manufacturer’s protocol.\u0026nbsp;We assessed RNA integrity by agarose gel electrophoresis as previously described for the other tissues.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBacterial plate counting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePseudomonas syringae\u003c/em\u003e pv. tomato DC3000 (kindly provided by Dr. Nicolás Cecchini, CIQUIBIC – UNC/CONICET) was grown in Luria-Bertani (LB) agar medium supplemented with rifampicin 100 μg/ml and kanamycin 75 μg/ml for 48h at 28ºC. Then, we resuspended the bacteria in MgCl\u003csub\u003e2\u003c/sub\u003e 10 mM to O.D\u003csub\u003e600\u003c/sub\u003e = 0.01. Afterwards, we treated bacteria with our Fast-lyzer (2000 RPM) with one 2.8 mm bead, two 2.8 mm beads or one 4 mm bead. Finally, we evaluated bacterial growth by serial dilution followed by droplet plating and counting on the same culture medium than before (CFU/mL). The experiments consisted of three biological replicates (different days) with at least three replicates each day.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBacterial counting from pathogen infection assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlants were grown in long-day conditions (16h/8h light/darkness) at 21 ºC with a light intensity of 100-120 μmol s\u003csup\u003e−1\u003c/sup\u003e m\u003csup\u003e−2\u003c/sup\u003e and 50% relative humidity for 25-28 days. On the other hand, \u003cem\u003eP. syringae\u003c/em\u003e pv. tomato DC3000 was grown as previously described for 48h at 28 ºC. Then, we re-suspended the bacteria and diluted to OD\u003csub\u003e600\u003c/sub\u003e=0.0005 in MgCl\u003csub\u003e2\u003c/sub\u003e 10 mM for infection. We infected the abaxial surface of leaves by pressure-mediated infiltration with a needleless syringe. To assess possible differences with classical methods, we cut 8 mm leaf discs to grind them either with a piston during 60 seconds per sample, or with our Fast-lyzer, both in MgCl\u003csub\u003e2\u003c/sub\u003e 10 mM at 72 hours post-infection. For the case of Fast-lyzer, we ground the samples with two 2.8 mm beads, 2000 RPM and for 15 seconds three times. Then, we assessed\u003cem\u003e\u0026nbsp;in planta\u0026nbsp;\u003c/em\u003ebacterial growth (CFU/mL) by droplet plating as previously described.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eDevice mounting:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe adapter consists of a tube holder and a steel flat bar (Figure 1C). The tube holder (consisting of a tube rack and a lid) is firmly attached to the wood jigsaw by the custom-cut blade-shaped steel flat bar with an integrated jigsaw shank (model available at Supplementary files, Device Building Files). This piece fits precisely into the jigsaw’s standard blade-mounting slot, allowing the entire adapter module to be attached or removed using the machine’s normal locking mechanism. Because 3D printing is inexpensive, multiple adapters can be produced to enable rapid sample processing. Loaded samples can be stored at the desired temperature (−20 °C or even −80 °C) until use. The holder can be printed in polyethylene terephthalate glycol (PETG) or polylactic acid (PLA) which are the most commonly used 3D printing materials\u0026nbsp;[19].\u003c/p\u003e\n\u003cp\u003eOur current design accommodates 25 tubes of 1.6 ml or 2 ml volume, but this can be easily modified depending on experimental needs. Before assembly, each tube must be filled with the sample, beads, and buffer if required. It is very important to balance the weight by placing pairs of tubes with equal weight symmetrically around the central position in order to avoid vibrations perpendicular to the one generated by the jigsaw. The type of beads used depends on the characteristics of the tissue being processed. To prevent tubes from opening during grinding, the tube holder and lid are secured together using bolts and locking nuts. Depending on the protocol, the assembled adapter can be placed on ice, or the tubes can be flash frozen in liquid nitrogen (ensuring that only the tubes — not the 3D printed adapter — are submerged).\u003c/p\u003e\n\u003cp\u003eOnce everything is prepared, the holder is mounted onto the jigsaw in the same way as a standard blade (Figure 1D). For safety, we placed the jigsaw inside a PET made protective container box before powering it on. It is also necessary that the operator wear safety glasses and hearing protection (see Supplementary Video 1 for full procedural details). The jigsaw must be used with the blade along the gravity axis. Because oscillation speed is critical for reproducibility across different jigsaw models, we implemented an optocoupled sensor system connected to an Arduino to directly measure and calibrate the jigsaw’s RPM. This allowed us to establish a precise relationship between the speed dial position and the actual oscillation frequency, ensuring consistent performance across experiments. In our setup, we routinely used speed setting 4 out of 6, corresponding to approximately 2000\u0026nbsp;RPM.\u003c/p\u003e\n\u003cp\u003eGiven that the weight of the tube holder can change substantially when loaded with tubes, buffers and stainless-steel beads, we first evaluated whether the total mass (samples + holder) affected the final RPM of the machine at a fixed speed setting. Our results showed that the weight variation introduced by the number of samples (Considering that the sum of the mass of microtubes, stainless steel beads, samples and up to 1 ml of buffer per tube, will not exceed 50 grams of total load in the holder) did not significantly alter the machine’s actual RPM and therefore does not represent an additional parameter that needs to be controlled (Figure 1E and Supplementary Table 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGrinding procedure:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDue to their structure, different plant tissues have distinct mechanical properties. We optimized the grinding conditions separately for whole seedlings\u0026nbsp;[20], leaf discs\u0026nbsp;[21], stem segments\u0026nbsp;[22]\u0026nbsp;and imbibed seeds\u0026nbsp;[23]. We adjusted size and number of metal beads, total grinding time and RPMs. We initially optimized all conditions using DNA extraction efficiency as the main indicator. We chose DNA because, due to its large molecular size, it is easily sheared and therefore serves as a sensitive proxy for assessing grinding harshness\u0026nbsp;[24]. To avoid sample heating and subsequent DNA degradation (Supplementary Figure S1), every 30 seconds, we chilled the samples by submerging the tube bottoms in liquid nitrogen for 10 seconds. Once the optimal parameters were established, we also validated them for RNA and protein extraction. We determined the time needed to process samples manually and with Fast-lyzer (considering that 25 tubes are loaded into the device). For manual grinding, we employed a Kimble Kontes plastic piston for DNA, protein and bacteria extractions, which required a grinding time of 181,0 +/- 7,1 sec per sample. For RNA extraction, we used mortar and pestle, requiring 409.2 +/- 33.7 sec per sample. In all cases, batch processing with Fast-lyzer significantly reduced the grinding to 25,5 +/- 0,9 sec per sample (Supplementary Figure S2) (One-way ANOVA P\u0026lt; 0,0001, Tukey’s post-test: mortar and pestle vs Fast-lyzer P\u0026lt;0.0001, piston vs Fast-lyzer P=0,001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWhole seedling:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInitially, we set the shaking speed to 2000 RPM and the grinding time to 1 minute and evaluated different bead numbers and sizes to identify the condition that maximized the recovery of intact genomic DNA. In all cases, bead-assisted extraction clearly outperformed manual grinding, yielding significantly higher concentrations of a high–molecular weight (HMW) DNA band on agarose gels, while maintaining comparable DNA integrity percentages (Figure 2A).\u003c/p\u003e\n\u003cp\u003eUsing two 4 mm beads and a fixed frequency of 2000 RPM, we next evaluated different grinding durations, reasoning that longer processing times might improve tissue disruption but could also increase DNA fragmentation. Extended grinding times indeed resulted in higher yields of the HMW band but contrary to our initial thoughts it also improved DNA integrity indices (Figure 2B).\u003c/p\u003e\n\u003cp\u003eHaving established that 120 seconds and two 4 mm beads were optimal, we then optimized frequency, again considering that higher frequencies could potentially fragment DNA. Contrary to this expectation, both DNA concentration and integrity peaked at 2000 RPM of frequency (Figure 2C and Supplementary Table 2). Frequencies exceeding 2000 RPM are not recommended because centrifuge tubes tend to fracture when operated above this threshold.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHaving determined these optimized conditions -two 4 mm beads, 2000 RPM, and 120 seconds of grinding- we proceeded to perform RNA and protein extractions. When we tested RNA extractions performed with TRI-reagent according to manufacturer’s instructions, we did not observe differences neither on integrity (at least by running standard Agarose gels) nor on yield (Figure 2E and Supplementary Table 3). Protein extractions were performed with urea extraction buffer (see methods) and also showed that the method can speed up the grinding process improving the extraction outcome (Figure 2D and Supplementary Table 4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFully developed leaf discs:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, we performed the same set of experiments using 8 mm discs cut from fully developed true leaves. As before, we began with 2000 RPM for 1 minute while testing different bead combinations. The optimal setup was one 4 mm bead and one 2.8 mm bead, which produced slightly higher -yet not statistically significant- yield in comparison to the manual extraction (Figure 3A). Adjusting the grinding duration did not affect either the yield or the integrity of the recovered DNA. We observed a slightly higher –not significant- yield at 60 seconds (Figure 3B). Finally, when we lowered the RPM while using one 4 mm beads and one 2.8 mm bead for a 60 second grind, we observed clear reductions in both DNA yield and integrity indices (Figure 3C).\u003c/p\u003e\n\u003cp\u003eOur optimal settings for leaves were set at one 4 mm and one 2.8 mm beads, 2000 RPM, and 60 seconds of grinding (Supplementary Table 2).\u003c/p\u003e\n\u003cp\u003eUsing the optimal grinding conditions for leaf discs established for DNA extraction, we proceeded to extract RNA with Tri-Reagent (Figure 3E and Supplementary Table 3) and proteins (Figure 3D and Supplementary Table 4) with an 8 M urea buffer, as described for whole seedlings. For proteins, we observed non-significant differences between manual extraction and Fast-lyzer mediated extraction under the same conditions employed for DNA extraction.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStems:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor stems we started with 2000 RPM and 1 minute grinding and adjusted bead size and amounts. Manual grinding outperformed bead grinding giving statistically higher and less fragmented DNA in comparison to beads (Figure 4A). We continued using two 4 mm beads since they gave better -yet not significant- parameters compared to the other bead combinations. With two 4 mm beads and 2000 RPM we tested grinding time and found that more grinding time resulted in more yield and better DNA integrity and fixed the time at 120 seconds (Figure 4B). Similarly, grinding at higher frequencies gave progressively higher and less fragmented DNA. Therefore, we determined that our optimal settings for stems were 2000 RPM, 2 4 mm beads for 120 seconds (Figure 4C). These later settings gave slightly higher but not significant yields compared to the manual extraction. See Supplementary Table 2 for full data.\u003c/p\u003e\n\u003cp\u003eUnder the conditions used for DNA extraction, we carried out total protein extraction with urea buffer. We observed that, in this case, manual grinding yields more proteins than Fast-lyzer grinding. 36% less protein is obtained when grinding using Fast lyzer compared to manual grinding; however, the amount of protein obtained is sufficient for further applications while time saving is considerable (Figure 4D and Supplementary Table 4).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFinally, we extracted RNA using Tri-Reagent and the same conditions for grinding used for DNA extraction (Figure 4E and Supplementary Table 3). We did not observed statistically differences between manual and mechanical disruption. Therefore, the use of the device is advantageous due to the time saved in sample processing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOther samples:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further extend our trials we also explored extraction on seeds, and bacteria inside of plant leaves.\u003c/p\u003e\n\u003col\u003e\n \u003cli\u003eSeeds.\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eGrinding conditions in the other tissues were initially evaluated using genomic DNA extraction, as high-molecular-weight DNA is highly susceptible to mechanical shearing and therefore provides a sensitive indicator of grinding harshness. While this approach proved effective for several vegetative tissues as shown before, assays for imbibed seeds of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e was performed using total RNA extraction. Seed tissues are particularly challenging for DNA purification because they are rich in storage compounds, including proteins, lipids, polysaccharides, and secondary metabolites that can interfere with nucleic acid extraction by promoting co-precipitation or increasing extract viscosity\u0026nbsp;[25,26]. In contrast, imbibed seeds contain relatively abundant RNA derived both from transcripts stored during seed maturation and from early transcriptional activity following imbibition\u0026nbsp;[27,28]. Many of these stored mRNAs are rapidly recruited for translation immediately after imbibition, supporting early metabolic reactivation before large-scale \u003cem\u003ede novo\u003c/em\u003e transcription occurs\u0026nbsp;[29,30]. The small size of \u003cem\u003eArabidopsis\u003c/em\u003e \u003cem\u003ethaliana\u0026nbsp;\u003c/em\u003eseeds further increases the sensitivity of nucleic acid extraction protocols to incomplete tissue disruption, making efficient homogenization a critical step for obtaining reproducible molecular analyses. Consequently, RNA yield and integrity provided a more robust proxy for evaluating grinding efficiency than genomic DNA recovery during optimization of the custom-built Fast-lyzer used to disrupt small seed samples. For efficient tissue disruption and optimal RNA recovery, seeds should be briefly blotted on filter paper prior to freezing to remove excess surface moisture. Seed disruption by means of Fast-lyzer was obtained using three 2.8 mm beads and a sequential grinding program consisting of three 20 seconds cycles at 1600 RPM followed by four 30 seconds cycles at 2000 RPM.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe imbibed seeds for three days at 4°C prior to sampling, and then, we performed the grinding as mentioned above. Even though we did not observed statistically differences between manual and mechanical disruption in terms of RNA yield, the use of the device is advantageous due to the time saved in sample processing (Figure 5A and Supplementary Table 3)\u003c/p\u003e\n\u003col start=\"2\"\u003e\n \u003cli\u003eBacterial extraction from plants.\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003ePlant immunity studies frequently rely on phenotyping approaches based on the quantification of bacterial growth, assays that are inherently variable and therefore require large numbers of samples and biological replicates\u0026nbsp;[31]. We evaluated whether the Fast‑lyzer could be used to accelerate these procedures while preserving experimental outcomes. First, we cultured \u003cem\u003ePseudomonas syringae pv. tomato DC3000 (Pst)\u003c/em\u003e to assess whether mechanical grinding negatively affects bacterial viability. Under all tested conditions, we consistently recovered equivalent total bacterial counts (Figure 5B), indicating that the grinding procedure does not compromise cell viability. Having established this, we next infected adult leaves with Pst to determine whether bead‑based leaf disc grinding enables recovery of bacterial populations comparable to those obtained with the classical manual piston method. No differences in bacterial counts were observed between the two approaches (Figure 5C), demonstrating that bead‑based grinding is as efficient as manual grinding for the extraction of viable bacterial cells from plant tissues.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur device outperformed manual extraction across most tested tissues with respect to processing time, nucleic acid yield, and integrity of the recovered material. In general, higher grinding frequencies and extended grinding durations increased both DNA yield and integrity. In the case of leaves and seedlings, Fast-lyzer optimal grinding conditions yielded better results than manual processing (Student´s T test, p=0.0047 and p=0.0022, respectively). Even in the case of floral stems where our initial trials gave poor results, after increasing the grinding time, we obtained better yields (Student´s T test, p=0.0188). The inability of the Fast-lyzer to outperform manual grinding in stems in short times, may be due to the specific action of the pestle, which crushes the tissue against the tube walls by applying direct pressure to the stem tips, thereby enhancing disruption. Conversely, the beads strike the stems randomly, which reduces the efficiency of tissue rupture in these tougher samples. By increasing the grinding time, this effect gradually disappears, eventually reaching levels higher than manual grinding. For protein extraction, whose result was worse than in manual grinding and considering that the conditions are the same as for DNA extraction, we hypothesize that further extraction with urea buffer may not be as efficient with that level of tissue grinding.\u003c/p\u003e\n\u003cp\u003eOur main scope is to validate the developed device, but we also provide optimized conditions for tissue grinding along three different tissues in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. Combination with smaller beads or different kinds of beads -zirconium, or glass- might further enhance the yield or quality of the extractions and may need further exploration for non-standard extraction needs like Oxford Nanopore Technology sequencing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eElectrophoretic profiles obtained through all the assays showed that the low‑molecular‑weight smear remained relatively constant across conditions, while harsher grinding predominantly increased the abundance of high molecular weight (HMW) DNA, resulting in superior integrity indices. We hypothesize that once genomic DNA is fragmented into relatively large pieces, it becomes less susceptible to additional mechanical shearing. Under this scenario, DNA retains its integrity while the grinding beads continue to disrupt remaining intact cells, thereby releasing additional nucleic acids\u0026nbsp;[32].\u003c/p\u003e\n\u003cp\u003eRNA proved less sensitive to mechanical disruption, consistent with its shorter average fragment length; RNA integrity is perhaps more dependent on the prevention of RNase activity rather than on the intensity of grinding. Proteins also demonstrated substantial resistance to bead-induced mechanical forces.\u003c/p\u003e\n\u003cp\u003eWhen performing bacterial extractions, we did not observe any reduction on bacterial counts. This is likely because bacterial cells, due to their small size relative to the steel beads, are not efficiently crushed between the beads or against the tube walls.\u003c/p\u003e\n\u003cp\u003eTemperature control was crucial. Continuous grinding for 120 seconds without cooling intervals markedly reduced DNA integrity and led to substantial heating of the tubes and samples. These observations emphasize the importance of incorporating cooling pauses during high intensity grinding to preserve nucleic acid quality.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRegarding the quality of nucleic acids or proteins required for downstream applications such as PCR, qPCR, RT-PCR, sequencing, or Western blotting, many studies focus on extraction buffers or procedures following tissue disruption regardless of the grinding methodology used. Often, tissue grinding is performed using either manual methods (mortar and pestle) or commercial grinders\u0026nbsp;[33]. Other studies employ a mortar and pestle followed by various strategies to ensure high-quality yields, such as specialized extraction buffers, commercial kits, or a combination of both\u0026nbsp;[34][35]. Notably, these works lack specific protocols for the grinding process itself. Therefore, a methodology that mimics manual grinding while reducing processing time would be highly desirable for any laboratory requirement involving nucleic acid or protein extraction.\u003c/p\u003e\n\u003cp\u003eTube quality is a critical variable: stainless-steel beads exert a considerable mechanical stress, and lower grade tubes are prone to rupture. Tubes rated certified for centrifugal forces up to 21500 ×\u003cem\u003e\u0026nbsp;g\u003c/em\u003e can usually withstand our experimental conditions, whereas tubes with a maximum rate of 15000 ×\u003cem\u003e\u0026nbsp;g\u003c/em\u003e frequently failed during grinding, particularly when samples were first flash frozen in liquid nitrogen and subsequently processed at 2000 RPM.\u003c/p\u003e\n\u003cp\u003eThe parameters used for 3D printing as well as the structural design of the tube holder were also critical determinants of overall device robustness. Additively manufactured plastic components are particularly vulnerable at the interfaces between printed layers. After testing multiple iterations, we identified the joint between the holder lid and the metal adapter as the principal mechanical weak point. Redesign of this interface, including the incorporation of a logarithmic conical geometry and reinforcing with 80% infill in critical areas, prevented holder fractures. The final configuration resulted in a robust plastic component capable of withstanding shear forces generated at 2000\u0026nbsp;RPM while holding 25 tubes, each containing two 4 mm metal beads and 1 ml of extraction buffer.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eWe present Fast-lyzer, a low-cost method for tissue grinding suitable for various types of plant tissues. The device performs tissue disruption with significant time savings and yields results that are superior, or at least similar, to manual grinding for most tissues tested, specifically for DNA, RNA, or protein extraction. We also propose optimal grinding configurations (bead number and quantity, time, and oscillation frequency) for different tissues that may be applied to available commercial devices. These details are often omitted in studies focused on nucleic acid or protein extraction, typically centered on buffers or centrifugation parameters. The developed device is an excellent alternative for laboratories with limited resources, as it provides an efficient way to homogenize tissues quickly and at a minimal cost.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eDNA: Deoxyribonucleic acid\u003c/p\u003e\n\u003cp\u003eRNA: Ribonucleic acid\u003c/p\u003e\n\u003cp\u003e3D: Three-dimensional\u003c/p\u003e\n\u003cp\u003eUSD: United States Dollar\u003c/p\u003e\n\u003cp\u003emm: millimeter\u003c/p\u003e\n\u003cp\u003eMS: Murashige and Skoog\u003c/p\u003e\n\u003cp\u003emg: milligram\u003c/p\u003e\n\u003cp\u003ecm: centimeter\u003c/p\u003e\n\u003cp\u003e°C: degrees Celsius\u003c/p\u003e\n\u003cp\u003eml: milliliter\u003c/p\u003e\n\u003cp\u003eCTAB: Cetyltrimethylammonium bromide\u003c/p\u003e\n\u003cp\u003ex g: times gravity\u003c/p\u003e\n\u003cp\u003eµl: microliter\u003c/p\u003e\n\u003cp\u003eV: Volt\u003c/p\u003e\n\u003cp\u003ekb: kilobase\u003c/p\u003e\n\u003cp\u003eng: nanogram\u003c/p\u003e\n\u003cp\u003emin: minute\u003c/p\u003e\n\u003cp\u003eh: hour\u003c/p\u003e\n\u003cp\u003ev/v: volume per volume\u003c/p\u003e\n\u003cp\u003ew/v: weight per volume\u003c/p\u003e\n\u003cp\u003emM: millimolar\u003c/p\u003e\n\u003cp\u003eSDS: Sodium Dodecyl Sulfate\u003c/p\u003e\n\u003cp\u003eDTT: Dithiothreitol\u003c/p\u003e\n\u003cp\u003eHCl: hydrochloric acid\u003c/p\u003e\n\u003cp\u003ePAGE: Polyacrylamide Gel Electrophoresis\u003c/p\u003e\n\u003cp\u003eRUBISCO: Ribulose-1,5-bisphosphate carboxylase/oxygenase\u003c/p\u003e\n\u003cp\u003eCol-0: Columbia-0\u003c/p\u003e\n\u003cp\u003eMgCl\u003csub\u003e2\u003c/sub\u003e:Magnesium chloride\u003c/p\u003e\n\u003cp\u003eOD\u003csub\u003e600\u003c/sub\u003e:Optical Density at 600 nanometers\u003c/p\u003e\n\u003cp\u003eRPM: Revolutions Per Minute\u003c/p\u003e\n\u003cp\u003eHz: hertz\u003c/p\u003e\n\u003cp\u003eµmol: micromole\u003c/p\u003e\n\u003cp\u003es: second\u003c/p\u003e\n\u003cp\u003em: meter\u003c/p\u003e\n\u003cp\u003eCFU: Colony Forming Unit\u003c/p\u003e\n\u003cp\u003eM: molar\u003c/p\u003e\n\u003cp\u003ePCR: Polymerase Chain Reaction\u003c/p\u003e\n\u003cp\u003eq-PCR: quantitative Polymerase Chain Reaction\u003c/p\u003e\n\u003cp\u003eRT-PCR: Reverse Transcription Polymerase Chain Reaction\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors give consent to the publication of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data supporting the findings of this study are available within the paper and its Supplementary Information.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAgencia Nacional de Promoción de la Investigación, el Desarrollo Tecnológico y la Innovación. PICT grant to Martiniano María Ricardi.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eR.M.G. performed DNA extractions, optimized grinding conditions, and contributed to the figures and the writing and proofreading of the manuscript.\u003c/p\u003e\n\u003cp\u003eC.A. performed RNA extractions from seedlings, leaves, and stems.\u003c/p\u003e\n\u003cp\u003eF.E.A. performed RNA extractions from seedlings, leaves, and stems, conducted all experiments involving \u003cem\u003ePseudomonas syringae\u003c/em\u003e and contributed with the writing of the manuscript.\u003c/p\u003e\n\u003cp\u003eC.C. performed protein extraction and SDS-PAGE electrophoresis.\u003c/p\u003e\n\u003cp\u003eF.S.R. and R.S.T performed RNA extractions from imbibed seeds and contributed with the writing of the manuscript.\u003c/p\u003e\n\u003cp\u003eF.C. and F.R. conducted prototype testing for the Fast-Lyzer, evaluating early versions through to the final iteration.\u003c/p\u003e\n\u003cp\u003eS.L. developed the RPM meter and evaluated device performance under sample-load conditions.\u003c/p\u003e\n\u003cp\u003eE.P. contributed to and supervised all RNA experiments.\u003c/p\u003e\n\u003cp\u003eM.M.R. conceived the project, designed the device, produced the 3D prints and the metal holder, and contributed to the figures and the writing of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll authors read and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors wish to thank Dr. Nicolás Cecchini for providing \u003cem\u003ePseudomonas syringae\u003c/em\u003e pvar. tomato DC3000 for the assays with bacteria. We also wish to thank the interns Tyara Alburquenque and Ignacio del Valle for contributing with plant growth, agarose gel electrophoresis and SDS-PAGE running.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGoldberg S. Mechanical/physical methods of cell disruption and tissue homogenization. Methods Mol Biol [Internet]. 2008;424:3\u0026ndash;22. https://doi.org/10.1007/978-1-60327-064-9_1\u003c/li\u003e\n\u003cli\u003eAlsenz J, Haenel E. Precellys(R) Evolution Homogenizer - a versatile instrument for milling, mixing, and amorphization of drugs in preformulation. Eur J Pharm Biopharm [Internet]. 2023;189:1\u0026ndash;14. https://doi.org/10.1016/j.ejpb.2023.05.002\u003c/li\u003e\n\u003cli\u003eJones AM, Van de Wyngaerde MT, Machtinger ET, Rajotte EG, Baker TC. Choice of Laboratory Tissue Homogenizers Matters When Recovering Nucleic Acid From Medically Important Ticks. 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Short Communication An efficient method for simultaneous extraction of high-quality RNA and DNA from various plant tissues. Genet Mol Res [Internet]. 2015;14:18828\u0026ndash;38. https://doi.org/10.4238/2015.December.28.32\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-methods","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plme","sideBox":"Learn more about [Plant Methods](http://plantmethods.biomedcentral.com/)","snPcode":"13007","submissionUrl":"https://submission.nature.com/new-submission/13007/3","title":"Plant Methods","twitterHandle":"@PlantMethods","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Tissue disrupter, 3D printing, Jigsaw, Nucleic Acid Extraction, Protein Extraction, Seedlings, Leaves, Stems, Seeds, Bacterial Extraction","lastPublishedDoi":"10.21203/rs.3.rs-9440300/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9440300/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground:\u003c/p\u003e\n\u003cp\u003eSample disruption and homogenization are critical steps in numerous biological extraction procedures (i.e., DNA, RNA, or protein isolation). These steps are often performed manually which can yield highly variable results depending on operator expertise. Several companies offer dedicated tissue-processing machines that enable high reproducibility and high-throughput sample preparation using various bead types and shaking mechanisms. However, the high cost of this equipment makes it inaccessible to many laboratories worldwide. To address this limitation, we developed an open‑source, 3D‑printable adaptor that enables the use of a low‑cost wood jigsaw as the source of mechanical agitation.\u003c/p\u003e\n\u003cp\u003eResults:\u003c/p\u003e\n\u003cp\u003eWe validated our device, Fast-lyzer, by extracting DNA, RNA, and proteins from different plant tissues, obtaining results comparable — if not superior — to traditional, time‑consuming manual grinding. We also demonstrated the effectiveness of the Fast-lyzer and bead‑based disruption for extracting live bacteria from plant tissues for leaf‑colonization assays.\u003c/p\u003e\n\u003cp\u003eConclusion:\u003c/p\u003e\n\u003cp\u003eFast-lyzer provides an open‑source, low‑cost solution that allows resource‑limited laboratories to access grinding and homogenization methods typically reliant on equipment costing thousands of dollars. The estimated cost of our system is approximately 200 USD, representing at least a 100‑fold reduction in price. More importantly, the Fast-lyzer speeds up sample grinding process by 8-fold if used for batch processing in comparison to manual procedure. Additionally, we optimized extraction conditions for several \u003cem\u003eArabidopsis thaliana \u003c/em\u003etissues and for bacterial cell counts to facilitate adoption by the research community.\u003c/p\u003e","manuscriptTitle":"“Fast-lyzer”: A low-cost 3D printable device for plant tissue disruption and homogenization","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-07 10:04:17","doi":"10.21203/rs.3.rs-9440300/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-08T22:59:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"172189086507107696576438187487438826908","date":"2026-05-08T20:58:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"3347641170595272022214900574424963334","date":"2026-05-04T16:46:14+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-29T08:02:33+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-21T03:12:01+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-21T03:11:40+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Methods","date":"2026-04-16T15:45:39+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-methods","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plme","sideBox":"Learn more about [Plant Methods](http://plantmethods.biomedcentral.com/)","snPcode":"13007","submissionUrl":"https://submission.nature.com/new-submission/13007/3","title":"Plant Methods","twitterHandle":"@PlantMethods","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4d25032c-ef22-4ead-a8be-8dec3e094a1f","owner":[],"postedDate":"May 7th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-08T22:59:47+00:00","index":33,"fulltext":""},{"type":"reviewerAgreed","content":"172189086507107696576438187487438826908","date":"2026-05-08T20:58:49+00:00","index":32,"fulltext":""},{"type":"reviewerAgreed","content":"3347641170595272022214900574424963334","date":"2026-05-04T16:46:14+00:00","index":22,"fulltext":""},{"type":"reviewersInvited","content":"16","date":"2026-04-29T08:02:33+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-07T10:04:17+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-07 10:04:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9440300","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9440300","identity":"rs-9440300","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}