Introduction

Protoplasts, which are plant cells devoid of cell walls, have a unique capability to efficiently uptake various macromolecules (such as DNA, RNA, and proteins). This ability makes them highly suitable for transient gene expression analysis, providing valuable insights into gene expression dynamics and regulatory mechanisms in plant biology (Shen et al. 2017; Xu et al. 2020). In addition, protoplast transfection using polyethylene glycol (PEG) is a simple and cost-effective method for studying gene function, subcellular localization, and protein–protein interaction in various plant species (Yu et al. 4 and supplementary Fig. 1), and the tube was incubated on a benchtop for 15–30 min to facilitate the accumulation of more protoplasts. Using a 200 µL pipette with cut tips (see supplementary note 4), a band of protoplasts was transferred from the interface to a new round-bottom tube (typically ~ 1 mL of protoplasts) and checked under the microscope using a Hemocytometer. The visible concentrated round protoplasts at this step were further washed by adding 5 mL of W5 buffer using centrifugation at 100 g for 5 min. After that, the supernatant was carefully removed leaving 0.25 mL, and further counted using a hemocytometer. Finally, MMG buffer (Supplementary Table S4) was added to obtain a titer of 2 × 106 protoplasts per mL. A detailed protocol is given in supplementary file.

Protoplast isolation and density evaluation from Arabidopsis and Chickpea

Protoplast isolation from Arabidopsis and chickpea leaves was performed as detailed below. Three-week-old leaves of Arabidopsis (15 leaves) and chickpea (30 leaves) were chopped with single-edge razor blades into 0.5–1 mm strips. The strips were transferred in a sterile 100 mL conical flask containing 10 mL of 0.6 M mannitol and kept in the dark for 10 min. Mannitol was removed, and 10 mL protoplast isolation buffer was added to the conical flask. The conical flask was incubated for 5 h at 25 °C in the dark with gentle shaking on an orbital shaker at 40 rpm with vacuum infiltration. The following steps are similar to those described for rice. Composition of different solutions, buffers and reagent set up are given in the supplementary file.

Protoplast viability check

The viability of protoplasts from rice, Arabidopsis, and chickpea was evaluated separately by using two different dyes. Firstly, 1% (w/v) Evans Blue staining was performed following a previously described protocol (Gaff and Okong’o-ogola 1971). Secondly, 0.01% of fluorescein diacetate (FDA) was used to stain protoplasts (Williams and Co 1972). Briefly, 5 µL of 1% Evans Blue was added to 20 µL of the protoplast solution. Evans Blue-stained protoplasts were visualized under a bright field using a Leica DMi8 fluorescence microscope with N.A. 1.25 (Leica Microsystems Inc., Buffalo Grove, IL). LAS X software was utilized for image acquisition and analysis. The protoplast viability was also assessed by adding 1 µL of 0.01% of FDA dissolved in acetone to 20 µL of protoplasts. The samples were visualized using a Leica DMi8 fluorescence microscope with a 480–510 nm excitation filter and a 535–585 nm emission filter. Data were recorded for five consecutive fields along with their corresponding bright field images (Grey). The green fluorescence was observed using a FITC filter excited at 488 nm with emission at 505–525 nm. The percentage of viable and non-viable protoplasts was calculated by dividing the number of unstained and stained cells by the total number of cells visible in each microscopic field.

PEG-mediated protoplasts transfection for rice, Arabidopsis and chickpea

The isolated protoplast solution was diluted with MMG buffer to obtain a working stock of 2 × 106/mL for transfection. 200 µL rice protoplast solution was transferred into 2 mL round-bottom microcentrifuge tubes. 30 µL of plasmids at a concentration of 1000 ng/µL were added and mixed gently by tap**. Different amounts of total DNA were used to standardize the optimum concentration and different sets were prepared for pRGE32-GFP, pRGE32-BFP, and pRGEB32-GFP reporter plasmids, and pRGE32-SWEET14-GG, pRGE32-SD1-GG, and pRGE32-Gn1-GG plasmids with three biological replicates. Plasmids were prepared using a Qiagen Plasmid plus midi kit (Qiagen, Cat. No. 12943). The protoplast and plasmid mixtures were incubated at room temperature for 10 min. Subsequently, 230 µL of freshly prepared PEG-calcium chloride transfection buffer (see solution composition, Supplementary Table S5) was added to each tube wall in a dropwise manner, gently mixed by inversion, and incubated for 20 min at room temperature.

Meanwhile, a 12-well culture plate (NEST®, Cat. No. 712001) was made ready by adding 80 µL of 5% calf serum (Gibco, Cat. No. 16170–086) in each well and spreading it using a 100 µL pipette throughout the inner wall of each well. Then, 1 mL of W5 buffer was added to each well. After the 20-min incubation, 900 µL of W5 buffer was added to each 2 mL round bottom tube, and the solution was gently mixed by inverting four times. The tubes were centrifuged at 100 g for 5 min to pellet the protoplasts using a swing bucket rotor. Then, half of the supernatant (680 µL) was carefully removed using a 1 mL pipette, and the remaining solution was mixed by gentle inversion for 4 times. The protoplast mixture was transferred to the 12 well culture plates. The plates were sealed with parafilm, wrapped in aluminum foil, and incubated at 32 °C in the dark for 72 h with gentle shaking at 25 rpm. The transfection procedure for Arabidopsis and chickpea was similar to that used for rice with two modifications: (1) the use of a higher plasmid concentration, 45 µg, and (2) a shorter 15-min incubation after mixing the protoplasts with the plasmid DNA. The protoplasts isolated from Arabidopsis were transfected with pRGEB31-GAT, eCaMV-GFP, and AtUbi10-GFP plasmids. Similarly, chickpea protoplasts were transfected with the constructs viz. pRGEB31-LCY, pRGEB31-M41, and eCaMV-GFP.

GFP and BFP detection using fluorescent microscopy

Green and blue fluorescent signals were seen after 16–18 h post-transfection and gained peak at 48 h. GFP and BFP fluorescence was visualized using a Leica DMi8 fluorescence microscope (Leica Microsystems Inc., Buffalo Grove, IL) from five consecutive fields as well as their corresponding bright field images (Grey) from each replicate. GFP fluorescence was excited at a 488 nm filter and detected using a 505–525 nm emission filter. In the case of BFP, a 351–363 nm was used for excitation, and emission was recorded using a 460 nm filter.

Storage of protoplasts for including a breakpoint

Rice protoplasts obtained from the sucrose gradient were preserved using MMG solution in a sealed 14 mL centrifuge tube (Thermo Scientific, Cat. No. 150268). The tube was covered with Parafilm and enveloped in aluminium foil for storage at room temperature (RT, 25 °C) and on ice. At intervals of 24 h (1, 24, 48 h), protoplasts underwent viability testing and transfection with a GFP-containing vector using PEG-CaCl2-mediated transfection, as previously described. Viability was assessed through FDA staining, and transfection efficiency was determined by counting GFP-positive protoplasts.

Validation of CRISPR-Cas9 vectors in rice, Arabidopsis, and chickpea protoplasts with a CRISPR deletion approach

Genomic DNA was extracted from the transfected protoplasts after three days. Target regions were amplified using high-fidelity Q5 DNA polymerase (New England Biolabs, USA). CRISPR deletions were visualized via agarose gel electrophoresis of PCR products. PCR amplicons were purified, cloned into a TA vector and then transformed in E. coli. The transformants were screened by colony PCR using M13F and M13R and also with gene-specific primer sets described earlier. The positive clones were subjected to CAPS assay and Sanger sequenced for verification.