Plant materials and growth conditions
Delinted cotton (Gossypium hirsutum cv. ND601) seeds were surface-sterilized in 75% ethanol for 10 min followed by 3% sodium hypochlorite treatment for 10 min and rinsed five times in sterile water. After soaking the seeds in sterile water for 7 h, they were placed between two layers of wet towels to retain moisture and air, and then placed in a 25 °C incubator for about 36 h. When the length of the radicle root reached about 1 cm, seeds showing normal germination were transferred to a hydroponic container for culture at 28/25 °C (day/night), with a 16 h/8 h (day/night) photoperiod (600 μmol·m−2·s−1).
Isolation of protoplasts from cotton taproots
Protoplasts were isolated following the methods of Yoo , Li , and Wu  with modifications as shown below.
Taproots of cotton plants grown in hydroponics for 72 h after germination were used to isolate protoplasts.
Note: The timing of hydroponics is critical; we found that taproots after 65–75 h of hydroponic culture were suitable for protoplast isolation, whereas hydroponic culture for < 48 h resulted in increased tissue fragments in the cell suspension. However, if the time exceeded 96 h, the cell harvest rate was significantly reduced.
Tapoots from 25–50 seedlings were cut into 0.5–1-mm slices and dipped in 10 ml of enzyme solution in a 50 ml conical flask.
Note 1: The enzyme solution was freshly prepared. It contained 1.5% (w/v) Cellulase R10 (Yakult, Tokyo, Japan), 0.75% (w/v) Macerozyme R10 (Yakult), 0.4 M mannitol, 20 mM KCl, and 20 mM MES (pH 5.7). Once prepared, the solution was warmed at 55 °C for 10 min. Upon cooling to room temperature, 10 mM CaCl2 and 0.1% bovine serum albumin were added, and the solution was filtered with a 0.45 μm Millipore filter (MilliporeSigma, Burlington, MA, USA).
Note 2: The slices must be thoroughly immersed in the enzyme solution. Sample cutting can be performed on plastic cultures or seed germination pouches (CYG-38LB; PhytoTC, Shanghai, China). In our experience, cutting 5–7 roots together is efficient and does not affect the isolation process. Slice the sample using a Gillette razor blade (Boston, MA, USA) from one side to the other. The cutting speed should not be overly rapid. The thickness is appropriate when the slice is translucent. It is necessary to switch the blade during slicing. Normally, for 25 roots you will use two blades.
Samples were incubated for 3 h in the enzyme solution with shaking on a shaker at a speed of 40–50 rpm at 25 °C in the dark.
Note: The number of protoplasts released can be estimated based on the turbidity of the enzyme solution; however, microscopic examination is the most accurate method.
An equal volume of W5 solution [154 mM NaCl, 125 mM CaCl2, 5 mM KCl, and 2 mM MES (pH 5.7)] was added to the enzyme mixture, which was shaken vigorously for 10 s to release the protoplasts and then filtered using four layers of Miracloth (MilliporeSigma).
Note: To maximize the protoplast yield, gently transfer the tissue residue back to the conical flask and add 10 ml of W5 solution. Incubate the mixture for 1 h on a shaker at a speed of 40–50 rpm at 25 °C in the dark, and then proceed to step (4).
The filtrate was filtered into a 50 ml centrifuge tube using a 40 μm cell strainer.
Note: In our experience, the range of cell sizes in root tissue is relatively large. Due to technical limitations, the cell size for scRNA-seq cannot be > 40 μm. In some cases, it is necessary to use a 30 μm cell strainer. The strainer should be moistened with W5 solution before use. We suggest using a round-bottomed tube throughout the experiment.
The mixture was centrifuged horizontally at 25 °C at 100 g for 5 min to pellet the protoplasts. The supernatant was discarded gently without disturbing the pellet.
Note: We recommend setting the centrifuge’s accelerate and decelerate controls to 1 or using the soft key. A centrifuge with a swinging-bucket rotor is well-suited for protoplast collection.
The protoplasts were resuspended in 5 ml of pre-chilled W5 solution. The suspension was kept on ice for 30–60 min.
Note: If the protoplasts are to be used for scRNA-seq, the cell suspension should not contain MgCl2 or CaCl2. Therefore, the protoplasts must be resuspended in 5 ml of pre-chilled 0.5 M mannitol.
The supernatant was carefully removed without touching the protoplast pellet. The protoplasts were then resuspended to a final concentration of 1 × 106 with MMG solution (4 mM MES, 0.4 M mannitol, and 15 mM MgCl2). The viability was determined using 0.01% (w/v) FDA staining .
Note: If the protoplasts are to be used for scRNA-seq, they should be resuspended in 0.5 M mannitol. If LSCs (≥ 40 um) are observed by microscopic examination, a 30 μm cell strainer should be used for filtration in step (5). All pipette tips used for protoplast isolation should be cut with scissors as the protoplasts are extremely fragile.
Vector construction and plasmid preparation
We used the CRISPR/Cas9 system as described previously . One reported active target site  was used to test the protoplast transient expression system. Two sites targeting PDS and CLA were also designed, respectively. Pairs of oligonucleotides including the targeting sequences (Additional file 1) were synthesized, annealed, and cloned into BsaI-digested pKSE401 . The targeting vectors were verified by sequencing and extracted using a Fastpure DNA Isolation Mini Kit (Vazyme Biotech, Shanghai, China). The 35S-GFP sequence, amplified from pBI221-CaMV35S-GFP , was cloned into pEASY-Blunt (TransGen Biotech, Beijing, China) to obtain pEASY-35S:GFP. pUC18-Man49-mCherry was used for protein subcellular localization analysis [29, 30]. For bimolecular fluorescence complementation (BiFC) assays, the coding sequences of GhBIN2 and GhBZR3 were cloned into the BiFC expression vectors p326-YFPN and p326-YFPC , respectively, to obtain p326-GhBIN2-YFPN and p326-GhBZR3-YFPC. The plasmids were extracted using a commercial kit (Wizard® Plus Midipreps DNA Purification System; Promega Biotech, Beijing, China) to yield quality DNAs (> 1,000 ng/μl).
PEG-mediated protoplast transfection
PEG-mediated protoplast transfection was carried out as described previously with modifications . For each transformation, 20 μl of DNA (10–20 μg of plasmid) were gently mixed with 200 μl of protoplasts in a 2-ml microfuge tube. Then, 220 μl of freshly prepared PEG solution [40% (w/v) PEG4000, 200 mM mannitol, and 50–300 mM CaCl2] were added and mixed completely by gently tapping the tube. The transfection mixture was incubated at 25 °C in the dark for different time periods (5, 10, 15, 20, or 25 min). It was stopped by adding 880 μl of W5 solution and mixed well by gently inverting the tube. After centrifugation at 100 g for 3 min, the supernatant was removed and the protoplasts were resuspended in 1.5 ml of W5 solution and then transferred to a 2 ml tube. For transient expression of the genome editing reagents or proteins (genes), transfected protoplasts were incubated for 12–48 h at 25 °C in darkness. Based on the experimental purpose, the materials used in the transfection system may be scaled up or down.
Verification of CRISPR/Cas9-mediated mutations in cotton protoplasts
A PCR/restriction enzyme (RE) assay was used to assess the activity of the targeting vectors. Genomic DNA was extracted from pooled protoplasts transformed with the targeting vectors, and the sequence (~ 700 bp) encompassing the CRISPR target site was amplified by PCR using a high-fidelity DNA polymerase. The amplicon was then digested with the appropriate RE and analyzed by gel electrophoresis. Mutations induced by non-homologous end joining (NHEJ) were resistant to RE digestion, resulting in an uncleaved band. Uncleaved bands were purified and then cloned into the cloning vector T-Blunt. The resulting transformants were identified by colony PCR. Subsequently, positive clones were sequenced using T7 primer to obtain the mutated sequences.
RNA extraction and gene expression analysis
Total RNA was isolated from roots tissue or protoplasts using TransZol reagent (TransGen Biotech); genomic DNA contamination was eliminated with DNase I (Roche). M-MLV reverse transcriptase (Thermo Scientific) was used for first-strand cDNA synthesis. Real-time PCR was conducted in technical triplicates using SYBR Green PCR master mix (DBI Bioscience). GhACTIN14 was used as the reference gene. Three to four biological replicates were performed; the results were analyzed with SPSS statistics 17.0 (IBM).
The protoplast yield was determined using a standard hemocytometer and light microscopy. The plasmid pEASY-35S:GFP was used to calculate the transformation efficiency of protoplasts and assess protein subcellular localization. GFP fluorescence was observed under a Nikon Ti-2U Fluorescence Microscope (Nikon Corp., Tokyo, Japan). The transformation efficiency (%) was calculated by dividing the protoplast number with bright green fluorescence by the total protoplast number. Images were acquired with an Olympus FV10i Confocal Microscope (Olympus Corp., Tokyo, Japan) using excitation wavelengths of 488 nm (GFP and YFP) and 561 nm (mCherry).