Protoplasts

Leaves (width: 2 cm, length: 5 cm in optimal light condition; width: 0.5 cm; length: 2.5 cm in low light conditions) were collected from 3 to 5-week-old plants grown under optimal light (ca. 150 μE·m^-2·s^-1) or low light (ca. 50·μE m^-2·s^-1) conditions. Arabidopsis protoplasts were isolated in two ways. First, to recreate the current technique, protoplasts were made according to the procedure of Yoo et al. [4 (link)]. Second, in a new technique, selected leaves were used in a 'Tape-Arabidopsis Sandwich' experiment. The upper epidermal surface was stabilized by affixing a strip of Time tape (Time Med, Burr Ridge, IL) while the lower epidermal surface was affixed to a strip of Magic tape (3 M, St. Paul, MN). The Magic tape was then carefully pulled away from the Time tape, peeling away the lower epidermal surface cell layer. The peeled leaves (7 to 10 optimal-light-growth leaves, about 1-2 g, up to 5 g), still adhering to the Time tape, were transferred to a Petri dish containing 10 mL of enzyme solution [1% cellulase 'Onozuka' R10 (Yakult, Tokyo, Japan), 0.25% macerozyme 'Onozuka' R10 (Yakult), 0.4 M mannitol, 10 mM CaCl₂, 20 mM KCl, 0.1% BSA and 20 mM MES, pH 5.7]. The leaves were gently shaken (40 rpm on a platform shaker) in light for 20 to 60 min until the protoplasts were released into the solution. The protoplasts were centrifuged at 100 × g for 3 min in an Eppendorff A-4-44 rotor (Hamburg, Germany), washed twice with 25 mL of pre-chilled modified W5 solution (154 mM NaCl, 125 mM CaCl₂, 5 mM KCl, 5 mM glucose, and 2 mM MES, pH 5.7) and incubated on ice for 30 min. During the incubation period, protoplasts were counted using a hemocytometer under a light microscope. The protoplasts were then centrifuged and resuspended in modified MMg solution (0.4 M mannitol, 15 mM MgCl2, and 4 mM MES, pH 5.7) to a final concentration of 2 to 5 × 10⁵ cells/mL.

Dehulled seeds of rice (Oryza sativa L.) cultivar Nipponbare were sterilized with 75% ethanol for 1 min. These seeds were further sterilized with 2.5% sodium hypochlorite for 20 min, washed at least five times with sterile water and then incubated on 1/2 MS medium with a photoperiod of 12 h light (about 150 μmol m^-2 s^-1) and 12 h dark at 26°C for 7-10 days. Green tissues from the stem and sheath of 40-60 rice seedlings were used. A bundle of rice plants (about 30 seedlings) were cut together into approximately 0.5 mm strips with propulsive force using sharp razors. The strips were immediately transferred into 0.6 M mannitol for 10 min in the dark. After discarding the mannitol, the strips were incubated in an enzyme solution (1.5% Cellulase RS, 0.75% Macerozyme R-10, 0.6 M mannitol, 10 mM MES at pH 5.7, 10 mM CaCl₂ and 0.1% BSA) for 4-5 h in the dark with gentle shaking (60-80 rpm). After the enzymatic digestion, an equal volume of W5 solution (154 mM NaCl, 125 mM CaCl₂, 5 mM KCl and 2 mM MES at pH 5.7) was added, followed by vigorous shaking by hand for 10 sec. Protoplasts were released by filtering through 40 μm nylon meshes into round bottom tubes with 3-5 washes of the strips using W5 solution. The pellets were collected by centrifugation at 1,500 rpm for 3 min with a swinging bucket. After washing once with W5 solution, the pellets were then resuspended in MMG solution (0.4 M mannitol, 15 mM MgCl₂ and 4 mM MES at pH 5.7) at a concentration of 2 × 10⁶ cells mL^-1, determined by using a hematocytometer. The viability of protoplasts was determined by the FDA staining method as described [44 (link)]. All manipulations above were performed at room temperature.
For isolating protoplasts from etiolated rice seedlings, the sterilized seeds were germinated under light for 3 days, and then moved to the dark for another 4-7 days. The isolation procedure was the same as that for isolation of green tissue protoplasts described above.

The full-length coding DNA sequences (CDS) of RCC1-3A (TraesCS3A02G362800), RCC1-3B (TraesCS3B02G395200), RCC1-3D (TraesCS3D02G356500), Myb-7B (TraesCS7B02G188000), and Myb-7D (TraesCS7D02G295400) were inserted into pCambia1300-35S-GFP, creating RCC1-3A::GFP, RCC1-3B::GFP, RCC1-3D::GFP, Myb-7B::GFP, and Myb-7D::GFP fusion vectors. The recombinant plasmids were mixed with the nuclear marker NLS-mCherry and transfected into wheat mesophyll protoplasts as previously described by Yoo et al. (2007) (link). The transfection mixture was induced by PEG-Ca²⁺, and the protoplasts were cultured for 12 h at 25°C. The protoplasts were observed and photographed with a fluorescence microscope (Zeiss Imager A2, Germany).

GmHMGR4 and GmHMGR6 cDNA sequences were cloned into a pC1300s vector with the CaMV 35S promoter and eGFP (enhanced green fluorescent protein). The pC1300S-35S:GmHMGR4-GFP and pC1300S-E.R-mCherry-HDEL (plant endoplasmic reticulum markers) were transfected into A. thaliana mesophyll protoplasts, pC1300S-35S:GmHMGR6-GFP and pC1300S-E.R-mCherry-HDEL also go through the same transfection. On the second day following transfection, confocal laser scanning microscopy (OLYMPUS FV 1200) was used to detect the fluorescence from fusion proteins and organelle markers.

Ppddm1 was obtained through the replacement of the gene coding region with Zeocin resistance antibiotic cassette through homologous recombination. Homologous flanking 5’ (1068bp upstream to ATG site) and 3’ (252 bp upstream to stop site and 536bp downstream to stop site) were amplified using KOD polymerase (Sigma-Aldrich) and cloned into a PMBL5-Zeo vector. Integration of flanking regions was validated using PCR. Linearized plasmid was introduced to moss via PEG-mediated transformation protocol [43 (link)]. After three days, the regenerated protoplasts were transferred to the BCDAT medium. After seven days the moss was subjected to selection using Zeocin (25ng\μl). After three weeks, surviving plants were subjected to PCR to validate the correct integration of the construct into the genome and gene replacement. This was done through primers targeting the endogenous gene sequence and primers targeting regions flanking the 5’ and 3’ homologous regions and Zeocin resistance cassette.