Sucrose

1,2-Dioleoyl-sn-glycerol-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt) (red; Rh–PE) lipids in chloroform were obtained from Avanti Polar Lipids (Alabaster, AL). Rh–PE has excitation/emission wavelengths of 560/583 nm. Lauric acid (LA) was obtained from Sigma-Aldrich (St. Louis, MO). The aqueous buffer used in all experiments was 10 mM tris(hydroxymethyl)aminomethane (Tris) buffer containing 150 mM NaCl (pH 7.5), prepared using Milli-Q water (Millipore Sigma, Burlington, MA). Sucrose was obtained from Affymetrix (Cleveland, OH), and Sucrose solutions were prepared by dissolving defined concentrations of Sucrose in Tris buffer.

The FT stress assay was performed to investigate the impact of freezing and thawing on the survivability of the cells post-harvest. At the end of each fermentation, the culture was harvested by centrifugation (Eppendorf 5810R, Germany) at 3000×g for 10 min. The supernatant was carefully discarded and the cells were concentrated 20-fold by resuspending the cell pellet in phosphate buffer saline (PBS) (Merck, Germany). An equivalent volume of PBS containing 20% (w/v) sucrose (Acros organics, Belgium) was added, thus reaching a final sucrose concentration of 10%. Cell samples were frozen at − 80 °C for 5–10 days, and carefully thawed under controlled conditions by incubating the sample vials in a water bath at room temperature for 15 min. Cells were subsequently used directly for further analysis by flow cytometry.

Embryos/larvae were fixed in 4% paraformaldehyde (Electron Microscopy Sciences) for minimum of 1 h at room temperature (RT). For each time point, a small piece of caudal tissue was excised for genotyping and the remaining rostral tissue was embedded in 1.5% agarose (Fisher) produced in 5% sucrose (Fisher) and embryo medium. Embedded blocks were incubated overnight in 30% sucrose (Fisher) and then snap frozen with dry ice and cryosectioned (12–20 μM). Sections were washed twice in 1× phosphate buffered saline (PBS) pH 7.4 at RT for 30 min each and blocked for 1 h in blocking buffer (2 mg/ml bovine serum albumin (Fisher), 2% goat serum (Fisher) diluted in 1× PBS). Primary antibody [1:200 anti-Sox2 (Abcam) or 1:500 anti-HuC/D (Fisher)] was incubated overnight at 4 °C and then washed twice in 1× PBS for 30 min each at RT. Alexa fluor antibodies (Fisher) were diluted 1:200 and incubated on each slide for 1 h at RT. All slides were cover slipped using Vectashield (Vector Laboratories) and imaged on a Zeiss LSM 700 at 20×–63× magnification. For cell proliferation, larvae were pulsed in 20 mM 5-ethynyl-2′-deoxyuridine (EdU) (Fisher) diluted in 10% dimethyl sulfoxide (DMSO) (Fisher) for 30 min at RT prior to fixation. EdU was detected using the EdU Click-It technology (Fisher) according to manufacturer protocol.

Seeds were washed with a solution of 0.05% Triton and 70% Ethanol then stratified for 4 days at 4°C. For the assay on solid plates, seeds were placed in sterile 0.1% agar solution and pipetted onto solid Murashige Skoog (MS) growth medium (Phytotechnology Lab, Overland Park, KS, USA) with 1.2% sucrose (Fisher) and 50 mg/L ampicillin (Fisher) and allowed to grow for 5 days at 22°C under a 16 h light/8 h dark (for the initial dose-response assay) or 12 h light/12 h dark (remaining experiments) photoperiod. 15 seeds were transferred to solid MS medium containing either 0.15 or 0.3 μg/mL Tm, (Sigma-Aldrich T7765) and allowed to grow for 3 days at 22°C. The treated seeds were then transferred back to solid MS medium plates and allowed to grow for 3 days before collection for chlorophyll analysis. For the assay in liquid media, seeds were placed in sterile 0.1% agar solution and 15–20 seeds were pipetted into each well of a 12-well polystyrene plate (Fisher) containing 4 mL 0.5 × MS medium with 0.6% sucrose and ampicillin and grown for 5 days at 22°C. The MS medium was removed and replaced with fresh MS media or media containing either 0.15 or 0.3 μg/mL Tm and allowed to grow for 3 days at 22°C. The medium was again replaced with fresh MS medium and plants were allowed to grow for 3 days before collection for chlorophyll analysis. The experimental design is outlined in Figure 1.

The pBluescript RN3 plasmid containing the full-length coding sequence of human PLCζ (generous gift from Dr. Rafael Fissore, University of Massachusetts-Amherst) was linearized at a site beyond the 3′ end of the coding sequence, and in vitro transcribed using the T3 mMessage mMachine kit (Ambion, Austin, TX). mRNA was purified according to manufacturer’s instructions. Prior to microinjection, concentrated cRNA (2 mg/ml) was heated for 3 min at 85 °C to reduce secondary structure and diluted as needed in nuclease free water. hPLCζ cRNA was microinjected in the cytoplasm of human eggs final concentrations of 0.05 mg/ml and 0.2 mg/ml. For microinjections, human eggs were first incubated in 1μM Fluo4-AM and 0.02% Pluronic F-127 in SAGE oocyte washing medium (Origio) for 30 minutes at 37 °C. Eggs were then microinjected with hPLCζ cRNA. For microinjection, human MII eggs were transferred to drops of L-15 medium containing 0.05% (w/v) polyvinyl alcohol and 0.1% (w/v) sucrose (Invitrogen) under light mineral oil on a heated stage set to 37 °C. hPLCζ cRNA was back-loaded into glass micropipettes and delivered by a XenoWorks Digital Microinjector (Sutter Instrument, Novato, CA). The injection volume was ~28–48 pl (1–3% of the total volume of the egg). The eggs were then washed through dye-free medium and placed in 50 μM FluoZin-3 in hCZB as described above.