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Abstract Polyethylene glycol calcium (PEG-Ca 2+) transfection-mediated analysis allows rapid and efficient examination of gene function. To investigate the diverse cellular functions of genes of interest in plant cells, macromolecules, such as DNA, RNA, and proteins, are delivered into protoplasts prepared from somatic tissues or calli using a PEG-Ca 2+ transfection procedure. To take advantage of this macromolecule delivery system in the reproductive and developmental biology of angiosperms, this study established a PEG-Ca 2+ transfection system with isolated egg cells and zygotes.

The conditions for PEG and plasmid DNA concentrations for transfection of rice egg cells were first addressed, and ~30% of PEG-Ca 2+-transfected egg cells showed exogenous and transient expressions of fluorescent proteins from plasmid DNA delivered into the cells. Interestingly, a dual expression of two different fluorescent proteins in the same egg cell using two kinds of plasmid DNAs was also observed. For PEG-Ca 2+ transfection with maize zygotes, ~80% of zygotes showed expression of GFP proteins from plasmid DNA. Importantly, PEG-transfected zygotes developed normally into cell masses and mature plants. These results suggest that the present PEG-Ca 2+-mediated transient expression system provides a novel and effective platform for expressing and analyzing genes of interest in egg cells and zygotes. Moreover, combined with the CRISPR/Cas9 approach, the present transient expression system in zygotes will become a powerful and alternative tool for the preparation of gene-edited plants.

• • Effect of PEG concentration on expression level of SP-GFP-HDEL in rice egg cell (a–c) and maize egg cell (d). (a–c), Rice egg cells were placed in a droplet of MMG solution containing pGFP-ER on a coverslip, and a droplet of the same volume of PEG solution containing 20% (a) 30% (b) or 40% (c) PEG and 100 mM CaCl 2 was merged with the MMG droplet holding the egg cell. After washing and 19-hr culture of the cells, fluorescent signals in egg cells were observed. After washing and 19-hr culture of the cells, fluorescent signals in egg cells were observed. (d), Maize egg cells were placed in a droplet of MMG solution containing pGFP-ER on a coverslip, and a droplet of the same volume of PEG solution containing 30% PEG and 100 mM CaCl 2 was merged with the MMG droplet holding the egg cell.

Left and right panels indicate fluorescence and bright-field images, respectively. N in (b) and (c) indicates egg nucleus. Bars = 20 μm Next, the effect of plasmid DNA concentration on the level of expression of SP-GFP-HDEL was examined. Egg cells were placed in MMG droplets containing plasmid DNA at the concentrations of 17, 68, or 272 ng/μl, and PEG-Ca 2+ transfection was conducted. For the egg cells located in the 272 ng/μl plasmid droplets, a strong GFP-derived signal was detected in two of the five treated cells at 13 hr after PEG-Ca 2+ transfection, and the fluorescent profile in these five cells did not change at 19 hr after transfection (Table ).

When eight egg cells were PEG-Ca 2+-transfected using an MMG droplet containing 68 ng/μl plasmid DNA, one and three egg cells showed strong and weak fluorescent signals, respectively, at 13 hr after PEG-Ca 2+ transfection. Interestingly, at 19 hr of PEG-Ca 2+ transfection, a strong GFP signal became detectable in three egg cells in which weak GFP signal was detected at 13 hr (Table ). In the case of 17 ng/μl plasmid DNA, two of the five treated egg cells showed weak fluorescent signals at 13 hr after PEG-Ca 2+ transfection, and the signal in these cells become stronger at 19 hr after PEG-Ca 2+ transfection. These results suggest that transient expression of SP-GFP-HDEL by PEG-Ca 2+ transfection of egg cells with plasmids will occur in a plasmid DNA concentration-dependent manner in the range of 17–272 ng/μl plasmid DNA. Therefore, we employed a plasmid concentration of 68 or 272 ng/μl in further analyses. Using the above-mentioned conditions, maize egg cells were also transfected.

Of the nine transfected egg cells, two cells showed SP-GFP-HDEL-derived signal (Fig. d and Table ). Table 1. Efficiency of PEG-Ca 2+ transfection of egg cells and zygotes with plasmid DNAs Cell (plant) Plasmid No. Of transfected cells Fluorescent signal Efficiency (%) + − Egg cell (rice) pGFP-ER 17 6 11 35.3 pDsRed 18 6 12 33.3 pH2B-GFP 26 5 21 19.2 Egg cell (maize) pGFP-ER 9 2 7 22.2 Zygote (rice) pGFP-ER 23 0 23 0 Zygote (maize) pGFP-ER 19 15 4 78.9 2. Digipan User Guide on this page. 2 Intracellular localization of exogenously expressed proteins and expression of multiple plasmid DNAs in rice egg cells by PEG-Ca 2+ transfection When rice egg cells transfected with pGFP-ER were observed with a confocal laser scanning microscope, the endoplasmic reticulum around the nucleus and at the cytoplasm/cell cortex was visible (Figure a).

Next, pH2B-GFP, into which DNA encoding histone H2B-GFP fusion protein was placed under the 35S promoter-HSP70 intron, was constructed and PEG-transfected with rice egg cells. A fluorescent signal was detected in the nucleus and a bright fluorescent signal was observed in the nucleoli (Figure b), and the intracellular localization pattern of H2B-GFP in PEG-Ca 2+-transfected egg cells was same as in egg cells isolated from transgenic rice plants expressing H2B-GFP proteins under the control of ubiquitin promoter (Figure c; Abiko et al., ). These results indicated that transiently expressed proteins in egg cells through PEG-Ca 2+ transfection were correctly translated, folded, and targeted to their destination organelle. In addition, when egg cells were PEG-Ca 2+-transfected with pDsRED, ~33% of transfected cells showed the RFP signal (Figure d and Table ). The efficiency of PEG-Ca 2+ transfection of pGFP-ER and pH2B-GFP was ~35% and 20%, respectively (Table ). • • Transient expression of fluorescent proteins in rice egg cells by PEG-Ca 2+ transfection with plasmid DNAs. (a), Rice egg cell was PEG-Ca 2+-transfected with pGFP-ER and observed with a confocal laser scanning microscope.

(b), Rice egg cell was PEG-Ca 2+-transfected with pH2B-GFP and observed with a fluorescent microscope. (c), Egg cell isolated from a flower of a transgenic rice plant expressing H2B-GFP was observed with a fluorescent microscope. (d), Rice egg cell was PEG-Ca 2+-transfected with pDsRed and observed with a fluorescent microscope. (e), Rice egg cell was cotransfected with pGFP-ER and pDsRed and observed with a fluorescent microscope. Left and right panels in (b-d) represent fluorescence and bright-field images, respectively.

Left, middle, and right panels in (e) represent images of a GFP-derived signal, RFP-derived signal, and bright-field, respectively. N in (a) and (e) represents egg nucleus. Arrowheads in (b) and (c) indicate nucleoli in egg nucleus. Bars = 20 μm We next tested the dual expression of two kinds of fluorescent proteins. Rice egg cells located in a MMG droplet containing pGFP-ER and pDsRED were PEG-transfected and fluorescent signals from both fluorescent proteins in cells were observed. Approximately 43% of transfected egg cells showed fluorescent signals.

Notably, of 16 fluorescent-positive cells, 15 showed signals from both SP-GFP-HDEL and DsRED (Figure e and Table ). These suggest that two kinds of plasmid DNAs were delivered into the same egg cell via PEG-Ca 2+ transfection and functioned in the cell. This tendency, dual expression in one egg cell, would be highly useful to analyze functions of genes of interest in the cells. When egg cells were transfected with two kinds of plasmids, one is harboring the target gene and the other is plasmid DNA of a fluorescent marker, such as pGFP-ER, pH2B-GFP, and pRFP, it can be expected that cells showing a fluorescent signal express the gene(s) of interest and the effect of ectopic and/or overexpression of the target gene in egg cells can be monitored in such fluorescent cells. Table 2. Efficiency of PEG-Ca 2+ transfection of rice egg cells with dual plasmids Plasmid No. Of transfected egg cells Fluorescent signal Efficiency (%) GFP + RFP GFP only RFP only None pGFP-ER + pDsRed 37 15 1 0 21 43.2% 2.3 PEG-Ca 2+ transfection of isolated zygotes and subsequent culture of the zygotes Although rice zygotes were mechanically isolated from pollinated rice flowers by dissection of pollinated ovaries as described by Abiko et al.

() and subjected to PEG-Ca 2+ transfection with pGFP-ER, no fluorescent signal was detected in the zygotes (Figure a and Table ). Maize zygotes were isolated by treating dissected ovaries with an enzyme mixture of cellulase, macerozyme, and pectolyase as described in Materials and Methods, and subjected to PEG-Ca 2+ transfection. In contrast with rice zygotes, PEG-Ca 2+ transfection of the maize zygotes with pGFP-ER resulted in expression of SP-GFP-HDEL with high efficiency (Figure b and Table ).