Abstract
Polyethylene glycol calcium (PEG‐Ca2+) 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‐Ca2+ transfection procedure. To take advantage of this macromolecule delivery system in the reproductive and developmental biology of angiosperms, this study established a PEG‐Ca2+ 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‐Ca2+‐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‐Ca2+ 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‐Ca2+‐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.
Highlights
There are several ways to introduce macromolecules, such as DNA, RNA, and proteins, into plant cells (Davey, Rech, & Mulligan, 1989)
For monitoring expression derived from plasmid DNA which was delivered into rice egg cells by polyethylene glycol (PEG)-Ca2+ transfection, we primarily used the pGFP-ER, in which DNA sequence encoding GFP protein tagged with signal peptide (SP) and carboxy terminal His-Asp-GluLeu (HDEL) sequence was located under the 35S promoter and HSP70 intron, as C-terminal HDEL tetrapeptide sequence constitutes an endoplasmic reticulum (ER) retention signal (Munro & Pelham, 1987; Pelham, 1989) and the SP-GFP-HDEL proteins targeted to the ER are effectively detected in plant cells (Haseloff, Siemering, Prasher, & Hodge, 1997; Hayashi et al, 2001; Ridge, Uozumi, Plazinski, Hurley, & Williamson, 1999)
Four egg cells were placed in a droplet of MMG solution (4 mM MES-KOH pH 5.7, 15 mM MgCl2 in mannitol solution of 450 mOsmol/kg H2O) containing pGFP-ER at a concentration of 68 ng/ll, and the same volume of a droplet of PEG solution (20% PEG and 100 mM CaCl2 in mannitol solution of 450 mOsmol/kg H2O) was merged with the MMG droplet where egg cells were held
Summary
There are several ways to introduce macromolecules, such as DNA, RNA, and proteins, into plant cells (Davey, Rech, & Mulligan, 1989). The functions of most genes expressing in a gamete-specific or fertilization-induced/suppressed manner have not been analyzed This is most likely because the experimental approach for addressing their molecular functions in gametes and zygotes usually consists of making several transgenic plants and subsequent observation of embryo sacs deeply embedded in ovaries. To establish an efficient experimental platform for investigating gene functions in gametes and zygotes, we combined the isolation procedures of female gametes and zygotes from rice and maize flowers (Kranz, Bautor, & Lo€rz, 1991; Uchiumi, Komatsu, Koshiba, & Okamoto, 2006) with the PEG-Ca2+-mediated DNA-delivery approach (Yoo et al, 2007; Zhai et al, 2009), which resulted in the transient expression of introduced DNA in egg cells and zygotes. This transient expression system in zygotes can be employed for gene editing procedures using the CRISPR/Cas system (Belhaj, Chaparro-Garcia, Kamoun, & Nekrasov, 2013; Jinek et al, 2012; Mikami, Toki, & Endo, 2015)
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