Figure S1. Flowers of possible triploid rice plants which were derived from three independent
triploid zygotes. Three possible triploid plantlets produced in the study (Table 1) were grown
into mature plants, and images of flowers from each three plants were presented in panels A-C.
Flowers from wild-type diploid plants were also presented in each images as control. Scale bars
= 1 cm.
Figure S2. Developmental defects of polyspermic zygotes at the step of karyogamy (A, B) and
first cell division (C). An egg cell was fused with two sperm cells expressing H2B-GFP, and the
resulting zygote was observed. A, Failure of fusion of two sperm nuclei with an egg nucleus
during karyogamy. Two sperm nuclei were detectable in the polyspermic zygote after fusion
(panels a to c). Two sperm nuclei were detected at 15 h after fusion (panels d to f), and no
nuclear fusion was observed in the zygote at 1 day after fusion (panels g to i). B, Failure of
fusion of one of the two sperm nuclei with a diploid zygote nucleus during karyogamy. Two
sperm nuclei fluorescently labeled with H2B-GFP were observed in the egg cell after its fusion
with sperm cells (panels a to c). At 50 min after fusion, one sperm nucleus fused with egg
nucleus, and the sperm chromatin began to decondense in the fused nucleus (panels d to f).
Although one sperm nucleus fused with egg nucleus, fusion of another sperm nucleus with egg
nucleus was not observed in the polyspermic zygote at 70 min (panels g to i), 280 min (panels j
to l), and 1 day (panels m to o) after fusion. C, Failure of first cell division in polyspermic
zygote. Two sperm nuclei fluorescently labeled with H2B-GFP were observed in fused egg cell
(panels a1, a2, b and c). At 15 h after fusion, H2B-GFP signal was detected in zygotic nucleus
which is possibly derived from fusion of two sperm nuclei with an egg nucleus (panels d to f).
However, the zygote did not divide at 1 day after fusion (panels g to i), and degenerated
thereafter. Upper panels are fluorescent images, middle panels are merged fluorescent and
bright-field images, and lower panels are bright-field images. Arrowhead in panel Bd indicates
sperm chromatin which is decondensing in fused nucleus. Scale bars = 20 µm.
Figure S3. Microtubule organization in diploid zygotes during mitotic division. Polyspermic
zygotes produced by fusion of an egg cell with a sperm cell expressing H2B-GFP were cultured,
and zygotes at each mitotic phase were fixed for analysis. Microtubule structure and
chromosome organization were visualized by immuno-fluorescent staining with anti-α-tubulin
antibody and DAPI staining, respectively. At prometaphase, chromosomes were arranged at
equator and microtubule structure was present around chromosomes (A–C). Microtubule
spindle was observed in the zygote at metaphase (D–F). At telophase, phragmoplast and
possible microtubule spindle remnants were present (G–I). Upper and middle panels are images
of immuno-fluorescent staining and DAPI staining, respectively. Lower panels represent
merged images. Scale bars = 10 µm.
Figure S4. Actin organization in diploid and polyspermic triploid zygotes during cell plate
formation. A–I, An egg cell expressing Lifeact-tagRFP was sequentially fused with two sperm
cells expressing H2B-GFP, and resulting polyspermic zygote was cultured. Actin organization
in zygote at telophase was observed under a fluorescence microscope. Throughout telophase,
intense signals from actin filaments were detected around putative phragmoplast between two
daughter nuclei. Images in upper and middle panels show H2B-GFP and Lifeact-tagRFP signals,
respectively. Lower panels represent merged images. J–L, An egg cell expressing
Lifeact-tagRFP fused with a sperm cell expressing H2B-GFP, and resulting zygote was cultured.
Signals from actin filaments were detected around putative phragmoplast between two daughter
nuclei in dividing zygote throughout telophase. Scale bars = 10 µm.