For examining the heterosis phenotype of early developmental
stages, plants were grown in plastic dishes containing Murashige and
Skoog (MS) agar medium supplemented with 1.0% sucrose (pH 5.7) in
growth chambers under a 16-h/8-h light/dark cycle at 22 °C. Cotyledons
at 6 days after sowing (DAS), and 1st and 2nd leaves at 14 DAS were fixed
in a formalin/acetic acid/alcohol solution (ethanol:acetic acid:formalin =
16:1:1). The image of the whole cotyledon or leaf was photographed
under a stereoscopic microscope, and sizes were determined with
Image-J software (http://rsb.info.nih.gov/ij/).
For examining the yield under field conditions, seeds were sown
on multi-cell trays on 21st August 2014 and grown in a greenhouse.
At 6 DAS, cotyledons were photographed, and the area of the cotyledons
was determined with Image-J software (http://rsb.info.nih.gov/ij/). On
3rd September 2014, seedlings were transplanted to the field at Osaki,
Miyagi, Japan (38°57′N, 141°00′E). Thirty plants per plot were
transplanted and plot size was 13.5 × 0.7 m. Row spacing is 70 cm
and planting distance is 40 cm. At 21 DAS, leaf lengths and widths of
the first and second largest leaves were measured. On 13th and 14th
November 2014, plants were harvested. Statistical comparisons of cotyledon area, leaf size, fresh weight of total biomass and harvested biomass were performed using Student's t-test (p b 0.05).
The ratio of heterosis performance between F1 and mid parent value
(MPV) (termed rMPV) is calculated as follows, rMPV = F1 (mean)/MPV
(mean of two parents). The ratio of heterosis performance between F1
and better parent value (BPV) (termed rBPV) is calculated as follows,
rBPV = F1 (mean)/BPV (mean of better parent).