Fig. 1: Phenotypes of pollen tubes and ovaries after pollination and at maturity validate the effect of pollination treatments.

a At 28 HAP, aniline blue-stained pollen tubes were observed in the transmitting tract of silks in CP and HP (both apical region outside of the bract and basal region adjacent to the ovary), while absent in NP and HNP, validating the manual pollination treatments and entry of pollen tubes into ovaries for fertilization. b At 28 HAP, in CP and HP, zygote and fertilized polar nuclei with enlarged embryo sac were observed, note that the nucellus was degrading. In NP and HNP, the embryo sac and nucellus remained intact. c At 96 HAP, endosperms underwent cellularization in both CP and HP, whilst embryo sac (antipodal cells and synergid) in NP and HNP was intact and still. d The grains in CP and HP were visually enlarged compared to that in NP and HNP. e–f Significant differences in ovary/grain fresh weight were observed between NP vs CP, NP vs HNP, and CP vs HP at 96 HAP. g At maturity, compared with normal dent grains in CP, some of the grains in HP were with round shape due to lack of physical extrusion from adjacent grains (HNP). h–k At maturity, approximate 40% ovaries were unfertilized in the HNP treatment, which resulted to a promotion in weight, volume and density of HP grains on the other side of ear. Values are mean with standard error. n represents numbers of biologically independent samples. Student’s t-test, significant differences are indicated by asterisks, *p < 0.05; **p < 0.01; ***p < 0.001; ns no significance, HAP hours after pollination, NP non-pollinated ovaries, CP completely pollinated ovaries, HP half pollinated ovaries, HNP half non-pollinated ovaries, Tt transmitting tract, Pt pollen tube, Ac antipodal cells, AL aleurone, Betl basal endosperm transfer cell, Pc placento-chalazal, Ec embryo sac, Fpn fertilized polar nuclei, S synergid, Se starchy endosperm, Z zygote, Nu nucellus, End endosperm, D dent, R round.