Fig. 5: The analysis of PaOAF-VIGS Phalaenopsis.

a A flower of wild-type Phalaenopsis Sogo Yukidian “V3”. The close-up views of the anther cap, pollinia and column are circled in the box. LP lips, P petals, DS dorsal sepals, LS lateral sepals. Bars = 20 mm; 5 mm (in box). b The back side of the Phalaenopsis V3 flowers revealed the organ of the ovary. Bars = 20 mm. c Close-up of the anther cap (Ac), pollinia (Po), column and ovary of the Phalaenopsis V3 flowers in which the sepal/petal/lips were detached. Bars = 4 mm. d Close-up of the pollinia (Po) and column after the removal of the anther cap from the Phalaenopsis V3 flowers. Bars = 2 mm. e Analysis of the expression of PaOAF in the sepal (S), petal (P), lips (L), anther cap (Ac), pollinia (Po), column (Col) and ovary (Ov) of the Phalaenopsis V3 30 mm flower bud and mature flowers (MF). f Alexander’s staining of developing seeds 130 DAP in control ovary. Normal viability of the embryos with dark purple staining (red arrow) and seed coats with purple staining (black arrow) was observed. Bar = 100 µm. g Close-up of the developing seeds from (f), which showed normal developed embryos (red arrow) and seed coats (black arrow). Bar = 50 µm. h Alexander’s staining of developing seeds 130 DAP in PaOAF-VIGS ovaries. Defective seeds with abnormal viability of the embryos that were either significantly reduced in size or completely absent (red arrow) were observed. Bar = 100 µm. i Close-up of the defective seeds from (h), which showed defective embryos (red arrow) and seed coats (black arrow). Bar = 50 µm. j Detection of PaOAF expression in control mock (C-1 to C-3) and PaOAF-VIGS Phalaenopsis V3 (V-1 to V-3) flowers by real-time quantitative RT–PCR. k Measurement of the size of the seeds from control mock and PaOAF-VIGS Phalaenopsis V3 ovaries. A total of 470 seeds from the control mock and 459 seeds from PaOAF-VIGS ovaries were used for measurement. l Normally germinated seedlings for the control mock (pCymMV) and the nongerminated seedlings from PaOAF-VIGS-deficient seeds. l leaf, r root. Bar: 0.2 mm. m Comparison of the total number of seeds successfully germinated from 200 seeds each for the control mock (pCymMV) and PaOAF-VIGS ovaries. +: germinated, −: nongerminated. Mäule staining of lignin for control mock (pCymMV) (n, o) and PaOAF-VIGS (p, q) ovules (arrowed) 80 DAP. o, q are close-up images from (n, p), respectively. Bars: 200 µm (n, p), 100 µm (o, q).