Extended Data Fig. 5: Various side effects of the DkRAD over-expression Arabidopsis and Nicotiana tabacum transgenic lines.

a-c, Ectopic over-expression of DkRAD resulted in failure of self-fertilization in A. thaliana. Siliques developed normally after self-pollination in the control, while DkRAD ox. lines were self-sterile. Phenotype of the control line and DkRAD over-expression line #12 were shown. DSd, deficient seeds; Sd, seed; Si, silique; SS, sterile silique; Sty, Style. d, Number of seeds in control and the DkRAD over-expression lines (n = 3–9 biological replicates, see detail in Supplementary Table 8). P-values were detected in comparison to the control #1 using two-sided Student’s t-test. Data were expressed as mean values ± SD. f, Cross-pollination with control lines could produce fertile silique, suggesting that female organs are functional in the DkRAD transgenic lines (n = 3-4 biological replicates). Comparison of the fertile seed numbers between open-pollination and artificial self-pollination or reciprocal crossing with the control in DkRAD ox. #12. All the artificial self-pollination and reciprocal cross-pollination exhibited recovery of the seed fertility from the open-pollination, supporting that physical distance between anthers and stigma would be the cause of sterility in the DkRAD ox. lines. P-values were detected in comparison to DkRAD ox. #12 using two-sided Student’s t-test. Data were expressed as mean values ± SD. g, The control and DkRAD ox. lines showed no substantial differences in ovule development (immediately before the anthesis). Self-sterility of the DkRAD ox. lines could be due to the physical distance between stigma and anthers, caused by the hyper-growth of carpel. h, DkRAD ox. lines showed severe growth retardance, although their flowering timing was not substantially delayed from the control lines. i-j, Precocious flowering in the transgenic lines in comparison to the control. The DkRAD and DkRAD2 over-expression lines often formed flower buds even in selection media immediately after the regeneration in vitro (i). k, The DkRAD2 over-expression lines exhibit enlarged carpel before flowering in comparison to the control. Cp, carpel; FB, flower bud; St, stamen.