*2.5. Both Tissue Culture and Transgenic Process Can Change the Phenotype of Plants*

We planted all the wild-type and transgenic lines in the field and estimated their gene expression, grain yield, and seed setting rate (Figure 4). For the lines used in the WGBS analysis, the phenotype showed that the plant height was significantly shorter in the CK line and significantly taller in the *pOsNAR2.1-OsNAR2.1* line compared with WYJ7, whereas there was no significant change in the SDWT line (Figure 4a, Supplementary Table S2). The total tiller number was higher in *pOsNAR2.1-OsNAR2.1* line with no significant difference in the CK and SDWT lines (Supplementary Table S2). For the expression of *OsNAR2.1*, while there were no significant differences between the WYJ7, CK and SDWT lines, expression was significantly higher in the *pOsNAR2.1-OsNAR2.1* line (Figure 4b). The grain yield and seed setting rate were significantly lower in the CK line and significantly higher in the *pOsNAR2.1-OsNAR2.1* line compared with WYJ7, with no significant difference in the SDWT line (Figure 4c,d). For the lines used in MeDIP analysis, both the plant height and tiller number (Figure 4e, Supplementary Table S3) were higher in the *pUbi-OsNAR2.1* line and lower in the RNAi line compared with NP. The relative expression of *OsNAR2.1*, grain yield, and seed setting rate were also higher in the *pUbi-OsNAR2.1* line and lower in the RNAi line, compared to NP (Figure 4f–h). As a summary of the global methylation, genetic and phenotype changes status of all materials Table 2 shows that global methylation changes of transgenic lines are dependent on the genetic background. For the CK line, the trend of changes of the phenotypes is same as global methylation, and for all the transgenic lines, the trend for phenotypic change appears the same as the expression of the transgene, but the percentages of the increases in overexpression lines are quite different. We consider that methylation

change is one of the reasons causing the difference. These results suggest that both regeneration and the transgenic process can change the phenotype of plants, with or without gene insertion.

**Figure 4.** Characteristics of phenotype (**a**) Gross morphology of WYJ7, CK, SDWT and *pOsNAR2.1-OsNAR2.1.* (**b**) Real-time quantitative RT-PCR analysis of *OsNAR2.1* expression in WYJ7, CK, SDWT and *pOsNAR2.1-OsNAR2.1* lines. Error bars: SD (*n* = 3 plants). (**c**) Grain yield for WYJ7, CK, SDWT and *pOsNAR2.1-OsNAR2.1* plants grown in the field. Error bars: SD (*n* = 5 plants). Significant differences between WYJ7 and transgenic lines are indicated by different letters (P < 0.05, one-way ANOVA). (**d**) Seed setting rate for WYJ7, CK, SDWT and *pOsNAR2.1-OsNAR2.1* plants grown in the field. Error bars: SD (*n* = 5 plants). Significant differences between WYJ7 and transgenic lines are indicated by different letters (*p* < 0.05, one-way ANOVA). (**e**) Gross morphology of wild-type of NP, *pUbi-OsNAR2.1* and RNAi. (**f**) Real-time quantitative RT-PCR analysis of *OsNAR2.1* expression in NP, *pUbi-OsNAR2.1* and RNAi lines. Error bars: SD (*n* = 3 plants). (**g**) Grain yield for NP, *pUbi-OsNAR2.1* and RNAi plants grown in the field. Error bars: SD (*n* = 5 plants). Significant differences between NP and transgenic lines are indicated by different letters (*p* < 0.05, one-way ANOVA). (**h**) Seed setting rate for NP, *pUbi-OsNAR2.1* and RNAi plants grown in the field. Error bars: SD (*n* = 5 plants). Significant differences between NP and transgenic lines are indicated by different letters (*p* < 0.05, one-way ANOVA).


**Table 2.** Characteristics of methylation and genetics in samples.

Note: NS means no significant difference. NA means not applicable. + means have, 1 means gene number, arrowhead indicates upregulate and downregulate, red indicates upregulate and green indicates downregulate.
