*2.2. Segregation of T-DNA in the T1 Generation*

The presence of CRISPR/Cas9 DNA and marker genes in gene-edited plants may cause adverse effects, such as an increased risk of off-target changes, and may trigger regulation concerns when these plants are used in crop breeding [22,31,32]. To test whether the T-DNA fragment carrying the CRISPR/Cas9 construct could be segregated out in the progeny of T0 mutants, the presence of *HPT*, sgRNA, and *Cas9* transgenes in T1 plants derived from one of the homozygous or bi-allelic T0 mutants of each target (except Osfwl3a) was examined (Figure S2). For target Osfwl3a, the progeny of a chimeric T0 plant (Osfwl3a#4) were used. The genotype of the T0 mutant for each T1 line used is shown in Table S1. Transgene-free plants were obtained in several randomly selected T1 progeny for all lines (Figure S2), suggesting that the number of T-DNA insertion loci was low in T0 plants.

In most T1 lines (13 out of 15) analyzed, consistent segregation patterns were observed for all the *HPT*, sgRNA, and *Cas9* transgenes (Figure S2). However, inconsistent segregation patterns were detected in two lines (Osfwl3a#4 and Osfwl4b#6). In line Osfwl3a#4, *HPT* and *Cas9* sequences were not detected in some plants, although the sgRNA sequence was present (Figure S2). Interestingly, the eight T1 plants of this line that lacked the complete sgRNA/*Cas9* expression cassette were all homozygotes, whereas the other 12 plants containing the complete sgRNA/*Cas9* expression cassette were all chimeras (Figures S2 and S3; Tables S4 and S5). In line Osfwl4b#6, sgRNA and *Cas9* sequences were not detected in all examined T1 plants, but the *HPT* sequence was present in some of the plants (Figure S2). Next, we

examined the T0 plant of this line for the presence of a T-DNA fragment and found that it also lacked sgRNA and *Cas9* sequences. This indicates that mutations of this line were generated by transient CRISPR/Cas9 expression.
