Download tpj12630-sup-0001-FigureS1

P
K/K X H/H
F1
K/-;H/-
F2
K/K;H/H
F3
K/K;H/H
HygR GUS+
M1 (F4)
K/K;H/H
EMS
M2 (F5)
K/K;H/H
KanR or KanR HygR
M3 (F6)
K/K;H/H
NPTII ELISA, ProNOS DNA meth., ProNOS siRNAs;
Figure S1: Outline of the mutagenesis approach
The parental lines (P) homozygous for the TARGET (Kchr1-1) or the SILENCER (H) transgene were crossed with each other
(Fischer et al., 2008). Obtained F1 hemizygous for both transgenes (K/-;H/-) were grown on soil and allowed to set seeds by selfpollination. To identify lines homozygous for both transgenes, the resulting F2 progeny was grown on soil again and again allowed
to set seeds by self-pollination. Resulting F3 seeds were harvested from individual F2 plants and were germinated on medium
containing 20 mg/l hygromycin. If the parental F2 plant had been homozygous for the SILENCER transgene, 100% of the F3
derived from this F2 were HygR. The F3 seedlings from such batches were then subjected to a GUS activity test, as, if the parental
F2 plant had been homozygous for the TARGET transgene, 100% of the F3 should be GUS+. F3 plants from such batches
showing 100% HygR GUS+, and thus containing only individuals homozygous for both transgenes (K/K;H/H), were allowed to set
seeds by self-pollination and resulting F4 seeds were harvested in bulk. Bulks of F4 seeds were treated with mutagen ethyl
methanesulfonate (EMS) and then allowed to germinate on soil to give rise to mutagenized M1 plants. M1 plants were allowed to
set seeds by self-pollination and resulting M2 seeds were harvested in bulk. M2 (equal to F5) are the first generation in which
EMS-induced mutations can become homozygous and thus, if recessive, result in selectable phenotypes. Thus, M2 progeny was
tested for resistance on medium containing 200 mg/l kanamycin (or 200 mg/l kanamycin and 20 mg/l hygromycin) and obtained
KanR or KanR HygR mutant candidates were counterchecked again in the M3 (F6) next generation obtained by self-pollination.