Figure 4.

Representative 1% agarose gel for the selection of T₂ candidates lacking the Cas9 transgene. Multiplex polymerase chain reaction (PCR) was used to confirm the presence/absence of the Cas9 transgene, using the constitutively expressed house‐keeping gene EF1a as a loading control. L denotes a 100‐bp DNA ladder. A Cas9 transformant (T₁ generation) and a non‐transformed wild‐type plant were used as positive and negative controls for Cas9 amplification, respectively. Samples #41–#49 are a representative selection of T₂ progeny from T₁ plant number 1 (as shown in Fig. 2). Candidates #44 and #45 represent two of a total of 55 candidates lacking the Cas9 transgene, which were selected by this method.