The method of McNeece et al. (2019) has been followed for cDNA synthesis [50]. G. max root mRNA has been isolated using the UltraClean® Plant RNA Isolation Kit (Mo Bio Laboratories®, Inc.) according to the manufacturer’s instructions. Genomic DNA has been removed from the mRNA with DNase I (Invitrogen®) according to the manufacturer’s instructions. The cDNA has been synthesized from RNA using SuperScript® First Strand Synthesis System for RT-qPCR (Invitrogen®) with oligo d(T) as the primer according to the manufacturer’s instructions. Assessment of gene expression in G. max has been accomplished by RT-qPCR using Taqman® 6-carboxyfluorescein (6-FAM) probes and Black Hole Quencher (BHQ1) (MWG Operon) (S1 Table) [50]. A ribosomal S21 protein coding gene (Glyma.15G147700) has been used as the RT-qPCR control [50]. The 2-ΔΔCT method of Livak and Schmittgen (2001) has been used to calculate the gene expression fold change caused by the genetic engineering event from three independent replicates [50,64]. The p-values have been calculated using a Student’s t-test for the replicated RT-qPCR reactions [65]. The results are significant if p ≤ 0.05.

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