Figure 4.

Evaluation of S. bicolor RNAi Transformant Events.

(A) Relative ARS1 and ARS2 endogenous transcript levels in 10-d-old S. bicolor hpRNA “+” and hpRNA “−” seedlings (representing six independent transformant events) were determined by quantitative real-time RT-PCR using gene-specific primers. Data were normalized to an internal control (18S rRNA), and the ΔΔC_T method was used to obtain the relative expression levels for each sequence, expressed as mean ± sd from assays performed in triplicate. Events numbered 1, 3, and 4 were generated using the vector pARS1-RNAi, and events numbered 2, 5, and 6 were generated using pARS2-RNAi (see Supplemental Figure 1 online).

(B) Genomic DNAs isolated from the six S. bicolor RNAi transformant events, and control seedlings (genotype Tx430) were digested with either BamHI or SphI and then probed using radiolabeled Arabidopsis thaliana FAD2 gene intronic sequences present in the RNAi cassette. C, control; B, BamHI; S, SphI.

(C) Sorgoleone levels were determined by GC-MS analysis of root exudates prepared from 10-d-old hpRNA “+” and hpRNA “−” seedlings representing the six RNAi transformant events. Data are expressed as mean ± sd from four measurements. The limit of quantitation (LOQ), determined to be ∼0.003 μg/mg fresh weight, is also indicated by a dashed line.