4.3. qRT-PCR Analysis

Material derived from four Liriodendron hybrid seedling tissues (leaf, shoot apex (1 cm), shoot (1 cm), and root (1 cm); Figure 3) was collected for the analysis of the LhWOX5 gene expression pattern. Total RNA was obtained using a Bioteke plant total RNA extraction kit (RP3301). Quantitative real-time PCR (qRT-PCR) reactions were performed using the Vazyme AceQ qPCR SYBR Green Master Mix (without ROX) (Q121-02) on a LightCycler 480 II (Roche). For each sample, three technical and biological replicates were used, and the result was normalized with 18S rRNA as a reference.

The same qRT-PCR protocol was applied for expression analysis in LhWOX5 overexpressing lines. Rosette leaves with flower buds and leaves and curly stems with undetermined hyperplasia were collected, using UBQ10 as an internal reference. The primers used for qRT-PCR are listed in Table S1. Expression data were calculated using the Livak calculation method [35] and visualized by GraphPad Prism 8 (https://www.graphpad-prism.cn/) (accessed on 25 Octorber 2022).