The transgenic lines were characterized for transcript abundance, protein abundance, enzyme activity, wood properties, and growth.
Transcript level
qRT-PCR was applied for first round of screening for transgenic trees of different lines of the same construct, and was the basis for selecting three to four transgenic lines with different levels of target gene knockdown.
RNA-Seq was applied for selected clones propagated for ~6 months to grow transgenic trees.
Protein level
Using Protein Cleavage-Isotope Dilution Mass Spectrometry (PC-IDMS), and a comprehensive set of stable isotope labelled surrogate peptides as internal standards, we quantified the absolute abundance of all monolignol biosynthetic proteins in stem differentiating xylem of the wildtype and transgenic trees.
The enzyme activities of the ten monolignol biosynthetic enzyme families were measured for total protein extracts of stem differentiating xylem in the transgenic trees.
Wood properties
Sugar content and composition, and lignin content of the transgenic trees were determined by the Klason method and HPLC. Lignin S/V ratio was determined by nitrobenzene oxidation and HPLC.
Characterization of lignin subunits and interunit linkage distribution for the wildtype and transgenic lines were carried out at the University of Wisconsin by John Ralph et al., using a 750 MHz (DMX-750) Bruker Biospin (Rheinstetten, Germany) instrument.
Modulus of Elasticity (MOE) measurements were made for the transgenic lines to investigate changes in tensile elasticity or the tendency of the stem to deform when opposing forces are applied.
Others
The height and diameter of the transgenic trees were measured one month before harvest, and measured again during harvest.