Poplar with modified lignin

The genetically modified trees have an altered lignin composition resulting from the downregulation of the cinnamyl alcohol dehydrogenase (CAD) enzyme through RNA interference. CAD catalyzes the last step in the monolignol synthesis. The remaining CAD activity in the modified trees is about 15% of that in wildtype trees. The altered lignin composition has a positive effect on the ease with which the lignin can be broken down to gain access to the valuable sugar content in the cellulose and hemicellulose in the wood of the trees. A lowered CAD activity has been found to naturally occur in the U.S. in loblolly pine (Pinus taeda) and in wild black poplar in Europe with comparable effects on the wood composition.

The modified trees also carry the NPT-II selection marker gene allowing an easy selection of transformed plants using kanamycin.

pHellsgate8

RNA interference cassette:
Transcription of the hairpin RNA (HpRNA) is under control of the Cauliflower Mosaic Virus (CaMV) 35S promoter and the Agrobacterium tumefaciens octopine synthase terminator. The transcript is expected to contain an inverted repeat of the Populus tremula x Populus alba cinnamyl alcohol dehydrogenase (CAD) gene separated by a segment sourced from Flaveria trinervia pyruvate orthophosphate dikinase intron 3, which is essential to form a loop that allows the inverted repeats to base pair and form the RNA secondary structure after transcription. Then the hpRNA is sufficient to trigger an RNAi response. Due to the RNAi response, the hpRNA is processed into small interfering RNA (siRNA) and thus no translation to protein is expected to occur.

Selectable marker:
The pHellsgate8 vector also contains a selectable marker for kanamycin resistance. The Escherichia coli neomycin phosphotransferase II gene is under transcriptional control of the A. tumefaciens nopaline synthase promoter and terminator.

Post-transcription, the inverted repeat segments of the poplar cinnamyl alcohol dehydrogenase (CAD) basepair to form a double stranded region separated a loop derived from the Flaveria trinervia pyruvate orthophosphate dikinase intron 3. The double strandedness of the hpRNA is recognized by host cell RNA interference machinery; DICER processes the hpRNA into siRNA molecules (21-24 base pairs in length), which then associate with ARGONAUTE to form the RISC complex. The siRNA are then used to guide host machinery to degrade transcripts with sequence homology. Thus, the host machinery targets the endogenous CAD transcripts, resulting in silencing of CAD gene expression.

Three lines have been created with the same genetics: pHG8-CAD4, pHG8-CAD19 and pHG8-CAD24. Thus, the transformation event listed above represents a placeholder and will be updated should more information become available.