Hygromycin

Genomic regions of RPP1-like Ler genes were amplified from 200 ng of freshly extracted genomic DNA (BioSprint Workstation, Qiagen) of the Ler accession (code number N20), using primer combinations listed in Table S2 and LA Taq DNA polymerase (Takara). PCR conditions were: 94°C 5 min, followed by 30 cycles (94°C, 15 s; 55°C, 30 s; 68°C, 4 min), 68°C 10 min. PCR products were separated on 1% agarose gel stained with ethidium bromide, and purified by gel scission (Gel extraction Kit, Qiagen). Purified fragments were cloned into pGEM T-easy (Promega), and the clones sequenced using T7, SP6 and primers listed in Table S2. Sanger sequencing was performed at the Max Planck Genome Center Cologne (Cologne, Germany). The RPP1-like Ler genes were released from pGEMT-easy by digestion with Not I and cloned into the pCambia1300 binary vector (www.cambia.org) modified to contain the PspOMI site in the MCS. The resulting clones were transformed into Agrobacterium tumefaciens GV3101 pMP90 strain [58] (link) for transformation of Col-0 plants [57] (link). T₀ seeds were selected on MS ½ media supplemented with hygromycin 15 µg/ml and 200 µg/ml ticarcillin/clavulanic acid 15∶1 (Duchefa). Homozygous lines with single T-DNA insertions were determined by segregation analyses and selected for further analyses.

Strain AKR1 was created using the GeneBridges ‘Quick & Easy Conditional Knockout Kit’ following the manufacturer’s protocol. In order to make the linear DNA for chromosomal insertion, the FRT-PGK-gb3-hyg-FRT cassette, containing hygromycin resistance, was cloned adjacent to a constitutively expressed mRFP1, and the resulting DNA was further flanked by insulating terminators. The final insertion cassette was amplified by PCR with 75-bp DNA primers containing 50 bp ends designed to be homologous to a chromosomal region within the arsB locus, a previously characterized insertion site (24 (link)).
For the chromosomal insertion, E. coli TOP10 cells were transformed with plasmid pRedET, which harbored inducible Lambda Red machinery. The cells were induced and electroporated with purified DNA containing the insertion cassette. After recovery, transformants were plated onto LB plates containing 100 μg ml⁻¹ hygromycin (Duchefa Biochemie). Chromosomal insertion was verified by Sanger sequencing of linear DNA amplified from 200 bp up- and downstream of the insertion site (Source Biosciences). hygromycin resistance was subsequently removed with the pCl-FLPe plasmid to produce the final strain AKR1, which showed constitutive expression of mRFP1. Correct antibiotic resistance removal was verified by Sanger sequencing of the genomic region.

Agrobacterium-mediated genetic transformations of poplar plants were performed by applying a leaf-disc transformation protocol [58 (link)] or with an advanced leaf disc method as described in Bruegmann et al. [59 ]. In summary, for the latter, plant tissue was infected with Agrobacterium and washed with Cefotaxime (500 mg/L, Duchefa Biochemie) and cultivated on WPM medium as described in Fladung et al. [58 (link)] and Bruegmann et al. [59 ], with thidiazuron added to a final concentration of 0.0022 mg/L ([60 (link)], Duchefa Biochemie). For selection, the regeneration medium was supplemented with Cefotaxime (500 mg/L, Duchefa Biochemie) and kanamycin (50 mg/L, Duchefa Biochemie) or hygromycin (20 mg/L, Duchefa Biochemie), depending on the selection marker gene. Until beginning stem regeneration, the batches were cultivated in the dark. Once regenerating shoots began to appear, the regenerants were transferred for one month to low light condition with 2.5 µmol photons m⁻² s⁻¹ until initial multiple stem formation and later on under standard conditions as described above. Selected regenerants were used for molecular analyses. After verification of transgenic status, the plants were propagated on WPM medium without supplements [38 ].