Supplementary Text S1 Generation of Potato Activation Tagged Mutants

Supplementary Text S1 – Generation of potato activation tagged mutants

The mutant lines were generated as follows: Potato (Solanumtuberosum L. cv.Bintje) plants were grown on plantlet growth medium [PGM; MS (Murashige and Skoog 1962) minimal organic medium, sucrose 3%, pH 5.7 and solidified with 0.7% agar, sterilized] from 1 cm nodal stem sections and kept under 16 h photoperiod, 20˚C day/18˚C night and 70-100 µE m-2s-1light intensity. For transformation, internodal stem segments from 4-week-old plants were excised and placed on infiltration medium (IM; MS minimal organic medium, sucrose 3%, MES 0.5g/L, mannitol 20g/L, pH 5.5, filter-sterilized) to prevent the explants from drying out.

The activation tagging vector pSKI074 (Weigel et al. 2000) was transformed into Agrobacterium tumefaciensstrain GV3101 (pMP90RK) by electroporation. The T-DNA of vector pSKI074 encompasses left and right borders, kanamycin selectable marker, T3 RNA polymerase promoter, T7 RNA polymerase promoter and four CaMV 35S enhancer repeats (Weigel et al. 2000). The transformed colonies were selected on ampicillin-containing Luria broth (LB) plates. For potato transformation, Agrobacterium was grown from freshly streaked LB plates supplemented with 100mg/L ampicillin, 50 mg/L kanamycin and 100mg/L rifampicin and plates were incubated for 2 days at 28°C in the dark. A single colony of Agrobacterium was picked and cultured overnight in liquid LB medium containing 100mg/L ampicillin, 50 mg/L kanamycin and 100mg/L rifampicin, at 28°C in the dark. Agrobacterium inoculum was grown to an OD600 of 0.6-0.8. Bacterial cells were pelleted by centrifugation at 3,000 rpm for 15 min, re-suspended in IM with 200 µM acetosyringone at a final concentration corresponding to an OD600 of 0.3.

Excised internodal explants (placed on IM medium) were transferred to a 50 ml tube and IM medium containing the re-suspended Agrobacterium and acetosyringone was added. Tubes were inverted gently for 2 to 5 min. For co-cultivation, explants were put on sterile cheesecloth or filter paper to remove excess Agrobacterium before transfer to petriplates containing 25 ml of callus growth medium [CGM; MS minimal organic medium, glucose 2%, adenine sulfate 40mg/L, MES 0.5g/L, polyvinylpyrrolidone (PVP) 0.5g/L, glutamine 200mg/L, pH 5.7 and solidified with 0.22% gelrite, sterilized and supplemented with 0.1mg/L trans-zeatin and 0.1mg/L IAA] for 2 days. Petri plates were sealed with micropore tape and incubated at 24°C with 16 h photoperiod under low-light (60-80 µE m-2sec-1) in a plant growth chamber.

After 2 days of co-cultivation, explants were washed 2-3 times with sterile distilled water containing 300 µg/ml claforan, blotted on sterile filter paper and placed on callus selection medium(CSM = CGM + kanamycin 100 µg/ml and claforan 300µg/ml). Petriplates were sealed with micropore tape and incubated as above. Explants were transferred after 10 days to fresh CSM and higher light intensity (150-200 µEm-2sec-1) and incubated for 10 days. A second transfer was done onto fresh CSM and explants incubated for 10 days for callus induction (total 30 days on CSM) before the explants were transferred to shoot growth medium. Calli were transferred to shoot growth medium (SGM; MS minimal organic medium, glucose 2%, adenine sulfate 40mg/L, MES 0.5 g/L, polyvinylpyrrolidone(PVP) 0.5 g/L, glutamine 200 mg/L, pH 5.7 and solidified with 0.22% gelrite, sterilized and supplemented with 0.1 mg/L trans-zeatin, 50mg/L kanamycin and 300 mg/L claforan) and incubated for 6-8 weeks, transferring to fresh SGM every 7-10 days to facilitate shoot elongation. Shoots were transferred to root growth medium (RGM; MS minimal organic medium, sucrose 2%, pH set to 5.7 and solidified with 0.7% agar, sterilized and supplemented with 50mg/L kanamycin and 300mg/L claforan ) or to PGM when 2 cm long with several leaves.

Transformed bacterial colonies were screened for the presence of the plasmid before infection of internodal explants and also after 2 days of co-cultivation on medium by colony PCR. Bacteria were transferred to fresh LB plates with antibiotics (ampicillin, kanamycin and rifampicin) and incubated for 2 days at 28°C in dark. Colony PCR with 074 Red and Fred primers (Supplementary Table S1) was done to check plasmid sequences. For screening of transformed plantlets, total DNA was extracted from leaves of putative transgenic plants and PCR was done with the same set of primers (074 Red and Fred) to confirm the presence of the plasmid.