In all cases, petri plates containing plant tissue were sealed with a single layer of micropore paper tape (3M, Maplewood, MN). Transformation of S. lycopersicum and S. pennellii LA0716 was performed using AGL0 using a modification of published protocols (59, 60). Fifty to sixty seeds were incubated in 40% bleach and agitated for 5 min. Seeds were rinsed six times, each with 40 ml of sterile double-distilled H2O with 5 min of rocking and decanting of wash solution. A flame-sterilized spatula was used to distribute the seeds onto the surface of 1/2× MSO medium (Murashige and Skoog media with no sucrose) (59) in a Phytatray II (Sigma-Aldrich, St. Louis, MO). Containers were incubated at 25°C on a 16-hour light/8-hour dark cycle with a light intensity of 70 μmol m−2 s−2 PPFD.

At day 8 for S. lycopersicum or day 11 for S. pennellii LA0716, the seedlings were removed from the 1/2× MSO medium jar. The hypocotyl and radicle were excised and discarded. The cotyledon explant was placed on a sterile petri dish. One to two millimeters was removed from the base and tip of the cotyledon. An autoclaved piece of Whatman #1 filter paper (GE Healthcare, Uppsala, Sweden) was placed on the surface of a sterile D1 media plate (59) on which the cotyledons were placed adaxial side up. Approximately 100 explants were added per plate. The plates were placed under the same conditions for 2 days until day 10.

For cocultivation, the Agrobacterium containing the construct was streaked out onto an LB plate containing the appropriate antibiotic. A single colony was inoculated into a 25-ml LB culture with the same antibiotic plus rifampicin (50 mg/liter) in a 250-ml Erlenmeyer flask. The culture was incubated at 30°C in a shaking incubator (225 rpm) for 2 days. The culture was transferred to a sterile 50-ml conical tube and centrifuged at 3200g for 10 min at 20°C. The supernatant fluid was decanted, and 10 ml of MSO media (59) was added to the tube (with no pellet resuspension). The cell pellet was centrifuged at 2000g for 5 min, and this washing step was then repeated. The cell pellet was resuspended in 10 to 20 ml of MSO liquid media. Absorbance of the culture was measured at 600 nm. The suspension was diluted with MSO to OD600 = 0.5. Acetosyringone dissolved in dimethyl sulfoxide (DMSO) was added at a final concentration of 375 μM, and 5 ml of the Agrobacterium suspension was pipetted onto the cotyledons on the plate and incubated with swirling at room temperature for 10 min, at which point the excess culture was pipetted off. Using a scalpel, cotyledons were transferred to a fresh D1 medium plate containing autoclaved Whatman paper. Approximately 50 cotyledons per plate were placed abaxial side up. Plates were incubated at 24°C for 2 days with a 16-hour light/8-hour dark cycle at 70 μmol m−2 s−2 PPFD.

For transgenic callus selection, 2 days after cocultivation, the cotyledons were transferred directly onto sterile 2Z media plates (60) containing kanamycin (100 μg/ml) and timentin (200 μg/ml; no filter paper). Explants were placed abaxial side up with 20 to 30 cotyledons per plate. Plates were incubated under the same growth conditions for 10 days. Cotyledons were then transferred to a sterile petri dish and, using a scalpel, calluses were cut and then placed onto fresh 2Z media plates with the same selection. Subsequently, explants were transferred to new 2Z plates every 2 weeks. Throughout the process, dying tissue was removed, and growing tissue was placed on the media. Five to eight weeks after cocultivation, shoots were harvested from the explants and placed into Phytatray II (Sigma-Aldrich, St. Louis, MO) containing 100 ml of Murashige and Skoog salts, 3% sucrose, and Nitsch Vitamins, 0.8% bactoagar, pH 6.0 (MSSV) media (Murashige and Skoog salts, 3% sucrose, and Nitsch Vitamins, 0.8% bactoagar, pH 6.0) (60) supplemented with timentin (100 μg/ml), kanamycin (50 μg/ml), and indole-3 butyric acid (1 μg/ml). MSSV-containing Phytatrays were incubated under the same growth conditions (16-hour light/8-hour dark cycle at 70 μmol m−2 s−2 PPFD). Shoots were monitored for leaf and root production, and shoots with roots and leaves were placed into pots containing Redi-Earth soil. Flats were covered with a plastic dome under the same growth conditions. Domes were removed from flats after 3 to 4 days.

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