4.6. Generation of Tomato Transgenic Plants

The full length of the SlSNAP33.2 CDS was replaced for the GUS sequence in the pBI121 vector by using the XmaI and XbaI sites on the SNAP33_Fw and SNAP33_Rv primers, generating after all the 35S::SlSNAP33 cassette. The new construct was introduced by a chemical transformation in Agrobacterium tumefaciens GV3101 pmp 90 strains [58]. The transformed bacteria were grown in solid YM medium (0.04% w/v yeast extract, 1% w/v mannitol, 1.7 mM NaCl, 0.8 mM MgSO₄ • 7H₂O, 2.2 mM K₂HPO₄, and 1.5% w/v agar) with rifampicin 100 (mg/L), gentamicin (25 mg/L), and kanamycin (50 mg/L). After 48 h at 28 °C, colonies were analyzed by PCR. Recombinant clones were used to transform cotyledon tomato explants, according to the method of Fillati et al. (1987) [59]. Transgenic plants were selected with kanamycin (50 mg/L) and micropropagated continuously to maintain a stock of plant biomass. The T0 transgenic lines were analyzed by PCR and qPCR. Several transgenic lines carrying the 35S::SlSNAP33 cassette and expressing the SlSNAP33.2 were identified. The tomato plants transformed with the empty vector were used as a control (vector).