Transgenic lettuce ( Lactuca sativa L.) harboring chitinase gene expressed resistance against a devastating fungus, Sclerotinia sclerotiorum

Lettuce ( Lactuca sativa L.) is the world’s most popular leafy salad vegetable cultivated throughout the temperate region of the globe. However, its quality and yield are severely constrained by Sclerotinia sclerotiorum , a destructive fungal pathogen. In this study, rice chitinase ( chi ) gene was transferred to lettuce cv. Solan Kriti via Agrobacterium -mediated gene transfer technique to obtain resistance against S. sclerotiorum . To enhance the transformation rate, critical determinant variables including explant type, pre-culturing and co-cultivation time and acetosyringone effect were optimized. A combination of 72 h pre-culturing followed by 72 h co-cultivation period resulted in the highest transformation frequency of 8.60% and 3.31% in leaf and petiole explants, respectively. Furthermore, the transformation frequency was enhanced in leaf (23.22%) and petiole (16.44%) explants when the selection medium was fortified with acetosyringone in a concentration of 100 µM, respectively. Transgene ( chi ) integration was confirmed through PCR and Southern blotting. Expression of the chi gene in transgenic lettuce plants was confirmed by RT-PCR and qRT-PCR analysis. Fungal bioassay of transgenic lettuce proved the effectiveness of chi gene against invasion of Sclerotinia sclerotiorum by showing delay in symptom appearance. Lesions appeared within 12 h after incubation with the fungus in control plants, while leaves of transgenic plant formed lesion after 72 h, but no colonization of the foliar tissue was observed.