Mulberry (Morus spp.) is a perennial tree with many commercial applications. It is cultivated across the globe for foliage to feed monophagous mulberry silkworms (Bombyx mori L.). Abiotic stresses, especially drought, reduce mulberry foliage production at a significant level. Silkworm growth, development, and cocoon production depend on the quality of mulberry leaf, which is attributed to sugar, protein, moisture content, and moisture retention capacity. This study developed three transgenic mulberry lines, co-expressing AtDREB2A and AtSHN1 genes, through Agrobacterium-mediated genetic transformation using cotyledon and hypocotyl explants of a high-yielding mulberry cv. G4. These transgenic lines had 1 to 2.01-fold expression of the AtDREB2A and AtSHN1 genes, regulated by a constitutive CaMV35S promoter. Co-expressing transgenic lines showed enhanced cuticular resistance and reduced post-harvest water loss from the leaves. Transgenic mulberry lines exhibited significantly higher photosynthetic rate (26-46%), instantaneous water use efficiency (9-15%), and relative water content (15-18%) and proline content (1.8 to 1.9-fold) than the wild-type plants under 60% field capacity. Leaf disc assays under 25% polyethylene glycol and 200 mM NaCl stresses revealed significantly less chlorophyll degradation and electrolyte leakage in transgenic lines than in wild-type plants. These co-expressing transgenic mulberry lines could be further tested in a confined field trial to evaluate their leaf yield, yield attributes, leaf quality, root traits, the composition of epicuticular wax, silkworm rearing performance, and tolerance to soil-moisture deficit stress.
