Genetic modification of Water spinach (<i>Ipomoea aquatica</i>), a genoprotective perennial leafy green

Improvement of leafy greens, especially perennials with year-round harvesting, is binding to the food security drive. "Food for All" by WHO demands the improvement of regional crops due to the agroclimatic specificity to ensure regional food security. Water spinach (Ipomoea aquatica) is a perennial nutritious leafy green with regional/ethnic cultivation. We accomplished organogenesis and somatic embryogenesis from different explants of I. aquatica, and transgenesis and genome editing through Agrobacterium-mediated transformation. The Ipomoea Basal (CLC-CP) medium was superior to the Murashige and Skoog medium. Hypocotyl explants produced a mean of 12.4 shoots on CLC-CP containing 4.5 μM thidiazuron and 8.7 μM gibberellic acid (GA₃), 50 mg l^-1 ascorbic acid (AA), and 100 mg l^-1 adenine hemi-sulfate (AdS). Leaf and root explants induced the highest somatic embryos on a medium containing AdS, AA, 4.4/4.7 μM 6-benzyladenine/kinetin (KIN), and 0.45 μM 2,4-dichlorophenoxyacetic acid. CLC-CP medium with 4.7 μM KIN, 8.7 μM GA₃, AA, and AdS exhibited elongation of hypocotyl-derived shoots and maturation of somatic embryos. A. tumefaciens-mediated transformation of hypocotyl developed a mean of 3.7 GFP expressing shoots per explant; leaf and root produced 4.3 and 3.1 somatic embryos, respectively. A. rhizogenes infection induced a mean of 4.1 and 3.4 hairy roots from leaf and root explants, respectively. Western blotting of the GFP protein validates water spinach to express human therapeutic proteins. Genome editing of IaNAP1 using hypocotyl explants confirmed the reproducibility of transformation. The plantlets exhibited 100% survival in soil. The present protocol is useful for improving this ethnic leafy green with traits-of-interest.