In vitro regeneration of mulberry plants from seedling explants of Morus indica cv. G4 through direct organogenesis

An efficient, high-frequency, and robust in vitro regeneration protocol was developed using cotyledon and hypocotyl explants from mulberry (Morus indica cv. G4) seedlings. Mulberry (Morus) is a perennial tree species with a wide range of commercial applications. Its leaves are predominantly used for feeding the monophagous silkworm (Bombyx mori L.) globally. In this study, the effects of plant growth regulators, additives and elevated levels of macronutrients, on in vitro adventitious shoot induction, shoot elongation, and rooting, were investigated. It was found that modified Murashige and Skoog (MS) medium supplemented with thidiazuron (0.5 mg/L) provided the most suitable medium for adventitious shoot bud induction, with a regeneration frequency of 88.62%, and yielded adventitious shoot buds of 10.60 ± 0.30 per cotyledon explant. Additionally, the MS medium fortified with 6-benzylaminopurine (1.0 mg/L), gibberellic acid (1.5 mg/L), silver nitrate (2 mg/L), putrescine dihydrochloride (1 mg/L), activated charcoal (AC, 0.2%), and supplementary dosage of calcium chloride (515 mg/L) resulted in the highest frequency of shoot elongation, spontaneous root induction, and the longest shoot length. In this medium, no hyperhydricity of regenerated shoots/leaves was observed. We observed the longest adventitious root length and secondary root length of the shoots grown on MS medium supplemented with indole-3-butyric acid (2 mg/L) and AC (0.2%). The frequencies of ex vitro survival of plantlets after hardening were 90–95% and 95–100%, under laboratory and field-like conditions, respectively. Even though in vitro regeneration protocol in mulberry is genotype-dependent and explant-specific, the robust regeneration protocol developed in this study could find its applications in genome editing and genetic transformation using cotyledon and hypocotyl explants of other cultivars.