A high throughput plant regeneration system from shoot stems of Sapium sebiferum Roxb., a potential multipurpose bioenergy tree

Abstract

Sapium sebiferum Roxb. is an economically important multipurpose woody plant in terms of its biodiesel-producing, medicinal and ornamental values. In the present study, a high throughput in vitro regeneration system of S. sebiferum through direct shoot organogenesis from shoot stems was developed. The effects of plant growth regulators (PGRs), explant orientation, culture vessel type, explant type and explant source on adventitious shoot induction were investigated. Adventitious shoots were induced from cut ends of shoot stems with or without axillary buds when they were cultivated on the Murashige and Skoog (MS) medium fortified with 6-benzylaminopurine (6-BA). The highest rate of shoot induction (100 %) with mean number of shoot buds (21.4 ± 0.2) per explant was obtained when in vitro shoot stems without axillary buds were placed horizontally on the medium supplemented with 0.5 mg l−1 6-BA in glass petri dish. The induced shoot buds were transferred to MS medium supplemented with 0.5 mg l−1 6-BA and 0.1 mg l−1 gibberellic acid (GA3) for shoot proliferation and elongation. The elongated shoots were continuously propagated through shoot stem sectioning until used for rooting. For in vitro adventitious root induction, the elongated shoots were cultivated on the MS basal medium supplemented with different strengths of macronutrient, various concentrations of auxins and sucrose. The highest root induction (100 %) was achieved when the elongated shoots were cultured on 1/8 MS medium fortified with 2.0 % sucrose and 0.5 mg l-1 indole-3-butyric acid (IBA). For ex vitro rooting, 98.3 % shoots rooted when they were pretreated with IBA (1000 mg l-1) for 60 s. The plantlets rooted by both methods were successfully acclimatized and showed normal development with 91.4 % and 98.1 % survival rate, respectively. The genetic fidelity of the regenerants was confirmed by inter simple sequence repeat (ISSR) markers. The present in vitro regeneration protocol will play an important role in large-scale propagation, germplasm conservation as well as genetic improvement of this economically important multipurpose plant species.