Effect of plant growth regulators, explants type and efficient plantlet regeneration protocol through callus induction in Naringi crenulata (Roxb.) Nicolson and its biochemical investigation

Abstract

Naringi crenulata (Roxb.) Nicolson, is a rare medicinal plant belonging to the family Rutaceae. It is a spinous tree and has great medicinal value. N. crenulata (Roxb.) Nicolson has in recent years suffered over-exploitation and has therefore been listed in the Red Data list of International Union for Conservation of Nature as a vulnerable species. A callus induction and in vitro plantlet regeneration system for N. crenulata (Roxb.) Nicolson was optimized by studying the influence of explants type (leaf, nodal segment and shoot tip) and different concentrations of plant growth regulators. Callus formation and shoot differentiation was initiated on Murashige and Skoog’s (MS) medium containing different concentrations of auxin and cytokinin. The best result was obtained using leaf explants and callus production was maximum at 0.5 mg/L BAP (6-benzylaminopurine) and 2.0 mg/L NAA (α- naphthaleneacetic acid) and for nodal and shoot tip explants, callus production was maximum at 2.0 mg/L BAP and 0.5 mg/L NAA. Highly organogenic callli with maximum number of shoots (25±0.3) were obtained on MS medium supplemented with BAP (2.0 mg/L) and NAA (0.5 mg/L) from leaf explants. Elongation and further development of shoot buds into shoots was achieved on MS medium fortified with 0.5 mg/L BAP and 0.5 mg/L Kn. However, the result reflected the existence of high inter-explant variability in response to growth regulators. In vitro rooting of shoots was achieved on ½ strength MS medium supplemented with IBA. Best rooting was achieved on ½ strength MS medium supplemented with 1.0 mg/l IBA (indole-3-butyric acid). The highest total soluble protein contents and peroxidase activity was observed in the four weeks old callus cultures derived from leaf explants and this changing pattern can be used as biochemical marker for differentiation. So, this protocol can be used for the rapid regeneration of Naringi crenulata through indirect organogenesis using a wide range of explants.

Key words: Naringi crenulata, callus, regeneration, leaf explants, peroxidase, total soluble protein.