Molecular and phytochemical analyses to assess genetic stability in alginate-encapsulated microshoots of Ocimum gratissimum L. following in vitro storage

Abstract

An efficient protocol was developed for short term storage and conservation of Ocimum gratissimum L., using encapsulated micro shoots. The encapsulation of micro shoots was significantly affected by the concentrations of sodium alginate and calcium chloride. A gelling matrix of 3 % sodium alginate and 75 mM calcium chloride was found most suitable for formation of ideal calcium alginate beads. Re-growth ability of encapsulated shoot tips explant was evaluated after storage at 4 and 25 °C for 30, 45, 60, 75 and 90 days. Preservation at 25 °C being significantly more resistant compared to that stored in 4 °C. Maximum shoot re-growth (98.62 %) was recorded on 0.8 % agar solidified full strength MS basal medium supplemented with 1.0 mg/l BA. Micro shoots, recovered from encapsulated shoot tips (capsule) were best rooted on ½ MS medium containing 1.5 mg/l IBA. Complete plantlets (with shoot and root) were transferred to plastic pots containing soil: vermiculite (1:1) showed 83.31 % survival rate in field condition. The genetic stability of synseed-derived plantlets (following 4 and 25 °C of storage) was assessed and compared with mother plant using two PCR based molecular markers namely random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR). Both types of markers revealed high degree of monomorphism, similar to those of the mother plants. Essential oil composition of the synseed-derived plants and the mother plant were determined by GC-FID analysis and insignificant differences were found in the oil composition, thereby confirming the stability of plants derived from synthetic seeds following short term storage.