An improved in vitro encapsulation protocol, biochemical analysis and genetic integrity using DNA based molecular markers in regenerated plants of Withania somnifera L

Abstract

Non-embrogenic, synthetic seeds were produced by encapsulating nodal segments (containing axillary buds) of Withania somnifera L. in calcium alginate hydrogel containing Murashige and Skoog (MS) medium. A 3% sodium-alginate with 100. mM calcium cloride found to be the optimum concentration for the production of uniform synthetic seeds. Effect of different treatments (M1-M5), i.e. MS medium containing different concentrations of cytokinins (0.5, 1.0, 2.5, 5.0 & 10.0. μM) along with optimal level of auxins NAA (0.5. μM) on in vitro morphogenic response of synthetic seeds was evaluated. The maximum frequency (86.2%) of conversion of encapsulated beads into plantlets was achieved on MS (M3) medium containing 6-benzyladenine, BA (2.5. μM) and α-naphthalene acetic acid, NAA (0.5. μM) after 4 weeks of culture. Rooting in plantlets was achieved on 1/2. MS. +. NAA (0.5. μM). Plantlets obtained from stored synthetic seeds were hardened, acclimatized and established in field, where they grew well without any detectable malformation. Significant enhancement in the pigment contents (chlorophyll, carotenoids and net photosynthetic rates) with an increase in acclimatization days may be attributed to chlorophyll biosynthesis. Activities of antioxidant enzymes i.e. superoxide dismutase, catalase and peroxidase activity) were significantly increased which suggests their preventive role in membrane oxidation and damage to biological molecules. Also, an enhanced level of lipid peroxidation, as indicated by MDA content, a sensitive diagnostic index of oxidative injury clearly indicating its positive determining role in combating oxidative stress during acclimatization of plantlets. The generated RAPD and ISSR profiles from regenerated plantlets with mother plant were monomorphic which confirms the genetic stability among the clones. This synthetic seed technology could possibly paves the way for the conservation, short-term storage, germplasm exchange with potential storability and limited quarantine restrictions. © 2013 Elsevier B.V.