Callus mediated in-vitro plant regeneration and clonal fidelity assessment of Lilium longiflorum cv. “Pavia” by ISSR markers

Abstract

Aim: Developing a protocol for mass multiplication of Lilium (Lilium longiflorum) bulbs via indirect organogenesis and somatic embryogenesis for in-vitro plant regeneration and ISSR marker-based mapping to assess the fitness of true to type. Methodology: To induce organogenic callus, six combinations of 2,4-D and BAP were tested; while nine combinations of picloram and NAA were used for somatic embryogenesis. NAA and IBA were tested for root induction. The clonal fidelity of the in-vitro regenerated plantlets from both calluses were tested using polymerase chain reaction (PCR)-based ISSR markers analysis. All experiments were arranged in a complete randomized block design and replicated three times. Results: Two methods (direct and indirect organogenesis) were investigated to assess the best callus mediated plant regeneration for Lilium bulb multiplication. The maximum organogenic callus induction and the highest regeneration percent was recorded with BAP (0.5 mg l-1) + 2,4-D (3.0 mg l-1). However, MS medium containing 0.50 mg l-1 picloram and 0.20 mg l-1 NAA was found best for initiation of embryogenic callus and proliferation and was found superior over direct organogenesis. Clonal fidelity was assessed through ISSR markers comparing the mother plant and regenerated plantlets. Interpretation: Both organogenic callus andembryogenic callus are capable of developing true to type in-vitro plants and can be explored for mass multiplication of Lilium bulbs. Embryogenic callus can further be utilized in liquid suspension based bioreactor system. The present protocol has potential applications in micropropagation and genetic transformation studies in other Lilium spp.