An efficient method for direct shoot organogenesis and plantlet regeneration from leaf explants of Piper colubrinum link and evaluation of genetic fidelity by DNA based markers

Johnson K George, Neema Malik, KU Revathy

Division of Crop Improvement and Biotechnology, Indian Institute of Spices Research, Marikunnu P O, Kozhikode 673012

Received: 28 January 2017                              Revised Accepted: 18 March 2017

ABSTRACT

An efficient regeneration protocol was standardised for rapid multiplication of the multiple disease resistant woody plant Piper colubrinum Link from greenhouse grown leaf explants via direct shoot bud formation. The effect of basal medium composition, the concentration of growth regulators (6-benzyladenine and α-naphthalene acetic acid) and inclusion of activated charcoal for direct organogenesis from leaf explants was evaluated. Half strength Murashige and Skoog (MS) media (macro and micro nutrients at half strength) supplemented with BA 2 mg/l + NAA 0.005 mg/l, BA 2 mg/l + NAA 0.01 mg/l and BA 1 mg/l + NAA 0.05 mg/l gave plantlet regeneration by direct shoot bud formation. The maximum number of shoots (13.2 per explant) and highest frequency of regeneration (83 %) was observed from leaf discs cultured on half strength MS medium supplemented with 2 mg/l 6-benzyladenine and 0.01 mg/l α-naphthaleneacetic acid. The regenerated shoot buds were elongated on full strength Murashige and Skoog medium with same hormone concentration and rooted in hormone free MS medium. The in vitro raised plantlets successfully hardened in sterile potting mix, were morphologically similar to the mother plant. The genetic fidelity of the in vitro raised plants was confirmed by subjecting them to two DNA based finger printing techniques; Inter Simple Sequence Repeat (ISSR) and Random Amplified Polymorphic DNA (RAPD).

Key Words: P. colubrinum Link, adventitious shoot, direct organogenesis, leaf explants, Piper spp. ISSR, RAPD