Peroxidase polymorphism for fingerprinting of black pepper (Piper nigrum L.) varieties and micropropagated clones

Abstract

The comparison of field performance of the micropropagated clones with those of conventionally propagated clones by band-to-band comparison of isozyme profiles is useful in assessing the real applicability of in vitro propagation [1, 2, 3]. In the present investigation, the clonal fidelity and varietal diversity was assessed in black pepper (Piper nigrum L.) using peroxidase polymorphism exhibited by 20 conventional clones (CC-raised by rooted cuttings) and 38 tissue culture derived clones (TCC) of the varieties Panniyur 1 (P1), Panniyur 2 (P2), Panniyur 4 (P4) and Subhakara (Su). Enzyme was extracted from 4g mature leaf pieces (5th to 7th dark green leaves from tip) using 5ml Tris buffer (0.15M, pH 7.5-7.6) containing sodium metabisulfite and L-ascorbic acid (0.1 M). The supernatant obtained by centrifugation (15000 rpm for 15 minutes at 4°C) was stored in aliquots at -20°C up to four days without any significant decrease in activity. Best resolution was obtained when electrophoresis was carried out using polyacrylamide gel (8.5%) prepared with Tris buffer (0.0375 M for resolving gel and 0.0125M for stacking gel, pH 8.9) and Tris-Glycine as tank buffer (0.0125 M Tris-0.096 M Glycine, pH 8.3). The staining was done by the standard procedure [4]. P. colubrinum sample was loaded in the last well of every gel in order to act as an internal marker for comparison. The relative mobility (Rm) value of individual isozyme band was calculated by measuring the distance travelled by the band relative to that of the marker band.