Abstract:
Piper nigrum L. is traditionally propagated by stem cuttings and seeds, but seeds tend to produce progenies with
higher variations due to cross-pollination habits. Plant tissue culture technique is the most efficient and reliable
method for rapid clonal multiplication, however, endophyte microbial contamination limits the success. Twelve
surface sterilization protocols were tested on two bud types (apical and axillary buds) and three different maturity
stages of the leaf (first, third and fifth leaves) to optimize the conditions for culture initiation. The Completely
Randomized Design was used with 60 explants. Maximum likelihood analysis of variance was conducted using the
Proc CatMod procedures of PC-SAS to analyse the count data. The continuous data were analysed using Analysis
of Variance and the mean separation was done using Least Significant Difference. Results revealed that the
optimal sterilization protocol was specific to the explant type. The third leaf from the top of the plant and the
apical bud was the best explants giving minimum tissue contamination and browning. Fungal contamination was
frequent in leaf explants whereas bacteria in bud explants. The protocols containing 70% ethanol (30s), 0.1%
HgCl2 (5 min) and sterile distilled water with activated charcoal (1 gL-1
; 25 min), and 20% sodium hypochlorite
(NaOCl) (15 min) with 70% ethanol (1 min) were comparable for the third leaf. In apical buds, the protocols of
0.1% HgCl2 (10 min) and 70% ethanol (1 min), and 10% NaOCl (15 min) with 70% ethanol (1 min) provided
comparable performances with the highest survival and least contamination rates. The potential of replacing
hazardous HgCl2 with non-toxic NaOCl by manipulating the concentration and the exposure time in combination
with 70% ethanol was highlighted.
