Surface sterilization protocols of leaf and bud explants for initiating in vitro cultures of Piper nigrum L. (Pepper)

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 (1gL^-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.