Development of a protocol for in-vitro propagation of black pepper (Piper nigrum L.) local selections

Abstract

Black pepper (Piper nigrum L.) belongs to family Piperaceae and it is one of the most economically important spice crops in the world (Srinivasan, 2007; Mathew et al., 2001). Unavailability of sufficient mother plant stock in the field, obtaining basal runners for propagation and less success and multiplication rate of the high yielding local pepper cultivars are the major problems faced by the farmers who cultivate. Being in vitro propagation a promising option, this study was focused to develop a suitable protocol for in vitro propagation of black pepper local selections.

Methodology

This research was carried out at Central Research Station, Department of Export Agriculture, Matale. Four experiments were conducted during the research period. Experiment one was conducted to find out the suitable surface sterilization method for the sterilization of black pepper shoot tips. Selecting of appropriate media for the culture establishment of black pepper shoot tips were carried out in second experiment using 1/3Murashige and Skoog (MS) medium and 1/2 Woody Plant Medium (WPM).Experiment three was conducted to find out suitable combination of auxin and cytokinin for the shoot multiplication of black pepper local selections. In fourth experiment, priority was given for the selection of best media and hormonal combination for the callus initiation of TG7 black pepper local selection. Full and half strength MS media were used as the culture media and two different concentration levels of kinetin and NAA were used as the growth regulators. Complete Randomized Design (CRD) was used as the experimental design. ANOVA was used to analyze the statistical difference of parametric data and non-parametric data were subjected for logarithmic transformation. SAS statistical software was used to analyze the data and mean separation was performed using Least Significant Difference (LSD).

Results and Discussion

As the results summarized in Table 1, sterilization using 10%- 20% Clorox for five to ten minutes (T1 to T5) showed higher percentages of bacterial contamination (40 to 80 %). Lower percentages of fungal contamination was observed in T4 to T8 within the period of three to five days (3% to 7%).The highest survival percentage (66.6 %) was reported in T8, 0.04 % HgCl2 for five minutes. Similarly, the lowest percentages of bacterial and fungal contamination were observed in T8. The highest percentage of phenolic browning (80%) was shown in T6 and lowest percentage of phenolic browning (10.0%) was observed in T2, i.e. 10% Clorox for 10 minutes within four to seven days.