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The study entitled “Efficacy of silver nanoparticles in eliminating systemic
contamination in the in vitro culture of black pepper (Piper nigrum L.) was carried
out at the Department of Plant Biotechnology, College of Agriculture, Vellayani,
Thiruvananthapuram during 2019 to 2021. The objective of the study was to evaluate
the efficacy of silver nanoparticles for eliminating the systemic contamination and
improving the efficacy of micropropagation protocol in black pepper Piper nigrum L.
Single nodal cuttings of Piper nigrum L. var. Panniyur 1 collected from
Instructional Farm, College of Agriculture, Vellayani were used as explants. Among
the five surface sterilization treatments tried with different concentrations of mercuric
chloride, ethyl alcohol and pretreatments with bavistin, surface sterilization with 70 %
ethyl alcohol for 1 min followed by 0.2 % mercuric chloride for 10 min showed
maximum percentage of uncontaminated cultures for a period of two weeks (26.6 %),
Phenolic exudation was observed in all the cultures within a period of two weeks. No
contamination was observed when the uncut surface sterilized nodes were stabbed on
Nutrient Agar (NA) and basal Murashige and Skoog (MS) media, thereby confirming
that surface sterilization treatment was effective and contamination observed in the
cut nodes after a period of two weeks was endophytic in nature. All the cultures were
incubated on the illuminated racks at 25 ◦C with 16 h photoperiod.
Tissue indexing by pricking the tissue with a sterile needle at three different
positions (portions below medium, above the medium, and also at the tip of the shoot)
followed by stabbing the needle on NA and MS media did not show any
contamination. Plant tissue homogenate indexing by grinding one-gram tissue
homogenate in one millilitre of sterile distilled water followed by serial dilution and
plating showed the presence of 5.84 x 106
cfu in NA medium on the second day of
inoculation and 4.88 x 106
cfu colonies in MS medium on the fourth day of
inoculation. On medium indexing by placing the two vertical halves of the node in
horizontal position on NA and MS media, endophytic contamination was observed on
seventh day and fourteenth day of inoculation on NA and MS media respectively.
Serial dilution of the endophytic contamination was done and the colonies picked
from 10-6
dilution were used for bacterial in vitro inhibition assay.
In vitro inhibition assay was carried out using sterile discs loaded with
different concentrations (10 mgL-1
and 20 mgL-1
) of 20 nm and 100 nm silver
nanoparticles alone and in combination with streptopencillin, 150 mgL-1 of
streptopenicillin alone, and sterile distilled water as control. No inhibition zone was
observed on treatments with silver nanoparticles alone whereas an inhibition zone of
28 ± 2 mm was observed for streptopenicillin (150 mgL-1
), 21 ± 1 mm for
streptopenicillin with 20 nm silver nanoparticles and 18 ± 1mm for combination of
streptopenicillin with 100 nm silver nanoparticles.
To conclude, surface sterilization with 70 % alcohol for 1 min followed by
0.2 % mercuric chloride for 10 min was effective in controlling surface contaminants
in black pepper var. Panniyur 1. Endophytic contaminants could not be effectively
removed by 20 nm and 100 nm of silver nanoparticles at concentrations of 10 and
20 mgL-1
and hence is not recommended as an alternative to incorporation of
150 mgL-1
of streptopencillin in controlling the systemic contamination of black
pepper in tissue culture.