1.
Name of Institute:- PAU Ludhiana
Project 1:Title:- Development of dsRNA based Nanoinsecticides: Novel applications for
insect control
Name of the PI:- Dr Satnam Singh
Main objectives:
1. Identification and designing of appropriate nanoparticle (encapsulated/ tethered),
nanoconjugate to be used as an active agent for the delivery of dsRNA into the
insect system
2. Delivery of suitable nanaoparticle tethered/encapsulated dsRNA/synthetic
organic molecule into the insect system through surface contact.
3. Quantification of efficacy of the nanoparticle mediated delivered target molecule
directed towards disruption of specific biological processes in the insect system.
Report:
Characterization of the Nano particles of chitosan: The protocols for the preparation
of nano particles of chitosan has been standardized and the nanoparticles have been
characterized. The conjugation protocols for dsRNA and nanoparticles has been
standardized (Fig. 1 and Fig 2). Preparation of dsGFP (double stranded RNA of green
fluorescent protein) for its conjugation with the chitosan nanoparticles is being carried
out. The GFP dsRNA will also serve as control for various bioassay on insects. The
purified dsGFP (30-40 µg) was mixed with 100 µl of Chitosan nanoparticles and 100
mM of Sodium phosphate buffer. The mixture was tapped and heated at 57 ˚C for 1
min. The mixture was kept at room temperature for two hours followed by overnight
exposure at 4˚C (refrigerator) for desirable conjugation. The mixture was centrifuged
at 18000g for one hour to pellet down the nanoconjugate. The pellet was resuspended
in 40 µl sterile deionized/ triple distil water. The Nanoconjugate was tested for
presence of dsRNA on agarose gel retardation, 1 µl of the conjugate was loaded on
1 % Agarose-EtBr gel. (Fig 3) along with dsGFP and chitosan alone. The gel image
clearly depicted the conjugation of dsRNA with chitosan which was stuck in the well
due to higher molecular weight. Cloning and characterization of the some potent genes
such as aquaporin, SNF7, IAP is in progress. The vital genes from the available
database are such as HSPs, Actin, tubulin, VATPase are being used for the in vitro
synthesis of the dsRNA and their conjugation with nanoparticles and feeding will be
taken up this year. The protocols for the synthesis of other nanoparticles such as
nanosilica, CQD have been in progress and their conjugation compatibility with dsRNA
is in progress.
Fig 1 Chitosan nanoparticles+ RNA observed under phase contrast microscope (40X) to check
the polydispersity of the particles
a
b
c
d
Fig 2SEM images of Chitosan nanoparticles+ RNA(image d possibly depicting the clumped
RNA coming out of the burst chitosan capsule)
M
1
2
3
4
5
Fig 3 Gel retardation assay for the confirmation of conjugation of dsRNA and chitosan
nanoparticles. Lane 23 and 4 showing the conjugate stuck in well (M-marker; 1Chitosan 2µl; 2-Chitosan 1µl; 3 Chitosan +dsGFP 2µl;4 Chitosan +dsGFP 1µl; 5-dsGFP
1µl)
Project 2:Title:- Nanosynthesis and application of micronutrients for enhanced nutrient
use efficiency
Name of the PI:- Dr. S.S. Mukhopadhyay,
Main objectives:
Fabricating Zn2+ and Cu2+ in clay-mineral receptacles in nanoforms - with and
without encapsulation - for their use as fertilizer in the neutral to alkaline soils
Report:
 The Principle Investigator had a NAIP funded project on nanotechnology till
31 March 2014, which was one of the three projects rated as “Over
Performed” by ICAR. During this period, 4 products on Zn 2+ and
nanofabrication processes were developed (Theme I and Theme 8.1). The
title of the project was, “Nano-technology for enhanced utilization of native
phosphorous by plants and higher moisture retention in arid soils”.
 During the tenure of the current project, Indian and PCT International Patent
Applications have been filled. (Please see Annexure for details).
 Commercialization process of the technologies is looking after by
ZTM&BPD Unit, ICAR-IARI, New Delhi. Presentations are made to some
corporate companies including M/s Corromandel International, and M/s ICL
Innovation.
 Receptacle: During the project period, mining source kaolin minerals are
characterized, and one paper is in press (Clay Research). (Theme I and
Theme 8.1).
 Novel Copper Nano product: Apatite as receptacle has been synthesized
and reacted with copper following an alternate route, and its
characterization is in progress. (Theme I and Theme 8.1)
 Capping material: Soil humus that are bound on clays could be the best
ecologically sustainable material to be used as capping material. A doctoral
work has been done towards this goal. (Theme 8.4; Programme iii).
Project 3:Title:- Enhancement of shelf life of fruits using nanocomposite based active
packaging.
Name of the PI:- Dr. Anirudh Thakur
Main objectives:
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
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To develop suitable nanocomposite based polymer films
Testing the effect of nanocomposite based active polymer films on
postharvest stability
and storage life of kinnow mandarin, guava, litchi and peach.
To study the effect of nanocomposite based active polymer films on kinnow
mandarin, guava, litchi and peach.
Report:
The silver and zinc oxide nanoparticles were synthesized by chemical and biological
routes synthesis (using bacterial and fungal cell free extracts). The synthesized
nanoparticles were characterized by UV-Vis spectroscopy, Scanning and
Transmission EM. Different working concentrations, shaking/ static and light and dark
conditions were standardized for the synthesis of these nanoparticles. The
synthesized nanoparticles were assessed for their antimicrobial activity using agar well
technique using known amount of formulation on both plant and human pathogenic
and beneficial microbes. The standard Kirby disc diffusion assay was also performed
for assessing the antimicrobial spectrum of the test cultures using reference
antibiotics. These nano-formulations were incorporated at different rates for synthesis
of biodegradable chitosan cling films for enhancing shelf life of wrapped fruit.
Fig 1. Synthesis, characterization and antimicrobial activity of silver and zinc
oxide nanoparticles and chitosan supplemented cling films. a. Scanning EM of
silver nanoparticles, b. Scanning EM of zinc oxide nanoparticles, c. Antimicrobial
activity of different concentrations of silver nanoparticles on Escherichia coli, d.
Transmission EM of silver nanoparticles, e. Transmission EM of zinc oxide
nanoparticles, f. Antimicrobial activity of different concentrations of zinc oxide
nanoparticles on Escherichia coli, g. Biodegradable chitosan cling film, h. Scanning
EM of chitosan supplemented with 8 ppm silver nanoparticles, i. Antimicrobial activity
of chitosan films supplemented with different concentrations of silver and zinc oxide
nanoaprticles on Escherichia coli.