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Research & Reviews in Biotechnology & Biosciences ISSN No: 2321-8681
29
©2018 The author(s). Published by National Press Associates. This is an open access article under CC-BY License
(https://creativecommons.org/licenses/by/4.0/),
Foldscopebased methods to detect in-tissue antioxidant activity
and secondary metabolites in pollen and stomata of Lantana
camara
Priya Nischal & ArunDev Sharma
PG Department of Biotechnology, Lyallpur Khalsa College, GT Road, Jalandhar- 144001,
Punjab, India
DOI: https://doi.org/10.6084/m9.figshare.8977313.v1
Abstract
Foldscope is the ultra-affordable, paper microscope designed to be
extremely portable, durable, and to give optical quality similar to
conventional research microscopes. However it’s potential as in tool
in the area of agricultural sciences is still not known. In this study,
how foldscope can be used as an efficient tool to detect in –tissue
activities of antioxidants and secondary metabolites in invasive alien
plant Lantana camara.Foldcsope images revealed viable stained areas
in the respective seeds, indicating live activities. Based on data the
use of foldscope was recommended.
Keywords: Foldscope, pollens, stomata, Lantana
INTRODUCTION
Foldscope, is an paper based, portable
microscope with magnification power of
140 X. it was invented by Prakash lab at
Stanford University of America in 2014 [1].
Thereafter, Department of Biotechnology
(DBT), Government of India and Prakash
Lab at Stanford University, USA earlier
signed an agreement to bring the Foldscope
to India to encourage curiosity in science.
Invasive Alien Species are basically non-
native plant species that displace native
species and pose adverse effects to
environment, ecosystem, economy and
human health by diminishing the growth of
native plants and also possess higher stress
tolerance. They are either introduced to a
new environment from their natural habitat
by sudden or accidental escape. Lantana
camara (belongs to Verbenaceae), is an
ornamental and noxious weed as it harms
other plants, crop productivity, animals,
organisms and others [2]. Stomata and
pollen represents are two important organs
present in plants, that are challenged by
unfavourable conditions including biotic
and abiotic stress like high and low
temperature, light, nutrients, etc.
Fluctuations in all these factors have large
impact on plant performance during their
regular life cycle. Stomata mainly helps in
exchange of gases and water balance
between the environment and intracellular
spaces of plants which results in changing
environment around the plant species and
involved in adaptation of plants under
adverse abiotic conditions. Pollen grains
contain the male reproductive cells which
need to be transferred to the female
reproductive structure of the plant for
further fertilisation process.However the
Research & Reviews in Biotechnology &
Biosciences
Homepage: www.biotechjournal.in
Volume: 5, Issue: 1, Year: 2018 PP: 29-33
Short
Communication
Article History
Received: 10/02/2018
Revised: 02/03/2018
Accepted: 03/04/2018
*Corresponding Author:
E-Mail: arundevsharma47@gmail.com
Lyallpur Khalsa College
Jalandhar-144001, Punjab
Research & Reviews in Biotechnology & Biosciences ISSN No: 2321-8681
30
©2018 The author(s). Published by National Press Associates. This is an open access article under CC-BY License
(https://creativecommons.org/licenses/by/4.0/),
role of foldscope as an efficient tool in plant
biology is not known worldwide so far.
With this background, the present study
was carried out to assess the usefulness of
portable foldscope for in–situ detection of
some antioxidant metabolites in pollens
and stomata of Lantanacamara. Due to its
less cost, environmental friendly, small
size, and portable in nature, can be widely
used even by Plant Biologists in the field
conditions.
MATERIALS AND METHODS
Plant materials
Leaves and flowers of Lantanacamara were
washed with distilled water for further
analysis. Epidermis from leaves and pollens
from flowers was then peeled out carefully
from fully expanded leaves at reproductive
stage, immediately prior to each experiment.
Image detection
Foldscope was used in current study, was
supplied by Department of Biotechnology
(DBT), Government of India. Thin section of
tissues were taken on glass slides and
covered with transparent cello tape/ cover
slip. The slide was inserted into the
foldscope in such a way that sample side
was close to lens of foldcope. A LED light
supplied with foldscope instruments was
used a light source. The clear images under
foldscope for each sample were
photographed using cell phone camera
(Samsung, Galaxy Tab A) by adjusting zoom
and focusing of camera and foldscope.
In-situ localisation of ROS and
antioxidants
H2O2 content
The tissue samples were incubated in 1
mg/ml of 3,3'-Diaminobenzidne (DAB)-HCl
,pH 3.8 for 6-7 hours at room temperature.
H2O2 was visualized as deposits of dark
Brown insoluble formazan compounds.
O2- content
Plant tissues were incubated in 6mM Nitro
Blue Tetrazolium (NBT) in 10mM Tris-HCl
buffer, pH 7.4 at room temperature for 24 h.
O2- was visualized as deposits of dark blue
insoluble formazan compounds.
Glutathione (GSH) content
The samples were incubated in reaction
mixture containing 50 mMTris–HCl buffer
(pH 7.0) and 0.3 mM 5,5´-dithiobis(2-
nitrobenzoic acid (DTNB) in a total volume
of 5 ml at 37oC for 24 hours. The activity can
be visualized as yellow colored stain in the
tissue.
Ascorbate (AsA) content
The tissue samples were incubated in
reaction mixture contained 2 ml of 2% Na-
molybdate, 2 ml of 0.15 N H2SO4, 1 ml of 1.5
mM Na2HPO4 bringing the total volume to 5
ml with buffer for 24 hours. The activity can
be visualized as yellow colored stain in the
tissue.
Thioredoxinreductase (TRx.R) activity
Plant tissues were incubated in assay
mixture contained 50 mMTris-HCl (pH 7.0),
1 mM EDTA, 0.2 mM NADPH, 0.2 mg/mL
bovine serum albumin and 5 mM
5,5´(dithiobis-(2-nitrobenzoic) acid at 25oC in
a total volume of 5 mL for 24 hours. The
activity can be visualized as yellow colored
stain in the tissue.
NADPH Oxidase activity
Tissues were incubated in dark for 24 hours
in a reaction mixture solution containing 50
mMTris-HCl buffer (pH 7.4), 0.2 mM NBT,
0.1 mM MgCl2 and 1 mM CaCl2, 0.2 mM
NADPH for 3-4 hours and the appearance of
blue formazancolor was monitored.
Catalase (CAT) activity
Research & Reviews in Biotechnology & Biosciences ISSN No: 2321-8681
31
©2018 The author(s). Published by National Press Associates. This is an open access article under CC-BY License
(https://creativecommons.org/licenses/by/4.0/),
The tissues were immersed in 50 mM K-
PO4, pH 7.0 for 20 min and then in 0.03%
H2O2 solution in H2O (1/1000 dilution of
30% stock) for 30 min. The tissue was rinsed
twice with H2O and then incubated in a
mixture (1:1) of freshly prepared 2%
K3Fe(CN)6 and 2% FeCl3 for 15 minutes. The
tissue became greenish-blue while zones of
catalase activity were yellow.
Superoxide dismutase (SOD) activity
The tissues were first soaked in 10 ml of 1.23
mM NBT for 15 min, briefly washed with
water, then soaked in the dark in 10 ml of
100 mM potassium phosphate buffer (pH
7.0) containing 28 mM TEMED and 2.8 x 10-
2mM riboflavin for another 4 hours. The
reaction was stopped by briefly washing the
tissue with water and illuminating it under
white light to initiate the photochemical
reaction. The regions of SOD activity
appeared as colourless areas on a purple
background.
Peroxidase (POD) activity
The tissue was washed three times in 50 mM
sodium acetate buffer (pH 5.0). Peroxidase
activity was visualised by incubating the
tissue in 50 ml of a solution containing 50
mM sodium acetate buffer (pH 5.0), 330 µl of
guaiacol and 330 µl of 6.6 % H2O2 for 20
minutes. The tissue was incubated at room
temperature in the dark until reddish brown
bands appeared.
Monodehydroascorbatereductase (MDAR)
activity
The plant tissue was immersed in a solution
of 1.3 mM NADH, 1.2 mM 3-[4,5-
dimethylthiazol-2-yl]-2,5-
diphenyltetrazolium bromide and 0.06 mM
2,6-dichlorophenol-indophenol in 0.25 M
potassium phosphate buffer (pH 8.0) for 2
hours in the dark. The spots appeared dark
on a blue background.
Histo-chemical detection of secondary
metabolites
Lignin Detection (Potassium permanganate)
Samples were flooded with a solution of 1%
KMnO4 for 4 hours and rinsed with water.
Precipitated KMnO4 appears very dark and
is claimed to be quite specific for lignin.
SuberinDetection (Gentian Violet)
Samples of plant tissues were flooded for 4
hours with a solution of 1% (w/v) gentian
violet in 70% ethanol plus 1 or 2 drops of
concentrated ammonium hydroxide. The
tissues were rinsed with 9% HCl for 2 min
or until clear. The sample were then flooded
with 95% ethanol for 10 min and rinsed with
ethanol and water. Suberized areas stain
blue or purple.
Phenolics and Tannins Detection (Ferric
chloride)
A few drops of 10% (w/v) ferric chloride
solution were added to the tissue for upto 4
hours. A blue-green precipitate will develop
in the presence of phenolic compounds and
tannins.
Lipids Detection (Nile Blue)
Tissues were incubated in 10 ml of saturated
aqueous solution of Nile blue sulfate
containing 1 ml of 0.5% H2SO4 for 4 hours.
Lipid stain blue.
Cellulose or hemicellulose Detection (Iodide-
sulphuric)
The tissues were treated with upto 1 ml of
potassium tri-iodide solution for 3 hours. A
cover slip was added and a drop of 65%
H2SO4 introduced and allowed to diffuse
under the glass. Cellulose or hemicellulose
stains dark blue, lignin stains orange to
yellow.
Cellulose or hemicellulose Detection (Zinc-
chloride-iodide)
Research & Reviews in Biotechnology & Biosciences ISSN No: 2321-8681
32
©2018 The author(s). Published by National Press Associates. This is an open access article under CC-BY License
(https://creativecommons.org/licenses/by/4.0/),
The samples were treated with a few drops
of chloro-iodide (50 g of zinc chloride plus
16 g of potassium iodide in 17 ml of water)
for 4 hours. Large amounts of cellulose or
hemicellulose stain blue. Lignin, cutin,
suberin or chitin stain yellow to orange and
may mask cellulose.
Suberin and Cutin Detection (Sudan IV)
Several drops of a saturated solution of
Sudan IV in 70% ethanol were added to the
tissues for 4 hours following by quick rinses
in three changes of 50% ethanol. Lipid
containing material such as suberin and
cutin stain pink to orange.
Callose Detection (Aniline Blue)
Five to ten drops of 0.5% (w/v) aniline blue
in 50% ethanol were placed on the sample
for 4-8 h and rinsed with 50% ethanol.
Callose deposits were stained blue.
RESULT AND DISCUSSION
Foldscope based in-tissue activity analysis
was carried out to detect antioxidants and
secondary metabolites in pollens and
stomata of lantanacamara. As compared to
control (unstained) conditions, In-situ
localisation and histochemical staining both
in pollens and stomata indicated the activity
stained area of various antioxidant enzymes
(including glutathione, NADPH oxidase,
SOD, POD, MDAR etc.) and secondary
metabolites (including lignin, lipid, cellulose
and hemicellulose, etc.) in pollens and
stomata when incubated in respected
staining solutions (Fig1 and 2).
Many investigators reported that increase in
activity of antioxidant enzymes and level of
secondary metabolites in stressful conditions
are the major factors that lead to stress
tolerance in various plant species under
various adverse abiotic conditions like high
and low temperature, drought, salinity, etc.
[3].
Research & Reviews in Biotechnology & Biosciences ISSN No: 2321-8681
33
©2018 The author(s). Published by National Press Associates. This is an open access article under CC-BY License
(https://creativecommons.org/licenses/by/4.0/),
Based on this data it was concluded that
foldcope can be a cheap, portable and
powerful efficient biological tool to evaluate
plant biology based studies. Further studies
are underway to find out effect of various
abiotic stresses and plant hormones like
ABA on enzymatic activities and metabolites
in stomata and pollens of Lantanacamara.
Acknowledgement
ADS want to thank DBT, Govt of India for
giving Foldscope project grant.
REFERENCES
1. Cybulski JS, Clements J, Prakash M. 2014.
Foldscope: origami-based paper microscope. PLOS
ONE., 2014, 9, pp: e98781.
2. Van Oosterhout, E. 2004. Lantana Control Manual:
Current Management and Control Options for
Lantana (Lantana camara) in Australia, Brisbane:
Dep. Nat. Resour.,Mines, Energy.
3. Gill SS, Tuteja N. 2010. Reactive oxygen species
and antioxidant machinery in abiotic stress
tolerance in crop plants. Plant
PhysiolBiochem48:909–930.
Source of Support: Authors want to Thank
DBT Govt of India for this project work
Conflict of interest: No
