Nutrient deficiency in food crops is seriously affecting human health, especially those in the rural areas. There are several ways of fortifying the nutrients in food such as dietary diversification, use of drugs and industrial fortification. One of the most intensively consumed metal oxide nanoparticles (NPs) worldwide, titanium dioxide nanoparticles (nTiO₂) is applied in many widely used products, such as in food production, in personal care products, in electronics and pharmaceuticals, and in environmental remediation. To date, little information is available on whether nTiO₂ amendment can enhance vegetable nutritional quality and alter spatial distribution of the important nutrient elements in the edible tissues. To address this knowledge gap, the vegetable coriander was selected as a model plant species. Coriander is an aromatic annual herb in Apiaceae family and possesses significant nutritional and medicinal properties. In this study, coriander (Coriandrum sativum L.) was treated with 0, 50, 100, 200, and 400 mg/L nTiO₂ to evaluate their possible benefit to plant growth and nutritional quality under hydroponic conditions. Observations showed that 50 mg/L nTiO₂ significantly increased the root and shoot fresh biomass by 13.2 % and 4.1 %, respectively, relative to the control. nTiO₂ at this level promoted shoot K, Ca, Mg, Fe, Mn, Zn, and B accumulation, while spatial distribution of K, Ca, Fe, Mn, Cu and Zn in coriander leaves was not affected. No nTiO₂ internalization or translocation to shoots occurred. 400 mg/L nTiO₂ significantly reduced root fresh biomass by 15.8 % and water content by 6.7%. Moreover, this high dose induced root cell membrane wrinkling, attributable to their aggregation and adsorption on root surfaces. At 100–400 mg/L concentration, antioxidant defense systems (SOD, CAT and APX) in plant were triggered to alleviate oxidative stress. At an appropriate dose (50 mg/L), nTiO₂ can improve nutrient quality of edible tissues without exerting toxicity to plant or posing health risk to consumers.

1. Improvement in nutritional quality of spices
through potential use of titanium dioxide (TiO₂)
nanoparticles application
Credit Seminar (BIO 691)
Shreya Mandal
PhD 1st Year (11733)
Division of Biochemistry
ICAR-IARI
2021

2. Introduction
Nutrient deficiency is a serious threat to human
health, especially in the rural areas.
Several ways of fortifying the nutrients in food
such as dietary diversification, use of drugs and
industrial fortification.
Most conventional fertilizers have low nutrient use
and uptake efficiency.
Metal oxide nanoparticles (NPs) nTiO₂ is widely
used
 food production
 personal care products
 electronics and pharmaceuticals
 environmental remediation
TEM of TiO₂ NPs
Non toxic, inexpensive,
highly photoactive, easily
synthesized, handled

3. Introduction
In 2020, production volume of TiO₂ across India around 140 thousand MT, 2
fold rise in production volume in 2018.
Nanomaterials - materials of which a single unit small sized (in at least one
dimension) between 1 and 100 nm (the usual definition of nanoscale)
High surface area of a material in nanoparticle allows heat, molecules, ions to
diffuse into or out of the particles at a very large rates
Small particle diameter allows whole material to reach homogeneous
equilibrium with respect to diffusion in a very short time
Number and share of nanoscience
articles of different countries in
2019

4. Introduction
Coriander is an aromatic annual herb
in Apiaceae family and possesses
significant nutritional and medicinal
properties.
This plant is mainly cultivated for
flavoring as an herb since its fresh
shoots and dry powder of seeds have
notable organoleptic properties.
Vegetable coriander was selected as a
model plant species

5. Reference articles

6. Materials and Methods
Preparation of nTiO₂
Chemical method
Hydrothermal method

7. Materials and Methods
Characterization of nTiO₂ nanoparticles (nTiO₂, 99.9 % in purity)
with an anatase crystalline structure
Size and morphology of nTiO₂ were determined
by transmission electron microscopy
Particle size in the incubation medium of nTiO₂
powder was dispersed by ultrasonication in 50 %
Hoagland’s nutrient solution and Milli-Q water (pH
5.7) for 1 h.
Hydrodynamic diameter of nTiO₂ was determined
with Zetasizer Nano ZS90

8. Materials and Methods
Application of nTiO₂
nanoparticles
nTiO₂ was suspended in
50 % Hoagland’s
nutrient solution (2 g/L
NPs) and dispersed by
ultrasonic vibration (100
W, 40 kHz) for 1 h
coriander seedlings
were cultivated in the
50 % nutrient solution
amended with 0, 50,
100, 200, 400 mg/L
nTiO₂ respectively for
one week
3 plants were
chosen for each
analysis and all
parameters were
measured with
three replications
To prevent photocatalysis
activity of nTiO₂ on uptake
of nutrient elements by
plants, the pot for
seedling cultivation was
covered with an aluminum
foil

9. Materials and Methods
Sample preparation
Samples (including roots and leaves) were
pre-fixed in 2.5 % glutaraldehyde
Washed in 0.1 M phosphate buffer (pH 7.0)
Post-fixed in 1 % osmium tetroxide
Dehydrated in graded concentrations of
ethanol, then infiltrated, embedded in epoxy
resin
Root tips and leaves were crosscut using a
Leica EM UC6 microtome, and were observed
using a FEI TecnaiSpiri 120 kV TEM
TEM SEM
Root tips and leaves treated with 400
mg/L nTiO₂ were cut into small pieces
Prefixed in 2.5 % glutaraldehyde
Standard dehydration treatment
Cross sections were observed using a FEI
Quanta 200 F field emission
environmental Scanning electron
microscopy with energy dispersive X-ray
spectroscopy(SEM-EDS)

10. Materials and Methods
1. Determination of Fresh Biomass (root and shoot) CPA 1003 P electronic analytical
balance (Sartorius AG., Germany)
2. Determination of Dry Biomass (root and shoot)
Tissues with nTiO₂ amendment at all
levels were then dried for 48 h at 60°C
and were re-weighed
3. Determination of water content (FB − DB)/FB × 100 %
4. Determination of plant pigment (chlorophyll
and carotenoids)
Extraction with 95 % ethanol
(Lichtenthaler, 1987)
5. Determination of Malondialdehyde (MDA)
content
Thiobarbituric acid method (Heath and
Packer, 1968)
6. Determination of soluble protein content Staining method with Coomassie brilliant
blue G-250 as described by Ren et al.,
(2011)

11. Materials and Methods
7. Determination of SOD activity Nitrobluetetrazolium (NBT) (Wang et
al., 2004)
8. Determination of CAT activity Method by Gallego et al., 1996
9. Determination of APX activity Method of Dixit et al., 2001
10. To map the distribution of Ti and other
nutritional elements (K, Ca, Fe, Mn, Cu, and Zn)
μ-XRF mapping; data reduction and
processing were performed using PyMca
package (Solé et al., 2007)
11. Concentration of Ti in coriander roots and
shoots, and the nutritional elements Na, K, Ca,
Mg, Fe, Mn, Zn, B, Cu, Co, and Ni in coriander
leaves
Prodigy 7 inductively coupled plasma
atomic emission spectrometer (ICP-AES)
(Leeman, USA)

12. Materials and Methods
μ-XRF analysis
Cleaned leaf samples were dissected and prepared
Leaf tissues were frozen in liquid nitrogen and embedded into Tissue Tek
resin
Cut into 40 μm thick sections using a cryomicrotome and mounted onto a
Kapton tape
Sections from 4 plants including two from the control and the rest two
from 400 mg/L nTiO₂ treatment were used for μ-XRF mapping
Distribution of Ti and other elements in leaves were measured at the
4W1B beamline. The electron energy in the storage ring was 2.5 GeV with
a current ranging from 200 to 300 mA. The incident beam was focused
with the size of 50 μm × 50 μm. A monochromatic X-ray with a photon
energy of 15 keV was used to excite the sample and the count time was
10s per pixel

13. Materials and Methods
Element content
analysis
Coriander roots were sonicated for 5s (Ultrasonic Cleanser
KQ500DE, 500 W, 40 kHz in Milli-Q water to remove the
surface-adhered NPs (Hu et al., 2017)
Root and shoot tissues of all treatments were thoroughly
rinsed with Milli-Q water and dried for 48 h at 60°C.
Samples were digested with hydrofluoric acid (HF) (Ohno et
al., 2001)
50 mg of shoot tissue or 10 mg of root tissue were transferred
to a digestion tube containing 2 mL concentrated H₂SO₄,
Mixture was heated for 1 h at 120 °C and was then cooled
down to ambient temperature
2 mL H₂O ₂ was added for further digestion for additional 30
min at 120 °C
2 mL HF was added to the mixture to thoroughly digest nTiO₂
and the samples were stored for approximately 1 day at room
temperature

14. Results and Discussion

15. Effects of nTiO₂ at different concentrations on coriander growth
Phenotypic images of coriander grown in nTiO₂-
amended system

16. The effects of nTiO₂ at different concentrations on coriander’s Fresh biomass (Left
side); Water content (Right side).
Data are shown as mean ± SD of three replicates. Within a tissue type, values
followed by different lowercase letters are significantly different at p ≤ 0.05 in
Duncan’s test.
Effect of nTiO₂ on the growth of coriander

17. (A). The effects of nTiO₂ at different concentrations on photosynthetic pigment
content in coriander leaves
Data are shown as mean ± SD of three replicates. Values followed by different
lowercase letters are significantly different at p ≤ 0.05 in Duncan’s test. (Ca:
chlorophyll a; Cb: chlorophyll b; Ca+Cb: total chlorophyll; Cx.c: carotenoid)
Photosynthetic pigment content in nTiO₂-
amended coriander leaves

18. (B). The effects of nTiO₂ at different concentrations on soluble protein
content in coriander roots and shoots
Data are shown as mean ± SD of three replicates. Values followed by different
lowercase letters are significantly different at p ≤ 0.05 in Duncan’s test
Soluble protein content in nTiO₂- amended
coriander shoots and roots

19. A.
B.
C. Table. A. B, C
Accumulation of macro and micro
nutrient (mg/kg) in coriander shoots
amended with nTiO₂ at different
concentrations

20. μ-XRF images from
crosscut coriander leaves
amended with 400 mg/L
nTiO₂ showing K, Ca, Fe,
Mn, Cu and Zn intensities
Colors from dark blue to
red indicate the intensity of
element from low to high
Distribution pattern of macro- and micro-nutrient in
coriander shoots

21. Uptake and translocation of nTiO₂
SEM image and corresponding EDS analysis of the root vascular cylinder and leaf cross
section in coriander plants amended with 400 mg/L nTiO₂

22. Coriander ultrastructure analysis
TEM images of roots and leaves from coriander plants without NP treatment (control) (A &
C) or treated with 400 mg/L nTiO₂ (B & D)
Cw: cell wall; cm: cell membrane; chl: chloroplast; sg: starch grain

23. Effect of nTiO₂ amendment on antixidant enzymes
in coriander shoots and roots
The effects of nTiO₂ at different
concentrations on the activities of
various antioxidant enzymes.
Data are shown as mean ± SD of
three replicates. Values followed by
different lowercase letters are
significantly different at p ≤ 0.05 in
Duncan’s test

24. Effect of nTiO₂ amendment on lipid peroxidation
in coriander shoots and roots
The effects of nTiO₂ at different concentrations on lipid peroxidation in
coriander shoots and roots
Data are shown as mean ± SD of three replicates. Values followed by different
lowercase letters are significantly different at p ≤ 0.05 in Duncan’s test

25. Conclusion
Application of
nTiO2 at
different
concentration
in coriander
demonstrates
50mg/L
significantly improve nutritional quality of coriander
plants without causing significant oxidative stress
50-200mg/L
increased fresh biomass of root (7.9-13.2%) and shoot
(4.1-12.3%)
100-200mg/L
exhibited evident oxidative stress
400mg/L
nutrient contents in edible tissues generally sharply
decreased; growth inhibited as indicated by decreased
root length, fresh biomass, water content

26. Summary
nTiO₂ at a low con. enhanced nutritional quality
of coriander
nTiO₂ did not change spatial distribution in
coriander leaves
No nTiO₂ accumulation in edible parts
suggests minimal risk to food safety
nTiO₂ at a high conc. may damage root cell
membrane and inhibit growth

27. Advantages
Potential
strategy to
enhance crop
nutritional
quality
Minimal risk
to food safety
Use of nTiO₂
as a nano-
enabled
growth
regulator

28. Challenges and Future Prospects
Assessment of
safety and toxicity
level
Nutritional
security,
Production
sustainability
International Agency for
Research on Cancer (IARC)
placed nTiO₂ into Group 2
carcinogen, “possibly
carcinogenic to humans
(Baan et al., 2006)
nTiO₂ - potential to
positively stimulate plant
growth, could pose threats
to human health especially
at a high amendment level
Suitable Particle
size < 20nm
(penetrate root
cell easily)

29. Pathahead
Plant mediated biological methods of synthesis
utilising raw materials such as waste vegetables,
plant barks, roots should be exploited and
expanded (Phytonanotechnology/ green
nanotechnology)
Additional work to fully characterise underlying
mechanism
Regulations on nanoproducts to protect environment
and entire public health
Product information for the nanomaterials should be
provided

30. I would like to acknowledge the support of Dr. Vinutha
mam as seminar leader, Professor -Dr. Anil Dahuja Sir,
HOD- Dr. Shelly Praveen mam and all my respected
faculties.
My special thanks to Dr. Archana Singh mam, as my
chairperson.
I would like to extend sincere thanks to IARI PG School.
Last but not the least I thank you batchmates, beloved
seniors and juniors.