Medicinal Plant Research 2015, Vol.5, No. 6, 1-9
7
control (EPC) injector. Helium was used as the carrier
gas with a flow rate 1.2 ml/min through Restek DB-5
capillary column (30 m long x 0.53 µm diameter and
film thickness 0.5 µm). One µl oil sample was
injected, diluted in hexane (0.05 ml of oil into 0.95 ml
of GC standard grade hexane) and the samples were
analyzed by GC. Analysis was programmed for 15
min at 60
o
C, rising up to 240
o
C at the rate of 5ºC/min.
EO components were identified by comparing their
retention times with those of authentic standards of
linalool, 1,8- cineol, methyl cinnamte, methyl eugenol
and camphor (CDH and Merck, India) run under the
same conditions (Kovats, 1965). Concentration of
each component in oil sample was calculated using
the following equation (Lee et al., 2005).
Concentration (mg/g) = Weight of extract (without
solvent) x GC peak area % 100 (mg) / Weight of plant
material (g)
3.4.
Trichome study
Micromorphological observations were carried out on
fresh basil leaves by light microscopy (LM), scanning
electron microscopy (SEM) and transmission electron
microscopy (TEM). TEM was used to reveal
ultrastructural details of trichomes.
3.4.1.
Light microscopy (LM):
Hand-peel mounts
were prepared to study trichome density per unit leaf
area on both adaxial and abaxial surfaces. For paraffin
wax sectioning, plant material was fixed in FAA
(formaldehyde: glacial acetic acid: ethanol, 1:1:18,
v/v/v) for 24 h. For dehydration the fixed plant parts
were passed through tertiary butyl alcohol (TBA)
series. Embedding was done in paraffin wax following
the procedure of O’Brien and McCully (1981). Wax
blocks containing embedded material were trimmed
and mounted on wooden blocks for sectioning with
the help of Riechert rotary microtome. Sections of 8
µm thickness were cut, dewaxed and dehydrated through
ethyl alcohol-xylene series and stained with 1% safranin
and 1% astra blue combination. Mounting was done
in DPX and observed under Nikon photomicroscope.
3.4.2.
Scanning electron microscopy (SEM):
Leaf
segments were fixed in Karnovsky’s fixative, buffered
with 0.1 M sodium phosphate buffer at pH 7.4 for 6-8
h at 4
o
C. After washing in the buffer the material was
dehydrated in a graded ethanol series, critical point
dried with CO
2
, mounted in stubs and coated with a
thin layer of gold (Serrato-Valenti et al., 1997).
Sections were observed under LEO 435 VP scanning
electron microscope at 15 KV.
3.4.3.
Transmission electron microscopy (TEM):
For ultrastructural investigation, leaves were trimmed
and small pieces from margin up to midrib were fixed
in Karnovsky’s fluid, buffered with 0.1 M sodium
phosphate buffer at pH 7.4 for 6-8 h at 4
o
C and post
fixed in 1% osmium tetraoxide. After dehydration in
ethanol series, the material was embedded in
Epon-Araldite resin. Sections were conventionally
stained with uranyl and lead citrate (Ascensão et al.,
1997) and examined under Philips 200 transmission
electron microscope at 80 KV.
3.5. Statistical analysis
Data were subjected to statistical analysis using
software package SPSS 10.0 (Statistical Package
for Social Sciences). One-way analysis of variance
(ANOVA) followed by multiple comparison least
significance difference (LSD) was employed to check
the significance of the differences between the
treatments at
p
≤ 0.05.
Conclusions
As in growth medium decreased both growth and
biomass of aerial parts in
O. basilicum
. EO yield
showed an up regulation at 10 and 50 mg/kg As, but it
decreased at 150 mg/kg As. Linalool, the main
component was augmented in all As treatments.
Methyl cinnamate and camphor diminished under
higher As concentrations. A strong positive correlation
between trichome density and EO yield was noticed.
Alteration in the structure and ultrastructure of
glandular trichomes demonstrated that trichomes
under As stress achieved precocious senescence,
which further led to malfunctioning of secretory
machinery. However, there is further need to carry out
experiments in natural and controlled conditions to
understand the exact mechanism by which the
metabolic pathways are altered. Data provides an
early indication that environmental pollutants like As
affect the medicinal quality of
O. basilcum
. The
development of optimized agricultural practices is
essential for the sustainable cultivation of
O. basilicum
and
adequate yield of EO.
References
Abedin M.J., and Meharg A.A., 2002, Relative toxicity of arsenite and
arsenate on germination and early seedling growth of rice (
Oryza
