ARTICLE IN PRESS

Journal of Plant Physiology 165 (2008) 755—765

www.elsevier.de/jplph

Oxidative metabolism-related changes

in cryogenically stored neem
(Azadirachta indica A. Juss) seeds
Boby Varghesea, Subhash Chandra Naithanib,

a
African Centre for Plant Germplasm Conservation Research, School of Biological & Conservation Sciences,
University of KwaZulu-Natal, Durban 4041, South Africa
b
Seed Biology Lab, School of Life Sciences, Pt. Ravishankar Shukla University, Raipur 492010, Chhattisgarh, India

Received 25 December 2006; received in revised form 28 March 2007; accepted 31 March 2007

KEYWORDS Abstract
AOS;
The seeds of Azadirachta indica were successfully cryopreserved for 12 months with
Antioxidant
45% survival following drying to 0.16 g H2O g1 dry mass (DM). Highest survival
enzymes;
(94–96%) was recorded during the first month of cryostorage. Subsequent
Cryopreservation;
cryopreservation up to 12 months resulted in decreasing germination. Post-thawing
Neem seeds;
pre-heat treatment enhanced the recovery marginally in seeds cryopreserved from 3
TBRS
to 12 months. Viability of cryostored seeds was negatively correlated with leachate
conductivity and accumulation of thiobarbituric acid reactive substances (TBRS)
estimated in cotyledons and axes. Leachate conductivity of imbibed seeds was low
during the first month of cryostorage but increased gradually with the duration of
cryostorage to a maximum after 12 months. TBRS accumulation was gradual
throughout cryostorage. Relatively low amounts of active oxygen species (AOS)
detected during the first month of cryostorage were closely associated with very high
activities of superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase
(APX) in seeds (cotyledons and axes). Marked accumulation of AOS from 3 to 12
months was associated with decrease in antioxidant enzyme activity.
& 2007 Elsevier GmbH. All rights reserved.

Corresponding author. Tel.: +91 771 2263038;
fax: +91 771 2262631.
E-mail address: naithani_nib@sancharnet.in (S.C. Naithani).
Introduction
Although cryopreservation is the preferred, most
suitable and safest means of conserving plant
genetic resources in seed banks, only a few studies
have evaluated the nature of cryoinjury and its

0176-1617/$ - see front matter & 2007 Elsevier GmbH. All rights reserved.
doi:10.1016/j.jplph.2007.03.009
 ARTICLE IN PRESS
756
relationship with the longevity of the germplasm
(Benson and Bremner, 2004; Walters et al., 2004).
Oxidative stress and related metabolism occur in
and are detrimental to several plant and animal
tissues during low and cryotemperatures (Dussert
et al., 2003, Benson and Bremner, 2004). Synthesis
and accumulation of active oxygen species (AOS:
1
O2, O 
2 , OH , H2O2), which are highly toxic and
reactive chemical species, is central to all oxida-
tive stress-related metabolism (Benson and Brem-
ner, 2004). These are apparently generated during
desiccation, chilling and cryogenic treatments of
plant tissue. Studies on cryopreserved tissues of
Brassica napus (shoot tip) and Daucus carota (cell
suspension) revealed an accumulation of singlet
oxygen that was involved in post-thawing injury
(Benson and Bremner, 2004). Similarly, enhanced
levels of OH radical that were reported in cryoex-
posed carrot cells, oil seed rape meristems, Daucus
carota cells, Euglena gracilis and Haematococcus
pulvialis (Benson and Bremner, 2004) have been
suggested to occur during early post-thawing. Free-
radical-mediated injury during post-thawing has
been suggested for cryopreserved rice and Daucus
carota cell suspensions (Benson and Bremner, 2004)
that showed higher recovery rates by addition of
the iron-chelating drug, desferrioxamine, used to
suppress oxidative stress through preventing AOS
accumulation.
Membrane lipids are primary targets for AOS
attack and oxidized fatty acid reaction products
like conjugated dienes, hydroperoxides and mal-
ondialdehyde (MDA) are frequently used as markers
of oxidative stress (Benson and Bremner, 2004).
Studies on Daucus carota tissue and rice cells
showed increased lipid peroxidation, measured by
MDA production, as a result of cryopreservation/
freezing (Benson and Bremner, 2004). These work
suggested that lipid peroxidation plays a significant
part in cryoinjury and that the early post-thaw
recovery period may be especially damaging,
leading to the conclusion that oxidative injury is
the major factor in destabilization of membranes.
Membrane damage due to imbibitional stress during
rapid rehydration is a prime cause of loss of
germination in frozen Coffea arabica (Dussert
et al., 2003) and dried Azadirachta indica (Sacande
et al., 1998) seeds. It was proposed that the plasma
membrane in the dried and/or frozen seeds is in a
gel phase, which is disrupted during imbibition
under the pressure of the penetrating water, i.e.
rapid rehydration (Dussert et al., 2003). The
imbibitional injury could be prevented by pre-
hydration treatments such as osmoconditioning,
pre-heating and pre-humidifying the seeds prior to
a germination assay (Dussert et al., 2003; Dussert
B. Varghese, S.C. Naithani
and Engelmann, 2006; Sacandé et al., 1998). These
authors have attributed the beneficial effect of
pre-hydration treatments to the melting of mem-
branes, which were in the gel phase as a conse-
quence of drying/drying–freezing.
Protection against free radical damage is the
major means of preventing oxidative stress (Bailly,
2004; Kibinza et al., 2006). Protective mechanisms
against AOS during seed storage are predominantly
enzymic (Bailly, 2004). The activities of superoxide
dismutase (SOD), catalase (CAT) and ascorbate
peroxidase (APX) change markedly during seed
germination in embryonic axes of soybean in
response to cytoplasmic increase in O 2 and H2O2
(Puntarulo et al., 1991). AOS metabolism is the
characteristic feature of fresh mature non-ortho-
dox seeds of Shorea robusta (Chaitanya and
Naithani, 1994, 1998), Azadirachta indica (Sacande
et al., 2000b; Varghese and Naithani, 2002), and
was also suggested for Avicienia marina (Greggains
et al., 2001) seeds. Decreasing antioxidant enzyme
activity is closely associated with the loss of
germinability in recalcitrant seeds like Shorea
robusta (Chaitanya and Naithani, 1994, 1998) and
Avicennia marina (Greggains et al., 2001). This
provides further evidence that disturbance in AOS
metabolism and the differential response of anti-
oxidants should be considered more fully in low/
cryotemperature plant stress physiology.
Loss of viability in Azadirachta indica (neem)
seeds during cryostorage for one year has been
reported (Varghese and Naithani, 2001). The pre-
sent study was therefore undertaken to investigate
the possible cause(s) of cryoinjury by monitoring
AOS, possible associated membrane perturbations
through lipid peroxidation, and antioxidant en-
zymes during long-term (1-year) conservation of
neem seeds at cryogenic temperature.
Materials and methods
Seed collection and extraction
In the third week of June, mature ripe yellow fruits of
Azadirachta indica A. Juss (neem) 12 weeks after
flowering (Varghese and Naithani, 2002) were harvested
manually from trees growing in Pt. Ravishankar Shukla
University campus, Raipur, and brought to the laboratory
immediately. A few seeds from the lot were immediately
depulped mechanically to determine the seed water
content, which is hereafter referred to as the initial
water content. Seeds were extracted from the fruit
tissue by soaking them in 20% H2SO4 (Merck, India) for
30 min after depulping by rubbing with hands over a wire
mesh and washing repeatedly under tap water as
described elsewhere (Varghese and Naithani, 2002).
 ARTICLE IN PRESS
Oxidative stress in cryo-stored neem seeds
The hard endocarp was then broken gently by hand to
extract the seeds, hereafter referred to as isolated seeds
(or simply as seeds). The isolated seed samples were
ready for drying within 18 h after collection.
Seed drying
Uniform, healthy and uninfected seeds with IWC
1.770.04 g H2O g1 dry mass (DM) were dried using
silica gel to various water contents down to 0.127
0.02 g H2O g1 DM. Desiccation was achieved by mixing
seeds and silica gel (1:20 w w1) in a glass desiccator,
maintained under ambient conditions. Separate desicca-
tors (four desiccators for four replicates of each target
water content) were used for seeds with six different
target water contents (treatments). The silica gel was
changed regularly when the color started fading in all six
containers at the same time. Seeds were mixed at least
twice daily for aeration. Seeds were dehydrated as
separate batches (200 seeds in each batch) for increasing
time intervals to 40 h, to achieve a series of decreasing
water contents. At the end of each drying period, seeds
from each desiccator were sampled for germination
assessment and water content.
Water content determination
Water contents were determined gravimetrically for
six replicates of 10 seeds/axes/cotyledons, after drying
for 48 h at 96 1C (for tropical seeds, ISTA, 1993), and
expressed as g H2O g1 DM. Since neem seeds are rich in
oil (51%, data not shown), the water content was
recalculated for zero amount of lipid (Sacandé et al.,
2000a). Similarly, water content was also corrected for
zero oil for axes (15% oil, data not shown) and cotyledons
(54% oil, data not shown) unless otherwise stated.
Germination
Seeds were surface sterilized with HgCl2 (0.1%) for
10 min, thoroughly washed with distilled water (DW) 4 or
5 times, allowed to imbibe DW and germinated in the
dark at 26–28 1C on a DW-saturated filter paper in 10 cm
Petri dishes (20 seeds per dish, 4 replicates). Germination
was scored daily for 11 days as the radicle elongation to
5 mm.
Cryopreservation
Neem seeds, fresh or dried to various water contents,
were placed in screw-capped 20 ml polypropylene cryo-
tubes (50 seeds per tube) and directly plunged into liquid
nitrogen (LN) within a cryocan (Jumbo Size; IBP, India).
The cryotubes containing seeds were retrieved from
cryostorage at various intervals and immediately thawed
by rapidly immersing them in a water bath at 40 1C, under
agitation. After thawing, seeds were immediately used
for germination assay and various biochemical analyses.
The germination test was performed either by placing the
seeds under germination conditions (after thawing, these
757
are referred to as control or untreated seeds) or by
preheating the seed by soaking in DW at 35 1C for 4 h prior
to germination assay (Sacandé et al., 2001). The pre-
heating treatment effectively prevents the imbibitional
damage to neem seeds (Sacandé et al., 1998).
Electrolyte leakage
Solute leakage from dried seed cryopreserved for
various intervals was estimated by placing 4 replicates
of 20 seeds each in 12 ml of DW at 26–28 1C. The
leachates were collected after 24 h of imbibition in DW
by the seeds and the electrolyte leakage loss by the seeds
was recorded using a conductivity meter.
Lipid peroxidation
Lipid peroxidation was measured as the concentration
of thiobarbituric acid reactive substances (TBRS) equated
with MDA according to Heath and Packer (1968) as
described by Varghese and Naithani (2002). Weighed
amounts of embryonic axes (approximately 50 mg) and
cotyledons (approximately 500 mg) were homogenized in
1 ml of DW with sterilized silica in a cold pestle and
mortar. Two ml of 0.5% thiobarbituric acid (in 20%
trichloroacetic acid) were added to the homogenate
and incubated in a boiling water bath for 30 min. The
reaction tubes were immediately placed in a freezer (at
0 1C) for 15 min to stop the reaction. The supernatant was
used for MDA determination. The concentration of the
MDA was calculated using the extinction coefficient of
155 mM cm1. Each data point represents the means7SD
of eight measurements carried out with four extracts.
Determination of AOS
Superoxide radical (O 2 ) liberation in the embryonic
axes and cotyledons of neem seeds was determined by
the method of Sangeetha et al. (1990). Weighed amounts
of sample (approximately 50 mg embryonic axis and
500 mg cotyledon) were homogenized in 3 ml of chilled
sodium phosphate buffer (0.2 M, pH 7.2) containing
0.18 M diethyl dithiocarbamate (Sigma). The supernatant
thus collected was used as the .O-2 source and was
detected by recording blue formazan (reduced NBT) at
540 nm. The calibration curve for pyrogallol oxidation
was obtained by determining the rate of NBT reduction at
540 nm. The superoxide formation in the embryonic axes
and cotyledon extract was estimated by recording the
kinetics of the reaction mixture containing 2.7 ml sodium
phosphate buffer, 200 ml extract and 100 ml NBT solution.
Superoxide production rate was calculated using an
extinction coefficient of 2.16  104 M1 cm1.
The content of H2O2 was measured in cryopreserved
seeds following the method described by O’Kane et al.
(1996). Seed samples (approximately 50 mg embryonic
axis and 500 mg cotyledon) were homogenized with 10 ml
of perchloric acid (0.2 N) in a chilled pestle and mortar
and then centrifuged for 20 min at 14,000g at 4 1C. The
supernatant was adjusted to pH 7.5 with 4 M KOH and
 ARTICLE IN PRESS
758
then re-centrifuged at 1000g for 3 min at 4 1C.
The supernatant was used immediately for estimation
of H2O2 using peroxidase-based assay. The reaction
mixture contained 12 mM 3-dimethylaminobenzoic acid
in 0.375 M phosphate buffer (pH 6.5), 1.3 mM 3-methyl-
2-benzothiazolidone hydrazone, 20 ml (0.25 units)
horseradish peroxidase (HRP, Sigma) and 1 ml of the
supernatant, for a total volume of 1.5 ml. The reaction
was started on the addition of HRP. The increase in
absorbance was read at 590 nm after 5 min. Absorbance
values were calibrated to a standard curve generated
with known concentrations of H2O2. Each value corre-
sponds to the mean7SD of eight measurements obtained
from four different extracts.
Enzyme assays
Weighed amounts of embryonic axes (50 mg) or
cotyledons (500 mg) were homogenized in cold borate
buffer (0.2 M, pH 7.4) containing 1 mM EDTA and 4% (w/v)
polyvinyl pyrollidone (PVP 40), with the addition of 1 mM
ascorbic acid in the case of APX assay, in a cold pestle and
mortar. The homogenate was centrifuged at 4 1C for
30 min at 15,000g and the supernatant was used for the
enzyme assays.
SOD (EC 1.15.1.1) activity was determined by the
method of Marklund and Marklund (1974). One unit of
SOD activity was defined as the amount of enzyme
required to cause 50% inhibition of the pyrogallol auto-
oxidation monitored at 420 nm. Auto-oxidation of pyr-
ogallol was monitored at 420 nm by recording the kinetics
of the reaction mixture containing 2.94 ml of 50 mM Tris-
HCl buffer (BDH), pH 8.2, 1 mM DETAPAC (Sigma) and 60 ml
pyrogallol, prepared in 10 mM HCl. For SOD assays, 0.2 ml
of enzyme extract was added to 2.74 ml of Tris-HCl buffer
and the absorbance was adjusted to zero. The reaction
was initiated by adding 60 ml of pyrogallol and change in
absorbance was recorded at 420 nm. Enzyme activity was
calculated and expressed on the basis of soluble protein
as units of SOD mg1 DM.
CAT (EC 1.11.1.6) activity was determined by following
the consumption of H2O2 (e ¼ 39.4 mM1 cm1 at 240 nm
for 3 min) (Aebi, 1984). The reaction mixture contained
50 mM potassium phosphate buffer (37.5 mM, pH 7),
10 mM H2O2 (60 mM) and 200 ml of enzyme extract in 3 ml.
APX (EC 1.11.1.11) assays were performed according to
Nakano and Asada (1981). The rate of hydrogen peroxide-
dependent oxidation of ascorbate, as an electron donor,
was determined in a reaction mixture that contained
50 mM potassium phosphate buffer (pH 7.0), 0.1 mM
hydrogen peroxide and 0.5 mM ascorbic acid and enzyme,
in a total volume of 1 ml. The reaction was started by
addition of hydrogen peroxide. The oxidation rate of
ascorbate was estimated by monitoring the decrease in
absorbance at 290 nm (e ¼ 2.8 mM1 cm1) between 30 s
and 2 min after the start of the reaction.
The activities of SOD, CAT and APX of each extract of
cotyledons and axes were measured twice; the results
correspond to the means of the values obtained with five
different extracts 7SD.
B. Varghese, S.C. Naithani
ANOVA and LSD (least significant difference) were
performed to determine the effect of drying and
cryostorage period on seed viability and related bio-
chemical analyses and to simultaneously determine the
significant difference for each data point. Prior to
analysis, the original percentage data (the germination
data obtained after drying and cryostorage of neem
seeds) were arcsine transformed. Arcsine transformation
(y0 ¼ arcsin y1/2) was necessary to stabilize the variance
of data that were proportions. Similarly, the chi-square
test (w2) was performed to analyze the effect of preheat
treatment over control (untreated) on germination of
cryostored seeds.
Results
Seed drying and germination
Seed drying using silica gel required 40 h to
reduce the water content of the freshly har-
vested neem seeds from 1.69 to 0.12 g H2O g1 DM
(Table 1). The seeds exhibited 100% germination
when sampled during dehydration from 1.69 to
0.23 g H2O g1 DM (Table 1). Further drying to 0.21,
0.16 and 0.12 g H2O g1 DM led to a slight decline
(significant as the LSD ¼ 2.56*, F ¼ 37.454**) in
percent germination (97–87%) (Table 1). The water
content was relatively higher in the cotyledons
than in the axes (Table 1) due to higher amount of
lipid (54%) that is inaccessible to water but
contributes to the DM (Sacandé et al., 2000a).
These results are in agreement with those of
Sacandé et al. (2000a).
Cryostorage
Fresh seeds and those dried to water contents of
1.84–0.09 g H2O g1 DM were tested for their cryo-
tolerance (Figure 1). No seeds in the water content
range 1.84–0.64 g H2O g1 DM survived 24 h of
cryoexposure. Further reductions in the water
content to 0.35 g H2O g1 DM resulted in 15% survi-
val, which was enhanced to 95% after 24 h exposure
to the cryotemperature for seeds dried (control) to
0.15 g H2O g1 DM. The germination percentage
of cryoexposed seeds (control) declined to 83%
and 55% (highly significant as the LSD ¼ 5.80*,
F ¼ 118.115**) as the water content was reduced
to 0.12 and 0.09 g H2O g1 DM, respectively. The
pre-heating before germination testing did not
prevent imbibitional injury to cryoexposed seeds,
suggesting that the dried neem seeds cryoexposed
for 24 h were not possibly suffering from imbibi-
tional injury per se. Seeds dried to approxi-
mately 0.15 g H2O g1 DM (drying period: 16 h)
 ARTICLE IN PRESS
Oxidative stress in cryo-stored neem seeds 759

Table 1. Water content in seeds, axes and cotyledons of Azadirachta indica during drying

Drying time Seed (51% oil) Axes (15% oil) Cotyledon (54% oil) Germination
(h) (g H2O g1 DM) (g H2O g1 DM) (g H2O g1 DM) (%)

0 1.6970.08 1.5370.06 1.8970.08 100

1 1.2270.09 1.3470.05 1.4670.06 100
4 0.8670.06 0.9170.05 1.170.05 100
8 0.4770.04 0.6170.04 0.6270.07 100
12 0.2670.04 0.3970.04 0.3870.06 100
16 0.2370.02 0.2770.03 0.2270.04 100
20 0.2170.03 0.1770.02 0.1670.02 9770.98
30 0.1670.01 0.1270.02 0.1270.04 9471.48
40 0.1270.01 0.0870.02 0.1170.01 8770.85

Water content was calculated for zero oil for the respective tissue. The values are means 7SD for six replicates of 10 seeds/axes/
cotyledons. Change in seed germination (%) is a function of water content. Each measurement is the mean of four replicates (from four
different desiccators) of 20 seeds. The germination data are significantly different when they exceed the LSD value (LSD ¼ 2.56*,
F ¼ 37.454**). The original percentage data were arcsine transformed before LSD calculation.

100 100
Germinatiion [%]

80
60
40
20
0
0.09 0.12 0.15 0.22 0.35 0.64 0.98 1.37 1.84
Water content [g H2O g-1 DM]
Figure 1. Effect of cryoexposure (24 h) on germination of
Azadirachta indica seeds of various water contents.
Germination was tested either after thawing (control
seeds) of cryoexposed seeds (white bars) or after thawing
followed by pre-heating treatment (black bars) at 35 1C
for 4 h before the germination assay. Each measurement
is the mean of four replicates (from four separate
desiccators) of 20 seeds. Vertical bars represent 7SD.
The germination data are significantly different when
they exceed the LSD values calculated for control
(LSD ¼ 5.80*, F ¼ 118.115**) and treated (preheat)
(LSD ¼ 6.01*, F ¼ 104.072**) cryoexposed seeds. The
original percentage data were arcsine transformed for
LSD calculation. The effect of preheat treatment over
control was not significant (w2-test).
Germinatiion [%] 80
60
40
20
0
0h 10h 1D 5D 1M 3M 6M 9M 12M
Cryostorage Period [hours /days /months]
Figure 2. Changes in germination percent of dried
(0.16 g H2O g1 DM) Azadirachta indica seeds cryostored
up to 12 months. Germination was tested either after
thawing of cryoexposed seeds (white bars) or after
thawing followed by pre-heat treatment at 35 1C for 4 h
before germination assay. Each measurement is the mean
of four replicates of 20 seeds each. Vertical bars
represent 7SD. The LSD values for control (LSD ¼ 5.45*,
F ¼ 62.643**) and treated (preheat) (LSD ¼ 4.95*,
F ¼ 31.629**) cryoexposed seeds are shown. The original
percentage data were arcsine transformed for LSD
calculation. The germination data on preheat treatment
over control are significantly different (Pp0.05) when
they differ by 17% or more (w2-test).

were selected for long-term cryopreservation
(12 months). The dried seeds with 0.16 g H2O g1 DM
(prior to cryoexposure) exhibited 92–96% survival
when cryopreserved for one month (Figure 2).
Subsequent cryopreservation up to 12 months
resulted in lower germination. Improved survival
was evinced in seeds cryopreserved for a longer
period by rehydrating the thawed seeds at elevated
temperature (35 1C) before germination. The pre-
heating treatment had an insignificant effect on
the germination percent at least up to 6 months of
cryostorage, but was significant (w2 test) in cryos-
tored seeds of 9 (73% G) and 12 (71% G) months.
Electrolyte leakage
Leachate conductivity increased two-fold
(0.21–0.47 mmhos ml1) as the fresh seeds were
dried to 0.16 g H2O g1 DM (Table 2). The dried
seeds did not show significant enhancement in
leakage, as measured by leachate conductivity
for at least 1 month in cryostorage (Figure 3), but
 ARTICLE IN PRESS
760 B. Varghese, S.C. Naithani

0.08370.00 0.01870.00 0.9570.11 0.2270.09 0.4870.016 0.1770.04 71.6578.13 19.1272.6

61.471.3
Cotyledon
(mmol min1 mg1 DM)
Ascorbate peroxidase
1.6

Leachate Conductivity
Comparison of leachate conductivity, lipid peroxidation, AOS content and antioxidant enzyme activity in fresh and dried seeds of Azadirachta indica

[m Mhos ml-1]
1.2

0.3170.09 151.7716.2
0.8

Axis
0.4
Cotyledon
(mmol min1 mg1 DM)

0
0h 10h 1D 5D 1M 3M 6M 9M 12M
Cryostorage Period [hours /days /months]

1.370.34 Figure 3. Effect of cryostorage on electrolyte leakage

Catalase

during imbibition (four replicates of 20 seeds each) of

Axis

Azadirachta indica seeds. Leachates were collected from

the cryostored seeds after thawing, which were then
3.870.79 0.5370.21

subjected to 24 h of imbibition. Means of four measure-

Cotyledon

ments 7SD. Data are significantly different (Pp0.05)

SOD (U mg1 DM)

when they are greater than the calculated LSD

(LSD ¼ 0.19*, F ¼ 29.976**).
Axis

a gradual increase was discernible (LSD ¼ 0.19*,

0.2670.19

F ¼ 29.976**) in the seeds cryopreserved longer for

Cotyledon
H2O2 (mmol mg1 DM)

up to 12 months.
0.9470.11

Lipid peroxidation
Axis

TBRS were comparatively higher in the cotyledons

than in the embryonic axes of cryoexposed seeds.
(mmol O2 min1 mg1 DM)

Cotyledon

3.670.28

Dehydration of fresh seeds to 0.16 g H2O g1 DM

8.270.7

resulted in more than 2–3.5-fold increment in

both axes (1.38–5.63 mmol MDA mg1 DM) and cotyle-
dons (0.26–0.58 mmol MDA mg1 DM (Table 2). The
Superoxide

5.370.41

11.171.2

accumulation of peroxidized lipid products was

low in dried seeds cryopreserved for 12 months
Axis

(Figure 4A). Cryostorage for 12 months enhanced

the lipid peroxidized products to 1.76 and
1.3870.128 0.2670.029
Cotyledon

0.5870.3

10.95 mmol MDA mg1 DM in cotyledon and axes,

(mmol MDA mg1 DM)

respectively.
Lipid peroxidation

5.6370.5

Superoxide
Axis

Desiccation of seeds from 1.69 to

0.16 g H2O g1 DM was accompanied by increased
(mmhos ml1)
conductivity

superoxide both in the cotyledon (3.6–8.2 mM O2 -

0.2170.05

0.4770.04

min1 mg1 DM) and in the embryonic axes

Leachate

(5.3–11.1 mM O2 min1 mg1 DM) (Table 2). Cryopre-

servation was accompanied by gradual increase in
superoxide accumulation, showing 27.12 and
H2O g1 DM)

H2O g1 DM)

Neem seed
Table 2.

38.4 mM O2 min1 mg1 DM in cotyledons and em-

content)

(1.69 g

(0.16 g
(water

bryonic axes, respectively, in seeds cryopreserved

Fresh

Dried

for up to 12 months (Figure 4B).

 ARTICLE IN PRESS
Oxidative stress in cryo-stored neem seeds 761

Hydrogen peroxide
14
The pattern of H2O2 accumulation in cryopre-
12
served dried seeds was similar to that of superoxide.
µm ol MDA mg-1 DM

10 An almost ten-fold increment was registered in the

8 cotyledons (0.018–0.26 mmol mg1 DM) and axes
(0.083–0.94 mmol mg1 DM) of neem seeds dehy-
6
drated from 1.69 to 0.16 g H2O g1 DM (Table 2).
4 A gradual rise in H2O2 levels was discernible after
2
cryopreservation for 1 month, with levels estimated
after 12 months of cryostorage being 1.39 for
0 cotyledons and 5.12 mmol mg1 DM for axes, respec-
0h 10h 1D 5D 1M 3M 6M 9M 12M
tively (Figure 4C).
Cryostorage Period [hours /days /months]

Superoxide dismutase
50

Drying of fresh neem seeds to 0.16 g H2O g1 DM

40
[µM o2 min-1 mg-1 DM]

elevated the SOD activity from 0.22 to 0.53

(LSD ¼ NS) and from 0.95 to 3.8 U mg1 DM (signifi-
Superoxide

30
cant promotion, LSD ¼ 0.99*) in the cotyledon and
embryonic axes, respectively (Table 2). A gradual
20
enhancement in SOD levels with the advancement in
cryoexposure period occurred for cryopreserved dried
10 seeds, with the highest activity both in the cotyle-
dons (1.2 U mg1 DM) and in the axes (6.3 U mg1 DM)
0 after 1 month of cryopreservation (Figure 5A).
0h 10h 1D 5D 1M 3M 6M 9M 12M
Thereafter, a gradual decline in SOD activity occurred
Cryostorage Period [hours /days /months]
in the cotyledons (0.91–0.13 U mg1 DM) and axes
(4.5–0.74 U mg1 DM) of the dried seeds cryopre-
6 served for 3–12 months.
5
Hydrogen peroxide

Catalase
[µm ol mg-1 DM]

3 CAT activity was promoted significantly both

2 don (0.17–0.31 mmol min1 mg1 DM) and in axis
1
0 seeds showed a gradual increase in CAT activity with
0h 10h 1D 5D 1M 3M 6M 9M 12M
Cryostorage Period [hours /days /months]
Figure 4. MDA (A), superoxide (B) and hydrogen peroxide
(C) content in extracts of cotyledons (white bars) and
axes (black bars) of dried (0.16 g H2O g1 DM) Azadirachta
indica seeds cryopreserved for various periods (10 h to 12
months). Means7SD of eight measurements carried out
with four extracts. The calculated LSDs in cotyledon
(0.33*) and axis (1.56*) (F ¼ 11.259** and 13.469** for
cotyledon and axis, respectively) for MDA (A), cotyledon
(5.21*) and axis (9.2*) (F ¼ 10.216** and 9.520** for
cotyledon and axis, respectively) for superoxide (B), and
cotyledon (0.24*) and axis (0.48*) (F ¼ 22.950** and
94.109** for cotyledon and axis, respectively) for hydro-
gen peroxide (C) are shown.
(cotyledon: LSD ¼ 0.13*, axis: LSD ¼ 0.46) in cotyle-
(0.48–1.3 mmol min1 mg1 DM) after desiccating the
fresh seeds to 0.16 g H2O g1 DM (Table 2). The dried
the advancement in cryostorage period over one
month, when maxima were reached for both
cotyledons (1.06 mmol min1 mg1 DM) and axes
(3.92 mmol min1 mg1 DM) (Figure 5B). Extending
the cryopreservation period beyond 1 month was
accompanied by a sharp decline in CAT activity
reaching the lowest levels after 12 months in
cryostorage for both cotyledons (0.12 mmol -
min1 mg1 DM) and axes (0.53 mmol min1 mg1 DM).
Ascorbate peroxidase
Similar to other antioxidant enzymes (SOD and
CAT), the APX increased with drying (Table 2 and
Figure 5C). Desiccation of fresh isolated seeds to
0.16 g H2O g1 DM enhanced (2-fold) the APX in
 ARTICLE IN PRESS
762 B. Varghese, S.C. Naithani

servation to the lowest activities in both

7 cotyledons (27.8 mmol min1 mg1 DM) and axes
6 (47.5 mmol min1 mg1 DM).
SOD [Units mg-1 DM]

5
4
3 Discussion
2
Fresh neem seeds that are intermediate in
1
storage physiology (Varghese and Naithani, 2000,
0 2001, 2002; Sacandé et al., 1998, 2000b, 2001) and
0h 10h 1D 5D 1M 3M 6M 9M 12M
highly intolerant to cryotemperature (Varghese and
Cryostorage Period [hours /days /months]
Naithani, 2001) became cryotolerant after drying
to 0.15–0.16 g H2O g1 DM. Reduction in water con-
5 tent has been shown to enhance the potential for
cryopreservation for embryonic axes of Aesculus,
[µmol min-1 mg-1 DM]

4
Castanea, Quercus (Pence, 1990) and seeds of
3 Azadirachta indica (Berjak and Dumet, 1996).
CAT

Presently dried neem seeds (0.16 g H2O g1 DM)

2 were storable with significantly high percentage
1
(LSD ¼ 5.45*) of survival (94–96%) for at least up to
1 month at cryotemperature as reported previously
0 (Berjak and Dumet, 1996). This water content is in
0h 10h 1D 5D 1M 3M 6M 9M 12M the range at which no freezable water is expected
Cryostorage Period [hours /days /months] in neem seeds (Sacandé et al., 2000a). Further
cryostorage of these seeds, however, resulted in
considerable loss of viability. Almost 50% loss in
175
germination was recorded over a period of 12
150 months of cryostorage (Figure 2). Good survival of
[µmol min-1 mg-1 DM]

Najas flexilis dried (0.14 g H2O g1 DM) seeds, a

125
desiccation sensitive, was reported during cryoex-
APX

100 posure for 1 day, but significant decline was

75
50
25
0h 10h 1D 5D 1M 3M 6M 9M 12M
Cryostorage Period [hours /days /months]
Figure 5. Changes in SOD (A), CAT (B) and APX (C)
activities in extracts of cotyledons (white bars) and axes
(black bars) of dried (0.16 g H2O g1 DM) Azadirachta
indica seeds cryostored for 10 h to 12 months. Means7SD
of ten measurements carried out with five extracts are
shown. The calculated LSD in cotyledon (NS) and axis
(0.99*) (F ¼ 1.478 NS and 7.930** for cotyledon and axis,
respectively) for SOD (A), cotyledon (0.13*) and axis
(0.46*) (F ¼ 49.158** and 49.40** for cotyledon and axis,
respectively) for CAT (B), and for cotyledon (1.30*) and
axis (12.85*) (F ¼ 638.74** and 80.238** for cotyledon
and axis, respectively) for APX (C) are shown.
cotyledon (19.12–61.4 mmol min1 mg1 DM) and
axes (71.65–152 mmol min1 mg1 DM). The dried
seeds showed a gradual fall in the levels of APX
analyzed over the 12-month duration of cryopre-
discernable when stored for 1 week (up to 59%)
(Hay and Muir, 2000). Recently, Walters et al.
(2004) reported measurable reduction even in
orthodox seed (that are naturally cryotolerant
compared with intermediate and recalcitrant
seeds) germination of 12 species stored at cryo-
temperature for a relatively long period (410
years). They suggested that cryogenic tempera-
tures were not sufficient to stop seed deteriora-
tion, especially if the initial stages of ageing were
allowed to progress at higher storage tempera-
tures. Our results corroborate their suggestion as
the percent germination of dried neem seeds
employed for long-term cryopreservation in the
present study was slightly (96% germination) but
significantly reduced (LSD ¼ 2.56*) after drying in
comparison to fresh seeds (100% germination). The
slight reduction in the viability may perhaps be
indicative of commencement/establishment of
deterioration-related changes (like enhanced elec-
trolyte leakage and lipid peroxidation that precede
loss of seed viability, as discussed below) in these
dried seeds. In the current study, the dried neem
seeds with 0.16 g H2O g1 DM were purposefully
 ARTICLE IN PRESS
Oxidative stress in cryo-stored neem seeds
chosen for long-term cryopreservation as they
(0.16 g H2O g1 DM) exhibited highest survival
(95%) after 24 h of cryoexposure (Figure 2). It is in
contrast to the seeds that were dried to 0.35 and
0.22 g H2O g1 DM, which registered 15% and 54%
survival, respectively, after 24 h of cryostorage.
Hence, despite the fact that neem seeds can be
dried to lower water contents (0.26 g H2O g1 DM)
with no loss in viability, this could not be used for
long-term cryopreservation studies.
Our results indicate that, at the cryotemperature,
maintenance of initial viability (94–96%) of dried
seeds was facilitated only up to 1 month, and
cryostorage for up to 12 months resulted in a
progressive viability loss (50% germination after
12 months). Hence, it is suggested that cryopreser-
vation studies conducted with the aim of determin-
ing the cryotolerance for long-term conservation in
non-orthodox seeds such as Camellia sinensis,
Theobroma cacao, Artocarpus heterophyllus and
Piper nigrum (Chandel et al. (1995) and references
therein) for a short period (up to 24 h) may not
provide adequate indicators of the potential for
long-term conservation (see also Hay and Muir,
2000). Ensuring the long-term stability (i.e. reten-
tion of high viability levels) of cryopreserved
germplasm in seed banks is an important aspect of
cryogenic storage. Therefore, long-term cryostorage
studies such as this one on non-orthodox seeds might
also offer clues and possibilities for developing
newer cryopreservation protocols that are based
on minimizing the cryoinjury.
In the present study, the decline in viability after
one month in cryostorage of the dried neem seeds
was accompanied by an increase in electrolyte
leakage and lipid peroxidation. The increase in
leakage was approximately proportional to viability
loss during cryostorage. It is possible that the loss
of viability in response to cryotemperature stress in
dried neem seeds (from 3 to 12 months of
cryostorage) may be linked to enhanced accumula-
tion of TBRS that is mediated by AOS and
membranes are one of the major targets of injury
(Benson and Bremner, 2004). The strong negative
correlation obtained between viability and lea-
chate conductivity (R2 ¼ 0.98) and lipid perox-
idation (cotyledon: R2 ¼ 0.92; axis: R2 ¼ 0.89)
supports the argument for their implications in
cryoinjury in neem seeds. The membrane damage
as indicated by leachate conductivity and/or
accumulation of TBRS following desiccation in
Shorea robusta (Chaitanya and Naithani, 1994,
1998), axes of Landolphia kirkii (Pammenter
et al., 1997) and intermediate Azadirachta indica
seeds/axes (Sacandé et al., 2001; Varghese and
Naithani, 2002) and cryopreservation in tea, cocoa
763
and jackfruit during cryopreservation (Chandel
et al., 1995) has been frequently associated with
loss of viability in non-orthodox seeds. Membrane
damage in dried seeds (0.14 g H2O g1 DM) of Aza-
dirachta indica that results in poor survival has also
been linked to imbibitional stress occurring due to
rapid uptake of water during rehydration (Sacande
et al., 2001). Cryopreservation of dried neem seeds
over 9 and 12 months presently showed imbibitional
injury as survival of these seeds was significantly
(417%, w2, PX0.05) improved by rehydrating the
seeds at elevated temperature (pre-heating treat-
ment). The promotory effect of pre-heat treatment
can be explained by the melting at elevated
temperatures of membranes that were in the gel
phase (due to drying), thus allowing establishment
of functional membrane prior to placing the seed
for germination assay in water (Sacandé et al.,
1998; Dussert et al., 2003). The pre-heat treatment
was ineffective (w2 test: non-significant) in dried
seeds (0.09–0.35 g H2O g1 DM) that were cryoex-
posed for 24 h (Figure 2). It is suggested that the
nature of cryoinjury in the short- and long-term
cryostorage may be different, perhaps due to the
ageing effect (additionally) in the latter type of
storage. Also, the argument that the imbibitional
injury could well be prevented in the desiccation-
sensitive seeds by rehydration treatment may not
hold true if the seeds are inherently desiccation
sensitive (Pammenter et al., 2004).
Disturbances in oxidative metabolism can lead to
unregulated AOS production in plant tissue exposed
to low and ultra-low temperatures. Studies on
cryopreserved carrot cells and oilseed rape mer-
istems (Benson and Bremner (2004) and references
therein) provided evidence for the production of
AOS during early post-thaw recovery. In our studies,
measurable amounts of AOS, such as superoxide
and hydrogen peroxide, accumulated as a result of
desiccation and cryoexposure in neem seeds. The
dried neem seeds that retained very high viability
(above 94–96%) showed negligible promotion in AOS
accumulation both in the cotyledons and in the
axes for at least up to one month of cryostorage.
Later AOS accumulation did increase in seeds after
3 months of cryostorage and constantly progressed
as the survival reduced over the 12-month period.
AOS are themselves highly toxic; however, their
damaging effects are further enhanced by second-
ary oxidative reactions (Benson and Bremner,
2004). It appears that the drying of neem seeds to
0.15–0.16 g H2O g1 DM (water contents suitable
for cryostorage) affords the potential for tolerating
cryotemperature at least for 1 month. These
frozen-thawed seeds accumulated remarkably low
levels of AOS and lipid-peroxidized products during
 ARTICLE IN PRESS
764
cryostorage for 1 month and survived with high
viability percentage. But the gradual loss of
viability in these seeds later during cryostorage
(3–12 months) corresponds to an equally slow rate
of AOS and TBRS accumulation, both in the axes and
in the cotyledons.
Retention of viability of neem seeds during
dehydration to intermediate water contents has
been associated with reduced accumulation of
AOS and TBRS associated with relatively high
levels of free radical processing enzymes (Vargh-
ese and Naithani, 2002). In the present study,
activities of antioxidant enzymes such as SOD and
CAT were enhanced in response to desiccation and
cryotemperature stress in dried neem seeds that
maintained a high percentage of germination, thus
providing protection against oxidative damage due
to accumulation of AOS in these seeds. Levels of
SOD and CAT exhibited 2 (cotyledon) and 3 (axis)
fold promotions upon drying of fresh seeds to
0.16 g H2O g1 DM (Table 1). These dried seeds
showed further enhancement in SOD and CAT
levels after retrieval from cryostorage up to 1
month. Enhanced levels of antioxidants have been
reported during cold acclimation (O’Kane et al.,
1996) and/or associated with freeze tolerance and
cryo-tolerance (Touchell and Walters, 2000). Chil-
ling tolerant genotypes of maize showed higher
antioxidant enzyme activity than the cold intoler-
ant genotype (Pinhero et al., 1997). Significant
loss of survival on further storage (from 3 to 12
months) of neem seeds at cryotemperature was
accompanied by gradual decrease in SOD and CAT
activities.
Unlike SOD and CAT, the activity of APX was
promoted by desiccation but declined after cryos-
torage of the dried seeds. Functionality of these
antioxidant enzymes is compromised at freezing
and chilling temperature in sensitive plant tissues
(Guy, 1990). We conclude that the loss of
cryotolerance, especially in neem seeds stored
for longer period (more than a month), is a
consequence of increased accumulation of AOS
concomitant with low levels of APX activity and
decreasing activities of SOD and CAT. The protec-
tive roles of SOD, CAT and APX have been
extensively discussed in relation to free radical
damage of cellular membranes (Touchell and
Walters, 2000; Bailly, 2004; Kibinza et al., 2006)
and failure of these antioxidant enzymes leads to
impairment of cellular metabolism (Chaitanya and
Naithani, 1994, 1998). Our results indicated
differential responses of antioxidant enzymes in
relation to desiccation and cryo stress. SOD, CAT
and APX activities were enhanced as a result of
desiccation of neem seeds, whereas this occurred
B. Varghese, S.C. Naithani
only for SOD and CAT upon exposure to cryotem-
perature. Cryopreservation has been shown to
regulate the oxidative stress-related scavenging
enzymes in yeast cells (Odani et al., 2003). Those
authors have further shown that the tolerance of
yeast cells to freezing and thawing was increased,
perhaps due to induction of oxidative scavenging
enzymes (actively participating in the repair
process), in response to heat-shock treatment
prior to cryopreservation, thus supporting the
significance of antioxidant enzymes during the
recovery after cryotreatment.
It is concluded that AOS generation and lipid
peroxidation occurred in the dehydrated neem
seeds. These reactions (partly of chemical nature)
may proceed unabated once initiated (during
drying) and can proceed even at cryotempera-
ture, as in the case of the dried neem seeds that
were slightly deteriorated (94–96%) prior to
cryopreservation, for 1 year. It is suggested that
accumulation of AOS is counteracted, thus avert-
ing cellular damage, due to efficient removal of
these cytotoxic chemical species in the presence
of high levels of antioxidant enzymes operating at
the time of thawing at least up to 1 month of
cryostorage. Measurable molecular mobility in
seeds at cryogenic temperatures has been con-
sidered apparently sufficient for ageing reactions
to proceed, albeit slowly (Walters et al., 2004). In
the present case, accumulation of AOS and their
damaging effects were favored in dried seeds
from 3 months of cryostorage onwards, probably
as a consequence of reduced activities of detox-
ifying enzymes, thus allowing all deteriorative
reactions to proceed, causing loss of viability
after thawing.
Acknowledgements
We thank the Head, School of Life Sciences,
Pt. Ravishankar Shukla University, Raipur, for provid-
ing the laboratory facilities. Statistical analyses by
Dr. M.L. Lakhera, Department of Statistics, Mathe-
matics and Computer Science, IGKV, Raipur (CG), is
thankfully acknowledged. Financial assistance by
the University Grants Commission [Project No. F.3-
32/94 (SR-II)], New Delhi, Council of Scientific and
Research Development, New Delhi [38(937)97-EMR-
II], and Ministry of Environment and Forest [Project
No. 14/41/98-ERS/RE], New Delhi, funding under
the FIST (DST, New Delhi) and SAP (UGC, New Delhi)
programmes, and cryostorage facility provided by
the DBT [BT/PR5168/BCE/08/342/2004], New Delhi,
are also gratefully acknowledged.
 ARTICLE IN PRESS
Oxidative stress in cryo-stored neem seeds
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