J. HoTt. Sci. 
Vol. 1 (1): 15-18, 2006 

Studies on physiological and biochemical changes in relation to 
seed viability in aged onion seeds 

K. Bhanuprakash, H. S. Yogeesha, L. B. Naik and M. N. Arun 
Section of Seed Science & Technology 

Indian Institute of Horticultural Research 
Hessaraghatta Lake Post, Bangalore-560 089, India 

E-mail: kbp_iihr@yahoo.co.in 

ABSTRACT 

Rapid loss in viability of onion seeds during seed storage is a major problem. Not much information concerning 
the physiological and biochemical changes is available. In the present investigations, seeds were aged artificially 
by exposure to 45*C +75% RH for a period of fifteen days. Samples were collected at three day intervals and 
physiological and biochemical changes in the aged seeds were compared to those in fresh seeds. Results revealed 
that ageing affected seed viability and vigour significantly and this effect was more pronounced with increase in 
duration of exposure to artificial ageing. Marked reduction in germination to an extent of 4,16 and 75% was 
noticed in three, six and nine day artificially aged (DAA) seeds, respectively, when compared to fresh seeds. 
Further increase in ageing duration to twelve and fifteen days resulted in total loss of germination. Increase in 
ageing duration decreased a amylase and dehydrogenase activities but increased peroxidase activity up to nine 
days of ageing. Lipid peroxidation increased consistently with increase in duration of ageing. At 15 DAA, 26.2% 
increase in malondialdehyde content over the control was observed. SDS PAGE protein profile and esterase 
zymograms of aged seeds showed alteration in banding pattern when compared to that of fresh seeds. 

Key words: Onion, accelerated ageing, protein profiles, enzymes 

INTRODUCTION up to 15 days at 45" C +75 % RH as per ISTA (1999) 

Onion {Allium cepa L.) seeds lose vigour and ^^^^ard procedure. Samples were collected every third day. 
viability at faster rates than seeds of most other vegetables ^ ^ e r agemg, the seeds of all the treatments were dned back 
(Choudhari and Basu, 1988). The relation between ^ the original moisture content for further studies. 

temperature, moisture content and viability period is similar Germination and growth parameters 
under the conditions used in hot air drying or at long-term 

, , , .̂ . T u *u.i- »u .. Four replicates of fifty seeds of each sample, planted 
storage at sub zero temperature. In both the cases, the pattern *̂  •' i- 't-
of deterioration preceding death is also the same, whether ^̂  moistened roll paper towels, were germinated in a growth 
the seed survives for seconds or decades (Roberts, 1981). ^^^^^er at 20''C under dark. Observations were recorded 
Artificial ageing techniques are employed to elucidate the "'^ germination (ISTA, 1999), germination rate (GR), 
mechanisms of deterioration during storage because gemiination speed (GS) and standard germination (StG). 
physiological changes in the seed occurring during artificial These parameters were computed, as given below, using 

and natural ageing are thought to be similar (Chen et al, the formula given by Mugnisjah and Nakamura (1986). 

1999). Hence, an experiment was conducted to examine GR=100/no. planted (normal seedlings day 6/ 
changes due to artificial ageing in relation to seed viability g+normal seedlings day 12/12) 
and vigour in onion cv. Arka Bindu. MATERIAL AND METHODS 

GS=100/no. planted (normal seedlings day 1/ 
1+normal seedlings day 2/2.... day 12/12) 

Agemg treatment StG=100/ no. planted (normal seedlings day 6 + 

Fresh seeds of cv. Arka Bindu were aged artificially normal seedlings day 12) 

mailto:kbp_iihr@yahoo.co.in


Bhanuprakash et al 

Lipid peroxidation, electrical conductivity and total 
soluble sugars 

Lipid peroxidation in fresh and aged seeds was 
studied by TBA colour reaction as outlined by Bemheim et 
a/ (1948). The methodology followed for studying electrical 
conductivity (EC) was that described by Rudrapal and Basu 
(1979). The amount of sugars in seed leachate was 
determined using the method of Dubois et al (1951). 
Leaching of sugar was expressed as glucose equivalent per 
ml of leachate. 

Enzyme activity 

a-amylase and peroxidase activities were estimated 
as per Sadasivam and Manickam (1996). Dehydrogenase 
activity was estimated as per the procedure outlined by 
Agarwal and Dadlani (1987). 

Protein electrophoresis 

Seeds of each sample (0.5g) were crushed to fine 
powder using a pestle and mortar and the flour was defatted 
by soaking in a mixture of chloroform, methanol and 
acetone in 2:1:1 ratio for 3 h. The supernatant was poured 
out and the process was repeated three times. The samples 
were dried at room temperature and a sub-sample of 0.1 g 
was homogenized in an eppendorf tube by adding 400 |J.l 
of the extraction buffer (Tris Glycine, pH 8.3). Initially, 
the tubes were left at room temperature for 1 h and then 
shifted to A°C until the next day. The samples were 
centrifuged at 10,000 X g for 10 min. and the supernatant 
was collected. To 10 |J,1 of this supernatant, 10 |a,l of working 
sample buffer was added and boiled at \00°C for 5 min. 
Protein content in the samples was quantified as per the 
procedure outlined by Lowry et al (1951). Protein 
characterization of seeds was carried out using SDS PAGE 
(Laemmli, 1970). 

Isozyme analysis 

Seeds of each sample (O.lg) were crushed to a fine 

powder using liquid nitrogen in pre- chilled pestle and 
mortar and the contents were transferred to eppendorf tubes 
containing 75|xl of O.IM Tris HCl, pH 7.5. The contents 
were homogenized using micropestle and left overnight at 
10°C for extraction. Samples were centrifuged at 4°C at 
10000 rpm and subjected to alkaline PAGE by loading 
25p,l in each well. The gel was run at 15°C for a period of 
six hours under a constant current of 1(X) V until samples 
crossed the stacking gel and later at 250 V. The gels were 
stained immediately in a staining solution (50 mg Fast blue 
RR salt in 2 ml of 50 mg Naphthyl acetate in 50 % acetone 
added to 100 ml of 0.5 M sodium phosphate buffer, pH 
6.2) for one hour. Data were statistically analyzed using 
Completely Randomized Block Design as outlined by Pause 
and Sukhatme (1978). 

RESULTS AND DISCUSSION 

Results revealed that ageing affected seed viability 
and vigour significantly and this effect was more 
pronounced with increase in duration of ageing. Marked 
reduction in germination of 4, 16 and 75% was noticed in 
three, six and nine day artificially aged (DAA) seeds, 
respectively, compared to fresh seeds. Further increase in 
ageing duration to 12 andl5 days resulted in total loss of 
germination. Ageing also showed significant deleterious 
effects on other quality traits. Three, six, nine, twelve and 
fifteen days of artificial ageing resulted in significant 
reductions in speed of germination, rate of germination, 
total seedling dry weight and seedling vigour index (Table 
1). Reduction in seedling vigour index was 14.7,42.9,91.2, 
1(X) and 100% in comparison to fresh seeds (Table 1). 
Similar loss in seed viability and vigour when subjected to 
artificial ageing has also been reported in various crops 
earlier (McDonald, 1999). 

Besides physiological changes, many biochemical 
changes were noticed due to ageing. Wide differences in 
membrane permeability between aged and fresh seeds were 
noted. Except at three DAA, there was progressively higher 

Table 1. Effect of duration of artificial ageing on onion seed vigour-physiological parameters 

S.No. 

1 
2 
3 
4 
5 
6 

Treatment 

Fresh 
3DAA 
6DAA 
9DAA 
12DAA 
15DAA 
SEd+ 
CD@5% 

% Germination* 

100(89.8) 
96(79.9) 
84(67.1) 
25(29.9) 
0 
0 
2.56 
5.38 

Total Seedling 
Length (cm) 

15.80 
14.00 
10.70 
5.50 
0 
0 
0.81 
1.69 

Total Dry 
Weight (mg) 

1.216 
1.296 
1.320 
0.600 
0 
0 
0.017 
0.035 

Shoot Vigour 
Index 

1575 
1343 
900 
139 

0 
0 

75.5 
158.7 

Germination 
Speed 
192.0 
145.0 
120.0 

19.7 
0 
0 
2.43 
5.10 

Germination 
Rate 
24.30 
23.30 
20.50 

4.08 
0 
0 
0.34 
0.72 

Standard 
Germination 

196 
188 
165 
37 
0 
0 
3.23 
6.79 

*Values in parentheses represent angular transformed values 

J. Hon. Sci. 
Vol. 1 (1): 15-18,2006 

16 



Seed viability in onion 

Table 2. Effect of duration of artificial ageing on onion seed vigour-biochemical parameters 

S.No. 

1 
2 
3 
4 
5 
6 

Treatment 

Fresh 
3DAA 
6DAA 
9DAA 
12DAA 
15DAA 
SEcl± 
CD@57c 

Electrical 
Conductivity 

(us/cm) 
70.4 
70.3 
74.4 
83.8 
91.8 

101.8 
1.92 
4.03 

Dehydrogenase 
activity (% change 

over control) 
100.0 
90.4 
62.3 
44.3 
35.6 
34.9 

1.55 
3.25 

Malondialdehyde 
content (% change 

over control) 
100.0 
103.9 
114.5 
119.4 
121.4 
126.2 

0.85 
1.80 

Total Soluble 
Sugars 
(Hg/ml) 

127.5 
137.5 
168.5 
470.0 
490.0 
530.0 

17.4 
36.7 

Amy!; 
(ug 

produc 

ise activity 
maltose 

ed/ml/min.) 
95.1 
97.1 
82.4 
62.4 
38.5 
37.6 

2.10 
4.41 

Peroxidase 
activity (Change in 

OD/ min.) 
0.009 
0.018 
0.022 
0.024 
0.023 
0.023 
0.002 
0.005 

leaching of electrolytes and sugars with increasing duration 
of ageing. Increase in ageing duration from six to fifteen 
days increased the EC values by 5.7 to 44.6% and sugar 
levels 1.07 to 4.16 times when compared to fresh seeds. 
These results clearly demonstrate that the aged seeds lost 
integrity of cell membranes. Lipid peroxidation, estimated 
by production of malondialdehyde, increased consistently 
with increase in duration of ageing. Increase in 
malondialdehyde content was noticed to the tune of 3.9, 
14.5, 19.4, 21.4 and 26.2% in comparison to fresh seeds. 
Pammenter et al (1974) also noticed more lipid peroxides 
in aged seeds than in the corresponding controls. These 

15DAA 

i -I a-sli seeds DAA-lJa>s alter Artificial Ageing 

Plate 1. SDS-PAGE Tris soluble protein profile of fresh and aged 
onion seeds showing alteration in banding pattern 

15DAA 12 9 6 .̂  F 

- ' ? i — . 

Plate 2. Esterase isozyme profile of fresh and aged onion seeds showing 
alteration in banding pattern 

results, when taken along with reduction in germination 
speed and seedling growth, indicate that increase in lipid 
peroxidation may be partly responsible for reduced onion 
seed vigour at supra-optimal temperature of 45" C when 
coupled with high humidity (Table 2). 

In the present investigation, activities of a-amylase, 
dehydrogenase and peroxidase were also examined in 
relation to seed deterioration. Although there was no 
significant decrease in a- amylase activity at three DAA, a 
sharp decline in activity of 12.7 to 57.5 |ig with increase in 
ageing duration from six to fifteen days was observed 
when compared with fresh seeds. It was further noted that 
following ageing, the activity of dehydrogenase declined 
more rapidly than in the fresh seeds, where as peroxidase 
activity increased significantly until nine DAA and 
thereafter, remained unaltered (Table 2). The sudden 
increase in peroxidase activity at the beginning of ageing 
may be due to activation of antioxidative mechanism to 
suppress the high levels of peroxides that were produced 
under supra-optimal ageing conditions. Age associated 
reduction in activity of key enzymes in seeds has been 
reported (Wilson and McDonald, 1986). Thus, reduction 
in enzyme activity may be a reflection of changes in protein 
synthesis (Nandi et al, 1995). 

SDS PAGE protein profile of accelerated aged 
seeds showed an alteration in twelve and fifteen DAA where 
the characteristic protein pattern was lost (Plate 1). The 
loss of subunits in six, nine and twelve DAA seeds indicate 
that protein degradation occurred due to prolonged ageing. 
Nautiyal et al (1985) reported that in Shorea robusta seeds, 
proteins with higher electrophoresis mobility deteriorated 
first during ageing. Analysis of esterase isozymes also 
revealed alteration in aged seeds. Results obtained for 
twelve and fifteen DAA seeds, particularly, showed no 
resolution into discrete bands (Plate 2). Aung and McDonald 
(1995) also noticed a decrease in some esterase isozymes 
in peanuts during storage, due to ageing. Based on these 
findings, it is concluded that seed ageing in onion is the 

J. Hort. Sci. 
Vol. 1 (1); 15-18,2006 

17 



Bhanuprakash et al 

result of slowdown processes in synthesis. This may be 
attributed to degradation of proteins, inactivation of 
enzymes and higher lipid peroxidation in onion seeds during 
ageing. 

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{MS Received 3 April, 2006 Revised 26 June. 2006) 

J. Hon. Sci. 
Vol. 1 (I): 15-18. 2006 

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