International Journal of Agricultural

Science and Research (IJASR)

ISSN(P): 2250-0057; ISSN(E): 2321-0087
Vol. 6, Issue 5, Oct 2016, 371-374
TJPRC Pvt. Ltd

BIOCHEMICAL CHANGES OF BIOPRIMED SNAKKEGOURD

Cv. CO2 SEEDS DURING GERMINATION
GOWTHAMY. U & P. SELVARAJU
Department of Seed Science and Technology, Seed Centre Tamil Nadu
Agricultural University, Coimbatore, Tamil Nadu, India
ABSTRACT
Seed priming is a controlled hydration process that involves exposing seeds to low water potentials that restrict
germination, but permits pregerminative physiological and biochemical changes to occur. Biopriming is a process of
biological seed treatment that refers combination of seed hydration (physiological aspect of disease control) and
inoculation (biological aspect of disease control) of seed with beneficial organism to protect seed. In order to utilize the
biopriming influence on seedling growth in snakegourd (Trichosanthes cucumerina),this experiment was conducted to
study the biochemical changes of bioprimed snakegourd seeds cv. CO2 germination. The results showed that the seeds
bioprimed with Pseudomonas fluorescens 80% concentration for 24h in equal volume of seeds was found to be the best
germination and Protease activity (0.522 OD value) at 48h of germination.
KEYWORDS: Snakegourd (Trichosanthes Cucumerina), Pseudomonas. Fluorescens, Seed Biopriming, Biochemical
Changes, -Amylase Activity, Protease Activity

Received: Sep 18, 2016; Accepted: Oct 06, 2016; Published: Oct 15, 2016; Paper Id.: IJASROCT201645

Original Article

and suitable biopriming treatment. This treatment showed a higher -Amylase activity (0.915mg maltose min-1) at 48h of

INTRODUCTION
Snake gourd (Trichosanthes cucumerina) also known as Chinese cucumber belongs to the family
Cucurbitaceae. It is called potlakaaya in Telugu, pudalankaai in Tamil, dhundul in Assamese, paduvalakaayi in
Kannada and padavalanga in Malayalam. It occupies an important place among vegetables in India. It is an annual
plant with rapid growth and of climbing habit. The fruits are large and greenish white. They often reach upto 150
cm. in length and 8 cm. in thickness. There is also a short-fruited type. Tender fruits are used as vegetables.
Snake gourd is a very nutritious vegetable. An analysis of this vegetable shows it consists of moisture
94.6 percent, protein 0.5 percent, fat 0.3 percent, fibre 0.8 percent and carbohydrate 3.3 percent per 100 grams of
edible portion. Snakegourd is cultivated in Tamil Nadu in larger area with an average productivity of 18 tonnes ha1

.
For any crop production, seed is the basic input and if the seed is not having good germination, the

optimum population in the field cant be maintained which ultimately affect the crop yield. In snakegourd,
normally the germination is below 60 per cent and by any presowing treatment if the germination is improved it
would help in maintaining the required population in the field. Biopriming is one of the presowing treatments that
can improve the germination and vigour of the seedling.
Research on biochemical changes of bioprimed seeds during germination of -amylase activity and
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Gowthamy. U & P. Selvaraju

Protease activity is lacking. Hence the present study was designed to investigate the beneficial effects of biopriming on
snakegourd seeds.

MATERIALS AND METHODS

Genetically pure seeds of snakegourd cv.CO2, a short duration variety obtained from Susi seeds international,
Mylapore, Chennai of Tamil Nadu, formed the base material for this study. The laboratory studies were carried out at the
Department of Seed Science and Technology, Tamil Nadu Agricultural University, Coimbatore during 2011-2013. Eight
treatments viz., non primed seed (control), hypropriming, biopriming with Humic acid, Trichoderma viride, Pseudomonas
fluorescens, Azotobacter, Azospirillum, phosphobacteria were taken up in this study. The experiment was carried out with
four replications in Completely Randomized Block Design (CRD). In order to standardize the optimum concentration of
biopriming agent and duration, the commercially available humic acid was diluted 5, 10, 15 and 20 percent. Biocontrol
agents were prepared at 40, 60 and 80 percent concentration and liquid biofertilizers were prepared in three different
concentration viz., 10, 15 and 20 percent. Seeds were soaked in equal volume of solution in different concentrations in each
of the biopriming agents. For hydropriming, simple water is used for soaking. The nonprimed seeds formed the control.
After soaking, the seeds were removed from the solutions and shade dried at room temperature for assessing the seed
quality parameters. The number of seeds germinated on each day was recorded daily upto 14th day. After 14th day
germination their performance were evaluated for germination (%), dry matter production (g seedlings-10) and vigour
index.
The best six treatments one each from the humic acid, biocontrol agents, liquid biofertilizers) along with
hydropriming and unprimed seeds were subjected to germination and the germinating seeds were collected at 12, 18, 24,
and 30 h. During different stages of germination the bioprimed and unprimed seeds were analyzed for the following
biochemical characterization. The study was conducted adopting factorial completely randomised design with four
replications.
-Amylase Activity
Four replicates of 500 mg of pregerminated seed samples were homogenised in 1.8 ml of cold 0.02M sodium
phosphate buffer (pH 6.0) and centrifuged at 20,000 rpm for 20 min. to extract enzymes. To 0.1ml of enzyme extract, one
ml 0.067 per cent starch solution was added. The reaction was stopped after 10 min. of incubation at 25oC by the addition
of one ml of iodine HCl solution (60mg KI and 6mg I2 in 100ml of 0.05N HCl).
Change in colour was measured at 620nm. The activity was calculated and expressed as mg maltose min-1
(Paul et al., 1970).

Protease Activity
Endopeptidase activity was measured by using chromogenic substrate, azocasein, following by the method, with
slight modifications. 250 l of 1% azocasein (prepared in 0.02 M sodium acetate buffer pH 5.5 containing
2mM -mercaptoethanol) was mixed with 150l of enzyme extract. The reaction mixture was incubated at 40C for 1h.
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Biochemical Changes of Bioprimed Snakkegourd Cv. Co2 Seeds during Germination

373

The reaction was arrested by adding 1.2ml of 10 % trichloro acetic acid and mixed thoroughly. The contents were
centrifuged,1.2 ml of supernatant was transferred to a tube containing 1.4ml of 1M NaOH, mixed and the absorbance was
read at 440nm against the reagent blank using UV / Vis Spectrophotometer model OPtize 2120 UV plus. One unit of
protease activity is defined as the amount of the enzyme required to produce an absorbance change of 1.0 in 1cm cuvette
under conditions of the assay. The OD value obtained was calculated as protease activity.
Statistical Analysis
The data obtained from different treatments were analysed for the F test of significance following the methods
described by Panse and Sukatme (1985). Wherever necessary, the per cent values were transformed to angular (Arc-sine)
values before analysis. The critical differences (CD) were calculated at 5 per cent probability level. The data were tested
for statistical significance. If the F test is non-significant, it was indicated by the letters NS.

RESULTS AND DISCUSSIONS

Significant variations were observed on between non priming and priming treatments and Pseudomonas
fluorescens 80% for 24 h at 48h of germination showed increase in the -amylase activity ( 0.915mg maltose min-1 ) over
nonprimed seeds ( 0.894 mg maltose min-1) and Protease activity also showed more at 48h (0.522 OD value) over
nonprimed (0.494 OD value) of germination.
During germination of several species, amylase activity increases in the cotyledons and starch content decreases
(Juliano and Varner, 1969; Yomo and Varner, 1973; Tarrago and Nicolas, 1976; Umezuriket and Numfor, 1979).
Kalaivani (2010) reported 370 times increased -amylase activity of maize seeds bioprimed with Pseudomonas
fluorescens 80% for 12h over the nonprimed seed which resulted in higher reducing sugars.
Afzal et al. (2008) who reported an enhancement of -amylase activity in primed seed which may be attributed to
proper hydration during imbibition that increased starch hydrolysis. This suggested that starch being converted into
reducing sugars. In sweet corn seeds, osmo and matrix priming increased and - amylase activity.

CONCLUSIONS
The documentation of biochemical event of bioprimed seed during germination (0 to 48h) revealed that the
Pseudomonas fluorescens 80% bioprimed seeds for 24h was found to imbibe faster and initiate quick and early metabolic
activities compared to nonprimed seeds. This is revealed by increased -amylase activity during the period of germination
and higher defence enzymes activity. Finally, increasing metabolic events helped the bioprimed seeds for efficient
degradation of endosperm and mobilization of food materials to the growing embryo which resulted in early radicle
emergence, increased radicle length and ultimately higher germination.
Table 1: Effect of Seed Biopriming on Biochemical Changes During Germination in
cv.CO2 Snake Gourd - - Amylase Activity (mg Maltose Min-1)
Biopriming
Treatments (T)
T0
T1
T2
T3
T4
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0
0.503
0.507
0.521
0.524
0.528

Time Course of Germination (H)

12
24
36
0.506
0.601
0.702
0.509
0.605
0.711
0.525
0.618
0.731
0.526
0.622
0.734
0.532
0.625
0.738

48
0.894
0.897
0.911
0.913
0.915

Mean
0.653
0.658
0.661
0.664
0.668

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Gowthamy. U & P. Selvaraju

T5
T6
T7
Mean
SEd
CD (P=0.05)

0.517
0.514
0.511
0.515
T
0.0043
0.0086**

Table 1: Contd.,
0.519
0.614
0.518
0.611
0.515
0.607
0.518
0.612
H
0.0034
0.0068**

0.727
0.724
0.721
0.739

0.907
0.904
0.901
0.905
TH
0.0097
0.0192**

0.657
0.654
0.651

Table 2: Effect of Seed Biopriming on Biochemical Changes During

Germination in Cv.CO2 Snake Gourd - Protease Activity (OD Value)
Biopriming
Treatments (T)
T0
T1
T2
T3
T4
T5
T6
T7
Mean
SEd
CD (P=0.05)

Time Course of Germination (H)

0
12
24
36
48
0.204
0.206
0.301
0.321
0.494
0.207
0.209
0.304
0.324
0.497
0.221
0.225
0.318
0.338
0.510
0.224
0.226
0.322
0.352
0.516
0.228
0.232
0.325
0.363
0.522
0.217
0.219
0.314
0.334
0.508
0.214
0.217
0.311
0.331
0.505
0.211
0.215
0.307
0.327
0.503
0.216
0.219
0.313
0.336
0.507
T
H
TH
0.004
0.003
0.009
0.008**
0.006**
0.017**

Mean
0.305
0.308
0.322
0.328
0.334
0.318
0.316
0.313

Treatment Details
T0 - Nonprimed seed

T3 -Trichoderma 40% 24h

T6 - Azospirillum 20% 18h

T1 - Hydropriming 24h

T4 - pseudomonas 80% 24h

T7 - phosphobacteria 15% 18h

T2 - humic acid 5% 30h

T5 - Azotobacter 20% 30h

REFERENCES
1.

Afzal, I., S.M.A. Basra, M. Shahid, M. Farooq and M. Saleem. 2008. Priming enhances germination of spring maize
(Zea mays L.) under cool conditions. Seed Sci. & Technol., 36: 497-503.

2.

Kalaivani, S. 2010. Seed biopriming studies with biocontrol agents and liquid biofertilizers in COH(M) 5 maize hybrid. M.Sc.
(Ag.) Thesis, Tamil Nadu Agricultural University, Coimbatore.

3.

Juliano, B.O. and J.E. Varner. 1969. Enzymic degradation of starch granules in the cotyledons of germinating peas.
Plant Physiol., 44: 886-892.

4.

Panse, V.G. and P.V. Sukatme. 1985. Statistical methods for agricultural workers. ICAR publication, New Delhi, 359.

5.

Paul, A.K., S. M. Erji and S.M. Sircar. 1970. Metabolic changes in rice seeds during storage. Ind. J. Agric. Sci., 40(12):
1031-1036.

6.

Tarrago, J.F. and G. Nicolas. 1976. Starch degradation in the cotyledons of germinating lentils. Plant Physiolo., 58: 618-621.

7.

Umezuriket, G.M. and F.A. Numfor. 1979. Changes in the content of starch and activities of some enzymes of carbohydrate
metabolism in cotyledons of germinating seeds of Voandzeia subterranean. J. Exp. Bot., 30: 583-588.

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