Hydrogen peroxide pre-treatment for seed enhancement in Cotton (Gossypim hirsutum L.)

Seed germination and plant stand is a major problem in cotton especially under rain-fed cultivation. Various seed hardening treatments have been recommended in cotton for combating the moisture stress during germination. The role of H2O2 as a seed treatment for enhancing cotton seed performance has not been determined. The present study was therefore undertaken to study the effect of H2O2 on germination under moisture stress in comparison with the reported KCl treatment and water treatment with untreated control. The results suggested that seed priming with H2O2 at 80 mM is an effective method for obtaining superior germination and seedling growth under moisture stress condition. Lower values for electrical conductivity of seed leachates as well as malonaldehydes released during germination in the H2O2 treated seeds revealed the improved membrane stability of treated seeds. Parallely, an increase in activities of anti-oxidants viz. Peroxidase and Catalase as well as Malate dehydrogenase was also observed supporting the positive role of H2O2 in enhancing the seed germination of cotton.


INTRODUCTION
Cotton is an important commercial crop of global significance and is cultivated under both irrigated and rain-fed conditions.Incomplete/improper plant population due to scanty/erratic rainfall which is a serious concern in rain-fed cultivation.Nearly 65% of the Indian cotton is grown under rain-fed which is a major cause for low cotton productivity in the country.Obtaining complete germination and establishing good plant stand is a prime requirement for cotton in India where high value seeds such as hybrids incorporated with transgene are sown in almost complete cotton acreage.Various seed hardening treatments to combat moisture stress during germination have been reported in cotton as reviewed extensively by Solaimalai and Subburamu (2004).These include soaking cotton seeds for various duration in water, succinic acid, potassium chloride, potassium dihydrogen phosphate, sodium or calcium salts etc. Seed priming is a technique widely used to overcome the germination related problems in different crops (Afghani et al., 2012;Dahamarudin and Arivin, 2013;Demir et al., 2012).Considerable interest have focused on seed priming since it also ameliorate stress tolerance by improving the activity of antioxidant enzymes which in turn decrease the adverse effects of reactive oxygen species (ROS).Hydro priming, osmopriming (using mannitol/PEG 6000) and halopriming (using KCl/KNO 3 /Calcium salts) have been found effective for cotton seed establishment under unfavourable field conditions (Toselli and Casenave, 2005;Casenave and Toselli, 2007;Papstyliano and Karamanos, 2012;Mohammad et al., 2012).
It has been known for long that pretreatment of seeds with oxidants such as H 2 O 2 (in a dose-dependent manner) leads to breaking, primary seed dormancy (Jann and Amen, 1977), secondary dormancy provoked by salinity, high temperature stresses (Kürsat and Kabar, 2010), or dormancy due to presence of germination inhibitors (Ogawa and Masaki, 2001).Hydrogen peroxide has been known to function as a stress signal in plants (Hung et al., 2005) and hence exogenously applied H 2 O 2 in a dose-dependent manner has been reported to ameliorate seed germination in many crops (Patade et al., 2012;Gregario et al., 2010;Liheng et al., 2009;Azevedo et al., 2005).However, in cotton, Saeed (1974) reported that other than enhancing the germination rate, H 2 O 2 pre-treatment of seeds neither increased the germination percentage nor the root length.There is limited information available on role of H 2 O 2 in cotton seed germination.Therefore, the present study was carried out with a primary objective to evaluate seed treatment effect of H 2 O 2 in comparison with the already reported KCl along with hydration on cotton seed germination and seedling growth under moisture stress condition.The second objective was to determine the biochemical changes occurring in treated seeds.

Seed material
The premium cotton seeds with boll guard insect protection gene incorporated (Bt gene) into it and treated with imidacloprid was used.These seeds were further treated (primed) with KCl (2%), water and hydrogen peroxide (80 mM) by spraying over seeds uniformly followed by quick drying and used for further studies.

Germination under controlled condition
Germination test was carried out using 50 seeds each in four replicates placed on rolled paper towels and incubated at 25°C for 7 days.The parameters observed during and after the experiment were 3 rd , 5 th and 7 th day count.10 normal seedlings selected on 7 th day were subjected to data on shoot length, root length, fresh weight and dry weight.

Germination under moisture stress condition (pot study)
The pots filled with potting mixture (FYM + Sand and clay soil) were watered (quantity required was noted) till complete saturation for one day.The seeds were carefully placed in the soil at 15 seeds per pot during extreme summer month of the year.Subsequent watering was restricted to 40% of initial amount of water required for saturation in pots subjected to moisture stress and 80% in pots subjected to normal soil moisture condition.The seed germination was noted daily for two weeks.After 20 days, the experiment was terminated and seedling length, fresh weight and dry weight of five randomly selected seedlings in each treatment were recorded.

Seed quality assessment on the basis of solute leakage
Primed and non primed seeds (2.5 g) were soaked in 10 to 25 ml of distilled water and incubated at 25 to 30°C for 17 h (Hampton and Tekrony, 1995).The leachate was decanted.The electrical conductivity of leachate was observed in a bench conductivity meter (Eutech Instruments, Singapore).

Volatile aldehydes
Volatile aldehyde released was determined spectrophotometrically. 20 seeds (primed and non primed) were kept in 150 ml conical flask over moist blotter.Test tubes containing 5 ml of 0.2% MBTH was inserted inside the flask and incubatred at 25°C for 48 h in dark.After 48 h the flask was removed and 2.5 ml of 0.23% FeCl 3 solution was added to 1 ml of MBTH.This solution was incubated for 5 min, 6.5 ml of acetone was added to it and absorbance was measured spectrometrically at 635 nm (Wilson and Mc Donald, 1986).

Activity assessment of major enzymes involved in seed germination
Extraction and enzyme activity assays were carried out with both primed and non primed seeds.The seeds were kept for germination in Petri plate over moist blotter at 25°C in dark for 2 to 3 days.The germinated seeds (hypocotyls emerged) were taken as sample for enzyme extraction (Sadasivam and Manikam, 1992).
Peroxidase: The fresh plant tissue was ground in pre chilled mortar and pestle in 0.1 M phosphate buffer (pH-7.0).The homogenate was centrifuged at 18000 g at 4°C for 15 min and supernatant was used as enzyme extract.The reaction mixture containing phosphate buffer (0.1 M, pH 7.0), guiacol solution (20 mM), H 2 O 2 solution (0.042%) and enzyme extract was made.The absorbance was recorded at 436 nm.
Catalase: The fresh plant tissue was homogenized in blender with phosphate buffer (pH 7.0) at 1-4°C and centrifuged.The supernatant was used as a source of enzyme extract.The reaction mixture containing phosphate buffer and enzyme extract was made as per the protocol and absorbance was recorded at 240 nm.
Malate dehydrogenase: The fresh plant tissue was ground in pre chilled mortar and pestle in grinding medium containing 50 mM Tris TCl (pH-8.0),50 mM MgCl 2 , 5 mM 2-mercaptoethanol and 1 mM EDTA and the filterate centrifuged at 3000 g for 20 min at 4°C and the supernatant was used as enzyme source.The reaction mixture was made as per the protocol and absorbance was recorded at 340 nm

Statistical analysis
All the data was subjected to statistical analysis with WEB AGRI STAT PACKAGE (WASP).Analysis of variance (ANOVA) was done to test different priming techniques.Multiple comparison tests were used to separate significant differences among all treatments at the 0.01 level.Standard error (SE) was calculated and results were shown in figure and tables.

RESULTS
Total seed germination, seedling lengths (shoot and root length) as well as seedling dry weight was investigated after seed treatments under normal (laboratory and pots) as well as moisture stress condition.

Effect on germination (Laboratory condition)
Data pertaining to the rate of germination observed on 3 rd , 5 th and 7 th day under laboratory condition are provided in Figure 1.The time required to reach 50% germination tended to be lower for H 2 O 2 treated seeds observed as 3 rd day count.The germination uniformity was also found higher for H 2 O 2 treated seeds (data now shown).The superiority of H 2 O 2 treatment was evident in observations on 5 th and 7 th days too with significantly highest germination percentage over others.

Effect on seedling growth (Laboratory condition)
The significant effect of treatments on seedling growth was evidenced by observations on seedling shoot, root length, fresh and dry weights (Table 1).The root length increased to 14 cm after H 2 O 2 treatment and 12 cm after KCl treatment from 10 cm in untreated control.Though significant difference was observed among the treatments for shoot length, the treatments varied minimally for their effect.
Paralleling with the improvement in shoot and root lengths, the seedling fresh and dry weight also was higher in the treated seeds especially, H 2 O 2 treated compared to control.The trend followed a similar pattern with H 2 O 2 treatment giving maximum values followed by KCl and water.The untreated control had least values for both parameters.

Germination/emergence under normal and moisture stress condition (Pot study)
The seedling emergence studied in pots under normal moisture (80% FC) and under moisture stress condition (40% FC) also showed the enhanced effect of treatment over untreated control Figure 2).Notably, the effect on final emergence was clear under moisture stress   condition where the H 2 O 2 treated seeds had significantly high emergence.
The values for shoot and root length showed a similar pattern under both the soil conditions with H 2 O 2 treated seeds giving the highest values followed by KCl and water (Table 2).To address whether these enhanced effects of treatment on germination and seedling growth is due to improvement in cellular membrane stability and antioxidant enzyme system, further study on electrical conductivity of seed leachates and malonaldehyde levels were determined.

Electrical conductivity
The membrane stability observed through electrical conductivity of seed leachates was maximum in H 2 O 2 treated seeds which had lowest values for electrical conductivity (Figure 3).The untreated control had the minimum membrane stability shown by the highest values for conductivity of seed leachates.

Malonaldehyde content
It was observed that treated seeds released fewer amounts of volatile aldehydes as compared to untreated control seeds.The best results were obtained in H 2 O 2 treated seeds which gave the lowest result where as the highest volatile aldehyde content was observed in control (Figure 4).

Activities of major anti-oxidant enzymes
Data showed that the activity of catalase, one of the major antioxidant enzyme as significantly high in treated seeds as compared to control (Figure 5a, b, c).The highest enzyme activity was observed in H 2 O 2 treatment followed by KCl and water.The lowest activity was observed in untreated control.Similarly peroxidase enzyme activity was highest for H 2 O 2 and KCl treated seeds compared to others.

DISCUSSION
Improved seed invigoration techniques play significant role in reducing the germination time, obtaining synchronized germination, improving germination rate and seedling stand in all major crops including cotton (Bradford et al., 1990; Rathinavel and Dharmalingam,  2000; Amjed et al., 2002) and field crops like wheat, maize and rice (Basra et al., 2006;Janmohammadi et al., 2008;Farooq et al., 2004).These invigoration techniques include seed soaking/seed priming/pelleting treatments.Spraying of a water mist over the seeds is considered a simple hydro priming technique which allows the moisture to equilibrate (Van Pijlen et al., 1996) and enhance early germination.Various organic seed treatment preparations (biodynamic and herbal) have been recommended to be applied as spray on seeds followed by quick drying to encourage germination and seedling establishment (Courtney, 1994).
Similar method was applied in the present study also where required concentration of H 2 O 2 and KCl as well as water was sprayed over the seeds followed by drying.The process allows limited entry of moisture just enough to initiate the germination process unlike soaking treatment where there is no limitation of water availability.Moreover, the technique is farmer friendly with no loss of imidachloprid insecticide coating provided on cotton seeds.
The results on seed germination, seedling growth and root length showed the positive effect of treatments, H 2 O 2 and KCl in enhancing the cotton seed performance under moisture stress, compared to water treated and untreated control.The influence of seed priming in improving the germination rate, germination uniformity and total seed germination has been well documented in rice and wheat (Basra et al., 2002(Basra et al., , 2004)).In cotton, seed priming with KCl enhanced the speed of germination and activities of antioxidant enzyme (Aghani et al., 2012).KCl has been shown as an effective osmotic agent for enhancing seed germination of wheat (Leila et al., 2010;Mehrdad and Hosseini, 2012) and rice (Mohammed et al., 2006).Greater efficiency of seed priming with KCl is possibly related to the osmotic advantage that K+ has in improving cell water saturation, and that they act as co-factors in the activities of numerous enzymes (Taiz and Zeiger, 2002).
The treatment with H 2 O 2 proved the best among all the seed treatments with regard to germination and growth parameter studied in the experiment.This is in contrary with earlier finding in cotton by Saeed (1974) (Hameed et al., 2004;Yushi et al., 2008), barley (Kürsat and Kabar, 2010;Kabar, 2010), maize (Azevedo et al., 2005) etc.The treated seeds revealed higher activity for antioxidant enzymes such   (Djanaguiraman et al., 2005) were observed in earlier studies on cotton.These enzymes are known to scavenge the reactive oxygen species produced during imbibitions and decrease their adverse effects (Del Ryo et al., 2002).Similar effect of seed treatments on activity of scavenging enzymes such as catalase, peroxidase and superoxide dismutase resulting in enhanced germination has been reported in other crops too (Zaheer et al., 2012;Papassorn et al., 2012;Adnan et al., 2012;Mustafa et al., 2010).
Membrane repair could be ascribed to evoke activities of free-radical scavenging enzymes (Adnan et al., 2012).This was confirmed by the low values obtained for electrical conductivity of seed leachates from treated seeds especially those from H 2 O 2 treated ones.Present results indicate the higher membrane stability of treated seeds.Correspondingly, there was decrease in malonaldehyde content in the treated seeds compared to untreated control as observed in Jatropha by Feng et al. (2011).Enhanced membrane stability due to reduced membrane damage rate and MDA content was observed in wheat after pretreatment of seeds with H 2 O 2 (Liheng et al., 2009).

Conclusion
The results suggest that seed priming (uniform spraying on seeds) with H 2 O 2 at 80 mM is a convenient method of seed priming to improve germination and better moisture stress tolerance in cotton.The improved germination and stress tolerance may be attributed to the enhanced activity of anti-oxidant enzymes and improved membrane stability.

Figure 1 .
Figure 1.Effect of treatments on germination at different intervals.

Figure 2 .
Figure 2. Effect of treatments on germination under normal and moisture stress condition.
same letters are not significantly different Comparison of Treatment means with Critical Difference (0.05).

Figure 3 .
Figure 3.Effect of seed treatment on electrical conductivity of seed leachates.

Figure 4 .
Figure 4. Effect of seed treatment on volatile aldehydes released from cell membrane.

Figure 5 .
Figure 5. a, b, c, Comparison of enzyme activities in treated and untreated seeds.

Table 1 .
Effect of treatment on seedling growth traits under controlled condition means followed by same letters are not significantly different.

Table 2 .
Effect of treatment on seedling growth traits under normal and moisture stress.
as peroxidase and catalase with H 2 O 2 pretreatment giving the highest values Increase in activities of these enzymes after seed priming with KCl 2%(Mohammed et al., 2012)and after seed treatment with Atonik