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Germination and Physiological Response of Wheat (Triticum aestivum) to Pre-soaking with Oligochitosan.

Byline: Lian-Ju Ma Yue-Ying Li Lan-Lan Wang Xue-Mei Li Ting Liu and Ning Bu


The effect of seed soaking in different concentrations of oligochitosan (1.0 0.5 0.25 0.125 0.0625 0.03125 and 0%) was studied on seed germination seedling photosynthetic capacity chlorophyll (Chl) contents root activity and antioxidant enzymes activities of wheat. The result indicated that seed germination rate was increased. The root length was elongated and root activity was increased. The seedling length was improved with increase in chlorophyll content photosynthesis rate (Pn) and stomatal conductance (Gs). Antioxidant enzymes activities (SOD CAT and POD) in leaves were increased. Variation in response of wheat to given oligochitosan concentration was found. Effect of low concentration oligochitosan was better than high concentration in the growth-regulatory activity among different oligochitosan concentrations.

The most suitable concentration found was 0.0625% followed by 0.125% oligochitosan. In conclusion pre-soaking with oligochitosan can be used to improve wheat growth and development via modification in physiological and biochemical process. Copyright 2014 Friends Science Publishers.

Keywords: Oligochitosan; Wheat; Seed germination; Antioxidant enzymes.


Various exogenous compounds are applied to promote plants growth and induce abiotic resistance (Koc 2013; Rehman et al. 2013; Yasmeen et al. 2013). For example foliar application of ascorbic acid kinetin and glutamic acid promoted growth of Codiaeum variegatum L. plants (Mazher et al. 2011). Exogenous application of 20 mM proline as seed treatment increased growth photosynthesis and antioxidant enzymes activities on Brassica juncea L. under natural conditions (Wani et al. 2012). Root application of 0.75 ppm selenium enhanced growth in mungbean (Phaseolus aureus Roxb.) through up-regulation of enzymes of carbohydrate metabolism (Malik et al. 2011). Biologically active oligosaccharides as signal molecules are one of exogenous compounds which can exert plant growth and regulate gene expression to enhance defense reactions (Albersheim and Darvill 1985).

For example alginate-derived oligosaccharide could promote maize seed germination (Hu et al. 2004) and alleviate Vicia faba root tip cells exposed to cadmium stress (Ma et al. 2010a). Oligochitosan obtained by enzymatic hydrolysis of chitosan is similar to plant growth regulators (PGRs) consists of AY-1 4-linked 2-amino-D-glucose units and contains only small amounts of 2-acetamido-D-glucose units. It is reported that oligochitosan possesses multifunctional properties because of biocompatible biodegradable and a sustainably renewable resource.

Oligochitosan application excites a series of defense responses in rice to enhance the disease resistance to Magnap or the Grisea (Ning et al. 2003) reduced decrease of photosynthetic rate (Pn) in B. napus seedlings under drought stress (Li et al. 2008) induced resistance to Sclerotinia scleraotiorum on B. napus (Yin et al. 2008) and ameliorated the adverse effects of wheat seedlings exposed to salt stress (Ma et al. 2012). It is also reported to regulate plant growth and development (Fry et al. 1993). Therefore oligochitosan has a strong potential application value in agriculture. Reactive oxygen species are related to light-dependent events produced in plants even under optimal conditions. Therefore photosynthetic cells are easily damaged by oxidative stress due to producing and consuming oxygen in metabolic processes.

The superoxide radical (O. -) singlet oxygen (1O2) and H2O2 are major source of activated oxygen which injures the cellular components of proteins nucleic acids and membrane lipids (Foyer et al. 1994). It is reported that oligochitosan induced accumulation of H2O2 in rice cells (Lin et al. 2005) and plants. It is involved in the oxidative burst and the induction of the reactive oxygen species (ROS) scavenging system (Agrawal et al. 2002). Enzymatic systems consisting of superoxide dismutase (SOD) catalase (CAT) and peroxidase (POD) are responsible for scavenging these active oxygen species in plants growth and development (Ahmad et al. 2013). Superoxide dismutase and CAT were found to be induced by oligochitosan treatment (Guo et al. 2003).

Nonetheless very little information is known about effects of oligochitosan on wheat seedlings growth and development. The present study evaluated the effect of different concentrations of oligochitosan on wheat seed germination and seedling physiological responses.

Materials and Methods

Plant Culture and Treatments

Wheat (Triticum aestivum L.) seeds were surface sterilized in 2% sodium hypochlorite solution for 10 min and rinsed three times with distilled water. Subsequently rinsed seeds were soaked in 1 0.5 0.25 0.125 0.0625 and 0.03125% oligochitosan solution (supplied by Ocean University of China) for 5 h. Seeds soaked in distilled water were taken as control. Germinated seeds were cultivated in a 500 ml beaker containing full Hoagland's nutrient solution and seedlings were grown in a controlled chamber. The average day/night temperature was kept at 25oC/ 20oC respectively with a mean photoperiod of 12 h relative humidity 80% and light intensity of 800 mol m-2 s-1. The Hoagland's nutrient solution was renewed every third day.

Growth Parameters

Daily counts of germinated seed were made. At 3-leaf stage seedling height root length and seedling fresh weight was recorded. Plant dry weight was obtained by oven-dried at800C until a constant weight.

Root Activity

Root activity was measured by triphenyl tetrazolium chloride (TTC) method (Steponkus and Lanphear 1967). Root samples of 0.5 g were fully immersed in solution containing 5 mL of 4% 2 3 5-triphenyl tetrazolium chloride (TTC) and 5 mL phosphate buffer. The mixtures were keptat 37 under dark conditions. After 2 h 2 mL of 1 M H2SO4 was immediately added to mixtures to terminate thereaction. Roots were taken out and blotted with the filter paper then fully grinded with 3-4 mL ethyl acetate. Redextract was exhaustively collected in test tube. The residue was washed 2 to 3 times with a small amount of ethyl acetate and graduated into test tube. Finally a total of 10 mL was made with ethyl acetate. Color intensity measured at485 nm. The reduction of TTC content was calculated according to standard curve.

Chlorophyll Content and Photosynthetic Characters Fresh leaves 0.1 g were collected and extracted with 80% acetone and ethanol (v/v =1:1) for 24 h in the dark. Chlorophyll content followed spectrophotometrically as described by Lichtenthaler (1987). The photosynthetic rate (Pn) and stomatal conductance (Gs) were measured with a portable photosynthesis system (LI-6400 Lincoln NE USA).

Antioxidant Enzyme Activities

About 0.5 g samples was homogenized with 5 mL of extraction buffer (0.1 M phosphate buffer at pH 7.8) 0.1 mM EDTA 1 g PVP). The homogenate were centrifuged at10000 A- g for 15 min and the supernatants were as crude to determine SOD POD and CAT activity. Superoxide dismutase CAT and POD activity were measured as described by Costa et al. (2002) Cakmak and Horst (1991) and Kochba et al. (1977) respectively. Protein concentration was estimated according to Lowry (1951) using bovine albumin as standard.

Statistical Analysis

Each treatment was conducted with three replicates. All data were analyzed according to Duncan's multiple range test using the SPSS 11.0 software package.


Plant Growth and Biomass Accumulation

Oligochitosan promoted wheat growth in terms of increased germination capacity seedling fresh and dry weight and enhanced root length and seedling height (Table 1). Except for seeds treated by 1% oligochitosan germination capacity reached above 90% than control with germination of 85.4%. There was apparent increase in seedling height when oligochitosan concentrations varied from 0.0625 to 0.125%. Seedling height treated by 0.0625% oligochitosan increased by 26.4% of the control. Root length with 0.0625% or0.125% oligochitosan application was significantly longer than control. Seedling fresh and dry weight treated by0.0625% oligochitosan increased by 17.1 and 13.9% of the control respectively.

Root Activity

Triphenyl tetrazolium chloride (TTC) indicated roots dehydrogenase activity as a proton receptor (Steponkus and Lanphear 1967). Root activity was significantly increased in comparison to control (Fig. 1) when oligochitosan concentration varied from 0.0625 to 0.5% and a maximum of increase was obtained at 0.0625% oligochitosan in root activity.

Chlorophyll Contents and Photosynthetic Characters

Chlorophyll content of plants treated with oligochitosan increased (Fig. 2). Chlorophyll content showed a significant difference (p 0.05 in comparison to control when oligochitosan concentration varied from 0.03125 to 0.25%.

Chlorophyll content of 0.0625% oligochitosan was the highest with increase of 111.2% of the control. Chlorophyll content gradually decreased with oligochitosan concentration varying from 0.0625 to 1%. Positive

Table 1: Effects of oligochitosan on seed germination percentage and growth of wheat seedlings

Oligochitosan concentration###Germination percentage###Seedling height###Root length###Fresh weight###Dry weight


0###85.4d###15.120.46b###11.440.32b###2.2510.12b###0.2210.01 b

0.03125###92.5b###16.040.52b###12.930.29b###2.480.15b###0.2300.01 b

0.0625###96.1a###19.110.41a###15.880.53a###2.6010.1a###0.2490.02 a

0.125###95.7a###18.620.63a###14.530.48a###2.5420.22a###0.2410.01 a

0.25###93.1ab###15.630.47b###12.710.37b###2.3060.25b###0.2260.03 b

0.5###91.2bc###15.240.57b###12.120.42b###2.2960.31b###0.2240.02 b

1###89.8cd###15.180.34b###11.980.33b###2.2730.17b###0.2230.01 b

Table 2: Correlation coefficient (r) between seedling and chlorophyll (Chl) content photosynthesis rate (Pn) and stomatal conductance (Gs)

Parameter###Chl content###Pn###Gs

Seedling height###0.86###0.83###0.89

correlation was found between seedlings and chlorophyll content (r=0.86) (Table 2). Oligochitosan induced a remarkably increase in photosynthetic rate (Pn) (Fig. 3a). Significant differences were noted in Pn from 0.03125 to 0.25% (p less than 0.05). Photosynthetic rate with 0.0625% oligochitosan reached the highest to 14.6 mol m-2 s-1. Stomatal conductance (Gs) response tendency to oligochitosan were consistent with Pn (Fig. 3b). A linear positive correlation existed between seedlings and Pn (r=0.83) and Gs (r=0.89) respectively (Table 2).

Antioxidant Enzymes Activities

Superoxide dismutase activity was higher than control under different oligochitosan concentrations (Fig. 4a). A peak in SOD activity was observed at 0.0625% but decreased gradually afterward. Superoxide dismutase activity variation of different concentrations of oligochitosan was not apparent. The response trend of CAT activity was similar to SOD activity (Fig. 4b). Except for 0.03125 and 1% oligochitosan CAT activity of wheat leaves showed significant difference compared with control. Peroxidase activity (POD) increased when oligochitosan concentrations was above 0.0625% but gradually decreased below0.0625%. Thus POD activity of 0.0625% oligochitosan treatment showed the highest value (Fig. 4c).


Oligochitosan promoted wheat growth in terms of germination capacity root length and seedling height (Table 1) and increase in root activity (Fig. 1). Li et al. (2008) reported increased dry weight of B. napus Zhang and Lin (2010) improved seedling vigor and uniform roots in Salvia miltiorrhiza Bge. Chlorophyll content of wheat seedlings treated with oligochitosan increased (Fig. 2) and increased photosynthetic rate (Pn) and Gs were found (Fig. 3). Increase of Pn might promote wheat leaves to enhance its assimilation which increased dry matter accumulation (Li et al. 2008). Increased root activity (Fig. 1) chlorophyll content Pn and Gs led to improve the growth and interestingly increased biomass after all the oligochitosan concentrations treatment was found (Table 1).

Different concentrations of oligochitosan induced oxidative burst in 20-30 min and antioxidant enzymes activities (SOD CAT) were increased in 60-90 min on suspended cotton cells (Guo et al. 2003). Increase of antioxidant enzymes activities might be beneficial to eliminate excess of reactive oxygen species. In present study we also found that antioxidant enzymes activities were changed in wheat leaves treated by different concentrations of oligochitosan. Superoxide dismutase (SOD) activity in wheat leaf significantly increased from 0.5 to 0.0625% oligochitosan in comparison with control (Fig. 4a). Superoxide dismutase activity increased protection of wheat seedling from oxidant damage is consistent with our earlier findings in wheat under salinity suggesting the participation of SOD in the defense mechanism during seedlings development (Ma et al. 2010b). Catalase subsequently scavenged H2O2 produced by SOD against oxidative stress. Except for 0.03125 and 1% oligochitosan CAT activity in wheat leaves showed significant difference compared with control after oligochitosan treatment (Fig.4b). Peroxidase is also considered to scavenge H2O2 and keep H2O2 balance in plant tissues. It was shown that POD activities of 0.0625 and 0.125% oligochitosan increased in wheat leaves compared with control (Fig. 4c). Thus increased CAT and POD activity helped wheat seedling development. Yin et al. (2008) and Yafei et al. (2009) found that oligochitosan upregulated the activities of phenylalanine ammonialyase (PAL) polyphenoloxidase POD CAT and SOD in tobacco and B. napus.Nonetheless variation was found in wheat seedling response to given oligochitosan concentrations. Effect of low concentration oligochitosan was better than high concentration in the growth-regulatory activity which acts as an antioxidant and stimulated the plant growth. The character of oligochitosan was similar to plant hormone IAA. Oligochitosan can promote growth by effectively induced the increase of IAA concentration in tobacco (Guo et al. 2009). Among different oligochitosan concentrations0.0625% oligochitosan was the most suitable to promote wheat seedling growth than 0.0325 and 1%. As high oligochitosan concentration induced accumulation of H2O2 (Lin et al. 2005) led to plant growth being inhibited. Low concentration oligochitosan had less effect on plant growth. The presence of oligochitosan triggers a wide range of cellular responses including changes in gene expression involved in different processes such as primary metabolismtranscription defense and signal transduction (Yoon et al.1999; Yin et al. 2010). Nonetheless oligochitosan functions as signals to promote the growth of wheat seedling and triggers expression of antioxidant enzymes gene (Yafei et al. 2009) and eliminate excess of ROS enhancing antioxidant enzymatic activation under optimal concentration.In conclusion oligochitosan effected growth and development defense and other interactions of wheat plants with the environment via changing physiological and biochemical process. The most suitable oligochitosan concentration was 0.0625% when wheat (Triticum aestivum L.) seeds were pre-soaked with different oligochitosan levels.


This work was supported by the National Natural Science Foundation of China (31070285) the Liaoning province Natural Science Foundation (20102205) and the Director Foundation of the Experimental Center at Shenyang Normal University (SY201004 SY201102).


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Publication:International Journal of Agriculture and Biology
Date:Aug 31, 2014
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