Seed priming with chitosan improves maize germination and seedling growth in relation to physiological changes under low temperature stress

Seed priming with chitosan improves maize germination and seedling growth in relation to physiological changes under low temperature stress 1 first-author 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:jhu@dial.zju.edu.cn †E-mail:jhu@dial.zju.edu.cn jhu@dial.zju.edu.cn 1 1 2 Seed Science Center, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310029, China Seed Science Center, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310029, China Yuan Longping High-Tech Agriculture Co. Ltd., Changsha 410001, China Yuan Longping High-Tech Agriculture Co. Ltd., Changsha 410001, China Low temperature stress during germination and early seedling growth is an important constraint of global production of maize. The effects of seed priming with 0.25%, 0.50%, and 0.75% (w/v) chitosan solutions at 15 °C on the growth and physiological changes were investigated using two maize ( Zea mays L.) inbred lines, HuangC (chilling-tolerant) and Mo17 (chilling-sensitive). While seed priming with chitosan had no significant effect on germination percentage under low temperature stress, it enhanced germination index, reduced the mean germination time (MGT), and increased shoot height, root length, and shoot and root dry weights in both maize lines. The decline of malondialdehyde (MDA) content and relative permeability of the plasma membrane and the increase of the concentrations of soluble sugars and proline, peroxidase (POD) activity, and catalase (CAT) activity were detected both in the chilling-sensitive and chilling-tolerant maize seedlings after priming with the three concentrations of chitosan. HuangC was less sensitive to responding to different concentrations of chitosan. Priming with 0.50% chitosan for about 60~64 h seemed to have the best effects. Thus, it suggests that seed priming with chitosan may improve the speed of germination of maize seed and benefit for seedling growth under low temperature stress. Seed priming Chitosan Low temperature stress Germination Physiological changes Maize INTRODUCTION 1 Low temperature is one of the most important limiting factors in the productivity of plants. Maize ( Zea mays L.) is a thermophilic crop. Low temperature frequently causes injuries to maize seed germination and seedling growth, thus being detrimental to early spring planting (Parera and Cantliffe, 1994 1994 ). Previous reports indicated that the most conspicuous changes occurred in cellular membranes and enzymes under low temperature stress conditions (Zhou and Leul, 1998 1998 ; Lukatkin, 2003 2003 ). Seed priming has been shown to improve seed performance under sub-optimal temperature conditions (Lin and Sung, 2001 2001 ). Priming increases the environmental range suitable for germination, and provides faster and synchronous seedling emergence (McDonald, 1999 1999 ). Common priming methods such as polyethylene glycol (PEG) treatment are not suitable for large scale cereal crop production because farmers in developing countries could not cover the costs. PEG priming is frequently applied to vegetable seeds in laboratory and field experiments. Other priming methods such as drum priming need special equipment, and some methods are not easy to operate or not cheap enough to be used in large scale (Hu et al., 2005 2005 ). Chitosan is an abundant and comparatively cheap organic compound in China. It is a large cationic polysaccharide mainly obtained from waste materials from seafood processing. Chitosan treatment of wheat seeds induced resistance to certain disease and improved seed quality (Reddy et al., 1999 1999 ). Seed soaked with chitosan increased the energy of germination, germination percentage, lipase activity, and gibberellic acid (GA 3 ) and indole acetic acid (IAA) levels in peanut (Zhou et al., 2002 2002 ). Seed coated with chitosan may accelerate seed germination and improve the tolerance to stress condition of hybrid rice seedlings (Ruan and Xue, 2002 2002 ) and restrain the growth and reproduction of sclerotinia rot in carrot (Cheah et al., 1997 1997 ). Seed priming with two different acidic chitosan solutions improved the vigor of maize seedlings (Shao et al., 2005 2005 ). Thus, it seems that chitosan is a promising material for seed treatments. In the present study, the effects of seed priming with different concentrations of chitosan solutions were investigated. For better understanding the results, chilling-tolerant and chilling-sensitive maize inbred lines were used to determine germination and the seedling growth in relation to physiological changes under low temperature stress after the seed priming. MATERIALS AND METHODS 1 Materials 2 Seeds of two maize ( Zea mays L.) inbred lines, ‘HuangC’ (chilling-tolerant) and ‘Mo17’ (chilling-sensitive) (Zheng et al., 2006 2006 ), from Zhangye Seed Company (Gansu, China) were used. Their original moisture contents were 12.3% and 11.7%, respectively. Seed priming 2 Chitosan solutions (Yuhuan Chemicals, Zhejiang, China) of 0.25%, 0.50%, and 0.75% (w/v) concentrations (pH 5.1) were used for seed priming. Four replicates of 50 seeds each were used for each treatment. Fifty seeds were placed on a 3-layer blotter wetted with 12 ml chitosan solution in a 12-cm diameter petri dish. The petri dishes were sealed with parafilm. The seeds were primed at 15 °C in darkness for 58 h (0.25%) and 64 h (0.50% and 0.75%) for Mo17, and 48 h (0.25%) and 60 h (0.50% and 0.75%) for HuangC, respectively. Following the priming, seeds were dried back to their original moisture contents at room temperature. The un-priming dry seeds were used as control (CK). Measurement of germination and physiological characteristics of seedlings 2 After priming, seed germination tests were carried out. Fifty seeds each for each treatment were placed in a plastic germination box (12 cm×18 cm) with sand bed, and each experiment was replicated four times. Then, seeds were incubated in a germination chamber at 25 °C under alternating cycle of 12 h illumination and 12 h darkness for 5 d (Zheng et al., 2006 2006 ), followed by low temperature stress at 5 °C for 3 d. After that, the seedlings were transferred to 25 °C for recovering growth for 3 d. Seeds were considered germinated when there was a visible coleoptile protrusion through the sand bed. The germinated seeds were counted daily for 11 d. Then, the germination percentage was calculated on Day 11. The germination index and mean germination time were calculated as GI =∑( G t / T t ) and MGT =∑( G t × T t )/∑ G t , respectively (Zhang et al., 2007 2007 ; Kaya et al., 2008 2008 ), where G t is the number of germinated seeds on Day t , T t is time corresponding to G t in days. Root length and shoot height were measured manually with a ruler and dry weights of shoot and root were determined after drying at 80 °C for 24 h. Relative electrolyte leakage as a measurement for membrane permeability was determined according to the method of Li ( 2001 2001 ). Leaves of seedlings were cut into segments of about 5 mm. Each replication (0.2 g fresh weight (FW)) was collected and washed with deionized water, and then placed in the test tube with 10 ml deionized water and covered with a plug. After incubation at 25 °C for 6 h, the relative electrolyte leakage of the leaves was measured using a conductometer (DDS-11A, Shanghai, China), and the value was named E 1 . Subsequently, the test tube was kept in water at 100 °C for 30 min, and then cooled to 25 °C, and the second relative electrolyte leakage was measured as E 2 . The relative electrolyte leakage of deionized water was named E 0 . The relative electrolyte leakage (REL) was calculated as follows: REL (%)=( E 1 − E 0 )/( E 2 − E 0 )×100. The concentrations of malondialdehyde (MDA), proline, and soluble sugar in leaves were measured using thiobarbituric acid (TBA) reaction method, colorimetric method, and anthrone colorimetric method, respectively (Li, 2001 2001 ). Leaf peroxidase (POD) activity was determined by guaiacol method (Zhu and Zhong, 1990 1990 ), and leaf catalase (CAT) activity was determined by the method described by Fu and Huang ( 2001 2001 ). All measurements mentioned above were made after seed germination for 8 d (at 25 °C under alternating cycle of 12 h illumination and 12 h darkness for 5 d, followed by low temperature stress at 5 °C for 3 d) on 20 randomly selected seedlings per measurement. Statistical analysis 2 All data were analyzed by means of analysis of variance (ANOVA) using Statistical Analysis System (SAS) software; percentage data were arcsin-transformed before analysis according to $$\hat y = \arcsin \sqrt {x/100}$$ . Significant level P =0.05 was used. RESULTS 1 Effects of seed priming with chitosan on germination 2 There were no significant differences in germination percentage among treatments irrespective of variety (Table 1 1 ). All the three concentrations of chitosan significantly increased the GI and reduced the MGT when compared with controls in both lines tested. The GI and the MGT of 0.50% chitosan priming treatment were significantly higher and lower than those of 0.75% chitosan treatment, respectively, but were not significantly different from those of 0.25% chitosan treatment in Mo17; however, there were no significant differences among the three priming treatments with chitosan in HuangC. Different letter(s) following the values indicated significant difference ( P =0.05, LSD) among treatments within the same variety Variety Treatment GP (%) GI MGT (d) Mo17 CK 98a 68.22c 7.17a 0.25% chitosan 97a 77.57ab 6.91c 0.50% chitosan 98a 80.09a 6.89c 0.75% chitosan 96a 73.23b 7.01b 5 HuangC CK 97a 69.09b 7.13a 0.25% chitosan 98a 81.18a 6.89b 0.50% chitosan 99a 80.63a 6.88b 0.75% chitosan 97a 80.67a 6.85b Effects of seed priming with chitosan on seedling growth 2 All priming treatments with chitosan significantly increased the shoot height and shoot dry weight compared with the control in Mo17 (Table 2 2 ). Seed priming with 0.50% and 0.25% chitosan significantly increased the root dry weight and the root length as compared with the control, respectively. The most pronounced effect was recorded in 0.50% concentration. Different letter(s) following the values indicated significant difference ( P =0.05, LSD) among treatments within the same variety Variety Treatment SH (cm) RL (cm) SDW (mg/shoot) RDW (mg/root) Mo17 CK 7.23c 12.47b 16.03c 18.15b 0.25% chitosan 10.80a 13.48a 19.23b 17.75b 0.50% chitosan 10.61a 12.49ab 21.43a 21.05a 0.75% chitosan 9.32b 12.69ab 19.67b 17.90b 6 HuangC CK 10.50b 15.26b 26.13b 15.60b 0.25% chitosan 12.99a 18.61a 32.40a 18.33ab 0.50% chitosan 12.65a 17.53ab 35.07a 20.43a 0.75% chitosan 12.88a 19.17a 32.53a 17.57ab For HuangC, seed priming with all the three concentrations of chitosan significantly increased the shoot height and shoot dry weight compared with the control, and there were no significant differences among the three treatments with chitosan (Table 2 2 ). The three priming treatments with chitosan increased root length, and the increase of root length by priming with 0.25% and 0.75% chitosan reached a significant level compared with the control. Seed priming with 0.50% chitosan significantly increased the root dry weight compared with the control, and there were no significant differences among 0.25% and 0.75% chitosan treatments and the control in both lines. Effects of seed priming with chitosan on relative permeability of plasma membrane and MDA concentration 2 The relative permeability of the plasma membrane after the 0.50% and 0.75% chitosan treatments was significantly lower than that of the control in Mo17 (Table 3 3 ). For HuangC, only the relative permeability of plasma membrane of 0.50% chitosan treatment was significantly lower than that of the control. Different letter(s) following the values indicated significant difference ( P =0.05, LSD) among treatments within the same variety Variety Treatment RPPM C MDA (nmol/g FW) Mo17 CK 0.364a 4.08a 0.25% chitosan 0.258ab 3.74ab 0.50% chitosan 0.207b 3.64ab 0.75% chitosan 0.198b 3.36b 4 HuangC CK 0.207a 5.55a 0.25% chitosan 0.184ab 4.67b 0.50% chitosan 0.172b 4.35b 0.75% chitosan 0.188ab 4.25b All the three priming treatments with chitosan reduced MDA concentration and the decrease of MDA by priming with 0.75% chitosan reached a significant level compared with the control in Mo17 (Table 3 3 ). For HuangC, the MDA concentrations of all the three treatments with chitosan were significantly lower than that of the control, and there were no significant differences among the three treatments. Effects of seed priming with chitosan on concentrations of soluble sugar and proline 2 Priming treatments with 0.25% and 0.50% chitosan significantly increased the soluble sugar concentration compared with the control in Mo17 (Table 4 4 ). Seed priming treatment with 0.50% chitosan significantly increased proline concentration compared with the control and 0.75% chitosan treatment, and there was no significant difference between 0.25% and 0.50% chitosan treatments in Mo17. Different letter(s) following the values indicated significant difference ( P =0.05, LSD) among treatments within the same variety Variety Treatment C SS (mg/g FW) C P (μg/g FW) Mo17 CK 18.37b 15.58b 0.25% chitosan 22.74a 18.73ab 0.50% chitosan 22.46a 21.61a 0.75% chitosan 20.41ab 17.61b 4 HuangC CK 32.47b 13.14b 0.25% chitosan 36.20a 14.84a 0.50% chitosan 37.09a 15.60a 0.75% chitosan 39.18a 15.38a For HuangC, all treatments with chitosan significantly increased the concentrations of soluble sugar and proline compared with the control, but there were no significant differences among all the three treatments with chitosan (Table 4 4 ). Effects of seed priming with chitosan on POD and CAT activities 2 In Mo17, the POD activity of the 0.25% chitosan treatment was higher than that of the control but higher concentrations of chitosan did not significantly affect POD activity (Table 5 5 ). There was no significant effect of chitosan treatment on the CAT activity of this variety either. Different letter(s) following the values indicated significant difference ( P =0.05, LSD) among treatments within the same variety Variety Treatment POD activity (U/(g FW·min)) CAT activity (U/(g FW·min)) Mo17 CK 1063b 16.0a 0.25% chitosan 1259a 15.3a 0.50% chitosan 1216ab 19.3a 0.75% chitosan 1108ab 19.0a 4 HuangC CK 732ab 16.5b 0.25% chitosan 606b 19.5a 0.50% chitosan 849a 21.0a 0.75% chitosan 732ab 24.0a In HuangC, seed priming with 0.50% chitosan had no significant effect on POD activity, but significantly increased CAT activity in comparison with the control (Table 5 5 ). Between the three chitosan treatments there was no significant difference in CAT activity. DISCUSSION 1 Priming is used to control the hydration level within seeds so that the metabolic activity necessary for germination can occur, but radicle emergence is still prevented. There are many factors associated with the effects of seed priming, but the concentrations of priming solutions as well as the time and temperature during priming were crucial (Khan, 1992 1992 ). Sometimes, priming effects and conditions varied with varieties (Adegbuyi et al., 1981 1981 ; Khan, 1992 1992 ). In the present study, we found that the combinations of priming time and concentration were different between the two maize lines, Mo17 and HuangC. Seeds of the two inbred maize lines showed different responses to chitosan priming under coldstress. Among the three different concentrations, priming with 0.50% chitosan had the best enhancement effects for chilling-sensitive Mo17, while no significantly different effects among the three concentration treatments were observed for chilling-tolerant HuangC. This difference may be related to the differences in antioxidant enzyme activities, proline concentration, and plasma membrane permeability in these two maize inbred lines (Gao et al., 2006 2006 ). Low temperature induces damage to cell membranes and also affects physiological functions of plants. Relative permeability of the plasma membranes has been used to illustrate the damage degree of low temperature stress (Lukatkin, 2003 2003 ). The lower relative permeability of plasma membrane under low temperature stress means less damage from low temperature stress. Priming with chitosan reduced the relative permeability of the plasma membranes of the two lines under low temperature stress, indicating that chitosan priming alleviated the chilling injury of the seedlings. Similar observations have been made previously by seed film coated with a cold-tolerant agent on maize subjected to low temperature stress (Li et al., 2004 2004 ). Proline and soluble sugar are small molecules, which are synthesized after suffering intimidation action (Hu et al., 2006 2006 ). These molecules are strongly hydrophilic, and alleviate stress damage in plant cells by reducing the water potential and keeping the activities of some biological macromolecules (Rathinasabapathi, 2000 2000 ). Accumulation of organic compounds such as proline in the cytoplasm also plays an important role in osmotic adjustment in plants (Watanabe et al., 2000 2000 ). After sand priming, seed germination and seedling growth of alfalfa were improved, and soluble sugar and proline concentrations under high salt concentration stress were also increased (Hu et al., 2006 2006 ). Similar effects were also achieved in this experiment, which suggests that priming with chitosan improved chilling tolerance in maize seeds. Cell membrane stability was affected by lipid peroxidation caused by active oxygen species under various stress conditions (Fu and Huang, 2001 2001 ), and the concentration of MDA was an indicator of lipid peroxidation in plant cells (Feng et al., 2003 2003 ). The decline of the MDA concentrations in the two primed maize lines under low temperature stress suggested that seed priming with chitosan reduced lipid peroxidation. MDA was produced when polyunsaturated fatty acids in the membrane underwent peroxidation (Meloni et al., 2003 2003 ). The MDA accumulation was reduced after seed film was coated with a cold-tolerant agent in maize under low temperature stress (Li et al., 2004 2004 ), and the decline of the MDA accumulation was achieved after sand priming in alfalfa seedlings under salt stress condition (Hu et al., 2006 2006 ). Our observations are consistent with these earlier reports. The accumulation of MDA may be related to the POD and CAT enzyme activities, the lesser degree of membrane damage (as indicated by low MDA concentration), and the higher activities of antioxidative enzymes (Meloni et al., 2003 2003 ). Chilling conditions may cause an increase in reactive oxygen species (ROS), starting oxidative damage to the membrane system of plants (Wise, 1995 1995 ). Cooperation of protective enzymes such as POD and CAT could eliminate ROS and keep a homeostasis between producing and cleaning of ROS and reduce the level of free radicals. Finally, injury to cells could be declined or avoided (Hu et al., 2006 2006 ). The three concentrations of chitosan used here increased the POD activity of Mo17 and significantly increased CAT activity of HuangC. It seems that for the chilling-sensitive maize seedlings, POD plays a key role in protecting seedlings from low temperature stress, and for the chilling-tolerant maize seedlings, CAT plays a key role. 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Plant Physiological Experiment 10.1631/jzus.B0820373 Project supported by the Major Science and Technology Special Project (priority subject) of Zhejiang Province (No. 2008C12005-1) and the Key Project of Education Department of Zhejiang Province (No. 20070147), China 2008-11-25 2009-04-28 2009-02-23 2022-01-05 Copyright © 2009, Journal of Zhejiang University Science 2009 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 06 10