Maize seed coatings and seedling sprayings with chitosan and hydrogen peroxide: their influence on some phenological and biochemical behaviors

Maize seed coatings and seedling sprayings with chitosan and hydrogen peroxide: their influence on some phenological and biochemical behaviors 1 first-author 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:spmcunam55@gmail.com †E-mail:spmcunam55@gmail.com spmcunam55@gmail.com 2 1 3 Laboratorio de Biotecnología, Facultad de Estudios Superiores Cuautitlán, Campo 1, Universidad Nacional Autónoma de México, Estado de México 54740, México Laboratorio de Biotecnología, Facultad de Estudios Superiores Cuautitlán, Campo 1, Universidad Nacional Autónoma de México, Estado de México 54740, México Unidad de Investigación en Granos y Semillas, Facultad de Estudios Superiores Cuautitlán, Centro de Asimilación Tecnológica, Universidad Nacional Autónoma de México, Estado de México 54740, México Unidad de Investigación en Granos y Semillas, Facultad de Estudios Superiores Cuautitlán, Centro de Asimilación Tecnológica, Universidad Nacional Autónoma de México, Estado de México 54740, México CA Ingeniería de Biosistemas, División de Investigación y Posgrado, Facultad de Ingeniería, Universidad Autónoma de Querétaro, Querétaro 76010, México CA Ingeniería de Biosistemas, División de Investigación y Posgrado, Facultad de Ingeniería, Universidad Autónoma de Querétaro, Querétaro 76010, México Objective: 2 To evaluate the effect of chitosan (CH) and hydrogen peroxide (H 2 O 2 ) seed coatings and seedling sprinklings on two different maize varieties by measuring their phenology, the H 2 O 2 presence, the catalase (CAT) activity, and the protein quantity. Methods: 2 Seven groups of ten seeds for each maize variety were treated with CH (2% (20 g/L) and 0.2% (2 g/L)) or H 2 O 2 (8 mmol/L) by coating, sprinkling, or both. Germination and seedling growth were measured. One month after germination, the presence of H 2 O 2 in seedlings in the coated seed treatments was evaluated. Protein content and CAT activity were determined under all treatments. Results: 2 H 2 O 2 seed coating enhanced the germination rate and increased seedling and stem length in the quality protein maize (QPM) variety. Seedlings had a higher emergence velocity under this treatment in both varieties. CH and H 2 O 2 sprinklings did not have an effect on seedling phenology. Exogenous application of H 2 O 2 promoted an increase of endogenous H 2 O 2 . CH and H 2 O 2 seedling sprinkling increased the protein content in both maize varieties, while there was no significant effect on the CAT activity of treated seeds and seedlings. Conclusions: 2 CH and H 2 O 2 enhance some phenological and biochemical features of maize depending on their method of application. Zea mays Chitosan Hydrogen peroxide Phenology Catalase Protein Introduction 1 Worldwide cereal crops are currently threatened by adverse environmental conditions and pathogen attacks which impede optimum seed germination, diminishing significantly seedling growth and productivity. Specifically, these conditions can be observed in the production of maize ( Zea mays ), one of the most important crops in the world. Both maize production and nutritional quality have been seriously affected. According to the Food and Agriculture Organization, during 2010, 844 405 181 tons of maize were produced globally, a mere 3.01% increase over 2009 production levels (FAOSTAT, 2010 2010 ). With these threats to human and animal food security in mind, researchers have been looking for new methods to enhance maize production and improve its quality. In order to increase seed germination and plant phenology, the application of some chemical and biochemical substances has been tested in different crops, focusing on the use of salicylic acid (Khodari, 2004 2004 ), abscisic acid (Sarath et al., 2007 2007 ), and more recently, hydrogen peroxide (H 2 O 2 ). Although H 2 O 2 has been used for years to disinfect seeds prior planting (Piernas and Guiraud, 1997 1997 ; Weissinger and Beuchat, 2000 2000 ; Miché and Balandreau, 2001 2001 ), it has recently been found that the exogenous application of vital cellular component to seeds and plants has positive effects over them. H 2 O 2 treatment increases seed germination rates (Korystov and Narimanov, 1997 1997 ; Amjad et al., 2004 2004 ; Çavusoglu and Kabar, 2010 2010 ), coleoptile emergence percentages, radicle and coleoptile elongation, and fresh weights of the seedlings (Çavusoglu and Kabar, 2010 2010 ). H 2 O 2 is coupled with important functions in metabolism, homeostasis of plants and reactive oxygen species (ROSs) generation too. It has been reported that soaking seeds in H 2 O 2 induces a pronounced increase in enzymes activity levels, including catalase (CAT), ascorbate peroxydase (APX), and superoxide dismutase (SOD) (Gondim et al., 2010 2010 ; Li et al., 2011 2011 ). These enzymes play an important role on naturally over-expressing stress responses and signal activation at biochemical level. Some authors have reported negative aspects in the use of H 2 O 2 . For example, exogenous H 2 O 2 poisons seedlings and reduces seed germination due to excessive activation of ROSs (Edwards and Sutherland, 1979 1979 ; James and Genz, 1981 1981 ; Pernezny et al., 2002 2002 ). Chitosan (CH) is another chemical that has recently been used in plant protection. This biopolymer is a large cationic polysaccharide mainly obtained from waste materials from seafood processing (Guan et al., 2009 2009 ), with antiviral, antibacterial, and antifungal properties (El-Hadrami et al., 2010 2010 ). When CH is applied to plant seeds, their germination index is enhanced, the mean germination time is reduced, shoot height, root length, and seedling vigor are increased (Bhaskara Reddy et al., 1999 1999 ; Ruan and Xue, 2002 2002 ; Shao et al., 2005 2005 ; Guan et al., 2009 2009 ; Kananont et al., 2010 2010 ; Ziani et al., 2010 2010 ; Zeng et al., 2012 2012 ), vegetative growth is increased, time to flowering is reduced (Ohta et al., 1999 1999 ), and fresh weight is increased (Asghari-Zakaria et al., 2009 2009 ). CH has been applied not only to seeds but also to seedlings. CH foliar application on strawberry crops increases plant height and number of leaves, augments leaf fresh and dry weights, and enhances the number and weight of seedlings (Abdel-Mawgoud et al., 2010 2010 ). It has been reported that CH has effects not only at the phenological level but also at the enzymatic level. This is evidenced by peroxidase (POD) and CAT activity level increases in the protein extract of some edible cultivars like tomato, guava, and sweet wormwood that were treated with CH (Ortega-Ortíz et al., 2007 2007 ; Guan et al., 2009 2009 ; Lei et al., 2011 2011 ; Hong et al., 2012 2012 ). The inhibition of CAT activity (Zeng et al., 2010 2010 ) is important in plant development, defense, aging, and senescence too (Yang and Poovaiah, 2002 2002 ). Although, there are multiple reports of CH application in food production, there are not reports for the effect of CH application on crop nutritional value. Research into the effects of CH and H 2 O 2 has targeted application at seed and seedling levels, because in these early stages most of the biochemical and enzymatic mechanisms are initiated, having continuing effects on growth as the plant continues to develop. The aim of this investigation was to evaluate the effects of CH and H 2 O 2 seed coatings, and CH and H 2 O 2 sprinklings on seedlings of two different varieties of maize by measuring their phenology, H 2 O 2 presence, enzymatic activity, and protein quantity under both chemical treatments. To carry out this investigation, we evaluated in vivo, during short periods of time and under greenhouse conditions, the effect of coating corn seedlings with CH and H 2 O 2 on their germination and growth (thickness of stems, total length of the leaves and stems) of seedlings. After one month of growing, the presence of H 2 O 2 was evaluated in treated plants in order to determine if the use of H 2 O 2 and CH enhance H 2 O 2 production. Then, CAT activity was determined in seedling leaves from treated plants. Finally, plant proteins were quantified in order to know if the application of CH and H 2 O 2 increased protein content. Materials and methods 1 Materials 2 Two maize seed varieties were used in this experiment: a normal variety (N-279, with a moisture content of 11.5% and a germination rate of 100%), and a high quality protein maize variety (QPM-374, with a moisture content of 11.7% and a germination rate of 100%) obtained from the Maize Breeding Program of the Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP) based in the Campo Experimental del Bajío, México. CH for maize treatment was obtained from chitin shrimp exoskeletons, with a molecular weight of 125 kDa and a deacetylation degree of 92%, and it was produced and characterized in our laboratories without further purification (Miranda, 2000 2000 ). H 2 O 2 (30%) of reagent grade was obtained from Fermont, Productos Químicos de Monterrey, SA de CV, Monterrey, NL, Mexico, with a molecular weight of 34.01 g/mol. Preparation of coating and sprinkling solutions 2 A 2% (20 g/L) CH solution was prepared, dissolving 10 g of the biopolymer in 500 ml of water acidified with acetic acid (Meyer Chemical SA de CV, Tlahuac, DF, Mexico), and kept under constant agitation for 24 h before its pH was adjusted to 5.0 with a 12% (120 g/L) sodium hydroxide solution (JT Baker, Xalostoc, Edo., de Mexico, Mexico). An 8 mmol/L H 2 O 2 solution was prepared, diluting 4.074 ml of commercial reagent in 500 ml of distilled water, reaching a pH of 7.8. For seedling sprinkling, a 0.2% (2 g/L) CH solution was prepared, dissolving 1 g of the biopolymer in 500 ml of acidified water, under the same conditions as the CH seed coating preparation. The same H 2 O 2 seed coating preparation was used for seedling sprinkling. Seed and seedling treatments 2 Fourteen groups were formed (each one of ten seeds, sowing one seed per pot): seven groups of the normal variety and seven groups of QPM maize. The first group was considered to be a control (with no treatment); in the second group, seeds were coated with CH; in the third group seedlings were sprinkled with CH; the fourth group was submitted to a combined treatment (seed coating and seedlings sprinkled with CH); in the fifth group seeds were coated with H 2 O 2 ; in the sixth group seedlings were sprinkled with H 2 O 2 ; and the seventh group was submitted to a combined treatment (seeds coating and seedling sprinkling with H 2 O 2 ). CH treated seeds were soaked in the 2% CH solution for 12 h and then they were dried in an oven at 29 °C for 24 h to counteract the effects of excessive moisture on the seeds by treatment with the biopolymer solution. H 2 O 2 -treated seeds were soaked in the 8 mmol/L solution for 12 h and were not dried. Maize seedlings were sprinkled with the CH and H 2 O 2 solutions, finely the entire seedling canopy. The study was conducted in the greenhouse of the Grains and Seeds Research Unit in Facultad de Estudios Superiores, Cuautitlán, UNAM, which was previously sanitized in order to carry out the planting under protected conditions (ISTA, 1993 1993 ; Albajes et al., 1999 1999 ; Sonneveld and Voogt, 2009 2009 ) to avoid interference in the experimentation results. The substrate on which seeds were seeded was prepared using Sunshine mixture #3® (Sun Gro Horticulture Inc., Canada CM, Ltd.) enriched with perlite Hortiperl® (Termolita SA de CV, México) in a proportion of 10:1. Seeds were sown at a depth of 3 cm, and pots were irrigated with municipal tap water. Evaluation of maize seedling germination 2 All seeds were sown at the same time. Germination was evaluated until seeds reached the final stages of germination (Monasterio et al., 2007 2007 ). Evaluation of maize seedling growth 2 First, seedling emergence velocity was evaluated nine days after sowing. Different numbers were given to each emergency phase: 0 for non germinated seeds, 1 for coleoptiles, 2 for plumules, 3 for seedlings with one leaf seedlings, and 4 for seedlings with two leaves. Then, seedling growth was assessed in two stages: 19 d after planting (where the seeds were only coated but not sprinkled with CH and/or H 2 O 2 ), and on the 13th day (because at this stage seeds were coated and seedlings were sprinkled 5 d before). Stem length, sheet length, overall seedling length, and stem diameter were measured. Diaminobenzidine (DAB) staining for determining the presence of H 2 O 2 2 This test was carried out only with seedlings grown from coated seeds. The DAB solution was prepared by homogeneously mixing 25 mg of 3,3′-diaminobenzidine and 25 ml of 3-( N -morpholino) propanesulfonic acid (MOPS) 10 mmol/L, and adjusting it to pH=3.8. The plant material (maize leaves) was cut into short pieces and immersed in DAB solution for 8 h and then, faded in several washes of methanol in a heated water bath. Once faded, the plant material was stored in 50% glycerol for preservation, and the presence of H 2 O 2 was determined according to Thordal-Christensen et al. ( 1997 1997 ). Quantification of total proteins 2 Maize leaf samples were ground in liquid nitrogen with a mortar pre-cooled to 4 °C, obtaining a fine tissue dust, which was transferred to Falcon tubes in order to clean the sample with acetone powders, adding 5 volumes of acetone per volume of tissue. The system was stirred by inversion and stored for 24 h at 4 °C. After this time, the system was transferred to Eppendorf tubes and was centrifuged at 4 000 r/min for 15 min to ensure tissue precipitation. The supernatant (which contained chlorophyll and other organic compounds) was discarded. Then, the tissue pellet was resuspended in 0.05 mol/L phosphate buffer at pH 7, and was centrifuged at 13 000 r/min for 15 min to ensure the protein extraction; the supernatant (enzyme extract) was recovered (Castro-Rivera et al., 2006 2006 ; Ortega-Ortíz et al., 2007 2007 ). Protein quantification was made by the Bradford method, adjusting it to micro-plate technique. Each reaction mixture was prepared using 200 μl of 1:5 Bradford ( 1976 1976 ) reagent and 10 μl of the enzyme extract, carrying them directly into the micro-plate wells. The blank was prepared with 200 μl of Bradford reagent 1:5 and 10 μl of phosphate buffer used for extraction. Samples and blank were incubated during for 5 min at room temperature and the corresponding absorbance values were read on a spectrometer (Spectronic 20D + Digital, Sargent-Welch) at a wavelength of 620 nm. The calibration curve was prepared from solutions of bovine serum albumin (Sigma Aldrich, Saint Louis, Missouri, USA) of known concentrations. Determination was made by triplicate. Determination of total CAT 2 The activity of CAT was assayed as described by Ortega-Ortíz et al. ( 2007 2007 ) with the following modifications: the reaction mixture was prepared using 6 ml of the enzyme extract and 1 ml of 0.022 mol/L H 2 O 2 . The phosphate buffer solution was used as a blank. Sample absorbance values were read immediately (to prevent oxidation of H 2 O 2 ) in an UV-Vis spectrophotometer (Spectronic 20D + Digital, Sargent-Welch) at a wavelength of 240 nm. The calibration curve was prepared from reagent grade H 2 O 2 solutions (Fermont, Productos Químicos de Monterrey, SA de CV, Monterrey, NL, Mexico) of known concentrations. Determination was made by triplicate. Data analyses 2 Differences between treatments were analyzed by analysis of variance (ANOVA) for experiments with one factor and a completely randomized design, with a post hoc analysis by Tukey test using the computer program JMP™ Release 5.0.1.2 (SAS Institute Inc., USA). Mean values with statistical difference of p < 0.05 were considered to be significant. Results 1 Effect of coatings on seed germination 2 A percentage of 98.57% of planted seeds reached their final phase of germination nine days after sowing. As shown in Fig. 1 1 , it was found that there was a significant difference between germination percentages by maize variety, namely, normal variety seeds had higher germination rates ( p =0.0017). It was found that H 2 O 2 was an effective seed coating in QPM variety seeds, promoting a higher germination percentage than the control ( p =0.0028). There were no significant differences in germination percentages among coatings in the normal variety. Different letter(s) above the bars indicate significant difference ( p =0.05, Tukey) among treatments within the same variety. Different letter(s) below the bars indicate significant difference ( p =0.05, Tukey) among varieties regardless of treatment Effect of coatings on seedling emergence velocity 2 Seeds from both varieties under H 2 O 2 coating emerged quicker than either those coated with CH or the control group ( p < 0.0001). The behavior of the control and the CH-coated seedlings was the same. According to varieties, normal seedlings emerged significantly quicker than the QPM ones ( p =0.6576) (Fig. 2 2 ). Different letter(s) above the bars indicate significant difference ( p =0.05, Tukey) among treatments within the same variety. Different letter(s) below the bars indicate significant difference ( p =0.05, Tukey) among varieties regardless of treatment Phenological seedling evolution under CH and H 2 O 2 treatments 2 Nineteen days after planting but before sprinkling, seedlings from coated seeds were measured. There were no significant differences in the response variable measurements irrespective of treatment for the normal variety (Table 1 1 ). In the QPM variety, H 2 O 2 coating promoted increased stem length when compared with CH coating and the control. Similarly, the length of seedlings from H 2 O 2 coating was greater than the corresponding control. QPM leaf length and stems thickness did not present significant differences by treatment. Values are expressed as mean±standard error (SE). Different letter(s) following the values indicate significant difference ( p =0.05, Tukey) among treatments within the same variety Variety Coating treatment Seedling length (cm) Leaf length (cm) Stem length (cm) Stem thickness (mm) 3 Normal Control 38.19±2.35 a 28.46±2.31 a 9.73±0.22 a 5.25±0.14 a CH 42.66±2.88 a 32.99±2.83 a 9.67±0.27 a 5.54±0.17 a H 2 O 2 40.00±2.86 a 29.83±2.81 a 10.17±0.27 a 5.76±0.17 a 6 3 QPM Control 36.97±0.57 b 27.26±0.48 a 9.71±0.19 b 5.96±0.14 a CH 37.40±0.61 ab 27.84±0.59 a 9.56±0.23 b 5.91±0.17 a H 2 O 2 39.25±0.89 a 28.75±0.60 a 10.49±0.23 a 5.81±0.18 a Phenological measurements performed on Day 30 were necessary to determine whether the seedling applications of CH and H 2 O 2 coatings and sprinkling promoted a change in physical characteristics. As shown in Table 2 2 , neither normal variety nor the QPM one present significant differences between dimensions in any of the growth responses measured. Values are expressed as mean±standard error (SE). Different letter(s) following the values indicate significant difference ( p =0.05, Tukey) among treatments within the same variety Variety Coating/sprinkling treatment Seedling length (cm) Leaf length (cm) Stem length (cm) Stem thickness (mm) 7 Normal Control 65.68±1.90 a 49.57±1.36 a 16.11±2.84 a 6.45±1.21 a CH/none 65.96±1.76 a 49.64±1.36 a 16.32±1.85 a 6.68±1.21 a None/CH 69.53±1.73 a 52.89±1.34 a 16.64±2.47 a 6.71±1.19 a CH/CH 67.16±1.73 a 51.08±1.34 a 16.07±3.43 a 6.69±1.19 a H 2 O 2 /none 67.36±1.73 a 51.08±1.34 a 16.27±2.18 a 9.71±1.19 a None/H 2 O 2 67.45±1.73 a 51.05±1.34 a 16.40±2.00 a 7.11±1.19 a H 2 O 2 /H 2 O 2 71.63±1.73 a 54.47±1.34 a 17.16±2.76 a 7.27±1.19 a 6 7 QPM Control 67.53±1.60 a 50.76±1.34 a 16.76±0.43 a 7.52±0.26 a CH/none 67.47±1.60 a 49.86±1.34 a 17.61±0.43 a 7.34±0.26 a None/CH 69.78±1.60 a 52.59±1.34 a 17.19±0.43 a 7.48±0.26 a CH/CH 68.40±1.63 a 51.56±1.37 a 16.84±0.43 a 7.57±0.26 a H 2 O 2 /none 69.51±1.69 a 52.34±1.42 a 17.17±0.45 a 7.64±0.27 a None/H 2 O 2 67.83±1.60 a 50.85±1.34 a 16.98±0.43 a 7.55±0.26 a H 2 O 2 /H 2 O 2 68.75±1.60 a 50.99±1.34 a 17.76±0.43 a 7.17±0.26 a Presence of H 2 O 2 2 In this qualitative test, the presence of H 2 O 2 was manifested by a red-brown coloration in leaves. H 2 O 2 was positive in all groups. As described in Table 3 3 , the variety with the highest presence of H 2 O 2 was QPM because the brownish color resulting from DAB coloration was evident in the cleavage site forming a wider colorful perimeter than the corresponding normal sample. The normal variety showed more coloration under H 2 O 2 treatment, followed by CH and finally the control. QPM showed more presence of H 2 O 2 in the control, followed by H 2 O 2 and then those treated with CH. Dark areas in the leaf images indicate the presence of endogenous H 2 O 2 Normal group 2 Stereoscopic observation OPM group 2 Stereoscopic observation Control Presence of H 2 O 2 in the ribs and in the middle of the sheet http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=23552861&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=23552872&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=23552866&type= Control Presence of H 2 O 2 in the entire sheet with great intensity mainly in ribs and cleavage http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=23552877&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=23552887&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=23552882&type= H 2 O 2 Presence of H 2 O 2 in the whole leaf, mainly in ribs and cleavage site, with an intense color http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=23552889&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=23552896&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=23552894&type= H 2 O 2 Presence of H 2 O 2 in secondary veins, but mainly in cleavage sites. The midrib acquired subdued colors http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=23552897&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=23552900&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=23552899&type= CH Slight presence of H 2 O 2 in the main rib; little coloration in the edges, despite the cleavage http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=23552901&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=23552903&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=23552902&type= CH Same manifestations of presence of H 2 O 2 in secondary veins, midrib and cleavage sites http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=23552904&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=23552906&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=23552905&type= Seedling protein content 2 This assessment, performed on tissue samples taken from seedlings 30 d after planting, showed that the highest protein content was present in the leaves of plants sprinkled with CH or H 2 O 2 independent of the variety used (Table 4 4 ). Values are expressed as mean±standard error. Different letter(s) following the values indicate significant difference ( p =0.05, Tukey) among treatments within the same variety Variety Coating/sprinkling treatment Protein content (μg) 7 Normal Control 21.317±3.538 c CH/none 16.104±3.538 c None/CH 51.470±3.538 a CH/CH 40.440±3.538 a H 2 O 2 /none 24.610±3.538 bc None/H 2 O 2 42.281±3.538 a H 2 O 2 /H 2 O 2 37.484±3.538 ab 3 7 QPM Control 14.761±4.595 b CH/none 34.213±4.595 ab None/CH 41.483±4.595 a CH/CH 31.469±4.595 ab H 2 O 2 /none 15.831±4.595 b None/H 2 O 2 42.535±4.595 a H 2 O 2 /H 2 O 2 39.334±4.595 a Specific activity of CAT 2 There was no statistically significant difference in terms of CAT specific activity in any treatment or in any variety, indicating that this enzyme does not enhance its activity when either CH or H 2 O 2 is applied to maize seeds as coatings (Table 5 5 ). Values are expressed as mean±standard error. Different letter(s) following the values indicate significant difference ( p =0.05, Tukey) among treatments within the same variety Variety Coating/sprinkling treatment CAT activity (μmol of destroyed H 2 O 2 /(min·mg protein)) 7 Normal Control 1720.640±447.96 a CH/none 2819.153±447.96 a None/CH 2834.457±447.96 a CH/CH 3203.963±447.96 a H 2 O 2 /none 1879.146±447.96 a None/H 2 O 2 2804.873±447.96 a H 2 O 2 /H 2 O 2 3280.392±447.96 a 3 7 QPM Control 3985.62±47741 a CH/none 3109.18±47741 a None/CH 3266.59±47741 a CH/CH 3728.48±47741 a H 2 O 2 /none 3774.02±47741 a None/H 2 O 2 128795.27±47741 a H 2 O 2 /H 2 O 2 2906.41±47741 a Discussion 1 Germination is a key step when sowing seeds, because if we do not have a good germination, productivity will decrease since the beginning. In this experiment, although maize seed technical data indicated that the germination rate was 100% for both varieties, experimentally and under greenhouse conditions, we found that the germination rate was 85% in all cases, indicating good quality seeds in general. In the case of QPM maize seeds, the application of H 2 O 2 coating promoted an increased germination when compared with the control; nevertheless, the normal variety did not show improvement on germination rates under any seed coating regime. These results match the pioneer reports of Hameed et al. ( 2004 2004 ) and Msanga and Maghembe ( 1989 1989 ), which showed that exogenous application of H 2 O 2 to seeds enhanced germination. Normal variety germination rates were higher than those for the QPM variety. CH coating did not promote higher seedling germination rates. This result contradicts some findings, because it has been reported that maize seed priming with CH enhances germination index; nevertheless, this finding was made under low temperatures (about 15 °C) using CH concentration from 0.25% (2.5 g/L) to 0.75% (7.5 g/L), and in previous studies the characteristics of CH have not been reported (Guan et al., 2009 2009 ). It also contradicts what has been found in other cultivars, because germination ability is improved in wheat when priming seeds with CH at a concentration of 2–8 mg/ml (Bhaskara Reddy et al., 1999 1999 ), peanuts (Zhou et al., 2002 2002 ) and rice seeds coated with 1.5% (15 g/L) CH solution (Ruan and Xue, 2002 2002 ). We chose to use a 2% (20 g/L) CH solution for coating seeds because at this concentration, CH not only enhances maize phenological characteristics, but also protects the crop against biotic and abiotic stresses (Lizárraga-Paulín et al., 2011a 2011a ; 2011b 2011b ). Variations on the response of CH on seed germination can be due to the biopolymer concentrations used, the specific seed and crop features, the conditions under which the cultivar is produced (specially temperature) and even the characteristics of CH, such as, the molecular weight or the deacetylation degree, which can vary drastically depending on the biopolymer extraction method or even the prime matter from which it is obtained. Seedlings from H 2 O 2 -coated seeds emerged quicker than the control and the CH-coated ones (between which there were no significant differences in emergence). It could be because H 2 O 2 stimulates seed germination and sprouts growth due to the oxidative stress caused by ROS at cellular level (Korystov and Narimanov, 1997 1997 ). A large portion of the maize plants grown in the field is destined to become forage (stubble), and is the basis of animal alimentation; that’s why researchers are looking for producing a larger amount of the cereal. According to the phenological variables evaluated in this experiment, we found that 19 d after planting, QPM seedlings from H 2 O 2 -treated seeds showed greater physical growth than the others. Principally, this was demonstrated by stem length, because stems from H 2 O 2 -treated seeds were higher than the other treatments and the other variety. Thirty days after planting, no significant differences were found between treatments, even when seeds were not only coated, but their seedlings were also sprinkled with the corresponding treatments. This indicates that CH and H 2 O 2 sprinklings do not favor phenological characteristics; only seed coating alters some physical characteristics on the plant. We expected to find a response when applying by a foliar way the CH or H 2 O 2 solutions, because it has been reported that this treatment affects the photosynthetic rate of some crops, increases the stomatal conductance and transpiration rate, and promotes plant development, specially leaves length (Khan et al., 2002 2002 ). Maybe sprinkling solution concentrations need to be increased in order to promote a higher conductance inside the plant. ROSs are involved in many important plant processes, principally in those involved in defending plants against stress. H 2 O 2 can be produced either directly or as a result of superoxide dismutation. It can diffuse into cells activating some enzymes, especially CAT and POD (Apel and Hirt, 2004 2004 ). Evaluating the presence of H 2 O 2 in plants, (qualitatively POD), it was found that when applying H 2 O 2 by seed coating, endogenous H 2 O 2 was produced in the leaves, particularly in stressed sites (cleavages sites). The application of CH by seed coating promoted the endogenous manifestation of H 2 O 2 , too. Nevertheless, it was qualitatively lesser than the coloration acquired by H 2 O 2 . The respective controls showed almost no coloration. This indicates that the POD activity increases when applying exogenous H 2 O 2 to the seeds, making the plant able to resist pathogens attacks and allowing it to initiate defense mechanisms against stresses (Bradley et al., 1992 1992 ; Camarena-Gutiérrez and de la Torre-Almaráz, 2007 2007 ), and generating major concentrations of ROSs. CH and H 2 O 2 sprinkling favored the production of proteins in maize seedlings, turning this treated cereal into a high quality food for animal consumption. Both varieties showed an increase in protein content according the treatments, because independently of being coated or not, those seedlings which were sprinkled with any of the substances, promoted more protein generation. In the case of QPM seedlings, sprinkling treatments with H 2 O 2 and CH are encouraging, because they have the potential to turn this crop into an even higher quality “QPM”, with not only excellent lysine and triptophane levels, but also a high protein content. There are still no reports of the effect of sprinklings on cereals for enhancing their protein quantity. Digestibility and well-use tests must be performed in order to give a better understanding of the benefits of this enriched cereal for animals. According to some reports, it was expected that CAT activity would increase with the application of H 2 O 2 and CH as reported by Ortega-Ortíz et al. ( 2007 2007 ), Guan et al. ( 2009 2009 ), Lei et al. ( 2011 2011 ), and Hong et al. ( 2012 2012 ). However, no significant differences were found when evaluating the specific activity of CAT in all treated plants. 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Application of bioactive coatings based on chitosan for artichoke seed protection To CONACyT for the scholarship 252089/214631. To PAPIIT program No. IT220411-3 “Estudio fitopatológico, bioquímico y molecular de la respuesta contra estreses bióticos y abióticos en plántulas de maíz”, for financial support. 10.1631/jzus.B1200270 2012-10-05 2013-01-02 2012-12-18 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2013 2013 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 02 14