Introduction

México is the largest maize (Zea mays L.) market in the world consuming 11 % of world production. From 2014 to 2015 maize imports into Mexico were 10 million Mg, and currently ranks second as an importing country in the world. Total maize production amounted to 37.1 million Mg, around 33 % of the total production value of the agricultural sector (SIAP-SAGARPA, 2015)².

In maize cultivation weeds compete for water, soil nutrients and sunlight mainly in the early stages of development, when maize seedlings are more sensitive to competition due to their reduced root system and scarce leaf area. The early vigor of maize seedlings leads to the faster establishment of the crop and better possibilities in the face of stress caused by competition with weeds (^{Costar et al., 2013}).

The root system of maize is divided into embryonic roots (primary root and seminal roots) and postembrionary roots (nodal roots and crown roots). In the first two weeks, the main root and the seminal roots make up most of the root system.

The nodal and crown roots that form the backbone of the maize root system appear after the embryonic roots. During the first phase of the root development, the lateral roots carry out most of the water and nutrient transport to the aerial parts of the maize plant because their branches have the ability to do so. The transition between the first and second stages of root development of the maize plant (early - late stage) begins 3 to 4 weeks after germination, along with the formation of crown nodal roots (^{Hochholdinger et al., 2004}).

Soil nutrient uptake by the plant depends on: an adequate soil pore space (pores that can be penetrated by the roots) to store the water that forms the soil solution, sufficient amount of nutrient reserves, a healthy root system in the plant and the efficiency of nutrient uptake or proportion of nutrients absorbed by the plant from the total applied. It depends on factors such as the volume of the root system, the physical and chemical form of the applied fertilizer, and the reactions in the soil (^{Etchevers, 2008}).

The germination process of the seed has three phases: hydration, sprouting and growth, which depend on the content of hydratable compounds and the permeability of the seed cover to water and oxygen. The growth phase comprises the mobilization of seed reserves and seedling development (^{Koornneef et al., 2002}). The growth and development of the plant after the germination process is determined by the existing reserves in the seed and secondly by the environmental conditions.

^{Revilla et al. (1999)} mention that three weeks after emergence, endosperm reserves are consumed and the seedling depends on its ability to generate assimilates, produce leaf area and continue its growth. The success of planting is largely determined by the physiological and biochemical characteristics of the seed, its reaction to the environment and the rate with which it uses its reserves to initiate and sustain seedling growth in the early stages of development, before being an autotrophic organism (^{Soltani et al., 2006}).

Covered seeds established under different environmental conditions reach percentages and emergency rate close to 100 %, an adequate initial seedling development and high final yields (^{Tillmann and Miranda, 2006}). The seed coating is based on the encapsulation process to make pellets, and aims to improve seedling growth and development characteristics and protect them from harmful external biotic aggressions (^{Lizárraga et al., 2011}). The animal and plant polysaccharides frequently used in microencapsulation are alginate, arabic gum and chitosan (^{Lupo et al., 2012}).

Chitosan is a biodegradable hydrophilic polysaccharide derived from the deacetylation of chitin which is used in the coating of seeds to preserve them during storage. Chitosan stimulates the growth of seedlings and has a bactericidal (^{Kong et al., 2010}), fungicide and antiviral effect (^{Niquette et al., 2004}; ^{Lárez, 2006}; ^{Lárez, 2008}). Alginate is a natural hydrophilic polysaccharide obtained from brown algae and is used in the microencapsulation of seeds for being biocompatible, non-toxic and degradable (^{Reddy and Reddy, 2010}; ^{Lupo et al., 2012}). Spray drying (Parra, 2010) and ionic gelling are coating techniques of seeds practically and economically suited (^{Gouin, 2004}; ^{Lakkis, 2007}; ^{Guevara and Jiménez, 2008}). Ionic gelling consists of the formation of three-dimensional networks by non-completely stable bonds (physical hydrogels) and weaker than covalent bonds (chemical hydrogels) (^{Lupo et al., 2012}).

The objective of this study was to evaluate the effect of chitosan, sodium alginate, salicylic acid and nutrients on the emergence, radical development, and absorption of water and nutrients in two lines of maize with contrasting root development. The hypothesis was that the seed coating with one or more of the materials mentioned favors a hydric and nutritional status of the seedling, which stimulates an efficient emergence, an increase in the production of root biomass and a greater absorption of water and nutrients.

Materials and Methods

This study was conducted in greenhouses of the Colegio de Postgraduados, Montecillo, Mexico. Before evaluating corn seed coatings, we performed the following tests: 1) kinetics of the consumption of seed reserves to determine the time in which maize seedlings are autotrophic and no longer depend on seed reserves in a native maize race; 2) selection of two maize lines with contrasting root development; 3) comparison of techniques to coat corn seeds.

The time required by the plant to become independent of the seed reserves was determined with seeds of native white maize. All the seeds used in this experiment were obtained from the Seed Program of the Colegio de Postgraduados and sown in conic pots (25 cm in diameter, 25 cm height) filled with red tezontle (<5 mm in diameter) in greenhouse. The pots were watered with 0.5 L d^-1 well water. The nutrient concentration (mg L^-1) of the irrigation water was analyzed: NO₃0.003; NH₄ + 0.001; P 0.025; K 9.334; Ca 29.210; Mg 9.334; Na 39.663; Fe 0.023; Cu 0.028; Zn 0.164; Mn 0.027.

Corn seeds were weighed and sown on January 27, 2014. On days 7, 15, 22, 29 and 36 after sowing (DAS) the dry matter (DM) of the remaining seed, above-ground and root were evaluated in six seedlings. The seedlings were manually extracted from the pot to obtain all the root biomass, washed with running water and dried 72 h in an oven at 70 °C to measure the dry matter and were weighed up on an analytical balance with an approximation of 1 x 10^-4 g. Data were processed with Microsoft Office 2010 Excel and plotted with CurveExpert Professional version 2.0.4.

For selecting two maize lines with contrasting production of root biomass, eight lines or varieties (five replicates in each one) were used: CMS939083, C13x11, CS14x15, CPV-20, HS-2, HT-PRECOZ, PROMESA and VS22 . The seeds were weighed before planting and those weighing 0.21 to 0.3 g were selected to obtain homogeneous stock quantities and seeded in pots filled with fine red tezontle in greenhouse on March 25, 2014; and were irrigated with 0.3 L d^-1 of water.

Seedlings were harvested 20 DAS and dried in an oven at 70 °C for 72 h. Dry weight per plant was measured on an analytical balance with an approximation of 1 x 10^-4 g. Data were processed using Excel of Microsoft Office 2010.

The comparison of coating techniques comprised the solvent selection, concentration and coating methodology for each polymer to produce a solid, uniform and homogeneous layer on the seed surface. The evaluated materials were: corn seeds, low molecular weight chitosan (50-190 kDa) with a degree of deacetylation between 75 and 85 %, sodium alginate (Sigma Aldrich), salicylic acid, concentrated glacial acetic acid, calcium chloride and sodium hydroxide. The suspensions evaluated had concentrations of 1, 3 and 5 % from each polymer and were stirred for 60 min at medium speed on a magnetic stirrer.

From the proposed fertilization dose for field corn cultivation with a cycle of 180 d and a population density of 60 000 plants ha-1 that was 120-40-60 kg ha^-1 N-P₂O₅-K₂O, we calculated the dose 26.77.5-13.3 kg ha^-1 for maize seedlings for 15 d. The nutrient amounts for each maize seedling during this period were: 0.44 g N, 0.05 g P and 0.18 g K per plant. The fertilizers used to cover the nutritional requirements of maize plants were: ammonium nitrate, potassium sulfate, monohydrate calcium phosphate, potassium nitrate and analytical grade monobasic potassium phosphate.

The suspensions were prepared by first solubilizing the polymers and then the fertilizers to avoid precipitates. Both were ground in a mortar to facilitate their integration. In the final suspensions, we added salicylic acid because it favors plant growth for being involved in physiological processes: thermogenesis, resistance to pathogens, induction of flowering, root growth and nutrient absorption (^{Larqué and Martín, 2007}).

Chitosan is a polycation and forms complexes with molecules of opposite charge to obtain colloidal coatings (^{Rodríguez et al., 2010}). The technique for gelling chitosan was proposed by Trasviña and Louvier (2010)⁴, which was modified to determine the appropriate concentration of NaOH because the alkalinity of this reagent can damage the seed surface. Maize seeds were immersed in the chitosan suspension, then in 0.5 and 1N NaOH solutions, for 5, 60 and 180 min for evaluation. Sodium alginate gelling was performed with 0.05 M calcium chloride by using the external gelation technique described by ^{Lupo et al. (2012)}. The coated seeds were dried in the shade outdoors for 24 h.

We evaluated the effect of the proposed coatings with the emergence, biomass produced and nutrient concentration in the maize plants obtained. The maize seeds of the selected lines were weighed on an analytical balance and those weighing 0.21 to 0.30 g were selected. The interaction between the two maize lines and the six seed coat suspensions gave rise to the 12 treatments evaluated (Table 1), to which two control treatments (uncoated seeds) were attached.

Seeds of the two maize lines were coated with the final suspensions and dried in the shade outdoors for 24 h (Figure 1). For seed coating we used chitosan (30 g L^-1), sodium alginate (50 g L^-1), salicylic acid (1 x 10^-3 M) and fertilizers (N-P₂ O₅K₂O: 26.7-7.5-13.3) as promoters of root development.

Figure 1 Coated seeds. A) Seeds of variety HS-2 uncoated, B) Seeds of variety HS-2 coated with chitosan, C) Seeds of variety HS-2 coated with sodium alginate, D) Seeds of line CMS939083 uncoated, E) Seeds of line CMS939083 coated with chitosan and, F) Seeds of line CMS939083 coated with sodium alginate. 

The coated seeds were sown in pots filled with fine red tezontle (<5 mm in diameter) in a greenhouse and watered daily with 0.3 L of modified Steiner solution (without macronutrients). Water for irrigation was analyzed to adjust the macro and micronutrient concentrations of the nutrient solution. The nutrient concentration of the nutrient solution according to Steiner (1961) without macronutrients was: 2 ppm Fe, 0.7 ppm Mn, 0.5 ppm B, 0.04 ppm Mo, 0.16 ppm Zn and 0.02 ppm Cu. This solution was prepared with: 0.50 mg L^-1, H₃ BO₃, 1.98 mg L^-1 chelate-Fe 13.2 %, 0.67 mg L^-1 MnSO₄ * H₂O and 0.04 mg L^-1 Na₂ MoO₄ * 2H₂O.

Sowing was done on December 21, 2014 and two harvests were made 30 and 50 DAS, in which the entire plant was extracted and the aerial part of the root was removed. The variables measured in plants 30 DAS were above-ground biomass and root, and the root length; in plants 50 DAS we measured only the biomass of the above-ground and root biomass. The weight of the biomass was measured with an approximation of 1 ×10^-4 g. The root length of the seedlings 30 DAS was evaluated with the modified Newman method (^{Böhm, 1979}). In both crops chemical analyzes of the plant tissue were carried out using the methodology of the ^{Walinga et al. (1995)} manual , and N, P and K totals of composite samples of the aerial part and root were determined.

The experimental design was completely randomized with a 2x6 factorial arrangement (2 maize lines and 6 treatments) and two control treatments (seeds without coating) for the comparison test of means. The experimental units were pots with fine red tezontle (<5 mm in diameter). The treatments had 10 replicates, with a total of 140 experimental units. In the first harvest (30 DAS) we collected the above-ground and root parts of five plants per treatment. In the second crop the plants per treatment were variable, where treatment 3 was completely lost. Results were subjected to a normality test (Shapiro-Wilks) and a homoscedasticity test (Bartlett). The ANOVA of data were performed using the GLM procedure and the comparison of means with the LSD test (p ≤ 0.05). All results were processed with SAS 9.0 and plotted with Microsoft Office 2010 Excel.

Results and Discussion

After thedevelopment of the roots, leaves and stem, the seedlings began to photosynthesize and produce their own food or obtain it from the soil through their root system, which made the plant independent of the seed reserves. During this stage, the DM of the seed decreased due to the consumption of the reserves. With an average between 87 and 92 %, the consumption of seed reserves stabilized between 15 and 22 DAS (Figure 2). These results showed that the maize seedlings were independent of the seed reserves at approximately 20 DAS.

Figure 2 Consumption of seed reserves of native white maize. 

The evaluation of maize lines was done 20 DAS, when the plant was already independent of seed reserves. The maize varieties with higher fresh and dry root values were: HT-Precoz, PROMESA, HS-2 and CPV-20, compared to lines CMS939083, CS13x11 and CS14x15 (Table 2). The variety HS-2 and line CMS939083 were chosen to evaluate the coatings in seeds because they presented contrasting root development.

Manual spray coating and ionic gelation techniques are common in seed treatment because they are low cost and methodogically easy to use. Two percent acetic acid was used to solubilize chitosan, and distilled water for sodium alginate. The concentrations leading to an adequate adhesion to the seed without interference in the emergence were 30 g L^-1 of chitosan and 50 g L^-1 of sodium alginate. Ammonium nitrate, potassium nitrate and monobasic potassium phosphate were the fertilizers chosen for the seed coating tests, since they did not generate precipitates in the suspension. The fertilizer concentrations were: 57 g ammonium nitrate (NH₄NO₃), 15.5 g potassium nitrate (KNO₃) and 11 g monobasic potassium phosphate (KH₂PO₄) per liter of suspension. Final suspensions were prepared by adding 10 mL of salicylic acid 1x10^-3 M per 100 mL of suspension before the fertilizers addition.

The retention of any product with the techniques of seed treatment depends on the adhesion of the products applied, the compatibility between the different formulations used and the characteristics of the seed cover (^{Avelar et al., 2012}). The manual spray technique did not achieve a uniform coating on both sides of the seed. Ionic gelling was chosen for coating corn seeds since it caused no problems of adhesion or homogeneity on the seed surface. Chitosan was gelled in both 0.5 and 1N NaOH solutions with no notable physical differences in the coating. However, the seed surface was affected by 1N NaOH, whereby the 0.5N NaOH solution was chosen. The minimum time to obtain stable and homogeneous coatings of chitosan was 5 min.

Root development

In maize plants 30 DAS there was an effect (p≤0.05) of the fertilization dose on most response variables (Table 3). The 50 DAS maize plants showed significant effects (p≤0.05) only on the fresh root matter, therefore the information from ANDEVA was omitted.

Table 3 Statistical difference of factors and interactions in the biomass produced and root length of maize plants 30 days after sowing ^† . 

Fuente de variación	MFPA	MFR	MSPA	MSR	LR
Variedad (V)	ns	**	ns	**	*
Polímero (P)	ns	ns	ns	ns	ns
Dosis de fertilización (F)	**	**	**	*	**
Interacción V*P	ns	ns	ns	ns	ns
Interacción V*F	ns	ns	ns	ns	ns
Interacción P*F	ns	ns	ns	ns	ns
Interacción V*P*F	ns	**	ns	ns	*

ns: not significant; * p≤0.05; ** p≤0.01; ^†MFPA: fresh matter of the aerial part; MFR: fresh root matter; MSPA: shoot dry matter; MSR: root dry matter; LR: root length.

Brown and Scott (1984) mentioned that the soil or substrate where the seed is placed, as well as the climate and the own crop influence the physical qualities of the roots developed in the plants. The variety HS-2 evaluated 30 DAS showed an initial seed vigor greater than the line CMS939083, because it produced greater root biomass. However, the root length was higher in the line CMS939083, which can be explained by the use of seed reserves for root elongation instead of dry matter production (Figure 3).

Figure 3 Effect of race on the root development of maize plants 30 days after sowing (LSD, p≤0.05, root dry matter, DMS=0.0057 and root length, DMS=50.756). 

The variables measured in plants 30 DAS were susceptible to the fertilization dose, evidencing a marked negative effect of fertilizers and salicylic acid (Table 4). These results are similar to those by ^{Gawade and Somawanshi (1979)}, who show that the application of fertilizers in insoluble forms does not affect the emergence, so they can be used in seeds for initial stages of cultivation. However, ^{Scott et al. (1985)} and ^{Scott (1975)} state that insoluble fertilizers are ineffective in promoting initial plant growth.

Table 4 Comparison of biomass means and root length in maize plants 30 days after sowing depending on the fertilization dose. 

	Materia fresca			Materia seca			Longitud radical
Tratamiento†	P. aérea	Raíz		P. aérea	Raíz		cm
			g
Testigo	1.772 a	1.051 a		0.155 a	0.057 a		370.4 a
D1	1.571 ab	0.833 b		0.132 b	0.047 b		359.9 a
D2	1.380 b	0.755 b		0.122 b	0.049 b		269.2 b
DMS¶	0.228	0.160		0.019	0.007		63.3

^†Different letters indicate significant dif ferences (LSD, p≤0.05). Check: no fertilizer, D1=2.2 g N, 0.25 g P and 0.9 g K; D2=4.4 g N, 0.5 g P and 1.8 g K. ^¶DMS = significant minimum difference.

The effect of fertilizers will depend on the type and amount of fertilizer added, crop species, and the texture and moisture of the substrate. The highest amount of fertilizer produced the lowest values in aerial and root biomass, and root length in plants with 30 days of development. However, 50 DAS plants did not show significant differences between treatments due to a likely decrease in the effect of fertilizers.

These results can be explained by what has been reported by Melaj and Daraio (2012), who found that chitosan releases fertilizer in short times since its swelling capacity is high, the previous phase for the release of fertilizer. Therefore, fertilizers probably generated saline conditions that affected the growth and development of the plant.

Conclusions

Chitosan had lower average emergence percentages than the control in both maize genetic lines when applied to the seed alone or combined with fertilizers. Sodium alginate reduced the percentages of emergence to a lesser extent than chitosan in the variety HS-2 and the line CMS939083, although without fertilizers it obtained germination percentages similar to the control. The root development of plants was not stable in both genetic lines. The variety HS-2 race produced dryer root matter, but the line CMS939083 generated greater root length. The addition of fertilizers and salicylic acid to the seeds decreased biomass production and root length of maize plants. The concentration of nitrogen, phosphorus and potassium in maize plants was variable, so there is no conclusive effect of the coatings on the accumulation of nutrients in the plant.