agronomy Effects on Germination and Plantlet Development of Sesame ( Sesamum indicum L.) and Bean ( Phaseolus vulgaris L.) Seeds with Chitosan Coatings

: In seed technology, the use of biocompatible materials, such as chitosan, has been demon-strated to improve the germination process and establishment of seedlings. This research is focused on the effect of a chitosan coating on the germination and development of sesame and bean plantlets. The seeds were treated with different coating techniques and combinations of chitosan: chitosan solutions at 0.1, 0.5 and 1% were used in ﬁlm coating, chitosan ﬂakes with particle sizes of 1.19 mm and 0.71 mm were used as a crusted coating, and chitosan ﬂakes with a size of 1.19 mm were used for coating with acrylic resin. Images of the coatings were obtained by means of scanning electron microscopy; the effect on germination, germination speed, vigor index, length and root area of plantlets were also determined. Chitosan treatments increased germination by 26% in bean and 16% in sesame compared with the control; the germination speed index showed an increase of 61% in bean and 58% in sesame. The treatments with chitosan increased the length of the root in bean by 77%, and in sesame four times more, compared with the control treatments. Different forms of chitosan coatings improve germination and seedling establishment; however, the response to the type of coating at a given stage of seedling development will depend on the crop species.


Introduction
Chitosan (Ch) is a widely used biopolymer in the agricultural industry; its application is determined according to its properties, such as the degree of deacetylation and molecular weight. Chitosan has been used in agronomy for different purposes, including roles as an antimicrobial [1], antiviral [2], elicitor [3], and adjuvant [4] agent; in the gradual release of active ingredients [5]; as a soil amendment [6], abiotic stress reliever [7], promoter of growth [8], and absorbent [9]; and in agricultural water treatment [10] and postharvest coatings [11]. Advances in seed technology associated with the use of Ch have highlighted applications for agricultural uses, which must consider the origin, nature, application form and physical characteristics of Ch, as these could influence the functional responses and compatibilities of the seed's physiological performance [12][13][14][15][16]. The application of Ch as a coating might influence the way in which the seed comes into contact with Ch, for example, Ch applied to wheat seeds using the priming technique increased their germination by 18%, germination rate by 53% and seedling vigor by 27% [17]. In broad bean seeds, it was reported that the priming treatment with nanoparticle chitosan showed harmful effects on germination and seedling growth [18]. In rice, an increase was reported in both germination rate and growth parameters [19]. In pearl millet seed primed with chitosan, an increased germination rate and vigor were reported (13% and 18%, respectively) [20]. Additionally, in chitosan film coatings on bean seeds, a 10% increase in germination and a 7% germination speed increase were reported [21]. In artichoke seed, the germination percentage increased by 11% when coated with chitosan film [22]. In soybeans, an increase in germination of 13% was shown [23].
In recent years, seed coating technology has incorporated the application of ingredients that are biocompatible with the environment to ensure a positive impact on the germination and establishment of crops [24,25]. The use of Ch in seed technology is part of the strategies that take advantage of its properties to formulate coatings that are part of the integral management of crops; its use is based on the improvement in the physiological and functional responses at the initial stages of crop development. Therefore, the objective of this research was to identify the structure and functionality of seed coatings with chitosan and to determine the effect of coatings on the germination and development stage of sesame seedlings (Sesamum indicum L.) and beans (Phaseolus vulgaris L.).

Materials and Methods
The sesame seed cultivar 'Zirándaro' was obtained from the 2016 harvest in Michoacán, Mexico, and the common bean cultivar 'Pinto Saltillo' was obtained from the 2016 harvest in San Luis Potosí, Mexico; germination tests were carried out in 2019. Chitosan was obtained from commercial chitin (Sigma-Aldrich, St. Louis, MO, USA), with the methodology of Ruiz-de-la-Cruz et al. [15] with some modifications. A first deacetylation was carried out with 1.75 M NaOH; subsequently, it was washed and left to dry at 50 • C for 24 h, before a second deacetylation with 17.5 M NaOH. The obtained product (flake) was washed and allowed to dry at 50 • C for 24 h. Once the chitosan was obtained, the degree of deacetylation (DD) was determined using the methodology of Yuan et al. [26]. To obtain different flake sizes, the chitosan was ground in a blender; subsequently, the different particle sizes were obtained using sieves with a mesh size of 0.71 and 1.19 mm.

(b) Seed coatings
In the case of film coating (CS0.1, CS0.5, CS1, and A), seeds were immersed in their respective treatment under agitation for 30-45 s; once coated, seeds were drained to remove the excess coating solution and then dried at room temperature (25 • C) for 48 h. The control treatment (W) seeds were immersed in distilled water under agitation for 30-45 s.
For the crust coating treatments (CSD and CDA), seeds were immersed in 1% chitosan in distilled water for CSD, or in acrylic adherent for CDA under stirring for 30-45 s; then, seeds were allowed to drain for 10 min to eliminate excess liquid. Later, seeds were mixed with the 0.71 mm chitosan flakes for 5 min until a uniform crust was obtained, and were left to dry at room temperature (25 • C) for 48 h.
Finally, for treatments CD071 and CD119, seeds were mixed in the respective chitosan flake sizes for a period of 5 min.

Scanning Electron Micrograph
To obtain the images with a scanning electron microscope (SEM), the coated seeds were subjected to dehydration in a critical point desiccator (Samdri-795, Tousimis, Rockville, MD, USA), then seeds were covered with a layer of gold with an Ionizer Fine Coat (Jeol JFC-1100) to later obtain the images with the SEM (JEOL-JSM-6360LV) at the Institute of Marine Sciences and Limnology, Universidad Nacional Autonoma de Mexico (UNAM).

Seed Germination, Germination Speed and Vigor Index
To evaluate the germination of treated seeds, a completely randomized design was established. The technique, on paper, was used according to ISTA [27]. Germination was managed under controlled conditions, with a temperature of 25 ± 4 • C, in the growth chamber (Model 818, Thermo Scientific ® Lab-Liner, Marietta, OH, USA). Germination was evaluated considering root protrusion, which was recorded every 24 h until 7 days after sowing. Germination speed was evaluated as proposed by Maguire [28]. Vigor index was obtained by multiplying the accumulated germination percentage by the total height of the seedling (14 days after sowing) divided by 100 [29].

Plantlet Evaluation
To evaluate the effect of treatments on the development of plantlet root and stem, germinated seeds were transplanted into pots with peat moss and vermiculite in a 2:1 v/v ratio (Green Forest México©, Premier ® , Puebla, Mexico) to allow for development. Seven days after transplantation, the length of the main root was measured, starting from the neck to the root apex. Stem height was obtained by measuring from the root neck to the stem apex, and the total length of plantlets was obtained by measuring from the stem apex to the root apex. To determine the root area, digital images were taken at a resolution of 1280 × 720 pixels; the images were processed and analyzed to obtain the area using Java (ImageJ) software (ver. 1.50).
The obtained data were subjected to an analysis of variance and comparison of means using the Tukey test (p ≤ 0.05) using the statistical package Statistical Analysis System version 9 (SAS ® Institute, Inc., Cary, NC, USA).

Micrographs of Chitosan-Coated Seeds
The chitosan used had a deacetylation degree of 89%. Regarding the images of sesame seeds obtained by SEM (Figure 1), treatments CS0.1, CS0.5, CS1, CSD and CDA showed differences in testa texture with respect to the control (W). A thin film was observed, that filled the rough texture that characterizes sesame seeds. On the other hand, CSD and CDA treatments showed chitosan flakes that were adhered in a dispersed way; this was attributed to the size of chitosan flakes. On the contrary, in CD119 and CD071 treatments, adhesion of chitosan to seed surface was scarce.
The SEM images of bean seeds ( Figure 2) showed that the coatings formed a continuous film for treatments CS0.1, CS0.5, CS1 and A, and in these treatments the porosities in the margin of the hilum were fully filled by the coating. On the other hand, the CSD and CDA treatments showed that chitosan flakes were adhered in a dispersed way. Finally, for the CD119 and CD071 treatments, the images showed no adhesion of chitosan flakes to seed surface. The SEM images of coatings showed the ability of chitosan in solution to form firm, homogeneous and continuous films. The SEM images of bean seeds ( Figure 2) showed that the coatings formed a continuous film for treatments CS0.1, CS0.5, CS1 and A, and in these treatments the porosities in the margin of the hilum were fully filled by the coating. On the other hand, the CSD and CDA treatments showed that chitosan flakes were adhered in a dispersed way. Finally, for the CD119 and CD071 treatments, the images showed no adhesion of chitosan flakes to seed surface. The SEM images of coatings showed the ability of chitosan in solution to form firm, homogeneous and continuous films.

Germination Capacity and Germination Speed
In the germination test for bean seeds, all the treatments were superior to the control; CS0.1, CS0.5 and CD119 treatments showed a 26% increase in germination; CS1 and CDA treatments were superior by 18 and 16%, respectively, and the CD071, CSD, CDA and A treatments were higher by 12% compared with the control ( Figure 3A). On the other hand, for sesame seeds, the best treatments compared with the control were CS0.1, CS0.5 and CD119 with an increase of up to 16% in the germination percentage; CS1, CD071, CSD, CDA and A treatments showed a similar germination to the control ( Figure 3B).

Germination Capacity and Germination Speed
In the germination test for bean seeds, all the treatments were superior to the control; CS0.1, CS0.5 and CD119 treatments showed a 26% increase in germination; CS1 and CDA treatments were superior by 18 and 16%, respectively, and the CD071, CSD, CDA and A treatments were higher by 12% compared with the control ( Figure 3A). On the other hand, for sesame seeds, the best treatments compared with the control were CS0.1, CS0.5 and CD119 with an increase of up to 16% in the germination percentage; CS1, CD071, CSD, CDA and A treatments showed a similar germination to the control ( Figure 3B). In bean seeds, the CS1 and CS0.5 treatments were superior by 57% and 62%, respectively, in terms of germination speed compared with the control; CDA and CS0.1 treatments presented increases of 40 and 33%, respectively; while the CD119 and CSD treatments showed an increase of 28 and 19%, respectively, compared with the control treatment; and treatments CD071 and A did not show statistical differences when compared with the control (Figure 3A). For sesame seeds, the best results for germination speed were for the CS0.1 treatment, with an increase of 58% compared with the control. The CD119, CS0.1 and CDA treatments increased by 43, 37 and 25%, respectively, compared with the control. In contrast, the CS1, CD071, CSD and A treatments did not show any differences compared with the control treatment ( Figure 3B). In bean seeds, the CS1 and CS0.5 treatments were superior by 57% and 62%, respectively, in terms of germination speed compared with the control; CDA and CS0.1 treatments presented increases of 40 and 33%, respectively; while the CD119 and CSD treatments showed an increase of 28 and 19%, respectively, compared with the control treatment; and treatments CD071 and A did not show statistical differences when compared with the control ( Figure 3A). For sesame seeds, the best results for germination speed were for the CS0.1 treatment, with an increase of 58% compared with the control. The CD119, CS0.1 and CDA treatments increased by 43, 37 and 25%, respectively, compared with the control. In contrast, the CS1, CD071, CSD and A treatments did not show any differences compared with the control treatment ( Figure 3B).

Root and Stem Growth and Vigor Index
Regarding the root length of bean plantlets, treatments CS0.1, CS0.5, CS1, CDA, CD119 and CD071 showed a growth-promoting effect, registering increases from 50% to 77% with respect to the control, while treatment A presented a lower root length, similar to the control treatment. Regarding root area in bean plantlets, CS0.5 treatment caused an increase of 2.6 times the area compared with the control; CDA, CSD, CD119 and CS071 treatments increased root area by between 68 and 90%; CS1 and A treatments did not show significant differences compared with the control ( Figure 4A). In sesame, the CDA treatment increased root length of plantlets four times more than control treatment (W), CS0.5 and CSD treatments showed an increase of 2.4 times the root length of plantlets, while the CS0.1 treatment increased by 2.1 times. Regarding root area, all treatments, except treatment A, were superior to the control, but the CS0.5 and CDA treatments increased root area by 24 times more than the control ( Figure 4B). 77% with respect to the control, while treatment A presented a lower root length, similar to the control treatment. Regarding root area in bean plantlets, CS0.5 treatment caused an increase of 2.6 times the area compared with the control; CDA, CSD, CD119 and CS071 treatments increased root area by between 68 and 90%; CS1 and A treatments did not show significant differences compared with the control ( Figure 4A). In sesame, the CDA treatment increased root length of plantlets four times more than control treatment (W), CS0.5 and CSD treatments showed an increase of 2.4 times the root length of plantlets, while the CS0.1 treatment increased by 2.1 times. Regarding root area, all treatments, except treatment A, were superior to the control, but the CS0.5 and CDA treatments increased root area by 24 times more than the control ( Figure 4B). Regarding the stem length of bean plantlets, treatments formulated with chitosan stimulated stem growth by up to 30% compared with the control; only treatments W and Regarding the stem length of bean plantlets, treatments formulated with chitosan stimulated stem growth by up to 30% compared with the control; only treatments W and A were statistically lower than coatings containing chitosan (Table 1). On the other hand, for sesame, the CDA treatment increased the length of the stem by up to double the size of the control, the CSD, CS0.1 and CD119 treatments achieved an increase from 81 to 77%, and, finally, the CS0.5 and CS1 treatments promoted an increase of 70 and 50%, respectively. Treatments CD071 and A did not show a statistical difference compared with the control (Table 1). For all treatments, the stem width showed no significant differences in both bean and sesame. In relation to the vigor index, for beans, treatments CS0.5, CS0.1, CS1, CDA and CD119 surpassed the control with an increase from 58% to 71%; in the CSD and CD071 treatments, the vigor index increased by up to 34% compared with the control. In the case of sesame, all treatments with chitosan were statistically superior to the control, CDA was the best treatment, promoting an increase in the vigor index of three times more compared with the control, followed by the treatments CS0.5, CS0.1, CSD and CD119, which were from 1.9 to 2.25 times higher than the control. Finally, the CS1 and CD071 treatments achieved an increase of 55 and 34%, respectively, compared with the control. The data from this study allowed us to determine that there was a greater sensitivity to stimulation by chitosan for the cultivation of sesame.

Scanning Electron Microscopy (SEM) of Seeds Coated with Chitosan
The importance of the uniformity of coatings is essential to ensure seed protection, the permanence of active ingredients and their mechanical properties. In this sense, studies such as SEM provide data on the characteristics of continuity and structure of coatings; as has been reported for the characterization of chitosan coatings in artichoke seeds where a smooth, homogeneous cover is reported, in addition to a shine given by the chitosan film [22]. On the other hand, Zeng et al. [23], report that chitosan coatings on seeds using the film technique generate a uniform protective layer, which covers the characteristic reliefs of the seed coat; these results agreed with the records in this study. Seed chitosan coating conferred homogeneity, firmness, and uniformity, as well as being colorless and compatible [22,30]; these types of coatings have shown their usefulness in postharvest storage by extending the life of the seed [21]. In turn, the film allows for water absorption, retention and the gradual release of compounds of agricultural interest mixed with chitosan, due to the physicochemical characteristics of chitosan [31].
The surfaces of the seeds with film coatings (CS0.1, CS0.5, CS1) were observed to be smooth, colorless, without fissures, homogeneous and firm. The coverage capacity of the film is related to the surface texture of the seed coat, since the greater the roughness and porosity, the greater the adhesion of the chitosan [32]. In this way, the coatings applied using the crusting technique (without adherents) in this research proved to stimulate germination and germination speed.

Germination and Germination Speed
It has been reported that the application of 0.25% chitosan in solution as a seed coating, in addition to osmoconditioning, have managed to increase germination by more than 15% [17]. There are several factors that affect or diversify the success of chitosan coating technologies, including origin, degree of deacetylation, concentration, crop stress level, agricultural species, and even varietal responses at the genetic level [7,14,[33][34][35][36][37], and that represent key points for these technologies. In this study, different responses were observed between agricultural species, since some treatments promoted germination or germination speed, while others did not show differences with respect to the control. In contrast, negative effects on germination speed have been reported in synthetic polymers, such as by de Barros et al. [38], where the super absorbent polymer treatment was exposed to sorghum seeds, resulting in the reduction of germination speed, while in another study [39], there was no effect on the speed of germination in onion seeds coated with chitosan using the priming technique. On the contrary, in this investigation, there was a stimulating response for germination speed, which agrees with that stated by Tovar et al. [40], who reported a positive effect on germination speed in treatments with moringa extract and chitosan mixed with iron oxide nanoparticles in corn seeds using the film technique. Similarly, Samarah et al. [41] found positive effects on germination speed in treatments with chitosan to bell pepper seeds using the film technique. It was thought that chitosan is processed at the cellular level in oligomers that have the chance to activate defense genes and other metabolic pathways. Considering this and the chance of increasing water availability due to the chitosan seed coating, there is a high probability of an increase in germination rate [42].

Root and Stem Development of Plantlets and Vigor Index
At present, advances in coating technology focus on the study of active ingredients that stimulate growth and high vigor in the seedling stage. Acharya et al. [43] used a film coating with silver nanoparticles on watermelon seeds (Citrullus lanatus L.), improving seedling growth by up to 35% in terms of stem length. Accinelli et al. [44] observed a stimulation of stem and root growth associated with the activation and survival of B. subtilis, in a study with bioplastics consisting of modified starch and chitin mixed with Bacillus subtilis, used as a coating for corn seeds. On the other hand, techniques such as pelleting with cellulose, diatomaceous earth and soy protein on broccoli seeds (Brassica oleracea L.) showed a 36% increase in stem and root growth. In addition, the vigor index exceeded the control by 30% [45]. Coatings with MWCNT have been shown to increase root growth and density in wheat [46]. The results of this study demonstrate chitosan's ability to stimulate the germination and germination speed of seeds, in addition to stimulating seedling development, with a higher potential than that reported for other polymers.
The results of this study also correlate with other findings, such as the stimulating effect on stem or root growth in the plantlet development stage, as reported in other studies [16,47,48]. This study shows the advantages and diversity of the responses to the application of chitosan as a seed coating, with different coating techniques being used to improve the physiological quality of seed in the germination process and plantlet development stage. There are also different degrees of sensitivity for each crop, acting as an active ingredient and additive.

Conclusions
In the present study, chitosan was highlighted as a functional agent for seed coating. In film treatments with low chitosan concentrations, germination and germination speed were improved for both species, while the pelletized coating technique for sesame showed better results in terms of plantlet development; thus, there is a different degree of sensitivity to the presence and concentration of chitosan for each crop species. Therefore, recommendations for the application of chitosan coatings, in addition to coating application technique, should be based on specific need; that is, the species to be worked on should be defined, in addition to the stage of crop development for which improvement is required.