Seed priming with chitosan alleviates salinity stress by improving germination and early growth parameters in common vetch (Vicia sativa)

Soil salinity is a serious environmental threat with varied effects on several aspects of growth and production of plants. Recently, however, the utilization of chitosan in agriculture has increased as it enhances the resistance of crop plants to different stresses factors such as salinity. In this experiment, the effects of chitosan seed-priming on alleviation of salinity stress was investigated in common vetch (Vicia sativa L.) using a completely randomized design with six replications. Chitosan pretreatment was applied at three levels (2, 4, and 8 g/l) and salinity of sodium chloride (NaCl) was applied at four levels (50, 100 and 200, and 300 mM). Different variables have been measured, including germination percentage, hypocotyl length, radical length, hypocotyl dry weight and radical dry weight. Salinity is found to affect the germination percentage of vetch by decreasing germination with increasing in NaCl concentration (50, 100 and 200, and 300 mM). However, the results showed an increase in germination percentage upon priming seeds with different levels of chitosan (2, 4, and 8 g/l) even with increasing the concentration NaCl salinity. Additionally, all salinity levels caused a significant reduction in vetch hypocotyl and radical dry weight and resulted in a decrease in their length when compared with the water control. However, chitosan at all levels of concentration improved all of these growth parameters (hypocotyl length, radical length, hypocotyl dry weight and radical dry weight) irrespective of salinity level indicating a successful role of chitosan in alleviating salinity stress. In general, these results may suggest that in saline soil seed pretreatment in chitosan could be utilized successfully for alleviating the effect of salinity stress on germination and on some growth parameters of common vetch.


Introduction
Soil salinity is one of the most serious environmental threat of agriculture, which increasingly exacerbates in different countries worldwide by human activities. Salinity causes a decrease in water potential of a plant and leads to the accumulation of Na + and Clions that eventually limit germination and seedling establishment and reduce the growth of plants [1,2]. To deal with salinity, plants have evolved varied physiological and structural strategies that increase the utilization of water and the elimination of toxic ions from the cell [3]. Moreover, Moreover, it is well documented that salinity stress raises free radicals and reactive oxygen species (ROS), damaging macromolecules and cell integrity [4,5]. Several studies indicated that salinity develops a series of morphological and physiological changes trought reduced germination percentage and affect the early growth parameters, such as hypocotyl and radical length and biomass by increased the accumulation of salt in leaves and to decreased water use efficiency as well as reducing seed viability and resource allocation for germination [6]. One option is currently being in an increased use to alleviate the effect of salinity stress on different plants is by application of biopolymersbased chemical compounds such as chitosan [7]. Chitosan is a linear oligosaccharide composed of a beta-(1-4)-linked N-acetyl-2-amino-2-deoxy-D-glucose (acetylated) and 2-amino-2-deoxy-D-glucose [8]. Chitosan is obtained from chitin, which is a structural component of insect and crustacean cuticle and exoskeleton, the cell wall of fungi, and some algae [9]. Chitosan is a naturally occurring, inexpensive, and environmentally safe biopolymer with diverse beneficial biological properties, such as biodegradability, biocompatibility, and non-allergenicity [10]. Chitosan has been demonstrated to enhance varied biological responses in plants, dependent on its concentration and on plant species. Additionally, chitosan has been proved to increase defence responses of the plant to different abiotic and biotic stresses [11]. For example, chitosan has shown to able to act as germination elicitor of Oryza sativa L. [12], and to positively increase seed germination and early seedling growth of wheat (Triticum aestivum L.) [13]. In addition, nanoparticles-based chitosan was able to improve the expression of pathogenesis-related proteins, thus increasing the resistance of tomato to F. andiyazi pathogen [14].
Common vetch (Vicia sativa L.) is an economically important self-pollinating annual forage legume. Common vetch has gained valuable attention in different countries throughout the world as fodder, a cover crop, hay, green manure, and also for silage production [15]. As a legume crop, it establishes successfully in mixtures with varied cereal crops and has the excellent ability for nitrogen fixation [16]. The main goal of this study was to determine the effect of seed priming with chitosan on common vetch under different salinity stress.

Materials and methods
Seven different chitosan concentrations, ranging from 0 to 6 g/L (pH 6.1), were used to prime seeds. Six replicates of 60 vetch seeds were used for each treatment in this experiment. Every treatment included wetting the seeds with 12 ml of chitosan solution in petri dishes. The seeds were exposed to 25°C for 48 hours in the dark. Once the priming was complete, the seeds were allowed to completely dry. 50, 100, 200, and 300 mM NaCl were added to the seeded vetch seeds, and the four levels of salinity were labelled "control," "50," "100," "200," and "300" mM. For this experiment, various variables have been measured, including the rate of germination, the length of the hypocotyl, the length of the radical, the weight of the hypocotyl, and the weight of the radical.
The MSTAT software was used for analysis of variance and LSD mean separation (Michigan State University, East Lansing, MI).

Results and discussion
We found that germination percentage of common vetch are declined significantly with increasing NaCl indicating that salinity of different levels may affect plant growth by reducing most likely seed viability, plant growth and establishment (Table 1). This result agrees with other studies that suggested that salinity reduces seed germination by delaying germination rate and or seed viability [11]. The lowest germination percentage was found at the NaCl concentration of 300 mM of NaCl, due to a reduction in water absorption into the seeds as they soak and swell. In addition, our results show significant increases in germination percentage, regardless of salinity concentration ( In fact, the results revealed that all the three concentrations of chitosan (2, 4 and 8 g/l) significantly enhanced germination percentage under all salinity levels of concentration (50, 100, 200, 300) suggesting that chitosan have the potential of alleviating the stressful condition of salinity [3,14]. Additional findings have proven that the highest germination percentage was reached when vetch seed was treated with 8 g/l chitosan in accordance with other studies which have shown that chitosan coating can increase germination percentage [7]. Generally, the results on germination effect of vetch may be demonstrated that the effects of chitosan are possibly attributed to its ability in enhancing germination rate, increasing seed viability and improving the energy allocation for germination. Additionally, germination enhancement by chitosan could be attributed to the fact that chitosan is an ideal film-forming property. Furthermore, as for germination, seeds priming with different concentrations of chitosan resulted in an increase in both hypocotyl and radicle length and also in their dry weight compared to control ( Table 2, 3, 4, 5).  These results agree with several studies on the effect of chitosan on different plant species. Additionally, these results confirm the findings of those of Chandrkrachang [15] who found that the application of chitosan could increase the growth parameters of cucumber, chilli, pumpkin, and cabbage. The exact mechanism of the effect of chitosan on plant growth is currently not well document. Yet, the effect of chitosan we found on common vetch could be attributed to it is able in the induction of plant defence enzymes, increasing the synthesis of secondary metabolites, such as polyphenolics, lignin, flavonoids, and phytoalexins as observed in many plant species treated with this oligopolymer substance [17]. Droughts can happen where very dry soils and reduced plant cover may decrease precipitation in a dry environment [18][19][20][21][22]. Plants and their production is limited by environmental factors such as salinity and drought in many parts of the world [23][24][25][26][27][28][29][30]. The lack of water in dryland farming is the only significant obstacle to crop production [25][26][27][28]. Many of these studies have found that low productivity is primarily associated with dryland agriculture management practices . Chitosan or chitin is the most common natural polysaccharide present in the cell walls of fungi, crabs, shrimp, insect exoskeletons,  [56][57][58]. Many scientists have stated that chitin can improve plant growth and suppress plant disease.

Conclusions
Soil salinity is a significant environmental problem that affects various aspects of plant growth and development. The use of chitosan in agriculture was, however, recently increased by increasing the resistance of crop plants to various stress factors, such as salinity. In general, these results may indicate that chitosan could successfully be used to mitigate the effect of salinity stress on germination and certain growth parameters of common vetch in saline soil seed pretreatment.