Effect of Interaction Between Ag Nanoparticles and Salinity on Germination Stages of Lathyrus Sativus l

The aim of the study was to effect of interaction between Ag nanoparticles and salinity on Germination Stages of Lathyrus sativus L. Treatments included in the study were viz. To 3 levels of salinity (0 as control, 8 and 16 dS/m NaCl), 8 and 16 dS/m and four levels of silver nanoparticles (0, 5, 10 and 15 ppm) on grass pea seed were tested. An experiment was conducted to evaluate the effects of silver nanoparticles (AgNPs), on the seed germination factors, root and shoot length (RL and SL) and proline content of grass pea Survival under Salinity Levels. Results showed a significant reduction in growth and development indices due to the salinity stress. The salt stress impaired the germination factors of grass pea seedlings. The application of Ag in combination improved the germination percentage, shoot and root length, seedling fresh weight and seedling dry weight and seedling dry contents of grass pea seedlings under stressed conditions. The results suggest that Ag nanoparticles enhancement may be important for osmotic adjustment in grass pea under salinity stress and application of Ag mitigated the adverse effect of salinity and toxic effects of salinity stress on grass pea seedlings.


Introduction
High salinity is a common abiotic stress factor that causes a significant reduction in growth. Germination and seedling growth are reduced in saline soils with varying responses for species and cultivars [1]. Soil saltiness may impact the germination of seeds either by causing an osmotic potential outside to the seed averting water uptake, or the poisonous effects of Na + and Cl − ions on germinating seed [2]. Salt and osmotic stresses are responsible for both inhibition or delayed seed germination and seedling establishment [3]. The majority of our present-day crops are adversely affected by salinity stress [4]. NaCl causes extensive oxidative damage in different legumes, resulting in significant reduction of different growth parameters, seed nutritional quality, and nodulation [5,6]. To mitigate and repair damages triggered by oxidative stress, plants evolved a series of both enzymatic as well as a non-enzymatic antioxidant defense mechanism. Ascorbate and carotenoids are two important non-enzymatic defenses against salinity, whereas proline is the most debated osmoregulatory substances under stress [7].

Lathyrus sativus L. (Grass pea) is an annual pulse crop belonging
to the Fabaceae family and Vicieae tribe [8]. Grass pea has a long history in agriculture. The crop is an excellent fodder with its reliable yield and high protein content. This plant is also commonly grown for animal feed and as forage. The grass pea is endowed with many properties that combine to make it an attractive food crop in drought-stricken, rain-fed areas where soil quality is poor and extreme environmental conditions prevail [9]. Despite its tolerance to drought it is not affected by excessive rainfall and can be grown on land subject to flooding [10,11]. Compared to other legumes, it is also resistant to many insect pests [12][13][14][15]. Nanoparticles (NPs) are wide class of materials that include particulate substances, which have one dimension less than 100 nm at least [16]. The importance of these materials realized when researchers found that size can influence the physiochemical properties of a substance e.g. the optical properties [17]. NPs with different composition, size, and concentration, physical/ chemical properties have been reported to influence growth and development of various plant species with both positive and negative effects [18]. Silver nanoparticles have been implicated in agriculture for improving crops. There are many reports indicating that appropriate concentrations of AgNPs play an important role in plant growth [19,20]. The application of Nano silver during germination process may enhance germination traits, plant growth and resistance to salinity conditions in basil seedlings [21]. The use of Silver Nanoparticle on Fenugreek Seed Germination under Salinity Levels is a recent practice studied [22]. Nanomaterials have also been used for various fundamental and practical applications [23]. Although the potential of AgNPs in improving salinity resistance has been reported in several plant species [24,25], its role in the alleviation of salinity effect and related mechanisms is still unknown. Therefore, the main objective of this work was to study the effect of Silver Nanoparticles on salt tolerance in Lathyrus sativus L. These all were washed with deionized water. Seeds were sterilized in a 5% sodium hypochlorite solution for 10 minutes [26], rinsed through with deionized water several times. Their germination was conducted on water porous paper support in Petri dishes (25 seed per dish) at the controlled temperature of 25 ± 1°C. After labeling the Petri dishes, seed were established between two Whatman No.

Material and Methods
2 in Petri dishes. Silver nanoparticles in different concentration silver nanoparticles (0, 5, 10 and 15ppm) were prepared directly in deionized water and dispersed by ultrasonic vibration for one hour.
Each concentration was prepared in three replicates. Every other day supply with 0.5 ml silver nanoparticles per every test plantlet was carried out for 21 days along with control. Germination counts were recorded at 2 days' intervals for 21 days after sowing and the seedlings were allowed to grow. The germination percentages of the seeds were finally determined for each of the treatments. After 21 days of growth, the shoot and root lengths were long enough to measure using a ruler. The controlled sets for germinations were also carried out at the same time along with treated seeds ( Figure   2).

A. Germination Stages
Total germination percentage (GT) was calculated as Gt = (n/N ×100), where n = total number of germinated seeds (normal and abnormal) at the end of the experiment and N = total number of seeds used for the germination test.

B. Germination Speed Index (GSI)
Conducted concomitantly with the germination test, with a daily calculation of the number of seeds that presented protrusion of primary root with length ≥2 millimeter, continuously at the same

Seedling Vigour Index
The seedling vigor index was determined by using the formula given by Abdul baki and Anderson [28].

Fresh and Dry Mass
The fresh mass was quantified through weighing on precision scale, and the dry mass was determined through weighing on a precision scale after permanence of the material in a kiln with air forced circulation, at a temperature of 70°C, until indelible weight.
At the ending of the experiment, At the end of the experiment, radical and plumule length and fresh weight measured. Plants were placed in the oven at 70°C for 48 h and weighted with sensitive scale.

Proline Contents
Proline was determined spectrophotometrically following the ninhydrin method described, using L-proline as a standard [29].
Approximately 300 mg of dry tissue was homogenized in 10ml of 3% (w/v) aqueous sulphosalicylic acid and filtered. To 2ml of the filtrate, 2ml of acid ninhydrin was added, followed by the addition of 2ml of glacial acetic acid and boiling for 60 min. The mixture was extracted with toluene, and the free proline was quantified spectrophotometrically at 520nm from the organic phase using a spectrophotometer. Statistical analysis each treatment was conducted, and the results were presented as mean ± SD (standard deviation). The results were analyzed by one-way ANOVA with used Minitab Version 16. The present study showed clearly that salinity had a negative effect on the yield and its components of grass pea. It is well known that seed germination provides a suitable foundation for plant growth, development, and yield [30]. Increased salt concentration caused a decrease in germination percent (Table 1).

Results and Discussion
Seed germination decreased as the doses increased. The Strong reduction in germination (-47%) was observed mainly at the highest level of salt concentration as compared to control treatment.

Delayed germination causes increased irrigation cost and irregular
and weak seedling growth in the establishment of legume crops.

Relevant results were reported by Gunjaca and Sarcevic [31] and
Almansouri et al. [32]. They reported that increasing osmotic potential decreased water uptake and slow down germination   (Tables 1 & 2). The results showed that the impact of Ag NPs was significant on germination percentage in P≤0.  conditions. The results of this study showed that Ag can be involved in the metabolic or physiological activity in higher plants exposed to abiotic stresses.