Genotypic response of barley to exogenous application of nanoparticles under water stress condition

Beneicial nanoparticles (SiO2 and TiO2) can have various profound effects on the crop physiological, biochemical and morphological characteristics. here, we evaluated the mitigation of drought stress in barley genotypes by foliar application of SiO2 and TiO2 nanoparticles under iled condition in North West of Iran. Nanoparticles were foliar applied in late vegetative phase and during reproductive stages. Drought was imposed at by irrigation withdrawals during the dry months in the end of the growing season. We measured parameters related morphological growth, yield, and yield component. The genetic diversity between the genotypes was quite evident and the highest seed yield and yield component were recorded for G1, G2, G4, G11, G12 and G13. Foliar application of nanoparticles considerably affected the plant height, thousand seed weight, biological and seed yield. The best performance was observed for plant treated with SiO2 nanoparticles. Spike length of G2, G6, G13 and G20 considerably responded to nano silicone foliar application. however, the best results for G8, G11 and G20 were obtained by foliar application TiO2 nanoparticles while this treatment decreased the seed yield components in G1, G5, G9, G10, G15 and G20. This could be due to genetic variation between the evaluated genotypes and high sensitivity of some genotypes to the applied concentration. The results of current study showed that application of SiO2 nanoparticles under water stress condition could have more beneicial effects on yield component of barley genotypes.


INTRODUCTION
Barley (Hordeum vulgar L.) is one of the most important crops in irrigated and rainfed areas and it is one of the four important cereals of the world.Despite the high importance of wheat, barley in semi-arid regions is strategic crop and this is partly due to higher drought resistance in the barley plant (27).
According to FAO, cultivated area of barley in Iran during 2016 growing season was about 1.61 million hectares and harvested production was estimated at 2.90 million tons, which is equivalent to 65 percent of domestic demand (3).Barley is a dual-purpose plant and it is very important for livestock industry.These statistics indicate that there are still steps to self-suficiency in the production of barley.The importance of this issue becomes even greater when it has been recognized that its forage shortages are very evident in North West semi-arid areas of Iran.The major part of the barley cultivated area (60%) is rainfed (1).Water is a major restraining aspect for the world's economy because of its reducing quality and quantity and changes in distribution (22).
Food security for the hundreds of millions of rural poor necessitates improved crop productivity through breeding for enhanced drought tolerance (21).Furthermore, the importance of agronomic management in improving production in extreme conditions should not be ignored, so that drought stress is limiting factor in semi-arid region.Droughts stress can be categorized into meteorological drought, hydrological drought and agricultural drought.Time-series of annual rainfall, number of rainy days per year and monthly rainfall in semi-arid region are very variable (20).The occurrence of all three stresses is possible in terms of dry periods.however, in most of the years, due to reduced rainfall during reproductive growth, rising temperatures, and increasing amount of evapotranspiration of the barley plants experience terminal drought and heat stress.Terminal drought of varied intensities is, therefore, a primary constraint to barley productivity.
Nanoparticles are proposed to be the materials for the new millennium.Nanoagriculture involves the employment of nanoparticles in agriculture.These particles will impart some beneicial effects to crops.The emergence of nanotechnology and the development of new nanodevices and nanomaterials open up potential novel applications in agriculture and biotechnology (6,8,10,23).however, with the advancement of science the ability to construct and manipulate materials at the nanoscale has increased dramatically in the last decade.
Although it seems that there is a widespread potential of nanoparticles applications in agricultural sector, nanoparticles are still unexplored, especially their mechanism and role on plant growth and development (24).Despite the prominence of Si as a mineral constituent of plants, Si is not considered as "essential" nutrient, for any terrestrial higher plants (2,18).The beneicial elements are not deemed essential for all crops but may be vital for particular plant taxa.Silicon (Si) has not been proven to be an essential element for higher plants, but its beneicial effects on growth have been reported in a wide variety of crops, including rice, wheat, barley (11,16).In plants, silicon is deposited in cell walls in the form of amorphous silica (SiO 2 -nh 2 O) and enhances cell wall rigidity and strength, interacting with pectins and polyphenols.Marschner (18) demonstrated that Si4+ is deposited in epidermal cells of leaves, hence improving leaf exposure to light by keeping leaves more erect; in roots, it increases cell elongation thus enhancing cell wall elasticity.As the beneicial effect of silicon has been proved as shown above, the application of nanosilicon can be more effective than the large applied particles, which means a more eficient input use (26).Si as a physical-mechanical barrier can prevent penetration of pesticides or pathogens into the plant cell.Silicon can also deposit on the walls of epidermis and vascular tissues of the stem and leaf surface in most plants, especially monocots, and also controls physiological properties of plants (9).Considering the increasing importance of nanoscience in agriculture and the remarkable effects of beneicial nanoparticles such as nano-SiO 2 , evaluating their effect on plants is very important.

U M C S
In this regard, there is not much information in the literature indicating whether nano-SiO 2 application may have similar beneicial effects on barley genotypes under drought stress.This information will help scientists choose genotypes that are more tolerant to hydric deiciency for breeding.
The present study was carried out to evaluate exogenous application of nano-SiO 2 and nano-TiO 2 on the yield and yield components of barley genotypes under water stress condition in northwest of Iran.

MATERIALS AND METhODS
Field experiments were carried out at the Research Farm of the Moghan College of Agriculture and Natural Resources, Parsabad, Ardabil, Iran, during the growing season of 2016.The ield was located at 46°46' east longitude and 39°36' north latitude, at an altitude of 32 meters above the sea level.Based on Koppen's classiication, this region has a semi-arid temperate climate.Moghan has warm and humid summers and temperate winters with dry winds and a short freezing period.Top 0-30 cm soil samples were randomly collected from ield and analyzed for physicochemical properties.The soil type was clay loam, ph 7.22 and EC 2.35 dS.m -1 , organic matter 0.85%, potassium 306.4 mg kg -1 , phosphorous 15.8 mg kg -1 .Moghan is located in the plain of the Aras River and its level areas with deep, well-drained soils, and cropping in this region is most reliable.The mean annual temperature was 15 °C while the mean maximum and minimum temperatures were 31.4 and 1.4 °C, respectively.The mean temperature during the growth season was 21°C and average annual rainfall was about 335 mm.
The germplasm of 20 genotypes was studied in the current experiment.Genotypes were obtained from Seed and Plant Improvement Institute (SPII), Iran and they were differing in growth and morphological characters.The experiment was laid out as factorial (3×20) based on Randomized Block Design with three replications.The irst factor was foliar treatment included control (Check; no treatment), nano-Ti (20 ppm) and nano-Si (20 ppm).The second factor was twenty genotypes of barley.Each genotype was sown in a double row with spaces apart at 30 v 10 cm in a plot of 5×3 m 2 size.The middle two rows were used for data collection.The ield was mouldboard-ploughed and twice disked before seed sowing.After primary and secondary tillage, planting was done by hand drilling using a seed rate of 80 kg/ha for each variety at the second week of November.Nitrogen and phosphorous fertilizers were applied at the rate of 100 kg/ha urea and100 kg/ ha triple superphosphate at planting.All other management practices were uniformly applied to all plots at planting.There was no incidence of pest or disease on plants during the experiment.Weeds were controlled by systemic selective chlorophenoxy herbicides including 2, 4-D and MCPA.Plants were grown under rainfed condition that received natural rainfall.
The nano-SiO 2 and TiO 2 were procured from Nano-Pishgaman (Iran).The shape of SiO 2 and TiO 2 nanoparticles was spherical.Their average size and purity were 100 nm and 99.5%, respectively.Characterization of SiO 2 and TiO 2 nanoparticles by Scanning electron microscopy (TSCAN, Czech Republic) image (SEM) are shown in Figure 1.NPs of TiO 2 and SiO 2 solutions were prepared at concentrations of 20 ppm with iltered, double-distilled water.Working solutions were made by vigorous vortexing (using ultrasonic) before the applications.Foliar application was carried out during leaf development (BBCh-scale=17; seven leaves unfolded), tillering (BBCh-scale=21; beginning of tillering: irst tiller detectable), stem elongation (BBCh-scale=34; node 4 at least 2 cm above node 3) and heading (BBCh-scale=55; middle of heading: half of inlorescence emerged).
Genotypic and phenotypic correlation coeficients of yield with the contributing characters and among themselves were calculated by using the genotypic and phenotypic variances and covariances as described by Singh et al. (25).19), yield components and morphological traits, such as plant height, number of fertile tillers, straw mass, spike length, number of grains/spike, 1000-seed weight and seed yield, were evaluated.The data were analyzed using the SAS statistical software.Statistical signiicance was accepted when the probability of the result assuming the null hypothesis, p is less than 0.05 (level of probability).Correlation analysis and principal component analysis (PCA), based on the rank correlation matrix and biplot analysis were performed by SPSS ver.16, STATISTICA ver.8 and Minitab ver.16.

RESULTS AND DISCUSSION
The results of variance analysis revealed that there are signiicant effects of nanoparticles spray and genotypes on plant height (Ph).Mean comparison of Ph between foliar treatments showed that nanoparticles spray increased this trait by 14% over control (Table 1).The highest Ph was recorded for G11, G15, G17, G18, G19 and G7 (Table 2).The interaction effect of nanoparticles spray × genotypes was signiicant for spike length (P < 0.01).Although the application of nanoparticles, especially SiO 2 , caused signiicant increase in spike length in most genotypes, spray of nano-TiO 2 signiicantly reduced spike length in G3, G4, G5, G18 and G12 (Fig. 2).Number of tillers and number of fertile tillers signiicantly was different between the evaluated genotypes and the highest tiller number was recorded for G11, G2, and G17.Furthermore, the comparison between total tiller and fertile tiller number revealed that G6 and G9 had the highest survival rate for produced tillers.The main effect of the nanoparticles spray and genotypes as well  Ph: plant height (cm), SL: spike length (cm), TN: tiller number, FTN: fertile tiller number, TSW: 1000-seed weight (g), SNP: seed number per plant, SWP: seed weight per plant, STy: straw yield (kg ha -1 ), By: biological yield (kg ha -1 ), Sy: seed yield (kg ha -1 ).G: genotype, T: foliar treatment.NS = Not signiicant, *= Signiicant at 5% level of probability, ** = Signiicant at 1% level of probability.Means followed by the same lower case letters in a column and capital letters on the lines do not differ signiicantly to the level of 5% probability.
ANOVA showed that seed number per plant (SNP) was not affected by foliar treatments.however, there were signiicant differences between SNP of genotypes and the highest SNP was recorded for G6, G7, G8, G14, G16, G17 and G19.Evaluation of seed yields revealed that the main effect of the nanoparticles spray and genotypes as well as their interaction effects were on this trait (Table 1).Mean comparison of seed yield between the foliar treatment showed that the best performance was obtained by nano-SiO 2 .however, the response of genotypes seed yield was signiicantly different against the foliar treatments.The highest seed yield was recorded for G8 under nano-TiO 2 foliar application.however, other genotypes like G1, G4, G2, and G12 produced the highest seed yield under nano-SiO 2 foliar application (Fig. 4).
Cluster analysis divided the genotypes into four clusters (Fig. 5).The irst group included G1, G2, G4, G6, G7, G8, G9, and G10, where the stimulating effect of nanoparticulate treatments, especially nano-SiO 2 , was somewhat evident.The second cluster included G5, where the foliar spray of nano-TiO 2 signiicantly GENOTyPIC RESPONSE OF BARLEy TO EXOGENOUS APPLICATION OF NANOPARTICLES... Ph: plant height (cm), SL: spike length (cm), TN: tiller number, FTN: fertile tiller number, TSW: 1000-seed weight (g), SNP: seed number per plant, SWP: seed weight per plant, STy: straw yield (kg ha -1 ), By: biological yield (kg ha -1 ), Sy: seed yield (kg ha -1 ).Means followed by the same lower case letters in a column and capital letters on the lines do not differ signiicantly to the level of 5% probability.decreased most of the traits.The third group includes G3, G12 and G15, where the effect of nano-SiO 2 on the growth characteristics and seed yield was very poor and in some cases foliar treatments were ineffective.The fourth group includes G11, G13, G14, G16, G17, G18, and G19, where the effect of silicon was greatly positive and the titanium effect was negligible (Fig. 5).

Pobrane z czasopisma
A number of central physiological processes contribute to the formation of grain in crops.Major ones are photosynthesis and the translocation of photosynthate to the grain, cell division and enlargement, and the accumulation and transport of nutrient elements for storage in the grain and for the general functioning of cell metabolism.These processes must occur during the appropriate stages of development, and consequently the timing of each contribution is important.Superimposed on this set of circumstances is the suitability of the environment for supplying light, water, and nutrients for the completion of each stage of growth (28).Some of the beneicial effects of nanoparticles, especially silicon, on plant regeneration can be attributed to the increase and excitation of defensive systems under abiotic stress conditions.Current experiment has been carried out under rainfed condition in semi-arid region, where plants are faced with terminal drought stress.Water content is an important property of soils, inluencing soil solution chemistry and nutrient uptake by plants.Morphology and other speciic properties of the root, nutrient concentration in the soil solution, the mobility of nutrients in the soil, and supply from solid phases, affect nutrient uptake.Therefore, when the terminal dry period and water stress emerged, vitality of plants was weakened, their growth reduced, and mortality increased.Drought stress, as a multidimensional abiotic stress, strongly effects growth, development and yield of plants.Under drought condition, the plants initiate two strategies for survival -avoidance or tolerance; the strategies include morphological and/or physiological adjustments.Finding resistance genotypes to biotic and abiotic stress is very important for plant research.The role of silica in plants under stress conditions is more pronounced, but there is no report for using of silica as nanoparticles in plant drought stress.Silicon is also known as an anti-stress agent and can reduce cuticle transpiration (17) or increase water eficiency (4).
Likewise, foliar application of nanoparticles can also increase the plant growth and seed yield through improving the function of photosynthetic apparatus (source activities) and photo-assimilate translocation.These results are consistent with those of other studies and suggest that the application of Si may represent an approach to improve the growth of this crop and increase its production in arid or semi-arid areas where water is at a premium; this technique, however, would not fully substitute for an adequate water supply (7,12).
Improvement of plant growth by foliar application of nano-SiO 2 and nano-TiO 2 can be due to modiication of source and sink relationships.Sources are plant organs such as leaves that produce sugars.Sinks are plant organs such as roots, bulbs (swollen leaves) and illing seeds that consume or store sugars.It has been revealed that SiO 2 and TiO 2 play a role in enhancement of source size by increasing the photosynthesis rate, increasing the strength of leaves, chlorophyll concentration per leaf area and leaf area duration (5,14).On the other hand, the activity and size of the reservoir are largely inluenced by the proportion of phytohormones.In this case it has been showed that Si reduced endogenous concentration of jamonic acid (JA) and salicylic acid (SA), while abscisic acid (ABA) irst increased and then decreased two weeks after exogenous application (13).however, it was recently demonstrated that Si increases cytokinin biosynthesis in Sorghum and Arabidopsis and that such an increase may strongly contribute  (15).Phytohormones communications are responsible for a complex biochemical and physiological network and a deep understanding of nanopartices inluence on hormonal properties can facilitate the breeding process.
This also accords with our earlier observations, which showed that foliar application nano-SiO 2 improved canopy spread, ground cover, number of capitula in main branch and accelerated canopy closure; however, it did not signiicantly affect the achene yield under different fertilizer system (6,8).
In addition, the principle component analysis (PCA) described a suitable amount of the total variation; the correlation coeficient between any two traits is approximated by the cosine of the angle between their vectors.In Figure 6, the most prominent relations are: a strong positive association among seed, tiller number, fertile tiller number and 1000-seed weight as indicated by the small obtuse angles between their vectors (r =cos 0=+1).There was no correlation between plant height, spike length and seed as indicated by the near perpendicular vectors (r =cos 90=0).This state also was conirmed by correlation curve for phenotypic and genotypic characters (Fig. 7).Crop plants in semi-arid region are faced with different challenge such as drought and heat stress.however, responding to these and other challenging issues in these regions will require new, impactful technologies.In current study we evaluated that possibility to exogenous use of nano-scaled beneicial elements on barley genotypes in semi-arid region.Nanotechnology is one of the exciting new ields of research that holds great promise in addressing many of the pressing needs in the food and agriculture sectors.Nanomaterials typically have at least one dimension that is in the size range of 1-100 nm.Our results showed that application of nanomaterial signiicantly affected plant height, seed weight, biological yields and economic seed yield.however, the response of the genotypes against the applied nanoparticles was somewhat different.In general, the stimulating effects of nano-SiO 2 on growth and yield components were much more pronounced than that of nano-TiO 2 .Application of nano-TiO 2 in some genotypes caused a signiicant reduction in the evaluated traits.The most obvious reactions to the use of nano-SiO 2 were recorded for G1, G8, G2, G6, G7, G4, G9, and G10.Findings revealed that application of nano-SiO 2 solutions in semi-arid region can improve barley seed yield and can be introduced as beneicial fertilizer for foliar application.

Fig. 2 .
Fig. 2. The effect of beneicial nanoparticles (C: control, S: nanosilicon dioxide and T: nanotitanium dioxide) on spike length of barley genotypes under rainfed condition in northwest of Iran.Vertical bar is SE and the dark line present the mean value of replications.

Fig. 5 .
Fig. 5. Cluster analysis of agronomic traits of barley (Hordeum vulgare L.) genotypes according to similarity in response to foliar application of silicon and titanium nanoparticles under rainfed condition in northwest of Iran.

Table 1 .
The effect of foliar spray of nano-SiO 2 and nano-TiO 2 on some yield components of barley (Hordeum vulgare L.) genotypes under water stress condition

Table 2 .
Mean comparison of seed yield components between the barley (hordeum vulgare L.) genotypes under rainfed condition in northwest of Iran GENOTyPIC RESPONSE OF BARLEy TO EXOGENOUS APPLICATION OF NANOPARTICLES...