On the basis of previous studies, an antioxidant enriched high yield potential genotype (Accession VA13) was selected for this investigation. This genotype was grown in pots of a rain shelter open field of Bangabandhu Sheikh Mujibur Rahman Agricultural University, Bangladesh (AEZ-28, 24° 23′ north latitude, 90° 08′ east longitude, 8.4 m.s.l.). The seeds were sown in plastic pots (15 cm in height and 40 cm length and 30 cm width) in a randomized complete block design (RCBD) with three replications. N: P₂O₅:K₂O were applied @92:48:60 kg/ha as a split dose. First, in pot soil, @46:48:60 kg ha 1 N: P₂O₅:K₂O and second, at 7 days after sowing (DAS) @46:0:0 kg/ha N: P₂O₅:K₂O. The genotype was grouped into three sets and subjected to four salinity stress treatments that are, 100 mM NaCl, 50 mM NaCl, 25 mM NaCl, and control or no saline water (NS). Pots were well irrigated with fresh water every day up to 10 days after sowing (DAS) of seeds for proper establishment and vigorous growth of seedlings. Imposition of salinity stress treatment was started at 11 DAS and continued up to 40 DAS (edible stage). Saline water (100 mM NaCl, 50 mM NaCl and 25 mM NaCl) and fresh water were applied to respective pots once a day. At 40 DAS the leaves of Amaranthus tricolor were harvested. All the parameters were measured in six samples.

At Moderate salinity stress (MSS) and Severe salinity stress (SSS) conditions, leaf color parameters and pigments, vitamins, phenolic acids, flavonoids and antioxidant capacity of A. tricolor leaves were very high compared to control condition. Hence, salt-stressed A. tricolor leaves had a good source of natural antioxidants compared to plant grown in normal cultivation practices.

AMF inocula [Funneliformis mosseae (T.H. Nicolson & Gerd.) C. Walker & A. Schubler] consisting of spores, soil, hyphae and infected clove (Trifolium repens L.) root fragment from a stock culture of F. mosseae.The inoculated dosage was 10 g of inocula per pot containing approximately 720 spores calculated by microscopy before experiment. There was 2100 infective propagules/g in the inoculum as determined by MPN assay . Seeds were surface sterilized by immersion in 70% ethanol for 5 min, rinsed four times with distilled water, and placed on wet filter paper in Petri dishes at 28 ℃ for germination. After 3 days, the germinate seeds were transplanted into 13 cm × 13 cm plastic pots containing 0.88 kg organized soil substrate (organic manure, soil and decomposed straw = 1:2:1). Half of the pots (AM plants) were inoculated with 10 g of F. mosseae per pot. Non-AM plants received the same weight of autoclaved inocula. The inocula were placed adjacent to each seeding root. The organic substrate was collected from the greenhouse of Institute of Vegetables and Flowers, CAAS and sterilized for 4 h at 160 ℃ , with the chemical properties as follows: pH 7.26, 11.1% organic matter, 150 mg/kg available phosphorus, 451 mg/kg available nitrogen and 518 mg/kg available potassium. The experimental pots were placed in solar greenhouse at an average temperature of 28 ℃ /20 ℃ (day/night) with photon flux density of 600 µmol m^-2 s^-1 and 85% relative humidity.The seedlings uniformed in size were transferred to a growth chamber subjected to different temperature conditions at 20 days after inoculation. The experimental design consisted of four treatments crossing two mycorrhizal inoculations levels (non-AMF and F. mosseae) with two temperature levels with photon flux density of 100 µmol m^-2 s^-1 (25 ℃ /15 ℃ , 15 ℃ /10 ℃ , day/night). (1) Normal temperature (NT): 10 g of sterilized inoculua, 25 ℃ /15 ℃ (day/night); (2) AMF-inoculation (AMF): 10 g of inocula, 25 ℃ /15 ℃ (day/night); (3) low temperature (LT): 10 g of sterilized inocula, 15 ℃ /10 ℃ (day/night); (4) AMF-inoculation under low temperature (AMF + LT): 10 g of inocula, 15 ℃ /10 ℃ (day/night).The experimental design was a completely randomized block design and thirty plants were arranged in each replication. On 45 days after inoculation, phenolic compounds contents, enzymes and gene transcription were determined.

AMF-inoculated cucumber seedlings had significant higher fresh weight and dry weight than non-AMF inoculated control plants under both normal (25/15 ℃ ) and low temperature (15/10 ℃ ) treatment. Under chilling stress, AMF inoculation significantly improved the content of related secondary metabolites including phenols, flavonoids, lignin, DPPH activity and phenolic compounds compared with the non-AMF control. Furthermore, large increments were observed in a number of enzymatic activities related to secondary metabolism and antioxidant system in AMF-inoculated seedlings under low temperature, such as glucose-6-phosphate dehydrogenase (G6PDH), shikimate dehydrogenase (SKDH), phenylalanine ammonia-lyase (PAL), cinnamyl alcohol dehydrogenase (CAD), polyphenol oxidase (PPO), guaiacol peroxidase (G-POD), caffeic acid peroxidase (CA-POD) and chlorogenic acid peroxidase (CGA-POD). As well, the expression of stress-related marker genes was enhanced in AMF-inoculated seedlings in comparison with the non-AMF control. Furthermore, AMF symbiosis decreased hydrogen peroxide (H₂O₂) content under low temperature.

Consistent with the changes of enzyme activities, the relative transcriptional level of genes related to secondary metabolism increased significantly in response to AM fungi inoculation in cucumber roots . Moreover, under chilling stress, the expression levels of WRKY30, PR-1, C4H, CCOMT, CAD, G6PDH, PAL, LPO, and POD genes in the AMF-inoculated seedling were 2.46, 2.90, 1.84, 2.47, 1.96, 2.52, 1.89 and 1.93 folds respectively compared with those under low temperature alone.

Seeds were treated with sulfuric acid (98%, 10 min) and then surface sterilized with 70% ethanol (v/v) for 1 min and sodium hypochlorite solution (10%, at 10 min). After sterilization, seeds were germinated on MS media (Murashige and Skoog, 1962) containing 7 g/L agar (Duchefa, Netherlands). Cultures were maintained under 16/8 h light/dark. Explants were taken from 4-week-old leaves for inoculation with bacteria strain.ATCC15834 strain of A. rhizogenes was supplied by microbial unit of the National Research Center for Genetic Engineering and Biotechnology, Tehran-Iran. Bacterial cells cultivated on LB (Luria-Bertani) culture medium (Bertani, 1952) on rotary shaker (at 26 ℃ and 180 rpm for 48 h) in the darkened state.The leaves were wounded and inoculated with bacterial suspension for 5 min and transferred to MS media containing 7 g/L agar in darkness at 25 ℃ . After 48 h treated Explants were cultured on the 1/2 MS media containing cefotaxime (500 mg/L) and indole-3-butyric acid (IBA) (2 mg/L). Hairy roots emerged at wounded sites, after 4-weeks of incubation, and then each hairy root line was isolated from explants tissue and was subcultured weekly in new media (1/2 MS hormone-free media) with appropriate antibiotic. The concentration of cefotaxime was decreased gradually and eliminated from the culture medium after 8 subcultures and axenic root cultures were obtained. Then hairy root lines were transferred to the 250 mL Erlenmeyer flasks containing 30 mL hormone- free 1/2 MS liquid medium and incubated on a rotary shaker (120 rpm) at 25 ℃ and subcultured every two week. Hairy root line, which showed sufficient growth in 1/2 MS liquid medium, was selected for further investigations.The genomic DNA was extracted from transformed hairy root lines and plant intact roots with CTAB method . Gene-specific primers from rol B were used for amplification of the 780-bp segment in PCR analysis. The primers sequences were, F:5'-ATGGATCCCAAATTGCTATTCCCCCACGA-3'and R:5'-TTAGGCTTCTTTCATTCGGTTTACTGCAGC-3'. Thirty-five PCR cycles were performed with 5 min initial denaturation at 94 ℃ , annealing steps at 60 ℃ for 80 s, extension at 72 ℃ for 90 s, and final extension step of 72 ℃ for 10 min. The amplimer were analyzed by 1% agarose gel electrophoresis.To investigate the effects of SiO₂ NPs, various concentrations (0, 25, 50, 100 and 200 mg/L) of this elicitor were added to the hairy roots culture medium (1/2 MS + 3% sucrose, pH = 5.7) at the end of log phase of growth stages (21-days-old cultures). Hairy roots were incubated with elicitor for 24 and 48 h of exposure time. Hairy roots were harvested 7 days after elicitation and dried on sterile filter paper to remove excess surface moisture and were weighed before freezing by liquid nitrogen and stored at -80℃ until used to measure growth, biochemical and phytochemical analysis.

The effect of silicon dioxide nanoparticles on production of phenolic compounds and expression rate of pal and ras genes involved in rosmarinic acid biosynthesis pathway has been investigated in D. kotschyi. SiO₂ nanoparticles, used as an abiotic elicitor in our study, has appropriate optical, electrical and catalysts properties and has many applications in various industries as well as agriculture. This study clearly suggested that, in the presence of this nanoparticle, induction, production and accumulation of valuable compounds and corresponding antioxidant activity increased in hairy roots.

According to the results, expression levels of the pal and ras genes were influenced by elicitor concentration and exposure time. The elicitation by SiO₂ NP of 100 mg/L after 48 h of exposure time dramatically increased pal expression compared to the control. Briefly, with increasing SiO₂ NP concentrations after 48 h of exposure time, the expression level of pal was also significantly induced . Similarly, ras expression was significantly raised at 48 h after treatment by increasing SiO₂ NP concentration and enhanced to the greatest extent in 50 mg/L concentration. After 24 h of exposure time, the minimum level of ras expression was observed in the 200 mg/L SiO₂ . Amplification products of real-time PCR were assessed with 1.8% agarose gel which was corresponded to the predicted size.

Different concentrations of TiO₂ NPs (0, 10, 20, 30, and 50) were prepared for hairy root treatments. 0.5 g of .S. officinalis hairy roots were transferred to 250 mL Erlenmeyer flasks containing 15 mL of liquid MS culture medium with three replicates. Then, they were placed in an incubator shaker at 110 rpm and 25 &#8451 in dark conditions. On the 22nd day, the liquid MS culture media containing different concentrations of nano titanium dioxide was added to Erlenmeyer flasks. 24 and 48 h after treatment, the hairy roots were taken out and transferred to the MS culture medium lacking elicitor.

The highest rate of total phenol (9.79 mg GLA/g FW) and total flavonoid contents (1.06 mg QE/g FW) were obtained in the treated hairy roots with 50 and 30 mg/L of nano elicitor in 24 and 48 h of treatments, respectively. The maximum level of most polyphenols, such as rosmarinic acid, cinnamic acid, and rutin, was produced in 24 h of treatment. The use of TiO₂ NP for 48 h with 50 mg/L concentration showed the highest production level of SO6 protein.