**2. Drought stress and experimental design**

Two replicate experiments with potted *F. arundinacea* were conducted. The treatment for each genotype had four replicates (four pots). The control plants were maintained in an optimal soil water condition (FC) during the whole experimental period. The experiment was carried out in loamy soil that was collected at a height of 20 cm from the top of the department's research field in plastic pots (45 cm in height). Potted plants grown under greenhouse conditions were subjected to drought by withholding irrigation for 8 days, whereupon leaves withered and started to discolor in most genotypes. Mini-mustang, Pixie, Jaguar, and h–d were selected in this experiment to capture the range of genetic and phenotypic (aesthetic value) diversity observed in the 11 cultivars. Fully expanded youngest mature leaves of the aforementioned genotypes were collected at days 2, 4, 6, and 8 after the start of the drought treatment for biochemical experiments. Well watered pots (irrigated daily) served as a control for collecting leaf tissue. Leaf samples were collected at midday and were immediately frozen in liquid nitrogen and stored at −80°C until analysis.

### **2.1. Biochemical experiment**

### *2.1.1. Enzyme assay*

The methods used to determine the activities of catalases (CAT) and ascorbate peroxidase (APX) were those reported by Ref. [18]. Superoxide dismutase (SOD) activity was estimated by measuring the decrease in absorbance of superoxide nitro blue tetrazolium complex by the enzyme [19]. The activity of peroxidase (POX) was determined using the method of Ref. [20], with minor changes. Proline content was quantified using the protocol described by Ref. [21].

### **2.2. qRT-PCR analysis**

Total Ribonucleic acid (RNA) extraction, cDNA synthesis, and qRT-PCR analysis were performed as described previously [22].

### **2.3. Tall fescue tissue culture and callus regeneration**

cultivation in urban landscapes throughout transitional climates. Tall fescue is a coarsetextured vigorous perennial bunchgrass. This species reproduces through tillering and seed. Its desirable agronomic characteristics encompass high yields of herbage, excellent persistence, adaptation to a wide range of soil conditions, compatibility with various management practices, long grazing season, and low incidence of pest problems [16, 17]. Given the contin‐ uous drought stress in many areas of the world, the necessity of highly tolerant turf species for landscaping, xeriscaping, and other usage is inevitable. Selection and/or production of high-tolerant transgenic turfgrass would be one of the key ways that leads to ameliorate their

Two replicate experiments with potted *F. arundinacea* were conducted. The treatment for each genotype had four replicates (four pots). The control plants were maintained in an optimal soil water condition (FC) during the whole experimental period. The experiment was carried out in loamy soil that was collected at a height of 20 cm from the top of the department's research field in plastic pots (45 cm in height). Potted plants grown under greenhouse conditions were subjected to drought by withholding irrigation for 8 days, whereupon leaves withered and started to discolor in most genotypes. Mini-mustang, Pixie, Jaguar, and h–d were selected in this experiment to capture the range of genetic and phenotypic (aesthetic value) diversity observed in the 11 cultivars. Fully expanded youngest mature leaves of the aforementioned genotypes were collected at days 2, 4, 6, and 8 after the start of the drought treatment for biochemical experiments. Well watered pots (irrigated daily) served as a control for collecting leaf tissue. Leaf samples were collected at midday and were immediately frozen in liquid

The methods used to determine the activities of catalases (CAT) and ascorbate peroxidase (APX) were those reported by Ref. [18]. Superoxide dismutase (SOD) activity was estimated by measuring the decrease in absorbance of superoxide nitro blue tetrazolium complex by the enzyme [19]. The activity of peroxidase (POX) was determined using the method of Ref. [20], with minor changes. Proline content was quantified using the protocol described by Ref. [21].

Total Ribonucleic acid (RNA) extraction, cDNA synthesis, and qRT-PCR analysis were

drought tolerance threshold in the realm of landscaping.

**2. Drought stress and experimental design**

nitrogen and stored at −80°C until analysis.

**2.1. Biochemical experiment**

*2.1.1. Enzyme assay*

66 Water Stress in Plants

**2.2. qRT-PCR analysis**

performed as described previously [22].

Tall fescue (*F. arundinacea*) seeds were submerged into 25% sulfuric acid for 30 min and then prewashed in tap water overnight. These seeds were moved into the airflow cabinet hood; then they were soaked into 25–50% Clorox (containing 5.25% sodium hypochlorite) solution containing 0.02% household detergent for 30 min for surface sterilization and then rinsed six times with sterilized distilled water. Finally, the dehusked seeds were cut longitudinally and placed onto MS [23] basal medium supplemented with 0–16 mg/l 2,4-Dichlorophenoxyacetic acid (2,4-D), in which explants gained the ability of callus induction at dark in 2 weeks. These calli were subcultured consequently for more than a year, every 4 weeks, on MS media containing 5–8 mg/l 2,4-D. We tried to keep the embryogenic calli in subcultures which were identifiable easily by the yellowish color and firm texture compared to the nonembryogenic calli. In all these experiments, the calli were kept at dark. BAP (0.1, 0.5 mg/l) and kinetin (0.1 and 0.2 mg/l), W/O 2,4-D, have been considered for plant regeneration of tall fescue calli. The pH of all media was adjusted to 5.8 by 0.1 N HCl before autoclaving for 15 min at 121°C and 1.5 kg/cm2 pressure. Cultures were kept at 25 ± 2°C temperature under cool white fluorescent light (30 μmol/m2 /s), with 16/8 h day/night photoperiods.

### **2.4.** *Agrobacterium***-mediated transformation**

pGV3101 strain of *Agrobacterium tumefaciens* harboring pBI121 plasmid as a binary vector contains the *gus* gene under the control of the cauliflower mosaic virus' (CaMV) 35S promoter and the selectable marker neomycin phosphotransferase II (nptII) under the control of the CaMV promoter. Aforesaid bacteria were grown and selected in rotator (200 rpm) in LB liquid media (tryptone 10 g/l, NaCl 5 g/l, yeast extract 5 g/l) supplemented with 50 μg/ml kanamycin for 24 h at 28°C. The cells were harvested by centrifugation (8000 rpm for 10 min at 4°C) and further resuspended in 10–15 ml of MS medium. Acetosyringone (AS) was added to the medium up to 100 μM. For transformation, 4 × 4 mm of calli grown at dark were considered for inoculation with *A. tumefaciens* (OD600 = 0.5–1) under 400 mg Hg pressure for 10–15 min; then, the callus pieces and *Agrobacterium* were incubated together for 20 min with gentle shaking. Excess bacteria were removed after the incubation. The infected callus pieces were transferred onto filter papers for a few minutes and then placed onto cocultivation MS medium supplemented with 100 μM acetosyringone (AS) and 5 mg/l 2,4-D for almost 3 days (in dark) at 25°C. Sometimes, we used to use an empty petri dish containing a Whatman filter paper, moisturized with sterile water, for cocultivation. After cocultivation, the explants were washed with sterile distilled water once; for the second time, the explants were incubated with either 500 mg/l cefotaxime or 400 mg/l Timentin for about 30 min, along with gentle shaking to prevent *Agrobacterium* overgrowth later on (in regeneration MS media); then they were blotted onto sterilized filter papers and placed on MS medium supplemented with 5–8 mg/l 2,4-D, 150–200 mg/l G418 for the selection of transformed explants and 200 mg/l Timentin to prevent *Agrobacterium* overgrowth for 2 weeks. For plant regeneration, explants were transferred to regeneration media supplemented either with BA (6-Benzylaminopurine) or kinetin in light. Cultures were kept at 25 ± 3 °C under cool white fluorescent light (30 μm/m2 /s), for 16 h each day.

### *2.4.1. PCR confirmation and gus histochemical staining*

The presence of *uidA* gene in embryogenic calli was confirmed by PCR amplification of tall fescue-calli genomic DNA with gene-specific primers: *gus*-F (GCTGTGCCAGGCAGTTT‐ TAAC) and *gus*-R (ATATCGTCCACCCAGGTGTTC). The predicted size of the amplified DNA fragments of *uidA* was 425 bp. DNA amplifications were performed in a total volume of 20 μl containing 1 μl of 10 μM forward pimer, 1 μl of 10 μM reverse primer, 2 μl of 10× Ex Taq Buffer, 0.5 μl of dNTP mixture (2.5 mM each), 0.1 μl of TakaRa Ex Taq enzyme (5 unit/μl) (Takara, Shuzo, Kyoto, Japan), by Thermocycler (Bio-Rad, United States). PCR was carried out for screening of regenerated transformed plantlets with an initial denaturation at 94°C for 5 min, followed by 30 cycles of 94°C, 30 min; 58°C, 30 min; 72°C, 30 s; and a final extension, 75°C for 10 min. The PCR products were separated in 1% agarose gel containing 0.5 μg/ml ethidium bromide. The size of the amplification products was estimated using a 100 bp DNA ladder (GeneRuler DNA Ladder Mix # SM 0331, Fermentas). GUS histochemical staining was performed as described previously [24].
