2.6. Analysis of Morphological, Physiological, and Biochemical Plant Parameters

Six replicates of each treatment were collected and growth parameters like shoot length (SL), root length (RL), plant fresh weight (PFW), plant dry weight (PDW) were recorded for each soybean seedling under well-watered and drought stress conditions. Plant dry weight was measured after drying the plant samples (shoot, root, and leaves) in a hot air oven at 70 °C until a constant weight was achieved [58]. To study the effect of endophyte inoculation on the stomatal development of the host plant, a common stomatal imprint technique was used [59]. The leaves were collected from the youngest trifoliate that had formed before the drought stress and after the imposed drought period. The imprints were collected from the abaxial surface of the leaves and numbers of stomata were counted per unit area of a leaf by using a standard compound microscope. Three fields of view were observed and the variables were counted for each sample [60]. LRWC (leaf relative water content) was calculated using the following formula, where FW is the fresh weight of the leaf, DW is the dry weight of the leaf after drying, and TW is the turgid weight of the leaf soaked in distilled water for 4–5 h [61,62].

A sample of fresh leaves was taken from each treatment for estimation of photosynthetic pigments like chlorophyll a, chlorophyll b, and total chlorophyll was measured using the standard protocol [63]. For estimation of total proline, 500 mg of leaf tissue was homogenized in 3% sulfosalicylic acid (w/v) followed by centrifugation for 10 min at 10,000 rpm. Then, the supernatant was mixed with glacial acetic acid and acidic ninhydrin and incubated in a water bath at 100 °C for about 1 h. Then, the reaction was terminated by keeping samples in an ice bath. Proline was extracted with toluene, and absorbance was recorded at 520 nm using the standard protocol [64]. The concentration of proline was determined by using the standard curve of proline and expressed as µmol proline g⁻¹ FW. Total soluble sugar content was measured using an anthrone reagent [65].

H₂O₂ (hydrogen peroxide) content was determined by homogenizing 500 mg of the leaf sample in 0.1% trichloroacetic acid (TCA) (w/v) followed by centrifugation for 15 min at 12,000 rpm; then, 0.5 mL of the supernatant was added to 1 mL of potassium iodide (1 M) and 0.5 mL potassium phosphate buffer (10 mM, pH 7.0). The absorbance was recorded at 390 nm and the concentration of H₂O₂ was calculated from the standard curve [66]. The obtained values were expressed as nmol g FW. However, the MDA or lipid peroxidation in leaves was measured by estimating the formation of the thiobarbituric acid (TBA) reactive substance. MDA content, a measure of lipid peroxidation, was determined as described by Hodges et al. [67]. Absorbance was recorded at 532 nm and 600 nm and MDA was expressed as nM MDA formed using an extinction coefficient of 155 mM⁻¹/cm.

The roots of 21-day-old bacterial inoculated soybean seedlings were rinsed 4–5 times with a sterile HEPES buffer (0.1 M) and then cut into 3–4 mm pieces. The root pieces were dehydrated at 4 °C in a graded series of alcohol (30–70%) and then fixed in 4% glutaraldehyde for 3 h at 4 °C. Root sections were then critical point-dried, mounted on a metal stub, sputter-coated with gold/palladium, and imaged using an FEI NOVA SEM 450 scanning electron microscope (United States).