The Reinforcement of Early Growth, Extract, and Oil of Silybum marianum L. by Polymer Organic Cover and Bacteria Inoculation under Water Deficit

Abstract

Early growth water stress reduces the extract and fresh oil of Silybum marianum L. (S. marianum) shoots. Two experiments were conducted to reduce the effects of early growth drought. Treatments in the first experiment were organic seed cover fillers at three levels (control, vermicompost, and peat moss), hydrogel at seven levels (control, 2, 4, and 6 g hydrogelF1 per kg OSC, and 2, 4, and 6 g hydrogelA200 per kg organic seed cover), and water deficit at three levels (100, 50, and 25% of field capacity), and in the second experiment, seeds were inoculated with bacteria at four levels (control, Pseudomonas fluorescens, Pseudomonas putida, and their combination) and water deficit at four levels (100, 50, and 25% of field capacity). Our results showed that milk thistle seeds are sensitive to water deficit at the emergence stage. Covering milk thistle (S. marianum) seeds with organic seed cover increased water retention around the seeds and improved emergence percentage. Use of organic seed cover with hydrogel increased relative water content (RWC), leaf area, and shoot length, and increased extracts and oils in fresh shoots. Bacterial inoculation also improved initial growth and reduced the effect of water stress on the plant, and increased leaf number, extract, and oil. The combination of bacteria had a positive effect on initial growth and inoculation of seeds, P. fluorescens and P. putida increased relative water content (RWC), shoot height, and specific leaf area, and increased extract and oil under water deficit conditions. A comparison of the results showed that seed inoculation is a simple method without new culture medium, and improves extract and oil under water deficit conditions.

1. Introduction

Silybum marianum L. (S. marianum) is the superlative medicinal plant for the production of silymarin, which is used for liver protection in humans and animals [1,2]. Seeds and fresh seedlings (leaves and stems) of S. marianum have the highest silymarin content [3,4]. Considering the volume and weight of leaves and stems, which is several times higher than that of seeds, it is very economical to extract silymarin from young stems and leaves of the plant for use as an active ingredient. On the other hand, the leaves and stems are widely used to treat liver disease in livestock through feeding. However, S. marianum is sensitive to water stress during emergence and early vegetative stages [5]. Water deficit reduces emergence rate, initial growth, and dry weight of seedlings. Water deficit is one of the abiotic factors that reduce the desirability of crop production in water-stressed ecosystems [6,7,8,9,10,11,12,13]. About 41% of the Earth’s surface is covered by drylands, where nearly 2 billion people live. About 5.45 billion hectares are arid, and 1 billion hectares are semi-arid and semi-humid. About 70% of the world’s drylands are used for low-productivity agriculture, and the severity of water deficits is the main reason for yield reduction in these areas [14]. Dryland soils are characterized by low organic matter content, low water-holding capacity, low infiltration, and low fertility, which reduces the percentage of seedling establishment due to low water maintenance. In this area, the addition of organic matter, such as around the seed as a seed cover [15], can improve water management in the rhizosphere for a longer period of time. Thus, improving irrigation water management in the early stages of growth mitigates the water deficit effect and increases emergence rate and seedling dry weight [16]. One way to improve seedling emergence and early seedling development is to use seed coating fillers [17] to increase the water stored in the soil for a longer period of time [18]. Organic materials such as peat moss and vermicompost can be used as soil water stores to increase moisture around the seed. The use of water-absorbing modifiers such as organic matter in the soil has become a common solution to improve agricultural sustainability and increase water use efficiency in arid regions. In addition, adding these substances to the soil in this way can significantly improve soil fertility [4] and reduce the effect of abiotic stress such as salinity and water stress in plants [1]. There is little information on the use of organic fillers for water absorption around seeds in arid regions. Our hypothesis is that absorbing and storing water around the seed in arid areas can increase water absorption and emergence percentage and improve the growth of aerial parts and the amount of extract and oil in milk thistle.

Hydrogel polymers are hydrophilic polymer gels or hydrogels that can absorb hundreds of times their weight in water. Once absorbed, the water within the polymer is gradually released by the drying environment [19]. This allows the soil to remain wet for long periods of time without the need for re-watering. Superabsorbent polymer (fabricated from polyacrylamide and acrylic acid) is very effective in increasing seed germination, emergence, and survival of grasses and woody plants in sandy soils [20]. The use of hydrogel polymers and organic materials together improves soil water absorption and retention [21]. The use of seed coatings such as polyacrylamide improves the water-holding capacity of the soil, and by holding moisture longer, improves moisture uptake and increases germination rates [22]. Other researchers have investigated the effects of superabsorbents on water retention in sandy loam soils on the growth of wheat [23] and barley [24] seeds. They reported that adding 1, 2, and 3% of the hydrogel to the soil linearly increased the soil water-holding capacity and delayed the wilting point of the plant by 4–5 days [25,26]. Seed organic matter together with super absorbent polymers (SAPs) are some of the new essential methods [27]. However, no report has been published on the use of organic seed cover with super absorbent polymers (SAPs) to reduce the effect of water deficit in the early growth stage of plants. Increasing the amount of water and keeping it close to the seed is one method to reduce the effects of desiccation due to water deficit after germination. SAPs can improve soil water retention [28], reduce leaching of nutrients from the soil, and optimize water and nutrient use through significant water uptake and long-term retention [29,30]. Hydrogels absorb significant amounts of water, up to several times their own weight [31]. They are used in agriculture and horticulture to absorb and hold water in the soil [32]. The use of 60 to 90 kg per hectare of hydrogel in rainy conditions increased the yield of potatoes [33,34] and wheat [35], improving the efficiency of water use. Other researchers found that the use of superabsorbents in clay and sandy loam soils increased the dry matter of maize [36]. However, no report has been published on the use of organic fillers with SAPs to cover seeds to reduce the effect of water deficit in the early growth stage of plants, especially milk thistle. We also hypothesize that the use of organic matter along with the superabsorber may aid in water uptake and retention during the greening and early growth stages of milk thistle. Plant growth promoting bacteria (PGPBs), such as Pseudomonas spp. [37,38], are found in the rhizosphere and are able to enhance plant development and protect plants from abiotic stresses [39,40,41,42]. PGPBs maintain leaf water content under water deficit conditions, increase main and lateral root growth, and increase water uptake [43]. Other researchers have found that treatment with bacteria promotes water deficit tolerance in rice [44,45] and soybean [46,47]. S. marianum is a medicinal plant grown in arid ecosystems. It has also been documented that chlorophyll content, chlorophyll fluorescence, and stomatal conductance were increased when plants were treated with PGPR compared with untreated plants [48]. On the other hand, there is no information on the inoculation of milk thistle seeds to improve growth under drought stress conditions. Our other hypothesis is that the use of bacteria in the form of seed inoculation can improve the percentage of emergence, growth of aerial parts, and amount of extract and oil of milk thistle under drought stress conditions. S. marianum seeds have glossy and hydrophobic coats and hard shells and are sensitive to water deficit during the germination stage [49]. Drought stress at this stage reduces longitudinal growth of the shoot and root and delays the time of emergence [5], and reduces the uniformity of seedling emergence and density in the field. If we can reduce drought stress in the germination stage until emergence, we can increase the uniformity of seedling emergence and emergence rate to obtain optimal plant densities. Providing moisture in the early stages of vegetative growth increases the amount of oil and extract of fresh seedlings (leaves and stems).

It appears that by using coatings of hydrogels and organic materials, we can increase the water uptake of S. marianum seeds. This increases the emergence rate and the dry length of the shoot. In addition, these materials can provide seedlings with the necessary moisture for a considerable time after emergence, reduce the effects of water deficit, and increase the amount of silymarin in the shoot (stem and leaf). It also appears that seed inoculation with bacteria can increase emergence rate, leaf area, and shoot length [50] and improve silymarin in the shoot (stem and leaf) under water deficit conditions. The present study aims to investigate the organic coating, hydrophilic polymers and Pseudomonas bacteria on early growth and development, extract, and oil of S. marianum under water deficit conditions.

2. Materials and Methods

In these studies, the Bajgah seed cultivar of S. marianum Gaertn. var. purple was used, which was provided by Dr. Mansour Taghvaei of the Department of Plant Production and Genetics, School of Agriculture, Shiraz University, Shiraz, Iran. The seeds were stored at 5 °C with minimum permeability and kept until the beginning of the experiment. Then, homogeneous, and uniform seeds were selected and the physiological characteristics of seeds such as moisture, 1000 seed weight and viability were evaluated and disinfected for 2 min in carboxinthiram (2 g L⁻¹) fungicide solution.

2.1. Experiment I

The study was conducted in factorial experiments based on randomized complete block design (RCBD) with three replications in greenhouse. The factors studied include organic seed cover (OSC) fillers at three levels (control or seed without organic cover filer (C1), vermicompost of manure-based from the company Golbaran Sabz (C2), and peat moss from the company Sphagnum Ltd., Moscow, Russia (C3)), hydrogel at seven levels ((S1 or OSC without hydrogel, S2, S3, and S4 including (2, 4, and 6 g hydrogel F1 per kg OSC), S5, S6, and S7 including (2, 4, and 6 g hydrogel A200 per kg OSC)), and water stress at three levels (100% field capacity (D1), 50% FC or moderate water deficit (D2), and 25% FC or severe water deficit (D3)). The field capacity of the soil was determined by the pressure plate method by the Water Engineering Department of the Faculty of Agriculture, Shiraz University [51].

Hydrogel F1 (a cationic amphoteric surfactant, white crystalline fine powder, produced by German Stockosorb company, Berlin, Germany) and Hydrogel A200 (an acrylamide, white crystalline granular powder, produced by Iranian Nano company under license Polymer and Petro-chemical of Iran) were ground and passed through 0.75 mm sieve. The pots were weighed daily to apply the filed capacity and the water treatments were applied based on the weight of the pots to achieve the target soil moisture. Plants were grown in greenhouses with a day/night (16 h/8 h) temperature of 25/18.2.1.1. SAPs F1 and A200 Water Absorption Measurement.

To measure water absorption, a certain weight of SAPs (about 5 g) was immersed in a certain amount of water at room temperature for 2 h, and after removing the excess water, the sample was weighed again and the water absorption was measured using the following formula [52]:

$$\mathrm{WA}=\frac{\left(\mathrm{M}-\mathrm{M}0\right)}{\mathrm{M}0}$$

where M is the weight of hydrogel after water absorption, M0 is the dry weight of SAPs, and WA is the water absorption per gram of dry SAPs.

2.1.1. Measurement of the Amount of Water Absorbed by the Mixture of Superabsorbent Polymer and OSC

To measure the water absorption, 4 levels (0, 2, 4, and 6 g) of hydrogel per kg of OSC were immersed in a certain amount of water at room temperature for 2 h. After removing the excess water, the sample was reweighed according to the following formula and the water absorption was measured:

$$\mathrm{WAabs}=\frac{\left(\mathrm{Msap}-\mathrm{Mh}\right)}{\mathrm{Mh}}$$

where Msap is the weight of a mixture of SAPs and organic matter after water absorption, Mh is the dry weight of the mixture of hydrogel and organic matter, and WAabs is the water absorption per gram of dry mixture of hydrogel and organic matter.

2.1.2. Organic Seed Cover (OSC) Fillers Preparation and Plantation

Organic compounds (vermicompost and peat moss) were dried in an oven and passed through a 1 mm mesh sieve. These compounds were then ground in a mill and combined with water (100 mL per kg of organic matter) and hydrogel polymers in 4 levels of 0, 2, 4, and 6 g per kg of organic matter to form a dough. Then the amount of 2 g of each dough covering material was injected into the plastic semicircle and the molds were dried at room temperature (Figure 1).

After preparing the half ball, two seeds were placed between two half balls and finally the balls were transferred to 5 L volume pots (with a hole drilled in the bottom of each pot for drainage) filled with 4 kg of field soil whose soil properties are described in

Table 1. Physico-chemical characteristics of soil used in the present.

Soil Texture	Bulk Density (g cm−3)	ECe (dS m)	pH	CEC (cmol + kg−1)	N (Total Nitrogen) %	P (Absorbable Potassium)	K (Absorbable Potassium)	* Fe (mg kg−1)	* Mn (mg kg−1)	* Zn (mg kg−1)	* Cu (mg kg−1)	OM (%)
Clay loam	1.28	0.35	7.7	15	0.06	18	620	3.5	11.5	0.4	1.5	1.3

* Extractable with DTPA.

. Balls containing seeds were planted at a depth of 5 cm. The plants were grown in greenhouses with a day/night (16 h/8 h) temperature of 25/18. After 15 days of sowing, water deficit treatments were carried out for three months and then plants were harvested to determine indices (emergence rate, leaf area, root length, shoot length, free leaf proline content, seedling length vigor index, activities of enzymatic antioxidants, seedling weight vigor index, fresh leaf extract, and oil).

2.2. Experiment II

The study was conducted in factorial experiments based on a completely randomized design (CRD) with three replications in the greenhouse. The treatments in the second experiment were growth promoting bacteria (PGPB) in four levels (control (no inoculation), Pseudomonas fluorescens, Pseudomonas putida, and combined inoculation of Pseudomonas fluorescens and Pseudomonas putida) and water stress in four levels (100%, 75%, 50%, and 25% FC). The bacteria were obtained from the Microbiology Laboratory of the Soil Science Department of Shiraz University and purified by the Iranian Gene Laboratory and Tehran University. The seeds were planted at a depth of 5 cm in 5 L volume pots (with a hole drilled in the bottom of each pot for drainage) filled with 4 kg of field soil, the characteristics of which are described in Table 1. The plants were grown in greenhouses with a day/night (16 h/8 h) temperature of 25/18. Water treatments were applied to the weight base and water was added to achieve the target soil moisture. After 15 days of sowing, water deficit treatments were carried out for three months and then the plants were harvested to determine indices (emergence percentage, leaf area, and relative water content (RWC)), seedling weight vigor index (SWVI), shoot length, SPAD, chlorophyll meter reading, extract, and oil of the fresh shoot.

2.2.1. Seed Inoculation with Bacteria

S. marianum seeds were surface sterilized with 60% ethanol for 2 min and 8% calcium hypochlorite solution for 8 min and then washed 5 times with sterile distilled water. The seeds were soaked in the bacterial suspension (pure culture of P. fluorescens, P. putida) and incubated with equal proportions of P. fluorescens and P. putida for 30 min.

2.2.2. Emergence Rate (Day)

The emergence count was performed simultaneously each day, and the following formulas were used to measure the emergence rate (ER).

ER = 1/MET

MET = Σ(n_i × d_i)/N

where n_i is the number of seedlings that emerged that day, d_i is the number of days until counting, and N is the total number of seedlings that emerged in the treatment.

2.2.3. Shoot and Root Length (cm)

Root and shoot lengths were measured with a ruler to an accuracy of one millimeter, then the average shoot and root size was recorded for each treatment of each replicate.

2.2.4. Seedling Length and Weight Vigor Index

Seedling length vigor index (SLVI) and seeding weight vigor index (SWVI) were measured by the below formula:

$$\mathrm{S}\mathrm{L}\mathrm{V}\mathrm{I}=\frac{\left(\mathrm{R}\mathrm{L}+\mathrm{S}\mathrm{L}\right)\times \mathrm{E}\mathrm{P}}{100}$$

$$\mathrm{S}\mathrm{W}\mathrm{V}\mathrm{I}=\frac{\left(\mathrm{R}\mathrm{W}+\mathrm{S}\mathrm{W}\right)\times \mathrm{E}\mathrm{P}}{100}$$

where RL, SL, RW, and SW are the shoot length (cm), root length (cm), root dry weight (g), and shoot dry weight (g), respectively, and EP is the emergence percentage. The root and shoot were dried in an oven at 70 °C for 24 h, and then the weight was determined with an accuracy of 0.0001 g.

2.2.5. Leaf Area (LA)

Leaf area measurements were fabricated with a one-meter leaf area meter, model MK2 (Delta Company, Cambridge, UK).

2.2.6. Relative Water Content (RWC)

To calculate RWC, the fresh leaf samples were weighed, then soaked in distilled water, dried at 70 °C for 48 h, and weighed again, and RWC was calculated using the following equation.

RWC = (FW − DW/TW − DW) × 100

where FW is fresh weight, DW is dry weight, and TW is turgor weight of leaf samples.

2.2.7. SPAD Chlorophyll Meter Reading (SCMR)

The SCMR was measured for the fully expanded third leaf from the top at 10:00–12:00 a.m. SPAD chlorophyll meter (Minolta SPAD-502 m, Tokyo, Japan).

2.2.8. Leaf-Free Proline Content

Fresh leaf samples (0.5 g) were mixed with 10 mL of a 3% solution of sulfosalicylic acid. Two ml of the extract was mixed with 2 mL of ninhydrin acid reagent (2, 2-dihydroxyindane-1,3-dione) and 2 mL of acetic acid, and the mixture was incubated at 100 °C for 60 min. After incubation, the mixture was transferred to a water bath; 4 mL of toluene was added, and the absorbance was read at 520 nm using a spectrophotometer (Bio wave II model, UK). The proline content of the leaves was determined from the standard curve [53].

2.2.9. Enzymatic Antioxidant Activities

Fresh leaf samples (0.5 g) were ground with liquid nitrogen in a Chinese mortar; 1 mL of 50 mM phosphate buffer containing 0.5 M EDTA-polyvinyl and 2% polypyrrolidone was added to the extracted tissue and centrifuged at 14,000× g for 20 min at 4 °C. The supernatant was used for the determination of ascorbate peroxidase (APX) and catalase (CAT) activities [54]. APX activity was estimated as described by Nakano and Asada [55], Beauchamp and Fridovich [56].

2.2.10. Reduction Percentage (RP)

Reduction percentage (RP) of emergence percentage (E), seedling length (SL), leaf area (La), relative water content (RWC), SPAD chlorophyll meter reading, seedling weight vigor index (SWVI), reduction percentage (PR) compared with control were calculated by the following equation.

$$\mathrm{RP}=(1-\left(\frac{\mathrm{Nx}}{\mathrm{Mc}}\right)\times 100$$

where Nx is the number of seedling characteristics under water deficit and Mc is the seedling characteristic under control conditions.

2.2.11. S. marianum Extraction

We performed exhaustive extractions in the Soxhlet apparatus using 2.0 g portions of the material. Accurately weighed S. marianum shoot samples were transferred to a paper thimble, and the loaded thimble was placed in a 100 mL Soxhlet extractor. The extractions were performed in a two-step procedure. In the first step of the procedure, the plant material was defatted for 6 h using 200 mL of n-hexane. During this stage, the heater was set at 60 °C and in the next step, the heater was fixed at 80 °C. At the end of the oil extraction process, the defeated samples were used for S. marianum extraction using Soxhlet and 200 mL of methanol for 5 h [57]. Finally, the solvents were evaporated using a vacuum rotary evaporator system and dry S. marianum extract was obtained.

2.3. Statistical Analysis

Data were analyzed using the statistical software SAS, version 9.3, after testing for normality using the Kolmogorov–Smirnov normality test, and graphs were created using Excel Software. Means were compared using the Duncan test at the 5% level.

3. Results

3.1. Experiment I

3.1.1. Emergence Rate

The interaction of OSC fillers and water deficit significantly affected the emergence rate. By comparing the means, it was observed that the emergence rate increased at all levels of water deficit, so that the highest emergence rate (0.132 seeds per day) observed in the control, or 100% field capacity (D1), was with the use of vermicompost (C2) (

Table 2. Effect of different organic seed cover (SOC) fillers on emergence rate, proline contents, catalase activity, ascorbate peroxidase, shoot oil, and shoot extract under different soil moisture regimes.

	Soil Moisture Regimes	D1	D2	D3
SOC
Emergence rate
C1		0.132 ± 0.02 a	0.117 ± 0.03 b	0.109 ± 0.03 c
C2		0.09 ± 1.01 d	0.082 ± 0.01 e	0.082 ± 0.02 e
Proline contents (μmol g−1 FW)
C1		3.06 ± 0.02 c	5.54 ± 0.09 b	10.75 ± 0.05 a
C2		4.11 ± 0.03 c	7.6 ± 0.09 b	12.21 ± 0.06 a
Catalase activity (U g−1 FW)
C1		22.1 ± 0.04 c	24.05 ± 0.06 b	26.61 ± 0.09 a
C2		29.21 ± 0.04 c	32.15 ± 0.01 b	38.71 ± 0.09 a
Ascorbate peroxidase activity (U g−1 FW)
C1		843 ± 0.08 c	935.61 ± 0.07 b	985.4 ± 0.09 a
C2		943.93 ± 0.01 c	1089.4 ± 0.04 b	1237.32 ± 0.03 a
Shoot oil
C1		2.18 ± 0.02 a	1.98 ± 0.03 b	1.95 ± 0.01 b
C2		2.10 ± 0.04 a	1.20 ± 0.04 c	1.15 ± 0.01 c
Shoot extract
C1		0.82 ± 0.02 a	0.80 ± 0.04 a	0.76 ± 0.05 ab
C2		0.80 ± 0.04 a	0.76 ± 0.03 ab	0.71 ± 0.02 ab

Mean values with the same letter are not significantly different from each other (p > 0.05). D1: field capacity; D2: 75% of field capacity; and D3: 50% of field capacity; C1: vermicomposting; C2: peat moss. Data are the average of three replicates.

). Vermicompost seed cover was more effective in offsetting the effect of water deficit as the highest emergence rates of 0.17 and 0.109 (seeds per day) in moderate water deficit (D2) and severe water deficit (D3) were obtained with vermicompost (C2), respectively (Table 2). The interaction of water deficit and the amount of hydrogel used in OSC significantly affected the emergence rate. The use of organic hydrogel seed cover increased the emergence rate by reducing the effect of water deficit.

The highest emergence rate (0.146 seeds per day) was obtained in the control condition or 100% field capacity (D1) using 6 g of hydrogel F1 per kg of cover material (S4). Although the emergence rate decreased with increasing water deficit in all treatments, the highest emergence rate (0.115 seeds per day) under severe water deficit conditions (D3) was observed in the S4 treatment (

Table 3. Effect of different superabsorbent levels on emergence rate, leaf area, and root length under different soil moisture regimes.

	Soil Moisture Regimes	Emergence Rate (Seed per Day)			Leaf Area (cm2)			Root Length (cm)
Superabsorbent		D1	D2	D3	D1	D2	D3	D1	D2	D3
S1		0.93 ± 0.02 g	0.80 ± 0.02 h	0.80 ± 0.01 h	133.79 ± 11 d	85.11 ± 16 ef	66.30 ± 12 f	21.37 ± 0.9 ef	17.94 ± 1.3 hi	15.60 ± 0.9 j
S2		1.1 ± 0.02 d–g	0.92 ± 0.02 g	0.80 ± 0.03 h	173.24 ± 7 c	124.76 ± 26 d	96.42 ± 27 e	22.43 ± 1.2 de	18.29 ± 1.1 hi	17.18 ± 1.3 i
S3		1.27 ± 0.03 b	1. ± 0.02 c–e	0.98 ± 0.02 e–g	228.77 ± 29 b	138.39 ± 24 d	122.56 ± 12 d	23.32 ± 2.4 cd	23.29 ± 1.7 cd	20.530 ± 0.8 fg
S4		1.4 ± 0.1 a	1.2 ± 0.1 b	1.1 ± 0.09 cd	314.84 ± 19 a	190.05 ± 23 c	144.41 ± 38 d	26.01 ± 4 a	25.24 ± 1.1 ab	24.94 ± 3.1 a–c
S5		0.480 ± 0.09 h	0.77 ± 0.07 h	0.78 ± 0.06 h	134.42 ± 19 d	90.41 ± 30 e	79.62 ± 14 ef	22.63 ± 1.6 de	20.60 ± 2.2 fg	18.13 ± 1.7 hi
S6		1.1 ± 0.05 c–f	0.97 ± 0.03 fg	1 ± 0.1 cf	192.33 ± 18	133.24 ± 21 d	96.64 ± 39 e	23.13 ± 1.6 cd	22.56 ± 2.2 de	19.09 ± 1.7 gh
S7		1.18 ± 0.1 b	1.1 ± 0.1 cd	1.1 ± 0.09 cd	241.26 ± 13 b	197.80 ± 37 c	142.27 ± 55 d	24.75 ± 1.9 a–c	23.83 ± 0.9 b–d	23.94 ± 1.4 b–d

Mean values with the same letter are not significantly different from each other (p > 0.05). D1: field capacity; D2: 75% of field capacity; and D3: 50% of field capacity, (S1 = (cover without superabsorbent), S2, S3, and S4 included (2, 4, and 6 g of superabsorbentF1 per kg of coating materials), S5, S6, and S7 included (2, 4, and 6 g of superabsorbentA200 per kg coating materials)), data are the average of three replicates.

).

3.1.2. Leaf Area

The interaction of hydrogel and water deficit level significantly affected leaf area (Table 3). The use of hydrogel increased leaf area at all levels of water deficit. The highest leaf area (314.84, 190.05, and 144.41 cm²) in D1, D2, and D3 water deficit conditions were obtained in the S4 treatment (6 g hydrogel F1 per kg OSC fillers), but it was not significantly different from the S7 treatment (6 g hydrogel A200 per kg OSC fillers) (Table 3).

3.1.3. Root Length

The interaction of hydrogel and water deficit level significantly affected root length (Table 3). The highest root lengths (26.01, 25.24, and 24.941 cm) were obtained in D1, D2, and D3 water deficit conditions, in the S4 treatment (6 g hydrogel F1 per kg OSC) (Table 3).

3.1.4. Shoot Length

The shoot length was significantly affected by organic seed cover and hydrogel (

Table 4. Effect of different organic seed cover (SOC) fillers and superabsorbent levels on shoot length (cm).

	Superabsorbent	S1	S2	S3	S4	S5	S6	S7
SOC
C1		17.80 ± 3.3 gh	19.69 ± 2.9 fg	22.24 ± 3.2 de	25.55 ± 3 bc	20.53 ± 4 ef	22.49 ± 3.4 de	26.79 ± 2.7 b
C2		17.47 ± 4 h	20.01 ± 3.7 f	26.42 ± 3.5 b	29.66 ± 2.5 a	21.67 ± 4 d–f	23.72 ± 3.2 cd	26.03 ± 3.1 b

Mean values with the same letter are not significantly different from each other (p > 0.05). C1: vermicompost; C2: peat moss; (S1 = (cover without superabsorbent); S2, S3, and S4 included (2, 4, and 6 g of superabsorbentF1 per kg of coating materials), S5, S6, and S7 included (2, 4, and 6 g of superabsorbentA200 per kg coating materials)); C1: vermicomposting; and C2: peat moss. Data are the average of three replicate.

). The lowest shoot length (16.30 cm) was observed in seeds without organic cover (C1). Shoot length increased with an increasing amount of hydrogel. The maximum shoot length (29.66 cm) was observed in peat moss cover (C3) with 6 g hydrogel F1 per kg OSC (S4) (Table 4).

3.1.5. Free Leaf Proline Contents

The interaction of water deficit and OSC fillers had a significant effect on leaf free proline content (Table 2). Water deficit significantly increased leaf proline accumulation. However, the use of organic fillers as a seed cover, by reducing the effect of water deficit, reduced leaf proline accumulation under moderate and severe water deficits (D3). Thus, the lowest amount of free leaf proline content (10.75) was obtained in the treatment with vermicompost (C2) (Table 2).

3.1.6. Enzymatic Antioxidant Activities

The interaction of water deficit and OSC fillers significantly increased CAT and APX under moderate and severe water deficit conditions, while the use of organic polymer seed cover decreased CAT and APX under severe water deficit (D3) (Table 2). The lowest CAT and APX were presented in the control condition or 100% of field capacity (D1) when vermicompost (C2) was used. Vermicompost (C2) seed cover was more effective in reducing the effect of water deficit. The lowest of the activities of CAT, (24.05 and 26.61) were obtained using vermicompost cover in D2 (moderate water deficit) and D3 (severe water deficit), respectively (Table 2). The lowest of the activities of APX, (935.61 and 985.4) were obtained with vermicompost cover in D2 (moderate water deficit) and D3 (severe water deficit), respectively (Table 2).

3.1.7. Seedling Length Vigor Index (SLVI)

Seedling length vigor index decreased with increasing water deficit. The seedling length vigor index of the plant decreased from 43.53 at control or 100% field capacity (D1) to 28.17 at severe water deficit (D3) (Figure 2 and Figure 3). The hydrogel had a significant effect on SLVI (Figure 3). Seedling length vigor index increased from 24.76 at the first hydrogel level (no hydrogel cover) to 43.5 with 6 g of hydrogel A200 per kg of cover material (S7) (Figure 3).

3.1.8. Seedling Weight Vigor Index

Seedling weight vigor index is significantly influenced by OSC fillers. The lowest (0.85) and highest (2.01) seedling weight vigor index were observed in seeds without organic amendments and with the use of vermicompost (C2), respectively (Figure 4). Hydrogel used with organic amendments had a significant effect on seedling weight vigor index. Seedling weight vigor index increased with increasing amounts of both hydrogel F1 and A200 (Figure 5). The highest seedling weight vigor index (2.45) was observed in the S7 treatment (6 g hydrogel A200 per kg OSC), and the lowest seedling weight vigor index (1.1) was observed in the S1 treatment (no hydrogel cover) (Figure 5).

3.1.9. Fresh Leaf Extract and Oil

The extracts and oil of fresh shoots are significantly affected by water deficit and OSC fillers. (Table 2). The extract and oil of the shoot decreased with increasing water deficit. Comparing the means showed that the highest of the extract and oil was observed in treatment vermicompost (C2) with 100% field capacity (D1), but with the increase in the water deficit from control or 100% field capacity (D1) to 30% of field capacity (D3) the extract and oil of the fresh shoot were decreased (Table 2). The use of vermicompost cover (C2) was more effective in reducing the effect of water deficit. The highest oil content of shoots (1.98 and 1.95) were obtained using vermicompost cover (C2) in moderate water deficit (D2) and severe water deficit (D3), respectively (Table 2), and the highest extract (4.17 and 4.02) were obtained using vermicompost (C2) in moderate water deficit (D2) and severe water deficit (D3), respectively (Table 2).

3.2. Experiment II

3.2.1. Emergence Percentage

The interactions of water deficit and seed inoculation had a significant effect on emergence percentage (

Table 5. Effect of inoculation milk thistle (Silybum marianum L.) seed with the bacterium of on seedling traits under different soil moisture regimes.

Inoculation	Seedling Traits	D1	D2	D3	D4	Inoculation	Seedling Traits	D1	D2	D3	D4
I0	EP	95.33 ± 4 ab	90.67 ± 0.5 bcd	84.00 ± 4.7 defg	76.67 ± 3.2 g	I0	SL (cm)	40.95 ± 0.8 b	33.30 ± 1.2 bcdef	31.45 ± 0.4 def	26.19 ± 0.5 fg
	RP	0.00	4.90	7.35	8.73		RP	0.00	18.68	23.19	36.04
I1	EP	97.67 ± 4.7 ab	93 ± 2.8 abc	90.33 ± 2.3 bcde	86 ± 3 cdef	I1	SL (cm)	40.31 ± 1.2 b	34.09 ± 1.4 bcdef	31.69 ± 1.6 def	22.22 ± 0.3 g
	RP	0.00	4.78	2.87	4.80		RP	0.00	15.44	21.38	44.88
I2	EP	97.67 ± 2.3 ab	85 ± 1.8 def	82.33 ± 1.4 efg	79.78 ± 2 fg	I2	SL (cm)	36.27 ± 1.3 bc	34.79 ± 1.9 bcde	31.27 ± 1.6 def	23.64 ± 0.2 g
	RP	0.00	12.97	3.14	3.10		RP	0.00	4.13	13.84	34.85
I3	EP	99.67 ± 0.4 a	94.67 ± 1.4 ab	90.33 ± 4.7 bcde	86.33 ± 2.8 def	I3	SL (cm)	41.08 ± 0.2 b	35.79 ± 0.8 bcdef	29.05 ± 0.7 ef	28.21 ± 0.4 fg
	RP	0.00	5.02	4.58	4.43		RP	0.00	12.86	29.26	31.32
I0	LA (cm2)	91.57 ± 1.9 bcd	84.11 ± 2.6 def	78.17 ± 2.9 fg	69.50 ± 2.3 h	I0	SPAD	96.67 ± 4 abc	83.73 ± 0.5 abcd	50.67 ± 4.7 fg	35.67 ± 3.2 g
	RP	0.00	8.33	14.80	24.25		RP	0.00	13.37	47.58	63.10
I1	LA	97.14 ± 1.01 ab	91.97 ± 2.8 bc	84.14 ± 3.3 def	80.31 ± 3.2 efg	I1	SPAD	100.47 ± 4. 7 a	92.67 ± 2.8 abc	64.00 ± 2.3 def	57.23 ± 3 ef
	RP	0.00	5.33	13.38	17.33		RP	0.00	7.76	36.29	43.03
I2	LA	96.58 ± 1.9 ab	86.35 ± 1.7 cde	77.83 ± 1.2 fg	73.33 ± 1.9 gh	I2	SPAD	98.77 ± 2.3 ab	77.60 ± 1.8 bcde	65.67 ± 1.4 def	49 ± 2 fg
	RP	0.00	10.59	19.41	24.07		RP	0.00	21.43	33.51	50.38.
I3	LA	101.08 ± 0.01 a	93.02 b ± 0.3 c	89.07 ± 2.8 cd	86.67 ± 3.3 cde	I3	SPAD	102.73 ± 0.4 a	100.70 ± 1.4 a	76.33 ± 4.7 cde	60 ± 2.8 ef
	RP	0.00	7.90	11.81	14.19		RP	0.00	1.98	25.70	41.59
I0	RWC	0.69 ± 0.04 bcd	0.67 b ± 0.03 cdef	0.66 ± 0.04 bcdef	0.56 ± 0.02 ef	I0	Shoot extract	0.88 ± 0.5 a	0.87 ± 0.04 a	0.85 ± 0.09 ab	0.80 ± 0.07 ab
	RP	0.00	1.47	3.58	18.20		RP	0.0	0.36	1.77	4.55
I1	RWC	0.78 ± 0.04 ab	0.72 ± 0.03 bc	0.68 ± 0.12 bcde	0.63 ± 0.23 cdef	I1	Shoot extract	0.95 ± 0.02 a	0.90 ± 0.04 a	0.86 ± 0.02 a	0.84 ± 0.08 ab
	RP	0.00	7.55	13.25	19.37		RP	0	2.28	4.48	4.33
I2	RWC	0.68 b ± 0.03 cde	0.65 ± 0.02 cdef	0.59 ± 0.03 def	0.57 ± 0.4 ef	I2	Shoot extract	0.87 ± 0.07 a	0.87 ± 0.03 a	0.81 ± 0.04 ab	0.75 ± 0.04 b
	RP	0.00	2.49	12.62	15.31		RP	0	0.24	3.10	5.11
I3	RWC	0.88 ± 0.07 a	0.71 ± 0.06 bc	0.70 ± 0.09 bcd	0.70 ± 0.1 bcd	I3	Shoot extract	0.86 ± 0.04 a	0.76 ± 0.03 b	0.78 ± 0.03 b	0.63 ± 0.04 c
	RP	0.00	18.14	19.08	20.22		RP	0	5.96	4.96	8.26
I0	SWVI	917.87 ± 4.7 cd	799.93 ± 3.2 cde	490 ± 5.2 ghij	250.96 ± 6.2 j	I0	Shoot oil	2.18 ± 0.05 a	2.13 ± 0.05 a	1.98 ± 0.01 b	1.87 ± 0.01 b
	RP	0.00	20.60	27.49	50.47		RP	0	1.26	4.58	6.99
I1	SWVI	1167.54 ± 5.1 ab	796.09 ± 9.8 cde	550.43 ± 7.8 fghi	364.73 ± 15 ij	I1	Shoot oil	2.2 ± 0.02 a	2.14 ± 0.05 a	2.11 ± 0.09 a	2.03 ± 0.01 a
	RP	0.00	18.13	33.13	45.97		RP	0	1.25	1.93	3.75
I2	SWVI	1017.40 ± 3.1 bc	629.23 ± 10.1 efgh	555.03 ± 6.8 efghi	276 ± 7.3 j	I2	Shoot oil	2.1 ± 0.04 a	2.05 ± 0.02 a	2 ± 0.04 a	1.83 ± 0.01 b
	RP	0.00	15.97	25.03	47.31		RP	0	1.19	2.38	6.31
I3	SWVI	1342 ± 5.6 a	939.68 ± 9.3 bc	689.71 ± 3 bc	431.66 ± 11 hij	I3	Shoot oil	2.06 ± 0.03 a	1.99 ± 0.06 a	1.76 ± 0.03 b	1.73 ± 0.01 b
	RP	0.00	21.25	37.63	41.88		RP	0	1.81	7.26	7.99

Mean values with the same letter are not significantly different from each other (p > 0.05). D1: field capacity; D2: 75% of field capacity; D3: 50% of field capacity; D4: 25% of field capacity; I0: without inoculation; I1: inoculation with Pseudomonas fluorescens; I2: inoculation with Pseudomonas putida; and I3: inoculation with combined of Pseudomonas fluorescens and Pseudomonas putida. EP: emergence percentage; LA: leaf area; RWC: relative water content; SWVI: seedling weight vigor index; SL: shoot length; SLA: specific leaf area; SPAD: SPAD chlorophyll meter reading; RP: reduction percentage. Data are the average of three replicate.

). Emergence percentage decreased with increasing water deficit even in all seeds inoculated with the bacterium. Seed inoculation increased emergence percentage at all levels of water deficit compared with the control. The lowest percentage reduction (4.43) in emergence percentage was observed in the I3 treatment (equal proportions of two bacteria, P. fluorescens and P. putida) compared with the control (Table 5).

3.2.2. Leaf Area

The interaction of water deficit and inoculation has a significant effect on leaf area (Table 5). Leaf area decreased with increasing water deficit in all seeds inoculated with the bacterium. However, the percentage of reduction was not the same. The lowest percentage of reduction (14.19) was related to the seeds inoculated with equal proportions of two bacteria, P. fluorescens and P. putida (I3), in severe water deficit (Table 5).

3.2.3. Relative Water Content (RWC)

The interaction of water deficit and inoculation had a significant effect on relative water content (Table 5). With increasing water deficit, the relative water content decreased in all inoculated seeds, but the percentage decrease was not the same. The highest relative water content (0.88) was associated with seeds inoculated with equal proportions of two bacteria, P. fluorescens and P. putida (I3), at the control level of water deficit (D1) but decreased to 0.70 with increasing water deficit (Table 5).

3.2.4. Seedling Weight Vigor Index (SWVI)

The interaction between water deficit and inoculation had a significant effect on SWVI (Table 5). SWVI, as an indicator of dry weight produced by the plant, decreased with increasing water deficit in all treatments (Table 5). The highest SWVI (1342) was associated with seeds inoculated with equal proportions of two bacteria, P. fluorescens and P. putida, at the control level of water deficit (D1). Seed inoculation increased SWVI at all levels of water deficit. The low percentage of reduction (41.88) was associated with seeds inoculated with equal proportions of two bacteria, P. fluorescens and P. putida (I3), at severe water deficit (D4) (Table 5).

3.2.5. Shoot Length

Shoot length was significantly affected by the interaction of water deficit and inoculation (Table 5). Seed inoculation increased shoot length at all levels of water deficit. Shoot length decreased with increasing water deficit in all bacterially inoculated seeds. However, the percentage of reduction was not the same, such that the lowest percentage of reduction (31.32) was associated with seeds inoculated with equal proportions of two bacteria, P. fluorescens and P. putida (I3), at severe water deficit (D4) (Table 5).

3.2.6. SPAD Chlorophyll Meter Reading

The interactions of water deficit and inoculation significantly affected the SPAD chlorophyll meter reading (Table 5). Water deficit reduced leaf chlorophyll content from 96.67 under normal conditions (no water deficit) to 35.67 (without bacterial seed inoculation). Seed inoculation increased the SPAD chlorophyll meter at all levels of water deficit (the highest SPAD chlorophyll meter reading (102.73) was associated with inoculated seeds with an equal proportion of two bacteria, P. fluorescens and P. putida (I3) at the control level of water deficit (D1)). However, there was no significant difference between seeds inoculated with P. fluorescens bacteria (Table 5).

3.2.7. Extract Fresh Shoot

Water deficit and bacterial inoculation had a significant effect on fresh shoot extract (Table 5). Fresh shoot extract decreased with increasing water deficit in all treatments. Fresh shoot extract decreased with increasing water deficit (Table 5), especially in all seeds inoculated with bacteria (Table 5). However, the percentage reduction was not the same, so that the lowest percentage reduction in the extract of the fresh shoot (2.69%) was related to inoculated seeds with an equal proportion of two bacteria, P. fluorescens and P. putida (I3), in the severe water deficit (D4). Seed inoculation increased the extract of fresh shoots at all levels of water deficit. The extract of fresh shoot decreased from 2.23 at the control level (D1) only to 2.11 at the level of severe water deficit (D3) (Table 5).

3.2.8. Fresh Shoot Oil

Leaf oil was significantly affected by water deficit and bacterial inoculation (Table 4). Leaf oil decreased with increasing water deficit (Table 5), especially in all seeds inoculated with the bacterium. However, the percentage reduction was not the same. The lowest percentage reduction, the oil of the shoot (1.937%), was related to seeds inoculated with equal proportions of two bacteria, P. fluorescens and P. putida (I3), at severe water deficit (D4) (Table 5). Seed inoculation increased leaf oil at all levels of water deficit (Table 5). The oil of fresh shoots decreased from 10.97 at the control level (D1) to 10.55 at the severe water deficit level (D3) (Table 5).

4. Discussion

One of the problems of semi-arid areas is the inability of the soil to retain the water needed for germination due to the lack of soil organic matter. Covering the seed with vermicompost (C2) and peat moss (C3) can store a lot of water to absorb and retain moisture for a longer time under water deficit and, in our study, improve the seedling emergence rate (Table 2). A possible consequence of water deficit is a reduction in the longitudinal rate of cell growth [58], resulting in poor shoot development and ultimately reduced emergence rate. Vermicompost positively affects the emergence of pine (Pinus pinaster Ait.) seedlings by increasing the water content, reducing the water deficit effect, providing plant hormones [59,60], and increasing the nutrient uptake of chamomile (Matricaria chamomilla L.) seedlings [61]. Another problem in semi-arid areas with regard to germination and seedling emergence is the maintenance of water around the seed for a sufficiently long time after spring and summer rains. The emergence rate was significantly affected by the interaction of water stress and the amount of hydrogel used in the OSC fillers (Table 3). The use of OSC fillers with polymer coatings increased the emergence rate by reducing the effect of water deficit (Table 3). Other researchers reported that incorporation of hydrogel polymer improved seed germination and emergence of wheat (Triticum aestivum L.) [23]. The addition of hydrogel to vermicompost (C2) and peat moss (C3) had a more positive effect on plant growth. The leaf area decreased with increasing water deficit, which is in agreement with the results of other researchers [62,63,64,65,66]. Water deficit reduces the RWC of the leaves (RWC) and reduces cell division [67], the transition between cell division and cell elongation results in reduced leaf area [1]. One of the most obvious physiological symptoms of soil water deficit is a decrease in leaf RWC [68]. With increasing amounts of hydrogel, shoot length (Table 4) and seedling length vigor index (Figure 3) increased. Seedling length vigor index increased from 24.76 at the first hydrogel level (no hydrogel cover) to 43.5 at 6 g hydrogel A200 per kg cover (S7) (Figure 3). Reduction in turgor pressure, cell division, and cell enlargement are the main reasons for stem length reduction under water deficit [9,12,62]. Other researchers have investigated the effect of hydrogel on seedling growth of Haloxylon [69]. They reported that the use of hydrogel had a significant positive effect on seedling height by increasing the plant’s chances of survival by maintaining the water supply around the plant’s roots in the soil [35]. Water deficit reduced root length. These results were consistent with those of other researchers on Quercus leucotrichophora [70], Thymus volgaris [44], and Tritipyrum lines [71]. Previous researchers have reported that root cell final lengths are smaller under water deficit [72] due to a decrease in root apical cell elongation under water stress [58]. The use of Hydrogel A200 increased soil water retention, reduced water deficit, and increased shoot and root length. Absorbent materials affect soil density, water, and air infiltration into the soil, increase water retention, and increase aeration and microbial activity. Other researchers investigated the effects of hydrogel on water storage in sandy loam soils and the growth of wheat, barley, and pea seeds and reported that adding 1, 2, and 3% hydrogel to the soil linearly increased soil water-holding capacity and delayed plant wilting point by 4–5 days [24]. Seedling weight vigor index, which is representative of the dry weight produced by the plant, decreased with increasing water deficit. Seedling weight vigor index was increased by organic matter (Figure 4) and hydrogel (Figure 5). Other researchers have shown that superabsorbent hydrogels increased the dry weight of Ocimum basilicum [18]. This is because lack of water in the soil causes a decrease in leaf photosynthesis, carbohydrate accumulation, and total dry matter in the plant [59,73]. The use of organic seed covers can enhance the activities of the antioxidant system and help plants cope with the oxidants produced under water deficit conditions (Table 2). When plants are exposed to biotic and abiotic stresses, a variety of reactive oxygen species (ROS) induce oxidative stress within the cells, which can lead to cell death [74]. In response to this condition, cells normally generate some kind of antioxidant defense, such as ascorbate peroxidase activity, peroxidase, and catalase. In this study, it was observed that the application of both OSC fillers can increase the efficiency of antioxidant defense in S. marianum seedlings. The same results were shown by researchers [75]. The application of organic seed coating was found to be a suitable and practical method for antioxidative stress under water shortage conditions [76]. An increase in proline content and antioxidants under water deficit indicates the tendency of S. marianum to survive even under limited moisture when successfully established but with the least growth. An increase in leaf proline content and enzymatic antioxidant activities is an index of the plant’s defense mechanism against abiotic stresses, including water deficit, and protects plant systems from oxidative damage [8,37,77]. The use of superabsorbent polymer caused a decrease in proline content in all moisture regimes (Table 2). This indicates that the application of organic seed cover helped plants to overcome stress by improving soil moisture availability. Growth and yield of essential oil per hectare in medicinal plants are affected by water deficit [78]. The effect of water deficit depends on the amount, duration, and rate of deficiency [62]. Water deficit via a decrease in leaf chlorophyll, an increase in proline content [39], catalase (CAT), ascorbate peroxidase (APX) decreased yield, oil, and silymarin extract (Table 1). However, the use of OSC fillers increased the percentage of extracts and oils under severe water deficit conditions (Table 2). The use of OSC fillers along with hydrogel F1 and A200 reduced the deleterious effects of water stress on germination and accelerated early growth of S. marianum for several reasons. The first is due to the increased water retention around the seeds caused by the organic filler seeds where the rainfall rate is low. Moreover, accelerated leaf formation and leaf area, shoot and root growth take place due to high moisture availability. In addition to maintaining relative water content, leaf chlorophyll content and accelerated seedling dry weight improve water uptake in the rhizosphere.

Bacterial application improves water relations with host plants in the rhizosphere and reduces the minor effects of water stress on morphophysiological parameters of the plant [40,79,80]. Seed inoculation with bacteria improved the seedling emergence percentage of S. marianum seeds under severe water deficit (Table 5). After seed inoculation with equal proportions of two bacteria, P. fluorescens and P. putida, leaf area was significantly increased under severe water deficit (Table 5). Water stress reduces cell division and leaf area [67]. Seed inoculation with Pseudomonas putida improves growth characteristics, including leaf number and leaf area index [81]. Decreased leaf relative water content (RWC) is one of the most obvious physiological symptoms of soil moisture deficiency [68]. Water deficit decreased RWC (Table 5). The use of bacteria protects plants against salinity and water deficit by controlling proline accumulation in leaves [82,83]. Water stress is one of the major abiotic stresses that prevents cell enlargement, reduces cell division, and affects uptake and transport [59], reducing growth and dry matter in plants. Seedling weight vigor index, as a representative of the dry weight produced by the plant, was decreased by increasing water deficit in a total of treatments (Table 5). Growth promoting bacteria release essential elements in the soil, make the host plant tolerant to pathogens, improve nutrient uptake, and thus increase plant biomass [4]. One of the most commonly used parameters to characterize the performance of the photosynthetic apparatus of plants under water deficit is chlorophyll [39]. Water deficit affects leaf chlorophyll and reduces its amount. Water deficit reduces root activity and ultimately nitrogen uptake, which can reduce leaf chlorophyll content [84]. Seed bacterial inoculation can prevent the deterioration of total chlorophyll in leaves under water deficit; thus, the chlorophyll content is maintained [31,85]. According to the basic reports, Pseudomonas increases the activity of enzymes such as CAT, and APX increases the level of leaf chlorophyll [22]. The effect of water deficit depends on the level of longevity and the rate of deficiency [70]. Water deficit via a reduction in leaf area, leaf chlorophyll relative water content (RWC) of leaves decreased yield, oil and silymarin extract. However, the use of bacteria reduced the percentage of extracts and oils at severe water deficit levels (Table 5). It is concluded that low water potential inhibits germination and emergence of S. marianum in arid and desert areas. The use of organic fillers in seeds along with hydrogel F1 and A200 and seed inoculation with combination of P. fluorescens and P. putida reduced the adverse effects of water stress on germination and accelerated the early growth of S. marianum for several reasons: The first is caused by the increased water maintenance around the seeds, by organic cover fillers seeds where the rainfall rate is low. Moreover, accelerated leaf formation and leaf area, shoot and root growth take place due to high moisture availability. In addition to maintaining RWC, leaf chlorophyll content, and accelerating the dry weight of seedlings through inoculation improve water absorption in the rhizosphere.

5. Conclusions

Our results showed that milk thistle seeds are sensitive to water deficit during germination and emergence. Water deficit at this stage delays the time of emergence and decreases the number of leaves, extract, and oil. Covering the milk thistle seeds with vermicompost (C2) and peat moss (C3) can increase the emergence rate, early growth, and extract and oil. Adding hydrogel to OSC fillers such as vermicompost (C2) and peat moss (C3) increases water retention around the seed during early growth. These further increase emergence rates and are appropriate methods of providing adequate moisture during the early stages of milk thistle growth in arid areas. Bacterial inoculation improved early growth, reduced the effects of water stress on the plant, and increased the number of leaves, extract, and oil of milk thistle in arid areas. It is recommended that both methods be used to accelerate the initial growth and rapid development of milk thistle seedlings to produce a greater yield of extract and oil under arid conditions. However, if possible, bacteria are much easier to use, although they are more expensive. These methods can be used for seedling establishment of other plants that have tiny seeds in arid regions.