Application of Silicon Iron and Silver Nanoparticles Improve Vegetative Development and Physiological Characteristics of Boysenberry Plants Grown under Salinity Stress In Vitro Cultivation Conditions

Abstract

Salinity is one of the most important abiotic stress factors that affect plant growth and limit agricultural productivity. In this study, the effects of iron (FeNP), silver (AgNP), and silicon dioxide (SiNP) nanoparticles on the morphological and physiological parameters of in vitro boysenberry plants grown under salinity stress (NaCl) were investigated. According to our study results, higher values were obtained from SiNP application in terms of shoot development parameters; FeNP application was found to be more successful for root development; AgNP application was effective in terms of SPAD, leaf relative water content (LRWC), and relative growth rate (RGR); and FeNP application increased superoxide dismutase (SOD) and catalase (CAT) enzyme activities. Salt stress significantly affected root development, SPAD values, LRWC and RGR, and SOD and CAT enzyme activities. As a result, under salt stress conditions, SiNP, FeNP, and AgNP applications can significantly reduce the negative effects of stress and promote the vegetative development of the plant compared to control conditions.

1. Introduction

Boysenberry, which is included in the order Rosales, the Rosaceae family, and the Rubus genus, is a shrub-shaped plant with high economic value that is traded all over the world due to its fruit’s pleasant taste and high nutritional value [1,2,3]. The cultivation of Boysenberry, which was obtained by crossing Rubus baileyanus and Rubus loganobaccus in the USA between 1921 and 1923, is increasing rapidly due to its suitability for fresh consumption and processing in the food industry, as well as its higher fruit quality compared to other blackberry varieties [4]. Boysenberry fruit is preferred by the food industry due to its strong aroma and appeal [5]. While boysenberry cultivation is a new agricultural production area for Turkey, it is produced at a tremendous rate in the world, especially in Mexico and the United States [6].

One of the most important problems in today’s agricultural production, salinity, also poses a serious problem in berry production, and many scientific studies in the literature have sought solutions to this problem [7,8]. Boysenberry is considered a salt-sensitive plant because of its ability to tolerate increased salt concentrations in the cytosol [9]. Salinity limits the yield and quality of agricultural products and causes morphological, biochemical, physiological, and molecular changes in the plant [10,11,12,13]. High salt concentrations in the growth medium restrict the water intake of plants, causing a loss of turgor, the closure of stomata, and a decrease in photosynthesis [14]. In addition, the detrimental effects of salt stress include dehydration; the inhibition of photosynthesis; the disruption of protein synthesis; nitrogen deficiency (chlorosis and necrosis); a slowing of plant growth; and the production of highly reactive oxygen species (ROS); as well as ionic, osmotic, and oxidative stress [15,16]. The accumulation of ROS in the cell induces antioxidative enzymes (such as CAT, SOD, nonspecific peroxidase (POD), and ascorbate peroxidase (APX)), significantly reducing superoxide and hydrogen peroxide levels, resulting in nonspecific oxidation of proteins and nucleic acids [9,17,18,19]. Such conditions can cause plant growth arrest, increased stress, and tissue death [8,17]. Although the salt tolerance of plants varies according to genetic differences, it can also be affected by environmental factors. Therefore, various methods have been used to prevent the negative effects of salinity. However, these methods are time-consuming and expensive [20]. Therefore, studies on applications that can increase plant tolerance to salt stress are gaining importance [21]. The rapid growth and development of nanotechnology have resulted in the rapidly increasing production and use of nanoparticles, and these technological products have begun to be used extensively in agricultural production [22]. The extensive amount of chemicals used, which cause various problems in agricultural production, can be reduced with NPs [23], and agricultural product productivity can also be increased [24,25,26]. NP applications in plants generally aim to increase the adaptation and resistance of plants to stress factors [27,28,29,30,31]. It has been demonstrated that the exogenous application of NPs to plants helps to regulate antioxidant system activity and cellular water balance in plants [27,32]. SiO₂ nanoparticles (SiNPs) are single particles of silica dioxide, which is an inorganic metal oxide with a diameter of less than 100 nm. The low-cost and easily produced SiO₂ form of Si, which is considered one of the most valuable elements for plant life, has an effective ability to increase resistance against diseases and stress factors in plants [33,34,35]. It has also been reported that SiO₂ is a plant growth inducer that strengthens the antioxidant system under salinity stress, increases silicification in the root endodermal layer, improves tolerance to abiotic stresses, and improves cell water balance [32]. Researchers have reported that fresh weight, chlorophyll concentration, photosynthesis rate, and leaf water content of plants exposed to salinity stress are affected by their use. They stated that Si application mitigated these effects in tomato plants and thus protected photosynthetic activity against the harmful effects of salinity [30]. The intake of Fe [36], which is a necessary and limiting micronutrient for plant growth, can be affected by many factors, such as soil and climatic conditions, fertilizer application, and plant growth period [37]. Therefore, the application of this element as an NP can increase the solubility and distribution of insoluble nutrients in the soil, reduce the inactivation of nutrients, and increase their bioavailability [38]. As FeNPs are a rich source of Fe, plants may be more useful because of the gradual release of Fe over a wide pH range [39,40]. In a study investigating the effects of different Fe sources on the growth, development, and yield parameters of wheat grown in normal and salt-affected soils, it was reported that the application of iron nanoparticles (FeNPs) and Fe sources (FeSO₄, Fe-EDTA) increased the yield of wheat exposed to salt stress and that both Fe sources improved the nutrient uptake of plants [40]. It has been shown that AgNPs [41,42,43], which are metallic nanoparticles, can be effective in promoting plant growth and development [27,44,45,46,47] and increasing the chlorophyll content and photosynthesis rate [27,44,48]. Some studies have reported that the application of NPs, such as silicon, is very effective in improving resistance to salinity in various plants [49,50]. Many different effects of Si applications have been evaluated in the literature. It is thought that the application of silver, a metallic nanoparticle, plays an important role in alleviating the effects of stress. Şener and Saygı [27] reported that AgNP application improved the vegetative development of boysenberry plants grown under drought stress and had a positive effect on their antioxidant system. Research is needed on the role of nanoparticles in improving plant tolerance to environmental stresses such as drought and salinity. Although there are many reports on the effects of NPs in vegetables and field crops and their microbiological activities [51,52,53,54,55], there are limited studies investigating their effects on berries, especially on boysenberry fruits. This study aimed to determine the effectiveness of some nanomaterials (SiNP, FeNP, AgNP) on the morphological and physiological properties of boysenberry plants grown under in vitro culture conditions and salinity stress (15 and 35 mM NaCl).

2. Materials and Methods

2.1. Materials

In the study, boysenberry was used as plant material, which was obtained by crossing Rubus baileyanus and Rubus loganobaccus. Certified saplings obtained from a commercial company were placed in the greenhouse, and explants from these plants were transferred to an in vitro culture. Sigma-Aldrich brand NPs were used as nanomaterials. SiNPs were used as a dry white powder (silica nanopowder) with a particle size of 10–20 nm, surface area of 590–690 m²/g, and purity of 99.8%. FeNPs (iron nanopowder) were used as black-colored and high-purity iron powders with a particle size of 35–45 nm, a bulk density of 0.5 g/cm³, and a true density of 7.9 g/cm³. AgNPs (silver nanopowder) with an average particle size of <10 nm, an average surface area of 10.0–15.0 m²/g, and a purity of 99.9% were used. Emission Scanning Electron Microscope (SEM) (ZEİSS-LEO 1430) images are shown in Figure 1.

2.2. Method

Two different concentrations of NaCl (15 and 35 mM NaCl) were used for salt stress application in the experiment. SiNP, FeNP, and AgNPs were applied at two different concentrations (7.5 and 15 mg L⁻¹ SiNP, 0.025 and 0.05 mM FeNP, and 0.2 and 0.4 mg L⁻¹ AgNP) [27,35]. The NaCl concentrations used in the experiment were determined based on the results of a preliminary study. Shoot tips and nodal cuttings taken from the boysenberry plant were kept in 70% ethanol for 1 min for surface sterilization, then passed through distilled water three times and sterilized for 15 min in a 20% sodium hypochlorite solution with a few drops of Tween 20 [56]. The explants were then rinsed thrice with distilled water in a sterile cabinet and transferred to the media. MS [57] basic medium composed of 3% sucrose and 0.7% agar was used as the nutrient medium, and the pH was adjusted to 5.8. Approximately 40 mL of medium was added to 190 cc glass jars and sterilized at 121 °C at 1.5 psi pressure for 20 min. Disinfected plant materials were cut in a sterile cabinet with a length of 0.5–0.7 cm, and a single seat bud was prepared for planting. The prepared plant materials were planted with four explants per jar. The plant materials taken into the environment were cultured for 4 weeks at 25 ± 1 °C, in a 16 h photoperiod, under a fluorescent lamp (30–35 μmol m⁻² S⁻¹) in the climate chamber.

2.2.1. NP Applications

Explants cultured in vitro for 4–5 weeks were then transferred to media containing different salt and NP concentrations, the contents of which are presented in

Table 1. Media used in the research.

Media	Applications
MS1	0 mM of NaCl
MS2	15 mM of NaCI
MS3	35 mM of NaCI
MS4	7.5 mg L−1 of SiNP
MS5	15 mM of NaCI + 7.5 mg L−1 of SiNP
MS6	35 mM of NaCI +7.5 mg L−1 of SiNP
MS7	15 mg L−1 of SiNP
MS8	15 mM of NaCI + 15 mg L−1 of SiO2 NP
MS9	35 mM of NaCI + 15 mg L−1 of SiO2 NP
MS10	0.025 mM of FeNP
MS11	15 mM of NaCI + 0.025 mM of FeNP
MS12	35 mM of NaCI + 0.025 mM of FeNP
MS13	0.05 mM of FeNP
MS14	15 mM of NaCI + 0.05 mM of FeNP
MS15	35 mM of NaCI + 0.05 mM of FeNP
MS16	0.2 mg L−1 of Ag
MS17	15 mM of NaCI + 0.2 mg L−1 of Ag
MS18	35 mM of NaCI + 0.2 mg L−1 of Ag
MS19	0.4 mg L−1 of Ag
MS20	15 mM of NaCI + 0.4 mg L−1 of Ag
MS21	35 mM of NaCI + 0.4 mg L−1 of Ag

. A total of 0.5 mL of BAP + 0.5 mL of IBA was also added to all of these media.

A probe-type sonicator was used to prepare NP stock suspensions. Solutions containing different doses of NPs were prepared using deionized water and sonicated for 30 min (Misonix, QSonica LLC., Newton, CT, USA) [58]. The deionized water in which the NPs were suspended was also used to prepare the MS. Two different doses of sodium chloride (15–35 mM of NaCl) were added to the solution prepared by adding 3% sucrose, 0.7% agar, and 0.5 mL of BAP + 0.5 mL of IBA (pH of 5.8).

Boysenberry plantlets were obtained via micropropagation under in vitro conditions in culture rooms at a temperature of 23 ± 1 °C, 16 h of daylight, 8 h of darkness, and a light intensity of 3000 lx. The research was conducted in three replicates, and 10 jars containing 1 plant were used for each repetition.

2.2.2. Morphological Parameters

Plantlets were cultured in the prepared nutrient media for 4–5 weeks; after that, shoot fresh weight (SFW) (g), shoot dry weight (SDW) (g), shoot length (SL) (mm), stem diameter (SD) (mm), root fresh weight (RFW) (g), root dry weight (RDW) (g), root length (RL) (mm), number of leaves (LN) (per plant), leaf width (LW) (mm), and leaf length (LL) (mm) were determined. To determine the dry weights of shoots and roots, the samples were placed in paper packages and kept in an oven at 70 °C for 48 h, and their weights were determined.

2.2.3. Physiological Parameters

Leaf relative water contents (LRWC%) were determined according to the method proposed by Sanchez et al. [59]. It was calculated using the following equation:

RWC = [(FW − DW)/(TW − DW)] × 100

FW: fresh weight, DW: dry weight, TW: turgor weight.

The SPAD index was measured using a chlorophyll content meter (SPAD–502, Konica Minolta Sensing, Inc., Tokyo, Japan).

Relative growth rate (RGR): plants were weighed in terms of total dry weight before exposure to salt stress (three true leaf stages) and after the stress period was completed, and the difference between the two measurements was divided by the number of days to determine the growth rates of varieties exposed to different treatments during the stress period in g dry weight/day [60].

Superoxide dismutase (SOD) and catalase (CAT) enzyme extraction was performed at 4 °C. Spectrophotometric analysis was conducted using a Shimadzu 2401 UV/visible-light spectrophotometer. Leaf samples used for the extract were stored at −80 °C. The extract was prepared by homogenization of a frozen 1 g leaf sample containing 0.1 M of phosphate buffer (pH of 7.8). The homogenate was centrifuged at 10,000 rpm for 10 min at 4 °C, and the supernatant was collected. Supernatants were used to assay for superoxide dismutase (SOD) and catalase (CAT).

Catalase activity (CAT) was measured spectrophotometrically by the disappearance of H₂O₂ by measuring the decrease in absorbance at 240 nm for 1 min due to H₂O₂ [61]. The reaction mixture contained 25 mM of phosphate buffer (pH 7.0), 10 mM of H₂O₂, and enzyme.

SOD activity was defined as measuring the inhibition in the photoreduction of nitroblue tetrazolium (NBT) by SOD enzyme [62]. The reaction mixture contained 50 mM sodium phosphate buffer (pH 7.8), 0.66 mM EDTA, 10 mM L-methionine, 33 µM NBT, 3.3 µM riboflavin. For the SOD reaction, absorbance was recorded at 560 nm after incubating the mixture at room temperature for 15 minutes under white light.

2.2.4. Statistical Analysis

The study data were evaluated in the “SPSS 23.0” statistical program. Differences between applications were evaluated using the Duncan Multiple Comparison test at p ≤ 0.05 significance level.

3. Results

In this study, we examined the effects of different doses of NaCl and NP on the physiology and morphological characteristics of boysenberry plants under in vitro growth conditions. To determine the effect of the application, the parameters SFW, SDW, SL, SD, RFW, RDW, RL, NL, LW, LL; chlorophyll index (SPAD); LRW; RGR; SOD; and CAT activity of the boysenberry plants were examined. Within the scope of the study, the significant differences between the applications were evaluated at the p ≤ 0.05 significance level in the Duncan Multiple Comparison test, and the results are presented in the tables below.

3.1. Vegetative Growth Parameters

The data obtained regarding shoot development are presented in

Table 2. Shoot growth parameters of the boysenberry plants grown in vitro conditions containing different types and concentrations of NP and NaCl applications.

NP Applications	NaCl Concentrations	SFW (g)	SDW (g)	SL (mm)	SD (mm)
0 mg L−1 of NP	0 mM	0.423 cd	0.137 b	33.62 b	0.95 b
	15 mM	0.617 abcd	0.186 ab	40.42 ab	1.06 ab
	35 mM	0.526 bcd	0.258 ab	29.56 b	1.12 ab
7.5 mg L−1 of SiNP	0 mM	0.614 abcd	0.137 b	46.14 ab	1.21 ab
	15 mM	0.487 bcd	0.180 ab	51.30 ab	0.83 b
	35 mM	0.610 abcd	0.230 ab	51.71 ab	2.20 ab
15 mg L−1 of SiNP	0 mM	0.651 abc	0.356 ab	52.19 ab	2.46 a
	15 mM	0.537 bcd	0.311 ab	47.39 ab	1.65 ab
	35 mM	0.376 d	0.250 ab	48.88 ab	1.60 ab
0.025 mM of FeNP	0 mM	0.516 bcd	0.132 b	35.19 b	1.39 ab
	15 mM	0.841 a	0.446 a	35.01 b	1.29 ab
	35 mM	0.522 bcd	0.355 ab	39.09 ab	1.19 ab
0.05 mM of FeNP	0 mM	0.688 ab	0.453 a	49.46 ab	1.18 ab
	15 mM	0.565 bcd	0.293 ab	29.78 b	1.09 ab
	35 mM	0.477 bcd	0.328 ab	27.08 b	1.29 ab
0.2 mg L−1 of AgNP	0 mM	0.537 bcd	0.168 b	29.41 b	1.60 ab
	15 mM	0.519 bcd	0.232 ab	61.10 a	1.14 ab
	35 mM	0.572 bcd	0.136 b	42.47 ab	1.42 ab
0.4 mg L−1 of AgNP	0 mM	0.478 bcd	0.287 ab	44.83 ab	1.07 ab
	15 mM	0.528 bcd	0.176 ab	46.14 ab	1.24 ab
	35 mM	0.520 bcd	0.120 b	36.88 ab	1.73 ab

SFW: shoot fresh weight, SDW: shoot dry weight, SL: shoot length, SD: shoot diameter. Each value is the mean of 3 replicated samples of 10 plants each. For each factor, values in a column followed by the same letter are not significantly different according to the Duncan test; significance is at p < 0.05.

. When these data were examined, the findings were statistically significant (p ≤ 0.05). The obtained data were evaluated, and it could be said that NP application had a significant effect on the shoot development of boysenberry plants grown in vitro. The highest SFW (0.841 g) value was obtained with the application of 0.025 mM of FeNP + 15 mM of NaCl, and the lowest SFW (0.376 g) was obtained with the application of 15 mg L⁻¹ of SiNP + 0 mM of NaCl. Similarly, the highest SDW values (0.453 g and 0.446 g) were determined for FeNP applications. On the other hand, it is possible to say that the most significant effect on SL was made by the application of 0.2 mg L⁻¹ of AgNP + 15 mM of NaCl (61.10 mm). When the effects of applications on the plant SD were examined in numerical terms, the highest plant SD was obtained from the application of 0 mM of NaCl + 15 mg L⁻¹ of SiNP (2.46 mm), and the lowest plant stem diameter was obtained from the application of 15 mM of NaCI + 7.5 mg L⁻¹ of SiNP.

As seen in Figure 2, while there was no statistically significant difference between NP applications on SFW, in terms of other parameters, it was seen that NP applications and their different doses affected the shoot development of boysenberry. According to the obtained data, it can be said that the SiNP application was significantly effective in increasing the shoot length, stem diameter, and shoot dry weight. In addition, the FeNP application was effective in increasing the shoot dry weight. Salt stress is known to negatively affect shoot development. Although it can be said that the development of boysenberry plants grown in vitro under different salt concentrations was generally negatively affected as a result of our study, it is also shown in Figure 2 that this effect of salt stress applied at 15–35 mM concentrations was not statistically significant.

In our study, we also examined the effects of salt stress and NP application on the root development of in vitro plantlets. According to our study results, when the root development data in

Table 3. Root growth parameters of boysenberry plants grown in vitro conditions containing different types and concentrations of NP and NaCl applications.

NP Applications	NaCl Concentrations	RFW (g)	RDW (g)	RL (mm)
0 mg L−1 of NP	0 mM	0.495 abcde	0.027 cde	15.64 ef
	15 mM	0.483 abcde	0.029 cde	15.76 ef
	35 mM	0.103 f	0.021 de	11.09 ef
7.5 mg L−1 of SiNP	0 mM	0.513 abcd	0.090 b	47.16 ab
	15 mM	0.429 abcde	0.035 cde	11.87 ef
	35 mM	0.371 cde	0.041 cde	8.50 f
15 mg L−1 of SiNP	0 mM	0.475 abcde	0.066 bcd	29.28 bcde
	15 mM	0.508 abcd	0.045 cde	19.47 def
	35 mM	0.402 bcde	0.018 e	30.10 bcde
0.025 mM of FeNP	0 mM	0.355 de	0.099 ab	36.89 abcd
	15 mM	0.613 a	0.088 b	54.07 a
	35 mM	0.552 abc	0.015 e	14.34 ef
0.05 mM of FeNP	0 mM	0.596 bc	0.135 a	15.56 ef
	15 mM	0.587 ab	0.060 bcde	36.11 abcd
	35 mM	0.489 abcde	0.069 bc	41.95 abc
0.2 mg L−1 of AgNP	0 mM	0.529 abcd	0.043 cde	21.80 def
	15 mM	0.519 abcd	0.057 bcde	19.37 def
	35 mM	0.347 de	0.070 bc	20.72 def
0.4 mg L−1 of AgNP	0 mM	0.483 abcde	0.042 cde	24.28 cdef
	15 mM	0.452 abcde	0.040 cde	30.53 bcde
	35 mM	0.313 e	0.044 cde	28.20 bcde

RFW: root fresh weight, RDW: root dry weight, RL: root length. Each value is the mean of 3 replicated samples of 10 plants each. For each factor, values in a column followed by the same letter are not significantly different according to the Duncan test; significance is at p < 0.05.

were examined, the findings were found to be statistically significant (p ≤ 0.05), and it was determined that there were statistically significant differences between the applications. In the data obtained, the highest average values in terms of root development among all applications were determined in the 15 mM of NaCl + 0.025 mM of FeNP application (RFW 0.613 g, RL 54.07 mm) and 0 mM of NaCl + 0.05 mM of FeNP application (RDW 0.135 g). The highest average value in terms of RFW was determined to be 0.613 g (15 mM of NaCl + 0.025 mM of FeNP), and it was found that there was a difference of approximately six times between this value and the lowest average value (35 mM of NaCl + 0 mg L⁻¹ of NP; 0.103 g) obtained from plants without NP application. A similar situation was observed in the RL parameter (Table 3).

When the graphs in Figure 3 were examined, the following situation was observed: salt stress negatively affected the root development of boysenberry plants grown under in vitro conditions. SiNP, FeNP, and AgNP added to the growth medium under salt stress can eliminate the negative effects of this stress and, at the same time, promote root development of the plant compared to control conditions. In this study, the effects of different concentrations of NPs on root development were compared. As a result, it was determined that 0.05 mM of FeNP application added to the medium was significantly effective compared to other NPs. It was also found that the application of 35 mM of salt to the medium stressed the plants and that the presence of salt at these concentrations weakened the root development of the plants. The lowest RFW (0.368 g) and RDW (0.040 g) mean values were found in plants treated with 35 mM of salt.

The development of leaves that meet the nutrient requirements of plants through photosynthesis is an important criterion among the plant growth and development parameters. In

Table 4. Leaf development of boysenberry plants grown in vitro conditions containing different types and concentrations of NP and NaCl applications.

NP Applications	NaCl Concentrations	NL (per Plant)	LL (mm)	LW (mm)
0 mg L−1 of NP	0 mM	8.75 cdef	14.85 abc	12.85 ab
	15 mM	13.25 bcde	7.19 bcd	6.43 bc
	35 mM	4.50 f	5.34 e	5.21 c
7.5 mg L−1 of SiNP	0 mM	12.50 bcdef	11.44 abcd	11.54 abc
	15 mM	17.50 ab	13.26 abcd	11.75 abc
	35 mM	12.75 bcdef	10.66 abcd	10.60 abc
15 mg L−1 of SiNP	0 mM	10.75 bcdef	14.56 abc	13.26 ab
	15 mM	12.75 bcdef	13.66 abc	10.75 abc
	35 mM	17.25 abc	12.75 abcd	11.62 abc
0.025 mM of FeNP	0 mM	23.25 a	14.24 abc	13.34 ab
	15 mM	17.50 ab	13.82 abc	11.93 abc
	35 mM	14.50 bcd	10.43 bcd	11.59 abc
0.05 mM of FeNP	0 mM	10.75 bcdef	6.61 cd	7.58 abc
	15 mM	11.00 bcdef	12.46 abcd	11.84 abc
	35 mM	13.75 bcde	14.40 abc	11.89 abc
0.2 mg L−1 of AgNP	0 mM	8.25 def	12.32 abcd	10.65 abc
	15 mM	9.00 bcdef	18.70 a	14.46 a
	35 mM	9.00 bcdef	9.97 bcd	9.70 abc
0.4 mg L−1 of AgNP	0 mM	11.50 bcdef	10.14 bcd	9.08 abc
	15 mM	5.25 ef	15.37 ab	11.55 abc
	35 mM	13.50 bcde	14.45 abc	11.77 abc

NL: number of leaves, LL: leaf length, LW: leaf width. Each value is the mean of 3 replicated samples of 10 plants each. For each factor, values in a column followed by the same letter are not significantly different according to the Duncan test; significance is at p < 0.05.

, the NL, LL, and LW data of the boysenberry plants of the applications are presented. Based on our findings, we determined the highest number of leaves per plant in the 0 mM of salt + 0.025 mM of FeNP application (23.25 per plant). The lowest value (4.50 per plant) was determined in the 35 mM of salt + 0 mg L⁻¹ of NP application, where no NP application was applied and 35 mM of salt was applied. As a different result from the findings we obtained using the criterion of the NL per plant, the highest average values in terms of LL (18.70 mm) and LW (14.46 mm) parameters were determined in the 15 mM of salt + 0.2 mg L⁻¹ of AgNP application. However, as in the NL criterion, the lowest values in these parameters were determined in the plants without NP application (LL; 35 mM of salt + 0 mg L⁻¹ of 5.34 mm NP, LW; 35 mM of salt + 0 mg L⁻¹ of 5.21 mm NP).

Leaf growth and development data obtained as a result of the study belonging to NP and salt stress applications are shown in Figure 4. According to the results obtained, statistically significant differences were detected between NP applications at the p ≤ 0.05 significance level. As shown in Figure 4, the effects of different NPs on the number of leaves of plantlets in vitro were evaluated, and it was determined that the best result was obtained with the 0.025 mM of FeNP application (18.43 per plant). Among the averages of LL and LW measurements performed to determine the size of the leaves, the highest values were obtained as a result of SiNP application. Although it is seen in the graph in Figure 4 that salt applications at different concentrations had a negative effect on the leaf development of plantlets, it was determined that this difference was not significant.

3.2. Physiological Parameters

In

Table 5. Physiological growth parameters of boysenberry plants grown in vitro conditions containing different levels of NaCl and NPs.

NP Applications	NaCl Concentrations	SPAD Value	LRWC (%)	RGR (%)
0 mg L−1 of NP	0 mM	65.35 cd	46.05 g	0.011 ef
	15 mM	87.48 ab	32.06 ı	0.008 fgh
	35 mM	55.04 d	35.23 hı	0.006 h
7.5 mg L−1 of SiNP	0 mM	69.16 cd	58.33 ef	0.020 d
	15 mM	63.98 cd	92.87 a	0.008 fgh
	35 mM	58.62 d	63.92 d	0.008 fgh
15 mg L−1 of SiNP	0 mM	58.52 d	54.35 ef	0.024 c
	15 mM	58.30 d	52.71 f	0.011 ef
	35 mM	72.36 bcd	45.67 g	0.007 gh
0.025 mM of FeNP	0 mM	78.70 abc	62.83 d	0.013 e
	15 mM	64.95 cd	78.89 c	0.010 efg
	35 mM	63.12 cd	53.33 e	0.006 h
0.05 mM of FeNP	0 mM	65.81 cd	52.74 f	0.007 gh
	15 mM	57.58 d	32.25 ı	0.011 ef
	35 mM	56.73 d	44.95 g	0.008 fgh
0.2 mg L−1 of AgNP	0 mM	69.85 cd	52.59 f	0.056 a
	15 mM	57.03 d	77.42 c	0.034 b
	35 mM	77.25 abc	36.75 h	0.013 e
0.4 mg L−1 of AgNP	0 mM	69.20 cd	84.71 b	0.025 c
	15 mM	89.63 a	65.17 d	0.010 efg
	35 mM	65.00 cd	64.60 d	0.013 e

LRWC: leaf relative water content, PGR: relative growth rate. Each value is the mean of 3 replicated samples of 10 plants each. For each factor, values in a column followed by the same letter are not significantly different according to the Duncan test; significance is at p < 0.05.

, the SPAD value, LRWC, and RGR of the boysenberry plants of the applications are presented. One of the questions sought to be answered within the scope of this research was whether salt stress affects the chlorophyll content of plantlets in vitro, the water content of leaves, and the growth rate of plants. We also tried to determine whether the negative effects, if any, caused by salt stress could be eliminated with different NP applications. When the data in Table 5 were examined, the following situation was observed: NP application of different types and concentrations to plants under salt stress had a significant effect on the SPAD, LRWC, and RGR content of plantlets. Among all applications, the highest SPAD value (89.63) was determined in the 15 mM of salt + 0.4 mg L⁻¹ of AgNP application, the highest LRWC value was determined in the 0 mM of slat + 7.5 mg L⁻¹ of SiNP (92.87) application, and the highest RGR value was determined in the 0 mM of salt + 0.2 mg L⁻¹ of AgNP (0.056) application.

One of the aims of the study was to determine the superiority of different NPs over each other in eliminating the effects of salt stress, which is an important abiotic stress factor. If we compare different types of NP applications, it can be seen in Figure 5 that FeNP and AgNP applications are more successful than SiNP and control in terms of increasing the SPAD value. A similar situation occurs when we examine the RGR parameter. The 0.2 mg L⁻¹ AgNP application was the application that obtained the highest average value (0.034) compared to all other applications. When the situation is examined in terms of LRWC, it is seen in Figure 5 below that the 0.4 mg L⁻¹ AgNP application is successful in increasing the average value. However, there is a difference here, which is that the 7.5 mg L⁻¹ SiNP (71.71) application and the 0.4 mg L⁻¹ AgNP (71.49) application give similar average results in this parameter and are statistically in the same group (Figure 5).

3.3. Biochemical Parameters

At the end of the study, CAT and SOD activity levels were determined in the leaves of plants obtained from all applications. According to our study results, when the data of the CAT and SOD enzyme activity of the boysenberry plants in

Table 6. CAT and SOD enzyme activity of boysenberry plants grown in vitro conditions containing different types and concentrations of NP and NaCl applications.

NP Applications	NaCl Concentrations	CAT Activity (U/g TA)	SOD Activity (U/g TA)
0 mg L−1 of NP	0 mM	16.81 h	59.20 h
	15 mM	17.42 bcd	96.34 fgh
	35 mM	17.67 ab	101.54 fgh
7.5 mg L−1 of SiNP	0 mM	17.27 cdef	89.19 fgh
	15 mM	16.98 fgh	146.07 defg
	35 mM	17.43 bcd	200.22 bcd
15 mg L−1 of SiNP	0 mM	16.86 gh	53.93 h
	15 mM	17.57 abc	263.49 ab
	35 mM	17.45 abcd	61.85 h
0.025 mM of FeNP	0 mM	17.44 bcd	94.61 fgh
	15 mM	17.37 bcde	101.71 fgh
	35 mM	17.45 abcd	68.23 gh
0.05 mM of FeNP	0 mM	16.99 fgh	112.98 efgh
	15 mM	17.42 bcd	162.75 cdef
	35 mM	17.82 a	311.55 a
0.2 mg L−1 of AgNP	0 mM	17.38 bcde	88.04 fgh
	15 mM	17.23 cdefg	239.31 abc
	35 mM	17.50 abcd	184.79 bcde
0.4 mg L−1 of AgNP	0 mM	17.03 efgh	198.63 bcd
	15 mM	17.13 defgh	155.70 def
	35 mM	17.19 cdefg	212.81 bcd

Each value is the mean of 3 replicated samples of 10 plants each. For each factor, values in a column followed by the same letter are not significantly different according to the Duncan test; significance is at p < 0.05.

were examined, statistically significant differences were determined between the applications at the significance level of p ≤ 0.05. For CAT and SOD, enhanced activities were seen in all plants grown under salinity stress conditions and treated with NP. Among all applications, the highest values for both CAT (17.82 U/g TA) and SOD (311.55 U/g TA) activity were reached in the 0.05 mM of FeNP + 35 mM of NaCl application. A striking and expected situation in Table 4 was that enzyme activities increased with increasing salt application levels in almost all applications. The lower values were obtained from the 0 mM NaCl application without salt application. The lowest CAT activity values among all applications were determined in the 0 mg of NP + 0 mM of NaCl (16.81 U/g TA) application.

According to our study results, when the data of the plant CAT and SOD enzyme activity in Figure 6 were examined, statistically significant differences were determined between the applications at the significance level of p ≤ 0.05.

Within the scope of the study, the effects of different NP types and their use on different doses on CAT and SOD enzyme activities of boysenberry plants grown in vitro under salt stress conditions were evaluated. At the same time, whether salt stress affected these enzyme activities was also the subject of the study. The enzyme activity data obtained from the study are given in Figure 6. When the graphs in Figure 6a are examined separately, it is seen that the most important applications that caused the increase in CAT enzyme activity among different NP applications are 0.025 mM and 0.050 mM FeNP and 0.2 mg AgNP applications.

In the SOD enzyme activity data, the effects of 0.050 mM FeNP and 0.2 mg AgNP applications were also found to be significant. However, here, the effectiveness of the 0.025 mM FeNP application was lower, while the 0.4 mg AgNP application was found to be significantly effective (Figure 6c). The effects of salt stress on enzyme activities were investigated, and as a result, the highest CAT and SOD values were determined as a result of the analysis made from the leaves of plants with 35 mM of NaCl applied (Figure 6b,d).

4. Discussion

Within the scope of the study, the effects of applications on boysenberry plant growth parameters, which are the fresh shoot weight; fresh root weight; shoot length; root length; stem diameter; leaf number; leaf length; leaf width; dry stem weight; dry root weight; chlorophyll index; LRWC, RGR, and SOD activity; and CAT activity, were evaluated.

4.1. Vegetative Growth Parameters

Shoot development is an important indicator of plant growth and production. Turhan and Eriş [63] examined the effects of NaCl applications on two different strawberry cultivars grown under greenhouse conditions and reported that the fresh root weight was affected by NaCl applications, and 17 and 34 NaCl applications reduced the fresh shoot weight. Kalteh et al. [50], in their study investigating the effects of Si NPs on the basil plant, reported that different NaCl concentrations reduced the shoot weight. In the findings obtained in our study, it was determined that there were significant increases in shoot development with FeNP and SiNP applications (Table 2). In the study conducted by Abdoli et al. [64], the responses of Ajowan (Trachyspermum ammi L.) to exogenous salicylic acid and iron oxide nanoparticles under salt stress were investigated, and at the end of the study, it was reported that exogenous applications improved root and shoot growth, similar to our findings. Yavuzlar et al. [65] investigated the effects of different glycine concentrations on shoot growth in explants at different NaCl levels in vitro cultivation and reported that salt concentrations had an effect on the shoot length and reduced the shoot length with NaCl concentrations. Mirmazlum et al. [66] investigated the effects of exogenous melatonin application in different salinity environments and concentrations on buttercup plants and reported that salinity stress had a negative effect on the growth parameters of buttercup plants and reduced the shoot length. Şener and Kurt [56], in their study examining the effect of IBA and BAP applications on the micropropagation of the shoot tip under in vitro conditions in boysenberry plants, reported the highest stem diameter of 1.44 mm. This value is lower than our study findings due to the difference in plant nutrients. Haghighi and Pessarakli [49] reported that the plant stem diameter decreased with the addition of NaCl and that salinity had detrimental effects on plant stem diameters. However, there were no significant differences between 25 and 50 mM NaCl applications in their research using a hydroponic growing system, in which they examined the curative effects of different levels of Si and nanosilicon on plant growth in cherry tomatoes under NaCl stress. However, our findings showed that different NP applications had a positive effect on the plant stem diameter. In addition, similar to our study, a decrease in plant stem diameter was observed in NaCl dose compared to control plant groups. The dry weight can be a very useful and reliable indicator to evaluate the results of any application carried out to increase crop yield or quality in the agricultural production process [67]. Similar to our study findings, Azimi et al. [68] examined the effects of six different concentrations of SiO₂ nanoparticles and three seed pre-cooling treatments on the germination and growth of wheatgrass and reported that SiO₂ nanoparticle applications increased the shoot dry weight in wheatgrass. Tahir et al. [69], in their study examining the effects of silicon application in salinity and stress growing medium on the growth and ionic composition of different wheat genotypes, reported that the addition of silicon to the nutrient solution increased the amount of shoot dry matter in all genotypes. Koçak [70], in his study examining the determination of salt tolerance levels of 32 local green bean genotypes, reported that the shoot dry weight decreased in all genotypes compared to the control. Şener et al. [35] reported that the shoot development of strawberry plants grown in vitro under drought stress could be increased via SiNP applications. The positive effects of NP applications on plant development have been reported in various studies. NPs, which can penetrate small systems owing to their properties, can reach distant regions within the plant to trigger different biochemical and physiological pathways [71]. Plants are often exposed to stress factors such as salinity, drought, high and low temperatures, and heavy metals in the environments in which they are grown. This can trigger cell toxicity, leading to the breakdown of macromolecules and disruption of the membrane structure. Reactive oxygen species, known for their growth-inhibiting properties, proliferate under stress conditions, resulting in cytotoxicity and genotoxicity [72]. It has also been suggested that nanoparticles significantly affect the production of bioactive compounds in different plant and plant cell cultures and therefore can be used as standard elicitors in plant cell, tissue, and organ cultures to produce valuable metabolites [73]. In this study, the effect of NPs on shoot development in the micropropagation of berries, whose propagation by tissue culture is becoming more widespread daily, was also investigated. When the obtained results were evaluated, it was determined that the use of different types and concentrations of NPs, in line with the information in the literature, stimulated the shoot development of boysenberry grown in vitro. As a result of comparing different NPs with each other, it was determined that SiNPs were more successful in increasing SD, SDW, and SL in vitro. Silicon (Si), which is not included in the category of essential elements of higher plants and whose effects have not yet been fully understood, is considered one of the most beneficial elements for plant life [74]. In some studies, it is reported that Si application increases growth and yield by improving plant water status, changing the ultrastructure of leaf organelles, activating plant defense systems, and reducing free radicals [35,75,76,77].

Plants need a strong root structure to live healthily [78]. Roots, as in all plants, are the most important organs of the plant that take the water and nutrients required for plant growth and yield from the soil or media [78]. Hwang et al. [79] consider that high-quality planting of strawberries should be performed with well-grown and well-developed plant roots and is valid for all plant production processes. Similar to our findings, it has been reported to have inductive effects on plant growth and development, such as shoot/root development, seed germination, biomass production, and physiological/biochemical activities [80]. Turhan and Eriş [63], in their study examining the effects of NaCl application on two different strawberry cultivars grown under greenhouse conditions, reported that the fresh root weight was affected by NaCl application, and 17 and 34 mM NaCl applications reduced the fresh root weight. Haghighi and Pessarakli [49] investigated the ameliorative effects of different levels of Si and nanosilicon on plant growth in cherry tomatoes under salt stress using a hydroponic cultivation system. They reported that the fresh root weight and root volume were significantly affected by stress, and the lowest values were obtained with 25 mM NaCl applications. They also reported that silicon positively contributed to the fresh shoot weight in applications at 25 mM NaCl levels [49]. Siddiqui et al. [32] investigated the effects of Na SiO₂ on the growth development of zucchini plants grown under salt stress conditions and reported that Na SiO₂ alleviated the negative effects of salinity, improved the growth characteristics of pumpkin plants, and increased the fresh root weight. Badawy et al. [81] studied the effects of nanoparticle (Si and Se) applications on two different rice varieties grown in saline conditions and reported that the root length decreased significantly as a result of the high inhibitory effect of NaCl. Salih et al. [82] investigated the effects of biogenic AgNPs on parameters such as the germination rate and growth of tomatoes and reported that Ag-containing nanoparticles had a significant effect on the development of stem and root systems. Regarding the root dry weight, Azimi et al. [68] reported that SiO₂ nanoparticle applications increased the root dry weight in wheatgrass. Similarly, Tahir et al. [69] reported that the addition of Si to the salinity stress growing medium increased the shoot dry matter in all genotypes. Siddiqui et al. [32] reported that Na SiO₂ improved the growth characteristics of zucchini plants by alleviating the negative effect of salinity in their study examining the curative effect of Na SiO₂ under salt stress conditions in zucchini plants. In another study, Doğan [83] examined the effect of humic acid and silicon applications on eliminating the negative effects of heavy metals on strawberry cultivation and reported that humic acid and silicon applications had positive effects on the dry root weight of plants. According to the data obtained from our study, NP application was able to protect the root development of boysenberry plants grown under in vitro conditions due to the negative effects of salt stress. NP applications resulted in significant increases compared to the control in all parameters examined to monitor root development. In the present study, we determined that the use of FeNP had the best effect on the root development of plants grown under salt stress under in vitro conditions. Leaves are important organs that ensure plant survival; therefore, the leaf number, leaf width, leaf length, chlorophyll index, and LRWC parameters are important indicators of healthy plant growth [84]. Şener and Kurt [56] reported the highest leaf number (48.20 mm) in boysenberry plants. This value was higher than our study findings due to the differences in plant nutrients. Kalteh et al. [50], in their study examining the effect of silicon nanoparticle applications on salinity stress in basil, reported that silicon nanoparticle applications increased the amount of photosynthesis of the plant by decreasing the salinity stress, increasing the leaf number and leaf area. Bora [85], in his research examining the effects of NaCl stress applications on the Jalapeno pepper cultivar in a hydroponic growing system, reported that the lowest number of leaves was obtained with NaCl applications. Similar results were obtained in this study. Bernal et al. [86] studied the effect of paclobutrazol and AgNPs on the in vitro regeneration of potato (Solanum tuberosum L.) and reported that the leaf number, root number, stomata density, and chlorophyll content increased when PAC was combined with AgNPs compared to when PAC was used alone. Avestan et al. [87] reported that NaSiO₂ application to strawberries reduced the negative effects of salinity and improved plant vegetative growth. Regarding the leaf area (length and width), Şener and Kurt [56] reported the highest leaf length of 14.09 mm and the highest leaf width of 11.72 mm in the boysenberry plant. These values were lower than our study findings due to the difference in plant nutrients. Akhoundnejad et al. [88], in their study examining the effects of different doses of AgNP applications on the growth and development of onion plants, reported that the leaf width was higher than the control, while the leaf length was lower. Iqbal et al. [51] reported that different doses of AgNP applications in wheat significantly increased the leaf area. In their study, Abou-Shlell et al. [89] examined the effect of foliar application of different growth-promoting nanoparticles at different concentrations of salinity on the growth, biochemical, and anatomical properties of Moringa oleifera L. plants and reported that FeNP applications increased the leaf area. In our study, the effect of salt stress on NL, LL, and LW was not significant. It was expected that the effect of stress on this green part of the plant, which contains a large amount of water, would be significant. Numerical differences were detected between the average values. The effects of stress may not have been measured significantly due to reasons such as the fact that the study was conducted with in vitro conditions, was more controlled than in vivo conditions, and had a shorter duration. One of the hypotheses of this study was that NPs alleviate the effects of stress and have a positive effect on plant growth and development. These findings support our hypothesis. The use of different concentrations of FeNP and SiNP significantly affected the leaf development of boysenberry plants grown under in vitro conditions. A striking point in the findings is that AgNPs fell behind FeNPs and SiNPs. The inhibitory properties of AgNPs have been reported in several studies [90,91,92]. When the data obtained from our study were evaluated, it would be incorrect to mention the inhibitory properties of AgNPs. For some parameters, lower average values were recorded for AgNP applications than for other NPs or the control application. However, because the study was conducted under in vitro cultivation conditions and on a plant from a botanically different family and species, the results obtained are more diverse and cannot be directly compared with the results of other studies. This indicates that the same nanoparticles have a multifaceted effect.

4.2. Physiological Parameters

Regarding the chlorophyll index (SPAD value), Al-aghabary et al. [93] investigated the effects of different nanoparticle applications and salt stress on the chlorophyll content, chlorophyll fluorescence, and malondialdehyde concentration in tomatoes and reported that salt stress significantly reduced the chlorophyll content, but added SiNP increased the chlorophyll content under salt stress 10 days after the application. Similar to our findings, Kuşvuran et al. [94], in their study examining the changes in the amount of Na⁺, K⁺, and Cl⁻ ions and the amount of chlorophyll in Cucumis genotypes treated with 100 mM of NaCl, reported a decrease in the amount of chlorophyll in plant leaves under NaCl stress. Ghafariyan et al. [39] examined the effects of FeNP applications in soybeans on chlorophyll variations and the possibilities of reducing iron deficiency in soybeans and reported that even low concentrations of FeNP treatments under hydrophobic conditions significantly increased the chlorophyll content in soybean leaves. In another study, Bora [85] examined the effects of different salt concentrations applied during different vegetation periods on the physiological, morphological, and chemical properties of pepper plants and reported that NaCl stress applications in pepper plants decreased chlorophyll values. These research results are similar to the NaCl applications in our study findings, which show that the negative effect of salt stress reduces AgNPs and FeNPs. Another study with similar results was conducted by Gökçe [95]. Gökçe, in his study examining the effects of AgNPs on tomato seeds, determined that the increase in AgNP concentrations was statistically significant regarding pigment values [95]. LRWC is an important indicator of leaf water status and reflects the balance between water uptake and transpiration rate in plants [96]. Fan et al. [97] evaluated the effects of Si and K applications on plant growth and ion-selective absorption under saline-alkali stress at different concentrations in ryegrass and reported that Si and K applications increased LRWC. Zahedi et al. [98], in their study examining the effects of drought on plant growth and fruit yield of SiO₂ and Se nanoparticle applications in strawberries, reported that SiO₂ and Se nanoparticle applications increased the LRWC. Bora [85], in his study examining the effects of NaCl stress applications on the Jalapeno pepper cultivar in the hydroponic growing system, reported that LRWC at a 0 mM dose was 96.87%, whereas LRWC at a 100 mM dose decreased to 67.29% in NaCl stress applications. Similarly, according to our study findings, LRWC decreased under the effect of NaCl stress, but the effect of stress was alleviated by NP application. NP application can lead to changes in the transcription of genes involved in plant hormone biosynthesis or signal transduction [45,99]. Transcriptomic studies have revealed that the expression of genes related to abiotic and biotic stress responses is also increased by nanoparticle application [100,101]. In some cases, various NPs can increase the activity and expression of phytohormone-related genes, such as ABA and cytokinin, which promote root growth. Thus, water and nutrient uptake by plants can be accelerated and increased [102,103,104]. RGR is an important indicator used to evaluate plant growth performance and productivity and is an important determinant of plant competitiveness [105]. Abou-Shlell et al. [89] reported that NP applications (Zn, Fe, Cu, and Si) decreased the negative effects of the salty environment and improved vegetative growth in Moringa oliefera L. Siddiqui et al. [106], in their literature review study to emphasize the key role of nanoparticle applications in plants, reported that the effect of NPs varies from plant to plant, depending on the application concentration and shape. Accordingly, they reported that SiNPs increased germination and antioxidant activity in different plants, Zn NPs increased plant growth and development, and AgPNs increased plant growth. In various studies, the inhibitory effects of AgNPs, which are metal-based NPs, have been mentioned [90,91,92]. The results obtained in our study are the opposite, indicating that AgNPs promote growth and development. It is thought that this is because AgNPs, when compared to AgNO₃, release ions (Ag⁺) that can block the ethylene receptor by changing the copper ion cofactor of the ethylene binding site [86]. According to Elatafi and Fang [107], AgNPs have a larger surface area-to-volume ratio than other forms of silver, such as AgNO₃, which can make them more effective. The ions (Ag⁺) of AgNPs, which have lower toxicity effects, can inhibit the formation of 1-aminocyclopropane-1-carboxylic acid and thus the effect of ethylene.

4.3. Biochemical Parameters

Antioxidants (such as SOD and CAT) are the first defense blocks against free radical damage caused by biotic and abiotic environmental factors that affect plant growth, development, and productivity. NP application often results in increased levels of reactive oxygen species (ROS) and subsequent activation of the antioxidant system [108]. Regarding CAT enzyme activity, Zahedi et al. [98] reported that SiO₂ and Se nanoparticle applications increased CAT enzyme activity. Al-aghabary et al. [93] reported that salt stress inhibited CAT activity, but added Si improved CAT activity under salt stress. Günalp [109], in a study examining the effects of Jasmonic Acid (JA) application on salt tolerance in eggplant embryo culture, reported that CAT enzyme activity, an antioxidative enzyme, was at very low levels compared to the control group, while on the other hand, it increased catalase enzyme activities in very high amounts. Similarly, in our study, we concluded that CAT activity in boysenberry plants was lower than that in the control group. According to our study findings, the high CAT activity in the 35 mM NaCl + 0.05 mM FeNP application shows that stress factors activate the defense mechanisms by activating the antioxidant defense system. Another study with similar findings was conducted by Marček et al. [19], in which they examined the effects of different concentrations of NaCl (15 and 35 mM) applications on blackberry plants in vitro; the 35 mM NaCl application caused a significant increase in CAT activities, which proved to be an effective antioxidant defense mechanism in blackberry plants. Regarding SOD enzyme activity, Zahedi et al. [98] reported that SiO₂ and Se nanoparticle applications increased SOD enzyme activity. Al-aghabary et al. [93] reported that salt stress significantly reduced SOD activity, but added Si improved SOD activity under salt stress. Günalp [109] examined the effects of Jasmonic Acid (JA) application on salt tolerance in eggplant embryo cultures. According to Günalp’s study findings, the control group determined that SOD activity was found in applications containing JA and applications containing only NaCl, and SOD activity was reported to be significantly increased in all JA or salt treatments compared to the control group [109]. Our study showed the same results, showing that SOD activity increased with increasing salt concentrations, as in the reference studies. In contrast, Gökçe [95] reported a decrease in SOD activity in the stem tissues of tomato plants, in which he examined the physiological, morphological, and biochemical effects of AgNPs on tomato seeds. Nanoparticles are known to play an important role in antioxidant defense systems and trigger other important metabolic activities in plants under salt stress [110]. In our study, the roles of NPs against oxidative stress were investigated under in vitro cultivation and salinity conditions, and significantly increased activities of SOD and CAT enzymes in the leaves of boysenberry plants were determined. Antioxidant enzymes such as SOD and CAT are induced in plants in response to abiotic stress. Therefore, these levels are expected to increase under stressful conditions. These enzymes are also a vital part of the antioxidant defense system to prevent hydroxyl radicals responsible for lipid peroxidation in cell membranes and biomarkers of oxidative stress [111].

5. Conclusions

The agricultural sector, which is continuously dynamic and develops in parallel with technological developments, produces solutions to the basic problems encountered in the continuation of human existence. It is an important research area to develop new solutions to the problems that arise in agricultural production activities using the latest developments in science and technology, such as nanotechnology. One of these problems is salinity, caused by factors such as excessive irrigation and fertilization. In this study, we investigated the effects of different concentrations of NaCl and different doses of NP on the morphological and physiological properties of boysenberry plants under in vitro growth conditions. According to our findings obtained from the parameters in which the effects of the applications were monitored, the 7.5 and 15 mg L⁻¹ SiNP application was significantly effective in increasing SL, SD, and SDW. When the effects of different concentrations of NPs on root development were compared, it was determined that the addition of FeNP to the medium was significantly more effective on RFW and RDW compared to other NPs. While the highest results in terms of NL of in vitro plants were obtained from the 0.025 mM FeNP application, the highest average LL values were determined in the 0.2 mg L⁻¹ AgNP and 15 mg L⁻¹ SiNP application, and the highest average LW value was determined in the 7.5 mg L⁻¹ SiNP application. While the 0.4 mg L⁻¹ AgNP application was found to be more effective than other applications in terms of the SPAD value, the highest RGR average value was determined in the 0.2 mg L⁻¹ AgNP application. The highest LRWC values were determined in 0.4 mg L⁻¹ AgNP and 7.5 mg L⁻¹ SiNP applications. The application of 0.2 mg L⁻¹ of AgNP and 0.025 and 0.05 mM of FeNP was found to be significantly effective regarding CAT enzyme activity. The highest SOD enzyme activity values were recorded when using 0.4 mg L⁻¹ AgNP and 0.05 mM FeNP treatments. In conclusion, when the results obtained from the study were evaluated as a whole, it was determined that the application of SiNPs, FeNPs, and AgNPs under in vitro cultivation conditions and salinity stress reduced the negative effects of salinity and had a positive effect on vegetative development and the physiological and biochemical parameters of boysenberry plants.