# Improvement of Polyethylene Glycol, Sorbitol, Mannitol, and Sucrose-Induced Osmotic Stress Tolerance through Modulation of the Polyamines, Proteins, and Superoxide Dismutase Activity in Potato

Authors: Zahoor Ahmad Sajid; Faheem Aftab
Journal: International Journal of Agronomy
 (2022)

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## Abstract

The present study was planned to investigate the changes in morphological and biochemical parameters ofin vitro-grown potato (cultivar Cardinal and Desiree) plants under osmotic stress conditions induced by various concentrations of sorbitol, mannitol (0, 0.025, 0.05, 0.10, or 0.15 M), sucrose (0, 2, 3, 4, 6, and 8%), and polyethylene glycol (PEG: MW-4000: 0, 5, 10, 15, and 20%). Nodal segments (ca. 1.0 cm) from healthy in vitro-grown potato plantlets were inoculated on Murashige and Skoog’s medium consisting of various levels of above mentioned drought stress-inducing agents. Data was recorded on 60th day of incubation exhibited a severe reduction in most of the growth parameters at 0.10 and 0.15 M of sorbitol and mannitol, respectively, and at 5–10% PEG. Similar results were observed when the sucrose level varied from 3% except for the number of roots and plant dry weight, which exhibited an increase in increasing the sucrose level. Data collected for total soluble protein content and activity of an antioxidant enzyme (superoxide dismutase) unveiled an overall increasing trend in osmotically stressed potato plants suggesting their major action in detoxification of active oxygen species produced under osmotic stress. Polyamines (putrescine, spermidine, and spermine) increased significantly in both the cultivars of potato by using osmotic stress-inducing agent in the present investigation indicating their positive role in stress alleviation. Overall results indicated that potato cultivar Desiree was more stress-tolerant than the cultivar Cardinal.


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## Body

## 1. Introduction

Yield reduction of major staple food crops under abiotic stresses such as drought stress is becoming a serious problem worldwide affecting food security. It has been a catalyst for great famines of the past and is reported to affect important crops such as soybean and maize in dry Savanna; chickpea and groundnut in Mexico and Central America; and potatoes in various countries of Asia [1]. Drought stress impairs plant growth and ultimately the crop yield by affecting cellular processes such as mitosis, cell expansion, and enlargement [2]. It also creates oxidative stress in plants by generating reactive oxygen species (ROS) including hydroxyl radicals (OH−), superoxide anions (O2−), hydrogen peroxide (H2O2) and singlet oxygen (O12) [3]. These ROS cause lipid peroxidation, denaturation of cell membrane, damaging protein structure, and destruction of DNA [4, 5]. Plants have a very strong antioxidant defense mechanism (enzymatic as well as nonenzymatic) to lessen the harmful effects of these ROS. Enzymatic antioxidants include catalase (CAT), peroxidase (POX), ascorbate peroxidase (APX), glutathione reductase (GR), and superoxide dismutase (SOD) [6]. Amongst these, SOD has a central role in the antioxidant defense network as it catalyzes the conversion of superoxide radicals (O2−) to hydrogen peroxide and molecular oxygen thus acting as the first line of detoxification against ROS [7]. It has also been observed that an increase in its activity is correlated with increased protection against the damaging effect of environmental stress [8]. Polyamines (PA’s) are important low molecular weight compounds involved in various metabolic processes in plants under both normal and extreme environmental stress conditions [9]. PA’s play’s regulatory role in plants under osmotic stress conditions by increasing the Ca, K, Fe, Mn, Zn, and NO3− ions in Lettuce leaves and reduced stomatal aperture [10]. Involvement of polyamine was also reported by Elsayed et al. [11] under salt stress by enhancing antioxidant enzymes in wheat.Potato (Solanum tuberosum L.) of family Solanaceae ranks first amongst nongrain staple food crops with a worldwide production of 388 million tons [12]. It is a good source of dietary starch which is having an adhesive property, binder, texture agent, and filler. Starch content of potato peel is used to produce fuel-grade ethanol. Although, there is a considerable increase in cultivation and production of this crop worldwide, the average yield is still considered as far below the existing potential owing to drought stress as a major limiting factor [13]. Potato has been considered as a drought-sensitive plant because plants have shallower root systems [14]. Its growth and productivity decline sharply with an increase in water stress in countries like Pakistan, where a total cropped area is some 23.63 million hectares. Out of this, 170.3 thousand hectares are being used for potato cultivation with an annual production of 23.4 tons per hectare [15]. In Pakistan, most of the land area is classified as arid and semiarid and water scarcity is a limiting constraint for agricultural production. Improvement in agricultural productivity is, therefore, imperative to ensure higher crop yield under such unfavorable environmental stresses and limited resources.Plant tissue culture is quite an amenable technique to understand the various aspects of drought tolerance in potatoes [16, 17]. Plants when grown under abiotic stress conditions accumulate various compatible solutes or osmolytes in their cells which include sorbitol and mannitol, polyethylene glycol, etc. [18]. These are highly soluble and low molecular weight organic molecules and mostly accumulate in the cytosol without disturbing the metabolism of cells even at high concentrations. In addition to this, these osmolytes also perform the function of stabilizing protein and structure of cell membranes under drought stress. Unlike drought stress that causes osmotic stress in plants, accumulation of these compounds decreases osmotic potential and hence maintains the cellular turgidity and increases uptake of water. Apart from this they also play an important role in protecting cells against oxidative stress by scavenging reactive oxygen species in plants [19]. Similarly, sucrose has also been found to accumulate in response to water stress conditions in plant tissues [20]. In addition, polyethylene glycol (PEG) has also been reported as a suitable osmoticum to modify the osmotic potential of plants. PEG is not being used up in the cellular metabolism of plants, but it increases water stress by decreasing the water potential of nutrient solutions and has thus been found to be effective in reducing the in vitro growth of plants [21].Several researchers have used different concentrations of abovementioned osmotica to understand the mechanism of drought stress in different plants [22–24]. However, the information about the precise level suitable for effective in vitro screening of potatoes is scanty. Considering this, the present study was undertaken with an objective to partially characterize biochemically the effect of some selected osmotica (sorbitol, mannitol, sucrose, and PEG) on in vitro cultures of two potato cultivars (Cardinal and Desiree). In doing so, the relative efficacy of abovementioned osmotica was also determined to induce in vitro drought like conditions. Considering its significance, work was also carried out on changes in polyamines activity (putrescine, spermidine, and spermine) on exposure to drought stress in potato cultivars used in the present investigation.


## 2. Materials and Methods

### 2.1. Plant Material and Experimental Layout

MS basal medium [25] was used with various concentrations of sorbitol, mannitol, sucrose, and polyethylene glycol (PEG; Sigma-Aldrich, St Louis, and MO) to mediate drought stress to potato plants. Potato germplasm of both cultivars (Cardinal and Desiree) was procured from seed center, University of the Punjab, Lahore, Pakistan. For osmotic stress treatments on potato plants, five different concentrations of sorbitol and mannitol (0, 0.025, 0.05, 0.10, or 0.15 M); and six concentrations of sucrose (0, 2, 3, 4, 6, or 8%) were used. In case of PEG: MW-4000, five different levels (0, 5, 10, 15, and 20%) were added to MS liquid medium directly. Single nodal portions of approximately 1.0 cm from earlier raised 30 days-old potato plantlets under in-vitro conditions were inoculated on MS medium containing various concentrations of abovementioned osmotica. Ten culture vessels with single nodal segments were inoculated for each treatment. The cultures were incubated for 16 hours under cool white florescent light (40 μmole m−2·s−1) at 25 ± 2°C. All the experiments were set up in a complete block design and repeated thrice with 10 replicates to study the effect of sorbitol, mannitol, sucrose, and PEG on potato plants.


### 2.2. Morphological and Biochemical Analyses

After 60 days of explants inoculation, data were recorded for various growth attributes, i.e., root/shoot length, root, shoot, number of nodes, and fresh/dry weight of plantlets. Estimation of protein content and SOD activity was also carried out at the conclusion of the experiment. Growth parameters were recorded by taking out plants from the culture tubes with intact roots and washing with tap water to remove all the traces of agar. The numbers of shoots/roots, nodes, and tubers were counted carefully. Shoot and root lengths were measured by using a suitable scale (excluding 1 cm; size of explants). Fresh/dry weight of plantlets was recorded by using an electric balance (Scientech 5220) and then by drying in an oven at 60°C for 72 hours.For the estimation of protein content and superoxide dismutase activities, 1 gm of plant material was ground in liquid nitrogen with 0.01 mL of Tritone X-100 (Sigma-Aldrich, St Louis, MO) and 0.1 gPolyVinyl Polypyrrolidone (PVP; Sigma-Aldrich). The resulting fine powder was dissolved in 2 mL of Phosphate buffer (0.1 M, pH 7.2) to make a slurry and centrifuged (Sorval RB-5) at 15400 × g for 30 minutes at 4°C. The supernatant was used for the estimation of protein contents and SOD activities.Total protein contents were estimated by following Biuret method of Racusen and Johnstone [26] with few modifications. Control and experimental samples were prepared. The experimental or reaction mixture in a tube consisted of 2 mL of Biuret reagent and 0.2 mL of supernatant or plant extract. Control comprises of 0.2 mL water and 2 mL of Biuret reagent. Both tubes were kept at room temperature for 20 minutes for the completion of the reaction. Finally, the absorbance was observed at 550 nm on a spectrophotometer (Hitachi U1100). Bovine serum albumin was used for the preparation of the standard curve. The amount of total protein contents was calculated by following formula (Eq. 1):(1)protein contentsmg/g=CV×TEEU×Wt×1000,where CV is the curve value, TE is the total extract, EU is the extract used, and Wt is the fresh weight of tissue.Superoxide dismutase (1.15.1.1) activity was measured spectrophotometrically as purposed by Maral et al. [27] with few modifications. Assay of SOD was based on recording its capability to inhibit the reduction of nitro blue tetrazolium (NBT; Sigma-Aldrich, St Louis, MO) by O2− produced photochemically. Experimental and control samples were prepared in separate test tubes. The experimental tube consisted of 2.0 mL of the reaction mixture (50 mM phosphate buffer (pH 7.8), 13 mM methionine, 75 μM nitro blue tetrazolium, 0.1 mM ethylenediaminetetraacetate (EDTA), and 2 μM riboflavin) with 20 µL enzyme extract. The control sample consisted of only a reaction mixture without enzyme extract. Both tubes (experimental and control) were placed under the white fluorescent light of two 30-W tube lights (Philips Pakistan) for 15 minutes to irradiate simultaneously. The absorbance was recorded at 560 nm on a spectrophotometer (Hitachi U1100). SOD activity was calculated by using the following formula (Eq. 2):(2)%inhibition=absorbance of control sample−absorbance of experimental sampleabsorbance of experimental sample×100.


### 2.3. Polyamine Extraction and Estimation

Fresh leaves samples (0.5 g) of potato were used for the estimation of polyamine contents. Polyamines were extracted in 1.5 mL ice chilled perchloric acid (5%) as described by Redmond et al. [28]. After centrifugation at 14,000 rpm, 500 μL of supernatant was mixed in Benzoyl chloride (10 μL) followed by adding 1 mL of NaOH (2 molL−1) and vertexing for 20 seconds. After 20 minutes of reaction at 37°C, 2 mL NaCl was added. This mixture was extracted again with 2 mL ether and then centrifuged at 15,000 rpm for 5 minutes at 4°C. The organic phase 1 mL was dried under nitrogen stream and the dried extract was redissolved in 1 mL methanol and filtered (0.22 μm) and stored at −20°C until analysis by high-performance liquid chromatography using an Agilent 1200 Infinity LC on a C18 column (250 × 4.6 mm) (Agilent Technologies, USA). The mobile phase was methanol: water (60 : 40) at a flow rate of 0.7 mL·min−1, column temperature 30°C, and detection wavelength 230 nm. The standard curves were created for the derivatives of putrescine, spermidine, and spermine (put, spd, and spm) standards (Sigma USA) between 1 and 100 μgmL−1.


### 2.4. Statistical Analysis

Univariate analysis was employed by using SPSS Version 22.0.0 to analyze the data. The standard error of means was calculated for each treatment. To compare the means values, Duncan’s multiple range test was used.


## 2.1. Plant Material and Experimental Layout

MS basal medium [25] was used with various concentrations of sorbitol, mannitol, sucrose, and polyethylene glycol (PEG; Sigma-Aldrich, St Louis, and MO) to mediate drought stress to potato plants. Potato germplasm of both cultivars (Cardinal and Desiree) was procured from seed center, University of the Punjab, Lahore, Pakistan. For osmotic stress treatments on potato plants, five different concentrations of sorbitol and mannitol (0, 0.025, 0.05, 0.10, or 0.15 M); and six concentrations of sucrose (0, 2, 3, 4, 6, or 8%) were used. In case of PEG: MW-4000, five different levels (0, 5, 10, 15, and 20%) were added to MS liquid medium directly. Single nodal portions of approximately 1.0 cm from earlier raised 30 days-old potato plantlets under in-vitro conditions were inoculated on MS medium containing various concentrations of abovementioned osmotica. Ten culture vessels with single nodal segments were inoculated for each treatment. The cultures were incubated for 16 hours under cool white florescent light (40 μmole m−2·s−1) at 25 ± 2°C. All the experiments were set up in a complete block design and repeated thrice with 10 replicates to study the effect of sorbitol, mannitol, sucrose, and PEG on potato plants.


## 2.2. Morphological and Biochemical Analyses

After 60 days of explants inoculation, data were recorded for various growth attributes, i.e., root/shoot length, root, shoot, number of nodes, and fresh/dry weight of plantlets. Estimation of protein content and SOD activity was also carried out at the conclusion of the experiment. Growth parameters were recorded by taking out plants from the culture tubes with intact roots and washing with tap water to remove all the traces of agar. The numbers of shoots/roots, nodes, and tubers were counted carefully. Shoot and root lengths were measured by using a suitable scale (excluding 1 cm; size of explants). Fresh/dry weight of plantlets was recorded by using an electric balance (Scientech 5220) and then by drying in an oven at 60°C for 72 hours.For the estimation of protein content and superoxide dismutase activities, 1 gm of plant material was ground in liquid nitrogen with 0.01 mL of Tritone X-100 (Sigma-Aldrich, St Louis, MO) and 0.1 gPolyVinyl Polypyrrolidone (PVP; Sigma-Aldrich). The resulting fine powder was dissolved in 2 mL of Phosphate buffer (0.1 M, pH 7.2) to make a slurry and centrifuged (Sorval RB-5) at 15400 × g for 30 minutes at 4°C. The supernatant was used for the estimation of protein contents and SOD activities.Total protein contents were estimated by following Biuret method of Racusen and Johnstone [26] with few modifications. Control and experimental samples were prepared. The experimental or reaction mixture in a tube consisted of 2 mL of Biuret reagent and 0.2 mL of supernatant or plant extract. Control comprises of 0.2 mL water and 2 mL of Biuret reagent. Both tubes were kept at room temperature for 20 minutes for the completion of the reaction. Finally, the absorbance was observed at 550 nm on a spectrophotometer (Hitachi U1100). Bovine serum albumin was used for the preparation of the standard curve. The amount of total protein contents was calculated by following formula (Eq. 1):(1)protein contentsmg/g=CV×TEEU×Wt×1000,where CV is the curve value, TE is the total extract, EU is the extract used, and Wt is the fresh weight of tissue.Superoxide dismutase (1.15.1.1) activity was measured spectrophotometrically as purposed by Maral et al. [27] with few modifications. Assay of SOD was based on recording its capability to inhibit the reduction of nitro blue tetrazolium (NBT; Sigma-Aldrich, St Louis, MO) by O2− produced photochemically. Experimental and control samples were prepared in separate test tubes. The experimental tube consisted of 2.0 mL of the reaction mixture (50 mM phosphate buffer (pH 7.8), 13 mM methionine, 75 μM nitro blue tetrazolium, 0.1 mM ethylenediaminetetraacetate (EDTA), and 2 μM riboflavin) with 20 µL enzyme extract. The control sample consisted of only a reaction mixture without enzyme extract. Both tubes (experimental and control) were placed under the white fluorescent light of two 30-W tube lights (Philips Pakistan) for 15 minutes to irradiate simultaneously. The absorbance was recorded at 560 nm on a spectrophotometer (Hitachi U1100). SOD activity was calculated by using the following formula (Eq. 2):(2)%inhibition=absorbance of control sample−absorbance of experimental sampleabsorbance of experimental sample×100.


## 2.3. Polyamine Extraction and Estimation

Fresh leaves samples (0.5 g) of potato were used for the estimation of polyamine contents. Polyamines were extracted in 1.5 mL ice chilled perchloric acid (5%) as described by Redmond et al. [28]. After centrifugation at 14,000 rpm, 500 μL of supernatant was mixed in Benzoyl chloride (10 μL) followed by adding 1 mL of NaOH (2 molL−1) and vertexing for 20 seconds. After 20 minutes of reaction at 37°C, 2 mL NaCl was added. This mixture was extracted again with 2 mL ether and then centrifuged at 15,000 rpm for 5 minutes at 4°C. The organic phase 1 mL was dried under nitrogen stream and the dried extract was redissolved in 1 mL methanol and filtered (0.22 μm) and stored at −20°C until analysis by high-performance liquid chromatography using an Agilent 1200 Infinity LC on a C18 column (250 × 4.6 mm) (Agilent Technologies, USA). The mobile phase was methanol: water (60 : 40) at a flow rate of 0.7 mL·min−1, column temperature 30°C, and detection wavelength 230 nm. The standard curves were created for the derivatives of putrescine, spermidine, and spermine (put, spd, and spm) standards (Sigma USA) between 1 and 100 μgmL−1.


## 2.4. Statistical Analysis

Univariate analysis was employed by using SPSS Version 22.0.0 to analyze the data. The standard error of means was calculated for each treatment. To compare the means values, Duncan’s multiple range test was used.


## 3. Results

### 3.1. Effect of Various Concentrations of Sorbitol on Potato Plants

A significant decrease was observed in shoot length, number, and root length in both potato cultivars (Cardinal and Desiree) by increasing the concentration of sorbitol (Table1). In case of Cardinal plants, a decrease in shoot length from 14.833 to 6.094 cm was recorded by a gradual increase of sorbitol from 0 to 0.15 M in MS medium. Similarly, root length was reduced from 10.294 to 4.766 cm and this reduction in root length was significant at 0.10 and 0.15 M concentration of sorbitol. As in the case of cv. Cardinal, both shoot and root length in Desiree decreased from 13.527 to 4.244 cm and 10.444 to 5.794 cm, respectively, with an increasing sorbitol concentration in MS medium. Number of roots and nodes also showed a decreasing trend in both the cultivars. Number of shoots, however, exhibited an increase in Cardinal, and a decrease in Desiree. Sorbitol treatment also significantly decreased the total fresh/dry masses of Desiree plants. Fresh weight was decreased from 0.910 to 0.293 g and dry weight from 0.072 to 0.039 g at 0.15 M concentration. However, the effect of different concentrations of sorbitol was nonsignificant in the case of dry weights of Desiree plants. Overall, plants that were grown on MS medium without any sorbitol treatment were healthier compared with the plants grown over various sorbitol treatments. Furthermore, a change in leaf morphology was also observed at higher sorbitol levels (0.15 M), where leaves appeared relatively smaller in size and yellowish green in color (Figure 1).Table 1
Growth parameters of potato cultivars in response to osmotic stress induced by various levels of sorbitol.
MediumSorbitol (M)Shoot length (cm)No. of shootsRoot length (cm)No. of rootsNo. of nodesFresh wt. (g)Dry wt. (g)CarDesCarDesCarDesCarDesCarDesCarDesCarDes∗S1014.83 ± 0.41a13.52 ± 0.71a1.83 ± 0.18c3.11 ± 0.26a10.29 ± 0.51a10.44 ± 0.69a7.44 ± 0.47a8.72 ± 1.02a20.33 ± 0.41a16.27 ± 0.49a0.42 ± 0.03ab0.91 ± 0.09a0.03 ± 0.00a0.07 ± 0.01aS20.02514.68 ± 0.61a10.69 ± 0.81b3.44 ± 0.23a2.50 ± 0.28ab9.63 ± 0.49a9.92 ± 0.41ab6.33 ± 0.596.72 ± 0.66a20.27 ± 0.7313.33 ± 1.08b0.54 ± 0.05a0.83 ± 0.08ab0.03 ± 0.00a0.06 ± 0.00aS30.0512.250 ± 0.77b8.58 ± 1.10b2.50 ± 0.18b2.33 ± 0.32bc8.92 ± 0.80a9.21 ± 0.69ab6.55 ± 0.67a6.61 ± 0.87a19.72 ± 0.83a9.77 ± 0.96c0.29 ± 0.02b0.68 ± 0.10ab0.05 ± 0.03a0.05 ± 0.01aS40.108.58 ± 0.87c9.07 ± 0.73b3.33 ± 0.25a1.72 ± 0.22cd7.14 ± 0.68b8.53 ± 0.32b5.61 ± 0.81ab6.38 ± 0.55a15.94 ± 0.90b13.27 ± 0.90b0.32 ± 0.05b0.59 ± 0.08b0.03 ± 0.00a0.05 ± 0.01aS50.156.09 ± 0.69d4.24 ± 0.60c2.33 ± 0.22bc1.05 ± 0.05d4.76 ± 0.54c5.79 ± 0.84c3.94 ± 0.59b3.88 ± 0.61b13.11 ± 0.90c7.83 ± 0.88c0.29 ± 0.05b0.29 ± 0.04c0.02 ± 0.00a0.03 ± 0.00aSignificance (P≤0.05)∗∗∗∗∗∗∗∗∗∗∗∗NSNSResults are mean from at least ten to replicate cultures ± S.E. Similar letters within the column do not differ significantly (P≤0.05) according to DMRT. Significant (∗) or nonsignificant (NS) at P≤0.05. ∗MS [25] contains different levels of sorbitol (designated as S1 to S5; sorbitol level as shown in the next column).Figure 1
Growth of CVS. Cardinal (a)–(e) and Desiree (f)–(j) at different sorbitol concentrations. Scale bar. (a) = 1.5 cm, (b)–(c) = 2.0 cm, (d) = 1.5 cm, (e) = 1.2 cm, (f)–(g) = 2.0 cm, (h) = 1.5 cm, (i) = 1.7 cm, and (j) = 1.0 cm.There was a significant difference in sorbitol treatment on total protein contents in both the cultivars. Proteins increased gradually by a value of 0.23 mgg−1 to 0.85 mgg−1 at 0 to 0.10 M sorbitol concentration. However, at a higher concentration of sorbitol (0.15 M), a decrease (0.79 mgg−1) in its content was recorded as compared to 0.10 M. SOD activity was also found to increase significantly in sorbitol-treated Cardinal plants as compared to control. However, the increase was not gradual as the maximum value for SOD activity (32.25 Umg−1 of protein) was observed at 0.025 M sorbitol concentration, which was followed by a progressive reduction in SOD activity with increasing sorbitol concentration in the medium. As indicated in Figures 2(a) and 2(b), SOD activity increased gradually by increasing sorbitol concentration in case of Desiree plants as compared to control ones. However, its value was decreased to 14.29 Umg−1 of protein at 0.15 M sorbitol level as compared to plants without any sorbitol treatment, i.e., 19.51 Umg−1 of protein.Figure 2
Protein contents and superoxide dismutase activity in potato cultivars cardinal (Car) and Desiree (Ds) in response to sorbitol (a) and (b) mannitol (c) and (d), sucrose (e) and (f) and PEG (g) and (h) induced osmotic stress. Results are the means ± S.E. of ten replicates cultures. Bars with similar letters are not significantly different atP≤0.05 by Duncan’s multiple range test.
(a)(b)(c)(d)(e)(f)(g)(h)The contents of endogenous polyamines in both cultivars of potato increase significantly by adding various concentrations of sorbitol in MS medium and the highest contents of all the investigated polyamines were observed in plants growing on 0.15 M of sorbitol. putrescine contents increased from 125.23 (control) to 150.89 nmol·g−1 FW (10 M) in cv. Cardinal, however, in Desiree this increase in putrescine contents was not as higher as in case of cardinal. Spermidine also showed an increasing trend by increasing concentrations of sorbitol from 2.5 to 10 M. Similarly, spermine also showed an increasing trend but its amount was less as compared to putrescine and spermidine. Potato cv. Cardinal showed less increase in polyamine contents as compared to Desiree (Table2).Table 2
Effect of various levels of sorbitol, mannitol, sucrose, and PEG on polyamines contents (putrescine, spermidine, and spermine) of potato plants.
MediumPutrescine (nmol·g−1 FW)Spermidine (nmol·g−1 FW)Spermine (nmol·g−1 FW)CultivarCarDesCarDesCarDes(Control) without sorbitol145.13 ± 3.42b143.56 ± 3.67a132.62 ± 3.23c122.45 ± 3.24c118.75 ± 2.78e119.28 ± 2.33dMS + 0.025 M sorbitol135.35 ± 2.07c148.34 ± 3.25a141.30 ± 2.27b131.73 ± 3.27b124.45 ± 1.15d124.46 ± 3.79cMS + 0.05 M sorbitol147.22 ± 1.18b142.28 ± 2.42a142.27 ± 2.14b133.65 ± 2.12b130.25 ± 3.13c129.33 ± 2.53bMS + 0.10 M sorbitol155.80 ± 2.27a145.39 ± 2.23a151.13 ± 2.28a142.35 ± 2.38a139.29 ± 3.20b130.13 ± 1.21bMS + 0.15 M sorbitol158.23 ± 1.37a147.32 ± 2.53a155.15 ± 2.78a143.05 ± 2.38a149.29 ± 3.20a140.03 ± 1.31a(Control) without mannitol115.23 ± 3.42e113.26 ± 3.67c122.20 ± 3.23c122.15 ± 3.34b128.95 ± 2.28d119.18 ± 2.23cMS + 0.025 M mannitol119.15 ± 1.27d118.34 ± 1.35b131.30 ± 2.27b124.72 ± 3.27b124.95 ± 1.13d114.27 ± 3.19cMS + 0.05 M mannitol127.29 ± 1.28c121.28 ± 2.12b129.25 ± 2.14b131.35 ± 1.22a130.15 ± 1.12c124.73 ± 2.53bMS + 0.10 M mannitol133.89 ± 2.37b121.29 ± 2.23b132.35 ± 2.28b131.15 ± 2.18a139.20 ± 3.20b129.35 ± 1.34aMS + 0.15 M mannitol140.89 ± 1.47a132.99 ± 1.33a134.25 ± 2.28a132.05 ± 2.28a149.20 ± 1.31a130.15 ± 1.22a(Control) without sucrose145.29 ± 2.42c143.26 ± 1.67d132.10 ± 3.43e132.25 ± 3.24c128.15 ± 2.78c129.08 ± 2.63cMS + 2% sucrose151.25 ± 1.07b144.34 ± 1.25bc134.20 ± 2.37d131.72 ± 2.97c129.25 ± 1.95c124.31 ± 2.79dMS + 3% sucrose157.21 ± 2.18b147.38 ± 2.42b139.05 ± 2.24c134.35 ± 2.12c130.35 ± 3.33c129.93 ± 2.53cMS + 4% sucrose159.80 ± 2.27a140.29 ± 1.23bc141.15 ± 2.08b142.15 ± 2.08b139.22 ± 3.30b140.22 ± 1.34bMS + 6% sucrose160.82 ± 2.27a150.29 ± 2.23a139.35 ± 2.28d142.15 ± 1.98b139.91 ± 3.32b140.23 ± 1.24bMS + 8% sucrose162.83 ± 2.27a152.19 ± 2.23a151.15 ± 1.28a152.25 ± 2.08a149.23 ± 3.31a150.21 ± 1.14a(Control) without PEG125.23 ± 3.42c123.76 ± 3.67c112.60 ± 3.23c102.35 ± 3.24b110.95 ± 2.78bc109.28 ± 2.63bMS + 2.5% PEG135.15 ± 2.07b128.64 ± 3.25bc121.90 ± 2.27b111.70 ± 3.27ab115.95 ± 1.15b114.37 ± 3.79bMS + 5% PEG137.29 ± 1.18b132.78 ± 2.42b122.25 ± 2.14b121.65 ± 2.12a122.15 ± 3.13b121.93 ± 2.53aMS + 10% PEG150.89 ± 2.27a140.99 ± 2.23a131.15 ± 2.28a122.05 ± 2.38a129.20 ± 3.30a120.25 ± 1.24aResults are mean from at least ten to replicate cultures ± S.E. Similar letters within the column do not differ significantly (P≤0.05) according to DMRT. Significant (∗) or non-significant (NS) at P≤0.05. ∗MS [25] contains different levels of sorbitol (designated as S1 to S5; sorbitol level as shown in the next column).


### 3.2. Effect of Various Concentrations of Mannitol on Potato Plants

Maximum shoot/root length (12.565 and 9.145 cm, respectively) was observed at 0 M and minimum (2.360 and 2.896 cm, respectively) at 0.15 M mannitol concentration. Similar results were observed for a number of roots and nodes. However, the results were different in case of number of shoots, where an increase in their number was observed with increasing stress levels in MS medium (Figure3). It is evident from the data given in Table 3 that mannitol treatment to Desiree plants had a significant effect on all the studied growth parameters. Reduction in the growth of several parameters including shoot/root length, shoot/root, and number of nodes was recorded as the concentration of mannitol was higher in the MS medium. Table 3 also indicates that mannitol treatment had a significant effect on the fresh weight of both the cultivars. Dry weight of plants was also affected by mannitol applications; however, it was not as significant as was in case of fresh weights in cardinal but significant in case of Desiree.Figure 3
Growth of Cardinal (a)–(e) and Desiree (f)–(j) at different mannitol concentrations. Scale bar (a)–(j) = 1 cm.Table 3
Growth parameters of potato cultivars in response to osmotic stress induced by various levels of mannitol.
MediumMannitol (M)Shoot length (cm)No. of shootsRoot length (cm)No. of rootsNo. of nodesFresh wt. (g)Dry wt. (g)CarDesCarDesCarDesCarDesCarDesCarDesCarDes∗M1012.56 ± 1.02a14.21 ± 0.51a1.65 ± 0.23a3.22 ± 0.19a9.14 ± 0.86a11.63 ± 0.70a6.35 ± 0.70a10.27 ± 0.73a16.90 ± 1.40a16.72 ± 0.35a0.36 ± 0.03a0.99 ± 0.06a0.03 ± 0.00ab0.06 ± 0.01abcM20.02510.36 ± 0.96a12.59 ± 0.72a2.05 ± 0.23a2.61 ± 0.14ab7.23 ± 0.78ab9.86 ± 0.51ab5.05 ± 0.60ab8.38 ± 0.61b17.20 ± 1.49a15.94 ± 0.51a0.35 ± 0.04a0.91 ± 0.08a0.03 ± 0.00a0.09 ± 0.01aM30.056.80 ± 0.99b10.08 ± 0.65b2.10 ± 0.28a2.88 ± 0.26ab6.82 ± 0.87b10.67 ± 0.61ab4.95 ± 0.88ab7.44 ± 0.53bc13.65 ± 0.69ab11.16 ± 0.60b0.28 ± 0.05a0.81 ± 0.07a0.03 ± 0.00ab0.08 ± 0.01abM40.104.49 ± 0.66bc5.35 ± 0.51c2.25 ± 0.27a2.27 ± 0.33b4.26 ± 0.54c9.56 ± 0.64b5.10 ± 0.03ab5.94 ± 0.80c10.30 ± 0.23bc9.33 ± 0.70c0.24 ± 0.04ab0.40 ± 0.05b0.03 ± 0.00ab0.05 ± 0.01bcM50.152.36 ± 0.38c5.13 ± 0.42c1.90 ± 0.27a2.16 ± 0.25b2.89 ± 0.54c7.25 ± 0.58c3.35 ± 0.75b3.77 ± 0.29d7.25 ± 0.97c10.27 ± 0.51c0.13 ± 0.02b0.31 ± 0.02b0.02 ± 0.00b0.04 ± 0.01cSignificance (P≤0.05)∗∗NS∗∗∗NS∗∗∗∗∗NSNSResults are mean from at least ten to replicate cultures ± S.E. Similar letters within 7the column do not differ significantly (P≤0.05) according to DMRT. Values are significant (∗) or nonsignificant (NS) at P≤0.05. ∗MS [25] was supplemented with different levels of mannitol (designated as M1 to M5).An overall increase in total proteins was recorded at all mannitol levels in both the cultivars of potato. In case of control Cardinal plants, protein content (0.26 mgg−1) increased gradually to 0.85 mgg−1 at 0.15 M treatment. Similar increase in its value was also observed for Desiree where the change in its values was from 0.16 (in control plants) to 1.15 mgg−1 at the highest (0.15 M) mannitol level. SOD activity was greatly influenced by the addition of mannitol in the MS medium. In case of Desiree, mannitol treatment resulted in an increase of SOD activity, with its highest value (66.6 Umg−1 of protein) observed at 0.15 M. With some minor variations, osmotic stress (induced by mannitol) also resulted in increased activity of SOD in Cardinal plants (Figures 2(c) and 2(d)). In this case, SOD activity was decreased (24.14 Umg−1 of protein) at 0.025 M mannitol as compared to control ones (27.64 Umg−1 of protein).All the tested polyamines were increased by increasing concentrations of mannitol in MS medium. Both cultivars of potato plants showed a significant (P≤0.05) increase in polyamines however, in Desiree plants, this increase was higher than in the cardinal plants. The highest putrescine contents were observed in plants treated with 0.15 M of mannitol. Spermidine contents was 122.20, 131.30, 129.25, 132.35, and 134.25 nmol·g−1 FW in cardinal plants at 0, 0.025, 0.5, 0.1, and 0.15 M of mannitol, respectively. In Desiree plants, this increase in spermidine contents was 122.15, 124.72, 131.35, 131.15, and 132.05 nmol·g−1 FW at 0, 0.025, 0.5, 0.1, and 0.15 M of mannitol, respectively. Spermine contents also showed a similar increasing trend by increasing mannitol concentration in MS medium (Table 2).


### 3.3. Effect of Sucrose-Induced Osmotic Stress on Potato Plants

The effect of sucrose was statistically significant on shoot growth, its number, and on number of roots in both the cultivars of potato with a maximum value at 3% sucrose level; and a gradual decrease as the concentration varied. Highest value of root length was recorded at 4% sucrose in Cardinal followed by 2% sucrose concentration (Table4). All the abovementioned parameters showed a decreasing trend by increase or decrease in sucrose concentration from 3% except for the root number, where an increase was recorded at 8% sucrose. For Cardinal, number of nodes decreased significantly with a gradual increase in sucrose concentration from 3%. Fresh weight exhibited in general a decrease, while dry weight exhibited an increasing trend in both the cultivars as the concentration of sucrose varied from 3 to 8%.Table 4
Growth parameters of potato cultivars in response to osmotic stress induced by various levels of sucrose.
MediumSucrose (%)Shoot length (cm)No. of shootsRoot length(cm)No. of rootsNo. of nodesFresh wt.(g)Dry wt.(g)CarDesCarDesCarDesCarDesCarDesCarDesCarDes∗S103.16 ± 0.47d7.06 ± 0.93c1.11 ± 0.07b0.55 ± 0.12c0.18 ± 0.18b4.48 ± 0.92b0.05 ± 0.05b1.44 ± 0.42b11.61 ± 0.93c11.22 ± 0.85a0.05 ± 0.00c0.09 ± 0.01c0.00 ± 0.00c0.00 ± 0.0bS228.98 ± 0.51bc11.64 ± 0.90ab5.30 ± 1.18a6.51 ± 1.91ab5.03 ± 0.91a8.35 ± 1.66a3.77 ± 0.28a3.94 ± 0.59b13.38 ± 0.78bc11.61 ± 0.74a0.178 ± 0.01b0.32 ± 0.03b0.01 ± 0.0bc0.01 ± 0.0bS3313.31 ± 0.68a13.47 ± 0.57a6.04 ± 1.30a10.20 ± 2.48a4.13 ± 0.63a5.46 ± 1.01ab5.11 ± 0.46a7.83 ± 0.74a17.44 ± 0.77a12.50 ± 0.60a0.35 ± 0.02a0.81 ± 0.09a0.02 ± 0.0ab0.05 ± 0.0aS4411.37 ± 0.15ab11.80 ± 0.84ab4.23 ± 0.90a5.00 ± 0.88b5.49 ± 0.90a6.04 ± 1.06ab4.88 ± 0.52a8.33 ± 1.49a15.16 ± 1.05ab10.33 ± 0.63a0.34 ± 0.05a0.84 ± 0.11a0.03 ± 0.0a0.08 ± 0.1aS568.42 ± 1.36c11.15 ± 0.85ab4.43 ± 0.78a4.28 ± 0.82bc4.08 ± 0.63a4.82 ± 0.75b3.83 ± 0.64a7.61 ± 1.08a12.33 ± 1.18bc11.11 ± 0.79a0.28 ± 0.04ab0.75 ± 0.09a0.03 ± 0.0a0.07 ± 0.1aS686.96 ± 0.90c9.83 ± 0.83b3.82 ± 0.73a5.17 ± 1.07b3.95 ± 0.85a4.88 ± 0.77b6.44 ± 1.13a9.83 ± 1.30a11.88 ± 1.24c10.55 ± 0.77a0.35 ± 0.06a0.69 ± 0.05a0.04 ± 0.01a0.08 ± 0.0aSignificance (P≤0.05)∗∗NS∗∗NSNS∗∗NS∗∗NSNSResults are mean from at least ten to replicate cultures ± S.E. Similar letters within the column do not differ significantly (P≤0.05) according to DMRT. Significant (∗) or nonsignificant (NS) at P≤0.05. ∗MS [25] medium supplemented with different levels of sucrose (designated as S1 to S6).In Cardinal plants, total protein contents decreased gradually at 2, 4, and 6% sucrose concentration and exhibited an increase at 0 and 8% sucrose levels while in Desiree, protein content increased at all the stress levels induced by sucrose. SOD activity was also significantly increased in both potato cultivars at different sucrose concentrations except at 2%, where a decrease in its level was observed. Highest SOD activity was observed at 8% sucrose concentration in Cardinal and at 6% in Desiree (Figures2(e) and 2(f)).Polyamines (putrescine, spermidine, and spermine) contents increased significantly (P≤0.05) in potato plants by increasing sucrose concentration in MS medium however, this increase was less sharp in case of Desiree as compared to cultivar cardinal. The highest tested polyamines contents were observed when 8% sucrose was added in MS medium and the lowest contents of polyamines were observed at control in both cultivars of potato (Table 2).


### 3.4. Effect of Polyethylene Glycol (PEG) on Potato Plants

All the tested PEG concentrations greatly influenced most of the morphological growth parameters in both the cultivars. In Cardinal, a significant reduction was recorded in shoot/root length as the concentration of PEG was increased gradually in the MS medium from 0–10%. Shoot/root and node numbers also reduced gradually with increasing PEG concentrations (Table5). Growth responses were similar in Desiree plants where a continuous reduction in shoot/root length was recorded by increasing PEG concentrations in the medium. Fresh/dry weights of potato plants also exhibited a decreasing trend with increasing PEG concentration. There was a statistically significant difference in total protein contents of both the cultivars at various PEG treatments. Maximum values of protein content (2.157 and 2.419 mgg−1) were observed at 5% PEG concentration in Cardinal and at 10% concentration in Desiree, respectively. SOD activity also showed an overall increase with some exceptions in Cardinal, where 2.5% PEG treatment reduced the activity of SOD (Figures 2(g) and 2(h)).Table 5
Growth parameters of potato cultivars in response to osmotic stress induced by various levels of polyethylene glycol.
MediumPEG (%)Shoot length (cm)No. of shootsRoot length(cm)No. of rootsNo. of nodesFresh wt. (g)Dry wt. (g)CarDesCarDesCarDesCarDesCarDesCarDesCarDes∗P1010.20 ± 0.42a13.76 ± 0.65a2.60 ± 0.22a2.35 ± 0.20a10.65 ± 0.46a8.38 ± 0.90a8.85 ± 0.78a9.23 ± 0.53a16.25 ± 0.91a12.95 ± 0.74a0.67 ± 0.06a1.02 ± 0.06a0.06 ± 0.00a0.07 ± 0.0aP22.55.15 ± 1.07b8.64 ± 1.24b1.90 ± 0.17b1.70 ± 0.17b3.20 ± 0.73b3.83 ± 0.51b4.35 ± 1.15b4.36 ± 0.79b10.80 ± 1.26b9.45 ± 0.75b0.24 ± 0.07b0.24 ± 0.04b0.01 ± 0.00b0.02 ± 0.0bP351.19 ± 0.18c2.78 ± 0.41c1.25 ± 0.14c1.65 ± 0.22b0.36 ± 0.33c2.40 ± 0.58bc0.15 ± 0.10c1.90 ± 0.53c4.15 ± 0.58c6.50 ± 0.75c0.05 ± 0.01c0.09 ± 0.02c0.00 ± 0.00b0.01 ± 0.0bP4100.89 ± 0.07c0.99 ± 0.13c1.15 ± 0.08c1.05 ± 0.08c0.17 ± 0.17c0.84 ± 0.29c0.20 ± 0.20c0.65 ± 0.24c3.75 ± 0.31c3.60 ± 0.71d0.04 ± 0.00c0.05 ± 0.00c0.00 ± 0.00b0.01 ± 0.0bSignificance (P≤0.05)∗∗NS∗∗∗NS∗∗∗∗∗NS∗Results are mean from at least ten to replicate cultures ± S.E. Similar letters within the column do not differ significantly (P≤0.05) according to DMRT. Significant (∗) or nonsignificant (NS) at P≤0.05. ∗ MS [25] basal medium supplemented with different levels of polyethylene glycol (designated as P1 to P5).All the tested polyamines were increased by increasing concentrations of Polyethylene glycol (PEG; MW-4000) in MS medium. The highest putrescine contents (150.89 nmol·g−1 FW) were observed in plants treated with 10% of PEG. Spermidine contents were 112.60, 121.90, 122.25, and 131.15 nmol·g−1 FW in cardinal plants at 0, 2.5, 5, and 10 M of PEG, respectively. In Desiree plants, this increase in spermidine contents was 102.35, 111.70, 121.36, 131.15, and 122.05 nmol·g−1 FW at 0, 2.5, 5, and 10 M of PEG, respectively. Spermine contents also showed a similar increasing trend by increasing PEG concentrations in MS medium. Spermine contents were 110.95, 115.95, 122.15, and 129.20 nmol·g−1 FW in cardinal plants, and in Desiree plants, this increase in spermidine contents was 109.28, 114.37, 121.93, and 120.25 nmol·g−1 FW at 0, 2.5, 5, and 10 M of PEG, respectively (Table 2).


## 3.1. Effect of Various Concentrations of Sorbitol on Potato Plants

A significant decrease was observed in shoot length, number, and root length in both potato cultivars (Cardinal and Desiree) by increasing the concentration of sorbitol (Table1). In case of Cardinal plants, a decrease in shoot length from 14.833 to 6.094 cm was recorded by a gradual increase of sorbitol from 0 to 0.15 M in MS medium. Similarly, root length was reduced from 10.294 to 4.766 cm and this reduction in root length was significant at 0.10 and 0.15 M concentration of sorbitol. As in the case of cv. Cardinal, both shoot and root length in Desiree decreased from 13.527 to 4.244 cm and 10.444 to 5.794 cm, respectively, with an increasing sorbitol concentration in MS medium. Number of roots and nodes also showed a decreasing trend in both the cultivars. Number of shoots, however, exhibited an increase in Cardinal, and a decrease in Desiree. Sorbitol treatment also significantly decreased the total fresh/dry masses of Desiree plants. Fresh weight was decreased from 0.910 to 0.293 g and dry weight from 0.072 to 0.039 g at 0.15 M concentration. However, the effect of different concentrations of sorbitol was nonsignificant in the case of dry weights of Desiree plants. Overall, plants that were grown on MS medium without any sorbitol treatment were healthier compared with the plants grown over various sorbitol treatments. Furthermore, a change in leaf morphology was also observed at higher sorbitol levels (0.15 M), where leaves appeared relatively smaller in size and yellowish green in color (Figure 1).Table 1
Growth parameters of potato cultivars in response to osmotic stress induced by various levels of sorbitol.
MediumSorbitol (M)Shoot length (cm)No. of shootsRoot length (cm)No. of rootsNo. of nodesFresh wt. (g)Dry wt. (g)CarDesCarDesCarDesCarDesCarDesCarDesCarDes∗S1014.83 ± 0.41a13.52 ± 0.71a1.83 ± 0.18c3.11 ± 0.26a10.29 ± 0.51a10.44 ± 0.69a7.44 ± 0.47a8.72 ± 1.02a20.33 ± 0.41a16.27 ± 0.49a0.42 ± 0.03ab0.91 ± 0.09a0.03 ± 0.00a0.07 ± 0.01aS20.02514.68 ± 0.61a10.69 ± 0.81b3.44 ± 0.23a2.50 ± 0.28ab9.63 ± 0.49a9.92 ± 0.41ab6.33 ± 0.596.72 ± 0.66a20.27 ± 0.7313.33 ± 1.08b0.54 ± 0.05a0.83 ± 0.08ab0.03 ± 0.00a0.06 ± 0.00aS30.0512.250 ± 0.77b8.58 ± 1.10b2.50 ± 0.18b2.33 ± 0.32bc8.92 ± 0.80a9.21 ± 0.69ab6.55 ± 0.67a6.61 ± 0.87a19.72 ± 0.83a9.77 ± 0.96c0.29 ± 0.02b0.68 ± 0.10ab0.05 ± 0.03a0.05 ± 0.01aS40.108.58 ± 0.87c9.07 ± 0.73b3.33 ± 0.25a1.72 ± 0.22cd7.14 ± 0.68b8.53 ± 0.32b5.61 ± 0.81ab6.38 ± 0.55a15.94 ± 0.90b13.27 ± 0.90b0.32 ± 0.05b0.59 ± 0.08b0.03 ± 0.00a0.05 ± 0.01aS50.156.09 ± 0.69d4.24 ± 0.60c2.33 ± 0.22bc1.05 ± 0.05d4.76 ± 0.54c5.79 ± 0.84c3.94 ± 0.59b3.88 ± 0.61b13.11 ± 0.90c7.83 ± 0.88c0.29 ± 0.05b0.29 ± 0.04c0.02 ± 0.00a0.03 ± 0.00aSignificance (P≤0.05)∗∗∗∗∗∗∗∗∗∗∗∗NSNSResults are mean from at least ten to replicate cultures ± S.E. Similar letters within the column do not differ significantly (P≤0.05) according to DMRT. Significant (∗) or nonsignificant (NS) at P≤0.05. ∗MS [25] contains different levels of sorbitol (designated as S1 to S5; sorbitol level as shown in the next column).Figure 1
Growth of CVS. Cardinal (a)–(e) and Desiree (f)–(j) at different sorbitol concentrations. Scale bar. (a) = 1.5 cm, (b)–(c) = 2.0 cm, (d) = 1.5 cm, (e) = 1.2 cm, (f)–(g) = 2.0 cm, (h) = 1.5 cm, (i) = 1.7 cm, and (j) = 1.0 cm.There was a significant difference in sorbitol treatment on total protein contents in both the cultivars. Proteins increased gradually by a value of 0.23 mgg−1 to 0.85 mgg−1 at 0 to 0.10 M sorbitol concentration. However, at a higher concentration of sorbitol (0.15 M), a decrease (0.79 mgg−1) in its content was recorded as compared to 0.10 M. SOD activity was also found to increase significantly in sorbitol-treated Cardinal plants as compared to control. However, the increase was not gradual as the maximum value for SOD activity (32.25 Umg−1 of protein) was observed at 0.025 M sorbitol concentration, which was followed by a progressive reduction in SOD activity with increasing sorbitol concentration in the medium. As indicated in Figures 2(a) and 2(b), SOD activity increased gradually by increasing sorbitol concentration in case of Desiree plants as compared to control ones. However, its value was decreased to 14.29 Umg−1 of protein at 0.15 M sorbitol level as compared to plants without any sorbitol treatment, i.e., 19.51 Umg−1 of protein.Figure 2
Protein contents and superoxide dismutase activity in potato cultivars cardinal (Car) and Desiree (Ds) in response to sorbitol (a) and (b) mannitol (c) and (d), sucrose (e) and (f) and PEG (g) and (h) induced osmotic stress. Results are the means ± S.E. of ten replicates cultures. Bars with similar letters are not significantly different atP≤0.05 by Duncan’s multiple range test.
(a)(b)(c)(d)(e)(f)(g)(h)The contents of endogenous polyamines in both cultivars of potato increase significantly by adding various concentrations of sorbitol in MS medium and the highest contents of all the investigated polyamines were observed in plants growing on 0.15 M of sorbitol. putrescine contents increased from 125.23 (control) to 150.89 nmol·g−1 FW (10 M) in cv. Cardinal, however, in Desiree this increase in putrescine contents was not as higher as in case of cardinal. Spermidine also showed an increasing trend by increasing concentrations of sorbitol from 2.5 to 10 M. Similarly, spermine also showed an increasing trend but its amount was less as compared to putrescine and spermidine. Potato cv. Cardinal showed less increase in polyamine contents as compared to Desiree (Table2).Table 2
Effect of various levels of sorbitol, mannitol, sucrose, and PEG on polyamines contents (putrescine, spermidine, and spermine) of potato plants.
MediumPutrescine (nmol·g−1 FW)Spermidine (nmol·g−1 FW)Spermine (nmol·g−1 FW)CultivarCarDesCarDesCarDes(Control) without sorbitol145.13 ± 3.42b143.56 ± 3.67a132.62 ± 3.23c122.45 ± 3.24c118.75 ± 2.78e119.28 ± 2.33dMS + 0.025 M sorbitol135.35 ± 2.07c148.34 ± 3.25a141.30 ± 2.27b131.73 ± 3.27b124.45 ± 1.15d124.46 ± 3.79cMS + 0.05 M sorbitol147.22 ± 1.18b142.28 ± 2.42a142.27 ± 2.14b133.65 ± 2.12b130.25 ± 3.13c129.33 ± 2.53bMS + 0.10 M sorbitol155.80 ± 2.27a145.39 ± 2.23a151.13 ± 2.28a142.35 ± 2.38a139.29 ± 3.20b130.13 ± 1.21bMS + 0.15 M sorbitol158.23 ± 1.37a147.32 ± 2.53a155.15 ± 2.78a143.05 ± 2.38a149.29 ± 3.20a140.03 ± 1.31a(Control) without mannitol115.23 ± 3.42e113.26 ± 3.67c122.20 ± 3.23c122.15 ± 3.34b128.95 ± 2.28d119.18 ± 2.23cMS + 0.025 M mannitol119.15 ± 1.27d118.34 ± 1.35b131.30 ± 2.27b124.72 ± 3.27b124.95 ± 1.13d114.27 ± 3.19cMS + 0.05 M mannitol127.29 ± 1.28c121.28 ± 2.12b129.25 ± 2.14b131.35 ± 1.22a130.15 ± 1.12c124.73 ± 2.53bMS + 0.10 M mannitol133.89 ± 2.37b121.29 ± 2.23b132.35 ± 2.28b131.15 ± 2.18a139.20 ± 3.20b129.35 ± 1.34aMS + 0.15 M mannitol140.89 ± 1.47a132.99 ± 1.33a134.25 ± 2.28a132.05 ± 2.28a149.20 ± 1.31a130.15 ± 1.22a(Control) without sucrose145.29 ± 2.42c143.26 ± 1.67d132.10 ± 3.43e132.25 ± 3.24c128.15 ± 2.78c129.08 ± 2.63cMS + 2% sucrose151.25 ± 1.07b144.34 ± 1.25bc134.20 ± 2.37d131.72 ± 2.97c129.25 ± 1.95c124.31 ± 2.79dMS + 3% sucrose157.21 ± 2.18b147.38 ± 2.42b139.05 ± 2.24c134.35 ± 2.12c130.35 ± 3.33c129.93 ± 2.53cMS + 4% sucrose159.80 ± 2.27a140.29 ± 1.23bc141.15 ± 2.08b142.15 ± 2.08b139.22 ± 3.30b140.22 ± 1.34bMS + 6% sucrose160.82 ± 2.27a150.29 ± 2.23a139.35 ± 2.28d142.15 ± 1.98b139.91 ± 3.32b140.23 ± 1.24bMS + 8% sucrose162.83 ± 2.27a152.19 ± 2.23a151.15 ± 1.28a152.25 ± 2.08a149.23 ± 3.31a150.21 ± 1.14a(Control) without PEG125.23 ± 3.42c123.76 ± 3.67c112.60 ± 3.23c102.35 ± 3.24b110.95 ± 2.78bc109.28 ± 2.63bMS + 2.5% PEG135.15 ± 2.07b128.64 ± 3.25bc121.90 ± 2.27b111.70 ± 3.27ab115.95 ± 1.15b114.37 ± 3.79bMS + 5% PEG137.29 ± 1.18b132.78 ± 2.42b122.25 ± 2.14b121.65 ± 2.12a122.15 ± 3.13b121.93 ± 2.53aMS + 10% PEG150.89 ± 2.27a140.99 ± 2.23a131.15 ± 2.28a122.05 ± 2.38a129.20 ± 3.30a120.25 ± 1.24aResults are mean from at least ten to replicate cultures ± S.E. Similar letters within the column do not differ significantly (P≤0.05) according to DMRT. Significant (∗) or non-significant (NS) at P≤0.05. ∗MS [25] contains different levels of sorbitol (designated as S1 to S5; sorbitol level as shown in the next column).


## 3.2. Effect of Various Concentrations of Mannitol on Potato Plants

Maximum shoot/root length (12.565 and 9.145 cm, respectively) was observed at 0 M and minimum (2.360 and 2.896 cm, respectively) at 0.15 M mannitol concentration. Similar results were observed for a number of roots and nodes. However, the results were different in case of number of shoots, where an increase in their number was observed with increasing stress levels in MS medium (Figure3). It is evident from the data given in Table 3 that mannitol treatment to Desiree plants had a significant effect on all the studied growth parameters. Reduction in the growth of several parameters including shoot/root length, shoot/root, and number of nodes was recorded as the concentration of mannitol was higher in the MS medium. Table 3 also indicates that mannitol treatment had a significant effect on the fresh weight of both the cultivars. Dry weight of plants was also affected by mannitol applications; however, it was not as significant as was in case of fresh weights in cardinal but significant in case of Desiree.Figure 3
Growth of Cardinal (a)–(e) and Desiree (f)–(j) at different mannitol concentrations. Scale bar (a)–(j) = 1 cm.Table 3
Growth parameters of potato cultivars in response to osmotic stress induced by various levels of mannitol.
MediumMannitol (M)Shoot length (cm)No. of shootsRoot length (cm)No. of rootsNo. of nodesFresh wt. (g)Dry wt. (g)CarDesCarDesCarDesCarDesCarDesCarDesCarDes∗M1012.56 ± 1.02a14.21 ± 0.51a1.65 ± 0.23a3.22 ± 0.19a9.14 ± 0.86a11.63 ± 0.70a6.35 ± 0.70a10.27 ± 0.73a16.90 ± 1.40a16.72 ± 0.35a0.36 ± 0.03a0.99 ± 0.06a0.03 ± 0.00ab0.06 ± 0.01abcM20.02510.36 ± 0.96a12.59 ± 0.72a2.05 ± 0.23a2.61 ± 0.14ab7.23 ± 0.78ab9.86 ± 0.51ab5.05 ± 0.60ab8.38 ± 0.61b17.20 ± 1.49a15.94 ± 0.51a0.35 ± 0.04a0.91 ± 0.08a0.03 ± 0.00a0.09 ± 0.01aM30.056.80 ± 0.99b10.08 ± 0.65b2.10 ± 0.28a2.88 ± 0.26ab6.82 ± 0.87b10.67 ± 0.61ab4.95 ± 0.88ab7.44 ± 0.53bc13.65 ± 0.69ab11.16 ± 0.60b0.28 ± 0.05a0.81 ± 0.07a0.03 ± 0.00ab0.08 ± 0.01abM40.104.49 ± 0.66bc5.35 ± 0.51c2.25 ± 0.27a2.27 ± 0.33b4.26 ± 0.54c9.56 ± 0.64b5.10 ± 0.03ab5.94 ± 0.80c10.30 ± 0.23bc9.33 ± 0.70c0.24 ± 0.04ab0.40 ± 0.05b0.03 ± 0.00ab0.05 ± 0.01bcM50.152.36 ± 0.38c5.13 ± 0.42c1.90 ± 0.27a2.16 ± 0.25b2.89 ± 0.54c7.25 ± 0.58c3.35 ± 0.75b3.77 ± 0.29d7.25 ± 0.97c10.27 ± 0.51c0.13 ± 0.02b0.31 ± 0.02b0.02 ± 0.00b0.04 ± 0.01cSignificance (P≤0.05)∗∗NS∗∗∗NS∗∗∗∗∗NSNSResults are mean from at least ten to replicate cultures ± S.E. Similar letters within 7the column do not differ significantly (P≤0.05) according to DMRT. Values are significant (∗) or nonsignificant (NS) at P≤0.05. ∗MS [25] was supplemented with different levels of mannitol (designated as M1 to M5).An overall increase in total proteins was recorded at all mannitol levels in both the cultivars of potato. In case of control Cardinal plants, protein content (0.26 mgg−1) increased gradually to 0.85 mgg−1 at 0.15 M treatment. Similar increase in its value was also observed for Desiree where the change in its values was from 0.16 (in control plants) to 1.15 mgg−1 at the highest (0.15 M) mannitol level. SOD activity was greatly influenced by the addition of mannitol in the MS medium. In case of Desiree, mannitol treatment resulted in an increase of SOD activity, with its highest value (66.6 Umg−1 of protein) observed at 0.15 M. With some minor variations, osmotic stress (induced by mannitol) also resulted in increased activity of SOD in Cardinal plants (Figures 2(c) and 2(d)). In this case, SOD activity was decreased (24.14 Umg−1 of protein) at 0.025 M mannitol as compared to control ones (27.64 Umg−1 of protein).All the tested polyamines were increased by increasing concentrations of mannitol in MS medium. Both cultivars of potato plants showed a significant (P≤0.05) increase in polyamines however, in Desiree plants, this increase was higher than in the cardinal plants. The highest putrescine contents were observed in plants treated with 0.15 M of mannitol. Spermidine contents was 122.20, 131.30, 129.25, 132.35, and 134.25 nmol·g−1 FW in cardinal plants at 0, 0.025, 0.5, 0.1, and 0.15 M of mannitol, respectively. In Desiree plants, this increase in spermidine contents was 122.15, 124.72, 131.35, 131.15, and 132.05 nmol·g−1 FW at 0, 0.025, 0.5, 0.1, and 0.15 M of mannitol, respectively. Spermine contents also showed a similar increasing trend by increasing mannitol concentration in MS medium (Table 2).


## 3.3. Effect of Sucrose-Induced Osmotic Stress on Potato Plants

The effect of sucrose was statistically significant on shoot growth, its number, and on number of roots in both the cultivars of potato with a maximum value at 3% sucrose level; and a gradual decrease as the concentration varied. Highest value of root length was recorded at 4% sucrose in Cardinal followed by 2% sucrose concentration (Table4). All the abovementioned parameters showed a decreasing trend by increase or decrease in sucrose concentration from 3% except for the root number, where an increase was recorded at 8% sucrose. For Cardinal, number of nodes decreased significantly with a gradual increase in sucrose concentration from 3%. Fresh weight exhibited in general a decrease, while dry weight exhibited an increasing trend in both the cultivars as the concentration of sucrose varied from 3 to 8%.Table 4
Growth parameters of potato cultivars in response to osmotic stress induced by various levels of sucrose.
MediumSucrose (%)Shoot length (cm)No. of shootsRoot length(cm)No. of rootsNo. of nodesFresh wt.(g)Dry wt.(g)CarDesCarDesCarDesCarDesCarDesCarDesCarDes∗S103.16 ± 0.47d7.06 ± 0.93c1.11 ± 0.07b0.55 ± 0.12c0.18 ± 0.18b4.48 ± 0.92b0.05 ± 0.05b1.44 ± 0.42b11.61 ± 0.93c11.22 ± 0.85a0.05 ± 0.00c0.09 ± 0.01c0.00 ± 0.00c0.00 ± 0.0bS228.98 ± 0.51bc11.64 ± 0.90ab5.30 ± 1.18a6.51 ± 1.91ab5.03 ± 0.91a8.35 ± 1.66a3.77 ± 0.28a3.94 ± 0.59b13.38 ± 0.78bc11.61 ± 0.74a0.178 ± 0.01b0.32 ± 0.03b0.01 ± 0.0bc0.01 ± 0.0bS3313.31 ± 0.68a13.47 ± 0.57a6.04 ± 1.30a10.20 ± 2.48a4.13 ± 0.63a5.46 ± 1.01ab5.11 ± 0.46a7.83 ± 0.74a17.44 ± 0.77a12.50 ± 0.60a0.35 ± 0.02a0.81 ± 0.09a0.02 ± 0.0ab0.05 ± 0.0aS4411.37 ± 0.15ab11.80 ± 0.84ab4.23 ± 0.90a5.00 ± 0.88b5.49 ± 0.90a6.04 ± 1.06ab4.88 ± 0.52a8.33 ± 1.49a15.16 ± 1.05ab10.33 ± 0.63a0.34 ± 0.05a0.84 ± 0.11a0.03 ± 0.0a0.08 ± 0.1aS568.42 ± 1.36c11.15 ± 0.85ab4.43 ± 0.78a4.28 ± 0.82bc4.08 ± 0.63a4.82 ± 0.75b3.83 ± 0.64a7.61 ± 1.08a12.33 ± 1.18bc11.11 ± 0.79a0.28 ± 0.04ab0.75 ± 0.09a0.03 ± 0.0a0.07 ± 0.1aS686.96 ± 0.90c9.83 ± 0.83b3.82 ± 0.73a5.17 ± 1.07b3.95 ± 0.85a4.88 ± 0.77b6.44 ± 1.13a9.83 ± 1.30a11.88 ± 1.24c10.55 ± 0.77a0.35 ± 0.06a0.69 ± 0.05a0.04 ± 0.01a0.08 ± 0.0aSignificance (P≤0.05)∗∗NS∗∗NSNS∗∗NS∗∗NSNSResults are mean from at least ten to replicate cultures ± S.E. Similar letters within the column do not differ significantly (P≤0.05) according to DMRT. Significant (∗) or nonsignificant (NS) at P≤0.05. ∗MS [25] medium supplemented with different levels of sucrose (designated as S1 to S6).In Cardinal plants, total protein contents decreased gradually at 2, 4, and 6% sucrose concentration and exhibited an increase at 0 and 8% sucrose levels while in Desiree, protein content increased at all the stress levels induced by sucrose. SOD activity was also significantly increased in both potato cultivars at different sucrose concentrations except at 2%, where a decrease in its level was observed. Highest SOD activity was observed at 8% sucrose concentration in Cardinal and at 6% in Desiree (Figures2(e) and 2(f)).Polyamines (putrescine, spermidine, and spermine) contents increased significantly (P≤0.05) in potato plants by increasing sucrose concentration in MS medium however, this increase was less sharp in case of Desiree as compared to cultivar cardinal. The highest tested polyamines contents were observed when 8% sucrose was added in MS medium and the lowest contents of polyamines were observed at control in both cultivars of potato (Table 2).


## 3.4. Effect of Polyethylene Glycol (PEG) on Potato Plants

All the tested PEG concentrations greatly influenced most of the morphological growth parameters in both the cultivars. In Cardinal, a significant reduction was recorded in shoot/root length as the concentration of PEG was increased gradually in the MS medium from 0–10%. Shoot/root and node numbers also reduced gradually with increasing PEG concentrations (Table5). Growth responses were similar in Desiree plants where a continuous reduction in shoot/root length was recorded by increasing PEG concentrations in the medium. Fresh/dry weights of potato plants also exhibited a decreasing trend with increasing PEG concentration. There was a statistically significant difference in total protein contents of both the cultivars at various PEG treatments. Maximum values of protein content (2.157 and 2.419 mgg−1) were observed at 5% PEG concentration in Cardinal and at 10% concentration in Desiree, respectively. SOD activity also showed an overall increase with some exceptions in Cardinal, where 2.5% PEG treatment reduced the activity of SOD (Figures 2(g) and 2(h)).Table 5
Growth parameters of potato cultivars in response to osmotic stress induced by various levels of polyethylene glycol.
MediumPEG (%)Shoot length (cm)No. of shootsRoot length(cm)No. of rootsNo. of nodesFresh wt. (g)Dry wt. (g)CarDesCarDesCarDesCarDesCarDesCarDesCarDes∗P1010.20 ± 0.42a13.76 ± 0.65a2.60 ± 0.22a2.35 ± 0.20a10.65 ± 0.46a8.38 ± 0.90a8.85 ± 0.78a9.23 ± 0.53a16.25 ± 0.91a12.95 ± 0.74a0.67 ± 0.06a1.02 ± 0.06a0.06 ± 0.00a0.07 ± 0.0aP22.55.15 ± 1.07b8.64 ± 1.24b1.90 ± 0.17b1.70 ± 0.17b3.20 ± 0.73b3.83 ± 0.51b4.35 ± 1.15b4.36 ± 0.79b10.80 ± 1.26b9.45 ± 0.75b0.24 ± 0.07b0.24 ± 0.04b0.01 ± 0.00b0.02 ± 0.0bP351.19 ± 0.18c2.78 ± 0.41c1.25 ± 0.14c1.65 ± 0.22b0.36 ± 0.33c2.40 ± 0.58bc0.15 ± 0.10c1.90 ± 0.53c4.15 ± 0.58c6.50 ± 0.75c0.05 ± 0.01c0.09 ± 0.02c0.00 ± 0.00b0.01 ± 0.0bP4100.89 ± 0.07c0.99 ± 0.13c1.15 ± 0.08c1.05 ± 0.08c0.17 ± 0.17c0.84 ± 0.29c0.20 ± 0.20c0.65 ± 0.24c3.75 ± 0.31c3.60 ± 0.71d0.04 ± 0.00c0.05 ± 0.00c0.00 ± 0.00b0.01 ± 0.0bSignificance (P≤0.05)∗∗NS∗∗∗NS∗∗∗∗∗NS∗Results are mean from at least ten to replicate cultures ± S.E. Similar letters within the column do not differ significantly (P≤0.05) according to DMRT. Significant (∗) or nonsignificant (NS) at P≤0.05. ∗ MS [25] basal medium supplemented with different levels of polyethylene glycol (designated as P1 to P5).All the tested polyamines were increased by increasing concentrations of Polyethylene glycol (PEG; MW-4000) in MS medium. The highest putrescine contents (150.89 nmol·g−1 FW) were observed in plants treated with 10% of PEG. Spermidine contents were 112.60, 121.90, 122.25, and 131.15 nmol·g−1 FW in cardinal plants at 0, 2.5, 5, and 10 M of PEG, respectively. In Desiree plants, this increase in spermidine contents was 102.35, 111.70, 121.36, 131.15, and 122.05 nmol·g−1 FW at 0, 2.5, 5, and 10 M of PEG, respectively. Spermine contents also showed a similar increasing trend by increasing PEG concentrations in MS medium. Spermine contents were 110.95, 115.95, 122.15, and 129.20 nmol·g−1 FW in cardinal plants, and in Desiree plants, this increase in spermidine contents was 109.28, 114.37, 121.93, and 120.25 nmol·g−1 FW at 0, 2.5, 5, and 10 M of PEG, respectively (Table 2).


## 4. Discussion

Drought stress is usually found to reduce morphological characteristics of the plants including leaf area, stem height, root number, and tuber yield under field and in vitro conditions [24, 29]. This is because cell expansion and growth are suppressed due to loss of turgor pressure or osmotic imbalance, which in turn reduces the plant growth and activity of all the metabolic processes [30]. It is evident from the literature that plant maintains the osmotic equilibrium by producing and enhancing the level of many osmotica for instance pinitol, mannitol, sucrose, sorbitol, trehalose, etc., [31, 32]. According to them, accumulation of these omotica resulted in an increase in solute concentration significantly. Furthermore, they also play a vital role in the protection of the cells from damage caused by dehydration [33]. However, it was also observed that when these osmotica accumulate in higher concentrations, they impair the growth in plants [34] and work as stressing agents [35]. Results of the present study indicate that all the studied osmotica (sorbitol, sucrose, mannitol, and PEG) induce osmotic stress in potato plants under in vitro conditions in both cultivars. These osmotic agents are known to produce drought like conditions as prevailing under field conditions [36]. It is well evident from the literature that sorbitol, sugar alcohol, was used in various in vitro experiments to induce osmotic stress in the medium [37–39]. In the present study, treatment of potato plants with various levels of sorbitol resulted in a decrease in most of the growth parameters in both the tested potato cultivars. This decline in growth due to sorbitol-induced stress was also reported by Gopal and Iwama [24], who studied the same pattern in several growth parameters. The growth reduction of in vitro-grown plants at higher concentrations of sorbitol could also be due to the accumulation of phenolic compounds in the medium [40, 41]. This may be due to the fact that drought can lead to disruption of metabolism and cell structure and ultimately to the termination of enzymatic reactions of the plants [42]. Sucrose is an important carbohydrate source in tissue culture medium besides being an osmoticum [43, 44]. Significant differences in growth parameters of potato plants grown on sucrose stressed media exhibited a similar picture as previously reported by Custodio et al. [43] those high concentrations of sucrose as compared to other carbon sources were responsible for an increased rooting index of Ceratonia siliqua. An increase in fresh/dry weights of plants in response to sucrose-induced osmotic stress might be due to the accumulation of sucrose in the plant tissues with its increase in the medium. Sucrose acts as a fuel source for sustaining photo mixotrophic metabolism [45]. This increase in root number at high sucrose level might be due to the fact that high sucrose level favors the formation of storage roots at high frequencies [46].In the present study, the addition of PEG to the culture medium affected most of the growth parameters negatively in both cultivars. A similar effect of PEG on growth has also been reported in other plant species, e.g., mulberry [47], Lycopersicon esculentum Mill [48], and cherry plants [49]. This decrease in growth parameters of plants in the present study might be due to the low water content at high PEG treatments, which results in less availability of water for cell expansion. During the present study, tuber formation was observed in osmotic-stressed potato plants, especially when higher levels of sorbitol, mannitol, and sucrose were added to MS medium. Maximum number of tubers was recorded at 0.10 and 0.15 M sorbitol and mannitol and at 6 and 8% sucrose concentrations in Desiree and Cardinal, respectively. Tuber formation at higher concentrations of these osmotica is a general phenomenon because of their absorbing nature in plant tissue.Quantitative analysis of the protein content revealed an increasing trend generally in both the potato cultivars (Cardinal and Desiree) when subjected to osmotic stress induced by various osmotic. The reason for an increased level of protein content under stressed conditions induced by different osmotica in the present study might be due to the synthesis of some stress-associated proteins in response to drought stress. These stress-associated proteins have been reported to be soluble in water, which contributes to stress tolerance by cellular dehydration [50]. Zang and Komatsu [51] reported the expression of some new proteins in rice in response to mannitol-induced drought stress. Under various stress conditions, it was observed that several reactive oxygen species (ROS) are also formed in plant cells, for instance, superoxide radicals (O2-), singlet oxygen, hydroxyl radicals (OH), and hydrogen peroxide. SOD plays a vital role in the scavenging of superoxide radicals [48]. Previously, several workers have reported that the activity of SOD increases significantly in plants as a result of various stresses, especially water stress [52–55]. In our work, an overall increase in SOD activity was recorded under sorbitol, mannitol, sucrose, or PEG-induced water deficit. This increase in SOD activity was also reported in mannitol or sorbitol-treated apple plants by Molassiotis et al. [19]. Wang and Li [56] also reported an increase in total leaf and chloroplastic SOD activity in white clover, which were grown on a medium containing different concentrations of PEG. Similar results were also observed by Wang et al. [57] in some plant species of Trifolium, where the application of PEG to the medium resulted in an enhancement of SOD activities and its various isozymes. On the basis of our results, it might be suggested that SOD is one of the major scavenger enzymes within the antioxidant defense system of potatoes under drought stress induced by sorbitol, mannitol, sucrose, or PEG. During the present investigation polyamines increase significantly in both cultivars of potato by using various osmotica. Polyamines have a protective role on plants under various abiotic stress by regulating and increasing the uptake of various inorganic ions directly, which in turn enhances stress tolerance in plants [10, 58]. Furthermore, an increase in the activity of SOD under various osmotic stress-inducing agents during the present study may justify the role of polyamines in modulating the homeostasis of reactive oxygen species.


## 5. Conclusions

In conclusion, all measured growth parameters were sensitive to osmotic stress induced by sorbitol, mannitol, sucrose, or PEG with severe reduction observed at higher levels of these osmotica. This reduction was more in the case of PEG-treated potato plants as compared to others. The increase in the level of protein content and superoxide dismutase activity suggested their major role in the detoxification of reactive oxygen species and compensation of drought stress conditions. Desiree plants performed comparatively better under osmotic stress by showing less reduction in growth. The use of compounds that do not interact with plants in any other way than lowering the water potential of the medium like PEG is more valuable to study the drought stress mechanism as compared to the compound having absorbing nature like sorbitol, mannitol, or sucrose. Viscous solutions of PEG limit the movement of oxygen ultimately resulting in oxygen deficiency and other toxic effects. Keeping all these points in view, one may conclude that the use of sorbitol or mannitol might be a better choice for further studies on drought stress mechanisms in potatoes. However, there is a need for further studies on these and related parameters before conclusive evidence could be presented.



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*Source: 5158768-2022-09-10.xml*