**2.2. Squirting cucumber (***Ecballium elaterium* **(L.) A. Rich.) fruit juice**

2105.74 μg of chlorophyll/g fresh tissue, respectively, for the treatment with a magnetic field

**Figure 2.** The effect of a magnetic field strength of 150 mT applied to potato tubers for different periods (a. 0 h, b. 24 h, c.

48 30.75 d 40.25 d 1327.69 e 72 29.25 d 42.45 d 1321.12 e

48 21.25 b 88.27 a 2000.27 ab 72 17.00 a 90.78 a 2105.74 a

48 26.25 c 77.27 b 1558.34 d 72 20.50 b 78.00 b 1789.46 c

**Table 3.** The effect of different magnetic field strengths applied to potato tubers for different periods of time on the day

**sprouts Plant height (cm) Total chlorophyll content (μg** 

**chlorophyll/g fresh tissue)**

**Day of emergence of** 

0—control 0 39.50 e 25.56 e 1127.46 g 75 24 31.25 d 39.00 d 1214.56 f

150 24 26.00 c 80.35 b 1927.36 b

300 24 31.00 d 75.76 c 1500.48 d

Each value is the mean of five replications. All experiments were repeated two times. Values within a column followed by different letters are significantly different at the 0.01 level.

of emergence of sprouts, plant height, and total chlorophyll content in cv. "Marabel".

strength of 150 mT for 72 h (**Table 3** and **Figure 2**).

48 h and d. 72 h) on plant development in cv. "Marabel".

**Treatment period (h)**

**Magnetic field strength (mT)**

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Squirting cucumber (*Ecballium elaterium* (L.) A. Rich.), which is commonly found in Turkey, is an important medicinal plant [29]. It contains different compounds such as α-elaterin (cucurbitacin E), β-elaterin (cucurbitacin B), elatericine A [30], and elatericine B (cucurbitacin I) in different plant organs [31]. It also contains sterols, phenolic compounds, vitamins, flavonoids, alkaloids, resin, starch, amino acids, and fatty acids [31]. *In vitro* regeneration was affected significantly by squirting cucumber fruit juice [32]. It was also reported that squirting cucumber fruit juice stimulated rapid germination and seedling growth in rapeseed [33].

Mature squirting cucumber fruits were collected from their natural growing areas around Ankara. Fruit juice was extracted and then filtered in order to remove the bigger parts. The fruit juice was subject to sterile filtration and kept in the refrigerator at −20°C.

In a study conducted for overcoming dormancy in cv. "Marabel" potato tubers using squirting cucumber mature fruit juice, tubers at 40–60 g in weight were rinsed for 6 h at 180 rpm in bottles containing water with different concentrations of mature squirting cucumber fruit juice (0-control, 200, 400, 800, and 1600 μl/L). Then, the tubers were planted in pots. Ten pots were used for each concentration, and one tuber was put in each pot. A pot was considered an experimental unit. The study used two parallel treatments according to the Completely Randomized Design concept. Data were statistically analyzed by Duncan's multiple range test using IBM SPSS Statistics 22 software. Values presented in percentages were subjected to arcsine (√*X*) transformation before statistical analysis [19].

Results clearly showed that squirting cucumber fruit juice had a significant role in overcoming dormancy in potato tubers. The best result was recorded at a concentration of 800 μl/L of fruit juice as 16.25 days. That meant that the sprouts of tubers treated with 800 μl/L of fruit juice emerged above the ground 16.25 days after planting. In contrast, in the control treatment where no fruit juice was used, sprouts emerged above ground later than the other treatments. From the results of the study, it could be concluded that squirting cucumber fruit juice stimulated sprout development by overcoming dormancy in potato tubers (**Table 4**).

In **Figure 3**, plant development was recorded for tubers treated with different concentrations of squirting cucumber fruit juice at the end of the 45th day. For a concentration of 800 μl/L of fruit juice, the plants grew better than any of the others. Squirting cucumber fruit juice at a concentration of 800 μl/L encouraged plants to develop faster and with more energy by overcoming dormancy in tubers. At this concentration, plants had more branches and leaves.

#### **2.3. Sodium hypochlorite solutions**

Sodium hypochlorite (NaOCl) has been most widely used for surface sterilization. NaOCl is highly effective against all kinds of bacteria, fungi, and viruses [34, 35]. Moreover, NaOCl has strong oxidizing properties that make it highly reactive with amino acids [36, 37], nucleic acids [38], amines, and amides [39, 40].

The most important treatment prior to culture initiation is perhaps surface-sterilization of the explant. Since *in vitro* conditions provide bacteria and fungi with an optimal growth


Each value is the mean of five replications. All experiments were repeated two times. Values within a column followed by different letters are significantly different at the 0.01 level.

**Table 4.** The effect of different concentrations of squirting cucumber fruit juice on the day of emergence of sprouts in potato cv. "Marabel".

**Figure 3.** Plant development at the 45th day for potato tubers of the cv. "Marabel" treated with different concentrations of squirting cucumber fruit juice.

environment, unsuccessful sterilization hinders the progress of tissue culture studies. On the one hand, sterilization of the tissue aims to eliminate all microorganisms that can easily grow *in vitro* conditions; on the other hand, it should guarantee the explant's viability and regeneration capacity, which are known to be affected by the concentration, treatment period [41], and temperature of the disinfectant [42].

NaOCl can also be used for overcoming dormancy [43–45] by decomposing germination inhibitors [46], scarifying the seed coat [42, 47], and increasing α-amylase activity [48].

In the study conducted by Telci et al. [49], sodium hypochlorite (NaOCl) solution was successfully used to overcome dormancy in the seeds of *Lathyrus chrysanthus* Boiss., which is used as an ornamental plant with its big, colorful flowers. In the study, *L. chrysanthus* Boiss. seeds of an ecotype (Diyarbakir) from southeast Turkey were treated with a 3.75% NaOCl solution at three different temperatures (25, 35, and 45°C) for 15 min with continuous stirring. This was followed by rinsing three times with sterile water. Seeds were then germinated on a basal medium containing Murashige and Skoog's (MS) mineral salts and vitamins [50], 3% sucrose, and 0.7% agar in Magenta vessels (15 × 15 cm). The pH of the medium was adjusted to 5.8 prior to autoclaving. For seed germination, cultures were incubated at 15±1°C in the dark for 5 days. Then, all cultures were transferred to a growth chamber for incubation at 25±1°C under cool white fluorescent light (27 μmol m−2 s−1) with a 16-h light/8-h dark photoperiod. Seed germination and seedling growth percentages were recorded after 5 and 14 days following culture initiation, whereas seedling height and root length, seedling fresh and dry weights, chlorophyll a, chlorophyll b, and total chlorophyll contents were noted 28 days after culture initiation. The chlorophyll contents were determined in the leaves of seedlings according to the protocol of Curtis and Shetty [21]. All statistical analyses were performed using SPSS for Windows software. Three replicates were tested. Petri dishes (100 × 10 mm) were considered the units of replication, and there were 30 seeds per replication. All experiments were repeated twice. One-way Analysis of Variance (ANOVA) was used to test the effect of the 3.75% NaOCl solution at different temperatures. Duncan's multiple range test was used for comparing the means. Data given in percentages were subjected to arcsine (√*X*) transformation before statistical analysis [19].

The lowest values were recorded in a 3.75% NaOCl solution at a 45°C temperature. Low results at 45°C could be attributed to the fact that the activity of NaOCl increases [51], and it penetrates more easily through the seed coat [52]. The highest results in all characteristics examined were obtained from a 3.75% NaOCl solution at 35°C. Seed germination and seedling growth percentages decreased to 67.76 and 53.53% at 45°C, while they were 88.74 and 77.74% in a 3.75% NaOCl solution at 35°C. Seedling height and root length were 6.77 and 9.26 cm in a 3.75% NaOCl solution at 35°C temperature (**Table 5** and **Figure 4**). These findings were parallel to those of Hsiao and Hans [53], Hsiao and Quick [54], and Yildiz and Er [42] who reported that disinfectants at high concentrations and high temperatures affected seed germination and seedling growth negatively.

The highest seedling fresh and dry weights and tissue water content were recorded when seeds were treated with a 3.75% NaOCl solution at 35°C for 15 min (**Table 5**). The fresh weight


environment, unsuccessful sterilization hinders the progress of tissue culture studies. On the one hand, sterilization of the tissue aims to eliminate all microorganisms that can easily grow *in vitro* conditions; on the other hand, it should guarantee the explant's viability and regeneration capacity, which are known to be affected by the concentration, treatment period

**Figure 3.** Plant development at the 45th day for potato tubers of the cv. "Marabel" treated with different concentrations

**Table 4.** The effect of different concentrations of squirting cucumber fruit juice on the day of emergence of sprouts in

**Concentration of squirting cucumber fruit juice (μl/L) Day of emergence of sprouts**

0—control 31.50 c 200 23.00 bc 400 22.00 bc 800 16.25 a 1600 20.00 b

potato cv. "Marabel".

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Each value is the mean of five replications. All experiments were repeated two times. Values within a column followed by different letters are significantly different at the 0.01 level.

NaOCl can also be used for overcoming dormancy [43–45] by decomposing germination

In the study conducted by Telci et al. [49], sodium hypochlorite (NaOCl) solution was successfully used to overcome dormancy in the seeds of *Lathyrus chrysanthus* Boiss., which is used as an ornamental plant with its big, colorful flowers. In the study, *L. chrysanthus* Boiss. seeds of an ecotype (Diyarbakir) from southeast Turkey were treated with a 3.75% NaOCl solution at three different temperatures (25, 35, and 45°C) for 15 min with continuous stirring. This was followed by rinsing three times with sterile water. Seeds were then germinated on a basal medium containing Murashige and Skoog's (MS) mineral salts and vitamins [50], 3%

inhibitors [46], scarifying the seed coat [42, 47], and increasing α-amylase activity [48].

[41], and temperature of the disinfectant [42].

of squirting cucumber fruit juice.

Values in a column followed by different letters are significantly different at the 0.01 level in germination percentage, root length, seedling fresh, and dry weights and chlorophyll a content, while significantly different at the 0.05 level in seedling growth, seedling height, chlorophyll b, and total chlorophyll contents. 1 Seedling growth means seedlings developed out of the total germinated seeds.

**Table 5.** The effects of a 3.75% NaOCl solution at different temperatures on *in vitro* seed germination, seedling growth, seedling height, root length, seedling fresh and dry weights, chlorophyll a, chlorophyll b, and total chlorophyll contents in the leaves of *L. chrysanthus* Boiss. seedlings.

**Figure 4.** *In vitro* seedling growth from *L. chrysanthus* Boiss. seeds treated with a 3.75% NaOCl solution at temperatures of (a) 25°C, (b) 35°C and (c) 45°C for 15 min.

increase could be attributed to cell enlargement [55]. The increase in dry weight was due to cell division and new material synthesis [56]. Higher results in seedlings grown were from seeds treated with 3.75% NaOCl solution at 35°C for 15 min and could be caused by higher tissue water content as reported that *in vitro* explant growth and plantlet establishment have been affected significantly by tissue water content [57].

In the study, the highest chlorophyll a, chlorophyll b, and total chlorophyll contents were seen with a 3.75% NaOCl solution at 35°C temperature (**Table 5**).

#### **2.4. Gamma radiation**

Gamma rays have an ionizing radiation effect on plant growth and development by inducing cytological, biochemical, physiological, and morphological changes in cells and tissues by producing free radicals in cells [58–60]. Higher doses of gamma radiation have been reported to be inhibitory [61, 62], whereas lower doses are stimulatory. Low doses of gamma rays have been reported to increase seed germination and plant growth, cell proliferation, germination, cell growth, enzyme activity, stress resistance, and crop yields [63–69]. Stimulation of plant growth at low gamma radiation doses is known as hormesis [70]. The hormesis phenomenon is described as a stimulating effect on any factor in the growth of an organism [71].

In the study conducted by Beyaz et al. [72], the effects of gamma radiation on overcoming dormancy in seeds of *L. chrysanthus* Boiss. under *in vitro* conditions were examined. In the study, *L. chrysanthus* Boiss., seeds of an ecotype "Diyarbakir" were first irradiated with different doses (0-control, 50, 100, 150, 200, and 250 Gy) of 60Co γ rays at 0.8 kGy h−1 at the Sarayköy Nuclear Research and Training Center of the Turkish Atomic Energy Authority at Sarayköy, Ankara. Seeds were surface-sterilized with a 3.75% NaOCl solution at 35°C temperature for 15 min. as reported by Telci et al. [49]. The seeds were then placed between filter papers in Petri dishes each containing 6 ml of distilled water. The Petri dishes were incubated for 7 days at 15±1°C in the dark for seed germination. The pre-germinated seeds were then transferred to Magenta vessels (12 × 12 cm) containing a basal medium of Murashige and Skoog's (MS) mineral salts and vitamins [50], 3% sucrose, and 0.7% agar 14 days after the study initiation. The pH of the medium was adjusted to 5.8 prior to autoclaving. Then, all cultures were transferred to a growth chamber for incubation at 25±1°C under cool white fluorescent light (27 μmol m−2 s−1) with a 16-h light/8-h dark photoperiod. The seed germination percentage was determined at the end of the 7th day, while seedling growth percentage, seedling height, and root length were recorded 14 days after culture initiation [20]. All statistical analyses were performed using SPSS for Windows software. Three replicates were tested, and there were 30 seeds per replication. All experiments were repeated twice. One-way Analysis of Variance (ANOVA) was used to test the effect of different doses of gamma radiation on seed germination and seedling growth. Duncan's multiple range test was used for comparison of the means. Data given in percentages were subjected to arcsine (√*X*) transformation before statistical analysis [19].

The stimulatory effect of low gamma doses was observed in the study at a radiation dose of 150 Gy. The best results in seed germination percentage at the end of the 7th day and in seedling growth percentage, seedling height, and root length at the end of the 14th day were observed at a dose of 150 Gy of gamma radiation (**Table 6** and **Figure 5**). In doses over 150 Gy, the inhibitory effect of gamma radiation was seen. Seed germination percentage was 62.4% at a gamma radiation dose of 150 Gy, while it was 14.3% for a gamma radiation dose of 250 Gy (**Table 6**). The highest seedling growth percentage, seedling height, and root length were again recorded for a 150 Gy gamma radiation dose as 75.7%, 1.2 cm, and 2.9 cm, respectively.

increase could be attributed to cell enlargement [55]. The increase in dry weight was due to cell division and new material synthesis [56]. Higher results in seedlings grown were from seeds treated with 3.75% NaOCl solution at 35°C for 15 min and could be caused by higher tissue water content as reported that *in vitro* explant growth and plantlet establishment have

**Figure 4.** *In vitro* seedling growth from *L. chrysanthus* Boiss. seeds treated with a 3.75% NaOCl solution at temperatures

In the study, the highest chlorophyll a, chlorophyll b, and total chlorophyll contents were seen

Gamma rays have an ionizing radiation effect on plant growth and development by inducing cytological, biochemical, physiological, and morphological changes in cells and tissues by producing free radicals in cells [58–60]. Higher doses of gamma radiation have been reported to be inhibitory [61, 62], whereas lower doses are stimulatory. Low doses of gamma rays have been reported to increase seed germination and plant growth, cell proliferation, germination, cell growth, enzyme activity, stress resistance, and crop yields [63–69]. Stimulation of plant growth at low gamma radiation doses is known as hormesis [70]. The hormesis phenomenon

In the study conducted by Beyaz et al. [72], the effects of gamma radiation on overcoming dormancy in seeds of *L. chrysanthus* Boiss. under *in vitro* conditions were examined. In the study, *L. chrysanthus* Boiss., seeds of an ecotype "Diyarbakir" were first irradiated with different

is described as a stimulating effect on any factor in the growth of an organism [71].

been affected significantly by tissue water content [57].

**2.4. Gamma radiation**

of (a) 25°C, (b) 35°C and (c) 45°C for 15 min.

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with a 3.75% NaOCl solution at 35°C temperature (**Table 5**).


Values in a column followed by the different letters are significantly different at the 0.01 level. 1 Seedling growth percentage means seedlings developed out of the total seed number.

**Table 6.** Effects of different gamma doses on *in vitro* seed germination, seedling growth, seedling height, and root length in *L. chrysanthus* Boiss.

**Figure 5.** *In vitro* seed germination and seedling growth in *L. chrysanthus* Boiss. seeds irradiated with (a) 0, (b) 50, (c) 100, (d) 150, (e) 200, and (f) 250 Gy gamma doses (white vertical bar = 1 cm).

The root length obtained from seeds irradiated with 150 Gy of gamma radiation was significantly increased by 63.2% from 1.8 cm in the control treatment (0 Gy) to 2.9 cm, which has been confirmed by Melki and Marouani [73].
