Acta agriculturae Slovenica, 121/3, 1–11, Ljubljana 2025 doi:10.14720/aas.2025.121.3.22753 Original research article / izvirni znanstveni članek Optimizing germination protocols for Citrullus colocynthis (L.) Schrad.: A study on seed dormancy breakthrough through chemical and physical treatments Nasir ABADDAR 1, Hassanali NAGHDI BADI1, 2 , Majid AMINI DEHAGHI 1, Elias SOLTANI 3, Alireza REZAZADEH 1 Received May 20, 2025, accepted September11, 2025 Delo je prispelo 20. maj 2025, sprejeto 11. september 2025 1 Department of Agronomy and Plant Breeding, Faculty of Agriculture, Shahed University, Tehran, Iran. 2 Corresponding author e-mail: Naghdibadi@shahed.ac.ir 3 Department of Agronomy and Plant Breeding (Abureyhan); University of Tehran, Tehran, Iran. Optimizing germination protocols for Citrullus colocyn- this (L.) Schrad.: A study on seed dormancy breakthrough through chemical and physical treatments Abstract: Bitter apple (Citrullus colocynthis (L.) Schrad.), a desert plant with medicinal properties, typically exhibits low seed germination due to dormancy. This study investigated physical and chemical treatments to improve C. colocynthis seed germination. A completely randomized design with fac- torial arrangement and three replications was used to test the effects of gibberellic acid (GA3) at concentrations of 0, 10, 50, 100, and 150 ppm, under four diurnal temperature and light (TL) regimes: (1) 20 °C (12 h light/dark), (2) 25 °C (12 h light/ dark), (3) 35 °C (12 h light) / 15 °C (12 h dark), and (4) 30 °C (12 h light) / 20 °C (12 h dark). Results showed that TL regime, GA3, and their interaction significantly affected all measured germination parameters. The highest germination percentage, relative germination percentage, mean daily germination, ger- mination index, germination value, and seedling dry mass were achieved with 100 ppm GA3 under the 35-15 °C regime. The study concluded that average germination speed, germination time variation coefficient, germination speed coefficient, and synchronization index were unreliable for assessing seed quality under low germination conditions, suggesting that physiological dormancy in bitter apple seeds may stem from embryo immaturity, inhibitory factors, or both. Key words: seed dormancy, physiological dormancy, di- urnal temperature alternation, gibberellic acid. Optimiziranje protokolov kalitve kolokinte (Citrullus colo- cynthis (L.) Schrad.): Študija o prekinitvi mirovanja semen s kemičnimi in fizikalnimi obdelavami Izvleček: Kolokinta (Citrullus colocynthis (L.) Schrad.)), puščavska rastlina z zdravilnimi lastnostmi, ima zaradi dor- mance semen majhno kalivost. V raziskavi so bila preučevana fizikalna in kemična obravnavanja za izboljšanje kalitve semen te rastline. Izveden je bil popolni naključni faktorski poskus s tremi ponovitvami za preučevanje učinkov giberilinske kisline (GA3) v koncentracijah 0, 10, 50, 100 in 150 ppm, v diurnal- nih temperaturno-svetlobnih režimih (TL): (1) 20  °C (12  h svetloba/tema), (2) 25 °C (12 h svetloba/tema), (3) 35 °C (12 h svetloba) / 15 °C (12 h tema), in (4) 30 °C (12 h svetloba) / 20 °C (12 h tema). Rezultati so pokazali, da so ti režimi in GA3, ter njihove interakcije značilno vplivali na vse merjene parameter kalitve. Največje vrednosti parametrov kot so največji odstotek kalivosti, relativni odstotek kalivosti, poprečna dnevna kali- vost, indeks kalivosti, vrednost kalivosti in suha masa sejank so bile dosežene pri 100 ppm GA3 in režimu 35-15 °C. Na os- novi raziskave je bilo ugotovljeno, da poprečna hitrost kalitve, koeficient spreminanja časa kalitve, koeficient hitrosti kalitve in sinhronizacijski indeks niso primerni za ocenjevanje kakovosti semen v razmerah majhne kalivosti, kar nakazuje, da fiziološka dormanca semen kolokinte lahko izvira iz nezrelosti embrija ali inhibitornih dejavnikov ali obeh skupaj. Ključne besede: dormanca semen, fiziološka dormanca, diurnalno menjavanje temperatur, giberilinska kislina Acta agriculturae Slovenica, 121/3 – 20252 N. ABADDAR et al. 1 INTRODUCTION Citrullus colocynthis (L.) Schrad., commonly known as bitter apple, is a perennial herb in the Cucurbitaceae family. This plant features a small prostrate or climb- ing stem with perennial rootstocks and can reproduce through seeds and vegetative buds (Li et al., 2022). Bitter apples exhibit anti-inflammatory and antibacterial prop- erties that are utilized in the treatment of various dis- eases, including type II diabetes (Jafarizadeh et al., 2022) and breast cancer (Perveen et al., 2021). Additionally, C. colocynthis produces a significant quantity of oily seeds, which can be converted into inexpensive biodiesel (Aziz et al., 2023). The essential oil derived from C. colocynthis has also been used in biological pest control to enhance resistance to whitefly infestations in watermelon cultivars (Kahrom et al., 2022). Seed germination and dormancy are critical factors in the cultivation and development of C. colocynthis (El-Keblawy et al., 2017). Seed dormancy is a temporary block to germina- tion, even in the presence of favorable environmental conditions. It is an adaptation that allows seeds to delay germination until conditions are suitable for the seed- ling’s survival (Klupczyńska and Pawłowski 2021). In dry climates, germination can be hindered by adverse con- ditions such as drought and high temperatures (Vicente et al., 2020). Many desert plants, such as C. colocynthis, produce seeds that exhibit various types and levels of dormancy, which can be effectively broken by exposure to appropriate environmental cues (Al-Turki et al., 2022). Environmental factors such as photoperiod and temperature significantly regulate the physiological pro- cesses that induce germination and break seed dormancy (Yan and Chen 2020). G.).))For many species, an optimal light duration signals the right time for seeds to awaken from dormancy, ensuring their growth aligns with fa- vorable seasonal conditions (Kharshiing et al., 2019). Similarly, temperature influences enzymatic activity and metabolic rates within the seed, facilitating the break- down of growth inhibitors and the activation of growth- promoting hormones (Zhang et al., 2021). Research has shown that specific combinations of light and warmth can enhance germination rates and synchronize seedling emergence, contributing to plant diversity and ecosystem stability (Bhatla and Lal 2023, Zheng et al., 2005). Growth regulators like GA3 are phytohormones used to enhance germination and seed establish- ment (Silva Edvan Costa Da et al., 2021). As a natural regulator, GA3 significantly influences plant physiology, resulting in various agricul- tural and horticultural applications (Joshi et al., 2023). Notably, GA3 promotes seed germina- tion by being released from the embryo during the germination process, which stimulates the production of mRNA and alpha-amylase (Ne- dunchezhiyan et al., 2023). Numerous studies highlight the importance of light, temperature, and GA3 in the germination of various Cu- curbitaceae species. For instance, darkness is essential for the germination of Citrullus lanatus (Thunb.) Matsum. & Nakai, Cucurbita maxima Duchesne, Lagenaria siceraria (Molina) Standl., Benincasa hispida (Thunb.) Cogn., and Momordica harantia L. (Nakamura et al., 1955), as well as for Citrullus lanatus ‘Sugar Baby’ (Thanos and Mitrakos 1992) and C. lanatus var. citroides (Ramirez et al., 2014). Additionally, germination temperature significantly af- fects other Cucurbitaceae species. For example, the ger- mination rate of melon dropped from nearly 100  % to zero at sub-optimal temperatures (Edelstein and Kigel 1990). The Sugar Baby watermelon germinated almost entirely in darkness at temperatures between 20 and 40 °C, but there was a significant decrease at 15 °C and 42.5 °C (Ramirez et al., 2014). No germination occurred in Citrullus lanatus var. citroides at day/night tempera- tures of 10/5 and 15/10 °C, regardless of light conditions (Ramirez et al., 2014). Mature seeds of C. colocynthis from the Iranian desert failed to germinate without treat- ments (Gharehmatrossian et al., 2014, Saberi M. et al., 2011). The dormancy of this species was attributed to a mechanical barrier in the testa (physical dormancy), rather than allelochemicals that might inhibit germina- tion (El-Keblawy et al., 2017). To optimize the germination and emergence of C. colocynthis while ensuring adequate field density, it is crucial to identify effective treatments for breaking seed dormancy. This study aimed to evaluate factors that might trigger the germination of C. colocynthis seeds, in- cluding light and temperature cycles, as well as GA3 lev- els. The research hypotheses propose that a combination of physicochemical factors—specifically light, diurnal temperature alternation, and GA3—can effectively break seed dormancy and enhance key parameters during ger- mination. 2 MATERIALS AND METHODS 2.1 SEED COLLECTION Fully ripened yellow fruits of large, uniform sizes were collected from a wild population of C. colocyn grow- ing around Masjed Soleyman City in Khuzestan Prov- Acta agriculturae Slovenica, 121/3 – 2025 3 Optimizing germination protocols for Citrullus colocynthis (L.) Schrad.: A study on seed dormancy ... chemical and physical treatments ince, southern Iran (48°, 24' east longitude and 31.93°, 49.30' north latitude). Immediately after collection, seeds were manually separated from the fruits, washed with water for 48 hours, dried at room temperature, and disinfected in a 10 % sodium hypochlorite solution for one minute. Fifty seeds were placed in each Petri dish, which were then transferred to the germinator. To deter- mine seedling dry mass, samples were dried at 70 °C and weighed on a digital scale. 2.2 STATISTICAL SCHEME AND TREATMENTS The experiment was conducted using a completely randomized design with a factorial arrangement across three replications. Seeds were exposed to different con- centrations of gibberellic acid (GA3) (0, 10, 50, 100, and 150 ppm) under alternating diurnal temperatures and light (TL) conditions. The light and temperature condi- tions for the seeds in this experiment were as follows: (1) 20°C with a 12-hour light/dark photoperiod, (2) 25 °C with a 12-hour light/dark cycle, (3) a 12-hour light cycle at 35 °C followed by a 12-hour dark cycle at 15 °C, and (4) a 12-hour light cycle at 30 °C followed by a 12- hour dark cycle at 20 °C. 2.3 SOWING AND GERMINATION Seeds were sown in 10 cm diameter Petri dishes lined with filter paper discs, each containing 10 ml of distilled water or varying concentrations of GA3, accord- ing to the respected treatment. Germination was record- ed daily for 10 days at 11 AM, and seedling length was measured using a digital caliper. A seed was considered as germinated when complete germination occurred. 2.4 STUDIED TRAITS Seedling length (SL) was measured using a digital caliper. For seedling dry mass (SDM) calculations, sam- ples were dried in an oven at 70  °C and weighed on a digital scale with an accuracy of 0.001 g. The germination parameters were calculated as fol- lows: 2.4.1 Germination percentage (GP) This criterion is “a measure of the survival of a col- lection of seeds” (Guragain et al., 2023). (1) where N is the total number of seeds utilized, and ni is the number of seeds that germinated on the ith day. 2.4.2 Relative germination percentage (RGP) As indicated in Eq. 2, “relativizing germination per- centage enables comparisons between treatments that are equivalent when the quantity of dormancy disruption varies” (Guragain et al., 2023). (2) 2.4.3 Mean germination time (MGT) As expressed in Eq. 3, MGT is defined as “the aver- age time it takes for a seed to germinate or emerge” (Gu- ragain et al., 2023). (3) where niti is the number of seeds germinated in the ith time interval, and ni is the number of seeds germi- nated at the ith time. 2.4.4 Mean daily germination (MDG) As stated in Eq. 4, MDG is defined as “the average number of seeds that germinate each day during a speci- fied period” (Sarwar et al., 2024). (4) where GP is the final cumulative germination per- centage and tn represents the total time intervals. 2.4.5 Mean germination rate (MGR) “MGR was calculated as the reciprocal of the MGT” (Guragain et al., 2023). (5) 2.4.6 Germination value (GV) “The GV reflects the performance of the seed lot, indicating the health and potential growth rate of the seeds” (Sarwar et al., 2024). (6) Where MDG represents the mean daily germination Acta agriculturae Slovenica, 121/3 – 20254 N. ABADDAR et al. and PV indicates the peak value, which is the final per- centage of germination. 2.4.7 Germination index (GI) “The germination index measures the number of days needed for a specific percentage of seeds to germi- nate.” (Sarwar et al., 2024). (7) Where ni and ti are defined as described above. 2.4.8 Synchrony of the germination process (Z) “This criterion indicates the degree of overlap among members of a specific demographic. The Z index yields a value only when two seeds complete the sprout- ing process simultaneously. This criterion can be estimat- ed using Eq. 8 (Guragain et al., 2023)” (8) Where Cni,2 represents the combination of seeds ger- minated in pairs at the ith time, ni is the total number of seeds germinated at that time. The value of Z equals one when all seeds sprout simultaneously and equals zero when at least two seeds sprout at the same time. 2.4.9 The coefficient of the velocity of germination (CVG) As indicated in Eq. 9, the CVG reflects the speed of germination and increases as the number of germinated seeds rises (Sarwar et al., 2024). (9) 2.4.10 Coefficient of variation of germination time (CVT) CVt was estimated using Equation 10 (Sarwar et al., 2024). (10) where: St represents the standard deviation of ger- mination time and represents MGT. 2.5 DATA ANALYSIS The data were analyzed using SAS statistical soft- ware, version 9.4. A factorial analysis of variance was conducted to examine the impact of treatments on germination indices and measured traits. Means were compared using Duncan’s multiple range test at a 5 % significance level. Bar charts were created using Micro- soft Excel (2019). The correlation diagram was generated using the corrplot package in R Studio 2024.090 with R version 4.4.2, while the heatmap was prepared in Minitab version 22.1. 3 RESULTS 3.1 GERMINATION PERCENTAGE AND RELA- TIVE GERMINATION PERCENTAGE Analysis of variance on traits related to C. colocyn- this seed germination revealed that both TL and GA3, along with their interaction, significantly impacted all examined indicators at the 1 % significance level (Data not shown). The treatment with 100 ppm GA3 at an al- ternating temperature of 15–35 °C (12 hours at 35 °C in light and 12 hours at 15  °C in darkness) produced the highest GP of 57.33 % and RGP of 84.31 %. Addition- ally, GP and RGP at this alternating temperature were consistently higher across all GA3 concentrations than at other temperature levels. In contrast, the control treat- ment with distilled water showed the lowest germination percentage across all conditions (Table 1). 3.2 MEAN GERMINATION TIME The longest MGT recorded was 4.41 days, occurring with the treatment of 100 ppm GA3 at an alternating tem- perature of 20-30 °C. While no significant difference was observed between the 50 ppm and 150 ppm GA3 treat- ments at the same alternating temperature, the control Acta agriculturae Slovenica, 121/3 – 2025 5 Optimizing germination protocols for Citrullus colocynthis (L.) Schrad.: A study on seed dormancy ... chemical and physical treatments Table 1: Effect of temperature and gibberellic acid concentrations on the germination indices of bitter apple (Citrullus colocynthis) seeds. GA3 (ppm) T (°C) G (%) R (%) MGT MGR CVt 0 20 0.67 ± 0.07 n 0.98 ± 0.04 k 0.67 ± 0.04 h 0.17 ± 0.01 j 0 ± 0 m 10 4 ± 0.58 kl 5.88 ± 0.03 i 2.17 ± 0.19 cdef 0.58 ± 0.05 b 20.54 ± 0.71 j 50 6.67 ± 0.67 ij 9.8 ± 0.27 h 2.03 ± 0.17 def 0.5 ± 0.01 c 55.35 ± 0.63 b 100 11.33 ± 0.67 fg 16.67 ± 0.27 f 2.69 ± 0.17 bc 0.38 ± 0.01 fg 50.13 ± 0.83 c 150 11.33 ± 1.2 fg 16.67 ± 0.2 f 2.81 ± 0.1 b 0.36 ± 0.01 g 48.09 ± 0.67 d 0 25 1.33 ± 0.67 mn 1.96 ± 0.09 k 1.67 ± 0.09 fg 0.28 ± 0.004 h 0 ± 0 m 10 4.67 ± 0.67 jk 6.86 ± 0.53 i 2.44 ± 0.29 bcd 0.42 ± 0.01 def 31.82 ± 1.07 h 50 8 ± 0.58 hi 11.76 ± 0.25 gh 1.78 ± 0.22 ef 0.58 ± 0.02 b 26.22 ± 1.24 i 100 12 ± 0.58 f 17.65 ± 0.03 f 2.39 ± 0.3 bcd 0.43 ± 0.01 de 47.97 ± 0.64 d 150 9.33 ± 0.67 gh 13.73 ± 0.1 g 2.05 ± 0.39 def 0.53 ± 0.01 c 63.05 ± 0.55 a 0 20-30 2 ± 0.58 lmn 2.94 ± 0.08 jk 1 ± 0.06 h 0.5 ± 0.03 c 0 ± 0 m 10 6.67 ± 0.88 ij 9.8 ± 0.27 h 2.67 ± 0.09 bc 0.17 ± 0.01 j 0 ± 0 m 50 30 ± 1.15 d 45.1 ± 0.88 d 4.31 ± 0.33 a 0.24 ± 0.01 hi 15.87 ± 1.07 k 100 21.33 ± 0.88 e 31.37 ± 1.01 e 4.41 ± 0.05 a 0.23 ± 0.002 i 15.94 ± 0.39 k 150 20 ± 0.58 e 29.41 ± 0.7 e 3.9 ± 0.05 a 0.26 ± 0.003 hi 13.71 ± 0.41 l 0 15-35 3.33 ± 0.33 klm 4.9 ± 0.1 ij 1.17 ± 0.09 gh 0.89 ± 0.03 a 15.71 ± 0.35 k 10 41.33 ± 0.88 b 60.78 ± 1.75 b 2.75 ± 0.03 bc 0.38 ± 0.01 efg 34.77 ± 0.7 g 50 40.67 ± 1.45 b 59.8 ± 2.47 b 2.29 ± 0.12 bcde 0.44 ± 0.01 d 38.66 ± 0.89 f 100 57.33 ± 0.88 a 84.31 ± 1.68 a 2.44 ± 0.2 bcd 0.41 ± 0.01 def 44.2 ± 0.68 e 150 38 ± 1.15 c 55.88 ± 1.63 c 2.63 ± 0.11 bcd 0.38 ± 0.01 efg 31.75 ± 0.77 h GA3 (ppm) T (°C) CVG GI Z MDG GV 0 20 16.67 ± 0.34 h 0.17 ± 0.01 i 0 ± 0 g 0.05 ± 0.005 i 0.05 ± 0.005 g 10 57.78 ± 0.62 b 0.97 ± 0.04 hi 0 ± 0 g 0.29 ± 0.02 fghi 0.44 ± 0.11 g 50 50 ± 0.66 c 2.14 ± 0.09 fg 0.28 ± 0.03 e 0.48 ± 0.05 efgh 1.62 ± 0.37 fg 100 37.5 ± 2.41 e 2.69 ± 0.25 ef 0.19 ± 0.05 f 0.81 ± 0.05 e 2.81 ± 0.27 fg 150 35.73 ± 0.4 e 2.49 ± 0.26 efg 0.16 ± 0.02 f 0.81 ± 0.01 e 2.44 ± 0.67 fg 0 25 27.78 ± 0.72 f 0.28 ± 0.01 i 0 ± 0 g 0.1 ± 0.01 hi 0.08 ± 0.04 g 10 42.06 ± 1.13 d 1.11 ± 0.02 hi 0.44 ± 0.01 c 0.33 ± 0.01 fghi 0.6 ± 0.14 g 50 58.33 ± 1.01 b 2.44 ± 0.24 efg 0.54 ± 0.03 b 0.57 ± 0.03 efg 2.29 ± 0.08 fg 100 43.3 ± 1.19 d 3.23 ± 0.05 de 0.2 ± 0.004 f 0.86 ± 0.03 e 3.43 ± 0.33 f 150 52.6 ± 1.43 c 3.31 ± 0.69 de 0.36 ± 0.04 de 0.67 ± 0.01 ef 3.71 ± 0.28 f 0 20-30 50 ± 1.15 c 0.67 ± 0.04 hi 0.33 ± 0.02 de 0.14 ± 0.004 hi 0.29 ± 0.01 g 10 16.67 ± 0.38 h 0.83 ± 0.05 hi 0.67 ± 0.03 a 0.48 ± 0.01 efgh 1.24 ± 0.04 fg 50 23.52 ± 0.65 g 4.07 ± 0.32 d 0.61 ± 0.03 ab 2.19 ± 0.53 c 16.55 ± 0.24 d 100 22.68 ± 0.25 g 2.51 ± 0.12 efg 0.36 ± 0.01 de 1.52 ± 0.01 d 8.38 ± 1.39 e 150 25.67 ± 0.34 fg 2.68 ± 0.85 ef 0.54 ± 0.03 b 1.43 ± 0.04 d 6.97 ± 0.51 e 0 15-35 88.89 ± 1.25 a 1.5 ± 0.1 gh 0.33 ± 0.02 de 0.24 ± 0.005 ghi 0.67 ± 0.05 g 10 38.31 ± 1.06 e 8.58 ± 0.26 c 0.37 ± 0.02 cd 2.95 ± 0.02 b 37.97 ± 2.63 b 50 43.93 ± 1.44 d 10.86 ± 0.24 b 0.32 ± 0.01 de 2.9 ± 0.03 b 40 ± 0.38 b 100 41.47 ± 1.73 d 14.83 ± 0.62 a 0.29 ± 0.03 de 4.1 ± 0.04 a 72.51 ± 1.66 a 150 38.13 ± 0.29 e 8.07 ± 0.46 c 0.34 ± 0.04 de 2.71 ± 0.07 b 31.94 ± 0.8 c Acta agriculturae Slovenica, 121/3 – 20256 N. ABADDAR et al. In each column, means that share at least one common letter are not significantly different at the 0.05 probability level, according to Duncan’s mul- tiple range test. GA3: Gibberellic acid concentration, T: Temperature, G (%): Germination percentage, R (%): Relative germination percentage, MGT: Mean Germi- nation Time, MGR: Mean germination rate, CVt: Coefficient of variation of germination time, CVG: Coefficient of velocity of germination time, GI: Germination index, Z: Synchrony of germination process, MDG: Mean daily germination, GV: Germination value. treatment showed the lowest average germination time of 0.67 days at a constant temperature of 20 °C (Table 1). 3.3 MEAN GERMINATION RATE The highest MGR (0.89) was observed in the con- trol treatment at an alternating temperature of 15- 35 °C, significantly different from the other treatments. Conversely, the lowest MGR (0.17) was recorded in the control treatment at a constant temperature of 20 °C and in the treatment with 10 ppm GA3 at an alternating tem- perature of 20-30  °C, both of which were significantly lower than the other treatments (Table 1). 3.4 COEFFICIENT OF VARIATION OF GERMINA- TION TIME The treatment of 150 ppm GA3 at a constant temper- ature of 25 °C resulted in the highest CVt (63.05), which was significantly different from the other treatments. In contrast, the lowest CVt, recorded as zero, was observed in the 10 ppm GA3 treatment at an alternating tempera- ture of 20–30  °C, as well as in the control treatment at constant temperatures of 20 °C and 25 °C (Table 1). 3.5 THE COEFFICIENT OF VELOCITY OF GER- MINATION The highest CVG (88.89) was observed in the con- trol treatment at an alternating temperature of 15-35 °C. In contrast, the lowest CVG (16.67) was associated with the control treatment at a constant temperature of 20 °C, as well as with the 10 ppm GA3 treatment at an alternat- ing temperature of 20-30 °C (Table 1). 3.6 GERMINATION INDEX The highest GI (14.83) was observed in the treat- ment with 100 ppm GA3 at an alternating temperature of 15-35 °C, and this result was statistically significantly different from the other treatments. Conversely, the lowest GI was recorded in the control treatment across all temperature levels (Table 1). 3.7 SYNCHRONY OF THE GERMINATION PRO- CESS The highest Z value (0.67) was observed in the treat- ment containing 10 ppm GA3 at an alternating tempera- ture of 20-30 °C. In contrast, the Z values in treatments with an alternating temperature of 15-35  °C exhibited a consistent pattern across all GA3 concentrations. The lowest Z values were recorded in the control and 10 ppm GA3 treatments at a constant temperature of 20  °C, as well as in the control treatment at a constant temperature of 25 °C (Table 1). 3.8 MEAN DAILY GERMINATION The treatment with 100 ppm GA3 at an alternating temperature of 15–35  °C exhibited the highest MDG Figure 1: Effect of temperature and gibberellic acid concentra- tions on the seedling dry mass of bitter apple (Citrullus colo- cynthis) seeds. Columns sharing common letters are not sig- nificantly different at the 0.05 probability level, according to Duncan's multiple range test. Figure 2: Effect of temperature and gibberellic acid concen- trations on the seedling length of bitter apple (Citrullus colo- cynthis) seeds. Columns sharing common letters are not sig- nificantly different at the 0.05 probability level, according to Duncan’s multiple range test. Acta agriculturae Slovenica, 121/3 – 2025 7 Optimizing germination protocols for Citrullus colocynthis (L.) Schrad.: A study on seed dormancy ... chemical and physical treatments (1.4), which was significantly different from the other treatments. In contrast, the control treatment, which did Figure 3: Graphical Pearson correlations of germination char- acteristics of bitter apple (Citrullus colocynthis) influenced by temperature periods and varying concentrations of gibberellic acid. Each circle represents a significant correlation at the 0.05 probability level. The circle’s diameter reflects the correlation’s magnitude, with blue indicating a positive correlation and red a negative one. The non-significant correlations, with the p-value above 0.05 are indicated with a cross. not contain GA3, recorded the lowest MDG across all temperature treatments (Table 1). 3.9 GERMINATION VALUE The highest GV of 51.72 was observed in the treat- ment involving 100 ppm GA3 at an alternating tempera- ture of 15–35 °C. In contrast, the control group and the treatment with 10 ppm GA3 exhibited the lowest germi- nation values across all temperature levels (Table 1). 3.10 SEEDLING DRY MASS AND SEEDLING LENGTH The highest SDM (0.33 g) was observed in the treat- ment with 100 ppm GA3 at an alternating temperature of 15–35 °C. Conversely, the lowest SDM was noted in the control treatment across all temperature levels (Fig. 1). Additionally, the maximum SL of 9.49 mm was recorded in the treatment with 150 ppm GA3 at an alternating tem- perature of 20–30 °C, while the minimum SL was again found in the control treatment across all temperature lev- els (Fig. 2). 3.11 PEARSON CORRELATIONS BETWEEN GER- MINATION INDICES Pearson’s coefficient was calculated to evaluate the correlation between various germination indices, as il- lustrated in Figure 3. MGT and SL were negatively and significantly correlated with MGR and CVG, but posi- tively and significantly correlated with GP, RGP, MDG, and Z parameters. This indicates that along with healthy seedling growth, rapid and synchronized germination is essential for optimal germination. 4 DISCUSSION One of the most critical strategies for uniform and successful crop production under agronomic conditions is the rapid and high germination of planted seeds. For certain crops exhibiting seed dormancy, it is essential to break dormancy appropriately. The findings indicated that, although the seeds exhibited dormancy, the appli- cation of appropriate treatments significantly enhanced their germination rate and effectively overcame this dor- mancy. Notably, the highest values for GP, RGP, MDG, GI, GV, SDM, and the shortest MGT were observed at alternating temperatures of 15-35 °C with a GA3 concen- Figure 4: Heat map of germination indices of bitter apple (Cit- rullus colocynthis) seeds in response to temperature periods and varying concentrations of gibberellic acid. All traits were standardized for comparability. Values less than 1 appear in blue, while values greater than 1 appear in red. Color intensity reflects the magnitude of values. Acta agriculturae Slovenica, 121/3 – 20258 N. ABADDAR et al. tration of 100 ppm. However, interaction effects between temperature and GA3 have been observed on the germi- nation indices of seeds from regions with hot, dry sum- mers and cold winters. During the germination stage, GA3 is one of the most important factors influencing the release of food reserves, including starch, in the seeds of these species (Baskin and Baskin 2014). A study on egg- plant seeds found that the germination percentage was significantly higher at alternating temperatures (20-30 °C or 20-35 °C) compared to a constant temperature of 25 °C (Ozden et al., 2021). Specifically, alternating tempera- tures, which mimic natural daily fluctuations, are more effective in enhancing seed germination rates (Hung et al., 2004). Several studies have attributed the positive effects of alternating temperatures on germination to a decrease in abscisic acid (ABA) synthesis and a reduc- tion in the ABA to GA3 ratio (Ali-Rachedi et al., 2004, Huarte and Benech-Arnold 2010). Asaadi and Heshmati (2015) reported the highest germination percentage with 100 ppm GA3 when breaking the dormancy of Khorasani thyme (Thymus transcaucasicus Ronn.), which aligns with our results. They linked this outcome to GA3’s role in the synthesis of auxins and cytokinins, necessary for inducing dormancy break. Our findings are consistent with those of Shahmoradi et al., (2015), who investigated the mechanisms of dormancy breaking in wild barley (Hordeum spontaneum (K. Koch) Thell.). This study indicates that MGT is not a reliable met- ric for evaluating the impact of treatments on germina- tion across all conditions. In scenarios where the seed germination rate is low, MGT fails to effectively reflect seed quality (Omidi et al., 2012). In this investigation, although the control treatment at a constant tempera- ture of 20 °C produced the shortest MGT (0.67 days), it was considered an unsuitable treatment. Conversely, the treatment with 100 ppm GA3 under an alternating tem- perature regime of 15-35 °C, which achieved the highest germination percentage, was identified as the most effec- tive treatment, despite its lower MGT. The GI is a significant indicator of the relationship between the percentage and rate of germination (Afzal et al., 2022). Begum et al., (2022) reported the highest GP and GI during high-temperature cycles (25-35  °C) compared to moderate-temperature cycles (20-30  °C), which aligns with the findings of the present study. Similarly, an experiment involving two cultivars of sage (Salvia verbenaca L.) found that increased temperatures correlated with enhanced germination (Javaid M. M. et al., 2018). Furthermore, studies on sweet sorghum seeds (Sorghum bicolor L. Moench) (Zhu et al., 2019) and Al- lium stracheyi Baker (Payal et al., 2014) demonstrated that GI also increased with higher GA3 levels, confirming our findings. Germination value serves as an index that integrates both the speed and completeness of germination (Czaba- tor 1962). A higher GV serves to indicate a more favora- ble germination process (Rath et al., 2023). Consequent- ly, the treatment involving 100 ppm GA3 combined with an alternating temperature of 15-35 °C demonstrated a superior germination process compared to other treat- ments, as evidenced by the highest GV recorded. The MDG rate is defined as the number of seeds that germinate per day concerning the maximum germi- nation rate (Rath et al., 2023). The treatment involving 100 ppm GA3 combined with alternating temperatures of 15-35 °C produced the highest MDG rate. A study in- vestigating the germination of jatropha (Jatropha curcas Linn.) seeds demonstrated a significant positive effect of elevated temperatures and temperature fluctuations on MDG (Gairola et al., 2011). Additionally, another study indicated that increasing concentrations of GA3 correlat- ed with an enhancement in the MDG of yarrow (Achillea millefolium L.) seeds, which is consistent with the find- ings of the present experiment (Nejad et al., 2022). The beneficial impact of GA3 on the enhancement of SDM has been documented in various plant species (Banerjee and Roychoudhury 2020, Chauhan et al., 2019, Kumari et al., 2017, Tsegay and Andargie 2018). An in- crease in GA3 concentration facilitates the augmenta- tion of SDM by promoting the hydrolysis of seed starch, thereby converting it into accessible materials for the embryo (Esanejad et al., 2015). This phenomenon elu- cidates the observed increase in SDM under the treat- ment of 100 ppm GA3 combined with an alternating temperature regime of 15-35 °C. Additionally, numerous studies have reported the positive influence of alternat- ing temperature on the enhancement of SDM (Kumar et al., 2016, Nogueira et al., 2014, Pellizzaro et al., 2019). Seeds exhibiting high germination capacity demonstrate an enhanced ability to synthesize materials with greater efficiency and to transport these materials more rapidly to the developing embryonic axis. This process contrib- utes to increased dry matter accumulation and greater seedling length (Omidi et al., 2012). Treatment with 150 ppm GA3, in conjunction with alternating temperatures of 20-30 °C, significantly improved these outcomes, with the maximum SL recorded under this treatment condi- tion. The beneficial effects of varying concentrations of GA3 on SL have been documented in multiple studies (Amini et al., 2019, Payal et al., 2014, Saberi Morteza et al., 2020). Additionally, research on Jeffersonia dubia (Maxim.) Benth. & Hook. f. ex Baker & Moore seeds has indicated a positive influence of alternating temperatures on the enhancement of SL (Rhie et al., 2015). Similarly, an investigation involving sunflower seeds (Helianthus annuus L.) revealed that the greatest SL occurred at alter- Acta agriculturae Slovenica, 121/3 – 2025 9 Optimizing germination protocols for Citrullus colocynthis (L.) Schrad.: A study on seed dormancy ... chemical and physical treatments nating temperatures of 20-30 °C, aligning with the find- ings of the present study (Yari et al., 2014). The positive influence of GA3 on seed germination is primarily associated with the stimulation of hydrolyz- ing enzyme synthesis within the seed. This mechanism aids in the degradation of starch, proteins, and other nutrients, thereby promoting the transfer of these es- sential substances from the endosperm to the developing embryo. Moreover, GA3 enhances the activity of the cat- echol oxidase enzyme, which reduces seed phenolic com- pounds, further facilitating germination. Additionally, it promotes the synthesis of DNA and proteins, which con- sequently impacts the phospholipid composition of the embryo’s cell membrane (Yousefi et al., 2021). Among the various environmental factors influenc- ing seed germination, temperature is recognized as the most critical determinant (Javaid Muhammad Mansoor et al., 2022). Research indicates that alternating tem- peratures significantly contribute to alleviating seed dor- mancy and stimulating germination (Ozden et al., 2021). Seeds that respond to alternating temperatures possess an enzymatic mechanism that functions optimally un- der varying thermal conditions, likely due to ecological adaptation to their environment (Silva Dandara Yasmim Bonfim de Oliveira et al., 2018). Furthermore, alternating temperatures enhance seed germination by reducing the concentration of growth inhibitors present in the seed coat of C. colocynthis (El-Keblawy et al., 2017). Under alternating temperature conditions, the levels of germi- nation-promoting hormones increase relative to those of germination-inhibiting hormones, thereby facilitat- ing the breakdown of the seed’s physiological dormancy (Ozden et al., 2021). The findings of the experiment indicate that the physiological dormancy observed in the C. colocynthis seed can be attributed to either the immaturity of the em- bryo, the presence of inhibitory factors within the seed, or a combination of both (Asaadi and Heshmati 2015). The mass production and economic viability of the C. colocynthis plant, which holds medicinal, edible, and in- dustrial significance, depend on the implementation of appropriate treatments aimed at enhancing germination and overcoming seed dormancy (Saberi Morteza et al., 2017). The results of this study suggest that alternating temperatures of 15-35 °C, in conjunction with a gibber- ellic acid concentration of 100 ppm, represent the most effective treatment for alleviating the dormancy of the C. colocynthis seed. 5 REFERENCES Afzal, O., Hassan, F., Ahmed, M., Shabbir, G., & Ahmed, S. (2022). Temperature affects germination indices of saf- flower (Carthamus tinctorius L.). Journal of Animal & Plant Sciences, 32(6), 1691-1702. https://doi.org/:10.36899/ JAPS.2022.6.0577 Al-Turki, T.A., Davy, A.J., Al-Ammari, B.S., & Basahi, M.A. (2022). Seed germination characteristics of some medici- nally important desert plants from the Arabian Peninsula. 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