Determination of Germination Parameters of Safflower ( Carthamus tinctorius L.) Cultivars Under Salt Stress

: The present study was carried out to characterize germination capacity of 10 safflower cultivars under saline conditions. Five salt (NaCl) levels (0, 60, 120, 180, 240 mM) were used to test safflower seeds. Germination of seeds was counted every day for 14 days and germination percentage, Timson’s germination index, mean germination time, mean germination rate and germination stress index were calculated. Cultivar and salinity treatments were important for all parameters; however mean germination time and mean germination rate interactions were not important. Germination percentage, Timson’s germination and germination stress indices decreased significantly with increased salt concentrations. However, mean germination time increased with higher salt concentrations. The most significant reductions in germination percentage were observed at 180 and 240 mM salt concentrations. Correlation coefficients were all important for germination indices. Based on germination indices, Leed and FO2 were more sensitive to salt stress at germination stage and Royal was more resistant than the other cultivars tested. Germination percentage, Timson’s


Introduction
Soil salinity is a major problem of agricultural lands, and it is caused by accumulation of salts in the soil through over watering and evaporation, irrigation with saline water and lack of adequate drainage systems. It is especially problematic in arid and semiarid parts of the world [1,2]. Soil salinization affects approximately 800 million hectares of agricultural land in the world. Over 1.5 million hectares of land is affected by salinity and alkalinity in Turkey [3].
Salinity affects many physiological processes in plants, such as sodium accumulation, reduction in water and nutrient absorption, production of reactive oxygen species leading to cellular damage, toxicity, water loss and decreased photosynthesis. As a result of these changes, plant growth is reduced and yields are decreased [4].
Safflower is an important oil seed crop and it has been grown for its olieferous seeds and flowers since ancient times. Safflower is cultivated more than 60 countries with production area of more than 650.000 hectares around the world. Turkey ranks as the 8th largest producer and the safflower is cultivated more than 15.000 hectares with over 21 million tons of seed production [5]. Oil demand and consumption exceeds the production in Turkey and oil deficit covered by import of oils and oliferous seeds. Therefore it is necessary to increase oil seed crop production in Turkey. In order to increase crop production, it is also necessary to alleviate the effects of environmental stress conditions on crops for increased yields [6].
Drought and salinity are the major causes of yield reduction in crop plants [2]. Therefore, it is necessary to investigate germination, growth and adaptation capabilities of crop plants required for food production under stress conditions. Safflower is known to be tolerant to drought and salinity stress and could be used as an alternative crop suitable to be grown under drought and saline conditions [7,8]. Application of stress conditions and their timing are important screening factors to determine tolerant and susceptible genotypes. Germination and seedling development phases of safflower are the most sensitive development periods to salinity stress [4]. Therefore, the aim of the present study was to evaluate diverse safflower cultivars under varying salt concentrations to determine their germination abilities and to calculate germination indices under salinity stress.

Material and Method
Seed of 10 safflower cultivars from 4 different countries were evaluated for their germination behaviors under different salt concentrations. Five safflower cultivars UC-1(PI 572434), Royal (PI 537694), US-10 (PI 572414), Leed (PI 572436) and Gila (PI 537692) were from United States, FO2 (PI 506426) was from China, Lesaf 414 (PI 603206) and AC Sunset (PI 592391) were from Canada, Quiriego 88 (PI 537110) and San Jose 89 (PI 561703) were from Mexico. Cultivars oil contents ranged from 25% to 31% in field trails and they were linoleic type with the exception of US-10, which was oleic type [9].
Seed surface sterilization was carried out by soaking seeds in 1% sodium hypochlorite solution for 10 min. After sterilization, all seeds were rinsed under running tap water for 5 min and dried at room temperature. Seeds were sown in 15 cm wide petri dishes and 5 different concentrations (0, 60, 120, 180, 240 mM) of NaCl solution was added on filter papers. Control groups were wetted with distilled water. Germination tests were carried out in a germination cabinet under 25 °C for 14 days [10]. Each petri dish contained 40 seeds and germination tests were conducted with 3 replications. Number of germinated seeds was counted every day and germinated seeds were removed from petri dishes.
Germination data were logit transformed for normality of results. Germination percentage, mean germination time, Timson's germination index [11], mean germination rate [12] and germination stress index [13] were calculated according to formulas in Table 1. Results were subjected to analysis of variance (ANOVA) using IBM SPSS Statistics 22.0 software (SPSS Inc., Chicago, IL, USA). Duncan's multiple range test (p≤0.05) was used to discriminate the differences between the means. To show the relationship between measured parameters, Pearson linear correlation analysis (heatmap correlation) was calculated using OriginPro software (version 2021, OriginLab, Northampton, MA).  (Table 3). Gila, UC-1 and Lesaf 414 showed significant decline for germination percentage for 240 mM salt concentration as well.
Timson's germination index values based on germination data were similar to germination percentage. Higher germination index values were found for control seeds. As salt concentrations increased, germination index values started to decrease and the lowest germination index values were found in seeds germinated in 240 mM salt concentrations (Table 3).
Mean germination times of cultivars belong to control group were generally lower than 2 days and only 4 cultivars had mean germination times higher than 2 days. Increased salt concentrations affected Leed, Royal, AC Sunset and FO2 mean germination times more those of other cultivars. Salt stress increased mean germination times of cultivars. Even though Royal and AC Sunset germination percentages were not greatly reduced by increased salt concentrations, their mean germination times increased as much as Leed and FO2, and these cultivars had the highest increase in mean germination time in the study.
Mean germination rate ranged from 0.28 to 0.73 among the genotypes under different salt concentrations. Control groups had the highest mean germination rates in the study and the lowest mean germination rates were found in seeds germinated under high salt concentrations (Table 3).
Since control groups was not exposed salt stress conditions, it was not possible to calculate germination stress index for them.

Discussion and Conclusion
Salinization of agricultural soils is an important environmental stress factor that limit seed germination, plant growth and yield. In addition, salinization exacerbates soil conditions for plant growth by increasing soil sodium content and soil pH, creating suboptimal conditions for plant growth and  [14][15][16][17]. Different treatments were tested to alleviate these environmental conditions, such as chitosan application to seeds [18], seed priming [19] and foliar applications to plants [20]. Another way to increase crop productivity is to find suitable genotypes resistant to abiotic stress factors through screening [21,22] and to incorporate necessary genes to elite lines through breeding [23].
In the present study, diverse safflower cultivars were screened for their germination capacities under 5 different salt concentrations. Different seed parameters were calculated using germination data. Seed germination parameters could be used to identify salinity tolerance of different genotypes at germination stage [24]. All tested cultivars had more than 50% germination up to 120 mM salt concentration (Table 3). However, increasing salt concentration to 180 and 240 mM differentiated  Timson's germination index uses daily cumulative germination percentage and the higher value of Timson's index correlates to speedy germination. Consequently, control seeds, which have not been exposed to salt stress, had higher values for Timson's index, hence speedier germination as observed by mean germination times as well. As germination percentage decreased and mean germination time increased with higher salt concentrations, Timson's germination index, speed of germination, decreased.
The lowest values for Timson's germination index was found for FO2 and Leed, which had the lowest germination percentages and high mean germination times at 240 mM salt concentrations.
Mean germination time is another germination parameter used to calculate relative germination speed. It is used to compare effects of different treatments on speed of germination but it does not always correlate with time to germinate to reach a specific germination percentage [25]. Increased salt concentrations caused an increase in mean germination times of cultivars. However, mean germination times of FO2 (3.26) and Leed (2.77) was lower than AC Sunset (3.62) at 240 mM salt concentration, whose germination percentage was higher than FO2 and Leed. Mean germination time did not correspond to germination percentages at 240 mM salt concentration may have overestimated speed of germination in FO2 and Leed. Similarly, San Jose 89 had high mean germination time at 0 mM but mean germination time increase for 240 mM was 0.62, lower than Royal's mean germination time.
Mean germination rate is based on mean germination time and they have inverse relationship to each other. As mean germination time is higher, mean germination rate gets lower. Consequently, higher mean germination rates were observed in control seeds, but mean germination rate values did not correspond to germination percentages. Both San Jose 89 and Lesaf 414 had similar germination percentages for their controls, their mean germination rates were very different (Table 3) due to differences in mean germination times, and negative correlation (-0.95) observed between the two parameters (Table 3).
Germination stress index was also calculated to determine effects of salt stress on germination capacity of seeds. Leed showed 29% decrease in germination stress index from 60 mM to 120 mM salt concentration. The highest decreases after Leed at the same salt concentrations was observed in Quiriego 88 and Gila by 13% and 12%; respectively. At 180 and 240 mM salt concentrations, both Leed and FO2 had the lowest germination stress index values compared to other genotypes. Based on germination stress index, Leed was more susceptible than FO2 and Royal was the most tolerant cultivar to salt stress at germination stage.
Based on germination indices, it could be concluded that the germination percentage, Timson's germination index and germination stress index were the best indices for the assessment of the germination capacity compared to mean germination time and mean germination rate. These two indices might be affected by the seed quality and seed features, such as imbibition time. These indices might reflect differences related to those characters rather than the differences in germination capacity under stress conditions. Based on these indices, Leed and FO2 were sensitive and Royal and US-10 were resistant to salt stress at the germination stage.