Variation for Salinity Tolerance During Seed Germination in Diverse Carrot [Daucus carota (L.)] Germplasm

in HortScience

Global carrot production is limited by the crop’s high susceptibility to salinity stress. Not much public research has been conducted to screen for genetic salinity stress tolerance in carrot, and few resources exist to aid plant breeders in improving salinity tolerance in carrot. The objectives of this study were to evaluate the response of diverse carrot germplasm to salinity stress, identify salt-tolerant carrot germplasm that may be used by breeders, and define appropriate screening criteria for assessing salt tolerance in germinating carrot seed. Carrot plant introductions (PIs) (n = 273) from the U.S. Department of Agriculture (USDA) National Plant Germplasm System representing 41 different countries, inbred lines from the USDA Agricultural Research Service (n = 16), and widely grown commercial hybrids (n = 5) were screened for salinity tolerance under salinity stress and nonstress conditions (150 and 0 mm NaCl, respectively) by measuring the absolute decrease (AD) in the percent of germination, inhibition index (II), relative salt tolerance (RST), and salt tolerance index (STI) of germinating seeds. All salt tolerance measurements differed significantly between accessions; AD ranged from −4.2% to 93.0%; II ranged from −8.0% to 100.0%; RST ranged from 0.0 to 1.08; and STI ranged from 0.0 to 1.38. Broad sense heritability calculations for these measurements were 0.87 or more, indicating a strong genetic contribution to the variation observed. Six accessions identified as salt-tolerant or salt-susceptible were evaluated in a subsequent experiment conducted at salt concentrations of 0, 50, 100, 150, 200, and 250 mm NaCl. Variations between mean AD, II, RST, and STI of tolerant and susceptible lines were greatest at 150 mm NaCl, validating the use of 150 mm NaCl concentrations during salt tolerance screening of carrot seed. Wild carrot accessions displayed little tolerance, and PI 256066, PI 652253, PI 652402, and PI 652405 from Turkey were most salt-tolerant.

Abstract

Global carrot production is limited by the crop’s high susceptibility to salinity stress. Not much public research has been conducted to screen for genetic salinity stress tolerance in carrot, and few resources exist to aid plant breeders in improving salinity tolerance in carrot. The objectives of this study were to evaluate the response of diverse carrot germplasm to salinity stress, identify salt-tolerant carrot germplasm that may be used by breeders, and define appropriate screening criteria for assessing salt tolerance in germinating carrot seed. Carrot plant introductions (PIs) (n = 273) from the U.S. Department of Agriculture (USDA) National Plant Germplasm System representing 41 different countries, inbred lines from the USDA Agricultural Research Service (n = 16), and widely grown commercial hybrids (n = 5) were screened for salinity tolerance under salinity stress and nonstress conditions (150 and 0 mm NaCl, respectively) by measuring the absolute decrease (AD) in the percent of germination, inhibition index (II), relative salt tolerance (RST), and salt tolerance index (STI) of germinating seeds. All salt tolerance measurements differed significantly between accessions; AD ranged from −4.2% to 93.0%; II ranged from −8.0% to 100.0%; RST ranged from 0.0 to 1.08; and STI ranged from 0.0 to 1.38. Broad sense heritability calculations for these measurements were 0.87 or more, indicating a strong genetic contribution to the variation observed. Six accessions identified as salt-tolerant or salt-susceptible were evaluated in a subsequent experiment conducted at salt concentrations of 0, 50, 100, 150, 200, and 250 mm NaCl. Variations between mean AD, II, RST, and STI of tolerant and susceptible lines were greatest at 150 mm NaCl, validating the use of 150 mm NaCl concentrations during salt tolerance screening of carrot seed. Wild carrot accessions displayed little tolerance, and PI 256066, PI 652253, PI 652402, and PI 652405 from Turkey were most salt-tolerant.

Salinity stress is considered one of the most important abiotic factors that limits the productivity of crop plants (Flowers and Yeo, 1995), and the estimated global cost due to salinity is more than $12 billion annually (Qadir et al., 2008). Annually, ≈10 million hectares of land are becoming salinized to a point where the land can no longer sustain adequate crop production. This is due to the extensive use of irrigation and high rates of evapotranspiration, which result in increased salt accumulating in the soil (Rozema and Flowers, 2008). Most crops, including cultivated carrot (Daucus carota var. sativus), are categorized as glycophytic plants. The growth of glycophytes is greatly reduced in saline soils because they lack physiological mechanisms such as salt glands and bladders that allow halophytes, which are salt-loving plants, to thrive in high salinity (Flowers et al., 2010). One approach to combating the negative effects of salinity stress in glycophytic crops is identifying new genetic sources of tolerance and efficient phenotypic methods to develop salinity-tolerant cultivars (Munns, 2005).

Data collected from many crop species suggest that the level of salinity tolerance is highly dependent on the developmental stage of the plant (Chinnusamy et al., 2005). This life stage-specific tolerance means that a genotype that has tolerance at one life stage may not be tolerant at earlier or later stages. Therefore, to more effectively identify tolerant genotypes, evaluation needs to occur throughout the varying stages of ontogeny of the plant, from germination through the reproductive phase. This type of extensive evaluation is needed to develop varieties that are considered fully tolerant at each developmental stage for carrots.

Screening for salt tolerance at the germination stage is the first step in identifying tolerant genotypes because it is a critical stage for plant development. A number of studies of several crop species, including alfalfa, barley, corn, red kidney bean, and sugar beet (Abel and MacKenzie, 1964), tomato (Cuartero and Fernández-Muñoz, 1998; Foolad and Lin, 1997), grain sorghum (Francois et al., 1984), sugar beet, cabbage, amaranth, and pak choi (Jamil et al., 2006), corn (Maas et al., 1983), cowpea (Ravelombola et al., 2017), and lettuce (Xu and Mou, 2015) have demonstrated that the percent of germination is adversely affected as salinity increases, decreasing the osmotic potential and thus lowering the water potential. Fortunately, screening for salinity tolerance at the germination stage is one of the most rapid and economical stages of development to evaluate a large number of diverse germplasm accessions for tolerance.

Carrot (Daucus carota L.; 2n = 2x = 18) is an economically important root vegetable crop worldwide; every year, 38 million metric tons of carrots are produced on 1.2 million hectares globally. Carrot is widely produced throughout Asia and in the United States, where it is the sixth most consumed fresh vegetable, with more than 80% of U.S. production under irrigation in California (FAO, 2017). Carrot is an important crop for world nutritional security because it is one of the leading dietary sources of provitamin A carotenoids (α-carotene and β-carotene) in the human diet. In the United States, carrots account for 30% of dietary β-carotene and 62% of dietary α-carotene (Simon et al., 2009).

Cultivated carrot (Daucus carota var. sativus) is one of the most salt-sensitive vegetable crops (Bernstein and Ayers, 1953; Maas and Hoffman, 1977). To date, there have been few evaluations of the salinity tolerance of carrot during the seed germination stage (Kahouli et al., 2014; Rode et al., 2012; Schmidhalter and Oertli, 1991). These studies suggest that carrot seed germination suffers greatly from increased salt concentrations having both total seed germination and rate of germination decrease under salinity stress with these effects becoming more drastic as the concentration of salts increases. The results of these evaluations indicated that concentrations in the range of 125 mm to 150 mm NaCl differentiate the tolerant and sensitive accessions of carrot. In the most expansive evaluation, Kahouli et al. (2014) evaluated 10 carrot accessions from Tunisia. The current lack of information regarding salinity tolerance during the germination stage for carrot suggests the need for a large germplasm evaluation to identify potentially tolerant accessions that could be used for breeding or other studies. The objectives of this study were to evaluate the responses of 294 diverse carrot germplasm accessions, inbred lines, and commercial cultivars to salinity stress during the germination stage, to identify the best measurement for assessing salt tolerance in carrot at the germination stage, and to identify salt-tolerant accessions of carrot to be used in breeding programs and other research.

Materials and Methods

Germplasm.

A total of 294 carrot accessions consisting of 273 accessions (210 cultivated and 63 wild) from the USDA National Plant Germplasm System (NPGS) collection of plant introductions (PIs) in Ames, IA, 16 inbred lines from the USDA carrot breeding program, and 5 widely grown commercial carrot hybrids were included in this analysis. Commercial cultivars were categorized by root color (Supplemental Table 1). The 294 carrot accessions originated from 41 countries and were classified into 14 geographic regions based on their origin (Eastern Africa, Northern Africa, South Africa, North America, South America, Central Asia, Eastern Asia, Southern Asia, Western Asia, Eastern Europe, Northern Europe, Southern Europe, Western Europe, and Oceania). These PIs represented much of the world’s genetic diversity for carrot (Iorizzo et al., 2013).

Germination assay.

Twenty seeds from each carrot accession were placed on P5 filter paper in 60-×15-mm petri dishes (Fisher Scientific, Waltham, MA). Each petri dish was filled with 7 mL of a 150-mm NaCl solution or deionized water (0 mm NaCl) for the salt stress and control treatments, respectively. Petri dishes were stacked in large plastic bins and placed in complete dark at 23°± 1°. Each accession–treatment combination was present in six total replications in a randomized complete block design, with each bin acting as a replication. The design and conditions were similar to those used in other studies (Maas et al., 1983; Ravelombola et al., 2017).

Evaluation using 150 mm NaCl.

Seed germination data were collected for a total of 18 d, with measurements taken 2, 4, 6, 10, 14, and 18 d after sowing. A seed was classified as germinated when the radicle had emerged and was longer than 1 mm. At each measurement time, any seed that had germinated was counted and removed from the petri dish. Standard criteria for determining the performance of carrot accessions under salinity included: 1) final percent of germination under nonstress conditions (PGControl); 2) final percent of germination under salt stress (PGNaCl); 3) absolute decrease (AD) due to salt; 4) inhibition index (II); 5) relative salt tolerance (RST); and 6) salt tolerance index (STI) (Ravelombola et al., 2017). These measurements were calculated using the following equations: AD = PGControl – PGNaCl; II = 100 * (PGControl – PGNaCl)/(PGControl); RST = PGNaCl/PGControl; STI = (PGNaCl*PGControl)/(PGAverage)2, where PGAverage is the average percent of germination of all carrot accessions evaluated under no salt stress.

Evaluations using varying salt concentrations.

After the initial germplasm evaluation, a second experiment was conducted to evaluate the germination of a subset of both tolerant and sensitive accessions with a range of NaCl concentrations. Using the same design as described, six carrot accessions (three tolerant and three sensitive) (Table 1) were tested to determine the percent of seed germination using six concentrations of salt (0, 50, 100, 150, 200, and 250 mm NaCl) for 7 d. These concentrations were obtained by dissolving 1.46, 2.92, 4.38, 5.85, and 7.3 g of sodium chloride (Fisher Scientific) in 500 mL of distilled water.

Table 1.

Three tolerant and three susceptible carrot accessions with the percent of germination without salt stress (Nonstress) using 150 mm salt stress (Stress), relative salt tolerance (RST), absolute decrease (AD), inhibition index (II), and salt tolerance index (STI).

Table 1.

Data analysis.

The statistical model used to analyze each of the six measurements was as follows: Yij = µ + Ri + Aj + Ɛij, where Yij is the value of the measurements for the jth carrot accession in the ith replication, where i = 1, …, 6 and j = 1, …, 296, µ is the total mean (constant), Ri is the effect of the ith replication (random effect) on the response measurement, Aj is the effect of the jth accession (fixed effect) on the response measurement, and Ɛij is the effect of the experimental error associated with ijth observation. All analyses were performed using R.3.4.4 (R Core Team, 2018). The analysis of variance (ANOVA) test was performed using the lmer function in the lme4 package (Bates et al., 2018). The least significant difference (lsd) test, with α = 0.05, of the mean separation was performed using the lsd test function found in the agricolae package (De Mendiburu, 2014). Pearson rank correlations of measurements were calculated using the cor function found in the stats package (R Core Team, 2018).

The variance values of the among-accession and within-accession measurements were used for calculating broad-sense heritability (H2), as derived from the work of Falconer and Mackay (1996). H2 = (σ2G2P) = [σ2G/(σ2G + (σ2E/r) + (σ2R/r)], where σ2G = genotypic (accession) variance, σ2P = phenotypic variance, σ2E = variance due to experimental error, σ2R = variance due to replication, and r is the number of replications for each treatment.

Variance components were derived using the following formulas: σ2G = (MSA – MSE)/r, σ2E = MSE, σ2R = (MSR – MSE)/n, where MSA is the mean square accession, MSE is the mean square error, MSR is the mean square replication, r is the number of replications, and n is the number of accessions.

Results and Discussion

Germination assay.

The average percent of germination for the 294 carrot accessions under nonstress conditions ranged from 50.0% to 100.0%, with a mean of 81.6% (sd, 12.6%). Germinating seeds in 150 mm NaCl solution reduced the average percent of germination to 37.0% (range, 0.0% to 95.0%; sd, 25.7%) (Fig. 1). These data indicated that salinity stress significantly reduced the percent of germination in most, but not all, carrot germplasm, as indicated by previous studies. For all salt stress-related traits, there was a significant replication effect (P < 0.0001) that may have been attributable to human error in phenotyping germination and to variations in the microenvironment within the boxes where the seed was germinated. The average percent of germination varied significantly among carrot accessions under control conditions (F = 8.24; P < 0.0001) (Table 2). PI 632391, PI 642756, PI 643119, and PI 652374 all had the maximum percent of germination values (100.0%), whereas PI 502914, PI 508473, PI 652154, and PI 652253 had the lowest percent of germination (50.0% to 52.2%) under nonstress conditions (Table S1). Percent germination under salt stress also varied significantly among carrot accessions (F = 12.42; P < 0.0001) (Table 2). Under salt stress conditions, B493B, Nb6526, PI 177381, PI 279764, PI 652344, and PI 652380 all had the lowest percent of germination (0.0%), indicating that they were especially salt-sensitive accessions, whereas PI 652374, PI 652402, and PI 652405 (91.7% to 95.0%) all performed well under salt stress conditions, indicating that they are salt-tolerant accessions.

Fig. 1.
Fig. 1.

Distribution and mean (dotted line) percent of seed germination among 294 carrot accessions without salt stress (dark gray) and with salt stress (light gray).

Citation: HortScience horts 54, 1; 10.21273/HORTSCI13333-18

Table 2.

ANOVA for six measurements related to seed germination among 294 carrot accessions.

Table 2.

Interestingly, several cultivated PIs from Turkey displayed the highest level of tolerance. PI 652402, PI 652403, and PI 652405 all had low AD and II values (1.6% to 3.4%), high RST values (0.97 to 0.98), and high STI values (1.28 to 1.38). In fact, most cultivated carrot from Turkey and other countries with low rainfall and, consequently, very reliant on irrigation for carrot production were relatively salt-tolerant. These results suggest that carrot cultivated in more saline soils over time were selected for higher levels of salt tolerance. The most sensitive carrot accessions were wild PIs or inbred lines. Inbred lines B493B and Nb6526B, along with wild carrot from Turkey, Syria, and Libya (PI 177381, PI 279764, PI 652344, and PI 652380), all had 0.0% germination under salt stress; therefore, they had II values of 100.0%, AD values ranging from 56.0% to 83.0%, and the lowest possible RST and STI values (0.0). Many of the most susceptible accessions were wild, which is contrary to what has been observed in many other species in which abiotic stress tolerance has been identified in wild relatives. It should be noted that not all wild carrot types were salt-susceptible. In fact, several wild carrots evaluated were relatively salt-tolerant, perhaps because they were collected from naturally saline nonagricultural land.

The AD measures the decrease in the percent of germination between nonstress conditions and salt stress (PGControl – PGNaCl). For the carrot accessions evaluated in this experiment, salt stress reduced the percent of germination from −4.2% to 93.0% (Fig. 2A). AD varied significantly among carrot accessions (F = 7.87; P < 0.0001) (Table 2). PI 515990 (93.0%) had the highest AD, indicating it is a salt-sensitive accession, and PI 256066 (−4.2%) had the lowest and only negative AD, indicating that salt stress increased the percent of germination.

Fig. 2.
Fig. 2.

Distribution and mean (dotted line) percent of seed germination among 294 carrot accessions with four different measures of salt tolerance: (A) absolute decrease; (B) inhibition index; (C) relative salt tolerance; and (D) salt tolerance index.

Citation: HortScience horts 54, 1; 10.21273/HORTSCI13333-18

The II for germination (100*AD/PGControl) was significantly different among accessions (F = 8.51; P < 0.0001) (Table 2) and ranged from −8.0% to 100.0% (Fig. 2B). The six sensitive accessions mentioned all had the highest II values of 100.0%, which followed the same trends as the salt stress percent of germination and AD values, indicating that they were highly salt-sensitive accessions. PI 256066 and PI 652253 had the lowest II values (−8.0% to −1.4%), indicating high levels of salt tolerance during the seed germination stage.

Relative salt tolerance (RST = PGNaCl/PGControl) was significantly different among accessions (F = 8.46; P < 0.0001) (Table 2) and ranged from 0 to 1.08, with a mean of 0.56 (Fig. 2C). The four wild PIs and two inbred lines previously mentioned had the lowest RST value of 0, indicating high salt sensitivity. PI 256066 (1.08) and PI 652253 (1.00) had the highest RST values, indicating that they were salt-tolerant when considering their percent of germination under nonstress conditions.

The STI [STI = PGNaCl*PGControl)/(PGAverage)2] is an important measurement of salt tolerance when comparing the tolerance of one accession to the rest of the collection (Saad et al., 2013). STI in this analysis ranged from 0 to 1.38, with a mean of 0.47 (sd, 0.36) (Fig. 2D), and it was significantly different among accessions (F = 14.04; P < 0.0001) (Table 2). The six accessions with the lowest RST value also had the lowest STI value of 0, indicating low salt tolerance, whereas the tolerant cultivated accessions from Turkey, PI 652402 and PI 652405, both had the highest STI value of 1.38.

These multiple criteria for quantifying salt tolerance demonstrated wide phenotypic variations in salt tolerance during the seed germination stage among diverse carrot accessions. Exposure to 150 mm salt solution significantly reduced the percent of germination for most of the carrot diversity panel, thus agreeing with the results of similar studies of other species (Abel and MacKenzie, 1964; Cuartero and Fernández-Muñoz, 1998; Ravelombola et al., 2017) and with smaller studies involving carrots (Rode et al., 2012; Schmidhalter and Oertli, 1991). This evaluation found a wider range of variations for salt tolerance in carrot during the germination stage than did previous studies. For example, Kahouli et al. (2014) identified a percent of germination range of 45.0% to 85.0% at 137 mm NaCl and of 15.0% to 50.0% at 171 mm NaCl for 10 carrot accessions. We observed a range of 0.0% to 95.0% at 150 mm NaCl, thus reinforcing the value of evaluating a large diverse germplasm collection for salt tolerance traits.

Salt tolerance according to geographic origin.

Significant differences in the percent of seed germination under nonstress conditions and for all salt tolerance germination measurements were observed among the 14 different regions of carrot accession origin (P < 0.0001) (Table 3). When evaluating all cultivated, wild, inbred, and hybrid accessions together, accessions from Southern and Eastern Asia displayed higher salt tolerance during the germination stage. Accessions from Southern Asia had a mean AD of 30.4% (range, −4.2% to 82.4%), a mean II of 40.0% (range, −8.0% to 100.0%), mean RST value of 0.6 (range, 0.00 to 1.1), and mean STI value of 0.56 (range, 0.00 to 1.22). Accessions from Eastern Asia had similar values and ranges as those of Southern Asia, with a mean AD of 33.1% (range, −5.0% to 70.0%), a mean II of 33.5% (range, 6.2% to 95.5%), mean RST value of 0.61 (range, 0.04 to 0.94), and mean STI value of 0.59 (range, 0.04 to 1.12). Oceania and South America each had only one accession, but they were among the most sensitive to salt stress during the germination stage (Table 4). Although accessions from Southern and Eastern Asia were more salt-tolerant than those of other regions, it is still important to note the wide range of variations among accessions from a single geographical region. For example, PI 256065 from Afghanistan and PI 274297 from Pakistan only had 2.5% germination under salt stress. These results reflect the importance of evaluating multiple accessions from within a single region.

Table 3.

ANOVA for six measurements related to seed germination among carrot accessions from 14 geographic regions of origin.

Table 3.
Table 4.

Mean separation of four salt tolerance measurements across 14 geographic regions of origin for all 294 accessions (63 wild PIs, 210 cultivated PIs, 16 inbred lines, and 5 hybrids).

Table 4.

When evaluating the cultivated PIs, accessions from Northern Africa, Western Asia, and North America displayed higher tolerance, with mean II and AD values ranging from 19.80% to 20.02%, along with RST and STI values ranging from 0.70 to 0.94. Accessions from Northern Europe, Eastern Africa, and Oceania had lower tolerance, with mean II and AD values ranging from 55.04% to 89.72%, as well as RST and STI values ranging from 0.10 to 0.33 (Table 5). These results suggest that the geographic origin significantly influenced salt tolerance, and it can be speculated that geographic origin is a factor influencing carrot adaptation to sub-optimal growing conditions, as has been seen in other plant species (Baxter et al., 2010; Khoury et al., 2015). Interestingly, although Central Asia is the center of origin of cultivated carrots (Iorizzo et al., 2013), greater diversity has been observed in salinity tolerance in other geographic regions, likely reflecting germplasm response to varying local conditions. To identify salt-tolerant germplasm at later stages of development, and to identify other abiotic stress tolerance traits, it will be critical to screen multiple accessions from diverse geographic regions of the world to best determine the full range of phenotypic variation.

Table 5.

Mean separation for four salt tolerance measurements based on region of origin among 210 cultivated carrot accessions.

Table 5.

Among wild PIs, accessions from Eastern Asia displayed the highest tolerance, with mean II, AD, RST, and STI values of 33.49%, 24.74% 0.66, and 0.51, respectively. Wild PIs from South America, Western Asia, and Central Asia had low tolerance, with mean II and AD values ranging from 67.76% to 91.76%, along with RST and STI values of 0.2 or less (Table 6).

Table 6.

Mean separation for four salt tolerance measurements based on region of origin among 63 wild carrot accessions.

Table 6.

Salt tolerance according to domestication status and root color.

No significant difference was observed between cultivated PIs, wild PIs, and inbred lines regarding the average percent of germination under nonstress conditions (Table 7). In contrast, cultivated PIs demonstrated significantly greater average salt tolerance during the germination stage than the wild PIs and inbred lines, with a mean percent of germination of 45.4% (range, 0.8% to 95.0%) under salt stress, an AD of 36.7% (range, −4.1% to 93.0%), II of 44.5% (range, −8.0% to 98.8%), RST of 0.55 (range, 0.1 to 1.1), and STI of 0.58 (range, 0.01 to 1.38). Most wild PIs and inbred lines demonstrated a higher level of sensitivity to salt stress than cultivated PIs, with no significant difference between the mean of these two groups. Both groups had a mean percent of germination under stress less than 17%, but with a range from 0.0% to 71.1%, mean AD and II values ranging from 59.9% to 82.0%, and RST and STI values of 0.22 or less (Table 7). Although both domestication status groups were not equally represented in this study, these results suggested that cultivated PIs are an especially promising source of salt tolerance during the germination stage for carrot breeding programs.

Table 7.

Mean (± SE) for the percent of germination without salt stress (Nonstress), percent of germination with 150 mm salt stress (Stress), absolute decrease (AD), inhibition index (II), relative salt tolerance (RST), and salt tolerance index (STI) separated by domestication status (DS) and primary root color (excluding wild PIs) with the number of accessions found in each category.

Table 7.

Analyzing the mean of each salinity response trait according to the primary root color of cultivated carrot demonstrated that all five color categories had relatively similar average percent of germination values under nonstress conditions. Although there were significant differences between some colors for the various parameters measured, all traits of the red and purple roots were similar and demonstrated the highest average level of salt tolerance, with germination more than 48.0% under salt stress, AD values less than 26.0%, II values less than 34.0%, RST values of 0.70, and STI values of 0.66 (Table 7). Given the relatively few samples of white and red accessions in this study, trends reported here should be confirmed with larger sample sizes.

Evaluations using varying concentrations of NaCl.

ANOVA for the percent of seed germination of three tolerant and three sensitive accessions displayed significant treatment (F = 123.5; P < 0.0001) and accession (F = 15.68; P < 0.0001) effects on the percent of seed germination (Table 8), which was not unexpected given the previous findings of this evaluation. The most significant difference between accessions was observed with the 150 mm treatment (F = 90.27; P < 0.0001), confirming that 150 mm was the optimum concentration for screening carrot germplasm for salt tolerance. Treatments using 0 and 50 mm differed only slightly among accessions (P = 0.03 and P = 0.02, respectively) (Table 9). With increased salt concentrations, there was a decrease in the percent of germination for both tolerant and sensitive accessions, and this trend was more noticeable in sensitive accessions (Fig. 3). The pool of sensitive accessions demonstrated a 37.5% reduction in germination when the concentration of salt was increased from 50 to 100 mm. Increasing the NaCl concentration to 150 mm resulted in a further reduction of 42.9%, thus supporting results from the larger accession evaluation, with no germination at 200 and 250 mm NaCl. Tolerant accessions had a minimal reduction (4.6%) in germination when the NaCl concentration was increased from 0 to 100 mm; however, they had a large reduction (64.6%) when it was increased from 150 to 200 mm. No accession had any germination with the 250 mm treatment (Fig. 3), indicating that a concentration between 200 and 250 mm NaCl is the threshold at which germination is completely suppressed. The results of this experiment indicated that future germplasm screening for salt tolerance in diverse carrot accessions should be performed with a concentration of 150 mm NaCl. The identification of carrot germplasm capable of germinating at 200 mm NaCl would be an outstanding discovery.

Table 8.

ANOVA for the percent of seed germination among six carrot accessions using six salt concentrations of NaCl.

Table 8.
Table 9.

ANOVA for seed germination among six carrot accessions under six concentrations of NaCl.

Table 9.
Fig. 3.
Fig. 3.

Pooled percent of seed germination for three sensitive (solid line) and three tolerant (dotted line) carrot accessions using six NaCl concentrations (0, 50, 100, 150, 200, and 250 mm).

Citation: HortScience horts 54, 1; 10.21273/HORTSCI13333-18

Broad-sense heritability.

High broad-sense heritability (H2) was identified for all germination-related measurements. Seed germination under nonstress conditions had an H2 of 0.88, which increased to 0.92 under salt stress. H2 values were 0.87, 0.88, 0.88, and 0.93 for AD, II, RST, and STI, respectively. The heritability of traits estimated for the carrot accessions evaluated in this analysis was higher than that displayed by cowpea (0.84) (Ravelombola et al., 2017) and by tomato (0.76) (Foolad and Jones, 1992), indicating a significant genetic basis for these traits. These genetic factors warrant further investigation, as has been performed for cowpea (Ravelombola et al., 2018), which was found to have a significant quantitative trait locus (QTL) associated with salt tolerance during the germination stage in mapping populations.

Correlation among salt tolerance parameters and seed weight.

Pearson correlation coefficients calculated for each of the salt tolerance measurements and for the hundred seed weight (HSW) of each accession (Table 10) demonstrated that germination under nonstress conditions had a moderate positive linear correlation with STI (r = 0.54) and germination under stress conditions (r = 0.4). This is not surprising because the percent of germination under nonstress conditions sets the upper limit for germination under stress conditions. There was no strong correlation between the nonstress percent of germination and the other four measurements (r range, −0.17 to 0.15), indicating that there was no relationship between the percent of germination under nonstress conditions and salt tolerance. The HSW had a weak positive linear correlation with the percent of germination under salt stress (r = 0.25) and a negligible negative correlation (r = −0.17) under nonstress conditions. These results suggest that seed weight had minimal effects on the ability of an accession to tolerate salt stress during germination.

Table 10.

Correlation among seven salt stress parameters: absolute decrease (AD), hundred seed weight (HSW), inhibition index (II), under nonstressed condition (Nonstress), relative salt tolerance (RST), salt tolerance index (STI), and under salt stress (Stress).

Table 10.

Conclusions

This study identified a wide range of phenotypic variations for salt tolerance during the germination stage in a collection of diverse carrot accessions. Five cultivated carrot accessions, all from Turkey (PI 509433, PI 652374, PI 652402, PI 652403, and PI 652405), were identified as salt-tolerant accessions, whereas inbred lines B493B and Nb6526B and four wild accessions (PI 177381, PI 279764, PI 652344, and PI 652380) were identified as highly salt-sensitive accessions. These accessions could serve as potential parents for creating mapping populations to identify the QTL associated with salt tolerance during the germination stage of carrot. The discovery of tolerant cultivated accessions is promising for breeders because they may be used to develop salt-tolerant cultivars. The development of carrot cultivars with tolerance to salt stress will provide growers everywhere with additional tools for growing on salt-affected soil. This evaluation of salt tolerance of diverse carrot germplasm provides valuable information for future studies of salt tolerance of carrots.

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    • Search Google Scholar
    • Export Citation
  • FlowersT.J.YeoA.R.1995Breeding for salinity resistance in crop plants: Where next?Austral. J. Plant Physiol.22875884

  • FooladM.R.JonesR.A.1992Parent offspring regression estimates of heritability for salt tolerance during germination in tomatoCrop Sci.322439442

    • Search Google Scholar
    • Export Citation
  • FooladM.R.LinG.Y.1997Genetic potential for salt tolerance during germination in lycopersicon speciesHortScience32296300

  • FrancoisL.E.DonovanT.MaasE.V.1984Salinity effects on seed yield, growth, and germination of grain sorghumAgron. J.765741744

  • IorizzoM.SenalikD.A.EllisonS.L.GrzebelusD.CavagnaroP.F.AllenderC.BrunetJ.SpoonerD.M.DeynzeA.V.SimonP.W.2013Genetic structure and domestication of carrot (Daucus carota subsp. sativus) (Apiaceae)Amer. J. Bot.1005930938

    • Search Google Scholar
    • Export Citation
  • JamilM.LeeD.B.JungK.Y.AshrafM.LeeS.C.RhaE.S.2006Effect of salt (NaCl) stress on germination and early seedling growth of four vegetable speciesJ. Cent. Eur. Agr.7273281

    • Search Google Scholar
    • Export Citation
  • KahouliB.BorgiZ.HannachiC.2014Effect of sodium chloride on the germination of the seeds of a collection of carrot accessions (Daucus carota L.) cultivated in the region of Sidi BouzidJ. Stress Physiol. Biochem.1032836

    • Search Google Scholar
    • Export Citation
  • KhouryC.K.Castañeda-AlvarezN.P.AchicanoyH.A.SosaC.C.BernauV.KassaM.T.NortonS.L.van der MaesenL.J.G.UpadhyayaH.D.Ramírez-VillegasJ.JarvisA.StruikP.C.2015Crop wild relatives of pigeonpea [Cajanus cajan (L.) Millsp.]: Distributions, ex situ conservation status, and potential genetic resources for abiotic stress toleranceBiol. Conserv.184259270

    • Search Google Scholar
    • Export Citation
  • MaasE.V.HoffmanG.J.1977Crop salt tolerance - current assessmentJ. Irrig. Drain. Div.103115134

  • MaasE.V.HoffmanG.J.ChabaG.D.PossJ.A.ShannonM.C.1983Salt sensitivity of corn at various growth stagesIrrig. Sci.414557

  • MunnsR.2005Genes and salt tolerance: Bringing them togetherNew Phytol.1673645663

  • QadirM.TubeilehA.AkhtarJ.LarbiA.MinhasP.S.KhanM.A.2008Productivity enhancement of salt-affected environments through crop diversificationLand Degrad. Dev.19429453

    • Search Google Scholar
    • Export Citation
  • R Core Team2018R: A language and environment for statistical computing. R Foundation for Statistical Computing Vienna Austria

  • RavelombolaW.ShiA.WengY.MouB.MotesD.ClarkJ.ChenP.SrivastavaV.QinJ.DongL.YangW.BhattaraiG.SugiharaY.2018Association analysis of salt tolerance in cowpea (Vigna unguiculata (L.) Walp) at germination and seedling stagesTheor. Appl. Genet.13117991

    • Search Google Scholar
    • Export Citation
  • RavelombolaW.S.ShiA.WengY.ClarkJ.MotesD.ChenP.SrivastavaV.2017Evaluation of salt tolerance at germination stage in cowpea [Vigna unguiculata (L.) Walp]HortScience5211681176

    • Search Google Scholar
    • Export Citation
  • RodeA.NothnagelT.KampeE.2012Developing methods to evaluate salt stress tolerance in carrot cultivarsActa Hort.960393400

  • RozemaJ.FlowersT.2008Crops for a salinized worldScience32214781480

  • SaadF.F.El-MohsenA.A.A.El-ShafiM.A.A.Al-SoudanI.H.2013Effective selection criteria for evaluating some barley crosses for water stress toleranceEgypt. J. Plant Breed.1757998

    • Search Google Scholar
    • Export Citation
  • SchmidhalterU.OertliJ.J.1991Germination and seedling growth of carrots under salinity and moisture stressPlant Soil132243251

  • SimonP.W.PollakL.M.ClevidenceB.A.HoldenJ.M.HaytowitzD.B.2009Plant breeding for human nutritional qualityPlant Breed. Rev.31325392

  • XuC.MouB.2015Evaluation of lettuce genotypes for salinity toleranceHortScience5014411446

Supplemental Table 1.

Carrot accession, root color, country of origin, domestication status (DS), mean percent germination without salt stress (Nonstress) ± standard error, mean percent germination with 150 mM salt stress (Stress) ± standard error, mean absolute decrease (AD), mean inhibition index (II), relative salt tolerance (RST), mean salt tolerance index (STI), hundred seed weight (HSW), and rank based on STI.

Supplemental Table 1.

If the inline PDF is not rendering correctly, you can download the PDF file here.

Contributor Notes

We thank the Global Crop Diversity Trust Project GS14014 for providing financial support. We also thank Kathleen Reitsma and the USDA National Germplasm System for their capable assistance and for providing the plant introduction carrot collection accessions.

Corresponding author. E-mail: psimon@wisc.edu.

  • View in gallery

    Distribution and mean (dotted line) percent of seed germination among 294 carrot accessions without salt stress (dark gray) and with salt stress (light gray).

  • View in gallery

    Distribution and mean (dotted line) percent of seed germination among 294 carrot accessions with four different measures of salt tolerance: (A) absolute decrease; (B) inhibition index; (C) relative salt tolerance; and (D) salt tolerance index.

  • View in gallery

    Pooled percent of seed germination for three sensitive (solid line) and three tolerant (dotted line) carrot accessions using six NaCl concentrations (0, 50, 100, 150, 200, and 250 mm).

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  • FlowersT.J.GalalH.K.BromhamL.2010Evolution of halophytes: Multiple origins of salt tolerance in land plantsFunct. Plant Biol.377604612

    • Search Google Scholar
    • Export Citation
  • FlowersT.J.YeoA.R.1995Breeding for salinity resistance in crop plants: Where next?Austral. J. Plant Physiol.22875884

  • FooladM.R.JonesR.A.1992Parent offspring regression estimates of heritability for salt tolerance during germination in tomatoCrop Sci.322439442

    • Search Google Scholar
    • Export Citation
  • FooladM.R.LinG.Y.1997Genetic potential for salt tolerance during germination in lycopersicon speciesHortScience32296300

  • FrancoisL.E.DonovanT.MaasE.V.1984Salinity effects on seed yield, growth, and germination of grain sorghumAgron. J.765741744

  • IorizzoM.SenalikD.A.EllisonS.L.GrzebelusD.CavagnaroP.F.AllenderC.BrunetJ.SpoonerD.M.DeynzeA.V.SimonP.W.2013Genetic structure and domestication of carrot (Daucus carota subsp. sativus) (Apiaceae)Amer. J. Bot.1005930938

    • Search Google Scholar
    • Export Citation
  • JamilM.LeeD.B.JungK.Y.AshrafM.LeeS.C.RhaE.S.2006Effect of salt (NaCl) stress on germination and early seedling growth of four vegetable speciesJ. Cent. Eur. Agr.7273281

    • Search Google Scholar
    • Export Citation
  • KahouliB.BorgiZ.HannachiC.2014Effect of sodium chloride on the germination of the seeds of a collection of carrot accessions (Daucus carota L.) cultivated in the region of Sidi BouzidJ. Stress Physiol. Biochem.1032836

    • Search Google Scholar
    • Export Citation
  • KhouryC.K.Castañeda-AlvarezN.P.AchicanoyH.A.SosaC.C.BernauV.KassaM.T.NortonS.L.van der MaesenL.J.G.UpadhyayaH.D.Ramírez-VillegasJ.JarvisA.StruikP.C.2015Crop wild relatives of pigeonpea [Cajanus cajan (L.) Millsp.]: Distributions, ex situ conservation status, and potential genetic resources for abiotic stress toleranceBiol. Conserv.184259270

    • Search Google Scholar
    • Export Citation
  • MaasE.V.HoffmanG.J.1977Crop salt tolerance - current assessmentJ. Irrig. Drain. Div.103115134

  • MaasE.V.HoffmanG.J.ChabaG.D.PossJ.A.ShannonM.C.1983Salt sensitivity of corn at various growth stagesIrrig. Sci.414557

  • MunnsR.2005Genes and salt tolerance: Bringing them togetherNew Phytol.1673645663

  • QadirM.TubeilehA.AkhtarJ.LarbiA.MinhasP.S.KhanM.A.2008Productivity enhancement of salt-affected environments through crop diversificationLand Degrad. Dev.19429453

    • Search Google Scholar
    • Export Citation
  • R Core Team2018R: A language and environment for statistical computing. R Foundation for Statistical Computing Vienna Austria

  • RavelombolaW.ShiA.WengY.MouB.MotesD.ClarkJ.ChenP.SrivastavaV.QinJ.DongL.YangW.BhattaraiG.SugiharaY.2018Association analysis of salt tolerance in cowpea (Vigna unguiculata (L.) Walp) at germination and seedling stagesTheor. Appl. Genet.13117991

    • Search Google Scholar
    • Export Citation
  • RavelombolaW.S.ShiA.WengY.ClarkJ.MotesD.ChenP.SrivastavaV.2017Evaluation of salt tolerance at germination stage in cowpea [Vigna unguiculata (L.) Walp]HortScience5211681176

    • Search Google Scholar
    • Export Citation
  • RodeA.NothnagelT.KampeE.2012Developing methods to evaluate salt stress tolerance in carrot cultivarsActa Hort.960393400

  • RozemaJ.FlowersT.2008Crops for a salinized worldScience32214781480

  • SaadF.F.El-MohsenA.A.A.El-ShafiM.A.A.Al-SoudanI.H.2013Effective selection criteria for evaluating some barley crosses for water stress toleranceEgypt. J. Plant Breed.1757998

    • Search Google Scholar
    • Export Citation
  • SchmidhalterU.OertliJ.J.1991Germination and seedling growth of carrots under salinity and moisture stressPlant Soil132243251

  • SimonP.W.PollakL.M.ClevidenceB.A.HoldenJ.M.HaytowitzD.B.2009Plant breeding for human nutritional qualityPlant Breed. Rev.31325392

  • XuC.MouB.2015Evaluation of lettuce genotypes for salinity toleranceHortScience5014411446

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