Abstract
Drought is one of the major environmental challenges constraining the production of agricultural crops, including carrot. Seed germination is the initial and most critical stage of crop establishment, and it is very sensitive to drought stress because water scarcity affects the enzymatic solubilization of stored metabolites in seeds that provide energy for the growth of germinating embryo. Few studies evaluating the effect of drought stress on carrot seed germination of more than a few cultivars grown under stress have been published. Therefore, the present study was designed to define the appropriate osmotic potential for evaluating drought tolerance of carrot, evaluate the response of diverse carrot germplasm to drought stress during seed germination to identify drought-tolerant accessions that may be used by plant breeders, and evaluate the relation between amylase activity and germination rate of drought-tolerant and drought-sensitive accessions. To identify an appropriate screening osmotic potential, two commercial cultivars and two United States Department of Agriculture inbreeds were evaluated at six osmotic potentials (00, −0.30, −0.51, −0.58, −0.80, and −1.05 MPa); −0.58 MPa was identified as the optimal osmotic potential for screening the drought tolerance of carrot seed. Cultivated and wild carrot plant introductions (PIs) (n = 200 and n = 50, respectively) from the National Plant Germplasm System were evaluated for drought tolerance under nonstress and simulated drought stress conditions (00 MPa and −0.58 MPa, respectively) by calculating the absolute decrease (AD) in percent germination, inhibition index (II), relative drought tolerance (RDT), and drought tolerance index (DTI). All measurements of drought tolerance identified significant differences among accessions; the AD in seed germination ranged from 0.0% to 69.3%, II ranged from 0.0% to 80.2%, RDT ranged from 0.2 to 1.0, and DTI ranged from 0.13 to 1.47. All wild carrot accessions displayed low levels of drought tolerance, but PI 652387 and PI 177381 (both from Turkey) and PI 274297 (Pakistan) were most drought-sensitive, whereas cultivated accessions PI 643114 (United States), PI 652208 (China), and PI 502347 (Uzbekistan) were most drought-tolerant. Tolerant accessions displayed much higher α-amylase activity under nonstress conditions than sensitive accessions, and α-amylase activity of tolerant accessions was also reduced less with seed germination under increasing osmotic potential (range, 0.0 to −1.05 MPa) than sensitive accessions over 24, 48, and 72 hours of seed germination. This is the first evaluation of drought stress tolerance during seed germination and the enzymatic response of diverse carrot germplasms under simulated drought stress.
Global warming has altered the climate and rainfall pattern, thus constraining the water resources and causing severe drought in temperate and tropical regions of the world (Bai et al. 2020; Silva de Almeida et al. 2018). Drought is a global challenge because it causes severe yield losses in agricultural crops (Fahad et al. 2017; Yadav and Yadav 2018). The current global scenario is making this situation more extreme (King et al. 2017) because high temperatures cause actual shortages of water in the soil, termed physical or meteorological drought, whereas the state when water is present in soil but is unavailable to plants because of soil salinity is termed physiological drought (Hasanuzzaman et al. 2018; Vicente-Serrano et al. 2020). Drought is one of the most serious environmental constraints limiting the growth, development, and especially the morphological, biochemical, and physiological processes that typically cause more than 50% yield reductions of agricultural crops (Ali et al. 2019; Shah et al. 2019).
Carrot (Daucus carota L., 2n = 2x = 18) is a biennial, cool season, glycophytic root vegetable (Bolton and Simon 2019) that performs poorly under physical/meteorological and physiological drought conditions (Ali et al. 2019; Shah et al. 2019, 2020). Drought conditions drastically weaken the stand establishment by limiting the seed germination. Nevertheless, these yield losses in response to drought stress can be minimized in many crops by the development of cultivars tolerant to a lack of water (Vadez et al. 2013). D. carota germplasm varies widely, and tolerant sources of drought tolerance would be valuable for creating new cultivars. A breeding program requires at least 5 years for the intermediate selection of accessions of interest and to release a new carrot cultivar derived from cultivated germplasm (Simon et al. 2008); however, his prolonged time could be reduced if the selection of accessions is performed during an early stage of development, starting with seed germination. Furthermore, in breeding programs, seed germination responses have been of primary importance to the selection of accessions that are tolerant and/or sensitive to biotic and abiotic stresses (Almaghrabi 2012). The identification of salt-tolerant, salt-sensitive, heat-tolerant, and heat-sensitive carrot accessions at the seed germination stage has been reported (Bolton and Simon 2019; Bolton et al. 2019); however, a diverse collection of germplasms has not yet been examined for the drought tolerance of carrot.
Seed germination is a complex process that involves a series of physical and metabolic events such as water imbibition, reactivation of stored macromolecules, cell division, seedcoat rupturing, and radicle and plumule protrusion into new seedlings (Nonogaki et al. 2010; Xiao et al. 2019). It is the initial, but most critical, stage of seedling establishment, and it is very sensitive to drought stress (Ibrahim 2016) because water availability is essential for enzymatic solubilization of metabolites, such as carbohydrates, in storage tissues of seeds (Ma et al. 2017). Amylase enzymes have a vital role during seed germination because they hydrolyze the endospermic carbohydrates into simple sugars, which provide the energy for the growth of embryonic roots and shoots (Hajihashemi et al. 2020). The activity of amylase enzymes is reduced by water stress, which negatively affects carbohydrate metabolism and seed germination in wheat (Ali et al. 2020), thus making amylase activity a metabolic monitor of stress. Drought-tolerant lentil accessions retain much more of their amylase enzyme activity under stress than drought-sensitive accessions relative to amylase activity under nonstressed conditions during seed germination (Muscolo et al. 2014).
Screening of drought-tolerant accessions in dry, controlled environment conditions is critical because uniformly repeated controlled simulations of drought during complete the life cycle of crops cannot be easily achieved in open field conditions (Ahmad et al. 2020). Moreover, in vitro screening of drought tolerance is an efficient approach that facilitates studies of drought tolerance traits and enables the selection of drought-tolerant accessions (Khakwani et al. 2011). Crop breeders reported that exposure to osmotic solutions, such as polyethylene glycol (PEG-6000), has been proven as an effective method to mimic drought stress conditions during seed germination and subsequent growth stages in wheat (Khakwani et al. 2011), sweet potato (Agili et al. 2012), lentils (Muscolo et al. 2014), coffee (Silva de Almeida et al. 2018), barley (Hellal et al. 2018), ryegrass (Guo et al. 2020), maize (Badr et al. 2020), and carrot (Zhang et al. 2021). Because screening of diverse carrot germplasm for drought tolerance at seed germination has not yet been reported, the identification of drought-tolerant carrot germplasm will be valuable for carrot breeding programs.
The objectives of this study were to identify the threshold level of PEG-6000 for simulating drought stress during carrot seed germination and evaluate variations in drought tolerance in a diverse carrot germplasm collection during seed germination using measurements of the absolute decrease (AD) in germination, inhibition index (II), relative drought tolerance (RDT), and drought tolerance index (DTI). With this information, the relationship between the domestication status and root color in regard to drought tolerance was evaluated; furthermore, in a subset of tolerant and sensitive carrot accessions, the relationships among amylase enzyme activity, seed germination, and drought tolerance were measured.
Materials and Methods
Germplasm.
A total of 250 carrot genetic stocks consisting of 200 cultivated and 50 wild accessions from the United States Department of Agriculture (USDA) National Plant Germplasm System collection of Plant Introductions (PIs) were evaluated during this study. Carrot accessions were selected for the drought tolerance evaluation based on the diversity of their response during recent evaluations of salinity and heat tolerance during the seed germination stage (Bolton et al. 2019; Bolton and Simon 2019). The 250 carrot accessions originated from 41 countries and were classified into 14 geographic regions based on their origin (East Africa, North Africa, South Africa, North America, South America, Central Asia, East Asia, South Asia, West Asia, East Europe, North Europe, South Europe, West Europe, and Oceania). Genotypic data of these accessions will be published in Carrot Omics when additional data collection and analyses are completed.
Determination of optimal drought tolerance under a range of osmotic pressure conditions.
A preliminary experiment was conducted to determine the optimal osmotic potential causing physiological drought stress to evaluate drought tolerance of carrot. For this experiment, two carrot cultivars known for their abiotic stress tolerance (Brasilia, T-29) and two inbreeds (R6636 and Nb6526) were evaluated to test seed germination under six osmotic potentials (0.0, −0.30, −0.51, −0.58, −0.80, and −1.05 MPa) for 10 d. These osmotic potentials were obtained by dissolving 0, 10, 15, 18, 21, and 25 g of PEG-6000 in 100 ml of distilled water (Table 1). These PEG (MW 6000) solutions are also osmotically comparable in MPa (−0.30, −0.51, −0.58, −0.80, and −1.05 to 50, 100, 150, 200, and 250 mM) concentrations of sodium chloride (NaCl), which we tested during a previous evaluation of salinity tolerance of diverse carrot germplasms during the germination stage (Bolton and Simon 2019). The osmotic potential at which the most statistically significant differences with the lowest P values were observed among these four genetic stocks was chosen as the optimal osmotic potential or osmotic pressure to evaluate drought tolerance of carrot germplasm at the seed germination stage.
Osmotic pressure of polyethylene glycol concentrations used to evaluate drought stress in carrots at the seed germination stage in this study and NaCl concentrations that induce comparable osmotic pressure.
Germination assay under nonstress (0.0 MPa) and drought stress (−0.58 MPa).
After the preliminary evaluation, a second experiment was conducted using a randomized complete block design with six replications and two treatments [i.e., nonstress (0.0 MPa) and drought stress (−0.58 MPa)]. Twenty seeds from each carrot accession were placed on P5 filter paper in each of six 60- × 15-mm petri dishes (Fisher Scientific®). In each petri dish, 10 ml of either distilled water or 18% osmotic solution of PEG was added for control and drought stress treatments, respectively. Petri dishes were placed in complete darkness at room temperature (range, 22 to 25 °C) in plastic bins.
Data collection.
Seed germination data were collected for a total of 10 d, with measurements performed after 2, 4, 6, 8, and 10 d of sowing. At each measurement time during this study, a seed was scored as germinated when the radicle had emerged and had a length greater than 1 mm (Bolton and Simon 2019); then, that seed was removed from the petri dish. Standard criteria for determining the performance of carrot accessions under drought stress included the final germination percent under nonstress conditions (FGPControl), final germination percent under drought stress (FGPDrought), AD in seed germination, II, RDT, and DTI; these were used to evaluate salinity and heat tolerance in carrot as well (Bolton et al. 2019; Bolton and Simon 2019).
Pooled evaluation under varying osmotic potentials.
After the initial evaluation of diverse germplasms under nonstress (0.0 MPa) and drought stress (−0.58 MPa) conditions, a second experiment was conducted to determine the range of drought tolerance for both sensitive and tolerant accessions. Six carrot accessions, including three tolerant [PI 643114_C (United States), PI 652208_C (China), and PI 502347_C (Uzbekistan)] and three sensitive [PI 652387_W (Turkey), PI 177381_W (Turkey), and PI 274297_W (Pakistan)], were tested to determine the seed germination percent under six osmotic potentials (0.0, −0.30, −0.51, −0.58, −0.80, and −1.05 MPa) that were obtained by dissolving 0, 10, 15, 18, 21, and 25 g of PEG-6000 in 100 ml of distilled water (Table 1). C refers to cultivated carrot accessions and W refers to wild accessions.
Amylase activity.
Enzymatic activity of α-amylase in imbibed seeds of the three most drought-tolerant and the three most drought-sensitive accessions was determined at 24, 48, and 72 h by the method of Bernfeld (1955), with slight modifications. Reaction mixtures included 1.0 ml of the seed enzyme extract, which included 1.0 g of imbibed seed ground in liquid nitrogen, 0.5 ml of soluble starch (2% volume/volume), and 0.5 ml of pH 6.5 acetate buffer (0.1 M). Mixtures were incubated for 2 h at 37 °C in a water bath; thereafter, 2.0 ml of 3,5-dinitrosalycylic acid (alkaline color reagent) was added. Then, the reaction mixture was boiled at 80 °C for 20 min. Reaction mixtures were cooled at room temperature. The volume comprised 25 ml of distilled water. Absorbance of the solution was read at 560 nm using an ultraviolet spectrophotometer.
Data analysis.
Results and Discussion
Optimal osmotic pressure for the evaluation of drought tolerance at the seed germination stage.
During our preliminary study, under the nonstress condition (0.0 MPa), the seed germination percent of all four genetic stocks ranged from 70% to 90%, with a mean of 82.5%. All were germinated at −0.30 MPa, and the germination percent was reduced to an average of 65% (range, 42% to 90%). Increasing the osmotic potential to −0.51 MPa completely inhibited the seed germination of inbred Nb6526, but the seeds of two cultivars (Brasilia and T-29) and the other inbred (R6636) germinated at rates of 70%, 30%, and 43%, respectively. At the osmotic potential of −0.58 MPa, the mean seed germination percent of all three genetic stocks was further reduced to an average of 10.5% (range, 8% to 24%). With a further increase in the osmotic potential to −0.80 MPa, no germination occurred in seeds of R6636, and the seed germination percent was drastically reduced to less than 10% in Brasilia and T-29. No seed germination was observed in any entry at −1.05 MPa (Fig. 1).
Mean seed germination percent and SE for two commercial cultivars and two inbreeds under six different PEG-induced drought stress levels (osmotic potentials: MPa).
Citation: HortScience 58, 2; 10.21273/HORTSCI16806-22
Mean seed germination percent and SE for two commercial cultivars and two inbreeds under six different PEG-induced drought stress levels (osmotic potentials: MPa).
Citation: HortScience 58, 2; 10.21273/HORTSCI16806-22
Mean seed germination percent and SE for two commercial cultivars and two inbreeds under six different PEG-induced drought stress levels (osmotic potentials: MPa).
Citation: HortScience 58, 2; 10.21273/HORTSCI16806-22
Distribution and mean (dotted line) of the seed germination percent among 250 carrot accessions without drought stress (dark gray) and with drought stress (light gray).
Citation: HortScience 58, 2; 10.21273/HORTSCI16806-22
Distribution and mean (dotted line) of the seed germination percent among 250 carrot accessions without drought stress (dark gray) and with drought stress (light gray).
Citation: HortScience 58, 2; 10.21273/HORTSCI16806-22
Distribution and mean (dotted line) of the seed germination percent among 250 carrot accessions without drought stress (dark gray) and with drought stress (light gray).
Citation: HortScience 58, 2; 10.21273/HORTSCI16806-22
Distribution and mean (dotted line) among 250 carrot accessions with four different measures of drought tolerance: (A) absolute decrease; (B) inhibition index; (C) relative drought tolerance; and (D) drought tolerance index.
Citation: HortScience 58, 2; 10.21273/HORTSCI16806-22
Distribution and mean (dotted line) among 250 carrot accessions with four different measures of drought tolerance: (A) absolute decrease; (B) inhibition index; (C) relative drought tolerance; and (D) drought tolerance index.
Citation: HortScience 58, 2; 10.21273/HORTSCI16806-22
Distribution and mean (dotted line) among 250 carrot accessions with four different measures of drought tolerance: (A) absolute decrease; (B) inhibition index; (C) relative drought tolerance; and (D) drought tolerance index.
Citation: HortScience 58, 2; 10.21273/HORTSCI16806-22
An analysis of variance of seed germination of the two carrot cultivars and two inbreeds displayed a highly significant treatment effect (F = 144.38; P < 0.0001) and moderately significant accession effect (F = 6.38; P = 0.0025) on seed germination (Table 2). A significant difference (F = 2.13; P = 0.0007) was observed between accessions in the control (0.0 MPa), whereas the most significant difference in the germination rate between accessions was recorded at −0.58 MPa (F = 9.0; P = 5.20E−16) (Table 3). Using this statistical approach to characterize variations in the abiotic stress tolerance of plant germplasm collections (Bolton and Simon 2019; Bolton et al. 2019; Ravelombola et al. 2017), these results suggest that −0.58 MPa with 18% PEG-6000 is optimal for inducing osmotic pressure to screen diverse carrot germplasms for drought tolerance at the seed germination stage, which, incidentally, is comparable to a 150 mM concentration of NaCl that was identified as optimal for evaluating the salinity tolerance of carrot (Bolton and Simon 2019). Because of this coincidence, an evaluation of carrot accessions at −0.58 MPa with 18% PEG-6000 would be expected to be reliable for identifying differences among both drought-tolerant and salt-tolerant accessions.
Analysis of variance of seed germination of four carrot accessions under six different treatments of polyethylene glycol-induced drought stress.
Analysis of variance of seed germination of four carrot accessions under six different treatments (MPa or osmotic potentials) of polyethylene glycol (PEG)-induced drought stress.
Germination assay.
The average seed germination percent under control conditions ranged from 50.0% to 100.00% (mean, 82.2%; SD, 12.3%) for the 250 carrot accessions (including 200 cultivated PIs and 50 wild PIs). When seeds were subjected to osmotic pressure conditions (−0.58 MPa), the average seed germination percent was reduced to 54.5% (minimum value, 0.0%; maximum value, 99.2%; SD, 16.9%). These results indicated that osmotic pressure (Fig. 2) significantly reduced the seed germination percent of the majority of carrot accessions (Table 4). This reduction in the seed germination potential of sensitive carrot accessions under osmotic pressure might be in response to phytochrome-interacting factors that are directly associated with the regulation of abscisic acid (ABA) encoding genes (Li et al. 2021). Recent studies also reported that phytochrome-interacting factors (DcPIF3) lower the drought stress tolerance of carrot by the overproduction of ABA (Wang et al. 2022), and that a DREB-binding transcription factor from carrot enhances drought tolerance and regulates lignin biosynthesis in Arabidopsis (Li et al. 2020).
Descriptive statistics of the final germination percent without (FGP_NS) and with (FGP_DS) drought stress, absolute decrease (AD), inhibition index (II), relative drought tolerance (RDT), and drought tolerance index (DTI) of 250 carrot accessions.
Significant variation was recorded for the average seed germination percent among carrot accessions under control conditions (F = 2.13; P < 0.0001) (Table 5). PI 652347 (Turkey, C), PI 643119 (France, C), and PI 642756 (the Netherlands, C) all had the maximum values (100.0%) for seed germination percent under control conditions, whereas PI 652253 (India, C, 50.0%), PI 502914 (Germany, C, 51.1%), and PI 652154 (the Netherlands, C, 51.2%) had the three lowest values for seed germination percent (Supplemental Table S1). The seed germination percent also varied significantly under drought stress among carrot accessions (F = 9.96; P < 0.0001) (Table 5). PI 643114 (United States, C) and PI 652208 (China, C) had the highest seed germination percent value (97.5%), followed by PI 264234 (France, C) and PI 502347 (Uzbekistan, C), which both had the second highest value (93.3%), indicating a high level of drought tolerance. Nb6526 (United States, inbred), PI 176563 (Turkey, C), PI 652387 (Turkey, W), and PI 652152 (United Kingdom, C) had the lowest values for seed germination percent under drought stress (0.0%, 13.3%, 14.3%, and 15.0%, respectively), indicating that they were highly sensitive to drought stress.
Analysis of variance of seed germination in 18% polyethylene glycol and drought tolerance measurements among 253 carrot accessions.
Most of the accessions with a high seed germination percent under drought stress were cultivated PIs from diverse geographic regions. Cultivated PIs from the United States (PI 643114), China (PI 652208), Uzbekistan (PI 502347), and Turkey (PI 264234 and PI 652400) had lowest AD in seed germination and II values (0.0% to 6.9%), high RDT values (0.93–1.0) and high DTI values (1.27–1.47). The most drought-sensitive carrot accessions were wild PIs from Turkey (PI 652387, PI 177381, and PI 279764) and an inbred from the United States (Nb6526), which all had seed germination percent values less than 15.0% under drought stress, and RDT and DTI values that ranged from 0.0 to 0.25 (Supplemental Table S1). Interestingly, the most drought-tolerant and drought-sensitive accessions were also among the most salt-tolerant, salt-sensitive, heat-tolerant, and heat-sensitive accessions. The seven PIs with drought, heat, and salt tolerance at germination were PI218076_C (Pakistan), PI226310_C (Mexico), PI502347_C (Uzbekistan), PI643114_C (United States), PI652248_C (Russia), PI652400_C (Turkey), and PI642403_C (Turkey), whereas the five PIs with drought, heat, and salt sensitivity at germination were PI163234_C (India), PI177381_W (Turkey), PI264232_C (France), PI274297_W (Pakistan), and PI502914_C (Germany). These observations suggest that these accessions could be especially useful for carrot breeding for abiotic stress tolerance. Another interesting observation of salt (Bolton and Simon 2019), heat (Bolton et al. 2019), and drought tolerance evaluations during seed germination of carrot germplasm was that cultivated accessions with diverse geographic origins are the main sources of abiotic stress tolerance in carrot. This is unlike other species in which wild germplasm is typically more tolerant than cultivated germplasm (Hajjar and Hodgkin 2007). However, it should be noted that several wild carrot accessions were reported previously (Bolton and Simon 2019; Bolton et al. 2019) as both salt-tolerant and heat-tolerant, and that several cultivated PIs were sensitive, contrary to general trends.
The AD in seed germination in response to abiotic stress is a useful parameter for evaluating the effect of stress (FGPControl − FGPStress) because it measures the actual reduction in seed germination. During this evaluation, the mean AD in seed germination for all accessions evaluated was 26.9% (range, 0.0% to 82.5%) (Table 4; Fig. 3), with significant variation among 250 carrot accessions (F = 7.59; P < 0.0001) (Table 5). PI 643114 (United States, C, 0.0%), PI 652208 (China, C, 1.7%), and PI 264234 (France, C, 4.3%) had the lowest AD in seed germination values, indicating drought tolerance. These accessions with low AD in seed germination values might be useful sources of drought tolerance for breeders. Nb6526 (USDA, inbred, 82.5%), R6636 (USDA, inbred, 80%), PI 234621 (South Africa, C, 69.3%), and Ames 26383 (Portugal, W, 60.8%) had the highest AD in seed germination values (Supplemental Table S1), indicating that drought stress greatly reduced their germination and that they were very drought-sensitive. Increased osmotic potential disrupted the enzyme activities and decreased the seed imbibition rate and moisture level required for germination in sensitive accessions (Billah et al. 2021). Moreover, osmotic and drought stresses trigger the overproduction of reactive oxygen species that damage the cellular structural components and hinder the several metabolic processes; for example, they decrease the hydrolysis of stored contents of seeds and their transportation to growing embryos (Basal et al. 2020).
The II [II = 100 × (FGPControl − FGPHeat) /FGPControl] values were significantly different among carrot accessions (F = 8.25; P < 0.0001) (Table 5; Fig. 3) (range, 0.0% to 100%). PI 643114 (United States, C, 0.0%) had the lowest II, followed by PI 652208 (China, C, 1.7%), and PI 264234 (France, C, 4.3%), which were in agreement with the AD, indicating that drought stress reduced the seed germination of these accessions, but at very low rate. Nb6526 (USDA, inbred) had a maximum II value of 100%, followed by R6636 (USDA, inbred, 90.5%), PI 274297 (Pakistan, W, 80.2%), PI 652387 (Turkey, W, 77.4%), and PI 177381 (Turkey, W, 74.7%), indicating that they are highly drought-sensitive accessions (Supplemental Table S1).
The RDT (RDT = FGPDrought/FGPControl) is a useful criterion for evaluating drought stress because it accounts for the seed germination percent relative to the control. Relative drought tolerance was also significantly different among carrot accessions (F = 8.24; P < 0.0001) (Table 5; Fig. 3), with a population mean of 0.65 and range from 0.0 to 1 (Table 4). PI 643114 (United States, C, 1.0), PI 652208 (China, C, 0.98), and PI 264234 (France, C, 0.96) had the highest relative drought-tolerance values, whereas Nb6526 (USDA, inbred, 0.0), R6636 (USDA, inbred, 0.1), PI 274297 (Pakistan, W, 0.2), PI 652387 (Turkey, W, 0.23), and PI 177381 (Turkey, W, 0.25) had the lowest values, indicating that these are sensitive to drought tolerance.
The DTI [DTI = (FGPDrought × FGPControl)/ (FGPAverage)2] is an important trait to consider because it accounts for the seed germination percent under both control and drought stress conditions while comparing each accession to the population average, thus providing a ranking among all accessions evaluated. An accession with a high DTI will have a higher seed germination percent under both conditions, making it useful for a commercial growing setting and breeding programs. The DTI was significantly different among carrot accession evaluations (F = 8.20; P < 0.0001) (Table 5; Fig. 3). PI 643114 (United States, C, 1.47), PI 652208 (China, C, 1.44), and PI 502347 (Uzbekistan, C, 1.40) had the highest DTI values, suggesting that they were highly drought-tolerant, whereas Nb6526 (USDA, inbred, 0.0), PI 652387 (Turkey, W, 0.13), PI 177381 (Turkey, W, 0.14), and PI 274297 (Pakistan, W, 0.15) had the lowest DTI values (Supplemental Table S1). The seed germination potential is controlled by the genetic potential of accession and growth condition; therefore, the stress tolerance index of the accession is significantly affected by the interaction between accession genetics and growth conditions (Basu et al. 2021).
The accessions used in this study demonstrated a wide range of phenotypic variations for each of the drought stress parameters measured. Exposure to osmotic pressure conditions with an osmotic potential of −0.58 MPa significantly reduced the seed germination for the majority of the carrot accessions, in agreement with the findings of previous evaluations of salt and heat tolerance in carrot (Bolton et al. 2019; Bolton and Simon 2019). Because of our observations that some carrot seeds will germinate even at −0.80 MPa with 21% PEG-6000, additional screening under greater stress and recovery of viable seedlings among more drought-tolerant germplasm identified in this study may provide an approach to genetic selection to improve carrot abiotic stress tolerance at germination in field conditions. Interestingly, drought-tolerant accession PI 643114 (United States, C) was also heat-tolerant and salt-tolerant during seed germination, suggesting that this accession could be a useful source of abiotic stress tolerance in carrot breeding programs.
Drought tolerance according to the geographic origin.
Significant differences were observed for seed germination under control conditions and for all drought germination parameters during comparisons of 14 different geographic origins of carrot accessions (P < 0.0001) (Table 6). A comparison of accessions according to the geographic origin showed that accessions from North America demonstrated higher drought tolerance at the germination stage, with a value of 1.0. Accessions from Eastern Europe had a mean DTI of 0.77. Accessions from East Asia, South Europe, and Central Asia had mean DTI values of 0.72, 0.71, and 0.70, respectively. The remaining accessions from other geographic regions had DTI values ranging from 0.47 to 0.68 (Table 7). Few accessions from East Africa, South Africa, South America, South Europe, and Oceania were evaluated for drought tolerance. It is interesting to note that, like evaluations for salt and heat tolerance, a wide phenotypic variation was observed within a single region among accessions from each of the geographic regions with more than 10 accessions, perhaps reflecting variations in drought, heat, and salt stress during seed germination across production environments within regions and also across climate changes during the domestication history of cultivated carrots. Variations in environmental parameters such as temperature and precipitation at the collection location of wild carrot germplasm were associated with heat and drought tolerance observed in carrots growing in field trials (Simon et al. 2021).
Analysis of variance of seed germination and drought tolerance measurements among 250 carrot accessions originated from 14 geographical regions.
Descriptive statistics of the drought tolerance index (DTI) of 253 carrot accessions originated from 14 different geographic regions.
Drought tolerance according to domestication status and root color.
No significant difference was observed for the seed germination percent under nonstress conditions when comparing cultivated PIs (81.2%) and wild PIs (81.1%) (Table 8). However, cultivated PIs demonstrated significantly higher drought tolerance than the wild PIs, with mean seed germination rate of 57.5% under drought conditions (range, 13.3% to 99.2%) and a mean AD in seed germination of 24.8%, mean II of 31.1%, mean RDT of 0.69, and mean DTI of 0.73; however, wild PIs had a mean germination rate 45.6% under drought conditions (range, 13.3% to 99.2%), a mean AD in seed germination of 35.5%, mean II of 43.9%, mean RDT of 0.56, and mean DTI of 0.55. However, inbreeds (Nb6526 and R6636) were highly drought-sensitive, with much lower relative drought tolerance and drought tolerance index values (0.04 and 0.09, respectively). Similar trends for abiotic stress tolerance were reported for carrot germplasm after previous evaluations for salt tolerance during seed germination (Bolton and Simon 2019), heat tolerance during seed germination (Bolton et al. 2019), and heat tolerance during early and late seedling stages (Nijabat et al. 2020).
Mean (±SE) of seed germination without drought stress (nonstress), seed germination with drought stress (stress), absolute decrease (AD), inhibition index (II), relative drought tolerance (RDT), and drought tolerance index (DTI) separated by domestication status (DS) and primary root color (RC) of accessions.
The mean values of drought stress tolerance traits for cultivated carrots of various root colors were not significantly different from those under nonstress conditions for orange, yellow, red, and purple accessions (germination range, 53.1% to 58.7%; DTI range, 0.62–0.75). In contrast, white rooted carrots were significantly more sensitive than carrots of other colors (47.2% germination under drought stress compared with 81.6% germination without stress) (Table 8). It is important to note that the number of samples for each color category were not equal and were not equally distributed across geographic origins; therefore, the trends reported here should be confirmed by larger sample sizes when possible.
Variation in abiotic stress tolerance and potential mechanisms.
Although the osmotic pressure induced by 18% PEG-6000 is comparable to 150 mM NaCl that we used to evaluate salt tolerance during our previous studies, the mechanisms for drought and salt tolerance may not be the same. To compare the trends in tolerance to these two abiotic stressors among carrot germplasm evaluated by Bolton and Simon (2019), we evaluated the correlation between DTI values in this study to STI values in the previous study. Pearson’s correlation indicated that seed germination under nonstress conditions was positively, but not significantly, correlated to the DTI (r = 0.41), whereas the correlation with seed germination under drought stress conditions was positive and highly significant (r = 0.97). Germination without stress was not strongly correlated to the other parameters evaluated (r = −0.01 to 0.32). These results indicate that the seed germination percent under nonstress conditions does not predict drought tolerance in carrot seed germination in carrot. Correlations were observed among all drought stress parameters and the salt tolerance index (r = 0.41–0.89 (Table 9). These data suggest that drought tolerance parameters and salt tolerance parameters follow similar trends, with the DTI having slightly stronger correlations among those parameters. The Pearson rank correlation of the DTI in this study and STI values calculated by Bolton and Simon (2019) was strong (0.89), suggesting that, although there were exceptions, many of the accessions tolerant to one stress are also tolerant to the other stress. These results are not surprising because drought stress and salt stress have similar effects on seed physiology and often cause similar stress responses.
Correlation among drought stress (−0.58 MPa) and salt stress (150 mM) parameters of 253 carrot accessions.
Salt and drought stresses both induce oxidative damage by overproduction of reactive oxygen species that disrupt protein biosynthesis and stability in plants. Salt-tolerant and drought-tolerant species counteract the damaging effects of cellular overaccumulation of reactive oxygen species by increased production of various antioxidants (catalase, superoxide dismutase, polyphenol oxidase, peroxidase) and transcription factors that upregulate the synthesis of proteins involved in defense mechanisms (Hu et al. 2012; Huang et al. 2015; Wang et al. 2012). ABA is also an important secondary messenger involved in abiotic stress tolerance (Fujita et al. 2011). The increased ABA level hastens stomatal closure, which leads to less water loss in drought-tolerant crop species, but salt tolerance in germinating seeds is not related to ABA (Osakabe et al. 2013; Shohat et al. 2021). The specific mechanisms observed for salt stress tolerance in plants are minimization of salt ion entry in cells and their amelioration in cell cytoplasm. Generally, glycophytes, including carrot, have very low potential to exclude salt ion entry in cells and accumulate salt toxicity in leaves (Basu et al. 2021; Hafeez et al. 2021; Venkataraman et al. 2021). Therefore, for those accessions with the widest variations in DTI and STI, studies examining mechanisms of drought tolerance and salt tolerance would be of particular interest to gain a better understanding of the differences in mechanisms.
Seed germination and amylase activity in drought tolerant and sensitive accessions.
In several other crops, variation in the seed enzymatic activity of α-amylase is associated with drought tolerance, inferring that the scarcity of water hinders the activity of amylase enzyme, which, in turn, limits the solubilization of endospermic carbohydrates into simple sugars, which provide the energy for the growth of embryonic roots and shoots (Hajihashemi et al. 2020; Ibrahim 2016; Ma et al. 2017). Therefore, enzymatic activity of α-amylase was evaluated in the germinating seeds of the three most tolerant [PI 643114_C (United States), PI 652208_C (China), and PI 502347_C (Uzbekistan)] and three most sensitive [PI 652387_W (Turkey), PI 177381_W (Turkey), and PI 274297_W (Pakistan)] accessions at 24, 48 and 72 h of germination under six PEG-induced drought stress conditions. As noted, the seed germination rates of both tolerant and sensitive accessions were significantly decreased as the osmotic pressure was increased from 0.0 MPa to −1.05 MPa, and the reduction in the germination rates were more pronounced at lower PEG concentrations in sensitive accessions than in tolerant accessions (Fig. 4). Interestingly, the activity of α-amylase in germinating seeds of tolerant accessions was 20-fold higher under nonstress condition (range, 17.13–36.37; mean, 26.51 µmole starch hydrolyzed min−1 mg−1 protein) at 24 h than that in germinating seeds of sensitive accessions. Under drought stress in germinating seeds of tolerant accessions, α-amylase activity was decreased by 15% at −0.30 MPa and by 73% at −1.05 MPa. Similar reductions in α-amylase activity were observed in sensitive accessions at 24 h of germination, and in both tolerant and sensitive accessions at 48 and 72 h of germination (Fig. 5). Similar observations of reduced α-amylase activity in drought-stressed germinating seeds were documented in other plant species, including barley (Guoxiong et al. 2002), lentils (Muscolo et al. 2014), and wheat (Bajji et al. 2000; Haouari et al. 2013); these drought-tolerant accessions exhibited a less pronounced decrease in amylase enzyme activity during seed germination than sensitive accessions. These results suggest that amylase enzymes may significantly contribute to drought stress tolerance and could be used as an effective stress indicator in carrot. Moreover, it will be interesting to identify the genes responsible for amylase activity in diverse carrot germplasms by performing genetic analyses.
Pooled seed germination percent of three sensitive (solid line) and three tolerant (dotted line) accessions under six PEG-induced drought stress levels (osmotic potentials: MPa).
Citation: HortScience 58, 2; 10.21273/HORTSCI16806-22
Pooled seed germination percent of three sensitive (solid line) and three tolerant (dotted line) accessions under six PEG-induced drought stress levels (osmotic potentials: MPa).
Citation: HortScience 58, 2; 10.21273/HORTSCI16806-22
Pooled seed germination percent of three sensitive (solid line) and three tolerant (dotted line) accessions under six PEG-induced drought stress levels (osmotic potentials: MPa).
Citation: HortScience 58, 2; 10.21273/HORTSCI16806-22
Activity of α-amylase in germinating seeds of three tolerant (black) and three sensitive (gray) accessions under six PEG-induced drought stress levels (osmotic potentials: MPa) after 24, 48, and 72 h of imbibition.
Citation: HortScience 58, 2; 10.21273/HORTSCI16806-22
Activity of α-amylase in germinating seeds of three tolerant (black) and three sensitive (gray) accessions under six PEG-induced drought stress levels (osmotic potentials: MPa) after 24, 48, and 72 h of imbibition.
Citation: HortScience 58, 2; 10.21273/HORTSCI16806-22
Activity of α-amylase in germinating seeds of three tolerant (black) and three sensitive (gray) accessions under six PEG-induced drought stress levels (osmotic potentials: MPa) after 24, 48, and 72 h of imbibition.
Citation: HortScience 58, 2; 10.21273/HORTSCI16806-22
Conclusions
This study identified a wide range of phenotypic variations in drought tolerance at the seed germination stage in a diverse collection of carrot germplasms. Three cultivated carrot accessions [PI 643114 (United States), PI 652208 (China), and PI 502347 (Uzbekistan)] were identified as the most drought-tolerant accessions, whereas all wild accessions displayed low levels of drought tolerance; however, PI 652387 and PI 177381 (Turkey) and PI 274297 (Pakistan) were the most drought-sensitive cultivated accessions. These results indicate that there are numerous genetic sources of drought tolerance during carrot seed germination for breeders to improve this trait, and the results suggest that drought tolerance has been under selection in carrot cultivating regions worldwide. This study adds to the current body of research of drought tolerance in carrot seed germination by identifying an optimal osmotic potential for screening and by providing evidence of a large genetic component underlying drought tolerance based on the wide variation of tolerance observed in a diverse collection of carrot germplasm. DTI in germinating seeds was found to be an effective measure of stress tolerance. Variations in the α-amylase activity were also associated with variations in drought tolerance in a small subset of the germplasms evaluated, indicating the need for additional studies evaluating the role of α-amylase and other enzymes in carrot seed germination beyond these preliminary observations. This study and the recent salt and heat tolerance studies that evaluated the same diverse germplasm collection provides a valuable foundation for future research and breeding for abiotic stress in carrot.
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Carrot accession, root color, country of origin, domestication status (DS), mean percent germination without drought stress (nonstress) (±SE), mean percent germination with drought stress (stress) (±SE), mean absolute decrease (AD), mean inhibition index (II), relative drought tolerance (RDT), mean drought tolerance index (DTI), and rank based on DTI.
