Evaluation of variability to drought and saline stress through the germination of different ecotypes of carob (Ceratonia siliqua L.) using a hydrotime model

Ecological Engineering 95 (2016) 557–566

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Evaluation of variability to drought and saline stress through the germination of different ecotypes of carob (Ceratonia siliqua L.) using a hydrotime model Valeria Cavallaro a , Antonio C. Barbera b , Carmelo Maucieri b,c,∗ , Greta Gimma a , Clara Scalisi a , Cristina Patanè a a

Trees and Timber Institute (IVALSA), Consiglio Nazionale delle Ricerche (CNR) Via Paolo Gaifami 18, Catania, Italy Department of Agriculture, Food and Environment (Di3A), University of Catania, Via Valdisavoia 5, 95123 Catania, CT, Italy c Department of Agronomy, Food, Natural Resources, Animals and Environment (DAFNAE), University of Padua, Agripolis Campus, Viale dell’Università 16, 35020 Legnaro, PD, Italy b

a r t i c l e

i n f o

Article history: Received 2 January 2016 Received in revised form 12 May 2016 Accepted 16 June 2016 Keywords: Carob Seed germination Scarification Salinity stress Drought stress

a b s t r a c t Carob is a valuable leguminous species for its productions but also for forestation of arid and degraded areas threatened by soil erosion and desertification processes. However, large-scale cultivation of this species is hampered by the difficult propagation by cuttings or seeds. Researches to evaluate genotype tolerance to saline and drought stress during germination and first plant establishment, are also inhibited by the scarce seed germination. In this study, carob seeds, from wild and domesticated carob genotypes collected in a representative area of the Mediterranean basin (Sicily, Italy), were subjected to germination tests under isotonic solutions of polyethylene glycol (PEG) and sodium chloride (NaCl) at −0.5, −1.0, −1.5 MPa. Before germination tests, a 20 min pre-treatment with 96% sulphuric acid was necessary to overcome seed coat dormancy which does not permit germination. Differences in drought and salinity tolerance were determined in the different genotypes during germination. All genotypes exhibited a higher sensitivity to drought stress as compared to salinity stress. The observed differences in some genetically inherited germination characteristics (i.e. base b(50)s and hydrotimes) suggest genetic differences even in seeds of individual old trees situated in close areas, as previously found in other countries of the Mediterranean basin. A greater sensitivity to stress was determined during early radicle growth, an essential feature for early plant establishment in marginal dry areas. In this paper, a protocol to test germination under simulated stress conditions (drought and salinity stress) was established. Including in the analysis other parameters representative of early embryo growth (i.e. radicle elongation), this protocol may be useful to select genotypes for breeding programs towards the selection of tolerant cultivars or rootstocks. It may be also applied to other leguminous species with a difficult germination. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Carob (Ceratonia siliqua L. of the Fabaceae family) is a long living tree, domesticated and grown since ancient times in most countries of the Mediterranean basin for its large fruits with high sugar content (Zohary, 2002). It is a drought resistant fruit species,

∗ Corresponding author at: Department of Agriculture, Food and Environment (Di3A), University of Catania, Via Valdisavoia 5, 95123 Catania, CT, Italy. E-mail address: [email protected] (C. Maucieri). http://dx.doi.org/10.1016/j.ecoleng.2016.06.040 0925-8574/© 2016 Elsevier B.V. All rights reserved.

which grows well on poor, sandy, calcareous and limestone soils (Batlle and Tous, 1997). For these characteristics it is particularly suitable to cultivation in arid and semiarid regions of southern Mediterranean basin, where irrigation is impractical (Haq, 2008). Nowadays, it is also found in most areas of Americas, Africa, Asia and Australia with Mediterranean-like climate (Müller et al., 2010). Carob is an economically important multipurpose tree, whose legumes (pods) have traditionally been used as animal feed and in the past for human consumption for their high energetic content and food value (Catarino, 1993).

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However, currently, the most economically important product extracted from seed, is the Locust Bean Gum (LBG), widely used as a natural food additive (E 410), instead of artificial gums (Rizzo et al., 2004), but also with increasing applications in the pharmaceutical industry (Sandolo et al., 2007). Nowadays, natural antioxidants contained in the seed coat and pulp fruit are potential new products for the food industry (Durazzo et al., 2014). Carob pods are actually also explored as material for the production of bioethanol (Ercan et al., 2013). Carob is also being increasingly used as an ornamental tree in Mediterranean countries. Moreover, this species has also an important ecological significance. It is recommended for forestation of arid and degraded areas threatened by soil erosion and desertification (Barwick, 2004) and, due to its ability to preserve and enrich the fertility of the soil, its cultivation facilitates the establishment of other species. It is particularly useful in the rehabilitation of marginal areas of the Mediterranean basin not adapted to other agricultural uses (Batlle and Tous, 1997), contributing to the protection of the environment. For these characteristics and being a long living tree, it is a species able to adapt to climatic changes but also to exert a long time carbon sequestration. However, at present, the large-scale cultivation of carob and the growth of a specialized nursery activity are limited by its difficult propagation. Up to now, in fact, the propagation of this tree is mainly carried out by grafting scions of selected productive female individuals on ‘wild’ rootstocks. The propagation by hardwood or semi wood cuttings is strongly limited by the low rooting ability of the species (Gubbuk et al., 2011). Even seed propagation for rootstocks production is particularly difficult. Like other legumes, carob seeds show a scarce and irregular germination due to the physical dormancy determined by the hard seed coat, an useful feature to form a persistent seed bank in the soil (Ortiz et al., 1995). MartinsLoucao et al. (1996), Tsakaldimi and Ganatsas (2001) and Piotto and Di Noi (2003) showed that the best pre-sowing treatments for enhancing carob seeds germination are chemical scarification with acids, soaking in warm water and mechanical scarification. However seed pretreatments have mainly been carried out on wild carob genotypes (Gunes et al., 2013) with no comparable work being conducted on domesticated ones. Moreover, to our knowledge, with the only exception of the work of Spyropoulos and Lambiris (1980) who studied the water stress (induced by a range of PEG 4000 solutions) effects, no other reports are available upon carob seed germination under water or salinity stress. Considering the productive but also the environmental role of the carob (Manaut et al., 2015) after individuating the best treatment to remove seed dormancy, a series of studies were carried out on the germination responses to salinity and drought stress of domesticated and wild carob genotypes collected in different marginal areas in Sicily. These studies may be helpful to identify the best performing genotypes for the production (in vivo or in vitro) of rootstocks, suitable for revegetation of marginal areas or to be cultivated in nurseries or open field under irrigation with scarce quality waters. The study was carried out using iso-osmotic concentrations of NaCl or PEG to assess the effects of the two components of salinity stress (osmotic stress or specific ion toxicity) on the germination of eight genotypes. The hydrotime analysis (Gummerson, 1986; Bradford and Still, 2004) has been adopted to describe the relationship between water potential (␺) and seed germination characteristics and to compare the different carob genotypes on the basis of their stress tolerance (␺b, the base potential that allows germination to be completed), their germination uniformity (the standard deviation in ␺b values among individual seeds −␴␺b ) and the speed of germination (hydrotime costant −␪H ).

2. Materials and methods 2.1. Plant material Seeds of 8 carob genotypes were collected in different cultivation areas in Sicily (Italy), at full pods ripening (August 2012). Sites and genotypes characteristics are illustrated in Table 1. When the experiments were conducted, seeds were 6 months old. 2.2. First experiment: treatments to overcome seed dormancy The purpose of this experiment was to determine the most effective dormancy-breaking treatments to be utilized in the subsequent experiments. On the first five collected genotypes (wild TDP and domesticated TDP, Croce, Cavette, Calamiccio), the effects on germination of the following pretreatments were tested: 1) no treatment (control); 2) mechanical scarification by seed micropile piercing (24 h after soaking in water) at the chalazal side of the seeds, with about 2 mm of the hard seed coat removed to facilitate seed imbibition; 3) chemical scarification by seed soaking in 96% sulfuric acid for 10, 15, 20 min. 4) liquid nitrogen scarification by three-fold seed soaking in liquid nitrogen (−196 ◦ C) for 5 min. On the remaining three genotypes (Sorba, Margitello and Pignato), only the effectiveness on germination of the best pretreatment was tested. After the treatment and prior to incubation, seeds were thoroughly washed under running tap water. All seed samples were placed on moist filter paper in Petri dishes during incubation. Germination was assessed when the radicle reached approximately 2 mm of length, and data were collected daily until no additional germination occurred for 72 h. At the end of the experiment, the final percentage germination was calculated. 2.3. Second experiment: testing seed germination under salt and drought stress The experiment was carried out on all carob genotypes (Table 1). Before testing, seeds were chemically scarified for twenty minutes in sulfuric acid, the best effective seed treatment in removing dormancy, according to the first experiment results. Four water potentials (␺) of the imbibition solution, in NaCl or PEG to induce saline and water stress respectively, were studied: 0 (control), −0.5, −1.0 and −1.5 MPa. Osmotic potentials (␺) in NaCl solutions were verified using an automatic cryoscopic osmometer (Gonotec Osmomat 030 model, Berlin, Germany). PEG solutions were prepared dissolving different concentrations of PEG 6000 in deionized water according to the water potential to induce, as described by Michel and Kaufmann (1973). Seeds were germinated at a constant temperature of 25 ± 1 ◦ C, in a thermostatically controlled incubator. Samples of 100 seeds (four replicates of 25 seeds each) were placed in 9-cm Petri dishes containing a single filter paper moistened with 7 mL of each NaCl or PEG solution. Petri dishes were hermetically sealed with Parafilm® to prevent evaporation and then randomized in an incubator in the dark. Germination (radicle protrusion of approx. 2 mm) was assessed at 24 h intervals until for three consecutive days no germination was observed. Germination temperature was 25 ◦ C since it is indicated as optimum for carob seed germination (De Michele et al., 1988).

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Table 1 Altitude and soil classification of the origin area, type and seed weight of the examined genotypes. Code

Origin area

Altitude (m asl)

Soil classification (USDA Texture Class)

Type

1000 seed weight (g)

Sorba TDP dom. TDP wild Margitello Pignato Croce Cavette Costa

Catania plain Zisola (Noto-SR) Zisola (Noto-SR) C.da Margitello (Ispica-RG) C.da Pignato (Ispica-RG) C.da Scorsone (Ispica-RG) C.da Cavette (Ispica-RG) C.da Calamiccio (Ispica-RG)

36 110 110 78 44 141 60 88

Sandy clay Loam Sandy loam Sandy loam Loam Loam Silt loam Silt loam

Domesticated Domesticated Wild Domesticated Domesticated Domesticated Domesticated Domesticated

202.7 209.9 214.5 215.8 209.2 201.8 215.3 215.0

2.4. Seed water uptake

The following equation describes the basis of the hydrotime model defining the hydrotime constant (␪H ):

This parameter was measured on four (Sorba, Costa, Cavette and domesticated TDP) out the eight studied genotypes. For each treatment, seed moisture at 24, 48, 72, 96 h of imbibition was measured. For this purpose, three replications of 20 seeds per replicate were placed in Petri dishes as described for the germination experiments, removed every 24 h after the imbibition initiation, blotted with absorbent paper and weighed. Seed moisture content was determined as: [(total weight − initial weight)/initial weight] × 100

(1)

2.5. Root length measurements Radicle length measurements were also carried out for each treatment during the germination test. To this purpose, 10 germinated seeds were chosen randomly from each Petri dish, 2 days after the beginning of germination. The radicle was excised from the seed and measured.

The data of the four replicates of each treatment were combined, and a mean germination curve was plotted against time. The time course of cumulative values of seed germination was modeled for each solution using the Weibull function (Yang et al., 1978; Dumur et al., 1990), which is formulated as follows: (2)

In this equation, y = cumulative germination at time t, M = final total germination, k = germination rate, z = interval between the start of incubation and the start of germination (lag in germination) and c = shape parameter. Weibull parameters and their standard errors were calculated using SIGMAPLOT® 9.0 software (Systat software Inc., San Jose, California, USA). Pooled observations that had a reported cumulative germination of zero or below 30% in the studied solutions were excluded from the analysis. The time (days) to 50% germination (T50) was calculated on the Weibull function. The linear regression of the GR (1/t50 ) of germination time vs. water potential (␺) was used to estimate ␺b(50) at the optimal germination temperature (25 ◦ C). According to that assumed for the estimation of theoretical minimum temperature of germination (Scott et al., 1984), the abscissa intercept on the ␺ axis may be considered as an estimate of the theoretical minimum ␺ at which seed germination is reduced to 50% (␺b(50) ) (Patanè et al., 2009; Patanè and Tringali, 2011). Probit regression analysis is used to fit the germination courses to the hydrotime model (Bradford, 1990; Bradford and Still, 2004; Windauer et al., 2007). By this method, a single regression is calculated to describe the progress of seed germination at all the studied constant osmotic potentials and a complete comparison among genotypes may be carried out independently of the osmotic potentials.

(3)

where ␪H is the hydrotime constant (MPa h) required to germination of percentage g, ␺ is the actual seed water potential of the germination medium, ␺b(50) is the base or theoretical threshold or base water potential ␺ at which seed germination is reduced to g and tg is the time (h) to radicle emergence of fraction g of the seed population. Pooled observations that had a reported cumulative germination below 30% in the studied solutions were excluded from the analysis. Observed cumulative germination percentages (on a probit scale) were, thus, plotted as functions of log ␪H in order to linearize cumulative germination time courses. Different values of log ␪H were used in repeated probit regressions until the optimal fit to all data was obtained. According to Bradford (1990), the equation for the regression line is the following: probit(g) = [␺ − (␪H /tg ) − ␺b(50) ]/␴␺b(g)

2.6. Data analysis

y = M {1 - exp[-k (x - z)]ˆc}

␪H = (␺ − ␺b(g) )tg

(4)

The midpoint of the regression line (probit = 5, 50% germination) gives the value of ␪b(50) (␺b of 50% germination). The inverse of the slope of the probit regression line was equal to the standard deviation ␴ ␺b(g) of seed population. ␪H , ␺b(50) and ␴␺b(g) thus estimated, were assumed as hydrotime parameters of the seeds of the eight genotypes of carob. The significance of the differences between slopes (b coefficients) and x-axis intercepts (corresponding to ␺b(50) ) of linear regressions were determined by the Student’s t-test and used to compare the different cultivars within each osmotic potential (water stress in PEG or salt stress in NaCl). The confidence intervals of the intercept were determined by the GraphPad Prism 6 XML. Total percentage germination and water uptake data were √ arcsine % transformed and then analyzed by a randomized two way analysis of variance (ANOVA) using COSTAT version 6.003 (CoHort 3Software). Water uptake data were analyzed separately for each hour of measurement. Means were separated on the basis of the Least Significant Difference (LSD) when the F test of the ANOVA was significant P < 0.05 or P < 0.01 (Snedecor and Cochran 1989). Actual data are shown in the text. 3. Results 3.1. First experiment: treatments to overcome seed dormancy Overall seed germination of intact seeds ranged from 1.3 to 27% (Table 2). Seed germination of mechanically scarified (pierced seeds) fluctuated from 85 to 97%, mostly within 6 days. Chemical scarification in sulfuric acid (96%) for 15 min resulted in germination values exceeding 90% in all the tested genotypes, if excluded the wild one. The same treatment for 20 min resulted in a germination rate from 90 to 100% in all tested genotypes (Table 2).

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Table 2 Influence of scarification on the seed germination (%) of carob genotypes 10 days from the beginning of the test. Genotypes

TDP dom. TDP wild Croce Cavette Costa Sorba Pignato Margitello

Control (intact seed)

1.7 ± 7.1 6.7 ± 7.2 15.0 ± 12.4 10.0 ± 9.0 3.3 ± 6.8 26.7 ± 15.2 1.3 ± 1.4 4.0 ± 5.2

Mechanical scarification

97.5 ± 6.2 97.0 ± 6.2 96.0 ± 12.4 97.5 ± 6.2 85.0 ± 8.6

Chemical scarification 10 min

15 min

20 min

88.3 ± 14.3 15.0 ± 12.4 78.3 ± 14.3 88.7 ± 8.9 85.0 ± 12.4

93.3 ± 7.2 30.0 ± 5.2 95.0 ± 12.4 98.3 ± 7.2 96.7 ± 14.3

98.3 ± 6.9 100.0 ± 0.0 90.0 ± 11.9 98.3 ± 7.1 96.5 ± 8.3 98.5 ± 14.3 100.0 ± 0.0 95.3 ± 9.6

Liquid nitrogen treatment did not determine significant enhancements in seed germination as compared to the control (data not shown). 3.2. Second experiment: testing seed germination and early root growth of different carob genotypes under salt and drought stress Seed water uptake, measured every 24 h after the imbibition, was significantly affected by water potential and genotype (Fig. 1). Whatever the genotype, as compared to water imbibition at 0 MPa (control), increasing reductions in water uptake over time were ascertained even at the lowest level of salinity stress (−0.5 MPa) in the NaCl solution (Fig. 1 upper graph). Moreover, water uptake in isotonic solutions of PEG was always significantly lower than that registered in NaCl solutions. It is worthy of mention, that whereas in most cases undifferentiated levels of water uptake were observed in seeds germinating in the solution of NaCl at the lowest potential (−1.5 MPa) and in PEG solution at −0.5 MPa, germination percentage in the first case was almost null, in the other, higher germination percentages (ranging from 34 to 90% in relation to the genotypes) were observed (Table 3). On average of the studied germination solutions, Sorba and to a lesser extend Pignato (up to 48 h) showed the highest water imbibition (Fig. 1 lower graph). Final germination percentage of carob seed was significantly influenced by the osmotic stress, the genotype and their interaction (Table 3). As concerns germination in NaCl, the effects on germination were significantly different in relation to the genotypes. At −1.0 MPa, two genotypes Costa (−22.2%) and domesticated TDP (−34.4%) showed the highest decrease in germinability. The most resistant genotypes resulted Sorba, the wild genotype TDP, Cavette, Pignato and Margitello with germination percentages ranging between 87 and 93%. At −1.5 MPa germination of all the tested genotypes was almost completely inhibited. As concerns the germination in PEG, a noticeable decline in germination was observed already at −0.5 MPa again in the genotypes Costa (−64.0%), and domesticated TDP (−48%) and to a less extent in Margitello (−42.7%). The genotypes wild TDP (90%), Pignato (77.5%), Sorba (73.3%) and Cavette (71.7%), gave the highest germination percentages. Germination was completely inhibited at −1.0 MPa in all genotypes. PEG was therefore the most detrimental solute. Cumulative germination time courses in NaCl are illustrated in Fig. 2. Weibull function fitted very closely to the raw data of cumulative germination time courses at each treatment with rsqr coefficients of determination ranging from 0.95 to 0.99. Stress intensities up to −1.0 MPa in most genotypes resulted only in a delayed germination, whereas at −1.5 MPa, NaCl germination percentage was almost completely inhibited. Data sets of the germination rates (GR50 i.e. = 1/T50 ), which are the inverse of the time to 50% germination (T50 ) derived from germination time-courses in the NaCl solutions (according to the

Fig. 1. Influence of osmotic solutions (upper graph) and genotype (lower graph) on seed water uptake. In the upper graph, a vertical bar indicate the LSDs at p ≤ 0.05. In the lower graph, different letters on each bar indicate significant differences for p ≤ 0.05.

Weibull function) were plotted against water potential, within each cultivar. The linear regression of GR50 vs.  allowed the calculation of the minimum b or base water potential  at which seed germination is reduced to 50% (Table 4). The analysis of the confidence intervals of the intercept revealed the significant higher  b(50) value of Cavette as compared to Pignato, Sorba, Costa, and to a lesser extend wild and domesticated TDP.

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Fig. 2. Germination time courses for seed population of each genotype at 0 MPa (black circle), at −0.5 MPa (white circle), at −1.0 MPa (black triangle down) and −1.5 MPa (white triangle down).

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Fig. 3. The cumulative germination curves on a hydrotime scale [(␺ − ␺b (g)) tg ] at all potentials for each genotype after transforming percentages into probit units and expressing hydrotime on a log scale.

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Table 3 Effects of salinity and osmotic stress on carob seed germination (%) in relation to the genotypes. Genotypes

Sorba TDP wild Croce Cavette Pignato Margitello Costa TDP dom. Means

Control

93.3 (77.7) 100.0 (90.0) 90.0 (75.7) 98.3 (85.7) 100.0 (90.0) 95.5 (79.9) 93.3 (77.7) 98.3 (85.7) 96.10 (82.74)

NaCl

PEG

Overall means

−0.5 MPa

−1.0 MPa

−1.5 MPa

−0.5 MPa

−1.0 MPaa

−1.5 MPaa

96.7 (83.8) 96.7 (83.8) 86.7 (68.8) 91.6 (73.7) 100.0 (90.0) 93.3 (77.7) 91.1 (76.1) 91.6 (76.8) 93.45 (78.52)

93.3 (77.7) 93.3 (77.7) 78.3 (68.2) 87.5 (69.9) 92.2 (73.8) 91.1 (76.1) 72.6 (58.4) 64.4 (58.7) 84.93 (69.84)

0.0 0.0 13.3 (17.6) 13.3 (17.6) 11.3 (18.8) 10.0 (18.0) 0.0 0.0 7.64 (11.22)

73.3 (58.9) 90.0 (75.7) 66.7 (54.8) 71.7 (58.2) 77.5 (61.7) 54.7 (47.7) 33.6 (35.4) 51.0 (45.7) 68.96 (53.36)

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

ANOVA EFFECT

71.33 (59.64) 76.00 (64.96) 73.67 (64.04) 73.81 (62.79) 76.17 (66.88) 68.91 (59.37) 58.12 (49.57) 61.06 (51.41) 70.22 (60.30)

L.S.D. SS

DF

MS

Genotype (G) 2421.04 7 488.72 80082.16 4 20020.53 Osmotic Solut. (OS) 8091.09 28 288.97 G x OS Residual 7058.69 80 88.23 98652.97 119 Total √ In brackets arcsin % transformed percentages (Bliss transformation) to be confronted by L.S.D. a Values not included in the ANOVA and in the calculation of the genotype means.

F

P ≤ 0.05

P ≤ 0.01

5.54** 226.90** 3.27**

6.83 5.40 15.26

9.05 7.15 20.24

Table 4 b(50) from linear regression, confidence intervals and b(50) from probit analysis. Code

b(50) fromlinearregression (MPa)

95% Confidence Intervals (Y = 0)

b(50) fromprobitanalysis (MPa)

Pignato Cavette Margitello TDP wild Sorba Croce TDP dom. Costa

−1.24 −1.71 −1.47 −1.44 −1.26 −1.47 −1.43 −1.31

−1.49 to −1.06 −1.88 to −1.51 −1.68 to −1.28 −1.53 to −1.35 −1.43 to −1.14 −1.76 to −1.28 −1.59 to −1.30 −1.43 to −1.21

−1.28 −1.74 −1.60 −1.40 −1.28 −1.47 −1.42 −1.31

The cumulative germination over a hydrotime scale [(␺ − ␺b (g)) tg ] at all potentials for each genotype is shown in Fig. 3. Base  (Table 5) which best fitted to probit analysis varied with genotypes, suggesting a genetic variability in salt tolerance to salinity stress during germination. Among genotypes, Cavette (−1.7 MPa) and Margitello (−1.6 MPa) exhibited the highest tolerance to salinity stress in terms of b s base. Base s calculated by the linear regression of GR50 vs.  did not present noticeable difference with those obtained by the probit analysis (Table 4) with the only exception of Margitello where, however, the correction of the  b(50) operated by the probit analysis seem to better justify the good tolerance observed in the germination of this genotype under salinity stress. Mean hydrotime estimated by the probit regression showed a larger variability (Table 5) than the base s ranging from 114.8 (genotype Sorba) to 229.0 MPa h (genotype Cavette). Two genotypes (Pignato and Sorba), giving a high mean germination over the three osmotic potentials in NaCl (67.7 and 63.2), showed the lowest hydrotime even if they did not show the lowest base s. 3.3. Root growth In all genotypes, root growth was negatively affected by salt stress, as significant reductions were registered with the more negative osmotic potential (Table 6). At 0 MPa (control) significant differences in root length were observed in relation to the different genotypes, showing Sorba (3.4 cm) and Pignato (3.3 cm) the highest values, followed by Margitello (3.0 cm), wild TDP and Costa (1.9 cm) the lowest ones. Total root length decreased with increasing concentration of the NaCl solutions.

Consistent decreases in root length were observed in almost all the genotypes already at −0.5 MPa. The inhibiting effect on seed germination and following embryo growth was stronger in PEG than in NaCl solution at the equivalent water potential (−0.5 MPa). In NaCl, as compared to the control, among the genotypes, Margitello showed the highest root lengths at −0.5 MPa (2 cm), followed by Pignato (1.7 cm), the wild genotype and Sorba (1.5 and 1.4 cm respectively). The same genotypes gave the best results also at −1.0 MPa in the NaCl solution and at −0.5 MPa in the PEG one. In most cases, these results may be attributed to less consistent decreases induced by drought and saline stress on root growth of the best performing genotypes (Table 6). 4. Discussion Only a small fraction (from 3 to a max of 27%) of seeds from the studied carob genotypes could germinate without any pre-sowing treatment and these seeds are probably the ones that would germinate shortly after they are dispersed. Ceratonia siliqua in fact, has been previously demonstrated, produces seeds with different degrees of physical dormancy and the degree of dormancy varies among individual trees. This could be due to the existence of a considerable variability in the toughness of the seed coat (Pérez-García, 2009). It is probable that this variation, can be attributed to genetic differences among individual parent plants, even within a small geographic area (Pérez-García, 2009). An impermeable seed coat, as occurs in many Fabaceae taxa (Baskin and Baskin, 2004; Eisvand et al., 2006; Finch-Savage and Leubner-Metzger, 2006; Silveira and Fernandes, 2006; PérezGarcía et al., 2008), is the cause of the physical dormancy of carob seeds and hinders the uptake of water preventing germination.

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Table 5 Mean germination percentage in NaCl and estimated hydrotime parameters for predicting germination time-course of carob genotypes. Different letters in the first column indicate significant differences for p ≤ 0.05 according to the LSD test. Code

Mean germination in NaCl (%)

Log ␪H (MPa h)

␪H (MPa h)

b(50) fromprobit analysis (MPa)

␴␺b(g) (MPa)

Degrees of freedom

rsqr

Pignato Cavette Margitello TDP wild Sorba Croce TDP dom. Costa

67.73 a 66.36 ab 64.80 ab 63.33 ab 63.23 ab 56.43 ab 54.27 b 54.56 b

2.12 2.36 2.28 2.23 2.06 2.32 2.28 2.25

131.8 229.0 190.5 169.8 114.8 208.9 190.5 177.8

−1.28 −1.74 −1.60 −1.40 −1.28 −1.47 −1.42 −1.31

0.109 0.115 0.165 0.068 0.130 0.161 0.146 0.259

24 17 19 13 18 20 23 27

0.91 0.82 0.76 0.97 0.90 0.79 0.87 0.86

Table 6 Root length (cm) of germinated seeds in NaCl and PEG solutions, in brackets the percentage reduction. Genotypes

Pignato Cavette Margitello TDP wild Sorba Croce TDP dom. Costa Overall Means

Control

NaCl

PEG

Overall Means

0 MPa

−0.5 MPa

−1 MPa

−0.5 MPa

3.3 (100%) 2.4 (100%) 3.0 (100%) 1.9 (100%) 3.4 (100%) 2.1(100%) 2.8 (100%) 1.9 (100%) 2.60 (100%)

1.7 (−48%) 1.0 (−58%) 2.0 (−33%) 1.5 (−21%) 1.4 (−59%) 0.7 (−67%) 1.1 (−61%) 1.2 (−37%) 1.33(49%)

0.8 (−76%) 0.6 (−75%) 1.0 (−67%) 0.9 (−53%) 0.9 (−73%) 0.5 (−76%) 0.8 (−71%) 0.6 (−68%) 0.76 (−70%)

0.9 (−73%) 0.5 (−79%) 0.8 (−73%) 0.6 (−68%) 1.3 (−61%) 0.7 (−67%) 0.9 (−68%) 0.7 (−63%) 0.81 (−69%)

1.68 1.15 1.69 1.22 1.77 1.01 1.41 1.10 1.38

ANOVA EFFECT

SS

DF

MS

F

L.S.D. (0.05)

L.S.D. (0.01)

Genotype (G) Osmotic Solution (OS) G x OS Residual Total

7.55 52.92 5.90 1.68 68.05

7 3 21 64 95

1.08 17.64 0.28 0.03

40.99** 670.58** 10.68**

0.13 0.09 0.26

0.17 0.12 0.35

Physical dormancy resulting in the production of seeds which are released from dormancy and germinate at different times over a determinate period is one of the most important survival strategies for wild species growing under variable and unpredictable environmental conditions (Qaderi and Cavers, 2000) and facilitates the germination of seeds over time and space, increasing the probability of resulting in an adult plant (Baskin and Baskin, 2001). During domestication in plant species, artificial selection may determine changes in physiological aspects such as loss of seed dormancy, faster and synchronous germination, and variations in periods of fruit maturation (Frary and Do˘ganlar, 2003). However, domestication does not appear to have influenced the germination behavior in this species since most seeds of domesticated and wild genotypes do not germinate unless the seed coat is scarified (Gunes et al., 2013). The results obtained on different genotypes collected in Sicily (Italy) showed that germination of untreated seeds can be drastically improved by mechanical (piercing seed coat) or chemical scarification (sulfuric acid treatment) and confirm the results obtained on wild and domesticated genotypes in Turkey (Gunes et al., 2013) and in Spain by Pérez-García (2009). In carob, under natural conditions, a number of factors (mechanical friction with soil particles, microbial action, passage through the digestive tract of mammals that feed on them, etc.) can alter seed coat (PérezGarcía, 2009) thus permitting germination. As concerns in general carob germination, optimum temperature for carob seed germination was found to be 25 ◦ C by De Michele et al. (1988) and 27.5 ◦ C by Mitrakos (1981). However, in the work of Pérez-García (2009) no significant differences were found among the different temperature regimes assayed since the seeds of the population studied reached similar final germination percentages at all incubation temperatures. Therefore, this species

seems to show an opportunistic strategy for seed germination. It was hypothesized that in the semi-arid natural habitats in which this species grows, soil moisture conditions would be the most decisive factor for germination and for seedling establishment. Our paper seem to confirm that carob is very sensitive to water stress simulated by the germination in PEG solutions. PEG resulted in fact the most detrimental solute both on germination and on root growth even at moderate stress conditions (−0.5 MPa) whereas when the same stress was induced by NaCl, germination was only slowed down and only high stress intensities (−1.0 MPa) had an impact on the final germination percentages of some of the studied genotypes. However at −0.5 MPa PEG solution, a different behavior of the eight genotypes was evidenced, since a noticeable decline in germination was observed in four out the eight tested genotypes whereas the remaining (Sorba, wild TDP, Pignato and Cavette) resulted particularly resistant giving percentages >70%. In the same osmotic compound, germination was almost completely inhibited at −1.0 MPa in all genotypes. Similar effects were recently observed on seed germination and shoot and root growth on sorghum by Patanè et al. (2013), on durum wheat by Almansouri et al. (2001), and on sunflower by Kaya et al. (2006). These last authors suggested an osmotic effect of PEG rather than an ion accumulation, because when PEG stress was removed, the seeds were able to germinate but should not be considered as a general rule since an opposite trend was also reported. It can be hypothesized that the presence of PEG, which is a non-penetrating solute, drastically compromised water uptake. Seed imbibition proceeds slower due to osmotic pressure, thus seed metabolism activation is delayed and seed germination takes place later. Indeed, in sorghum it has been demonstrated that seed respiration, as a result of seed metabolic activity, at the early stages of imbibition is highly corre-

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lated with seed water uptake (Patanè et al., 2006). A threshold level of hydration is required for the initial steps of germination and for the subsequent radicle elongation (Ramagopal, 1990). In NaCl solutions, the ions may penetrate the cell wall, determining a decrease in the internal osmotic potential of the germinating structures as suggested by Thomas et al. (1995), Dodd and Donovan (1999) and Almansouri et al. (2001) and leading to water uptake and initiation of germination processes (Cavallaro et al., 2014). The ‘osmotic adjustment’, by ensuring turgor maintenance, may reduce sensitivity to water stress or allow the growth at a slow rate under stress conditions (Meyer and Boyer, 1981). Our results confirm that water imbibition in isotonic solutions of NaCl is often higher than that registered in PEG ones. However at the lowest potential in NaCl (−1.5 MPa) a reduced and erratic germination is registered even if water adsorption is similar to that registered at the highest PEG potential (−0.5 MPa). In this last solution, a higher seed germination in all genotypes has been registered. It seems therefore that at the highest stress levels induced by NaCl, germination is not unpaired by the external osmotic barrier preventing water uptake (which is still high) but probably by the toxic effect due to Na+ and Cl− ions accumulation. An absorption of toxic ions has been demonstrated to occur during seed imbibition of cowpea in salt solution (Murillo-Amador et al., 2002), which may affect some enzymatic activities of the seed, resulting in an extended germination time and a consequent reduction in root and shoot elongation (Barbera et al., 2014). Moreover, it is likely that, carob should be classified as a species moderately tolerant to salinity since at the studied levels of  , the injuring effect of NaCl ions are negligible up to −1 MPa while the osmotic effects due to reduced  in NaCl were evident only at its greatest concentrations as reported in sorghum by Patanè et al. (2013), a species classified as moderately tolerant to salinity (Maas, 1985). The hydrotime analysis permitted to highlight some important features of the tolerance to salinity stress (determined by NaCl solutions) during germination of the studied genotypes. These differences seem to be tied not only to different base water potentials (b ) but also to lower hydrotimes. Two of the salinity tolerant genotypes (Pignato and Sorba) did not show very low (b ) but lower hydrotimes as compared to the other genotypes. According to Mayer and Poljakoff-Mayber (1989) results like these could be attributed to absence in the low tolerant genotypes of the energy to start the germination process, since energy is obtained by increments in the respiratory pathway after the imbibition which is tied to the levels of the soluble sugars. At low levels of water potential determined by the osmotic stress, water absorption and soluble sugars demolition proceeds slowly. In these conditions, seeds need therefore longer or too long times (hydrotimes) to accumulate the energy necessary to begin the germination process. Moreover, in the two genotypes (Pignato and Sorba) showing a good tolerance to water and salinity stress, during the first imbibition phase, whatever the osmotic potential of the germination solution, a higher water absorption capability was observed when compared to the less tolerant genotype Costa. This feature probably leads these two genotypes to a more rapid and complete germination (lower hydrotimes) even at high salinity levels (−1.0 MPa). Based on our experience, a possible hypothesis supporting this observation may be that each genotype may possess a different gelling ability (water absorption) of the galactomannans, characteristic compounds of the carob seed endosperm and widely used for their gelling properties. Moreover, in the work of Spyropoulos and Lambiris (1980) on carob germination under water stress (induced by range of PEG 4000 solutions), during germination plumule axis growth, total dry weight decrease, and starch formation in the embryo were closely correlated to galactomannan depletion, the latter being inhibited by lower external water potential. Soluble sugar content was higher in cotyledons of seeds germinated under stress conditions, mainly

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due to higher sucrose accumulation. The higher sugar content in the endosperm was mainly due to higher galactose and mannose content. Water stress effected a delay in the raffinose-type oligosaccharide depletion. It seems therefore that the role of these compounds is still a matter of debate and investigation in order to determine their influence on carob germination under different water or salinity stress conditions Root growth is an important parameter to evaluate genotypes tolerance soon after germination. In all the carob genotypes, however, seedling development was significantly reduced already at a higher osmotic potential (−0.5 MPa) than that affecting germination so that the inhibition of root growth, during the earliest phases after germination, may inhibit the emergence and a better exploitation of the water reserves in the soil. A higher tolerance to salt stress as compared to the drought one was observed also in root elongation, since the reductions observed in this parameter were lower in NaCl than in PEG solutions, in almost all genotypes. Among the genotypes so far studied, Margitello, Pignato, Sorba and wild TDP showed higher root lengths upon all the studied solutions. Cavette root growth was severely inhibited by all the salinity and osmotic stresses even showing a good germination at the same stress levels. Results obtained on three cultivars of soybean by Machado Neto et al. (2004) showed that NaCl water deficit affected germination just below −0.6 MPa, but seedling development parameters were negatively affected already below −0.3 MPa, especially in relation to root length and shoot dry weight. Kramer (1974) reported that the first effect measurable due to water deficit was the growth reduction, caused by the declining in the cellular expansion. The cellular elongation process and the carbohydrates wall synthesis were very susceptible to water deficit and the growing decrease was a consequence of the turgescence laying down of those cells (Hsiao, 1973; Shalhevet et al., 1995).

5. Conclusions A protocol was designed to assess the germination response to saline and drought stress of different genotypes of carob collected in a representative Mediterranean area. Sulphuric acid treatment is necessary to overcome seed dormancy which does not permit germination. Germination was carried out in isotonic solutions of PEG and NaCl to simulate water and salinity stress. Differences were observed in the germination response of the different genotypes to such stresses. All genotypes exhibited a higher sensitivity to water stress as compared to salinity stress. The observed differences in some genetically inherited germination characteristics (i.e.in base b(50) s and hydrotimes determined by the hydrotime analysis) suggest genetic differences even in seeds from individual old trees situated in close areas as previously ascertained in other countries of the Mediterranean basin. Water stress and salinity affected seed germination and root growth development, however, root growth was already affected at higher osmotic potential (−0.5 MPa) than that affecting germination. Due to the greater sensitivity to stress, root growth must be taken in consideration to forecast plantlets behavior under both abiotic stresses in field conditions. These findings suggest that, there is a noticeable genetic variability to be explored in many areas of old carob cultivation in the Mediterranean. Germination tests under different simulated salinity or water stress conditions may be helpful for an earlier exploration of genetic variability but other parameters representative of early embryo growth (i.e. radicle elongation) must be included in the analysis as helpful indicators in breeding programs towards the development of water or salt stress-tolerant rootstocks.

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