Journal of Applied Botany and Food Quality 90, 224 - 231 (2017), DOI:10.5073/JABFQ.2017.090.028 1Research Institute for Biotechnology and Bioengineering, Isfahan University of Technology, Isfahan, Iran 2 Department of Agronomy and Plant Breeding, College of Agriculture, Isfahan University of Technology, Isfahan, Iran The effects of salt stress on physio-biochemical traits, total phenolic and mucilage content of Plantago ovata Forsk under in vitro conditions Pooran Golkar1, Farzad Amooshahi2, Ahmad Arzani2* (Received January 13, 2017; Accepted April 19, 2017) * Corresponding author Summary Plantago ovata Forsk (psyllium) is an important source of mucilage which is an ingredient in certain drugs and foodstuffs. The calli of 14 genotypes of psyllium were cultured on MS (Murashige and skoog, 1962) medium containing 0, 100, and 200 mM NaCl, for four weeks, and the effects of salt stress on the following callus traits were evaluated: growth rate (CGR), relative growth rate (RGR), relative water content (RWC), Na+ concentration, K+ concentration, K+/Na+ ratio, proline content, total phenolic compounds (TPC), and muci- lage content. A reducing trend was observed in GR, RGR, RWC and K+/ Na+ of the callus cultured in the medium with 100 mM NaCl, comparing to NaCl-free medium, while an increasing trend was observed in Na+ content, proline content, and TPC under the same conditions. Mucilage content of callus was found to increase in the medium containing 100 mM NaCl (0.13 g g-1 DW) but decreased afterwards at 200 mM NaCl (0.117 g g-1 DW), albeit with significant variations among genotypes. The results showed that among evalu- ated genotypes, Isfahan-1 was the most salt tolerant genotype at cel- lular level. In addition, the highest mucilage content was obtained in Khor-Biabanak genotype when the calli grown at 100 mM NaCl. It was postulated that mucilage content likely to be associated with salt tolerance and could be exploited to counteract the negative osmotic potential in callus affected by salt stress in P. ovata. Keywords: callus induction, medicinal plant, mucilage, psyllium, tissue culture Introduction Global agricultural and food production are seriously threatened by high soil salt levels (Parvaiz and satyawati, 2008; Arzani and Ashraf 2016), which affects many of the plant biochemical and physiological processes at the cellular or whole plant level (Ashraf and Harris, 2004; GuPta and Huang, 2014; Arzani and Ashraf, 2016). Plants’ response to salt stress comprises numerous processes in cells that coordinately operate to alleviate hyperosmolarity and re- establish ionic homeostatic conditions (Arzani and Ashraf, 2016). Development of salt tolerance in plants is, hence, a complex pheno- menon (rai et al., 2011). Saline-induced osmotic stress triggers a wide range of perturbations ranging from growth and development disruption to modification of the ion transport and uptake systems (Basu et al., 2002; Parihar et al., 2015). Moreover, intracellular solutes and certain inorganic ions like K+ and Na+ play intrinsic roles in plant response to salt stress (Basu et al., 2002; Arzani and Ashraf, 2016). The synthesis and accumulation of compatible os- molyte such as proline is one of the effective mechanisms of adap- tation to salt stress by stabilizing membranes and macromolecular structures (Ashraf and foolad, 2007), detoxifying reactive oxygen species, and adjusting cellular osmosis (zhu, 2002).To this may be added the fact that actively growing cells, like those experiencing salt stress, may be characterized by a high phenolic content (liM et al., 2012). Moreover, many studies have shown that changes in the levels of secondary metabolites, including phenolic compounds, enhance plant defense mechanisms against stress, particularly against oxida- tive stress induced by high salt concentrations (Matkowski, 2008; liM et al., 2012). The study of salt tolerance mechanism is preferably conducted at the cellular level because it provides the means to study plant physio- logical and genetic processes independently of the regulatory mecha- nisms occurring at the whole plant level (Arzani, 2008; lokhande et al., 2010; shibli et al., 2011). The in vitro salt stress has been stu- died in such different species as rice (basu et al., 2002), Medicago sativa (ehsanPour and fatahian, 2003), wheat (Arzani, 2008), Cucurbitacea (Cano et al., 1998; shibli et al., 2011), date palm (al- khairy, 2002), oil seeds (alvarez et al., 2003; soheilikhah et al., 2013), sugar cane (gandonou et al., 2006), and medicinal plants such as Foeniculum vulgare (khoraMi and safarnejad, 2011) and Thymus vulgaris (zia et al., 2010). Plantago ovata Forsk, commonly known as psyllium, is an annual medicinal herb plant with beneficial health properties, whose seeds have found important medicinal ap- plications in treating high blood pressure, high cholesterol, diabetes, and hemorroid (sharMa, 2004). The leaves, seeds, and seed husks of psyllium are rich sources of phytochemical substances such as muci- lage (talukder et al., 2015). The plant is native to the Mediterranean region and is found in India, Pakistan, and Iran (SharMa, 2004). Plantago genus is generally considered to be moderately tolerant to salt, though depending on the genotype and salt stress level (kariMi and haghighi-Pak, 2012). To the best of the authors’ knowledge, no published work has been reported on the biochemical and physio- logical characterization and mucilage production of psyllium, espe- cially with respect to its mechanisms for combating salt stress at the cellular level. Moreover, the efficient and economical production of mucilage from the psyllium calli through in vitro salt stress culture optimization was one of our desired goals. We tested this hypothesis in a series of experiments, in which the types of growth regulators combinations and concentrations have already been tested. Thus, the experiment described here is the final experiment of the series to in- vestigate the physiological and biochemical adaptive mechanisms of different genotypes of psyllium involved in responding to in vitro salt stress. In addition, the effects of NaCl treatments on the phenolic and mucilage contents produced as secondary metabolites were assessed at the callus level in response to exogenous elicitor “NaCl”. Materials and methods Plant material and growth conditions Fourteen Plantago ovata genotypes comprising 12 Iranian (origi- nated from different geographical regions of Iran) and two exotic (from Pakistan and India) genotypes were used in this study. The seeds were obtained from Seed and Plant Improvement Institute, Karaj, Iran. The seeds of 14 genotypes were surface sterilized in 20% (v/v) sodium hypochlorite solution containing 0.1 ml of tween-20 per 100 ml for 15 min, followed by treatment in 95% ethanol for Effects of in vitro salt stress on Plantago ovata 225 1-2 min. The Murashige and skoog (1962) basal salt medium (Sig- ma-Aldrich) adjusted to pH 5.8, supplemented with 3% sucrose and 0.8% agar (Sigma -Aldrich), and then autoclaved for 20 min at 121 °C and 1.1 Kg cm-2. The sterilized seeds were place on the medium and incubated in a growth chamber in the 16/8 day/night photoperiod at a temperature of 25±2 °C for up to two weeks until full germination. Callus induction and in vitro salt stress treatment The calli were induced from hypocotyl explants cultured in MS medium (Sigma- Aldrich) that supplemented with 3% sucrose, 0.8% agar (w/v), 2,4D (0.5) mgL-1 and Kin (1) mgL-1. The hypocotyl de- rived calli were incubated at 24±2 °C for 4 weeks under dark con- ditions. Then, the calli were transferred onto solid MS medium con- taining the same concentrations of growth regulators as above and different concentrations of NaCl: 0 (control), 100 and 200 (mM) [0, 0.6 and 1.2% NaCl (w/v)] for 4 weeks (rai et al., 2011). The cultures were transferred to fresh medium every 2 weeks. After 4 weeks of salt treatment, different physiological and biochemical traits were measured. Physiological traits Relative growth rate (RGR) of callus was calculated as (W1-W0)/W0 × 100, where W0 is the initial callus fresh weight and W1 was con- sidered as the final fresh weight of callus after 4 weeks of salt treat- ment (errabii et al., 2007). Relative water content (RWC) was cal- culated as [(callus fresh weight – callus dry weight)/callus dry weight × 100] (lutts et al., 2004). Callus samples of known fresh weight were dried in an oven set at 65 ºC for 48 h, after which they were re- weighted and the difference in the initial and final mass determined. The callus growth rate (CGR) was calculated by measuring callus growth (mm/day) at 0, 15 and 30 days after callus transferring to salt containing media induction according to CoMPton (1994). The callus diameter (di) was calculated by root square of (callus length × callus width) (CoMPton, 1994). Biochemical traits Ion assay (Na+ and K+) Flame photometry method was used for measuring sodium and potassium concentration of the treated calli based on the method described by skoog et al. (2007). First, calli were collected and dried at 60 °C for at least 5 days, then 10 mg of each powder of dried cal- lus was digested with 10 ml 3% (w/v) aqueous sulfosalicylic acid for 24 h at 4 °C, sample extract purified with Whatman No. 1 filter paper and Na+ and K+ concentrations were measured. The standard solu- tions of NaCl and KCl prepared from reagent grade salts were used to estimate the ions concentrations by flame photometer (Jenway Model PEP7, UK). Proline content The modified bates et al. (1973) method was used for proline deter- mination. In this method, 40 mg of callus tissue was homogenized in 1.7 ml of 3% (w/v) aqueous sulfosalicylic acid. The extractions were transferred to centrifuge tubes and centrifuge at 14000 g for 20 min. Then 1 ml of each supernatant transferred into a 10 ml test tube, 1 ml of glacial acetic acid and 1 ml of the ninhydrin reagent added to each tube and heated for 1 h at 100 °C. Tubes were then cooled using cold water and 2 ml of toluene added to each tube, sealed with aluminum foil and vortexed at 30 rpm for 2 min. The phase separation was completed after 30 min and the upper phase was used for measuring the absorbance at 520 and 490 nm by spectrophotometer (Shimadzu UV-120-02, Japan). Total phenolic compound The extracts was prepared from 25 g 4 weeks old friable calli pow- dered in liquid nitrogen and immediately suspended in 50 ml of methanol and kept at room temperature for 24 hrs with periodic shaking. The solution was then filtered through Whatman filter paper no. 41, the methanolic extract was concentrated until dry. The dried methanolic extract was dissolved in least amount of methanol and kept at refrigerator temperature 2-4 °C till further use (uMesh, 2014). Total soluble phenolic compounds (TPC) were determined by using Folin-Ciocalteu reagent according to the method of Makkar et al. (1997). Aliquots of extract (200 μl) was taken from each sample, and the volume was made up to 1 ml with methanol. Then, 0.5 ml of Folin-Ciocalteu reagent (1:1 with water) and 2.5 ml of so- dium carbonate solution (20%) were added sequentially in each tube. After vortexing, all the tubes were incubated at room temperature in the dark for 40 min. Absorbance was measured at 725 nm against the reagent blank. The results are means of three repetitions expressed in the form of gallic acid equivalents (GAE) per gram of dry mass. Mucilage assay Mucilage was extracted from callus according to guPta et al. (2015) with minor modification in psyllium. Statistical analysis The experiments were conducted as either completely randomized design (CRD) or factorial experiment based on CRD with five repli- cations for each treatment. Five calli per replication were randomly selected and analyzed for free proline, total phenolic compounds, mucilage content, ion concentrations (Na+ and K+) and physiological traits. The data was analyzed using analysis of variance (ANOVA) by PROC GLM of SAS (SAS institute, 2011). Mean comparisons were conducted according to Fisher’s least significant difference (LSD) test at the 0.05 level of probability. Principal component analysis was performed using PROC FACTOR (method = prin) with, that is, Eigen value ≥ 1.0 in SAS 9.3 (SAS institute, 2011). Then, a genotype- by-trait biplot was constructed by using the first two trait-focused scaling principal components (PC1 and PC2) derived from principal component analysis (PCA) of a genotype-by-trait matrix containing standardized trait data. A simple regression and a multivariate back- ward stepwise regression analyses were also performed using PROC REG of SAS version 9.3 (SAS institute, 2011). Results and discussion Physiological traits The results of analysis of variance showed significant differences among the genotypes investigated and the significant effects of salt treatments on the physiological traits (Tab. 1). The genotype × salini- ty interaction was not significant for RWC, CGR and RGR (Tab. 1). Addition of NaCl to psyllium callus cultures led to a significant de- cline in the callus physiological traits RWC, RGR and CGR under in vitro salt stress (Fig. 1a, b) in P. ovata calli. kariMi and haghighat- Pak (2012) reported the non-significant reduction of seedling-related traits of psyllium genotypes at 200 mM NaCl under NaCl stress. The inconsistency between the in vitro and in vivo responses may be ex- plained by the multiplicity of the mechanisms involved in whole plant salt tolerance (arzani, 2008; rai et al., 2011). It has been established that the water potential gradient between the cell and the nutrient medium caused by NaCl results in dehydrated cells and reduced cal- lus fresh weight (al-khayri, 2002; rafiq et al., 2008; lokhande et al., 2010; shibli et al., 2011) as well as declining callus growth rate (Cano et al., 1998; ashraf and ahMad, 2000; ehsanPour and fatahian, 2003; lutts et al., 2004). 226 P. Golkar, F. Amooshahi, A. Arzani RWC decreased significantly with increasing salt stress, but no sig- nificant differences were observed in this parameter between the two 100 and 200 mM NaCl levels (Fig. 1a). The changes observed as a result of the reducing relative water content at 100 mM NaCl might have been due to the fast reaction of callus cells to the de- clining water availability in cells and the resulting loss of turgor pressure (al-khayri, 2002). Reduced RWC values due to in vitro salt stress have been previously reported for different glycophytes (gandonou et al., 2006) and halophytes such as Sesuvium portu- lacastrum (lokhande et al., 2010) and Suaeda nudiflora (Cherian and reddy, 2003). Callus growth started to be appreciably inhibited at 100 mM NaCl compared to that in the absence of NaCl. The re- duced callus growth could be compromised from a reduction in the osmotic potential of the medium culture that enhances the require- ment for maintaining the turgor of the growing callus cells, a process that consumes energy and, eventually, leads to the declining callus growth (shibli et al., 2011). To gain a better understanding of salt-tolerance and salt-sensitivity, some researchers have used the ionic selectivity and osmoprotectant accumulation as traits to classify genotypes regarding their response to salt and also to select tolerant cell lines. The genotypes used in this study showed different physio-biochemical responses to in vitro salt stress (Tab. 2). The mean comparisons of the genotypes evaluated for their callus growth and water related traits showed that salt stress had its greatest inhibitory effect on reducing callus growth in all the genotypes investigated at 200 mM concentration of NaCl (Fig. 2 and Tab. 2). The highest (0.61 mm day-1) and the lowest (-0.19 mm day-1) values of CGR averaged over all the salt levels tested were obtained with Behbahan and Ghazvin genotypes, respectively (Tab. 2). The highest values of RWC (35.55%) and RGR (82%) were obtained with Isfahan-1, whereas the least RWC (11%) and RGR (47%) values be- longed to Ghazvin and Isfahan-3, respectively (Tab. 2). On the other hand, the genotypes with sensitive callus had severely reduced callus RWC and RGR under salt treatments relative to the control. Simi- lar results have been reported for other species (basu et al., 2002; ehsanPour and fatahian, 2003; gandonou et al., 2006). RWC was significantly correlated with CGR, RGR and mucilage content under 200 mM NaCl conditions as evident by their vector angles in the biplot presented in Fig. 3. A similar relationship was previously observed between RWC and CGR (basu et al., 2002; ahMad et al., 2009). This relationship could be explained by the osmotic effect of NaCl which results in reduction of water content in cytosol causing in turn decline in CGR (Arzani and Ashraf, 2016). In addition, the increase in salt concentration may lead to the reduc- tion of osmotic potential of the medium, results in reduced turgor of the growing cells, and eventually leads to a decrease in callus growth (CGR) (HaMedi et al., 2016). Biochemical traits Ion content (K+ and Na+) K+ ion reportedly plays an important role in enzyme activation (tester and davenPort, 2003) although the relationship between K+ content and salt stress may vary from one species to another (al- khayri, 2002). The results of analysis of variance showed signifi- cant differences among the genotypes and the significant effects of salt treatments for ion content (K+, Na+ and K+/Na+) (Tab. 1). The genotype × salt interaction was only significant for K+ concentra- tion (Tab. 1). In the present study, increased salt at 100 and 200 mM (NaCl) led to a significant decrease in K+ concentration (Fig. 2a). Similar to these findings, K+ reportedly declined steadily in response to increasing salt concentration of callus cultures (Chauhan and PrathaPasenan, 2000; basu et al., 2002; gandonou et al., 2006; lokhande et al., 2010; soheilikhah et al., 2013). Other studies, however, have reported callus cultures to exhibit an initial increase in their K+ levels in response to low NaCl levels (such as 25 mM) which later declined steadily at higher NaCl levels (al-khayri, 2002). The reduction in K+ concentration in callus cells under salt stress could be explained by the alterations in expression and/or function of transporters as well as the ion channels especially those related to K+ Tab. 1: The results of analysis of variance for callus related traits of psyllium genotypes subjected to salinity stress. Trait¥ Genotype Salinity G × S Residual (G) (S) df: 13 2 26 168 RWC 778.99** 5117.9** 160.03 96.21 CGR 0.93** 14.77** 0.142 0.10 RGR 0.23** 2.39** 0.031 0.021 K+ 7222.9** 2492.1** 2182.4** 288.75 Na+ 2194.7** 701025 ** 1459.8 960.6 K+/Na+ 1.65** 76.23** 1.12 0.86 Proline 0.047** 18.07** 0.033** 0.005 TPC 6.2** 32.7** 1.31** 0.009 Mucilage 0.003** 0.005** 0.0017* 0.0009 ** and * Significant at P < 1% and 5%, respectively. ¥ df: degree of freedom; RWC: relative water content; CGR: Callus growth rate; RGR: relative growth rate; TPC: total phenolic compound. Fig. 1: Effect of in vitro NaCl stress on the callus RWC and RGR (a) callus growth rate (b) of Plantago ovata. Means by common letter do not significantly differ at the LSD5%. Effects of in vitro salt stress on Plantago ovata 227 Ta b. 2 : T he m ea n co m pa ri so ns o f d iff er en t g en ot yp es o f P . o va ta fo r d iff er en t p hy si o- bi oc he m ic al tr ai ts a nd m uc ila ge c on te nt u nd er in v itr o sa lt st re ss . R W C C G R R G R N a+ K + K + / N a+ Pr ol in e T PC M uc ila ge (% )* (m m (% ) (m m ol (m m ol g -1 D W ) (m g g- 1 FW ) (m g G A E g- 1 D W ) (g g -1 D W ) da y- 1 ) g- 1 D W ) N aC l ( m M ) A vg . A vg . A vg . A vg . 0 10 0 20 0 A vg . 0 10 0 20 0 0 10 0 20 0 0 10 0 20 0 G en ot yp e Is fa ha n- 1 35 .5 5a ** 0. 29 b- e 82 a 13 5. 5d 24 2a 10 4b 78 a 1. 84 ab 0. 05 cd e 0. 59 a 1. 4a 1. 90 e 2. l0 f 2. 32 g 0. 11 a- d 0. 11 bc 0. 11 ab c M as hh ad 22 .2 0d 0. 21 d- g 75 ab 17 0. 6 a 20 8b c 11 0a 68 ab 1. 41 ab c 0. 08 a- e 0. 40 c 1. 06 c 2. 00 de 2. 10 f 3. 30 d 0. 14 a 0. 14 b 0. 14 a Sh ir az 12 .3 4e f 0. 05 fg h 48 c 14 0. 6c d 19 2c de 80 cd e 50 bc d 1. 66 ab c 0. 10 a- e 0. 50 b 1. 08 c 2. 80 b 3. 8a 4. 04 b 0. 12 ab c 0. 13 bc d 0. 13 ab G ha zv in 11 .0 f -0 .1 9i 53 c 14 2. 6b cd 21 8b 90 bc 52 bc d 2. 47 a 0. 10 a- e 0. 50 b 0. 90 d 1. 90 e 2. 50 e 3. 60 c 0. 09 cd 0. 10 c 0. 10 bc A ra n- B id go l 26 .0 1b c 0. 47 ab c 69 b 15 2. 8 a- d 15 2h 80 cd e 52 bc d 1. 09 bc 0. 10 a- e 0. 50 b 1. 20 b 2. 10 cd 2. 8d 3. 04 e 0. 13 ab 0. 14 b 0. 14 a K ho r- B ia ba na k 26 .1 4b c 0. 42 a- d 50 c 15 8. 8 ab c 10 4i 74 c- f 54 bc d 0. 97 bc 0. 13 ab c 0. 40 c 1. 02 c 2. 0d e 1. 9g 2. 80 f 0. 08 d 0. 20 a 0. 09 cd Pa ki st an 18 .4 4d e 0. 25 c- f 50 c 16 5. 4 a 20 0b cd 72 c- f 56 bc d 1. 53 ab c 0. 03 e 0. 40 c 1. 00 c 1. 01 g 1. 08 i 4. 80 a 0. 10 bc d 0. 12 bc 0. 11 ab c A hv az 22 .6 3b cd 0. 50 ab 51 c 15 1. 5a d 16 0g h 70 de f 38 de 1. 15 bc 0. 14 ab 0. 50 b 1. 20 b 1. 40 f 1. 7h 2. 40 g 0. 10 bc d 0. 11 bc 0. 11 ab c In di a 27 .4 0b 0. 01 ef g 51 c 14 1. 7 cd 12 4i 64 ef 48 cd 0. 79 bc 0. 04 de 0. 40 c 1. 20 b 2. 20 c 3. 08 c 4. 00 b 0. 12 ab c 0. 14 b 0. 13 ab B eh ba ha n 19 .1 4c de 0. 61 a 76 ab 17 3. 2a 10 8i 56 f 28 e 0. 74 c 0. 16 a 0. 40 c 1. 07 c 2. 20 c 3. 81 a 4. 30 b 0. 10 bc d 0. 12 bc 0. 12 ab c C ha ha rm al - B ak ht ia ri 13 .6 6e f 0. 02 gh i 72 ab 16 5. 93 a 11 2i 70 de f 38 de 1. 01 bc 0. 07 b- e 0. 55 ab 1. 20 b 3. 00 a 3. 5b 3. 90 b 0. 11 a- d 0. 11 bc 0. 11 ab c Is fa ha n- 2 24 .4 5b cd -0 .0 01 gh i 52 c 16 4. 8 ab 16 4f gh 88 bc d 64 ab c 0. 98 bc 0. 10 a- e 0. 50 b 0. 90 d 2. 00 de 2. 9d 3. 00 e 0. 12 ab c 0. 13 bc d 0. 13 ab Is fa ha n- 3 12 .7 1e f -0 .1 6i 47 c 15 7. 8a bc 18 5d ef 10 4b 64 ab c 1. 65 ab c 0. 07 b- e 0. 40 c 0. 80 d 1. 50 f 2. 4e 2. 80 f 0. 12 ab c 0. 14 b 0. 13 ab Is fa ha n- 4 13 .8 2e f 0. 29 b- e 71 ab 16 2. 3a bc 17 4e fg 84 cd 54 bc d 1. 51 ab c 0. 12 a- d 0. 40 c 1. 01 c 1. 0g 1. 1i 2. 00 h 0. 10 bc d 0. 11 cb 0. 06 d G ra nd m ea n 16 7. 3 81 .6 9 53 .1 4 0. 10 0. 46 1. 07 1. 93 2. 47 3. 30 0. 11 0. 13 0. 12 *M ea ns in e ac h co lu m n fo llo w ed b y th e sa m e le tte r a re n ot s ig ni fic an tly d iff er en t a t t he 5 % p ro ba bi lit y le ve l. ** R W C : R el at iv e w at er c on te nt ; C G R : C al lu s G ro w th R at e; R G R : R el at iv e gr ow th ra te ; T PC : T ot al p he no lic c om po un ds . 228 P. Golkar, F. Amooshahi, A. Arzani (arzani and ashraf, 2016). The patterns of K+ content in response to increasing NaCl levels (Fig. 2a) were parallel to the trends of callus growth and callus water related traits. For instance, the highest callus growth was achieved with a culture medium containing no NaCl, that is, the same concen- tration at which the highest K+ uptake was observed. Furthermore, the inhibitory concentration of psyllium callus growth was identified to be 100 mM NaCl (Fig. 1b), which is the same concentration at which potassium concentration significantly reduced relative to the control (Fig. 2a). Na+ concentration in the callus was observed to increase significant- ly with increasing salt concentration (Fig. 2a). The sharpest increase in Na+ concentration was observed when the callus was cultured on a medium containing 100 mM NaCl (Fig. 2a). The rising trend of Na+ concentration observed in this study was similar to those reported for such other plants as safflower (soheilikhah et al., 2013), Foe- niculum vulgare (khoraMi and safarnejad, 2011), and date palm (al-khayri, 2002). Experimental NaCl concentrations (i.e., 100 and 200 mM) concurrently led to an increase in plant Na+ content (Fig. 2a) although no significant changes were observed in the values of callus RGR (Fig. 1a). This suggests that the elevated nutritional uptake of both Na+ and K+ might have led to the retention of water in the callus (Chaudhary et al., 1997). Maintaining the cellular K+/Na+ homeostasis is pivotal for plant survival in saline environments (arzani and ashraf, 2016). As a result of increasing Na+ concentration in the medium, Na+ ions com- pete with K+ ones during salt stress for the transporter as they both share the same transport mechanisms, thereby decreasing the uptake of K+. Accordingly, the K+/Na+ ratio was observed in this study to de- crease significantly from 3.25 (the control) to 0.24 (at 200 mM NaCl) (Fig. 2b). This result is in agreement with those reported elsewhere (Chaudhary et al., 1997; al-khayri et al., 2002). Since the K+/ Na+ ratio is critical for salt tolerance, increasing this ratio at 100 mM NaCl in Plantago ovata will be a promising area of future research. The genotypes examined were found to differ with respect to their intracellular ions under control conditions (Tab. 1); all the genotypes, however, accumulated more Na+ ions than did the control. The Na+ content averaged over the salt level tested varied from 173.1 (mmol g-1 DW) in the Behbahan genotype to 135.5 (mmol g-1 DW) in Isfa- han-1 (Tab. 2). However, compared to those with salt-sensitive callus Fig. 3: Biplot drawn based on the first (PC1) and second (PC2) components obtained from principal component analysis using CGR, RWC, RGR, TPC, Mucilage Na+, K+, and K+/Na+ ratio of psyllium genotypes under 200 Mm (NaCl) under in vitro conditions. Fig. 2: Effect of in vitro NaCl stress on the K+ and Na+ content (a) and K+/Na+ ratio of Plantago ovata callus. Mean with one or more letter in common are not significantly different at the 5% probability level as tested by LSD5% test. Effects of in vitro salt stress on Plantago ovata 229 (i.e., Indian and Pakistani), the genotypes with salt-tolerant calli (i.e., Isfahan-1) accumulated less Na+ but more K+ ions. The imposition of NaCl-shock caused an injury to the tissue leading to excessive leaching and poor retention of K+ in the callus in genotypes with lower salt stress tolerance, demonstrating a very sharp reduction in K+ content with increasing salt from control to higher levels. This confirms that the K+ inclusion mechanism, which is an indicator of salt tolerance at the whole plant level (kariMi and haghighat-Pak, 2012), was also expressed at the cellular level in psyllium. Further- more, it is seen that, compared to those with susceptible calli, the genotypes with tolerant calli (such as Isfahan-1) accumulated less Na+ and maintained higher levels of K+, rather than the genotypes with sensitive calli (such as Ahvaz). This may be one reason under- lying the better growth of the Isfahan-1 calli under NaCl. The different genotypes and their calli exhibited significant diffe- rences in their intracellular ion accumulation (Tab. 2). The evalu- ated genotypes showed variations in their K+/Na+ ratios from 0.74 (in Behbahan) to 2.47 (in Ghazvin) (Tab. 2). The highest K+/Na+ ratio recorded for the Ghazvin genotype demonstrated its high salt tole- rance and capacity for maintaining an ionic equilibrium between Na+ and K+ ions. The harmful effect of Na+ depends on its accumulation site. Indeed, its accumulation is toxic to salt-sensitive callus in which it is carried into the cytoplasm; it is, however, nontoxic to salt-tole- rant callus since it accumulates in their cell vacuoles (Volkov, 2015). In this case, the vacuolar sequestration in different genotypes would play an important role in maintaining the water balance in the callus by increasing the osmotic pressure in the cells and keeping a high K+/Na+ ratio. The K+ content and K+/Na+ ratio was found to be correlated with proline content in callus affected by salt stress (Fig. 3). These results were in agreement with those of other researchers who reported a positive and significant correlation between K+ content with K+/Na+ ratio (BASU et al., 2002; ehsanPour and fatahian, 2003). Accord- ing to Cherian and reddy (2003), the reduction in K+ concentration is capable of inhibiting growth as a result of reducing plant capacity for osmotic adjustment and turgor maintenance, or alternatively, by adversely affecting metabolic functions. In the current study, no sig- nificant relationship was found between K+ and other physiological traits (CGR, RWC and RGR) (Fig. 2). On the other hand, positive relationship was observed between Na+ with some of these traits. It is expectable that the enhanced Na+ content would not only disturb plant nutrient balance and osmotic regulation but also reduce growth (guPta and huang, 2014). Proline content Proline acts as osmotica that protects the cytosol from dehydration as symptomatic salt stress damage (Ashraf and foolad, 2007). The analysis of variance showed significant differences between evalu- ated genotypes, salt treatments and genotype × salt interaction for proline content (Tab. 1). In psyllium callus, the proline content ave- raged over all the genotypes was found to increase dramatically with increasing salt from 0.1 mg g-1 FW in control to 1.1 mg g-1 FW in 200 mM (NaCl) (Tab. 2). The increase in proline content practically occurred at 100 mM NaCl, which indicates that salt stress was prob- lematic to cellular functions at this point. Endogenous, free proline accumulation with increasing medium salt concentration has been corroborated in various in vitro culture systems subjected to salt stress (al-khayri, 2002; ehsanPour and fatahian, 2003; alva- rez et al., 2003; lokhande et al., 2010). There was a significant dif- ference among the genotypes for proline accumulation, as reported in Tab. 2. Clearly, proline content increased sharply in all the geno- types as the elevated concentration of NaCl. Genotypic differences in proline accumulation under salt stress have also been reported in Medicago sativa (ehsanPour and fatahian, 2003), safflower (soheilikhah et al., 2013). Proline content did not correlate with CGR, RWC and RGR under 200 mM NaCl conditions (Fig. 2). Although the role of proline accu- mulation in tolerance to salt stress remains controversial, it is thought play role in osmotic phase of salt stress at either early stages of salt treatment or at mild salt stress conditions (arzani, 2008). Nonethe- less, some authors reported a negative correlation between proline accumulation and callus growth traits in tomato (Cano et al., 1998) and date palm (al-khayri, 2002). There was a negative and signifi- cant correlation between K+ and proline content (Fig. 2). It could be explained that proline and K+ involve in different pathways influ- encing salt tolerance in plant cells as stated in part in the preceding sentence. Total phenolic compound The synthesis and accumulation of polyphenols in in vitro cultures are generally stimulated in response to salt stress (ksouri et al., 2007). The reduction in growth induced by salt stress might have resulted in a new pattern of resource providing additional car- bon skeletons for phenolic biosynthesis (ksouri et al., 2007). The analysis of variance showed significant differences between evalu- ated genotypes, salt levels and genotype × salt interaction for TPC content (Tab. 1). Total phenolic content increased in term of general mean of the genotypes with increasing salt from control 1.93 (mg GAEg-1 DW) to higher levels including 100 mM (2.47 mg GAEg-1 DW) and 200 mM (3.3 mg GAEg-1 DW) (Tab. 2). These results could be explained by the hypothesis that salt stress might be associated with increasing total phenolic compounds due to antioxidant activi- ties (GIRI et al., 2012; LiM et al., 2012). Elevated phenol contents under salt stress have also been reportedly observed in the callus cultures of other species such as Salvadora persica (SharMa and RaMawat, 2014). However, accumulation of phenolic compounds in plants due to salt stress may be species depend (LiM et al., 2012). In addition, it depends on the salt concentrations, i.e. while phenolic compounds has not affected by moderate salt concentration; high salt stress caused a remarkable reduction in TPC in the seedlings of rice (Chunthaburee et al., 2014). It was found a co-ordinate change in proline content and TPC of callus in this study, while this relationship was not significant (Fig. 3). It has been demonstrated that accumulation of high amounts of proline in the cell increases TPC through the biosynthetic path- way of proline-linked pentose phosphate, which, in turn, enhances the phenyl propanoid synthesis (giri et al., 2012). Mucilage content The variation in mucilage content was evaluated under in vitro salt stress. The analysis of variance showed significant differences be- tween evaluated genotypes, salt levels and genotype × salt interac- tion for mucilage content (Tab. 1). Grand mean comparison of muci- lage content showed slight increase from control (0.11 g g-1 DW) to 0.13 g g-1 DW at 100 mM NaCl (Tab. 2). The increase in mucilage (as a secondary metabolite) content under salt stress has been reported in such few species as Salicornia brachiate (Jaiswar and Kazi, 2016) under in vivo conditions. A decrease in mucilage content at 200 mM NaCl (0.115 g g-1 DW), rather than 100 mM NaCl (0.13 g g-1 DW), may suggest the inhibitory effect of NaCl at 200 mM on in vitro mucilage synthesis in psyllium. No steady trend was observed in the variations of mucilage content in the callus subjected to different salt treatments (Tab. 2). Mucilage content increased in all the genotypes as salt increased from con- trol (0 mM) to 100 mM NaCl and decreased afterward at 200 mM NaCl (Tab. 2). There were significant variations among genotypes for in vitro mucilage content of callus grown at control, 100 mM and 230 P. Golkar, F. Amooshahi, A. Arzani 200 mM NaCl treatments. The highest mucilage content at 100 mM (NaCl) (0.20 g g-1 DW) was produced by Khor-Biabanak genotype. Mucilage is a polysaccharide mixture with highly variable chemical constituents, which may have played an important role in the salt tolerance through modulating water retention and ion homeostasis in plants (GhaneM et al., 2010). A strong and positive relationship was found between mucilage content and Na+ content affected by 200 mM NaCl at cellular level (Fig. 3). This finding may be a clue to the physiological role of mu- cilage in salt tolerance of mucilage-containing plants and deserve further investigation. Furthermore, it is in keeping with our goal of the eliciting effects of salt stress on the mucilage content at the cel- lular level. In vitro mucilage extraction and preparation could be an alternative and possible way to produce this medicinal substance. Conclusions Substantial variations were detected in the cellular responses of the different genotypes of psyllium to in vitro NaCl stress. Although, our data provides evidence of increasing mucilage content under in vitro mild salt stress in a mucilage-containing plant, dissecting the phy- siological and molecular role of mucilage in regulating ion and water status deserves further attentions. Acknowledgments The financial support of this research was provided by Research In- stitute for Biotechnology and Bioengineering at Isfahan University of Technology, Iran under grant #93-02-20. 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