Evaluation Of Salt Tolerance In Eruca Sativa Accessions .
Evaluation of salt tolerance in Eruca sativaaccessions based on morpho-physiologicaltraitsSadia Afsar1, Gulnaz Bibi1, Raza Ahmad1, Muhammad Bilal2,Tatheer Alam Naqvi1, Ayesha Baig1, Mohammad Maroof Shah1,Bangquan Huang3 and Jamshaid Hussain11Department of Biotechnology, COMSATS University Islamabad, Abbottabad Campus,Abbottabad, Pakistan2Department of Environmental Sciences, COMSATS University Islamabad, Abbottabad Campus,Abbottabad, Pakistan3State Key Laboratory of Biocatalysis and Enzyme Engineering, College of Life Sciences,Hubei University, Wuhan, ChinaABSTRACTSubmitted 20 May 2020Accepted 27 July 2020Published 13 August 2020Corresponding authorJamshaid Hussain,jamshaidhussain@cuiatd.edu.pkAcademic editorRenato BenesperiAdditional Information andDeclarations can be found onpage 21DOI 10.7717/peerj.9749Copyright2020 Afsar et al.Distributed underCreative Commons CC-BY 4.0Background: Salinity is one of the most lethal abiotic stresses which affect multipleaspects of plant physiology. Natural variations in plant germplasm are a greatresource that could be exploited for improvement in salt tolerance. Eruca sativa(E. sativa) exhibits tolerance to abiotic stresses. However, thorough evaluation of itssalt stress tolerance and screening for traits that could be reliably applied for salttolerance needs to be studied. The current study was designed to characterize 25E. sativa accessions, originating from diverse geographical regions of Pakistan, for thesalt stress tolerance.Methods: Salt stress (150 mM NaCl) was applied for 2 weeks to the plants at fourleaf stage in hydroponics. Data of the following morpho-physiological traits werecollected from control and treated plants of all the accessions: root length (RL), shootlength (SL), plant height (PH), leaf number (LN), leaf area (LA), fresh weight (FW),dry weight (DW), chlorophyl content (SPAD), electrolyte leakage (EL), relativewater content (RWC), gas exchange parameters and mineral ion content. Salttolerance was determined based on membership function value (MFV) of the testedtraits.Results: Compared with control, the salt-stressed group had significantly reducedmean SL, RL, PH, LN, LA, FW, DW and SPAD. NaCl treatment triggered a slightincrease in EL in few accessions. Mean RWC of control and treated groups were notsignificantly different although few accessions exhibited variation in this trait.Salt stress caused a significant reduction in photosynthesis rate (PR), transpirationrate (TR) and stomatal conductance (SC) but intercellular CO2 (Ci) was notsignificantly different between control and treated groups. Compared with control,the salt-stressed plants accumulated significantly higher Na , K and Ca2 whilesignificantly lower Mg2 . K /Na ratio was significantly decreased in salt-stressedplants compared with control. Importantly, significant inter-accession variationswere found for all the tested traits. The principal component analysis identifiedSL, RL, PH, LN, LA, FW, DW and PR as the most significant traits for resolvinginter-accession variability. Based on MFV of the tested traits, accessions werecategorized into five standard groups. Among 25 accessions, one accession wasHow to cite this article Afsar S, Bibi G, Ahmad R, Bilal M, Naqvi TA, Baig A, Shah MM, Huang B, Hussain J. 2020. Evaluation of salttolerance in Eruca sativa accessions based on morpho-physiological traits. PeerJ 8:e9749 DOI 10.7717/peerj.9749
ranked as highly tolerant, four as tolerant while 15 accessions were ranked asmoderately tolerant. Of the remaining five accessions, four were ranked as sensitivewhile one accession as highly sensitive.Conclusion: E. sativa accessions were found to exhibit significant genetic diversity inall the tested traits. A few most significant traits for dissecting the genetic variabilitywere identified that could be used for future large-scale germplasm screening inE. sativa. Salt tolerant accessions could be a good resource for future breedingprograms aiming to improve salt stress tolerance.Subjects Agricultural Science, Biodiversity, Plant Science, Biosphere Interactions, EcotoxicologyKeywords Environment, Abiotic stress, Salinity, Diversity, Chlorophyl, Photosynthesis,Morpho-physiological traitsINTRODUCTIONSalt stress is one of the major abiotic stresses affecting the productivity of cultivated soils.The salt-affected areas are increasing, and the irrigated soils are more prone to damage bysalinity. Presently about 45 million hectares of irrigated land is affected by salinity.Furthermore, it is estimated that about half of the world’s arable land could becomesalinized by 2050 (Li et al., 2014; Machado & Serralheiro, 2017). For addressing challengesto the global food security, genetic engineering to create salt tolerant species is consideredas a promising strategy (Bhardwaj et al., 2010; Shokri-Gharelo & Noparvar, 2018).Salt stress is one of the most common abiotic stresses which affect multiple aspects ofplant physiology (Munns & Tester, 2008; Fageria, Stone & Santos, 2012). It disruptsnutrient ion balance, decreases stomatal conductance (SC), and negatively affects thephotosynthetic activity. Excessive salt accumulation severely impairs the membraneintegrity, water relations and pigment content of plants (Acosta-Motos et al., 2017;Munns & Tester, 2008). The plants undergo morphological alterations which include areduction in root length (RL) and shoot length (SL), plant height (PH) and leaves size andnumber (Munns & Tester, 2008). As most of the important crops are glycophytes hencetheir growth is seriously hindered by salt stress (Yang & Guo, 2018).Natural variations in plant germplasm are a great resource that could be exploitedfor improvement in salt tolerance without affecting valuable agronomic traits. Geneticvariations could be exploited by plant biologists to identify the physiological mechanisms,sets of genes and proteins involved in stress tolerance. Subsequently, these genes can beincorporated in suitable plant species to yield salt stress tolerant varieties (Gupta & Huang,2014; Ismail & Horie, 2017; Singh, Singh & Sharma, 2018). Moreover, a reliable andextensive phenotypic evaluation of germplasm is important for the identification oftolerance-associated traits (Ismail & Horie, 2017).Several factors are responsible for the lack of success in developing salt tolerantgenotypes. These include low efficiency of morphological, biochemical and physiologicalparameters being used as screening criteria, limited availability of genetic diversity forbreeding programs, and the lack of efficient evaluation methods for identifying saltAfsar et al. (2020), PeerJ, DOI 10.7717/peerj.97492/26
tolerant genotypes through multivariable screening criteria (Zeng, Shannon & Grieve,2002; Oyiga et al., 2016).Eruca sativa (also known as rocket or arugula, and locally as taramira) is a diploidherbaceous plant from Brassicaceae family. It is an important industrial crop that cangrow in poor fertility lands and diverse climatic conditions. E. sativa exhibits tolerance toabiotic stresses like salt, drought, and temperature stress (Ashraf, 1994; Shannon &Grieve, 1999; Garg & Sharma, 2014). The seed oil of E. sativa possesses antimicrobial,anti-cancerous and antioxidant properties (Khoobchandani et al., 2010; Azarenko,Jordan & Wilson, 2014). Furthermore, due to high erucic acid content, E. sativa isconsidered as a potential source for industrial oil (Pignone & Gomez-Campo, 2011).E. sativa exhibits significant genetic diversity and a broader market range. There ishuge potential for germplasm improvement; however, limited work on this crop has beencarried out so far (Slater, 2013). The genetic variability in E. sativa is a valuable resourcethat could be exploited in order to screen for high salt tolerance. However, a thoroughevaluation of its salt stress tolerance or screening for traits that could be reliably applied forsalt tolerance needs to be carried out.Keeping in mind the above mentioned research gaps, major objectives of the currentresearch were as follows: (1) to understand the effect of salt stress on growth anddevelopment of E. sativa accessions, (2) to determine the extent of variability in salinitytolerance among E. sativa accessions, (3) to identify morpho-physiological traits with amaximum contribution towards variability and (4) to identify the salt tolerant and saltsensitive accessions for further studies and future breeding programs.MATERIALS AND METHODSPlant material, growth conditions and salt stress treatmentTwenty five E. sativa accessions were used in this study. The seeds of these accessions werekindly provided by Bio-Resources Conservation Institute, National Agricultural ResearchCentre, Islamabad, Pakistan. The list of accessions along with other details is given inTable S1. The seeds were surface sterilized and sown in a thick moist sheet of foam whichwas then placed in 72-cell seed starter trays in a growth chamber under controlledconditions of 16 h/8h day night (350 µmol/ m2/s2) at 23 C and 60% relative humidity.After one week of growth, uniform sized seedlings were transferred to the Hoagland-typesolution (Hoagland & Arnon, 1938) in 4-liter plastic containers. The set up was placed in agrowth chamber under controlled conditions (as mentioned above). The solution waschanged every 2–3 days. The experiment was carried out in a completely randomizeddesign (CRD). Plants were divided into two groups; one as control (0 mM NaCl) and otheras treated (150 mM NaCl). At the four-leaf stage, the salt stress was applied for 2 weeks.Before the determination of gas exchange parameters plants were kept in full sunlightfor 2 h (9.00 a.m.–11.00 a.m.).Determination of plant growth and development traitsData from control and treated plants was collected after 14 days of growth. Morphologicaltraits like, SL, RL, PH and leaf attributes i.e., leaf number (LN) and leaf area (LA) wereAfsar et al. (2020), PeerJ, DOI 10.7717/peerj.97493/26
recorded. The experiment was performed in six replicates for each parameter. SL, RL andPH were measured from digital images by using image analysis software Digimizer.The LA was manually measured using a grid paper. Fresh weight (FW) of control andtreated plants was determined immediately after harvesting by using a digital lab scale.For determination of dry weight (DW), whole plants (root shoot) were dried in anincubator at 75 C for 72 h, and then weight was determined by using a digital lab scale.Plant biomass assay was carried out in three replicates.Electrolyte leakageElectrolyte leakage (EL) was determined following the previously described method(Yildirim, Karlidag & Turan, 2009). Briefly, ten equal size leaf discs (10 mm diameter)from fully expanded leaves of control and treated plants were prepared and washedwith deionized water to remove electrolytes adhered to the surface. These were thenincubated at 10 C for 24 h in glass tubes filled with 10 ml of deionized water and the firstelectrical conductivity reading (EC1) was recorded. The tubes were heated at 95 C in awater bath for 20 min to release all the electrolytes. After cooling at room temperature,final electrical conductivity reading (EC2) was recorded. The EC readings were determinedby using a portable conductometer (HI-98129 Pocket EC/TDS and pH Tester).The experiment was performed in three replicates. Following equation was used for thecalculation of EL: EL ¼ EC1 100EC2Determination of chlorophyl contentRelative chlorophyl content was measured by using Chlorophyll Meter (CCM-200plus;Opti-Sciences, Hudson, NH, USA). Fully expanded fourth leaf from all the plants (controland treated) was selected for the reading. The data are presented in the form of chlorophylcontent Index (CCI). All the measurements were recorded in six replicates.Relative water contentRelative Water Content (RWC) was determined from the data of fresh, dry and turgidweight of control and treated plants, as described previously (Loutfy et al., 2012).The experiment was performed in three replicates. RWC was calculated by using theformula:RWCð%Þ ¼FW DW 100TW DWwhere FW, stands for fresh weight TW for turgid weight and DW for dry weight.Mineral ion contentFor the determination of mineral ions, 100 mg of dried whole plant samples were placedin a furnace for 5 h at 520 C for ash formation. It was then mixed with the nitricacid-perchloric acid mixture (5:1) and the final volume was raised to 15 ml with distilledAfsar et al. (2020), PeerJ, DOI 10.7717/peerj.97494/26
water. The filtrate was used to determine the concentrations of Na , K , Ca2 and Mg2 by atomic absorption spectrometry. For standard curves, different concentrations of Na ,K , Ca2 and Mg2 were prepared by diluting stock solutions of CaSO4, KCl, NaCland MgSO4. The standard curve was used to determine the content of each element andthe values were expressed in mg/g DW. The experiment was performed in three replicates.Leaf gas exchange parametersThe photosynthesis rate (PR), intercellular CO2 concentration (Ci), transpiration rate(TR), and stomatal conductance (SC) were measured with a portable gas exchange systemiFL (ADC BioScientific Ltd., Hoddesdon, UK). All the measurements were conducted on asunny day with full light intensity (11.00 a.m.–4.00 p.m.). Young fully expanded leaves(third and fourth) were used in situ for recording the above mentioned gas exchangeparameters. All the measurements were recorded in four replicates. The followingconditions were applied for the assay: Leaf surface diameter 6 cm, ambient atmosphericCO2 concentration (Cref) 352 mmol mol 1, PAR (Qleaf): 1,200 mmol/m2s and wide-rangeof chamber water vapor pressure 4.4–6.6 mbar. Three plants from each accession andtreatment were analyzed for leaf gas exchange parameters.Salt tolerance evaluationThe data of all morpho-physiological parameters (control and treatment) for eachaccession was converted to the salt-tolerance index (STI) which is the ratio of the value forthe NaCl-treated plant/value for the control. For the categorization of E. sativa accessionsaccording to their salt tolerance, membership function value (MFV) was applied aspreviously described (Chen et al., 2012). The MFV was calculated according to thefollowing formula:Xp ¼X Xmin 100%Xmax XminFor the traits inversely related to salt tolerance (e.g., EL), following formula for MFVcalculation was used:Xp ¼ 1 ðX Xmin Þ 100%ðXmax Xmin Þwhere Xp is the MFV value of the salt stress parameter “P” in a specific accession, X is theactual value of salt tolerance parameter while Xmin and Xmax represent the minimumand maximum MFV values, respectively, for that parameter in all accessions. A singleMFV value (Xc) was obtained for each accession by taking the mean of MFV values of alltested morpho-physiological traits. E. sativa accessions were divided into five standardgroups according to the average MFV value (Xa) and S.D. The accession was considered ashighly tolerant if Xc Xa 1.64 S.D., tolerant if the Xa 1 SD Xc Xa 1.64 S.D.,moderately tolerant if Xa 1 S.D. Xc Xa 1 S.D., sensitive if Xa 1.64 S.D. Xc Xa 1S.D., and highly sensitive if Xc Xa 1.64 S.D.Afsar et al. (2020), PeerJ, DOI 10.7717/peerj.97495/26
Statistical analysisDifferences among accessions and treatments were considered statistically significant atp 0.05 by Duncan’s multiple-range test performed using IBM SPSS Statistics forWindows, V.20 (IBM Corporation, Armonk, NY, USA). Principal component analysis(PCA), cluster analysis, and correlation matrix analyses were performed on MFV values ofstudied salt tolerance traits using STATISTICA Statsoft (version 10).RESULTSMorphological traits and leaf attributesShoot length was significantly reduced in the salt-stressed plants compared with thecontrol (p 0.05). The mean SL values for the control and treatments groups were 2.30 0.56 and 1.10 0.48 inches, respectively (Table 1). To determine the salt stress responseof accessions based on shoot growth, SL of each accession was expressed as stressindex, as described in the material and methods section. Accessions showing the higherstress index were considered more salt tolerant and vise versa. SL stress index of accessionsvaried from 16.74 to 88.96. Es-1 and Es-15 exhibited the highest and the lowest SL stressindex, respectively (Fig. 1A).Salt stress significantly inhibited RL as compared to control (p 0.05). Mean RL valuesin control and treated groups were 2.17 0.42 and 0.98 0.43 inches, respectively(Table 1). RL stress index ranged from 15.88 to 73.47. The highest RL stress index wasexhibited by Es-12 while the lowest by Es-18 (Fig. 1B).Plant height was also significantly reduced in salt-stressed plants compared with control(p 0.05). Mean PH values for control and treated groups were 4.43 0.77 and 2.18 0.76inches, respectively (Table 1). PH stress index varied from 19.80 to 73.98; accessionsEs-1 and Es-18 showed the highest and the lowest stress index, respectively (Fig. 1C).Leaf number and LA were also significantly reduced in salt-stressed plants comparedto control (p 0.05). Mean LN for control and treatment groups were 4.22 0.78 and2.43 0.82, respectively (Table 1). LN stress index varied from 28 to 94.12; Es-19 exhibitedthe highest stress index while Es-17 the lowest (Fig. 1D). Mean LA was also significantlyless in the treated group compared with control (p 0.05) (Table 1). Mean LA forcontrol and treatment groups was 0.07 0.025 and 0.03 0.016 inch2, respectively(Table 1). LA stress index varied from 7.6 to100; Es-25 and Es-3 showed the highest andthe lowest stress index, respectively (Fig. 1E). To summarize, these data show that saltstress significantly inhibited morphological traits in all the accessions. Moreover, the extentof inhibition of these traits, by salt stress, was variable in E. sativa accessions.Plant biomassSalt stress significantly reduced the FW and DW of E. sativa plants (p 0.05). MeanFW values in control and treatment groups were 241 164 mg and 96 66 mg,respectively (Table 2). FW stress index ranged from 10.2 to 83.92 (Fig. 2A). Cultivar Es-9showed the highest FW stress index while Es-3 the lowest (Fig. 2). Mean DW values forcontrol and treated groups were 10.6 6.9 and 6.29 3.9 mg, respectively (Table 2).Afsar et al. (2020), PeerJ, DOI 10.7717/peerj.97496/26
Table 1 Morphological traits determined in Eruca sativa accessions grown at 0 mM NaCl and 150 mM NaCl.AccessionsSL (inch)ControlRL de0.45BaEs-230.
tolerance among E. sativa accessions, (3) to identify morpho-physiological traits with a maximum contribution towards variability and (4) to identify the salt tolerant and salt sensitive accessions for further studies and future breeding programs. MATERIALS AND METHODS Plant material, growth conditions and salt stress treatment
Literature on Tolerance Design The relationship between the functional requirements and entities of the mechanical part can be derived and expressed as F 1 ¼ fðE 1;E 2;.;E nÞ. Tolerance design consists of tolerance analysis and tolerance synthesis. In tolerance analysis, the goal is to ensure the tolerance of functional require-
Van Oosten et al. Chem. Biol. Technol. Agric. Page 3 of 12 Table 1 Summary of species, biostimulant, and stress effect Type of BE Crop Stress and effect Reference A. brasilense T. aestivum Drought tolerance [16, 17] A. brasilense C. arietinum Salt tolerance [18] A. brasilense V. faba Salt tolerance [18] A. brasilense L. sativa Salt tolerance [19, 20]
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plant Stress tolerance of transgenic plants References AtCBF1/2/3 Brassica napus Constitutive overexpression enhanced both basal and acquired freezing tolerance (22) AtCBF1 Tomato Constitutive overexpression enhanced oxidative stress tolerance under chilling stress; enhanced tolerance to water-deficit stress (23, 24) AtDREB1A/ CBF3
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