Comparative Salt Tolerance Study of Some Acacia Species at Seed Germination Stage
Khalil Cherifi, El Houssein Boufous, Hassan Boubaker, Fouad Msanda
OPEN ACCESS
Asian Journal of Plant Sciences
ISSN 1682-3974DOI: 10.3923/ajps.2016.66.74
Research ArticleComparative Salt Tolerance Study of Some
Acacia
Species at SeedGermination Stage Chérifi Khalil, Boufous El Houssein, Boubaker Hassan and Msanda Fouad Laboratory of Biotechnology and Valorization of Natural Resources, Faculty of sciences, Ibn Zohr University, P.O. Box 8106, 8000 Agadir,Morocco Department of Biochemistry and Microbiology, Laval University, Quebec City (Quebec), Canada
Abstract
Objective:
The purpose of this study was to assess and compare the seed germination response of six Acacia species under differentNaCl concentrations in order to explore opportunities for selection and breeding salt tolerant genotypes.
Methodology:
Germinationof seeds was evaluated under salt stresses using 5 treatment levels: 0, 100, 200, 300 and 400 mM of NaCl. Corrected germination rate (GC),germination rate index (GRI) and mean germination time (MGT) were recorded during 10 days.
Results:
The results indicated thatgermination was significantly reduced in all species with the increase in NaCl concentrations. However, significant interspecific variationfor salt tolerance was observed. The greatest variability in tolerance was observed at moderate salt stress (200 mM of NaCl) and thedecrease in germination appeared to be more accentuated in A. cyanophylla and A. cyclops. Although, A. raddiana, remains the mostinteresting, it preserved the highest percentage (GC = 80%) and velocity of germination in all species studied in this study, even in thehigh salt levels. This species exhibited a particular adaptability to salt environment, at least at this stage in the life cycle and could berecommended for plantation establishment in salt affected areas. On the other hand, when ungerminated seeds were transferred fromNaCl treatments to distilled water, they recovered largely their germination without a lag period and with high speed. This indicated thatthe germination inhibition was related to a reversible osmotic stress that induced dormancy rather than specific ion toxicity.
Conclusion:
This ability to germinate after exposure to higher concentrations of NaCl suggests that studied species, especially the mosttolerant could be able to germinate under the salt affected soils and could be utilized for the rehabilitation of damaged arid zones.
Key words: Acacia species, osmotic stress, salt tolerance, germination recovery, glycophyte, halophyte, seed germination, variability, plant breeding,rehabilitation, arid zones, salt areasReceived: June 12, 2016 Accepted: August 02, 2016 Published: September 15, 2016Citation: Chérifi Khalil, Boufous El Houssein, Boubaker Hassan and Msanda Fouad, 2016. Comparative salt tolerance study of some Acacia species at seedgermination stage. Asian J. Plant Sci., 15: 66-74.Corresponding Author: Chérifi Khalil, Laboratory of Biotechnology and Valorization of Natural Resources, Faculty of Sciences of Agadir,Ibn Zohr University, BP 8106, 8000 Agadir, MoroccoCopyright: © 2016 Chérifi Khalil et al. This is an open access article distributed under the terms of the creative commons attribution License, which permitsunrestricted use, distribution and reproduction in any medium, provided the original author and source are credited. Competing Interest: The authors have declared that no competing interest exists.Data Availability: All relevant data are within the paper and its supporting information files. sian J. Plant Sci., 15 (3-4): 66-74, 2016
INTRODUCTION
Salinity of soils is one of the most environmental factorslimiting agricultural production and has significant effects oncrop productivity and biodiversity. It has more severe impactin arid and semi-arid environments and combined with thewater constraint presents a serious threat to food stability inthese areas . Indeed, salinization already has affected morethan 800 million hectares throughout the world or 6% of totalland . In the North Africa and the Middle East, salinity affects15 million hectares of arable lands and this area is incontinuous progression . In Morocco, more than 5% of areasare already affected by salinity to various degrees . Theycorrespond especially to arid and semi-arid regions where80% of available water for irrigation contains salinity higheror equal to 5 g L G . These wide geographical area are notexploited to a great extent, except when they occasionallyconstitute a poor pasture land or irrigated domain with lowyield.The best economic approach for exploitation andrehabilitation of these marginal regions is selection of salttolerant species and varieties capable of sustaining areasonable yield within salt-affected soils . Theeffectiveness of such approach depends on the availability ofgenetic variation in relation with salt tolerance and itsexploitation by screening and selection of the powerful plantsunder saline stress . For many crop species, intraspecificvariability for salt tolerance have been identified amongcultivars and wild species . These rustic resources are welladapted and constitute a potential reservoir useful to provideinteresting materials in order to diversify and increase theproductivity, particularly in pasture land affected by salinity.Acacia species have the ability to survive in a diverserange of habitat and environments. They are well adapted tothe arid and semi-arid regions and are known for themtolerance to high pH and salinity as well . These species areable to stabilize and fertilize soils via nitrogen fixing andmycorrhizal symbioses and constitute sources of wood,fodder, medicine and gum . Acacia trees may thus includeideal candidates for enabling saline land reclamation with apotential for financial returns because of their combinedproduction and soil improvement .In Morocco, the genus Acacia is represented by fourspontaneous species including one Moroccan endemic(Acacia gummifera Wild.); the other three are Acaciaraddiana Savi, Acacia ehrenbergiana Hayne., Acacia albidaDel. Otherwise, about 10 species are introduced forornamentation, reforestation or the fight againstdesertification . Successful establishment of plants often depends ongermination success, especially those that grow in saltaffected areas. Thus, seeds must remain viable for longperiod in high salinity conditions and germinate when salinitydecreases . Various halophytic or glycophytic species, showan important variability with their ability to germinate underdifferent salt concentration . The effects on germinationdepend on the concentration of NaCl and varied among theplant species . To overcome salt stress effect, plants haveevolved various mechanisms that help them to adapt theosmotic and ionic stress caused by high salinity . Otherwise,salts can affect germination of seeds either by restricting thesupply of water (osmotic effect) or by causing specific injurythrough ions to the metabolic machinery (ionic effect) .The purpose of this study was to assess and compare theseed germination response of 6 Acacia species (Acaciagummifera, Acacia raddiana, Acacia eburnea, Acaciacyanophylla, Acacia cyclops and Acacia horrida) underdifferent NaCl concentrations in order to explore opportunitiesfor selection and breeding salt tolerant genotypes that can beutilized in future land reclamation projects. The study will alsoassess to determine whether salt stress is induced by osmoticconstraints or by toxic effect of NaCl. MATERIALS AND METHODSPlant material:
Six Acacia species were studied: Towspontaneous species (Acacia gummifera and Acaciaraddiana) and four introduced species (Acacia eburnea,Acacia cyanophylla, Acacia cyclops and Acacia horrida).Mature, dry seeds were collected from trees growing underfield conditions in semi-arid region of South Moroccan region.Seeds collected from pods were generously provided by theRegional Forest Seeds Station of Marrakech and stored in acold chamber at 6 E C.Acacia gummifera is an endemic species to Morocco andAcacia tortilis (Forssk.) Hayne subsp., raddiana (Savi) Brenan,commonly named A. raddiana, considered as a keystonespecies is more prevalent in the inland part of the ecoregionand are widely distributed throughout the Sahara desert .The other four species were introduced in Morocco fromSouth-western Australian in the 18th century. Theseplantations were created for several purposes such as their useas ornamental plant, in the fight against desertification and fordune stabilization . Germination:
Seeds from different pods were manuallyscarified, to overcome hard seed coat dormancy and sterilizedwith 0.5% sodium hypochlorite solution (NaOCl) for 10 min,67sian J. Plant Sci., 15 (3-4): 66-74, 2016then rinsed with sterile distilled water several times and brieflyblotted on filter paper. Three replicates of 20 seeds from eachaccession were placed in plastic petri dishes (90 mm diameter)on filter paper wetted with distilled water (control) and foursalinity concentrations (100, 200, 300 and 400 mM NaCl).Petri dishes were randomized in a precision incubator andmaintained in the dark at 25±0.5 E C. Seeds were consideredto have germinated when their radicle reached at least 3 mmlong. Germination response was recorded daily for 10 days.Several germination parameters were calculated tocharacterize the salt tolerance, including the correctedgermination rate (GC), germination rate index (GRI) and meangermination time (MGT): C Corrected germination rate (GC) was expressed as thenumber of seeds germinated in a concentration of saltdivided by the number of germinated seeds in distilledwater (control) for 10 days C The germination rate index (GRI) was calculated by usingthe following formula:
G1 G2 GxGRI = + +...+1 2 x where, G is the germination percentage at each day aftersowing and 1, 2, … and x is the corresponding day ofgermination. The value of GRI was higher when seedsgerminated earlier. This parameter described by Weng andHsu and Mirzamasoumzadeh et al. is a measure ofseedling vigor and should involve not only germination butemergence characteristics.The Mean Germination Time (MGT) is a measure of therate and time-spread of germination (lower values indicatingfaster germination). It was estimated as: ni-tiMGT = N where, t is time from the beginning of the germination test interms of days and n is the number of newly germinated seedsat time t .To test germination recovery performance after saltexposure, ungerminated seeds in severe salt stress (300 and400 mM of NaCl) were transferred to distilled water andincubated for 6 days. The recovery germination percentagewas calculated by dividing the number of germinated seedsin recovery test by the number of seeds transferred to distilledwater . Statistical analysis:
All values expressed as a percentagewere arcsine square root transformed before performingstatistical analysis to normalize the data and improvehomogeneity of variance . These measures were submittedto a two ways analysis of variance (ANOVA) with species andsalinity treatments as factors followed by a Student-Newman-Keuls post hoc test. A difference was considered to bestatistically significant when p<0.05. All statistical analysiswere performed with Statistica software Version 6.1 forWindows . RESULTS AND DISCUSSIONEffect of salinity on seed germination:
For the 6 species, thetwo-way ANOVA revealed highly significant main effect ofboth species and salinity regarding final germinationpercentage and germination rate index (p<0.001) (Table 1, 2).However the existence of a significant interaction betweenthese two effects (F = 34.00 at (GC) and F = 6.385 at (GRI))indicated that species studied did not similarly respond to theeffect of salt at a given concentration of NaCl.For each concentration of NaCl, a significant interspecificvariation in both potential of seed germination (Fig. 1) andseedling vigor (Fig. 2) among the 6 examined species wasobserved. Mean comparison at different salinity levelsindicated that increase of salinity causes a decrease in seedgermination capacity, which was higher in distilled water thanin any NaCl concentration. Moreover, all the species showedan increase in Mean Germination Time (MGT), indicating thatseeds germinated more slowly as salinity increased (Table 3).At the lowest stress (100 mM), the decrease ingermination seemed to be more accentuated inA. cyanophylla (GC never fell above 30%). However, all the
Table 1: Two way analysis of variance (ANOVA) for final germination percentage(GC) of the 6 species under different concentrations of NaClSource of variation df MC FSpecies (Sp.) 5 0.618 270.110**Salinity (Salt.) 4 5.113 2233.421**Sp.×Salt 20 0.077 34.000**Error 60 0.002df: Degree of freedom, MC: Mean square, F: Ratio of variances, **Significantat 1% probability levelTable 2: Two way analysis of variance (ANOVA) for germination rate index (GRI)of the 6 species under different concentrations of NaClSource of variation df MC FSpecies (Sp.) 5 0.570 283.194**Salinity (Salt.) 4 4.149 2064.370**Sp.×Salt 20 0.040 20.010**Error 60 0.002df: Degree of freedom, MC: Mean square, F: Ratio of variances, **Significantat 1% probability level C o rr ec t e d g e r m i n a ti on r a t e ( % ) Species
A. cyanophyllaA. cyclopsA. eburneaA. gummifera
A. horridaA. raddiana
Fig. 1: Corrected germination rate of different species invarious NaCl concentrations, at each concentration ofNaCl, values in the same ellipse are not significantlydifferent (p<0.05) (Newman-Keuls test)
Table 3: Mean Germination Time (MGT) of different species in various NaClconcentrationsNaCl concentrations (mM)-----------------------------------------------------------------------------Species 0 100 200 300 400A. cyanophylla 2.48 a a b NC NCA. cyclops 2.58 a a b c NCA. eburnea 3.20 a a a b c A. gummifera 3.05 a a a c c A. horrida 3.03 a ab b c NCA. raddiana 2.66 a a a b c Values followed by the same letters in a row are not significantly different atp<0.05, (Newman-Keuls test), NC: MGT was not calculated because of insufficientgermination other species are distinguished by forming a homogeneousgroup which is little affected by salinity compared with control(GC = 84% on average). At this saline level, seeds germinatedrapidly and no significant change was noticed in germinationspeed (MGT not differing from that of control) (Table 3).At 200 mM, the results revealed considerable interspecificvariation in the response of seed germination to salinityamong the studied species. Thus, four groups weredistinguished, the first was formed by A. raddiana whichshowed very high germination percentage (GC = 80%) andrevealed earlier seed germination MGT = 3.29, followed byA. eburnea (GC and GRI close respectively to 67 and 51%),while A. gummifera and A. horrida occupied anintermediate position between the tolerant species and therest of the others, considered as the most sensitive (GC = 15%in the case of A. cyanophylla and 19% for A. cyclops).Otherwise, from this level, seed germination was relativelyslower in A. cyanophylla and A. cyclops (high MGT).The elevated doses of salt (300 and 400 mM) inducedsignificant reduction in seed germination and retarded their initiation of all the species. The A. cyanophylla, A. horrida and A. cyclops appeared to be sharply affected in the samemanner by these two concentrations, their correctedgermination percentage does not exceed 6% and the time togermination gradually lengthened (MGT >6 days) at 300 mM.Moreover, no germination took place in this group at 400 mM.Seeds of A. gummifera and A. eburnea could be regardedas moderately tolerant to salt stress and reacted in thesame way at these two concentrations (GC and GRI closerespectively to 32 and 12% in the case of 300 mM). In this casegermination was significantly delayed at the tow highest NaCllevels.Whereas, A. raddiana continued to record highestpercentage and velocity of germination in all species studiedin this study, even in the high salt levels with GC reached 55%at 300 mM and 41% at 400 mM of NaCl. These speciesexhibited a particular adaptability to salt environment, at leastat this stage in the life cycle and could be recommended forplantation establishment in salt affected areas.The effect of salinity on germination has been addressedby several researchers and in different species . Thisstage is very important for the development of the plants,particularly those that live in environments affected by salinity.In this study, the monitoring of germination process revealedthat salinity was notably affected germination in Acaciaspecies but also delayed the time needed to completegermination, especially with increasing salinity level. This isconsistent with present result particularly for sensitive speciesand is explained by the time required for the seeds to developmechanisms allowing to adjust them internal osmoticpressure .Moreover, the results showed significant interspecificvariation in salt tolerance during germination of the speciesstudied in the range of concentrations of sodium chloridefrom 100-400 mM. This variability, required to start a breedingprograms for salt tolerance, have been also observed inseveral species including halophyte or glycophytespecies .The Moroccan Acacia raddiana particularly, toleratedsalinity until 400 mM (probably also at higher levels) with agermination rate that exceeded 40%. Indeed, according toDanthu et al. , Acacia raddiana is among the African Acaciaspecies whose germination is less affected by the presence ofsalt. Its seed germination could be blocked only up to saltconcentrations close to seawater (35 g L G ). Previous studieshave also reported that this species were the most tolerantand could be used in increasing forage production in saltaffected areas . Furthermore, the observed germinationpercentages in the current study were relatively well higherthan those published by Jaouadi et al. in Tunisian species69sian J. Plant Sci., 15 (3-4): 66-74, 2016 G e r m i n a ti on r a t e i nd e x ( % ) a aa a a a a a a a a a a bbb bb abab a b d e ccccc d A . cy anoph y ll a A . cyc l op s A . e bu r n e a A . gu m m if e r a A . ho rr i da A . r add i ana NaCl0 mM100 mM200 mM300 mM400 mMSpecies
10 11 12 13 141009080706050403020100 Species
A. cyanophyllaA. cyclopsA. eburneaA. gummifera
A. horridaA. raddiana R ec ov e r y g e r m i n a ti on ( % ) Fig. 2: Germination rate index of different species in various NaCl concentrations, at each concentration of NaCl, means ofpopulations having the same letter are not significantly different (p<0.05) (Newman-Keuls test)Fig. 3: Reversible effect of high NaCl concentration on thegermination kinetics in the studied species(maximum germination rate of 21% under 22 g L G of NaCl)and by Abari et al. in Iranian taxa (germination was stoppedat 300 mM). El Nour et al. reported that no germinationtook place in four Yemeni Acacia (A. cyanophylla, A. seyal,A. tortilis and A. tumida) at salt concentration above20 dS m G (around 200 mM of NaCl).Among the rest of the 5 Acacia species examined in thepresent study, A. gummifera and A. eburnea was shownto possess the medium level of salt tolerance, whereasA. cyanophylla, A. horrida and A. cyclops were categorizedas the least tolerant group. Several studies that havecompared salt tolerance in some Acacia species havesuggested relatively the same ranking .Moreover, previous studies have reported that salttolerance was positively correlated with seed size . The A. raddiana, A. gummifera and A. eburnea seeds arelarger than those of A. cyanophylla, A. horrida and A. cyclops and according to Croser et al. , larger seeds maycontain more food reserves, which could be used to overcomeosmotic effects of salts and greater energy reserves makingthem less dependent on photosynthesis for early growth.Similar observation which made on Triticum aestivum onAtriplex species as well as on Acacia longifolia , which alsoshowed that larger seeds had greater success in overcomingosmotic constraints during the initial stages of germination. Recovery of germination:
After transfer of the seeds thatfailed to germinate under high concentrations of salt todistilled water, they recovered relatively their aptitude ofgermination at all the species studied (Fig. 3). The delay ingermination speed tended to be relatively rapid than thatobserved in distilled water.This, revealed that NaCl had no toxic effect, because saltstress did not damage the embryo as verified by not only thegermination recovery but also pink embryos in the tetrazoliumstaining test. The NaCl concentrations did not destroy seedgermination ability, it had only repressed the germinationmomentarily and the viability was preserved. These results areconsistent with those obtained in several other salt tolerantspecies including Atriplex halimus , Medicago ruthenica ,Medicago polymorpha and Medicago ciliaris , Panicumturgidum , Triticum aestivum , Brassica napus and Lolium perenne and Bromus tomentellus .An important characteristic of salt tolerant seeds, whichdifferentiate them from seeds of glycophytes is their aptitude70sian J. Plant Sci., 15 (3-4): 66-74, 2016to maintain seed viability for lengthy period of time duringexposure to hyper-saline conditions and then initiategermination when salinity stress is decreased . Thepresent study revealed that the recovery of germination is nota criterion of salt tolerance which distinguishes halophytesfrom glycophytes. It was maybe due to a reversible osmoticeffect that induced dormancy, as revealed in findings of Khanand Ungar and Tilaki et al. . Consequently, a highproportion of seeds remained viable and had the ability togerminate when salinity stress was alleviated .Reduction in mechanisms of germination by osmoticstress may be related to the lower diffusion of waterthrough the seed coat caused by the increased osmoticpressure environment, preventing the seed imbibition and mobilization of reserves for embryoʼs growth . Thus,dormancy decrease the risk of seedling mortality whenmoisture is limited and salinity is augmented . Highrecovery germination speed observed in the studied species,particularly in A. raddiana, indicated that seeds have theability to avoid deterioration caused by prolonged exposureto unfavorable biotic factors . This situation constitutes anecophysiological adaptive strategy to take full advantages offavorable conditions, available for a short time, during thegermination stage . It also, secure the long-term subsistenceof seed bank helping the species in dispersal germination andseedling establishment over years . Tilaki et al. reportedthat under saline environments, seed survival may be asuitable condition for success instead of germination capacity,since recovery germination does occur in the seeds whenhyper-saline conditions are alleviated. CONCLUSION
In the light of the above result, it is concluded that: C Sodium chloride caused a reversible osmotic effect ofgermination rather than ion specific toxicity and exerts atemporary inhibition of germination which is eliminatedwith the removal of the constraint C The ability to germinate after exposure to higherconcentrations of NaCl suggests that Acacia species,especially the most tolerant, could be able to germinateunder the salt affected soils and could be utilized for therehabilitation of damaged arid zones C The interspecific diversity in salt tolerance duringgermination is useful to start a breeding programs for salttolerance C The native A. raddiana species remained the mosttolerant and conserve its aptitude to germinate until400 mM, probably also at higher levels, consequently itwas behaved as a halophyte plant C More ambitious programs, including Acacia species arenecessary, not only at germination but also at the otherstages of the life cycle. This opens the possibility tocontinue this study to verify correlation between salttolerance during seed germination and early stage ofplant development which will be most useful in abreeding programs for selecting salt-tolerant in Acaciaspecies
ACKNOWLEDGEMENT
We express gratitude to the Regional Forest Seeds Station(R.F.S.S.M) of Marrakech (Morocco) for their information andguidance during prospecting and pods harvesting.
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