Research ArticleA Novel Method to Overcome Coat-Imposed Seed Dormancy inLupinus albus L and Trifolium pratense L

Iskender Tiryaki1 and Mustafa Topu2

1 Department of Agricultural Biotechnology Faculty of Agriculture Canakkale Onsekiz Mart UniversityTerzioglu Campus 17020 Canakkale Turkey

2 Kahramanmaras Anadolu Ogretmen Lisesi 46100 Kahramanmaras Turkey

Correspondence should be addressed to Iskender Tiryaki itiryakicomuedutr

Received 7 May 2014 Revised 18 July 2014 Accepted 19 July 2014 Published 3 August 2014

Academic Editor Bhagirath S Chauhan

Copyright copy 2014 I Tiryaki and M Topu This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

We have developed a novel method to overcome coat-imposed seed dormancy in legume plants Seeds of Lupinus albus L andTrifolium pratense L were stored in a freezer at minus80∘C for a period of time and then immediately treated with or without hot waterat 90∘C for 5 seconds Germination tests were carried out in darkness at 20plusmn10∘Cwith four replications in a completely randomizeddesign Final germination percentage (FGP) germination rate and synchrony of seeds were evaluatedThe results showed that newapproach of freeze-thaw scarification provided high percentage of germinations in white lupin (8416) and red clover (7450)seeds while control seeds had FGPs of 33 and 260 respectively The immediate thawing of frozen seeds in hot water for 5seconds was found not only an effective and reliable but also the quickest seed treatment method to prevail against coat-imposedseed dormancy in legume species and may become operationally applicable to other plant species

1 Introduction

Dormant seeds which are unable to germinate underfavourable environmental conditions remain hard and unger-minated for a period of time [1] However the mechanisms ofseed dormancy are still to be elucidated and therefore theclassification of the different types of dormancy is entirelybased on its expression under various conditions [1ndash4]The coat-imposed seed dormancy (hardseededness) is dueto either the impermeability of the coat to water andorgases the mechanical prevention of radicle extension orthe seed coat preventing inhibitory substances from leavingthe embryo or by supplying inhibitors to the embryo [5 6]Water impermeable testa prevents the entry of water into seedand seed remains hard even when appropriate moisture andtemperature conditions are provided [5] The presence of ahard and impermeable seed coat is regarded as a widespreadcause of seed dormancy in several important legume speciessuch as in lentil [7] faba bean [8] common bean [9] soybean[10 11] cowpea [12] common vetch [13] alfalfa and clover[14ndash16] and in some other important plant families such as

Geraniaceae (Pelargonium sp) [17] Oleaceae (Fraxinus sp)[18] andMalvaceae (Abelmoschus sp) [19]

Although hard seed coat improves the survival of seeds inthe soil especially in adverse environmental conditions andhelps to avoid extinction of species in nature it may preventuse of plant cultivars or wild-type relatives for agricultural orbreeding purposes [20 21]There are however several factorsor events that reduce or partially overcome coat-imposedseed dormancy in which the seed coat is usually disruptedby usingmechanical or chemical applications such as nickingor sandpaper acid scarification or heat treatments such asboiling water soak [13 22 23] The objective of the presentstudy was to develop an alternative and effective as well asreliable seed treatment method to defeat coat-imposed seeddormancy in Fabaceae

2 Material and Methods

21 Material Lupinus albus L (white lupin) and Trifoliumpratense L (red clover) seeds were used in this studyWhite lupin seeds were collected from natural flora of Aksu

Hindawi Publishing CorporationJournal of BotanyVolume 2014 Article ID 647469 6 pageshttpdxdoiorg1011552014647469

2 Journal of Botany

(37∘3010158403310158401015840N 36∘5310158404310158401015840E) Kahramanmaras Turkey and redclover seeds collected from natural flora of Ordu Turkeywere generously provided by Dr Asci et al [16] White lupinand red clover seeds were 5 and 26 months old respectivelySeed moisture contents of white lupin and red clover seedswere determined as 111 and 107 respectively basedon dried sample mass by using moisture analyzer (MX-50AampD Company Limited Tokyo Japan) at 200∘C as per themanufacturerrsquos instructions

22 Methods

221 Seed Treatments White lupin seeds were stored in afreezer at minus80∘C for 0 1 2 4 or 7 days in plastic zip lackbags and then immediately treated with or without hot water(HW) at 90∘C for 5 seconds Based on the results of lupinexperiment redclover seeds were stored in a freezer at minus80∘Cfor 0 or 1 day and then were immediately treated with orwithout HW at 90∘C for 5 seconds

222 Germination Test Germination tests were carried outin darkness in a temperature-controlled incubator held at20 plusmn 10∘C Seeds were placed on two layers of filter papermoistened with 3mL of deionized water in covered 55 cmglass petri dishes Four replications of 40 and 50 seeds werearranged in a completely randomized design for lupin andred clover seeds respectively Shortage of lupin seeds forcedus to use 40 seeds per replication rather than 50 seeds as weused in red clover To be able to calculate rate and spreadof germination the seeds germinated (radicle visible) wereremoved from petri dishes daily until the numbers stabilized(for 7 days) From the total number of seeds germinated finalgermination percentage (FGP) and its angular transforma-tion (arcsine radicFGP) days to 50 of FGP and days between10 and 90 of FGP were calculated [24] Time to 50 ofFGP (G

50) is an inverse measure of germination rate while

time between 10 and 90 of FGP (G10ndash90) is considered to

be an estimate of the spread of germination the inverse ofgermination synchrony

223 Data Analysis Data were subjected to analysis of vari-ance using SAS statistical software [25] and mean separationwas performed by Fisherrsquos least significant difference (LSD)test if 119865 test was significant at 119875 lt 005

3 Results

The preexperimental trials including boiling water acidscarification storage of seeds in a deep freezer at minus20∘C andat minus80∘C and immersing seeds in liquid nitrogen indicatedthat white lupin seeds stored in deep freezer at minus80∘C ortreatedwithHWhadpromising results compared to the othermethods tested (data not shown) Therefore we have set anew experiment to evaluate storage time of seeds in deepfreezer at minus80∘C for 0 1 2 4 or 7 days in plastic zip lack bagsand treatment of those seeds with or without HW at 90∘C for5 seconds

0102030405060708090

100

Ger

min

atio

n (

)

Treatments

F1on

ly

F2on

ly

F4on

ly

F7on

ly

HW

onl

y

UT

LSD005

F1+

HW

F2+

HW

F4+

HW

F7+

HW

Figure 1 The effects of various treatments on final germinationpercentage of Lupinus albus L seed germination HW hot waterF1ndash7 storage of seed in deep freezer for 1ndash7 day(s) UT untreatedcontrol seed Error bars indicate standard deviation (119899 = 4)

Table 1 Angular transformation [degree] of final germinationpercentage (FGP) days to 50 of FGP (G

50

) and days between 10and 90 germination (G

10ndash90) of Lupinus albus L seed germinationat 20∘C following various treatments

Treatments FGP G50

G10ndash90

[Degree] (Days) (Days)F1 only [1983] 504 338F2 only [2036] 531 236F4 only [2409] 400 382F7 only [2184] 400 403F1 + HW [6685] 358 231F2 + HW [6531] 368 329F4 + HW [6280] 346 341F7 + HW [7018] 344 269HW only [2529] 310 282UT [882] 150 105LSD005 [729] 090 134Significance lowastlowast lowastlowast lowastlowast

lowastlowastSignificant at 119875 lt 001 HW hot water F1ndash7 storage of seeds in deepfreezer for 1ndash7 day(s) UT untreated control seed

The FGPs of this experiment were presented in Figure 1Speed and span of germination and angular transformation ofFGP were given in Table 1 The results revealed that all seedtreatments tested significantly improved FGP of white lupincompared to untreated (UT) control seeds The results alsoindicated that storage of seeds in deep freezer for any giventime or seeds treated with HW only had significant effecton germination parameters in comparison to UT controlseeds which had an FGP of 333 Significant differenceswere also determined between storage of seeds in deepfreezer and treatment of seeds with HW only Seeds storedin deep freezer only had lower FGPs than seeds treated with

Journal of Botany 3

Table 2 Angular transformation [degree] of final germinationpercentage (FGP) days to 50 of FGP (G

50

) and days between10 and 90 germination (G

10ndash90) of Trifolium pratense L seedgermination at 20∘C following various treatments

Treatments FGP G50

G10ndash90

[Degree] (Days) (Days)F1 + HW [5972] 161 244F1 only [3772] 251 370HW only [3410] 163 308UT [3050] 194 255LSD005 [516] 058 150Significance lowastlowast lowast nslowastlowastlowastSignificant at 119875 lt 005 and 119875 lt 001 respectively ns not significant at119875 lt 005 HW hot water F1 storage of seeds in deep freezer for 1 day UTuntreated control seed

HW per se (Table 1) Although not significantly differentextending the storage time in deep freezer led to a progressiveincrease in FGP (Figure 1) The highest FGPs (8833 and8416) were however obtained from seeds treatedwithHWfollowing storage of seeds in deep freezer for 1 or 7 daysrespectively Germination rate and synchrony (higher G

50

and G10ndash90) were worsened when seeds were stored in deep

freezer only (F1ndash7) compared to UT control seeds and F1ndash7+ HW combination (Table 1) Seeds treated with HW onlyhad faster (G

50= 31 days) germination rate than seeds of the

other treatments while the lowest rate of germination (G50

= 15 days) was obtained from UT control seeds Speed ofgerminationwas the slowest (G

50= 504 days) for seeds stored

in deep freezer for 1 day only (Table 1)Seeds stored in deep freezer for 7 days resulted in the least

synchronous germination (G10ndash90 = 403 days) while themost

uniform germination (G10ndash90 = 105 days) was obtained from

UT control seeds (Table 1) The F1 + HW treatment had thelowest number of days for germination synchrony (G

10ndash90 =231 days) compared to the other treatments tested (Table 1)Considering storage time and its effect on germinationparameters immediate immersing of seeds in HW at 90∘Cfor 5 seconds following storage in a deep freezer at minus80∘Cfor one day (F1 + HW) was not only an effective but also thequickest seed treatment method to defeat coat-imposed seeddormancy in white lupin compared to the other treatmentsThe effectiveness of F1 + HW treatment was further tested onred clover seeds

The treatment of F1 + HW on red clover seeds signif-icantly enhanced FGP and rate of germination while nobeneficial effect was determined on span of germinationcompared to UT control seeds (Table 2 and Figure 1) Thehighest FGP (745) was obtained from seeds treated withF1 + HW method compared to UT control seeds whichhad an FGP of 26 (Figure 2) The results also indicatedthat storage of red clover seeds in deep freezer at minus80∘Cfor 1 day (F1 only) was more effective in defeating coat-imposed seed dormancy (375) than treatment of seedswith HW only (315) (Figure 2) All the seed treatmentsworsened germination rate and synchrony (higher G

50and

G10ndash90) except F1 + HW (G

50= 161 days) which resulted

0102030405060708090

TreatmentsUT F1 HW

Ger

min

atio

n (

)

LSD005

F1+ HW

Figure 2 The effects of various treatments on final germinationpercentage of Trifolium pratense L seed germination HW hotwater F1 storage of seed in deep freezer for 1 day UT untreatedcontrol seed Error bars indicate standard deviation (119899 = 4)

in slight decrease on germination rate compared to the restof the treatments tested including UT control seeds (G

50=

194 days) however this improvement was not significant(Table 2) The treatments showed no significant effect on thegermination synchrony (Table 2)

4 Discussion

Fabaceae comprise one of the most important agriculturaltaxa worldwide providing a major source of protein formankind as well as animals [14 26ndash29] However mostspecies of these genus seeds present coat-imposed seeddormancy [2 30 31] which may cause a seed lot to germinateovermonths or years Although genetic factors determine theproportion of seed coat hardnesses [10 31] there are severalother factors such as the geographical location [32 33] theearliness of the seeds [11 15] the ecological differences inrelation to temperature and relative humidity [32 34ndash36] andseed storage conditions [37 38]

Previous reports indicated that genotypes and cultivarsof Lupinus and Trifolium species have similar seed coatdormancy [22 39 40] Studies on Lupinus species showedthat mechanical or chemical scarification is needed to obtainuniform and rapid germination and hot water or boilingtreatments can improve germination [22] Although scarify-ing seeds of L varius in sulfuric acid were found as alternativeto mechanical scarification [22] boiling and scarifying inconcentrated sulfuric acid did not provide sufficient ger-mination [41] suggesting that mechanical scarification wasthe most adequate treatment to obtain uniform and rapidgermination

It was previously reported that ultralow temperature(minus196∘C) exposure in liquid nitrogen enhanced final germi-nation and germination speed in seeds of Medicago orbic-ularis while it did not improve germination parameters ofAstragalus hamosus seeds [42] In addition to mechanicalor chemical scarification storage of Persian clover seeds

4 Journal of Botany

in deep freezer at minus5∘C for 15 days significantly improvedthe germination ratios of seeds in different colours [43]Asci et al [16] also reported that mechanical scarificationwas the most effective method to remove the hardness ofred clover seeds relative to other methods tested includingprecooling preheating hot water and potassium nitrate[16] The importance of the mechanical scarifying techniquehowever lies not just in the possibility that it improves thegerminability of seed but also in how much it can acceleratethe process and how practically it can be applied In additionmechanical scarification may damage other parts of the seedincluding the embryo and may reduce seed viability dueto the pathogenic and saprophytic organisms to gain entryinto the seed and thus reduce the shelf life of the seedsalthough intention of this technique is to provide a minordamage on seed coat for water permeability [44 45] Acidscarification on the other hand is commonly not preferreddue to its cost safety risk and environmental precautionsinvolved and not reliable or lacking the requisite qualities onseeds of other important plant species [43 44 46]Thereforesuch precautions have great importance whether or not thetechnique becomes operationally applicable in large seed lotsof various plant species In agreement with previous reports[46] treatment of white lupin or red clover seeds with HWper se significantly improved FGPs even though the beneficialeffects of these treatments were limited in comparison to F1+ HW method (Figures 1 and 2) The effectiveness of F1 +HW method can also be judged by the high percentage ofgerminations resulting in white lupin (8416) and red clover(7450) seeds while UT control seeds had FGPs of 33 and260 respectively In addition germination improvement ofred clover seed with F1 +HWmethod was found to be higherthan that ofmechanical scarification reported previously [16]

Freeze-thaw scarification method has been known andbeen used to improve germination of hard seeds in severallegume species for a long time [47ndash51] Midgley [47] reportedthat germination of alfalfa seeds was not improved whenthe seeds were subjected to freeze-thaw scarification withcooling temperatures at minus5∘C or at minus15∘C for 36 h and withwarming at room temperatures for six days Similarly freeze-thaw scarification of milk vetch seeds at minus22∘C for 2 4 730 60 90 or 180 days did not improve seed germinationmore than 78 [50] Both Stout [48] andBusse [49] suggestedthat repeated freeze-thaw cycles were needed to improvegermination of alfalfa seeds although Rutar et al [51] foundno influence on germination of alfalfa seeds when they usedthe same seed treatments as the study of Stout [48] Freeze-thaw scarification using liquid nitrogen was also previouslytested and showed no effect on hard seeds of several legumespecies including alfalfa red clover soybean common vetch[52] and white lupin Taken together the results of thisstudy revealed that thawing temperatures or conditions ina freeze-thaw scarification technique would be more criticalthan freezing temperatures and immediate or rapid thawinglike in hot water would be more beneficial to remove coat-imposed seed dormancy in legume plants

It has been suggested that cracks in the seed coat pro-duced by heat treatment result from seed coat expansionand contraction due to the increase of room temperature

to minus80∘C (hot water) [23 53] while cracks in acid-scarifiedseeds may be attributed to chemical erosion The F1 + HWmethod provides cracks on seed coat and results may beattributed to seed coat expansion or contraction due to theheat shocked on testa of frozen seed Depending on thespecies seed coat thickness and the size of the seeds usedit is even possible to hear a hatch sound when the seedswere immersed in HW following storage in a deep freezerAs indicated before storage of white lupin seeds at minus20∘C fora period of time or immersing the seeds in liquid nitrogenwas not sufficient to meet a need for practical applicationsuggesting that combining freezing temperature with HW atgiven conditions provided a surface stress on seed coat whichcan easily be broken and made seeds permeable to waterPrevious reports indicated that lens or hilum sides of the testaare the physically weakest part and thus can more easily bebroken by treatments [6 23 53] We speculate that the F1 +HW method may also provide cracks on lens or hilum sidesof the testa on white lupin and red clover seeds

In conclusion we developed a novel and reliable combi-nation of freeze-thaw scarification method (F1 + HW) whichis apparently the quickest and simplest method available forreleasing coat-imposed seed dormancy of white lupin and redclover seeds and has a great potential in other plant species inoperational applications if the coat-imposed seed dormancyis due to either the impermeability of the coat to water or themechanical prevention of radicle extension

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors would like to thank Dr Adem Erol for his help incollecting L albus seeds and Dr Ozlem Onal Asci for kindlyproviding T pratense seeds

References

[1] M Koornneef L Bentsink and H Hilhorst ldquoSeed dormancyand germinationrdquo Current Opinion in Plant Biology vol 5 no1 pp 33ndash36 2002

[2] J M Baskin and C C Baskin ldquoA classification system for seeddormancyrdquo Seed Science Research vol 14 no 1 pp 1ndash16 2004

[3] R FinkelsteinWReeves TAriizumi andC Steber ldquoMolecularaspects of seed dormancyrdquo Annual Review of Plant Biology vol59 pp 387ndash415 2008

[4] E Copete J M Herranz P Ferrandis C C Baskin and J MBaskin ldquoPhysiology morphology and phenology of seed dor-mancy break and germination in the endemic Iberian speciesNarcissus hispanicus (Amaryllidaceae)rdquo Annals of Botany vol107 no 6 pp 1003ndash1016 2011

[5] K M Kelly J van Staden and W E Bell ldquoSeed coat structureand dormancyrdquo Plant Growth Regulation vol 11 no 3 pp 201ndash209 1992

Journal of Botany 5

[6] JM Baskin C C Baskin andX J Li ldquoTaxonomy anatomy andevolution of physical dormancy in seedsrdquo Plant Species Biologyvol 15 no 2 pp 139ndash152 2000

[7] G Ladizinsky ldquoThe genetics of hard seed coat in the genusLensrdquo Euphytica vol 34 no 2 pp 539ndash543 1985

[8] H Weber L Borisjuk and U Wobus ldquoControlling seeddevelopment and seed size in Vicia faba a role for seed coat-associated invertases and carbohydrate staterdquo Plant Journal vol10 no 5 pp 823ndash834 1996

[9] P M Erdmann R K Lee M J Bassett and P E McClean ldquoAmolecular marker tightly linked to P a gene required for flowerand seedcoat color in common bean (Phaseolus vulgaris L)contains the Ty3-gypsy retrotransposon Tpv3grdquo Genome vol45 no 4 pp 728ndash736 2002

[10] C Tinius ldquoVariable expression of seed coat permeabilityrdquoSoybean Genetics Newsletter vol 18 pp 256ndash259 1991

[11] K Ranathunge S Shao D Qutob M Gijzen C A PetersonandMA Bernards ldquoProperties of the soybean seed coat cuticlechange during developmentrdquo Planta vol 231 no 5 pp 1171ndash1188 2010

[12] W M Lush and L T Evans ldquoThe seed coats of cowpeas andother grain legumes structure in relation to functionrdquo FieldCrops Research vol 3 pp 267ndash286 1980

[13] H N Buyukkartal H Colgecen N M Pinar and N ErdoganldquoSeed coat ultrastructure of hard-seeded and soft-seeded vari-eties of Vicia sativardquo Turkish Journal of Botany vol 37 no 2 pp270ndash275 2013

[14] E Can N Celiktas R Hatipoglu and S Avci ldquoBreaking seeddormancy of some annual Medicago and Trifolium species bydifferent treatmentsrdquo Turkish Journal of Field Crops vol 14 no2 pp 72ndash78 2009

[15] F R Hay R D Smith R H Ellis and L H Butler ldquoDevel-opmental changes in the germinability desiccation tolerancehardseededness and longevity of individual seeds of Trifoliumambiguumrdquo Annals of Botany vol 105 no 6 pp 1035ndash10522010

[16] O O Asci Z Acar I Ayan U Basaran and H Mut ldquoEffectof pretreatments on seed germination rate of red clover (Tri-folium pratense L) populationsrdquoAfrican Journal of AgriculturalResearch vol 6 no 13 pp 3055ndash3060 2011

[17] A Meisert D Schulz and H Lehmann ldquoStructural featuresunderlying hardseededness in geraniaceaerdquo Plant Biology vol1 no 3 pp 311ndash314 1999

[18] I A Takos and G S P Efthimiou ldquoGermination resultson dormant seeds of fifteen tree species autumn sown in aNorthern Greek nurseryrdquo Silvae Genetica vol 52 no 2 pp 67ndash71 2003

[19] M M A El Balla A I Saidahmed and M Makkawi ldquoEffectof moisture content and maturity on hardseededness andgermination in okra (Abelmoschus esculentus L Moench)rdquoInternational Journal of Plant Physiology and Biochemistry vol3 pp 102ndash107 2011

[20] J D Bewley ldquoSeed germination and dormancyrdquo Plant Cell vol9 no 7 pp 1055ndash1066 1997

[21] R M Nair A D Craig T D Rowe S R Biggins and CH Hunt ldquoGenetic variability and heritability estimates forhardseededness and flowering in balansa clover (Trifoliummichelianum Savi) populationsrdquo Euphytica vol 138 no 3 pp197ndash203 2004

[22] O Karaguzel S Cakmakci V Ortacesme and B AydinogluldquoInfluence of seed coat treatments on germination and early

seedling growth of Lupinus varius Lrdquo Pakistan Journal ofBotany vol 36 no 1 pp 65ndash74 2004

[23] X W Hu Y R Wang Y P Wu and C C Baskin ldquoRole ofthe lens in controlling water uptake in seeds of two Fabaceae(Papilionoideae) species treated with sulphuric acid and hotwaterrdquo Seed Science Research vol 19 no 2 pp 73ndash80 2009

[24] S R Wiley R J Kraus F Zuo E E Murray K Loritz and J EMertz ldquoSV40 early-to-late switch involves titration of cellulartranscriptional repressorsrdquo Genes and Development vol 7 no11 pp 2206ndash2219 1993

[25] SAS I SASSTAT Software Changes and Enhancements throughRelease 612 SAS Inst Cary NC USA 1997

[26] M J Messina ldquoLegumes and soybeans Overview of theirnutritional profiles and health effectsrdquoThe American Journal ofClinical Nutrition vol 70 no 3 1999

[27] M Carbonaro G Grant MMattera A Aguzzi and A PusztaildquoInvestigation of the mechanisms affecting Cu and Fe bioavail-ability from legumes role of seed protein and antinutritional(nonprotein) factorsrdquoBiological Trace Element Research vol 84no 1-3 pp 181ndash196 2001

[28] D M Roth and B R Paulicks ldquoContents of nutrients andfeed value of blue and yellow lupins (Lupinus angustifoliusL Lupinus luteus L) for pigsrdquo Proceedings of the Society ofNutrition Physiology vol 11 p 137 2002

[29] L W Bell M H Ryan R G Bennett M T Collins andH J Clarke ldquoGrowth yield and seed composition of nativeAustralian legumes with potential as grain cropsrdquo Journal of theScience of Food and Agriculture vol 92 no 7 pp 1354ndash13612012

[30] C C Baskin and J M Baskin ldquoEcology and evolution of spe-cialized seed dispersal dormancy and germination strategiesrdquoPlant Species Biology vol 15 no 2 pp 95ndash96 2000

[31] J A Van Assche and F E A Vandelook ldquoCombinationaldormancy in winter annual Fabaceaerdquo Seed Science Researchvol 20 no 4 pp 237ndash242 2010

[32] P J Argel and L R Humphreys ldquoEnvironmental effects onseed development and hardseededness in Stylosanthes hamatacv Verano I Temperaturerdquo Australian Journal of AgriculturalResearch vol 34 no 3 pp 261ndash270 1983

[33] F H Dubbern De Souza and J Marhos-Filho ldquoThe seed coatas a modulator of seed-environment relationships in FabeceaerdquoRevista Brasileira de Botanica vol 24 pp 365ndash375 2001

[34] J J Mott S J Cook and R J Williams ldquoInfluence of shortduration high temperature seed treatment on the germinationof some tropical and temperate legumesrdquo Tropical Grasslandsvol 16 pp 50ndash55 1982

[35] H C Norman P S Cocks and NW Galwey ldquoHardseedednessin annual clovers variation between populations from wet anddry environmentsrdquo Australian Journal of Agricultural Researchvol 53 no 7 pp 821ndash829 2002

[36] A Balkaya ldquoModelling the effect of temperature on the germi-nation speed in some legume cropsrdquo Journal of Agricultural vol3 pp 179ndash183 2004

[37] R L Benech-Arnold R A Sanchez F Forcella B C Kruk andC M Ghersa ldquoEnvironmental control of dormancy in weedseed banks in soilrdquo Field Crops Research vol 67 no 2 pp 105ndash122 2000

[38] K M G Gehan Jayasuriya J M Baskin C C Baskin andM T R Fernando ldquoVariation in seed dormancy and storagebehavior of three liana species of derris (Fabaceae Faboideae)in Sri Lanka and ecological implicationsrdquo Research Journal ofSeed Science vol 5 no 1 pp 1ndash18 2012

6 Journal of Botany

[39] G S Valenti LMeloneM Ferro andA Bozzini ldquoComparativestudies on testa structure of ldquohard-seededrdquo and ldquosoft-seededrdquovarieties of Lupinus angustifolius L (Leguminosae) and onmechanism of water entryrdquo Seed Science and Technology vol 17pp 563ndash581 1989

[40] T D Davis S W George A Upadhyaya and J ParsonsldquoImprovement of seedling emergence of Lupinus taxensisHookfollowing seed scarification treatmentsrdquo Journal of Environmen-tal Horticulture vol 9 pp 17ndash21 1991

[41] O Karaguzel I Baktir S Cakmakci V Ortacesme BAydinoglu and M Atik ldquoEffects of scarification methods tem-perature and sowing date on some germination characteristicsof Lupinus varius Lrdquo inProceedings of the 2ndNational Congresson Ornamental Plants pp 40ndash47 Citrus and GreenhouseResearch Institute Antalya Turkey October 2002

[42] C Patane and F Gresta ldquoGermination of Astragalus hamosusand Medicago orbicularis as affected by seed-coat dormancybreaking techniquesrdquo Journal of Arid Environments vol 67 no1 pp 165ndash173 2006

[43] E Ates ldquoInfluence of some hardseededness-breaking treat-ments on germination in Persian clover (trifolium resupinatumssp typicum Fiori et Paol) seedsrdquo Romanian AgriculturalResearch no 28 pp 229ndash236 2011

[44] DS Loch and GL Harvey ldquoComparison of methods for reduc-ing hard seed levels in three subtropical legumesrdquo inProceedingsof the 4th Australian Seeds Research Conference pp 243ndash246Broadbeach Australia 1992

[45] S N Acharya D G Stout B Brooke and D ThompsonldquoCultivar and storage effects on germination and hard seedcontent of alfalfardquo Canadian Journal of Plant Science vol 79no 2 pp 201ndash208 1999

[46] A Alderete-Chavez L Aguilar-Marın N de la cruz-Landeroet al ldquoEffects of scarification chemical treatments on thegermination of Crotalaria retusa L seedsrdquo Journal of BiologicalSciences vol 10 no 6 pp 541ndash544 2010

[47] A R Midgley ldquoEffect of alternate freezing and thawing onthe impermeability of alfalfa and dodder seedsrdquo Journal of theAmerican Society of Agronomy vol 18 pp 1087ndash1098 1926

[48] D G Stout ldquoEffect of freeze-thaw cycles on hard-seededness ofalfalfardquo Journal of Seed Science and Technology vol 14 pp 47ndash55 1990

[49] W F Busse ldquoEffect of low temperatures on germination ofimpermeable seedsrdquo Botanical Gazette vol 89 pp 169ndash1791930

[50] T Shibata and Y Hatakeyama ldquoBreaking of dormancy in theseeds ofAstragalus mongholicus Bunge (Leguminosae)rdquo Journalof Plant Physiology vol 146 no 3 pp 366ndash368 1995

[51] R Rutar M Stjepanovic S Popovic Z Bukvic and D PacekldquoEffect of temperature on germination and hard alfalfa seedrdquoCIHEAM vol 2 pp 137ndash139 2001

[52] P C Stanwood ldquoTolerance of crop seeds to cooling and storagein liquid nitrogen (-196∘C)rdquo Journal of Seed Technology vol 5no 1 pp 26ndash31 1980

[53] L W Zeng P S Cocks S G Kailis and J Kuo ldquoTherole of fractures and lipids in the seed coat in the loss ofhardseededness of six Mediterranean legume speciesrdquo Journalof Agricultural Science vol 143 no 1 pp 43ndash55 2005

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Microbiology

2 Journal of Botany

(37∘3010158403310158401015840N 36∘5310158404310158401015840E) Kahramanmaras Turkey and redclover seeds collected from natural flora of Ordu Turkeywere generously provided by Dr Asci et al [16] White lupinand red clover seeds were 5 and 26 months old respectivelySeed moisture contents of white lupin and red clover seedswere determined as 111 and 107 respectively basedon dried sample mass by using moisture analyzer (MX-50AampD Company Limited Tokyo Japan) at 200∘C as per themanufacturerrsquos instructions

22 Methods

221 Seed Treatments White lupin seeds were stored in afreezer at minus80∘C for 0 1 2 4 or 7 days in plastic zip lackbags and then immediately treated with or without hot water(HW) at 90∘C for 5 seconds Based on the results of lupinexperiment redclover seeds were stored in a freezer at minus80∘Cfor 0 or 1 day and then were immediately treated with orwithout HW at 90∘C for 5 seconds

222 Germination Test Germination tests were carried outin darkness in a temperature-controlled incubator held at20 plusmn 10∘C Seeds were placed on two layers of filter papermoistened with 3mL of deionized water in covered 55 cmglass petri dishes Four replications of 40 and 50 seeds werearranged in a completely randomized design for lupin andred clover seeds respectively Shortage of lupin seeds forcedus to use 40 seeds per replication rather than 50 seeds as weused in red clover To be able to calculate rate and spreadof germination the seeds germinated (radicle visible) wereremoved from petri dishes daily until the numbers stabilized(for 7 days) From the total number of seeds germinated finalgermination percentage (FGP) and its angular transforma-tion (arcsine radicFGP) days to 50 of FGP and days between10 and 90 of FGP were calculated [24] Time to 50 ofFGP (G

50) is an inverse measure of germination rate while

time between 10 and 90 of FGP (G10ndash90) is considered to

be an estimate of the spread of germination the inverse ofgermination synchrony

223 Data Analysis Data were subjected to analysis of vari-ance using SAS statistical software [25] and mean separationwas performed by Fisherrsquos least significant difference (LSD)test if 119865 test was significant at 119875 lt 005

3 Results

The preexperimental trials including boiling water acidscarification storage of seeds in a deep freezer at minus20∘C andat minus80∘C and immersing seeds in liquid nitrogen indicatedthat white lupin seeds stored in deep freezer at minus80∘C ortreatedwithHWhadpromising results compared to the othermethods tested (data not shown) Therefore we have set anew experiment to evaluate storage time of seeds in deepfreezer at minus80∘C for 0 1 2 4 or 7 days in plastic zip lack bagsand treatment of those seeds with or without HW at 90∘C for5 seconds

0102030405060708090

100

Ger

min

atio

n (

)

Treatments

F1on

ly

F2on

ly

F4on

ly

F7on

ly

HW

onl

y

UT

LSD005

F1+

HW

F2+

HW

F4+

HW

F7+

HW

Figure 1 The effects of various treatments on final germinationpercentage of Lupinus albus L seed germination HW hot waterF1ndash7 storage of seed in deep freezer for 1ndash7 day(s) UT untreatedcontrol seed Error bars indicate standard deviation (119899 = 4)

Table 1 Angular transformation [degree] of final germinationpercentage (FGP) days to 50 of FGP (G

50

) and days between 10and 90 germination (G

10ndash90) of Lupinus albus L seed germinationat 20∘C following various treatments

Treatments FGP G50

G10ndash90

[Degree] (Days) (Days)F1 only [1983] 504 338F2 only [2036] 531 236F4 only [2409] 400 382F7 only [2184] 400 403F1 + HW [6685] 358 231F2 + HW [6531] 368 329F4 + HW [6280] 346 341F7 + HW [7018] 344 269HW only [2529] 310 282UT [882] 150 105LSD005 [729] 090 134Significance lowastlowast lowastlowast lowastlowast

lowastlowastSignificant at 119875 lt 001 HW hot water F1ndash7 storage of seeds in deepfreezer for 1ndash7 day(s) UT untreated control seed

The FGPs of this experiment were presented in Figure 1Speed and span of germination and angular transformation ofFGP were given in Table 1 The results revealed that all seedtreatments tested significantly improved FGP of white lupincompared to untreated (UT) control seeds The results alsoindicated that storage of seeds in deep freezer for any giventime or seeds treated with HW only had significant effecton germination parameters in comparison to UT controlseeds which had an FGP of 333 Significant differenceswere also determined between storage of seeds in deepfreezer and treatment of seeds with HW only Seeds storedin deep freezer only had lower FGPs than seeds treated with

Journal of Botany 3

Table 2 Angular transformation [degree] of final germinationpercentage (FGP) days to 50 of FGP (G

50

) and days between10 and 90 germination (G

10ndash90) of Trifolium pratense L seedgermination at 20∘C following various treatments

Treatments FGP G50

G10ndash90

[Degree] (Days) (Days)F1 + HW [5972] 161 244F1 only [3772] 251 370HW only [3410] 163 308UT [3050] 194 255LSD005 [516] 058 150Significance lowastlowast lowast nslowastlowastlowastSignificant at 119875 lt 005 and 119875 lt 001 respectively ns not significant at119875 lt 005 HW hot water F1 storage of seeds in deep freezer for 1 day UTuntreated control seed

HW per se (Table 1) Although not significantly differentextending the storage time in deep freezer led to a progressiveincrease in FGP (Figure 1) The highest FGPs (8833 and8416) were however obtained from seeds treatedwithHWfollowing storage of seeds in deep freezer for 1 or 7 daysrespectively Germination rate and synchrony (higher G

50

and G10ndash90) were worsened when seeds were stored in deep

freezer only (F1ndash7) compared to UT control seeds and F1ndash7+ HW combination (Table 1) Seeds treated with HW onlyhad faster (G

50= 31 days) germination rate than seeds of the

other treatments while the lowest rate of germination (G50

= 15 days) was obtained from UT control seeds Speed ofgerminationwas the slowest (G

50= 504 days) for seeds stored

in deep freezer for 1 day only (Table 1)Seeds stored in deep freezer for 7 days resulted in the least

synchronous germination (G10ndash90 = 403 days) while themost

uniform germination (G10ndash90 = 105 days) was obtained from

UT control seeds (Table 1) The F1 + HW treatment had thelowest number of days for germination synchrony (G

10ndash90 =231 days) compared to the other treatments tested (Table 1)Considering storage time and its effect on germinationparameters immediate immersing of seeds in HW at 90∘Cfor 5 seconds following storage in a deep freezer at minus80∘Cfor one day (F1 + HW) was not only an effective but also thequickest seed treatment method to defeat coat-imposed seeddormancy in white lupin compared to the other treatmentsThe effectiveness of F1 + HW treatment was further tested onred clover seeds

The treatment of F1 + HW on red clover seeds signif-icantly enhanced FGP and rate of germination while nobeneficial effect was determined on span of germinationcompared to UT control seeds (Table 2 and Figure 1) Thehighest FGP (745) was obtained from seeds treated withF1 + HW method compared to UT control seeds whichhad an FGP of 26 (Figure 2) The results also indicatedthat storage of red clover seeds in deep freezer at minus80∘Cfor 1 day (F1 only) was more effective in defeating coat-imposed seed dormancy (375) than treatment of seedswith HW only (315) (Figure 2) All the seed treatmentsworsened germination rate and synchrony (higher G

50and

G10ndash90) except F1 + HW (G

50= 161 days) which resulted

0102030405060708090

TreatmentsUT F1 HW

Ger

min

atio

n (

)

LSD005

F1+ HW

Figure 2 The effects of various treatments on final germinationpercentage of Trifolium pratense L seed germination HW hotwater F1 storage of seed in deep freezer for 1 day UT untreatedcontrol seed Error bars indicate standard deviation (119899 = 4)

in slight decrease on germination rate compared to the restof the treatments tested including UT control seeds (G

50=

194 days) however this improvement was not significant(Table 2) The treatments showed no significant effect on thegermination synchrony (Table 2)

4 Discussion

Fabaceae comprise one of the most important agriculturaltaxa worldwide providing a major source of protein formankind as well as animals [14 26ndash29] However mostspecies of these genus seeds present coat-imposed seeddormancy [2 30 31] which may cause a seed lot to germinateovermonths or years Although genetic factors determine theproportion of seed coat hardnesses [10 31] there are severalother factors such as the geographical location [32 33] theearliness of the seeds [11 15] the ecological differences inrelation to temperature and relative humidity [32 34ndash36] andseed storage conditions [37 38]

Previous reports indicated that genotypes and cultivarsof Lupinus and Trifolium species have similar seed coatdormancy [22 39 40] Studies on Lupinus species showedthat mechanical or chemical scarification is needed to obtainuniform and rapid germination and hot water or boilingtreatments can improve germination [22] Although scarify-ing seeds of L varius in sulfuric acid were found as alternativeto mechanical scarification [22] boiling and scarifying inconcentrated sulfuric acid did not provide sufficient ger-mination [41] suggesting that mechanical scarification wasthe most adequate treatment to obtain uniform and rapidgermination

It was previously reported that ultralow temperature(minus196∘C) exposure in liquid nitrogen enhanced final germi-nation and germination speed in seeds of Medicago orbic-ularis while it did not improve germination parameters ofAstragalus hamosus seeds [42] In addition to mechanicalor chemical scarification storage of Persian clover seeds

4 Journal of Botany

in deep freezer at minus5∘C for 15 days significantly improvedthe germination ratios of seeds in different colours [43]Asci et al [16] also reported that mechanical scarificationwas the most effective method to remove the hardness ofred clover seeds relative to other methods tested includingprecooling preheating hot water and potassium nitrate[16] The importance of the mechanical scarifying techniquehowever lies not just in the possibility that it improves thegerminability of seed but also in how much it can acceleratethe process and how practically it can be applied In additionmechanical scarification may damage other parts of the seedincluding the embryo and may reduce seed viability dueto the pathogenic and saprophytic organisms to gain entryinto the seed and thus reduce the shelf life of the seedsalthough intention of this technique is to provide a minordamage on seed coat for water permeability [44 45] Acidscarification on the other hand is commonly not preferreddue to its cost safety risk and environmental precautionsinvolved and not reliable or lacking the requisite qualities onseeds of other important plant species [43 44 46]Thereforesuch precautions have great importance whether or not thetechnique becomes operationally applicable in large seed lotsof various plant species In agreement with previous reports[46] treatment of white lupin or red clover seeds with HWper se significantly improved FGPs even though the beneficialeffects of these treatments were limited in comparison to F1+ HW method (Figures 1 and 2) The effectiveness of F1 +HW method can also be judged by the high percentage ofgerminations resulting in white lupin (8416) and red clover(7450) seeds while UT control seeds had FGPs of 33 and260 respectively In addition germination improvement ofred clover seed with F1 +HWmethod was found to be higherthan that ofmechanical scarification reported previously [16]

Freeze-thaw scarification method has been known andbeen used to improve germination of hard seeds in severallegume species for a long time [47ndash51] Midgley [47] reportedthat germination of alfalfa seeds was not improved whenthe seeds were subjected to freeze-thaw scarification withcooling temperatures at minus5∘C or at minus15∘C for 36 h and withwarming at room temperatures for six days Similarly freeze-thaw scarification of milk vetch seeds at minus22∘C for 2 4 730 60 90 or 180 days did not improve seed germinationmore than 78 [50] Both Stout [48] andBusse [49] suggestedthat repeated freeze-thaw cycles were needed to improvegermination of alfalfa seeds although Rutar et al [51] foundno influence on germination of alfalfa seeds when they usedthe same seed treatments as the study of Stout [48] Freeze-thaw scarification using liquid nitrogen was also previouslytested and showed no effect on hard seeds of several legumespecies including alfalfa red clover soybean common vetch[52] and white lupin Taken together the results of thisstudy revealed that thawing temperatures or conditions ina freeze-thaw scarification technique would be more criticalthan freezing temperatures and immediate or rapid thawinglike in hot water would be more beneficial to remove coat-imposed seed dormancy in legume plants

It has been suggested that cracks in the seed coat pro-duced by heat treatment result from seed coat expansionand contraction due to the increase of room temperature

to minus80∘C (hot water) [23 53] while cracks in acid-scarifiedseeds may be attributed to chemical erosion The F1 + HWmethod provides cracks on seed coat and results may beattributed to seed coat expansion or contraction due to theheat shocked on testa of frozen seed Depending on thespecies seed coat thickness and the size of the seeds usedit is even possible to hear a hatch sound when the seedswere immersed in HW following storage in a deep freezerAs indicated before storage of white lupin seeds at minus20∘C fora period of time or immersing the seeds in liquid nitrogenwas not sufficient to meet a need for practical applicationsuggesting that combining freezing temperature with HW atgiven conditions provided a surface stress on seed coat whichcan easily be broken and made seeds permeable to waterPrevious reports indicated that lens or hilum sides of the testaare the physically weakest part and thus can more easily bebroken by treatments [6 23 53] We speculate that the F1 +HW method may also provide cracks on lens or hilum sidesof the testa on white lupin and red clover seeds

In conclusion we developed a novel and reliable combi-nation of freeze-thaw scarification method (F1 + HW) whichis apparently the quickest and simplest method available forreleasing coat-imposed seed dormancy of white lupin and redclover seeds and has a great potential in other plant species inoperational applications if the coat-imposed seed dormancyis due to either the impermeability of the coat to water or themechanical prevention of radicle extension

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors would like to thank Dr Adem Erol for his help incollecting L albus seeds and Dr Ozlem Onal Asci for kindlyproviding T pratense seeds

References

[1] M Koornneef L Bentsink and H Hilhorst ldquoSeed dormancyand germinationrdquo Current Opinion in Plant Biology vol 5 no1 pp 33ndash36 2002

[2] J M Baskin and C C Baskin ldquoA classification system for seeddormancyrdquo Seed Science Research vol 14 no 1 pp 1ndash16 2004

[3] R FinkelsteinWReeves TAriizumi andC Steber ldquoMolecularaspects of seed dormancyrdquo Annual Review of Plant Biology vol59 pp 387ndash415 2008

[4] E Copete J M Herranz P Ferrandis C C Baskin and J MBaskin ldquoPhysiology morphology and phenology of seed dor-mancy break and germination in the endemic Iberian speciesNarcissus hispanicus (Amaryllidaceae)rdquo Annals of Botany vol107 no 6 pp 1003ndash1016 2011

[5] K M Kelly J van Staden and W E Bell ldquoSeed coat structureand dormancyrdquo Plant Growth Regulation vol 11 no 3 pp 201ndash209 1992

Journal of Botany 5

[6] JM Baskin C C Baskin andX J Li ldquoTaxonomy anatomy andevolution of physical dormancy in seedsrdquo Plant Species Biologyvol 15 no 2 pp 139ndash152 2000

[7] G Ladizinsky ldquoThe genetics of hard seed coat in the genusLensrdquo Euphytica vol 34 no 2 pp 539ndash543 1985

[8] H Weber L Borisjuk and U Wobus ldquoControlling seeddevelopment and seed size in Vicia faba a role for seed coat-associated invertases and carbohydrate staterdquo Plant Journal vol10 no 5 pp 823ndash834 1996

[9] P M Erdmann R K Lee M J Bassett and P E McClean ldquoAmolecular marker tightly linked to P a gene required for flowerand seedcoat color in common bean (Phaseolus vulgaris L)contains the Ty3-gypsy retrotransposon Tpv3grdquo Genome vol45 no 4 pp 728ndash736 2002

[10] C Tinius ldquoVariable expression of seed coat permeabilityrdquoSoybean Genetics Newsletter vol 18 pp 256ndash259 1991

[11] K Ranathunge S Shao D Qutob M Gijzen C A PetersonandMA Bernards ldquoProperties of the soybean seed coat cuticlechange during developmentrdquo Planta vol 231 no 5 pp 1171ndash1188 2010

[12] W M Lush and L T Evans ldquoThe seed coats of cowpeas andother grain legumes structure in relation to functionrdquo FieldCrops Research vol 3 pp 267ndash286 1980

[13] H N Buyukkartal H Colgecen N M Pinar and N ErdoganldquoSeed coat ultrastructure of hard-seeded and soft-seeded vari-eties of Vicia sativardquo Turkish Journal of Botany vol 37 no 2 pp270ndash275 2013

[14] E Can N Celiktas R Hatipoglu and S Avci ldquoBreaking seeddormancy of some annual Medicago and Trifolium species bydifferent treatmentsrdquo Turkish Journal of Field Crops vol 14 no2 pp 72ndash78 2009

[15] F R Hay R D Smith R H Ellis and L H Butler ldquoDevel-opmental changes in the germinability desiccation tolerancehardseededness and longevity of individual seeds of Trifoliumambiguumrdquo Annals of Botany vol 105 no 6 pp 1035ndash10522010

[16] O O Asci Z Acar I Ayan U Basaran and H Mut ldquoEffectof pretreatments on seed germination rate of red clover (Tri-folium pratense L) populationsrdquoAfrican Journal of AgriculturalResearch vol 6 no 13 pp 3055ndash3060 2011

[17] A Meisert D Schulz and H Lehmann ldquoStructural featuresunderlying hardseededness in geraniaceaerdquo Plant Biology vol1 no 3 pp 311ndash314 1999

[18] I A Takos and G S P Efthimiou ldquoGermination resultson dormant seeds of fifteen tree species autumn sown in aNorthern Greek nurseryrdquo Silvae Genetica vol 52 no 2 pp 67ndash71 2003

[19] M M A El Balla A I Saidahmed and M Makkawi ldquoEffectof moisture content and maturity on hardseededness andgermination in okra (Abelmoschus esculentus L Moench)rdquoInternational Journal of Plant Physiology and Biochemistry vol3 pp 102ndash107 2011

[20] J D Bewley ldquoSeed germination and dormancyrdquo Plant Cell vol9 no 7 pp 1055ndash1066 1997

[21] R M Nair A D Craig T D Rowe S R Biggins and CH Hunt ldquoGenetic variability and heritability estimates forhardseededness and flowering in balansa clover (Trifoliummichelianum Savi) populationsrdquo Euphytica vol 138 no 3 pp197ndash203 2004

[22] O Karaguzel S Cakmakci V Ortacesme and B AydinogluldquoInfluence of seed coat treatments on germination and early

seedling growth of Lupinus varius Lrdquo Pakistan Journal ofBotany vol 36 no 1 pp 65ndash74 2004

[23] X W Hu Y R Wang Y P Wu and C C Baskin ldquoRole ofthe lens in controlling water uptake in seeds of two Fabaceae(Papilionoideae) species treated with sulphuric acid and hotwaterrdquo Seed Science Research vol 19 no 2 pp 73ndash80 2009

[24] S R Wiley R J Kraus F Zuo E E Murray K Loritz and J EMertz ldquoSV40 early-to-late switch involves titration of cellulartranscriptional repressorsrdquo Genes and Development vol 7 no11 pp 2206ndash2219 1993

[25] SAS I SASSTAT Software Changes and Enhancements throughRelease 612 SAS Inst Cary NC USA 1997

[26] M J Messina ldquoLegumes and soybeans Overview of theirnutritional profiles and health effectsrdquoThe American Journal ofClinical Nutrition vol 70 no 3 1999

[27] M Carbonaro G Grant MMattera A Aguzzi and A PusztaildquoInvestigation of the mechanisms affecting Cu and Fe bioavail-ability from legumes role of seed protein and antinutritional(nonprotein) factorsrdquoBiological Trace Element Research vol 84no 1-3 pp 181ndash196 2001

[28] D M Roth and B R Paulicks ldquoContents of nutrients andfeed value of blue and yellow lupins (Lupinus angustifoliusL Lupinus luteus L) for pigsrdquo Proceedings of the Society ofNutrition Physiology vol 11 p 137 2002

[29] L W Bell M H Ryan R G Bennett M T Collins andH J Clarke ldquoGrowth yield and seed composition of nativeAustralian legumes with potential as grain cropsrdquo Journal of theScience of Food and Agriculture vol 92 no 7 pp 1354ndash13612012

[30] C C Baskin and J M Baskin ldquoEcology and evolution of spe-cialized seed dispersal dormancy and germination strategiesrdquoPlant Species Biology vol 15 no 2 pp 95ndash96 2000

[31] J A Van Assche and F E A Vandelook ldquoCombinationaldormancy in winter annual Fabaceaerdquo Seed Science Researchvol 20 no 4 pp 237ndash242 2010

[32] P J Argel and L R Humphreys ldquoEnvironmental effects onseed development and hardseededness in Stylosanthes hamatacv Verano I Temperaturerdquo Australian Journal of AgriculturalResearch vol 34 no 3 pp 261ndash270 1983

[33] F H Dubbern De Souza and J Marhos-Filho ldquoThe seed coatas a modulator of seed-environment relationships in FabeceaerdquoRevista Brasileira de Botanica vol 24 pp 365ndash375 2001

[34] J J Mott S J Cook and R J Williams ldquoInfluence of shortduration high temperature seed treatment on the germinationof some tropical and temperate legumesrdquo Tropical Grasslandsvol 16 pp 50ndash55 1982

[35] H C Norman P S Cocks and NW Galwey ldquoHardseedednessin annual clovers variation between populations from wet anddry environmentsrdquo Australian Journal of Agricultural Researchvol 53 no 7 pp 821ndash829 2002

[36] A Balkaya ldquoModelling the effect of temperature on the germi-nation speed in some legume cropsrdquo Journal of Agricultural vol3 pp 179ndash183 2004

[37] R L Benech-Arnold R A Sanchez F Forcella B C Kruk andC M Ghersa ldquoEnvironmental control of dormancy in weedseed banks in soilrdquo Field Crops Research vol 67 no 2 pp 105ndash122 2000

[38] K M G Gehan Jayasuriya J M Baskin C C Baskin andM T R Fernando ldquoVariation in seed dormancy and storagebehavior of three liana species of derris (Fabaceae Faboideae)in Sri Lanka and ecological implicationsrdquo Research Journal ofSeed Science vol 5 no 1 pp 1ndash18 2012

6 Journal of Botany

[39] G S Valenti LMeloneM Ferro andA Bozzini ldquoComparativestudies on testa structure of ldquohard-seededrdquo and ldquosoft-seededrdquovarieties of Lupinus angustifolius L (Leguminosae) and onmechanism of water entryrdquo Seed Science and Technology vol 17pp 563ndash581 1989

[40] T D Davis S W George A Upadhyaya and J ParsonsldquoImprovement of seedling emergence of Lupinus taxensisHookfollowing seed scarification treatmentsrdquo Journal of Environmen-tal Horticulture vol 9 pp 17ndash21 1991

[41] O Karaguzel I Baktir S Cakmakci V Ortacesme BAydinoglu and M Atik ldquoEffects of scarification methods tem-perature and sowing date on some germination characteristicsof Lupinus varius Lrdquo inProceedings of the 2ndNational Congresson Ornamental Plants pp 40ndash47 Citrus and GreenhouseResearch Institute Antalya Turkey October 2002

[42] C Patane and F Gresta ldquoGermination of Astragalus hamosusand Medicago orbicularis as affected by seed-coat dormancybreaking techniquesrdquo Journal of Arid Environments vol 67 no1 pp 165ndash173 2006

[43] E Ates ldquoInfluence of some hardseededness-breaking treat-ments on germination in Persian clover (trifolium resupinatumssp typicum Fiori et Paol) seedsrdquo Romanian AgriculturalResearch no 28 pp 229ndash236 2011

[44] DS Loch and GL Harvey ldquoComparison of methods for reduc-ing hard seed levels in three subtropical legumesrdquo inProceedingsof the 4th Australian Seeds Research Conference pp 243ndash246Broadbeach Australia 1992

[45] S N Acharya D G Stout B Brooke and D ThompsonldquoCultivar and storage effects on germination and hard seedcontent of alfalfardquo Canadian Journal of Plant Science vol 79no 2 pp 201ndash208 1999

[46] A Alderete-Chavez L Aguilar-Marın N de la cruz-Landeroet al ldquoEffects of scarification chemical treatments on thegermination of Crotalaria retusa L seedsrdquo Journal of BiologicalSciences vol 10 no 6 pp 541ndash544 2010

[47] A R Midgley ldquoEffect of alternate freezing and thawing onthe impermeability of alfalfa and dodder seedsrdquo Journal of theAmerican Society of Agronomy vol 18 pp 1087ndash1098 1926

[48] D G Stout ldquoEffect of freeze-thaw cycles on hard-seededness ofalfalfardquo Journal of Seed Science and Technology vol 14 pp 47ndash55 1990

[49] W F Busse ldquoEffect of low temperatures on germination ofimpermeable seedsrdquo Botanical Gazette vol 89 pp 169ndash1791930

[50] T Shibata and Y Hatakeyama ldquoBreaking of dormancy in theseeds ofAstragalus mongholicus Bunge (Leguminosae)rdquo Journalof Plant Physiology vol 146 no 3 pp 366ndash368 1995

[51] R Rutar M Stjepanovic S Popovic Z Bukvic and D PacekldquoEffect of temperature on germination and hard alfalfa seedrdquoCIHEAM vol 2 pp 137ndash139 2001

[52] P C Stanwood ldquoTolerance of crop seeds to cooling and storagein liquid nitrogen (-196∘C)rdquo Journal of Seed Technology vol 5no 1 pp 26ndash31 1980

[53] L W Zeng P S Cocks S G Kailis and J Kuo ldquoTherole of fractures and lipids in the seed coat in the loss ofhardseededness of six Mediterranean legume speciesrdquo Journalof Agricultural Science vol 143 no 1 pp 43ndash55 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Signal TransductionJournal of

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BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Nucleic AcidsJournal of

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Journal of Botany 3

Table 2 Angular transformation [degree] of final germinationpercentage (FGP) days to 50 of FGP (G

50

) and days between10 and 90 germination (G

10ndash90) of Trifolium pratense L seedgermination at 20∘C following various treatments

Treatments FGP G50

G10ndash90

[Degree] (Days) (Days)F1 + HW [5972] 161 244F1 only [3772] 251 370HW only [3410] 163 308UT [3050] 194 255LSD005 [516] 058 150Significance lowastlowast lowast nslowastlowastlowastSignificant at 119875 lt 005 and 119875 lt 001 respectively ns not significant at119875 lt 005 HW hot water F1 storage of seeds in deep freezer for 1 day UTuntreated control seed

HW per se (Table 1) Although not significantly differentextending the storage time in deep freezer led to a progressiveincrease in FGP (Figure 1) The highest FGPs (8833 and8416) were however obtained from seeds treatedwithHWfollowing storage of seeds in deep freezer for 1 or 7 daysrespectively Germination rate and synchrony (higher G

50

and G10ndash90) were worsened when seeds were stored in deep

freezer only (F1ndash7) compared to UT control seeds and F1ndash7+ HW combination (Table 1) Seeds treated with HW onlyhad faster (G

50= 31 days) germination rate than seeds of the

other treatments while the lowest rate of germination (G50

= 15 days) was obtained from UT control seeds Speed ofgerminationwas the slowest (G

50= 504 days) for seeds stored

in deep freezer for 1 day only (Table 1)Seeds stored in deep freezer for 7 days resulted in the least

synchronous germination (G10ndash90 = 403 days) while themost

uniform germination (G10ndash90 = 105 days) was obtained from

UT control seeds (Table 1) The F1 + HW treatment had thelowest number of days for germination synchrony (G

10ndash90 =231 days) compared to the other treatments tested (Table 1)Considering storage time and its effect on germinationparameters immediate immersing of seeds in HW at 90∘Cfor 5 seconds following storage in a deep freezer at minus80∘Cfor one day (F1 + HW) was not only an effective but also thequickest seed treatment method to defeat coat-imposed seeddormancy in white lupin compared to the other treatmentsThe effectiveness of F1 + HW treatment was further tested onred clover seeds

The treatment of F1 + HW on red clover seeds signif-icantly enhanced FGP and rate of germination while nobeneficial effect was determined on span of germinationcompared to UT control seeds (Table 2 and Figure 1) Thehighest FGP (745) was obtained from seeds treated withF1 + HW method compared to UT control seeds whichhad an FGP of 26 (Figure 2) The results also indicatedthat storage of red clover seeds in deep freezer at minus80∘Cfor 1 day (F1 only) was more effective in defeating coat-imposed seed dormancy (375) than treatment of seedswith HW only (315) (Figure 2) All the seed treatmentsworsened germination rate and synchrony (higher G

50and

G10ndash90) except F1 + HW (G

50= 161 days) which resulted

0102030405060708090

TreatmentsUT F1 HW

Ger

min

atio

n (

)

LSD005

F1+ HW

Figure 2 The effects of various treatments on final germinationpercentage of Trifolium pratense L seed germination HW hotwater F1 storage of seed in deep freezer for 1 day UT untreatedcontrol seed Error bars indicate standard deviation (119899 = 4)

in slight decrease on germination rate compared to the restof the treatments tested including UT control seeds (G

50=

194 days) however this improvement was not significant(Table 2) The treatments showed no significant effect on thegermination synchrony (Table 2)

4 Discussion

Fabaceae comprise one of the most important agriculturaltaxa worldwide providing a major source of protein formankind as well as animals [14 26ndash29] However mostspecies of these genus seeds present coat-imposed seeddormancy [2 30 31] which may cause a seed lot to germinateovermonths or years Although genetic factors determine theproportion of seed coat hardnesses [10 31] there are severalother factors such as the geographical location [32 33] theearliness of the seeds [11 15] the ecological differences inrelation to temperature and relative humidity [32 34ndash36] andseed storage conditions [37 38]

Previous reports indicated that genotypes and cultivarsof Lupinus and Trifolium species have similar seed coatdormancy [22 39 40] Studies on Lupinus species showedthat mechanical or chemical scarification is needed to obtainuniform and rapid germination and hot water or boilingtreatments can improve germination [22] Although scarify-ing seeds of L varius in sulfuric acid were found as alternativeto mechanical scarification [22] boiling and scarifying inconcentrated sulfuric acid did not provide sufficient ger-mination [41] suggesting that mechanical scarification wasthe most adequate treatment to obtain uniform and rapidgermination

It was previously reported that ultralow temperature(minus196∘C) exposure in liquid nitrogen enhanced final germi-nation and germination speed in seeds of Medicago orbic-ularis while it did not improve germination parameters ofAstragalus hamosus seeds [42] In addition to mechanicalor chemical scarification storage of Persian clover seeds

4 Journal of Botany

in deep freezer at minus5∘C for 15 days significantly improvedthe germination ratios of seeds in different colours [43]Asci et al [16] also reported that mechanical scarificationwas the most effective method to remove the hardness ofred clover seeds relative to other methods tested includingprecooling preheating hot water and potassium nitrate[16] The importance of the mechanical scarifying techniquehowever lies not just in the possibility that it improves thegerminability of seed but also in how much it can acceleratethe process and how practically it can be applied In additionmechanical scarification may damage other parts of the seedincluding the embryo and may reduce seed viability dueto the pathogenic and saprophytic organisms to gain entryinto the seed and thus reduce the shelf life of the seedsalthough intention of this technique is to provide a minordamage on seed coat for water permeability [44 45] Acidscarification on the other hand is commonly not preferreddue to its cost safety risk and environmental precautionsinvolved and not reliable or lacking the requisite qualities onseeds of other important plant species [43 44 46]Thereforesuch precautions have great importance whether or not thetechnique becomes operationally applicable in large seed lotsof various plant species In agreement with previous reports[46] treatment of white lupin or red clover seeds with HWper se significantly improved FGPs even though the beneficialeffects of these treatments were limited in comparison to F1+ HW method (Figures 1 and 2) The effectiveness of F1 +HW method can also be judged by the high percentage ofgerminations resulting in white lupin (8416) and red clover(7450) seeds while UT control seeds had FGPs of 33 and260 respectively In addition germination improvement ofred clover seed with F1 +HWmethod was found to be higherthan that ofmechanical scarification reported previously [16]

Freeze-thaw scarification method has been known andbeen used to improve germination of hard seeds in severallegume species for a long time [47ndash51] Midgley [47] reportedthat germination of alfalfa seeds was not improved whenthe seeds were subjected to freeze-thaw scarification withcooling temperatures at minus5∘C or at minus15∘C for 36 h and withwarming at room temperatures for six days Similarly freeze-thaw scarification of milk vetch seeds at minus22∘C for 2 4 730 60 90 or 180 days did not improve seed germinationmore than 78 [50] Both Stout [48] andBusse [49] suggestedthat repeated freeze-thaw cycles were needed to improvegermination of alfalfa seeds although Rutar et al [51] foundno influence on germination of alfalfa seeds when they usedthe same seed treatments as the study of Stout [48] Freeze-thaw scarification using liquid nitrogen was also previouslytested and showed no effect on hard seeds of several legumespecies including alfalfa red clover soybean common vetch[52] and white lupin Taken together the results of thisstudy revealed that thawing temperatures or conditions ina freeze-thaw scarification technique would be more criticalthan freezing temperatures and immediate or rapid thawinglike in hot water would be more beneficial to remove coat-imposed seed dormancy in legume plants

It has been suggested that cracks in the seed coat pro-duced by heat treatment result from seed coat expansionand contraction due to the increase of room temperature

to minus80∘C (hot water) [23 53] while cracks in acid-scarifiedseeds may be attributed to chemical erosion The F1 + HWmethod provides cracks on seed coat and results may beattributed to seed coat expansion or contraction due to theheat shocked on testa of frozen seed Depending on thespecies seed coat thickness and the size of the seeds usedit is even possible to hear a hatch sound when the seedswere immersed in HW following storage in a deep freezerAs indicated before storage of white lupin seeds at minus20∘C fora period of time or immersing the seeds in liquid nitrogenwas not sufficient to meet a need for practical applicationsuggesting that combining freezing temperature with HW atgiven conditions provided a surface stress on seed coat whichcan easily be broken and made seeds permeable to waterPrevious reports indicated that lens or hilum sides of the testaare the physically weakest part and thus can more easily bebroken by treatments [6 23 53] We speculate that the F1 +HW method may also provide cracks on lens or hilum sidesof the testa on white lupin and red clover seeds

In conclusion we developed a novel and reliable combi-nation of freeze-thaw scarification method (F1 + HW) whichis apparently the quickest and simplest method available forreleasing coat-imposed seed dormancy of white lupin and redclover seeds and has a great potential in other plant species inoperational applications if the coat-imposed seed dormancyis due to either the impermeability of the coat to water or themechanical prevention of radicle extension

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors would like to thank Dr Adem Erol for his help incollecting L albus seeds and Dr Ozlem Onal Asci for kindlyproviding T pratense seeds

References

[1] M Koornneef L Bentsink and H Hilhorst ldquoSeed dormancyand germinationrdquo Current Opinion in Plant Biology vol 5 no1 pp 33ndash36 2002

[2] J M Baskin and C C Baskin ldquoA classification system for seeddormancyrdquo Seed Science Research vol 14 no 1 pp 1ndash16 2004

[3] R FinkelsteinWReeves TAriizumi andC Steber ldquoMolecularaspects of seed dormancyrdquo Annual Review of Plant Biology vol59 pp 387ndash415 2008

[4] E Copete J M Herranz P Ferrandis C C Baskin and J MBaskin ldquoPhysiology morphology and phenology of seed dor-mancy break and germination in the endemic Iberian speciesNarcissus hispanicus (Amaryllidaceae)rdquo Annals of Botany vol107 no 6 pp 1003ndash1016 2011

[5] K M Kelly J van Staden and W E Bell ldquoSeed coat structureand dormancyrdquo Plant Growth Regulation vol 11 no 3 pp 201ndash209 1992

Journal of Botany 5

[6] JM Baskin C C Baskin andX J Li ldquoTaxonomy anatomy andevolution of physical dormancy in seedsrdquo Plant Species Biologyvol 15 no 2 pp 139ndash152 2000

[7] G Ladizinsky ldquoThe genetics of hard seed coat in the genusLensrdquo Euphytica vol 34 no 2 pp 539ndash543 1985

[8] H Weber L Borisjuk and U Wobus ldquoControlling seeddevelopment and seed size in Vicia faba a role for seed coat-associated invertases and carbohydrate staterdquo Plant Journal vol10 no 5 pp 823ndash834 1996

[9] P M Erdmann R K Lee M J Bassett and P E McClean ldquoAmolecular marker tightly linked to P a gene required for flowerand seedcoat color in common bean (Phaseolus vulgaris L)contains the Ty3-gypsy retrotransposon Tpv3grdquo Genome vol45 no 4 pp 728ndash736 2002

[10] C Tinius ldquoVariable expression of seed coat permeabilityrdquoSoybean Genetics Newsletter vol 18 pp 256ndash259 1991

[11] K Ranathunge S Shao D Qutob M Gijzen C A PetersonandMA Bernards ldquoProperties of the soybean seed coat cuticlechange during developmentrdquo Planta vol 231 no 5 pp 1171ndash1188 2010

[12] W M Lush and L T Evans ldquoThe seed coats of cowpeas andother grain legumes structure in relation to functionrdquo FieldCrops Research vol 3 pp 267ndash286 1980

[13] H N Buyukkartal H Colgecen N M Pinar and N ErdoganldquoSeed coat ultrastructure of hard-seeded and soft-seeded vari-eties of Vicia sativardquo Turkish Journal of Botany vol 37 no 2 pp270ndash275 2013

[14] E Can N Celiktas R Hatipoglu and S Avci ldquoBreaking seeddormancy of some annual Medicago and Trifolium species bydifferent treatmentsrdquo Turkish Journal of Field Crops vol 14 no2 pp 72ndash78 2009

[15] F R Hay R D Smith R H Ellis and L H Butler ldquoDevel-opmental changes in the germinability desiccation tolerancehardseededness and longevity of individual seeds of Trifoliumambiguumrdquo Annals of Botany vol 105 no 6 pp 1035ndash10522010

[16] O O Asci Z Acar I Ayan U Basaran and H Mut ldquoEffectof pretreatments on seed germination rate of red clover (Tri-folium pratense L) populationsrdquoAfrican Journal of AgriculturalResearch vol 6 no 13 pp 3055ndash3060 2011

[17] A Meisert D Schulz and H Lehmann ldquoStructural featuresunderlying hardseededness in geraniaceaerdquo Plant Biology vol1 no 3 pp 311ndash314 1999

[18] I A Takos and G S P Efthimiou ldquoGermination resultson dormant seeds of fifteen tree species autumn sown in aNorthern Greek nurseryrdquo Silvae Genetica vol 52 no 2 pp 67ndash71 2003

[19] M M A El Balla A I Saidahmed and M Makkawi ldquoEffectof moisture content and maturity on hardseededness andgermination in okra (Abelmoschus esculentus L Moench)rdquoInternational Journal of Plant Physiology and Biochemistry vol3 pp 102ndash107 2011

[20] J D Bewley ldquoSeed germination and dormancyrdquo Plant Cell vol9 no 7 pp 1055ndash1066 1997

[21] R M Nair A D Craig T D Rowe S R Biggins and CH Hunt ldquoGenetic variability and heritability estimates forhardseededness and flowering in balansa clover (Trifoliummichelianum Savi) populationsrdquo Euphytica vol 138 no 3 pp197ndash203 2004

[22] O Karaguzel S Cakmakci V Ortacesme and B AydinogluldquoInfluence of seed coat treatments on germination and early

seedling growth of Lupinus varius Lrdquo Pakistan Journal ofBotany vol 36 no 1 pp 65ndash74 2004

[23] X W Hu Y R Wang Y P Wu and C C Baskin ldquoRole ofthe lens in controlling water uptake in seeds of two Fabaceae(Papilionoideae) species treated with sulphuric acid and hotwaterrdquo Seed Science Research vol 19 no 2 pp 73ndash80 2009

[24] S R Wiley R J Kraus F Zuo E E Murray K Loritz and J EMertz ldquoSV40 early-to-late switch involves titration of cellulartranscriptional repressorsrdquo Genes and Development vol 7 no11 pp 2206ndash2219 1993

[25] SAS I SASSTAT Software Changes and Enhancements throughRelease 612 SAS Inst Cary NC USA 1997

[26] M J Messina ldquoLegumes and soybeans Overview of theirnutritional profiles and health effectsrdquoThe American Journal ofClinical Nutrition vol 70 no 3 1999

[27] M Carbonaro G Grant MMattera A Aguzzi and A PusztaildquoInvestigation of the mechanisms affecting Cu and Fe bioavail-ability from legumes role of seed protein and antinutritional(nonprotein) factorsrdquoBiological Trace Element Research vol 84no 1-3 pp 181ndash196 2001

[28] D M Roth and B R Paulicks ldquoContents of nutrients andfeed value of blue and yellow lupins (Lupinus angustifoliusL Lupinus luteus L) for pigsrdquo Proceedings of the Society ofNutrition Physiology vol 11 p 137 2002

[29] L W Bell M H Ryan R G Bennett M T Collins andH J Clarke ldquoGrowth yield and seed composition of nativeAustralian legumes with potential as grain cropsrdquo Journal of theScience of Food and Agriculture vol 92 no 7 pp 1354ndash13612012

[30] C C Baskin and J M Baskin ldquoEcology and evolution of spe-cialized seed dispersal dormancy and germination strategiesrdquoPlant Species Biology vol 15 no 2 pp 95ndash96 2000

[31] J A Van Assche and F E A Vandelook ldquoCombinationaldormancy in winter annual Fabaceaerdquo Seed Science Researchvol 20 no 4 pp 237ndash242 2010

[32] P J Argel and L R Humphreys ldquoEnvironmental effects onseed development and hardseededness in Stylosanthes hamatacv Verano I Temperaturerdquo Australian Journal of AgriculturalResearch vol 34 no 3 pp 261ndash270 1983

[33] F H Dubbern De Souza and J Marhos-Filho ldquoThe seed coatas a modulator of seed-environment relationships in FabeceaerdquoRevista Brasileira de Botanica vol 24 pp 365ndash375 2001

[34] J J Mott S J Cook and R J Williams ldquoInfluence of shortduration high temperature seed treatment on the germinationof some tropical and temperate legumesrdquo Tropical Grasslandsvol 16 pp 50ndash55 1982

[35] H C Norman P S Cocks and NW Galwey ldquoHardseedednessin annual clovers variation between populations from wet anddry environmentsrdquo Australian Journal of Agricultural Researchvol 53 no 7 pp 821ndash829 2002

[36] A Balkaya ldquoModelling the effect of temperature on the germi-nation speed in some legume cropsrdquo Journal of Agricultural vol3 pp 179ndash183 2004

[37] R L Benech-Arnold R A Sanchez F Forcella B C Kruk andC M Ghersa ldquoEnvironmental control of dormancy in weedseed banks in soilrdquo Field Crops Research vol 67 no 2 pp 105ndash122 2000

[38] K M G Gehan Jayasuriya J M Baskin C C Baskin andM T R Fernando ldquoVariation in seed dormancy and storagebehavior of three liana species of derris (Fabaceae Faboideae)in Sri Lanka and ecological implicationsrdquo Research Journal ofSeed Science vol 5 no 1 pp 1ndash18 2012

6 Journal of Botany

[39] G S Valenti LMeloneM Ferro andA Bozzini ldquoComparativestudies on testa structure of ldquohard-seededrdquo and ldquosoft-seededrdquovarieties of Lupinus angustifolius L (Leguminosae) and onmechanism of water entryrdquo Seed Science and Technology vol 17pp 563ndash581 1989

[40] T D Davis S W George A Upadhyaya and J ParsonsldquoImprovement of seedling emergence of Lupinus taxensisHookfollowing seed scarification treatmentsrdquo Journal of Environmen-tal Horticulture vol 9 pp 17ndash21 1991

[41] O Karaguzel I Baktir S Cakmakci V Ortacesme BAydinoglu and M Atik ldquoEffects of scarification methods tem-perature and sowing date on some germination characteristicsof Lupinus varius Lrdquo inProceedings of the 2ndNational Congresson Ornamental Plants pp 40ndash47 Citrus and GreenhouseResearch Institute Antalya Turkey October 2002

[42] C Patane and F Gresta ldquoGermination of Astragalus hamosusand Medicago orbicularis as affected by seed-coat dormancybreaking techniquesrdquo Journal of Arid Environments vol 67 no1 pp 165ndash173 2006

[43] E Ates ldquoInfluence of some hardseededness-breaking treat-ments on germination in Persian clover (trifolium resupinatumssp typicum Fiori et Paol) seedsrdquo Romanian AgriculturalResearch no 28 pp 229ndash236 2011

[44] DS Loch and GL Harvey ldquoComparison of methods for reduc-ing hard seed levels in three subtropical legumesrdquo inProceedingsof the 4th Australian Seeds Research Conference pp 243ndash246Broadbeach Australia 1992

[45] S N Acharya D G Stout B Brooke and D ThompsonldquoCultivar and storage effects on germination and hard seedcontent of alfalfardquo Canadian Journal of Plant Science vol 79no 2 pp 201ndash208 1999

[46] A Alderete-Chavez L Aguilar-Marın N de la cruz-Landeroet al ldquoEffects of scarification chemical treatments on thegermination of Crotalaria retusa L seedsrdquo Journal of BiologicalSciences vol 10 no 6 pp 541ndash544 2010

[47] A R Midgley ldquoEffect of alternate freezing and thawing onthe impermeability of alfalfa and dodder seedsrdquo Journal of theAmerican Society of Agronomy vol 18 pp 1087ndash1098 1926

[48] D G Stout ldquoEffect of freeze-thaw cycles on hard-seededness ofalfalfardquo Journal of Seed Science and Technology vol 14 pp 47ndash55 1990

[49] W F Busse ldquoEffect of low temperatures on germination ofimpermeable seedsrdquo Botanical Gazette vol 89 pp 169ndash1791930

[50] T Shibata and Y Hatakeyama ldquoBreaking of dormancy in theseeds ofAstragalus mongholicus Bunge (Leguminosae)rdquo Journalof Plant Physiology vol 146 no 3 pp 366ndash368 1995

[51] R Rutar M Stjepanovic S Popovic Z Bukvic and D PacekldquoEffect of temperature on germination and hard alfalfa seedrdquoCIHEAM vol 2 pp 137ndash139 2001

[52] P C Stanwood ldquoTolerance of crop seeds to cooling and storagein liquid nitrogen (-196∘C)rdquo Journal of Seed Technology vol 5no 1 pp 26ndash31 1980

[53] L W Zeng P S Cocks S G Kailis and J Kuo ldquoTherole of fractures and lipids in the seed coat in the loss ofhardseededness of six Mediterranean legume speciesrdquo Journalof Agricultural Science vol 143 no 1 pp 43ndash55 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

4 Journal of Botany

in deep freezer at minus5∘C for 15 days significantly improvedthe germination ratios of seeds in different colours [43]Asci et al [16] also reported that mechanical scarificationwas the most effective method to remove the hardness ofred clover seeds relative to other methods tested includingprecooling preheating hot water and potassium nitrate[16] The importance of the mechanical scarifying techniquehowever lies not just in the possibility that it improves thegerminability of seed but also in how much it can acceleratethe process and how practically it can be applied In additionmechanical scarification may damage other parts of the seedincluding the embryo and may reduce seed viability dueto the pathogenic and saprophytic organisms to gain entryinto the seed and thus reduce the shelf life of the seedsalthough intention of this technique is to provide a minordamage on seed coat for water permeability [44 45] Acidscarification on the other hand is commonly not preferreddue to its cost safety risk and environmental precautionsinvolved and not reliable or lacking the requisite qualities onseeds of other important plant species [43 44 46]Thereforesuch precautions have great importance whether or not thetechnique becomes operationally applicable in large seed lotsof various plant species In agreement with previous reports[46] treatment of white lupin or red clover seeds with HWper se significantly improved FGPs even though the beneficialeffects of these treatments were limited in comparison to F1+ HW method (Figures 1 and 2) The effectiveness of F1 +HW method can also be judged by the high percentage ofgerminations resulting in white lupin (8416) and red clover(7450) seeds while UT control seeds had FGPs of 33 and260 respectively In addition germination improvement ofred clover seed with F1 +HWmethod was found to be higherthan that ofmechanical scarification reported previously [16]

Freeze-thaw scarification method has been known andbeen used to improve germination of hard seeds in severallegume species for a long time [47ndash51] Midgley [47] reportedthat germination of alfalfa seeds was not improved whenthe seeds were subjected to freeze-thaw scarification withcooling temperatures at minus5∘C or at minus15∘C for 36 h and withwarming at room temperatures for six days Similarly freeze-thaw scarification of milk vetch seeds at minus22∘C for 2 4 730 60 90 or 180 days did not improve seed germinationmore than 78 [50] Both Stout [48] andBusse [49] suggestedthat repeated freeze-thaw cycles were needed to improvegermination of alfalfa seeds although Rutar et al [51] foundno influence on germination of alfalfa seeds when they usedthe same seed treatments as the study of Stout [48] Freeze-thaw scarification using liquid nitrogen was also previouslytested and showed no effect on hard seeds of several legumespecies including alfalfa red clover soybean common vetch[52] and white lupin Taken together the results of thisstudy revealed that thawing temperatures or conditions ina freeze-thaw scarification technique would be more criticalthan freezing temperatures and immediate or rapid thawinglike in hot water would be more beneficial to remove coat-imposed seed dormancy in legume plants

It has been suggested that cracks in the seed coat pro-duced by heat treatment result from seed coat expansionand contraction due to the increase of room temperature

to minus80∘C (hot water) [23 53] while cracks in acid-scarifiedseeds may be attributed to chemical erosion The F1 + HWmethod provides cracks on seed coat and results may beattributed to seed coat expansion or contraction due to theheat shocked on testa of frozen seed Depending on thespecies seed coat thickness and the size of the seeds usedit is even possible to hear a hatch sound when the seedswere immersed in HW following storage in a deep freezerAs indicated before storage of white lupin seeds at minus20∘C fora period of time or immersing the seeds in liquid nitrogenwas not sufficient to meet a need for practical applicationsuggesting that combining freezing temperature with HW atgiven conditions provided a surface stress on seed coat whichcan easily be broken and made seeds permeable to waterPrevious reports indicated that lens or hilum sides of the testaare the physically weakest part and thus can more easily bebroken by treatments [6 23 53] We speculate that the F1 +HW method may also provide cracks on lens or hilum sidesof the testa on white lupin and red clover seeds

In conclusion we developed a novel and reliable combi-nation of freeze-thaw scarification method (F1 + HW) whichis apparently the quickest and simplest method available forreleasing coat-imposed seed dormancy of white lupin and redclover seeds and has a great potential in other plant species inoperational applications if the coat-imposed seed dormancyis due to either the impermeability of the coat to water or themechanical prevention of radicle extension

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors would like to thank Dr Adem Erol for his help incollecting L albus seeds and Dr Ozlem Onal Asci for kindlyproviding T pratense seeds

References

[1] M Koornneef L Bentsink and H Hilhorst ldquoSeed dormancyand germinationrdquo Current Opinion in Plant Biology vol 5 no1 pp 33ndash36 2002

[2] J M Baskin and C C Baskin ldquoA classification system for seeddormancyrdquo Seed Science Research vol 14 no 1 pp 1ndash16 2004

[3] R FinkelsteinWReeves TAriizumi andC Steber ldquoMolecularaspects of seed dormancyrdquo Annual Review of Plant Biology vol59 pp 387ndash415 2008

[4] E Copete J M Herranz P Ferrandis C C Baskin and J MBaskin ldquoPhysiology morphology and phenology of seed dor-mancy break and germination in the endemic Iberian speciesNarcissus hispanicus (Amaryllidaceae)rdquo Annals of Botany vol107 no 6 pp 1003ndash1016 2011

[5] K M Kelly J van Staden and W E Bell ldquoSeed coat structureand dormancyrdquo Plant Growth Regulation vol 11 no 3 pp 201ndash209 1992

Journal of Botany 5

[6] JM Baskin C C Baskin andX J Li ldquoTaxonomy anatomy andevolution of physical dormancy in seedsrdquo Plant Species Biologyvol 15 no 2 pp 139ndash152 2000

[7] G Ladizinsky ldquoThe genetics of hard seed coat in the genusLensrdquo Euphytica vol 34 no 2 pp 539ndash543 1985

[8] H Weber L Borisjuk and U Wobus ldquoControlling seeddevelopment and seed size in Vicia faba a role for seed coat-associated invertases and carbohydrate staterdquo Plant Journal vol10 no 5 pp 823ndash834 1996

[9] P M Erdmann R K Lee M J Bassett and P E McClean ldquoAmolecular marker tightly linked to P a gene required for flowerand seedcoat color in common bean (Phaseolus vulgaris L)contains the Ty3-gypsy retrotransposon Tpv3grdquo Genome vol45 no 4 pp 728ndash736 2002

[10] C Tinius ldquoVariable expression of seed coat permeabilityrdquoSoybean Genetics Newsletter vol 18 pp 256ndash259 1991

[11] K Ranathunge S Shao D Qutob M Gijzen C A PetersonandMA Bernards ldquoProperties of the soybean seed coat cuticlechange during developmentrdquo Planta vol 231 no 5 pp 1171ndash1188 2010

[12] W M Lush and L T Evans ldquoThe seed coats of cowpeas andother grain legumes structure in relation to functionrdquo FieldCrops Research vol 3 pp 267ndash286 1980

[13] H N Buyukkartal H Colgecen N M Pinar and N ErdoganldquoSeed coat ultrastructure of hard-seeded and soft-seeded vari-eties of Vicia sativardquo Turkish Journal of Botany vol 37 no 2 pp270ndash275 2013

[14] E Can N Celiktas R Hatipoglu and S Avci ldquoBreaking seeddormancy of some annual Medicago and Trifolium species bydifferent treatmentsrdquo Turkish Journal of Field Crops vol 14 no2 pp 72ndash78 2009

[15] F R Hay R D Smith R H Ellis and L H Butler ldquoDevel-opmental changes in the germinability desiccation tolerancehardseededness and longevity of individual seeds of Trifoliumambiguumrdquo Annals of Botany vol 105 no 6 pp 1035ndash10522010

[16] O O Asci Z Acar I Ayan U Basaran and H Mut ldquoEffectof pretreatments on seed germination rate of red clover (Tri-folium pratense L) populationsrdquoAfrican Journal of AgriculturalResearch vol 6 no 13 pp 3055ndash3060 2011

[17] A Meisert D Schulz and H Lehmann ldquoStructural featuresunderlying hardseededness in geraniaceaerdquo Plant Biology vol1 no 3 pp 311ndash314 1999

[18] I A Takos and G S P Efthimiou ldquoGermination resultson dormant seeds of fifteen tree species autumn sown in aNorthern Greek nurseryrdquo Silvae Genetica vol 52 no 2 pp 67ndash71 2003

[19] M M A El Balla A I Saidahmed and M Makkawi ldquoEffectof moisture content and maturity on hardseededness andgermination in okra (Abelmoschus esculentus L Moench)rdquoInternational Journal of Plant Physiology and Biochemistry vol3 pp 102ndash107 2011

[20] J D Bewley ldquoSeed germination and dormancyrdquo Plant Cell vol9 no 7 pp 1055ndash1066 1997

[21] R M Nair A D Craig T D Rowe S R Biggins and CH Hunt ldquoGenetic variability and heritability estimates forhardseededness and flowering in balansa clover (Trifoliummichelianum Savi) populationsrdquo Euphytica vol 138 no 3 pp197ndash203 2004

[22] O Karaguzel S Cakmakci V Ortacesme and B AydinogluldquoInfluence of seed coat treatments on germination and early

seedling growth of Lupinus varius Lrdquo Pakistan Journal ofBotany vol 36 no 1 pp 65ndash74 2004

[23] X W Hu Y R Wang Y P Wu and C C Baskin ldquoRole ofthe lens in controlling water uptake in seeds of two Fabaceae(Papilionoideae) species treated with sulphuric acid and hotwaterrdquo Seed Science Research vol 19 no 2 pp 73ndash80 2009

[24] S R Wiley R J Kraus F Zuo E E Murray K Loritz and J EMertz ldquoSV40 early-to-late switch involves titration of cellulartranscriptional repressorsrdquo Genes and Development vol 7 no11 pp 2206ndash2219 1993

[25] SAS I SASSTAT Software Changes and Enhancements throughRelease 612 SAS Inst Cary NC USA 1997

[26] M J Messina ldquoLegumes and soybeans Overview of theirnutritional profiles and health effectsrdquoThe American Journal ofClinical Nutrition vol 70 no 3 1999

[27] M Carbonaro G Grant MMattera A Aguzzi and A PusztaildquoInvestigation of the mechanisms affecting Cu and Fe bioavail-ability from legumes role of seed protein and antinutritional(nonprotein) factorsrdquoBiological Trace Element Research vol 84no 1-3 pp 181ndash196 2001

[28] D M Roth and B R Paulicks ldquoContents of nutrients andfeed value of blue and yellow lupins (Lupinus angustifoliusL Lupinus luteus L) for pigsrdquo Proceedings of the Society ofNutrition Physiology vol 11 p 137 2002

[29] L W Bell M H Ryan R G Bennett M T Collins andH J Clarke ldquoGrowth yield and seed composition of nativeAustralian legumes with potential as grain cropsrdquo Journal of theScience of Food and Agriculture vol 92 no 7 pp 1354ndash13612012

[30] C C Baskin and J M Baskin ldquoEcology and evolution of spe-cialized seed dispersal dormancy and germination strategiesrdquoPlant Species Biology vol 15 no 2 pp 95ndash96 2000

[31] J A Van Assche and F E A Vandelook ldquoCombinationaldormancy in winter annual Fabaceaerdquo Seed Science Researchvol 20 no 4 pp 237ndash242 2010

[32] P J Argel and L R Humphreys ldquoEnvironmental effects onseed development and hardseededness in Stylosanthes hamatacv Verano I Temperaturerdquo Australian Journal of AgriculturalResearch vol 34 no 3 pp 261ndash270 1983

[33] F H Dubbern De Souza and J Marhos-Filho ldquoThe seed coatas a modulator of seed-environment relationships in FabeceaerdquoRevista Brasileira de Botanica vol 24 pp 365ndash375 2001

[34] J J Mott S J Cook and R J Williams ldquoInfluence of shortduration high temperature seed treatment on the germinationof some tropical and temperate legumesrdquo Tropical Grasslandsvol 16 pp 50ndash55 1982

[35] H C Norman P S Cocks and NW Galwey ldquoHardseedednessin annual clovers variation between populations from wet anddry environmentsrdquo Australian Journal of Agricultural Researchvol 53 no 7 pp 821ndash829 2002

[36] A Balkaya ldquoModelling the effect of temperature on the germi-nation speed in some legume cropsrdquo Journal of Agricultural vol3 pp 179ndash183 2004

[37] R L Benech-Arnold R A Sanchez F Forcella B C Kruk andC M Ghersa ldquoEnvironmental control of dormancy in weedseed banks in soilrdquo Field Crops Research vol 67 no 2 pp 105ndash122 2000

[38] K M G Gehan Jayasuriya J M Baskin C C Baskin andM T R Fernando ldquoVariation in seed dormancy and storagebehavior of three liana species of derris (Fabaceae Faboideae)in Sri Lanka and ecological implicationsrdquo Research Journal ofSeed Science vol 5 no 1 pp 1ndash18 2012

6 Journal of Botany

[39] G S Valenti LMeloneM Ferro andA Bozzini ldquoComparativestudies on testa structure of ldquohard-seededrdquo and ldquosoft-seededrdquovarieties of Lupinus angustifolius L (Leguminosae) and onmechanism of water entryrdquo Seed Science and Technology vol 17pp 563ndash581 1989

[40] T D Davis S W George A Upadhyaya and J ParsonsldquoImprovement of seedling emergence of Lupinus taxensisHookfollowing seed scarification treatmentsrdquo Journal of Environmen-tal Horticulture vol 9 pp 17ndash21 1991

[41] O Karaguzel I Baktir S Cakmakci V Ortacesme BAydinoglu and M Atik ldquoEffects of scarification methods tem-perature and sowing date on some germination characteristicsof Lupinus varius Lrdquo inProceedings of the 2ndNational Congresson Ornamental Plants pp 40ndash47 Citrus and GreenhouseResearch Institute Antalya Turkey October 2002

[42] C Patane and F Gresta ldquoGermination of Astragalus hamosusand Medicago orbicularis as affected by seed-coat dormancybreaking techniquesrdquo Journal of Arid Environments vol 67 no1 pp 165ndash173 2006

[43] E Ates ldquoInfluence of some hardseededness-breaking treat-ments on germination in Persian clover (trifolium resupinatumssp typicum Fiori et Paol) seedsrdquo Romanian AgriculturalResearch no 28 pp 229ndash236 2011

[44] DS Loch and GL Harvey ldquoComparison of methods for reduc-ing hard seed levels in three subtropical legumesrdquo inProceedingsof the 4th Australian Seeds Research Conference pp 243ndash246Broadbeach Australia 1992

[45] S N Acharya D G Stout B Brooke and D ThompsonldquoCultivar and storage effects on germination and hard seedcontent of alfalfardquo Canadian Journal of Plant Science vol 79no 2 pp 201ndash208 1999

[46] A Alderete-Chavez L Aguilar-Marın N de la cruz-Landeroet al ldquoEffects of scarification chemical treatments on thegermination of Crotalaria retusa L seedsrdquo Journal of BiologicalSciences vol 10 no 6 pp 541ndash544 2010

[47] A R Midgley ldquoEffect of alternate freezing and thawing onthe impermeability of alfalfa and dodder seedsrdquo Journal of theAmerican Society of Agronomy vol 18 pp 1087ndash1098 1926

[48] D G Stout ldquoEffect of freeze-thaw cycles on hard-seededness ofalfalfardquo Journal of Seed Science and Technology vol 14 pp 47ndash55 1990

[49] W F Busse ldquoEffect of low temperatures on germination ofimpermeable seedsrdquo Botanical Gazette vol 89 pp 169ndash1791930

[50] T Shibata and Y Hatakeyama ldquoBreaking of dormancy in theseeds ofAstragalus mongholicus Bunge (Leguminosae)rdquo Journalof Plant Physiology vol 146 no 3 pp 366ndash368 1995

[51] R Rutar M Stjepanovic S Popovic Z Bukvic and D PacekldquoEffect of temperature on germination and hard alfalfa seedrdquoCIHEAM vol 2 pp 137ndash139 2001

[52] P C Stanwood ldquoTolerance of crop seeds to cooling and storagein liquid nitrogen (-196∘C)rdquo Journal of Seed Technology vol 5no 1 pp 26ndash31 1980

[53] L W Zeng P S Cocks S G Kailis and J Kuo ldquoTherole of fractures and lipids in the seed coat in the loss ofhardseededness of six Mediterranean legume speciesrdquo Journalof Agricultural Science vol 143 no 1 pp 43ndash55 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Journal of Botany 5

[6] JM Baskin C C Baskin andX J Li ldquoTaxonomy anatomy andevolution of physical dormancy in seedsrdquo Plant Species Biologyvol 15 no 2 pp 139ndash152 2000

[7] G Ladizinsky ldquoThe genetics of hard seed coat in the genusLensrdquo Euphytica vol 34 no 2 pp 539ndash543 1985

[8] H Weber L Borisjuk and U Wobus ldquoControlling seeddevelopment and seed size in Vicia faba a role for seed coat-associated invertases and carbohydrate staterdquo Plant Journal vol10 no 5 pp 823ndash834 1996

[9] P M Erdmann R K Lee M J Bassett and P E McClean ldquoAmolecular marker tightly linked to P a gene required for flowerand seedcoat color in common bean (Phaseolus vulgaris L)contains the Ty3-gypsy retrotransposon Tpv3grdquo Genome vol45 no 4 pp 728ndash736 2002

[10] C Tinius ldquoVariable expression of seed coat permeabilityrdquoSoybean Genetics Newsletter vol 18 pp 256ndash259 1991

[11] K Ranathunge S Shao D Qutob M Gijzen C A PetersonandMA Bernards ldquoProperties of the soybean seed coat cuticlechange during developmentrdquo Planta vol 231 no 5 pp 1171ndash1188 2010

[12] W M Lush and L T Evans ldquoThe seed coats of cowpeas andother grain legumes structure in relation to functionrdquo FieldCrops Research vol 3 pp 267ndash286 1980

[13] H N Buyukkartal H Colgecen N M Pinar and N ErdoganldquoSeed coat ultrastructure of hard-seeded and soft-seeded vari-eties of Vicia sativardquo Turkish Journal of Botany vol 37 no 2 pp270ndash275 2013

[14] E Can N Celiktas R Hatipoglu and S Avci ldquoBreaking seeddormancy of some annual Medicago and Trifolium species bydifferent treatmentsrdquo Turkish Journal of Field Crops vol 14 no2 pp 72ndash78 2009

[15] F R Hay R D Smith R H Ellis and L H Butler ldquoDevel-opmental changes in the germinability desiccation tolerancehardseededness and longevity of individual seeds of Trifoliumambiguumrdquo Annals of Botany vol 105 no 6 pp 1035ndash10522010

[16] O O Asci Z Acar I Ayan U Basaran and H Mut ldquoEffectof pretreatments on seed germination rate of red clover (Tri-folium pratense L) populationsrdquoAfrican Journal of AgriculturalResearch vol 6 no 13 pp 3055ndash3060 2011

[17] A Meisert D Schulz and H Lehmann ldquoStructural featuresunderlying hardseededness in geraniaceaerdquo Plant Biology vol1 no 3 pp 311ndash314 1999

[18] I A Takos and G S P Efthimiou ldquoGermination resultson dormant seeds of fifteen tree species autumn sown in aNorthern Greek nurseryrdquo Silvae Genetica vol 52 no 2 pp 67ndash71 2003

[19] M M A El Balla A I Saidahmed and M Makkawi ldquoEffectof moisture content and maturity on hardseededness andgermination in okra (Abelmoschus esculentus L Moench)rdquoInternational Journal of Plant Physiology and Biochemistry vol3 pp 102ndash107 2011

[20] J D Bewley ldquoSeed germination and dormancyrdquo Plant Cell vol9 no 7 pp 1055ndash1066 1997

[21] R M Nair A D Craig T D Rowe S R Biggins and CH Hunt ldquoGenetic variability and heritability estimates forhardseededness and flowering in balansa clover (Trifoliummichelianum Savi) populationsrdquo Euphytica vol 138 no 3 pp197ndash203 2004

[22] O Karaguzel S Cakmakci V Ortacesme and B AydinogluldquoInfluence of seed coat treatments on germination and early

seedling growth of Lupinus varius Lrdquo Pakistan Journal ofBotany vol 36 no 1 pp 65ndash74 2004

[23] X W Hu Y R Wang Y P Wu and C C Baskin ldquoRole ofthe lens in controlling water uptake in seeds of two Fabaceae(Papilionoideae) species treated with sulphuric acid and hotwaterrdquo Seed Science Research vol 19 no 2 pp 73ndash80 2009

[24] S R Wiley R J Kraus F Zuo E E Murray K Loritz and J EMertz ldquoSV40 early-to-late switch involves titration of cellulartranscriptional repressorsrdquo Genes and Development vol 7 no11 pp 2206ndash2219 1993

[25] SAS I SASSTAT Software Changes and Enhancements throughRelease 612 SAS Inst Cary NC USA 1997

[26] M J Messina ldquoLegumes and soybeans Overview of theirnutritional profiles and health effectsrdquoThe American Journal ofClinical Nutrition vol 70 no 3 1999

[27] M Carbonaro G Grant MMattera A Aguzzi and A PusztaildquoInvestigation of the mechanisms affecting Cu and Fe bioavail-ability from legumes role of seed protein and antinutritional(nonprotein) factorsrdquoBiological Trace Element Research vol 84no 1-3 pp 181ndash196 2001

[28] D M Roth and B R Paulicks ldquoContents of nutrients andfeed value of blue and yellow lupins (Lupinus angustifoliusL Lupinus luteus L) for pigsrdquo Proceedings of the Society ofNutrition Physiology vol 11 p 137 2002

[29] L W Bell M H Ryan R G Bennett M T Collins andH J Clarke ldquoGrowth yield and seed composition of nativeAustralian legumes with potential as grain cropsrdquo Journal of theScience of Food and Agriculture vol 92 no 7 pp 1354ndash13612012

[30] C C Baskin and J M Baskin ldquoEcology and evolution of spe-cialized seed dispersal dormancy and germination strategiesrdquoPlant Species Biology vol 15 no 2 pp 95ndash96 2000

[31] J A Van Assche and F E A Vandelook ldquoCombinationaldormancy in winter annual Fabaceaerdquo Seed Science Researchvol 20 no 4 pp 237ndash242 2010

[32] P J Argel and L R Humphreys ldquoEnvironmental effects onseed development and hardseededness in Stylosanthes hamatacv Verano I Temperaturerdquo Australian Journal of AgriculturalResearch vol 34 no 3 pp 261ndash270 1983

[33] F H Dubbern De Souza and J Marhos-Filho ldquoThe seed coatas a modulator of seed-environment relationships in FabeceaerdquoRevista Brasileira de Botanica vol 24 pp 365ndash375 2001

[34] J J Mott S J Cook and R J Williams ldquoInfluence of shortduration high temperature seed treatment on the germinationof some tropical and temperate legumesrdquo Tropical Grasslandsvol 16 pp 50ndash55 1982

[35] H C Norman P S Cocks and NW Galwey ldquoHardseedednessin annual clovers variation between populations from wet anddry environmentsrdquo Australian Journal of Agricultural Researchvol 53 no 7 pp 821ndash829 2002

[36] A Balkaya ldquoModelling the effect of temperature on the germi-nation speed in some legume cropsrdquo Journal of Agricultural vol3 pp 179ndash183 2004

[37] R L Benech-Arnold R A Sanchez F Forcella B C Kruk andC M Ghersa ldquoEnvironmental control of dormancy in weedseed banks in soilrdquo Field Crops Research vol 67 no 2 pp 105ndash122 2000

[38] K M G Gehan Jayasuriya J M Baskin C C Baskin andM T R Fernando ldquoVariation in seed dormancy and storagebehavior of three liana species of derris (Fabaceae Faboideae)in Sri Lanka and ecological implicationsrdquo Research Journal ofSeed Science vol 5 no 1 pp 1ndash18 2012

6 Journal of Botany

[39] G S Valenti LMeloneM Ferro andA Bozzini ldquoComparativestudies on testa structure of ldquohard-seededrdquo and ldquosoft-seededrdquovarieties of Lupinus angustifolius L (Leguminosae) and onmechanism of water entryrdquo Seed Science and Technology vol 17pp 563ndash581 1989

[40] T D Davis S W George A Upadhyaya and J ParsonsldquoImprovement of seedling emergence of Lupinus taxensisHookfollowing seed scarification treatmentsrdquo Journal of Environmen-tal Horticulture vol 9 pp 17ndash21 1991

[41] O Karaguzel I Baktir S Cakmakci V Ortacesme BAydinoglu and M Atik ldquoEffects of scarification methods tem-perature and sowing date on some germination characteristicsof Lupinus varius Lrdquo inProceedings of the 2ndNational Congresson Ornamental Plants pp 40ndash47 Citrus and GreenhouseResearch Institute Antalya Turkey October 2002

[42] C Patane and F Gresta ldquoGermination of Astragalus hamosusand Medicago orbicularis as affected by seed-coat dormancybreaking techniquesrdquo Journal of Arid Environments vol 67 no1 pp 165ndash173 2006

[43] E Ates ldquoInfluence of some hardseededness-breaking treat-ments on germination in Persian clover (trifolium resupinatumssp typicum Fiori et Paol) seedsrdquo Romanian AgriculturalResearch no 28 pp 229ndash236 2011

[44] DS Loch and GL Harvey ldquoComparison of methods for reduc-ing hard seed levels in three subtropical legumesrdquo inProceedingsof the 4th Australian Seeds Research Conference pp 243ndash246Broadbeach Australia 1992

[45] S N Acharya D G Stout B Brooke and D ThompsonldquoCultivar and storage effects on germination and hard seedcontent of alfalfardquo Canadian Journal of Plant Science vol 79no 2 pp 201ndash208 1999

[46] A Alderete-Chavez L Aguilar-Marın N de la cruz-Landeroet al ldquoEffects of scarification chemical treatments on thegermination of Crotalaria retusa L seedsrdquo Journal of BiologicalSciences vol 10 no 6 pp 541ndash544 2010

[47] A R Midgley ldquoEffect of alternate freezing and thawing onthe impermeability of alfalfa and dodder seedsrdquo Journal of theAmerican Society of Agronomy vol 18 pp 1087ndash1098 1926

[48] D G Stout ldquoEffect of freeze-thaw cycles on hard-seededness ofalfalfardquo Journal of Seed Science and Technology vol 14 pp 47ndash55 1990

[49] W F Busse ldquoEffect of low temperatures on germination ofimpermeable seedsrdquo Botanical Gazette vol 89 pp 169ndash1791930

[50] T Shibata and Y Hatakeyama ldquoBreaking of dormancy in theseeds ofAstragalus mongholicus Bunge (Leguminosae)rdquo Journalof Plant Physiology vol 146 no 3 pp 366ndash368 1995

[51] R Rutar M Stjepanovic S Popovic Z Bukvic and D PacekldquoEffect of temperature on germination and hard alfalfa seedrdquoCIHEAM vol 2 pp 137ndash139 2001

[52] P C Stanwood ldquoTolerance of crop seeds to cooling and storagein liquid nitrogen (-196∘C)rdquo Journal of Seed Technology vol 5no 1 pp 26ndash31 1980

[53] L W Zeng P S Cocks S G Kailis and J Kuo ldquoTherole of fractures and lipids in the seed coat in the loss ofhardseededness of six Mediterranean legume speciesrdquo Journalof Agricultural Science vol 143 no 1 pp 43ndash55 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

6 Journal of Botany

[39] G S Valenti LMeloneM Ferro andA Bozzini ldquoComparativestudies on testa structure of ldquohard-seededrdquo and ldquosoft-seededrdquovarieties of Lupinus angustifolius L (Leguminosae) and onmechanism of water entryrdquo Seed Science and Technology vol 17pp 563ndash581 1989

[40] T D Davis S W George A Upadhyaya and J ParsonsldquoImprovement of seedling emergence of Lupinus taxensisHookfollowing seed scarification treatmentsrdquo Journal of Environmen-tal Horticulture vol 9 pp 17ndash21 1991

[41] O Karaguzel I Baktir S Cakmakci V Ortacesme BAydinoglu and M Atik ldquoEffects of scarification methods tem-perature and sowing date on some germination characteristicsof Lupinus varius Lrdquo inProceedings of the 2ndNational Congresson Ornamental Plants pp 40ndash47 Citrus and GreenhouseResearch Institute Antalya Turkey October 2002

[42] C Patane and F Gresta ldquoGermination of Astragalus hamosusand Medicago orbicularis as affected by seed-coat dormancybreaking techniquesrdquo Journal of Arid Environments vol 67 no1 pp 165ndash173 2006

[43] E Ates ldquoInfluence of some hardseededness-breaking treat-ments on germination in Persian clover (trifolium resupinatumssp typicum Fiori et Paol) seedsrdquo Romanian AgriculturalResearch no 28 pp 229ndash236 2011

[44] DS Loch and GL Harvey ldquoComparison of methods for reduc-ing hard seed levels in three subtropical legumesrdquo inProceedingsof the 4th Australian Seeds Research Conference pp 243ndash246Broadbeach Australia 1992

[45] S N Acharya D G Stout B Brooke and D ThompsonldquoCultivar and storage effects on germination and hard seedcontent of alfalfardquo Canadian Journal of Plant Science vol 79no 2 pp 201ndash208 1999

[46] A Alderete-Chavez L Aguilar-Marın N de la cruz-Landeroet al ldquoEffects of scarification chemical treatments on thegermination of Crotalaria retusa L seedsrdquo Journal of BiologicalSciences vol 10 no 6 pp 541ndash544 2010

[47] A R Midgley ldquoEffect of alternate freezing and thawing onthe impermeability of alfalfa and dodder seedsrdquo Journal of theAmerican Society of Agronomy vol 18 pp 1087ndash1098 1926

[48] D G Stout ldquoEffect of freeze-thaw cycles on hard-seededness ofalfalfardquo Journal of Seed Science and Technology vol 14 pp 47ndash55 1990

[49] W F Busse ldquoEffect of low temperatures on germination ofimpermeable seedsrdquo Botanical Gazette vol 89 pp 169ndash1791930

[50] T Shibata and Y Hatakeyama ldquoBreaking of dormancy in theseeds ofAstragalus mongholicus Bunge (Leguminosae)rdquo Journalof Plant Physiology vol 146 no 3 pp 366ndash368 1995

[51] R Rutar M Stjepanovic S Popovic Z Bukvic and D PacekldquoEffect of temperature on germination and hard alfalfa seedrdquoCIHEAM vol 2 pp 137ndash139 2001

[52] P C Stanwood ldquoTolerance of crop seeds to cooling and storagein liquid nitrogen (-196∘C)rdquo Journal of Seed Technology vol 5no 1 pp 26ndash31 1980

[53] L W Zeng P S Cocks S G Kailis and J Kuo ldquoTherole of fractures and lipids in the seed coat in the loss ofhardseededness of six Mediterranean legume speciesrdquo Journalof Agricultural Science vol 143 no 1 pp 43ndash55 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology