全 文 ：第21卷 第2期
Vol.21 No.2
草 地 学 报
ACTA AGRESTIA SINICA
2013年 3月
Mar. 2013
doi:10.11733/j.issn.1007-0435.2013.02.017
PhysiologicalCharacteristicsofAging-resistance
ofUltradriedCeratoidesarborescensSeeds
TONGLi-rong1,DONGKuan-hu1,HANJian-guo2,XUQing-fang1
(1.ColegeofAnimalScienceandTechnologyofShanxiAgriculturalUniversity,Taigu,ShanxiProvince030801,China;
2.InstituteofGrasslandScience,ChinaAgriculturalUniversity,Beijing100193,China)
AbstractPhysiologicalandbiochemicalindexesincludingseedgerminationpercentage,vigorin-
dex,antioxidantenzymeactivitiesandfreeradicalsweredeterminedtostudythedeterioration
processofCeratoidesarborescensseedstreatedbyartificialaging.Resultsindicatedthatthecriti-
calmoisturecontentofCeratoidesarborescensseedswas3.78%.Ultradringwithinthecritical
moisturecontentdidnotaffectseedvigorandsignificantlyenhancedaging-resistantcapabilityun-
deraccelerated-aging(45℃,2days).Theactivitiesofantioxidantenzymesincludingcatalaseand
superoxidedismutasewereremainedduringstorageinanultradrystate.FreeradicalsinCera-
toidesarborescensultradriedseedsincreased.Rehydrationhadafreeradicalscavengingactivity.
Therefore,moderateultradryingcouldmaintainthevigorofCeratoidesarborescensseedsanden-
hancestorageeffectiveness.
Keywords:Ceratoidesarborescensseeds;Ultradrying;Aging-resistance;Freeradical
华北驼绒藜超干种子抗老化生理特性的研究
佟莉蓉1,董宽虎1,韩建国2,许庆方1
(1.山西农业大学动物科技学院,山西 太谷 030801;2.中国农业大学草地研究所,北京 100193)
摘要:以华北驼绒藜(Ceratoidesarborescens)种子为材料,采用超干燥和人工加速老化的方法处理种子,测定其发芽率、活力指
数、抗氧化酶活性、自由基水平及其他指标,研究种子劣变的生理生化特性。结果表明:在一定含水量范围内,种子超干对种
子活力没有不良影响,而且种子在老化条件下(45℃,2d)能显著地提高种子的抗老化能力。华北驼绒藜超干种子最适含水量
为3.78%。超干种子经老化处理后抗氧化酶(过氧化氢酶、超氧化物歧化酶)仍保持较高的活性。超干种子老化处理后自由
基水平升高,但超干种子在萌发前经回水处理对自由基有清除能力,从而使自由基水平下降。因此,适度超干可以使华北驼
绒藜种子保持较高的活力,提高其耐贮藏性。
关键词:华北驼绒藜种子;超干;抗老化;自由基
中图分类号:S330.2 文献标识码:A 文章编号:1007-0435(2013)02-0322-05
Maintenanceofplantbiodiversityisaglobal
concern.Onestrategytopreservegeneticdiversity
withinaspeciesisthroughseedstorageinexsitu
genebank.Effectivepreservationofseedsdepends
ontheirmoisturecontentandstoretemperature[1],
butindevelopingcountries,thecostsofcoldstor-
ageareprohibitive[2].Ultradryseedstorageisa
techniquefordecreasingtheseedmoisturecontent
tolessthan5%thenstoredatambienttempera-
tures.Itcanreducethecostforconstructingand
maintainingthegenebankandhasbroughtatten-
tionbecauseofitspotentialeconomiceffectand
promisingapplicationingermplasmconservation.
Studiesshowthatultradryseedstoragenotonly
canbeusedtomaintainthequalityofseedsbutal-
soimprovesthestorabilityofseeds[3-4].
GenusCeratoidesL.belongstothefamilyChe-
nopodiaceae.Itiscomposedofapproximately7spe-
cies,including Ceratoidesarborescens,C.lateens,
C.ewersmanniana,C.compacta.C.arborescensisour
收稿日期:2012-07-29;修回日期:2012-09-20
基金项目:山西省青年科技研究基金(2011021033-2);教育部高等学校博士学科点专项科研基金(20111403120007);山西农业大学博士后
科学基金资助
作者简介:佟莉蓉(1976-),女,副教授,博士,主要从事牧草种子生理生化研究,E-mail:tlr122500@126.com
第2期 佟莉蓉等:华北驼绒藜超干种子抗老化生理特性的研究
specialtyxerophyticshrubofChina.However,lit-
tleresearchattentionhasbeengiventotheprotec-
tionofgermplasmresourceandbiochemicalbasis
ofseedstorageinC.arborescens.Ourobjective
wastoinvestigateseedgerminationabilityandvia-
bilityinC.arborescensseedsafterultradryingand
toexplorethephysiologicalcharacteristicsofag-
ing-resistanceofultradriedC.arborescensseeds
andtheefficiencyoffreeradicalscavengingsys-
tems.
1 MaterialsandMethods
1.1 Seedmaterials
SeedsofC.arborescenswereprovidedbyPro-
fessorYijinofInnerMongoliaAgriculturalUni-
versityinNovember2007.Theinitialgermination
percentage(GP)ofC.arborescenswas93.0%and
moisturecontent(MC)wasabout10.78%.
1.2 Ultradrytreatmentofseed
Seedswereultradriedbyusingsilicageldrying
method.Theseedswereputintoadesiccatorcontai-
ningsilicagelatnormalatmospherictemperature
(25℃)for56d.TheMCwasreducedto5.89%,
4.91%,3.78%and2.69%.Ultradriedseedswere
storedinsealedaluminumfoilpackages.
1.3 Prehumidification
Ultradriedseedswereputintoasealedcontai-
ningsaturatedCaCl2solutions(relativehumidityis
35%),thentransferredtoasealeddesiccatorcon-
tainingsaturatedNH4Clsolutions(relativehumid-
ityis70%),andfinalytransferredintoasealed
desiccatorcontainingwaterwith100%relativehu-
midityatnormalatmospherictemperature(20℃)
beforegermination.Eachsteplastedfor24h.
1.4 Measurementofseed MC,germinationand
vigortest
AccordingtoInternationalRulesforSeedTes-
ting[5].Seed MC wasdeterminedbytheoven
method(8hat110℃ ±1℃)andcouldbeex-
pressedonwetbasis(%,w.b.).Seedsurfaces
weresterilizedusing10% Na-hypochloritebefore
thegerminationprocess.Fourreplicateswith100
seedseachweregerminatedat25℃.Germination
wascheckedevery24h,andGPandradicleweight
wereestimatedat7d.Seedsvigorindex(VI)was
determinedaccordingtothefolowingequation:VI
=GI×Sx,GI=∑(Gt/Dt),whereGIisgermina-
tionindex,Sxisradiclemeanlengthxdaysafter
germination,GtisGPaftertdays,andDtisdays
ofgermination.
1.5 Acceleratedaging
Theultradriedandnon-ultradriedseeds(con-
trol)ofC.arborescensweresealedinaluminum
bagandplacedat45℃for2dtoacceleratedaging.
1.6 Freeradicalmeasurements
Thecontentsoffreeradicalweremeasuredimme-
diatelyafterseedsweresoakedin10 mmol·L-1
DMPOand1mmol·L-1POBNat25℃usingelec-
tronicspinresonance (ESR),instrumenttype:
JES-FEIXG,madebyJEOLCompany,JAPAN.
Measurementmethodwasconductedaccordingto
Huetal.[6]methodwithmeasurementconditions:
centralmagneticfield(3400±250)G;microwave
frequency9.24GHz;modulate3.2G;response
0.1s;scanning4min.
1.7 Malondialdehyde(MDA)test
Samplesof0.1gseedsweresoakedindistil
waterat25℃for24h,thenhomogenizedincold
50umol·L-1phosphatebuffer(pH7.0).The
homogenatewascentrifugedat15000×g4℃for
10minandthesupernatant(extractMDA)content
wasassayedaccordingtoBailyetal.[7].
1.8 Enzymeextractionandassays
Forenzymeextraction,1.0gseedsweresoaked
indistiledwaterat25℃for12handthenhomoge-
nizedonicewith50mmol·L-1phosphatebuffer
(pH=7.0).Thehomogenatewascentrifugedat
15,000gfor20minandthesupernatantwasused
forenzymeassay.
Superoxidedismutase(SOD)activitywasde-
terminedbymeasurementofinhibitionofphoto-
chemicalreductionofnitrobluetetrazolium(NBT)
at560nm[8].The3mLreactionmixturecontained
50mmol·L-1phosphatebuffer(pH=7.8),0.1
mmol·L-1ethylenediaminetetra-acetic-acid(ED-
TA),13mmol·L-1 methionine,75μmol·L-1
NBT,16.7μmol·L-1riboflavinandenzymeex-
tract.Riboflavinwasaddedlastandthereaction
wasinitiatedbyplacingthetubesundertwo9W
fluorescentlamps.Thereactionwasterminatedaf-
323
草 地 学 报 第21卷
ter15minbyremovalfromthelightsource.Anil-
luminatedblank withoutproteingavethemaxi-
mumreductionofNBT,therefore,themaximum
absorbanceat560nm.SODactivityispresentas
absorbanceofsampledividedbyabsorbanceof
blank,givingthepercentageofinhibition.1unit
ofSODisdefinedastheamountrequiredinhibiting
thephotoreductionofNBTby50%.Theactivity
ofSODwasexpressedasU·mg-1protein.
Catalase(CAT)activitywasestimatedbythe
methodofGoelandSheoran[9].Thereactionmix-
turecontained0.6mLenzymeextract,0.1mLof
10mmolH2O2and2mL30mmolphosphatebuffer
(pH=7.0).Theabsorbancewasrecordedat240
nmimmediatelyafteradditionofenzymeextractat
anintervalof15sfor2min.Theblankwaswith-
outenzymeextract.
1.9 Statisticalanalysis
Dataweresubjectedtoanalysisofvarianceu-
singSAS8.0system.IftheF-testforafactorwas
significantintheANOVA,theleastsignificant
difference (LSD) was calculated to compare
means.
2 Results
2.1 Ultradryingmarkedlyenhancestheaging-re-
sistantcapabilityofC.arborescensseeds
Itwasimportanttoestablishwhethertheul-
tradrytreatmentcauseddamageandwhetherthe
damagecouldbeamelioratedwithslowimbibition.
ThedataconfirmedthatseedsofC.arborescens
driedto2.69% MCwereslightlysusceptibleto
imbibitionaldamage,buttheinjurycouldbecom-
pletelyrepairedbypre-humitificationtreatment,
andtheextentofthedamagewasnotsufficientto
reduceviability(Table1).
Afteracceleratedagingtreatment (45℃,2
d),theGPandVIofnon-ultradriedseedsmarked-
lydecreasedfrom 93.0% to17.6% andfrom
192.25to9.04respectively,butforultradried
seedsonlyslightlydecreased,therangesofdrop
wereGP4.4%~26.6%,VI76.52~148.1re-
spectively(Table2),especialymoisturecontent
of3.78%.TheseresultsshowedthattheGPand
VIofcontrolweredecreasedmorethanthatoful-
tradriedseeds.Itmeantthatultradriedseedswith-
incertain MClimitshadnonegativeeffectson
C.arborescensseedvigorandultradriedC.arbo-
rescensseedsbecame moretolerantofagingat
45℃,howevertheC.arborescensseedscouldnot
bedriedtooseverely.
TheMDAistheproductsoflipidperoxida-
tion.Therefore,thecontentsofMDAinseedscan
indicateitsdeteriorationdegree.Afterartificialag-
ing,theMDAcontentsofultradriedseedswere
lowerthanthatofcontrol(Table2).Thisresult
indicatedthatultradriedseedshadefficientantioxi-
dantdefensivesystemandthedeteriorationoful-
tradriedseedswaslessthanthatofcontrol.
Table1 EffectofprehumitificationonGPandVI
ofultradriedC.arborescensseeds
MC/% GP/% VI
10.87(CK) 93.0b 192.25±9.53c
5.89 RH 94.1b 192.10±7.60b
N-RH 93.0ab 190.34±6.64c
4.91 RH 94.7a 194.87±8.62ab
N-RH 94.3ab 192.11±4.56b
3.78 RH 95.4a 197.63±6.81a
N-RH 95.1a 193.65±3.79ab
2.69 RH 92.8ab 190.19±7.49b
N-RH 91.8c 188.21±3.41d
Note:RH-Standforprehumidificationbeforegerminationtrial;
N-RH-Standfornoprehumidificationbeforegerminationtrial.Dif-
ferentsmallettersamongthesamespeciesinthesamecolumnmean
significantdifference(P<0.05),thesameasbelow
Table2 EffectofartificialagingonGP,VIand
MDAcontentsofultradriedC.arborescens
seedswithvariousmoisturecontents
MC/% GP/% VI MDAcontents/nmol·g-1
10.87(CK) 17.6d 9.04e 63.27a
5.89 67.5c 43.90d 51.16b
4.91 80.3b 82.40c 40.62c
3.78 91.0a 121.11a 30.43b
2.69 86.4ab 106.42b 34.09b
2.2 EffectofartificialagingonactivitiesofSOD
andCATinultradriedandnon-ultradriedseeds
SODandCATareimportantscavengersof
freeradicalsandactivatedoxygen[10].Afteraccel-
eratedaging,activitiesofSODandCATofnon-ul-
tradriedandultradriedseedsalsodropped,butul-
tradriedseeds(MC3.78%and2.69%)hadmuch
higheractivitiesofSODandCATthanthatofnon
ultradriedseeds(Fig.1).Thismeansthattheself-
defencesystemofultradriedseedswouldbekept
betterthanthatofcontrol.
423
第2期 佟莉蓉等:华北驼绒藜超干种子抗老化生理特性的研究
Fig.1 SODandCATactivitiesofC.arborescensseedsindifferentmoisturecontentsafteracceleratedagingat45℃for2d
2.3 Effectsofultradryingandagingonfreeradical
level
Resultsshowedthatfreeradicallevelsoful-
tradriedseedsweremuchhigherthanthatofnon-
ultradriedseeds(control)(Table3).Thelower
theseedMCis,thehighertheleveloffreeradical
is.However,comparingwithfreeradicallevelsof
ultradriedseedsafteracceleratedagingtreatment,
freeradicallevelsofthecontrolseedsincreased
morerapidly.Agingincreasedonly19.89%offree
radicalinultradriedseeds,butincreased77.25%
offreeradicalincontrolseeds.However,freerad-
icallevelsofultradriedseedswerestilhigherthan
thatofcontrolseeds.Aftersoakingtheseedsin
thewaterfor24h,theleveloffreeradicalgreatly
decreased.Freeradicallevelofultradriedseedsfel
to73.13%,butfreeradicallevelofcontrolseeds
decreased36.63%.Theleveloffreeradicaloful-
tradriedseedswasmarkedlyaltered,lowerthan
thatofcontrolseeds.Itisquiteevidentthatability
offreeradicalscavengingofultradriedseedswas
strongerthanthatofcontrol.
Table3 Comparisonoffreeradicalcontentsinultradried
(MC3.78%)andnon-ultradried(MC10.87%)C.arborescens
seedsafteracceleratedagingat45℃for2day ×1014Spin·g-1
MC/% Non-aged
Aged
Non-soaked Soakedfor24h
10.87 68.27b 121.01b 10.87b
3.78 153.66a 184.22a 49.50a
3 DiscussionandConclusion
LittleresearchattentionhasbeengiventoC.
arborescensseeds.Inourresearch,seedsofC.ar-
borescensweredriedtowatercontentsoflessthan
5%,theviabilityandvigorwerenotstatisticaly
significantlyinfluenced;onthecontrary,theag-
ing-resistantcapabilitywasgreatlyenhanced(Ta-
bles1and2).Afteracceleratedaging,theresults
showedthatthe3.78% moisturecontentwasmore
appropriateforC.arborescensseedultra-drystor-
age.GPandVIwerekepthigherthannon-ultrad-
riedseeds,implyingthatnobiochemicalandbio-
physicalreactionmighthaveoccurredtoinjurethe
seedcelsundertheconditionsoflow MC(Table
1).
Duringseedaging,thelipidperoxidationoc-
cursandacomplexseriesofreactionsgeneratea
varietyofpotentialtoxicproductssuchasfreerad-
ical.Butgeneraly,ascavengersysteminseeds,
includingSODandCAT,caneffectivelypreventor
terminatethesereactionswhichpossiblyhappen
duringstorage[10].Duringourresearch,theactivi-
tiesofSODandCATsignificantlyincreasedafter
acceleratedaging(Fig.1).Theseresultsshowthat
thechangesofactivitiesofantioxidantenzymesare
closelyrelatedtodesiccationtoleranceandtheul-
tradryingdoesnotdestroytheenzymes.Theultra-
dryingtreatmentcanprolongtheseedstoragelife
byincreasingSODactivity[11].Simiarpatternof
resultswereobservedinourotherexperiment[12].
Freeradical-induceddamageplaysakeyrole
inseeddeteriorationduringaging[13].Afteraccel-
eratedaging,thefreeradicallevelofultradried
seedshashigherthanthatofnon-ultradriedseeds,
523
草 地 学 报 第21卷
butfinalyviabilityandvigorofultradriedseeds
washigherthanthatofcontrol(Tables2and3).
Moreoversoakingtreatment,thefreeradicallevel
ofultradriedseedsislowerthanthatofnon-ultrad-
riedseeds(Table3).DeterminationofMDAisa
convenientmethodofquantifyingtheextentoflip-
idperoxidation.Inourresearch,thecontentof
MDA decreasedafteracceleratedagingandwas
correlatedwiththeincreaseofSODandCATac-
tivities(Fig.1).Theresultsofthisexperiment
showthatthelipidperoxidationwasgreatlysup-
pressedundertheultradriedcondition.Thisim-
pliedthattheenzymesystemswerenotdestroyed
andhighactivitiesofantioxidantenzymeswere
keptinultradriedseeds.
Theimbibitiondamageisinevitabletoultrad-
riedseeds[14].Evenifthereisimbibitiondamage,
theprehumitificationofultradriedseedscanbere-
paired.Theeffectsofprehumidificationwerecor-
relatedtotherecoveryofthemembraneanden-
zyme,whichdecreasethefreeradicallevelandim-
provetheaging-resistantcapabilityoftheultrad-
riedseed.
Insummary,asuitableextentofultradrying
lessinjuredtheseedsbutstronglyenhancedaging-
resistantcapabilityandremainedtherecoveryof
themembraneofC.arborescensseeds.Thistech-
niquehaspracticalpotentialforthepreservationof
C.arborescensgermplasm.
Acknowledgement
TheauthorsrealyappreciateProfessorYijin
ofInnerMongoliaAgriculturalUniversityforpro-
vidingC.arborescensseeds.Theauthorswishto
thankanonymousrefereesforsuggestivecomments
andmodification.
References
[1] TsouCH,SA Mori.Seedcoatanatomyanditsrelationship
toseeddispersalinsubfamilyLecythidoideaeoftheLecythi-
daceae(TheBrazilNutFamily)[J].BotanicalBuletinAcade-
miaSinica,2002,43(1):37-56
[2] ZhengGH,JingXM.Ultradryseedstoragecutscostofgene
bank[J].Nature,1998,393(4):223-224
[3] WangXF,JingXM,JLin.Starchmobilizationginultradried
seedofmaize(ZeamaysL.)duringgermination[J].Journalof
IntegrativePlantBiology,2005,47(2):443-451
[4] LiY,FengHY,ChenT,etal.PhysiologicalresponseofLi-
moniumaureumseedstoultradrying[J].JournalofIntegrative
PlantBiology,2007,49(4):569-575
[5] InternationalSeedTestingAssociation (ISTA).Internation
rulesforseedtesting[S].SeedScienceandTechnology,1993
[6] HuJS,ZhuC,ZengGW,etal.Effectofaging-resistanceof
ultra-driedsafflowerseedsanditsmechanism[J].ActaPhyto-
physiologicaSinica,1999,25(2):171-177
[7] BailyC,BenamarA,CorbineauF.Changesinmalondialde-
hydecontentandinsuperoxidedismutase,catalaseandgluta-
thionereductaseactibitiesinsunflowerseedsasrelatedtodete-
riorationduringacceleratedaging[J].PhysiologyPlant,1996,
97(4):104-110
[8] GiannoplitisCN,RiesSK.Superoxidedismutasepurification
andquantitativerelationship with water-solubleproteinin
seedlings[J].PhysiologyPlant,1977,59(3):315-318
[9] GoelA,SheoranIS.Lipidperoxidationandperoxide-scaven-
gingenzymeincottonseedsundernaturalaging[J].Biology
Plant,2003,46(2):429-434
[10]PriestleyDA.Morphologicalstructuralandbiochemicalchan-
gesassociatedwithseedaging[C]//PriestleyDA,ed.Seed
aging.IthacaandLondon:CornelUnivPress,1986:125-165
[11]McDonaldMB.Seeddeterioration:physiology,repairandas-
sessment[J].SeedScienceTechnology,1999,27(1):177-237
[12]TongLi-rong,HanJian-guo.IncreasingstorabilityofCera-
toidesarborescensseedsinultradrystorage[J].AfricanJournal
ofBiotechnology,2010,9(29):4601-4605
[13]PinheroRG,PaliyathG,YadaRY,etal.Modulationof
phospholipaseDandlipoxygenaseactivitiesduringchiling:
Relationtochilingtoleranceofmaizeseedlings[J].Plant
PhysiologyandBiochemistry,1998,36(3):213-224
[14]ZhengG H.Ultradryseedstorage:Improvestrategyand
technologyforgermplasmconservation[J].ChineseBiodiver-
sity,1994,20(2):61-65
(责任编辑 李美娟)
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