全 文 ：第 30卷 第 1期
2009年 1月 　　　　　　　　　　
华南农业大学学报
JournalofSouthChinaAgriculturalUniversity　　 　　　　　　　　
Vol.30, No.1
Jan.2009
　Receiveddate:2008-03-11
Biographies:DahanayakeNILANTHI(1969—), female, SriLankan, Ph.Dstudent, sponsoredbyChinesegovernment;Corre-
spondingauthor:YANGYue-sheng(1957—), male, professor, Ph.D., E-mail:ysyang@scau.edu.cn
Foundationitem:ThisresearchwassupportedbyagrantfromtheScienceandTechnologyBureauoftheGuangzhouMunicipal
Government(2004Z3-E5021)
EvaluationforPlantRegenerationPotentialofRoot
ExplantsinEchinaceapurpurea
DahanayakeNILANTHI1 , ZHAOFu-cheng1 , YANGYue-sheng1, 2 , WUHong1, 2
(1 ColegeofLifeSciences, SouthChinaAgriculturalUniversity, Guangzhou510642, China;
2 ResearchCenterforMedicinalPlants, SouthChinaAgriculturalUniversity, Guangzhou510642, China)
Abstract:Forevaluationoftheplantregenerationpotentialofrootexplants, explantsofroot, leafandpetioleweretaken
frominvitrogrownpurpleconeflower, EchinaceapurpureaL.plantletsandculturedonadventitiousbudinducingmediawith
differentcytokininsandauxinsatvariousconcentrations.Inmostofthecases, theregenerationpotentialofrootexplantswas
muchhigherthanthatofleafonesandsimilartothatofpetioleexplants, andacombinationof0.3mg/Lbenzyladeinewith
0.01 mg/Lnaphthaleneaceticacidwasthemostefectivecombinationandconcentrationsforinducingadventitiousbudregen-
eration.Althoughthebestresultofbudregenerationwasobtainedfromcultureofpetioleexplants, agoodresultinregenera-
tionrateof100% andahighnumberof1.75budsperexplantwereobtainedfromcultureofrootexplants.Budsregenerated
fromrootexplantsinitiatedrootsandbecameintactplantsreadilyupontransfertoamediumcontaining0.01mg/Lnaphthale-
neaceticacid.Resultsoftheexperimentsindicatedthatrootwasanidealexplantsourceforrapidpropagationbymeansoftis-
suecultureinthisplantspecies.
Keywords:Echinaceapurpurea;invitroculture;plantregeneration;root;explant
CLCnumber:S717.1　　　　　Documentcode:A　　　　ArticleID:1001-411X(2009)01-0051-04
松果菊根外植体植株再生能力的评价
DahanayakeNILANTHI1 , 赵福成 1 , 杨跃生1, 2 , 吴　鸿 1, 2
(1华南农业大学 生命科学学院 , 广东 广州 510642;2华南农业大学 药用植物研究中心 ,广东 广州 510642)
摘要:为了评价松果菊 EchinaceapurpureaL.根外植体的再生能力 , 将从松果菊无菌小苗得到的根外植体和叶片以及
叶柄外植体接种到含有不同种类和浓度的细胞分裂素和生长素的培养基上 ,诱导不定芽的再生.结果表明 ,在多数
情况下 , 根外植体的再生能力显著高于叶片 , 和叶柄类似.0.3mg/L的苄基腺嘌呤 和 0.01mg/L的萘乙酸是诱导根
外植体不定芽再生最合适的激素种类和质量浓度组合.根外植体培养的不定芽再生频率为 100%, 每个根外植体得
到再生芽 1.75个.当把这些由根再生的不定芽从母体组织切开并转移培养到含有 0.01 mg/L萘乙酸的培养基后 ,
很容易生根并成为完整的植株.可见根是组培快繁松果菊理想的外植体材料.
关键词:松果菊;离体培养;植株再生;根;外植体
　　Sincepurpleconeflower, EchinaceapurpureaL.,
isoneofthetopthreemostimportantherbaceousmedic-
inalplantsintheworld[ 1] rankingbesidegingkoand
ginseng, applicationofbiotechnicalmeansforpromoting
theproductionofthisplanthasbeenemphasizedrecently
[ 2] .Althoughpurpleconeflowerseedlingsareroutinely
propagatedbyseeds, quiteaportionoftheseedsare
pooringermination[ 3] andmostpopulationsarestrictly
self-incompatible[ 4] andcultivationusingtheseseedsre-
sultsinmanyproblemssuchasdificultyinmanagements
andunreliabilityoftheproductsduetothevariationin
thegrowingbehavioroftheseed-grownplants.Toover-
cometheseshortages, oneofthestrategiesistoproduce
geneticalyidenticalplantletsinlargescalebymass
propagationofplantletsusinginvitroculturemeans.
Plantregenerationinpurpleconeflowerhasbeen
reportedbyculturingleafexplants[ 5-6] andpetioleex-
plants[ 7] , andtheuseofroottissueassourceofex-
plantforplantregenerationpurposeshasnotbeenrepor-
tedinthisspeciessofar.Inourpreliminaryexperi-
ments, weobservedthatshootsofpurpleconeflower
rootedeasilyunderinvitrocultureconditionsandquite
aquantityofroottissuecouldbeproducedalongwith
thegrowthoftheshoots.Withtheseobservationsand
thefactsthatroottissuehasbeenusedsuccessfulyfor
regenerationinarangeofotherplantspecies[ 8-11] , re-
centlyweinvestigatedtheregenerationpotentialofpur-
pleconeflowerrootbycomparingwithleafandpetiole.
Detailsoftheexperimentsarepresentedinthispaper.
1　Materialsandmethods
1.1　Plantsource
Seedsofpurpleconeflowerwerepurchasedatasu-
permarketprovidedbytheCompanyofPlantationProd-
ucts(Norton, MA02766, USA)andcultivatedatthe
ChineseMedicinalPlantGardeninthecampusofSouth
ChinaAgriculturalUniversity.Seedswerecolectedfrom
theseseed-grownplantsandusedforthepresentstudies.
1.2　Establishmentofasepticseedlings
Seedsweresurface-sterilizedbyimmersingin
φ=70% ethanolfor1 minandsoakinginaφ=0.1%
mercuricchloridesolutionfor10 minfolowedbyφ=
1% sodiumhypochloritesolutioncontainingonedropof
Tween20 per50 mLfor10 min.Sterilizedseedswere
thenrinsedthreetimesinsteriledeionizedwaterandin-
oculatedonaPhytagel-solidifiedmediumcomprisedof
half-strengthMS(MurashigeandSkoog)[ 12] salts, 10
g/Lsucroseand500 mg/Llactalbuminhydrolysis.Af-
ter14dcultureunderdim-light, germinatedseedswere
transferedtoaPhytagel-solidifiedmedium containing
ful-strengthMSsaltsand10 g/Lsucrose, andthepH
wasadjustedto6.0.Cultureswerethenincubatedina
roomof25 -27 ℃ andwith12 hphotoperiodunder
cool-whitelight(about50 μmol·m-2·s-1).
1.3　Regenerationandrootingcultures
Leaf, petioleandrootexplantswereexcisedfrom2-
monthsoldseedlingsandculturedontotheMSbasalme-
diumwithvariouscombinationsofplantgrowthregula-
tors.Incultureofleafexplants, leafsectionsofabout
0.5cm2 wereplacedonmediawiththeadaxialsurface
towardthemedia.Petiolesandrootswerecutintoabout
5 mmandculturedbylayingonthemedia.Althetreat-
mentswerekeptinthesameroomasmentionedabove.
Asfortherootingcultureoftheregeneratedbuds, the
budswereexcisedfromthemothertissueandculturedon
Phytagel-solidifiedful-strengthMSmediawith15 g/L
sucroseand0.01 mg/Lnaphthaleneaceticacid(NAA).
1.4　Datacolectionandanalysis
Regenerationcultureswereevaluated40dafterin-
itiation.Alexperimentswererepeatedatleastonce
withaminimumoffourreplicates, eachwitheightex-
plantsperbotle.Statisticalanalysiswascariedoutu-
singDuncan sMultipleRangeTest.
2　Results
2.1　Comparisononplantregenerationpotentialof
diferentexplantsonthemediawithBA
　　Explantstakenfromleaf, petioleandrootwerein-
oculatedonMSbasalmediumwithbenzyladenine(BA)
atvariousmassconcentrations(0.1, 0.3, 0.9, 2.7
mg/L)andNAAat0.01mg/L.Mostexplantsformed
calusatthecutsurfaceintwoweeks, andthecalus
begantoproducebudprimordiainanotheroneweek.
Theprimodiadevelopedintoadventitiousbudsafter-
wards.Itwasfoundthatmedium supplementedwith
0.3mg/LBAyieldedthebestresults, alowingalthe
root-andpetiole-explantsandahigherpercentageof
leaf-explantstoregenerateadventitiousbuds(Tab.1).
LowerorhigherconcentrationofBAwaslessefective;
especialywhenhigherconcentrationofBAwasused,
notonlythefrequencyofregenerationdecreased, the
qualityoftheregeneratedbudsalsodroppedasthe
symptomsofvitrificationonthebudsbecameevident.
Althoughdiferenceinregenerationabilitywasobserved
amongthethreekindsofexplants, qualityoftheregen-
eratedbudsfromaltheexplantswerealalike.
52 　　 华　南　农　业　大　学　学　报　　　 第 30卷　
Tab.1　Adventitiousbudregenerationfromdiferentexplantsonthemediawithvariousmassconcentra-
tionofBAand0.01mg/LNAA1)
Explant
0.1 mg· L-1 BA 0.3mg· L-1 BA 0.9 mg· L-1 BA 2.7 mg· L-1 BA
Regene-
ration/%
No.buds
perexplant
Regene-
ration/%
No.buds
perexplant
Regene-
ration/%
No.buds
perexplant
Regene-
ration/%
No.buds
perexplant
Leaf 37.5c 0.28c 75.0 b 1.05 b 12.5 b 0.13 b 22.5b 0.10c
Petiole 87.5b 0.80b 100.0 a 1.84 a 87.5 a 0.83 a 40.0a 0.25b
Root 100.0a 1.13a 100.0 a 1.75 a 87.5 a 1.00 a 40.0a 0.40a
　1)Meansfolowedbythesameleterineachcolumnarenotsignificantlydifferentat5% levelinDuncan sMultipleRangeTest
2.2　Comparisononplantregenerationpotentialof
diferentexplantsonthemediawithKTand
TDZ
　　Twoothercytokinins, KT(kinetin)andTDZ
(thidiazuron)weretestedseparatelyatvariousmasscon-
centrations(0.1, 0.3, 0.9, 2.7mg/L)incombination
with0.01 mg/LNAAfortheirefectsofinducingbud
regenerationfromexplants.Althoughrootandpetioleex-
plantshadhigherregenerationratesthanthoseofleafex-
plants, regenerationratesofalthetreatmentsweremuch
lowerthanthoseculturesappliedBA(datanotshown).
2.3　Comparisononplantregenerationpotentialof
diferentexplantsonthemediawithNAA
　　Onthebasesoftheaboveexperiment, BAwas
usedat0.3 mg/LandNAAwastestedatvariousmass
concentrations(0, 0.01, 0.05, 0.15, 0.75 mg/L).
ResultsoftheexperimentsaresummarizedinTab.2.It
isclearthatconcentrationofNAAalsoplayedavery
important role in regulating shoot regeneration.
Explantsofrootandpetiolewerefoundtopossesshigher
Tab.2　Adventitiousbudregenerationfromdiferentexplantsonthemediawithvariousmassconcentra-
tionofNAAand0.3 mg/LBA1)
Explant
0mg· L-1 NAA 0.01mg· L-1 NAA 0.05mg· L-1 NAA 0.15mg· L-1 NAA 0.75mg· L-1 NAA
Regene-
ration/%
No.buds
perexplant
Regene-
ration/%
No.buds
perexplant
Regene-
ration/%
No.buds
perexplant
Regene-
ration/%
No.buds
perexplant
Regene-
ration/%
No.buds
perexplant
Leaf 50.00a 0.71b 59.4b 1.01b 43.75a 0.63b 20.83b 0.21b 8.33c 0.08b
Petiole 56.25a 1.07a 91.66a 1.54a 45.83a 1.13a 40.00a 0.83a 29.16b 0.46a
Root 59.40a 1.04a 93.75a 1.73a 50.00a 1.27a 45.00a 0.83a 40.0a 0.44a
　1)Meansfolowedbythesameleterineachcolumnarenotsignificantlydifferentat5% levelinDuncan sMultipleRangeTest
budregenerationpotentialinaltheNAAconcentrations
tested, andunderthemostsuitableNAAconcentration
of0.01 mg/L, explantsofrootandpetiolehadatleast
30% higherregenerationpotentialthanthoseofleaf.
2.4　Comparisononplantregenerationpotentialof
diferentexplantsonthemediawithIBA
　　IBA(indo-butyricacid)isananalogtoNAAand
variousmassconcentrationsofIBA(0 , 0.01, 0.05,
0.15, 0.75mg/L)weretestedincombinationwithBA
at0.3 mg/L.FromtheresultsshowninTab.3 and
comparedwiththoseinTab.2, itisclearthatIBAhad
almostthesamefunctionasthatofNAAinregulating
budregeneration:0.01 mg/Lwasagainthemostsuit-
ableconcentrationandexplantsofrootandpetiolehad
significantlyhigherregenerationpotentialthanthoseof
leafinmostoftheIBAconcentrationstested.
Tab.3　Adventitiousbudregenerationfromdiferentexplantsonthemediawithvariousmassconcentra-
tionofIBAand0.3 mg/LBA1)
Explant
0mg· L-1 IBA 0.01mg· L-1 IBA 0.05mg· L-1 IBA 0.15 mg· L-1 IBA 0.75mg· L-1 IBA
Regene-
ration/%
No.buds
perexplant
Regene-
ration/%
No.buds
perexplant
Regene-
ration/%
No.buds
perexplant
Regene-
ration/%
No.buds
perexplant
Regene-
ration/%
No.buds
perexplant
Leaf 50.0b 0.68b 55.0b 0.94b 44.2a 0.69b 25.0b 0.13c 12.5b 0.06c
Petiole 55.0ab 0.94a 85.2a 1.31ab 46.0a 1.01a 37.5a 0.69b 26.0ab 0.36b
Root 60.0a 0.88a 91.5a 1.69a 48.0a 1.19a 37.5a 0.88a 36.0a 0.50a
　1)Meansfolowedbythesameleterineachcolumnarenotsignificantlydifferentat5% levelinDuncan sMultipleRangeTest
53　第 1期 　　　　　DahanayakeNILANTHI等:松果菊根外植体植株再生能力的评价 　　　
2.5　Rootingoftheregeneratedbudsandgrowth
oftheregeneratedplants
　　Budsregeneratedfromrootexplantswereisolated
fromthemothertissuesandculturedonful-strengthMS
mediawith0.01 mg/LNAA.Uponculture, thesebuds
developedrootsfromthebaseseasilyandbecameintact
plants.Theseplants, afterbeingtransplantedtothe
soil, grewandsetflowersnormaly.
3　Discussion
Therehavebeenafewreportsonplantregenera-
tioninpurpleconeflowerbyculturingtissuesofleafand
petioleasexplants[ 5-7, 13] ;Resultsofthepresentexperi-
mentsindicatedthatexplantsofroothadsignificantly
higherregenerationpotentialthanthoseofleafandare
comparablewiththoseofpetiole.Itwasfoundthatthe
respondofrootexplantstocultureconditionsforregen-
erationwasverysimilartothoseofleafandpetiole,
havingthehighestregenerationeficiencyonmediawith
0.3mg/LBAand0.01 mg/LNAAorIBA.
Recentlywehavesuccessfulyobtainedhaploid
plantregenerationinpurpleconeflowerbyanthercul-
tureforthefirsttime[ 14] , andasubsequentprogram
whichaimsatbreedingvarietieswithhybridvigorby
useofthehaploidmaterialsisundertaken.Propagation
ofelitegenotypesbytisueculturehasimportantappli-
cationvalueinpurpleconeflowerproduction, especialy
whenthegenotypesofthevarietiesareheterogenous.
Sincerootisoneimportantpartinpurpleconefloweroc-
cupying20 orevenhigherpercentageofthetotalfresh
massoftheplant, highregenerationpotentialofrootand
equalyhighqualityoftheregeneratedbudsformrootex-
plantsindicatingthatrootisanimportantsourceofex-
plants.Byusingrootasexplantmaterialtogetherwith
petioleandleaf, morenumberofplantletscanbepro-
ducedtomeetthedemandofincreasedcultivationarea.
References:
[ 1] 　BAUERR.Chemistry, analysisandimmunologicalinves-
tigationsofEchinaceaphytopharmaceuticals[ M] ∥WAG-
NERH.ImmunomodulatoryAgentsfrom Plant.Basel,
Switzerland:BirkhauserVerlag, 1999:41-48.
[ 2] 　ABBASIBH, SAXENAPK, MURCHSJ.Echinaceabi-
otechnology:Challengesandopportunities[ J] .InVitro
CelDevBiol-Plant, 2007, 43:481-492.
[ 3] 　LIPeng, WUHong, GENGShi-lei, etal.Germinationand
dormancyofseedsinEchinaceapurpurea(L.)Moench(As-
teraceae)[J] .SeedSciTechnol, 2007, 35:9-20.
[ 4] 　MCGREGORRL.ThetaxonomyofthegenusEchinacea
(Compositae)[ J] .UniversityKansasSciBul, 1968, 11
(8):113-142.
[ 5] 　KOROCHA, JULIANIHR, KAPTEYNJ, etal.Invitro
regenerationofEchinaceapurpureafromleafexplants[ J] .
PlantCelTissOrganCult, 2002, 69:79-83.
[ 6] 　MECHANDASM, BAUMBR.Directshootregeneration
fromleafsegmentsofmatureplantsofEchinaceapurpurea
(L.)Moench[ J] .InVitroCelDevBiol-Plant, 2003,
39:505-509.
[ 7] 　CHOFFEKL, VICTORJMR, MURCHSJ, etal.In
vitroregenerationofEchinaceapurpureaL.:Directsomat-
icembryogenesisandindirectshootorganogenesisinpeti-
oleculture[ J] .InVitroCellDevBiol-Plant, 2000, 36:
30-36.
[ 8] 　BHATSR, CHITRALEKHAP, CHANDELKPS.Re-
generationofplantsfromlong-termrootcultureoflime,
Citrusaurantifolia(Christm)Swing[ J] .PlantCellTiss
OrganCult, 1992, 29:19-25.
[ 9] 　KNOLLKA, SHORTKC, CURTISIS, etal.Shootre-
generationfromculturedrootexplantsofspinach(Spinacia
oleraceaL.):Asystemforagrobacteriumtransformation
[ J] .PlantCellRep, 1997, 17:96-101.
[ 10] VINOCURB, CARMIT, ALTMANA, etal.Enhanced
budregenerationinaspen(PopulustremulaL.)rootscul-
turedinliquidmedia[ J] .PlantCellRep, 2000, 19:
1146-1154.
[ 11] CHAUDHURIKN, GHOSHB, JHAS.Theroot:apo-
tentialsourceofcompetentcellsforhigh-frequencyregen-
erationinTylophoraindica[ J] .PlantCelRep, 2004,
22:731-740.
[ 12] MURASHIGET, SKOOGF.Arevisedmediumforrapid
growthandbioassayswithtobaccotissuecultures[ J] .
PhysiolPlant, 1962, 15:473-497.
[ 13] ZOBAYEDSMA, SEXENAPK.Auxinandadarkin-
cubationincreasethefrequencyofsomaticembyogenesis
inEchinaceapurpureaL.[ J] .InVitroCelDevBiol-
Plant, 2003, 39:605-612.
[ 14] ZHAOFu-cheng, NILANTHID, YANGYue-sheng, et
al.Anthercultureandhaploidplantregenerationinpurple
coneflower(EchinaceapurpureaL.)[ J] .PlantCellTiss
OrganCult, 2006, 86:55-62.
【责任编辑　李晓卉】
54 　　 华　南　农　业　大　学　学　报　　　 第 30卷