全 文 ：StudyonTissueCultureandEffectiveCloneEstablish-mentofHemisteptalyrataBunge
ZHANGWen, GUOXiao-dan, WANGYan, XUNa, JIANGChang-yang＊
ColegeofLifeScience, LiaoningNormalUniversity, Dalian116029
Abstract　[Objective] TheresearchaimedtostudytherapidpropagationtechnologyandestablishefectivecloneofHemisteptalyrataBunge.
[ Method] WithtenderstemofHemisteptalyrataBungeasmaterial, theconditionsneededincalusesinductionanddiferentiation, adventitiousbuddiferentiationandradication, testtubeseedlingcutingandtransplantationwerestudied.[Result] Theresultsshowedthattheoptimumme-
diumforgranulatedcalusesinductionfromtenderstemwasMS+BA0.3mg/L+2, 4-D1-1.5mg/L, forgranulatedcalusesandadventitious
buddiferentiationwasMS+AgNO3 1.5mg/L+BA0.4mg/L+NAA0.1mg/L.1/2MS+IAA0.6mg/Lwassuitablefortesttubeseedlingroo-tingandregeneration, andcinderwasusedastransplantationandcutingsubstrate.[ Conclusion] Thisstudywilprovidethescientificreference
forchoosingthefeasiblemediumintissuecultureofHemisteptalyrataBunge.
Keywords　HemisteptalyrataBunge;Caluses;Clone;Rapidpropagation
Received:December8, 2008　　Accepted:February24, 2009
＊Correspondingauthor.E-mail:changyangjiang@126.com
　　HemisteptalyrataBungeisbiyearlyvegetablethatbe-
longstoHemisteptaBungeinCompositaeanditusualygrows
inwasteland, roadside, undergrowthandcoastalsandyland
andsoon[1-3] .HemisteptalyrataBungecanbeusedasmed-
icineandhastheeficaciessuchasheat-clearinganddetoxif-
ying, eliminatingswelinganddispersingbloodclots, besides
itcantreatfracture, cataractandsoon[ 4-5] .Becausethe
medicinalvalueandediblevalueofHemisteptalyrataBunge
areemphasized, itseconomicvalueisincreasingyearby
year, sotheresourceofwildHemisteptalyrataBungeisde-
creasedgradualy.InordertoprotectwildHemisteptalyrata
Bunge, thecultureoftissueandestablishmentofcloneare
studiedinthisexperiment.Although, therearemanyreports
abouttissuecultureofmanyspeciesofCompositaeatpres-
ent[ 6-15] , therehasnoreportaboutHemisteptalyrataBunge
yet.
MaterialsandMethods
Materials
HemisteptalyrataBungewerecolectedfromShimenshan
inDalianCity, LiaoningProvince.
Methods
Acquirementofasepsismaterial　Thetenderstemandpet-
ioleofbasalleafwerecutintonodandstalknodaround4 or5
cm, thentheywereputinto500 mljarandfirstlywashedby
waterfor15 minthenwashed3timesby0.05%Amwayliquid
andtwicebyasepticwater, finalytransferredonsuper-clean
worktable.Underasepticcondition, 70%to75% ethanolwas
usedtodisinfectfor30 s, then0.05% HgCl2 wasusedtodis-
infectfor3min.Aftertheseoperations, 0.03%HgCl2 solution
wasusedtodisinfectfor13 min, finalyasepticwaterwas
usedtowash5 timestoobtainasepsismaterial.
Culturecondition　MSand1/2 MSweretakenasbasic
mediawithdifferentkindsofcytokininandauxinatdifferent
concentration.Theagarcontentinsolidmediumwas4.5g/L
while, sucrosecontentincalusesinductionanddifferentiation
mediaofcaluseswere30g/Landsucrosecontentinrooting
mediumwas15 g/L.Thelightintensitywasaround2 000 lx
andiluminationtimewas12 h/d, besides, pHwasfrom 5.6
to5.8 andculturetemperaturewas(25±1)℃.
Influencesofauxinatdifferentconcentrationoninducing
caluses　Theaseptictenderstemandpetiolewerecutinto
nodandstalknodaround0.1 or0.2 cmtheninoculatedinto
IBA, 2, 4-Dand0.3 mg/LMS+6-BAofNAAculturemediaat
diferentconcentrationstoconductinducingcultureofcalu-
ses.Everyculturemediumwasinoculated100 experimental
materialsthentheobservationandrecordwerecarriedout50
dlater.
Influencesofdifferentculturemediaonthedifferentiation
ofcaluses　Thegranularcalusesofsubculturewereinocu-
latedonbasicculturemediacontaining1.5 mg/LMS+
AgNO3 and1.5 mg/LMS+AgNO3 andNAAculturemediacontaining6-BAatdifferentconcentrationstoconductdiferen-
tiationculture.Everyculturemediumwasinoculated100 gran-
ularcalusesandhad5duplicatetests.Theexperimentalma-
terialswereculturedunderdarkandiluminationconditions.
Influencesofdifferentculturemediaonrootingcultureof
adventitiousbud　Thesubculturedadventitiousbudover
0.6 cmwithvigorousandplexiformgrowthwerecutofthen
inoculatedonIAAatdiferentconcentration(0.2, 0.6, 1.0, 1.4
mg/L)and1/2MSculturemediumof2, 4-Datdifferentcon-
centration(0.2, 0.6, 1.0 and1.4 mg/L)toconductrooting
culture.Everytreatmenthad100adventitiousbudand3 dupli-
catetests, thentherootedplantandrootnumberofperplant
wererecordedandtherootingratewascalculated.
Theplantletswiththehighestrootingrateandroot
numberwerecutinto1.5 cmstem withoneterminalbud
andoneortwolateralbud, thentheywereinoculatedin
sameculturemediatoconductrootingsubculture.Under
theunchangedcultureconditions, theneededtimeand
propagationcoefficientofeverygenerationwerecalculat-
ed.Everyexperimenthad100 explantsand3 duplicate
tests.
Transplantandcuttingofplantlet　①Transplant.Thepla-
ntletswereseedlingadaptationaround4 000 lxilumination
for3 dafteropeningbotlestopperofcuttlebottlethenthe
sewndhalfpartafterwashingoutculturemediarootswere
transplantedinnurserybedorflowerpotwhichcontained5to
7 cmfurnaceashandslaginsurfacelayerandfertilegarden
AgriculturalScience＆Technology, 2009, 10(1):47-50, 104
Copyright 2009, InformationInstituteofHAAS.Alrightsreserved. AgriculturalBiotechnology
mouldinunderlayer.Thesoilwaswateredwelandhumidity
wasabove90%, whiletemperaturewasaround25 ℃ and
directiluminationwasavoided, thenobservationandrecord
werecarriedout30dlater.Therewere4 transplantsand800
plantsweretransplantedeverytime.②cutting.Theupper
halfoftransplantedplantletswereputinto100 mg/LIAAaf-
tercutofthelowestleaffortreating4 min.Thecutings
weretransplantedonthenurserybedwhichhadFurnaceash
andslagonsurface, gardensoilatbotom and1 cm key
hole, besidesitwaswateredwel.Theconditionsofcutings
wereassameastheseoftransplantthentheobservation
andrecordwereconducted30 dlater.Therewere3 cutings
and800 plantsofeverycuting.
Transplantationofplantlet　 Thesurvivedplantletsof
HemisteptalyrataBungeaftertransplantandcutingwere
transplantedonhilsideofcolectionsitethentheywereplan-
tedaccordingtowildconditions, besidestheobservationand
recordwerecarriedoutatcertaintime.
ResultsandAnalysis
Influencesofauxinatdiferentconcentrationoncaluses
induction　15 dafterinoculation, therehadyelowcaluses
formedonculturemediawheretenderstemculture.50 daf-
terinoculation, itwasconcludedfromTable1 thatonculture
mediawithdiferentIBA, thecalusesofpetioleandtender
stemwerenotinduced, whileonculturemediawithdiferent
2, 4-DandNAA, thecalusesofpetioleandtenderstem
wereinduced, however, theinductionrateandgrowthpoten-
tialhadcertaindiference.Itwasconcludedfrominduction
rateandgrowthpotentialofcaluses, theefectofculture
mediacontainingdifferent2, 4-Dwasbeterthanthatofcul-
turemediacontainingdifferentNAA, especialy, whenthe
culturemediacontaining1.0 mg/Land1.5 mg/L2, 4-D
couldnotonlyhave100%inductionratebuthadgreengran-
ularinducedcaluses.Generalyspeaking, thiskindofcalu-
seshaddiferentiationability[ 16-17] .Thecalusesinducedon
culturemediumcontaining1.0 mg/Land1.5 mg/L2, 4-D
wereconductedsubcultureonsameculturemedia.After3
duplicatetests, theobservationof6 generationineachsub-
culturedemonstratedthatthecultureperiodofsubculture
wasshortento40 dandineverycultureperiod, eachcalus
aggregatecoulddiferentiate28.6 new granuleandtheir
growthwerebasicalysame.Thephenomenonshowedthat
MS+0.3 mg/L6-BA+1.0 to1.5 mg/L2, 4-Dwastheopti-
mumculturemediaforinducinggranularcalusesoftender
stemofHemisteptalyrataBunge.
Influencesofdifferentculturemediaonthedifferentia-
tionofgranularcaluses
15 dafterinoculation, calusaggregatesinsomeculture
mediawerestartingdiferentiation.Theobservationwascon-
ducted50dlateranditwasconcludedfromTable2 thatcalu-
seswerenotdiferentiationunderdarkconditionwhilethey
werealdifferentiationunderiluminationcondition;however,
theinductionrateoftakingMSasbasicmediumwashigher
thanthatoftaking1/2 MSasbasicmedium.Particularly,
thedifferentiationrateonculturemediacontainingMS+1.5
mg/LAgNO3 +0.4 mg/L6-BA+ 0.1 mg/LNAAwas
100%, thegrowthpotentialofdiferentiatedadventitiousbud
wasgood.Itwasconcludedfromobservationthatthedifer-
entiationrateofgranularcaluswas79% aftercultured30d,
besides, thereweremanyadventitiousbudsweregrownon
thebasalpartofdiferentiatedadventitiousbud.Aftercultured
for50 d, everycaluscoulddiferentiate2.1 adventitiousbuds
withabove0.5cmheightandgoodgrowthpotential.Thedifer-
entiationculturedadventitiousbudswerecutoffrombasalpart
theninoculatedonthesameculturemediatoconductdiferentia-
tionsubculture.After3duplicatetests5 timesofeverysubcul-
tureshowedthattheperiodofsubculturewasshortenedto40d.
Oneadventitiousbudineverysubculturecouldform 4.8 plexi-
formadventitiousbudswithvigorousgrowthandmorethan0.6
cm.Theresultsofexperimentmentionedabovedemonstrated
thatculturemediacontainingMS+1.5mg/LAgNO3 +0.4mg/L
6-BA+0.1 mg/LNAAwastheoptimumculturemediafordifer-
entiationofgranularcalusesandadventitiousbudsofHemistep-
talyrataBunge.
Table1　Influencesofauxinondiferentconcentrationsoncaluses
induction
Typeof
auxins
Concentration
mg/L
Typeof
materials
Induced
numberof
caluses
Induction
rate
%
Growth
potential
IBA 0.5 Petiole 0 0 -
0.5 Tenderstem 0 0 -
1.0 Petiole 0 0 -
1.0 Tenderstem 0 0 -
1.5 Petiole 0 0 -
1.5 Tenderstem 0 0 -
2, 4-D 0.5 Petiole 47 47 +
0.5 Tenderstem 76 76 ++
1.0 Petiole 56 56 +
1.0 Tenderstem 100 100 +++
1.5 Petiole 89 89 ++
1.5 Tenderstem 100 100 +++
NAA 0.5 Petiole 34 34 +
0.5 Tenderstem 63 63 ++
1.0 Petiole 42 42 +
1.0 Tenderstem 76 76 ++
1.5 Petiole 53 53 ++
1.5 Tenderstem 94 94 ++
+, ++and+++meantnormal, relativelygoodandgoodgrowth
potentialrespectively.-meantnogrowth.
Influencesofdifferentculturemediaonadventitiousroot
10 dafterculture, itwasconcludedfromtheobservation
thatcalusesgrewaroundsomeadventitiousbudsinculture
mediacontaining2, 4-Datdifferentconcentration, however,
rootprimordium couldnotbeformed, whileculturemedia
containingIAAatdiferentconcentrationcouldinducethe
formofrootprimordium.Whenculturedfor25 d, themateri-
alsinoculatedinculturemediacontaining2, 4-Dcouldnotin-
duceroots, whileinculturemediacontainingIAA, theycould
induceroots.Particularly, whentheIAAwas0.6 mg/Lin
culturemedia, notonlytherootingratewas100%, butalso
rootnumberwas7.2 perplant(Table3)andplantletwasa-
round5 cmheightwithvigorousgrowth.Theplantletscul-
turedonthismediawereconductedsubcultureunderthe
samecultureconditionandthefirstgenerationplantletswith
vigorousgrowthwereobtainedaftercultured23 d, besides,
thepropagationcoeficientofeverygenerationwas3.2 after
7 generationssuccessiveculture.Thegrowthpotentialand
propagationcoeficientofplantletswereunchanged.There-
sultsmentionedabovedemonstratedthat1/2MS+0.6 mg/L
IAAwastheoptimum culturemediaforrootingandrooting
subcultureofplantletsofHemisteptalyrataBunge.
48 AgriculturalScience＆TechnologyVol.10, No.1, 2009
Table2　Influencesofdiferentculturemediaoncalusesdifferentiation
Condition Culturemedium Diferentiatednumberofcaluses Diferentiationrate∥% Growthpotential
Ilumination 1/2MS+0.4mg/L6-BA+0.1 mg/LNAA 86 86 +++
1/2MS+0.4mg/L6-BA+0.3 mg/LNAA 51 51 +
1/2MS+0.8mg/L6-BA+0.1 mg/LNAA 45 45 ++
1/2MS+0.8mg/L6-BA+0.3 mg/LNAA 32 32 +
MS+0.4mg/L6-BA+0.1mg/LNAA 100 100 +++
MS+0.4mg/L6-BA+0.3mg/LNAA 56 56 +
MS+0.8mg/L6-BA+0.1mg/LNAA 77 77 ++
MS+0.8mg/L6-BA+0.3mg/LNAA 42 42 +
Darkness 1/2MS+0.4mg/L6-BA+0.1 mg/LNAA 0 0 -
1/2MS+0.4mg/L6-BA+0.3 mg/LNAA 0 0 -
1/2MS+0.8mg/L6-BA+0.1 mg/LNAA 0 0 -
1/2MS+0.8mg/L6-BA+0.3 mg/LNAA 0 0 -
MS+0.4mg/L6-BA+0.1mg/LNAA 0 0 -
MS+0.4mg/L6-BA+0.3mg/LNAA 0 0 -
MS+0.8mg/L6-BA+0.1mg/LNAA 0 0 -
MS+0.8mg/L6-BA+0.3mg/LNAA 0 0 -
+, ++and+++meantnormal, relativelygoodandgoodgrowthpotentialrespectively.-meantnogrowth.
Table3　Influencesofdiferentculturemediaonrooting
Typeofculturemedium Numberofrootedplant
Rooting
rate
%
Average
numberof
rootsper
shoot
Growth
potential
1/2MS+0.2mg/LIAA 53 53 3.6 +
1/2MS+0.6mg/LIAA 100 100 7.2 ++
1/2MS+1.0mg/LIAA 65 65 5.3 +
1/2MS+1.4mg/LIAA 23 23 3.1 +
1/2MS+0.4mg/L2, 4-D 0 0 0 -
1/2MS+0.6mg/L2, 4-D 0 0 0 -
1/2MS+1.0mg/L2, 4-D 0 0 0 -
1/2MS+1.4mg/L2, 4-D 0 0 0 -
+, ++and+++meantnormal, relativelygoodandgoodgrowth
potentialrespectively.-meantnogrowth.
Transplantandcuttingofplantlet
Theobservationdemonstratedthattheplantletsweresur-
vivedandgrown14 daftertransplant.Afterculturedfor30 d,
thevigorousplantletsandtheirsurvivalratewas95.5% while
theygrewvigorously50 dlater.17 daftercuting, theplant-
letsweresurvivedandaveragesurvivalratewas91.2%,
while60 dlater, theygrewvigorously.cuttingseedlingwas
1/3 smalerthantransplantseedlingatinitialstages, while70
dlater, theylookedthesame.Theresultsmentionedabove
demonstratedthatfurnaceashandslagwastheoptimumsub-
stratefortransplantandcutingofplantletsofHemisteptalyra-
taBunge.
Growthpotentialoftransplantedplantlet
Theregularobservationaftertransplantdemonstrated
thattherateofsuccessfultransplantwasnearly100%.Com-
paredwithwildseedlings, thecutingseedlingsandtransplant
seedlingsweresturdyandvigorous.InthemiddleofAprilnext
year, germinationwasstartedandthetimewas7 dearlier
thanthatofwildseedling., besides, thecuttingseedlingsand
transplantseedlingswerefloweredinJuneandJulyandfruit-
edinAugustandSeptember.Inaddition, theobservationalso
demonstratedthatplantletshadmuchrootsystemwhichwas
asaround2 timesasthatofwildseedlings.Throughtheob-
servationof3 consecutiveyears, itwasconcludedthatal
charactersofthetransplantedplantletsinhilsideweresame
totheseofwildplants, besides, theplantletsalsohadother
characterssuchasvigorousgrowth, earlygerminationin
springanddevelopedrootsystem.
ConclusionandDiscussion
ThetenderstemHemisteptalyrataBungewastakenas
anexperimentalmaterialinthisexperimenttosuccessfuly
createahigheficientcloneoftenderstemofHemisteptalyra-
taBunge.TheexperimenthasdemonstratedthatMS+0.3
mg/L6-BA+1.0 to1.5 mg/L2, 4-Distheoptimumculture
mediaforinducinggranularcalusesoftenderstem, whileMS
+1.5 mg/LAgNO3 +0.4 mg/L6-BA+0.1 mg/LNAAisthe
optimumculturemediafordiferentiationcultureofgranular
calusesandadventitiousbud, besides, 1/2 MS+0.6 mg/LI-
AAistheoptimumculturemediaforrootingandrootingsub-
cultureofplantlets, whilefurnaceashandslagistheideal
substratefortransplantandcuttingofplantlets.Thetrans-
plantedplantletshavenew characterssuchasvigorous
growth, earlygerminationinspringanddevelopedrootsystem
withoutchangesofothercharacters.
Theresearchalsohasfoundthatonecalusparticlecan
form 28.6 newparticlesin40 dandreproduce28.69 of-
spring.Oneadventitiousbudcandifferentiateinto4.8 new
adventitiousbudswithvigorousgrowthandreproduce4.89
offspring.Therootingtubeplantletscanproduceonegenera-
tionin23dandthepropagationcoefficientofeverygeneration
is3.2, besides, thereare3.216 ofspringinoneyear.There-
fore, nomaterwhichrapidpropagationmethodsareused,
massivetubeplantletswilbeproduced, sothehighefficient
cloneiscreated.However, fromtheperspectivesofgrowth
vigorandefectiveness, althoughrootingsubculturehasslow
reproductivespeed, thetubeplantletsculturedbythismethod
arenotonlyvigorouslygrown, butalsoarealmostefective
buds.Therefore, inlarge-scaledpropagation, rootingsubcul-
tureshouldbeused.
References
[ 1] FloraEditorialBoard, ChineseAcademyofSciences.Florareipu-
blicaepopularissinicae(Vol.78(1))[ M].Beijing:SciencePress,
1987:138-139.(inChinese).
[ 2] InstituteofBotany, ChineseAcademyofSciences.Plant(cormo-
phyte)iconographyofChina(part4)[M].Beijing:SciencePress,
1975:615.(inChinese).
[ 3] LISX.FloraofLiaoning(part2)[ M].Shenyang:LiaoningSci-
enceandTechnologyPress, 1991:586.(inChinese).
[ 4] JiangsuNewMedicalColege.DictionaryofChinesemateriamedi-
49ZHANGWenetal.StudyonTissueCultureandEfectiveCloneEstablishmentofHemisteptalyrataBunge
ca(part1)[M] .Shanghai:ShanghaiPeoplesPress, 1977:1458
-1459.(inChinese).
[ 5] ZHENGHC.EdibleandmedicinalplantsofChina[ M].Shanghai:
ShanghaiLexicographicalPress, 2003:311-312.(inChinese).
[ 6] WUYG.Studiesontheplantregenerationandinductionanddifer-
entiationofexplantofDendranthemamorifolium[ J].ChineseAgri-
culturalScienceBuletin, 2007, 23(12):63-67.(inChinese).[ 7] GAOYK, ZHAOB, DINGGX.Shootregenerationfromstemand
leafexplantofDendrathemagrandiflorum[ J].JournalofBeijing
ForestryUniversity, 2001, 23(1):32-33.(inChinese).
[ 8] LIYF.Floweringorgancultureofseveralchrysanthemumandstud-
yondiferentialfrequencyofcalus[J].Biotechnology, 1997, 7(2)
:24-26.(inChinese).
[ 9] LIY, LIUDX, FANGJ.Preliminaryreportonapplicationofhor-
moneintisurecultureofchrysanthemum[ J].JournalofHenan
Vocation-TechnicalTeachersColege, 1987, 15(1):25-28.(in
Chinese).[ 10] SIHJ, DAIZX, YUPH.Theinductionofcalusfromyoungpetal
ofchrysanthemumanditsregeneration[J] .JournalofGansuAg-
riculturalUniversity, 1998, 33(2):174-177.(inChinese).
[ 11] LIY, LIUDX, LICM, etal.Thetissuecultureofalabastrumof
chrysanthemum[J].JournalofHenanVocation-TechnicalTeach-
ersColege, 1992, 20(4):59-60.(inChinese).
[ 12] GAOCY, SHANGHQ.Astudyontissuecultureofletuce[ J].
JournalofHezeTeachersColege, 2004, 26(4):61-62.(inChi-
nese).
[ 13] LIPF.Tissuecultureofapicalbudsandleafsectionsofheadlet-tuce[ J].JournalofSouthChinaAgriculturalColege, 1980, 1
(3):39-42.(inChinese).
[ 14] ZHONGZX.Tissuecultureofletuce(Lactucasativa)[J].Plant
PhysiologyCommunications, 1988, 24(6):37 -38.(inChi-
nese).
[ 15] LIUXM.Studyofleaforganogenesisofheadletuce[ J].Journal
ofHunanAgriculturalColege, 1990, 1(3):233-240.(inChi-
nese).
[ 16] JIANGCY.Discussionondoseofagarmedium[ J].PlantPhysi-
ologyCommunications, 1992, 28(2):155.(inChinese).[ 17] ANLJ, JIANG CY.Introductiontoplanttissueculture[ M].
Dalian:LiaoningNormalUniversityPress, 1996:68-72.(inChi-
nese).
Responsibleeditor:JINPing　　　　Responsibletranslator:LIZhu-le　　　Responsibleproofreader:WUXiao-yan
泥胡菜的组织培养及高效无性系建立
张 文 ,郭晓丹 ,王艳 ,徐娜 ,姜长阳 ＊　(辽宁师范大学生命科学学院 , 辽宁大连 116029)
1　材料与方法
1.1　无菌材料获得　将泥胡菜(采自大连石门山)嫩茎和基生
叶叶柄切成长 4 ～ 5cm的茎段和柄段后 ,分别放入 500 ml的磨
口广口瓶中 ,用清水冲洗约 15min,再用 0.05%的安利液振荡洗
涤 3次 ,接着用无菌水洗涤 2次 ,然后将材料移到超净工作台
上 。在无菌条件下用 70% ～ 75%的乙醇灭菌 30 s后 ,用 0.05%
的 HgCl2溶液振荡灭菌 3min,再用 0.03%的 HgCl2溶液振荡灭
菌 13 min,最后用无菌水振荡洗涤 5次即获得无菌材料 。
1.2　培养条件　以 MS、1 /2 MS为基本培养基 ,附加不同种类、
不同浓度的细胞分裂素和生长素。固体培养基琼脂含量为 4.5
g/L,诱导愈伤组织和愈伤组织分化培养基含蔗糖 30 g/L,生根
培养基含蔗糖 15g/L;光照强度 2 000lx左右 ,光照时间 12h/d,
pH值 5.6 ～ 5.8,培养温度(25±1)℃。
1.3　不同浓度生长素对愈伤组织诱导的影响　把无菌嫩茎和
叶柄切成长 0.1 ～ 0.2cm的茎段和柄段后 ,分别接种到含不同
浓度 IBA、2, 4-D、NAA的 MS+6-BA0.3 mg/L的培养基上 ,进行
愈伤组织诱导培养 。每种培养基接种 100个材料 , 50 d后进行
观察统计。
1.4　不同培养基对愈伤组织分化的影响　将上述继代培养的
颗粒状愈伤组织 ,接种到以 MS+AgNO3 1.5 mg/L和 1/2MS+AgNO3 1.5 mg/L为基本培养基 ,附加不同浓度 6-BA和 NAA的培养基上 ,进行分化培养。每种培养基接种 100个颗粒 , 5次重
复 。材料分别放在光照和无光 2种条件下进行培养。
1.5　不同培养基对不定芽生根培养的影响　将上述分化继代
培养的高 0.6cm以上 、生长旺盛的 、呈丛生状的不定芽从基部
剪下 ,接种到附加不同浓度 IAA(0.2、0.6、1.0、1.4 mg/L)和 2,
4-D(0.2、0.6、1.0、1.4mg/L)的 1/2 MS培养基上进行生根培
养 。每处理接种 100个不定芽 , 3次重复 , 25 d后统计生根株数
和每株生根数量 ,计算生根率。
将生根率和生根数量最多的试管苗剪成长 1.5cm、具有 1
个顶芽和 1 ～ 2个侧芽的茎段 ,接种到相同的培养基上进行生根
继代培养。在培养条件不变的情况下 ,统计每代培养所需时间
并计算繁殖系数。连续培养 7代 ,观察生根试管苗的长势并计
算繁殖系数 。每次试验接种 100个外植体 , 3次重复 。
表 1　 不同浓度生长素对愈伤组织诱导的影响
生长素种类 浓度mg/L 材料类别
愈伤组织
诱导数 诱导率∥% 长势
IBA 0.5 叶柄 0 0 -
0.5 嫩茎 0 0 -
1.0 叶柄 0 0 -
1.0 嫩茎 0 0 -
1.5 叶柄 0 0 -
1.5 嫩茎 0 0 -
2, 4-D 0.5 叶柄 47 47 +
0.5 嫩茎 76 76 ++
1.0 叶柄 56 56 +
1.0 嫩茎 100 100 +++
1.5 叶柄 89 89 ++
1.5 嫩茎 100 100 +++NAA 0.5 叶柄 34 34 +
0.5 嫩茎 63 63 ++
1.0 叶柄 42 42 +
1.0 嫩茎 76 76 ++
1.5 叶柄 53 53 ++
1.5 嫩茎 94 94 ++
注:+为长势一般;++为长势较好;+++为长势好;-为不生长。
表 3 　不同培养基对生根的影响
培养基种类 生根株数∥株
生根率
%
平均每株生
根数∥条 长势
1/2MS+IAA0.2mg/L 53 53 3.6 +
1/2MS+IAA0.6mg/L 100 100 7.2 ++
1/2MS+IAA1.0mg/L 65 65 5.3 +
1/2MS+IAA1.4mg/L 23 23 3.1 +
1/2MS+2, 4-D0.4mg/L 0 0 0 -
1/2MS+2, 4-D0.6mg/L 0 0 0 -
1/2MS+2, 4-D1.0mg/L 0 0 0 -
1/2MS+2, 4-D1.4mg/L 0 0 0 -
注:++长势旺盛;+长势一般;-为不生长。
(下转第 104页)
50 AgriculturalScience＆TechnologyVol.10, No.1, 2009
数最大值为 13条 ,泥沙质的为 12条 ,沙质中的最少 ,只有 9条;
淤泥质 、泥沙质 、沙质中平均每穗种子数分布范围分别为 14 ～
42、15 ～ 35、21 ～ 43粒;第一小穗种子数范围分别为 4 ～ 48、3 ～
34、8 ～ 46粒;第二小穗种子数范围分别为 8 ～ 50、6 ～ 38、18 ～ 48
粒 。方差分析发现 ,生长在淤泥质与泥沙质中的互花米草小穗
数无显著差异 ,而二者均与沙质中的小穗数有显著差异;3种生
境中的平均每穗种子数均有显著差异;淤泥质和沙质中的互花
米草第一小穗种子数 、第二小穗种子数无显著差异 ,二者与泥沙
质中的均有显著差异;饱满种子百粒重是淤泥质中的最大 ,其次
是沙质 ,泥沙质中的最小 , 3者间均有显著差异(表 2)。
表 1 　不同生境中互花米草结实器官外部形态特征 cm
生境 种穗长 穗颈长 穗长 平均每穗长 第一小穗长 第二小穗长
淤泥质 49.625±0.838a 23.085±0.635a 26.540±0.591a 10.284±0.221a 9.560±0.370a 10.137±0.241a
泥沙质 49.491±0.591a 24.767±0.482b 24.724±0.347b 9.355±0.176b 6.753±0.167b 8.546±0.169b
沙质 53.593±0.454b 26.502±0.644c 27.091±0.576a 13.347±0.241c 10.879±0.366c 12.955±0.280c
　注:不同英文字母表示在 0.05水平上差异显著(t检验)。
表 2 　不同生境中互花米草结实器官数量特征
生境 小穗数∥条 平均每穗种子数∥粒 第一小穗种子数∥粒 第二小穗种子数∥粒 饱满种子百粒重∥g
淤泥质 7.300±0.312a 29.583±0.830a 29.033±1.392a 30.500±1.110a 0.432±3.33×10-6 a
泥沙质 7.011±0.154a 25.544±0.442b 20.272±0.705b 24.609±0.596b 0.358±4.05×10-5 b
沙质 5.482±0.167b 32.071±0.691c 28.107±1.116a 32.393±0.829a 0.415±4.73×10-5 c
3　结论与讨论
在淤泥质 、泥沙质和沙质 3种生境中 ,淤泥质土质肥沃而透
气性不佳 ,沙质的透气性较好 ,泥沙质的肥沃性和透气性处于二
者之间 ,属于较优生境 。研究结果发现 ,单株互花米草以泥沙质
中的长势最好 ,沙质中生长的互花米草结实器官各形态因子均
大于淤泥质和泥沙质 ,其中种穗长达到了 53.593 cm,穗颈长为
26.502cm,穗长为 27.091cm,平均每穗长 13.347cm,除了穗长
和淤泥质无差异外 ,其余各形态因子均与其他 2个生境中的有
显著差异。 3种生境中第二小穗的长度均大于第一小穗的长度 ,
这与米佳等认为当所处环境条件较好 ,植株较高 ,植物表现为利
用营养器官的生长来获得更多养分 ,从而提高个体在种群中的
竞争力的结论一致 。
3种生境中的互花米草结实器官的形态因子与数量特征呈
负相关关系 。结实器官的数量特征是淤泥质中的互花米草占优
势 ,沙质中的其次 ,泥沙质中的最低 ,且泥沙质中饱满的种子极
少 。淤泥质 、泥沙质 、沙质中饱满种子的百粒重均值分别为
0.432、0.358、0.415 g。其反映了生境条件较好时 ,互花米草为
了保证个体在种群中的竞争力 ,营养生长相对旺盛 ,植株较高 ,
此时分配给结实器官的营养物质相对较少 ,导致小穗的结实率
下降;当生境条件较差时 ,个体获得的营养物质有限 ,为繁衍后
代 ,限制对营养器官的供应 ,将相对较多的营养分配给种子 。另
外 ,研究结果发现 3种生境中小穗顶部种子的饱满度远高于小
穗底部的种子 ,说明在顶端的小花受粉较易 。
在一个稳定的群落中植物常常采取营养繁殖的方式来占据
领地 ,而在干扰环境中植物常常采取种子繁殖的方式来扩大空
间。研究样地上的互花米草群落周围生长的是红树幼苗 ,二者
的生态位相接近 ,但是由于互花米草的繁殖力强 ,而红树幼苗的
生长缓慢 ,在二者的竞争中互花米草处于优势 ,该区域的互花米
草群落已经属于较稳定的群落 ,因此营养繁殖占种群扩大的主
要地位 。
基金项目　国家自然科学基金项目(30660036);广西自然科学基金项
目(桂科自 0728096);广西研究生教育创新计划项目
(2008106020907M266)。
作者简介　覃盈盈(1983-),女 ,广西贵港人,硕士研究生,研究方向:繁
殖生态学。 ＊通讯作者。
收稿日期　2008-12-29　　修回日期　2009-03-10
(上接第 50页)
1.6　试管苗的移栽和扦插　①移栽。打开生根试管苗培养瓶
的瓶塞 ,置于 4 000 lx左右的光照下炼苗 3d后 ,用镊子将试管
苗从培养瓶中取出 ,剪下上半段 ,洗净根部的培养基后 ,将具有
根的试管苗移栽到表层为 5 ～ 7cm厚的炉灰渣、下层为肥沃园
土的温室苗床或花盆中 。然后弥雾浇透水 , 保持湿度 90%以
上 、温度 25℃左右 、防止直射光照 , 30 d后进行观察统计 。共
移栽 4次 ,每次移栽 800株 。 ②扦插 。将移栽试管苗剪下的上
半段从培养瓶中取出 ,剪去最下面一个叶片后 ,将下部切口放
到 100mg/L的 IAA溶液中处理 4 min左右 ,扦插到浇透水并
打上深约 1 cm小孔的与移栽相同的温室苗床的炉灰渣(下面
为园土)中 ,随即弥雾喷浇淤闭插孔 ,再按照与移栽试管苗相同
的条件进行管理 , 30 d后观察统计 。共扦插 3次 , 每次扦插
800株 。
1.7　试管苗的移植　将在温室中移栽和扦插成活的泥胡菜试
管苗移植到试验材料采集地山坡上 ,按照野生的条件进行栽
培 ,并定期观察统计 。
2　结论与讨论
该研究以泥胡菜嫩茎为材料 ,成功建立起泥胡菜高效无性
系 。研究结果表明 , MS+6-BA0.3 mg/L+2 , 4-D1.0 ～ 1.5
mg/L是诱导泥胡菜嫩茎形成颗粒状愈伤组织的理想培养基
(表 1);MS+AgNO3 1.5 mg/L+6-BA0.4 mg/L+NAA0.1
mg/L是颗粒状愈伤组织和不定芽分化培养的理想培养基(表
见第 49页 Table2:不同培养基对愈伤组织分化的影响);1 /2
MS+IAA0.6mg/L是试管苗生根和生根继代培养的理想培养
基(表 3);炉灰渣是试管苗移栽和扦插的理想基质。移植的试
管苗在其他各种性状保持不变的情况下 ,出现了长势旺盛 、春
天发芽早 、根系发达的性状特点 。
研究还发现 ,用继代培养的方法进行快速繁殖 , 1个愈伤
组织颗粒 40d能形成 28.6个新颗粒 , 1年能繁殖出 28.69个
后代;1个不定芽 40 d可分化形成 4.8个生长旺盛的不定芽 , 1
年能繁殖出 4.89个后代;生根试管苗 23d可繁殖培养 1代 ,每
1代的繁殖系数为 3.2 , 1年能繁殖出 3.216个后代 。可见不论
采用哪种方法进行快速繁殖 ,每年都能产生大量的试管苗 ,达
到建立高效无性系的目的。但是 ,从长势和有效性看 ,生根继
代的方法虽然繁殖速度较慢 ,但所培养的试管苗不仅生长旺
盛 ,而且几乎没有无效苗 。所以 ,在进行规模繁殖中 ,应采用生
根继代的方法 。
作者简介　张文(1986-),男 ,辽宁大连人 ,本科 ,专业:生物科学。
＊通讯作者 ,教授。
收稿日期　 2008-12-08　　修回日期　 2009-02-24
104 AgriculturalScience＆TechnologyVol.10, No.1, 2009