全 文 ：OptimizingEficientMicropropagationSystem of
RhododendronBrachycarpumD.DonbyUniform
DesignMethod
JIANGYun-tian1 , CHENYan-qiu2 , GUDi-zhou1 , LIYu-mei3 , QUBai-hong2＊
1.DepartmentofBiology, TonghuaNormalUniversity, Tonghua134002;2.HorticultureDepartment, AgriculturalColege, YanbianUniversi-
ty, Longjing133400;3.JilinNormalUniversity, Siping136000
Abstract　[Objective] ThestudywasconductedtoestablishahigheficientmicropropagationsystemforRhododendronBrachycarpumD.Don
andrealizethehighefficiencyinvitromicropropagationofR.brachycarpum.[Method] YoungstemsofR.brachycarpumwereusedasexplants,
suitablemediumcompositionsforaxilarybudgrowingandrootingwerescreenedthroughuniformdesignexperiments.[Result] MS(modified)
+IAA0.15mg/ L+IBA0.30mg/L+GA3 3.00mg/Lwasthemostsuitablemediumwiththeregenerationrateof92%.Stemseachwithonenodewerecutfromregeneratedshootsandculturedforpropagation, theproliferativemultiplewasover45 withinonecultureperiodof35 d.
[ Conclusion] HigheficientmicropropagationsystemofR.brachycarpumhasbeensuccessfulyestablished, whichprovidessomebasisforde-
velopmentandutilizationandindustrialseedlinggrowthofthealpinerhododendroninChangbaiMountainnortheastChina.
Keywords　RhododendronBrachycarpumD.Don;Micropropagation;UniformDesign
Received:July27, 2009　　Accepted:August20, 2009
SupportedbyFundedProjectofScienceandTechnologyDepart-
mentinJilinProvince(200705C05)andNaturalScienceFundsof
TonghuaNormalUniversity(XS060074).
＊Correspondingauthor.E-mail:bhqu@ybu.edu.cn
　　RhododendronBrachycarpumD.DoninthegenusRho-
dodendronofEricaceaeisanevergreenshrub.Itisastate-
protectedpreciousandrareplant, anddecidedastheprovin-
cialfirst-classprotectiveplantbyJilinProvisionalRegulations
onProtectionandManagementofWildFaunaandFlora.With
greenandshinyfoliagesincoldwinter, leaflengthof8 to20
cm, andwhiteflower, R.brachycarpumcanbeusedaspot-
tedflower.Inaddition, somehorticulturalcultivarscanbede-
velopedtoresistcoldanddisease, andtheycanbeusedas
germplasmresourcesofalpinerhododendron.However, itis
onlydistributedinsomeareasofNortheastChinawithanum-
beroflessthan200[ 1] .TheutilizationanddevelopmentofR.
brachycarpumisgreatlyrestrictedbecauseofitslowgermina-
tionrateandrootingratebysomeconventionalpropagation
methodssuchasseedandcutingreproduction.Therefore, it
isverynecessarytorealizethehigheficientinvitromicro-
propagationofR.brachycarpumtogetherwithwelprotecting
itswildresources.
Itisfoundthatthemethodofrediferentiatedmediumhas
theadvantageofhighpropagationcoefficient, anditstilhas
somedisadvantages:longperiodofexplantscalusinduction,
lowrateofadventitiousbudgrowth, degradationanddiferen-
tiationofcalus.Comparedwithcalusrediferentiation, axila-
rybudmultiplicationandrootingmeantimecanproduceyoung
plantverysoon, highpropagationcoeficientandgoodgenetic
stability, inthewaytheproductionefficiencyofseedlingisin-
creaseddramaticaly.Basedonorthogonaldesign, theuni-
formdesignmethod, combinedwithNumbertheoryandMulti-
variatestatistics, wascreatedbyamathematicianFANGKai-
taiinChina.Andthismethodissuitableforthemulti-factor
andmulti-levelexperiment.Themethodhasthefolowingad-
vantages:① Fewerexperimentnumbers.Eachfactoronly
needsonetestforitseachlevel.Andthenumberoftestsis
equaltothatofitslevels.② Theleveloffactorscanbeap-
propriatelyadjustedtoavoidmeetinghighlevelandlowlevel
factorstopreventanyunexpectedissueinexperimentortoo
slowreactionrate.Especialyundersomeconditionshardto
controlsuchasnotfiercereactionortoofastchange, the
technologicalconditionsandregularimpactingfactorsshould
benoticed.③Datatreatmentwithcomputerscanrapidlyand
accuratelygetthequantitativeregressionequation.Itiscon-
venienttoanalyzetheinfluencesofeachfactorandquantita-
tivelyforecasttheoptimizingconditionsandintervalestima-
tion.Therefore, suitablemediumcompositionsforaxilarybud
growingandrootingwerescreenedthroughuniformdesignex-
periments, aimingatprovidingabasisfortheutilizationand
industrialseedlinggrowthofR.brachycarpum.
MaterialsandMethods
Treatmentofexplants
InearlyApril, restingshootsweregotfrom themixed
broadleaf-coniferforestofChangbaiMountainatanelevation
of1 470 m.Theshootswerewater-culturedinlabtopromote
thegerminationofaxilarybuds.Theaxilarybudswerecut
whentheygrewto2.0cmlongandwashedfor60swith75%
alcoholonsuper-cleanbench.Thentheyweresoakedinsodi-
umhypochloritesolutionfor10 min, washedfor10 timeswith
sterilewater, andsurfacewaterwasabsorbedbysterilefilter
paper.Aftercutingoffthepartdamagedbydisinfectant, the
pieceswereusedasexplants.
Axilarygerminationandscreeningofmedium
MSwasusedasthebasicmedium(1/4 macro-element,
1/3 traceelement, 1/3 malysite, and1/3 organiccompo-
nents)plussucrose20 g/L, agarpowder9.5 g/L, pH5.5,
temperature(24±2)℃, andlightintensity1100 lx, andlight
period10h/d.ExplantsofstemswereinoculatedintoMSme-
diumswithdifferentconcentrationsofIAA, IBA, NAAand
GA3.Andthemostsuitablemediumwasscreened.
AgriculturalScience＆Technology, 2009, 10(5):49-51
Copyright 2009, InformationInstituteofHAAS.Alrightsreserved. AgriculturalBiotechnology
DOI :10.16175/j.cnki.1009-4229.2009.05.032
Establishmentofeficiencymicropropagationsystem
Nodecultivationwasusedtomakemicropropagation[ 2].
Thestemofregeneratedplantswascutintopiecestobeinoc-
ulatedinthemodifiedmediumtomakenodepropagationand
rooting.Thepropagationperiodandproliferativemultiplewere
computed.
Dataanalysis
Theuniformdesignmethodwasusedtomakeinitialreg-
ularitydesigningexperiment, andUniformDesign3.0 Vwas
adoptedtomakedataanalysis.
ResultsandAnalysis
Axillarygerminationandscreeningofthebestmedium
Thepreliminaryexperimentresultsshowedthatamong
themodifiedMSmediums, theconcentrationrangesofmedi-
umforaxilarybudgrowingandstemrootingwereasfolows:
IAA0.15to0.35mg/L, IBA0.10to0.30mg/L, NAA0.07-
0.12 mg/LandGA3 2.50 to3.00 mg/L.Theplantgrowthregulatingsubstancewhichwaslowerthanthecontroling
rangecannotpromotethestemrootingandaxilarybudgrow-
ing;andtheplantgrowthregulatingsubstancewhichwas
higherthanthecontrolingrangecaninducebaseexpansion
orcalustumor, andinhibittheaxilarybudgrowing.Theroots
generatedfromcalustumorfeloffintransplanting, andthe
transplantingsurvivalratewasnearlyzero.Inordertopro-
moteregeneratingrate, theuniformdesignmethodwasadopt-
ed[3] .Thenumberforeachinoculationwas30, U10(108)uni-
formtablewasused(Table1).Atthesametime, theinflu-
encesofcrossmatchingofconcentrationsofIAA, IBA, NAA
andGA3ontheaxilarygrowingandrootingwerestudied.
Table1　U10(104)factorsandlevelsdesign mg/L
Levels FactorsX1 X2 X3 X4
1 0.15 0.10 0.07 2.50
2 0.20 0.15 0.08 2.60
3 0.25 0.20 0.09 2.70
4 0.30 0.25 0.10 2.80
5 0.35 0.30 0.11 2.90
6 0.15 0.30 0.12 3.00
7 0.20 0.25 0.10 2.70
8 0.25 0.20 0.09 2.80
9 0.30 0.15 0.08 2.90
10 0.35 0.10 0.07 3.00
X1 isIAAconcentration;X2 isIBAconcentration;X3 isNAAconcen-tration;X4 isGA3 concentration.
Table2　U10(104)uniformdesigntestplanandresult
Treatnum-
ber
Factors∥mg/L
X1 X2 X3 X4 Y∥%
1 0.15 0.20 0.10 2.90 86.00
2 0.20 0.30 0.09 3.00 88.50
3 0.25 0.15 0.07 2.80 80.00
4 0.30 0.10 0.11 2.90 78.50
5 0.35 0.25 0.08 2.70 79.00
6 0.15 0.25 0.08 2.80 86.00
7 0.20 0.10 0.12 2.60 77.50
8 0.25 0.15 0.07 2.70 78.50
9 0.30 0.30 0.09 2.50 79.50
10 0.35 0.20 0.10 3.00 81.00
Yregenerationrate, thegrowthofaxilarybudsandtherootingof
steminthesanetimeistakenasstandard.
　　Thedatawastreatedwiththeuniformdesignmethod, the
regressionequationwasgot:Y=53.4-29.9X1 +24.2X2 -
34.9X3 +12.3X4 , samplesizeN=10, significancelevelα=
0.01, multiplecorrelationcoefficientR=0.996 6, testvalueFt=182.2, criticalvalueF(0.01, 4 , 5)=11.39, Ft>F(0.01 , 4, 5), anditshowsthattheregressionequationissignificant.Throughsig-
nificanttesttoeachequationitem, testvalueF(3) =15.16,criticalvalueF(0.01, 1, 5) =16.26, F(3)≤F(0.01 , 1 , 5), anditshows
thatX3 regressionitemisnotsignificantanditneedstobere-jected.Thenotsignificantregressionitemisrejectedanda
newregressionequationisbuiltforcontinuouscomputation:Y
=50.4-29.6X1 +27.0X2 +12.0X4 , correlationcoeficientR
=0.986 2, testvalueFt=70.80, criticalvalueF(0.01, 3, 6) =
9.780, Ft>F(0.01, 3, 6), andtheregressionequationissignifi-
cant.Asforthesamereason, eachregressionitemissignifi-
cantlytested, andeachregressionitemhasanobviousim-
pactonY.Accordingtoregressionequation, thebestcombi-
nationofYisgot, X1 =0.15, X2 =0.30, X4 =3.00, andtheoptimalsolutiony=90.0.Itshouldbesolvedaccordingtothe
formulaY=y±uα· stocomputetheoptimalvalueintervalvalueY=90.0(±2.82):87.18% to92.80%.Theoptimal
combinationwasusedforverificationtest, andthestemwas
inoculatedintothemodifiedMSmediumofIAA0.15 mg/L+
IBA0.30mg/L+GA33.00 mg/L.20 dlater, thestembaseproducedroot;andaxilarybudbegantogerminateandgrow
after25 d;and5 to7 rootsdirectlygeneratedfromthestem
baseafter35d(Fig.1), andtheseedlingreachedaheightof
2.5 cmafter45d.Someseedlingsproducedlateralroots, the
rootsandseedlingsdevelopednormalywitharegeneration
rateofover92%.Therefore, MS(modified) +IAA0.15
mg/L+IBA0.30 mg/L+GA3 3.00 mg/Lwasthemostsuitablemedium.
Fig.1　AxilarybudgrowingandrootingofRhododendron
BrachycarpumD.Don
Establishmentofefficientmicropropagationsystem
Nodecultivationwasusedtomakemicropropagation.
Whentheseedlinggrewtoaheightofover3.0 cm, culture
flaskwasopenedatthesupercleanbench, andtheseedling
wascutuntilonly1leafwasleft.Thenthecutpieceswerein-
oculatedintotheMS(modified)+IAA0.15 mg/L+IBA
0.30mg/L+GA3 3.00mg/Lwhichwasforstemrootingand
axilarybudgrowingfornodespropagationandrooting.Itwas
foundthat35dwasacultureperiodandtheproliferativemulti-
plewasover45(Fig.2).
Seedlingadaptationandtransplanting
Whentherootgrewto2.0 cm, seedlingwasgotfromthe
cultureflask.Andtheagarwaswashedinoxadixyl.mancozeb
solution5.00 mg/L.Thentheseedlingwasplantedintothe
mixturemedium ofdecomposedpineneedles, peatsoiland
50 AgriculturalScience＆TechnologyVol.10, No.5, 2009
Fig.2　StemmultiplicationofRhododendronBrachycarpumD.
Don
thinriversandwhichweresterilizedbyoxadixyl.mancozeb20
times.Plasticfilm withgoodlightpenetrationwasusedfor
coveringtokeepgoodmoistandtemperatureconditionssuch
as.Humidity75%, temperature18 ±2 ℃, naturallight8 h
andairexchangefor10 mineachafternoon.10 dlater, the
plasticfilmwasmoved, andwaterwassprayedonceevery
morningandeverynight.Itwasfoundthatthesurvivalrate
reachedover95% withtheabovementionedmethods(Fig.3).
Fig.3　PlantlettransplantsurvivalofRhododendronBrachycarpum
D.Don
Conclusion
TheresultsshowedthatMS(modified)+IAA0.15 mg/L
+IBA0.30mg/L+GA3 3.00 mg/Lwasthemostsuitablemediumforinducingtheaxilarybudelongationandstemrooting.
IAAandIBApromotedtherootingrateofstems.AndGA3
3.00mg/Lacceleratedthegerminationandelongationofaxil-
larybuds.Stemseachwithonenodewerecutfromregenera-
tedshootsandculturedforpropagation, theproliferativemulti-
plewasover45 withinonecultureperiodof35 d.Thestem
nodesfromregeneratedshootswereusedasmaterialstobe
inoculatedinthemediumofMS(modified)+IAA0.15mg/L
+IBA0.30 mg/L+GA3 3.00 mg/Ltomakerootingculture.
Themethodhasthefolowingadvantages:goodgeneticsta-
bility, greatsurvivalrate, shortenedpropagationperiod, high
multiplication, andsimpleoperation.Applyinguniformdesign
methodtoanalyzedatagreatlyshortenstheexplorationperiod
ofmediumingredients.
Inrecentyears, thehorticulturalcultivarsofalpinerhodo-
dendronfrom German, ItalyandBelgium haveenteredthe
marketofChina.However, onlyafewcultivarsinGansuand
YunnanProvincearesemi-introducedinChina.AndR.
brachycarpuminChangbaiMountainhasnotbeenintroduced
untilnow.AhighefficientmicropropagationsystemofR.brachy-
carpumhasbeensuccessfulyestablished, whichisveryhelpful
fordevelopmentandutilizationandindustrialseedlinggrowthof
thealpinerhododendroninChangbaiMountain.
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Responsibleeditor:CHENJuan　　　　Responsibletranslator:LIPing-ping　　　　Responsibleproofreader:WUXiao-yan
均匀设计法优化短果杜鹃高效快繁体系(摘要)
姜云天 1 , 陈艳秋 2 ,顾地周 1 , 李玉梅 3 ,曲柏宏2＊　
(1.通化师范学院生物系 ,吉林通化 134002;2.延边大学农学院园艺系 ,吉林龙井 133400;3.吉林师范大学 ,吉林四平 136000)
[目的 ] 建立短果杜鹃高效快繁体系 ,实现短果杜鹃的高效离体快繁。
[方法] 以短果杜鹃嫩茎段为外植体 ,选用 U10(108)均匀表 ,考察IAA、IBA、NAA和GA3浓度交叉配比对短果杜鹃腋芽生长伸长及生根的影响 ,筛选最适合短果杜鹃腋芽萌发生长及生根的培养基。继代快繁采取节培法。
[结果 ] 最适合嫩茎段的腋芽萌发生长及生根的最佳培养基为MS(改良)+IAA0.15mg/L+IBA 0.30mg/L+GA3 3.00mg/L,再生率达
92%以上。以再生植株的茎节为材料进行快繁的结果表明 ,在 35d的 1个培养周期内增殖倍数平均达 45以上。待苗根长至 2.0cm以上时 ,
从培养瓶中取出试管苗 ,在含有 5.00mg/L杀毒矾溶液中洗去苗上残留的琼脂 ,然后将苗植入经 20倍杀毒矾消毒过的腐烂松针 、泥炭土和细
河砂混合(比例为 2∶2∶1)的基质中 ,用透光好的塑料薄膜覆盖以保湿保温 ,湿度保持在 75%,温度控制在(18±2)℃,每天自然光照 8h,每天
中午通风换气 10min。 10d后揭去薄膜 ,每天早晚喷洒清水各 1次。采用上述炼苗和移栽方法 ,短果杜鹃试管苗的成活率达 95%以上。
[结论 ] 该研究建立了短果杜鹃的高效快繁体系 ,为长白山高山杜鹃的开发利用和工厂化育苗提供了依据。
关键词　短果杜鹃;快繁;均匀设计
基金项目　吉林省科技厅资助项目(200705C05)和通化师范学院自然科学基金项目(XS060074)。
作者简介　姜云天(1975-),男 ,吉林四平人 ,硕士,助教 ,从事长白山区植物生理生化研究。 ＊通讯作者。
收稿日期　 2009-07-27　　修回日期　 2009-08-20
51JIANGYun-tianetal.OptimizingEficientMicropropagationSystemofRhododendronBrachycarpumD.DonbyUniformDesignMethod