全 文 ：EstablishmentofTissueCultureRegenerationSystems
ofStemSegmentsofEuphorbiatirucali
LIUZhao-liang, HEJue-min＊ , CHENBiao, LIANGJia-xian
AgriculturalBiotechnologyInstitute, GuangdongOceanUniversity, Zhanjiang524088
Abstract　[ Objective] TheaimwastostudytheconditionsoftissuecultureregenerationseedlingbyusingthestemsegmentsofEuphorbiatiru-
calianddeterminetheoptimumcultureconditionofeachculturestage, soastoprovidereferencesforthefactoryproductionandrelativestudyoftissuecultureseedlingofE.tirucali.[ Method] TakingthestemsegmentsofE.tirucaliasexplants, theefectsofvariousmediumsongermi-
nationrate, multiplicationcoeficientandrootingratewerestudied.[ Result] Theoptimuminductionmediumofgerminationculturewas1/2MS+
NAA0.02mg/L+6-BA1.0mg/L, withdiferentiationrateof89.7%;thebestsubculturemediumwas1/2MS+NAA0.02mg/L+6-BA0.60
mg/L+AD3.0mg/L, withmultiplicationcoeficientof5.70;theoptimumrootingculturemediumwas1/2MS+NAA0.40mg/L+IBA0.4mg/L,
withrootingrateof100%andtransplantingsurvivalrateof80%.[Conclusion] ThetissuecultureconditionsofstemsegmentsofE.tirucaliwere
determinedprimarily.
Keywords　Euphorbiatirucali;Stemsegment;Tissueculture
Received:March4, 2011　　Accepted:April11, 2011
SupportedbyKeyTechnologiesR＆DProgramofGuangdongProv-
ince(2009B0203030092006B20201007).
＊Correspondingauthor.E-mail:hejm@gdou.edu.cn
　　BelongingtoEuphorbia, Euphorbiaceae, Dicotyledone-
ae, Angiospermae, Euphorbiatirucaliisalsonamedmilk
bush, rubbereuphorbiaandsoon[ 1] .OriginatinginAfrica,
E.tirucaliwasintroducedinAsia, Europe, AmericaandAus-
traliaandmainlydistributesinGuangdong, Hainan, Yunnan
andotherprovincesinChina[ 2] .Withrichwhiteoryelowmilk
inbranches, E.tirucalihasmanyeficacies, suchascathar-
sis, diuresis, treatingedema, tumor, tuberculosis, psoriasis
andpromotinglactation[ 3-4].Inaddition, thereexistrichste-
rols, terpenes, estersandhydrocarboninthemilkofE.tiru-
cali, anditschemicalcompositionapproachesorexceeds
gasolinestandard, soitcanbeusedasgreenfueloilafter
mixingwithothermaterialsorbeingprocessedintocrude
oil[ 5-6].CalvinsuggestedthatE.tirucaliplantedinaridregion
unsuitabletograinproductioncouldproduce10-50 tubsofoil
perhectareeveryyear[7] .Moreover, thedregsofE.tirucali
afterjuicingcanbeusedasmethaneandpapermaterial, soit
hasimportantresearchvalue.
ThecutingpropagationofbranchesofE.tirucaliisslow
innon-originandneedsmanymaterials, buttissueculture
techniquecanspeedupreproductiveeficiencyandpromote
factorymassproduction[8] .Astudyonthegerminationand
multiplicationofE.tirucaliexplantbyJiangLijuanetal.[9]
showedthatthegerminationandmultiplicationofE.tirucali
dependedonplentyofexpensivezeatin, andthebreedingof
E.tirucaliwasrestricted, soitwasdifficulttocarryoutfacto-
ryproductionoftissuecultureseedling.Therefore, theefects
ofdifferentmediumsandconcentrationratiosofNAA, IBA,
6-BAandKT[10] onthetissuecultureofstem segmentsof
E.tirucaliwerestudied, soastofindouteconomicalyviable
methodtobreedtissuecultureseedlingofE.tirucali.
MaterialandMethods
Material
E.tirucaliintroducedfromXuwenCountyinGuangdong
ProvincewasastestmaterialandwasprovidedbyAgricultural
BiotechnologyInstitute, GuangdongOceanUniversityafter
germplasmresourcesselection.
Disinfectionmethodofexplants
One-yearstemsegments(3 -4 cm)ofE.tirucaliwere
selectedfromfieldinSeptember, afterwashedbyrunningwa-
terfor0.5 h, theywerepreprocessedin1% Xiaojiajingdisin-
fectionsolutionfor1 min, finalystemsegmentswhichwere
washedby75% ethanolfor30 s, treatedby0.1% mercuric
chlorideandTween-20for6minandwashed6 timesbyaseptic
watersuccessivelywereinoculatedinpreparedmediums.
Culturemethods
Germinationculture　Disinfectedstemsegments(3 -4cm)
ofE.tirucalicutinto1-2 cmlengthwereinoculatedingermi-
nationmediumsincludingMS+NAA(0.02-0.10mg/L)+6-BA
(0.6-3.0 mg/L), MS+NAA(0.10 mg/L)+KT(0.2 -1.0
mg/L)and1/2MS+NAA(0.02-0.10 mg/L)+6-BA(0.6-
3.0 mg/L), andthentheywereculturedunderthetempera-
tureof26-28 ℃, lighttimeof8 -10 h/d, lightintensityof
about3 000lx.Observationandstatisticswereconducedafter
inoculationfor24 d.
Proliferationculture　Thenewbudsfromgerminationmedi-
umswerecutandinoculatedin1/2MS+NAA(0.02 -0.10
mg/L)+6-BA(0.6-3.0mg/L)+AD3.0 mg/L.Afterinocu-
lationfor40 d, 20botlesofmediumswererandomlyselected
fromdiferenttreatmentsandculturedunderthesamecondi-
tionsofgerminationculture, finalythenumberofnewbuds
withthelengthof≥0.5 cmwascounted.
Rootingculture　Theplantletswith4-5 leavesweretrans-
ferredintotherootingmediumsof1/2MS+IBA(0.04 -0.40
mg/L)and1/2MS+NAA(0.04-0.40 mg/L), anditscul-
tureconditionswerethesameasgerminationculturecondi-
tions.Growthconditionofrootswascountedafter20d.
Domesticationandtransplantation　 Thetissueculture
seedlingofE.tirucaliwaseasytoroot, andplantletswhich
had3 rootswiththelengthofover2 cmwereputinoutdoor
nurserywithshadingnetfor3-5d, soastoadapttoexternal
environmentassoonaspossible.Plantletsafterwashingme-
diumweretransplantedintothedisinfectedculturecupswith
matrix(nutrientsoil∶Yekang=1∶4), andthentheywereputin
AgriculturalScience＆Technology, 2011, 12(3):379-381, 465
Copyright 2011, InformationInstituteofHAAS.Alrightsreserved. AgriculturalBiotechnology
basketencapsulatedbyfilmtokeepairhumidity.Transplan-
tingsurvivalratewascountedafter20 d.
ResultsandAnalysis
GerminationcultureofE.tirucalli
FromTable1, thegerminationrateofE.tirucaliwas
higherunderthemediumofMS+NAA0.02 mg/L+6-BA
1.0 mg/L(53.3%), whileitwentdownwiththeincreaseof
6-BAconcentrationfrom1.0 to3.0 mg/Lwhenmediumwas
MS+NAA0.1 mg/L+6-BA(1.0-3.0 mg/L), andbasal
partwasblockedbymorecaluses, whichaffectednormal
growth.UnderthemediumofMS+NAA(0.10 mg/L)+KT
(0.2 -1.0 mg/L), germinationratewaslowerandshowed
decreasetrendwiththeincreaseofKTconcentration.When
mediumswere1/2MS+NAA0.02 mg/L+6-BA0.6 mg/L
and1/2MS+NAA0.02 mg/L+6-BA1.0 mg/L, theirgermi-
nationrateswere80.0% and89.7% (Fig.1), respectively,
andgerminationtimeafterinoculationwasshort.Underthe
sameconcentrationratioofNAAand6-BA, germinationrate
under1/2MSmediumwashigherthanthatofMSmedium.
MultiplicationcultureofE.tirucali
FromTable2, theinducedbudsofE.tirucaliwerethe
mostunderthemediumof1/2MS+NAA0.02 mg/L+6-BA
0.6 +AD3.0 mg/L, withmultiplicationcoefficientof5.70,
andexplantsgrewrapidly;themediumof1/2MS+NAA0.02
mg/L+6-BA1.0 mg/L+AD3.0mg/Ltookthesecondplace
withmultiplicationcoeficientof4.35 (Fig.2), andexplants
grewslowly;themultiplicationeffectof1/2MS+NAA0.10
mg/L+6-BA1.0 +AD3.0 mg/Lwastheworstwithmultipli-
cationcoeficientof3.60, andcalusincreasedobviouslyat
basalpart, whichafectedplantgrowthinthelatestage.
Table1　EfectsofdiferentmediumsandplantgrowthregulatorsonthegerminationofE.tirucali
Basicmedium Plantgrowthregulator∥mg/L Numberofexplants Numberofbuddingexplants Germinationrate∥%
MS NAA0.02+6-BA0.6 26 12 46.2
MS NAA0.02+6-BA1.0 30 16 53.3
MS NAA0.02+6-BA3.0 29 8 27.6
MS NAA0.10+6-BA1.0 30 14 46.7
MS NAA0.10+6-BA3.0 31 11 35.5
MS NAA0.10+KT0.2 27 9 33.3
MS NAA0.10+KT0.6 29 6 20.7
MS NAA0.10+KT1.0 28 5 17.9
1/2MS NAA0.02+6-BA0.1 29 22 75.9
1/2MS NAA0.02+6-BA0.6 30 24 80.0
1/2MS NAA0.02+6-BA1.0 29 26 89.7
Table2　EfectsofdiferentconcentrationratiosofNAAand6-BAonthemultiplicationofE.tirucali
Plantgrowthregulator∥mg/L Numberofinocul-atedbotles Numberofnewbuds(≥0.5cm) Multiplicationcoeficient Growthstate
NAA0.02+6-BA0.6+AD3.0 20 114 5.70 Moremultipleshoot, stronggrowthvigor
NAA0.02+6-BA1.0+AD3.0 20 87 4.35 Morebuds, fewerefectivebuds, dificultextension
NAA0.10+6-BA1.0+AD3.0 20 59 2.95 Generatingcalus, slowgrowth
NAA0.10+6-BA3.0+AD3.0 20 72 3.60 Morecalusesblockedbasalpartandafectedplant
growth
Basicmediumis1/2MS.
Fig.1　Germinationstateofexplantsunderthemedium of
1/2MS+NAA0.02mg/L+6-BA1.0mg/L
Fig.2　MultiplicationstateofE.tirucaliunderthemediumof
1/2MS+NAA0.02 mg/L+6-BA1.0 mg/L+AD3.0
mg/L
RootingcultureofE.tirucali
ItwaseasytoinduceE.tirucalitoroot, butitsrooting
qualitywaspoor.FromTable3, E.tirucalicouldalsoroot
underthemediumof1/2MSwithoutanyplantgrowthregula-
tor, butrootingratewaslower, androotswereshortanddifi-
culttotransplant.TheadditionoflowconcentrationofIBAand
NAAinmediumcouldimproverootingquality, androotingrate
reachedabove80%.Amongvariousmediums, rootingeffect
wasthebestunderthemediumof1/2MS+NAA0.40mg/L+
IBA0.4 mg/L, anditsrootingratewasupto100% (Fig.3).
DomesticationandtransplantationofE.tirucali
Afterdomesticationandtransplantation, thetissueculture
380 AgriculturalScience＆TechnologyVol.12, No.3, 2011
seedlingsofE.tirucaligrewwel(Fig.4), andtransplanting
survivalratewasupto80%, butleavesbecameyeloworfel
inearlygrowthperiod, anditmightbecausedbylowair
humidity.
Table3　EfectsofdiferentmediumsontherootingofE.tirucali
Plantgrowthregulator∥mg/L Numberofrootingdays∥d
AverageLength
ofroot∥cm
Number
ofroots
Rooting
rate∥% Growthstate
1/2MS 13 0.3 1-3 75 Shortroots
1/2MS+NAA0.40 10 4.2 3-5 95 Longandthinwhiteroots
1/2MS+IBA0.4 10 3.2 2-3 80 Shortandthickdarkroots
1/2MS+NAA0.4+IBA0.4 8 4.6 3-6 100 Longandthickroot, morefibrousroots
Fig.3　RootingstateofE.tirucaliunderthemediumof1/2MS
+NAA0.40mg/L+IBA0.4mg/L
Fig.4　Growthstateoftissuecultureseedlingsinculturecups
ConclusionsandDiscussions
AstudyonthetissuecultureinvitroofE.tirucaliby
JiangLijuanetal.revealedthatthegerminationandmultipli-
cationcultureofE.tirucalidependedonplentyofexpensive
zeatin, whichwentagainstitslargescaleapplication[ 11].The
studyaimedatdiscussingtheregenerationsystemsofstem
segmentsofE.tirucalisuitabletofactoryproductionbyusing
diferentcombinationsofcommongrowthregulators.There-
sultsshowedthatgerminationrateunderthemedium of
1/2MS+NAA0.02 mg/L+6-BA1.0mg/Lwasupto89.7%
after24 d;whenthemedium ofmultiplicationculturewas
1/2MS+NAA0.02 mg/L+6-BA0.60 mg/L+AD3.0 mg/L,
explantsgrewrapidlywithmoreefectivebuds, andmultiplica-
tioncoefficientreached5.70;whenthemediumofrootingcul-
turewas1/2MS+NAA0.40 mg/L+IBA0.4 mg/L, there
weremanylongroots, andfibrousrootsweremore.Thecul-
tureefectfarexceededthatofLS+TDZ0.02 mg/Lstudied
byUchidaetal.(multiplicationcoefficientwas1.44)[ 12] , so
thestudycouldprovidereferencesforthefactoryproduction
oftissuecultureseedlingofE.tirucali.
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381LIUZhao-liangetal.EstablishmentofTissueCultureRegenerationSystemsofStemSegmentsofEuphorbiatirucali
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gentransformationinhorizontalsubsurfaceconstructedwetland(水
平潜流人工湿地氮转化模型研究)[J].JournalofHydroelectricEngi-
neering(水力发电学报), 2009, 28(6):151-156.
Responsibleeditor:YINJian-li Responsibleproofreader:WUXiao-yan
垂直潜流湿地对生活污水的净化效能研究
仝昭昭 1 ,王延华2＊ ,顾中铸 1　(1.南京师范大学能源与机械工程学院 ,江苏南京 210042;2.南京师范大学地理科学学院 ,江苏省环境
演变与生态建设重点实验室 ,江苏南京 210046)
摘要　采取间歇式进水和垂直潜流湿地结合的方法处理实际生活污水 , 构建了 4个人工湿地 ,即菖蒲湿地、芦苇湿地 、混栽湿地(菖蒲和芦
苇)及空白对照湿地 ,针对不同水力停留时间(HRT)及植物种属进行研究。结果表明:总氮(TN)和氨氮(NH+4 -N)的去除 ,种植植物的湿地以
HRT为 7d处理效果最好 , TN和NH+4 -N去除率最高分别达到 99.60%和 99.58%。空白湿地中 , TN去除率以 HRT为 6d和NH+4 -N去除率
以 HRT为 5d为最好 ,最高分别达到 87.90%、91.80%。对总磷(TP)的去除 , 4个湿地均以HRT为 6d为最佳 ,去除率均高于 93.00%。通过
对不同植物的研究 ,发现植物床对氮的去除效果优于空白湿地 ,芦苇湿地和混栽湿地的处理效果均优于菖蒲湿地。
关键词　垂直流湿地;生活污水;脱氮除磷;水力停留时间
基金项目　国家自然科学基金项目(50908116);南京师范大学 211基金项目(2009112XGQ0054);江苏高效优势学科建设工程资助项目;江苏省教育
厅重大项目(2009105TSJ0165)。
作者简介　仝昭昭(1987-),男 ,河南商丘人 ,硕士研究生,研究方向:污染生态修复研究。 ＊通讯作者,讲师 ,博士 ,硕士生导师,从事生态修复方面
的研究, E-mail:remanda@126.com。
收稿日期　 2011-02-08　　修回日期　 2011-04-06
(上接第 381页)
绿玉树茎段组织培养再生体系的建立(摘要)
刘召亮 ,何觉民＊ ,陈 彪 ,梁钾贤　(广东海洋大学农业生物技术研究所,广东湛江 524088)
[目的 ]研究绿玉树茎段组织培养再生苗的条件 ,确定各培养阶段的最佳培养条件 ,为绿玉树组培苗工厂化生产和相关研究提供参考。
[方法 ]以绿玉树茎段作为外植体试验材料 ,研究了不同培养基对萌芽率、增殖倍数、生根率的影响。
[结果 ]萌芽培养最佳的诱导培养基为 1/2MS+NAA 0.02 mg/L+6-BA1.0mg/L, 分化率为 89.7%;继代培养最佳培养基为 1/2MS+NAA
0.02mg/L+6-BA0.60mg/L+AD3.0mg/L,增殖倍数为 5.70;生根培养最佳培养基为 1/2MS+NAA0.40mg/L+IBA0.4mg/L,生根率达
100%,移栽成活率达 80%。
[结论 ]试验初步确定了绿玉树茎段组织培养的生长条件。
关键词　绿玉树;茎段;组织培养
基金项目　广东省科技攻关计划项目(2009B0203030092006B20201007)。
作者简介　刘召亮(1983-),男 ,山东临沂人 ,硕士研究生 ,研究方向:能源作物育种。 ＊通讯作者, 教授 ,从事作物遗传育种研究 , E-mail:hejm@
gdou.edu.cn。
收稿日期　 2011-03-04　　修回日期　 2011-04-11
(上接第 428页)
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[ 10] DINGXG(丁喜桂), YESY(叶思源), GAOZJ(高宗军).Methodsofheavymetalpolutionevaluationforofshoresediments(近海沉
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动态), 2005, 21(8):31-36.
Responsibleeditor:LIJing-wei Responsibleproofreader:WUXiao-yan
五指山茶园表层土壤重金属污染危害评价(摘要)
王 鹏 ,赵志忠＊ ,王军广 ,张忠伟 ,鲁双凤　(海南师范大学地理与旅游学院 ,海南海口 571158)
[目的 ]评价五指山茶园表层土壤重金属污染的危害程度。
[方法 ]采样分析五指山茶园表层土壤重金属(Cr、Ni、Cu、Zn、As、Cd、Pb)的平均含量及富集状况 ,分别以现代工业化以前沉积物中重金属的
全球最高背景值和我国 《土壤环境质量标准》的一级自然背景值为参比值 ,采用潜在生态危害指数法对五指山茶园表层土壤重金属的富集系
数和生态危害系数以及生态危害指数进行了评价。
[结果 ]以 2种背景值为参比得出的重金属污染水平相近 , Cd和 Pb是对五指山茶园表层土壤生态环境具有潜在影响的主要重金属元素。
[结论 ]该研究结果为五指山茶园重金属污染修复、茶园生态系统科学管理、保证五指山茶叶的优质高产提供了理论依据。
关键词　五指山;茶园;重金属;污染危害
基金项目　海南省自然科学基金项目(40879);海南省教育厅项目(HJKJ2010-28);教育部留学回国人员科研启动基金资助项目;海南师范大学 “地图
学与地理信息系统 ”、“自然地理学 ”重点学科联合资助项目。
作者简介　王鹏(1985-),男 ,陕西宝鸡人 ,硕士研究生 ,研究方向:热带海岛地表过程与环境评价。 ＊通讯作者,研究员。
收稿日期　 2011-01-26　　修回日期　 2011-04-01
465TONGZhao-zhaoetal.StudyonPurificationEficiencyofVerticalFlowWetlandsonDomesticWastewater