全 文 ：安徽省自然科学基金项目，项目编号：070411010
崔广荣 男，1964年生，硕士，副教授。研究方向：植物生物技术。 Tel：0550-6732029，E-mail：cuigr@ah163.com
收稿日期：2005-12-19 修回日期：2006-07-18
热 带 作 物 学 报
CHINESEJOURNALOFTROPICALCROPS
Vol.28No.1
Mar.2007
第 28卷 第 1期
2007年 3月
DirectSomaticEmbryogenesisonLeafExplantsof
Phalaenopsis,SubsequentDevelopment
andPlantRegeneration
CuiGuangrong LiaoLinlin LiuYuecheng ZhangZixue
PlantScienceDepartmentofAnhuiScienceandTechnologyUniversity Fengyang Anhui 233100 China
Abstract Youngleafsegmentsofanorchid (PhalaenopsisTsueiFoaLady)culturedinvitroproducedclustersofso-
maticembryosdirectlyfromupperepidermisandmesophylcelsofleafandwoundedsurfaceswithin45days.Embryonic
celproducedmulticelularproembryosbyceldivision.Multicelularproembryosweredevelopingsuccessivelyinto
globularembryoids,pear-shapedembryoids,heart-shapedembryoids,cotyledon-shapedembryoidsandwel-developed
embryos--protocorm-likebodies(PLBs).Highfrequencyofsomaticembryogenesiswasfoundon1/2MSbasalmedium
supplementedwithahighdosageofN6-benzyladenine(6-BA,4.0~8.0mg/L)andadeninesulphate(AdSO4,3.0~5.0mg/L).
Thehighestinducingratewasupto40percent.The6-BAandAdSO4atlowerconcentrationshadadvantagesinplant
regenerationfromPLBswhileattoolowerconcentrationsinfluencedthegrowthanddevelopmentofthetubeplantlets.
KeywordsPhalaenopsisleaf somaticembryogenesis protocorm-likebody plantregeneration
PhalaenopsisTsueiFoaLady,whichbelongstothegenusPhalaenopsisandthefamilyOrchidaceae,isa
commercialyimportantrepresentativespecies.Thiskindoforchid,havingsinglestemandpeculiarplantshape,
isoftencaledEmpressofexoticorchidsinChina[1].Themasspropagationwasmainlyconductedbyseedpro-
liferationunderasepticsituationsandplanttissuecultureinvitro.Plantletswereusualyobtainedfromproto-
corm-likebodies(PLBs)producedfromthecultureofshoot-tips[2,3],leaves[3~5],joint-pointsofflower-stems[6]
andotherorgansortissues[7,8]intheprocessofplanttissuecultureinvitro.Protocorm-likebodiescanbein-
ducedfromintermediatecalusproliferatedonundefinedmedia[9]ordirectlyfromshoottipandyoungleafcul-
turedonappropriatemedium[3,5,6].Manyorchidresearchershavesuggestedthattheprocessofsomaticembryoge-
nesisbeapartoftheearlystepsofPLBformationonorchids[9~11].TheinducingprocessofthePLBofPha-
laenopsisisjustaprocessofsomaticembryogenesis[3].Chineseresearchersreportedtheanatomicalstudieson
protocorm-likebodiesofphalaenopsis[12].IshietalreportedthatPLB-derivedpreliminarycaliofaPhalaenop-
siscultivarwereinducedonanundefinedmediumsupplementedwithcoconutmilk[9].SomeChineseresearchers
reportedtheirstudyresultsaboutcalusinductionaswel[13].Somaticembryogenesisisparticularlyadvantageous
instudiesonspontaneousvariationwhichoriginatedduringinvitrocultureorwasexperimentalyinducedby
mutagensortransformation[11,14,15].However,thestudyonorchidsomaticembryogenesisisattheinitialstage[8].
ThestudiesonhistologicalcharacteristicsofsomaticembryogenesisdirectlyfromPhalaenopsisyoungleafhave
notbeenweldocumented.Inthepresentstudy,theyoungleavesoftubeshootswereusedasexplantsforinitiat-
ingsomaticembryogenesis.Thehistologicalcharacteristicsofsomaticembryogenesisandtheirdevelopment
weredescribedaccordingtotheparafinwaxsectionofan8μmthickness.Meanwhile,anatemptwasmadeto
studytheefectsofplantgrowthregulatorsof6-BAandAdSO4onsomaticembryogenesis,PLBdevelopment,
1期 崔广荣等：蝴蝶兰叶片体细胞胚胎发生发育及其分化成苗的研究
plantregenerationandgrowth.ThepurposeofthisstudywastoclarifythePhalaenopsishistologicalcharacteris-
ticsofsomaticembryogenesisandthetissueculturebasedplantregenerationfromthePLB,inordertoprovide
withsomereferencesforfurtherstudyofphysiologicalandmolecularmechanismofsomaticembryogenesisand
orchidmutationbreeding.
1 Materialsandmethods
The3~4leavesplantletsofPhalaenopsisTsueiFoaLadywereobtainedfromflower-stalkbudculturedin
vitrointheCenterofBiotechnology,AnhuiScienceandTechnologyUniversity.Theleafsegmentswereexcised
asexplants,whoseareawasabout1.0cm2orso.Theexplantswereplacedonthesurfaceofbasalmediumwith
1/2strengthmacro-andmicro-elementsofMurashigeandSkoog(1962)supplementedwithdiferentconcentra-
tionsof6-BAandAdSO4(seeTable1),NAA(0.5mg/L,Naphthaleneaceticacid),sucrose(20000mg/L),agar
powder(4000mg/L),andcoconutmilk(100ml/L).Plantgrowthregulatorswereaddedpriortoautoclavingas
optionaladditives.ThepHofthemediawasadjustedto5.4with1mol/LNaOHorHClpriortoautoclavingfor
25minat121.3℃.Theexplantswereincubatedin280mLsaucebotleundera12:12-hphotoperiodat1500lx
and(25±1)℃.Eachtreatmenthadtensaucebotleswhereleaffragmentswereinoculated.Observationswere
madeafter30daysofgrowthwhilestatisticalanalysisofsomaticembryoinducingratewasmadeafter60days.
Thepercentageofembryosperexplantwasdeterminedforeachtreatment.Theinducedsomaticembryoswere
subculturedonthesameinducingmediuminordertoobservesomaticembryodevelopment,plantregeneration
andgrowth.ThestatisticalanalysisaboutplantletsregeneratedfromPLBandgrowthstateweremadeafter30
daysofplanting.Cultureswereexaminedandphotographedwithacamera(SEAGULL,DF-2).
TissuesforhistologicalobservationswerefixedinFAA (95%ethylalcohol:glacialaceticacid:formalde-
hyde:water,10:1:2:7),dehydratedinatertiary-butyl-alcoholseries,embeddedinparafinwax,sectionedatan8μm
thicknessandstainedwithSafranineTandFastGreen.Thesectionswereexaminedunderamicroscopeand
photographedwithadigitalcamera(Sony,1.3MEGAPLXELS).
2 ResultsandAnalysis
2.1 Characteristicsofsomaticembryoproliferationanddevelopment
Itcanbeseenthatsomaticembryoswereoriginatedfromleafupperepidermalcellayersandsomemeso-
phylcels(Figs1,2).Butthesomaticembryosstemmedfromlowerepidermalcellayerswerenotfound.The
somaticembryogenesisoriginatedfromsinglecellikeotherplants.Proembryocelsofepidermallayersormes-
ophylwereofdenselystainedcytoplasmandnucleiandhadobviousboundarywithothercels(Fig2).The
proembryoscelsproducedmulticelularproembryosrapidlythroughceldivision.Thelaterwerethendevel-
opedintoglobularembryoids,whichhadsmalcelsanddenselystainedcytoplasm,andhadmoreobvious
boundarywithothertissueorcels(Figs3,4).Theglobularembryoidswentondevelopingintopear-shapedem-
bryoidswhichwereeasilyfoundinthemonocotyledon (Fig5).Thepear-shapedembryoidswentondeveloping
intoheart-shapedembryoidswhichprotrudedfromtheupperepidermalcellayersorthewoundedsurfacesof
explants(Figs6,7).Themargincelsofheart-shapedembryoidsgrewfasterthanthecentercels,andtheheart-
shapedembryoidseventualyformedcotyledon-shapedembryoids(Figs8,9)andthecotyledon-shapedembry-
oidsbecamewel-developedyoungPLBs(Fig10,12,13).Embryogenicnodularmassesprotrudedfromtheepi-
dermalcellayersandthewoundedsurfacesofexplantswithininoculated45days(Figs11,12).Meanwhile,the
celslinkingsomaticembryoandtheepidermalcellayerswereturnedtoloosestructuretissuesinwhichcels
becamelongandbig(Fig10).Thistypeofstructuremayberelatedtomaterialstransport,facilitatingeasysepa-
rationofPLBsfromothertissues.
55
热 带 作 物 学 报 28卷
2.2 Efectsofplantgrowthregulatorsonembryoproliferation,developmentandplantregeneration
Table1showedthattheconcentrationsof6-BAandAdSO4afectednotonlysomaticembryogenesisand
developmentbutalsoplantregenerationgreatly.InthetreatmentsofI-1,I-1,II-1,IV-1withoutAdSO4,em-
bryoproliferationonlyoccuredonthemediawhichhadhighconcentrationsof6-BA.Therewasnoembryo
proliferationonthemediumcontaining2.0mg/L6-BA.
Whentheconcentrationof6-BAwaskeptdefinite,addingregulatorAdSO4stronglypromotedsomaticem-
bryogenesis.Fromthegeneraltrend,itcanbeseenthattheembryoinductionrateincreasedwiththeconcentra-
tionofAdSO4.Thehighestembryoinductionratecameupto40percentwhenthe6-BAwasappliedatthe
concentrationof6.0mg/LandAdSO4at3.0mg/L.ButiftheconcentrationofAdSO4exceeded3.0mg/L,the
embryoinductionratedecreasedunderthesamecondition.Therefore,thetreatmentII-3isfoundsuitablefor
somaticembryogenesisunderthisexperimentcondition.
Clumpsoftheleaf-derivedprimaryembryogenicnodulemasswereproliferatedtoproducemoreorless
embryos/PLBswhensubculturedonthesamemedia(Fig12).Someoftheprimaryembryogenicnodulemass
gradualyturnedbrownanddiedwhensubculturedonthemediawhichcontainedhigherconcentrationsof6-BA
andAdSO4(6-BA>6.0mg/L,AdSO4>3.0mg/L).Therefore,lessembryos/PLBswereformed.Onthecontrary,
theprimaryembryogenicnodulemasssubculturedonthemediacontaininglowerconcentrationsof6-BAand
AdSO4producedmoreembryos/PLBsandfurtherregeneratednewplants(Fig14).Inaddition,plantletsregener-
atedfromPLBssubculturedonthemediathatcontainedhigherconcentrationsof6-BAandAdSO4became
swolenandmalformed (Fig15).However,plantletsregeneratedfromPLBssubculturedonthemediathatcon-
tainedlowerconcentrationsof6-BAandAdSO4formedsomerootsandbecamesmalandweak.Therefore,the
toohighortoolowconcentrationsof6-BAandAdSO4arenotsuitableforshootgrowthanddevelopment.
Ⅰ-1 2.0 0 0 0e - - - -
Ⅰ-2 2.0 1.0 4 10.0d 4 100 100 Smal,thinleaf,rooting
Ⅰ-3 2.0 3.0 4 10.0d 4 100 100 Smal,thinleaf,rooting
Ⅱ-1 4.0 0 6 15.0cd 6 100 100 Thickleaf,rooting
Ⅱ-2 4.0 1.0 10 25.0bc 10 100 100 Thickleaf,rooting
Ⅱ-3 4.0 3.0 12 30.0b 11 91.7 100 Thickleaf,norooting
Ⅲ-1 6.0 0 9 22.5c 8 88.9 100 Thick,broadleaf,norooting
Ⅲ-2 6.0 1.0 10 25.0bc 8 80.0 100 Thick,broadleaf,norooting
Ⅲ-3 6.0 3.0 16 40.0a 13 81.3 100 Thick,broadleaf,norooting
Ⅳ-1 8.0 0 7 17.5cd 6 85.7 83.3 Thick，broad，fat,leaf,norooting,malformation
Ⅳ-2 8.0 1.0 9 22.5c 5 55.6 80.0 Thick,broad,fat,leaf,norooting,malformation
Ⅳ-3 8.0 3.0 12 30.0b 6 50.0 66.7 Thick,broad,fat,leaf,norooting,malformation
Ⅰ-4 2.0 5.0 5 12.5d 5 100 100 Smal,thinleaf,rooting
Ⅱ-4 4.0 5.0 13 32.5b 12 92.3 100 Thick,broadleaf,norooting
Ⅲ-4 6.0 5.0 11 27.5bc 8 72.7 100 Thick,broadleaf,norooting
Ⅳ-4 8.0 5.0 12 30.0b 3 25.0 0 -
Note:①AddingNAA0.5mg/Landcoconutmilk100ml/Lineachmedium;②No.offormingembryoid/totalNo.ofinoculatedleaves;③Leaf
No.offormingPLB/LeafNo.offormingprotrudingembryoid;④PlantregenerationNo.ofPLBs/totalNo.ofPLBs.
No.of
treatment①
Concentration
of6-BA
Concentration
ofAdSO4
LeafNo.of
formingembryoid
Embryoid
inducing
rate/%②
PieceNo.of
formingPLBs
PLBforming
rateof
subculture③
Plantregeneration
ratefromPLBs④
Plantletsgrowth
situation
Table1 Efectsofplantgrowthregulatorsonembryoproliferation,developmentandplantregeneration
56
1期 崔广荣等：蝴蝶兰叶片体细胞胚胎发生发育及其分化成苗的研究
3 DiscussionandConclusion
Therehavebeenmorethan150plantspeciesfoundtoinducesomaticembryofrominvitroculture.These
speciesareunderover40familieswhichincludealmostimportantfamiliesofAngiospermaeandsomeGym-
nospermae[15].Thisindicatedthatsomaticembryogenesisisacommonphenomenonamonghigherplants[14,15].
Therearethreewaystoproliferatesomaticembryo:directlyfromplantorgans,fromcalus,andfromsinglefree
cel.IshietalreportedthatPLBswerederivedfrompreliminarycalusofaPhalaenopsiscultivar[9].Somedo-
mesticresearchersreportedtheirstudyresultsaboutPLBsinduction,propagationandplantletsregenerationfrom
PLBs[3,6,16,17].Butnodetailedstudiesonthehistologicalcharacteristicsofsomaticembryogenesisaredocument-
ed.ThepresentstudyshowedthattheprocessofsomaticembryogenesisanddevelopmentintoPLBofPha-
laenopsiswassimilartothatofotherMonocotyledonplants.Thisprocessincludessingleembryoniccel,multi-
celularproembryos,globularembryoid,pear-shapedembryoid,heart-shapedembryoid,cotyledon-shapedem-
bryoidandwel-developedembryos---protocorm-likebodies(PLBs).However,asfarasembryoniccelori-
ginisconcerned,theprocessofsomaticembryogenesisofPhalaenopsisyoungleafisdiferentfromthatofother
plantsinthatthesomaticembryosstemmedfromlowerepidermalcellayershavenotbeenfound.Thereasons
ofthisphenomenonremaintobefurtherstudied.
Thesomaticembryogenesis,whichisakindofexpressionofplantceltotipotency,indicatedthattheplant
celhasnotonlyalgeneticinformationbutalsorecurtheproliferationprocessofzygoticembryo[6,9~11,15].Al-
thoughthisstudywasconductedwithonlyonecultivar,thereshouldbeimportantreferencevalueforrelatedre-
searchesofotherorchids.Itisvaluabletorevealthemechanismofceldiferentiation,developmentandmorpho-
genesistomakeathoroughinvestigationandstudyonthehistologicalcharacteristicsofsomaticembryogenesis
anddevelopmentofPhalaenopsis.Thisstudyonsomaticembryogenesislaysafoundationforcultivarimprove-
ment,genetransformationandmolecularbiologydevelopment.
Manyfactorsarefoundtoinfluencesomaticembryogenesisanddevelopment.Plantgrowthregulatorsand
theirconcentrationsplayanimportantroleinregulatingsomaticembryogenesisanddevelopment[3,6,9~11,15].Qin
Fanetalreportedthat6-BAplayedakeyroleintheprocessofsomaticembryogenesisofPhalaenopsisleafand
thatlowerconcentrationof2,4-Dimprovedsomaticembryogenesis[3].Ourstudyshowedthathigherconcentra-
tionsof6-BAcombiningwithAdSO4canresultinahighersomaticembryoinductionrate.Theefectsofdifer-
entexplants,mediumandcultureconditiononsomaticembryogenesisofPhalaenopsisyoungleafneedafurther
study.
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蝴蝶兰叶片体细胞胚胎发生发育
及其分化成苗的研究
崔广荣 廖玲玲 刘跃成 张子学
安徽科技学院植物科学系 安徽 凤阳 233100
摘 要 蝴蝶兰幼叶离体培养直接诱导体细胞胚胎的发生并进一步发育成原球茎和分化成苗。体细胞胚胎
发生起源于上表皮细胞或上表皮下方的叶肉细胞，为单细胞起源。单细胞原胚分裂形成多细胞原胚，历经球
形胚、梨形胚、心形胚和子叶胚的发育过程，最终成为较大颗粒状的原球茎。较高浓度的苄基腺嘌呤（6-BA）
和腺嘌呤硫酸盐（AdSO4）配合使用能有效诱导体细胞胚胎的发生，最高诱导率可达 40%。适当降低 6-BA和
AdSO4浓度有利于原球茎分化成苗，但两者浓度过低苗的生长发育会受到影响。
关键词 蝴蝶兰 叶片 体细胞胚胎发生 原球茎 植株再生
中图分类号 S682.31 Q343.5
58
1期 崔广荣等：蝴蝶兰叶片体细胞胚胎发生发育及其分化成苗的研究
崔广荣等：蝴蝶兰叶片体细胞胚胎发生发育及其分化成苗的研究 图 版
ExplanationofplateI
PlateI:Fig1、2.Somaticembryooriginatedfromleafupperepidermalcellayersandsomemesophylcels;Fig3、4.Globularembryoid;
Fig5.Pear-shapedembryoid;Fig6、7.Heart-shapedembryoid;Fig8、9Cotyledon-shapedembryoid;Fig10.Wel-developedyoungPLBsand
loosen-structuretissues(arow);Fig11,12.Embryogenicnodular;Fig13.PLBsformationfromleafsegments;Fig14,15.Plantletsdeveloped
fromPLBs.
41
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876
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5
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