Table Of Content云 南 植 物 研 究 , ():
暋2010 321 25~31
ActaBotanicaYunnanica : /
暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋DOI10灡3724SP灡J灡1143灡2010灡09140
基质对中国特有濒危蕨类中国蕨配子体
形态发育和繁殖的影响
王卫清 , ,成 晓 *,焦 瑜 ,陈贵菊
1 2 1 1 3
暋 暋
中国科学院昆明植物研究所 云南 昆明 中国科学院研究生院 北京
(1 , 暋650204;2 , 暋100049;
济宁市农业科学研究院 山东 济宁
3 , 暋272031)
摘要:采用 种培养基质对中国蕨 孢子进行培养 结果显示 不同基质上中国
4 (Sinopterisgrevilleoides) , :
蕨配子体和幼孢子体生长发育存在差异 尤其是心形原叶体的形态差别明显 腐叶土基质上的心形原叶体
, ;
上未见颈卵器的发生 因而其有性繁殖过程不能完成 原生境土和腐叶土 混合土基质最适合中国蕨
, ; 1暶1
配子体生长发育及有性繁殖 改良 琼脂培养基上要保证幼孢子体正常生长需追加培养液
; Knop曚s 。
关键词:中国蕨 配子体 形态发育 繁殖 培养基质
; ; ; ;
中图分类号: 文献标识码: 文章编号:
Q945暋暋暋暋 暋暋 A暋暋暋暋 暋暋暋 0253灢2700(2010)01灢025灢07
SubstratumAffectstheGametophyteDevelopmentandReproduction
ofSinopterisgrevilleoidesSinopteridaceae
( ),
anEndangeredFernEndemictoChina
1,2 1* 1 3
WANGWei灢Qing ,CHENGXiao ,JIAOYu,CHENGui灢Ju
(1BotanicalGarden,KunmingInstituteofBotany,ChineseAcademyofSciences,Kunming650204,China;
2GraduateUniversityofChineseAcademyofSciences,Beijing100049,China;
3JiningInstituteofAgriculturalSciences,Jining272031,China)
Abstract
:SinopterisgrevilleoidesisanendangeredfernendemictotheregionofYunnanandSichuanin
southwestChina.Inthisstudy,weinvestigatedtheeffectsoffourdifferentculturesubstrataonthedevelop灢
mentofgametophyteandreproductionofthisfern.Culturedondifferentculturesubstrata,thefernshowed
differentmorphologyofgametophyte.Inhumussoil,cordateprothallididnotbeararchegonia,andthusthe
processofsexualreproductionofS灡grevilleoidescouldnotbefinished.Thesubstratumcomposedofhumus
soilandoriginalsoilinratioof1暶1wasthemostsuitablesubstratumforgametophytedevelopmentandsex灢
ualreproductionofthisspecies.Otherwise,infantsporophytesneededextranutrientsolutiontosustainits
growthonimprovedKnop曚sagarmedium.
Keywords
:Sinopterisgrevilleoides;Gametophyte;Morphologicaldevelopment;Reproduction;Culturesub灢
stratum
暋暋Sinopterisgrevilleoides(Christ)C.Chr.et (ChingandShing,1990).Decliningrapidlyin
Ching(Fig灡1),arareandendangeredspecies populationsizeandonthevergeofextinction,it
endemictoChina,hasascattereddistribution hasbeenlistedasaplantofsecond灢classprotec灢
onlyinSichuanandYunnaninsouthwestChina tioninTheListofChineseUrgentlyProtected
* Authorforcorrespondence;E灢mail:xcheng@mail灡kib灡ac灡cn;Tel:+86灢871灢5223903
Receiveddate:2009灢07灢28,Accepteddate:2009灢11灢23
作者简介 王卫清 女 在读硕士研究生 主要从事蕨类植物保育生物学研究
: (1982-) , , 。
云 南 植 物 研 究 第 卷
2暋6暋暋暋暋暋暋暋暋暋暋暋 暋暋暋暋暋暋暋 暋 暋 暋 暋 暋 暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋 32
WildPlants (Yu,1999).Previousstudiesof Table1暋Theresultsofchemicalpropertyanalysis
ofthesoil(mean暲SE)
thisspeciesmostlyfocusedonitssystemposi灢
Testingitems Originalsoil Humussoil
tion(Guoetal灡,1992;Zhangetal灡,2007),
pH 6灡58暲0灡01 4灡23暲0灡03
whilelittleworkhasbeendoneonitsgameto灢 Organicmatter(%) 3灡736暲0灡03 36灡08暲0灡06
TotalN (%) 0灡199暲0灡00 0灡93暲0灡01
phytedevelopmentandreproductionstrategy.
TotalP(%) 0灡071暲0灡00 0灡14暲0灡00
Theeffectsofculturesubstratumongameto灢 TotalK(mg/kg) 1灡747暲0灡00 0灡23暲0灡00
phytemorphologicaldevelopmentandreproduc灢 AvailableN(mg/kg) 124灡76暲4灡93 231灡87暲3灡01
AvailableP(mg/kg) 7灡01暲0灡21 56灡77暲2灡36
tionofSinopterisgrevilleoideswereinvestigated
AvailableK(mg/kg) 157灡66暲2灡89 404灡73暲2灡97
here.Gametophyteandinfantsporophytedeve灢 PotentCa(mg/kg) 4462灡65暲0灡01 468灡96暲4灡18
lopmentandthereproductioncharacteristicsof AvailableMg(mg/kg) 264灡30暲0灡01 175灡89暲0灡54
AvailableZn(mg/kg) 1灡77暲0灡01 16灡09暲0灡20
thespeciesonfourdifferentculturesubstrata AvailableFe(mg/kg) 9灡53暲0灡01 18灡71暲0灡22
werereported.Theinformationobtained will Available(mg/kg)B 0灡81暲0灡03 1灡10暲0灡00
AvailableS(mg/kg) 33灡80暲1灡54 13灡85暲0灡12
provideabasisforitsartificialpropagationand
(MeanandSEnumbersintheabovetablewerecalculatedwith
conservation.
theresultsofthreeparalleltests)
MaterialsandMethods
ImprovedKnopsagarmediumculture
1暋 1灡3暋 '
Sporecollection
1灡1暋 ForCulture4,thesporesweresterilizedwith4%so灢
Sporeswereobtainedfromfertilefrondsofsporo灢 diumhypochloritefor5minthenrinsedwithsterilized
phytes.Pinnaewerelefttodryatroomtemperaturein waterfourtimes;betweenrinsessporeswerecentrifuged
paperenvelopestofacilitatetheopeningofthesporangia at3500r/minforfivetimes(AD灢72centrifuge).After
andexpulsionofthespores.Sporeswereseparatedfrom that,thesporesweresownevenlyatanaveragedensityof
fragmentsoffrondsandsporangia,thenstoredinrefrig灢 200-300sporespercm2in6cmdiameterpetridishes.
eratoratabout4曟.Voucherspecimensaredepositedin ThemediumwasimprovedKnop曚sagarmedium(Liuet
theHerbariumofKunmingInstituteofBotany,Chinese al灡,1991)andthepHofthismediumtypewas6灡8.
AcademyofSciences(KUN). Everyculturewassetfifteenrepetitions.Allcultures
Soilculture
1灡2暋 werekeptinthedarkat25曟for24hthentransferredto
ForCulture1-3,sporesweresowninthreedifferent thelabwithartificiallight(pinkfluorescentillumination)
soilsubstrata:humussoil,mixturesoilcomposedofhumus at1000-1500lxunderadiurnalcycleof12/12h.The
soilandoriginalsoilinratioof1暶1andoriginalsoil,which temperaturewas22-28曟duringilluminationand14-
weremarkedasCulture1,Culture2andCulture3respec灢 18曟indark.Throughtheentireprocess,soilcultures
tively.Thehumussoilwascollectedunderthemixedconifer灢 weremoistenedwithcoolboiledwatertopreventdesicca灢
ousbroadleavedforestinYunnanProvince.Theoriginalsoil tion,andtohelptheopeningofantheridiaandthemove灢
wascollectedfromthehabitatofSinopterisgrevilleoides灢 mentofantherozoidinthelaterstage.Ontheoriginalsoil
Dayao,Yunnan.Theresultsofchemicalpropertyanalysisof (Culture3),prothalli,whichstoppedgrowingafterbear灢
thetwokindsofsoilarepresentedinTable1.ThepHofthe ingantheridiaandarchegonia,weretransplantedfrom
mixturesoilwas6灡0-6灡5.Thesoilsubstrataweresterilized threepetridishestoonepetridish,andthenweresprayed
bysaut湨ingintheChinesewayforfortyminutesatthetem灢 withwatertwiceaday.
peratureof140-160曟,andthensub灢packagedin12cmdi灢 Morphologicaldevelopmentwasobservedunderthe
ameterpetridishesaftercooled.Thedepthofsoilinthepetri opticalmicroscopes(OLYMPUSBX51)andanatomical
disheswasabout0灡5cm.Thesurfaceofthesubstratumwas lens (OLYMPUSSZ61).Forobservations,gameto灢
keptsmoothandsubstantial,andthenthesoilwassoakedby phytesandinfantsporophyteswereremovedfromculture
coolboiledwater.Finally,thesporeswererinsedwithsteri灢 andmountedinwater.Morphologicalcharacteristicsof
lizedwaterforfourtimes,andthensownevenlyatanaverage freshgametophyteswererecordedwithopticalCANNON
densityof200-300sporespercm2. POWERSHOTA640camera.
期
1 暋 暋WANGWei灢Qingetal灡:SubstratumAffectstheGametophyteDevelopmentandReproduction...暋暋2暋7
Results
2暋 ontheoriginalsoil(Culture3)wassimilarto
Thegametophytemorphologicaldevelopmentof thatonthe mixturesoilbutalittlelonger
Sinopterisgrevilleoidesondifferentculturesubstra灢 (Fig灡4).OntheKnop'sagarmedium,thepro灢
tavariedandthedetailswereshowedinTable2. thalliarebutterfly灢shapedwithflexuouswing
Thesporesonthemixturesoilcomposedofhumus margins(Fig灡5).
soilandoriginalsoilinratioof1暶1(Culture2)be灢 Theprothalliwerebisexualalthoughthe
gantogerminateat8-12daysaftersown,similar antheridiumandarchegoniumoccurredoneach
tothesporesontheKnop曚sagarmedium (Culture prothallusasynchronously.Theprothallionthe
4),8-15daysearlierthanthatonthehumussoil humussoilnolongerdevelopedarchegoniaafter
(Culture1),and1-9daysearlierthanthatonthe theyhadgivenbirthtoantheridiaandthepro灢
originalsoil(Culture3).Theratesofsporegermi灢 thalligrew continuouslyintolargeirregular
nationinCulture2andCulture4wereover67% greenlamina(Fig灡6)bykeepingthesoilsub灢
(Table2). stratum曚smoistureatmorethan60%.
Filamentsallhad3-7cellsandrarelybranched Ontheoriginalsoil(Culture3),mostpro灢
onthethreekindsofsoilmedia,whilebifurcationof thallistoppedgrowingafterbearingantheridia
filamentsontheKnop曚sagarmedium(Fig灡9)was andarchegonia;theformationrateofyoungspo灢
apparent灢therateofbranchedfilamentreachedupto rophyteswasonly20%,eventhoughtheculture
25%-30% (Table2). moisturewaskeptatmorethan90%;whenall
Themorphologyofcordateprothalliondif灢 theprothalliinthreepetridishesweretransplan灢
ferentculturesubstrata differed significantly tedtoonepetridish,theratewasimproved
fromeachother.Thecordateprothallielongated from20%to60%.Theprothallihadbornan灢
andthe marginsofthe wings wereslightly theridiabeforetheygrewintocordateonthe
curvedonthehumussoil(Culture1)(Fig灡2). Knop'sagarmedium (Fig灡8).Theprothallion
Thecordate prothalligrew wider with the themixturesoilmediumusuallyproducedmore
smoothwingmarginsonthemixturesoil(Cul灢 antheridiaandarchegoniathanthoseontheagar
ture2)(Fig灡3).Theshapeofcordateprothalli medium.
Table2暋GametophytemorphologicaldevelopmentofS灡grevilleoidesonfourculturesubstrata
Culture2
Culture1 Culture3 Culture4
Gametophytemorphogenesis (humussoil:
(humussoil) (originalsoil) (Knop'sagar)
originalsoil=1:1)
Sporegermination(d) 20-23 8-12 13-15 7-11
Rateofsporegermination(%) 10-15 67-73 18-20 68-75
Numberoffilamentcells(unit) 3-7 3-7 3-7 4-8
Ratioofbranchedfilament(%) 0 0 0 暋25-30
Seconddimensionalgrowth(d) 29 14 20 12
Cordateprothallus(d) 37 29 32 25
Ratioofthelengthandwidthoftheprothallus 1灡4-1灡6 0灡6-0灡8 1灡1-1灡3 曑0灡5
Antheridiaappearance(d) 46 30 45 23
Antheridianumber(u/p) 14-16 20-57 16-20 17-24
Archegoniaappearance(d) Non 50 74 46
Archegonianumber(u/p) Non 11-20 7-13 6-14
Infantsporophyteappearance(d) Non 93 122 114
﹪
Rateofprothallusdevelopingintosporophyte( ) 0 70 20 60
d=day(numberofdaysfromseedingsporestofirstlyobservingeverystageofgametophytemorphologicaldevelopment),
u=unit,p=prothallus.(Allvaluesintheabovetableweremeanvaluesofresultsofatleasttencyclicaltests)
云 南 植 物 研 究 第 卷
2暋8暋暋暋暋暋暋暋暋暋暋暋 暋暋暋暋暋暋暋 暋 暋 暋 暋 暋 暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋 32
Figs灡1-5暋Cordateprothalli
1.HabitatofS灡grevilleoides(Dayao,Yunnan,China,alt.1350m,Aug灡4,2008);2.(Culture1,37d);3.(Culture2,25d);4.(Culture
3,32d);5.(Culture4,29d).d=day(daysfromseedingsporestofirstlyobservingcordateprothalli).(1:bar=3cm;1-5:bar=150毺m)
暋暋OntheKnop曚sagarmedium (Culture4), caseofoneprothalluswithmorethanonemeristem灢
somesporesdevelopedintotwofilamentsandthe aticzonefrequentlyoccured(Fig灡9);infantsporo灢
filamentsbothgrewintoprothalli(Fig灡10);the phytesstoppedgrowingafterthefirstprimary
期
1 暋 暋WANGWei灢Qingetal灡:SubstratumAffectstheGametophyteDevelopmentandReproduction...暋暋2暋9
leaforthesecondonecameout(Fig灡11)and lution;therateofprothallidevelopingintosap灢
theirconstantgrowthneededextranutrientso灢 rophytesreachedupto70%.
Figs灡6-11暋GametophytemorphologyofS灡grevilleoides
6.Largeirregulargreenlamella(Culture1,48d,bar=160毺m);7.Prothalluswithseveralmeristematiczones(Culture4,38d,
bar=120毺m);8.Non灢heart灢shapedprothalluswithantheridias(Culture2,20d,bar=120毺m);9.Bifurcatefilament
(Culture4,25d,bar=50毺m);10.Filamentandlamellarstructuredevelopingfromonespore(Culture4,30d,bar=70毺m);
11.Infantsporophytes(Culture4,155d,bar=200毺m).
d=day(daysfromseedingsporestofirstlyobservingthemorphologyshown);an=antheridium;MZ=meristematiczone
Discussion
3暋 sexualreproductionofS灡grevilleoidescouldn曚t
Ourobservationshowedthatthematurecor灢 finished.Obviously,thehumussoilwasnot
dateprothallionthehumussoildidn曚tdevelop suitableforthesporepropagationofthisfern.
archegonia,and,thus,thenormalprocessof ThatthepHofthehumussoilwastoolowwas
云 南 植 物 研 究 第 卷
3暋0暋暋暋暋暋暋暋暋暋暋暋 暋暋暋暋暋暋暋 暋 暋 暋 暋 暋 暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋 32
probablythereason.Onthemixturesoilcom灢 pendentofsubstratetype.Themalegameto灢
posedofhumussoilandoriginalsoil(1暶1) phytesdevelopedinitiallyandthefemaleones
(Culture2),thesporesgerminatedanddevel灢 later,whichbroughtintocorrespondencewith
opedearlierthanthatonthehumusandoriginal mosttaxa(HauflerandRanker,1985).
soil,andtheratesofsporegerminationandpro灢 Ontheoriginalsoil,therateofprothallus
thallusdevelopingintosporophytewereboththe developingintosporophyteimprovedgreatlyaf灢
highestamongthefourcultures.Themixture tertransplanting;accordingly,itwasmorethan
soilprovidedthefernnotonlywithsuitablepH likelythepreviouslowformationrateofsporo灢
6灡0-6灡5,butalsowithabundantorganicmat灢 phytewascausedbythelowdensityofcordate
terandavailablemineralelementssuchasN,P, prothallionthesubstratum,andthelowdensity
andK.Thusthemixturesoilismostfavorable ofprothalliwasresultedfromthelowrateof
forthesporepropagationofthisspeciesamong sporegerminationontheoriginalsoil.
thethreeexaminedsoilsubstrata. Stokey(1951)reportedthatunfavorablecon灢
GametophytesofS灡grevilleoidesallformed ditionspromotedfilamentousgrowth.Inthis
nearlysymmetrical,cordateprothalliatayoung study,ontheKnop曚sagarmedium,thespores
stagethoughtheshapesofprothallioneachme灢 ofS灡grevilleoidesgerminatinglatertendedto
diumwerenotallthesame.Itwasreportedthat growlongfilamentsoronesporedevelopedinto
thegrowingdensityinfluencedtheshapeofpro灢 two filaments and some filaments always
thallus(Caoetal灡,2003).Itwasalsoshowed branched.Thepossiblereasonwasthespores
thatthecontentsofN灡P灡K,andCainthesoil germinatinglaterlackedadequatenutrientsand
affecttheearlydevelopmentofthefernrepro灢 space.AccordingtoTable2,theantheridiaand
ductionbyspores(Suzukietal灡,2005).Inour archegoniaontheKnop曚sagarmedium(Culture
study,thoughthesporesallweresownevenlyat 4)appearedearlierthanthoseonthemixture
2
anaveragedensityof200-300sporespercm soil(Culture2),however,infantsporophytesin
foreachculture,therateofsporegermination Culture4appearedlaterthanthoseinCulture2.
onfourculturesubstratawasdifferent,which Thisindicatedthatthehigherdensityofan灢
madethedensityofcordateprothallioneach theridiaandarchegoniacouldpromotetheemer灢
culturesubstratumdiffer.Itwasunclearthat genceofinfantsporophytes.Unliketheinvesti灢
whichfactor,differentdensityofcordateproth灢 gationofChenetal灡 (2008)—theprothallion
alliordifferentphysicalandchemicalnatureof theimprovedKnop曚sagarmediumrarelypro灢
eachsubstratum,resultedindifferentshapesof ducedinfantsporophytes,about60%ofprothal灢
cordateprothalli. liontheagarmediuminthisstudyproducedin灢
Themostcommonsequenceofsexualdevel灢 fantsporophytes.Thismaybecausedbythe
opmentinhomosporousfernsinvolvesthefor灢 differencesbetweenspecies.Theinfantsporo灢
mationofantheridiafollowedbyarchegonia(At灢 phytesstoppedgrowingunlessthenutritionwas
kinsonandStokey,1964).However,Pradaet sufficientinCulture4.
al灡 (2008)reportedthatinthecaseofPterisin灢 Prothalliofmostferntaxaarecapableofregen灢
completaprogressionofsexexpressionvaried eratingnewprothallifromoldones(Atkinsonand
dependingontheculturemedium,namely,fe灢 Stokey,1964;NayarandKaur,1971).Inthis
malegametophytesdevelopedearlierthanmale study,thelargeirregulargreenlaminaonthehu灢
onesonsoilcultures.Inthisresearch,these灢 mussoil(Fig灡6)andtheprothalluswithseveral
quenceofsexualexpressionappearedtobeinde灢 meristematiczonesontheKnop曚sagarmedium
期
1 暋 暋WANGWei灢Qingetal灡:SubstratumAffectstheGametophyteDevelopmentandReproduction...暋暋3暋1
(Fig灡7)suggestedthatS灡grevilleoidesmayregen灢 AnumericaltaxonomyofthefamilySinopteridaceae[J].
广西植物
Guihaia( ),12(3):235—242
eratenewprothallifromoldones,butadditionalob灢
HauflerCA,RankerTA,1985.Differentialatheridiogenre灢
servationwasneededtoproveit.
sponseandevolutionarymechanismsinCystopteris[J].A灢
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:WethankProf.WeimingZhufrom 刘保东 包文美 敖志文
LiuBD( ),BaoWM( ),AoZW( ),1991.
YunnanUniversityandMr.YuChenfromKunmingIn灢
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