全 文 ：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1,2,成暋晓1
*,焦暋瑜1,陈贵菊3
(1中国科学院昆明植物研究所,云南 昆明暋650204;2中国科学院研究生院,北京暋100049;
3济宁市农业科学研究院,山东 济宁暋272031)
摘要:采用4种培养基质对中国蕨 (Sinopterisgrevileoides)孢子进行培养,结果显示:不同基质上中国
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1暶1混合土基质最适合中国蕨
配子体生长发育及有性繁殖;改良Knop曚s琼脂培养基上要保证幼孢子体正常生长需追加培养液。
关键词:中国蕨;配子体;形态发育;繁殖;培养基质
中图分类号:Q945暋暋暋暋 暋暋文献标识码:A暋暋暋暋 暋暋暋文章编号:0253灢2700(2010)01灢025灢07
SubstratumAffectstheGametophyteDevelopmentandReproduction
ofSinopterisgrevileoides(Sinopteridaceae),
anEndangeredFernEndemictoChina
WANG Wei灢Qing1,2,CHENGXiao1* ,JIAOYu1,CHENGui灢Ju3
(1BotanicalGarden,KunmingInstituteofBotany,ChineseAcademyofSciences,Kunming650204,China;
2GraduateUniversityofChineseAcademyofSciences,Beijing100049,China;
3JiningInstituteofAgriculturalSciences,Jining272031,China)
Abstract:SinopterisgrevileoidesisanendangeredfernendemictotheregionofYunnanandSichuanin
southwestChina.Inthisstudy,weinvestigatedtheeffectsoffourdifferentculturesubstrataonthedevelop灢
mentofgametophyteandreproductionofthisfern.Culturedondifferentculturesubstrata,thefernshowed
differentmorphologyofgametophyte.Inhumussoil,cordateprothalididnotbeararchegonia,andthusthe
processofsexualreproductionofS灡grevileoidescouldnotbefinished.Thesubstratumcomposedofhumus
soilandoriginalsoilinratioof1暶1wasthemostsuitablesubstratumforgametophytedevelopmentandsex灢
ualreproductionofthisspecies.Otherwise,infantsporophytesneededextranutrientsolutiontosustainits
growthonimprovedKnop曚sagarmedium.
Keywords:Sinopterisgrevileoides;Gametophyte;Morphologicaldevelopment;Reproduction;Culturesub灢
stratum
暋暋Sinopterisgrevileoides(Christ)C.Chr.et
Ching(Fig灡1),arareandendangeredspecies
endemictoChina,hasascattereddistribution
onlyinSichuanandYunnaninsouthwestChina
(ChingandShing,1990).Decliningrapidlyin
populationsizeandonthevergeofextinction,it
hasbeenlistedasaplantofsecond灢classprotec灢
tioninTheListofChineseUrgentlyProtected
云 南 植 物 研 究暋2010,32(1):25~31
ActaBotanicaYunnanica暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋DOI:10灡3724/SP灡J灡1143灡2010灡09140
* 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 (1982-)女,在读硕士研究生,主要从事蕨类植物保育生物学研究。
WildPlants (Yu,1999).Previousstudiesof
thisspeciesmostlyfocusedonitssystemposi灢
tion(Guoetal灡,1992;Zhangetal灡,2007),
whilelittleworkhasbeendoneonitsgameto灢
phytedevelopmentandreproductionstrategy.
Theeffectsofculturesubstratumongameto灢
phytemorphologicaldevelopmentandreproduc灢
tionofSinopterisgrevileoideswereinvestigated
here.Gametophyteandinfantsporophytedeve灢
lopmentandthereproductioncharacteristicsof
thespeciesonfourdifferentculturesubstrata
werereported.Theinformationobtained wil
provideabasisforitsartificialpropagationand
conservation.
1暋MaterialsandMethods
1灡1暋Sporecolection
Sporeswereobtainedfromfertilefrondsofsporo灢
phytes.Pinnaewerelefttodryatroomtemperaturein
paperenvelopestofacilitatetheopeningofthesporangia
andexpulsionofthespores.Sporeswereseparatedfrom
fragmentsoffrondsandsporangia,thenstoredinrefrig灢
eratoratabout4曟.Voucherspecimensaredepositedin
theHerbariumofKunmingInstituteofBotany,Chinese
AcademyofSciences(KUN).
1灡2暋Soilculture
ForCulture1-3,sporesweresowninthreedifferent
soilsubstrata:humussoil,mixturesoilcomposedofhumus
soilandoriginalsoilinratioof1暶1andoriginalsoil,which
weremarkedasCulture1,Culture2andCulture3respec灢
tively.Thehumussoilwascolectedunderthemixedconifer灢
ousbroadleavedforestinYunnanProvince.Theoriginalsoil
wascolectedfromthehabitatofSinopterisgrevileoides灢
Dayao,Yunnan.Theresultsofchemicalpropertyanalysisof
thetwokindsofsoilarepresentedinTable1.ThepHofthe
mixturesoilwas6灡0-6灡5.Thesoilsubstrataweresterilized
bysaut湨ingintheChinesewayforfortyminutesatthetem灢
peratureof140-160曟,andthensub灢packagedin12cmdi灢
ameterpetridishesaftercooled.Thedepthofsoilinthepetri
disheswasabout0灡5cm.Thesurfaceofthesubstratumwas
keptsmoothandsubstantial,andthenthesoilwassoakedby
coolboiledwater.Finaly,thesporeswererinsedwithsteri灢
lizedwaterforfourtimes,andthensownevenlyatanaverage
densityof200-300sporespercm2.
Table1暋Theresultsofchemicalpropertyanalysis
ofthesoil(mean暲SE)
Testingitems Originalsoil Humussoil
pH 6灡58暲0灡01 4灡23暲0灡03
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
TotalP (%) 0灡071暲0灡00 0灡14暲0灡00
TotalK (mg/kg) 1灡747暲0灡00 0灡23暲0灡00
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
AvailableK (mg/kg) 157灡66暲2灡89 404灡73暲2灡97
PotentCa(mg/kg) 4462灡65暲0灡01 468灡96暲4灡18
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
AvailableFe(mg/kg) 9灡53暲0灡01 18灡71暲0灡22
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
(MeanandSEnumbersintheabovetablewerecalculatedwith
theresultsofthreeparaleltests)
1灡3暋ImprovedKnopsagarmediumculture
ForCulture4,thesporesweresterilizedwith4%so灢
diumhypochloritefor5 minthenrinsedwithsterilized
waterfourtimes;betweenrinsessporeswerecentrifuged
at3500r/minforfivetimes(AD灢72centrifuge).After
that,thesporesweresownevenlyatanaveragedensityof
200-300sporespercm2in6cmdiameterpetridishes.
ThemediumwasimprovedKnop曚sagarmedium (Liuet
al灡,1991)andthepHofthismediumtypewas6灡8.
Everyculturewassetfifteenrepetitions.Alcultures
werekeptinthedarkat25曟for24hthentransferredto
thelabwithartificiallight(pinkfluorescentilumination)
at1000-1500lxunderadiurnalcycleof12/12h.The
temperaturewas22-28曟 duringiluminationand14-
18曟indark.Throughtheentireprocess,soilcultures
weremoistenedwithcoolboiledwatertopreventdesicca灢
tion,andtohelptheopeningofantheridiaandthemove灢
mentofantherozoidinthelaterstage.Ontheoriginalsoil
(Culture3),prothali,whichstoppedgrowingafterbear灢
ingantheridiaandarchegonia,weretransplantedfrom
threepetridishestoonepetridish,andthenweresprayed
withwatertwiceaday.
Morphologicaldevelopmentwasobservedunderthe
opticalmicroscopes(OLYMPUSBX51)andanatomical
lens (OLYMPUS SZ61).Forobservations,gameto灢
phytesandinfantsporophyteswereremovedfromculture
andmountedinwater.Morphologicalcharacteristicsof
freshgametophyteswererecordedwithopticalCANNON
POWERSHOTA640camera.
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2暋Results
Thegametophytemorphologicaldevelopmentof
Sinopterisgrevileoidesondiferentculturesubstra灢
tavariedandthedetailswereshowedinTable2.
Thesporesonthemixturesoilcomposedofhumus
soilandoriginalsoilinratioof1暶1(Culture2)be灢
gantogerminateat8-12daysaftersown,similar
tothesporesontheKnop曚sagarmedium (Culture
4),8-15daysearlierthanthatonthehumussoil
(Culture1),and1-9daysearlierthanthatonthe
originalsoil(Culture3).Theratesofsporegermi灢
nationinCulture2andCulture4wereover67%
(Table2).
Filamentsalhad3-7celsandrarelybranched
onthethreekindsofsoilmedia,whilebifurcationof
filamentsontheKnop曚sagarmedium (Fig灡9)was
apparent灢therateofbranchedfilamentreachedupto
25%-30% (Table2).
Themorphologyofcordateprothaliondif灢
ferentculture substrata differed significantly
fromeachother.Thecordateprothalielongated
andthe marginsofthe wings wereslightly
curvedonthehumussoil(Culture1)(Fig灡2).
The cordate prothali grew wider with the
smoothwingmarginsonthemixturesoil(Cul灢
ture2)(Fig灡3).Theshapeofcordateprothali
ontheoriginalsoil(Culture3)wassimilarto
thatonthe mixturesoilbutalittlelonger
(Fig灡4).OntheKnopsagarmedium,thepro灢
thaliarebutterfly灢shaped withflexuouswing
margins(Fig灡5).
Theprothaliwerebisexualalthoughthe
antheridiumandarchegoniumoccurredoneach
prothalusasynchronously.Theprothalionthe
humussoilnolongerdevelopedarchegoniaafter
theyhadgivenbirthtoantheridiaandthepro灢
thaligrew continuouslyintolargeirregular
greenlamina(Fig灡6)bykeepingthesoilsub灢
stratum曚smoistureatmorethan60%.
Ontheoriginalsoil(Culture3),mostpro灢
thalistoppedgrowingafterbearingantheridia
andarchegonia;theformationrateofyoungspo灢
rophyteswasonly20%,eventhoughtheculture
moisturewaskeptatmorethan90%;whenal
theprothaliinthreepetridishesweretransplan灢
tedtoonepetridish,theratewasimproved
from20%to60%.Theprothalihadbornan灢
theridiabeforetheygrewintocordateonthe
Knopsagarmedium (Fig灡8).Theprothalion
themixturesoilmediumusualyproducedmore
antheridiaandarchegoniathanthoseontheagar
medium.
Table2暋GametophytemorphologicaldevelopmentofS灡grevileoidesonfourculturesubstrata
Gametophytemorphogenesis
Culture1
(humussoil)
Culture2
(humussoil:
originalsoil=1:1)
Culture3
(originalsoil)
Culture4
(Knopsagar)
Sporegermination(d) 20-23 8-12 13-15 7-11
Rateofsporegermination(%) 10-15 67-73 18-20 68-75
Numberoffilamentcels(unit) 3-7 3-7 3-7 4-8
Ratioofbranchedfilament(%) 0 0 0 暋25-30
Seconddimensionalgrowth(d) 29 14 20 12
Cordateprothalus(d) 37 29 32 25
Ratioofthelengthandwidthoftheprothalus 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
Rateofprothalusdevelopingintosporophyte(﹪) 0 70 20 60
d=day(numberofdaysfromseedingsporestofirstlyobservingeverystageofgametophytemorphologicaldevelopment),
u=unit,p=prothalus.(Alvaluesintheabovetableweremeanvaluesofresultsofatleasttencyclicaltests)
721期暋 暋WANG Wei灢Qingetal灡:SubstratumAffectstheGametophyteDevelopmentandReproduction...暋暋暋
Figs灡1-5暋Cordateprothali
1.HabitatofS灡grevileoides(Dayao,Yunnan,China,alt.1350m,Aug灡4,2008);2.(Culture1,37d);3.(Culture2,25d);4.(Culture
3,32d);5.(Culture4,29d).d=day(daysfromseedingsporestofirstlyobservingcordateprothali).(1:bar=3cm;1-5:bar=150毺m)
暋暋OntheKnop曚sagarmedium (Culture4),
somesporesdevelopedintotwofilamentsandthe
filamentsbothgrewintoprothali(Fig灡10);the
caseofoneprothaluswithmorethanonemeristem灢
aticzonefrequentlyoccured(Fig灡9);infantsporo灢
phytesstoppedgrowingafterthefirstprimary
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leaforthesecondonecameout(Fig灡11)and
theirconstantgrowthneededextranutrientso灢
lution;therateofprothalidevelopingintosap灢
rophytesreachedupto70%.
Figs灡6-11暋GametophytemorphologyofS灡grevileoides
6.Largeirregulargreenlamela (Culture1,48d,bar=160毺m);7.Prothaluswithseveralmeristematiczones(Culture4,38d,
bar=120毺m);8.Non灢heart灢shapedprothaluswithantheridias(Culture2,20d,bar=120毺m);9.Bifurcatefilament
(Culture4,25d,bar=50毺m);10.Filamentandlamelarstructuredevelopingfromonespore(Culture4,30d,bar=70毺m);
11.Infantsporophytes(Culture4,155d,bar=200毺m).
d=day(daysfromseedingsporestofirstlyobservingthemorphologyshown);an=antheridium;MZ=meristematiczone
3暋Discussion
Ourobservationshowedthatthematurecor灢
dateprothalionthehumussoildidn曚tdevelop
archegonia,and,thus,thenormalprocessof
sexualreproductionofS灡grevileoidescouldn曚t
finished.Obviously,thehumussoilwasnot
suitableforthesporepropagationofthisfern.
ThatthepHofthehumussoilwastoolowwas
921期暋 暋WANG Wei灢Qingetal灡:SubstratumAffectstheGametophyteDevelopmentandReproduction...暋暋暋
probablythereason.Onthemixturesoilcom灢
posedofhumussoilandoriginalsoil(1暶1)
(Culture2),thesporesgerminatedanddevel灢
opedearlierthanthatonthehumusandoriginal
soil,andtheratesofsporegerminationandpro灢
thalusdevelopingintosporophytewereboththe
highestamongthefourcultures.Themixture
soilprovidedthefernnotonlywithsuitablepH
6灡0-6灡5,butalsowithabundantorganicmat灢
terandavailablemineralelementssuchasN,P,
andK.Thusthemixturesoilismostfavorable
forthesporepropagationofthisspeciesamong
thethreeexaminedsoilsubstrata.
GametophytesofS灡grevileoidesalformed
nearlysymmetrical,cordateprothaliatayoung
stagethoughtheshapesofprothalioneachme灢
diumwerenotalthesame.Itwasreportedthat
thegrowingdensityinfluencedtheshapeofpro灢
thalus(Caoetal灡,2003).Itwasalsoshowed
thatthecontentsofN灡P灡K,andCainthesoil
affecttheearlydevelopmentofthefernrepro灢
ductionbyspores(Suzukietal灡,2005).Inour
study,thoughthesporesalweresownevenlyat
anaveragedensityof200-300sporespercm2
foreachculture,therateofsporegermination
onfourculturesubstratawasdifferent,which
madethedensityofcordateprothalioneach
culturesubstratum differ.Itwasunclearthat
whichfactor,differentdensityofcordateproth灢
aliordifferentphysicalandchemicalnatureof
eachsubstratum,resultedindifferentshapesof
cordateprothali.
Themostcommonsequenceofsexualdevel灢
opmentinhomosporousfernsinvolvesthefor灢
mationofantheridiafolowedbyarchegonia(At灢
kinsonandStokey,1964).However,Pradaet
al灡 (2008)reportedthatinthecaseofPterisin灢
completaprogressionofsexexpressionvaried
dependingontheculturemedium,namely,fe灢
malegametophytesdevelopedearlierthanmale
onesonsoilcultures.Inthisresearch,these灢
quenceofsexualexpressionappearedtobeinde灢
pendentofsubstratetype.The malegameto灢
phytesdevelopedinitialyandthefemaleones
later,whichbroughtintocorrespondencewith
mosttaxa(HauflerandRanker,1985).
Ontheoriginalsoil,therateofprothalus
developingintosporophyteimprovedgreatlyaf灢
tertransplanting;accordingly,itwasmorethan
likelythepreviouslowformationrateofsporo灢
phytewascausedbythelowdensityofcordate
prothalionthesubstratum,andthelowdensity
ofprothaliwasresultedfromthelowrateof
sporegerminationontheoriginalsoil.
Stokey(1951)reportedthatunfavorablecon灢
ditionspromotedfilamentousgrowth.Inthis
study,ontheKnop曚sagarmedium,thespores
ofS灡grevileoidesgerminatinglatertendedto
growlongfilamentsoronesporedevelopedinto
two filaments and some filaments always
branched.Thepossiblereasonwasthespores
germinatinglaterlackedadequatenutrientsand
space.AccordingtoTable2,theantheridiaand
archegoniaontheKnop曚sagarmedium (Culture
4)appearedearlierthanthoseonthemixture
soil(Culture2),however,infantsporophytesin
Culture4appearedlaterthanthoseinCulture2.
Thisindicatedthatthehigherdensityofan灢
theridiaandarchegoniacouldpromotetheemer灢
genceofinfantsporophytes.Unliketheinvesti灢
gationofChenetal灡 (2008)—theprothalion
theimprovedKnop曚sagarmediumrarelypro灢
ducedinfantsporophytes,about60%ofprothal灢
liontheagarmediuminthisstudyproducedin灢
fantsporophytes.This maybecausedbythe
differencesbetweenspecies.Theinfantsporo灢
phytesstoppedgrowingunlessthenutritionwas
sufficientinCulture4.
Prothaliofmostferntaxaarecapableofregen灢
eratingnewprothalifromoldones(Atkinsonand
Stokey,1964;Nayarand Kaur,1971).Inthis
study,thelargeirregulargreenlaminaonthehu灢
mussoil(Fig灡6)andtheprothaluswithseveral
meristematiczonesonthe Knop曚sagar medium
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(Fig灡7)suggestedthatS灡grevileoidesmayregen灢
eratenewprothalifromoldones,butadditionalob灢
servationwasneededtoproveit.
Acknowledgements:WethankProf.WeimingZhufrom
YunnanUniversityandMr.YuChenfrom KunmingIn灢
stituteofBotanyfortheirhelpinthecolectionofspores
inthewild,andthankMs.MeijingSongandProf.Dagan
Jifrom KunmingInstituteofBotanyfortheiraidtothe
experiments.
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131期暋 暋WANG Wei灢Qingetal灡:SubstratumAffectstheGametophyteDevelopmentandReproduction...暋暋暋