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The25thJnternauonalSymposiumonTransportPhenomenα 5-7November2014，Krabi，Thailand
THEDIFFERENCEINTHECHANGEOFCYTOSOLlCPHOFPLANTCELL DUETOCOOLlNGRATE
TakakoNinagawa1，AkiraNarumi2，YukioKawamura3 andTadashiKonishi4 1GraduateStudent，KanagawaInstituteofTechnology，1030Shimo-Ogino，Atsugi，243・0292Japan 2KanagawaInstituteofTechnology，1030Shimo-Ogino，Atsugi，243・0292Japan 3IwateUniversity，子 18-8Ueda，Morioka，020-850Japan 4OitaNationalColegeofTechnology，16Maki，Oita，870・0152Japan
ABSTRACT CIγopreservationplaystheesentialroleinthevarious
fields，suchasmedicalandfodenginering.Consideringthe potential 白 旬 re fo od shOliage， cryopreservation technique mustdevelopfurtherfromnowon.Forachievingthisgoal，it isimpOlianttounderstandfrezinginjurymechanisms.Ithas benwidelybelievedthatextracelularfi'ezingcausesthe damage to the plasma membrane which leads to lethal damages，suchasintracelular企ezing(l)ー(3)
Under the above background，we investigated the behaviors ofintracelular frezing using the high sped camera in order to discus s the pos sibility of decrease in damagesleadingtotheintracelularfrezingandreported thediferencesintheformingbehaviorsoficecrystal，the deformationofcelandthetempera同 reattheocurenceof intracelularfrezing，duetocolingrate.Bytheway，itis welknownthatintracelularpHistheoneofindicatorsof cel l activity and membrane integrity. In the case of low- temperaturesensitiveplants，colingcausestheintracelular acidification，whichmeansthatmembraneintegritydecreases duringcolingandconsequentlyrf10wsintocytoplasm.
On the basis ofour previous research，this research focusedonthechangeofintracelularpHandinvestigatedthe diference in the change ofintracelular 合ezing due to coling rate. The experiment was perfOlmed using cryomicroscopyequipedwiththehighspedcameraandthe coledCCDcamera.HelianthustuberosusandAlliumcepa L.wereusedforplanttobetested.Fluorescenceintensity ratiometlγwasusedforthemeasurementofintracelularpH. Consequently，it was clear that the coling rate distinguished the decrease ofintracelular pH with the progresofcoling.
INTRODUCTION CIγopreservationplaystheesentialroleinthevarious
fields，suchasmedicalandfodenginering.Atpresent，itis fearedthatthereisthehighposibilityoffodcrisisinnear 白加reduetoanexplosiveincreaseinworldpopulation.To avoid this crisis， CIγopreservation technique has at tracted specialatention，andmustbedevelopedfuliherfromnowon.
Bytheway，becausegeneralbiomaterialiscomprisedof mostwater，intracelular企ezingspontaneouslyocursafter extracelularwaterfi'ezesifthecolingisrapidlyprogresed. IthasbenreportedthatextracelulariceClγstalscausethe disruptiontotheplasmamembraneandleadstolethalcel damages，including intracel lular fre ezing.(l)ー(3) Under the
above background，we have studied the way in which injuries of the plasma membrane are decreased in extracelular frezing，which provides hints of new CIγopreservationtechnique.First，usingtheepidermaltisue of Strawber ry geranium (8似 折 aga stolon件 ra) leaf， w e investigatedtheintracelular‘Ifezingbehaviorsusingthe high sped camera，and reported the diferences in the characteristicsoftheformationofintracelularicecrystal， the cel's defOlmation and temperatures when the intracelular frezing ocured(4). The results are summarizedasfolows:withmoreincreaseincolingrate， (1)thefOlmationrateandthegrainsizeofintracelularice Clγstalarehigherandfinerrespectively(Figure1)，(2)the deformationdegreofacelissmaler，(3)thetemperature whenintracelularfrezingocursislower(Figure2).
Ontheotherhand，itiswelknownthatintracelularpHis
oneofimportantindicatorsofcel'sactivityandmembrane
integrity. In the case of low-temperature sensitive plants，
intracelular pH drops with the progres ofcoling and
changes from slightly alkaline to acidic pH. Fmihelmore，
MuraiandYoshidahavereportedthattherewastheclose
relationbetweencytoplasmicpH(hereafterpHi)andsurvival rate(5)
Onthebasisofthementionedabove，thisresearchfocused onthediferenceinthechangeofpHiduetocolingrateand
Figure1.Diferenceinicecrystalformingbehaviorat intracelularfi'ezingduetocolingrate.