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frezingtoleranceundergovariouschangesintheirplasmamem- branes[1，15，26，27，30，34]aswelasinsidethecel[2，13]andcan facilitate intracelular supercoling to prevent intracelular frezing.Thus，theauthorsaimedtoinvestigateacryopreservation techniquethatcanpreventorreduceintracelularfrezingevenat lowertemperaturesaslivingmaterialsarefrozen.
Yoshidaeta.lreportedthatprotoplastsisolatedfromnon- aclimatedplanthadreducedcytoplasmicpHandcelsurvival withtemperaturelowering，althoughprotoplastsisolatedfrom cold-aclimatedplantscouldreducethedeclineofcytoplasmic pHandcelsurvival[31].IntracelularpH(pHi)，whichisalso caledcytoplasmicpH，isanindicatorofcelactivity.Theirarticle ledustotheideathatareductionofthedeclineofcelactivity duetotemperatureloweringmayfacilitateintracelularsuper- coling.Thus，theauthorshypothesizedthatsomeoutsidestim- uluscouldreducethedeclineofcelactivityduetotemperature lowering，and adopted electrical stimulation as an external stimulation. When the experiments had been performed to observeocurenceofintracelularfrezinginplanttisuesusing highvoltageandchangingelectriccurentvalues，amicroelectric curent load lowered the temperature at which intracelular frezingocured.
Inapreviousstudyof口yopreservationtechniqueusingelectric stimulation，Xanthakiseta.linvestigatedtheicenuc1eationtem- peraturesandicecrystalsizesinporkmeatsthatwerefrozenusing astatichigh-voltageelectricfieldtoreducethedeclineoffod qualityduetofrezing[29].Moreover，inapreviousstudyusing electriccurent，Kojimaeta.lreportedthatanextremelyweak electriccurentsysteminducesanti-apoptoticefectsandanti- necroticefectsinlivingcels[12.]Althoughusingamicroelectric cur rent load has high potential in the development of a new cryopreservationtechnique，therehasbeennoreporttoinvestigate thistechnique.
ThisstudywasperformedtoevaluatetheIFandpHiofplant celsatlowtemperatureswithorwithoutamicroelectricalcurent load.Theplanttisuesofasinglecelayerwereusedasspecimens becausetheywereeasytoobtaininalivingstate.TheIFbehavior withelectriccurentloadingwasevaluatedbasedonthedegreof celdeformationandthegrainsizeandgrowin
Jerusalemartichoke(Helianthus印 berosusL.)tubertisueswere usedtoevaluatepHiduringcolingwithorwithoutanelectric curentload.TheJerusalemartichoketuberswereharvestedatthe KanagawaInstituteofTechnologyfromFebruarytoMarch2014. TheonionswerepurchasedfromavegetablestoreinAtsugi，Japan fromApriltoJune2014.
2.2. Oyomicroscopicsystem
Thecryomicroscopysystemconsistedofaninvertedmicroscope (IX7，1 Olympus)，whichwasequipedwithacolingstage (H仁D30，1 IN5TEC)， ahigh-spedcamera(PhantomV4.2，Vision Research)，acoledCCDcamera(DXM1200C，Nikon)，andanordi- nary仁Dcamera(5HC-721A，5amsung)(Fig.1).
Thehigh-spedcameracouldcaptureimagesatamaximum framerateof2000framespersecond(fps)andaresolutionof 512x512pixelsusingPhantomCameraControlsoftwarepackage (Version9.0.640.0-CPhCon:640)andwasusedtodocumenttheIF behaviorduringintracelularfrezing.Then，theplantspecimens were iradiated with light from a 100-W halogen lamp (12V100WHAL-L，Olympus) pased through a condenser (IX2- LWUCD， NA: 0.55， Olympus). Intracel lular fre ezing in the plant specimenswasthencapturedbythehigh-spedcamerathroughan objectivelens(5LCPlanFl40x，Olympus).
ThecoledCCDcamerawasusedtocaptureimagesofspeci- mensdyedwithafluorescentreagenttomeasurepH.iTheordinary CCD camera was used to observe the range in specimen morphologyfromthestartofcolingthroughthecompletionof thawing.
Colingwasperformedbypouringcombinationairusingliquid nitrogenandaheater(5TC20，Instec)ontothecolingstage，with thecolingratemaintainedat-1oC/minbyacomputer.Nitrogen gaswasflowedontothecolingstagetopreventtheobservation windowfromc1ouding.
2.3. Electriccurentloadonaspedmen
Themethodtoloadanelectriccurentonaspecimenbetween coverglasesonthecolingstageisshowninFig.2A.Twoelectric wireswererespectivelyatachedtothealuminumseals，which wereadheredtobothedgesofacoverglas，andthespecimenwas placedontothecoverglaswithdistiledwatertopreventdrying. Then，anothercoverglastowhichsilicongreasehadbeenaplied attheedgewassetuponthecoverglaswiththespecimenand distiledwaterwasadded(Fig.28).Electriccurentloadingwas performedusinganelectricstimulator(5EN-3401，NihonKohden) andloadedat1~A or10ドAofanisolator(5-203J，NihonKohden). The electric curent loading patern was for 0.1 msec every 0.5msec，withthepolarityreversedevery60s(Fig.2C).
2.4.Experimentα， 1method
Theheatgenerationinsidecoverglasesduetoloadingelectric curentwasvalidatedusingathermocouple，afiberopticther- mometer，andaninfrared(IR)camera.
Next，theIFbehaviorintheepidermaltisuesofstrawbery geranium leaves with or without an electric cur rent load was evaluatedacordingtothedegreofsupercoling，thedegreof celdeformation，thegrainsizeandgrowingrateofintracelularice crystalthatwereobtainedfromhigh-spedcameraimages.10m m2 specimenswereplacedontoacoverglaswithdistiledwaterto preventdrying，andthenanothercoverglaswithsilicongreaseat theedgewasplacedontop.Thecoverglasesweresetonthe colingstageandwerecoledfromroomtemperature(aproxi- mately22oC)untiltheocurenceofintracelularfrezingwithor
2. Materialsandmethods 2.1. Plantmα terials
Epidermal tisues on the abaxial surface ofthe leaves of strawberygeranium(Sax折qα stoloniferaCurtis)plantswereused asspecimenstoevaluateIFbehaviorwithorwithoutamicro electriccurentload.Theseleaveswereobtainedatthecampusof KanagawaInstituteofTechnologyfromlateDecember2014tomid-
January2015.
Epidermal tis sue of onion (Al lium cepα L.) bulb scales and
工Ninagawaeta.l/Cryobiology73ρ01旬 30-39 31