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。 寸一一一一。
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0μA
国 lμA
_10μA
thepHiat-100OCjminremainingataproximately7.7evenat -4o.cInadition，thepHitransitionat-10OCjminwasbetween -1OCjminand-100oCfmin.Itisindicatedthatthedec1ineofthe pHiwithtemperatureloweringreducedwithanincreaseinthe colingrate.
Next，thepHitransitioninthecolingofspecimenswithelec- triccurentloadingwasmeasured(Fig.10).Fig.lOAshowsthepHi transitionsinthecolingof]erusalemartichoketubertisueswith orwithoutelectriccurentloadingat-1OCjminandthevaluesof thepHiaretheaveragepHivaluesfrom12celsobtainedfrom4or moreexperimentsforeachelectriccurentloadingvalue.Fig.10B showsthepHitransitionsinthecolingofonionbulbscaleswith orwithoutelectriccurentloadingat-1OCjminandthevaluesof thepHiaretheaveragepHivaluesfrom9celsobtainedfrom3or more experiments for each electric cur rent loading value. The values ofthe pHi before coling and electric curent loading indicated ap proximately 8.1 in Fig.lOA or ap proximately 7.5 in Fig.10B，andwerethesamerespectively.However，thedeclineof the pHi with lowering temperature was reduced with electric curentloadinginbothcases，ascomparedwithnoelectriccurent loading. In the coling of]erusalem artichoke tuber tisues (Fig.lOA)，althoughthepHiwithelectriccurentloadingslightly decreasedwithloweringtemperature，itremained7.5at1ドAor 7.7at10同， evenat-12oc.Eveninthecolingofonionbulbscale epidermaltisues(Fig.10B)，althoughthepHiwithelectriccurent loadingdecreasedwithloweringtemperature，itremained6.6at 10lIAor7.2at1lIA，evenat-80仁 ThepHiwithoutelectriccurent loadingreducedwithloweringtemperatureandacidifiedfinalyin bothcases.Itisindicatedthatelectriccurentloadingcouldreduce the decline of the pHi in the co oling of plant specimens， as comparedtosampleswithoutelectriccurentloading.Inadition， itappearedthat1μAelectriccurentloadingwasmoreefectivein theonionbulbscaleepidermaltisuesthan10μA，ascompared withthe]erusalemartichoketubertisues.Itisindicatedthatthe efectiveconditionofelectriccurentloadingtoreducethedecline of pHi with temperature lowering might vary between plant speCles.
4. Discusion
Xanthakiseta.linvestigatedacryopreservationtechniqueusing electricstimulationandreportedthatanincreaseinelectricvoltage causedareductioninicecrystalsizebutanincreaseintheice nuc1eationtemperaturewhentheporkmeatswerefrozenina staticelectric白eld[29J.Ontheotherhand，thisstudyusedamicro electriccurentastheelectricstimulationandshowedthatanin- creaseinthevalueofelectriccurentloaddecreasedthegrainsize of intracel lular ice crystal (Fig.7D). These results indicate that electricstimulationcoulddecreasethesizeoficecrystalsformed whensomeorganismswerefrozen.
Theauthorspreviouslyshowedthatthecharacteristicgrainsize ofintracelularicecrystalsdecreaseswithrespecttoanincreasein thecolingrateandthatadecreaseinthecharacteristicgrainsize causesadecreaseinthedegreofceldeformationjustafterthe completionofintracelularfrezing[21.JItisconsideredthatthe densityoficecrystalsformedinsideacelincreaseswithadecrease inthegrainsize.Consequently，thedegreofceldeformationde- creases.Boththecharacteristicgrainsizeofintracelularicecrystal (Fig.7D)andthedegreofceldeformation(Fig.6)weredecreased withmicroelectriccurentloadinginthisstudy.Therefore，itis consideredthatadecreaseinthedegreofceldeformationat microelectriccurentloadingwascausedbecauseofadecreasein thegrainsizesofintracelularicecrysta.l
Incontrast，themicroelectriccurentloadincreasedthedegre of supercoling (Fig.5)，namely，the temperature at which
Time[ms]
Fig.8.Growingratesofintracelularicecrystalswithorwithoutelectriccurent loadingat-1oCfmin.Thegrowingratewasexaminedusingthearealvelocity.Sta- tisticalanalyseswereperformedineachelapsedtime.Lower-casealphabeticcharac- tersshowthestatisticalysignificantdiferencesbetwentreatmentsbyone-way
ANOVAwithTukey-Kramermethod(Pく
0.05).Bars:SE(n= 10).
comparedwithnoelectriccurentloading.Itwasindicatedthatthe growingrateofintracelularicecrystalwithelectriccurentloading wasslowerthanwithoutelectriccurentloading.
3.3.pHitransitionincolingofplantspecimen
Toexaminetheefectofelectriccurentloadingoncelactivity undercoling，intracelularpH(pHi)wasmeasuredusing]erusa- lemartichoketubertisuesandonionbulbscaleepidermaltisues.
ThepHitransitioninthecolingof]erusalemartichoketuber tis sues without electric cur rent loading was measured first at varyingcolingrate(Fig.9).ThevalueofeachpHishowsthe averagevalueofpHiin12celsobservedduringfourormoreex- perimentspercolingrate.AlthoughthepHiinspecimensbefore colingshowedaproximately8.0irespectiveofthecolingrate， thepHiatacolingrateof-1OCjminbecameaproximately7.0at -4OCandthenfinalyacidified.However，thepHiatthecoling ratesof-10oCfminand-100OCjmindecreasedmoregently，with
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任 100C/min
合調100C/min
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24 20 16 12
4 0 -4 ・8 ・12 -16 Temperature[oC]
Fig.9.pHitransitionsin]erusalemartichoke(HelianthusωberosusL.)tubertisuesat diferentcolingrates.Diamond:ー 1oC/min;square:-10oCfmin;triangle:ー 1000Cf min目 ThepHivaluesarethemean土 SE(n= 12)
主 Ninagaw口 et日1./Cryobiology73ρ016)30-39 37
10