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Cryobiology40, 311–322 (2000)doi:10.1006/cryo.2000.2251, available online at http://www.idealibrary.com on
Extended Alternating-Temperature Cold Acclimation and Culture DurationImprove Pear Shoot Cryopreservation
Yongjian Chang* and Barbara M. Reed†,1
*Department of Horticulture, Oregon State University, Corvallis, Oregon 97331, U.S.A.; and†USDA ARS, NationalClonal Germplasm Repository, 33447 Peoria Road, Corvallis, Oregon 97333-2521, U.S.A.
Meristems of many pear genotypes can be successfully cryopreserved following 1 week of cold acclimatibut an equal number do not survive the process or have very little regrowth. This study compared commoused cold acclimation protocols to determine whether the cold acclimation technique used affected the chardiness of shoots or the regrowth of cryopreserved meristems.In vitro-grown pear (PyrusL.) shoots were coldacclimated for up to 16 weeks, then either the shoot tips were tested for cold hardiness or the meristems wcryopreserved by controlled freezing. Cold acclimation consisted of alternating temperatures (22°C wlight/21°C darkness with various photo- and thermoperiods) or a constant temperature (4°C with an 8photoperiod or darkness). Compared with nonacclimated controls, both alternating- and constant-temperaacclimation significantly improved postcryopreservation regrowth ofP. cordataDesv. andP. pashiaBuch.-Ham. ex D. Don meristems. Alternating-temperature acclimation combined with either an 8-h photoperioddarkness was significantly better than constant-temperature acclimation. Alternating-temperature shoot amation for 2 to 5 weeks significantly increased postcryopreservation meristem regrowth, and recovery remaihigh for up to 15 weeks acclimation. Postcryopreservation meristem regrowth increased with 1 to 5 weeksconstant-temperature acclimation and then declined with longer acclimation. Shoot cold hardiness varied wthe acclimation procedure. The LT50 of shoots acclimated for 10 weeks with alternating temperatures was225°C; that with constant temperature was214.7°C; and that of the nonacclimated control was210°C. Lessfrequent transfer of cultures also improved acclimation of shoots. Shoots grown without transfer to fresh medifor 6–12 weeks had higher postcryopreservation recovery with shorter periods of acclimation than shoots wa 3-week transfer cycle.© 2000 Academic Press
Key Words: Pyrus;germplasm; meristems; cryopreservation; cold hardiness; photoperiod; osmotic potentilow temperature.
Pear is an important temperate tree-fruit cropes
ionnte,
eldrestlntetheenteldryo
preservation is considered an ideal method forela-tictionryo-lantlledla-are
2,
edl fortedat-
.re-ing
00.58-
ma
throughout the world. Nearly 1500 genotypare available in the NCGR world pear collectin Corvallis, Oregon, U.S.A. (U. S. Departmeof Agriculture, Agricultural Research ServicNational Clonal Germplasm Repository). Ficollections are the most common way to pserve tree-fruit germplasm, but can be codue to the need for land and constant mainance (16). In addition, the susceptibility ofplants to insects, diseases, and environmstress is a major constraint to long-term fipreservation of these genetic resources. C
Received November 22, 1999; accepted April 7, 20This work was supported by USDA/ARS CRIS 53
21000-014.1 To whom correspondence should be addressed. E-
311
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-
long-term base storage of germplasm in a rtively small space, free from biotic/abiostresses, and amenable to rapid multiplicaon demand (41). In the past two decades cpreservation techniques developed for pcells, callus, and organs include controfreezing, vitrification, and alginate encapsution–dehydration (2). These techniquesavailable for severalPyrus genotypes (10, 227).
A standard controlled-freezing protocol usto screen 60 pear genotypes was successfuabout half of the species and cultivars tes(28). This protocol applied 1 week of alterning-temperature cold acclimation.Pyrus cor-data Desv. andP. pashiaBuch. -Ham. ex DDon are two low-chilling pear species thatspond poorly to the standard controlled-freezil:
0011-2240/00 $35.00Copyright © 2000 by Academic PressAll rights of reproduction in any form reserved.
technique. Improvements are needed to developdi-
orld
ryol-tol-ma
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climation with alternating and constant temper-and
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312 CHANG AND REED
cryopreservation protocols suitable for theverse pear germplasm available in the wpear collection.
Growth and nutritional conditions are veimportant for the development of freezing terance in field-grown plants (40). Freezingerance is induced by low, above freezing teperatures (35). Most hardy plants, suchwoody perennials, cannot tolerate23°C duringhe growing season; however, when fully ccclimated in winter, they tolerate temperatus low as2196°C (42). Low temperaturehort photoperiod, drought, or abscisic aABA) treatment may influence cold acclimion and thus the freezing tolerance of pla21, 23, 35, 38). Cold acclimation is associaith many physiological and biochemical alttions, including membrane alterations, chan
n protein composition, increases in sugar cent, changes in plant hormone concentratind alterations in gene expression (5, 8, 142, 35). Cold acclimation and increased fre
ng tolerance ofin vitro-grown shoots and meistems are not well understood.
Dehydration techniques and plant dehydtion tolerance are two of the most importfactors in cryopreservation pretreatmentscryoprotectant regimes. Increasing osmolaof pretreatment solutions causes cell dehytion and reduction of freezable water. Sucrand other disaccharides used to dehydratesues have interstitial osmotic effects and ethe cells to stabilize proteins and membraduring desiccation (11, 35). Metabolic chancaused by desiccation and low temperaturesimilar and may be the result of similar meanisms (21).
Cold acclimation is used as a pretreatmencryopreservation of manyin vitro-grown plantsConditions used for cold acclimation incluconstant low temperature with short photoriod (9, 10, 22), total darkness (4), or alternatemperatures with short photoperiod (26,29). There are no reports directly comparingeffects of these different cold acclimation cditions.
This study compared the effects of cold
-s
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atures, several photoperiods or darkness,the length of cold acclimation on meristemgrowth of pear shoot tips following cryopresvation. In addition, culture duration and plawater content were studied relative to cold hdiness and cryopreservation.
MATERIALS AND METHODS
Plant Material
Micropropagated shoots of eightPyrus ac-cessions,P. koehneiSchneider (PYR 818.001P. communisL. cvs. Beurre d’Amanlis Panachee (PYR 61.001), Bosc-OP-5 (P1165.001), and Monchallard (PYR 397.001)P.calleryanaDecne. (PYR 661.001),P. communis 3 P. pyrifolia (Burm. F) Nakai cv. GooChristian (PYR250.003),P. pashia (PYR871.001), andP cordata (PYR 750.001) fromhe NCGR collection were cultured in MageA7 boxes (Magenta Corp., Chicago,.S.A.) on Cheng medium (7) with 4.9mM
N6–benzyladenine (BA), 3% sucrose, 0.3agar (Bitek agar, Difco, Detroit, MI, U.S.Aand 0.18% Gelrite (Kelco, San Diego, CU.S.A.). The pH was adjusted to 5.2 befautoclaving. Growth room conditions we25°C with a 16-h photoperiod (25mmol z m22 zs21) and relative humidity at 40%. Shootscm) were transferred every 3 weeks, excepthose in the culture duration experiment.
Response of Genotypes to Cold Acclimatio
Eight genotypes (P. koehnei, P. pashia,cordata, Beurre d’Amanlis Panachee, BoOP-5, Monchallard,P. calleryana,and ‘GoodChristian’) were tested to determine the effeof acclimation on regrowth following cryopreservation. Three-week old shoots were amated at alternating temperatures 22°C withlight (10 mmol z m22 z s21)/21°C 16 h darknes(AL8) for 1 week. We extended the acclimatto 6 weeks for some of the genotypes thatformed poorly with only 1 week at AL8. Fnally, we extended the acclimation to 16 wefor P. pashiaand P. cordatato determine theffect of extended cold acclimation on the
growth of meristems following cryopreserva-e
c ksw ngt ksT ol iuw wac
A ts
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;( ith8 re( m-p
P
era o6 .S (1)( )( )( )( )2 )(
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itha ms anp ar0 e( a
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scribed by Reed (27). Meristems were trans-s-c,), a
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ated
313ALTERNATING TEMPERATURES FOR COLD ACCLIMATION
tion. Cultures were not transferred to new mdium during acclimation.
Extended Culture Effects
Shoots of P. pashia and P. cordata wereultured in the growth room for 3 or 12 weeithout transfer and then placed in alternati
emperature acclimation (AL8) for 0–15 weehe osmotic potential and water content
eaves and stems of shoots and the medere measured and the meristem regrowthompared following cryopreservation.
lternating- vs Constant-Temperature Effec
Six-week old shoots ofP. pashia and P.ordatawere cold acclimated for 1 to 16 weeithout transfer. Five treatments of alternatr constant temperatures combined with sr no photoperiod were used: (1) (AL8) 22ith 8 h light (10mmol z m22 z s21)/21°C 16 h
dark; (2) (AD8) 22°C 8 h dark/21°C 16 h dark3) (CL8) constant low temperature (4°C) w
h light; (4) (CD) constant low temperatu4°C) dark; (5) control—constant warm teerature (25°C with 16 h light).
hoto- and Thermoperiod Effects
The effect of long, neutral, and short tempture and photoperiod cycles was tested-week oldP. pashiaand P. cordata shootshoots were acclimated for 5 weeks with
AL16) 22°C 16 h light/21°C 8 h dark; (2AL8) 22°C 8 h light/21°C 16 h dark; (3AD16) 22°C 16 h dark/21°C 8 h dark; (4AD8) 22°C 16 h/21°C 8 h dark; (5) (AL122°C 12 h light/21°C 12 h dark; and (6AD12) 22°C 12 h/21°C 12 h dark.
ryopreservation Procedure
After cold acclimation 25 meristems wttached leaf primordia (0.8–1.0 mm) frohoots of each treatment were dissectedrecultured on a firmer medium (0.35% ag.185 gelrite) with 5% dimethyl sulfoxidMe2SO) for 48 h under the same cold acclim-
tion conditions as for the parent shoots. Tbasic controlled-freezing procedure was
-
-.fms
rt
-n
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-
ferred to 0.25 ml liquid medium in 1.2-ml platic cryo-vials (Cryovial, Beloeil, QuebeCanada) on ice. The cryoprotectant PGD (12mixture of 10% each polyethylene glycol (M8000), glucose, and Me2SO in liquid mediumwas added dropwise up to 1.2 ml over 30 mAfter 30 min equilibration on ice, the meristewere frozen to240°C at 0.1°C/min in a programmable freezer (Cryomed, Forma ScientMt. Clemens, MI, U.S.A.) and immersedliquid nitrogen for at least 1 h. Vials wethawed in 45°C water for 1 min and then23°C water for 2 min. The cryoprotectant wremoved and replaced with liquid mediuMeristems were plated in 24-cell plates withml medium per cell (Costar, Cambridge, MU.S.A.) for recovery. Regrowth data were ta4 weeks after thawing. Each experiment usemeristems per vial for each treatment anmeristems for unfrozen controls, with at lethree replications per experiment.
Cold Hardiness Tests
A piece (11.43 11.5 cm) of moist sterilKimwipes tissue paper (Kimberly-ClaCorp., Roswell, GA, U.S.A.) was folded aplaced into each glass test tube (103 75 mm)with 10 pear shoots (1–1.5 cm). The sampwere cooled in the programmable freeze0.1°C/min and nucleated at22°C. Five tube
er treatment were removed at 2.5°C intervrom 0 to 250°C. Shoots were thawed at 4or 3 h and recultured for 4 weeks on Cheedium in the growth room to determi
iability. Viability was expressed as LT50, thetemperature at which 50% of the shoots wkilled.
Fresh Weight (FW)/Dry Weight (DW)
The water content of shoots ofP. cordatawas determined as the difference between fweight and dry weight. Dry weights were dtermined after heating the samples in an ove95°C for 24 h. Percentage water was calculas [(FW2 DW)/FW] 3 100%.
Osmotic Potential Measurement
thedgcoasforofmlghtses
ra-traorola
ist ths tesi ast
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ndm iplr( c.R
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1B). P. cordata meristem recovery remainedl ).
E
shm o-p ith-o m-b ds hp (1w eks)( er,l x.T medd otss avesd de-v on-t
ss cul-t 2-w thew andt oren ew ratea ofl ins Fig.4 msd s ofAM
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urtta ).T nop olda to5 re-c es.A ofm ing-
314 CHANG AND REED
The water potential of the medium andshoots ofP. cordataat different culture or colacclimation durations was measured usinvapor pressure osmometer (5100C; WesInc., Logan, UT, U.S.A.). The medium wfrozen to220°C overnight and then thawed5 min at room temperature (22°C). Aliquotsthe medium (0.5 ml) were centrifuged in 1.5-microfuge tubes (Fisher Scientific, PittsburPA, U.S.A.) at 3000 rpm for 5 min. Shoo(1–1.5 cm) were frozen in the microfuge tubovernight, thawed for 3 min at room tempeture, and then pressed with a glass bar to exthe sap. Sap was centrifuged at 3000 rpm fmin to obtain a clear supernatant (13). Osmlities were converted to MPaC by multiplyingby 2.48 (According to Van’t Hoff’s equation:2C 5 2RTSCj, where R is the gas constant, The temperature in degrees Kelvin, and Cj isummation of the concentrations of all solu
n the solution). Each treatment had at lehree replications (n 5 30).
tatistical Analysis
The results were analyzed by ANOVA aeans were separated with Duncan’s mult
ange test (P # 0.05) using StatgraphicsplusStatistical Graphics Corp. and STSC Inockville, MD, U.S.A.).
RESULTS
The Response of Genotypes to AL8Acclimation
Pear genotypes screened with the stanAL8 cold acclimation procedure had signcantly improved postcryopreservation merisregrowth, but the degree of response variedgenotype (Fig. 1A). P. koehnei, ‘Beurre
’Amanlis Panachee,’ ‘Monchallard,’ andP.alleryanacultured for 3 weeks in the growoom and for 1 week in AL8 acclimation pruced.75% regrowth, compared to,25% re-rowth for control nonacclimated shoots. BoP-5, P. pashia, and ‘Good Christian
esponded poorly to 1-week acclimation aequired 4–6 weeks for good regrowth (F
ar,
,
ct5-
e
t
e
,
rd
h
-
ow with 1–6 weeks cold acclimation (Fig. 1B
xtended Culture Effects
Extended culture without transfer to freedium improved regrowth following cryreservation for all eight genotypes even wut acclimation (data not shown). When coined with AL8 acclimation, long-culturehoots ofP. pashiaandP. cordatareached higostcryopreservation recovery in less timeeek) than 3-week-cultured shoots (5–8 we
Fig. 2). After 12-week culture without transfittle growth medium was left in the culture bohe shoots stopped growing, and some forormant apical buds. In long-cultured shoome mature leaves abscised and young leied but the shoot tips remained alive andeloped higher freezing tolerance than the crols.
Water content ofP. cordatastems and leaveignificantly decreased during the extendedure experiment (Fig. 3A). Over the 3- to 1eek culture duration in the growth room,ater content of leaves decreased by 10%
he osmotic potential of leaves became megative (21.37 to22.22 MPa) (Fig. 3B). Thater content of stems declined at the sames that of leaves. In contrast to the effects
ong-term culture, AL8 acclimation resultedmaller changes in tissue water content (A). The osmotic potential of leaves and steecreased significantly after 8 or more weekL8-cold acclimation but remained above22Pa (Fig. 4B).
lternating-vs Constant-Temperature Effect
For the first 5 weeks of acclimation, all foreatments significantly (P # 0.05) improvedhe postcryopreservation regrowth ofP. pashiand P. cordatameristems (Figs. 5A and 5Bhe nonacclimated controls had little orostcryopreservation regrowth. One-week ccclimation increased regrowth from 0–40% for all acclimation treatments, butovery was still very low for these genotypfter 3 or 5 weeks acclimation, the regrowtheristems from shoots grown under alternat
hotteera
oncov
fm ight ovew rea
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itha nd5 temr rehpa coldh s to2 er-a ineds
n
315ALTERNATING TEMPERATURES FOR COLD ACCLIMATION
temperature treatments with either an 8-h ptoperiod or darkness was significantly bethan that of meristems from constant-tempture-acclimated shoots. At least 3 (P. pashia) or5 weeks (P. cordata) of AL8 acclimation werenecessary to obtain 80–100% regrowth. Cstant-temperature-acclimated meristem reery generally remained below 40% forP. cor-dataand below 80% forP. pashia.Regrowth o
eristems with AL8 acclimation remained hhroughout the 16-week test, while shoot recry declined after 5 weeks (P. cordata) or 8eeks (P. pashia) with constant-temperatucclimation (Figs. 5A and 5B).
FIG. 1. Regrowth ofPyrusmeristems cryopreserwithout cold acclimation (CA) as a control and witon four pear genotypes. Cold acclimation conditconditions 25°C with 16 h light. Results are expre
-r-
--
-
old Hardiness and CryopreservationRecovery
Cold hardiness of both species improved wll acclimation conditions tested (Figs. 5C aD). Freezing tolerance of shoots and merisegrowth following cryopreservation weighly correlated (R 5 0.9025**) for both P.ashia and P. cordata (Fig. 5). All four coldcclimation treatments increased shootardiness. All treatments produced hardines17°C or below by 3 weeks. Constant-tempture acclimation shoot cold hardiness remateady from 3 to 10 weeks (LT50 217°C).
by controlled freezing. (A) Four pear genotypes growweek CA. (B) Effects of 0–6 weeks of cold acclimation: AL85 22°C with 8 h light/21°C 16 h dark. Control
ed as means6 SD; n 5 60.
vedh 1ionsss
hess
5w
P
(08 nos e-s ng-t vedta n-aP ntd h a1 fob nific .
hel-
t iedap d o
t edf w-i olda -t (2)m ,p oldan ro-t reec iond y 1g reeC re-m ac-c n).
edt ryo-p ultss ma-t nt-t yo-p han1 g-t mayh gu-
hith
316 CHANG AND REED
Shoots grown with AL8 acclimation had tbest LT50 (225°C) at 5 weeks, and hardineremained below220°C for the remaining
eeks.
hoto- and Thermoperiod Effects
Changes in the length of the photoperiod, 12, 16 h) or thermoperiod (8, 12, 16 h) didignificantly affect regrowth of the cryoprerved meristems (Table 1). All six alternatiemperature treatments significantly improhe postcryopreservation regrowth ofP. pashiandP. cordatameristems compared to the nocclimated controls (0% forP. cordata,20% for. pashia); however, there were no significaifferences among them. Shoots grown wit2-h thermoperiod had the highest regrowthoth pear species tested, but it was not sigantly better due to the variability observed
DISCUSSION
Cold acclimation significantly improved tsurvival of cryopreservedPyrusmeristems, ahough the required length of acclimation varmong the genotypes. Reedet al. (28) dividedear genotypes into three categories base
FIG. 2. The interaction of culture duration andP. cordatameristems following cryopreservation broom at 25°C for 3 or 12 weeks without transfer to8 h light/21°C 16 h dark;n 5 20.
,t
r-
n
he amount of AL8 cold acclimation requiror moderate to high meristem regrowth follong cryopreservation: (1) short, 1 week of ccclimation, e.g.,P. koehnei(of 93 pear geno
ypes tested, 43% belong to this group);edium, 3–7 weeks of cold acclimation, e.g.P.ashia;and (3) long, more than 8 weeks of ccclimation, e.g.,P. cordata.All the Pyrusge-otypes tested with the controlled-freezing p
ocol in our present study fit in these thategories (Fig. 1). When the cold acclimaturation was extended for the four Categorenotypes included in Fig. 1A and the thategory 2 genotypes in Fig. 1B, regrowthained high, indicating that extended cold
limation was not detrimental (data not showThe type of cold acclimation protocol appli
o the pear shoots was important for postcreservation meristem recovery. Our reshowed that alternating-temperature accliion was much more effective than constaemperature acclimation for meristem crreservation for acclimation periods longer tweek (Figs. 5A and 5B). The alternatin
emperature conditions used in our studiesave provided strong stimulation of cold-re
cold acclimation on the recovery ofPyrus pashiaandontrolled freezing. Shoots were cultured in the growth medium before cold acclimation treatment at 22°C w
AL8y c
fres
oldenaftatr.
5 era res ralw r 5w s tt on
aseduedratethe
tureantionure,tis-nt inor-ts.in-
by
317ALTERNATING TEMPERATURES FOR COLD ACCLIMATION
lated gene expression similar to natural cacclimation. Constant-temperature treatminitially increased shoot cold hardiness, butter 3 weeks hardiness remained constan216°C, while hardiness of shoots in alterning-temperature treatments increased foweeks before stabilizing below220°C (Figs
C and 5D). During the long constant-tempture acclimation we observed that shoots glowly or stopped growing for the first seveeeks, but exhibited increased growth afteeeks (data not shown). Adaptation of shoot
he constant-temperature cold acclimation c
FIG. 3. Water content and osmotic potential ofwater content of leaves and stems ofP. cordataduricordatastems and leaves after 4–15 weeks in cult0.05), asindicated by capital letters for the leaveDuncan’s multiple range test.
ts-at-5
-w
o-
ditions may have reduced stress and decrecold-regulated gene expression. Contingrowth may also have depleted carbohydreserves that serve as cryoprotectants incells. Shoots held under alternating-temperaconditions grew little and some formed dormbuds (data not shown). During cold acclimatmany changes occur in the cellular structphysiology, and biochemistry of cells andsues (6, 14, 37). These changes were evidethe slow growth, compact structure, and dmant bud formation of cold acclimated plan
Several series of cold-regulated genes
ves and stems ofin vitro-culturedPyrusshoots. (A) The12 weeks in culture. (B) The osmotic potential ofP.. Bars with different letters are significantly different (P #
nd lowercase letters for the stems. Means separation
leangures a
andeneteanamlowffecellthedeo
n-
deddi-his
yo-ver,
forris-ure
re-iona-er-
by
318 CHANG AND REED
duced by cold acclimation are now clonedthere is clear evidence that cold-regulated gplay a role in plant cold hardiness (17). Wastress increases freezing tolerance for mplant species and induces some of the sgenes as does treatment with ABA andtemperature (20, 42). Water stress is an etive pretreatment for cryopreservation of ccultures (24, 25). During extended culturewater content of the pear shoots graduallycreased and the osmotic potential became mnegative (Fig. 3). The very low osmotic pote
FIG. 4. P. cordataleaves and stems during 0–1216 h dark). (A) Water content; (B) osmotic potenti0.05), asindicated by capital letters for the leaveDuncan’s multiple range test.
srye
-
-re
tial of pear leaves and stems following extenculture without transfer to fresh medium incated that the shoots were in water deficit. Tdrought stress slightly improved the postcrpreservation regrowth of meristems; howelow-temperature treatment was essentialdramatic increases in regrowth (Fig. 2). Metems from shoots grown with extended cultnot only had increased postcryopreservationgrowth, but also required less cold acclimatfor high regrowth than control shoots. Acclimtion of hardy species as a result of low-temp
eeks of AL8 cold acclimation (22°C with 8 h light/21°Cars with different letters are significantly different (P #
nd lowercase letters for the stems. Means separation
wal. Bs a
uctnein
lasdyion
mezvethrisano-t aion
otsx-on-Fig.-s ine inin
re1).ny
ex-or
s inularo-cell
hs
319ALTERNATING TEMPERATURES FOR COLD ACCLIMATION
ature treatments is associated with early indtion and de novo synthesis of distincpolypeptides that may stabilize membraagainst dehydration stress with resultingcreases in freezing tolerance (14, 35). The gforming tendency of the cytoplasm of wooplants is also increased with cold acclimat(15, 30).
Reduced water content induced by low teperature is correlated with an increase in freing tolerance in some species (6, 14); howethis was not the case for pears. Althoughwater content and osmotic potential of metems decreased during acclimation (Fig. 4)were correlated with regrowth following crypreservation, shoot water content was noimportant factor affecting postcryopreservat
FIG. 5. The effect of cold acclimation conditionthe recovery ofPyrusmeristems following cryoprescryopreservation. (B) Regrowth ofP. pashiameristecordatashoots. (D) Cold hardiness ofP. pashiashoolight/21°C 16 h dark; CD, constant low temperatulight. Not shown: Control, constant warm temperatSD. Regrowthn 5 60; LT50 n 5 50.
-
s-s-
--r,e-d
n
recovery in our procedure. Meristems of shogrown in growth-room conditions with etended culture duration had lower water ctents than those of cold acclimated shoots (3A) but still had less survival following cryopreservation. Desiccation causes increaseabscisic acid in plants, as well as a decreasprotein, RNA, and starch, but an increasesugar. InArabidopsiscold-regulated genes aboth ABA and desiccation responsive (2During low-temperature cold acclimation machanges occur such as cold-regulated genepression and the production of dehydrinsdehydrin-like proteins (1, 3, 14) and changethe plasma membrane structure (32), cellsugar composition (6), and cell wall compnents (39). These changes all contribute to
n cold hardiness ofPyrus in vitro-grown shoots and onation. (A) Regrowth ofP. cordatameristems followingfollowing cryopreservation. (C) Cold hardiness ofP.
(AD8, 22°C 8 h dark/21°C 16 h dark; AL8, 22°C 8 h4°C) dark; CL8, constant low temperature (4°C) with 8(25°C with 16 h light). Results are expressed as mean6
s oervmsts.re (ure
cet
or-lann-erjorm
term-due toanndn-ed
lart thiceda
theas
atendtiobe
ing
redof
the photoperiod had no significant effect on coldionis
er(34,esins
to-larar-,e-
ghtn-ingiodhe/12odary
ua-ell
earionem-cli-uir-g-orein
er-ul-
higheseer-
pesandlant
owde-
cli-
TABLE 1
er-
320 CHANG AND REED
cold hardiness and tolerance to freeze-indudehydration, and decreased water contenlikely a result of these processes (8).
Dehydration and intracellular ice crystal fmation are the most common causes of pdeath during freezing (6, 31, 38). Injury to noacclimated rye protoplasts resulting from sevcell dehydration is associated with machanges in the ultrastructure of the plasmembrane. Cold acclimation results in alations in the lipid composition of cellular mebranes that increase the membrane stabilitying freeze-induced dehydration (32, 36). Duacclimation, solutes increase in the cellscontribute to stabilization of membranes aproteins during dehydration (33). The cotrolled-freezing process that we used allowwater to move out of cells into the intracelluspaces and crystallize there. We believe thamain cause of meristem death was not fromformation in the cells, but from freeze-inducdehydration. Freeze-induced dehydration mresult in the precipitation of molecules anddenaturation of cellular protein (35), as wellirreversible membrane lesions (33). Shoot wcontent was well correlated with regrowth amay be important, but the freeze–dehydratolerance induced by cold acclimation maymore important during the controlled-freezprocedure.
In our study a photoperiod was not requifor improved cold acclimation and the length
Postcryopreservation Regrowth ofPyrusMeristems fromShoots Cold Acclimated for 5 Weeks with Various Thmoperiods and Photoperiods
Thermoperiod(22/21°C)
Photoperiod(h)
Regrowth (%)
P. cordata P. pashia
8/16 8 53.76 11.0 72.56 17.512/12 12 67.36 14.1 74.66 14.616/8 16 46.86 19.0 63.56 17.88/16 0 55.06 18.2 68.36 7.6
12/12 0 72.36 24.4 83.46 20.216/8 0 50.06 5.0 58.36 15.3
Note.Means6 standard deviation;n 5 60.
dis
t
e
a-
r-
d
ee
y
r
n
acclimation or recovery from cryopreservat(Fig. 5 and Table 1). A short photoperiodrequired for cold acclimation of many wintannuals, biennials, and perennials in nature38). InArabidopsisa short photoperiod inducsome proteins similar to cold-regulated prote(18). In poplar trees light is required for phosynthesis to supply sufficient energy for celluchanges or to signal the induction of cold hdiness (19). As with manyin vitro experimentssucrose was plentiful in the pear growth mdium and may have substituted for any lirequirement. Although all cold acclimation coditions tested were very effective for increasmeristem regrowth, the 12/12 h thermoperwith or without a photoperiod, resulted in tmost postcryopreservation regrowth. The 12h thermoperiod regime may provide a gobalance of a cold period for inducing necesscell products and a warm period for contintion of the metabolic activities required for csurvival under adverse conditions.
CONCLUSIONS
This study showed that the intensity of pcold hardening varies with the cold acclimatconditions used. Constant and alternating tperatures were equally effective for short acmation periods; however, for genotypes reqing longer acclimation, the alternatintemperature treatments were clearly meffective. Extended culture alone resultedslightly increased recovery. Alternating-tempature acclimation combined with extended cture produced deep cold hardiness andpostcryopreservation recovery in pears. Thcombined techniques allow for the cryopresvation of the complete range of genotypresent in important germplasm collectionsthe long-term base storage of irreplaceable pcollections.
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