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Characterisation of ‘Royal Gala’ and‘Cox's Orange Pippin’ apple (Malusdomestica) softening during controlledatmosphere storageJason W. Johnston a b , Errol W. Hewett a c & Maarten L.A.T.M.Hertog a da Institute for Food Nutrition and Human Health , MasseyUniversity , Private Bag 11 222, Palmerston North, New Zealandb The Horticultural and Food Research Institute of New ZealandLimited , Private Bag 92169, Auckland, New Zealandc College of Sciences , Massey University, North Shore Mail Centre ,Private Bag 102 904, Auckland, New Zealand E-mail:d Flanders Centre/Laboratory of Postharvest Technology ,Katholieke Universiteit , Leuven, W. de Croylaan 42, Leuven,B‐3001, BelgiumPublished online: 22 Mar 2010.

To cite this article: Jason W. Johnston , Errol W. Hewett & Maarten L.A.T.M. Hertog (2006)Characterisation of ‘Royal Gala’ and ‘Cox's Orange Pippin’ apple (Malus domestica) softening duringcontrolled atmosphere storage, New Zealand Journal of Crop and Horticultural Science, 34:1, 73-83,DOI: 10.1080/01140671.2006.9514390

To link to this article: http://dx.doi.org/10.1080/01140671.2006.9514390

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New Zealand Journal of Crop and Horticultural Science, 2006, Vol. 34: 73-830014-0671/06/3401-0073 © The Royal Society of New Zealand 2006

73:

Characterisation of 'Royal Gala' and 'Cox's Orange Pippin' apple(Malus domestica) softening during controlled atmosphere storage

JASON W.JOHNSTON1

ERROLW. HEWETT2

MAARTEN L. A. T. M. HERTOG3

Institute for Food Nutrition and Human HealthMassey UniversityPrivate Bag 11 222Palmerston North, New Zealand

Present address: The Horticultural and FoodResearch Institute of New Zealand Limited,Private Bag 92169, Auckland, New Zealand.

2Present address: College of Sciences, MasseyUniversity, North Shore Mail Centre, PrivateBag 102 904, Auckland, New Zealand, email:E.W.Hewett@massey.ac.nz

3Present address: Flanders Centre/Laboratory ofPostharvest Technology, Katholieke UniversiteitLeuven, W. de Croylaan 42, B-3001 Leuven,Belgium.

Abstract Firmness is an important trait ofpostharvest quality in apple fruit (Malus domestica).This study characterises softening rates of earlyseason cultivars before, during, and after controlledatmosphere (CA) storage. 'Royal Gala' and 'Cox'sOrange Pippin' apples in CA had a triphasic softeningcurve during storage similar to that of fruit in regularair storage (RA). Fruit in CA had a longer initial slowsoftening phase, and a slower rapid softening phase,than fruit in RA. Firmness benefits in the marketarising from CA were attained only when CA wasapplied to fruit before the onset of rapid softening.Fruit in CA also exceeded an internal ethyleneconcentration (IEC) of 1.5 µl litre-1 later, and had alower maximum IEC, than fruit in air. Thus, CA mayextend the initial slow softening phase, and reducesoftening in the rapid softening phase by decreasingethylene production in both cultivars.

H05059; Online publication date 13 March 2006Received 7 June 2005; accepted 4 November 2005

Keywords Malus domestica; firmness; softeningrate; ethylene; controlled atmospheres; temperature;empirical modelling; quality

INTRODUCTION

Pioneering storage studies with apples (Malusdomestica Borkh.) found softening was slower incontrolled atmospheres (CA) than in a regular airatmosphere (RA) (Magness & Diehl 1924; Kidd& West 1936). Subsequently, years of intensiveresearch resulted in the successful commercialimplementation of CA storage for apples in manycountries, including New Zealand. Despite thecommercial success of CA, the mechanisms by whichCA reduce softening and maintain other aspects ofquality are not completely understood (Hertog et al.2001). The first step to improve understanding of themechanisms involved in softening is to accuratelycharacterise the softening curve in CA. In most appleCA studies, firmness is measured only 1-5 timesduring storage, which is insufficient to accuratelycharacterise the softening curve. Detailed knowledgeof the softening profile shape in CA could be usedto identify critical times in CA when importantphysiological and biochemical changes occur thatinfluence the storage potential of apples.

Wholesale and retail marketers are using firmnessthresholds increasingly as a basis for accepting orrejecting shipments of fruit (Harker et al. 2002).Failure to comply with these standards can causeshipment rejections and reduced returns to growers.Sensory studies suggest minimum firmness thresholdsfor consumer acceptability of apples varies betweencultivars, with thresholds for some cultivars beingdependent on storage atmosphere (Konopacka &Plocharski 2004). Characterisation of softening ratesbefore, during and after CA would enable a moreaccurate estimate of the market life of apples in relationto minimum firmness thresholds. Detailed knowledgeon the patterns of softening in CA could also be used todevelop models that estimate the firmness consequencesof changes to postharvest handling procedures.

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74 New Zealand Journal of Crop and Horticultural Science, 2006, Vol. 34

'Royal Gala' and 'Cox's Orange Pippin' areimportant early-season apple cultivars in NewZealand that soften rapidly after harvest. Softeningof these cultivars has been characterised in RA, andwas found to have an initial slow softening phase,followed by a phase of more rapid softening, anda final slow softening phase (Johnston et al. 2001).Factors such as storage temperature, fruit size, andharvest maturity influenced the initial slow softeningphase and rate of rapid softening (Johnston et al.2001, 2002). It is possible that softening in CA mayalso be triphasic for these cultivars, and like theseother factors, CA may influence duration and/or rateof softening in these phases.

Despite CA being effective in reducing applesoftening, it is logistically difficult to maintain applesin CA throughout the entire postharvest handlingchain. Freshly harvested apples are often stored inRA until a sufficient volume of fruit is obtained tofill aCA store. Once delivered to wholesale or retailoutlets, fruit are usually removed from CA. Thus theinfluence of time in RA before CA storage and timein CA before RA storage needs to be characterised forapples in relation to the softening curve. Reducingthe time between harvest and establishment of CAconditions in storage improves post-storage firmnessof several apple cultivars (Smock &Blanpied 1963;Lau&Looney 1982; Lau 1983; Ficaetal. 1985; Liu1986; Dilley et al. 1989), including 'Cox's OrangePippin' (Sharpies & Munoz 1974). However, inthese studies firmness was only measured 1-5 timesduring storage, making it difficult to determine theeffect of time in RA on the shape of the subsequentsoftening curve in CA. Limited information is alsoavailable on the effect of time in CA on subsequentapple softening in RA. Softening rates of 'Mclntosh'apples in RA at 0°C were similar after differenttimes at 3.0%O2:5.0%CO2, but were slower afterlonger time at 1.0%O2:1.5%CO2 (Lidster 1982).Further research is required to determine if time inCA influences the subsequent softening profile ofapples in RA at low and shelf-life temperatures.

It has been suggested that CA improves post-storage firmness in apples predominantly by reducingbiosynthesis and action of ethylene (Fica et al. 1985;Dilley et al. 1989), and/or by reducing respirationrate (Beaudry 1999; Hertog et al. 2001). Apples inatmospheres with reduced O2 concentrations arefirmer, induce rapid ethylene production later, andhave lower ethylene production after prolongedstorage than apples stored in air (Knee 1980; Stow1989; Fanetal. 1997). Likewise, fruit in atmosphereswith higher CO2 concentrations are firmer and have

lower internal ethylene concentrations (IEC) thanfruit in lower CO2 concentrations (Ben-Arie et al.1993). Studies with inhibitors of ethylene actionhave shown that ethylene has an important role inpromoting apple softening (Fanetal. 1999; WatMnsetal. 2000). Thus, the effects of CA on loss of firmnessin each phase of the apple softening may reflectchanges in endogenous ethylene concentrations.

Despite extensive and numerous studies on theeffects of CA on post-storage firmness in many applecultivars, the effects of CA on softening in eachof the three phases of apple softening is currentlynot known. This study characterises the effect ofconstant CA, time in RA before CA storage, and timein CA before RA storage on softening of the 'RoyalGala' and 'Cox's Orange Pippin' apple cultivars. Italso determines if softening responses to CA and/orRA treatments are related to changes in IEC for thesecultivars.

MATERIALS AND METHODS

Fruit supply, treatments, and measurementsExport quality apple fruit were harvested fromorchards in Hawke's Bay ('Royal Gala') andNelson ('Cox's Orange Pippin') in February 2000 atcommercial maturity, graded, packed and transportedto Palmerston North, New Zealand within 72 h ofharvest. Fruit size was 160-180g for 'Royal Gala'and 130-150g for 'Cox's Orange Pippin'.

'Royal Gala' fruit were randomly allocated to eachof the following treatments (120 fruit per treatment):constant RA at 0.5±0.5°C and 20.0±0.5°C; constantCA at 0.5±0.5°C; RA at 0.5±0.5°C for 7, 20, 50,and 80 days before transfer to CA at 0.5±0.5°C; andCA at 0.5±0.5°C for 7, 20, 50, and 80 days beforetransfer to RA at 0.5±0.5°C (80 fruit) and 20.0±0.5°C(40 fruit). The same treatments were used for 'Cox'sOrange Pippin', except that 3.0±0.5°C was usedinstead of 0.5±0.5°C to avoid the low-temperaturebreakdown disorder.

CA treatments were performed using a flow-through system in 50-litre plastic barrels. Atmospheresof 2.0+0.2% O2 and 1.8+0.3% CO2 were generatedusing cylinders of N2 (O2-free grade; B.O.C. GasesNew Zealand Ltd) and CO2 (food grade; B.O.C.Gases New Zealand Ltd), and compressed air. Thebarrels were purged with N2 for 2 h after sealing torapidly reduce O2 concentrations, after which thehumidified (c. 95% relative humidity (RH)) gasmixture was applied at a flow-rate of 10 ml min"1

per barrel. Initially, "Soda-lime" was required to

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Johnston et al. —Controlled atmospheres and apple softening 75

reduce CO2 concentrations, and "Purafil" to reduceethylene concentrations, in barrels containing fruitrecently transferred from air treatments (fruit withhigh respiration rates and IEC). Where fruit washeld in CA for different times before transfer toRA, each treatment was in a different barrel (fivebarrels), otherwise the CA fruit was in one barrel.Concentrations of O2 and CO2 were equilibrated ineach barrel 18hafter sealing, and gas concentrations,including ethylene, were monitored regularly every1-2 days.

Apples for RA treatments were placed inperforated polyethylene bags (35//m thickness;50 x 5 mm diam. perforations per in2) and packedinto commercial cardboard cartons for storage. Anadditional RA control was stored in a barrel withsimilar conditions (temperature, RH, and flow-rate)to those in CA barrels, except that only humidifiedair was passed through the barrel. Softening and IECin fruit from the barrel RA control was similar to fruitfrom the static RA control for both cultivars (datanot shown), allowing direct comparison betweenfruit in flow-through CA and fruit in static RA.

Concentrations of CO2 and O2 were determinedby injecting 1 ml headspace gas samples from eachbarrel into a gas chromatograph (Shimadzu GC-8A) fitted with a thermal conductivity detector (setat 60°C; current of 90 milliamps), a CTR1 columncontaining activated molecular sieve and porouspolymer mixture (set at 30°C with H2 as the carriergas at 30 ml min"1), and a Hewlett Packard integrator(model 3390A) calibrated with external O2 andCO2 standards (certified as p-standard by B.O.C.Gases New Zealand Ltd). Ethylene concentrationwas determined by injecting 1 ml headspace gassamples from each barrel into a gas chromatograph(Pye Unicam GCD) fitted with a flame ionisationdetector (set at 140°C with H2 and air flow ratesof 30 ml min"1 and 300 ml min"1, respectively), anactivated alumina column (set at 100°C with N2

as the carrier gas at 30 ml min"1), and a HewlettPackard integrator (model 3390A) calibrated withexternal ethylene standards (certified as p-standardby B.O.C. Gases New Zealand Ltd).

For each treatment, flesh firmness and IEC weremeasured on 20 fruit at harvest, and subsequentlyon 10 fruit immediately before and after transfer todifferent atmospheres, and at 20-30-day intervalsin RA fruit at 0.5-3°C, 2-5-day intervals in RAfruit at 20°C, and 30-50-day intervals in CA fruit.Flesh firmness and IEC were measured as previouslydescribed (Johnston et al. 2001). Fruit with rots anddisorders were omitted from firmness and IEC data

sets. Market life of apples (minimum acceptablefirmness accepted by the market) was reached whenfruit firmness declined to 63.7 N (6.5kg/), a valuedictated by commercial supermarket clients.

Data analysisFirmness (f, N) after different times (t, day) inconstant RA and CA at 0.5-3°C, and constant RAat 20°C, were fitted with the following empiricalsigmoidal function (Johnston et al. 2001) usingnon-linear regression:

/=

(1)where model parameters were an initial firmnessasymptote (/_«,, N), a minimum firmness asymptote(f+0o, N), and rate of firmness change (k, day"1).Softening rates of fruit in CA that had different timesin RA were estimated using linear regression. Non-linear regression was performed using the NLINprocedure, and linear regression performed usingthe REG procedure, in the SAS statistical software(version 8.0, SAS Institute Inc, Cary, NC 27513,United States).

RESULTS

Fruit from both cultivars had a triphasic softeningcurve in RA and CA, consisting of an initialslow softening phase immediately after harvest,followed by a phase of more rapid softening, andthen a slower final softening phase (Fig. 1). Themain effect of CA storage was to delay onsetof rapid softening and slow the overall rate ofsoftening. The rate of firmness change (k) in CAwas 10 and 2 times slower than in RA for 'Cox'sOrange Pippin' and 'Royal Gala', respectively(Table 1). When comparing the two cultivars, kvalues were consistently lower for 'Royal Gala'than for 'Cox's Orange Pippin' regardless of thestorage atmosphere.

The duration of the experiment was too shortto accurately characterise the effect of CA on thefinal slow softening phase in either cultivar (Fig.1). However, the softening curve for 'Cox's OrangePippin' fruit in CA appeared to be converging withthe final slow softening phase for fruit in RA after c.200 days at 3°C, with final firmness in CA being c. 7N higher than in RA. In contrast, 'Royal Gala' fruitin CA had only softened by half of that in RA at thecompletion of the experiment (275 days), making it

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76 New Zealand Journal of Crop and Horticultural Science, 2006, Vol. 34

'Cox's Orange Pippin' 'Royal Gala'

cE

80 -1

70

60

50

40

100

10

oLLJ -I

0.1

0-1 ' T"

100 200 0 100 200 300

Fig. 1 Flesh firmness and inter-nal ethylene concentration (IEC,using a non-linear scale) of' RoyalGala' and 'Cox's Orange Pippin'apples (Malus domestica) at 0.5°Cor 3°C respectively in: • , regularair atmosphere (RA); • , control-led atmosphere (CA); and in RAfor • 7, O 20, • 50, and A 80days before transfer to CA. CAconditions were 2.0% O2:1.8%CO2. Treatment means (n = 10)and standard errors of the differ-ence (P < 0.001, d. f. = 463 for bothcultivars) are shown. IEC standarderror of the difference values are9.8 JA\ litre"1 for 'Cox's OrangePippin' and7.5^1 litre"1 for'RoyalGala' (P < 0.001, d.f. = 463 forboth cultivars). Firmness data fromcontinuous RA and CA treatmentswere fitted with Equation 1 usingnon-linear regression.

Days at 3°C Days at 0.5°C

Table 1 Rate of firmness change (k) for 'Royal Gala' and 'Cox's OrangePippin' apples (Malus domestica) in regular air atmosphere (RA) and a controlledatmosphere (CA) of 2.0% O2:1.8% CO2. Estimates of k and associated standarderrors were obtained from non-linear regression analysis of firmness data (Fig.1) with Equation 1.

Cultivar

Cox's Orange PippinCox's Orange PippinRoyal GalaRoyal Gala

Temperature (°C)

33

0.50.5

Atmosphere

RACARACA

*(da;Estimate

0.5600.0550.0660.033

SE

0.1470.0020.0070.003

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Johnston et al. —Controlled atmospheres and apple softening 77

0.25- O— 'Cox's Orange Pippin1

•— 'Royal Gala'

40 60 80

Days in RA before transfer to CA

Fig. 2 Softening rates in controlled atmospheres (CA)and market life (time to soften to 63.7 N) for' Royal Gala'apples (Mains domestica) at 0.5°C and 'Cox's OrangePippin' apples at 3°C after different times in regular airatmosphere (RA) before transfer to CA. CA conditionswere 2.0% O2:1.8% CO2. Softening rates and associatedstandard errors were estimated from linear regression offirmness data in CA, and market life estimated from firm-ness data in Fig. 1.

difficult to extrapolate confidently the effect of CAon the final slow softening phase in this cultivar.

CA affected the rate at which IEC increased atthe beginning of storage, and maximum IEC attainedthereafter for both cultivars (Fig. 1). IEC of 'Cox'sOrange Pippin' fruit increased rapidly from 0.25 jAlitre"1 to 50 ]A litre"1 during the first 10 days of RAstorage, whereas in CA, IEC remained <5 jA litre"1

for 20 days before increasing to 40 jA litre"1 after60 days. CA reduced the maximum IEC attainedfor 'Cox's Orange Pippin' fruit from 90 jA litre"1

in RA to 50 ]A litre"1 in CA. Similarly for 'RoyalGala' fruit, IEC exceeded 1 ]A litre"1 later in CA (60days) than in RA (20 days), and the maximum IECattained in fruit was lower in CA (20 jA litre"1) thanin RA (90 ]A litre"1).

Effectiveness of CA in reducing softening at0.5-3°C was lessened when fruit were previouslystored in RA (Fig. 1). 'Cox's Orange Pippin' and' Royal Gala' fruit transferred to CA within 7 days ofRA storage were placed into CA while in the initialslow softening phase. 'Cox's Orange Pippin' fruittransferred to CA after 20 days in RA, and 'RoyalGala' fruit transferred to CA after 20-80 days inRA, were placed into CA after rapid softening hadbegun.' Cox's Orange Pippin' fruit were in the finalslow softening phase when transferred to CA after50 and 80 days in RA. Softening rates of 'Cox'sOrange Pippin' fruit increased slightly when delayedapplication of CA increased from 0 to 20 days, butthen decreased thereafter for fruit placed into CAwhen in the final slow softening phase (Fig. 2).' Royal Gala' fruit in CA also softened slightly fasterwith delayed application of CA from 0 to 50 days,but softening then decreased from 50 to 80 days.

The commercial implications of delayedapplication of CA storage were determined byestimating the market life (time to soften to 63.7N; 6.5kg/) for each delayed CA treatment (Fig. 2).For' Cox's Orange Pippin' fruit, reducing time in RAfrom 20 to 0 days before transfer to CA increasedmarket life by 45 days. Similarly, reducing the time' Royal Gala' fruit were in RA before transfer to CAfrom 38 days to 0 days increased market life by 37days. Application of CA to 'Cox's Orange Pippin'fruit after 20 days in RA, and to 'Royal Gala' after40 days in RA, had no beneficial increase in marketlife over continuous RA storage.

Delayed application of CA influenced IEC atthe start of, and throughout CA storage (Fig. 1).For 'Royal Gala', IEC was low (<1 jA litre"1) atthe time of transfer to CA for fruit that had 0 and 7days in RA, whereas fruit from longer delayed CAtreatments were transferred to CA during the rapidincrease in IEC that occurred in RA. The changein IEC through CA storage was similar for 'RoyalGala' fruit that had 0 and 7 days in RA. IECs of'Royal Gala' fruit from the longer RA treatmentsdecreased by 5-20 ]A litre"1 once transferred toCA, but then attained greater maximum IECs inCA than fruit that previously had less time in RA.For' Cox's Orange Pippin', transfer from RA to CAimmediately reduced the IEC for the 20-80-daydelayed CA treatments. In contrast the 7-day delayedCA treatment had an increased IEC, although therate of increase was slower than for fruit maintainedcontinuously in RA.

In the reciprocal experiment, time in CA slightlyinfluenced softening of both cultivars once transferred

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78 New Zealand Journal of Crop and Horticultural Science, 2006, Vol. 34

'Cox's Orange Pippin' 'Royal Gala'

ILI

80

70

60

50

40

100

£ 10

oLLI

0.1

0 100 200 0Days at 3°C

100 200 300Days at 0.5°C

Fig. 3 Flesh firmness and inter-nal ethylene concentration (IEC,using a non-linear scale) of' RoyalGala' and 'Cox's Orange Pippin'apples (Mains domestica) at 0.5°Cor 3°C respectively in: • , regularatmosphere (RA); • , controlledatmosphere (CA); and in CA for• 7, O 20, • 50, and A 80 daysbefore transfer to RA at 0.5°C or3°C. CA conditions were 2.0%O2:1.8% CO2. Treatment means(n = 10) and standard errors of thedifference (P < 0.001, d.f. = 440for both cultivars) are shown. IECstandard error of the differencevalues are 12.2 ml litre"1 for' Cox'sOrange Pippin' and 9.6 ml litre"1

for 'Royal Gala' (P < 0.001, d.f.= 440 for both cultivars). Firm-ness data from continuous RAand CA treatments were fittedwith Equation 1 using non-linearregression.

to RA at 0.5-3°C (Fig. 3) and 20°C (Fig. 4). Onceboth cultivars were transferred from CA to RA,rapid softening was initiated either immediately orwas delayed 2-10 days. Rapid softening occurredimmediately in RA at 3 °C for' Cox's Orange Pippin'fruit that had less than 50 days in CA, and for' RoyalGala' fruit that had 0-80 days in CA (Fig. 3). Incontrast, 'Cox's Orange Pippin' fruit that had 50and 80 days in CA softened slowly in RA at 3°Cfor c. 10 days, but then softened more rapidly thanall other RA treatments. Softening rates of 'RoyalGala' in RA at 0.5°C were similar regardless of priorCA treatment. 'Cox's Orange Pippin' fruit exposedto CA for 80 days, and 'Royal Gala' fruit that had

been in CA for 50 and 80 days, softened slowly forc. 2 days once transferred to shelf life conditionsat 20°C (Fig. 4). In contrast, rapid softening wasinitiated immediately at 20°C for fruit exposedto CA for shorter times (7-20 days). Regardlessof whether softening was delayed 1-2 days orinitiated immediately on transfer from CA to RA,the subsequent softening rates of fruit at 20°C weresimilar.

Transfer of fruit from CA to RA at 0.5-3°C (Fig.3) and 20°C (Fig. 4) caused an immediate increasein IECs for both cultivars. Duration of CA storagebefore transfer to RA did not affect the maximumIEC attained for 'Cox's Orange Pippin' at 3 and

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Johnston et al. —Controlled atmospheres and apple softening 79

Fig. 4 Flesh firmness and inter-nal ethylene concentration (IEC,using a non-linear scale) of' RoyalGala' and 'Cox's Orange Pippin'apples (Malus domestica) at 0.5°Cor 3°C respectively in • , regularatmosphere (RA); • , controlledatmosphere (CA); and in CA for• 7, O 20, • 50, and A 80 daysbefore transfer to RA at 20°C. CAconditions were 2.0% O2:1.8%CO2. Treatment means (n = 10)and standard errors of the differ-ence (P< 0.001, d.f. = 350 for bothcultivars) are shown. IEC standarderror of the difference values are65.9 ml litre"1 for 'Cox's OrangePippin' and 54.8 ml litre"1 for' Royal Gala' (P < 0.001, d.f. = 350for both cultivars). Firmness datafrom continuous RA and CA treat-ments were fitted with Equation 1using non-linear regression.

c

80

70

60

50

40

100-

2 10

oy 1

0.1

'Cox's Orange Pippin' 'Royal Gala'

0 100 200 0Days at 3°C

100 200 300Days at 0.5°C

20°C, nor for' Royal Gala' at 20°C. However,' RoyalGala' fruit had a lower maximum IEC in RA at 0.5°Cwhen previously stored in CA for 50-80 days thanfruit from shorter CA duration treatments (Fig. 3).

DISCUSSION

Softening of 'Royal Gala' and 'Cox's OrangePippin' apples in CA was characterised and foundto have a triphasic softening curve similar to thatpreviously found in RA for these cultivars (Johnstonet al. 2001). Apples in CA had a longer initial slowsoftening phase and slower rapid softening phase

than fruit in RA. Firmness data from Lau & Looney(1982) indicated that' Golden Delicious' fruit in CAalso had a longer initial slow softening phase andslower rapid softening phase than fruit in RA. Asimilar pattern also occurs in kiwifruit (McDonald& Harman 1982; Arpaia et al. 1984) and avocadofruit (Meir et al. 1995).

The mechanism by which CA slowed softeningof 'Royal Gala' and 'Cox's Orange Pippin' applesat 0.5-3°C may have been mediated by ethylene, asapples from both cultivars had a slower increase inIEC, and a lower maximum IEC in CA than in RA.Studies with inhibitors of ethylene action have shownthat this growth regulator has an important role in

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New Zealand Journal of Crop and Horticultural Science, 2006, Vol. 34

promoting apple softening (Fan et al. 1999; Watkinset al. 2000). It has been suggested if the IEC of applesis maintained below 0.1 pd litre"1 (Stow et al. 2000),and external ethylene concentrations below 1 //Ilitre"1 (Liu 1977), then apple softening is reduced inCA. In previous softening studies on 'Royal Gala'and 'Cox's Orange Pippin' in RA, onset of rapidphase softening in both cultivars was associatedwith IEC exceeding 1.5 //I litre"1, regardless oftemperature or harvest date (Johnston et al. 2001,2002). A similar response was observed in this studyfor both 'Royal Gala' and 'Cox's Orange Pippin' inCA, as both duration of the initial slow softeningphase, and time before IEC exceeded 1.5 pd litre"1,were longer for fruit in CA than in RA. An importanteffect of ethylene in these apple cultivars may be toinitiate rapid phase softening under both RA and CAconditions. Stow et al. (2000) found that ethyleneappeared to initiate but not regulate the rate of 'Cox'sOrange Pippin' softening in CA. A slower rapidsoftening phase in CA than in RA could be becauseof a reduced respiration rate and/or reduced activityof cell wall degrading enzymes, rather than a directeffect of reduced IEC (Siddiqui etal. 1996; Beaudry1999; Hertog et al. 2001).

Lower IECs for 'Royal Gala' and 'Cox's OrangePippin' fruit in CA than in RA are most likely tobe the result of the antagonistic effects of low O2and elevated CO2 on ethylene biosynthesis andaction (Burg & Burg 1967). Elevated CO2 (17%O2:20% CO2) and low O2 (0.25% O2:0% CO2)reduced the expression of key ethylene biosyntheticenzymes in 'Golden Delicious' apples, including1-aminocyclopropane-l-carboxylic acid (ACC)synthase and ACC oxidase (Gorny & Kader1997).

Another mechanism for reduced softening of'Royal Gala' and 'Cox's Orange Pippin' applesin CA may involve slower cell wall disassembly.'Golden Delicious' apples from CA were firmer, hada slower loss of total pectin and hemicellulose, aslower increase in free pectin, and a slower decreasein covalently bound pectin in the cell walls thanin RA fruit (Siddiqui et al. 1996). Tijskens (1979)suggested that loss of texture consisted of rapiddegradation of some cellular components in both CAand RA, slower degradation of a second componentin CA than in RA, and the slow decay of a thirdcomponent in both CA and RA. It was subsequentlysuggested that some of these components could bedifferent forms of pectin, which may degrade ineither the presence or absence of oxygen (Tijskensetal. 1999). More research is required to determine

what biochemical changes occur in the cell wall inrelation to the three phases of softening for applesin CA and RA, and whether or not these effects aredriven by ethylene.

Delayed application of CA resulted in fruit thatwas less firm at the beginning of and throughoutCA storage. For each additional day in RA beforeCA storage, subsequent market life of fruit in CAwas reduced by 2 days for 'Cox's Orange Pippin'and 1 day for 'Royal Gala'. Fruitfrom several otherapple cultivars were firmer after storage when CAconditions were established rapidly after harvest(Smock & Blanpied 1963; Lau & Looney 1982; Lau1983; Ficaetal. 1985; Liu 1986; Dilleyetal. 1989).However, when 'Cox's Orange Pippin' was storedin ultra-low O2 (1.25%) CA, rapid establishmentof CA did not improve post-storage firmness whencompared to a delay of 10-15 days in RA (Stow1986; Stow & Genge 1990). Stow (1986) suggestedthat long-term storage of 'Cox's Orange Pippin' inultra-low O2 (1.25%) may nullify the effect of delayedCA after harvest, as ultra-low CA is more effectivein retaining firmness than atmospheres (O2 >1.5%)used in the studies referred to above. Softening of'Mclntosh' apples is completely inhibited in ultra-low CA (Dewey & Bourne 1982), indicating thatultra-low CA may inhibit or substantially delay theonset of rapid softening.

S oftening rates in CA varied depending on the rateof softening at the time of transfer from RA. ' RoyalGala' and 'Cox's Orange Pippin' fruit transferred toCA while in the initial slow softening phase softenedmore slowly in CA than fruit transferred afterrapid softening had been initiated. 'Cox's OrangePippin' fruit transferred to CA when in the finalslow softening phase, as expected, had the slowestsoftening rates in CA. Similar results were foundin another study on 'Cox's Orange Pippin', whererate of softening in CA increased as the precedingtime in RA increased from 0 to 20 days, and thendecreased as time in RA increased further to 40 and80 days (Sharpies & Munoz 1974). The softeningrates of 'Golden Delicious' (Lau & Looney 1982),'Mclntosh' and 'Empire' (Dilley et al. 1989) cultivarsin CA also increased when previously stored in RAfor increased time. From a commercial perspective,these results indicate that maximum firmness benefitsare attained from CA when applied to fruit in theinitial slow softening phase, with minimal firmnessbenefits attained if CA is applied to fruit alreadysoftening rapidly.

The effect of delayed application of CA onsubsequent softening rates may have been mediated

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Johnston et al. —Controlled atmospheres and apple softening 81

by ethylene, as the longer the fruit were in RA beforetransfer to CA, the greater the IECs were at thestart of CA storage. Rapid establishment of CAconditions after harvest reduced ethylene productionand softening in 'Mclntosh' (Fica et al. 1985; Liu1986; Dilley et al. 1989) and 'Golden Delicious'(Lau & Looney 1982) apples, and slowed theincrease of ACC in CA (Lau et al. 1984; Fica etal. 1985). Improved retention of firmness by rapidestablishment of CA conditions after harvest maybe mediated by a reduction in ethylene biosynthesis(Lau & Looney 1982; Fica et al. 1985; Liu 1986)the consequences of which are to extend the initialslow softening phase and slow loss of firmness inthe rapid softening phase.

CA storage for 50-80 days had a slight residualeffect on softening of 'Cox's Orange Pippin' whentransferred to RA at 3°C, and for both cultivars whentransferred to RA at 20°C. This consisted of a short2-10-day period of slow softening immediately aftertransfer to RA. Once rapid softening was initiated,softening rates in RA were similar regardless of priortime in CA for both cultivars. Results with otherapple cultivars and different types of fruit suggestthat a more pronounced residual effect on softeningin RA may occur after ultra-low O2 (<1.25% O2)CA than at the higher O2 concentrations used in thisstudy. For pears, the short period (1-3 days) of slowsoftening after transfer to RA is more pronouncedafter storage at lower O2 concentrations (Stow 1984).'Mclntosh' apples in RA at 0°C have no periodof slow softening and have similar softening ratesafter different times in3.0% O2:5.0% CO2, but haveslower softening rates when previously stored inlower O2 and CO2 concentrations (1.0% O2:1.5%CO2) (Lidster 1982). Kiwifruit softening rates areincreased in RA at 0°C when previously stored in CAforlongertime(Arpaiaetal. 1984). For strawberries,the residual effects of CA on reduction of respirationand ethylene production of strawberries in RA weremore pronounced when O2 concentrations weredecreased and CO2 concentrations increased (Li &Kader 1989).

CONCLUSION

This study demonstrated that a similar softeningpattern occurs in both RA and CA for 'Cox's OrangePippin' and 'Royal Gala', and that ethylene mayhave an important role in regulating the onset ofrapid phase softening in either atmosphere. CAimproved firmness retention in both cultivars by

extending the duration of the initial slow softeningphase and slowing the rate of rapid phase softening.The effectiveness of CA in reducing softening andethylene production was reduced when fruit werepreviously stored in RA, and CA was ineffective atreducing softening once rapid softening had beeninitiated. Thus, to maximise the market life of applesfor firmness, post-harvest handling systems needto place fruit in CA while still in the initial slowsoftening phase.

ACKNOWLEDGMENTS

Thanks to New Zealand Pipfruit Limited for fundingthis research, and Dr J. R. Benge for comments on thismanuscript.

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