HORTSCIENCE 39(6):1185–1188. 2004.

Evaluation of Differences in Softeningof Apple Genotypes by Linear
Regression
Hiroshi Iwanami, Makoto Ishiguro,1 Nobuhiro Kotoda, Sae Takahashi,
and Junichi Soejima2
Department of Apple Research, National Institute of Fruit Tree Science,
Morioka 020-0123, Japan
Additional index words. Malus ×domestica, breeding
Abstract. The firmness of the flesh in 27 apple (Malus ×domestica Borkh.) cultivars andselections (genotypes) was measured as an indicator of storage potential at 20 days after
harvest under 20 ± 2 °C, 80% ± 5%relative humidity storage conditions. Softening ranged
from 9% to 58% of initial values among genotypes after 20 days of storage. In somegenotypes, softening was not continuous, a minimum firmness being reached before day
20. After a period of rapid softening, firmness declined to at least 20% of that at harvest.
For each genotype, linear regression analysis of firmness changes from harvest until whenfirmness decreased by 20% was carried out. In genotypes in which firmness did not drop
>20% within 20 days of storage, the entire dates to 20 days were used for analysis. The
homogeneity of the regression residual variances and their normal distribution was not
rejected at P = 0.05, and the linear regression analysis was assumed to be applicable tothe change in firmness for each genotype. Results of the regression analysis showed thatthe regression was significant for all genotypes except one. Therefore, storage potential
could be evaluated by comparing the regression coefficient of each genotype.
Texture, particularly firmness, is often used
as an indicator of maturity, ripeness, and qualityof apples (Abbott, 1994). Many studies haveused changes in firmness during storage to investigate different ripening types and the effectsof storage technologies (Drake, 1993; Gussman
et al., 1993; Ingle and D’Souza, 1989; Plotto
et al., 1995, 1997; Tong et al., 1999; Watkins
et al., 2000). Moreover, several studies havepredicted changes in fruit firmness from thatat harvest time to after or during storage using
regression equations by measuring quality at
harvest (Blankenship et al., 1997; Evensen etal., 1993; Ingle and Morris, 1989; Ingle et al.,
2000; Johnson and Ridout, 1998). However,these studies used only one or a few commercially important cultivars and the data were notused to compare cultivar-related differences insoftening rates during storage.
Many fruit are needed to evaluate storage
potential of apple cultivars and selections.
However, in breeding programs, young seedlings usually do not bear enough fruit to permit
extensive sampling for storage tests, and as aconsequence, this important part of the seedlingscreening is ignored in many apple-breeding
programs (Alston, 1988).
The objective of this study was to develop
a method to evaluate changes in fruit firmness
that could be used as an indicator of storage
potential, using restricted fruit samples fromapple breeding programs.

Received for publication 7 May 2003. Accepted for
publication 31 Dec. 2003.
1
Current address: Yamagata Prefectural Horticultural
Experiment Station, Sagae, Yamagata 991-0043.
2
Current address: National Institute of Fruit Tree
Science, Fujimoto, Tsukuba, Ibaraki 305-8605.

HORTSCIENCE VOL. 39(6) OCTOBER 2004

Materials and Methods
Fruit materials. Twenty-seven apple cultivars and selections from the orchards at theApple Research Center, National Institute ofFruit Tree Science (NIFTS) in Japan wereselected based on the diversity of harvest date,rootstock, and years since planting/grafting(Table 1). The genotypes consist of 16 cultivars
that originated in Japan and have been usedas parents in breeding, six cultivars originating from North America, and five promising
selections from the NIFTS apple-breeding
programs. Twenty-five fruit samples from each
cultivar and selection were picked randomly
from a tree when most of the fruit was judged
to be mature based on sensory evaluations from
August to November 2001. Fruit of similar
size (within 15% of average fruit weight) was
picked from each cultivar/selection because
fruit size affects fruit firmness (Blankenship,1997) and a strong relationship has been shown
to exist between fruit size and fruit firmness
after storage (Marmo et al., 1985).
Storage conditions. Five fruit from eachsample of the 27 cultivars or selections wereused for measurement of firmness at harvest.
The remaining 20 fruit were stored in 25-L
containers, which were arranged on racks,
in a chamber controlled at 20 ± 2 °C, 80%± 5% relative humidity (RH) with constant
air circulation. Firmness of four to five fruit
was measured at 5-d intervals until 20 d after
harvest. Fruit displaying rot were immediately
removed from the containers during storage.
Firmness measurement. A fruit pressure
tester (FT327; McCormick Fruit Technology,
Wash.), mounted in a drill press, and fittedwith an 11.1-mm probe, was used on the paredsurfaces of the sunny and shady sides of eachfruit. Data were expressed as Newtons (N).
Statistical analysis. Firmness measurements of each cultivar/selection (genotype)were subjected to linear regression analysis andto analysis of variance (ANOVA). Individual
fruit data for each sampling, not mean valueof each sampling fruit, and only the reduction
periods in firmness of 20 d of storage wereused for the analyses. The model of ANOVA
was yijk = µ+ gi + bi x + eijk, where yijk isthe firmness measurement as the dependent
variable of the kth fruit at jth days after harvestof the ith genotype, µ is the overall mean, gi isa random effect contributed by the ith genotype, bi is the regression coefficient of the ith
genotype, x is the kth fruit of ith genotype jthdays after harvest, and eijk is the residual of
the regression line of the kth fruit of jth days
after harvest of the ith genotype. ANOVA was
performed using a Statistical Analysis System(SAS Institute, Cary, N.C.).
Results and Discussion
Firmness of the flesh at harvest and thepercentage firmness reduction after 20 d ofstorage varied among genotypes (Table 2).
Softening ranged from 9% to 58% of initial
firmness values. ‘Red Gold,’ ‘Starking Delicious’ and ‘Orin’ had relatively firmer fruit atharvest (77.7, 72.2, and 74.5 N, respectively),
but fruit became very soft, after 20 d of storage (40.5, 33.9, and 40.9 N, respectively). In
contrast, ‘Santaro’ and ‘Toko’ had relatively
soft fruit at harvest (59.8 N), but their fruit
were firmer after 20 d of storage (42.7 and46.1 N respectively) than those of ‘Red Gold,’‘Starking Delicious’ and ‘Orin.’
Fruit did not always soften continuously
during storage. The flesh of some genotypes
softened rapidly and by at least 20% of harvest firmness until 10 d after harvest but thensoftened slowly day 10 to 20 (Fig. 1B). The
minimum firmness of ‘Tsugaru’ was 52.2 N(27% loss of harvest firmness), was muchhigher than that of ‘Red Gold,’ which was
40.5 N (48% loss of harvest firmness). These
results are consistent with those of Johnston
et al. (2002) who suggested that apples only
soften by 25% to 50% to a final firmness of 35
to 50 N. On the other hand, some genotypes
continued to soften for 20 d. Firmness of‘Megumi’ and ‘Starking Delicious’ declined
to 31.9 N (49% loss) and 33.9 N (53% loss)
on day 20, respectively (Fig. 1A), in contrast
to that of ‘Fuji’ and ‘Kitaro,’ which was 67.5 N(13% loss) and 62.9 N (13% loss), respectively
at this time (Fig. 1D).
The reduction in firmness in ‘Tsugaru’(27%) and ‘Kinsei’ (22%) was similar on day
20, but different on day 10, being, 27% and6%, respectively (Fig. 1B and D). In contrast,
the reduction in firmness of ‘Kitaro’ was 7% on
day 10 and almost identical to that of ‘Kinsei,’but that of ‘Kitaro’ on day 20 (13%) and lessthan that of ‘Kinsei’ (22%) (Fig. 1D). Therefore,
the extent of softening after harvest is greatly
influenced by the storage period, and the genotypic differences in the storage potential cannot
be evaluated by total changes in firmness overa single ripening period. However, determining

1185

Relative firmness (% of harvest firmness)

120

120

120

A

120

B

C

D

100

100

100

100

80

80

80

80

60

60

60

60

40

40

Starking Delicious
McIntoshMegumiOrin
Hokuto20

Mutsu
Kitakami
Morioka57
Tsugaru
Morioka59
Akane
Sansa
Golden Delicious

20

0

0
0

5

10

15

20

40

40

SilkenHimekami
KotaroMorioka60
JonathanSantaroRed Gold

20

20

0
0

5

10

15

20

Morioka58
Fuji
Ralls Janet
Toko
Kinsei
KitaroMiki Life

0

0

5

10

15

20

0

5

10

15

20

Storage duration (day)Fig. 1 Relative flesh firmness of 27 apple cultivars and selections during 20±2 °C, 80% ± 5%RH storage. The relative firmness was calculated as the averagefirmness of four or five fruit of each sampling compared with the average harvest firmness of five fruit of each genotype. Flesh firmness (A) decreased rapidly
and constantly during 20 d; (B) decreased rapidly during 10 d and changed little from 10 to 20 d; (C) decreased during 15 d and changed little from 15 to 20d; (D) decreased gradually during 20 d.

how long it takes for fruit to become too softfor market acceptance requires regular evaluation until firmness reaches a certain value. For
this purpose, many samples of fruit are needed
when a genotype is likely to have long storage
capability. In this study, the firmness of ‘Miki
Life’ decreased only 9% by day 20, and more
fruit would be needed to continue measuring
firmness (Fig. 1D).
Since it is unnecessary for evaluating storage potential to observe changes in firmness
after a minimum value has been reached, alinear regression of firmness against a specified loss of firmness (20% of harvest values)
was applied. When firmness did not decrease>20% within 20 d of storage, the full length ofstorage was used for linear regression analysis.
Homogeneity of the variances of the regression residuals among sampling dates for eachgenotype was tested by Bartlett’s test, and thenormal distribution of the residual was tested
using the Kolmogorov-Smirnov one-sample
test. The results of these tests showed thatthe homogeneity of the variance and normal
distribution was not rejected at P = 0.05, andtherefore, linear regression analysis was assumed to be applicable to this data.
Regressions were significant (P < 0.05) for
all genotypes except one (Table 2). This result
indicated that the regression coefficient (b),which is the firmness reduction rate and its
standard error, could be calculated within at least
20 d of harvest and that the regression coefficient
could be compared among genotypes.
Johnston et al. (2002) reported that the
reduction in firmness in many apple cultivars
can be characterized by a nonlinear curve1186

consisting of three distinct phases; i.e., fruit
softening slowly in the first phase (I), more
rapidly in the second phase (II), and slowly
in the final phase (III). Johnston et al. (2001),
moreover, used a nonlinear regression modelwith three parameters to describe the change
in firmness during storage.
In our studies, the regression residual vari-ability could be divided into two components:
within-sampling variability and deviationfrom linearity. If the relationship between thefirmness and length of storage is a straight
line, then the deviation-from-linearity MS and
the within-sampling MS will be estimates ofthe same variance. Thus, the F value for thefitness for a straight line was calculated as F

Table 1. Twenty-seven apple cultivars and selections and tree age, rootstock, and harvest date evaluatedin this study.
Cultivar or selection
Tree agez
Rootstock
Kitakami(3)
Jonathan/Marubakaido
Sansa
15
M26E
Tsugaru
7
JM7
Akane
(3)
Jonathan/Marubakaido
Miki Life9
M9
McIntosh41
Marubakaido
Silken11
M9
Himekami(17)
Starking Delicious/MM106
Santaro12
M26
Red Gold
7
M9
Morioka57
12
M26
Morioka59
10
M9
Morioka60
10
M9Jonathan43
Marubakaido
Kitaro10
JM2
Starking Delicious
7
JM7
Kotaro11
JM2
Hokuto7
JM7Golden Delicious
7
M9
Mutsu
7
JM7
Orin
7
M9
Morioka58
11
M9
MegumiUnknown
Unknown
Kinsei
15
CG57
Fuji
7
M9
Ralls Janet
7
M9
Toko
15
CG57
z
Numeral in parenthesis indicates years since top-grafting on intermediate stock.

Harvest date
31 Aug.
7 Sept.
14 Sept.
14 Sept.
14 Sept.
20 Sept.
20 Sept.
20 Sept.
28 Sept.
5 Oct.
5 Oct.
5 Oct.
11 Oct.
11 Oct.
11 Oct.
20 Oct.
26 Oct.
26 Oct.
26 Oct.
26 Oct.
2 Nov.
2 Nov.
9 Nov.
9 Nov.
9 Nov.
17 Nov.
17 Nov.

HORTSCIENCE VOL. 39(6) OCTOBER 2004

= the deviation-from-linearity MS divided bywithin-sampling MS and tested by the F test.
The F test for the fitness for a straight line
showed that the linearity was not rejected ingenotypes in which firmness decreased gradually, i.e., those in which firmness decreased
20% at 15 d or more than 20 d (Table 3).
Johnston et al. (2001) also observed that the
rapid-phase of softening (phase II) was notapparent at storage temperatures of 20 and 30°C in some slow-softening cultivars and they
could not fit the nonlinear regression model
they proposed to the curves for the firmness
reduction of the cultivars. In our studies, whenfirmness measurements continued for 40 d after
harvest in some genotypes in which firmness
decreased gradually, the phase changes were
not observed (data not shown).
In genotypes in which firmness decreased
20% by day 10, the deviation-from-linearity
MS was significantly large (143.6) (Table 3).
These genotypes appeared to have distinct
phase changes from phase I to phase II. ‘Starking Delicious’ and ‘Silken’ were the genotypes

in which firmness decreased 20% by day 10
and the firmness of the cultivars changed little
within 5 d of harvest and decreased rapidly
within 5 to 10 d of harvest (Fig. 1A and C). The
regression residual variances (Ve) of the cultivars were large (84.08 and 52.96, respectively)(Table 2). However, linear regression analysis
was also applicable to these genotypes having
distinct phase change because the variances infirmness of individual fruit within sampling
were relatively large and the regression residuals were not significantly imbalanced from theregression line.
When linear regression equations were
calculated for two sets of sampling, i.e., whenfirmness decreased 20% at 5 d, the Ve indicatesthe firmness variance for each sampling time.
The firmness of ‘McIntosh’ did not decrease (P= 0.076) because of the large residual variance(Ve = 91.8), although the average firmness
rapidly decreased during the first 5 d after
harvest. The large Ve of ‘McIntosh’ resulted
from the large variances in firmness at eachsampling point.

The average firmness of ‘Miki Life’ washigher than that at harvest for 10 d, and it thenbegan to decrease for the next 15 d. The firmness variance within sampling was also large
in the cultivar (data not shown). Therefore,
the large Ve of the cultivar resulted from the
combined effect of the large residual from theregression line and the large firmness variances
within the sampling.
In breeding situations, many cultivars andseedlings are needed to effectively evaluate
storage potential and select superior genotypes.
Alston (1988) evaluated the storage potential
in many cultivars and seedlings in his applebreeding program. However, he evaluated thestorage potential by measuring firmness at only
one time after storage. Although the curves
showing the reductions in firmness were notalways linear, linear regression analysis is still
very effective for evaluating storage potential
because the regression was significant for atleast 20 d after harvest in almost all genotypes
under the conditions used in this study; furthermore, regression analysis could be adapted

Table 2. Flesh firmness changes during 20-d storage and results of linear regression analysis on the firmness during storage of each of the 27 cultivars andselections.

Firmness
Cultivarator
harvestselection
(N)
Mutsu
79.09
Kitakami
54.40
Megumi
62.36
McIntosh
58.36
Red Gold
77.66
Starking Delicious
72.24
Silken63.43
Morioka57
72.90
Himekami
69.92
Morioka59
59.51
Orin
74.50
Morioka60
81.31
Tsugaru
71.08
Kotaro
75.13
Akane
61.56
Hokuto
71.26
Sansa
59.34
Golden Delicious
64.10
Jonathan
64.58
Ralls Janet
77.97
Santaro59.83
Kinsei
75.39
Toko
59.83
Morioka58
74.46
Fuji
77.62
Kitaro72.37
Miki Life
61.16
zRegression residual variance

Firmnessdecreaseafter
20-d
storage
(%)
36.7
35.8
48.9
42.1
47.9
53.1
58.1
37.3
42.4
42.2
45.1
29.5
26.5
34.5
36.9
36.9
33.7
32.3
29.3
25.2
28.6
21.5
22.9
16.5
13.0
13.1
9.4

20% Firmness
decrease
periods
fromharvest(d)
Sampling (no.)
5
2
5
2
5
2
5
2
10
3
10
3
10
3
10
3
10
3
10
3
10
3
10
3
10
3
10
3
10
3
10
3
10
3
10
3
15
4
15
4
15
4
20
5
20
5
>20
5
>20
5
>20
5
>20
5

Linear regression analysis (firmness vs. storage periods )
n
r2
P
Vez
b ± SE100.852<0.001
15.620–3.389 ± 0.500
10
0.842<0.001
13.914–3.078 ± 0.472
10
0.729
0.002
22.767–2.802 ± 0.604
10
0.342
0.076
91.789–2.473 ± 1.212
15
0.849<0.00135.205–3.203 ± 0.375
15
0.610<0.00184.077–2.615 ± 0.580
15
0.684<0.00152.958–2.442 ± 0.460
15
0.766<0.00131.368–2.309 ± 0.354
15
0.874<0.00114.427–2.286 ± 0.240
15
0.837<0.00119.357–2.273 ± 0.278
15
0.773<0.00126.855–2.180 ± 0.328
14
0.702<0.00127.694–1.873 ± 0.352
14
0.731<0.00123.514–1.856 ± 0.325
15
0.690<0.00129.614–1.850 ± 0.344
15
0.799<0.001
16.113–1.824 ± 0.254
15
0.810<0.00114.985–1.824 ± 0.245
15
0.884<0.0017.861–1.761 ± 0.177
15
0.609<0.00135.887–1.704 ± 0.379
20
0.786<0.001
15.610–1.285 ± 0.158
20
0.679<0.00126.019–1.258 ± 0.204
20
0.649<0.001
20.172–1.035 ± 0.180
25
0.642<0.00123.937–0.888 ± 0.138
25
0.503<0.00118.711–0.590 ± 0.122
25
0.582<0.00118.587–0.689 ± 0.122
25
0.719<0.001
5.762–0.520 ± 0.068
25
0.267
0.008
27.382–0.429 ± 0.148
25
0.160
0.048
48.897–0.414 ± 0.198

Table 3. Analysis of variance for firmness and mean square and significances of linear regression in all studied genotypes except ‘McIntosh’ and in three groups
divided according to firmness decrease periods.

Included all genotypes
Source of variationdf
Mean squares
Cultivar or selection (genotype)25
937.42**
Linear regression
26
913.24**
Residual
396
25.84
Deviation from linearity
38
68.44**
Within sampling
358
21.32
NS,**
Nonsignificant and significant at P < 0.01, respectively, using F test.

HORTSCIENCE VOL. 39(6) OCTOBER 2004

df
13
14
180
14
166

Genotypes in which the firmness
decreased 20% of harvest firmness at
10 d
15 d
Mean squares
df
Mean squares
df
660.99**21644.27**51175.34**3
897.29**
6
30.04
54
20.60
138
143.60**624.84NS
18
20.47
48
20.07
120

>20 d
Mean squares
1194.79**
466.09**23.88
24.52NS
23.78

1187

to genotypes in which reductions in firmness
occurred either rapidly or gradually in the sameway. As a consequence, superior genotypes
can be selected to compare the regression
coefficients of seedlings with those of control
cultivars. When the nonlinear regression modelthat was proposed by Johnston et al. (2001)
is applied to softening curve during storage,
genotypic differences can be also evaluated tocompare the rate of firmness change per day,which is one of the parameters in the model.
The nonlinear regression model, however,requires three parameters to fit the softening
curve, and one of those parameters is a final
firmness asymptote. Therefore, firmness must
be measured until it no longer decreases if
the nonlinear model is to be used. Since theyield of young seedlings is limited, their fruit
are not very appropriate for use in storage
potential evaluations. In this study, 25 fruit
were harvested, and five were used to determine firmness at 5 d intervals. Measurementsof firmness continued from harvest until theaverage firmness decreased 20% of harvest
firmness or until 20 d had elapsed.
Ingle and Morris (1989) observed that
there were good correlations between firmnesschanges at 20 and that at 0 °C in ‘Rome’ apple.
However, Johnston et al (2001) observed that
rapid softening of some genotypes occurred
only when the fruit had been stored at cold
temperature. Further studies are required todetermine the genotypic differences of softening responses to the storage temperature.

1188

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