Scientia Horticulturae, 46 ( 1991 ) 215-223 215

Elsevie:r Science Publishers B.V., Amsterdam

Removal of embryonic dormancy in apple

(Malus × domestica Borkh ) by
6-benzylaminopurine

Yong Xiang Zhang and Yves Lespinasse

LN.R.A., Station d'AmOlioration des Espkces Fruiti~res et Ornementales, Beaucouze, 49000 Angers
(France)
(Accepted for publication 25 September 1990)

ABSTRACT

Zhang, Y.X. and Lespinasse, Y., 1991. Removal of embryonic dormancy in apple (Malus × domes-
tica Borkh ) by 6-benzylaminopurine. Scientia Hortic., 46:215-223.

Embryos excised from non-stratified cultivar 'Golden Delicious' apple seeds germinated soon after
soaking in a solution of 6-benzylaminopurine (BAP) at 12.5-25 mg 1-~ for 6-24 h or at 50-100 mg
1- ~for 1-24 h. The low BAP concentrations ( 12.5-25 mg 1- ~) resulted in more normal development
of the plants than the high concentrations (50-100 mg 1-~). Compared with BAP, gibberellic acid
(GA3) (lid not consistently stimulate the germination of dormant embryos. The possibility of remov-
ing embryonic dormancy of apple seeds by a BAP treatment could be very useful in plant breeding by
shortening the breeding cycle.

Keywords: apple; 6-benzylaminopurine; embryo germination; embryonic dormancy;

Malu,:× domestica.

Abbreviations: BAP=6-benzylaminopurine; EDTA=ethylenediaminetetraacetic acid; GA 3=

gibberellic acid; MS = Murashige and Skoog's (1962) basal medium.

INTRODUCTION

Apple embryos cannot germinate normally, or fail to develop into normal

plants, immediately after harvesting when placed under conditions consid-
ered adequate for germination. This phenomenon is referred to as embryonic
dormancy, but its mechanisms are not well known (Th6venot and C6me,
1983 ). To remove embryonic dormancy in apple, the method most often em-
ployed is to place seeds at low temperature (2°C) and under moist condi-
tions tbr 2-3 months (a process commonly known as stratification) (De-
courtye and Brian, 1967 ). However, alternative methods are also available: a
cold treatment of the fruit at 0 °C or of the bare embryos on a moist cotton
medium at 5 °C (Th6venot and C6me, 1973). Some growth regulators, espe-

0304-4238/91/$03.50 © 1991 - - Elsevier Science Publishers B.V.

216 Y,X. ZHANG AND Y. LESPINASSE

cially gibberellic acid ( G A 3 ), are known to stimulate the germination of dor-

mant embryos, but do not completely replace the usual stratification in most
cases. Renard (1960) and Decourtye and Brian (1967) obtained some em-
bryo germination by treating non-stratified apple seeds with 100 mg 1-1 GA3.
Durand (1974) obtained complete germination by treating dormant apple
embryonic axes with 35 mg 1-' GA3 after removal of their cotyledons. An-
other growth regulator, 6-benzylaminopurine (BAP), was also employed by
Rouskas et al. (1980) to break embryonic dormancy in peach; soaking peach
seeds in 200 mg 1- ' BAP for 24 h allowed a normal germination and the de-
velopment of plants without previous stratification. Nevertheless, BAP has
not been tested as an embryonic dormancy eliminator in apple.
The purpose of this study was to determine whether or not a BAP treatment
could remove apple embryonic dormancy, replace the usual long stratifica-
tion by low temperature and cause embryo germination with normal plant
development.

MATERIALS AND METHODS

Plant material. - Three experiments, one in 1988 and two in 1989, were car-
ried out. Seeds used in 1988 were taken from fully mature fruits of open-
pollinated cultivar 'Golden Delicious' apples and stored, after their extrac-
tion, under dry conditions and at room temperature (16-20 ° C) until used
( ~ 3 months). Seeds used in 1989 were from a controlled pollination of
'Golden Delicious'. Fruits were collected on 17 July (90 days after pollina-
tion ) and 6 September ( 140 days after pollination ); seeds and embryos were
extracted from fruits 2 weeks after collection and stored in darkness at 16-
22 ° C for the two experiments.

Embryo extraction and treatment. - Embryos were aseptically extracted from

seeds sterilized by serial immersions in Mercryl Lauryl6 (mercurobutol at
0.01 g 1-1, Labaz) for 30 s, in 70% ethanol for 30 s, and in 1.25% sodium
hypochlorite for 15 min, followed by rinsing three times with sterilized water.
Then, they were immediately soaked in a BAP or GA3 solution. The details
of the treatments are shown in Table 1 for the experiment undertaken in 1988,
and in Tables 2 and 3 for the 1989 experiments. Twenty embryos were used
per treatment. For all the experiments, three controls were used: embryos not
treated with any solution, embryos treated with cold for 2 months and em-
bryos treated with sterilized water. The duration of soaking was 24 h for Ex-
periments 1 and 2, and from 1 to 24 h for Experiment 3. Following treat-
ments, the embryos were cultured in vitro in a test tube ( 150 × 22 m m ) , two
embryos per tube, on an em~bryo germination m e d i u m composed of half Mu-
rashige and Skoog's (1962) basal m e d i u m (MS) (macro- and micro-salts,
Fe-Na2 EDTA ) + micro-organic supplements (all in mg I- ~: myo-inositol, 100;
REMOVALOF EMBRYONICDORMANCYIN APPLE 217

TABLE 1

Effects of cold, BAP and GAs treatments on the removal of embryonic dormancy of 'Golden Deli-
cious' seeds after 3 months of dry storage at 16-20°C

Embryo treatment Percentage of Percentage of germinated Percentage Mean length of

germinated embryos developing into of abnormal plants
embryos plants plants (cm) _+SE

Non-treated embryos 50 d 100 a 20 a b 3.1 ± 1.0

One month of cold 95 a b 100 a 0a 3.1 + 1.3
Two months of cold 95 a b 100 a 0a 3.3+ 1.0
Three months of cold 100a 100a 0a 3.4+0.8
Sterile H20 70cd 93 a b 8 ab 2.4_+ 1.0
BAP (mg1-1 )
12.5 95 a b 100 a 0a 2.3_+0.7
25.0 90abc 94ab 30b 2.3_+0.7
50.0 95 a b 100 a 37 b c 1.6 ± 0.8
100.0 80b c d 100a 63 c 1.7_+0.9
200.0 65 c d 77b 100 d 1.5±0.7

GA3(mg1-1 )
25.0 85abc 94ab 13ab 2.0±0.9
50.0 90abc 94ab 12ab 2.0±0.9
100.0 80bcd 100a 13ab 2.8±1.0

Percent~,ges in each column followed by the same superscript are not significantly different at the 5%
level by the Student's t-test.

TABLE 2

Effects of cold, BAP and GA3 treatments on the removal of embryonic dormancy of 'Golden Deli-
cious' immature seeds collected 90 days after pollination

Embryo treatment Percentage of Percentage of germinated Percentage of

germinated embryos developing into abnormal
embryos plants plants

Non-treated embryos 55 bc 64 b 43 b
One month of cold 85 ab 82 ab 57 b c
Two months of cold 100 a 95 a 5a
Sterile E[20 60 bc 67 b 13 a b
BAP (mg1-1 )
12.5 85 a b 88 a b 20 a b
25.0 80bc 88ab 71 c
50.0 80bc 81 a b 69c
100.0 50c 90ab 89 c

GA 3 (mg1-1 )
50.0 65 b c 85 a b 73 c

Percentages in each column followed by the same superscript are not significantly different at the 5%
level by the Student's t-test.
218 Y.X. ZHANG AND Y. LESPINASSE

TABLE3

Influence of concentrations and soaking durations of BAP and GA 3 on the germination and growth of
fully mature embryos of non-stratified 'Golden Delicious' seeds collected 140 days after pollination

Embryo treatment Percentage of Percentage of Percentage of Mean length of

germinated germinated embryos abnormal plants plants
embryos developing into ( c m ) _+SE
plants

Non-treated embryos 50 b 90 a b 78 e f g 1.1 _+ 1.2

Two months of cold 100 a 100 a 0a 3.6 _+0.9
Sterile H20 60 b 100 a 50 c d 1.5 -+ 1.3
BAP
12.5 m g l l
6h 100 a 100 a 15 a b 3.2-+0.8
12h 100a 100a 10ab 3.1 _+ 1.
24 h 100 a 100 a 25 b c 3.2+0.8

25.0 m g l i
6h 100a 100a 25bc 3.1_+0.8
12 h 100a 100 a 35 b c d 3.0_+0.9
24 h 100a 100 a 55 c d e 3.0_+0.6

50.0 mg 1-1
lh 100a 100a 65def 2.5_+0.6
3h 100a 100 a 65 d e f 2.3_+0.8
6h 100 a 95 a b 84e fg 2.3+0.8

100.0 mg 1-I
1h 100a 100a 90fg 2.8_+0.8
3h 100 a 100 a 95 g 2.2_+0.7
6h 100a 80b 100g 2.3_+0.7

GA3
50.0 mg 1
6h 65b 85ab 27bc 2.2+1.4
12h 55b 100a 36bcd 1.8_+1.4
24 h 55 b 100 a 36 b c d 1.9_+ 1.2

Percentages in each column followed by the same superscript are not significantly different at the 5%
level by the Student's t-test.

glycine, 2; nicotinic acid, 0.5; pyridoxin, 1; thiamine, 1 ) + 5 8 . 4 mM su-

crose+ 0.65% agar (Difco). The embryos destined for stratification by cold
were then put in a cold chamber (3 °C) in darkness. The others were imme-
diately placed in a culture room for germination.

Embryo germination and parameters studied. - Germination of embryos was

carried out at 18-22°C in darkness for the first week and then in the light
with a 16-h photoperiod at 40-60/zMol m - 2 s - i. The embryos stratified by
cold were transferred directly from the dark cold chamber to the light culture
REMOV/~,L OF EMBRYONIC DORMANCY IN APPLE 219

room for germination. The embryo germination rate was recorded 1 month
later. An embryo was considered to have germinated when it showed marked
elongation of the radicle (Luckwill, 1952; Th6venot and C6me, 1973). Four
parameters were adopted to assess the effects of BAP and GA3 on the removal
of apple embryonic dormancy: the embryo germination rate (%), the per-
centage of germinated embryos developing into plants, the percentage of ab-
normal plants, and the mean length of plants. The plants considered abnor-
mal were those with abnormal leaves, or poorly developed roots, or multi-
shoots or abnormally thick stems.

RESUkTS

Effects o f water and cold treatments on embryo germination. - For all three
experiments (Tables 1-3), nearly half of non-treated embryos germinated
and the percentage of abnormal plants was relatively high, especially for the
experiments carried out in 1989 (Tables 2 and 3). Soaking embryos in steri-
lized water for 24 h did not significantly improve the embryo germination
rate. Two months of cold treatment gave nearly complete embryo germina-
tion, regardless of the type of experiment. Moreover, almost all of the germi-
nated embryos developed into normal plants with good plant growth.

Effects o f B A P treatments on embryo germination. - In Experiment 1 (Table

1 ), the germination rate reached 90-95% when embryos were treated with
BAP at a concentration of 12.5-50 mg 1-~, which is comparable with the ger-
mination rate obtained with cold treatment. Thereafter, it decreased when
the BAP concentration was > 50 mg 1- ~. Nearly all the germinated embryos
developed into plants, except those treated with 200 mg 1- l BAP, where only
77% developed. The percentage of abnormal plants increased markedly with
increasing BAP concentration: from 0% at 12.5 mg 1-1 to 100% at 200 mg l-
Abnormal plants developing from BAP treatments principally presented ab-
normally thick stems (Fig. 1 ), poorly developed root systems or multi-shoots.
Moreover, treatments with a high BAP concentration had a tendency to re-
duce plant growth. In this respect, the mean length of the plants from the
embryos treated with BAP was shorter than that of the plants from the em-
bryos treated with cold. Based on the results obtained from this preliminary
experiment, 200 mg 1- ~ BAP was considered too strong and was eliminated
from later experiments.
For ]Experiment 2 (Table 2), with embryos more or less immature, 80-85%
of embryos germinated after being treated with BAP at 12.5-50 mg 1-~.
Treatment with BAP at 100 mg 1- l caused only 50% of embryos to germinate.
With 81-90% of germinated embryos developing into plants for the four
treatments, these results were not significantly different from those of the cold
220 Y.X. ZHANG AND Y. LESPINASSE

Fig. 1. Plants obtained 3 weeks after BAP treatment in Experiment 1 (in 1988). On the right,
plants showing normal development from the 12.5 mg 1-i treatment; on the left, plants from
the 100 mg 1- ~treatment showing reduced growth, an abnormally thick stem and a poorly de-
veloped root system.

treatments. However, increasing BAP concentrations increased the percent-

ages of abnormal plants.
In Experiment 3 (Table 3) for all 12 BAP treatments (concentra-
tion X soaking duration ), all embryos germinated, whatever the combination.
In addition, almost all germinated embryos developed into plants, except those
treated at 50 and 100 mg 1-1 for 6 h. The differences observed between the
different treatments mainly concerned the percentage of abnormal plants ob-
tained and the mean length of plants. Increasing the BAP concentration and
prolonging the duration of soaking increased the percentage of abnormal plants
from 10% at 12.5 mg 1-1 for 12 h to 100% at 100 mg 1- ~for 6 h. In this respect,
increasing the BAP concentration had much more influence than prolonging
the duration of soaking. In fact, more than half of the plants obtained were
more or less abnormal with treatments at 50 and 100 mg 1-~ BAP, regardless
of the duration of soaking. The percentage of abnormal plants was the same
REMOVAL OF EMBRYONIC DORMANCY IN APPLE 221

with 12.5 mg 1-' BAP (6-24 h) and 25 mg 1-I BAP (6-12 h). Again, the
mean stem length was reduced with increasing BAP concentration. The influ-
ences of the different durations of soaking for each BAP concentration tested
on stem length were not very clear even if the shortest duration of soaking for
the same concentration seemed to favour plant growth.

Effects o f GA3 treatments on embryo germination. - In Experiment 1 (Table

1 ), treatments with 25-100 mg 1-l GA3 caused 80-90% of embryos to ger-
minate and 94-100% of germinated embryos to develop into plants. These
result,; were comparable with those obtained after cold and BAP treatments.
About 12% of the plants were abnormal and abnormality primarily involved
leaves. By comparing the three concentrations, the results observed were al-
most 1;he same. Consequently, only 50 mg 1-1 GA 3 was chosen to compare
with BAP for the experiments carried out in 1989. For Experiments 2 and 3
(Tables 2 and 3), only 55-65% of embryos germinated after being treated
with G A 3 at 50 mg 1- l for 6-24 h; these results were no different from those
of non-treated embryos. The percentage of germinated embryos developing
into plants was comparable with those observed in cold and BAP treatments.
In Experiment 2, the percentage of abnormal plants was significantly higher
than those of non-treated and water-treated embryos. However, the embryos
treated with GA3 in Experiment 3 included fewer abnormal plants than the
non-treated embryos, the water-treated embryos or the embryos treated with
BAP at 50-100 mg 1-I for 1 or 6 h.

DISCUSSION AND CONCLUSIONS

The present study, to our knowledge, is the first that demonstrates the pos-
itive effect of BAP soaking on the removal of embryonic dormancy of non-
stratified apple seeds.
The germination rate achieved for non-treated embryos in this study con-
firms that apple embryos are partially dormant at harvest (Krugman et al.,
1974 ). Nevertheless, embryos with their coats (i.e. intact seeds ) failed to ger-
minate under the same conditions, regardless of experiments and materials
(results not shown ).
Results obtained in our experiments showed that the embryonic dormancy
of 'Golden Delicious' mature seeds could be completely removed by soaking
dorm~.nt embryos in a BAP solution at a suitable concentration for an appro-
priate duration. Using the same BAP concentration and same duration of
soaking, the embryos excised from immature seeds (Experiment 2) gave ger-
mination rates inferior to those from mature seeds (Experiment 3). This
meant that the immaturity of the embryos limited the effect of BAP treat-
ments on the removal of embryonic dormancy. Another cytokinin, kinetin,
was tested by Durand (1974) for removing apple embryonic dormancy, but
222 Y.X. ZHANG AND Y. LESPINASSE

no positive effect was observed. Rouskas et al. (1980) obtained normal ger-
mination by soaking peach rootstock seeds with their coats in 200 mg 1- ~BAP
for 24 h. However, for apple embryos without their coats, the high concentra-
tions of BAP ( 50-200 mg 1- ~) induced much more abnormal plants than the
low concentrations ( 12.5-25 mg 1-~ ).
For GA3, our results indicated that its effect on the removal of apple em-
bryonic dormancy varied with experiment and year. Treatments with GA3 in
Experiment 1 gave germination rates comparable to those of cold and BAP
treatments, but were not comparable in Experiments 2 and 3. By in vitro cul-
ture of dormant embryos of 'Golden Delicious' on a liquid m e d i u m contain-
ing 35 mg 1-1 GA3, Durand (1974) observed embryo germination rates vary-
ing from 55 to 90% according to year. This annual variation in results was
also observed by the same author when treating embryonic axes with the same
GA3 concentration. Thus, GA3 treatments were less effective and consistent
than BAP treatments on the removal of embryonic dormancy in 'Golden
Delicious'.
Usually, chilling is required to break dormancy in apple seeds. Our results
demonstrated that BAP treatment could replace, at least under our condi-
tions, the long cold treatment of apple embryos or seeds to obtain a normal
germination. This is especially useful to plant breeding programmes, for ex-
ample disease resistance breeding. In fact, the results of our experiments have
already been applied to the fire blight breeding programme in our laboratory.
By treating dormant embryos with BAP at 12.5 or 25 mg 1-1 during 12-24 h,
germination occurred and plants quickly obtained from several crosses were
inoculated and pre-selected in vitro at least 2 months earlier than usual in the
glasshouse (results not published). The breeding cycle could thus be short-
ened. For this purpose, slightly abnormal plants could be used as well. The
possibility of removing the embryonic dormancy of dormant apple seeds with
BAP should offer a new way to study apple embryonic dormancy mechanisms.

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REMOVAL OF EMBRYONIC DORMANCY IN APPLE 223

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