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Improvement of seed dehiscence and germination in ginseng by

stratification, gibberellin, and/or kinetin treatments

Article  in  Horticulture, Environment and Biotechnology · May 2018

DOI: 10.1007/s13580-018-0039-6

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Horticulture, Environment, and Biotechnology (2018) 59:293–301
https://doi.org/10.1007/s13580-018-0039-6

RESEARCH REPORT

Cultivation Physiology

Improvement of seed dehiscence and germination in ginseng

by stratification, gibberellin, and/or kinetin treatments
Jung‑Woo Lee1 · Ick‑Hyun Jo2 · Jang‑Uk Kim2 · Chi‑Eun Hong2 · Young‑Chang Kim2 · Dong‑Hwi Kim2 ·
Young‑Doo Park1

Received: 26 May 2017 / Revised: 7 December 2017 / Accepted: 11 December 2017 / Published online: 4 May 2018
© Korean Society for Horticultural Science and Springer-Verlag GmbH Germany, part of Springer Nature 2018

Abstract
Korean ginseng (Panax ginseng C.A. Meyer) is a largely sought after resource due to its substantial health benefits. However,
large-scale culture of ginseng seeds is limited because of its long maturation time and triple-dormancy. To develop new
strategies to improve the dehiscence, germination, and breaking dormancy, we investigated the effects of stratification, gib-
berellin ­(GA3), and/or kinetin treatments on the dehiscence, development and germination of P. ginseng. Indehiscent seeds
for the dehiscence test and dehiscent seeds for the germination test were immersed in a solution containing 288.7 μM ­GA3
and/or 232.3 μM kinetin for 24 h, respectively, at 25 °C. After dehiscence, a cold stratification experiment was conducted at
2 °C for 15, 30, 45, 60, and 90 days in the dark. Exogenous ­GA3 and kinetin treatments significantly improved the dehiscence
rate. In addition, both plant growth regulators (PGRs) appeared to enhance the zygotic embryo development of ginseng
seeds. ­GA3 on its own had a slight effect on breaking dormancy and germination, but the effect was not sufficient to serve
as a complete substitute for the cold stratification requirement due to the deep physiological dormancy characteristic of P.
ginseng. Kinetin treatment had a significant effect on germination and breaking dormancy, although a short period of cold
stratification was also required. The combination of ­GA3 and kinetin treatments was more effective at enhancing both seed
dehiscence and germination than either PGR treatment alone.

Keywords  Dehiscence · Germination · Dormancy · Panax ginseng

1 Introduction (Lee et al. 2005; Min et al. 2006). Germination is an impor-

Korean ginseng (Panax ginseng C.A. Meyer) is a major
medical and perennial herbal plant in the family Araliaceae.
Ginseng is grown and cultivated under shaded conditions in
the northeast of Asia and primarily in the Korean peninsula.
The root is thought to have many human health benefits,
such as anti-fatigue, anti-cancer, and anti-oxidant properties
Jung-Woo Lee, Ick-Hyun Jo have contributed equal to the work.
* Young‑Doo Park
ydpark@khu.ac.kr
1
Department of Horticultural Biotechnology, Kyung Hee
University, Yongin 17104, Republic of Korea
2
Department of Herbal Crop Research, National Institute
of Horticultural and Herbal Science (NIHHS), Rural
Development Administration (RDA), Eumseong 27709,
Republic of Korea
tant process for most plants that reproduce via seeds. Korean
ginseng is propagated by seeds and requires 3–4 years to
produce mature seeds from the mother plants. Therefore,
the reproduction rate of ginseng is very slow under natural
life cycle conditions. Another challenge associated with the
large-scale culture of ginseng seeds is their complicated dor-
mant characteristic of “triple dormancy”. Seed dormancy is
defined as a pause prior to the completion of germination
even in a favorable environment (Finch-Savage and Leub-
ner-Metzger 2006). There are various types of dormancy,
which include physical, physiological, morphological, and
morpho-physiological dormancy (Baskin and Baskin 2004).
A ginseng seed separated from the sarcocarps are sur-
rounded by a hard and impermeable seed coat that acts
as a mechanical inhibitor that must be overcome by the
embryo growth potential. It takes approximately 21 months
to achieve complete germination owing to delayed embryo
development once the seeds naturally detach from their

13
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294
mother plants. To shorten this period, the seeds can be
subjected to warm stratification known as dehiscence, in
which they are placed at 15–20 °C in wet sand for more than
90 days with periodic watering. In a completely dehisced
seed, the zygotic embryo is still immature and additive
embryo development is required to overcome the state of
morphological dormancy. Moreover, physical dormancy pre-
vents the seeds from germinating at 25 °C, even with a fully
matured embryo; therefore, the seeds also require a chilling
treatment known as cold stratification for over 90 days in
at 4 °C or exposure to fluctuating temperature for 75 days
outdoors (Kwon et al. 2001).
Due to the long time required for seed setting and the con-
sequent high price of ginseng seeds, new seed dehiscence
and germination methods are needed to enhance ginseng
production at a low cost. In addition, strategies to break
early dormancy would help to reduce the breeding cycle
period. Numerous studies have been carried out to improve
dehiscence, germination, and breaking dormancy using
treatments with exogenous plant growth regulators (PGRs).
Among many PGRs, ­GA3 is known to play an important role
in early embryonic development as a plant growth regulator
(Alpi et al. 1975; Picciarelli et al. 1984) that participates
in breaking dormancy and improving germination (Finch-
Savage and Leubner-Metzger 2006; Toh et al. 2008). Specifi-
cally, ­GA3 has been shown to increase the germination rate
and shorten the germination period in P. ginseng (Son and
Reuther 1977). Cytokinins are essential for plant growth and
are known to be involved in cell differentiation, migration
of inorganic and organic nutrients, and aging (Skoog and
Armstrong 1970). Some studies have also shown that plant
growth substances related to cytokinin activity could affect
seed germination and breaking dormancy in some plant spe-
cies (Prasad et al. 1983; Kabar 1998). In particular, kinetin
alone was able to break seed dormancy (Cohn and Butera
1982). Park et al. (1979) showed that kinetin stimulated bud
emergence and the breaking of bud dormancy in 1-year-old
ginseng. Although ­GA3 treatment was reported to improve
the seed dehiscence rate (Kim et al. 2014), germinating char-
acteristics (Lee et al. 1986) and breaking dormancy (Lee
et al. 2016), Kwon et al. (1986) showed that cytokinins had
a greater effect than ­GA3 on improving germination and
breaking dormancy in P. ginseng. Therefore, further study
is required to evaluate the precise and relative effects of G
­ A3
and cytokinins on ginseng seeds. In this study, we investi-
gated the effects of stratification, G
­ A3 and/or kinetin treat-
ments on seed dehiscence, development and germination in
Korean ginseng. We also evaluated the synergistic effect of
these two PGRs.
13
Horticulture, Environment, and Biotechnology (2018) 59:293–301
2 Materials and methods
2.1 Seed material
Ginseng berries (Panax ginseng C.A. Meyer) were har-
vested at complete maturation from a research field at the
Department of Ginseng Research, National Institute of Hor-
ticultural and Herbal Science (NIHHS), Rural development
administration (RDA), Eumseong, Korea (latitude 36°94,
longitude 127°75) in July 2016. To completely remove the
sarcocarp from the seeds, the berries were rubbed several
times by hand, washed with running water for 24 h, and then
dried in the shade.
2.2 GA3 and kinetin treatments
Indehiscent seeds for the dehiscence test and dehiscent
seeds for the germination test were immersed in a solution
containing 288.7 μM ­GA3 or 232.3 μM kinetin for 24 h,
respectively, at 25 °C. To confirm the potential effect of the
combination of ­GA3 and kinetin, the seeds were immersed in
a solution containing 288.7 μM G ­ A3 + 232.3 μM kinetin. As
a control, the seeds were immersed in distilled water ­(dH2O).
2.3 Dehiscence test
After 24 h PGRs treatment, the immersed seeds were mixed
with sand at a volume ratio of 3:1 and then stacked in gravel
and sand for 90 days. Other management practices includ-
ing the times and volume of periodic watering, complied
with the good agricultural practices (GAP) standard culti-
vation guide (RDA 2012). The dehiscence rate of the seeds
was examined observing whether or not the seed coats were
splited from each other (Fig. 1a, b). The dehiscence test was
carried out in three replicates with 150 seeds each.
2.4 Cold stratification
A total of 100 dehiscent seeds per treatment group were
placed in a plastic bag and incubated at 2 °C for 15, 30, 45,
60, and 90 days in the dark. The seeds in the control group
were sowed immediately without cold stratification. Three
replicates were conducted per treatment.
2.5 Germination test
The treated seeds were sown according to the end date of each
cold stratification. 10 seeds from each group were set aside
to investigate embryo and endosperm length, and the other
90 seeds were placed in 100 × 20 cm petri dishes containing
25 g of peat and perlite (7:3, v:v) with 55 mL distilled water
Horticulture, Environment, and Biotechnology (2018) 59:293–301 295
Fig. 1  Seeds of Panax ginseng C.A. Meyer. a Indehiscent seed. b
Dehiscent seed after 90  days cold stratification. c Sowed seeds in a
petri-dish. d Emerged radicle after 232.3  μM treatment and 15  days
cold stratification. e Cross and f longitudinal sections of an immature
(Fig. 1c). Each petri dish was then incubated in a plant growth
chamber kept in the dark at 20 °C for 24 h (VS-1203PFC-L,
Vision Scientific Co., Korea). Three replicates were conducted
for each treatment. The seeds were considered to be germi-
nated when the radicle had emerged from the seed covering
layers by more than 1 mm (Fig. 1d). The seeds were observed
daily for 50 days to obtain cumulative germination data for
each treatment. At the end of the incubation period, the ger-
mination percentage (GP) and mean germination time (MGT)
were calculated using the following equations:
n GI =
( )
GP(%) = × 100
N
MGT(days) = 𝜋
∑ (ni × ti)
n nized and considered to be ungerminted.
where n is the number of germinated seeds at the end of the
incubation period, N is the total number of sowed seeds,
and ni is the number of germinated seeds on survey day ti .
Time to 50% germination (­ T50) was calculated according to
the following equations described by Farooq et al. (2005):
( )(
N )
− n i tj − ti
2
T50 (days) = ti +
nj − ni
seed after cold stratification. Sc seed coat, Ra radical, En endosperm,
Em embryo, Co cotyledons, Hy-Ra hypocotyl-radical. Scale bar,
1 mm
where N is the final number of germinated seeds, ni and nj
are the cumulative number of seeds germinated by adjacent
seed count at times ti and tj , respectively, when ni < N/2 < nj.
The germination performance index (GPI) indicates the
degree of germination uniformity and was calculated with the
following equation described by Sundstrom et al. (1987):
GPI = GP × MGT
The germination index (GI), indicating the degree of seed
vitality, was calculated as:
∑ ni
ti
where ni is the number of germinated seeds on survey day ti .
Damaged seeds were removed as soon as they were recog-
2.6 Embryo‑to‑endosperm length ratio
Morphological characteristics such as embryo and endosperm
length were measured in each treatment group at intervals of
15 d for a total of 90 d using a light microscope (S8AP0, Leica,
Germany) after cutting each of the 10 seeds per group in half
(Fig. 1e). The embryo-to-endosperm length ratio was calcu-
lated using the following equation:
embryo length
Embryo−to−endosperm length ratio =
endosperm length
13

296
The ratio was considered to be ‘1’ when the embryo
length was longer than the endosperm length, which was
due to germination occurring before sowing. Measurements
were taken from seeds in each treatment group, with three
replicates per treatment.
2.7 Statistical analysis
All experiments were conducted using a completely rand-
omized design. When two subjects were compared, the t test
was used. The two-sample t-test was performed if variance
equality was proven; otherwise, the Welch two-sample t-test
was conducted. Multivariate data were subjected to one-way
analysis of variance followed by Duncan’s multiple range
test. Statistical analyses were performed using R version
3.3.2 (The R Foundation for Statistical Computing, Vienna,
Austria) software and assessed at a 0.05 level of probability.
3 Results and discussion
3.1 Effects of ­GA3 and/or kinetin treatment
on the seed dehiscence rate
Indehiscent seeds soaked with G ­ A3 and/or kinetin were
stacked in gravel and sand for 90 days and then observed
to determine whether the seed coat was splited (Fig. 1a, b).
The ­GA3 and/or kinetin treatments significantly affected the
dehiscence rate of ginseng seeds (Table 1). The percentage
of seed dehiscence was 94.5 ± 0.08% with 288.7 μM G ­ A3
treatment, representing a significant induction of dehiscence
compared to the seeds exposed to warm stratification alone
(90.6 ± 1.49%). In addition, 232.3 μM kinetin treatment
significantly improved the dehiscence rate (92.8 ± 0.54%).
These results suggest that ­GA3 has a more substantial effect
on improving the dehiscence rate in P. ginseng than kine-
tin. The highest dehiscence (96.2 ± 0.66%) was detected in
seeds treated with the combination of both 288.7 μM G ­ A3
and 232.3 μM kinetin, indicating that the combined treat-
ment with these PGRs was more effective than their separate
applications in the induction of dehiscence.
The dehiscence of ginseng seed is an essential process
required to overcome mechanical dormancy. After 90 days
of warm stratification, both the embryo and endosperm grow
Table 1  Effects of G
­ A3 and/or kinetin on dehiscence after 90 d of warm stratification in P.ginseng
Type Control (0 μM) GA3 (288.7 μM)
Seed dehiscence (%) 90.6 ± 1.49y ­dx 94.6 ± 0.08 b
z
 Significance was evaluated for the same stratification period: ns, *, **, *** Non-significant or significant at p < 0.05, 0.01, or 0.001, respectively
y
 Values indicate the mean ± standard error (n = 150)
x
 Different letters within rows indicate significant differences (p < 0.05)
13
Horticulture, Environment, and Biotechnology (2018) 59:293–301
considerably, which are potential components of overcoming
mechanical inhibitors of the dehiscence. Only a few studies
have reported the use of chemical treatments that improve
the dehiscence of ginseng seed. Yang et al. (1982) described
that 288.7 μM G ­ A3 + 378.33 μM abscisic acid (ABA) could
significantly increase ginseng seed dehiscence compared
with warm stratification alone; however, this result is contra-
dictory to the results showed that a single ­GA3 treatment was
actually the best for improving dehiscence. Kim et al. (2014)
reported that the dehiscence rate of ginseng seeds increased
with an increasing ­GA3 concentration up to 866.1 μM but
that treatment with 288.7 μM G ­ A3 was sufficient to show a
statistically significant improvement. Similarly, the dehis-
cence of Eleutherococcus senticosus Maxim, which is
also in the Araliaceae family, was positively influenced by
soaking seeds in ­GA3 before warm stratification (Lim et al.
2008). However, to our knowledge, this is the first report to
demonstrate a synergistic effect of G ­ A3 and kinetin on the
dehiscence rate of ginseng seeds.
3.2 Effects of cold stratification on seed
development and germination
The developmental characteristics of the dehiscent seeds
subjected to cold stratification were examined by determin-
ing the embryo-to-endosperm length ratio. Immediately
after dehiscence, the zygotic embryo was still immature;
the embryo grew gradually throughout the cold stratification
duration and then growth rapidly increased between 30 and
60 days (Fig. 2a). It was assumed that these periods repre-
sented a key breaking point in the morphological dormancy,
which is defined as an embryo that is undeveloped in size
but still differentiated into cotyledons and the hypocotyl-
radical (Fig. 1e). After 60 days in the 2 °C cold chamber, the
average embryo-to-endosperm length ratio of the seeds was
0.84, indicating that seed development was almost complete.
The germination characteristics of the seeds were inves-
tigated according to the cold stratification through compari-
son of the GP, MGT, T ­ 50, GPI, and GI values. The seeds
treated with cold stratification alone did not germinate at all
until 60 days, and the initial seed germination was observed
after 90 days of cold stratification. However, the germination
characteristics were generally unfavorable (Table 3) because
of the physiological dormancy, which is divided into three
 
Kinetin (232.3 μM) GA3 + Kinetin (288.7 + 232.3 μM) Significancez
92.8 ± 0.54 c 96.2 ± 0.66 a ***
Horticulture, Environment, and Biotechnology (2018) 59:293–301 297
Fig. 2  Development of ginseng seeds according to cold stratification
duration and PGR treatment. a Cold stratification alone. b ­GA3 treat-
ment with cold stratification. c Kinetin treatment with cold stratifi-
levels depending on the dormant depth (deep, intermediate,
and non-deep) (Baskin and Baskin 2004).
The duration of cold stratification is an important factor
for promoting seed germination (Baskin et al. 2001) and to
relieve the dormancy of many plant species (Conner 2008;
Tang et al. 2008). In this study, cold stratification for 90 days
was not enough for effectively breaking dormancy and most
seeds germinated only at the end of the cold stratification of
120 days (data not shown).
3.3 Effects of cold stratification and ­GA3 treatment
on seed development and germination
Similar to the cold stratification results (Fig. 2a), the embryo-
to-endosperm length ratio of ginseng seeds gradually
increased with exposure to cold stratification and 288.7 μM
­GA3 treatment, although the size of zygotic embryos was
affected by the small endosperm (Fig. 2b). After 45 days
of cold stratification, the embryo-to-endosperm length ratio
was 0.80, suggesting that seed development was almost
cation. d ­GA3 + kinetin treatments with cold stratification. The bars
indicate standard deviations from the means of three replicates per
treatment (n = 30)
complete. After 90  days of cold stratification, the ratio
reached 0.92, which was higher than that obtained with the
cold stratification alone (Table 2).
After treatment with 288.7 μM ­GA3, seed germination
was first observed at 45 days of cold stratification, but the
GP was very low (2.1%) (Fig. 3). The GP was still only
3.8% after 60 d of cold stratification, and the other germina-
tion characteristics (MGT, ­T50, GPI, and GI) showed also
unfavorable low figures (Table 3). However, after 90 days
of cold stratification, the seed GP was 95.0%, MGT was 2.3,
­T50 was 1.3, GPI was 0.41, and GI was 55.11, representing
significant stimulation in development compared to seeds
exposed to only cold stratification alone.
Although there is no statistically significant difference,
­GA3 might have a slight effect on embryo development
because the embryo-to-endosperm length ratio was higher
than cold stratification alone at all periods. Nevertheless,
this effect of G
­ A3 treatment was not sufficient for replacing
the cold stratification requirement to break dormancy and
promote germination in P. ginseng. These results suggest
13

298 Horticulture, Environment, and Biotechnology (2018) 59:293–301

Table 2  Embryo-to-endosperm length ratio following ­GA3 and/or kinetin treatment with cold stratification duration
Cold Type Control (0 μM) GA3 (288.7 μM) Kinetin (232.3 μM) GA3 + Kinetin Significancez
stratification (288.7 + 232.3 μM)
(days)

15 Embryo-to-endosperm length ratio 0.70 ± 0.01y ­ax 0.71 ± 0.03 a 0.73 ± 0.03 a 0.73 ± 0.03 a ns

30 0.70 ± 0.01 b 0.74 ± 0.03 ab 0.76 ± 0.02 a 0.78 ± 0.02 a *
45 0.76 ± 0.01 a 0.80 ± 0.01 a 0.80 ± 0.04 a 0.80 ± 0.02 a ns
60 0.82 ± 0.01 a 0.84 ± 0.01 a 0.85 ± 0.03 a 0.84 ± 0.03 a ns
75 0.86 ± 0.02 a 0.87 ± 0.02 a 0.92 ± 0.04 a 0.92 ± 0.04 a ns
90 0.86 ± 0.01 c 0.92 ± 0.02 b 0.97 ± 0.03 a 0.99 ± 0.02 a ***
z
 Significance was evaluated for the same stratification period: ns, *, **, *** Non-significant or significant at p < 0.05, 0.01, or 0.001, respectively
y
 Values indicate mean ± standard error (n = 10)
x
 Different letters within columns indicate significant differences (p < 0.05)

Fig. 3  Germination of ginseng seeds treated with ­GA3 and/or kinetin according to cold stratification duration. The bars indicate standard devia-
tions from the means of three replicates per treatment (n = 90)

that ginseng seeds undergo a state of deep physiological
dormancy such that several months of cold stratification are
necessary for an effective breaking dormancy and successful
germination.
3.4 Effects of cold stratification and kinetin
treatment on seed development
and germination
Similar to the results from the ­GA3 treatment group, seeds
exposed to cold stratification and 232.3 μM kinetin treatment
had a significant increase in embryo development (embryo-
to-endosperm length ratio) with increasing cold stratifica-
tion (Fig. 2c). The embryo length gradually increased with
prolonged cold stratification, and the embryo-to-endosperm
length ratio was 0.80 when the seeds were cold stratified for
45 days, which was similar to the observation made in the
­GA3 treatment group; however, the ratio reached 0.97 after
90 d of cold stratification, which is higher than that detected
with the ­GA3 treatment (Table 2).
The GP was 2.1% with 232.3 μM kinetin treatment with-
out cold stratification, which was too low to evaluate ger-
mination characteristics (Table 3). The GP increased from
3.3% at 15 days to 87.5% at 60 days with a gradual increase
in the cold stratification duration (Fig. 3). Most of the seeds
germinated after 90 d of cold stratification (97.9%). GI and
GPI are also increased as the cold stratification duration
increased, whereas MGT gradually decreased throughout
the cold stratification duration. Compared to the MGT,
which was 0.24 at 90 days of cold stratification, most of the
seeds had already germinated before they were sown on the
petri dish. Thus, kinetin clearly had a significant effect on
promoting germination and breaking dormancy in P. gin-
seng, although a short period of cold stratification was still
required.
Overall, the embryo-to-endosperm length ratio of kinetin-
treated seeds was higher than that of ­GA3-treated seeds at
most stratification periods (except at 45 days). Furthermore,
kinetin treatment significantly improved germination char-
acteristics of seeds more than ­GA3 treatment for the same
period, suggesting that exogenous kinetin was more effective

13
Horticulture, Environment, and Biotechnology (2018) 59:293–301 299

Table 3  Germination Cold stratifica- Treatment GPz MGT T50 GPI GI

characteristics of ginseng tion (days)
seeds after G­ A3 and/or
kinetin treatments during cold 0 Control (0 μM) 0y – – – –
stratification duration
GA3 (288.7 μM) 0 − − − −
Kinetin (232.3 μM) 2.1 43.8 38.5 0.0005 0.04
GA3 + Kinetin (288.7 + 232.3 μM) 5 42.4 40.1 0.0011 0.10
Significancex ns ns ns ns ns
15 Control (0 μM) 0 – – – –
GA3 (288.7 μM) 0 – – – –
Kinetin (232.3 μM) 3.3 36.2 32.2 0.0010 0.08
GA3 + Kinetin (288.7 + 232.3 μM) 25.8 36.3 35.4 0.0071 0.61
Significance ** ns ns ** **
30 Control (0 μM) 0 – – – –
GA3 (288.7 μM) 0 – – – –
Kinetin (232.3 μM) 24.6 32.8 29.8 0.007 0.68
GA3 + Kinetin (288.7 + 232.3 μM) 36.3 34.0 32.9 0.011 0.96
Significance ** ns ns ** *
45 Control (0 μM) 0 – – –
GA3 (288.7 μM) 2.1 b 12.0 b 12.5 b 0.002 c 0.15 c
Kinetin (232.3 μM) 72.9 a 21.2 a 21.3 a 0.035 b 5.51 b
GA3 + Kinetin (288.7 + 232.3 μM) 77.9 a 16.3 ab 13.7 b 0.048 a 8.83 a
Significance *** * ** *** ***
60 Control (0 μM) 0 – – – –
GA3 (288.7 μM) 3.8 b 4.1 b 2.0 b 0.01 c 1.02 c
Kinetin (232.3 μM) 87.5 a 8.7 a 2.4 a 0.10 b 27.04 b
GA3 + Kinetin (288.7 + 232.3 μM) 92.5 a 3.8 b 1.7 b 0.24 a 41.33 a
Significance *** ** ** *** ***
90 Control (0 μM) 17.5 b 3.4 a 2.0 a 0.05 c 5.39 c
GA3 (288.7 μM) 95.0 a 2.3 b 1.3 b 0.41 c 55.11 b
Kinetin (232.3 μM) 97.9 a 0.2 c 0.0 c 4.48 b 76.28 a
GA3 + Kinetin (288.7 + 232.3 μM) 98.3 a 0.1 c 0.0 c 9.19 a 77.50 a
Significance *** *** *** *** ***
z
 GP germination percentage, MGT mean germination time, T50 time to 50% germination, GPI germination
performance index, GI germination index
y
 No germination observed
x
 Statistical significance determined within the same stratification period: ns, *, **, *** Non-significant or
significant at p < 0.05, 0.01, or 0.001, respectively

for zygotic embryo development and germination than exog-
enous ­GA3.
3.5 Effects of cold stratification and a combined ­GA3
and kinetin treatment on seed development
and germination
Similar to the each PGR treatments, a prolonged cold strati-
fication significantly increased the embryo-to-endosperm
length ratio in seeds treated with 288.7 μM ­GA3 + 232.3 μM
kinetin (Fig. 2d). The ratio was 0.80 at 45 days of cold strati-
fication, as observed in the group treated with kinetin alone.
The maximum embryo-to-endosperm length ratio (0.99) was
observed for the G­ A3 + kinetin treatment group after 90 days
of cold stratification with no significant difference obtained
with kinetin treatment alone.
The GP of the G ­ A3 + kinetin-treated seeds was 5.0% with-
out cold stratification, but the other germination characteristics
such as MGT, T ­ 50, GI, and GPI were too low to evaluate those
(Table 3), similar to the group treated with kinetin only. After
15–30 days of cold stratification, the GP increased beyond
those observed in the other treatments at the same time points
(Fig. 3). However, after 45 days of cold stratification, there
was no significant difference in the GP compared to that of
the kinetin treatment alone. The maximum GP (98.3%) was
observed in the ­GA3 + kinetin treatment groups after 90 days
of cold stratification. MGT and T ­ 50 decreased with prolonged
cold stratification. GI and GPI improved to a significant extent

13

300
(p < 0.05) with the combined PGR treatment than obtained
with kinetin alone when combined with up to 60 days of cold
stratification.
Our results showed that the combination treatment of G ­ A3
and kinetin had a synergistic effect on cold stratified ginseng
seed germination. A previous report showed that the combi-
nation of ­GA3 and kinetin improved the germination rate and
alleviated salt stress and thermodormancy-induced seed ger-
mination inhibition in lettuce seeds (Kabar and Baltepe 1990).
Similarly, the combination of ­GA3 and cytokinin benzylami-
nopurine (BA) improved the number and length of sprouts of
potato tubers more than that achieved with the treatment of
each PGR alone (Njogu et al. 2015). Furthermore, the com-
bined treatment of ­GA3 and kinetin accelerated the conver-
sion of embryos into plantlets in Citrullus colocynthis during
in vitro differentiation (Ramakrishna and Shasthree 2016);
however, to our knowledge, this is the first report of a syner-
gistic effect of ­GA3 and kinetin on ginseng germination and
development.
4 Conclusion
In this study, we identified that both ­GA3 and kinetin improved
the dehiscence, embryo development, and germination of P.
ginseng. ­GA3 treatment significantly increased the ginseng
dehiscence rate and made a slight contribution to breaking dor-
mancy considering as germination characteristics was higher
than cold stratification alone after 45 days of cold stratification.
Kinetin treatment had a positive effect on dehiscence, although
not as much as ­GA3 treatment. Also, kinetin had a greater
effect on promoting germination and breaking dormancy than
­GA3. When used in combination, ­GA3 and kinetin treatments
were more effective than each PGR used alone in promoting
both seed dehiscence and germination. However, no combina-
tion of PGR treatments could completely substitute for the cold
stratification requirement to break dormancy and promote ger-
mination in P. ginseng due to its intrinsic deep physiological
dormancy. These results aim to help improve the understand-
ing of the respective and combined roles of stratification, G ­ A3,
and kinetin on seed dehiscence, development, and germination
in P. ginseng and enhance seed production in an economical
manner.
Acknowledgements  This work was carried out with the support of the
Cooperative Research Program for Agriculture Science and Technol-
ogy Development (Project No. PJ01267901) of the Rural Development
Administration, Republic of Korea.
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