全 文 ：Journal of Forestry Research (2009) 20(1):27−30
DOI 10.1007/s11676-009-0005-3





Variations in seed germination of Hippophae salicifolia with different pre-
soaking treatments

S. Airi, I.D. Bhatt *, A. Bhatt, R.S. Rawal, U. Dhar

G.B. Pant Institute of Himalayan Environment and Development, Kosi- Katarmal, Almora - 263 643 (UK), India

Abstract: Mature seeds of H. salicifolia, collected from five provenances (i.e. Hanuman Chatti, Helang, Lata, Rambara and Janggal Chatti)
in west Himalaya, India, were treated with stratification (at 4ºC for 15, 30 and 60 days) and in different concentrations of GA3 (5, 10, 20
mM), KNO3 (50, 100, 200 mM) and Thiourea (50, 100, 200 mM) solution to determine the variations in seed germination. Results reveal that
the germination rates of seeds from different provenances under different pre-sowing treatments are significantly increased compared to
those in control (24%–30%). The seeds treated with Thiourea (100 mM) have highest germination rate (76%–83% for different seed
sources), followed by those (63%–71% for different seed sources) pretreated with stratification (4oC, 30 days). GA3 treatment significantly
shortens the mean germination time (MGT) and improves seed germination percentage. Considering the practical applicability and cost ef-
fectiveness, thiourea (100 mM) and stratification (at 4oC) treatments for seed germination are recommended for mass multiplication through
seeds of H. salicifolia in village/forest nurseries of the west Himalaya, India.
Keywords: Seed germination percentage; Hippophae salicifolia; presoaking treatment; stratification; Thiourea



Introduction

Hippophae salicifolia D. Don (Vernacular- Chuk, Tarwa) is a
deciduous tree species restricted to the Himalayan region, be-
tween 1500−3500 m a.s.l. (Hooker 1990; Gaur 1999). The spe-
cies bear red and yellow berries, which are edible, a rich source
of vitamins, and used in preparations of various products includ-
ing local beverages (Gaur 1999). The flavanoids, fatty acids and
other bio-active compounds of H. salicifolia berries might be
capable of reducing the incidence of cancer (Mathews 1994).
The species is also considered as fine vegetation in improving
soil fertility and restoring degraded sites in high hills (Gamble
1972; Huxley 1992).
H. salicifolia generally propagates by root suckers. However,
extensive collection and short growing season can hamper mass
multiplication of the species. The natural regeneration of the
species through seed is also very scanty (Sankhyan et al. 2005).
Sankhyan et al. (2005) studied the seed germination of H. salici-

Received: 2008-05-29; Accepted: 2008-07-24
© Northeast Forestry University and Springer-Verlag 2009
The online version is available at http://www.springerlink.com
Biography: S. Airi (1966), male, Masters in Botany, Technician –B in
G.B. Pant Institute of Himalayan Environment and Development, Kosi-
Katarmal, Almora - 263 643 (UK), India.
(Email: airisubodh@rediffmail.com)
*Corresponding author: Bhatt I.D. (Email: idbhatt@gbpihed.nic.in;
id_bhatt@yahoo.com)
Responsible editor: Zhu Hong

folia collected from single population. However, to achieve
higher productivity and select suitable genotypes for future
breeding programmes, seed source testing is important (Mamo et
al. 2005). Therefore, in the present study we designed several
presoaking treatments for seeds of H. salicifolia collected from
different provenances, aimed at achieving a high germination
percentage and short mean germination time and assessing the
variation in seed germination among populations.

Methods

Mature seeds of H. salicifolia were collected during first and
second week of November (2004) from five provenances (Ha-
numan Chatti, Helang, Lata, Rambara and Janggal Chatti) in
west Himalaya, India (Table 1). Provenances were selected on
the basis of (1) distant localities at diverse altitude (1715–2965 m
a.s.l.), (2) association with diverse forest communities, and (3)
substantially large population with mature individuals. Five ap-
parently healthy mature trees located inside the population in
each provenance were identified for collection of seeds. Mar-
ginal plants were avoided. All the seeds collected were ensured
to be apparently at same stage of development and the infected
seeds were discarded. Immediately after collection, seeds were
cleaned manually, sunned to be dried for seven days, and stored
in brown paper bags at (25±2)ºC until the start of experiments.
Seed viability was tested using 1% (w/v) solution of Tetra-
zolium chloride TTC (2, 3, 5 triphenyl tetrazolium chloride,
C19H15N4Cl, Sigma) with pH adjusted at 6.0 (Hendry and Grime
1993). Fifty dry seeds from each provenance were soaked in 1%
aqueous solution of TTC for 24 h and kept in dark at room tem-
perature. Seeds were bisected longitudinally and examined under
RESEARCH PAPER
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a microscope to determine viability percentage. Seeds with a
red-stained embryo were considered to be viable.
Various pre-sowing experiments were performed to determine
the effects of different treatments on seed germination percent-
age and mean germination time (MGT). A set of seeds without
pre-sowing treatments was considered as control. Every treat-
ment with three replicates of 60 seeds was arranged from indi-
vidual provenance.

Table 1. Site characteristics of identified various provenances of H. salicifolia
Various provenances Altitude
(m, asl)
Aspect Seed weight
(g) n=10
Latitude Longitude Seed moisture
content (%)
Dominant associated tree species
Helang (P1) 1715 NE 0.14 (±0.010) 30º34′ 79º34′ 27 Alnus nepalensis
Lata (P2) 2310 NW 0.15 (±0.009) 30º29′ 79º44′ 31 Alnus nepalensis, Picea smithiana
Hanuman Chatti (P3) 2600 NE 0.16 (±0.014) 30º43′ 79º17′ 34 Alnus nepalensis, Abies pindrow, Taxus baccata
Rambara (P4) 2820 NW 0.13 (±0.004) 30º44′ 79º03′ 29 Betula utilis, Abies pindrow
Jangal Chatti (P5) 2965 NE 0.14 (±0.005) 30º35′ 79º30′ 33 Alnus nepalensis
Notes: Values in parenthesis represent standard deviation

Selected seeds were surface-sterilized by dipping in 0.5%
aqueous solution of mercuric chloride for 2 min to remove bacte-
rial and fungal contamination and then rinsed thoroughly (four
times) in distilled water. The sterilized seeds were soaked for 24
h in different concentration of GA3 (5, 10, 20 mM), KNO3 (50,
100, 200mM) and Thiourea (50, 100, 200mM) solutions. For the
cold stratification, seeds were kept at 4 ºC for 15, 30 and 60 days
and then placed at (25±2) ºC. A set of untreated seeds were used
for comparing the results. Treated seeds were washed thoroughly
with distilled water and placed in Petri plates (95 mm ×17mm)
containing moistened filter paper (Whattman No.1). Petri plates
were kept at 25±2ºC in growth chamber and moistened as needed
with distilled water. The date of first germination was recorded
for each treatment. The seed with emergence of radicle (>5 mm
long) was considered as seed germinated. The number of seeds
infected by fungi and bacteria was recorded each day and re-
moved from Petri plates. The whole experiment was monitored
up to 90 days. MGT was calculated as:

MGT = ∑(nd) / N (1)

where, n is the number of seed germination after each incubation
period in days d, and N is the total number of seeds germination
at the end of test (Hartmann and Kester 1989).

Statistical analysis

All the experiments were conducted in a completely randomized
block design. Analysis of variance (ANOVA) was performed
with the help of SYSTAT (Wilkinson 1986). Data on germina-
tion percentage were subjected to arcsine transformation before
being considered for ANOVA. Fishers Least Significant Differ-
ence (F-LSD) was estimated separately for the comparison of
treatment and provenance means (Snedecor et al. 1967).

Results

The viability pattern of the seeds stained by Tetrazolium test
shows that there is no significant difference in viability of the
seeds from different provenances (Fig.1). Invariably high
(84%−92%) viability of H. salicifolia seeds indicates that seeds
would largely be capable of germination under favorable condi-
tions and that failure of germination implies dormancy of seeds.
The seed germination percentage (Fig. 1 and Table 2) for un-
treated seeds (control set) is low (i.e. 24.7% to 30.7%). The per-
centage of seed germination, however, is significantly higher
(p<0.05) from two provenances (P2 and P5) compared to others.
Under various pre-sowing experiments, seeds from different
provenances do not exhibit uniformity in responses (Table 2, 3).


Fig. 1 Percentage of seed viability and seed germination in control
set of Hippophae salicifoliaa
P1----Hanuman Chatti, P2----Helang, P3----Lata, P4----Rambara,
P5----Janggal Chatti

Under all the pre-sowing treatments, cold stratification and
Thiourea treatments show maximum improvement in mean per-
centage of seed germination over control (Table 2). In case of
cold stratification, seed germination percentage is maximum
(58%−71.3%) under 30-day stratification. Considering the re-
sponses across provenances, significantly (p<0.05) more germi-
nation of seeds is revealed from P5 (71.3%) and significantly less
(58.0%) from P4 provenance. In case of Thiourea, seed germina-
tion percentage increases with increase in Thiourea concentration
up to 100mM. Further increase in concentration of Thiourea
significantly (p<0.05) reduces the germination percentage in
most cases. The highest mean seed germination (83.3%- Helang
(P1)) under 100-mM thiourea treatment is significantly (p<0.05)
better than that of P4 and P5 provenances. As compared to cold
Journal of Forestry Research (2009) 20(1):27−30

29
stratification and thiourea, seeds soaked in GA3 (different Conc.)
resulted in lesser improvement in germination. Responses under
KNO3 treatment are comparable with GA3 treated seeds.

Table 2. Seed germination percentage of Hippophae salicifolia from different provenances and under different pretreatments
Seed germination rate (%)
Treatments
Helang (P1) Lata (P2) Hanuman Chatti (P3) Rambara (P4) Jangal Chatti (P5)
LSD (p<0.05) F-ratio
Control 27.34 35.34 25.33 24.66 30.66 4.50 9.46**
Stratification
15 days 44.00 62.00 54.67 50.66 55.33 4.60 20.52**
30 days 63.34 65.00 66.00 58.00 71.33 2.16 10.30**
60 days 55.34 61.67 58.67 56.00 60.66 3.85 5.18*
Thiourea
50mM 72.00 68.66 72.66 68.66 73.00 3.54 3.68*
100mM 83.33 81.34 80.00 77.33 76.66 5.54 2.49ns
200mM 62.66 62.66 78.00 74.44 70.00 3.95 30.24**
GA3
5mM 38.66 40.66 33.33 42.66 43.33 4.12 9.43**
10mM 38.00 36.00 26.00 56.33 46.66 4.79 57.98**
20mM 38.66 34.66 33.33 45.33 49.33 6.28 11.93**
KNO3
50mM 47.34 46.00 44.00 29.33 51.33 2.82 88.50**
100mM 66.00 64.66 64.67 31.33 68.66 5.71 73.89**
200mM 54.00 53.34 49.33 32.33 56.00 5.63 28.96**
LSD (p<0.05) 7.20 4.35 4.53 2.39 4.82
F-ratio 41.61** 99.82** 152.52** 332.64** 67.75**
Notes:* p<0.05, **p<0.01, ns : non significant.

The best responses were observed for KNO3 (100 mM). Fur-
ther increase in concentration of KNO3 (>100 mM) lowered the
mean seed germination significantly in most cases (except P4
provenance).
The mean seed germination time (MGT) for untreated seeds
(control set T1) across provenances varied from 15.2d to 19.5d.
Compared to control, all the treatments (except GA3 (10 mM)),
caused increase in mean germination time (Table 3).

Table 3. Effect of best pre-sowing treatment on mean germination
time (MGT) of Hippophae salicifolia seeds
Mean germination time of seed (d) Seed
source T1 T2 T3 T4 T5
Mean
P1 15.18 21.84 14.70 20.95 16.80 17.89
P2 18.27 23.81 13.86 22.49 17.47 19.18
P3 15.63 22.33 14.46 18.10 16.31 17.35
P4 18.96 24.67 13.36 18.28 17.67 18.59
P5 19.49 25.31 10.99 20.01 19.32 18.98
Mean 17.50 23.58 13.47 19.97 17.51
Notes: LSD (p<0.05) for treatment and seed source mean 2.09; 2.89.T1 –
Control; T2- Cold Stratification (30 days); T3-GA3 (10 mM); T4-KnO3 (100
mM) and T5- Thiourea (100 mM).

Discussion

Variability in seed germination ability for several species has
been reported among seed sources in different regions (Tewari
and Dhar 1996; Siril et al. 1998). Causes of such variations are
attributed to (1) genetic characters of different populations/plants
(Bewley and Black 1994) or (2) impact of mother plant envi-
ronment (Anderson and Milberg 1998), or (3) wider habitat con-
ditions (Friis 1992), or (4) diverse altitudinal gradients (Rawat et
al. 2007). These factors could be considered as important factors
affecting germination characteristics of H. salicifolia.
The high seed viability percentage (84%−92%) and poor ger-
mination (24.7%−30.7%) of untreated seeds (Fig. 1) would sug-
gest prevalence of dormancy among seeds of H. salicifolia. As
such, the members of family Elaegnaceae mostly have the fruits
with stony endocarp (Baskin et al. 1998) and embryo having
deep physiological dormancy, which require cold stratification in
long periods to overcome dormancy (Nikolaeva 1969). The Im-
proved germination responses under cold stratification for H.
salicifolia in our study are agreed with these reports.
Our study result indicates that seed germination of H. salicifo-
lia can be improved significantly through various pre-sowing
treatments. Thiourea and cold stratification treatments are proven
to be superior over GA3 and KNO3 treatments for seed germina-
tion of H. salicifolia. Improved seed germination under thiourea
treatment is in agreement with reports of many other species
(Chaudhary et al. 1996; Mclntyre et al. 1996; Pandy et al. 2000).
Likewise, improvement of germination in stratification at 4ºC is
in agreement with the report of Sankhyan et al. (2005) who re-
ported 53% germination in the cold water (2 d). However, strati-
fication at longer duration (30 d) had seed germination of
58%−71.33%. It is found that stratification at 2−5ºC for different
time duration is effective in number of other species for seed
germination (Stokes 1969, Khan et al. 1969) including H. rham-
Journal of Forestry Research (2009) 20(1):27−30

30
noides (Pearson et al. 1962).
The improved seed germination rates under Thiourea and cold
stratification treatments reveal that both the pretreatments are
quite simple and inexpensive. These two pre-sowing treatments
can be widely used by the village/forest nurseries in the Himala-
yan region and can be recommended for large scale plantlet pro-
duction of the species.

Acknowledgements
We thank the Director, GBPIHED for support and encourage-
ment. Thanks are due to Dr. S.S. Samant, and Mr. K. Gaira for
their help and support.

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