全 文 ：Journal of Forestry Research (2009) 20(4): 323–330
DOI 10.1007/s11676-009-0055-6





Effect of pre-soaking and pre-chilling treatments on seed germination of
Pinus roxburghii provenances from western Himalaya, India

Sunil Kumar Ghildiyal1, Chandra Mohan Sharma1, Vinod Prasad Khanduri2*
1Department of Botany, HNB Garhwal University, Srinagar Garhwal-246 174, Uttarakhand India
2Department of Forestry, Mizoram University, Aizawl -796001, Mizoram, India

Abstract: After subjecting the seeds to GA3, and H2O2 treatments for 24 h and chilling at 2–3°C for a period of 15 days, we conducted
the seed germination tests for 21 different seed sources of Pinus roxburghii from western-central Himalaya under laboratory conditions
at various temperatures viz., 20°C, 25°C and 30°C inside a seed germinator. The results reveal that the soaking of seeds in H2O2 (1%
v/v) and GA3 (10 mg·L-1) solutions manifested 82.39% and 78.19% germination, respectively whereas untreated seeds exhibited 70.79%
average germination. Both GA3 and H2O2 treatments caused an appreciable shortening of the germination period by 8 days and 10 days,
respectively. Moist-chilling did improve the rate and percentage of germination when germinated at 20°C over 21 days; however total
germination was not affected at temperatures 25ºC and 30°C. Although the seeds of P. roxburghii germinate well due to lack of dor-
mancy, the increasing demand for large quantities of seeds of P. roxburghii for reforestation programmes make pre-sowing treatments
useful in improving the rate and percentage of germination.
Keywords: seed germination; seed sources; provenances; temperature; GA3; H2O2; chilling; chir pine; Pinus roxburghii; Himalaya



Introduction

Pinus roxburghii Sargent, commonly known as the chir pine or
Himalayan long needle pine is an indigenous, fire resistant tree
species of western-central Himalaya, which occurs naturally
between 450 m to 2 300 m elevation (Sharma and Baduni 2000).
This species constitutes about one-third of the total forest area of
Uttarakhand Himalaya, India (Tewari 1994). Qualities such as a
straight cylindrical bole, rapid growth, high volume returns,
yielding of timber and resin, and capacity to colonize in de-
graded habitats make this species a precious resource of Himala-
yan region., The P. roxburghii is being planted on a massive
scale in different parts of the country as a source of wealth due to
its multifarious uses particularly the resin of commercial value.
Anemophily (Khanduri and Sharma 2007) and wide geographic
distribution of this species manifested considerable natural varia-

Foundation project: This work was suportted by the Indian Council of
Forestry Research and Education (ICFRE) Dehradun
Received: 2008-11-11; Accepted: 2009-02-05
© Northeast Forestry University and Springer-Verlag 2009
The online version is available at http://www.springerlink.com
Biography: S. K. Ghildiyal (1972- ). Male, Ph.D. in Forestry, Research Asso-
ciate, Department of Botany, HNB Garhwal University, Srinagar Garhwal-
246 174 (Uttarakhand) India. (E-mail: skghildiyal@gmail.com)
*Corresponding author, Khanduri VP. E-mail: khandurivp@yahoo.com
Tel: 91-9436199694; Fax:91-0389-2330416
Responsible editor: Chai Ruihai

tion. To explore this variability, provenance testing is essential to
obtain quality traits for afforestation programmes and genetic
improvement of specific traits.
Selection of the best provenances of the desired species for a
given site or region is necessary for achieving maximum produc-
tivity. In those species for which there are data on individual tree
variability, genetic differences are associated with place of ori-
gin, which have often been several times as great as those among
individual trees in the same stand. Plus trees chosen for selective
breeding, without regard to provenance performance might give
birth to an inferior race. Therefore, it is particularly necessary to
do provenance testing prior to more intensive breeding work.
Temperature affects the percentage and rate of germination
(Baskin and Baskin 1992; Landis et al. 1998) through its effects
on seed deterioration, loss of dormancy (Wang and Berjak 2000)
and the germination process itself (Roberts 1988; Bewley and
Black 1994; Ghildiyal and Sharma 2005). Some chemicals, like
hydrogen peroxide and other growth promoting substances such
as gibberellic acid have been found helpful in enhancing germi-
nation rate of the seeds in many species (Vogt 1970; Krishna-
murthy 1973; Chandra and Chauhan 1976; Shafiq 1980; Thapli-
yal et al. 1985; and Ghildiyal 2003), through which losses in
seed germination could be minimised (Quarberg and Jahns
2000). Cold moist stratification is a common practice to enhance
the rate and percentage of germination of dormant seeds of most
conifers (Mergen 1963; Fowler and Dwight 1964; Roberts and
Ellis 1982; Wang and Berjak 2000; Rawat et al. 2008). The ef-
fective temperatures for cold stratification are from about 0ºC to
10ºC, but 5ºC being optimal for many species (Stokes 1965;
Baskin and Baskin 2001). Keeping in view the all above facts in
RESEARCH PAPER
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324
mind, the present study was aimed at understanding the efficacy
of hydrogen peroxide, gibberellic acid, and moist chilling treat-
ments on the germination of seeds of different provenances of P.
roxburghii.

Materials and methods

Mature seeds of P. roxburghii were collected during the months
of February – April, 2000 from 21 different seed sources of two
states, i.e. Uttarakhand (U.K.) and Himachal Pradesh (H.P.),
situated in the Western-Central Himalaya, India. The details of
the seed sources/study areas are presented in Table 1& Fig. 1.
The studies pertaining to seed germination after pre-soaking
(at room temperature, 25°C) and pre-chilling (3–5°C) treatments
were carried out at various temperatures viz., 20°C, 25°C and
30°C inside a seed germinator (Model No. 8LT-SGL CALTAN).
The seeds of all the provenances of P. roxburghii were germi-
nated at similar specified temperatures after applying following
treatments to each set:
Treatment 1: Soaking of the seeds in distilled water at room
temperature (25°C) for 24 h.
Treatment 2: Soaking of the seeds in Gibberellic acid (GA3 10
mg/L) at room temperature (25°C) for 24 h.
Treatment 3: Soaking of the seeds in Hydrogen peroxide
(H2O2 1% v/v) at room temperature (25°C) for 24 h.
Treatment 4: Seeds treated with Gibberellic acid (GA3 10
mg/L) as above at room temperature (25°C) for 24 h and then
chilled for 15 days at 3–5°C.


Fig. 1 Location Map of the Study Area.

The seeds of different provenances having same level of ripe-
ness were collected and subjected to viability test by floating
method to select the viable seeds. For germination, the seeds in
five replicates of 100 seeds each were placed in Petri dishes (di-
ameter- 10 cm) containing two filter papers, kept in the germina-
tor, and maintained at desired temperature. Observations were
recorded daily regarding germinated /non-germinated seeds up to
21 days. Radicle emergence was taken as the criteria for germi-
nation.
Chilling of seeds was carried out in folded-over-polythene
bags at 3–5°C for a period of 15 days (as per method suggested
by Tompsett and Pritchard 1998), after soaking the seeds in 10
mg/L Gibberellic acid for 24 h followed by brief drying. The
chilled seeds were then subjected to germination tests in all
specified temperatures.
Percent germination is the ratio of seeds germinated at the
completion of the germination period, whereas, germination
value is an index, combining both speed and completeness of
germination; which according to Czabator (1962) can be ex-
pressed as:
MDGPVGV ×=
where, GV is the germination value, PV is the peak value of ger-
mination, and MDG is the mean daily germination
The statistical analysis of each parameter was carried out on
mean values and the analysis of variance (ANOVA) was per-
formed using SPSS package. The critical difference (CD) was
calculated as:
01.0tSEdCD ×=
Where, SEd is the standard error of difference calculated as SEd
= √(2×Me/r), Me = Mean sum of square due to error, r = Number
of replicates. Pearson correlation was calculated between germi-
nation parameters and geographic factors (latitude, longitude,
altitude and rainfall) to rectify the effect of geographic factor on
seed germination.
Journal of Forestry Research (2009) 20(4): 323–330

325
Table 1. Geographic and climatic descriptions of the selected seed sources of Pinus roxburghii
Temperature (°C) Provenance District (State) Latitude (N) Longitude (E) Altitude (m)
Min. Max.
Mean Annual
rainfall (mm)
Agustmuni Rudraprayag (U.K.) 30° 23 79° 02 875 4.31 36.59 833
Badiyargarh Tehri (U.K.) 30° 17 78° 50 1080 7.5 36.3 930
Chhoti Singri Mandi (H. P.) 31° 49 76° 59 1220 0.3 32.5 1025
Dhulcheena Almora (U.K.) 29° 42 79° 49 1850 -0.14 26.4 1125
Gallu Mandi (H. P.) 31° 42 77° 01 1520 -0.2 31.4 1100
Kaligad Almora (U.K.) 29° 38 79° 25 1800 0.42 26.86 1060
Khamlekh Pithoragarh (U.K.) 29° 47 80° 04 1450 3.1 31.2 1230
Matiyal Nainital (U.K.) 38° 10 69° 20 1740 3.8 23.4 2270
Matnoh Hamirpur (H. P.) 31° 45 76° 43 980 0.8 33.6 1150
Mayali Tehri (U.K.) 30° 23 78° 47 1400 2.6 25.1 1030
Nagali Solan (H. P.) 30° 54 77° 12 1545 0.5 34.1 1000
Nihari Hamirpur (H. P.) 31° 29 76° 28 800 1.2 35.4 1125
Pabo Pauri (U.K.) 30° 15 79° 01 1640 1.8 32.4 875
Patwadangar Nainital (U.K.) 29° 16 79° 20 1500 7.4 28.5 2850
Pauri Pauri (U.K.) 30° 09 78° 48 1660 -0.48 26.3 1792
Pokhal Tehri (U.K.) 30° 25 78° 59 820 5.7 37.63 800
Ranital Kangra (H. P.) 32° 10 76° 05 960 0.2 32.5 1350
Seshan Shimla (H. P.) 31° 07 77° 45 1540 0.5 30.3 1075
Seuri Mandi (H. P.) 31° 50 77° 02 1460 0.15 33.2 925
Soni Almora (U.K.) 29° 12 79° 24 1650 2.3 28 1040
Thalisain Pauri (U.K.) 30° 02 79° 03 1640 1.9 31 1025
* U. K.= Uttarakhand, H. P.= Himanchal Pradesh

Results

Germination of seeds of various provenances after pre-soaking
treatments (H2O2 1% v/v and GA310 mg/litre) under different
temperature regimes, (i.e., 20°C, 25°C and 30°C) have yielded
significant differences in seed germination. The data analysed for
its variance indicated much variation between different seed
sources, which are presented in Table 2. The detailed treatment-
temperature interactions are given below.

Soaking of seeds in distilled water (as control)

At 20°C the maximum germination percentage was recorded for
Pokhal provenance (98.0%±2.00%) and minimum for Chhoti
Singri provenance (46.0%±4.64%) (Table 2). However, the
maximum germination value was recorded for Mayali prove-
nance (18.74±1.83), and minimum for Chhoti Singri provenance
(0.73±1.23). On the other hand, at 25°C, the maximum germina-
tion percentage was recorded for Dhulcheena provenance
(97.8%±0.66%), and minimum for Chhoti Singri provenance
(33.2%±1.10%). The higher germination value was recorded for
Mayali provenance (24.43±1.99); whereas, the lowest germina-
tion value was again recorded for Chhoti Singri provenance
(0.59±0.17). At 30°C, Dhaulcheena provenance showed maxi-
mum germination percentage (86.5% ±0.87%) and minimum
was recorded for Kaligad provenance (20.6% ±1.97%).

Soaking of seeds in GA3

Seeds treated with gibberellic acid reflected lesser germination
percentage as compared to seeds treated with H2O2 1% v/v. Sub-
stantial germination after GA3 treatment was recorded in Pokhal
(94.0%±2.45%), Dhulcheena (92.6%±1.78%), Kaligad
(88.2%±3.25%), Soni (86.6%±4.46%) and Thalisain
(86.0%±6.00%) provenances, whereas minimum germination
was recorded for Chhoti Singri provenance (56.2%±3.92%).
Remarkable germination values were observed for Mayali
(16.27±0.36), Soni (12.56±1.48) and Agustmuni (12.16±0.48)
provenances, as compared to the value recorded for Chhoti Sin-
gri provenance (1.94±1.54). At 25°C, higher germination per-
centages were recorded for Dhulcheena (98.4%±1.75%), Nagali
(97.6%±1.47%), Seshan (96.8%±1.86%), Matnoh
(93.6%±0.75%) and Kaligad (93.2%±1.60%) provenances and
lowest germination percentage was recorded for Khamlekh
provenance (60.8%±1.86%). It is interesting to point out that the
seeds treated with gibberellic acid have shown better germination
percentage at 30°C as compared to 20°C and 25°C. The prove-
nances that showed better germination percentages at 30°c were
Matiyal (97.2%±0.86%) and Pokhal (94.0%±1.23%). Poor ger-
mination was recorded for Nihari (45.9%±1.51%) and Chhoti
Singri (46.1%±1.65%) provenances (Table 2). It was also ob-
served during the experiment that on an average this treatment
reduced the germination period by eight days in all the tempera-
ture regimes.

Soaking of seeds in H2O2 1% v/v

At 20°C, the highest and lowest germination percentages were
recorded for Pokhal (98.0%±2.00%), and Chhotisingri prove-
nances (68.4% ±2.72%), respectively. Similarly the highest and
lowest germination values were recorded for Mayali provenance
(21.66 ±1.62), and Chhoti Singri provenances (2.76±0.62), re-
spectively. At 25°C, higher germination percentages were re-
corded for Soni (98.4%±0.85%), Patwadangar (98.0%±0.63%),
Journal of Forestry Research (2009) 20(4): 323–330

326
Thalisain (96.0%±0.90%), Matiyal (94.8% ±1.10%), Nihari
(93.6 ±2.17) and Matnoh (93.6 ±1.17%) provenances. The seeds
from Chhoti Singri provenance had the lowest germination per-
centage (34.4%±1.33%). The seeds of all provenances can be
germinated at 30°C, and some of the provenances i.e., Seuri
(96.8 ±1.46%), Matnoh (96.2 ±1.53%), Thalisain (96.2 ±0.86%),
and Agustmuni (93.6 ±1.44%), have reflected higher germination
percentages, whereas Chhoti Singri (26.5 ±0.87%) and Nihari
(46.6 ±1.19%) provenances have shown lower germination per-
centages (Table 2). Further, the experiment revealed that this
treatment shortened the germination period in all the aforesaid
temperatures averagely by 10 days.

Table 2. Effect of different treatments and temperatures on seed germination percentage and germination value of various provenances of Pinus
roxburghii
Seed germination percentage (data in standardized form) and germination value (data in italics form)
20°C 25°C 30°C

Provenance
control H2O2 (1%v/v) GA3
(100ppm)
control H2O2
(1%v/v)
GA3
(100ppm)
control H2O2
(1%v/v)
GA3
(100ppm)
Agustmuni 78.4 ±2.62 88.2 ±2.54 85.2 ±2.10 75.2 ±1.02 83.2 ±1.02 81.2 ±1.10 63.4 ±1.08 93.6 ±1.44 87.8 ±1.50
16.32 ±1.42 12.72 ±0.83 12.16 ±0.48 18.39 ±2.57 14.97 ±0.94 14.08 ±1.41 7.09 ±1.28 21.24 ±0.97 14.79 ±0.97
Badiyargarh 52.2 ±1.58 78.2 ±2.42 69.6 ±5.62 39.2 ±1.36 83.6 ±1.33 61.2 ±1.10 51.6 ±1.21 64.2 ±1.72 81.0 ±1.18
4.62 ±1.76 12.68 ±1.42 2.18 ±0.32 2.84 ±0.74 17.85 ±2.33 6.38 ±1.13 1.59 ±0.36 2.67 ±0.55 4.47 ±0.96
Chhoti Singri 46.0±4.64 68.4 ±2.72 56.2 ±3.92 33.2±1.10 34.4 ±1.33 73.2 ±1.20 32.9 ±0.91 26.5 ±0.87 46.1 ±1.65
0.73 ±1.23 2.76 ±0.62 1.94 ±1.54 0.59 ±0.17 0.88 ±0.10 3.72 ±1.57 0.24 ±0.01 0.13 ±0.01 0.48 ±0.04
Dhulcheena 90.0 ±1.48 86.2 ±0.73 92.6 ±1.78 97.8 ±0.66 86.6 ±0.81 98.4 ±1.75 86.5 ±0.87 92.7 ±1.17 65.5 ±1.31
4.68 ±1.32 10.24 ±0.83 11.39 ±1.73 5.74 ±0.38 10.03 ±0.93 11.14 ±1.41 3.08 ±0.59 7.49 ±0.46 1.70 ±0.38
Gallu 75.6 ±3.55 78.4 ±3.34 72.6 ±6.65 86.0 ±0.71 73.2 ±1.10 66.0 ±1.30 65.7 ±1.46 86.3 ±1.66 65.9 ±1.41
6.34 ±0.92 9.47 ±0.93 7.25 ±0.89 7.81 ±0.86 7.02 ±1.06 6.19 ±0.66 1.96 ±0.37 4.44 ±0.69 2.66 ±0.47
Kaligad 82.2 ±1.26 90.6 ±2 .38 88.2 ±3.25 80.4 ±0.75 93.2 ±1.10 93.2 ±1.60 20.6 ±1.97 72.3 ±1.67 73.1 ±1.85
5.14 ±1.67 8.59 ±1.44 6.67 ±0.96 5.09 ±0.70 16.70 ±1.17 5.98 ±0.98 0.22 ±0.02 3.88 ±0.87 1.83 ±0.34
Khamlekh 60.4 ±1.55 84.2 ±0.57 75.4 ±0.85 53.2 ±1.10 85.8 ±0.58 60.8 ±1.86 53.2 ±1.14 79.6 ±1.72 52.1 ±1.32
3.52 ±1.52 9.42 ±0.64 4.23 ±0.36 2.29 ±0.88 6.19 ±1.42 1.80 ±0.67 1.44 ±0.24 4.80 ±0.47 1.67 ±0.32
Matiyal 64.2 ±1.34 87.2±0.92 83.2 ±1.93 60.0 ±1.42 94.8 ±1.10 85.8 ±1.36 84.9 ±1.71 65.5 ±1.52 97.2±0.86
2.29 ±1.74 6.61 ±1.24 3.07 ±1.55 2.85 ±0.64 8.89 ±1.42 5.25 ±1.45 6.35 ±0.40 5.42 ±0.57 9.16 ±0.96
Matnoh 84.0±2.72 90.2 ±1.48 87.4 ±2.74 80.0 ±1.42 93.6 ±1.17 93.6 ±0.75 53.4 ±1.12 96.2 ±1.53 79.4 ±2.02
8.27 ±0.76 6.93 ±0.70 4.19 ±0.52 6.29 ±1.26 11.27 ±0.61 5.38 ±1.49 0.72 ±0.05 2.18 ±0.50 1.74 ±0.08
Mayali 76.2±4.23 80.4 ±4.16 72.2 ±5.27 81.6 ±1.17 88.0 ±0.90 79.6 ±1.17 63.8 ±1.50 62.2 ±1.50 64.2 ±1.99
18.74 ±1.83 21.66 ±1.62 16.27 ±0.36 24.43 ±1.99 66.62 ±2.87 43.81 ±1.42 6.99 ±0.90 7.41 ±0.39 4.64 ±0.82
Nagali 76.2 ±5.51 75.4 ±4.63 79.4 ±5.56 80.4 ±0.95 79.6 ±0.75 97.6 ±1.47 76.2 ±1.77 80.6 ±1.86 85.8 ±1.56
8.96 ±0.37 6.16 ±0.43 9.36 ±2.19 4.33 ±1.23 12.63 ±0.86 10.95 ±1.29 2.29 ±0.38 6.33 ±1.07 8.81 ±1.22
Nihari 62.4 ±4.12 80.6 ±0.97 74.8 ±1.21 66.6 ±0.93 93.6 ±2.17 85.4 ±1.66 26.7 ±0.93 46.6 ±1.19 45.9 ±1.51
4.21 ±1.42 5.68 ±1.80 8.71 ±1.46 3.46 ±0.96 9.12 ±0.50 5.14 ±1.23 0.39 ±0.04 0.90 ±0.04 0.28 ±0.03
Pabo 86.4 ±3.67 90.6 ±4.35 72.4 ±7.28 84.0 ±2.42 85.2 ±1.02 65.2 ±1.50 75.2 ±1.43 84.2 ±2.20 81.4 ±1.36
7.25 ±0.84 9.21 ±2.67 5.92 ±1.42 5.49 ±0.94 24.38 ±1.69 7.21 ±1.54 17.98 ±2.00 13.60 ±0.93 13.93 ±1.09
Patwadangar 84.6 ±2.14 94.0 ±1.23 70.0 ±2.82 86.6 ±0.51 98.0 ±0.63 66.0 ±1.82 66.3 ±1.01 86.7 ±1.28 81.0 ±1.73
7.23 ±0.48 11.75 ±1.86 4.24 ±1.64 6.04 ±1.69 17.44 ±1.19 3.36 ±1.07 1.80 ±0.41 5.26 ±0.79 1.95 ±0.38
Pauri 82.2 ±1.92 80.8 ±3.56 82.6 ±2.16 85.6 ±1.17 90.4 ±1.17 90.0 ±1.62 55.2 ±1.86 65.4 ±1.78 58.0 ±1.64
8.68 ±1.32 11.33 ±1.86 10.71 ±0.77 4.81 ±1.65 32.19 ±2.35 17.46 ±1.17 3.53 ±0.99 10.76 ±0.83 2.30 ±0.56
Pokhal 98.0 ±2.00 98.0 ±2.00 94.0 ±2.45 82.2 ±2.06 88.4 ±0.75 65.2 ±1.40 83.2 ±1.86 80.2 ±1.80 94.0 ±1.23
3.91 ±0.53 8.91 ±0.73 3.43 ± 0.32 10.88 ±1.18 18.29 ±1.61 5.29 ±1.36 6.03 ±0.88 11.12 ±0.37 7.18 ±0.73
Ranital 68.2 ±6.28 83.0 ±2.72 75.0 ±4.32 73.2 ±1.16 86.2 ±0.86 73.6 ±0.75 40.9 ±1.62 73.1 ±1.79 72.9 ±1.50
3.88 ±1.78 7.25 ±2.07 2.63 ±1.21 5.75 ±1.81 12.48 ±0.68 4.03 ±1.33 0.33 ±0.06 3.70 ±0.86 0.25 ±0.02
Seshan 70.8 ±2.39 78.0 ±1.86 80.2 ±3.84 73.2 ±1.10 92.2 ±1.28 96.8 ±1.86 70.4 ±1.44 83.8 ±2.06 82.2 ±1.72
6.43 ±0.79 7.85 ±0.81 5.54 ±1.70 6.51 ±1.36 9.36 ±0.70 9.32 ±1.33 2.21 ±0.45 4.86 ±1.01 6.35 ±0.55
Seuri 80.2 ±7.36 84.0 ±0.65 80.0 ±8.39 87.0 ±1.00 80.0 ±2.42 85.8 ±0.68 73.1 ±0.86 96.8 ±1.46 86.4 ±0.82
5.86 ±0.54 8.32 ±1.28 6.26 ±0.79 4.83 ±0.68 8.12 ±0.84 5.24 ±0.86 1.04 ±0.15 2.98 ±0.55 1.85 ±0.07
Soni 86.2 ±0.86 97.4 ±1.06 86.6 ±4.46 86.6 ±0.93 98.4 ±0.85 86.2 ±1.69 60.1 ±1.46 71.7 ±1.55 85.2 ±1.37
8.76 ±0.93 14.37 ±1.32 12.56 ±1.48 8.12 ±1.16 17.06 ±0.93 12.36 ±2.15 2.63 ±0.54 4.62 ±0.60 5.05 ±1.85
Thalisain 90.0 ±5.48 94.0 ±4.60 86.0 ±6.00 90.0 ±1.42 96.0 ±0.90 83.2 ±1.10 80.0 ±1.92 96.2 ±0.86 89.4 ±1.36
2.74 ±0.53 7.83 ±1.31 3.79 ±0.60 7.26 ±0.86 43.57 ±1.95 6.10 ±1.55 3.33 ±1.02 40.66 ±1.51 5.32 ±0.89
Mean 75.92 84.86 79.22 75.33 85.92 80.38 61.11 76.40 74.98
6.60 9.51 6.79 6.85 17.38 9.06 3.39 7.83 4.59
Range 46.0-98.0 68.4-98.0 56.2-94.0 33.2-97.8 34.4-98.4 60.8-98.4 20.6-86.5 26.5-96.8 45.9-97.2
0.73-18.74 2.76-21.66 1.94-16.27 0.59-24.43 0.88-66.62 1.80-43.81 0.22-17.98 0.13-40.66 0.25-14.79
C.D at 1% 5.44 4.16 6.12 5.76 4.24 3.72 5.81 6.19 7.23
2.28 6.1 8.17 4.67 8.89 4.58 1.18 3.86 3.51

Pre-chilling effects on germination

It has now been well established that presowing treatments of
seeds by cold stratification enhances the rate and percentage of
germination of seeds of many tree species. Therefore, we com-
pared the germination of pre-chilled and unchilled seeds of vari-
ous provenances (Table 3). Great similarity in seed germination
percentages were recorded between pre-chilled and unchilled
seeds. Nevertheless, germination values were recorded higher in
unchilled seeds as compared to the pre-chilled seeds. A perusal
Journal of Forestry Research (2009) 20(4): 323–330

327
of Table 3 further revealed that the number of seed sources, par-
ticularly Agustmuni, Pabo, Gallu, Ranital and Nihari, excelled
over other seed sources.

Table 3. Seed source variations, mean germination percentages (data in standardized form) and Germination values (data in italics form) of
pre-chilled and unchilled seeds of Pinus roxburghii at different temperatures
Pre-chilled Unchilled Provenance
20°c 25°c 30°c 20°c 25°c 30°c
Agustmuni 86.2 ±4.94 84.5 ±0.91 79.2 ±4.64 85.2 ±2.10 81.2 ±1.10 87.8 ±1.50
8.38 ±1.12 8.76 ±1.70 10.28 ±0.92 12.16 ±0.48 14.08 ±1.41 14.79 ±0.97
Badiyargarh 73.4 ±2.19 67.0 ±1.25 72.4 ±4.23 69.6 ±5.62 61.2 ±1.10 81.0 ±1.18
2.27 ±0.68 5.37 ±1.43 6.25 ±0.84 2.18 ±0.32 6.38 ±1.13 4.47 ±0.96
Chhoti Singri 62.0 ±4.26 78.4 ±0.86 51.3 ±2.56 56.2 ±3.92 73.2 ±1.20 46.1 ±1.65
1.67 ±2.42 5.77 ±1.39 2.14 ±0.36 1.94 ±1.54 3.72 ±1.57 0.48 ±0.04
Dhulcheena 94.6 ±2.56 98.8 ±0.92 63.2 ±3.24 92.6 ±1.78 98.4 ±1.75 65.5 ±1.31
9.32 ±1.84 9.75 ±1.87 2.06 ±0.54 11.39 ±1.73 11.14 ±1.41 1.70 ±0.38
Gallu 78.6 ±8.23 70.4 ±1.48 68.4 ±2.83 72.6 ±6.65 66.0 ±1.30 65.9 ±1.41
8.16 ±1.25 3.72 ±0.75 4.64 ±1.37 7.25 ±0.89 6.19 ±0.66 2.66 ±0.47
Kaligad 90.2 ±3.68 94.5 ±1.36 75.6 ±2.48 88.2 ±3.25 93.2 ±1.60 73.1 ±1.85
6.44 ±1.12 4.63 ±0.74 2.35 ±1.07 6.67 ±0.96 5.98 ±0.98 1.83 ±0.34
Khamlekh 83.2 ±4.63 66.0 ±1.24 58.2 ±2.52 75.4 ±0.85 60.8 ±1.86 52.1 ±1.32
4.39 ±0.67 2.47 ±0.66 1.92 ±0.74 4.23 ±0.36 1.80 ±0.67 1.67 ±0.32
Matiyal 87.5 ±2.27 86.2 ±1.39 88.6 ±1.82 83.2 ±1.93 85.8 ±1.36 97.2 ±0.86
6.78 ±1.19 6.54 ±1.72 5.68 ±1.28 3.07 ±1.55 5.25 ±1.45 9.16 ±0.96
Matnoh 90.3 ±5.45 94.0 ±0.96 72.0 ±3.16 87.4 ±2.74 93.6 ±0.75 79.4 ±2.02
5.63 ±0.69 6.48 ±1.36 2.26 ±0.54 4.19 ±0.52 5.38 ±1.49 1.74 ±0.08
Mayali 77.2 ±6.22 84.2 ±0.96 60.2 ±2.63 72.2 ±5.27 79.6 ±1.17 64.2 ±1.99
3.78 ±0.56 11.36 ±1.66 7.46 ±1.32 16.27 ±0.36 43.81 ±1.42 4.64 ±0.82
Nagali 81.6 ±6.23 97.8 ±1.56 81.3 ±1.76 79.4 ±5.56 97.6 ±1.47 85.8 ±1.56
7.13 ±0.98 10.62 ±1.46 6.74 ±1.28 9.36 ±2.19 10.95 ±1.29 8.81 ±1.22
Nihari 77.5 ±6.46 88.0 ±1.72 52.8 ±2.94 74.8 ±1.21 85.4 ±1.66 45.9 ±1.51
6.86 ±1.54 6.23 ±1.17 1.68 ±0.39 8.71 ±1.46 5.14 ±1.23 0.28 ±0.03
Pabo 76.2 ±6.66 69.2 ±1.62 84.2 ±2.68 72.4 ±7.28 65.2 ±1.50 81.4 ±1.36
5.52 ±1.28 3.25 ±0.88 11.08 ±1.72 5.92 ±1.42 7.21 ±1.54 13.93 ±1.09
Patwadangar 75.0 ±2.38 72.2 ±1.68 78.0 ±4.15 70.0 ±2.82 66.0 ±1.82 81.0 ±1.73
5.98 ±0.89 4.39 ±1.19 2.60 ±0.92 4.24 ±1.64 3.36 ±1.07 1.95 ±0.38
Pauri 88.6 ±4.73 90.6 ±1.26 44.0 ±5.09 82.6 ±2.16 90.0 ±1.62 58.0 ±1.64
7.25 ±1.33 10.42 ±1.21 4.56 ±0.78 10.71 ±0.77 17.46 ±1.17 2.30 ±0.56
Pokhal 96.0 ±7.48 71.4 ±2.14 86.0 ±5.10 94.0 ±2.45 65.2 ±1.40 94.0 ±1.23
2.00 ±0.54 4.34 ±0.76 5.21 ±0.82 3.43 ± 0.32 5.29 ±1.36 7.18 ±0.73
Ranital 80.0 ±2.17 75.8 ±1.19 75.6 ±4.28 75.0 ±4.32 73.6 ±0.75 72.9 ±1.50
4.62 ±1.77 2.08 ±1.26 1.52 ±0.42 2.63 ±1.21 4.03 ±1.33 0.25 ±0.02
Seshan 82.2 ±4.92 96.0 ±1.64 78.6 ±3.48 80.2 ±3.84 96.8 ±1.86 82.2 ±1.72
5.92 ±1.27 8.76 ±0.98 4.25 ±0.64 5.54 ±1.70 9.32 ±1.33 6.35 ±0.55
Seuri 84.8 ±7.67 86.2 ±1.12 80.7 ±1.84 80.0 ±8.39 85.8 ±0.68 86.4 ±0.82
4.34 ±0.94 6.32 ±0.84 1.94 ±0.33 6.26 ±0.79 5.24 ±0.86 1.85 ±0.07
Soni 90.8 ±6.59 88.4 ±1.45 80.4 ±2.91 86.6 ±4.46 86.2 ±1.69 85.2 ±1.37
8.75 ±1.36 10.08 ±2.03 3.21 ±1.15 12.56 ±1.48 12.36 ±2.15 5.05 ±1.85
Thalisain 88.0 ±5.83 87.6 ±1.21 78.0 ±6.63 86.0 ±6.00 83.2 ±1.10 89.4 ±1.36
2.48 ±0.40 6.29 ±1.75 4.37 ±0.69 3.79 ±0.60 6.10 ±1.55 5.32 ±0.89
Mean 83.04 83.20 71.84 79.22 80.38 74.98
5.60 6.55 4.39 6.79 9.06 4.59
Range 62.0-96.0 66.0-98.8 44.0-88.6 56.2-94.0 60.8-98.4 45.9-97.2
1.67-9.32 2.08-11.36 1.52-11.08 1.94-16.27 1.80-43.81 0.25-14.79
C.D at 1% 7.3 7.72 6.7 7.14 7.18 8.46
1.32 1.18 0.9 0.72 1.1 1.7

The analysis of variance shows that there were significant dif-
ferences in germination percentage and germination value of pre-
chilled and unchilled seeds of all the seed sources at various
temperatures and treatments (Table 4 and Table 5). The results of
correlation between germination parameters and geographic
factors (latitude, longitude, altitude and rainfall) were presented
in Tables 6, 7, and 8. Statistically significant negative correlation
was observed between latitude and various germination parame-
ters; however significant positive correlation was recorded be-
tween longitude and germination parameters of various seed
sources.
Journal of Forestry Research (2009) 20(4): 323–330

328
Table 4. Analysis of variance for germination percentage and germination value of different seed sources of Pinus roxburghii
Germination percentage
Control H2O2 1% v/v GA3 100 ppm Source of Variation df
20°c 25°c 30°c 20°c 25°c 30°c 20°c 25°c 30°c
Temperature 2 1229.63 1530.39 3255.78 489.53 634.77 441.55 190.54 247.05 1444.26
Provenance 20 429.53 664.92 481.32 225.97 336.10 219.90 370.26 262.68 515.26
Error 40 123.88 133.71 162.66 34.70 115.64 129.24 57.72 142.92 118.66
Total sum of Square 62 16004.96 21707.56 22644.47 6886.36 12617.07 10450.58 10094.97 11464.28 17939.89
Probability 0.000316 0.000117 <0.00001 <0.00001 0.007824 0.04267 0.047102 0.19052 <0.00001
0.000401 <0.00001 0.001716 <0.00001 0.002001 0.075339 <0.00001 0.05013 <0.00001
Germination value
Control H2O2 1% v/v GA3 100 ppm Source of Variation df
20°c 25°c 30°c 20°c 25°c 30°c 20°c 25°c 30°c
Temperarure 2 795.92 7.22 10.06 86.63 547.32 9.37 56.81 692.56 104.70
Provenance 20 160.41 62.60 44.78 24.01 287.33 213.05 28.33 177.93 76.93
Error 40 51.68 31.02 2.44 7.30 49.14 11.08 5.24 44.80 24.27
Total sum of Square 62 6867.16 2507.04 1013.32 945.41 8806.88 4723.06 889.81 6735.60 2718.61
Probability <0.00001 0.793357 0.023568 <0.00001 0.000143 0.436945 0.000173 <0.00001 0.020099
0.001125 0.029068 <0.00001 0.000662 <0.00001 <0.00001 <0.00001 0.000101 0.00093
All parameter are significant at 5% level

Table 5. Analysis of variance for Pre-chilled and Unchilled seeds germination percentages and germination value of different seed sources of
Pinus roxburghii
Germination percentages
Pre-chilled Unchilled Source of variation Degree of Freedom
df 20°c 25°c 30°c 20°c 25°c 30°c
Temperarure 2 345.18 467.99 1097.41 176.48 222.75 1355.78
Provenance 20 253.59 331.29 635.32 394.72 275.83 544.95
Error 40 33.32 112.16 129.66 50.17 139.85 127.11
Total 62 7094.90 12048.21 20087.50 10254.28 11555.94 18694.96
Probability 0.000237 0.022603 0.00086 0.039157 0.215972 0.000194
<0.00001 0.001743 <0.00001 <0.00001 0.0334 <0.00001
Germination value
Pre-chilled Unchilled Source of variation
Degree of Freedom
(df)
20°c 25°c 30°c 20°c 25°c 30°c
Temperarure 2 86.63 799.35 113.87 0.25 34.16 10.06
Provenance 20 24.01 134.54 55.70 45.19 165.38 46.27
Error 40 7.30 59.36 23.65 2.49 10.53 2.44
Total 62 945.41 6663.99 2287.80 1004.00 3797.14 1043.07
Probability <0.00001 <0.00001 0.013377 0.9061 0.049479 0.023568
0.000662 0.013647 0.010417 <0.00001 <0.00001 <0.00001
All parameter are significant at 5% level

Table 6. Correlation coefficient between germination percentages and geographic parameters of the seed sources of Pinus roxburghii
20°C 25°C 30°C Geographic parame-
ters control H2O2 (1%v/v) GA3 (100ppm) control H2O2 (1%v/v) GA3 (100ppm) control H2O2 (1%v/v) GA3
(100ppm)
Latitude -0.2813 -0.5168 -0.2866 -0.1889 -0.5229 0.0482 -0.2605 -0.0689 -0.2467
Longitude 0.3075 0.5012 0.3196 0.1389 0.3786 -0.1395 0.3375 0.1613 0.1849
Altitude (m) 0.2830 0.0859 0.1424 0.3886 0.1680 0.2969 0.3607 0.1604 0.0868
Rainfall (mm) -0.0433 0.1139 -0.1687 0.0437 0.2917 -0.0510 0.0753 -0.0659 0.0179

Table 7. Correlation coefficient between germination values and geographic parameters of the seed sources of Pinus roxburghii
20°C 25°C 30°C Geographic
parameters control H2O2
(1%v/v)
GA3
(100ppm)
control H2O2
(1%v/v)
GA3
(100ppm)
control H2O2
(1%v/v)
GA3
(100ppm)
Latitude -0.2059 -0.5698 -0.2901 -0.2505 -0.4444 -0.3190 -0.3116 -0.2523 -0.2490
Longitude 0.0173 0.4387 0.1426 0.0836 0.3032 0.1398 0.3332 0.2842 0.2227
Altitude (m) 0.0325 0.2498 0.2930 -0.0297 0.3002 0.2803 0.2036 0.1298 0.0405
Rainfall (mm) -0.0567 0.0723 -0.1230 -0.1530 0.0349 -0.0386 -0.0976 -0.1453 -0.1928
Journal of Forestry Research (2009) 20(4): 323–330

329
Table 8. Correlation coefficient between the germination percentages and germination values of Pre-chilled, Unchilled seeds and the geographic
parameters of the seed sources of Pinus roxburghii
Germination percentages Germination values
Pre-chilled Unchilled Pre-chilled Unchilled
Geographic
parameters
20°c 25°c 30°c 20°c 25°c 30°c 20°c 25°c 30°c 20°c 25°c 30°c
Latitude -0.3181 0.0216 -0.1769 -0.2866 0.0482 -0.2467 -0.1832 -0.2046 -0.2207 -0.2901 -0.3190 -0.2490
Longitude 0.3619 -0.1148 0.1140 0.3196 -0.1395 0.1849 0.0647 0.0870 0.1825 0.1426 0.1398 0.2227
Altitude (m) 0.1923 0.2930 0.0563 0.1424 0.2969 0.0868 0.2865 0.3098 0.0488 0.2930 0.2803 0.0405
Rainfall (mm) -0.1068 -0.0590 -0.0399 -0.1687 -0.0510 0.0179 0.1854 -0.0420 -0.1981 -0.1230 -0.0386 -0.1928

Discussion

Seed germination test in the present study demonstates that seed
source variation has significant effect on seed germination per-
centage and germination value. Germination value varied con-
siderably among seed sources and exhibited a random pattern.
Germination value is an index of combining speed and com-
pleteness of germination, which in itself is a function of seed size
and weight (Czabator 1962 and Dunlap and Barnett 1983). Seeds
of Mayali, Pokhal, Pabo, Dhulcheena, and Pauri sources, which
have higher vigour, took minimum time to complete germina-
tion. Significant variation in germination value among seed
sources is in conformity with those found in fir and spruce
(Singh and Singh 1981), Acacia spp. (Mathur et al 1984) and
Albizia falacataria (Bahuguna et al 1989). Khalil (1986) was of
the opinion that germination energy (vigour) and germination
capacity are valid criteria for early selection of fast growing
provenances.
Under favourable conditions the seeds of P. roxburghii germi-
nated well (60%–80%) within 7–21 days (Thapliyal 1986). Be-
cause the seeds are non-dormant, it is often assumed that pre-
conditioning treatments are unnecessary in this species. How-
ever, Barnett (1971) found that soaking seeds in aerated water,
promotes the germination of non-dormant seeds of South Pine.
Stratification or chilling under moist conditions has long been
recognised as a useful method of treating seeds to improve the
germinability (Outcall 1991). Heydecker and Coolbear (1977),
suggested many other presowing treatments that increased ger-
mination rate.
Soaking the seeds for 24 h in a solution of H2O2 (1% v/v) or
GA3 (10 mg·L-1) had a significant effect on average germination
percentage. About 82.39% germination was revealed by the
seeds treated with H2O2, while the mean average germination
percentage of untreated (control) seeds was just 70.79%. Simul-
taneously, GA3 and H2O2 treatments caused an appreciable de-
crease, in shortening of germination period by 8 and 10 days,
respectively. Similar results were recorded by Chandra and
Chauhan (1976) for Picea smithiana and Safiq (1980) for Not-
hofagus obliqua and Nothofagus procera seeds. Thus, soaking of
chir-pine seeds in H2O2 (1% v/v) should be preferred, since it
involves the saving of expenditure as compared to GA3. The cost
involved in case of H2O2 will be approximately half the cost of
GA3. Differences in the rate of germination from provenance to
provenance have also been documented by Webb and Farmer
(1968) and Wilcox (1968). The sexual reproductive efficiency
that can be assessed by determining germination capacity may
vary with altitude. Thus, altitudinal provenances of a species
may differ not only in seed germination but also in their repro-
ductive efficiency.
The practical implication of this study is that seeds of this
commercially important tree species should be pre-treated with
H2O2 1% v/v for 24 h to optimize germination. However, it
seems that the seed source is also as important as pre-treatments,
as the best germination was recorded in Thalisain, Dhulcheena,
Soni, Pokhal and Matnoh provenances. Hence, seed collection
should be carried out from these seed sources because seed qual-
ity has definite relations to some selective range of sources.
Responses of untreated vs moist chilled seeds in germination
trials carried out under the same photoperiod at different constant
temperatures, demonstrated the non-dormant condition of P.
roxburghii seeds. Similar results have also been reported by
Wang and Berjak (2000) for Picea mariana. In the the present
investigation, the differences in germination were statistically
significant (p=0.05) between the 15 days moist-chilled and non-
chilled seeds those were subjected to different constant tempera-
tures. In terms of the totality of germination, moist chilling for
15 days had no effect on rate and percentage of germination after
being subjected to 10 mg/L Gibberellic acid treatment (Wang
and Berjak 2000).
Although there was still an improvement in germination of P.
roxburghii seeds that were moist-chilled, after a 24 h GA3 pre-
treatment, but the final germination was low, which could have
otherwise the practical value. Moist-chilling is acknowledged to
be an effective treatment for overcoming dormancy and improv-
ing the rate as well as the percentage of germination of dormant
tree seeds (Baskin and Baskin 2001). The treatment may also
facilitate germination at sub-optimal temperatures (20°C), which
is particularly important for spring sowing in nurseries in tem-
perate climates. For the shallowly-dormant seeds of Douglas-fir
(Pseudotsuga mengeiesii), Lodgepole pine (Pinus contorta) and
Stika spruce ( Picea sitchensis), moist-chilling is not only a re-
quirement to alleviate dormancy, but a prolonged period for
chilling is necessary to achieve rapid and uniform germination
under the low temperature in early spring (Jones and Gosling
1994; Jinks and Jones 1996). Moist-chilling for 15 days did im-
prove the rate and percentage of germination of the non-dormant
P. roxburghii seeds at 20°C over 21 days, but total germination
was not affected at temperatures 25°C and 30°C. The effect of
moist-chilling on the activation of germination in the P. rox-
burghii seeds has not been previously reported and, therefore, is
a new facet in understanding of the benefits of short-term main-
tenance of seeds in a moistened condition at 3–5°C. The present
results have emanated from the P. roxburghii seed lots, which
are considered important for forestry practices on the basis of
Journal of Forestry Research (2009) 20(4): 323–330

330
their maximum germination.

Acknowledgements
Authors are thankful to the Indian Council of Forestry Research
and Education (ICFRE) Dehradun, for providing financial sup-
port.

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