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Standardization of seed storage conditions in chilgoza pine (Pinus gerardiana

Wall.): An endangered pine of Hind Kush Himalaya

Article in Trees · October 2013

DOI: 10.1007/s00468-013-0868-y

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Trees (2013) 27:1497–1501
DOI 10.1007/s00468-013-0868-y

SHORT COMMUNICATION

Standardization of seed storage conditions in chilgoza pine (Pinus

gerardiana Wall.): an endangered pine of Hind Kush Himalaya
A. R. Malik • G. S. Shamet • Jitendra S. Butola •

G. M. Bhat • A. A. Mir • Gowher Nabi

Received: 19 August 2012 / Revised: 16 February 2013 / Accepted: 21 February 2013 / Published online: 7 March 2013
Ó Springer-Verlag Berlin Heidelberg 2013

Abstract The seeds of chilgoza pine (Pinus gerardiana) development and sustainable utilization of P. gerardiana in
show moderate germination and not retain better germi- Himalayan region, has been suggested.
nability under normal ambient storage. In the present study,
five storage containers [polythene bags (C1), plastic jars Keywords Pinus gerardiana
Chilgoza
Edible

(C2), canvas bags (C3), earthen pots (C4) and tin boxes Endangered
Germinability
Storage and Himalaya
(C5)] and four temperature regimes (19–22 °C) (room
temperature, T1), 0 ± 1 °C (T2), -4 ± 1 °C (T3) and
4 ± 1 °C (T4) were tested and the suitable seed storage Introduction
conditions of chilgoza pine to retain viability were stan-
dardized. The storage devices preserve and retain viability Pinus gerardiana Wall. (Family-Pinaceae), popularly
trend of C4 [ C3 [ C1 [ C2 [ C5 and T2 [ T3 [ T4 [ T1 known as Chilgoza or Neoza pine, is an important tree
in the species throughout the storage period. However, the species, near endemic to Himalayan region. Its distribution
interaction treatment (C4T2) of earthen pots under is confined only to Hindu Kush Himalaya, i.e., eastern
0 ± 1 °C temperature regime maintained significantly Afghanistan, Pakistan and India. In India, it is reported
(P \ 0.05) maximum germinability after 9 months from dry temperate zone of Jammu and Kashmir (J and K)
(58.3 %) followed by C3T2 and C4T3 (47.5 %) as com- and Himachal Pradesh (H.P.) (Sehgal and Khosla 1986).
pared to other storage conditions. Notably, a sharp decline The species is called as Jhalgoza in Afghanistan, Chiri or
in germinability was recorded in seeds stored in tin boxes Chilgoza in Kashmir, Rhi or Neoza in Kinnaur, Chujin in
placed at room temperature. The implementation of these Chitral and Mirri in Pangi (H.P.). The species grows
results for conservation management, especially nursery between 1,600 and 3,300 m above sea level and is sparsely
distributed in H.P. covering a total area of about 2,060 ha
with most of the area falling in Kinnaur district (2,040 ha)
Communicated by U. Luettge. and a small portion (20 ha) in Chamba district (Troup
1921). The species has aptly been described as the
A. R. Malik (&)
G. M. Bhat
A. A. Mir
G. Nabi
‘‘Champion of Rocky Mountains’’ as it grows under
Faculty of Forestry, Sheri-Kashmir University of Agricultural
Science and Technology (SKUAST-Kashmir), extremely rough site conditions and can withstand the
191 121 Jammu and Kashmir, India extremes of cold climate and aridity.
e-mail: malikrashid2@gmail.com Chilgoza pine is an excellent source of fine turpentine
resin. The seeds and seed oil of the plant have medicinal
G. S. Shamet
Department of Silviculture and Agroforestry, Dr. Y. S. Parmar properties; the seeds are used as anodyne and stimulant and
University of Horticulture and Forestry, Nauni, the oil against wounds and ulcer. It is the only conifer
Solan-173 230 Himachal Pradesh, India species in North West Himalayas which provides edible
kernels/nuts rich in carbohydrate, fat, fiber and mineral
J. S. Butola
Herbal Research and Development Institute, matter. The fresh seeds also contain sugars (4.07 %), pro-
Mandal-Gopeshwar, Chamoli-246 401 Uttarakhand, India teins (13.03 %), oils (52.15 %) and moisture (25.36 %)

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1498 Trees (2013) 27:1497–1501

(Malik and Shamet 2009). Being nutritious and delicious, appropriate seed storage conditions for Chilgoza pine.
the seeds have high demand in local, national and inter- Therefore, an experiment was undertaken to standardize
national markets and fetch very good prices (Peltier and storage practices, i.e., regulation of temperature and use of
Dauffy 2009). Ecologically, the species is an excellent soil storage device to preserve and retain viability of the seeds
binder and prevent large scale soil erosion from the to meet plantation target, and thus conservation and sus-
otherwise lose and fragile strata particularly in dry tem- tainable utilization of the species.
perate and semi-arid regions. The natives have rights of
seed collection from natural forests growing in their
jurisdiction. Overall, the species has high potential and Materials and methods
keeps the promises live for the socio-economic develop-
ment of rural communities residing nearby Chilgoza Seed collection
forests.
Due to high demand, almost every cone of Chilgoza The present study was conducted in the laboratory of
pine is lopped retaining a very few inaccessible cones for Department of Silviculture and Agroforestry, Dr. YS Par-
natural regeneration (Singh et al. 1973). Moreover, some mar University of Horticulture and Forestry, Nauni-Solan,
seeds which fall on the ground are eaten by insects, birds H.P., India. During November 2004 and 2005, seeds of
and termites and also get easily infested by various fungi P. gerardiana were collected from the dry temperate zone
especially during transportation and normal ambient stor- ranging from 1,700 m to 2,900 m asl in District Kinnaur of
age (Singh and Gupta 1989). According to Peltier and H.P. The seeds were dried at room temperature for a week.
Dauffy (2009), the rural communities have become less Damaged and deformed seeds were eliminated.
dependent on the Chilgoza trade and sell the nut harvest
contracts to private contractors who employ foreign Experimental design
workers, cut many branches, ‘‘consequently’’, natural
regeneration of the species is being fatally hampered; the The seeds were stored in five storage containers: polythene
current mean natural regeneration status being only 15 % bags (C1), plastic jars (C2), canvas bags (C3), earthen pots
(Malik et al. 2012). Besides, the species has erratic and (C4) and tin boxes (C5). The containers were kept under
infrequent seed years and dormancy related problems four storage temperatures: 19–22 °C (room temperature,
which also prevents its regeneration in natural habitat T1), 0 ± 1 °C (T2), -4 ± 1 °C (T3) and 4 ± 1 °C (T4).
(Malik and Shamet 2008; Malik et al. 2008). Long years The experiment, thus, consists of 20 treatments each rep-
back, Singh et al. (1973) estimated that the area under licated thrice in a split plot design with temperature as
Chilgoza forest has shrunk to about 2,000 ha in H.P. The main plot and container as sub plot, their being 1,800 seeds
species is thus facing higher risk of extinction and cate- per plot. Prior to storage, viability, moisture content and
gorized as endangered species (Sehgal and Sharma 1989). germination attributes of the seeds were tested using 50
It has already been included in The Red Data Book (Dogra seeds per replication (in triplicate) for each test following
1964). guidelines of ISTA (2011). The viability was determined
Considering limited distribution, failure of natural by immersing seeds in 0.5 % aqueous solution of 2,3,5,
regeneration, endangered status and over all, social, eco- triphenyltetrazolium chloride (Tz). After storage, the via-
nomical and ecological significance of the species, there is bility, moisture content and germinability of the seeds were
an urgent need to undertake its artificial regeneration in ex- determined at every 3 months’ intervals, i.e., 0, 3, 6, 9 and
situ conditions. Developing vigorous seedlings in nursery 12 months for 1 year. To examine viability and moisture
and their plantation in suitable habitats has been viewed content 15 seeds per replication (in triplicate) were used.
one of the viable options for its conservation and effective For germination test, seeds were disinfected with
management. Efforts have been made by the authors to 0.04 % HgCl2 (1 min), washed thoroughly with double
develop mass scale nursery of the species (Malik et al. distilled water (DW), and placed in Petri dishes lined with
2009). Although, seeds of the species show moderate Qualigens (615 A) filter paper (3 replications/treatment, 50
germination but do not retain better germinability under seeds/replication). The Petri dishes were placed inside
normal ambient storage (Singh 1982; Singh et al. 1992; germinator (25.0 ± 1.0 °C) in complete randomized
Bhardwaj and Gupta 1998; Malik and Shamet 2009). Seed design and monitored daily. The filter papers were moist-
storage is an important aspect of any sound management ened daily using DW. Seeds were considered germinated
programme, especially in conifers species where seed upon the radical emergence reached twice the seed size.
supply is often limited due to low production and long seed The data were recorded daily up to 28 days to calculate
years (Allens 1962; Robbins 1984). A perusal of literature germination per cent and other parameters (AOSA 1981–
revealed that there is hardly any information available on 1984). Various parameters of seed germination, viz., mean

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Trees (2013) 27:1497–1501 1499

germination, germination value (GV) and germination germination parameters. The seeds stored at T2 (0 ± 1 °C)
index (GI) were calculated. The GV which is an index showed better results in terms of higher mean germination
combining speed and completeness of seed germination (73.7 %, Fig. 1), GV (9.5) and GI (1.01) at 3 months of
was calculated as GV = PV 9 MDG following Czabator storage. This was, however, followed by T3 (-4 ± 1 °C)
(1962), where, PV = peak value of germination and with T1 (room temperature) showing least germinability
MDG = mean daily germination or final daily germination with respect to all parameters. The better performance of
speed, while the GI was analyzed as the total number of T2 storage temperature might be attributed to improvement
seeds germinated at the end of experiment with the time in biological processes and moisture content for the
taken for 50 % germination. The moisture content was development of embryo thereby giving better germinability
determined by toluene distillation method given by Inter- in the seeds (Malik and Shamet 2009). Seed stored at low
national Seed Testing Association (2011). The whole temperature maintained seed viability and also may reduce
experiment was repeated once to confirm consistency of seed metabolism including respiration, decreasing oxida-
the results in next season. tion and minimizing issue damage (Robbins 1984). There
was, however, a steady decline in almost all germinability
Data analysis parameters as the storage period was extended from 3 to
12 months. The findings are in agreement with the work of
The data were analyzed statistically using MS Excel 2000. Gordon et al. (1972) who reported that Pinus merkusii seed
The 2 years results (2005 and 2006) were subjected to the storage responds well to 2 °C, shown 80 % germination
analysis of variance (ANOVA) technique. To determine even after 3 years of storage, while seed storage at room
significant difference among mean values of the various temperature exhibits significant loss in germination only
treatments, the Duncan test (P \ 0.05) and (P \ 0.01) was after 3–4 months in the species. Likewise, Butola and Sa-
used. Data as percentages were transformed to arcsine mant (2006) experimenting with Saussurea costus seeds
(9/100)0.5. reported 18-months storage in air tight plastic containers at
4 °C as suitable practice giving 100 % germination. On the
other hand, Effendi and Sinaga (1996) while experimenting
Results and discussion with red wood reported that 4 months storage at 4 °C
proved best resulting in maximum germinability in the
The freshly collected seeds of P. gerardiana showed species. He further concluded that germination capacity,
95.0 % viability, 25.4 % moisture content, and 85.3 % germination value and mean daily germination decreased
mean germination, 1.7 GV and 0.40 GI under laboratory rapidly after 4 months of storage. The results are also in
conditions. The viability was significantly (P \ 0.05) harmony with the findings of Yap and Wang (1983) in
decreased to 92.5, 90.0, 80.0 and 64.2 % after 3, 6, 9 and pinus caribaea and Tectona grandis. The loss of germi-
12 months of storage, respectively under room temperature nability at room temperature might be due to wide tem-
Malik (2007). perature fluctuation, activity of mycoflora and fauna
present in the seeds and depletion of food reserves ren-
Effect of storage temperatures on germination dering the seeds as non-viable. The results are in agreement
with the findings of Gupta and Raturi (1975) who con-
The statistical analysis of the data revealed that storage ducted viability tests in a number of forest tree seeds.
temperature exerts significant (P \ 0.05) influence on
Effect of storage containers on germination

The seeds stored in different types of storage containers

showed significant (P \ 0.05) difference in germinability.
Notably, C4 (earthen pots) showed the highest values with
respect to mean germination (78.0 %, Fig. 2), GV (10.2)
and GI (1.04) at 3 months of storage. There was an
increase of 42.0 % in mean germination and 63.9 % GV
over the respective least values obtained in C4 (tin boxes).
There was a steady decline in germination as the storage
Fig. 1 Effect of storage temperatures (T) on mean germination in was extended from 3 to 12 months. The findings are in line
P. gerardiana seeds. Symbols used: T1 room temperatures, T2
with those of Barton (1954) in Pinus pseudotsuga,
0 ± 1 °C, T3 -4 ± 1 °C, T4 4 ± 1 °C, 3 m 3 months after storage, 6
m 6 months after storage, 9 m 9 months after storage, 12 m 12 months P. ponderosa and Tsuga heterophylla and Donald and
after storage Jacobs (1990) in Pinus elliottii, P. patula, P. radiate and

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1500 Trees (2013) 27:1497–1501

Harrington 1969 and Malik and Shamet 2009), besides

entry of mycoflora and fauna in the seeds (Bhardwaj and
Gupta 1998). The results, thus, get support from the work
of Bhardwaj and Gupta (1998) who reported that cloth bag
storage of P. gerardiana seeds exhibits minimum per cent
incidence of fungi during 9 months storage. The maximum
spoilage was recorded on seeds stored in tin boxes, plastic
jars and polythene bags. The results are also in line with
those of Barton (1954) and Donald and Jacobs (1990) in
forest tree seeds.
Fig. 2 Effect of storage container (C) on mean germination in P.
gerardiana seeds. C1 polythene bags, C2 plastic jars, C3 canvas bags,
C4 earthen pots, C5 tin boxes, 3 m 3 months after storage, 6
m 6 months after storage, 9 m 9 months after storage, 12 m 12 months Interaction effect of storage temperatures
after storage and containers (T 9 C) on seed moisture content
and germination
P. taeda seeds. The better performance of earthen pots’
storage might be due to the less reduction in moisture, better The appraisal of data revealed that interaction between
exchange of gases and of biochemical parameters in the storage temperatures and containers had a significant
seeds (Yap and Wang 1983 and Malik and Shamet 2009). (P \ 0.05) influence on moisture content and germinability
The low germinability registered in the seeds stored in (Table 1) in P. gerardiana seeds. The maximum moisture
tin boxes might be ascribed to more reduction of moisture, content was observed in the seeds stored in T2C4
resulting in the loss of seed viability (Koostra and (0 ± 1 °C 9 earthen pots), i.e., 13.9, 12.2, 8.2 and 6.6 %,

Table 1 Interaction effect of storage temperatures (T) and containers (C) on various parameters of seed attributes in P. gerardiana
Treatments Mean germination (%) Germination value Germination index Moisture content (%)
(T 9 C)
3m 6m 9m 12 m 3m 6m 9m 12 m 3m 6m 9m 12 m 3 m 6m 9m 12 m

T1C1 60.8 50.8 29.2 6.7 6.0 4.5 1.4 0.1 0.74 0.66 0.34 0.05 8.96 6.47 4.41 2.72
T1C2 47.5 45.0 20.0 4.2 4.1 3.5 0.7 0.1 0.65 0.54 0.20 0.05 9.14 5.93 4.45 3.11
T1C3 70.8 48.3 19.2 10.0 7.6 4.1 0.6 0.2 0.85 0.60 0.17 0.10 9.67 6.96 3.79 2.84
T1C4 75.0 55.0 20.8 10.8 12.3 5.0 0.7 0.2 1.18 0.62 0.21 0.08 10.67 7.68 4.83 3.02
T1C5 36.7 35.0 14.2 4.2 2.5 2.5 0.4 0.04 0.60 0.49 0.14 0.04 8.55 5.75 4.01 2.09
T2C1 70.0 61.7 32.5 18.3 8.6 6.9 1.7 0.9 1.02 0.84 0.40 0.25 11.06 7.86 6.21 3.32
T2C2 80.0 60.8 31.7 29.2 9.2 7.2 2.0 1.5 0.65 0.92 0.44 0.36 12.14 8.33 6.05 3.29
T2C3 85.3 84.2 72.5 47.5 11.4 11.5 7.8 3.8 1.10 1.10 0.84 0.58 11.84 10.20 8.00 6.54
T2C4 88.3 86.7 73.3 58.3 12.3 12.0 8.3 5.2 1.22 1.09 0.89 0.71 13.87 12.17 8.24 6.55
T2C5 49.2 47.5 31.7 23.3 4.1 4.2 2.1 1.0 1.07 0.60 0.44 0.27 10.73 8.10 5.04 2.98
T3C1 57.5 54.2 40.8 30.0 6.0 4.8 3.0 1.6 0.86 0.68 0.55 0.34 12.23 10.25 6.15 3.61
T3C2 63.3 46.7 38.3 30.8 6.4 3.8 2.5 1.6 0.82 0.59 0.44 0.36 13.72 10.15 6.50 3.70
T3C3 81.2 76.7 50.8 34.2 11.2 9.4 4.0 1.9 1.05 0.97 0.62 0.37 12.17 10.04 7.75 6.39
T3C4 84.2 82.5 71.7 47.5 11.2 11.0 7.5 3.3 1.18 1.13 0.83 0.51 13.48 11.42 7.45 6.21
T3C5 45.8 48.3 36.7 25.0 3.6 4.5 2.5 1.0 0.56 0.69 0.47 0.27 12.00 8.76 5.20 3.05
T4C1 79.2 47.5 34.2 19.2 11.0 4.2 1.9 0.9 1.05 0.76 0.37 0.21 10.83 7.63 5.41 2.33
T4C2 65.8 57.5 39.2 30.0 7.4 5.8 2.6 1.5 0.92 0.82 0.41 0.34 10.04 7.48 5.65 2.12
T4C3 56.7 50.8 41.7 29.2 5.7 4.7 2.7 1.4 0.78 0.67 0.42 0.33 9.90 6.49 5.50 2.30
T4C4 58.3 46.7 39.2 25.0 6.2 4.1 2.4 1.0 0.85 0.65 0.43 0.25 12.42 7.61 5.57 2.83
T4C5 49.2 45.0 30.0 13.3 4.4 3.4 1.5 0.3 0.62 0.56 0.22 0.08 9.98 5.80 4.30 2.14
SE 2.22 1.46 2.34 1.76 0.74 0.47 0.53 0.23 0.06 0.03 0.04 0.03 0.52 0.35 0.36 0.20
CD0.05 4.81* 3.16* 5.07* 3.81* 1.60* 1.01* 1.15* 0.49* 0.13 0.06 0.08 0.06 NS 0.75* 0.78* 0.43*
M months after storage, T1 room temperatures, T2 0 ± 1 °C, T3 -4 ± 1 °C, T4 4 ± 1 °C, C1 polythene bags, C2 plastic jars, C3 canvas bags, C4
earthen pots, C5 tin boxes, SE standard error, CD critical difference
* Significant difference between means; NS = non significant

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Trees (2013) 27:1497–1501 1501

it was significantly (P \ 0.05) higher than that in T1C5 Gupta BN, Raturi AS (1975) Tetrazolium staining of seeds for
(room temperature 9 tin boxes) after 3, 6, 9 and 12 months interpretation of viability of six Indian tree species. Ind For
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58.5 % in mean germination and 76.6 % GV over T1C5 membrane lipids of Cucumis sativus seeds. Inter Seed Test Asso
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