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Natl. Acad. Sci. Lett. (July–August 2015) 38(4):301–304
Effect of Sulfuric Acid Treatment on Breaking of Seed Dormancy
and Subsequent Seedling Establishment in Zanthoxylum armatum
DC: An Endangered Medicinal Plant of the Himalayan Region
Sumit Purohit . S. K. Nandi
Lalit Giri . Arun Bhatt
. L. M. S. Palni .
Received: 21 January 2014 / Revised: 13 June 2014 / Accepted: 2 January 2015 / Published online: 6 May 2015
Ó The National Academy of Sciences, India 2015
Abstract An attempt has been made to influence seed
germination in Zanthoxylum armatum DC, a medicinally
important plant that produces seeds with hard coat and low
natural regeneration. Pretreatment of seeds with concentrated (98 %) or diluted (50 %) sulfuric acid (H2SO4) for
various time periods (1, 5, 10, 15, 20 and 25 min) and
subsequent sowing in the soil (under polyhouse conditions)
resulted in considerable improvement in germination as a
result of some treatments. Treatment with diluted (50 %)
H2SO4 (15 min) resulted in maximum germination
(93.3 %) along with mean germination time (MGT) of
149.5 days. The other treatments (50 % H2SO4 for 5, 10,
20 and 25 min) also improved germination, but to a lesser
extent (15–40 %) with MGT of 130–160 days. The values
were significantly (P \ 0.05) lower than the most effective
treatment (50 % H2SO4, 15 min). No germination was
recorded in other treatments including the untreated control. The growth of seedlings was monitored and showed
rapid increase in height and leaf numbers with 100 %
survival up to 2 years. The results clearly indicate a simple,
S. Purohit S. K. Nandi (&) L. M. S. Palni (&) L. Giri
G.B. Pant Institute of Himalayan Environment and
Development, Kosi-Katarmal, Almora 263 643, Uttarakhand,
L. M. S. Palni
L. M. S. Palni
Flat 1-A/B, Riturain Woldorf Compound, Nainital 263001,
College of Forestry and Hill Agriculture, Ranichauri,
Tehri Garhwal 249199, Uttarakhand, India
inexpensive and convenient method of improving and obtaining uniform seed germination for nursery raising.
Keywords Endangered Himalaya Seed germination
Sulfuric acid Zanthoxylum armatum
The genus Zanthoxylum (Family: Rutaceae), a dioecious
shrub to large tree, and characterized by sharp thorns on
either the stem or foliage , is distributed worldwide from
tropics to temperate regions. Zanthoxylum armatum DC
(syn. Z. alatum Roxb.; Hindi/Local name: Timur), is one of
the six species reported from the Indian Himalayan region
and out of eleven species found in India . The extract of
its bark, fruits and seeds is used in the Indian folklore
medicine, and has recently received much attention. It
grows up to 2.5–3.5 m occurring in hot valleys of the
subtropical Himalayan region from Jammu and Kashmir to
Khasi hills in Meghalaya between 1,000–2,100 m, and in
Eastern Ghats in Orissa and Andhra Pradesh around
1,200 m .
The medicinal properties of this genus, including Z.
armatum, are well known for the treatment of stomach and
tooth ache, intestinal worms, snake bites, rheumatism,
scabies, fever and cholera throughout Asia . The most
commonly used part is the seed pericarp, used as spice.
Moreover, on account of deodorant, disinfectant and antiseptic properties it is used in dental hygiene. Analysis of
Zanthoxylum extracts has revealed compounds with antibacterial and antioxidant properties along with those used
in the treatment of cancer, etc. [5–7]. High levels of linalool, an essential oil, have been reported from Z. armatum seeds .
Due to multiple utility of Z. armatum and its high cultural value for the locals, unsustainable harvest of above
ground parts, and low regeneration in nature , the plant
has been categorized as endangered . Although it
propagates through seed, germination is poor . Seeds of
other Zanthoxylum species also have low germination ,
attributed to hard seed coat, improper seed setting, seed
predation by insects, and presence of oily seed coat.
Although scarification (by physical or chemical means) and
treatment with plant growth regulators are known to influence seed germination in several species [12–16], such
treatments have not been reported for Zanthoxylum spp.
This study, therefore, examined the effect of acid treatment
for enhanced and uniform seed germination for raising
healthy seedlings of Z. armatum.
Ripe fruits (red colour) of Z. armatum DC were collected from Dummar in Munsyari (District Pithoragarh,
Uttarakhand; 1663 m; 30o060 16.000 N, 80o140 57.100 E) during
November, 2010. The seeds were carefully removed from
the pericarp, air dried (shade, 3 days; moisture content:
52 %), placed in air tight plastic bags and stored
(4 ± 1 °C). One hundred air dried seeds weighed 2.22 g.
After a month of storage at 4 ± 1 °C, seeds were taken
out, and 3000 seeds were placed in a 5-liter beaker containing
water (3L, 22 ± 1 °C, 24 h). Immediately the submerged
seeds (sinkers) were removed and the floaters (76 %) were
discarded. The sinkers were blotted dry, air dried
(22 ± 1 °C, 2 days) and treated either with concentrated
(98 %; sp. gr. 1.84; Merck) or diluted (50 %, v/v) sulfuric
acid for 1–25 min as follows: concentrated H2SO4 for 1, 5
and 10 min (T1, T2 and T3, respectively); diluted H2SO4
(50 %, v/v) for 5, 10, 15, 20 and 25 min (T4, T5, T6, T7 and
T8, respectively); only water (25 min) as control (T9).
Following treatment the seeds were washed thoroughly
with water (93) and sown in thermocole trays (32 cm
length, 32 cm width, 8 cm height; 16 cells/tray; cell diameter 11 cm, depth 7 cm; 3 seeds/cell) containing sieved
soil (forest soil: farm yard manure: sand; 1:1:1 by vol.) at
2.0 cm depth. The trays were placed inside a polyhouse
(9.2 m length, 3.7 m width, 1.8 m height on sides and
2.5 m height in the middle) in the Institute campus at KosiKatarmal, Almora (1150 m; 29°380 1500 N and 79°380 1000 E).
Germination was considered when the plumule emerged
above the soil); data was monitored daily up to 190 days
after sowing. The trays were regularly watered. The experiment was repeated with freshly collected seeds with
Three and a half months after germination, the seedlings
were transplanted into polythene bags (16 cm height; 8 cm
dia) containing sieved soil (as above) and kept in the same
polyhouse for 1 month. Subsequently these were transplanted into pots (20 cm height; 24 cm dia) containing
sieved soil and maintained in polyhouse for further 2 years.
During the entire period, growth (seedling/saplings height,
in cm) was monitored at 6 months interval.
S. Purohit et al.
Germination percentage (GP) and mean germination
time (MGT) were calculated for each treatment as follows:
GP % ¼ No: of germinated seeds
=Total no: of seeds sown 100
MGT ðdÞ ¼ Rðti ni Þ =Rni
where ti is the number of days starting from the day of
sowing and ni is the number of seed completing germination on day ti. Data were subjected to analysis of variance
(ANOVA) using SPSS version 7.5. Significance level was
determined (at P \ 0.05) and significant difference was
separated using Duncan’s multiple range test (DMRT).
Majority of seeds (76 %) collected from Dummar
floated on water indicating empty seeds. Seeds collected
from other populations in Munsyari region also exhibited
high percentage (70–80 %) of emptiness. Germination (%)
and MGT following different treatments is depicted in
Fig. 1. Among various treatments, T6 (50 % H2SO4 treatment for 15 min) resulted in highest germination (93.3 %)
with MGT of 149.5 days (Fig. 1). The other treatments
(T4, T5, T7 and T8) exhibited significantly (P \ 0.05)
lower germination (15–40 %) than T6, with MGT ranging
between 130 and 160 days. However, significant differences in percentage germination were not found between
T4, T5, T7 and T8. Germination failed to occur in remaining treatments including control, even after 190 days.
The seedlings (T6) initially raised in thermocole trays
(Fig. 2.1, 2.2), were transplanted into polythene bags and
then to pots (Fig. 2.3). The growth showed appreciable
increase in height along with leaf number. A mean height
of 51.73 cm (n = 20) was recorded with 100 % survival
even after 2 years (Fig. 2.4).
Z. armatum trees were found to bear male and female
flowers on separate individuals. Frequently, there is poor
Fig. 1 Seed germination (%) and mean germination time recorded in
Z. armatum seeds following different treatments. T1 Conc. H2SO4,
1 min; T2 Conc. H2SO4, 5 min; T3 Conc. H2SO4, 10 min; T4 50 %
H2SO4, 5 min; T5 50 % H2SO4, 10 min; T6 50 % H2SO4, 15 min; T7
50 % H2SO4, 20 min; T8 50 % H2SO4, 25 min, and T9 Control
(water, 25 min). Values are mean ± standard error; mean values
(n = 48) with the same letter over the graph/bar are not significantly
different (P \ 0.05) based on DMRT
Effect of Sulfuric Acid Treatment
Fig. 2 Seed germination and
seedling establishment in Z.
armatum. 1 Seed germination in
soil following 50 % H2SO4
treatment for 15 min
(bar = 1 cm), 2 Seedlings (as
in Fig. 2.1) after 30 days of
growth in a 16 cell thermocole
tray (bar = 3 cm), 3 Seedlings
growing in pots (bar = 7.5 cm),
4 A seedling after 2 years
growth (developed from T6
treated seeds; bar = 7.5 cm)
synchronization in flowering of male and female trees;
fruits formed without pollination result in empty seeds.
Further, seed infestation with insect larvae also results in
poor germination in several Zanthoxylum species .
Physical dormancy due to hard and impermeable seed coat
hinders uptake of water and diffusion of oxygen into the
embryo. A variety of pre-soaking treatments, namely sulfuric acid, hot water and mechanical scarification are
known to overcome coat imposed dormancy [14, 15, 18].
In this study scarification with 50 % sulfuric acid (15 min)
was most effective. In Entada africana (legume), exposure
of seeds to conc. sulfuric acid for 15 or 20 min resulted in
significantly higher germination (60 and 80 %, respectively) compared to 7 % in control . Moreover, conc.
sulfuric acid treatment (15 min) significantly improved
germination in Vigna spp. . Disintegration of the seed
coat as well as micropylar plug do facilitate subsequent
imbibition and germination of seeds treated with concentrated sulfuric acid .
Pretreatment of Z. armatum seeds with gibberellic acid
was ineffective (unpublished results) in improving germination. Various other treatments improved germination in
other species of Zanthoxylum, for example, cold stratification in Z. piperitum from 28 to 74 % [21, 22], washing
with soap solution in Z. mayarum from 5 to 100 % ,
and insecticide spray (just before seed collection) in Z.
flavum up to 70 % .
A major constraint in seedling establishment even after
successful germination is poor survival of seedlings, and
hence decline in the natural populations of Z. armatum. In
this study, a simple and inexpensive method of achieving
over 90 % seed germination is being reported with subsequent hardening and seedling growth under polyhouse
conditions; 100 % seedling survival was found even up to
2 years of growth. This method can, therefore, be adopted
for nursery raising of this multiutility species for large
Acknowledgments The authors are thankful to the Director, G.B.
Pant Institute of Himalayan Environment and Development, KosiKatarmal, Almora, for facilities; Ministry of Environment & Forests,
Govt. of India, New Delhi and UCOST, Dehradun are thanked for
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