Int. J. Med. Arom. Plants, ISSN 2249 – 4340
RESEARCH ARTICLE
Vol. 2, No. 3, pp. 468-475, September 2012
Effect of light and temperature on seed germination of important medicinal and aromatic plants in north western Himalayas
Rakesh KUMAR*, Saurabh SHARMA
Natural Plant Products Division, CSIR-Institute of Himalayan Bioresource Technology, (Council of Scientific
and Industrial Research), PO Box No. 6, Palampur, 176 061 (H.P.) India
*Corresponding Author, Tel.: +91-1894-233341, Fax: +91-1894-230433
Article History: Received 27th June 2012, Revised 22nd August 2012, Accepted 23rd August 2012.
Abstract: Experiments were conducted during January 2009 at CSIR-Institute of Himalayan Bioresource Technology,
Palampur, (HP) India to study the effect of light and temperature on seed germination of important medicinal and aromatic plants viz., Stevia rebaudiana, Salvia sclarea and Tagetes minuta in sand and filter paper. The treatments comprised of two factors viz., light and temperature regimes under room temperature, 20° C and 10° C. Maximum seed germination was observed in the seed which were placed for 2 days open in room temperature and then placed at 20° C in
continuous light in all the three plant species in both media except salvia seeds which recorded maximum germination
(76%) when placed in room temperature under light condition in sand. S. rebaudiana recorded 58.7 % and 54.7% germination in sand and paper media, whereas, T. minuta recorded 45.3% and 22.7% germination in both medias, respectively
when placed in room temperature for 2 days and then incubated at 20° C in continuous light. Among three plant species,
maximum seed germination was recorded by the seeds of S. sclarea in paper (89.3%). Seeds placed in sand medium recorded higher germination percentage than the seeds placed on paper in case of Stevia and Tagetes, whereas, in Salvia the
trend was reverse. Light is not required for the seed germination of tagetes.
Keywords: Light; temperature; germination; Stevia rebaudiana; Salvia sclarea; Tagetes minuta.
Introduction
Stevia rebaudiana Bertoni is a perennial
plant of family Asteraceae and popularized as
sweet herb of Paraguay (Brandle and Rosa
1992). The leaves of stevia have commercial
importance due to the presence of non-caloric
diterpenes and sweet glycosides, especially
stevioside and rebaudioside-A which are ~300
times sweeter than sugar without any sideeffects. Chemical constituent which is responsible for sweetness is stevioside and is extracted
from leaves in the form of white powder
(Kinghorn and Soejarto 1985). Seeds are contained in slender achenes, about 3 mm in length.
Each achene has about 20 persistent pappus
bristles (Figure 1A). Weight of 1000 seeds was
0.39 g. Seed germination of stevia is often poor
(Miyazaki and Wantenabe 1974).
Salvia sclarea L. is an important aromatic
plant commonly called clary sage belongs to
family Lamiaceae. It is a xerophyte, biennial
plant and native to southern Europe (Carruba et
al. 2002). Plant is used worldwide as a source of
essential oil and other compounds derived from
different parts of the plant (Lawrence 1992). It
is also used in folk medicines, pharmaceuticals,
cosmetics, chemicals, food and many other industries. Generally, oil is obtained from inflorescence of salvia plant and the main oil constituent are linalool and linalyl acetate known
for anti-bacterial (Deans and Svoboda 1988) and
accaricidal (Perrucci et al. 1994) properties,
while Germacrene-D is known for its pheromone activity (Bruneton 1995). After the essential oil extraction, the crude material left behind
is a source of sclareol which is converted to
sclareolide which is used to flavor tobacco
(Werker et al. 1985; EINECS 199o). The
sclareol and ambrox derived from this plant are
sold at premium prices in international market.
It is mainly propagated by seeds. Seeds of S.
*Corresponding author: (E-mail) rakeshkumar <@> ihbt.res.in
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Int. J. Med. Arom. Plants
sclarea are oval in shape having 2.03 mm diameter (Figure 1B) and weight of 1000 seeds was
4.27 g.
A
B
469
Effect of light and temperature on seed germination
for its essential oil (Sastri 1982). It is an aromatic, branched, erect, annual herb. Essential oil
obtained from T. minuta is known as Tagetes oil
which is now a days finds a good market in perfumery industry, flavor industry and food products including cola, alcoholic beverages, candy,
baked goods etc. Oil also possesses biological
activity, therefore, has better scope in pharmaceutical industry. Achenes are brown 10-12 mm
long, with a pappus of 1-4 tiny scales and 0–2
retrosely serrulate, awns 1-3 mm long (Figure
1C). Weight of 1000 seeds was 0.57 g.
Germination is regarded as phenomenon
which commence with uptake of water by a quiescent dry seed and terminates with the elongation of the embryonic axis (Bewley 1997). Information on seed germination and viability is
important for selection of methods (direct sowing or transplants) for crop establishment, since
it can influence further crop uniformity in the
field (Zutic and Dudai 2008). Effect of light
and temperature on seed germination of many
plant species has been studied earlier by many
workers (Taylorson and Hendricks 1972; Baskin
and Baskin 1998; Schutz et al. 2002; Kambizi,
et al. 2006; Ortega-Baes and Rojas-Arechiga
2007; Kandari et al. 2008) but no efforts have
been made to study the effect of light and temperature on S. rebaudiana, S. sclarea and T.
minuta. Understanding the light requirements
for seed germination is also important for these
species. The objective of this study was, therefore, to investigate germination response of
seeds of these plant species to different temperature regimes and light conditions.
Materials and methods
Seed collection
C
Figure 1: Seeds of A, Stevia rebaudiana, B.
Salvia sclarea, C. Tagetes minuta
Tagetes minuta L. generally known as wild
marigold belongs to family Asteraceae, valued
Kumar and Sharma
The mature seeds of S. rebaudiana, S.
sclarea and T. minuta were collected from experimental fields of the Institute of Himalayan
Bioresource Technology (CSIR) located at
Palampur (32° 06′ N, 76° 33′ E), at 1300 m amsl
during September-October 2008. After collection, immature seeds and those damaged by insects were discarded. They were air dried and
stored in closed plastic bags at room temperature. Before the start of the experiment in January 2009, the seeds were surface sterilized by
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Effect of light and temperature on seed germination
Int. J. Med. Arom. Plants
soaking in 1% mercuric chloride (HgCl2) for 1
minute and subsequently rinsed thoroughly with
sterilized water and dried in shade.
Viability test
To ensure that the seeds used for the experiment were viable and of high quality, the sample lot of three plant species was subjected to
viability test using the tetrazolium technique
(Grabe 1970). The seeds were imbibed for 24 h
in water, cut along the margin without damaging
the embryo and soaked in colourless 0.1% solution of 2,3,5-triphenyltetrazolium chloride
(TTC) solution for 16 h at 25° C in the dark.
The seeds were then removed from TTC solution, washed with distilled water and soaked in
10 ml of 95% ethyl alcohol to permit direct observation of the embryo. The embryos of viable
seeds appeared reddish in colour. Three replicates of 50 seeds each were examined.
en method (ISTA 1985). Moisture content for
the seeds of S. rebaudiana, S. sclarea and T.
minuta was 32%, 6% and 15%, respectively.
Filter papers and sand were moistened daily using the distilled water. Germinating seeds were
counted daily. A seed was considered germinated when the tip of the radical had grown out of
the seed coat (Wiese and Binning 1987; Auld et
al. 1988).
Table 1: Treatments used in the germination
test
S.No.
T1
T2
T3
T4
T5
T6
T7
T8
Temperature & Light conditions
Room temperature (RT) open
Room temperature (RT) continuous dark
2 days open (RT) then continuous dark
2 days open (RT) then 20° C
3 days open (RT) then continuous dark
3 days open then (RT) 20° C
3 days open (RT) then 10° C
3 days dark (RT) then 10° C
Germination experiments
Statistical analysis
Seeds of the target plant species were exposed to different light and temperature treatments (Table 1) placing them in Whatman No.1
filter paper moistened with distilled water in
sterilized petri dishes (9 cm diameter). Seeds
were also grown in sterilized river sand placed
in the petri dishes for simulating the pattern and
pace of germination under field conditions. This
experiment was completed within 18 days after
sowing in paper and sand medium. There were
total 8 treatments (Table 1) for all plant species
and each treatment had 3 replications and each
one contain 25 seeds. Seeds were sown on 19
January 2009 in the petri dishes for all treatments. The treatments comprised of two factors
viz., light and temperature regimes. Under light
conditions; room temperature, 20° C (constant
temperature) and 10° C were studied. Besides,
the effect of dark conditions (room temperature)
was also studied. To avoid exposure of seeds to
light in treatments T2, T3 and T5, Petri plates
were covered with black polythene sheets from
all sides. Average daily maximum temperature
of the lab during the experiment was 15.2° C.
The moisture content of the seeds of each species was determined as per high constant air ov-
The germination was calculated according to
the following formula;
Kumar and Sharma
Germination (%) = (Number of germinating
seeds/ number of viable seeds initiated) X 100
The data were tested at 5% level of significance to interpret the significant findings. The
data were compounded for analysis of variance
(ANOVA) by software SYSTAT-12 of Systat
Software Inc., USA. The percentage data from
the different germination tests were arcsine
transformed.
Results and discussion
Stevia rebaudiana
A cursory glance of Table 2 reveals that
minimum 4 days were taken to first seed germination of stevia in both sand and paper, while
maximum 5.7 and 8 days were taken in sand and
paper method respectively. Stevia seeds germinated fast when they were placed in room temperature in open (T1) as compared to continuous
dark conditions in room temperature (T1).
When the seeds were incubated for 2 days open
in room temperature and then placed under dark
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conditions (T3), germination of first seed was
enhanced as compared to continuous dark in
room temperature (T2). Low temperature (10°
C) significantly delayed the germination of first
seed as compared to other treatments. This implies that stevia seed germination is hampered
by low temperature. When the seeds were
placed under room temperature and dark conditions (T2), the seed germination was fast as
compared to seed, which were placed for 3 days
in room temperature and then at 10° C continuous dark conditions (T8). Maximum germination in sand (58.7%) and paper (54.7%) methods
were recorded when the seeds were placed in 2
days open in room temperature and then placed
at 20° C in continuous light (Figure 2).
Kawatani et al. (1977) also reported that optimum temperature for seed germination of stevia
was 20° C and seeds germinated better in light
than darkness. Takahashi et al. (1996) compared
germination performance of stevia under different temperatures (20, 25, 30° C) and reported
that at 25° C, 63.2% of maximum germination
(90.3%) occurred after 101.4 hours. Minimum
germination percentage in sand (24.0%) was
recorded in treatment T5 i.e. 3 days open then
continuous dark in room temperature while in
paper method, minimum germination (31.3%)
was recorded in T8 treatment i.e. 3 days in dark
at room temperature and then at 10° C continuous dark (Table 3). This shows that both light
and temperature are essential for enhancing seed
germination in stevia seeds and for seed germination 20° C is ideal temperature. At 10° C
though the seed germinated but rate of germination was low and seed took longer time to germinate. Aamlid and Arntsen (1998) reported
that dark germination was inferior to germination in light/ dark cycles at constant temperatures of 15° C and higher, but light showed no
advantage at alternating temperature in Poa
pratensis. Goettemoeller and Ching (1999)
while using two types of stevia seeds (black and
tan) reported that germination in the dark was
higher for black as compared to tan seed. Seeds
of halophyte shrub Halocnemum strobilaceum
showed higher germination in light than in
darkness and at high than at low temperatures
(Qu et al. 2008).
Kumar and Sharma
471
Effect of light and temperature on seed germination
Salvia sclarea
S. sclarea is a temperate plant and grows
naturally in places above 2000 m amsl. It is evident from the data presented in Table 2 that
minimum 4 days were taken to first seed germination in paper, while 24 hours more were taken
by sand medium. Likewise, seeds took 24 h
more when they were incubated at low temperature (10° C) as compared to room temperature
and 20° C. Light had no impact on first seed
germination of salvia but low temperature elongates seed germination by 24 h. Maximum germination percentage (89.3%) of salvia seeds
was recorded when they were placed in paper at
2 days open in room temperature and then incubated at 20° C under continuous light, whereas,
minimum (30.7%) in T8 when seeds were
placed in dark conditions for 3 days in room
temperature and then placed at 10° C in continuous dark. However, in sand, maximum germination (76.0%) was recorded when the seeds
were placed at room temperature (15° C) open
under light (T1) followed by T8 (73.3%) when
seeds were placed for 3 days in dark conditions
and then at 10° C under continuous dark conditions. However, minimum germination (57.7%)
was recorded by T7 when the seeds were placed
3 days open and then at 10° C continuous dark
(Table 3). Salvia seed germination was not
hampered by light but it is the temperature,
which influences germination. Light is one of
the most important environmental factors that
interact with temperature to regulate seed germination in many plant species (Baskin and
Baskin 1998), but light requirement for germination may vary with temperature (El-Keblawy
and Al-Rawai 2005). This result implies that the
seeds of S. sclarea are light-dependent for germination but exposure to regimes of alternating
temperatures may probably suppress the effect
of the photoperiod. In a similar work, Zaia and
Takaki (1998) observed that the seeds of
Tibouchina pulchra and T. granulose did not
germinate in darkness at constant temperatures,
while 40% germination was recorded under a
regime of alternating temperatures in the dark.
Senel et al. (2007) also reported that maximum germination percentage was obtained at
20° C for Salvia dicroantha. While studying the
effect the effects of 12 temperature treatments
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Effect of light and temperature on seed germination
Int. J. Med. Arom. Plants
(between 5 and 40° C) on germination in light
(12 h) and darkness on S. officinallis and S.
sclarea, Oberczian and Bernath (1988) reported
that the best temperature for S. sclarea were
15-20° C. Alternating day/night temperatures of
30° C /20° C were as effective for S. officinalis
as a constant 25° C, but for S. sclarea they were
less satisfactory than a constant 15-20° C. Zutic
and Dudai (2008) tested germination of 5-year
old seed of S. officinalis at temperatures of 20,
25 or 30° C under continuous light or in dark
and reported that the highest germination rate
(63%) was observed at 25° C with continuous
light.
Stevia seed germ ination in sand
T1
T2
T3
T4
T5
T6
Stevia seed germ ination in paper
T7
T8
T1
70
T2
T4
T5
T6
T7
T8
60
60
50
50
40
40
30
30
20
20
10
10
0
0
0
2
4
6
8
10
12
14
16
0
18
Salvia seed germ ination in sand
T1
T2
T3
T4
T5
T6
2
4
6
8
10
12
14
16
18
Salvia seed germ ination in paper
T7
T8
T1
Germination (%)
T3
80
100
60
80
T2
T3
T4
T5
T6
T7
T8
60
40
40
20
20
0
0
2
4
6
8
10
12
14
16
0
18
0
Tagetes seed germ ination in sand
T1
T2
T3
T4
T5
T6
T7
T8
T1
25
40
20
30
15
20
10
10
5
0
0
2
4
6
8
10
12
14
4
6
8
10
12
14
16
18
Tagetes seed germ ination in paper
50
0
2
16
T2
0
18
2
T3
4
T4
6
T5
8
10 12
T6
T7
T8
14 16 18
Days
Figure 2: Effect of light and temperature on seed germination of stevia, salvia and tagetes in sand
and paper.
Kumar and Sharma
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Effect of light and temperature on seed germination
Int. J. Med. Arom. Plants
Table 2: Days taken to first seed germination of stevia, salvia and tagetes
Treatments
T1
T2
T3
T4
T5
T6
T7
T8
CD 5%
Sand
4.0
4.3
4.0
5.0
4.0
5.0
5.3
5.7
NS
Stevia
Paper
4.0
8.0
4.3
4.0
5.7
4.0
7.3
8.0
1.8
Pooled
4.0
6.2
4.2
4.5
4.8
4.5
6.3
6.8
1.3
Sand
5.0
5.0
5.0
5.0
5.0
5.0
5.3
5.0
NS
Salvia
Paper
4.0
4.0
4.0
4.0
4.3
4.0
5.0
5.0
0.4
Pooled
4.5
4.5
4.5
4.5
4.7
4.5
5.2
5.0
0.2
Sand
5.0
5.0
4.7
4.0
4.7
5.0
8.0
11.7
2.5
Tagetes
Paper
Pooled
5.3
5.2
8.0
6.5
8.7
6.7
4.7
4.3
8.0
6.3
6.0
5.5
8.0
8.0
14.7
13.2
3.1
1.9
NS- Not significant
Table 3: Effect of light and temperature on seed germination (%) of stevia, salvia and tagetes
Treatments
T1
T2
T3
T4
T5
T6
T7
T8
CD (5%)
Stevia
Sand
Paper
Pooled
37.6
46.1
41.9
(37.3)* (52.0)
(44.7)
37.3
43.0
40.2
(37.3)
(46.7)
(42.0)
41.5
47.7
44.6
(44.0)
(54.7)
(49.3)
50.0
47.7
48.9
(58.7)
(54.7)
(56.7)
29.0
45.4
37.2
(24.0)
(50.7)
(37.3)
38.3
43.8
41.1
(38.7)
(48.0)
(43.3)
32.8
38.3
35.5
(29.3)
(38.7)
(34.0)
37.6
31.0
34.3
(37.3)
(26.7)
(32.0)
9.8
9.9
9.5
Salvia
Sand
Paper
Pooled
61.0
46.2
53.6
(76.0)
(52.0)
(64.0)
58.6
54.7
56.7
(72.0)
(66.7)
(69.3)
50.8
62.4
56.6
(60.0)
(77.3)
(68.7)
51.5
71.0
61.3
(61.3)
(89.3)
(75.3)
54.8
52.4
53.6
(66.7)
(62.7)
(64.7)
57.3
67.5
62.4
(70.7)
(85.3)
(78.0)
50.0
40.0
45.0
(58.7)
(41.3)
(50.0)
59.0
33.2
46.1
(73.3)
(30.7)
(52.0)
NS
10.3
9.5
Sand
34.4
(33.3)
35.2
(33.3)
35.9
(34.7)
42.3
(45.3)
40.7
(42.7)
27.5
(21.3)
26.5
(20.0)
22.5
(14.7)
10.2
Tagetes
Paper
Pooled
14.6
24.5
(9.3)
(21.3)
26.5
30.8
(20.0)
(26.7)
20.7
28.3
(13.3)
(24.0)
28.3
35.3
(22.7)
(34.0)
25.6
33.1
(18.7)
(30.7)
28.0
27.8
(22.7)
(22.0)
20.1
23.3
(12.0)
(16.0)
14.8
18.6
(6.7)
(10.7)
NS
10.0
Values in parenthesis are the original means
Tagetes minuta
Tagetes seeds took significantly highest
number of days for first seed germination in
both sand and paper when they were placed in
continuous dark conditions in room temperature
for 3 days and then incubated at 10° C (T8),
whereas, significantly lower number of days
were taken by treatment T4 when the seeds were
placed in continuous light at 20° C in both sand
and paper Table 2. At low temperature (10° C)
when placed in continuous dark, seeds did not
germinate up to 11.7 and 14.7 days in sand and
paper, respectively. T. minuta seeds germinated
best at a constant temperature 20° C in sand and
paper. It is observed that maximum and minimum germination percentages in sand were
Kumar and Sharma
found to be 45.33 % in treatment T4 and 14.7 %
in treatment T8, respectively. On the other hand
maximum and minimum germination percentage in paper are 22.7% in treatment T4 and T6
and 6.7% in treatment T8 i.e. 3 days under dark
conditions in room temperature and then at 10°
C for continuous dark. At room temperature under light conditions (T1) germination was poor
(33.3%) as compared to 3 days open and then
continuous dark (42.7%). These results shows
that germination of tagetes would not be inhibited under dark conditions but low temperature
below 15° C hamper the germination. Forsyth
and Van Staden (1983) also reported similar results. Drewes and Ataden Van (1990) reported
that GA3 and GA4+7 accelerated germination
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of tagetes seeds at 25° C than 35° C. When the
seeds were kept under dark they recorded germination. These results show that germination
of tagetes would not be inhibited under dark
conditions suggesting that light is not a requirement for germination of tagetes seeds.
Conclusion
Maximum seed germination was observed in
the seed which were placed for 2 days open in
room temperature and then placed at 20° C in
continuous light in all the three plant species in
both media except salvia seeds which recorded
maximum germination (76%) when placed in
room temperature under light condition in sand.
Light is not required and temperature is limiting
factor for seed germination of tagetes.
474
Effect of light and temperature on seed germination
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J.
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Pharmacognosy,
phytochemistry,
medicinal
plants.
Lavoisier: Paris, 401.
Carruba, A. Torre, R., Piccaglia, R., Marotti, M.
2002. Characterization of an Italian biotype
of clary sage (Salvia sclarea L.) grown in a
semi-arid Mediterranean environment. Flavour and Fragrance Journal, 17:191-194.
Deans, S.G., Svoboda, K.P. 1988. Antibacterial
activity of French tarragon (Artemisia
dracunculus Linn.) essential oil and its
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Horticulture Science, 63: 503-508.
Drewes, F.E., Ataden Van, J. 1990. Germination
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Acknowledgement: Authors are grateful to the
Director, CSIR- IHBT, Palampur, HP, India for
providing necessary facilities and also thankful
to DST, New Delhi, India for financial support
to undertake this study. This is IHBT Publication No. 1000.
EINECS, 1990. Annex to the official journal of
the European Communities, No. C 146 A.
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