African Journal of Biotechnology Vol. 10(83), pp.

19407-19414, 21 December, 2011

Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB11.1311
ISSN 1684–5315 © 2011 Academic Journals

Full Length Research Paper

Studies on seed germination and in vitro shoot

multiplication of Satureja khuzistanica Jamzad, an
important medicinal plant
Parvin Ramak1, Mozafar Sharifi1*, Shahrokh Kazempour Osaloo1, Hassan Ebrahimzadeh2 and
Mehrdad Behmanesh3
1
Department of Plant Biology, Faculty of Biological Sciences, Tarbiat Modares University, P. O. Box 14115-154, Tehran,
Iran.
2
Department of Botany, School of Biology, College of Science, University of Tehran, P. O. Box 14155-6455, Tehran,
Iran.
3
Department of genetics, Faculty of Biological Sciences, Tarbiat Modares University, P. O. Box 14115-154, Tehran, Iran.
Accepted 12 October, 2011

Satureja khuzistanica Jamzad is an important multipurpose medicinal plant in Iran. The essential oil of
S. khuzistanica is characterized by high concentration of carvacrol (93%). The seeds of S. khuzistanica
are dormant and have a very low germination under normal conditions. This is the first protocol for in
vitro seed germination and plantlet regeneration of this plant. Experiments were done to determine the
effects of cold stratification (two, four and six weeks), chemical treatments including various levels of
gibberellic acid (GA3) (50, 100, 200 and 500 ppm), potassium nitrate (0.2, 0.4 and 0.6%), sulfuric acid
(50%) and also the synergistic effect between cold stratification and other factors on seed germination
of S. khuzistanica. The most germination percentage was achieved when six weeks stratification was
followed by 200 ppm GA3. For shoot proliferation, the node explants were cultured on Linsmaier and
Skoog (LS) medium supplemented with different concentrations of benzylaminopurine (BAP) and
kinetin (Kn) within the range of 0.5 to 2 µM and combinations of indole-3-butyric acid (IBA: 2 and 5 µM)
with BAP (2 and 5 µM). Multiple shoots were obtained from the nodal explants, the higher frequency
(77%) of shoot formation was observed in the LS that contained BAP (5 µM) in combination with IBA (2
µM). The best condition for rooting were LS medium plus 2.5 µM of IBA. The rooted plants were
successfully transferred to garden soil, exhibiting a normal development.

Key words: Germination, gibberellic acid, growth regulators, node explants, Satureja khuzistanica,
stratification.

INTRODUCTION

Satureja khuzistanica Jamzad is an endemic plant and of under the names ‘saatar’, ‘nadgh’ and ‘marzeh kouhi’ (a
a dispersed distribution in the southern of Iran. Satureja montane variety of savory). These herbs are described
belongs to the tribe mentheae of lamiaceae-nepetoideae as stomachic, sedative and analgesic, especially in
(Jamzad, 1994). This plant has been used as analgesic toothache (Avicenna, 1985; Amiri, 1974).
and antiseptic among the inhabitants of southern parts of The main constituents of S. khuzistanica are mono-
Iran (Abdollahi et al., 2003). In the classical Iranian terpenoids such as carvacrol (93%), γ-terpinene (2.6%)
medical books, a few varieties of herbs are described and p-cymene (1.7%) (Majd et al. 2009). Carvacrol as
the main component of the wild S. khuzistanica has been
found to have significant antioxidant properties (Abdollahi
et al., 2003). In recent years, antimicrobial (Amanlou et
*Corresponding author. E-mail: [email protected]. Fax: al., 2004), anti-inflammatory and anti nociceptive
98-21-82884717. (Amanlou et al., 2005) as well as plasma glucose
19408 Afr. J. Biotechnol.

lowering (Shahsavari et al., 2009) activities were reported carried out in completely randomized design with four replications
for the species. The seeds of S. khuzistanica are dormant and 50 seed per each replication. Data were subjected to analysis
of variance using SPSS version 18. Duncan's multiple range test
and have a very low germination rate under the normal
(DNMRT) was used to detect significant differences among the
conditions. Hitherto, despite the potential commercial treatments with a probability of 99% (p ≤ 0.01). Germination
interest of this important multipurpose medicinal plant, percentage values were correlated using linear regressions (p =
there has been no report on the seed germination and 0.05).
propagation for this species.
In vitro culture techniques can be an alternative method In vitro propagation
for the continuous provision of plantlet stocks for the
large scale field cultivation. More and more medicinal Establishment of cultures
plant species of Lamiaceae are now propagated via in
Treated seeds (six weeks stratification + 200 ppm GA3) were placed
vitro culture techniques, just to mention a few, such as on MS medium (Murashige and Skoog, 1962) containing 3%
Lavandula stoechas (Nobre, 1996), Cunila galioides sucrose and 0.7 % (w/v) agar. The 3-week-old seedlings provided
(Fracaro and Echeverrigaray, 2001), Salvia fruticosa material for in vitro cultures used in this study. Four basal media
(Arikat et al., 2004), Salvia brachyodon (Misic et al., were used: LS (Linsmaier and Skoog, 1965), B5 (Gamborg et al.,
2006), Ocimum gratissimum (Gopi et al., 2006) and 1968), MS and half-strength MS. All the media were supplemented
Ocimum basilicum (Begum et al., 2002). with 1 µM of BAP and except half-strength MS, other media
contained 3% sucrose as the carbon source. The pH of the media
Moreover, in vitro propagation of S. khuzistanica is was adjusted to 5.8 prior to the addition of 0.7% agar and
considered to be important for chemical analysis of autoclaving (1 atm, 127°c, 20 min). Glass tube cultures were
essential oils, physiological studies as well as pharma- incubated in a culture room at 25 ± 2°C with 16 h photoperiod under
cological and genetic transformation programs. To the standard cool white fluorescent tubes (35 µmol s-1 m-2).
best of our knowledge, this paper is the first report on the
seed germination and in vitro multiplication of S. Shoot multiplication
khuzestanica.
For shoot multiplication, single node segment, approximately 1 cm
in length were transferred to LS medium supplemented with
MATERIALS AND METHODS different concentrations of BAP and kinetin (Kn) within the range of
0.5 to 2 µM and combinations of indole-3-butyric acid (IBA: 2 and 5
Plant material and seed germination µM) with BAP at two concentrations (2 and 5 µM). After six weeks,
the efficacy of each treatment on shoot proliferation and growth was
S. khuzistanica seeds were collected from Paalam region, located determined. Hormone-free medium was used as control.
in the south-eastern part of Lorestan province in Iran. The mature
plants were identified according to Jamzad, (1994) and a voucher
specimen was deposited at the Tarbiat Modarse University Shoot rooting and acclimatization
Herbarium. Seed viability was determined by tetrazolium (TZ) test
(Perry, 1987). To initiate rooting and elongation, the micro-shoots (around 3 cm
Seeds were placed in a sealed plastic box in a refrigerator at height) were placed on LS medium containing 30 g/L sucrose and
temperature of 4°C, for two, four and six weeks. The stratified (six three different auxins, indoleacetic acid (IAA), naphthaleneacetic
weeks) and non-stratified seeds were surface sterilized by soaking acid (NAA) and IBA, at three concentrations (2.5, 5 and 10 µM).
in 1% sodium hypochlorite (NaOCl) for 5 min and subsequently The medium was solidified with 7 g L-1 agar and dispersed at 40 ml
rinsed thoroughly with sterilized water. After sterilization, the per glass tube cultures. Four shoots were cultured in each tube and
stratified (six weeks) and non-stratified seeds were soaked in four were incubated in dark condition for three days at 25 ± 2°C in a
GA3 (SIGMA) concentrations (0, 50, 100, 200, and 500 ppm) in dark growth chamber, afterwards the culture tubes were set under
at room temperature for 48 h. Stratified (six weeks) and non- standard cool white fluorescent tubes (35 µmol s-1 m-2) with a 16 h
stratified seeds were soaked in different concentrations [0, 0.2, 0.4 photoperiod for 45 days. After 45 days, plantlets with well-grown
and 0.6 (w/v%)] of KNO3 (MERCK ) for 72 h in dark at room roots were removed from the culture tubes; the roots were washed
temperature. in running tap water and the plants were transferred to pots
Stratified and non-stratified seeds after sterilization, were soaked containing sterilized garden soil, covered with polythene bags to
in H2 SO4 (80% v/v) for 5 min then washed several times by distilled maintain humidity and kept at 25°C in a growth chamber for one
water. The seeds were then placed in 10 cm diameter Petri-dishes week. The bags were removed gradually and acclimatized plants
with 10 ml of water agar (7%). All dishes were sealed with a trip of were transferred to the greenhouse.
parafilm to reduce water loss. The seeds were transferred to All the experiments were conducted in randomized design with
germinators with continuous darkness, constant temperature of 25 explants per treatment and each was repeated twice. The data
20°C and relative humidity between 70 and 75%. Germinated were submitted to statistical analysis by ANOVA and the means
seeds were counted and removed every 24 h for 25 days. A seed compared by the Tukey´s test.
was considered germinated when the tip of the radicle had grown
free of the seed coat (Wiese and Binning, 1987).
The germination rate was calculated as follows (Wiese and RESULTS
Binning, 1987): Germination rate = ∑ (number of germinating since
n-1) / n; n = the days of incubation. The seed vigor index was Seed germination
calculated as follows (Abdul-baki and Anderson, 1973): Vi = (Ls ×
Pg) / 100, where Vi is vigor index, Ls is the mean of seedling length Seed viability of S. khuzistanica was 77% according to
and Pg is the germination percentage. The experiments were TZ staining. As shown in Table 1, stratification had a
 Ramak et al. 19409

Table 1. Mean values (Duncan means test; p≤0.01) of seed germination and seedling characters for S. khuzistanica under
stratification, chemical and combined stratification (6w) and chemical treatments.

Germination Germinationrates Shoot length Root length

Treatment Vigor index
percentage (%) (seeds per day) (cm) (cm)
Stratification
(week)
2 14h 0.63 g 1ab 2ab 0.423e
gh gh ab ab d
4 18 0.53 1.1 2.3 0.621
g h ab ab d
6 21.5 0.38 1.08 2.18 0.699

GA3 (ppm)
f f ab b c
50 30.5 0.93 1.18 1.6 0.844
e d ab ab bc
100 39.75 1.37 0.93 2.35 1.30
e e ab b bc
200 38.5 1.215 0.9 1.6 1.06
500 44.5d 1.198 ef 0.95 ab 1.95ab 1.25bc

KNO3 (v/v%)
0.2 22.25g 0.645g 0.83ab 1.15de 0.44e
0.4 29.75f 1.010ef 0.85as 1.5c 0.69d
0.6 33.75f 0.937f 1.13ab 1.13e 0.76d

H2SO4 (50%) 4i 0.107i 0.98ab 2.58ab 0.14f

Stratification for six weeks

+ GA3 (ppm)
50 53c 1.697c 1.025ab 1.750ab 1.485b
100 56c 1.805bc 1.075ab 2.725a 2.147a
200 72.5a 2.313a 1.025ab 2.275ab 2.398a
500 66.75b 2.065ab 1.025ab 2.200ab 2.150a

KNO3 (v/v%)
0.2 44.25d 1.357de 0.825b 1.175d 0.889c
0.4 56.25c 1.795bc 0.800b 1.475cd 1.293bc
0.6 62.5b 1.995b 1.050ab 1.050f 1.313bc

H2SO4 (50%) 21.75g 0.920f 1.200a 2.050ab 0.706cd

Control 2.5i 0.055it 0.77b 1.88ab 0.087f

Means with different letters in each column differ significantly according to Duncan’s multiple range tests at p≤ 0.01.

significant effect on seed germination of S. khuzistanica germination percentage (44%) (Table1).

(p≤0.01). Regressions indicate that the germination percentage
Further improvement of germination through stratifi- had significant positive correlations with the GA3
cation was tried, but it was not a complete success. concentrations (r = 0.67, p≤0.05). Seedling characters
Seeds stratified for six weeks recorded a moderate such as germination rate, root length, and vigor index
increase in germination (21%) with high vigor index were also affected by GA3 and showed a significant
(0.699). Except root length, other traits such as difference at various concentration of GA3.The combined
germination rate and vigor index were also affected by treatment with GA3 and stratification (6w) had a
stratification (Table1). Treatment with GA3 significantly statistically significant effect on germination (Table1).
improved germination. Also, increasing the concentration Seeds treated with 200 ppm GA3 without stratification,
of GA3 resulted in an increase in germination percentage gave 38% germination, whereas seeds treated with 200
where, at 500 ppm, concentrations caused the highest ppm GA3 preceded by six weeks of stratification gave
19410 Afr. J. Biotechnol.

Table 2. Influence of culture medium composition on in vitro response of S. khozistanica

Culture Number of shoots Shoot height Number of Viability

media per explant (cm) leaf (%)
a a a
LS 6.8 ± 1 6.9 ± 0.6 25 ± 3 86
MS 5.5 ± 0.8a 5.4 ± 1b
20 ± 2a 65
a c b
1/2MS 5.0 ± 0.6 4 ± 0.5 b 17 ± 2 54
b c c
B5 2.8 ± 0.9 3.2 ± 0.6 8±1 19
Values represent the mean ± SD; means followed by same letters within a column are not significantly different
(p< 0.05) using Tukey test. All media were supplemented with 1 µM BAP.

72% germination. Rooting and acclimatization

The results also show that potassium nitrate can
effectively release seeds dormancy in S. khuzistanica. As can be observed in Table 4, three types of auxines
Soaking of seeds in 0.6% (w/v) KNO3 for 72 h increased (IAA, NAA and IBA, at three concentrations: 2.5, 5 and 10
the germination to 33% (Table1). Although stratification µM) had different effects on rhyzogenesis. The most
produced much better results compared with separate effective auxin for root number and root length was IBA
application of these two factors, the combination of (2.5 µM) (Figure 1b). Shoots exposed to higher con-
stratification and KNO3 significantly enhanced centrations of NAA and IAA (10 µM) became necrotic,
germination percentage at 0.6% KNO3 (62%) (Table 1). lost leaves, and died gradually. For acclimatization,
Moreover, the results of the study reveal that plantlets were removed from rooting medium after 45
germination rate, root length, and vigor index were also days of incubation and transferred to plastic pots
affected through various levels of potassium nitrate. containing sterilized garden soil, covered with polythene
Recorded experimental findings (Table1) show that bags, to maintain humidity and were kept under culture
scarification with sulphuric acid did not promote room conditions for one week (figure 1c).
germination of S. khuzistanica seeds. The root length After one week, polythene bags were removed and
(2.58 and 2.05 cm) and shoot length (0.98 and 1.2 cm) transferred to green house. The survival rate was 80%
were recorded to be in their maximum length in H2SO4 and the appearance of acclimatized plants was normal
and a combination of six weeks of stratification and without any morphological abnormalities or variations
H2SO4 treatment. (figure 1d).

In vitro regeneration DISCUSSION

The effects of four salt formulations on the Stratification was found to be effective in increasing
micropropagation of S. khuzestanica are shown in Table germination and in breaking dormancy in several species
2. The number of shoots were not significantly different of Labiatae, for example in Stachys alpina by Pinfield
among MS, 1/2MS, and LS media, but the high explants (1971); in Isanthus brachiatus by Baskin and Baskin
viability (86%) and shoot formation with the most number (1975), and in Salvia columbariae by Hashemi and Estilai
of nodes and leaves were observed on the LS media (1994). The reports and the results of this study show
(Figure 1a). Nodal explants cultured on B5 medium that stratification was successful in breaking seed
produced the shortest shoot with abnormal leaves. dormancy, though the duration of treatment may vary
The presence of both BAP and Kn on the LS media with the species. At cold temperatures, more oxygen is
positively influenced shoot proliferation, where among soluble in water, so the oxygen requirements of the
cytokinins, BAP responded better when compared with embryo are better satisfied. Cold-moist stratification
Kn regarding shoot proliferation (Table 3). The synergistic imitates overwintering in a field seed bed (Young and
effect of BAP with IBA for direct plant regeneration was Young, 1986). The actions of low temperatures in
evaluated and the best response and the maximum terminating dormancy may promote a fall in the level of
induction of multiple shoots (9.5 ± 0.5) were achieved inhibitors and it automatically increases the level of
from medium supplemented with 5 µM BAP and 2 µM promotive hormones (Bewley and Black, 1982).
IBA. Although the treatment of 5 µM BAP and 5 µM IBA It has been reported that germination can be induced
resulted in lower shooting compared with 5 µM BAP and by gibberellic acid in Salvia glutinosa, Lycopus
2 µM IBA, it did not show any significant difference in europaeus, and Scutetlaria galericulata (Thompson,
terms of number of shoots, shoot height, and number of 1969), S. columbariae (Hashemi and Estilai, 1994), and
leaves. Stachys alpine (Pinfield, 1971). It has also been reported
 Ramak et al. 19411

Figure 1. Regeneration of S. khuzistanica from the nodal explants. (a) Multiple shoots regeneration from the nodal explants; (b)
rooted plantlets; (c) plantlets covered with polythene bags; (d) hardened plants.

Table 3. Effect of BA, Kn and combination of IBA with BAP on shoot induction and proliferation from the Nodal Segments of
S.khozistanica in LS medium after 6 weeks in culture.

Growth regulator (µM) Shooting (%) Number of shoot Shoot height (cm) Number of leaf
BAP
0.5 50 7.8 ± 1.3ab 6.0 ± 1.8c 24 ± 7c
1 65 9.0 ± 0.8a 7.3 ± 0.5ab 29 ± 2abc
2 25 2.8 ± 1.2de 3.2 ± 0.6d 13 ± 2d

Kn
0.5 37 3.0 ± 0.8cde 4.8 ± 1.2cd 19 ± 5cd
1 40 4.0 ± 0.8cde 3.3 ± 0.9d 13 ± 3d
2 27 2.8 ± 0.9de 3.1 ± 1.3d 12 ± 5d

BAP + IBA
2 2 65 5.5 ± 0.6bc 6.8 ± 0.5bc 27 ± 2bc
2 5 58 5.3 ± 1bcd 6.5 ± 1.9bc 26 ± 8bc
5 2 77 9.5 ± 0.5a 9.5 ± 0.6a 38 ± 2a
5 5 52 7.3 ± 2ab 9.0 ± 0.8ab 36 ± 3ab

Control 7.5 1.5 ± 0.6e 2.5 ± 0.5d 10 ± 2d

Means followed by same letters within a column are not significantly different (p< 0.05) using Tukey test.
Values represent the mean ± SD.
19412 Afr. J. Biotechnol.

Table 4. Effect of different types and concentrations of auxin on root induction in LS media.

Auxin (µM) Rooting (%) Number of root Root length (cm)

IBA
a a
2.5 91 7.5 ± 1.3 4.5 ± 1.7
a a
5 62 4.8 ± 1.9 4.2 ± 1.5
b ab
10 29 3.3 ± 1.2 2.5 ± 0.6

NAA
b ab
2.5 25 3.2 ± 1 3.3 ± 0.5
ab ab
5 28 4.5 ± 1.2 2.8 ± 0.6
c c
10 0.0 0.0 0.0

IAA
a ab
2.5 26 4.3 ± 2 3.2 ± 0.6
a ab
5 52 5.5 ± 1.2 3 ± 0.8
c c
10 0.0 0.0 0.0
Control 25 1.2 ± 1.5c 0.9 ± 1c
Means followed by same letters within a column are not significantly different (p< 0.05) using Tukey
test. Values represent the mean ± SD.

that treatment with GA3 can increase the formation of khuzistanica seeds; therefore, seed coat cannot be
rough endoplasmic reticulum and polyribosomes (Evins, considered as a dormancy factor in S.khuzistanica .
1971). Moreover, it is found that GA3 stimulates the H2SO4 scarification has a positive effect on root and
synthesis of mRNA which is specific for α-amylase shoots length of S.khuzistanica seeds (Table1). Positive
(Higgins et al., 1976). Effects of GA3 preceded by strati- effect of H2SO4 on the length and height of the root and
fication were found to be successful for Prunus mahaleb shoot was also reported in Aframomum corrorima (Eyob,
L. (Gercekcioglu and Cekic, 1999) and Morus nigra L. 2009) and Lupinus varius (Karaguzel et al., 2004).
(Koyuncu, 2005). These reports are in consistent with our According to the results found in this study, S.
results showing that stratification followed by GA3 khuzistanica species probably exhibits a deep
enhance germination of S. khuzistanica seeds. physiological dormancy. Seeds with intermediate and
The results of this study are similar to other findings deep physiological dormancy are characterized by a
demonstrating that potassium nitrate treatment was an requirement for a one to three month period (sometimes
important factor in breaking seed dormancy and increa- more) of chilling, while in an imbibed and aerated state
sing seed germination (Brandel, 2005; Pupala and (Crocker 1948). Seeds of this type ripen in the fall
Fowler, 2003). Nitrogen containing compounds are overwinter in the moist leaf litter, and germinate in the
considered to be effective in breaking seed dormancy spring.
and thus inducing seed germination (Tang et al., 2008). This requirement led to the horticultural practice of
Nitrate plays the role of a co-factor in phytochrome action “stratification”, in which seeds are placed between layers
(Grubisic and Konjevic, 1990). Potassium nitrate may be of moist sand or soil in boxes (or in the ground) and
helpful for reactivation of metabolic process of seeds. exposed to chilling temperatures, either out-of-doors or in
This compound may cause biosynthesis of auxin, which refrigerators. Temperature is the most important factor
ultimately triggers the growth of the embryo (Khan et al., controlling stratification. The most effective temperature
1999). is next to freezing (1 to 10°C). The time required to
Although Satureja species do not have thick seed coat, stratify seeds results from the interaction of the genetic
the strong inhibitory effect of seed coat on the seed characteristics of the seed population, seed development
germination may be caused by several possible environment, and the stratification environment
mechanisms, including mechanical constraint, prevention (Wartidininghsih and Geneve 1994).
of water and oxygen uptake, and retention or production The results related to the effects of four salt
of chemical inhibitors (Taiz and Zeiger, 2002). The formulations, which can be observed in Table 3, can be
3- 4+
integument breaking or softening is, therefore, necessary attributed to the NO /NH ratio on LS and MS media,
to remove dormancy imposed by seed coat hardness or both with 66 : 34, compared with the B5 media (80 : 20).
impermeability. Nitrate/ammonium relation is essential for the nitrogen
Results in Table1 indicate that H2SO4 scarification did supply and pH control during in vitro culture, which
not increase the germination percentage of S. drastically affects culture performance (George, 1993).
 Ramak et al. 19413

Based on these results, LS medium was used in shoot and accumulation of essential oils in wild sage (Salvia fruticosa Mill.).
Sci. Hort. 100: 193-202.
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