Stojičić, D. et al. Factors influencing axillary bud induction on nodal…
BIOLOGICA NYSSANA 8 (1) September 2017: 93-98
DOI: 10.5281/zenodo.964247
8 (1) • September 2017: 93-98
Original Article
Received: 16 June 2017
Revised: 29 June 2017
Accepted: 15 July 2017
Factors influencing axillary bud induction on nodal segments of
Micromeria pulegium (Rochel) Benth.
Dragana Stojičić*, Svetlana Tošić, Jovana Pavlović, Aleksandra Golubović, Jelena
Simonović, Bojan Zlatković
University of Niš, Faculty of Science and Mathematics, Department of Biology and Ecology,
Višegradska 33, Niš, Serbia
* E-mail: draganadstojicic@gmail.com
Abstract:
Stojičić, D., Tošić, S., Pavlović, J., Golubović, A., Simonović, J., Zlatković, B.: Factors influencing axillary
bud induction on nodal segments of Micromeria pulegium (Rochel) Benth. Biologica Nyssana, 8 (1),
September 2017: 93-98.
Micromeria pulegium (Rochel) Benth. is an endemic species from family Lamiaceae. Plants from this family
are characterized by presence of secondary metabolites and antioxidant components. Micromeria pulegium
contains pulegone which is a potential bio-insecticide and a bio-pesticide. Natural populations of this species
are so small that there is a need for an alternative way of propagate and proliferation of individuals. Method of
micropropagation was used with the goal of mass production of plants with the chemical composition of
essential oils as similar as possible to that in wild-harvested plants. This paper presents the study on influence
of concentration of mineral salts, carbon sources (sucrose and maltose) and nitrogen source (casein
hydrolysate) on process of in vitro regeneration of plants through induction of axillary buds on the nodal
segments of Micromeria pulegium. The greatest number of axillary buds was formed in explants grown on MS
culture medium with 3% sucrose and 500 mg/L casein hydrolysate.
Key words: axillary bud induction, shoot culture, biomass production
Apstrakt:
Stojičić, D., Tošić, S., Pavlović, J., Golubović, A., Simonović, J., Zlatković, B.: Faktori koji utiču na
indukciju aksilarnih pupoljka na nodalnim segmentima vrste Micromeria pulegium (Rochel) Benth..
Biologica Nyssana, 8 (1), Septembar 2017: 93-98.
Micromeria pulegium (Rochel) Benth. je endemična vrsta iz familije Lamiaceae. Ovu familiju karakteriše
prisustvo sekundarnih metabolita i antioksidativnih komponenti. Micromeria pulegium sadrži pulegon koji je
potencijalni bio-insekticid i biopesticid. Prirodne populacije ove vrste imaju mali broj jedinki pa se nameće
potreba za pronalaženjem načina njihovog gajenja i umnožavanja. U cilju masovne produkcije biljaka čiji će
hemijski sastav etarskih ulja biti najsličniji onome koji sadrže biljke iz prirode primenjena je metoda
mikropropagacije. U ovom radu ispitan je uticaj jačine mineralnih soli, izvora ugljenika (saharoze i maltoze) i
izvora azota (kazein hidrolizata) na proces regeneracije biljaka in vitro putem indukcije aksilarnih pupoljaka
na nodalnim segmentima Micromeria pulegium. Najveći broj aksilarnih pupoljaka je formiran na eksplantatima
gajenim na MS hranljivoj podlozi sa 3% saharoze i 500 mg/L kazein hidrolizata.
Ključne reči: indukcija aksilarnih pupoljaka, kultura izdanaka, produkcija biomase
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Stojičić, D. et al. Factors influencing axillary bud induction on nodal…
BIOLOGICA NYSSANA 8 (1) September 2017: 93-98
Introduction
Micromeria
pulegium
(syn.
Clinopodium pulegium) belongs to
section Pseudomelissa within the
genus Micromeria Benth. (family
Lamiaceae). This genus includes
four
sections:
Pseudomelissa,
Micromeria,
Cymularia
and
Pineolentia (H a r l e y et al., 2004).
According to molecular evidence,
emphasizing similarity of selected
morphological traits, B r ä u c h l e r
et al. (2006) have included species of
genus
Micromeria,
sect.
Pseudomelissa, into Clinopodium L.
M. pulegium is an endemic species
of southern Carpathians. Its range
includes Romania, Serbia and
Federation
of
Bosnia
and
Fig. 1. Wild-growing plant M. pulegium, Svrljiški Timok George
Herzegovina. In Serbia it was
recorded in the east, in the area of
2013). Through micropropagation it is possible to
Svrljiški Timok gorge, while previously it used to be
multiply selected genotypes and chemotypes of
present at mountain Tara (Š i l i ć , 1979). Gorge of
different plants, avoiding collection from their
Svrljiški Timok is situated at low altitude, so
natural habitat. In vitro propagation from field-grown
summers are very warm and dry, while winters are
plants through multiplication of nodal segments
characterized by strong winds and snow. Habitats are
(axillary shoot formation) is a good method of
rocky and steep, mostly gorges at 1000-1200 m
producing a large number of plants without changing
above sea level. Micromeria pulegium is a perennial,
the chemical composition (S a n t o s -G o m e s &
medium-sized, erect plant (Fig. 1), and its leaves are
F e r n a n d e s - F e r r e i r a 2003; A f f o n s o et al.
densely covered in small glands, rendering a pleasant
2007). This study was initiated in order to improve
aroma to the plant. Species of genus Micromeria are
the reliable protocol for rapid propagation of M.
characterized by presence of secondary metabolites,
pulegium through axillary bud induction from nodal
serving as the foundation of their diverse biological
explants.
activity (V l a d i m i r - K n e ž e v i ć et al., 2000).
These species have antimicrobial properties (S a r a c
& U g u r , 2007), mostly based on phenol
Material and methods
compounds, flavones and flavonoids, terpenoids and
Plant material and source of explants
alkaloids (C o w a n et al., 1999; C o s e n t i n o et al.,
1999). These aromatic plants have been traditionally
Aerial parts of M. pulegium plants, at the vegetative
used as spices and in alternative medicine (A l stage of development, were collected from natural
H a m w i et al., 2011; D u d a i et al., 2001; T e l c i
populations in Svrljiški Timok gorge, in August 2012
et al., 2007). Antioxidant and antimicrobial activities
(Fig. 1). Voucher specimen (Nº 6912) was deposited
were recorded in both micropropagated plants of M.
in the Herbarium collection of the Faculty of Science
pulegium and those collected from natural habitats
and Mathematics, University of Niš (HMN). Nodal
(T o š i ć et al., 2015). Chemical composition of M.
segments (one-node stem segments, 1 cm long and
pulegium essential oils from native and
bearing two axillary buds) were surface-sterilized for
micropropagated plants was also studied (S t o j i č i ć
30 min with 25% solution of sodium hypochlorite
et al., 2016). Endemic species with small populations
(6% active chlorine) containing two drops of liquid
such as those of M. pulegium, situated in vicinity of
detergent. After three rinsing in sterile distilled water,
urban environment, are often under negative
the explants were treated with 5% solution of nystatin
anthropogenic influence. In order to preserve
for 24 hours in order to eliminate possible fungal
biodiversity, various biotechnological methods have
infections. After that nodal segments were rinsed
been developed, and among them micropropagation
three times with sterile distilled water, and they were
of plants in vitro is highly important (P a u n e s c u ,
placed in different variations of culture medium.
2009; R e e d et al., 2011; S h a r m a & S h a r m a ,
Every jar closed with polycarbonate cover.
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Stojičić, D. et al. Factors influencing axillary bud induction on nodal…
BIOLOGICA NYSSANA 8 (1) September 2017: 93-98
Table 1. Effect of medium strength on Micromeria pulegium shoot proliferation, number of shoots per explant,
shoot length and shoot fresh and dry weight after 28 days of culture
MS
0MS
0.25MS
0.5MS
1MS
2MS
Explants
producing
shoots (%)
0
80.0 ± 0.1a
85.0 ± 0.1b
93.3 ± 0.2c
81.7 ± 0.2a
Number of
shoots per
explant
0
8.15 ± 0.32a
8.52 ± 0.51a
12.32 ± 0.71b
12.12 ± 0.81b
Shoot length
(mm)
Explant fresh
weight (g)
Explant dry
weight (g)
0
6.25 ± 0.40a
8.48 ± 0.46b
9.31 ± 0.80c
9.88 ± 1.11d
0
0.09 ± 0.01a
0.11 ± 0.01b
0.21 ± 0.02c
0.22 ± 0.05c
0
0.009 ± 0.001a
0.010 ± 0.001a
0.017 ± 0.002b
0.019 ± 0.003b
Values are mean ± SE, n = 60. Means in the column followed by different letters are different according to LSD multiple
range test (P ≤ 0.05)
Table 2. Effect of carbon source on Micromeria pulegium shoot proliferation, number of shoots per explant,
shoot length and shoot fresh and dry weight after 28 days of culture
Carbon
source
(%)
0
Maltose
0.5
1.0
3.0
5.0
Sucrose
0.5
1.0
3.0
5.0
Explants
producing
shoots (%)
51.7 ± 0.1a
Number of
shoots per
explant
2.33 ± 0.93a
Shoot length
(mm)
Explant fresh
weight (g)
Explant dry
weight (g)
4.93 ± 0.33a
0.06 ± 0.02a
0.002 ± 0.001a
70.0 ± 0.1b
75.0 ± 0.1b
76.7 ± 0.2bc
68.3 ± 0.2b
4.84 ± 0.46b
4.94 ± 0.66b
4.60 ± 0.66b
6.09 ± 0.21c
6.41 ± 0.35ab
6.43 ± 0.48ab
6.05 ± 0.48ab
6.07 ± 0.44ab
0.10 ± 0.02ab
0.15 ± 0.03bc
0.11 ± 0.03ab
0.24 ± 0.01e
0.005 ± 0.001b
0.005 ± 0.001b
0.006 ± 0.001b
0.020 ± 0.001d
80.0 ± 0.1c
85.0 ± 0.1c
93.3 ± 0.2d
81.7 ± 0.2c
9.09 ± 0.21d
12.52 ± 0.21e
12.32 ± 0.71e
10.09 ± 0.21de
7.43 ± 0.44c
7.00 ± 0.44c
9.31 ± 0.80d
6.33 ± 0.44ab
0.21 ± 0.01d
0.18 ± 0.01c
0.21 ± 0.02d
0.21 ± 0.01d
0.017 ± 0.001c
0.016 ± 0.001c
0.017 ± 0.002c
0.019 ± 0.001c
Values are mean ± SE, n = 60. Means in the column followed by different letters are different according to LSD
multiple range test (P ≤ 0.05)
Effects of carbohydrates - to determine the
influence of different carbon sources, MS medium
was supplemented with one of two carbohydrates
(sucrose or maltose), each at five different
concentrations (0; 0.5; 1; 3; or 5%).
Effect of enzymatic casein hydrolyzate (CH) as a source of organic nitrogen, CH was tested at the
following concentrations 0; 125; 250; 375; or 500
mg/L.
Culture medium and culture conditions
Isolated nodal segments were placed horizontally on
basal Murashige and Skoog (MS) medium (1962)
supplemented with 3% sucrose (w/v) and 0.7% (w/v)
agar (Torlak, Belgrade) in 250-mL glass jars
containing 25 mL of the medium, if not stated
otherwise. Ten explants were placed in each jar. The
pH of the media was adjusted to 5.8 prior to
autoclaving at 114 °C for 25 min. Cultures were
maintained at 25 ± 2°C under conditions of a 16 h/8 h
photoperiod, with a photon flux density 45 µE m-2 s-1
provided by cool white fluorescent lamps, at
25 ± 2 °C.
Measured parameters
After 4 weeks in culture, the explants formed axillary
buds. The explants reacting positively to treatment
were recorded and following parameters were
measured. The number of explants producing shoots
and number of shoots per explant, as well as explant
fresh and dry weight were recorded in order to
evaluate the effect of nutritive factors on shoot
multiplication. The dry weight of shoots was
recorded after drying in separate paper containers for
24 h. Buds shorter than 1 mm were disregarded. The
Variations of media
Medium strength – the effects of five different
medium strengths were tested: 0; 0.25; 0.5; 1.0; or 2.0
times those of MS. The other components in all five
media were the same as in MS medium.
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BIOLOGICA NYSSANA 8 (1) September 2017: 93-98
Table 3. Effect of casein hydrolyzate on Micromeria pulegium shoot proliferation, number of shoots per
explant, shoot length and shoot fresh and dry weight after 28 days of culture
CH
(mg/L)
0
125
250
500
750
Explants
producing
shoots (%)
93.3 ± 0.2b
80.0 ± 0.1a
90.0 ± 0.1b
93.3 ± 0.2b
81.7 ± 0.2a
Number of
shoots per
explant
12.32 ± 0.71a
12.11 ± 0.45a
12.27 ± 0.24a
16.93 ± 0.38c
15.06 ± 0.42b
Shoot length
(mm)
Explant fresh
weight (g)
Explant dry
weight (g)
9.31 ± 0.80a
9.90 ± 0.70a
9.51 ± 0.51a
15.36 ± 0.57b
15.01 ± 1.10b
0.21 ± 0.02b
0.19 ± 0.02a
0.19 ± 0.02a
0.25 ± 0.04c
0.25 ± 0.02c
0.017 ± 0.002a
0.018 ± 0.002a
0.017 ± 0.002a
0.023 ± 0.002b
0.024 ± 0.002b
Values are mean ± SE, n = 60. Means in the column followed by different letters are different according to LSD multiple
range test (P ≤ 0.05)
when concentration of salts increased. The maximum
axillary bud proliferation was obtained at 1MS and
2MS, in contrast to results by F a d e l et al. (2010)
with Mentha spicata L. and M i š i ć et al. (2006)
with Salvia brachyodon. They achieved the best
results by using medium with MS salts reduced to one
half (0.5MS). Supplementation of the medium with
salts and vitamins promoted elongation of the shoot.
The greatest average length of axillary buds was
developed on explants grown at medium with 2MS.
The explants with the greatest average fresh and dry
weight were grown on 1MS and 2MS medium.
efficiency of different treatments on growth rate was
determined by comparing biomass increase and in
vitro proliferation rates, using different methods.
Biomass increase was calculated on both fresh and
dry weight basis. Proliferation rate was assessed by
counting the number of shoots at subculture and
following 4-week treatment under previously defined
conditions.
Statistical methods
For each treatment, a total of 60 nodal segments (ten
explants per jar) were used, divided into two
replicates. Data collected from experiments were
calculated and statistically analyzed, and differences
were tested for significance using ANOVA Multiple
range test at the significance level of P ≤ 0.05.
Results and discussion
Micropropagation is a good method for achieving
uniform plant material, and at the same time use of
nodal segments for plant regeneration with the goal
of mass production is considered a reliable method
for many Lamiaceae species (D o d e et al., 2003). In
most species of Lamiaceae family, shoot
proliferation demands presence of cytokinin in the
nutrient medium, with or without auxin (S a h a et al.,
2012; B a k h t i a r et al., 2014). However, the nodal
explants of M. pulegium cultured on MS medium
without plant growth regulators have produced
shoots (S t o j i č i ć et al., 2016). These results were
used as a foundation for determining the influence of
nutritive factors of nutrient substrate on production of
axillary buds on nodal segments of M. pulegium.
Research on the effects of five different medium
strengths has shown that medium without salts and
vitamins was not sufficient for development of
axillary buds, and necrosis of whole explants
happened already in the second week (Tab. 1). On
medium supplemented with salts and vitamins, the
percentage of explants developing shoots was greater
Fig. 2. In vitro plantlets cultured on medium with 750
mg/L caseine hydrolysate (left), flowering in vitro
plantlets on same medium (right)
Explants of M. pulegium grown on culture
medium MS without a source of carbon were light
green, with sporadically developed axillary buds in
leaf axils. The percentage of explants with
developing axillary buds was the smallest in the
whole study (52%). The values of number of buds,
length of buds, fresh and dry mass were also
significantly smaller than in axillary buds grown in
medium supplemented with sugar (Tab. 2). Sucrose
is often the best source of carbon in micropropagation
of various species from family Lamiaceae (S u j a n a
& N a i d u , 2011). After adding maltose or sucrose
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BIOLOGICA NYSSANA 8 (1) September 2017: 93-98
essential oils rich in pulegone that potentially may be
used as bio-insecticide and bio-pesticide (K o u l et
al. 2008). Specimens collected in the wild and grown
in vitro both produced essential oil of relatively stable
composition (S t o j i č i ć et al., 2016). Careful
selection of the culture conditions may increase
accumulation of biomass and production of
secondary metabolites, which may be employed to
obtain essential oils for commercial use. Results
indicate that micropropagation of M. pulegium is
influenced by composition of nutritive medium.
Selection of proper mineral salt concentration,
carbon source and especially presence of nitrogen
source were shown to be important for stimulation of
development and growth of axillary buds in this
species. The presented protocol may be used as a
foundation in further research in order to produce M.
pulegium plants with desired characteristics,
especially for production of secondary metabolites.
sugars to the nutrient medium, the percentage of M.
pulegium explants with axillary buds has
significantly increased (68-93%). The explants
grown on medium with sucrose were healthy, with
good branching, green, with elongated internodes and
a large number of developed axillary buds. The
results of our study show that sucrose was a better
source of carbon for M. pulegium than maltose. In all
studied concentrations sucrose caused a greater
number of axillary buds than when the medium
contained maltose. The maximum number of buds
developed on medium with 1% and 3% sucrose,
while the greatest length was recorded in axillary
buds growing on medium with 3% sucrose. The
explants grown on medium with maltose had bushlike form with shorter internodes. Therefore the
greatest values of average dry and fresh biomass were
recorded in explants grown on medium with the
greatest concentration of maltose.
Casein hydrolysate is commonly used in
micropropagation as a source of nitrogen. Most of the
recorded effect was stimulative (S t o j i č i ć et al.,
2008). Explants of M. pulegium grown on MS
medium with or without casein hydrolysate showed
no morphological differences. They were elongated,
branching explants, dark green in color, with a large
number of developed axillary buds (Fig. 2). The
stimulative effect of casein hydrolysate was
manifested in increase of length of axillary buds and
their fresh and dry biomass (Tab. 3). On medium
with the lowest concentrations of casein hydrolysate
this increase was not statistically significant.
However, use of casein hydrolysate in concentrations
of 500 and 750 mg/L is justified as it leads to
statistically significant increase in bud length and
biomass. The maximum length of axillary buds was
recorded in explants grown on medium with 500 and
750 mg/L of casein hydrolysate. Some of the explants
grown on this medium produced flowers (Fig. 2). In
some of the explants grown on medium with the
greatest concentration of casein hydrolysate
adventive roots have formed spontaneously without
addition of auxin to the medium. Root formation
without auxin was observed in a number of
Lamiaceae species (Z u z a r t e et al., 2010;
Bassolino et al., 2015), where roots were induced on
plant growth regulator free medium. However, in M.
pulegium shoots rooted spontaneously on the plant
growth regulator free medium; hence, the auxin
treatment was used to promote rooting (S t o j i č i ć et
al., 2016).
Acknowledgements. This research was supported by the
Ministry of Education, Science and Technological
Development of the Republic of Serbia (grant 173015,
173030).
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