J. Appl. Environ. Biol. Sci., 7 (9) 115-126, 2017
© 2017, TextRoad Publication
ISSN: 2090-4274
Journal of Applied Environmental
and Biological Sciences
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*Corresponding Author: Ghada Abd El-Moneim Hegazi, Department of Genetic Resources, Desert Research Center, El-
Matareya, Cairo, Egypt. Email: [email protected], (+20122)7550486
Micropropagation of Bacopa monnieri
and Enhancing Bacoside A Production in Shoot Cultures
Ghada Abd El-Moneim Hegazi1*, Hussein Sayed Taha2, Abdel Monem Mohamed Sharaf3,
Saad Ramzy Elaish1
1Department of Genetic Resources, Desert Research Center, El-Matareya, Cairo, Egypt 2Department of Plant Biotechnology and Genetic Engineering, National Research Center, Giza, Egypt
3Department of Botany and Microbiology, Faculty of Science, Al-Azhar University, Cairo, Egypt
Received: April 3, 2017
Accepted: July 19, 2017
ABSTRACT
An efficient protocol for micropropagation of the rare medicinal herb Bacopa monnieri (L.) Pennell was described.
Bacoside A is one of the active principles in the plant, responsible for improving memory. Enhancing bacoside A
production in shoot cultures was also investigated. Establishment of in vitro cultures of B. monnieri was induced by
culturing shoot tips and stem nodal segments, collected from mature plants grown in damp and marshy places in the
Eastern Mediterranean coastal region of Egypt, on Murashige and Skoog (MS) medium supplemented with different
combinations of growth regulators. The maximum mass multiplication of shoots was achieved when explants were
subcultured for six successive subcultures on MS medium supplemented with 2.45 µM indole-3-butyric acid (IBA)
and 2.3 µM kinetin (KIN). Axillary shoots were developed 100% of rooting using different concentrations of NAA,
IAA or IBA, in addition to MS medium without growth regulators. The highest mean number and length of roots with
the optimum mean shoot height were obtained on MS medium containing 4.9 µM IBA. Plantlets were successfully
transferred to the greenhouse into peatmoss: sand mixture (1:1 v/v) with a 100% survival rate. The effect of mevalonic
acid, as a precursor of bacoside A and the elicitors; chitosan and methyl jasmonate, on the stimulation of biomass
growth and bacoside A production in shoot cultures of B. monnieri was investigated. The greatest biomass was
achieved when shoots were elicited by methyl jasmonate at 100 µM. Bacoside A accumulation was maximally
increased by 8.26-fold, in comparison to control, when mevalonic acid was added at 10 mM. This paper reports a
simple, reproducible procedure for micropropagation of B. monnieri that can be used for its conservation and to form a
stock of elite plant material for large-scale cultivation.
KEYWORDS: Brahmi, microporopagation, bacoside A, mevanolic acid, chitosan, methyljasmonate
INTRODUCTION
Bacopa monniera (L.) Pennell is commonly knowm as ‘Brahmi’ and belongs to family Scrophulariaceae. It
is a creeping herb and its habitat includes wetlands and muddy shores. Brahmi is an ancient medicinally important
plant. Traditionally, it is used as a nervine tonic, and to treat rheumatism, asthma, epilepsy and spleen enlargement.
It has anti-inflammatory, analgesic and antipyretic activities (Tiwari and Singh, 2010). The plant is mainly used as a
brain tonic to enhance learning skills, memory development and concentration and to relief anxiety or epileptic
disorders (Kaur et al., 2013 and Kunte and Kuna, 2013). Besides, the antioxidant properties of brahmi have also
been studied, which account for its anticancer activity (Sudharani et al., 2011 and Sundriyal et al., 2013). B.
monnieri extract is a potential cognitive enhancer and neuroprotectant against Alzheimer’s disease (Uabundit et al.,
2010). It was estimated that by 2020, neurodegenerative diseases (like Alzheimer’s disease) will be the eighth
leading cause of death in developed countries and by mid-century neuro degenerative diseases will be the world’s
second leading cause of death overtaking cancer (Menken et al., 2000).
B. monnieri contains different types of saponins; such as bacosides A, B, C, and D, which are the active
triterpenoid saponins known as "memory chemicals" (Sivaramakrishna et al., 2005). These bacosides are
responsible for improving memory and cognition through enhancing the efficiency of nerve impulses transmission
(Anon, 2004).
The medicinal importance of B. monnieri and its over-collection from natural habitat (Sharma et al., 2012)
are the mean reasons to apply in vitro techniques for conserving the plant and for bacosides production. To increase
the amount of saponins in B. monnieri, various strategies have been applied in in vitro cultures of the plant (Prasad
et al., 2008; Prakash and Nikam 2009 and Parale et al., 2010).
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Unlike the conventional methods of plant propagation, micropropagation could be carried out throughout
the year, resulting in large scale production of disease free and superior quality plants (Rohit, 2014).
Micropropagation and regeneration protocols from different explants of B. monnieri have been reported previously
by various scientific and research groups. Among the recent published work on micropropagation of B. monnieri;
plantlets have been successfully regenerated from various explants; such as leaves (Bhusari et al., 2013; Karatas et
al., 2013; Sharma et al., 2013; Koul et al., 2014; Dharishini et al., 2015 and Nandhini et al., 2015), nodal explants
(Begum and Mathur, 2014; Jain et al., 2014; Mohanta and Sahoo, 2014; Behera et al., 2015 and Nagarajan et al.,
2015) and internodal explants (Bhusari et al., 2013; Karatas et al., 2013 and Subashri and Koilpillai, 2013).
Production of bacosides from plant tissue culture is of great importance as their content in the intact B.
monnieri plant is very low, i.e. 0.2%, which limits their optimal utilization (Tejavathi and Shailaja, 1999). The
exogenous supply of a precursor of the desired compound to culture medium could increase its accumulation (El-
Nabarawy et al., 2015). Triterpoinds like bacosides are biosynthesized through mevalonate pathway and regulated
by various enzymes (Kumari et al., 2015). Mevanolic acid (MVA) is transformed into C5 isoprene units, which
combine in the form of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) to produce
terpenoids and steroids (El-Nabarawy et al., 2015).
Chitosan (CH) has been shown to have elicitor effects on the biosynthesis of a number of secondary
metabolites in a wide range of plant cell and organ cultures (Putalun et al., 2010; Lei et al., 2011 and Udomsuk et
al., 2011). Also, jasmonates are elicitors known to be efficient for a variety of secondary metabolites from different
plants (Memelink et al., 2001). Methyl jasmonate (MJ) enhanced the production of a number of secondary
metabolites including triterpenoid saponins in various plant species (Sharma et al., 2013). It is a volatile organic
compound used in plant defense and many diverse developmental pathways.
The present study aimed to develop a micropropagation protocol for the Egyptian rare plant; B. monnieri from
stem nodal segments and shoot tips and enhancing bacoside A production in shoot cultures using MVA, MJ and CH.
MATERIALS AND METHODS
The experiments of the present study were carried out in Tissue Culture Unit, Desert Research Center, El-
Matareya, Cairo, Egypt.
1. Plant Material and Surface Sterilization
Explants of B. monnieri were obtained from damp and marshy places in the Eastern Mediterranean Coastal
Region, North Sinai, Egypt (Fig. 1). Actively growing shoots with terminal buds were collected, moistened and
wrapped. Shoot tips (0.5-1 cm long) and stem nodal segments (1.5-2 cm long) and were dissected out of the cuttings
and washed thoroughly under running tap water for 2-3 hours. Surface sterilization was carried out using 2%
aqueous solution of sodium hypochlorite for five minutes followed by washing in sterilized distilled water for three
times. Subsequently, explants were treated with 0.1% (w/v) mercuric chloride solution for two minutes, followed by
washing in sterile distilled water for three times.
Fig. 1. B. monnieri grown naturally at Eastern Mediterranean Coastal Region, North Sinai, Egypt.
2. Culture Medium and Conditions
Murashige and Skoog (MS) medium (Duchefa, Netherlands) (Murashige and Skoog, 1962) was used for
micropropagation of B. monnieri, supplemented with 30 g/L sucrose and 100 mg/L myo-inositol. Plant growth
regulators (PGRs) (Duchefa, Netherlands). α-Naphthaleneacetic acid (NAA), indole-3-acetic acid (IAA), indole-3-
butyric acid (IBA), 6-benzylaminopurine (BAP) and N6-furfuryladenine (Kinetin; KIN) were added independently
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or in combinations at different concentrations for the micropropagation stages of B. monnieri. The pH of the media
was adjusted to 5.7-5.8 before gelling with 2.7 g/L phytagel. Fifteen ml volumes of media were dispensed into
25×150 mm culture tubes or 50 ml volumes into 12×350 mm large jars. Then, closed with polypropylene caps and
autoclaved at 121°C at a pressure of 1.1 kg/cm² for 20 minutes, then left to cool (IAA was sterilized by filtration in
Millex syringe driven filter unit of 0.45 µm diameter). The sterilized explants were cultured on the prepared media
under complete aseptic conditions in the laminar air flow hood. Tissue cultured tubes and jars were incubated in an
air-conditioned incubation room at a temperature of 26±2°C and 70±10% relative humidity, under a photoperiod of
16 hour with a light intensity of 2 klux, provided by cool white light fluorescent tubes (F 140t9d/38, Toshiba).
3. Culture Establishment
MS medium was supplemented with different concentrations of NAA, IAA, IBA, BAP and KIN,
individually or in combinations, in addition to the control treatment (MS nutrient medium without PGRs) for the in
vitro establishment of B. monnieri explants. Percentage of explants forming growth (%), mean number of axillary
shoots/explant and mean length of axillary shoots (cm) were recorded after six weeks of culture.
4. Multiple Shoots Induction
Established shoots of B. monnieri were subjected to be multiplied on MS medium containing IBA (2.45
µM) in combination with KIN (2.3 µM) and NAA (5.4 µM) in combination with IAA (5.7 µM). For further
multiplication, the explants were subcultured six successive subcultures on the best medium to obtain stock
materials to be used in the following experiments. Mean number and length (cm) of axillary shoots/explant were
recorded after six weeks from each subculturing.
5. Rooting and Acclimatization
The healthy in vitro produced shoots of B. monnieri were tested for roots induction on MS medium
supplemented with auxins; NAA, IAA or IBA in addition to the control treatment (MS medium without PGRs).
Rooting percentage (%), mean number of roots/explant, mean length of roots (cm) and mean shoot height (cm) were
recorded after six weeks of culture.
Rooted shoots (at least 3 cm long) were washed from medium residues and treated with 0.2% topsin (w/v)
solution as a fungicide. Then, hardened off inside the growth room in soilrite for two weeks, then transplanted into
pots filled with a mixture of sand and peat moss (1:1 v/v) and irrigated with tap water. Pots (5 cm in diameter) were
covered with transparent polyethylene bags and placed in the greenhouse. One week later, the covers were removed
gradually within a month. The percentage of survived transplants (%) was recorded.
6. Precursor Feeding
B. monnieri multiple shoots were cultured on MS medium supplemented with 2.45 µM IBA and 2.3 µM
KIN, and augmented with the precursor MVA at 0, 2.5, 5.0, 7.5 and 10 mM. Aqueous solution of MVA (Sigma-
Aldrich, United Kingdom) was prepared by adding 50 ml distilled water to 1 g of MVA powder. MVA was filter
sterilized by filtration in Millex syringe driven filter unit (0.22 µm).
7. Elicitation
The multiple shoots of B. monnieri were cultured on MS medium supplemented with 2.3 µM KIN and 2.45
µM IBA, and augmented with the elicitors; CH (Fluka, Japan) at 0, 50, 100, 200 and 250 mg/L and MJ (Fluka,
Japan) at 0, 25, 50, 100 and 200 µM.
CH treatments were prepared from a stock solution obtained by dissolving 500 mg CH in about 30 ml of
glacial acetic acid and the solution was titrated with 1 N NaOH to give a final pH of 5.7 before autoclaving. MJ was
filter sterilized by filtration in Millex syringe driven filter unit (0.22 µm). The mean number and length (cm) of
axillary shoots/explant and bacoside A content (µg/g dry weight) were recorded after six weeks of culture.
8. Determination of Bacoside A
8.1. Extraction of Bacoside A
Oven-dried plant tissues (at 40ᵒC for three days until constant weights were obtained) from mother plant;
multiplied shoots and in vitro produced plantlets; which were subjected to precursor and elicitors feeding; were
finely powdered and extracted with 10 ml of 80% methanol for 24 hours at room temperature. The extracts were
filtered through Whatmann no. 1 filter paper, kept in the vacuum desiccator for one week and the residue was then
dissolved in 1 ml of methanol and filtered through 0.22 µM membrane filters (Largia et al., 2015).
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8.2. Estimation of bacoside A content
Bacoside A content was determined in the samples by Dionex UltMate 3000 HPLC system equipped with
quaternary pump LPG3400SD, a WPS 3000 SL analytical autosampler, and a DAD – 3000 photodiode array
detector (Thermo Dionex, Germany). Samples were run on an analytical column C18 using gradient elution. The
mobile phase was a mixture of Milli Q water containing 0.2% phosphoric acid and acetonitrile (65:35, v/v; pH 3) at
a flow rate of 1 ml/min and column temperature was maintained at 30ᵒC. The detection wavelength was set at 205
nm. The injection volume was 20 µl and the chromatography system was equilibrated by the mobile phase. Data
were analyzed and integrated by Chromeleon 7 software (Largia et al., 2015).
9. Experimental Design and Statistical Analysis
The experiments were subjected to completely randomized design. Each experiment was repeated twice
and treatments consisted of at least 10 replicates. Variance analysis of data was carried out using ANOVA program
for statistical analysis. The differences among means for all treatments were tested for significance at 5% level by
using Duncan´s multiple range test (Duncan, 1955).
RESULTS AND DISCUSSION
1. Culture Establishment
Five PGRs (NAA, IAA, IBA, BAP and KIN) at different concentrations and combinations were evaluated
for in vitro establishment of B. monnieri, in addition to the control (MS medium without PGRs), from shoot tip and
stem nodal segment explants. The two explant types gave the same response. Data in Table 1 reveal that 100% of
explants survived after culturing on the different tested media. Shoot initiation started after 8-10 days of culture and
multiple shooting started after 15-20 days. This confirms the earlier reports on shoot induction from different
explants of B. monnieri. With respect to the percentage of explants forming growth, it was 100% for all PGRs
combinations, except treatments of BAP at 1.1 µM individually, or at 13.2 and 17.6 µM either individually or in
combination with NAA at 0.54 µM, in addition to MS medium without PGRs.
Among tested concentrations of PGRs; 2.45 µM IBA and 2.3 µM KIN gave the maximum response
concerning the number of axillary shoots/explant, followed by 5.4 µM NAA and 5.7 µM IAA. Both PGRs
combinations were the most effective for inducing maximum mean axillary shoot number of 19.1 and 13.6
shoots/explant, respectively (Fig. 2).
Concerning the mean length of axillary shoots, it is clear that MS medium supplemented with NAA at the
lowest concentrations (2.7 and 5.4 µM) individually or in combination with IAA, gave the highest mean length of
axillary shoots/explant. The maximum mean length of axillary shoots was 10.2 cm on MS medium supplemented
with 5.4 µM NAA and 5.7 µM IAA.
The superior effect of MS medium supplemented with 2.45 µM IBA in combination with 2.3 µM KIN on
growth formation and number of axillary shoots is in harmony with the results of Ceasar et al. (2010), who found
that the addition of auxin to the cytokinin improved the frequency of shoot induction, where the adventitious shoot
buds of B. monnieri were obtained on MS medium supplemented with 6.75 µM TDZ and 2.7 µM NAA. This may
be due to the synergistic effect of auxin and cytokinin on in vitro shoot induction. It is clear that the addition of two
different PGRs is essential for increasing shoot induction of B. monnieri (Ceasar et al., 2010). Also, Showkat et al.
(2010) supported the use of two auxins for the culture initiation of B. monnieri, they successfully used MS medium
supplemented with 5.7 µM IAA and 4.9 µM NAA.
2. Multiple Shoots Induction
IBA with KIN and IAA with NAA at their optimum concentrations (from the establishment stage)
regarding the number of axillary shoots, were studied for their effect on in vitro shoot multiplication for six
successive subcultures. Incorporation of IBA and KIN into MS medium supported multiplication of axillary shoots
and gave the best response, which proved to be a better choice than NAA and IAA (Table 2).
The highest axillary shoots multiplication was observed on MS medium supplemented with 2.45 µM IBA
and 2.3 µM KIN (Fig. 3a). On the other hand, the length of axillary shoots were higher on MS medium
supplemented with 5.4 µM NAA and 5.7 µM IAA (Fig. 3b).
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Table 1. Effect of different PGRs on the in vitro establishment of B. monnieri explants cultured on MS medium.
Results were taken after six weeks of culture.
Means followed by the same letter within a column are insignificantly different at P ≤ 0.05.
Fig. 2. Axillary shoots of B. monnieri established in vitro on MS medium supplemented with a. 2.45 µM IBA + 2.3
µM KIN and b. 5.4 µM NAA + 5.7 µM IAA.
Concentration of PGRs (µM) % of explants
forming growth
Mean number of
axillary
shoots/explant
Mean length of
axillary shoots
(cm) NAA IAA IBA BAP KIN
0.00 0.00 0.00 0.00 0.00 de40 hij3.20 j1.50
0.00 0.00 0.00 1.10 0.00 b80 defghij5.60 fghij2.30
0.00 0.00 0.00 2.20 0.00 a100 fghij3.50 fghij2.50
0.00 0.00 0.00 4.40 0.00 a100 defghi5.80 fghij2.40
0.00 0.00 0.00 8.80 0.00 a100 defghi6.10 fghij3.00
0.00 0.00 0.00 13.20 0.00 c60 j2.30 ij1.60
0.00 0.00 0.00 17.60 0.00 d45 j2.20 hij1.80
0.54 0.00 0.00 1.10 0.00 a100 defghij4.50 efghij3.20
0.54 0.00 0.00 2.20 0.00 a100 defghij5.60 cdefghij3.70
0.54 0.00 0.00 4.40 0.00 a100 cdef6.90 cdefghij3.70
0.54 0.00 0.00 8.80 0.00 a100 cdefg6.80 cdefghij3.90
0.54 0.00 0.00 13.20 0.00 c60 defghij4.80 fghij2.50
0.54 0.00 0.00 17.60 0.00 e30 fghij3.70 fghij2.60
0.00 2.85 0.00 2.20 0.00 a100 ij3.00 bcdefg5.20
0.00 2.85 0.00 4.40 0.00 a100 cdefghi6.20 cdefghij4.60
0.00 2.85 0.00 6.60 0.00 a100 defghij5.50 cdefghij3.90
0.00 0.00 0.00 2.20 2.30 a100 defghij5.00 fghij2.20
0.00 0.00 0.00 4.40 2.30 a100 defghij4.40 ghij2.00
0.00 0.00 0.00 6.60 2.30 a100 efghij4.10 fghij2.30
0.00 0.00 0.00 2.20 4.60 a100 ghij3.40 fghij3.00
0.00 0.00 0.00 4.40 4.60 a100 fghij3.70 fghij2.60
0.00 0.00 0.00 6.60 4.60 a100 fghij3.50 fghij2.30
0.00 0.00 0.00 2.20 6.90 a100 efghij4.00 defghij3.50
0.00 0.00 0.00 4.40 6.90 a100 hij3.30 fghij3.00
0.00 0.00 0.00 6.60 6.90 a100 fghij3.70 fghij2.60
2.70 0.00 0.00 0.00 0.00 a100 c9.50 bcd6.50
5.40 0.00 0.00 0.00 0.00 a100 cde7.20 ab8.00
8.10 0.00 0.00 0.00 0.00 a100 cdefghi6.40 bcdef5.40
2.70 5.70 0.00 0.00 0.00 a100 cd7.70 bc6.80
5.40 5.70 0.00 0.00 0.00 a100 b13.60 a10.20
10.80 5.70 0.00 0.00 0.00 a100 cdefghi6.40 bcdefgh5.10
0.00 0.00 2.45 0.00 2.30 a100 a19.10 bcde6.30
0.00 0.00 4.90 0.00 2.30 a100 cdefgh6.50 bcdefghi4.90
0.00 0.00 9.80 0.00 2.30 a100 cd7.80 cdefghij4.60
a b
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Table. 2. Effect of the best establishment PGRs combination on in vitro multiplication of B. monnieri axillary
shoots cultured on MS medium. Results were taken after six weeks of culture. Subcultures 2.45 µM IBA + 2.30 µM KIN 5.40 µM NAA + 5.70 µM IAA
MNS MLS MNS MLS
1st 24.0a 5.6b 18.3b 10.2a
2nd 26.4a 5.4b 17.9b 9.60a
3rd 32.6a 6.6b 20.0b 10.4a
4th 30.7a 4.8b 20.4b 9.50a
5th 28.1a 5.8b 16.9b 8.50a
6th 30.5a 5.9a 18.2b 6.20a
MNS: mean number of axillary shoots
MLS: mean length of axillary shoots Means followed by the same letter within a column are insignificantly different at P ≤ 0.05.
Fig. 3. Multiple shoots of B. monnieri cultured on MS medium supplemented with a. 2.45 µM IBA + 2.3 µM KIN
and b. 5.4 µM NAA + 5.7 µM IAA.
The results show that on MS medium supplemented with 2.45 µM IBA and 2.3 µM KIN, the number of
axillary shoots increased until reached the maximum value in the third subculture (32.6 axillary shoots/explant), then
declined during further subculturing. Also, the length of axillary shoots recorded its highest value in the third
subculture and reached 6.6 cm, then decreased. On the other hand, the results show that on MS medium
supplemented with 5.4 µM NAA and 5.7 µM IAA, the number of axillary shoots increased until reached the
maximum value in the fourth subculture (20.4 axillary shoots/explant), then declined during further sub-culturing.
Although, the length of axillary shoots recorded its highest value in the third subculture and reached 10.4 cm, then
decreased. There was a reverse correlation between shoot number and length in the two treatments. Axillary shoots
cultured on MS medium supplemented with 2.45 µM IBA and 2.3 µM KIN, produced the maximum number of
axillary shoot, but it produced short shoots. In contrast, axillary shoots cultured on MS medium supplemented with
5.4 µM NAA and 5.7 µM IAA produced the minimum number of axillary shoots accompanied by longer shoots.
Several workers have reported multiple shoots induction of B. monnieri with auxins and cytokinins in the
growth medium (Praveen, 2009; Joshi et al., 2010 and Tanveer et al., 2010). Also, addition of IBA and NAA gave
high response of multiple shoots regeneration of B. monnieri as previously described (Ali et al., 1999; Praveen, 2009
and Tanveer et al., 2010).
3. Rooting and Acclimatization
Data in Table 3 represent the rooting of axillary shoots of B. monnieri. The excised shoots showed 100%
rooting at the basal end of the shoots within two weeks in all tested treatments, even the medium without PGRs.
However, the best results were obtained with MS medium supplemented with IBA. IBA at 4.9 µM produced the
maximum mean number (8.05 roots/ shoot) and length (7.6 cm) of roots/shoot, in addition to the maximum shoot
height of 11 cm (Fig. 4). Moreover, the increasing or decreasing in IBA concentration decreased the mean number
and length of roots/shoot in addition to the mean shoot height.
The minimum mean number and length of roots/shoot was recorded on the control MS medium without
PGRs. In general, IBA recorded the best rooting response, followed by IAA and the minimum response was
obtained by NAA. Similar observations were recorded by several workers, who proved that IBA at 4.9 µM was the
a b
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most efficient treatment for the development of healthy root system for B. monnieri (Ceasar et al., 2010; Tiwari and
Singh, 2010; Kaur et al., 2013 and Jain et al., 2014).
Table. 3. Effect of different auxins on in vitro rooting of B. monnieri axillary shoots cultured on MS medium.
Results were taken after six weeks of culture. Auxin concentration (µM) Rooting (%) Mean number of
roots/shoot
Mean length of
root (cm)
Mean shoot
height (cm) NAA IAA IBA
0.00 0.00 0.00 100 2.20g 2.8f 5.5d
2.70 0.00 0.00 100 2.50f 4.5e 5.0d
5.40 0.00 0.00 100 4.89d 5.7bc 5.5d
10.80 0.00 0.00 100 5.00d 5.7bc 5.0d
0.00 2.85 0.00 100 3.20e 4.2e 6.0cd
0.00 5.70 0.00 100 6.80b 5.8b 6.5bcd
0.00 11.40 0.00 100 5.40c 5.3d 6.5bcd
0.00 0.00 2.45 100 6.85b 5.7bc 7.5bc
0.00 0.00 4.90 100 8.05a 7.6a 11.0a
0.00 0.00 9.80 100 5.15cd 5.4cd 8.0b
Means followed by the same letter within a column are insignificantly different at P ≤ 0.05.
Fig. 4. Rooted shoots of B. monnieri cultured on MS medium supplemented with 4.9 µM IBA.
IBA is a highly effective auxin for rooting of in vitro regenerated shoots in several plant species (Gururaj et
al., 2007). It has been widely used as a roots induction PGR under in vitro and in vivo conditions (Begum and
Mathur, 2014). Although the exact method of how IBA works is still exactly unknown, genetic evidence has been
found that IBA may be converted into IAA, which suggests that IBA works as a storage sink for IAA in plants
(Zolman et al., 2008). There is another evidence that suggests that IBA acts as an auxin on its own (Ludwing-
Müller, 2000).
Plantlets of B. monnieri were survived when they were hardened off inside the growth room in soilrite for
two weeks. Hundred percent of the acclimatized transplants were survived after two weeks of transferring into the
peatmoss: sand mixture (1:1 v/v) in the greenhouse. After one month, the transplants were transferred to bigger pots
supplemented with the same soil composition, covered with polyethylene bags, and were irrigated with tap water.
Transplants were kept in greenhouse for twelve months (Figs. 5 and 6).
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Fig. 5. Hardened and acclimatized in vitro derived plantlets of B. monnieri in the greenhouse after: a. two weeks, b.
six weeks, c. two months, d. four months, e. six months and f. twelve months.
Fig. 6. Mass hardened and acclimatized in vitro derived plantlets of B. monnieri in the greenhouse after six months.
a
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4. Effect of Precursor and Elicitors Feeding on Shoot Culture
Data in Table 4 show the effect of various concentrations of MVA, as a precursor and CH and MJ, as
elicitors, on biomass and bacoside A content in shoot cultures of B. monnieri after six weeks of culture. Addition of
exogenous MVA, CH and MJ enhanced the mean number of axillary shoots, except for 2.5 mM MVA and 25 µM
MJ, compared to the control treatment (32.6 axillary shoots/ explant). However, among the concentrations used, the
highest mean number of axillary shoots was obtained when feeding with 100 µM MJ (42 axillary shoots/explant)
(Fig. 7). Concerning the mean length of axillary shoots, the highest mean length of axillary shoots was obtained
when feeding with MVA at 2.5 mM (8.2 cm), followed by 10 mM MVA (8 cm).
Table 4. Effect of precursor feeding and elicitation on biomass and bacoside A content of shoots of B. monnieri
cultured on MS medium supplemented with 2.3 µM KIN + 2.45 µM IBA. Data were recorded after
six weeks of culture. Precursor Elicitors Mean number of
axillary shoots/
explant
Mean length of
axillary shoots
(cm)
Bacoside A
content (µg/g
dry weight)
Bacoside A content
(fold) compared to
intact plant
(1.13 mg/g dry weight)
MVA
(mM)
CH
(mg/L)
MJ
(µM)
0.0 0 0 g32.6 6.6b 1.27 1.12
2.5 0 0 g32.0 8.2a 7.73 6.84
5.0 0 0 34.0ef 7.5ab 9.70 8.58
7.5 0 0 35.0de 7.9a 10.19 9.01
10.0 0 0 33.0fg 8.0a 10.49 9.28
0.0 50 0 33.0fg 6.5b 4.98 4.40
0.0 100 0 38.0b 6.2bc 5.18 4.58
0.0 150 0 38.0b 6.9ab 6.17 5.46
0.0 200 0 36.0cd 7.2ab 5.18 4.58
0.0 250 0 34.0ef 6.4bc 4.93 4.36
0.0 0 25 25.0de 5.2cd 6.20 5.48
0.0 0 50 37.0bc 4.8d 6.95 6.15
0.0 0 100 42.0a 4.2d 6.33 5.60
0.0 0 200 38.0b 4.6d 6.37 5.64
Mean followed by the same letter within a column are insignificantly different at P ≤ 0.05.
Fig. 7. Shoots of B. monnieri grown on MS medium supplemented with
2.3 µM KIN + 2.45 µM IBA + 100 µM MJ.
The positive effect of MJ on shoot growth could be explained by a synergistic effect of MJ with the used
PGRs combinations. Similar effect of MJ on shoot growth has been reported with micropropagation of Pistacia
vera, where MJ was added at concentrations of 0.3, 1, or 3.2 μM and improved shoot multiplication rates (Sanjuan
and Claveria, 1995). On the other hand, this result is in contrast with those of Sharma et al. (2013), where MJ treated
shoots of B. monnieri had decreased growth compared to the control.
The results of HPLC chromatograms of bacoside A content in oven-dried plant tissues obtained from
mother plant, multiplied shoots and in vitro produced plantlets subjected to precursor and elicitors feeding cultures
are represented in Table 4. All concentrations of the precursor and elicitors enhanced bacoside A accumulation in
shoot cultures of B. monnieri. Addition of 10 mM MVA induced the highest accumulation of bacoside A (10.49
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Hegazi et al., 2017
µg/g dry weight) after six weeks of MVA feeding, representing a 8.26-fold increase in bacoside A accumulation as
compared with that in the control cultures (1.27 µg/g dry weight) and 9.28-fold increase as compared to the intact
plant (mother plant). The MVA pathway initiates with the acetyl-coenzyme A, which condenses into the
acetoacetyl-CoA by the catalyzing action of acetyl-CoA acetyltransferase. Acetyl-coenzyme A is converted to
isopentenyl diphosphate (IPP) through MVA, and HMGR (HMG-CoA reductase), which finally supplies carbon for
the bacoside biosynthesis (Miziorko, 2011 and Vishwakarma, 2015).
Concerning the effect of MJ on bacoside A accumulation, it was observed that the elicitation with 50 µM
MJ gave the highest accumulation of bacoside A (6.95 µg/g dry weight) representing a 5.47-fold increase as
compared with that in the control cultures and 6.15-fold increase comparing to the intact plant. The concentrations
of 25, 100 and 200 µM of MJ were produced less amount of bacoside A of 6.20, 6.33 and 6.37 µg /g dry weight,
representing a 4.88-, 4.98- and 5.01-fold increase, respectively, comparing to the control and 5.48-, 5.6-and 5.63-
fold increase, respectively, as compared to the intact plant. MJ and its derivatives are key signal compounds
efficiently used as an elicitor for eliciting secondary metabolites production in various plant species (Yu et al., 2002
and Qian et al., 2005; Zhao et al., 2005).
Similar effects of MVA on saponins production have been reported with other plant species. The optimal
concentration of mevalonate for increased saponin production in Panax ginseng callus was found to be 50 ppm
(Furuya et al., 1983). Also, a maximum sesquiterpene lactone (artemisinin) accumulation was produced on
supplementation of MVA and MJ as selected precursor and elicitor, respectively, which was 4.79 times higher in
productivity than control callus cultures of Artemisia annua (Baldi and Dixit, 2008).
The lowest increase in accumulation of bacoside A was achieved with the elicitation with CH. It was
found that the maximum production of bacoside A in shoot cultures of B. monnieri using CH after six weeks of
treatment was recorded with 150 mg/L CH and gave 6.17 µg bacoside A/g dry weight, representing a 4.86-fold
increase as compared with that in the control cultures and 5.46-fold increase as compared to the intact plant.
Followed by 100 and 200 mg/L CH (5.18 µg bacoside A/g dry weight) representing a 4.07-fold increase as
compared to the control treatment and 4.58-fold increase comparing to the intact plant. The least accumulation of
bacoside A was observed after elicitation with 50 and 250 mg/L CH.
In conclusion, the micropropagation protocol represented in this study could be commercially applied in
Egypt for the mass production of this medicinally important rare herb to meet the ever-increasing demands of the
pharmaceutical industries, as well as to save this species from extinction due to over exploitation of natural
populations. Also, using MVA as a precursor of bacoside A in the multiplication stage could produce plantlets rich
in the memory enhancing compound.
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