micropropagation of bacopa monnieri and enhancing …. appl. environ. biol. sci., 7... · bacopa...

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J. Appl. Environ. Biol. Sci., 7 (9) 115-126, 2017 © 2017, TextRoad Publication ISSN: 2090-4274 Journal of Applied Environmental and Biological Sciences www.textroad.com * 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 Hegazi 1* , Hussein Sayed Taha 2 , Abdel Monem Mohamed Sharaf 3 , Saad Ramzy Elaish 1 1 Department of Genetic Resources, Desert Research Center, El-Matareya, Cairo, Egypt 2 Department of Plant Biotechnology and Genetic Engineering, National Research Center, Giza, Egypt 3 Department 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). 115

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J. Appl. Environ. Biol. Sci., 7 (9) 115-126, 2017

© 2017, TextRoad Publication

ISSN: 2090-4274

Journal of Applied Environmental

and Biological Sciences

www.textroad.com

*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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Hegazi et al., 2017

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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Hegazi et al., 2017

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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Hegazi et al., 2017

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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J. Appl. Environ. Biol. Sci., 7 (9) 115-126, 2017

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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Hegazi et al., 2017

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

122

b c

d e f

J. Appl. Environ. Biol. Sci., 7 (9) 115-126, 2017

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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