UserMicrosoft Word - JBASR-3144-1 (revised)J. Basic. Appl. Sci. Res., 6(12)35-43, 2016 |
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ISSN 2090-4304 |
Journal of Basic and Applied Scientific Research www.textroad.com |
In vitro Preservation of Bacopa monnier (L.) Pennell as a Rare |
Medicinal Plant in Egypt |
Ghada Abd El-Moneim Hegazi |
Tissue Culture Unit, Department of Genetic Resources, Ecology and Dry Land Agriculture Division, |
Desert Research Center, 11753 El-Matareya, 1 Mathaf El-Matareya St., Cairo, Egypt |
Received: August 18, 2016 |
Accepted: November 29, 2016 |
ABSTRACT |
Bacopa monnieri (L.) Pennell, commonly known as ‘Brahmi’, belonging to family Scrophulariaceae, is a |
rare medicinal herb with global interest, and is one of the natural plants in the flora of Egypt. The plant grows in damp and marshy places of the Eastern Mediterranean coastal region in North Sinai. It is mainly used for the treatment of neurological disorders. With increasing demand for B. monnieri in pharmaceutical industries, there is a need to conserve the wild stocks of this plant through biotechnological approaches. A successful protocol for synthetic seeds production is developed in the present study for conservation of B. monnieri. Shoot tips excised from in vitro-derived multiple shoots were encapsulated in calcium alginate beads. Uniform spherical beads were obtained using 2.5% sodium alginate and 100 mM calcium chloride (CaCl2.2H2O) solutions. Shoot tips were stored for six months in calcium alginate beads containing different matrixes; water, Murashige and Skoog medium and Murashige and Skoog medium containing 3% sucrose, and were tested for storage at 24±2 and 4ºC. The efficiency of regrowth of B. monnieri encapsulated shoot tips was investigated. Hundred percent viability and high regrowth percentage and performance of recovered shoot tips, on Murashige and Skoog medium, were observed in all tested alginate matrixes at 4°C. However, Murashige and Skoog medium with sucrose was significantly superior. Rooting was achieved on the same medium and 93% of regenerated plantlets were successfully hardened under greenhouse conditions. The regeneration ability and regrowth performance of encapsulated shoot tips were declined monthly during storage duration. The protocol derived can be efficiently used for the preservation of encapsulated shoot tips of B. monnieri for six months at 4°C without any treatments with growth regulators. It could be cost-effective and useful in germplasm conservation and delivery of tissue cultured |
B. monnieri plants. |
KEY WORDS: Brahmi, in vitro conservation, encapsulation, synthetic seed, storage, regrowth |
INTRODUCTION |
Medicinal plants serve as traditional medicine and natural raw material for drug preparations; |
therefore, they play a key role in world health. According to world health organization (WHO), more than 80% of the world population relies on plants as a source of traditional medicines for their primary health care needs, due to their fewer side effects in comparison to artificial drugs. The conservation of medicinal plants is a must. Natural medicinal plants are facing a combination of factors; such as over-collection, urbanization, pollution, habitat destruction, degradation, spread of invasive alien species and climate change. A large number of medicinal plants are already at the risk of extinction. Plant tissue culture is an emerging tool for the propagation and conservation of plants that are listed as endangered or rare. In vitro propagation and preservation of medicinal plants, especially natural plants, and their extracts are efficient and the most suited alternative solution to the problems faced by the phytopharmaceutical industry. They provide a ready source of sterile, uniform and compatible plant material (Banerjee and Shrivastava, 2008). |
Synthetic seed technology is an emerging and broadly used technique in the field of plant biotechnology to conserve germplasm of the endangered and economically important plants (Ikhlaq et al., 2010). It greatly aids in germplasm exchange programs and storage of viable and productive tissues (Mehrotra et al., 2012). Synthetic seeds (synseeds) produced through encapsulation technique have the ability to produce an entire plant after storage at room temperature or under low temperatures; above 0ºC (Gantait et al., 2015). They have great advantages in the field of plant conservation, because of their small size, minimum requirements of space, time and care, virus free germplasm, low cost of production, ready |
Corresponding Author: Ghada Abd El-Moneim Hegazi, Tissue Culture Unit, Department of Genetic Resources, |
Ecology and Dry Land Agriculture Division, Desert Research Center, 11753 El-Matareya, 1 Mathaf El-Matareya St., Cairo, Egypt. E-mail: ghada.hegazi1211@gmail.com |
Hegazi, 2016 |
availability, ease of handling and transporting, and also produce viable and genetically identical plants even |
after long term storage (Mishra et al., 2011). In the beginning, synthetic seeds for germplasm conservation were prepared from somatic embryos. However, recently synthetic seeds were mostly prepared from shoot tip explants, because of their effectiveness for in vitro regeneration and having great mitotic activity of the meristem, when compared to other parts of the plants (Ballester et al., 1997). |
Bacopa monniera (L.) Pennell, commonly known as “Brahmi”, is a member of family Scrophulariaceae. It is a rare perennial creeping herb grows in wetlands and muddy shores and found in Egypt; mainly in wet and marshy areas of the eastern Mediterranean coast in North Sinai (Boulos, 2009). B. monnieri has a great medicinal value. It is considered the main rejuvenating herb for nerve and brain cells; therefore, has a very important role for the treatment of the neurological and cognitive disorders of aging (Russo and Borrelli 2005 and Aguiar and Borowski, 2013), especially, Alzheimer's disease (Goswami et al., 2011). It is the second most important medicinal plant in India according to its medicinal importance, commercial value and potential for further research and development (Anonoymous, 1997). It contains many active compounds; mainly saponins such as bacosides A, B, C and D, which are the active triterpenoid principles that are known as ‘‘memory chemicals’’, alkaloids (nicotine, brahmine and herpestine), and other chemicals; such as stigmastanol, bsitosterol and stigmasterol (Russo and Borrelli, 2005). |
The plant is extensively investigated for its pharmacological and therapeutic effects; mainly |
memory, learning and intelligence improvement (Singh et al., 2015), and in treatment of neurological diseases like epilepsy, insomnia, asthma, insanity, depression, psychosis and stress (Russo and Borrelli, 2005 and Pandareesh et al., 2016). It is reported to have anti-inflammatory, analgesic, antipyretic, sedative (Hossain et al., 2014), antioxidant (Simpson et al., 2015 and Sabina et al., 2016) and hepatoprotective (Sumathi and Devaraj, 2009) effects. The plant has also been used to treat cardiac, digestive and respiratory disorders (Channa et al., 2003). Moreover, it is known to have anticancer properties (Patil et al., 2014). Beside its various medicinal uses, B. monnieri has also been utilized in phytoremediation programs, due to its ability to remove heavy metals (Mehta et al., 2012). |
Because of the rapid expansion of the pharmaceutical industry coupled with the popularity of Brahmi based drugs, the pharmaceutical requirement of this plant has increased dramatically. Therefore, it is very important to conserve this already rare herb. Furthermore, their natural regeneration is limited by death at two-leaf stage and submerged shoots can hardly attain the required growth and multiplication. Its ex situ conservation in the seed bank is limited due to insufficient seed availability, and propagation through seeds is low due to short seed viability and frequent seedling death. Also, vegetative propagation of |
B. monnieri is slow (Tiwari and Singh, 2010). Therefore, there is a need to conserve the wild stocks of this medicinally important herb to reduce the exploitation of its natural populations and increase the supply of plant materials for pharmaceutical industry. |
From the literature, there are many studies that have reported in vitro multiple shoot induction and regeneration of B. monnieri, using various explants and different culture media and growth regulators; such as the recent studies of Behera et al. (2015), Gupta et al. (2015), Nagarajan et al. (2015), Karatas et al. (2016), Pothiaraj et al. (2016) and Wangdi and Sarethy (2016). Although attempts towards its in vitro conservation are limited. There are few detailed reports on B. monnieri synthetic seeds production, although no studies were carried out without the application of growth regulators or other elicitors. B. monnieri regeneration was assessed after storage of alginate encapsulated seeds from nodal segments (Sharma et al., 2012 and Gantait et al., 2015) and shoot tips (Bansal and Pandey, 2011 and Rency et al., 2016) or both (Muthiah et al. 2013) with the use of growth regulators or elicitors, either in the alginate matrix or the conversion medium. Attention should be paid to conserve this plant and improve the technology to achieve more success with the least cost, to meet the growing demand of its raw material. Thus, this study aimed to in vitro preserve B. monnieri through encapsulation technique, to conserve the wild stocks of this medicinally important rare plant in Egypt. It reports the effect of synthetic seeds production without growth regulators, for cost reduction, on viability and regrowth performance of alginate-encapsulated shoot tips for six months. |
MATERIALS AND METHODS |
1. Plant material |
Actively growing shoots of B. monnieri were collected from a wild population grown in the |
eastern Mediterranean coast of North Sinai in Egypt. In vitro cultures of B. monnieri, from shoot tips and stem nodal segments, were initiated and subcultured every four weeks for multiplication on Murashige and |
J. Basic. Appl. Sci. Res., 6(12)35-43, 2016 |
Skoog (MS) (Murashige and Skoog, 1962) medium (Duchefa, Haarlem, the Netherlands) containing 0.1 |
mg/L myo-inositol and 3% sucrose and supplemented with 4.44 μM 6-benzylaminopurine (BAP) and 0.53 μM naphthalene acetic acid (NAA) (Sigma Cell Culture, min. 90%, USA). The pH of the medium was adjusted to 5.7-5.8 prior to addition of 2.7 g/L phytagel (Duchefa, Haarlem, the Netherlands) as a solidifying agent. Medium was dispensed into glass jars and autoclaved under a pressure of 1.06 kg/cm2 and 121°C for 20 min. Cultures were maintained at 24±2°C with a 16 h photoperiod under white cool fluorescent tubes (F140t9d/38, Toshiba). |
2. Encapsulation of shoot tips and storage conditions |
In vitro-derived shoot tips, obtained from the proliferated shoots, were used for encapsulation. Shoot tips (≤ 5 mm) were encapsulated using 2.5% sodium alginate (Na-alginate; CDH, India) and 100 mM calcium chloride (CaCl2.2H2O; Merck, Germany) solutions. Encapsulation was carried out by mixing the shoot tips into Na-alginate solution. The alginate-covered shoot tips were then dropped into the CaCl2.2H2O solution with an autoclavable micropipette. The beads (each usually containing one or two shoot tips) were left for 20-30 min to ensure polymerization of calcium alginate. After hardening, the encapsulated beads were rinsed with sterilized distilled water for three times. The capsules had an average diameter of about 7 mm (Photo 1). In order to achieve efficient storage, encapsulation solution was prepared using different Na-alginate matrix compositions; 2.5% Na-alginate in distilled water, MS medium and MS medium + 3% sucrose. |
Encapsulated shoot tips with different Na-alginate matrixes were stored in Petri dishes containing water agar medium (0.7% w/v agar; Duchefa, Haarlem, the Netherlands) for six months. Two different temperatures of 24±2 and 4°C (in the dark) were tested for storage. Non-encapsulated shoot tips were treated as control. |
3. Recovery of encapsulated shoot tips |
Every one month, encapsulated shoot tips were cultured on MS medium without growth regulators for the recovery. Percentage of viability, regrowth, mean number and length (cm) of shoots per explant, in addition to rooting percentage were evaluated, for a period of six months, after four weeks from culturing. Rooted plantlets were carefully separated from the medium and washed with distilled water to remove excess medium. The healthy root induced plantlets were transferred to small pots containing a mixture of peatmoss and sand at a 1:1 ratio. The pots were covered with transparent plastic bags with minute holes to maintain humidity and placed inside the greenhouse. After 2 weeks, covers were removed for further growth at natural conditions and plantlets were irrigated with tap water every two days. The survival rate of plantlets was calculated a month after transfer to the greenhouse. |
4. Experimental design and data analysis |
The experiment was subjected to the completely randomized design. Treatments were represented by twenty replicates and the experiment was repeated twice. Variance analysis of data was carried out using ANOVA program for statistical analysis. Means were compared by using Duncan´s multiple range test (Duncan, 1955) at p ≤ 0.05. |
RESULTS AND DISCUSSION |
1. Effect of storage temperature and alginate matrix on the recovery of encapsulated shoot tips |
The encapsulation of in vitro regenerated shoot tips via Na-alginate has been proved to be the most efficient technique for mass propagation of plants as well as short term conservation of important germplasms (Kulus and Zalewska, 2014). Based on the previous reports, shoot tip explants are the best source for in vitro regeneration of B. monnieri (Rency et al., 2016), and are the most effective explants for synthetic seeds preparation, because of the great mitotic activity of the meristem (Ballester et al., 1997). |
Suitable storage temperature plays a vital role in preservation of synthetic seeds. Two different temperatures of 24±2 and 4°C were tested for storing the encapsulated shoot tips. Results showed that the beads stored at 24±2°C started sprouting within a week, and thus this temperature was found to be inefficient for storage. This observation was not occurred in the beads stored at 4°C. According to this observation, further storage of encapsulated shoot tips was carried out at 4°C for six months. Previously, |
Hegazi, 2016 |
4°C was very promising for low-temperature in vitro preservation of B. monnieri (Muthiah et al., 2013), |
and other plants (Faisal and Anis, 2007; Adhikari et al., 2014 and Saha et al., 2015). |
The composition of the alginate matrix used for encapsulation is a crucial factor in plant regeneration after storage. The proper optimization of the alginate matrix composition improves the regrowth performance of encapsulated shoot tips and consequently the technique could be more efficient and applicable. Non-encapsulated and encapsulated shoot tips of B. monnieri in 2.5% Na-alginate and 100 mM CaCl2.2H2O exhibited shoot formation within one week after transferring them to regeneration medium (Photo 2). All tested Na-alginate matrix compositions demonstrated 100% viability of encapsulated shoot tips after six months of storage (Table 1). However, regrowth percentage and the number and length of regenerated shoots were lower in encapsulated shoot tips compared to non- encapsulated ones (Photo 3a), because the non-encapsulated shoot tips are directly in contact with the nutrient medium. |
The Na-alginate matrix containing MS medium demonstrated significant superiority over that containing distilled water (Photo 3b) with respect to regrowth percentage and shoot number and length. This treatment gave a percentage of regrowth of 85.2% of encapsulated shoot tips, produced 7 shoots per explant with a length of 5.27 cm, after six months of storage (Photo 3c). This result confirms that MS nutrients are essential ingredients of Na-alginate matrix for regrowth and regeneration. Gelling matrix containing nutrient ingredients served as “artificial endosperm”, that provides nutrients to the encapsulated explants for regrowth (Bapat and Rao, 1992). |
Table 1. Effect Na-alginate matrix composition on the viability and regrowth performance of encapsulated |
shoot tips of Bacopa monnieri. Data was recorded after four weeks from culturing following six months of storage (non-encapsulated shoot tips were served as a control). |
Alginate matrix composition Viability % Regrowth % Mean no. of Mean shoot Rooting %
shoots/explant length (cm) |
Non-encapsulated shoot tips | 100 | 100.0a | 18.00a | 7.30a | 100 |
Distilled water | 100 | 73.7d | 1.33d | 4.27d | 100 |
MS medium | 100 | 85.2c | 7.00c | 5.27c | 100 |
MS medium + 3% sucrose | 100 | 93.3b | 10.00b | 6.00b | 100 |
The regrowth percentage and shoot number and length gave their maximum values when sucrose |
was supplemented in the nutrient medium (Photo 3d). Using the MS medium containing normal sucrose concentration for encapsulation, 93.3% of encapsulated shoot tips regrew 10 shoots per explant with a mean length of 6 cm, after six months of storage. These findings proved that sucrose is an essential component in the alginate matrix. The nutrient ingredients and sugar of the alginate matrix of the encapsulated explants are of key importance for their storage and regrowth efficiency (Tsvetkov et al., 2006). Sucrose is a carbohydrate and energy source in plant tissue culture systems (Bhojwani and Razdan, 1996). It is known to provide a carbon source for in vitro propagated tissues, and its incorporation in the alginate matrix of encapsulated explants enhanced plant recovery and regrowth performance (Danso and Ford-Lloyd, 2003). |
2. Effect of storage duration on the recovery of encapsulated shoot tips |
The most important feature of the encapsulated explants is their ability to remain viable and regrow after storage. Low temperature and high humidity were essential for retention of the viability of encapsulated shoot tips and thus for their storage. Response for the regrowth of encapsulated shoot tips was slightly affected by increasing storage duration at 4˚C, until six months. Regrowth percentage and the mean number and length of shoots produced from the encapsulated shoot tips declined following storage at 4˚C, during the storage duration for each tested Na-alginate matrix composition (Figure 1-4). |
J. Basic. Appl. Sci. Res., 6(12)35-43, 2016 |
120 100 100 100 100 100 97 93.3 |
100 |
80 |
60 |
40 |
20 |
0 |
87 79 |
85.2 |
73.7 |
Na-alginate in |
water |
Na-alginate in MS medium |
0 1 2 3 4 5 6 |
Storage duration (month) |
Figure 1. Effect of different storage durations on regrowth percentage of encapsulated shoot tips of Bacopa |
monnieri in Na-alginate of different tested matrixes, after four weeks from culturing. |
8 7.5 7 7 |
6 |
4 |
6.2 5.8 |
5.3 |
4.27 |
Mean no. of |
2 2 2 1.5 1.33 1.33 1.33 |
0 |
0 1 2 3 4 5 6 |
Storage durage (month) |
shoots/explant |
Mean length of shoots (cm) |
Figure 2. Effect of different storage durations on regrowth performance of encapsulated shoot tips of |
Bacopa monnieri in Na-alginate matrix containing water after four weeks from culturing. |
15 12.7 12.3 12 12 |
10 |
10.4 |
9.2 |
6.7 6.6 6.6 6 5.7 5.7 |
5 |
0 |
7 |
5.27 |
Mean no. of |
shoots/explant |
Mean length of shoots (cm) |
0 1 2 3 4 5 6 |
Storage duration (month) |
Figure 3. Effect of different storage durations on regrowth performance of encapsulated shoot tips of |
Bacopa monnieri in Na-alginate matrix containing MS medium after four weeks from culturing. |
Hegazi, 2016 |
20 18.3 17 |
15.3 |
15 13 |
11 10 |
Mean no. of |
10 7.5 7 6.8 6.5 6.5 6.33 6 |
5 |
0 |
0 1 2 3 4 5 6 |
Storage duration (month) |
shoots/explant |
Mean length of shoots (cm) |
Figure 4. Effect of different storage durations on regrowth performance of encapsulated shoot tips of |
Bacopa monnieri in Na-alginate matrix containing MS medium with 3% sucrose after four weeks from culturing. |
It is supposed that the decline in the growth parameters, observed among encapsulated shoot tips |
during storage for longer durations, may be a result of the inhibition in the respiration of tissues by the alginate cover due to oxygen deficiency; or a loss of moisture content due to partial desiccation of tissues (Danso and Ford-Lloyd, 2003 and Faisal and Anis, 2007). Also, Bazinet et al. (1992) found that plant regeneration rate after in vitro preservation was reduced by loss of viability of tissues caused by mechanical constraints or diffusional limitation. |
All the plantlets had the potential to produce roots and they were not affected by the storage duration. The rooting of the plantlets regrown from encapsulated shoot tips after storage proved the efficiency of the in vitro preservation potential. Hardening in the greenhouse was carried out after one month from culturing. About 93% of the acclimatized plantlets remained healthy in natural conditions (Photo 4). |
Conclusion |
The present study describes a detailed promising and efficient germplasm preservation method for |
conserving the rare multipurpose B. monnieri plant. In order to obtain about 93% of regrown B. monnieri plants, with high regrowth performance, from encapsulated shoot tips after storage at 4°C for six months, 2.5% Na-alginate solution (containing MS nutrient medium with 3% sucrose) and 100 mM calcium chloride (CaCl2.2H2O) should be used as encapsulation matrix. |
This protocol can be applied as an effective solution for the mass conservation of the plant with minimum requirements, without any subculturing, for the period of six months. This could facilitate germplasm exchange between laboratories and transportation to different parts of the world, while maintaining their viability and growth can resume immediately after culture. This research opens new prospects for further studies on this valuable Egyptian plant to preserve its elite tissues for longer periods of time. |
Photo 1. Calcium alginate encapsulated shoot tips of Bacopa monnieri. |
J. Basic. Appl. Sci. Res., 6(12)35-43, 2016 |
a b |
Photo 2. Sprouting of encapsulated shoot tips Bacopa monnieri after a. one week and |
b. two weeks from culturing. |
a b c d |
Photo 3. Regrown plantlets with healthy shoots and roots from shoot tip-derived synthetic seeds of Bacopa |
monnieri in different Na-alginate matrix compositions after four weeks from culturing following six months of storage at 4°C. a. non-encapsulated shoot tips (control), b. encapsulated shoot tips in distilled water, c. encapsulated shoot tips in MS medium, d. encapsulated shoot tips in MS medium containing 3% sucrose. |
Photo 4. Acclimatized plantlets from encapsulated shoot tips of Bacopa monnieri after two months from |
transfer to the greenhouse following six months of storage at 4 °C. |
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