In vitro clonal propagation and evaluation of antibacterial activity of Benghal dayflower-Commelina benghalensis L

An efficient protocol for in vitro propagation of Commelina benghalensis was developed. Nodal segments were showed the superb explants in performance for shoot proliferation than other explants. On the other hand, BAP with auxin was better combination. The maximum (90.52) number of explants response and the highest (23.25) number of shoots per plant were obtained from nodal segments on MS medium fortified with 3.0 mg/l BAP+0.1 mg/l IBA. The highest shoot length (17.25 cm) was achieved on MS medium containing 3.0mg/l BAP+0.5mg/l IBA. In vitro proliferated shoots were transferred to full and half strength of MS media where 1.5 mg/l IBA on full strength of MS media was the best to fit for the maximum number (12.69) of roots formation per micro-shoot. Well rooted plantlets were transferred to soil and successfully acclimatized with 97% survival rate. Three extracts i.e. methanol, ethanol, Petroleum ether of C. benghalensis L and four concentrations of each extracts were used against five gram (+ve) and five gram (-ve) bacterial species for the screening of antibacterial activity. Ethanol extracts was the superior in performance. The susceptibility of tested pathogenic bacterial species was increasing compare with increasing of extracts concentration with few exceptions. The highest zone of inhibition was obtained against S. aureus (17.50 mm) and P. aeruginosa (17.44 mm) at 800 mg/l dose level of extracts. It was also noticed that Gram (+ve) bacterial species are more susceptible to Benghal dayflower crude extracts than Gram (-ve) bacterial species.


Introduction
The species Commelina benghalensis L. (2n = 22) belongs to the family Commelinaceae includes large number of species that differ widely in their characteristics. Generally, it is usually known as "Benghal dayflower". It has several scientific synonym names such as C. kilimandscharica, C. obscura, C. pyrrhoblepharis, C. rufociliata and C. uncata. This plant is a prostrate or scrambling ascending herb of about 1m in height. In the Kalangala district of Uganda, it is used as a wound ecbolic on skin or over a wound by crashing whole fresh

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Notulae Scientia Biologicae plant with mortar and pestle (Hamill et al., 2003). Traditionally, the people of china use this plant as a diuretic, an anti-inflammatory agent. Hong and Defillipps (2000) Where Pakistani eat as a vegetable. Besides, according to Manandhar and Sanjay (2000) the people of Nepal eat the young leaves as a vegetable, use a paste of plant for burns and the fluid of roots is also use to treat indigestion. In Bangladesh, for the treatment of otitis media, burns and conjunctivitis, this plant is ethno-used. The plant is also used for skin disease such as eczema, acne, warts and scabies etc. But the responsible compounds of these medicinal properties have not been identified yet. It also contains alkanols, sterols, stigma sterol, beta-sito sterol and compesterol (Ghani, 2003). In these connections, plant tissue culture has a potential to introduce genetic variability in Commelina genotypes through somaclonal variants, somatic hybrids or transgenic plants. It's considered as a promising technology to get these subjects and it's also played a major role in the mass multiplication, conservation, secondary metabolites production and sustainable use of medicinal plants (Hasan and Roy, 2005). In the beginning of ethno-use of plants, people were used plants or plants derived substance to treat their health illnesses which is existing in modern health practices in almost all ancient civilizations, the Egyptian, the Chinese and even Greek and Roman civilizations (Aftab and Sial, 1999). However, although antibiotic considered as an important weapon for fighting against pathogen, it shows less efficiency on certain illnesses due to emergence of drug resistant bacteria. Plants can be used for various pharmacological purpose whereas it's known as a reservoir of secondary metabolites. Antibiotics is continuously decreasing its resistance power which is insufficient to control some pathogen due to non-prescribed use. In these circumstances, it is very urgent necessary to induce newer drug with high resistance (Sarkar et al., 2003). In the present study, plant regeneration and antibacterial activity investigation using the extracts (methanolic, ethanolic and petroleum etheric) were done.

Materials and Methods
Explants collection and sterilization The juvenile explants of Commelina benghalensis L. were collected from Rajshahi university campus, Rajshahi, Bangladesh. Shoot tips, nodes, internodes and leaf segments were used. Explants were washed thoroughly under running tap water for 15 minutes and then washed with continuous agitation in a few drops Savlon™ containing water for 15 minutes. The pre-treated explants were then treated with 0.1% HgCl2 for 5 minutes under laminar air flow to disinfect them. Finally, explants were washed 3 to 5 times with sterile distilled water.

Culture media preparation and inoculation
Explants were cultured on MS medium (Murashige and Skoog, 1962) supplemented with different concentrations and combination of cytokinin i.e. BAP (6-benzyl amino purine), KIN (kinetin) and auxin i.e. IBA (Indole-3 butyric acid), IAA (Indole-3 acetic acid) and NAA (Napthalene acetic acid) for axillary shoot proliferation. The pH of the medium was adjusted to 5.80 ± 2 before autoclaving at 121 °C for 20 minutes at 1.2 kg/cm 2 pressure. For the root formation different types of auxin were used with half strength and full strength of MS media. In vitro elongated shoots (5-6 cm) bearing at least 3-4 internode were excised from the mass of proliferated shoots and transferred to rooting medium. The explants in all experiments were sub cultured at 2-week intervals.

Hardening
Rooted plantlets were carefully washed with tap water and transferred to polycups containing sterile soil and vermiculite (1:1) and covered with plastic bag to maintain humidity. Finally, plantlets were acclimatized under field condition.

Data analysis
The data pertaining to number of inoculated explant response, number of multiple shoots per plant, length of shoots per microshoot and rooting were subjected to analysis of variance (ANOVA) test. Mean separation was done using Duncan's Multiple Range Test (DMRT) (P< 0.05).
Sample collection and culture media preparation In the present investigation, five-gram (+ve) bacteria (Staphylococcus aureus, Bacillus cerus, Streptococcus phaemolytica, Bacillus rubbitis, Sarcina lutea) and five-gram (-ve) bacteria (Klebsiella strain, Pseudomonas aeruginosa, Salmonella typhi, Shigella dysentriae, Klebsiella pneumoniae) were used to investigate the antibacterial activity of the plant extracts of Commelina benghalensis L. Those bacteria were collected from the international Centre for Diarrhoeal Diseases Research of Bangladesh (ICDDR, B). In this study, nutrient LB (Laury broth) medium was used for antibacterial screening. For bacterial growth 28 g nutrient was taken into two separate 500 ml autoclaved conical flask and the media were properly dissolved with distilled water then sterilized. Sterilized media were allowed to cool and poured into the autoclaved petri discs in laminar airflow cabinets.
Bacterial suspension preparation and plant extract preparation 10 ml of distilled water was taken into the screw capped test tube and pure colony of freshly cultured bacteria which earlier prepared were added into the tube and vortexed. The OD (optical density) was measured with the colorimeter and microbial population was also confirmed to be within in 10 7 ml -1 to 10 8 ml -1 . This suspension was used as inoculums. On the other hand, properly drying plant materials were pulverized into a coarse power. 30 g of studied plant powder was weighted with the electric balance and 60 ml each of the solvents (ethanol, methanol, petroleum either) was added in each conical flask. The samples with solvents were placed in water bath shaker for 5-6 hours. The extracts (ethanol, methanol, petroleum either) were filtered using filter paper than air dried after filtration to concentrate. The experiment was performed with three replications. Three concentrations i.e. 400 mg/l, 600 mg/l, and 800 mg/l) of plant extracts were used on paper disc to create zone of inhibition which was measured in terms of millimetre.

Disc preparation, incubation and measurement
In vitro antibacterial activities of the extracts were measured by employing standard agar disc diffusion method (Bauer et al., 1966). In this method, the filter paper was punched with the punching machine for preparing disc and sterilized. The discs were placed aseptically over the bacterial culture which was earlier prepared by striking with sterile transfer loop on nutrient agar plates and incubated at 37 ºC temperature for 20 h. After incubation, the zone of inhibition around the disc was measured by millimetre scale. Discs were impregnated with each treatment and control was assayed on duplicate agar medium plate for all bacterial species. Blank paper discs were impregnated with only solvent (petroleum ether, methanol and ethanol) and used as negative control and Tetracycline (30 µg/disc) contained paper disc were used as positive control each time. In the present study, it was determined following the serial dilution technique according to Reiner (1982).

Results and Discussion
The present investigation was carried out to standardize some of the aspects of in vitro plant regeneration from the excised tissues and antibacterial screening of Benghal dayflower. For easy understanding and precise assessment of the results, the present investigation has been carried out under two parts.

Micro propagation
The experiments were conducted with four different types of explants viz. shoot tips, nodes, internodes and leaf segments. Differences were taken into account regarding to regeneration system, explants type and response to nutrient media, growth regulator type and concentration as well as other chemical and physical factors, in regenerating the complete plantlets of Benghal dayflower subsequently, tested for antibacterial activity against a number of bacterial strains. For the best explant's selection, different concentration of cytokinin like, BAP or KIN either alone or in combination with auxin i.e. IAA, NAA or IBA were used on MS basal medium and observed on morphogenetic response of field grown surface sterilized juvenile explants (shoot tips, internodes, leaf segments and nodal segments) of C. benghalensis. Observing phenomena denoted that nodal segments were found to be the best source of multiple shoot buds' inductions. This result similar with other medicinal plants Holarrhena antidysenterica (Ahmed et al., 2001) and Enicostemma hyssopifolium (Seetharam et al., 2002). It also indicates that single node explants elicited more numbers of multiple shoots as compared to shoot tip explants in cytokinin media. Similar results were also noted in Zehneria scabra (Anand and Jeyachandran, 2004) and Eclipta alba (Borthakur et. al., 2000). Plant proliferation by in vitro process from nodal segments is simple and it is also economically more efficient technology. In this study, we also found that shoot buds' induction and auxiliary shoots proliferation were more effective and better on MS media supplemented with BAP than others cytokinin as also reported earlier Zehneria scabra (Anand and Jeyachandran, 2004), Alternantrer sessilis (Boro et al., 1998).  As shown in Table 1 the maximum (90.52%) response of explant and number of shoots (23.25) per plant were showed from nodal segments on MS medium fortified with BAP 3.0 mg/l + IBA 0.1 mg/l but the highest length (17.25 cm) of shoots were obtained whilst IBA 0.5 mg/l was applied with BAP 3.0 mg/l. These results disparity with Sarwar et al. (2009) have reported that BAP 1.0 mg/l with NAA 0.5 mg/l, 75 % and 70% shooting response was observed respectively from shoot tip and nodal segments of Commelina benghalensis L.
These contrast results may be occurred due to strength of MS and media combinations. On the other hand, the lowest (72.09%) explant response, minimum number (10.44) of shoots per plant and the lowest (9.13 cm) length of shoot were found at NAA 0.1 mg/l, IBA 0.5 mg/l and NAA 0.5 mg/l with BAP 3.0 mg/l respectively. For root induction three types of auxin i.e IBA, IAA and NAA were used where four concentration of each auxin (0.5-2.0 mg/l) and two strength of MS media were tested as shown in Table 2. In this study, two types of MS strength i.e. full strength and haft strength of MS were used. Among these media, we found that full strength of MS media was better for root formation than half strength of MS media. The maximum (99.12%) response of micro-shoot and highest (12.69) number of roots per micro-shoot were obtained on full strength of MS medium having 1.5 mg/l IBA. The highest (6.32 cm) average length of roots was found at IBA 1.0 mg/l. For acclimatization, well rooted plantlets were gently removed from the test tubes and thoroughly washed with running tap water to remove traces of medium and transferred to plastic pots having soil and compost (1:1). Through this process of acclimatization, regenerated plantlets were established under ex-vitro conditions. About 97% of plantlets survival under ex vitro environment was observed after hardening of C. benghalensis as shown in Figure 1.

Evaluation of antibacterial activity
For the assessment of antibacterial activity of C. benghalensis, three types of organic solvent i.e. methanol, ethanol and petroleum ether were used to prepare plant extract.
In this study, ethanol and methanol extract were more active because of consistent more antimicrobial activity against the gram (+ve) bacteria than on gram (-ve) bacteria. This is in agreement with previous reports (Vlietinck and Vanden, 1991;Rabe and Van, 1997). The experimental data was obtained from the disc diffusion method (Table 3) indicate that all of three extracts exhibited a variable degree of antibacterial activity by producing zone of inhibition with few exceptions on different tested bacterial strains. These extracts of leaf and stem of studied plant were shown zone of inhibition at three concentrations i.e. 400 mg/ml, 600 mg/ml, and 800 mg/ml against all tested bacterial stain except E. coli and S. typhi (Table 3). These results indicate that plant extracts were showed its activity against tested gram-positive and gram-negative bacteria that may lead to active with broad spectrum. It's also denoted that it would be performance against multidrug-resistant organisms for the probability of developing therapeutic substances. Range of zone of inhibition was found from 08.05 mm to 15.75 mm at all tested concentrations of petroleum ether extract of studied plant. Highest zone of inhibition was produced against S. aureus (12.28, 14.08 and 15.75) mm and Pseudomonas aeruginosa (12.75, 13.84 and 15.05) mm at all tested dose levels respectively. S. β-phaemolytica bacterial species was showed minimum 08.05 mm zone of inhibition at 400 mg/ml concentration level. On the other hand, minimum (10.55 and 11.75) mm zone of inhibition was obtained at 600 mg/ml and 800 mg/ml concentration against S. lutea and B. subbitis. From these observations, it was indicated that the producing zone of inhibition was higher against gram (+ve) bacteria than gram (+ve) bacteria. This result is similar with (Jigna and Sumitra, 2006) as reported earlier.  In case of methanol extract it produces the maximum zone of inhibition was showed against S. aureus (13.58, 14.67 and 16.44) mm and Pseudomonas aeruginosa (13.34, 14.75 and 16.21) mm at all concentration levels respectively. S. lutea bacterial species was showed lowest (10.05 and 12.05) mm zone of inhibition at 400 mg/ml and 800 mg/ml dose level whilst 10.87 mm zone of inhibition was found at 600 mg/ml against S. β phaemolytica tested bacteria. As shown in (Table-2), Ethanol soluble fraction of leaf and stem of Benghal dayflower was found the highest inhibitory activity under in vitro condition against tested bacterial species comparing the methanol and petroleum ether extract due to allow the extraction of chemical components that were showed activity against those bacteria. Highest zone of inhibition was obtained against S. aureus (14.47, 15.52 and 17.50) mm and Pseudomonas aeruginosa (14.25, 15.42 and 17.44) mm at all concentrations respectively. Besides, S. aureus was the most sensitive bacterial strain with the strongest inhibition zones (17.50 mm) at 800 mg/ml dose level followed by K. pneumonia (16.31 mm), P. aeruginosa (17.44 mm), S. dysenteriae (16.75 mm), B. cereus (15.58 mm), S. β-phaemolytica (14.84 mm), B. subbitis (16.03 mm) and S. lutea (14.75 mm). Negative control (disc containing only solvent) exhibited no zone of inhibition against all tested bacterial strain. But positive control (disc containing tetracycline antibiotic) exhibited zone of inhibition against almost all bacteria. Besides, these data indicate that zone of inhibition were increasing compare with increasing of dose level of extracts against all studied bacterial stain except S. dysentriae, K. pneumoniae bacterial stain on methanol extract and S. β-phaemolytica on methanol and petroleum either extracts. It may be occurred due to exist inadequate active components in crude extracts as also reported earlier (Taylor et al., 2001).
Comparatively, Gram-negative bacteria exhibited low susceptibility to the extract which is in accordance with the fact that those have high level of intrinsic resistance to virtually all known antimicrobials and antibiotics due to a combination of a very restrictive outer membrane barrier, and it is highly resistant even to synthetic drugs. Considering the three extracts of Benghal dayflower ethanol extract showed the best antibacterial activity.