In-vitro Callogenesis and Screening of Antimicrobial Activity of Callus and Seed of Caesalpinia bonducella F.: A Threatened Medicinal Plant of Western Ghats

Aim: Caesalpinia bonducella Flem is a dioecious scrambling woody liana of Caesalpinoideae, a subfamily of Leguminosae. The plant is threatened and distributed in the deciduous forests of the Western Ghats of India. Being an important medicinal plant Caesalpinia bonducella F. attracted many scientists to exploit various activities associated with a number of phytoconstituents. The present study was undertaken to evaluate the most suitable media and suitable concentrations of plant growth regulators for in vitro Callogenesis and screening of antimicrobial activity of callus and seed of Caesalpinia bonducella. Materials and Methods: Callus was initiated from stem explants, on 1x and 0.5x MS medium plus supplements. The effects of plant growth regulators on callus cultures were studied and observations were made. The in vitro antibacterial activity was performed by using extracts of callus and seed of Caesalpinia bonducella in petroleum ether and methanol against multidrug resistance organisms. The organic extracts of seed and callus of the plant at concentrations of 0.02 mg/ml 0.1 mg/ml were taken and their activities were measured. Original Research Article Shirsat et al.; JPRI, 33(31A): 76-89, 2021; Article no.JPRI.69393 77 Results: The combination of 2.5 mgL -1 2,4-D with 2 mgL -1 BAP, resulted in the highest frequency and the highest mean percentage of callus formation (2.35 ± 0.294) with yellow friable callus. The results revealed that all the extracts had a variable degree of antibacterial activity. Conclusions: It was observed that 2,4-D at 2.5 mgL -1 in combination with BAP,2.0 mgL -1 BAP resulted in early initiation, highest induction percentage, with frequency highest mean percentage of callus formation, Antimicrobial tests with methanol and diethyl ether extract of Caesalpinia bonducella seed powder against the clinical isolates showed the zone of inhibition for all the pathogens tested with concentration of methanolic extract of C. bonducella seed powder.


INTRODUCTION
Caesalpinia bonducella Flem is a dioecious scrambling woody liana of Caesalpinoideae, a subfamily of Leguminosae. The plant has spiny stem and bipinnate leaves. The plant is threatened and distributed in the deciduous forests of the Western Ghats of India [1] Sri Lanka, etc In India it is especially found in tropical regions [2]. In the Ayurvedic system of medicine, the plant is popularly known as fever nut.The plant contains biologically active compounds like phenols, diterpenes, flavonoid, alkaloids and tannins. Caesalpinia bonducella is well known for its several medicinal properties. It has been reported that different parts of the plants are used for several ailments like diabetes [3,4,5], anti-inflammatory [6,7], anthelmintic [8], anti-estrogenic [9], antimalarial [10,11,12], and memory enhancer [13]. Overutilization of the plant for medicinal purposes and the destruction of natural habitat makes the species to be endangered [14]. In recent years, In vitro culture has achieved major industrial importance in the production of secondary metabolites, and callus cultures have been employed in the study of secondary metabolism. The In Vitro Callogenesis technique provides an appealing alternative for the production of valuable secondary metabolites, and they have been used throughout the years as a tool for the elucidation of the biosynthesis of metabolites [15].
There are reports of in vitro culture of Caesalpinia bonducella [16,17,18,19] dealing with callus studies as well as direct and indirect organogenesis using different explants. Since Caesalpinia bonducella has an immense medicinal value, the present paper attempted to derive in vitro callogenesis using stem explants. To evaluate the effect of different strength of Murashige and Skoog (MS) media (1x and 0.5x MS medium) and Plant growth regulators on callusing was the main objective of this study.
Microbes are responsible for many infectious diseases [20,21]. Although antibiotics are one of the most important weapons in fighting bacterial infections and have greatly benefited the healthrelated quality of human life for a long time, their continuous use makes clinical pathogens drugresistant [22,23]. The increasing clinical implications of drug-resistant fungal and bacterial pathogens has prompted antimicrobial drug research. Kachhawa et al. [24] suggested that there is a need to search for new antimicrobial agents from plants to control microbial infections. Among the potential sources of new agents, plants have long been investigated, as they contain many bioactive compounds that can be of interest in therapeutics. Literature states that plants are the natural reservoirs of many antimicrobial agents [25,26]. Shula et al. [27] observed Caesalpinia bonducella (defatted and non-defatted) extracts with different solvents possess potent antibacterial activity. They reported that acetone and ethanol extracts exhibited the highest antibacterial activity against all the tested bacteria used. They mentioned the antibacterial activity increased with the increased proportion of the extract concentration. Tambekar et al. [28] reported that methanol extract of Caesalpinia bonducella proved antibacterial to Staphylococcus aureus, Shigella flexneri, and Enterobacter aerogenes.
The difference in the antibacterial potentials of different extracts suggested that the solubility of various phytochemicals in various solvents made it different from the others. As the literature suggests Caesalpinia bonducella (defatted and non-defatted) extracts with different solvents possess potent antibacterial activity. Therefore, in the present study, an attempt has also been made to test the antimicrobial activity of callus and seed powder of Caesalpinia bonducella against gram-positive and gram-negative bacteria.

Surface sterilization of explants
Internodal segments of the stem were used as explants for callus induction. After the collection, the stem were excised into small segments (1 to 1.5 cm). These explants were kept under running tap water for 30 minutes and then rinsed with Dettol for 10 minutes. Tween-20 (a detergent and antiseptic) was used as a surfactant for 10 minutes. Bacillocid at a concentration of 1% (v/v) and Bavistin (1% w/v) were used for 15 minutes as antibacterial and antifungal agents respectively. The explants were washed thrice with sterilized distilled water, followed by treatment with 70% alcohol for 30 seconds. The explants were swirled for 1-2 minutes in 0.07% HgCl 2 (w/v) followed by repeated washings with sterilized distilled water. The explants were trimmed. Surface-sterilized explants were blot dried with sterilized tissue paper. Such explants were aseptically inoculated onto Murashige and Skoog(MS 1962) mediums supplemented with different combinations of auxins and cytokinins.
After callus induction; the calli were transferred to the fresh medium of the same combination. Every 15 days of incubation the cultures were maintained for further proliferation. The inoculated culture tubes were observed at an interval of 3 to 4 days and the results were recorded. Callus was sub-cultured at regular intervals in the appropriate medium for further studies.
The chemicals for these experiments were used of analytical grade and procured from HiMedia Pvt Ltd Mumbai. Sigma Chemical Co., USA, and E-merck, (India).

Preparation of extract
The seed powder and callus of Caesalpinia bonducella was used to screen their antibacterial potential. The extracts were prepared by cold infusion method, suggested by Handa [30]. Five grams of callus was homogenized in a minimum quantity of methanol and diethyl ether at a concentration of 40%. It was kept for 48 hours. The volume was made to 50 ml using respective solvents. After 48 hours, the extracts were filtered using Whatman grade 1 filter paper. The extracts were evaporated to dryness. Prior to use, the residue were dissolved in known quantity of solvents (stock solutions) so as to maintain the concentrations (Table A1). To get the seed extract, the seeds of Caesalpinia bonducella were separated from the testa and were sun-dried. The kernels of seeds were ground into fine powder. A procedure which was used for preparation of callus extract was followed in a similar way to get the extract from seed powder. Similarly, methanol and diethyl ether were used as organic solvents.

Bacterial strains
The Gram-positive bacteria Bacillus subtilis, Staphylococcus aureus and Gram-negative bacteria Escherichia coli, Klebsiella pneumonia were selected because of their highly pathogenic nature. The required bacterial cultures were obtained from Department of Pharmacology, Bombay college of Pharmacy, Kalina, Santacruz, (Mumbai). These clinical isolates as pure cultures were maintained by regular sub culturing on nutrient agar slants. The bacteria were sub cultured 48 hours prior to use so as to get vigorously growing microorganisms.

Antibacterial bioassay
Antibacterial activity of callus of Caesalpinia bonducella was studied by the agar well diffusion method [31]. The Methanolic and Diethyl Ether extracts were tested in dose levels of 0.02 to 0.1 mg/ml. Stock solutions of extracts were prepared in Saline solution (Table A1). The Mueller-Hinton's [32] agar medium was prepared. The suspension was prepared from 48-hour old cultures. One ml of culture suspension added in sterile molten agar butts of MH media, having an approximate temperature of 45 o C. The butts were mixed thoroughly and the media was poured into sterile petri plates and inoculated with one ml of culture suspension of the above-cited test organisms. The Mueller-Hinton agar medium was allowed to set at room temperature for about 10 minutes. The plates were left undisturbed for media to set and allowed to solidify in a refrigerator for 30 minutes. With the help of sterile cork borer wells of 6mm diameter were made in each petri plate. Wells were filled with 20µl of different concentrations of each extract. The petri plates were incubated for 48 hours at 37°C. After 48 hours, the zone of inhibition was observed and results were recorded by measuring the diameter in millimeter. Inhibition zones are the average values calculated from three replicate. The results were tabulated. Positive control, negative control, and medium control were also maintained as per Table A2. All the experiments were carried out in triplicate.

Induction of Callus
In this study, the two different media composition were used. Murashige and Skoog (MS) and h strength MS and the result indicated that both MS and half Strength MS with auxins alone, and in combination with cytokinins responded for induction of callus using intermodal segment of stem.

Effect of auxins on stem internode as explants
The two auxins used to analyse the potency for callus induction are as follows.

Effect of 2, 4-D on stem internode as explants
Results indicated that explants dedifferentiated and initiated calli at the cut surfaces.

RESULTS AND DISCUSSION
In this study, the two different media composition were used. Murashige  day, with only 40%, explants responded for callus initiation, and 45 ± 0.0531g of callus formed (Table 1).
Out of these two media used half strength MS medium with both 2,4-D and IBA responded for induction of callus in stem internode, however MS medium was found to be better than half strength MS culture medium.

Effect of BAP on stem internode as explants
Effect BAP initiated callus on 5 th day. The response was 60% and the appearance of callus was white and brittle. The concentrations 1.0 mgL -1 and 2.0 mgL -1 of BAP did not show significant results, as the response was 30% and 20% respectively with moderate callus induction. It was observed that the hard callus did not proliferate. However, soft callus proliferated at much faster than rate.

Effect of kinetin on stem internode as explants
Effect  Table 4). The callus obtained was white and hard. BAP at 2.0 mgL -1 concentrations in the media showed callus initiation on 12 th day with only 10% response. The callus obtained was white and was hard.  Kinetin produced callus but the response was 60% and callus was initiated after 10 days, the obtained callus was whitish cream and hard. Out of these two media used half-strength MS medium with both BAP and Kinetin responded for induction of callus in stem internode but MS medium was found to be better than MS half strength culture medium.Out of cytokinins used, BAP was found to be more suitable than Kinetin ,in terms of early initiation and percentage of explant in callus formation.

The combined effect of BAP and 2,4-D on callogenesis
The combined effect of BAP with both auxins was also evaluated, The BAP fortified media which responded optimal for callus induction and proliferation, MS +2.0 mgL -1 BAP combined with both 2,4-D/IBA as shown in ) and 2,4-D at different concentrations were studied. Morphologically the callus was greenish-white or cremish and brittle at all the concentrations. It was observed that 2,4-D at 2.5 mgL -1 in combination with BAP,2.0 mgL -1 resulted in early initiation, highest induction percentage, with highest mean percentage of callus formation 2.35 ± 0.2941 (Table 5). This observation was also recorded by Meena K C, et al [17]. According to them, the addition of 1.0 mgL -1 2, 4-D, or NAA in combination with BAP crucial for callus induction in the same species. Meena K. Cheruvathur et al [33] obtained optimum callus induction when the pulvini were cultured on MS medium fortified with 6.0 mgL -1 2,4-D and 1.0 mgL -1 BAP. Santosh Kumar S.E et al [18] found that callogenic media for Caesalpinia bonducella consisted of a range of 0.25 mgL   (Fig.3).

Antimicrobial Activity of Seed of Caesalpinia bonducella
The effect of methanolic and diethyl ether extracts from the callus of C. bonducella pathogens is depicted in Table 6. All the bacterial cultures were found to be susceptible to the methanol extract of C. bonducella aureus. It was observed that no inhibition zone was obtained at lower concentrations (0.2 for E. coli and K. pneumonia. concentrations such as 0.04 ml, 0.06 and 0.1ml were found to be effective with increasing antimicrobial activity for Hence, it can be interpreted that the higher concentration inhibited the growth of the microbial culture. K. pneumoniae and showed 13 mm zone of inhibition at 0.1mg/ml concentration (Fig. 4). It was observed that coli and K. pneumonia both Gram bacteria were susceptible to the C. bonducella callus in methanol extract. For Gram bacteria only B. subtilis showed zone of inhibition

of Callus and Caesalpinia bonducella
The effect of methanolic and diethyl ether C. bonducella on four 6. All the bacterial cultures were found to be susceptible to the C. bonducella except S. . It was observed that no inhibition zone was obtained at lower concentrations (0.2 mg/ml) . Rest of the ml, 0.06 ml 0.08 ml, and 0.1ml were found to be effective with increasing antimicrobial activity for E. coli. Hence, it can be interpreted that the higher concentration inhibited the growth of the and B. subtilis mm zone of inhibition at 0.1mg/ml . It was observed that E. both Gram-negative C. bonducella For Gram-positive showed zone of inhibition (0.06 mg/ml, 0.08 mg/ml and 0.1 aureus did not get affected at any concentrations of the extracts used. The extract in diethyl ether did not show any inhibitory effect against the selected pathogens ( Table 6).
The results for antimicrobial tests with methanol and diethyl ether extract of bonducella seed powder against the clinical isolates are depicted in Table 7 concentration (0.1mg/ml) of methanolic extract of C. bonducella seed powder showed the zone of inhibition for all the pathogens tested ( pneumonia, B. subtilis, and S. aureus The lower concentrations such as 0.02 mg/ml, 0.04 mg/ml, 0.06 mg/ml and 0.08 mg/ml did not show any significant activity. It was ob B. subtilis was the most susceptible amongst all the tested pathogens. The zone of inhibition was found to be 18mm diameter. The extracts with diethyl ether also inhibited the growth of subtilis by producing a zone of inhibition (15 mm in diameter at 0.1mg/ml concentration) mg/ml and 0.1 mg/ml). S. did not get affected at any concentrations of the extracts used. The extract in diethyl ether did not show any inhibitory effect against the The results for antimicrobial tests with methanol and diethyl ether extract of Caesalpinia seed powder against the clinical isolates are depicted in Table 7. Highest concentration (0.1mg/ml) of methanolic extract of wder showed the zone of inhibition for all the pathogens tested (E. coli, K. pneumonia, B. subtilis, and S. aureus) Fig. 5. The lower concentrations such as 0.02 mg/ml, 0.04 mg/ml, 0.06 mg/ml and 0.08 mg/ml did not show any significant activity. It was observed that was the most susceptible amongst all the tested pathogens. The zone of inhibition was

Zone of inhibition (mm) Gram-negative
The extracts with diethyl ether also inhibited the growth of B.
by producing a zone of inhibition (15 mm in diameter at 0.1mg/ml concentration) (Fig. 6 Bushra and Ganga, [35] reported that the solvents like ethanol, hexane, and methanol are used to extract plant chemicals and most of them can exhibit inhibitory effects on bo positive and gram-negative bacteria. Taraquzzaman et al. [36] observed that there was no significant activity with methanol extract of Pterospermum semisagittatum against gram positive and gram-negative bacterial cultures. There are several reports in the literature indicating that Gram-positive bacteria are more susceptible to plant extracts as compared to Gram-negative bacteria (Linn et al. [40,41], suggested that these differences may be attributed to fact that the cell wall in Gram-positive bacteria is of a single layer, whereas the Gramnegative cell wall is a multilayered structure. Nostro et al. [42] and Hodges [43] explained the reason for the differences in sensitivity of Grampositive and Gram-negative bacteria towards plant extracts. They suggested that it is due to the morphological constitutions between the organisms. Gram-negative bacteria have an outer phospholipidic membrane carrying the structural lipo-polysaccharide components. This makes the cell wall impermeable to antimicrobial chemical substances. The gram-positive bacteria on the other hand have only an outer peptidoglycan layer which is an effective permeability barrier. Therefore, the cell walls of gram-negative organisms which are more complex than the gram-positive ones act as a diffusional barrier and make them less susceptible to antimicrobial agents than the gram-positive bacteria. This may be the reason in the present study as the zone of inhibition was high in Gram-positive bacteria than the Gram-negative bacteria.
Several reports are indicating multidrug resistance in bacteria, among them E. coli is the most prominent [44,45]. It can be concluded that C. bonducella callus possesses antibacterial properties with methanolic extract. Most of the methanolic extracts inhibited the growth of E. coli and B. subtilis. Hence; callus obtained from Caesalpinia bonducella can be a good source of antimicrobial agent.

CONCLUSION
This investigation reports that, among MS and half strength MS culture medium was found suitable for callus induction in Caesalpinia bonducella.
In the case of all tested concentrations and combinations of plant growth regulators, It was observed that 2,4-D at 2.5 mgL -1 in combination with BAP,2.0 mgL -1 BAP resulted in early initiation, highest induction percentage, with highest mean percentage of callus formation of 2.35 ± 0.2941g in 12 days for stem explants. These studies again proved medium containing both auxin and cytokinin was capable of inducing the callus formation. Hence the production of secondary metabolites from Caesalpinia bonducella on large scale by callogenesis is possible. The results for antimicrobial tests with methanol and diethyl ether extract of Caesalpinia bonducella callus show that the bacterial cultures were found to be susceptible to the methanol extract of C. bonducella except for S. aureus. The results for antimicrobial tests with methanol and diethyl ether extract of Caesalpinia bonducella seed powder against the clinical isolates that a high concentration of methanolic extract of C. bonducella seed powder showed the zone of inhibition for all the pathogens tested.

DISCLAIMER
The products used for this research are commonly and predominantly used products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.