The hypocotyl explants of R. communis L. cv. Roktima were used as experimental materials in the present investigation. Seeds were collected from the research field of the Institute of Biological Sciences, Rajshahi University, Rajshahi, Bangladesh. Seeds were washed throughly under running tap water and then treated with 1% savlon and 2-3 drops of Tween- 80 for about 10 min. This was followed by successive three washing with distilled water to make free the seeds from savlon and Tween-80. Surface sterilization was carried out with 0.1% HgCl2 for 6-7 min followed by gentle shaking. After this treatment, the seeds were rinsed 4-5 times in sterile distilled water to make free the seeds from HgCl2. Sterilized seeds were partially decoated and aseptically germinated in glass bottle containing 50 mL of autoclaved (121ºC temperature for 20 min at 1.1 Kg/cm² pressure) MS medium, fortified with BAP (1.0 mg/L), sucrose (30 g/L) and agar (8.0 g/L). The pH of the medium was adjusted to 5.7-5.8 before autoclaving and the germinating seeds were kept in a growth room maintained at 25 ± 2ºC temperature and 60ºC RH. The experiment was conducted in the biotechnology laboratory, Institute of Biological Sciences, Rajshahi University, Rajshahi, Bangladesh.
2.1 Callus Culture Hypocotyl segments, as explants were taken from 12 d old in vitro growing seedlings of the plant. The explants were cultured in 9 cm petridish and placed horizontally in the callus induction medium. The MS medium supplemented with 3% sucrose and different concentrations (0.1-1.0 g/L) of NAA (α-Napthalene acetic acid), 2,4-D (2,4-Dichlorophenoxy acetic acid), IAA (Indole-3-acetic acid) and BAP 0.5-3.0 g/L (6-Benzylaminopurine) were compared in combination for the induction of callus.The medium was adjusted to pH 5.7 and autoclaved. The data for callus initiation were scored after 4 weeks of culture. 2.2 Cell Culture Rapidly proliferating friable calli subcultured for 18 d, were aseptically transferred to MS liquid medium supplemented with 2.0 g/L BAP + 0.05 g/L NAA, 2.0 g/L BAP + 0.2 g/L NAA and 2.0 g/L BAP + 0.5 g/L NAA in three lots in 250 mL flasks. The flasks were placed on a rotary shaker (100 rpm). After 4 d, the liquid medium containing cells and micro calli were filtered through a 500 µm sieve. The filtrate containing cell suspension culture was maintained in the laboratory. To observe the growth efficiency, flasks containing the liquid medium with cell culture were kept in a rotary shaker. The growths of the cells were measured by weighting the cells in 5 mL liquid medium taken in every after two d. On the other hand, to obtain callus, some cells were distributed to petridishes (4 cm) containing the fresh semi solid medium at 25ºC in dark for 35-42 d of incubation. Micro calli were appeared in the plates initiating induction of callus from cell aggregates. 2.3 Preparation of Extracts The filtrate containing cell suspension culture was maintained in the laboratory. Every after two d, 5 mL liquid medium with growing cells were centrifuged at 4000 rpm for 10 min. The cells were discarded and the clear 15 µL of the supernatant was applied on the test disc. 2.4 Microorganisms Five gram positive bacteria viz., Sarcina lutea, Staphylococcus aureus, Bacillus megaterium, Bacillus subtillus, Bacillus halodurans; and six gram negative bacteria viz., Shigella sonnei, Klebsiella species, Proteus species, Escherichia coli, Pseudomonas aeruginosa and Salmonella typhi were employed in this test. These species were obtained from the mother stock of the Molecular Biology Laboratory, Institute of Biological Sciences, University of Rajshahi, Bangladesh.
2.5 Media Nutrient broth and Nutrient agar (Mast Diagnostics, Mast Group Ltd. Merseyside, UK) were used for bacterial culture and conducting antibacterial test.
2.6 Disc Diffusion Assay Antibacterial activity was determined as diameter of inhibition zone using disc diffusion method. Nutrient agar (NA) was distributed in sterilized petridishes. This was accomplished by placing 15 µl of the extract on a small paper disc. This disc was placed on an agar growth medium containing a confluent lawn of microorganism. The concentration of the organism was also 10 µL/petridish.
2.7 Preparation of the Test Discs Sterile test discs were prepared by punching and saturating filter paper (Whatman no1) discs (each disc is 6 mm in diameter) in cell extracts solution using sprit flame sterilized forceps. These discs were dried in the sterilized petridishes. The name (code) of the plant extract was written at the bottom of the petridishes. Inhibition zone of all extracts was compared to the standard antibiotic (Ciprofloxacin 05 µg/disc).
2.8 Culture Media and Inoculums Solid media of nutrient agar was prepared by dissolving 28 g/L water. About 25 mL of media was poured into a petridish. The inoculum was prepared by culturing a large number of organisms in a test tube containing 10 mL liquid media for bacterial strains and incubating over night at 37ºC. The agar plates for the assay were prepared by labeling them with the date, the name of the microorganism and the name (code) of the discs. The inoculi of bacteria were transferred into petridish containing solid nutrient media of agar using a sterile swab. The swab was used to spread the bacteria on the media in a confluent lawn. It was done by rotating the petridish at 90ºC and continuing the spread of bacteria. One swab was used for one species of bacteria.
2.9 Placing Test Discs Dried test discs were transferred on bacterial lawn under aseptic conditions using spiritflame sterilized forceps each time. Each disc was placed gently on the agar surface and plated with the forceps so that it sticks. The petridish was incubated upside down at 37ºC for 24 h. Resulting zones of inhibition were observed and measured in millimeters.