Leaf discs of local garlic variety were used as explants. Garlic was collected from elite farmer at Khulna region in Bangladesh. The cloves were washed 3 times with tap water and subsequently with distilled water and then immersed into 70% ethanol for one minute and washed thoroughly. The ethanol treated cloves were sterilized in 0.1% HgCl2 solution for 5 min with agitation followed by 3-4 rinses in sterile water to remove trace of HgCl2. The cloves were then cultured onto MS basal media (Murashige and Skoog, 1962). The explants were collected from cloves germinated of MS basal medium. Leaf disc from each germinated clove was separated by sterile scalpel. Each disc was divided into 3-4 pieces (0.2-0.3 cm) and then placed into the culture vessel.
The concentration of callus induction media were MS and supplemented with different concentration of 2, 4-D (0.0, 0.5, 1.0, 1.5, 2.0, 2.5 mg L-1) and MS and supplemented with different concentration of 2,4-D (0.0, 0.5, 1.0, 1.5, 2.0, 2.5 mg L-1) in combination with a constant 10% CW.
The concentration of regeneration media were a) MS and supplemented with different concentration of BA (0.0, 5.0, 10, 15, 20 mg L-1) and MS and supplemented with different concentration of NAA (0.5, 1.0, 1.5, 2.0 mg L-1) plus constant concentration of 1.0 mg L-1 BA.
Incubation
Cultures were incubated in dark (10 days) and then transferred to light conditions under controlled temperature (25 ± 2°C). The photoperiod was maintained 16 h light intensity of 2000-3000 lux provided from white cool fluorescent tube. Observations were carried out daily to note the response.
Subculture
When the calli attained a convenient size they were removed aseptically from the culture vessels and placed on a sterilized petridish inside the laminar airflow cabinet. The calli were cut into small pieces and were placed into freshly prepared media with appropriate combinations and concentrations. These were again sub-cultured to freshly prepared medium containing different hormonal supplements BA and NAA plus BA for regeneration. The culture vessels showing signs of contamination were discarded. Repeated sub-culturing was done at 15 days interval.
Data Collection and Analysis
Seven replications were used per treatment for callus induction. Visual observation of culture was taken every week. Duration of callus induction and its color, texture was recorded after 8 weeks. Also seven replications were used per treatment for regeneration. Regeneration initiation, percentage were recorded after 6-7 weeks.