iii. Establishment of cell suspension cultures

After 7-8 passages of callus subcultures, healthy, green, friable and soft calli maintained on responding semi-solid medium were utilized to establish suspension cultures. Cultures were initiated in Erlenmeyer flasks of 250 ml capacity, containing 50 ml of liquid medium and inoculated with 0.2 g of fresh calli. The cultures were incubated in an orbital shaker under shaking conditions at 120 rpm at 25°C ± 2°C and maintained in diffuse light (1000-1600 lux) with 16 h photoperiod or in continuous darkness. The cell biomass was subcultured regularly into fresh medium at every three weeks.

 

vi. Batch kinetics of cell suspension cultures

a. Batch kinetics studies

To determine the specific growth rate, cells were harvested from liquid medium at an interval of two days, washed and dried. The pH and conductivity of the suspension cultures were monitored after every two days. Phosphate was estimated by the standard calibration curve made from dihydrogen sodium phosphate (NaH₂PO₄ ); to 0.5 ml of standard or sample solution, 4 ml of reagent [Acetone (CH₃COCH₃), Sulphuric acid (H₂SO₄)2.5 M and Ammonium molybdate tetrahydrate (NH₄)₂MoO₄.4H₂ O) 10 mM, mixed in the ratio of 2:1:1] was added. After mixing the solutions thoroughly, 0.4 ml of 1M citric acid was added and absorbance was taken at 355 nm. Similarly, for nitrate estimation, standard curve was made from 0.01N stock solution of Potassium nitrate (KNO₃), preserved in chloroform. After acidification of samples with hydrochloric acid, absorbance was recorded at 275 nm in a UV visible spectrophotometer. All measurements and results are average readings obtained from three flasks.

 

b. Agitation speed and cell viability

Effect of agitation speeds was evaluated on fresh and dry weight of cells and their viability, at the end of each passage. Callus cells weighing approximately 0.2 g were harvested at the end of growth period and re-inoculated in 50 ml of fresh medium of the same composition. The cultures were incubated in shaking conditions at 60, 120 and 240 rpm, under darkness, for a period of three weeks and their fresh and dry weights were recorded. The viability of cells under each condition was checked with 1% fluorescein diacetate (FDA) solution. FDA is a cell permeant dye. Within the cells, the molecule is cleaved by esterase activity to fluorescein which is unable to pass through the cell membrane of live cells while it leaches out from the dead cells. Hence, only the live, intact cells take up the stain and fluoresce green.