Li L. and Zhang C.R., Journal of Environmental Biology 27: 21-26 (2006)
Callus induced from leaf explants of Pueraria lobata seedlings were suspended in Gamborg B5 medium supplemented with 1 mg l-1 2,4-dichlorophenoxyacetic acid, 1 mg l-1 naphthalene acetic acid, 0.5 mg l-1 kinetin and 30 g l-1 sucrose. The effects of coconut milk and casein hydrolysate (CH) on cell growth and yields of puerarin and isoflavones in cells suspension were studied. The contents of total isoflavones and puerarin in suspension cultures were determined by spectrophotometry and HPLC. Coconut milk (10%, filter sterilized) decreased the growth of cell cultures and the accumulation of total isoflavones, while 0.2% CH promoted the growth of cell cultures and the accumulation and release of puerarin and total isoflavones. The total yield of puerarin and isoflavones were 34% and 40.8% higher than in the control, respectively. The optimum medium for cell cultures of leaves of P. lobata seedlings was B5 liquid medium supplemented with 2% sucrose, 1.0 mg l-1 2,4-D, 1.0 mg l-1 NAA, 0.5 mg l-1 kinetin and 20 mg l-1 CH. The procedure use is a potentially useful for the production of isoflavones.
The aim of this study was to determine the effects of CM and CH on cell growth and on isoflavone levels in suspension cell cultures of P. lobata.
P. lobata growing in the Botanical Garden (South China Normal University).
Leaves of seedlings that were germinated aseptically.
(Callus formation) Unripe green pods of P. lobata were rinsed in water, 70% EtOH for 1 min and 0.2% HgCl2 for 20 min, and finally rinsed three times in sterile water. The leaves of aseptic seedlings with four leaves developed after 8 days were excised and placed on MS medium supplemented with 1mg l-1 NAA, 0.5 mg l-1 4-PU and 30 gl-1 sucrose to induce callus formation. Cultures were maintained under a 16 hrs photoperiod regime with fluorescent light at 25ºC without light.
(Callus proliferation) Pieces of callus were excised and transferred to 0.8% agar gel with Gamborg B5 medium supplemented with 1mg l-1 2,4-D, 1mg l-1 NAA, mg l-1 kinetin and 30 g l-1 sucrose for culture. The callus was subcultured every 15 days.
(Suspension culture) After 6 subcultures, a light-yellowish coloured, friable callus (1.5 g fresh wt) was transferred to a 150 ml flask containing 50 ml of liquid Gamborg B5 medium supplemented with 1mg l-1 2,4-D, 1mg l-1 NAA, mg l-1 kinetin and 30 g l-1 sucrose and also contained either 20 mg l-1 CH or 10% (v/v) fresh CM. The friable callus rapidly disintegrated to single cells and developed into small cell aggregates after incubation at 25ºC on an orbital shaker (130 rpm).
Traints of regenerants
(Extraction for UV-VIS spectrophotometer analysis) Dry powder (25 mg) was blended with 30 ml 95% EtOH and placed in a water bath at 70°C for 6 hr. After cooling, the crude extract was diluted with 95% EtOH to 50 ml. Samples (1.0 ml) of the extract were diluted with distilled H2O upto 25.0 ml.
(Extraction for HPLC analysis) 100 mg dry powder samples were extracted in 1.0 ml absolute EtOH for 24 h. Extracts were combined, filtered, and diluted with absolute EtOH to 5 ml.