The study included initiation and growth of callus from stem Nigella sativa L. using Murashige and Skoog (MS) medium containing 106- 1010 molar 2,4 -dichlorophenoxy acetic acid (2,4-D) or pentadienoic acid (PDA). Glutamate dehydrogenase (GDH) was partially isolated from the initiated callus on the nutrient medium containing variable concentrations of potassium and ammonium nitrates. In addition, a comparative study of the enzyme activity was performed in the presence of 2,4-D or PDA.
The results revealed that best medium for initiation and growth of callus was obtained in the presence of 106- M of 2,4-D or PDA. The fresh weight of the callus grown on MS medium in the presence of 2,4-D or PDA was 5.72 gm and 2.81 gm respectively after 45 days of culturing. However, the protein content of the callus was 1.050 and 1.116 mg / gm fresh weight grown on media containing 2,4 –D or PDA, respectively.
The results also predicted that the specific activity of GDH was increased by 23 folds after partial purification. The optimum conditions for GDH activity were obtained using: 100 mmole of Tris – HCl buffer, at 35º, pH of 8.6, 1 µmole of NAD, 20 µg of protein as a source for the enzyme and 30 µmole of glutamate as a substrate. Using Linweaver – Burk plot, the value of Michaelis – Menten constant (km) was 27 µmole. The results also revealed that the specific activity of GDH isolated from the callus where 2,4–D or PDA was added is 21.3 and 22.3 µmole / min / mg protein, respectively using the optimum conditions.
The comparative molecular weight of GDH was determined using gel filtration chromatography and found to be in the range of 302000 dalton.
Based on the results from the current study it was concluded that PDA could be used instead of 2,4-D in plant tissue culture of Nigella sativa L. Its action is similar somehow to the standard auxin. Therefore, PDA could be considered as a synthetic auxin and used in plant tissue culture for several plants.