2.5. Soluble BMY 7378
and starch estimation
The grains were dissected out from spikelets on day 28 after anthesis and dried in oven at 70 °C for 72 h. The material was crushed to fine powder and approximately 100 mg of the powder was immersed into 3 mL of boiling 80% aqueous methanol for 5 min. The extract was removed to a 10 mL volumetric flask and the residue was boiled again with 50% aqueous methanol. Both the extracts were pooled together and made to 10 mL with distilled water. The extract was used for estimation of soluble carbohydrates . The residue after methanolic extraction was digested in 3% HCl and used for estimation of starch .
2.6. Estimation of ACC synthase and ACO oxidase enzyme activities
ACC oxidase assay was done following Vriezen et al. . Approximately 0.5 g of plant material was ground to powder in liquid nitrogen followed by addition of 1.5 mL of chilled extraction
buffer (300 mM Tris–Cl, pH 7.2, 30 mM sodium ascorbate, 10% v/v glycerol). The homogenate was centrifuged at 15000 × g for 10 min at 4 °C and the supernatant was used for the assay. The reaction mixture consisted of 1.7 mL of incubation buffer (100 mM Tris–Cl, pH 7.2, 30 mM sodium ascorbate, 10% v/v glycerol), 50 ?L of 80 mM ACC, 50 ?L of 3 mM FeSO4, 100 ?L of 1 M NaHCO3 and 200 ?L of the enzyme extract. Ethylene produced was measured as above. Protein in the enzyme extracts was determined by Bradford , and the activities of the enzymes
were expressed as pmol ethylene produced mg−1 protein h−1 mL−1 air.