Developing caryopsis of the apical and basal spikelets

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 [34]. The residue after methanolic extraction was digested in 3% HCl and used for estimation of starch [34].
2.6. Estimation of ACC synthase and ACO oxidase enzyme activities
ACC oxidase assay was done following Vriezen et al. [36]. Approximately 0.5 g of plant material was ground to powder in liquid nitrogen followed by addition of 1.5 mL of chilled extraction image 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 [37], and the activities of the enzymes were expressed as pmol ethylene produced mg−1 protein h−1 mL−1 air.
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