**4.1 Materials and methods**

Commercialized artificial soil (pH, 5.2; EC, 1240 mS m–1; NO3 ––N, 280 mg Kg–1; available P2O5, 1020 mg Kg–1) was mixed with 15N labeled potassium nitrate (10 atom % 15N) and incubated at room temperature for 14 days at 60% of their maximum water–holding capacity. Finally, the high nitrate soil (pH, 5.0; EC, 3230 mS m–1; NO3 ––N, 1906 mg Kg–1; available P2O5, 1060 mg Kg–1) was obtained and used for this experiment. Seeds of radish were sown into 100 mL pots filled with the incubated soil and grown in a glasshouse.

The mixed amino acids (MAA) solution contained equal concentrations of amino acids viz., alanine (Ala), β–alanine (β–Ala), aspartic acid (Asp), asparagines (Asn), glutamic acid (Glu), glutamine (Gln) and glycine (Gly). From 17 or 24 days after sowing, seedlings of radish were sprayed with 0.2 or 0.5 mM MAA solution for 2 or 4 times, as indicated in Table 8. The pH of the MAA solutions was maintained between 6.0–6.1 by adding 1.0 M KOH appropriately.


\* Same amount of distilled water sprayed

Table 8. Composition of the treated solutions and application times for radish in pot experiment

Fresh leaves were collected at 28 days after sowing to determine the NO3– content and enzyme activities and at 30 days after sowing to determine the NO3–, amino acids and protein contents. Plant shoots were harvested at 30 days after sowing to determine crop yield and N assimilation. After harvest the soils were collected for chemical analysis.
