This research aims to determine growth and deficiency patterns as well as antioxidative potentials of Japanese mint (Mentha arvensis) hydroponically grown under limited macronutrients and micronutrients. The experiment was conducted for 60 days after transplanting in an evaporative greenhouse (avg temp = 28–30 °C, 60–65 %RH), using deep water culture technique. Plants were grown in nutrient solution consisting of complete Hoagland’s solution (CTRL), and nutrient solutions lacking one of the following macronutrients and micronutrients: nitrogen (-N), phosphorus (-P), potassium (-K), iron (-Fe), manganese (-Mn), and copper (-Cu). The deficiency symptoms, growth patterns, and stress response mechanism were followed. All treatments except for the CTRL induced deficiency symptoms and physiological changes. Macronutrient deprivation reduced growth determined by the morphological parameters while micronutrient omission had no effect except for no iron treatment. The result showed that potassium and iron deficiencies had foremost adversely effect on growth of Japanese mint. Under nutrient stress conditions, plant only gave antioxidative responses to phosphorus and potassium deficiencies. However, the negative plant-stress relationship was found for no iron treatment indicating the detoxification mode of plant for lacking of micronutrient.

The present protocol described staining protocol for Reactive Oxygen Species (ROS) in aromatic crop grown under nutrient stress through DAB and NBT histochemical method.  Spearmint (Mentha spicata) were grown under manganese and salt toxicity stresses and after 10 and 20 days of the stress treatments, plants showed stunted growth. The morphological characteristics of leaves under stresses were observed. Manganese toxicity and salt stress induced the production of ROS.  Accumulation of hydrogen peroxide was characterised as brown spots from the DAB polymerisation which were emerged and clearly observed in leaves from plant grown under 2.5 and 5 mM concentrations of manganese as well as 300 mM concentration of salt.  Furthermore, accumulation of superoxide anion was characterised as blue pigments based upon the ability of cells to reduce NBT.  Spearmint leaves showed the distribution of the blue pigment which was obviously observed under the 5 mM of manganese and 300 mM of salt. DAB and NBT staining method can be the rapid method to characterise ROS accumulation in plant cell under the abiotic stresses.

Graphical AbstractSchematic diagram of physiological and biochemical responses of Japanese mint grown under the limit supplies of water and nitrogen fertilizer.

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