2.3 Low-nitrate treatment
A hydroponic system was utilized to evaluate the tolerance of apple plants to N deficiency using. Initially, healthy and consistently growing plants were selected for hydroponic culture using half-strength Hoagland’s nutrient solution. An air pump was used to supply oxygen to the plant roots and maintain the dissolved oxygen concentration in the solution at 8.0-8.5 mg L-1. After 15 days of pre-cultivation, both WT and MdASMT9 -OE plants were divided into half-strength Hoagland’s nutrient solution with 6 mM NO3 (control) and with 0.2 mM NO3 (low-N conditions). After 35 d of treatment, leaves and roots were harvested. Before sampling, the nutrient solution was replaced with a new solution containing15N-KNO3 (98% atom, Sigma Aldrich, USA) as the sole N source for 3 h.
M. hupehensis were used to screen the concentration of MT in the hydroponic culture. After 10 d of pre-cultivation, the plants were designated to receive half-strength nutrient solutions containing 0, 0.05, 0.5, or 2.5 μM MT, respectively. After 10 d of MT treatment, a nutrient deficiency (0.2 mM NO3) was induced. The deficiency trials spanned 30 d. The transgenic apple plants mediated by A. rhizogenes were pre-cultured with or without MT and subjected to low-nitrate treatment according to the method described above.