Physiological and molecular mechanisms of cytokinin involvement in nitrate-mediated adventitious root formation in apples

Potassium nitrate (KNO3) promotes adventitious root (AR) formation in apple stem cuttings. However, evidence for the possible involvement of cytokinin (CK) in KNO3-mediated AR formation in apples is still lacking. In this study, we used GL-3 apple microshoots and cultured them in different treatment combinations. T1 (KNO3 9.4 mmol L-1 + 6-benzyl adenine, 6-BA 2.22 μmol L-1) and T3 (6-BA 2.22 μmol L-1) treatments completely inhibited AR formation. However, control, T2 (KNO3 9.4 mmol L-1), and T4 (KNO3 9.4 mmol L-1 + lovastatin, Lov 1.24 μmol L-1) treatments developed ARs. But T4 developed fewer and shorter ARs, indicating that optimum CK synthesis was needed for normal AR growth. This also suggests that these fewer and shorter ARs were developed because of the KNO3 presence in the same medium. The stem basal part anatomy indicated that inhibition of CK biosynthesis delayed AR primordia formation. The endogenous levels of indole‐3‐acetic acid (IAA) and zeatin riboside (ZR) were higher in T2-treated microshoots, and abscisic acid (ABA), gibberellic acid 3 (GA3), and brassinosteroid (BR) levels were higher in T4-treated microshoots. The expression levels of MdNRT1.1 and MdNRT2.1 were higher in T2-treated microshoots at 3 and 8 days, and MdRR2 and MdCKX5 were higher at 8 and 16 days, respectively. Furthermore, higher IAA levels increased MdWOX11 expression, which in turn increased MdLBD16 and MdLBD29 expressions in response to T2. The combined expression of these genes stimulated adventitious rooting by upregulating cell cycle-related genes (MdCYCD1;1 and MdCYCD3;1) in response to T2-treated microshoots. This study shows that specific genes and hormonal pathways contribute to KNO3-CK-mediated adventitious rooting in apples.