Nitrogen is regarded as an important macronutrient that is needed for plant development and higher yields [103]. Nitrogen assimilation in soil occurs in two ways, such as organic and inorganic [104]. Nitrate is a vital nitrogen source that also functions as a signaling molecule for controlling flowering time, AR, and LR formation, as well as prompting auxin-related gene expressions [28,105,106]. Nitrate levels in the soil are relatively low due to its high solubility, leaching capabilities, and fast absorption by bacteria and fungus [107]. In higher plants, such as apples, there are two types of nitrate transport mechanisms: low-affinity transport systems (LATS) and high-affinity transport systems (HATS), which are responsible for uptake, distribution, and storage of nitrate [108]. The nitrate supply is immediately and strongly detected by the plant cells. Following this, the nitrate signaling system changes the relative expression levels of several gene sets to control cell and organ metabolism. Furthermore, the availability of nitrate has a significant effect on AR formation. In general, the external concentration of nitrate generated binary effects on the formation of AR depending on their concentrations, with an activated impact at low levels; however, a limiting effect at high levels was seen in Arabidopsis [109]. A similar phenomenon was also seen in a recent study, where different nitrate concentrations (9.4 mM/L, 18.8 mM/L, 28.1 mM/L, 46.9 mM/L, and 84.5 mM/L) were exogenously treated to B9 apple rootstock stem cuttings during adventitious rooting, and 28.1 mM/L was found to be the most favorable nitrate level for adventitious rooting, and 46.9 mM/L and 84.5 mM/L were found to be inhibitors [28]. High nitrate inhibits AR in apples by elevating the endogenous levels of ABA, ZR, JA, BR, and GA₃, which may create a hormonal imbalance in the plant. In addition, the high ratios of IAA/ABA and IAA/ZR promote ARs under nitrate treatments. Furthermore, transcriptome analysis showed that hormone signaling pathway-related genes were upregulated, and root development and cell cycle-related pathways were repressed by the application of high nitrate [31]. Moreover, auxin and ABA signaling miRNAs (miR390a, miR160a, miR167, miR169a, and miR394) were activated, and miRNAs related to cell fate transformation, expansion, and enlargement (miR166, miR171, miR319, miR156, and miR396) were repressed by high nitrate [30]. The same cited authors explained the mechanism of a different set of genes how 28.1 mM/L treatment promotes AR formation in B9 compared with 46.9 mM/L (inhibiting treatment). The results showed that treatment with 28.1 mM/L noticeably upregulated the relative expression levels of nitrate related genes (NRT1.1, NRT2.1, NIA1, and ANR1) and auxin biosynthesis (IAA14 and IAA23), which enhances the AR development-related gene expression (WOX11, ARRO1, and SHR) and collectively induces the expression of cell cycle related genes (CYCD1;1, CYCD3;1, and CYCP4;1) in comparison with 46.9 mM/L nitrate treatment [29]. NRT2.1 a high affinity nitrate transporter showed the highest response to nitrate availability, indicating that NRT2.1 may play a key role in forming AR in apples, and the overexpression of MdNRT2.1 gene in tobacco produced superior roots compared to WT plants.

Ammonium, such as nitrate, played an important role in root development in apple and other crops [110,111,112]. The effects of nitrate and ammonium were studied in apples, where significant differences were detected in root morphology within a week of application. The roots were thin and long in response to nitrate treatment, although they were thick and short in response to ammonium application, with prominent enlarged areas behind the tip. Furthermore, nitrate-treated roots were nearly devoid of root hairs, but those treated with ammonium were entirely covered in thick, long root hairs, and the root hair cylinder diameter in the ammonium treated was around three times that of the nitrate treated [111]. Hilo and colleagues [112] found that ammonium-treated cuttings (without nitrate) had a higher total nitrogen content, which was indicated by enhanced glutamine and asparagine levels. These authors point to faster ammonium assimilation in the stem base, which could have resulted in a lower expression level of N-regulated genes such as the ammonium transporter AMT1 [112]. Moreover, the effects of ammonium nitrate (NH₄NO₃) and potassium nitrate (KNO₃) were also studied in three apple scion cultivars [110]. The percentage ARs of Gala and Royal Gala rose dramatically when the level of NH₄NO₃ in the medium was reduced from full strength to 1/4 strength, but not in Jonagold. A further decrease in NH₄NO₃ concentration from 1/4 strength to zero considerably decreased the rate of ARs in Gala but not in Royal Gala. However, Jonagold rooted optimally in the absence of NH₄NO₃. Moreover, without NH₄NO₃, adventitious rooting for all three cultivars was as high as 100% when KNO₃ was given at full strength. These results suggest that the effect of NH₄NO₃ was cultivar-specific, but KNO₃ treatment at full strength promoted ARs in all cultivars.

Potassium (K) is an indispensable macronutrient, and it is the most abundant cation absorbed by higher plants. It comprises more than 10% of the plant’s total dry weight [113]. Its decline to below 10 g/kg of dry weight leads to serious growth issues in various plant species. Indeed, despite not being an important element of any structural and functional molecules, it is involved in various key physiological processes, such as metabolism and plant growth and development [114], flowering [115], improved fruit quality [116], and AR formation in apples and other plants [32,117]. In addition, under K deficiency in soil, plant root growth is weakened. As a result, the yields and outcomes are often limited [118]. The uptake and translocation of K is mediated by several K channels and transporters [113,119].

The underlying physiological and molecular mechanisms regulating AR by KCl application were studied in B9 apple rootstock. KCl-treated cuttings produced a significantly higher number of ARs and increased AR length than control cuttings. At most time points during AR formation, KCl promoted several hormone levels, including IAA, ZR, JA, and GA, and decreased the ratios of ABA/JA, ABA/ZR, and ABA/IAA. Moreover, transcriptome analysis revealed that KCl promoted ARs through the auxin signaling pathway and sugar metabolism and by increasing the genes related to AR development and cell cycle [32]. Z.R. Zhao and colleagues found the promotive effect of K on the formation of ARs in cucumber cotyledons and mung bean hypocotyls, as well as in kidney beans [117]. The results of these studies indicated that the K application promotes adventitious rooting in apples and other crops.

The effect of nitrate and potassium treatments on the various endogenous hormones during adventitious rooting in apples is shown in Figure 2.