4.1. Auxin Combinations Positively Affect Rooting Performance of M. wufengensis
Adventitious root (AR) formation is a complex process influenced by a large set of external and internal factors [
34,
35,
36]. It is generally accepted that the application of exogenous hormones has a certain role in adventitious root initiation. Auxins are a response to the induction of the meristems of root primordia, redistribution of nutrients, and biosynthesis of endogenous hormones in the cuttings, thereby stimulating adventitious root formation [
15,
37]. Synthetic auxins such as IBA and NAA have been used to promote rooting in plant cuttings [
38,
39,
40]. Unfortunately, for some hard-to-root plants, application of single auxins usually failed to stimulate meristematic activity and root initiation, or had only a slight rooting effect [
39,
41]. In the case of
M. wufengensis stem cuttings, a study on the role of auxins showed NAA and IBA exhibited better rooting results compared with IAA and GGR
6 (a synthetic plant growth regulator, active ingredient: amino acids ≥ 20%, trace elements ≥ 2%; Beijing Erbitux Biological Technology Co., Ltd., Peking, China), as in the different sensitivity of
M. wufengensis rhizomes to these two plant growth regulators, NAA was more stable and effective than IBA [
13]. In the present study, combined applications of NAA and IBA were found to be more effective than using NAA separately to increase the rooting ability of
M. wufengensis, and when treated with the median level (1000 mg/L NAA:IBA (2:1) solutions), we achieved the best rooting performance (
Table 6), which had significant differences to single auxin application and CK. The rooting percentage was about 16.16 times higher than CK and 1.39 times higher than NAA:IBA (1:0) treatment. Complementary application of NAA and IBA is found to be synergetic in terms of cells division and root formation [
15,
42]. These findings are also supported by B. Kaviani, who studied the effects of combinations of NAA and IBA on softwood cuttings of
Buxus hyrcana and found that 1000–2000 mg/L NAA and IBA treatments provided greater consistencies in rooting and shooting [
17]. Shahram [
43] investigated the effect of auxin combinations on stem cuttings of Mugo pine; when compared with CK, the rooting percentage of cuttings under NAA and IBA application increased from 14% to 55%, which confirms the positive effect of auxin combination.
In addition, the promoting effect of auxin combination on stem cuttings was closely related with the proportions of auxins. With the continuous increase of IBA proportion, the higher concentration of IBA may have a negative impact on stem cuttings and even suppress the activity of NAA [
17,
44,
45]. In this study, when cuttings were immersed in NAA:IBA (1:1), NAA:IBA (1:2), or NAA:IBA (0:1) (the proportion of IBA was 50%, 66.7%, or 100%, respectively), the rooting percentage was reduced by 45.3%, 35.9%, or 49.9%, compared to NAA:IBA (2:1) (the proportion of IBA was 33.3%). Similar results were found in the findings of Shahram [
43] who studied different concentrations of IBA and NAA on stem cutting of Mugo pine; the results indicated NAA:IBA (4:1)(the proportion of IBA was 20%) treatment exhibited the highest rooting percentage and dry weight. When cuttings were immersed in NAA:IBA (1:2) and NAA:IBA (1:4) (the proportion of IBA was 66.7% and 80%), the rooting percentage of these two treatments were decreased [
43]. Taken together, combined application of exogenous hormones is very important for the formation and improvement of root system in
M. wufengensis.
4.2. Anatomical Evaluation and Root Primordia Development during Adventitious Root Formation
The origin of adventitious roots in stem cuttings has been reported in various tissues and varies among species to species [
11,
46,
47]. There are two cell sources from which adventitious root differentiate: (a) various existing cells, for example the fascicular cambium; (b) callus tissue formed at the base of cuttings in response to wounding occurring during cutting collection [
48]. In this study, we firstly reported the formation of adventitious roots in
M. wufengensis. As shown in
Figure 4 and
Figure 6, little callus tissue was formed on the epidermis of
M. wufengensis cuttings, as a natural reaction of stems to wounding caused by cutting both in woody [
49,
50,
51] and herbaceous plants [
52]. At first glance, the roots of
M. wufengensis seemed to originate from the callus developed on the epidermis. However, the cells of callus generated from
M. wufengensis cuttings did not acquire the rooting competence (
Figure 6c and
Figure 7b), as they expressed different genetic routes with root-forming meristemoids which can initiate the establishment of a root development program [
53]. Furthermore, it was observed from the sections that the adventitious roots of
M. wufengensis arose directly from the stem vascular cambium and xylem parenchyma (
Figure 6d,e), which have been reported to have an enormous potential for the formation of root primordia [
54]. The histological structure was not sequentially performed, the function of callus on epidermis was only to heal the wound and emergent root, and it had no physical association with the adventitious root primordia in this species (
Figure 8d). Similar results were obtained in other difficult-to-root species such as
Diospyros virginiana and chestnut [
11,
53,
55]. Some studies indicated that the excessive growth of callus may inhibit the production of adventitious roots in cuttings, as a high level of cytokinin and a low level of auxin in callus would lead to a continuous formation of callus and to a large consumption of nutrients and energy, thus inhibiting the formation of adventitious roots [
51,
56,
57]. Fortunately, in NAA:IBA (2:1) treatment, callus on the epidermis did not present the phenomenon of overgrowth, and the small amount of callus could also protect against losses of water and nutrients of cuttings.
4.3. Changes of Metabolic Process in Response to Application of Auxin Combination during Adventitious Root Formation
The initiation, induction, and growth of adventitious root is an intensive metabolic process. Promoted by auxins and other plant growth regulators, these processes will lead to the increase of enzyme activities and the synthesis of RNA and proteins [
22,
54,
58,
59].
Plant peroxidases (POD) influence rooting by stimulating the production of iso-2-tyrosin in hydroxyproline-rich glycoproteins (HRGP, extensin) and catalyzing the biosynthesis of lignin and phellem layer of specific cells which will then be differentiated as root primordial cells [
24,
60,
61]. In cuttings, total POD activity has been considered as a biochemical marker of AR development [
27,
62,
63]. In this study, we observed two peak values of the POD activity synchronized well with the critical rooting points in NAA:IBA (2:1) treatment: one at the beginning of initiation phase, while the other was at the beginning of extension phase (
Figure 9a). The increase in POD activity at these two stages indicated that the pre-treatment with combination of NAA and IBA facilitated a lot in scavenging for H
2O
2 molecules. It can also increase cell wall strength, and subsequently increase resistance to stress. Similar results were reported in Persian walnut and
Malus hupehensis [
24,
64].
Polyphenol oxidases (PPO), which contained four atoms of copper per molecule and bound sites for two aromatic compounds and oxygen, are a group of copper enzymes localized in plastid tylacoids; they were able to catalyze the oxidation of aromatic compounds by oxygen [
26]. In this study, the trends that PPO activities exhibited were totally different in NAA:IBA (2:1) and CK. When cuttings were immersed in NAA:IBA (2:1) solutions, PPO activity increased continuously during induction, initiation, and expression phase but declined slowly after extension phase (
Figure 9b). As a previous study reported, PPO played an important role during root differentiation and root primordia formation and development. This might be due to its ability to participate in regulating synthesis of phenolic precursors during lignin biosynthesis [
64,
65,
66,
67,
68]. The continuous increase of PPO activities in NAA:IBA (2:1) treatment might have promoted adventitious root formation in the entire process of induction to development. The same results were obtained by Zhang [
24].
IAA oxidase (IAAO) affects rooting due to the IAA oxidation and regulates levels of IAA [
26,
69,
70]. In this study, the activity trend of IAAO in NAA:IBA (2:1) treatment showed less remarkable changes than that of CK. At 0–8 days after planting, IAAO activities of NAA:IBA (2:1) treatment was higher than CK (
Figure 9c). As previous study, an increase in the IAAO activity and the high contents of endogenous IAA and their derivatives could stimulate the root primordia induction and development on the auxin-supplemented medium [
26], this was in accord with our results. High activity of IAAO enzyme will inhibit rooting, as was found in previous studies; high IAAO activity reduced contents of endogenous IAA, which inhibited root development [
32,
70,
71]. In NAA:IBA (2:1) treatment, the low amount of IAAO activity 8–13 days after planting may be conducive to the initiation of root primordia. In expression and extension phase (13–28 dap), although IAAO activities of NAA:IBA (2:1) treatment continued to rise, the inhibitory effect on rooting was not exerted as there was no significant difference with the value on the 8th day. In Control, the activities of IAAO showed a rapid increase in the initiation, expression, and extension phases, which might induce low rooting percentage. A similar relationship was found during rooting of poplar,
Populus tomentosa, and smoke trees [
26,
71,
72].
Soluble protein content is relevant for both structural and regulatory/metabolic processes [
27]. Studies have shown that nucleic acid and protein synthesis are necessary for adventitious root formation [
64]. Presence of the auxin increases protein content in the base of a stem [
34]. In this study, the soluble protein contents showed stabilization and modest increase during 8–28 days in NAA:IBA (2:1) treatment. Nutrient substance was adequate throughout the initiation, expression, and extension phases. It suggested that the pre-treatment with auxin combination was beneficial to the sufficient nutrient supply during the rooting process (
Figure 9d). While in control, the soluble protein exhibited a continuously decrease trend; it was speculated that cuttings of control might suffer a defect of nutrient substance, and this finally led to the decline of rooting percentage (
Figure 9d). Taking into account the obtained results, we can speculate that the positive effect of auxin combination on stem cuttings might be linked to the progressive transition from heterotrophic metabolism to the autotrophic metabolism and the attaining of functionality of the water and nutrient balance system. These factors jointly promoted the development of the roots [
11].
4.4. Changes of Endogenous Hormones in Response to Application of Auxin Combination during Adventitious Root Formation
Plant hormones play an important role in the control of AR formation as they respond to the changing environment, provide a signaling network within the plant, and are decisive for cell fate determination and specification [
35]. 3-indoleacetic acid (IAA) is common in plants as a main hormone of the auxin group. In this study, endogenous IAA showed a modest increase in NAA:IBA (2:1) treatment in the first 3 days (
Figure 10a). It was speculated that the isolation of stem cuttings may induce the polar auxin transport, the basipetally transported auxin from shoot tips and young leaves may accumulate at the cutting base, and then create a local concentration gradient that drives the induction of adventitious root formation [
27,
34,
56]. The early peak of endogenous IAA appeared at 8 days after planting, which synchronized well with the induction phase. It was reported that IAA could affect the root formation by acting directly in the cambium cells that initiate root primordia or indirectly through its engagement in the overall metabolism [
73,
74]. Our results confirmed the promotion role of IAA in the induction phase (0–8 days), the results were in accord with the findings obtained by Ahkami [
74]. In the initiation and expression phases (8–18 days), cuttings showed a decrease of IAA (
Figure 10a). This might be reduced by catabolism and the physiologically active conjugate of jasmonic acid after the induction phase [
35,
75,
76].
Abscisic acid (ABA), which is a stress-related hormone, has been shown to inhibit adventitious root emergence and lateral root development in deepwater rice,
Arachis hypogaea, and tomato, possibly by interfering with ethylene (ET) and gibberellic acids (GAs) signaling pathways and blocking cell cycle progression [
23,
34,
77,
78]. In this study, ABA contents of cuttings immersed in NAA:IBA (2:1) treatment increased slightly in the first 3 days after planting (
Figure 10b). This was speculated to be related to the isolation of the cuttings. It was reported that isolation of the cuttings from donor trees broke the soil–plant–atmosphere continuum and led to numerous stresses caused by the interruption of water and nutrient uptake, and then altered transport of endogenous phytohormones and the activation of wound responses [
47,
79]. At 3–8 days, ABA contents showed a decreased trend both in NAA:IBA (2:1) and CK, while after 8 days, ABA contents of cuttings immersed in NAA:IBA (2:1) solutions increased slightly (
Figure 10b). This coincided with the stage when root primordia are determined but their vascular connection with the stem has not been established yet. The same results were observed in the stem cutting of carnation [
47]. In these conditions, the incipient root primordia might suffer some stress such as water shortage, and that leads to a transitory increase in their ABA contents [
47]. When the root development was gradually completed, the contents of ABA decreased.
Gibberellins (GAs) are a complex family of tetracyclic diterpenoid plant growth regulators, some of which, such as GA
1, GA
3, GA
4, and GA
7, are thought to function as bioactive hormones [
25]. These bioactive hormones control diverse aspects of plant growth and development, including seed germination, flower initiation, fruit development, stem elongation, leaf expansion, and trichome differentiation [
80,
81,
82,
83]. In cutting propagation, GA
3 is generally considered an inhibitor of adventitious root formation [
34]. In poplar plants, in which defects in GA production or perception cause the growth of lateral root to be promoted, higher root mass and highly branched roots are produced [
82], while according to some findings, GAs are assumed to have an adventitious root phase-dependent effect [
34]. In our study, the results indicated that GA
3 contents in cuttings immersed in NAA:IBA (2:1) solutions exhibited an increase trend in the initiation and extension phases (
Figure 10c), which confirmed the phase-dependent effect of GAs. Similar results were observed in deep water rice,
Malus hupehensis, and chestnut [
23,
24,
55]. The effect of GAs being inhibitory to root induction and stimulatory to root formation might contribute to their synthesis and transportation characteristics. Previous studies indicated that GAs synthesized in the meristem and cortical and epidermal cell layers [
25,
84,
85], then specifically accumulated in the endodermis of the root elongation zone [
86].
Cytokinins play key roles in regulation of root development [
87]. The major cytokinins in leaves, stems, and roots of plants have been rigorously identified by several physicochemical methods [
88]. Zeatin (ZT) and zeatin riboside have been identified as two major cytokinins in root exudate [
89]. Zeatin is considered more active than its riboside [
47]. In this study, contents of ZT showed a W-shaped trend with the presence of NAA and IBA. In the first 3 days, the contents of ZT showed a slight decrease, this was speculated to be related to the isolation from the stock plants (
Figure 10d). As the previous study reported, the root primordia cells required a dedifferentiation process before adventitious root induction [
35]. Early wounds in cuttings induced accumulation of ethylene and jasmonic acid, these two hormones together with cytokinins which exhibited a transient decrease in levels contributed to the dedifferentiation process by enhancing auxin responsiveness [
35]. During days 3–8, ZT contents of cuttings immersed in NAA:IBA (2:1) increased, while there were no significant differences with the initial value (
Figure 10d). According to the previous reports, cytokinins regulated adventitious root formation in a stage-specific manner, the low level of cytokinins combined with high auxin levels was thought to be involved in the contribution of adventitious root induction [
34,
47,
90,
91,
92,
93]. In the present study, cuttings immersed in NAA:IBA (2:1) solutions revealed a high level of IAA and low level of ZT at induction phase (0–8 days), which confirmed the theory that a rise in both auxin and cytokinin depletion controls induction of adventitious roots in cuttings [
47,
91]. At 8–18 days, contents of ZT continuously increased in NAA:IBA (2:1) treatment (
Figure 10d), the increasing cytokinin could regulate the longitudinal zonation and radial patterning of root vasculature by controlling cell differentiation, promoting protophloem cell identity, and spatially inhibiting protoxylem formation in root meristem [
93]. Owing to the increasing ZT contents, massive root primordia emerge, the vascular reconnection between newly formed roots and shoot was fully established, allowing root nutrition, hydration, and growth [
47,
94]. At the same time, the requirement for ZT further expanded with the cell differentiation and proliferation in the phloem, xylem, and cambium zones, which could explain the increase of ZT contents in the extension phase of NAA:IBA (2:1) treatment.