The Dwarfing Effects of Different Plant Growth Retardants on Magnolia

Article The Dwarﬁng Effects of Different Plant Growth Retardants on Magnolia wufengensis L.Y. Ma et L. R. Wang

Xiaodeng Shi , Siyu Chen and Zhongkui Jia *

Key Laboratory for Silviculture and Conservation of the Ministry of Education, College of Forestry, Beijing Forestry University, Beijing 100083, China; [email protected] (X.S.); [email protected] (S.C.) * Correspondence: [email protected]

Abstract: The effects of varieties, concentrations, and number of applications of plant growth retardants (PGRs) on the morphological, physiological, and endogenous hormones of Magnolia wufengensis L.Y. Ma et L. R. Wang were assessed to obtain the most suitable dwarfing protocol for M. wufengensis and to provide theoretical support and technical guidance for the cultivation and promotion of this species. One-year-old M. wufengensis ‘Jiaohong No. 2’ grafted seedlings served as the experimental materials. In the first part of the experiment, three PGRs (uniconazole, paclobutrazol, prohexadione calcium), three concentrations (500, 1000, 1500 ppm), and three applications (one, three, and five applications) were applied in dwarfing experiments to perform L9 (34) orthogonal tests. In the second part of the study, dwarfing experiments were supplemented with different high uniconazole concentrations (0, 1500, 2000, 2500 ppm). Spraying 1500 ppm uniconazole five times achieved the best M. wufengensis dwarfing effect, related indicators of M. wufengensis under this treatment were better than other treatment combinations. Here, M. wufengensis plant height, internode length, scion diameter, and node number were significantly reduced by 56.9%, 62.6%, 72.8%, and 74.4%, respectively, compared with the control group. This treatment increased superoxide dismutase (SOD) activity by 66.0%, peroxidase (POD) activity by 85.0%, soluble protein contents by 43.3%, and soluble sugar contents by 27.6%, and reduced malondialdehyde (MDA) contents by 32.1% in leaves of M. wufengensis compared with the control. The stress resistance of M. wufengensis was enhanced. The treatment also reduced gibberellin (GA3) levels by 73.0%, auxin (IAA) by 58.0%, and Citation: Shi, X.; Chen, S.; Jia, Z. zeatin (ZT) by 70.6%, and increased (abscisic acid) ABA by 98.1% in the leaves of M. wufengensis. The Dwarfing Effects of Different The uniconazole supplementation experiment also showed that 1500 ppm was the optimal unicona- Plant Growth Retardants on zole concentration. The leaves exhibited abnormalities such as crinkling or adhesion when 2000 or Magnolia wufengensis L.Y. 2500 ppm was applied. Given the importance of morphological indicators and dwarfing for the Ma et L. R. Wang. Forests 2021, 12, 19. ornamental value of M. wufengensis, the optimal dwarfing treatment for M. wufengensis was spraying https://dx.doi.org/10.3390/f12010019 1500 ppm uniconazole five times.

Received: 25 November 2020 Keywords: Magnolia wufengensis; dwarf; plant growth retardant; endogenous hormones; morphol- Accepted: 24 December 2020 ogy; physiology Published: 26 December 2020

Publisher’s Note: MDPI stays neu- tral with regard to jurisdictional claims in published maps and institutional 1. Introduction afﬁliations. Magnolia wufengensis L.Y. Ma et L. R. Wang and its variant Magnolia wufengensis var. multitepala are a new species of genus and subgenus Magnolia that were discovered by Professor Ma Luyi of Beijing Forestry University in Wufeng County, Hubei Province, China in 2004 [1,2]. M. wufengensis is a tall tree with a height of 20 m, and the crown is Copyright: © 2020 by the authors. Li- beautiful [1,2]. Its most distinctive feature is that it is the only species of Magnolia that is censee MDPI, Basel, Switzerland. This completely red inside and outside the perianth [3]. Its ﬂowers are rich in morphological article is an open access article distributed under the terms and conditions of the variations and exhibit different types, such as rose and lotus. There are also various tepal Creative Commons Attribution (CC BY) colors, ranging from purple-red to red to pale red and even close to white, and the number license (https://creativecommons.org/ of petals in the perianth varies from nine to 46 [3]. Therefore, M. wufengensis exhibits a licenses/by/4.0/). very high ornamental value and is an excellent ornamental tree species [4]. At present,

Forests 2021, 12, 19. https://dx.doi.org/10.3390/f12010019 https://www.mdpi.com/journal/forests Forests 2021, 12, 19 2 of 17

eight new varieties have been selected and authorized, among which the new variety Magnolia wufengensis ‘JiaoHong 1’ has been widely introduced as a city garden greening tree species to cities such as Yunnan, Guangxi, Guizhou, Henan, Jiangsu, Shandong, Beijing, Hebei, and other provinces in China [5]. Similar to a large number of Magnoliaceae plants, M. wufengensis has strong foliage and properties that improve its viewing and can also be used for garden greening [6]. However, M. wufengensis is too tall for indoor cultivation. Therefore, from the perspective of garden viewing, the dwarfing of M. wufengensis into a small garden tree or indoor potted plant can save space and allow adaptation to limited indoor heights. After dwarfing, M. wufengensis can integrate harmoniously with other plants and improve their overall ornamental effect. Furthermore, it is also convenient to be able to move potted plants, and dwarfing can extend its industrial chain to further increase its ornamental and economic value. Dwarfing is an important process and is a hot topic in current plant work. Miniatur- ized and compact potted flowers and bonsai are becoming increasingly popular. Therefore, dwarfing is an important aspect of the ideal plant type of higher plants [7]. Common dwarfing methods can be roughly divided into chemical and physical methods. The physical methods mainly include dense dwarfing [8], grafting dwarfing anvil [9], and pruning dwarfing [10]. The chemical methods mainly include the application of plant growth retardants [11], virus dwarfing [12], and radiation-induced dwarfing [13]. Among these methods, the application of plant growth retardants (PGRs) effectively dwarfing ornamental plants and having the advantages of being inexpensive, easy and rapid, with high efficiency and minimal side effects, has become one of the important measures for modern agriculture [14]. There are many studies on the application of PGRs (Plant growth retardants) in tree species at home and abroad, and these methods have been successfully used to dwarf apple, pear, peach, arbutus, and other trees, achieving good modeling effects [15,16]. At present, PGRs are mainly quaternary ammonium salts (chlormequat chloride, mepperidone), tri- azoles (paclobutrazol, uniconazole), and cyclohexanecarboxylic acids (prohexadione calcium, trinexapac-ethyl). PGRs operate by inhibiting the synthesis of plant gibberellin (GA3), cell division, and elongation and growth of the lower part of the top of the stem, thereby inhibiting the vegetative growth of plants to a certain extent and achieving dwarfing [17]. PGRs promote plant photosynthesis and metabolism, significantly increase crop yield, and even promote flower bud differentiation and increase flower diameter [17]. Among PGRs, paclobutrazol has a remarkable dwarfing effect and minimal side effects on fruit trees, vegetables, and ornamental plants, which has attracted international attention [18,19]. In the nineteenth century, Aron applied 1 g·L−1 paclobutrazol to ‘Minneola’ tangelo (Citrus reticulata Blanco × Citrus paradisi Macf.) before the rapid growth of new shoots in summer, and the length of the shoots was reduced by approximately 50% on average [20]. The use of 100 ppm paclobutrazol in the mid-leaf development period of Sapindus mukorossi (Gaertn.) can achieve the best dwarfing effect, inducing vigorous growth in S. mukorossi, significantly reducing plant height, thickening the stalk, and increasing the chlorophyll content [21]. Wang Fang studied the influence of different concentrations of paclobutrazol (PP333) on some physiological characteristics in leaves of Rhododendron hybrides ‘Cosmopolitan’ and found that paclobutrazol can significantly increase the activity of superoxide dismutase (SOD) and peroxidase (POD) enzymes in leaves and petals and reduce the content of malondialdehyde of R. hybrids [22]. Furthermore, paclobutrazol alters the endogenous hormone content in young leaves, inhibiting GA content and increasing abscisic acid (ABA) content [23]. Although paclobutrazol can be absorbed by various parts of the plant, its mobility in the plant is not good [20]. Therefore, the specific application method needs to be considered when using paclobutrazol, and the commonly used methods include foliar spraying and soil irrigation [24,25]. Uniconazole is a highly effective PGR that can change plant morphology and cause a series of physiological responses [26]. Repeated foliar application of uniconazole on Citrus reticulata fruit can effectively reduce shoot growth and the number of nodes, re- Forests 2021, 12, 19 3 of 17

ducing internode length [27]. Under stress conditions, uniconazole increases the activity of SOD, POD, and catalase (CAT) in rice seedlings, reduces the content of malondialdehyde (MDA), reduces electrolyte permeability, and increases the free proline content of metabolites in seedlings to improve stress resistance [28]. Spraying uniconazole under salt stress can increase the accumulation of ABA, cytokinin (CTK), crude protein, total soluble protein, proline, auxin (IAA), and GA3 in barley plants [29]. Compared with paclobutrazol, uniconazole is more easily degraded, and its soil residue is only one tenth of the former [30]. Prohexadione calcium works through foliar treatment and can inhibit the synthesis of gibberellin in plants, especially3β-hydroxylation of GA1 to GA20, thereby inhibiting the stem elongation of many plants [31]. Kang treated Chinese cabbage with prohexadione calcium and found that the contents of GA1 and GA4 both decreased [32]. The application of 100 ppm prohexadione calcium can effectively reduce the growth of pear (Pyrus communis L.) shoots and enlarge the fruit [33]. Under conditions of low temperature stress, spraying prohexadione calcium results in a slow increase in the membrane lipid peroxidation product MDA; increases proline content; weakens the inhibition of SOD, POD, and CAT activity under cold stress, and enhances the enzyme activity [34]. Compared with paclobutrazol and uniconazole, the residual amount of prohexadione calcium is reduced, which is more friendly to the human body and the environment, and has no significant impact on subsequent crops [31]. Therefore, the effects of spraying PGR at different concentrations a different number of times on plant growth vary greatly [21–31]. The purpose of this study is to dwarf M. wufengensis. Based on previous experience with other tree species, we mainly chose to explore the effects of the PGRs paclobutrazol, uniconazole, and prohexadione calcium at different concentrations on the morphological and physiological properties and endogenous hormones of M. wufengensis. It is hoped that the most suitable PGR variety, concentration, and number of applications can be identified through experiments to obtain a cost-effective method for dwarfing M. wufengensis plants, thus providing theoretical support and technical guidance for the cultivation and promotion of M. wufengensis.

2. Materials and Methods 2.1. Study Area The experiment started on July 1, 2016 and was performed in Wufeng County, Yichang City, Hubei Province, China (30◦12’ N, 110◦40’ E). This area is located in the southwest of Hubei Province and borders Hunan Province in the north. The region has a subtropical monsoon climate with sufﬁcient light, four distinct seasons, high temperatures and rain in summer, and low temperatures and snow in winter.

2.2. Study Design 2.2.1. Dwarﬁng Experiment of the PGRs We selected one-year-old grafted seedlings of M. wufengensis ‘JiaoHong 2’. These seedlings were provided by Wufeng Bo Ling Magnolia wufengensis Technology Development Co., Ltd., Hubei, China; exhibited good growth and uniform scion diameters and plant heights; and served as the experimental material. Uniconazole, paclobutrazol, and prohexadione calcium were selected as the experimental PGRs. Uniconazole (5% WP) and paclobutrazol (15% WP) were provided by Sichuan Guoguang Agrochemical Co., Ltd., Sichuan China. Prohexadione calcium (5% WP) was provided by Anyang Quanfeng Biological Technology Co., Ltd., Henan China. The 3 PGRs were used at 3 concentrations (500, 1000, and 1500 mg·L−1) and applied 1, 3, or 5 times. An orthogonal (L9 (34)) experimental design was adopted in the experiment to yield a total of 9 treatments as shown in Table1. Using the treatment without PGRs as a control group (CK), there were 10 treatments in total, and each treatment con- tained 15 seedlings repeated 3 times. The experiment was conducted from June 2016 to October 2016. Forests 2021, 12, 19 4 of 17

Table 1. L9 (34) orthogonal experimental design.

Treatment Concentration Number of Empty Variety Number (mg·L−1) Application (Times) Column 1 Uniconazole 500 1 1 2 Uniconazole 1000 3 2 3 Uniconazole 1500 5 3 4 Paclobutrazol 500 3 3 5 Paclobutrazol 1000 5 1 6 Paclobutrazol 1500 1 2 Prohexadione 7 500 5 2 Calcium Prohexadione 8 1000 1 3 Calcium Prohexadione 9 1500 3 1 Calcium

There are two rapid growth periods in the one-year growth season of M. wufengensis grafted seedlings: June and August. The PGRs were generally applied before the rapid growth of plants, i.e., in the early stage of rapid growth of M. wufengensis grafted seedlings (early June 2016). The application method was foliar spray. A pressure-type sprayer was used to spray water on the front and back of the foliage. Plants were not watered within 1 week after the application of the reagent to prevent the solution from being diluted. The interval between applications was 10 days until the number of applications used in the experiment was reached. If the plant encountered rain within two days of applying the PGR, the experiment was extended and sprayed at 10-day intervals.

2.2.2. Supplemental Dwarfing Experiments at Different Uniconazole Concentrations According to the results of the PGR dwarfing experiment, the best dwarfing treatment was spraying 1500 ppm uniconazole 5 times, and the number of applications was the highest value set. However, spraying 5 times crossed from the first to the second growth peaks of M. wufengensis, so no additional experiments were applied to test the number of applications. The optimal PGR concentration obtained in the experiment was also the highest value set in the experiment. To determine the optimal concentration of uniconazole, this supplemental dwarfing experiment was added. In this experiment, the M. wufengensis ‘JiaoHong 2’ 1-year-old grafted seedlings with uniform scion diameters and plant heights that exhibited superior growth were selected as the experimental materials. The experiment was performed in a completely random block design, and a total of 4 treatments were employed, i.e., spraying uniconazole at 0, 1500, 2000, and 2500 ppm. Each treatment comprised 9 seedlings in plots and was repeated thrice. The experiment was conducted from June 2017 to October 2017.

2.3. Method 2.3.1. Morphological Investigation In each plot, seedlings were randomly selected and morphologically investigated [7]. The plant height and scion base diameter were measured before the start of the experiment, and the number of nodes and internodal length were recorded. The scion diameter was measured 5 cm above the graft joint.

2.3.2. Determination of Physiological and Biochemical Indicators Sampling was performed before the start of the experiment (early June) and two months after the experiment. The sampling time was 8 o’clock in the morning. For each treatment, 10 seedlings were randomly selected to pick 1–3 functional leaves (the 2nd–4th leaf from the top) in each plot, wrapped with tin foil, marked, ﬁxed in liquid nitrogen and stored in a refrigerator at −80 ◦C. Forests 2021, 12, 19 5 of 17

The measured indexes included SOD, POD, soluble protein, MDA, and soluble sugar. SOD activity was determined using the NBT reduction method [35]. POD activity was determined using the guaiacol chromogenic method [36]. MDA content was determined using the thiobarbituric acid method [37]. The content of soluble sugar was determined using anthrone colorimetry [35], and soluble protein was determined using the Coomassie blue method [38].

2.3.3. Determination of Endogenous Hormones The sampling time, sampling method, and storage method were consistent with those used in the determination of physiological and biochemical indicators reported in Section 2.3.2. The samples were analyzed by high-performance liquid chromatography (HPLC) [39]. The measurement indicators included IAA, GA3, ABA, and ZT content [39].

2.4. Data Processing In this study, EXCEL (2007) and SPSS 19.0 statistical software were used for data statistics and analysis. A factorial analysis of variance (ANOVA) and the Duncan test (p < 0.05) were performed to analyze the variance and for multiple comparisons.

3. Results The conclusion is divided into two parts. Sections 3.1–3.3 report the dwarﬁng experiment of the PGRs, and Sections 3.4–3.6 describe the supplemental dwarﬁng experiment at different uniconazole concentrations.

3.1. Dwarfing Effects of PGRs on Morphology The PGR variety, concentration, and number of applications could significantly affect plant scion diameter, plant height, number of nodes, and internodal length. The experimental results are shown in Table2 and Figure1. In the range analysis, the larger the range value, the greater is the effect of this factor on the index. As shown in Table2, the degree of influence of the three factors on the plant height of M. wufengensis exhibits the following order: concentration (43.1) > number of application (33.6) > variety (28.3). All factors had extremely significant effects (p < 0.01). The concentration and number of applications of PGRs had a significant impact on the internodal length (p < 0.01). Specifically, the concentration (2.5) had a greater impact, whereas the variety had no significant impact on internodal length (p > 0.05). The concentration and variety of PGRs had a significant effect on the scion diameter of M. wufengensis (p < 0.01), whereas the concentration (2.0), variety (1.9), and number of applications had no significant effect on the scion diameter (p > 0.05). The variety of PGRs had a significant effect on the node number (p < 0.01), and the concentration also had a significant effect (p < 0.05). However, the number of applications had no significant effect on the node number (p > 0.05). In summary, the concentration of PGRs has the strongest effect on the plant height, internodal length and scion diameter of M. wufengensis, and it plays a leading role in the morphological indicators of M. wufengensis. According to Figure2, the plant height (Figure2a), internodal length (Figure2b), scion diameter (Figure2c) and node number (Figure2d) of M. wufengensis in the 10 treatments were significantly different (p < 0.05). The minimum plant height was 115.8 cm for treatment No. 3, which was only 56.9% of the CK (203.4 cm) (Figure2a). The minimum internodal length was noted for treatment No. 3 (6.2 cm), which was 62.6% of the CK (9.8 cm) (Figure2b ). The smallest scion diameter was noted for treatment No. 3 (13.5 mm), which was 72.8% of the largest scion diameter in CK (18.6 mm) (Figure2c). Figure2d shows that among the 10 treatments the node number of M. wufengensis was significantly lower than that of CK (p < 0.05). The minimum node number was noted for treatment Nos. 1, 2, and 3, and the difference between them was not significant (p > 0.05). The node number for treatment No. 3 (18.9) was 74.4% of the maximum node number of CK (25.4). In summary, No. 3 treatment (spraying 1500 ppm uniconazole five times) exhibited the best effect on Forests 2021, 12, 19 6 of 17 Forests 2021, 12, x FOR PEER REVIEW 6 of 18

the(p dwarﬁng > 0.05). In morphology summary, M.the wufengensisconcentration. Compared of PGRs withhas the CK, strongest plant height, effect internodal on the plant length,height, scion internodal diameter length and node and scion number diameter were signiﬁcantly of M. wufengensis reduced, and (56.9%, it plays 62.6%, a leading 72.8%, role andin 74.4% the morphological of CK, respectively). indicators of M. wufengensis.

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FigureFigure 1. The1. The effects effects of differentof different PGR PGR treatments treatments on theon the height height (a), ( internodala), internodal length length (b), (b scion), scion diameter diameter (c), ( andc), and node node numbernumber (d) of(d)Magnolia of Magnolia wufengensis wufengensisL.Y. MaL.Y. et Ma L. R. et Wang.L. R. Wang. Treatment Treatment No. 10 isNo. the 10 control is thegroup control (CK). group Different (CK). smallDifferent letters small indicateletters significant indicate significant differences differences among treatments among treatments as assessed as by assessed the Duncan’s by the test Duncan’s (p < 0.05). testBras (p < are0.05). means Bras ±areSDs means (n = ± 3). SDs (n = 3). The same is shown below. The same is shown below. Table 2. Range analysis of the L9 (34) orthogonal experimental design assessing morphological Tablefactors. 2. Range analysis of the L9 (34) orthogonal experimental design assessing morphological factors. Range Height/cm Internodal Length/cm Scion Diameter/cm Node Number RangeVariety Height/cm 28.3 ** Internodal Length/cm 0.4 Scion Diameter/cm 1.9 ** Node 3.6 Number ** Concentration 43.1 ** 2.5 ** 2.0 ** 1.2 * VarietyTimes 28.3 33.6 ** ** 0.4 2.1 ** 1.9 1.0 ** 3.6 0.9 ** Concentration 43.1 ** 2.5 ** 2.0 ** 1.2 * Note: * indicates the different varieties, concentrations, and times of PGRs significantly affect plant Times 33.6 ** 2.1 ** 1.0 0.9 height, internodal length, scion diameter, and node number of Magnolia wufengensis. *, p < 0.05; **, Note:p < 0.01. * indicates The same the different parameters varieties, are concentrations,reported in Tables and times 3 and of 4. PGRs significantly affect plant height, internodal length, scion diameter, and node number of Magnolia wufengensis. *, p < 0.05; **, p < 0.01. The same parameters are reported in Tables3 and4. According to Figure 2, the plant height (Figure 2a), internodal length (Figure 2b), 3.2.scion Dwarfing diameter Effects (Figure of PGRs 2c) on and Physiology node number (Figure 2d) of M. wufengensis in the 10 treatments were significantly different (p < 0.05). The minimum plant height was 115.8 cm for The PGR variety, concentration, and number of applications affected the SOD and treatment No. 3, which was only 56.9% of the CK (203.4 cm) (Figure 2a). The minimum POD activities and soluble protein, MDA, and soluble sugar content of M. wufengensis as internodal length was noted for treatment No. 3 (6.2 cm), which was 62.6% of the CK (9.8 shown in Table3 and Figure2. cm) (Figure 2b). The smallest scion diameter was noted for treatment No. 3 (13.5 mm), As shown in Table3, the variety of PGRs had a significant impact on SOD activity which was 72.8% of the largest scion diameter in CK (18.6 mm) (Figure 2c). Figure 2d in M. wufengensis (p < 0.05). The PGR concentration (84.2) had a greater impact on SOD shows that among the 10 treatments the node number of M. wufengensis was significantly activity than the number of applications (74.3), but it was not significant (p > 0.05). The lower than that of CK (p < 0.05). The minimum node number was noted for treatment Nos. number of applications of PGRs had a significant impact on POD activity (p < 0.05), 1, 2, and 3, and the difference between them was not significant (p > 0.05). The node num- whereas the variety and concentration had no significant impact (p > 0.05). The variety and ber for treatment No. 3 (18.9) was 74.4% of the maximum node number of CK (25.4). In

Forests 2021, 12, 19 7 of 17

number of applications of PGRs had a significant impact on the soluble protein content of M. wufengensis (p < 0.01). In addition, the number of applications (14.4) had a greater impact than the variety (11.6), but the concentration of PGRs had no significant effect on the soluble protein content (p > 0.05). The 3 factors had no significant influence on the MDA Forests 2021, 12, x FOR PEER REVIEWcontent of M. wufengensis (p > 0.05). The 3 factors of PGRs on the content of soluble sugar,7 of 18

which is arranged as follows: concentration (0.7) > variety (0.6) >number of applications (0.5). The concentration reached an extremely signiﬁcant level (p < 0.01), and the variety p andsummary, number of No. applications 3 treatment reached (spraying a signiﬁcant 1500 ppm level uniconazole ( < 0.05). five In summary, times) exhibited the variety the best of PGRs has the strongest effect on the SOD activity and MDA content of M. wufengensis. effect on the dwarfing morphology M. wufengensis. Compared with CK, plant height, in- The concentration has the strongest effect on the soluble sugar content, and the number of ternodal length, scion diameter and node number were significantly reduced (56.9%, applications has the strongest effect on the POD activity and soluble protein content. 62.6%, 72.8%, and 74.4% of CK, respectively).

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Figure 2. The effects of PGRs on SOD activity (a), POD activity (b), soluble protein (c), MDA content (d), and soluble sugar Figure 2. The effects of PGRs on SOD activity (a), POD activity (b), soluble protein (c), MDA content (d), and soluble sugar (e) of(eM.) of wufengensis M. wufengensis. .

3.2. Dwarfing Effects of PGRs on Physiology The PGR variety, concentration, and number of applications affected the SOD and POD activities and soluble protein, MDA, and soluble sugar content of M. wufengensis as shown in Table 3 and Figure 2. As shown in Table 3, the variety of PGRs had a significant impact on SOD activity in M. wufengensis (p < 0.05). The PGR concentration (84.2) had a greater impact on SOD activity than the number of applications (74.3), but it was not significant (p > 0.05). The number of applications of PGRs had a significant impact on POD activity (p < 0.05), whereas the variety and concentration had no significant impact (p > 0.05). The variety and number

Forests 2021, 12, 19 8 of 17

Table 3. Range analysis of the L9 (34) orthogonal experimental design assessing physiological factors.

Range SOD POD Soluble Protein MDA Soluble Sugar Variety 137.4 * 12.8 11.6 ** 1.5 0.6 * Concentration 74.3 34.9 4.7 1.1 0.7 ** Times 84.2 131.6 * 14.4 ** 1.4 0.5 * Note: * indicates the different varieties, concentrations and times of PGRs that signiﬁcantly affect SOD and POD activity and the content of soluble protein, MDA and soluble sugar. *, p < 0.05; **, p < 0.01.

As shown in Figure2, the effects of different treatment combinations on the SOD and POD activities and soluble protein, MDA, and soluble sugar content of M. wufengensis were significantly different (p < 0.05). The SOD activities with treatment Nos. 3 (780.6 U·mg−1) and 2 (757.5 U·mg−1) were significantly increased compared with CK. The SOD activity of M. wufengensis with treatment No. 3 was the strongest and was 66.0% higher than that in CK (470.1 U·mg−1) (Figure2a). Figure2b shows that the POD activity of M. wufengensis with treatments No. 4 (316.5 U·g−1·min−1), No. 6 (335.3 U·g−1·min−1), and No. 8 (285.9 U·g−1·min−1) was not significantly different from CK (306.4 U·g−1·min−1)(p > 0.05), and the other treatments yielded results that were significantly increased compared with CK (p < 0.05). The POD activity of M. wufengensis with treatment No. 3 was the strongest (566.9 U·g−1·min−1), which was 85.0% higher than CK. Figure2c shows that the soluble protein content of M. wufengensis with treatments No. 1 (10.6 mg·g−1) and No. 6 (17.8 mg·g−1) was not significant compared with CK (15.5 mg·g−1)(p > 0.05), and the re- maining treatments yield significantly higher values than CK (p < 0.05). The soluble protein content with treatment No. 9 was the highest (39.5 mg·g−1), which was 154.8% higher than CK. The soluble protein content treatment No. 3 (27.8 mg·g−1) was significantly increased by 43.3% compared with CK (p < 0.05). Figure2d shows that compared with CK (17.6 umol·g−1), different treatment combinations could significantly reduce the MDA content of M. wufengensis (p < 0.05). Treatment No. 3 (11.9 umol·g−1) exhibited the lowest MDA content, which was 32.1% lower than the CK. Figure2e shows that treatment No. 1 (5.2 mmol·g−1), No. 4 (5.2 mmol·g−1) and No. 7 (5.5 mmol·g−1) were not significantly different from CK (5.0 mmol·g−1)(p > 0.05), and the other treatments were significantly higher than CK (p < 0.05). The soluble sugar content was the highest treatment No. 3 (6.4 mmol·g−1), which was increased by 27.6%, compared with CK. In summary, compared with CK, spraying 1500 ppm uniconazole five times with treatment No. 3 increased superoxide dismutase (SOD) and peroxidase (POD) activities in grafted seedlings of M. wufengensis by 66.0% and 85.0%, respectively. The soluble protein content increased by 43.3%, soluble sugar content increased by 27.6%, and malondialdehyde (MDA) content decreased by 32.1%.

3.3. Dwarﬁng Effects of PGRs on Endogenous Hormones The variety, concentration, and number of applications of PGRs had different effects on the four endogenous hormones GA3, IAA, ABA, and ZTs as shown in Table4 and Figure3.

Table 4. Range analysis of the L9 (34) orthogonal experimental design for endogenous hormones.

Range GA3 IAA ABA ZT Variety 13.6 3.1 * 0.9 0.6 Concentration 70.1 ** 7.2 ** 3.0 ** 5.0 ** Times 63.0 ** 6.3 ** 3.6 ** 2.0 ** Note: * indicates the different varieties, concentrations and times of PGR treatments that signiﬁcantly affect the content of GA3, IAA, ABA and ZT. *, p < 0.05; **, p < 0.01. Forests 2021, 12, x FOR PEER REVIEW 9 of 18

3.3. Dwarfing Effects of PGRs on Endogenous Hormones The variety, concentration, and number of applications of PGRs had different effects on the four endogenous hormones GA3, IAA, ABA, and ZTs as shown in Table 4 and Fig- ure 3.

Table 4. Range analysis of the L9 (34) orthogonal experimental design for endogenous hormones.

Range GA3 IAA ABA ZT Variety 13.6 3.1 * 0.9 0.6 Concentration 70.1 ** 7.2 ** 3.0 ** 5.0 **

Forests 2021, 12, 19 Times 63.0 ** 6.3 ** 3.6 ** 2.0 ** 9 of 17 Note: * indicates the different varieties, concentrations and times of PGR treatments that significantly affect the content of GA3, IAA, ABA and ZT. *, p < 0.05; **, p < 0.01.

24 200 (b) (a) 20 a a a )

a −1 ) 160 ab −1 b 16 b b 120 12 c c d cd cde 80 e e 8 content(μg·g de de e 3 f g IAA content(μg·g GA 40 4 0 0 12345678910 12345678910 Treatment number Treatment number

16 20 (c) (d) 14 a a ) 16 a )

−1 12 −1 b b b b 10 b 12 b c cd cd 8 cd bc cd de cd 8 e de 6 d

ZT content(μg·g 4

ABA content(μg·g 4 2 0 0 12345678910 12345678910 Treatment number Treatment number

FigureFigure 3. The 3. The effects effects of PGRs of PGRs on GAon 3GA(a),3 ( IAAa), IAA (b), (b ABA), ABA (c) and(c) and ZT ZT (d) ( ind)M. in wufengensisM. wufengensis..

BasedBased on on the the range range analysis analysis results results shown shown in in Table Table4, 4, the the concentration concentration and and times times of of PGR treatments had extremely significant significant effe effectscts on on GA3, GA3, IAA, IAA, ABA, ABA, and and ZT ZT levels levels (p < (p <0.01), 0.01 ),and and the the effects effects of ofboth both variables variables were were greater greater than than that that noted noted for forthe thevariety variety of PGRs, of PGRs,which which only only had hada significant a significant effect effect on IAA on content IAA content (p < 0.05). (p < 0.05).Among Among them, the them, influence the influenceof PGR of concentration PGR concentration on GA3, on IAA GA3, and IAA ZT and content ZT content was greater was greater than thanthat thatnoted noted for the fornumber the number of applications, of applications, and and the theinfluence influence of ofZT ZT content content was was less less than than that that noted noted for for the thenumber number of of applications. applications. As noted in Figure3a, different PGR treatments had different effects on the GA As noted in Figure 3a, different PGR treatments had different effects on the GA3 con-3 content of M. wufengensis. Among them, the GA content of M. wufengensis with treat- tent of M. wufengensis. Among them, the GA3 content3 of M. wufengensis with treatment ment Nos. 2 (34.5 µg·g−1), 3 (78.1 µg·g−1), and 9 (47.0 µg·g−1) was significantly reduced Nos. 2 (34.5 μg·g−1), 3 (78.1 μg·g−1), and 9 (47.0 μg·g−1) was significantly reduced compared compared with the other treatments (p < 0.05). The GA3 content of M. wufengensis with with the other treatments (p < 0.05). The GA3 content of M. wufengensis with treatment No. treatment No. 3 was the lowest, which was 73.0% lower than CK (128.0 µg·g−1). Figure3b 3 was the lowest, which was 73.0% lower than CK (128.0 μg·g−1). Figure 3b shows that shows that different PGR treatment combinations had different effects on the IAA content different PGR treatment combinations had different effects on the IAA content of M. of M. wufengensis. The IAA content with treatment Nos. 3 (5.7 µg·g−1), 5 (5.2 µg·g−1), 6 (4.9 µg·g−1), and 9 (7.8 µg·g−1) was significantly reduced by 58.0%, 61.9%, 63.5%, and 42.1%, respectively, compared with that of the CK (13.5 µg·g−1), and the difference between them was not significant (p > 0.05). Figure3c shows that different PGR treatment combinations had different effects on the ABA content of M. wufengensis. With the exception of the ABA content of M. wufengensis with treatment Nos. 1 (6.4 µg·g−1) and 4 (7.2 µg·g−1), which was not significantly different from CK (7.1 µg·g−1), the ABA contents of the other treatments were significantly greater compared with CK. Treatment No. 3 had the highest ABA content (14.0 µg·g−1), which was 98.1% higher than CK. Figure3d shows that different PGR treatment combinations had different effects on the ZT content of M. wufengensis. Compared with CK (11.9 µg·g−1), except for treatment No. 1 (12.1 µg·g−1), which was not significantly different, the others were significantly lower than the value noted for CK. Treatment No. 3 (3.5 µg·g−1) had the lowest ZT content, which was 70.6% lower than that noted for CK. In summary, compared with CK, No. 3 treatment (spraying 1500 ppm Forests 2021, 12, x FOR PEER REVIEW 10 of 18

wufengensis. The IAA content with treatment Nos. 3 (5.7 μg·g−1), 5 (5.2 μg·g−1), 6 (4.9 μg·g−1), and 9 (7.8 μg·g−1) was significantly reduced by 58.0%, 61.9%, 63.5%, and 42.1%, respectively, compared with that of the CK (13.5 μg·g−1), and the difference between them was not significant (p > 0.05). Figure 3c shows that different PGR treatment combinations had different effects on the ABA content of M. wufengensis. With the exception of the ABA content of M. wufengensis with treatment Nos. 1 (6.4 μg·g−1) and 4 (7.2 μg·g−1), which was not significantly different from CK (7.1 μg·g−1), the ABA contents of the other treatments were significantly greater compared with CK. Treatment No. 3 had the highest ABA content (14.0 μg·g−1), which was 98.1% higher than CK. Figure 3d shows that different PGR treatment combinations had different effects on the ZT content of M. wufengensis. Com- pared with CK (11.9 μg·g−1), except for treatment No. 1 (12.1 μg·g−1), which was not signif- Forests 2021, 12, 19 10 of 17 icantly different, the others were significantly lower than the value noted for CK. Treat- ment No. 3 (3.5 μg·g−1) had the lowest ZT content, which was 70.6% lower than that noted for CK. In summary, compared with CK, No. 3 treatment (spraying 1500 ppm uniconazole

uniconazole5 times) reduced 5 times) GA3 reduced levels in GA leaves3 levels of M. in wufengensis leaves of M. by wufengensis 73.0%, IAAby by 73.0%, 58.0%IAA and byZT 58.0%content and by ZT 70.6%, content and by increased 70.6%, and the increasedABA content the ABAby 98.1%. content by 98.1%.

3.4.3.4. EffectsEffects ofof DifferentDifferent ConcentrationsConcentrations ofof UniconazoleUniconazole onon MorphologyMorphology ToTo determine determine the the optimal optimal concentration concentration of uniconazole, of uniconazole, a supplementary a supplementary experimental experi- testmental was test conducted was conducted to examine to examine the effects the ofeffe differentcts of different concentrations concentrations of uniconazole of unicona- on M.zole wufengensis on M. wufengensis. According. According to the experimental to the experimental results, different results, concentrationsdifferent concentrations of unicona- of zoleuniconazole had different had different effects on effects the plant on height,the plan sciont height, diameter, scion nodediameter, number, node and number, internodal and lengthinternodal of M. length wufengensis of M. .wufengensis The detailed. The results detailed are shown results in are Figure shown4. in Figure 4.

40 (a) 8 (b) a a a ab ab 30 6 b a a 20 4 Height (cm) Height

10 Scion diameter (mm) 2

0 0 0 1500 2000 2500 0 1500 2000 2500 10 (c) 6 (d) 5 a 8 a a a a a 4 6 a a 3 4 2 Node number Node

2 (cm) length Internodal 1

0 0 0 1500 2000 2500 0 1500 2000 2500

FigureFigure 4. 4.The The effectseffects ofof differentdifferent concentrations concentrations of of uniconazole uniconazole on on height height ( a(a),), internodal internodal length length ( b(b),), scion scion diameter diameter ( c(c),), and and nodenode number number ( d(d)) in inM. M. wufengensis wufengensis. Different. Different small small letters letters indicate indicate signiﬁcant significan differencest differences among among treatments treatments as assessed as assessed by theby Duncan’sthe Duncan’s test test (p < ( 0.05).p < 0.05). Bars Bars represent represent means means± SDs ± SDs (n =(n 3). = 3). The The same same is shownis shown below. below.

According to the experimental results shown in Figure4, the height (Figure4a), scion diameter (Figure4b), node number (Figure4c) and internodal length (Figure4d) of the grafted seedlings of M. wufengensis gradually decreased with increasing uniconazole concentrations. When the sprayed uniconazole concentration was 2500 ppm, the average

plant height was the shortest (20.7 cm), the scion diameter was the smallest (5.1 mm), the node number was the smallest (5.3), and the internodal length was the shortest (3.9 cm) at 70.5%, 81.8%, 80.3%, and 88.1% of the average CK of the control group, respectively. Based on the above indicators, the higher the concentration of uniconazole sprayed, the better is the dwarfing effect. However, most of these morphological indicators were not significantly different (p > 0.05). Therefore, it can be determined that further increases in the concentration of uniconazole would have no significant effect on the morphological indicators. The use of a high concentration of uniconazole for foliar spraying of M. wufengensis caused leaf deformities. When the concentration of uniconazole was 2000 ppm, the newly formed leaves shrank and were lighter and more uneven in color than normal leaves. When 2000 ppm uniconazole was sprayed, adhesions appeared in the front part of some Forests 2021, 12, 19 11 of 17

newborn leaves, and the ornamental nature of M. wufengensis was weakened. In summary, the optimal dwarﬁng concentration of uniconazole is 1500 ppm.

3.5. Effects of Different Concentrations of Uniconazole on Physiology Foliar spraying of different concentrations of uniconazole had different effects on the physiology of grafted seedlings of M. wufengensis. This part of the experiment focused

Forests 2021, 12, x FOR PEER REVIEWon the effects of different concentrations of uniconazole on SOD, POD, soluble protein,12 of 18 malondialdehyde (MDA), and soluble sugar as shown in Figure5.

1000 1000 (a) (b) a ） −1 b c 800 a ） 800 b ·min −1 c −1 d 600 600

d u·mg （ 400 400

200 200 SOD activity/(U·mg POD activity 0 0 0 1500 2000 2500 0 1500 2000 2500

30 12 a a ) (c) (d) −1

25 ) 10 ab −1 b b b b 20 c 8

15 6

10 4

5 MDA content (umol·g 2 Soluble protein content (mg·g content protein Soluble 0 0 0 1500 2000 2500 0 1500 2000 2500

8 (e) ) −1 6 a a b c 4

Soluble sugarcontent ( mmol·g ( sugarcontent Soluble 0 0 1500 2000 2500 Figure 5. The effects of different concentrations of uniconazole on SOD activity (a), POD activity (b), soluble protein content Figure 5. The effects of different concentrations of uniconazole on SOD activity (a), POD activity (b), soluble protein con- (c), MDA content (d) and soluble sugar content (e) in M. wufengensis. tent (c), MDA content (d) and soluble sugar content (e) in M. wufengensis.

3.6. Effects of Different Concentrations of Uniconazole on Endogenous Hormones Foliar spraying with different concentrations of uniconazole had different effects on endogenous hormones in grafted seedlings of M. wufengensis. These experiments explored the effects of different concentrations of uniconazole on GA3, IAA, ABA, and ZT as shown in Figure 6.

Forests 2021, 12, 19 12 of 17

According to the experimental results shown in Figure5, a high concentration of uniconazole caused the SOD activity (Figure5a) and soluble sugar content (Figure5e) of the grafted seedlings of M. wufengensis to initially increase and subsequently decrease, whereas POD activity (Figure5b), soluble protein (Figure5c) and MDA content (Figure5d ) increased. These effects were significant (p < 0.05). When 2000 ppm was applied, the highest SOD activity (855.2 U·mg−1) and soluble sugar content (5.7 mmol·g−1) were noted (182.1% and 128.6% of the control group, respectively). The POD activity (723.7 U·mg−1·min−1), soluble protein (26.5 mg·g−1), and MDA content (9.8 umol·g−1) reached the greatest levels when plants were sprayed with 2500 ppm uniconazole (124.4%, 159.1%, and 125.7% of the control group, respectively). Although the results of the physiological indicators indicated that M. wufengensis was more tolerant to uniconazole, spraying 2000 ppm uniconazole caused the leaves of M. wufengensis to be deformed in the experiment assessing the concentration of uniconazole. Therefore, considering the morphological indicators and given that dwarfing was mainly performed for the ornamental value of M. wufengensis, the optimal dwarfing concentration of uniconazole for M. wufengensis was 1500 ppm in this study.

3.6. Effects of Different Concentrations of Uniconazole on Endogenous Hormones Foliar spraying with different concentrations of uniconazole had different effects on endogenous hormones in grafted seedlings of M. wufengensis. These experiments explored Forests 2021, 12, x FOR PEER REVIEWthe effects of different concentrations of uniconazole on GA3, IAA, ABA, and ZT as13 shown of 18 in Figure6.

300 14 a (a) a (b) 12 250 ) )

−1 b

−1 10 200 c b 8 d 150 c 6 content (μg·g content

3 100 4 IAA content (μg·g content IAA GA d 50 2

0 0 0 1500 2000 2500 0 1500 2000 2500

4 16 (c) a (d)

) a )

−1 3 12 −1 b b 2 8 c c d d

1 (μg·g content ZT 4 ABA content (μg·g ABA content

0 0 0 1500 2000 2500 0 1500 2000 2500

FigureFigure 6. 6.The The effects effects ofof differentdifferent concentrationsconcentrations of uniconazole on on GA GA3 3(a(),a ),IAA IAA (b), (b ABA), ABA (c) (andc) and ZT ZT(d) (contentd) content in M. in M.wufengensis wufengensis. .

AccordingAccording toto the the experimental experimental results results shown shown in Figure in Figure 6, the6, contents the contents of GA of3 (Figure GA 3 (Figure6a), IAA6a), (Figure IAA ( 6b),Figure and6 bZT), (Figure and ZT 6d) (Figure in M.6 wufengensisd) in M. wufengensis gradually decreasedgradually as decreased the con- ascentration the concentration of uniconazole of uniconazole increased, increased,whereas the whereas content the of contentABA (Figure of ABA 6c) (Figuregradually6c) graduallyincreased increased as the concentration as the concentration of uniconazole of uniconazole increased. increased. The difference The difference betweenbetween the different concentrations of uniconazole was significant. Using 2500 ppm uniconazole, the contents of GA3 (49.9 μg·g−1), IAA (6.6 μg·g−1), and ZT (4.7 μg·g−1) were the lowest, whereas the contents of ABA (2.9 μg·g−1) were the highest (20.2%, 55.2%, 33.6%, and 221.8% of the average content of the control group, respectively). Further increases in the uniconazole concentration had similar effects on endogenous hormones as the original concentration. These findings illustrate that the lower the GA3, IAA, and ZT content, the greater is the ABA content. However, when 2000 ppm uniconazole is applied, it caused the leaves of M. wufengensis to become deformed. Therefore, the optimal dwarfing concentration of uniconazole for M. wufengensis was 1500 ppm.

4. Discussion 4.1. Dwarfing Effects of PGRs on Morphology The dwarfing of tree species is an important method that is used for the commercial production of ornamental plants [40]. Plant dwarfing technology is a comprehensive and complex system that involves multiple factors, and the use of plant growth retardants can promote the dwarfing of many plants [15,16]. Plant dwarfing is not only related to the genetic characteristics of the plant itself, but also to the variety, concentration and number of applications of PGRs [22,32,33]. M. wufengensis is a tall tree species that is typically difficult to dwarf [1,2]. This study shows that the variety and concentration of PGRs have a significant impact on plant height, internode length and scion diameter of M. wufengensis. After spraying 500 ppm uniconazole five times, the optimal plant dwarfing effect was obtained. Compared with CK, the plant height, internode length, scion diameter and node

Forests 2021, 12, 19 13 of 17

the different concentrations of uniconazole was significant. Using 2500 ppm uniconazole, −1 −1 −1 the contents of GA3 (49.9 µg·g ), IAA (6.6 µg·g ), and ZT (4.7 µg·g ) were the lowest, whereas the contents of ABA (2.9 µg·g−1) were the highest (20.2%, 55.2%, 33.6%, and 221.8% of the average content of the control group, respectively). Further increases in the uniconazole concentration had similar effects on endogenous hormones as the original concentration. These findings illustrate that the lower the GA3, IAA, and ZT content, the greater is the ABA content. However, when 2000 ppm uniconazole is applied, it caused the leaves of M. wufengensis to become deformed. Therefore, the optimal dwarfing concentration of uniconazole for M. wufengensis was 1500 ppm.

4. Discussion 4.1. Dwarfing Effects of PGRs on Morphology The dwarfing of tree species is an important method that is used for the commercial production of ornamental plants [40]. Plant dwarfing technology is a comprehensive and complex system that involves multiple factors, and the use of plant growth retardants can promote the dwarfing of many plants [15,16]. Plant dwarfing is not only related to the genetic characteristics of the plant itself, but also to the variety, concentration and number of applications of PGRs [22,32,33]. M. wufengensis is a tall tree species that is typically difficult to dwarf [1,2]. This study shows that the variety and concentration of PGRs have a significant impact on plant height, internode length and scion diameter of M. wufengensis. After spraying 500 ppm uniconazole five times, the optimal plant dwarfing effect was obtained. Compared with CK, the plant height, internode length, scion diameter and node number were reduced by 56.9%, 62.6%, 72.8%, and 74.4%, respectively, and these effects were also better than those noted with the paclobutrazol and prohexadione calcium. Cohen found that uniconazole more effectively reduced the height of tree trunks than paclobutrazol during the process of spraying two PGRs on date palm seedlings, thereby dwarfing plants for intensive management [41]. Gao Shurui studied the effects of different concentrations of uniconazole on the growth of Salvia miltiorrhiza and found that S. miltiorrhiza plants were significantly dwarfed after foliar sprays of uniconazole, which mainly manifested in a decrease in plant height, plant width, and height-to-diameter ratio [42]. Roux and GH Barr used different concentrations of paclobutrazol, uniconazole, and prohexadione calcium to spray the grafted seedlings of Citrus lemon, and the results showed that spraying of uniconazole can shorten the internode length by 50% and new shoots by 34%, and the plants were obviously dwarfed [42]. The results of this paper are similar to the above studies. The plant growth retardant mainly reduced the plant height and shortened the internode length to achieve plant dwarfing.

4.2. Dwarfing Effects of PGRs on Physiology Plant growth retardants effectively increase the content of osmotic adjustment sub- stances, increase the activity of antioxidant enzymes, delay the peroxidation of membrane lipids, and delay protein decomposition, thereby improving plant resistance and disease resistance [43,44]. In this study, treatment No. 3 showed the best treatment combination for dwarfing grafted seedlings of M. wufengensis obtained in the PGR dwarfing experiment is (spraying 1500 ppm uniconazole 5 times). Compared with the control, this treatment increased the activity of SOD by 66.0%, the activity of POD by 85.0%, the content of soluble protein by 43.3%, and the content of soluble sugar by 27.6%, and it decreased MDA content by 32.1%. In the supplementary experiment assessing the uniconazole concentration, it was also found that an excessively high concentration of uniconazole resulted in further increases in SOD and POD activity as well as the content of soluble protein and soluble sugar and reduced the MDA content. SOD is a common biological enzyme that primarily functions to scavenge oxygen free radicals. Therefore, the level of SOD activity reflects the resistance of a plant to a certain extent. Higher SOD activity indicates that the plant is more capable of coping with environmental stress, disease, and senescence [45–47]. POD is an oxidoreductase that is closely related to plant respiration and photosynthesis. According Forests 2021, 12, 19 14 of 17

to previous research results, the higher the POD activity, the stronger is a plant’s ability to cope with adverse environments [48]. Soluble protein and soluble sugar have important effects on plant physiology and are closely related to plant stress resistance [49]. When plants experience stress, they can adapt by increasing the soluble protein content [50]. Increasing the soluble sugar content can improve plant cold resistance [51]. Excessive MDA content indicates a high degree of damage to the plant membrane system [52]. These ﬁve physiological indicators can jointly explain the strong ability of M. wufengensis to cope with adverse environmental conditions. Zhou Xiulin et al. used uniconazole to treat Chlorophytum capense within a certain concentration range and at various application times. These researchers demonstrated that uniconazole enhances antioxidant enzyme activity (including CAT, SOD, POD), reduces MDA content, and increases soluble protein and chlorophyll contents, thereby enhancing the stress resistance of C. capense [53]. Many studies have also demonstrated that spraying an appropriate concentration of uniconazole can delay plant growth, increase chlorophyll levels, increase leaf soluble sugar and soluble protein contents, promote antioxidant enzyme activity, enhance the cell’s ability to remove active oxygen, reduce the relative permeability of the plasma membrane, and improve plant resistance to stress [54–56]. Therefore, in this study, the application of uniconazole to the grafted seedlings of M. wufengensis not only dwarfed the plant but also improved the stress resistance of M. wufengensis.

4.3. Effects of PGRs on Endogenous Hormones PGRs are a class of artificially synthesized hormones that are antagonists of GA, which can delay plant growth. The mechanism of PGRs mainly involves hindering the activity of certain enzymes during the synthesis of GA, which reduces GA levels in plants and thus inhibits cell elongation in the elongation zone between the stems, shortens the internode length, and achieves the effect of dwarfing in plants [57]. Some of the precursors of GA are closely related to IAA, ABA, CAT, and ethylene (ETH), so changes in GA content will affect the overall endogenous hormone levels of the plant [58]. Studies have demonstrated that uniconazole can reduce the formation of GA synthesis precursor materials by reducing the activity of kaurene oxidase, thereby inhibiting the biosynthesis of GA, reducing the level of endogenous GA, and reducing the content of IAA while increasing the content of ABA [59]. Yang Weiru’s research shows that the plant growth retardant chlormequat effectively inhibits the synthesis of gibberellin GA3, IAA, and ZT and simultaneously affects the ABA content, thereby dwarfing the plant height of Sanguisorba officinalis [60]. Li Ningyi and Liu Bing used uniconazole to treat Lilium Asiatic Hybrids and found that it could significantly reduce the content of IAA and GA3 and increase the content of zeatin nucleoside (ZR) and ABA. In addition, the effect gradually increased with an increasing treatment concentration, thereby inhibiting the elongation of plant cells and resulting in lower plant height [61]. Therefore, in this study, spraying uniconazole on M. wufengensis reduced the content of GA3, IAA, and ZT and increased the content of ABA in leaves, which is consistent with previous studies. Changes in endogenous hormone levels in plants are related to physiology. For example, a large amount of ABA in a plant can cause the stomata of the plant to close, reduce transpiration, enhance the water retention capacity of leaves, improve the survival rate of the plant under stress, affect the contents of SOD, POD, MDA, soluble protein and soluble sugar, and reduce damage caused by drought or low-temperature stress [62,63]. The ABA content is different for different types of plants, and plants with strong cold resistance have a higher ABA content than plants with weak cold resistance. Moreover, studies have found that GA3 content is related to plant cold resistance, plants with strong cold resistance have lower GA3 levels, and plants with weak cold resistance have higher GA3 content [64]. SOD, POD, MDA, soluble protein, and soluble sugar levels are widely considered to be related to cold and drought resistance. Furthermore, plant endogenous hormones dynamically change, and the content is different at different times and in different parts in different growth stages. Concurrently, there are complex antagonistic and promoting effects between Forests 2021, 12, 19 15 of 17

different hormones that are affected by external conditions. The growth and development of plants are mainly affected by the synergistic effects of various hormones [58]. The changes in content of the four endogenous hormones in this experiment are consistent with the changes in morphological and physiological indicators of M. wufengensis. Therefore, it can be concluded that PGRs change the endogenous hormone level in M. wufengensis grafted seedlings, thereby changing the physiology and the consequent morphology and dwarfing the plant. The seedlings used in this experiment were all 1-year-old grafted seedlings of M. wufengensis JiaoHong No. 2. The rootstock of the grafting seedlings was Magnolia biondii with the same two-year-old specification. It should be noted that grafting is also a dwarfing measure [9]. Although M. biondii is a tree, it exhibits a certain dwarfing effect as a M. wufengensis grafting rootstock. Therefore, according to the characteristics of graft dwarfing, M. wufengensis can choose appropriate rootstock grafting to achieve further dwarfing. Graft dwarfing has also been studied by researchers, and a dwarfing approach that is suitable for M. wufengensis will be found in the future. Judging from the current results, the dwarfing effect of PGRs is significant.

5. Conclusions The optimal combination for dwarfing found in the PGR experiments was spraying 1500 ppm uniconazole five times. This treatment combination successfully dwarfed the grafted seedlings of M. wufengensis and enhanced its stress resistance. Uniconazole supplementation results showed that the optimal uniconazole concentration was 1500 ppm. When the uniconazole concentration was 2000 ppm or greater, the new leaves were deformed, and shrinkage or adhesion deformation occurred. Therefore, considering the morphological indicators and given that dwarfing is mainly performed for the ornamental value of M. wufengensis, the best measure for dwarfing M. wufengensis is to spray 1500 ppm uniconazole five times.

Author Contributions: Data curation, X.S. and S.C.; Formal analysis, X.S.; Funding acquisition, Z.J.; Investigation, X.S. and S.C.; Writing—original draft, X.S.; Writing—review & editing, Z.J. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by Special Fund for Forest Scientific Research in the Public Welfare under grant no. 201504704 and the Transformation and application of forestry intellectual property project under grant no. Intellectual property transformation 2017-11. Data Availability Statement: The data presented in this study are available on request from the corresponding author. The data are not publicly available due to ready to combine other data for further analysis and research. Conflicts of Interest: The authors declare that they have no conflict of interest.

References 1. Ma, L.Y.; Wang, L.R.; He, S.C.; Liu, X.; Wang, X.Q. A new species of Magnolia (Magnoliaceae) from Hubei, China. Bull. Bot. Res. 2006, 26, 4–7. 2. Ma, L.Y.; Wang, L.R.; He, S.C.; Liu, X.; Wang, X.Q. A new variety of Magnolia (Magnoliaceae) from Hubei, China. Bull. Bot. Res. 2006, 26, 516–519. 3. Liang, D.W. Select on Superior Tree Magnolia wufengensis and Cultivar Classification; Forestry University: Beijing, China, 2010. 4. Sang, Z.Y.; Ma, L.Y.; Chen, F.J.; Zhang, P.; Zhu, Y.C. Protection status and utilization countermeasure of germplasm resources of the Magnolia wufengensis in Wufeng County. Hubei Agric. Sci. 2011, 50, 1564–1567. 5. Shi, X.D.; Ma, L.Y.; Duan, J.; Sang, Z.Y.; Zhu, Z.L.; Yao, N.; Zhou, M.M.; Jia, Z.K. Potential climatically adaptive regions for the introduction of Magnolia wufengensis. J. Zhejiang A F Univ. 2018, 35, 705–715. 6. Wang, B.; Ma, Y.; Chen, G.; Li, C.; Dao, Z.; Sun, W. Rescuing Magnolia Sinica (Magnoliaceae), a critically endangered species endemic to Yunnan, China. Oryx 2016, 50, 446–449. 7. Bai, L.J.; Yin, S.X. Progress on Origins and Mechanism of Dwarfish Plants Mutant. Biotechnol. Bull. 2014, 6, 34–39. 8. Qin, S.Q.; Yang, J.J. High-yield cultivation techniques for Juglans regia dwarfing and dense planting. Deciduous Fruits 2015, 47, 46–48. 9. Yang, F.Y.; Guan, H.C.; Zhang, Z.; Hao, R.M.; Dong, S.J. Introduction to several apple dwarf rootstocks. Deciduous Fruits 2014, 46, 53–54. Forests 2021, 12, 19 16 of 17

10. Huang, J.S.; Wu, Z.W.; Yu, H.G. Dwarfing effect of large branch pruning of Myrica tinctorius in Dingdai county. South China Fruits 2009, 38, 16–17. 11. Chen, Z.M.; Du, G.J.; Hu, W.B.; Chen, W.; Zhang, Y.X.; Ying, Y.D. Effect of Plant Growth Regulators on Dwarfing of Potted Osmanthus fragrans. J. Zhejiang For. Sci. Tech. 2012, 32, 53–56. 12. Zhang, Y.Y.; Yu, M.L.; Ma, R.J.; Cai, Z.X.; Shen, Z.J.; Xu, J.L. Identification of SRAP markers linked to peach gene with the character of tall normal/brachytic dwarf. J. Fruit Sci. 2011, 28, 586–590. 13. Zhao, Y.L.; Li, C.C.; Chen, X. Effect of 60Co-γ Radiation on the Dwarfing Mechanism of Rhododendron decorum. North. Hortic. 2012, 19, 48–50. 14. Huang, H.Y.; Zhong, F.L.; Zhang, F.Y.; Cao, M.H.; Yang, B.Y.; Lin, Y.Z. Paclobutrazol application in horticultural plants. Subtrop. Agric. Res. 2011, 7, 37–41. 15. Cui, H.Y.; Huang, X.G.; Xin, B.J.; Zhang, Q.Y.; Yu, H.X.; Liu, Q.D. The use of Paclobutrazol on the young Apple trees. J. Fruit Sci. 1996, 13, 84–87. 16. Wei, M.B.; Wei, R.X. Paclobutrazol Application in Fruit-tree. Beijing Agric. 2009, 9, 51–54. 17. Pan, R.C. Physiological and applications of the plant growth retarders. J. S. China Norm. Univ. Nat. Sci. 1984, 2, 121–129. 18. Kumawat, G.L.; Swaroop, K.; Janakiram, T.; Jain, R. Induction of dwarfing in bougainvillea cv Mahara by use of growth regulators. Indian J. Agric. Sci. 2014, 84, 802–807. 19. Porlingis, I.C.; Voyiatzis, D.G. Influence of paclobutrazol plant growth regulator on vegetative and reproductive growth of olive (Olea Europaea L.). Acta Hortic. 1986, 179, 587–588. 20. Aron, Y.; Monselise, S.P.; Goren, R.; Costo, J. Chemical control of vegetative growth in Citrus trees by paclobutrazole. HortScience 1985, 20, 96–98. 21. Chen, S.H.; Lin, X.P.; Chen, Y.; Zheng, Y.N. The influence of dminozide and paclobutrazol of Sapindus mukorossi Gaertn growth. J. Ningde Normal Univ. Nat. Sci. 2014, 1, 7–10. 22. Wang, G.; Xiao, J.Z.; Li, Z.B.; Bai, X.X. The influence of paclobutrazol on physiological characteristics of Rhododendron hybrides leaves. J. Agric. Univ. Hebei 2010, 33, 50–53. 23. Wang, X.Y.; Ma, C.Z.; Li, X.D. Effects of paclobutrazol on content of polyamines in peanut leaves and leaf senescence. Acta Bot. Boreal. Occident. Sin. 2001, 21, 323–328. 24. Xu, K.; Li, S.Z. The effects of irrigation and PP333 on the growth and seed yield of Agropyron smithii. Pratacult. Sci. 2011, 28, 1291–1295. 25. Wei, S.L.; Chang, L.B.; Wang, Z.J.; Wang, X.N. Inhibitory effect of the preliminary test of Paclobutrazol on the growth of new shoots of persimmon. Sci. Tech. Vis. 2015, 2, 385–386. 26. Gao, S.R.; Zhao, Z.G.; Hou, J.L.; Wang, W.Q.; Song, Y.; Yan, B.B.; Jin, Y.Q. Effects of plant growth regulator uniconazole on plant morphology and biomass allocation of Salvia miltiorrhiza. China J. Chin. Mater. Med. 2015, 40, 1925–1929. 27. Wheaton, T.A. Triazole bioregulators reduce internode length and increase branch angle of Citrus. Acta Hortic. 1989, 239, 277–280. [CrossRef] 28. Yang, W.Y.; Han, H.F.; Ren, W.J.; Zhao, L.; Fan, G.Q. Effects of Uniconazole Waterless-dressing Seed on Endogenous Hormones and C/N Ratio at Tillering Stage of Wheat. Acta Agron. Sin. 2005, 31, 760–765. 29. Bakheta, M.A.; Hussein, M.M. Uniconazole effect on endogenous hormones, proteins and proline contents of barley plants (Hordium vulgare) under salinity stress (NaCl). Nusant. Biosci. 2014, 6, 2087–3948. 30. Tao, L.X.; Wang, X.; Yu, M.Y.; Huang, X.L.; Xu, R.S. Effects of environmental conditions on degradation of S-07 and PP333 in soil. Acta Agric. Zhejiangensis 1997, 9, 246–250. 31. Ouzounidou, G.; Ilias, I.; Giannakoula, A.; Papadopoulou, P. Comparative study on the effects of various plant growth regulators on growth, quality and physiology of Capsicum annuum L. Pak. J. Bot. 2010, 42, 805–814. 32. Kang, S.M.; Kim, J.T.; Hamayun, M. Influence of prohexadione calcium on growth and gibberellins content of Chinese cabbage grown in alpine region of South Korea. Sci. Hortic. 2010, 125, 88–92. [CrossRef] 33. Andreotti, C.; Costa, G.; Bucchi, F.; Bazzi, C. Growth regulation and reduced fire blight incidence in pear induced by Prohexadione- Ca [Pyrus communis L.-Emilia-Romagna]. Atti V Giornate Sci. SOI 2000, 10, 71–86. 34. Pan, M.J.; Yin, Y.Q.; Shen, F.K.; Luo, B.X.; Tang, X.L.; He, H.H.; Chen, D.K. Effects of prohexadione calcium on physiological index of Tobacco seedlings during cold stress. Southwest China J. Agric. Sci. 2016, 29, 288–293. 35. Zhao, S.J.; Liu, H.S.; Dong, X.C. Plant Physiology Experiment Guide; China Agricultural Press: Beijing, China, 1997. 36. Zhang, Z.L.; Qu, W.J. Experiment Guide for Plant Physiology, 3rd ed.; Higher Education Press: Beijing, China, 2003. 37. Gao, J.F. Experimental Guidance for Plant Physiology; Higher Education Press: Beijing, China, 2006. 38. Li, H.S. Principles and Techniques of Plant Physiological Biochemical Experiment; Higher Education Press: Beijing, China, 2000; pp. 194–197. 39. Xiao, A.H.; Chen, F.J.; Jia, Z.K.; Sang, Z.Y.; Zhu, Z.L.; Ma, L.Y. Determination of 4 plant hormones in Magnolia wufengensis by gradient elution high performance liquid chromatography. Chin. J. Anal. Lab. 2020, 39, 249–254. 40. Wójcik, P. Response of ‘red delicious’ apple trees drip-fertigated with ammonium nitrate to application of silicic acid. Sci. Hortic. 2019, 249, 15–21. [CrossRef] 41. Cohen, Y.; Aloni, D.D.; Adur, U.; Hazon, H.; Klein, D.J. Characterization of growth-retardant effects on vegetative growth of date palm seedlings. J. Plant Growth Regul. 2013, 32, 533–541. [CrossRef] Forests 2021, 12, 19 17 of 17

42. Roux, S.L.; Barry, G.H. Vegetative growth responses of citrus nursery trees to various growth retardants. Hortic. Tech. 2010, 20, 197–201. [CrossRef] 43. Mao, Y.Q.; Zheng, Q.S.; Chen, J.M.; Liu, Z.P.; Liu, G.H.; Jiang, C.Q. Physiological mechanism of foliage spraying paclobutrazol on increasing salt tolerance of Jatropha curcas Seedlings. Acta Ecol. Sin. 2011, 31, 4334–4341. 44. Xiang, J.L.; Huang, Y.L.; Yin, K.D.; Liu, Z.B.; Xu, Y.M.; Feng, J.S.; Ren, H.J. Effect of S3307 on anti-oxidant enzyme activity and DNA methylation level of coix seedlings under drought stress. Chin. J. Biol. 2017, 29, 264–270. 45. Qin, P.; Liu, F.H.; Liang, X.N. Superoxide dismutase and plant resistance to the environmental stress. Heilongjiang Agric. Sci. 2002, 1, 31–34. 46. Du, X.M.; Yin, W.X.; Zhang, H.; Zhao, Y.X. The Researching Progress of Superoxide Dismutase. J. Chin. Biotechnol. 2003, 23, 48–50. 47. Dou, J.H.; Yu, S.X.; Fan, S.L.; Pang, C.Y.; Song, M.Z. Sod and plant stress resistance. Mol. Plant Breed. 2010, 8, 359–364. 48. Chen, Y.L.; Cao, M.; Li, Y.Y.; Zhou, Y. Effect of Fulvic Acid on ABA, IAA and Activities of Superoxide Dismutase and Peroxydase in Winter Wheat Seedling Under Drought Condition. Plant Physiol. Commun. 2000, 36, 311–314. 49. Zuo, W.B.; Wu, J.L.; Yang, Q.; Zhang, J.N.; Liu, G.R. Study on the influence of root of different wheat varieties under drought stress. Acta Agric. Boreal. Occident. Sin. 2010, 25, 191–193. 50. Zhang, M.S.; Xie, B.; Tan, F.; Zhang, Q.T. Relationship Among Soluble Protein, Chlorophyll and ATP in Sweet potato Under Water Stress with Drought Resistance. Sci. Agric. Sin. 2003, 36, 13–16. 51. Lin, Y.; Guo, W.Z.; Xu, Z.H.; Jia, Z.X. Cold Resistance and Changes on MDA and Soluble Sugar of Leaves of Ligustrunlucidum Ait in Winter. Chin. Agric. Sci. Bull. 2012, 28, 68–72. 52. Li, Y.B.; Yang, S.Q.; Ren, G.X.; Feng, Y.Z.; Zhang, Q.; Li, P. Changes analysis in physiological properties of several gramineous grass species and cold -resistance comparison on under cold stress. Acta Ecol. Sin. 2009, 29, 1341–1347. 53. Zhou, X.L. Studies on the Dwarfing Effect of PP333 and SS3307 to Chlorophytum Capense ‘Vittatum’; Zhejiang University: Hangzhou, China, 2017. 54. Nie, L.; Xie, J.B.; Liang, Y.M. Improve cold-resistance of Zoysia tenuifolla in winter by plant growth retardants. Pratacul. Sci. 2003, 20, 63–65. 55. Wang, H.S.; Sun, H.M. The Research on Plant Growth Retardants Improving Drought Resistance of Solanum Integrifolium Poir. Chin. Agric. Sci. Bull. 2012, 28, 126–132. 56. Liu, G.; Fei, Y.J.; Tu, M.; Ding, Z.L. Effects of CCC and DPC on the cold resistance of phoebe bournei. Hubei Agric. Sci. 2015, 54, 1403–1406. 57. Grossmann, K. Plant growth retardants as tools in physiological research. Physiol. Plantarum. 1990, 78, 640–648. [CrossRef] 58. Wang, Z. Plant Physiology; China Agricultural Press: Beijing, China, 2000; pp. 265–290. 59. Wan, Z.; Kothare, M.V. Uniconazole-induced tolerance of rape plants to heat stress in relation to changes in hormonal levels, enzyme activities and lipid peroxidation. Plant Growth Regul. 1999, 27, 99–104. 60. Yang, W.R.; Zhang, Y.G.; Shi, X.X. Effects of chlormequat on height and endogenous hormones of Sanguisorba officinalis L. J. Agric. Univ. Hebei 2006, 29, 12–15. 61. Li, N.Y.; Liu, B. Effects of Plant Growth Retardant S3307 on the Growth and Endogenous Hormones Contents of Lily and the Correlation Analysis. Acta Agric. Boreal. Occident. Sin. 2010, 19, 153–156. 62. Li, C.Y.; Xu, W.; Liu, L.W.; Yang, J.; Zhu, X.K.; Guo, W.S. Changes of endogenous hormone contents and antioxidative enzyme activities in wheat leaves under low temperature stress at jointing stage. Chin. J. Appl. Ecol. 2015, 26, 2015–2022. 63. Morgan, P.W.; Drew, M.C. Ethylene and plant responses to stress. Physiol. Plantarum. 1997, 100, 620–630. [CrossRef] 64. Luo, Z.R. Relationship between plant hormones and cold resistance. Plant Physiol. Commun. 1989, 3, 1–5.