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Article

Ortet Age Effect, Anatomy and Physiology of Adventitious Rooting in Tilia mandshurica Softwood Cuttings

by
Huaizhi Mu
,
Xuhong Jin
,
Xinyu Ma
,
Anqi Zhao
,
Yuting Gao
and
Lin Lin
*
Key Laboratory of State Forestry Administration on Conservation and Efficient Utilization of Precious and Rare Forest Resource in Changbai Mountain, Forestry College, Beihua University, Jilin 132013, China
*
Author to whom correspondence should be addressed.
Forests 2022, 13(9), 1427; https://doi.org/10.3390/f13091427
Submission received: 19 July 2022 / Revised: 17 August 2022 / Accepted: 2 September 2022 / Published: 5 September 2022
(This article belongs to the Section Forest Ecophysiology and Biology)
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Abstract

:
Tilia mandshurica is recognized as one of the most valuable timber, ornamental and nectariferous tree species, and its cutting propagation is very difficult. To evaluate the influence of ortet age on the rooting of T. mandshurica cuttings and the relationship between the variations of nutrients, enzyme activities, endogenous hormones and the formation of adventitious roots, a series of softwood cuttings of T. mandshurica were collected from 2-, 7-, 15- and 20-year-old healthy ortets, respectively. The rooting percentage, mean root number and total root length were investigated after 60 days of cultivation. Phenotypic and anatomical changes and the variations of organic nutrients, enzyme activities and endogenous hormones were measured during adventitious root formation. The results showed that ortet age effect existed in T. mandshurica softwood cuttings. The adventitious rooting of 2-year-old ortets was superior to that of 7-, 15- and 20-year-old ortets. No root primordium was visible in T. mandshurica softwood cuttings before cultivation, and the root primordia were induced after planting. The adventitious roots derived from vascular cambium and callus in cuttings. Soluble sugar increased during root primordium induction and decreased in adventitious root elongation. Soluble protein decreased during root primordium induction and increased in adventitious root elongation. Polyphenol oxidase (PPO) activity increasing and peroxidase (POD) and 3-indoleacetic acid oxidase (IAAO) activities decreasing were conducive to root primordium induction. High 3-indoleacetic acid (IAA) and gibberellin (GA3) level could promote root primordium induction and low IAA and GA3 level was beneficial to adventitious root elongation. Abscisic acid (ABA) and zeatin riboside (ZR) inhibited root primordium induction and adventitious root elongation. IAA, ABA and ZR coordinated with each other in the process of root primordium induction and adventitious root formation. IAA/ABA increasing could promote root primordium induction, and IAA/ZR increasing was beneficial to root primordium induction and adventitious root elongation. The soluble sugar content, PPO activity, IAA/ABA and IAA/ZR of softwood cuttings from 2-year-old ortets were relatively high, which may be the reason for the better adventitious rooting. The present study provides a reference to build a propagation by cuttings technology of linden trees.

1. Introduction

Tilia mandshurica Rupr. et Maxim., a perennial arbor species belongs to the Tiliaceae family, is mainly distributed in northeastern China, Korean peninsula and southeastern Siberia. This species is recognized as one of the most valuable timber, ornamental and nectariferous plant [1]. Long-term unreasonable logging has led to serious damage to T. mandshurica resource. In recent years, the demand for T. mandshurica saplings is increasing with the rapid development of forestry [2]. Nevertheless, the cultivation of T. mandshurica is seriously limited due to its unique biological characteristics, such as seed deep dormancy and rooting ability of cuttings [3]. Consequently, it is of great significance to research the vegetative propagation of T. mandshurica.
Propagation by cuttings is a method that inserts the isolated plant vegetative organs into a certain substrate. The isolated vegetative organs generate a completely new plant under appropriate conditions according to plant cell totipotency [4]. Compared with grafting and tissue culture, propagation by cuttings has the advantages of high rooting coefficient, simple operation and low cost. Compared with sowing, propagation by cuttings can maintain the excellent characteristics of varieties and early flowering. Therefore, propagation by cuttings is of great significance to the production of forest saplings [5]. Propagation by cuttings can be divided into hardwood cutting and softwood cutting according to the lignification degree of cuttings. Due to the cell division rate is higher in softwood than in hardwood cuttings, the survival rate of softwood cutting is often higher than that of hardwood cutting [6,7]. In recent years, research on softwood cutting has shown that ortet age and nutrients, enzymes and endogenous hormones in cuttings are closely related to adventitious root formation [8,9].
The genus Tilia cuttings hardly produce adventitious roots under natural conditions, and need to be stimulated by external conditions to induce root primordia [10,11,12,13]. In the early stage, we carried out the softwood cutting test of T. mandshurica. The results revealed that the rooting rate of softwood cuttings can reach more than 40% after being treated with exogenous hormones. Nevertheless, the influence of ortet age on the rooting of T. mandshurica cuttings and the relationship between the variations of nutrients, enzyme activities, endogenous hormones and the formation of adventitious roots are not clear. In response to this problem, softwood cuttings from ortets of different ages were collected. By investigating the anatomy and physiology of adventitious rooting, our study deepens our understanding of the relationship between physiological changes and cutting rooting, provides a reference to build a propagation by cuttings technology of linden trees.

2. Materials and Methods

2.1. Plant Materials

The softwood cuttings of T. mandshurica were collected from 2-, 7-, 15- and 20-year-old healthy ortets, respectively, in Jiangmifeng Town Forest Farm (44.03 °N, 126.76 °E), Jilin Province, China, and about 400 cuttings of individuals of the same age use for the experiment in early July 2020. The softwood cutting was a semi-lignified branch with a terminal bud and a half leaf, which were not affected by pests and diseases. The cuttings were cut into 10 cm lengths with diameters of 0.5 cm. Furthermore, the polarity of the cuttings was considered. The upper part should be flat, and the surface of cut should be 1 cm above the bud. The lower part should be an oblique cut and the surface of cut should be 9 cm below the bud. To prevent cuttings from dehydrating by adverse circumstances, 20 cuttings were bundled together and stored in a cellar (temperature: 8 °C, humidity: 50%) to keep them moist for no more than 24 h.

2.2. Experimental Design

The experiment was conducted as a randomized block design at a greenhouse (temperature: 28 °C, humidity: 95%, light conditions: 500 lx in the daytime and dark at night) in Xinshan Nursery Stock Cooperative. The basal regions (2 cm above the cutting site) of the cuttings were submerged in the rooting solution of ABT1 (a mixture containing 0.1 mol/L 1-naphthlcetic acid (NAA) and 0.17 mol/L 3-indoleacetic acid (IAA)) for 2 h. Afterwards, the cuttings were cultivated in a seedbed filled with perlite as growth substrate. During cultivation, 1.25‰ (v/v) carbendazim sprayed and sterilized on the seedbed once a week. Substrate water content (10 cm in depth) was maintained at 80% by auto-spraying device (sprayed water for 40 s per 6 min) during the experimental period. The nursery site was divided into two zones: one zone was subdivided into three similar blocks under the same site conditions, and each treatment consisted of three plots containing 20 cuttings each. After 60 days, surviving cuttings of each treatment were dug out for adventitious root growth analysis; the other zone was used for anatomical and physiological analysis of cuttings.

2.3. Adventitious Root Growth Measurement

Adventitious root growth was determined using routine methods. The total root length was measured using a vernier caliper with 0.01 mm precision. The rooting percentage and mean root number were calculated, respectively, as described by Kang et al. [14].

2.4. Anatomical Analysis

Phenotypic and anatomical changes in cuttings from 7-year-old ortets were investigated in the process of adventitious root formation. Approximately 1 cm segments were taken from the cuttings base at the 0th-, 10th-, 20th-, 30th-, 40th-, 50th- and 60th day during cultivation, respectively, and were immediately fixed in FAA fixative (70% (v/v) ethanol:acetic acid:formalin = 90:5:5) for 48 h. Afterwards, samples were cut into 8 μm slices using the paraffin section method [15]. The slices were stained with safranin O and fast green, and visualized under a microscope (Axioimanger A1, ZEISS, Jena, Germany) using 40× magnification.

2.5. Organic Nutrient Measurements

The basal cortex, phloem and vascular cambium tissues (lowest 1 cm portion) from cuttings were scraped at the 0th-, 10th-, 20th-, 30th-, 40th-, 50th- and 60th day during cultivation, respectively, and were immediately frozen in liquid nitrogen and stored at −80 °C. The frozen cortex tissues were used for organic nutrients, enzyme activities and endogenous hormones measurements. All experiments were conducted with three biological replicates for each sample.
Soluble sugar was extracted with boiling distilled water for 1 h, and was stained with anthrone, ethyl acetate and concentrated sulfuric acid for 1 min. Soluble protein was extracted with distilled water at room temperature for 10 min, and was colored with coomassie brilliant blue G-250 for 3 min. The spectrums of soluble sugar and soluble protein in the extracts were scanned with a spectrophotometer (722, TP, Shanghai, China). Soluble sugar and soluble protein quantifications were performed as described by Zhang and Huang [16] and Mailafia et al. [17].

2.6. Enzyme Activity Measurements

The tissues were ground to homogenate in cold phosphate buffer (50 mmol/L, pH 5.8), and the supernatants were collected by centrifugation (1790× g, 15 min, 4 °C) as enzyme solution. To analyze peroxidase (POD), enzyme solution was added to the reaction solution (phosphate buffer: 0.3% (v/v) hydrogen peroxide: 0.2% (w/v) guaiacol = 1:1:0.95). To analyze polyphenol oxidase (PPO), enzyme solution was added to the reaction solution (phosphate buffer:50 mmol/L pyrocatechol = 3.9:1). To analyze 3-indoleacetic acid oxidase (IAAO), enzyme solution was added to the reaction solution (1 mmol/L manganese chloride: 1 mmol/L 2,4-dichlorophenol: 1 mmol/L 3-indoleacetic acid:phosphate buffer = 1:1:2:5) at 25 °C for 0.5 h, and was colored with ferric chloride and perchloric acid at 30 °C for 0.5 h in dark. The spectrums of POD, PPO and IAAO in the enzyme solution were scanned with a spectrophotometer. The unit of enzyme activity is U·g−1·min−1, which means 1 μmol of substrate was converted in 1 min per gram of sample. Enzyme activities quantifications were performed as described by Zhu and Liao [18].

2.7. Endogenous Hormone Measurements

The tissues were ground to powder in liquid nitrogen, and were extracted in methanol containing 0.01% (w/v) butylated hydroxytoluene overnight at 4 °C. The supernatants were collected by centrifugation (1370× g, 8 min) and dried under a stream of air. Samples were purified using C-18 columns that were flushed with methanol. Enzyme linked immunosorbent assay (ELISA) (Multiskan MK3, Thermo, Waltham, USA) was used to assay phytohormones for 3-indoleacetic acid (IAA), abscisic acid (ABA), zeatin riboside (ZR) and gibberellin (GA3) according to Zeng et al. [19].

2.8. Statistical Analysis

All data were analyzed by analysis of variance, Duncan test and t test. These tests were performed using the SAS software (v9.21, SAS Institute Inc., Raleigh, NC, USA).

3. Results

3.1. Adventitious Rooting in Cuttings from Ortets of Different Ages

Analysis of variance showed extremely significant variations in different ortet ages with regard to measures of rooting percentage, mean root number and total root length (Table 1). The adventitious rooting of 2-year-old ortets was superior to that of 7-, 15- and 20-year-old ortets. The rooting percentage of 2-year-old ortets was 50.00% and 574.66% higher than that of 7- and 15-year-old healthy ortets, respectively (Figure 1a). The mean root number of 2-year-old ortets was 57.78% and 162.32% higher than that of 7- and 15-year-old healthy ortets, respectively (Figure 1b). The total root length of 2-year-old ortets was 78.65% and 472.72% higher than that of 7- and 15-year-old healthy ortets, respectively (Figure 1c). The adventitious rooting of 20-year-old ortets was the worst, and the rooting percentage, mean root number and total root length were all 0. These results indicate that ortet age effect existed in T. mandshurica softwood cuttings. The adventitious rooting of younger ortets was superior to that of elder ortets.

3.2. Phenotypic and Anatomical Changes in Adventitious Root Formation

It takes about 60 days in adventitious root formation. The phenotypic changes in cuttings were continuously monitored during the whole rooting process. Compared with the cuttings before cultivation (Figure 2a), the first macroscopic evidence of root initiation was the appearance of bulges at the surface of cuttings bases after 10 days of cultivation (Figure 2b). Ivory calluses were found at the excision site after 20 days of cultivation (Figure 2c). Young white roots emerged from cortices and calluses 40 days after planting, some of which had grown to about 1 cm long (Figure 2d). During the following weeks, the roots continue growing along with the production of secondary lateral roots. After 60 days of cultivation, the cuttings had developed complete root systems (Figure 2e).
To determine the kinetics of adventitious root formation, anatomical analyses were performed. The softwood cutting was composed of epidermis, cortex, phloem, vascular cambium, xylem and pith. No root primordium was visible in the cuttings before cultivation (Figure 3a), which suggest that root primordia were induced after planting. Transverse sections showed numerous closely arranged root primordium initial cells were formed at the junction of pith rays and vascular cambium after 10 days of cultivation (Figure 3b), and these dividing cells gradually formed root primordia through mitosis (Figure 3c). After 30 days of cultivation, the root primordial cells divided continuously to form adventitious roots (Figure 3d). The adventitious roots elongated and grew out of surrounding tissues 40 days after planting (Figure 3e). In addition, calluses were visible on the inner and surface of cutting bases (Figure 3f,g), which formed adventitious roots after 40 days of cultivation (Figure 3h). These results suggest that adventitious roots derived from vascular cambium and callus in T. mandshurica softwood cuttings.

3.3. Organic Nutrient Variations in Adventitious Root Formation

The cuttings from 15- and 20-year-old ortets showed low adventitious rooting which did not meet the materials requirements for physiological analysis. Consequently, we only measured organic nutrients, enzyme activities and endogenous hormones of cuttings from 2- and 7-year-old ortets. The soluble sugar variation of cuttings from 2- and 7-year-old ortets was similar in rooting process. Nevertheless, the soluble sugar content of cuttings from 2-year-old ortets was significantly higher than that from 7-year-old ortets at the 10th-, 20th-, 30th-, 40th- and 50th day during cultivation. The first drop occurred at the 10th day, and the soluble sugar level of cuttings from 2- and 7-year-old ortets was 21.80 mg·g−1 and 11.58 mg·g−1, respectively. The soluble sugar level continued to rise thereafter and peaked at the 40th day (58.42 mg·g−1 and 51.75 mg·g−1, respectively). The soluble sugar content decreased gradually again at the 50th- and 60th day (Figure 4a).
Regarding to the time zero, the first drop of soluble protein content occurred at the 10th day. There was a gradually decrease in soluble protein content from the 0th- to 30th day, and which from 2-year-old ortets was always significantly lower than that from 7-year-old ortets. The soluble protein level of cuttings from 2- and 7-year-old ortets declined the minimum value at the 30th day (7.39 mg·g−1 and 8.33 mg·g−1, respectively). There was a high increase from the 40th- to 50th day and peaked at the 60th day (14.30 mg·g−1 and 15.67 mg·g−1, respectively) (Figure 4b).

3.4. Enzyme Activity Variations in Adventitious Root Formation

In the rooting process, the POD activity of cuttings from 2-year-old ortets was significantly higher than that from 7-year-old ortets at the 0th-, 10th-, 20th-, 30th-, 40th- and 50th day during cultivation. The POD activity of cuttings from 2- and 7-year-old ortets rose and peaked at the 10th day (881.71 U·g−1·min−1 and 798.65 U·g−1·min−1, respectively; the unit of enzyme activity is U·g−1·min−1). Thereafter, the first drop occurred at the 20th day, and the POD activity declined the minimum value at the 30th day (451.77 U·g−1·min−1 and 435.23 U·g−1·min−1, respectively). The POD activity increased gradually again from the 30th- to 60th day (Figure 5a).
The PPO activity of cuttings from 2-year-old ortets was significantly higher than that from 7-year-old ortets at the 0th-, 10th-, 20th-, 30th-, 50th- and 60th day during cultivation. The PPO activity of cuttings from 2- and 7-year-old ortets peaked at the 30th day (185.69 U·g−1·min−1 and 171.39 U·g−1·min−1, respectively), after gradually increasing from the 0th- to 30th day. After this, the POD activity decreased gradually from the 30th- to 60th day (Figure 5b).
Regarding the time zero, the first drop of IAAO activity occurred at the 10th day. There was a gradually decrease in IAAO activity from the 0th- to 30th day, and which from 2-year-old ortets was always extremely significantly lower than that from 7-year-old ortets. The IAAO activity of cuttings from 2- and 7-year-old ortets declined the minimum value at the 30th day (5.47 U·g−1·min−1 and 6.13 U·g−1·min−1, respectively). The IAAO activity continued to rise afterwards and peaked and at the 60th day (13.33 U·g−1·min−1 and 16.54 U·g−1·min−1, respectively) (Figure 5c).

3.5. Endogenous Hormone Variations in Adventitious Root Formation

The IAA content of cuttings from 2-year-old ortets was always significantly higher than that from 7-year-old ortets. The IAA level of cuttings from 2- and 7-year-old ortets peaked at the 30th day (149.16 ng·g−1 and 138.37 ng·g−1, respectively), after gradually increasing from the 0th- to 30th day. After this, the IAA level decreased gradually from the 30th- to 60th day (Figure 6a).
Regarding to the time zero, the first drop of ABA content occurred at the 10th day. There was a gradually decrease in ABA content from the 0th- to 30th day, and which from 2-year-old ortets was significantly lower than that from 7-year-old ortets at the 0th-, 10th-, 20th-, 30th-, 40th- and 50th day during cultivation. The ABA level of cuttings from 2- and 7-year-old ortets declined the minimum value at the 30th day (63.73 ng·g−1 and 77.98 ng·g−1, respectively). Thereafter, the ABA level was increasing fast at the 40th day and decreased slightly again at the 50th- and 60th day (Figure 6b).
The ZR content showed a downward trend during the first 30 days of rooting, and which from 2-year-old ortets was significantly lower than that from 7-year-old ortets at the 0th-, 10th-, 20th-, 30th- and 40th day during cultivation. The ZR level of cuttings from 2- and 7-year-old ortets decreased gradually 30 days after planting, and rapid rose at the 40th day (13.33 ng·g−1 and 15.62 ng·g−1, respectively). After that, the ZR level was decreasing fast from the 40th- to 50th day and declined the minimum value at the 60th day (4.86 ng·g−1 and 4.80 ng·g−1, respectively) (Figure 6c).
The GA3 content of cuttings from 2-year-old ortets was significantly lower than that from 7-year-old ortets only at the 40th-, 50th- and 60th day during cultivation. There was no significant variation between 2- and 7-year-old ortets 30 days after planting. The GA3 level of cuttings from 2- and 7-year-old ortets peaked at the 30th day (7.62 ng·g−1 and 7.69 ng·g−1, respectively), after gradually increasing from the 0th- to 30th day. Henceforth, the GA3 level decreased gradually and declined the minimum value at the 60th day (3.06 ng·g−1 and 3.65 ng·g−1, respectively) (Figure 6d).
The IAA/ABA increased from the 0th- to 30th day, and which from 2-year-old ortets was always extremely significantly higher than that from 7-year-old ortets. The IAA/ABA level of cuttings from 2- and 7-year-old ortets peaked at the 30th day (2.34 and 1.77, respectively), after gradually increasing from the 0th- to 30th day. After this, the IAA/ABA level decreased gradually from the 30th- to 60th day (Figure 6e).
The IAA/ZR of cuttings from 2-year-old ortets was always extremely significantly higher than that from 7-year-old ortets. The IAA/ZR level of cuttings from 2- and 7-year-old ortets peaked at the 30th day (16.82 and 15.02, respectively), after gradually increasing from the 0th- to 30th day. The IAA/ZR level was decreasing fast afterwards at the 40th day and rapid rose again at the 50th- and 60th day (Figure 6f).

4. Discussion

4.1. Ortet Age Effect on Adventitious Rooting

Ortet age is one of the key factors affecting adventitious rooting. With aging and tree maturation, meristematic cells lose competence for de novo regeneration of roots, and endogenous hormones, enzymes and organic nutrients that promote adventitious rooting are greatly reduced, while the rooting inhibitors are gradually increased, and the rooting percentage of cuttings shows a downward trend. This phenomenon is called the ortet age effect of cuttings [20,21,22]. Research on Picea crassifolia Kom. reported that the rooting percentage of softwood cuttings from 25-year-old ortets was inferior to that of 10- and 15-year-old ortets [14]. In Dalbergia melanoxylon Guill. and Perr., callus formation and adventitious root development of cuttings from younger ortets were superior to those of elder ortets [23]. In pine hybrid, adventitious roots in juvenile cuttings developed faster than those in mature cuttings and the juvenile cuttings had a much higher rooting percent [24]. Similar results were also shown in Pinus banksiana Lamb. and Grewia optiva Drumm. [25,26]. In the present study, ortet age effect of adventitious rooting in T. mandshurica softwood cuttings was obvious. With the increase in ortet age, the adventitious rooting became worse. No adventitious root was found in softwood cuttings from 20-year-old ortets.

4.2. Initiation of Root Primordia in Softwood Cuttings

Previous research showed that adventitious root primordium could be divided into latent root primordium and induced root primordium according to the origins [27]. The latent root primordia were originally in dormancy, which existed in the phloem, vascular cambium and pith rays before cutting. The dormancy of latent root primordia would be released when the suitable cutting environment appeared, and then the adventitious root would be formed [28]. The de novo root primordia were induced by certain stimulation after cutting, which located in pith ray, phloem ray, vascular cambium, phloem parenchyma and callus [29]. The locations of induced root primordia were different according to the tree species. The induced root primordia originated from vascular cambium in Morus alba var. multicaulis (Perrott.) Loud. [30]. The root organogenesis of Juglans nigra L. only originated in juvenile phloem parenchyma cells within phloem fiber gaps [31]. The induced root primordia of Tilia mongolica Maxim. were produced at vascular cambium, callus and the junction of pith ray and cortex [32]. This present study discovered that no latent root primordium was found in softwood cuttings, and the adventitious roots were formed by the induced root primordia in T. mandshurica. The induced root primordia of T. mandshurica originated from callus and the junction of pith rays and vascular cambium, and differentiated into adventitious roots which grew through cortex and epidermis. For the past few years, analyses of Corylus avellana L., Carpinus betulus L. and Betula platyphylla Suk. showed that the formation of callus and adventitious roots were independent of each other. The callus could not form adventitious roots, which prevented the invasion of pathogens and the loss of effective substances, and transported water and nutrients in cuttings. For some species, such as Picea abies (L.) Karst., Platanus acerifolia (Aiton) Willd. and Rosa chinensis Jacq., callus was the premise for adventitious root formation [33]. Our previous study found that exogenous hormones such as NAA, IBA and IAA were beneficial to the induction of callus and promoted the rooting of callus. Consequently, callus was profit to the formation of adventitious roots in T. mandshurica. Furthermore, research on Cunninghamia lanceolata (Lamb.) Hook. and Chamaecyparis obtusa (Sieb. et Zucc.) Endl. reported that adventitious roots induced from cortex absorbed water and nutrients to maintain the cuttings life, and gradually decayed after the callus taking root. The adventitious roots induced from callus would develop into the main root system [34]. For T. mandshurica, the effects of adventitious roots from vascular cambium and callus on cutting growth need to be further studied.

4.3. Effects of Organic Nutrients on Adventitious Rooting

It has been known for decades that organic nutrients especially carbohydrate and nitrogen compound, were necessary for adventitious rooting. Carbohydrate was the main energy source for adventitious rooting [35]. The soluble sugar level of T. mandshurica cuttings decreased in early days, indicating that the cuttings needed to consume stored soluble sugar to maintain metabolic needs. After that, the soluble sugar level continued to rise, which may be the recovery of photosynthesis in the leaves retained on the cuttings, resulting in the soluble sugar increasing. Adventitious root elongation needed to consume nutrients, which led to the soluble sugar decreasing again [36]. Soluble protein was closely related to the formation and differentiation of root primordium [37]. Calluses and adventitious roots formed 30 days after planting, which required a large amount of nutrients, resulting in the soluble protein decreasing. After adventitious root formation, due to photosynthesis and nutrients accumulation, the soluble protein finally increased [38]. Moreover, previous research showed that the adventitious rooting ability was related to the ratio of carbon to nitrogen in cuttings. The greater the ratio of carbon to nitrogen, the stronger the adventitious rooting ability of cuttings [39]. During the rooting process, the soluble sugar level of cuttings from 2-year-old ortets was mostly significantly higher than that from 7-year-old ortets, and the soluble protein level of cuttings from 2-year-old ortets was significantly lower than that from 7-year-old ortets, which may be the reason for the adventitious rooting of 2-year-old ortets was superior to that of 7-year-old ortets.

4.4. Effects of Enzyme Activities on Adventitious Rooting

POD, PPO and IAAO affected adventitious root formation by regulating IAA level in cuttings. POD was not only involved in plant stress resistance, but also closely related to adventitious root induction and elongation and lignin biosynthesis [40]. In early days, the T. mandshurica cuttings were subjected to stress due to shearing, which resulted in POD activity increasing. Afterwards, the POD activity decreasing was beneficial to IAA increasing, and adventitious roots were observed 30 days after planting. The POD activity increased again after 30 days of cultivation, resulting in IAA decreasing, which was conducive to adventitious root elongation [41]. Furthermore, POD was a key enzyme involved in lignin biosynthesis. In the adventitious root elongation period, a large amount of lignin was required for root lignification, resulting in POD activity increasing [42]. During the root primordia formation period, PPO activity increasing was conducive to the formation of IAA-phenol complex, which promoted adventitious root formation. PPO activity decreasing afterwards was beneficial to adventitious root elongation [43]. IAAO was a specific enzyme that decomposed IAA, which affected adventitious root formation by regulating IAA level in cuttings [44]. In the root primordium induction period, IAAO activity decreasing resulted in IAA increasing, which was conducive to adventitious root formation. IAAO activity increasing afterwards led to IAA decreasing, which promoted adventitious root elongation [45]. Compared with 7-year-old ortets, cuttings from 2-year-old ortets had better rooting effect because of their higher PPO activity and lower IAAO activity. This result was identical to that of Eucommia ulmoides Oliv. [46].

4.5. Effects of Endogenous Hormones on Adventitious Rooting

Endogenous hormones are important factors for adventitious root formation in cuttings, among which auxin is a central player in the hormone cross-talks that control adventitious rooting [47,48]. In the root primordium induction period, IAA level increasing could transport carbohydrate to the phloem at the base of T. mandshurica cuttings, which was conducive to the initiation of cell division and callus formation. IAA level decreasing afterwards was beneficial to adventitious root elongation. Similar results were also shown in Robinia pseudoacacia L. [49]. ABA is a plant growth inhibitor. The formation of adventitious roots is inhibited by the high ABA level in some plants [50]. The ABA content was generally in a downward trend, indicating that a low ABA level was conducive to the adventitious rooting in T. mandshurica. ZR is a cytokinin that can affect the growth and differentiation of plant cells. Low ZR level could promote adventitious root formation and high ZR content could inhibit the adventitious rooting of cuttings [51]. The variation of ZR level in T. mandshurica cuttings was similar to that of ABA, indicating that the formation and elongation of adventitious roots needed a low ZR level. Previous research showed that GA3 had inhibitory effect on adventitious rooting [52]. In the present study, the GA3 level of T. mandshurica cuttings peaked at the 30th day during cultivation, indicating that GA3 was involved in the root primordium induction. This result was identical to that of Juglans regia L. [53]. During the adventitious root elongation period, the GA3 content of T. mandshurica cuttings decreased rapidly, indicating that a low GA3 level was conducive to the adventitious root elongation. However, no differences were found in GA3 content of cuttings between 2- and 7-year-old ortets during the first 30 days of cultivation, but the mean root number of 2-year-old ortets was 57.78% higher than that of 7-year-old ortets. This issue should be investigated further.
It can be seen that the effect of endogenous hormones on adventitious rooting is no single, but interaction, which jointly participate in root primordium induction and adventitious root elongation. Therefore, the ratio of endogenous hormone can better reflect the adventitious rooting ability of in cuttings [54]. Research on Zizyphus jujuba Mill. reported that the IAA/ABA increasing was beneficial to the root primordium induction [55]. This present study discovered that the IAA/ABA of T. mandshurica cuttings peaked at the 30th day during cultivation, indicating that callus formation and root primordium induction were promoted under the joint action of IAA and ABA. IAA/ZR also affected the adventitious rooting. The IAA/ZR of T. mandshurica cuttings showed a rising trend during the rooting process, and the IAA/ZR of cuttings from 2-year-old ortets was significantly higher than that from 7-year-old ortets, indicating that the IAA/ZR increasing was beneficial to root primordium induction and adventitious root elongation. Similar result was also shown in Tilia miqueliana Maxim. [56].
In conclusion, adventitious root formation is a relatively complex process, and physiological studies cannot fully reveal the rooting mechanism in T. mandshurica. Molecular biological studies are also needed for discovering the ortet age effect of adventitious rooting in T. mandshurica. At present, the difficulty of adventitious rooting is still one of the problems that restrict the large-scale propagation of T. mandshurica. Establishing a fast and efficient cutting propagation technology system will become an important research interest for the promotion of T. mandshurica improved varieties.

Author Contributions

Conceptualization, H.M. and L.L.; Formal analysis, X.M., A.Z. and Y.G.; Funding acquisition, L.L.; Investigation, X.J., X.M. and A.Z.; Methodology, X.J. and Y.G.; Resources, H.M.; Supervision, L.L.; Writing—original draft, X.J. and Y.G.; Writing—review and editing, H.M. and L.L. All authors have read and agreed to the published version of the manuscript.

Funding

This work was financially supported by a grant from the Central Finance Forestry Science and Technology Promotion Demonstration Program of China (JLT2021-35) and the Science and Technology Development Program of Jilin Province (20210202124NC).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Acknowledgments

We thank Jiangmifeng Town Forest Farm and Xinshan Nursery Stock Cooperative for support related to the plant material and greenhouse facilities.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Multiple comparisons of different ortet ages with respect to adventitious rooting. (a) Rooting percentage; (b) mean root number; (c) total root length. Different lower-case letters indicate significantly differences under 0.05 levels.
Figure 1. Multiple comparisons of different ortet ages with respect to adventitious rooting. (a) Rooting percentage; (b) mean root number; (c) total root length. Different lower-case letters indicate significantly differences under 0.05 levels.
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Figure 2. Phenotypic properties of adventitious root development in the cutting bases from 7-year-old ortets at the 0th- (a), 10th- (b), 20th- (c), 40th- (d) and 60th day (e) during cultivation, respectively.
Figure 2. Phenotypic properties of adventitious root development in the cutting bases from 7-year-old ortets at the 0th- (a), 10th- (b), 20th- (c), 40th- (d) and 60th day (e) during cultivation, respectively.
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Figure 3. Anatomical properties of adventitious root development in the cutting bases from 7-year-old ortets. (a) Transverse sections of cutting bases before cultivation; (b) Root primordium initial cells were formed in vascular cambium at the 10th day in cultivation; (c) Root primordia were formed in vascular cambium at the 20th day in cultivation; (d) Adventitious roots were formed in vascular cambium at the 30th day in cultivation; (e) Adventitious root elongation from vascular cambium at the 40th day in cultivation; (f) Calluses were formed on the inner of cutting bases at the 20th day in cultivation; (g) Calluses were formed on the surface of cutting bases at the 20th day in cultivation; (h) Adventitious roots were formed in calluses of cutting bases at the 40th day in cultivation. E—epidermis; C—cortex; Ph—phloem; VC—vascular cambium; X—xylem; PI—pith; RPIC—root primordium initial cell; RP—root primordium; AR—adventitious root; Cal—callus.
Figure 3. Anatomical properties of adventitious root development in the cutting bases from 7-year-old ortets. (a) Transverse sections of cutting bases before cultivation; (b) Root primordium initial cells were formed in vascular cambium at the 10th day in cultivation; (c) Root primordia were formed in vascular cambium at the 20th day in cultivation; (d) Adventitious roots were formed in vascular cambium at the 30th day in cultivation; (e) Adventitious root elongation from vascular cambium at the 40th day in cultivation; (f) Calluses were formed on the inner of cutting bases at the 20th day in cultivation; (g) Calluses were formed on the surface of cutting bases at the 20th day in cultivation; (h) Adventitious roots were formed in calluses of cutting bases at the 40th day in cultivation. E—epidermis; C—cortex; Ph—phloem; VC—vascular cambium; X—xylem; PI—pith; RPIC—root primordium initial cell; RP—root primordium; AR—adventitious root; Cal—callus.
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Figure 4. Organic nutrients of cuttings from 2- and 7-year-old ortets during cultivation. (a) Soluble sugar; (b) Soluble protein. Each data point contains three replicates, and error bar means standard deviation. * and ** indicate significantly differences under 0.05 and 0.01 level, respectively. The same below.
Figure 4. Organic nutrients of cuttings from 2- and 7-year-old ortets during cultivation. (a) Soluble sugar; (b) Soluble protein. Each data point contains three replicates, and error bar means standard deviation. * and ** indicate significantly differences under 0.05 and 0.01 level, respectively. The same below.
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Figure 5. Enzyme activities of cuttings from 2- and 7-year-old ortets during cultivation. (a) Peroxidase (POD); (b) polyphenol oxidase (PPO); (c) 3-indoleacetic acid oxidase (IAAO). The unit of enzyme activity is U·g−1·min−1, which means 1 μmol of substrate was converted in 1 min per gram of sample.
Figure 5. Enzyme activities of cuttings from 2- and 7-year-old ortets during cultivation. (a) Peroxidase (POD); (b) polyphenol oxidase (PPO); (c) 3-indoleacetic acid oxidase (IAAO). The unit of enzyme activity is U·g−1·min−1, which means 1 μmol of substrate was converted in 1 min per gram of sample.
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Figure 6. Endogenous hormones of cuttings from 2- and 7-year-old ortets during cultivation. (a) 3-indoleacetic acid (IAA); (b) abscisic acid (ABA); (c) zeatin riboside (ZR); (d) gibberellin (GA3); (e) IAA/ABA; (f) IAA/ZR.
Figure 6. Endogenous hormones of cuttings from 2- and 7-year-old ortets during cultivation. (a) 3-indoleacetic acid (IAA); (b) abscisic acid (ABA); (c) zeatin riboside (ZR); (d) gibberellin (GA3); (e) IAA/ABA; (f) IAA/ZR.
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Table
Table 1. Analysis of variance of different ortet ages on rooting percentage, mean root number and mean root length.
Table 1. Analysis of variance of different ortet ages on rooting percentage, mean root number and mean root length.
Dependent VariableSourceSum of SquaresDegree of FreedomMean SquareF
Rooting percentageBlock0.00320.0011.004
Ortet age0.97430.325231.262 **
Error0.00860.001
Total0.98511
Mean root numberBlock0.48220.2410.532
Ortet age256.057385.352188.392 **
Error2.71860.453
Total259.25711
Total root lengthBlock208.6202104.3101.619
Ortet age47,369.459315,789.820245.085 **
Error386.555664.426
Total47,964.63411
** indicates significantly differences under 0.01 level.
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Mu, H.; Jin, X.; Ma, X.; Zhao, A.; Gao, Y.; Lin, L. Ortet Age Effect, Anatomy and Physiology of Adventitious Rooting in Tilia mandshurica Softwood Cuttings. Forests 2022, 13, 1427. https://doi.org/10.3390/f13091427

AMA Style

Mu H, Jin X, Ma X, Zhao A, Gao Y, Lin L. Ortet Age Effect, Anatomy and Physiology of Adventitious Rooting in Tilia mandshurica Softwood Cuttings. Forests. 2022; 13(9):1427. https://doi.org/10.3390/f13091427

Chicago/Turabian Style

Mu, Huaizhi, Xuhong Jin, Xinyu Ma, Anqi Zhao, Yuting Gao, and Lin Lin. 2022. "Ortet Age Effect, Anatomy and Physiology of Adventitious Rooting in Tilia mandshurica Softwood Cuttings" Forests 13, no. 9: 1427. https://doi.org/10.3390/f13091427

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