Study on the Rooting Physiological Mechanism of Schisandra chinensis (Turcz.) Baill. Green-Branched Cuttings

Abstract

Schisandra chinensis (Turcz.) Baill. is an important medicinal plant in northeast China. Cutting propagation is an effective method for the rapid propagation of many tree species. This research aimed to determine a suitable growing medium and appropriate root hormone type, concentration and treatment time through the utilization of different substrates and hormones to treat one-year-old branches of S. chinensis. The optimal treatment achieved a rooting rate of 60% with 225 ppm ABT and 75 ppm NAA for 2.5 h. The substrate of vermiculite/perlite = 1:1 (urea+potassium) was beneficial to the adventitious root formation, which reached 79%. The adventitious root primordium of S. chinensis originated from the junction of the xylem and cambium. ABT and NAA treatments altered the peak timing of POD, PPO and IAAO in S. chinensis cuttings. During the adventitious root formation of S. chinensis cuttings, the high endogenous IAA concentration promoted the occurrence of adventitious roots in the early stage and the lower endogenous IAA and GA₃ concentrations promoted the elongation and growth of adventitious roots in the later period. Low ABA and ZR concentrations enabled adventitious root formation and elongation. An efficient cutting propagation system would enable the mass propagation of S. chinensis seedlings.

1. Introduction

Schisandra chinensis (Turcz.) Baill. is a perennial deciduous woody vine native to the forests of northern China. The dried fruit of S. chinensis has been used as a traditional herb in China for thousands of years. It was first recorded in the Shennong Materia Medica Classic medical text, and it has a long history of use in medicine. It is also used as a traditional tonic for medical treatments in Russia and other countries due to its potential to treat tumors, gastrointestinal dysfunction, and liver diseases, reduce inflammation, and delay aging [1,2]. The fruit of S. chinensis is mainly used for treating coughs, palpitations, and nocturnal emissions. Modern pharmacological research has revealed the antitussive activity, cardioprotective activity, and prostate relaxing activity of S. chinensis, which provides a basis for traditional application. Additionally, S. chinensis can protect the liver and nerves, resist oxidation, treat cancer and inflammation, and has hypoglycemic properties and other medicinal values [3].

The reproduction modes of S. chinensis include sexual reproduction (i.e., seed reproduction) and asexual reproduction. An orchard is built with seedlings. There are significant differences in yield characteristics due to the high mutation probability of seed breeding progeny traits, inability to stably inherit excellent traits, low germination rate, and different sizes at emergence. The asexual rapid propagation of S. chinensis is the primary problem that needs to be solved for its successful cultivars promotion. Asexual reproduction is an important way to cultivate excellent individual clones. Cutting is the main technique used to ensure asexual reproduction, and this regeneration mechanism will ensure plant cell totipotency [4]. Seedlings produced by cutting propagation will retain the excellent characteristics of the mother plant. The culture time is short, and culturing is easy; therefore, cutting propagation is an excellent rapid propagation method.

In this study, we established an efficient cutting propagation system and revealed the physiological mechanism of rooting, which can quickly produce large numbers of seedlings of S. chinensis with excellent genetic characteristics and promote the industrial development of superior varieties of S. chinensis.

2. Materials and Methods

2.1. Test Materials

This experiment was conducted in the greenhouse of Jilin Agricultural University in Changchun. A small arch shed was set on a seedbed and sealed with non-dripping film. To ensure the growth of cutting seedlings, the air temperature in the greenhouse was 20−30 °C, the temperature and soil temperature in the small arch shed were controlled to about 30 °C, and the relative humidity was above 90%.

Cuttings were taken from a two-year-old plant from the superior ‘Zaohong’ line. Semi-lignified branches with no mechanical damage, vigorous growth, no pests and diseases, consistent internode length, and a full bud head were selected as cuttings. They were cut into stems with a length of 10 cm with one leaf stalk. Cutting and post-cutting management were conducted in the seedbed. The upper 1 cm was cut away from the bud. and half of the leaves were removed. Then the cuttings were treated with growth regulators, and then inserted into the substrate. The experiment was repeated for three years, and the propagated plants in the first year had partially begun to bear fruit.

2.2. Experimental Design and Methods

2.2.1. Cutting Experiment with Different Exogenous Growth Regulators

An L9(33) orthogonal design was established (

Table 1. The L9(33) orthogonal experimental design for cutting experiment with different exogenous growth regulators and treatment time.

	Combination	NAA (ppm)	ABT (ppm)	Time (h)
Treatment A	A1B1C1	25	225	1.5
Treatment B	A1B2C2	25	250	2.0
Treatment C	A1B3C3	25	275	2.5
Treatment D	A2B1C2	50	225	2.0
Treatment E	A2B2C3	50	250	2.5
Treatment F	A2B3C1	50	275	1.5
Treatment G	A3B1C3	75	225	2.5
Treatment H	A3B2C1	75	250	1.5
Treatment I	A3B3C2	75	275	2.0
Treatment J	Control	0	0	0

). The growth regulators were ABT rooting powder (ABT-1, bought from the Chinese Academy of Forestry) and NAA (All the abbreviations are explained in the Abbreviation). The ABT concentrations were set to 225 ppm (A1), 250 ppm (A2), and 275 ppm (A3). The NAA concentrations were set to 20 ppm (B1), 50 ppm (B2), and 75 ppm (B3). The treatment times were 1.5 h (C1), 2.0 h (C2), and 2.5 h (C3). Nine orthogonal experiments and a control were conducted using the same substrate (perlite/vermiculite) = 1:1.

2.2.2. Cutting Experiment with Different Substrates

Six different matrix combinations were prepared using perlite, vermiculite, sand, and other cutting substrates. The fertilizers applied were urea+potassium dihydrogen phosphate diluted 300 times and a Greencare universal water-soluble fertilizer (Total nitrogen 20%, nitrate nitrogen 12%, ammonium nitrogen 8%, available phosphorus 10%, available potassium 20%, Fe 0.1%, Mn 0.05%, Zn 0.05%, Cu 0.025%, P 0.025%, Mo 0.01%, Mg 0.4% and S 0.5%) 600 times solution containing nitrogen, phosphorus, and potassium. The substrate treatments were carried out as followes: A. perlite/vermiculite = 1:1 (urea+potassium dihydrogen phosphate); B. vermiculite/sand = 1:1 (urea+potassium dihydrogen phosphate); C. perlite/sand = 1:1 (urea+potassium dihydrogen phosphate); D. perlite/vermiculite = 1:1 (water-soluble fertilizer); E. vermiculite/sand = 1:1(water-soluble fertilizer); F. perlite/sand = 1:1 (water-soluble fertilizer). The exogenous growth regulator applications were ABT 225 ppm and NAA 75 ppm for 2.5 h.

2.2.3. External Morphology and Anatomical Structure of the Rooting Process of S. chinensis Cuttings

The cuttings were treated with ABT 225 ppm and NAA 75 ppm for 2.5 h and then inserted into the substrate of perlite/vermiculite = 1:1 with urea+potassium dihydrogen phosphate diluted 300 times. Cuttings without any treatment were inserted into the same substrate as the control. Samples were taken every seven days from the initial cutting to the time that adventitious roots emerged. The time when the cutting base began to bulge, the time and quantity of callus formation, and the time and quantity of adventitious root formation were recorded. Stem segments were cut approximately 3 cm from the base of the cuttings, and fixed with FAA fixing solution. Cross sections of stem segments were used to make paraffin sections. They were dyed with saffron fixation green, and changes in cell morphology and structure were observed under the microscope. The external appearance of the cuttings was photographed and recorded.

2.2.4. Determination of Physiological Indicators

The cuttings were treated with Section 2.2.3. The IAA, ABA, GA₃ and ZR contents with IAA, POD and PPO activities in the cortex of stem segments at the base of the cuttings were determined every week for up to 42 days. The IAA, ABA, GA₃, and ZR contents were determined by an enzyme-linked immunosorbent assay (ELISA) [5]. The IAAO, POD, and PPO activity were determined by a colorimetric method [6] and catechol colorimetry [7].

2.2.5. Determination of Rooting Status

The rooting status was investigated two months after cutting. The comprehensive rooting quality was evaluated by determining the rooting rate, average root length and average root number (taking five cutting seedlings as the average).

2.2.6. Data Processing and Analysis

Each experiments was conducted with 21 seedlings for each group and set for three repetitions. The data were processed using the EXCEL package in Microsoft Office and analyzed by the SPSS24.0 statistical analysis software. The Duncan method was used to analyze the significance of differences among multiple groups of samples.

3. Results and Analysis

3.1. Effects of the Different Exogenous Growth Regulators on Rooting Ability

The rooting rate, root number, and rooting length of cuttings soaked with different ABT and NAA concentrations were better than those of the control group (Figure 1), indicating that the rooting rate of S. chinensis cuttings was significantly improved under the action of exogenous growth regulators (

Table 2. Comparison of the rooting rate, length, and number of S. chinensis treated with different exogenous growth regulators and time.

Treatment	Rooting Rate	Average Root Length (cm)	Average Number of Roots (Strip)
A1B1C1	0.30 ± 0.09 c	2.95 ± 1.35 b	3.00 ± 2.00 b
A1B2C2	0.40 ± 0.08 b	3.33 ± 0.50 ab	3.00 ± 1.00 b
A1B3C3	0.34 ± 0.14 b	3.25 ± 0.46 ab	3.00 ± 1.00 b
A2B1C2	0.30 ± 0.15 c	2.15 ± 0.20 c	2.67 ± 0.58 c
A2B2C3	0.27 ± 0.15 d	2.78 ± 1.12 b	4.00 ± 1.73 a
A2B3C1	0.33 ± 0.07 b	3.72 ± 1.11 a	3.67 ± 2.08 ab
A3B1C3	0.60 ± 0.11 a	3.54 ± 0.79 a	3.67 ± 0.58 ab
A3B2C1	0.34 ± 0.09 b	3.30 ± 1.11 ab	3.67 ± 2.08 ab
A3B3C2	0.23 ± 0.01 d	3.16 ± 1.36 b	2.67 ± 1.16 c
Control	0.00 ± 0.00 e	0.00 ± 0.00 d	0.00 ± 0.00 d

Note: Different lowercase letters in the same column indicate significant differences at the level of 0.05.

). Additionally, when the substrate was consisted of vermiculite/perlite = 1:1, the treatment of A3B1C3 with 75 ppm NAA, 225 ppm ABT and a soaking time of 2.5 h achieved the best results, with the highest rooting rate of 60%, average root length of 3.54 cm, and average root number of 3.67. The treatment with 225 ppm ABT, 50 ppm NAA, and a soaking time of 2 h achieved a rooting rate of 40%, an average root length of 2.95 cm, and an average root number of 3. The lowest rooting rate and root length were achieved in the control. There were significant differences in average root length and average root number. The average Number of Roots under the A2B2C3, A2B3C1, A3B1C3 and A3B2C1treatment was significantly better than that under the other treatments.

3.2. Effects of Different Matrix Fertilizers on Rooting

Different matrix fertilizers had effects on rooting, and the vermiculite/perlite = 1:1 (urea+potassium) treatment achieved the best rooting rate of 79%, average root length of 3.79 cm, and average root number of 8 among the six treatments (

Table 3. Comparison of the rooting rate, length, and number of S. chinensis treated with different substrates and fertilizers.

Matrix Number	Rooting Rate	Average Root Length (cm)	Average Number of Roots (Strip)
A	0.79 ± 0.05 a	3.79 ± 0.16 a	8.00 ± 0.58 a
B	0.32 ± 0.03 c	2.28 ± 0.35 c	3.67 ± 0.33 c
C	0.74 ± 0.09 a	3.15 ± 0.42 b	5.67 ± 0.20 b
D	0.42 ± 0.10 b	3.11 ± 0.70 ab	4.00 ± 1.15 c
E	0.49 ± 0.01 b	3.32 ± 0.39 ab	5.33 ± 0.88 b
F	0.74 ± 0.12 a	2.84 ± 0.35 bc	3.67 ± 0.33 c

Note: Different lowercase letters in the same column indicate significant differences at the level of 0.05.

). For perlite/sand = 1:1 (urea+potassium) and perlite/sand = 1:1 (water-soluble fertilizer) the rooting rate was 74%. The vermiculite/sandy soil = 1:1 (urea+potassium) treatment had the lowest rooting rate of 32%, the lowest average root length of 2.28 cm, and the lowest average root number of 3.67. The rooting ability under the vermiculite/perlite = 1:1 (urea+potassium) treatment was significantly better than the other treatments and achieved the optimal rooting ability (Figure 2). A comprehensive evaluation of rooting ability using the rooting index showed that the five treatments followed the order of: B: vermiculite/sand = 1:1 (urea+potassium) > C: perlite/sand = 1:1 (urea+potassium) > F: perlite/sand = 1:1 (water-soluble fertilizer) > E: vermiculite/sand = 1:1 (water-soluble fertilizer) > D: perlite/vermiculite = 1:1 (water-soluble fertilizer).

3.3. Observation of Rooting External Morphology

The morphological characteristics of the bases of S. chinensis cuttings of control and the treatment were observed (Figure 3 and Figure 4). As shown in Figure 3, after 42 days, the cuttings treated with clean water only formed a callus and did not take root, but the cuttings treated with exogenous growth-regulators formed roots from 28 days. Every 7 days of cuttings were photographed, and the initial cutting of treatment was shown in Figure 4A. After seven days, the lenticels at the base of the cuttings expanded with white protrusions (Figure 4B). After 14 days, the phloem and xylem interbedded to form a milky tender tissue, i.e., the callus (Figure 4C). The callus expanded continuously from 14 to 21 days (Figure 4D). After 28 days, the callus no longer grew, some tissues began to brown after the callus was cut, and adventitious roots elongated and grew after the callus appeared (Figure 4E). After 28–42 days, the root system of the newly born S. chinensis was relatively well developed, and the root length of some cuttings was 5–10 cm (Figure 4F,G).

In summary, the rooting process of S.chinensis cuttings could be divided into three stages: callus formation stage (Figure 4B,C), root induction stage (Figure 4D,E), and root extension stage (Figure 4F,G). It was difficult for S. chinensis cuttings to take root, and only callus was formed unless a treatment with exogenous growth regulators was applied.

3.4. Anatomical Characteristics of the Cutting Roots

The cross-section of the cuttings showed that the stem was mainly composed of a periderm, cortex, phloem, cambium, xylem, vascular bundle, and pith (Figure 5). The periderm cells at the outermost end of the stem were closely arranged and formed protective tissue. The inner layer had the fewest cells, which were irregularly distributed. The tissue near the periderm was the cortex. The cells were loosely arranged with large spacing, and the cells were rich in storage materials. Both the cell wall near the pith and the cytoplasm were thick. There were vascular tissues in the cortex, including the phloem, xylem, and pith. The phloem was thin and the cells were densely arranged. The xylem is largely composed of woody fibers and vessels. The tissue near the phloem was the secondary xylem, and the tissue near the pith was part of the primary xylem. The pith was composed of many parenchyma cells, which were closely arranged without gaps (Figure 5A).

On the seventh day after cutting, the xylem and cambium cells changed and the cell volume increased, which split and grew outward (Figure 5B). The cell clusters (root primordia) were formed with the continuous division of xylem and cambium cells on the 14th day (Figure 5C) and the root primordia continued to expand on the 21st day (Figure 5D). the root primordia grew outward to reach the cortex from the 28th (Figure 5E) to the 35th day (Figure 5F) after cutting. The adventitious roots kept protruding outward, breaking through the periderm to complete the rooting process and forming adventitious roots with a complete structure until the 42th day (Figure 5G).

3.5. Dynamic Changes of Enzyme Activity

As shown in Table 2 and Table 3, the rooting rate of the treatment and control were 79% and 0, respectively. The treatment with exogenous growth regulators improved POD, PPO, and IAAO activity, thus increasing the rooting rate (Figure 6). The POD activity of the treatment group exhibited two peaks, occurring at 14 and 42 days post-treatment, respectively, which were earlier than those of the control group. In contrast, the POD activity of the control group gradually increased to its maximum value within 28 days and subsequently declined. The PPO activity in the treatment group continued to increase and peaked on the 35th day, which maybe promote the condensation of phenolic compounds and IAA, and formed rooting auxiliary factors. The comprehensive rooting auxiliary factors gradually increased and promoted the formation of a root primordium and adventitious roots. In the control group, PPO activity reached its peak on the 14th day, gradually decreased until the 21th day, and then slowly increased again, reaching its maximum on the 42th day. The IAAO activity in the control group was consistent with the trend of the treatment group, but reached a maximum on the 21st day and then decreased and stayed at a stable level during the 35~42th day. In the treatment group, the IAAO activity gradually increased the early stage after green branch cutting, which reached a peak on the 28th day and then decreased before increasing again during the 28~42th day.

3.6. Dynamic Changes of Endogenous Growth Regulators

As shown in Figure 7A, the IAA content in the treatment group displayed an upward trend, with slight fluctuations in the middle of the treatment and peaked on day 14 (58.81 ng/g) and day 35 (138.98 ng/g). However, the IAA content in the control group remained at a low level with no obvious change before day 21, the peak of which was delayed to day 42. These results indicated that IAA is a key endogenous hormone for the formation of adventitious roots in S. chinensis cuttings. The ZR content of cuttings in both the treatment and control groups generally displayed an “increasing-decreasing” trend (Figure 7B). Except for the sudden sharp increase in the control group on the 28th day, the ZR content in the treatment group was higher than that in the control group at other stages and reached the highest value of 23.3 ng/g on the 21st day. This may be related to the application of an early exogenous hormone treatment.

As shown in Figure 7C, the ABA content in cuttings of both groups all displayed a downward trend compared to the initial samples, which reached a small peak on the 14th day, were 128.95 ng/g (control group) and 76.98 ng/g (treatment group), respectively. The ABA content in the treatment group remained lower than that in the control group during the whole period of adventitious root growth and development, The ABA content in the treatment group was low during this period. The GA₃ content in both groups displayed a “rising-falling-rising” trend (Figure 7D) and was higher in the treatment group than in the control group. In the treatment group, the GA₃ content increased significantly in both the induction and elongation periods of adventitious root and peaks on the 14th (43.6 ng/g) and 42nd day (53.94 ng/g), which were higher than the control group.

4. Discussion

4.1. Effects of Different Plant Growth Regulators and Substrates on the Rooting of Plant Cuttings

Cutting propagation is a widely used technique, but it is easily influenced by the type of exogenous growth regulators used, soaking time, substrate and fertilizer formula. In the process of cutting propagation, the difficulty of rooting and seedling growth of different plants are not only affected by the growth characteristics of the plants themselves, but also by the cutting environment, and the use of exogenous growth regulators to induce rooting is also an important factor [8,9]. ABT and NAA are widely used as plant growth regulators, which significantly promotes the rooting of cuttings, but their optimal concentrations are greatly different for various species [10,11]. ABT is a new type of broad-spectrum and highly efficient series of green plant growth regulators developed by the Chinese Academy of Forestry. The main components of ABT rooting powder are indole-3-butyric acid potassium and naphthaleneacetic acid sodium, which can improve the survival rate of seedlings. The cuttings of Manglietia conifera Dandy were treated with either ABT, IBA, or IAA solutions at different concentration gradients and the most effective hormone treatment for rooting was 1000 mg/L IBA with a treatment time of 10 s [12]. In this study, the results showed that exogenous growth regulators can improve the rooting ability of S. chinensis cuttings taken from green branches. The treatment with 225 ppm ABT, 75 ppm NAA, and soaking for 2.5 h was the optimal treatment, with the highest rooting rate of 60% and the best roots length and number.

The nutrient content and the physical and chemical characteristics of substrates are the most important factors affecting plant growth [13,14]. Previous studies have shown that there are differences in cutting rooting number, root length, and root activity of micro-roses in different substrates, and the volume ratio of peat:GWC:soil:perlite of 4:1.5:1:2 was the best substrate ratio for S. sempervirens [15]. The results of this experiment showed that the cutting substrate had an obvious rooting effect on the cuttings from green branches of S. chinensis. The perlite/vermiculite = 1:1 (urine+potassium) mixed culture substrate had the best cutting effect, with obvious advantages in rooting rate, average root number, and rooting length compared with other substrates. This may be because vermiculite/perlite = 1:1 (urea+potassium) is well mixed, it has good water retention and air permeability, and the nutrient content in the matrix is relatively high, which is beneficial to the rooting and growth of cuttings.

4.2. Anatomical Changes during Plant Cutting and Rooting

The origin of adventitious roots in different plants is different. Some plant root primordium only comes from one part, and some tree root primordium appears in several parts [16]. Some plants have a single origin of root primordium, such as P. tremula × P. tremuloides, whose adventitious roots are only produced near the cambium, which is one of the reasons why it is difficult for P. tremula × P. tremuloides to form root by cutting [17]. The adventitious root of some tree species could be induced by callus and other parts of the stem segment at the same time, and the origin of adventitious root primordium is multi-site. For example, the adventitious root primordium of peach can be formed in the cortex, phloem, xylem and callus [18]. In this study, the cross sections of the S. chinensis cutting stem were observed by the paraffin method and revealed that there was no root primordium before cutting, which indicated that S. chinensis is an induced rooting plant with difficult rooting. By dissecting and observing the rooting parts of S. cuttings in different stages, it can be seen that the root primordium may come from the intersection of the xylem and cambium. Based on anatomical observations, the process of S. chinensis cutting rooting could be divided into three periods: the callus formation period, root induction period and root elongation period.

4.3. Relationship between Physiological Indexes and Rooting of S. chinensis Cuttings

The PPO, POD, and IAAO activities are significantly related to plant-cutting rooting. Suitable exogenous growth regulators can promote the activities of key enzymes for rooting. Peroxidase can destroy some inhibitors that hinder the rooting process and is considered to be one of the indicators of rooting. There are two peaks of POD activity during root formation of Eucommia ulmoides Oliv cuttings: the induction and extension periods [19]. In this experiment, the PPO and POD activities increased in the induction and formation stages of the root primordium and decreased in the elongation and growth stages of adventitious roots. This was consistent with the reported effect of POD in Torreya grandis cuttings [20], and indicates that some exogenous growth regulators can activate POD activity. The IAAO enzyme could oxidize IAA. IAAO activity decreased is beneficial to callus formation and root formation [21]. In this experiment, the IAAO activity initially increased and then decreased. In the initial stage of cutting, the IAAO activity was maintained at a low value and displayed a slow-increasing trend. During the induction period of the callus, the low level of IAAO activity promoted the callus. The IAAO content then decreased, which was the expression period of adventitious roots. With the growth of roots, the cuttings developed into complete plants, and their IAAO activity decreased. This was consistent with the results of the study on the softwood cutting technology of Pteroceltis tatarinowii [22].

The endogenous hormone content in plants has an important influence on the formation and occurrence of adventitious roots in cuttings. In the cutting process, several hormones do not play an independent role, but rather cooperate with each other to participate in the differentiation and development of the root primordium. The changes in the endogenous hormones during the treatment of cuttings can be regulated within the cuttings and under the action of hormones, therefore inducing the cuttings to produce a callus and then promoting the emergence of lateral roots [23]. IAA is involved in regulating the induction of the plant root primordium, the formation of the callus, and the development and growth of adventitious roots [6,7]. The IAA content in this experiment peaked on the14th and 35th day, which coincided with the time of occurrence of the root primordium and root elongation in morphological observations. The total IAA level in the treatment group was higher than that in the control group, which may be an important reason for the root morphogenesis of cuttings. As an endogenous hormone, the sharp decrease of the ABA content is not only beneficial to the hydrolysis of starch in cuttings into sugar, but is also beneficial to the formation of IAA receptors, thus promoting the root elongation of S. chinensis cuttings. During the whole period of the experiment, the ABA content in the treatment group decreased sharply and remained lower than that in the control group. This was similar to the conclusion of An et al. [24], who found that exogenous hormone treatment can reduce the level of endogenous ABA, thus promoting rooting and improving the rooting rate.

5. Conclusions

In this study, we established an efficient green branch-cutting propagation system for S. chinensis and studied its rooting mechanism. The optimal treatment was 225 ppm ABT, 75 ppm NAA, and a soaking time of 2.5 h, which achieved a rooting rate of 60%. The highest rooting rate (79%) of cuttings was achieved in a substrate of vermiculite/perlite = 1:1 (urea+potassium). The root primordium originated from the intersection of the xylem and cambium in cuttings of S. chinensis. The high endogenous IAA concentration was conducive to the formation of adventitious roots in cuttings, and the low level of endogenous IAA and GA₃ in the later stage was conducive to the elongation of the adventitious roots. A low ABA and ZR concentration was beneficial to adventitious root formation and elongation. The findings confirm the possibility of vegetative propagation for mass production of this species in S. chinensis.