Wintersweet (Chmonanthus praecox (L.) Link), a deciduous ornamental shrub belonging to the Chimonanthus of the Calycanthaceae family, is endemic to China (Liu et al., 2014). Due to its outstanding flowering time and unique aroma in deep winter, it is becoming more and more popular in mainland China and has been widely introduced into Japan, Korea, Europe, Australia, and the United States, becoming commercially important cut flowers and garden plants (Liu et al., 2017; Li et al., 2018). Due to extensive commercial development and deforestation by people, its wild germplasm resources have been continuously destroyed or even lost (Dai et al., 2012).Moreover, wintersweet is also one of the precious relic plants that can be traced back to the Tertiary Period, and it is listed as a second class of endangered plants on the Red List of China (Wang et al., 2014; Lu et al., 2015). Therefore, it is necessary to establish a complete set of high-efficiency breeding technology to realize the introduction and protection of the wintersweet resources.

Because the rootstock used has a complete and developed root system, it can supply enough water and nutrients to the scion, so that the growth and development of the grafted plant is much faster than that of the cutting plant, so that the grafted plant will bloom and bear fruit earlier (Zhang et al., 2010; Cassol et al., 2017). Due to the variation of seedlings of wintersweet, and the ornamental value of seedlings after flowering is poor, people often use sowing methods to cultivate grafted rootstocks and select new varieties (Zhao and Zhang, 2007; Wei et al., 2010). Wintersweet grafting commonly used cutting method and docking method, the cutting method is generally carried out in the spring (March to April) when the buds are just sprouting, and the docking method is suitable for spring and summer (May to July), and performed better in May (Zhao and Cao, 2012; Ji et al., 2013). Although wintersweet grafting is generally carried out in spring and summer, the branches of wintersweet are too tender and short in spring, the mother tree can provide fewer available scions and the temperature in summer is higher. Due to actual demand, people often need to introduce and expand the excellent wintersweet germplasm resources in autumn and winter.

At present, there have not been reports on the related experimental research on autumn and winter for wintersweet grafting, and the effect of rootstock age on wintersweet grafting. Therefore, this study explored the effects of different grafting times, different scion positions, and different rootstock ages on the survival percentage of wintersweet grafting through grafting experiments in two consecutive years, in order to provide technical guidance for the introduction and rapid propagation of excellent wintersweet resources in autumn and winter.

1 Results

1.1 The effect of different grafting time on the survival percentage of wintersweet grafting

Through grafting experiments in two consecutive years, the results showed that with the passage of time, the survival percentage of wintersweet grafting showed a downward trend. In 2018, the graft survival percentage was the highest in late September at 73.25%, which was significantly higher than the graft survival percentage in mid-October and late October; the survival percentages in mid-October and late October was 36.71% and 39.16%, respectively, and there was no significant difference between the two. In 2019, the graft survival percentage was the highest in late September at 80%, which was significantly higher than that in late October and mid-November. The graft survival percentage in late October (39.76%) was higher than that in mid-November (12.22%), but there was no statistically significant difference between the two (Figure 1).

1.2 The effect of different scion position on the survival percentage of wintersweet grafting

In autumn and winter, there is no obvious rule for the effect of scion position on the survival percentage of wintersweet grafting (Figure 2). In late September, the influence of the scion position on the survival percentage of the wintersweet grafting was the middle of the branch (100%)>the bottom of the branch (80%)>the upper part of the branch (60%). In late October, the effect of the scion position on the survival percentage of wintersweet grafted was the upper of the branch=the bottom of the branch (40%)>the middle of the branch (20%). In mid-November, only the wintersweet with the upper branch as the scion survived the grafting, and the survival percentage was 23.07%. The analysis of variance showed that different scion positions had no significant effect on the survival percentage of wintersweet grafting (p=1).

1.3 The effect of different stock age on the survival percentage of wintersweet grafting

Both annual seedlings and perennial seedlings can be used as rootstocks for grafting wintersweet, and the survival percentage varies with species (Figure 3). Except for the variety 9 that was grafted with perennial seedlings as rootstocks, the survival percentage of all the other varieties grafted with perennial seedlings as rootstocks were significantly higher than that of annual seedlings. The results of t-test showed (Table 1) that the survival percentage of grafting with perennial seedlings as rootstocks (56.03%) were significantly higher than that of annual seedlings (21.41%).

2 Discussion

A large number of studies have shown that the grafting time has a great influence on the graft survival percentage, which mainly affects the physiological state of the stock and scion and the formation of callus by affecting the weather conditions, such as temperature, humidity, and other environmental conditions, thereby affecting the survival of grafting, and the best grafting period for different tree species is different (Sheng et al., 2014; Zhang et al., 2017; Swierczynski et al., 2020). Zhao et al. (2007) studied the grafting of wintersweet at different times in spring and summer. Their results showed that wintersweet can be grafted from mid-April to late August, but the survival percentage of wintersweet grafted from mid-May to mid-June is the highest, which the survival percentage can be as high as 90%. The results of our study showed that in autumn and winter, with the decrease of temperature, the survival percentage of wintersweet grafted gradually decreased. Grafting in late September is more conducive to the survival of wintersweet grafting. At this time, the ambient temperature is about 20℃, which is more conducive to the formation and combination of callus, and the survival percentage can also be as high as 80%.

With the growth and development of the branches, the water content, fullness, lignification and cell activity of different position of the branches are often different at different stages, and the survival percentage of grafting as a scion is also different (Mao et al., 2018). Zhao and Zhang (2007) found that in spring and summer, the survival percentage of wintersweet grafting for the middle position of the branches as scion was the highest, followed by the bottom position, and the lowest survival percentage for the upper position of the scion grafting. The results of our study shown that there is no obvious rule for the effect of scion position on the survival percentage of wintersweet grafting in autumn and winter. The reason for this difference may be that the upper position of the branch generally matures in autumn, and the development status of each position of the branches tend to be consistent in autumn and winter. Grafting should be based on the actual situation, try to choose full buds, full and robust branches as scions.

Choosing appropriate rootstocks can greatly increase the survival percentage of tree species grafting (Ma et al., 2012; Qi et al., 2019). Wintersweet grafting commonly used 2-3 years old ‘Gouying’ wintersweet as rootstock (Zhao and Cao, 2012; Chen et al., 2018, Xiang Cun Keji, (25): 74-75). This experiment tried to use annual seedlings as rootstocks for grafting. The results showed that it is feasible to use annual seedlings as rootstocks. However, the grafting survival percentage of seedlings at least 3 years old as rootstocks were significantly higher than that of annual seedlings. This may be due to the more developed root system of perennial seedlings, stronger growth potential, thicker branches, higher maturity, strong physiological metabolism, and sufficient nutrients, which are more conducive to the growth of callus at the grafted incision (Sheng et al., 2014). Therefore, in actual production, the rootstocks should be cultivated in advance according to actual needs, and water and fertilizer management should be strengthened to promote thickening of stocks.

The new plants formed after the rootstock and scion are connected together by callus marks the survival of grafted seedlings (Figure 4F) (Yun et al., 2011; He et al., 2017). Many other factors affect the survival of grafting, such as meteorological factors (average daily temperature, sunshine duration, relative humidity, rainfall and rainfall days, etc.), proficiency of grafting technology, and management level after grafting (You et al., 2015). Grafting requires fast and accurate action, flat and smooth interface, so as to ensure that the contact area between the rootstock and the scion cambium is maximized, and then enough parenchyma cells can be produced between the contact surfaces. These parenchyma cells have strong meristematic ability and can significantly accelerate the speed of the rootstock healing. The binding is required to be tight, and even, and must be completed before the surface is transfer brown and yellow. During the wound healing period, it is necessary to do a good job of protection to prevent infections caused by lesions at the interface (Li, 2019).After the grafted seedlings survive, a series of field management measures such as removing buds and shoots, cutting stocks, weeding, fertilization and pest control should be carried out in time. In addition, when grafting in autumn and winter, it should be bagged or covered with a film for heat preservation when the temperature is low so that the rootstocks can survive the winter safely.

Due to the limitation of the number of scions and stocks, this study only explored the grafting time, stock age and scion position, and initially obtained the conditional factors to increase the survival percentage of wintersweet grafting in autumn and winter, but the experiment was less repeated, the number of grafts is small, which has certain one-sidedness and limitations. In the future, a more scientific and reasonable experimental design should be carried out, and the factors affecting the grafting of wintersweet should be studied more systematically, in order to increase the survival percentage of grafting to a greater extent.

3 Materials and Methods

3.1 Test site

The test site is located in the Wintersweet Germplasm Resource Nursery, College of Horticulture and Landscape Architecture, Southwest University, Xiema Town, Beibei District, Chongqing City (29°83' N, 106°43' E, elevation 360 m). Chongqing has a humid subtropical monsoon climate, with an average annual rainfall of 1000-1350 mm and an average annual temperature of 16-18 ℃.

3.2 Experiment material

Grafted rootstock：The rootstocks used in the experiment are all seedlings of ‘Gouying’ wintersweet, which are divided into annual seedlings and perennial seedlings. The annual seedlings are sown in the summer of the year before grafting, and the perennial rootstocks are cultivated for at least 3 years. Rootstocks are required to grow robustly, without mechanical damage, pests and diseases, frost damage, sunburn, and dried top.

Grafted scion: In this experiment, the wintersweet variety 1 and variety 2 used as scion came from the Wintersweet Germplasm Resource Nursery of the College of Horticulture and Landscape Architecture, Southwest University(29°83' N, 106°43' E, alevation360 m); the variety 3 is a wild wintersweet from Wuxi County, Chongqing City (31°40' N, 109°40' E, elevation 580 m); Varieties 4, 5, 6, 7, 8, and 9 were derived from the excellent wintersweet varieties in the Wintersweet Resource Nursery of the Horticulture Institute, Sichuan Academy of Agricultural Sciences (30°78' N, 104°21' E, elevation 479 m). When grafting, select robust and nutritious branches of various varieties as scion branches. All scions were grafted within 1 day after collection.

3.3 Grafting method

The experiment mainly uses the cutting method for grafting. Firstly, make a short cut 10-20 cm from the lower end of the morphology of the rootstock used (Figure 4A), and cut a smooth incision of 2-3 cm longitudinally on the smooth side of the short cut, deep to the xylem (Figure 4B). Select the branches that have been prepared for use as scions and cut them into scions. Each scion is about 3-4 cm long, with a pair of buds remaining, and the lower part is cut into a flat wedge shape, and the cambium is exposed (Figure 4C). Then insert the shaved scion into the incision of the rootstock, align the cambium of the two to make the rootstock tightly connected (Figure 4D), and finally tie the rootstock and scion tightly with a plastic tape (Figure 4E).

3.4 Influence of different grafting times and different scion positions on the survival percentage of grafting

Experiment 1: The grafting trials were conducted for two consecutive years in late September, mid-October, and late October in 2018, and late September, late October, and mid-November in 2019. The specific time, details and number of grafts are shown in Table 2.

Experiment 1: The upper, middle, and bottom position of the annual semi-lignified branches of wintersweet were selected as scions and divided into 3 groups for comparative experiments. The specific time, details, and number of grafts are shown in Table 2.

Among them, the upper branches are about 20 cm in the upper position of the morphology, the lower branches are about 20 cm in the bottom position of the morphology and the rest are the middle branches.

3.5 Influence of different stock ages on the survival percentage of grafting

Experiment 2: Grafting different wintersweet varieties, using perennial seedlings and annual seedlings as rootstocks for comparative experiments. The specific time, details and number of grafts are shown in Table 2.

3.6 Statistics on the survival percentage of wintersweet grafting

When the buds on the scion turn green or elongate into new shoots, it is considered that the scion survives. Count the survival percentage of each treatment 120 days after grafting.

3.7 Data analysis

The experiment uses Excel 2016 and SPSS.23 for statistical analysis. ANOVA analysis of variance was used to compare whether the grafting results at different times and different scion positions were significantly different. The t-test was used to compare whether the grafting results of the two rootstock ages were significantly different.

Authors’ contributions

Hu Huan is the experiment designer and executor of this study. Hu Huan and Yang Ximeng completed the data analysis and the writing of the first draft of the manuscript. Wang Xia, Zhu Ting, Li Guixiang and Li Tao participated in the collection and processing of grafting materials, and analysis of experimental results. Li Mingyang and Sui Shunzhao are the project creators and leaders, directing experimental design, data analysis, manuscript writing and revision. All authors read and approved the final manuscript.

Acknowledgments

This work was supported by National Natural Science Foundation of China (Grant nos. 31971711), and the Chongqing Science and Technology Bureau (cstc2018jscx-mszdX0004).

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