Study on the Efficient Cutting Propagation Technology for Ilex “China Girl”

In order to realize the efficient industrial production of Ilex “China Girl”, an orthogonal experiment with 4 factors and 3 levels was designed. Firstly, the optimal orthogonal cutting scheme was selected from 9 treatments. Then through the systematic analysis of the effects of cutting position, substrate, exogenous hormones type and concentration on rooting indexes, such as rooting rate, root number, root length and root effect index, the theoretical optimal scheme was predicted and verified. The results showed that the theoretical optimal scheme (3000 mg/L IBA treatment for 15 s, cutting in mixed matrix with peat soil, perlite and vermiculite ratio of 2:1:1) was the optimal cutting rooting scheme of Ilex “China Girl”. After the treatment of this scheme, the rooting rate of Ilex “China Girl” reached 100%, the average root number was 51.67 per plant, and the average root length was 6.13 cm. The rooting time was greatly shortened, the rooting rate and rooting effect were greatly improved. In this study, the efficient cutting propagation technology system of Ilex “China Girl” was established, which laid a foundation for the popula-rization and application of Ilex “China Girl”, and also provided reference for further improving the cutting propagation efficiency of other evergreen holly. This study laid a foundation for the application of Ilex “China Girl”, and also provided a reference for further improving the cutting propagation efficiency of other evergreen holly.

ornamental value, such as evergreen leaves, beautiful tree shape, bright fruit that does not fall in winter [1]. However, the cold resistance of most evergreen holly is poor, cultivated areas in China are mainly concentrated in Jiangsu Province and the south of Jiangsu Province [2] [3] [4] [5]. A few evergreens holly such as Ilex cornuta, Ilex cornuta "fortunei" and Ilex crenata "convexa" can be used in gardens in Shandong Province and surrounding areas. However, their cold resistance is average, and they all suffered severe freezing damage in the extreme low temperature of −18˚C in winter of Shandong Province in 2015.
Ilex "China Girl" is an evergreen holly with strong cold resistance selected by our research group after years of adaptation studies. It is a hybrid of Ilex rugosa and Ilex cornuta. It is an evergreen shrub with beautiful leaf shape, bright green leaf color, beautiful fruit, full plant type and rapid growth. It can tolerate extreme low temperature of −18˚C in Shandong Province without freezing injury, and is especially suitable for application in Shandong Province and surrounding areas. Therefore, it is of great significance to establish an efficient cutting propagation technology system for Ilex "China Girl" to produce a large number of highquality seedlings as soon as possible and apply them to improve the current situation of winter landscape depression in Shandong Province and surrounding areas.
The cutting propagation techniques of more than 40 evergreen holly species were systematically studied. It was found that the cutting difficulty of different holly species varied greatly, and the survival rate was 20% -100% [6] [7] [8] [9]. At present, only Tian Weili et al. conducted a preliminary study on the cutting survival rate of Ilex "China Girl". After 60 days, the rooting rate was 61.75% -82.75%, and the average rooting number was 5.63 -9.33 per plant [6]. However, in order to realize the efficient industrial production of Ilex "China Girl" and produce a large number of high-quality seedlings rapidly, it is necessary to further shorten the rooting time of cutting and improve the rooting rate and root number of cuttings. Therefore, Ilex "China Girl" was taken as the research object, and the effects of cutting position, substrate, exogenous hormone type and concentration on rooting indexes were systematically studied. The efficient cutting propagation technology system of Ilex "China Girl" was established, which laid the foundation for the application of Ilex "China Girl", and also provided reference for further improving the cutting propagation efficiency of other evergreen holly.

Materials
The materials were Ilex "China Girl" introduced from Nanjing in April 2019, and planted in the seedling base of Fanzhen, Daiyue District, Tai'an City, Shandong Province.

Materials Drawn
In early September 2020 and early June 2021, the current-year-old branches of Ilex "China Girl" with strong growth, no pests and diseases, consistent length and diameter were collected.

Cutting Treatment
Each branch was cut into 9 cuttings, with 3 cuttings in each upper, middle and lower part. Each cutting contained 3 axillary buds and retained 2 upper leaves. Cut horizontally at 0.3 cm above axillary bud and obliquely at lower incision. The cuttings were immersed in 1000 times of 80% carbendazim WP for 15 min. Align the biological lower ends of sterilized cuttings downwards, each 60 into a bundle. The 2 cm base of the bundled cuttings was dipped in hormone solutions of different types and concentrations for 15 s (Table 1).

Matrix Preparation
The perlite, peat soil and vermiculite were prepared according to Table 1 and placed in the cutting bed. The day before cutting, sprayed water with a sprayer. On the day of cutting, 80% carbendazim 800 times liquid was sprayed by sprayer.

Post-Cutting Management
After cutting, 1000 times of carbendazim was sprayed once. After that, 1000 times of carbendazim was sprayed once a week for three times. Automatic timing spray equipment was used to adjust the temperature and air humidity during cutting, so that the temperature in the cutting shed was maintained at 20˚C -38˚C, and the humidity was maintained at more than 80%.

Rooting Index Observation
The base of cuttings treated by different schemes was observed every 10 days after cutting. The rooting rate, root number, root length and root effect index (average root length × average root number/number of cuttings) of 30 cuttings randomly selected were counted at 50 d and 60 d after cutting. It should be noted that 9 treatments (T 1 -T 9) in September 2020 were only counted once at 60 d after cutting, and 2 treatments (T 3 and T L ) in June 2021 were counted once at 50 d and 60 d after cutting.

Data Statistical Analysis
Data statistical analyses were performed using SPSS 26 software.

Selection of Optimal Orthogonal Scheme
As shown in Table 3 and Figure 1, the rooting rates of T 3 , T 5 , T 7 and T 9 treatments were 100%. The average root number of T 3 treatment was the highest (49.33 ± 6.17/plant), and the root effect index was the highest (16.20 ± 2.97).
Although the average root length of T 3 treatment was only 4.95 ± 0.36 cm, the  difference was not significant compared with that of T 7 treatment (5.90 ± 0.57 cm/plant) with the longest average root length. Therefore, T 3 treatment (A1B3-C3D3), i.e., the lower cuttings were dipped in 3000 mg/L NAA solution for 15 s, and cut in mixed matrix with peat soil, perlite and vermiculite ratio of 2:1:1, was the optimal solution in 9 treatments (T 1 -T 9 ).

Effects of Hormone Type on Rooting Indexes
As shown in Table 4, the rooting rate of cuttings treated with IBA alone was the highest and significantly higher than that treated with NAA alone. The average number of roots treated with NAA alone was the highest and significantly higher than that treated with IBA alone and NAA: IBA (2:3). Many researchers generally believed that rooting rate was the most important in four rooting indexes. So IBA was the best rooting hormone.

Effects of Hormone Concentration on Rooting Indexes
As shown in Table 4, the rooting rate and average root number of cuttings treated with 3000 mg/L hormone were the highest and significantly higher than those treated with 1000 mg/L hormone. Therefore, the optimal rooting hormone concentration was 3000 mg/L.
In  index, followed by hormone concentration. In short, according to the results of multiple comparison and range analysis of orthogonal experiment, A 2 B 3 C 3 D 2 , that was, using 3000 mg/L IBA to treat middle cuttings for 15 s, cutting in peat soil: perlite: vermiculite (2:1:1) mixed matrix, was the best theoretical scheme for cutting rooting.

Verification of Optimal Theoretical Scheme
From June to August 2021, the rooting indexes of the optimal theoretical scheme T L and the optimal orthogonal scheme T 3 were observed regularly. It was found that all the rooting indexes of T L treatment were higher than those of T 3 , and the rooting indexes of 50 d after treatment were higher than those of 60 d after treatment (Table 5). Therefore, T L treatment (A 2 B 3 C 3 D 2 ) was the best cutting rooting scheme.

Discussion
There are many factors affecting plant cutting propagation, including internal factors such as plant species (varieties), cutting age, sampling site, cutting specification, and external factors such as matrix, temperature, humidity, light, plant growth regulators [10] [11] [12] [13]. The results showed that substrate type was  [15]. In this study, four rooting indexes in the mixed matrix containing 50% peat soil were significantly higher than those in perlite for Ilex "China Girl". This may be because Ilex "China Girl" requires relatively high substrate humidity, but the water retention of perlite cannot meet its requirements.
Exogenous hormone treatment can significantly improve the rooting rate and rooting effect of cutting, but the optimal hormone types and concentrations of different plants are different [16] [17]. In this study, it was found that IBA was the optimal rooting hormone for Ilex "China Girl". When other treatments were the same as those in this study, the highest rooting rate of Ilex "China Girl" treated with ABT by Tian et al. was only 61.75%, and the average root number was only 8.61/plant [6]. In this study, the rooting rate was up to 100% and the number of roots was up to 45.67/plant after IBA treatment, which greatly improved the rooting rate and rooting effect of Ilex "China Girl". In addition, this study also found that the effect of 2000 -3000 mg/L hormone treatment was significantly better than 1000 mg/L hormone treatment, and with the increase of hormone concentration, the rooting rate and average root number were further improved. However, whether further increasing hormone concentration can further improve rooting rate and average root number still needs experimental verification.

Conclusion
In this study, the optimal cutting rooting scheme of "Chinese young girl" was developed, that is, using 3000 mg/L IBA to treat middle cuttings for 15 s, cutting in peat soil:perlite:vermiculite (2:1:1) mixed matrix. After the treatment, the cutting rooting time of Ilex "China Girl" was greatly shortened, and the rooting rate and rooting effect were greatly improved.