Effect of Activated Carbon on Rooting of Tissue Culture Seedlings of Qiuzi Pear

In order to improve the rooting rate of Qiuzi pear tissue culture seedlings, this study took ‘sorb’ as experimental material, based on Qiuzi pear rooting tissue culture medium (1/2MS+IBA 0.5 mg/L), and added different concentrations of activated carbon (AC): 0.25 g/L, 0.50 g/L, 0.75 g/L, 1.00 g/L. The results showed that the rooting rate, rooting number and root length of tissue culture seedlings cultured with activated carbon were significantly higher than those of the control group. The most suitable medium for rooting Qiuzi pear was 1/2MS+IBA 0.5 mg/L+AC 1.00 g/L, and the rooting rate of the treated ‘sorb’ tissue culture seedlings reached 90%, which could obviously promote the growth of Qiuzi pear tissue culture. The results provide a theoretical basis for improving the rooting rate and survival rate of tissue culture seedlings of pear plants, and provide a reference for the establishment of rapid propagation system of woody plants.


Effects of different AC concentrations on the growth of tissue culture seedlings of 'sorb'
On the 14 th day, red young roots appeared in the roots of 'sorb' tissue culture seedlings. With the increase of activated carbon concentration, the number of red young roots increased obviously. On the 21 st day, the roots grew Figure 1 Rooting of 'sorb' seedlings with different AC concentrations on the 28 th day Note: when the concentration of activated carbon is 0.00 g/L, the rooting status and Length of 'sorb' seedling; T1, when the concentration of activated carbon is 0.25 g/L, the rooting status and Length of 'sorb' seedling; T2, when the concentration of activated carbon is 0.50 g/L, the rooting status and Length of 'sorb' seedling; T3, when the concentration of activated carbon is 0.75 g/L, the rooting status and Length of 'sorb' seedling; T4, when the concentration of activated carbon is 1.00 g/L/ The rooting status and Length of 'sorb' seedlings Figure 2 Rooting of 'sorb'seedlings with different AC concentrations on the 35 th day Note: CK, activated carbon concentration is 0.00 g/L; T1, activated carbon concentration is 0.25 g/L; T2, activated carbon concentration is 0.50 g/L; T3, activated carbon concentration is 0.75 g/L; T4, activated carbon concentration is 1.00 g/L Figure 3 Rooting of 'sorb'seedlings with different AC concentrations on the 41 st day Note: CK, activated carbon concentration is 0.00 g/L; T1, activated carbon concentration is 0.25 g/L; T2, activated carbon concentration is 0.50 g/L; T3, activated carbon concentration is 0.75 g/L; T4, activated carbon concentration is 1.00 g/L 1.2 Effects of different AC concentrations on rooting rate and rooting number of 'sorb' seedlings in tissue culture As shown in Figure 4, on the 41 st day of culture, the rooting rates of T2 and T3 were both 50%, T4 was 90%, CK was the lowest, which was 40%. The rooting rates of rooting seedlings cultured with activated carbon were higher than those of the control group. The rooting rate of T1 was 80%. The rooting rate of T4 was significantly higher than that of CK, T2 and T3.
In different treatments, there were significant differences in the rooting number of pear tissue culture seedlings, which were 2.2 for T1, 2.2 for T2, 3.6 for T3, 6.2 for T4 and 0.8 for CK. As shown in Figure 5, with the increase of activated carbon concentration, the number of rooting strips increased. T4 was significantly higher than CK, T1 and T2. Figure 4 Effects of different AC concentrations on rooting rate of 'sorb' seedlings Note: CK, activated carbon concentration is 0.00 g/L; T1, activated carbon concentration is 0.25 g/L; T2, activated carbon concentration is 0.50 g/L; T3, activated carbon concentration is 0.75 g/L; T4, activated carbon concentration is 1.00 g/L Figure 5 Effects of different AC concentrations on rooting number of 'sorb' seedlings Note: CK, activated carbon concentration is 0.00 g/L; T1, activated carbon concentration is 0.25 g/L; T2, activated carbon concentration is 0.50 g/L; T3, activated carbon concentration is 0.75 g/L; T4, activated carbon concentration is 1.00 g/L

Effects of different AC concentrations on root morphological indexes of tissue culture seedlings of 'sorb'
Measure the average root length of 'sorb' seedlings under different treatments on the 41 st day ( Figure 6). It can be seen from the figure that the average root length of T4 is significantly different. Compared with the other four groups of results, the total root length is far greater than other treatments.
As shown in Figure 7, with the increase of AC concentration, the root surface area of 'sorb' seedlings increased. The root surface area of T4 was significantly higher than that of other treatments.
According to Figure 8, with the increase of AC concentration, the volume of Qiuzi pear seedlings increased. Molecular Plant Breeding 2021, Vol.12, No.10, 1-7 http://genbreedpublisher.com/index.php/mpb 4 Figure 6 Effects of different AC concentrations on total length of 'sorb' seedlings Note: CK, activated carbon concentration is 0.00 g/L; T1, activated carbon concentration is 0.25 g/L; T2, activated carbon concentration is 0.50 g/L; T3, activated carbon concentration is 0.75 g/L; T4, activated carbon concentration is 1.00 g/L Figure 7 Effects of different AC concentrations on the surface area of 'sorb' seedlings Note: CK, activated carbon concentration is 0.00 g/L; T1, activated carbon concentration is 0.25 g/L; T2, activated carbon concentration is 0.50 g/L; T3, activated carbon concentration is 0.75 g/L; T4, activated carbon concentration is 1.00 g/L Figure 8 Effects of different AC concentrations on seedling volume of 'sorb' Note: CK, activated carbon concentration is 0.00 g/L; T1, activated carbon concentration is 0.25 g/L; T2, activated carbon concentration is 0.50 g/L; T3, activated carbon concentration is 0.75 g/L; T4, activated carbon concentration is 1.00 g/L

Discussion
During the growth and development of plants, hormones in the body are in a dynamic process, and plant growth regulators can regulate the growth of tissue culture seedlings. Different varieties have different responses to the types and concentrations of growth regulators, and only by choosing appropriate hormone ratio can better proliferation effect be achieved . Qiu et al. (2015) thought that the most suitable rooting medium for North American pear was MS+TDZ 0.5 mg/L+ABT1 # 1.5 mg/L+Sucrose 20 g/L+Agar 4.5 g/L+AC 2.0 g/L, and the rooting rate was 52.03%. Zhang et al. (2009) thought that the most suitable rooting medium for Huangguan pear was 1/2MS+IBA 1.0 mg/L+ sucrose 20 g/L, and the rooting rate was 33%. Wen and Zhang (2007) thought that the most suitable rooting medium was 1/2 ms+NAA 1.0 mg/L+IBA 1.0 mg/L+AC 3.0 mg/L, and the rooting rate was 95%. Sun et al. (2001) thought that the medium for strong seedling of Fengshui pear was 1/2 MS+BA 0.5 mg/L+GA 0.5 mg/L+3% sucrose. The experiment found that 1/2MS+IBA 0.5 mg/L+AC 1.0 g/L was suitable for rooting culture of 'sorb', and the rooting rate was 90%. It is difficult to take root in tissue culture of Pyrus. One of the important factors affecting rooting of tissue culture seedlings is the type and concentration of auxin. IBA, IAA and NAA are commonly used growth regulators to promote rooting. IBA was used for rooting and tissue culture seedlings of seedless Rosa roxburghii (Jiang et al., 2017) and Douli (Li et al., 2012), but the suitable concentration was different. 0.2 mg/L IBA is suitable for seedless Rosa roxburghii and 1.5 mg/L IBA is suitable for bean pear. In this experiment, the rooting of 'sorb' seedlings in tissue culture was studied. The results showed that the most suitable rooting medium for 'sorb' seedlings was 1/2 ms+IBA 0.50 mg/L+AC 1.00 g/L.
Light conditions and culture methods also have effects on adventitious root induction of tissue culture seedlings. Tang et al. (2006) found that early dark culture promoted the rooting induction of 'Zaosu' and 'Shenbuzhi', but had poor rooting induction effect on 'Bali' and 'Komisi'. In this experiment, active carbon was added to provide a dark environment and placed under light conditions, which improved the rooting rate and rooting number of 'sorb'. Yang et al. (2008) induced Qiuzi pear to take root, the medium was 1/2MS+IBA 0.5 mg/L, the rooting rate was 45%, and the number of rooting strips was 3. When Wang et al. (2018) induced Qiuzi pear to take root, the medium was 1/2MS+IBA 0.5 mg/L, the rooting rate was 45.5%, and the number of rooting strips was 2.24. In this experiment, based on the rooting media used in Yang Fang and Wang Defen, activated carbon was added to promote the rooting of 'sorb' seedlings. The effect of the medium added with activated carbon was higher than that without activated carbon in rooting rate, rooting quantity, average root length, surface area and volume. When the concentration of activated carbon was 1 g/L (1/2MS+IBA 0.5 mg/L+AC 1.0 g/L), the rooting rate of 'sorb' seedlings was as high as 90%, and the number of rooting strips was 6.2. In this study, the efficiency and quality of tissue culture of 'sorb' seedlings were significantly improved by adding activated carbon, which provided reference for the tissue culture technology of fruit trees.

Test materials
The tissue culture seedlings of 'sorb' cultivated in Key Laboratory of Fruit Tree, College of Horticulture, Anhui Agricultural University were used as test materials.

Culture conditions
The activated carbon used in this experiment is black powder. When the culture medium is prepared, if it is added in advance, it will agglomerate and adhere to the pot wall. Therefore, it is advisable to add activated carbon after the culture medium melts and before sterilization. It is necessary to constantly shake the culture medium before solidification, so that it can be evenly distributed during solidification. Rooting medium was 1/2MS+IBA(0.5 mg/L)+AC (activated carbon concentration gradients were 0.25 g/L, 0.50 g/L, 0.75 g/L and 1.00 g/L, respectively). The medium was used after sterilization (sterilized in a sterilization pot at 121℃ for 20 minutes). The culture temperature is 25℃, the illumination is 2000 lx, and the illumination time is 24 h/d.

Test design
The experiment was conducted in November, 2020, with a single factor randomized block design. Take the tissue culture seedlings of 'sorb' in the same growth condition in the same period, and carry out increment culture on the tissue culture seedlings of 'sorb', and inoculate 2 plants in each bottle, totally inoculating 50 bottles. Taking tissue culture seedlings of 'sorb' as materials, more than one third of petiole was cut, and the petiole was beveled at 45 with a scalpel on an ultra-clean workbench. The cut seedlings of 'sorb' were put into rooting medium containing different concentrations of activated carbon for culture.

Index determination
Electronic vernier caliper and instruments regent scanner calibrated for image analysis with regent instruments software were used for determination and analysis. Rooting rate(%)=(number of rooted stem segments/number of inoculated stem segments)×100% Rooting number(number)=total root number of stem segment/stem segment number of rooting Average root length(cm)=total root length/total number of rooted plants

Data analysis
SPSS software (IBM SPSS statistics 26) was used for data statistical analysis, Duncan method was used for variance analysis and multiple comparison (p<0.05).

Authors' contributions
Luan Xiaolong is the executor of this experimental study, finishing data and writing the first draft of the paper; Shi Hao participated in experimental design and analysis of experimental results, Xu Bo and Zhang Qiannan participated in some experimental studies, and Li Liu was the project leader, guiding the design of experimental studies and the revision of papers. All authors read and approved to the final manuscript.