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Article

Study on the Establishment of Efficient Leaf Regeneration System in ‘Yuluxiang’ Pear

by
Xiaohua Cheng
1,
Weilong Zhang
1,
Yarui Wei
2 and
Yuxing Zhang
1,*
1
College of Horticulture, Hebei Agricultural University, Baoding 071000, China
2
Institute of Plant Protection, Hebei Academy of Agricultural and Forestry Sciences, Integrated Pest Management Innovation Centre of Hebei Province, Baoding 071000, China
*
Author to whom correspondence should be addressed.
Horticulturae 2025, 11(1), 77; https://doi.org/10.3390/horticulturae11010077
Submission received: 12 December 2024 / Revised: 5 January 2025 / Accepted: 9 January 2025 / Published: 13 January 2025
(This article belongs to the Section Propagation and Seeds)
Browse Figures
Versions Notes

Abstract

:
The ‘Yuluxiang’ pear is a key cultivated variety in China, celebrated for its high quality. However, it exhibits a low leaf regeneration frequency of only 35.0%, which hinders its transgenic breeding process. To establish an efficient regeneration system, we utilized tissue culture seedlings of the ‘Yuluxiang’ pear and investigated various factors influencing leaf regeneration: plant growth regulators, natural organic materials, leaf wounding and positioning methods, duration of dark culture, ages and lines of plantlets, as well as culture containers. Our results indicated that the optimal medium for leaf regeneration consisted of MS supplemented with 6-BA (6-Benzyl Aminopurine) 1.5 mg/L, NAA (α-Naphthalene acetic acid) 0.4 mg/L along with 10% (v/v) coconut water. Suitable wounding involved ensuring no damage to leaves while placing the abaxial side facing down on the medium; the ideal duration for dark culture was determined to be 21 days; optimal plantlet age was found to be 20 days; both plantlet line 1 and line 2 demonstrated effectiveness; triangle bottles were identified as appropriate culture containers. In summary, we successfully established an efficient leaf regeneration system for the ‘Yuluxiang’ pear that achieved a maximum regeneration frequency of 96.70% with an average bud number of 5.15 per explant. This system also proved effective for the ‘Qiuyue’ pear, yielding a regeneration frequency of 88.89% and an average bud number of 3.44 per explant. After investigating the germination methods of 200 leaves from this screened leaf regeneration system of ‘Yuluxiang’, it was found that there were both direct and indirect regeneration methods, and the germination rate of direct and indirect regeneration was 68.00% and 70.50%, respectively. Therefore, this study also laid a solid foundation for the future genetic transformation of ‘Yuluxiang’ pear.

1. Introduction

Pear is an important temperate fruit, which comprises at least 22 primary species and 10 naturally occurring interspecific hybrid taxa worldwide, with a wide distribution in Asia and Europe [1,2]. Like other woody fruit species, pears exhibit the disadvantages of high levels of heterozygosity and a long juvenile period, which means making genetic improvement by conventional is time-consuming and difficult [3,4].
Transgenic engineering breeding is widely used at present, which is a faster breeding process. The commonly used genetic transformation method is Agrobacterium-mediated leaf disk transformation, which has been successfully applied to various species, including tobacco [5], apples (‘Royal Gala’) [6], etc. An efficient and stable leaf regeneration system is fundamental for successful genetic transformation. It was reported that the regeneration frequency should be at least 80% to meet the requirement of genetic transformation [7]. However, it is widely recognized that pear leaf regeneration has significant challenges due to its inherent difficulty and instability, with considerable variation observed among different plant species. There are many factors that influence pear regeneration, including genotypes, plant growth regulators, explants, dark culture, etc. [8,9], in which the genotype is considered the most important factor [10,11]. In 1979, Lane researched regeneration in pear [12]; there were many pears reported, and their leaf regeneration frequency was from 0 to 100%. The regeneration rates of pears such as ‘Duli’ [13], ‘Korla Fragrant’ [14], ‘Farold®87’, ‘Conference’ [15], and ‘Shanli’ [16] were 33.0%, 84.9%, 53.3%, 87.3%, and 100.0%, respectively. Some varieties have difficulty with leaf regeneration, with a minimum regeneration rate 0 [8]. Another problem is that there is no medium widely applied to other plant species. So, it is still necessary to establish an efficient regeneration protocol for a new genotype.
The ‘Yuluxiang’ pear is an excellent medium-late maturing cultivar, which has a good quality of thin skin, delicate flesh, sweet taste and aroma, etc. [17]. The ‘Yuluxiang’ pear is the mainly cultivated cultivar, which is promoted in China and has been exported to lots of countries around the world [18]. However, the research on the ‘Yuluxiang’ pear is mainly focused on physiological and biochemical aspects [19,20], and studies about leaf regeneration were few and had a low frequency of 35.0% [21], which is not enough for the transgenic breeding of ‘Yuluxiang’ pears. Therefore, it is necessary to establish an efficient leaf regeneration system for the ‘Yuluxiang’ pear. At present, the research on pear leaf regeneration primarily focuses on the screening of basic media, plant growth regulators, and culture conditions. However, there were few reports about the impact of natural organic matter on pear leaf regeneration. In this study, we compared the effects of these factors on the ‘Yuluxiang’ pear leaf regeneration and extended our findings to other pear genotypes, aiming to establish an efficient and widely applicable system for pear leaf regeneration; we then laid the foundation for its study of genetic breeding.

2. Materials and Methods

2.1. Plant Materials and Culture Condition

The plant materials comprised tissue culture seedlings of ‘Qiuyue’((‘Niitaka’ × ’Housui’) × ‘Kosui’), ‘Xinli7’ (‘Kuerlexiangli’ × ‘Zaosu’), ‘Xueqing’ (‘Xuehuali’ × ‘Shinseiki’), ‘Huangjin’ (‘Niitaka’ × ‘Nijisseiki’), and 3 lines of ‘Yuluxiang’ (‘Kuerlexiangli’ × ‘Xuehuali’), which were subcultured every 30 days.
The following is the procedure obtained for the tissue culture seedling for 3 lines of ’Yuluxiang’ pear: In mid-April 2021 (after flowering for 5~10 days), the young, healthy branches from the same ‘Yuluxiang’ pear tree were collected from the specimen garden of our laboratory. The leaves of the explants were removed, leaving only the petiole. The single bud stem segment was cut to about 1.0 cm with pruning scissors and placed in a clean triangular bottle. It was rinsed with running water for 30 min, sterilized with 0.1% HgCl2 for 6 min, and then with 75% alcohol for 1 min. After rinsing three times with sterile water, it was dried using sterile filter paper. The explant was inoculated onto the subculture medium and cultured in the tissue culture room. The surviving plants were obtained after about 10 days. Proliferation of seedlings began after 3 months and we subcultured the seedlings every 30 days. There were 30 single bud stem explants that were collected in this test, and 6 (6 lines) survived in the end. Three robust lines were selected for propagation, which were the three plant lines used in this experiment.
The subculture medium was MS (Murashige and Skoog 1962), supplemented with 6-BA1.5 mg/L, IBA (Indole-3-butyric acid) 0.2 mg/L, sucrose 30.0 g/L, agar powder 6.0 g/L, PVA (Polyvinyl alcohol) 2.0 g/L (to inhibit the seedlings vitrification). The pH value for the medium was 5.8–6.0. The container used was a 100 mL triangle bottle, and each bottle had 40 mL of culture medium added. The test materials were the 1 to 3 young leaves from the upper part of the pear seedlings that had been cultured for about 20 days. The culture temperature was 25 ± 2 °C, with the light intensity at 25.0–37.5 µmol/m2/s1 (the light source was a white fluorescent lamp) and a photoperiod of 16 h/8 h (light/dark). The medium was sterilized in a 121 °C autoclave cooker for 20 min.

2.2. Drug Information

This part contains all the drug companies and product origin used in the study, which can achieve the same leaf regeneration effect when used by others later. MS (Coolaber, Beijing, China), Agar power (Haiyan, Qingdao, China), Sucrose (Solarbio, Beijing, China), 6-BA (6-Benzyl Aminopurine) (Solarbio, Beijing, China), Thidiazuron (TDZ) (Solarbio, Beijing, China), α-Naphthalene acetic acid (NAA) (Solarbio, Beijing, China), IBA (Indole-3-butyric acid) (Solarbio, Beijing, China), Polyvinyl alcohol (PVA) (Solarbio, Beijing, China), NaOH (Sodium hydroxide) (Solarbio, Beijing, China). Coconut water and meat were from the Aromatic Coconut (Cocos nucifera L.), which was purchased from Joyvio Corporation (Beijing, China). Ripe banana (Musa acuminata ×Musa balbisiana (AAA Group)‘Dwarf Cavendish’) fruits. Potatoes (Solanum tuberosum L.) were the Xiaotangshan potatoes. The bananas and potatoes were purchased from Jingdong Mall (Beijing, China) online.

2.3. The Screened of Plant Growth Regulators on the Leaf Regeneration of ‘Yuluxiang’ In Vitro

The screened mediums were MS with 0.2 mg/L IBA, sucrose 30.0 g/L, agar powder 6.0 g/L and PVA 2.0 g/L, added with 6-BA (1.5, 2.0, 3,0, and 5.0) mg/L or TDZ (0.2, 0.5, 1.0, 2.0, 3,0, 4.0, and 5.0) mg/L, and MS with 1.5 mg/L 6-BA, added with (0.4, 0.6) mg/L IBA or (0.2, 0.4, 0.6) mg/L NAA, 16 treatments total. The container used was a 100 mL triangle bottle, each bottle had 40 mL of culture medium added, and 15 leaves were placed in each bottle, totalling 4 bottles with 60 leaves.
Leaves with no wound were placed backside down on the medium, and 60 leaves were inoculated per treatment. These explants were incubated in the dark for 21 days and then moved to the light. The experiment was biologically replicated three times, and the regeneration frequency and number of adventitious shoots per explant were evaluated and recorded after 50 days total.

2.4. The Screened of Organic Matters on the Leaf Regeneration of ‘Yuluxiang’ In Vitro

In this experiment, the selected organic matters were potato, banana, coconut meat and coconut water, with concentrations set to 0%, 5%, 10%, 15%, 20%, 30%, and 50% (v/v) being added to the medium of MS with 6-BA 1.5 mg/L, and 0.2 mg/L IBA, sucrose 30.0 g/L, agar powder 6.0 g/L, and PVA 2.0 g/L. The container used was a 100 mL triangle bottle, each bottle had 50 mL culture medium added, and 15 leaves were placed in each bottle, totaling 4 bottles with 60 leaves per treatment. Leaves with no wound were placed backside down on the medium, and 60 leaves were inoculated per treatment. These explants were incubated in the dark for 21 days and then moved to the light. The regeneration frequency and number of adventitious shoots per explant were evaluated and recorded after 50 days total.
The steps for adding organic matter were as follows:
Added the prepared medium (MS + 6-BA1.5 mg/L + IBA 0.2 mg/L + sucrose 30.0 g/L + agar powder 6.0 g/L + PVA 2.0 g/L) into the 100 mL triangular bottle, and measured 50 mL medium to each bottle with a measuring cylinder. Then, weighed the organic matter at a concentration of 0%, 5%, 10%, 15%, 20%, 30%, and 50% (v/v), added it to the bottles, and adjusted the pH to 5.8–6.0 using 1 M NaOH one by one. Finally, sealed the bottles and autoclaved at 121 °C for 20 min for later use. Specific procedures are as follows:
Coconut water: Cut the coconut and obtained the fresh coconut water, directly took it into a measuring cylinder and poured it into each bottle according to the volume ratio.
Coconut meat: After removing the coconut water, chopped the white flesh and added it according to the volume ratio into each bottle.
Bananas: Used ripe bananas flesh, cut them up, weighed according to the volume ratio, and added them to each bottle.
Potatoes: Peeled and chopped potatoes, weighed them based on volume ratio, then added accordingly to each bottle.

2.5. The Sccreened of Leaf Wounding and Positioning Method, Dark Culture Time, Plantlet Ages and Lines on the ‘Yuluxiang’ Leaf Regeneration In Vitro

To further improve the leaf regeneration efficiency, dark culture time, plantlet ages and lines were screened. The leaf regeneration medium was determined based on the results of 2.3 and 2.4, the medium was MS with 6-BA 1.5 mg/L, 0.4 mg/L NAA, 10% coconut water (v/v), sucrose 30.0 g/L, agar powder 6.0 g/L, and PVA 2.0 g/L. The container used was a 100 mL triangle bottle, each bottle had 40 mL culture medium added, and 15 leaves were placed in each bottle, totaling 4 bottles with 60 leaves per treatment. The young, tender and unfolded leaves were chosen as the test materials. The leaf wounding methods were set to no wound, two wounds, and four wounds. The leaf positioning methods were set to the abaxial or adaxial side facing downwards on the medium. The dark culture time was set to (0, 7, 14, 21, 28, 35, 42, and 50) days; plantlet ages were set to (10, 15, 20, 25, 30, and 35) days; and three plantlet lines were selected for the experiment. Except for the leaf wounding and positioning method treatments, all leaves with no wound were placed backside down on the medium. Except for the dark culture time treatments, other treatments were incubated in the dark for 21 days and then moved to the light. Except for the plantlet ages and line treatments, all leaves were from the plantlets of 20 d age and line 2. The regeneration frequency and number of adventitious shoots per explant were evaluated and recorded after 50 days total.

2.6. The Screened of Cultured Containers on the Leaf Regeneration of ‘Yuluxiang’ In Vitro

The medium containers of the canning jar, 100 mL triangle flask, and petri dish (90 × 90 mm) were selected to carry out this experiment, and the leaf regeneration among them was compared; the medium was MS with 6-BA 1.5 mg/L, 0.4 mg/L NAA, 10% coconut water, sucrose 30.0 g/L and agar powder 6.0 g/L. In total, 40 mL of culture medium was added to each container, and 15 leaves were placed in each one, resulting in a total of 4 containers and 60 leaves per treatment. Leaves with no wound were placed backside down on the medium. These explants were incubated in the dark for 21 days and then moved to the light. The regeneration frequency and number of adventitious shoots per explant were evaluated and recorded after 50 days total.

2.7. The Screened ‘Yuluxiang’ Leaf Regeneration System Applied to Other Pear Genotypes

To determine the applicability of the screened ‘Yuluxiang’ leaf regeneration system to other pear genotypes, leaves from ‘Qiuyue’, ‘Duli’, ‘Xinli7’, ‘Xueqing’, and ‘Huangjin’ pears were selected for this experiment. The control was ‘Yuluxiang’ pear, and all the plantlet ages were 20 d. The leave regeneration medium was MS with 6-BA 1.5 mg/L, 0.4 mg/L NAA, 10% coconut water, sucrose 30.0 g/L, agar powder 6.0 g/L, and PVA 2.0 g/L. The container used was a 100 mL triangle bottle, each bottle had 40 mL culture medium added, and 15 leaves were placed in each bottle, totaling 4 bottles with 60 leaves per treatment. Leaves with no wound were placed backside down on the medium. These explants were incubated in the dark for 21 days and then moved to the light. The regeneration frequency and number of adventitious shoots per explant were evaluated and recorded after 50 days total.

2.8. Observation and Statistics of Leaf Regeneration Mode in the Screened ’Yuluxiang’ Leaf Regeneration System

To ensure the leaf regeneration mode in the screened ’Yuluxiang’ leaf regeneration system, we selected 200 leaves from 4 treatments applied to this ‘Yuluxiang’ leaf regeneration system in the experiment (including 21 d of dark culture treatment, treatment no damage to leaves while placing the abaxial side facing down on the medium, treatment with line 2, and treatment with 20 d plantlet age) to make the statistics on the leaf regeneration mode. That included the leaf number and frequency of regeneration of direct or indirect organogenesis, or both having direct and indirect organogenesis.
This screened ’Yuluxiang’ leaf regeneration system was as follows: the medium for leaf regeneration was (MS + 6-BA 1.5 mg/L + 0.4 mg/L NAA + sucrose 30.0 g/L + agar powder 6.0 g/L + PVA2.0 g/L + 10% coconut water (v/v) (pH = 5.8–6.0)); no damage to leaves while placing the abaxial side facing down on the medium; the dark culture time was 21 days; the plantlet age was 20 days; the materials were plantlet line 2; 100 mL triangle bottles were used culture containers.

2.9. Statistical Analysis

All experiments followed were arranged in a completely randomized design with three replicates, and each treatment contained 60 leaves. The data were analyzed using one-way ANOVA, followed by Duncan’s multiple range test. The significance level was set at p < 0.05. Data are expressed as the mean ± standard error (SE). All statistical analyses were conducted using SPSS ver. 22.0 (IBM, Armonk, NY, USA). The following were calculated:
Regeneration frequency (%) = number of regenerated adventitious bud explants/number of inoculated explants × 100%;
Adventitious shoots per explant = number of regenerated adventitious buds/number of inoculated explants.

3. Results

3.1. Effects of Plant Growth Regulators on the ‘Yuluxiang’ Leaf Regeneration In Vitro

During the process of subgeneration, we found an interesting phenomenon that the ‘Yuluxiang’ leaves could induce buds with no wound on the media (MS + 6-BA1.5 mg/L + IBA0.2 mg/L + agar powder 6.0 g/L + sugar 30 g/L + PVA2.0 g/L, pH 5.8–6.0) (Figure 1A), so it was considered that this subculture medium may be conducive to the leaf regeneration and budding of ‘Yuluxiang’ tissue culture seedlings. However, after using this medium for regeneration, the leaf regeneration frequency only was 30% (Figure 1B). Therefore, it was concluded that the leaves of ‘Yuluxiang’ tissue culture seedlings could regenerate buds on this subculture medium, but the regeneration frequency was low, so the hormone ratio should be optimized. According to our observation, we suspected that the reason for the low regeneration efficiency was the low concentration of hormones. Therefore, a single-factor experimental design was carried out in this experiment according to the hormones 6-BA1.5 mg/L and IBA0.2 mg/L. So, based on this, we started this experiment to screen the suitable plant growth regulators for the leaf regeneration of the ‘Yuluxiang’ pear; the results are presented in Table 1 and Figure 2.
When IBA concentration was at 0.2 mg/L, the 6-BA 2.0 mg/L treatment was significantly higher than the 1.5 and 5.0 mg/L 6-BA treatments, and the TDZ 1.0 mg/L treatment was significantly higher than 0.2, 0.5, 2.0, 4.0 and 5.0 mg/L TDZ treatments in regeneration frequency. There was no significant difference between 6-BA 2.0 mg/L and 6-BA 3.0 mg/L treatments in the regeneration frequency and bud number per explant, and both of them were significantly higher than other 6-BA and TDZ treatments. In general, compared with TDZ treatments, the 6-BA 2.0 mg/L, 3.0 mg/L, and IBA 0.2 mg/L were beneficial to the ‘Yuluxiang’ pear leaf regeneration, and the regenerate buds of most 6-BA treatments were longer than the TDZ treatments. When the concentration of 6-BA was 1.5 mg/L, the NAA was 0.4 mg/L, which was significantly higher than other treatments in regeneration frequency and the number of adventitious shoots per explant. Therefore, among the 16 treatments, the leaf regeneration effect of treatment 13 was better than others, whose 6-BA was 1.5 mg/L, and NAA was 0.4 mg/L, in which the leaf regeneration efficiency reached the peak, the regeneration frequency was 65.0%, and bud number per explant was 1.6.

3.2. Effects of Organic Matters on the ‘Yuluxiang’ Leaf Regeneration In Vitro

The results are shown in Figure 3. As the concentration of potato and coconut meat increased, both regeneration frequency and average bud number initially rose before declining. At a 5% concentration, peak values were reached: regeneration frequency of 66.67% and 57.78%, with average bud numbers of 2.33 and 1.34, respectively. Compared to the control, regeneration frequency increased by 0.31 and 1.89 times, while average bud numbers rose by 1.49 and 4.48 times. When the banana concentration was at 5% to 20%, leaf regeneration frequency and average bud regeneration number were higher than the control. At concentrations of 30–50%, both metrics significantly decreased compared to the control. The peak regeneration frequency (66.67%) and average bud number (1.76) occurred at a 5% concentration, which were 1.73 and 3.53 times higher than those of the control, respectively. For coconut water, when concentrations were at 5% to 30%, regeneration frequency and average bud number significantly exceeded control levels; however, at a concentration of 50%, its regeneration frequency dropped below control levels with no significant difference in average bud number compared to the control. At a concentration of 10%, peak values were achieved with a regeneration frequency of 90.94% and an average bud number of 5.85, which were 1.92 and 8.41 times higher than the control, respectively. In summary, adding an appropriate concentration of natural organic matter in the medium can promote the ‘Yuluxiang’ leaf regeneration, but it will significantly inhibit the leaf regeneration over a certain concentration. The addition of 5% organic matter of potato, banana, and coconut meat or 10% coconut water in the medium had a peak effect on the leaf regeneration of ‘Yuluxiang’ pear; however, when the concentration of potato exceeded 15%, coconut meat exceeded 20%, banana exceeded 30% and coconut water exceeded 50%, the leaf regeneration of ‘Yuluxiang’ pear was significantly inhibited. The inhibitory effect of different organic matters on the leaf regeneration was different in concentration. Compared with other organic matters, the inhibitory effect of coconut water on leaf regeneration was relatively small. Therefore, the suitable organic matter for the ‘Yuluxiang’ leaf regeneration was coconut water, and the suitable concentration was 10%, whose regeneration frequency was 90.94%, and the average number of regeneration buds was 5.85.

3.3. Effects of Leaf Wounding and Positioning Method, Dark Culture Time, Plantlet Ages and Lines on the ‘Yuluxiang’ Leaf Regeneration In Vitro

For the leaf wounding and positing method, when the leaf was with abaxial or adaxial side facing downwards on the medium, there was no significant difference in the leaf regeneration frequency between the no-wound and two-wound treatments, both of them significantly higher than the four-wound treatment (Figure 4 and Figure 5A). When the leaf wounding method was no-wound, the average number of regenerated buds of the leaf with the abaxial side facing downwards was significantly higher than other treatments (Figure 5B). In summary, the appropriate leaf wounding and positioning method for leaf regeneration was no-wound and the leaf with abaxial side facing downwards on the medium (Figure 5A,B). For the dark culture time, the regeneration frequency and bud number initially increased and then decreased extending from 0 d to 42 d. The treatment of 0 d had significantly lower regeneration frequency and bud number than other treatments, and the treatment at 42 d showed the second lowest. The peak regeneration frequency (93.33%) and bud number (3.98) occurred at 21 d of dark culture, indicating that the optimal dark culture time was 21 d (Figure 5C,D). Regarding plantlet ages, there were no significant differences in regeneration frequency among treatments at 15 d, 20 d, and 35 d; however, adventitious shoots per explant were highest at 20 d. Thus, the suitable plantlet age was 20 d, whose leaf regeneration frequency and average bud number were 91.63% and 5.15 (Figure 5E,F). For plantlet lines, all three exhibited a leaf regeneration frequency exceeding 85%, with no significant difference between line 1 and line 2 (both above 3.5), which was notably higher than line 3. Therefore, lines 1 and 2 were suitable for ‘Yuluxiang’ leaf regeneration (Figure 5G,H). In summary, the suitable leaf wound and position method was no-wound and the abaxial side facing downwards on the medium; the suitable dark culture time, plantlet ages and lines for ‘Yuluxiang’ leaf regeneration were 21 d, 20 d, and plantlet line 1 and line 2, respectively.

3.4. Effects of Culture Containers on the ‘Yuluxiang’ Leaf Regeneration In Vitro

There was no significant disparity between the triangular bottle and canning jar in the regeneration frequency and adventitious shoots per explant, both significantly higher than the plastic dish treatment, exceeding 90% and 3.5, respectively (Figure 6A,B). However, the leaves and regenerated bud vitrification in the canning jar were more severe than the triangular flask treatment (Figure 6C–E). In conclusion, the triangular flask was the suitable culture container for the ‘Yuluxiang’ leaf regeneration, whose leaf regeneration frequency was 96.70%, and the average bud number was 3.98.

3.5. The Adaptability of Screened ‘Yuluxiang’ Leaf Regeneration System to Other Pear Genotypes

The screened ‘Yuluxiang’ pear regeneration system was the result of 3.1 to 3.4. The results of applying this system to other pear genotypes are shown in Figure 7. There was no significant difference between ‘Qiuyue’ and ‘Yuluxiang’ in leaf regeneration frequency; both exceeded 80%, which was significantly higher than other treatments. Adventitious shoots per explant of ‘Qiuyue’ were significantly lower than ‘Yuluxiang’, but also over 3.0. There was no significant difference between ‘Xinli7’ and ‘Duli’ pears in regeneration frequency; both of them were low, about 10%, and the regeneration frequency of ‘Xueqing’ and ‘Huangjin’ pears was even lower at 0. Apart from ‘Yuluxiang’ and ‘Qiuyue’, the regenerated bud number was significantly lower than 1.0 for the other four varieties. In conclusion, the ‘Yuluxiang’ leaf regeneration system had a positive effect on the leaf regeneration of ‘Qiuyue’, with a frequency of 88.89%, and an average bud number of 3.44 (Figure 7). So, this system is suitable for ‘Qiuyue’ pear leaf regeneration but unsuitable for most pear cultivars.

3.6. Leaf Regeneration Mode in the Screened ’Yuluxiang’ Leaf Regeneration System

From Table 2 and Figure 8, it can be seen that both direct and indirect organogenesis exist in this ‘Yuluxiang’ leaf regeneration system, and the frequency of regeneration of direct organogenesis was 68.00%. The frequency of regeneration of indirect organogenesis was 70.50%. The frequency of regeneration of both direct and indirect organogenesis was 39.00%.
These data mean that this leaf regeneration system can achieve direct regeneration of buds, so it can be said that this system laid a good foundation for future transgenic breeding of ’Yuluxiang’.

4. Discussion

4.1. 6-BA and NAA Were the Suitable Plant Growth Regulators for the ‘Yuluxiang’ Leaf Regeneration In Vitro

6-BA and TDZ are the cytokinins that commonly being used for leaf regeneration, often in combination with auxins like NAA, IBA, or IAA; however, different varieties require specific hormone ratios [22]. In this experiment, we compared the effect of 6-BA and TDZ on ‘Yuluxiang’ leaf regeneration. As hormone concentrations are increased, vitrification worsens. Compared to 6-BA, buds of TDZ treatments were relatively smaller, and 6-BA promoted seedling elongation better than TDZ. Similar results have been observed in other varieties; for example, a bud regeneration rate of 55% was achieved with 6-BA while TDZ failed to induce bud formation in ‘Yali’ pear [23]. In wild pears as well, 6-BA outperformed TDZ for leaf regeneration with fewer vitrified seedlings [24]. However, TDZ was more effective than 6-BA in some varieties, like ‘Zhongli 1’ [25] and ‘Jinhua’ Pear [23]. Thus, the effectiveness of these cytokinins varies by variety. Generally speaking, TDZ has stronger activity than 6-BA and is suitable for hard-to-regenerate varieties, although it may not be universally applicable; therefore, screening for appropriate cytokinins is essential.
NAA is widely utilized in western pear species such as ‘Conference’, ‘Farold®87’, and ‘Abate Fetel’ [15], and IAA and IBA are widely used in Oriental pears, such as ‘Shanli’ [16] and ‘Dangshan’ pears [26]. However, we examined the regenerative effects of NAA and IBA at varying concentrations for the Oriental pear ‘Yuluxiang’, and the results indicated that NAA promoted better leaf regeneration compared to IBA; thus, NAA is deemed suitable for its leaf regeneration.

4.2. Natural Organic Matters Significantly Promoted the ‘Yuluxiang’ Pear Leaf Regeneration

In this experiment, we compared the leaf regeneration effects of four types of natural organic matter: potato, banana, coconut water, and coconut meat. It was found that adding an appropriate concentration (5%) of these organic matters to the medium significantly promoted the ‘Yuluxiang’ leaf regeneration. However, higher organic matter concentrations inhibited leaf regeneration at varying levels: 15% for potato, 20% for coconut meat, 30% for banana, and 50% for coconut water. Among them, coconut water required the highest concentration to show inhibition, and concentrations between 5% and 30% significantly enhanced leaf regeneration; notably, the coconut water outperformed other organic matters for ‘Yuluxiang’ pear leaf regeneration. The coconut types used in tissue culture are coconut water and coconut milk. Natural coconut water is derived from coconuts, while coconut milk is a mixture of coconut water and meat [27]. In 1941, Overbeek found that the ‘heart-shaped’ embryo of Datura grew well when supplemented with coconut milk [28]; nowadays, coconut milk and water are utilized in various species in tissue culture. For instance, adding 10% coconut milk to the medium aids embryos of ‘Ginkgo biloba’ developing into seedlings [29]. Incorporating coconut water during sugarcane callus differentiation can achieve a 100% differentiation rate [30]. Coconut milk or water is commonly used for orchids, particularly for secondary proliferation, rooting, or ball embryo induction [31], while its use in fruit trees is rarely reported, only in Japanese pear [32]. Natural organic matter can supply essential minerals, nitrogen sources and auxins like indole-3-acetic acid (IAA), gibberellins (GAs), cytokinin as 6-benzylaminopurine (6-BA) [33,34,35], and other uncertain organic composition, which is the reason why the organic matter can promote ‘Yuluxiang’ pear leaf regeneration in this experiment. In addition to the coconut milk or water, yeast extract, hydrolyzed casein (CH), apple [31,36,37], and orange juice [35] also have been investigated. However, these applications for leaf regeneration in fruit trees remain unreported, so we can also try to apply these organic matters to fruit tree regeneration in the future. The composition of natural organic matter is intricate due to unclear specific components. Future research can focus on the isolation and identification of organic components that promote leaf regeneration; then, these components can be commercially produced to improve the species that have difficulty in regeneration.

4.3. No-Wound Was the Optimal Wounding Method for the ‘Yuluxiang’ Leaf Regeneration

We often damage leaves to promote budding in leaf regeneration. A common method involves making two to three wounds in the leaf perpendicular to the vein, as in ‘Shanli’ [16], or making the leaf square, as in ‘Fupingqiuzi’ [38] or ‘Diospyros oleifera’ [39]. There are also some reports of cutting leaves into two upper and lower parts for regeneration, such as ‘Harrow delight’ and ‘Bartlett and Dargazi’ [40]. Leaf injury enhances the secretion of phenols and hormones, aiding callus formation. During wound healing, substances like AS (Acetylsyringone) are produced that promote callus differentiation; thus, wounds play a crucial role in plant leaf regeneration [36]. However, interestingly, prior to this experiment, we observed that ‘Yuluxiang’ leaves could regenerate buds with no wound on the subculture medium, which means that making wounds is not always essential for leaf regeneration. Based on these findings, we compared the regeneration effects of three methods including no-wound, two or four wounds (cut leaves into squares), results also showed that the leaf regeneration with no wound was better than other treatments, which was the suitable way for ‘Yuluxiang’ leaf regeneration; however, there was no report in other pear cultivars. The reason may be that the leaf structure of this variety is special because its leaves are less leathery than ‘Duli’, ‘Huangjin’, ‘Xinli7’, and ‘Xueqin’ leaves, and it is also easier to cut than them, which also was observed in ‘Qiuyue’ pear leaves. Presumably, this is the reason why ‘Qiuyue’ pears applied to the ‘Yuluxinag’ leaf regeneration system well, and both are easy to regenerate, but there is no report on this. Maybe we can estimate the difficulty level of leaf regeneration according to the leaf structure.

4.4. The Plantlet Lines Had a Significant Effect on the ‘Yuluxiang’ Leaf Regeneration

This experiment on three ‘Yuluxiang’ lines showed significant differences in the average number of shoots during leaf regeneration, indicating that the plant lines affected pear leaf regeneration. Research about the impact of different strains on leaf regeneration is limited; we only found a similar study in Pyrus betulifolia Bunge. For instance, Yang et al. [41] compared the effects of leaf regeneration across 18 strains of Pyrus betulifolia Bunge. and discovered there were significant variations in regeneration frequency among these strains, which ranged from 0 to 80%, with most exhibiting around 50%. In summary, there were significant differences in leaf regeneration capabilities among various strains within the same variety. For varieties that exhibit difficulty in leaf regeneration, it could be considered to screen different strains and to find the superior regenerative potential lines from them. Furthermore, it could be inferred that the issue of poor repeatability in pear leaf regeneration is associated with the specific strain utilized.

4.5. Culture Container Had a Significant Effect on the ‘Yuluxiang’ Leaf Regeneration

In this experiment, we found that the culture container had a significant effect on the ‘Yuluxiang’ leaf regeneration. The leaf regeneration effect of three culture containers from high to low was triangle bottle = can jar > plastic dish. Among them, the regeneration bud in the jar and plastic dish had a vitrification phenomenon, and the vitrification degree of the jar was more serious than others. Vitrification is related to many factors, which mainly are humidity and temperature; when the temperature and humidity in the bottle are too high, the vitrification seedlings will be produced [7]. During the culture process, there was heavier water vapor in the wall of the can bottle and the upper lid of the plastic dish, while there was no water vapor in the triangular bottle. So, it is more difficult to produce vitrification seedlings in the triangular bottle than in the can jar and plastic dish. Plants competed for space and air. In the plastic dish, there is smaller space than the other two containers and the sealing material of the plastic dish is film, which has the worst air permeability and least gas exchange with the outside. The inner space of the triangle bottle and jar is larger than the plastic dish. The sealing material for the triangle bottle is a plastic sealing film and for the jar is a plastic cap, both of which have a breathable 0.22 μm micron aperture aseptic film on the upper side, which has better air permeability and more gas exchange with the outside. The light transmittance of the jar is not as good as the triangle bottle, so the leaf regeneration sprouts are more robust in the triangle bottle [36]. In summary, the culture container has an important impact on leaf regeneration. If the leaf regeneration effect of a certain variety is not good, it may be the problem of the culture container. There are many kinds of culture containers in tissue culture, and three commonly used containers were selected in this experiment; perhaps other containers will have similarly good regeneration effects. This is a limitation of this experiment.

4.6. Applicability of the Leaf Regeneration System of ‘Yuluxiang’ to Other Genotypes

In this experiment, the ‘Yuluxiang’ leaf regeneration system was applied to four varieties of ‘Xinli7’, ‘Xueqing’, ‘Huangjin’ and ‘Qiuyue’ pear, along with one rootstock, ‘Duli’ pear. Results showed that this system effectively promoted the leaf regeneration of ‘Qiuyue’ pear whose regeneration frequency exceeded 80%, and the average number of regenerated buds was 3.0 to 4.0. But other varieties were less effective, and even the regeneration rate was 0, as in the ‘Xueqing’ and ‘Huangjin’ pears. There were few reports about leaf regeneration of multiple varieties on the same medium, but it did not work well. Xu et al. [42] reported a regeneration frequency of 77.8% for ‘Bayuehong’, while lower rates were observed for ‘Suisho’ (33.3%), ‘Dangshansu’ (38.9%), and ‘Zaosu’ pears (33.3%) in the same leaf regeneration medium. Additionally, bud numbers across these varieties were also low, with averages of 1.83, 0.83, 0.67, and 0.50, respectively. These findings indicated significant differences among genotypes in their adaptation to the same media. However, the ‘Yuluxiang’ leaf regeneration system demonstrated excellent adaptability specifically in ‘Qiuyue’ pear, confirming it had some applicability to other pear varieties, which also provided important references for the development of pear leaf regeneration technology.

5. Conclusions

Results revealed that the suitable leaf wounding method was no-wound; the optimal medium for ‘Yuluxiang’ leaf regeneration was (MS + 6-BA1.5 mg/L + NAA 0.4 mg/L + 10% (v/v) coconut water + sucrose 30.0 g/L + agar powder 6.0 g/L + PVA 2.0 g/L); the most suitable dark culture time was 21 d; the most suitable leaf regeneration plantlet age was 20 d; the most suitable plantlet lines were line 1 and line 2; the most suitable culture container was a triangle bottle. Based on these findings, an efficient leaf regeneration system for the ‘Yuluxiang’ pear was successfully established. In this system, the maximum regeneration frequency of ‘Yuluxiang’ reached 96.70%, and the maximum average number of regeneration buds was 5.15. When this system was applied to other pear genotypes, it was also suitable for the ‘Qiuyue’ pear leaf regeneration, whose regeneration frequency reached 88.89% with an average of 3.44 regeneration buds per explant. After investigating the germination methods of 200 leaves from this screened leaf regeneration system of ‘Yuluxiang’, it was found that there were both direct and indirect regeneration methods, and the germination rates of direct and indirect regeneration were 68.00% and 70.50%, respectively. Therefore, this study also laid a solid foundation for the future genetic transformation of the ‘Yuluxiang’ pear.

Author Contributions

Conceptualization, X.C. and Y.Z.; methodology, X.C. and Y.Z.; writing—original draft preparation, X.C.; writing—review and editing: W.Z., Y.W. and Y.Z.; supervision, W.Z., Y.W. and Y.Z.; project administration, W.Z., Y.W. and Y.Z.; funding acquisition, Y.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Pear Technology Innovation Center of Hebei Province and the Pear Industrial Technology Engineering Research Center of the Ministry of Education.

Data Availability Statement

All data are available in the manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. The regeneration of no-wound leaf when falling onto the medium during the subculture process and the effect of using this medium for regeneration. (A) The regeneration of no-wound leaf when falling onto the medium during the subculture process. (B) The effect of using this medium for regeneration. The medium was MS + 6-BA 1.5 mg/L + 0.2 mg/L IBA + sucrose 30.0 g/L + agar powder 6.0 g/L + PVA 2.0 g/L (pH = 5.8–6.0).
Figure 1. The regeneration of no-wound leaf when falling onto the medium during the subculture process and the effect of using this medium for regeneration. (A) The regeneration of no-wound leaf when falling onto the medium during the subculture process. (B) The effect of using this medium for regeneration. The medium was MS + 6-BA 1.5 mg/L + 0.2 mg/L IBA + sucrose 30.0 g/L + agar powder 6.0 g/L + PVA 2.0 g/L (pH = 5.8–6.0).
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Figure 2. Effects of plant growth regulators on the ‘Yuluxiang’ leaf regeneration in vitro; 1–4: MS + 6-BA (1.5, 2.0, 3.0, 5.0) mg/L + IBA 0.2 mg/L; 5–11: MS + TDZ (0.2, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0) mg/L + IBA 0.2 mg/L; 12–14: MS + 6-BA 1.5 mg/L + NAA (0.2, 0.4, 0.6) mg/L; 15–16: MS + 6-BA 1.5 mg/L + IBA (0.4, 0.6) mg/L.
Figure 2. Effects of plant growth regulators on the ‘Yuluxiang’ leaf regeneration in vitro; 1–4: MS + 6-BA (1.5, 2.0, 3.0, 5.0) mg/L + IBA 0.2 mg/L; 5–11: MS + TDZ (0.2, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0) mg/L + IBA 0.2 mg/L; 12–14: MS + 6-BA 1.5 mg/L + NAA (0.2, 0.4, 0.6) mg/L; 15–16: MS + 6-BA 1.5 mg/L + IBA (0.4, 0.6) mg/L.
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Figure 3. Effect of different kinds of organic matters on ‘Yuluxiang’ leaf regeneration. (A,B) Regeneration frequency and adventitious shoots per explant in different kinds of organic matter treatments. The statistical significance of the differences was determined by Duncan’s multiple range test, and the different letters in rows represent significant differences (p < 0.05). (C–F) The types of organic matter used are Potato, Banana, Coconut meat, Coconut water, in order. 0–50%: The concentration of different kinds of organic matter (v/v).The medium was MS + 6-BA 1.5 mg/L + 0.2 mg/L IBA + sucrose 30.0 g/L + agar powder 6.0 g/L + PVA 2.0 g/L + different concentrations organic matters (v/v). (pH = 5.8–6.0).
Figure 3. Effect of different kinds of organic matters on ‘Yuluxiang’ leaf regeneration. (A,B) Regeneration frequency and adventitious shoots per explant in different kinds of organic matter treatments. The statistical significance of the differences was determined by Duncan’s multiple range test, and the different letters in rows represent significant differences (p < 0.05). (C–F) The types of organic matter used are Potato, Banana, Coconut meat, Coconut water, in order. 0–50%: The concentration of different kinds of organic matter (v/v).The medium was MS + 6-BA 1.5 mg/L + 0.2 mg/L IBA + sucrose 30.0 g/L + agar powder 6.0 g/L + PVA 2.0 g/L + different concentrations organic matters (v/v). (pH = 5.8–6.0).
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Figure 4. Picture of leaf wounding and positioning method.
Figure 4. Picture of leaf wounding and positioning method.
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Figure 5. Effects of different plant lines on the ‘Yuluxiang’ leaf regeneration. (A,B) Regeneration frequency and adventitious shoots per explant in different kinds of wounding and positing method treatments; (C–H) regeneration rate and adventitious shoots per explant in different kinds of dark culture time, plantlet ages and line treatments. The statistical significance of the differences was determined by Duncan’s multiple range test, and the different letters in rows represent significant differences (p < 0.05). The medium was MS + 6-BA 1.5 mg/L + 0.4 mg/L NAA + sucrose 30.0 g/L + agar powder 6.0 g/L + PVA2.0 g/L + 10% coconut water (v/v) (pH = 5.8–6.0).
Figure 5. Effects of different plant lines on the ‘Yuluxiang’ leaf regeneration. (A,B) Regeneration frequency and adventitious shoots per explant in different kinds of wounding and positing method treatments; (C–H) regeneration rate and adventitious shoots per explant in different kinds of dark culture time, plantlet ages and line treatments. The statistical significance of the differences was determined by Duncan’s multiple range test, and the different letters in rows represent significant differences (p < 0.05). The medium was MS + 6-BA 1.5 mg/L + 0.4 mg/L NAA + sucrose 30.0 g/L + agar powder 6.0 g/L + PVA2.0 g/L + 10% coconut water (v/v) (pH = 5.8–6.0).
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Figure 6. Effects of culture containers on the ‘Yuluxiang’ leaf regeneration. (A,B) Regeneration frequency and adventitious shoots per explant in different kinds of culture containers. (C) The 100 mL triangular flask; (D) canning jar; (E) plastic dish. The statistical significance of the differences was determined by Duncan’s multiple range test, and the different letters in rows represent significant differences (p < 0.05). The medium was MS + 6-BA 1.5 mg/L + 0.4 mg/L NAA + sucrose 30.0 g/L + agar powder 6.0 g/L + PVA2.0 g/L + 10% coconut water (v/v) (pH = 5.8–6.0).
Figure 6. Effects of culture containers on the ‘Yuluxiang’ leaf regeneration. (A,B) Regeneration frequency and adventitious shoots per explant in different kinds of culture containers. (C) The 100 mL triangular flask; (D) canning jar; (E) plastic dish. The statistical significance of the differences was determined by Duncan’s multiple range test, and the different letters in rows represent significant differences (p < 0.05). The medium was MS + 6-BA 1.5 mg/L + 0.4 mg/L NAA + sucrose 30.0 g/L + agar powder 6.0 g/L + PVA2.0 g/L + 10% coconut water (v/v) (pH = 5.8–6.0).
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Figure 7. Effects of ‘Yuluxiang’ leaf regeneration system on other pear genotypes leaves. (A,B) The leaf regeneration frequency and the number of adventitious shoots per explant from various pear genotypes. The statistical significance of the differences was determined by Duncan’s multiple range test, and the different letters in rows represent significant differences (p < 0.05). (C–H) Leaf regeneration of ‘Yuluxiang’, ‘Qiuyue’, ‘Duli’, ‘Xinli7’, ‘Xueqing’, and ‘Huangjin’ pears, respectively. The medium was MS + 6-BA 1.5 mg/L + 0.4 mg/L NAA + sucrose 30.0 g/L + agar powder 6.0 g/L + PVA2.0 g/L + 10% coconut water (v/v) (pH = 5.8–6.0).
Figure 7. Effects of ‘Yuluxiang’ leaf regeneration system on other pear genotypes leaves. (A,B) The leaf regeneration frequency and the number of adventitious shoots per explant from various pear genotypes. The statistical significance of the differences was determined by Duncan’s multiple range test, and the different letters in rows represent significant differences (p < 0.05). (C–H) Leaf regeneration of ‘Yuluxiang’, ‘Qiuyue’, ‘Duli’, ‘Xinli7’, ‘Xueqing’, and ‘Huangjin’ pears, respectively. The medium was MS + 6-BA 1.5 mg/L + 0.4 mg/L NAA + sucrose 30.0 g/L + agar powder 6.0 g/L + PVA2.0 g/L + 10% coconut water (v/v) (pH = 5.8–6.0).
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Figure 8. Statistical situation of leaf regeneration mode in ’Yuluxiang’ leaf regeneration system. (A) Direct organogenesis; (B) indirect organogenesis; (C) both direct and indirect organogenesis. The medium was MS + 6-BA 1.5 mg/L + 0.4 mg/L NAA + sucrose 30.0 g/L + agar powder 6.0 g/L + PVA2.0 g/L + 10% coconut water (v/v) (pH = 5.8–6.0).
Figure 8. Statistical situation of leaf regeneration mode in ’Yuluxiang’ leaf regeneration system. (A) Direct organogenesis; (B) indirect organogenesis; (C) both direct and indirect organogenesis. The medium was MS + 6-BA 1.5 mg/L + 0.4 mg/L NAA + sucrose 30.0 g/L + agar powder 6.0 g/L + PVA2.0 g/L + 10% coconut water (v/v) (pH = 5.8–6.0).
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Table 1. Effects of plant growth regulators on the ‘Yuluxiang’ leaf regeneration in vitro.
Table 1. Effects of plant growth regulators on the ‘Yuluxiang’ leaf regeneration in vitro.
TreatmentThe Concentration of Plant Growth
Regulators		Regeneration Frequency (%)Adventitious Shoots per Explant
6-BA (mg/L)TDZ (mg/L)IBA
(mg/L)		NAA
(mg/L)
11.5-0.2-43.3 ± 3.3cde1.0 ± 0.1cd
22.0-0.2-56.7 ± 3.3ab1.4 ± 0.2b
33.0-0.2-51.7 ± 7.6bc1.5 ± 0.1b
45.0-0.2-36.7 ± 2.9ef0.7 ± 0.1ef
5-0.20.2-27.8 ± 6.9f0.5 ± 0.1fg
6-0.50.2-37.0 ± 3.4ef0.9 ± 0.2cd
7-1.00.2-50.6 ± 2.7bcd0.9 ± 0.1cd
8-2.00.2-30.0 ± 0.0f0.5 ± 0.2g
9-3.00.2-45.5 ± 1.5cde0.8 ± 0.1de
10-4.00.2-18.9 ± 5.1g0.4 ± 0.1g
11-5.00.2-30.4 ± 8.3f0.7 ± 0.0ef
121.5--0.247.1 ± 2.8cd1.0 ± 0.1c
131.5--0.465.0 ± 8.7a1.6 ± 0.0a
141.5--0.645.0 ± 5.0cde0.7 ± 0.0ef
151.5-0.4-51.7 ± 2.9bc1.4 ± 0.0b
161.5-0.6-43.3 ± 2.9de0.8 ± 0.1de
The statistical significance of the differences was determined by Duncan’s multiple range test, and the lowercase letters indicated the statistically significant differences between each treatment (p < 0.05).
Table 2. Statistical situation of leaf regeneration mode in the ’Yuluxiang’ leaf regeneration system.
Table 2. Statistical situation of leaf regeneration mode in the ’Yuluxiang’ leaf regeneration system.
Total Leaves NumberLeaf Number of Direct OrganogenesisFrequency of Regeneration of Direct Organogenesis (%)Leaf Number of
Indirect Organogenesis		Frequency of Regeneration of Indirect Organogenesis (%)Leaf Number of Both Direct and Indirect OrganogenesisFrequency of Regeneration of Both Direct and Indirect Organogenesis (%)
20013668.00%14170.50%7839.00%
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Cheng, X.; Zhang, W.; Wei, Y.; Zhang, Y. Study on the Establishment of Efficient Leaf Regeneration System in ‘Yuluxiang’ Pear. Horticulturae 2025, 11, 77. https://doi.org/10.3390/horticulturae11010077

AMA Style

Cheng X, Zhang W, Wei Y, Zhang Y. Study on the Establishment of Efficient Leaf Regeneration System in ‘Yuluxiang’ Pear. Horticulturae. 2025; 11(1):77. https://doi.org/10.3390/horticulturae11010077

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Cheng, Xiaohua, Weilong Zhang, Yarui Wei, and Yuxing Zhang. 2025. "Study on the Establishment of Efficient Leaf Regeneration System in ‘Yuluxiang’ Pear" Horticulturae 11, no. 1: 77. https://doi.org/10.3390/horticulturae11010077

APA Style

Cheng, X., Zhang, W., Wei, Y., & Zhang, Y. (2025). Study on the Establishment of Efficient Leaf Regeneration System in ‘Yuluxiang’ Pear. Horticulturae, 11(1), 77. https://doi.org/10.3390/horticulturae11010077

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