In Vitro Immature Embryo Culture of Paeonia ostii ‘Feng Dan’

in HortScience
Authors:
Li XuBeijing Advanced Innovation Center for Tree Breeding by Molecular Design, Beijing Forestry University, Beijing, China; Peony International Institute, School of Landscape Architecture, Beijing Forestry University, Beijing, China; Beijing Key Laboratory of Ornamental Plants Germplasm Innovation and Molecular Breeding, Beijing Forestry University, Beijing, China; Key Laboratory of Genetics and Breeding in Forest Trees and Ornamental Plants of Ministry of Education, Beijing Forestry University, Beijing, China; and National Engineering Research Center for Floriculture, Beijing Forestry University, Beijing, 100083, China

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Fangyun ChengBeijing Advanced Innovation Center for Tree Breeding by Molecular Design, Beijing Forestry University, Beijing, China; Peony International Institute, School of Landscape Architecture, Beijing Forestry University, Beijing, China; Beijing Key Laboratory of Ornamental Plants Germplasm Innovation and Molecular Breeding, Beijing Forestry University, Beijing, China; Key Laboratory of Genetics and Breeding in Forest Trees and Ornamental Plants of Ministry of Education, Beijing Forestry University, Beijing, China; and National Engineering Research Center for Floriculture, Beijing Forestry University, Beijing, 100083, China

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Yuan ZhongBeijing Advanced Innovation Center for Tree Breeding by Molecular Design, Beijing Forestry University, Beijing, China; Peony International Institute, School of Landscape Architecture, Beijing Forestry University, Beijing, China; Beijing Key Laboratory of Ornamental Plants Germplasm Innovation and Molecular Breeding, Beijing Forestry University, Beijing, China; Key Laboratory of Genetics and Breeding in Forest Trees and Ornamental Plants of Ministry of Education, Beijing Forestry University, Beijing, China; and National Engineering Research Center for Floriculture, Beijing Forestry University, Beijing, 100083, China

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Paeonia ostii ‘Feng Dan’ is an economically important, multipurpose woody plant in terms of its medical, ornamental, and oil values; however, there is a noticeable contradiction between the increasing demands and the lack of excellent germplasm resources because of traditional breeding and propagation approaches. In vitro embryo culture is an attractive option for this issue. This study presents a protocol for in vitro immature embryo culture in P. ostii ‘Feng Dan’, which involves two steps: 1) immature seeds at 30 days after anthesis (DAA) (cellularization stage of endosperm, proembryo stage) or after being cultured in vitro for cotyledon embryo formation (upward micropyle with placenta was the best inoculation method with the highest ratio of seed with cotyledon embryo of 66.67%); and 2) seedling establishment was realized within 7 months via embryo (at 40 DAA or after) germination, shoot induction, rooting, and acclimatization. The multiplication potential was increased with embryo maturity. This protocol provides an available reference for embryo rescue and propagation of tree peony and will be beneficial to shortening the breeding cycle.

Abstract

Paeonia ostii ‘Feng Dan’ is an economically important, multipurpose woody plant in terms of its medical, ornamental, and oil values; however, there is a noticeable contradiction between the increasing demands and the lack of excellent germplasm resources because of traditional breeding and propagation approaches. In vitro embryo culture is an attractive option for this issue. This study presents a protocol for in vitro immature embryo culture in P. ostii ‘Feng Dan’, which involves two steps: 1) immature seeds at 30 days after anthesis (DAA) (cellularization stage of endosperm, proembryo stage) or after being cultured in vitro for cotyledon embryo formation (upward micropyle with placenta was the best inoculation method with the highest ratio of seed with cotyledon embryo of 66.67%); and 2) seedling establishment was realized within 7 months via embryo (at 40 DAA or after) germination, shoot induction, rooting, and acclimatization. The multiplication potential was increased with embryo maturity. This protocol provides an available reference for embryo rescue and propagation of tree peony and will be beneficial to shortening the breeding cycle.

Tree peony (Paeonia sect. Moutan) is an economically important woody plant used for medical, ornamental, and oil production purposes (Yu et al., 2016). In 2014, the seed oil of tree peony was approved as a new food resource by the Chinese Ministry of Health because of its high levels of unsaturated fatty acids, especially α-linolenic acid (Shi et al., 2014). P. ostii ‘Feng Dan’, one of the landraces of tree peonies, has been widely cultivated for oil production because of its high seed yield and climatic and regional adaptability. However, there is a noticeable contradiction between the increasing demands and the lack of excellent germplasm resources. It usually takes more than 10 years to develop a new tree peony variety through traditional breeding methods because of cross-incompatibility, embryo dysplasia, dormancy, scanty germination rates, and low efficiency propagation (Cheng, 2007). However, tissue culture seems to be an attractive tool for overcoming these bottlenecks for suitable applications in breeding and propagation programs.

For P. ostii ‘Feng Dan’, the differentiation rate (45.83%), rooting rate (43.33%), and, consequently, survival rate (45.83%) were recorded via meristematic nodules culture using cotyledons at 90 DAA as explants to induce callus. However, it takes more than 1 year to obtain survival plantlets because of the long induction time of the nodules from callus (Xu et al., 2022). The frequency of 48% somatic embryogenesis and mean number of five somatic embryos were obtained for the flare tree peony P. rockii ‘Jing Hong’, but the high deformity resulted in lower germination (45%), and no survival plantlets were obtained (Du et al., 2020). Efficient micropropagation methods for tree peony have been established through nearly three decades of research, and the high multiplication rate (≥3.0), rooting rate (≥50%), and survival rate (≥60%) for several varieties were realized through the optimization of cultivation conditions, but only using buds as explants (Wen et al., 2020). Embryo culture is a tissue culture intervention usually adopted to overcome the postfertilization developmental barriers of the growing embryos; on the basis of the stage and histological origin of isolated embryos, the embryo culture is classified into mature embryo culture and immature embryo culture (embryo rescue). This tissue culture method has been widely applied for rescuing rare hybrids of Ziziphus jujuba Mill. (Ren et al., 2019), Rosa L. (Abdolmohammadi et al., 2014), Ilex crenata Thunb. (Yang et al., 2015), and Vitis vinifera L. (Li et al., 2014), and for realizing mass propagation using embryos as explants of Camellia oleifera Abel. (Li et al., 2016), Castanea henryi Rehd. (Xiong et al., 2018), Abelmoschus esculentus L. (Irshad et al., 2018).

Some advancements in embryo culture of peony have been made recently. Rapid seedling formation within 40 d was reported by in vitro germination of mature embryos (90 DAA) of P. ostii ‘Feng Dan’, but propagation cannot be achieved (Xu et al., 2017). It was found that the explant type (immature vs. mature embryos) could influence the successful embryo culture of herbaceous peony, with higher germination rates and seedling rates for mature embryos (>90 DAA) than for immature embryos (50–70 DAA) (Shen et al., 2015). Among other factors, immature embryo culture could provide prerequisites for successful embryo rescue (Uma et al., 2011; Varshney and Johnson, 2010). No literature regarding the success of embryo rescue or in vitro immature embryo (before 65 DAA) culture of the tree peony is available (He, 2006). However, the protocol for shoot multiplication for tree peonies with buds, but not with embryos as explants, has been reported (Beruto et al., 2004; Wen et al., 2016a, 2016b, 2016c).

Therefore, the objective of this study was to achieve successful in vitro immature embryo culture of P. ostii ‘Feng Dan’. The first step was to obtain more cotyledon embryos through in vitro immature embryo culture with seeds carrying young embryos as explants. Then, the effects of the inoculation methods and development stage were evaluated. The second step was to establish the seedlings via embryo germination, shoot induction and multiplication, rooting, and acclimatization. Our results will provide an available reference for embryo rescue of the tree peony and for achieving mass propagation with embryos as explants. Furthermore, these results will be beneficial to shortening the breeding cycle.

Materials and Methods

Plant material and sterilization.

Experiments were performed over the course of 2 consecutive years (2017 and 2018) to highlight the best method of seed germination that could be considered for explant excision and to determine how the explant orientation and the presence of placenta could affect further embryo development. In 2017, follicles of P. ostii ‘Feng Dan’ with immature seeds at 50 DAA (globular embryo) were collected (Fig. 1A and B), and different inoculation methods (upward micropyle with or without placenta, downward micropyle with or without placenta) were tested. In 2018, follicles were collected at different DAA (5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, and 60 DAA) (Fig. 1C and D). The length and width of immature seeds were measured with a ruler. Robust donor plants (nearly 10 years old) have been grown in Beijing Guose Peony Garden in Beijing, China (lat. 40°45′N, long. 115°97′E).

Fig. 1.
Fig. 1.

Explants of Paeonia ostii ‘Feng Dan’ used for this study. (A) Follicles of P. ostii ‘Feng Dan’ [50 days after anthesis (DAA)] collected in 2017. (B) Anatomical observation of P. ostii ‘Feng Dan’ seeds (50 DAA, globular embryo stage) collected in 2017. (C) Follicles of P. ostii ‘Feng Dan’ from 5 to 60 DAA at intervals of 5 d collected in 2018. (D) Anatomical observation of P. ostii ‘Feng Dan’ seeds from 30 to 60 DAA at intervals of 5 d collected in 2018.

Citation: HortScience 57, 5; 10.21273/HORTSCI16477-21

Follicles were washed under running tap water for 15 min before exposure to ultraviolet lamp for 30 min. Then, they were sterilized by dipping in ethanol (70% v/v; 3 min), followed by dipping in a solution of NaOCl (0.2% v/v; 10 min) and rinsed three times with sterile distilled water. Immature seeds were aseptically isolated from follicles and transferred in the inoculation medium.

Media and culture conditions.

All media were supplemented with 3% sucrose and 0.7% agar, and the pH was adjusted to 5.8 to 6.0 before autoclaving (at 118 kPa and 121°C for 20 min). All reagents were supplied by Biodee (Beijing, China).

The initiation medium consisted of modified Murashige and Skoog (mmS, half-strength macroelements and full-strength Ca2+) medium (Murashige and Skoog, 1962) supplemented with 1.29 μM 6-benzyladenine (BA) + 0.72 μM gibberellin (GA3). After 30 d of culture, the seedcoats and endosperm were removed using sterilized scalpels and tweezers, and the ratio of seeds with cotyledon embryo (%) was calculated. Each experiment consisted of three replicates of 300 explants per treatment.

The cotyledon embryos obtained from seeds from 30 to 60 DAA immature seeds were inoculated on germination medium (mmS + 1.29 μM BA); after 30 d of culture, the germination rate (%), height (cm), and shoot length (cm) of seedlings were calculated. Each experiment consisted of three replicates of 65 explants per treatment.

To assess the multiplication efficiency, cotyledon nodes (≈1.0 cm long) were excised from the germinated embryos and cultured in modified woody plant medium [mWPM, double-strength Ca(NO3)2⋅4H2O] (Lloyd and McCown, 1980) supplemented with 2.57 μM BA + 0.58 μM GA3 for shoot proliferation. Shoot cluster was developed after 40 d, and the axillary shoots (≥1 cm) induced were excised together with the rest of the shoot clusters and transferred to new medium with the same composition with a subculture cycle of 40 d. Height (cm), number of leaves (n), and number of shoots (n) were calculated after the first, second, and third subcultures, respectively. Each experiment consisted of three replicates of 35 explants per treatment.

Cultures, unless otherwise stated, were maintained at 24 ± 1 °C under a 16-h photoperiod of 50 μmol⋅m−2⋅s−1 illumination intensity provided by LED light (70% red light + 30% blue light) (TLD 36 W; Philips, Beijing, China).

At the end of the multiplication phase, shoots were rooted according to the protocol described by Wang et al. (2016). Shoots (≥1 cm) were cultured on root induction medium [1/2 MS (all macroelements at half-strength) + 4.92 μM indolebutyric acid + 11.34 μM putrescine) for 38 d in the dark; then, they were transferred to root expression medium [PGR-free 1/2 MS medium containing 0.4% activated carbon (AC)] for 20 d in the light. During the root induction phase, the shoots were subjected to cold treatment (4 °C, 8 d) in the dark and then cultured at 24 ± 1 °C for another 30 d. The rooting rate (%) was calculated. Each experiment consisted of three replicates of 35 explants per treatment.

The rooted plantlets were washed thoroughly with tap water to remove gelled medium residues. Finally, they were transferred in vivo to pots containing a mix of an autoclaved vermiculite, peat, and perlite (1:1:1, v/v/v) substrate. The plantlets were grown in a culture chamber at 20 ± 1 °C under a 16-h photoperiod of 50 μmol⋅m−2⋅s−1 photosynthetic photon flux density provided by fluorescent lamps. Survival rates (%) were recorded 2 months after acclimatization. There were three replicates of five rooted plantlets per treatment.

Histological analysis.

Immature seeds at different DAA (5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, and 60 DAA) were excised from follicles and fixed in a mixture of formaldehyde, glacial acetic acid, and 50% alcohol (1:1:18) for 48 h. The fixed samples were dehydrated in a series of graded alcohol (50%, 70%, 85%, 95%, and 100%) at 1 h per level, submerged in absolute ethanol and xylene (1:1) for 2 h, and dipped in pure xylene for 2 h. Samples were embedded in paraffin wax overnight and sectioned at 8 to 10 μm on a rotary microtome. The sections were dried overnight and exposed to a xylene-ethanol series to remove the paraffin. Then, they were stained with 0.1% safranin and 0.1% fast green. A Leica model DM500 microscope (Germany) was used for histological analysis.

Statistical analysis.

Before data analysis, arcsine transformation was performed for any percentage data. The statistical analysis was performed with SPSS 17.0 (SPSS Inc., Chicago, IL). Microsoft Excel 2013 software (Microsoft Corp., Redmond, WA) was used for data statistics and charts. The data were subjected to an analysis of variance, followed by Duncan’s multiple range test at P ≤ 0.05, and expressed as the mean ± se.

Results

Development of immature embryos (seeds) used as explant.

The endosperm development of P. ostii ‘Feng Dan’ is of the nuclear type. The free nucleus was divided continuously without the cell wall around the periphery of the embryo sac during 5 to 25 DAA (Fig. 2A and B). The cellularization of endosperm was noticed during 30 to 45 DAA, and the walls formed between the free nuclei, separating them into cells (Fig. 2C). Sections revealed a centripetal increase in endosperm cellularization (Fig. 2D and E) corresponding to the conversion from fluid endosperm and gradually into the solid state phenotypically; the endosperm in the solid state filled the entire embryo sac at 45 DAA (Fig. 2F). In our observation, the proembryo stage lasted 5 to 45 DAA, and the proembryo sequentially developed into the globular embryo (50 DAA) (Fig. 2G), torpedo-shaped embryo (55 DAA) (Fig. 2H), and cotyledon embryos (60 DAA) (Fig. 2I).

Fig. 2.
Fig. 2.

Histological observation of immature seeds of Paeonia ostii ‘Feng Dan’. (A–I) Immature seeds at 5, 25, 30, 35, 40, 45, 50, 55, and 60 days after anthesis (DAA), respectively. (A) Free nuclear endosperm (black arrow). (B) Free nuclear endosperm (black arrow) divided continuously around the periphery of the embryo sac. (C) Cellularization of endosperm (black arrow). (D, E) Centripetal increase of cellularization endosperm cells. (E) Endosperm filled the entire embryo sac. (G) Globular embryo. (H) Torpedo-shaped embryo. (I) Cotyledon embryo.

Citation: HortScience 57, 5; 10.21273/HORTSCI16477-21

Initiation stage of in vitro embryo culture.

Table 1 shows that inoculation methods significantly influenced the success of the culture initiation. Immature seeds (embryos) in P. ostii ‘Feng Dan’ collected after 50 DAA at the globular stage developed into phenotypically visible cotyledon embryos only when upward micropyle was used as the inoculation method. However, significant differences were scored with this method whether the placenta was included or not (66.7% or 48.89% conversion percentage from the globular stage to the cotyledonary stage of embryos whether the placenta was included or not, respectively).

Table 1.

Effects of inoculation methods on the initial culture of immature seeds of Paeonia ostii ‘Feng Dan’.

Table 1.

When the inoculum was performed with immature seeds at different DAA, the results indicated that the ratio of seeds with the cotyledon embryo after the initial culture was significantly affected by embryo maturity (Fig. 3). No immature seeds (embryos) before 30 DAA at the endosperm cellularization stage (proembryo stage) can develop into the cotyledon embryo, and the conversion percentage exhibited a gradual increase up to 100% from immature seeds (embryos) at 50 to 60 DAA after completion of endosperm cellularization (organ differentiation stage of the embryo). Correspondingly, the length and width of seeds gradually increased with development and did not present a significant difference between 50 and 60 DAA after completion of endosperm cellularization.

Fig. 3.
Fig. 3.

Effect of the development stage on the length and width of seeds and the ratio of seeds with cotyledon embryos after initial culture.

Citation: HortScience 57, 5; 10.21273/HORTSCI16477-21

Embryo germination.

It was demonstrated that cotyledon embryos obtained from immature seeds (embryos) at different DAA had significant differences on germination (Table 2). The germination rate of embryos and height and shoot length of seedlings exhibited a gradual increase. The germination rates of cotyledon embryos at 30 to 35 DAA (early stage of endosperm cellularization, proembryo stage) were 53.33% and 66.67%, respectively, and reached 100% after 50 DAA (mature growth stage of endosperm, globular embryo stage); the former seedlings were weak (low height and shoot length), but the latter were robust.

Table 2.

Effects of the development stage on the germination of immature embryos of Paeonia ostii ‘Feng Dan’.

Table 2.

Shoot proliferation.

The number of shoots and leaves of shoot clusters induced from cotyledon nodes of seedlings was enhanced gradually with embryo maturity with the first, second, and third subculture, respectively (Fig. 4). Shoot proliferations (numbers of shoots) with the second and third subculture were better than those with the first subculture.

Fig. 4.
Fig. 4.

Proliferation of shoots induced from cotyledon nodes of germinated embryos during 30 to 60 days after anthesis (DAA) at the first, second, and third subcultures.

Citation: HortScience 57, 5; 10.21273/HORTSCI16477-21

Rooting and acclimatization.

The rooting and survival rates were enhanced gradually for seedlings proliferated from cotyledon nodes of embryos during 30 to 60 DAA (Table 3). No survival plantlets were obtained from seedlings at 30 to 35 DAA. Seedlings at 30 DAA had no formed roots.

Table 3.

Rooting and acclimatization of shoots obtained from immature embryos at different days after anthesis (DAA) of Paeonia ostii ‘Feng Dan’.

Table 3.

Discussion

Histological observation of seeds (embryos) development.

Successful in vitro embryo culture largely depends on embryo maturity. The number of days after pollination (Cheng and Aoki, 2008; Uma et al., 2011) or the seed/embryo size (Promchot and Boonprakob, 2007; Varshney and Johnson, 2010) is usually used to indicate maturity. However, histological observation is necessary to confirm the development stage of seeds (embryos) in tree peony because discrepancies exist during same period with morphological observation due to distinctness of peony varieties, planting regions, and climates. For instance, the development stage of seeds (embryos) of P. rockii (Cheng and Aoki, 1999) and that of P. ostii (Dong, 2010) were inconsistent, which is similar to what occurs with the same variety in different regions. Free nuclear, cellularization, and mature growth stages of endosperm of P. ostii ‘Feng Dan’ collected from Shanghai (lat. 31°08′N, long. 121°18′E) were distinct from our observations in Beijing. The key to successful embryo rescue is the determination of the optimal sampling time. Whether the hybrid embryos could be rescued by transferring them to fresh medium before they started to degenerate was usually considered. Hence, this study revealed the stage of successful in vitro immature embryo culture at the histological level and recorded the corresponding length and width of seeds at the phenotypic level, which could provide a more accurate reference for embryo rescue of tree peony or other plants in breeding programs with selected parent lines.

Key factors for immature embryo culture.

A two-step method of immature embryo culture was developed during this research (Figs. 5 and 6). Seeds at 30 DAA (cellularization stage of endosperm, proembryo stage) or after were cultured in vitro to obtain cotyledon embryos; then, the cotyledon embryos were cultured for seedling establishment. A similar method was also reported for other plants (Li et al., 2015, 2018). The first step, cotyledon embryo formation, is a prerequisite for the protocol. The inoculation method and embryo maturity were key factors in the first step. During this study, no cotyledon embryos were developed with the inoculation method of downward micropyle. We attributed this phenomenon to the airtight condition and loss function of the endosperm aspirator observed at the chalazal position in seeds of P. rockii (Cheng, 1996) and P. ostii (Dong, 2010), which was presumed to be related to the function of nutrient absorption from the surroundings. Similar results and speculations were reported for Ziziphus jujuba; the rates of embryos and germination with the inoculation method of upward micropyle (63.20% and 82.60%) were higher than those with the inoculation method of downward micropyle (0% and 0%) (Liang et al., 2014). Additionally, upward micropyle with placenta had better effects than upward micropyle without placenta, which may be related to its functions of nutrient absorption and transportation.

Fig. 5.
Fig. 5.

In vitro immature embryo culture of Paeonia ostii ‘Feng Dan’. (A) Immature seeds were cultured with the inoculation method of upward micropyle with placenta. (B) Immature seeds with early-stage embryos developed into phenotypically visible cotyledon embryos after initial culture. (C) Germination of cotyledon embryos. (D) Shoots induced from cotyledon nodes after subculture. (E) Rooted shoots. (F) Survival plantlets after 6 months of acclimatization.

Citation: HortScience 57, 5; 10.21273/HORTSCI16477-21

Fig. 6.
Fig. 6.

Schematic diagram of the proposed protocol for in vitro immature embryo culture of Paeonia ostii ‘Feng Dan’.

Citation: HortScience 57, 5; 10.21273/HORTSCI16477-21

Nutrients are mainly absorbed from integument cells and other maternal cells in the early stage of embryonic development; however, it is the endosperm that mainly reserve sufficient nutrients in the later stage (Dong, 2010). In the present experiment, the earliest time when cotyledon embryos could be obtained was 30 DAA during the endosperm cellularization stage; moreover, the ratio of seeds with cotyledon embryos gradually increased among endosperms during the cellularization stage (30–45 DAA). There was no significant difference during the organ differentiation stage (50–60 DAA), when endosperm cellularization was completed. Hence, we can speculate that the transition of seeds from the heterotrophic state to the autotrophic state, which guaranteed the success of in vitro immature embryo culture, is closely related to endosperm cellularization.

The second step, or embryo germination and seedling establishment, of the protocol is also important. Usually, tree peony seeds are sown in the fall and sprout in the spring of the following year with traditional methods (Cheng, 2007). Therefore, we have established a method of rapid seedling establishment via direct germination of cotyledon embryos (90 DAA) of P. ostii ‘Feng Dan’ by inoculating embryos in the 1/2 MS medium supplemented with GA3 and AC, which will dramatically shorten the period of seedling establishment to 40 d (Xu et al., 2017). On the basis of obtaining cotyledon embryo in the first step, this method can also be considered for seedling establishment in immature embryo culture. However, only one plantlet was obtained with one embryo. In contrast, propagation from one embryo was achieved in this study via shoot induction, rooting, and acclimatization within 7 months. These two approaches can be chosen according to specific goals (Fig. 6).

When in vitro immature embryo culture is used early, the breeding time becomes shorter and there is a higher probability of successful embryo rescue. The protocol advanced the time of successful in vitro embryo culture to 30 DAA (cellularization stage of endosperm, proembryo stage); however, the proliferation, rooting, and survival rates were increased with embryo maturity. The sampling time had a significant effect on embryo rescue efficiency. Cotyledon embryos have difficulty forming if the sampling time is too early; however, they will degenerate if the sampling time is too late. This study provides an available reference for determining the optimal sampling time when breeding using embryo rescue techniques. Moreover, the multiplication efficiency of this system could be further optimized with the next step through the adjustment of medium and PGRs during selected embryo stages.

Conclusion

This study reports the success of in vitro immature embryo culture in P. ostii ‘Feng Dan’, which involves two steps: 1) culturing immature seeds in vitro for cotyledon embryo formation; and 2) establishing seedlings via embryo germination, shoot induction, rooting, and acclimatization. The protocol advanced the time of successful in vitro immature embryo culture to 30 DAA (cellularization stage of endosperm, proembryo stage). The seeds at 30 DAA or after were cultured to obtain cotyledon embryos; then, the embryos at 40 DAA or after could realize propagation within 7 months and survival plants could be obtained. This procedure provides the means for producing plants from the very early embryo stage, thus expanding the prospects for tree peony breeding.

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  • Shen, M.M., Tang, Z.J., Teixeira da Silva, J.A. & Yu, X.N. 2015 Induction and proliferation of axillary shoots from in vitro culture of Paeonia lactiflora Pall. mature zygotic embryos N. Z. J. Crop Hortic. Sci. 43 42 52 https://doi.org/10.1080/01140671.2014.944548

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  • Shi, G.A., Jiao, F.X., Jiao, Y.P., Yang, H.A., Han, M.W., Wu, Y.Q. & Shi, B.Y. 2014 Development prospects and strategies of oil tree peony industry in China J. Chinese Cereals and Oils Association 29 124 127 (In Chinese)

    • Search Google Scholar
    • Export Citation
  • Uma, S., Lakshmi, S., Saraswathi, M.S., Akbar, A. & Mustaffa, M.M. 2011 Embryo rescue and plant regeneration in banana (Musa spp.) Plant Cell Tissue Organ Cult. 105 105 111 https://doi.org/10.1007/s11240-010- 9847-9

    • Search Google Scholar
    • Export Citation
  • Varshney, A. & Johnson, T.S. 2010 Efficient plant regeneration from immature embryo cultures of Jatropha curcas, a biodiesel plant Plant Biotechnol. Rep. 4 139 148 https://doi.org/10.1007/s11816-010-0129-0

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  • Wang, X., Cheng, F.Y., Zhong, Y., Wen, S.S., Li, L.Z.M. & Huang, L.Z. 2016 Establishment of in vitro rapid propagation system for tree peony (Paeonia ostii) Scientia Silvae Sinicae 52 102 110 (In Chinese), https://doi.org/10.11707/j.1001-7488.20160512

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    • Export Citation
  • Wen, S.S., Chen, L. & Tian, R.N. 2020 Micropropagation of tree peony (Paeonia sect. Moutan): A review Plant Cell Tissue Organ Cult. 141 15 https://doi.org/10.1007/s11240-019-01747-8

    • Search Google Scholar
    • Export Citation
  • Wen, S.S., Cheng, F.Y., Zhong, Y., Wang, X., Li, L.Z.M., Zhang, Y.X. & Qiu, J.M. 2016a Efficient protocols for the micropropagation of tree peony (Paeonia suffruticosa ‘Jin Pao Hong’, P. suffruticosa ‘Wu Long Peng Sheng’, and Plemoinei ‘High Noon’) and application of arbuscular mycorrhizal fungi to improve plantlet establishment Scientia Hort. 201 10 17 https://doi.org/10.1016/j.scienta.2016.01.022

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  • Wen, S.S., Cheng, F.Y. & Zhong, Y. 2016b Micropropagation of tree peony (Paeonia × lemoinei ‘High Noon’) and the assessment of genetic stability by SSR analysis Propag. Ornam. Plants 16 19 27

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  • Wen, S.S., Cheng, F.Y., Zhong, Y., Wang, X., Li, L.Z.M. & Huang, L.Z. 2016c Protocol for the micropropagation of tree peony (Paeonia × Lemoinei ‘High Noon’) Plant Sci. J. 34 143 150 (In Chinese), https://doi.org/10.11913/PSJ.2095-0837.2016.10143

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  • Xiong, H., Sun, H., Zou, F. & Fan, X.M. 2018 Micropropagation of chinquapin (Castanea henryi) using axillary shoots and cotyledonary nodes HortScience 53 1482 1486 https://doi.org/10.21273/HORTSCI13292-18

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  • Xu, L., Cheng, F.Y. & Zhong, Y. 2022 Efficient plant regeneration via meristematic nodule culture in Paeonia ostii ‘Feng Dan’ Plant Cell Tissue Organ Cult. https://doi.org/10.1007/s11240-021-02216-x

    • Search Google Scholar
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  • Xu, L., Cheng, F.Y. & Zhong, Y. 2017 Study on rapid seedling-raising technology of tree peony embryo culture Bull. Bot. Res. 5 690 699 (In Chinese)

    • Search Google Scholar
    • Export Citation
  • Yang, Y.J., Zhang, D.L., Li, Z.H., Jin, X.L. & Dong, J.Y. 2015 Immature embryo germination and its micropropagation of Ilex crenata Thunb HortScience 50 733 737 https://doi.org/10.21273/HORTSCI.50.5.733

    • Search Google Scholar
    • Export Citation
  • Yu, S., Du, S., Yuan, J. & Hu, Y. 2016 Fatty acid profile in the seeds and seed tissues of Paeonia L. species as new oil plant resources Sci Rep UK 6 26944 https://doi.org/10.1038/srep26944

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Contributor Notes

This study was supported by the National Key R&D Program of China (2020YFD1000500).

This article does not contain any studies with human participants or animals performed by any of the authors.

F.C. is the corresponding author. E-mail: Chengfy8@263.net.

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    Fig. 1.

    Explants of Paeonia ostii ‘Feng Dan’ used for this study. (A) Follicles of P. ostii ‘Feng Dan’ [50 days after anthesis (DAA)] collected in 2017. (B) Anatomical observation of P. ostii ‘Feng Dan’ seeds (50 DAA, globular embryo stage) collected in 2017. (C) Follicles of P. ostii ‘Feng Dan’ from 5 to 60 DAA at intervals of 5 d collected in 2018. (D) Anatomical observation of P. ostii ‘Feng Dan’ seeds from 30 to 60 DAA at intervals of 5 d collected in 2018.

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    Fig. 2.

    Histological observation of immature seeds of Paeonia ostii ‘Feng Dan’. (A–I) Immature seeds at 5, 25, 30, 35, 40, 45, 50, 55, and 60 days after anthesis (DAA), respectively. (A) Free nuclear endosperm (black arrow). (B) Free nuclear endosperm (black arrow) divided continuously around the periphery of the embryo sac. (C) Cellularization of endosperm (black arrow). (D, E) Centripetal increase of cellularization endosperm cells. (E) Endosperm filled the entire embryo sac. (G) Globular embryo. (H) Torpedo-shaped embryo. (I) Cotyledon embryo.

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    Fig. 3.

    Effect of the development stage on the length and width of seeds and the ratio of seeds with cotyledon embryos after initial culture.

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    Fig. 4.

    Proliferation of shoots induced from cotyledon nodes of germinated embryos during 30 to 60 days after anthesis (DAA) at the first, second, and third subcultures.

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    Fig. 5.

    In vitro immature embryo culture of Paeonia ostii ‘Feng Dan’. (A) Immature seeds were cultured with the inoculation method of upward micropyle with placenta. (B) Immature seeds with early-stage embryos developed into phenotypically visible cotyledon embryos after initial culture. (C) Germination of cotyledon embryos. (D) Shoots induced from cotyledon nodes after subculture. (E) Rooted shoots. (F) Survival plantlets after 6 months of acclimatization.

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    Fig. 6.

    Schematic diagram of the proposed protocol for in vitro immature embryo culture of Paeonia ostii ‘Feng Dan’.

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    • Search Google Scholar
    • Export Citation
  • Shi, G.A., Jiao, F.X., Jiao, Y.P., Yang, H.A., Han, M.W., Wu, Y.Q. & Shi, B.Y. 2014 Development prospects and strategies of oil tree peony industry in China J. Chinese Cereals and Oils Association 29 124 127 (In Chinese)

    • Search Google Scholar
    • Export Citation
  • Uma, S., Lakshmi, S., Saraswathi, M.S., Akbar, A. & Mustaffa, M.M. 2011 Embryo rescue and plant regeneration in banana (Musa spp.) Plant Cell Tissue Organ Cult. 105 105 111 https://doi.org/10.1007/s11240-010- 9847-9

    • Search Google Scholar
    • Export Citation
  • Varshney, A. & Johnson, T.S. 2010 Efficient plant regeneration from immature embryo cultures of Jatropha curcas, a biodiesel plant Plant Biotechnol. Rep. 4 139 148 https://doi.org/10.1007/s11816-010-0129-0

    • Search Google Scholar
    • Export Citation
  • Wang, X., Cheng, F.Y., Zhong, Y., Wen, S.S., Li, L.Z.M. & Huang, L.Z. 2016 Establishment of in vitro rapid propagation system for tree peony (Paeonia ostii) Scientia Silvae Sinicae 52 102 110 (In Chinese), https://doi.org/10.11707/j.1001-7488.20160512

    • Search Google Scholar
    • Export Citation
  • Wen, S.S., Chen, L. & Tian, R.N. 2020 Micropropagation of tree peony (Paeonia sect. Moutan): A review Plant Cell Tissue Organ Cult. 141 15 https://doi.org/10.1007/s11240-019-01747-8

    • Search Google Scholar
    • Export Citation
  • Wen, S.S., Cheng, F.Y., Zhong, Y., Wang, X., Li, L.Z.M., Zhang, Y.X. & Qiu, J.M. 2016a Efficient protocols for the micropropagation of tree peony (Paeonia suffruticosa ‘Jin Pao Hong’, P. suffruticosa ‘Wu Long Peng Sheng’, and Plemoinei ‘High Noon’) and application of arbuscular mycorrhizal fungi to improve plantlet establishment Scientia Hort. 201 10 17 https://doi.org/10.1016/j.scienta.2016.01.022

    • Search Google Scholar
    • Export Citation
  • Wen, S.S., Cheng, F.Y. & Zhong, Y. 2016b Micropropagation of tree peony (Paeonia × lemoinei ‘High Noon’) and the assessment of genetic stability by SSR analysis Propag. Ornam. Plants 16 19 27

    • Search Google Scholar
    • Export Citation
  • Wen, S.S., Cheng, F.Y., Zhong, Y., Wang, X., Li, L.Z.M. & Huang, L.Z. 2016c Protocol for the micropropagation of tree peony (Paeonia × Lemoinei ‘High Noon’) Plant Sci. J. 34 143 150 (In Chinese), https://doi.org/10.11913/PSJ.2095-0837.2016.10143

    • Search Google Scholar
    • Export Citation
  • Xiong, H., Sun, H., Zou, F. & Fan, X.M. 2018 Micropropagation of chinquapin (Castanea henryi) using axillary shoots and cotyledonary nodes HortScience 53 1482 1486 https://doi.org/10.21273/HORTSCI13292-18

    • Search Google Scholar
    • Export Citation
  • Xu, L., Cheng, F.Y. & Zhong, Y. 2022 Efficient plant regeneration via meristematic nodule culture in Paeonia ostii ‘Feng Dan’ Plant Cell Tissue Organ Cult. https://doi.org/10.1007/s11240-021-02216-x

    • Search Google Scholar
    • Export Citation
  • Xu, L., Cheng, F.Y. & Zhong, Y. 2017 Study on rapid seedling-raising technology of tree peony embryo culture Bull. Bot. Res. 5 690 699 (In Chinese)

    • Search Google Scholar
    • Export Citation
  • Yang, Y.J., Zhang, D.L., Li, Z.H., Jin, X.L. & Dong, J.Y. 2015 Immature embryo germination and its micropropagation of Ilex crenata Thunb HortScience 50 733 737 https://doi.org/10.21273/HORTSCI.50.5.733

    • Search Google Scholar
    • Export Citation
  • Yu, S., Du, S., Yuan, J. & Hu, Y. 2016 Fatty acid profile in the seeds and seed tissues of Paeonia L. species as new oil plant resources Sci Rep UK 6 26944 https://doi.org/10.1038/srep26944

    • Search Google Scholar
    • Export Citation
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