Efficient Plant Regeneration via Meristematic Nodule Culture in Paeonia Ostii ‘Feng Dan’

doi:10.21203/rs.3.rs-986175/v1

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Efficient Plant Regeneration via Meristematic Nodule Culture in Paeonia Ostii ‘Feng Dan’

https://doi.org/10.21203/rs.3.rs-986175/v1

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published 10 Jan, 2022

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Tree peony (Paeonia sect. Moutan) is an economically important multipurpose woody plant in terms of its medical, ornamental and oil values, but its breeding and industrial development are severely limited due to inefficient traditional propagation methods and existing in vitro regeneration systems. Meristematic nodules (MNs) are an attractive alternative to solve this problem. This study first presented a protocol for in vitro regeneration of P. ostii ‘Feng Dan’ via MN culture with four consecutive steps, including embryogenic callus (EC) formation, MN induction and leaf cluster differentiation, shoot elongation, rooting and acclimatization. The highest EC induction rate (81.25%) was achieved when cotyledons were cultured on modified Murashige and Skoog (mMS) medium with 4.04 µM N-(2-chloro-4-pyridyl)-N-phenylurea (CPPU)+5.37 µM α-naphthylacetic acid (NAA) for 30 days. The optimal MN induction rate (100%) and leaf cluster differentiation rate (45.83%) were obtained when ECs were cultured on modified woody plant medium (mWPM) supplemented with 2.02 µM CPPU+2.27 µM thidiazuron (TDZ) for a subculture time of 10 days. The combination of 1.29 µM 6-benzyladenine (BA)+0.58 µM gibberellin (GA₃) yielded the best shoot elongation (13.40 shoots per nodule), rooting rate (43.33%) and survival rate (45.83%). The protocol will play an important role in mass propagation, breeding and genetic improvement of tree peony.

Horticulture

Tree peony

Paeonia ostii ‘Feng Dan’

Organogenesis

Meristematic nodule

Tree peony (Paeonia sect. Moutan) is well-known in China because of its medical, ornamental and oil value (Yu et al. 2016). P. ostii is main option for oil tree peony because of the high yield and adaptability. However, the low efficiency and long cycle of conventional propagation methods, such as grafting and division, severely constrain its breeding and are insufficient to address the increasing commercial demands. Thus, an efficient and stable in vitro regeneration system is urgently needed. In general, no regeneration system published to date can meet the needs for the propagation and genetic manipulation of tree peonies due to various obstacles, such as vitrification, low multiplication, poor rooting and difficult acclimatization in micropropagation (Beruto et al. 2004; Wen et al. 2020), rare differentiation in callus culture (Zhu et al. 2018), and high deformity rates and low germination in somatic embryogenesis (Du et al. 2020); hence, innovative breakthroughs are needed to overcome this problem.

Meristematic nodules (MNs) are a special structure comprising organization centers (OCs), a cortical-like area of parenchymatous cells and an epidermal-like area under histological observation, which have high regeneration potential, genetic stability and long-term cellular dynamics, making them an attractive alternative for plant regeneration via organogenesis, in vitro phytochemical production and plant transformation (McCown et al. 1988; Batista et al. 2008). Successful in vitro regeneration through MN culture has been reported in several woody and herbaceous plants, such as Eucalyptus globulus (Dobrowolska et al. 2017), Liquidambar orientalis (Bayraktar et al. 2015), Populus euphratica (Ferreira et al. 2009), Humulus lupulus (Fortes and Pais 2000), Sclerocarya birrea (Moyo et al. 2009) and Billbergia zebrina (Dal Vesco et al. 2011). The regeneration patterns were significantly affected by genotype, plant growth regulators (PGRs) and subculture time (Xie and Hong 2001; Trindade and Paris 2003). Therefore, the optimal culture conditions should be screened with genotype changes.

There are scarce reports of MNs in Paeonia. Zhong (2011) established a nodule induction and multiplication system of P. rockii through calli induced from petiole sections. Subsequently, Qin et al. (2012) further optimized the system in P. lemoinei ‘Golden Era’ and P. itoh ‘Barzella’ but only found a 16% differentiation rate in P. itoh ‘Barzella’, and no rooted shoots and surviving plantlets were obtained. Overall, efficient in vitro shoot organogenesis via MN culture was lacked in tree peony. Thus, the aim of the present study was to establish a protocol for in vitro regeneration of P. ostii ‘Feng Dan’ based on an MN culture system. This protocol will be beneficial to the large-scale propagation, genetic improvement, gene identification and functional analysis of tree peony.

Plant material and sterilization

Mature seeds of P. ostii ‘Feng Dan’ at 90 days after anthesis (DAA) were collected in August 2018 from living adult plants grown in Beijing Guose Peony Garden in Beijing, China (40°45′N, 115°97′E) (Fig. 1a and b) and washed under running tap water for 15 mins before soaking in commercial liquid detergents (1% v/v; 5 min). Then, seeds were sterilized by dipping in ethanol (70% v/v; 30 s), followed by dipping in a solution of NaOCl (0.2% v/v; 5 min) and three rinses with sterile distilled water.

Medium and culture conditions

The basal media was modified Murashige and Skoog medium (mMS, half-strength macroelements and full-strength Ca²⁺) (Murashige and Skoog 1962),1/2 MS (all macroelements at half-strength) and modified woody plant medium (mWPM, double strength of Ca²⁺) (Lloyd and McCown 1980). All media were supplemented with 3% sucrose and 0.7% agar, and the pH was adjusted to 5.8–6.0 before autoclaving (at 118 kPa and 121°C for 20 min). All reagents were supplied by Biodee (Beijing, China). The cultures, without additional description, 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).

Callus induction

Zygotic embryos were aseptically isolated from seeds and inoculated on germination medium [mMS+2.57 µM 6-benzyladenine (BA)+2.89 µM gibberellin (GA₃)] for 15 days. The expanded cotyledons (Fig. 1c) were cut into 1×1 cm pieces and inoculated on callus induction medium (CIM) [mMS+5.37 µM α-naphthylacetic acid (NAA)+ 2.02 µM N-(2-chloro-4-pyridyl)-N-phenylurea (CPPU)] with the abaxial side touching the medium, under the dark conditions. The embryogenic callus (EC, yellow compact calli with densely arranged clumps) (Fig. 1d) induction rate (%) and browning rate (%) were calculated after cotyledons were inoculated on CIM with different induction times (20, 30, 40 and 50 days). Each experiment was conducted in three replications with 16 explants in each replication.

To screen optimal PGRs for callus induction, two experiments were carried out in chronological order. The EC induction rate (%), browning rate (%) and callus biomass (g) were calculated after 30 days. Each experiment was conducted in three replications with 16 explants in each replication. Step 1: Two auxins alone, 5.37 µM NAA, 4.52 µM 2,4-dichlorophenxyacetic acid (2,4-D), and in combination with three kinds of cytokinin (CK), including 2.27 µM thidiazuron (TDZ), 2.02 µM CPPU and 2.57 µM BA, were used to screen suitable PGRs. Step 2: Based on the suitable PGRs obtained in step 1, the combination of NAA (1.34, 2.69, 5.37, 10.74 µM) with CPPU (1.00, 2.02, 4.04 µM) was used to select the best concentration of PGRs.

Meristematic nodule induction and leaf cluster differentiation

The ECs induced on optimal CIM after 30 days of culture were inoculated into MN induction medium (MIM) (mWPM+2.02 µM CPPU) to optimize the subculture time for MN induction and leaf cluster differentiation. The MN induction rate (%), leaf cluster differentiation rate (%) and browning rate (%) were determined after 12 subcultures with different subculture times (10, 15, 20, 25 d), respectively. Each experiment was conducted in three replications with 16 explants in each replication.

To examine the effects of PGRs and callus induction time on MN induction and leaf cluster differentiation, two experiments were designed. The MN induction rate and leaf cluster differentiation rate were determined after 12 subcultures. Each experiment was conducted in three replications with 16 explants in each replication. Experiment 1: ECs induced on optimal CIM with different days of culture (20, 30, 40, 50 d) were inoculated into MIM containing different PGR combinations (2.02 µM CPPU, 2.02 µM CPPU+2.27 µM TDZ, 2.02 µM CPPU+2.57 µM BA, 2.02 µM CPPU+2.69 µM NAA, 2.02 µM CPPU+0.58 µM GA₃), with a subculture time of 20 days. Experiment 2: ECs induced on optimal CIM with 30 days of culture were used. Based on experiment 1, an orthogonal test with three factors and three levels of CPPU (1.00, 2.02, 4.04 µM), TDZ (0, 2.27, 4.54 µM) and BA (0, 2.57, 5.15 µM) was established, with a subculture time of 10 days.

Shoot elongation, rooting and acclimatization

Nodules with leaf clusters cultured on the optimal MIM after 12 subcultures with a subculture time of 10 days, were cultured on mWPM containing 1.29 µM BA and (0.29, 0.58, 0.87, 1.15 µM) GA₃ for 5 months with a subculture time of 30 days. Shoots induced from leaf clusters and elongated to 1–3 cm were successively excised and cultured for rooting. The mean number of shoots per nodule (n) was calculated, and each experiment included three replications of 5 nodules per treatment. According to Wang et al. (2016), shoots were cultured on root induction medium [1/2 MS+4.92 µM indole-3-butyric acid (IBA)+11.34 µM putrescine (Put)] for 38 days in the dark and then transferred to root expression medium [PGR-free 1/2 MS+0.4% activated carbon (AC)] for 20 days in the light. During the root induction phase, the shoots were subjected to cold treatment (4°C, 8 days) in the dark and then cultured at 24 ± 1°C for another 30 days. The rooting rate (%) was calculated, and each experiment included three replications of 30 shoots per treatment. The rooted plantlets were removed carefully from the medium, washed thoroughly with tap water, and finally transferred 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^-1photosynthetic photon flux density provided by fluorescent lamps, and the survival rate (%) was recorded two months after acclimatization with three replications of 30 rooted plantlets per treatment.

Statistical analysis

Prior to data analysis, arcsine transformation was carried out for any percentage data. Statistical analysis was performed with SPSS 22.0 (SPSS Inc., Chicago, USA), and Microsoft Excel 2013 software (Microsoft Corp., Richmond, ca., USA) was used for data statistics and charts. The data were submitted to analysis of variance (ANOVA) followed by Duncan’s multiple range test at p ≤ 0.05 and expressed as the mean ± standard error.

Callus induction

A small proportion of calli (33.33%) expanded into clumps (ECs) after 20 days, and the browning rate was 6.25% (Fig. 2). The EC induction rate and browning rate gradually increased with induction time and yielded no significant difference between 30–50 days of treatment. The selection of the optimal induction time should comprehensively consider the EC induction rate, browning rate and differentiation potential (following in this paper).

The EC induction rate (12.50%, 8.33%) and biomass (1.20 g, 1.05 g) under NAA and 2,4-D alone were significantly lower than those under the combination with CKs, but the browning rate (93.75%, 97.92%) was significantly higher. There were few differences between the 3 combinations of auxin and CKs in the three parameters. The highest EC induction rate (81.25%) and biomass (1.73 g) were obtained under 2.02 µM CPPU+5.37 µM NAA with a low rate of browning (12.50%) (Fig. 3).

Based on the suitable PGR combinations obtained, the optimal concentrations are described in step 2.The highest EC induction rate was 87.50%, with the highest biomass (1.58 g) and lowest browning rate (10.42%) when 4.04 µM CPPU+5.37 µM NAA was used (Fig. 4).

Meristematic nodule induction and leaf cluster differentiation

Morphological study revealed the development of MN and leaf cluster differentiation was asynchronous and chronologically included four main stages: ①Pre-nodular structures: During 1–4 cycles of subculture in MIM, ECs turned green and visible small protuberances were gradually observed on the surface (Fig. 5a). ②MNs: After subculture for 5–6 times, protuberances greatly increased in diameter (Fig. 5b). ③Nodular clusters: During 7–10 subcultures, MNs proliferated with small grooves that appeared on the surface of nodules, and then progressively deepened, yet nodule break-up was never observed. Several MNs displaying different levels of development were loosely attached to each other and developed into nodular clusters in appearance (Fig. 5c). ④Leaf cluster differentiation: After 11–12 subcultures, differentiated leaf clusters started to develop and elongate (Fig. 5d).

Figure 6 showed that the MN induction rate and leaf cluster differentiation rate decreased, while the browning rate significantly increased with prolonged subculture time. The optimal subculture time for MN induction and leaf cluster differentiation was 10 days, with the highest MN induction rate (68.75%), leaf cluster differentiation rate (54.17%) and the lowest browning rate (16.67%).

Figure 7 demonstrated that different combinations of PGRs and induction times significantly influenced MN induction and leaf cluster differentiation. Among all the media tested, no MN or differentiation was observed under the combinations of CPPU and GA₃. The promoting effect of CPPU+NAA treatment on the MN induction rate and differentiation rate was conspicuously lower than that with CPPU alone or in combination with CKs, while the combinations of CPPU+TDZ and CPPU+BA resulted in significantly higher rates than that of CPPU alone with no significant difference between them, which indicated combinations of CPPU+BA+TDZ might be conducive to MN induction and differentiation. Based on an orthogonal combination test (Table 1), the optimal combination of factors and levels for MN induction and leaf cluster differentiation was A3B2C2 and A2B1C2, respectively (Table 2). In other words, the suitable medium for MN induction was mWPM+4.04 µM CPPU+2.57 µM 6-BA+2.27 µM TDZ, and the optimal medium for enhancing leaf cluster differentiation was mWPM+2.02 µM CPPU+ 2.27 µM TDZ, with the highest MN induction rate (100%) and leaf cluster differentiation rate (45.83%).

The rate of MN induction and leaf cluster differentiation rate significantly decreased with induction time among all PGR treatments (Fig. 7). Comprehensively considering the effects of induction time on the EC induction rate and browning rate, 30 days was selected as the best induction time in this paper.

Shoot elongation, rooting and acclimatization

Nodules with clusters of leaves were not conductive to promote shoot elongation when cultured in the same MIM (Fig. 8a), but efficiently achieved by transferring to medium containing BA and GA₃(Fig. 8b). There were elongated shoots successively induced and mean number of shoots per nodule was increased with GA₃ concentration and obtained the best at 0.58uM, with 13.40 shoots per nodule. Reversely, the rooting and survival rate of elongated shoots were gradually declined with GA₃concentration, and no significant difference was found between 0.29-0.58uM GA₃ treatment (Table 3). Therefore, mWPM+1.29uM BA+ 0.58uM GA₃ was the optimal medium for shoots elongation. The rooted seedlings (Fig. 8c) were successfully transferred into pots and developed well in the chamber (Fig. 8d).

To our knowledge, this is the first report of MNs culture system capable of de nova shoot organogenesis in tree peony. The MNs was initiated indirectly form callus derived from cotyledons, which was consistent with pioneering work (Batista et al. 2000; Fortes and Pais 2000; Xie and Hong 2001). Our study demonstrated that all differentiation was preceded by a MNs stage with three developmental phases, including pre-nodular structure, MNs and nodular clusters in sequence, subsequently, differentiation occurred only in these nodular areas, suggesting the nodules was prerequisites of de nova shoot organogenesis in tree peony. Similar phenomenon was reported in Humulus lupulus that the production of nodules on the callus had been described as a sign of shoot initiation and the development stage of nodules was analogously divided into 3 stages, consisting of pre-nodular structures, MNs and ‘polycenter nodules’ (McCown et al. 1988; Batista et al. 1996, 2000).

In this report, the acquisition of ECs was a prerequisite for de nova shoot organogenesis. Combination of auxin and CKs had a better effect on the induction of ECs than auxin alone. Besides, NAA was better than that of 2,4-D. The results in agreement with those reported in Eucalyptus grandis (Warrag et al. 1991) and Eucalyptus botryoides (Ito et al. 1996) that NAA was appropriate auxin in nodule systems. In addition, the appropriate CKs type for MNs induction and differentiation was genotype dependent, for example, BA was recommended for MNs induction of Radiata pine (Aitken-Christie et al.1988) and Eucalyptus globulus (Trindade and Pais 2003), TDZ for Pelargonium×hortorum, Pelargonium×domesticum (Haensch 2004) and Acacia mangium (Xie and Hong 2001), CPPU for Eucalyptus botryoides (Ito et al.1996). In this study, CPPU efficiently promoted MNs induction and differentiation rate in tree peony, while Trindade et al. (2003) showed contradictory results in Eucalyptus globulus that BA enhanced both nodule induction and shoot organogenesis, but in the presence of CPPU, explants showed vitrification and no morphogenesis. Simultaneously, the MNs induction and differentiation rate of CKs combinations treatment was higher than that of CPPU alone, far beyond combination of CPPU and auxin treatment in the present article, which was in accordance to results in Garcinia mangostana that mixed use of CKs presented better response than single one in MNs induction (Te-chato and Lim 2000), but contrasted with an earlier report in Ananas comosus that a certain number of shoots can be regenerated from nodules in medium supplemented with BA and NAA (Teng 1997).

Our results showed that not all the nodular were able to induce shoots, also, the best efficiency of MNs induction and subsequent differentiation required different exogenous hormonal combinations according to variance analysis. Similar result was described by Ito et al. (1996) who found nodules were induced preferentially with CPPU, but shoot organogenesis occurred on the medium with BA in place of CPPU. Therefore, a two-step procedure for MNs induction and differentiation should be studied in the future in order to optimize the regeneration system, instead of a single-step protocol in this experiment. Additionally, shoots elongation from nodules differentiated with leave clusters on the SIM was slowly, whereas a plenty of newly formed shoots were elongated efficiently with GA₃ application. The necessity of shoot elongation step in medium supplemented with GA₃ had been emphasized in previous findings (Scherer et al. 2013; Kongbangkerd and Wawrosch 2003; Xie and Hong 2001). Concurrently, only after the elongated shoots were excised did the nodules continue mass differentiation. The inhibition of shoots organogenesis by preexisting shoots had also been reported inFerreira et al. (2009) that continuous organogenesis of new shoots from nodules of Populus euphratica only occurred in regions where previous shoots had been removed. This behavior resembled an apical dominance mechanism, that further shoot organogenesis might be suppressed by the auxins produced in the first shoots(Gurriarán et al. 1999).

Similar morphological response pattern between effect of induction time and subculture days was performed. In detail, the differentiation rate was gradually decreased while the browning rate aggravated with the prolong of time, indicating the decline of organogenic potential. This phenomenon might be result from depletion of hormone content in medium, accumulation and oxidation of phenolic compounds in tissues resulting in the inhibition of growth and a decrease in the regeneration ability of plant cells with long time culture (Jones and Saxena 2013).

In the present study, an efficient in vitro regenerative system was developed for the first time via MN culture in P. ostii ‘Feng Dan’. The protocol was divided into four consecutive steps, including EC formation, MN induction and leaf cluster differentiation, shoot elongation, rooting and acclimatization. De novo shoot organogenesis and surviving plantlets were realized through screening of optimal culture conditions, which revealed that this protocol is feasible for mass propagation and genetic transformation of tree peony.

AC, activated carbon; BA, 6-benzyladenine; CIM, Callus induction medium; CK, Cytokinin; CPPU, N-(2-chloro-4-pyridyl)-N-phenylurea; DAA, Days after anthesis; EC, Embryogenic callus; GA₃, Gibberellin; IBA, Indole-3-butyric acid; MIM, Meristematic nodule induction medium; MN, Meristematic nodule; mMS, Modified Murashige and Skoog; mWPM, Modified woody plant medium; NAA, α-naphthylacetic acid; Put, Putrescine; PGR, Plant growth regulator; TDZ, Thidiazuron; 2,4-D, 2,4-Dichlorophenxyacetic acid

Compliance with ethical standards

Funding The study was supported by National key R&D Program of China (2020YFD1000503).

Conflict of interest The authors declare that they have no conflict of interest.

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

Author contribution LX conducted the experiments and written the manuscript. FYC and YZ revised the manuscript. All authors read and approved the final manuscript.

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Table 1 Screening of optimal PGRs concentration for the MN induction and leaf cluster differentiation rates in Paeonia ostii ‘Feng Dan’
CPPU (µM)	BA (µM)	TDZ (µM)	The MN induction rate (%)	The leaf cluster differentiation rate (%)
1.00	0	0	95.83±3.61^b	31.25±0.00^b
1.00	2.57	2.27	100.00±0.00^a	20.83±3.61^de
1.00	5.15	4.54	87.50±0.00^c	18.75±6.25^e
2.02	0	2.27	100.00±0.00^a	45.83±3.61^a
2.02	2.57	4.54	100.00±0.00^a	12.50±0.00^f
2.02	5.15	0	85.42±3.61^c	25.00±0.00^cd
4.04	0	4.54	100.00±0.00^a	29.17±3.61^bc
4.04	2.57	0	97.92±3.61^ab	10.42±3.61^fg
4.04	5.15	2.27	100.00±0.00^a	6.25±0.00^g
Different letters within a column show significant differences by Duncan’s multiple range tests (p ≤ 0.05). Each data represent mean ± standard error.

Table 2 Duncan test and range of the MN induction and leaf cluster differentiation rates in Paeonia ostii ‘Feng Dan’
	The MN induction rate (%)			The leaf cluster differentiation rate (%)
	CPPU	BA	TDZ	CPPU	BA	TDZ
	94.44^b	98.61^a	93.06^c	23.61^a	35.42^a	22.22^a
	95.14^b	99.31^a	100.00^a	27.78^a	14.58^b	24.31^a
	99.31^a	90.97^b	95.83^b	15.28^b	16.67^b	20.14^a
R	4.87	8.34	6.94	12.5	20.84	4.17
Different letters within a column show significant differences by Duncan’s multiple range tests (p ≤ 0.05).

Table 3 Effect of GA₃ on shoot elongation, rooting and acclimatization in Paeonia ostii ‘Feng Dan’
GA₃ (µM)	Mean number of shoots per nodule (n)	The rooting rate (%)	The survival rate (%)
0.29	7.93±0.42d	46.67±3.34a	50.00±0.00a
0.58	13.40±0.20a	43.33±3.34a	45.83±7.22a
0.87	10.47±0.31b	34.44±5.09b	41.67±7.22ab
1.15	9.27±0.31c	27.78±1.92b	33.33±7.22b
Different letters within a column show significant differences by Duncan’s multiple range tests (p ≤ 0.05). Each data represent mean ± standard error.

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published 10 Jan, 2022

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Efficient Plant Regeneration via Meristematic Nodule Culture in Paeonia Ostii ‘Feng Dan’

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