induction and proliferation of axillary shoots from in vitro culture of paeonia...

This article was downloaded by: [Queensland University of Technology]On: 22 November 2014, At: 09:35Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registeredoffice: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

New Zealand Journal of Crop andHorticultural SciencePublication details, including instructions for authors andsubscription information:http://www.tandfonline.com/loi/tnzc20

Induction and proliferation of axillaryshoots from in vitro culture of Paeonialactiflora Pall. mature zygotic embryosMiaomiao Shena, Zhengjiao Tanga, Jaime A. Teixeira da Silvab &XiaoNan Yua

a Beijing Key Laboratory of Ornamental Plants, GermplasmInnovation and Molecular Breeding, National Engineering ResearchCenter for Floriculture, College of Landscape Architecture,Beijing Forestry University, Beijing, PR Chinab PO Box 7, Miki-cho Post Office, Ikenobe 3011-2, Kagawa-ken761-0799, JapanPublished online: 10 Sep 2014.

To cite this article: Miaomiao Shen, Zhengjiao Tang, Jaime A. Teixeira da Silva & XiaoNan Yu(2014): Induction and proliferation of axillary shoots from in vitro culture of Paeonia lactifloraPall. mature zygotic embryos, New Zealand Journal of Crop and Horticultural Science, DOI:10.1080/01140671.2014.944548

To link to this article: http://dx.doi.org/10.1080/01140671.2014.944548

PLEASE SCROLL DOWN FOR ARTICLE

Taylor & Francis makes every effort to ensure the accuracy of all the information (the“Content”) contained in the publications on our platform. However, Taylor & Francis,our agents, and our licensors make no representations or warranties whatsoever as tothe accuracy, completeness, or suitability for any purpose of the Content. Any opinionsand views expressed in this publication are the opinions and views of the authors,and are not the views of or endorsed by Taylor & Francis. The accuracy of the Contentshould not be relied upon and should be independently verified with primary sourcesof information. Taylor and Francis shall not be liable for any losses, actions, claims,proceedings, demands, costs, expenses, damages, and other liabilities whatsoever orhowsoever caused arising directly or indirectly in connection with, in relation to or arisingout of the use of the Content.

This article may be used for research, teaching, and private study purposes. Anysubstantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,

http://www.tandfonline.com/loi/tnzc20

http://www.tandfonline.com/action/showCitFormats?doi=10.1080/01140671.2014.944548

http://dx.doi.org/10.1080/01140671.2014.944548

systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions

Dow

nloa

ded

by [

Que

ensl

and

Uni

vers

ity o

f T

echn

olog

y] a

t 09:

35 2

2 N

ovem

ber

2014

http://www.tandfonline.com/page/terms-and-conditions

http://www.tandfonline.com/page/terms-and-conditions

RESEARCH ARTICLE

Induction and proliferation of axillary shoots from in vitro culture of Paeonialactiflora Pall. mature zygotic embryos

Miaomiao Shena, Zhengjiao Tanga, Jaime A. Teixeira da Silvab* and XiaoNan Yua*aBeijing Key Laboratory of Ornamental Plants, Germplasm Innovation and Molecular Breeding, NationalEngineering Research Center for Floriculture, College of Landscape Architecture, Beijing Forestry University,Beijing, PR China; bPO Box 7, Miki-cho Post Office, Ikenobe 3011-2, Kagawa-ken 761-0799, Japan

(Received 22 March 2013; accepted 1 July 2014)

Zygotic embryos of three herbaceous peony (Paeonia lactiflora Pall.) cultivars (‘Fen Yu Nu’, ‘ZhongSheng Fen’ and ‘Zhu Sha Pan’) were used to study embryo germination and optimum plant growthregulator (PGR) combinations for in vitro propagation and root initiation. Mature zygotic embryos(>90 days after flowering [DAF]) gave better germination and survival than immature zygotic embryos(50–70 DAF). A protocol for initiating shoot growth and axillary shoot proliferation was establishedusing mature embryos. The best results were obtained when using excised zygotic embryos (EZEs)obtained by removing the testa and endosperm from the seed. The best medium for EZE germination(an embryo with cotyledons) was half-strength Murashige and Skoog (MS) medium (with double-strength CaCl2) supplemented with 0.5 mg L−1 6-benzyladenine (BA) and 0.5 mg L−1 gibberellic acid(GA3). The best medium for axillary shoot proliferation was half-strength MS medium (includingdouble-strength CaCl2) supplemented with 1 mg L−1 BA and 1 mg L−1 GA3. PGR-free half-strengthMS was the best medium for promoting root development on seedlings (a germinated EZE with tenderleaves) and for robust in vitro seedling establishment. The acclimatization of herbaceous peonyremains the most challenging step of the in vitro protocol.

Keywords: embryo rescue; herbaceous peony; micropropagation; tissue culture; zygotic embryo

Introduction

Herbaceous peony (Paeonia lactiflora Pall.) is along-lived perennial of the Paeoniaceae that hasbeen cultivated in China for more than 3900 years(Wang & Zhang 2005). Chinese people favour her-baceous peony because of its diverse forms, colou-rs and applications, from garden plant, to pottedplant, or dry or cut flower use. Plant division andseed breeding are the main propagation techniques(Kamenetsy & Dole 2012), although these methodsrequire a considerable amount of propagation ma-terial and can only be carried out during the vegeta-tive growth season (Qin 2004). When propagatingby plant division, in which the tuberous root is

divided into several small plants each of whichcontains three to five dormant vegetative buds(Shannon & Kamp 1959), the coefficient of propa-gation is low and the ornamental value of stockplants declines because of changes in shape of thetuberous root and because of the decline in thevigour of underground buds. Seed germinationis limited by epicotyl dormancy and is stronglydependent on season; peony has a slow reproduct-ive cycle of 5–10 years (Krekler 1962; Qin 2004).The slow propagation rates mean it is difficult tomeet the increasing demand of Chinese and inter-national markets with herbaceous peony being afocus for exports from China (Wang et al. 2011).

*Corresponding authors. Email: XiaoNan Yu: [email protected]. Email: Jaime A. Teixeira da Silva:[email protected]

New Zealand Journal of Crop and Horticultural Science, 2014http://dx.doi.org/10.1080/01140671.2014.944548

© 2014 The Royal Society of New Zealand

Dow

nloa

ded

by [

Que

ensl

and

Uni

vers

ity o

f T

echn

olog

y] a

t 09:

35 2

2 N

ovem

ber

2014

mailto:[email protected]

mailto:[email protected]

http://dx.doi.org/10.1080/01140671.2014.944548

Use of tissue culture could increase propagationrates, which would be significant for the productionof popular or newly selected cultivars (Shen et al.2012). Although various explants have been used toinitiate in vitro cultures of herbaceous peony, in-cluding underground buds (Wu & Yu 2010; Wu, Yuet al. 2011; Wu, Shen et al. 2011), stems, petioles andleaves (Wu, Shen et al. 2011; Yu, Wu & Pan 2011),problems continue to be encountered with tissueculture (Yu et al. 2005; Yu, Wu et al. 2011).

Peony seeds have a complex sequential dorm-ancy and may take up to 2 years to germinate(Krekler 1962; Griess & Meyer 1976). In peonybreeding, embryo culture can be used to overcomehybrid embryo abortion, bypass seed dormancy,shorten the breeding cycle and improve germina-tion rates. Work in embryo culture of herbaceouspeony has made some advances, including the iden-tification of basal medium, choice of plant growthregulators (PGRs) and breaking epicotyl dormancy.The seed coat (testa), endosperm extract and gib-berellic acid (GA3) influence the germination ofP. ostii var. lishizhenii embryos (Wang & van Staden2002). In their study, epicotyl dormancy was bro-ken by soaking intact seeds in GA3 or by growingexcised zygotic embryos (EZEs) on woody plantmedium (WPM; Lloyd &McCown 1980) containing1mg L−1 GA3.Wang and van Staden (2002) reportedthat 0.1 mg L−1 indole-3-butyric acid (IBA) induceda strong root system in in vitro germinated seed-lings. Lan (2003) overcame seed dormancy in intactseeds using Murashige and Skoog (MS) medium(Murashige & Skoog 1962) supplemented with1 g L−1 lactoalbumin hydrolysate (LH), 0.1 g L−1

vitamin C (VC), 3 g L−1 active charcoal (AC) and3 mg L−1 GA3. Somatic embryos could be inducedfrom P. anomala L. EZEs in the presence of 0.5 mgL−1 6-benzyladenine (BA), and also from callus inthe presence of 1 mg L−1 1-naphthyleneacetic acid(NAA) and 1 mg L−1 BA (Brukhin & Baatygina1994). High concentrations of NAA increased callusformation, but caused browning and a decrease inleaf differentiation.

Adventitious buds can be induced on cotyle-dons on MS medium containing 0.5–1.0 mg L−1

GA3 (Qiu et al. 2009). Gao (2010), using matureherbaceous peony EZEs as explants, found that

GA3 at 0–1.0 mg L−1 could replace low temper-ature (4 °C) to break epicotyl dormancy, while1.0 mg L−1 BA with 1.5 mg L−1 kinetin (Kin)promoted axillary shoot proliferation. Epicotyl dor-mancy was broken in herbaceous peony EZEs afterculture on modified Linsmaier and Skoog (LS)(Linsmaier & Skoog 1965) medium containing1 or 10 µM BA, and leaf number and petiolelength increased in response to 1.5 mg L−1 GA3

although roots that formed on EZEs growing on1 mg L−1 BAwere stunted and did not form lateralroots (Buchheim et al. 1994).

In these experiments, mature seeds of threevarieties of P. lactiflora were used to study EZEgermination, and to explore the optimum level andcombination of PGRs necessary to proliferate androot seedlings derived from EZE culture.

Materials and methods

Plant material

Paeonia lactiflora ‘Fen Yu Nu’ (Fig. 1A), ‘ZhongSheng Fen’ (Fig. 1B) and ‘Zhu Sha Pan’ (Fig. 1C)were used in the experiment. Open-pollinated seeds(50, 70 and >90 days after flowering [DAF]) werecollected from July to September 2011 from theSociety of Forestry Experimental Station (Beijing).Seeds older than 90 DAF were considered to bemature, while those at 50 and 70 DAF were con-sidered immature.

Reagents

All chemicals and reagents (tissue culture grade)were purchased from Beijing Biodee Biotechnol-ogy Co Ltd, Beijing, China.

Surface sterilization

Before initiating in vitro cultures, the explants—mature seeds whose testa had been either removed(i.e. EZEs) or retained—were washed in tap waterfor 30 min. Explants were dipped in 75% ethanolfor 30 s, immediately followed by 8 min steriliza-tion with a dilute solution of HgCl2 (0.1% w/v),then rinsed five times (5 min each time) in auto-claved distilled water. Following sterilization, the

2 M Shen et al.

Dow

nloa

ded

by [

Que

ensl

and

Uni

vers

ity o

f T

echn

olog

y] a

t 09:

35 2

2 N

ovem

ber

2014

Figure 1 Culture of mature Paeonia lactiflora Pall. zygotic embryos. A, Mature flowers of ‘Fen Yu Nu’; B, Matureflowers of ‘Zhong Sheng Fen’; C, Mature flowers of ‘Zhu Sha Pan’; D, Excised zygotic embryos (EZEs) of ‘ZhongSheng Fen’ on BM + 0.2 mg L−1 BA + 0.05 mg L−1 NAA + 0.5 mg L−1 GA3 10 days after initiation; E, Growth ofmature EZE of ‘Zhong Sheng Fen’ after culture in BM + 1.0 mg L−1 BA + 1.0 mg L−1 GA3 for 30 days; F, Growthof mature EZE of ‘Zhu Sha Pan’ after culture in BM + 1.0 mg L−1 BA + 1.0 mg L−1 GA3 for 35 days; G, Normalseedlings of ‘Fen Yu Nu’ formed in the presence of 0.2 mg L−1 BA + 0.05 mg L−1 NAA + 0.5 mg L−1 GA3 after 30days (normally expanded cotyledons); H, Axillary shoot induction of ‘Fen Yu Nu’ on BM + 0.5 mg L−1 BA + 0.3mg L−1 GA3 after 35 days; I–J, Shoot proliferation of ‘Fen Yu Nu’ on BM + 0.5 mg L−1 BA + 0.5 mg L−1 GA3

after 35 days; K, Root length of ‘Fen Yu Nu’ (L > 5 cm; 2 cm ≤ L ≤ 5 cm; and (L) < 2 cm) on PGR-free BM after35 days. Scale bars = 1 cm.

Induction and proliferation of Paeonia lactiflora axillary shoots 3

Dow

nloa

ded

by [

Que

ensl

and

Uni

vers

ity o

f T

echn

olog

y] a

t 09:

35 2

2 N

ovem

ber

2014

endosperms were removed from some EZEs toleave isolated embryos that were cultured on agar-solidified medium containing different PGR com-binations. Mature surface-sterilized seeds with testaand endosperm intact, seeds with testa removed

but with endosperm attached, and EZEs wereinoculated on to the basal medium (BM) supple-mented with 1.0 mg L−1 BA and 1.0 mg L−1 GA3

(initiation medium), based on trials conducted andexplained below.

Figure 1 (Continued)

4 M Shen et al.

Dow

nloa

ded

by [

Que

ensl

and

Uni

vers

ity o

f T

echn

olog

y] a

t 09:

35 2

2 N

ovem

ber

2014

Culture medium and growing conditions

The BM for all experiments was 1/2 MS supple-mented with double-strength calcium chloride (Ca2+),30 g L−1 sucrose and 7 g L−1 agar (≥98% purity)(Wu, Yu et al. 2011). Different combinations andconcentrations of PGRs, including BA, GA3, NAAand Kin, were added to this BM. The explants werecultured for 30 days when effects of experimentaltreatments were assessed and then plants weretransferred to fresh medium as required.

The pH of all media was adjusted to 5.8 prior toautoclaving at 118 °C for 18 min. The culturevessels comprised 100 mL Erlenmeyer flasks with40 mL of medium. Plants were grown in a tissueculture room at 25 ± 2 °C and a 16 h photoperiodwith 50 μmol m−2 s−1 photosynthetic photon fluxdensity (PPFD) supplied using cool white fluorescenttubes. All data (contamination percentage, germina-tion percentage [GP] and percentage seedling con-version to plantlets, in which cotyledons expandednormally) were recorded after 30 days of culture.

Effect of embryonic age on young embryoculture

EZEs of ‘Fen Yu Nu’, ‘Zhong Sheng Fen’ and ‘ZhuSha Pan’ of different maturities (i.e. 50, 70, 90DAF) were inoculated on to initiation medium.After 30 days, the level of contamination (%),germination (%) and seedling conversion to plant-lets (the number of seedlings/the number of explantsinoculated) (%) were calculated.

Initiation of zygotic embryo culture

EZEs of ‘Fen Yu Nu’ were used for this experi-ment. The effects of BA (0.2, 0.5, 1.0 mg L−1) incombination with NAA (0, 0.05, 0.1 mg L−1) andGA3 (0, 0.5, 1.0 mg L−1) on induction of growthwas studied. No zero value of BA was includedbecause a previous study (Wu, Yu et al. 2011) hadalready proved that BA had an effect on EZEinduction. Thus, the objective here was to confirmand investigate a wider range of effective BA con-centrations. This experiment was designed as anorthogonal test (L9 [33] in which explants werecultured for 30 days.

Axillary shoot induction and proliferation

Two experiments were carried out in chronologicalorder. In the first experiment, uncontaminated seed-lings obtained from the initiation of zygotic embryoculture and following germination of EZEs invitro were transferred to BM supplemented with0.5 mg L−1 BA and 0.3 mg L−1 GA3 for axillaryshoot growth; this medium had previously been op-timized for axillary shoot outgrowth by Wu (2011).In a subsequent experiment, the effect of differenttreatments on axillary shoot proliferation was tested.Shoots obtained from axillary shoot growth weretransferred to BM supplemented with (screenedfrom media reported in the literature; Shen et al.2012): 1.0 mg L−1 BA and 1.0 mg L−1 GA3, or BMsupplemented with 1.0 mg L−1 BA and 1.5 mg L−1

Kin, or BM supplemented with 1.0 mg L−1 BA, 1.0mg L−1 GA3 and 1.5 mg L−1 Kin (following theresults of Shen et al. 2012). The appearance of newleaves (number of leaves/number of leaves beforetransfer to medium) was recorded after 30 days.Explants were subcultured on to fresh mediumevery 30 days, and there were no more than fivetransfers for any seedling as seedling vigour de-clined after this.

Root initiation and seedling establishment

In this experiment, seedlings showing normal growthwere transferred to seedling medium in an attemptto improve acclimatization success. The media testedcomprised BM, BM supplemented with 2 mg L−1

BA and 0.2% AC, and BM supplemented with2 mg L−1 BA, 2 mg L−1 IBA and 0.2% AC (thesemedia were selected on the basis of results fromShen et al. 2012). The numbers of seedlings withdefined root lengths (L < 2 cm, 2 cm ≤ L ≤ 5 cm,or L < 2 cm) were determined after 35 days.

Acclimatization and transplant of rootedplantlets

Parafilm, which covered the flasks, was half openedfor 7 days, and then completely opened for 7 daysprior to deflasking. At deflasking, the seedling-derived plantlets (roots having already emerged)were removed from the flask using forceps and


Dow

nloa

ded

by [

Que

ensl

and

Uni

vers

ity o

f T

echn

olog

y] a

t 09:

35 2

2 N

ovem

ber

2014

rinsed with warm water to detach agar from theroots. Then they were dipped in 0.3% (w/v) potas-sium permanganate solution medium to providesome protection against fungi. Finally they weretransferred to an autoclaved soil-less medium com-prising perlite and vermiculite (1:1) and wateredwith 1/2 MS nutrient solution every day. The green-house conditions were identical to in vitro condi-tions: 25 ± 2 °C in a 16 h photoperiod with50 μmol m−2 s−1 PPFD supplied by cool whitefluorescent tubes.

Experimental design and statistical analyses

Experiments were laid out in a completely rando-mized design. Means were separated by one-wayanalysis of variance and significant differenceswere assessed using Duncan’s multiple range testat P = 0.05 using SPSS (Statistical Product andService Solutions; USA) software version 13.0.Each experiment included at least three replica-tions of 10 explants per treatment.

Results

When mature seeds of ‘Fen Yu Nu’ with or withoutthe testa were inoculated on to initiation medium,

the contamination level was high (37.67% and62.91%, respectively) (Table 1) and GP and seed-ling conversion to plantlets were close to zero. Bycomparison, EZEs showed lowest contamination(6.67%) while GP and seedling conversion to plant-lets were highest (89.43% and 61.33%, respectively)(Table 1). Moreover, about 5 days after transfer toinitiation medium, cotyledons expanded. Seedswith the testa (i.e. intact seeds) only germinatedafter 20 days. Thus, EZEs were the most suitableexplants for culture initiation.

GP and seedling conversion to plantlets weresignificantly higher in ‘Fen Yu Nu’ EZEs harvested90 DAF (90.54% and 71.29%, respectively; Table 2)than 50- and 70-day-old EZEs. Similar trends wereobserved for ‘Zhong Sheng Fen’ and ‘Zhu ShaPan’. GP and seedling conversion to plantlets werehigher (95.33% and 81.29%, respectively) (Table 3)in 90-day-old ‘Zhong Sheng Fen’ EZEs than in‘Zhu Sha Pan’ EZEs (95.00% and 66.67%,respectively; Table 4) (Figs 1D, 1E). In summary,mature (i.e. 90-day-old) EZEs plated on to initiationmedium had better and more effective germinationand development into seedlings than less matureEZEs (i.e. 50 or 70 days old).

The highest GP of EZEs (70%) (Table 5) wasobserved onBM supplementedwith 0.2mgL−1 BA,

Table 1 Assessment of three parameters using explants derived from zygotic embryos of P. lactiflora ‘Fen Yu Nu’(n = 30).

Treatments Contamination (%) Germination (%) Seedling conversion to plantlets (%)

Seeds with testa retained 37.67 ± 2.36b 0.00 ± 0.00c 0.00 ± 0.00b

Seeds with testa peeled off 62.91 ± 1.96a 6.67 ± 2.68b 0.00 ± 0.00b

ESEs 6.67 ± 1.88c 89.43 ± 2.18a 61.33 ± 1.35a

Different letters within a column indicate significant differences according to Duncan’s multiple range test (P ≤ 0.05). Explantswere inoculated on to BM supplemented with 1.0 mg L−1 BA and 1.0 mg L−1 GA3.

Table 2 Effect of age on culture of young embryos of ‘Fen Yu Nu’ (n = 30).

Sampling time Germination (%) Seedling conversion to plantlets (%)

50 d after flowering 51.67c 41.67c

70 d after flowering 72.00b 59.33b

90 d after flowering 90.54a 71.29a


6 M Shen et al.

Dow

nloa

ded

by [

Que

ensl

and

Uni

vers

ity o

f T

echn

olog

y] a

t 09:

35 2

2 N

ovem

ber

2014

0.05 mg L−1 NAA and 0.5 mg L−1 GA3 (Fig. 1F).The highest rate seedling conversion to plantlets(Fig. 1G), 40% (Table 5), was observed fromtreatment 4 (Table 5). The highest rate of root initi-ation (23.33%) was on initiation medium (Table 5).The presence of GA3 was the most important factorensuring germination of EZEs and seedling for-mation, while NAA was the most important factorfor root initiation. As the concentration of NAAincreased, the percentage of rooting decreased, and

there was increased callus formation on the cotyle-dons. As GA3 concentration increased, the frequencyof deformities in seedlings increased, particularly tothe epicotyls, which showed symptoms of hyperhy-dricity. Based on the average seedling rate (Table 6),the most suitable medium for initiation of in vitrocultures from EZEs was BM supplemented with0.5 mg L−1 BA and 0.5 mg L−1 GA3.

In the transitional stage from germinationto axillary branching, the cotyledons had fully

Table 3 Effect of age on culture of young embryos of ‘Zhong Sheng Fen’ (n = 30).


50 d after flowering 61.67b 49.73c

70 d after flowering 86.67a 63.33b



Table 4 Effect of age on culture of young embryos of ‘Zhu Sha Pan’ (n = 30).


50 d after flowering 76.79c 54.72c

70 d after flowering 87.88b 58.79b



Table 5 The influence of different PGRs on seedling production of P. lactiflora ‘Fen Yu Nu’ EZEs (L9 (33)) (n = 30).

TreatmentsBA

(mg L−1)NAA

(mg L−1)GA3

(mg L−1)Germination

(%)Seedling conversion to

plantlets (%)

1 1 (0.2) 1 (0) 1 (0) 40.00 ± 1.06d 0.00 ± 0.00d

2 1 (0.2) 2 (0.05) 2 (0.5) 70.00 ± 3.87a 30.68 ± 2.38b

3 1 (0.2) 3 (0.1) 3 (1.0) 46.67 ± 0.00c 0.00 ± 0.00d

4 2 (0.5) 1 (0) 2 (0.5) 60.00 ± 10.00b 40.00 ± 10.00a

5 2 (0.5) 2 (0.05) 3 (1.0) 26.67 ± 1.89e 10.00 ± 0.00c

6 2 (0.5) 3 (0.1) 1 (0) 13.33 ± 0.00g 0.00 ± 0.00d

7 3 (1.0) 1 (0) 3 (1.0) 23.56 ± 1.76e,f 5.56 ± 5.09c,d

8 3 (1.0) 2 (0.05) 1 (0) 17.72 ± 1.00f,g 3.33 ± 5.77c,d

9 3 (1.0) 3 (0.1) 2 (0.5) 30.00 ± 0.00e 0.00 ± 0.00d

Different letters within a column indicate significant differences according to Duncan’s multiple range test (P ≤ 0.05). EZEs wereinoculated on to BM supplemented with different concentrations of PGRs. Values in parenthesis = level of concentration of thedifferent factors. The PGR combination in treatments 1, 6 and 9 were unsuitable for tender/true leaves, so seedlings were unable toconvert into plantlets even though they developed roots. Seedling conversion to plantlets is different from germination. This isbecause in the process of embryo growth, many cotyledons are unable to expand normally and cannot form healthy seedlings, sothere is some difference between both processes, hence the use of two terms.


Dow

nloa

ded

by [

Que

ensl

and

Uni

vers

ity o

f T

echn

olog

y] a

t 09:

35 2

2 N

ovem

ber

2014

expanded, the taproot extended normally, and seed-lings were growing well after 30 days on 1/2 MSmedium supplemented with 0.5 mg L−1 BA and0.3 mg L−1 GA3 (Fig. 1H). Significantly moreaxillary shoots grew on initiation medium than onthe two other media with a concomitantly high

number of expanded leaves produced (Table 7).Thus, the optimal medium for axillary shoot pro-liferation was initiation medium (Figs 1I, 1J).

During establishment, most seedlings developeda taproot that was slender and weak. In an attemptto form a stronger root system and seedling—thought essential for effective acclimatization—shoots were transferred to media expected to promoteroot initiation. The highest rooting percentage(100%) was observed on PGR-free BM, on whichthe frequency of seedlings with roots longer than 5cm was 41.9% which was higher than that observedon the other two media (BM + 2 mg L−1 BA +0.2% AC and BM + 2 mg L−1 BA + 2 mg L−1 IBA+ 0.2% AC), at 30.79% and 37.23%, respectively(Table 8). The frequency of roots with lengthbetween 2 cm and 5 cm was highest (47.53%) onBM supplemented with 2 mg L−1 BA and 0.2% AC(Fig. 1K). Thus PGR-free BMwas the best mediumfor seedling growth and establishment (Table 8).

Plantlet acclimatization was generally poor.Approximately 7 days after deflasking, most weak

Table 6 Analysis of average rate of seedling conversionto plantlets.

TreatmentsBA

(mg L–1)NAA

(mg L–1)GA3

(mg L–1)

R (seedling) 13.71 15.19 22.45x̄1 (seedling) 10.23 15.19 1.11x̄2 (seedling) 16.67 14.67 23.56x̄3 (seedling) 2.96 0 5.19

Range = the detection of that concentration of PGR that wasmost suitable for the experiment. X1, X2 and X3 are the averagevalues of each concentration of PGR for three tests whosemaximum minus minimum represents the range, R. Forexample, for BA: X1: 10.23 = (0.00 + 30.68 + 0.00)/3; X2:16.67 = (40.00 + 10.00 + 0.00)/3; X3: 2.96 = (5.56 + 3.33 +0.00)/3; R: 13.71 = 16.67–2.96.

Table 7 Impact of three PGR treatments on axillary shoot proliferation and growth of P. lactiflora ‘Fen Yu Nu’seedlings (n = 30).

Treatment (mg L−1) Leaf expansion (number of times that leaves expanded) Number of axillary shoots

1.0 BA + 1.0 GA3 3.76 ± 0.28a 10.98 ± 0.85a

1.0 BA + 1.5 Kin 2.69 ± 0.09b 7.43 ± 1.15b

1.0 BA + 1.0 GA3 + 1.5 Kin 2.72 ± 0.16b 7.38 ± 1.29b

Different letters within a column indicate significant differences according to Duncan’s multiple range test (P ≤ 0.05). EZEs wereinoculated on to BM supplemented with different concentrations of PGRs.

Table 8 Treatments used to increase lateral root induction and strengthen seedlings (n = 10).

Seedlings with defined root length (L) (%)

Treatments (mg L−1) Rooting (%) L < 2 cm 2 cm ≤ L ≤ 5 cm L > 5 cm

1/2 MS (control) 100.00 ± 0.00a 25.26 ± 1.72a 32.82 ± 2.52c 41.92 ± 0.73a

2.0 BA + 0.2% AC 98.89 ± 1.92a 21.68 ± 2.05b 47.53 ± 1.48a 30.79 ± 1.51c

2.0 BA + 2.0 IBA + 0.2% AC 96.67 ± 0.00b 21.41 ± 2.38b 41.36 ± 2.77b 37.23 ± 0.84b

Different letters within a column indicate significant differences according to Duncan’s multiple range test (P ≤ 0.05). BMsupplemented with different concentrations of PGRs.

8 M Shen et al.

Dow

nloa

ded

by [

Que

ensl

and

Uni

vers

ity o

f T

echn

olog

y] a

t 09:

35 2

2 N

ovem

ber

2014

seedlings gradually yellowed, wilted and perished,while some perished from bacterial or fungal con-tamination. Even though the strong seedlings per-sisted for 3 weeks, and leaf area and plant heightincreased, acclimatized plants did not produce newleaves. These plants also eventually died.

Discussion

The seed coat or testa is not a significant barrierto water uptake in peony seed but does providea degree of mechanical resistance, thereby inhibit-ing germination (Wang & van Staden 2002). Ourstudy confirms previous findings for P. ostii thatEZEs are the best explants to ensure high germina-tion (Wang & van Staden 2002). He et al. (2006)also used zygotic embryos (equivalent to EZEs) ofdifferent ages (20, 28, 48, 66 and 90 days afterpollination) of ‘Shu Sheng Peng Mo’ and ‘FengDan Bai’ as explants. In their study, 90-day-oldEZEs grew into seedlings 5 weeks after culture onMS medium + BA (0–2 mg L−1) + IAA (0–1 mgL−1) + GA3 (0–1 mg L−1) for 3 to 4 months, with aseedling percentage of 7.4% and 13.5% for ‘ShuSheng Peng Mo’ and ‘Dan Feng Bai’, respectively.Their results showed that successful germinationand seedling establishment depend on embryo ma-turity (>90 days for best germination), with poorgermination and seedling establishment observedwhen immature embryos (50–70 days) were used.GA3 is frequently used to replace low temperatureto break epicotyl dormancy in herbaceous peonyzygotic embryos (Zhang & Wang 2008; Gao2010). The seed structures surrounding the embryoimpose mechanical restraints on germination whilethe embryo itself appears to not have sufficientPGRs to promote epicotyl growth as exogenousGA3, which functions to promote epicotyl growth(Wang & van Staden 2002). In the present study,GA3 was the most important factor in promotingzygotic embryo germination and seedling devel-opment. NAA is also a good inducer of embryodevelopment in tree peony (Jia et al. 2006; Zhouet al. 2009). However, in our study, as the concen-tration of NAA increased, so too did the amountof callus at the base of seedlings. Conversely, in

the absence of added NAA, many seedlings(23.33%) grew roots directly, indicating eitherthat BA could directly induce embryos and formtaproots without any specific and separate rootingstep being required, or that the level of BA wastoo low to interfere with root development. BAand NAA are usually combined during peony(‘Shu Sheng Peng Mo’) seedling induction fromzygotic embryos (Gao 2009; Zhou et al. 2009),although both their experimental results show thatleaving out NAAwas most favourable for formingseedlings when the concentration of BAwas 0.2 or0.5 mg L−1. However, in the present study, thecombination of BA and GA3 was best for ‘Fen YuNu’, supporting the notion that protocols remaingenotype-dependent and conditions need to beoptimized for each cultivar. Buchheim et al.(1994) noted a highly significant increase in leafnumber when embryos were cultured on mediumcontaining BA and usually treated with GA3,which resulted in good rates of axillary shootproliferation.

Roots developed spontaneously from EZEs,as expected. Using media with double the Ca2+

concentration is useful for the in vitro growth ofboth herbaceous peony (Guo 2001) and tree peony(Bouza et al. 1994). The use of BA, IBA and ACresulted in more roots 2–5 cm in length, but fewerroots less than 2 cm or longer than 5 cm in length,compared with the control treatment (Table 4),indicating the need for more focus on root initi-ation and growth in P. lactiflora in future studies.The development of a weak root system appears toremain to be the main limiting factor when defla-sking herbaceous peony (Shen et al. 2012). At theend of these experiments, no seedlings were suc-cessfully acclimatized. Possible reasons for poorsurvival may be few lateral roots on the seedlings,the need to adjust the balance of porosity andmoisture in the growing medium after deflasking,or a requirement for better environmental control,including temperature, humidity and sterility fol-lowing transfer to the greenhouse. Special attentionneeds to be paid in the future to these factors toachieve a higher level of survival of acclimatized


Dow

nloa

ded

by [

Que

ensl

and

Uni

vers

ity o

f T

echn

olog

y] a

t 09:

35 2

2 N

ovem

ber

2014

plantlets. Germinating peony normally results inabout 70% GP but can only be done after seedmaturation (5–6 months after pollination) and canonly be carried out in the autumn (Xiaonan Yu,pers. obs.). We thus believe that this in vitromethod can help shorten the breeding cycle.

Acknowledgements

This work was financially supported by the NationalNatural Science Foundation of China (31400591) andFundamental Research Funds for the Central Universit-ies (No. YX2014-20). The authors wish to thankthe valuable comments by the editor and anonymousreviewers.

References

Bouza L, Jacques M, Miginiac M 1994. In vitro propaga-tion of Paeonia suffruticosa Andr. cv. ‘Mme deVatry’: development effects of exogenous hormonesduring the multiplication phase. Scientia Horticul-turae 57: 241–251.

Brukhin VB, Baatygina TB 1994. Embryo culture andsomatic embryogenesis in culture of Paeoniaanomala. Phytomorphology 44: 151–157.

Buchheim JAT, Burkhart LF, Meyer MM 1994. Effectof exogenous gibberellic acid, abscisic acid, andbenzylaminopurine on epicotyl dormancy of cul-tured herbaceous peony embryos. Plant Cell, Tis-sue and Organ Culture 36: 35–43.

Gao CY 2009. Study on the embryo culture of peony(Paeonia suffruticosa) in vitro. Journal of AnhuiAgricultural Science 37: 8844–8865.

Gao CY 2010. Study on embryo culture of Chineseherbaceous peony in vitro. Northern Horticulture16: 157–158.

Griess JL, Meyer MM 1976. Dormancy and survival ofperennial plants. American Peony Society Bulletin220: 21–24.

Guo FY 2001. Study on tissue culture of Paeonia lacti-flora. MSc thesis. Beijing, Beijing Forestry Uni-versity. Pp. 37–38.

He GM, Chen FY, Li P 2006. Preliminary studies onculture in vitro of ovule and immature embryo oftwo tree-peony cultivars. Acta Horticulturae Sinica33: 185.

Jia WQ, Wang GY, Liu Y 2006. The study on the peonyembryo culture in vitro. Journal of Anhui Agricul-tural Science 34: 6441–6444.

Kamenetsy R, Dole J 2012. Herbaceous peony (Paeonia):genetics, physiology and cut flower production. In:Van Tuyl J, Arens P eds. Bulbous Ornamentals.Floriculture and Ornamental Biotechnology 6(Special Issue 1): 62–77.

Krekler WH 1962. Peony culture: uses and propagation.In: Wister JC ed. The peony. Washington, DC,American Horticultural Society. Pp. 100–111.

Lan HY 2003. Studies on embryo culture of Paeonialactiflora Pall. and paeoniflorin content in the roots.MSc thesis. Hohhot, Inner Mongolia University.

Linsmaier EM, Skoog F 1965. Organic growth factorrequirements of tobacco tissue. Physiologia Plan-tarum 18: 100–127.

Lloyd G, McCown B 1980. Commercially-feasiblemicropropagation of mountain laurel, Kalima latifo-lia, by use of shoot tip culture. Proceedings of theInternational Plant Propagators Society 30: 421–427.

Murashige T, Skoog F 1962. A revised medium for rapidgrowth and bioassays with tobacco tissue cultures.Physiologia Plantarum 15: 473–497.

Qin KJ 2004. Herbaceous peony. Beijing, China For-estry Press. 76 p.

Qiu DK, Zhang SR, He XD 2009. The tissue culture ofherbaceous peony. China Flowers and Horticulture2: 32–33.

Shannon J, Kamp JR 1959. Trials of various possiblepropagation methods on herbaceous peonies. Illi-nois State Florists Association Bulletin 197: 4–7.

Shen MM, Wang Q, Yu XN, Teixeira da Silva JA2012. Micropropagation of herbaceous peony(Paeonia lactiflora Pall.). Scientia Horticulturae148: 30–38.

Wang H, van Staden J 2002. Seeding establishmentcharacteristics of Paeonia ostii var. lishizhenii.South African Journal of Botany 68: 386–388.

Wang JG, Zhang ZS 2005. Herbaceous peony of China.Beijing, China Forestry Press. 1 p.

Wang LH, Zheng LW, Yu XN 2011. Chinese andwestern cut flower application of herbaceous peonyand market trend analysis. Heilongjiang Agricul-tural Science 2: 147–149.

Wu HJ 2011. Rapid propagation in vitro and callus cul-tivation of Paeonia lactiflora. MSc thesis. Beijing,Beijing Forestry University.

Wu HJ, Shen MM, Yu XN 2011. Multiple shoot induc-tion and rooting of Paeonia lactiflora Dafugui. Jour-nal of Northeast Forestry University 39: 20–22.

Wu HJ, Yu XN 2010. The effect of different cultivarson buds inducing of Paeonia lactiflora Pall. andclustered shoots inducing of ‘Zhu Sha Pan’. Jour-nal of Inner Mongolia Agricultural University 31:29–33.

10 M Shen et al.

Dow

nloa

ded

by [

Que

ensl

and

Uni

vers

ity o

f T

echn

olog

y] a

t 09:

35 2

2 N

ovem

ber

2014

Wu HJ, Yu XN, Teixeira da Silva JA, Lu GP 2011. Dir-ect shoot induction of Paeonia lactiflora ‘ZhongSheng Fen’ and rejuvenation of hyperhydric shoots.New Zealand Journal of Crop and HorticulturalScience 39: 271–278.

Yu HM, Lu M, He XG 2005. The techniques of planttissue culture and its frequent problems and resolu-tions. Journal of Shandong Education Institute 20:101–103.

Yu XN,WuHJ, Cheng FY, Teixeira da Silva JA, ShenMM2011. Studies on multiple shoot induction and proli‐feration of Paeonia lactiflora Pall. ‘Zhong ShengFen’. Propagation of Ornamental Plants 11: 144–148.

Yu XN, Wu HJ, Pan T 2011. Callus induction anddifferentiation of four peony cultivars. Journal ofHunan Agricultural University (Natural Sciences)37: 166–171.

Zhang RR, Wang KC 2008. Activity of intrinsic inhib-itor in seed of Paeonia lactiflora. Chinese Tradi-tional and Herbal Drugs 39: 1880–1883.

Zhou XM, Cheng FY, Zhong Y, Qi LW 2009. Induce-ment and development of somatic embryos inPaeonia rockii ‘Shu Sheng Peng Mo’. Journal ofBeijing Forestry University 31: 151–154.


Dow

nloa

ded

by [

Que

ensl

and

Uni

vers

ity o

f T

echn

olog

y] a

t 09:

35 2

2 N

ovem

ber

2014

induction and proliferation of axillary shoots from in vitro culture of paeonia...

Documents