the effects of glucose and growth regulators on the … · 2013-05-15 · the effects of glucose...

Journal of Fruit and Ornamental Plant Research Vol. 18(2) 2010: 309-320

THE EFFECTS OF GLUCOSE AND GROWTHREGULATORS ON THE ORGANOGENESIS OF

Paeonia lactiflora Pall. IN VITRO

Eleonora Gabrysz ewska

Research Institute of Pomology and FloriculturePomologiczna 18, 96-100 Skierniewice, POLAND

e-mail: [email protected]

(Received November 4, 2010/Accepted November 29, 2010)

A B S T R A C T

Herbaceous peony plants successfully propagated in vitro do not survive thetransfer to the ex vitro environment. For other species, storage organ formation invitro can limit the loss of plants during acclimatization. In the natural conditions, therenewal buds for the following year originate on the underground crown (metamor-phosed underground shoot, rhizome) of herbaceous peony. A perennial crown androots serve for the accumulation of the storage products and plant renewal.

The aim of the experiment was to investigate the influence of glucose (30, 6090 g l- 1) and growth regulators (kinetin, IBA, GA3) on the shoot, renewal bud, rootgrowth and development of Paeonia lactiflora ’Jadwiga’ in vitro. Excision of allleaves from isolated explants inhibited production of new shoots and leaves, andevidently induced formation of renewal buds. Increasing the glucose supply, espe-cially in the absence of growth regulators, decreased production of shoots and out-growth of leaves. The stronger inhibition of shoot growth by glucose was observed onthe explants without leaves. By contrast, the beneficial effect of glucose on renewalbud formation was observed. A single supply of kinetin, IBA or GA3 stimulatedshoots and leaf growth and inhibited renewal bud formation, on the explants isolatedwith leaves. Interaction of kinetin, GA3 and IBA (added together) and the highestglucose level enhanced the growth of shoots on the explants containing leaves, andincreased the number of renewal buds, on the explants without leaves. Increasingglucose level enhanced the number of roots in the absence of growth regulators on theexplants containing leaves. The supply of IBA in the medium containing 30 g l -1

glucose, stimulated the root production on the explants without leaves. The additionof GA3 or kinetin (singly or simultaneously with IBA) to the medium with differentconcentrations of glucose, strongly inhibited rooting.

The results presented here, show that a high level of glucose and exogenousgrowth regulators (kinetin, GA3 , IBA) together stimulate shoot and renewal bud for-

E. Gabryszewska

J. Fruit Ornam. Plant Res. vol. 18(2) 2010: 309-320310

mation but the way of organogenesis depends on the presence or absence of leaves.The interaction between auxin (exogenous or endogenous) and glucose regulate rootformation on the peony shoots but the final effect depends on the type of explants(with or without leaves). It is possible that leaves have a very important hormonalfactors, which stimulate shoot growth or rooting and inhibit renewal bud formation.

Key words: Paeonia lactiflora, tissue culture, glucose, growth regulators, organo-genesis, renewal bud formation

Abbreviations: GA3 – gibberellic acid, IBA – indole-3-butyric acid, BAP – 6-benzylaminopurine, ABA – abscisic acid, MeJA – methyl jasmonate, TDZ –thidiazuron, MS – Murashige and Skoog medium

INTRODUCTION

Peonies are perennial ornamentalplants of the genus Paeonia, whichbelongs to the family Paeoniaceae.Herbaceous peonies are grown suc-cessfully in moderate, cold-winterclimatic zones. The renewal buds forthe following year originate on theunderground crown, at the base ofthe annual stems. After the floweringof the aboveground annual stems (atthe end of June), the new monocarpicshoot is initiated in the renewal bud.The renewal bud contains central andaxillary (4-6) lateral shoots. It is pro-tected by cover leaves (Barzilayet al., 2002; Czekalski and Jerzy,2003). A perennial crown (metamor-phosed underground shoot, rhizome)and roots serves for the accumulationof the storage products and plantrenewal (Kapinos and Dubrov, 1993;Walton et al., 2007). The main stor-age carbohydrate is starch. The con-centration of starch declines withresumption of the growth in spring(Walton et al., 2007).

In herbaceous peony, the promot-ing effect of growth regulators onaxillary and adventitious bud or root

induction was demonstrated by a fewauthors (Hosoki et al., 1989; Albersand Kunneman, 1992; Tian, 2008;Gabryszewska, 2009). The plants suc-cessfully propagated in vitro do notsurvive the transfer to the ex vitro envi-ronment. The success of acclimatiza-tion has been reported by only a fewauthors (Hosoki et al., 1989; Albersand Kunneman, 1992; Gabryszewska,2009; Gabryszewska and Kawa-Miszczak, 2010). However, aftertransferring to soil in the greenhouse,the peony microcuttings survive ata low percentage because of infectionfrom pathogens (Albers and Kunne-man, 1992; Tian, 2008; Gabryszewska,2009; Gabryszewska and Kawa-Miszczak, 2010). Also, the growth ofyoung plants is slow or stopped be-cause of bud dormancy (Gabryszew-ska and Kawa-Miszczak, 2010).

Storage organ formation in vitrocan limit the loss of plants duringacclimatization. In the case of herba-ceous peony, there are no studiesconcerning the in vitro formation ofunderground crown with renewalbuds and/or roots. For other species,there are a number of factors thatinfluence the induction and growth

The effects of glucose and growth regulators…

J. Fruit Ornam. Plant Res. vol. 18(2) 2010: 309-320 311

of storage organs in vitro: environ-mental conditions (temperature, pho-toperiod, light quality), mediumcomposition (type and concentrationof growth regulators, carbohydrates,activated charcoal, gelling agents)and the genetic makeup of the plant(Ascough et al., 2008).

Plant growth regulators and theirinteraction with sugars play an im-portant role in the storage organ in-duction of many plants propagated invitro. Jasmonic acid and methyl jas-monate were capable of inducing invitro tuberization of storage organ inmany plant species. Jasmonic acidstimulated bulb formation of garlic(Ravnikar et al., 1993) and inductionof tuber formation of potato (Koda etal., 1991) propagated in tissue cul-ture. An increase in the level of jas-monates and a decrease in the levelof gibberellins influenced the initia-tion of potato tuberization (Koda andKikuta, 2001). Exogenous gibberel-lin added to the medium inhibitedbulbing of Allium cepa. On the otherhand, the inhibitors of gibberellinbiosynthesis (ancymidol, flurprimi-dol, paclobutrazol) promoted bulbformation (Le Guen-Le Saos et al.,2002). The interaction between gib-berellin and ABA and sucrose regu-lates tuber formation of potato (Xu etal., 1998). Sucrose plays an impor-tant role in the storage organ forma-tion of many species propagated invitro. The increase of the sucroseconcentration in the medium stimu-lated the lily and onion bulb forma-tion (Takayama and Misawa, 1980;Gerrits and De Klerk, 1992; Keller,1993; Kästner et al., 2001), the corm

induction of Watsonia vanderspuyiae(Ascough et al., 2008), the microtubersformation of Solanum tuberosum(Gopal et al., 2001), Xanthosoma sag-ittifolium (Omokolo et al., 2003), Dio-scorea cayenensis – D. rotundatacomplex (Ovono et al., 2009) and themicrorhizome production of Zingiberofficinale (Zheng et al., 2008).

Sugars also act as signaling mole-cules whose transduction pathwaysinfluence developmental and metabolicprocesses. Some of the effects of sug-ars on plant growth and developmentsuggest an interaction of sugar signalswith hormonal regulation (Smeekens,2000, Rolland et al., 2002, 2006). Glu-cose has emerged as a key regulator ofmany vital processes (Moore et al.,2003).

The influence of glucose andgrowth regulators on the Paeonialactiflora renewal bud, shoot and rootformation in vitro was investigated. Inaddition, the interactions between theglucose and growth regulators in thegrowth and development of differentorgans were studied.

MATERIAL AND METHODS

The study was carried out onPaeonia lactiflora ‘Jadwiga’. Plantmaterial used for the experiment waspropagated on the MS (Murashigeand Skoog, 1962) medium contain-ing: kinetin 1 mg l-1 + 2iP 1 mg l-1 +BAP 1 mg l -1 + TDZ 0.01 mg l-1 andsucrose 30 g l-1. In the experimenttwo different peony explants wereused: the pieces of crown with oneshoot (with or without leaves). Theinfluence of glucose (30, 60, 90 g l-1)

E. Gabryszewska


and kinetin 1 mg l-1, GA3 1 mg l-1,IBA 1 mg l-1 (added to the mediumalone or together) on the shoot, re-newal bud and root formation wasinvestigated. Each treatment con-sisted of 3 jars with 5 explants. Theexperiment was repeated twice (2series). Culture conditions werea 16 h photoperiod provided by cool-white fluorescent lamps (PhilipsTLD 36W/95) at 80 µmol m -2s -1 anda temperature of 25 °C.The observa-tions and measurements were re-corded after two months of culturing.The number of shoots, leaves, buds,and roots per explant were recorded.The statistical analysis of the treat-ments was tested using analysis ofvariance and means were comparedby Duncan’s multiple range test.

RESULTS AND DISCUSSION

Plant growth regulators influencein vitro organogenesis in differentspecies, in diverse ways. Thesevariations are due to the fact thatdifferent species, organs or section oforgans differ in their ability to pro-duce endogenous growth substances.Also, the different species and organsshow various abilities to recognize,take up, transport, metabolize andrespond to the exogenous plantgrowth regulators (Cheesman et al.,2010). Studies in vitro seem to bea very useful tool for gaining a betterunderstanding of the hormonal con-trol of the development and growthof different organs.

After two months of culturing, themorphogenic response of Paeonialactiflora ‘Jadwiga’ explants isolated

with or without leaves were evalu-ated. Excision of all leaves stronglyinhibited production of new shootsand leaves on the explants (Fig. 1 A,B; Fig. 3). Increasing the glucose(30-90 g l-1) supply in the medium,especially in an absence of growthregulators, decreased production ofshoots and outgrowth of leaves onthe both kinds of explants. Thestronger inhibition of shoot growthby glucose was observed on the ex-plants without leaves. Presence ofleaf and simultaneous supply of theexogenous growth regulators (ki-netin, GA3, IBA), added to the me-dium singly or together, had a stimu-latory effect on the shoot and leafgrowth of peony. It is possible, thatexogenous growth regulators canovercome the sugar-derived inhibi-tion and stimulate the shoot forma-tion and growth. Some authors dem-onstrated the promoting effect ofexogenous cytokinins on axillary oradventitious bud induction and mul-tiplication of herbaceous peony (Ho-soki et al., 1989; Albers and Kunne-man, 1992; Tian, 2008; Gabryszewska,2009). The axillary shoot formationwas induced by BAP, TDZ or the addi-tion of a few cytokinins together (Ho-soki et al., 1989; Albers and Kunne-man, 1992; Tian, 2008; Gabryszewska,2009). The mixture of TDZ and cyto-kinins (BAP, 2iP, kinetin) in the me-dium was more effective comparedto TDZ used alone (Gabryszewska,1998). An increased level of TDZ(0.1-3 mg l-1) in the medium stronglystimulated the adventitious shootregeneration of herbaceous peony(Thian, 2008). The combination of



cytokinins with a high level of GA3

(10 mg l-1) and sucrose (60-90 g l-1)significantly increased multiplicationrate (8 shoots/explant) of Paeonialactiflora ‘Jadwiga’ by axillarybranching (Gabryszewska, 2009). Amodel has been proposed in whichsugar inhibits the peony axillary budgrowth by negative regulation of theproduction and/or signal transductionof gibberellin. Exogenous gibberel-lins can overcome the sugar-derivedinhibition and stimulate the axillaryshoot growth in the presence of cyto-kinins. However, the addition of ex-ogenous auxin, to the above mentionedmedium, inhibited the shoot growthand influenced the progression ofshoots from a juvenile to adult phase.Application of gibberellin does notovercome the auxin-derived inhibition(Gabry-szewska, 2009).

At present, there is no informa-tion on the mechanism of in vitroformation of renewal buds on under-ground crown (rhizome) of Paeonialactiflora. The data indicate that ex-cision of all leaves promoted therenewal bud formation in the absenceof exogenous growth regulators aswell as in their presence in the me-dium (Fig. 2 A; Fig. 3, 4). The addi-tion of 1 mg l -1 kinetin, IBA or GA3

(singly or together) inhibited renewalbud formation on the explants iso-lated with leaves. Similarly, in tissueculture of Dioscorea spp., cytokinininhibited tuberization. But a highlevel of sugar in the medium canovercome the cytokinin-derived in-hibition and stimulate the tuber for-mation (Lauzer et al., 1995). On theother hand, the highest tuberization

of Xanthosoma sagittifolium wasobtained on medium with BAP anda high concentration of sucrose(80 gl-1) (Omokolo et al., 2003). Therenewal bud formation of peony‘Jadwiga’ was induced by glucose ona growth regulator-free medium(Fig. 2 A). It is known that solublesugars could affect the formation ofadult structures, such as leaves andtubers (Gibson, 2005). Interaction ofall growth substances (kinetin, GA3,IBA added together) and high glu-cose level induced renewal bud for-mation (about 4.5/explants), on thepeony explants isolated withoutleaves (Fig. 2 A). On the same kindof explants, the single addition ofGA3 to the medium with glucose,resulted in a similar growth of budsas that on the medium which had anabsence of growth regulators. Re-cently, it was reported that interac-tion of gibberellin and sugars affectstorage organ formation in variousspecies of plants in vitro (Xu et al.,1998; Ascough et al., 2008; Zheng etal., 2008). In the case of Watsoniavanderspuyiae, interaction betweenGA3, sucrose and reduced tempera-ture, promoted corm formation (As-cough et al., 2008). Also, the addi-tion of GA3 together with a highlevel of sucrose (80 gl -1) markedlyincreased rhizome production ofZingiber officinale (Zheng et al.,2008). For this species, the effect ofgibberellin on rhizome inductionwas larger than that kinetin orNAA. The dominant role of GA inthe potato tuber formation wasdemonstrated by Xu et al. (1998).In addition, sucrose regulates

E. Gabryszewska


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Explanations: G – glucose, GA – gibberellic acid, KIN- kinetinMeans marked with the same letter are not significantly different at p ≤0.05, according to Duncan’smultiple range test

Figure 1. The influence of glucose (30, 60, 90 g l-1) and growth regulators (kinetin,GA3, IBA – 1 mg l-1) on the number of shoots (A) and leaves (B) of Paeonia lactiflora‘Jadwiga’ in vitro



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Explanations: G – glucose, GA – gibberellic acid, KIN- kinetinMeans marked with the same letter are not significantly different at p ≤0.05, according to Duncan’smultiple range test

Figure 2. The influence of glucose (30, 60, 90 g l-1) and growth regulators (kinetin,GA3, IBA – 1 mg l-1) on the number of renewal buds (A) and roots (B) of Paeonialactiflora ‘Jadwiga’ in vitro

E. Gabryszewska


tuberization of potato by influencingthe endogenous gibberellin level.Other plant growth regulators areinvolved in the regulation of tuberformation but their effects seem todepend on the final GA content inthe tissue.

The present results indicate, thatthe leaves as the source of endoge-nous growth regulators, also influ-enced root formation on the peonyshoots. Explants isolated with leavesproduced roots on the growth regula-tors free-medium but with the pres-ence of glucose (Fig. 2 B, Fig. 3).Increasing glucose level stronglyenhanced the number of roots (from0.4 to 2.6 roots/explant) in the ab-sence of growth regulators, on theexplants containing leaves. But, theaddition of GA3 or kinetin to themedium with different concentra-tions of glucose strongly inhibitedrhizogenesis. Also, the presence ofGA3 and kinetin (added together) inthe IBA-containing medium signifi-cantly suppressed the rooting on theboth kinds of explants. The excisionof leaves and supply of IBA (1 mg l-1)promoted rhizogenesis (3 roots/explant)on the shoots growing at the lowestconcentration of glucose (30 g l-1).Thus, it seems that the interactionbetween auxin (exogenous or en-dogenous) and glucose, regulate rootformation on the peony explants invitro. In a previous study, it wasfound that exogenous IBA addedalone had no effect on IAA produc-tion but added together with a highlevel of sucrose stimulated IAA pro-duction in peony ‘Jadwiga’ shoots invitro (Gabryszewska, 2009). In the

Zingiber officinale rhizome culture,the influence of sucrose concentrationon the endogenous level of IAA wasalso very little. In contrast, the pro-duction of gibberelins and ABAchanged sharply when the sucroselevel was increased (Zheng et al.,2008). The promoting effect of theincreasing sucrose concentration onadventitious root formation was dem-onstrated for apple microcuttings(Calamar and De Klerk, 2002). Inaddition, there was an interactionbetween sucrose and auxin. For manyothers plant species it was stated thatsoluble sugars could affect the forma-tion of adventitious roots and can actas signaling molecules in this process(Gibson, 2005).

Among all the plant growth sub-stances, jasmonates play an importantrole in various morphogenic events inplants including tuberization, tuberousroot or bulb formation (Tampe et al.,2001). In tissue culture of the peony’Jadwiga’, a simultaneous supply ofMeJA with IBA inhibited root forma-tion on the shoots growing at hightemperatures. In contrast, the additionof indomethacin (inhibitor of jas-monic acid biosynthesis) to the me-dium with IBA stimulated rooting andleaf formation (Gabryszewska, 2009).Previously, MeJA had been shown toinduce bulb (lily, garlic) and tuber(potato) formation or to inhibit adven-titious shoot (radiata pine) regenera-tion (Koda et al., 1991; Ravnikar etal., 1993; Shimasaki and Fukumoto,2000, Tampe et al. 2001). The role ofjasmonates in the organogenesis ofherbaceous peony should be studiedin detail.



glucose (g l-1) glucose (g l-1)Figure 3. The influence of glucose at different concentrations and growth regulators (IBA,GA3, kinetin) on the shoot, renewal bud and root formation of Paeonia lactiflora ‘Jadwiga’in vitro

Figure 4. The renewal buds (arrows) formed on the crown of Paeonia lactiflora‘Jadwiga’ (explant without leaves) grown on the MS medium containing glucose90 g l - 1 and IBA, GA 3, kinetin (1 mg l - 1) added to the medium together

Explants with leaves

Explants without leaves

E. Gabryszewska


In conclusion, the results pre-sented here show that a high level ofglucose and exogenous growth regu-lators (kinetin, GA3, IBA) together,stimulate shoot and renewal budformation but the way of organo-genesis depends on the presence orabsence of leaves. The interactionbetween auxin (exogenous or en-dogenous) and glucose regulate rootformation on the peony shoots butthe final effect depends on the typeof explants (with or without leaves).It is possible that leaves have veryimportant hormonal factors, whichstimulate shoot growth or rootingand inhibit renewal bud formation.

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E. Gabryszewska


WPŁYW GLUKOZY I REGULATORÓW WZROSTU NAORGANOGENEZĘPIWONII CHIŃSKIEJ

(Paeonia lactiflora Pall.) IN VITRO

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S T R E S Z C Z E N I E

Sadzonki piwonii chińskiej pochodzące z rozmnażania in vitro bardzo słaboaklimatyzująsięw szklarni i nie podejmujądalszego wzrostu. U innych gatunków roślinformowanie in vitro organów spoczynkowych, zawierających materiały zapasowe,wpływa korzystnie na aklimatyzację roślin rozmnażanych in vitro. W warunkachnaturalnych piwonia chińska wytwarza na kłączu pąki odnawiające, z którychw następnym sezonie rozwijająsięjednoroczne pędy nadziemne. Kłącze i zgrubiałekorzenie gromadząsubstancje zapasowe. Przeprowadzone badania miały na celuokreślenie wpływu glukozy i regulatorów wzrostu (kinetyna, IBA, GA3) oraz ichwspółdziałania w procesie formowania oraz wzrostu pędów, pąków odnawiającychi korzeni u piwonii chińskiej odmiany ‘Jadwiga’ rosnącej in vitro. Doświadczenieprzeprowadzono na fragmentach kłącza pochodzących z rozmnażania in vitro.

Odcięcie liści z izolowanych eksplantatów wpływało hamująco na powstawaniei wzrost pędów, natomiast stymulowało formowanie pąków odnawiających. Wzroststężenia glukozy w pożywce ograniczałformowanie pędów i wzrost liści, szczególnieu eksplantatów rosnących na pożywce bez regulatorów wzrostu. Glukoza silniej hamo-wała wzrost pędów u eksplantatów pozbawionych liści. Stymulujące działanie tegocukru obserwowano w procesie formowania pąków odnawiających. Pojedyncze zasto-sowanie regulatorów wzrostu (kinetyna, IBA, GA3) sprzyjało powstawaniu pędów orazhamowało formowanie pąków odnawiających na eksplantatach zawierających liście.Łączne zastosowanie wszystkich regulatorów wzrostu (kinetyna, IBA, GA3) i glukozyw najwyższym stężeniu (90 g l-1) stymulowało powstawanie i wzrost pędów na eksplan-tatach izolowanych z liśćmi, a pąków odnawiających na eksplantatach bez liści. Wzroststężenia glukozy w pożywce zwiększałliczbępowstających korzeni na eksplantatachzawierających liście, rosnących na pożywce bez regulatorów wzrostu. Dodanie IBA dopożywki z najniższym stężeniem glukozy (30 g l-1) najsilniej stymulowało ukorzenianieeksplantatów z odciętymi liśćmi. Egzogenna giberelina i kinetyna, zastosowane łącznielub odzielnie z IBA, silnie hamowały powstawanie korzeni.

Uzyskane wyniki wskazująna współdziałanie glukozy w wysokim stężeniu i łącz-nego zastosowania egzogennych regulatorów wzrostu (kinetyna, IBA, GA3) w procesieformowania zarówno pędów, jak i pąków odnawiających, jednakże kierunek organoge-nezy uzależniony jest od obecności lub braku liści na izolowanych eksplantatach.Współdziałanie auksyn (egzogennych, endogennych) z glukoząreguluje powstawaniekorzeni, ale ostateczny wynik ukorzeniania zależy równieżod zachowania lub odcięcialiści. Można przypuszczać, że liście zawierająistotny hormonalny czynnik, który sty-muluje rozwój pędów i korzeni oraz hamuje powstawanie pąków odnawiających.

Słowa kluczowe: Paeonia lactiflora, kultury tkankowe, glukoza, regulatory wzrostu,organogeneza, powstawanie pąków odnawiających

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