Maejo International Journal of Science and Technology

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1 Mejo Int. J. Si. Tehnol. 2011, 5(02), Full Pper Mejo Interntionl Journl of Siene nd Tehnology ISSN Aville online t Dynmi hnges in enzyme tivities nd phenoli ontent during in vitro rooting of tree peony (Peoni suffrutios Andr.) plntlets Zhenzhu Fu 1, Pnpn Xu 1, Songlin He 1,*, Jime A. Teixeir d Silv 2 nd Mihio Tnk 2 1 Fulty of Forestry, Henn Agriulturl University, No.95 Wenhu Rod, Zhengzhou , Chin 2 Deprtment of Hortiulturl Siene, Fulty of Agriulture nd Grdute Shool of Agriulture, Kgw University, Miki-ho, Ikenoe 2393, Kgw-ken , Jpn * Corresponding uthor, e-mil: hsl213@163.om Reeived: 15 April 2011 / Aepted: 4 July 2011 / Pulished: 28 July 2011 Astrt: The dynmi hnges of phenoli ontent nd peroxidse (POD), polyphenol oxidse (PPO), indole-3-eti id oxidse (IAAO) nd phenyllnine mmoni lyse (PAL) tivities were ssessed during the in vitro rooting proess of three ultivrs of tree peony (Peoni suffrutios Andr.). These hnges in enzyme-relted tivity nd phenoli ontent oserved t the level of the whole plnt differed during the first 20 dys of the rooting proess in esy-to-root Feng Dn Bi ultivr nd diffiult-to-root Wu Long Peng Sheng nd Ti Ping Hong ultivrs, nd in most ses they were tully opposite. The ese with whih Feng Dn Bi ws le to root ws losely relted to the tivity of ll four enzymes (POD, PPO, IAAO, PAL) s well s to the phenoli ontent. Keywords: tree peony, Peoni suffrutios, rooting, enzyme tivities, phenoli ontent, dynmi hnges INTRODUCTION Tree peony (Peoni suffrutios Andr, Peoniee) is perennil woody flowering ornmentl plnt nd fmous trditionl flower in Chin [1-2]. Tree peony tissue ulture is in vogue sine trditionl reeding methods hve limittions [3]. However, it hs not yet een le to meet the requirements for mss prodution due to diffiulty in rooting nd the low root qulity of plntlets in vitro [4]. Therefore, solving the prolem of rooting in vitro my e key for rekthrough in tree peony tissue ulture.

2 Mejo Int. J. Si. Tehnol. 2011, 5(02), There is lose reltionship etween the ourrene of dventitious roots in plnts nd their peroxidse (POD, EC ), polyphenoloxidse (PPO, EC ), indole-3-eti id oxidse (IAAO, no EC numer) nd phenyllnine mmoni lyse (PAL, EC ) tivities s well s their phenoli ontent. These enzymes hve different funtions during rooting [5-6]. Inresing POD tivity is rooting signl in the indution nd formtion of root primordi [7-8]. POD is involved in uxin metolism nd in the lignifition of ell wlls in the presene of phenoli ompounds [9]. PPO is the min enzyme using the oxidtion of phenoli ompounds; it n lso tlyse phenoli ompounds nd IAA to form IAA-phenoli omplexes, whih promote the ourrene nd development of dventitious roots [10]. IAAO ffets the ourrene of dventitious roots in plnts y oxidising IAA nd hnging its levels [11-12]. PAL is key enzyme involved in the synthesis of phenoli ompounds [13-14]. PAL tivity nd totl phenoli ontent of lettue plnts nd Phlenopsis orhids were signifintly orrelted with rowning [5-6,15]. There is yet-to-edisproved trin of thought tht n importnt differene etween esy-to-root nd diffiult-to-root ultivrs lies in the differene in the ontent of phenoli ompounds: the former is thought to ontin less polyphenols thn the ltter [16]. In order to provide theoretil nd mehnisti sis for the estlishment of effiient rooting during the tissue ulture of tree peony, this experiment ims to determine the dynmi hnges in POD, PPO, IAAO nd PAL tivities nd totl phenol ontent during the in vitro rooting ulture of tree peony ultivrs in order to estlish whether reltionship etween the tivities of these enzymes nd the phenoli metolism exists. If orreltion etween rooting ility nd enzyme tivity ould e estlished, this would open up gtewy for moleulr mnipultion of the plnts to inrese the level of those enzymes tht would permit greter nd more suessful in vitro rooting, nd hene for suessful miropropgtion of this ornmentl plnt. Sine rooting nd root formtion re oth poorly understood nd eqully poorly hieved for this ornmentl, n understnding of the enzymti nd iohemil dynmis is expeted to provide vitl lues to how etter to try nd improve its in vitro rooting. MATERIALS AND METHODS Chemils nd Regents All hemils nd regents used in this study were of tissue ulture or HPLC grde. Murshige nd Skoog (MS) medium [17] nd woody plnt medium (WPM) [18] were mde fresh from MS stoks nd WPM stoks respetively. Other more importnt hemils nd regents were purhsed from the following ompnies: Phytgel, Folin-Ciolteu s regent nd gr powder from Solrio Siene nd Tehnology Co. Ltd., Beijing, Chin; 1-nphthleneeti id (NAA) from Huixing Chemil Regents Ltd., Shnghi, Chin; 6-enzylminopurine (6-BA), indole-3-eti id (IAA) nd indole-3-utyri id (IBA) from Boo Bioteh Co. Ltd., Shnghi, Chin; guiol nd L-phenyllnine from Gungfu Chemil Institute, Tinjin, Chin; tehol, glli id nd polyvinylpyrrolidone-30 (PVP-30) from Ke Miou Chemil Regents Development Centre, Tinjin, Chin; trihloroeti id, ferri hloride nd surose from Gunghu Chemil Regents Ltd., Gungzhou, Chin; mngnese hloride from Xilong Chemil Ftory, Shntou, Chin; 2,4-

3 Mejo Int. J. Si. Tehnol. 2011, 5(02), dihlorophenol from Chin Ntionl Phrmeutil Industry Corp. Ltd., Shnghi, Chin; β- merptoethnol from Tinrui Chemil Co. Ltd., Shnghi, Chin. Phosphte nd orte uffers were mde fresh. Plnt Mteril nd Culture Medi The xillry uds of tree peony from uniform, lonlly propgted in vitro stok mother plnts, originlly derived from 5-yer-old ex vitro plnts, were used. Three tree peony ultivrs ( Feng Dn Bi, Wu Long Peng Sheng nd Ti Ping Hong ) were gthered from Luoyng City in Ferury, Axillry uds were pled on MS medium supplemented with 0.3 mg/l of NAA, 0.3 mg/l of 6-BA nd 3% of surose nd solidified y 7.5 g/l of gr power in 100-ml flsks (three xillry uds per flsk) fter sterilistion of the ulture t 24±1 C. The flsks were pled under 12-h photoperiod t 36 μmol m -2 s -1. After 35 dys, stndrdised shoots formed (4-5 leves, ~5 m in height, nd no roots), nd these were used s explnts for ll susequent experiments. Assy Methods Adventitious shoots (one per flsk) were trnsferred to WPM supplemented with 4 mg/l of IBA nd 3% of surose, nd solidified y 2.0 g/l of Phytgel for ulture under the sme onditions s for shoot indution. No ntioxidnt ompounds were dded to the medium. The enzyme tivities nd totl phenoli ontent of whole plntlets were determined fter ulturing for 0, 1, 2, 3, 4, 5, 7, 9, 12, 15 nd 20 dys. Ten plntlets were ut into smll piees nd mixed, nd 0.5-g liquots were used in enzyme ssys. Eh tretment ontined 10 replites nd ws repeted three times. All medi used were djusted to ph 5.8 (mesured with phs-3b meter, Hongyi Instrument Co., Shnghi, Chin) with 1 M NOH efore utolving t 121 C for 15 min t 121 psi. POD tivity The plntlets (0.5 g) from different rooting ulture periods were ground immeditely on ie y dding 5 ml of phosphte uffer (ph 7.0). The rude enzyme extrt ws otined fter entrifugtion t g for 15 min t 4 C. POD tivity ws mesured following the Guiol method [19]. Briefly, 0.3 ml of rude enzyme, 2.0 ml of phosphte uffer (ph 7.0), 0.5 ml of 0.2% queous guiol nd 0.5 ml of 0.15% H 2 O 2 were mixed. POD sorne vlues were reorded immeditely t 470 nm using UV-3200 spetrophotometer (Mei Pud Instrument Co., Shnghi, Chin). One unit of POD tivity is equivlent to n inrese in 0.01 times the mount of enzyme for 1 g of fresh weight/min. PPO tivity The plntlets (0.5 g) from different rooting ulture periods were ground immeditely on ie y dding 5 ml of phosphte uffer (ph 6.0). The rude enzyme extrt ws otined fter entrifugtion t g for 15 min t 4 C. PPO tivity ws mesured ording to Zhu et l. [20]. Briefly, 0.1 ml rude enzyme, 3.9 ml phosphte uffer (ph 6.0) nd 1 ml 0.l M queous tehol

4 Mejo Int. J. Si. Tehnol. 2011, 5(02), were mixed in 30 C wter th for 10 min. Then 2 ml of 20% trihloroeti id ws dded quikly to stop the retion. PPO sorne vlues were reorded immeditely t 525 nm. One unit of PPO tivity is equivlent to n inrese in 0.01 times the mount of enzyme for 1 g of fresh weight/min. IAAO tivity The tivity of IAAO, extrted y the sme method s tht for PPO, ws mesured ording to Zhng [21]. Briefly, 1 ml rude enzyme, 2 ml 1 mm queous MnCl 2, 1 ml 1 mm queous 2,4-dihlorophenol, 2 ml 1 mm queous IAA nd 5 ml phosphte uffer (ph 6) were mixed in 30 C wter th for 30 min. Then 2 ml of this mixture nd 4 ml of retion solution (1.0 ml 0.5M FeCl ml 35% perhlori id) were mixed in the drk in 30 C wter th for 30 min. IAAO sorne vlues were reorded t 530 nm. One unit of IAAO tivity ws expressed in terms of μg of IAA degrded/g fresh weight/h. PAL tivity The plntlets (0.5 g) from different rooting ulture periods were ground immeditely on ie with ddition of 5 ml pre-ooled 0.1 M orte uffer (ph 8.8, ontining 5 mm β-merptoethnol nd 0.25 g PVP-30). The superntnt ws used for the determintion fter entrifugtion t g for 15 min t 4 C. PAL tivity ws mesured y the method of Li [22] with minor modifitions. Briefly, 1 ml rude enzyme, 1 ml 0.02 M L-phenyllnine in orte uffer (ph 8.8) nd 2 ml distilled wter were mixed in 30 C wter th for 30 min. PAL sorne vlues were mesured t 290 nm using wter s ontrol. One unit of PAL tivity is equivlent to n inrese in 0.01 times the mount of enzyme for 1 g of fresh weight/h. Totl phenoli ontent Polyphenols were extrted y methnol nd wter extrtion method [23]. Plntlets (0.5 g) from different rooting ulture periods were pled in 55 C wter th for 30 min fter grinding in 5 ml of 40% methnol. A rude extrt of polyphenols ws otined fter entrifugtion t 3000 g for 10 min. Polyphenols ontent ws determined y the Folin-Ciolteu method [24]. Briefly, 1.0 ml of rude polyphenol extrt, 1.0 ml of distilled wter, 0.5 ml of Folin-Ciolteu s regent nd 0.5 ml of 7.5% N 2 CO 3 were mixed for the retion to tke ple t room temperture for 1 h. Asorne vlues were then reorded t 765 nm using wter s ontrol. Glli id ws used s stndrd. The totl phenoli ontent ws expressed s μg/g fresh weight. Morphologil prmeters Rooting perentge, verge numer of roots/plnt, nd root length (i.e. totl length of ll roots/totl root numer) of the three ultivrs were determined fter 60 dys of rooting sine root tips would emerge, on verge, within dys while ll roots ppered within dys of ulture. The rooting index (RI) [25] ws lulted s: RI = verge numer of roots/plnt root length % rooting (where % rooting = (numer of plnts tht formed roots / totl numer of plnts)

5 Mejo Int. J. Si. Tehnol. 2011, 5(02), ). RI is omposite inditor for the mesurement of root onditions tht n fully explin the degree of diffiulty of in vitro rooting. Experimentl design nd sttistil nlyses In ll experiments, eh tretment hd 10 smples per tretment (tissue ulture nd iohemil experiments) nd ws repeted in triplite. Mens were seprted y one-wy nlysis of vrine nd signifint differenes were ssessed using Dunn s multiple rnge test t P = 0.05 using DPS softwre version RESULTS AND DISCUSSION Rooting The rooting perentge nd RI were signifintly different (P < 0.05) mong the three tree peony ultivrs. The rooting perentge of Feng Dn Bi ws highest (51.72%) with RI of 5.2, while rooting perentges of Wu Long Peng Sheng nd Ti Ping Hong were lowest, i.e nd 14.29% respetively (RI = 1.94 nd 1.61 respetively; Tle 1). From the results it n e onluded tht Feng Dn Bi is n esy-to-root ultivr while Wu Long Peng Sheng nd Ti Ping Hong re diffiult-to-root ultivrs. Tle 1. Rooting in three tree peony ultivrs (n = 30) Cultivr Root numer Root length (m) Rooting perentge Rooting index (RI) Feng Dn Bi 2.8 ± ± ± ± 0.71 Wu Long Peng Sheng 2.0 ± ± ± ± 1.14 Ti Ping Hong 2.25 ± ± ± ± 0.7 Note: Eh vlue is the men ± SD of triplite. Different letters within olumn indite signifint differene t P < 0.05 ording to DMRT. POD Ativity POD tivity hnged during the in vitro rooting of the three tree peony ultivrs (Figure 1). The hnge in POD tivity of Feng Dn Bi showed jgged trend, peking twie on the 3 rd nd 9 th dys. The tivity ws signifintly different (P < 0.05) from tht of Ti Ping Hong nd Wu Long Peng Sheng in the erly dys of rooting ulture. The tivity of Ti Ping Hong nd Wu Long Peng Sheng ws not signifintly different, lthough similr jgged trend ws oserved, leit with lower mplitude.

6 Mejo Int. J. Si. Tehnol. 2011, 5(02), POD tivity (u g -1 FW min -1 ) Culture dy Culture dy Ti Ping Hong Feng Dn Bi Wu Long Peng Sheng Figure 1. Trends in POD tivity hnge in plntlets of different ultivrs during the rooting proess. Different letters in eh olumn indite signifint differene etween the three ultivrs on the sme dy t P < 0.05 using DMRT. Non-signifint differenes on some dys re not indited y ny letters. Eh tretment hd 10 smples nd ws repeted in triplite. Some studies hve shown tht there is lose reltionship etween the tivity of POD, PPO nd IAAO nd the ourrene nd growth of dventitious roots in plnts nd tht the hnges in these enzymes differ t different periods of rooting [26]. Other studies indited tht the inrese in POD tivity ws signl of rooting ility in the periods of root indution nd expression [7, 27], implying tht POD tivity would reh two peks in the indution nd expression periods. Pheo et l. [28] lso oserved two peks in the vitro rooting of Eulyptus gloulus uttings, the first on the first dy of rooting ulture, whih might hve een relted to root indution, nd the seond pek on the 10 th dy when the root hd roken through the epidermis. In our study, the POD tivity of esy-to-root Feng Dn Bi ultivr hd two peks on the 3 rd nd 9 th dy, orresponding to possile root primordium indution nd expression. However, POD tivity of Ti Ping Hong nd Wu Long Peng Sheng did not hve the sme ehviour s tht of Feng Dn Bi. Molssiotis et l. [29] oserved tht the rooting stems of rootstok GF-677 (Prunus mygdlus P. persi) showed mximum solule POD tivity on the 9 th dy nd peked in ionilly-ound POD to ell wll POD tivity on the 6 th nd 12 th dys on the rooting medium; similr ehviour ws not oserved in nonrooting stems. In the se of the KIBA (potssium slt indole-3-utyri id) tretment of A. unedo genotype D, POD tivity showed one pek on dy 10, ut in the ontrol tretment there ws no pek in tivity [30]. PPO Ativity PPO tivity hnged during the in vitro rooting of the three tree peony ultivrs (Figure 2). The tivity showed jgged trend with peks nd troughs similr for ll three ultivrs. The tivity in Feng Dn Bi peked on the 4 th dy nd ws signifintly higher thn in Ti Ping Hong nd

7 Mejo Int. J. Si. Tehnol. 2011, 5(02), Wu Long Peng Sheng. The tivity ws lso signifintly different (P < 0.05) mong the three tree peony ultivrs on the 7 th nd 15 th dy. 30 PPO tivity (u g -1 FW min -1 ) Culture Cuture dy dy Ti Ping Hong Feng Dn Bi Wu Long Peng Sheng Figure 2. Trends in PPO tivity hnge in plntlets of different ultivrs during the rooting proess. Different letters in eh olumn indite signifint differene etween the three ultivrs on the sme dy t P < 0.05 using DMRT. Non-signifint differenes on some dys re not indited y ny letters. Eh tretment hd 10 smples nd ws repeted in triplite. Bhtthry [31] proved tht PPO n tlyse the metolism of uxin, promoting the genertion nd development of dventitious roots. Moreover, PPO n lso tlyse phenoli ompounds nd IAA to form IAA-phenol omplexes, whih would e type of rooting oftor tht n promote the ourrene nd development of dventitious roots [27]. In this experiment on tree peony, fter 3-4 dys of ulture, the PPO tivity of ll three ultivrs inresed, the inrese eing signifintly greter, however, in esy-to-root Feng Dn Bi thn in diffiult-to-root Ti Ping Hong nd Wu Long Peng Sheng, whih my e more dvntgeous to the formtion of IAA-phenol omplexes tht promote the indution of root primordi. During dys of rooting, the PPO tivity of Ti Ping Hong nd Wu Long Peng Sheng ontinued to rise, while tht of Feng Dn Bi egn to drop nd eme signifintly lower thn tht of the other two ultivrs on the 15 th dy. This redution in PPO tivity my hve prtiipted in or een responsile for the formtion of root primordi. Molnr nd Lroix [32] found tht when Hydrnge mrophyll formed dventitious roots from stem tissue, PPO tivity inresed drmtilly when the root tip emerged. Hguhi [33] oserved the sme result in rrot llus ulture: when root tips emerged from llus, PPO tivity inresed shrply. In this study on tree peony, fter rooting for 20 dys, the PPO tivity of ll three ultivrs inresed, whih my e relted to the emergene of root tips. IAAO Ativity The hnges in IAAO tivity during rooting of the three tree peony ultivrs re shown in Figure 3. Similrly to POD nd PPO, IAAO tivity ws jgged for ll three ultivrs nd ws higher in Ti Ping Hong nd Wu Long Peng Sheng thn in Feng Dn Bi t ll times, prtiulrly on

8 Mejo Int. J. Si. Tehnol. 2011, 5(02), the 2 nd, 3 rd, 7 th nd 9 th dys. The tivity ws similr in Ti Ping Hong nd Wu Long Peng Sheng on the 2 nd, 3 rd, 4 th, 5 th, 9 th nd 12 th dys. It ws signifintly different (P < 0.05) mong the three tree peony ultivrs on the 7 th dy. IAAO tivity (u g -1 FW h -1 ) Culture Cuture dy dy Ti Ping Hong Feng Dn Bi Wu Long Peng Sheng Figure 3. Trends in IAAO tivity hnge in plntlets of different ultivrs during the rooting proess. Different letters in eh olumn indite signifint differene etween the three ultivrs on the sme dy t P<0.05 using DMRT. Non-signifint differenes on some dys re not indited y ny letters. Eh tretment hd 10 smples nd ws repeted in triplite. IAAO n degrde IAA nd modify its level in plnts, therey ffeting the rooting of plntlets, nd it is theoretilly likely tht there is high IAAO tivity (in roots, leves nd stems) in diffiult-to-root ultivrs, whih n strongly degrde IAA [34-35]. In this se, more IAA is destroyed nd onsequently little IAA would e trnsported downwrds to the roots, resulting in few or no roots eing indued. Conversely, n esy-to-root ultivr would hve low IAAO tivity nd hene lower ility to degrde IAA tht filittes rooting [36]. In this study, IAAO tivity of Feng Dn Bi ws onsistently lower thn tht of Ti Ping Hong nd Wu Long Peng Sheng. Consistent with the results of our study, Hu et l. [26] found tht the IAAO tivity in Corylus velln uttings ws higher in the ontrol (0% of rooting) thn in the plnts treted with IBA (60% of rooting). The IAAO tivity of Cmelli sinensis (L.) Kuntze ws lso higher in the ontrol (0% rooting) uttings s ompred to IBA-treted uttings (92.6% rooting) [9]. IAAO tivity in Vign rdit L. v. 105 remined higher in ontrols thn in uttings treted with PUT nd IBA [37]. PAL Ativity The PAL tivity followed pttern similr to tht of POD, PPO nd IAAO during the in vitro rooting of the three tree peony ultivrs (Figure 4). The tivity is similr mong the three tree peony ultivrs on the 0, 1 st, 2 nd, 4 th, 5 th nd 12 th dys, lthough there were signifint differenes on the 3 rd dy. PAL tivity of Feng Dn Bi ws lso signifintly lower thn tht of Ti Ping Hong nd Wu Long Peng Sheng on the 7 th nd 9 th dys.

9 Mejo Int. J. Si. Tehnol. 2011, 5(02), PAL tivity (u g -1 FW h -1 ) Ti Ping Hong Feng Dn Bi ong Wu Peng Long Sheng Peng Sheng Culture Cuture dy Figure 4. Trends in PAL tivity hnge in plntlets of different ultivrs during the rooting proess. Different letters in eh olumn indite signifint differene etween the three ultivrs on the sme dy t P < 0.05 using DMRT. Non-signifint differenes on some dys re not indited y ny letters. Eh tretment hd 10 smples nd ws repeted in triplite. PAL is key enzyme in the synthesis of phenoli ids nd its tivity is ffeted y externl nd internl ftors in ells [38]. Highly positive orreltions were oserved etween phenoli ontent nd PAL tivity or free phenyllnine ontent in uds nd sles in Lilium dvidii vr. uniolor uls [39]. The totl phenoli ontent of three vrieties of Phlenopsis ws positively orrelted with their PAL tivity during tissue ulture [40]. Smith-Beker et l. [41] oserved tht the onstitutive tivity of PAL in stems nd petioles of uumer ws pproximtely 20-fold higher thn in leves. However, no differene in PAL tivity ws deteted etween ontrol nd plnts inoulted with Pseudomons syringe pv. syringe in the lef diretly ove the inoulted lef. In this study, there were hnges in PAL tivity during the rooting proess, implying tht PAL prtiipted in the rooting proess of tree peony plntlets. However, there ws no signifint differene in the tivity mong the three tree peony ultivrs, nd there ws no orreltion with the totl phenoli ontent, whih seemed to indite tht PAL did not ply leding role in the rooting of tree peony plntlets. Other ftors whih my e t ply would hve to e further studied. Totl Phenoli Content Figure 5 shows hnges in the totl phenoli ontent during the in vitro rooting of the three tree peony ultivrs. The jgged ptterns of Ti Ping Hong nd Wu Long Peng Sheng were similr to eh other, ut different from tht of Feng Dn Bi. Exept for the 1 st nd 12 th dys of rooting, the phenoli ontent of Feng Dn Bi ws signifintly higher thn tht of Ti Ping Hong nd Wu Long Peng Sheng during dy 2-7 nd dy of rooting.

10 Mejo Int. J. Si. Tehnol. 2011, 5(02), Totl phenol ontents(ug.g -1 ) Culture dy dy Ti Ping Hong Feng Dn Bi Wu Long Peng Sheng Figure 5. Trends in totl phenoli ontent hnge in plntlets of different ultivrs during the rooting proess. Different letters in eh olumn indite signifint differene etween the three ultivrs on the sme dy t P<0.05 using DMRT. Non-signifint differenes on some dys re not indited y ny letters. Eh tretment hd 10 smples nd ws repeted in triplite. The funtion of phenoli ompounds in rooting is well estlished. Hrtmn et l. [16] proposed one view: differenes in the ontent of phenols indite differenes etween esy-to-root nd diffiult-to-root uttings, the former hving higher ontent of phenols thn the ltter. The ontent of some flvonoids in Eulyptus seedlings ws positively orrelted with rooting ility [42]. In Rhizoium isolte of Vign mungo (mung en), the root nodules ontined higher mount of IAA nd phenoli ids thn non-nodulted roots [43]. Another study showed tht exogenous phenoli ompounds nd IAA, when used in onjuntion, ould synergistilly promote rooting. Penthydroxyflvone plus IBA, for exmple, signifintly inresed the rooting rte of wlnut [44]. In this experiment, the totl phenoli ontent of esy-to-root Feng Dn Bi ws lso generlly higher thn tht of diffiult-to-root Wu Long Peng Sheng nd Ti Ping Hong, whih is in line with the ove oservtions nd implies n tive role of phenoli ompounds in the promotion of rooting. CONCLUSIONS Enzyme-relted tivities nd totl phenol ontent, whih were relted to the rooting of tree peony, hnged in the first 20 dys (erly phse of in vitro rooting) for ll three tree peony ultivrs. These hnges differed over the rooting period nd there were signifint differenes etween esyto-root Feng Dn Bi nd diffiult-to-root Wu Long Peng Sheng nd Ti Ping Hong ultivrs s determined initilly y RI vlues. POD tivity nd totl phenoli ontent were higher in esy-to-root Feng Dn Bi ultivr thn in diffiult-to-root Ti Ping Hong nd Wu Long Peng Sheng ultivrs on the whole, while IAAO tivity ws lower in Feng Dn Bi thn in Ti Ping Hong nd Wu Long Peng Sheng throughout the entire experimentl period. However, no ler onlusions ould e drwn for PPO nd PAL tivities in reltion to the rooting ility of the three tree peony ultivrs. The hnges in the tivity of the POD nd IAAO enzymes nd phenol ontent n thus e used s mrker nd preditor of rooting ility in tree peony during the erly dys of in vitro rooting s depited in Figure 6.

11 Mejo Int. J. Si. Tehnol. 2011, 5(02), Condut rooting experiment to differentite esy-to-root nd diffiult-to-root ultivrs Medium: Axillry uds on MS medium mg/l NAA mg/l 6-BA + 0.3% surose Growth onditions: 12-h photoperiod t 36 µmol m -2 s -1 RI is HIGH Esy-to-root ultivr Feng Dn Bi Rooting RI is LOW Diffiult-to-root ultivrs Wu Long Peng Sheng; Ti Ping Hong Biohemil ssys: POD, PPO, IAAO, PAL tivities nd phenoli ontent Esy-to-root ultivr Feng Dn Bi POD is HIGH PPO? IAAO is LOW PAL? Phenoli ontent is HIGH NOTE: PPO nd PAL tivities nnot e desried s HIGH or LOW euse those of esy-to-root ultivr were higher or lower only on some dys nd not t ll times thn those of diffiult-to-root ultivr. Diffiult-to-root ultivrs Wu Long Peng Sheng; Ti Ping Hong POD is LOW PPO? IAAO is HIGH PAL? Phenoli ontent is LOW Predit if n untested tree peony ultivr is n esy-to-root or diffiult-to-root ultivr Figure 6. Proposed sheme for lssifying tree peony ultivr into esy-to-root or diffiult-to-root ultivr sed on RI vlue nd other iohemil prmeters. Note tht RI, POD nd IAAO represent stle prmeters while PPO nd PAL re unstle prmeters for prediting the nture of in vitro rooting of ultivr. REFERENCES 1. D. F. Lu, Floriulture, Chin Agriulture Press, Beijing, M. Z. Bo, Floriulture, 2 nd Edn., Chin Agriulture Press, Beijing, Y. L. Li, D. Y. Wu, S. L. Pn nd S. L. Xu, The study on miropropgtion tehnology of tree peony plntlets in vitro, Chinese Si. Bull., 1984, 8, L. Bouz, M. Jques, B. Sott nd E. Migini, The retivtion of tree peony (Peoni suffrutios Andr.) vitro plnts y hilling is orrelted with modifitions of sisi id, uxin nd ytokinin levels, Plnt Si., 1994, 97, M. Murt, M. Nishimur, N. Muri, M. Hrut, S. Homm nd Y. Itoh, A trnsgeni pple llus showing redued polyphenol oxidse tivity nd lower rowning potentil, Biosi. Biotehnol. Biohem., 2001, 65, H. Hisminto, M. Murt nd S. Homm, Reltionship etween the enzymti rowning nd phenyllnine mmoni-lyse tivity of ut lettue, nd the prevention of rowning y inhiitors of polyphenol iosynthesis, Biosi. Biotehnol. Biohem., 2001, 65, C. Monousin nd T. H. Gspr, Peroxidse s mrker for rooting improvement of Cynr solymus L. ulivted in vitro, Biohem. Physiol. Pflnz., 1983, 178,

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