Optimized conditions for callus induction, plant regeneration and alkaloids accumulation in stem and shoot tip explants of Phyla nodiflora

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1 Instituto Nionl e Investigión y Tenologí Agrri y Alimentri (INIA) Aville online t Spnish Journl of Agriulturl Reserh 2 9(4), ISSN: X eissn: Optimize onitions for llus inution, plnt regenertion n lklois umultion in stem n shoot tip explnts of Phyl noiflor A. B. A. Ahme,2 *, R. Pllel 4, A. S. Ro 3, M. V. Ro 2 n R. Mt Th Institute of Biologil Sienes, Fulty of Siene, University of Mly, 563, Kul Lumpur, Mlysi 2 Deprtment of Plnt Siene 3 Deprtment of Biotehnology. Bhrthisn University. Tiruhirpplli-6224, Tmil Nu, Ini 4 Mrine Bioproess Reserh Center, Pukyong Ntionl University, Busn-68737, South Kore Astrt The present stuy esries llus inution n the susequent plnt regenertion with lklois umultion in stem n shoot tip explnts of Phyl noiflor. Both explnts were ulture on ifferent mei (MS, B5, SH n WPM) for llus inution. explnts showe etter llus iomss (ry weight) thn shoot tip explnts with green ompt llus when ulture on MS meium ontining.5 mg L α-nphthlene eti i. Shoots were regenerte from the llus on MS meium with.5 mg L α-nphthlene eti i n. mg L enzyl enine. The rooting of ll regenerte shoots ws suessfully performe on hlf-strength MS meium with. mg L inole-3-utyri i. The plntlets were limtize n estlishe in soil (9%) n exhiite morphologil hrteristis similr to those of the mother plnt. In ition, the lklois ontent ws higher in regenerte llus thn intt stem n shoot tip explnts, whih were nlyze y grvimetri metho, TLC (thin lyer hromtogrphy) n HPTLC (high performne thin lyer hromtogrphy). The propose metho oul effetively e pplie for the onservtion n lonl propgtion to meet the phrmeutil emns of this meiinlly importnt speies. Aitionl key wors: llus iomss; grvimetri metho; growth urve; HPTLC; rooting; TLC. Resumen Coniiones optimizs pr inuión e llos, regenerión e plnts y umulión e lloies en explntes e tllos y rotes e Phyl noiflor El presente estuio esrie l inuión e llos y l posterior regenerión e plnts on l umulión e lloies en los explntes e tllos y rotes e Phyl noiflor. Pr l inuión e llos, mos explntes fueron ultivos en iferentes meios (MS, B5, SH y WPM). Los explntes e tllos tuvieron mejor ioms (peso seo) e llo que explntes e ápies e nturlez vere ompt uno se ultivron en un meio MS on,5 mg L e áio α-nftlén étio. Los rotes fueron regeneros prtir e los llos en meio MS on,5 mg L e áio α-nftlén étio y, mg L e enilenin. El enrizmiento e toos los rotes regeneros se relizó on éxito en un meio ½ MS on, mg L e áio inol-3-utírio. Ls plántuls fueron limts y estleis en suelo (en un 9%) y mostrron rterístis morfológis similres ls e l plnt mre. Aemás, el ontenio e lloies fue myor en los llos regeneros que en los explntes e tllo y rotes inttos, que fueron nlizos por un métoo grvimétrio, TLC y HPTLC. El métoo propuesto se porí plir on efii pr l onservión y propgión lonl, fin e stisfer l emn frméuti e est espeie meiinlmente importnte. Plrs lve iionles: ioms e llo; urv e reimiento; enrizmiento; HPTLC; métoo grvimétrio; TLC. *Corresponing uthor: r.krueenlihme@yhoo.o.in Reeive: Aepte: Arevitions use: BA (enzyl enine); BFC (rown frile llus); DMRT (Dunn s multiple rnge test); GCC (green ompt llus); GFC (green frile llus); HPTLC (high performne thin lyer hromtogrphy); IAA (inole eti i); IBA (inole- 3-utyri i); KN (kinetin); NAA (nphthlene eti i); PGR (plnt growth regultor); WFC (white frile llus); WPM (wooy plnt meium); WWC ( white wtery llus); 2,4-D (2,4-ihlorophenoxy eti i).

2 Cllus regenertion n lkloi umultion 263 Introution Phyl noiflor (L.) Greene (= Lippi noiflor (L.) Mihex) elongs to Verenee fmily, whih is wiely istriute in South Afri n Centrl Ameri (Terlnhe n Kornelius, 996). It is runner plnt with snty roots possessing vrious ethnootnil n meil pplitions in enopthy, hroni inolent ulers, et. (Kirtikr n Bsu, 975). It hs een use s tritionl n Unni system of meiine to tret irrhe, pin in knee joints, lithisis, ol, n fever, whih lso hs een use for inuing owel moments in the stomhi ptients (Nkrni n Nkmi, 954). P. noiflor ontins flvonois, sugrs, sterols, essentil oils, resins, tnnins n other meiinlly vlule onstituents. In ition, the ompouns hllerione n hllerone serve s nti-ner, ntitumor, nti-mlril, nti-fungl n other ytotoxi tivities (Rviknth et l., 2). The eril prts of this plnt re use s noyne, ntiteril, iureti, emmengogue, prsitiie, refrigernt n ferifuge gents (Agrwl, 997). Severl reserhers hve reporte vrious phrmologil properties inluing ntispsmoi, hypotensive, nti-inflmmtory, nlgesi, ntipyreti (Forestieri et l., 996), ntiteril, ntinoieptive, ntifungl, ntioxint n free ril svenging tivities (Shukl et l., 29). P. noiflor extrts hve een use to ure multiple skin iseses n hir fflitions (Asi et l., 2). This plnt is over-exploite ue to its high meiinl vlue n hene, propgtion of this plnt y tissue ulture my e mntory, whih offers greter potentil to eliver lrge quntities of isese-free, true-totype helthy stok within short spn of time (Hussin et l., 2). While, the systemti mnipultion of mei, phytohormone onentrtions n seletion of suitle explnts re mong severl key ftors tht ontrol the proess of shoot regenertion from llus; the llus-meite regenertion protool is ritil requirement s it llows exploittion of in vitro seletion, somlonl vrition n geneti engineering tehniques, whih ims t the geneti improvement of plnts (Georges et l., 993). Sine the lst ee, very few in vitro stuies hve een one in this genus; the ltest stuies hve een performe on the propgtion of Lippi junellin n Lippi l (Julini et l., 999; Gupt et l., 2). Diret shoot propgtion using xillry noe explnts hs een implemente in our previous stuies n somti emryogenesis ws oserve to e very suessful in this meiinl plnt (Ahme et l., 25 n 2). The present stuy is n vnement over the erlier protool, euse it esries the hormonl regultion, llus initition, regenertion n lkloi umultion from in vivo stem n shoot tip explnts of P. noiflor. In ition, this stuy els with the evelopment of rpi regenertion system, the quntifition of lklois from stem n shoots tip llus (iniret orgnogenesis) n the susequent trnsplnttion of the plntlets to nturl environmentl onitions. Mteril n methos Plnt mteril n steriliztion Helthy, young stem (shoot internol segment without us) n shoot tips of P. noiflor were ollete from fiel grown plnts n mintine in the Deprtment of Plnt Siene Gren t Bhrthisn University, Tiruhirpplli, Tmil Nu, Ini. First, oth type of explnts were wshe thoroughly in running tp wter 3-5 times, inluing 2% (v/v) Teepol (Rekitt Benkiser, Ini) for min; then wshe with 7% ethnol for 3 min followe y nother wsh with.% HgCl 2 for 2 min. Prior to inoultion, explnts were wshe three times with sterile istille wter. Cllus inution n shoot tip explnts of P. noiflor were grown in MS meium (Murshige n Skoog, 962), SH meium (Shenk n Hilernt, 972), B5 meium (Gmorg et l., 968) n WPM (wooy plnt meium) (Lloy n MCown, 98) supplemente with.-2.5 mg L of NAA (nphthlene eti i), 2,4-D (2,4-iholorophenox eti i), IBA (inole utyri i) n IAA (inole eti i);.-3. mg L of BA (enzyl enine) n KN (kinetin) were use for llus inution. The llus ulture ws mintine t 25 ± 2 C, 6/8 h (light/rk) of photoperio with 25 µmol m 2 s of light intensity. The role of mei on the nture n iomss of llus ws stuie in stem n shoot tip explnts of P. noiflor. In vitro llus growth urve Cllus ultures were optimize n evlute quntittively for their nture, iomss n lkloi ontent t

3 264 A. B. A. Ahme et l. / Spn J Agri Res (2) 9(4), the en of their respetive growth yle se on previous protool (Ahme et l., 2). By treting with vrious onentrtions of uxins, fresh n ry weights of the lli were etermine t 5, 25, 35, 45 n 55 ys. At regulr intervl for ll the tretments, eh llus ws hrveste y reful seprtion from mei using metl sptuls, n fresh n ry weight ws promptly reore. Extrtion n isoltion of lklois For the extrtion of lklois, 5 g of rie intt stem n shoot tips of P. noiflor n in vitro regenerte lli were extrte with methnol 5 times (Rehmn et l., 23). The ollete methnol extrt ws entrifuge t 5 g for min t room temperture, n the methnol superntnt refully pipette out into fresh eppenorf tues n sujete to the etermintion of lklois. For grvimetri nlysis, 5 g of the intt stem, shoot tip n llus were tken in 25 ml eker iniviully, then 2 ml of ethnol ontining % eti i ws e, overe n llowe to stn for 4 h. After filtrtion, the filtere extrt ws onentrte on wter th n then onentrte mmonium hyroxie ws e rop wise to the extrt until the preipittion ws omplete. The preipitte ws wshe with ilute mmonium hyroxie, filtere one gin n rie. The rie resiue ws weighe n use s lkloi stnr (Hrorne, 973). TLC n HPTLC nlysis Methnol extrt of intt plnt n regenertive llus rise from stem n shoot tip explnts were nlyze y TLC (thin lyer hromtogrphy) n HPTLC (high performne thin lyer hromtogrphy). TLC ws performe using sili gel G with MeOH:CHCl 3 (2:8) s solvent system. After loing the extrt, Drgenorff s regent ws pplie to the TLC plte for the etermintion of lklois. The isolte grvimetri lkloi resiue stnr ws ompre with respetle R f (rtio-to-front) vlue of the smple. In ition, methnol extrts (2 µl) were pplie in the Cmg (Switzerln) HPTLC system ssiste with smple pplitor Linomt IV for qunti fition of lklois. The moile phse of hloro form:me thnol:eti i (8:.5:.5) ws suitle for seprtion of lklois n snne t 2 nm y the use of TLC snner III n the supporting softwre CAT 4.. In vitro llus regenertion For orgnogenesis, fresh stem n shoot tip lli were ple in respetive ulture tues ontining MS meium supplemente with ifferent onentrtions of BA (.-3. mg L ) or KN (.-3. mg L ) with NAA (.5 mg L ) n 2,4-D (2. mg L ) ws use. During the experiment, to voi phenoli exretion from the llus, mg L of sori i ws e to the MS mei n suulture every week up to llus regenertion. Inution of rooting n limtiztion Cllus regenertion plntlets (6-7 mm) were kept in either qurter or hlf or full strength MS meium with IAA n IBA (.-3. mg L ) for in vitro rooting. Six-week-ol well-roote plntlets were remove from the ulture tues n wshe to mke it free of gr. The plntlets were trnsferre to plsti pots (5 m imeter) ontining re soil n vermiulite (:3) n mintine t 25 ± 2 o C, 6 h y length (35-5 m EM 2 s ) n t 75-8% reltive humiity. Then the grown plnts were trnsplnte to erthenwre pots ( m imeter) ontining nturl soil, kept uner she for 2 weeks n finlly move to the gren. Sttistil nlysis All the experiments were repete thrie n use 3 replites. The effet of ifferent tretments ws quntifie s men ± SE n the t were sujete to sttistil nlysis using Dunn s multiple rnge test (DMRT) t 5% level signifine (Gomez n Gomez, 976). Results n isussion Cllus inution n growth urve nlysis In ll mei teste, the llus initition i not our without PGRs (plnt growth regultors, ontrol) in stem n shoot tip explnts (t not shown). The suitle llus inution ws otine in 2,4-D n NAA on MS, B5, SH n WPM whih inue GCC (green ompt llus), GFC (green frile llus), WFC (white frile llus), WWC (white wtery llus) n BFC (rown frile llus) in stem n shoot tip explnts (Fig. - n Fig. 2-h). For suessful llus

4 Cllus regenertion n lkloi umultion 265 ). mg L.5 mg L.5 mg L.5 mg L.5 mg L NAA. mg L 2 2. mg L Cllus inution frequeny (%) (WWC) (BCC) (BCC) (BCC) (WWC) Cllus inution frequeny (%) ) 2. mg L 2,4-D B5 MS SH WPM Figure. Effet of ifferent mei (B5, MS, SH, WPM) supplemente with ) NAA (nphthlene eti i) n ) 2,4-D (2,4-ihlorophenoxy eti i) on inution of ifferent llus nture n their frequeny on stem n shoot tip explnts of Phyl noiflor fter 45 ys inution. BCC: rown ompt llus; BFC: rown frile llus; GCC: green ompt llus; GFC: green frile llus; WFC: white frile llus; WWC: white wtery llus. g m h n i o j p q e f k l r Figure 2. -) White wtery llus (WWC). -) White frile llus. e-f) Brown frile llus in stem-shoot tip explnts. g) Green frile llus in shoot tip explnt. h) Green ompt llus in stem explnt. i-j) Shoot regenertion in shoot tip explnt. k-l) Shoot regenertion in stem explnt. m) Shoot elongtion in stem explnt. n) Rooting in MS with IBA (inole-3-utyri i). O) ½ MS with IBA. p) ¼ MS with IBA in stem explnt. q) Hrening. r) Fiel grown plnts.

5 266 A. B. A. Ahme et l. / Spn J Agri Res (2) 9(4), inution, ftors suh s type of explnts, PGRs, ulture mei n ulturl onitions re very importnt (Yeomn n Yeomn, 996). The stem explnts proue GCC with higher inution frequeny (92%) thn shoot tip explnts (83%) (Fig. -). Among the vrious onentrtions of uxins trie in llus inution, NAA (.5 mg L ) n 2,4-D (2. mg L ) signifintly inue GC nture llus with mximum iomss t 45 ys in terms of fresh n ry weight (Fig. 2h; Fig. 3-). In our stuy, llus iomss progressively inrese with n inrese in the NAA onentrtion (>.5-3. mg L ) n espeilly when the ulture meium ontine mg L NAA, WFC n WWC were inue signifintly (t not shown). In ition, it ws oserve tht IAA n IBA rstilly reue the llus iomss resulting in WFC nture lli ll the mei. These results re well uthentite with the previous reports of Ahme et l. (2), who emonstrte tht NAA n 2,4-D were suitle in the llus inution of P. noiflor. P. noiflor llus growth urve ws sigmoi type n four growth phses n e istinguishe in ifferent ys (5, 25, 35, 45 n 55 ys). In the lg phse (5-25 ys), llus initition n prolifertion were oserve y profoun ell ivision (Letii et l., 2). At ys (exponentil phse), iomss of the GCC nture ws signifintly inrese. The high level of llus iomss in the sttionry phse (45 ys) of the llus growth urve suggests the ellulr memrne stiliztion. It hs een previously reporte tht the sttionry phse llus eviently emonstrte n inrese in the umultion of ggminine in the llus (GC) of Cynnhum wilforii (Shin et l., 23). At the eline phse (55 ys) the llus iomss ws rstilly reue s ompre to other phses. Grvimetri, TLC n HPTLC nlysis The ientifition n quntifition of lklois ws one in grvimetri, TLC n HPTLC stuies. Gr vimetri nlysis showe tht the lkloi ontent (26.2 mg g ) ws higher in the mei ontining NAA (.5 mg L ) with BA (2. mg L ), whih ws greter thn intt stem explnts followe y shoot tip explnts (Tle ). However, the lkloi ontent ws signifintly reue in other PGRs tretments for llus (Tle ). Methnol extrts of intt n regenertive stem n shoot tip explnts showe n itionl n (.75) with Rf vlue nering the stnr lkloi (.78) (figure not shown). The ove Rf vlue ws onforme in the presene of yellow fluoresene, when the TLC plte ws sprye with Drgenorff s regent. On the other hn, intt n regenertive llus from stem n shoot tip explnts were further nlyze y HPTLC for the quntifition lklois n ompre with the lklois in grvimetry (Tle ). In HPTLC, the smple urve for the lkloi ontent ws liner, where the orreltion oeffiient inite goo linerity etween onentrtion n re. To sertin the purity of smple peks, the test smple ws nlyze in refletne spetr. The lkloi ontent ws signifintly inrese in regenertive llus of stem explnts (5.5 mg g ) followe y shoot tip explnts ) ) 3 4 Fresh weight (g L ) Dry weight (mg L ) NAA (mg L ) 2,4-D (mg L ) NAA (mg L ) 2,4-D (mg L ) NAA (mg L ) 2,4-D (mg L ) NAA (mg L ) 2,4-D (mg L ) B5 MS SH WPM Figure 3. Effet of ifferent mei (B5, MS, SH, WPM) supplemente with 2,4-D (2,4-ihlorophenoxy eti i) n NAA (nphthlene eti i) on inution of llus iomss [() fresh n ) ry weight] on stem n shoot tip explnts of Phyl noiflor fter 45 ys inution.

6 Cllus regenertion n lkloi umultion 267 Tle. Effet of plnt growth regultors on shoot regenertion n lkloi ontent from inue lli of stem n shoot tip explnts of Phyl noiflor ulture on MS meium fter 6 ys Plnt growth regultors (mg L ) NAA + BA NAA + BA NAA + BA NAA + KN NAA + KN NAA + KN Perentge of response (%) Shoot tip Regenerte shoot numer Men ± S.E Shoot length (m) Men ± S.E 6.6 ± ± ± ± ± ± ± ± ± ± ± ± ± ±. 5.6 ±.2 6. ± ±. 5.8 ± ± ±.8 7. ± ± ± ± ± ± ± ±.2 7. ± ±.9 4. ± ± ± ± ± ± ± ±.9 2. ± ± ± ± ±. 3.4 ± ± ± ± ±.6.8 ± ± ±.9.2 ±.4.8 ±.22.4 ± ±.6 3. ± ± ± ± ±. 2.8 ± ± ±.6.8 ± ±. 2. ±.6.9 ±. 3.6 ± ± ± ±.2.6 ±.22 Grvimetri nlysis (mg g ) Alkloi ontent Shoot tip HPTLC (mg g ) NAA: nphthlene eti i; BA: enzyl enine; KN: kinetin. Vlues re men of 3 replites per tretment n repete trie. Vlues with the sme supersript re not signifintly ifferent t 5% proility level oring to Dunn Shoot tip (4. mg g ); the results were ompre to the intt stem n shoot tip explnts (Tle ; Fig. 4-). However, few llus extrts showe the ition peks whih evite from intt plnt extrts n tht hve een ientifie for the presene of other ompouns. These results were orrelte with the grvimetri, HPTLC n HPLC nlysis of poly-herl omintions n ompre with respetive iomrker ompouns (Joshi et l., 27). In vitro llus regenertion Suessful llus regenertion ws otine y the use of BA (2. mg L ) n NAA (.5 mg L ), whih inrese the shoot numer (2.3 shoots stem ) n shoot length (4.2 m) followe y shoot tip explnts (9.6 shoots shoot tip ) n shoot length (3.5 m) erive llus from stem of P. noiflor fter 6 ys (Tle ; Fig. 2i-k). However, BA + 2,4-D n KN + 2,4-D omintions rstilly reue the llus regenertion in stem n shoot tip explnts (t not shown). It ws oserve tht the frequeny of regenerte shoots per ulture vrie y % in stem n % in shoot tip erive lluses respetively (Tle ). In ition, vrious omintions of BA n KN were trie for llus regenertion, whih resulte in less shoot numers n stunte shoot length thn with other uxins n ytokinins omintion (figure not shown). During the llus regenertion, the

7 268 A. B. A. Ahme et l. / Spn J Agri Res (2) 9(4), ) Intensity (AU) Intensity (AU) ) Rf (snne t 2 nm) Rf (snne t 2 nm) ) Intensity (AU) Intensity (AU) ) Rf (snne t 2 nm) Rf (snne t 2 nm) Figure 4. Alklois etermine y HPTLC (high performne thin lyer hromtogrphy) in intt n in vitro regenerte llus from stem n shoot tip explnts of Phyl noiflor: ) intt stem explnts; ) in vitro regenerte llus (MS +.5 mg L NAA + 2. mg L BA) from stem explnts; ) intt shoot tip explnts; ) in vitro regenerte llus (MS +.5 mg L - NAA + 2. mg L BA) from shoot tip explnts. BA: enzyl enine; NAA: nphthlene eti i; Rf: rtio-to-front; AU: sorption unit. th llus ulture ws ontinuously exmine y tking suulture t weekly intervls to prevent the ell eth n rowning of mei. These finings re well supporte y Ptel n Shh (29), who reporte the inue llus regenertion y NAA with BA in the nol explnts of Stevi reuin. In vitro rooting n limtiztion Regenerte lefy shoots erive from llus of oth stem n shoot explnts i not show ny root inution, when they were trnsferre to MS mei without PGRs (t not shown). Roots were forme on exise shoots grown on hlf-strength MS mei ontining either IAA or IBA (Fig. 5). A high perentge of root inution (72.7%) ws otine in MS meium with. mg L IBA in stem explnts fter 35 ys (Fig. 2o; Fig. 5-), in omprison to IAA. The present results inite tht the stem explnts showe etter root inution thn shoot tip explnts when the ulture meium ws supplemente with.4-.4 mg L of IAA n IBA. Further, the root length of the stem explnts ws higher in hlf-strength MS meium ontining. mg L IBA, when ompre to full n qurter strength MS meium (Fig. 2n-p; Fig. 5). However, stronger inhiition of root length y IAA tretment my e responsile for the poorer rooting responses respet to

8 Cllus regenertion n lkloi umultion 269 ) Rooting responses (%).4 mg L.6 mg L i.8 mg L. mg L.2 mg L.4 mg L.4 mg L.2 mg L. mg L.8 mg L.6 mg L.4 mg L ) Rooting responses (%) ) Root length (m) MS + IBA /2 MS + IBA /4 MS + IBA MS + IAA /2 MS + IAA /4 MS + IAA mg L.6 mg L.8 mg L. mg L.2 mg L.4 mg L.4 mg L.2 mg L. mg L.8 mg L.6 mg L.4 mg L Figure 5. Effet of MS, ½ MS n ¼ MS meium with IBA ( n ) n IAA ( n ) on rooting response n root length per explnt in the stem explnts of Phyl noiflor. IBA: inole-3-utyri i; IAA: inole eti i. ) Root length (m) IBA tretment (Fig. 5). Blrju et l. (28) oserve suessfully roote plntlets of Vitex gnus-stus (Verenee) on hlf-strength MS meium ontining IBA (. mg L ). The present finings of rooting n limtiztion were similr to our previous stuies of P. noiflor (Ahme et l., 25). After one month of trnsfer, the roote plntlets were trnsferre into pots ontining soil, sn n well rotte ow-ung mnure, n shifte to green house for limtiztion (Fig. 2q), where they grew normlly (Fig. 2r). Conlusions Suessful llus regenertion ws optimize from stem explnts in the omintions of BA n NAA. The est GCC inution on MS meium with NAA (.5 mg L ) in the stem explnts were optimum for plnt regenertion. In this stuy, the lklois ontent ws superior in regenertive llus thn intt stem, shoot tip explnts, s onfirme y grvimetri n HPTLC. Our t on the effets of PGRs in P. noiflor th ulture will e useful in enhning the umultion of lkloi ompouns in suh ells n n lso e further extene to other plnt ulture moels. Aknowlegements The first uthor is thnkful to Dr. K. Mriul, Dr. V. Kumresn, Dr. T. Gouthmn n Dr. A. R. Lvny for their help uring the stuy; n to Dr. Mnimrn, Leturer, JSS College of Phrmy, Ooty, for the help renere uring HPTLC nlysis. Referenes ABBASI A.M., KHAN M.A., AHMAD M., ZAFAR M., JAHAN S., SULTANA S., 2. Ethnophrmologil pplition of meiinl plnts to ure skin iseses n in folk osmetis mong the tril ommunities of northwest frontier provine. Pkistn J Ethnophrmol 28,

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