Effects of Explant Position and Polarity on Callus Induction and Shoot Regeneration of Gladiolus (Gladiolus hybridus Hort.)

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1 Floriulture n Ornmentl Biotehnology 212 Glol Siene Books Effets of Explnt Position n Polrity on Cllus Inution n Shoot Regenertion of Gliolus (Gliolus hyrius Hort.) Fouzi Houl * Emn Chïe Déprtement es Sienes Hortioles et u Pysge, Institut Supérieur Agronomique, 442 Chott Mriem, Sousse, Tunisi Corresponing uthor: * fouzi.houl@lposte.net ABSTRACT Different types of explnts were use for llus inution in Gliolus tissue ulture of ultivrs ChCh n Prisill. Different onentrtions of 2,4-ihlorophenoxyeti i (2,4-D) or -nphthleneeti i (NAA) were teste. Explnt polrity ws stuie for leves y pling frgments of these orgns horizontlly n vertilly on the ulture meium. Also, explnts were tken from the pil, mile n sl prts of leves n petls. Apil us, leves n flower stlks showe exellent llus formtion (1%). However, petls were hrterize y low llus formtion ility (1%) while florl stems, rts n florl spikes showe no llus formtion. The rte of llus evelopment ws signifintly higher for horizontlly ulture leves n erese from sl explnts to mile n pil ones. The highest rte of llus formtion ws otine on mei ontining either 2,4-D (1 or 3 mg l -1 ) or NAA (2 or 5 mg l -1 ). Cllus uing ws signifintly higher on meium supplemente with 1 mg l -1 BA. The uing rte of llus otine from pil us ws 93.3 n 1%, respetively, for ultivrs Prisill n ChCh. For llus erive from lef frgments, the rte of uing ws 1% for oth ultivrs. Keywors: llus formtion, uing, in vitro, polrity Arevitions: BA, N6-enzylenine; 2,4-D, 2,4-ihlorophenoxyeti i; KIN, kinetin; MS, Murshige n Skoog sl meium; NAA, -nphthleneeti i INTRODUCTION Speies of Gliolus (Iriee) re of gret hortiulturl vlue. Moern ultivte glioli evolve through suessive nturl n rtifiil hyriiztion of Gliolus speies originting from the Meiterrnen re n South Afri (Buh 1972; Wilfret 198) inluing over 18 speies with more thn 1, ultivrs. Approximtely 2 ultivrs re grown for ommeril flower proution (Nhut et l. 24). This genus hs een sujet of in vitro propgtion experiments (Ziv et l. 197; Hussey 1977; Bjj et l. 1983; Kmo et l. 199; Ziv n Lilien-Kipnis 199; Sen n Sen 1995). Gliolus miropropgtion ws hieve from xillry us (Begum n Hiuzmn 1995; Boonvnno n Knhnpoom 2), pex (Hussin et l. 21; Aft et l. 28), orms (Ngrju n Prthsrthy 1995; Emek n Erg 27) n infloresene (Ziv n Lilien-Kipnis 2). Mny uthors (Bjj et l. 1983, 1992; Kmo 1995; Remotti n Löffler 1995; Tori-Giglou n Hjieghrri 28; Prgy et l. 212) stuie Gliolus llus formtion from ifferent explnts: infloresene, flower stems, erly flower, rt, perinth, orm, ormel n lef frgments. The est llus formtion ws oserve in segments of flower stems n the est shoot proution ws hieve on meium ontining.5-1 mg l -1 N6-enzylenine (BA) n 6-9% surose, n mintine t 15 C. In this pper we stuy the effets of the position n the polrity of lef explnts on llus formtion in Gliolus using ifferent ulture mei. Shoot regenertion is lso stuie from ifferent explnts erive llus. MATERIALS AND METHODS Plnt mteril The strting mteril ws glioli (Gliolus hyrius Hort. ChCh n Prisill ) orms hving 1/12 m irumferene. Explnts use were: - pil us 4-5 mm long tken from uls, - lef frgments of 25 mm 2 tken from in vitro plnts 4-weeks ol; explnts were tken from the pil, mile n sl prts of leves, - flower stlk setions 5 mm long n imeter of 2-3 mm, - petls of 25 mm 2 tken from pil, mile n sl prts, - florl stems 5 mm long n imeter of 4-6 mm, - rts of 25 mm 2, - florl spikes 5 mm long n 2-4 mm in imeter. All florl prt explnts were otine from plnts ultivte in n open fiel in the region of Sousse (ltitue North), Tunisi. Explnts were isinfete with 7% ethnol. After wshing them in sterile istille wter, they were soke in 1% hyrogen peroxie (H 2 O 2 ) for 1 min, rinse twie (15 min eh) with sterile istille wter then soke gin in.1% HgCl 2 for 1 min, rinse four times (15 min eh) with sterile istille wter n use for ulturing. Explnt polrity ws stuie for leves: frgments were li horizontlly or stuk vertilly with the sl en into the meium t 2 mm epth. Culture mei n experimentl onitions Culture meium ws Murshige n Skoog (1962; MS) whih ws supplemente with 3% surose n 8% gr (Bto Difo, Pnre Quimi) n the ph ws juste to 5.8. Explnts were ulture iniviully in tues of 24 mm imeter n 15 mm length, ontining 15 ml of MS meium. Cultures were onute in growth hmer t 24 1 C, uner 16-h photoperio uner ool white light t 35 μmol m -2 s -1 provie y white fluoresent tues (18W/54 Philips). Cllus formtion ws onute in rkness (Bettïe 23). Inution of llus Cllus formtion mei were MS supplemente with 2,4-ihlorophenoxyeti i (2,4-D) t ifferent onentrtions (, 1, 2, 3 Reeive: 16 Septemer, 21. Aepte: 2 Deemer, 212. Originl Reserh Pper

2 Floriulture n Ornmentl Biotehnology 6 (Speil Issue 2), Glol Siene Books 'Prisill' 'ChCh' Cllus proution rte (%) Vertil leves Horizontl leves Cllus proution rte (%) ,4 D (mg l 1 ) ,4 D (mg l 1 ) Cllus proution rte (%) Cllus proution rte (%) NAA (mg l 1 ) Fig. 1 Polrity ifferenes in llus proution from lef frgments of gliolus (Gliolus hyrius Hort. Prisill n ChCh ) when ple horizontlly or vertilly on MS meium with 2,4-D or NAA. Durtion of ulture: 8 weeks. Numer of explnts per tretment: 24. Different letters inite signifint ifferenes t P =.5 (Dunn s multiple rnge test). mg l -1 ) (Kim et l. 1988; Ziv n Lilien-Kipnis 2; Giglou n Hjieghrri 28), or -nphthleneeti i (NAA) t ifferent onentrtions (, 1, 2, 5, 1 mg l -1 ) (Bettïe 23; Aft et l. 28). Cultures were mintine for 8 weeks with suulture fter 4 weeks. Shoot regenertion Eight weeks ol llus ws put on regenertion mei; MS with ifferent onentrtions of BA (,.5, 1, 2 mg l -1 ). Eh meium tretment (BA onentrtion) ontine 15 explnts. A B Dt nlysis Severl prmeters were reore: inution of llus (+/-); rte of uing: numer of lli whih forme us/totl numer of lli; u (numer of newly-forme us per llus) n shoot prolifertion (numer of shoots per llus); morphologil pperne of lli n shoots (struture, olor, et.). Anlysis of vrine (ANOVA) ws performe for ll t using SPSS softwre. Sttistilly signifint ifferenes etween mens were etermine using Dunn s multiple rnge test t =.5. RESULTS Cllus formtion 1. Inution of llus from leves In ultures without plnt growth regultors (ontrol), there ws no llus formtion in either ultivr for leves li horizontlly or vertilly on the ulture meium (Fig. 1). Cllus formtion rte inrese with the onentrtion of 1 mm Fig. 2 Formtion of llus on lef frgments of gliolus (Gliolus hyrius Hort. Prisill ) ulture for 4 (A) or 8 weeks (B) on MS meium with 3 mg l -1 2,4-D. uxin n ws the highest using 3 mg l -1 2,4-D: 6 n 7% for horizontl explnts, respetively, for Prisill n ChCh, 3% for vertil explnts of oth ultivrs. For NAA, the est onentrtion ws 5 mg l -1, whih h llus formtion rte of 1% for horizontl leves n 5, 7% for vertil leves, respetively, for Prisill n ChCh (Fig. 1). At higher onentrtion of NAA (1 mg l -1 ), inution of llus ws very low n i not exee 1% for oth ultivrs. Explnts li horizontlly were etter for llus formtion thn those li vertilly, ut ifferenes were signifint only for the onentrtion of 3 mg l -1 for 2,4-D n 5 mg l -1 for NAA. Clli forme were noulous or rumly with some ells 134

3 Shoot regenertion from Gliolus llus. Houl n Chïe 'Prisill' 'ChCh' 1 1 Cllus proution rte (%) Apil position Mein position Bsl position Cllus proution rte (%) Cllus proution rte (%) Cllus proution rte (%) Fig. 3 Effet of explnt position on llus proution from lef frgments of gliolus (Gliolus hyrius Hort. Prisill n ChCh ) ulture on MS meium with 2,4-D or NAA. Durtion of ulture: 8 weeks. Numer of explnts per tretment: 24. Different letters inite signifint ifferenes t P =.5 (Dunn s multiple rnge test). 1 1 Cllus proution rte (%) ChCh Prisill Cllus proution rte (%) Fig. 4 Cllus proution from flower stlks of gliolus (Gliolus hyrius Hort. Prisill n ChCh ) ulture on MS meium with 2,4-D or NAA. Durtion of ulture: 8 weeks. Numer of explnts per tretment: 24. Different letters inite signifint ifferenes t P =.5 (Dunn s multiple rnge test) ontining hlorophyll (yellow-greenish olor: Fig. 2). For explnt position on the lef, inution of llus ws possile only with sl n mile explnts. Bsl explnts h the highest rte of llus formtion whih ws 7% for ChCh n 5 to 7% for Prisill, oring to the type of uxin. There ws no llus formtion with pil explnts (Fig. 3). 2. Inution of llus from flower stlks Without hormones, there ws no llus formtion on flower stlks (Fig. 4). The presene of 2,4-D ws very fvourle for llus formtion n its rte rehe 1% for oth ultivrs using 1 mg l -1 2,4-D. On mei ontining higher onentrtions of 2,4-D, llus formtion rte ws unhnge for Prisill (1%) ut erese to 7% for ChCh. Inution of llus inrese with NAA onentrtion rehing its mximum (1%) with 2 or 5 mg l -1 NAA, respetively, for Prisill n ChCh. Using 1 mg l -1 NAA, llus formtion rte erese to 5 n 7%, respetively, for ChCh n Prisill. Clli were gloulr, whitish n hrterize y ompt struture (Fig. 5). 135

4 Floriulture n Ornmentl Biotehnology 6 (Speil Issue 2), Glol Siene Books A B 1 mm Fig. 5 Formtion of llus on flower stlks of gliolus (Gliolus hyrius Hort. Prisill ) ulture for 8 weeks on MS with 1 mg l -1 2,4-D. Cllus proution rte (%) Cllus proution rte (%) Prisill ChCh Inution of llus from pil us Fig. 6 Cllus proution from pil us of gliolus (Gliolus hyrius Hort. Prisill n ChCh ) ulture on MS with 2,4-D or NAA. Durtion of ulture: 8 weeks. Numer of explnts per tretment: 15. Different letters inite signifint ifferenes t P =.5 (Dunn s multiple rnge test). e e Fig. 7 Formtion of llus from pil us of gliolus (Gliolus hyrius Hort. Prisill ) ulture on MS with 1 mg l -1 2,4-D. Swelling n erly llus formtion (A), llus fter 4 weeks of ulture (B). With these types of explnts, meium without hormones llowe llus formtion ut its rte ws low: 16.6 n 2.8%, respetively, for ChCh n Prisill (Fig. 6). Using either 1 mg l -1 2,4-D or 2 mg l -1 NAA permitte mximl (1%) llus inution for oth ultivrs. On mei supplemente with higher onentrtions of 2,4-D, llus formtion rte ws unhnge for Prisill (1%) ut erese sensily for ChCh to 8 n 7% with 2 n 3 mg l -1 2,4-D, respetively. Using higher onentrtions of NAA, this prmeter erese for oth ultivrs ut i not fll uner 6%. Cllus ws noulous, rumly, not hlorophyllous n yellowish for oth ultivrs (Fig. 7). 4. Inution of llus from petls Petls were tken from pil, mile n sl prts of these orgns. Apil n mile explnts of oth ultivrs i not evelop llus on ny ulture mei. On meium ontining 1 mg l -1 2,4-D, only sl explnts showe swelling fter 4 weeks while spongy, rk rown llus ws forme fter 8 weeks. The llus formtion rte ws very low ( 1%) for oth ultivrs. For the other types of explnts (florl stems, rts, florl spikes), there ws no llus initition t ll. Shoot regenertion 1. From lef frgments erive llus On the ontrol meium (without BA), llus uing ourre t 2 n 47%, respetively, for ChCh n Prisill (Tle 1). Rte of uing inrese with BA onentrtion n ws the highest on meium ontining 1 mg l -1 BA rehing 1% for oth ultivrs fter 4 weeks of ulture. The numer of newly-forme us ws signifintly the est on the sme meium n ws 2.4 n 2.6 us per llus, respetively, for ChCh n Prisill. Using higher onentrtion of BA (2 mg l -1 ), the numer of us per llus ws reue to 1.3 n 1 us, respetively, for ChCh n Prisill. The numer of shoots per llus inrese with onentrtion of BA n ws the highest with 1 mg l -1 BA (Tle 1): 1.9 n 2.5 shoots per llus for ChCh n Prisill, respetively. On meium ontining 2 mg l -1 BA, shoot regenertion erese to.94 for oth ultivrs. Shoot regenertion from lef llus ws generlly etter for Prisill ut results were not signifintly ifferent (Tle 1). 2. From pil us erive llus The numer of newly-forme shoots per llus epene on ultivr n BA onentrtion. Inee, on ontrol meium without BA, shoot regenertion ws possile, ut its rte i not exee 13.3 n 26.7%, respetively, for ChCh n Prisill, signifintly lower thn the tretments with BA (Tle 2). Buing rte of pil us erive llus ws the highest on meium ontining 1 mg l -1 BA (Tle 2). In ition, uing on this meium ws the fstest n rehe for oth ultivrs 53.3% fter 1 ys of ulture n 93.3 n 1% fter 4 weeks of ulture, respetively, for Prisill n ChCh (Tle 2). 1 mm 136

5 Shoot regenertion from Gliolus llus. Houl n Chïe Tle 1 Rte of uing (%), numer of us/llus n numer of shoots/llus from gliolus ChCh n Prisill lef frgment erive lli. Culture onitions: regenertion meium = Murshige n Skoog (MS) ± BA; 16-h photoperio; light intensity 35 μmol m -2 s -1 ; 24 1 C. Numer of explnts per tretment: 15. Regenertion meium Cultivr Rte of uing (%) Numer of us per llus Numer of shoots per llus MS (ontrol) ChCh ± ±.11 Prisill ±.31.4 ±.2 MS +.5 mg l -1 BA ChCh ±.54.9 ±.5 Prisill ± ±.63 MS + 1 mg l -1 BA ChCh ± ±.9 Prisill ± ± 1.2 MS + 2 mg l -1 BA ChCh ± ±.5 Prisill ± ±.48 Vlues in the sme olumn followe y ifferent letters re signifintly ifferent (P.5) oring to DMRT. Tle 2 Rte of uing (%), numer of us/llus n numer of shoots/llus from gliolus ChCh n Prisill pil u erive lli. Culture onitions: regenertion meium = Murshige n Skoog (MS) ± BA; 16-h photoperio; light intensity 35 μmol m -2 s -1 ; 24 1 C. Numer of explnts per tretment: 15. Regenertion meium Cultivr Rte of uing (%) Numer of us per llus Numer of shoots per llus MS (ontrol) ChCh ±.9.27 ±.12 Prisill ±.23.2 ±.11 MS +.5 mg l -1 BA ChCh ± ±.51 Prisill ± ±.43 MS + 1 mg l -1 BA ChCh ± ±.91 Prisill ± ± 1.34 MS + 2 mg l -1 BA ChCh ± ±.46 Prisill ± ±.48 Vlues in the sme olumn followe y ifferent letters re signifintly ifferent (P.5) oring to DMRT. Tle 3 Rte of uing (%) n numer of us/llus from gliolus ChCh n Prisill flower stlk erive lli. Culture onitions: regenertion meium = Murshige n Skoog (MS) ± BA; 16-h photoperio; light intensity 35 μmol m -2 s -1 ; 24 1 C. Numer of explnts per tretment: 15. Regenertion meium Cultivr MS (ontrol) MS +.5 mg l -1 BA MS + 1 mg l -1 BA MS + 2 mg l -1 BA Rte of uing (%) ChCh Prisill ChCh Prisill ChCh Prisill ChCh Prisill The numer of us per llus ws signifintly higher thn ll other tretments on the meium with 1 mg l -1 BA: 2.2 n 2.5 us, respetively, for ChCh n Prisill (Tle 2). For higher onentrtions of BA (2 mg l -1 ), the numer of us/llus ws reue to only 1 u for oth ultivrs. The numer of shoots/llus inrese with the onentrtion of BA n ws signifintly higher t 1 mg l -1. For higher onentrtions of BA (2 mg l -1 ), the numer of shoots erese y more thn hlf. Anlysis of vrine showe signifint ifferenes etween the ontrol n ll BA onentrtion, however, ifferenes etween the two onentrtion of BA (.5 n 2 mg l -1 ) were not signifint. Differenes etween ultivrs were not lso signifint. 3. From flower stlks erive llus Numer of us per llus For lli erive from flower stlks, shoot regenertion ws ompletely inhiite, n egenertion ffete ll lli fter four weeks of ulture (Tle 3). There ws no shoot regenertion from petls erive llus. DISCUSSION Cllus formtion Cllus inution ws hieve on MS meium supplemente with vrious onentrtions of 2,4-D or NAA, similr to previous results using the sme uxins (Bettie 23; Emek n Erg 27; Aft et l. 28). For inrese llus inution, ultures were ple in rkness t 24 ± 1 C (Bettie 23). Cllus oul e inue from vrious explnts: leves, pil us, flower stlks n petls, ut not other types of explnts (florl stems, rts n florl spikes). These results re not in greement with those of Bjj et l. (1983) who reporte tht llus formtion ws otine for Gliolus Snow Priness n Osr from vrious explnts: infloresenes, florl stems, rts n perinths on 1 mg 1-1 NAA n.5 mg.1-1 KIN. These ifferenes oul e ttriute to the llus inution mei n/or genotype. Cllus oul e otine lso from orm setions in MS meium with 8.5 mg 1-1 NAA (Emek n Erg 27) n from ormels on 4.44 μm BA n 2.32 μm KIN n 2.68 μm NAA (Prgy et l. 212). For lef explnts, mei ontining 3 mg l -1 2,4-D or 5 mg l -1 NAA ppere most fvourle to llus formtion n oth ultivrs were omprle (Fig. 1). Aft et l. (28) showe tht inution of llus from lef explnts ws possile on MS with 3 mg l -1 2,4-D or 2 mg l -1 NAA. Lower or higher NAA onentrtions i not permit llus inution. However, Bettie (23) reporte tht lef explnts of Gliolus Peter Pers n White frienship on ifferent mei ontining 2,4-D or NAA i not inue llus. Clerly, ultivr (genotypi) ifferenes wrrnt testing of vrying plnt growth regultors onentrtions to mximize llus formtion. The explnts of oth ultivrs ple horizontlly on the mei showe etter ility to llus inution thn those ple vertilly (Fig. 1). This is proly relte with the surfe of the explnt in ontt with the ulture meium. Explnts tken from the sl prt of the lef gve the est results (Fig. 3). This is in greement with Ksumi et l. (1999) on Gliolus Topz, where lef explnts tken from the sl prt n ultivte in rkness for 6 ys on MS with 5 mg l -1 NAA proue 1% llus. Wernike n Milkovits (1987) showe for young leves segments of whet (Tritium timopheevi Zukh) tht the most sl ells 137

6 Floriulture n Ornmentl Biotehnology 6 (Speil Issue 2), Glol Siene Books ivie reily in ulture n omplete the mitoti yle. Flower stlks showe strong ility for llus proution, rehing 1% on most mei (Fig. 4). Petls i not generte llus, eing inhiite for explnts erive from pil n mile prts of the petl. For sl explnts, inution of llus ws very low ( 1%) on meium ontining 1 mgl -1 2,4-D. Ksumi et l. (21) inite tht llus ws not inue for the pil prts of the perinth, ut it ws possile for the sl prts (56% on MS supplemente with 5 mg l -1 NAA). Shoot regenertion The formtion n numer of us on lli ws possile in presene of BA (Tle 1). After 2 weeks of ulture, meristemti protuernes ppere on the lli n evolve in shoots (Fig. 2). Shoot formtion ws lso possile without BA t signifintly lower rte thn 1 mg l -1 BA (Tles 1, 2). Inee, Mrgr (1984) note tht the formtion of new us in monoots from lli on meium without plnt growth regultors ws foun. Aoring to our results, shoot regenertion from llus ws possile on mei with or without BA (,.5, 1, 2 mg l -1 ) (Tles 1, 2). There were signifint ifferenes etween BA onentrtions ut not etween ultivrs for llus proution (Tles 1, 2). The highest numer of shoots per llus ws otine on meium ontining 1 mg l -1 BA (Tles 1, 2). De Bruyn n Ferreir (1992) inite tht the est proution of shoots per explnt ws hieve on meium ontining.5 to 1 mg l -1 BA. The highest shoot inution (98.3%) n numer of shoots ws otine from ormel sprouts on MS with 4 mg l -1 BA (Memon et l. 21). Shheenuzzmn et l. (211) reporte tht the highest perentge of shoot regenertion (91.6%), for lli erive from lef iss, ws oserve in MS meium ontining 3 mg l -1 BA n.5 mg l -1 KIN. However, Bettie (23) showe tht the est formtion of us on llus ws otine using.5 mg l -1 BA n 1 mg l -1 AgNO 3. Thus, the onentrtion of BA oul epen on ultivr n the ition of metl ions suh s Ag + is neee in some ses to stimulte the formtion of us (De Blok 1988; Hnnhi 1996; Houl 1999). AgNO 3 ts y inhiiting ethylene, unfvourle ftor for regenertion (Rethmeier et l. 1991). CONCLUSIONS Cllus proution in Gliolus epens on type of explnts, polrity n position, plnt growth regultors type, onentrtion n genotype. Inee, llus inution ws possile from leves, flower stlks, pil us n petls. However, florl stems, rts n florl spikes i not permit llus initition t ll. Lef explnts li horizontlly were more fvourle for llus formtion thn those li vertilly. A sipetl grient ws oserve in lef explnts n llus formtion ws possile only with sl n mile ones while only sl petl explnts forme llus. Inution of llus ws possile either with 2,4-D or NAA t vrious onentrtions oring to type of explnts. However, only 2,4-D proue llus from petls. Genotype-speifi llus proution ws not signifintly ifferent in this experiment. Shoot regenertion ws possile for lli erive from lef frgments n pil us ut ompletely inhiite from tht of flower stlks. The rte of uing, numer of newlyforme us n numer of shoots per llus were highest on meium ontining 1 mg l -1 BA. Shoot regenertion from lef llus n pil us i not iffer etween genotypes. ACKNOWLEDGEMENTS The uthors woul like to thnk Dr. Messou Mrs for lnguge revision. REFERENCES Aft F, Alm M, Afrsi H (28) In vitro shoot multiplition n llus inution in Gliolus hyrius Hort. Pkistn Journl of Botny 4 (2), Bjj YPS, Sihu MMS, Gill APS (1983) Some ftors ffeting the in vitro propgtion of Gliolus. Sienti Hortiulture 18, Bjj YPS, Sihu MMS, Gill APS (1992) Miropropgtion of Gliolus. In: Bjj YPS (Es) Biotehnology in Agriulture n Forestry 2, High-Teh n Miropropgtion IV, Springer Verlg, Berlin, pp Begum S, Hiuzzmn S (1995) In vitro rpi shoot prolifertion n orm evelopment in Gliolus grniflorus v. Rern. Plnt Tissue Culture 5, 7-11 Bettie T (23) Régénértion in vitro e vrints somlonux e glïeul (Gliolus grniflorus Hort.) tolérnts ux sses tempértures. PhD thesis, Tunis, Tunisi, 136 pp Boonvnno K, Knhnpoom K (2) In vitro propgtion of Gliolus. Surnree Journl of Siene n Tehnology 7, Buh PO (1972) The speies. In: Koenig N, Crowely W (Es) The Worl of Gliolus, North Amerin Gliolus Counil, Egerton Press, MD, pp 2-7 De Blok M (1988) Genotype-inepennt lef is trnsformtion of potto (Solnum tuerosum) using Agroterium tumefiens. Theoretil n Applie Genetis 76, De Bruyn MH, Ferreir DI (1992) In vitro orm proution of Gliolus lenii n G. tristis. Plnt Cell, Tissue n Orgn Culture 31, Emek Y, Erg B (27) In vitro propgtion of Gliolus ntolius (Boiss.) Stpf. Pkistn Journl of Botny 39 (1), 23-3 Giglou M, Hjieghrri B (28) In vitro stuy on regenertion of Gliolus grniflorus orm lli s ffete y plnt growth regultors. Pkistn Journl of Biologil Sienes 11, Hnnhi C (1996) Améliortion e l tolérne e l pomme e terre (Solnum tuerosum L.) à l slinité (NCl) pr voie iotehnologique. PhD thesis, Gn, Belgium, 152 pp Houl F (1999) Régénértion in vitro et tolérne à l slinité e l œillet (Dinthus ryophyllus L.). PhD thesis, Tunis, Tunisi, 171 pp Hussin I, Mohme H, Rshi Qurishi A (21) In vitro multiplition of Gliolus (Gliolus rssifolius). Plnt Tissue Culture 11, Hussey G (1977) In vitro propgtion of Gliolus y preoious xillry shoot formtion. Sienti Hortiulture 6, Kmo K (1995) A ultivr omprison of plnt regenertion from suspension ells, llus n ormel slies of Gliolus. In Vitro Cellulr n Developmentl Biology Plnt 31, Kmo K, Chen J, Lwson R (199) The estlishment of ell suspension ulture of Gliolus tht regenerte plnts. In Vitro Cellulr n Developmentl Biology Plnt 26, Ksumi M, Tktsu Y, Tomotsune Skum T, Ii S (1999) The isoltion of vrie flower olor plnts y ovry ulture of setoril himer of Gliolus. Journl of the Jpnese Soiety for Hortiulturl Siene 68 (1), Ksumi M, Tktsu Y, Mne T, Hyhi M (21) The effet of irriting gliolus (Gliolus grniflorus Hort.) ormels with gmm rys on llus formtion, somti emryogenesis n flower olor vritions in the regenerte plnts. Journl of the Jpnese Soiety for Hortiulturl Siene 7 (2), Kim KW, Choi JB, Kwn KY (1988) Rpi multiplition of Gliolus plnts through llus ulture. Journl of the Koren Soiety for Hortiulturl Siene 29, Mrgr J (1984) Néoformtion e méristèmes et emryogenèse somtique. In: INRA (Es) Bses e l Multiplition Végéttive. Les Méristèmes et l Orgnogenèse, Pris, pp Memon N, Qsim M, Jskni MJ, Ahm R (21) In vitro ormel proution of Gliolus. Pkistn Journl of Agriulturl Sienes 47 (2), Murshige I, Skoog F (1962) A revise meium for rpi growth n iossys with too tissue ultures. Physiologi Plntrum 15, Ngrju V, Prthsrthy VA (1995) Effet of growth regultors on in vitro shoots of Gliolus hyrius. Foli Hortiulture 7/2, 93-1 Nhut DT, Teixeir Silv JA, Huyen PX, Pek KY (24) The importne of explnt soure on regenertion n miropropgtion of Gliolus y liqui shke ulture. Sienti Hortiulture 12, Prgy R, Singh SK, Misr RL, Rnjn JK (212) In vitro shoot regenertion from ormel erive llus of gliolus n io-hrening of plntlets. Inin Journl of Biotehnology 11, Remotti PC, Löffler HJM (1995) Cllus inution n plnt regenertion from gliolus. Plnt Cell, Tissue n Orgn Culture 42, 2, Rethmeier NOM, Jnsen CE, Snel EAM, Nijkmp HJJ, Hille J (1991) Improvement of regenertion of Lyopersion pennellii protoplsts y eresing ethylene proution. Plnt Cell Reports 9, Sen J, Sen S (1995) Two-step u ulture tehnique for high frequeny regenertion of Gliolus orms. Sienti Hortiulture 64, Shheenuzzmn M, Hque MS, Krim MM, Noor ZU (211) In vitro shoot prolifertion n evelopment of miropropgtion protool from lef is of Gliolus. Journl of Bnglesh Agriulturl University 9 (1), Tori-Giglou M, Hjieghrri B (28) In vitro stuy on regenertion of Gliolus grniflorus orm lli s ffete y plnt growth regultors. 138

7 Shoot regenertion from Gliolus llus. Houl n Chïe Pkistn Journl of Biologil Sienes 11 (8), Wernike W, Milkovits L (1987) Effet of uxin on the mitoti ell yle in ulture lef segments t ifferent stges of evelopment in whet. Physiologi Plntrum 69, Wilfret GJ (198) Gliolus. In: Lrson RA (Es) Introution to Floriulture, Aemi Press, New York, pp Ziv M, Hlevy AH, Shilo R (197) Orgn n plntlet regenertion of Gliolus through tissue ulture. Annls of Botny 34, Ziv M, Lilien-Kipnis H (199) Gliolus. In: Ammirto PV, Evn DA, Shrp WR, Bjj YPS (Es) Hnook of Plnt Cell Culture (Vol 5), MGrw- Hill, New York, pp Ziv M, Lilien-Kipnis H (2) Bu regenertion from infloresene explnt for rpi propgtion of geophytes in vitro. Plnt Cell Reports 19,