Regeneration of Broccoli (Brassica oleracea L. var. bejo) from Hybrid Mature Seed and Molecular Analysis of Regenerants

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1 4 n Interntionl Conferene on Agriulture n Biotehnology IPCBEE vol. 79 (4) (4) IACSIT Press, Singpore DOI:.776/IPCBEE. 4. V79. Regenertion of Brooli (Brssi olere L. vr. ejo) from Hyri Mture See n Moleulr Anlysis of Regenernts Titin Hnyni Institute for Environmentl Tehnology, Ageny for the Assessment n Applition of Tehnology, BPPT Buil. II 9 th Floor, MH Thmrin Street No. 8 Jkrt 4, Inonesi Astrt. The ojetive of this reserh ws to otin rooli plnts through iret regenertion in vitro. Hypootyls n shoot tips were use s explnts in vitro plnt regenertion of rooli. The explnts were exise from sterile germinte seelings n ple on shoot inution meium ontining sl slts of Murshige n Skoog (MS) n vrious onentrtions of 6-enzylminopurine (BAP) n α- nphthleneeti i (NAA). The highest perentge of hypootyls explnts prouing shoot (9.4%) on MS + mgl - BAP +. mgl - NAA n the highest men numer of shoots proue per hypootyls explnts (5.76) were otine on mgl - BAP. Menwhile, the highest perentge of shoot tip explnts proue shoot (%) n highest numer of shoot proue per shoot tip explnts (.56) were reore on mgl - BAP. For rooting of shoots, NAA t - mgl - were pplie. RAPD nlysis ws use to ientify the ifferenes mong regenernts from tissue ultures. The results of PCR prouts showe no ifferene etween ultures n emonstrte tht iret orgnogenesis is the most effetive wy to proue true-type regenernts in rooli hyri. A suessful limtiztion to the free onitions ws otine y trnsferring of plntlets into pots ontine mixture of petmoss n vermiulite (:) uner high reltive humiity onitions. Key wors: Brooli, in vitro ulture, hypootyl, enzylminopurine (BAP), npthleneeti i (NAA), RAPD.. Introution Brooli (Brssi olere L. vr. ejo) whih originte from Holln n is relte to the ge n uliflower, is very importnt vegetle rop. It is well known for its high vitmin, lium n sulforphne ontent.[] In vitro regenertion offers gret opportunity for rpi proution of esirle n essentilly genetilly ientil plnts. [] [] An effiient in vitro regernertion system is lso ruil tool in geneti engineering of the rop for improve hrteristis.[4] Brooli, Brssi olere is one of the mny vlule Brssi speies, whih is still less ulture uner in vitro onition.[5] Some experimentl results showe suessful in vitro ulture of Brssi speies from hypootyls segments, root segments, primry lef iss, otyleons n nther. [4] Among the Brssi speies inlue kle, Brussels sprouts, ge n uliflower.[6] In most Brssi speies, the suessful pplition of in vitro ulture is mostly epenent on the genotype n the influene of growth regultors. The ition of ytokinins, suh s kinetin or enzylenine woul enhne shoot prolifertion n root formtion.[7] Vrious onentrtions of uxins suh s nphthleneeti i (NAA), inoleutyri i (IBA) n inoleeti i (IAA) hve een evlute for rooting of in vitro regenerte shoots of rooli n uliflower.[5], [8] This pper reports on the influene of BAP either singly or in omintion with NAA, on ventitious shoot prolifertion from hypootyls n shoot-tips explnts of rooli v. ejo, n the effet of of α- nphthleneeti i (NAA) on rooting of the in vitro regenerte shoots. Corresponing uthor. Tel.: E-mil ress: htitin557@gmil.om. 57

2 . Mterils n Methos.. Plnt Mteril n Steriliztion Protool Sees of rooli were surfe sterilize for min in 7% ethnol solution followe y ontinuous gittion for 5 min in % Clorox solution (.5% soium hypohlorite) e with two rops of Tween. The sees were rinse three to four times in sterile istille wter n ulture on germintion meium ontining hlf-strength MS slts supplemente with.5 gl - gr n gl - surose... Meium Composition n Tretment Hypootyl n shoot tip explnts, 5-8 mm in size, were exise from 6-y-ol rooli seelings. The explnts were ulture on MS (Murshige ns Skoog, 96) meium inorporte with ifferent onentrtions of plnt growth regultors for shoot prolifertion n root formtion.[9] For shoot inution n multiplition from hypootyls explnts vrious onentrtions of BAP (,,, 5, 7 n mg L - ) were test. While for shoot tip explnts, BAP t,,, 5 n 7 mgl- either lone or in omintion with.5 n mgl - NAA were ssesse NAA t,.,.5 n mgl - were use for inuing root formtion. The mei were soliifie with.5 gl - gr n the ph juste to 5.8 prior to utolving t o C for 5 min. MS meium without ny growth regultor (MS ) ws onsiere s ontrol... Prmeter Reore In the shoot inution n multiplition stuy from hypootyls n shoot tip explnts, prmeters reore were the perentge of explnts prouing shoots (%). An the men numer of shoots proue per explnt. Dt were ollete fter eight weeks of ulture, while growth hrteristis were oserve every week. In the rooting stuy, the prmeters reore were the perentge of explnts prouing root (%), the men numer of roots proue per explnts n the foot length ttine (m). the t on rooting were ollete fter four weeks of ulture. The experiments were rrnge in Rnomize Complete Blok Design (RCBD), with three replitions n eh replition per tretment ontine explnts. Dt were nlyze using the Anlysis of Vrine (ANOVA) n Dunn New Multiple Rnge Test (DNMRT) t α = 5% for omprison etween tretment mens.. Results n Disussion.. Multiple shoot formtiom from hypootyls explnts In the first week of ulture the hypootyls explnts egn to expn n swelle in ll BAP onsentrtions teste. Within three to four weeks of ulture shoot ppere in the mile n istl en of the hypootyls explnts. By the eight week of ulture, signifint ifferenes in the perentge of explnts with shoot n men numer of shoot proue per explnts were oserve mong the BAP tretments. The highest perentge of hypootyls explnts prouing shoot (9.4%) on MS + mgl - BAP +. mgl - NAA. BAP t mgl - inue the highest perentge of explnts prouing shoots (9.4%) followe y 5 mgl - BAP (8.%). There ws no signifint ifferene oserve etween the two tretments ( n 5 mgl - BAP) on perentge of explnts prouing shoots. However, tretment mgl- BAP showe signifint ifferene ompre to the ontrol n the rest of the tretments. BAP t mgl - inue the highest men numer of trnsferle shoots per explnt fter eight weeks of ulture. It is iffere signifintly ompre to the rest of tretments (Fig. ). BAP is most effetive in enhning shoot multiplition n triggering shoot elongtion.[] BAP lso promotes ifferentition of ell in to shoot initils followe y the formtion of shoots. Aove 5 mgl - BAP the min numer of shoots forme per explnts erese n eome toxi to the shoot growth. 58

3 Men numer of shoot per explnt e e e e e e e e e e e e e e e Tretment Fig. : Effet of ifferent onentrtions of BAP n NAA on men numer of shoot proue per hypootyl explnts fter eight weeks of ulture. Men with the sme letter were not signifintly ifferent t.5 proility level oring to DNMRT test... Multiple shoot formtion from shoot tip explnts Fig. shows the stges of multiple shoot formtion from shoot tip explnts of rooli. It ws oserve tht the shoot tips elongte fter the first week of ulture. By the four week of ulture multiple shoots were forme, showe signifint ifferenes with shoot ulture on the ontrol meium. The highest perentge of explnts forming shoots (%) ws otine in tretment ontining mgl - BAP whih showe signifintly ifferene ompre to the ontrol meium (MS meium without plnt growth regultor, MS ), 5 mgl - BAP, mgl - NAA n 7 mgl - BAP + mgl - NAA BAP t mgl - signifintly enhne the men numer of shoots proue per explnts n ws signifintly ifferent ompre to the ontrol meium tretments. The highest perentge of shoot tip explnts proue shoot (%) n highest numer of shoot proue per shoot tip explnts (.56) were reore on mgl - BAP (Fig. ). The itions of.5 n mgl - NAA in the BAP ontining mei erese the men numer of shoot proue. Aoring to Wiiynto n Erytin () highest shoot formtion in rooli ws foun in ulture ontining μm ( mgl - ) BA. Cytokinin t reltively high onentrtions promote u formtion.[] BAP lone ws more effetive on inuing shoot prolifertion from the rooli hypootyl n shoot-tip explnts.[] The wie rnge of BAP onentrtions without NAA influene uxiliry shoot prolifertion on shoot tips of rooli. Cllus formtion ws lso oserve on mei ontining omintions of BAP n NAA. Men numer of shoot per explnt e e e e e e e e e Tretment Fig. : Effet of ifferent onentrtions of BAP n NAA on men numer of shoot proue per shoot tip explnts fter eight weeks of ulture. Men with the sme letter were not signifintly ifferent t.5 proility level 59

4 oring to DNMRT test. Men numer of shoot of lef explnt Tretment Fig. : Effet of ifferent onentrtions of BAP n NAA on men numer of shoot proue per lef explnts fter eight weeks of ulture. Men with the sme letter were not signifintly ifferent t.5 proility level oring to DNMRT test... Multiple shoot formtion from lef explnts In the first week of ulture the lef explnts egn to expn n swelle in ll BAP onsentrtions teste. Within three to four weeks of llus ulture ppere in the ege of the lef explnts. It ws oserve tht the ventious shoot showe fter the first week of ulture. By the ten weeks of ulture multiple shoots were forme, showe signifint ifferenes with shoot ulture on the ontrol meium. The highest perentge of explnts forming shoots (%) ws otine in tretment ontining 5 mgl - BAP whih showe signifintly ifferene ompre to the ontrol meium, 5 mgl - BAP, mgl - NAA n 7 mgl - BAP + mgl - NAA (Fig. ).4. Effet of NAA on rooting of rooli shoots Shoot, m in height n with three or more leves, were trnsferre to MS meium ontining ifferent onentrtion of NAA. By the fourt week of ulture, it ws oserve tht tretment ontining. mgl - NAA n ontrol (MS ) gve the highest perentge of explnt prouing roots n men numer of roots proue per explnts. Aoring to Ke Hung et l () the est regenertion reipe for rooli hypootyl explnt is on meium MS + NAA.7 μm + BAP 7.76 µm + % surose +.8% gr. Roote plntlets were suessfully limtize in potting meium ontining petmoss n vermiulite (:) n grew nturlly in the greenhouse. The survivl rte of regenerte plnts ws %..5. Moleulr Anlysis Moleulr nlysis y using RAPD (Rpi Amplifie Polymorphi DNA) tehnique showe tht UBC5, ROTH 48., n ROTH 48. primer hve polymorphi n tht n e use for linkge nlysis. All polymorphisms regenernts inepenently of eh ther. Spesifi RAPD mrker of Roth n UBC primers for hyri of Brssi olere vr. Bejo regenernts. RAPD nlysis ws use to ientify the ifferenes mong regenernts from tissue ultures. The results of PCR prouts showe no ifferene etween ultures n emonstrte tht iret orgnogenesis is the most effetive wy to proue true-type regenernts in rooli hyri. 4. Conlusions The results inite tht hypootyls, shoot-tips, n lef re potentil explnts for in vitro shoot multiplition of rooli (Brssi olere vr.ejo). The highest perentge of hypootyls explnts prouing shoot (9.4%) on MS + mgl - BAP +. mgl - NAA n the highest men numer of shoots proue per hypootyls explnts (5.76) were otine on mgl - BAP. Menwhile, the highest perentge 6

5 of shoot tip explnts proue shoot (%) n highest numer of shoot proue per shoot tip explnts (.56) were reore on mgl - BAP. For rooting of shoots, NAA t - mgl - were pplie. Highest perentge of shoots with roots (%) n highest men numer roots proue per shoot (5.5) ourre on meium with. mgl - NAA, while the mximum root length (.9 m) ws ttine on MS meium without plnt growth regultor (MS ). BAP lone ws more effetive on inuing shoot prolifertion from the rooli hypootyls, shoot-tip n lef explnts. The est regenertion reipe for the rooli is on meium MS + BAP mgl -. NAA. mgl - is selete s the most suitle onentrtions fot the root initition. Regenerte plnts survive n grew normlly in the green house. Bse on this menu, the regenertion frequeny of rooli rehe %. 5. Aknowlegements This work ws supporte y Plnt Biotehnology Reserh Projet of Inonesin Government. I woul like to thnk PT Agrout Bnung Inonesi who hve given support rooli sees. 6. Referenes [] Henzi, MX. Christey MC, MNeil DL (). Ftors tht influene Agroterium rhizogenes-meite trnsformtion of rooli (Brssi olere L. vr. itli). Plnt Cell Rep. 9: [] Lzzeri PA, Dunwell JM (986). In vitro regenertion from seeling orgns of Brssi olere vr. Itli PLenk v. Green Comet. I. Effet of plnt growth regultors. Ann. Bot. 58: [] Msikit W, Skirvin RM (989). In vitro regenertion from Hypootyl n seeling otyleons of trohun (Brssi olere L. vr. trohun Biley). Plnt Cell Tissue Orgn Cult. 9: [4] Co J, Erlie ED (). Trnsgene expression in rooli (Brssi olere L. vr.itli) lones propgte in vitro vi lef explnts. Plnt Cell Rep. : [5] Wiiynto SN n Erytin D (). Clonl propgtion of rooli (Brssi olere L. vr. itli) through in vitro shoot multiplition. J. Me. Si 6(): -. [6] Qin Y, Li HL, Guo YD (6). Hogh-frequeny emryogenesis, regenertion of rooli (Brssi olere vr. itli) n nlysis of geneti stility y RAPD. Si. Hort. :-8. [7] Arnison PG, Donlson P, Jkson A, Semple C, Keller W (99). Genotype-speifi response of ulture rooli (Brssi olere vr. itli) nthers to ytokinins. Plnt Cell Tissue Orgn Cul. :7-8. [8] Vnemoortele JL, Billir JP, Bouu J, Gspr T (999). Eviene for n intertion etween sl meium n plnt growth regultors uring ventious or uxiliry shoot formtion of uliflower. In Vitro Cell. Dev. Biol. Plnt. 5:-7. [9] Murshige T, Skoog F (96). A revise meium for rpi growth n iossys with too tissue ultures. Physiol Plnt. 5(): [] Ke Hung, Qiuyun Wu, Junheng Lin, Jingui Zheng (). Optimiztion of plnt regenertion protool for rooli. Afrin Journl of Bioteh. Vol.(): [] Hrtmnn DE, Dvis FT, Geneve FL (7). Plnt Propgtion. Priniples n Prties. Lonon: Prentie Hll, In. [] Rvnfr SA, Aziz MA, Kir MA, Rshi AA, Sirhi MHT (9). Plnt regenertion of Brssi olere susp.itli (Brooli) CV Green Mrvel s ffete y plnt growth regultors. Afrin Journl of Bioteh. Vol. 8():