ELIMINATION OF CITRUS TRISTEZA CLOSTEROVIRUS (CTV) AND PRODUCTION OF CERTIFIED CITRUS PLANTS THROUGH SHOOT-TIP MICROGRAFTING

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1 Pk. J. Bot., 40(3): , ELIMINATION OF CITRUS TRISTEZA CLOSTEROVIRUS (CTV) AND PRODUCTION OF CERTIFIED CITRUS PLANTS THROUGH SHOOT-TIP MICROGRAFTING M. ABBAS 1, M.M. KHAN 1*, B. FATIMA 1, Y. IFTIKHAR 1, S.M. MUGHAL 1, M.J. JASKANI 1, I.A. KHAN 1 AND H. ABBAS 2 1 Institute of Hortiulturl Sienes, University of Agriulture, Fisld-38040, Pkistn. 2 Dr. A.Q. Khn Institute of Biotehnology nd Geneti Engineering, University of Krhi, Krhi-75270, Pkistn Astrt Kinnow mndrin (Citrus retiult) nd Musmi sweet ornge (C. sinensis) re the predominnt itrus fruits ultivted in Pkistn. Citrus speies re highly vulnerle to mny types of pthogens. Among the viruses, Citrus tristez virus (CTV) is the most devstting whih n use the deth of millions of itrus trees if one estlished. CTV is grft-trnsmissile nd n e indvertently propgted through infeted udwood. The prodution of ertified nd CTV-free itrus plnts ould e helpful to restrit the widespred of CTV. The study ws designed to optimize the mirogrfting tehnique for the propgtion of CTV free Kinnow mndrin nd Musmi sweet ornge plnts under septi onditions. The MS medium supplemented with 5 mgl -1 BA ws found etter for suessful mirogrfting of oth itrus ultivrs on rough lemon seedlings. The survivl rte of mirogrfted itrus plntlets ws 88% when trnsferred to soil. The ELISA results showed tht more tht 90% Kinnow nd Musmi plnts either grfted on rough lemon or sour ornge rootstok were found free from CTV. The foundtion lok of mndrin nd sweet ornge free from CTV ws suessfully estlished using shoot-tip mirogrfting tehnique. Introdution Citrus represents one of the most importnt nd widely grown fruits in the world nd Pkistn is one of the mjor itrus produing ountries. Citrus ws ultivted on hetres with nnul prodution of tonnes during (Anon., 2006). Sine the introdution of Kinnow mndrin (Citrus retiult Blno.), its prodution hs een inresing stedily to fulfill the growing demnds in the ountry nd rod (Khn, 1992). Citrus speies re highly vulnerle to mny types of pthogens. The itrus groves in generl re fing serious prolem of deline tht is ttriuted to different uses. The mjor use, however, is the prevlene of itrus virus nd virus-like diseses whih hve signifint impt nd often eome mjor onstrint in itrus prodution. In Pkistn, the itrus plnttion hs hd shorter produtive life whih ulmintes in low yields nd deteriortion in fruit qulity. Chpot (1970) reported virl diseses in Pkistn for the first time nd oserved diek nd deline symptoms in lrge numer of itrus trees ut he ws not sure out the use of these symptoms. Lter on susequent surveys of itrus groves t numer of lotions reveled the presene of severl virus nd viruslike diseses of itrus in Pkistn (Ctr et l., 1988; Bove, 1995). Anwr & Mirz (1992) onfirmed the presene of Citrus tristez losterovirus (CTV) in itrus trees on the sis of survey nd the enzyme-linked immunosorent ssy (ELISA). * Corresponding uthor: mumtz59pk@hotmil.om

2 1302 M. ABBAS ET AL., The strtegies to ontrol CTV my differ ording to the inidene nd severity of its strins in eh re nd with the ultivrs nd rootstoks used. There is no single ontrol strtegy for the CTV tht will e pplile in ll situtions (Grnsey et l., 2005).The pproh to use CTV tolernt rootstoks n lso help to ontrol the CTV indued losses. Tolerne to CTV is seen in the ility of rootstok phloem to withstnd the presene of the virus without dverse effets on norml ellulr nd physiologil funtions (Bordignon et l., 2004). The most effetive mens of limiting CTV-indued disese is through ertifition progrmme in whih ud wood soure trees re tested nd mintined free of CTV (Grnsey et l., 2005). The shoot-tip mirogrfting is known tehnique for the potentil elimintion of virus nd viroid pthogens from itrus germplsm. It is generlly utilized in qurntine nd ertifition progrms using smll trees mintined under glss or sreenhouses (Krueger et l., 2003). Mirogrfting tehnique is importnt minly for woody speies euse in suh plnts meristem-tip ulture is often diffiult (Fioli & Mrni, 2005). This tehnique ws used for elimintion of different viruses from itrus speies (Murshige et l., 1972; Nvrro et l., 1975). The spets of shoot-tip grfting nd its pplition on itrus virus reserh were omprehensively reviewed y Nvrro (1981). The proedure onsists of preprtion of rootstok nd sion mterils, grfting, ulture in vitro of grfted plntlets nd trnsfer to soil. The study hs een tken up to stndrdize the different ftors ffeting shoot tip grfting in C. retiult v. Khsi mndrin (Mdhv et l., 2001). Higher rte of suess (60%) ws otined in shoot tip grfting when 2- week old etiolted seedling of trifolite ornge ws used s rootstok. Sion size of mm (meristem dome plus three lef primordil) ws found to e optimum for grfting. The suessful grfted plntlets were susequently trnsferred to soil nd 90% plntlets were estlished in the soil. These plntlets were mintined in phid proof ge to prevent re-infetion of virus through phids. Preliminry results of indexing indited reovery of plntlets free from CTV nd exoortis. Similrly, the shoot-tip grfting tehnique ws lso found 100% effiient in eliminting the CTV, Citrus exoortis viroid (CEVd) nd hexi-xiloporosis viroid from vrieties of the itrus germplsm nk t the Centro de Citriultur Sylvio Moreir CCSM-IAC (Crvlho et l., 2002). However, 60% effiieny ws oserved in eliminting the itrus psorosis virus omplex. The use of mirogrfting lone without indexing will not gurntee freedom from viruses in propgtive udwood. This n e sertined only when the tehniques of pthogen elimintion re omined with omprehensive indexing progrmme (Roisther, 1988). The present study ws designed to optimize the shoot-tip mirogrfting tehnique for rpid propgtion of itrus plntlets in vitro to estlish foundtion lok of ertified itrus plnts of Kinnow mndrin (C. retiult) nd Musmi sweet ornge (C. sinensis). The indexing of STG plntlets were onduted through diret ntiody sndwih enzyme-linked immunosorent ssy (DAS-ELISA). Mterils nd Methods Shoot-tip mirogrfting: Mirogrfting ws employed for the prodution of CTV-free itrus plnts to develop len foundtion lok for future propgtion. Shoot-tip grfting ws used s desried y Nvrro et l., (1975) nd Nvrro (1981). Rough lemon (C. jmhiri) nd sour ornge (C. urntium) were used s rootstok, nd sions were tken from CTV-infeted tissues of mndrins nd sweet ornges. The sterilized seeds of rough lemon nd sour ornge were germinted in test tues ontining MS medium (Murshige & Skoog, 1962) solidified with 1% togr. One seed ws sown

3 ELIMINATION OF CTV THROUGH SHOOT-TIP MICROGRAFTING 1303 per tue nd mintined t 25±2 o C for two weeks under ontinuous drkness. The shoot tips were otined from xillry uds of CTV infeted Kinnow mndrin (C. retiult) nd Musmi sweet ornge (C. sinensis). The leves were removed from twigs whih were ut into setions ontining 2-3 xillry uds. The setions were rinsed with 95% ethnol followed y wshing under running tp wter to remove dust. Then dipped for 10 minutes in 0.5% Sodium hypohlorite (NOCl) followed y 3 times wshing with sterilized distilled wter. The sions were exised from these sterilized setions. Two weeks old rough lemon nd sour ornge rootstok seedlings were removed from test tues nd depitted to remove ll leves. The otyledons nd their xillry uds were lso dethed. The upright ut ws mde on the entrl prt of the rootstok nd the sion with V shpe se exised from the sterilized twig setions ws fit into the ut. The grfted plntlets were ultured in MS medium supplemented with different levels (0, 1, 3 nd 5 mg) of Benzyl Adenine (BA). The grfted plnts were exposed to 16 hour dily 1000-lux illumintion nd mintined t the temperture of 25±2 o C. The grfted plnts hving t lest two expnded leves in the sion were trnsferred to plsti pots ontining medium of grden soil, snd nd ompost (3:1:1). The pots were overed with polythene gs to mintin humidity nd to void ny dmge from desition. One week lter the gs were opened nd fter seond week gs were removed. The grfted plnts were llowed to ontinue growth under glsshouse onditions (25±2 o C). The suessfully estlished plnts were trnsferred to plsti ontiners, filled with grden soil only nd fter hrdening the plnts were trnsferred to sreenhouse. CTV indexing: The grfted plnts in sreenhouse were ssyed through doule ntiody sndwih enzyme-linked immunosorent ssy (DAS-ELISA) for CTV infetion. The leves of survived itrus plnts were hrvested t the ge of 4-6 months for virus indexing. The ELISA proedure using CTV speifi polylonl ntiodies (Ctlogue No. 1102, Adgen, UK) ws performed s desried y Clrk & Adms (1977). The 96- well mirotiter pltes were oted with 200 µl of ntiodies (diluted t 1:200). The pltes were then overed nd inuted t 4 o C in refrigertor for over night. The pltes were wshed with PBS-Tween uffer three times t five minute intervls using ELISA plte wsher (Model: EL X 50, BIO-TEK USA). The tissues were extrted in extrtion uffer nd 200 µl of plnt extrt ws dded to duplite the wells. The pltes were inuted over night t 4 o C nd wshed ordingly. After wshing wells were hrged with 200µl of lkline phosphtse enzyme onjugte nd the pltes were inuted t 4 o C for over night. After wshing the pltes 200 µl liquots of freshly prepred sustrte uffer were dded to eh well nd the pltes were inuted t room temperture (25±2 o C) for minutes. The retion ws stopped y dding 50 µl of 3M NOH in eh well nd the sorne ws mesured t 405 nm in Universl Miroplte Reder (Model: EL X 800, BIO-TEK USA). The sorne vlue reorded three times higher ompred to helthy tissues ws onsidered s CTV positive (Lid et l., 2005). Dt olletion nd nlysis: The dt ws olleted for suessful grfts, dys to estlish union, dys to sprout 1 st lef nd perent of CTV elimintion (ELISA). The experiment for shoot-tip grfting ws lid out under ompletely rndomized design nd three replitions (100seeds/repet) with four tretments were used for eh rootstok. The dt olleted ws nlyzed sttistilly nd mens ws ompred using Dunn s Multiple Rnge (DMR) test (Steel et l., 1997).

4 1304 M. ABBAS ET AL., Results Mirogrfting suess: Highly signifint (p<0.05) differene were oserved mong the tretments for mirogrfting suess of mndrin nd sweet ornge sions on rough lemon rootstok (Fig. 1). The results reveled tht MS medium supplemented with 5 mgl -1 BA vried signifintly (p<0.05) from the remining tretments for suessful mirogrfting of oth itrus ultivrs on rough lemon seedlings. Among other tretments, MS medium supplemented with 3 mgl -1 BA ws found sttistilly signifint (p<0.05) for mirogrfting suess ompred to other tretments (Fig. 1). The MS medi supplemented with 1, 3 nd 5 mgl -1 BA were found sttistilly similr for sweet ornge sion grfted on sour ornge ut showed signifint (p<0.05) differene with ontrol (Fig. 2). The results were highly signifint (p<0.05) for mirogrfting suess of mndrin sions grfted on sour ornge rootstok seedlings (Fig. 2). The grfting suess ws signifintly (p<0.05) high in MS medium ontining 5 mgl -1 BA for mndrin sions grfted on sour ornge. The 3 mgl -1 of BA in MS medium ws found seond est tretment for suessful grfting of mndrin sion on sour ornge rootstok seedlings (Fig. 2). Dys to estlish union of sions on rootstok seedlings: The results were sttistilly signifint (p<0.05) for the numer of dys tken y sweet ornge sions for suessful union with rough lemon rootstok seedlings (Fig. 3). The MS medium without BA (ontrol) signifintly (p<0.05) took higher numer of dys for suessful grft union of sweet ornge sions with rough lemon. The sions tken from sweet ornges lso took more dys (sttistilly similr with ontrol) for suessful union with rough lemon rootstok, ultured in MS medium ontining 5 mgl -1 of BA. The sweet ornge grfted plntlets took less numer of dys for suessful sion-stok union when ultured in MS medium supplemented with 1 nd 3 mgl -1 of BA (Fig. 3). The results were nonsignifint for numer of dys tken y sions of oth ultivrs when grfted on sour ornge rootstok seedlings (Fig. 4). Dys to sprout first lef of sions grfted on In vitro grown rootstok: The results showed highly signifint (p<0.05) differenes mong the tretments for dys tken y sions of oth ultivrs mirogrfted on rough lemon rootstok to sprout first lef fter suessful union. The grfted mndrins ultured in MS medium (ontrol) nd MS medium supplemented with 3 mgl -1 of BA took mximum dys to sprout first lef (Fig. 5). The grfted mndrins took minimum numer of dys to sprout first lef ultured in MS medium ontining 5 mgl -1 BA. The sweet ornge sions grfted on rough lemon rootstok seedlings took minimum dys to sprout first lef when ultured in MS medium supplemented with different onentrtions of BA. The ulture medi supplemented with 1, 3 nd 5 mgl -1 BA showed signifint (p<0.05) differene from ontrol (Fig. 5). The results were highly signifint (p<0.05) for the numer of dys tken y sions of oth ultivrs grfted on sour ornge rootstok to sprout first lef. The mndrin sions took mximum dys to sprout first lef when ultured in MS + BA (3 mgl -1 ) nd MS + BA (5 mgl -1 ) medi (Fig. 6). The mndrin plntlets ultured in MS medium (ontrol) nd MS medium ontining 1 mgl -1 BA took minimum dys to sprout first lef. The differenes were highly signifint (p<0.05) for the sweet ornge sions grfted on sour ornge rootstok seedlings to produe their first lef (Fig. 6). The in vitro grown sweet ornge plnts took higher numer of dys to sprout first lef fter suessful grft union when ultured in MS medium (ontrol) nd MS medium ontining 3 mgl -1 BA. The sweet ornge plnts ultured in MS medium supplemented with 1 mgl -1 BA took minimum dys to sprout the first lef (Fig. 6).

5 ELIMINATION OF CTV THROUGH SHOOT-TIP MICROGRAFTING Mndrin Sweet ornge Mirogrfting sues (%) Control MS+BA(1 mgl ¹) MS+BA(3 mgl ¹) MS+BA(5 mgl ¹) Tretments Fig. 1. Suessful grfting of oth ultivrs on Rough lemon rootstok. Brs with different letters re signifintly different y DMRt (5% level) 100 Mndrin Sweet ornge Mirogrfting sues (%) d 0 Control MS+BA(1 mgl ¹) MS+BA(3 mgl ¹) MS+BA(5 mgl ¹) Tretments Fig. 2. Suessful grfting of oth ultivrs on Sour ornge rootstok. Brs with different letters re signifintly different y DMRt (5% level)

6 1306 M. ABBAS ET AL., 20 Mndrin Sweet ornge Numer of dys Control MS+BA(1 mgl ¹) MS+BA(3 mgl ¹) MS+BA(5 mgl ¹) Tretments Fig. 3. Dys to estlish union of oth ultivrs mirogrfted on Sour ornge. 20 Brs with different letters re signifintly different y DMRt (5% level) Mndrin Sweet ornge 15 Numer of dys Control MS+BA(1 mgl ¹) MS+BA(3 mgl ¹) MS+BA(5 mgl ¹) Tretments Fig. 4. Dys to estlish union of oth ultivrs mirogrfted on Rough lemon.

7 ELIMINATION OF CTV THROUGH SHOOT-TIP MICROGRAFTING Mndrin Sweet ornge 25 Numer of dys Control MS+BA(1 mgl ¹) MS+BA(3 mgl ¹) MS+BA(5 mgl ¹) Tretments Fig. 5. Dys to sprout first lef of oth sions grfted on Rough lemon rootstok. Brs with different letters re signifintly different y DMRt (5% level) Mndrin Sweet ornge Numer of dys Control MS+BA(1 mgl ¹) MS+BA(3 mgl ¹) MS+BA(5 mgl ¹) Tretments Fig. 6. Dys to sprout first lef of oth sions grfted on Sour ornge rootstok. Brs with different letters re signifintly different y DMRt (5% level)

8 1308 M. ABBAS ET AL., Sion Tle 1. Sttus of CTV in shoot-tip mirogrfted itrus plnts under sreenhouse. No. of Perentge Helthy No. of mirogrfted plnts Exmined (sion tree) Infeted/ Infeted Rootstok survived (%) of CTV (mirogrfted plnts free Plnts plnts) Mndrin (Kinnow) Sweet ornge (Musmi) RL / SO /89 91 RL / SO /50 94 Totl (88%) 26/ * Asorne vlue reorded on ELISA miroplte Reder RL: Rough lemon SO: Sour ornge Indexing of mirogrfted itrus plnts through ELISA: The mirogrfted itrus plnts with seven to nine fully expnded leves were trnsferred in inset proof netted sreenhouse. A totl of n=300 in vitro grown itrus plnts grfted on rough lemon rootstok omprising of mndrin (n=200) nd sweet ornge (n=100) were trnsferred to soil under sreenhouse. The in vitro grown itrus plnts (100 mndrin nd 50 sweet ornge) grfted on sour ornge rootstok were lso trnsferred to soil under sreenhouse. Out of 450 trnsferred itrus plnts 396 were survived whih showed survivl rte of 88% (Tle 1). The survived itrus plnts were indexed for CTV through ELISA. The results reveled tht more thn 90% mndrin nd sweet ornge plnts either grfted on rough lemon or sour ornge rootstok were found free from CTV. The overll perentge of CTV-free itrus plnts ws 93.43% (Tle 1). The foundtion lok of mndrin nd sweet ornge free from CTV ws suessfully estlished using shoot-tip mirogrfting tehnique. Disussion The present study ws designed to optimize the mirogrfting tehnique for the propgtion of CTV-free itrus plnts under septi onditions. The ojetives of optimizing this tehnique were to produe virus free itrus plnts, estlishment of foundtion lok of helthy plnts nd to provide helthy udwood to itrus growers, s reported y Nvrro et l., (1975) nd Nvrro (1976). The shoot-tip mirogrfting protool ws stndrdized keeping in view the limited ville resoures (hemils nd tissue ulture filities). Two rootstoks (rough lemon nd sour ornge) nd two sions (kinnow mndrins nd musmi sweet ornge) were evluted for mirogrfting tehnique. The results suggested tht inresing onentrtion of BA in MS medium signifintly effet the mirogrfting perentge of mndrin nd sweet ornge sions grfted on rough lemon rootstok. The grfting suess ws signifintly inresed with the inrese of BA onentrtion in MS medium for the mndrin sions grfted on sour ornge rootstok. However, mirogrfting suess ws not onsiderly ffeted y different onentrtions of BA in MS medium in se of sweet ornge grfted on sour ornge rootstok. The results reported in this study for mirogrfting suess in mndrin grfted on rough lemon nd sour ornge nd sweet ornge grfted on rough lemon re ontrry to Nvrro et l., (1975). However, in the present study, BA did not influene the grfting suess in sweet ornge grfted on

9 ELIMINATION OF CTV THROUGH SHOOT-TIP MICROGRAFTING 1309 sour ornge whih is in ordne with Nvrro et l., (1975). The mirogrfting suess perentge reported here ws high in sweet ornge ompred to mndrin grfted on rough lemon rootstok nd this is in ordne with Vijykumri et l. (2006) who reported the mximum grfting suess in sweet ornge followed y mndrin. The present findings suggested tht ddition of BA in MS medium did not signifintly ffet the numer of dys tken y oth ultivrs either grfted on rough lemon or sour ornge. However, minimum dys were tken y mndrin nd sweet ornge grfted on rough lemon to sprout first lef with the inrese in level of BA in MS medium ut when mndrin sions were grfted on sour ornge numer of dys tken to sprout first lef were inresed with the inrese in BA onentrtion. Mny ftors ffet the grfting suess, dys to estlish grft union nd sprouting of first lef in mirogrfetd itrus plnts. The frequeny of grfting suess nd other ftors n e ffeted y type of sion nd rootstok used. The grfting suess lso depends on rootstok ge nd on extent of tissue differentition. All these onditions n e vried with different rootstoks used in mirogrfting of itrus plnts (Nvrro, 1981). In ddition genotype n lso signifintly ffet the suessful mirogrfting of itrus plnts whih rnged from 5-40% (Murshige et l., 1972). In this study, the onentrtion of BA signifintly effet the dys tken y sions to produe their first lef nd it hs een well reported tht ytokinins like BA re often used to stimulte growth nd development of in vitro grown plntlets (Chwl, 2000). The mirogrfting tehnique ws suessfully pplied for the elimintion of CTV in mndrins nd sweet ornges. This tehnique ws suessfully nd effiiently used for elimintion of different viruses from itrus speies (Murshige et l., 1972; Nvrro et l., 1975; Nz et l., 2007). Similrly, Vijykumri et l., (2006) used mirogrfting in qurntine greenhouse to produe the len stok of diverse itrus germplsm to strengthen the itrus gene nk. The shoot-tip grfted itrus plnts were found 76 nd 86.95% negtive for CTV nd greening disese, respetively. However, perentge of CTV free itrus plnts reported in this study ws 93.43%. The present study suggested tht 88% mirogrfted itrus plnts were suessfully estlished under sreenhouse onditions, theses results re ontrry to Mdhv et l., (2001) who reported 90% mirogrfted mndrin plntlets were estlished in the soil. Reently, Rez et l., (2007) reported tht 100% CTV elimintion in sweet ornges nd 90% survivl rte when trnsferred to greenhouse. In present studies less suess rte for survivl of trnsferred plntlets ws oserved, whih suggested tht the utmost re should e tken t the stge of limtiztion nd doption under sreenhouse onditions. Similrly, our results re similr to Crvlho et l., (2002) tht the mirogrfting tehnique ws found effiient to suessfully eliminte CTV. They reported tht 100% mirogrfted plnts were CTV free, however, in our se this perentge ws 93.43%. This is euse the shoot-tips used in this study were of lrge size, whih n ffet the perentge of virus elimintion in mirogrfted plnts (Surez et l., 2005). The reson for use of lrge size shoot-tips is tht it enhned the ud uptke with etter mirogrfting suess rte for the people who re less experiened in this tehnique (Shrm et l., 2007). The CTV ws suessfully eliminted through this tehnique without the omintion of thermotherpy with mirogrfting. However, it is well reported tht the proess of elimintion of some viruses nd viroids n e improved y using thermotherpy oupled with shoot-tip grfting. The omintion of thermotherpy nd shoot-tip grfting (STG) is reommended for the elimintion of ll known mjor itrus

10 1310 M. ABBAS ET AL., pthogens (Roisther, 1988). The re-indexing of 427 plnts reovered y shoot tip grfting ssoited with thermotherpy showed 100% suess in eliminting Citrus tristez virus, exoortis, psorosis nd hexi/xyloporosis (Crvlho et l., 2001). However it is importnt tht udwood e thoroughly indexed efore nd fter therpy. The results re lso similr to Sertky (2004) tht is good numer of itrus plnts rehed to the size of indexing for virus diseses in short time y mirogrfting method. Mny tehniques esides mirogrfting re now ville whih n effiiently e used for the propgtion of virus free itrus plnts. Miroudding tehnique ws lso found to e effiient for prodution of CTV-free sweet ornge (As et l., 2006). However, perentge of virus free itrus plntlets is low euse the sion wood used in miroudding ws of lrge size. Ghzvini et l., (2001) produed CTV-free itrus plnts using unfertilized ovules of suspeted CTV-infeted plnts y under septi onditions. The In vitro itrus plnts when udded on lime (inditor host) did not show ny CTV positive retion. Similrly, somti emryogenesis ws found to e very promising tehnique for the prodution of virus-free itrus plnts (D'Onghi et l., 2001). Citrus psorosis virus CPsV ws not deteted in ny of the mndrin nd sweet ornge plnt, otined using somti emryogenesis even fter 24 months regenertion. The use of thermotherpy or STG lone without indexing will not gurntee freedom from viruses in propgtive udwood. This n e sertined only when the tehniques of pthogen elimintion re omined with omprehensive indexing progrmme (Roisther, 1988). The shoot-tip mirogrfting (STG) is well estlished tehnique for the potentil elimintion of virus nd viroid pthogens from itrus germplsm. It is generlly utilized in qurntine nd ertifition progrms using smll trees mintined under glss or sreen (Krueger et l., 2003). Shoot-tip mirogrfting tehnique ws suessfully optimized nd employed for the rpid propgtion virus-free itrus nursery plnts. This tehnique suessfully produed the CTV-free itrus plnts for the estlishment of foundtion lok of helthy mndrin nd sweet ornge plnts to provide helthy udwood to itrus growers. Aknowledgement The uthors highly knowledge the finnil support of Ministry of Siene nd Tehnology, Government of Pkistn for the ompletion of this study. Referenes As, M., M.M. Khn, S.M. Mughl, M.J. Jskni nd H. As Propgtion of CTV-free sweet ornge [Citrus sinensis (L.) Osek] plnts through miroudding tehnique. Pk. J. Bot., 38(3): Anonymous Fruit, vegetles nd ondiments sttistis of Pkistn Government of Pkistn, Ministry of Food, Agriulture & Livestok (Eonomi Wing), Islmd. pp Anwr, S.M. nd S.M. Mirz Survey of itrus tristez virus in Punj. In: Proeedings 1 st Int. Seminr on Citriulture in Pkistn. University of Agriulture Fisld, Pkistn, Bordignon, R., H.P. Medin-Filho, W.J. Siqueir nd J.T. Sorinho The genetis of tolerne to tristez disese in itrus rootstoks. Gen. Mol. Biol., 27(2): Bove, J.M Virus nd virus-like disese of itrus in the Ner Est region. FAO, Rome Crvlho, S.A., F.A. Sntos nd M.A. Mhdo Psorosis virus omplex elimintion from itrus y shoot-tip grfting ssoited to thermotherpy. Fitoptol. Brsileir., 27(3):

11 ELIMINATION OF CTV THROUGH SHOOT-TIP MICROGRAFTING 1311 Crvlho, S.A., M.A. Mhdo, G.W. Müller nd H.D. Colett Filho Bsi udwood for prodution of helthy itrus nursery-trees in São Pulo Stte. Lrnj., 22(1): Ctr, A., A. Azzro, S.M. Mughl nd D.A. Khn Virus, viroids nd prokryoti diseses of itrus in Pkistn. In: Proeedings 6 th Int. Congress of IOCV., pp Chpot, H Les prolèmes de l prodution des grumes en Prohe-Orient t en Afrique du Nord. Prog. Des Ntions Unies le Develop. FAO, Rome, Chwl, H.S Nutrition medium. Introdution to Plnt Biotehnology. Oxford & IBH Pulishing Co. Pvt. Ltd. New Delhi, Indi. pp Clrk, M.F. nd A.N. Adms Chrteristis of the miroplte method of enzyme-linked immunosorent ssy for the detetion of plnt viruses. J. Gen. Virol., 34: D'Onghi, A.M., F. Crimi, F. depsqule, K. Djelouh nd G.P. Mrtelli Elimintion of Citrus psorosis virus y somti emryogenesis from stigm nd style ultures. Plnt Pthol., 50(2): Fioli, G. nd F. Mrni Virus elimintion y meristem tip ulture nd tip mirogrfting. In: Plnt Virus Disese Control. (Eds.): A. Hdidi, R.K. Khetrpl, H. Kognezw. Seond Printing, APS Press, St. Pul. Minn. pp Grnsey, S.M., T.R. Gottwld nd R.K. Yokomi Control strtegies for itrus tristez virus. In: Plnt Virus Disese Control. (Eds.): A. Hdidi, R.K. Khetrpl, H. Kognezw. Seond Printing, APS Press, St. Pul. Minn. pp Ghzvini, R.F., Y. Hmmidoghli nd Y. Erhimi Prodution of virus-free lones from unfertilized ovules of Stsum mndrin ultivrs. J. Si. Teh. Agri. Nturl Resoures, 5(2): Khn, I.A Virus nd virus-like diseses of itrus. In: Proeedings 1 st Int. Seminr on Citriulture in Pkistn. University of Agriulture Fisld, Pkistn, Krueger, R.R., T.L. Khn, D.J. Gumpf, P.M. Bllne, O.J. Bier nd J.A. Bsh Shoot-tip miro grfting for resue or therpy of field-grown itrus germplsm essions. In: Proeedings 9 th ISC Congress (Astrt). Lid, B., A. Bennni, M.N. Serrhini nd M. Zemzmi Biologil, serologil nd moleulr hrteriztion of three isoltes of Citrus tristez losterovirus introdued into Moroo. EPPO Bulletin, 35(3): Mdhv, M.S., B.N Ro, S. Singh nd P.C. Dek In vitro shoot tip grfting in Citrus retiult Blno v. Khsimndrin. Res. Crops, 2(3): Murshige, T. nd F. Skoog A revised medium for rpid growth nd iossy with too tissue ulture. Physiol. Plnt., 15: Murshige, T., W.P. Bitters, E.M. Nver, C.N. Roisther nd P.B. Holidy A tehnique of shoot tip grfting nd its utiliztion towrds reovering virus-free itrus lones. Hort. Si., 7: Nvrro, L The itrus vriety improvement progrm in Spin. In: Proeedings 7 th Int. Congress of IOCV., pp Nvrro, L Citrus shoot-tip grfting In vitro (STG) nd its pplitions: review. Pro. Int. So. Citriulture., pp Nvrro, L., C.N. Roisther nd T. Murshige Improvement of shoot-tip grfting in vitro for virus-free itrus. J. Amer. So. Hort. Si., 100: Nz, A.A., M.J. Jskni nd H. As In vitro studies on mirogrfting tehnique in two ultivrs of itrus to produe virus free plnts. Pk. J. Bot., 39(5): Rez, F., B. Golein, H. Theri nd Y. Tdjvr Elimintion of Citrus tristez virus of Wshington Nvel ornge (Citrus sinensis [L.] Osek) through shoot-tip grfting. Int. J. Agri. Biol., 9(1): Roisther, C.N Conepts in detetion nd ontrol of itrus virus nd virus-like diseses. In: Proeedings 6 th Int. Citrus Congress Middle-Est. Int. So. Cit., pp Sertky, G Effets of different rootstoks in mirogrfting on growing of wshington nvel ornge plnts otined y shoot tip grfting. Biotehnology & Biotehnologil Equip., 18(2):

12 1312 M. ABBAS ET AL., Shrm, S., B. Singh, G. Rni, A.A. Zidi, V. Hlln, A. Ngpl nd G.S. Virk Prodution of Indin itrus ringspot virus-free itrus plnts of Kinnow mndrin (Citrus noilis Lour x C. deliios Tenor) employing phytotherpy oupled with shoot tip grfting. In Vitro Cell Dev. Biol., 43: Steel, R. G.D., J.H. Torrie nd D. Dikey Priniples nd proedures of sttistis: iometril pproh. 3rd Ed. MGrw Hill Book Co. In., New York. Surez, I.E., R.A. Shnell, D.N. Kuhn nd R.L. Litz Mirogrfting of ASBVd-infeted Avodo (Perse merin) plnts. Plnt Cell, Tissue Orgn Cult., 80: Vijykumri, N., D.K. Ghosh, A.K. Ds, A. Singh nd S. Singh Elimintion of itrus tristez virus nd greening pthogens from exoti germplsm through in vitro shoot tip grfting in itrus. Ind. J. Agri. Si., 76 (3): (Reeived for pulition 31 Mrh 2008)