EMBRYO CULTURE TO ENHANCE EFFICIENCY OF COLCHICINE INDUCED POLYPLOIDIZATION IN GRAPEFRUIT

Transcription

1 Pk. J. Bot., 44: , Speil Issue Mrh EMBRYO CULTURE TO ENHANCE EFFICIENCY OF COLCHICINE INDUCED POLYPLOIDIZATION IN GRAPEFRUIT MUHAMMAD USMAN*, BILQUEES FATIMA, MUHAMMAD USMAN, WAQAR ABDUS SAMAD AND KARIM BAKHSH Institute of Hortiulturl Sienes, University of Agriulture, Fisl * Corresponing uthor s e-mil: musmn74@gmil.om Astrt Different ultivrs of grpefruit (Citrus prisi L.) were explore for olhiine inue polyploiiztion using emryo ulture on MS meium. Emryo germintion ws foun to e highly genotype epenent on olhiine ontining MS meium. The highest numer of germinte emryos per see ws oserve in vs. Shmer n Frost Fresh 2.89 n 2.27, respetively, ompre to other ultivrs. Tretment of ntimitoti gent olhiine rreste shoot growth in most of the ultivrs wheres numer of leves ws signifintly higher in v. Foster n Re Mx Cne Foster (6.15 n 4.87, respetively) ompre to ontrol (2.17 eh). Rtio lef lmin ws lso higher in se of trete plnts ompre to ontrol. Numer of stomt erese (11) while length n with of the stomt inrese (2.9 n 2.4, respetively) in leves of trete plnts ompre with ontrol (2.3 n 1.7, respetively) plnts in v. Shmer. These puttive hyperploi plnts will e further onfirme for ploiy using hromosoml stuies. The plnts were shifte to greenhouse fter limtiztion for further geneti hrteriztion of ploiy n nuellr or zygoti origin of the plnts. Introution Citrus inustry of Pkistn is ontriuting 1% of fresh fruit in totl proution from 2% re uner Citrus ultivtion roun the worl yieling merely 12.7 tons per hetre. Citrus provies mjor stke to the fruit inustry of this ountry ontriuting 25% of totl fruit proution out of whih 95% shre is oming from Punj (Anon., 2010). Citrus inustry is minly epenent upon Mnrins inluing Kinnow n Feutrell s Erly overing 80% of totl re uner Citrus (Usmn et l., 2002). Grpefruit is n importnt memer of Citrus n offers plenty of vitmin C, higher mount of β-rotene tht oy onverts to vitmin A n hnful mount of spermiine tht prolongs ell geing proess. Progress in Citrus inustry is hnippe y nrrow geneti se, lternte ering, high numer of sees, post hrvest losses n numer of ioti n ioti stress. Puli likeliness towrs seeless ultivrs is rising n to e ompetitive in the glol Citrus inustry we nee to evelop our own seeless ultivrs for instne in Grpefruit whih is one of the lest explore n unerutilize Citrus memer in Pkistn. Suess stories of seelessness in the worl inlue Mrsh seeless, Thiti lime n mny others. Seelessness through triploiy is ommonly inue y interploi hyriiztion followe y emryo resue In vitro (Soost & Cmeron, 1985; Usmn et l., 2002; Grosser & Chnler, 2004; Reforgito et l., 2005; Zhng et l., 2007). For interploi rossing we nee utotetrplois whih re not ville in our germplsm resoures n less likely ville in the worl s well (Zeng et l., 2006; Usmn et l., 2008). In Pkistn, nrrow Citrus germplsm se is further onfine to reserh sttions only. There is no report of vilility of utotetrploi grpefruit tht is require for triploi grpefruit evelopment n for further grpefruit rop improvement progrms. These fts suggest ire nee to exploit potentil of seeless Citrus reeing (Ro et l., 1992) n to evelop genetilly iverse hyperploi popultion of grpefruit germplsm. Citrus polyplois re importnt for their hortiulturlly useful hrteristis, soure of vriility n use s prents in reeing progrms. Aitionlly hyperplois re vigorous n helthy plnts. In vitro hromosome ouling is populr soure of prouing tetrploi plnts rtifiilly in Citrus n other fruit trees (Preieri, 2001). Tetrplois often generte vrints hving fvorle hortiulturl hrteristis s inrese in orgn size like leves, flowers, fruits leing to high proution n y ouling genome tetrplois provie wier germplsm se for reeing progrms (Arn et l., 1997; Tho et l., 2003; Zhng et l., 2007). Conventionl methos of inuing tetrploiy inlue soking roots or whole plnts in olhiine solution, ulturing shoots in olhiine-ontining meium in greenhouse, injeting olhiine into seonry us n pplying olhiine-soke otton sw to uxiliry us. These methos re less effiient in polyploi plnt proution with higher frequeny of himers tht nee severl propgtion yles to seprte himers (Pei, 1985). Colhiine forms tuulin-olhiine omplex preventing mirotuule ssemly n rrests spinle formtion uring mitosis resulting in uplite hromosomes whih sty within the sme nuleus leing to tetrploiy (Khn & Rhmn, 1994; Pn et l., 1995; Srsn & Roerts, 2004; Cpert et l., 2006). Plnt ells survive on olhiine n resulting olhiplois hve plye signifint role in vriety evelopment progrms (Brrett, 1974). Non-himeri utotetrplois using olhiine tretment In vitro hs een evelope in Citrus (Wu & Mooney, 2002; Zhng et l., 2007). Use of multiellulr somti tissues or orgns hs le to himeri polyploi plnt evelopment y olhiine (Sho et l., 2003; Tho et l., 2003) ompre to emryo ulture (Yng et l., 2006). Therefore emryo ulture of grpefruit ultivrs ws preferly use to minimize the hne of evelopment of himerism in evelope hyperploi germplsm. The stuy ws initite to exploit vntges of polyplois n to wien germplsm se for future rop improvement in grpefruit. Mterils n Methos Plnt mteril, steriliztion n inoultion proeures: Fruit of grpefruit ultivrs viz., Shmer,

2 400 MUHAMMAD USMAN ET AL., Frost Fresh, Re Mx Cne Fster, Foster, J BC 430 Mrsh, n Ree were ollete from Experimentl Fruit Gren, Institute of Hortiulturl Sienes, University of Agriulture, Fisl. Fruits were ut in to two hlves with shrp knife, squeeze n sees were extrte from fruit y twisting. Extrte sees were wshe y tp wter to remove the pulp. Unerevelope n immture sees were isre. Develope sees were surfe sterilize with 70% ethnol for 5 minutes n then with 10% soium hypohlorite plus 1-2 rops of Tween 20 for minutes followe y 3-4 rinses with sterilize wter. See ot ws remove n emryos were exise with sterile neele n foreps. These emryos were ulture on MS meium supplemente with vrious levels of olhiine (0.0, 0.001, 0.005, 0.01, 0.02, 0.03, 0.05 n 0.1mgL -1 ). Mei steriliztion n ulture onitions: Mei were sterilize in utolve t temperture of 121 o C n 15 Psi for 20 minutes. Cultures were ple in growth room filitte with 2500 lux light intensity n mintine t temperture 25 C ± 2. Morphologil n ytologil hrteriztion of puttive hyperplois: These puttive hyperploi plnts evelope from emryo germintion In vitro were ientifie n sreene out on the sis of morphologil n ytologil hrteriztion. Dt were ollete for lef re, root n shoot length (m) n survivl (%) fter trnsplnting. Stomtl stuies: This inlue etermintion of stomtl size, numer n frequeny of stomt in iploi (prent) n puttive hyperploi plnts. Frequeny of stomt: Numer of stomt ws ounte y removing thin lyer from the lower epiermis. A rop of istille wter ws put on glss slie n mounte the epierml lyer on the rop of istille wter. The slie ws overe with the fine slie over very refully to voi ny ir ules uner the slie over. Fine tissue pper ws use to sor extr wter y pling it sie the over slip. The slie ws then juste on the stge of mirosope n ws oserve t ifferent mgnifying powers. Numer of stomt ws ounte uner mgnifition of 1250x. Size of stomt (µ): The length n with of stomt were mesure in mirons (µ). The stge mirometer ws ple uner pproprite mgnifition of mirosope. The oulr mirometer hs 50 smll ivisions in the sle from 0 to 1, while in the se of stge mirometer there were 100 smll ivisions in the sle from 0 to 1. The fous ws juste in suh position tht the zero of oth mirometers oinie. Then the ivisions of stge mirometer whih oinie with the fifty of oulr mirometer were note. The ivisions of stge mirometer were 5 whih were use to lulte vlue of eh ivision of oulr piee in miron y putting vlues in the formul: Size of Stomt = ivision of stge x 10 ivision of oulr = 5/50 x 10, = 50/50 = 1 Eh ivision of stge mirometer is equl to 1µ. To etermine the length n with, ivisions were multiplie with 1µ thus otining mesurement of ny prtiulr stomtl imension uner the mirosope (1250x mgnifition). Length n with of stomt: Length n with of the stomt ws mesure y removing the stge mirometer n pling the slie on the stge of the mirosope. Then the oulr mirometer ws juste length/with wise n the ivisions were ounte n then multiplie with the ftor 1µ. Length/With = Counts of oulr ivision x 1µ Size of stom (µ): Size of stom (Stomtl opening) ws mesure y revolving the eye tue tht h the oulr mirometer n y justing the ivision in ross setionl position from the entre of stomt, the numer of ivisions were ounte n then multiplie with the ftor 1µ. Aperture = Counts of oulr ivisions x 1µ Results n Disussion Genotypi response for emryo germintion, shoot length n root length: Highly signifint genotypi response ws oserve for emryo germintion n shoot length ompre to root length. In emryo germintion, signifintly (p>0.005) higher germintion (2.9 emryos) ws oserve in grpefruit ultivr Shmer n the genotypi tren ws oserve s Shmer >Frost Fresh >Re Mx Cne Fster > J BC Mrsh 430 >Ree >Foster (Fig. 1). Regring shoot length, v. Ree showe signifintly (p>0.005) more shoot growth (3.6 m) n the rest of the ultivrs showe shoot growth s Ree >Shmer >Frost Fresh, J BC Mrsh 430 >Re Mx Cne Fster > Foster (Fig. 2). Mximum root length ws oserve in v. Shmer while rest of the ultivrs showe non-signifint ifferenes for root length rnging from 20ms -25ms (Fig. 3). Effet of In vitro Colhiine pplition on emryo germintion, shoot length n root length: Emryo germintion ws foun inversely proportionl to the inrese in olhiine level in the meium n emryo germintion rnge from 2.06 to 2.39 from mgL -1 of olhiine (Fig. 4) n further inrese in its onentrtion showe eline in the emryo germintion suggesting the 0.03mgL -1 of olhiine s the lethl level for emryos (Tle 1). Regring shoot n root length oth were foun inversely proportionl to the olhiine level in the meium n signifintly higher shoot n root length ws oserve on ontrol (4.08m n 26.66ms, respetively) while the highest onentrtion of olhiine in the meium showe minimum shoot n root growth (2.67ms n 17.98ms, respetively). Lef emergene n growth showe vrile response to olhiine levels s signifintly higher numer of leves 6.15 were oserve t 0.02mgL -1 while oth lower n higher levels of olhiine showe erese numer of leves per plnt suggesting tht olhiine tretment h mrker inhiitory effet on lef evelopment in shoots (Tle 1). However, the vriility oserve might e ttriute to the vrition in evelopment, helth n ge of emryos in polyemryoni Citrus see.

3 EMBRYO CULTURE TO ENHANCE POLYPLOIDIZATION IN GRAPEFRUIT 401 e f olhiine onentrtions n see germintion. These results gree with the work of Duren et l., (1996) n Chkrorti et l., (1998) who foun negtive effet of higher olhiine onentrtions on germintion of explnts. Root n shoots rising from ontrol tretments h higher growth rte thn those from olhiine trete ones. Vrile effet of olhiine tretment ws oserve on root n shoot length initing n verse n suppressing effet of olhiine on oth root n shoot length of plnts. Seelings rising from trete emryos showe slow growth ompre to ontrol. Root n shoot length erese with inrese in olhiine onentrtion leing to more root n shoot length thn ontrol. The growth retrtion in trete plnts might e ttriute to reue rte of ell ivision use y olhiine. Similr oservtions in whih root n shoot length erese ue to growth retrtion re lso reporte y Skir & Jolly (1994). High onentrtion of olhiine ouple with long exposure time proue plnts with stunte growth (Ruuluz et l., 2007; Zhng et l., 2007) n reovery of triplois with reue growth ompre to iplois (Sho et l., 2003). Figs Genotypi response for 1) emryo germintion 2) shoot length n 3) root length on MS meium with olhiine in grpefruit. Emryo germintion perentge is n importnt prmeter while pplying olhiine to emryos for polyploi inution. Colhiine elye germintion with inresing levels n eing n ntimitoti gent use eth of emryos t higher levels inuing more stress. Results showe n inverse reltionship etween vrious Genotypi response for frequeny n size of stomt (µ) in puttive hyperploi grpefruit plnts: Strong genotypi vriility ws oserve for frequeny of stomt n length n with of stomt (Fig. 5). Signifintly (p>0.005) higher numer of stomt were foun in ultivrs Frost Fresh n Foster ( ) followe y Re Mx Cne Fster, Ree n J BC Mrsh 430 while the lowest numer of stomt were oserve in v. Shmer (11.85; Fig. 6). Stomtl length ws signifintly higher in Shmer n Foster (2.8µ-2.9µ) while rest of the ultivrs showe similr stomtl length (2.70µ-2.75µ; Fig. 7). Higher stomtl with ws oserve in vs. Ree n Shmer (2.08µ-2.10µ) followe y J BC Mrsh 430 n Foster while the lowest stomtl with ws oserve in Frost Fresh n Re Mx Cne Fster (1.98µ-1.99µ; Fig. 8). The t suggeste mximum size of stomt in vs. Shmer n Foster with mximl length n with ompre to other ultivrs. Vrile genotypi ifferenes were oserve for stomtl pore size. Aperture size ws signifintly higher (0.45µ) in vs Shmer n Foster (0.43µ) followe y Re Mx Cn Fster (0.42µ) n J BC Mrsh 430 (0.42µ) wheres minimum stomtl pore size ws foun in Frost Fresh (0.41µ) n Ree (0.40µ) s shown in (Fig. 9). Tle 1. Effet of In vitro olhiine pplition on emryo germintion n grpefruit plnt growth MS meium + Colhiine No of emryos Shoot length Root length No. of leves (mgl -1 ) germinte (m) (m) D 4.08 A A B D 3.87 B AB C B 3.76 C ABC D C 3.55 D BCD E A 3.22 E CD A F F DE G G 2.80 G BCD F E 2.67 H E F

4 402 MUHAMMAD USMAN ET AL., A B Fig. 4. Emryo germintion of Grpefruit v. Shmer on MS mei with Colhiine 0.1% (A) n 0.01% (B). () () (A) (B) Fig., 5. Stomtl ensity in puttitive polyploi plnts ) iplois n ) polyplois.

5 EMBRYO CULTURE TO ENHANCE POLYPLOIDIZATION IN GRAPEFRUIT 403 Figs Genotypi response in stomtl stuies for 6) numer, 7) length n 8) with of stomt (µ). Fig. 9. Genotypi response for stomtl opening (µ). Effet of In vitro Colhiine pplition on frequeny n size of stomt (µ) in puttive hyperploi grpefruit plnts: Frequeny of stomt ws foun inversely proportionl to numer of stomt n the numer erese (13.89 to 9.63) with inrese in the olhiine onentrtion (0-0.1 mgl -1 ) in the MS meium. Length n with of stomt were foun iretly proportionl to the olhiine level in the meium n oth length (2.47µ-3.18µ) n with (1.77µ-2.34µ) inrese with inrese in olhiine onentrtion (0-0.1 mgl -1 ) in the meium (Tle 2). These results suggest tht olhiine pplition hve iret impt on stomtl ensity n size. Size of stom (perture) inrese with inrese in the level of olhiine in the meium n mximum opening ( µ) ws foun in ll the ultivrs on 0.10 mgl -1 of olhiine. Signifintly higher stomtl size (0.51µ) ws foun on the higher level of olhiine 0.10 mgl -1 wheres it ws less (0.35µ) in ontrol meium (MS evoi olhiine). Stomtl size hs een iretly proportionl to ploiy n it inrese with the inrese in the ploiy level of plnts (Cutter, 1986; Cost et l., 2004). This tren hs een reporte in mny fruit rops like Citrus vs. mnrin (Khn et l., 1992., Usmn et l., 2002, Usmn et l., 2008), Vitis (Yng et l., 2006) n Ziziphus (Gu, 2005). Vnehout et l., (1995) oserve greter stomtl length of olhiine trete Citrus ultivrs s well. Gu (2005) oserve tht the stomt size of tetrploi plnts ws signifintly lrger thn those of iploi plnts, while the frequeny of stomt ws reue signifintly in Ziziphus. Our results suggeste tht oth genotype n olhiine tretment hs signifint impt on stomtl size in grpefruit ultivrs. Higher onentrtion of olhiine hs een more useful in inuing hyperploiy ompre to low oses. Low onentrtion of the olhiine inues epolymeriztion of mirotuulr ytoskeleton uring ell yle n insuffiient spinle isruption ourre leing to inution of polyploi ells with ell nomlies. Higher onentrtion ws foun more effetive in formtion of polyploi ells s it my le to proution of new tuulin strutures llowing evelopment of tetrploi nulei (Cpert et l., 2006). Zeng et l., (2006) reporte evelopment of tetrplois on 0.1% of olhiine tretment for 4-8 ys in grpefruit ultivr Frost ell lines. We lso oserve less stomtl ensity n more stomtl size on these levels of olhiine. However, we iffer in use of explnt (emryo) ompre to protoplsts use y them. Stomtl stuies prove to e useful tool in initil sreening of iploi n polyplois in grpefruit ultivrs n re strengthene y similr reports of Crvlho et l., (2005) in Citrus n Stnys et l., (2006) in Jpnese Quine. Similr results were reporte y See et l., (2004) n Ftim et l., (2010) who reovere spontneous polyplois in mnrins, sweet ornges n grpefruit ultivr Foster. We iffer, however, in inution of polyploiy in grpefruit ultivrs y emryo ulture In vitro on olhiine. This hyperploi germplsm hs een trnsplnte in pots n shifte to green house fter limtiztion (Fig. 10). In our stuies, emryo ulture of grpefruit ultivrs on olhiine ontining mei prove to e n effiient metho of polyploiy inution n suessful reovery s reporte in triploi ornge (Minoru & Nrio, 2006). Confirmtion of hyperploi germplsm is suggeste through hromosome ounting or flow ytometery. Geneti hrteriztion of the germplsm is suggeste to sreen mixoplois, himers n the origin of emryos (zygoti/nuellr). These utotetrplois oul e useful in rop improvement progrms in grpefruit.

6 404 MUHAMMAD USMAN ET AL., Fig. 10. Puttive grpefruit polyplois in greenhouse fter limtiztion. Tle 2. Effet of In vitro olhiine pplition on stomtl ensity n size. MS mei + Colhiine (mgl -1 ) No. of stomt Length of stomt (µ) With of stomt (µ) Stomtl pore size (µ) A 2.47 H 1.77 H 0.35 E B 2.53 G 1.79 G 0.35 E C 2.63 F 1.91 F 0.39 D D 2.74 E 2.02 E 0.41 CD E 2.85 D 2.08 D 0.42 C F 2.95 C 2.17 C 0.45 B G 3.06 B 2.24 B 0.48 A H 3.18 A 2.34 A 0.51 A Referenes Anonymous Foo n Agriulture Proution (FAO) Yer Book. Rome, Itly. ( Arn, R.R., B.R. Bon, J.P. Syvertsen n J.W. Grosser Lef hrteristis n net gs exhnge of iploi n utotetrploi Citrus. Annls of Bot., 79: Brrett, H.C Colhiine-inue polyploiy in Citrus Bot. Gz., 135: Cpert, A.D., M. Delgo, F. Ressurreição, A. Meister, R.N. Jones, W. Viegs n A. Houen Colhiineinue polyploiiztion epens on tuulin polymeriztion in -metphse ells. Protoplsm, 227: Crvlho, R., W.S. Filho, A.C. Brsileiro-Vil n M. Guerr The reltionships mong lemons, limes n itron: hromosoml omprison. Cytogen. & Genome Res., 109: 1-3. Chkrorti, S.P., K. Vijyn, B.N. Roy n S.M.H. Qri In vitro inution of tetrploiy in mulerry (Morus l L.). Plnt Cell Rep., 17: Cost, M.A.P., W.A.B. Almei, A.A.A.M. Filho, B.M.J. Menes n A.P.M. Roriguez Stomtl nlysis of Citrus hyris otine y protoplst fusion. Pesq. Agrope. Brs. Brsili., 39: Cutter, E.G Epierme. In: Antomi vegetl. 2 n.e. São Pulo: Ro, pp. 2. Duren, M.R., J. Dolezel n R. Afz Inution n verifition of utotetrplois in iploi nn (Mus umint) y In vitro tehniques. Euphyti, 88: Ftim, B., M. Usmn, I.A. Khn, M.S. Khn n M.M. Khn Exploring Citrus ultivrs for unerevelope n shrivele sees: A vlule resoure for spontneous polyploiy. Pk. J. Bot., 42: Grosser, J.W. n J.L. Chnler Proution of twelve new llotetrploi somti hyri Citrus reeing prents with emphsis on lte mturity n ol-hriness. Am. Pomol. So., 58: Gu, X.F In vitro inution of tetrploi plnts from iploi Zizyphus juju Mill. v. Zhnhu. Plnt Cell Rep., 24: Khn, M.D n H. Rhmn In: Crop improvement in Hortiulture. (Es.): E.Bshir n R. Bntel. Ntionl Book Fountion. pp Khn, M.M., I.A. Khn n A.H. Mughl Growth n morphologil omprison of iploi n tetrploi strins of Kinnow mnrin. Pro. Int. So. Citriulture., pp 151. Minoru, K. n K. Nrio Effet of olhiine tretment on Tetrploi proution of nuellr emryos in trifolite ornge (Ponirus trifolit Rf.). Bull. Fukuok Agri. Res. Cen., 25: Pn, D., BL. Gooe, S.C. Feinstein n L. Wilson Kineti stiliztion of mirotuule ynmis t stey stte y tu n mirotuule-ining omins of tu. Biohem., 34: Pei, X.S Polyploiy inution n reeing. Shnghi Si. n Teh., Press, pp Preieri, S Muttion inue n tissue ulture in improving fruits. Plnt Cell Tiss. Org. Cult. 64: Ro, A.R., M.D. Khn, M. Hmee n T. Mhmoo Polyploiy in Citrus speies n iotypes in Punj. Pro. Ist. Int. Sem. Pk., pp Reforgito, R., G. Russo n S. Reupero New promising Citrus triploi hyris selete from rosses etween monoemryoni iploi femle n tetrploi mle prents. Hort. Siene., 40: Ruuluz, T., R.V. Nikolov, M.T. Smith n K. Hnnweg In vitro inution of tetrplois in Colophospermum mopne y olhiine. S. Afrin J. Bot., 73:

7 EMBRYO CULTURE TO ENHANCE POLYPLOIDIZATION IN GRAPEFRUIT 405 See, T., M. Usmn, M.M. Khn, B. Ftim n I.A. Khn Reovery of polyplois from unerevelope sees. Pro. Int. So. Citriulture, Srsn, V. n A.V. Roerts In vitro hromosome ouling. In: Enylopei of plnts n rops. (E.): M. Dekker. Mrel Dekker In., New York, pp Sho, J.Z., C.L. Chen n X. Deng In vitro inue of tetrploi in pomegrnte (Puni grntum). Plnt Cell Tissue Orgn Cult., 75: Soost, R.K. n J.W. Cmeron Melogol, triploi pommelo-grpefruit hyri. Hort. Siene., 20: Sikr A.K. n Jolly M.S Inue polyploiy in mulerry (Morus spp.): inution of tetrplois. Seriologi, 34: Stnys, V., A. Wekmn, G. Stniene n P. Duhovskis In vitro inution of polyploiy in jpnese quine (Chenomeles jponi). Plnt Cell Tissue Orgn Cult., 84: Tho, N.T.P., K. Ureshino, I. Miyjim, Y. Ozki n H. Okuo Inution of tetrplois in ornmentl Alosi through olhiine n oryzlin tretments. Plnt Cell Tissue Orgn Cult., 72: Usmn, M., B. Ftim, K.A. Gillni, M.S. Khn n M.M. Khn Exploittion of potentil trget tissues to evelop polyplois in Citrus. Pk. J. Bot., 40: Usmn, M., M. Rmzn, B. Ftim, M.J. Jskni n M.M. Khn Citrus Germplsm Enhnement By Interploi Hyriiztion 1: Reiprol Crosses of Kinnow & Suri. Int. J. Agri. Biol., 4: Vnehout, H., R. Ortiz, D. Vuylsteke, R. Swennen n K.V. Bi Effet of polyploiy on stomtl n other quntittive trits in plntin n nn hyris. Euphyti, 83: Wu, J.H. n P. Mooney Autotetrploi tngor plnt regenertion from In vitro Citrus somti emryogeni llus trete with olhiine. Plnt Cell Tissue Orgn Cult., 70: Yng, M.X., Z.Y. Co, L.Z. An, Y.M. Wng n X.W. Fng In vitro tetrploi inution vi olhiine tretment from iploi somti emryos in grpevine (Vitis vinifer L.). Euphyti, 152: Zeng, S.H., C.W. Chen C.W, H. Liu, J.H. Liu n X.X. Deng In vitro inution, regenertion n nlysis of utotetrplois erive from protoplsts n llus trete with olhiine in Citrus. Plnt Cell Tissue Orgn Cult., 87: Zhng, J., M. Zhng n X. Deng Otining utotetrplois In vitro t high frequeny in Citrus sinensis. PInt Cell Tissue Orgn Cult., 89: (Reeive for pulition 12 Ferury 2011)