The Effects of Different Sucrose, Agar and ph Levels on In Vitro Shoot Production of Almond (Amygdalus communis L.)

Transcription

1 Tr. J. of otny (199) TÜİTK Reserh rtile The Effets of Different Surose, gr nd ph Levels on In Vitro Shoot Prodution of lmond (mygdlus ommunis L.) Songül GÜREL Sugr Institute, Plnt reeding Deprtment, Etimesgut, 679 nkr-turkey Yüel GÜLŞEN nkr University, Fulty of griulture, Deprtment of Hortiulture, Dışkpı, 611 nkr-turkey Reeived: epted: strt: In this study the possiilities of in vitro vegtitve propgtion of lmond (mygdlus ommunis L.) v. Texs nd v. Nonpreil y shoot-tip ulture were investigted. Different levels of surose, gr nd ph were tested nd shoot prolifertion, shoot development nd growth were oserved during three suessive stges, nmely initition, trnsplnttion nd multiplition. During the initition stge, prolifertion ws hieved only with 5 nd 6% surose nd the est explnt development oured with.5% gr nd ph 5.5. During the seond stge, gin 5 nd 6% surose nd ph 5.5 gve the est results with respet to shoot prodution nd growth. lthough the medium with low gr onentrtions (.5%) ws fvourle for shoot formtion nd growth,.7% gr ws determined to e the most pproprite level for this stge sine low gr levels (.5 nd.6%) used vitrifition. During the multiplition stge, the highest rte of shoot prodution ws hieved with 3 nd % surose,.7% gr nd ph5.5, nd higher surose nd gr levels inresed llus formtion t the se of the shoots. Key Words: lmond, tissue ulture, miropropgtion, shoot-tip ulture Frklı Skkroz, gr ve ph Düzeylerinin demde (mygdlus ommunis L.) İn Vitro Sürgün Verimine Etkileri Özet: u rştırmd, Texs ve Nonpreil dem (mygdlus ommunis L.) çeşitlerinin sürgün uu kültürü ile in vitro vejettif çoğltım olnklrı rştırılmıştır. u mçl frklı skkroz, gr ve ph düzeyleri, tkip eden üç frklı kültür şmsınd (ilk dikim, şşırtm ve çoğltm) yrı yrı test edilmiş ve sürgün prolifersyonu, sürgün gelişmesi ve üyümesi inelenmiştir. İlk dikim şmsınd, sdee %5 ve 6 skkroz dozlrı prolifersyonu uyrmıştır, ve %.5 vey 6 skkroz ile ph 5.5 ise en iyi eksplnt gelişmesini sğlmıştır. Şşırtm şmsınd, yine %5 vey 6 skkroz ile ph 5.5 içeren ortmlrd sürgün gelişmesinin ve üyümesinin en iyi olduğu görülmüştür. Her ne kdr, düşük gr dozlrınd sürgün verimi ve gelişmesi iyi olmuşs d, u dozlrd sürgünlerde mlşm meydn geldiğinden, u şm için.7% gr en uygun doz olrk elirlenmiştir. Çoğltm şmsınd ise, en yüksek sürgün verimi %3 ve skkroz, %.7 gr ve ph 5.5 düzeylerinde sğlnmıştır. Yüksek skkroz ve gr dozlrının, sürgünlerin dip kısımlrınd kllus oluşumunu rtırdığı gözlenmiştir. nhtr Sözükler: dem, doku kültürü, mikroçoğltım, sürgün-uu kültürü Introdution lmond (mygdlus ommunis L. [syn. Prunus mygdlus tsh]) hs long een grown in Turkey, mostly long the ost of the egen nd Mediterrnen regions, s ntoli hs een one of the min germplsm ses of this fruit speies (1). Turkey rnks in seventh ple mong the lrgest produers of lmond in the world with.5% ontriution (). Sine it is ross-pollinted speies, ontinuous geneti vrition hs ourred in lmond through the yers, whih hs led to the formtion of very different types in terms of rop development, fruit yield nd qulity, nd tolerne to environmentl stresses. To minimize the prolem of enormous geneti vrition nd to otin genetilly identil popultions, vegettive propgtion vi lyerge or utting is ineffiient due to the gret prolems of this fruit speies in rooting in vivo. This, therefore, mkes the plnt tissue ulture tehniques more vlule for the lonl propgtion of lmond trees. * To whom orrespondene should e ddressed. 363

2 The Effets of Different Surose, gr nd ph Levels on In Vitro Shoot Prodution of lmonds mygdlus ommunis L. Tissue ulture tehniques hve een intensively used for the lonl propgtion of mny tree speies, of whih the fruit nd timer trees re of gret eonomi importne (3-5). However, mny tree speies re not suitle for vegettive multiplition nd in those whih re suitle, to ertin extent, the proess is rther slow. There hve een few reports on the propgtion of lmonds through tissue ulture. Mehr & Mehr (6) were the first to report orgnogenesis nd plnt regenertion from llus otined from vrious prts of the lmond seedlings. More reports hve sine ppered on shoot regenertion from llus (7-9) nd emryo ultures in lmonds (1-1) ut they hieved very limited suess in rooting these shoots. In vitro plnt regenertion of lmonds through shoot-tip ulture lso gined muh ttention in the lte 1971s, with severl reserhers reporting on lonl propgtion y ulturing isolted shoot-tips on medium ontining -3% surose,.7-.9% gr nd.7-1. mg/l P (13-15). The present study ws rried out to determine the optimum levels of surose, gr nd ph essentilly used in the ulture medium with the im of developing relile protool for in vitro propgtion of two lmond ultivrs (v. Texs nd v. Nonpreil), whih re ommonly grown in the US, the lrgest produer of lmonds in the world, nd were found to e very dptle to Turkey s onditions (1). The effets of different uxin nd ytokinin onentrtions nd omintions on in vitro shoot prodution in the sme ultivrs were lso investigted nd the results will e presented in future report (16). Mterils nd Methods In spring, 5-1 m long shoots, whih were still tively developing, were tken from lmond (Prunus mygdlus tsh) trees v. Texs nd v. Nonpreil, grown in nkr nd wshed thoroughly, first with tp wter nd then with sterile wter m long shoottips exised from these shoots were then surfesterilized in % sodium hypohloride for 1 min followed y rinses (5 min eh) with sterile-distilled wter. Then, - mm long shoot-tip meristems with -5 lef primordi were exised under disseting mirosope in lminr flow hood nd then inuted in either glss ulture tues (16x15 mm) or in 5 ml Erlenmeyer flsks, ontining MS (17) sl medium supplemented with.1 mg/l G 3,.1 mg/l I nd 1. mg/l P, whih hd een sterilized y utolving for 15 min t 13.5 kp. For testing the effets of surose, the medium solidified with.7% gr t ph 5.5 ws supplemented with, 3,, 5 nd 6% surose. The effets of gr were epmined y keeping the surose onentrtion t 3% t ph5.5 while hnging the gr onentrtion from.5 to.9% t.1 intervls. Finlly, different levels of ph (.5, 5., 5.5, 6. nd 6.5) t 3% surose nd.7% gr were used for testing the effets of ph on shoot prolifertion. Explnts were inuted in ontrolled environment (5±1 o C, 16 h photoperiod, lux light intensity, 75±5% humidity) nd kept in ulture for 1 weeks, suulturing every 3 weeks. The experiments involed three ulture stges; i) the initition stge (the first three weeks of ulture), ii the trnsplnttion stge (the next three weeks of ulture following the initition stge) nd iii) the multiplition stge (the remining 1 weeks of ulture with four suultures t three week intervls). t the end of eh suulture during the lst (multiplition) stge, multiple shoots were singled out nd then the individul shoots were suultured. 1- soring system ws developed for the determintion of the growth rte of shoots tht developed (Tle 1). Men shoot numer per explnt nd the men growth rte of the shoots were reorded t the end of eh stge, exept for tking the men of four suultures for the multiplition stge sine this stge lsted 1 weeks. The experiments were lid out ording to rndomised plot design with 1 replites per tretment nd the results were evluted using the method of nlysis of vrine, nd sttistilly signifint differenes were indited in letters ording to the Dunn Test t p=.1 (1). Shoot Length (m) Inititition Stge Trnsplnttion Stge Multiplition Stge Growth Rte (-3 weeks) (-6 weeks) (7-1 weeks) 1. (Poor) up to.5 up to 1. up to 1.. (Medium) (Good) (Very Good) 1.6 or longer.1 or longer 5.1 or longer Tle 1. The soring system developed for mesuring the growth rte of the shoots developed. During the inititition stge, the growth rte of oth initil nd newly proliferting shoots ws reorded while during the trnsplnttion nd multiplition stges only the growth rte of the newly developing shoots ws 36

3 S. GÜREL, Y. GÜLŞEN Results Effets of Different Surose Conentrtions on Shoot Development Initition Stge Shoot prolifertion ws oserved only with 5 nd 6% surose onentrtions in oth the ultivrs tested, with men of 3.6 nd 3.3 shoots per explnt with 5% surose, nd. nd.1 shoots per explnt with 6% surose in v. Texs nd v. Nonpreil, respetively. The differenes etween vrieties nd surose tretments (i.e., etween 5 nd 6% surose) were not found to e signifint t p=.1 (Figure 1). The first shoot prolifertion ourred on the 6th dy of inution with 5 nd 6% surose onentrtions nd y dy 1, nerly 1% of the shoottip explnts hd lredy proliferted in oth ultivrs. No shoots were produed with other surose onentrtions (i.e.,, 3 nd %). Shoot growth pity, expressed s the growth rte of the developing shoots (Tle 1), showed stedy inrese with inresing onentrtions, with 5 nd 6% surose onentrtions produing more vigorous shoots thn the lower onentrtions (Figure 1). In ddition, the growth of those explnts, whih did not produe ny shoots t ll, ontinued to inrese with inresing surose onentrtions. It ws lso oserved tht tissue of the new explnts nd of the newly forming shoots remined green throughout the ulture ut smll mounts of rown llus fromde t the se of the shoots with higher surose levels. Trnsplnttion Stge s seen in Figure, it is pprent tht during this stge tht there were no ler-ut differenes etween the effets of different onentrtions of surose on shoot development in either ultivr, with the exeption of % surose whih signifintly lower numers of shoots per explnt thn the other onentrtions. However, the highest shoot prodution (7.3 shoots per explnt) ws otined in v. Nonpreil when 3% surose ws used in the ulture medium while onentrtions of 3-6% surose indued similr numers of shoots in v. Texs, rnging from men of 5.3 to 6.1 shoots per explnt. In terms of the growth rte of the shoots, s in the initition stge, there ws stedy inrese s the surose level inresed, with 5 nd 6% surose onentrtions resulting in signigintly higher growth rte, from. (Medium) to 3.5 (Good-Very Good), in oth of the ultivrs. The shoots tht developed on Men Shoot Growth Rte Men Numer of Shoots per Explnt d Texs Nonpreil d d Figure 1. The effets of different surose onentrtions on shoot explnts () nd their susequent growth () during the initiiton stge in v. Texs nd v. Nonpreil Surose Conentrtion (%) 365

4 The Effets of Different Surose, gr nd ph Levels on In Vitro Shoot Prodution of lmonds mygdlus ommunis L. Men Shoot Growth Rte Men Numer of Shoots per Explnt Texs Nonpreil Figure. The effets of different surose onentrtions on shoot explnts () nd their susequent growth () during the trnsplnttion stge in v. Texs nd v. Nonpreil Surose Conentrtion (%) medium ontining % surose hd greenish-yellow pperne wheres the others, ultured with 3-6% surose, remined green nd helthy throughout the suulture. Smll mounts of rown llus lso formed during this stge with only 5 nd 6% surose onentrtions. Multiplition Stge During this stge, 3 nd % surose levels were found to e signifintly etter thn the other onentrtions used in oth ultivrs with respet to oth shoot prodution, with men rnging from 6.3 to 7. shoots per explnt, nd growth rte of the developing shoots, with men rte of.-3. ompred with otined with, 5 nd 6% surose levels (Figure 3). % surose produed the lest numers of shoots nd led to muh lower shoot growth rte in oth ultivrs. The shoots tht developed on medi ontining, 5 or 6% surose onentrtions were mostly greenish-yellow wheres those tht developed whih 3 nd % surose were green. Lrge mounts of llus developed with 5 nd 6% surose during only this stge. Effets of Different gr Conentrtions on Shoot Development Initition Stge No new shoot prolifertion ws oserved during this stge nd the explnts were, therefore, evluted for their growth rtes only (Figure ). It ws ler tht the inresing onentrtions of gr used stedy derese in the growth of shoot tip explnts in oth ultivrs, with.5% gr resulting in signifintly higher shoot growth rte thn the other gr onentrtions, exept for.6% gr whih ws s good s.5% gr in v. Nonpreil. No visile differenes in the explnt olour were oserved etween different gr onentrtions s ll the explnts were green. However,. nd.9% gr onentrtions produed smll mounts of llus t the proximl ut ends of the shoots, nd no vitrifition ourred in the explnts during this stge. Trnsplnttion Stge During this stge,.5-.% gr onentrtions seemed to hve similr effets on shoot prodution in 366

5 S. GÜREL, Y. GÜLŞEN Men Shoot Growth Rte Men Numer of Shoots per Explnt Texs Nonpreil Figure 3. The effets of different surose onentrtions on shoot explnts () nd their susequent growth () during the trnsplnttion stge in v. Texs nd v. Nonpreil Surose Conentrtions (%) Men Shoot Growth Rte 3 1 Texs Nonpreil Figure. The effets of different surose onentrtions on shoot explnts () nd their susequent growth () during the trnsplnttion stge in v. Texs nd v. Nonpreil gr Conentrtion (%) 367

6 The Effets of Different Surose, gr nd ph Levels on In Vitro Shoot Prodution of lmonds mygdlus ommunis L. oth ultivrs, with men of shoots per explnt nd the highest gr onentrtion (.9%) redued the numer of shoots in signifint mnner (Figure 5). No ler pttern ws oserved in the growth rte lthough onentrtions of.5,.6 nd.7% seemed notiely etter thn. nd.9% onentrtion, whih is rodly onsistent with the effets on shoot prodution during this stge (Figure 5). ll explnts ultured were green ut the lower gr onentrtions (i.e.,.5 nd.6%) used low frequeny of vitrifition whih ws not oserved during the initition stge. On the other hnd,. nd.9% gr (i.e higher onentrtions), indued smll mount of rown llus t the proximl ends of the shoots. Multiplition Stge onsiderle differene etween the ultivrs ws oserved in terms of their response ptterns to different gr onentrtions. Signifintly more shoots were otined with.5% gr in v. Texs thn t the other onentrtions whih led to grdul derese, up to men of.3 shoots per explnt, s the gr level inresed (Figure 6). In v. Nonpreil, onentrtions of.5-.% gr produed similr numers of shoots with men rnging from 5.9 to.5 shoots per explnt. s in v. Texs,.9% gr ws lso the lest effetive for shoot prodution in v. Nonpreil. For the growth rte of the shoots,.5,.6 nd.7% gr onentrtions were signifintly etter thn. nd.9% gr with eh group induing similr growth rte. high level of shoot vitrifition ourred with lower gr onentrtions (i.e.,.5 nd.6%) in oth ultivrs while high gr levels (. nd.9%) used n inresed mount of rown llus development t the proximl end (se, of the shoots whose leves were greenish yellow in pperne. Effets of Different ph Levels on Shoot Development Initition Stge s in the gr tretments, no new shoot prolifertion ourred during this initil stge nd the shoot explnts were, therefore, evluted for their growth rtes only (Figure 7). The results lerly indited tht ph 5.5 ws the optimum level for shoot growth in oth v. Texs nd v. Nonpreil. s the ph level deresed from 5.5 to.5 or inresed from 5.5 to 6.5, there were signifint dereses in the growth rte of the shoots in oth Men Shoot Growth Rte Men Numer of Shoots per Explnt Texs Nonpreil Figure 5. The effets of different gr onentrtions on shoot explnts () nd their susequent growth () during the trnsplnttion stge in v. Texs nd v. Nonpreil gr Conentrtion (%) 36

7 S. GÜREL, Y. GÜLŞEN Men Shoot Growth Rte Men Numer of Shoots per Explnt Texs Nonpreil d Figure 6. The effets of different gr onentrtions on shoot explnts () nd their susequent growth () during the trnsplnttion stge in v. Texs nd v. Nonpreil gr Conentrtion (%) Men Shoot Growth Rte 3 1 Texs Nonpreil Figure 7. The effets of different ph levels on shoot development from shoot-tip explnts () nd their susequent growth () during the trnsplnttion stge in v. Texs nd v. Nonpreil ph 369

8 The Effets of Different Surose, gr nd ph Levels on In Vitro Shoot Prodution of lmonds mygdlus ommunis L. ultivrs. No detetle differenes were seen etween the different ph tretments with regrd to the olour of the shoots, ll hving green nd helthy pperne. It is importnt to point out here tht no llus formtion or vitrifition ourred during this stge. Trnsplnttion Stge In oth ultivrs, ph 5.5 nd 6. produed signifintly more shoots thn the other ph levels, with men shoots prodution rnging from.5 to 6. shoots per explnt (Figure ). With respet to shoot growth rte, ph 5.5 ws gin signifintly etter thn the other ph levels tested. t ll ph levels, explnts were green with no vitrifition ut t ph 6.5, smll mounts of greenish-white llus fromed t the se of the shoots. Multiplition Stge gin ph 5.5 ws found to e signifintly more effetive thn the other ph levels for oth shoot prodution nd shoot growth rte in oth ultivrs (Figure 9). Unlike during the initittion nd trnsplnttion stges, low ph levels, espeilly ph.5, used shoots to turn yellow in oth ultivrs. In ddition, the lowest (.5) nd the highest (6.5) ph levels resulted in the development of shorter shoots with nrrow, urled nd shrp-pointed leves (i.e., lef mlformtion). Lrge mounts of greenish-white llus developed t the se of the shoots gin t ph 6.5 only ut no vitrifition ourred. Disussion Sine most fruit trees nd ornmentl plnts re highly heterozygous, their seed progeny is not true-totype. sexul (vegettive) reprodution, on the other hnd, gives rise to plnts whih re genetilly identil to the prent plnt nd thus permits the perpetution of the unique hrters of the ultivrs (19). The lmond is ross-pollinted speies nd relile method for the lonl propgtion would e of gret vlue for its Men Shoot Growth Rte Men Numer of Shoots per Explnt Texs Nonpreil Figure. The effets of different ph levels on shoot development from shoot-tip explnts () nd their susequent growth () during the trnsplnttion stge in v. Texs nd v. Nonpreil ph 37

9 S. GÜREL, Y. GÜLŞEN MenShoot Growth Rte Men Numer of Shoots per Explnt d Texs Nonpreil Figure 9. The effets of different ph levels on shoot development from shoot-tip explnts () nd their susequent growth () during the trnsplnttion stge in v. Texs nd v. Nonpreil ph reeding. We therefore ttempted to develop n effetive protool for the lonl propgtion of two lmond ultivrs (v. Texs nd v. Nonpreil) through shoot-tip ulture, exmining the effets of surose, gr nd ph during three suessive ulture stges. ll the ftors studied were found to e highly effetive on shoot development nd growth in oth ultivrs. During the prolifertion nd trsplnttion stges, stedy inrese in the men shoot prodution nd men shoot growth rte per explnt ws oserved with inresing surose (5 nd 6%) whih ws used s ron (energy) soure. This my e minly due to the ft tht high sugr levels vlile in the ulture medium my speed up ell division thus leding to n inrese in the volume nd weight of tissues ultured, s suggested y other reserhers (). In ontrst with the prolifertion nd trnsplnttion stges, lower surose onentrtions (3 nd %) were signifintly etter during the multiplition stge in terms of shoot prodution rte nd their susequent growth, whih ws onsistent with the findings of Hisjim (11, 1), Rugini & Verm (15) nd Rugini () who suggested tht 3% surose should e used in ll ulture stges for the in vitro multiplition of lmond. However, our results differed in tht younger ultures (i.e., during the prolifertion nd trnsplnttion stges) required higher surose level, 5 or 6% nd s ultures proeeded further (i.e., during the multiplition stge), the need for surose deresed onsiderly to 3 or %, suggesting diret reltionship etween the rte of ell division nd energy (surose) onsumption in the ultured tissues sine ell division in geing tissues dereses with time (1). We tested rnge of gr onentrtions from.5 to.9% nd our oservtions demonstrted tht gr levels of.5-. were rodly effetive in promoting erly shoot prolifertion, shoot development nd growth during ll stges. Nevertheless, lower levels (.5 nd.6%) used severe vitrifition during the trnsplnttion nd multiplition stges. Some reserhers hve reported tht the leves or lef explnts 371

10 The Effets of Different Surose, gr nd ph Levels on In Vitro Shoot Prodution of lmonds mygdlus ommunis L. ultured t low gr levels were le to uptke more wter from the medium nd, therefore, exhiited muh higher vitrifition (15,, 3). On the other hnd, higher gr onentrtions my result in derese in shoot prodution nd growth s gr would inrese the solidness of the medium from whih nutrient uptke would e limited. Our results demonstrted tht shoot prolifertion ws onsiderly delyed, nd shoot prodution nd growth were gretly inhiited when.9% gr ws used. In ddition,.9% gr inresed llus formtion whih then proly eme inhiitory for shoot development. sed on our experimentl results nd oservtions, we n suggest tht.7% gr is the most suitle onentrtion for shoot-tip ulture of the lmond ultivrs tht we used sine lower or higher levels hd dverse effets. However, the use of gr t.5 my lso e suggested during the initition stge s vitrifition my not neessrily our t limiting level t this erly stge of the ulture. The other ftor we exmined ws ph. It hs een reported tht the ph of the medium hs regultory effet on nutrient uptke y the explnt tissue (3, ). Our results indited tht ph 5.5 ws the optimum during ll stges in oth ultivrs nd either low or high ph levels used serious normlities, produing shorter shoots with nrrow, urled nd shrp-pointed leves. There re onfliting reports on the optimum ph level for lmond tissue ulture, with some determining ph 5.5 s optimum (11, 1), whih is onsistent with our findings, while others hve reported tht higher level (ph 5.9) produed the highest numer of regenernts (13). This might e ttriuted to gnetypi vrition s it n e serious prolem in tissue ulture work with mny plnt speies. s mtter of ft, in our experiments we oserved differenes etween the ultivrs in terms of shoot prodution, with v. Nonpreil usully produing 1-3% more shoots thn v. Texs ultured on the sme medium lthough no sttistil nlysis ws rried out to onfirm this oservtion. In onlusion, it n e summrised tht high surose level (5-6%) should e used during the first nd seond stges of the ulture ut redued to low level (3- %) during the finl stge. s for gr, unlike surose, the first stge needs low gr level (.5%) while the seond nd third stges require n inrese up to.7%. The ph seems to e the most onsistent vrile s ll stges produed their highest yield t ph 5.5. knowledgement The uthors wish to thnk The Reserh Fund Diretorte of nkr University for their finnil support. Referenes 1. Dokuzoğuz M., Güln R., dem Yetiştiriiliği ve Sorunlrı. TÜİTK XV. Kuruluş Yılı ilimsel Yyınlrı, nkr, Turkey (1977).. nonymous. F..O. Yerook Prodution. Volume 1, Rome, Itly (197). 3. Henry P.H., lzih F.., Hinesley L.E., Vegettive propgtion of estern rededr y stem uttings. HortSi. 7(1), (199).. Gjulev V., tnssov., Miropropgtion of Pltnus erifoli in vitro. Silve Geneti 3(), 15-1 (199). 5. Gomez M.P., Segur J., xillry shoot prolifertion in ultures of explnts from mture Juniperus oxyedrus trees. Tree Physiol. 15(9), 65-6 (1995). 6. Mehr., Mehr P.N., Orgnogenesis nd plntet formtion in vitro in lmond. ot. Gzet. 135(1), (197). 7. Kester D.E., Thnik L., Negueroles J., Use of miropgtion nd tissue ulture to investigte geneti disorders in lmond ultivrs. t Hortiul. 7, (1977).. Rugini E., Progress in studies on in vitro ulture of lmonds. In: Plnt Tissue Culture nd Its griulturl pplitions, 1 st Conferene in the Ester Shool Series in griilturl Siene, Englnd (19). 9. Rugini E., lmond. In: Evns D.., Shrp W.R., mmirto V. (eds.), Hndook of Plnt Cell Culture, Tehniques nd pplitions, Volume, MMilln Pulishing Compny, New York, US (196). 1. Hnsmn D., Novo C.O., Miropropgtion of temperte nut trees. Hortiulturl strts 56(6), 3-16 (196). 11. Hisjim S., Multiple shoot formtion from lmond emryos. iol. Plnt (3), 35-3 (19). 1. Hisjim S., Multiple shoot formtion from lmond seeds nd n exised single shoot. griul. iol. Chem 6(), (19). 13. Thnik L., Kester D.E., Shoot ulture for lmond nd lmondpeh hyrid lones in vitro. HortSi. 1(6), (1977). 37

11 S. GÜREL, Y. GÜLŞEN 1. Rugini E., Verm D.C., Miropropgtion nd ell suspensions of diffiult to propgte lmond (Prunus mygdlus tsh) ultivr. In: Fujiwr. (ed.), Plnt Tissue Culture 19, The Proeedings of the 5 th Interntionl Congress on the Plnt Tissue nd Cell Culture, Tokyo, Jpn (19). 15. Rugini E., Verm D.C., Miropropgtion of diffiult-topropgte lmond (Prunus mygdlus tsh) ultivr. Plnt Si. Let., 73-1 (193). 16. Gürel S., Gülşen Y., The effets of I nd P on in vitro shoot prodution of lmond (Prunus mygdlus tsh). Tr. J. ot., (this issue). 17. Murshige T., Skoog F., revised medium for rpid growth nd iossys with too tissue ultures. Physiol. Plnt. 15, (196). 1. Dniel W.W., iosttistis: Foundtion for nlysis in the Helth Sienes, 6 th Edition, John Wiley & Sons, In., Singpore (1995). 19. hojwni S.S., Rzdn M.K., Plnt Tissue Culture: Theory nd Prtie, Elsevier Siene Pulishers.V., msterdm, The Netherlnds (199). 1. Gürel E., Wren M.J., In Vitro Development from lef explnts of sugr eet (et vulgris L.): Rhizogenesis nd the effet of sequentil exposure to uxin nd ytokinin. nn. ot. 75, 31-3 (1995).. Gspr T., Kevers C., Deergh P., Meng L., Pques M., oxus P., Vitrifition: Morphologil, physiologil nd eologil spets. In: ong J.M., Durzn J. (eds.), Cell nd Tissue Culture in Forestry, Generl Priniples nd iotehnology, Volume 1, Mrtinus Nifhoff Pulitions, Dordreht, The Netherlnds (197). 3. Hussey G., Vegettive propgttion of plnts y tissue ulture. In: Yeomn M.M. (ed.), Plnt Cell Culture Tehnology, lkwell Sientifi Pulitions, Oxford (196).. Quk F., Meristem ulture nd virus-free plnts. In: Reinert J., jj Y.P.S. (eds.), pplied nd Fundmentl spets of Plnt Cell, Tissue nd Orgn Culture, pp , Springer-Verlg, erlin, Germny (1977). 5. Pennell D., Miropropgtion in Hortiulture, Grower ooks, London, UK (197).. Chong C., Tper C.D., Mllus tissue ulture. I. Soritol (D-gluitol) s ron soure for llus initition nd growth. Cn. J. ot. 5, (197). 373