International Journal of Green and Herbal Chemistry

Transcription

1 IJGHC, June 215 August 215; Sec. B; Vol.4, No.3, Interntionl Journl of Green nd Herl Chemistry E-ISSN: Reserch Article An Interntionl Peer Review E-3 Journl of Sciences Aville online twww.ijghc.com Section A: Green Chemistry CODEN (USA): IJGHAY In Vitro Micropropgtion nd Conservtion of Prunus Ntive Stone Fruit Trees Dorin (Bode) Xhulj 1, Efigjeni Kongjik 2 nd Skerdilid Xhulj 3 1 Genetic Resources Centre, Agriculture University of Tirn, Alni 2 Alnin Science Acdemy, Tirn 3 Fculty of Nturl Sciences, Tirn, Alni Received: 1June 215; Revised: 3June 215; Accepted: 6 July 215 Astrct: The present study imed to evlute the in vitro micropropgtion nd conservtion potentil of wild ntive stone fruit trees, prt of the genus Prunus. The results otined suggest tht the type of explnts used cn ffect the estlishment nd in vitro propgtion of these wild species. Shoot tips cn dpt esier compred to other type of explnts used. The presence of BAP hormone, is necessry to promote the prolifertion of our four plnt species explnts during the first stge of in vitro propgtion. Plntlets development nd their micropropgtion coefficient vlues suggest their dption to the in vitro multipliction phse. In this study the use of BAP in low levels, induct shootformtionwith higher vlues of length, numer of leves nd dditionl uds compred to higher concentrtions of it. The results otined on the in vitro rooting process, indicte tht the reduction of minerl concentrtion of MS medi nutrient, in hlf of their norml vlues, nd the presence of uxin in high levels cn ffect the stimultion nd formtion of new roots. Applying the drkness regime during the rooting phse gve positive effect in rhizogenesis process of. Using slow growth techniques (low tempertures) we could storge our Prunus stone fruits for period from 3.5 months to7.5 months. Keywords: Prunus sp., micropropgtion, shoot tips, hormones, rooting 43 IJGHC, June 215 August 215; Sec. A; Vol.4, No.3,

2 INTRODUCTION Micro propgtion is the process of vegettive growth nd multipliction from plnts tissues or seeds. It is crried out in septic nd fvorle conditions on growth medi, using vrious plnt tissue culture techniques 1. Tissue culture is sed on concept of totipotency; the ility of plnt cells nd tissues to develop into whole new plnt 2. Alni represent one of the Europen countries with rech flor, due to relief vritions, fvorle climtic condition, geogrphicl position different geologicl nd soil lyers. Aout 3 % of Europen flor is grown in Alni s gret ntionl sset with economic nd Alno logicl vlues 3. Prt of this vlule reservoir of germplsm is ntive nd cultivted s: P. spinos, P.cersifer, P. persic, P. rmenic etc. Mny of the ntive cultivrs of Prunus gener geogrphiclly distriuted in ll the Prunus re of the country cll for ttention ecuse they constitute n essentil genetic resource which is very precious for hyridiztion work nd for stone fruit reeding. Socil economic nd environmentl impcts justify the need to preserve these genetic resources 4. Tissue culture techniques re of gret interest for the colleting, multipliction nd storge of plnt germplsm 5,6. Thus, gret effort is eing mde to get pproprite regenertion protocols for these plnts nd recent reports hve een pulished for different Prunus species, s in pricot 7, cherry 8 nd pech 9. Regenertion of stone fruit trees is usully difficult to chieve nd, while success hs een otined using juvenile tissues, few reports exist from mture plnts 7. Some of these works, s well s other previous reports on pricot nd plum 1 11 involved multiple steps nd mnipultions tht include trnsfers to different medi, successive chnges in environmentl conditions, nd preconditioning of source plnt mteril, suggesting tht orgnogenesis is ffected y severl fctors, some of them not precisely defined. The present study imed to evlute the in vitro estlishment, prolifertion nd multipliction potentil of ntive stone fruit trees, prt of the genus Prunus. MATERIAL AND METHOD Explnt desinfection: Active shoot tips nd lterl uds of 4 Prunus popultion suject of our study, efore the inocultion process under lminr flux, were treted with ) 7º ethnol solution (1 minute) nd rinsed three times in distilled wter, followed with.1% HgCl 2 solution for severl minutes (gin rinsed three times in distilled wter); ) shoot tips tht preliminry were rinsed with run wter (3-6 minutes), were immerse in NOCl.3% solution (1 minutes) thn merged with mphiciline ntiiotic (5 minutes), t the end rinsed severel time with distilillted wter under lminr flux. Type of nutrient medium used: under this study were inoculted in two different nutrient medium; sic nutrient medium MS 12 nd the modified nutrient medium for wood plnts species WP 13. Bsed on the initil results otined for ech Prunus sp. the pproprite medium is selected for the prolifertion nd multipliction of plntlets. In vitro micropropgtion: Stge 1. Explnt prolifertion of Prunus rmenic: Both types of nutrient medium MS nd WP were used for this first stge. In the sic solutions of mcro nd microelements were dded: MS medium: (i) mio-inositol 1 mg l -1 (ii) BAP.7 mg l -1, GA 3.1 mg l -1, ANA.1 mg l -1. Type of crohidrte 44 IJGHC, June 215 August 215; Sec. A; Vol.4, No.3,

3 used, shroze 3%, for nutrient solidifiction ws used gr.6% nd nutrient ph t the level of 5.9. WP medium: (i) mio-inositol 1 mg l -1 (ii) BAP 1 mg l -1. Type of crohidrte used, shroze 3%, for nutrient solidifiction ws used gr.6% nd nutrient ph t the level of 5.6. Stge 2. Suculture. Medium used for this stge is the sme oftht of prolifertion one, WPM. Shoot tips with minimlly two leves were inoculted in the nutrient medium. Stge 1. Explnt prolifertion of woody plnts: P. cersifer, P. persic nd P.spinos. Explnts were tested in oth MS nd WP nutrient medium. MS first medium: (i) mio-inositol 1 mg l -1 (ii) BAP.7 mg l -1, GA 3.1 mg l -1 nd ANA.1 mg l -1. Type of crohidrte used, shroze 3%, for nutrient solidifiction ws used gr.6% nd nutrient ph t the level of 5.9. MS second medium: (i) mio-inositol 1 mg l -1 (ii) BAP 1 mg l -1, GA 3 1 mg l -1 sence of ANA. Type of crohidrte used, shroze 1% for nutrient solidifiction ws used gr.3% nd nutrient ph t the level of 5.9. WP modified medium: (i) mio-inositol 1 mg l -1 (ii) BAP 1 mg l -1. Type of crohidrte used, shroze 3%, gr ws dded t the level of.6% nd the ph ws precisenned on 5.6. Stge 2. Suculture: Nutrient medium used for this stge ws MS supplemented with BAP in different mounts: (i).3 mg l -1, (ii).7 mg l -1 nd (iii) 1 mg l -1. Also is dded different type of uxine, AIB.1 mg l -1 nd t the nd phitohormone GA 3.3 mg l -1. Shrose s crohidrte (3%) nd gr for medium solidifiction (.6%). Nutrient medium ph ws 5.6. Stge 3. Plntlets rooting inittion nd development: Bsed on similir studies on rooting process on species of genus Prunus 14-17, t this stge in vitro developed shoots with n verge of 4-6 leves were plced in different nutrient medium. Plntlets lso were tested for the impct of light/drkness effect on rooting promotion. Specificlly plntlets were plced for period of 12 dys in complete drkness followed y regime of photoperiode 16 hour lightness/8 hours drkness. Terms of cultivtion in the cultured room. After leling with the nme nd type of explnts, dte of culturing, ll the cultures were plced in culture chmer with controlled physicl prmeters (temperture 23⁰C ±2⁰C, lighting intensity 2 lux nd photoperiod 16 hour lightness/24 hours). Plnt mteril in vitro storge y using miniml growth method: After sucultering, in vitro plntlets were hels for period of two weks in norml conditions of in vitro culture (temp. 23⁰C ±2⁰C, photoperiod 16 hour lightness/24 hours). Susequently this period the plnts were storge in refrigirtor t 4 C of temperture in drkness. Experimentl results elortion: The performnce of the experiments nd the determintion of the rigenertion nd propgting potencil is determinted y iometric indictors such us: Survil rte in %; min shoot length; numer of uds nd lterl shoots developed per explnt; numer of leves, root length etc. In vitro plnt mteril were photogrphed with digitl photogrphed cmer. RESULT AND DISCUSSION Results of desinfecttion method: Among two tretments of desinfecttions used on our Prunus explnts, the one of NOCl comined with mphiciline ntiiotic, resulted unpropieded for the in vitro 45 IJGHC, June 215 August 215; Sec. A; Vol.4, No.3,

4 estlishment (1% of Prunus explnts were infected). Desinfecttion method of.1% HgCl 2 resulted succesfull with high survivl rte of 88.46% for Prunus spinos explnts, stisfctory for plums explnts on level of 58.97%, menwhile 66.6% nd 89.74% were the survivl rte respectitvely for P.rmenic nd P.persic explnts used in our study. Results otined coincide nd re within the limits of results reported from other uthors 14,18,where the survivl rte of Prunus sp. vry from 27-93%. Results of the prolifertion phse: Type of medium used effected the prolifertion process of Prunus explnts, emphizising the ide tht the composition with micro nd mcro elements nd the presence nd type of hormones, cn effect the prolfertion process of plnt mteril. MS medi were pproprite for the prolifertion of our three Prunus explnts, plum, lckthorn nd pech repectitvely proliferted in 7%, 57.14% nd 47.82% (Fig.1). WP medi ws promotive for the estlishment nd the prolifertion of pricot explnts in the level of 62%, while in the sme medi P.spinos exlpnt rected negtively (5.36% of prolifertion). Reports from other studies suggest the use of MS medi in comperison with different nutrient medi for the estlishment nd in vitro multipliction of Prunus spieces. Results otined in our study coincide with those pulished in similir studies for Prunus sp 7,19,2. According to other reserches 19 the prolifertion level of plum explnts on sl MS medi ws 2-8%. Prunus persic explnts rect positively on MS full medi (6 % prolfertion) in comperison with hlf MS medi 21. According to other studies 22 the reson why full strength MS ws not eing successful for culturing pricot explnts might e ttriuted to high concentrtion of nitrogen nd/or high totl slts. Nitrogen concentrtion in WPM is less thn tht of MS medium. Figure 1: Buds nd shoot tips of Prunus sp. during prolifertion. Shoot length results of our 4 Prunus sp. during prolifertion phse originted from ctive shoot tips (Figure 2). Plntlets originted from pricot shoot tips, presented men shoot length higher in vlue (1.6 mm), thn other plntlets. 46 IJGHC, June 215 August 215; Sec. A; Vol.4, No.3,

5 12 ± Shoot length c de c P.cersifer P.persic P.spinos P.rmenic Plnt Species Figure 2:Dt of Prunus sp. length plntlets originted from shoot tips Figure 3:Prunus cersifer plntlets during in vitro prolifertion Plums plntlets were the one with the higher numer of leves (8.6 leves/explnt) nd Punus persic tht with the lower results (3.65 leves/explnts, Fig.3). 1 ± nr.of leves c P.cersifer P.persic P.spinos P.rmenic Figure 4:Dt on numer of leves Prunus sp. plntlets originted from shoot tips during prolifertion 47 IJGHC, June 215 August 215; Sec. A; Vol.4, No.3,

6 We otined different results from Prunus plntlets originted from lterl uds. Dt show tht in this cse Prunus rmenic plntlets presents the higher results (7.25 leves/explnts) nd Prunus spinos the lower one (4.8) for the sme prmeter (Figure 5). 8 ±nr.of leves c P.cersifer P.persic P.spinos P.rmenic Figure 5: Dt on numer of leves Prunus sp. plntlets originted from lterl ctive uds during prolifertion Plntlets of pricot nd plums originted from lterl uds show the higher vlues in shoot length prmeter menwhile peches plntlets re those with the lower vlue (3.65mm, Figure 6). ± Shoot length P.cersifer P.persic P.spinos P.rmenic Figure 6: Dt of Prunus sp. length plntlets originted from lterl ctive uds during prolifertion Type of explnts used ffected the dynmic of development of our four Prunus spieces. Plntlets originted from ctive shoot tips presented men shoot length higher (for 4 Prunus sp.) during the prolifertion stge, in comprison with plntlets originted from lterl uds. Within the sme species WP medi resulted positive for shoot tip explnts of pricot eside lterl ctive uds; we recorder the sme for Prunus spinos plntlets ut inoculted in MS medi, menwhile plntlets of two other Prunus species in our study originted from lterl uds presented lightly higher vlues in in vitro multipliction thn shoot tips inoculted in the sme type of medi. 48 IJGHC, June 215 August 215; Sec. A; Vol.4, No.3,

7 12 1 Shoot tips Buds ± Shoot length Pr.cersifer Pr.persic Pr.spinos Pr.rmenic Figure 7: Comprison of plntlets shoot length during prolifertion mong types of explnts used ± numer of leves Shoot tips Buds Pr.cersifer Pr.persic Pr.spinos Pr.rmenic Figure 8: Comprison of ± numer of leves mong Prunus sp. during prolifertion sed on type of explnts used. Dt recorded for nr. of leves prmeter show tht plntlets originted from lterl uds, presents lightly higher vlues (fig.8) in compre with plntlets formed from shoot tips explnts, for the sme terms of experiment. Type of medi used in this cse doesn t influence in essentil distinctions for nr. of leves prmeter for ll of our Prunus sp. Results of multipliction phse: During the second phse Prunus sp. plntlets presented higher vlue in numer of leves prmeter (±1.55) in comprison of first phse (±5.65). Prunus plntlets show high coefficient of vrition for the sme prmeter. As it is shown (fig.9) does not exist ny significtiv difference in nr. of leves prmeter mong plntlets originted from shoot tips explnts nd type of medi used. 49 IJGHC, June 215 August 215; Sec. A; Vol.4, No.3,

8 ± nr. of leves c P.cersifer P.persic P.spinos P.rmenic Figure 9: Comprison of ± numer of leves mong Prunus sp. during suculture originted from shoot tips. Type of medi used ffected the development of Prunus plntlets for the shoot length prmeter. As it is shown (fig.1) Prunus rmenic plntlets cultured in WP medi( sence of GA 3 ) presented higher vlues (± 21.65mm) within the sme levels of proility in compre of three other Prunus sp. cultured in MS medi (presence of GA 3 ). In vitro shoot elongtion is criticl process within the micro propgtion system; it considerle depends from the composition of nutrient medi 23. The use of shroze s source of crohydrte (3 g) in WP medi in our study resulted in plntlets with height more thn 2 cm. This result is in ccordnce with those pulished from other uthors 24, for the sme type nd mount of crohydrte used. During this phse our Prunus plnts originted from shoot tips represented duplicted in men shoot length prmeter in comprison with the vlues mesured during the first phse of in vitro micropropgtion. ± shhot length c c cd P.cersifer P.persic P.spinos P.rmenic Figure 1: Comprison of ± shoot length mong Prunus sp. during suculture originted from shoot tips. Plnts originted from lterl uds presented high coefficient of vrition 39.48% for the nr. of leves prmeter. Type of medi ffected the results of nr. of leves iometric prmeter for the sme terms of experiment used for ll our Prunus spieces. MS medi used hs fvoured the development of leves in plums plntlets s well s in peches nd lckthorn(fig.11, 12), menwhile we couldn t sy the sme for WP medi used for pricot plntlets. 41 IJGHC, June 215 August 215; Sec. A; Vol.4, No.3,

9 Prunus plntlets in our study presented n verge of shoot length duplicted (±13.77 mm), in compre of the first phse of in vitro micropropgtion mesured for the sme trit (±5.98 mm). Dt recorded show (fig.13) n predominnce in shoot length of Prunus persic plntlets (±19.55 mm) in compre of three other, their vlues re qudruplicted during this phse. Plntlets presents 49.51% coefficient of vrition mong them for the sme trit. 15 ± Nr.of leves 1 5 cd P.cersifer P.persic P.spinos P.rmenic Figure 11: Comprison of ± numer of leves mong Prunus sp. during suculture originted from lterl uds Figure 12:() Plums plntlets; () Peches plntlets during suculture 411 IJGHC, June 215 August 215; Sec. A; Vol.4, No.3,

10 ± shoot length c P.cersifer P.persic P.spinos P.rmenic Figure 13:Comprison of ± shoot length mong Prunus sp. during suculture originted from lterl uds Although plums nd pricot plntlets re cultured in different type of medi, they presented similr vlues for the sme trit, suggesting tht the composition of nutrient medi doesn t ffect the expression of the trit. Type of explnts used for Prunus persic nd Prunus rmenic influenced the results during the second phse for the shoot length prmeter. In vitro multipliction of Prunus persic plntlets, cultured in MS medi (in the presence of BAP.3 mg l -1, AIB.1 mg l -1 GA 3.3 mg l -1 ) fvored the development of plntlets originted from lterl uds, with higher vlues of shoot length, in comprison of shoot tips explnts used in the sme type of nutrient medi (fig.14). Length/mm Pr.cersifer Pr.persic Pr.spinos Pr.rmenic Shoot tips Buds Figure14: Comprison on shoot length mong Prunus sp. sed on type of explnt Menwhile pricot plntlets originted from shoot tips explnts inoculted in WP nutrient medi ( only in the presence of BAP 1 mg l -1 ), presented higher vlues of shoot length prmeter (Fig.15 & Fig.16) in comprison with plntlets originted from lterl uds. Even in this cse, the trit under study (type of explnts) is different not only etween species ut lso within the sme specie for the sme terms of condition, suggesting tht the micropropgtion of pricot plntlets is most fvored if s initil explnts re used ctive shoot tips eside lterl uds. 412 IJGHC, June 215 August 215; Sec. A; Vol.4, No.3,

11 Figure 15: Apricot plntlets during suculture Type of initil explnts inoculted in nutrient medi, ffected the results mesured for the numer of leves prmeter during the multipliction stge of our Prunus in vitro propgtion (Fig.16). As it is shown, there re no significnt differences in Prunus spinos plntlets originted from two types of explnts for the nr. of leves prmeter, menwhile dt recorded suggest tht for plums plntlets type of explnt used ply n importnt role, fvored shoot tips explnts. There is diminutive distinct for the sme trit in the cse of Prunus persic plntlets, in fvor of lterl ctive uds. ± nr.of levs Shoot tips Buds Pr.cersifer Pr.persic Pr.spinos Pr.rmenic Figure 16: Comprison of ± numer of leves mong Prunus sp. from two types of explnts used, during suculture RESULTS OF ROOTING PHASE Results otined on in vitro rooting process show tht the reduction of the minerl concentrtion in the MS medium (1/2 MS medium) is ssoccited with the rootlets genesis. The pliction of β-indol utiric cid (AIB 1 mg l -1 ) ply positive role on root genesis, its sence in the nutrient medium ws ccompnied with no root development on our Prunus plntlets, emphzising the ide tht the dministrtion of this phitohormone from the plnts explnts is necessry for the stimultion nd development of rooting process. According to the otined results from the 5 rooting medium used in our study, the only one with the hlf concentrtion on microelements in their vlues, the presence of phitohormone ANA 2 mg l -1, photoperiode 16 light/ours nd 8 ours/drkness develop rootlets in percent of 1 % for pricot plntlets. The reminder ws suject of cllusgenesis, reching up to 6 % (Fig.17.). 413 IJGHC, June 215 August 215; Sec. A; Vol.4, No.3,

12 Lower concentrtion of uxine hormone in the nutrient medium ccompnied with lower concentrtions of shroze didn t led to the cllus genesis process, in the plnts shoots. Our root inittion results on pricot shoots, goes within the limits reported 25 on in vitro rooting of 11 peches cultivrs (6-95% rooting explnts). We didnt record ny significnt difference in iometric prmeters (shoot length nd nr. of leves) etween our sudied Prunus sp.. Similir results on increse in percentge of cllusgenesis t higher concentrtions of ANA mg l -1 in hlf MS medium is reported in in vitro propgtion of Prunus persic cultivrs in percentge of 2-4% 21. In our study the use of uxine phitohormone in high concentrtion stimultes the orgnogenesis, menwhile its sence or low levels inhiits root genesis. Figure 17. () Plntlets in cllus: () Prunus cersifer in rooting;( c) Prunus rmenic in rooting The ppliction of 2 weeks of drkness regime followed from 8 weeks cultured in 16 our light regime (1/2 MS medium, AIB 1 mg l -1 ), effected positivily rooting process (Fig. 17.c). As result pricot exlnts incresed the percente of rooting in the level of 2%, mentime wild plums explnts rooted in percentege of 18%. Similir results re reported 22 where plnts of control in the sinence of AIB didn t root, while the use of AIB 1-2 mg l -1 effect positively the rooting of pricots cultivrs from 26.7% to 53.3%. The presence of drkness during prolifertion stge, influence positivily the rooting process ut ws followed y clorozes nd necrozes signs on our leves plntlets. Results of in vitro storge: Prunus plntlets under study were stored y using low temperture technique (4⁰C in refrigirtor), in period of 7.5 months (Prunus rmenic) nd 3.5 months (Prunus cersifer). The rigenertion level of our plnts from the storge ws high. Our results is higher of tht reported from other uthors 26 on Prunus rmenic sp., ccording to whom, the storge in low tempertures riched up till 4.5 months. Intersting to mention ws the insignifictiv morphologicl differences etween plntlets tht were under storge. The ilitty of plnts in low temperture to hve no chnges in shoot length prmeter might e the result of the cumultion of scisic cid, which couses the slow growth of shoots in this terms 27, lso might e due to the overproduction of ethylene s result of osmotic process, low lightning level nd stress to tempertures 28. The ppliction of miniml growth method pperes the pproprite wy for the interntionl exchnge of Prunus sp. plnt germplsm. 414 IJGHC, June 215 August 215; Sec. A; Vol.4, No.3,

13 CONCLUSION The results otined suggest tht the type of explnts used cn ffect the estlishment nd in vitro propgtion of these wild species. Shoot tips cn dpt esier compred to other type of explnts used. The presence of BAP hormone, is necessry to promote the prolifertion of our four plnt species explnts during the first stge of in vitro propgtion. Plntlets development nd their micropropgtion coefficient vlues suggest their dption to the in vitro multipliction phse. In this study the use of BAP in low levels, induct shoot formtion with higher vlues of length, numer of leves nd dditionl uds compred to higher concentrtions of it. The results otined on the in vitro rooting process, indicte tht the reduction of minerl concentrtion of MS medi nutrient, in hlf of their norml vlues, nd the presence of uxin in high levels cn ffect the stimultion nd formtion of new roots. Applying the drkness regime during the rooting phse gve positive effect in rhizogenesis process of. Using slow growth techniques (low tempertures) we could storge our Prunus stone fruits for period from 3.5 months to 7.5 months. REFERENCES 1. L.G.Zhou nd J.Y.Wu; Development nd Appliction of Medicinl Plnt Tissue Cultures for Production of Drugs nd Herl Medicinls in Chin. Nturl Product Reports; 26, 23: M.R.Fowler, F.W.Ryns nd C.F.Hunter (Ed.); The lnguge nd ims of plnt cell nd tissue culture: In Vitro Cultivtion of Plnt Cells, Butterworth-Heinemnn Ltd., Oxford; 1993, J. Vngjeli, B. Ruci nd A. Mullj; Bimët e kërcënur dhe të rrll të Shqipërise.Liri i Kuq; E.Kongjik, V.Sot, A.Bcu,Zh.Zekj, D. Bode, F.Bni nd A.Myrt; Use of tissue culture methods for the conservtion of vlule germplsm of Alnin flor species,buletini I ShkencveNtyrore; 211,1: V.A.Srsn, R. Cripps, M.M. Rmsy, C. Atherton, M.McMichen, G. Prendergst,nd J.K.Rowntree;Conservtion in vitro of thretened plnts-progress in the pst decde, In vitro cell Dev.Biol.Plnt;26, 42: M.Cpunnd F.Ponti; In vitro medium term conservtion of Myrtuscommunis L., Propgtion of ornmentl plnts; (28) 8 (2): O. Perez-Tornero nd L. Burgos; Different medi requirements for micropropgtion of pricot cultivrs. Plnt Cell, Tissue nd Orgn Culture, Dordrecht;(2)vol.63: N.J. Grnt nd N.Hmmt;Adventitious shoot development of wild cherry (Pr.vium) leves. New For.2: Y.W.Sindhu; Invitromicropropgtion of medicinl plnts y tissue culture. The Plymouth Student Scientist; 21, 4(1): A. Gentile, S. Monticcelli nd C.Dmino; Adventitious shoot regenertion in pech (Prunuspersic L.)Plnt Cell Report; 22, 2: M.Antonelli nd Ph. Drurt;The use of rief 2,4-D tretment to induce lef regenertion on PrunuscnescensBois. ActHortic 199, 28: V.Esclettes nd F.Dos;In vitro dventitious shoot regenertion from leves of Prunusspp. Plnt Science 23, 9: IJGHC, June 215 August 215; Sec. A; Vol.4, No.3,

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