Propagation of Blue Honeysuckles (Lonicera caerulea L.) in In Vitro Culture

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1 164 Journl of Bsi & Applied Sienes, 214, 1, Propgtion of Blue Honeysukles (Lonier erule L.) in In Vitro Culture Krup-M kiewiz Mrelin 1,* nd Ohmin Ireneusz 2 1 Deprtment of Plnt Genetis, Breeding nd Biotehnology, West Pomernin University of Tehnology in Szzein, S owkiego 17, Szzein, Polnd 2 Deprtment of Hortiulture, West Pomernin University of Tehnology in Szzein, S owkiego 17 str., Szzein, Polnd Astrt: The im of this study ws to develop miropropgtion protool for Lonier erule L., nd Br zow, three importnt invsive woody hortiulturl plnts. Atively growing shoots from the shrus grown in the field were used for initition of ulture. Shoots were surfe sterilized with ethnol, then with sodium hypohlorite nd merury sulfte. medium supplemented with ytokinin BAP t onentrtions of mg dm -3 hd no sttistilly signifint effet on the shoot initition of seleted lue honeysukle genotypes. Multiplition rte vried depending on the genotype nd plnt growth regultor onentrtions. The highest numer of miroshoots produed per explnt of nd ws otined t using 2. mg dm -3 BAP, while of ultivr Br zow 1. mg dm -3 BAP. Shoots were rooted in vitro in the presene of IBA nd IAA. Miroshoots hve rooted differently depending on the tretment nd genotype. In the se of 58% rooting ws hieved t mg dm -3 IBA nd sl nutrient medium tretment. Keywords: In vitro, Lonier erule L., medi, miropropgtion, plnt growth regultors. INTRODUCTION Lonier elongs to the fmily Cprifoliee nd grow extensively in Europe, Asi nd North Ameri. The finely shrus or woody plnts my e used for providing food, ornmentl purposes nd lso s shelter for wildlife [1]. Lonier (honeysukle) speies re populr plnts suitle for vegettive mss propgtion vi tissue ulture [2]. The germintion rte of lue honeysukle seeds is very low. Therefore tissue ulture n e useful tool for rpid lonl propgtion nd distriution of good helth plnt mteril. In vitro ulture is used to otin more plnts in short time with less lor input nd t lower prodution osts. An dditionl feture of the method is independene of the vegettive seson [3, 4]. Severl Lonier spp. hve een mnipulted in in vitro ulture. In most reports, dventitious shoots hve een indued from lef explnts [5], llus ultures [6, 7] or xillry shoot prodution hs een hieved using L. erule [2]. Unfortuntely, it ws found tht these methods n not e rodly pplile euse of the effetiveness of the miropropgtion whih is highly genotype-speifi [8]. Hene, the im of this reserh in vitro propgtion ws to provide n effiient plnt prodution system of three genotypes of Lonier erule L. *Address orrespondene to this uthor t the Deprtment of Plnt Genetis, Breeding nd Biotehnology, West Pomernin University of Tehnology in Szzein, S owkiego 17, Szzein, Polnd; E-mil: mkrup@zut.edu.pl, Mrelin.Krup-Mlkiewiz@zut.edu.pl ISSN: / E-ISSN: /14 MATERIAL AND METHODS Initil explnts were tken from four young shoots derived from pproximtely 4-yer-old Polish lue honeysukle from the experimentl orhrd of the Pomology Deprtment. The leves were removed prior to soking the shoots for 15 minutes in wter with detergent (Ludwik wshing-up liquid), then immersed in 7% (v/v) ethnol solution for 3 s. After the preliminry disinfetion, the explnts were disinfeted with 1% (v/v) solution of sodium hypohlorite (NOCl) for 1 minutes nd with.2% (v/v) solution of merury sulfte (HgSO 4 ) for 1 minutes. Under sterile lminr flow hood, the shoot tips were rinsed three times with sterile distilled wter. Shoot frgments of size m, with n pex or node with lterl meristems were used s primry explnts for in vitro ulture. From 59 to 94 explnts were ultured in n Erlenmyer flsks, eh with 2 ml of medium [9] supplemented with 1., 2. nd 4. mg dm enzylominopurine (BAP). Plnts pled on medium without the ddition of growth regultors ws the ontrol t ll stges of the experiment. The initition stge lsted 6 weeks. The ontmintion rte nd survivl of the explnts fter steriliztion were nlyzed. Explnts initited for growth were put onto multiplition medi differed onentrtion of miro- nd mroelements (full strength, nd miro- nd mroelements), supplemented with BAP t onentrtions of 1. nd 2. mg dm -3, 25 explnts per medium. 214 Lifesiene Glol

2 Propgtion of Blue Honeysukles (Lonier erule L.) Journl of Bsi & Applied Sienes, 214 Volume Proliferted shoots were pled on rooting medi (full strength nd miro- nd mroelements supplemented with uxins: indole-3-utyri id (IBA) t the onentrtions of 2. nd mg dm -3 nd indole-3-eti id (IAA) t the onentrtion of 5. mg dm -3. At ll stges of experiments the medi were supplemented with 8. g dm -3 gr (Bioorp), 3 g dm -3 surose nd 1 mg dm -3 inositol nd their ph ws djusted to 5.7 y dding.1m of NOH or HCl. After dding growth regultors, the medi were utolved for 2 minutes t the temperture of 121 C. The ultures were mintined in growth room t temperture of 24 ± 1 º C under 16h photoperiod from fluoresent lmp (photosyntheti photon flux density 4 mol m -2 s -1 ). The men vlues of mesurements t initition nd prolifertion stges (shoot length, numer of: xillry shoots per explnt, internodes nd leves), nd t the rooting stge (plnt height, root length, numer of roots nd leves) otined in the experiments re presented in tles. The results were nlyzed sttistilly. The signifine of differenes ws determined y mens of vrine nlysis nd Tukey s test, t level of signifine of =.5. RESULT AND DISCUSSION Steriliztion of plnt explnts is one of the mjor prolems in tissue ulture. Chemil disinfetnts suh s ethnol, meruri hloride, sodium hypohlorite nd hydrogen peroxide re generlly used for surfe steriliztion of explnts for rising shoot ultures in vitro [1, 11]. Sedlák nd Ppr tein [8] used.15% solution of meruri hloride (HgCl 2 ) for disinfetion of Lonier kmtshti (Sevst.), Dziedzi [3] for disinfetion of vegettive xillry uds of lue honeysukle ultivrs Czelink nd Duet used 1% solution of lium hypohlorite (C(OCl) 2 ), nd [2,12] pplied sodium hypohlorite (NOCl) solution. The results of steriliztion proedures nd development of Lonier shoots from initil explnts re reorded in Tle 1. In this pper, two disinfetnts with different disinfetion potenies were used. For nd etter results were otined fter the using of.2% HgSO 4 for disinfetion (Tle 1). Out of the 29 explnts of nd of, only 1 explnts were ontminted with fungl infetion fter the first four weeks of ulture. Out of the left 19 unontminted explnts of nd 19 of, 16 nd 18 explnts developed shoots (respetively). After using 1% NOCl solution for disinfetion, 7 (23.3%) explnts of nd 1 (28.6%) explnts of were sujeted for further growth. However, for the ultivr Br zow, fter the pplition of.2% HgSO 4 for disinfetion, 27 explnts were infeted nd 23 explnts were seleted for further growth (Tle 1). Less infeted plnts (3 explnts) were oserved fter using 1% NOCl for the disinfetion, wheres out of the remining 2, only 8 explnts 1were seleted for further growth. The ddition of ytokinin BAP to medium did not signifintly ffet the initition of shoots from the exmined lones nd the ultivr of lue honeysukle (Figure 1). The highest plnts (5.m) were otined for the ultivr Br zow in medium supplemented with 2. mg dm -3 BAP, while the lowest (1.5m) were reported for in medium supplemented with 4. mg dm -3 BAP (Tle 2). A high numer of new shoots (2.) were formed y nd Br zow on medi with BAP t onentrtions of 4. nd 2. mg dm -3 (respetively). Krhu, Sedlák nd Ppr tein nd Dziedzi [2, 3, 8] lso hieved effiient prodution of high qulity miroshoots of L. erule f. erule y Tle 1: Surfe Steriliztion with 7% Alohol Followed y 1% NOCl nd.2% HgSO 4 for 1 min Cultivr ontminted explnts unontminted explnts tht developed shoots unontminted explnts tht did not develop shoots numer % numer % numer % Br zow 1% NOCl % HgSO % NOCl % HgSO % NOCl % HgSO

3 166 Journl of Bsi & Applied Sienes, 214 Volume 1 Mrelin nd Ireneusz Figure 1: Comprison of shoot prodution of three lue honeysukle genotypes in response to BAP onentrtions: ) Clone 44, ), ) ultivr Br zow. Tle 2: Shoot Prodution of Lonier erule L. in Response to BAP Conentrtions Genotype shoot length [m] new shoots per explnt numer of internodes numer of leves + 1. mg dm -3 BAP + 2. mg dm -3 BAP + 4. mg dm -3 BAP * men LSD mg dm -3 BAP mg dm -3 BAP mg dm -3 BAP men LSD Br zow mg dm -3 BAP mg dm -3 BAP mg dm -3 BAP men LSD *Mens in the sme olumn followed y the sme letter re not signifintly different (p <.5; Lest Signifint Differenes test LSD). supplementing nutrient medi with 2. mg dm -3 BAP. Aording to Sedlák nd Ppr tein [8] inreses in the onentrtion of BAP in the medium hd negtive impt on the numer of new shoots oserved in the ultivr Altj. Explnts of, nd Br zow honeysukle remined live in medium without PGRs (Plnt Growth Regultors), ut the prolifertion rte ws low (Tle 3). A greter numer of miroshoots were produed on medium ontining 1. mg dm -3 BAP for Br zow nd 2. mg dm -3 BAP for oth

4 Propgtion of Blue Honeysukles (Lonier erule L.) Journl of Bsi & Applied Sienes, 214 Volume Tle 3: Prolifertion of Shoot Tips of Lonier erule L. Cultured on Vrious Medi Cultivr shoot length[m] numer of new shoots per explnt numer of leves mg dm -3 BAP mg dm -3 BAP mg dm -3 BAP mg dm -3 BAP mg dm -3 BAP mg dm -3 BAP men LSD mg dm -3 BAP mg dm -3 BAP mg dm -3 BAP mg dm -3 BAP mg dm -3 BAP mg dm -3 BAP men LSD Br zow mg dm -3 BAP mg dm -3 BAP mg dm -3 BAP mg dm -3 BAP mg dm -3 BAP mg dm -3 BAP men LSD *Mens in the sme olumn followed y the sme letter re not signifintly different (p <.5; Lest Signifint Differenes test LSD). Clones. Aording to Krhu [13] onentrtions of BAP strongly ffeted the prolifertion nd growth of the form of L. erule shoots. Suzuki et l. nd Osurn et l. [12, 14] showed tht the shoots otined y primry ulture were proliferted effetively on norml strength medium supplemented with 5 M BAP. Mirouttings rooted differently depending upon the tretment nd speies [12]. Sedlák nd Ppr tein [8] rooted lue honeysukle shoots on medium supplemented with mg dm -3 IBA hieving 1% rooting nd roots of good qulity. In our study, the mjority of shoots (58%) whih developed roots were oserved for, nd the lest (1%) for the ultivr Br zow (dt not shown). Morphologilly, roots were very vrile. Roots produed in medi with supplemented with 2. mg dm -3 IBA nd 5. mg dm -3 IAA were thiker nd shorter (2.m) thn roots formed in medi with mg dm -3 IBA (4.83m) (Tle 4). developed roots only in nd medium supplemented with 2. mg dm -3 IBA nd 5. mg dm -3 IAA. In ontrst, the ultivr Br zow exhiited the wekest rooting in vitro. Dziedzi [3] suggested tht the est rooting of the ultivrs Duet nd Czelink ws hieved on WPM medi supplemented with 2. mg dm -3 IBA nd 5. mg dm -3 IAA. On the other hnd, Krhu [15] suggested tht for estlishing plnts ex vitro fous should e

5 168 Journl of Bsi & Applied Sienes, 214 Volume 1 Mrelin nd Ireneusz Tle 4: Rooting on Vrious Medium Supplemented with Different Conentrtions of IBA nd IAA Cultivr shoot length [m] root length [m] numer of roots per one shoot numer of leves + mg dm -3 IBA + mg dm -3 IBA + 2. mg dm -3 IBA+ 5. mg dm -3 IAA + 2. mg dm -3 IBA+ 5. mg dm -3 IAA men LSD mg dm -3 IBA + mg dm -3 IBA + 2. mg dm -3 IBA+ 5. mg dm -3 IAA + 2. mg dm -3 IBA+ 5. mg dm -3 IAA men LSD Br zow + mg dm -3 IBA + mg dm -3 IBA + 2. mg dm -3 IBA+ 5. mg dm -3 IAA + 2. mg dm -3 IBA+ 5. mg dm -3 IAA men LSD *Mens in the sme olumn followed y the sme letter re not signifintly different (p <.5; Lest Signifint Differenes test LSD). pled on the qulity of rooted plnts rther thn on the root numer. CONCLUSION 1. Among the solution hosen for the disinfetion of shoots from nd, the est results were otined fter the pplition of.2% HgSO 4 solution, while for the ultivr Br zow it ws 1% NOCl solution. 2. medium supplemented with ytokinin BAP t onentrtions from 1. to 4. mg dm -3 hd no sttistilly signifint effet on the shoot initition of seleted lones nd the ultivr Br zow. 3. The multiplition rte vried depending on the genotype nd the onentrtions of BAP. The highest multiplition rte ws otined on medium supplemented with 2. mg dm -3 BAP for nd nd for the ultivr Br zow with 1. mg dm -3 BAP. 4. The est miroshoot rooting rtes (58%) were hieved for when sl nutrient medium with mg dm -3 IBA ws used. REFERENCES [1] Plios N, Christou P, Leeh M. Regenertion of Lonier ttri plnts vi dventitious orgnogenesis from ultured stem explnts. Plnt Cell Rept 22; 2: [2] Krhu ST. Axillry shoot prolifertion of lue honeysukle. Plnt Cell, Tissue nd Orgn Cult 1997; 48: [3] Dziedzi E. Propgtion of lue honeysukle (Lonier erule vr. kmthti Pojrk.) in in vitro ulture. J Fruit Ornm Plnt Res 28; 16: [4] Hui JX, Wen SCh, Hu ZY, Ming LX. Comprtive study on different methods for Lonier jponi Thun. miropropgtion nd limtiztion. J Med Plnt Res 212; 6: [5] Georges D, Chenieux JC, Ohtt SJ. Plnt regenertion from ged-llus of the woody ornmentl speies Lonier jponi v. Hll s Prolifi. Plnt Cell Rep 1993; 13:

6 Propgtion of Blue Honeysukles (Lonier erule L.) Journl of Bsi & Applied Sienes, 214 Volume [6] Cmeèdes J, Duron M, Deourtye L. Adventitious ud regenertion from lef explnts of the shruy ornmentl honeysukle, Lonier nitid Wils v. Migrün: effets of thidizuron nd 2,3,5-triiodoenzoi id. Plnt Cell Rep 1991; 1: [7] Ohtt SJ. Requirements for plnt regenertion from protoplsts of the shruy ornmentl honeysukle, Lonier nitid v. Migrün. Plnt Cell, Tissue Orgn Cult 1991; 25: [8] Sedlák J, Ppr tein F. In vitro propgtion of lue honeysukle. Hort Si 27; 34: [9] Murshige T, Skoog F. A revised medium for rpid growth nd iossys with too tissue ultures. Physiol Plnt 1962; 15: [1] Yildiz M, Er C. The effet of sodium hypohlorite solutions on in vitro seedling growth nd shoot regenertion of flx (Linum usittissimum). Nturwissenshften 22; 89: [11] Tiwri AK, Tripthi S, Ll M, Mishr S. Sreening of some hemil disinfetnts for medi steriliztion during in vitro miropropgtion of sugrne. Sugr Teh 212; 14: [12] Osurn LD, Yng X, Li Y, Cheng Z-M. Miropropgtion of Jpnese honeysukle (Lonier jponi) nd Amur honeysukle (L. mkii) y shoot tip ulture. J Environ Hort 29; 27: [13] Krhu ST. Rooting of lue honeysukle miroshoots. Plnt Cell, Tissue nd Orgn Cult 1997; 48: [14] Suzuki T, Uenoht M, Oosw K. Polyploidy reeding of lue honeysukle nd lk hokeerry y utilizing in vitroultures treted with olhiine. At Hort 26; 76: [15] Krhu ST. Performne of Lonier miroutting s ffeted y minerl nutrients nd genotype. At Hort 23; 616: Reeived on Aepted on Pulished on Mrelin nd Ireneusz; Liensee Lifesiene Glol. This is n open ess rtile liensed under the terms of the Cretive Commons Attriution Non-Commeril Liense ( whih permits unrestrited, non-ommeril use, distriution nd reprodution in ny medium, provided the work is properly ited.