Characteristics of micro-propagated banana (Musa spp.) cultures stressed with NaCl and polyethylene glycol

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1 Afrin Journl of Biotehnology Vol. 10(1), pp , 3 My, 011 Aville online t DOI: /AJB ISSN Aemi Journls Full Length Reserh Pper Chrteristis of miro-propgte nn (Mus spp.) ultures stresse with NCl n polyethylene glyol Ikrm-ul-Hq 1,*, Mih Ftim 1, Hin Shikh 1, Muhmm Umr Dhot 1, Muhmm Thir Rjput, Furqn Memon 1, Ali Muhmm Dhri 1, Shh Nwz 3 n Aul Ltif 4 1 Institute of Biotehnology n Geneti Engineering (IBGE), University of Sinh, Jmshoro-76080, Pkistn. Institute of Plnt Sienes, University of Sinh, Jmshoro-76080, Pkistn. 3 Deprtment Plnt Breeing n Genetis, Sinh Agriulture University, Tnojm, Pkistn. 4 Byer Institute of Crop Sienes, Hyer, Pkistn. Aepte 15 April, 011 The effet of NCl n PEG ws ssesse on plnt miro-propgtion rte in nn (Mus spp.) v., Bsri. Well miro-propgte plntlets were ulture on four ifferent stresses of NCl n PEG-4000 inluing ontrol level: MS (MS mg l BAP), MS (MS mol m NCl), MS (MS 0 + 5% PEG) n MS e (MS mol m NCl + 5 % PEG) for 6-weeks. Effiieny of plnt miro-propgtion ws reue signifintly mong the stresse ultures. Similrly, photosyntheti pigments like hl ws erese non-signifintly ut hl, hl were erese signifintly. Totl rotenois were inrese in the sline s well s PEG stresse ultures. Cell size of epiermis n erenhym ws inrese (p < 0.05), while prenhym erese. Proline n glyineetin ontents were inrese (p < 0.05) in eh stresse ulture ut were high in MS thn in MS 3 n MS 4 ultures. Menwhile, proteins, sugrs, phenolis n nitrtes were oserve to e in the reverse (p < 0.05) phenomen. In onlusion, NCl tretment ws oserve to e most toxi thn the PEG or PEG with NCl on the nn miro-propgtion. Key wors: Mus spp., miro-propgtion, NCl (soium hlorie), PEG (polyethylene glyol), hlorophyll ontents, proline, reuing sugrs. INTRODUCTION Bnn (Mus spp.) rop is eliious fruit with 5% rohyrtes. Being n importnt stple foo rop, it provies vlule soure of inome for omesti s tre. Toy, the rte of the worl s nn proution hs een pproximtely 44 tons hetre (Frison et l., 1997; Roux et l., 001; FAO, 005). Commeril yiel of nn is eing lost y numer of vrile environmentl stresses. *Corresponing uthor. E-mil: ro.ikrm@yhoo.om. Tel: Arevitions: MS, Murshige n Skoog; NCl, soium hlorie; PEG-4000, polyethylene glyol 4000; Chl, hlorophyll; IAA, inole eti i; BA, enzyleminopurine; NOCl, soium hypohlorite; B5 vit, B5 vitmins. Among the ioti stresses, exessive sline onitions of the soil uses loss of oth vegettive potentil n reproutive plnt growth in rops (Neumnn, 1997). Inherent tolerne in rops n goo mngement prties my enle prtil meliortion of mge from soil slinity (Cherin n Rey, 003; Bine et l., 004; Flowers, 004; Dvenport et l., 005). Similrly, rought requires proper unerstning for etter plnt growth. It uses efiit wter supply to the growing plnts. Menwhile, slinity uses inhiitory effets on plnt growth euse of the toxiity of exessive N + n Cl - to the sorne of wter. It n e ttriute to erese in vilility of the wter require or impose y evelope osmoti stress (Kefu et l., 003; Djiril et l., 005; Munns et l., 006). However, oth rought n slinity re involve in limiting plnt growth. These re eoming wiespre in

2 4388 Afr. J. Biotehnol. the worl n my onvert more thn 50% rle soil to sline soil in 050 (Alm et l., 00; Wng et l., 003; FAO, 005). With the pssge of time, mny rops hve evolve ertin mehnisms in metolism to omt wter efiit stresse onitions (Seghin n Yvri, 004). Among the stresse plnt ultures, one of suh mehnism is uiquitous, tht is the umultion of vrious orgni metolites espeilly uring see germintion thn in vegettive growth (Bewley n Blk, 1994). Similrly, polyethylene glyol (PEG) inues wter stress when supplie in plnt growth ultures (Ruf et l., 1967; Kufmn n Ekr, 1971). It reues the osmoti potentil in nutrient ulture meium. This inert osmotium is not tken in the plnt ell n phyto-toxiity (Lwlor, 1970). Presently, septi plnt ultures is gining muh importne for the evelopment of ioti stress resistnt plnts n lso in eluiting physiologil n iohemil operting mehnisms tht involve uner stresse onitions. These spets re to keep plnts survivl uner severe ioti stresses t the ellulr level ginst rought n slinity (Jin, 001). Plnt tissue ulture provie ontrol n homogeneity stresse onition for the mesurements of ell ehviors uner stress onitions t the whole plnt level; not for single regultory system (Lutts et l., 004). During this stuy, effets of oth ioni n wter efiit stresses uner in-vitro onition through the pplition of NCl n PEG or oth were oserve in miropropgting plntlets of nn (Mus spp.). After 6- weeks of stress, numer of morpho-io-omponents were stuie. Differentil phenomen were oserve in the stresse ultures thn in the ontrol. This onute work my e useful in the future for the improvement of slt or rought resistnt plnts or their seletion. MATERIALS AND METHODS Apprently, helthy sukers of Bsri ultivr (Mus spp.) were ollete from open-ir nn fiels. Inner portions of the suker (meristem) were exise. They were sterilize with ethnol (90%) tretment for 1 min. They were wshe with wter n thn stirre for 30 min in 30% Roin leh (5.5% NOCl). Orgnogenesis in the sterilize explnts ws inue y ulturing them on MS (MS (Murshige n Skoog, 196) sl meium, B 5 vitmins (Gmorg et l., 1968) n 3% surose plnt nutrient meium, supplemente with 10 µm IAA n 8 µm BA. After 3-weeks of ulture, they were su-ulture on MS [MS, BA (15 µm), phytgel 1.0 g l )] meium for shoot inution for -weeks. This orgnogenesis ulture ws su-ulture on MS [MS, BA (15 µm), phytgel (.0 g l )] nutrient meium for multiplition of inue shoots (Hq n Dhot, 007). Plntlets from (MS ) ultures were su-ulture on ifferentilly stresse miro-propgtion ultures. They were represente s MS (ontrol), MS (MS mol m NCl), MS (MS + 5% PEG-4000) n MS e (MS mol m NCl + 5% PEG-4000) ultures. Eh estlishe ultures were supplie with 3% surose (s ron soure) n 0 µm L-ystein. The 5.7 to 5.8 ph ws juste efore utolving (11 C n 0 ls for 15 min). Among the estlishe ultures, eh tretment ws ompromise on 7- replites. These ultures were mintine uner light onitions with ~000 lux light intensity for 18/6 h for 6 weeks t 5 ± 1 C. The miro-propgte plntlets from ll the ultures exise n entngle meium ws wshe with tp-wter. They were rie with filter-pper n t ws ollete. Numers of plntlets propgte in the single explnt were ounte. Shoot height n its iomss were lso mesure. Photosyntheti pigments were etermine s y Lihtenthler (1987). Antomy of lef ws lso stuie oring to Gielwnowsk et l. (005) n Johnsen (1940) methos. Both glyine-etin (Grieve n Grtter, 1983) n proline (Btes et l., 1973) ontents were etermine spetrophotometrilly, y using fresh lef tissues. Phenol ontents were etermine y using fresh leves from eh ulture (Ozyigit et l., 007). Plnt mteril ws rie t 7 C in eletri oven for -ys. It ws use to stuy vrious io-hemil hrters. Protein ontents (Brfor, 1976) Totl rohyrtes (Duois et l., 1956; Cih n Brun, 1978), reuing sugrs (Miller, 1959) n nitrte ontents (Morris n Riley, 1963) were etermine. Dt signifine ws lulte using COSTAT omputer sttistil softwre, inluing LSD t 5% level of ifferene mong the ultures. RESULTS AND DISCUSSION Verstile soil omposition hs een oserve in nture. Some environmentl ftors re ville in soils tht re involve in plnt growth inhiition. Similr experiment ws onute to ssess the effet of NCl n PEG seprtely s well s in omintion on plnt miropropgtion effiieny of nn (Mus spp.) v., Bsri. In this experiment, 4-weeks ol plntlets of the 3r suulture with well propgtion on MS nutrient were suulture on three ifferentilly stresse ultures whih were MS (100 mol m NCl), MS (5% PEG) n MS e (100 mol m NCl + 5% PEG) inluing MS (est shoot multiplition ulture) for 6 weeks. A numer of vritions mong the io-metris of the multiplying plntlets were oserve in omprison to the ontrol ulture (MS ). Mximum shoot multiplition ws oserve on MS meium supplemente with BA (Hq n Dhot, 007, ), while, multiplition rte ws erese signifintly in ll the stresse ultures in the orer, MS > MS > MS e. These stresses not only reue the multiplition shoots mong the ultures, ut lso the shoot height, shoot fresh weight s well s the shoot ry weight were lso erese (p < 0.05). Inluing growth rte of the ultures, photosyntheti pigments were lso ffete (Hq et l., 008; Ch-um et l., 010). Chlorophyll ws erese non-signifintly, while hlorophyll n totl hlorophyll ontents were lso erese ut signifintly. Totl rotenois were inrese signifintly in ll the stresse ultures (Tle 1; Figure 1). A signifint inrese in the epierml ells n erese in erenhymtous ells (p < 0.05) ws oserve, while non-signifint hnge in the size of mesophyll ells ourre (Tle 1; Figure ). Generlly, ll the ioti stresses on plnts use injury in the hloroplsts tht result to erese in the mount of photosyntheti pigments uner oth slinity n/or

3 Ikrm-ul-Hq et l Tle 1. Comprtive growth-relte hrteristis uner ifferent NCl n PEG stresse onitions in the miro-propgte nn (Mus spp.) v., Bsri. #s Chrters MS (0 mol m NCl) A. Miro-propgtion effiieny. # of plntlets / explnt 8.33± Plnt height (m) 3.57±0.10. Fresh weight (g) 6.391± Dry weight (g) 0.50±0.04 B. Chlorophyll ontents (mg g F.Wt). Chlorophyll 0.978± Chlorophyll 0.399±0.006 Chlorophyll ± Totl rotenois 58.6±8.88 C. Antomil fetures. Epierml ells (µm) ±1.74. Prenhym ells (µm) 58.4±.4. Aerenhym ells (µm) 89.15±1.999 D. Biohemil ontents. Protein ontents (mg g ).517±0.01. Totl sugrs (mg g ) 0.643±0.49. Reuing sugrs (mg g ) 0.794± Proline (mg g ) 0.833±0.006 e. Glyineetine (mg g ) 0.635±0.01 f. Phenol (mmol g ) 0.116±0.007 g. Nitrtes (µ mol g ) 1.16±0.061 A MS (100 mol m NCl) MSe(5% PEG+ 100 mol m NCl MS (5% PEG) Signifine 5.33± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±13.60 ns * 114.6± ± ± ± ± ± ± ± ±3.534 * ns.051± ± ± ± ± ± ±.006 B C.039± ± ± ± ± ± ± ± ± ± ± ± ± ±0.047 D Figure 1. Almost 6-weeks ol miro-propgte plntlets of nn (Mus spp.) v., Bsri on ifferent stresse ultures uner in vitro onitions. A, MS (ontrol); B, MS (100 mol m NCl); C, MS (5% PEG); D, MSe, (100 mol m NCl + 5% PEG). wter efiit onitions onentrtions of the free mino is like s proline n glyineetin. Within the mesophyll ells, oth proline n photosyntheti pigments re synthesize from the sme preursor. Uner stresse onitions, this inter-onnetion is performe in n opposite mnner; photosyntheti pigments ereses, while proline ontents inrese uner slinity (NCl), wter efiit (PEG) n slinity + wter efiit onitions (Aspinl n Pleg, 1981). Reuing sugr ontents were lso inrese uner the stresse onitions, while totl proteins n rohyrtes erese (p < 0.05). Phenolis inrese

4 4390 Afr. J. Biotehnol. Epierml ells Aerenhym ells Mesophyll ells MS MS MS MS e Figure. Antomil spets (ell sizes) of nn (Mus spp.) v., Bsri ulture on ifferent plnt nutrient mei supplemente with ifferent slt n rought stressing gents. Epierml, erenhymtous n mesophyll ells in the plntlets ulture on MS (ontrol), MS (100 mol m NCl), MS (5% PEG) n MS e, (100 mol m NCl + 5% PEG). (p < 0.05), while nitrte ontents were erese signifintly. The umultion of ertin ontents in the stress ultures my e for the sfety of the ell t tht moment or for the future. After the pplie stress, the umulte io-omponents inite soure of ron, nitrogen n energy for reovering stresse tissues, when stress-es were relxe (Blum n Eeron, 1976; Slm et l., 007). All the ioti stresses suh s wter efiit stress (rought) or sline stress re proly involve in limiting

5 Ikrm-ul-Hq et l plnt growth signifintly (Hrtmnn et l., 005). Both stresses (slinity n rought) re metolilly s well s physiologilly relte phenomen, s oth inue osmoti stress on the ffete plnts (Djiril et l., 005; Vnen n Zeng, 006). In this experiment, lmost ll the prmeters were onsiere to e ffete signifintly in the miro-propgting plntlets of nn (Mus spp.) ultivr Bsri. Both photosyntheti pigments n orgni ontents evelope ifferentil hrteristis in the stresse ultures in omprison with the ontrol. REFERENCES Alm MZ, Stuhury T, Nylor RE (00). Effet of NCl n PEG inue osmoti potentils on germintion n erly seeling growth of rie ultivrs iffering in slt tolerne. Pk J. Biol. Si. 5: Aspinl D, Pleg LG (1981). Proline umultion. In: Physiology n Biohemistry of Drought Resistne in Plnts (Physiology Aspets), Aemi Press, New York, pp Bine FA, Diouf D, Sné D, Diouf O, Gouiy V, Dillo N (004). Sreening owpe [Vign unguiult (L.) Wlp.] vrieties y inuing wter efiit n RAPD nlyses. Afr. J. 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