-tdz

V Vo l. 13(35), p p . 3581-3590, 27 7 Aug ust, 2014 4 D DOI: 10.5897/ AJB2014.13762 A 2 A Artic le Numb e r: 95327824705 55 IS SSN 1684-5315 5 C Co p yrig ht © 20 014 A Autho r(s) re ta in n the c o p yrig ht h o f this a rtic le e h http:/ / www.a c a d e mic jo urna ls.o rg / AJB Africcan Journal of Bioteechnology Fu ull Length Research h Paper T Thidiaz zuron-iinduce ed in vitro v bu ud orga anoge enesis of the da ate palm (Pho oenix dactyllifera L L.) CV. Hillaw wi Ah hmed Mad di Waheed A Al-Mayahi Depa artment of Pla ant Tissue Culture, Date Pa alm Research h Centre, University of Bassra, Basra, Ira aq. Rec ceived 27 Februa ary, 2014; Accep pted 3 July, 2014 4 The objective of the pre esent was to o enhance th he frequency y of plant reg generation in n date palm (Phoenix dactylifera L.) cv. Hiillawi. Expla ants were incubated on Murashiige and Sk koog (MS) medium zyladenine (BA) ( and diffferent conc centrations (0.1 to 2.0 mgl-1) of supplementted with 1 mgl-1 6-benz thidiazuran (TDZ), or frree of BA an nd TDZ (con ntrol treatme ent). The res sults indicatte that the M Maximum s observed on medium m supplemen nted with 1..0 mgl-1 BA and 0.5 mg response (66.67%) was gl-1 TDZ, producing an a average of o 4.2 and 18 8.2 buds perr culture afte er 16 and 24 week from c culture, resp pectively. TDZ at conc centrations higher h than 0.5 0 mgl-1 resu ulted in supp pressed bud ds formation,, where a dec crease in the numberr of buds was w noticed when w the co oncentration of TDZ was s increased from 0.5 to o 2 mgl-1. Regarding the activity of o antioxidan nt enzyme peroxidase p d during budding of date p palm cv. Hilla awi, (The chemical analyses a of peroxidase e compound ds were spe ectrophotome etrically perrformed) the e applied concentratiion of 0.5 mg gl-1 TDZ with h 1 mgl-1 BA enhanced pe eroxidase ac ctivity, where peroxidase e activity was assoc ciated with increased number of buds form mation. Histtological stu udies revea aled that adventitious buds were e formed dire ectly from ep pidermal cellls without ca allus formatiion, and adv ventitious f meriste ematic cells in i shoot tip tissues. Sho oots were elongated on 0.5 mgl-1 buds were developed from d on MS m media supplemented w with 0.2 mg gl-1 of αGA3+ 0.1 mgl-1 NAA MS media and rooted naphthaleneacetic acid d (NAA). Rooted shoots s were succ cessfully acc climatized a and establish hed in a p moss an nd perlite (2::1) with 80% success. mixture of peat t (T TDZ), adventittious buds, pe eroxidase (PO OD), histologyy. Key words: Date palm, thidiazuran INT TRODUCTION N Datte palm (Pho oenix dactylife era L.) the dioecious, mon nocotyyledon specie es belonging to the family Arecaceae is sa multipurpose tre ee having food, medicinal and ornamen ntal imp portance. Witth the prese ent uncertaintty in the wo orld food supply and d the expecte ed increase in demand, the t date palm could b be a good sou urce of food o of high nutria ationall value (Khan n and Bi, 2012 2). It has long g been one of o the m most importan nt fruit crops in the arid rregions of the e Arabiian Peninsula a, North Africa, and the e Middle Eas st o, 2007). Iraq q was one of the top ten date producers (Chao *Co orresponding au uthor. E-mail: hng_1988@y yahoo.com. Author(s) agree th hat this article remain permanently open ac ccess under th he terms of the e Creative Com mmons Attribution License 4.0 Inte ernational Licen nse 3582 Afr. J. Biotechnol. producers in the world between 1991 and 2001, where it contributed a 7.5% of the total world dates production (FAO, 2011). The vegetative multiplication of date palm is traditionally achieved by offshoots. This offshoot propagation has limitations such as slow propagation rate, transmission of disease-causing pathogens and insects and production of offshoots in a limited number for a certain period in the lifetime of a young palm tree (Gueye et al., 2009). Date palm readily grows from seeds but half of the seedlings may turn out to be males and high proportion of inferior quality segregates (Al-Khalifah and Shanavaskhan, 2012; Mohammad, 2013). Furthermore, seedlings take 6 to 10 years to fruit, so male and female trees are not identifiable until flowering (Othmani et al., 2009). Hence, in vitro propagation is the only available alternative to produce disease free, uniform and good quality planting material to establish large scale cultivation within a short period of time (Zaid and De- Wet, 2002). In vitro production is applied through two main protocol, one of them is the somatic embryogenesis and the second is via meristim apexes or buds in the axil bottom of the leaves (Bekheet et al., 2001; Eke et al., 2005). Micropropagation through direct organogenesis lacking callus phase, has the advantage of producing highly identical plants to the mother plants in their vegetative characteristics (Khan and Bi, 2012). Shoot tips are most appropriate explant used for date palm in vitro multiplication (Al-Mayahi, 2013). Thidiazuron (TDZ) is a cytokinin-like substance that has often been used for shoot regeneration in recent years (Erisen et al., 2011). Also, it is a powerful regulator of in vitro plant regeneration and subsequent growth (Murthy et al., 1998). There are many reports showing that the application of thidiazuron (TDZ; N-phenyl-N'-1,2,3-thiadiazol-5-ylurea) results in a better shoot regeneration capacity in comparison with other cytokinins (Srikandarajah et al., 2001; Zhang et al., 2001; Thomas, 2003; Husain et al., 2007). TDZ is used as a plant growth regulator to stimulate high rate of axillary shoot proliferation in many woody plant species, and its releases the lateral bud dormancy and stimulates shoot formation in wide variety of plant species (Malik and Saxena, 1992; Anandan et al., 2011). In date palm, N-phenyl N'-1,2,3-thidiazol-5-ylurea (TDZ) is used in the stimulation of direct somatic embryo regeneration from shoot tip explants (Sidky and Zaid, 2011). The plant cells possess highly efficient defence systems for elimination of the harmful effect of oxidative stress. Guaiacol- peroxidase (EC 1.11.1.7), catalase (EC 1.11.1.6) and ascorbate-peroxidase (EC 1.11.1.11) are among enzymes expressing antioxidative functions, where peroxidase (EC 1.11.1.7) is considered among enzymes expressing antioxidative functions (KapchinaToteva and Yakimova, 1997). There are still limited data concerning plant regeneration using thidiazuron (TDZ), and the peroxidase activity for date palm is through direct budding. The overall objectives of this study were to determine the optimal concentration of TDZ for adventi- tious buds regeneration from shoot tip and investigate the relationship between TDZ and peroxidase through budding in date palm tissues cultured cv. Hillawi in vitro. Cultivar Hillawi is a good soft date with good quality and famous in Iraq. It early ripens early and yields about 94.3 kg/palm and it is best for raw eating at rutab stage (Vij et al., 2005). But this cultivar is suffers from some problems; the ageing of the adult plants as well as study the histological events related to direct regeneration from shoot tips. MATERIALS AND METHODS Plant material The experiments were conducted in the Laboratory of Plant Tissue Culture at the Palms and Dates Research Centre, University of Basra. The success of tissue culture largely relies on the selection of suitable explants for use as the starting material for the experiment. The selected offshoots cv. Hillawi were 3 to 4 years old. Cleaning of explants Cleaning of the explants was done according to Junaid and Khan (2009). In short, the offshoots were washed with tap water to remove the attached soil and other debris. The outer large leaves and fibers were carefully removed with a sharp knife until the shoot tip zone was exposed. Sterilization efficient The explants were kept in 1% sodium hypochlorite solution mixed with one drop/100 ml of Tween-20 for 20 min followed by 4 to 5 rinses in distilled water. It is recommended to give sterilization for 10 min with fresh sodium hypochlorite solution. Then, the explants were immersed in 0.1% mercuric chloride solution for 5 min, followed by 4 to 5 washes in distilled water. Sterilized explants were kept in a cold sterilized solution of ascorbic and citric acid (150 mgl1 ) to avoid browning (Al-Khalifah and Shanavaskhan, 2012). Establishment of initial cultures In this study, shoot tips were used for culture initiation (Figure 1). The shoot tip terminal, about 1 to 1.5 cm long, was sectioned longitudinally into four sections. Several media were tested. Murashige and Skoog (MS) (1962) basal medium supplemented with 1.0 mgl-1 NAA, 1.0 mgl-1 Naphthoxy acetic acid (NOA), 1.0 Indol butyric acid (IBA), 1.0 mgl-1 kinetin (K), 100 mgl-1 glutamine, 5 mgl-1 thiamine HCl, 1 mgl-1 biotin, 30 gl-1 sucrose, 2.0 gl-1 activated charcoal, and solidified with agar at 5.0 mgl-1 were used. All the media were adjusted to pH 5.8 with 0.1 N NaOH or HCl, before the addition of agar. Media were dispensed into culture jars. All jars with media were autoclaved at 121°C and 1.04 kg/cm² for 15 min. Cultures were kept under complete darkness at 27±2°C which provide the cultivation on initiation medium that enhanced the percentage of explant survival which was 60%, where explants started exhibiting signs expansion after 3 to 4 weeks, as well as reduced browning. Similar reports are given by (Al-Maarri and Al-Ghamdi, 1997; AlMayahi, 2014a). Al-Maya ahi Figure 1. Apica al buds used in this t study. 3583 3 Figure 3B. Roo oted plantlets ready for tran nsplanting to F p lastic pots. media a (MS) contain ning 1.0 mgl-1 BA and Thidiiazuron TDZ at a ent concentrations (0.1, 0.5, 1.0 0, 1.5 and 2.0 m mgl-1), or used as s differe free o of 6-benzylamino opurine (BA) and TDZ (contro ol treatment). All A the cu ultures were inccubated in a cu ulture room maintained at 27 ± 2°C u nder 16/8 h, an nd light/dark wiith 55 to 60% rrelative humidity plicated 6 times s. (RH). Every treatment in the experriment was rep ubcultured on to Buds formed on this medium were divided and su obtaining enoug gh number from m fresh media every 6 weeks until o buds tto complement this study (Figure 2). The datta was recorded as folllows: The perccentage of resp ponse of culture es on direct bud formattion after 16 we eek; number of d direct buds/explant after 16 and 24 we eks. Shoott elongation, rooting and a acclimatization n of date palm m plantl ets Figure 2. Subcultured buds on n to fresh media. Figure 3A. Shoo ots formed on th he elongation me edium. Bud ds initiation and multiplicatio on The e developing exp plants were shiffted on initiation and multiplication Develo oped buds werre isolated and d transferred to o the elongation mediu um supplemente ed with 0.5 mg gl-1 Gibberellic Acid GA3+ 0.1 mgl-1 α α-Naphthalene acetic acid (NA AA). Well-deve eloped shoots (5 cm lon ng) (Figure 3A)) were separate ed from each o other, and trans sferred individually to rooting medium m supplemented d with NAA (0.2 d Al-Ghamdi, 1997). The rootted plants were mg-1) (Al-Maarri and m the vesselss, washed initiially to remove gentlyy removed from ed agar and trraces of the m medium to avoid d contamination adhere (Figure e 3B). Then, th he plantlets we ere washed with distilled wate er -1 and trreated with fung gicide (Benlet 5 500 mg ) for 20 0 min and trans soclaved a mixtu ure of peat moss ferred to plastic pots containing auto erlite (2:1) (AL-Mayahi, 2014b)). The plants we ere covered with and pe glass bottles to mai ntain humidity (Figure 3C). T The plants were h inorganic saltss of MS medium m initiallyy irrigated with q quarter-strength for 2 w week followed by tap water. P Potted plantletss were grown in culture e room (25±2°C C, 55±5% RH, under 16 h of ph hotoperiod with a light in ntensity of 40 μm mol m-2 s-1) for 4 45 to 60 days. T The glass bottles were gradually remo oved upon em mergence of new leaves and matized plantletss were transferre ed to the greenh house. acclim Estim mation of peroxidase at a budd ding stage To ext xtract the enzym me, 1.0 g of ho omogenized buds tissues were ground d in 20 ml. cold d distilled water in a mortar at 0 0°C. The extrac ct was o obtained by filttering off the d debris with a cclean cloth and centriffuging at 3000 rrpm for 15 min in a refrigerated centrifuge. The e 358 84 Afr. J. J Biotechnol. Figure 3C. 3 Coverage off plant by glass bottle. Table 1. Effect E of differen nt concentrations of TDZ in com mbination with 1 1.0 BA on perce entage of culturres responding and number of buds for date palm cv. Hillawi. Treatmen nt (mgl-1) 0 0.1 TDZ 0.5 TDZ 1.0 TDZ 1.5 TDZ 2.0 TDZ Percentage e of explants responding (A After 16 weeks s) 1 BA+ 1 BA+ 1 BA+ 1 BA+ 1 BA+ 0.0±0.0e 33.34±2.06c a 66.67±1.11 b 50.0±1.05 33.34±2.06c d 16.67 ±0.0 Average n number of Bud ds / explants ((S.E)* After 16 weeks After 24 wee eks e e 0.0±0..0 ** 0.0±0.0 2.0±0 0.2c 0.29 c± 4.0 4.2±0 0.14a 2 1.2a±18.2 2.8 ±0 0.7b 0.14b±7.2 2 1.5±0 0.4cd 0.75 d±2.6 6 1.0 ±0 0.0d 0.0 d±2.0 0 * ± Standarrd error (n = 6). ** Values followed d by the same le etter are not signiificantly differentt at P<0.05. supernatants were recovered and kept in a tube in an ice bath until u ayed. POD activity was assay yed spectrophotometrically Mo odel assa CEC CEIL CE-2021 at a 470 nm using g guaiacol as a phenolic substrrate with h hydrogen perroxide (Díaz et al., 2001). The e reaction mixtture conttained 0.15 mL of 4% (v/v) guaiacol, 0.15 mL L of 1% (v/v) H2O2, 2.66 6 mL of 0.1 M phosphate p buffe er pH 7 and 40 µL of the enzy yme extrract. The blank k sample conta ained the same e mixture soluttion with hout the enzyme e extract. Histtological analy ysis Histtological examinations during bud formation were carried out usin ng a freezing microtome. Microtome M slide preparation and a observation were made following g the methods as described by Sarkker and Awal (1999) Exp perimental design and statistical analysis mpletely random mized design wa as used. The data was subjec cted Com to th he analysis of variance v and mean values werre compared us sing revised LSD at 5% (Snedecor and Cochran, 1989 9). RESU ULTS Buds s initiation an nd multiplica ation The a adventitious b buds develop ped from shoot tips on MS S mediu um suppleme ented with a combination n of TDZ and d BA affter 4 to 6 months of cultu ure without b basal callusing g (Tabl e 1). The op ptimal response percentag ge of explants ucing buds w with the high hest number of buds pe er produ expla ant was recorrded on MS m medium supplemented with h 1 mg gl-1 BA+ 0.5 m mgl-1 TDZ. On this medium, 66.67% of o the ccultures respo onded (Figurre 4A) with a an average of o 4.2±0 0.14 and 18.2 2±1.2 buds per explant affter 16 and 24 4 weekk, respectivelyy (Table 1, F Figures 4A a and B), which h was sstatistically significant com mpared with th he other treattmentss, followed b by 1 mgl-1 BA A + 1.0 mgl-1 TDZ (Figure e 4C). The response percentag ge of explan nts producing g buds and frequenccy of direct bu ud regeneratiion decreased d signifificantly when the concenttration of TDZ Z was increa ased o over 0.5 mgll-1, whereas TDZ at high her concentra a- Al-Maya ahi 3585 5 Fig gure 4. Bud pro oliferation and multiplication m A)) Bud induction from shoot tip on MS media ssupplemented w with 0.5 mgl-1 TDZ + 1.0 mgl-1 -1 -1 -1 BA A after 16 week ks. B and C) Bu ud proliferation on o 0.5 mgl TD DZ + 1.0 mgl B BA and 1.0 mgll TDZ +1.0 mg gl-1 BA media a after 24 weeks, resspectively. Table 2. Efffect of differentt concentrations s of TDZ in combination n with 1.0 BA on Activity off guaiacolperoxidase through direct budding b in in vitrro cultured date palm cv. Hillawi. Treatment (mgl-1) 0 0.1 TDZ 1 BA+ 0.5 TDZ 1 BA+ 1.0 TDZ 1 BA+ 1.5 TDZ 1 BA+ 2.0 TDZ 1 BA+ POD activitty (U/ml) 14.74 48d 19.79 94c 33.15 59a 24.16 66b 21.03 39c 16.89 99d * Values follo owed by the sam me letter are not significantly s different at P<0.05. P tion ns, specially 1.5 1 and 2.0 mgl m -1, resulted d in suppress sed bud ds formation. In the absen nce of BA an nd TDZ (conttrol trea atment) there e was not an ny response for direct bu uds form mation, imply ying that these compounds s are critical for bud d regeneration n in date palm m cv. Hillawi. Perroxidase "PO OD" activity On the basis of the obtained results in the e present stud dy, ble 2, illustrattes a measurre of the activ vity of guaiac colTab perroxidase "PO OD" regarding g the effect of o TDZ on bud b devvelopment off in vitro culttured date pa alm cv. Hillawi. Perroxidase activ vity in date palm buds wa as stimulated by TDZ Z with BA. Thus, cytokinin ns increase peroxidase p ac ctivityy whereas, lo owest activity was observed in conttrol bud ds. In buds grown on med dium containing 1 mgl-1 BA+ -1 2.0 mgl TDZ, the activity of peroxidase e did not difffer ase was exhiisignifficantly from control buds. More increa bited in buds culttured on med dium supplem mented with 1 mgl-1 BA and 0.5 mgl-1 TDZ, w where activity of peroxidase e ed significanttly compared with the othe differe er treatments. Histo ological origiin of adventitious buddin ng The anatomy of buds regen nerated in viitro by direc ct nogenesis fro om shoot tipss was investigated in date e organ palm cv. Hillawi. T The repeated cultivation on bud-forming g mediu um in the presence of ccytokinin, pro oduced tissue e massses which rap pidly propaga ated and divided, and can n alwayys produce n new buds which grow or appear at the e surfacce of the ma asses or insid de them. Hisstological sec ctions showed that the epidermal cells were the source of o organ nogenesis. T The structure exhibited la arge cells no ot uniforrm in size an nd compactne ess. Also, the ere were scatttered cell clusterss near the ep pidermis. The ese cells were e distin nguished by ttheir finenesss and compactness, which h was cconsidered a as the reason n of the protru usions' forma ation, and consequently the fo ormation of tthe promeris stemattic tissue. Sim multaneouslyy, with the diffferentiation of o epide ermal cells a and the subssequent adve entitious buds s forma ation, meriste ematic cells (MC) becam me more and d more abundant du ue to the conttinuing divisio on in the shoo ot tip tisssue (Figure 5 5A). Cell diffe erentiation be ecame eviden nt throu gh appearance of large nucleus underrgoing division n conta aining dense cytoplasm. The meristem matic isolates emerrged from larrge and stron ngly vacuolatted parenchy ymatou us cells. The ere meristem matic zones ccould develop p into m meristems (F Figure 5B). F From the MC C cells, apica al merisstem (AM) a and leaf prim mordia (LP) differentiated d underr the same co onditions in cculture (Figure e 5C). Severa al merisstematic reg gions differen ntiated and these were e 358 86 Afr. J. J Biotechnol. o an adventitiou us bud develope ed from shoot tip of date palm cv. Hillawi cultu ured on MS Figure 5. Trransverse histological section of + 0.5 mg/l TDZ T + 1 mg/l BA A. A) Mristematiic center (MC) originating o from the shoot tip, cconsists of very fine active cellss, scale bar 100 μm. B)) Formation of meristematic which w constituted d by small cellss, scale bar 50 0 μm. C) Apica al meristem (AM M) and leaf primordia (L LP), scale bar 50 0 μm. D) Adven ntitious buds with h apical meriste em (AM), leavess (L) and vascullar bundles (VB), scale bar 50 μm. responsible for the formation n of adventitio ous buds. Co ontinu uity of cells division led to vascular bun ndles which can c be either induce ed to form oth her independ dent nodules, or erentiating in nto a bud. It is worth men ntioning the ada diffe ven ntitious buds induction i proc cess showed that it emerg ged succcessively from basal supe erficial of node e. Initially, sm mall bulg ges raised fro om the epidermal cell of th he node (Figu ure 5D)). Also, the nodules develo oped into bud ds when mov ved to a an auxin free e medium. Such nodules developed in nto disttinct buds orr leaf primord dium. Figure 5D shows well w devveloped buds s with apicall meristem, leaves (L) and a vasscular bundles s (VB). ebuds were found from shoots (Figure 6A).. The regene rated shoots were e transferred to rooting m medium MS + 0.2 m mgl-1 NAA (Figure 6B), and rooted d successfully y (80% %) with rapid e elongation, with an averag ge of 4.4 roots per s hoot and sho oot of an averrage root leng gth of 5.2 cm m. After 6 weeks off culture (Figure 6C). Complete plants s nerated planttwere obtained 6 tto 8 weeks after the regen were transferrred to this m medium. The rooted plants lets w were acclimatized successfullyy in a mixture of peat moss perlite (2:1) w with 80% afterr 10 weeks of transferred to o and p plastiic pots (Figu ure 7). All th he micropropa agated plants were free from extternal defectss. Sho oot elongatio on, rooting and a acclimattization of da ate palm plantlets DISC CUSSION The e budding tis ssues formed d in the seco ond step, we ere tran nsferred to th he elongation medium sup pplemented with w 0.5 mgl-1 GA3 + 0.1 mgl-1 NA AA. After 8 to o 10 weeks, the t Direcct regeneratio on is the usefful means of production of o plantllets with a lo ower risk of genetic insta ability than by y otherr routes (Kha an and Bi, 2 2012). The composition of o inducction media is importan nt for adventitious buds s Al-Maya ahi 3587 7 gure 6. A) elon ngation of shoo ots on MS med dia supplemente ed with 0.5 mg gl-1 GA3+ 0.1 mgl-1 NAA. B)) Shoots on roo oting medium Fig supplemented with 0.2 mgl-1 NAA A. C) Rooting of shoots on same media after 6 . Figure 7. Date e palm plantlets s transplanted in plastic pots ffilled with mixture of peat moss s and perlite 2:1 ratio (v/v). devvelopment, the presence of o cytokinin is critical for bu uds induction and for f differentia ation from ex xplants of da ate palm, where ad dventitious buds b were not observed in cyto okinin-free medium (contro ol treatment).. Cytokinin (B BA) and d cytokinin-lik ke compound d (TDZ) brea ak apical dom minan nce (Tawfik and a Mohamed d, 2006). Thiidiazuron (TD DZ) hass gained a considerable e attention during d the pa ast deccades due to o its efficient role in plant cell and tiss sue cultture. The hig ghest number of buds (18 8.2 per expla ant afte er 24 weeks) was induced d from shoot tip explants on MS m medium with 0 0.5 mgl-1 TDZ Z and 1.0 mgl-1 BA (Table 1 and F Figure 5B). Such a respon nse may perhaps be due to o the in ncrease in th he levels of endogenous cytokinins by y the e effect of the e TDZ used, which brin ngs about an n increa ase in the levvel of naturally occurring ccytokinins, and d it is liikely to have a common ssite of action with the natu urally occurring cyytokinins (R Ruzić and Vu ujović, 2008)). Also, Casanoval e et al. (2004) determined the effects of o ous plant gro owth regulato ors in organo oTDZ on endogeno TDZ levels. Also, the T TDZ mediated d genessis as low T altera ation in the ccytokinin biosyynthetic pathway might be e respo onsible for the e depletion of the endogenous 2iP poo ol and the elevated d concentrations of the other purine e metab bolites (Zhan ng et al., 2005). TDZ can highly induce e synth hesizing, gath hering and m modifying oth her produced d cytokkinins (Visserr et al., 199 92). It is well known tha at cytokkinins stimula ate plant cell division and participate in n the re elease of late eral bud dorm mancy, in the e induction of o adven ntitious bud fformation, in the growth o of lateral buds s and i n the cell cyycle control (G Gaspar et al., 2003). TDZ Zed plant tissu ues enhanced d endogenouss auxin meta atreate bolism m and transp port (Murch and Saxena,, 2001). Also o, Nabil a et al. (2003) found thatt TDZ had be een useful fo or of economically importan nt secondary y the p production o metab bolites in som me plant speccies. The othe er possibilities includ de the mod dification in cell membra anes, energy y levelss, nutrient up ptake, or nuttrient assimillation (Murthy y and S Saxena, 1998 8; Murthy et al., 1998; Guo et al., 2011).. Mo reover, Abba asi et al. (201 11) reported tthat TDZ inhiibits ssynthesis of abscisic acid d according ((Li and Yang g, 1998)), where the frequency o of induction a and growth of o buds was found to vary signiificantly depe ending on the e conce entrations of T TDZ. It mightt be due to the e low concen- 3588 Afr. J. Biotechnol. trations of TDZ inhibiting synthesis of abscisic acid was more than high concentrations. These results agree with the reports of Lincy and Sasikumar (2010) which suggested that combinations of TDZ and other plant growth regulators could be more effective than TDZ used alone. Also, these results are similar to those reported by Husain et al. (2007) and Husaini and Abdin (2007) who confirmed that the frequency of shoot regeneration ability declined markedly at higher concentrations of TDZ. Also, higher concentrations of TDZ hindered further growth and development of the regenerates (Shirani et al., 2010). Generally, explants cultured on media supplemented with low levels of TDZ were positive for adventitious buds regeneration. TDZ has been shown to stimulate buds regeneration at low concentrations and was used mainly in combination with other plant growth regulators. These results suggest that the response of explants to buds formation ensure the development of budding, necessary to modify the hormonal balance in favour of the cytokinins. Moreover, it is reported that TDZ is the best choice compared with other phytohormones for protoplast proliferation (Murthy et al., 1998; Xiao et al., 2007). The acceleration of peroxidase activity was associated with increased number of buds under TDZ effect, and had more stability and important role in POD synthesis. The stimulation of peroxidase in the present experiments in accordance to enhancement of bud growth and development at 0.5 mgl-1 TDZ, supports the view that TDZ might make this enzyme active, thus controlling the level of H2O2 and the rate of cell division (KapchinaToteva and Yakimova,1997). In addition, we suggest that the activity of peroxidase could be used as a biochemical marker of development in the plant object studied. Moreover, TDZ promotes the activities of POD, which may be one reason for budding. Sharifi and Ebrahimzadeh (2010) and Mamaghani et al. (2010) reported that antioxidant enzymes play an important role in the organogenesis of 20 plants as confirmed by analysis of POD which was also, reported by Ezaki et al. (1996). This enzyme has high activity as a marker during stressful conditions. In addition, such an enhancement of peroxidase activity can be caused by stress due to changes in media composition; where the TDZ is the induction of a stress response (Murthy et al., 1998; Abbasi et al., 2011), and stress has been considered to be a stimulus to developmental switch by reprogramming gene expression and reorganizing cellular state (Fehér et al., 2003). During this process, some defense-related genes may be induced to adapt the stress conditions. The ability of the explant tissue to survive the applied stresses of the culture process seems to be an integral part of the morphogenic phenomena and some studies provide indication of the factors involved in the regulation of plant regeneration, with various active forms of peroxidase involved in growth regulation, development and organogenesis. The acceleration of enzyme activity was associated with formation of more shoots (Kapchina-Toteva et al., 2005) since plant peroxidases are involved in many functions such as growth, vegetative development, resistance to biotic and abiotic stresses (Gonzalez-Verdejo et al., 2006). The results of the present study are in agreement with the results of other studies related to using cytokinins on peroxidase activity (Synková et al., 2006), and with Wang et al. (1991) who reported that many of the TDZstimulated enzymes were associated with cell walls membranes and membrane fluidity was modified. Also, this result is in accordance with earlier reportof an enhancement of peroxidase activity in response to TDZ (Todor and Iordanka, 1995). As the overall result, it seems that changes in POD activity, is an index for regeneration. TDZ is resistant to oxidases, is stable, but biologically more active at low concentrations. These properties may enhance future use in tissue culture manipulations. These findings may promote further investigations of the physiological properties and selectivity of phenylurea cytokinins. The importance of the cytokinins in releasing the process of meristems and, consequently, formation, is well-known; these formations may be caused by the cytokinins that enhance the multiplication of the DNA, and the chromosomes separation which encourages the cell division (Auge, 1984). The formation of the organs in the moncotyledonae is generally, enhanced by addition of the cytokenines (Duhoux, 1988). Also, the nodules developed into buds when moved to an auxin free medium. Moreover, organogenesis, due to the influence of growth regulators present in the medium, is the result of dedifferentiation of certain cells showing a mitotic activity. This also showed that cell division is initiated in the epidermal layers and that from a multiplelayered epidermis occasional meristematic bulges are produced. Regeneration of adventitious bud meristems formed directly on explants in vitro is often initiated by cell divisions beginning in the epidermal. Adventitious buds primordial is initiated as a result of organized directional growth of cells from meristematic cells. Although mitotic activity was found throughout the explant, activity was concentrated in the epidermis regions that were in close contact or adjacent to the nutrient media. Clusters of cells began to appear in the subepidermal region of the explant. Regeneration in this manner is widespread, occurring in monocotyledons (Crinum macowanii) (Slabbert et al., 1995). The initial cell divisions result in a mass of small cells forming new meristematic primordia. Continuity of division cells led to vascular bundles, where development of the vascular bundles is essential to guarantee the transport between tissues and distant organs, assuring the growth. Conclusion In the present study, the in vitro protocol for regenerating plantlets of date palm cv. Hillawi using shoot tip explants was described. Since the plantlets were developed direct- Al-Mayahi ly without intervening of callus phase, it can be concluded that results obtained in this study, permit the development of a mass propagation protocol with a good budding rate and a high regeneration percentage. Data suggest that TDZ and BA were indispensable for in vitro propagation of date palm since no excisable shoots were produced on MS-0 medium (control). 0.5 mgl-1 TDZ with 1 mgl-1 BA are recommended as a component of culture media. Stimulation of number of buds was accompanied by an enhancement of guaiacol peroxidase activity. Histological studies revealed the development of meristemmatic regions, which later developed into buds meristems. Conflict of Interests The author(s) have not declared any conflict of interests. 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