Pk. J. Bot., 5(5): 1871-1876, 218. EFFECT OF DIFFERENT GROWTH REGULATORS ON IN VITRO PROPAGATION OF BRASSICA NAPUS L. SARWAT NAZ 1, MUHAMMAD FAHEEM SIDDIQUI 1 *AND SABOOHI RAZA 2 1 Deprtment of Botny, University of Krhi, Krhi-7527, Pkistn 2 Deprtment of Agriulture nd Agriusiness Mngement, University of Krhi, Krhi-7527, Pkistn * Corresponding uthor s emil: mfsiddiqui@uok.edu.pk Astrt A lrge mount of foreign exhnge is expending to import the edile oil in Pkistn. The improvement of lol oil produing vrieties suh s nol my ply vitl role for etter soio-eonomi ondition of Pkistn. Plnt tissue ulture tehnique is one of the methods for rop improvement through somlonl vrition. The present study ws onduted to evlute the response of different explnts inluding hypootyl, lef, node nd otyledon of nol (Brssi npus L.) on Murshige nd Skoog medium supplemented with 2,4-D, NAA, IBA, BAP, 2,4-D+BAP t vrious onentrtions. Eh explnt responds differently in the presene of phytohormones. The est llus ws otined t.1 mg/l 2,4-D with hypootyl explnts. The optimized lli ws further shifted to different shooting medi supplemented with BAP (-3mg/l), without or with silver nitrte (5mg/l) nd BAP (2mg/l) with or without Kinetin (-3mg/l). The mximum numer of shoots ws formed on MS medi ontining 5mg/l silver nitrte nd 2mg/l BAP. The elongted shoots were inoulted on different rooting medi ontining hlf MS medi ugmented with or without IBA t different onentrtions. The mximum root regenertion ws notied on hormone free hlf MS medi. The present study will e useful for indiret propgtion of Brssi npus, whih ould e utilised for rop improvement of vi somlonl vrition. Key words: Brssi npus L, Cllus indution, Regenertion, Auxins, Cytokinins. Introdution Brssi npus L. elongs to fmily Brssiee (Cruifere) ommonly known s rpeseed or Cnol. It is found in ountries with temperte limte or s winter rop in sutropil limte (Vughn nd Geissler, 29). Brssi npus ws improved through the onventionl reeding for low erui id (<2%) nd gluosinoltes (<3 umol/g) for humn onsumption in form of Cnol. Cnol ws identified s helthy oil produing rop with low level of sturted ftty id (7%). It ontins high level of olei id, linolei id, nd lph-linoleni id, whih re good for rdiovsulr helth (Lin et l., 213). Animl feed nd iodiesel my lso e produed from nol (Ry et l., 21). It lso ers mediinl importne s it is used s emollient, diureti nd ntiterrhl (Khre, 27). The oil of nol seed ws lso reported to ure gllstone (Khre, 27). Pkistn imports out 1.79 million tonnes of edile oil/ oil seeds tht ost pproximtely US $ 1.37 illion (Pkistn Bureu of Sttistis, 215). Brssi npus L. is the seond most importnt soure of edile oil in Pkistn (Munir et l., 28). Pkistn produed 27 thousnd tonnes of rpe seed in 216 y ultivting it on n re of 226 thousnd hetre. The world verge rpe seed prodution is 2.23tonnes/hetre. However in Pkistn the verge prodution of rpe seeds per unit hetre is only.8548 tonnes/hetre, sine lst twenty yer. In 216 only.9144 tonnes/hetre yields ws produed (FAO sttistis, 218). The redued yield potentil of the ultivted vrieties needs n improvement of this rop for etter yield. Plnt tissue ulture tehniques offer n opportunity for the geneti improvement of the rop (Khr et l., 22). Plnt tissue ulture tehnique hs een suessfully utilized for the onservtion of germplsm of endngered nd rre speies, prodution of pthogen free plnts (Fy, 1992) nd to indue vriility in mny rops (Lrkin & Sowroft, 1981; Jin et l., 199; Ktiyr, 1997 nd Khr et l., 22). In Brssi npus L. vrious explnts were suessfully used for llus indution y different workers; stem ws used s explnts y Shrm & Gupt, 212. Mny reserhers used hypootyls for llus indution (Duey & Gupt, 214; Borjin & Ark, 213; Burulis et l., 28; Munir et l., 28; Qmrunnis et l., 215), while otyledon ws used s explnts y Chmndostiet l., 26; nodl stem ws used y Duey & Gupt, 214 nd nther y Hssn et l., 1994. The present work ws rried out in order to estlish n optimized protool for llus indution of Brssi npus L. vriety Hyol 41 using different explnts. Hypootyls, otyledons, nodl stems nd leves were used s explnts with different growth regultors nd to regenerte plntlets through llus. Mteril nd Methods Plnt mteril: Seeds of Brssi npus L. vriety Hyol 41 provided y ICI life Sienes, Shiwl were used in the urrent study. The seeds were sterilized with 2.5 % sodium hypohlorite hving 3-4 drops of tween-2 for 2 minutes nd followed y rinsing severl times with sterile distilled wter (Qmrunnis et l., 215). The seeds were inoulted on Murshige & Skoog (MS, 1962) sl medium nd kept t 25±2 C under drk onditions for 2 dys then trnsferred in growth hmer under 16-h light/8-h drk yle. Different prts of the germinted seedlings were used s explnts for llus indution. Hypootyls nd otyledons were exised from 6-7 dys old seedlings. Wheres nodl stems (.3-.5 m) nd leves were exised from 27-3 dys old seedlings.
1872 SARWAT NAZ ET AL., Cllus indution: For llus indution, different formultions of MS medi with phytohormones were used. Eighteen formultion of llus indution medi were prepred with MS mediumugmented with vrious onentrtions (,.1,.5, 1, 1.5 nd 2 mg/l)of 2,4- dihlorophenoxy eti id (2,4-D), Indole-3-utyri id (IBA), 6-Benzylminopurine (BAP), 1-Nphthlene eti id (NAA) lone.other medi formultions inlude the omintions of BAP nd 2,4-D (Tle 1). All medi were ugmented with 3g/l surose nd solidified with 2.5g/l gelrite t ph 5.8. Explnts inluding hypootyls, otyledons, nodl stems nd leves were inoultedon different formultions of llus indution medi in 1 replitions. The ultured explnts were kept t 25±2ºC temperture under drk. Shoot regenertion nd multiplition: Different regenertion medium ws formulted y ugmenting the MS medium with vrying onentrtions of BAP (- 3mg/l) with/ without silver nitrte (5mg/l) nd lso BAP (2mg/l) with/without kinetin (-3mg/l). After 4weeks of llus indution,.7 gm of llus produed from the hypootyls ws trnsferred to shoot regenertion medium (Tle 1). The lli were pled for photoperiodi regime16-h light/8-h drk yle t 25±2 C. Eh tretment ontined 2 replites. The shoots otined from optimized shoot regenertion medi were su ultured in sme medi for shoot multiplition. Root indution of regenerted shoots: In vitro regenerted nd elongted shoot were seprted nd trnsferred to rooting medi ontining hlf strength MS medi (hormone free) or ugmented with IBA (.1,.5,1mg/l). Eh tretment ontined 1 replites. Rooting ws reorded fter 3 dys of ulture. Sttistil nlysis: Anlysis of vrine (ANOVA) ws done using SPSS version 17. The effet of eh tretment ws evluted in different explnts nd the signifint differene y vrying onentrtion ws oserved y Dunn Multiple Rnge Test (DMRT) t p<.5. Sme lphets in one explnt represent non-signifint differene t p<.5. Result Cllus indution: Different explnts i.e. hypootyl, otyledon, lef nd nodl stem were showed initil swelling of tissuesfollowed y llus formtion within week fter inoultion on llus indution medi. In hypootyl nd nodl stem, llus indution ws strted from the ut edges. However there ws no llus formtion when otyledon, lef nd nodl stem were used s explnts on hormone free medium, even fter 48 dys. Wheres, smll mount of llus ws formed from hypootyls on hormone free medium. Sttistilly different responses of llus indution were oserved with different growth regultors nd lso with different explnts t p<.5. Cllus formtion ws oserved from the hypootyls, otyledons nd from the nodl stem y the pplition of NAA (Fig. 1).Highest mount of white nd trnsprent llus (.52 g) ws formed on hypootyl, fter 48 dys. This llus lso showed some dventitious roots long with llus. Similrly, MS medium ugmented with IBA lso ws not indued llus in ny explnts exept in hypootyls nd nodl explnt (Fig. 2). Nodl stem explnt showed sustntilly lowest mount of llus only t.5 mg/l. The highest mount of trnsprent nd white llus ws oserved in hypootyl t 1mg/l of IBA (.31 g) fter 48 dys of ulture. Different onentrtions of 2, 4-D positively influenes the weight of llus in ll explnts used. Sustntilly inresed mount of llus ws oserved in ll tretments s ompre to ontrol. In hypootyl explnt, the remrkle inrese in weight of llus ws oserved t.1 mg/l of 2,4-D (Fig. 3 nd ). Higher onentrtions of 2,4-D (.5 mg/l or ove) inhiited the weight of llus in hypootyl.in lef explnt the growth of llus ws redued y inresing the onentrtion of 2,4- D from.1 (Fig. 3). Growth of llus ws lso redued in llus otined from otyledon t higher onentrtion of 2,4-D (.5 to 2mg/l); however in nodl explnt lli ws only otined t.1 mg/l 2,4-D (Fig. 3). BAP ws found to e effetive for llus indution t ll onentrtions irrespetive of the explnts (Fig. 4). Hypootyl showed the highest mount of llus t 1 mg/l BAP, wheres higher onentrtions of BAP inhiited the growth of llus in hypootyls ulture. In otyledons the highest mount of llus (8 g) ws found t 1.5 mg/l of BAP. The tretment of lef explnts with BAP resulted in highest indution of soft rown llus (.58 g) t 1mg/l onentrtion. Nodl explnts indued llus ws only found t.5mg/l onentrtion. Tle 1. Effet of different phytohormones nd AgNO3 on llus regenertion of Brssi npus L. fter 28 dys of ulture. BAP Kinetin AgNO 3 Men weight of Numer of shoots Length of shoots Numer of leves Con. Con. Con. llus (g)* Men±SE Men±SE Men±SE (mg/l) (mg/l) (mg/l) 2.5 ±.32.1 ±.6 ±.1 ±.6 5 2.7 ±1 5 ±.9.2 ±.1 5 ±.18 1 5 1.61 ±.18 ±.11.2 ±.1 ±.11 2 5 2.3 ±.12 2.7 ± 3.86 ± 4 1.35 ± 3 5 2.51 ± 2.85 ±.19.89 ± 6 1. ± 2 2.83 d ±.2.7 ±.17 1.12 ±.35 1.4 ±.33 2 1.75 d ± 5 ±.15.85 ± 2 5 ±.18 2 2.77 d ±.4.8 ± 1.52 ±.16 1.5 ± 2 2 3.91 d ±.2.55 ±.11.5 ±.1.55 ±.11 *Dt represented s men vlue ± stndrd error; different letters in the olumn re signifintly different (Dunn Multiple Rnge Test p<.5)
EFFECT OF GROWTH REGULATORS ON CANOLA 1873 WEIGHT OF NODAL STEM CALLUSING AFTER 48 DAYS WEIGHT OF NODAL STEM CALLUS AFTER 48 DAYS.35.5.3.1.1.5 1 1.5 2.3 5.15.1.5.1.5 1 1.5 2 NAA Con. (mg/l) IBA Con. (mg/l) Fig. 1. Effet of different onentrtions of NAA on weight of llus fter 48 dys of ulturing. Fig. 2. Effet of different onentrtions of IBA on llus weight fter 48 dys of ulturing. 1.8 1.6 1.4 1.2 1.8 WEIGHT OFNODAL STEM CALLUS AFTER 48 DAYS.1.5 1 1.5 2 2,4-D Con. (mg/l) Fig. 3. Effet of different onentrtions of 2,4-D on llus weight fter 48 dys fter ulturing. Fig. 3. Cllus derived from hypootyl explnt of Brssi npus L. in MS medi ontining.1 mg/l 2,4-D..8.7.5.3.1 WEIGHT OF NODAL STEM CALLUS AFTER 48 DAYS.1.5 1 1.5 2 BAP Con. (mg/l) 1.4 1.2 1.8 WEIGHT OF NODAL STEM CALLUS AFTER 48 DAYS.1.5 1 1.5 2 2,4-D + BAP Con. (mg/l) Fig. 4. Effet of different onentrtion of BAP on llus weight fter 48 dys of ulturing. Fig. 5. Effet of equl onentrtions of 2,4-D + BAP on llus weight fter 48 dys of ulturing.
1874 SARWAT NAZ ET AL., Fig. 6. Regenertion of Brssi npus L.vr. Hyol- 41 from llus: ) Shoots initition from llus on MS medi ontining ugmented with 5 mg/l AgNO3 long with 2mg/l BAP. ) Shoot elongted on llus. ) Multiple shoots rising from hypootyl derived lli on the sme medi. d) Rooting initited fter 1 dys of inoultion on hormone free hlf MS medi in In vitro regenerted shoots. e nd f) Rooted plntlets fter 3 dys of inoultion on rooting medi. The omine tretment of 2,4-D nd BAP lso indued llus ut less thn 2,4-D lone (Fig. 5). In hypootyl explnt there ws signifint inrese in llus growthwith inrese in onentrtions of omine tretment up to.5 mg/l further inrese in the mount of growth regultors redued the llus formtion. Cotyledon explnts ws not muh responsive y omined tretment. Cllus indution in lef explnts nd nodl stem ws mximum t omine tretment of 2,4-D nd BAP t.1 mg/l. Cllus indution ws redued y the inrese in onentrtions of lef explnts. Similrly in nodl stem explnt llus indution ws inhiited with inrese of onentrtions ut t 2mg/l (BAP+2,4-D) yellowish rem llus ws indued. Regenertion from lli: On shifting the lli to hormone free medium fter 4 weeks the olour of lli hnged to yellow rown nd few to green. Inresed in weight of llus ws oserved nd sustntilly few shoots roused. AgNO 3 lone in MS medi did not show ny remrkle inrese in the numer of shootlets s ompre to ontrol, however n inrese in llus weight ws oserved within 4 weeks. The effet of AgNO 3 long with BAP ppered to e more signifint thn AgNO 3 lone (Tle 1). The most responsive medium for shoot regenertion ws MS medium ugmented with 5 mg/l AgNO 3 long with 2mg/l BAP (Fig. 6) s the numer of shoots formed ws more s ompre to other omintions of BAP (Tle 1; Fig. 6 nd 6). Culturing on BAP independently ws found to inrese the length of shoots s well s numer of leves, wheres the numer of shoot nd mount of llus formed ws less s ompred to other omintions. The omintion of BAP with Kinetin in MS medium ws found less responsive s ompred to inorportion of BAP with AgNO 3 in MS medium. Regenerted shoots were shifted to hlf strength MS medi with vrious onentrtions of IBA nd hormone free medi. From the ll trils used the est rooting medi ws hlf strength (hormone free) medi. When elongted regenerted shoots were shifted to hlf strength MS rooting medi roots indued in ll regenerted plntlets fter 1-12 dys (Fig. 6d). 6-1 roots per explnt were oserved with n verge length of 7.98 m (Fig. 6e nd f). Disussion The results of present study reveled tht hypootyl ws the est explnt for llus indution in MS medium ontining.1 mg/l 2,4-D s ompred to other growth regultors like NAA, BAP, IBA or even the omintion of 2,4-D with BAP. This result supports Ali et l., (27), where the highest llus ws otined t.5mg/l of 2,4-D. Wheres Borjin nd Ark (213) reported intertion etween BAP, NAA nd 2,4-D hormones were more effetive thn 2,4-D lone in Brssi npus L. (SLM-46 vr.).
EFFECT OF GROWTH REGULATORS ON CANOLA 1875 Duey & Gupt (214) reported the est llus indution on MS medi ontining.5 mg/l 2,4-D with.5 mg/l NAA. Shrm nd Gupt (212) lso used the higher mount of 2,4-D (1.5 mg/l)s ompre to the present work for llus indution from Brssi npus L. hypootyl segments on B5 medium. Alm et l., (213) reported llus indution in Brssi npus L. hypootyl nd otyledon on medium supplemented with.5 mg/l 2,4-D,.5 mg/l BAP nd o.5 mg/l AgNO 3 nd etter llus prodution in hypootyls thn otyledons. Similrly these finding re lso not in onsistent with Muniret l., (28) who oserved est llus indution in MS medium ontining.5 mg/l IAA, 1 mg/l BAP,.5 mg/l NAA nd 1 mg/l Kinetin in Brssi npus hypootyls. Neh & Ashtutosh (214) reported 1% llus prodution in Brssi june on MS medium supplemented with BAP t 2-2.5 mg/l nd NAA.5 mg/l. Josen (1983), Nz & Khlid (214) reported tht higher effiieny of 2,4-D depends on its greter vilility in tissues. Aumultion of 2,4-D in tissue ours euse of its higher moility nd of its restrited oxidtion nd onjugtion rte s ompre to other hormones like NAA hving higher degrdtion rte nd lower moility justifying higher effiieny of 2,4-D. Cytokinins ply vitl role in regulting growth nd morphogenesis in plnts. Its min funtion is to indue shoots (Leopold, 1987). For the diret or indiret regenertion of Brssi npus L. BAP hs een widely used (Burulis et l., 29). In the present experiment the regenertive pity of Brssi npus L. into plntlets vi hypootyl derived llus ws estimted y trying nine different omintions of phytohormones in MS medi. Shoot regenertion ws oserved t ll onentrtions of BAP. The mximum numer of shoot regenertion ws notied in the medium fortified with 2mg/l BAP nd 5mg/l AgNO 3. Present results re in onformity with Burulis et l., (28) who reported mximum numer of dventitious uds from hypootyl derived llus of Brssi npus L. on MS medium supplemented with 4mg/l BAP nd.5 mg/l NAA. Hussin et l., (214) investigted shoot regenertion through hypootyl derived llus in MS medium ontining 5 mg/l BAP nd.5 mg/l IAA. Shoot regenertion ws lso reported in other speies of Brssi. Arh et l., 214 exmined suessful shoot regenertion from llus on MS medium ugmented with 2mg/l BAP in two verities of Brssi rint i.e. yellow Dodol nd Holet-1. Lone et l., 217 ws suessful in regenerting Brssi june through hypootyl derived llus on MS medium supplemented with.5 mg/l 2,4-D nd 5mg/l BAP in vriety RSPR 3 nd on MS medium supplemented with.5 mg/l 2,4-D nd 4mg/l BAP in ultivr RSPR 1.This is not in ordne with Munir et l., (28) who otined est results when llus ws ultured on MS medium ontining 1mg/l Zetin nd.1mg/l IAA in Brssi npus L. Inorportion of Adjuvnts AgNO 3 to MS regenertion medium used ws signifintly effetive to Brssi npus L. Its stimulting effet on shoot regenertion wsreported y different workers. Shrm & Gupt (212) reported mximum shoot regenertion on MS medium ontining 3mg/l Kinetin,.5 mg/l NAA nd 1mg/l AgNO 3. Ali et l., (27) demonstrted shoot regenertion from llus using NAA, 2mg/l BAP nd 5mg/l AgNO 3 in MS medium. Alm et l., (213) reported est shooting medi for Brssi npus L. ws 3mg/l BAP,.1mg/l NAA nd 5 mg/l AgNO 3. For induing roots different tril ws used nd the est result rooting medi used ws hlf MS medi (hormone free). These results re in onformity with erlier reports. Shrm & Gupt (212) oserved mximum numer of roots from medi fortified with hlf strength. These results re not in onformity with Alm et l., (213) who otined est root system in MS medi supplemented with 2.5 mg/l NAA nd 1 mg/l IBA. Hussin et l., (214) used MS medi ontining.125 mg/l IAA nd 5 mg/l IBA for rooting of In vitro regenertion shoots. Burulis et l., (28) hieved rooting of In vitro Brssinpus L. on MS medi ugmented with.1 mg/l NAA. Ali et l., (27) used hlf strength medi supplemented with.3 mg/l IBA in 3ultivrs of Brssi npus L. (str, Westr nd ylone). Chmndosti et l., (26) oserved rooting on MS medi ontining 1mg/l IBA in Cnol. In further reserh this stndrdized llus indution protool might e used for regenertion of slt tolernt Brssi npus L. vi indiret regenertion method. Conlusion On the sis of present study it my e onluded tht the effiieny of llus indution in hypootyl explnt ws est hieved in MS medium ontining.1 mg/l 2,4- D nd regenertion of plntlet through llus ws est hieved in MS medium ontining 2mg/l BAP nd 5mg/l AgNO 3. Hene y using these protools, the extensive use of high mount of growth regultors ould e redued for In vitro propgtion of elite ultivrs of Brssi npus L. Referenes Arh, G.T., F. Meki nd B. Amssu. 214. In vitro propgtion of Ethopin mustrd (Brssi rint A. Brun). Afr. J. Biotehnol., 13(4): 4438-4448. Alm, S.S., L. Khled nd M. Al-Forkn. 213. Estlishment of regenertion protool for nol (Brssi npus L.). 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