Ksetsrt J. (Nt. Sci.) 46 : 9-6 (1) In Vitro Regenertion of Africn Ym Ben (Sphenostylis stenocrp (Hochst ex. A. Rich.) Hrms y Direct Orgnogenesis Adesoye A.I. 1,*, Emese A. 1 nd Olyode O.M. ABSTRACT Studies on in vitro orgnogenesis of Africn ym en (Sphenostylis stenocrp) were crried out with the im of developing rpid regenertion system for this crop. Emryo nd lef explnts were cultured on Murshige nd Skoog (MS) contining vrying concentrtions nd comintions of 6-enzyl minopurine (BAP), kinetin nd α-nphthlene cetic cid (NAA). The mximum numer of shoots per explnt (4.) nd percentge multiple shoot induction (1%) were otined in MS supplemented with. mg.l -1 NAA nd. mg.l -1 BAP. The mximum shoot length (13 mm) ws otined on medium with 1. mg.l -1 kinetin nd.1 mg.l -1 NAA. When cotyledonry node explnts nd shoot tip explnts were cultured on medi with BAP nd kinetin singly, ech t 1., 1. nd. mg.l -1, oth explnts produced the mximum numer of shoots (4.7) nd shoot length ( mm) on. mg.l -1 BAP while the lest responses were otined on 1. mg.l -1 kinetin. There ws no orgn formtion from leves s they ll produced clli. Multiple shoots from the emryo produced roots directly on shoot induction medium while shoot tip-derived multiple shoots rooted when tested on oth. nd. mg.l -1 NAA. Shoots from cotyledonry nodes did not produce roots. Successfully rooted plntlets otined from this study is the first report of in vitro plnt regenertion in Africn ym en. This procedure for direct orgn differentition would fcilitte micropropgtion nd improvement of this species through genetic trnsformtion. Keywords: Africn ym en, orgnogenesis, in vitro regenertion, multiple shoot, Sphenostylis stenocrp. INTRODUCTION Sphenostylis stenocrp (Hochst ex. A. Rich.) Hrms, commonly known s Africn ym en (AYB), is memer of the fmily Fcee, sufmily Ppilionoidee, trie Phseolee, sutrie Phseoline nd genus Sphenostylis (Okigo, 1973; Allen nd Allen, 1981; Potter, 199). The plnt is found growing either wild or in cultivtion in much of centrl (Gon, Congo) nd western (Nigeri, Cmeroon, Togo, Ghn nd Ivory Cost) Afric. Nigeri is very significnt for AYB production where extensive cultivtion hd een reported in the estern, western nd southern prts (Okigo, 1973). AYB is hrdy protein-rich underutilized tropicl tuerous legume tht hs een proven to hve high nutritionl vlue (Azeke, 3). The seeds nd tuers re the two orgns of economic importnce, providing food for 1 Deprtment of Botny, University of Idn, Nigeri. Ntionl Centre for Genetic Resources nd Biotechnology, Idn, Nigeri. * Corresponding uthor, e-mil: desoye@yhoo.com Received dte : 1/1/11 Accepted dte : 3//1
Ksetsrt J. (Nt. Sci.) 46(4) 93 humns nd livestock in Afric. The protein in the tuer of AYB is more thn twice tht in sweet potto (Ipome tts) or Irish potto (Solnum tuerosum) nd higher thn in ym nd cssv (Amotey et l., ). Moreover, the mino cid vlues in AYB seeds re higher thn those in pigeon pe, cowpe nd mr groundnut (Uguru nd Mdukife, 1). On verge, the protein content is up to 19% in the tuer nd 9% in seed grin. Despite its high protein content, AYB is unpopulr compred to other legumes (Njoku et l., 1989) nd hs een underutilized s result of the chrcteristic prolems of eing hrd to cook, the long cycle of reproduction nd the presence of nti-nutritionl secondry metolites which re known to e found in the seed nd vegettive prts of this plnt (Asuzu nd Undie, 1986). The high incidence of seed-orne fungl pthogens hs een reported to significntly reduce seed germintion nd seedling emergence s well s the nutritionl qulities of the seeds (Nwchukwu nd Umechuru, 1991, 1997). Therefore in vitro micropropgtion for the production of clen nd disese-free plnts is essentil. Moreover the vilility of n pproprite in vitro regenertion technique is prerequisite to the use of trnsgenic technology for this species for introgression of desirle genes for trits like disese resistnce, erly flowering nd modifiction of the seed cot for incresed permeility nd fster cooking. As first step towrds septic in vitro culture, Aliyu nd Adesoye (7) estlished steriliztion conditions for tissue culture seed germintion nd cllus initition in AYB. They found.1% mercuric chloride to e the most suitle chemicl for steriliztion of AYB explnts. Aknde et l. (9) lso reported on the effect of vrious phytohormones nd explnt types on cllus induction in this species. Presently there is no informtion on in vitro orgnogenesis in this crop. Plnt regenertion in pulses, like in other plnts, cn occur through three pthwys nmely, de novo orgnogenesis, somtic emryogenesis or through prolifertion of shoot meristems from res surrounding shoot ud (Jiwl nd Singh, 3). The current study ws crried out with the im of developing rpid regenertion system for this crop. MATERIALS AND METHODS Explnt preprtion Emryos were excised from disinfected seeds of locl vriety of AYB. Disinfected seeds were germinted septiclly on Murshige nd Skoog (MS; Murshige nd Skoog, 196) sl medium to initite seedlings. Shoot tips, cotyledonry nodes nd leves were otined from seedlings fter 14 d of culture. The MS sl slt medium used in this study contined 3% sucrose, nd.8% cto-gr. The medium ph ws djusted to.7 nd utoclved t 11 C for 1 min. The plnt growth hormones were dded to the MS sl medium for emryo, lef, cotyledonry node nd shoot tip explnts s explined elow. Emryo nd lef culture The responses of emryos nd leves to growth regultors were studied y culturing on MS supplemented with 6-enzylminopurine (BAP) in comintion with α-nphthlenecetic cid (NAA) t vrious concentrtions, nd 6-furfurylminopurine (kinetin) in comintion with NAA t vrious concentrtions. Ten different tretments (Tle 1) were otined from the vrious hormonl comintions. Cotyledonry node nd shoot tip culture Cotyledonry nodes nd shoot tips were cultured in MS supplemented with BAP nd kinetin singly ech t 1., 1. nd. mg.l -1 mking totl of six tretments. Twenty emryos, immture leves, cotyledonry nodes, nd shoot tips per tretment were incuted t ± C under 16 hr photoperiod of cool-white fluorescent light (3 μ mol.m -.s -1 ). The cultures were mintined for totl of 8
94 Ksetsrt J. (Nt. Sci.) 46(4) Tle 1 Hormonl concentrtions nd comintions of five tretments (NB1 NB, where N = α-nphthlene cetic cid nd B = 6-enzylminopurine) nd five tretments (NK1 NK, where N = α-nphthlene cetic cid nd K = kinetin) used for emryo nd lef culture. Hormone (mg.l -1 ) NB1 NB NB3 NB4 NB NK1 NK NK3 NK4 NK NAA....1...1. 1. 1. BAP 4. 3. 1. 1.. - - - - - Kinetin - - - - - 1. 1...1. wk ut were sucultured t 4 wk. Dt on the percentge shoot induction, multiple shoot induction, numer of shoots per explnt nd shoot length were sujected to nlysis of vrince nd the mens were seprted where pproprite, using the lest significnt difference t the % level for significnce. RESULTS AND DISCUSSION Since legumes re notoriously reclcitrnt to regenerte from tissue culture, much effort hs een devoted to developing nd optimizing the efficiencies of in vitro regenertion. Direct in vitro orgnogenesis from explnts is rpid shoot multipliction method for elite strins of legumes nd is preferred for developing trnsgenic plnts to void somclonl vrition (Chndr nd Penti, 3). Shoot orgnogenesis The current work is elieved to e the first report of orgnogenesis in Africn ym en. Shoots were induced from emryo, shoot tip nd cotyledonry node explnts within 3 wk of culture nd complete plntlets were derived susequently (Figure 1). Intct emryo explnts produced multiple shoots under the influence of NAA nd kinetin s well s NAA nd BAP comintions. New shoots ppered to originte from the hypocotyl region of the emryo. Although ll ten tretments produced shoots, three did not produce multiple shoots (Figures nd ). The mximum numer of shoots per explnt (4.) ws otined in MS supplemented with. Figure 1 () Multiple shoots derived from Africn ym en shoot tip explnts cultured on mg.l -1 6-enzyl minopurine; () plntlets derived from shoot tip explnts of Africn ym en.
Ksetsrt J. (Nt. Sci.) 46(4) 9 % Shoot induction 1 8 6 4 LSD=17.88 % Multiiple shoots induction 1 8 6 4 LSD=19.1 c No. of shoots 4 3 1 LSD=. Shoot length (mm) 1 1 d LSD=39.64 NB1 NB NB3 NB4 NB NK1 NK NK3 NK4 NK Hormonl concentrtion (mg.l -1 ) NB1. NAA + 4. BAP NB. NAA + 3. BAP NB3. NAA + 1. BAP NB4.1 NAA + 1. BAP NB. NAA +. BAP NK1. NAA + 1. KIN NK.1 NAA + 1. KIN NK3. NAA +. KIN NK4 1. NAA +.1 KIN NK 1. NAA +. KIN Figure Influence of α-nphthlene cetic cid (NAA) + 6-enzyl minopurine (BAP), nd NAA + kinetin comintions on: () shoot induction; () multiple shoot induction; (c) shoot numer; nd (d) shoot length from Africn ym en emryo explnts. Verticl rs represent lest significnt difference (LSD) vlues.
96 Ksetsrt J. (Nt. Sci.) 46(4) mg.l -1 NAA nd. mg.l -1 BAP (NB) s well s.1 mg.l -1 NAA nd 1. mg.l -1 BAP (NB4) s shown in Figure c. However, the former, which contined hlf the hormonl concentrtions of the ltter, ws considered etter ecuse it gve the higher percentge of multiple shoot induction. The lowest shoot numer ws produced in medium contining. mg.l -1 NAA nd 1. mg.l -1 kinetin (NK1). The NAA + kinetin comintions gve higher shoot growth thn the NAA + BAP comintions (P <.) nd the optiml shoot length per explnt (13mm) ws otined in.1 mg.l -1 NAA nd 1. mg.l -1 kinetin medium (NK) s shown in Figure d. In other legumes, NAA comined with BAP hs een effective in inducing high numer of multiple shoots. For exmple in Vign rdit, NAA nd BAP yielded etween six nd seven shoots per explnt (Ro et l., ). In the sme species, Ydv et l. (1) reported tht mong the different uxins nd cytokinins tested, the presence of BAP + NAA in the shoot ud induction medium gve the est regenertion response, producing 19 shoots per explnt. Similrly, regenertion-competent explnts were induced using comintion of NAA nd BAP in other plnt species such s Citrus urntifoli, Limonium werightii nd pinepple (Moore, 1986; Tripepi, 1997; Hung et l., ; Al-Bhrny, ; Al-Sif et l., 11). In other cses, NAA nd kinetin comintions hve yielded the optiml shoot numer. According to Uddin et l. (), NAA + kinetin comintions showed the est shoot multipliction in Peltophorum pterocrpum mong different comintions of BAP, kinetin nd NAA. In the current study, shoot tip nd cotyledonry node explnts were cultured on MS supplemented with three concentrtions of BAP nd kinetin. Kinetin induced single shoots in oth explnt types wheres BAP induced multiple shoots (Figures 3 nd 4). The numer of shoots nd the percentge shoot induction incresed with the BAP concentrtion. BAP is % Shoot induction No. of shoots Shoot length (mm) 1 8 6 4 8 6 4 % Multiple shoots induction1 4 3 1 1 1 8 6 4 c d LSD=1.66 LSD=9.9 LSD=.1 LSD=3.1 Figure 3 Influence of three concentrtions ech of 6-enzyl minopurine (BAP) nd kinetin on: () shoot induction; () multiple shoot induction; (c) shoot numer; nd (d) shoot length from Africn ym en cotyledonry node explnts. Verticl rs represent lest significnt difference (LSD) vlues. BAP 1 1. KINETIN Hormonl concentrtion (mg.l -1 )
Ksetsrt J. (Nt. Sci.) 46(4) 97 % Shoot induction 1 8 6 4 BAP KINETIN LSD=9.9 % Multiple shoots induction 1 8 6 4 LSD=9.9 c LSD=.8 4 No. of shoots 3 1 3 d LSD=4.48 Shoot length (mm) 1 1 1 1. Hormonl concentrtion (mg.l -1 ) Figure 4 Influence of three concentrtions ech of 6-enzyl minopurine (BAP) nd kinetin on: () shoot induction; () multiple shoot induction; (c) shoot numer; nd (d) shoot length from Africn ym en shoot tip explnts. Verticl rs represent lest significnt difference (LSD) vlues.
98 Ksetsrt J. (Nt. Sci.) 46(4) very importnt cytokinin most widely used for shoot ud initition, shoot development nd elongtion. In cses where BAP comines with other cytokinins to induce shoot regenertion, it is found to e the most essentil component, s its sence inhiits shoot ud development s ws found in Phseolus ngulris (Mohmed et l., 6). BAP-supplemented medi produced significntly higher numer of shoots thn those from kinetin medi (P <.) in oth explnts. Geeth et l. (1998) reported similrly higher shoot induction nd numers for BAP thn kinetin in pigeon pe. However they noted tht comintion of BAP with NAA gve higher shoot numer nd elongtion thn BAP lone. The mximum numer of shoots for cotyledonry nodes (4.) nd for shoot tip explnts (4.7) were otined in medi contining. mg.l -1 BAP (Figures 3 nd 4). Optiml shoot production nd multipliction hve een reported using. mg.l -1 BAP for severl explnts in vrious species. For exmple hypocotyl nd epicotyl explnts of Vign suterrne gve 3.7 shoots per explnt (Mongomke et l., 9). Other concentrtions of BAP hve lso een optiml in other plnt species. Rekh nd Thiruvengdm (9) reported tht 1. mg.l -1 BAP for cotyledonry nodes nd 1. mg.l -1 BAP for xillry uds gve the optiml shoot numer, nd of the two different explnts tested, cotyledonry nodes produced the greter numer of shoots. In ddition, Uddin et l. () reported tht shoot tip explnts of Peltophorum pterocrpum gve mximum of 3.61 shoots per explnt in MS supplemented with 4. mg.l -1 BAP. In the current work, shoot growth ws significntly higher in BAP thn kinetin medi t ll concentrtions tested in oth explnts. The mximum shoot length ws mm nd 1 mm in shoot tip nd cotyledonry node cultures, respectively. Root orgnogenesis Rooting of dventitious in vitro shoots is normlly induced on medi contining uxin. These hormones ffect the in vitro rooting of vrious species differently (Al-Bhrny, ). Emryo explnts were le to root directly on shoot regenertion medi. Figure shows tht. mg.l -1 NAA nd. mg.l -1 BAP gve the est response with 1 roots nd 1% root induction. Emryo explnts were oserved to root within 1 d fter shoot induction while still on the shoot induction medium. Microshoots from shoot tip explnts were excised from the sl end nd tested on two concentrtions of NAA. Both produced roots, however the numer of roots induced in. mg.l -1 ws significntly higher thn in. mg.l -1. The initil concentrtion of shoot regenertion medium did not influence the numer of roots nd root length induced. However the percentge root induction from shoots otined on kinetin ws significntly lower thn tht otined on BAP (Figure 6). The progress from shoot induction to rooting nd plntlet formtion occurred within 3 d. The entire process of in vitro plntlet production, prticulrly from the shoot tip explnts, could e possile in n verge of 4 d. The verge percentge root induction of AYB mounted to 81.67%. Micro-shoots from cotyledonry node explnts did not respond to the rnge of NAA tested. Cllus induction from lef explnts Lef explnts were only le to induce cllus formtion on the sme hormonl tretments used for emryo culture. Aknde et l. (9) hd similrly reported cllus initition from lef, stem nd root explnts on medi with vrious comintions of BAP, NAA, indole cetic cid nd kinetin. The est medium for cllus growth ws 1. mg.l -1 ech of NAA nd kinetin. Clli otined in this study with different comintions of NAA, BAP nd kinetin were fresh, greenish nd frile (dt not shown). Further hormonl,
Ksetsrt J. (Nt. Sci.) 46(4) 99 sl medi nd gel-type mnipultions nd vitmin supplementtion could result in indirect orgnogenesis or yield somtic emryos which might e excellent trgets for gene trnsformtion in AYB. CONCLUSION The protocol developed from the current investigtion provides good strting point for micropropgtion nd genetic trnsformtion of AYB explnts s hd previously een estlished for other lrge-seeded legumes like Cjnus cjn (Dyl et l., 3; Thu et l., 3), Cicer rietinum (Srmh et l., 4), Vign mungo (Sini et l., 3) Pisum stivum (Pniewski nd Kpust, ) nd Vign unguicult (Popelk et l., 6; Solleti et l., 8). 1 LSD=.33 % Root induction 8 6 4 LSD=.33 No. of roots 1 1 NB1 NB NB3 NB4 NB NK1 NK NK3 NK4 NK Hormonl concentrtion (mgl -1 ) NB1. NAA + 4. BAP NB. NAA + 3. BAP NB3. NAA + 1. BAP NB4.1 NAA + 1. BAP NB. NAA +. BAP NK1. NAA + 1. KIN NK.1 NAA + 1. KIN NK3. NAA +. KIN NK4 1. NAA +.1 KIN NK 1. NAA +. KIN Figure Influence of α-nphthlene cetic cid (NAA) + 6-enzyl minopurine (BAP), nd NAA + kinetin comintions on: () root induction; nd () numer of roots from Africn ym en emryo explnts. Verticl rs represent lest significnt difference (LSD) vlues.
6 Ksetsrt J. (Nt. Sci.) 46(4) 1 8 LSD=1.13 % Root induction 6 4 LSD=.44 No. of roots 1. 1. c LSD=4.9 Root length (mm) 1 1 1.BAP. BAP 1. KN. mg.l -1 NAA. mg.l -1 NAA Hormonl concentrtion (mg.l -1 ) Figure 6 Influence of two α-nphthlene cetic cid (NAA) concentrtions long with 6-enzyl minopurine (BAP) nd kinetin on: () root induction; () numer of roots; nd (c) root length of Africn ym en in vitro shoots. Verticl rs represent lest significnt difference (LSD) vlues.
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