Afrin Journl of Biotehnology Vol. 1(46), pp. 9396-941, 22 August, 211 Aville online t http://www.demijournls.org/ajb DOI: 1.5897/AJB1.2115 ISSN 1684 5315 211 Ademi Journls Full Length Reserh Pper Effet of 2,4-D, explnts type nd ultivr on the llogenesis expression of ssv (Mnihot esulent Crntz) in Ghn Eugene K. A. Flether, Theres N. E. Amoko nd Peter Twumsi* Deprtment of Biohemistry nd Biotehnology, Kwme Nkrumh University of Siene nd Tehnology, Kumsi, Ghn. Aepted 22 June, 211 Modern iotehnologil pplitions in reeding progrmmes for eonomilly-importnt rops like ssv egins with suessful estlishment of stle nd tive llus ultures for somti emryos whih n e genetilly mnipulted nd indued into plnting mterils. We hve investigted the effets of explnts (leves, stem petiole nd xillry uds), uxin (2, 4-D) onentrtions (8, 12 nd 15 mg/l) nd ultivr in the indution of llus ultures from lol ssv vrieties in Ghn - Tuk, Afisifi, Afenkye nd Doku. Cllus formtion ws suessfully indued in ll explnts (lef, petiole nd uxiliry uds) from the four ssv ultivrs. Generlly, there were no signifint differenes in llogenesis of the different explnts t the three different 2,4-D onentrtions (p <.5) produing 75 for explnts. The stem petiole or stem rk produed the lest perentge of llus per ultivr t ll the uxin onentrtions. However, the lef explnts were superior in llus formtion in ll the ssv vrieties. Although, sttistilly insignifint, the 8 mg/l 2,4-D onentrtion ws visully the est in llus formtion from explnts oth within nd ross the ssv ultivrs. This study hd shown tht different ssv explnts respond differently to tissue ulture onditions estlished for llus ulture formtion. Key words: Auxin, llogenesis, llus indution medi, ssv Mnihot esulent Crntz, 2,4- dihlorophenoxyeti id (2,4-D), somti emryogenesis. INTRODUCTION Among ll the tuer rops grown in Ghn nd other prts of Afri, ssv (Mnihot esulent) is the most lrgely grown nd therefore, serves s the mjor tuer food rop for humn onsumption nd lso s niml feed (Ky, 1973). In su-shrn Afri, ssv is onsidered s life-lood euse the strhy tuer is onsumed s stple nd lso supports the strh, textile, pint nd phrmeutil industries (Srinivs, 27). Thus, nything tht ffets the prodution of the ssv rop my hve serious onsequenes in food vilility nd the eonomy of the people in suh growing res. It is therefore of no surprise tht every yer millions of dollrs re lloted y governments in *Corresponding uthor. E-mil: twumsipeter@gmil.om Tel: +233-245-1318 these growing res to omt pests nd diseses whih ffet the yields of the rop. Some hllenges of ultivtion inlude low nutritive vlue of ssv (1.2 to 1.8% rude protein,.1 to.8% rude lipid, 1.5 to 3.5% rude fire nd 1.3 to 2.8% sh) (Alert et l., 25) nd osionlly umultions of toxi ynogeni gluosides (White et l., 1998). This therefore, neessittes the doption of reeding progrmmes to introdue new vrieties of higher nutritionl qulity. In Ghn, nd to dte, trditionl reeding methods whih involve sexul hyridiztions etween lol nd exoti vrieties of desired trits remin the only option in generting new vrieties. Unfortuntely, suh methods re ineffetive in eliminting ynogeni ompounds, diseses nd pests nd lso, they re time-onsuming (Smith nd Drew, 199). As lterntives, modern methods of the ell nd tissue ultures llow the overoming of suh rriers. These methods llow prodution of genetilly improved
Flether et l. 9397 vrieties y trnsfer of trits of interest (Shopke et l., 1996; Ye et l., 2). The use of ssv ell nd tissue ulture tehniques inluding mirouttings, somti emryogenesis, ndrogenesis (nthers ulture), gynogenesis (ovry ulture), mong others hs gret potentil in generting disese-free plnting mterils for ultivtion (Smith nd Drew, 199). A stle nd tive ell or tissue ulture is n essentil step for ny suessful plnt reeding progrmme (Guo nd Zhng, 25). Beuse different plnt speies nd different explnts of some speies respond differently to ulture tretments (Preek nd Kothr, 23), speifi sets of onditions re neessry for eh explnts per plnt. Although, mny lortories hve generted ssv plntlets from somti emryogenesis using different vrieties of ssv (Stmp nd Henshw, 1982; Smith nd Drew, 199), s fr s we know, there hs een no suh work on ssv vrieties in Ghn. Even in the se of those tht hve een exploited elsewhere, not muh informtion is ville on the vritions different ssv explnts undergoing llogenesis. Cllus is n importnt mteril for estlishing emryogeni ulture nd suessful prodution of it from Ghnin ssv would offer n opportunity for ultivr genertion nd germplsm storge. This study imed to test the effet of ftors 2,4-D onentrtion, explnts type nd ultivr on the llogenesis expression of ssv. MATERIALS AND METHODS Study site, explnts preprtion nd ulture onditions Four seleted ssv ultivrs ommonly grown in Ghn, nmely Doku, Afisifi, Afenkye nd Tuk, were otined from the frms of the Crop Reserh Institute (CRI), Fumesu in the Ashnti Region. Fresh stem uttings of 2 to 3 m with 5 to 8 nodes were prepred from mture plnts. The uttings were plnted in the lomy soil of the Kwme Nkrumh University of Siene nd Tehnology (KNUST) experimentl fields in Novemer 28 (verge dy temperture of 3 C; mient verge night temperture of 24 C, verge dy light of 12 h nd reltive humidity of 55%) nd repeted in Jnury 29 (verge dy temperture of 27 C; verge night temperture of 22 C, verge dy light of 12 h nd reltive humidity of 55%). The plnts were wtered dily with sprinkler. Six-week old shoots were severed from the plnts nd trnsported in vse of wter to the lortory. The plnts were surfe-sterilized with 2% household leh (L Croix) ontining 3.5% sodium hypohlorite nd.15% Triton-X1 for 2 min (lef explnts) or 3 min (stem petiole explnts) nd rinsed t lest three times with sterile wter. All stges of steriliztion were done in the lminr flow hood. Sterile leves nd stem petioles were ut into diss nd piees of out 1 nd 5 mm, respetively. 5 mm of pil nd uxiliry were lso prepred. The ontrol ultures onsisted of ll expllnts ultured in medi without phytohormonl supplementtion. Cllus ulture The ssv explnts were ultured on Murshige nd Skoog (MS) medium modified y ddition of 2% surose,.5 mg/l CuSO 4 nd.6% gr (gelling gent) s indited y Atehnkeng et l. (25). The ph of the medium ws djusted to 5.7 nd medi ontining one of the uxin onentrtions (8, 12 nd15 mg/l) were prepred then utolved t 121 C for 15 min. The medi were st in sterile glss ulture pltes nd solidified in sterile lminr flow hood. The prepred explnts (lef diss, stem petiole nd uds) were septilly trnsferred to the MS medi pltes nd seled with prfilm. The ultures were done in triplites nd inuted in the drk t 25 C nd monitored dily over 21-dy period to oserve llus formtion. Mesurement of llusing explnts nd initition of somti emryos At the end of eh ulture yle of 21 dys, the llogeni explnts numer nd initited somti emryo numer ws sored. Prior, the length of shoots grown in university KNUST soil ws mesured in ex-vitro onditions. Experimentl design A 3 3 4 ftoril omintion in ompletely rndomized design with three replitions ws uilt. In ll, 18 tretments were otined t the end of eh ulture. A tretment ws onstituted of the omintion of 2,4-D onentrtion, explnt type nd ultivr. Sttistil nlysis The olleted dtset were proessed y Prism v.4 (GrphPd Softwre, In.). The effet of three ftors, 2,4-D onentrtion, explnt type nd ultivr, ws tested through the ANOVA. The verges were seprted ording to Newmn-Keuls, Dunnett, Dunn or Bonferoni test t 5% signifine level. Prior, the normlity of mesured distriutions nd vrines equlity of nlyzed smples were verified. RESULTS Effet of ultivr on the expression growth in length of shoots in ex-vitro onditions Two sttistilly different groups of ultivrs were identified. First, omposed of Afisifi (.714 m/dy) nd Tuk (.748 m/dy) ultivrs, ws hrterized y high growth in length of shoots (Figure 1). Seond, omprising Doku (.547 m/dy) nd Afenkye (.645 m/dy), ultivr differed from three previous ultivrs y low growth in length of shoots. The reorded verges rnged from.547 m/dy (Duku) to.748 m/dy (Tuk). Effet of 2,4-D on the llogenesis expression Clli were suessfully produed from explnts from eh of the four ssv ultivrs ommonly grown in Ghn (Figure 2). On the verge, the formtion of llus from explnts of the ssv vrieties used ourred 7 dys
9398 Afr. J. Biotehnol. 2 Cultivr Afisifi Afisifi Cultivr Tuk Height (m) height (m) 1 1 14 18 22 26 33 Time time (dys) (dys) Figure 1. Growth rtes (stem inrese in m/dy) of ssv ultivrs grown in the first 35 dys fter sprouting. The grph shows growth rtes for ssv ultivrs Afisifi (filled irles) nd Tuk (filled squres). Eh represents n verge stem height. Vertil trits represent the rs of stndrd errors of men. A B Figure 2. Cllogeni explnts deriving from stem (A) nd ud (B) formed onto MS medium supplemented with 8 mg/l 2,4-D. The ud nd stem llus were formed 14 nd 21 dys, respetively fter phytohormone tretment. Br =.5 mm. fter trnsfer to ulture medium. The llusing explnts ppered swollen, yellowish-green to rown in olor nd showed initil growth round the exised portions efore enlrging into llus mss. Cllusing of lef explnts egn with urling of the tissue followed y swelling t the ut edges, while in the se of petioles nd uds, the llus ws initited t their terminls. Brk explnts from ultivr Afisifi ultured in the medium with 12 mg/l 2,4-D onentrtion gve the highest perentge of per explnts (Figure 2). The stem petiole derived-derived llus, unlike the lef nd ud-llus, were hrd llus. Different perentges of llus formtion, rnging from to 75% were oserved for different explnts (leves, rk nd uds) t the three different uxin onentrtion used (8, 12 nd 15 mg/l 2,4-D). The ultivr Tuk did not show ny signifint differene etween the perentge llus produed from the explnts (lef, stem petiole nd uds) t ll the three 2,4-D onentrtions
Flether et l. 9399 4 3 2 1 A 1 5 C leves ud stem petiole 8 mg/l 12 mg/l 15 mg/l Conentrtion of 2,4-D 8 mg/l 12mg/l 15 mg/l Conentrtion of 2,4-D 1 5 B 1 75 5 25 D 8 mg/l 12 mg/l 15 mg/l Conentrtion of 2,4-D 8 mg/l 12 mg/l 15 mg/l Conentrtion of 2,4-D Figure 3. The perentge of llogeni explnts of four ssv ultivrs. A. Cultivr Tuk; B, ultivr Afisifi; C, ultivr Afenkye; ultivr Doku. (N = 36; r = SEM; different letters indite signifint differenes). used (Figure 3). Generlly, the lef explnts were most suessful in forming llus nd the rk explnts were the lest. However, the results from Afisifi ultivr show signifint inrese in the men perentge of llus formed y the lef explnts when ompred with petiole nd uds in the 8 mg/l 2,4-D-ontining medium (Figure 3). Effets resulting from the 12 nd 15 mg/l 2,4-D were not signifintly different mong the ultivrs. In ultivr Afenkye nd ultivr Doku, the lef, petiole nd uds produed the sme mounts of llus upon tretment with the vrious 2,4-D onentrtions (Figure 3 nd 3d). Compring the effiieny in forming llus, the lef explnt, irrespetive of the ultivr gve the highest perentge of llus in the tissue ulture (Figure 3). Effet of uxin onentrtion on llus indution The three 2,4-D onentrtes pplied did not signifintly ffet indution of llus from the explnts otined from Tuk ultivr (Figure 4). In the Afisifi ultivr, however, the 8 mg/l 2,4-D onentrtion signifintly produed higher llus from lef explnts thn the other onentrtions (Figure 4). There ws however, no signifint differene (p <.5) etween the three 2,4-D levels for the other explnts for the Afisifi ultivr. Results for Afenye nd Doku ultivr followed similr trend (Figure 4 nd 4d). Bud explnt in medi ontining 12 nd 15 mg/l 2,4-D filed to yield llus. DISCUSSION The responses to llogenesis nd somti emryogenesis of four ultivrs grown in Ghn were investigted. Mny works hve een rried out on the estlishment of llus ulture from ssv in different lortories (Fietos et l., 27; Atehnkeng et l., 25, Shopke et l., 1996). But this is the first time one is done on Ghnin ssv vrieties. Our works rried out on four ultivrs grown in Ghn displyed tht t 8 mg/l onentrtion of uxin in ulture medium, explnts from Afisifi ultivr generted signifintly underwent llogenesis when ompred with explnts from the other three ssv vrieties used. Although, ssv hs genertion period of nerly six months, mterils for the tissue ulture work were produed from 2 to 4 week old plnts (Figure 1). Thus, unlike the lssil plnt reeding whih requires pollintion nd seed development, genertion time for
94 Afr. J. Biotehnol. 4 3 2 1 A 1 5 B 12 mg/l 15 mg/l 8 mg/l Lef lef Brk rk ud Bud Cssv explnts lef Lef rk Brk ud Bud Cssv explnt 1 5 C d d 1 75 5 25 D lef rk ud Cssv explnt lef rk ud Cssv explnt Lef Brk Bud Lef Brk Bud Figure 4. The effet of 2,4-D onentrtions on perentge of llogeni explnts (llus) indued from ssv explnts. The perentge of llus formed from ultivr Tuk (A); ultivr Afisifi (B), ultivr Afenkye (C.) nd ultivr Doku (D) y 8 mg/l (filled rs), 12 mg/l (rossed rs) nd 15 mg/l (open rs) re shown in the grph. (N = 36; r = SEM; different letters indite signifint differenes). new vrieties y emryogeni ulture vi llus ulture n e signifintly redued. This lso llows preise geneti modifition to produe progeny of desirle phenotype. We hve shown in this study tht lthough, the ssv explnts used (tht is, leves, stem petiole nd uds) n produe llus; there is signifint differene in the perentge of llus formed per explnts (Figure 3). This rod use of ssv explnts for estlishing tissue ulture with miniml onstitution of medi provides gret opportunity for inorporting the rop in tissue ulture for reeding of vrieties of desired gronomi importne. This will lso provide lterntive mterils for storing germplsm. Cllus is n undifferentited ell mss produed from differentited tissues nd thus, importnt mteril for either diretly regenerting plnts or vegettive emryogenesis suspension ulture. Every differentited plnt tissue is totipotent, ut the onditions to de-differentite them vry from speies to speies nd even from tissue to tissue within sme plnt (Ezhov, 23). Among the three ssv explnts used in the experiment, the leves were the most totipotent with the highest perentge (75 t 8 mg/ml) of llus formed in ultures. The lef prenhym ells, espeilly mesophylls, re esily reprogrmmed through dedifferentition into undifferentited ells with hrteristis similr to meristemti tissues (Twumsi et l., 2). With the exeption of lef epidermis, lef ells hve thin ell wlls whih re less lignified (Shädel et l., 21). These ells re reltively less relitrnt to differentition in ulture medium due to their high responsiveness to phytohormones (Sn-José et l., 21). One of the four ssv ultivrs used in this study, Afisifi, ws most llus-indutive t ll the three 2,4-D onentrtions nd explnts used (Figure 3) nd therefore, inditing high totipoteny of the ultivr. Suh
Flether et l. 941 vritions mong explnts tissues of some plnt speies hve een well doumented. Irrespetive of the ulture medi nd ultivr, lef tissues gve the est yield of llusing. Leves re known to e most totipotent, ple of eoming de-differentited tissues (Dhr nd Joshi, 25). In Zinni ell ultures, perfet model for ell wll synthesis nd progrmmed ell deth in plnts, mesophyll ells of young leves re known to e highly totipotent. These ells esily eome de-differentited into undifferentited ells like mersitemti ell mss whih re lter differentited into trhery elements (Twumsi et l., 29). Buds re mostly mde of meristemti ells whih re undifferentited. Cssv uds s ws oserved mong the four ultivrs used in this study, showed the lowest perentge of llus in the medi used. It is onluded from this study tht ssv lef explnts re 24.7 nd 47.2% higher in the formtion of llus when ompred with the ud nd petiole explnts. This trend ws similr mong ultivrs Afisifi, Afenkye nd Doku tested. It ws lso relized tht 2,4-D onentrtions used hve no signifint differenes in the indution of llus using explnts from Tuk, Afenkye nd Doku ssv ultivrs. However, the 8 mg/l 2,4-D onentrtion produed the highest numer of lli per explnts from Afisifi ultivr. REFERENCES Alert CL, Klnrong S, Tzou-hi H (25). Proximte omposition, minerl ontents, hydrogen ynide nd phyti id of 5 ssv genotypes. Food Chem. 92: 615-62. Atehnkeng J, Adetimirin VO, Ng SYC (25). Exploring the Afrin ssv (Mnihot esulent Crntz) germplsm for somti emryogeni ompetene. Afr. J. Biotehnol. 5: 1324-1329. Fietos T, Bstos JLP, Ponte LFA, Juá TL, Cmpos F (27). Somti Emryogenesis in Cssv Genotypes from the Northest of Brz. Arh. Biol. Tehnol. 5: 21-26. Dhr U, Joshi M (25). Effiient plnt regenertion protool through llus for Sussure ovllt (DC.) Edgew. (Asteree): effet of explnt type, ge nd plnt growth regultors. Plnt Cell Rep., 24: 195-2. Ezhov TA (23). Geneti ontrol of totipoteny of plnt ells in vitro. Ontogenez, 34: 245-52. Guo Y, Zhng Z (25). Estlishment nd plnt regenertion of somti emryogeni ell suspension ultures of the Zingier offiinle Ros. Sienti Horti. 17: 9-96. Ky DE (1973). Root rops. Tropil Produts Institute, London. p. 245. Preek A, Kothr SL (23). Diret somti emryogenesis nd plnt regenertion from lef ultures of ornmentl speies of Dinthus. Sienti Horti. 98: 449-459. Sn-José MC, Corredoir E, Mrtínez MT, Vidl N, Vlldres S, Mllón R, Vieitez AM (21). Shoot pex explnts for indution of somti emryogenesis in mture Querus rour L. trees. Plnt Cell Rep. 29(6): 661-671. Shädel C, Blöhl A, Rihter A, Hoh G. (21). Quntifition nd monoshride omposition of hemielluloses from different plnt funtionl types. Plnt Physiol. Biohem. 48(1):1-8. Shopke C, Tylor N, Crmo R, Konn NDK, Mrmey P, Henshw GG, Behy RN, Fuquet C (1996). Regenertion of trnsgeni ssv plnts (Mnihot esulent Crntz) from miroomrded emryogeni suspension ultures. Nt. Biotehnol. 14: 731-735. Smith MK, Drew RA (199). Current Applitions of Tissue Culture in Plnt Propgtion nd Improvement Aust. J. Plnt Physiol. 17(3): 267-289. Srinivs T (27). Industril demnd for ssv strh in Indi. Strh/Stärke 59: 477-481. Stmp JA, Henshw GG (1982). Somti emryogenesis in ssv. Plzenphysiology, 15: 183-187. Twumsi P, Shel JH, vn Ieperen W, Woltering E, vn Kooten O, Emons AM (29). Estlishing in vitro Zinni elegns ell suspension ulture with high trhery element differentition. Cell Biol. Int. 33: 524-533. Vines, R (21). Plnt Biotehnology. Biotehnology Informtion, Virgini Teh. p. 443. White WLB, Aris-Grzon DI, MMhon JM, Syre RT (1998). Cynogenesis in Cssv - The Role of Hydroxynitrile Lyse in Root Cynide Prodution. Plnt Physiol. 116: 1219-1225. Ye X, Al-Bili S, Kloti A, Zhng J, Lu P, Beyer P, Potrykus I (2). Engineering the Provitmin A (et-crotene) Biosyntheti Pthwy into (Crotenoid-Free) Rie Endosperm. Si. 287: 33-35.